M WWW“ ”MIN ‘ 1 *4— — F_ — — — _‘_— :1 __,—d — — 116 104 HTHS fi‘QME FACTQM WCTENG ZOGS?GRES PROQUCTION BY APHANOMYCES EUTEECHE$ QEECHS. Thesis for the Degree o§ M. S. MICHEGAN STAYE UNWERSITY Carmen Llano: M. 1959 Trim LIBRARY Michigan State University .6 4 .- 1 u o . . . . . ,. . . '74 » Q. s ‘ . . . .. .n ..b »\ Abstract SOME morons AFFECTING ZOOSPORE Pnonuc'rxon B! W mummy-Q Dawns. Cor“ genes K. Testing of brooding material in the greenhouse for the selection of plants rosistant to root rot caused by Aghanomgs gteichcs Drochs. requires large numbers of soospores of the casual organist. The Mess when growing nndor laboratory conditions is variable in its ability to prodnco tho soospores. The present experiments were oriented towards a study of the factors affecting soospors production using I. isolatos of the fungus. The hotbed employed to induce tho hmgus in the production of the scospores was s lodification of that described by Slith and Walker in 1941. The liquid mdiul in which the fungus was growing was replaced by tap water, then after 2 hours changed to dietillod water for 15 to 17 hours. Four different media, pea and corn deooctions, mltose-peptone hath, and potato-dextroso broth were used to grow tho isolates. We. of isolates 50. 72, and 103 growing in mltoso-peptone broth prodncod as new or more soospores than in on other medial. Isolato 102 spornlstod better in corn decoction. Msltoso-peptone broth, which was silply and uniformly prepmd, was used in all the subsequent experiments. The most favorable tonporatnre for maxim soosporo production of isolates 50, 72, and 103 was 24° aftor cultures had been grown at rcou tuperaturo. when the menu- was induced to sporulste at tempera- tures 4.degrees higher or lower, the socepore concentration decreased consistently; Isolate 102, possibly a different strain of the pathcgol, seemed to have a slightly higher Optimum temperature, about 28°. Vege- tative growth of isolate 50 increased with temperature from 160-2180 at which the maxi-n was reached. However, maxim noospore production did not correspond with nnxinnn.dry'weight of IvcoliUIh cultures grown.st a temperature range fro-.200-280 produced optilnltnnihcrs of loosporoe. Aeration.ef the distilled replacement water containing the ngceltml ofieolato 50 induced the production.of eeospores at concentration 2 and I. tiles higher than in flasks containing non-seated water. Culture 102,usually'yicldod very'few soospores, produced a 10 fold or more increase in soosporo ”bore (MOS/I1) when air was bubbled through the distilled replacement water. Light intensity'on the other hand, had no effect on soospore discharge. Ruin sooepore discharge oocm‘ed 8—10 hours after medial nets were put in the distilled replacement water. Tap water, followed 14-2 hours later by distilled water, induced maxi." production of sooepores. Tap water clone produced very few soospores. The influence of the age of the culture and volume of distilled replacement water on sccspore production was studied by growing isolate 50 for 3, 5, 7, 9, and 11 days before sporulaticn. 24mm concentra- tions and total nulbors of soospores were obtained fron.cu1turo 5 day: old when 30 and 50 Id of distilled replacement water were used. is the cultures grew older fewer soospores were obtained even though vegetative growth increased. When 20 ll of distilled replacemut water was used, highest concentrations and total nunbers of soospores were obtained fro: Hay-old cultures. Zoospcres from cultures of different ages had different capacities to infect Miragreen pea seedlings. , Four and 6-day-cld cultures of isolate 50 were significantly lore pathogenic than cultures 11 days old. Zoospores from A—dayuold cultures of isolates 72 and 103 were not: here pathogenic than cultures 6 or 11 days old. Isolate 102 consistently showed a very low capacity to produce the sworn cells. Furthermore, it was very mildly pathogenic on urcgroon pee seedlings as covered with isolates 50, ‘72, or 103. Other morpho- logical and biological dissinilaritics of isolato 102 in relation to the other isolates imply that it is possibly e. different strain of the pathogen. some Morons mums zoospoaas momma a! menses M Dances. Carmen Llanos ll. ATHBSIG thod to the college of Science and Arts of nohigsn State University of Agriculture and Applied science in partial ful- fillnnt of the roquironents for the degree of ILSTER 01' some! Dopartnent of Botany and Plant Pathology 1959 W The author wishes to express her sincere gratitude to Dr. John L. Lockwood whose guidance and constructive criticism nade possible the completion of this investi- gation, and to Dr. Willis: .1. Hooker and Dr. Henry A. Inshaug for reviewing the manuscript. TABLE 01" COWS INTROD’ICTION..................... REVIEW OF LITERATURE . . . . . . . . . . . . . . . . . Importance of the disease . . . . . . . . . . . . The fungus. . . . . . . . . . . . . . . . . . . . Persistence of the parasite . . . . . . . . . . . Culture of the fungus under laboratory conditions Temperaturerelations.............. MATERIALS AND METHODS . . . . e . . . . . . . . . . . media. . . . . . . . . . . . . . . . . . . . . . Temperature. . . . . . e . . . . . . . . . . . . Light . e . . . . e . . . e . . e e . . s . . . Aeration .......... ......... Waterrequirements....... ........ Ageeftheculture....... ........ Amount of distilled replacement water . . . s . . Pathogenicity of soospores from cultures of different RESULTS. . . . . . . . . . . . . . . . . . . . . . . . Ebdia . . . . . . . . . . . . . . . . . . . . . . TBMPGI‘Etmee'eeeeeeeeeeeeeeeee Light . . . . . . . . . . . . . . . . . . . . . . Aeration. . . . . . . . . . . . . . . . . . . . . Time interval for production of zoospcres . . . . Waterrequirements.................. Age of the culture and volume of distilled replacement mterseeeeeeeeeeeeeeeeeeeeeeeee Utitifit-‘E'b' 88 25 Pathogenicity of soospores from cultures of different ages. . 32 Comparison Of 13019.t88. e e e e e e e e e e e e e e e e e e e 36 DImTtSSION. O O O O O O 0 O O O O O O O O O O O O O O O O O I O O 37 LITERAWCITED00.0..OOOOOOOOOOOOOOOOOCu r13. Pig. Pig. rig. rmsorncuass Effect of temperature of distilled replacement water on soospore production by isolates 50, 72, 102, and 103 of gphanomes euteiches. 17 Effect of growth temperature of Aphanomes ggteioheg isolate 50 on weight of the sqceliun and soospore production. 1? Concentration of soospcres of Aphsnogzggs euteiches isolate 50 obtained when the weeliun was in distilled replacement water for varying periods. 2!. Concentration of scospores of Aphanomes ggteiches isolate 50 obtained from melial mats placed in tap replacement water for varying periods. 2'7 West of age of the culture and values of distilled replacement water on soospore concentrations produced by ghanoms cuteiches isolate 50. 31 Effect of the age of the culture and volume of distilled replacement water on the total number of soospores pre- duced by _A_phanomes euteiches isolate 50. 33 Pathogenicity on Hiragreen pea seedlings of soospores from cultures of W euteichee isolate 50 of different ages. A: culture 1. days old. B; culture 6 days old. 03 cultun 11 days old. D: “inoculated conmlo 35 m morons Arr-norm zoosrons moccnos s! W mom was. INTRODWTIOII Conn root rot of pea caused by Aphanoms euteiches Drechs. is considered to be one of the most destructive diseases affecting peas in the principle growing areas of the United States. losses as high as 25% and 1.5% have been reported in recent years (2, 13). In the development of commercial varieties resistant to the disease, the search for resistant parent varieties, as well as testing of resistant and susceptible segregating breeding lines, requires artificial inoculation in the greenhouse. Such inoculations require large numbers of socspores in order to secure a consistently high disease index for effective selection of the resistant plants and elimination of the susceptible ones (5). The specific casml organism when groping under laboratery condi~ tions, presents a great variability in its ability to produce an adequate concentration of scospcres. In an attempt to find the possible sources of this variation, factors affecting soospore pro- duction were stalled. ‘ REVIEW OF LITERATURE We of the disease. Aphanosyces root rot of pea is considered to be the limiting fac- tor in pea production. It is present in nearly all the growing areas of the United States with a vaivlng severity which depends upon the degree to which intensive culture of pea permits the accumulation of the fungus in the soil (1). In this country it has been found in the hetero and central States; it has also been reported in Utah, Ibntana, California, New York (1), mchigan (5), Wisconsin (12), New Jersey (6, 7) and other states. The disease was found to be far more ilportent than all other parasitic diseases combined in Wisconsin in a survey nude in 1921. by Jones and Idnford (2); losses were found anounting to 8% of the yield of the total acreage inspected. In some of the older districts in unfavorable years as mach as 25% of the acreage was infected. Several thousand acres of peas were rendered unprofitable or destroyed in the United States every year by this root rot. In 191.2 Walker and flare (12) pointed out that 455 of the fields inspected in Wisconsin were affected, 29% severely. nam- ‘l‘he organic: was first mu; described by Jones and Drechslsr in 1925 (1). It belongs to the family Saprolegniaceae and although the parasite is not strictly aquatic in habit, the fungus requires periods of submergence in water to produce its asexual biflsgellate seospcres. Its presence in the host tissue is determined by the presence of its characteristic subspherical oospores, 18-19 microns in diamter, which are surrounded by unusually thick oogonial walls (1). The parasite in the host tissue has abundant intracellular mceliun which remains for only a short time in a living condition since its mutants are rapidly transferred to the numerous soap-ores. The nycelium is quite difficult to distinguish from other species of Phyconwcetes especially from Pythiun which very often accompanies it. In the host tissue the oospores have never been found to germinate but the ones produced in artificial mdiunl germnats and produce nen-septate meliun in a mist substrate or in water with adequate nutrients. The asexual stage of the organism is obtained when the lyceliun is put in water for a certain period of time. It is represented by the morons soospores which are discharged and encyst at the tip of undifireneiatéd sporangia usually arising from the man tips. After encystnent thi spores become biflallelate and motile. Following s. period of motility they come to rest and germinate giving rise to a coenocitic uieliun. The hyphal structures from germinating oospores are able to pinstrate'pea roots and it is possible that soospores formed when the soil is filled with water may also be a source of infection (1). sistenc f the site. The cospores formed on decaying roots are mainly responsible for the survival of the parasite in the soil. Jones and Drechsler (l) thought it possible that the wcsliul may persist for a time in t1- soil as a saprcpwte. Walker and Hare (12) found that the parasite remains in the soil more or less indefinitely when once thoroughly infested, but nevertheless it seems to decline definitely in the absence of pea as a host. However when root rot is severe, at least a six-year rotation is necessary before peas can be planted with reasonable safety. I In 1927 Linford (3) found that the fungus is not limited in its parasitism to Pisuu sativum but may live as a parasite in the roots of a wide range of plants, such as Medicagg sativa, Me otus alba, three species of Lathyrus, and eight species of Vicia'. Through these hosts the parasite nay also survive in the soil from season to season. Sherwood (10) in a recent paper found that species of the felily Leguninosae we 9 slightly to moderately susceptible and that Spinafig oleracea and spehies of Linux: and Chenopodiun were mildly parasitised. mun-e of the m under laboratory concutions. When the higus is grown under laboratory conditions in corn-lea]. agar, the eyceliun is less abundant than in most substrate, but there is a moderately abundant production of cospores. For this reason cul- tures in this substratu- can survive longer than those in other media. Bali-send nedia such as corn-meal with varying arounts of water added also give good growth but the oospores are less perfectly for-ed as the nediun becomes more moist (1);. Smith and Walker (1.2) grew the fungus in potato-dextrose agar to manure increase of‘ radial growth of colonies due to temperature. fl. Jones and Drechsler ”(1) reported a pee decoction of 10-20 pass of a wrinkled variety to 75 m1 of water as a useful liquid medium for production of wcelium and sporangia. After 7-10 days at room tempera- ture on this media: oospores began to torn and seen most of the lyceliul werefeundteesptyanddie. Inesisilsrwaygoodgrowthofthefimgus was obtained by growing the organism in a pee decootion prepared fro: . 24 wrinkled type pea seeds in 100 ml of water (11). The production of soospores was obtained by removing the nycelial mats from the ”dial, washing in tap water for two hours, rinsing in distilled water, then placing then is shallow pens with just enough water to cover the syne- lius; sesspores were romd within 6-8 hours. Schneider and Johnson (9) adapted a nethod of obtaining soospores of Aphanoms Qchflgidgg Drechs. and A, enteigheg to be used in test- ing resistance of sugar beats and peas, respectively, to these fungi. Cultures'were grown for 5-7 days in flasks containing a decoction of miss kernels or Pablus in tap water. The broth was then decanted, the «11th were rinsed twice and the flasks half-filled with sterile tap water. Within 12 hours spores were produced, often 11105 per I]. as determined by a eemating chamber. in a similar way Schneider (8) obtained a good amount of inoculun of A, cochlioides by growing the fungus in 250 I1 flasks on a sterile deeoction of 5 corn grains in 50 ll of tap water. After one week broth was decanted and flasks with the syoelial mats were half filled with sterile tap water. Zoospore production occurred after 12-36 hours at the optima temperature range of 20°45" O. Successive crops of spores could be obtained by adding more water to the wceliun in the flasks. Sherwood (10) found that growth of the fungus in agar or liquid media in equilibrium with mixtures of air and nitrogen wee more rapid with 5% mgen than with air, slow with lfi engen, and was prevented when ongcn was absent. The fungus did not grow in aqueous inorganic ealt mdiun but grow slowly in glutamic ecid-cysteine—glucoce medium, and rapidly in this medium supplemented with yeast extract. The cane author used naCl, Egg)“ sucrose eolutione or distilled water to obtain the swarm cells from the meliun and found all the above cubctratu to he better than tap water. Emit-attire rela_t_ig_n_g. Amrding to Jones and Linford (2) vigorous growth of the tongue wee obtained in e uni-solid corn meal nediun at 34° but no growth wee obtained at 37°. The crganien had not grown after 6 day: at 8°~lO°.i A linin- growth was obeerved at temperatures between 9° and 11°; the optimal wae eonewhere between 15° and 34°. After 18 hours the firet coup of eooepcree wee observed at temperaturee between 15° end 21°. m the tongue wae growing in petri platen of potato-dextrose agar and the radial increment neaeured daily, eteady increaee in rate of growth wee obtained with increase of temperature tron 120-280. Above 28° growth rate decreased (11). - N ‘ ‘ comic» and Johnson (9) reported that tenmeraturee between 15° and 20° were optima for the production of eooeporec. Recent]: Sherwood (10) etated that equal amber of primary coocporea were discharged at eeverel temperaturee from 40-320. He reported that eecondary ewerung wee inhibited at 32°. Optim temperature for infection ranged fron 150-300 which is more or less the same found by Jones and Drecheler (l) for growth of the organism. Smith and Walker (11) found the maxim disease index at coil temeratures of 240.280 which is near the temperature they reported to he optim for growth of the fungus in culture media. MATERIALS ANDMETEDS Four different isolates of A, euteiches obtained from different localities were used: isolate 50 from Michigan, isolate 72 from New Iork and isolates 102 and 103 fron California. In some experiments the I. isolates were tested sinultaneously for certain variables; in others they were tested separately. Sometimes only isolate 50 was concerned. Honosporic cultures of each of the isolates were obtained and used in all the experiments. Inoculun was prepared by growing the fungus in petri plates on potato-dextrose agar (extract from 200 g potatoes, 20 g glucose, 20 g agar) for ly-S days. Uniforl pieces of wceliun obtained by means of a cork borer were used to inoculate flasks of liquid media. The method used to stimulate the fungus in the production of the soospores was similar to that described by Smith and Walker (11) in which the myoelial nets were removed from the mdiun, washed in tap water for 2 hours, rinsed in distilled water, and then placed in shallow pens with Just enough distilled water to cover them. In the present experiments the organism was usually gram in neltose-peptone broth at 230-250 for 5 days. The wcelial salts were left in the same 250 erlenneyer flasks in which they were grown, the nediun was poured off and about 60 ll of tap water added. This water was replaced after two hours by [.0 ll of distilled water and flasks were then placed in a 24° incubator. Both the distilled and the tap water used in the pro- duction of the soospores were stored in large bottles to avoid possible variations in the water sanples. The number’of soospores was recorded after 15-17 hours. To esti- Iate the number of soospores produced seven fields of a sample frcn.each flask were counted in a standard haemscytcmeter. To avoid difficulties in counting the soospores due to their great mobility, cellulose gum (type 120 high, Hercules Powder Co.) was used at the rate of 0.3 g per 40 ml of soospore suspension. aegis. four media were tested to select the one most favorable for soospore production: 1) pea decoction (per liter: extract from.il g of Miragreen pea seeds prepared by autoclaving for one hour, then steaming for 30 [minutes)] 2) corn.deccction (per liter: extract of 41 g of field corn kernels prepared by autoclaving for one hour, than steaming for 30 minutes); 3) maltose-peptone broth (per liter: 1 g peptone, 3 g maltose); L) potato-dextrose broth (per liter: extract from 200 g potatoes, 10 g dextrose). e reture Using maltose-peptone broth the four isolates were induced to sporulate in incubators at temperatures of 16°, 20°, 24°, 28°, and 320. Temperature requirements of the fungus for growth and for subsequent production of the soospcres were also studied. Cultures in maltose- peptons broth were grown at the temperature range mentioned above for 5 days. After the mediuntwas poured fron the flask and myoelial mats were in the replacement water, flasks were placed at 24°. The myoclial 'nats were transferred to aluminum.foil containers, dried for 24.hours in a 60° oven and weighed. 10 me. To investigate the possible effect of light intensity on the develop- ment of the soospores by isolate 50 the mycelial mats in open containers were placed in two 280 incubators and submitted to the following condi- tions of light intensity: a) dark incubator, b) myselium placed 10 inches below 1!. watt flourescent lamp, c) container wrapped in aluminun foil forming an epen cylinder 7-8 inches long and placed in darkest part of illuninated incubator. Aeration, Aeration requirements for discharge of soospores of isolates 50 and 102 of 5, euteiches were studied by subjecting awcelial mats in the distilled replacement water to various conditions of aeration. Vacuu- was applied by placing the flasks in a desiccator connected to a water pulp, reducing the pressure to 8 m of mercury. Air was mbbled into the water by closing flasks with a rubber stopper fitted with 2 glass tubes of 3 no inside diameter, one connected to the vacuun line, the other exposed to the air at one end and below the surface of the water at the other. The rate of bubbling was 6 bubbles per second. In some experiments air was discharged at the surface of the water. Other flasks were closed tightly with rubber steppers. In another test 200 ll of distilled water was cooled to 6°; while the water was mintained at this tennaerature, air was bubbled into the water for about 5 hours before being used to induce sporulation. Water at 24° was given the same aeration treatment. ygter reguirements. The quality of the replacement water was varied in several tests. Treatments included washing the myoclium first in distilled water for 2 hours and then in tap water or vice versa, tap or distilled water alone, tap water followed by tap water, and distilled water followed by distilled water. For the purpose of investigating the effect of the length of time of the tap water treatment myoclium.of isolate 50 was allowed to stand in tap water for 1 minute, 15 minutes, 1 hour, 2 hours, or 6 hours before being transferred to the distilled replacement water. Age of the gglture, Isolate 50 was allowed to grow for different periods of time before inducing soospore production. The time intervals were 2, L, and 6 days in one experiment and 3, 5, 7, 9, and 11 days in 2 other tests. The flasks were inoculated at such a time that all the mats were harvested the same day} nwcelial sets were ovenpdried at 60° for 24 hours. Amount of ggplacement water, Effect of the volume of the distilled replacement water‘wes studied in 3 experiments in which 20 ml, 30 ml, and 50 m1 of water were used to induce production of soospores by isolate 50. gathogenicitz;of soospgres from.cultures of different ages. Differences in infectivity of the soospores obtained from cultures of isolates 50, 72, and 103 allowed to grow for 4, 6, and 11 days was 12 investigated. Pea seedlings of the variety Mdragrsen were soaked in 0.5% sodiun.hypoclorite (10$ Clorox) for 5 minutes, then.p1anted in rows in white silica sand in galvanized metal pans. Twenty seeds were planted in each row and there were 4.replioations per treatment. Seedling were inoculated 5 days after planting and pans were then placed in temperature tanks at 249 C. The concentration of the soospore suspension for all treatments was adjusted to 5:104 spores per ml and each row was inoculated with 10 ll of this suspension; imnediately after inoculation the pans were saturated with water (5). Disease index was estimated in tops, epicotyls and roots 11 days after inoculations using the method of Lockwood and Ballard (5). Tops of plants with only the lower one or two leaves wilted were rated 1, coupletely wilted tops were rated 3 and intermediate stages were rated 2. Water soaked epicotyls were rated 1 and collapsed ones, 2. Slightly decayed roots were rated 1, completely decayed roots were rated 3, and intermediate stages, 2. The ratings were made for each row as a whole, not for individual plants. In all the experiments A replications per treatment were used. Analysis of variance was applied to all data, and statistically significant results are at either the 1% or 5% levels. RESULTS see; Comparison was made between 4 different lituid media for growth and spornlation of the fungus in order to select the most convenient Indium in which to culture the fungus for all the following experiments. One experiment showed that myceliun of isolates 50, 72, and 103 produced I significantly higher average number of soospores when the organise was growing in maltose-peptone broth than in pea decoction, corn decoc- tion, or potato—dextrose broth (Table 1); no significant differences were found for the cane isolates when growing in the latter 3 media. In another emeriment pea decoction was better than the other mdia, although the difference from mltose—peptone broth was not largo. In all the subsequent experiments naltose-peptone broth.was used since its preparation was simple and most of the isolates sporulated well when grown in it._ Isolate 102 produced the greatest mmbers of zoospores when grown in corn decoction in test 1 and in potato-dextrose broth in test 2. However this isolate was also grown in.naltose-peptone broth to mks possible comparison with the other isolates although with this Indian very few spores were counted in every test. When an average of each of the isolates was taken (Table l), the number of zoospores per ml for isolates 50, ‘72, and 103 did not differ significantly in either test. Isolate 102, in the 2 experiments produced a significantly smaller number of socspores per ml than any of the others. o rature In an attempt to determine the optimum temperature for soospore discharge, melial nets grown for 5 days at 230-250 were induced to Table 1. - Zoospore production'by 4 isolates of‘é. euteiches when grown in 4.different liquid media. Number of soospores per :1 (10,000's) for indicated isolatea Test Ifledflml F50 72 102 103 Average 1 P08 dwoction 22.2 17.5 009 1408 13.8 Corn decoction 6.7 9.1 3.8 9.5 7.3 Maltese-peptone 12.9 13.6 0.4 9.4 9.0 broth Potato-dextrose 9.1 7.7 0.1 4.7 5.4 broth Average 12.8 12.0 1.3 9.6 2 POI dCGOOt-ion 7.1 5.2 306 505 5.5 Corn decoction 8.5 9.6 5.3 4.1 6.8 muBO-Pepton. 2109 230‘ ‘ 0.3 23.7 17.3 broth hmwem.. 6s8 6.7 903 300 ‘01 broth Average 11.2 11.2 2.3 9.1 aLeast significant differences: Test 1: Medium.and isolates averages; 5% level, 4.1; If level, 1.3. Individual values per isolate medium; 5% level, 8.2; 1% 167.1, loege Test 2: Medina: and isolates averages; 5% level, 2.9; 1% level, 3.9. Other values; 5% level, 5.8; 1% level. 7.8. sporulate at temperatures of 16°, 20°, 24°, 28°, and 32°. In 9 tests the maximum number of soospores was constantly reached at 24° for isolates 50, 72, and 103 (Table 2, Fig. 1). Culture 102 was a very poor spore producer at all the temperatures considered; however from the limited data obtained it seems likely that for this isolate a slightly higher optimum temperature is required for the fungus to sporulate properly. As the temperature decreased from 24°, smalls: soospore mbers with isolate so were counted, differences being statistically signifi- cantinrelationtonnnbersat20°intests2and4, sndto16°ina11 4 tests. it temperatures above 24° a steady decrease in the soospore concentration also occurred. Statistically significant differences wersfoundinrelationto28°intests2and3,andto32°inallthe experiments. Result i similar to those with isolate 50 in tests 2 and 4 were observed wh ' culture 103 was assayed in experiments 1 and 2. In test 3 this isol showed no significant difference when sporulated at 24° or 28° but significant decrease in the countings was observed at still higher or letter temperatures. The greatest average number of soospores was at 24°. Culture 72 produced more soospores than isolates 50 or 103. Maxi- nun where were obtained at 24° in 2 experiments, the differences being significantly greater as compared with 20° or 28° in both experi- ments. is with the other isolates soospore numbers decreased at higher or lower temperatures. Table 2. zoospore production by A, euteiches. l6 - Effect of temperature of distilled replacement water on Number of zoospores produced by wcelis in replacement water at indicated temperature‘l Isolate Test 16° 20° 24° 28° 32° LSD 50 1 007 100‘ 15e6 Melt 3e3 5e8’8e0 2 7.6 18.1 29.1. 17.5 9.9 6.639.1 3 2.8 - 30.1. 17.1. 7.1 5.8;8.o I. 1.1 10.5 25.9 21.1 10.? 10.23114 _ i, m. 3.0 13.0 25.3 17.6 7.6 72 1 10.1. 23.8 32.6 18.1 9.8 5.5;7.5 2 11.9 18.8 1.2.5 22.3 7.9 11.3315.7 m. 11.1 21.3 37.5 20.2 8.8 i; 103 1 2.5 8.7 29.8 20.0 3.5 9.8;13.5 2 2.7 12.1. 30.7 16.2 7.5 3.5;5.1 AVG. 2e5 1303 3060 18.5 [no 102 1 o o 0.1 0.1 o 2 O 001 0.5 100 x 001 aAverage number of .soosporee per m1, 10,000's bLoaat significant difference at the 5% and 1% level respectively. l7 72 w 0 03 ZOOSPORES PER ML. IO OOO'S - N o o I1 IO 0 —-——— L IO 20 24 28 32 TEMPERATURE. °C Fig. 1. — Effect of temperature of distilled replacement water on zoospore production by isolates 50, 72, 102, and 103 of Aphanomyccs entcichos. * The values for isolate 102 have been multiplied 10 times. studied with respect to subsequent production of weapons. 18 The effect of temperature at which the culture was grown was When i, euteichgg isolate 50 was grown at 16°, 20°, 24°, 28°, and 32° and the myceliun induced to for! zoospores at 24°, mats grown at 20°, 240 and 28° produced the highest numbers of soospores (Table 3, Fig. 2). when these values were compared no significant differences were found except in test 1 in which a significantly higher concentration was obtained at 24°. when the readings obtained at the 3 temperatures men- tioned above were compared with those obtained when the fugue was grown at either 16° or 32°, the differences were significant statisti- cally in each test. So it seems that the optimm temperature range for growth of the culture for soospore production is 200-9230. Table 3. - Effect ‘of temperature at which culture was grown on production of soospores of A, euteiches isolate 50. Ember of soospores produced by culture grown at indicated temperaturea Test 16°C 2090 21m 28°C 3200 web 1 7.6 25.2 35.2 19.5 14.6 11.53125 2 16.1. 34.2 26.? 40.3 18.0 15.3321.0 3 1.1 23.6 24.6 17.2 0.1. 9.0;15.5 A“. 9e3 27e6 28.8 25e5 11.0 °Average soospores per m1, 10,000“ bLeast significant difference at 5% and l$ levels respectively. l9 ZOOSPORES PER ML. '0 OOO'S L . . . It 20 24 as so TEMPERATURE. °C Fig. 2. - Effect of growth temperature of Aphanomyces euteichns isolate 50 on weight of the mycelium and ZOOSpOTe production. WEIGHT OF MYCELIUM. MG 20 Weight of the wceliun increased with temperature reaching its peak at 28°. As the temperature increased from this point growth was - retarded (Fig. 2). When the average values for weight of wceliun grown at the 5 temperatures and zoospore concentration were compared, a high weight of myceliun did not correspond to the highest number of soospores obtained. chlial nets grown at 28° reached the highest vegetative growth and produced a rather high number of soospores which did not differ statistically from the number obtained at 24° in 2 experiments. In e third test the svceliun grown at 21.0 produced a soospore concentration which was significantly higher than that obtained from nyceliun grown at 28° (Table 3)., In the‘same way myceliun growing at 20° was even less abundant than at 24° but soospore production was as good as that produced by the organism grown at either 24° or 28°. nan. Different light intensities to which the fungus was subjected during sporulation did not increase or decrease the number of zoospores pro- duced. There were no statistically significant differences in numbers of soospores produced by melis in darkness, low light or under fluorescent light. amazes... Aeration proved in several experiments to be a very important factor affecting soospore production. Bubbling air into the distilled replace- ment water containing the wceliun of isolate 50 increased greatly the nunber of soospores. ‘ Spore concentrations 2 and 1. times greater than 2.1 nornal were obtained. is shown on Table l. the differences were signi- ficant if compared with soospores produced in the open flasks. The number of soospores was even less when the flasks were closed with a rubber stopper. When air was applied at the surface of the water significantly fewer soospores were produced than when air was bubbled through the water. If compared with the open controls, no significant difference was obtained when air was applied on the surface, but when compared with stoppered flasks, a significantly higher number of soospores was obtained. 2 With the intention of simplifying equipment and work it was thought that by bubbling air into the total amount of water before the wceliun was placed in it one may obtain the same increase in the lumber of soospores as when bubbling the air individually into each flask during spcrulation. When water at 6° and water at 24° were aerated for 5 hours and then used to induce sporulstion, no signifi- cant increaee in the number of soospores was observed; bubbling air into the distilled replacement water containing the meliun again gave significantly higher number of soospores than the other treatments. Since isolate 102 was a very poor spore producer the air bubble method was applied to induce the fungus to sporulate in 2 experiments. In both tests aerated 5-day-old cultures produced a very good number of spores, 1.3 x 105 and 2.3 x 105 per :11 as compared with 2.1 x 104 and 2.1. x 10” per I1, respectively, in the controls (Table 5). Table 4. - Effect of aeration of distilled replacement water on zoospore production by A, euteiches isolate 50. number of soospores produced at indicated treatmentsa air air at cpen. closed aerated aerated Open bubble, surface, flasks, flasks, water, water, flasks, Test vacuum 24° 24° 24° 24° 6° 24° 6° 1 0.0 108.5 34.2 22.4. 14.2 - - - 2 0.0 4.9.9 .. 2198 fl 1900 25.7 léel ‘Average number of soospores per ml, 10,000's Least significant differences: Test 1: 17.1 and 24.1 for 5% and 1% levels respectiveLy. Test 2: 8.5 and 11.8 for 5% and 1% levels respectively. Table 5.1- Effect of aeration of distilled replacement water on soospore production'hy é, euteiches isolate 102. Number of soospores produced by indicated treatmentsa Culture Test age, days aeration open flasks l 5 12.6 2.1 2 5 22.5 2.5 3 4. 15.3 0.1 10 1.0 0.0 'hverage number of soospores per m1, 10,000's 23 Time interva1_for production of soosporea. . In order to determine when the soospores are discharged countings were made at 2—hour intervals after mycelial mats were submerged in the distilled replacement water. No soospores were present until 6 hours when an average of 5.6 x 10"per ml was counted (Table 6, Fig. 3). Eight hours after the mycelium.was put in distilled water 2.1.: 105 - 2.5 x 105 soospores per ml.were counted. When countings were nude 2 hours later no significant increase was found. Two more read- ings at intervals of 2 hours likewise showednc significant increase in the numbers in test 1 but in a similar experiment a significant increase was noticed in 10 hours as compared with the reading made after 8 hours. Table 6. - Zeospore production following immersion of mwoelinn.ef A, euteiches isolate 50 in distilled water. Huiber of soospores produced at the indicated intervals, hoursa Test 4 6 s 10 12 13 23 L52" 1 o 5e3 21e3 25e‘ 24e8 *" 25.4 5e6,7e8 2 O 5e9 25cc 37e2 32e0 31e5 "" ue93‘6e6 ‘Average number of soospores per ml, 10,000's. bLeast significant difference at the 5% and 1% levels respectively. There was no further increase in the numbers of soospores. It can be said, therefore, that all the zoospores in one single crop are {\TJ {,\ a 0 fl U 0 V 5 fl.— ZOOSPORES PER ML IO 0005 N O l l l 4 6 8 IO l2 I4 23 HOURS IN DISTILLED WATER 9 Fig. 3. - Concentration of zoospores of Anhsnomyces euteiches isolate 50 obtained when the mycelium was in distilled replacement water For varying periods. 25 produced 8-10 hours after the mycelium is imersed in the distilled replacement water. Water requirements, The usual method to induce the fungus in the production of the scospores was that in which the liquid medium was replaced by tap water, let stand for 2 hours and then changed to distilled water for about 15-17 hours. Experiments were planned with the purpose of deter- mining whether the actual change from tap water to distilled water was necessary or not. The greatest number of soospores was obtained by the usual method in 2 experiments (Table 7). This treatment gave significant differences in relation to the other treatments in test 1, except when the distilled water was followed by tap water. In test 2 tap water followed by distilled water differed significantly from all other treatments except distilled water alone. Table 7. - Effect of different combinations of water changes on spore- lation of A, euteiches isolate 50. W Huber of soospores at indicated treatments. Test T—Db r—r 13-1 M n 1' 153° 1 18.6 4.9 14.1 9.6 10.7 1.7 7.2.9.? 2 ”so 3e2 Be, 14.5 18s? le‘r 508’8e1 ‘Average number soospores per ml, 10,000's. bf. tap water] I) a: distilled water. °Lsast significant difference at 5% and 1% levels respectively. 26 When the treatments distilled followed by tap water, distilled followed by distilled water, or distilled water only were compared, there was no significant differences between the average number of weepores produced- per ml; this number was fairly good when compared with that obtained by the standard method, However when tap water was used without Eany change or 2 changes of tap water were made, the average mmbgr of soospores was significantly stellar than in any other treatment. Experiments were devised to determine how long the mycelial mats should be washed in tap water after pouring off the medium. The mats were allowed to stand in tap water for 1 minute, 15 minutes, 1 hour, 2 hours, and 6 hours before replacing with distilled water. When the fungus was left in tap water for l or 15 minutes a very low number of soospores was obtained as compared with those in tap water for 1 hour (Table 8, Fig. 1.). There were no significant differences in soospores numbers when the organism was left for l, 2, or 6 hours in tap water. Table 8. - Zoospore production by A, euteiches isolate 50 as affected by the length of time in tap water. aEaB======EE============================================================ number of soospores for indicated time interval“ I fest lain. 15m. 1hr. 2hr. 6hr. rash 1 13.1 19.5 2.4.6 23.3 22.3 8.23113 2 13s]. 17e6 23e3 23e3 26e6 9e6’12e1 ‘Zoospores per ml, 10,000's. hLeast significant difference at 5% and 1% levels respectively. -_30 .J 2 s 20L I 0- U) 200.39an U) " CONCENTRATION w 8 g 0 C) 3' IO- N [:3 House IN ___Q’l7 DISTILLED warts g 3: Z a 9: 3: g '2:::::; a h\\ ('3' pp A Fig. A. — Concentration of zoos:ores of Aphanomzces outcichcs isolate 50 obtained from mycelial msts placed in tap replacement water for varying periods. 28 Therefore, it seems that the initial tap water wash, for at least one hour, is necessary to secure a high number of accepores. However to leave the fungus indefinitely in tap water inhibited 2003pore discharge. Age of the culture and volume of distilled replacement water. The influence of the age of the culture when the soospores were to be produced was studied by'growing the fungus for 3, 5, 7, 9, and 11 days in 2 experiments (Table 9) and for 2, A, and 6 days in another (Table 10) before the production of the soospores. In test 1 when 30 ml of distilled replacement water was used, 3-dayhold cultures produced significantly fewer soospores than cultures 5 days old at which a laxinum was reached. As the age of the culture increased the ability of the fungus to produce the soospores decreased. Differences between 5-day-old cultures and 7-, 9—, or ll-day-old cultures were highly sigdfie cant. The older cultures did not differ statistically from one to another in their ability to eporulato. Similar results were observed when 50 ml of water was used in experiment 1. However in test 2 when 50 ml of distilled replacement water was used, a culture 3 days old produced a concentration of soospores similar to that obtained from cultures 5 days old. In general it seems that a 5-daybold culture is the Optimum for production of a maxinnn.concentration of zoospores. However, if only 20 ml of distilled replacement water is used with a 3-dayaold culture, the concentration of zoospores obtained was as good as or higher than with a Hay-old culture using 20, 30, or 50 ml (Table 9, Fig. 5). Table 9. - Influence of age of the culture and volume of distilled replacement water on zoospore production by A._euteiches isolate 50. 29 Concentration of soospores for indicated age of culture‘1 Vblwne of Test water, ml 3 days 5 days 7 days 9 days 11 days Ave. 30 16.3 24.6 9.0 6.5 4.7 12.2 SC 3.3 21.5 3.3 5.5 2.8 7.3 Ave. 15.5 23.5 9.0 6.2 2 20' 42.2 26.5 25.7 20.6 11.1 25.2 30 24.1 27.2 23.5 14.8 7.5 19.4 50 27.9 23.1 12.6 15.8 7.0 17.3 t". 31“ 25.6 20.6 17. 1 8. 5 ‘Zoospores per ml, 10,000'e. Least significant differences at 5% and 1% respectively: feet 1: Test 2! individual.valnas per age-volume, 7.0 and 9.1. other values, 12.6 and 7.5. age of culture, L.O and 5.4: amount of water, 3.1 and 4.23 age of culture, 7.3 and 9.8; amount of water, 5.6 and 7.5; 30 Table 10. - Influence of age of culture and volume of distilled replacement water on accepore production by A, euteiches isolate 50. Concentration of zoospores for indicated age cf’culture‘ volume of ‘ueter, m1 2 days 4.ddye 6 days Awerage 20 19.0 17.4 23.6 19.7 30 4.1 20.7 34.7 19.8 50 1.4 6.0 21.8 9.5 A“. 708 14.7 26.? aZoospores per'ml, 10,000'8. Least significant differences at 5% and 1% levels respectively: Age of culture and amount of water, 5.3; 7.1. Individual values per age-volume, 9.2; 12.4. In one experiment in uhich.2—,4~,2nd 6-day—old cultures were com- pared (Table 10) for their ability to discharge zoosporee the results were similar to those of test 1 in Table 9; highest readings were obtained from.cultures 6 days old. The zOOSpore concentration decreased as the culture was younger when 30 ml or 50 m1 of distilled replacement water were used. when mycelial mate as young as 2 days were induced to eporulete in 20 ml of water, slightly higher countings were noted as compared with 4-dayhold cultures. 31 50 300 (3 ES ZOIIL 3" \ 3¢>IA a «29 zoo- 33 ' 50> :5 IA 3 2 x,» s 9 Q IOO- r”" m S MYceLlugx 8 #4 1" O I l I l 3 5 7 9 ll AGE OF CLLTURE. DAYS Fig. 5. - Epfect of age of +he culture and volume of distilled replacement water on the zoosbore concentrations produced by Anhanonyces euteichos isolate 50. 32 When the total number of soospores per flask was considered, the maximum number was reached from cultures 5 days old when 30 or 50 ml of spcrulating water was used (Fig. 6). The number of soospores decreased as the culture grew older. Three-day—cld cultures produced a total number of zoospores greater than 5-, 7-, 9., or ll-day-cld cultures only when put in 20 ml of distilled water. Vegetative growth was least with a 3-day—old culture. Cultures at this age produced a smll concentration of soospcres when 30 ml or 50 ll of water were employed, and to least total numbers of weapons at any volume (Fig. 5, 6). Myocliun increased considerably in weight at 5 days. At this «point the highest concentration of soospores for all volumes of water and the maxim total author of soosreree for 30 or 50 ml of water was obtained. After 5 days, increase in gicwth of meliun did not correspond to an increase in concentrations or total numbers of soospores. On the contrary the ability of the fungus to epcrulate began to decrease at this point, although the growth was more abundant. figthogenicitLof accepores from cultures of different ageg‘ Correlation between the age of the sweeliun when the soospores were produced and the pathogenicity of isolates 50, 72, and 103 was studied. Zooepores from cultures of different ages showed different capacities to infect the seedlings. Diseases indexes as high as 6.3 and 5.3 (mximml disease index was 8.0) were obtained from 1..- and 6- day—old cultures respectively of isolate 50. However when an 11-day- old culture was used disease index was 3.0; this value was significantly IO CD ZOOSPORES PER FLASK. MILLIONS N U A U. J l l l I Fig. 6. - 3 5 7 9 n AGE OF CULTURE. DAYS Effect of the age of the culture and volume of distilled replacement water on the total number of zoospores produced by Anhanomycos euteiches isolate 50. MYCELIUM. MG 34 lower than that obtained wit. 4- or 6—dayhold cultures (Table 11, Fig. 7). Zoosnores from L—dsy-cld cultures of isolate 72 were significantly'more pathogenic than cultures 6 or 11 days old. There were no significant differences between 6- and 11-daybold cultures. The same response was Obtained with isclete 133. Table 11. -vPethogenicity of soospores from 3 isolates of A, euteiches of different ages on root rot of Hiregreen pea. Disease index of plants inoculated with cultures at indicated agesa Isolate 4 days 6 days 11 days Average 50 6.3 5.3 3.0 4.9 72 5.7 3.7 3.0 4.1 103 4.2 1.3 1.1 2.2 Average Sele 3e 5 2.3 aDisease index based on scale from.0 to 8 with 0 indicating healthy plants and 8, dead plants. Least significant differences: Isolate and age averages: 5% level, 0.93 13 level, 1.3. Individual values for isolate-age: 1$ level, 1.7; 5% level, 2.4. If the total average of disease indexes is compared, a Andayhold culture of the pathogen was significantly better than a culture 6 or 11 days old as a source of soospores for artificial inoculations. Fig. 7. - Fathogenicity on Miragreen pea seedlings of zoospores from cultures of éphanomyces euteiches isolate 50 of dif°erent ages. A: culture 4 days old. B: culture 6 days old. C: culture ll days old. D: uninoculated control. 36 Spores from Hay-old cultures were still mch more effective than those from 11-day-old cultures which gave the lowest disease index. Isolates 50 and 72 were appmximtely equally pathogenic whereas isolate 103 was significantly less pathogenic than either of those formerly mentioned. Cameos of isolates. In several biological and morphological aspects isolates 50. 72, and 103 were quite different from isolate 102. Isolate 102 is premnnably a distinct strain of the pathogen. The capacity of the first 3 isolates to produce soospores under different conditions was sinilar. Their optimum temperature for sporulation was 21.0, whereas that for isolate 102 was somewhat higher, probably 28° (Pig. 1). The sise of the soosporos of isolates 50, 72, and 103 ranged from 5-6 microns in diameter while those of isolate 102 were 9—10 micron and showed such less 35th than the others. Furthermore under all conditions isolate 102 showed a very limited capacity to sporulate, usually producing only I. x 103 soospores per ml whereas the other 3 isolates under the same conditions produced concentrations 20 or 30 times higher. Growth of isolates 50, 72, and 103 in potato-dextrose agar was similar in nature and in rate, being very uniform and without aerial muslin. Evidence of a difference in pathogenicity of isolate 102 as ‘ compared with isolates 50, 72, and 103 has been found (4). The variety Mix-agrees was susceptible to isolates 50, 72, and 103 and was highly resistant to isolate 102 while the variety Early Perfection was. ‘ susceptible to all 1. isolates. DISCUSSION Tho preceding experiments demonstrated the influence of several enviromnental factors on soospore production by A euteic e , and the importance of standardizing these variables at optimum levels to pro- duce consistently high concentrations of soospores. By using the information reported herein for the production of soospores of g, euteicheg for greenhouse inoculations of pea seedlings in breeding work mch fewer cultures are required with important savings in labor and materials, and the danger of wasting valuable plant mterial is practically slindnated. Maltese-peptone broth was a very simple median to prepare and was favorable for sporulation of isolates 50, 72, and 1033 isolate 102 apex-slated at a somewhat higher rate in corn decoction or potato- dextrose troth. Pee. decoction in agreement with results obtained by other workers (1, 11), was a good sporulating medium In the present work it was better, in one orperiment, than maltose-peptone broth, but because of the advantage of its very easy preparation and reproducibia lity the latter was selected as the growing medium for the fungus in all the subsequent tests. Schneider and Johnson (9) and Schneider (8) recommended a con decoction as a good median for growth and sporulation of cultures of A, euteiches and A, cochlioides. Schneider (8) obtained with this mediun a concentration of soospores of mm as high as 1 x 105 per :1. In the present experiments a corn decoction was always inferior to maltose-peptone broth, except for isolate 102. For example concentrations of l x 105 soospores per ml were obtained with isolate 38 '72 using corn decoction whereas with mltose-peptone broth concentrations of 2 x 105 or more per ml were obtained in many cases. Various authors have reported a wide range of temperatures as optimise for soospore discharge. Schneider and Johnson (9) reported this optimum range as being 15°-20°. More recently Sherwood (10) stated that equal numbers of soospores were obtained at a series of temperatures from l.°-32°. The peak for the maxim soospore concentration in the present experiments with isolates 50, 72, and 103 was always 24°. Steady decrease in the numbers always occurred at either higher or lower tenperatures. Vegetative growth of isolate 50 increased steadily with temperature, reaching its mxinmn at 28°. Smith and Walker (11) also found the greatest increase in the total colony growth at this temperature. Maidm melial growth however did not necessarily correspond to the greatest ability of the fungus for the production of soospores. At 20° the vege- tative growth of the organism was much less than at 21.0 or 28° but at these termeratures concentrations of zoospores as high as that at 28° were obtained when the fungus was induced to sporulate. Therefore the optimum temperature range of growth of the pathogen for subsequent production of the swarm cells was 20°-28°. Light intensity proved was not a factor in soospore discharge, while aeration was a very important one. Sherwood (3.0) obtained in- creases in the rate of growth of the fungus cultured in agar or liquid medium with 5% oxygen as . compared with air. He found that growth was slower with only 1% oxygen and that it was prevented when oxygen was 39 absent. Sherwood's experiments were not concerned with soospore pro- duction. Bubbling air into the sporulating water increased the soospore concentration of isolate 50 2-4 times in the present work. Isolate 102 which was usually a very poor spore producer, especially when growing in maltose-poptone broth reached concentrations 6 and 9 times higher when air was bubbled into the distilled replacement water than without air. By means of supplementary air in the distilled replacement water very high concentrations of zoospores can be obtained using only a few colonies of the fungus. By diluting the suspensions to the desired con- centrations, the soospores can be used for greenhouse inoculations. Zoospore discharge began 5-6 hours aft}!- the myoclial mats were placed in the distilled replacement water. In 8-10 hours the maximum number of soospores of one single crop was 95me since further read- ings did not show any increase in the countings. These results more or less agree with those obtained by Smith and Walker (11) who stated that formation of soospores occurred within 6-8 hours. Schneider and Johnson (8) on the other hand reported soospore production of g. ggchligideg after 12-36 hours. The usual method to induce the fungus to sporulats, a modification of the method of Smith and Walker (ll), was to replace the liquid media by tap water, then after 2 hours, change to distilled water for 15-17 hours. This method was better than several variations tried. When only tap water was used, making either one or 2 changes a much lower number of soospores was obtained. It is possible that some of the salts or the chlorine present in the tap water may inhibit the sOOSpore discharge. 40 Similarly, e harmful effect of the tap water on zooepore production was obtained by Sherwood (10) in which NaCl, M3804, or sucrose solution: (in distilled water”, or distilled water alone supported a better zoospore discharge than tap water. However, in the present eXperimente an initial tap water wash for at least one hour was necessary for lexi- m sooepore production. when nets were left in the tap water for only 1 or 15 minutes the zooepore concentration was lowered (Fig. A). Cultures of isolate 50, 5 days old produced higher concentrations and higher total number of acceporee when 30 or 50 ml of distilled re- placement water were used than cultures younger or older. 0n the other hand when Hey-old cultures were induced to eporulete in 20 ml of die- tilled replacement water e. greater concentration of sooeporee was obtained than with older cultures. Increase in weight of the mycelium from cultures 3 day! old to thoee 5 days old wee correlated with an increase in number of :ooeporee per ml. Cultures older than 5 days continued increasing in total dry weight but in this case the increase was not proportional to greater zooepore nmnber. On the contrary the capacity of the cultures to diecharge the were celle was diminished no they grew older than 5 days. Lockwood and Ballard (5) found that zoosporee from n Hay-old cul- ture of ieolate 50 were more pathogenic than those from 8- or 11-day- old culturee. As the cultme grew older the zooeporee showed less ability to infect the plants. Lowest disease indexes were obtained with zooeporee from the oldest cultures. In the present work similar reenlte were found for isolates 50, '72, and 103. Four and 6-dey-old cultures of Ll isolate 50 were significantly more pathogenic than cultures 11 days old. Zoospores from isolates 72 and 103 behaved in a similar manner and were equally'pethogenic when obtained from 4. or 6-day—old cultures as comp pared with cultures 11 days old which showed a much lower disease index. In summary a Apday-old culture as a source of soospores for artificial inoculations is mmch more pathogenic than a culture 6 or 11 days old. Isolate 102 showed under all conditions a very low capacity to pro- duce the swarn.cells. It was very mildly pathogenic on Hiragreen pea seedlings as compared with the other isolates (4). Some other morpho— logical and biological diesimilarities between isolate 102 and isolates 50, 72, and 103 imply that isolate 102 is possibly a different strain of the parasite. 1. 3. 4. 5. 7. 9. LITERATUREIOITED Jones, F. R., and O. Drechsler. 1925. Root rot of peas in the United States caused by Aphanommces euteiches (n. 8p.). J. Agr. Res. 30:293-325. Jones, F. R., and M. B. Linford. 1925. Pea disease survey in Wisconsin. Wisconsin Agr. Exp. Sta. Res. Bull. 6411-30. Linford, H. B. 1927. Additional hosts of sphanoggpes euteiches, the pea root rot fungus. Phytopathology 17:133-134. _ Lockwood, J. L. Unpublished results. Lockwood, J. L., and J. G. Ballard. 1959. A seedling test for evaluating resistance to Aphanomyces root rot of pea. Phytopathclogy (in press). New Jersey State Agricultural Experiment Station Annual Report. 1925. 36-37. New Jersey State Agricultural EXperinlnt Station Annual Report. 1936. 73-74. Schneider, 0. L. 1954. Method of inoculating sugar beets with Aphanogyces goehlioides Drechs. Proc. Amer. Soc. Sugar Beet Tech. 83247-251. Schneider, 0. Lu, and H. G. Johnson. 1952. The production of soospore inoculun.of Aphanonyces. (Abstr.) Phytopathology442318. 10. 11. 13. Sherwood, R. T. 1958. Aphanomyces root rot of garden pea. Dissertation Abstracts. University microfilms, Ann Arbor, Mich. 18:751-752. Smith, P. Go, and J. C. Walker. 1941. Certain enmnal. and nutritional factors affecting aphanomyces root rot of garden pea. J. Agr. Res. 6381-20. Walker, J. 0., and W. W. Here. 1943. Pea diseases in Wisconsin in 1942. Wisconsin Agr. Exp. Sta. Res. Bull. 145:33p. Walker, J. 0., and‘w. C. Snyder. 1933. Pea'wilts and root rota. Wisconsin Agr. Exp. Sta. Res. Bull. 424:16p. (revised 1942).