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1988 A Toxic Metabolite Produced by Diaporthe Phaseolorum Var. Caulivora, the Causal Organism of Stem Canker of Soybean. Lalitha R. Burra Louisiana State University and Agricultural & Mechanical College

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Recommended Citation Burra, Lalitha R., "A Toxic Metabolite Produced by Diaporthe Phaseolorum Var. Caulivora, the Causal Organism of Stem Canker of Soybean." (1988). LSU Historical Dissertations and Theses. 4558. https://digitalcommons.lsu.edu/gradschool_disstheses/4558

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A toxic metabolite produced byDiaporthe phaseolorum var. caulivora, the causal organism of stem canker of soybean

Burra, Lalitha R., Ph.D.

The Louisiana State University and Agricultural and Mechanical Col., 1988

UMI 300 N. ZeebRd. Ann Arbor, MI 48106

A TOXIC METABOLITE PRODUCED BY Diaporthe phaseolorum var. caulivora, THE CAUSAL ORGANISM OF STEM CANKER OF SOYBEAN.

A Dissertation Submitted to the Graduate Faculty of the Louisiana State University and Agricultural and Mechanical College in partial fulfilment of the requirements for the degree of Doctor of Philosophy in The Department of Plant Pathology and Crop Physiology

By LALITHA BURRA B.S., Osmania University, India, 1975 M.S., Osmania University, India, 1977 August 1988 ACKNOWLEDGMENTS

I express my sincere gratitude to my major advisor Dr. J.P. Snow and Co­ advisor Dr. G.T. Berggren for their guidance and suggestions in conducting the investigation and in preparation of the manuscript. I also thank Dr. D.R. Mackenzie, Head, Department of Plant Pathology and Crop Physiology for his support and encouragement and for making the facilities of the department available.

I am grateful to the members of my committee Dr. K.E. Damann, Dr. L.L. Black, Dr. J.P. Jones, Dr. J.M. Larkin and Dr. R.J. Siebeling for their suggestions and critical comments on the manuscript. I would also like to thank Dr.K.E. Damann for making the facilities of his lab available and for his valuable suggestions in the investigations.

I would like to thank Mr. Dana Berner and Mr. K.V. Subba Rao for helping me with the graphs and analyses of data. I also thank Dr. R. Minard, Dept, of Chemistry, Pennsylvania State University and Dr. P. Krishna, Dept, of Biochemistry, Louisiana State University for their help in conducting mass spectroscopy and Mr. Raphael Cueto, Biodynamics Lab, Louisiana State University, for his help with IR spectroscopy. Finally, I thank all my colleagues, particularly those in the cotton lab, for their help and company which made my stay here enjoyable. TABLE OF CONTENTS

Chapter Page

Acknowledgements ii List of Tables iv List of Figures v Abstract v ii

CHAPTER I Review of Literature 1 Literature Cited 14

CHAPTER II Properties of a Phytotoxin Produced byDiaporthe phaseolorum var. caulivora. 21

CHAPTER TTT Histochemical Changes in Soybean Trifoliates Treated with a Toxic Metabolite of Diaporthe phaseolorum var. caulivora. 48

Appendix 65 Vita 69 Approval Sheet 70

iii LIST OF TABLES

Table CHAPTER n

1. Reaction of various plant species toDiaporthe phaseolorum var. caulivora and a toxin extracted from culture filtrate of the fungus.

2. Effect of various reagents on the toxigenic spot extracted from the culture filtrate of Diporthe phaseolorum var. caulivora and separated by thin layer chromatography.

3. Reaction of soybean cultivar RA 606 to Diaporthe phaseolorum var. caulivora and culture filtrate from four isolates of the fungus.

4. Response of soybean cultivars to culture filtrates of four isolates of Diaporthe phaseolorum var. caulivora.

5. Reaction of soybean trifoliates of cultivar RA 606 to toxic metabolites.

CHAPTER TTT

1. Metabolites produced in the soybean plants in response to treatment with toxic metabolite produced byDiaporthe phaseolorum var. caulivora and the fungus. LIST OF FIGURES.

Ei&um Page CHAPTER n

1. Soybean trifoliates excised under water and cut ends dipped in 1:10 dilution of culture filtrate for 36 hours A, Red discoloration on the petiole B, Lesions on the petiole and red discoloration of midrib and veins. Lamina showing few patches of discolored cells. C, Interveinal chlorosis of leaflets 48 hours after treatment. 43

2. Relationship between the toxin dose (dilution series of 1:10,1:20, 1:40,1:80 and 1:160) and disease severity rated on a 0-5 scale (0= no symptoms, 5=lesions on petiole, midrib and veins discolored). Excised trifoliates were dipped in dilutions of culture filtrate and symptoms rated after 36 hours. 44

3. Effect of incubation temperature on growth ofDiaporthe phaseolorum var. caulivora and toxin production. The amount of toxin produced was measured in terms of symptom severity and rated on a 0-5 scale (0=no symptoms, 5=Lesions on petiole, midrib and veins showing discoloration) after 36 hours of treatment Dry weight was obtained by drying the mycelium at 45 C for 3 days. 45

4. Intact soybean plants (RA 606) grown in the greenhouse and treated with partially purified toxin.Toxin (100 |ig) dissolved in acetone was placed on a puncture made with a needle at the node. Symptoms appear as dark lesions on stem, petiole and discoloration of midrib and veins after 3-4 days. 46

5. Infrared spectrum of purified toxic metabolite produced Diaportheby phaseolorum var. caulivora. 47

CHAPTER III.

1. Soybean trifoliates treated with 1:10 dilution of culture filtrate of Diaporthe phaseolorum var. caulivora for 36 hours. A, Leaflet showing symptoms of reddish discoloration of midrib and veins while the lamina remains healthy B, Discoloration of midrib and veins showing the toxin action on the vascular tissue. 60

v Eigam Page

2. Autofluorescence of soybean trifoliates treated with a 1:10 dilution of culture filtrate of Diaporthe phaseolorum var. caulivora for 36 hours. A, Yellow fluorescence around the lesions on the petiole (56X) B, Cross section of petiole showing yellow fluorescence of vascular bundles which correspond to the lesion seen externally (64X). 61

3. Autofluorescence of leaf and petiole sections of toxin treated samples. A,Cross section of leaf through midrib and veins showing discoloration. Note fluorescence of the midrib and vascular bundle of one of the veins (160X). B, Vascular bundles of petiole showing autofluorescence (200X) 62

4. Leaf sample treated with the toxin and stained with 10% aqueous ferric chloride for 12 hours. A, Top: Control, Bottom: midrib and veins showing staining. B, Lamina showing stained discolored cells. 63

5. Thin layer chromatogram of fluorescent and phenolic compounds produced by soybean plants in response to toxin or fungus inoculation. Chromatogram was developed in benzene:ethylacetate:acetic acid (75:24:1) and viewed under short wave UV lamp. 1,Control treated with acetone solution. 2, Purified toxin treated. 3, Partially purified toxin treated. 4, Culture filtrate treated. 5, Fungus inoculated by toothpick. 6, Control inoculated with toothpick. A= yellow fluorescence (2-5) B=yellowish orange fluorescence (2-3), C and D gave positive reaction for phenols (4-5). . 64

APPENDIX

1. Thin layer chromatogram (Silica gel GF 254) developed in chloroform:methanol (90:10) and veiwed under short wave UV lamp A and C are partially purified toxins, B is purified toxin 66

2. Ultra violet spectrum of purified toxic metabolite produced in the culture filtrates of Diaporthe phaseolorum var caulivora. The spectrum was taken in methanol. 67

3. Mass spectrum of purified toxic metabolite produced by Diaporthe phaseolorum var. caulivora showing molecular ion peak m +at 349.1. 68 ABSTRACT

The role of a toxic metabolite in the stem canker disease of soybean was investigated. Isolates ofDpc produced a metabolite in the culture filtrate which produced symptoms similar to stem canker on soybean plants. The severity of symptoms was dose dependent. The isolate from Opelousas, Louisiana produced significantly higher amounts of the toxin than the other isolates. A positive correlation was seen between the amount of toxin produced and the severity of disease produced by theDpc isolates. Among twelve plant species tested, only soybean and lima bean, were sensitive to the toxin. Reaction of five soybean cultivars to the culture filtrates of the Dpc isolates did not reflect the resistance of the cultivars to the pathogen as seen in the field. Spectral analyses indicated that the toxic metabolite is a low molecular weight aromatic compound, has one or more hydroxyl groups, methyl groups and C=C, and has a reducing nature. The trifoliates were not sensitive to cytochalasins H and B, but were sensitive to a toxic metabolite produced by aPhomopsis sp. isolated from wilted pine trees. Some of the chemical properties ofDpc toxin are similar to the Phomopsis/pine wilt toxin. Preliminary studies indicated a similar molecular ion peak of the two toxins. The initial symptom of toxin action was a red discoloration of midrib and veins before chlorosis was observed. The toxin induced yellow autofluorescence of vascular bundles. The fluorescence was also associated with lesions on the plant parts. The discoloration of the midrib and veins, and lesions on the plant were due to accumulation of phenols which are alkali soluble, can be extracted and separated by thin layer chromatography, and react with Folin-Ciocalteu reagent and ferric chloride. Two phenolic compounds, and a compound showing yellow fluorescence, not seen in untreated samples were observed in the toxin treated and fungus inoculated samples suggesting a common host response to toxin action and fungal inoculation. The role of toxin in the stem canker disease appears to be as a virulence factor.

viii I. REVIEW OF LITERATURE REVIEW OF LITERATURE

Soybean (Glycine max (L.)Merr.) is an important food crop with approximately 24 x 10^ ha under cultivation in the United States in 1985 (4). Production has undergone tremendous expansion in tropical as well as in temperate areas of the world, with an estimated production of 100 million metric tons in 1979 (3). Soybeans are grown primarily as a source of vegetable oil and protein, the United States being the world's foremost producer and exporter. In the years 1985- 86, the United States produced 57 x 10^ metric tons of soybean, 60% of the world's output of about 94 x 10^ metric tons. In Louisiana the total area under cultivation was estimated to be 8.8 x 10^ ha in 1985 (4). As soybean acreage has expanded throughout the world, diseases have increased in number and severity. In 1985, losses due to soybean diseases in the southern United States were estimated at $ 490 million of which 40% were due to foliar and stem diseases (36). Soybean is subject to a number of serious diseases caused by viruses, bacteria, fungi and nematodes which limit production. The fungal diseases of major economic importance in Louisiana are aerial blight, stem canker, pod and stem blight, red crown rot and anthracnose. Stem canker and pod and stem blight together with soybean seed decay constitute a disease complex caused by the fungusDiaporthe and its anamorphPhomopsis sp. (31,16). During the past decade there has been a substantial increase in research on the Diaporthe/Phomopsis disease complex which is prevalent throughout the soybean growing areas of the world. This complex causes more losses than any other soybean disease with the possible exception of root rot (48). Three fungi are associated with the disease complex:Diaporthe phaseolorum (Cke. & Ell.) var. sojae Wehm. (Dps) the causal agent of pod and stem blight,D. phaseolorum (Cke. & Ell.) Sacc. var. caulivora. Ath. & Cald. (Dpc) the causal organism of stem canker andPhomopsis sp. causing seed decay. Of the three diseases, pod and stem blight was the most severe of all stem and foliar diseases during the years

2 3

1985 and 1986 (36). Though Dps andDpc are associated with seed decay, Phomopsis sp. is the most prevalent and could be a source of introduction of the pathogen to new areas (31).Phomopsis sojae and/orDpc were present in 89% of seed lots examined (1) and significant negative correlation was found between seed infection and their germination, vigor and field emergence (32). STEM CANKER: The third component of theDpc-Dps disease complex and the subject of the present study is stem canker of soybean. Losses caused by the disease in 1983 were valued at $ 36.5 x 10^ in seven states in the southeast in the United States (42). The disease was first identified by Welch in Iowa in 1947. Subsequently there were reports from Michigan (2), Iowa (11), Minnesota (30) and Ontario, Canada (24). Stem canker was wide spread throughout the midwest in the early 1950's but by 1956, the disease disappeared due to the reduced planting of susceptible cultivars Blackhawk and Hawkeye. However, stem canker has recently become a major problem in the southeastern United States. The earliest reports were in 1975 in Mississippi, followed by reports from Alabama in 1977, Tennessee in 1981, South Carolina and Georgia in 1982, Florida in 1983 (42), Arkansas in 1983 and Texas in 1984 (6). In Louisiana, stem canker was first observed in 1981 near Rosa in St. Landry parish on the cultivar Bragg (50). In 1983, stem canker was found in 24 soybean producing parishes. Observations of disease reaction on susceptible varieties grown in Louisiana indicated significant negative correlation between yield and disease ratings (22). Severe outbreaks were observed at Burden Research Plantation, Baton Rouge, La in 1983 and 1984 (50). Symptoms: The first symptom of stem canker is the appearence of reddish- brown lesions on one or both cotyledons. The disease generally is observed during the later half of the growing season. Infection starts as a small superficial reddish- brown lesion on the leaf scar usually evident on the lower part of the stem. The lesion rapidly enlarges to form a slightly sunken, reddish-brown canker up to 4

several centimeters long that girdles the stem and kills the plant while older lesions turn brown or black. When plants are killed, green stem tissue above and below the canker distinguishes this disease from other diseases. Invaded stem tissues are brittle and plants break easily at the canker (47). Foliar symptoms are characterized by interveinal chlorosis and necrosis. Leaves on the petiole attached to girdled stems exhibit necrotic areas between veins (50). Foliar symptoms are manifest only after the host vascular system is disrupted. Stem canker may thus be inconspicuous in the field unless foliar symptoms are evident. Late season die of soybean was confirmed to be caused Dpcby infection in later stages of growth in Ohio and Michigan in 1979 and 1980 (25). The fungus: Diaporthe is the largest genus of the family Diaporthaceae belonging to the sub-class Hymenoascomycetidae of the class Ascomycetes. The anamorph belongs to the form genusPhomopsis. Lehman in 1922 first described Diaporthe sojae as the cause of pod and stem blight of soybean. Later, Wehmeyer, in 1933, based on morpohological considerations, reducedD. sojae on soybean andD. batatatis on sweet potato to varietal status underD. phaseolorum causing pod and stem blight of lima bean, thus distinguishingD. phaseolorum var. sojae and£>.phaseolorum var. batatatis (24). Welch and Gilman studied the two fungi and found that/).phaseolorum var. batatatis is homothallic, produced perithecia in cespitose clusters and actively attacked soybean stems girdling them and killing the plants. D. phaseolorum var. sojae is heterothallic, produces perithecia singly and is weakly pathogenic on soybeans. Athow and Caldwell retained the nomenclature ofD. phaseolorum var. sojae for the weakly-pathogenic organism causing pod and stem blight but differentiated the stem canker causing organism as Diaporthe phaseolorum var. caulivora based on morphological characters and the absence of an anamorph (5). Differences in morphological characters and symptomatology have been observed between northern and southern isolates Dpc. of Cankers in southern grown soybean are more delimited and unilateral in appearence and the variations 5 seen in canker appearence and multiple infections in southern stem canker are rarely seen with northen isolates. In addition, the southern isolates are more aggressive and attack a wider range of susceptible cultivars than northen isolates (6,49). There are also reports of different pathogenic biotypes within the southeastern isolates (35). Hobbs and Phillips (26) suggested that differences in symptom development and cultural characters were significant enough to warrant designation of the disease as "southern stem canker" and the isolates as "southernD. phaseolorum". In addition to symptomatological and morphological differences, differences in pathogenecity have also been reported. Differential reaction of soybean cultivars to stem canker infection was reported from nearly immune Tracy M and Braxton to highly susceptible Hutton, Coker 237 and the breeding line J77-339 (58,29). An artificial inoculation technique was developed by Keeling (27) using toothpick inoculations and good agreement was found between the seedling response of artificially inoculated field grown plants and the incidence of naturally occurring disease. Severity of stem canker incidence varied greatly at different locations. This variation may be due to environmental conditions or variations in the virulence of the pathogen. Keeling (29) evaluated twelve isolates recovered from diseased plants from different locations and found significant differences in virulence. He also identified six differential cultivars and six corresponding races using the toothpick inoculation technique (28). The differential pathogenecity ofDpc isolates on soybean cultivars also demonstrated that two races ofDpc exist among the three isolates tested from Mississippi and Iowa (23). Tracy M which was resistant in many earlier reports (58,29) was susceptible to the Iowa isolate but resistant to the Mississippi isolate (23). THE TOXIN CONCEPT: The idea that toxins play a causal role in some plant diseases dates to the time of de Bary who succeeded in reproducing soft rot by applying sterile extracts from rotted carrots to healthy carrot tissues (21). This report and results of other early physiological investigations were the beginning of 6 the evolution of the toxin concept (21). The involvement of a toxin in a disease can be determined by examining symptoms developing on the plant. Yellowing, wilting, brightly colored-lesions and necrosis may be associated with phytotoxins. If symptoms are produced at sites away from the site of infection of the pathogen, the involvement of a phytotoxin can be strongly suspected (56). However, reproducible symptoms do not necessarily mean the involvement of a toxin since many compounds in culture filtrates produce similar symptoms. Microbial toxins substituted for the microbes which produce them hold great promise as tools of investigations of the nature and cause of diseases. The use of toxins would permit exclusion of the pathogen and allow a more quantitative approach than is possible when two living organisms and their interactions are to be considered. Before such substitutions are made, it is essential to be sure that the toxin plays a causal role in the disease concerned. To distinguish the different roles a toxin might have in disease expression several terms have been suggested. Dimond and Waggoner (12) have proposed the term vivotoxin, which was defined as a substance produced in the infected host by the pathogen and/or its host which functions in the production of disease, or is a disease producing entity. They have also proposed criteria for a vivotoxin which included purification of the metabolite, separation from the affected plant, and reproduction of at least a part of the disease syndrome on the healthy plant Wheeler and Luke did not consider these criteria ideal and adequate because isolation of a toxic compound from a diseased plant does not prove that it played a causal role. The toxin could be a product of disease. They proposed that (a) the toxin applied at concentrations reasonably expected in the diseased plant should produce in a susceptible host all the symptoms characteristic of the disease; (b) the pathogen and the toxin should exhibit similar specificity (c) the ability of pathogen to produce toxin varies directly with its ability to cause disease and (d) a single toxin is required. They also proposed the term "pathotoxin” for those toxins which 7 play an important causal role, thus dividing toxins into three classes: pathotoxins, which have a causal role in the disease, vivotoxins which are producedin vivo in host plant and phytotoxins which do not have any specific role in the disease (61). Later Scheffer (43) classified toxins into host-specific (or selective) and non­ specific (or non-selective). Host-specific toxins are those which are toxic to hosts of producing organisms, while non-host genotypes and species are tolerant to the toxin. Several of the host-specific toxins are known tc recognize plants which cany a single gene for sensitivity which also gives susceptibility to the fungus. Thus, host-specific toxins are those that are required for the pathogenecity of the producing fungus while non-specific toxins are not required for pathogenecity but may contribute to virulence and are responsible for certain symptoms. While highly specific and non-specific toxins can be recognized as such, there may be several intermediate types. Toxins that are non-specific may show differences in toxicity to plant species which may not match the selectivity of the pathogen (14). Likewise non-specific toxins may have specific target sites (51). Toxins can also be distinguished as primary determinants which are essential for pathogenicity and secondary determinants which are not required for pathogenicity but contribute to virulence (61). Yoder proposed the terms "pathogenicity factors" required for pathogenicity which are usually host-specific toxins and "virulence factors" which are required for virulence and are usually non specific (63), while these terms are more related to the toxin involvement in the disease, pathogenicity pertains to the ability of the microorganisms to induce disease and virulence refers to the relative severity of the disease. Earlier it was believed that phytotoxic metabolites of plant pathogens were of limited importance in pathogenesis and lead only to visible injuiy on host plants but it is now clear that host-specific toxins are important determinants in pathogenesis. Among the toxins for which evidence supporting a causal role is strongest are the host-specific toxins. Many of the host-specific toxins are related to serious epidemics of economically important crops. The first well known example is the 8

blight of oats which appeared when cultivars with the Vb gene for resistance to the rust Puccinia coronata were introduced. These cutivars were susceptible to the HV toxin produced byHelminthosporium victoriae. Another serious epidemic for which a toxin was responsible was the HmT toxin produced byH. maydis race T which was responsible for the southern com leaf blight epidemic. Race T was indistinguishable from race O except in its capacity to produce the toxin to which maize with male sterile cytoplasm was susceptible. In addition to the HV and HmT toxins, two other Helminthosporium toxins play important roles in disease- the HC toxin produced byH. carbonum causing blight of maize and HS toxin, produced by H. sachari, causing eye spot of sugarcane (44). Another example of how toxin involvement can explain the seasonal outbreaks of epidemics is shown by the HS andPericonia circinata (PC) toxins. Temperatures above approximately 34 C cause sorghum tissues to become insensitive toP. circinata and sugarcane insensitive to H. sachari while temperatures below 34 C make them sensitive. In the southwest United States, milo disease of sorghum was evident in spring but disappeared in summer and again appeared in fall, while the eye spot disease of sugarcane was evident in Hawaii and Florida in winter and spring and disappeared in summer (10). The toxin produced by the pathogen thus plays a very crucial factor and may result in epidemics. Knowledge of these metabolites could lead to practical applications in plant pathology. In addition to these toxins for which there is considerable evidence for disease involvement,H. oryzae, has been recently reported to produce a host-specific toxin which differentiated rice cultivars susceptible to brown spot disease (57). Another group of toxins which have been shown to be host-specific are those produced byAlternaria sp. Species of Phyllosticta, Periconia andCorynespora are also reported to produce host-specific toxins responsible for the yellow blight of com, milo disease of sorghum and target spot of tomato, respectively (44). The biological role of host-specific toxins extends from being uniquely required to incite disease (pathogenecity factor) to situations in which infection can occur in the 9

absence of toxin but toxin contributes to the severity of the disease (virulence factor). The host specific Alternaria toxins includeA. kikuchiana (AK toxin) affecting Japanese pear,A. mali (AM toxin) affecting apple,A. citri (AC toxin) affecting citrus, A. alternata f. sp. lycopersici (AAL toxin) affecting tomato,A. fragariae affecting strawberry andA. longipes affecting tobacco. Each of these is known to produce a host-specific toxin responsible for specific pathogenecity (38). In addition to the above mentionedAlternaria toxins, A. brassicae also produces a toxic metabolite which is specific to species of Brassicae. The sensitivity of the Brassicae sp. to the toxin was similar to the susceptibility to the fungus and the metabolite was identified as Destruxin B (7). Nishimura et al used the term "release toxin" to denote a host-specific toxin from germinating spores of the pathogen. All known host-specificAlternaria sp. produce the release toxin. The importance of the release toxin was first proposed by Nishimura and Scheffer (37) when they observed HV toxin in germinating spores. Wheeler (61) proposed that the toxin producing plant pathogens may be "telepathogenic", capable of causing disease at a distance without being in physical contact. Non-host specific toxins are generally known as virulence factors. Though not required for pathogenecity these toxins enhance the virulence of the pathogen. One classic example of non-host specific toxin which has contributed to virulence is the tabtoxin in wildfire disease of tobacco caused byPseudomonas syringae pv. tabaci (62) causing chlorosis of leaves. Production of tabtoxin leads to symptoms in tobacco, oats, beans and soybean. The toxin plays a major role in the alteration of biochemical processes leading to the expression of virulence. Natural Tox-- mutants which did not produce detectable levels of tabtoxin when injected into tobacco plants colonized the tissue but did not produce the extensive chlorosis and necrosis typical of the Tox+ strains (34). In contrast,H. maydis occurs as two races, race T which produces HmT toxin and race O which does not. Race T is 10 more virulent than race O on maize genotypes with Texas male sterile cytoplasm. Both races are equally virulent on genotypes with normal cytoplasm. Thus HmT toxin determines virulence which is a quantitative trait (44). Other non-specific toxins which have a role in disease are coronotine produced by P. syringae pathovars, phaseolotoxin, produced by P. syringae pv. phaseolicola and tentoxin produced A.by tenuis. Coronotine causes chlorosis and browning of leaves of soybean and rye grass. The toxin also induced, in addition to chlorosis, hypertrophic growth of rice seedlings (34). Phaseolotoxin is associated with chlorosis of many plant species, particularly Phaseolus sp. infected byP. syringae pv. phaseolicola causing halo blight of bean. Production of phaseolotoxin contributes significantly to virulence and halo production while avirulent strains which did not produce the halo did not produce the toxin. Alternaria tenuis causes seedling albinism of the cotton seedcoat and citrus seedlings which was later found to be a result of the action of tentoxin (17). In addition to the above-mentioned toxins, a number of non-specific toxins play a role in diseases. These include fusicoccin produced Fusicoccum by amygdali, tagetitoxin produced byP. syringae pv. tagetis, syringomycin produced byP. syringae pv. syringae, rhizobitoxin produced byRhizobium japonicum, and ceratoulmin produced byCeratocystis ulmi (34). Studies with host-specific and non-specific toxins have potential economic and scientific value. Toxins functioning as pathogenecity factors may select for high levels of resistance whereas an intermediate level may be selected by those which are virulence factors An example of a host-specific toxin which has a causal role in stem canker disease is the metabolite produced byAlternaria alternata f. sp. lycopersici (AAL) on tomato. The disease is characterized by brown to black cankers on the stem, interveinal necrosis of leaves and epinasty of petioles. The pathogen is confined to the canker and cannot be isolated from leaf tissue which suggested that the foliar symptoms are due to a translocatable toxin (20). Susceptible genotypes are a thousandfold more sensitive to the toxin than resistant cultivars. A preliminary 11 genetic analysis using Fj and2 F progeny from a cross between homozygous resistant and susceptible cultivars showed that host reaction to both fungus and toxin was governed by a single gene with two alleles (19). The AAL toxins which occurred as two closely related esters (9) were also recovered from infected tomato leaves (46). Extracts from both culture filtrates and extracts of necrotic leaves of tomato plants infected with the fungus had equal specific activity and were equally host specific (20). Toxins function as a chemical interface between pathogens and their host plants and structural information is an important prerequisite for the study of mode of action. Structures of several of the host-specific and non-specific toxins have been elucidated. The chemical nature of these toxins is diverse. Even host specific toxins though biologically very narrowly defined, represent widely different chemical categories. The chemical nature varies from sesquiterpene glycosides (HS toxin), linear polyketals (HmT toxin), cyclic tetrapeptide (HC toxin, AM toxin) and esters of epoxydecatrienoic acid (AK toxin) (18). Phytotoxins have been categorized in regard to their organismal origin, similarity and differences in chemical structure, symptoms, and role in disease. None of these characterizations have provided clues to the molecular mode of action. The ultimate objective is to understand the complete sequence of the metabolic and physiological alterations due to infection which arise from the primaiy interaction, and pathotoxins have been chosen to study this sequence of physiological events in pathogenesis. The pathotoxins avoid alterations due to actual growth of the pathogen in diseased tissues subsequent to determining event, permit reduction in time of the stress determining events from days to hours and allow experimental use of tissues (18). The objective is to learn the precise site of interaction of the toxin with the host cell and what amplified sequences lead to the disease symptoms. In spite of the characterization of many host-specific toxins, there are very few host/toxin systems for which the primary target site has been conclusively shown. 12

A rapid and nearly universal response of susceptible plants or tissues to toxin treatment is an alteration of membrane permeability (43,60), detected as depolarization across the plasmamembrane (41). This has led to the hypothesis that susceptible genotypes contain a toxin receptor which is absent in resistant genotypes (45,55). AAL toxin did not cause ion leakage like other host-specific toxins (13) but inhibited aspartate carbomyltransferase which is an important step in pyrimidine biosynthesis (18). This enzyme was suggested to have a binding site for the toxin. With each toxin having a specific biochemical mode of action, understanding the role of enzymatic processes appears central to the resolution of the molecular mode of action and ultimately the role of the pathotoxin in disease. Specific binding of Fusicoccin, a toxin producedFusicoccum by amygdali to plasma membrane enriched fractions is thought to be associated with ATPase (54). ATPase activity was also stimulated by syringomycin, produced by P. syringae (8). In addition to the above-mentioned toxins, there are examples of a number of potential target sites where a pathotoxin could alter the metabolism of the plant cell. Changes in dark carbon dioxide fixation is seen in the case of HC toxin, HS, PC and HV toxins. Tentoxin inhibits photophosphorylation by binding to CF1-ATPase complex (52). Tabtoxin produced byP. syringae pv. tabaci inhibits glutamine synthase leading to accumulation of ammonia which may uncouple photophosphorylation and cause chlorosis (15). Non-specific toxins have a high biochemical specificity yet little specificity between plant species, presumably because the targets are enzymes common to most plants (34). Apart from the role of a host-specific toxin in tomato, no other toxins are reported to be involved in canker-causing diseases.Phomopsis sp., the anamorph of Diaporthe, produces metabolites called cytochalasins which are generally toxic to mammalian cells (59,40) and cause cytological changes in plants and eucaiyotic microbes (53). A recent report on the production of a trihydroxy lactone by a species of Phomopsis isolated from pine trees and its capacity to cause red 13

discoloration of pine callus suggest a role in pine wilt (39). Culture filtrate of Diaporthe phaseolorum var. sojae, the causal organism of pod and stem blight of soybean inhibited germination of cabbage, onion, wheat, cantaloupe, and soybean seed and wilted soybean seedlings (33). No evidence for its role in the disease has been suggested. In diseases in which a toxin plays a major role in pathogenicity, disease resistant individuals can be selected from those in a population (18). Radio- or fluoroscent-labelled toxins can be used to define cellular location of the site of action and for biocontrol of other pathogens. The possibility for host-specific toxins and closely-related phytotoxins in practical application are many. Advances are being made in determining the role of host-specific toxins as initiation factors of pathogenesis at the site of host-pathogen interaction. Studies on the symptomatology and distribution of stem canker of soybean give an indication of the possible involvement of a metabolite. There are indications of the asymptomatic occurrence of Dpc in soybean plants and also the production of symptoms without the association of a fungus. Other phenomena such as the differences in disease severity in southeastern and mid-western states and the sudden outbreak of the disease in southern states in the 1980's (31) suggest the role of a toxin. The present investigation was conducted to determine if a toxic metabolite is produced byDpc and if so, whether it has any role in stem canker of soybean. LITERATURE CITED

1. Anderson, T.R. 1985. Seed molds of soybean in Ontario and the influence of production area on the incidenceDiaporthe of phaseolorum var. caulivora andPhomopsis. Can. J. Plant. Pathol. 7: 74-78. 2. Andrews, E.A.S. 1950. Stem blight of soybean in Michigan. Plant Dis. Rep. 34: 214. 3. Anonymous, 1980. Agricultural Statistics, 1980. Pages 129-135. U.S. Govt. Print. Off. Washington D.C., 603 pp. 4. Anonymous 1986. Agricultural statistics. 1986. Pages 124-129. U.S. Govt. Print. Off. Washington D.C. 551 pp. 5. Athow, K.L., and Caldwell, R.M. 1954. A comparative study ofDiaporthe phaseolorum stem canker and pod and stem blight of soybean. Phytopathology 44: 319-325. 6. Backman, P.A., Weaver, D.B., and Morgan-Jones, G. 1985. Soybean stem canker: an emerging disease problem. Plant Dis. 69: 641-647. 7. Bains, P.S., and Tiwari, J.P. 1987. Purification, chemical characterization and host specificity of the toxin produced byAlternaria brassicae. Physiol. Mol. Plant Pathol. 30:259-271. 8. Bidwai, A.P., Lei Zhang, Bachman, R.C., and Takemoto, J.V. 1987. Mechanism of action ofPseudomonas syringae phytotoxin syringomycin. Plant Physiol. 83: 39-43. 9. Bottino, A.T., Bowen, J.R., and Gilchrist, D.G. 1981. Characterization of a phytotoxin fraction fromAlternaria alternata f. sp. lycopersici. Tetrahedron Lett. 29: 2723-2726. 10. Bronson, C.R., and Scheffer, R.P. 1977. Heat and aging induced tolerance of sorghum and oat tissues to host-selective toxins. Phytopathology 67:1232- 1238.

14 15

11. Crall, J.M. 1950. Soybean diseases in Iowa in 1949. Plant Dis. Rep. 34: 96- 99. 12. Dimond, A.E., and Waggoner, P.E. 1953. On the nature and role of vivotoxins in plant diseases. Phytopathology 43: 229-234. 13. Dunkel, L.D., and Wolpert, T.J. 1981. Independence of milo disease symptoms and electrolyte leakage by the host specific toxins from Periconia circinata. Physiol. Plant Pathol. 18: 315-323. 14. Durbin, R.D., and Uchytil, T.F. 1977. A survey of plant insensitivity to tentoxin. Phytopathology 67: 602-603. 15. Frantz, T., Peterson, D., and Durbin, R. 1982. Sources of ammonium in oat leaves treated with tabtoxin or methionine sulfoximine. Plant Physiol. 69: 345-348. 16. Frosheiser, F.I. 1957. Studies on the etiology and epidemiology of Diaporthe phaseolorum var. caulivora, the cause of stem canker of soybean. Phytopathology 47: 87-94. 17. Fulton, N.D., Bollenbacher, K., and Templeton, G.E. 1965. A metabolite from Alternaria tenuis that inhibits chlorophyll production. Phytopathology 55: 49-51. 18. Gilchrist, D.G. 1983. Molecular modes of action. Pages 81-136 in: Toxins and Plant Pathogenesis. J.M. Daly and B.J. Deverall, eds. Academic Press, New York. 181 pp. 19. Gilchrist, D.G., and Grogan, R.G. 1976. Production and nature of host- specific toxins from Alternaria alternata f. sp. lycopersici. Phytopathology 66: 165-171. 20. Gilchrist, D.G., Clouse, S.D., McFarland, B.L., and Martensen, A.N. 1985. Phytotoxins as molecular determinants of pathogenecity and virulence. Pages 405-420 in: Molecular genetics of filamentous fungi. W.Timberlake, ed. Alan R. Liss. Inc, New York. 465 pp. 16

21. Graniti, A. 1972. The evolution of toxin concept in plant pathology. Pages 1- 18 in: Phytotoxins in plant diseases. R.K.S. Wood, A. Ballio and A. Graniti, eds. Academic Press, New York. 530 pp. 22. Harville, B.G., Berggren, G.T., Snow, J.P., and Whitam, H.K. 1986. Yield reductions caused by stem canker in soybean. Crop Science 26:614- 616. 23. Higly, P.M. and Tachibana, H. 1987. Physiologic specialization of Diaporthe phaseolorum var. caulivora in soybean. Plant Dis. 71:815-817. 24. Hildebrand, A.A. 1956. Observations on stem canker and pod and stem blight of soybean in Ontario. Can. J. Bot. 34: 577-599. 25. Hobbs, T.W., Schmitthenner, A.F., Ellett, C.W., and Hite, R.E. 1981. Top dieback of soybean caused by Diaporthe phaseolorum var.caulivora. Plant Dis. 65:618-620. 26. Hobbs, T.W., and Phillips, D.B. 1985. Identification ofDiaporthe and Phomopsis isolates from soybean. (Abstr.) Phytopathology 75:500. 27. Keeling, B.L. 1982. A seedling test for resistance to soybean stem canker caused by Diaporthe phaseolorum var. caulivora. Phytopathology 72: 807- 809. 28. Keeling, B.L. 1984. Evidence for physiologic specialization inDiaporthe phaseolorum var. caulivora. J. Miss. Acad. Sci. Suppl. 29:5. 29. Keeling, B.L. 1985. Soybean cultivar reactions to soybean stem canker caused by Diaporthe phaseolorum var. caulivora and pathogenic variation among isolates. Plant Dis. 69: 132-133. 30. Kemkamp, M.F., and Gibler, J.W. 1951. Diseases of soybean new to Minnesota. Plant Dis. Rep. 35: 509-510. 31. Kulik, M.M. 1983. The current scenario of pod and stem blight -stem canker- seed decay complex of soybean. Int. J. Tropical Plant Dis. 1:1-11. 32. Kulik, M.M. and Schoen, J.F. 1981. Effect of seed borneDiaporthe phaseolorum var. sojae on germination, emergence and vigor of soybean 17

seedlings. Phytopathology 71:544-547. 33. Kunwar, I.K., Malfon-Meiri, A., Manandhar, J.B., and Sinclair, J.B. 1987. Possible phytotoxic metabolites in culture filtrates of Diaporthe phaseolorum var. sojae. Mycopathologia 99: 71-75. 34. Mitchell, R.E. 1984. The relevance of non-host specific toxins in the expression of virulence by pathogens. Annu. Rev. Phytopathol. 22: 215-245. 35. Morgan-Jones, G., and Backman, P.A. 1984. Characterization of southeastern biotypes of Diaporthe phaseolorum var. caulivora, the causal organism of soybean stem canker. (Abstr.) Phytopathology 74: 815. 36. Mulroony, R.P. 1988. Soybean disease loss estimate for southern United States in 1985 and 1986. Plant Dis. 72:364-365. 37. Nishimura. S., and Scheffer, R.P. 1965. Interaction between Helminthosporium victoriae spores and oat tissues. Phytopathology 55: 629-634. 38 Nishimura, S., and Kohmoto, K. 1983. Roles in pathogenesis. Pages 137- 158 in: Toxins and Plant Pathogenesis. J.M. Daly and B J. Deverall, eds. Academic press, New York. 181 pp. 39. Nobuhisi Morooka., Takashi Tatsuno., Hiroshi Tsunoda., Kimiko Koboyashi and Tsio Sakurai. 1986. Chemical and toxicological studies of the phytotoxin 6a, 7|3,9a, trihydroxy-8(14)15-isopimaradiene-20, 6y-lactone, produced by a parasitic fungusPhomopsis sp. in wilting pine trees. Agric. Biol. Chem. 50: 2003-2007. 40. Patwardhan, S.A., Pandey, R.C., Sukhdev., and Pendse, G.S. 1974. Toxic cytochalasins of Phomopsis paspali, a pathogen of kodo millet. Phytochemistry 13: 1985-1988. 41. Payne, G.A., Knoche, H.W., Kono, Y., and Daly, J.M. 1980. Biological activity of purified host specific pathotoxin produced byBipolaris (Helminthosporium) maydis race T. Physiol. Plant Pathol. 16: 227-240. 42. Ploetz, R.C., Sprenkel, R.K., and Shokes, F.M. 1986. Current status of 18

soybean stem canker in Florida. Plant Dis. 70: 600-602. 43. Scheffer, R.P. 1976. Host-specific toxins in relation to pathogenesis and disease resistance. Pages 247-269 in: Physiological Plant Pathology. E. Heitefuss and P.H. Williams, eds. Springer-Verlag, New York. 890 pp. 44. Scheffer, R.P. 1983. Toxins as chemical determinants of plant disease. Pages 1-40 in: Toxins and Plant Pathogenesis. J.M .Daly and BJ. Deverall, eds. Academic Press, New York. 181 pp. 45. Scheffer, R.P., and Samaddar, K.R. 1970. Host specific toxins as determinants of pathogenecity. Recent Advances in Phytochemistry 3: 123- 142. 46. Siler, D.J., and Gilchrist, D.G. 1983. Properties of host specific toxins produced byAlternaria alternata f. sp. lycopersici in culture and in tomato plants. Physiol. Plant Pathol. 23: 265-274. 47. Sinclair, J.B. 1982. Compendium of soybean diseases. Pages 39-40. American Phytopathological Society, St. Paul, Minnesota. 104 pp. 48. Sinclair, J.B. 1988. Diaporthe!Phomopsis complex of soybean. Pages 96- 101 in: Soybean diseases of North Central Region. T.D. Wyllie and D.H. Scott, eds. American Phytopathological Society, St. Paul, Minnesota. 149 pp. 49. Smith, E.F., and Backman, P.A. 1988. Soybean stem canker: An overview. Pages 47-55 in: Soybean diseases of North Central Region. T.D. Wyllie and D.H. Scott, eds. American Phytopathological Society, St. Paul, Minnesota. 149 pp. 50. Snow, J.P., Berggren, G.T., Harville, B.G., and Whitam, H.K. 1984. Stem canker: a soybean disease recently found in Louisiana. Louisiana Agric. 27:8- 9, 24. 51. Steele, J.A., Uchytil, T.F., Durbin, R.D., Bhatnagar, P., and Rich, D.H. 1976. Chloroplast coupling factor 1: a species specific receptor for tentoxin. Proc. Natl. Acad. Sci. (US) 73:2245-2248. 19

52. Steele, J., Uchytil, T., and Durbin, R. 1977. The binding of tentoxin to a tryptic digest of chloroplast coupling factor 1. Biochim. Biophys. Acta. 459: 347-350. 53. Stoessel, A. 1981. Structure and biogenetic relations: Fungal non host- specific toxins. Pages 209-220 in: Toxins in Plant Disease. R.D. Durbin, ed. Academic Press, New York. 514 pp. 54. Stout, R.G., and Cleveland, R.E. 1980. Partial characterization of Fusicoccin binding to receptor sites on oat root membranes. Plant Physiol. 66:353-359. 55. Stroebel, G.A. 1973. The Helminthosporoside binding protein of sugarcane. Its properties and relationship to susceptibiltiy to eye spot disease. J. Biol. Chem. 248:1321-1328. 56. Stroebel, G.A. 1982. Phytotoxins. Annu. Rev. Biochem. 51: 309-333. 57. Vidyasekharan, P., Borromeo, E.S., and Mew, T.W. 1986. Host specific toxin production byHelminthosporium oryzae. Phytopathology 76: 261- 266. 58. Weaver, D.B., Casper, B.H., Backman, P.A., and Crawford, M.A. 1984. Cultivar resistance to field infestation of soybean stem canker. Plant Dis.68 : 877-879. 59. Wells, H.M., Cutler, H.G., and Cole, RJ. 1976. Toxicity and plant growth regulator effect of cytochalasin H isolated from Phomopsis sp. Can. J. Microbiol. 22: 1137-1143. 60. Wheeler, H. 1978. Disease alteration in permeability of membranes. Pages 327-347 in Plant Disease. Vol. III. J.G. Horsefall and E.B. Cowling, eds. Academic Press Inc., New York. 487 pp. 61. Wheeler, H. 1981. Role in Pathogenesis. Pages 477-494 in: Toxins in Plant Disseae. R.D. Durbin, ed. Academic Press, New York. 515 pp. 20

62. Wooley, D.W., Pringle, R.B., and Braun, A.C. 1952. Isolation of the phytopathogenic toxin from Pseudomonas tabaci. J. Biol. Chem. 197: 409- 417. 63. Yoder, O.C. 1980. Toxins in Pathogenesis. Annu. Rev. Phytopathol. 18: 103-129. n. PROPERTIES OF A PHYTOTOXIN PRODUCED BY DIAPORTHE PHASEOLORUM VAR. CAULIVORA, THE CAUSAL ORGANISM OF STEM CANKER OF SOYBEAN

21 PHYSIOLOGY AND BIOCHEMISTRY

Properties of a Phytotoxin ProducedDiaporthe by Phaseolorum var. caulivora, the causal organism of stem canker of soybean.

B. Lalitha, J.P. Snow, and G.T. Berggren

Graduate Research Assistant, Professor and Associate Professor, respectively, Department of Plant Pathology and Crop Physiology, Louisiana Agricultural Experiment Station, Louisiana State University Agricultural Center, Baton Rouge, LA 70803. We thank Dr. RJ. Cole, National Peanut Research Laboratory, USDA, Georgia, for providing the cytochalasins H and B and Dr. N. Morooka, Science University of Tokyo, Japan for providing the Phomopsis toxin. We also thank Mr. Raphael Cueto for assisting with the IR spectroscopy. Accepted for publication______

ABSTRACT Lalitha, B., Snow J.P., and Berggren, G.T. 1988. Properties of a phytotoxin produced byDiaporthe phaseolorum var caulivora, the causal organism of stem canker of soybean. Phytopathology: Diaporthe phaseolorum var. caulivora isolated from stem canker infected soybean plants produced a toxin which caused symptoms characteristic of stem canker when introduced into the plant. The amount of toxin and symptom production were linearly related. Four fungal isolates produced the toxin in significantly different amounts, when measured in terms of severity of symptoms and dilution end point. The amount of toxin produced by the isolates correlated to the length of cankers produced by the fungus upon inoculation. Of the twelve plant species evaluated, only soybean and lima bean were sensitive to the toxin and also

22 23

susceptible to the fungus. The purified toxin produced symptoms very similar to stem canker and some of the chemical properties are similar to a phytotoxin suggested to have a role in pine wilt caused byPhomopsis sp. The toxin appears to play a role in stem canker of soybean.

Additional Key words', soybean, stem canker.

Stem canker of soybean (Glycine max (L.) Merr.) is caused by Diaporthe phaseolorum (Cke. & Ell.) Sacc. var. caulivora Ath. & Cald. (Dpc). The disease was originally described in Iowa in the 1940's. In the past decade, stem canker has caused severe losses in the southeastern United States and yield losses of 20-50% have been reported in individual counties of some states. Stem canker was found in 24 soybean producing parishes in Louisiana in 1983 with 16 parishes reporting severe losses (20). The disease is characterized by long brown lesions on stems usually beginning at the nodes and spreading to the intemodes and petiole. The lesions enlarge, become sunken and eventually may girdle the stem. Leaf symptoms appear as interveinal chlorosis and necrosis followed by plant death in highly susceptible cultivars. Dpc was isolated in previous studies from mature soybean plants that were asymptomatic throughout the growing season and also from resistant and susceptible cultivars apparently infected too late in the season to produce symptoms (1,18). In some cases the fungus could not be isolated from parts of plants showing symptoms which suggested the possible involvement in disease expression of a metabolite which advances before the fungus and produces symptoms. Several phytotoxins have been reported to have a role in disease and act as either pathogenecity or virulence factors (23) and some are involved in other stem canker diseases. A host-specific toxin is produced byAlternaria alternata f. sp. lycopersici causing stem canker of tomato (6). Phomopsis sp., the anamorph of Diaporthe sp., produces toxic metabolites, cytochalasins H and B (17,22), which 24 were not implicated in any plant diseases. Recently, culture filtrates Diaportheof phaseolorum var. sojae were reported to contain a toxic metabolite which caused wilting of soybean seedlings (14). In this study, we investigated the possibility of involvement of a toxin in stem canker and evaluated some of the fundamental chemical characters of the toxin.

MATERIALS AND METHODS

Isolates of Dpc. Cultures of Dpc were obtained from cankers on soybean plants collected from four locations in Louisiana: Opelousas, St Joseph, Slaughter and Lawtell. Single spored cultures were derived from field isolates and maintained on potato dextrose agar (PDA). Soybean cultivar Ring Around 606 (RA 606) which is susceptible to stem canker was used in all studies. Culture conditions. Four isolates were inoculated separately to 50 ml of sterile potato dextrose broth (PDB) in 250 ml flasks with cotton plugs, and incubated in darkness for three weeks at 24 C as stationary cultures. The inoculum consisted of 10 mm plugs of the fungus from 10 day old PDA cultures. The medium was filtered through Whatman #1 (W and R Balston Ltd., England) filter paper and used for bioassay. The optimum temperature for toxin production was determined by inoculating the medium with the Opelousas isolate and incubating at 12, 16, 20, 24 and 28 C for 23 to 25 days. The culture medium was filtered through a Whatman #1 filter paper and the mycelium dried in an oven at 45 C for 3 days. Dry weight of the mycelium in each treatment in three replications was recorded. Bioassay. Cell-free culture filtrate was obtained by passing the medium through a sterile 0.45 |im millipore filter (Millipore Filter Corporation, Bedford, Massachusetts) under vacuum. Two-fold dilutions of the culture filtrate from 1:10 to 1:160 in sterile distilled water were used for the assay. Similar dilutions of sterile PDB were used as controls. Soybean plants were grown in the greenhouse 25

maintained at 22-30 C, in autoclaved 12.5 cm clay pots containing a mixture of silt, peat moss and perlite (3:2:1). Two grams of Osmocote plant food 14-14-14, (Sierra Chemical Co., Milpitas, California) was added to each clay pot after seedlings emerged. Trifoliates of nearly uniform size from 30-50 day old plants were excised under water using a razor blade. The cut end of each trifoliate petiole was immersed immediately in 3 ml of the toxin solution in vials (4.5 cm x 1.0 cm). Symptoms were rated on a 0-5 scale after 36 hours. (0- no symptoms, 5- all veins, midrib showing discoloration and elongated red lesions on the petiole). Three or more replications were used to overcome the slight differences in sensitivity of leaves of different ages. Stability of the toxin. The stability of the toxin to changes in temperature and pH were tested. Test tubes (2.2x17 cm) containing 10 ml of the culture filtrate of the Opelousas isolate were heated individually to the specified temperatures in a hot water bath. The temperature of the culture filtrate in the test tube was increased through the required range and the upper temperature maintained for 15 min. The temperatures were 40, 60, 80 and 95 C. Ten ml of the culture filtrate was also autoclaved for 15 min. The tubes were cooled to room temperature and the filtrate assayed. The pH of 10 ml aliquots of the culture filtate was altered using IN HC1 to pH 3 and 5 and with 0.5 M sodium bicarbonate to pH 9.0. The bioassays were conducted as mentioned earlier. Specificity of the toxin. Pathogenecity of the four Dpc isolates was determined using the toothpick inoculation technique (10) on 30 day old greenhouse grown plants. Toothpicks which were boiled, air dried and saturated with potato dextrose broth were autoclaved for20 min and inoculated with the four isolates separately and incubated for 3 weeks at 24 C. The pointed ends of the toothpicks were cut to a length of 5-6 mm with a razor blade and inserted into a puncture made with a sterile needle at the lowest plant node. Five plants per treatment were inoculated and the length of cankers above and below the point of inoculation was 26 measured after four weeks. Bioassay of the culture filtrates of the four isolates was conducted simultaneously. The Opelousas isolate was used to test several plant species for sensitivity to the toxin. The plant species listed in Table 1 were grown in the greenhouse at 22-30 C in autoclaved pots as described previously and leaves of 30-50 day old plants were used for bioassay. Some of the plant species were also tested for susceptibility to the Opelousas isolate using the toothpick inoculation technique. Varietal reaction to toxin treatment. The Opelousas, St. Joseph, Slaughter and Lawtell isolates were grown under conditions described previously and the culture filtrates of each bioassayed. Five cultivars, Bragg (susceptible), RA 606 (moderately susceptible), Davis and Asgrow 5474 (moderately resistant) and Tracy M (resistant) (2,3) were grown in the greenhouse. Symptom ratings at 1:10 dilution of the culture filtrate and dilution end point (the highest dilution at which symptoms are produced) were noted. The reaction of the cultivars to the culture filtrates of the four isolates was compared to their stem canker reaction in the field (2,3). Purification of the toxin.Culture filtrate (2-3 L) was acidified to pH 5.0 with IN HC1 and extracted with equal amounts of ethyl acetate. The combined organic phase was dried by passing through anhydrous sodium sulphate and evaporated to dryness under vacuum at 40 C using a Rotary Flash Evaporator (Buchi, Switzerland). Sephadex LH 20 (Pharmacia Fine Chemicals, Piscataway, NJ) was allowed to swell in methanol for 24 hours and packed in a column (1 x 30.5 cm). The column was washed with 200 ml methanol before loading the sample. The residue was dissolved in a small quantity of methanol, placed on the column and eluted with the same solvent. Fractions (10 ml) were collected and bioassayed. The fractions showing activity were pooled and further purified by preparatory thin layer chromatography using silicagel GF 254 (J.T. Baker Chemical Company, Phillipsburg, NJ) and chloroform : methanol (9:1) as the solvent system. The three major spots that separated were scraped off the plate, eluted with 27

100 ml chloroform : methanol (1:1) and bioassayed. The toxin was purified by repeated separation and elution on TLC. The crude residue and purified toxin spot on the TLC plate were sprayed separately with various reagents (Table 2) and the color reaction observed. A modified bioassay was used for routine detection of the toxin. The trifoliates were excised under distilled water and placed in a petri plate over moist filter paper. The toxin residue was dissolved in 5 ml acetone and a 10 pi aliquot was placed on a puncture made with a sterile needle at the base of the midrib of each leaflet. The presence of the toxin was indicated by the reddish discoloration of the midrib and veins within 24 h. Trifoliates treated in a similar manner with acetone served as controls. The purified toxin was dissolved in spectrophotometric grade methanol for the UV absorption spectrum which was recorded on a Gilford 'Response' UV-Vis spectrophotometer (CIBA-Coming Diagnistic Corp., Oberlin, Ohio), and in acetone for the infrared spectrum recorded on an IBM IR44 spectrophotometer. Reaction of soybean trifoliates to other toxic metabolites. Pure Phomopsis sp. toxins (cytochalasin H, cytochalasin B, and a toxin produced by Phomopsis sp. from pine wilt) (100 Jig) were dissolved in 100 pi of acetone and made up to 3 ml with distilled water. The cut end of each excised trifoliate was immersed in the solution. The compounds (100 pg) were also assayed by the leaf puncture method. Controls containing solutions of acetone in water were tested simultaneously.

RESULTS

Toxic activity of the culture filtrate: Excised trifoliates treated with the culture filtrate produced symptoms similar to stem canker symptoms observed in the field. Initial symptoms appeared on the petiole within 24-36 hours as small reddish brown lesions (Fig.1) which gradually increased in length. On the leaflets, 28

the midrib and veins became reddish brown. Leaf lamina began to exhibit prominent interveinal chlorosis and subsequent necrosis after 48-72 hours at a dilution of 1:10. No wilting was seen after 4-5 days at dilutions above 1:10. At a dilution of 1:10 the leaflets became brittle and dry. The mean disease rating ranged from 3.83 at 1:10 to 0 16 at 1:160, showing an inverse linear relationship (r2=0.81) with increasing dilutions (Fig.2). The pH of the culure filtrate in all cases ranged from 7.6 - 7.9. The control samples treated with similar dilutions of noninoculated PDB filtrate did not show any symptoms. All four isolates produced the toxin though they differed significantly in the amounts of toxin produced based on the severity of symptom expression. The Opelousas isolate produced greater amounts of the toxin than the other isolates and was therefore used for further studies. The dilution end point was 1:160 for the Opelousas isolate and 1:20 for the other three isolates.The symptom rating also gave similar results, the Opelousas isolate differing significantly from the other three isolates (Table 3). Mycelial dry weight and toxin production were greatest at 20 C. Fungal growth was similar at 20 C and 28 C, while toxin production measured in terms of symptom severity sharply declined at the higher temperature (Fig.3), indicating that greater fungal growth does not necessarily indicate greater toxin production. Specificity of the toxin. Among the different plant species only soybean and lima bean were sensitive to the toxin and susceptible to the fungus (Table 1). The symptoms of toxin treatment were similar on lima bean to those on soybean with reddish lesions on the petiole, midrib and veins, and interveinal chlorosis. Many of the other plant species were neither sensitive to the toxin nor susceptible to the fungus. RA 606 plants inoculated with the fourDpc isolates produced typical stem canker symptoms after four weeks. The length of the cankers produced by the four isolates varied significantly. The Opelousas isolate produced the longest cankers 29 and also caused chlorosis of the leaves and death of the plants. The Opelousas isolate differed significantly from the other three isolates in the length of the cankers produced and also in the symptom severity produced by the culture filtrate (Table 3). Three out of five plants inoculated with each fungus isolate produced cankers and only plants showing symptoms were included in analyses. A correlation of r= 0.82 was obained between the length of the cankers and the amount of toxin producedin vitro by the four isolates (Table 3). Varietal reaction to the toxin. The dilution end point of the Opelousas isolate for all the five cultivars treated with the culture filtrate was 1:160. The differences in the dilution end point were not definitive enough to distinguish differences in sensitivity of the cultivars to the culture filtrates of the isolates. The disease rating on a 0-5 scale at 1:10 dilution was therefore used for comparison of the cultivars (Table 4). The Opelousas isolate produced significantly more severe symptoms than the other isolates tested. However, the cultivars did not show any differences in symptom rating in response to the culture filtrates of the Opelousas isolate. Similar trend was observed at other dilutions. Tracy M and A 5474 which are resistant and moderately resistant respectively (2,3), to stem canker in the field showed significantly higher symptom ratings than the other cultivars when treated with the toxic culture filtrates (Table 4). Stability of the toxin. The dilution end point of culture filtrates subjected to heating was 1:160 at all temperatures except after autoclaving the toxin. In this case, the activity was reduced to a dilution end point of 1:40. However, symptoms produced by the culture filtrate at1:10 dilution were different in samples heated at 95 C or when autoclaved. The interveinal chlorosis and necrosis were not produced in samples heated to 95 C or autoclaved but the discoloration of midrib, veins and production of lesions were similar to that produced when samples were heated at other temperatures. The dilution end point at pH 5 was 1:160, the same as that of the original culture filtrate which had a pH of 7.6-7.9. At pH 3.0 and pH 9.0 the dilution end point was reduced to 1:40. 30

Characteristics of the Dpc toxin. Dialysis of the culture filtrate against distilled water for 24 hours removed all detectable toxic component from the culture filtrate indicating a low molecular weight compound. Ethyl acetate extracted most of the toxic component from the culture filtrate and the residue produced symptoms typical of stem canker. When the crude extract was separated on TLC, three major compounds appeared as purple quenched spots under UV light. All three spots reacted with ethanolic sulphuric acid (brown). The compounds also reacted with phosphomolybdic acid (bluish green), alkaline potassium permanganate (yellow), neutral potassium permanganate (yellow), Pauly's reagent (yellow), Folin-ciocalteu reagent (bluish grey) and acidic ferric chloride (blue). No reaction was seen with either Ninhydrin or lodoplatinate indicating that the toxin is not a protein and does not contain nitrogen (Table 2). When each spot was eluted and assayed, the majority of the toxic activity was detected in the spot with Rf of 0.4. When the purified extract was separated on TLC and sprayed with the reagents separately, no other spot was detected. The eluted compound produced reddish discoloration of veins when assayed by the leaf puncture technique. Lesions on petioles, discoloration of midribs and veins, and interveinal chlorosis were produced by the TLC purified toxin while control plants did not exhibit symptoms. The toxin produced reddish-brown lesions in 2-3 days on stems, petioles and midrib when applied to a puncture at the node of intact plants (Fig.4). Lesions were seen both above and below the point of toxin application. The minimum amount of TLC purified metabolite required to produce reddish veinal discoloration in 36 hours by leaf puncture was 4 |ig. The UV spectrum in methanol showed maximum absorption at 210 nm and the IR spectrum showed peaks at 3339, 2928, 2855, 1711, 1662 and 1599 (Fig.5). Reaction of soybean to other Phomopsis toxins. No symptoms were produced by 100 (ig of cytochalasins H or B after 72 hours. The toxin from Phomopsis sp. isolated from wilted pine trees however, produced red discoloration of the midrib and veins after 72 hours. The intensity of discoloration 31

was very low while 10 |ig of the the Dpc toxin produced severe discoloration in 24-36 hours (Table 3). DISCUSSION Dpc produces a toxinin vitro which causes symptoms typical of stem canker on excised trifoliates and intact plants. The severity of symptoms was linearly related to the toxin dose which may indicate a role in symptom production. The variation in disease rating and dilution end point seen in the two experiments (Table 3 and 4) is probably due to the differences in the capacity of the trifoliates to translocate the toxin. Assay by uptake of toxin solution by petioles, though not very efficient and sensitive, does provide valuable information and worked satisfactorily. The two commonly used assays which are the inhibition of seed germination and increase in electrolyte leakage could not be used in this study. Preliminary studies using the culture filtrates did not induce electrolyte leakage of soybean trifoliates or petioles and did not inhibit seed germination. However, a reddish blue discoloration was observed when the seed was treated with partially purified toxin. A more sensitive assay based on the site of action or viability tests using protoplasts (5) would probably resolve the differences more effectively. The minimum amount of toxic metabolite required for symptom production by the leaf puncture assay was 4 jig. It was concluded that the toxin action was on the vascular system, since inoculation on any part of the lamina produced symptoms on the midrib and veins before any chlorosis was seen. The reproduction of many of the disease symptoms by the culture filtrate and also by partially and completely purified toxic metabolite, though not an indication of a specific role of the toxin in disease does sugggest a role in symptom production. The correlation between the amount of toxin and the length of cankers produced by the isolates is also evidence for the involvement of the toxin in symptom expression. Similar correlation of virulence with the quantity of toxin produced has been reported in other cases (19). However, a non-toxigenic isolate Dpcof if 32 shown to be non-pathogenic, would give evidence that might determine the role of the toxin as a pathogenecity or virulence factor. The sensitivity of lima bean to the toxin which is also a host for Dpc (9) also gives an indication of specificity.D. phaseolorum is also reported to cause fruit rot of pepper and tomato (9) but both were insensitive to theDpc toxin. Eggplant, which has been reported to be a host forD. vexans (9) was also insensitive to the toxin. However, for a toxin to be host specific, varieties susceptible to the fungus should also be sensitive to the toxin and in addition, the toxin or its metabolite should be produced in the host in quantities sufficient to cause symptoms. In preliminary studies, using 1:10 dilutions of the culture filtrate, resistant and susceptible soybean cultivars reacted similarly to the toxin treatment Reaction to the toxin did not reflect the resistance of the cultivar to the pathogen. Tracy M, which is reported to be highly resistant to the pathogen 21( ), produced severe symptoms when treated with a1:10 dilution of culture filtrate of all isolates and was as sensitive as the susceptible cultivar Bragg to the Opelousas culture filtrate. Davis, which is moderately resistant in the field was not significantly different from the susceptible cultivar Bragg or the moderately susceptible RA 606 in its sensitivity to the culture filtrates of all the four isolates. One possibility for the lack of correlation of the effects produced by Dpc and the toxin is that, the toxin may not be a pathogenecity factor. Another possibility is based on earlier reports on the physiologic specialization by Dpc isolates. Significant differences were observed between some cultivars treated with culture filtrates from St. Joseph, Slaughter and Lawtell isolates, but the differences did not correspond to their reaction to the fungus. The differences in the reaction may be due to physiological differences in the isolates, as observed in the case of Tracy M which was reported to be susceptible to one pathogenic isolate from Iowa, resistant to isolates from Mississippi (7), but susceptible to some isolates from Mississippi (11). In addition, the 1:10 dilution of the culture filtrate may be too concentrated to allow proper functioning of resistance factors. However, these results do not give any conclusive 33 evidence on the role of toxin as a pathogenecity factor. Further studies using a larger number of isolates including a non-pathogenic isolate and cultivars with simultaneous inoculations with the fungus might lead to conclusions on the role of toxin in the disease. The role of a toxin in stem canker expression may explain many of the observations on the disease seen in nature. The presence of the fungus in symptomless plants could be due to a non-toxigenic isolate and conversely, the presence of symptoms without the association of the fungus could be due to the translocation of the metabolite ahead of the fungus. The stem canker pathogen in the southern United States has been reported to be more aggressive than the pathogen in the mid-western United States (1). Recent reports also suggest that there are differences in the pathogens causing stem canker in mid-western and southeastern states (1,8,15). The involvement of a toxin in the disease could explain these differences in terms of the varying toxigenic potential of the pathogen. Structural information on the toxin is necessary for mode of action studies and also to relate the presence of the toxinin vivo. The purification procedure was satisfactory to obtain quantities sufficient for experimental studies. Though there are no reports on the production of a toxic metabolite byDpc, the imperfect stage Phomopsis sp. produces a class of compounds known as cytochalasins (17,22). These compounds were, however, not implicated in plant diseases. Purified cytochalasins H and B were assayed on soybean trifoliates and no symptoms resembling stem canker were observed. The passage of the metabolite through dialysis membrane indicates a low molecular weight and the stability after heating to a temperature of 80 C indicate the compound is not a protein. The color reaction to spray reagents indicated that the toxic metabolite could have a reducing nature (phosphomolybdic acid, alkaline potassium permanganate), aromatic carboxylic groups (alkaline potassium permanganate), one or more phenolic groups (Pauly's reagent, Folin-ciocalteu reagent), and hydroxy groups (Ferric chloride), and heterocyclic ring (Pauly's 34

reagent). (13). However, the absence of nitrogen and a non-proteinaceous nature are indicated by the negative reactions with iodoplatinate and ninhydrin respectively (13). The presence of one or more -OH groups is also indicated by peaks at 3339, -CH3 groups is indicated by peaks at 2928 and 2855, and C=C by peaks at 1711,1662 and 1599. A species ofPhomopsis isolated from wilted pine trees produces a metabolite which also causes reddish discoloration of pine callus (17). The chemical properties of Dpc toxin which include the UV spectrum, color reaction to spray reagents, presence of -OH and -CH3 groups resemble the Phomopsis toxin. The production of reddish discoloration of midrib and veins and lesions on the petiole by thePhomopsis toxin after 72 hours at about 10 times the concentration of theDpc toxin indicates a lower activity than theDpc toxin but the possibility of a relationship. There are reports of closely related molecules which show differences in biological activity. Alternaria tenuis produces dihydroxytentoxin which is two protons less than tentoxin and with the activity reduced from 0.4-0.8 |ig/ml to 100 (ig/ml (4). Further characterization of theDpc toxin is in progress using an improved extraction procedure to obtain the metabolite in larger quantities. Our results indicate that a toxin may be involved in stem canker of soybean which may answer questions like the sudden increase of stem canker in the southern United States probably due to the appearence of toxigenic strains. The variation in severity of stem canker may be due to the appearence of toxigenic strains of Dpc. Many of the outbreaks of epidemics involved toxins (23). In a preliminary study, negligible amounts of the toxin were produced Dpcby isolates from Iowa (unpublished) in their culture filtrates. This observation in conjunction with reports of the greater aggressiveness of the southern isolates (1) may indicate that the increased severity of disease in the southeastern states could be caused by a toxin. The Dpc toxin may be related to the toxin produced byPhomopsis sp. Further studies on the possible role of a toxin in the disease are in progress. Toxins 35 functioning as pathogenecity factors may select for high levels of resistance and those functioning as virulence factors may select intermediate levels. The information regarding the presence and involvement of a phytotoxin in host parasite interaction may be helpful in the development of breeding programs to select for disease resistance based on the reaction of the host to the toxin. LITERATURE CITED

1. Backman, P.A., Weaver, D.B. and Morgan-Jones, G. 1985. Soybean stem canker: an emerging disease problem. Plant Dis. 69:641-647. 2. Berggren, G.T., McGawley, E.C., Snow, J.P., and Whitam, H.K. 1984. Soybean disease control annual report. Louisiana Agric. Expt. Station., Louisiana State Univ. Agric. Center, Louisiana. 42 pp. 3. Berggren, G.T., Snow, J.P., Damicone, J.P., and Whitam, H.K. 1987. Soybean disease control annual report. Louisiana Agric. Expt. Station., Louisiana State Univ. Agric. Center, Louisiana. 87 pp. 4. Cutler, H.G. 1988. Unusual plant growth regulators from microorganisms. CRC Reviews in Plant Science 6:323-342. 5. Gendloff, E.H., Scheffer, R.P. and Somerville, S.C. 1987. An improved bioassay for victorin based on the use of oat protoplasts. Physiol, and Mol. Plant Pathol. 31:421-427. 6. Gilchrist, D.G., and Grogan, R.G. 1975. Production and nature of a host specific toxin from Alternaria alternata f. sp. lycopersici. Phytopathology 66:165-171. 7. Higley, P.M., and Tachibana, H. 1987. Physiologic specialization of Diaporthe phaseolorum var caulivora in soybean. Plant Dis. 71:815-817. 8. Hobbs, T.W., and Philips, D.V. 1985. Identification ofDiaporthe and Phomopsis isolates from soybean. (Abstr.) Phytopathology 75:500. 9. Horst, R.K. 1979. Wescott’s plant disease hand book. Pages 121-122. Van Nostrand Reinhold CO., New York. 802 pp. 10. Keeling, B.L. 1982. A seedling test for resistance to soybean stem canker caused by Diaporthe phaseolorum var. caulivora. Phytopathology 72:807- 809. 11. Keeling, B.L. 1984. Evidence for physiologic specialization ofDiaporthe phaseolorum var. caulivora. J. Miss. Acad. Sci. Suppl. 29:5. 12. Krausz, J.P., and Fortnum, B.A. 1983. An epiphytotic ofDiaporthe stem canker of soybean in South Carolina. Plant Dis. 67:1128-1129. 13. Krebs, K.G., Heusser, D., and Wimmer, H. 1969. Spray reagents. Pages 854-905 in: Thin Layer Chromatography. A Laboratory Handbook. E. Stahl, ed. Springer-Verlag, New York. 1041 pp.

36 37

14. Kunwar, I.K., Halfon-Meii, A., Manandhar, J.B., and Sinclair, J.B. 1987. Possible phytotoxic metabolites in culture filtrates of Diaporthe phaseolorum var. sojae. Mycopathologia 99:71-75. 15. Morgan-Jones, G., and Backman, P.A. 1984. Characterization of southern biotypes of Diaporthe phaseolorum var. caulivora the causal organism of soybean stem canker (Abstr.) Phytopathology 74: 815. 16. Nobuhisa Morooka., Takashi Tatsuno., Hiroshi Tsunoda, Kimiko Kobayashi and Tosio Sakurai. 1986. Chemical and toxicological studies of the phytotoxin 6a, 7P, 9a,-trihydroxy-8(14),15-isopimaradiene-20,6y-lactone, produced by a parasitic fungusPhomopsis sp. in wilting pine trees. Agric. Biol. Chem. 50:2003-2007. 17. Patwardhan, S.A., Pandey, R.C., and Sukh Dev. 1974. Toxic cytochalasins of Phomopsis paspali, a pathogen of kodo millet. Phytochemistry 13: 1985- 1988. 18. Ploetz, R.C., and Shokes, F.M. 1985. Soybean stem canker incited by ascospores and conidia of the fungus causing the disease in the southeastern United States. Plant Dis. 9:990-992. 19. Smedegard-Peterson, V. 1977. Isolation of two toxins produced by Pyrenophora teres and their significance in disease development of net blotch of barley. Physiol. Plant Path. 10, 203-211. 20. Snow, J.P., Berggren, G.T., Harville, B.G., and Whitam, H.K. 1984. Stem canker : A soybean disease recently found in Louisiana. Louisiana Agric. 27:8-9, 24 21. Weaver, D.B., Casper, B.H., and Backman, P.A. 1984. Cultivar resistance to field infestations of soybean stem canker. Plant Dis. 68:877-879. 22. Wells, J.M. Cutler, H.G. and Cole, R.J. 1976. Toxicity and plant growth regulator effects of cytochalasin H isolated from Phomopsis sp. Can. J. Microbiol. 22: 1137-1142. 23. Yoder, O.C. 1980. Toxins in pathogenesis. Annu. Rev. Phytopathol. 18:103-129. 38

Table 1. Reaction of various plant species to Diaporthe phaseolorum var. caulivora and a toxin extracted from culture filtrate of the fungus.

Plant species Reaction to fungus x Reaction to toxin y

Soybean ( Glycine max) + + Lima bean (Phaseolus limemsis) + + Cow pea (Vigna sinensis) - - Snap bean Phaseolus ( vulgaris) - - Runner bean(Phaseolus vulgaris) NT - Cotton (Gossypium hirsutum) - - Okra (Hibiscus esculentus) -- Sorghum (Sorghum vulgare) -- Cabbage (Brassicae oleraceae) - - Eggplant (,Solarium melongina) NT - Tomato (Lycopersicon esculentum)NT - Bell pepper (Capsicum annuum) NT -

x Fungus inoculated by toothpick inoculation technique y Assays conducted using 1:10 dilution of culture filtrate and partially purified toxin NT = Not tested + = Symptoms produced - = No symptoms

/ 39

Table 2. Effect of various reagents on the toxigenic spot separated from culture filtrate of Diaporthe phaseolorum var caulivora by thin layer chromatography.

Reagent Color

1. 50% ethanolic sulphuric acid Grey * 2. 5% phosphomolybdic acid in ethanol bluish green 3. alk. Pot. permanganate (1% aq. KMnC>4+ Yellow 5% aq NaHC03) 4. 0.05% aq. Pot. permanganate Yellow* 5. Paulys reagent Yellow 6. Folin-Ciocalteu reagent blue* 7. Ferric chloride in 0.5N HC1. blue 8. Ninhydrin None* 9. Iodoplatinate None*

* TLC plate heated to 100 C for 5-10 min. 40

Table 3. Reaction of soybean cultivar RA 606 to Diaporthe phaseolorum var. caulivora and culture filtrate from four isolates of the fungus.

culture filtrate x Fungal inoculation Isolate Dilution Symptom y canker2 end point rating length

Opelousas 160 3.83 a 22.1 a St. Joseph 20 C.98 b 5.4 b Slaughter 20 1.83 b 12.2 b Lawtell 20 1.66 b 11.4 b

x Culture filtrate diluted to1 :10 y Symptoms rated on a 0-5 scale 36 hours after treatment of trifoliates (0= no symptoms, 5 = mid rib, all veins discolored; petiole showing reddish lesions) LSD .0 5 = 1 -4 8 z Length of canker measured four weeks after inoculation. L.S.Dq 5=9.39 41

Table 4. Response of soybean cultivars to culture filtrate of four isolates of Diaporthe phaseolorum var caulivora.

cultivar x Isolate Tracy M A 5474 Davis Bragg RA 606 Mean (R) (MR) (MR) (MS) (S)

Opelousas 4.50 4.50y 4.50 4.00 3.50 4.20a Lawtell 3.83 3.75y 1.66 1.00 1.66 3.05b Slaughter 3.83 4.25 2.00 2.33 2.16 2.91b St. Joseph 3.83 3.00 2.83 3.25 2.33 2.38c

Mean 4.00a 3.80a 2.75b 2.64b 2.41b x symptoms were rated on a 0-5 scale 36 hours after treatment of the trifoliates with 1:10 dilution of the culture filtrate (0- no symptoms, 5- midrib and veins discolored and red lesions on the petiole) Reaction of cultivars to stem canker: R=resistant, MR=moderately resistant MS=moderately susceptible, S=susceptible. yStandard Error (S.E.) for isolate/cultivar interaction = 0.40. S.E.for all other interactions= 0.32 Means followed by the same letter are not significantly different (P=0.05) 42

Table 5. Reaction of soybean trifoliates of cultivar RA 606 to toxic metabolites.

Metabolitex symptomy organism

Cytochalasin B - Phomopsis sp. Cytochalasin H - Phomopsis sp. Phomopsis toxin 2 + Phomopsis sp. from pine wilt Dpc toxin +++ Dpc from stem canker x 100 (ig of compound tested by leaf puncture technique. y symptoms appear as red discoloration of midrib and veins - = no symptoms + = slight discoloration of midrib and veins +++ = intense discoloration of midrib and veins. 2 6 a , 7(5, 9 a- trihydroxy-8(14), 15-isopimaradiene-20, 5y-lactone. 43

Fig. 1. Soybean trifoliates excised under water. Cut ends dipped in 1:10 dilution of culture filtrate. A, Reddish lesions on the petiole. B, Lesions on the petiole and red discoloration of midrib and veins. Lamina shows few patches of necrotic cells. C, Interveinal chlorosis of leaflets after 48 hours of treatment. 44

0 .7

0 .5 - Y = 0.70 - 0.18 X + 0.0007 X5 cn r3 = 0.81** c 0 .3 - a: v S 0.1- (L) OT

“ 0.0- CP O _l - . 3 -

20 6 0 8 0 1 0 040 Dilution

Fig. 2: Relationship between the toxin dose (dilution series of 1:10, 1:20, 1:40, 1:80, and 1:160) and disease severity rated on a 0-5 scale (0= no symptoms, 5= Lesions on petiole, midrib and veins discolored). Cut ends of excised trifoliates were dipped in dilutions of culture filtrate and symptoms rated after 36 hours. Each value is a mean of three replications. 45

3 .0 - - 0.6

-0.5 cn .E 2 .0 - o (Z <1) V) D a) V) lO S 1-°- -0 .3

0.0 b—b Diseose Rating • Dry Wt 18 22 26 30 Temperature (C)

Fig. 3. Effect of incubation temperature on growth ofDiaporthe phaseolorum var. caulivora and toxin production. The amount of toxin produced was measured in terms of symptom severity rated on a 0-5 scale (0=no symptoms, 5=Lesions on petiole, midrib and veins showing discoloration) after 36 hours of treatment of soybean trifoliates with 1:10 dilution of culture filtrate. Dry weight was obtained by drying the mycelium at 45 C for 3 days. 46

Fig. 4. Intact soybean plants (RA 606) grown in the greenhouse and treated with partially purified toxin. Toxin (100 |ig) was dissolved in acetone and placed on a puncture made with a needle at the node. Symptoms appear as dark lesions on stem, and petiole and as discoloration of midrib and veins after 3-4 days. NAVENUMERS 600.0

Fig. 5. Infrared spectrum of purified toxic metabolite produced Diaportheby phaseolorum var. caulivora.. HI. fflSTOCHEMICAL CHANGES IN SOYBEAN TRIFOLIATES TREATED WITH A TOXIC METABOLITE OF D1APORTHE PHASEOLORUM VAR. CAUUVORA.

48 Histochemical Changes in Soybean Trifoliates Treated with a Toxic Metabolite ofDiaporthe phaseolorum var. caulivora.

B. Lalitha, G.T. Berggren, and J.P. Snow.

Graduate Research Assistant, Associate Professor and Professor respectively, Department of Plant Pathology and Crop Physiology, Louisiana Agricultural Experiment Station, Louisiana State University Agricultural Center, Baton Rouge, LA 70803. We thank Dr. K.E. Damann, Department of Plant Pathology and Crop Physiology, Louisiana State University, for help in conducting autofluorescence investigations. Accepted for publication______

ABSTRACT Lalitha, B., Berggren, G.T., and Snow, J.P. 1988. Histochemical changes in soybean trifoliates treated with a toxic metabolite ofDiaporthe phaseolorum var. caulivora. Soybean trifoliates treated with the culture filtrate ofDiaporthe phaseolorum var. caulivora or the purified toxic metabolite exhibited browning of midrib and veins, reddish lesions on the petiole and interveinal chlorosis. The lesions and discoloration of midrib and veins were associated with an intense yellow autofluorescence. Autofluorescence was also associated with the vascular bundles of samples treated with the toxin. The discoloration of the midrib and veins and the lesions on the petiole were associated with accumulation of phenols which were alkali soluble and were separated on TLC. Two alkali-soluble phenolic compounds not seen in the control samples were present in the samples treated with culture filtrate but not in those treated with purified toxin. Similar phenols were also

49 50 produced in soybean plants inoculated with the fungus and showing canker symptoms. A compound showing yellow fluorescence was present in all treated samples including samples treated with the purified toxin and those inoculated with the fungus but not present in the control samples. Since the toxin does not fluoresce by itself, the yellow fluorescence is probably a host response. The presence of this metabolite and similar phenolic compounds in culture filtrate treated and fungus inoculated plants suggest that the toxin and fungus induce similar host responses and the toxin may have a role in the disease. Additional Key words-. Stem canker, autofluorescence, phenols

Stem canker of soybean (Glycine max (L.) Merr.) is caused by Diaporthe phaseolorum (Cke. & Ell.) Sacc. var. caulivora Ath. & Cald.(Dpc). The symptoms on the stem are characterized by dark reddish-brown cankers while the foliar symptoms which appear after the host vascular system has been disrupted are characterized by interveinal necrosis (13). Symptomless infection of soybean plants with Dpc and the presence of symptoms without the association of a fungus has been observed (2). The later observation suggests that a translocatable metabolite may be involved in symptom production. The culture filtrates ofDpc contain a toxic metabolite which produces symptoms similar to stem canker causing reddish lesions on stems and petioles and red discoloration of midrib and veins and interveinal chlorosis of both intact and excised trifoliates (10). Evidence from earlier experiments suggests that the metabolite may have a role in symptom production

( 11). Necrotic lesions in many instances have been shown to be associated with the accumulation of phenols both in susceptible and resistant host-pathogen interactions (7). Symptoms on soybean trifoliates appeared to be due to an accumulation of phenolic compounds in response to the toxic metabolite. Symptoms also indicated that vascular disruption may contribute to the disease (2). The present paper shows some of the histochemical changes in the soybean plants treated with the toxin and 51

provides evidence which indicates that the toxin may mediate symptoms expressed by fungus infection. MATERIALS AND METHODS Production of toxic metabolite. AD pc isolate from Opelousas, Louisiana was obtained from naturally-infected soybean plants showing stem canker symptoms. The fungus was grown on autoclaved potato dextrose broth (PDB) and incubated for 20-23 days in dark as stationary cultures. The medium was filtered through Whatman #1 filter paper (W and R Balston Ltd., England) and a 1:10 dilution of the filtrate was used in all experiments. The partially and completely purified toxic metabolite was obtained from the culture filtrate 11( ). Production of symptoms on soybean trifoliates. Soybean plants of the cultivar Ring Around 606 (RA 606) which are susceptible to stem canker (2,3) were grown in the greenhouse in autoclaved pots containing a mixture of silt, peatmoss and perlite (3:2:1) with 2g of Osmocote plant food 14-14-14, (Sierra Chemical Co., Milpitas, California) added to each pot after seedlings emerged. Trifoliates from 30-50 day old plants were excised under water and the cut ends were immersed in a beaker containing 50 ml of 1:10 dilution of the culture filtrate. Controls consisted of a 1:10 dilution of the sterile uninoculated PDB. Acetone (100 |il) containing 100 |ig of partially purified or purified compound was diluted to 3 ml with distilled water in a vial (4.5 x1 cm). The cut ends of excised trifoliates were placed in the acetone-toxin solution. Trifoliates treated in a similar manner with 100 |il of acetone served as controls. Fluorescence microscopy. Free-hand sections of petiole and leaflets, midribs with and without symptoms were placed on a microscopic slide and observed using a Leitz Ortholux II microscope (12V, 100W, Hg Lamp) fitted with a Ploemopak using the H2 filter block (exciting filter 390-490nm, beam splitting mirror 580 nm, supression filter 580nm) at a magnification of 62.5 x or 156.25 x. Histochemistry: Histochemical studies were conducted using various stains 52 on petioles with lesions, leaflets with discolored midrib and veins and control samples. One cm long pieces of the petiole and leaf material were dipped into 50% ethanol for 5 sec., washed in distilled water and immersed in the stain solutions in petri plates overnight. The stains used were: a) 0.5% Aniline blue in 50% ethanol which stains callose blue, b) 1% aqueous KMNO4 which precipitates and appears dark in the presence of lignin, c) 12N KOH, which gives yellow-red color in presence of suberin, d) saturated solution of Sudan IV in 70% ethanol which stains lipid containing material pink to orange, and10 e)% aqueous ferric chloride which gives blue green precipitate in the presence of phenolic compounds and tannins6). ( Extraction of phenols: Petioles (5 g) of trifoliates which showed reddish lesions after treating with the toxin for 36 hours were cut into1 cm pieces and extracted with 50 ml 10% aqueous KOH for 30 min to 1 hour. The extract was filtered through 2-3 layers of cheesecloth and the pH adjusted to 5.0 with 2N HC1 (8). The acidic solution was extracted with 20 ml ether and the ether phase was passed over anhydrous sodium sulphate and evaporated to dryness in a fume hood. The petioles of control samples were extracted in a similar manner. Fifty day old RA 606 plants were inoculated with the Opelousas isolate using the toothpick inoculation technique (9). Stems were removed from the cankered plants and from controls after four weeks. Stem material (5 gm) showing symptoms was sliced into 1 cm pieces and the phenols were extracted as previously described. Plants inoculated with sterile autoclaved toothpicks served as controls. The residue obtained after evaporating the ether was dissolved in 1 ml acetone. Twenty pi of the acetone solution was spotted on a TLC plate coated with silica gel 7G (Mallinckrodt Chemical Works, St. Louis, Missouri.) and developed in benzene: ethyl acetate: acetic acid (75:24:1) (8). The separated compounds were viewed under a short wave UV lamp and sprayed with Folin-Ciocalteu reagent (Sigma Chemical Co., St. Louis, Misssouri) or 10% aqueous ferric chloride. 53

RESULTS Symptoms on soybean trifoliates treated with the diluted culture filtrate, partially or completely purified toxin were produced in 24-36 hours. The symptoms on the petiole appeared as intense reddish-brown lesions while on the leaflets the midrib became red with the veins appearing as a fine red network. Most of the leaf tissue remained healthy with a few patches of lamina cells showing red discoloration (Fig.l). Interveinal chlorosis appeared after 48 hours in samples treated with culture filtrate, partially purified or purified toxin. Trifoliates treated with 1:10 dilution of PDB and 100 pi acetone solution which served as controls did not show symptoms. Plants inoculated with the fungus produced cankers on the stems, red discoloration of midrib and chlorosis of the leaf at the node immediately above the point of inoculation. Control plants produced no cankers or discoloration of midrib or veins. The lesions and other red discolored areas were surrounded by an area of intense yellow fluorescence when viewed with a fluorescence microscope. The lesions appeared dark on a background of red fluorescence produced by chlorophyll (Fig.2). Free-hand sections of petiole and midrib showing red discoloration showed similar yellow fluorescence of vascular bundles. Most of the fluorescence of the vascular bundles was restricted to the bundle sheath cells while the vascular bundles in general showed a more intense fluorescence than those of untreated samples. The fluorescence was however, limited to only those vascular bundles associated with the external lesion. Fluorescence of the vascular bundles of midrib and veins was observed only in tissues showing external discoloration (Fig.3). Stems inoculated with the fungus showed cankers and discoloration of the midrib and veins and interveinal chlorosis of a few leaves. Longitudinal sections of stems showed red discoloration of pith in the cankered areas and in areas where the canker had not developed. Of the stains tested, only ferric chloride gave a positive bluish black-reaction, which indicates the presence of phenols. All the discolored areas of the midrib and a 54 few discolored patches of cells on the lamina were stained (Fig.4). Stains other than ferric chloride gave negative reaction with all treated samples. Potassium hydroxide extracted most of the red pigment from tissues of toxin treated and fungus infected plants in a few minutes. Ether extractable compounds from samples were separated on TLC as fluorescent spots. Culture filtrate treated and fungus inoculated tissues exhibited comparable profiles of the compounds while the samples treated with partially purified and purified toxins showed comparable profiles (Fig.5). The control samples treated with noninoculated PDB and with acetone solution did not show any fluorescent compounds. The sample inoculated with a sterile toothpick which served as a control for fungus inoculated samples showed a fluorescent metabolite which was also present in treated samples. A compound showing bright yellow fluorescence (A) was present in all treated samples which included the culture filtrate, partially pure and pure toxin treatments and fungus inoculation, but was not present in any of the control samples. In addition to this compound, extracts from samples treated with the partially purified and purified toxins had a compound with yellowish orange fluorescence (B) which was not present in the culture filtrate treated or fungus inoculated samples. The fluorescence of compounds A and B was very unstable and faded within an hour. Spraying with Folin-ciocalteu reagent showed two phenolic compounds (C and D) as blue spots which were not present in the control samples (Fig. 5). These appeared as dark spots under short wave UV. The two compounds also gave a reddish-brown color with ferric chloride spray which is also an indication of their phenolic nature. These phenolic compounds were consistently associated with the samples treated with the culture filtrate and those inoculated with the fungus. Metabolite A showing yellow fluorescence which was seen in all the treated samples did not react with either Folin-Ciocalteu reagent or ferric chloride while, metabolite B gave a blue color with ferric chloride but did not react with Folin- Cicoulteau reagent (Table. 1.) 55

DISCUSSION Culture filtrate and fungus inoculation induced the production of similar phenolic and fluorescent compounds which were not present in control samples as indicated by TLC. Autofluorescence has been reported in response to infection mainly as localized lignification of cell wall in incompatible reactions (14). Similar fluorescence in response to translocated metabolites has not been previously observed. As seen in fluorescence microscopy, not all vascular bundles were affected by the toxin and this may be the cause of lack of wilting and unilateral canker formation seen in the infected soybean plants in the field. The restriction of symptoms initially to the midrib and veins and the autofluorescence of the bundle sheath indicate that the action of the toxin is mainly on the vascular system. The toxin treated extracts reacted with Folin-Ciocalteu reagent giving an intense blue color indicating an increased phenol accumulation in the diseased tissue compared to the control samples. Increased levels of phenols, polyphenoloxidases (PPO) and peroxidases (PO) have been reported in both susceptible reactions and in the early stages of the resistant host-pathogen interaction (7). However, comparisons of resistant and susceptible cultivars has not always resulted in a positive correlation between the increased phenolics and resistance (7).Fusarium oxysporum the vascular pathogen of muskmelon is also reported to increase PPO and PO activities causing vascular browning and disintegration of tissues (12). However, there are very few reports on similar activities by toxic metabolites though it is possible that such a response may be induced. Ophiobolin produced Cochliobolus by miyabeanus activates PPO of rice leaves (7) while a crude toxin produced by Botrytis cineria, activates peroxidases of cabbage (1). There is also evidence for the release of bound phenolics by the action of the P-glucosidases released by Fusarium oxysporum. causing wilt of tomato (5). Earlier evidence (11) suggested that the toxic metabolite produced byDpc may have a causal role as a virulence factor in the stem canker disease. The consistent 56 association of the metabolite A showing yellow fluorescence in all toxin treated samples including those treated with purified toxin and inoculated with the fungus suggest a common host reaction in response to both the toxin and the fungus. The additional fluorescent compounds in the samples treated with the culture filtrate which were not present in the samples treated with purified toxin are probably associated with the intermediate metabolites in the culture filtrate. Similar intermediates may also be produced in the host by the fungus. Since the toxin itself does not fluoresce, the compound seen in the toxin treated samples is probably a host response and may not be a phenol as indicated by the negative reaction with ferric chloride and Folin-Ciocalteu reagent. Preliminary studies (unpublished) also indicated that the crude phenol extract from the toxin treated samples slowed the growth of Dpc while the extract from the control samples did not. The phenols which accumulate in the cankers in response to the toxin may slow the growth of the fungus. This may explain the absence of association of the fungus with the lesions observed in nature. Though the increased levels of phenols in diseased tissue is not a new phenomenon, the production of similar compounds in response to the crude toxic filtrate, to fungus inoculation and to purified toxin treatments provide indirect evidence for the possible role of the toxin in the disease. LITERATURE CITED

1. Arzichowskaja, E.V. 1946. On the physiology of host parasite relations of Botrytis cineria-cabbage (Brassica oleraceae) complex. Mikrobiologiya 15: 47-56. 2. Backman, P.A., Weaver, D.B., and Morgan-Jones, G. 1985. Soybean stem canker: an emerging disease problem. Plant Dis. 69:641-647. 3. Berggren, G.T., McGawley, E.C., Snow, J.P., and Whitam, H.K. 1984. Soybean disease control annual report. Louisiana Agric. Expt. Station, Louisiana State Univ. Agric. Center, Louisiana. 42 pp. 4. Berggren, G.T., Snow, G.P., Damicone, J.P., and Whitam, H.K. 1987. Soybean disease control annual report. Louisiana Agric. Expt. Station., Louisiana State Univ. Agric. Center, Louisiana. 87 pp. 5. Davis, D., Waggoner, P.E., and Dimond, A.E. 1953. Conjugated phenols in Fusarium wilt syndrome. Nature,172: 959-961. 6. Faulkner, G., and Kimmins, W.C. 1985. Staining reactions of tissue bordering lesions induced by wounding, TMV and TNV in bean. Phytopathology 65: 1396-1400. 7. Goodman, R.N., Kiralay, Z., and Wood, K.R. 1986. Secondary metabolites. Pages 211-244 in: The Biochemistry and Physiology of Plant Diseases. Univ. of Missouri Press, Columbia. 433 pp. 8. Harbome, J.B. 1984. Phenolic compounds. Pages 37-99 in: Phytochemical Methods. A Guide to Modem Techniques of Plant Analysis. Chapman and Hall, New York. 288 pp. 9. Keeling, B.L. 1982. A seedling test for resistance to soybean stem canker caused by Disporthe phaseolorum var. caulivora. Phytopathology 72:807- 809. 10. Lalitha Burra, Snow, J.P., and Berggren, G.T. 1986. Phytotoxin production by Diaporthe phaseolorum var. caulivora and its role in the stem canker

57 58

disease of soybean. (Abstr.) Phytopathology 70:1063. 11. Lalitha Burra, Snow, J.P., and Berggren, G.T. 1988. Studies on the role of toxin produced byDiaporthe phaseolorum var. caulivora causing stem canker of soybean. Pages 8-9 in: Proc. Southern Soybean Disease Workers Meetings, 15th., Nashville, TN. 71 pp. 12. Mariate, H. 1972. Qualitative and quantitative changes induced in polyphenoloxidases and peroxidases of plants by several ecological factors. Pages 479-480 in: Phytotoxins in Plant Diseases. R.K.S. Wood, A. Ballio and A. Graniti, eds. Academic Press, New York. 530 pp. 13. Sinclair, J.B. 1982. Compendium of soybean diseases. American Phytopathological Society, St. Paul, Minnesota. 104 pp. 14. Stockwell, V., and Hanchey, P. 1987. Lignification of lesion borders in Rhizoctonia infected bean hypocotyls. Phytopathology 77:589-593. 59

Table. 1. Metabolites produced in soybean plants in response to treatment with toxin produced byDiaporthe phaseolorum var. caulivora and to fungus inoculation

Metabolite FC. Fluo. FeCl3 CF. P.Pure Pure Fungus Cont.

A - + - + + + + - B - + + - + + - - C + - + + -- + -

D + ~ + + - ■ + -

FC= Folin-Ciocalteu reagent, Fluo= Fluorescence in short wave UV lamp, P.Pure= partially purified, CF= Culture filtrate, Cont.= control + = positive reaction - = no reaction. 60

Fig. 1. Soybean trifoliates treated with 1:10 dilution of culture filtrate ofDiaporthe phaseolorum var. caulivora for 36 hours. A, Leaf let showing Symptoms of red discoloration of midrib and veins while much of the lamina remains healthy. B, Necrosis of midrib and veins of leaf showing the toxin action on vascular tissue. 61

Fig. 2. Autofluorescence of soybean trifoliates treated with 1 :10 dilution of culture filtrate of Diaporthe phaseolorum var. caulivora for 36 hours. A, Yellow fluorescence around the lesions on the petiole (56X) B, Cross section of petiole showing yellow fluorescence of vascular bundles which corresponds to the lesion seen externally (64X). 62

Fig. 3. A, Cross section of toxin treated leaf through midrib and veins showing discoloration. Note fluorescence of midrib and vascular bundle of one of the veins (160X). B, Vascular bundles of petiole showing autofluorescence (200X). 63

Fig. 4. Leaf sample treated with the toxin and stained with 10% aqueous ferric chloride. A, Top: Control, Bottom: showing stained midrib and veins B, Lamina showing discolored cells stained. 64

Fig. 5. Thin layer chromatogram of fluorescent and phenolic compounds produced by soybean plants in response to toxin or fungus inoculation. Chromatogram was developed in benzenerethyl acetate:acetic acid (75:24:1) and viewed under short wave UV lamp. 1, control treated with acetone solution, 2, Purified toxin treated, 3, Partially purified toxin treated, 4, Culture filtrate treated, 5, Fungus inoculated by toothpick, 6, control inoculated with toothpick. A=yellow fluorescence (2-5), B= yellowish orange fluorescence (2-3) C and D, gave positive reaction for phenols (4-5). APPENDIX 66

Fig. 1. Thin layer chromatogram (Silica gel GF 254) developed in Chloroform : Methanol (90:10) and viewed under short wave UV lamp. A and C are partially purified toxin, B is purified toxin 0.05 2.00 ABSORBANCE methanol. Fig. 2. Ultraviolet spectrum of the purified toxic metabolite produced in the culture culture the in produced metabolite toxic purified the of spectrum Ultraviolet 2. Fig. filtrates of of filtrates 2oo. iprh phaseolorum Diaporthe NANOMETERS var. var. caulivora. The spectrum was taken in in taken was spectrum The 67 400 ^ I 115.1 *E+86 100- 55.0

80- 133. 1 349. 1

-4 60-

91. 1

67.0 40 -

149. 1

161. 1 261.2 20- 350.2

207.0 307. 1 233. 1 365. 1

700 800 m ' T

Fig. 3: Mass spectrum of purified toxic metabolite produced byDiaporthe phaseolorum var. caulivora showing molecular ion peak at m+ 349.1 a co VITA

Name: Ms. Lalitha Burra

Bom: Andhra Pradesh (A.P.), India, May 27, 1956.

Marital Status: Married to Dr. B.L. Subba Rao.

Education: High School: 1970 Hyderabad, A.P., India. Junior College: 1970-1972 Hyderabad, A.P., India. B.Sc.: 1972-1975 Hyderabad, A.P., India. M.Sc.: 1972-1977 Osmania University, Hyderabad, A.P., India. Ph.D.: 1985-1988: Department of Plant Pathology & Crop Physiology, Louisiana State University, Baton Rouge, Louisiana, U. S. A.

Employment: 1979-1981: Technical A ssistant, G roundnut Improvement Program, International Crops Research Institute for Semi Arid Tropics (ICRISAT), Hyderabad, A.P., India.

1982-1984: Research Fellow, Central Institute of Medicinal and Aromatic Plants (CIMAP), Lucknow, Uttar Pradesh, India.

69 DOCTORAL EXAMINATION AND DISSERTATION REPORT

Candidate: Lalitha Rao Burra

Major Field: Plant Health

Title of Dissertation: A toxic metabolite produced by Diaporthe phaseolornm var. caulivora, the causal organism of stem canker of soybean.

Approved:

(/ Major Professor and Chairman -

/ i f £ 2^ ______Dean of the Graduat^SSftool

Date of Examination: