WM" \H \ , ’ \ 105 091 THS VHF. LEAF SPOT QISEASE CF MUSKMELUN Them for Degree cf M. S. John R Cole 3924 . --..-._ ._... -fi...».,-.._._.,‘ g V THE LEgF SPOT DISEASE OF XUSKLELOK. Submitted in partial fulfillment of the requirements for the degree of Easter of Science at the nichiean Agricultural 3011858‘ John R. Cole. ”I 1924 I' ""r"' r1::) '1‘7“7m:‘ AL J‘L-‘(_) ’J ‘ Ga“Jll‘-LLJO The writer is grateful to Dr. E. A. Bessey and to Dr. G. H. Coons for advice and suvgestions given throughout this study, and for their criticisms and corrections of this manuscript. 9 1305 TABLE OF CONTENTS. Introduction History onDisease Distribution Economic Importance Signs of Disease Etiology The Etiological Factors Cultural Studies AnaerobicVGrowth Change in Reaction of Kedia and Production of Ammonia Infection Phenomena Infection T«“{periments Inoculation upon Other Hosts Resistant Varieties Loss of Virulence Relation of Host and Parasite Temperature Relations Viability of Spores Attempts to find Perfect Stage of the Fungus Cause of Zonation of Cultures Dissemination by Wind Dissemination by Splashing of Water Control Summary Literature Cited THE LEAF SPOT DISEASE Ob‘ EIUSKTJELON. INTRODUCTION. The growing of muskmelons in Michigan on a commercial scale is a localized and more or less restricted industry. The climatic conditions favoring the plant have given certain areas marked advantage in melon cul— ture. The muskmelon is a plant requiring a rather long, warm growing sea— son and the best crops have come in seasons of light to moderate rainfall. The plants are usually grown in the greenhouse or cold frame and transferred to the field before they begin to run. An important factor in restrict— ing the utilization of this crop is to be found in the plant diseases which periodically cause a great loss. The disease problems of michigan growers, however, are not different from those confronting the growers of melons in other states. Wherever melons are grown, the fungous diseases play an important role in causing loss. The leaf Spot disease or "leaf blight" has been reported for many years as a cause of much damage. Little attention has been given to this disease by plant pathologists. But now that the acreage is increasing and the production is decreasing, the desirability of investigation is apparent. In 1920 there were 950 acres planted in muskmelons, and about 400 cars of melons were shipped. In 1923 there were about 1700 acres in melons and 300 cars were shipped. In about four years the acreage was quadrupled, but the total yield was actually decreased. macro- sporium alone has not caused this decrease, but it is the most import- IO ant disease of muskmelons in Michigan and has been an important factor in the decrease of production. During the season of 1923 it was the most important muskmelon disease. As its popular name implies, the leaf spot attacks the leaves and causes practical defoliation of the plant. While the growing melons are not directly attacked by the parasite causing this disease, the ef- fect of loss of leaf surface is to render the fruit unsalaole because of the lack of flavor and the delay in ripening which is brought about. The epidemic of 1923 developed following abundant rainfall in late August and early September. Helons in Berrien County, which is the largest producing area, were greatly injured, as were local plantings at Grand Rapids and East Lansing. A field of melons near Ovid, Michigan, showed on September 25th practically 100 per cent defoliation as a re- sult of this disease. The experience of 1923 was but a repetition of the epidemics of previous seasons, for periodically this disease has been known to pro- duce similar losses throughout the various melon-growing districts. HISTORY OF THE DISEASE. There has not been a great deal of investigation of this di- sease. The researches which have been carried on have dealt largely with the description of the causal organism and the methods of its con- .trol. The American investigations start with the work of Ellis (7) and Everhart in 1895. Whole fields of muskmelons around Las Cruces, New Mexico, were being destroyed by a "leaf blight," and some of the diseased leaves were sent to Ellis and Everhart for identification. -3- They gave a brief description of the disease and named the associated fungus Macrosporium cucumerinum. Since that time many names have been used for the disease and for the causal factor. The disease is commonly called "blight," "rust," or "leaf spot" of muskmelon, according to the stage noted or some peculiarity in its manifestation. Vhat seems to have been the same disease was described by Peglion (15) in Italy in 1892. He called the organism Alternaria brassicae variety nigrescens. The influence of Peglion's work has resulted in the general acceptance of the name by American authors. There is, therefore, considerable confusion in the literature as to the name and cause of this disease. E. F. Smith (18) reports that in "September, 1892, a wide- Spread disease of muskmelon leaves was observed in Southwestern Mich- igan. The foliage was destroyed almost completely over entire fields and the fruit failed to ripen. An Alternaria or macrosporium was the only organism present and the disease was reproduced in the field from a single spore culture." Blinn (1) reports that "rust" or "blight" has inflicted severe injury to muskmelons in the vicinity of Rocky Ford. He calls the causal organism hacrosporium cucumerinimn A severe "rust" or "blight" almost destroyed the muskmelons in Ohio (17) in 1896. The disease was reported in 1894, but as that was a very dry year, little damage was done. In September, 1896, there was an increase in rainfall and the plants were killed before -4— frost, shortening the crop considerably. The disease was probably caused by the same organism as the one reported by Smith (18) in Southwestern Michigan in 1892. Chester (4) reports the disease as being very destructive in Delaware thus: "During the season of 1907 the cantaloupe crop was cut down at least one-half in some parts of Delaware. The greatest trouble was Macrosporium cucumerinum (E. and 3.). Probably the same trouble has been described by a number of mycologists, but two distinct species have been confounded in referring to it." Chester found a number of Alternaria spores present on the rusty or spotted leaves, but he was un- able to produce the disease with any of them. He was able, however, to produce the disease with the Kacrosporium that was present. DISTRIBUTION. What seems to be the same disease as the one under consideration has been reported in Italy by Peglion and in all parts of the United States where muskmelons are grown extensively. The herbarium at the Michigan Agricultural College contains specimens collected by R. E. Smith in Massachusette, 1898, and C. E. Goodwin, Portland, Michigan. Experiments show that Hacrosporium was present and injurious in New Mexico in 1905, Rocky Ford, Colorado, in 1898, and in 1905 when it caused an enormous loss. It was also reported in massachusetts, 1898, Ohio,1896, and probably the same disease was reported in Michigan in 1892. It seems to have caused considerable damage about 1900. 3001301110 IILIPURTAIICE. As has been said, this is one of the most important diseases of muskmelons. Most of the work that has been done on the disease has been done at Rocky Ford, Colorado, which is one of the greatest melon— growing districts in the country. Very little work has been done on the disease in Michigan. SIGNS OF DISEASE. The disease usually begins in the latter part of the growing season, usually about the middle of August. The first evidence of the disease is the appearance of small black spots, after the organism has entered the host. These spots enlarge by concentric markings, usually one dark and one light ring, the black dot marking the center of infec- tion. At times the entire leaf is killed by the spots merging together, especially if the humidity is increased. The disease is always recog- nizable by the dark brown leaves, which crumble easily when handled, due to the discoloration and drying effect of the fungus. The characteris— tics of the disease vary according to the host, being light brown on muskmelon and darker brown on watermelon, squash, pumpkin and gourd. The writer has never observed infection on any parts of the plant other than the leaves. ETIOLOGY. A fungus of the Macrosporium type was isolated from diseased leaves collected at East Lansing and at Ovid. After the organism was obtained in pure culture, dilutions were made and plates were poured until a single spore was found. This spore was transferred to another Plate and examined closely to see that only one spore was present. From such spore isolations the different strains used in this work were obtained. The organisms thus obtained were grown in pure culture and these organisms were tested for pathogenicity in the following manner: Fifty inoculations were made with each organism. Twenty-five were made by wounding and the remaining twenty-five were made without wounding. A heavy suspension of spores was made and a drop of water containing the spores was placed on the leaf. Large leaves were used and three in— oculations were made on each leaf. The plants were then placed in a moist chamber for twenty—four hours. In three to four days typical spots began to appear. They appeared somewhat sooner on the plants that were wounded. After the Spots had appeared they were examined for the causal organism. Over 90 per cent infection was produced with each strain. Plates were again poured, the organism was re-isolated and ob- tained in pure culture. THE ETIOLOGICAL FACTOR. From a study of typical strains of the organisms thus tested for pathogenicity, the following descriptions have been made. The organisms used have consisted of five strains from various sources and, so far as could be determined, were the same. Mycelium: When the spore germinates it sends germ tubes from each of several cells of the spore. These germ tubes develop into the vegetative mycelium. The mycelium is at first finely granular and almost hyaline. In 48 hours it becomes dark brown, has many septa, occasionally branched, and is from 3 to 6 microns in diameter. Conidiophores: The conidiophores are borne at right angles to the mycelium. They are mostly solitary, out occasionally fasciculate. They are dark brown and on the host are from 25 - 50 x 5 - 7 microns, and usually have 1 to 4 septate. Conidia: The conidia are clavate, muriform, somewhat constricted, 6 to 9 septate, 40 - 70 x 15 - 25 microns. In culture they always possess a long beak which is from 25 to 50 microns long x 2 to 5 mi- crons wide. The conidia are borne on the end of the conidiOphore and are mostly solitary, but occasionally a second spore is produced on the beak of the primary conidium, depending on the age of the culture. If the culture is old there is usually a small spore produced on the tip of the beak of a large conidium, but no more than two conidia have ever been found so associated in culture on any of the laboratory media. On the host the spores are always solitary. At first the conidia are olivaceous but they grow darker with age. When they reach maturity they are fuliginous or crown. The following table sumarizes the measurements made for both Alternaria brassicao from cabbage and the pathogene from muskmelon. The former species was used throughout this study for a comparison with the muskmelon fungus. The sources of the fungi are indicated in the ta— ble. Those starred were obtained in pure culture. (See Table l.) .-3- .H apeam «mm flu. .oadpaso mafia a“ deafiMuno monomm . 0.0 u m u n a 0.0m” mm a ma "a 0m AOHH Jinn « ow "me " onwa a? 3“ Amme_ « EsaHaoESOSO “ u u u a u" a u u a “ «uwmuan moamflom «0 hfimdmow « Eswpoamoaomfi u n u u n «a a u a a u « oz «.Hwnm mo mwnfiemooopm " a u u a u a“ n u u u a u u.adam.m an donfipomoa « 0.5 u m u o « o.¢nu mm u ON u" Hm «0&H « mm a 0m who «mom a e « ”mama; mmeafioo « Efidfimmfidodo " u a u u "u a n a a n u « mama noaoEmmsE « Esfluoomonoam o.s “ m a mid o.Hmu mm a ma ad‘mHH aoefl « mu a on «ma «low a m a gamma. .HdeH canes " u a a u u" u a a a a u » aonEsmSE . mama u Edwaoamoaonfl u a a u n “u a u a u u u . taco .oaspHSO oasm u 0.5 u m u m q,m.muu on «.ma a«.Hm “HoH «tam « #0 «ea « mm «1 m « gammaq .mwanomwfiwo " adaaaefidodu‘ " u u u u a" u a a u a a « .mmamflan .aoHoEMmsa «seamaccmoaowa o.« u m a m « o.owdxflm «Ina ma be «do 4 mm " an "mm xdlom «1 ml." .aflw so « macawwmnn “ n a a a a" a u u u a “ ":mw:.onaomu pm apHEm « magmsampad u a a u a «u u u n u u u u .m .m he umpoeflaoo « u u u u u "u a u u u u a « .Ammmav emmnnwo « u a u u u a“ u a u n u a u me>meH no ommna Hm « u u a u u a" u u a u a u « Imam nwofineE<.zpanz . Com” " o u H " m.mJ. NH u m «u mm «mm u «CH .4 0H «Orv 41m d O u . thuHfio mhfim . u a u a a u" a a n u u u u « magmaaepaw m.¢ “ w u H a o.nHa >H aloH «a me “we a pm " am “we w H“ u m “ mm :deap mu .mme " auwm,sAp " u a u u "u a u u a a u « afioo Hwea omsno no u smegmaaepaw o.n " Ha “ a " o.mau mm u ea a“ mHH lama “ mm " noH "mam " ma " auua aT lanai: as clans " smeawmaae « a u u u “a u n u a a a « Iguana epanwo mHHmd " mannaaopaw “ a u n u a" u a u u u a « wuwmmmawm Hzmasm « a a u u u "a u a u u a “ .mmmav mama mmwppmo « ohm ”ESE; ”ENHEMJH meNJEEMAESEMAJ. may .Ed EJFTEEHJ emoafisu. 4&5le u u 4. nam>munana“unawunam>muuawfinnawsu"upm>wuuwaa.iewauuumsmnuxnannnH2HOpmHm" “ n "u xmmp msfic.lwil‘ Neon maoao. “ “ msmrsm macaw mud“: .moosam use neon macaw l.l.ll.- "I'- .| i. ' I'll. . O maoaoflfi 2w sumac: heave um douooaaoo Hmfiaoame gawk EsnwaeESOSO Ezwaoomopoam no . meadow mu omofimmnan mammaampfiw mo mpnosohswmmu mpfiuwammsoo .H 34fi£9 The original description of Macrosgorium cucumerinum as given by Ellis and Everhart was presumably made from spores taken from the host. From the table it is seen that the measurements given by them fit those found for the new isolations. The minimum spore length given by Ellis and Everhart is 30 microns and that from the pure culture is 40 microns; but the cultures used were grown for two weeks before any measurements were made. In making these measurements, the same difficulties that Rosen- baum (16) reports in making measurements of the Macrosporium of tomato were found. There is considerable variation in the conidia, and in the case of the Macrosporium there is more variation in the beaks. Alter- naria brassicae does not have the long beak. In must cases the apical segment was somewhat elongated, and fOllowing Bosenbaum's suggestion, measurements were made from the point of attachment of the conidiOphore to the last segment. This portion constitutes the spore body. Then the beak or last segment was measured. One hundred spores taken at random were measured in each case. (See Table 1.) It is the writer's Opinion that pending complete monographic working over of the entire genus, the name given by Ellis and Everhart should be used for the fungus found in Michigan. This means the dis- regarding, for the present, of the name suggested by Peglion for the fungus causing the disease of muskmelons. The reasons for this de- cision are, briefly, that Peglion was dealing vith a fungus of the Alternaria type, characterized by the formation of spores in chains. The spore measurements given are in no way comparable with the measure- .- 10-. ments from American material. Furthermore, the fungus isolated from the typical cabbage leaf spots from different sources in the United States are distinctly differ- ent from the muskmelon pathogene and are not like the organism described by Peglion. It is worthy of note that study of the organisms from cabbage leaves in Briosi and Cavara's collection‘ show typical macrosporium forms, only they occur in chains and do not contain as many longitudional septa as the Kacrosphorium from Michigan. They are also more irregular in size, being very much longer than they are wide. Alternaria brassicae forms a spot on cabbage similar to the spot on muskmelon, only it is somewhat darker. The spot begins with a black dot about the size of a pin head, and in two to four days a spot several millimeters (3 - 5) in diameter is formed. It also shows the typical con- centric markings. The spores are much more abundant than in the muskmelon leaf Spot, and are readily visible with a hand lens. Spots occur on leaves petioles, but not on the stems. Spore measurements of Alternaria from Louisiana in 1889 and from .Alabama in 1888 are within the range of the measurements of the Eichigan strains of Alternaria brassicae. Studies of a culture of Eacrosporium** from California, kindly fur- nished by Brisley (3) have shown that the conidia correspond very closely to the strain of Macrosporium isolated at East Lansing. It will be noted - that Brisley used the name EacrOSQOrium cucumerinum. (E. and E.) ‘ Funghi parassiti delle piante cultivarte od utili #87. *“This is an old dried culture which was still alive and which was induced to fruit by the method described by Bands - PhytopathOIOyy 7:316. 1917. CULTURAL STUDIES Cultural characteristics of the organisms were determined in the usual manner. The media used were of three types: nutrient agars, vege- table plugs, and liquid media. The purpose of the tests was to determine the differences in the cultural characters of Alternaria brassicae and macrosporium cucumerinum. (See tables 2 and 3.) From the results given in tables 2 and 3 it is observed that the two organisms will grow on all the ordinary laboratory media which are slightly acid in reaction. Macrosporium cucumerinum produced abnormal spores on rice medium, potato dextrose agar, and sweet potato plugs. On these media the Spores were "warty" in some cases and triangular in the others. On potato dextrose agar the spores were so dark that the septa 'were almost indistinguishable. When transferred back to cornmeal agar, the spores again became normal. The abnormal formation of the spores was probably due to malnutrition. Rice is rich in starch and poor in proteins, while the sweet potato and potato dextrose is rich in sugars and poor in proteins. This seems the probable explanation of the abnormal Spore forms. The spores of Kacrosporium cucumerinum were always solitary or in pairs, never in long chains. Alternaria brassicae likewise will grow well on the ordinary lab- oratory media, but there is a yr at difference in the Spores in culture and on tkm host. They do not show the abnormal forms as the HacrOSporium did. They form long chains, as many as 32 spores occurring in one chain. Spores were never found to have muriform septation in culture, although this type 0f structure is commonly found in spores grown on the host. -12- TABLE .. . Growth 0 Encrosporium cucumorinum and Alternaria brassicae on solidified media. Thirty- ay cultures at room temperature. Hacrosporium cucumerinum : : Spore : . . : : pro- :Aecial . : : : duc— :mycel- : Color : Color medium : Growth : tion : ium : Spores : Eycelium nutrient agar : .+++ : 4+ : + :Olivaceous : light brown Oatmeal agar : 4.4.4.4. : 4.4+ : 4...- :dark dark brown Tornmeal agar : 4.4+... : +444. : +4. :dark : dark brown Prune juice agar : 4.4.4.4. : +4.4. : +++ :darlzjwarigL : ark Potato dextrose 19° : 4.4.4.4. : +4. : 4-: (very warty : very dark : : : (hardly recog- : : : {nizable : Cooked rice : +4.4. : 4. . +++ :light brown : light : : : :and warty : Alternaria brassicag_ Nutrient agar : +4. : ++ : - :iight brown : light Oatmeal agar : 4.4.4.4. : 4.4.4. : 4. :light brown : dark Cornmeal agar : .++44. : +++q-: + :dark brown : dark brown Prune juice agar : 4.4.4.4. : ++++ : ++ :dark brown : dark brr vn Cooked rice : 4+ : + : ++1- :light brown : light brown ~ nutrient sol. Co rnmeal bro th Potato dextrose broth _§Qons' Synthetic sol. Liquid media - (250 0.0. flasks). Macrosporium cucumerinum : .++ : ++ : -’ :light brown : light brown : ++++ : +++ : ++++ :dark brown : dark brown : .444 : ++ : .+ :almond : dark brown : t1+'= 4¢+ : -tt++ :dark brown : dark brown Nutrient sol. Cornmeal broth Potato dextrose broth: ++++ Coons' Synthetic $01.: «++++ Alternaria brassicae : 4.4.4.4. : +++ : - Eli-“ht brown : light brown : +4.4. : 4+4. : +++ :dark brown : dark brown : ++++ : 4. :very dark : dark brown : 4+++ : - :light brown : light brown +l+ I! No growth .44 = Average growth Slight growth +++ = Good growth Poor growth 44.44. a Abundant growth -13- TABLE 5. Growth of Iiacrosporium cucumerinum and Alternaria brassicae on vegetable plug-gs and stems. Thirty-day cultures at room temperature. Nacrosporium cucumerinum : : Spore : : z : : pro— : Lecial : : : : duc- : mycel- : : Color Medium : Growth : tion : ium : Color Spores: mycelium Carrot plugs : ++++ : ++ .: ++ :light brown : brown Parsnip plugs : .444 : .+++4.; + :light brown : brown Potato plugs : ++~++ : +1- : + :light brown : light brown Sweet potato : ++++- : ++ :-r+++ :light brown : Plugs : : : : abnormal : light brown Keillotus stems : +++4' : +++’ : + :dark brown : dark brown Muskmelon stems :‘++++' : -++++-; ++ :dark brown : dark brown Alternaria brassicae light brown light brown Carrot plugs : 4.4.4.4. : 4.4.4.4.: 4. : : Parsnip plugs : 4+ : +4. : - :light brown : light brown Potato plugs : ++++ : ++++: +++ :dark brown : dark brown Sweet Potato : -+++ : ++ : + : : Plugs : z : :light brown : light brown Melilotus stems : +++- : +++- : - :light brown : light brown lguskmelon stems : ++4- : +++- : + :dark brown : dark brown - I No growth i '= Slight growth 4- 3 Poor growth ++ '- Average growth +++ ‘ Good growth ++++ " Abundant growth -14— AILLZIROR IC SRO/TH. A Giltner H—tube was used to grow the fungi under strongly an— aerobic conditions. In one arm of the tube corn meal agar was planted and solidified, while in the other pyrogallic acid and KOF were placed at once. Five tubes were inoculated with Alternaria brassicae and five with Macrosporium cucumerinum. The tubes were then sealed with rubber stoppers and modeling clay. In this condition they were set away at room temperature. Growth was very slow, out in ten days the organisms had covered a Space about 5 millimeters of the surface of the agar. Both organisms seemed restricted to the surface of the agar, neither penetrating the substratum very deeply. Few Spores were produced, which shows that both organisms require oxygen to fruit extensively. This ex- periment was repeated with nutrient and prune juice agar with the same results. CHANGE IN REACTION OF YEDIIL AND PRWDUCTION O F A} ELTONIA The organisms grow well on slightly acid medium, but change it slowly to neutral or alkaline. Coons' Synthetic solution (pH 5.5) was changed by Macrosporium cucumerinum and Alternaria brassicae to pH 7.4 in thirty days, while the uninoculated solution had a reaction of pH 5.7 at the end of thirty days. By tests with Nessler's solution ammonia was demonstrated, although no quantative tests were made. IITFEC T I 0N- i’H 31:01.31; .1 . An attempt was made to determine how the fungus entered the host by placing a leaf attached to the host between absorbent cotton and blotting paper and clamping it down on the stage of the microsc0pe. A -15- small hole was cut in the paper so that the leaf could be seen. A sus- pension of spores was placed on the leaf and the hole was covered with a cover glass. In this way a moist chamber could be kept by wetting the blotting paper occasionally. However, the method was not successful, as only a few of the spores germinated, and they were very slow in doing so. It is believed that the germ tubes bore into the leaf, as a few were seen in the leaf, but it was impossible to tell whether they entered through the stomata or not. The stomata are very small, and close observation is required to determine whether or not the fungus enters through them. Time did not permit the sectioning of inoculated leaves, which would have settled the question. INEBCTION EKPERIRECTS. The first experiments were conducted to determine if infection took place without wounding and to determine the parts of the leaf most easily infected. A heavy suspension of spores was sprayed on the plants with an atomizer. The plants were then placed in a moist chamber for twenty- four hours. The spots on the wounded plants appeared in two to three days and on the unwounded in three to five days. The percentage of in- fection was approximately the same in each case, but on the unwounded the spots were slower in appearing than on the wounded. Inoculations were then made on the upoer and lower surfaces of the leaves, on the edges and in the center. Infection develOped as rapidly in one place as in the other. The second method of infection was similar to that used by -15- Makemson (13) in his work with Cladosporium fulvum. This was done by plac- ing a mass of spores on the leaf, covering all with moist absorbent cotton and placing in a moist chamber for 24 hours. This was found to be a very convenient way to make the inoculations. The cotton seems to mark the place of inoculation better than India ink, and it has no harmful effect on the leaves. Attempts were also made to infect the stems and petioles, but they were unsuccessful, even by severe wounding. As all of this work was carried out in the greenhouse, very few blossoms were produced and no melons. Then inoculations were made on melons but without result. The writer made close observations on the fruit at the College farm during a severe epidemic of the disease, but was unable to find any spots on the fruits due to this fungus. IITUCIJL.”iTlUi?S U‘r‘ II OTHER HOST“ In these experiments comparisons of Alternaria brassicae and Macrosporium cucumerinum were made on a wide range of hosts. Fifty inoculations were made on each series of plants. The plants are arranged in the following table according to their susceptibility. (See table 4.) Some of the Spots on other hosts were very characteristic of the organisms mentioned in the table while others were not. This was especial— 1y true in the case of wounding. The spots produced on gourd, squash and cnunnnber by Macrosporium cucumerinum were very characteristic of this organism.and similar to the spot produced by this organism on muskmelon. The organism was isolated and the same plant was re-inoculated to see if the pathogenicity was increased in that plant, but this did not occur. Spore measurements were made and chain formations were observed very closely -17.. TABLE 4. Showing plants inoculated with Kacrosoorium cucumerinum and Alternaria brassicae and arranged accord- ing to their susceptibility. Organism : Host Leaf : Results of Infections Macrosporium = i ‘ cucumerinum 3 Nu skme lon ‘ Iornal 3 46/50“ * Repeated 44/ 50 " ;Watermelon ; " : 37/50 " 39/50 " ;Gourd ; " : 35/50 " 37/50 " ;Squash : u : 35/50 " 23/50 n ;Pumpkin ; " : 31/50 " 30/50 " ;Tomato :Vounded' : 11/50 " 13/50 " ;Radish ; u ; 7/50 " 10/50 " ;Turnip : " : 5/50 " 5/50 " :Potato : " : 0/50 " 0/50 n ;Cabbage : n : 0/50 " 0/50 " ;Cucumber f " : 0/50 " 0/50 Alternaria : : : brassicae :Cabbage : Normal : 48/50 " 46/50 " ;Radish ; " : 38/50 " 55/50 n 2 Turnip ; n : 31/50 " 29/50 " ;Tomato ; Wounded : 18/50 " 21/50 n ;Squash : r' : 14/50 " 11/50 " :GOurd : " : 9/50 " 10/50 " :Muskmelon : " ; 5/50 " 2/50 " ;Cucumber ; " : 0/50 " 0/50 .. gPotato ; " : 0/50. " 0/50 ‘ The figures in the numerators refer to successful inoculations. to The figures in the denominators refer to number of inoculations made. -18- on each host. In every case the spores of Alternaria brassicae from cabbage were produced in long chains and they were the same size as they were on the original host. The spores of Nacrosporium cuCumerinum were solitary and characteristic of those on muskmelon. No differences were noted in their sizes. RESISTAXT VARIDT ES. All varieties of cabbage that were used in this work were sus- ceptible to Alternaria brassicae. The varieties used were Early Wakefield, Express and Premier. The varieties of muskmelcns that were susceptible to Macrqsporium _cucumerinum were Osage, Edwards-Perfecto, Knight, Hoodoo, Bender's Surprise, Delicious Goldlined and Rocky Ford. Blinn (I) found two strains of Rocky Ford muskmelons that were somewhat resistant to muskmelon leaf spot. The strains were called Pol- lock 25, and 1035. The 25 and 1025 were both used with Alternaria brassicae and_gacrosporium cucumerinum. Authentic seed* of the original Pollock strains was used in this work. The 25 was resistant to Alternaria_b:assicae and somewhat resistant to fiacrosporium cucumerinum. Forty out of 100 in- oculations were positive. The 1025 was resistant to the Alternaria brassicae, but out of 100 inoculations with kacrosporium cucumerinum only 15 were positive. The spots that were produced were slow and they were not typical of the organ- ism. Some of the spots did not occur for 8 days. ‘ Furnished through the courtesy of Mr. George Starr of the Department of Herticulture, Michigan Agricultural College. LOSS OF VIRULBIICE. While working with old cultures isolated in September, 1925, one culture of HacrOSporium cucumerinum had apparently lost its pathogenicity. Under ideal conditions only a small percentage of infection took place. As a test, 100 inoculations were made on muskmelon leaves, 50 with a new culture and 50 with the old culture. Forty—three spots were produced with the new culture, while only 9 were produced with the old culture. The experiment was repeated with similar results. The organism loses its pathogenicity but partially. None of the old cultures of Alternaria brassicae had lost their pathogenicity. RELATION OF HOST AND PARASITE. After a study of the cultural characteristics of the organism had been made, further studies were made to determine the cause of the discol- oration of the leaves. most of the spores are produced in the central or older portion of the spot, and there is a portion around the edge of the discolored spot that is lighter and does not produce any spores. Very little of the mycelium has been found in this region. It was suspected that the organisms produced substances toxic to their respective hosts. It also seemed that these toxic substances differed outwardly from the growing'mycelium into a zone ahead of the fungus. deBary (5) (1886) working with Sclerotinia libertiana was the first to experiment with the substances produced by plant pathogenes. He showed that this fungus produced a substance which injured the host cells in advance of the fungus and finally killed them. He was unable to tell whether the fungus produced oxalic acid or an enzym. -20- Jones (9) (1909) working with Bacillus caratovorus found that in growing the bacteria in both cultures, a substance was produced with which he was able to reproduce the symptoms of the disease. This substance was anlnzym called pectinase, which killed the cells by dissolving out the middle lamella. Munn (14) in his work with Botrytis allii, which causes the neck rot of onions, found that oxalic acid and anlnzyym are produced. He was able to obtain the enzym from the fungus tissue but he was unable to dem- onstrate the particular roles the acid and enzyms play in disease produc- tion. Krakcver (11) while working with Macrogporium sarcinaeformae found that a toxic substance was formed in culture medium which discolored and finally killed the wounded leaves. He found that this substance was in- oculated by boiling. Ammonia was also found present in the culture, but he did not believe that it alone was responsible for the killing of the leaves. In the experiments which follow,the writer undertook to determine if the killing of the host tissue was caused by the organism working di- rectly in the cells, or if the metabolic by-products of the fungus cul— tures were responsible. The technique in these tests was similar to that used by Kran koner (11) in his work with Macrosporium sarcinaformae, as follows: The leaf'vwas held in a horizontal position by means of a glass slide which rested on a wire support. Van Tieghem cells were sealed to the leaf with a small amount of vaseline and held in place by paper clips. The clips . .2...“ ..I .Ic'r Eu!.! 'EE,E‘DEH I l1.i-.'..I . . ’7 . {Ill .il Ir. i..rflié.§‘yitlirflflu. 'Il 5.in 0.15. -;1_ were used because they could be hunt and made to fit any cell and they did not exert pressure enough to injure the leaf. Sufficient experiments were run with vaseline to prove that the quantity used had no injurious effects on the leaves. Both sides of the leaves were coated with vaseline, and empty Van Tieghem cells were fas— tened on the leaves, but the leaves suffered no injurious effects. Flasks containing 250 c.c. of Coons' Synthetic solution were inoculated with_flicrosporium cucumerinum and Alternaria brassicae res— pectively. The organisms were grown at room temperature for 30 days. The mycelial growth was then filtered through filter paper and a No. 5 Pasteur Chamberland filter. Petri dishes containing cornmeal agir were inoculated with a small amount of this filtrate, and observations were made for 48 hours to determine that the filtrates were free from the fungi under con- sideration. A few bacterial colonies were developed, but no Macrosporium or Alternaria growths appeared. The experiments were run with cabbage and muskmelon using uninjured and wounded leaves for compariSon, care being taken not to wound the leaf severely in each case. Two cells were used on each leaf. In some cases the same leaf was tested in both the wounded and unwounded condition. Cabbage and muskmelons were used interchangeably with each filtrate. About .5 c.c. of the filtrate was placed in the Van Tieahem cells, sealed with a cover glass and allowed to remain for 48 hours, when observations were As checks, sterile Synthetic solution was applied. (See table 5.) made. This table shows that the toxic substances from EacrOSporium cucum- erinum will produce a Spot on the muskmelon leaf in 48 hours. The spot is produced on the uninjured leaf as well as on the injured, but it is some- what slower to develop in the case of the uninjured leaf. This toxic sub- ‘.\.“ Tests of filtrates from Alternaria -22- TABLE 5. brassicae and Hacrosporium cucumerinum cultures on 60 hours. Muskmelon cabbage and muskmelon loaves; after 48 and Macrosporium cucumerinum filtrate Leaf Treatment 48 hours : 60 hours 1 : wounded : tissue discolored : dark brown 2 : wounded : tissue discolored : dark brown 3 : wounded : tissue dark brown : no change 4 : wounded : tissue dark brown : no change 5 : not wounded : slightly discolored : darker Q : not wounded : slightly discolored : darker 7 : not wounded : light brown : darker 8 : not wounded : light brown : darker Alternaria brassicae filtrate 48 hours : 60 hours 9 : wounded : dark around wound : light brown 10 : wounded : discolored : no change 11 : wounded : discolored : no change 12 : wounded : discolored : no change 13 : not wounded : slightly discolored : light brown 14 : not wounded : slightly discolored : light brown 15 : not wounded : slightly discolored : light brown 16 : not wounded : slightly discolored : light brown Sterile Synthethic medium 48 hours : 60 hours 17 : wounded : no change : no change 18 : not wounded : no change : no change (Table 5 continued on next page) -23—- TA‘LB LE 5 0 (Continued) Cabbage .Sterile Synthetic Kedium Leaf : Treatment : 48 hours : 60 hours 1 : wounded : no change : no change 2 : not wounded: no change : no change alternaria brassicae filtrate 48 hours : 60 hours 1 : wounded : complete discolora— : no change : : tion : 2 : wounded : comp. discoloration : no change 3 : wounded : comp. discoloration : dark brown 4 : wounded : comp. discoloration : no change 5 : not wounded : light brown : darker 6 : not wounded : light brown : darker 7 : not wounded : light brown : darker 8 : rot wounded : light brown : darker HacrosPQrium cucumerinum filtrate 48 hours : 60 hours 9 : wounded : sligitly discolored : darker lO : wounded : slightly discolored : darker ll : wounded : light brown : no change 12 : wounded : liaht brown : no change 13 : not wounded : slightly discolored : darker 14 : not wounded : slightly discolored : darker 15 : not wounded : slightly discolored : darker l6 : not wounded : slightly discolored : darker “-h‘ l" - t — N4 stance also discolors the cabbage leaves. The toxic substances from Alternaria brassicae discolor the cabbage leaves in like manner and also on muskmelons produce the same sort of results as the substances from Necrosporium cucumerinum. This sub- stance aiso discolors the muskmelon leaves. Both the Hacrosporium cucumerinum and Alternaria brassicae had the same effect on the Synthetic medium. In tha beginning of the experiment the medium was titrated, using Clark and Lubb as an indicator. It was found to have a reaction of pH 5.5. After the organism had grown for 30 days the pH was 7.4. A test with Nessler's solution gave a positive 5 test for ammonia. After finding ammonia present in the filtrates, experiments were begun to determine if the ammonia was the cause of the tissue discoloration. The following solutions were used in this experiment: Coons' Syn- thetic pH 5.5, KHBPO4 pH 5.2 and distilled water pH 6.7. Enough ammonia was added to make these solutions pH 7.4, or the pH of the active filtrates. These ammonia solutions were placed on the cabbage and muskmelon leaves, as the active filtrates had been, but all of the results were negative. These results are similar to those of Krakover (11), who was not able to show that ammonia was the factor producing the discoloration of leaves in his exper— iments. Krakovar (ll) had shown that a non-virulent culture of Kacrosporium sarcinaeformae did not produce toxic substances. Tests were undertaken to i See if the same condition held with the organisms of muskmelon. Flasks of Synthetic solution were inocalated with the culture of l macrosporium cucumerinum that had partially lost its virulence. The organ- 3 ism was grown for 50 days at room temperature and filtered as before. A series of experiments were run by placing the filtrate alone from the old culture on the leaves, and by placing tie filtrate plus ammonia on the leaves. The pH of the old culture was 6.1. The anmonia was added to make it pH 7.4, or the pH of the active toxic substance. (See table 8.) This experiment shows rather striking results, since by the ad- dition of ammonia to the old weakly virulent culture, the toxicity to the muskmelon leaves was intensified, producing results similar to those ob- tained from the toxic broth of the virulent culture. It seems that am- nmnia plays some part in leaf discolorations, although alone it will not . produce discoloration. a saprophytic Alternaria was grown on Coon's Synthetic solution for 50 days and filtered as before. It did not produce armonia and when placed on the leaf no discoloration occurred. This result seems signifi- cant in this connection. In further studies of this toxic substance, the writer undertook tocietermine if this toxic substance was thermolable. Broth which dis- colored melon and cabbage leaves was boiled for 2 minutes and placed on the leaves as before. Table 7 shows that the toxic substance is rendered non—toxic after 1 boiling for 2 minutes. It may be inactivated at a lower temperature, but 1 no experiments were conducted to determine the point. i 3 Further experiments were conducted to determine the effects of dilutions of the toxic filtration. A series of dilutions ranging from i to 5 vere made, using sterile Synthetic solution. .If-I- '- F‘, .1} .J Test of filtrate from old culture TAdLn 6. of Lacrosnorium Cucumerinum vhich had partially lost its virulence; after 48 and 60 hours. 311.15%? 1 021. : iacresnorium cuCumerinum filtrate Leaf : Treatmen : 48 hours : 60 hours L z a l : wounded : discolored around sound : no Change 2 : wounded : discolored around mound : no change 3 : nounded : discolored arouni wound : no change 4 : woundel : discolored around vound : no change 5 : not vounded : no change : siizhtly discolored 5 : not wounded : no change : slightly discolored 7 : not vounded : no Change : no Change 8 : not vounded : no change : no hangs inactive filtrate plus ammonia to brinz mirui7.4. _ .~__ 1 : woundei : camplataly discolored : no change 2 : vouniel : completely liscolore : no change 3 : wounded : completely discolored : no change 4 : wounded : completely discolored : no change 5 : not Voundei : slightly discolored : darker 6 : not rounded : slightly discolored : darker 7 : not wounded : light brown : darxer 8 : not wounded : light brown : darner -27- . . _ es oxlc trvf}, -r boii’z- fur L minutes; on cabbage and must- : : Ascrosporium cucumerinum filtrate : Treatrent : 48 hours : 60 hours 1 : wounded : light around wound : no change 2 : wounded : lijht around wound : no chaige 5 : not vounded : no change : no change 4 : not ”cunded : no charge : no change Cabbage 48 hours j 60 hours 1 : wounded : light brovn around vound: no change 2 : wounded : light brovn around Wound: no change 3 : not wounded : no change : no change 4 : not rounded : no change : no ~hange Alternaria brassicae filtrate 48 hours : 60 hours 1 : wounded : light around vound : no Cha‘ge 2 : wounded : light around wound : no change 3 : not vounded : no change : no change 4 : not wounded : no change : no change ihnflcu:lon 46 hsurs 1 60 hours 1 : wounded : discolored around Hound : no change 2 : wounded : discolored around wound : no change 3 : not vounded : no change : no change 4 not wounded : no change : no ~hange -23- As Table 8 shows, the toxic substance is not active in higher dilu- tions, although the lower dilutions produce the spot almost as quickly as the undiluted or normal toxic substances. The experiment was repeated with the same results. During the inoculation experiments, cucumbers and one strain of muskmelons (1035) were found to be resistant to this toxic substance. The next experiment was to determine if these plants that were resistant to the organisms were also resistant to their toxic substances. The active toxic substance was placed on the leaf in the same manner as before. (See Table 9.) From this table it is seen that both the muskmelon and cucumber are resistant to the toxic substances from Kacrosporium cucumerinum and Alternaria brassicae. From these tests it is evident that the organisms causing musk— melon leaf spot and cabbage leaf spot produce toxic substances, and these are responsible for the diseased Spots which characterize these diseases. The cabbage fungus will spot the muskmelon leaf after wounding, but the organism is not capable of attacking the leaf alone. The toxic sub- stance from either Alternaria brassicae or Hacrosporium cucumerinum will Spot either the cabbage or muskmelon leaves in the same manner, but it will not spot leaves of other plants that are resistant to the above organisms. The resistance of the plant to the toxic effects of the filtrate from culture seems correlated with infection. The toxic substance is a thermolable compound and while activated by ammonia, is not dependent on the ammonia for its effect. -29- TABLE 8 . Effect of dilution upon the toxicity of the filtrates from cultures of Xacrosporium cucumerinum, grown in Coons' Synthetic medium. Nuskmelon :Xacrosporium cucum. filtrate Leaf : Treatment : Dilutions : 48 hours : 60 hours 1 : wounded : 1:1 : dark brown : no change 2 : not wounded : 1:1 :(slightly : darker (discolored 5 : wounded : 1:2 : discolored : darker 4 : not wounded : 1:2 : light yellow: darker 5 : wounded : 1:3 :(sliahtly : light brown (discolored 6 : not wounded : 1:3 : no change : slight change 7 : wounded : 1:4 : no change : brown around wound 8 : not wounded : 1:4 : no change : no change 9 : wounded : 1:5 : no change : no change 10 : not wounded : 1:5 : no change : no change - 50- Tests of filtrates from toxic broth on muskmelon (1025) and cucumber leaves. } Euskmelon Macrosporium cucumerinum filtrate Leaf : Treatment : : 48 hours : 60 hours 1 : wounded : slightly discolored: n 0 change 2 : wounded : slightly discolored: no change 3 : not wounded : no change : no change 4 : not wounded : no change : no change Alternaria brassicae filtrate f" 5 : wounded : discolored around : no change f wound 6 : wounded : discolored around : no change wound 7 : not wounded : no change : no change 8 : not wounded : no change : no change Cucumber Xacr03)orium cucumerinum filtrate l : wounded : discolored artund : no change wound 2 : wounded : discolored around : no change wound 3 : not wounded : no change : no change 4 : not wounded : no change : no change Alternaria brassicae filtrate 5 : wounded : light around wound : no change 6 : wounded : light around wound : no change 7 : not wounded : no change : no change 8 : not wounded : no change : no change -51- TEMBERATURE RELATILKS. To determine the temperature at which the two organisms grow best, a series of temperature studies was made by placing four test tubes of cornmeal agar, containing two cultures of Hacrosporium cucumerinum, and two cultures of Alternaria brassicae in a differential thermostat. This experiment was run for 5 days, after which the results were taken. (See Table 10.) It was repeated with similar results, using organisms from different sources. There is some difference between macrosporium cucumerinum and Alternaria brassicae, in that the former is inhibited at 4° C, while the latter grows slightly. Both organisms failed to grow at 350 c. and did not revive when removed to room temperature. The death point of these fungi is between 28° C. and 35° C. The Optimum temperature for growth and spore production lies between 20° C. and 26° c. A series of germination tests were made in which a heavy suspen- sion of spores was tested in hanging drop cultures. They were placed in a thermostat for 48 hours, In table 11 it is seen that the optimum temperature for germination is the same as for growth — between 19° C. and 26° C. No germination took place between 2° C. and 4° C. The experiment to determine the thermal death point of the spores was run as fOllOW‘: Six water baths were kept constant within one degree of the following temperatures: 35, 40, 45, 50, 55 and 60° C. One c.c. of a heavy spore suspension was placed in a test tube and the tube was inserted into the water bath. Thermometers were placed in the test tubes. The period of exposure was 10 minutes; then a hanging drOp was made -32— Growth of Eacrosoorium cucumerinum and Alternaria brassicae at different temperatures. Organi sm : Temgerature : irowt h : Spo res 1.1. Jucumerinum : 4°C. - 5°C. : - * : - A. brassicae : " ” : + : - 1.1. v'ucumerinum : ll°LJ.- 1;: “C. : i. : — -i. brassicae : " " z + : i 1:1. o‘ucumerinum : 15°C.- 17%. . + : + A. brassicae : " " : ++ : +4. 2.1. Cucuzner’lnum : 19°L3.- 20°C. : ++ : ++ a. orass‘lcae : " " : +++ : +4.... 2.2. Cucumerinum : 21°C.— :32 °~J. : +++ = +++ ll. brassicae : " " : ++++ : ++++ 2.1. Gucumerinum : 132°C.— 24 “-3. : ++++ : ++++ A. brassicae : " " : ++++ : +4.4...- I.’.. I‘lucumerinum : 23°C.- 26".). : ++++ = +4.4...- A. brassicae : " " : +++ : +4.... LC. Cucumerinum : .lb G.- :38 C. : +++ = +++ A. brassicae : " " : ++ : ++ LI. ‘Jucumerinum : 35°C.- .59 CC. : " ** : - A. brassicae : " " : - : - * Revived at room temperature. ** Failed to revive at room temperature. - 'r' no growth 'H’ "‘ Average growth +|+ '3 Poor grovth ++++ 'Abundant growth TABLE 11. Germination of spores of macrosporium cucumerinum at different temperatures. One hundred Spores were counted in each case. Record made at the close of 48 hours. Temperature percentage_germination 2° c. - 4° c. 0 6° 0. - 8° c. 24% 10° C. -15° ‘. 72% 17° 0. -18° 0. 86% 19° c. -19° c. 94% 22° 0. -°2° 0. 96% 24° 0. -24° 0. 95% 26° 0. -27° 0. 94% 33° 0. —34° c. 2% TABLE 12. Thermal death point of the spores of Macrosporium cucumerinum, 10 minute exposure. Temperature percentage germination 550 0. 97% 400 0. 2372’, 450 c. 76% 500 c. 52% . 550 0. p 0 60° 0. 0 -34— of the suspension and placed at room temperature for 48 hours. The re- sults were taken by counting 100 spores. From the results shown in Table 12, it is noted that the death point lies between 50 and 550 C. for ten—minute exposures. This seems somewhat low considering the thick-walled spores, but they were taken from culture and not from the host, which might have something to do with the low thermal death point. VIABlLITY OF 3£ORES. While working with old herbarium specimens hanging drop slides were made of the spores and mycelium. Quite a number of them germinated. The first examination was made of a muskmelon* leaf collected in Massachu- setts in 1898. The technique was as follows: The specimen sheet was ta- ken into a closed culture room and Opened, a sterile scalpel being used to remove the pieces of diseased material. The experiments were of two kinds: first, spores were mounted in sterile water and a hanging drop was made. Second, small pieces of the tissue were placed in culture dishes on cornmeal agar. is a check, 5 petri dishes of cornmeal agar were left Open in the culture room during the setting up of the tests. Fifty-six per cent of the spores germinated in the hanging drop. All of the pieces of leaves showed an abundance of mycelium coming from their edges in 60 hours. A pure culture'of this organism was made and inoculation experiments were tried. No infections appeared. Other specimens showing germination were specimens collected in Alabama*' in 1889, and Louisiana“‘ in 1888. This was the Alternaria * Herbarium specimen NO. 515 - Collected by R. E. Smith. ** Herbarium specimen No.2045 - Collected by C. L. Newman. *** Herbarium specimen No.2483 - Collected by A. G. Langlois. -55- brassicae on cabbage. The spores from the leaf collected in 1889 showed 42 per cent germination; the one collected in 1888 showed 62 percent ger- mination. There was no growth on any of the check plates except for a few colonies of bacteria. This experiment was repeated several times with the same results. Not all of the cells of the Spores germinated; sometimes only one germ tube was sent from a spore. These organisms were also ob- tained in pure culture and inoculation experiments were made on cabbage. The culture from Alabama showed 22 per cent infection, and the one from Louisiana showed 14 per cent infection after the leaf was wounded. No in- fection was secured on muskmelon. Specimens from which spores did not germinate were cabbage* col- lected in Hisconsin in 1897 and caboage** collected in Italy in 1889. These specimens might have been poisoned to prevent insect injury before they were put in the herbarium. Brisley (3) found that Kacrosporium cucumerinum did not live over winter in the spore stage in California. Specimens of the cabbage and muskmelon leaves were collected by the writer in the fall of 1923 and placed out of dtnrs during the winter. They were examined the first of May, l924. Over 90 per cent of the spores had germinated. The fungus certainly lives over winter in the spore stage under Michigan conditions. ATTEEPTS TO FIND THE PERFECT STAGE OF THE FUKGUS. Kirby (10) found that monosporous cultures of Qphiobolus cariceti are of two distinct sexual types and when mixed in culture result in the formation of typical perithecia. He mixed two cultures of this organism * Herbarium specimen No. 3596 - Coll. by E. F. Smith. ** Herbarium specimen No. 87 - Coll. by Briosi e Cavara. \ III tall. . -36- and grew them on wheat agar and melilotus stems. The perfect stage re- sulted in two weeks. Leonian (12) was able to control pycnidial and porithecial form- ation in Valsa leucostoma by the audition of maltose and malt extract to nutrient solutions. .Some experiments were carried out with temperature and light con— ditions varying. Some cultures were kept in the ice box, while others were kept in the incubator. Other cultures were placed in the window, in the dark room and in continual electric light. These cultures were observed carefully, but showed no perithecia. Melilotus and muskmelon stems were next placed in large test tubes, distilled water was added and they were inoculated with single and mixed strains of Macrosporium cucumerinum and Alternaria brassicae. They grew readily but no perfect stage was produced. In the last attempts, cornmeal agar, prune agar and maltose agar were inoculated with different strains of the above organisms. The strains mixed readily on the plates, but no perfect stages were found. Old leaves that were collected the previous fall and left out all winter were covered with conidia but no perithecia were found. CAUSE ‘F ZOHATIOK IN CULTURES. On most laboratory media typical zonation occurs. Hedgcock (8) WDIflcing with such fungi as mucor, penicillium, etc., found that temperature is that the cause of zonation. He also found that zonation did not occur hi tcrtal darkness, but only in cultures grown in partial light. He then cormhacted an experiment to determine what colors of light affected zonation. He grwmn the organisms in orange, red, blue and ordinary lights. He also grew the cultures in a black-lined box. He discovered that the blue light affected zonation. He also discovered that the blue light inhibited spore production. In the writer's work two experiments were conducted with gacrosoorium cucumerinum and Alternaria brassicae. On the first one, two srall light wooden boxes were usedf One was lined with black paper and the petri dish cultures were placed inside. Cultures were also placed in the other box and a thick glass was placed across the lid. The two boxes were set side by side on a table in the laboratory. The temperature was approximately the same in both boxes. After ten days the boxes were opened and the cul— tures were examined. The cultures in the light showed typical zonation, while those in the dark did not show so great zonation as those in the light, but some zonation was present. (See Plate No. 8) The second experiment was conducted in the same manner by placing the boxes in the dark room and using electric light instead of sunlight. The light was left on 48 hours and off 48 hours. The line of growth was marked with a blue pencil each day. Results in ten days indicated that the cultures in the electric light showed some zonation, while those in the dark showed no zonation. This would indicate that heat and light are both factors in zonation. The greatest growth.and spore production took place 111 the presence of light. No attempt was made to find the kind of light that was best for this growth. DISSEiIIh‘QiOII BY '.'.'II.’D. Since this disease is very common in some of the western States where the rainfall is very light, it seems possible that the spores are * Ldicrbscope boxes were used for this test. -38- are disseminated by wind. An experiment was conducted to find the dis— tance that the spores may be blown. An electric fan was used in this ex- periment a the speed of the air current produced was measured with an anemometer. The experiment was carried out in a large laboratory, the doors and windows being closed to prevent draughts. The floor was sprinkled to avoid dust. The fan was placed on the floor with the blades 3 inches above the floor so that the current would easily hit the culture dish. A culture of Hacrosporium cucumerinum was placed 3 feet in front of the fan. Four petri dishes containing cornmeal agar and one muskmelon plant were placed every 3 feet for a distance of 21 feet. Petri dishes containing cornmeal agar were placed around the room as checks. The speed of the wind at the Culture dish was 11,040 feet per hour. The speed of the last petri dish, 24.feet, was 10,884 feet per heur. 1310308903 [1qu CU CLTERII‘TL 1'.- Dishes Colonies Plants infected l 19 - 2 l4 - 3 ll - 4 6 — 5 2 _ 6 2 — 7 — _. a - mw- )Q'Ir at) I ‘2’, vac? - ‘ :iLaJtlunilt. I'vuiu‘dICx‘lh l 16 — 2 21 l 3 15 - 4 10 - 5 B - 6 5 — 7 2 — -39.. Although the spores are not so numerous on leaves as they are on petri dishes, they are borne on t0p of the leaf and are easily blown. On- ly one plant in the two series developed the disease, although the plants, after the exposure, were placed in moist chambers in conditions favorable for infection. What few spores fell on the plants probably blew off. Only two of the check plates showed any colonies of Alternaria and they were near the path of the air current, one colony being found in each plate. The one positive result is significant, and the results with the plates show beyond question that even a light wind can carry these Spores a con- siderable distance. Wind dispersal of spores is certainly a factor in this disease. DISSEIiKnTlON BY SPLASNIK" OF VATER. Several cases of spread of the disease from plant to plant were noticed in the greenhouse during the writer's work, and these were thought to have been caused from improper watering. The plants were sometimes watered with the hose in a manner to cause splashing from leaf to leaf. Brandes (2) working with anthracnose of lettuce found that when one plant in the bed became infected, the disease spread rapidly over the entire bed when hose watering was used. He then planned an experiment by inoc- ‘ulating one row of plants and spattering them.with.water. Almost all of the plants were infected within a week, while those watered by irriga- tion remained healthy. An experiment was planned similar to his. Plants were grown in jpots and placed in moist chambers. As had already been found, Alternaria wouflxi only infect muskmelons by severe wounding. muskmelons and cabbage iwere pdaced in the same chambers. (See following diagrams.) One chamber —40- Plot I. Watered with hose. Cabbage 0 $ 0 O 9 0 0 9 G 0 0 9 O 6 9 O (D 6 O 0 9 0 9 63 muskmelons 9 0 0 0 9 0 O 0 O 0 9 0 O 0 6 9 9 0 0 6 O G) 0 9 0 :- Inoculated. 0 a Became infected O I: Remained healthy -41- Plot II. '.‘.’atered EL Sign-irritation. _____ . ” Cabbage O 6 O O O O O 0 9 O O Q 0 O O 9 O 0 O 0 O 0 0 O 0 Q 0 O Muskmelons O 0 O 0 9 O 0 0 0 0 O ‘0 O 0 0 0 0 Q 0 O O O O 0 O O O O 0 == Inoculated 0 = Became infected 0 == Renamed healthy -42- was watered with the hose (See Plot I), while the other (Plot II) was watered by irrigation. In the plot that was watered with the hose, 19 of the remaining 24 caboages became infected in 21 days. Sixteen of the remaining musk- melons were infected in the same time. In the plot watered by irrigation, 6 of the remaining 24 cabbages became infected, while only 4 of the muskmelons were infected in the same time. The disease had spread farther on the plants that had been inoculated. Bearing these two dissemination experiments in mind, it is easy to see how the fungus is disseminated. Wind and rain are probably the two main agents involved. In the tests reported wherever a drop of water remained for any length of time, on the cabbage especially, infection soon took place. The dew on plants is sufficient to allow infection to take place as germination usually occurs in 12 to 24 hours. CONTROL. The control measure with this disease as with similar leaf spot diseases of plants will fall under three types. The first method is by the use of resistant varieties. As has been mentioned previously, Rocky Fbrd 1025 is practically resistant and is recommended for the growers of Michigan. The only objection to this melon is its lateness. The second method is that of sanitation. The organism lives over the winter on the host, and the spores are viable for a long time. There- fore rotation would not be so profitable as it might be if the organism were easily killed, DUt it doubtless is very profitable. The third method is by the use of protective sprays. The writer -43- was unable to conduct any experiments in the field and the sprays were tested on slides, using the method proposed by Hesler (18), Blodgett and Wallace. The technique of this experiment was as follows: Ordinary slides were divided into two parts, transversely, with a wax pencil mark. One part was used as a check, and the fungicide was placed on the other. The check was covered with pasteboard, while the other side was sprayed with an atomizer. The slides were allowed to dry, then dusted with spores, and finally placed in the ice box for 30 minutes. Bell jars were saturated with moisture by spraying with an atomizer and then placing on a plate covered with moist filter paper. The slides were taken out of the box and placed under the Bell jars. Due to the coldness of the slide, a thin film of water was deposited on the slide, which made it comparable to a plant-leaf wet with dew. In this manner the spray mixture was not di- luted as much as it is when the spores are put in a drop of water. The sprays were tested with both Racrosporium cucumerinum and Alterraria brassicae. Two hundred spores were counted in each case. Table 13 shows that the fungicides did not completely prevent germination of the two fungi. However, Bordeaux mixture, lime sulphur enui soda Bordeaux decreased the germination of the spores markedly and any of’the three doubtless will prove efficient in reducing the amount of the disease, in comparison with unsprayed plants. All of the sprays used were sprayed on both old and young rmiskmelons to determine if any of them had any injurious effects on the foliage. There was no evidence of harming in any case. 13. TABLE Effect of fungicides on the fungi. : : Germination of spores Fungicide : Fungus : z :: Check : Treated Bordeaux mixture :racroSporiun; 94$ : 11% 4—4-50 :Alternaria : 88% z 8% Soda Bordeaux :Hacrosporium: 90$ : 9% 4-lgu50 :Alternaria : 86¢ : 7% Commercial Lime Sulphur :hacrosporium: 93$ : 5% 1-40 :Alternaria : 89% : 8% Sublimed Sulphur :Hacrosporium: as» : 10% . ¢ . ,,.‘7 (used as a dust) :Alternaria : 8’” : 14p Colloidal Sulphur‘ :hacrosporium: 96? : 14% 1-100 :Alternaria : as? : 12% 1-200 :Kacrosporium: 91$ : 20$ :Alternaria : 78% : 17% ~ . . . ,. ,7 l-4v0 :macrosporium: 94$ : 31p 0 ' "’ :Alternaria : 86$ : 263 1-600 :Eacrosporium: 88$ : 393 . . , .1 :Alternaria : 81% : 63p ‘ Prepared by Mr. L. E. Tisdale of the M . Bot. Garden. -45- SUXEARY. The leaf Spot disease of muskmelons, caused by Hacrosporium cucumerinum is widely distributed, out has oeen studied very little. It is one of the most important diseases of muskmeloas in Michigan. The fungus attacks only the leaves of the plant. The description of the fungus, as given by Ellis and Everhart, agrees very closely with the characters of the fungus studied by the writer and the two are undoubtedly identical. The fungus was isolated from several different sources during the series of experiments. The fungus grows well on a large variety of laboratory media. The fungus grows anaerobically, but few Spores are produced. The organism changes acid medium to a slightly alkaline condi- tion with the production of ammonia. The organism is believed to enter the host by sending the germ tubes through the epidermis. Macrosporium cucumerinum will attack other cucurbits, out will not attack cabbage. Alternaria brassicae will attack cruclfers out not cucuroits without wounding. Rocky Ford 1025 is resistant to both KacrOSporium cucumerinum and éiternaria brassicae. Macrospgrium cucumerinum partially loses its virulence in old cultures, but no Cultures of Alternaria brassicae lost their virulence. Both Kacrosporium cucumerinum and Alternaria brassicae produce a toxic substance that will discolor the leaf. This toxic substance is interchangeable on cabbage and muskmelons, but will not produce the spot on other hosts or on resistant varieties of muskmelons. Ammonia has something to do with the spot, out will not produce the spot alone. The Optimum temoerature for growth is between 19° C and 26° C. The thermal death point of the spores is between 500 C. and 550 C. for 10 minutes. The sgores retain their viability for a long tine, germination being obtained from herbarium specimens 35 years old. Attempts to find the perfect stave failed. Zonation in cultures is caused by temperature and light. The greatest growth and spore production takes place in the presence of light. The fungus is disseminated oy vind and rain. The spores live over vinter on the old leaves. The effects of various standard spray mixtures, as well as sul- phur dust and colioidal sulphur were determined. (1) Blinn, Philo, K. a Bust Lesisting dantaloupe 00100 ‘0'. JJ'Lpto Ctao 13:11. 104. 1905 -53 A [‘3 brandes, s. n. anthraenose of Lettuce Caused by marssonia :anattahiana. Journal of Apr. nesearch 13:Lel-hbo. 1918 (3) brisley, narold noy studies on the blight of churbits caused by “acrosnoninm cucumerinum. rnylepatnology 15:139-L04. 1943. (4) Chester, r Diseases of Centaloupes. Delavare anpt. station neport. 13:36-41. 19d1. (5) desary, A Uber Einige Sclerotien uni Sclerotian hrgnkyeiten. dot. Zeit 44:377-587, 533-404, 409-4L6, 435-441, 449-461, 4e5-474 1886. (6) Elliott, John, A Taxonomic Characters of the Genera, alternana, and necrosporium. American uournal of octany 4:4;3-476. 1917. (7) Allis, e. a. and averhart, so 4° £27 species of rungi from VariOus Localities. A“? free. iced. Lat. sci. of fhi1.. 4ie-414. 139V. (8) heivceck, deerge drant Zonation in Artificial Cultures of Cephalotneaium and other fungi. neport of the dissouri satanical larden 17:119-117. 1306. (9) (ones, L. E. fectinase, the Cytolytic ensym proiucei by bacillus Caratovorus, i certain other soft-rot organisms. i... Y. AW'I‘. -4.pto Sta. Dill. 11::-31-3080 1909. (10) derby, a. S. Heterethallism in oohiob’ol s dariceti. rhytOpathology 15:55. lane. (11) Krakever, L. J. The Leaf Spot Disease of had Clover caused by Macrosborium sarcinae- forme. deport of nichigan Acaiery'of science 19:274-3b5. 1917. (12) Leonian, L. H. Physiology Of Benthecial ani fycniiial fuffiatiun iE_T;.: PhytOpathOIOgy lE;Z57-L7z. 1955, (13) (14) (15) (16) (17) (18) (19) nakemson, U. K. Tie Leaf mold of fomatoes caused by Cladosporium fulvum. Report Mich. Academy or science 20:311-348. 1918. munn, a. T. be k-not Disease of onions 1:. Y. 1333‘. $9.9. Sta. 91.11. 457:504'4550 1917. Beglion, littoria Una nuona malittia del melons cagionata dall Alternana brassicae f. nigrescens. hivista di patalogic vegetale 1:Z9b-a9e. 13,2 Rosenbaum, J. etudies vith necrOSQOrium from fomatoes ‘\ I Bhytonatholowy 10:9-4 . 1920. Selby, it. D. Hiccus~s of Qantaloupes in bhio. UhiO ag. s p. Lta. nul. 73. 1336. Smith, Arvin, F. Diseases of nus melons in nichigan. Journal of flycology 7:373. 1892. “allace, sledgett and desler . Studies of the runfiiCidial values 0f lime-sulphur Pr993r3t10n5° Cornell Univ. dul. 230. 1911. -\ Plate 1. Spores of Alternaria brassicae_and Eacrosporium cucumerinum A. Cabbage leaf (1889) Funghi parassiti delle piante cultivarte 0d utili #87 B. Cabbage leaf - College, Segtember, 1925. C. Pure culture. D. North American fungi #3596 on leaves cabbage(18d7). Coll. by E. F. Smith at Racine, Wis. '. muskmelon, Brisley, California. (1924) F. Pure culture - college - muskmelon leaf (1923) G. Kusmneion leaf - College (1925) PLATE II Macrosgorium cucumerinum on muskmelon leaf. (Eight days after inoculation) PLATE III gafik Alternaria brassicae on cabbage leaf. (Eight days after inoculation) PLATE IV Alternaria brassicae on muskmelon. Leaf wounded. (Twelve days after inoculation) PLATE V A. B. A. Macrosporium cucumerinum B. Alternaria brassicae Comparison of macrosporium cucumerinum and alternaria brassicae on gourd leaves. (Ten days after inocalation) PLATE VI .-- - . ' 4- ;_ . ‘, .‘L... iv“ p———~———— -«-«)sb- e 0* w..- ,.. do BI A. Macrosporium cucumerinum B. Alternaria brassicae Toxic substances from macrosoorium cucumerinum and Alternaria brassicae on muskmelon leaves. (After sixty hours) PLATE VII '1 K *,~\73 A.BRASS|OAE M.CUCUMER)NUM Alternaria brassicae and Hacrosoarium cucumerinum on Coons' Synthetic Solution. (After thirty days' growth) PLATE VIII Comparison of nacrosoorium cucumerinum grown in darkness, and sunlight. A. Darkness B. Sunlight PLATE IX A. Darkness B. Light Comparison of Macrospgrium Cucumerinum grown in darkness and electric light ROQM USE ’0‘?"th ”'°)))))I))))))Milli) LIBRARIES 00