STUDIES ON EESISTANCE IN CERTAIN POTATO HYBRIDS TO BACTERIAL RINGROT CAUSED BY CORYNEBACTEF.IUM SEPEDONICUM (SPIECKERMANN AND KOTTHOEE) SNAPTASON AND BURKHOLDER Frank Vernon Stevenson A THESIS Submitted to the Graduate School of Michigan State College of Agriculture and Applied Science in partial fulfilment of the requirements for the degree of MASTER OP SCIENCE Department of Botany and Plant Pathology 19UH ProQuest Number: 10008720 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest ProQuest 10008720 Published by ProQuest LLC (2016). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code Microform Edition © ProQuest LLC. ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 Acknowl edgment The writer is indebted, to Dr. J. H. Muncie for his help and suggestions in handling the material and to him and Dr. E. A. Bessey for their valuable suggestions incorporated in the preparation of the manuscript. Table of Contents Page Introduction....................... ..................... ...... 1 Name of the Disease.............................. 1 ......... . 2 Economic Importance, Distribution.................................................. 2 Transmission and Spread*....... .................... ........ 2 Causal Organism..................... ......................... 3 Nutritional Requirements.............. U Burkholder1s Medium............. U 5 Colony Characteristics,...... ............. ............... Synonymy............. ....................... . 5 ......................... .......... 3 Host Range, Symptoms.............................. ..................... . 6 ........... 6 Foliar, Tuber.............. 6 7 Diagnosis. ........ ............................... . 10 Ultra Violet Light Diagnosis...................... Oram Stain. ................................. 10 Gram's Modif ic ations of Lugol'sI odine.............. . 11 Burke’s Modification of the GramStain..................... 11 Varietal Resistance. .... .................. 13 Inoculation Technic ...... lH Method of Reading. .............. l6 Results of 19^2 Experiments.................... 17 Emergence,................................................. 17 Michigan State Seedling Varieties.......................... 1J U.S.D.A. Seedling Varieties............. 18 Varieties Used in 19^3** .............. 20 ................ 21 Results.... * Discussion and C o n c l u s i o n s ......... 22 Summary. .... ..................................... ,2h 26 Literature Cited................ Table 1. Emergence and Presence or Absence of Ringrot in " Seedling Varieties of Two U.S.D.A. Crosses Pedigree Nos.185 and B186 in Tests at East Lansing, Michigan, in 19^3...... 29 Table 2. Presence or Absence of Ringrot Infection in 32...... Selections Prom 18 Hybrid Varieties and Selections Carried, at Michigan State College Inoculated and Tested at East.... Lansing, Michigan, in 19^-2............ 30 Table 3* Emergence and Type of Response to Ringrot Inoculatim of Seventeen Crosses or Varietal Selections, Mostly Obtained From the United States Department of Agriculture, as...... Determined in Field Trials at East Lansing, Michigan, in... 19U3............................................ 31 Figure 1. Prevalence of Ringrot in the United States of..... America in 19^0....... 33 Figure 2. Ringrot in Michigan Including I9UU Reports — by... Counties. .............. 3^ INTRODUCTION The potato occupies sixth place among the crops grown in the United States as determined by acreage and value of the crop pro­ duced, As a table food product, it undoubtedly is only second to that of wheat (28), The potato is a very large factor in the economic set-up of Michigan as it is the number one cash crop of the state (17). A considerable part of this acreage is in certified seed production both for use in the state and in adjacent areas. In 1931* Conners reported a serious new disease of potatoes in Canada (8). The first published report regarding this disease was by Spieckermann (2^-) in an article in a German illustrated farming paper in 1913* Donde stated in 1937 that he had seen it in this country as early as 1932 (l). It was first suspected to be present in Michigan by Muncie in 1939 and the suspicion was confirmed in I9H0 (18). Considerable attention has been given this disease because of its rapid spread and its heavy toll in those fields wherever it has taken hold. Name of the Disease In first publishing on this disease, Savile and Racicot (22) called it "Bacterial Wilt and Rot," terial Wilt and Soft Rot" (l). Bonde used the name "Bac­ Since "bacterial wilt" was already in use for a disease of potatoes caused by Phytomonas solanacearum and "soft rot" for one caused by Erwinia carotovora. it was de­ cided by the Committee to Coordinate Research on New and Unusual Potato Diseases to translate the descriptive German "Ringfaule" and call the new disease "Ringrot" or "Bacterial Ringrot" (lh) and thus avoid the confusion which would result from using names which were already in use for diseases caused by other organisms. Economic Importance Losses due to the disease have been extensive and severe in localities where it has become established. mittee states that: The above-named com­ "It is evident from the letters received by the Committee that ringrot is rapidly becoming one of the most de­ structive diseases of the potato in this country. — — The impera­ tive necessity for both extensive and intensive research has been emphasized by all collaborators" (10), Distribution The known distribution of ringrot in the United States is shown in Figure 1. This shows it to be present in greater or lesser degree in 37 of the US states; nine of them for the first time in 19^0 ( 29), Further search will probably show it to be in more of the remaining 11 states since many of those now reported as free are bounded by states already reporting the disease. In Michigan, the disease has been reported from 2U widely scattered counties. These are shown in Figure 2. These findings are based on stain tests of material received in the Botanical Section of the Experiment Station from county agents and potato inspectors. Transmission and Spread ProbabLy the principle method of spread is in diseased tubers although it may be carried along for considerable time on machin­ ery such as the grader, planter, and digger, in sacks, bins, crates, warehouses and other articles coming in contact with diseased potatoes, Dykstra, in discussing the means by which the pathogen sur­ vives from season to season, states that the California station found little if any over-wintering of the pathogen in the soil, Bonde showed, however, that in Maine diseased tubers may survive and give rise to volunteer plants (2) which may harbor the disease. Brentzel and Munro working in North Dakota reported that in­ sect transmission experiments, with grasshoppers as vectors, gave uniformly negative results (U), list and Kreutzer, of Colorado, indicate that grasshoppers, Colorado potato beetle, and black blister beetle are capable of transmitting the disease, but it is doubtful if insect transmission in the field is a problem (15). Experiments at various experiment stations wherein a cutting knife was contaminated and then used to cut a series of healthy tubers which were planted in the order cut have shown ringrot promiscuously throughout the lots and as high as the 2hth hill, Dykstra reported that California workers showed the cutting knife to be the most effective agent in the spread of bacterial ringrot (10), Kreutzer et al of Colorado (12) and Starr and Riedl (26) of Wyoming report good results in holding down spread by the cutting knife through the use of a power-driven revolving knife which is partially immersed in a disinfecting bath. Causal Organism The organism causing ringrot is a small (,~{2 p , ), by 1 25jj non-encapsulated, non-motile, non-sporulating, rod-shaped bac- - U~ terium occurring singly but rarely. Most often, the rods are paired and occasionally chains of four or more will be seen. The staining reaction is Gram-positive although variations are quite often present. Pleomorohism is frequent and enlarged, club- shaped cells result. According to Skaptason and Burkholder, divi­ sion is by "snapping" and V- or L-shaped pairs are fairly common ( 23). Nutritional requirements The organism is quite fastidious and this factor makes it difficult to grow on ordinary nutrient media as the rate of growth is very slow and colonies are quite small. The use of an enriched medium devised by Burkholder gives quicker growth and more certain results (5). Burkholder's Medium: 300 grams of sliced potatoes boiled, filtered and broth made up to one liter with distilled water. Peptone 5 grs N«gBP0 2 11 NaCl 2 H Sodium Citrate 1 II Asparagine 6 If Dextrose 6 II 12 II Agar Marten et al of West Virginia showed that the addition of a solution of C. P. potassium dichromate at the rates of 1:9000, 1:12,000 or l:15*000 to the above agar is very helpful in keeping - 5- down Gram-negative contaminants when making isolations (l6). Colony Characteristics Colonies average about 2mm. in diameter. They are cream colored, wet, glistening, and rather sticky in consistency. Synonymy The organism was first called Bacterium sepedonicum Spieck. and Kot., in 191^. E. F. Smith then named it Aplanobacter sepe­ donicum (Spieck. and Kot.) E. F, S., in 1920; Magrou put it in the genus Pbytomonas as Phytoroonas sepedonica (Spieck. and Kot.) Magrou in 1937* ond. later in 1937* Savile and Racicot again con­ sidered it to be a member of the Phytomonas group. The name Corynebacterlum sepedonicum (Spieck. and Kot.) Skap. and Burk, was given by Skaptason and Burkholder in 19^2 because the presence of numerous pleomorphic forms and the "snapping" type of division taken together with its other characteristics seemed to indicate the organism to be a member of the diphtheria group (23). Dowson places all Gram-positive plant pathogens in the genus Corynebacterium (9)» Host Range All commercial varieties of the potato are susceptible (10). Rupert (20) tried inoculations on eight other related hosts and of these he found the tomato (Lycopersicum esculentum) to be the only one showing definite infection. Stapp, working in Germany, reported infection of tomato, Pjsuffl arvense. and Phaseolus vulgaris by artificial inoculation (27). - 6- Symptoms Foliar Symptoms on the potato plant in the field are a slight graying with a later yellowing and wilting of the leaves. Frequently, not all branches are infected, but the infected branches droop or curl downward on the ends and the leaves roll upward. Often all that is affected is a part of a shoot or as little as one side of a single leaflet. and dry. Ag the condition advances, the parts turn brown There is no apparent browning of the stem although the organism may be found in abundance up to a height of three inches. Kreutzer and McLean found that a rapid decrease in numbers oc­ curred as the height above that point increased (13). Tuber In the tubers, the symptoms of severe infection are a shrink­ ing and cracking of the skin. These cracks may extend below the skin as far in as the vascular ring. If such a tuber is cut across, especially near the stem end, it will frequently show a yellowing of the vascular ring, although the disease may be present without showing this coloring. In advanced stages, a soft cheese­ like exudate may be squeezed out of the ring in a manner suggest­ ing toothpaste out of a tube. cortex from the vascular ring. It is often possible to "peel" the If there are no secondary rots, such as that caused by Erwlnla carotovora. the tuber will become a hard leathery mummy. Often the decay is more general than that described above and the crumbly condition extends far into and often entirely through the tuher# In such cases, a honey-combed mummy is all that is left# This mummy is also firm and dry. One striking difference between ringrot and other potato rots is that it is entirely devoid of a disagreeable odor. Many tubers having ringrot are a stinking, slimy mass, but this is due to the presence of secondary invaders. The disease is not readily identified by the field symptoms as they are not distinct and may be confused in certain stages by the presence of blackleg or the browning caused by Fusarium wilt. Because symptoms appear so late in the growing season, early and late blights, insect injuries and especially maturation of the plant tissue, serve to confuse the picture further. Many tubers do not break down, if at all, until they have been in storage for many weeks, but even though they may fail to show any symptoms, the presence of typical Gram-positive staining bacteria in the vascular ring proves them to be affected with the disease. Diagnosis Because of the uncertainty of identification of ringrot from symptoms visible on the tops and tubers,, due to its highly variable character, and through the presence of other conditions which serve to confuse the observer, it becomes necessaiy to rely on some other technic for certainty in diagnosing this disease. The slow growth and inactive biochemical nature of the organism make the use of fermentation indicator technics practically an impossibility. Gram-stain seems to be the method of choice. The This is unfortunate because the Gram-stain is not intended to be a final diagnostic test. The Gram-stain is considered to he reliable only for young actively growing cultures as it has been shown, according to Salle that older cultures become quite Gram-variable (2l). The age of the organism in a stored tuber can only be guessed at. Since the identification of the ringrot organism is usually made from smears of diseased tubers and because these bacteria are so slow-growing, much variation is bound to occur* According to Salle, "The change of Gram character with age is especially true of those organisms which are only weakly Grampositive and are cultivated in media containing fermentable sub­ stances that become acid in reaction as growth proceeds." He also shows that little of either stain is taken up at the iso-electric point and that the Gram character of many organisms can be shifted by the addition of suitable acid or basic substances to the medium One or more of these conditions may well explain such stained material as is found where every other condition except the Gramstain reaction indicates the presence of ringrot. The author has had stained material wherein all but a few pairs of bacteria were red, or one half of the pair stained red and one blue. In others which were all doubtfully reddish-blue or negative, all other con­ siderations such as size, shape, numbers, and condition- of the tuber were strongly indicative of ringrot infection. In this laboratory, most of these would be marked + indicat­ ing them to be, at most, suspicious. This author believes that seed lots containing a number of tubers unrecognized as infected, thus may be allowed to continue in growers 1 hands for a consider­ able period until conditions become such that the bacteria recover their Gram-positive expression and are then recognized. A definite indication of this being possible was to be seen in a case involv­ ing one grower's certified seed. The author stained some smears which gave a + reading because of their reddish-blue color, as above, and was told this same rating had been given this grower's potatoes in previous readings, later in the year, material was returned from a car lot of the same grower's seed that had been shipped out of state and refused because of ringrot. On staining, the organism was found in abundance and was clearly recognizable so that this lot which had merely been under suspicion was now proved to have the disease* The return to an active growing state of the organisms present or the influence of other more obscure balances of factors such as oxidation of products, or change in the metabolic rate, or some other conditions, had produced a condi­ tion more nearly optimum for the recovery of expression of the Gram character at some time subsequent to the first examination. It is also possible that in selecting rotted tubers to be sent in for diagnosis, the inspector did not originally select the tubers badly infected with the ringrot organism. The presence, however, of organisms, which were morphologically those of ringrot but doubt­ ful as to staining reaction strongly indicate some change in dye absorption, and such behavior, coupled with the uncertainties of the Gram^-stain technic mentioned before, may thus be accountable for failure of recognition in the stain test. It is possible, therefore, for an incorrect reading to be made if too much stress - 10- is laid on Grsm reaction color, "Ultra Violet Light Diagnosis A technic for use in the H0°F. seed storage at the time of cutting seed is based on the fact that ringrot tubers fluoresce a characteristic greenish color, in the vascular ring, under ultra violet black light (ll). It should be emphasized that thorough experience with the technic combined with a tendency toward a heavy discard of all suspicious looking tubers seems to give good results* It cannot, however, take the place of the stain technic for laboratory diagnosis. Burkholder reports that Skaptason found the fluorescing substance to be riboflavin, and he says that this is by no means a specific test as other bacteria synthesize this same vitamin. It is further pointed out that riboflavin is rapidly destroyed at high temperatures and especially under ultra violet light (6). Gram Stain In working on a new tecbnic for staining bacteria in tissues, Christian Gram (188H) discovered the differential principle of the stain which now bears his name (2l), "The usual procedure calls for anilin gentian violet for 1 to 2 minutes; blotting without washing; Gram's iodine solution for 1 to 2 minutes; blotting without washing decolorization in 95 per cent ethyl alcohol for 30 to SO seconds; blotting, and counterstaining briefly ip eosin, safranin, fuchsin, or Bismark brown( 7)•" Gram’s solutions: Anilin Gentian Violet Sat, Ale, Sol, gentian violet..........520 c.c. Anilin water (2 c.c, anilin shaken with 9S c.c. water and filtered).........100 c.c. — 11- Gram*s Modifications of Lugol*s Iodine Iodine... 1 g. Potassium Iodide..................... 2 g. Distilled Water.......................3^0 c.c. Counterstain Safranin (2.5 per cent solution in 95 P er cent ethyl alcohol).................. Distilled Water In this 10 c.c. 100 c.c. study,a modification of the Gram stain by Burke was used as a basis for further modification (25). Burke*s Modification of the Gram Stain •'Hood a thin film with a 1 per cent aqueous solution of methyl violet. (Add to and) Mis’with the dye 3 to 8 drops of 5 Pe** cent solution of HaHCO,* Allow to stand 2 to 3 minutes. Plush off excess^stain with Lugol's iodine solution. Cover with fresh solution and let stand one minute or longer. Wash in water briefly and remove practically all of the water by blotting, but do not allow smear to become dry. De­ colorize with acetone-ether (3 to l) until decolorizer flows from the slide almost uncolored (usually less than 10 seconds). Wash with water, blot or air-dry. Counter-stain, using 2 per cent solution of Safranin 0. Wash briefly with water, blot and dry quickly." This was somewhat changed in that the recommendations of Professor G. B. Reed as given by Racicot, Savile and Conners (19) were followed in making up the following solutions: 2.5 gms. Solution 1. Crystal violet............... .. Water................................ 1000 c.c. " 2. Sodium bicarbonate................... 12.5 gms. Water. .1000 c.c. " 3 * Iodine 20 gms. Sodium hydroxide (molarsolution)... 100 c.c. Water............. ..... ........... 900 c.c. " U. Ethyl alcohol,95 per cent.......... 750 c.c. Acetone...................... ..... 25O c.c. " 5* Basic fuchsin, saturated solution in 95 P er cent alcohol...... .. 100 c.c. Water........................ ....... 900 c.c. - 12- Tha last five solutions mentioned above were originally in­ tended to be used in a very rapid staining technic, but this author returned to more conventional periods of time for allowing the solutions to remain on the smears. T^e slide, with the smear affixed, was first flooded with the sodium bicarbonate solution because less trouble from crystallizing out of the crystal violet seemed to result than when the usual order of crystal violet followed by sodium bicarbonate was used. The crystal violet solution was then added drop by drop until the smears were well covered and this mixture was allowed to stand exactly two minutes. The slide was then drained and the iodine mordant flooded on and left for one minute. The slide was flushed using a washing bottle and then decolorized using a medicine dropper to apply the acetone-alcohol mixture. Decolorizing was continued for some time until the solution ran clear from the slide. The slide was washed thoroughly, using the wash bottle and then flooded with the counterstain. This was washed off almost immediately and the slide blotted and dried by holding it above the flame of a Bunsen burner. The stained smear was then examined under oil immersion using oil of cedar because of its excellent light conducting qualities. The smears were prepared by scraping out the whole of the vascular ring in the area exposed by cutting off the stem end of the tuber, as near the end as possible, with a sharp-pointed scalpel, and then pressing this material on the slide and squeezing out some of the juice until it appeared milky. Each slide was marked off - 13- into ten squares with a wax pencil, and a small area on one end was left to allow for identitying the slide and for gripping it during staining operations. Tor ease of examination, the order on the slide was from one through five across the "bottom from left to right, then up to the right-hand square above for six, and thence to the left through number ten, thus finishing above number one. Since so much stress in this method is placed on color, the microscope light is an important factor. In this study a Spencer 370 lamp, using a 100 watt projection type bulb, was used. an iris arrangement whereby the light can be focused. It has The dis­ tance from filament to the flat side of the mirror was ten inches. A blue filter was used. The beam of light was focused with the iris until it just filled the field using the low power objective. The substage condenser of the microscope was kept at its highest level. All readings of stained material were made with the light as above and using the oil immersion lens. Varietal Resistance Attempts so far made to control ringrot by treating the seed pieces have been too uncertain in results and have in many cases materially reduced the stand, and, as a consequence, emphasis is being placed on the problem of developing or finding varieties re­ sistant to ringrot. Results from Wyoming, based on a. reading of field symptoms, as reported by Dykstra (10), show only United States Department of Agriculture seedling as having no symptoms by the time of frost which occurred shortly after the September lgth reading. -lH~ Bonde et al also have reported, in preliminary observations of hybrid potato material, a possibility of inherited differences with regard to resistance to bacterial ringrot infection (3 ). In order to see if such differences do exist and to determine if some of these strains might be suitable for parent material, which could be used in developing higher resistance, it seemed desirable to test some of them more fully. In the au t h o r ^ test, stress was placed on the actual pre­ sence of the bacteria in the tuber rather than on the presence or absence of field symptoms. The potatoes used in the 19U 2 study were grown from tubers of United States Department of Agriculture seedlings from the crosses President x Earlaine (Pedigree No, B185) and President x 96 - 56 (Pedigree No, Blg6). There were 797 tubers of the first and 776 of the second cross. In addition, 32 selections of 18 hybrids carried at Michigan State College for scab resistance were in­ cluded. Chippewa was used as the check variety. Inoculation Technic In the Wyoming study cited above the inoculation technic was as follows: "The cut surface of the seed piece was smeared with bacterial ooze from a ringrot diseased tuber, and the whole seed piece was then submerged in a water suspension (of) bacterial ooze taken from diseased tubers." Bonde et al omitted the submersion in ooze and inoculation was affected by rubbing slices of infected Green Mountain tubers on the freshly cut surfaces of the healthy seed pieces. The needle method of inoculation was used in the present in­ vestigation because it had given 'quite a high incidence of in­ fection in previous tests at this station. All of the tubers tested in 19^2 were inoculated into each of two eyes per tuber. A "spear" point needle was used because it gives greater access to vascular tissue than does the stab of a straight needle. The needle was dipped into the exudate from a known ringrot tuber, just prior to inoculating each potato, and it was then thrust into the "eyebrow" and well into the flesh of the tuber in order to make certain that the vascular tissue had been penetrated and that an adequate amount of inoculum had. been placed sufficiently deep to secure infection. After inoculation, an incubation period of a week at room tem­ perature was given in order to allow the slow-growing bacteria to become established. The tubers were planted in the experimental plots at East Lansing on May 20, I9 U 2. The txibers were spaced 1^— 15 inches apart and covered with a hoe. The rows were three feet apart and were approximately 135 feet long. The plants were dusted as needed through the season using a fixed copper-calcium arsenate "Cuke and Melon" dust or "Talcla" with calcium arsenate added at the rate of one pound to twenty of dust to keep down the injury from early blight, hopperburn, and the Colorado potato beetle. The potatoes were dug the first week in October in separate hill lots. The tubers from each hill were put in a two-pound paper bag and these were collected in potato crates and stored in — a fruit cellar. 16— They were allowed to stand until Februaiy because it was thought that this would allow the disease to progress to a recognizable degree in those tubers having the organisms present, but not showing any symptoms. They were then removed to the labora­ tory and the readings made. Method of Reading To reduce the number of microscopic examinations necessary, each lot was examined macroscopically and the hill eliminated, if possible, by that means. The macroscopic inspection consisted of cutting off the stem ends of all the tubers from each hill and squeezing the m as they were cut to see if the "ooze" of bacteria would result. The yellow discoloration was not used as a basis for discarding a hill in this test as it was not thought to be con­ clusive enough. As soon as an oozing potato was found, that hill was eliminated and tabulated in the "Macroscopic positive" column. If none of the tubers showed this symptom, they were "apparently free" and a representative tuber looking the most suspicious with regard to ring color was used to make a stain test. These stains were made when ten "Apparently Free" hills had accumulated, A tuber found to have the disease on microscopic examination was classed "stain positive" and the remainder which had successfully passed both the macroscopic and microscopic tests were classed "Ringrot Free," See Table 1 . The Michigan State selections of seedling varieties were handled in the same manner except that one hill showing a ringrot tuber served to eliminate all hills of that selection. See Table 2 . -17Results of 19^2 Experiments Emergence Emergence was fairly low in the inoculated groups. Of the 797 tubers of Pedigree No. B 185 planted, ij-5^ or 57 Per cent emerged. Of the 77^ tubers of Pedigree No. Blg 6 planted, 501 or 65 per cent emerged. Seventy-three of the inoculated Chippewa checks emerged. Fifty tubers of each pedigree number, selected at random, and fifty of the Chippewa variety were planted uninoculated to check for the presence of ringrot so that the efficiency of the method of inoculation might be determined. All of these emerged and remained free of ringrot as determined by Gram-stain tests. Undoubtedly, much of the poor emergence in the inoculated groups was due to ringrot because in addition to the 27 per cent failure in the Chippewa check, h3 per cent of Pedigree No. Bl ?5 and 35 per cent of Pedigree No. Blg 6 failed to emerge or died before setting tubers as there was no evidence of them at digging time. Per cent of Disease-free Tubers Present. Michigan State Seedling Varieties. The selections of Michigan State seedling varieties are, re­ ported without percentages being figured as each selecti on number represents a variety. Those that were ringrot free were a four- hill lot of Selection 1 of the cross Eindenburg x 627-618, a twohill lot of Hindenburg x 8-1 selection unknown, a six-hill lot of the selection K 38- 8 , a six-hill lot of selection 3 of the cross 627-618 x Katahdin, an eigat-hill lot of selection 2 of cross - 18- 336-l^U x Ostragis, a six-hill lot of the variety K 38-7 and a three-hill lot of 528-118. (See Table ?) U. S, D. A, Seedling Varieties There is a noticeable difference in the ease with which the presence of ringrot may be detected macroscopically in the two U. S. D. A, groups. All but 11 of the 301 hills showing ringrot positive tubers were recognizable macroscopically in Pedigree Ho. B185, In contrast, 77 of the 33& positive for ringrot hills of Pedigree No. B186 passed the macroscopic test but showed in­ fection by ringrot organisms in the stain tests. The percentage of hills from emerging tubers free of the ringrot organism was the same in both of the U. S. D. A. groups being 33 P©r cent. Of the ^ V b i l l s of Pedigree No* B185 evident at the time of digging, 153 were free of the disease and of the 501 hills of Pedigree No. B186, 165 were also free of the disease. Since 35 P er cent of the inoculated check yielded negative Gram-stain reactions, it seems necessary to attribute at least a third of the ringrot free survivors, in the hybrid groups, to the failure of inoculati on. This would give 102 tubers of Pedi gree No. BI 85 and 110 tubers from Pedigree No. Blg 6 free of ringrot. In an effort to determine why such a high per cent of the check should give rise to apparently ringrot-free tubers, twentyfive Chippewa tubers used whole, cut, and in some cases as "hearts" (center portions containing no eyes which result when large tubers are cut for seed) were inoculated and allowed to stand in an un­ covered jar on the laboratory table until they had sprouted. - 19- It was interesting to note that of the three "hearts" included, one developed two sprouts. These were either from very inconspic­ uous eyes or adventitious buds which developed during the incubation period. These sprouts were free from the disease as the ringrot infection had not had sufficient time to progress from the point of inoculation to these buds before the shoots were big enough to have become firmly established. It might be possible, therefore, to have healthy plants, where all eyes were inoculated, through the agency of these very small insignificant eyes or adventitious buds. In a few cases, the sprout appeared to be healthy even though the eye had been inoculated and it thus seems possible that sprouts may become established and then softrot, or some other rots, would destroy the seed piece before the slow-growing ringrot organism could get into the daughter plant’s vascular system, again yield­ ing an "escape" plant. In some cases, sprouts were produced from uninoculated eyes of the inoculated tuber. Thus it may be seen that there are several possible ways in which healthy plants may arise from inoculated tubers and, considering these, the number of escapes does not seem too high. That ringrot may reduce emergence was shown by some inocu­ lated tubers in that they did not seen to be able to nroduce a viable sprout. The sprouts that attempted to grow were weak and the tips shrivelled and blackened and death occurred when they were about two inches in length. - 20- It was necessary to make further inoculations of the surviv­ ing material in order to determine if the survivors were escapes and to eliminate these from the material being tested. Four tubers of each Michigan State variety and one tuber from each ringrot-free hill of the U. D, A. seedlings were saved for testing in 19^3» Varieties Used in I9U3 In addition to the tubers just mentioned above, a number of seedling varieties from 15 U. S. D, A. crosses and selfed lines were included in the tests for the first time this year. Table 3« See Since all tubers, including those of the new accessions, were larger this year than last when two eyes were inoculated, they were inoculated into each of three eyes, incubated for a short time at room temperature, and planted on May 10, 19^3* The method of planting and cultural practices were essentially the same as in 19h2. The weather was unseasonably cold and rainy and very little growth was made early in the season. The ground was extremely moist, and, a.fter each rain, large puddles remained for consider­ able lengths of time at various points in the field. As a con­ sequence, there was a large reduction in stand, many tubers fail­ ing to emerge, and any attempts to compare most of the varieties according to the per cent of infection would necessarily lead to erroneous conclusions. However, even though the stand was re­ duced, where considerable numbers were present, the results give a fair indication of that variety's resistance because the sample is sufficiently large. That the inoculations were quite successful 21- *• is shown by the high proportion of tubers showing ringrot infection; there was a total of J20 out of the 753 plants giving that reading. The severe reduction in stand is shown in that, excluding the checks, of the 2631+ tubers inoculated and planted, only 753 hills or 29 per cent were evident at time of digging, The 50 Chippewa inoculated checks were very hard hit being on the low side of the field and only eighteen plants survived. eleven negative. Of these, seven were positive and The Michigan State varieties and selections were in this same portion of the field and it was only possible to identify and harvest a selection of Green Mountain and the cross 627- 6I8 x Katabdin Selection No, 3» ^he selection from the variety Green Mountain was obtained from H. C. Moore of the Farm Crops Department, as a suspected ringrot carrier which had been elimiated from bushel lots by the use of ultra violet light. It was included because it had failed to show a positive staining re­ action. The Chippewa uninoculated checks were favored in locati on, being on the high side of the field and J2 per cent or 36 out of 50 tubers survived. The results obtained with each different cross or varietal selection are shown in Table 3* Results The highest per cent of tubers apparently free from ringrot was from Cross B272 (President x 336-1M O which had 67.7 per cent showing negative Gram-stain reaction followed by Cross ®355 (96-56 x 336-IHU) with 66.9 per cent and the Cross B 27I (President x Earlaine) - with 63.0 per cent. 22- An intermediate group consisted of Cross BP 91 (Katahdin x 1+6952) with 57*1 per cent obtained from 7 tubers, Cross B 269 (Earlaine x 33^"^^) with 5^»7 per cent, and Cross B?7l+ (Presi­ dent x H 715- 6 ) with 55*1 P er cent of negatives. x ^ 6952), Crosses B 303 (Houma U 6 0 0 0 x U 6952) and B 3IK) (C. S. 1608 x Earlaine 2) had 27.7 per cent, 26.3 per cent and 21.U per cent tubers showing negative Gram-stain reaction respectively. The selfed line B1103 (President selfed) and the Cross B 316 (Friso x Katahdin) had too few survivors to warrant placing much confidence in their relatively high per cent of gram-negative re­ actions. This was also true of the Michigan State seedling variety and the varietal selection, and, to a certain extent, of the inocu­ lated check. Cross B? 6h (Irish Cobbler x Earlaine) had a very low survival rate and all of these plants gave positive Gram-stain re­ action for ringrot. Discussion and Conclusions The progenies of the two crosses in their second year of test­ ing have moved up from 33 P er cent showing negative gram-stain re­ action to 87.0 for Pedigree No. B 185, and 8U.7 for Pedigree No. Blg'6 . These are well above the averages of the other strains, which are present in sufficient numbers to warrant direct comparison, so it seems reasonable to attribute a large share of this rise to the elimination of susceptibles in the first year test. The high per­ centage of individuals negative for ringrot in the Blg 6 cross, being tested for the first time this year, is probahLy due to the fact that infected tubers were unable to survive in the field and so there was a preponderance of the disease-free tubers present at time of harvest. Probably the general tendency to a higher per cent of tubers showing negative Gram-stain reaction in all strains as compared to the rate of the first season is also due in part to the same response to adverse conditions, i. e., a low per cent survival due to disease and a correspondingly higher per cent of apparently ringrot-free tubers in the surviving plants. The frequency with which President appears as one parent, in those crosses having a high per cent of tubers testing negative for ringrot, shows it to possess some resistance and to be capable of transmitting this when used as a parent in making hybrids. Earlaine and the selections ?Bd also appear in crosses having a fair degree of resistance. On the other hand, Irish Cobbler, Strain C. S. 1608, and the varieties with numbers in the U 6000rs seem to be low or lacking in resistance. The results obtained on the tubers inoculated the second year strongly support the idea that resistance to ringrot is hetero­ zygous (3 ) and that progeny more resistant than either parent can be obtained from hybrids with resistant material. A final conclusion is that persons working with such material should examine their survivors microscopically because it is im­ possible to diagnose the disease by the plant symptoms or macroscopically in the ring of the tuber with the degree of accuracy that is possible with the stein test. This is shown by the number of stain nositives obtained from apparently ringrot-free material as .judged by the macroscopic test. — 2H— Summary The results obtained from two years* work on ringrot identi­ fication at Michigan State College are discussed with regard to the diagnostic technics used with recommendations regarding their use and showing a modification of the stain technic to give more certain results. The uncertainty of diagnosis of the presence of ringrot from symptoms on the tops or in the tubers forces the use of some other technic. In spite of its shortcomings, due to variability of rates of dye absorption under different conditions and in different strains of the bacteria, the Gram-stain seems to be the method of choice. A modification of this technic using stains intended for a fast technic but with more conventional periods of application is given. The importance of familiarity with the technic is discussed and the fact that improper readings may be made because of irregularities in dye absorption is stressed. The needle technic of inoculation was used and gave a high percentage of ringrot positives in the two years that this study was in progress. The survival rate was always lower in the inoculated groups, as compared to the uninoculated checks, and so a considerable portion of the failure is assigned to rotting by tie ringrot organism before the plants emerged or soon enough afterwards so that there were no tubers present at digging time. Cold wet weather in 19^3 materially reduced the stand, , but large enough numbers of several of the lines remained so that comparisons may be made. - 25- The seedling varieties used were from selections of hybrids carried at Michigan State College for scab resistance and from several crosses and selfed lines of U. S, D. A. material. Tables showing the results obtained from the inoculation of crosses and selfed lines of potatoes are given. The varieties President, Earlaine, and 336-lUU appeared frequently in resistant crosses. Materiel being tested the second year showed a tendency toward greater resistance because of the discard of susceptibles the preceding year. A comparison of macroscopic and microscopic readings showed a fairly high number of apparently ringrot-free tubers to give a positive stain test. Maps of Michigan and the United States of America are included and show the prevalence of ringrot by counties and states respec­ tively, - 26- Literature Cited 1. Bonde, Reiner. 2 ,_______________ 3* A Bacterial Wilt and Soft Rot of the Potato in Maine. Phytopath. 27:106- 108. 1937* Ring Rot in Volunteer Plants. Jour. 19:131-133, 19U2. , Amer. Potato P. J. Stevenson, C. I*. Clark, and R. V, Akeley. Resistance of Certain Potato Varieties and Seedling Progenies to Ring Rot. Phytopath. 32:813- 819. 19U2. 4. Brentzel, W. E. and J. A. Munro, Bacterial Ringrot of the Potato: Investigation on Possible Dissemi­ nation by Grasshoppers. N, Dak. Agr. Exp. Stat. Bull. ?95, 19UO. 5. Burkholder, W. H. The Occurrence in the United States of the Tuber Ring Rot and Wilt of the Potato. Amer. Potato Jour, 15:2^3-2^5. 1938. 6. Diagnosis of the Bacterial Ring Rot of the Potato. Amer. Potato Jour. 19:208- 212. 19^ 2 . 7. Committee on Bacteriological Technic of the Soc. of Amer. Bac­ teriologists. Staining Procedures. Pure Culture Study of Bacteria, 2(2): I V .-3 to IV , -2U. 101U. ^ 3>+ 8 . Conners, I* L. Canad, Plant Disease Survey Ann. Report 11:HP. 1932. 9 . Dowson, W. J. 10. Dykstra, On the Generic Name of the Gram-Positive Bac­ terial Plant Pathogens. Transactions of the British Mycologies! Society 19U1-U2 . T. P.Results of Experiments in Control of Bacterial Ringrot in IPUO. (A compilation). Amer. Potato Jour. 18:27-55* 19^1. 11. Iverson, V. B. and H. C, Kelly. A New Method of Identifying Potato Tubers Free from Bacterial Ringrot and Other Types of Tuber Decay. Mont, Agr. Exp, Stat. Mimeo, Cir. 20, 19^0 . 12. Kreutzer, W. A., D. P. Glick and J. G. McLean. Bacterial Ring Rot of Potato. Colo. Exp, Stat, Press Bull. 9U. 19U1. 17. and J. G. McLean. Location and Movement of the ~ ’ Causal Agent of Ring Rot in the Potato Plant, Col. Agr. Exp. Stat. Tech. Bull. 30* 19^3* - 27- lh, Leach, J, G, et al. Report of the Committee to Coordinate Research on New and Unusual Potato Diseases. Amer. Potato Jour. 17:81- 88. 19h 0 . 15- List, S, M. and W, A, Kreutzer. Transmission of the Causal Agent of the Ring-Rot Disease of Potatoes "by Insects, Jour, of Scon. Ent. 35:^55-^56* 19^2. 1 6. Marten, E. A,, C. V. Lowther, and J, G. Leach, A Differential Medium for the Isolation of Phytomonas senedonica. Phytopath. 33:h0S-h07. 19^-3 • 17. Moore, H, C. Better Potatoes for Michigan. U9» 1939. 18. Muncie, J. H, Bacterial Ringrot of Potato. 227. 19^1. 19. Rscicot, H. N.t D. B, 0 , Savile, end I, L, Connors. Bacterial Wilt and Rot of Potatoes — Some Suggestions for Its Detection, Verification, andControl. Amer. Potato Jour. 15:312-318. 1938. 20. Rupert, J. A. Investigations on Bacterial Ringrot of Potatoes caused by Phytomonas sepedonica (Spieckermann and KotthoffJ Bergey et al. Master's Thesis (Unpublished.) Michigan State College. 19hl. 21. Salley A. J. fundament al Princ iples of Bacteriology, p. 6l. McGraw-Hill Book Company. Hew York, 19h3» Mich, Ext. Bull. Mich. Ext. Bull. 22. Savile, D. B. 0 ., and H. N, Racicot. Bacterial % l t and Rot of Potatoes. Sci. Agric. 17:518-522. 1937. 23. Skaptason, J. B, and W. H. Burkholder. Classification and Nomenclature of the Pathogen Causing Bacterial Ring Rot of Potatoes. Phytopath. 32:h39-UUl. 19^2. 2h. Spieckermann, A, 25. Stafseth, H, J. A Laboratory Guide in Pathogenic Bacteriology. p. 6 . University Lithoprinters. Ypsilanti, Michigan. 19h2. 26 Starr, G. H. and. W. A, Riedl. Bacterial Rina-Bot of Potatoes. Wyo, Agr. Exp. Stat. Bull. 2hh. IP hi. Zur Kenntnis der in Deutschland auftretenden GefSsskrankheiten der Kartoffelpflanze. Illus. Landw. Zeitung 33:380-382. 1913* - 28- ?7* stapp, C, Beitrage zur Kenntnis des Bacterium sepedonicum Spieckerm. et Kotth. Zeitschr. f. Parasitenk. 2:756-822. 1929-30. 28. Stuart, W. The Potato, Company. 29. The Plant Disease Survey. 19^1. pp. 3“^» Bippincott pp. (New York.) 1937* Plant Disease Beporter 255 130. 50 50 50 0 0 0 © > H S n iH h- 1-1 43 0 «t£ fl fl I •H w CO 43 0 © to Ai 0 0 rH VO 60 oiqco pi 1 O iH CD O i-C 43 P fl p, *H S O O CO F 1 fl O O Scop, Pf H (o, U~\0 h-fl ir> rd fl ® © 43 P fl (0 9 rH P pi >3 43 © •ri fl cd !> r-vo 0 O' r— 0 r— 1 iH l P -d © ® O 43 fl CO 4H iH co > ® fl LflVO fl 60 60 1-1 iH iH id m pq 0 50 •d ® fl) fl ® S ® 50 50 fl CD ,£> H P 100 100 43 0 50 O' iH 1 CO •H ® 43 CO > O O 1-C E-t PM 43 fl o o •cJ fl ® 1-c 43 !§ Chippewa . KMPITi LTWD rH rH 50 50 © I—1f„d ♦ H © m C l ID O f l to p -h rH ! ® cO §n 43 fl 43 ® O •H O fl €4 Ai A B185 Bigg CD * c CD fl Ci a fl C w> ■fH l) l)•fH fl •H •d .d A © •0 A, iH «H *5* • O CO CD * © CO O CO s fl 0 •H © fl CO CO0 fl CO a •P <*{ +3 a • CO 0 CP 0 CD * fl03 +3 CD cd CO * © p CO fl 43 fl 0 CO £ © to • fl)6C fH fl rH 43 K\K36C 100 43 fl 43 CD O O fl fl) ffi fl fl © CD *H fl Ac pci «H 29- 100 100 - - 30- T a b le 2 Presence or absence of ringrot infection in 3^ selections from IS hybrid varieties and selections carried at Michigan State College inoculated and tested at East Lansing, Michigan, in 19^2. Ringrot Positive Cross or Selection Selection Ho, ■ Chippewa X Ostragis m 11 Chippewa X 8-1 ii X Katahdin ii X 627-618 Ogtragis X Katahdin ii 627-618 ii 11 X Ostragis if ii X Katahdin 11 X Katahdin X 627-618 Hindenburg x Katahdin ii ii 11 ii X 8-1 Russet Rural x Katahdin II 11 n 528-102 3c 336-lU^ :ic Ogtragis 336-lHU 528-118 K38-7 AKY-5 11 11 5 6 7 5 3 1 2 1 2 1 6 3 5 9 2 6 — 1 1 2 6 H 1 2 Ho, of hills 7 2 16 12 8 . — . 7 6 19 12 12 8 1 — 11 2 5 5 11 . 6 7 15 2 2 Ringrot Negative K 38-8 Hindenburg x 627—1 618 " x 8-1 627-618 x Katahdin 336-1UU x Ostragis 528-118 K38-7 .inr 1 — 3 2 1 1 6 U 2 6 8 3 - 31- 43 £ O KvO N H vo LT-fl ©V f t rk CTv m in cvj rk cvj m v o in inA i— r— r- © inm O O 60 -A cvi cu m in in h O h- cvivo CVJ CVJ rk rk m cvj c— f— cvj m A o h v d n r-vo cvvo incu cvj rk © O *H o P Ek Pd P «h 43 fl +> © rk 1 © fk © •P 03 > O O fk Ek P i P> ha fl fl fk © Td -P •H © © Pi to I •H © to !> c «H rk in-fl m in H 60 rk 0 6 0 rH 1— O Kv O Pi -p 0) > 1 o •H o fk p P i -H © P o o 0} © o c Ek 53 03 Pi «H O © 43 o fl fl © © t> *V0 p p © © E Pi © • * m m •d p © © 60 P © p E © nd p © © •p in m VO m 1 rk —I § rk h rk -A -fl rk © © fl fl-fl •ik •HJj VD A VD © © rk 1 in rk rk I m nd rk I p P VO rk © 1 H VD CO © mr— Vi VO cr.w pq m-fl rk m p © a fk VD © IT\rk I P vo cc O'.Pi M M 4> © •H P © N r ©i f ©l »d Ti *H *H © © © © p a © C rk © A j ini^i— rk CVJ m rk © to © m© K CO fk fk fk -jk © 03 03 0) © © © © P P P P Pi Pi Pi Pi Pi Pi fl © r-i cvj P © A in Hfl© rk VO CVJ cvj K tA in in © © vo to m vo vo -fl- -fl O m fl © o © vo flo fl rk K -H M M •H fk Td cti,fl © *v o .d © O r k 03 r k CO i n © a o P fk p 1 -p p o © • VO C O o vo (6 p M o CT M W A >s © © o X K © fk P o © fk © -p © *H O H +3 +3 -P 4> fl fl f ©l f ©l © © rfl nd nd rd 4> -p P P Pi Pi Pi |23 -fl- C VJ Pi © © P P 6fl© fk •Ci E © fl 60 CVI rk vo to rk pq in 60 rk pq •P fl m o p o *H p| Pi -H m VD in rk CVJ -fl O © A 60 60 l“— 1"- f— rk VO VD h i—i cvj Cvj cvi «—i cvj cvi p q p q p E |- p q p q p q p q p c | O in. rk m A o -fl - f l in © m m cvj m fl pq pq pq fq pq 32*^ "• 43 O 43 0) O of ° fl S3 © ® -ri fl P* rH 0 43 fl +> ffi Table 3 (continued) ^mergence and type of response to ringrot inoculation seventeen crosses or varietal selections, mostly obtained from the United States Department of Agriculture, as determined in field trials at East Lansing, Michigan, in 19^3* O rH • ■ O rH O VD rH O • K> I H rH rH VD IP I Cvj 1 1 aS tdS KV • K \ O I IP I I O «H O fl EH PC fl «H r-l 1 CD •H O O «H Eh Pi 43 P 1 •H •H CD m •d 43 O co EQ fti 0 I O O rH HV 0 > *rl 43 O I I 0) > O *rl O •H 43 0 fl *rl O O CO 0 O O Eh CO A, IP I cvi 60 o• 0■ 0 • LfS I— IT A D CVI K - , KV 60 O O P «fl o I 0 43 V P P 0 CD O W fl fl ffi 0) E Ph ffi vo o *d fl © III) fl B © a p 0 VD o xi fl © © +3 P vo ID O irv M M fit O 60 VD 60 VC CVI r- rH Pi 1^1 'd d M CD D *H 4C3 r-i CD ffi M OT 43 0) •H fl CC > M $ © O P 43 p O o CD p •H «H fl fii P o 0 0 © •H fit xi a © p Pi T* P P -H to u 1C P O H d VD ,3 P 60 *H O 0 fii O > 0 P P I CD 0 P r- 4> 0 ■rH •rH CVj CD fl p fl o CD .H © +3 r*3 CC 43 fl CO o fl o * rH ® 1 P tlO C D3-Pri 6J CVJ VD O rH IO H m m r-l •rH 1—1 I P 0 0 .P d -P O ffi © •rH -rH S £> © -rH to f l P d © 4> P © 1 —1 Figure 1. Brevalence of ringrot in the United States of America in I9 U0 Figure 2. □ Disease in field H Diseased potatoes on sale for seed Ringrot in Michigan including I9I& reports — hy counties.