. ‘. k. A ~. L - 'v ‘3 ‘ ;" 0‘ :‘ g . ru’ ,‘ s a “db/tama- This is to certify that the thesis entitled Some Studies On The Rotting Of Potato Tubers In Michigan In 19149 presented by Moustapha Ibrahim Zeidan 7'- has been accepted towards fulfillment "":, of the requirements for " Mdegree inm—Bathology ‘ ' r” 4... Q I ; ." Major pro r Date WM SOME STUDIES ON THE ROTTING OF POTATO TUBERS IN MICHIGAN B‘J MOUSTAPHA IBRAHIM ZEIDAN W A THESIS Submitted to the School of Graduate Studies of Michigan State College of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Botany and Plant Pathology 1950 THES¥S AC KN OW LED GMENTS The writer wishes to express his appreciation to Dr. J. Vaughn for advice and suggestions given during this study and for the criticism and the correction of the manuscript. The writer is also grateful to Dr. J. H. Muncie for help in supplying the specimens and for helpful suggestions and library facilities. The writer is also thankful to Dr. R. Nelson for his aid in the classification of Fusarium spp. Acknowledgment is also made to Dr. D. J. deZeeuw and Mr. E. A. Andrews for correction and criticism of the manuscript. 237566 CONTENTS Introduction........................................ Economic importance of potato crop............. Geographic distribution in Michigan............ Economic importance of the diseases............ Review of the literature....................... Object of this study................................ Materials and methods............................... Isolation...................................... Experimental results................................ Pathogenicity tests............................ Discussion and conclusions.......................... Summary............................................. Literature cited.................................... B'Iap Of P'IIiChiganOOOOOOOOOOOOOOOOOOOOOOOOO00.0.0000... 39 43 45 5o INTRODUCTION The potato is distributed nearly over the entire world. Consequently tuber diseases become more and more important. For that reason, an investigation was made during this study to find the organisms most responsible for these different diseases, the ultimate object being to help in the disease control. Economic Importance of the Potato Crop: The potato belongs to the family Solanaceae and is one of the most important vegetable crops. The potato tuber is a storage organ developed from a swelling at the end of the rhizome or underground stem. This crop is usually carried over from year to year as tubers called seed pieces (19). The potato crop is one of the most important food crops in the world. Many peoples, especially the Western ones, use the potato as a primary source of carbohydrate. The cereal grains such as wheat, corn and oats take place next to the potato and rice crops. The average annual production of potatoes in the world as determined for 1935 to 1939 is 8,126,500,000 bushels. The production in the United States for the same period is 355,513,000 bushels. This puts the U. S. potato production in fifth place - after U.S.S.R., Germany, Poland and France. The average annual yield for the U. S. for 19M6 to 1948 was 439,691,000 bushels 2. whereas for Michigan the average annual yield for the 1946 to 1949 period was 16,057,000 bushels. In 1949 the total yield of potatoes in Michigan brought Michigan an income of $20,935,000. The potato crop ranks sixth among the different crOps grown in the U. S. With respect to the yield of 1949, the potato crop ranks fourth in Michigan, coming after hay, corn and oats (45, 46, 34, 51, 47, 48, 49, 50, 19)- Geographic Distribution in Michigan According to the Crop Report for Michigan, January- February, 1950 (50) a potato crop is grown in almost all the counties of each district (see map, p. 50). l. U. P. District 2. Northwest District . Northeast District . West Central District . Central District . East Central District . Southwest District . Southern District . Southeast District In the northern counties, the growers usually harvest the potatoes in late September. In the southern counties the harvest is in early October. About one— third of the potato harvest is sold to truckers at harvest time; one—third is stored in nearby commercial or cooperative warehouses and the rest is stored on the farm (16). The northern and the southern growers use a common method of harvesting the potato crop: 3. 1. Pick up the potatoes from the soil and put them into the picking basket. 2. Transfer the potatoes from the baskets into crates. 3. Then tranSport the crates to storage where tubers are put in the bins. This makes three handlings to get the potato tubers from the field to the storage (16). Experiments were set up by Edger, Jefferson and Wheeler (16, 43) to show the loss caused by grading. The results obtained showed: For one handling the relative loss was 3%. For two handlings the relative loss was 15%. For three handlings the relative loss was 20%. Etc. Thus, as the number of handlings increases the damage will increase accordingly. Economic Importance of the Diseases The rotting organisms of potato tubers are very wide-spread. They are present in any area which grows potatoes: Asia, Europe, and America. The loss caused by these organisms is estimated to amount to many millions of dollars. In Michigan, Muncie (35) noted many organisms causing different kinds of rot of tubers such as: Bacteria: Erwinia carotovora (Jones) Holland Corynebacterium sepidonicum (Spekerman Nandkottoff) Skaptason and Burkholder) Fungi: Phytopthora infestans (Mont) DeBary Alternaria solani (E and M) Jones and Grout Armillaria mellea (Vahl) Quel and several types of storage dry rot caused by Fusaria such as E. trichothecioides Wr., E, eumartii Carp, E, oxysporum Schlecht, and E. radicicola Wellenw. These organisms cause a tremendous loss to the Michigan growers each year. Review of Literature Potato rot organisms are found in any place where a potato crop is grown. The investigation of 0. w. Carpenter (11) showed that the most important of these organisms are Phytophthora infestans, Fusarium sp., bacteria, Armillaria s2}, and other fungi such as Rhizopus nigricans. Of those organisms cited above after Phytophthora infestans, Fusarium spp. are undoubtedly the most important causes of tuber decay. Phytophthora infestans causes more or less damage to the tubers each year and when favorable conditions for the spread of this fungus occur, it appears in epidemic form. Many earlier investigators found that several species of the genus Fusarium (Link) caused rot to the tubers of Solanum tuberosum. Among these investigators were Clinton (12), Pethybridge and Browers (42), Carpenter (11) and others. Wollenweber did a lot of work with the genus Fusarium. He established the pathogenicity and the nature of the infection of Fusarium discolor var. sulphureum (Schlecht) Ap. Wollenw. and E. coeruleum (Lila) Sacc. Jamieson and Wollenweber (24) in 1912 first described a dry rot of the potato tuber caused by Fusarium trichothicioides. In their inoculation experiments they found the most rapid penetration of the tuber takes place at 10—120 C. in an atmOSphere of low humidity. In 1913, Wollenweber added three new Species of Fusarium causing rot to potato tubers as ‘E. ventricosum, E. rubiginosum, and E. subulatum. C. w. Carpenter (11) in 1915 investigated the effect of different species of Fusarium on potato tubers. (E. oxysporum, E. hyperoxysporum, E. vasinfectum, E. radicicola and E. eumartii). He reported that all those organisms caused rot to potato tubers. He also noted that under a constant storage temperature below 500 F. the action of E, radicicola and E. eumartii and E. oxysporum is prevented. He stated that most of the species of Fusarium which are able to destroy potato tubers belong to the sections Martiella, Elegans and Discolor which were already established provisionally by Wollenweber (53). R. W. Goss (20) investigated the ability of E, solani to cause a tuber rot as a wound parasite. He made many inoculations through a tangential cut of the tuber and kept this tuber in a moist chamber at about 220 C. After 6. twelve days he found that tissue affected by the fungus became a light brown color. C. B. Ramsey, J. S. Wiant, and M. A. Smith (1932 and revised in 1949) (39) discusSed the potato rot caused by the genus Fusarium. They stated that all Fusaria are soil inhabitants and infect potato tubers in two principal ways during the growth of the tuber: 1. By growing into the stem—end from infected stolons 2. By entering lenticels, eyes or wounds. Some species such as E. radicicola, E. oxysporum, E. eu- martii, E. solani, and E. avenaceum attack growing plants in the field and cause wilt diseases which show different symptoms on plants and on tubers. They also found that some species of Fusaria invade the central part of the tuber, others decay surface tissues. Other species invade the stem end where the surface of the tuber becomes brown and sunken. On a growing plant the external symptoms caused by species of Fusarium wilts is different. Some species such as E. oxysporum induce a wilt marked by yellowing around the margins of the lower leaves and finally the yellowing involves the entire tops. The wilt induced by E. eumartii causes a light variegation of the leaves accompanied by the irregular bronzing of the upper surface. The same authors (39) found also two kinds of rots, a dry rot which is formed by the slow work of 7. those Species and a watery soft rot of the entire tuber in which the causal organism decays the whole tuber in a short time. They also noted that Fusarium species require relatively high temperatures for optimum growth. They grow best at 780 to 860 F. and most of them grow slowly below 500 F. Armillaria Tuber Rot History W. Jones and H. S. MacLeod stated (27) that Armillaria tuber rot was reported first from Australia in 1910 by Johnson and from Washington State in 1914 by Bailey (2). It was first observed in British Columbia in 1934. This disease was reported in Michigan by Muncie (35) in 1942 who mentioned its frequent occurrence in the state. The causal organism is Armillaria mellea which belongs to the Order Agaricales, Class Basidiomycetes. The mycelium of this fungus forms structures known as rhizomorphs which give rise to a mushroom fruiting body. Signs of the Disease W. Jones and H. S. MacLeod (27) and Muncie (35) described the symptoms of the disease. They are hard, brown, roughened, shrunken areas which vary in size and are more or less corky in texture. Eventually dry 8. rot results. From the economic standpoint, Armillaria tuber rot disease is not considered an important disease and its presence is negligible. Late Blight: Phytopthora infestans (Mont) DeBary Historical data of potato diseases show that the Late Blight disease is the oldest one of the potato. L. R. Jones, N. J. Giddings, and B. F. Lutman (28) found out that there is no reference to any potato disease in Europe previous to 1830. They concluded that the disease appeared some time between 1830 and 1845 in Europe and North America. In North America, B. M. Watson of Boston (28) informed Jensen that the disease was found around the city in 1842 and in 1843 it was found around the city of New York and in 1844 it was spread in many parts of the United States and Canada. J. H. Muncie in 1941 (36) stated that the most severe occurrence of this disease in Michigan was in 1915 when the loss to the potato crop was estimated at $4,000,000. Jones, Giddings, Lutman and Muncie (28, 36, 39) and many other investigators studied the signs of the disease on the foliage and on the tubers. The results of these investigations proved that the first signs of potato blight appear near the tip of the leaf of the plant. The invaded area on the leaf appears water-soaked 9. or pale greenish, and later wilts and blackens. Under dry conditions the affected tissues become dry and brittle and under humid conditions they become soft and decayed and have a strong odor. In the earliest stages of the disease there appear small brownish or purplish discolorations of the skin on the tuber. Later the tissues under the affected areas change to a brownish color. Usually the blighted area becomes sunken because of the death and drying out of the underlying tissues. During a long period of wet weather the whole tuber affected with late blight softens and there is a secondary invasion by bacteria and fungi resulting in a foul, rotten mass. 0n the contrary, in dry cool conditions of soil or storage, infected tubers decay slowly resulting in a dry rot. Cause of the Disease The cause of potato blight is Phytophthora infestans which is in the order Peronosporales. Butler and Jones (10) cited that the fungus occurs on a number of other Solanaceous hosts of which the tomato crop is the most important. The mycelium is intercellular and the Sporangia are borne on aerial branched sporangiophores. Jones, Giddings and Lutman (28) cited that the germination of the sporangium (conidium) may take place in two ways: 1. Direct germination; i.e. by pushing out a germ tube through the apex of the sporangium. lO. 2. Indirect germination; i.e. the sporangia give rise to zoospores that swim for a short time, encyst, form a wall, and then germinate. They found that the method of Sporangium germina- tion is determined by the temperature. At 250 C. more than 50% of the germinations are by tubes while at 100 to 200 C. from 60% to 70% of the sporangia produce zoospores. Infection of the tuber in the field was demonstrated by deBary and Jensen (28) and others. They concluded that the tubers are infected by spores washed through the soil from the blighted foliage. The infections occur often at the tip and at the stem end. Jones, Giddings and Lutman (28) showed also that the fungus can move from tuber to tuber in the soil or in storage. They showed also that the tubers can be infected at harvest time before they are transported to the storage. For long distances the dissemination of this fungus is accomplished by wind or insects. Reiner Bonde and E. S. Schultz (5) investigated the dissemination of Phytopthora infestans. They con- cluded after five years of investigations that Late lBlight often develops early in the season on plants égrowing in potato refuse piles and from there dissemina- TSions take place by the wind to the environmental I?ie1ds. H. L. C. DeBruyn (8) investigated the over- Wfintering of the fungus. He concluded that Phytophthora iJnfestans can live saprophytically and can stand cold and drought. ll. ggflrfl§;g_9;nerea (Pers) Gray Mold Rot In 1916, Pethybridge failed to isolate Botrytis EEDEEEE' In 1933, Folsom repeated the experiment. He isolated the organism. Then he inoculated a fresh deep wound made in the tuber. After inoculation had been completed he closed the wounds tightly. He kept some of the tubers in a damp chamber at about 500 c. From the results that he obtained he concluded that Botrytis cinerea produced a similar disease when inoculated into the tuber under certain conditions. He stated that mycelial cultures were much more effective than spores in producing infection. In 1940, Glen Ramsey (40) investigated the effect of Botrytis cinerea on potato tubers. The strain of Botrytis was isolated from California potatoes. He selected twenty—four medium tubers free from disease and treated them for thirty minutes in a 1:240 solution of formaldehyde. After rinsing the tubers in sterile distilled water and allowing them to dry in a sterile chamber, he made a deep wound of three-eighths inches into which he introduced a mycelium of a week-old cul— ture of Botrytis. He closed the wound by the plug and kept the inoculated tuber in temperatures of 700 and 400 F. and at high humidity. He concluded that Botrytis cinerea caused typical brown rot. The rot of the tubers kept at 700 F. was rare and was evident in a Inonth. The rot of the tubers kept at 400 F. was more esvident at the end of the first week. 12. Because the infection of the tuber is accomplished through a wound, Ramsey, Wiant and Smith (39) suggested that infection of the tubers occurred at harvest time, when the mycelium grows through the stolons into the stem end. Early Blight: Algernaria Tuber Rot Early blight is caused by Alternaria solani (E and M) Jones and Grout. It was first described in 1925 by Donald Folsom and Reiner Bonde. Lesions on affected tubers appear as dark, sunken, variously shaped areas and some times they are surrounded by irregularly raised borders of a brown to purplish-brown color. G. H. Coons and J. E. Kotila (14) in 1923 as well as Muncie (35) in 1942 noted the presence of Alternaria solani in Michigan. They estimated the losses by early blight in the year when it is severe amount to 25%. In 1927, Gratz and Bonde (22) investigated the infection of potato tubers by Alternaria solani. The organism was isolated and grown in pure culture. Five thousand freshly dug tubers were used in this experiment. Some of them were inoculated by rubbing with the affected potato foliage; others were stored in contact with diseased foliage. From the foregoing observations they concluded that potato tubers were readily infected at digging time by stirring them among the affected foliage. Affected tubers may be invaded by secondary organisms which help to 13. increase the losses. Ramsey, Wiant, and Smith (39) noted that Alternaria tuber rot becomes a dangerous storage and market disease of potato tubers. Affected tubers are always invaded by secondary organisms such as Fusarium or other organisms. Bacterial Diseases Bacterial diseases are considered important because they damage the potatoes, either as tubers or plants in the field and as tubers in storage. These diseases are caused by varied organisms: Erwinia phytophthora (Appel) Bergey et a1, causes black leg. Erwinia carotovora (Jones) Holland causes soft rot. Bacillus mesentericus (Fluggi) Migula, Bacillus aroideae Townsend, Corynebacterium sepidonicum (Spekermann and Kotthoff) Skaptason and Burkholder, causes bacterial ring rot. Many studies have been completed to isolate these organisms and to prove their pathogenicity. P. Brierley (7) in 1918 investigated the pathogenicity of different bacterial organisms. He concluded that Erwinia carotovora, E. mesentericus and E. aroideae possessed the ability to cause rot to potato tubers. In Michigan in 1923, G. H. Coons and J. E. Kotila (l4) mentioned the presence of black leg in the Upper Peninsula before the summer of 1917. From their experi- Inental work made on varieties susceptible to the black 14. leg organism, they concluded that no variety is resistant enough to prevent the loss. Bacterial ring rot was first discovered in Germany by Appel in 1906. It appeared in Canada in 1931 where it was reported by Barleau as bacterial wilt. In 1937 the disease appeared in Maine in the United States. The presence and the prevalence of this disease in Michigan was first reported by Muncie in 1939 (35) when he reported a heavy loss caused by this disease. OBJECT OF THIS STUDY The objective of this investigation was to find out which organisms were rotting the potato tubers during the year 1949, to determine the prevalence of these organisms, and to estimate the proportional im- portance of such organisms. The results of this kind of research may indicate the importance of the various organisms and this could lead to development of the proper control measures. MATERIALS AND METHODS Isolation In this particular survey specimens were collected from as many different places as possible in Michigan, either from the fields or from the storages. Most of them were recorded from the following counties: Mbntcalm I, Presque Isle II, Oceana III, Cass IV, 15. Missaukee V, Allegan VI, Livingston VII, Newaygo VIII, Mid- land IX, and nine only from unknown places (see map, p. 50). The method used to isolate the organisms was that advised by A. J. Riker and R. S. Riker (41). Affected tubers were washed, dipped in a 1:1000 mercuric chloride solution for one to three minutes, and rinsed thoroughly in sterile distilled water. Eventually, small pieces of diseased tissue were taken aseptically from the advanced margin and transferred to a sterile petri dish of potato dextrose agar or soya bean agar. When the specimen was severely affected, one drop of 50% lactic acid was added to the medium to prevent growth of secondary bacterial organisms. The plates were kept in the laboratory at the temperature and the humidity of the room. When the growth of the organisms was obvious, each different organism obtained was transferred into a plate of water agar. After one to two days hyphal tips of each organism obtained were transferred to a slant of potato dextrose agar. These cultures were numbered and used in the research reported below. In the following tables the affected tubers are grouped with respect to their external symptoms: Table I. Tubers with large, irregular, shallow sunken, reticulated and brown colored lesions. Table II. Tubers with large, irregular, shallow sunken, reticulated, cracked and brown to dark brown colored lesions. 16. Table III. Tubers soft, watery, decayed, and with a foul smell. Table IV. Tubers with brown to dark brown, irregular, reticulated spots at the stem end. External Symptoms: TABLE I sunken, reticulated and brown colored lesions. Tubers with large, 17. irregular, shallow §§§% E§§§igf 323::3 variety Medium Sttziizi 53 10—1-49 Montcalm Sebago P.D.A. Bacteria 54 " " " " " 55 " " " " " 56 " " " " " 57 " " " " " 58 n Presque Russet " " Isle Rural 59 " " " " Fungus 60 " " " " " 61 " " " " " 62 " " " " " 63 " " " " " 64 " " " " " 65 " " " " " 66 " " " " " 67 " " " " " 68 " " " " " 69 " Montcalm Sebago " Bacteria 72 " Prigqge Unknown " " 73 " Montcalm Sebago " " 94 10-23—49 Oceana " " " ESoya.B.A. = Soya Bean Agar. I’.D.A. = Potato Dextrose Agar. TABLE I (continued) 18. ISO- late No. Date of isola- tion Source County Variety Medium Organism Obtained 98 99 100 101 102 121 122 123 124 125 126 128 131 132 133 131+ 135 136 138 139 140 141 143 144 145 12-25-49 ll Cass I! Missaukee Montcalm H Allegan Missaukee Livingston H H II H H ll Montcalm & Livingston H H Russet Rural H H Unknown I! ll Pontiac Russet Rural 529-2 ll II II ll ll Unknown H H Soya.B.A. ll P.D.A. + Acid :1 Fungus TABLE I (continued) Iso- Date df late isola- No. tion Source County Organism Variety Medium Obtained P.D.A. + Acid 147 H H H H ll 148 H H ll ll H 149 4-11—50 " " n n 146 4-6-50 Livingston Unknown Fungus 150 H H ll ll ll Russet " Bacteria 153 " Missaukee 154 " " " " Fungus 155 " " " " " 156 " " " " " 157 " " " " " 159 " " " " " 164 4-12—50 " " " " 165 " " " " " 167 " " " " " 169 " " " " " 17o " " " " " 171 " " " " " 173 " " "v " " 176 4—14-50 " 46-1 " " 177 " " " " " 178 " " " " " 179 4—21—50 " " " " 180 " " " " " 181 " " " “ " 182 " " Sebago " Bacteria TABLE I (continued) 20. Date of isola- tion Iso- late No. Source County Variety Medium Organism Obtained 183 4-21-50 185 II 187 H 199 " 200 " 201 " 205 4-22-50 206-1 " 209-1 " 210 " 211-1 4-27-50 212 4-28-50 214 5-8-50 216 " 217 " 218 " 220 " 222 223 H ¥ Missaukee Michigan n Livingston n n n n ll Sebago P.D.A. + Acid ll Soya.B.A. Fungus H H H II ll Bacteria Fungus Bacteria Fungus ll TABLE I (continued) 21. ISO- late No. Date of isola- tion Source County Variety Medium Organism Obtained 225 226 203 205 206 208 209 211 213 215 231 239 240 241 254 255 257 258 5-11-50 ll ll ll 6-14-50 Livingston It Michigan I! ll Missaukee Newaygo Presque Isle Sebago Unknown ll Katahdin ll ll ll ll Unknown ll Sebago ll P.D.A. Acid + Fungus 22. TABLE II External Symptoms: Tubers with large, irregular, shallow sunken, reticulated, cracked and brown to dark brown col- ored lesions. Iso- late No. Datefof isola- tion Source County Variety Medium Organism Obtained 77 78 80 83 84 85 88 95 151 152 158 160 161 166 168 172 184 189 191 194 196 10-8-49 Montcalm Livingston It Missaukee It It Sebago P.D.A. ll H H H Unknown ll P.D.A. + it It Russet " Rural Fungus Bacteria H H ll 23. TABLE 11 (continued) Iso— Date of late isola- No. tion Organism Obtained Source County Variety Medium 197 4—12-50 Missaukee Sebago Soya.B.A. Fungus " P.D.A.. + II Acid 227 " " Katahdin " H 221 5-9-50 Livingston 228 " Missaukee Sebago " " 229 " H n n n 230 " .. 1: n . n 232 " " it n n 233 " .. n .. n 234 " " " H n 235 " " " v! n 236 " " n z: n 237 " .. .. n u 242 5-11-50 " n n u 243 n .. .. .. .. 244 " " " N H 245 " " " " n 246 " " " " " 2247 " " " I' n 2248 " " " " H 249 " H n u u 225%) " .. n .. n EBEEL " " I' n n 25 2 II H H ll ll External Symptoms: TABLE III with a foul smell. 24. Tubers soft, watery, decayed, and §§§% §§§§iff SSEEE: variety Medium Sitiiiii 5O 10-1-50 Montcalm Sebago P.D.A. Bacteria 51 " " " " " 52 " " H n n 74 10-2-49 " n n n 75 " " " " " 76 " " H n u 79 10—8-49 " " n n 82 " " " " " 87 " " " " " 91 10-23—49 Michigan Unknown " Fungus 92 " " " " " 93 " " " " " 97 11-19-49 Midland " " N j162 4-12-50 Missaukee RESESI " H 2256 5-11-50 Michigan Unknown Burkholder Bacteria External Symptoms: TABLE IV irregular, reticulated spots at the stem end. Tubers with brown to dark brown, 180- late No. Date of isola- tion Source County Variety Medium Organism Obtained 70 71 103 104 105 106 107 108 110 111 112 113 114 :115 2116 3417 1L18 3419 :1220 :1227 10-1-49 11-16-49 ll 3-28-50 Presque Isle It H H H II Missaukee Russet Rural ll Unknown Chip- pewa P.D.A. ll ll Fungus ll Bacteria Fungus ll 26. TABLE IV (continued) Iso- Date of late Isola- Source Variet Organism No. tion county y medium Obtained 142 4_ _ Montcalm & P.D.A. + 4 50 Livingston Unknown Acid " 155 u n Russet " H Rural 202 1+-21-5O Missaukee Sebago " " 203‘]. 4-22-50 ll ll " H 204 H n n n n 208-1 " " " H n 213-1 5_4_50 n Russet n " Rural 215-1 5-8-50 Livingston Sebago " n 219 " " N u u 253 5-11-50 Montcalm 125-4 " u 27. EXPERIMENTAL RESULTS Pathogenicity Tests Proof of the pathogenicity of the foregoing isolated organisms was accomplished as recommended by Koch's postulates (41, pp. 44a). The potato tubers used in the experiments were picked from Michigan potatoes. There were only three varieties used of those certified in the state. Varieties Used Varieties Grown in Michigan (according to J. W. Muncie) Sebago Chippewa Russet Rural Sebago Katahdin Erie Pontiac Green Mountain Menominee Sequoia Irish Cobbler Russet Burbank Katahdin Russet Rural 125-4 (new variety of uncertified seed) 225-2 (new variety of uncertified seed) The isolates were tested at different times during ‘the year. The first test was made in November, 1949. fPubers of Sebago variety, sound and free from disease tvere selected. Surface disinfections were obtained by clipping the tubers in a 1:1000 mercuric chloride solution I?or 3-5 minutes. Then they were rinsed in sterile Ciistilled water. Four plugs of 5 mm square and 3-5 mm (iEEep were made in the tuber. Then a little piece of 28. potato dextrose agar which contained the inoculum, either bacterium or fungus, was transferred into each of the three wounds. The fourth was kept without inocula- tion as a control. The plugs were turned back to their place and were covered with a layer of paraffin wax to avoid any contamination coming from the air. The tubers inoculated with fungus were kept in an incubator of approximately 80% of humidity and of temp- erature ranging from 160 C. to 180 C. for thirteen to sixteen days. Those which were inoculated with bac- teria were kept in an incubator of approximately 80% of humidity and of temperature ranging from 210 C. to 250 C. for twenty-two days. The results obtained are indicated in the following tables. 29. TABLE V Results of the first pathogenicity test made in November, 1949 O Tuber No. Variety Kind of Inoculum Observation 6O Sebago Fungus - 61 " " - 62 " " - 63 " " - 64 " " - 65 " " - 66 " " - 67 " " — 68 " " - 7O " " - 91 " " + 92 n n _ 93 H H + 97 H H _ 203 It 11 + 205 " " - 206 " " + 208 " " - 209 " ' " + 211 " " + Pathogenic Organism. Non—pathogenic Organism. + II ll 30. TABLE VI Results of the first pathogenicity test made in November, 1949 (continued). Tuber No. Variety Kind of Inoculum Observation 5O . Sebago Bacteria - 5l " " - 52 " " - 58 " " - 72 " " - 74 " " - 76 " " - 77 " " ' 79 I! ll __ 82 ll ll _ 87 u n _ Pathogenic Organism. Non—pathogenic Organism. 1.- 31. The second test was made in December, 1949. Sound tubers of Katahdin variety, free from disease were used. Surface disinfection and inoculation procedure were accomplished in the same way as in the first test. Inoculated tubers were kept in an incubator at a temperature ranging from 250 C. to 280 C. and at 100% humidity during the experiment for 18 to 23 days. The results are shown in Table VII. 32. TABLE VII Results of the second pathogenicity test made in December, 1949. Tuber No. variety Kind of Inoculum Observation 50 Katahdin Bacteria - 69 " " - 73 " " - 77 " " - 78 " " - 84 " " . - 85 " " - 88 " " - + = Pathogenic Organism. = Non-pathogenic Organism. 33- The third, fourth and fifth tests were made suc- cessively in May, June, and July, 1950. In the three tests sound tubers of Russet Rural variety, free from disease were used. Surface disinfection and inoculation procedure were accomplished in the same way as in the first and second tests. Inoculated tubers of the last three tests were kept in the same incubator at 100% humidity but the tempera— ture differed in each of these tests. In the third test the temperature of the incubator ranged from 240 C. to 290 C. for twenty—five days. The results of this test are listed in Table VIII. In the fourth test the temperature of the incubator ranged from 240 C. to 290 C. for twenty-five days. The results are listed in Table IX. In the fifth test the temperature of the incubator ranged from 210 C. to 300 C. for fifteen days. The results are listed in Table X. 34. TABLE VIII Results of the third pathogenicity test made in May, 1950. Tuber No. Variety Kind of Inoculum Observation 111 Russet Rural Fungus + 115 " " + 117 " " + 118 " " + 121 " " - 122 " " - 123 " " + 124 " " - 125 ‘ " " + 126 " " + 127 " " + 128 " " + 131 " " - 133 " " - 134 " " + 135 " " + Pathogenic Organism. Non-pathogenic Organism. I + 35- TABLE VIII (continued) Tuber No. Variety Kind of Inoculum Observation 136 Russet Rural Fungus + 138 " " + 141 " " + 143 " " — 144 " " + 146 " " + 147 " " + 148 " " — 149 " " + 150 " " + 151 " " - 152 " " + 159 " " - 169 " " - 173 " " - 176 " " — 178 " " - 203-1 " " + 208 " " — 210 " " + Pathogenic Organism. a + Non-pathogenic Organism. TABLE IX 36. Results of the fourth pathogenicity test made in June, 1950. Tuber No. Variety Kind of Inoculum Observation 165 Russet Rural Fungus + 181 " " + 183 I! ll _ 187 n u _ 188 " " + 191 H II + 197 n n _ 213 H I! + 214 ll ll _ 220 ll ll _ 225 ll ll _ 227 II II _ 228 " " + 229 H H _ 231 H H _ 233 II II + 239 it I! + 254 H II + Pathogenic Organism. | + Non-pathogenic Organism. 37- TABLE X Results of the fifth pathogenicity test made in July, 1950. Tuber No. Variety Kind of Inoculum Observation 215 Russet Rural Fungus - 236 " " - 238 " " + 240 " " + 242 " " + 245 " " + 247 " " - 250 " " + 251 " " + 252 " " + 253 " " + 254 " " - 257 " " - 258 " " + athogenic Organism. + P Non—pathogenic Organism. The following method was used to prove the patho- genicity of the Phytophthora sp. in causing rot to the tubers. Because Phytophthora infestans is a delicate organism it is hard sometimes to make it grow on an artificial medium. To prove the pathogenicity of this organism, which was isolated from tubers No. 98 and N0. 99 collected in Cass County, the experiment was accomplished with respect to the following method. The surface of the tuber was sterilized by dipping it in a 1:1000 mercuric chloride solution for 3—5 minutes. Then it was rinsed in sterile distilled water. Each tuber was cut into two halves with a sterilized scalpel. Into each cut surface several criss-cross linear incisions two to three mm in depth were made. The inoculum which consisted of mycelium with abundant sporangia was transferred with a sterilized needle and Spread over the entire cut surface of the tuber. Then the two halves of the tubers were fastened tightly together again and put in a moist chamber and kept in the laboratory in which the temperature ranged from 250 c. to 280 c. After twelve days rot was observed in all the cut surfaces and microscopic examination showed that the causal organism was Phytophthora sp. 39. DISCUSSION AND CONCLUSIONS Two hundred and three isolates were made during the study as listed in the Tables I, II, III, IV. Many of these organisms were duplicates. Consequently, one hundred and seven apparently different organisms were used for the pathogenicity tests. According to the results listed in Tables V, VI, VII, VIII, IX, X, and XI, forty-eight organisms were pathogens. The identification of these different pathogenic organisms was accomplished by reference to the key made by Clements and Shear (13). It was found that most of them belonged to the genus Fusarium which is characterized by the sickle-shaped spores. Some of them (No. 99, No. 98) belonged to the genus Phytophthora, which according to the microscopic studies made by Jones, Giddings and Lutman (27), are probably of the species infestans. The other organism (N0. 239) belonged to the genus Alternaria which according to the description discussed by Stevens in Plant Disease Fungi (44) is probably of the Species solani. The other (No. 165), according to E. A. Bessey's classification and to the cultural comparison studies made by the writer, was found to be a Botrytis. Since the bacterial organisms isolated from the rotten tubers did not infect the sound tubers, they were not pathogenic. Consequently they were disregarded. 40. It was stated before that most of the pathogenic organisms belonged to the genus Fusarium. Because of the instability of the members of this genus, scientists divided it into different sections. Each section was divided into species. To classify these organisms which were already established as pathogenic, the writer followed the information and the key advised by H. W. Wollenweber, C. D. Sherbakoff, O. A. Reinking, Helen Johann, and Alice A. Bailey (55). The organisms were grown on different media; rice, sweet clover stems, and potato dextrose agar of 0.5% sugar for 15 days. On the rice medium the acid modification is of considerable help in the identification of the sections. On sweet clover stem, color of spore mass is well produced. On potato dextrose agar the macroconidia have more uniform shape. The temperature of the laboratory during the growth of the organisms ranged from 26.50 C. to 25.50 C. The humidity ranged from 80% to 52%. The results of this identification are listed in the following table. 41. TABLE XI Identification results of the Sections of the genus Fusarium. Section Elegans Section Martiella Section Ventricosum Pathogen No. Pathogen No. Pathogen No. 91 111 210 ? 93 115 203 117 206 134 209 135 211 136 118 138 123 238 125 240 126 242 127 245 128 250 141 251 144 252 146 253 147 149 150 152 203-1 181 188 191 213 228 233 254 258 42. From the foregoing data listed in the Tables I to XI the rotting of the potato tubers in the counties from which rotten specimens were collected was caused mostly by Fusarium, Phytophthora, Alternaria, and Botrytis organisms. The Fusaria were present in the greatest number. Within the genus Fusarium the greatest number of pathogenic species belonged to the Sections Elegans and Martiella (Table x1). The data listed in the above Tables do not repre- sent those for the whole state of Michigan, but only for certain counties, namely: Missaukee, Presque Isle, Montcalm, Newaygo, Livingston, Cass, Oceana, Allegan, Midland, and some miscellaneous areas. Hence, we can not assume as a valid conclusion the prevalence of these different tuber rotters over the entire state. On the other hand it was stated that the largest number of the pathogenic organisms belonged to the Sections Elegans and Martiella and those organisms were- found in most of the counties cited above. Consequently, they were responsible for the rotting of potato tubers in these areas for 1949 and according to these results we can conclude that continued studies must be made on these different species which will help to set up a control program in the future. 43. SUMMARY The potato crop is one of the most important crops in the world. Many people use it for their chief carbohydrate supply. Potatoes are grown in almost all the counties of Michigan. They are harvested in September of each year. About one-third of the crop is sold to truckers at harvest time, one—third is stored in cooperative warehouses and the rest is stored on the farm. Three handlings are made before the tubers reach the storage. Experiments (16, 43) showed that the increase in damage is proportional to the amount of handlings. Potato diseases are widespread in the world, being found in any area where potato plants occur. In Michigan potato tubers are affected by different organisms such as bacteria, fungi, etc. Some bacterial organisms are Erwinia carotovora (Jones) Holland, Corynobacterium sepetonicum (Speckermann and Kotthoff) Skaptason and Burkholder. Some fungal organisms are Fusarium gp., Phytophthora infestans (Mont) deBary, Alternaria solani, (E and M) Jones and Grout, and Armillaria mellea (vahl) Quel. Most of the organisms are soil inhabitants. Many of them start to infect the tuber in the field, especially at harvest time, and they continue their effects in storage. Some of them are purely storage rotters. Two 44. hundred three isolations were made from different varieties grown in the following counties: Montcalm, Presque Isle, Oceana, Cass, Missaukee, Allegan, Living- ston, Newaygo, Midland and miscellaneous areas in Michigan. Proof pathogenicity tests showed that forty-eight organisms were pathogenic. Most of them were Fusaria. Some of them belong to the following genera: Phytoph— thora, Alternaria, and Botrytis. All bacterial organisms isolated from the different specimens were non-pathogenic and were disregarded. The pathogenic organisms were found in the follow- ing counties: Missaukee, Livingston, Presque Isle, Montcalm, Cass, Newaygo, and miscellaneous areas in Michigan (see map, p. 50). The classification of the pathogenic Fusaria showed that they belong to the following Section: Elegans, Martiella and Ventricosum? Organisms belonging to these Sections were isolated from specimens collected in the following counties: Missaukee, Livingston, Presque Isle, Montcalm, Newaygo and miscellaneous areas of Michigan (see map, p. 50). Consequently, the organisms reSponsible for the rotting of potato tubers either in the fields or in storage, in the different areas stated above, were the organisms which belonged to the Sections Elegans and Martiella. 10. ll. 12. 45. LITERATURE CITED Artschwager, E. F. 1927. Wound periderm formation in the potato as affected by temperature and humidity. Jour. Agr. Research. 35: 995-1000. __ Bailey, F. D. 1914. Notes on potato diseases from the Northwest. Phytopath. fl:32l-322. Bennett, J. P. and E. T. Bartholonew. 1924. The respiration of potato tubers in relation to the occurrence of Black Heart. Cali- fornia Agr. Exp. Sta. Tech. paper 14, 40 pp. Bliss, D. E. 1946. The relation of soil tempera- ture to the development of Armillaria root rot. Phytopath. 36_: 302-318. Bonde, R. and Schultz, E. S. 1943. Potato refuse piles as a factor in the dissemination of late blight. Maine Agr. Exp. Sta. Bul. 416, pp (229)- 246. Bonde, R., 1939. Comparative studies of Bacteria associated with potato black leg and seed—piece decay. Phytopath. E9:831-851. Brierley, Ph. 1928. Pathogenicity of Bacillus mesentericus, E, aroiedeae, E, carotovorus, and E, phytophthorus to potato tubers. Phytopath. l§:819-938. Bruyn, H. L. G. de. 1926. The over-wintering of Phytophthora infestans (Mont) deBary. Phytopath. ll§:121-140. Burkholder, W. H. 1938. The occurrence in the United States of the tuber Ring Rot wilt of the potato. Amer. Potat. Jour. 15:243- 245. '7— Butler, E. J. and Jones, S. A. 1949. Plant pathology. London. Carpenter, C. W. 1915. Some potato tuber-rots caused by species of Fusarium. Jour. Agr. Res. 2:183-210. "' ' Clinton, G. P. 1895. Fungus diseases of potato. l O. l3. 14. 16. l7. 18. 19. 20. 21. 22. 23. 24. 25. 46. Clements and Shear. 1931. The genera of Fungi. The H. W. Wilson Company, New York. Coons, G. H. and Kotila, J. E. 1923. Michigan Potato disease. Agr. Exp. Stat. Mich. Agricultural College. Botanical section. Special Bulletin No. 125. DeBary, Anton. 1863. Recherches sur le developpe- ment de quelques champinions parasites. Annales des Sciences Naturelles, Botanique, sec. 4. vol. 20, 1863. pp. 5-148. Edger, A. D., C. H. Jefferson and E. J. Wheeler. 1942. Potato storages for Michigan. Michigan State College. Agri. Exp. Sta. Section of Agr. Engineering. Special Bulletin 320. Folsom, D. 1933. Botrytis cinerea as a cause of potato tuber rot. Phytopath. .33z993-999. and Reiner Bonde, 1925. Alternaria solani 7 as a cause of tuber rot in potato. Phytopath. V0 Ell-j“: 282’2860 Gardenburg, E. V. 1949. Potato Production. Comstock Publishing Company, Inc., New York. Goss, R. W. 1940. A dry rot of potato stems caused by Fusarium solani. Phytopath. .39:160- 165. . 1921. Temperature and humidity studies of some Fusaria rots of the Irish potato. Jour. Agr. Res. ‘EE:65-80. Gratz, L. O. and Bonde, R. 1927. Infection of potato tubers by Alternaria solani in relation to storage conditions. Fla. Agr. Exp. Sta. Bul. EEZ. pp(165)—182. Hilborn, M. T., and Bonde, R. 1942. A new form of low temperature injury in potatoes. Amer. Potato. Jour. ';9:24-29. Jamieson, Clara 0. and Wollenweber, H. W. 1912. 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The survival of the pathogen in the soil and some factors influencing infection. Phytopath. 29:215-228. Link, G. K. K. 1916. A physiological study of two - strains of Fusarium in their causal rela— tion to tuber rot and wilt of potato. Bot. Gaz. 62:169—209. (Reprinted as Nebr. Agr. EEE. Sta. Res. Bul. 9, 45 pp. illus. 1916). and Gardner, M. W. 1919. Market pathology and market diseases of vegetables. Phytopath. 9;497-520. Moore, H. C. 1939. Better potatoes for Michigan. Mich. Ext. Bul. 49. iuncie, J. H. 1942. Michigan potato diseases and their control. Michigan State C011. Ext. Div. Extension Bul. 162 (Revision). . 1941. Late blight of potato. Michigan State College Ext. Div. Extension Bul. 226. Nielsen, L. W. and F. A. Todd. 1946. Bacterial soft rot of Irish potatoes as influenced by sublethal temperatures. Amer. Pot. Jour. 23:73—87. Peters, E. J. 1943. Stem—end vascular discoloration of potatoes due to Fusarium oxysporum, F. tuberosi. Amer. Potato Jour. .gg;10-12. 48. 39. Ramsey, Glen, B. and J. S. Wiants and M. A. Smith. 1946. Market diseases of fruit and vege- table. U. S. Dept. of Agr. Miscellaneous publications No. 98. 40. . 1941. Botrytis and Sclerotinia as potato tuber pathogens. Phytopath. El: 439-448 0 41. Riker, A. J. and Riker, R. S. 1936. Introduction to research on plant diseases. John S. Swift 00., Inc., St. Louis. 42. Rose, D. H. and D. F. Fisher. 1940. Desiccation effects on skinned potatoes. Amer. Potato Jour. 'EZ:287-289. 43. . 1946. Handling and shipping early potato. U. S. Dept. Agr. Circ. 744. 44 pp. 44. Stevens, F. L. 1925. Plant disease fungi. Mac- millan 00., New York. 45. Stuart, W. 1937. The potato. J. B. Lippincott Co., New York. 46. U. S. D. A. and Michigan Dept. of Agr. 1940. Crop Report for Michigan. 47. . 1947. Crop Report for Michigan. Jan. Feb. 48. . 1948. Crop Report for Michigan. Jan. Feb. 49. . 1949. Crop Report for Michigan. Jan. Feb. 50. . 1950. Crop Report for Michigan. Jan. Feb. 51. . 1949. Agriculture Statistics. 52. Wiss, F. 1928. Factors in the inception and development of Fusarium rot in stored potatoes. U. S. Dept. Agr. Tech. Bul. EE. 36 pp- 53. Wollenweber, H. W. 1913. Studies on the Fusarium problem. In Phytopath. .;:(1). p. 24-50. 54. . 1914. Identification of species of Fusarium occurring on the sweet potato, Ipomea Eatatas. Jour. Agr. Res. E:251—286. 49. 55. Wollenweber, H. W., C. D. Sherbakoff, O. A. Reinking, H. Johann, and A. Bailey. 1925. Funda— mentals for Taxonomic Studies of Fusarium. Reprinted from Jour. of Agr. Res. 30:(9) 833-843. Washington, D. C. '—— 50.‘ Isle Royale Distribution of sample collected from Michigan . 4M 0 1949 L i r v . run / _ L. g- I _I .. | j ' Ontonagan r' 5,“ Samoa 3 l L-.. - 3| I" 1-.-. ! 2; i . . Gagebuc 7 l . .Morquetle r_ _____ J Luce | K I u— — —I.— — — J i | I K D ' I l ‘ . ChI ewo ‘ ' F'_‘JUOP0 I I pp \. D . - l I a I __l_ : O 3 O n r 3 0| 0 DJ lg ' O | ‘I I r I I l I I J I--—~--—-—-' --. ‘LSflchoolcraH iMOCkanC /'—- I - Ila I" T"’ / I r" H I i . I 9 ’ ‘Q ' 0 .4 C | 5 I ' Chebquonr 5 .9 i ‘5' I l=II I i Presque Isle ho—v—I‘ ICtsego iMonlmorenCyl Alpena O) E Oscoda IAIcona I I _ _ ’7‘: - _ — Crow—ford :— I I I l B Gr and I enZIei Traverse I ' i —————— *—--—-————‘——-—- —-—‘ Manistee. Wexlord IMIssaukee Roscommoniogemaw Ilosco I ; I . ' I I V I - J 1 Osceola IClare IGladmn I ——..___-_ _._-—-—_‘_____-—_ I—-—-—.__ Ionia TCIinIon IShiowasse i l i ! i ..... —L-——-- h-m-T-—-+-—-T-—-.L.-—.q . . ' Allegan Barry -Eaton ilnqharn Lwnngsloni I ‘ 9 ' VII ' @ ® I . I 7 ....... . --_-.L-._-_-T.- -_._-_._-_._-_..L- 7-..---_.._-- Van Buren -Kolomazoo Calhoun idackson Wash-lenaw iWayne I . . -1 ' l I I I . L ............... -i. ----- b-J '''''' L-—.“ iCOss St. Joseph“ Branch FHIIISCIOIG Lenowee Monroe I i i i I 00—.O'—IO_J‘ 1. Roman numerals represent the counties in which rotten specimens were collected. 2. The Arabic numerals represent the districts in Michigan. “‘1 AAAAA ”’II'I‘IIII IIIIIIIIIIIIIII’IIIII’IIIII 96985