fiSEES '8 ON” INFEBT§G§€ *3? VERTICEMUEQ W 1LT 0F WYATG‘ES thfl‘ fa: the Dagree 0f '93:. D. Mffifitfim STATE UMEVERSW C‘L‘EFéS'E‘fiZ’éE‘EfiE G. ‘fiLQWflEOEELGFC‘aiE-ifi 3&3? .HLSL‘ 0-169 Date This is to certify that the thesis entitled ASPECTS ON INFECTION OF Vb‘rt‘l'lCILLIUL‘. I‘v’ILT OF IOTATOL'JS presented by Constantine G. Thanassoulopoulos has been accepted towards fulfillment of the requirements for Ph.D. May in 1967 degree in Plant Pathology L 13 R A R Y Michign TS re University Qéé7éézifl?;;7éZ‘/W.J.Hooker / Major professor ASPECTS ON INFECTION OF VERTICILLIUM WILT OF POTATOES By Constantine C. Thanassoulopoulos AN ABSTRACT OF A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Botany and Plant Pathology 1967 ABSTRACT ASPECTS ON INFECTION BY VERTICILLIUM‘MILT OF POTATOES by Constantine C. Thanassoulopoulos Certain aspects of penetration and infection of potatoes by Verticillium albo-atrum Reinke and Berthold, and perpetuation of the fungus in seed tubers have been studied. X. albo—atrum, "microsclerotial" and "dark mycelial" types, grown on potato-dextrose agar or corn meal-Perlite media, were used for inoculation. Two inoculum types were used throughout: 1) a water suspension of Spores and mycelial fragments from scraped PDA cultures, and 2) a suspension obtained by crumbling a corn meal-Perlite culture and suspending in water. Sebago, Kennecbec, Cherokee, Russet Arenac, and Russet Burbank varieties were used in laboratory, greenhouse, and field experiments. Considerable evidence was obtained histologically and by isolations that the fungus penetrated leaves of potatoes through stomata whether leaves were detached or attached to the plants, and was followed by invasion of the vascular tissue of petioles. Further evidence of systemic spread of the fungus from the leaf lamina to the stem and the Constantine C. Thanassoulopcnilos tubers was also obtained by positive isolations from stems and tubers. High inoculum concentration, low light intensity and high relative humidity were important factors in increased severity of symptom appearance and infection of leaves. Tuber sprout infection caused the death of apical and leaf primordia meristems. Evidence was obtained suggesting direct penetration through epidermal cells of the sprouts, apparently at any location, followed by granulation and death of protoplasm. Abundant conidiophores and heavy sporulation was produced on dead sprout tissues. Of the different methods used for field inoculation, root inoculation and seed surface contamination were the most effective, as evaluated by vascular discoloration of the tubers, and positive isolations of the fungus. The leaf inoculation method resulted in a relatively high incidence of infection, as indicated by both vascular discoloration of the tubers, and positive isolations from the discolored areas. The cut tuber method in which a tuber was cut and the opening was filled with inoculum was completely ineffective. Inoculation either by injection of inoculum with a hypodermic syringe or by inserting a tooth pick carrying inoculum in the stem of the plants were intermediate in effectiveness. The root dip method, in which plants were dug out and dipped in inoculum, was not useful for field work because of the direct influence of transplanting injury on the plant growth. Constantine G. Thanassoulopoulos Seed of healthy appearing and vascular discolored tubers were evaluated in field trials. Isolations before planting gave no evidence of viable fungus within vascular regions of the seed. No differences between stands vigor, and general appearance were evident during the growing season. No significant differences were obtained at harvest time in vascular discoloration of the harvested tubers. Positive isolations made from vascular discolored areas of tubers were essentially similar whether tubers were grown from healthy or from vascular discolored seed lots. Considerable reduction of viable fungus in vascular discolored tubers was evident A months after harvest. A further reduction in viability of Verticillium in vascular discolored tubers was obtained 7 months after harvest. In 2 seed lots no viable Verticillium was obtained and in 2 seed lots viable fungus was obtained from approximately 10% of the tubers. This is in contrast with previous results when seed stored from the 1965 crop and used in isolation tests before planting in 1966 was completely free from viable Verticillium. This trend suggests that survival of_y. albo-atrum in seed tubers may be of relatively minor importance in transmission of the fungus in potato crops. ASPECTS ON INFECTION OF VERTICILLIUM WILT OF POTATOES By Constantine C. Thanassoulopoulos A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Botany and Plant Pathology 1967 ACKNOWLEDGMENTS The author is greatly indebted to Dr. W. J. Hooker for his valuable guidance, encouragement, and help during the course of this work and in the preparation of the manuscript. The author would also like to thank the other members of his committee, Drs. E. S. Beneke, E. H. Barnes, M° L. Lacy, and C. E. Peterson, for their real interest and assistance. Dr. Petersonfs assistance in helping the author's coming to this University is greatly appreciated. Special thanks are due Mr. Leon Alwood for his help in greenhouse and field work, Mr. P. Coleman for preparation of black and white photographs, and other associates of this laboratory for their kindness and help. To my wife Anastasia for here encouragement, help, and patience and to my daughter Maria for her encouraging smile, I am deeply indebted. ii TABLE OF CONTENTS Page ACKNOWLEDGMENTS . . . . . . . . . . . . . ii LIST OF TABLES . . . . . . . . . . . . . iv LIST OF FIGURES . . . . . . . . . . . . . v INTRODUCTION . . . . . . . . . . . . . . 1 PART I. LEAF AND SPROUT INFECTION . . . . . . 5 Materials and Methods . . . . . . . . . 5 ReSUltS O O O O O I O O 0 C O O O l 6 Symptoms of leaf infection . . . . . . 6 Infection of detached leaves . . . . . . 9 Infection of attached leaves . . . . . . 12 Penetration and subsequent progress of the fungus O 0 O O O O O O O I O O O O 19 Infection of sprouted tubers . . . . . . 19 Infection of detached Sprouts . . . . . . 24 PART II. COMPARISON OF METHODS FOR FIELD INOCULATION 31 Materials and Methods . . . . . . . . . 31 RESUILS o o o o o e o c o o 0 o o o 33 PART III. THE IMPORTANCE OF VASCULAR DISCOLORED SEED TUBER IN TRANSMISSION OF VERTICILLIUM WILT OF POTATOES . . . . . . . . . . . . . . 39 Materials and Methods . . . . . . . . . 39 Results . . . . . . . . . . . . . . U0 DISCUSSION . . . . . . . . . . . . . . A5 SUMMARY . . . . . . . . . . . . . . . 51 LITERATURE CITED . . . . . . . . . . . . 53 iii Table LIST OF TABLES Influence of inoculum type, light intensity, humidity, and wounding on infection of detached, inoculated potato leaves by X. albo-atrum . . . . . . . . . . . . Infection of attached leaves and petioles following inoculation of leaf lamina by 1. 31b O-atrum o o o o o o o o o o o o Systemic invasion of potato stems by I; albo-atrum folowing leaf inoculation. . . . Effectiveness of different inoculation methods for field infection of Sebago potato by X. alb O- atrum o o o o o a o o o o Effectiveness of different inoculation methods for field infection of Kennebec potato by X- 31b O-atrum o o o o o o o a o I Yield and vascular discoloration of potato tubers after harvest grown from healthy and vascular discolored seed . . . . . . . Frequency and type of y. albo-atrum isolated from potato tuber at different times . . iv Page 10 15 18 3A 37 Al U2 Figure ‘Jl IO. LIST OF FIGURES Page Bronzing and necroses of potato leaves 5 days after field inoculation with K. albo- atrum . . . . . . . . . . . . . Bronzing symptoms on potato leaves 3 days after greenhouse inoculation with K. albo- atrum O O 0 I O O O 0 O O 0 O O O Penetration of K. albo-atrum through leaf stomata A days after inoculation . . . . Mycelium in vascular tissue of potato leaf A days after inoculation . . . . . . . Reduction of tuber sprout growth by K. albo— atrum . . . . . . . . . . . . . Penetration of X. albo-atrum through tuber sprout surface . . . . . . . . . . Hyphae of X. albo—atrum within cells of tuber Sp rout 0 0 O O O O O O O O O O Apical meristem and leaf primordium of a tuber sprout following X. albo-atrum infection . . Conidiophores of X. albo—atrum developing on necrotic potato sprout after infection . . . Necrotic peripheral cells of apical meristem of a tuber sprout . . . . . . . . . . . 16 2O 21 23 25 27 28 29 BO INTRODUCTION Verticillium wilt is a major disease problem in potatoes. In Michigan, the chief loss is associated with poor tuber quality because of vascular discoloration. More information is needed concerning infection and inoculum survival. Verticillium albo-atrum Reinke and Berthold was described for the first time in 1879 as a potato parasite. Since that time it has been recognized as a soil borne pathOgen invading roots or root hairs (Reinke and Berthold, 1879). This observation was confirmed later by several authors (Roberts, 19A3, Ayers, 1952, van den Ende, 1958) and also it has been found recently to invade cotton through the root cap, between or within root epidermal cells, root hairs, and through hypocotyls (Garber and Houston, 1966). Reinke and Berthold (1879) reported brown spots on potato leaves infected with Verticillium wilt, but they did not report isolation of the fungus from these brown spots. They also reported brown patches on subterranean stems due to Verticillium infection, but also they did not state whether the fungus was isolated from them. In 1918, successful isolations of the fungus were effected from yellow Spots on the outer leaves of beets by Westerdijk 1 (from Rudolph, 1931). The first report of true leaf infection by Verticillium was made on tomato and eggplant in 1959 (Providenti and Schroeder, 1959). In their trials, the disease obtained by leaf infection was similar to the disease resulting from root infection. Sackston (1960), one year later, reported leaf infection of clover seedlings accomplished by spraying leaves with spore suspensions, but there are some doubts from his paper whether or not the fungus invaded through leaves or from lower portions of the plants. In 1963, Griffiths and Isaac confirmed the work of Providenti and Schroeder (1959) on tomato leaf infection. In spite of the fact that Verticillium has been reported in systemically invaded potato leaves even to the tips (Barrus and Chupp, 1926), no attention has been given to the possibility that leaves could also be primary sites of infection. In 1912, Dale, in England, in a brief paper described "Blindness" as a tuber disease of potatoes due to K. albo- atrum. She claimed that the fungus attacked the tubers by means of the eyes. Pethybridge (1916) suggested also that the mycelium of the fungus grows around the outside cork of the tubers, it reaches the bases of the young sprouts and probably penetrates through the cortical portion of the young roots. Pitt et_al. (196A) reported also that the abnormality of Sprouts known as "coiled Sprout" was caused by X. nubilum Peth. infection. The fungus also has been reported to invade stems of young potato plants by growing directly from infected tubers (Ayers, 1952, Robinson, gt_al., 1957, Robinson and Ayers, 1961). The same authors (1957, 1961) have also found that this type of inoculation is not as important in inciting disease as the inoculum on the tuber surface. Friedman and Folsom (1953) found no increase of vascular discoloration during storage of Kennebec tubers. Although it is generally recognized that X. albo- £2323 may be isolated from vascular discolored areas of tubers, Edson (1920) and Muncie (195A) have reported isolations of the fungus from apparently healthy tubers. Some method of stimulating severe incidence of X. albo—atrum in the field is highly desirable. For that reason, methods of inoculation have been attempted using among other methods those of several investigators working with different plant hosts (Brinkerhoff, 19A9, Keyworth and Bennett, 1951, Robinson, e£_al., 1957, Robinson and Ayers, 1961, Patil §£_Ei:: 196A, Erwin gt_al., 1965, Fronek, 1965). A critical evaluation of these methods to determine which of them is most efficient for increasing disease incidence and severity in field experiments with potatoes is necessary, in evaluating varietal resistance and in developing of methods of control. This study deals with certain aSpects of infection of potatoes by X. albo-atrum: 1) leaf and tuber sprout infection; 2) methods of inoculation in the field; and 3) perpetuation in seed tubers during the storage and incidence of disease in the subsequent crop. PART I LEAF AND SPROUT INFECTION Materials and Methods Isolates of X. albo-atrum from potato both microsclerotial (MS) and dark mycelial (DM) types were grown on potato agar (PDA), or on corn meal-Perlite which contained reSpectively A00 g and 250 g of each in 1000 ml water. Two types of innoculum were used throughout the experiments: 1) "spores"--from PDA culture, a suSpension of mycelial fragments and conidia, approximately l-l.2X105 per m1, prepared by scraping spores from the surface of colonies; and 2) "resting bodies"--from corn meal- Perlite culture, a suspension of mycelial fragments and spores adjusted to the same Spore concentration as l), but having in addition abundant resting bodies (microselerotia or dark mycelial structures), and also nutrients from the corn meal. The second type was prepared by crumbling and suspending in water a culture grown on corn meal-Perlite. Leaves or usually the upper 2/3 of the plants were dipped in an inoculum suspension. Sprouts of potato tubers were inoculated with "resting bodies" type innoculum 5 from an MS isolate of the fungus. Inoculations were made either on detached sprouts growing in a moist chamber, or surface infected tubers were planted between 2 layers of damp Sphagnum moss infested with inoculum Isolations from potato petioles and stems were made on PDA adjusted to approximately pH 5 with 25% lactic acid. All plant material used for isolations, leaf petioles, sprouts, stems etc., had been previously sterilized with 1% sodium hypochlorite for 1 min. Histological sections were cut with microtome for cutting fresh plant material (Hooker, in press) and mounted in distilled water without fixation. Photographs of fresh, unfixed, infected plant material are shown. Results Symptoms of leaf infection.--Similar symptoms developed in laboratory, greenhouse, and field inoculations (Fig. 1). In the field, typical symptoms appeared when the inoculation was made beneath the leaf canopy. Certain aspects of field trials are treated in greater detail in another section of this paper. Early symptoms became evident A8 hours after inoculation as small bronze spots on leaf lamina. These areas enlarged during the next 2-3 days and the center of the lesion became darker brown with a watersoaked appearance. The leaf became bronze to yellow in color within a few days after inoculation. In a few cases Fig. 1. Bronzing and necroses of potato leaves 5 days after field inoculation with "resting bodies" inoculum of 11:. albo—atrum. chlorosis was most prominent in the interveinal areas. The larger veins maintained the green color somewhat longer. By the fifth day after inoculation, some dead leaflets were present, and infection may have progressed sufficiently to kill the entire leaf. In some cases, the whole leaf was dead within 5-6 days. Usually most of the leaves were dead within 15-20 days after inoculation. Vascular discoloration following leaf infection was most common in the leaf petiole, but was also present in stems and in tuber stolon attachments. The fungus was readily isolated from vascular discolored areas, eSpecially from those of leaf petioles. Heavy sporulation of X. albo-atrum, evident macroscopically, formed on wounded detached leaves under conditions of low light and high relative humidity. It also developed on wounded attached leaves in greenhouse plants under Similar conditions. Macroscopic Sporulation did not develop under the other environmental conditions and apparently wounding was a prerequisite, under the conditions of the trials. Dead leaves in the field were very soon invaded by different saprophytic fungi, especially Botrytis and Alternaria, making diagnosis of verticillium in infected leaves difficult. Invasion by other fungi may possibly have accounted for the fact that Sporulation was not observed in the field. Abundant microsclerotia were formed on dead leaves in laboratory experiments if the leaves were kept for 1 month or more under moist chamber conditions. Infection of detached leaves.--In a preliminary laboratory trial in petri plates, infection of detached potato leaflets of the Russet Arenac variety was demonstrated by positive isolations from the petioles in 20 of 30 inoculated leaflets. A drop of "spores" inoculum was placed near the tip of leaflet, and A8-72 hours later a bronze colored spot around the inoculation point suggested some infection. Positive isolations made from the leaflet petioles, 3-10 days after inoculation, indicated that there had been infection as well as some fungus movement through the vascular system of the leaflets. In later inoculation tests with the Russet Arenac variety, green leaves with 5-7 leaflets were detached and the petioles were placed in small glass vials (1.5X5.5 cm) containing tap water. Water was added once or twice a day to replace transpiration loss. Care was taken during and following inoculation to avoid contamination of the water. Water was added carefully and the inoculated portions of the leaves were arranged so that the leaflets hung over the side of the vials. Factors studied in relation to infection (Table l) were: 1) inoculum type, "spores" or "resting bodies"; 2) light intensity, 100 ft-c approximately vs. 20-25% ft-c each at 12 hours per day; 3) humidity; and A) 10 .zpfiafinwnopo do Ho>ofi SH who am pcmofieficwfim mmz mLOpomw Lsom mo coapompmch .zpfiaflnwnosd do Hm>oH RH one pm Locpo some Sosa maucwofiuficmfim wcflsouofio poc muazmop omeHocH mpoppoa anfiEHm\m .moH co mEOerzm\m mm: am so: ms msfi mom mm .s@ smomazoocfi o 0 sun m o o 0 am Hops:00 .omezzox Soc mm: Sm. «mm :H mam as: o: oo smsmflsoocfi o c n ma m c o o co Hospcoo .oopczoz cofiuoomcfi wcfipcsoz mm: ma mmm HH 9 CH n ma 3H we oopmfizoocfi o o n ma w o o o om Hoppcoo .LonEmzo omHoE on mm: mm mam ms aim mmm am as smsmfisoocH o o s as s c o 0 am Hopscoo .Emsemco pmaoe :H suaeaszz m>fipmfimm mm: s 2 am e s m s :H as ms smpmfisooca o o s m o o o 0 mm Hopscoo .cmfin mm: c: aw: ma mom mom 2m mo ooomazoocfi o o 9 SH s o o o as Hopscoo .3oH pswfiq D m m D o m D m . D NH :H :w :wwcHOQw: ems as ems ma mam .msm Hm om =mmfisop mcapmmsz o o 9 ms m o o o FHA Hopscoo EnazooccH \Mm w \wm m \M& \Mm .oc .oc when om \Mmamo 0H cofiomH5002fi scope xoocfi powwow Lopmm mofioflpoa Ca Esnpmlonam mzmp om when OH mo>moq smog ommomwo mo>moa popcomcH wo>moq \mmo>mofi co wEOmemm UoLmQEoo mLOpomm Ensemlonam .M an mo>moa oumuoo. oopmazoocfi venomooo Lo cofipoomca co mcficcso3 0cm .mpacflezc .zpfimcopca pzwfia .oamp ESHSooca mo mocozaucth.H oHnt 11 influence of wounding the leaf surface. Treatments were planned in all possible combinations of these A factors. For each treatment, 3-7 leaves were used with 3 replications in a factorial design. Leaf infection was determined by isolations from the petioles. Isolations were made when the inoculated leaflets were dead or almost dead. At this time, the noninoculated basal leaflets and the lower part of the leaf petiole still appeared to be in good health. Significant differences were determined by the Tuckey test (Guenther, 196A). A disease index was developed by classifying diseased leaves into O-healthy, to 5-dead, and expressing this on a percentage basis. Intermediate values, 1, 2, 3, and A, were assigned according to increasing severity of symptoms on the leaflets. The leaf was considered systemically invaded when the fungus could be recovered from the petiole below the innoculated leaflets. More severe symptoms and higher infectivity was obtained with "resting bodies" type inoculum than with "Spores" inoculum. Leaf survival with "spores" after 30 days was good and was very similar to that of the controls. In contrast, leaf survival with "resting bodies" inoculum was low. Systemic infection after 10 or 30 days was high with "resting bodies" inoculum and was very low when "Spores" were used for inoculation. More severe symptoms and more frequent systemic infections (10 day) were obtained with low than with high 12 light intensity. After 30 days the amount of systemic infection was similar. Leaf survival in high light intensity after 10 and 30 days was significantly higher than in low light intensity and essentially similar to that of the noninoculated controls. Leaf symptoms were less severe in low than in high humidity. The number of dead leaves, however, was essentially similar after 10 or 30 days, and in either case the number of dead leaves following inoculation was higher than in the noninoculated controls. Systemic infection was lower outside the moist chamber at 10 days but after 30 days differences were not Significant. Wounding of leaves had Slight or no influence on symptom appearance, survival of leaves, or systemic invasion either 10 or 30 days after inoculation. The interaction of the above A factors was Significant. Most severe infection was obtained with heavy inoculum in low light, high relative humidity, and following wounding. Younger leaves seemed to be more easily infected, while older leaves were more quickly killed. Infection of attached leaves.--In preliminary green- house experiments, infection of attached potato leaflets of Russet Arenac variety by y. albo-atrum was readily obtained. This was evident by symptoms developing on 52 of 60 inoculated leaflets, each leaflet being on a separate plant. A drop of "spores" inoculum was placed on 13 30 apical leaflets and a small piece of "resting bodies" inoculum was placed on the other 30. Half of the plants inoculated with "spores" inoculum and half of those inoculated with "resting bodies" were placed in the dark and the other in 50—80 ft-c (1A hours light). All plants were covered by plastic bags. Similar treatment was given to control plants. The plants remained under these low light intensity and high humidity conditions for 5 days, and then they were placed under regular greenhouse conditions. Three to A days after inoculation a bronze area was evident around the point of inoculation on leaflets infected with both inoculum types. Leaflets inoculated with "resting bodies" inoculum type and held in the dark had more severe symptoms than leaflets kept in 50-80 ft-c light intensity. One week to 10 days after inoculation 10 of 30 leaves with inoculated leaflets held in the dark abscissed and 2 of 30 leaflets from the plants in 50—80 ft-c were also abscissed. No symptoms were evident in controls except that of a Slight chlorosis, from which the leaves soon recovered after exposure in full light. No leaves from controls abscissed within 10 days after inoculation. No isolations were attempted. Later experiments were designed to determine if systemic invasion of stems following leaf infection was common. Sebago, Kennebec, and Cherokee varieties were inoculated with "resting bodies" type inoculum of either 1A MS or DM culture types. Since the DM and MS culture types caused similar types and frequency of infection the data were combined (Table 2). Plants were kept in moist chamber conditions by placing them in plastic bags for 3 days after inoculation. Half of the plants were placed under the greenhouse bench with 10-50 ft-c light intensity during the day (1A hours light), while the other plants were kept in day light but not in direct sun light at 1000-5000 ft-c. Except for the 2 basal leaflets for each leaf, every other leaf of each plant was inoculated by dipping into inoculum suspension. The alternate noninoculated leaves served as controls. Random isolations from leaf petioles before inoculation established that X. albo-atrum was not originally present in the plant. Early symptoms on leaves were visible 2 days after inoculation and they became clearly evident by the third day (Fig. 2). Isolations from leaf petioles 3 days after inoculation were positive for X. albo-atrum in 6 of 9 leaves. Isolations from leaf petioles of inoculated leaves were also made 7 and 1A days after inoculation. These data are combined (Table 2). The fungus was isolated from 2A of A0 leaf petioles at low light intensity. At high light intensity petioles of only 2 of 36 leaves were systemically infected. Mycelium and some granulation of vascular elements were present within xylem in sections of leaf petioles, while such structures and textures were 15 Table 2.—-Infection of attached leaves and petioles following inoculation of leaf lamina by E. albo- atrum Variety and Leaves with Leaves with light Inoculated symptoms 2. albo-atrum intensity leaves after 3 days in petiolesE/ no. no. no. Sebago low light 12 10 9 high light 11 0 0 Kennebec low light l7 17 10 high light 16 O 2 Cherokee low light 11 9 5 high light 9 0 0 g/Presence of X. albo-atrum established by isolations to PDA after 7 and 1A days, and the data were combined. l6 Fig. 2. Bronzing symptoms on potato leaves 3 days after greenhouse inoculation with Z. albo-atrum. l7 absent in the controls. The influence of high light intensity on systemic invasion of petioles of attached leaves was quite similar to that obtained in detached leaves. This is evident by comparing the 10 day observation (Table l) of systemic infection with data obtained within 1A days after infection (Table 2). Resistance of the leaves to systemic infection evident on detached and attached leaves is apparently increased by light. Systemic invasion of potato stems by X. albo-atrum following infection of aerial part of plants was established (Table 3). Plants of the varieties of Sebago and Russet Arenac were inoculated by dipping the upper 2/3 of the plant into inoculum consisting of "resting bodies" of the MS type. To avoid soil contamination, pots were covered with an aluminum foil sheet tied around the plant stem. The plants were watered by subirrigation. Non inoculated controls were similarly dipped into tap water. Plants were kept under conditions favorable for infection, i.e., high relative humidity and low light intensity, for A days. Systemic infection of 11 of 32 plants was demonstrated by isolations made from different places on the main stem of the plant 2 months after inoculation. In 9 of 11 infected plants the fungus could only be isolated from the part of the plant above the inoculated leaves; in l 18 Table 3.--Systemic invasion of potato stems by V. albo- atrum following leaf inoculation Plants with Variety and treatment Plants V. albo-atrum In the stemé/ no. no. Sebago control . 8 0 inoculated l6 6 Russet Arenac control 8 0 inoculated l6 5 E/Significant differences at the 1% level of probability with t test. case it was isolated from both above and below, and in 1 plant it was recovered only from the lower portion of the stem but not from the roots. In 5 of these 11 infected plants, distinguishable vascular discoloration was evident. Similar isolations from control plants were negative and tissue was believed to be sterile. Infected leaves had a tendency to absciss early. This resulted in defoliation of plants following severe leaf infection. Isolations attempted from petioles of dead abscissed leaves were almost always negative, with respect to recovering of Verticillium. 19 Penetration and subsequent progress of the fungus.-- Detached or attached leaves inoculated with "resting bodies" inoculum of both types of the fungus and kept under low light intensity and high moisture were sectioned. Penetration of V. albo-atrum through leaf stomata (Fig. 3) of either the upper or lower surface was demonstrated. Infection of stomata was apparently accomplished by a number of hyphae growing between the guard cells. A heavy mass of hyphae was present in the substomatal chambers at least by the third to fourth day after inoculation. From this mass, hyphae spread intercellularly to the neighboring cells. Necroses of cells around infection points were microscopically evident about the same time that penetration had been completed by the fungus. When "spores" was used as inoculum the same pattern of penetration was observed histologically by mycelium within the stomata was not as abundant. Fungus hyphae were present in xylem and xylem parenchyma of vascular bundles of leaf lamina A days after inoculation (Fig. A). In control plants, the presence of fungus hyphae could not be demonstrated in vascular tissue of leaf lamina by histological examination, nor in that of petioles by histological examination or by isolation. Infection of sprouted tubers.--A stunting of young sprouts and roots was obtained in preliminary inoculation experiments. 20 Fig. 3. Penetration of V. albo-atrum through leaf stomata, above-upper surface, below-lower surface, A days after inoculation. (Reference line indicates 10 u)- 21 Fig. A. Mycelium in vascular tissue of potato leaf upper-cross section of vascular elements lower-lingitudinal section of xylem vessels, A days after inoculation. (Reference line indicates 10 u). 22 In more extensive trials, tubers from Sebago, Kennebec, and Russet Burbank varieties with sprouts approximately 0.5 cm long were placed on a 1 cm layer of damp Sphagnum, had been treated with abundant corn meal-Perlite inoculum. When the tips of sprouts of controls had grown above the layer of Sphagnum, 15—16 days later, tubers were removed carefully, washed thoroughly in running water, and observed. In all cases Sprouts from inoculated tubers were less vigorous than those from control tubers. In some cases, the number of Sprouts per tuber was decidedly reduced in infected tubers as compared to controls. Tips of sprouts which were present at the time of inoculation were usually dead. Sprouts grown from inoculated tubers grew almost exclusively as lateral or secondary branches from the original Sprout, and their length was markedly reduced in comparison with the noninoculated tubers (Fig. 5). In several tests, approximately a 20% reduction of tuber germination was obtained. These results are in good agreement with observations in field experiments with the Sebago variety and are described elsewhere. When tubers with 0.5 cm sprouts were inoculated, the length and fresh weight of roots were noticeably reduced. 0n the other hand, when tubers were completely dormant at the time of inoculation no root reduction was observed. This may suggest that within a short time, 23 Fig. 5. Infection of tuber sprouts-by V. albo-atrum and reduction of sprout growth. 2A viable inoculum was considerably reduced and that root primordia escaped contamination because they grew later than primordia of leaves and tips of growing sprouts. Infection of detached Sprouts.--Visual symptoms of infection on Sebago and Kennebec sprouts consisted of browning of the surface of the tips. Sprouts 2—3 cm long were detached from the tuber, disinfected in 1% sodium hypochlorite for l min and thoroughly washed twice in sterile water. The upper half of the sprouts was then dipped into inoculum and the portion bearing root primordia was inserted in water agar. Without exception apical meristems were necrotic when exposed to infection in this way. Lateral buds were usually killed but when they were not, they germinated producing secondary sprouts. There was no inhibition of root formation, as evidenced by comparison with the controls. Controls grew normally and Sprouts had vigorous green tips. The following sequence of events obtained in sprouts grown on water agar: Verticillium hyphae, either those growing from a mycelial mass or from the germ tube of germinating spores, were capable of penetrating epidermal cells of the sprouts apparently at any location (Fig. 6). Some evidence also was obtained that infection may have taken place through hair cells on the surface of the sprout developed a brown color and shortly after that 25 \ Sn . I Fig. 6. Penetration of V. albo-atrum through tuber sprout surface. (Reference line indicates 25 u). 26 protoplasm of the cells which the hypha had penetrated became granulated (Fig. 7), turned brown and died. This was fOllowed by death of neighboring cells. The fungus progressed intracellularly. In many cases necrotic tissues were limited to the superficial layer of the Sprout. Further growth of the Sprout was stopped since the growing cells of apical meristems and leaf primordia had been killed (Fig. 8). Soon after cells had been killed the fungus developed conidiophores on which abundant spores were produced (Fig. 9). Necrotic tissue extended into the sprout and was observed in close proximity to the vascular bundles. This suggested that systemic invasion by V. albo-atrum could be anticipated (Fig. 10). 0f 20 isolations attempted, 2 positive isolations were made from the central vessels of the sprouts as soon as 7 days after inoculation. 27 Fig. 7. Hyphae of V. albo-atrum within cells of tuber sprout. Note granulation of protoplasm. (Reference lines indicate, upper 25 u, lower 100 u). 28 albo—atrum infection. Apical meristem and leaf primordium of a tuber (Reference line indicates 100 u). sprout following V. 8. Fig. 29 Fig. 9. Conidiophores of V. albo—atrum developing on necrotic potato sprout after infection. (Reference line indicates 100 u). Fig. 3O 10. Necrotic peripheral.cells of apical meristem of a tuber sprout. Note proximity of necrotic tissue to vascular elements. (Reference line indicates 100 u). PART II COMPARISON OF METHODS FOR FIELD INOCULATION Materials and Methods Healthy appearing tubers of the Sebago (moderately susceptible) and Kennebec (susceptible) varieties were planted on muck soil at the Michigan State University Muck Experimental Station at Bath. Before planting, all tubers were cut at the stolon attachment, and if a suggestion of vascular discoloration was present, tubers were discarded. Sebago tubers were planted in a randomized block design with 25 tubers for each treatment in 6 replications, and with Kennebec 3 replications of 10 tubers for each treatment. For inoculum, V. albo-atrum, MS type, originally isolated from diseased potatoes from Michigan, was grown on corn meal-Perlite medium for about 2 months. Unless otherwise indicated, this inoculum, consisting of spores, mycelial fragments, microsclerotia, corn meal, and Perlite, was crumbled and shaken with seed tubers or suSpended in water for spraying and root dipping. Inoculation procedures were: 1) Control. No inoculation. 2) Root inoculation.‘ Soil containing roots near the plants was cut with a Shovel, inoculum poured into the 31 32 freshly cut soil and presumably over the cut ends of roots,and the soil pressed back into place. 3) Root dip. Plants were dug and roots dipped into inoculum suSpension and replanted. A) Seed surface contamination (5 day). Whole seed tubers were shaken in inoculum contained in a plastic bag, air dried, and planted 5 days later. 5) Seed surface contamination (0 day). Same as 3 but seed were planted immediately after inoculation. 6) Tuber cut. Seed tubers were out once between apical eyes with a sharp knife contaminated with inoculum, tuber opening filled with inoculum, and the tubers planted 5 days later. 7) Leaf inoculation. Inoculum mixed with A00 mesh carborundum Sprayed on leaves at close range with an atomizer at 70 lbs pressure. 8) Hypodermic syringe. 0.5 ml Spore and mycelial suSpension (12X10u spores /ml) injected with a hypodermic syringe into the first or the second above ground node of each plant. 9) Tooth pick inoculation. Inoculum grown in potato dextrose broth culture on tooth picks which were inserted into the stem of each plant. Inoculation 2),3L 7% 8L and 9 were made A0 days after planting, when plants were 15-20 cm tall. Treatments A), 5), and 6), were made before planting. 33 Stand counts were made 1 month after planting. Average height of plants from each inoculation treatment was expressed as a per cent ratio of the average height of control plants. The extent of tuber vascular discoloration after harvest was determined from a random sample of approximately 200 tubers by cutting the stolon end 0.5 cm below the point of attachment. Analysis of variance and the Duncan test were used for statistical analysis. V. albo-atrum infection of tubers was determined by isolations made by planting 3 pieces from each tuber into PDA adjusted to approximately pH 5 with 25% lactic acid. Results Stands of Sebago (Table A) were significantly reduced when seed were covered with surface borne inoculum 5 days before planting. Stands from tubers similarly inoculated just before planting were slightly reduced. No other treatments reduced stands. Plants which grew from seed which had been surface contaminated (0 day) were significantly smaller (1% level) in size 1 month after planting as compared to controls. Plants growing from surface contaminated seed (5 day) were slightly stunted. Root dip inoculation which entailed a transplanting type of treatment at an unfavorable time in plant development caused the greatest reduction in plant vigor and growth of any of the inoculation treatments. 3A .cocHELopoo some can mUCMpw scope poomHSQOQH one: mocmao peso mumOHch mwmmnpcosmq CH mmszwfim I .seflaseeeoee co He>eH .poHQ sod whens» mm .mpOHQ o Sosa meadow ommso>< &H.o one we moocosommflo osmofimflcmfim oc oomoflocfl whoppoa anHEam \o \m \m- s.sm ma eeew.afi mam em.m As.mmv mes eoaeefiseoea eons goose Am e.eH ma so o.ea Hem ee.m Ao.mmv ems eweaesm easemeoemm Am e.eH ma ee m.mm mom em.m Ae.mmv ems eoaemaeeoefl ewes As 0 ma we e.mH was mo.m mo.em SSH use sense As m.mm ms eem.mm ass eH.m eem.flm Hmfl flame ov Coaumcfiempcoo oomhhsm poem Am e.ea ms ee m.mm was em.m e m.mfi MHH Ammo my cospmcfiewpsoo oomMLSm comm A: H.HH ma mm.mm mam e ©.H Aw.mmv mma use poem Am H.HH SH es.mm so: as.m \mie.mmv ems eofieeflseoeH Boom Am 0 mm m e.m em: em.m \meo.mm mam Hoeeeoo AH R .0c Ms .od .moa |.oc .o: Umpooecfl coaumflomfi oopmoemw cocflEMxm ocean \mpoad Hmpob pcoEumoLB ESAHHHOHpLo> Lop msoczb msocse sod ocmpm messes Uses» see: coprsoHoomHQ smasomm> mpcmam I ESLpMIoch .> an oomQOQ ommoom no COHpoomcfl UHmHm Lou moonpoe cospmazoocfl pcostMHU mo mmoso>fipomMMMIl.: maomB 35 Yields from plants following root dip inoculation were significantly reduced as compared to controls. Yields from other treatments were not significantly different from yields of control plots. Except for the cut tuber method, vascular discoloration of tubers was significantly higher (0.1% level of probability) than that of control tubers with each of the inoculation procedures tested. The highest percentage of vascular discoloration was obtained by the methods of root dip, root inoculation and seed surface contamination (0 day). Isolations from vascular discolored areas of the tubers showed that Verticillium was present in tubers of all treatments except those of the control and cut tuber inoculation. In all cases of positive isolations, the same type of fungus used for inoculation was subsequently isolated. The least variation as determined by incidence of vascular discoloration was obtained following inoculations by the hypodermic syringe, leaf inoculation, and both seed surface contamination methods. Greatest variability was encountered with the tooth pick method of inoculation. The leaf inoculation method provided relatively high levels of infection as measured by both vascular discoloration of the tubers and positive isolations of the fungus from vascular discolored areas. Furthermore, results were quite uniform between replications. This method could be useful for further work. The hypodermic 36 syringe method was not so effective in stimulating vascular discoloration and the isolations of the fungus was relatively limited, but the method had the advantage of considerable uniformity. The tooth pick method, quite similar in some respects with the hypodermic syringe, gave better results than the hypodermic syringe in stimulating vascular discoloration and incidence of infection, but had the disadvantage of great variability. This may have been due to variation in quantities of inoculum inserted into each stem. The tuber cut method was completely ineffective for inoculation. Stands of Kennebec (Table 5), were not influenced significantly by any of the inoculation methods tested. In other reSpects, results were essentially Similar to those obtained with Sebago. Leaf inoculation, hypodermic syringe, and tooth pick inoculation methods were negative in isolation tests. Possibly differences between responses of Sebago and Kennebec have been due to the small number of tubers available for isolation. The root dip method for inoculating both varieties was the most effective as measured by incidence of vascular discoloration. Infection of tubers as measured by frequency of positive isolations was no more effective than that with certain other methods. Plants did not recover from the shock of inoculation and transplanting. Two to 3 days after inoculation plants were wilting. 37 .oocHELopoo some own mpCMum Loose oopmazoocfl one: mocmflm ownp opmofipcw mfimonpcopma CH mopswflm \w .uOHQ sod mLoQSB OH .mooaa m Eons mpcmum mmmno>¢\m .zpfiaflcmcosa do Ho>oa wa.o who he mooconomeflo oeaoflmflcwfim o: oumOHUCH msoppofl smHHEHm\m o m on m.ms em mfi.m no.9sv om eeflseflzeoefi ease eeooe Am G . m on m.m~ om mo.m AQ.QHV om owsflszm Sassoooom: AW 0 a o i.em ea mm.m no.3 V em ceaseseeoefl sees as m.mm e e S.HH Hm mo.m no.0s om use sense As H.HH o c m.sm Hm em.m mm.m gm Ammo ov :oHDSCHEmpcoo momuszm boom Am H.HH c on q.m~ om s~.m no.0H om Ammo mv coapmcflfimpcoo oommszm been A: m.mm e -e.se so eo.m Ae.s V mm use poem Am fi.HH o n m.om ms ma.m AQ.:HV om cowpwHSoocH poom Am 0 ma e e.o~ ems ec.m \m:.es so accocoo AH m .oc \mm .oc .mcH \l.oc .oc pcoecfi coflpmflomw pooooecm confiscxm ucmflo .rooHQ Hence pcoEpwose HHofluLo> soc wsosse msmcss Loo osmom msocze wfloww new; coflumsoflocmflv smasommb I Essomlocflm .> an cocoon oonoscox so :oaoooesfl Uflowu Lou moocomE coflpmasoocfl psosmeuflo no mmoco>fiuooQLMIl.m mapwe 38 This method is relatively undesirable for field work, because of the inherent injury to plants, large amount of inoculum needed, and relative high labor requirements. PART III THE IMPORTANCE OF VASCULAR DISCOLORED SEED TUBERS IN TRANSMISSION OF VERTICILLIUM WILT OF POTATOES Materials and Methods Tubers from 2 lots of Sebago, and 1 each from Kennebec, Cherokee, and Russet Burbank, were separated into 2 groups, those apparently healthy and those with vascular discoloration. For this, tubers were sectioned at the stolon attachment, 0.5 cm below the surface, and were separated by macroscopic observation. The extent of seed transmission of Verticillium wilt was determined by planting these 2 groups of seed in paired rows of 10 hills eachrwiththe Sebago seed lots and of 5 hills each with Kennebec, Cherokee, and Russet Burbank. Standard potato culture practices were followed throughout the growth period. At harvest, the yield of each plot was weighed and a random sample of 30-50 tubers from each plot were used for evaluating vascular discoloration. The presence of viable Verticillium in the tubers was established by isolating from the vascular region near the stolon at 3 random locations from each tuber. For this, a random sample of tubers from each group 39 AC was selected. Isolations were made from vascular discolored tubers to a petri plate of potato dextrose agar adjusted to approximately pH 5 by adding a few drops of 25% lactic acid. Isolations were made after harvest (Sept. 15) from a random sample and the balance of the tubers was held in a potato storage maintained under usual conditions of A C temperature. From stored tubers random samples were taken for isolations after A months (Jan. 5) and 7 months (April 5). Results Both seed lots were of similar seed quality. There were no missing hills with either seed lot, healthy or vascular discolored. Throughout the growing season, no differences were apparent between plants grown from healthy or grown from vascular discolored seed. Particular attention was given to differences in appearance, vigor, foliage color, etc. The frequency of vascular discoloration of tubers and tuber yields were similar from both vascular discolored seed and from seed healthy in appearance (Table 6). No positive evidence was obtained from isolations made before planting (April, 1966) that the seed tubers contained viable V. albo-atrum in the vascular region (Table 7). Over 600 isolations were attempted from over 200 tubers with vascular discoloration and an equal number A1 Table 6.--Yield and vascular discoloration of potato tubers after harvest grown from healthy and vascular discolored seed Tuber symptoms Variety and Tuber Average seed tuber seed / yield per Tubers Vascular appearance planted— plant examined discolored no. lbsg/ no. % Sebago, lot 1 healthy 180 2.7 710 27 vascular discolored 180 2.5 83A 29 Sebago, lot 2 healthy 130 2.8 626 29 vascular discolored 130 2.9 6A6 3A Cherokee healthy A5 2.3 A15 6A vascular discolored A5 2.A A52 69 Kennebec healthy 70 2.7 37A 12 vascular discolored 70 3.0 388 15 Russet Burbank healthy 25 3.5 2A1 6 vascular discolored 25 3.5 212 11 a/ — Hills per replication-10 for Sebago, 5 for other varieties. b/ — No significant differences were obtained. .oamu =defiecmc xsmu: mmpmofipca 2a ram .eaau eamfiuOLoHomopoae: mmomoauca mx.m \ .uc052omsum ceflcSm use: cease; seasons» an; no wounds sesame m Echo some one: mLSDSu Eopu mcoHpmHomH.m \ “canaaaa>a atone» one o o o o o s OH c e u e a a ma 0 mm umeefieenae . . . swazomm> 3.33.223 upon...» 05 o o o o o c ow c c . o o e e S 9 mm Page: . wapLJm nemmrm o o o o , o o mm o o o e e e ms 0 n m.: . o H 1 es a ma emeo cease u ruflHDUWM? o o o o o 0 mm o o o c o c ms 0 a r.: o m - a: 0 mm sunset: omnvucox oH . o oa m m m om m o m.o~ a H m es en EH n.ee a Li A as e em senescenae . 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