SB 601 .H3 Copy 1 DAMI'LNG-OFF IN FOREST NURSERIES A THESIS ACCEPTED IN PARTIAL SATISIW TION "I' THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY AT THE UNIVERSITY OF CALIFORNIA BY CARL HARTLEY December, 1919 << UNITED STATES DEPARTMENT OF AGRICULTURE BULLETIN No. 934 Contribution from the Bureau of Plant Industry WM. A. TAYLOR. Chief Washington, D. C. PROFESSIONAL PAPER June 16, 1921 DAMPING-OFF IN FOREST NURSERIES By CARL HARTLEY, formerly Pathologist, Office of Investigations in Forest Pathology CONTENTS Page Damping-Off in General 1 Damping-Off of Conifers 7 Causal Fungi 27 Relative Importance of the Damping-Off Fungi on Conifers 65 Damping-Off Fungi as Causes of Root- Rot and Late Damping-Off 70 Relation of Environmental Factors to Damping-Off 73 Page Density of Sowing 74 Moisture and Temperature Factors ... 75 Chemical Factors 79 Biologic Factors 82 Acknowledgments 86 Summary 86 Literature Cited 91 WASHINGTON GOVERNMENT PRINTING OFFICE 1921 ■ » "» W in m i ' "" tmmm^mt^mtttmi^Hmtt LIBRARY OF CQNQKtSS ilVIO SEP8"1Mlf pocumemt;-. UNITED STATES DEPARTMENT OF AGRICULTURE fe BULLETIN No. 934 $fjy| s^jtrs&ju Contribution from the Bureau of Plant Industry \VM. A. TAYLOR, Chief Washington, D. C. PROFESSIONAL PAPER June 16, 1921 DAMPING-OFF IN FOREST NURSERIES. By Cabl II\i:tm'. . formerly Pathologist, Office of Investigations in Forest Pathology. CONTENTS. Page. Damping-off in general 1 Damping-off of conifers 7 Causal fungi 27 Corticium vagum -7 Fusarium spp , 34 Pythium debaryanum 35 Rheospora ngi am aphani der- ma tus 55 Phytophthora spp 59 Miscellaneous phycomj c< tes >il Other fungi -.-, 64 Relative importance of the damping- off fvinsi on conifers 65 Page. Damping-off fungi as causes of root- rot and late damping-off 70 Relation of environmental factors to damping-off 7-". Density of sowing 74 Moisture and temperature factors-- 7.~> Chemical factors-. 70 Biologic factors 82 Acknowledgments S6 Summary ' S6 Literature cited 91 DAMPING-OFF IN GENERAL. Damping-off is the commonest English name for a symptomatic .rmip of diseases affecting great numbers of plant species of widely separated phylogenetic groups. It is commonly used for any disease which results in the rapid decay of young succulent seedlings or soft cuttings. Young shoots from underground rootstocks may also be damped-off before they break through the soil (Go). 1 The same term is even used for diseases affecting the prothallia of vascular crypto- gams (2). The name apparently originated in the fact that the dis- ease is usually m<»t prevalent under excessively moist conditions. In those cases in which the disease becomes serious without the pres- ence of unusual amounts of moisture the term is a misnomer. It is, however, so thoroughly established in practical use that it would be impossible, even if desirable, to establish any other name. 1 The serial numbers in parentheses refer to " Literature cited," at the end of this bulletin. 19651°— Bull. 934—2] 1 2 BULLETIN 934, CJ. S. DEPARTMENT OF AGRICULTURE. While the parasites reported as causing damping-off are probably not as numerous as the host species which are subject to it. a con- siderable number are known. Two quite different types of damping- off parasites may be recognized. In the first type we have fungi, such as Pythium debaryanum Hesse and Corticium vagum T>. and ('.. soil inhabiting and primarily saprophytic, which attack a great variety of hosts, and are at least better known, if not more destruc- tive, as damping-off organisms than as parasites on older plants. They are specialized as to the type and age of tissues which they at- tack rather than as to host. The second type includes fungi Less common as saprophytes and with a relatively limited, sometimes very closely limited, host range. Phoma betae, the systemic parasite of sugar beet (37). is an excellent example of the host-specialized para- site, transmitted in the seed and capable of seriously injuring various parts of the older plant at different stages of growth as well as at- tacking seedlings. Most damping-off parasites are intermediate in habit between the extremes of these two types. Of those which are somewhat host specialized, the following may be mentioned: Phomopsis reran*, the cause of foot-rot of eggplant, reported by Sherbakoff (128) as a frequent cause of damping-off of this host ami believed to ho carried on seed. Cribberclla sa-ubinetii (Mont.) Sacc. (29) and the imperfect fungi which kill grain seedlings as well as cause diseases of the older plants (80; 126, p. IMS). Species of Gloeosporium and Volutella named by Atkinson I 2 p. 269; 52) as able to kill seedlings or cuttings of particular host plants. Glomerella (Colletotrichum) gossypii, described by Atkinson (1) and Barre (4) as likely to cause damping-off of cotton (112). Fusarium lint, the Hax parasite, reported by Bolley (14) as destructive .to young seedlings. Phoma lingam, the cause of black-leg of cabbage, at least under inoculation conditions able to kill quickly seedlings of cabbage and other crucifers (72). Peranospora parasitica (Pers.) De Bary, a downy mildew attacking cabbage and various other crucifers. reported as killing thousands of very young cabbage plants in Florida seed heds (41). The entomophthoraceous Completoria complens, on fern prothallia i 1 ; 87, p. 203). Bacillus malvacearum, a parasite of the leaves of cotton plants, which can also cause damping-off of its favorite host (113) and the bacteria from diseased cucumber plants with which Halsted (53) caused typical damping-off of cucumbers. Damping-off fungi with wider host ranges include PhytopMTiora fagi, Aphanomyces levis (100), Rheosporangiwm aphanidermatus (38, 39), Botrytis cinerea, and certain Fusaria. The so-called prop- agation fitngiis. " vermehrungspilz," a sterile damping-off mycelium which Sorauer (133, p. 321) believed related to Sclerotinia and for which Ruhland (115) has erected a new genus, considered by both i ' l 3 DAMPING-OFF IH FOREST NUESERIES. 6 authors the most serious enemy encountered in growing softwood cuttings in Germany, if distinct would be a further addition to these generalized parasites. However, it is now believed (H4) to be identi- cal with Cart /(in in vagum. Common generalized parasites of older plants, such as Sclerotima libertiana, Sclerotium rolfsii (129), and Thielavia basicola (47), capable of attacking roots or other pails of older plants of numerous species, may also be considered among the damping-off fungi when they cause the death of small seedlings, as occurs, for example, in attacks by Sclerotinia libertiana on lettuce (20, p. 28) and celery (Id-"., p. 536) in seed beds. Further study will probably result in multiplying almost indefinitely the number of more or less important damping-off parasites, both of the specialized and uftspecialized groups, although the most important of the latter type are probably already known. Most of the references in literature to damping-off describe its occurrence in truck crops and the losses caused in these crops. Ac- cording to Halsted (53, p. 342), weed seedlings are also very com- monly attacked. Duggar (33) names lettuce, celery, cotton, sugar beet, cress, cucumber, and sunflower as especially susceptible to injury by the two most important damping-off organisms. Except for the plant species in which damping-off by seed-carried parasites is common, it appears that the economic damage from damping-off is serious only with plants whose culture involves the raising of the seedlings in crowded seed beds for subsequent transplanting. For example, tomatoes do not ordinarily suffer from damping-off in the field (TO), but the growing of seedlings in flats for subsequent trans- planting is sometimes seriously hampered as a result of the preva- lence of damping-off. This same principle holds in general for trees. Broad-leaved trees, which are usually not as crowded in the seedling stage as are the conifers, seldom give rise to complaint on the score of damping-off. The conifers, subject to serious losses in nursery beds, are not believed to be greatly affected in this country by the better known types of damping-off under forest conditions (68) except in the less common cases in which seedlings come up in close groups from squirrel hoards, artificial seed spots, or similar sources. A considerable number of broad-leaved trees have been reported at one time or another as injured by damping-off, though complaints of commercially serious losses are not common. The cases which have come to the writer's attention are listed below: Cause not determined : Orange (43, 108). Olive, in greenhouse at the University of California. Russian wild olive (Elaeagnus sp. ), serious at an Iowa nursery; oral re- port by Mr. C. R. Bechtle, formerly of the United Slates Foresl Service; at another nursery in the same region this plant was reported as very little subject to injury. ,.■ .. o, , I 4 BULLETIN 934, U. S. DEPARTMENT or AGRICULTURE. Cause not determined — Continued. Magnolia (31), troublesome if the pulp is not washed off the seed before planting. Eucalyptus spp. (88, i». 45; L31), serious under moist conditions. Bet uia spp. Communication by Dr. Perley Spaulding, of the Bureau of riant Industry; found especially susceptible in a Pennsylvania nursery. Carob, at United States Plant Introduction Garden, Chico; Calif. I>r. .Mel T. Cook states that damping-off is more serious in carob seedlings if the seed is removed from the pod than if pods and seeds are sown together. Robinia pseudacacia (13). Apple, in greenhouse at the Michigan Agricultural College. Sclerotinia sp. (Europe) : Betula (79), a disease of seed and germinating seedlings. Phytophthora fagi (Europe) : Fagus. Elartig (59) and many other writers; seriouslj affected, even in forest. Platanus (15). Acer (15), l. platanoides and .1. pseudoplatanus (SO, 104). Robinia ("»!•. 73). Fraxinus (73). Acacia (59). Cercospora acerina (Europe) : leer platanoides and A. pseudoplatanus (58). Pythium debaryanum: Tilia europea and 'I', ulmifolia (137). serious. Robinia (7."), p. 13-14), killing germinating seed. Catalpa (126). Rhizoctonia : Citrus seed beds (130) ; much loss. Catalpa (126). Botrytis cinerea: Catalpa (126). Fusariuvi sp. : Citrus seed beds (130) ; much loss. The sugar beet is apparently the only plant whose damping-off diseases have been investigated with any decree of eompleteness by modern methods. While there is a great mass of literature on damping-off, it is mainly descriptive and on control measures. Most of the reports of the causal relation between the different fungi and the disease in the various host plants have been based on demon- strations of the presence of the fungus in diseased seedlings. In a great number of these cases identification has been doubtful. Even when a fungus is known to belong to a parasitic species, it is by no means certain that the mycelium found belongs to a para- sitic strain. It has been found, for example, that only part of the strains of Corticium vagum occurring in sugar beets are able to attack that host vigorously (38, p. 154). Similar data for pine appear in figures 1 and 2. Furthermore, even parasitic strains of several of the damping-off organisms are so widely distributed as DAMPING-OFF IX FOREST NURSERIES. 5 saprophytes that one of them might easily get into a killed seedling after sonic other parasite had caused its death. Not only in the case of seedlings killed by fungi like Peronospora parasitica, but in HOST YErtR £XPT. P/HCS erz/OBas /V/VOiS &rtMH>S/ l 9A/# P/A/i/S po/yoepos* PINUS P£S/A/OS/9 19/3 /9/3 /9/f- /9/f 1917 /9/Q 28 R 28 B 31 36 T-7 IS 7/ 72 - R / \ / v /■ \ ■ ^\ y\ / \ **^" V \ A ^*\ V \ \ /^ \ l i i - \ • * \ v \ \ \ \ \ \ \ V // - ©— 1 1 -1 1 1 i i Fig. 1. — Diagram showing the relative activity of different strains of Corticium vagum in inoculations made at the time of sowing the seed. In experiments Nos. 36, 45, and 47 the values are plotted for the number of seedlings appearing above the soil. For the other experiments the number of seedlings surviving at the close of the experiment have been taken. Explanation of symbols : 0=Strain 147, from spruce seedlings, Washington, D. C, 1910; +=strain 50, from pine seedlings, Nebraska, 1900; D=strain 233, from Elaeagnus sp., Kansas, 1913; H=strain 230. from the same lesion as strain 233; • =strain 1S3, from bean, New York. 1910. HOST Y£f)R EXPT, %/SO I P/A/C/S STffOBUS W/VUS B/9A/H S^ D» -S^ \k \ \ / / / ' X '"•-a Fio. 2. — Diagram showing the relative activity of different strains of Corticium vagum, as indicated by the number of seedlings surviving in inoculated soil. Explanation of symbols: ©=Strain 1S9, from sugar beet, Michigan, 1910 (light-brown mycelium with few sclerotia) ; A=strain 211 and A=strain 212, from sugar beet, Colorado, 1910; B=strain 186, from potato, Ohio, 1910; D=strain 1S7, from potato. New York, 1910; +=strain 205, from Douglas fir, Colorado, 1911; X=straiu 192 and 0=«train 206, from pine, Nebraska, 1911. cases of true damping-off produced by the rotting type of parasite, much of the rapid decay of the seedling after death is brought about by bacteria and fungi other than the one causing death. BULLETIN 934, U. S. DEPARTMENT OF AGRICULTURE. Iii()cii]:ition experiments are therefore probably even more neces in \ in damping-off investigations than in studies of most other dis- eases in order to demonstrate etiological relationships. Unfortu Qately, most of the inoculation work with damping-off organisms prior to L900 was either crudely done by placing diseased seedlings against healthy ones or consisted of experiments in which purity of cultures and validity of controls did not receive sufficient eon sideration. Recent investigations not primarily directed toward damping-off, but which have decidedly increased our knowledge of the relation between Corticium and the disease, are those of Peltier (98) and Fred (43). The latter established a strong presumption that the difficulty in securing stands of various field crops having oily seeds in soil where green manures had been recently turned under is due to the killing of the sprouting seed by damping-off organisms. In tobacco, sugar beet, and pine, whose damping-off has received considerable attention, it has been found that the damping-off proper is commonly preceded by the killing of many of the sprouting seeds in the soil (38; 68, p. 522; 81, p. 5) and followed, after the plants become too large to be killed by the damping-off organisms, by root sickness and the death of small roots (38, p. 161 ; 64; 100). This latter has been reported also as a serious matter in the case of Corticw/m vagum for potato (51), a host on which damping-off is not important because of the lack of commercial propagation from seed. PytTwam debaryanurn further has been reported as continuing to work in the cortical tissues and leaves of tobacco plants which have been in- fected too late to result in death (81). The fact that a number of the damping-off fungi are able to attack young or soft tissues of so great a variety of plants and are much less able to kill older plants suggests that resistance to damping-off may be in part based on purely mechanical factors. Hawkins and Harvey (71) recently have extended to Pythium debaryanurn the idea, developed by Blackman and Welsford (12) and Brown (16) for Botrytis cinerea, of the importance of mechanical penetration in the fungous invasion of plant tissues. While for B. cinerea mechanical pressure was found to be the main factor only in cuticle penetration. with P. debaryanurn the penetration of the cell walls of all parts of the potato tuber was apparently largely dependent on mechanical puncturing by the hyphae, only tubers with mechanically weak cell walls being susceptible to decay by the fungus. The extreme sus- ceptibility to /'. debaryanurn and Corticium vagum of soft, thin- walled tissues and the resistance of older stems and root parts would fit in well with such a theory as to the method of wall penetration, as in the older tissues the thicker cell walls would obviously be a serious bar to the extension of a fungus dependent partly or en- DAMPING-OFF IX FOREST NURSERIES. 7 tirely on mechanical puncturing Bor its progress from cell to cell. Hartig (61, p. 1 17 L50) shows a fungus which he does no! name, luit which is evidently a species of Fusarium, dissolving the young un- cuticularized epidermis of pine seedlings; but he stall's that it can not so dissolve older epidermis. The increased protective value of the epidermis of older plants can only in part explain the immunity most of them develop against serious attack by damping-off organ- isms, as lesions already started or which may later develop from the infection of young roots are unable to extend into the older pa its of the plants. It may be mentioned here that the writer in a very preliminary test found strains of Corticiwn vagwm and Fusa/riwm mondliforme Sheldon which had been proved able to cause damping-off of pines also apparently able to destroy filter paper in inorganic salt solu- tion, while Pythium debaryanum seemed not so able. Ruhland (116), on the other hand, found the strain of the " vermehrungspilz " {Corfu-/ inn vagum) which he tested to be very weak in cellulose- destroying ability as compared with Botrytis cinerea. DAMPING-OFF OF CONIFERS. HISTORICAL. While the losses from damping-off in seed beds of dicotyledonous tree species are occasionally serious and in the case of beech in Europe have required considerable study, they have been so far overshadowed in this country by the losses in coniferous seed beds that practically all the attention thus far, both as to etiology and measures of prevention, has been devoted to the disease in conifers. The literature on the damping-off of conifers is considerable. A large part of it, because of the extensive early development of plant pathology and forest planting in Germany, has been writ- ten by Germans. A large portion of the German articles on it was either by foresters or by botanists in the day when most patho- logical work was of the reconnaissance type. Therefore, while the work of one of the best known of the parasites on coniferous seed- lings was noticed in Europe as early as the eighteenth century (21, p. 252-253) most of the European data available are observational. The only fungi which were at all definitely connected with the dis- ease on conifers seem to have been Fusarium {Fuxoma spp.) and Phytophthora fagi (P. omnivora De Bary in part). The damping- off Rhizoctonia was described in Germany in L858 and Pythium de- baryanum in L874; the fact that neither of these, important in conif- erous seed beds in both the eastern and western United States, has ever been reported from conifers in Europe is perhaps the best evi- 8 BULLETIN 934, V. S. DEPARTMENT OF AGRICULTURE. dence of the relatively small amount of actual investigation carried on there on this disease in the nurseries. A aumber of references to the damping-off of conifers in the English horticultural and botani- cal Literature yield even less definite information as to the causal fungi than do the German articles. • With the awakening of interest in reforestation in the United States between L5 and 20 years ago and the first efforts to grow pines in quantity for forestry purposes, attempts were made to de- termine the cause of the disease in this country and to develop direct- control methods. Duggar and Stewart (32) made what appears to be the first report of Rhizoctonia in- connection with the damping-off of conifers. Spaulding (136, 137), in work begun in L905, con- tributed much to our knowledge of the etiology of the damping-oil' of pine in this country, especially in relation to Fusarium, and origi- nated the sulphuric-acid method of control. The writer in L910 re- ported preliminary inoculations on conifers with both Rhizoctonia and Pythiwn debaryanum (62). The work of Gifford (46) and Hofmann (77) added to the information on the causal relation of Fusarium spp. and /'. debaryanum, respectively. Hartley. Merrill, and Rhoads (68) have recently established the parasitism of a num- ber of strains of the Corticium vagum type of Rhizoctonia on pine seedlings under inoculation conditions, have confirmed Spaulding's conclusions as to the parasitism of Fusarium moniliforme Sheldon, and have given preliminary data on other fungi. They consider /'. debaryanum and ('. vagum more important in pine seed bedsthan any single Fusarium species. Hartley and Halm (G9) have announced successful inoculations on pines with- P. debaryanum and Rheospo- rangium aphanidermatus Edson, with less satisfactory evidence of the parasitism of PhytopJithora sp. and a fungus tentatively referred to Pythium artotrogus. Hartley and Pierce (67) report the finding of P. debaryanum in Tsuga rrn rtensiana and Pseudotsuga taxifolia as well as in the pines. In damped-off pine seedlings they find P. debaryanum more commonly than ('. vagum, especially in beds which have received disinfectant treatments. Other considerations, how ever, keep them from concluding that the former is necessarily the more important of the two. Both of these latter papers and all of the reports of Pythium with the exception of Hofmann's are brief notes, presenting no evidence in support of the statements made. DESCRIPTION. The symptoms of damping-off in conifers have already been de- scribed in some detail (68). In the paper cited, injury due to exces sive heat of the surface soil and injury caused by high wind, both of which may easily be confused with damping-off, are described and accompanied by colored illustrations both of different types of damp- DAMPING-OFF in kokkst ni'kskimks. 9 ing-off and of these nonparasitic troubles. The detailed descriptions will not be repeated here. A brief summary of the different types of disease recognized as included in damping-off follows: (1) Germination loss: The radicles arc killed very soon after the seeds sprout and before the seedlings can appear above ground. This is an important type, which can be caused probably by any of the organisms commonly capable of causing the better known types of trouble (61, 63, <»S, 137). (2) Normal damping-off (figs. •".. 1, and 5) : The seedlings are killed by fungi invading either the root or hypocotyl after the seedling has appeared above the soil and while the stem is still dependent largely on the turgor of its cortical tis- sues for support. In sandy soils root infection is more common than hypocotyl infection, though the latter is the type most emphasized in the early horticul- tural descriptions. Ruttner (26) some time ago recognized the frequence of Fig. 3. — Normal type of damping-off of Plnus ponderosa. At the left is a dampeil-off seedling or root sprout of the southwestern ragweed (Ambrosia psilostachya) . (Photo- graphed by S. C. Brunei-.) root infections. Damping-off in beds out of doors is primarily in most cases a root rot, either of this type or of the types preceding and following. (3) Late damping-off includes cases of the root-rot type occurring only after the seedling stems have started to become woody and the cortex has begun to shrivel. The damping-off parasites, or at least part of them, continue to kill seedlings by rotting their roots for some time after the stems become too woody to be decayed. The seedlings affected do not fall over till a considerable time after death. For convenience, all cases of this sort up to the purely arbitrary a.ue of two months are classed as damping-off. However, in weather permitting of average speed of development the seedlings are usually able to resist attack before they reach this age. Seedlings at the marginal age between suscepti- bility and nonsusceptibility to killing infections are found often with the younger parts of their roots killed, but with the older portions apparently able to resist invasion by the fungus, recovery taking place by laterals. Dr. R. D. Rands and the writer in 1911 established the ability of seedlings from 4:;-day-old beds of Pin/us sylvestris, P: banksiana, P. vigra amtriaca, and P. nigra poiretiana to survive such infections, even when more than half of the root system lias been destroyed, by transplanting such root-sick seedlings and 10 BULLETIN 934, U. S. DEPARTMENT OF AGRICULTURE. observing their continued growth (fig. 6). An article recently found (2.""0 shows thai Biittner had earlier made the same sorl of demonstration of recovery of root-sick conifers. Observations on olive seedlings in 1916 showed cases of partially rotted roots which were recovering by sending out lateral root branches. (4) To]> damping: Tin- cotyledons or upper part of the stem are invaded by tiu 1 parasite, sometimes before the seedling breaks through the soil. The infec- tion may or may not be fatal. A special case of this type, probably caused by a different parasite from those most commonly active, is that which in a publica- tion above referred to was described and figured as " blacktop " (68). Tt is ■A*- ~* 'i^ftg*' (&4gMt& &*. Fiq. 4. — The ttosdiining of an epidemic in drill-sown Pinus banksiana. Black crosses (X^ indicate disease foci where the germinating seed were apparently killed and from which the disease is now spreading to adjacent seedlings. (Photographed by It. J. V. Hofmann.) distinguished from ordinary top damping by the very dark color of the invaded tissues and its apparent dependence on some unusual set of climatic factors for its progress in the seedling after infection. The killing- of dormant seed by fungi is a matter of some practical interest in seed beds, and possibly still more so in forests, as it may help to explain the failure of certain conifers to reproduce except on mineral or certain other special soil types (68). With sugar beets Pythium debaryanum (100) is said to attack dormant seed as well as seeds which have sprouted. It is to be presumed that with conifers some of the damping-off fungi will be found to attack dormant as well as sprouting seed. This matter is now under investigation. TUMPING-OFF IN FOREST NURSERIES. 11 Something is already known aboul the seed Fungi of herbaceous plants ("(!. 91), broad-leaved trees (7!». 92"), and juniper (95). RELATIVE IMPORTANCE OF THE DIFFERENT TYPES. Of the types of damping-off described in the foregoing pages the first two are ordinarily the most important. Late damping-off is rarely as serious as the normal type of damping-off. Top damping is only of importance in eases of excessive and unusual atmospheric moisture, so far as the writer's experience indicates. In the Middle West it has proved relatively insignificant. The three types which Fig. . — Nearly complete destruction of the seedlings of Pinua banksiona at an unusually early aye, at Garden City. Kans. (Photographed by Dr. J. V. Hofmann.) occur after the seedlings appear above the soil surface can, of course, be evaluated by frequent counts during the damping-off season. This has apparently not yet been done by anyone. However, in experi- ments on damping-off control by soil disinfection, data have been obtained on comparative emergence (number of seedlings appearing above the soil surface) in treated and untreated plats and on the total parasitic losses after the seedlings appear which permit a certain amount of analysis of the losses due to damping-off parasites. The data from five nurseries bearing on (his point are presented in Table I. 12 BULLETIN £>34, V. s. DEPARTMENT OF AGRICULTURE. Table I. -Relative importance of losses by damping-off before and after conifer seedlings emerge (rum the soil. Series. I; i I oss in control plal s. Nursery and species. Disinfectant. Number of plats. Emergi Pinus banksiana at the Bessey Nursery, the loss after emergeni e is slightly low and the ratio slighth high, because of the closing of count's on a few of the series before damping-off was entirely over. The procedure was to average the number of seedlings which emerged in the control plats in each series and subtract this number from the average number emerging (that is. appearing above the soil surface) in the treated plats in the same series. The treated plats chosen were the ones which allowed the averaging of the greatest number of plats treated with the same disinfectant. Only those plats were taken in which there was no evidence of injury to the seed or seedlings by the disinfectant and in which the amount of normal damping-off during the first few days after emergence was so slight as to indicate satisfactory initial control of the parasites by the treatment. In such plats it was assumed that the germination loss was unimportant, and the average number of seedlings appear- ing on them was taken as representing the number of viable seeds per plat. The difference between this emergence figure and the average emergence in the controls was taken as indicating the extent of para- sitic loss before the seedlings appeared, including any destruction of dormant seed by parasites which may have occurred as well as the killing of germinating seed. Both this figure and the number DAMPlXti-Ol F IX FOREST NURSERIES. 13 of seedlings which succumbed to damping-off after emergence were reduced to a percentage based on the indicated number of viable sec. Is. and they are directly compared in columns 6 and 7 of Table I. At three of the nurseries the data of the same species of pine and with the same treatment were averaged. The data in Table 1 do not indicate any regularity either in the extent of loss before emergence, the loss after emergence, or in the ratio between these two values. For ob- vious reasons, no reg- ularity is to be ex- pected in any of these items. The table is of some interest, however, in confirm- ing the evidence of the inoculation ex- periments, of obser- vation of sprouting seed dug up in the beds, and of the par- tial or complete fail- ure of emergence at the centers of large damping-off foci (figs. 4, 7, and 8) that the work of parasites before the seedlings appear may in some cases be of consider- able importance. It is obviously impos- sible to make any general quantitative statement of the se- riousness of such loss, in view of the varia- tion in its extent at different times and places and of the in- accuracy of any computations based on the relative emergence of hosts as irregular in their germination as the conifers are known to be. The case is complicated in addition by the fact that, despite careful avoidance of treated plats known to have suffered chemical injury, it is probable that a few seedlings were killed before emergence by the disinfectants used in some of the Fig. ti. — Root sickness in Pinus nigra poiretiana. The two seedlings at the right are healthy. The three at the lefl have had their taproots decayed to within li inches of the soil surface. All are putting out lateral roots from the lowermost sound point. Similarly injured seedlings when transplanted lived and made satisfactory growth. 14 BULLETIN !»::t. r. S. DEPARTMENT of agriculture. cases. It may furthermore be that in other cases the disinfectant had a stimulating effect, resulting in better germination in the treated plats, entirely aside from that resulting from parasite control. The number of disinfectant methods which concurred in giving apparent increases in germination, however, makes it seem reasonably cer- tain that no great part of the increase was d\w to this stimulation. In addition to the different disinfectants shown in the table, mer- curic chlorid, heat, hydrochloric acid, nitric acid, and ammonia all apparently resulted in approximately the same increases of germina- tion in tests at the Bessey Nursery as the sulphuric acid which was used as the standard for comparison in most of the series. Relative emergence in treated and untreated plats, as well as damping-off Fig. 7. — A clean-killed area in a bed of Pinus ponderosa, caused by Oorticium vagum. Inside a 12-inch circle at the center of this "patch" no seedlings appeared. It will be noted that the weeds as well as the pines have been killed with the exception of Salsola tragus. loss after the seedlings appeared, was determined at two nurseries in addition to those given in the table. The results at these nurseries in general confirmed those at the five nurseries covered by the table in showing lower emergence in the controls. Although it is impossible to draw positive conclusions, some idea of the seriousness of losses before the appearance of the seedlings above ground can be obtained by studying the data in Table I. The fact that such losses appear considerable, sometimes exceeding the losses from the damp- ing-off that occurs after emergence, is believed to explain the com- mon failure to secure satisfactory results from control measures taken after the seedlings have come up and the disease has become noticeable. It is somewhat interesting to note that the data in the T)A-MPIXC-OFF IX FOREST NURSERIES. 15 table tend to confirm field observations that, a> compared with other species. Pinus resinosa is more susceptible to the Later forms of damping-off than to germination loss. Further indication that the killing of germinating seed before emergence may be important enough to help explain eases of poor germination is obtained by an entirely different method, as follows: At the Wind River Experiment Station of the United States Forest Service counts of the seedlings emerging and of those which later died were made on a number of untreated plats by forest officers, who kindly permitted the writer to use the data obtained. The counts were made separately on 10 plats each of noble fir (Abies nobilis) and silver fir (Abies eoncolor). The plats of each species had been sown with equal quantities of seed. It appeared on in- spection of the figures that the plats which showed the poorest emer- Fig. 8. — The area shown in figure 7 after the bed had been weeded and damping-off had practically ceased. (Photographed by S. C. Bruner.) gence were also the ones which suffered the most subsequent loss. The coefficient of correlation between the number of seedlings emerging and the percentage of subsequent loss in the same plats was found to be — 0.49±0.16 for the noble fir, and — 0.50±0.16 for the silver fir, an average of — 0.49±0.11 for the two species, confirming the conclusion drawn from inspection of the figures. In other words, poor emergence and heavy subsequent loss were in general associated. The simplest explanation of this association appears to be to suppose that both poor emergence and subsequent loss were largely clue to the same cause, namely, the damping-off parasites. Another possible explanation of the correlation would be to neglect parasites as im- portant causes of the poor emergence in certain plats and to suppose that the higher subsequent loss in such plats was due to heat injury, the less dense stands affording less shade to the bases of the seedlings composing it. As damping-off is in general so much more important than heat injury as a cause of death after emergence and the dif- ference in the degrees of shade between the plats with the denser 16 BULLETIN 934, V. S. DEPARTMENT OF AGRICULTURE. and the plats with the thinner stands must have been very slight, this latter explanation has not much to support it. The data are believed to constitute further evidence of the importance of parasites in de- creasing the percentage of emergence in coniferous seed beds. That the effect of parasites on emergence should have been large enough in this case to make itself apparent on the face of the figures, despite the variations due to other sources, is especially interesting in view of the fact that the losses after emergence in these plats were not high. ECONOMIC IMPORTANCE OF DAMPING-OFF. The importance of damping-off in coniferous nurseries in Europe is indicated b}^ frequent reference to the disease in the literature. Biittner (25, 26) states that whole beds are frequently destroyed by it. Baudisch (9) speaks of the death of entire stands in many nurseries as the result of damping-off. In the United States Spauld- ing (137) considers damping-off a serious obstacle in forestation operations. Clinton (28, p. 348-349) reports serious damage to conifers in New England nurseries. The writer has found the dis- ease especially prevalent in nurseries in Nebraska and Kansas, a some- what unexpected situation in view of the relatively dry conditions prevailing there. A correspondent has reported heavy loss in seed beds in Texas. The economic importance of the disease in conifers is due in part to the rather heavy average losses experienced at many nurseries and in part to the irregular character of the losses. In one season losses may be negligible, while the next season the beds of certain species may be practically wiped out. Even without this element of uncertainty the losses experienced are expensive, because of the high cost of coniferous seed. The seed of some species costs from $3 to $5 a pound and seldom shows a germination of more than 60 per cent under nursery conditions. A loss of half of this 60 per cent from parasites, both before and after the seedlings break through the soil, is therefore a matter which deserves attention. The figures in column 8 of Table I, obtained by adding together those in columns 6 and 7, show that the loss is frequently higher than this. At the nurseries at which control experiments have been conducted, the percentage of the seedlings in untreated beds which have been found by actual count damped-off after emergence is frequently more than 50 per cent, in addition to the considerable but less accurately de- terminable loss indicated by the foregoing data as being caused by the parasites before the seedlings appear. It has been suggested by foresters and others that the net economic damage from damping-off is not as great as is indicated by the loss of seed and seedlings which it may cause. The argument is ad- DAMPING-OFF IX FOREST NURSERIES. 17 JOO / 2 eo / S\SULPHURIC ACID <£~~T 1 1 1 so ^0 ~<3 1 30 > 30 i zo 1 - — so - / a /0 ^/ CX?/=>PSR SULPHATE 1 1 1 10 zoo /eo /so / ^/COPPER SALTS L /OO k eo \ eo 3 '"' 1 1 t 1 *' 1 t I V 00 % S so »5 > 1 1 \/ FORMAL DC HYDE 30 a-o J y/ SULPHURIC ACID \ 1 Ui 30 kj ^ so _Jf 1 1 1 eo so r 1 1 1 7 / 1 / / y / a — 1 1 1 /o - / s //SULPHURIC AC/D S*^| 1 1 10 ISO 7 yT^ SULPHURIC ACID If 1 1 1 1 ISO 9 100 - j? /oo /' eo - eo t 1 eo - 4 / 60 1 1 90 » /I 1 J 40 1 / / S^SULPHURIC ACID zo 1 — f J r It I SULPHURIC ACID ' 1 1 1 1 zo 10 so &o so /o 20 30 0S SINCE GERMIN/9T/ON <5k? SO Fig. 9. — Diagram showing the progress of damping-off in treated plats (solid line) as compared with untreated plats (broken line). Graphs 1 to 4 represent Pinun ponderosa; graphs •"> to 8, /'. resmosaj graph 9, P. banksiana; and graph 10, plats each of which was half /'. nigra poiretiana and half P. sylvestris. Nurseries in Kansas, Nebraska. Minne- sota, and Michigan are represented. The relatively high total number damped off in the treated plats shown in two of the graphs is due to the fact that a large proportion of the seedlings in the untreated plats had been killed before they appeared above the soil surface. In both the eases in which the absolute number of seedlings killed was as great in the treated plats as in the controls, the percentage of the seedlings killed was nevertheless lower and the survival more than twice as good as in the controls. 19651°— Bull. 934—21 2 18 BULLETIN 934, U. S. DEPARTMENT OF AGRICULTURE. vanced that damping-off may be a valuable selective agent in nursery- grown stock for forest planting, eliminating the weaker individuals and thus insuring the vigor of the trees which go into the forest plantation. This is a possibility which must be considered. It is by no means certain, however, that escape from damping-off is correlate I with permanently superior vigor. It is believed that temperature, moisture, and other environmental factors, which as yet are very im- perfectly analyzed, together with the age of the seedlings and the presence or absence of virulent strains of the parasites, are much more important factors than inherent differences in individual re- sistance in determining whether or not seedlings are destroyed. Evi- dence from inoculation experiments and from field observation, sup- ported by data of the sort presented in Table I. indicate that damp- ing-off ordinarily does a considerable part of its damage by killing the sprouting seed before emergence from the soil, while the graphs in figure 9 show that of the loss which occurs after emergence in tin- treated beds a large part occurs very early in the life of the seedlings. Observation of the clean sweeps which the disease commonly makes in the immediate neighborhood of infection foci (figs. -\. 4. and 5) indicates that either before or just after the seedlings break through the soil none of them have any considerable resistance to the really virulent strains of the parasites, which are believed to be the ones responsible for the major share of the damping-off. Even if there should be found to be an appreciable selective value in damping-off, this would not be a valid argument against control by seed-bed disinfection for the following reason : The graphs show ing the course of damping-off in treated plats in figure 0. together with the decided differences in germination between treated and un- treated plats, indicate that the very early damping-off is more com- pletely controlled by disinfectants than the later damping-off. This early damping-off which the treatments so largely prevent is the part of the loss which has the least possibility of selective value. The later damping-off is rarely controlled at all thoroughly by disin- fectants. As shown by the graphs, it is often even heavier on the treated than on the untreated soil. It is the part of the loss which is most likely to have selective effect. At this stage beds are not taken clean, as earlier: only seedlings which are below normal re- sistance succumb. The damping-off in disinfected beds seems there- fore at least as likely to have true selective value as that which occurs in untreated beds. The only way in which the effect of damping-off as a selective agent can be positively determined will be to compare through sev- eral subsequent years the growth rate, or survival after transplant ing, of trees from beds which suffer seriously from damping-off with the growth of trees from the same lot of seed in seed beds in DAMPING-OFF IX FOREST NURSERIES. 19 which the disease is either accidentally absent or is artifically con- trolled. Such an experiment is within the silvical father than the pathological investigative field. If it be found that there is some selective value in the action of the disease and that it is greater in untreated than in treated beds, it would still seem that a much more desirable and dependable selection could be obtained by discarding weak plants at the time of transplanting than by letting damping- off run uncontrolled in the seed beds. Damping-off is sometimes negligible and sometimes destroys practically all the seedlings in a given area, in neither of which cases can it have any material selective value. RELATIVE SUSCEPTIBILITY OF DIFFERENT CONIFERS. Biittner (25) writing of European conditions, states that exotic conifers are especially subject to damping-off. He includes fir, spruce, pines, larches, and cypress in this statement. He mentions the same subject in a later paper (20). Neger and Biittner (91) give a long list of different species of conifers from various parts of the Avorld with statements as to their susceptibility to damping-off. Beissner (11. p. 6T>G-0r>7), Neger (93), Clinton (28), Bates and Pierce (T), Boerker (13), and Tillotson (139, p. 69) have all given information on the susceptibility of different conifers. The data re- ported by Tillotson are drawn from reports by various officers of the United States Forest Service which he has compiled. While it is probable that the nurserymen who are responsible for most of his records have not observed the disease as closely as Neger and Biittner, the fact that their observations are mostly based on repeated seasons' work with large-scale seed beds of the species they report on makes their observations in some respects more reliable than the other pub- lished data. Neger and Biittner presumably worked in most cases with small beds of the various conifers on which they report, and the variations which they attribute to differences in specific resistance might easily in such case be largely due to accidental variation. The error which nurserymen are most likely to make in their notes on susceptibility is to underestimate the loss, especially for the small- seeded species. The seedlings of small-seeded conifers decay and shrivel so quickly after they fall that in taking notes at any one time only a small proportion of the total loss is visible. Frequent counts of dead seedlings are the only way by which the loss after germina- tion in such species can be properly appreciated. The data given by the authors mentioned in the foregoing para- graph, together with unpublished data obtained by personal observa- tion or from commercial and other nurserymen in the United States, are summarized in Table II, the source of each report being shown by letters signifying the authority. The unpublished data on two 20 BULLETIN 934, U. S. DEPARTMENT OF AGRICULTURE. nurseries yi Illinois and Minnesota were obtained from the nursery- men by Mr. R. G. Pierce and are indicated by the initial "P." In- formation obtained directly from the nurserymen by the writer is in- dicated by " N." For nurseries where the statements are based on the writer's personal observation rather than on the authority of the nurserymen, his own initial ("H") is given. Most of the writer's own estimates of relative susceptibility are based on a comparison of detailed counts of the damped-off seedlings in a large number of untreated plats at different times, as well as on observation. The nurseries on which Tillotson reported were all west of the Missouri River, most of them being in the mountain region. The reports in- dicated by "H" and "N" were mostly from nurseries east of the Rocky Mountains. In cases in which the data permit it, the species are classified as most susceptible, intermediate, least susceptible, or immune. In a number of cases, however, it is only possible to classify them as "more" or "less" susceptible. Table II. — Relative susceptibility to damping-off of different conifer species. [Figures in parentheses in this table indicate the number of nurseries from which the susceptibility noted has been reported by the observer to whom the preceding letter refers.] Reports of relative susceptibility.* Host species." Not sus- cep- tible. Least sus- cep- tible. Less than average. Inter- medi- ate. More sus- ,, , ceotible M "\' than the ' s f,^" average. u R ' Pinaceae (Abietoidese): ('•) Nb.... Nb... Bu, Nb. T Nb. Nb.... XI... . Nb Mi... Nb. T Nb.... N (2) Nb. Larix occidentals T T Ne Nb.. Nb.. P N.. T (2) H, N, Ne, Nb. P Nb .. H, Bu.... T. Ne, N Ne Ne N(2) Nb... N (2). Nb.. Picea pungens H, P.. Ne Picca sitchensis ■ Nb. Ne Nb. Nb.. N .. . Nb.... Nb.... N T P B, P, H B.. H. T T (2), T. n Host names for American species follow the usage in the publications and a later verba] communication of Mr. George B. Sudworth, of the CJhil <1 States Forest Service. For exotic species the standard Cyclo- pedia of IIoi'iinil!iin,\c\v York, 1916, edited by 1-. E. Bailey, is taken as the standard. The classification follows Saxton (UN). 6 Symbols signifying the authority for the report: B=Boerker (13). Bu=Buttner (25, 26), Bp - Kales and Pierce (7), C=Clinton (28), H= Writer's estimate, N= Nurserymen s estimate (obtained by the writer I, Nb=Negerand Biittner (94), Ne=Neger (93), P= Nurserymen's estimate (obtained by Pierce), T=Forest officers' estimate (Compiled by Tillotson, 139). c Susceptibility to Phytophthorafagi. DAMPING-OFF IX FOREST NURSERIES. 21 Table II. — Relative susceptibility to dampmg-off of different conifer species < 'ontinued. Reports of relath e suscepl ibilitj . Host species. Not sus^ cep- tiblo. i ea i sus- cep- tible. Less than average. Inter- medi- ate. More ii - ceptible than the average. Most suscep- tible. Pinacese ( A.bietoidese) — Continued. Nb.... N T T(2) T P T T T Nb.-.. N, T(3).. Bp, N,T.. Pinus nigra poiretiana (Corsican pine) Bp . H... B Nb.... Pinus ponderosa (type not specified ) Pinus ponderosa (Eastern Rocky Moiui- T(3).. p Bp,N(4), T(6). II (1) Nb.... T(4) B B N (3) Bp, 11, T.. N B, H. P........ p 11, B.N.. T(2) N H, 1'.. C, N'T... Bp,T(3).. N. B Nb.... Pseudotsugataxifolia (type not specified). N(3)T(3) 11 N, T(5)... B Nb... Pseudotsuga taxifolia (Northwestern B Nb.... Nli, P. Bu 9 6 17 10 51 31 23 11 48 29 15 9 Sciadopitoidese: Nb. Cupressacete (Cupressoideae): Nb.... Nb. .. T Bu Nb.... Nb.... N. . Nb.... P N T... . II, P.. T... Nb... Nb.... Nb.... T Nb... Thuja orientalis P... Nb.... Nb.... Number of reports 9 39 7 30 2 9 4 17 1 4 Sequoidem: B T Taxaceoe: V The fact most evident in Table II is the extreme variation between reports, not only on closely related species but even on the same species. While it is, of course, possible that the obvious lack of a definite basis and method of comparison in most of the reports is responsible for most of this variation, it seems to the writer more probable that different species do actually vary in their relative sus- ceptibility to damping-off in different localities. Tn the first place. 22 p.ri.LKTix 934, r. s. department of agriculture. the conditions which might increase resistance of one host might very easily decrease its resistance for a host with different environ- mental requirements. To illustrate by an extreme example, the pinon (Pinus edulis) of the arid or semiarid regiou might remain resistant in soils in which Picea engelmanni of the high mountain- stream bottoms or Picea mariana of the northern swamps might be low in vigor and easily attacked. In the second place, it is to be expected that species with a certain order of relative susceptibility to the parasites which predominate at one nursery may exhibit a very different order of susceptibility to the different combination of para- sites which might be prevalent in another locality. The only individual species on which there are a sufficient number, of reports and a sufficient agreement between the reports are the two common western spruces, Picea pungens and P. engelmanni, which (at least as compared with Picea excelsa) seem rather susceptible, and Pinus ponderosa., which (as compared with most of the other species of the Abietoidese) is to be regarded as generally more re- sistant than the average. Within each of the larger genera of this group it seems evident that susceptibility is extremely varied and that no statement as to the relative susceptibility of the genera them- selves can therefore be made. The only group generalization that is perhaps permissible is derived from the consideration of the Cupressoidese. In this group, out of 23 reports, 16 are in the " not susceptible " or " least susceptible " columns and only one indicates more than intermediate susceptibility. Of 163 reports pertaining to the Abietoidese, only 26 place them in the " not susceptible " or " least susceptible " columns and 63 in the classes of more than inter- mediate susceptibility. The general feeling among nurserymen seems to be that serious damping-off need not be feared among the cedars and their relatives. The data at hand tend to justify this confidence. CONTROL OF DAMPING-OFF. Early efforts to prevent damping-off were chiefly directed to the avoidance of excessive moisture in either the air or soil. A means to this end, which has been observed more or less by nurserymen for many years, both in the United States and elsewhere, is the applica- tion of small quantities of dry sand to the seed beds after the disease becomes noticeable (18, p. 166; 83). This is sometimes applied hot (101, p. 43^44; 145), though even this procedure does not result in very great advantage. Surfacing with hot sand can not always be counted on to give any measurable advantage over untreated beds (67. p. 3). The use of sand (25) or sterile subsoil (101) instead of ordi- nary soil in covering seed at the time of sowing has been advised. Johnson (82) did not secure satisfactory results with sand in tobacco DAMPlXd-OIT IX FOREST NtJRSEfclES. 23 beds. Making the upper part of the bed to a depth of sex era] inches of recently dug subsoil appeared effective in a single test by Spaulding (137) and at four nurseries by cooperators of the writer in Inter tests, the results of which will be published elsewhere. The procedure is unfortunately rather expensive in large-scale work and under some conditions at least undesirable because of the poor subsequent growth on such soil. Excessive vegetable matter (45), imperfectly rotted manure (07), or green manures recently plowed under (43) have all been advised against as likely to favor the disease. The experience reported with conifers (07, 139) indicates that damping-off can be to a certain extent decreased by broadcast sowing as compared with sowing in drills. The usual recommendation of thin sowing to avoid the seed-bed disease of other plants lias also been made for conifers (67). Transplanting healthy seedlings from infected beds into new soil is recommended as a means of saving them from attack (11, 145). The writer's tests of transplanting at a Nebraska nursery gave no promise of economic value as a control method, although he is in- formed that it was successfully employed in a nursery in New Mex- ico. The time of sowing appears to have a relation to the amount of disease at some nurseries, but conditions in this regard evidently differ in different localities, so that' the best time to sow from the standpoint of avoiding damping-off must be determined separately by repeated tests at each nursery. For example, observations both by the nurserymen and the writer during several seasons at the Bessey Nursery, in Nebraska, indicate that fall sowing is an ex- cellent means of decreasing loss from damping-off in at least one pine species, and Retan (110) reports the same thing for a nursery in Pennsylvania, while at two Kansas nurseries and at nurseries men- tioned by Tillotson (139) fall-sown beds suffer more than those sown in the spring. Treatment of the seed with mercuric chlorid (25) or with copper sulphate (122) has been recommended. While it has been demon- strated (38) that a proper heat treatment of the seed will greatly decrease the damping-off in sugar-beet seedlings, this is explained by the fact that one of the most important parasites of the sugar beet is systemic and often present in the seed. There is no reason to believe that seed-carried infection is of any importance in conif- erous seed beds. The only advantage that could reasonably be expected from a seed treatment of conifers would be that which would come from the prevention of seed decay in the soil before germination starts, and this protection could be expected to be ef- fective only if a relatively insoluble disinfectant, such as Bordeaux mixture, was used. Soil treatment is the most direct and probably the most profitable method of attack on the disease. It is especially easy, lor tobacco 24 BULLETIN 034, V. s. DEPARTMENT OF AGRICULTURE. seedlings (82) as well as for pines, to prevent by soil disinfection losses U'l'ore the seedlings appear above the ground. Heat disinfec- tion of seed beds lias boen frequently mentioned. Burning wood or litter on the surface of the beds before sowing, said by Gilbert (47, p. 36) to be a common procedure in preparing tobacco seed beds both in Italy and in parts of this country, has been recommended for coniferous seed beds by Biittner (25). The disadvantageous results sometimes noticed following the application of wood ashes to pine seed beds may prove an objection to this type of treatment in some of the nurseries. At a Nebraska nursery (67) moist heat proved only partly satisfactory, unavoidable reinfection having serious results. Steam disinfection, using the inverted-pan method commonly advo- cated for tobacco seedlings (10, 47, 81), has been reported by Scott (123) as successful at a nursery in Kansas. Gilford (46) found steaming with the inverted pan only partly satisfactory. It is not believed that it is likely to pay to install the necessary apparatus for steam disinfection at nurseries in nonagricultural districts where steam tractors are not available for temporary use. The hot-water soil treatment as used by Byars and Gilbert (27) is probably worth a trial at any nursery where damping-off is serious and fuel cheap. It may be that in some localities where steam or hot-water treatment of the soil is not sufficients effective, its efficiency can be increased by reinoculating the soil immediately after treatment with sapro- phytic molds and bacteria to provide maximum competition for parasites which come in from the outside. Tests of this procedure will be described later in the present bulletin. The value of char- coal has been emphasized by Eetan (109, 110). Chemical disinfection of the soil has also been employed. Sulphur has long been in use as a soil treatment against the damping-off of various plants (45, 111) in addition to its use in combating potato scab and onion smut. It was tested on conifers by Spaulding (136, 137) in the form of light surface applications to the beds after ger- mination, but without decisive result. In later cooperative tests pow- dered sulphur raked into the soil before the sowing of the seed failed to indicate any large measure of value. Very finely divided forms of sulphur in various amounts and times of application are prob- ably worth some further test. Moller (90) and Sherbakoff (128) have reported the successful use of copper sulphate in combating attacks of Corticium on dicotyle- dons. In Johnson's experiments on tobacco seedlings (82, table 3) copper salts and Bordeaux mixture were the only chemicals for which any value was indicated. Sherbakoff apparently used copper sulphate and other strong disinfectants chiefly to stop the extension of vigorously spreading damping-off foci by local treatment rather than as a general treatment for use over the beds. Such treatment I>A.M1MX(;-(>FF IN FOREST NURSERIES. 25 would presumably kill all seedlings on the area treated, but would, of course. In- of considerable value in stopping at the outset sued mycelia as those which caused the damped-off area in figure 7. The procedure would be of practical value only in eases in which damp- ing-off was chiefly limited to a few large patches of this sort, a rather rare condition in conifers. Copper sulphate solutions have been used on pine seed beds at the time of sowing with considerable success at some nurseries (65, 67). Except in a nursery in which the soil contained carbonates, it has proved rather difficult to prevent injury to the pines. The trial of some such combination of copper sulphate and lime as was used by Spaulding (136) on the surface of pine beds before sowing, which apparently pi-evented the damping-off of lettuce in some unpub- lished pot experiments of Mr. J. F. Breazeale, is considered desir- able. Treating seed beds with ordinary Bordeaux mixture has also been recommended. Home (78) secured especially good results against Corticium vagum in tobacco seed beds by heavy applications of Bordeaux mixture, and Schramm (122) and Clinton (28) have obtained indications of its value as a spray in preventing the damp- ing-off of conifers. It is probably worth further tests in various amounts of application. In tests conducted by the writer in 1912 and still unpublished, some advantage was indicated for Bordeaux mixture as a surface treatment after soil disinfection with acid. Zinc chlorid as a soil disinfectant has also been found valuable in a number of cases (65, 67), but it is more expensive and apparently less dependable than copper sulphate. Formaldehyde and sulphuric acid have been tested more fre- quently than other disinfectants. The use of sulphuric acid on coniferous seed beds was originated by Spaulding (136). The first intensive experiments with this acid were reported by the writer (63) . The first experiments with formaldehyde on conifers seem to have been in the early greenhouse tests of Spaulding (137), repeated in forest nurseries in 1907 by Jones (83) and Spaulding (136). Most of the experiments with these two substances have already been sum- marized (67). A report not mentioned in this summary is that of Schaaf (119, p. 88), who obtained favorable results with the acid. The great trouble with formaldehyde is its tendency to kill dormant seed. The length of time which must be allowed to elapse between treatment and sowing in order to avoid this killing varies with con- ditions. Formaldehyde is more expensive than acid and seems on the whole to have been less effective in disease control. Acid, on the other hand (applied just after the seed is sown, which is found to be the best time), on a few soils has caused injury to radicles, which it was at first thought could be prevented only by very frequent watering during the germination period; while in a few cases, when cold £6 BULLETIN &34, l'. S. DEPARTS i:ST OF AGRICULTURE. weather resulted in a long germination period, it has killed or in- hibited the germination of some of the dormant seed. All injury can be prevented by treatment a few days before sowing, followed by the addition of lime just before sowing, but lime used in this way has apparently destroyed :i considerable part of the value of the acid treatment against the disease. Further consideration of the data on which earlier papers (6-'5, 67) were based indicates that the apparent need for frequent watering during the germination period, which was required at a few of the nurseries where the first tests of acid treatment were made, ;is well as practically all of the germina- tion reduction, was due to the use of unnecessarily large applications of acid and that the trouble can be eliminated by determining by test the minimum quantity of acid which will be reasonably effective in each locality. If this can be done it should establish the acid treatment as the most profitable for general use of any of the methods of damping-off control which have so far been extensively tested. In view of the various parasites which may cause damping-off at different times and places and which vary greatly both in their means of dissemination and in their physiological qualities, it is not believed that any single disinfectant will be found entirely satisfac- tory at all nurseries. It is also unfortunately true that no one strength of treatment can be recommended for all nurseries. The quantity of acid to be used at any specified nursery will have to be determined by test at that nursery. A single test, no matter how well conducted, is not sufficient to serve as a basis for conclusions. However, a number of small-scale tests, made at different times and in different parts of the seed-bed area, should determine the best treatment for any particular nursery with a reasonable degree of certainty and with very little work. If the plats are equal in size and receive equal quantities of seed, all the nurseryman needs to do to determine the value of the treatments is to count the number of living seedlings on the different plats at the end of the season. The decrease in the number of weeds as a result of the use of acid is itself sufficient at a number of nurseries to pay the entire cost of the treatment. Detailed methods of application have already been pub- lished (67). The differing proportions of acid between which the best treatment will ordinarily be found to lie are 2 and 7 c. c. (one- sixteenth and one-fourth of a fluid ounce) of the concentrated com- mercial acid per square foot of seed bed, applied just after the seed is sown and covered. It should be dissolved in 500 to 1,000 c. c. (1 to 2 pints) of water per square foot of bed before applying. The drier the soil before treatment, the more water should be used in dissolving the acid. No treatment applied after germination begins can have the maxi- mum value in controlling the disease, because the damping-off para- DAM H \i, nl'l' IX FOREST NURSERIES. 27 sites frequently, it' not usually, do part of their work before the seed- lings appeal' above the soil. Furthermore, any treatment at all effect- ive against the disease is almost certain to hurt the seedlings if applied after the seed starts to sprout. Both the acid and copper-sulphate treatments which have been found useful in pine seed beds are of very doubtful value for most hosts other than conifers, as the angiosperms on which observations have so far been made are too easily injured by the disinfectants. The weeds in the nurseries have been injured or entirely kept from appearing by treatments which caused no injury to the pines. CAUSAL FUNGI. CORTICIUM VAGUM. Occurrence and parasitism. — Tn a recent publication (68) Corti- cium vagum B. and C. (C. vagum solard Burt. Hypochnus solani Pril. et Del., the common damping-off Rhizoctonia) has been reported on a number of conifers. Inoculation, reisolation, and reinoculation on pine have established its parisitism on this host beyond a reason- able doubt, though in these inoculations, as in most, if not all, the work which has been done with the fungus on angiospermous hosts, the cultures employed have been from plantings of diseased tissue instead of from single spores. The inoculation experiments have confirmed the field observations indicating that this fungus is fully as able to cause loss by destroying germinating seed below the soil surface as to cause damping-off of the better known type after the seedlings appear above the soil surface. An extensive list of angiosperms on which the fungus has been reported is given by Peltier (98). Cross-inoculations between the pines (G8), on the one hand, and potato (40) and sugar beet (38) have shown the same strains to be parasitic on both conifers and angiosperms and established the physiological as well as the morpho- logical identity of the fungus attacking pines with the common Corticium vagum. Now that Duggar (34) has offered strong, though not yet entirely conclusive, evidence of the identity of C. vagum with the European " vermehrungspilz " (the MonUiopsis aderholdil of Ruhland; 115) it is to be presumed that it is a cause of damping-off of conifers in Europe as well as in Amer- ica, though no reports of it on conifers have been so far en- countered in European literature. The Rhizoctonia reported by Somerville (132) on Pinus sylvestris and the Rhizoctonia strobi de- scribed by Scholz (121) as killing young Pinus strobus w r ere both on trees more than 4 years old, so that they had no relation to damp- ing-off. Furthermore, the first of these was apparently the old Rhizoctonia violacea, now known as /?. crocorum (i?. medacaginis) , a fungus entirely distinct from Corticium vagum, probably belong- 28 BULLETIN 934, U. S. DEPARTMENT OF AGRICULTURE. ing to an altogether differenl group of fungi and not known to c;\u^' damping-off of any host. Rhizoctonia strobi is not sufficiently de- scribed to allow determination of its identity. Variations in virulence. — In the inoculations earlier reported on conifers, different strains of Corticium vagwm were said to vary greatly in virulence (68). Further examination of the data on which this statement Mas based yields confirmatory evidence. Part of this evidence is shown graphically in figures 1, 2, and 10. The experiments on which these graphs were based involved at the time of seed sowing the addition to the soil of apparently pure cultures of C. vagum. Throughout each experiment the different units received HOST YERR £XPT. 5 0:60 $60 %40 %20 S o MNUS BSWKG//9A//4 P/A/OS fi£S/NO&/9 1913 /9/S /9/S /9/Y /9/S 31 S3 S9 7/ 72. e ■ o o Fig. 10. — Diagram showing the relative activity of different strains of Corticium vagum, as indicated by the number of seedlings appearing in inoculated pots. Explanation of symbols : O =Strain 147, from spruce seedlings, Washington, D. C, 1910; V =strain 213, from sugar beet seedlings, Washington, D. C, 1911 ; H=strain 230, from Elaeagnus sp., Kansas, 1913 ; □ =strain 233, from the same lesion as strain 230. Strains re- isolated from these, the results of which appear in experiments Nos. 71 and 72, are indicated by the same signs as the original strains used in the inoculations from which they were taken. The original strains in experiments Nos. 71 and 72 are indicated by arrows. equal quantities of seed, and the culture substratum used in inoculat- ing was the same for all strains. Experiments 36, 45, 47, 49, and 51 were conducted on plats in out-of-door drill-sown beds, experiment 36 on an alkaline soil, all of which had been heated in a moist con- dition at a temperature of not less than 80° C. for not less than 10 minutes, 2 and experiments 45, 47, 49, and 51 on a sand which had 2 This temperature is probably high enough to eliminate dainping-off organisms. Tests by Dr. Theodore C Merrill indicate that the three most virulent parasites so far worked witli are killed by placing agar tube cultures for 10-minute periods in water ai the following temperatures : Pythium debaryanum, G~>° C. ; Corticium vagum, 50° C. for mycelium and (>o° c. for sclerolia; Fusarium moniUforme, 70° C. Both the Pythium and Fusarium cultures contained spores. The possibility of the survival of oospores which would not be capable of germination for several months was apparently eliminated by the writer, who retained Dr. Merrill's heated Pythium tubes and made final transfers from them 7i months after heating, still without securing growth. Plenty of typical oospores were present in the part of the heated culture from which transfers were made. DAMPIXC-OFF IX FOREST NURSERIES. 29 been treated with sulphuric acid followed later by lime. The other experiments included in the graphs were on autoclaved sandy loams in pots in the greenhouse. Tn these graphs are included all of the results in which the same groups of strains were used repeatedly in different experiments. In figure 1. the values plotted for experi- ments 36, 49, and 51 are for the number of seedlings which appeared above around, the heavy inoculations and favorable conditions for damping-off in these experiments being such that even weak strains caused heavy losses and the survivals therefore do not give differ- ential results. Comparison of the survival data in the other experi- ments in figure 1 with the emergence data for the same strains in that figure and in figure 10 indicates that the strains best able to reduce survival are also the ones best able to reduce emergence. While the data presented in the graphs are not entirely consistent, it is very evident from them that strains 147, 213, and in a lesser degree 206 were regularly more virulent than most of the strains in tests conducted several years apart on different species of Pinus. It is also evident that certain strains of 1S6 and ISO which appear in figure 2 are quite regularly of low or doubtful virulence. Strains 50, 183. 19-2. -ill. 212, 230, and 233. whose virulence is apparently inter- mediate, show a greater variability. In experiments 36. -45. 47, 49, and 51, in which conditions especially favor parasitism, they may cause practically as serious loss as the regularly virulent strains, the best differential results being shown in experiments in which the disease is less favored. The apparent variation in the relative viru- lence of such strains in different experiments may, of course, mean that their virulence is differently affected by different conditions. It seems rather more probable that the variation in relative activity is to be classed as accidental variation, necessarily great with small units which are subject to numerous uncontrollable variables. It seems entirely possible, however, that part of the observed differences in relative activity may lie due to differences, not in virulence, but in the ability of the different strains to maintain themselves saprophyt- ically in different soils during the period between inoculation and the commencement of germination. For example, strains 230 and 233 came from a nursery in southwestern Kansas in which the soil- acidity exponent, as determined by Dr. L. J. Gillespie, of the United States Bureau of Plant Industry, is 8.4. It seems entirely possible that these strains, rather strongly parasitic in some of the experi- ments, including an experiment on the soil from which they w r ere taken, might prove less able than strains from some other habitats to maintain themselves on some of the eastern soils used in the green- house tests. The source of strains 230 and 233 was furthermore a locality where high soil temperatures are to be expected. The fact 30 BULLETIN 934, U. S. DEPARTMENT OF AGRICULTURE. that experiments 71 and 72. in which they showed the least virulence, were conducted in a colder greenhouse than any of the other tests may have had something to do with the lower activity indicated for these strains. Variation in the temperature requirements of different strains in accordance with the temperature of the source locality has already been demonstrated by Edgerton (35) for one of the anthrac- noses. It is hoped later to determine the temperature and acidity preferences of these two strains as compared with the others used. It should be noted that the consistently weak strain No. L89 (fig. 2). was abnormal in habit, lighter brown, and produced fewer sclerotia than typical strains. The other strains appearing in the graphs showed no conspicuous morphological or cultural differences that were identical. The only other strain which was noticeably abnormal in culture was one from pine seedlings in Kansas, intermediate in habit and color between No. ISO and the typical strains and indicat- ing little more virulence than Xo. 180 in the lV\v experiments in which it was used. It does not appear in figures 1 and 2. but was included in the experiments reported in the following paragraph. Peltier (00) believes low sclerotium-forming capacity to be a sign of degeneration and low virulence; the writer's experience agrees with his as to virulence, but these two strains showed no other evi- dence of lack of vigor. As a further check on the reality of the apparent differences in virulence between different strains, all of the original strains avail- able at the time, a total of 20, were used in the practically duplicate experiments 71 and 72 and the relative survivals of the same strains in the two series mathematically and graphically compared (fig. 11). The very decided correlation between the two experiments indicated by the graph has a coefficient 3 of 0.813±0.042, nineteen times its 3 The correlation coefficient, a very useful thing for many kinds of biological work, which unfortunately has received little attention from plant pathologists, is explained by Secrist (124, p. 43 et seq.) and the process of computation described (124, p. 453—467). A shorter method of computation is given by E. Davenport (30, p. 465—467) : the example he gives is of a series with a large number of varieties, in which the correlation table is employed. Davenport's method is, however, just as useful in such a case as this, in which the number of varieties is too small to make the formal table advantageous. In such a case the computation should be arranged as by Secrist (124. p. 460—461), but the guessed rather than the lino mean used and Davenport's formula employed. If the coefficient is i- 1. the correlation is perfect: if it is there is no correlation,, and if — 1. perfect negative correlation. The significance of a coefficient less than 1 is judged from iis relation to its probable error. King (84), in an excellent discussion of cor relation, uives rules for judging the degree of significance of the coefficient. The correlation coefficient has its greatest potential usefulness in examining apparent causal relations. It is soused in connection with the relation between the hydrogen-ion exponent and damping-off in considering figure 1 2 of the present bulletin. Interexperimental, or, as Harris calls them, " interannnal " correlation coefficients of the sort used for these Corticium strains have been used by Norton (00, p. 51) in measuring the constancy of rust resistance of asparagus strains, by Harris 1 54 l in demonstrating the constancy of differences in various characters between strains or individuals, and they appear to be useful for this purpose jn the present case. DAMPING-OFF IX FOREST NURSERIES. 31 probable error. Peltier's results permit similar correlations for the 18 strains common to his experiments 1 and 1A on carnation cuttings and for the 22 strains common to experiment 1 on cuttings and ex- periment 2 on seedlings. The coefficients found are decidedly lower than those obtained from the experiments on pine. 0.51 ±0.117 for the experiments on cuttings and 0.36±0.124 for the comparison of the results on cuttings with the results on seedlings, hut neverthe- less indicate some interexperimental correlation for the same strains and therefore inherent differences in parasitic ability between the dif- ferent strains. STRftf/VS OFCORT/C/UM l/rfGUM Pig. 11. — Diagram showing the comparative virulence of 20 strains of Corticium vagum in two successive inoculation experiments on Pinus resinosa. The results in experiment Xo. 71 are shown by the solid line, the strains being arranged from left to right in the order of descending virulence indicated by the number of seedlings surviving in this experiment. The results from the use of the same strains in experiment Xo. 72 are shown by the broken line. The obvious correlation between the two curves (coefficient 0.81±0.04) indicates a real difference in virulence between the different strains. The strains indicated by the underscored numbers are original strains and those not under- scored reisolations from the original strains in earlier inoculation experiments on pine seedlings. In the work on pine seedlings, with the possible exception of strains 230 and 233, there was no evidence of attenuation in arti- ficial culture. Strains 147 and 213, isolated in 1910 and 1911, re- spectively, seemed as strongly parasitic in experiments 71 and 72 (1917 and 1918) as any of the five strains isolated in 1916 or the six strains isolated in 1915. Of the 20 strains above mentioned, three pairs were isolated under such conditions and showed such later agreement in performance as to indicate their individual identity. For the purposes of considera- tion in the following paragraph, the One of these probably duplicate strains which happens to have the higher number was eliminated from each pair. The survival figures in pots inoculated with the 17 strains 32 BULLETIN 934, r. s. DEPARTMENT OF AGRICULTURE. remaining, giving the mean of the results in experiments 71 and 72, are shown graphically in figure 13, together with the results of some of Peltier's experiments in which other strains were used. Per- centages of seedlings damped-off after germination are not included in these and most of the other data on pines because the most viru- lent strains often entirely prevent germination, and no value for sub- sequent loss is obtainable. The grouping of most of the writer's strains at the least virulent end of the register (that is, the one with the highest number of living seedlings) is of some interest. The distributions based on the two experiments considered separately - 9 • M 1 1 k ' » "£ 8 ~ » X \ % X » A Q. \ 1 \ \ 1 \ X % i V_ » / \ -UiT ♦— - — ' ^ .^/ * * \ 1 \ »w_ o X ' x ^^*- v V V 4 i \ ■$ 6 - \ ' _ \ A 5 - s - \ \ \ \ i \ i \ I -7 1 \ 1 \ \ -' \_ 1 \ % 1. I I &r/moN I IO II 12 13 /f- 15 16 17 Fig. 12. — Diagram showing the relation between damping-off of conifers (broken line) and soil acidity (solid line). The acidity of soil samples from the different. nurseries, determined by Dr. L. J. Gillespie, is reported as P H 7, indicating approximate neutrality while P a 6 indicates ten. and T H 5 one hundred times as great a hydrogen-ion concentra- tion as I'h" ; therefore the lower the hydrogen-ion exponent line, the greater the acidity. The seriousness of damping-off at each nursery is on an arbitrary scale in which nurseries with negligible loss are rated as 1, and the nursery which suffered most is rated as 10. These values are estimates, though for some of the nurseries extensive counts were available on which the estimates were based. agreed Aery well in this grouping. The minor group at the end of extreme virulence is not taken to indicate an actual grouping but, rather, an artificial one, due to the fact that both the strongest strains and some less strong were thrown into the same group by the lack of additional seedlings for the stronger strains to kill. This lack of additional seedlings constituted a limiting factor. In other words, conditions favored damping-off even in these two experiments too much to permit completely differential results for the more viru- lent strains. Despite this artificial limit preventing the full vari- ability becoming evident, the coefficient of variability of the survivals DAMPING-OFF IN FOREST NURSERIES. 33 had the high value of 63±9.7 per cent. The graph indicates also a decided, though less extreme, degree of variability for Peltier's strains on carnations; the survivals for the 18 strains which he used in both of his experiments on cuttings have a variability coefficient of 29±3.5 per cent and the , _ ) -' , > strains in experiment 2 on seedlings 55±G.9 per cent. /=>YTH/UM DEB/9RY/9/VI/M • oo CORT/C/UM VrtGUM Wtf/TEftS &TRP/NS ON R//VE D □ □ CD □ DD □ □ D [TDDD cP nP&T/ERS STR/9/NS O/V ' CfiftNtiT/ON CUTT/NGS an □ ODDD DD D DD D DD □ PELT/ER'S STR/9/NS ON CRRHRTIOM SEEDL/NGS a a DD D DDD DD DD DDDDDDD DDDD O 20 40 60 eo /oo PI/9MTS &ORI//V//V0 PER /OOJN CONTROLS Fig. 13. — Diagram showing the results of inoculations with 17 strains of Corticium vagum and 3,"> strains of Pythium debaryanum, arranged in decreasing order of virulence from left to right, as indicated by the survivals in pots of pine seedlings artificially inoculated with them. The Pythium results represent the mean survivals in 5 pots inoculated with each strain in each of experiments Nos. (!(!, o) that a Fusariumlike fungus was able to cor- rode the young epidermis of pine seedlings has already been men- tioned. PYTHIUM DEBARYANUM. Pythium debaryanum llesse (Artotrogus debaryanwm Atkinson, Luczdium pytJiioides Lohde) has been known since 1874 (74, 86) as a common cause of damping-off of various angiosperms. The first known observation was made by De Bary about 1864 (74). Despite the large number of hosts on which it has been listed, its parasitism has been definitely established on few. Peters (100) has successfully inoculated sugar beets with pure cultures ; at least part of his strains, including presumably part or all of those he used in inoculation tests, were obtained from single spores. Edson (38) working with the same host, reisolated the fungus from inoculated seedlings, and made reinoculations with it. Both find it able to cause root sickness of plants not attacked early enough to be killed outright. Johnson (82) and Knechtel (85) have caused damping-off of tobacco seed- lings with it, and the former reported it also able to persist in the cortex and kill the lower leaves of tobacco plants which survived attack. The fungus has long been reputed parasitic on potato tubers and has now been found by Hawkins (70) to be the chief cause of the rot known as "leak" in California. Peters (99) made success- ful inoculations with pure cultures on cuttings of Pelargonium. Most of the reports of parasitism, however, have been based on microscopic examination or more or less crude inoculation experi- ments. Noteworthy among the latter are those reported by Hesse (74) on Camelina sativa in the original description of the fungus. These were made before pure-culture technique had come into use with fungi, but were so thoroughly checked by microscopic obser- vations at every step in the process that they must be admitted as very good evidence of the parasitism of the fungus. A number of reported angiospermous hosts are listed by Butler (23), Voglino (143), and Johnson (82, p. 34, footnote, and p. 35). Reinking (107) recently reported Canica papaya as attacked. A host which the writer has not found in the literature is rice, found by Dr. Haven Metcalf seriously attacked in the seedling stage in a field in South Carolina. A second apparently new host for the fungus is fenu- greek {Trigonella foenum-graecum) . The writer found oospores tvpical of Pythium debaryanum in the tissues of damped-off seed- 36 BULLETIN 934, U. S. DEPARTMENT OF AGRICULTURE. lings received by Prof. William T. Home, from Sonoma County, Calif., with the statement that the disease was seriously affecting the stand. The fungus was easily isolated, and the results of suc- cessful inoculations on pine with the cultures obtained are included in Table V (p. 47). A fungus resembling P. debaryanum was also found in damped-off cowpea (Vigna sp.) seedlings grown in rotation with pines at a Nebraska nursery. Pythdwn equiseti Sadebeck, reported as parastic on the prothallia of Kijii'ix' turn arvenxt\ was successfully used by Sadebeck (117) in crude cross-inoculations direct from E. arvense to potato tubers. I>e Bary (5) reversed the direction of the experiment between eryptogamous and phanerogamous hosts by successfully inoculating prothallia of Equisetum arvense with Pythium debaryanum directly from diseased Lepidium seedlings. He also secured positive results on prothallia of the fern Todea africana by the same method. The Equisetum prothallia he found to be especially favorable media on which to develop Pythium debaryanum,. Fischer considers the fungus found by Bruchmann (17) and Goebel (49) on prothallia of Lycopodium sp. as probably identical with P. debaryanum. A care- ful reading of the original articles is sufficient to show that the sym- biotic fungus wdiich they described was an entirely different or- ganism. Saprolegnia schachtii and Sporodospora jungermanniae, re- ported on two of the Hepa'ticse, are of doubtful position (42, p. 403), though Butler ('23, p. 89), after a survey of the literature, apparently favors the view that the former is distinct from the damping-off fungus. De Bary (5) reported Vaucheria and Spirogyra apparently immune against P. debaryanum. Early references to Pythium debaryanum in connection with gynosperms seem to have been based on the probability that it would be found to be the cause of damping-off in conifers (6 ; 97 ; 134, p. 27). The first actual finding of the fungus in any gymnosperms of which the writer is aware is indicated by a label marked Pythium debaryanum in the handwriting of Mrs. Flora W. Patterson on a package of coniferous seedlings from a New York nursery collected in 1904. 4 The seedlings, judging from the several rather long cotyle- dons and the fact that both cotyledons and primary leaves are denticu- late, are probably of one of the species of Pinus having medium-sized seed. In 1908 Dr. R. J. Pool, of the University of Nebraska, and his student, Mr. H. S. Stevenson, obtained in culture from damped-off coniferous seedlings a nonseptate fungus which was probably Pythium debaryanum, but which formed no distinctive spores on the media on which it was grown. A year later the writer obtained the fungus from pine seedlings at the same nursery and reported it as 4 In the Office of Pathological Collections, United States Bureau of Plant Industry. DAMPING-OPE IX FOREST KTURSERIES. 37 parasitic on pines in preliminary inoculation experiments (62). In L910 Spaulding (b'57) found it on spruce in NeAv York, and Hof niann (76) later made successful inoculations on both pine and spruce seedlings, using P. debaryanum, cultures both from aerial trap plates and from recently damped-off seedlings of cabbage, radish, and Russian thistle {Salsola tragus). Hofmann's work, detailed notes of which the writer has been permitted to examine, was done with cul- tures which were contaminated by molds, but was checked up by microscopic examination of the lesions resulting, which showed the affected tissues filled with nonseptate hyphse. His results are taken as a rather strong indication that /\ debaryanwm, attacks spruce as well as pine and that the fungus attacking conifers is physiologically as well as morphologically identical with that causing the damping-off of angiosperms. There thus appears from the literature to be reason to believe that Py tit iu m debaryaw/um is parasitic on representatives of two groups of the Pteridophyta and on gymnosperms, as well as on various monocotyledons and dicotyledons, a range of hosts not only remark- able but perhaps unequaled in our present knowledge of plant parasites. Final published proof of parasitism seems to be available for three or four species of dicotyledons only. Additional inocula- tions on conifers with strains isolated from various other hosts are reported in the present bulletin. Some of the detailed evidence neces- sary for complete proof of the parasitism of the Pythium on conifers, lacking in experiments previously reported because of the doubtful purity of the cultures used and failure to reisolate and reinoculate with the organism, is also given here, together with evidence of the ability of the parasite to cause root sickness of pines too old to suffer from damping-off. Descriptive data of interest on Pythium debaryanwm have been supplied by Hesse (74),. De Bary (5), Ward (144), Miyake (89), Butler (23), and Butler and Kulkarni (24). An important contri- bution to the physiology of the fungus and the factors controlling its passage through the tissues of one of its hosts has recently been made in the previously mentioned paper of Hawkins and Harvey (71). IDENTITY AND ISOLATION. The fungus in the writer's cultures referred to Pythium debary- anum Hesse has been so called for the following reasons : (1) The morphological characters agree with those described and figured for Pythium debaryanum by other workers and with those of strains obtained from Dr. H. A. Edson under this name. (2) The absence of zoospores in the writer's cultures agrees with the experi- ence of others with PytMum debaryanum (2, 5, 23, 24, 38, 100), nil workers with pure cultures having obtained zoospores infrequently, if at all. The earliest work by Hesse (74) in which zoospores were apparently produced 38 BULLETIN 934, l\ s. DEPARTMENT OF AGRICULTURE. readily :ii certain times of the year was done before the development of pure- culture methods. Water cultures kepi in the dark and in the Light, at constant and at varying temperatures, with nutrient substrata consisting of steamed or outoclaved fragments of potato, carrot, sweet potato, turnip, sugar beet, corn meal or rice, nutrient agar, sugar-beet seedlings, and insects have all produced only sexual fruiting bodies and chlamydospores (the so-called conidia). (3) The successful cross-inoculations, those which Edson (38) used on sugar- beel strains and the writer had found parasitic on pine and had used on pine, strains winch Hawkins had found parasitic on potato tubers and Edson on' sugar beets, confirm the work of ELofmann (77) in indicating that the Pythium which causes the damping-off pine is a parasite on entirely unrelated species of host plants, a commonly recognized characteristic of Pythi/wm debaryanum. The organism is easily isolated from recently damped-off conifer- ous seedlings or from soil direct by placing the seedlings or a lump of soil at the edge of a Petri dish of solidified prune agar and transfer- ring to tubes mycelium from the advancing edge of the resulting growth. It has been found in or obtained from damped-off conifers in California. Kansas,Nebraska,Minnesota,and the District of Columbia, as well as in cultures made by Mr. Glenn G. Hahn in Michigan. Plcea engd niduni, P. sitcTu /<*is, Tsuga mertensiana, Pinus nigra austriaca, and Pseudotsuga taxifolia tire the coniferous hosts from which cul- tures of Pythium debaryanum have been obtained. It has been iso- lated directly from soil not only in coniferous seed beds but from open grassland in California not adjacent to any seed bed or culti- vated crop. Unless Mucor is abundant, Pythium is commonly ob- tained in apparently pure condition on the first transfer from the plate, as prune agar appears unfavorable for most bacterial growth while allowing rapid spread of the Pythium. On media made from prunes which taste sweet and with a total gross weight of not more than 40 or 50 grams per liter of medium, the Pythium will make a rapid growth, often extending radially 1 mm. per hour at tempera- tures in the neighborhood of 22° C. and produce both chlamydospores and oospores. A less valuable medium for isolation work, but more convenient for subcultures than any other which has been tested, is autoclaved corn-meal agar. The growth is not luxuriant, but spores are always formed and the cultures seem to be as long lived as those on any other medium, retransfer being rarely necessary more often than twice a year. Much stronger growth and more abundant fruit- ing is obtained on such media as sugar-beet or rice-stem agar, but the leathery surface of the culture on such media makes transferring difficult. On rice grains, corn-meal mush, beef agar, and on corn- meal agar plus 2 per cent dextose or sucrose no spores are formed and the cultures are short lived, though growth is heavy and on the last-named medium extremely rapid. On agar containing the juice from sour prunes or on corn-meal agar prepared without sub- jecting it to the high temperature of the autoclave, both growth and fruiting have been very poor or even lacking. DAMPING OFF IX FOREST NURSERIES. 39 In both artificial cultures and in the tissues of coniferous and dicotyledonous hosts the numerous strains observed showed no con- spicuous differences in the size or other characters of the spores pro- duced, though noticeable and constant abnormality was found in one strain in the readiness with which spores were produced and in two strains in the ratio between chlamydospores and oospores in agar cultures. In the first-mentioned strain, obtained from pine in Kansas, and in cultures reisolated from seedlings inoculated with it, both chlamydospores and oospores are produced tardily and so scantily that it is often difficult to find them. In most strains, on the other hand, almost the entire contents of the mycelium are promptly emptied into the spores as soon as the limits of rapid vege- tative growth are reached. In another abnormal strain from pine from the same locality, and in still another furnished by Hawkins from a California potato, chlamydospores are produced in large numbers, but oospores are few. In many other strains, including several from California, the opposite condition obtains, oospores in plate cultures being decidedly more numerous than chlamydospores. These peculiarities of particular strains seem to be fairly constant characters, the first abnormal strain mentioned having been under observation for more than three years without any change in its tend- ency to scanty fruiting, and the low ratio of oospores to chlamydo- spores having been constant during the shorter periods over which the observation of the other strains extended. In view of the small variation between different strains in the matter of speed of growth, a purely vegetative character, this variation in reproductive habit is somewhat surprising. The strain which produced spores infre- quently was unquestionably parasitic, though it killed fewer seed- lings than the average Pythlum deharycunum strains. The strains with the high ratio of chlamydospores were both of at least average virulence on pine. Oospores in the strains the writer has had in culture, whether ex- amined in agar, in water cultures, or in root tissues, have ordinarily been somewhat larger than the diameter of 14 or 15 to 18 pi given in a number of the descriptions. The maximum range has been 12.8 to 20.6 [j., the same strain sometimes being well down within the usual size range and sometimes ranging from 17 to 20 \i. The largest oogones observed were 26 \i in diameter. Various stages of fertili- zation are shown in Plate I, figures 2 to 4. Chlamydospores attain a maximum diameter in the case of the limoniform intercalary forms of 32 [x, and spherical chlamydospores sometimes reach a diameter of 28 [jl. There is no lower limit for these bodies, as under unfavor- able conditions — e. g., in sour-prune agar — they are sometimes all less than 15 [/. in diameter, and the smaller ones are little larger than the 40 BULLETIN 934, 0. S. DEPARTMENT OF AGRICULTURE. hyphse which bear them. Both oogones and chlamydospores may be either termina] or intercalary. The normal hyphse are large, varying from 3 to 7 \j. and sometimes more in diameter. Typical hyphse, showing the false septa developed at the boundary of the protoplasm and the portions of the hyphse which have been evacuated in the extension of the younger parts, are shown in Plate I, figure 1. At points at which the ends of hyphse come in contact with the glass of tin 1 culture dish, peculiar contact swell- ings are produced (PL I, figs. 5 to 7), much the shape and size of antheridia, but not walled off from the adjacent hyphse and having no apparent significance in the life history of the fungus. These are not always terminal (PL I, fig. 8). It is noteworthy that Hesse de- scribed contact swellings at the tips of the hyphse just before pene- trating the epidermis of Cameliria sativa. The asexual nonsporangial fruiting bodies of Pythium debaryanum are referred to as chlamydospores rather than as conidia, though in most of the previous literature the latter term has been used for them. Hesse called the terminal bodies conidia and the intercalary, gemmae (74). It is believed that the best terminology and the one which should be followed for all fungi, as it now is for most, is that which limits the term conidium to a spore which is adapted primarily for aerial distribution or which is at least readily separated as soon as it is mature from the parent hypha from which it arises. The most typical conidium, in fact, is a spore which is abstricted by the parent hypha at maturity. The asexual spores of this Pythium re- main attached to the parent hypha? indefinitely even after the hyphse are dead and empty. It is a common thing to find numbers of these bodies in water cultures, still attached to hyphse which are so com- pletely empty that it is only with favorable lighting that their thin colorless walls can be seen. So firm is the attachment that vigorous shaking is required to release any considerable proportion of the spores. It seems probable that in nature the spores are released chiefly as a result of the destruction of the hypha? walls by bacteria. While there is reason to think that Pythium debaryanum is some- times disseminated by wind, it is by no means certain that it is through the medium of these spores. It is true that these bodies have thinner walls than are commonly found in chlamydospores of some other fungi, but they have somewhat thickened walls as compared with the vegetative hyphse, and they are commonly intercalary. These facts, and (lie indications that they are better able to with- stand unfavorable conditions than are the hyphse, all tend to entitle them to rank as chlamydospores. De Bary (5) speaks of them as kv dauerconidia." Their ability to stand drying is not entirely demon- strated, but is indicated by the relative longevity of the fungus on different media. On beef agar and on rice, on which no spores are Bui, 934, U. S. Dept. of Agriculture. Plate I Pythium debaryanum from Artificial Cultures. Fig. 1.— Hyphae, showing old portions of hyph:« and false septa separating them from the portions still containing protoplasm. Figs. 2 to 4.— Various stages of oospore formation. Figs. 5 to 8.— Hyphal swellings at points of contact with glass. From camera-lucida drawings. DAMPING-OFF IX FOREST NURSERIES. 41 formed, a few tests indicate that the fungus is very short lived, sometimes dying in a month. On media on which spores are pro- duced, transfers any time before the sixth month, and often as late as the tenth month, start immediate growth on fresh media. This is true even for strains which produce few or no oospores. The im- mediate commencement of growth from cultures 3 or 4 months old is taken as an indication that the new growth results from the asexual spores, as oospores are commonly believed to require a rest- ing period of five or more months before they are able to ger- minate (5, 38). INOCULATION ON STERILIZED SOIL. Pythiwn debaryarwm has been used in inoculation in pots of recently autoclaved soil in 16 different series of tests. In 10 of these, fragments of agar cultures were scattered over about one- fourth of the area at the side of each pot when seed was sown; in 2 of these 10 and also in 2 other tests some pots were inoculated over their entire surface. In every one of these 12 heavily inoculated series positive results were indicated by smaller emergence and where any considerable number of sprouting seeds escaped the fungus by heavier damping-off loss in the inoculated pots than in the controls. In some cases the fungus killed all or practically all of the seed or seedlings in the inoculated pots before they emerged from the soil. In a total of 7 series, part or all of the pots received lighter in- oculations, consisting of one or two small fragments of an agar culture placed just below the surface of the soil at the edge of each pot. In 5 of these success was indicated. In the sixth and seventh also of these lightly inoculated sets, there was more damping-off in the inoculated pots than in the controls, but the difference was neg- ligible. The damping-off caused by light inoculations was in general distinctly less than that resulting from broadcast inoculations. To sum up the evidence : Sixteen separate experiments were conducted with Pythium debaryanum on pine seedlings in autoclaved soil, and in every one fewer seedlings survived in the inoculated pots than in the checks ; the difference in most of the experiments was large. Of the successful inoculation experiments — that is, those in which the difference between the inoculated pots and the checks seemed significant — 9 series included jack pine (Pinus banksiana) , 7 series western yellow pine (P. ponderosa, Colorado and New Mexico seed), and 3 series red pine (P. resinosa). In addition to the pines, Doug- las fir (Pseudotsuga taxifolia, Colorado seed) was grown in two large plats in one of the earlier series, one being inoculated over its entire surface with Pythium debaryanum. Because of the poor quality of the seed in the test on Douglas fir, too few seedlings were obtained to furnish a decisive test, but the difference in the emergence in the inoculated plat and the control affords preliminary evidence that 42 BULLETIN 934, U. s. DEPARTMENT OF A.GRICULT1 RE. Pythium can cause the "germination-loss" type of damping-off in Douglas fir as well as in specie's of Pinus. Of the seeds sown in the control plat !•"> produced seedlings which appeared above the soil, while only two seedlings appeared from an equal number of seeds sown in the inoculated plat. Altogether. 38 strains, excluding reisolations, have been tested on one or more of the 3 pine species. Strains from both the Pacific coast and the eastern United States and from a number of hosts other than pines were among those used. With the exception of two or three strains from a pine nursery in Michigan, the use of which was followed by so little damping-off as to leave their parasitism uncertain, all of the strains proved parasitic under favorable condi- tions, though some were more virulent than others. The positive re- sults in the 14 successful experiments are based on the comparison of a total of approximately 1,160 inoculated pots with 195 control pots. Table III. -Inoculation experiments with Pythium debaryawum in pots of steri- lized soil. Pythium strain. Num- ber of potS. Inoculation method. Results. Series, experiment number, and host. No. Initial st rain from which it wasreisolated. Emerged 1 per 5-pOl unit ). Damp- ing-oil after germi- nation (per cent). Sur- vival ( per .">- pot unit). Series A.— Initial inocula- tions: [218 5 5 5 5 5 5 5 5 6 5 5 5 5 5 5 Agarcultures broadcast atoneside of pot. Noinoculuni do Agarcultures at single point in each pot. Noinoculuni Agarcultures broadcast atoneside of pot. do Noinoculuni. do do Agarcultures broadcast atoneside of pot. do do No inoculum (>) 1 74 82 41 55 16 18 90 82 82 14 3 13 78 14 78 100 2S 81 89 72 67 67 72 No. 5S, Pinus banksiana'. . Controls. ..do 74 82 [295 30 No.5S, Pinus ponderosa.. 55 [258 3 ..do 2 Controls. do 90 82 .do 82 4 347« 1 348a 4 .Controla 1338 (controls. 78 SeeiesB.— Reisolatedstrains: >2, Pinus banksiana. . No. 295 (in P. ponderosa, expt. 58). 4 78 «.V different soil used in these pots from that used wit h strain 25S and the first three control units. J>A11 inoculations with fragments of agar cultures scattered broadcast at one side of the pot, including about one-fourth of its area. Nothing was added to the controls in experiment 62, but sterile agar was added to the controls in experiments 66, 67, and 68. DAMPING-OFF IX FOBEST NURSERIES. 43 Table III. — Inoculation experiments with Pythium debaryanum in pots of steri lized soil < 'ontinued. Pyth nun train. Num- te nil . Damp- Series, experiment number, and host. No. Initialstrain from which it ber of pots. Inoculat inn method. Emerged i per 5-pol ing-oil after germi- Sur- vival (per 5- wasreisolated. unit i. nal ion pof (per unit i. cent). Series B.— Heisolated strains- Continued. 338 No. 295 (in P. ponderosa, expl . 58). 5 (a) 9 67 3 345 No. 218 (in P. banksiana, expt. 58). 5 (a) 15 33 10 414 No. 258 (in P. banksiana. 5 (a) 36 25 27 No. 06, Pinus banksiana. . expt. 62, 2d unit). 415 do 5 (a) («) 59 10 54 419 No. 348 (in P. 5 25 100 banksiana, expt. 62). 450 No. 347 (in P. banksiana, expt. 62). 5 (") 41 72 11 ( (Jilt nils. 33S 25 5 75 7 14 57 64 No. 295 (in P. ( a ) 3 ponderosa, expt. 58). No. 218 (in P. banksiana, expt. 58). 5 (a) 24 37 15 414 No. 258 tin P. banksiana, expt. 62, 2d unit). 5 (a) 57 24 44 No. 67, Pinus banksiana. . No. 258 (in P. banksiana, expt. 62, 1st unit i. 5 (a) 30 37 19 419 No. 348 (in P. banksiana, expt. 62). 5 (a) 62 65 22 450 No. 347 (in P. banksiana, exnt. 62). 5 (a) 53 27 39 (.Controls 338 23 5 87 85 5 26 83 No. 295 (in P. (a) 63 ponderosa, expt. 58). 345 No. 218 (in P. banksiana, expt. 58). 5 (a) 76 24 58 414 No. 258 (in P. banksiana. expt. 62, 2d unit). 5 (») 98 14 84 No. 68, Pinus resinosa 415 No. 2.W (in P. banksiana, expt. 62, 1st unit ). 5 (•) 92 11 82 419 No. 348 (in P. banksiana, expt. 62). 5 («) 95 45 52 4.50 No. 347 (in P. banksiana, expt. 62). 5 (o) 84 40 51 [Controls 18 104 104 a All inoculations with fragments of agar cultures scattered broadcast at one side of the pot, including about one-fourth of its area. Nothing was added to the controls in experiment 62, but sterile agar was added to the controls in experiments 66, 67, and 68. As has been stated, securing positive results did not always mean that the control pots remain uninfected. Even with the most care- ful treatment and the use of boiled water throughout the experiment 14 BULLETIN 934, r. S. DEPARTMENT OF AGRICULTURE. it proved difficult to keep the control pots entirely free from damp- ing-off. Cultures from seedlings which damped-off spontaneously iu control pots indicated that Pythium us well as Fusarium may be introduced by accident, ever when insects, birds, and rodents are ex- cluded. This agrees with the evidence of Hofmann (77) that Pythium debanjanum is sometimes disseminated by wind, despite its apparent lack of adaptation to wind distribution. It is also in- dicated, however, that unheated tap water increases damping-off when used on control pots and probably carries this semiaquatic fungus. Notwithstanding infections in the controls of a number of the experiments, it is believed that the large number of pots whose results have been considered in drawing conclusions, the fact that the Pythium pots lost more heavily than the controls in every one of the 16 experiments, and the magnitude of the differences between both the emergence and subsequent damping-ofF figures for the inoculated pots and the controls in most of the experiments establish the parasitism of the fungus in inoculation on autoclaved soil without it being neces- sary to present all the evidence in detail. The pot series which in- volved reisolation and reinoculation (Table III), together with the results given for other purposes in Tables V and VI, seem sufficient by themselves to establish a parasitic relationship. REISOLATION AND REINOCULATION. In a number of the experiments dead seedlings in the inoculated pots were examined and typical Pythium hyphse and spores were found. In three of the experiments in which the controls remained entirely free from disease up to the time the experiment was closed, reisolations and reinoculations were made in accordance with the usual rules of proof. The results are given in Table III. From Table III it will be seen that five strains reisolated from Pinus banksiana and one strain reisolated from P. ponderosa gave positive results in pots of P. banksiana and P. resinosa. In addition to the reinoculations shown in the table, the strain reisolated from Pinus ponderosa (No. 338) was again reisolated in duplicate from P. banksiana in experiment G2, and both these secondary reisolations gave cultures which w T ere parasitic on P. banksiana and P. resinosa in subsequent inoculations. That the organisms reisolated were actually the same as those used in the initial inoculation is indicated not only by the absence of dis- ease in the control pots of experiments 58 and 62, but by the distinc- tive characters of some of the strains. In general, cultures reisolated from strongly parasitic initial strains were themselves strongly parasitic and vice versa. This is shown by comparing the figures for the initial and reisolated strains, as shown in Table IV. Each figure represents the average results in 10 pots of jack pine and 5 of red DAMPING-OFF IX FOREST NURSERIES. 45 pine in experiments <'»<;, 67, and 68 combined. The figures are rel- ative* the mean survival of 47 different strains used in all three ex- periments being taken as 10. A survival figure above 1 ( » therefore means that the strain was less destructive than the average Pythium, and a figure below 10 indicates more than average virulence. As strain 218 was not used in these three experiments, strain 345 can not be compared. Table IV. — Comparative virulence of original cultures and reisolated strains of Pythium dehunjunum in c.vperhuciit.s (Hi, 67, a in! 68. Pythium strain. Description. Rela- tive sur- vival. Pythium strain. Description. Rela- l i\ r sur- vival. No. 258 i Original culture 16 12 12 4 4 6 No 409 Reisolated from 338 No. 414 Reisolated from 258 do No. 347... No. 415 No. 450 Reisolated from 347 7 10 4 No. 295 < >riginal culture No. 348 No. 338 Reisolated from 338 No. 419 Reisolated from 34S No. 408 These figures are not absolutely consistent, but are to be viewed as contributing to the evidence furnished by the absence of damping- off in the control of experiments 58 and 62 that the cultures reiso- lated in those experiments were actually identical with the original strains. A further proof of this identity is in the fruiting tendencies of the strains. Both Nos. 414 and 415, the strains reisolated from original strain 258, exhibited the peculiarly sparse spore production which has been characteristic of strain 258 for the entire period dur- ing which it has teen in culture. The other reisolated strains, taken from pots inoculated with normally fruiting strains, all showed normal spore production. PURITY OF CULTURES. A slight deficiency in the evidence as to the parasitism of Pythium debaryanum both in the writer's work and apparently in all previous investigations except those of Peters (100) and possibly Knechtel 5 is the lack of single-spore cultures. The large number of strains which have remained apparently pure through numerous subcultures and have retained their individual characteristics as to virulence and fruiting tendencies (one strain having been carried on artificial media continuously for eight years without material change) give very strong justification for believing that the cultures used were pure. In three early inoculation tests the cultures used were after- wards found to have been contaminated *by bacteria carried by mites; the positive results obtained in these three were the basis of the ear- 5 Knechtel's work in Rumanian has been available to the writer only in the German abstract, which makes an ambiguous statement on this point. 46 BULLETIN 984, U. S. DEPARTMENT OF AGRICULTURE. liest report of pathogenicity (02), but have not been used as evidence in the present bulletin, though the contaminating bacteria in one of them, when tested independently, showed no evidence of parasitism. In all the experiments mentioned in the foregoing as giving positive results with Pythium the cultures used were apparently pure. Cultures from single chlamydospores should be reasonably easy to secure, part of the chlamydospores in water cultures being separa- ble from the mycelium by vigorous slinking, and further inoculation tests with cultures so obtained are probably desirable. The experi- ments so far conducted are believed to be sufficiently conclusive, how- ever, for all practical purposes. For isolation of absolutely pure lines of this or any other coenocytic fungus, it is evident, as pointed out by Dr. W. H. Weston (146), that isolations should be made from the uninucleate swarm spores. For the determination of the bare fact of pathogenicity such a refinement would be superfluous. CROSS-INOCULATIONS. The physiological identity of the Pythium attacking coniferous seedlings with the one which attacks dicotyledons is indicated by the results of several inoculation experiments. The last two experi- ments, one with jack pine and one with red pine for the host, are the most comprehensive and give results sufficiently decisive so that quotation of the corroborative evidence from earlier experiments is considered unnecessar}^. The results appear in Table V. Each unit consisted of five 3-inch pots except in the controls, in which 23 pots were used in the jack-pine experiment and 18 in that with red pine. In the second experiment, separate records were kept of the survival in each pot, and the probable error calculated from the controls was less than two seedlings per pot for a single pot, less than 0.9 for a mean of 5 pots, and less than 0.5 for the mean of the 18 control pots. While the number of controls was, of course, in- sufficient to furnish an exact basis for such a calculation, the small value found tends to confirm the impression gained from inspection of the table that considerable confidence can be placed in the results. The difference appearing in Table V between jack pine and red pine in point of susceptibility to germination loss from Pythium agrees with field observations in Nebraska, the red pine at the Bessey Nursery, though on the whole more susceptible than jack pine to dam ping-off losses, having given indication of more resistance to the disease for the first week or two. Inoculations in other experiments on western yellow pine indicate that the strains which attack it are identical \\ it li those attacking jack pine and red pine. The conclusion reached from the cross-inoculation results is that the Pythium causing damping-off of the three species of pine men- DAMIMNi; ul-'l IN L'ORKST N IT.KERIKS. 47 tinned is identical with PytMum debaryanvm, causing leak of potato tubers and the damping-off of seedlings of two dicotyledonous families. Table V. — Results j>( inoculations on jack pine and red pine with Pythium deoaryanum from various hosts. Host from which isolated. Inoculation results. On jack pine. On red pine. Emerged ( per 5-pot unit). Damp- ing-oir (per cent). Sur- vival (per 5-p ot unit). Emerged (per 5-pot unit). Damp- ing-off (per cent). Sur- vival (per pot). No. 131 a Dicotyledons: 45 45 36 34 30 30 101 62 23 11 15.6 No 8106 Do 7.4 45 35 30 82 32 11.6 No. 294 c 50 28 19 8 32 32 46 19 13 79 62 68 36 48 58 10.2 No. 295 c Originally potato strain 131, but 1 wire inoculated on and reisolatod from sugar-beet seedlings by Ed- 6.6 No. 296d 5.8 32 24 26 70 47 7.4 No. 529 36 1 31 60 49 25 31 102 108 26 22 15.0 No. 530' Do 16.8 4.8 40 28 105 24 16.0 Conifers: Western yellow-pine seedlings No. 258 58 9 53 109 17 18.0 No. 550 15 1 80 42 29 3 30 39 45 -IS 70 .2 No. 555 5.0 Average, spruces Controls 29 ' 55 17 42 84 2.6 87 5 83 104 20.9 n Furnished by Mrs. C. R. Tillotson- has been used b Furnished by Dr. L. A. Hawkins: cause of leak, successfully on sugar-beet seedlings by Dr. H. A. c Furnished by Dr. H. A. Edson. Edson. d Diseased material furnished by Prof. W. T. Home. VARIATIONS IN VIRULENCE OF PYTHIUM STRAINS ON PINE. In Pythium debaryanum strains, as in the case of Corticium vagum, there appeared to be a considerable difference in the parasitic activity of different strains used in the same experiment. Figures 14, 15, and 16 show graphically the results from inoculations with different strains of P. debaryanum in all the experiments in which it was possible to compare directly the activity of different strains. All the inoculations involved at the time of sowing the addition to the soil of cultures on nutrient media in recently autoclaved 3-inch pots. In experiment 31C the inoculum fragments were scattered over the whole pot, in 31D at only one point in each pot, and in the others were distributed over about one-fourth of the pot's area. As noted elsewhere, the variations observed in the results may have been due in part to differences in the ability of the different strains to main- 48 BULLETIN 934, l T . S. DEPARTMENT OF AGRICULTURE. tain themselves saprophytically in the soil used rather than entirely due to difference in virulence. The data shown in figures 15 and 1G indicate in the first place rather more accidental variations in the results with Pythium than with Corticiuin (see figs. 1, 2, 10, and 11). The agreement between original and reisolated strains from the same original source is de- cidedly less good than in the case of Corticium (see experiments 71 and 72, figs. 10 and 11). In general, there are only two strains of HOST Y£/>/? £XPT. 'c//VUS B/WrtS/rtA/rt P/NUS ff£SINOSP /9/3 /9/a /9/S /9/S /9/6 /9/6 /9/6 /9I7 /9/7 /9/S /9/7 31 C 3/D sa S9 62C 62D 66 67 63 7e 71 m \ *x is \ \ \#/ ' ya \\ + -"S^F =±3£- --* \ V* Fig. 14. Diagram showing variations in virulence as indicated by the living seedlings in pots of autoclaved soil inoculated with different strains of Pythium debaryemum. For experiments Nos. .'{1 and 66 to 72, inclusive, the surviving seedlings at the end of two or three weeks after germination are shown. For the other experiments daniping-off was so heavy in the inoculated pots that the survivals did not give differential results for the different strains, and the germinations are therefore shown. The reports are based for experiments Nos. 71 and 72 on 2 or 3 pots for each strain in each experiment, and for the other experiments on not less than 5 pots for each strain. In experiments Nos. (!G, 67, and //Vt/S &#NK&/#N# f?£3/A/0M /9/6 /9/6 1917 1917 620 66 67 63 m : HP E3£IG> °OC\ ■ * 40 hosa YEAR EXPT. ||* Ete Fig. 15. — Diagram showing the results of inoculations with strains of Pythium debaryanum. This figure supplements figure 14, giving the results for original and reisolated strains independently. Each point plotted is base. I on the results in five pots. The object of this diagram is to give an idea of the degree of variability in the success of inoculations. An explanation of the symbols used will be found in the legend of figure 14. 50 BULLETIN 934, U. S. DKI'AKTMKNT OK AC.KIGXJLTURE. the horizontal line showing the locution of the mean for experiment 68 are much larger below the moans than above it in the left-hand portion of the graph, while the reverse is true in the right-hand portion. To this extent the relative activity of the strains in this experiment agrees with the performance of the same strains in the two jack-pine experiments, as shown by the solid line. It can not be decided from an inspection of the graph whether there is a real agreement, in view of the large accidental variation present. How- ever, the correlation coefficient, 0.446+ 0.079, five and one-half times its probable error, indicates a considerable correlation, not as good as was found for the Cbrticium strains, but sufficient to establish a strong presumption that observed differences in activity of the dif- i I A /•% I A / S / i 1 J \ j^AYJrJff- M£#/V eXPTS. 66/7/VO 67 ill' I L_l l_ 'I'll' m\ Fig. 16. — Diagram showing the comparative virulence of 47 strains of Pythium debar yanum in successive inoculation experiments on species of Tinus. The results in experiments Nos. 66 and 67 (on Pinus banksiana) are shown by the solid line, the strains beinj; arranged from left to right in the order of descending virulence indicated by the number of seedlings surviving in those experiments. The results from the use of the same strains in experiment No. 68 (on Pinus resinosa) are shown by the broken line. Such correlation as there is between the two curves (coefficient 0.45±0.08) goes to indicate a real difference in virulence between the different strains. The strains indicated by (he underscored numbers are original strains, -and those not underscored are reisolations from the original strains in earlier inoculation experiments on pine seedlings. ferent strains in these inoculation experiments were in part actually due to differences in the capacity of the strains. It has been suggested in the foregoing that the difficulty in demon- strating constancy in the difference in virulence between the various strains of Pythium debaryanum is due in part to the lack of such extreme differences as were observed between the various Corticium strains. Figure 13 shows the distribution of the different original Pythium strains according to the virulence indicated in the three inoculation experiments of figure 16 (application to autoclaved soil at the time of sowing). Each value plotted is based on the average results in 15 pots. Of the strains used, 21 were from species of pine, 1 from spruce, 2 from potato tubers, 2 from fenugreek, 3 from sugar beet, and 6 from soil direct. Despite the considerable number of DAMPING-OFF IN FOREST NURSERIES. 51 strains, they are not much more representative than the smaller num- ber of Cortieium strains experimented with. All of the strains from soil direct and 11 of the strains from pine were taken at approxi- mately the same time from the same nursery in Michigan hy Mr. (Jlenn G. Halm; despite the fact that these were the most recently isolated of the strains used, nearly all of them proved weak in the inoculations. Of the 17 strains which proved the weakest (out of 35), all but 3 were from this Michigan nursery. The 18 strains from other sources (5 from California, 2 from Minnesota, 2 from Kansas, 1 from Wisconsin, 2 from an unknown locality, and 6 from Washing- ton, D. C, representing two coniferous and three dicotyledonous host genera), as shown by solid circles in figure 13, for the most part were rather closely grouped within the more virulent portion of the range. The coefficient of variability in the survivals allowed by the 35 Pythium debaryanum strains is 39±3.6 per cent, while for the smaller number of original strains of Cortieium vagum in experi- ments 71 and 72 it is 63 ±9.7 per cent. It is evident from figure 13 that if there had not been a disproportionately larger number of strains from the Michigan nursery the variability of the P. de- baryanum strains would have been much less than 39 per cent. The number of strains was, of course, altogether insufficient for either fungus to represent adequately a population as immense as the total number of strains of either of these omnipresent species. The above data, however, contain the only available information of which the writer is aware on variation in the virulence of different strains of P. debaryanum. The evidence as a whole, both from the results shown in figure 13 and the experience with 6 other strains Avhich were not used in the experiments on which figure 13 was based, lead the w r riter to believe that most strains of Pythium debaryanum taken from lesions in plants are ordinarily likely to prove rather virulent parasites on pine seedlings. It further appears that the variation in virulence between the different strains of P. debaryanum on pine seedlings is less than the variation in strains of Cortieium vagum. PYTHIUM INOCULATIONS ON TJNHEATED SOIL. Inoculations with Pythium debaryanum were made in western Kansas on a fine sand containing little humus after treating the soil with acid followed by lime. Commercial sulphuric acid was applied at the rate of 14.8 c. c. per square foot of bed, followed two days later by 25.5 grams of air-slaked lime raked into the soil (0.16 liter of acid and 0.274 kg. of lime per square meter). The acid was diluted before applying with 256 volumes of water. The seeds were sown in drills, and inoculum was placed in the drills at the time of sowing. Each unit involved approximately 11 linear inches of drill, and all 52 BULLETIN 934, U. S. DEPARTMENT OF AGRICULTURE. received equal quantities of seed. Three strongly parasitic strains of Pythium were used, and a total of 12 units of jack pine and an equal number of western yellow pine was inoculated with 12 inter- spersed units of each species as controls. The mean results arc as follows : Pirms banksiana. — Inoculated plats: Emerged, 64.2±4.9; died during the next 17 days. 25 per cent. Control plats: Emerged, 85.5±3.6; died during the next 17 days, 13 per cent. Pinus ponderosa. Inoculated plats: Emerged, 34.6±1.8; died during the aexl 9 days, 39 per cent. Control plats: Emerged, 45.4±1.3; died during the next 9 days, 25 per cent. The difference in emergence apparently due to the inoculation is for the first species three and one-half and for the second nearly five times its probable error. While, of course, 12-unit means are in- sufficient to allow the calculation of entirely reliable probable errors, they give some idea of the amount of variability of the results and the confidence which can be given them. It is impossible to give any such expression applying directly to the damping-off percentages and their differences, for the reason that averages for this item have been made in the writer's work not by averaging the percentages for the individual units but by totaling all the seedlings and the dead seedlings on the plats to be averaged and recalculating the percent- age from these figures. This seems the only safe method, as other- wise units in which germination is low by accident or by the action of parasites will be given an influence on the resultant mean entirely disproportionate to the number of seedlings which they contain. Average values for damping-off obtained by this method and by the method of averaging the percentages of the individual plats or pots are often very different; it not uncommonly happens that the units in which germination is lower than the average also have especially high damping-off percentages, both phenomena being caused by an unusual activity of parasites. In such case to average the percentages themselves usually gives a higher damping-off figure than to total the seedlings for the different units and redetermine the percentage, and the latter practice is considered the better. In the present case the differences in the damping-off percentages obtained by the two methods are not great. The figures obtained by averaging the per- centages of the ultimate units are as follows: Pinus banksiana. — Inoculated, loss 30.9±5.0 per cent; controls, loss 13.2±2.S pel- cent. Pinus ponderosa.— Inoculated, loss 40.0±5J per cent; controls, loss 24.1±:$.3 per per cent, as compared with •'!!' per cent in the controls; subsequent damping-off the same in both. Inoculations al tour points in each pot: Experiment 58B. .hick pine, 5 pots inoculated, 5 controls; inoculated, emergence 51 per cent, damping-ofE 10 per cent, survival 46; controls, emergence 48 per cent, damping-off 22 per cent, survival 84. Experiment 59A. Jack pine, 5 pots inoculated, 5 controls; inoculated, emergence 55 per cent, damping-ofl 2 per cent, survival 54; controls, emergence 50 per cent, damping-ofE S per cent, survival 4C. Of these experiments, No. 29 was in the original fine sandy soil of a nursery in Nebraska in which Pythium is commonly found native and damping-off losses are usually heavy. Experiments 58A and 59 were conducted on soil from the same source which had, been kept dry in the laboratory for five years; experiments 25, 27, 31, and 58A were on greenhouse mixtures of sand and soil. In experi- ments 31, 58 A, 58B, and 59 parallel inoculations were made on auto- claved portions of the same soil, with definitely positive results in three of the four cases. In the heated soil the results were positive. not only because of smaller losses in the controls but because the losses in the inoculated pots were actually heavier in the sterilized soil than in that untreated. Inoculations broadcast : Experiment 31. Jack pine, 8 pots inoculated, S controls; inoculated, emergence 31 per cent, damping-ofE 39 per cent, survival 129; con- trols, emergence 3S per cent, damping-ofE 26 per cent, survival 196. Experiment 59. Jack pine, 5 pots inoculated, 5 controls; inoculated, emergence 58 per cent, damping-ofE 22 per cent, survival 45 ; controls, emergence 44 per cent, damping-ofE 2 per cent, survival 43. Even with these broadcast inoculations the results on untreated soil were too indefinite to allow the drawing of positive conclusions. In both experiments much heavier losses than these resulted from inoculations on steamed soil. It is evident that experiments on steril- ized soil do not always show what can be expected on ordinary soil. The same tiling is indicated by the results of Edgerton with tomato wilt (36). CONCLVSIONS AS TO THE PARASITISM OF PYTHIUM DEBARYANUM. Pythium debarijanum has been found in low-altitude nurseries in all the species of conifers from which a serious effort has been made to obtain it, and its parasitism has been indicated in autoclaved soil on all of the conifers on which inoculation has been attempted. Therefore, although the work reported has been limited to a relatively small number of hosts, it seems likely that it will be found able to cause damping-off in most of the species of the Abietoidese which suffer seriously from the disease. Just how active as a parasite it is DAMPING-OFF IX FOREST NURSERIES. 55 under ordinary nursery conditions is yet to be proved. The results in inoculations on disinfected soil, together with the frequency with which the fungus has been isolated from seedlings in the nurseries, lead the writer to believe that it is an important cause of disease in the seed beds. Further experiments on unheated soil, however, are considered desirable. RHEOSPORANGIUM APHANIDERMATUS. CULTUBAL STRAINS. A culture of a parasite on radishes and sugar beets, described by Edson (39) under the above name, was obtained from him, and an- other strain, shown by Edson's records to be a subculture from the same original strain, was furnished by the department of plant pathol- ogy of the University of Wisconsin. In parallel cultures on solid media this fungus proved in many ways remarkably like Pythium deharyanum, reacting in practically the same way to the different media on which it was grown both in relative growth rate and in spore production. Mycelium, chlamydospores, oogones, antheridia, and oospores are not recognizably different from those of Pythium deharyanum. The oospores have seemed on the whole slightl} 7 larger and the mycelium a little more inclined to aerial growth than most of the Pythium deharyanum strains, but neither difference was suffi- cient to have diagnostic value. Swellings of the hyphse occurred at points in contact with glass, just as with Pythium deharyanum (PL I, figs. 5 to 7). In liquid cultures the Rheosporangium was readily distinguished from Pythium by the formation of the presporangia described by Edson. Autoclaved cylinders of turnip, 15 to 20 mm. long, cut with a 5-mm. cork borer, proved convenient bases for growth of both Rheosporangium and Pythium in water culture and quite as satis- factory as sterilized beet seedlings. Presporangia were also pro- duced in autoclaved soil, and in u single lot of corn-meal agar they were formed abundantly in the agar in Petri dish cultures. In none of the writer's cultures, either with flies, sugar-beet seedlings, or turnip cylinders as nutrient bases, were mature escaped sporangia or swarm spores commonly produced. The Rheosporangium was not obtained in any of the numerous cultures made from coniferous seedlings or from seed-bed soil. INOCULATION EXPERIMENTS. The Rheosporangium cultures above referred to, strain 229 fur- nished by Dr. Edson and strain 351 received from the University of Wisconsin, were tested on pine and red-beet seedlings, with parallel inoculations with Pythium deharyanum. The results appear in Table VI. 56 BULLETIN 934, CJ. S. DEPARTMENT OF AORTrrT/TURE. Tabi i VI.- Results of parallel inoculation with Rheosporangium aphanidermatut mid Pythium debaryanum on pine seedlings in autociaved soil. Experiment number, host, and inoculating fungus.' No, 30, Pinus ponderosa: Rheosporangium, strain 229. - Pythium, 2 strains Controls No. 31, Pinus banksiana: Rheosporangium, strain 229 Pythium, strain 225 Controls No. 58, Pinus banksiana: Rheosporangium, strain 229 Pythium, 8 strains Controls No. 59, Pinus banksiana : Rheosporangium, strain 229 Pythium, 8 strains ('nil Iri iN No. 61, Pinus banksiana: Rheosporangium, strain 35] Pythium, 2 strains Controls No. 61, Funis banksiana and beets in same pots: Beet? [Rheosporangium, strain :i5l No. 61, beets alone: Rheosporangium, strain 351 No. 62A, beets: Rheospora„gium{^;n^. ■-...-• --...-...- Pythium, 2 st rains Controls No. 62A, beets: Rheosporangium {^^ ;:;;;;:;:;:::;;;:: Controls No. 62A, beete and Pinus banksiana in same pots: g™^>Rheosporangium, strain 229 gj^?| /Rheosporangium, strain :i">i Be»ontrols No. 62B, Jack pine: d Rl,e,,spo,,n,i,,mgraiu2|9... ................ Pythium, strain 258 Controls No. 66, Jack pine: Rheosporangium^-;;;^;;;;;;;;;;;;;;;;; Pythium, 17 strains and substrains Cunt nils No. 67, Jack pine: Rbeosporangium^i?".-.:;;;;;;;;;;;;;;;; Pythium, 17 strains and substrains Controls No. 68, Red pine: Rheosporangium^ I?-;;;;;;;;;;;;;;;;;; Pythium, 17 strains and substrains Controls Number ol pots. 235 25 5 5 235 23 5 23S 18 Result; Emerged Damping- i per cent ofl i |>er ofseed). vent). /'< r pot. 23 II 20 45 12 43 Per 5-pot unit. 46 2.6 89 12 12 86 50 c27 c77 r,l 42 46 27 81 67 58 53 10 82 63 80 43 107 ss 51 87 105 124 86 nil Per pot. 3 (*) 88 72 1 :« 83 20 78 1 14 22 33 78 14 10 100 a Location of the inoculum: In experiment 30, at one point at the edge of each pot: in experiment 31, over the entire pot; in all other experiments, over one-quarter the area of each pot. b The breakage of the one seedling not killed while sprouting prevented the determination of results. c Double seed density in these pots: emergence and survival figures halved to allow direct comparison with other units. This high seed density may explain in part the higher loss in strain 351 than in strain 229. d Experiment 62B was conducted at the same time as 62A, but with a different soil. Table VI shows that in experiments 30 and 67 the loss was less in the Rheosporangium pots than in the controls and that in experiment 68 the results were entirely negative, while in the remaining seven DAMPINO-OFF TN FOREST NURSERIES. f)7 experiments the losses were heavier in the Rheosporangium puts. Especially in experiments <>l and 62A the evidence indicates very strongly that both germination loss and subsequent damping-off of the seedlings which come up can be caused by inoculation with Rheo- sporangium on jack pine under favorable inoculation conditions. It is, however, obvious that in all of the experiments the parallel in- oculations with Pythium debaryamMi gave much more positive re- sults. The Pythium was active under conditions in which the Rheo- sporangium gave no evidence whatever of parasitic capacity. It furthermore appears that the two strains of Rheosporangium, though probably identical originally, differed in virulence at the time of their comparison in these experiments. The greater virulence of strain 351 was quite distinct in most of the comparative tests on beets as well as on pines. The possibility that the original culture was really a composite of two or more strains, of which different ones survived in the subcultures kept at Washington and Madison, re- spectively, seems worth considering. Such an accident might also have been responsible for the divergence of Pythium strains 131 and 295 referred to in another section. Further evidence of the parasitism of Rheosporangium was ob- tained in inoculations with cultures reisolated from seedlings killed by the original strains in experiment 62. Typical Rheosporangium, identified by presporangium formation, was easily recovered from the damped-off seedlings in pots of pines only, those of beets only, and the pots in which both hosts were sown. The recovery of a virulent Pythium strain from a single one of the pots inoculated with the weaker Rheosporangium shows that despite the absence of dis- ease in the controls a slight amount of contamination did occur. However, the comparative ease w^ith which the Rheosporangium was isolated from seedlings in other pots inoculated with it and the fact that it has never been obtained in the numerous cultures made from controls and from pots inoculated with other organisms leave little room for doubt that the strains isolated were really recoveries of the Rheosporangium used in the original inoculations. The results of reinoculation w T ith these strains are shown in Table VII. From Table VII and by comparison with Table VI it appears — (1) That in one experiment each on jack pine and red pine the reisolated Rheosporangium strains gave positive results. In a second experiment on jack pine (No. 67) the difference between the Rheosporangium pots and the con- trols was not significant. (2) That, as in Table VI, the Pythium strains used proved on the whole decidedly more parasitic than the Rheosporangium strains. In experiment 66 this is not shown by the percentage of seedlings damped-off, but is sufficiently evident when the germination loss as well as the subsequent dainping-off per- centage is considered, the survival being here, as in most other cases in which 58 BULLETIN 334, U. S. DEPARTMENT OF AGRICULTURE. either of the groups of pots compared is seriously affected by parasites, the most convenient index of the comparative activity of the fungi used. In such a comparison as thai between the Rheosporangium pots ;inots w/thoc/t \ /=>YTHtUM 30 / 1 / % ' / X > / \. \ / / / /■ /! \ \ \ \ ' / \ \ SO / / • \ \ / / \ \ / / \ \ \ \ * / 1 / / 1 \ \ \ \ JO / * fc X \ 1 \\ # / I ^ s **~ B / 1 1 ! 1 1 ^***^vj 0-/4- /S-24- 30-4-4- 4-SS9 60-74- 76-89 L//V(?S S(/#{//y//VG P£/?POT 90-/04- Pig. 18.— Frequency of pots with different numbers of surviving seedlings of Pinus banksiana, Inoculation experiment No. 31. The solid lines represent pots to which cultures of saprophytic organisms were added. The broken lines are based on pots to which no saprophytes bad boon added. The solid lines are based on 78 pots in the upper graph and 80 pots in the lower one; the broken lines on 33 pots in the upper and 25 pots in the lower graph, Pythium debaryanum was added jus) after sowing the seed at a single point in each pot represented by the two upper linos. Cultures of saprophytes were applied broadcast two days before the Pythium inoculations were made. sprayed with a spore suspension. The pots were covered with glass to increase atmospheric moisture, and the seedlings were occasionally sprayed with an atomizer. The soil was an autoclaved mixture in which simultaneous inoculations in a different room with Pythium and Corticium proved successful. The failure of the Phytophthora may possibly have been due to the lower temperature at which the pots inoculated with it were kept (15° to 20° C.). DAMPING-OFF IN FOREST NURSERIES. 61 A species of Phytophthora was isolated by Mr. R. (1. Pierce from damped-off Pinus resinosa in Minnesota ami used in four inocula- tion experiments, the results of which appear in Table VIII. In the first of these experiments unboiled water was used on the pots, and mice obtained access to the pots of the second test. Probably as a result of these things infection occurred in the controls in both cases, and the results were inconclusive; in the later experiments these two sources of infection were eliminated, and in experiments 68 and 72B the controls were free from disease. Parasitic activity was in- dicated rather strongly in experiments 68 and 72 (on P. resinosa and P. banksiana) and to a certain extent in experiment 66. In experi- ment (>7 it was evident that the Phytophthora was nearly or entirely inactive. Comparison of the results in experiments 66 and 68 with the results from inoculations with Rheosporangium aphanidermatus in the same experiments (Table VII) suggests that the Phytoph- thora may be better able to attack the pine from which it was isolated than the Rheosporangium, while the latter fungus caused consider- ably more destruction to Pinus banksiama than the Phytophthora. Comparison of the results in the pots inoculated with Phytophthora and those inoculated with Pythium debaryanum in all the experi- ments indicates that the Phytophthora strains used were less virulent than most of the strains of P. debaryanum and very certainly less destructive than the most dctive strains of either P. debaryanum or Corticium vagum. This species of Phytophthora has been reisolated from damped-off Pinus ponderosa in experiment 72. Direct inoculations of the stems of seedlings of Pinus resinosa soon after they emerge from the soil have so far confirmed the lack of parasitism of Phytophthora cactorum and of the cultures of Phy- tophthora sp. grown by the writer. The identity of this species has not yet been determined. It is able to grow only about one-fourth as rapidly as Pythium debaryanum on the medium which has been used for isolation and may therefore be more common in the seed beds than the small number of isolations by the planted-plate method would indicate. However, its oospores, larger and darker than those of Pythium debaryanum (usually over 20 [x in diameter), should have been recognized in the routine microscopic examination of planted- plate cultures had this species been frequently present, even if it had not grown fast enough to get out ahead of the other organism and allow isolation. It is not believed that it is common enough in pine seed beds to be of importance, even if other strains should be found more virulent than those which have been available. MISCELLANEOUS PHYCOMYCETES. A fungus, apparently referable to the somewhat indefinite Pythium artotrogus (Mont.) De Bary, w r as isolated by Mr. Glenn (x. Hahn from Pinus resinosa in Michigan and from damped-off Pinus bank- 62 BULLETIN 934, 0*. S. DEPARTMENT OF AGRICULTURE. siana in pots of autoclaved soil which had received tap water at Washington, D. C. It agreed both in the appearance and measure- ments of its spiny oogones and smooth oospores with Pythw/m arto- trogus (P. hydnosporus) as described and figured by Butler (23). In addition to the spores which Butler describes, there appeared in apparently pure prune-agar cultures of different strains bodies with smooth walls, of somewhat irregular ovoid outline, and mostly larger than either oospores or oogones. They are very much less abundant than the sexual spore forms. Their greatest diameter varied from 11 \x. to over 40 \).. The germination of these bodies was not observed. Efforts to induce the fungus to produce swarm spores by growing them in liquid nutrient media and transferring them to pure water were unsuccessful. This failure to produce /oospores is further in- dication of the identity of the fungus with that described by Butler, who says that asexual reproduction is unknown. The strain from Michigan was a rather weak growing organism, difficult to maintain in tube cultures without rather frequent trans- fers. Its parasitic activity in the experiments reported in Table VIII is nil or negligible. Because of the poor seed and small number of seedlings involved in experiment 72B, the percentage of damping- off there given means only a single seedling dead. The Washington strains, on the other hand, though evidently not strong parasites, did apparently cause the death of a number of seedlings. The best evi- dence of this is in experiment OS. in which there was damping-off in each of the live 5-pot units containing the Washington strains and none in any of the IS control pots. The available strains were less active not only than PytMum debarymyu/m^ but less than the Rheo- sporangium and Phytophthora strains used. The fungus is be- lieved to be a potential parasite on [tine seedlings, but not one of any general importance. What is probably the same fungus had ap- peared in the writer's cultures from western nurseries in conjunc- tion with /'. (lelxtri/aitum, but not commonly, and it had not been isolated. While its growth rate is only about half that of P„ debartj- anum on prune agar, it is nevertheless so much faster than that of many fungi that it should have been more often obtained in culture were it at all common in damped-off seedlings. Another fungus, presumably an oomycete but producing only chlamydospores in the writer's cultures, was obtained from damped- off olive seedlings furnished by Prof. W. T. Home and from soil direct, both at Berkeley, Calif. The fungus is apparently the same as one which has been occasionally seen in cultures from pine seed- lings in the Middle West, but had not before been isolated. The hyphse are ordinarily nonseptate, and the growth on corn-meal agar is superficially much like that of Pythium debaryanum, but with greater tendency toward local zonation and aerial growth and less DAMPING-OFF IN FOREST NURSERIES. 63 than half as rapid. Chlamydospores are mostly intercalary, at first subspherical, soon becoming polygonal, and after a Eew days they shrivel and exhibit thick, angular walls. In size the unshrunken spores usually lie between 8 and L2 y. in diameter, but bodies as large as 20 \x occasionally occur. Antheridia have not been observed, and the shriveled bodies are not believed to be oospores, though the observations made have not been sufficient to exclude such a possi- bility. No other spore form was obtained in water culture, using various nutrient substrata. In inoculation the strain from olive (the "undetermined Phycomycete " included in Table VIII) has given negative or nearly negative results in three inoculation tests in which other fungi gave positive results. In a test not included in the table, in which Firms ponderosa was the trial host, damping-off was slightly higher in the inoculated pots than in the controls, but the difference was apparently due to accidental infection with Botrytis and Pythium debaryanum. As all the seedlings in pots inoculated with P. debaryam/um in this additional experiment were killed, the rela- tive unimportance of this strain of the small-spored fungus was further indicated. An additional test of both the olive strain and the strain from soil was made by inoculating seedlings of Pinus han/i'siana and P. ponderosa growing on filter paper in Petri dishes. Some of these were kept wet with water, some with an inorganic culture solution, and some with the inorganic solution plus peptone and dextrose. Agar cultures were applied directly to the seedlings. The seedlings inoculated with the small-spored fungus remained alive as long as the control seedlings, while parallel inoculations with Pythium debaryanum resulted in the early decay of the seedlings. Table VIII. — Results of inoculations with miscellaneous oomycetes on pines in autocluved soil at the time of soicing. [In all the experiments included in this table, the inoculum consisted of fragments of agar cultures distributed with the seed at one side of each pot over about one-fourth of the pot area. The controls received sterile agar in the same way.J Num- ber of pots. Results. Experiment number, host, and inoculating fungus. Emerged. Bamp- ing-on". Survival. No. 66, Pinus banksiana: Phytophthora sp.— 5 5 5 4 5 25 5 5 5 S 23 Per B-pot ii nil. 70 75 94 115 83 75 96 88 99 102 98 87 Percent. 30 28 16 14 9 14 1 3 2 5 Per S-pot unit. 40 Strain 372 54 79 99 76 64 No. 67, Pinus banksiana: Phytophthora sp. 95 85 Strain 375 99 Hid 98 83 64 Bl LLETI2S 934, U. S. DEPARTMENT OF AGRICULTURE. Table VIII. Results of inoculations with miscellaneous oomycetes on pines iii autoclaved soil at the time of solving Continued. Num- ber of pots. Results. Exp rimenl number, host, and inoculating fungus. Emerged. l>ani|>- ing-ofT. Sun i\ai No. fis, Finns resinosa: Phytophthora sp. — Strain 35s .") 5 5 5 5 5 5 5 5 5 IS 3 3 2 3 3 16 3 3 3 2 2 14 /■. i .' pot unit. 10! 109 98 121 122 120 96 110 94 84 104 Per S-pot n nil. 20 62 45 37 40 35 8 13 11 29 6 9 r, rc< in. 7 18 Per n-ix>t n nil. 97 RQ Pythium artotrogus (?), Michigan strain 1 9 5 6 1 2 35 7 25 5 50 8 6 121 Pythium artotrogus (?), Washington, l>. e. St rain vji 121 Strain 82S 109 Strain 83] in Strain 832 103 Strain RH3 93 Undetermined Phycomycete 82 Controls mi No. 72 A. Pinus resinosa: Phytophthora sp. — 'Strain:;:.'. Per .1-pot unit. 13 Pythium artotrogus (?), Michigan strain Pythium artotrogus (?), Washington, !>.('. Strain 821 62 42 St rain 831 37 Strain 833 30 Controls 33 No. 72B, Pinus pondcrosa: 4 12 Pythium artotrogus ('.'). Washington, D.C. Strain 821 11 Strain 833 27 6 9 OTHER FUNGI. Data on the possible relation between various other fungi and the damping-off of conifers have been already summarized by Hartley, Merrill, and Rhoads (68, p. 546-550). Pestalozzla funerea on the basis of the experiments of Spaulding (135), Botrytis cinerea on the basis of observation and very preliminary inoculations, and Tricho- derma koningi on cultural evidence only are all believed to be po- tential causes of damping-off, though not ordinarily important. Al- ternaria sp. is under a certain amount of suspicion on account of its frequent association with the damping-off of conifers, but it has never been used in experiments. Rhisopus nigricans (incorrectly re- ported as Mucor), Trichothecium roseum, RoseJlinia sp. from nursery soil, Chaetamium sp. from maple roots, strains of Penicillium and Aspergillus, Phoma betae, and Phoma spp. are all reported to have been used in inoculations with negative results. Since the publication of the above summary a preliminary success- ful inoculation experiment with Botrytis dnereaon recently emerged Pseudotsuga taxifolia has been found briefly mentioned in an article by Tubeuf (140) on another disease. Further experiments with va- DAMPING-OFF IN FOREST NURSERIES. 65 rious strains of Botrytis, both from conifers and from other hosts (the latter supplied by the departments of plant pathology of the California and New York (Cornell) Agricultural Experiment Sta- tions), have already yielded confirmatory evidence of the parasitism of B. cine red. "While a considerable number of fungi have been considered in the foregoing, it is entirely possible that there are still parasites which have received no consideration and that some of them may perhaps be important. The moist-chamber method of culturing parasites for isolation yields only those which produce spores readily; the planted-plate method is not well adapted to the isolation of slow- growing fungi or bacteria. It is suggested that in further culture work with damped-off conifers an attempt be made to secure slow- growing organisms by dilution plates of teased-up fragments of recent lesions. RELATIVE IMPORTANCE OF THE DAMPING-OFF FUNGI ON CONIFERS. The relative importance of the different damping-off parasites is something that has not been thoroughly investigated for any host. The most information on this point is that given by Busse, Peters, and Dlrich (22) for sugar beet. In this case they find the special- ized Phoma beto-e distinctly the most important, with Pythium de- baryanum second and Aphanomyces levin third. Peters (l ( "i) apparently considered Rhizoctonia unimportant as a cause of beet damping-off. The opposite was indicated by a small number of cultures by Edson (38) from beet seedlings on Kansas and Colorado soil. These yielded more Corticium vagum than any other parasite- and no Pythium at all. Johnson (81) states that most of the damping-off of tobacco seedlings is due to Pythium debaryanum and Corticium vagum. Atkinson (1), speaking for cotton in Alabama, and Sherbakoff (127, p. xcv; 128; 129), speak- ing for truck crops in Florida, make Corticium vagum the impor- tant damping-off parasite, with P. debaryanum negligible. Home (oral communication) found the same situation in tobacco seed beds in Cuba. Atkinson (3), in an article on trees, held that many of the cases of damping-off attributed to P. debaryanum are in real- ity due ta C. vagum. Peltier (98, pp. 336-337) has reported Rhi- zoctonia solani as the cause of damping-off of a large number of plants, recording his observation of the damping-off of seedlings of nearly 50 species of miscellaneous genera and cuttings of 13 different species, all of which he attributes to the Rhizoctonia. He does not state whether in this case he used diagnostic methods likely to de- tect Pythium debaryanum if it had been present. 19651°— Bull. 934—21 5 66 BULLETIN 934, U. S. DEPARTMENT OF AGRICULTURE. For the conifers, no very reliable data on relative importance have been published. Numerous European reports emphasize the damage due to Fusarium spp., while a smaller number attribute loss to Phytophthora fagi or to both. There seems to have been little effort to determine the presence or absence of Corticium or Pythium, so these reports can not be given great weight. Spaulding's evident belief (136) in the importance of Fusarium has more weight, as ho was on the lookout for the other fungi ; the moist-chamber diagnostic method employed in most of this work was, however, not Avell adapted to the detection of either one. The same is true of the work of Eathbun (106), in which dilution plates of seed-bed soil were employed. Rankin (105) attributes to Fusarium spp. the greatest importance in tree seed beds in this country, with Pythium debary- anum and Rhizoctonia spp. important in certain cases. Gifford (46) emphasizes the importance of Fusarium, while Clinton (28) appar- ently found Khizoctonia {Corticium vagum) especially prevalent in the examinations he made. On the basis of the data presented or summarized in this bulletin, it is believed that of the various organisms which have been con- nected with damping-off in coniferous seed beds Pythium, debary- anum, Corticium vagum, and Fusarium spp. include all of impor- tance. The others, either because of low indicated virulence or infrequent occurrence, and in most cases both, do not seem to merit extensive consideration. In order to form an idea of the relative frequency of the parasites named above as important, there have been brought together in Table IX the results of the examination of 438 damping-off foci in untreated beds and 304 foci which have appeared in beds which had received various disinfectant treatments. The data are presented by foci rather than by individual seedlings, as was done in the census reported by Busse and his coworkers. Most of the diagnoses were made by planting recently diseased seedlings in plates of solidified prune agar, all the seedlings taken from the same focus, or " patch," of damped-off seedlings being put into the same Petri dish. The resulting growth was in some cases transferred to a tube for later examination, but was usually examined directly in the plate. In a smaller number of foci the seedlings were macerated and examined directly without recourse to culture methods. As Pythium debary- < mum does not commonly fruit in diseased seedlings of pine or of tobacco (81) and its hyphse are both difficult to find and not in themselves considered a sufficient diagnostic character, this latter method of examination is not so satisfactory for the determination of Pythium as it is for Corticium, which is easily recognized by its DAMPlNC-oi-T IN FOREST NURSERIES. 67 thick-walled truncate-tipped hyphae and characteristic branching. A further difficulty in the direct-examination method, unless the seed- lings are sectioned, is in distinguishing between Corticium hyphse which are in the tissues and those outside. The well-known habit of the Corticium of sending hyphae superficially over the surface of plants which it is not appreciably injuring makes it evident that only hyphae actually found in the tissues have diagnostic value. Direct microscopic examination is. furthermore, very likely to fail to detect Fusarium. The planted-plate method therefore appears the better of the two. and the results of the culture diagnoses appearing in the lowest two lines of Table IX deserve probably more attention than the total occurring a few lines above, in which the results of direct examination of the seedlings are also included. The high proportion of Corticium reported from the Michigan and Minnesota nurseries is probably due in part to the fact that most of the examinations made there were of the direct microscopic type. Table IX. — Results of the examination of damping-off foci in coniferous seed beds for Pythium dciaryanum, Corticium vagum, and Fusarium xin>. Untreated beds. Beds of heated soil. Beds treated with strong acids. Grouping. •6 c a 03 a Cj o ft Number showing — •6 s . el a 03 X 'o O ft Number showing — •6 CO .9 9 '3 o ft Number showing — • 3 3 >> ft a 3 'P u o O 3 03 3 ft a 3 3 ft a 3 |3 u o O OS B ft i 3 3 >. ft a 3 ]3 o O a 3 *n 03 3 ft By locality: Berkeley, Calif 4 22 34 18 20 224 42 4 45 13 12 3 6 4 4 124 15 14 11 3 1 19 20 9 45 21 4 33 7 3 2 13 9 7 155 5 1 2 1 9 5 5 2 Garden City, Kaiis. — 14 15 28 5 2 10 7 6 17 28 16 99 1 8 9 34 2 2 2 q 8 61 n Dundee, 111 East Ta'was, Mich. — 1 13 1 7 6 o ? 7 2 19 30 3 33 52 64 39 6 38 58 39 12 5 8 50 4 3 Total: 438 100 184 42 1(12 37 204 47 64 100 163 100 82 50 By diagnostic methods: Direct examination — 156 100 2S2 100 39 25 145, 51 108 69 54 19 25 16 179 63 16 100 147 100' 4 ?•> l 'lanted-plate cultures- 64 100 19 30 33 52 7S 53 68 IHLLETIN 034, U. S. DEPARTMENT OF AlilU CULTURE. Table IX.- — Results of the examination of damping-off foci in coniferous seed beds for Pythium debaryanum, Cortieium vagum, and Fusarium spi>. — Con. Beds treated with formaldehyde. Hods treated with copper sulphate. [Beds i reated with zinc chlorid. All treated beds. Grouping. ■6 e a 1 y, Cj 'o o Number showing — •6 a/ | 1 03 y. oj *o o Pi Number showing — ■d o> 3 • 3 03 y. '3 o Pi Number showing — ■6 9 .3 S a o 'o o Pi Number showing — 3 Pi u o o 03 3 3 B PL) u o g .3 'C 03 9 Pi s Pn i u o 3 Pi a 3 2 5 £ 3 |o Ei o O a 3 03 3 Pi By locality: Berkeley, Calif 5 ? Garden Oily, Kans. — Garden City Nurseries . . Kansas Nurseries (sand) 6 6 3 1 1 3 1 3 52 31 175 1 14 11 67 8 2 3 23 14 31 20 1 24. 3 5 8 5 112 East Ta'was, Mich.— 1 32 7 1 20 2 13 East Tawas Nurseries. . . 8 5 4 6 5 3 3 5 3 2 7 3 Total: Nnmher ■ 48 100 25 52 5 (13 20 50 11 23 4 50 7 18 27 .56 27 08 13 100 8 62 4 80 4 50 3 23 2 40 1 13 9 69 2 40 7 88 16 100 4 25 3 CO 1 9 10 63 2 40 8 73 304 100 120 39 20 9 14 41 12 4 101 53 By diagnostic methods: Direct examination — 8 100 40 100 5 100 8 1(10 5 100 11 100 34 100 270 100 18 53 102 38 8 24 Planted-plate cultures — 153 57 The data on the different nurseries do not allow any generalizing on the basis of locality except to say that all of the fungi seem quite generally distributed in the Lake States and Great Plains region. In general, it appears that the Fusaria as a group are more common than either of the other fungi ; as they grow more slowly than either the Pythium or the Cortieium, they were probably rather more common relatively than even the plate-culture method indicated. It also appears that the Pythium occurred in more foci than the Cortieium in the beds examined. Further culture work, perhaps by the method of dilution plates of fragments of lesions, seems de- sirable, especially in the East and the Northwest, regions in which there are large coniferous nurseries and in which nothing like a parasite census has been attempted. Observations on the type of focus occurring in most of the nurseries in the Rocky Mountains leads the writer to believe that Cortieium will be found especially important there. While the data on the fungi in foci in disinfected beds are insuffi- cient to serve as a basis for much in the way of conclusions for any individual treatment, they in general agree with the assumption, which knowledge of the fungi would favor, that Cortieium is the DAMPING-OFF IN FOREST NURSERIES. 69 most easily controlled by soil disinfection (see the bottom line in the last four columns of Table IX). Its poor adaptation for aerial dissemination would lead one to expect to find it seldom in beds treated with efficient disinfectants. The entire absence of Corticium in heated soil therefore seems somewhat significant. The rather high Corticium yield in the formaldehyde plats is of some interest in view of the reported inefficiency of formaldehyde in destroying Corticium vagumon. potato tubers (48, 50). As will be noted from the data given, more than one suspected parasite was often found in what appeared to be a single focus. This was probably in some cases due to independent foci being nearly concentric ; it also in some cases undoubtedly means that one of the organisms found was only secondary. In the beet-seedling cultures by Pmsse and bis associates, individual seedlings yielded two or more potential para- sites in 100 of their nearly 1,300 examinations. It not infrequently happened in the work on pine seedlings that no fungus recognized as a likely parasite could be isolated. This was especially true in plate cultures when Rhizopus or Trichoderma happened to be abundant, as both are very fast growing and often suppress para- sites. This is an additional reason for the development of some method as a dilution plate of lesion fragments for diagnosing damp- ing-off. Even an accurate and complete census of the organisms present in the different foci could not be directly interpreted in terms of rela- tive importance. None of the parasites so far used in inoculation have been vigorously parasitic under all conditions. Of both Corti- cium vagum and Pythium dclmryanum some strains, microscopically indistinguishable from the others, are very weak as parasites. Only part of the Fusarium species are parasitic on pine, and data showing which are and which are not parasitic are known for only a very few. There is therefore no fungus which can be said positively to be the cause of any particular damping-off " patch " simply because it was found in some of the dead seedlings in the patch. In an occa- sional exceptional case, such as the large Corticium patch in figures 7 and 8, there is such a vigorous growth of the fungus that its pre- dominance is undoubted, but such cases are rather rare. A census throws light on the importance of the different fungi, but can be interpreted only in the light of inoculation results. For Pythium and Corticium the inoculation data do not permit any simple comparison between the two, for the reason that neither is uniform. Each has strains of high virulence and strains having practically no effect on pines. In the inoculations in autoclaved soil at sowing time the strongest strains of Corticium vagum have on the whole caused more damage than any of the Pj'thium strains, but, on the other hand, there has seemed to be a higher proportion of very 70 r.ru.KTix 034, U. s. DEPARTMENT ok AGRICULTURE. weak strains of C. vagum than in the case of Pythium. Tn inocula- tions on Pinus banksiana and P. ponderosa in Kansas sand treated with acid followed by lime, the average Corticium was very much more destructive than even the strongest Pythium strains, allowing practically no germination in most cases. On the other hand, in ex- periments in which the inoculum was applied directly to Phn/s resinosa and P. ponderosa seedlings, either immediately after germ- ination or after the older parts had become resistant, the Pythium has been the more effective. The inoculation evidence so far avail- able justifies so nearly equal emphasis on the two that it can prac- tically l>e eliminated from the calculations. It is the waiter's opinion that the Corticium strains are probably rather less virulent on the average than the Pythium strains, but perhaps better able to main- tain themselves and spread from one seedling to another in most soils. The evidence of Table IX that the Corticium seemed less fre- quent in the damping-off foci is more or less counterbalanced by the apparent larger size of many of the disease patches which it seems to cause in the seed beds. Nearly all the large clean areas such as are shown in figures 7 and 8 have been found to contain abundant Corticium hyphae. The evidence on the whole seems to indicate a very nearly equal importance for the two fungi. The Pythium is probably somewhat the more important for the stations at which most of the cultures in Table IX were made, but the Corticium has received more emphasis from other observers in this country and is indicated by the writer's observations to be more important in the western mountains than any other damping-off fungus. The inoculation evidence for Fusarium spp., though less complete than for Corticium and Pythium, is nevertheless rather helpful in indicating their importance rating. Xone of those so far tested in inoculations at sowing have shown the destructiveness of the aver- age strains of Pythium or of the stronger strains of Corticium ; while this is only in part a test of virulence and in part a test of the ability of the fungus to grow saprophytically in the soils used, the indication is that no one Fusarium species is the equal in destructive capacity of either Corticium vagum or Pythium debaryanum. How- ever, when all of the Fusarium species which occur in the seed beds are considered, the group as a whole may prove quite as important or even more important than either of the other two fungi. The data already at hand rather definitely indicate considerable importance for all three. DAMPING-OFF FUNGI AS CAUSES OF ROOT-ROT AND LATE DAMPING-OFF. As has been already stated, root-rot, often with frequent recovery, has been commonly observed in seedlings several weeks old. It has been especially common in the vicinity of old damping-off foci in DAMPING-OFF IN FOREST NURSERIES. 71 which Corticium vagv/m appeared to be the active parasite, but beyond this indication of the causal relation of C. vagurn it was not known which of the damping-off fungi were able to attack the roots of seedlings too old to be killed by damping-off. To throw light on this point, seedlings of Pinus ponderosa and P. resinosa grown in autoclaved soil in the greenhouse and approximately H months old were inoculated with different fungi. There had been a certain degree of early damping-off in these pots, but it had apparently ceased before the inoculations were made. The inoculum used con- sisted of cultures on rice introduced through the drainage holes at the bottoms of the pots. The strains of Pythium debaryanum and Corticium vagum used were the ones which had given maximum results in earlier inoculation experiments at the time of sowing. The strain of Fusarium ventricosum was the only one available, and the Fusarium monilifornu strains were all of approximately equal viru- lence, the three used having given as much evidence of parasitism as any of the strains of this species in the earlier damping-off experi- ments. Three pots of each pine were inoculated with each strain. Two 3-pot units of each pine were set aside as controls and inoculated with sterile rice. In addition, three pots of each pine were kept in the same bench without the addition of any inoculum, for comparison with the controls with rice. The results of this experiment, taken a month after the inoculations were made, with the seedlings averag- ing 2% months old, appear in Table X. The roots of the living seed- lings were washed out carefully with water to permit examination. The results in so far as they indicate root-rot of the oldest seedlings are. best shown by the figures in columns 4 and 5. These seedlings were so far advanced that the fungi had not been able to kill them, and nearly all would probably have recovered if they had not been dug up. It will be noted from column 4 of Table X that a consider- able portion of the Pinus ponderosa seedlings with root-rot had al- ready made their recovery apparent by pushing out adventitious roots above the decayed portion at the time they were examined. For Fusarium ventricosum, there was only the merest indication of ability to attack pine roots at this stage. For F. moniliforme the evidence is somewhat better, more pots being included and the dif- ference in healthy-topped seedlings with injured roots between the inoculated pots and the controls being approximately twice its indi- cated probable error for each species. The percentage of root-injured seedlings in the Pythium debaryanum pots exceeded that in the con- trols in each species by between three and four times the probable error of the difference, while the difference in percentage between the Corticium vagum pots and the controls is approximately four times its probable error in the case of Pinus ponderosa and five and one-half times its probable error in the Pinus resinosa pots. The 72 BULLETIN 934, U. S. DEPARTMENT OF AGRICULTUEE. weak point in the results is, of course, the insufficiency of the 6-pot and 9-pot groups as bases for probable-error determination. The indicated relative ability of these different fungi to cause root-rot is about the same as their relative ability to cause the damping-off of sprouting seed and young seedlings, as indicated by the results of the earlier experiments in which inoculations were made at the time of sowing. The fact that only the very strongest available strains were used and that the pots were rather heavily inoculated is to be kept in mind in considering these results. As in the seedlings examined in the nursery beds, when a root system was partly rotted it was only the younger portions of the roots that were affected. The evi- dence obtained from this experiment needs to be amplified by experi- ments with other coniferous hosts, other strains of the fungi, and under other conditions. The experiment just described furnishes the only evidence available on the relation of the important fungi Pytldum debaryanum and Corticium vagum, to the root-rot of conifers and is therefore presented as a preliminary contribution. Table X. -Results of root inoculations of older pine seedlings with damping-off fundi. Number of — Seedlings which developed root-rot (percent). Host and inoculating fungus. Pots. Seed- lings. Tops still healthy. Killed. Root recovery. Average of in- dividual pots. Total. Started. Not started. 1 2 3 4 5 (5 7 8 Finns ponderosa: Pythium debaryanum, strains 295, 550, and 810.a Corticium vagum, strains 147, 213, and 747. a Fusarium moniliforme, strains 249, 251, and 260.6 9 9 9 3 6 3 9 9 8 3 fi 3 71 56 64 18 41 18 140 146 128 39 115 51 27 16 19 17 2 3 3 25 34 27 39 15 17 16 16 11 5 3 53±4.5 51±3.5 42±6.2 50 22±6.5 23 18±4.0 21±2.4 12±3.7 4 4±2.0 4 4 .12 13 2 6 2 56 54 45 56 17 17 Pinusresinosa: 31 33 13 5 10 2 a For relative virulence of these strains on younger seedlings as compared with other strains of the same species, note their position in figures 11 and 14. t> For performance of these strains in inoculations at time of sowing, see an earlier publication (68, table 2). The figures in column 7 give information as to the percentage of late damping-off resulting from the inoculations. A certain per- centage of the early t}^pe of damping-off appeared in some of the DAMPING-OFF IX FOREST NURSERIES. 73 pots, as there were still present a number of soft-stemmed seedlings from seeds which were slow in germinating. These younger seed- lings were excluded in counting the dead, the rule being to include only plants which had developed a sufficiently rigid stem to remain upright alter death. Comparison of the percentage of killed with the total percentage attacked for the two pines is rather interesting. As has already been pointed out, while Pinus resinosa suffers very heavy damping-off losses at a number of nurseries it seems to be less susceptible than some other species to parasitic injury during the sprouting period, before the seedlings appear above the soil sur- face. Observation of beds of this species during different seasons has indicated that it has not a greater susceptibility, but rather the fact that its susceptibility lasts longer, which causes it to suffer as seriously as it does at certain nurseries. It is indicated in Table X that the succulent root tips of Pinus pondcrosa are just as easily attacked by damping-off parasites as those of P. resinosa — in fact, considerably more easily attacked, as indicated by the figures in col- umn 8. With the P. ponderosa seedlings, however, the older parts of the roots had become resistant at this age in nearly all cases, while of the affected P. resinosa seedlings more than one-third were still unable to limit the lesions, and death resulted. In general, this experiment indicates that Corticium vagum and Pythium debaryamim are able to cause the death of some pine seed- lings which have developed rigid stems and that both are also able, as has been found by other workers in the case of sugar beets, to cause " root sickness," the rot of the younger portions of the root systems, in seedlings which have developed too much resistance to be killed. The evidence for the parasitism of the two Fusarium species on these older root systems is not so good; as in the experi- ments on younger seedlings, their ability to attack the pines is prob- ably less than that of the other two fungi. Further inoculation ex- periments are desirable both with these fungi and with others on the roots of seedlings too old to succumb to the more ordinary types of damping-off. RELATION OF ENVIRONMENTAL FACTORS TO DAMPING-OFF. In the earlier section dealing with disease control, mention was made of the general belief on the part of men who have had experi- ence with seedling diseases that damping-off is favored by thick seed- ing, by much organic matter, especially by poorly rotted manure in the soil, and by excessive moisture in the air and soil. It is also commonly stated that high temperature favors the disease ; on this point there is perhaps a less general agreement. Practically all the evidence on these points is observational. 74 BULLETIN 934, U. S. DEPARTMENT OE AGRICULTURE. DENSITY OF SOWING. The relation between the disease and thick sowing was strikingly indicated for tobacco seedlings in a single experiment by Johnson (82). For pines the only available information is from four experi- ments on Pinus banksiana. The results of the first two appear in figure 19. In both experiments there is an indication of an increase in the percentage of diseased plants as the seed density is increased. There is, however, no such marked relation as in Johnson's work. As the pines were sown in drills, they were so close together even in the less dense plats that no very great increase in the ease of spread i ___^__^_ - ______________^^^^____ of the disease was to be expected from in- creasing the density. Greater differences should be expected in broadcast beds. That heavier losses have been found in drill-sown beds than in those sown broad- cast (69, 139) is pre- sumably explained by the fact that with equ al numbers of seed per square foot of seed bed the seed- lings are much closer together in drills than in broadcast beds, and thus the spread of the mycelium of para- sites from one seedling to another is facilitated. Two tests of different seed densities were also made in 3-inch pots of autoclaved soil in the greenhouse. Each regular pot was sown with 28 seeds (equivalent to 000 per square foot). The pots were inoculated by adding to each a single small fragment of an agar culture of PytMum debaryanum. Uninoculated pots showed an emer- gence of approximately 50 per cent of the seed and were entirely free from subsequent damping-off in both experiments. The results ap- pear in Table XI. In this case not only the damping-off after emergence but the loss before the seedlings appeared bore an apparent relation to sowing density. In the field experiments there was no evidence that the loss before the seedlings appeared was affected by seed density. 600 J.200 4300 Z/f-OO 3,000 A/OMBSff orseeos sotv/v /=£■/? square - /vot or bed Fig. 19. — Diagram showing the extent of damping-off in drill-sown Pinus banksiana in plats with different seed densities. The regular seed density at tins nursery was dint seeds per square foot. DAMTMXc OFF IX FOREST NTJRSERIES. 75 Table XT. — Results of modulation, ai tin time <>f sowing, nith Pythiwn deba/ryanum on Firms banlcsiana in different sun-inn densities in pots of (HI tori 1 1 red SOU. [The percent age of" Damping-off," columns 4 and :w i'ii iii columns 3, 5, 6, and 8 r,are based on the numbei of seedlings ii. IT, , ,\ ,,n i he number ol seed the percentages Num- ber of pol s in cx- prri- 11 HI 1 1 . Results (per ceni i. I lensil y of spoil sowing. Experiment . r >*. Experiment 59. Emerged . Damn- ing-oil. Sur- vival. Emerged. Damp- in "-1,11 7 Sur- vival. 1 2 3 4 5 6 8 10 5 5 5 15 8 1 6 43 G5 100 33 10 3 4 2G 8 11 17 13 91 34 37 •a Double l Triple Regular, l>ut in additional seeds near 7 11 MOISTURE AND TEMPERATURE FACTORS. The relation of damping-off to moisture and temperature are sub- jects less easily studied. In 11)07 and 1908 Mr. W. H. Mast, then supervisor of the Nebraska National Forest, conducted daily counts of the number of seedlings damped-off and compared these records with temperature and rainfall records. The writer in 1909 repeated his work, maintaining parallel records of damping-off, air and soil temperatures, soil moisture, atmospheric humidity, wind movements, and evaporation. The 1909 records of damping-off, temperature, soil moisture, and evaporation appear in figure 20. The damped-off seedlings were counted and removed in the morning and evening, the day period thus being in most cases 10 to 11 hours and the night period 13 to 14 hours. Because the period was not always the same length, the data are reduced to a per hour basis. Air temperature was recorded by a sheltered thermograph 3 feet above the soil sur- face. The evaporation graph represents the mean loss per hour from two porous cup atometers of the writer's own design, in which the rather long and slender Chamberlain filter bougie was used and supported in a horizontal position just above the soil surface so as to be under as nearly as possible the same atmospheric conditions as the seedlings. The two bougies were placed at right angles to each other in order to eliminate as far as possible the effect of change of wind direction on their mean loss. While the rain-correction mount- ing had not at that time come into use, the error due to rain absorp- tion appeared negligible; atometers filled shortly before rainfall were read immediately after without any gain being found in the water in the reservoir. The psychrograph and wind-movement rec- ords are not presented, as the evaporation valucsarc more easily inter- preted. Soil moisture was periodically determined in the soil of the 76 BULLETIN f)34, U. S. DEPARTMENT OF AGRICULTURE. (±H30d3c/) -0 -yflOHt/ld-O-D 3dnj.QI0mi09 3df)lVd3dH31. NO/J.b>6/Odl//!3 '■!) <3 o* G ci G ^ (j. N30 H3dJ dDOH if 3d AJO -OSdW&O 59NI1033S DAMPTNG-OFF IN FOREST NURSERTES. 77 plats on which the seedling counts were conducted, each determina- tion representing two, and in some cases four, points. The deter- minations for the upper one- fourth inch of soil, made more frequently than for lower levels, are connected in figure 20 by a dotted line, which gives some idea of the amount of moisture in the surface soil during the periods be! ween determinations. The determinations were too infrequent to permit anything more than an estimate of the moisture conditions between determinations, but the writer, having before him the records of the times and amounts of rainfall and artificial watering as well as the evaporation and soil-moisture de- terminations, is in a better position to make such an estimate than the reader. The dotted line which gives this estimate should not be depended on to show what the percentage of moisture was at any one time, but is believed reasonably reliable as showing whether in gen- eral the soil was wet or dry. In interpreting the soil-moisture rec- ords, it should be kept in mind that the soil was very sandy, the wilting coefficient of composite samples from various parts of the nursery, as determined by the indirect method of Briggs and Shantz in the Laboratory of Biophysical Investigations of the Bureau of Plant Industry, being only 3.4 per cent. The hygroscopic moisture in dry air for the soil of the plats actually under consideration was indicated by repeated determinations for the surface soil on dry days to be in the neighborhood of or slightly below 2 per cent. The nursery is located in a region of large temperature fluctuations, where the air during the day is generally dry, and consequently the dew is heavy at night. The first result of interest is the difference between the damping- off for the day and the night periods. In the records of every day but two, more seedlings went down during the day period than during the night, the differences in most cases being large. As the evaporation and temperature showed similar day and night fluctua- tions, it is difficult to say whether temperature or moisture condi- tions were responsible. The other interesting result brought out by the graphs is the sudden drop in the general level of the damping- off graph following the rains of June 15, June 19-20, and July 3. In each of these three cases the damping-off came up again only after the soil moisture came down. The fact that in the daily fluctuations the damping-off varied directly with the evaporation rather than inversely is an apparent contradiction of the generally accepted doctrine that moisture favors the disease. This contradiction is, however, only apparent. Dur- ing the first part of the damping-off period, when the seedlings are still soft, the recognition of damping-off depends on the decay of that part of the stem just above the soil surface which allows the seedling to fall over. This usually takes place at this nursery as 78 BULLETIN »34, U. S. DEPARTMENT OF AGRICULTURE. a result of the extension upward of Lesions which have started on the parts a little below the soil surface. It is supposed that such decay takes place most rapidly at high temperatures and that it is the temperature rather than the evaporation graph which the damp- ing-off is following in these early day and night fluctuations. Dur- ing the latter part of the damping-off period a dying seedling shows its first signs of distress in the drying up of its leaves, the stem being too stiff to go down until alter the infection has gone far enough in the roots to cut off most of the water supply. It is, of course, under dry conditions that such a sign of distress will be most in evidence. During the latter part of the damping-off period it is therefore altogether likely that the day and night fluctuations are caused, at least in part, by the higher evaporation rate which ob- tains during the day. This is a relation not to the rate of progress of the disease, but rather to the rate at tvhich the symptoms of dis- ease appear in plants already seriously affected. The drop in damping-off following the increased soil moisture of June 15, 19-20, and July 3 also apparently contradicts established doctrine. While it is ordinarily true that a wet soil is a cold soil and that in the rainy weather which causes wet soil the evaporation is usually low, it does not seem possible on inspection of the graphs for these items to attribute entirely the reduction of damping-off during these periods of wet soil either to low temperature or to low evaporating power of the air. Lowered soil temperature probably had something to do with the reduced loss following the rains. It is also suggested that a sudden change in moisture content may tem- porarily hinder a soil fungus by decreasing its air supply. In this samty soil the fungi can work at very considerable depths during dry periods. Initial lesions have been found as much as 12 inches below the surface. If this soil is as completely changed in its aera- tion qualities by wetting as the sandy soil with which Buckingham (19) worked, a rain might result in a rather sudden change in the level at which the fungus is able to operate. On the whole, the graphs tend to confirm the common statement that high temperature favors damping-off. It must, however, be borne in mind that in uncontrolled field plats several factors vary simultaneously, and it is impossible to definitely attribute any ob- served phenomenon to any one of them. Furthermore, it is not possible to say for the seedlings at different ages just how long it will take a factor to exert an effect on the damping-off curve. An addi- tional consideration is that a method of investigation which gives entirely reliable information on the speed with which the disease develops does not necessarily throw light on the conditions under which the greatest total amount of disease can be expected before the seedlings become old enough to resist attack. High temperatures, DAMPING-OFF IN FOREST NURSERIES. 79 within reasonable limits, are expected to increase the speed with which the disease works, but these should also hasten the develop- ment of the host to a point at which infections are unable to cause death. It is the total amount of damage in the beds rather than the damage per unit of time which is of practical importance. For a number of reasons, then, the method followed in obtaining the data for these graphs can not give information of maximum value. While data of the sort mentioned are of undoubted interest and would be of still more value if the records had been commenced when the first seedlings appeared instead of a few days later, the relation of any specific factor to the total extent of the disease can be better deter- mined by comparing plats in series in which the factors are as far as possible controlled and varied one at a time. To vary soil moisture and soil temperature independently will prove somewhat difficult, but it can be done with the proper facilities. Some work with en- vironmental factors should be done under conditions of artificial in- oculation in the greenhouse, in which the different damping-off parasites can be experimented with separately, as it is obvious that the factors which favor the activity of one may not be favorable for another. CHEMICAL FACTORS. Chemical factors are presumably also important, as the soil is in most cases the culture medium for both the parasite and the host. The much greater activity of Pythium debaryanum in autoclaved soil than in untreated soil may be due to the larger quantity of soluble organic matter commonly present in autoclaved soil. Pythium debaryanum has been found more sensitive to unfavorable substrata in artificial culture than Corticium vagum and is apparently more dependent on soil organic matter in the nurseries than is C. vagum. For example, in the normal humus-containing surface sand in the beds at Cass Lake, Minn., both Pythium and Corticium occurred frequently in the damped-off seedlings, while in beds a few feet distant, from which enough of the surface soil had been removed to leave no humus, nearly all the damping-off foci contained abundant Corticium, and no Pythium could be found. With both fungi and, in addition, with two species of Fusarium (68) heavy inoculation has been more successful in experiments at the time of sowing than light inoculation. This has been thought possibly due in part to the larger amount of nutrient substratum added in the heavy inocula- tions, allowing better saprophytic development of the fungus in the soil. In each of the two experiments with Pythium reported in Table XI, a 5-pot unit was treated with corn-meal infusion and another with prune infusion at the time of inoculation. In both experiments germination was lower, damping-off after germination higher, and the survival less than half as great in the pots with 80 BULLETIN 934, U. S. DEPARTMENT OF AGRICULTURE. infusion as in the inoculated pots not so treated. Tn the first experi- ment 5-pot units of unheated soil were also inoculated in the same way. In these also both the units which received infusions showed less germination and more loss after germination than the unit which received no infusion, though the differences were smaller than in the autoelaved soil. In the second experiment the light inoculation used failed to cause material loss in the unheated soil units, even though two of them were treated with the in fusion as in the previous test and two others received triple portions of the infusion. The experience in the nurseries, in which heavy applications of manure, and especially poorly rotted manure, in a number of cases have apparently resulted in increased disease, and the finding of Fred (43) that green manures recently plowed under favored the work of Corticium have already been mentioned.. The addition of dried blood at two nurseries in Kansas was in both cases followed by very much heavier loss than in the controlled plats. The only instances known to the writer in which the addition of organic matter to the soil has shown any indication of materially decreasing damping-ofT (with the exception, of course, of the organic disin- fectants) are the result reported by Gifford (46) with tankage, a single case in the writer's experience with bone meal, and the cases in which cane sugar has seemed to decrease losses somewhat (67). It is of some interest to note that the experience available also indi- cates increased disease as a result of the addition of inorganic nitrog- enous substances. Sodium nitrate and sodium nitrite have both given some indication of increasing damping-off. Ammonium sul- phate in six separate series has in every case resulted in decreased stands, though unfortunately in experiments in which the damped- off seedlings were not counted. Ammonium hydroxid, though ap- parently having some initial value as a disinfectant, as indicated by early damping-off losses, in a number, of cases has been followed by very heavy total losses. This experience is of some interest in view of the apparently rather general belief that plants on a soil rich in nitrogen are especially susceptible to disease. The chemical factor for which there is perhaps the most evidence of a relation to damping-off of conifers is acidity. The fact that sulphuric-acid soil treatment has been found to be one of the most effective means of controlling the disease, that its value is mainly lost if lime is later added to the soil, that soil treatment with sulphur in a number of cases has seemed to decrease the disease, and that lime alone and wood ashes have had either no effect or have appar- ently increased the damping-off whenever they have been tried, all suggest that soil acidity is not favorable to the disease. Additional indication of this appears in figure 12. The acidity determinations serving as the basis for the graph were made by Dr. L. J. Gillespie, DA.MJMNC (il I |\ FOREST NURSERIES. 81 of the Bureau of Plant Industry. The estimates of the relative riousness of damping-off arc very approximate, based in part on observation only. The stations ai which damping-off is rated as 1 are places at which it lias been reported by nurserymen or foresters as negligible or absent. The estimates for stations LO, 11, 11, and 15 are based entirely on the reports of others, and for station 5 on the basis of counts of damped-off seedlings made by Mr. R. G. Pierce and Mr. Glenn (J. Ilahn. The writer personally has made the esti- mates or checked the estimates of the nurserymen at the other sta- tions. A considerable degree of correlation between the hydrogen- ion exponent and the amount of damping-oil' appears on the face of the graphs, the coefficient being 0.75 ±0.07. If the correlation is calculated with the H + concentration itself instead of its negative exponent, the coefficient, in this case itself negative, is not so high (— 0.58±0.11). All of the above data on acidity relation have been picked up incidentally in connection with other work and are merely suggestive. The suggestion, however, seems sufficiently strong to warrant further experimental work directed specifically at the rela- tion between soil acidity and the disease. The indication in the graph that damping-off is not serious in soils in which the hydrogen-ion exponent (P H )' ; is less than 6 is of particular interest, in view of the experience of Hawkins and Har- vev (~1) with cultures of Pythivm debaryanum on potato juice. The} 7 obtained good growth through a range of acidity from P H 3.4 to 5.8, with no growth or practically none at 3.0G or 8.4. If this represents the acid tolerance of the fungus in the soil solution, it is evident that ordinarily acid soils can not be expected to remain free from damping-off because of inhibition of this particular fungus. This suggests that the apparently salutary influence of soil acidity in decreasing the damping-off of some of the conifers may be ex- erted in the direction of increasing the resistance of the host rather than of inhibiting the parasites. In any case, it must be kept in mind that as the numerous conifer hosts commonly grown in nurseries have many different habitat preferences and many very different parasites of potential importance, it is not to be expected that there will be found any such constant relation between any factor and the amount of disease as would be expected in a disease in which only a single parasite and a single host are involved. •Ph 6 is equivalent to a hydrogen-ion concentration, expressed in mols per liter, of lXlO- 6 or 0.000001. The higher the exponent, the smaller the hydrogen-ion concentra- tion. An exponent of 7 moans approximate neutrality. In dealing with this exponen- tial form of expression, it should be kept in mind that P H 6 means ten times and l' H 5 one hundred times the hydrogen-iron concentration indicated by P H 7. Conversely, the concentration of hydroxy I ions at I'm" is one hundred times as great as at 1*h5. 10651°— Bull. 934—21 6 82 l.l I.I.I.'I'IX 934, U. S. DEPARTMENT OF AGRICULTURE. BIOLOGIC FACTORS. Mention has already been made of two strictly biologic factors which may influence the amount of damping-off. Taylor (138) and Rathbun (IOC) have found Fusarium not only at considerable depths in the soil of pine seed beds, but viable Fusarium spores without hyphse in the alimentary canals of earthworms and insect larvae in the soil, and they attribute to the migrations of these and to the tunnels which various animal forms make in the soil a possible im- portance in the distribution of damping-off Fusaria. A likely rela- tion between Corticiurn vagv/m epidemics in pine seed beds and the character of the weed flora has also been considered (GG). The relation between the damping-off parasites and other micro- organisms in the soil is also a matter of some interest. The effect of the microfauna of the soil on the microflora in general has been considered by Russell and others in a number of papers. The effect of soil disinfection by heat in favoring the work of artificially intro- duced soil-inhabiting fungous parasites, apparently a rather frequent phenomenon and quite evident in the inoculation experiments with Pyihium debcuryanum reported in the present bulletin, has been in other cases attributed tcr the removal of bacteria and other fungi which might compete with the parasites (3G, 80). Heating soil is known to produce physical changes and also very considerable chemi- cal changes both in organic and inorganic substances. These must not be ignored in considering the effect of previous soil heating on para- site activity. With a view to determining whether all the difference noted in the behavior of P. debaryanum in heated soil is due to the direct effects of the heating or in part to the elimination of com- peting microorganisms, an experiment was conducted in 3-inch pots of antoclaved soil in which 111 of them were inoculated with agar cultures of the Pythium at one point in each pot shortly after seed sowing. The seeds sown in each pot approximated 136, considerably more than are used on equal areas of nursery seed bed. Of these, fifteen 5-pot units and one 3-pot unit had been inoculated broadcast with rice or nutrient agar cultures of various organisms supposed to be saprophytic on pines. These included Phoma betae, Phoma sp., Chaetomium sp. (from a maple root), Rhizopus nigricans, Tricho- thecium roseum, Trichoderma koningi, Aspergillus spp. (including one with black and one with bright-colored spore heads) , Rosellinia sp. (from soil) , Penicillium sp., an undetermined bacterium, and three undetermined higher fungi. The whole 78 pots inoculated with P. debaryanum and saprophytes, the percentages being based on the total number of seeds in the case of emergence and survival and on the number of seedlings which appeared above ground for damping- off loss, as compared with those which had received the parasite only, gave results as follows: DAMPING-OFF IN FOREST NURSERIES. 83 Pots with saprophytes: Emergence, I7.4±0.80 per cent; damplng-off, 9J per cent; survival, 41.0±1.23 per cent. Pots without saprophytes: Emergence, :'."'.7 per cent; damping-off, 14.3 per cciil ; survival. 20.2 per cent. It has been noted in the attempts to diagnose damping-off by planted-plate cultures that when Rhizopus appears Pythium debary- atiiun is not frequently obtained. It is therefore of some interest to note that in this case, in the two 5-pot units which received Rhizopus in addition, the parasite killed only 1.2 per cent and 3.3 per cent, respectively, of the seedlings which appeared above the soil. At the same time pots not inoculated with parasites were sown, 16 other 5-pot units were inoculated with the same saprophytes as those used in the Pythium inoculated pots, while 25 pots were left entirely without inoculation. A certain amount of damping-off occurred in these pots also as a result of accidental infection. The results were as follows : Pols wiih saprophytes: Emergence, 17.8±0.8 per cent; damping-off, 3.9 per cciil : survival, 43.7±0.95 per cent. Pots without saprophytes: Emergence, 43.0 per cent; damping-off, r>.2 per cent ; survival. 38.4 per cent. It is of some interest to note that in these pots also the 5-pot units inoculated with Khizopus suffered less from damping-off than the average of the saprophyte-inoculated pots. The probable-error values given above are based on the variability of the emergence and survival figures of the different 5-pot units. No individual figures are available to serve as a basis for the deter- mination of the variability of the pots without saprophytes. The 16 units which support the error determinations are, of course, not a sufficient number to give an entirely reliable index of variability, though these 16 units are respectively derived from the combination of a total of 78 and 80 ultimate units. The distribution of the data appears to be such as to justify the use of probability methods. Of the 64 items which went into the germination and survival calcula- tions, 34 showed a deviation equal to E s (probable error of a single unit), 9 to a deviation equal to 2E S , and only one a deviation equal to 3E S . All of the above figures are based on the results at the end of 10 days after average emergence in the pofcs. The pots were kept on the benches till practically all damping-off had ceased, 36 days after emergence. As additional accidental infection with saprophytes certainly, and probably with parasites, occurred during this period, the results at the end of the tenth day are considered to give a better indication of the effect of the original inoculations. It is of some interest, however, to note that during the period from the tenth to the thirty-sixth day the difference between the pots to which sapro- 84 BULLETIN 934, IT. s. DEPARTMENT OK AGRICULTURE. phytes had been added and those which received no saprophytes showed a slight increase, proportionally as well as in the absolute figures. At the end of the 3G days the survivals on all the pots were counted separately. The average number of seedlings per pot were as follows : Without Pythium : Without saprophytes, 42.8±2.3; with saprophytes, 52.1±1.1 With Pythium: Without saprophytes, o0.1±2.4; with saprophytes, 4S.1±1.4. The difference in the survivals in favor of the pots with sapro- phytes in the first case is 9.3 ±2.5, three and two-thirds times its probable error. In the second case it is 18.0±2.8, more than six times its probable error. In general, it appears that in this experiment the inoculation of ster- ilized soil with saprophytes gave the seedlings some protection both against damping-off due to accidental infection with unidentified parasites and from the additional loss caused by light inoculation with Pythium debaryanum. The indication is, as would be expected, that only part of the favoring influence of heat sterilization of soil on introduced P. debaryanum is immediately due to the elimination of competition with other fungi. If a mixture of different bacteria and fungi had been added to each of the pots instead of but one or tw T o organisms to each 5-pot unit, the effect might have been more marked. It will be noted that for all the groups (fig. 18), whether with or without Pythium inoculation and with or without added parasites, the frequency polygon is asymmetrical, indicating by its shape, as did the frequency polygon of survivals in pots inoculated with Rheo- sporangium (fig. IT), that with infections which do not kill all of the seedlings the selection tends to be by pots rather than by seed- lings. In other words, in pots in which the parasites succeed in kill- ing any of the seedlings, they usually kill a considerable number. The tendency is illustrated not only by inspection of the graphs, but by the variability of the different groups. The greater variability in survivals between different pots was in both cases in the groups in which both the damping-off after emergence and the survival per- centages indicated the largest loss. The percentages of seedlings damped-off during the entire 3G days following emergence and the coefficients of variability of the survivals of the individual pots at the end of that time are as follows : Without Pythium : Without saprophytes, 15.5 per cent damped-off; coefficient of variability, 39±4.2 per cent. With saprophytes, 11.1 per cent damped-off; coefficient of variability. 2S±1.6 per cent. DAMPING-OFF IN FOREST NURSERIES. 85 With Pythium: Without saprophytes, 27.5 per cenl damped-off; coefficient <>r variability, G7±7.S per cent. With saprophytes. 16.9 per rout ilainpoil-otT ; coclhcient of variability, 39±2.4 per cent. This tendency has been frequently observed in experiments in which inoculum is applied to the soil at the time of sowing. Even in experiments in which a relatively small proportion of the seed- lings are killed, some of the pots are nearly or entirely cleaned out. It is taken as an indication that failure of inoculation to give results is often due to the inability of the fungus to maintain itself in a vigorous condition till the germinating seed is far enough along to allow easy infection. Tt may also be in part due to lack of uni- formity of the soil in different pots affecting virulence of parasites or resistance of hosts. In addition to this experiment on autoclaved soil, a somewhat similar experiment was conducted in a nursery in the Kansas sand hills on soil which had been treated with sulphuric acid, followed by lime raked into the soil. Saprophytes, for the most part the same strains that had been used in the experiment in the greenhouse, were added to 2-4 plats, each of one-half square foot, of Pinus bank- siana and 24 of P. pondcrosa, with 16 interspersed plats of each species serving as controls. The saprophytes were growing on rice, part of which was added to the plat with the inoculum in addition to the fungous mycelium. Damping-off w T as rather heavy in this soil from accidental infection or from parasites which survived the initial acid treatment, no parasites having been artificially intro- duced. The loss was probably due to Corticium vagum, or Fusarium spp. rather than to Pythium debaryanum in this case. In both pines, emergence was slightly better in the control plats than in those to which the saprophytes had been added, the difference for Pinus banh- siana being less than half its probable error and for P. ponderosa slightly more than its probable error. Damping-off for the first few days after emergence was somewhat less in the controls in one species, but higher in the saprophyte-inoculated pots in the other. The saprophytes therefore gave no evidence of effective competition with the parasites on this acid-lime treated sand. While the competition for water which seems to be the form of competition most common among green vascular plants is not likely to be of significance between fungi such as those which cause damping- off, a very little observation of the growth of mixed cultures of the parasites and other organisms in Petri dishes is sufficient to make one realize that the latter may very considerably decrease the activity of certain of the parasites. In nutrient agar most of the fungi and bacteria introduced from the soil in attempting parasite isolations. 86 BULLETIN 934, U. S. DEPARTMENT OF AGRICULTURE. as well as to a less extent the paramenia, nematodes, and amoeba? which develop in such plates, exert a very considerable limiting in- fluence on the growth of most of the damping-off fungi. That they should also limit the growth of parasites in soil, whether by the production of toxic compounds, the exhaustion of food materials, or in other ways, seems entirely reasonable. The results in the writer's experiments on heated soil warrant the suggestion that further trials should be made of the introduction of vigorously growing bacteria or molds, preferably mixed cultures containing a number of different organisms, on seed beds which have been disinfected by some such method as steam or hot water, which leaves the soil in a favorable condition for the development of accidentally reintroduced parasites. If such treatment should be successful in improving the rather dis- appointing results with soil heating at some nurseries, it might easily become of practical value, as the cultivation of certain of the more easily grown saprophytes on a scale large enough to yield con- siderable quantities of bacterial or spore suspensions should be fairly easy and entirely practicable in an operation as intensive as that of raising coniferous seedlings. ACKNOWLEDGMENTS. The writer wishes to express 1 his obligations to Dr. H. A. Edson, of the Bureau of Plant Industry, United States Department of Agriculture, and to Prof. W. T. Home, of the University of Cali- fornia, for suggestions during the progress of this work; to Mr. Roy G. Pierce and Mr. Glenn G. Harm, of the Bureau of Plant Industry, for assistance in a number of the inoculation experiments; and to other members of 'the staff of the Office of Forest Pathology for assistance and suggestions at various times. SUMMARY. (1) Damping-ofT in nurseries is caused mainly by seedling para- sites which are not specialized as to host ; Pythium debaryanum and Corticium vagum are probably the most important of these. Damp- ing-off of various herbaceous hosts, including ferns, is often caused by specialized parasites which are limited to a particular host or group of hosts. Phoma betae is a rather extreme example of such specialization. For the conifers all the damping-off appears to be due to parasites of the generalized type. (2) Damping-off of trees, as of herbaceous plants (with the ex- ception of the cases caused by specialized seed-carried parasites), is ordinarily serious only in seed beds or cutting beds in which large numbers of plants are crowded together in a small space. In most of the forest nurseries it is a much more serious matter in conifers than in dicotyledonous seedlings. DAMPING-OFF TN FOREST NURSERI KS. 87 (3) The most serious losses in conifers are ordinarily from the root-rot type of damping-off, occurring soon after the seedlings appear above ground and while the hypocotyls are still soft. Losses due to the killing of dormant or sprouting seed by parasites before the seedlings appear above the soil are also frequently serious, some- times necessarily more so than the later types, as in extreme cases more than half of the seed or young seedlings are destroyed in this way. Damping-off due to infections of parts above the soil surface is serious only under extremely moist atmospheric conditions. The late type of damping-off, in which the roots are rotted after the stem becomes too rigid to be easily decayed, is ordinarily less im- portant than the early types. Seedlings more than 2 months old are ordinarily able to recover from infections by the damping-off fungi. Even after the first month, seedlings with part of their root system killed often recover. (4) It is possible that damping-off has a certain value as a selec- tive agent by eliminating weak individuals in the seed-bed stage and allowing only the best trees to go into forest plantations. This value, however, is believed to be slight. Disinfectant treatments of seed beds, even when controlling early parasitic losses very well, allow a considerable percentage of disease during the last part of the damping-off period, often as much as occurs at the same stage of development in untreated beds. As it is only this late damping-off in which differences in individual resistance of the seedlings seem to be of importance in determining whether or not they 'succumb, it is believed that whatever selective value the disease may "have will appear in a larger proportion of the damping-off in the treated than in the untreated beds. (5) Of the different conifers, reports are available as to the sus- ceptibility of 63 species. Species which are especially susceptible at some nurseries may prove more resistant than the average at others. Pinus resinosa, which is especially subject to loss at some nurseries, is believed to be so because its growth at these nurseries is slow and its period of susceptibility is therefore especially long. In its early stages it does not seem especially susceptible. Repre- sentatives of all the commonly grown genera of the Abietoidese have been reported by one observer or another as decidedly susceptible. The reports on junipers and other members of the Cupressoidea3, on the other hand, have indicated a considerable amount of group re- sistance to damping-off. (6) The best control method appears to be the disinfectant treat- ment of the seed-bed soil before or immediately after seed is sown. Sulphuric acid has been found very useful for conifers, as they are apparently especially tolerant of acid treatment. No method has yet been worked out to a point at which all of its details are entirely 88 lULl.KTlX 984, U. S. DEPARTMENT OF AGRICULTURE. satisfactory, though the acid treatment lias now boon in successful use for several years at some nurseries. At most nurseries, if the m in im um effective quantity of acid is used, there is no need of any special precautions to prevent injury to the seedlings. It is not expected that any single treatment can be found that can be uni- versally applied without change in details irrespective of differences in soil characters and in fungous flora. (7) Corticium vagum and Fusarium spp. have boon previously shown to be parasitic on pine seedlings. Different strains of C. vagum are found to vary considerably in their ability to cause damp- ing-off, certain strains being consistently destructive and others much less active in tests conducted on different species of pine and several years apart. The differences in activity between strains were greater, and apparently rather more constant from one ex- periment to the next, than with Peltier's strains in his carnation ex- periments. Comparison of the results on pine with those of Edson and Shapovalov on potato gives some indication that strains vigor- ously parasitic on one of these hosts are likely to be parasitic on the other also. (8) PytMum di baryanum, reported on many hosts and proved to be parasitic on few, is shown by repeated inoculation, reisolation, and reinoculation to be capable of causing the damping-off of seed- lings of pine species. The identity of the fungus causing the damp- ing-off of conifers with that attacking dicotyledons has been estab- lished by cross-inoculations as well as by morphological comparison. Inoculations on unheated soil are much less destructive than on heated soil. PytMum <1( baryanum has been obtained in culture from Picea engelmanni, P. sitcihensis, Tsuga merfo nsiana, Pinus banksiana, P. nigra austriaca, P. ponderosa] P. resinosa, and Pseudotsuga taxi- folia. In addition, fenugreek (TrigoneUa foenum-grm cum), cowpea (Vigna sp.), and rice (Oryza sativa) are reported as apparently new hosts among the dicotyledons. In inoculations the fungus has been successfully used on Pinus banksiana, P. ponderosa, P. resinosa, and in a preliminary experiment on Pseudotsuga taxifoMa. It had already been successfully used in preliminary inoculations on Picea canadensis by Hofmann (77). Differences in parasitic activity on pine are found between differ- ent strains of PytMum debaryanum. These differences are not as large and partly for this reason their constancy is not quite as con- clusively demonstrated as in the case of the strains of Corticium vagum. (9) Rheosponnu/ium aphanidermatus Edson, a parasite of radish and sugar beet, in many ways closely resembling PytMum debary- anum, has killed seedlings of Pinus banksiana and P. resinosa in certain experiments, and reisolations and reinoculations have been DAMPING-OFF IN FOREST NURSERIES. 89 made. The strain available is much less destructive to the pines than most of the P. debaryamirn strains used, and as the fungus has never been isolated from coniferous seed beds it is not believed to be of any great importance in forest nurseries. (10) Phytophthora sp. from Pinus resinosa seedlings has been suc- cessfully used in inoculation on Pi mis resinosa and in a preliminary test on P. ponderosa. The strains available have been less destruc- tive to the pines than Pythiwm debaryanwm and the stronger strains of Corticium vagum. It is not common. Its relation to Phytoph- f/iora fagi, the European damping-off parasite of both conifers and dicotyledons, which has not been reported in this country, is being investigated. (11) A fungus referred to Pythiwm artotrogus, also obtained from damped-off Pinus resinosa, has indicated a very low degree of par- asitism on this host, even less than that shown by the Rheospor- angium and Phytophthora strains. An addition is made to the statements in a previous paper concerning the ability of Botrytis cinerea to cause damping-off in conifers. (12) The results of the cultural or direct examination of 742 dis- ease foci in seed beds of various conifers are reported. Pythium debaryamirn, in the plate-culture examination method, considered more reliable than direct examination, appeared in 51 per cent of the foci from untreated beds, while Corticium vagum was found in 19 per cent. In foci in beds treated with various disinfectants, P. debaryanum was identified in 38 per cent of the foci and C. vagum in only 4 per cent. When direct microscopic examination was sub- stituted for isolation, C. vagum was found on a larger proportion of the seedlings. It was not found at all in soil which had been heated. The relative ease with which it appears to be controlled by soil disinfection is in agreement with its poor adaptation for aerial distribution. It was found more commonly in cases in which the seedlings were directly examined than when cultures were made. In view of the fact that at least some of the Corticium foci extend rapidly and include very large numbers of seedlings, it seems that the Corticium may be as important as P. debaryanum in causing the damping-off of pines. (13) Fusarium spp. have occurred more commonly in plate cul- tures than either of the above-mentioned fungi. Because little is known as to the parasitism of different species of this group on conifers, it is not possible to make any statement regarding the im- portance of the individual species. The evidence as a whole indi- cates so much importance for Pythiwm debaryanum, Corticium vagum, and for the Fusarium spp. considered as a group that no one of the three can be safely said to be more important than the others. None of the other fungi considered appear to be of real economic rank in the United States. 90 BULLETIN 934, U. S. DEPARTMENT OF AGRICULTURE. (14) In an inoculation experiment on the roots of pines 1 .', months old, CorUdv/m vagwm and Pythium debdryemu/m were found able to cause the death of seedlings which had already developed rigid stems and to destroy the younger parts of the roots of seedlings which they were unable to kill. Indications were also obtained of similar but less vigorous action by Fusarium monUiforme and F. ven- tricosuu). (15) Data are given confirming the general belief that thick sow- ing favors the disease and indicating that soil acidity is, in general, unfavorable. Preliminary data on the relation of temperature and moisture to the disease are also presented. The parasitic activity of Pythi/wm debaryanum in steamed soil was in one extensive test con-, siderablv decreased, following the inoculation of the soil with various saprophytes ; this indicates both that competition of different fungi is a factor to be considered and that the inoculation of treated soil with saprophytes may sometimes prove of value in increasing the efficiency of heat disinfection. It is pointed out that with such a com- plex of parasites capable of producing identical symptoms on a num- ber of different hosts, no relationship between environmental factors and the disease can be expected to hold in all cases. LITERATURE CITED. Atkinson, Gboege F. (1) 1S92. Some diseases of cotton. Ala. Agr. Exp. Sta. Bui. 41, 65 p., 25 fig., 1 pi. (2) 1895. Damping-off. N. Y. (Cornell) Agr. Exp. Sta. Bui. 94, p. 233-L'7L\ fig. 55, t; pi. (3) llMiL 1 . Studies of some tree-destroying fungi. In Trans. Mass. Hort. Soc, 1901, pt. 1, p. 109-130. (4) Babbe, H. W. L912. ('otton anthracnose. S. C. Agr. Exp. Sta. Bui. 1G4, 22 p., 7 pi. (5) Bast, A. de. 1SS1. Zur Kenntniss der Peronosporeen. In Bot. Ztg., Jahrg. 39, No. :VA. p. 521-530; No. 34, p. 537-544; No. 35, p. 553-5G3 ; No. 36, p. r.(;i>-r,7,S ; No. 37, p. 5S5-595 ; No. 3S, p. 601-G09; No. 39, p. 017-625, pi. 5. (6) Bates, Carlos G. 1907. Timber fungi, with special reference to the pines. In 38th Ann. Rpt. Nebr. Hort. Soc, 1907, p. 201-208. (7) and Pierce, Boy <;. 1913. Forestation of the sand hills of Nebraska and Kansas. U. S. Dept. Agr., Forest Serv. Bui. 121, 1!) p., 1 fig., 13 pi. Baumsch, Fbiedeich. (8) 1SSS. Ueber " Phytophthora omnivora " als Schiidling des Buchenauf- schlages. In Centbl. Gesam. Forstw., Jahrg. 14, Heft 8/9, p. 3S2-385. (9) 1903. Notizen fiber Septoria parasitica It. H., Fusoma Pini R. H. und Allescheria Laricis R. II. hi Centbl. Gesam. Forstw., Jahrg. 29, Heft 11. p. 401-404. ■ (10) Beinhabt, E. G. 1918. Steam sterilization of seed beds for tobacco and other crops. U. S. Dept. Agr., Farmers' Bui. 090, 15 p., 4 fig. (11) Bkissnek, Ludwic 1909. Handbuch der Nadelholzkunde. Aufl. 2, xvi, 742 p., illus. Berlin. (ill) Blackman, V. H., and Welsford, E. J. 1916. Studies in the physiology of parasitism. II. Infection by Botrytis cinerea. In Ann. Bot., v. 30, no. 119, p. 389-398, pi. 10. Literature cited, p. 397. (13) Boebkeb, Richard H. 1910. Ecological investigations upon the germination and early growth of forest trees. In Nebr. Univ. Studies, v. 16, no. 1/2, p. 1-90, illus., 5 pi. Bibliography, p. 88-89. (14) Bolley, H. L. 1901. Flax wilt and flax sick soil. N. Dak. Agr. Exp. Sta. Bui. 50, p. 27-58, [17] fig. (15) Brown, James. 1894. The Forester . . . ed. 6 enl., ed. by John Nesbit. Vol.2. Edin- burgh. London. 91 92 BULLETIN 934, 1 T . S. DEPARTMENT OF AGRICULTURE. (1G) Brown, William. L916. Studies in the physiology of parasitism. III. On the relation between the "infection drop" and the underlying host tissue. /// Ann. Bot, v. 30, no: 119, p. 399-400. (17) BlWCHMANN, H. 18S5. Das Prothallium von Lyeopodium. hi Bot. Oentbl., Bd. 21, No. 10, p. 309-313. (IS) Bryant, Abthxtr. 1871. Forest Trees for Shelter, Ornament, and Proiit . . . 247 p., [12] pi. New York. (19) Buckingham, Edgar. 1904. Contributions to our knowledge of the aeration of soils. U. S. Dept. Agr., Bur. Soils Bui. 2;".. r>2 p. (20) Burger, O. F. 1913. Lettuce drop. Fla. Agr. Exp. Sta. Bui. 116, p. [25]-32, 3 flg. (21) BuRcsnoRF, Friedrich August Ludwtg von. 17S3. Versuch einer vollstandigen Geschichte vorziiglicher Holzarten . . . Bd. 1, Theil 1. Berlin. (22) Busse, W., Feters, L., and Ulrich, F. 1911. Ueber das Vorkominen von Wurzelbranderregern im Boden. In Arb. K. Biol. Anst. Land- u. Forstw., Bd. 8, Heft 2, p. 260-302. (Untersuchungen fiber die Krankheiten der Rfiben, von W. Busse. 6.) Butler. E. J. (23) 1907. An account of the genus Pythium and some Chytridiacese. In Mem. Dept. Agr. India. Bot. Ser., v. 1. no. 5, 160 p., 1<» pi. Literature, p. 142-147. (24) 1913. Pythium debaryanum Hesse. In Mem. Dept. Agr. India. Bot. Ser., v. 5, no. 5, p. 262-267, pi. 5. (Studies in Peronosporacese, by E. J. Butler and G. S. Kulkarni.) Buttner, G. (25) 1903. Uber das Absterbon junger Nadelholzpflanzen im Saatbeete. In Mitt. Deut. Dendrol. Gesell., No. 12, p. 81-82. (26) 1906. Etwas fiber den Keimlingspilz, Fusoma parasitieuin v. Tub. In Mitt. Deut. Dendrol. (Jesell., No. 15, p. 282. (27) Byars. L. P., and Gilrert, W. W. 1919. Soil disinfection by hot water to control the root-knot nema- tode and parasitic soil fungi. (Abstract.) In Phytopathology, v. 9, no. 1, p. 49. (28) Clinton, G. P. 1913. Report of the botanist, 1911 and 1912. In Conn. Agr. Exp. Sta., 36th Ann. Rpt 1911-1912, pt. 5, p. [341]-453, pi. 17-2S. (29) Cook. Mel T., and Hei.yar, J. P. 1915. Diseases of grains and forage crops. N. J. Agr. Exp. Sta., Circ. 51, 8 p. (30) Davenport, E. 1907. Principles of Breeding . . . xiii. 727 p., illus. Boston, New York. (31) Dawson, Jackson. 18S5. The propagation of trees and shrubs from seed. In Trans. Mass. llorl. Sue. 1885, pt. 1. p. 145-166. DAMPING-OFF IN FOREST NURSERIES. 93 DUGGAS, B. M.. and Stewabt, F. C. (32) 1903. The sterile fungus Rhizoctonia as a cause of planl diseases in America. X. V. (Cornell) Agr. Exp. Sin. Bui. L86, p. 51-76, [fig.] 15 23. (33) 1909. Fungous Diseases of Plants . . . xii. 508 i>., illus., pi. Bos- ton, New York. (34) 1916. Rhizoctonia solani in relation to the "Mopilz" and the " Ver- mehrungspilz." In Ann. Mo. Bot. (lard., v. ::, no. 3, p. 1-10. Literature cited, p. : » l o. Edgerton, C. W. (35) 1915. Effeel of temperature on Glomerella. In Phytopathology, y. 5, no. 5, p. 247-259, I flg. (36) 1915. A new method of selecting tomatoes for resistance to the wilt disease. /// Science, n. s., \. 42, no. 1095, p. 914-915. EDSON, II. A. ( "7 i L915. Histological relations of SUgar-heet seedlings and I'hoina betae. J ii Jour. Agr. Research, v. 5, no. 1, p. 55-58, pi. 1-2. (38) 1915. Seedling diseases of sugar beets and their relation to root-rot and crown-rot. /// Jour. Agr. Research, v. 4, no. 2, p. 135-1GS, pi. 16-26. Literature cited, p. 165-168. (39) 191"). Rheosporangium aphanidermatus, a new genus and species of fungus parasitic on sugar beets and radishes. In Jour. Agr. Re- search, v. 4, no. -1, p. 279-292, pi. 44-48. (40) — and Siiapovalov, M. 1918. Potato-stem lesions. In Jour. Agr. Research, v. 14, no. 5, p. 213- 220, pi. 24-26. (41) Fawcktt, H. S. 1909. Report of planl pathologist. In Fla. Agr. Exp. Sta. Rpt. 108/09, p. xlvi-lxii, fig. 7-12. (42) Fischer, Alfred. 1892. Die Pilze Deutschlands, Oesterreichs und der Schweiz. IV. Ab- theilung: Phycomycetes. In Rabenhorst, L., Kryptogamen-Flora ... Aufl. 2, lid. 1, Pilze. Leipzig. (43) Peed, E. B. 1916. Relation of green manures to the failure of certain seedlings. In Jour. Agr. Research, v. 5, no. 25, p. 11G1-117G, pi. S3-S4. Litera- ture cited, p. 1175-1176. (44) Galloway, B. T. IS'.)'.). Report of the Chief of the Division of Vegetable Pathology. In U. S. Dept. Agr., 1892, p. 215-246, 1 fig., 4 pi. (45) Gardner, John. 1S90. [Daniping-off.] In Anier. (lard., v. 11, no. G, p. 347-348. (46) Gifford, C. M. 1911. The damping-off of coniferous seedlings. VI. A.u r r. Exp. Sta. Bui. 157, ]i. 1 13 171, 10 fig., 4 pi. Bibliography, p. 171. (47) Gilbert, W. W. 1909. The root-rol of tobacco caused by Thielavia basicola. U. S. Dept. Agr., Bur. Plant Indus. Bui. 158, 55 p., 5 pi. Bibliography, p. 14-4S. 94 r.ri.l.KTIX 934, l. S. DEPARTMENT OF AGRICULTURE. (48) Glot er, W. (>. L913. Tlie efficiency of formaldehyde in the treatment of seed pota- toes for Rhizoctonia. N. V. State Agr. Exp. Sta. Bui. 370, p. 417- 131. (49) GOEREL, K. 1SS7. Oeber Prothallieo und Keimpflanzen von Lycopodium inun- datnm. In Bot. Ztg., Jahrg. 15, No. 11, p. 161 168; No. L2, p. 177- 190, |»1. 2. Gtrssow, II. T. (50) 1912. Reporl of the Dominion botanist. /// Canada Exp. Farms Rpts., 1P11 12, p. 191-215, 4 Bg., pi. 0-7. (51) 1917. The pathogenic action of Rhizoctonia on potato, In Phyto- pathology, v. 7, no. 3, p. 209-213, 1 fig. Haimkii, BYBOH I >. (52) 1892. Fungous troubles in the cutting hods, in Gard. and Forest, v. 5, no. 209, p. 91-92. (53) 1S93. Fungi injurious to wood seedlings. hi N. J. Agr. Exp. Sta. 13th Ann. Up!.. 1891, p. :'. 12-345. (54) Harris, J. Arthxtr. 1915. The value of inter-annual correlations. In Amer. Nat., v. 49, no. 587, p. 707 712. Hartig, lv. (55) 1S76. Die Buchencotyledonen - Krankheit. hi Ztschr. Forst- u. Jagdw., V,d. 8, p. 117-123. (56) 1S79. Die Buchenkeimlingskrankheil erzeugt durch Phytophthora Eagi M. In Forstw. Centbl., Jahrg. 23, Hefl 3, p. 161-170. (•">") 1S80. Der Buchenkeimlingspilz, Phytophthora (Peronospora) Eagi M. In Unters. Forstbot. Inst., Miinchen, [Bd.].l, p. 33-57, pi. 3. (58) 1880. Der Ahornkeimlingspilz, Cercospora acerina INI. hi Unters. Foist hot. Inst., Miinchen, [Bd.] 1, p. 58-62, pi. 4. (59) 1883. Beschadigung der Nadelholzsaatbeete durch Phytophthora omnivora (Fagi). In Forstw. Centbl., Jahrg. 27, Heft 12, p. 593- 596. (GO) 1892. Ein neuer Keimlingspilz. hi Forstl. Naturw. Ztschr., Jahrg. 1, Heft 11, p. 432-436, 4 fig. (01) 1S94. Text-Book of the Diseases of Trees. Trans, by William Sbmerville . . . rev. and ed., by II. Marshal] Ward, xvi, 331 p.. illUS. London. Hartley, Carl. (02) 1910. Notes on some diseases of coniferous nursery stock. (Ab- stract.) In Science, n. s., V. 31, no. 799, p. 639. (63) 1912. Use of soil fungicides to prevent damping-off of coniferous seedlings. In Proc. Soc. Amer. Foresters, v. 7, no. 1, p. 96-99. (64) 1!)13. The blights of coniferous nursery stock. U. S. Dept. Agr. Bui. 44, 21 p. (65) and Merrtll, Theodore < !. 1914. Preliminary tests of disinfectants in controlling damping-off in various nursery soils. /// Phytopathology, v. 4, no. 2. p. 89 92. (66) and Bruner, S. C. 1915. Notes on Rhizoctonia. hi Phytopathology, v. 5, no. 1, p. 73-74. DAMPING-OFF IN FOREST NURSERIES. 95 if.Ti 1 1 m; ri.io. . <\\i;i,. and Pierce, Roy G. 1017. The control of damping-off of coniferous seedlings. U. S. Dept. Agr. Bui. 453, 20 p., 2 pi. (68) Merrill, T. C, and Rhoads, Arthur s. i«n,9. Seedling diseases of conifers. In Jour. Agr. Research, v. 15, ao. 1<>. p. 523 558, pi. B. Literature cited, p. 556 558. (69) and 1 1 \n \, ( rLENN (!. 1919. Oomycetes parasitic on pine seedlings. (Abstract.) In Phyto- pathology, v. 0, no. 1, p. 50. (70) 1 I AWKIN'S, Lon A. 191G. The disease of potatoes known as " leak." In Jour. Agr. Re- search, v. c», no. 17. p. 627 c.M), 1 fig., pi. 90. Literature cited, p. 639. (71 ) ;ind Harvey, Rodney B. 1919. Physiological study of the parasitism of Pythium debaryanum Hesse on the potato tuber. In Jour. Agr. Research, v. 18, no. 5, p. 275-298, 2 fig., pi. 35-37. Literature cited, p. 295> 297. (72) I Lenderson, M. P. 1918. The black-leg disease of cabbage caused by Phoma lingam (Tode) Desmaz. In Phytopathology, v. 8, no. 8, p. 370-431, 10 fig. Literature cited, p. 431. ! 7.". i I Ikss. Richard. 1000. Der Forstschutz. Anil. .'?, Bd. 2. Leipzig. (74) Hesse, Rudolph. 1874. Pythium debaryanum, ein endophytischer Schmarotzer 76 p., 2 pi. Halle. Inaug.-Dissert., GSttingen. (75) Hiltner, L. 1002. Die Keimungsverhaltnisse der Leguminosensamen und ihre Beeinflussung durch Organismenwirkung. hi Arb. K. Gsndhtsamt., Biol. Abt, Bd. 3, Heft 1, p. 1-102, 3 fig. (70) and PETERS, L. 1004. Untersuchungen fiber die Keimlingskrankheiten dor Zucker- immI Runkelruben. In Arb. K. Gsndhtsamt., Biol. Abt., Bd. 4, Heft 3, p. 207-253. (77) 1 1 oi. mann, Julius V. 1012. Aerial Isolation and Inoculation with Pythium debaryanum. In Phytopathology, v. -, no. 6, p. 273. (78) Hobne, William T. 1907. Algunos inconvenientes de los Semilleres de Tabaco. Cuba Estac. Cent. Agron. Circ. 28, 13 p. (79) JACZEWSKI, A. DE. 1911. O gribnekh holies makh liesnekh porod i mierakh borbi s nimi. (Les maladies cryptogamiques des essences forestieres, et la fagon de les comhattre.) [Russia.] Biuro po Mikologhii Etiopatologhii. (Bureau de Mycologie et de Phytopathologie.) St. Petersbourg. (Not seen. Title from Just's Bot. Jahresber., Jahrg. 39 (1911), Abt. 1, Heft 3, p. 1249. 1913.) (SO) Johnson, Edward C. 1014. A study of some imperfect fungi isolated from wheat, oat. and barley plants. In Jour. Agr. Research, v. 1, no. 6, p. 475-490, pi. 62-63. Literature cited, p. 487 489. 96 BULLETIN 934, 1. S. DEPARTMENT OF AGRICULTURE. Johnson, .Tajiks. (81 ) 1014. The control of diseases and insects of tobacco. Wis. Agr. Exp. Sta. Bui. 237, 34 p., 9 fig. Literature cited, p; 60-61. (S2) 1014. The control of damping-off disease in plant beds. Wis. Agr. Exp. Sta. Research Bui. 31, p. 20-G1, 12 fig. (83) Jones. L. K. 100,9. The damping-off of coniferous seedlings. In Vt. Agr. Exp. Sta.' 20th Ann. Rpt. 1906/07, p. 342-347. (84) Kl\C, WrLWOED I. 1012. The Elements of Statistical Method, xyi, 250 p., 20 fig. New- York. (85) Knechtel, Wilhelm K. 1014. Pythium do Baryanum Hesse ca provacator al unei boale de rasad do tutun. Supl. Bulet. Re.u r . Monop. Statul., Bueuresti, 48 p., pi. .VT. (Abstract in Ztschr. Pflanzenkrank., Bd. 28, p. 61-G2. 1918.) (86) Linuau, G. 190S. Die pflanzlichen Parasiten. In Sorauer, raid, Handbuch der Pflanzenkrankheiten. Aufl. 3, Bd. 2. Berlin. (87) Lohde, [G.] 1S74. Ueber einige neue parasitische IMlze. In Tagebl. 47, Versamml. Deut. Naturf. u. Arzte, Breslau, 1K74, p. 203-206. (88) McClatchie, Alfred James. 1902. Eucalypts cultivated in the United States. U. S. Dept. Agr., Bur. Forestry Bid. 35, 106 p., 91 pi. Bibliography, p. 90-101. (SO) Miyake, Kiicur. 1001. The fertilization of Pythium debaryanum. In Ann. Bot., v. 15, no. 60, p. 663-667, pi. 36. List of papers cited, p. G60-666. (90) Moller, F. 1909. Kupfervitriol gegen die Vermehrungspilz. In Moller's Deut. Gart. Ztg., Jahrg. 24, No. 7, p. 77. (91) Muth, Franz. 1908. Ueber die Infektion von Samereien im Keimbett. In Jahresber. Angew. Bot., Jahrg. 5 (1907), p. 49-S2. Neger, F. W. (02) 1909. Beobachtungen und Erfahrungen fiber Krankheiten einiger Geholzsamen. In Tharand. Forstl. Jahrb., Bd. 60. p. 222-252, 4 fig. (93) 1910. Pathologisehe Mitteilungen aus dem Botanisehen Institut der Kgl. Forstakademie Tharandt. III. Ueber bemerkenswerte, in sachsischen Forsten auftretende Baumkrankheiten. In Tharand. Forstl. Jahrb. Bd. 61, Heft 2, p. 141-167, 13 fig. (94) and Buttner, G. 1907. Ueber Erfahrungen mit der Kultur frerndlandischer Koniferen im akadennschen Forstgarten zu Tharandt. In Naturw. Ztschr. Land- u. Forstw., Jahrg. 5, Heft 4. p. 204-210. (95) Nestler, A. 1S00. Ueber das Vorkommen von Pilzen in Wachholderbeeren. In Ber. Deut. Bot. GeseU., Bd. 17, Heft S, p. 320-325, pi. 25. DAMPING-OFF IX FOREST NURSERIES. 97 (96) Norton, J. B. L913. Methods used in breeding asparagus for rusl resistance, U. S. Dept. Agr., Bur. Plant Indus. Bui. 263, 60 p., I Qg., 18 pi. (97) Pearson. G. W. 1906. Two diseases of pines. />/ 37th Ann. Rpt. ,Nebr. Hort. Soc, 1906, p. 230-231'. (98) Peltier, George L. 1016. Parasitic Rhizoctonias in America. 111. Agr. Exp. Sta. Bui. 189, p. 283-390, 24 fig. Bibliography, p. 386-390. Peters, L. (99) 1910. Eine haufige Stecklingskrankheil der Pelargonien. in Garteri- fiora, Jahrg. 59, Hefl LO, p. 209-213, pi. 1582. (100) 1911. Ueber die Erreger des Wurzelbrandes. In Arb. K. Biol. Anst. Land-u. Forstw., Bd. S, Hel't 2, p. 211-259, 12 abb. (TJnter- suchungen fiber 'lie Krankheiten der Itiiben, von W. Busse. 5.) (101) Pettis, Clifford Robert. 1909. Problems in nursery practice. In Proc. Soc. Amer. Forest i is, v. 4, no. 1 , p. 42-49. i 102) Pierce, Roy G., and Hartley, Carl. 1919. Relative importance of Pythium and Rhizoctonia in coniferous seed beds. (Abstract.) In Phytopathology, v. 9, no. 1, p. 50. (103) Poole, R. F. l'.tlS. Report of celery investigation. In N. J. Agr. Exp. Sta. 3Sth Ann. Rpt. 1916/17, p. 536-539. (104) Prillievx, Ed. 1S95. Maladies des Plantes agricoles et des Arbres fruitiers et fores- tiers causees par des Parasites vegetaux. T. 1. Paris. (105) Rankin, W. Howard. 191S. Manual of Tree Diseases, xx, 39S p., 70 fig. New York. (106) Rathbun, Annie E. 191S. The fungous flora of pine seed beds. In Phytopathology, v. S, no. 9, p. 469-4S3. Literature cited, p. 483. Reinking, Otto A. (107) 1918. Philippine economic-plant diseases. In Philippine Jour. Sci.„ v. 13, sect. A, no. 4-5, p. 165-274, 43 fig., 22 pi. (108) 1919. Philippine plant diseases. In Phytopathology, v. 0, no. 3, p. 114-140. Retan, George A. (109) 1915. Charcoal as a means of solving some nursery problems. In Forestry Quart, v. 13, no. 1, p. 25-30. (110) 1918. Nursery practice in Pennsylvania. In Jour. Forestry, v. 16„ no. 7, p. 761-769. (111) Rogers, Stanley S. 1913. The culture of tomatoes in California, with special reference- to their diseases. Cal. Agr. Exp. Sta. Bui. 239, p. 591-617, 13 fig.. (112) Rolfs, F. M. 1915. Angular leaf-spot of cotton. S. C. Agr. Exp. Sta. Bui. 184, 30 p., 9 pi. Literature cited, p. 30. (113) Rolfs, P. H. 1013. Tomato diseases. Fla. Agr. Exp. Sta. Bui. 117, p. [33]-4S, fig. 4-5. 19651°— Bull. 934—21 7 98 Bl I.U.TIN 934, U. S. DEPARTMENT OF ACKK'ULTURE. I 114) BttSENBAUM, J. 1917. Studies of the semis Phytophthora. hi Jour. Agr. Research, V. 8, No. 7, p. 233-27G, 13 lis., pi. 71-77. Literature cited, p. 273-27G. RVHLAND, W. (115) 1908. Beitrag zur Kenntniss des sog. " Yormehrimgspilzes." In Arb. K. Biol. Ansi. Land- u. Forstw., Bd. 0, Heft 1, p. 7l-7C>, 3 abb. (116) 1908. Beitrag zur Kenntniss des sog. " Vermehi angspilzes." In Mitt K. Biol. Anst. Land- u. Forstw., Heft 6, p. 24-25. (117) Sadebeck, R. 1874. Ueber Pythium Equiseti. In Verhandl. Bot. Ver. Brandenb., Jahrg. 1G, p. 11G-124. (118) Saxton, W. T. 1913. The cl ; i. ssiti cation of conifers. In New Phytol., v. 12, no. 7, p. 242-262, 1 lis. List of references, p. 259-262, (119) Schaaf, Marcus. 1915. Report of the State forester. In Rpt. Mich. Pub. Domain Com., 1913/14, p. 60-106, [27] fig. (120) SCHEIXE, E. 1909. Die winterharten Nadelholzer Mitteleuropas . . . viii, 356 p., 173 tig., map. Stuttgart. (121) SCHOT.Z, EdTMRI). 1S97. Rhizoctonia strobi, ein nener Parasit der Weymouthskiefer. In Verhandl. K. K. Zool. Bot. Gesvll. Wien, Bd. 47, Heft. 8, p. 54 1-557, G abb. <122) Schramm, R. 1004. Zuiu Absterben junger, unverholzter Nadelholzpflanzen im Saatbeet. In Mitt. Deut. Dendrol. Gesell., No. 13, p. 203. <123) Scott. Charles A. 1917. A practical method of preventing the damping-off of coniferous seedlings, hi Jour. Forestry, v. 15, no. 2, p. 192-196, 2 pi. (124) Secrist. Horace. 1917. An Introduction to Statistical Methods ... 482 p., 28 pi. New York. I L25) Selby, A. D. 1910. A brief handbook of the diseases of cultivated plants in Ohio. Ohio Alt. Exp. Sta. Bui. 214, p. 307-45G, i-vii, 105 fig. Literature of plant diseases, p. i-vii. (12G) and Manns, Thomas F. 1909. Studies in diseases of cereals and grasses. Ohio Agr. Exp. Sta. Bui. 203, p. 187-236, 7 fig., 14 pi. SlIERBAKOFK, 0. D. (127) 1916. Report of the assistant plant pathologist. In Fla. Agr. Esp Sla. Rpt. 1914/15, p. xciv-xcviii. <128) 1917. Reporl of the associate plant pathologist. In Fla. Agr. Exp. Sta. Rpt. L915/16, p. 80R-98R, fig. 12-16. (129) 1917. Some important diseases of truck crops in Florida. Fla. Agr. Exp. Sta. Bui. 139, p. [191]-277, fig. 75-112. (130) Smith, Ralph E. 190'.». Reporl of the plant pathologist and superintendent of southern California stations, July 1, 1906, to June 30, 1909. Cal. Agr. Esp. Sta. Bui. 203, 63 p., 23 fig. DAMPIXG-OFF IK FOREST NURSERIES. 99 (131) Smith, Ralph E., and Smith, Elizabeth II. 1911. California planl diseases. Cal. Agr. Exp. Sta. Bui. 218. p. [1039] L193, L02 fig. (132) SOMEBVILLE, WILLIAM. 1909. Rkizoctonia violacea, causing a new disease of trees. In Quart. Jour. Forestry, v. ::. no. 2, p. 134r-135. (133) SOBAUEB, P. 1S99. Der " Vermehrungspilz." In Ztschr. Pflanzenkrank., Bd. 9, Heft G. p. 321-328, pi. G. (134) Spalding, V. M. 1899. The white pine (Pinus strobus Linnaeus) rev. and enl. by B. E. Pernow. P. s. Dept. Agr., Div. Forestry Bui. 2^. 185 p., -10 fig., 13 pi. Spaulding, Pebley. (135) 1907. A blight disease of young conifers. In Science, n. s.. v. 26, do. 659, p. 220 221. (13G) 1908. The treatment of damping-off in coniferous seedlings. U. S. Dept. Agr., Bur. Plani Indus. Circ. 4. S p. (137) 1914. Tin* damping-off of coniferous seedlings. In Phytopathology, v. 1, no. 2, p. 73-SS, 2 fig., pi. 6. Bibliography, p. 85-87. (13S) Taylor. Minnie W. 1917. Preliminary report on the vertical distribution of Fusarium in soil. In Phytopathology, v. 7, no. 5, p. 374-378. (139) Tillotson, C. R. 1917. Nursery practice on the National Forests. U. S. Dept. Agr. Bui. 479. 86 p., 6 fig., 22 pi. TrrtEUF, C. von. (140) 1888. Eine neue Krankheit der Douglastanne. In Bot. Centbl., Bd. 33, No. 11, p. 347-348. (141) 1901. Fusoma-Infektionen. In Arb. K. Gsndhtsamt., Biol. Abt., Bd. 2, Heft 1, p. 167-168. 2 fig. MIL' i 1914. Hitzetot und Einschniirungskrankheiten der Pflanzen. In Naturw. Ztschr. Forst- u. Landw., Jahrg. 12. Heft 1, p. 19-36, 4 fig. (143) VOGLINO, PlEKO. 1908. I funghi parassiti delle piante osservati nella Provincia di Torino e regione vieine nel 1907. In Ann. R. Accad. Agr. Torino, v. 50 (1907), p. 247-271. (144) Ward. H. Marshall. 1883. Observations <>n the genus Pythium (Pringsh.). In Quart. •lour. Micros. Sci., n. s., v. 23, no. 92, p. 4^7-515. pi. 34-36. (145) Watson. B. M.. Jr. Win. [Damping-off.] In Amer. (lard., v. 11. no. 6, \>. 348. (146) Weston, William H. 1918. The development of Thraustotheca, a peculiar water-mould. In Ann. Bot., v. :\2, no. 125, p. .155-173, 2 fig., pi. 4-.~>. Literature cited, p. 171-172. 1 147) Wilson, Guy West. 1914. studies in North American Peronosporales. A'. A review of the genus Phytophthora. In Mycologia, v. 6. no. 2, p. 54-83, pi. 11'.). Bibliography, p. S0-S2. ADDITIONAL COPIES OF THIS PUBLICATION MAT BE PROCURED FROM THE SUPERINTENDENT OF DOCUMENTS GOVERNMENT PRINTING OFFICE ■WASHINGTON, D. C. AT 10 CENTS PER COPY LIBRARY OF CONGRESS 022 265 914