fllfliifiiilflflilWU]iiiflfl’flflflifliliWfifill V fi3 1293 01535 2275 PLACE ll RETURN BOX to remove this checkout from your record. TO AVOID FINES return on or before date due. DATE DUE DATE DUE DATE DUE MSU le An Affirmative MONEquel Opportunlty Institution W ; A MACRTSPORIUH LEAF SPDT DISEASE OF RED CLO 53. (mezzo smmmz QARCIIUxEFORIE CAV.) ******>¢~******* Thesis fiar the degree of’master of Science. lichigan Agricultural College Department of Botany ‘9 f} f . l”., L. J; I . k [Krakover Dec. 1915. .THELSI'S TABLE OF CO RTE HTS Page IntrOduCtion 0...000.......0..0.0...000000.00.0.0..000 1 The red Clover crop .0000...00..00.0..0.0000.000...00. 2 Clover diseases in general ........................... 5 Field Observations ....00..0.00000-...00000000000.000. 8 Historical .00.....00000...000.0....000I0.00......0000010 Economic importance .................................. 12 Distribution 000.000.00.000. 0.0.00.000000000000.0.000 12 EtiOlogy .0000....0..00.0.000..000.000.000.0000.00000. 15 Infection experiments ................................ 21 Inoculation upon other hosts ......................... 26 Symptoms of the disease .............................. 27 The morbid hOSt ....0.0.0..00.0.0.00000000000....0.0.0 29 Infection phenomena .................................. 32 Germination Studies 000....00....000.000.0000.00.00... 34 cultural Studies 0.0.0000.000....0...0.00..0.0.0.00.0. 38 IrIet3b0113m .000000.0.000.000.000000....0.0000 0.000000 48 Temperature relations ................................ 52 Burnidity relations 0.000..0.00.00.......0000000...0.00 57 Light relations 00000000000. 0000000000000.00.00000.00 62 Exper. dealing with the toxic nature of the fungus ... 65 methods of dissimination ............................. 78 contml 000000....0...00000..000.0..0.00....00000000O0 88 Summry 00..00...0000.000000.0.00.0000.000...0000.000. 90 Idea-1a form1ae .0.0.0.0.00..00.0.00.0.....0....00.00.. 94 Bibliography 0000.000...0000.00.00.0.0..0...000.000..0 96 Explanation of plates ................................ 98 -1- INTRODUCTION The diseases of red clover are so common that little attention seems to be given to them. Rarely is a specimen sent in to tne Botanical Dept. ior identification. These diseases h are widespread wherever clover is grown, and tho the lose they cause has not been definitely determined, it appears to be large The culture of red clover is rapidly being disconthnued because o1 Iungus diseases and other troubles, in favor of other legumes. The general abandonment of a crop oi such agricultural importance has given rise to apprehension. The questions to be settled are (l) whether red clover with its wide agricultural utility can be proiitably replaced by other legumes,- alfalfa for example; (¢)wnetner the troubles which are making the crop unproiitable can oe remedied. The very important hindrance to the culture . or red clover known as “clover sickness” may eventually be e- liminated by proper soil management. The fungus diseases of the crop, in order to be controllea,at once open up a big field of en- deavor. The possibility ox oreeoing resistant strains, the in- troouction and the testing of foreign strains which in themselves might oe resistant, the study of the methods by means of whicn the diseases are disseminated, and the modification of cultural meth- ods, suggest themselves when considering the problem of control. This paper deals with a leaf spot disease on red clo- ver, caused bngggrgsporium sarcinaeforme Cav. This disease was very serious in the vicinity of East lensing, Mich. during the entire growing season of l9l5gprhe various phases which enter into tne study of a plant disease will be taken up more or less in their usual order. -2... THE RED CLOVER CROP The importance of the red clover crop is too well known to require an extended discussion here. The following brie! summary is given because of its importance, distribution and commoner cultural methods are given because of their beer- ing on the disease and methods of control: Importance and distribution:- Red clover is by far the most important leguminous crop grown in America. The area devoted to it, according to Piper*, is about five times that devoted to alfalfa. The total acreage in 1909 reached almost 12,000,000. The greater part of this is grown in the northern states west of the great plains and on the Pacific coast, but it is of little importance in southern and semi- arid states. It is essentially a crop for humid regions without excessive summer or winter temperatures. Dry atmos- pheric conditions are very unfavorable as is high tempera- ture combined with humidity. Utility and culture:- Red clover is really a short lived perennial but as a crop is treated as biennial. It has a very high agricultural adaptability: as a forage crop, hay, pasture, silage, green feed, and as a cover crop for orchards; as a nurse crop it is especially valuable When sown with wheat, barley rye, or cats, and it also does well with corn or a root crop. If sown alone, a good stand may often be secured but this is seldom done unless all other methods prove a failure. *. Piper, p. 362. -3- If sown in a mixture with timothy or other grasses a good hay crop may be secured. Being a biennial it is adapted to a short rotation. A five-year rotation of corn, oats, wheat, clover, and timothy is commonest in the central states. Corn, clever, or corn, corn, clover are the simplest rotations. but the clever is repeated too often to give the best results. Sowing is best done early in the fall in order to avoid excessive winter-killing, or in the spring by broad casting unathe fall-sown grain. The seed should be planted shallow,- l - 1-1/2 in. deep,- in order to get the best results. The first cutting yields a heavy hay crop, while the second is usually allowed to mature for seed. ”If cut for hay, different authorities recommend cutting in full bloom, in young bloom, and when the heads are half brown. The contents of the digesti- ble nutrients is greatest in full bloom. Later cuttings, how» ever, cure more easily than the earlier ones, and it is possible that the better curing counterbalances largely the lower con- tent of nutrients."* If used for seed the clover should be cut . when all of the heads have turned brown and the seed is firm and shiny. The commonest method of harvesting is by mowing and raking into windows. Under favorable conditions the clover is ready to store within about four days. if out in the late dough stage. It is then piled into cocks or best kept under cover, so during wet weather there is danger of the seeds sprouting if kept continuously damp. * Piper, p. 380. -4- Clever diseases in general: The most common diseases 01 clover are those which attack the Ioliage. Since the leaves constitute the most valuable part of the hay, the defoliation which results from these disease may cause considerable less. Perhaps the most widespread of all diseases on plants of the genus TriIolium , is the rust caused by species “f.EEEEZE££' Uremyces is cosmopolitan, and the most susceptible hosts are important forage plants. Red clover, (T. pretense) alsike clover, (T. hybridum) white clover, (T. repens) and crimson clear ver, (T. incarnatum) are the most commonly infected. Theneis no exact knowledge of the relation of the disease to climatic conditions. It varies with the season, and is to a consider- aoie excent determined by spring conditions. Stevens and Ha11* state that it is most injurious to the second crop, to whicn it may cause a damage of 20-50% if the weather is cool and damp. A very common leaf spot is caused by Pseudopeziza tri- ‘£3;33(Pers.) Fckl,, and is at times the cause of much damage. This fungus is closely related to Pseudspeziza medicgginis(Lib) Sacc., the leaf spot of alfalfa., with which it may be identical. Tne sooty spot caused by Polythrincium trifslii Kze. (Phyiia- cncra trifolii(Pers.) Fckl.) is distributed upon species e: clove er in many parts of the world, especially on red clover. Anoth- er ieaf spot which is widely disseminated,'but seems to have attracted little attention, is that caused by the black mold, Macrqsysréumngazginasquss Cav., the disease to bew discussed in this paper. The most serious disease of the clover stem and petiole is the anthraonose caused by Cllglcevtotrichum tr'ifslii Bain. ‘vwvvrmvvv— vfi— vv 7 * Bioiiography given on page rf 91907 VI Illlllltxitlrt. [[11 -5- Rain and hssary,(l908) in investigating the cause of the failure 01 tne red clover crop in Tennessee found this to be the enter agent. It is found mostly on the stems near the ground or Just below the flower cluster., and but rarely on the leaves. The elongated, sunken spots which are formed, result, eventually, in the death of the plant. The fungus also attacks alfalfa, but alsike clov*er seems to be nearly immune. Thus far the disease has already been found in Tenn., Ohio,‘w. Va., Ky., Del., Ark.. Va., and My. Another anthracnose caused by Gloeosgqgium CEELEL, 12229 is said to cover nearly all of Europe where red clover is 3 grown. Plant grown from European and American seeddsuffer alike. The brown to black sunken spots on the stems and petioles cause the death of the more distal parts of the plant. The disease is said to be carried by the seed and wet weather is famorable to the spead. Seed tratment with one percent Bordeaux has been re- commended as a control. The Gloeospcrium may be identical with the Colletotrichum above mentioned, while both may eventually connected with Morals clgglgjjg (Stonem) S&: v . s . The stem rot of clover, a decay near the base of the s stool, resulting from an attack of Sclerotinigfitryfgliormipriks., has several times been reported as epidemic in the United States. It is not widely distributed, nor is occurence so frequent that it can be considered a serious disease. The fungus is occasional ly destructive to various species of clover in EurOpe. and has been reported upon clover and alfalfa in Michigan and Ohio. The damping off fungus, Pythium_de‘paryanum Hesse, v—v '— v—vv and Peroncspora trifoliorum de Bary have been reported on clover but are not ofmuch importance. h -6- Clover diseases in E. Lansing Mich. during 1215:The most serious disease of red clover was that caused by Macrospgrig um sagginaefsrme Cav. The next in importance was rust, Urogyces fallens Desm., which caused considerable damage during the late summer and early fall months, Pseudopeziza mrifolii first became common in August, but did little damage. The sooty spot,‘gg;y: thrincium trifolli, was quite common during tne rainy periods, upon many flan: species of Trifolium, especially on red clover (y: pretense) and white clover (T. repens) but not persistent. In one instance, a second crop was noticed which was practis- ally iree irom the disease, altho the first crop was badly infect so. This was perhaps due to the decreased rainfall. Anthrac— nose,stan rot, and the other clover diseases were notiound in this Vicinity. Hacrosporium diseases of other plants: This genus con- ttrimh-es toward one diiseases or several economic plantsoi’ im- portance. One of the most important is the early blight of potao toeanoeo by !4_gglggi E. a M. This disease is common tnruout the United States, Canadh, Europe, Asia, and Australia. Jones (1899), has conclusively proven the parisitism of the fungus, this depending upon sufficient moisture to allow germination and vigorous growth. The fungus occurs also upon tomato and Jimson weed (Datura stramonium). The control has been found possible by means of spraying. _ M; sarcinujILa ,Berk. var. Earisiwticum Thum, commonly assOciateu with Peronospora Schlaideniagg, e Iary on onion, is 01 secondary importance. It may be a wound parasite, while in other cases it may be the direct cause of a spot involving the seed stalks. In such cases it is very injurious, since ‘55“ -7- seed stalks affected seldom mature their product. Several other species have been reported upon onion. M. brassigge Berk. is the cause of a leaf spot on cab- bage, and is sometimes very destructive. M. cucumerinum is the cause of th4e so-called leaf blig blight or rust of the muskmellon. It attacks the older leaves and destroys the foliage of the central part of the hill. In Colorado, Georgia, and Delaware the disease has been very serious but is controlable by spraying with Bordeaux. Other'Macrosporium diseases which are more or less com- mon, but have not yet become of economic importance, are M. nig- ricanthum Atkinson on cotton, M. tabacigum Ell. & Ev. on to- bacco, and M. iriidis C. & E. on Iris. Anumber of the foregoing species exhibit a catenulate arrangement of the spores when grown in culture. and are, there- fore, sometimes referred to the genus Alternarig; -8- Field observagions: During the spring months a close watch was kept on all clover growing on, and in the vicinity of, the M.A.C. Campus. Since fungi of all kinds were unusually abun- dant, it was expected that the Macrosporium disease would appear. Nothing was found, however, until June 12, when it was observed in a large field of red clover, situated on the north side of the railroad spur which runs by the collegee forestry nursery. The plants were 32-45 cm. high, with a few beginning to bloom. only the lower leaves were infected, i.e. those leaves close to the ground and shaded by the upper leaves. When the plants were look ed at from.above, no diseased leaves could be seen. The lower leaves bore characteristic spots, with mature spores present. The number of spots varied from one to five per leaflet, andin some cases the spots were run together. The plder diseased leaf- lets were dried, shriveled, and lying upon the ground, but still clinging to the petiole. In the light of later knowledge, the disease must have been present at least two weeks. At this time no other fields of red clover , or volunteer plants were found to be infected. The field was revisited from time to time. By June 21 the disease had spread to the uppermost leaves, not a single plant escaping infection. Up to this time the plants had been practically free from other fungi, but now the rust(Uromyces fal- .;3gg (Desm)) was beginning to appear. The disease later spread to every clover field in the vicinity of East Lansing, and is still common at the time of this writing (Nov. 15). It is possible that the field first observed was the original source from which the disease spread to other fields. -9- The correlation between the spread of the disease to toward the upper parts of the plants in the field above describ- ed, and the precipitation during the month of June, is striking, and corroborates what was later proven by experiment. The total precipitation froh June 1 to June 10 inclusive was 0.46 inch, or a daily average of 0.046 inche._ The total precipitation from June 11 to June 21 inclusive was 3.28 inches, or a daily average of 0.298 inch. The fieald observations showed that the spread upward was most rapid between the last mentioned dates. This re- lation will be further discussed under the subject of dissemina- tion. -10- HISTORICAL , The first mention of this disease made in the litiga- ture was made by Oavara, F., (1890) who discovered it in the vi- cinity of Pavia, Italy. He gives a brief description of the caus- al organism and names it Eggrosporium sarcinaefqgme Cav. As the symptoms ne gives ”a leaf spot.“ Tubeuf and Smith.(1892n in their text book are the next to record the disease, but add neth ing new. Malkeff, v. K., (1902) found the disease at Gdttingen, Germany. He inoculated the organism upon red clover leaves kept damp under a bell jar, and observed characteristic spots within five to seven days. His description of the fungus differs in certain details from that of Oavara and the present author. A discussion of these differences will be taken up later. Volkart, A., (1903. 1904), first records the presence of the disease in Switzerland, remarking that heretofore the dis- ease was known only in Germany and in Italy. Orton, W. A., (1904), in discussing the American plant diseases for 1903, states, "Comment was also caused by the pres- ence of clover leaf spot ( Macrosporium sarcinaeforme Cav. and Phyllachora trifolii(Pers.)Fck1 J in Connecticut and New York.” Bain, S. M. and Essary S. H., (1905), note the disease in Tennessee this: “A rather destructive disease caused oy‘ggg: rosporium sarcinaeforme Cav. is very frequent and widely distrib- uted. It often appears on stray alsike plants associated with red clover. The Macrosporium disease appears to capable of destroy ing the plants unassisted tho the statement is made only on field observation.“ Uhder the name of 'Macrosporiose" it is feferred to as widely disseminated in America by Stevens and Hall (1910). -1 l- Milburn, F.,(and Bessey, E. A.) (1915), states that the fungus causes considerable damageron the leaves and stems of clo- ver anu lucerne, and that it ' has been found inside the seed causing non-germination. Such diseased seed is shrunk and wrink- led, and much darker than healthy seed . The mode of infection is not known, but in all probability it spreads from the stems and leaves to the seed.“ Dr. E. A. Bessey informs the author that he has confirmed the presence of the fungus within the seed sent to him from England by Mr. Milburn. ECONOMIC IMPORTANCE. Since this disease has been so little noticed, little or nothing has been recorded concerning the loss which it causes. However, during seasons which are favorable to its spread the damage caused may be very great. In East Lansing, during the past season fields have been observed where the damage ranged from a loss of 15 to 40 percent of the crop. The loss may be especially great when, as is often the case, the young crap is attacked. The nature of the damage caused in the United States is to’a certain extent given in the re- port as to its dissemination given below. DISTRIBUTION. The disease seems to be widely disseminated on red clover, in Europe and the United States. In Europe it has been reported from Italy, (Oavara, 1890), Germany, (Mal- koff. 1902), Switzerland, (Volkart, 1905, 1904), and England, (Milburn. 1915). In the herbarium of the U. 8. Dept. of Agriculture there are specimens collected in Moravia, Austria, and Saxony, Germany. The same herbarium also contains speci- mens collected in 1889 in Manhattan, Kansas, - probably the first specimens collected in this country. The M. A. C. herbarium contains specimens collected in the local Botanical Garden in 1898. Other specimens contained in the U. 8. Dept. of Agriculture herbarium were collected at Houlton, Me., (1906), Arlington Farm, Va.. (1907), Germantown, Md.. (1908), and Philadelphia, Pa.. (1909). besides one specimen on Trifolium 2p. collected at Potomac Flats, Va.. (1890). A report from the Plant Disease Survey of the Bureau of Plant Industry, from records kept between 1903 and 1912 reads as follows: "1903, Connecticut: Disease reported by G. P. 1905, 1906, 1910, 1910, 1912, Clinton. Common and injurious in certain fields. Tennessee: Reported by S. M. Bain as widely distributed in Tennessee. West Virginia: 'The cause of great loss of young clover.‘ Minnesota: Reported by E. C. Stakman, locally prevalent in the neighborhood of Minneapolis. Earliest reported, July 5. First time reported in this territory. West Virginia: Reported by J. L. Clinton, Monongalia and Ritchie Counties, occasionally serious in the northern part of the State. West Virginia: Unimportant. West Virginia: 'Rather destructive in patches at Morgantown this year.’ " The department of the herbarium of the U. S. D. A. also reports the disease on four specimens of alfalfa. Slides from some of this material were sent for examination. It at once became evident that the fungus on alfalfa is not the same as that on red clover. Slide mounts of spores on material collected in Philadelphia, Pa.. and Arlington Farm, Va.. (Turkestan alfalfa) contained spores which in shape and color are the same as the spores of Macrosporium sarcinaeforme Cav. on clover. but they are smaller in size and decidedly warty (eichinulate). On the strength of these morphological differ- ences, the author believes that the fungus on alfalfa is a new species of Macrosporium. and is surely not identical with E, sarcinaeforme Gav. on red clover. A search of the litera- ture has so far failed to reveal a species of Macrosporium which conforms to thagizzscribed on alfalfa. A more complete description of this fungus will be given at another time. ETIOLOGY. Macrosporium sarcinaeforme was isolated from the leaf spot several times during the course of this study. In a demonstration of’Koch's rules of proof, the fungus from one of these isolations was inoculated upon red clover leaves. and reisolated from one of the resulting disease sptts. The fungus from the second isolation was inoculated upon another plant, the disease produced, and the fungus again isolated, thus proving its constant association with the disease. The causal orggpism: The spores are muriform, sarcina-like (packet) in shape, constricted in the middle, and separated at that point into two distinct parts by a cross wall. These parts are subdivided by transverse and longitudinal partition walls which are ordinarily not as thick as the outer or the median division wall. A spore viewed from the point of attachment shows a small circular scar where it was attached to the conidiophore. The con- tents of the spore consists of a dense, hialin protoplasm ‘with many globules (mostly oil) which ooses out when the spore is crushed. The color ranges from hfialin in the very young to dark brown or fuliginous in the older spores. Occasionally a yellow-colored spore may be seen fully developed in form and size. but this darkens with age. The surface of the spore is smooth and no roughenings or prominences of any sort have ever been seen. The size ranges from 22.4 - 37.7 X 19.1 - 27.4 microns, with an average of 28.9 X 22.4 microns. The conidiophores are usually borne at an approximate right angle to the mycelium which gives rise to them. They -15- are dark brown to fuliginous in color, and on the host are . 23.2 - 74.7 microns long by 5.0 - 5.1 microns wide. The tip cell which is darkest in color is swollen and flattened, resembling in shape the knob of a pestle. The two or three cells nearest the tip of the conidiophore are more homogeni- ous and finely granular than the basal cells. These basal cells frequently bear knobs (ElateIIL fig. 2). Some times on the host, but rarely in culture, the tip cell of the conidiophore instead of at once giving rise to a spore, sends out another cell which is similar to it in shape and struc- ture. The spore is then produced upon this secondary tip cell (Platem Fig. 3). The young mycelium is more or less vacuolate and finely granular in structure, sparingly septate, branched, and 2.5 - 4.0 microns in diameter. As it grows older it darkens to a brown or fuliginous color and attains a diameter of 4.0 -5.1 microns. Within the tissues of a well developed spot the mycelium is sparingly branched and does not exhibit the modifications which appear in culture. The type of my- celium found when the fungus is grown on culture media of O the various sorts will be described later. A comparison of the type of pg sarcinaeforme studied by the author with those described by Oavara (1890) and Malkoff (1902):- In comparing the organism studied with those des- cribed by Oavara and Malkoff, several differences may be noted, especially with reference to the sise of spores and the length -17- of conidiophores. The following is a comparative table: Spores Conidiophores Oavara 24-28 x 12-18u 14-18u long 4. “alkOff. 25.2-5506 I 1.6.8.2204.-1 95.2-142.8 x 4.2“ Author 22.4-57.7 1 19.1-27.4u Avg. 28.9 x 22.4fl* - 30-154 x 4.9-5,lu Avg. 77 x 5.0m 0n host: 23.2-74.7:5-5.1u O. O. O. O. O. O. O. O. O. O. O. O. O. O. O. O. O. I. O. O. * These figures are the average of 217 measurements made upon spores grown on seven different media; age of cultures 1 - 3 weeks. ‘* Measurements are the average of 58 made from four of the above cultures. Since Oavara makes no mention of having cultured the fungus his measurements were evidently made from that grow- ing on the host. It will be noted that his spore and conidio- phore sizes are smaller than those of both Malkoff and the author, especially in regard to the width of the spores. A specimen issued by Briosi and Cavara* was available for examination. The few spores which were found when measured agreed fairly well with the also he gives. The measurement ‘ Briosi and Cavara - I Funghi parassiti delle piante coltivate od utili. 116. - MacrosEorium sarcinaeforme Cav., in Difesa dai P0338881 1 e e 1 ano 18 e -18- of only a few very old spores cannot be considered as a criterion, but there is no reason to believe that Oavara was mistaken in his measurements. It is possible, however, that he measured the very young spores. The figure which accompanies the herbarium specimens looks very much like that pf a young spore. The measurements of Malkoff and those of the author agree closely enough, so that at least in this respect they may be considered the same. The author has measured the spores from material collected in various parts of the U. S. and finds that they agree in size with those found in East Lansing, Mich. Malkoff believed that the fungus he observed was perhaps not exactly the same as that described by Oavara, for, he remarks, ”If my diagnosis is compared with the original diagnosis (Oavara), one may see that they entirely agree except for the size and form of conidiaI which are not entirely sarcina-like. Just what he meant by "not entirely sarcina-like" or whether his conception of the term sarcina was different, can not be determined. That the fungus studied by the pres- ent author, may, by anology with the coccus bacteria grouped in packets , (Sarcina lutea, for example) be considered as sarcina-form, is entirely within the common aoceptation of this term. This is evidently what Oavara had in mind when he named the fungus. While the average length of thy conidiophores when the fungus is grown on culture media is more than those growing on the host , the length of the conidiophores as given by Oavara is much less than that given by the author. According to the former's measurements, they arenonly a little over one half the length of the spore(12-18:24-28), while his figure represents the ratio as about 2:1, an appar= ant contradiction. One important point, wherein Malkoff differs with both Oavara and the author, would indicate that he was per- haps dealing with another species: He states that the spores are somewhat warty. Oavara does not mention this (a point he could hardly have overlooked) and the author has not found this to be the case with spores from amterial from various parts of the U. S. In this connection the warty spores on alfalfa already referred to, is of interest. Thar is also considerable difference in the appear- ance of the leaf spots on Cavara's material, ( herbarium specimen) and on his figure which accompanies it. The spots III as compared with American specimens, are smaller, far more numerous, irregular in shape, and do not bear the con- centric markings so typical of the spoton the latter speci- mens. The small size and greater number of the spots may be due to the smoothness of some European varieties of red clover. A smooth leaf surface has a better chance of retaining many small droplets of moisture than a hairy sur- face, whereon the droplets have a tendency to collect in one or several large drops on the hairs. Aphotograph of a diseased leaf accompanies the description given by Malkoff, but it is too blurred to be of any use for comparison. The specimens collected in Manhattan, Kas. in 1889 -20- and identified by G. H. Hicks, evidenly after Cavara's pub- lication (1890) bear a question mark after the name, (Maggg- sporium sarcinaeforme Cav. ? ) indicating that he had some doubt as to whether it was the same fungus . On the basis of these differences, the conclusion that the author is dealing with a fungus other than those previously described, may not be Justified. Only by a com- parative series of cultural and infection studies could this be determined. Again, these differences might be entirely within the limit of variation of a single species. -21- INFECTION EXPERIMENTS. In performing these experiments three method of inoculation were used. These will be referred to by number in the subsequent discussions, where convenient. Method 1 - The plants are first drenched with water (watering can) and then sprayed with a suspension of spores in sterile water. The plants are then kept in a humid atmos- phere under a bell Jar. It was found that the droplets con- taining the spores adhere much better if the leaves are first drenched with water. By this method, characteristic spots develop within five to seven days. Method 2 - Small masses of fungus growth (spores and mycelium) are placed upon the upper surface of the leaf- lets, the inoculum covered with a tuft of cotton, and the plants kept under a bell jar as above. .Method 3 - A drop of spore suspension is carefully placed upon the leaflets by means of a cappllary pipette and then covered with a tuft of cotton. This retains the mois- ture and prevents the droplet from rolling off the leaf. The plants are also kept under a bell Jar. When inoculations are made by either of the last two methods, typical spots are not produced. The leaf tissue surrounding the inoculum is progressively killed and the spores produced upon the dead area. When young leaves are so inoculated,the entire leaflet may be killed within twelve to fifteen days. During this study red clover plants were inocu- lated many times under a variety of conditions, using all the methods above described. Successful infection was -22- almost invariably secured. The following experiment was undertaken to deter- mine the relative susceptibility of red clover plants in the various stages of their development: Group l:- A lot of healthy, green, plants, about 5 cm. high, grown in the greenhouse in a large flower pot. Age 3 weeks. Group 2:- Healthy plants, about 88 cm. high, were transplanted to the greenhouse, care being taken not to in- Jure the roots. Group 3:- Two large, leafy plants, about ‘6 cm. high, transplanted to the greenhouse tcghther with some soil from the field in which it was growing. All diseased or otherwise weakened leaves were removed from the plant. All were drenched with water and sprayed with a suspension of spores. An effort was made to make the amount of spray as nearly as possible proportional to the quantity of foliage in each group. The plants of Group 3 were sprayed with 30 cc. of the spore suspension, those of Group 2 with 20 cc., and those of Group 1 received only 6 "squirts” from the atomizer. After eight days the plants showed infection in about the following proportion: Group l:- All but a few leaflets infected. Group 2:- About one-half of the total number of leaves infected, with the heaviest infection on the lower and central leaves. -23- Group 3:- About one-third of the total number of leaflets infected, with the heaviest infection on_the lower and central leaves. From these and similar infection experiments, be- sides observation in the field, it is concluded that the very young plants are more susceptible to the attack of the fungus than are the older plants. This is perhaps due to the more tender and succulent condition of the young phases. In West Virginia, in 1906, it was reported as causing a great loss of young clover. Stem and petiole infection:- Although numerous infection experiments by spraying a suspension of spores upon the plant were made, in no case was the infection of the stems or petioles observed as the result of such a method of infection. This may be due to the fact that a drop of spore-containing water adheres with difficulty to these parts of the plant, and may roll off before the fun- gus has a chance to penetrate. Infection of the petioles, however, is readily obtained by inoculating with a small mass of fungus growth and then tieing cotton around the »part inoculated. Within five to seven days dark brown to black, linear streaks are developed. Similar attempted inoculations made upon the stems have not been successful. In another experiment,longitudinal slits about 1 cm. in length, were made upon four old, woody stems, and the fungus inserted in the wound. Even after two weeks there was no sign of infection. Some of the inoculum was removed and -24- examined: the spores had merely germinated but seem to have been incapable of entering the tissue of the stem. This may be due to the comparative dryness of these older stems. Only in a few rare cases have infected petioles been seen in the field. Young leaflets when inoculated by Method 2 may be eventually destroyed and the fungus spread from them to the petiole. Petioles so infected have a black, pinched appearance near the base of the leaflets (Plate I, Figu 4, a). Floral infection:- Floral infections were under- taken with the idea of infecting the seed. Reference to the statement of Milburn (1915) concerning the presence of the fungus in the seed, has already been made. At various times during the summer flower heads of red clover in early, full, and late bloom respectively were inoculated by Method 1, but in no case was the infection of any floral part or of the seed obtained. Flowers from plants in the field whose leaves were badly infected with the disease were often examined but the fungus was never found growing upon them. Seed infection:- It was found that the fungus grows readily upon sterilised seed (Hg012, etc.) with Just enough water present to allow the seed to germinate. The growth of the fungus was rapid and abundant. Within two weeks the en- tire mass of seed was well overrun, and an abundance of spores produced. The seed coats were almost completely covered by the fungus and eventually destroyed. The cotyledons of germin- ating seeds could be seen emerging from a black fungus shell of what had been the seed coat. The cotyledons were not -25- observed growing in the midst of badly diseased red clover, but neither were these diseased. We may conclude, therefore, that the strain of alsike clover tested under East Lansing conditions is not so susceptible to the strain of M. sarcinaeforme on the red clover gfowing in this vicinity. Information from hr. Bain as to the nature of this disease on alsike clover in Tennessee was not forthcoming. Inoculations upon other hggtgz- Similar inoculation experiments were performed upon crimson clover (T. incarnatum), white clover (T. repens), Sweet clover (Mglotus alba), alfalfa (medicago sativa), and vetch (Vicia villosa), with- out success.‘ Among the non~legumes the following plants were tested and found to be not susceptible to the disease: Potato, tomato, cucumber, muskmelln, cabbage, rape, and lettuce. While there may be some conditions under which alsike clover and Ither legumes are susceptible to attack by the fungus, M5_ggrcinaeforme, for the present must be considered a parasite restricted to red clover.‘* * Pea (Pisum sativum) and also been (Phaseolus vulgaris) were also found to be unsusceptible to the disease. ** A discussion of the Macros orium found on specimens of alfalfa have already Seen referred to on page 13. -25- attacked, though some of them were slightly discolored, but the tip of the radical, which in some cases attained the length of 10 mm. was attacked by the fungus and killed, there- by preventing further growth. It is possible that as the seed dried out somewhat, the growing tip died and was attacked saprophytically. However, some of the radicals were attacked Just as they were emerging from the seed. The large bulk of the seed was so completely enveloped by the fungus that they could not even germinate. Some of these seeds were planted but failed to grow. INOOULATIONS UPON OTHER HOSTS. Alsike Olover:- Bain and Essary.(l905) state that M. sarcinaeforme was often found upon stray Alsike plants associated with red clover in Tennessee. Repeated attempts were made to infect alsike clover, but without success. The following experiment was repeated at various times during the study: A pot of red clover and a pot of alsike clover plants in about the same stage of development were sprayed thoroughly with a suspension of spores, and both pots kept under the same bell Jar. Within the usual time the red clover plants were infected, but never the alsike. None of the other methods of inoculating, using a heavy inoculum, produced infection, except for an occasional slight discolor- ation of the leaf, but never infection. Wounded leaves also failed to take the disease. Other inoculations were made upon young seedlings and plants in other stages of growthm without success. In the field alsike plants have often been -27- SYMPTOMS OF THE DISEASE On the leaves:- Usually flithin 24-36 hours after inoculation, a minute brown spot 3! Just visible with a hand lens, appears on the leaf surface as a result of the penetra- tion of the fungus. After three days, the spot has attained a size of 2-3 mm. diameter but has no definite shape. After this the spot enlarges rapidly, and the typical concentric markings begin to form. The center of the spot (where the spore has entered) is darkest and very distinct. Around this appear the alternately lighter and darker concentric rings. The darker rings are sepia to dark brown in color, the lighter ones ochre to light brown. Toward the center the dark rings form ridges and are narrow, while towards the margin of the spot they are broad and not raised. The celor contrast between the two outermost rings is very sharp. Conidiophores and spores usually appear first on the underside of the leaf, and can be seen with a hand lens as tiny black clusters, densest nearest the center of the spot., Spores on the upper side of the spot may appear simultaneously with those on the lower side, or later. The shape of the spot is oval to round, and after seven days attains the size of 4 - 7 mm. x 3 - 6 mm. The spot if isolated, may increase in size, the maximum noted being 13 x 8 mm. The spots resemble in general other Macrosporium or Alternaria leaf spots. They are easily distinguiehld from the spots caused by Pseudopezizd trifolii, which do not have well marked concentric rings and bear the characteristic central apothecium. In the early stages of infection, however, -28- it is difficult to distinguish between them. ‘ Spots seem to be most frequent toward the edge of the leaf. Those adjacent to each other, soon run together and lead to the death of the tissue lying beyond them, es- pecially that near the edge of the leaf (Pl. I, Fig. 1). Sometimes a large area is completely covered with small spots which have run together and shriveled up the leaf. (Pl.I, Fig.3) The spots become dry and brittle with age and eventually cause the death and drying out of the entire leaf. If the weather is dry such leaves may hang on for a few days and then fall to the ground. During wet weather they cling to the plant and in the case of the lower leaves, the petioles droop and leaves soon rot in the wet soil. On thegpetioles:- This form is very uncommon, and is restricted to the young, succulent petioles. Upon them the fungus appears in the form of dark brown to black linear streaks one to three mm. long. Little black clusters of spores may be seen on the surface of these streaks. (Pl. I, Fig. 5) The nature of the petiole infection which may spread from the leaves has already been described on page 24. (Pl. I, Fig. 4a) THE MORBID HOST l. Morbid Anatomy. For a cytological study of the disease tissue the material was treated in the following manner: Hilton's fix ng fluid, 6-8 hrs. Washed in 7 alcohol until odor of acetic acid disappeared. 70% alcohol 24 hrs. From this point, dehydration, embedding, until the sections were ready to stain, were pro- ceeded with in the usual manner. Staining:- Delafield's haemotoxylon, 4 - 6 hrs.; Wash in water 20 - 30 min. Eosin, 30-45 sec.; Clear in phenol-turpentine; mount in bllsam. The fixing and subsequent treatment failed to remove the brown color from either the dead tissues or the spores and conidiophores. Peroxide of hydrogen likewise failed to bleach this color. By this staining process the mycelium within the tissue was stained deep purple, the healthy tissua,red to light purple, and the diseased tissue either a reddish brown or not at all (retaining its original brown color). The diseased spots are completely collapsed and only about one-third to one-half as thick as the normal tissue. Within the spot proper the cells are almost entirely disorgan- ized and permeated with the older mycelium. This mycelium is brown in color, septate, branching, and of a more or less homogenious structure without any vacuoles. From both sides of the leaf, at irregular intervals, project conidiOphores, their length varying from 1-3 times that of the spore. Upon these conidiophores are borne the spores.* ‘ The fixing fluid did not, as it is reputed, serve to retain the spores upon the conidiophores. -30- Near the edge of the spot, the young mycelium may be seen spreading intercellularly and to a certain extent intracellularly,into the adjacent healthy tissue. The mycelium does not as a rule, grow straight up through the leaf, but ordi- narily spreads laterally among the cells and may grow for a considerable distance immediately underneath the epidermis. (P1.I, Figs. 6 & 7). Usually the hyphae begin-to come up through the leaf surface on about the fourth day after inocu- lation. The hyphae underneath the epidermis turn up and penetrate between the epidermal cells and in some cases a hypha may be seen coming up through a stoma. (Pl. I, Fig. 6) The former method is much more common. The hyphae once out side the leaf begin to swell at the tip and produce spores. The process of spore formation will be described later. 2. Physiology:- Experiment to determine whether or not the disease spots transpire. Because of their di'flsdand collapsed structure, the old and fully developed disease spots would not be expected to transpire. In order to determine this the following experiment was performed: A simple potometer (modification of Ganong's) the construction of which may be easily understood by referring to the diagram (P1. 11, Fig. ) was used. Into this was fitted a red clover leif bearing 6 - 8 large, old disease spots, the place where the petiole enters the tube being sealed by the application of lanolin. By careful manipula- tion, an air bubble was obtained in the horizontal arm of -31- the tube. A paper centimeter scale was then glued to the outside of the tube so that the zero point was opposite the bubble. Three such potometers were set up. As water evaporates from the leaf, the bubble moves forward, and the distance trav versed being read on the scale. By this means the relative transpiration may be determined. After three hours the dis- tance traversed by the bubble was recorded. The disease spots were then coated with lanolin on their upper and lower sur- faces in order to prevent the evaporation of water from these parts. After three hours the reading was again taken. The following table gives the results. Leaf No. E Distance traversed by the bubble. E Spots before covering E Spots after covering 1 E 21 mm. E 22 mm. 2 E 17 " E 17 " 3 E 24 " E as " Avg. E 20.67 " ; 20.67 " Hourly readings of the temperature were taken. This ranged from 190 to 20° 0. While this experiment was conducted upon a small scale, yet from the fact that the relative transpiration of the leaves with the spots uncovered checked so closely with that from the leaves with the spots covered, it is 'fairly certain that the disease spots,at least when old and ’dried,do not transpire. -52.. Whether the transpiration of the diseased leaves is less than that of healthy leaves, was not determined. If all other factors are eliminated, the transpiration of the diseased leaves would be less because of the reduction in the transpiration surface by the non-transpiring disease spots. INFECTION PHENOMENA The following method was used in determining how the fungus enters the host: A leaf attached to the living plant was inserted through the opening in the stage of the microspppe from which the sub-stage had been removed. One of the leaflets was clamped in place by means of clips and a small drop of spore suspension placed upon its surface. The germination of the spores could thus be observed under the low power of the microscope. A fresh drop of water was added from time to time to prevent drying. Usually germination begins within two to three hours after inoculation. Within ten to fourteen hours the germ tubes have attained a length of four to six times that of the spore, after which, penetration begins. This is best ob- served by focusing the high power directly into the drop, using for illumination a micro-ass focused on the mirror. The germ tube appears to enter between the epidermal cells. A few cases of stomatal entrance have been observed. but this is not characteristic. Where it was desirable to examine the means of penetration more closely the leaf was treated in the following -33- manner: The spores were permitted to dry down on the leaf and a piece of the infected tissue fixed in 96% alcohol for one hour. This treatment removes the chlorophyll and makes the tissue almost transparent. The material was then stained in aosin, 5-10 minutes, cleared in phenol-turpentine, and mounted in balsam. The germ tubes are stained a deep pink while the leaf tissue is stained a lighter shade of the same color. Stained in this manner, the place where the germ tube has entered, and the mycelium which has already begun to grow through the leaf tissue,may be plainly observed. The conditions which favor infection are discussed under the headings of humidity and light relations. -34- GERMINATION STUDIES The various studies in the germination phenomena of the fungus were made in the usual vantTieghem cells kept at room temperature (20° - 24° 0.). The spores of M. sarcinaeforme germinate readily in either tap or distilled water. If taken from a young culture, they begin to germinate within an hour. Usually within six hours every viable cell of the spore has sent out a germ tube. These are at first hyalin and finely‘ granular, but become much vacuolated as the tube elongates. The limit of growth in water is reached after 36-48 hours. At this stage, the mycelium is somewhat branched, septate, vacuolate, with a length of 500 to 700 microns. Spore formation in water has not been obserted. Malkoff (1902) mentions observing the formation of new spores in a hanging drop, but does not state what medium he used. In clover Juice,‘ the germ tubes are at first hyalin but more coarsely granular than those growing in water. They begin to swell at the base very early,and darken in color. This basal swelling is soon cut off by a cross wall and rounds up into a cell containing one or more large central vacuoles. These vacuoles enlarge much faster than the cells containing them(Pl. IV). From this time on the growth of the tubes is more rapid; the mycelium becomes coarsely granular, .' turning to a brown color which is darkest near the spore and shades off to hyalin at the growing tip. As the mycelium * Method of making this and other media given on page -35- becomes older, the cell walls thicken, and oil globules ap- pear. Within three days the colony attains a diameter of 2-4 mm. Spore formation:- The conidiOphores begin to dif- ferentiate about the fourth day. A short branch usually at right angles to the main thread, begins to swell at the tip, This branch may or may not elongate as the swelling progresses. The swollen cell darkens, and is further differentiated from the vegetative cells in that it is finely granular and con- tains no oil drOps or vacuoles. The first sign of the spore, which usually begins to appear on the fifth day, is a small, hyalin protuberance from the swollen tip cell. This enlarges rapidly, until it emerges as an oval, hyalin cell perched on the tip of the conidiOphore. A horizontal cross wall divides the cell in the middle, and soon the first longitudinal divi- sions appear. As the spore matures the color graduihly darkens to a depp brown or fuliginous color, the constriction at the middle becomes more prominent, and the remaining sub- divisions are produced. As a rule, the outer cell wall and the median division wall become more thickened than the partition walls of the spore. Sometimes the end of the mycel- ium which gives rise to the conidiophores, or even a primary branch becomes modified into a conidiOphore and bears a spore. This is the source of some of the abnormally long conidiOphores. The process of spore formation is deliniated on Plate II, Figs. 1-IX. The general method of spore formation is analogous to the process among many of the Alternarias, where a spore -35- sends out a little swelling from the beak end, which later enlarges, becomes muriform, and develops into a spore like that which gives rise to it. The method of spare formation as observed by the author does not agree with that described bytcavararor Malkoff, who state that the apical cell of the conidiophore swells, and itself becomes differentiated into the spore. The author has never witnessed this proceedure. In a 5% dextrose solution, growth prodeeds to the formation of spores, but the germination process develops abnormal swellings. In a few instances, a cell budding from the spore was seen to endarge to a size equal to that of the one or two spore which gave rise to it, and become divided by l.lateral cross walls, giving it the appearance of a young spore.(P1-IIIo5) Though this develop into a mature spore, it might possibly have done so under the proper conditions. This may be evi- dence of a tendency towards aniAltennaria-like habit. Some Macrosporiums are known to develop the Alternaria habit in culture. The cause of these abnormalities is not due entirely to the nature of the medium, since they did not appear in a series of 5% dextrose sol. cultures prepared at another time. The mature mycelium also germinated readily.In water, thin hyalin threads, similar to the germ tubes from the spores, are produced. Old conidiophores from which the spore has fallen often germinate from the tip cell, sending out a long vacuolate tube with occasional swellings. (Pl. III, Fig. 6). The percentage of germination of spores from young -37- cultures is practically 100%. The following table gives the time and amount of germination of five series of van Tieghen cells (6 to a series). The spores were sown in sterile tap water and kept at room temperature. Age of culture from which spores were taken was eight days. ? Series E 6 hours E 13 hours E 24 hours 1 x 90% : 99% plus : same 2 E 85 E 99 " E " s 95 99 .. .. 4 90 95 . .. 5 E 70 i 95 " E 99% plus In cases where old spores were used, the germination percentage was found to vary from 70 to 90%. -33- OULTURAL STUDIES The original culture of the fungus was obtained from the dried material, which at the time we a 18-20 months old. Dilution plates were poured, using corn meal agar. A single spore was marked, and when germination occurred it was removed together with a small block of agar containing it and transferred to prune Juice agar. From the colony which developed, transfers were made, originating the stock cultures. The comparative cultural study of M, sarcinaeforme has not revealed any striking morphological divergencies. The fungus grows readily on a large variety of media. Three groups of media were used: agare, vegetable plugs, and liquids. All cultures in each group were run simultaneously, and growth observed over a period of three weeks. In general growth proceeds as follows: The spores germinate within a few hours and after 24-28 hrs., small white tufts of aerial mycelium, 1-3 mm. long,appear. During the next two or three days the mycelium spreads gradually over the surface of the medium, the rate of spread depending upon the amount of moisture present and the amount of inoculum used. .Beginning with the third day, the mycelium usually darkens in color, at first a dull gray woolyyappearance which gradually darkens. Spore formation begins on the fifth or sixth day, and as the spores mature and increase in number, the cul- ture assumes a black, felt-like appearance. On agar media, the fungus usually begins to grow down below the surface after the first week. This submerged growth which may extend down -39- 5-10 mm., consists of a dark mycelium and spreads out more or_ less like the roots of a plant. The maximum amount of growth is reached within 16-20 days. Of the various agar media used, oat meal agar pro- duces the most abundant growth of mycelium and spores. Plain nutrient sad the synthetic agars used produced the smallest amount of growth. With the addition of glucose ordddxtrose to the nutrient agar, growth is materially increased. Of the vegetable media used, it is noteworthy that on potato, while a dense mycelial growth is produced, spores are few. The presence of sugar seems to favor spore produc- tion. Of these media sugar best and red table beet rank first Anithe amount of spores formed, carrot second, parsnip third, and potato last, which also represents the relative order of their sugar content. If glucose is added to potato, as in the case of hard potato agar, spores grow in abundance. On the other hand, wheat starch paste (Kahlbaum's) is an excellent medium for spores. A test of the substrat upon which the fungus had been growing for two weeks showed that much dex- trose had been formed. The sterile, uninoculated starch paste check gave no such test. Evidently the fungus secretes the enzyme invertase. On plain corn meal, growth is abundant, but the hypothetical ascus stage has thus far failed to appear on this medium. (Oldest culture, nine months). Clover stems, bean pods, and sorghum stems are very good media for obtain- ing an abundance of spores. The following tables give a comparAsOnnof the growth on the various media, under as uniform conditions as it was -40- possible to obtain. As a quantitative standard of growth, oat meal agar was used for the various agar media, sugar beet for the vegetables, and clover Juice for the liquids. 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