| IN TH _ ASSAY os smzmm CHEWCALS on ASCOSi’ORES or [sawing SWQUAEE Thufis for flu Dogma of 59L 5. MICHiGAN SKATE COLLEGE Néchoias Gene Vreéeveid W55 WHEflS This is to certify that the thesis entitled Assay of eradicant chemicals on ascospores of Venturia inaggualig presented by Nicholas Gene Vredeveld has been accepted towards fulfillment of the requirements for “S degree in km and P1811t Pathology A/fl/nux @412“, Major professor . Date W , 0-169 ASSAY 0F ERADICANT CHEMICALS ON AdCObPOth OF mq'mmi gamut-3&3 “‘ By Nicholas G. Vredeveld W {A TdLblS Submitted to the School of Graduate Studies of Michigan State College of Agriculture and Applied dcience in partial iulfillment of the requirements for the degree of mASTEH OF SCIENCE Department of Botany and Plant Pathology 1955 (<42 ’ 5K ASSAY or rumour CILKCICALS on ASCOELPORl-JS OF VLTITIQTA EULB'KWfing ABSTRACT Apple scab, a disease of apples, is caused by lEBEBEiflzifif aeoualis. Control proceedures for this prevalent disease have utilized protectant sprays. The inconsistent results and phyto- toxicity of these sprays, namely lime-sulfur and Bordeaux, led eXperimenters to reduce the primary inoculum potential by eradi- cant ground Sprays on\overwintering leaves. This would allow better control by milder foliage sprays. Elgetol, a dinitro produce. has been proven effective. The present experiment was carried out to test the preperties of other chemicals for eradi- cant ground Spraying and to test a laboratory method of assay. Tests showed that DN 289 was superior to the other materials .tested. Aerocyanamid dust showed some promise while the sodium . cyanamide salt. the mercury compounds, and Actidione were of less value according to this test. The results of spraying leaves in screens and dipping leaves in solutions showed correlation with the simulated ground Spraying application. These results corroborate the experiments of others showing the effectiveness of dinitro compounds for orchard floor sprays. Cyanamid dust may have considerable merit as a floor application, judging from one eXperiment, but further work is needed to deter— mine its real effectiveness. It is possible that it would need special moisture conditions following application to merit orchard use . 3542783 Nicholas G. Vredeveld Botany and Plant Pathology 1 LCKN 0 .U'LEUGEL‘LEN T S The writer would like to express his sincerest thanks to Professor D. Cation under whose guidance this experiment was accomplished. It is to him gratitude is extended for the suggestion of this problem and for assistance in its completion. Also, thanks are due to him for the constructive criticism offered in the preparation of this thesis.' Many thanks are also due to hrs. C.m. Strong for her help in correcting and criticizing this manuscript. To my wife, Ruth, I am indebted for ever ready encouragement, assistance in typing, and grammati— cal appraiSal Cf this paper. TABLE OF CONTENTS Page INTRODUCTION . . . . . . . . . . . . 1 REVIEW OF LITERATURE . . . . . . . . . . h MATERIALS AND METHODS . . . . . . . . . 13 ‘ Spraying leaves in screens . . . . . . 13 Dipping leaves in solution . . . . . . 15 Spraying leaves on ground . . . . . . 16 Method of determining eradicative effect. .. 18 Table of materials used . . . . . . . 21 EXPERIMENTAL RESULTS . . . . . . . . . 22 DISCUSSION . . . . . . . . . . . . . 28 SUMMARY . . . . . . . . . . . . . . 29 LITERATURE CITED . . . . . . . . . . . 31 INTRODUCTION Apple scab, a disease caused by ngturia inae ualis, is consistently the worst pest of apple orchards in the northern states. Infection by this disease is enhanced by cool, wet Springs common in‘Michigan and Wisconsin. Recommended spray proceedure in the early part of the 1930-50 decade was five sprays of lime-sulfur applied according to the changing bud stages through the primary spore discharge period. In most years this method held the disease in check; but in years when critical rain- fall did not coincide with timely applications, the dis- ease was not well controlled. Coupled with inconsistent control, the use of lime-sulfur caused much spray damage on the fruit. With the increased stress on good fruit quality and realization that lime-sulfur has been reduc- ing yields, it is desirous to use chemicals which are less phytotoxic. The primary inoculum of apple scab is the asco- spores. These ascospores are produced in perithecia which develop from.the mycelium overwintering on dead apple leaves under the tree. The dosage of the primary inoculum and suitable rainfall-temperature relationship are the major factors in determining the intensity of the disease develOpment (21). Keitt et al (19) showed that the ascospore quantity can be lowered by applying a dor- mant spray to the dead leaves on the ground. He found that Elgetol, the sodium salt of dinitro-ortho-cresol, if applied at 0.5% concentration and using 500-600 gallons of Spray per acre could reduce the primary inoculum by more than 90%. Such a reduction would allow the use of mild, less toxic fungicides to prevent primary infection by the few ascospores which do escape the ground spray. This eradicant system has been recommended in several stateS. There are several drawbacks to existing ground spray- ing techniques. The large amount of water required per acre is limiting in some areas. The need for Special equipment which can be used only once each year is an- other. The faet that a large boom is difficult to man- euver in the low branched trees of Michigan orchards is a hindrance and the method is uncertain of success where nearby adjacent areas are unsprayed. The published data dealing with ascospore eradica- tion have been obtained from orchard results. 'Under such conditions an entire season is expanded for each set of observations. In a situation where the primary inoculum is subject to dissemination by wind, consider~ able distances are necessary between the treated and the check plots, and between differently treated plots. It is nearly impossible to find isolated orchards with simi— lar environments which are subject to the same disease- influencing factors. These considerations lead us to consider a labora- tory approach for evaluating other chemicals as possible agents to be used against scab. It would be exception- ally desirable to have an acceptable laboratory method of assay which would correlate with field results. Other cheaper and possibly better eradicant fungicides that might replace Elgetol require evaluation for Best control. This study attempted to compare several possible mater- ials using laboratory techniques. REVIEW OF THE LITERATURE Apple scab occurs throughout the world wherever apples are grown with the exception of a few arid irri- gated regions. The causal fungus has been known since 1819 when Fries described the conidial stage in Sweden. In 1894 Aderhold (2) in Germany found the overwintering stage of the fungus and thus completed the knowledge of the life cycle. Clinton (7), a worker in Illinois,imi 1901 confirmed the findingsof Aderhold by isolation and inoculation methods. Realizing that part of the control measures could be aimed at the overwintering stage, Clin- ton recommended the practice of sanitation consisting of picking up or plowing under the leaves from.the previous season. Concerning the protective measures which were used against the disease, it can be said that Bordeaux was the early mainstay of the industry. Keitt and Palmiter (23) are of the Opinion that the success which.was obtain- ed with Bordeaux was responsible for the lack of compli- mentary control measures. In 1909, lime-sulfur was intro- duced as an apple scab Spray because it caused less rus- seting of the fruit than Bordeaux. heitt (21), however, noting the occasional failure of these protectant fungi- cides to control scab by following existing spraying schedules, began a study of the epidemiology of the dis- ease. .By observing the relationship between time, amount of ascospore discharge in the spring, the weather condi- tions (particularly rainfall and temperature), the sev- erity of the disease, and after considerable search for overwintering conidia, he concluded that the ascospores comprised the only important primary inoculum, and that the quantity of this inoculum was directly related to the amount of scab and its control. Spray recommenda- tions at that time were aimed at control on the fruit. Leaf infection, which can be initiated over a longer period of time than fruit infection, built up the inocu- lum potential to a dangerously high level for the follow- ing spring. Besides the inability to reduce the amount of overwintering fungus, the available protectant fungi- cides were too toxic to.the plant, causing objectionable visible and innate host injury. Concurrently rising standards of fruit quality made the use of milder mater- ials.mandatory for scab control during the growing season. Milder materials need more frequent applications-~an add- ed expense--or complimentary inoculum reduction for ade- quate disease control. 1.5” -' keitt turned his attention toward a method of com— batting the scab organism directly with attempts at erad- ication. It was noted already that an old practice of turning under the old leaves even when it was feasible did not materially reduce the threat of scab infection. Also, a so—called "clean up" spray of Bordeaux or copper sulfate applied after harvest did not cause a reduction in ascOSpore develOpment (23). The two most vulnerable points in the disease cycle for eradication were consid- ered to be: (1) the post harvest period before the in- fected leaves had fallen and when the tree could with- stand a more drastic treatment than would be possible during the growing season, and (2) during the dormant period when the mycelium is confined to the dead leaves on the orchard floor. V For the post harvest foliage sprays Keitt (23), dur- ing the period of 1924 to 1935, tried a number of mater- ials considered to have fungicidal value. Sprayed leaves were removed from the tree and placed in cloth.mesh bags. These were laid on the ground and held intact at the corners by Spikes. After overwintering on the ground they were brought into the laboratory and inspected for the number of perithecia that developed per unit area of leaf surface. Sprays consisting of certain arsenites mixed with Bordeaux proved most efficient in inhibiting the perithecial formation. The main disadvantage of such mixtures was their toxicity to the tree. keltt finally abandoned this method in favor of attacking the overwin- tering inoculum in the dead leaves on the ground. The mycelium in these leaves and the concurrent ascigerous stage offered the second vulnerable phase~ for eradicant sprays. The ascigerous stage of apple scab fungus was studied by Keitt and Wilson in 1926 (21). They gathered leaves off the trees at various times dur- ing the fall and placed them.in different environments. They noted that the earlier the leaves fell in the autumn the earlier the ascospores would mature and be ready for discharge in the Spring. The fungus required a period of cold weather after which it initiated its perithecia. The perithecia developed most rapidly when placed at 20°C. and kept alternately wet and dry. The awareness of the importance of perithecial de- velopment for the continued existence of the fungus also suggested to others the second method of eradicating the fungus--that of applying a Spray to the overwintered leaves on the ground. In 192h.Miss Curtis (9) in New Zeeland . tested the effects of Spraying the leaves on the ground in the Spring with lime-sulfur at the strength used on tree foliage. A definite reduction in foliage infection the following season occured where she had made three applications. One application was not significant. She also noticed that applications immediately after a rain were most effective in retarding subsequent infection in the tree. In 1935 Weismann working in Germany treated leaves in the same way with 5% Ubstbadnxerbolineum. Eighty- five percent of the ripe perithecia were killed. Keitt and Palmiter in 1937 applied a solution of ammonium sul- fate consisting of one pound of the material in one gallon of water to overwintering leaves. Investigation showed that the mature ascospores were killed and that further develOpment was prevented. In 1939 Keitt (17) tried a group of chemicals which were useful as weed killers in an attempt to find an efficient eradicant material. The compounds were sprayed on leaves which were placed in cloth mesh bags and allow- ed to remain in the field until the checks were discharg- ing. The treated leaves were then brought into the labor- atory. Those which bore the greatest number of perithecia were selected and a representative é inch disk was cut from each. The piece of leaf was placed on the inside surface of a petri dish cover with the dorsal surface ori- entated toward the water agar which was in the bottom. The spores which.were discharged from the fruiting bodies were counted and the results recorded as the number of Spores remaining per unit area of the leaf as an indica- tion of the effectiveness of the eradicant Spray material. Of a large number of compounds they found Elgetol, a prod- uct consisting of 3h% sodium dinitro-ortho-cresol, to be superior. A second part of the experiment involved the use of Elgetol in an actual orchard test. A ten acre sod orchard was sprayed with a 1% concentration. The closest unSpray- ed orchard was 0.3 mile distant. The application was made at 450 gallons per acre by using double nozzle guns at #00 pounds pressure.“ The result as determined by the discharge of ascOSpores per unit leaf area showed about a 96% inhibition. Tabulation of the amount of primary infection on the Spur leaves in both sprayed and non-Spray- ed orchards indicated a 98% inhibition of ascosporic in- fection. Other investigators working concurrently with and since the time of Keitt's experiments have run similar tests to compare the effectiveness of ground sprays. Ka- dow and Hepperstead (1A) in 1937 sprayed an orchard floor with sodiumpdinitro-ortho-cresylate at 0.5% concentration and a mixture of 100 pounds of sodium.nitrate plus A pounds of calcium arsenite in 100 gallons of water. They determined the effectiveness of the sprays by the amount of primary infection on the trees. A good deal of varia- tion was found both from season to season and between var- ieties of apples. Their conclusion was that the treatment was especially worthwhile when the amount of primary in- oculum was high, at which the this treatment aided a good foliage program of mild sulfur sprays to materially reduce the extent of infection. Similar mild sulfur foliage sprays applied to non-ground treated portions of the or- chard were less successful. Carpenter (5) compared Elgetol and Lignasan (ethyl mercuri phOSphate) in both in vitro and in vivo studies. Varying concentrations of the fungicides were added to {media in which monosporically-derived cultures of X,_ggé ae ualis, Qgcggmyces hiemalis, and Sclerctinia fructicola were placed. In all cases C. hiemalis was most suscepti- ble; V. inaegualig, second; and §Lflfructicola, most re- sistant. Lignasan was more toxic than Elgetol. However, when the same chemicals were applied to the scab fungus in the leaf, Elgetol was found to be more efficient. Elgetol killed the mature ascospores within three hours after application and prevented any further development 10 of immature Spores. When the leaves were.kept constantly wet, the killing time was extended to six hours. This re- sult was explained as a dilution of the fungicide. How- ever, Palmiter (29) found that if Elgetol was applied to dry leaves at 0.5% using 500 gallons per acre, it dried before leaf penetration was complete. Rain or dew further aided penetration and a more complete kill of ascospores was accomplished. In 1937 Henrick (12) in Tasmania Sprayed urea and lime-sulfur on overwintered leaves infected with scab. He found no ascospores to be ejected from treated leaves into his Spore trap boxes, but large numbers of spores were discharged from.the untreated leaves with each wetting. In 1942 Sharville (32) began a project in which a ground Spray of Elgetol was used in early spring in conjunction with a summer program in order to determine an acceptable summer spray schedule. Sulfur replaced lime-sulfur as the main material in the scab spray program and a reduction from 9.1% scab in the control orchards to 0.5% scab in _ ground treated orchards was demonstrated. The last found published work on ground spraying with Elgetol for apple scab was performed by Goldworthy et a1 (11) in 19h9. They also eXperimented with possible summer Spray pro- grams to compliment a ground spray in the early Spring. 11 Their results show a good scab control by using several mercurial sprays at pre-blossom and calyx in conjunction with the ground treatment. Calcium cyanamid, a fertilizer and weed killer and one of the chemicals tested in these eXperiments, has been shown by Crowther and Richardson (8) to be toxic to seeds of higher plants. .Bauer and Huber (A) used it as a dust application on the ground to prevent the emergence of the apothecia of Sclerotinia fructicglg (Wint) Rehm. This dust was more efficient if it was relatively dry after the application. Calcium cyanamide dust is also being currently studied by Fulton for eradicating the apothecia of S. vaccinium from the overwintering mummies - of blueberries. Among other chemicals available for these tests was Actidione, an antibiotic known to be toxic to cherry leaf spot (C. hiemalis)(30). Thanos (3h) Showed toxicity to spores of various common pathogenic fungi at rates from 0.5 p.p.m. to 100 p.p.m. Leben and Keitt (26), in experiments with V. inaequalis in culture, inhibited growth of the fungus with an antibiotic at a concentration of 1:8,000,000. The material was a fractionated ethanol extraction of an uni- dentified Streptomyces culture. 12 METHODS AND MATERIALS Leaves were collected on December 12 from.beneath one unSprayed he Intosh apple tree that was heavily infected with apple scab during the previous season. The leaves were laid out between layers of % inch mesh hardware cloth.with the dorsal side of the leaf facing upward. This permitted eXposure of the leaves while at the same time holding them in place. These screens were placed on the dirt floor beneath a greenhouse bench in a cool room, kept about 50° to 60° F. at night. It has been shown by Wilson (35) that perithecia will mature most quickly if placed at a temperature of 20°C. (689F.) and if subjected to alternating wet and dry periods. There- fore, the leaves were wet thoroughly with tap water from a garden hose at least every other day during the months of January, February, and March. The water quickly drain- ed through the screens into the soil allowing good air circulation around the leaves causing them to dry out completely before the next wetting. With these apparently Optimum conditions, the peri— thecia developed faster than those outdoors and were ready for chemical spray tests on February 27. Examin- ation of the perithecia at that time showed that many of 13 the asc03pores had differentiated, and about 5% of these had developed the cross septa. On screens 1, 2, 3, and A chemicals were applied while screen number 5 remained as a check. The treatments given to the different series of leaves were as follows: (1) A prepared dust (Aerocyanamid) con- taining 57% calcium cyanamide at the rate of 200 pounds per acre. (2) A commercial preparation contain- ing 65% sodium cyanamide in a spray at 0.35% concentration equivalent to 600 gallons per acre. (3) DN 289 at 0.5% concentration at 600 gallons per acre. (A) Coromerc at 0.06% concentration at 600 gallons per acre. The fluid quantity of spray striking the leaves was deter- .mined by measuring the amount of spray issuing from the nozzle of a knapsack sprayer at 20 pounds pressure per square inch per minute. This calculated to 10 seconds for 50 cc. of Spray. Thus, each square foot was sprayed (for 10 seconds. These Sprayed leaves in the screens weretnmm,rep1aced to their previous location on the ground and allowed to dry. The following day the usual treatment of watering was resumed to enhance perithecial development. By the mdddle of March the perithecia in the control leaves were developed and many aSCOSpores were mature and discharging. At this time watering was discontinued in order to inhibit excessive development and discharge. Leaves were then removed for laboratory testing. A second experiment was performed in which application was made by dipping the leaves into the spray mixture. For this method leaves were gathered from the orchard sev- eral times during the last of April and first of May after many of the ascOSpores were mature. Applying chemicals to these leaves containing mature spores would indicate the ability of a material to inhibit ascOSpore discharge or ascospore germination after discharge. The chemical solution was not Sprayed on the leaves at the rate of 600 gallons per acre, the amount necessary to thoroughly wet the leaves; but they were wetted by being dipped into the chemical which was prepared to the same concentration as the spray. The leaves were dried at room temperature for a day before treating to assure equal moisture content and allow for similar absorption of chemical. The leaves were dipped into the solution with agitation for about 10 seconds. The excess solu- 15 tion was drained off and they were set out on paper towel- ing to dry. Even though leaves which have weathered throughout the winter are very absorbent and wet easily, it was desir- ous to know if a wetting agent would increase the efficien- cy of a compound. A series of leaves were dipped in solu- tions with and without the addition of a wetting agent. Triton B 1956 at the rate of 2 drops per liter was the wetting agent used. The chemicals used in the dipping eXperiment were: (1) Sodium cyanamide at 0.35% con- centration. (2) Coromerc at 0.06% concentration. (3) DN 289 at 0.5% concentration. (4) Actidione at 2,6,8,10 p.p.m. (5) Calcium cyanamide was dusted on individual leaves by blowing an a- mount over the surface of the leaf until it was lightly covered. In the third experiment actual conditions in an or- chard were simulated. Five plots of one square yard each were marked out under a tree which had been infected the previous season. The ground was covered with a grass sod, onto which the leaves had fallen and lodged. Applya 16 ing Spray on the leaves here would duplicate normal or- chard conditions, where the effect of sod and leaves sit- uated in compact layers would be encountered. The time of spraying was determined by the amount of material it took to wet visibly the surface of the leaves and to approximate 600 gallons per acre. This spraying was carried out the last of April on a sunny afternoon when the leaves were relatively dry. The materials were applied with a small compressed air Sprayer with a pressure of about 20 pounds per square inch. The chemicals which were used were: (1) Sodium cyanamide at 18 pounds per acre with 600 gallons of water. (2) Sodium cyanamide at 36 pounds in 600 gallons per acre. (3) DN 289 at 0.5% concentration. (A) Tag at 0.37% concentration. (5) Coromerc at 0.06% concentration. After spraying, the leaves were allowed to dry for several hours before the first sample was collected. A few leaves from.the surface layer were then gathered at random from each plot. These were brought into the lab- oratory for discharge and germination counts. A second 7 17 collection was made after a simulated rain. It was thought possible that the toxic action of the chemical occurs only when the ostiole Of the perithecia was Open. A rain sub— sequent tO application would cause the ostiole to Open and permit more fungicide to enter. Since no rainfall occured in the three day interval, the leaves and surround- ing ground were Sprayed with water at 20 pounds pressure and in relatively small drOps several days after chemi- cal treatment tO simulate the effect of rainfall. After drying, the second sample Of leaves was collected. In all the experiments the effects Of the treatments were determined by the discharge and germination of asco- spores on agar plates. Petri dishes were used as the discharge chamber. Plain agar was prepared at 2% concen- tration and poured into the bottom plates. The apple leaves which were dry were then wetted and washed clean by running water to remove at least some of the remain- ing chemical and dirt. A piece Of paper toweling was soaked and placed on the inner surface of the Petri dish cover. The wet leaf was placed on the toweling so that the side which was up during development of the perithecia was facing down toward the agar in the lower plate. Thus, the ostiole Of the perithecia would be di- rected toward the agar. 18 With the perithecia wet, the mature ascospores were expelled from.the ostiole down on to the agar. The asco- 3pores of the check leaves were germinated in 24 hours and they were used as a basis Of comparison. Consequently, the readings were taken 24 hours after the plates were pre- pared. For each leaf a count Of 100 random Spores was made and the percent Of those which were germinated was recorded. In an effort to reduce the amount of time required for petri plate examination and for clearer Observation, sev- eral other methods were tried. One was to use a thin lay- er Of distilled water on the bottom of the plate in order to get a more random distribution Of spores. Several difficulties arose. If the layer was too thin, it would tend to dry out. If it was too thick, a good distribu- tion would occur but.movement of the dish during counting would interfere with the attempt to avoid counting the same spore twice. Also, the reflection Of light by the meniscus Of water which formed on the margin Of the spore increased the difficulty of observing the smaller germ tubes from the spore. Another attempt was made to use glycerin since it was clear but more viscous than water. Again, there was a more even distribution obtainable. However, the glycerin would 19 vaporize and collect on the leaf and paper toweling situ- ated on the upper surface of the dish, increasing the leaf weight and causing it to drOp into the glycerin the end of 24 hours. Scotch tape was applied to hold the leaf up, but the tape did not hold well. The glycerin appeared to inhibit the germination of the Spores, so it was discarded. In order to facilitate a random.count of the Spores on a petri plate, the Spores were counted throughout the entire area of the distribution pattern. One hundred spores were counted per plate. Mechanical counters were used, one for germinating Spores, another for non-germ- inating spores.) As the mechanical stage would not hold the petri plates, the plates were moved over the micro- scope stage by hand and as accurately as possible. When the distribution pattern on the agar was small, the agar was cut out and placed on a glass slide whicthas inserted in the.mechanical stage for more accurate manipulation. 2O .Oo manna oHSHOMHHmo .oo coaasoomo smeanoat .oo Hcoflaoao 309 .03 moses oooam mesooooem .ou oedemcmao cheapest .oo moan cacao: M24maoo compact anoomoa Hmooom ceasedoho Beacon coca mamas (Hooooooana Hoooom Haven -eomuoeooo-oeoeoee season moanedo ocoamnom «scones amcommuz owefidcmmo addoamo oeaeagoaoaoso ZOHBHnoAEOO Adoajmmo woe oafimcmzo Edfleon Open 0mm 2Q ohoaonoo pose cwsdnmmoonom occaowoom @313 madam. .m .4 .m .N V..m Honopomwdqms one comb mamanooea H Maria 21 -RESULTS The observed results of the emporimsnts performed on the perfect stage of V. inaequalis are presented in the following pugssg Within each test the various chem- icals will be viewed for their efficiency and their prac- tical use in ground spraying treatments of apple orchards. The resultSof spraying the leaves before spore matur- ity are presented in table II. This table shows a com- parison of the percentage germination of ascOSpores. The DN 289 treatment resulted in a characteristic reaction of plasmolysis of the spores, both mature and immature and also the undifferentiated material within the usous. The asci of these perithecia were so disorganized that they did not discharge any Spores down on to the agar surface. Calcium cyanamide materially inhibited Spore germination. .If one compares the inhibition of calcium cyanamide-in table II with that in table III where the chemical was applied to mature ascospores, it is seen that this compound has a greater effect in preventing the deveIOpment of the immature ascospore than it does in preventing germination Of the mature spore. It appears that the solution of sodium cyanamide is more effective if applied on immature aSOOSpores than on those that were mature. . 22 TABLE II Results obtained by spraying.materials on leaves in screens containing immature ascospores. Percentage Germination a b 0 Chemicals Calcium Cyanamide O ' 5 3 Sodium “Cyanamide L. 20 18 DN 289 P P P Coromerc V 12‘ 5h 56 Check 36 92 89 ’U I! no ejection of spores and visible plasmolysis 23 TABLE III Results obtained by dipping leaves containing mature aSCOSporeS into solution. Percentage Germination a b c d 6 Chemical Check 100 89 92 96 80 Sodium Cyanamide 69 56 50 6a Calcium Cyanamide 2h . 1% 20 12 Coromerc 62 51 65 69 DN 389 P P P P Actidione 2 p.p.m. #7 5h 27 37 25 Actidione 6 p.p.m. ' #5 77 55 Actidione 8 p.p.m. 62 Actidione 10 p.p.m. 53 P a no Spores ejected and visible plasmolysis * This was applied by dusting it on wet leaves. The results of the dipping process are presented in table III. DN 289 again completely inhibited discharge. as the time lapse between the dipping of the leaf and the rewetting of it for discharge of the aSCOSporBS was about two hours, it appears that the Speed with which DN 289 acts is very fast. Sodium cyanamide inhibited the germ- ination of less than 50% of the spores. Calcium cyana— mide showed better inhibition within the limits of this experiment, but all the dust may not have reacted before being washed from the leaves. Coromerc was more effective than sodium cyanamide. The Actidione series was apparent- ly ineffective and somewhat erratic in its response. In the fourth eXperiment the same chemicals used in Experiment 2 were used with a spreader added. The results given in table V may be compared with those in table III. The solutions to which the Spreader was added gave no more inhibition than that obtained without the Spreader. Experiment number III attempted to simulate orchard conditions. The data are shown in table IV. the shown in this table, sodium cyanamide used on the ground at 36 pounds per acre, gave fairly good results. DN 289 again gave a high inhibition. In series g and'g the number germinated for the DN 289 treatment were the only spores which were found on the agar. Tag and Coromerc, closely related compounds, gave comparable results. 25 Results obtained by Spraying leaves on the orchard floor. kercentage Germination before rain after rain a b c d 0 Chemical Check 82 91 73 65 85 Sodium Cyanamide 18 ll 13 16 2O ' 22 Sodium Cyanamide 36 3 1 7 20 60 IDN 289 6* P 3* P P Tag .14 19 13 1h 16 Coromerc ' I7 13 16 _ 18 27 P 3 no spores ejected and visible plasmolysis * Actual number discharged--not percentage 26 TABLE V Results obtained by dipping leaves into solutions with spreader (Triton B 1956) added compared with those with- out spreader. Percentage Germination a b c ' d Chemical Actidione 2 p.p.m. 47 54 27 37 Actidione 2 p.p.m. and spreader 41 05 23 4h Actidione 6 p.p.m. #5 77 actidione 6 p.p.m. and spreader - 24 68 Coromerc 62 51 65 69 Coromerc and spreader 67 71 62 6O 27 DISCUSSION The techniques of these exPeriments differ from those of other workers in that the percentage germination of discharged spores was observed instead of the number of ascospores which were discharged on to the agar. It would appear that the number of spores discharged from a unit area of leaf surface was not an exact criterion of the chemical effect on the fungus. Many of the Spores of treated leaves discharged but did not germinate, while those of the untreated leaves were exceptionally viable. The results, therefore, of this experiment are based on the percentage of germination of discharged spores. This is not the entire answer, however, since DN 289, the most effective chemical, completely inhibited discharge. Therefore, in future work it would appear desirable to determine the degree of inhibition of discharge as well as the inhibition of germination. The use of the chemicals, other than DN 289, as ground Sprays would need the Justification of orchard tests. Sodium cyanamide in this eXperiment did not give outstanding results, and it has been found by others to cause injury to the tree by absorption through the roots when applied to the ground. 28 SUMMARY Apple scab, a disease of apples, is caused by Venturia inaequalis. Control proceedures for this prevalent disease have utilized protectant sprays. The inconsistent results and phytotoxicity of these sprays, namely lime-sulfur and Bordeaux, led experimenters to reduce the primary inocu- lum potential by eradicant ground sprays on overwintering leaves. This would allow better control by milder foliage Sprays. Elgetol, a di nitro product, has been proven effective. The present experiment was carried out to test the preperties of other chemicals for eradicant ground spraying and to test a laboratory method of assay. Tests showed that DN 289 was superior to the other materials tested. Aerocyanamid dust showed some prom~ ise while the sodium cyanamide salt, the mercury compounds, and Actidione were of less value according to this test. The results of spraying leaves in screens and dipping leaves in solutions showed correlation with the simulated ground spraying application. These results corroborate the experiments of others showing the effectiveness of dinitro compounds for or- chard floor Sprays. Cyanamid dust may have considerable merit as a floor application, judging from one experiment, 29 but further work is needed to determine its real effective- ness. It is possible that it would need Special moisture conditions following application to merit orchard use. 30 5. 8. 9. 10. LITERATURE CITED adams, U.F. 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