INFLUENCE OF THE HERBICIDE. CHLORAMBEN. ON SEVERlTY OF ROOT ROT OF SOYBEAN CAUSED BY THIELAVROPSIS BASSCOLA PhD. MEQEIGAN STATE UNEVERSEIY MAY LEE 3975 2-1151015 Y i 5:1“ - v. C ‘~r - . twin-331154.12 5 F "HES S This is to certify that the thesis entitled Influence of the herbicide chloramben dn severity of root rot of soybean caused by Thielaviopsis basicola presented by May Lee has been accepted towards fulfillment of the requirements for Ph.D. degreein Plant Pathology w/ < 4; A f (/7‘5 AA/i‘f‘rv ,1\_/ 44-- ' / Major professor Date February 24, 1976 0-7639 . ABSTRACT INFLUENCE OF THE HERBICIDE, CHLORAMBEN, ON SEVERITY OF ROOT ROT OF SOYBEAN CAUSED BY THIELAVIOPSIS BASICOLA By May Lee The herbicide chloramben (3-amino—2,S-dichlorobenzoic acid) applied at rates equivalent to recommended field rates, caused enhanced necrosis of root and hypocoytls of soybeans infected by Thielaviopsis basicola. In the greenhouse, soybeans planted in soil infested with the pathogen and treated with 2 mg chloramben per kg soil (3 lb/acre) had more severe symptoms than soybeans grown in infested soil without chloramben and fresh weight of roots and foliage was reduced. On a scale increasing from 0—6, the disease index usually was 4 for plants grown ingnylWith 2. basicola only, and 6 for plants in soil with 1. basicola plus chloramben. Soybeans grown in artificially infested soil in the field which had been treated with 3 lb chloramben per acre also were more severely diseased, had plant stand reduced by 22%, were stunted, and had seed yield reduced by 17%, as compared with soybeans grown in infested soil without herbicidal treatment. May Lee Soybean varieties "Harosoy 63", "Corsoy" and "Hark" showed enhanced root rot in chloramben-treated infested soil, in the greenhouse. Both endoconidia and chlaymdo- spores of one isolate of the pathogen caused enhanced disease severity in infested soil treated with chloramben. Inocula consisting of mycelia with spores of four isolates of 2. basicola collected from different localities in Michigan also enhanced disease in chloramben-treated soil. Spraying the herbicide on the surface of the soil in pots, or mixing it with the soil were equally effec— tive in inducing more severe Thielaviopsis root rot. Chloramben at 2, 10, 30 or 100 ug/ml did not stimu- late the germination of chlamydospores or endoconidia in water or on soil. It had no effect on linear growth, dry weight or amount of sporulation of the pathogen in cul- ture. Longevity of both types of propagules in soil was not affected by chloramben. The virulence of E. basicola was not altered after being cultured in nutrient media containing 2 pg chloramben/ml. Other soil microflora did not play a role in disease enhancement, since chloramben—induced disease enhancement was expressed in pathogen-inoculated plants otherwise grown aseptically. Moreover, the fungal and actinomycetes population in chloramben-treated unsterilized soil was not significantly altered by the herbicide. Changes of May Lee bacterial populations were inconsistant. Soybeans grown in the presence of chloramben (2ng/kg soil) for one week then transplanted into T. basicola- infested soil lacking chloramben developed more severe symptoms than those first grown in untreated soil. Chlo- ramben at 2 mg/kg soil, did not decrease the resistance of soybeans after penetration of the pathogen, since the cortex of soybeans injected with endoconidia developed lesions of similar length with or without chloramben treatment. Chlamydospores and endoconidia germinated 3 and 5 times more, respectively, in the rhizospheres of soybeans first grown aseptically for 7 days in a 2 ug chloramben/ml salt solution, than in rhizospheres of plants without chloramben treatment. The root exudates of soybeans grown axenically in a dilute salt solution containingcflflxnwnflxniat 2 ug con- tained more than 5 times the amino acids and 3 times the electrolytes of those without the herbicide treatment. Nosignificantcfijference in total fatty acids, carbohy- drates and nucleic acids in root exudate was detected from plants untreated or treated with chloramben. The root exudates of soybeans grown with chloramben supported germination of u-fold greater numbers of T. basicola endoconidia than did exudates from soybeans lacking chlo- ramben—treatment. Soybeans inoculated with endoconidia suspended in a concentrated (l6 X)exudate from May Lee chloramben-treated soybeans developed lesions more than twice as long as those treated with exudates from soy- beans grown in the absence of chloramben. Casamino acids in an amount estimated to be similar to that exuded by soybeans under chloramben treatment, when added to sterile soil containing T. basicola, caused increased symptom severity on aseptically cultured soybeans as compared with soybeans not so treated. It is concluded that chloramben stimulated the exu- dation of nutrients particularly amino acids from roots of soybeans which intuuw1increased the nutrient status of T. basicola inoculum in the rhizosphere and that this resulted in increased severity of infection. INFLUENCE OF THE HERBICIDE, CHLORAMBEN, ON SEVERITY OF ROOT HOT OF SOYBEAN CAUSED BY THIELAVIOPSIS BASICOLA By May Lee Cf’H A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Botany and Plant Pathology 1975 © Copyright by May Lee Chen 1975 ii ACKNOWLEDGMENT I wish to express my gratitude to Dr. John L. Lockwood, my major professor, for his encouragement and support throughout the course of the study and in the preparation of this manuscript. I am also grateful to Dr. Norman E. Good, Dr. Melvyn L. Lacy and Dr. Arthur R. Wolcott for their interest in and helpful comments on the research. iii TABLE OF CONTENTS Page List of tables ............... .... .................... vii List of figures ...................................... Viii Introduction.... ............. ...... ....... . .......... 1 Literature review Fate of chloramben in soybeans.... .............. 3 Biochemical responses of plants to chloramben treatment.............. ..... ... ....... .......... Influencecyfherbicides on disease development... 7 Effect of chemical compounds on T. basicola and disease development ...... ....... ...... ..... ..... Materials and methods The pathogen ....................... ......... ..... 13 The herbicides............. ...... ... ......... ....13 Greenhouse tests ....... . ...... . ............ . ..... 15 Field experiment.................................l6 Spore germination tests ............. . ........... 17 Preparation of chlamydospores..... ..... ......... l9 Enumeration of the population of T. basicola in soil.............................T..TTTTTTTT.... 20 Enumeration of soil microorganisms .............. 20 Axenic culture of soybean plants ....... ......... 21 Collection and analysis of root exudates ........ 23 Spore germination at the rhizosphere.. ....... ... 2“ iv Page Results Effect of herbicides on Thielaviopsis root rot.. 26 Effect of chloramben on Thielaviopsis root rot.. 26 Effect of chloramben and linuron on Thielaviopsis root rot of soybeans in the field............... 29 Enhancement of root rot symptoms by chloramben on soybean varieties...... ......... . ............ 35 Effect of chloramben on disease severity with different isolates of the pathogen......... ..... 35 Effect of chloramben on the severity of disease using chlamydospores or endoconidia as inoculum. 35 Effect of rate of chloramben and application methods on disease development........... ....... 39 Effect of soil temperature on the development of Thielaviopsis root rot of soybeans in chlo- ramben-treated soil ..... ........................ 39 Possibility of direct stimulation of germination and growth of T. basicola...... ............ ..... ”1 Possibility of increased virulence of T. basi- cola due to chloramben.......................... “5 Effect of chloramben on microbial populations in soil ..... . ................ . ...... . ........... “5 Predisposition of soybeans to Thielaviopsis root rot by chloramben .......... ................ “7 Lesion development on chloramben-treated soy— beans by T. basicola introduced under the epidermiSOOOOOO000.00.00.00000000000000000000000 U8 Spore germination in soybean rhizosphere........ “9 Root exudation.................................. “9 Germination of endoconidia in root exudates of chloramben-treated soybeans..................... 50 Effect of root exudates from chloramben-treated soybeans on Thielaviopsis lesion development.... 57 Page Results (cont') Enhancement of root rot severity by Casamino acids... ....................... . ................ 58 Discussion ..................... .... ......... . ........ 60 Literature cited ..................................... 67 Vi LIST OF TABLES Table Page 1 Herbicides, their sources and rates applied in greenhouse experiments .......................... 1U 2 The effect of herbicides on Thielaviopsis basicola root rot of soybeans planted in steamed greenhouse soil and in naturally infested soil in the greenhouse ............................... 27 3 The effect of chloramben on Thielaviopsis basicola root rot of soybean in the greenhouse.. 29 A Effect of chloramben on Thielaviopsis basicola root rot of soybean in the field ................ 33 5 Effect of chloramben on severity of Thiela- viopsis basicola root rot in three varieties of soybeans ........................................ 36 6 Effect of chloramben on severity of disease induced by different isolates of Thielaviop- sis basicola .................................... 37 7 Effect of chloramben on severity of disease using endoconidia or chlamydospores as inoculum. 38 8 Effect of mode of application of chloramben on ThielaviOpsis basicola root rot of soybeans ..... MO 9 Effect of soil temperature on the development of Thielaviopsis root rot of soybeans in chlo- ramben-treated soil.......... .................. . Ml vii LIST OF FIGURES Figures Page 1. Effect of chloramben at 3 lb/acre on Thielaviopsis basicola root of soybeans in greenhouse mix.................. ..... .... 3O 2. Effect of chloramben at 3 lb/acre on Thielaviopsis basicola root rot of soybean in greenhouse mix (detail of roots).. ....... 31 3. Effect of chloramben at 3 lb/acre on Thielaviopsis basicola root rot of soybean in the field .......... ................ ...... 3A A. Electrolytes exuded in a 12 hour period by one soybean seedling (mean of 64 seedlings) cultured aseptically in a dilute salt solu- tion containing 2 ug chloramben/ml, acetone in an amount equivalent to that in the chloramben treatment, or in the salt solu- tion alone (water control)............. ..... 51 5. Amino acids exuded in a 12 hour period by one soybean seedling (mean of 6H seedlings) cultured aseptically in a dilute salt solu- tion containing 2 pg chloramben/ml,acetone in an amount equivalent to that in the chloramben treatment,or in the salt solu- tion alone (water control)........... ....... 52 6. Carbohydrates exuded in a 12 hour period by one soybean seedling (mean of 6A seedlings) cultured aseptically in a dilute salt solu— tion containing 2 ug chloramben/ml,acetone in an amount equivalent to that in the chloramben treatment,or in the salt solu- tion alone (water control)........... ....... 53 7. Fatty acids exuded in a 12 hour period by one soybean seedling (mean of 6A seedlings) cultured aseptically in a dilute salt solu- tion containing 2 ug chloramben/mlsacetone in an amount equivalent to that in the chlo- ramben treatment,or in the salt solution alone (water control).. ............... ...... 5U viii Figures (cont') Figures 8. Page Nucleic acids exuded in a 12 hour period by one soybean seedling (mean of 6A seedlings) cultured aseptically in a dilute salt solu- tion containing 2 ug chloramben/ml, acetone in an amount equivalent to that in the chlo- ramben treatment, or in the salt solution alone (water control) ........................ 55 Effect of chloramben on quantities of elec— trolytes, amino acids, carbohydrates, fatty acids and nucleic acids exuded by roots of soybeans seedlings. Exudates were collected during four, 12-hour periods over 10 days from seedlings cultured aseptically in a dilute salt solution containing 2 ug chlo- ramben/ml, acetone in an amount equivalent to that in the chloramben treatment, or in the salt solution alone (water control) ...... 56 ix INTRODUCTION The increased demand for plant protein has made soy- beans one of the most rapidly expanding crops in Michigan and in the United States. Between 19A8 and 1952 the aver- age harvested acreage of soybeans in Michigan was 128,000. This had increased to 630,000 in 197“. The nation's annu- al soybean production increased from 846 million bushels in 1965 to 1.6 billion bushels in 1973 (56). In Dickson's second edition of "Disease of Field Crops" published in 1956, he discussed 16 different di— seases of soybeans. Thielaviopsis basicola as a pathogen infecting soybeans in the field was first reported by Lockwood, Yoder and Smith (28). The disease was found in Michigan causing necrosis of roots and hypocotyls of soy- beans. The wide host range of Thielaviopsis basicola, and its wide geographical range suggest a widespread dis- tribution of this pathogen. Its ability to produce chla- mydospores allows the fungus to survive in soil for a long period of time. Herbicides have been more and more widely applied to field crops in recent years, and are indispensable for production in mechanized farming. Chloramben is one of several herbicides used on soybeans. It is recommended for use in most soybean-producing states (11,30,31,35). The estimated usage of chloramben in Michigan is 20% of the total acreage of soybeans (W.B. Meggitt, Personal communication). An increasing number of diseases is reported to occur more frequently or more severely after the application of herbicides (23). The purpose of this research was to de- termine if any of the herbicides commonly applied to soy- beans could cause increased T. basicola root rot; to esta- blish whether the herbicide-pathogen-soybean interaction resulting in disease enhancement is specific to certain soy- bean cultivars, isolates of the pathogen or environmental conditions; and to investigate the mechanism of disease enhancement. LITERATURE REVIEW Chloramben [3-amino-2,5-dichlorobenzoic acid] was first synthesized in 1957 by Amchem Products, Ambler, Penn- sylvania. Its biological activity was discovered at the Amchem Research Farm and its patent was issued December 19, 1961. It has been extensively used on soybeans, vegetable crops and ornamentals in the United States (30,31) and other parts of the world (37). Fate of chloramben in soybeans --— The absorption, translocation and molecular fate of chloramben in soybean, and physiological response of the plant were reviewed by Ashton and Crafts (3). Haskell and Rogers (16) soaked soybean and maize seeds in water for 12 to 72 hours before they were placed in radioactive chloramben for 3 hours. Soybean seeds presoaked for 12 hours absorbed chloramben readily throughout the seed in 3 hours, while maize seeds required 2“ hours presoaking for penetration of chloramben into the embryo and 72 hours of presoaking for penetration throughtout the seed. Reider et a1. (U0) showed that the uptake of chloramben, linuron and other herbicides by dry soybean seed was directly proportional to the herbicide concentration in the soaking solution. Increase in tem- perature from 10 C to 30 C also increased the rate of absorption. The rate of uptake of chloramben by viable seeds was similar to that by liquid nitrogen-killed seeds. They also found that the absorption of chloramben continued after imbibition of water had stopped. Swan and Slife (“8) showed that 32 days after preemmergent application of radio- active chloramben to soil, most of the radioactivity re- mained in the cotyledons and root system, and no signifi- cant amount of activity could be detected in the respired CO . Therefore, chloramben was not readily translocated 2 nor broken down to CO in the soybeans. 2 Colby (7) found that most of the chloramben in soybeans was present as N-glycosyl chloramben, which was later con- firmed to be N-(3—carboxy-2,5-dichloropheny1)-glucosy1a— mine by infrared spectroscopy by Swanson et a1. (A9). This conjugate was found both in plants tolerant or sus- ceptible to chloramben. Stroller and Wax (“7) found that the log precent N-glucosyl chloramben in plants treated with radioactive chloramben was highly correlated with log I50 (chloramben concentration required to give 50% reduc- tion of radicle extension); i.e., plants more tolerant to chloramben had higher percentages of N—glucosyl chloramben among the total radioactive compounds present in the plants. Decarboxylation of chloramben was reported by Freed at a1. (12) to occur in soybean roots to a limited degree, and was not a major degradation pathway. A struc— turally unclear chloramben conjugate, chloramben-X, was also found in various treated plants, including soybeans. Biochemical responses of plants to chloramben treat— ments --— Little is known about the mode of action of ben- zoic type herbicides except for their auxin-like properties. Chloramben did not inhibit or stimulate incorporation of ATP into RNA, nor of leucine into protein, in hypocotyl sections of soybeans (33). Moreland and his coworkers (33) reported that chloramben had no effect on gibberel- lic acid—induced d-amylase synthesis in deembryonated barley half—seeds. Penner (37) found chloramben inhibi- tion of d-amylase induction in intact barley seeds, and addition of gibberellic acid could not overcome this block. These results suggested that chloramben inter- fered with the normal function exerted by the embryo of aleuronecells signaling a—amylase production, and that secretion of gibberellic acid is merely one part of the function. Mann and Pu (29) reported that chloramben at l to 20 mg/l stimulated 20 to HU% more incorporation of malonic acid into lipid by excised hypocoytls of Sesbania wexaltata than occurred in the untreated control. Other herbicides with auxin activity, such as 2,U-D, 2,“,5-T, and picloram were also stimulatory to lipid synthesis. Johnson and Jellum (22) tested the effects of herbicides on the chemical composition of soybean seeds, and found that chloramben did not affect the protein and oil content of soybean seeds. Chloramben was the strongest stimulator of upward force of emerging soybean seedlings among 19 herbicides tested (A). Hubbeling and Chaudhary (21) reported a mutagenic effect of chloramben on Verticillium dahliae. After one week's incubation in 5% chloramben solution, one single spore culture out of fourteen survived. Subsequent single spore isolationscd‘the survivor culture yielded isolates with varying degrees of pigmentation and viru— lence to one susceptible and three resistant tomato varie- ties. The most virulent was a black microsclerotial type producing much white mycelium; next was a totally black microsclerotial type, followed by a white mycelial type. A hyaline microsclerotial type proved to be the least pathogenic isolate. All these variants were less virulent than the original isolate, since they produced a less severe symptoms on the resistant tomato varieties than on the susceptible variety, while the original isolate in— duced equally severe disease on both resistant and sus- ceptible hosts. The high concentration of chloramben used here is not normally encountered in the field. Soldatov et a1. (AM) found that chloramben applied at “-6 kg/ha 3 days before sowing of sugar beets decreased soil catalase activity during the first 10 days after application, but later the enzyme activity was similar to that in untreated soil. Chloramben applied at 5 kg/ha insignificantly sup- pressed the growth of Azotobactor and pea root nodules. However, the biological activity of nitrogen-fixing bac- teria was restored during the detoxification of the herbi- cide (43). There apparently are no other reports of changes in soil microbial activity caused by chloramben. Effects of chloramben in plant disease development and on the permeability of cells or plant tissues have not been reported. Influence of herbicides on disease development —-- Recently, Katan and Eshel (23) reviewed the interactions between herbicides and plant pathogens. Four possible mechanisms whereby disease severity could be enhanced were identified. These were 1) direct stimulation of the patho- gen, 2) increase in virulence of pathogen, 3) increase in host susceptibility, and A) an indirect effect of patho- gens through interaction with other affected microrganisms. A recent report by Percich and Lockwood (38) on increased populations of Fusarium and severity of pea root rot fol- lowing atrazine treatment of soil is an example of the first type of mechanism. Incidence of pea root rot was three times, and of corn seedling blight was twice, that in nonamended soil, when the soil was amended with 30 ug/g atrazine and infested with F. solani f. sp. pig; or F. roseum f. sp. cerealis "Culmorum". Germination of macro- conidia and subsequent chlamydospore formation of both fungi on soil were also increased by atrazine. Moreover, Fusarium populations in natural soil increased up to u— fold after amendment with atrazine at 10, 30 and 100 ug/g, both in the laboratory and in the field. Examples of unequivocal evidence of increases in virulence of the pathogen by herbicides are lacking. Hsia and Christensen (20) reported that wheat infected with washed spores and mycelia of Helminthosporium sativum grown in potato-dextrose broth containing 5,000 ppm 2,A-D had more severe root infection and lower* green weight than those infected by the same fungus grown in the absence of 2,A-D. However, the unwashed inoculum grown in 2,AaD inducedanieven lower stand, heavier infection and lighter green weight than the washed inoculum. It was not clear whether the enhancement of disease was due to a change in virulence of the fungus or to predisposition possibly by the residual herbicide in the inoculum. However, the same authors showed that monosporous isolates of H. sativum developed sectors on PDA containing 5,000 ppm 2,4—D. Iso- lates from the sectors showed varying degrees of virulence, but none seemed obviously more virulent than the parent. This result is inconsistent with the hypothesis of increa- ses of virulence by the herbicide. Deep and Young (8) reported increased incidence of crown gall on Mazzard cherry (Prunus avium L.) when dich- lone (2,3- dichloro- 1,4-naphthoquinone), a fungicide for control of Thielaviopsis and Chalaropis root infections was applied to the trees. Under dichlone treatment, young trees inoculated with an avirulent strain of Agrobacterium tumefacience were as heavily infected (38%) as those inoculated with a virulent strain under the same conditions, yet only 3% were infected by the avirulent strain without dichlone. Moreover, 27% of the trees became infected when treated with dichlone but not inoculated, and 11% of the trees were infected without any treatments. The authors speculated that dichlone suppressed the antagonists in the soil, but the disease enhancement could not be duplicated with captan when the avirulent strain was used. Whether the fungicide predisposed the plant, increased the viru- lence of the pathogen, or suppressed the antagonists was not determined. The virulence of the pathogen isolated from galls infected with the avirulent strain treated with dichlone was not tested. Crop plants are more closely related to the target organisms of the herbicides thantx)the target organisms of other pesticides e.g.,fungicides, nematocides, insecti- cides. Consequently, physiological effects on crop plants are more likely with herbicides than with other pesticides. The enhancement of disease in the host could be caused by morphological or physiological deviations which would in— crease the susceptibility oftfimahost and/or facilitate the infection of the pathogen. For instance, enhanced root exudation as a result of herbicide treatments could stimu- late the germination of pathogens and result in severe infection. Altman (2) found that Tillam [S-propyl butyl- ethylthiocarbamate] and Pyramin [5—amino-A-chloro-2-phenyl- 10 3—(2H)pyridazinone] increased Rhizoctonia damping-off of sugar beets in steamed soil beyond that caused by Rh}: zoctonia alone. Glucose in exudates of roots was greater in plants grown in herbicide —treated soil than in soil not so treated. Lai and Semeniuk (25) showed that piclo- ram at 500 ppm induced increased exudation of carbohydrates from corn seedlings in axenic culture. No increase in amino acid excretion was detected. It is interesting that picloram also stimulates lipid synthesis in hemp seedlings (29). Recently Wyse (60) found that EPTC [S-ethyl dipro- pylthiocarbamate] and chloramben at 10-5M stimulated the exudation of amino acids from excised roots and hypocotyls of 6-day-old navy beans. Fusarium root rot of navy beans was enhanced by these herbicides,possib1y by increased root exudation. The survival and development of a microorganism de— pends not only on the physical environment, but also on the biological make-up of its surroundings, i.e. the antag- onistic and synergistic interactions with other microflora or microfauna. Herbicide changes in the biological balance involving pathogens could either favor or inhibit the growth, multiplication and finally infection of the patho- gen. In spite of reports of the lack of pronounced effects of herbicides on the gross population of soil microorga- nisms, e.g.,picloram(55)andsimazine (1“), such herbicides may specifically inhibit certain groups or species of ll microflora while allowing the others to flourish, thus not altering the total population. A good example of a study of the specific effects of herbicides on interactions of microorganisms was the report on competitive colonization of paraquat—treated potato haulms by Trichoderma veride and Fusarium culmorum by Wilkinson and Lucas (59). Paraquat was sprayed on mature potato plants to desiccate the haulms before lifting, as a standard practice in the field. Paraquat-destroyed haulms were brought into the laboratory incubated in a mixture of sand-maize—meal cultures of T. viride and T. culmorum and unsterilized sand at 10 and 20 times the weight of inoculum. Disks were cut out from the potato leaves after incubation for 2, 7 and 14 days, sur- face sterilized and plated on acid PDA. Nearly 100% of untreated haulms were colonized by T. viride but the ratios of T. viride and T. culmorum dropped to about 60/“0 and A5/55 due to 5,000 and 10,000 ppm paraquat respectively. T. viride is antagonistic to many important plant patho- gens, such as Fomes annosus (Al), Armillaria mellea (13), Rhizoctonia solani (58) and Sclerotium rolfsii (A2). Although the experimental conditions did not simulate the complex species composition of microflora in nature, it provided a quantitative measurement of the competitive colonization under controlled conditions. The influence of an herbicide on the initial colonization of an organic substrate could change the population of the l2 colonizing pathogen or that of its antagonists and the rate of decomposition of the substrate, which could in turn affect the persistence of the pathogen or its antagonists. Effect of chemical compounds on Thielaviopsis basicola and disease development --- Linderman and Toussoun (26) found that benzoic, phenylacetic, 3-phenylpropionic and A-phenylbutyric acids from decomposing plant residues in soil increased susceptibility of cotton to Thielaviopsis root rot. The chemicals did not stimulate germination of the fungus. Increased spore germination on root surfaces of plants treated with these chemical led to the specula— tion that changes in root exudation resulted in the in- creased activity of the fungus. Toussoun and Patrick (51) showed that extracts of decomposing plant residues enhanced root rot of beans caused by ”Fusarium solani f. sp. pha- seoli, Thielaviopsis basicola and Rhizoctonia solani. Ninhydrin-positive substances and traces Cd'sugarswere exuded from the extract-treated area but not from water- treated area. The authors suggested that the alteration of cell permeability is one of the more important effects produced by toxic decomposition products. Toussoun et a1. (52) reported that benzoic acid and phenylacetic acid were the major components of the extract of decomposing plant residue. v\_ MATERIALS AND METHODS The pathogen --- Thielaviopsis basicola (Berk. & Br.) Ferr. isolate 157, originally from Petersburg Township, Monroe Co., Michigan, was used unless stated otherwise. Isolates 170, 171 and 172 were obtained from Saginaw Co., Shiawassee Co.,and Saginaw Co., respectively. Inoculum for greenhouse and laboratory experiments was grown for ten to fifty days at 2A C on potato—dextrose agar in petri dishes. For soil infestation for greenhouse experiments, the culture was blended with 100 m1 of water before mixing with soil. Inoculum used in field experiments was grown on soybean-sand medium. Twenty g dry soybean seeds were chopped in a blender into a coarse meal and mixed with 1,000 g sand and 500 ml of water. The mixture was steamed for one hour, broken up, distributed into A one-liter flasks and autoclaved for 30 minutes. The herbicides -—- The herbicides used in the green- house and the field experiments are listed in Table l with their common names, trade names, chemical names, sources amdconcentrationsapplied. Commercial formulations were used in the greenhouse and field experiments. A stock solution of 10 mg active chloramben/m1 was made by dilut- ing the commercial formulation of chloramben, which is 2 1b. active chloramben/gal. with distilled water. It was stored at A C in the dark. A technical preparation of 13 1A pcoflpohwcfl o>Hpoow EOpQEmm mafimmohocfi go oamom 0 co women was xopca ommomfia .mpcwaa 0H|w wcficfimpcoo comm mQOHQmOHHQop : Oo cmozo .ohom\oa m mo cums on» up wcfipoom oAOOoo aflom on wofiamam mm: Conemhoanom BHOOHme .H 0 0.m 0 0.m 0 0.NH 0 m.ma 0 m.m 0 0.0 + sooewpoano a m.o a m.o m m.om m :.:m m m.m m o.m «Hosammn .H m 0.m m 0.H m 0.0m m m.mm < 0.0 < 0.0 cooempoano < 0.0 < 0.0 < m.mm < 0.0m < 0.0 < 0.0 pcoEpmohp oz Ewoa was Emoa was Emoa xHE ho>ocoo omSOMQooEU so>ocob, omSOBCoop0. ho>ocoo omzoscoopw pcoEpmope muoonm mo wcfipczpm vampcmaa mo uanoB xoan ommomfim o o amoum o m.omdocsoopw osp CH smooxom go pop poog BHOOHme mHmQOH>mHoH£E so cooEmpoaco mo poommo one .m magma 3O Fig. 1. Effect of chloramben at 3 1b/acre on Thiela— viopsis basicola root rot of soybeans in greenhouse mix. Left, no treatment; center, T. basicola alone; right, chloramben + T, basicola. Chloramben alone was similar to the no treatment. 31 Fig. 2. Effect of chloramben at 3 lb/acre on basicola root rot roots). Left, no right, chloramben similar to the no Thielavio sis of soybean in greenhouse mix (detail of treatment; center, T. basicola alone; + T. basicola. Chloramben alone was treatment control. 32 in all three sampling periods. No treatment controls or chloramben alone had no or very light infections (Table A, Fig. 3). Linuron had no significant effect on disease development. A count taken 38 days after planting showed a reduction in plant population of about 10% due to T. basicola. Stands were reduced about 20% more in the chlo- ramben + T. basicola treatment. Chloramben alone did not significantly reduce stand count. T. basicola was often found colonizing and sporulating on ungerminated seeds, and forming discolored lesions on cotyledons all through the first three sampling periods. No signs of any elonga- tion of radicles or growth of these infected seedlings could be detected. These seeds were found in both chloramben- treated and untreated rows of soil infested with T. basicola. It is possible that T. basicola is important in causing pre-emergent damage to soybeans in the field, in addition to its pathogenesis to established stands (28). Chloramben + T. basicola also decreased height and yield of soybeans when compared with the treatment of T. basicola alone (Table A). Although the later was not sig- nificantly different in plant height when compared with no treatment or chloramben alone, its seed yield was 12 and lA% lower than the other two, respectively. 33 .Amo.oumv pcmsmggfle sapcwoagacwam ohm msmppma pcmemggae hp UoSOHHog owoE 0C0 zfiopmgmgom Bump go CECHoo Cups 00 CoHHCQm mm: umoulu m.mpCoCCpmo .wCHHQEmw mCOH>oCQ o» oCC ©o>oEoC mewCoH poogIoCo m Cog paooxo «mCoa poog 0m Como .mCogpmowaaoC 0 go Cmozp .gon povaogCC pmowCCog go owoC on o» oCHH HHom 0C0 Eogg pogsmmoe mm: prHoC 0C8 .zog pooglom 0 Song Comoco zHEOUCwC mpCmHC 0H go Como mCOHCBOHHCoC 0 go mCmoE ogm sumo .mCHUoom Conga mgmp no CoCCmmoE mm; prHoC prHgo .mCoa poog 0m Como mzoC CopmoHHCoC 0 CH mpCHHC go CoCECC HBCOC go maoE ogm mama .mCHooom Copgm mzmp mm 6008 mm: pCCoo UCmpmo .010 Eogg mpflgm>om SOCCEmm wCHmmoCoCH go mamom 0 Co popmg ego: mEouCEmm .zog go CmeoH poogIoCo ComOCo mHEOUCmC m Eogg mpCmHC 0m 00 0: go mCOHCCOHHCoC 0 go CmoE on mg oCHw> Comm .mCHUoom Cough mmww mm 0C0 ma .0H wepmsam>o egos mEogCEmm ommomfimw 0 mmma m 0.:H 0 :0 0 00 0 w.m 0 0.m 0 :.m maoogmmn .H + CooEmCoaCo m :Hma < 0.0a m 0HH m :HH m m.m m :.m m w.a mHoogmmC .H < momH < m.gH < wma < 2mg < m.0 ¢ m.0 < H.0 CooEmCoaCo < mesa < m.mH C 02H < gma C m.0 < m.0 < H.0 pCoEuwon oz pmo>Cwm mzmp 0m , pmo>Cwm wasp mm wasp mm mmmp ma mzmo 0H uCoEpmogB va AmoCoCHV CpCsoo CCmpm mxopCH ommomgo enemas emmm opswaom pcmfim e.gaowg on Cg CmoCBOm go Cog poop CHOOfimmC mHmQOH>mHoHCE Co CooewgoaCo go pooggm .2 oHnt 3A hfiongoo pCoEpmogp 0C oCC eplmmwwmwm mm: mCon CoCEmCoHCo .maoogmmo .9 + CoCEmCoHCo Apgoa moCon mgoogmwn .9 0CopCoo mpCoEpmogp 0C nucmgm .Uaogg on CH Cwonzom go Cog poop .m wfloogmmn mwmmofl>0HoHCE Co ogom\oa m 00 CcoEBCoHCo go pooggm .mflm 35 Enhancement of root rot symptoms by chloramben on 3 soybearrvarieties ——— The soybean varieties "Corsoy" and "Hark" were compared with "Harosoy-63" for development of disease caused by T. basicola in chloramben-treated soil in the greenhouse. All three varieties showed typical symptoms in soil infested with T. basicola. Severity was enhanced by application of chloramben (Table 5). The ex- f“ periment was repeated and similar results were obtained. I Effect of chloramben on disease severity with diffe- rent isolates of the pathogen --- Four isolates of T. basicola from different localities were tested in thegmeen- Fry house for their virulence in soil treated with chloramben. All the isolates were pathogenic to soybean. Disease in— duced by all isolates was increased in soil treated with chloramben (Table 6). Effect of chloramben on the severity of disease using chlamydospores or endoconidia as inoculum -—— Endoconidia or chlamydospores of isolate 157 of T. basicola were used as inocula in chloramben-treated or untreated soil in the greenhouse. Endoconidia were applied at the rate of 6 X 107 spores per pot (A X 10” spore/g soil) and chlamydo- 3 propagules/g spores at 3 X 106 propagules per pot (2 X 10 soil). Soybeans grown in chloramben-treated soil devel- oped much more severe disease than controls with either form of inoculum (Table 7). 36 Table 5. Effect of chloramben on severity of Thiela- viopsis basicola root rot in three varie- ties of soybeana Diseaseindexb Treatment Harosoy-63 Corsoy Hark No treatment 0.0 A 0.0 A 0.0 A Chloramben 0.0 A 0.0 A 0.0 A T. basicola 3.A B 3.7 B 3.A B Chloramben + A.8 C 5.1 C 5.5 C T. basicola aChloramben was applied to soil before seeding at the rate of 3 lb/acre. b Mean of A replications each containing 8-10 plants. Disease index was based on a scale of in- creasing symptom severity from 0-6. Student's t- test was applied to each column of data separately, and means followed by different letters are signifi— cantly different (3 = 0.05). f T ‘4': T-‘n'. "-._' 5? 37 Table 6. Effect of chloramben on severity of disease induced by different isolates of Thiela- viopsis basicolaa Diseaseindexb Isolate Isolate Isolate Isolate Treatment 157 170 171 172 7 No treatment 0.0 A 0.0 A 0.0 A 0.0 A Chloramben 0.0 A 0.0 A 0.0 A 0.0 A .‘ g. basicola 5.A B 5.2 B 5.2 B 5.0 B Chloramben + 6.0 c 5.8 c 5.8 c 5.6 c i T. basicola aChloramben was applied to soil before seeding at the rate of 3 lb/acre. Isolates 157, 170, 171 and 172 of T. basicola were originally obtained from soybean fields in different localities in Michigan. bMean of A replications, each containing 8—10 plants. Disease index was based on a scale of increa- sing symptom severity from 0-6. Student's t-test was applied to each column of data separately, and means followed by different letters were significantly diffe- rent (2 = 0.05). There was no difference in the viru— lence among isolates, with or without chloramben appli— cation, as determined by analysis of variance. 38 Table 7. Effect of chloramben on severity of disease using endoconidia or chlamydospore as inocu- lum.a . . b Disease index Treatment Endoconidia Chlamydospores No treatment 0.0 A 0.0 A Chloramben 0.0 A 0.0 A T. basicola 3.9 B A.0 B Chloramben + T. basicola 5'0 C 5'3 C aChloramben was applied to soil before seeding at the rate of 3 1b/acre. bMean of A replications each containing 8-10 plants. Disease index was based on a scale of in- creasing symptom severity from 0—6. Student's t—test was applied to each column of data separately, and means followed by different letters were signifi- cantly different (2 = 0.05). \ 39 Effect of rate of chloramben and application methods on disease development --— Chloramben applied at half of the usual rate, 1.5 mg/pot (ca. 1.5 lb/acre) did not re- sult in enhanced disease development. Disease indiceswere 5.3 for this treatment and the T. basicola alone. In the same experiment the disease index for 3.0 mg chloramben/ pot in the presence of T. basicola wasgueater than u, that of the T. basicola control according to the t-test (g = 0.05). '5' Chloramben was either incorporated into soil as in the : usual greenhouse experimental treatments, or an equal amount was sprayed with an atomizer onto the soil surface after sowing to simulate field practice. The chloramben solution used for spraying was diluted to 5 ml per pot so the volume of liquid received per unit surface area was close to that of a field application. One hundred ml of water was added to the saucer under each pot daily except that the same amount of water was sprinkled onto the soil surface to simulate natural rainfall once every six days. Application of chloramben by spraying enhanced disease to a similar level as application by incorporation into soil (Table 8). Similar results were obtained in another ex- periment. Effect of soil temperatureon the development of Thielaviopsis root rot of soybeans in chloramben-treated soil -—— Pots of soybeans in soil treated with chloramben I“ A0 Table 8. Effect of mode of application of chloramben on Thielavippsis basicola root rot of soybeans. O O a Disease index Treatment Spray applicationb Soil incorporationc ‘1 No treatment 0.0 A 0.0 A ’ Chloramben 0.0 A 0.0 A T. basicola 3.3 B 3.3 B r Chloramben + 5.2 C 5.5 C T. basicola aMean of A replications each containing 8-10 plants. Disease index was based on a scale of increasing symptom severity from 0-6. Student's t-test was applied to each column of data separately, and means followed by diffe- rent letters were significantly different (T = 0.05). bFive ml of a suspension containing 0.6 mg chloram- ben/ml was atomized onto the soil surface in each potafter seeds were sown. CTwenty ml of a suspension containing 0.15 mg chlo— ramben/ml was atomized onto soil (ca 1A00 g) with mixing. The soil was then potted and seeded. Al and/or T. basicola, were set in temperature-regulated water baths at 1A, 18, 22 and 26 C. The lower temperatures, 1A and 18 C, were most favorable for disease development (Table 9). However, at 1A C, the plants germinated and grew much slower. Even so, chloramben enhanced disease at all temperatures. Possibility of direct stimulation of germination and growth of T. basicola by chloramben --- The possibility that the pathogen was directly stimulated by chloramben in the soil and that this would account for enhanced disease, was investigated. The criteria used to test the possible stimulation were the germination of spores in soil and in water, mycelial growth as linear extension on agar media and dry weight, and the sporulation and longevity in soil of the pathogen in the presence of chloramben. Neither endoconidia nor chlamydospores germinated more frequently when incubated in technical chloramben solution than in water or acetone solution. Endoconidia germinated less than 1% and chlamydospores about 10% in water, acetone (10 ul/ml water) solution, and in all con— centrations (2, 10, 30 and 100 ug/ml) of chloramben. Similar results were obtained in repeated experiments. Analysis of variance showed no significant differences with either spore type. Neither endoconidia nor chlamydospores showed in- creased germination during incubation on Conover loam k A2 Table 9. Effect of soil temperature on the development of Thielaviopsis root rot of soybeans in chlo- ramben—treated soila Disease indexb Treatment 1A C 18 C 22 C 26 C No Treatment 0.0 A 0.0 A 0.0 A 0.0 A Chloramben 0.0 A 0.0 A 0.0 A 0.0 A T. basicola 5.3 B A.8 B A.2 B 1.5 B Chloramben + 6.0 C 6.0 C 5.3 C 2.5 C T. basicola aPots of soybeans with the various treatments were set in temperature-regulating water baths at 1A, 18, 22 and 26 C. Chloramben was applied to soil before seeding at the rate of 3 lb/acre. bMean of A replications, each containing 8au3plants. Disease index was based on a scale of increasing symptom severity from 0-6. Student's t-test was applied to each column of data separately, and means followed by diffe— rent letters were significantly different. (2 = 0.05). A3 amended with chloramben as compared with that on non- amended soil. Endoconidial germination on natural soil was nearly zero regardless of the chloramben amendment; yet germination was 70% on sterilized soil. About 10% of the chlamydospores germinated on soil with or without chloram- ben, and no difference was detected with analysis of vari— ance. Growth of T. basicola was measured in 0.1 strength PDA containing 0, l, 3, 10, 30 or 100 ppm chloramben. The center of the petri dish was inoculated with a disk cut with a cork borer from the edge of a young colony of T. basicola. Plates were incubated at 2A 0. Colony diameters were mea- sured daily from the third to the 10th day. No significant differences in colony diameters were detected. No sector- ing was observed in any treatment during the experiment. Dry weight and sporulation were measured in colonies of T. basicola in liquid culture. Five flasks containing 50 m1 modified potato-dextrose broth (per liter: extract of 200 g potato, 30 g dextrose and 5 g yeast extract) were supplemented with technical chloramben at 2 ug/ml. The other five flasks did not contain chloramben and were used as controls. One—tenth ml of an endoconidial suspension was added to each flask, and the cultures were incubatedzfi: 2A C for 21 days. The mycelia in each flask were fragmented in a Waring blender, and four 2 m1 samples were taken for dry weight determination by drying at 70 C overnight in pre—weighed pans. Sporulation was determined by counting AA the endoconidia in samples of the fragmented tissue with a haemocytometer. Mycelia grown in chloramben-amended or control media had 106 to 107 endoconidia per m1. Mean dry weight of mycelia from the media containing chloramben was A8.9 mg and that of the controls was A9.2 mg. No signifi- cant differences in sporulation or dry weight were detec- ted between the chloramben treatments and controls when paired t—tests were applied to the data. The possibility that chloramben caused the spores of T. basicola to survive longer or to multiply in the soil was tested by comparing the populations of the pathogen in soil with or without chloramben treatment. Washed endo— conidia or chlamydospores were mixed into 120 g sieved 5 Conover loam at 10 and 1.5 X 10“ spores/g soil, respec- tively. A chloramben solution was atomized into half of the quantity of each infested soil to give a final concen— tration of 3 ug/g; this was incubated in three 6 cm dia— meter petri dishes (20 g each) at 18 C. The remaining soil was treated in the same manner except that the chloramben solution was replaced with water. Population of T. pagi- ppTa determined after 1, 6 and 16 days' incubation showed no change due to chloramben amendment. The populations stayed within the confidence limits of either treatment throughout the incubation period. The two types of propa- gules gradually declined in numbers at a similar rate. Fl A5 Possibility of increased virulence of T. basicola due to chloramben --- The effect of chloramben on virulence of T. basicola was tested by growing the pathogen in modified potato-dextrose broth amended with chloramben. The washed and fragmented mycelia were mixed (3 ml/pot) into vermicu- lite into which soybean seeds were sown. Pots were repli- cated 3 times and all pots were kept in a greenhouse for A weeks. Disease index was the same (A.A) for plants grown in vermiculite infested with T. basicola cultured in chloramben—amended media as with the fungus grown with- out chloramben. From the above experiments it was concluded that chloramben did not affect the germination, growth, sporu— lation,survivia1 in soil or virulence of T. basicola. Effect of chloramben on microbial_populations in spTT --- The possibility that chloramben altered the micro- bial population which in turn influenced disease develop- ment was investigated. Conover loam (ca. 1500 g) was moistened with an aqueous suspension of chloramben until the soil moisture content was about 12% and the concen- tration of chloramben was 2 ug/g soil (3 1b/acre). Con- trol soil contained 12% moisture, but no chloramben. Two additional treatments included glucose added to the chlo- ramben solution or to water to make a concentration of 0.1% (1.5 g/1500 g soil) to provide an energy source for the microorganisms. The soils were incubated in \— . A6 closed. plastic bags at 2A C. Total populations of fungi, bacteria, and actinomycetes were determined one and three weeks after treatment with the dilution plate method using selective media. The same experiment was repeated once. Fungal and actinomycete populations were not affected by chloramben application, as compared with their controls. The bacterial population was increased by chloramben in the first experiment,but was decreased in the second experi- § ment. Thus, the data were inconclusive regarding changes 3 in bacterial populations. Glucose amendment increased the population of all microbial groups slightly as compared i with treatments lacking glucose amendment, but no diffe- rential effects due to chloramben on fungi, bacteria and actinomycetes were revealed. An experiment was designed to bypass the influence of other microorganisms by testing whether chloramben would increase the disease in axenically-grown plants. Soy- bean seedlings grown for 8 days in the presence or absence of chloramben were transferred to autoclaved soil (15 g) in each of 32 test tubes (16 X 120 mm). Two ml of washed endoconidial suspension (5 X 10“ spores/ml) were trans- ferred into each test tube with a sterilized pipette. Thirty ug chloramben (2 ug/g soil) from a stock solution in acetone were addeth>half of the tubes containing chlo— ramben-treated seedlings. The tubes containing seedlings that had no chloramben treatment received water instead A7 of the chloramben suspension. All procedures were done aseptically. The tubes were incubated for 1A days at 2A C about 6 inches below continuous fluorescent light (6.3 X 103 lux). After incubation, 2 m1 of sterilized water were transferred into each tube, the tube was shaken, and 1-2 ml of the soil suspension that did not settle was poured immediately into a petri dish in which molten PDA (A2 C) was mixed. Colonies observed after 2 and 5 days were T. basicola only. The average disease index of 16 control plants was 3.8 and that of 16 chloram- ben-treated plants was 5.7. Student's t—test indicated the two treatments differed significantly (3:0.05). The same experiment was repeated with similar results. The result indicated that, in the absence of other microorganisms, chloramben increased disease to a similar extend as it did in the greenhouse and in the field. Consequently, other microroganisms did not play a major causal role in disease enhancement. Predisposition of soybeans to Thielaviopsis root rot by chloramben --- Since chlorambencfltlnot stimulate the pathogen directly nor influence microbial activities that could indirectly result in more severe disease, the effect of chloramben on the host was studied by allowing the herbicide and the host to interact before inoculation. Soybeans were grown in chloramben-treated greenhouse mix (2 mg/kg soil) for seven days, washed and transplanted A8 into T. basicola-infested soil with no chloramben. The roots were examined 2 weeks after transplanting. The disease index of such plants was A.0 as compared with 2.8 for plants transplanted into infested soil from soil without chloramben. Student's t—test indicated the two treatments were significantly different (T=0.05). This experiment indicated that soybeans became more susceptible *5 when exposed to chloramben without the presence of the pathogen, and that the effect persisted when chloramben was removed. The experiment was repeated with similar results. The nature of the predisposition of soybeans to Thielaviopsis root rot by chloramben was investigated by determining changes in spore germination in the rhizo- sphere, chemical composition of root exudates and altera- tion of host resistance. Lesion development on chloramben-treated soybeans by T. basicola introduced under the epidermis —-- The en- hanced susceptibility of soybeans brought about by chlo- ramben could be due to decreased host resistence. To differentiate this possibility from any change in the ex- ternal environment of the root, such as altered root exu— dation, approximately 5 ul of an endoconidia suspension containing 300 spores/pl, were injected into the cortex of the hypocotyls of 9-day-old soybeans grown in chloram- ben-amended greenhouse mix or greenhouse mix without chloramben in the greenhouse. An alcohol-sterilized A9 microsyringe was used to deliver the spore suspension to soybeans removed from the soil. The inoculated soybeans were replanted into their original pots and incubated 2 weeks in a controlled temperature water bath at 18 C. The mean lesion lengths for Al chloramben-treated and 37 untreated soybeans were 1.9 and2.0cn1respectively. The two figures were not significantly different when com- pared by the t-test. Plants injected with sterile water or those uninjected showed no symptoms. The experiment was repeated with similar results. The experiment indicated that chloramben treatment did notainxn°host resistance after penetration of the fungus. Spore germination in spybean rhizospheres -—- An average of 39% of chlamydospores and 17% of endoconidia germinated in the rhizospheres of chloramben-treated plants, whereas lA% of chlamydospores and A% of endoconi— dia germinated in rhizospheres of untreated soybeans. The results were significantly different (3:0.05) for both kinds of spores. The experiment was repeated with similar results. The experiment indicated that chloram- ben had caused quantitative or qualitative changes in the nutrient level of the rhizosphere. A study of root exu- dates followed. Root exudation --- Since spores germinated more fre- quently in rhizospheres of chloramben-treated soybeans, the chemical nature of the root exudates of plants grown 50 aseptically in a salt solution in the presence or absence of chloramben (2 ug/ml) was investigated. The data were based on four replications, each of which was the pooled contents of four test tubes. Chloramben-treatedseedlings exuded significantly more amino acids and electrolytes than seedlings exposed to either acetone or water in mea- surements made on the A th, 6th, 8th and 19th days (Figs. A and 5). Chloramben had no significant effect on the overall exudation of carbohydrates, fatty acids and nucleic acids. (Figs. 6, 7 and 8). However, the amount of fatty acids in exudates collected on the 8th day was significantly higher than that in the controls. Exuda- :5 tion of amino acids, carbohydrates and fatty acids was more abundant in the 8th and 10th days' collection than in earlier collections. The total material exuded by plants cultured in chloramben in excess of that exuded by plants cultured in the absence of chloramben were as follows: AAO% of amino acids, 105% for electrolytes, 23% for fatty acids and 32% for mucleic acids (Fig. 9). The increases in exudates from chloramben-treated soybeans over acetone controls were 268% for amino acids, 113% for electrolytes and insignificant amounts of carbohydrates, fatty acids, and nucleic acids. Germination of endoconidia in root exudates of chlo- ramben-treated soypeans ——- Washed endoconidia were sus- pended in sterile distilled water and 50 ul of this suspen- sion were placed in each well of a sterilized depression slide . 51 .AHoppCoo Copmzv oCOHm CoHoCHOm pHmm me CH Co .pCoEpmon ConEmCOHCo on CH 00C» 0» pCon>HCUo pCCoEm Cm CH ocouoom .HE\CoCEmCoHCo”w: m mCHCHmpCoo COHpCHom pHmm oCCHHC m CH mHHCOHpComm CoCCpHCo AmeHHUoom :0 go Cmoev mCHHpoom Cmongom oCo mo COHCmC CCOC NH 0 CH Coosxm mopmHogpoon .2 .ng $52: 00.3.. 295332. ... 5 1.. i. . 49:28 .2 m mzomo< \ N COEZOU \ .u M 1 «3.35 \ 40 3 u I \ In." 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M IL 9) . 12. m; P P ” 55 .AHOCpCoo Copmzv 0C0Hm C0HpCHOm uHmm 0C0 CH C0 .pC0Epmep CoCEmCOHCo 0C0 CH pde Op 0C0H0>Hsdo quoem Cm CH 0Couoom .HE\C00E0C 10HC0 m: N wCHCHmpCoo COHCCHOm pHmm ouCHHp 0 CH mHHmoHpaomm UoCCpHso AmeHHcmom :0 go C0080 wCHHpoom Cmonzom 0C0 mo 00HC00 CCOC NH 0 CH Copsxo mpHom 0H0Hosz .0 .ng .223 00:2... 20.2302. 0.. a o v d A . d J L 0 I N v ..OngOU fl 0 0 «I «0.715 . O t 11111111<.I11 .onflnnunuullllllo 1mm mm 2:33.920 Ivy/\I I”... m 1 olll Io‘\ m..O~_._.ZOU 1c. N wZOth< W O T in. M 56 ELECTROLYTES AMINO ACIDS C A RBOHYDRATES FATTY ACIDS .. NUCLEIC ACIDS CONTROL OF °/o A B C D E A B C D E CHLORAMBEN CHLORAMBEN WATER ACETONE Fig. 9. Effect of chloramben on quantities of electrolytes, amino acids, carbohydrates, fatty acids and nucleic acids exuded by roots of soybean seedlings. Exudates were collec- ted during four, l2-hour periods over 10 days from seedlings cultured aseptically in a dilute salt solution containing 2 ug chloramben/ml, acetone in an amount equivalent to that in the chloramben treatment, or in the salt solution alone (water control). 57 Concentrated (0.25 of the original volume) root exudate collected from soybeans cultured aseptically in the pre- sence or absence of chloramben were filtered through Millipore filters (0.22 pm) and 10 ul of each ofthe fil— tratcswereadded aseptically to each of 6 wells. The wells were then covered with sterilized coverslips and the slides were incubated in a moist petri dish for 22 hours at 2A C. Two hundred spores per well were then examined for germi- nation. The exudate from chloramben-treated soybeans supported a mean of 20% germination, whereas the exudate from water and acetone controls supported an average of 5% germination. None of the spores germinated in water alone. The experiment was repeated once with similar results. Effect of root exudates from chloramben—treated soy- beans on Thielaviopsis lesion development --- Root exudates from soybeans grown aseptically in chloramben solution (2 ug/ml), acetone of the same concentration as in the chloramben solution, or without either, were concentrated A X. An equal volume of concentrated exudate was mixed with a washed endoconidialsuspension. Five ul of each of these mixtures were applied to the hypocotyls of each of 20 axenically grown 6 day-old soybean seedlings, keptnmdst in petri dishes. The inoculated plants were incubated at 2A C under fluorescent light (12 hours/day) for 7 days. The mean length of lesionswas 20.8 mm for seedlings recei- ving exudates from chloramben-treated soybeans. By 58 contrast, hypocotyles receiving exudates from untreated and acetone-treated soybeans, and water alone, had lesion lengths of 8.1, 8.A and A.2 mm, respectively. The experi- ment was repeated with similar results. Enhancement of root rot severity by Casamino acids --- To verify that amino acids are the ingredient of the root exudate mainly responsible for the increased severity of symptoms, vitamin-free Casamino acids (Difco) were added to tubes of sterilized soil (approximately 12 g) containing an axenically grown soybean seedling and endoconidia of T. basicola. The tubes were incubated at 2A C for 21 days. Since the mean quantity of amino acids exuded during four 12 hours periods over 10 days by a single soybean seedling cultered with chloramben was approximately 71 ug, the amount of Casamino acid used was 200 pg per tube. This was a conservative estimate of that exuded during an incu— bation period for 21 days. The mean disease index of 18 plants in tubes supplemented with Casamino acids was 2.7 and that ofifluainoculated control without Casamino acids was 2.0. The difference was significant at the 5% level. N0 disease symptoms were detected on the uninoculated control. Plant grown at the same time in chloramben- amended soil (2 ug/g soil) infested with T. basicola also had an average of disease index of 2.7, whereas controls without chloramben had a disease indexcfi‘2.0.Similarresults 59 were obtained when the experiment was repeated. The results indicated that amino acids are a component in the root exudates important in the increased infection. DISCUSSION Soybeans grown in the greenhouse and in the field in soil containing herbicide chloramben had more severe symp- toms of Thielaviopsis root rot than those grown in soil without chloramben treatment. Enhancement of the disease was found to be caused by increased nutrient levels in root exudates from soybean grown in the presence of chlo- ramben. Possibilities of disease enhancement due to the direct stimulation of the pathogen, to increased virulence of the pathogen, or to population changes of other soil microorganisms were ruled out. Chloramben-induced enhancement of Thielaviopsis root rot of soybean was shown in the greenhouse using different soybean cultivars, various isolates of the pathogen, and with endoconidia or chlamydospores as inoculum. It occurred over a range of soil temperatures from 1A to 26C. Applications of the herbicide to soil by spraying the soil surface or by incorporation into the soil both resulted in increased disease. Disease enhancement was also shown when soybeans were planted in naturally-infested soil brought from the field, then treated with chloramben in the greenhouse. The possible economic importance of this effect was shown in a field experiment, where chloramben plus T. basicola significantly reduced plant stand, height and seed yield as compared with plants grown in the 60 61 presence of the pathogen alone. The wide host range and geographical distribution, and long survival time of the pathogen in soil suggest that chloramben be avoided when there is evidence of the presence of fungus in the field. Chloramben did not stimulate germination of endoco- nidia or chlamydospores of T. basicola in water or on soil, and did not increase mycelial growth or sporulation. More- over, population changes following infestation of soil with endoconidia or chlamydospores of T. basicola were not altered due to chloramben amendment. These results are in contrast to those of Percich and Lockwood (38) on the en- hancement of Fusarium root rot of pea and corn by atrazine (38). Atrazine increased the population of the Fusaria in the soil up to lO-fold. It increased the germination of macroconidia and subsequent chlamydospore formation of Fusarium solani f. sp. pisi and T. roseum f. sp. cerealis "Culmorum" on soil. T. basicola grown in the presence of chloramben was not more virulent than when grown in the absence of chemi— cal. No sectors were observed when the fungus was grown on chloramben—amended media, indicating that mutation rate was not increased. Chloramben and 2,A-D were reported to induce variants of Verticillium dahliae (21) and Helmin— thosporium sativum (20) in cultures,tnn;none of the vari- ants was more virulent than the original isolate. The rates applied (a 5% solution of chloramben and 5,000 ppm 2,A-D) were far beyond the rates used in the field and in 62 this research. Herbicides that inhibit antagonists of the pathogen may cause increase in disease (23). Chloramben increased Thielaviopsis root rot in axenically cultured soybeans to an extend similar to that occurring in soil. Moreover, soil treatment with 3.3 kg/ha chloramben caused no signi- ficant displacements in populations of actinomycetes and fungi in soil. Consequently, chloramben—enhanced disease did not appear to involve inhibition of other organisms. The study of total microbial populations is useful as a preliminary step to search for drastic population shifts, yet it may not be very helpful in understanding interactions which are more specific. Therefore, the lack of detectable population changes does not imply that its influence on other organisms was negligible. Soldatov et a1. (AA) reported that chloramben at A—6 kg/ha was one of several herbicides that decreased soil catalase activity during the first 10 days after application. Later, the enzyme activity returned to the level of untreated soil. Catalase is one of the enzymes responsible for detoxifica- tion of hydrogen peroxide, which is speculated to play a key role in microbial community composition (2A,50). If catalase—producing microorganisms were suppressed in the present work, this p0pulation shift does not seem to be crucial to disease enhancement. Soybeans first grown in soil amended with chloramben, 63 then transplanted into T. basicola-infested soil without chloramben,showed more severe disease than those grown in soil without the chemical and similarly transplanted. This experiment indicated that the soybean itself was the element in the system that was altered by the herbicide. The nature of the predisposition was sought first by determination of whether the change affected pre- or post- penetration stages. Since lesion development was not dif- ferent when the inoculum was injected into the cortex of plants in chloramben-treated or untreated soil, chloramben apparently did not influence the post-penetration defense mechanisms in the host. Therefore, the response of the pathogen in the rhizosphere of chloramben—treated soybeans was studied. Chlamydospores and endoconidia germinated two or four times more frequently in the rhizospheres of chloramben-treated soybean seedlings than in thosecfl‘ untreated seedlings, indicating that nutrient levels in the rhizosphere were raised due to chloramben treatment of the host. Application of exudates from chloramben—treated plants to seedlings inoculated with T. basicola endoconi— dia resulted in development of much longer lesions than occurred with exudates from control plants. Chemical analyses of exudates from soybeans showed large increases in amino acids from chloramben-treated seedlings. Moreover, the addition of Casamino acids to T. basicola infested soil caused the soybeans gnmmnltherein 6A to develop more severe symptoms than those without amend- ment. These experiments indicated that increasedwaxudation of amino acids is the cause of the enhancement of the disease. Wyse (60) found similar effects of chloramben andEEfiC on excretion of amino acids from excised hypocotyls and roots of navy beans. The application of EPTC to soil en- hanced Fusarium root rot of navy beans. Toussoun and Patrick (51) reported that ether extracts of decomposing plant materials, later found to contain benzoic acid, caused increased exudation of ninhydrin—positive substan- ces from bean stems, and increased the pathogenesis of T. basicola, Fusarium solani f. sp. phaseoli and Rhizoctonia solani. Linderman and Toussoun (26) showed that ether extracts of decomposing barley did not stimulate the germination of T. basicola when applied to non-sterilized soil, but germination of chlamydospores of T. basicola was stimulated on root surfaces of cotton seedlingfi‘treated with these ether extracts. Chloramben itself [3-amino, 2,5 dichloro benzoic acid] is a substituted benzoic acid; its enhancement of Thielaviopsis root rot seems to share the same mechanism as its analogue, benzoic acid. These related researches support the hypothesis that chloramben stimulated the exudation of amino acids which intAHW1in— creased the inoculum potential of the pathogen resulting in increased severity of T. basicola root rot in soybeans. 65 Papavizas and Kovacs (36) reported that germination of chlamydospores and endoconidia of T. basicola was in- creased in soil amended with unsaturated fatty acids, soy- bean lecithin, and was partially increased by soybean pro— tein. Although the total fatty acids exuded by soybeans treated with chloramben was 23% higher than those exuded by the control seedlings, only on the 8th day, were the fatty acids exuded from chloramben-treated soybeans sig— nificantly higher than in control plants. It is possible that during that short period of increased fatty acidlevels, the inoculum potential was further increased. Little is known about the mode of action of substi- tuted benzoic acid-type herbicides except for their auxin- like properties in promoting proliferation of planttissue. These herbicides, e.g.,2,A—D have been shown to interfere with the metabolism of nucleic acids. However, according to Moreland et a1. (33), chloramben does not stimulate or inhibit RNA and protein synthesis, and therefore does not share this mode of action with the phenoxy-type herbicides. The increase in electrolytes lost from roots of soybeans treated with chloramben indicated that chloramben altered the permeability of cell membranes. Whether the permea- bility change is a cause of herbicidal activity merits investigation. It is concluded that chloramben stimulated the exu- dation of nutrients, particularly amino acids, from roots 66 of soybeans, which in turn increased the nutrient status of T. basicola inocula in the rhizosphere resulting in in- creased severity of infection. 10. ll. 12. LITERATURE CITED Adams, P. B., and G. C. Papavizas. 1969. Survival of root-infecting fungi in soil. X. Sensitivity of propagules of Thielaviopsis basicola to soil fungistasis in natural and alfalfa-amen- ded soil. Phytopathology 59:135—138. Altman, J. 1969. Predisposition of sugar beets to Rhizoctonia solani damping-off with herbicides. Phytopathology 59:1015 (Abstr.). Ashton, F. M.,and A. S. Crafts. 1973. Mode of action of herbicides. John Wiley & Sons, Inc., New York. 50A?- Burnside, O. C. 1971. Effect of herbicides on seedling emergence force. Weed Sci. 19:182-18A. Cochran, W. C. 1950. Estimation of bacterial densi- ties by means of the "most probable number". Biometrics 6:105-116. Chen, T., R. A. Kilpatrick,and A. E. Rich. 1961. Ste- rile culture techniques as tools in plant nema— tology research. Phytopathology 51:799—800. Colby, S. R. 1965. Herbicide metabolism: N-glycoside of amiben isolated from soybean plants. Sci- ence 150:619-620. Deep, 1. W. and R. A. Young. 1965. The role of pre- planting treatments with chemicals in increas- ing the incidence of crowngall. Phytopathology 55:212-216. Dickson, J. G. 1956. Diseases of field crops. McGraw- Hill Inc., New York. 517 p. Farley, J. D.,and J. L. Lockwood. 1969. Reduced nutri- ent competition by soil microorganisms as a possible mechanism for pentachloronitrobenzene- induced disease accentuation. Phytopathology 59:718-72A. Fletchall, O. H. 1970. Steps to profitable weed con- trol. Farm Technology 36:26—28. Freed, V. H., M. Montgomery,and M. Kief. 1961. The metabolism of certain herbicides by plants - a factor in their activity. Proc. 15th North- east Weed Control Conf. pp. 6-16. 67 13. 1A. 15. 16. 17. 18. 19. 20. 21. 22. 23. 68 Garrett, S. D. 1958. Inoculum potential as a factor limiting lethal action by Trichoderma viride Fr. on Armillaria mellea Fr. Trans.Ehn Mycol. Soc., Al:l57-16A. Guillemat, J., M. Charpentier, P. Tardieux, and J. Pochon. 1960. Chloroaminotriazine herbicide. Interaction with fungus and soil bacteria. Ann. Inst. Natl. Recherche Afron. Ser. C 11:261-296. Halvorson, H. D.,and N. R. Ziegler. 1933. Application of statistics to problems in bacteriology. J. Bacteriol. 25:101-121. Haskell, D. A.,and B. J. Rogers. 1960. The entry of herbicides into seeds. Proc. 17th North Cen— tral Weed Control Conf. p. 39. Hawthorne, B. T. 1969. Germination of chlamydospores of Thielaviopsis basicola: physiology, ecology in soil and nature of soil fungistasis. Ph.D. Thesis, University of California, Riverside. 129 p. Hawthorne, B. T.,and P. H. Tsao. 1970. Effects of separation of spores from the chain and of culture age on germination of chlamydospores of Thielaviopsis basicola. Phytopathology 60:891-895. Hawthorne, B. T.,and P. H. Tsao. 1969. Inadequacy of the nutrient hypothesis to explain soil fungi- stasis in relation to chlamydospores of Thielaviopsis basicola. Phytopathology59zlo30 (Abstr.). Hsia, Y. T.,and J. J. Christensen. 1951. Effect of 2,A-D on seedling blight of wheat caused by Helminthosporium sativum. Phytopathology Al: 1011-1020. Hubbeling, N., and K. C. Basu Chaudhary. 1970. Mutagenic effect of a herbicide on Verticilli- um dahliae Kleb. Meded. Fac. Landbouwweten- schappen Gent 35:627—635. Johnson, B. J.,and M. D. Jellum. 1969. Effect of pesticides on chemical composition of soy- bean seed. Agron. J. 61:379-380. Katan, J.,and Y. Eshel. 1973. Interaction between herbicides and plant pathogens. Residue Re- views A5:lA5—177. 2A. 25. 26. 27. 28. 29. 30. 31. 32. 33. 3A. 35. 69 Kraus, F. W., J. F. Nickerson, W. I. Perry,and A. P. Walker. 1957. Peroxide and peroxidogenic bac- teria in human saliva. J. Bacteriol. 73: 727-735. Lai, M. T.,and G. Seminiuk. 1970. Picloram-induced increase of carbohydrate exudation from corn seedling. Phytopathology 60:563—56A. Linderman, R. G. and T. A. Toussoun. 1968. Predis- position to Thielaviopsis root rot of cotton by phytotoxins from decomposing barley resi- dues. Phytopathology 58:1571-157A. Lloyd, A. B.,and J. L. Lockwood. 1962. Precautions in isolating Thielaviopsis basicola with carrot discs.Phytopathology 52:131A-l315. Lockwood, J. L., D. L. Yoder,and N. A. Smith. 1970. Thielaviopsis basicola root rot of soybeans in Michigan. P1. Dis. Reptr. 5A:8A9-850. Mann, J. D.,and M. Pu. 1968. Inhibitioncfl‘lipid syn- thesis by certain herbicides. Weed Sci. 16: 197-198. Meggitt, W. B. 1970. Amiben provides long—lasting weed control in Michigan-Farm Tech. 36:33-37. Miller, G. R. 1970. How we get optimum results with amiben in Minnesota. Farm Tech. 36:31-3A. Moore, S.,and W. H. Stein. 195A. A modified ninhydrin reagent for the photometric determination of amino acids and related compounds. J. Biol. Chem. 211:907-913. Moreland, D. E., S. S. Malhotra, R. D. Gruenhagen, and E. H. Shokraii. 1969. Effect of herbicides on RNA and protein synthesis. Weed Sci. 16: 519-522. Morris, D. L. 19A8. Quantitative determination of carbohydrates with Dreywood's anthrone rea- gent. Science 107:25A-255. Mullendore, G. P. 1970. Amiben and Butyrac effective "one-two" punch. Farm Tech. 36:35. 36. 37. 38. 39. A0. A1. A2. A3. AA. A5. A6. A7. 70 Papavizas, G. C.,and M. F. Kovacs, Jr. 1972. Stimu- lation of spore germination of Thielaviopsis basicola by fatty acids from rhizosphere soil. Phytopathology 62:688—69A. Penner, D. 1968, Herbicidal influence of amylase in barley and squash seedlings. Weed Sci. 16: 519-522. Percich, J. A.,and J. L. Lockwood. 1975. Influence of atrazine on the severity of Fusarium root rot in pea and corn. Phytopathology 65:15A-158. Rapport, M. M. and N. Alonzo. 1955. Photometric deter- mination of fatty acid ester group in phospho- lipides. J. Biol. Chem. 217:193-198. Reider, G., K. P. Buchholtz,and C. A. Kust. 1970. Uptake of herbicides by soybean seed. Weed Sci. 18:101-105. Rishbeth, J. 1951. Observations on the biology of Fomes annosus with particular reference to East Anglian pine plantations. III. Natural and experimental infection of pines, and some factors affecting severity of the disease. Ann. Bot. 15:221-2A7. Rodriguez-Kabana, R., E. A. Curl, and H. H. Funderburk. 1967. Effect of a atrazine on growth response on Sclerotium rolfsii and Trichoderma viride. Can. J. Microbiol. 13:13A3-13A9. Shkola, A. I. 1970. Effect of prometryne and amiben on development of nitrogen-fixing bacteria. Vestu. Sel'skokhoz. Nauki. 13:3A-37. Soldatov, A. B., N. Barbut'ko,and L. Latushkin. 1971. Effect of herbicides on the biological activity of the soil. Tr. Beloruss. Sel'skokhoz. Akad. 76:131-137. Sommerville, D. N.,and L. M. Wax. 1971. Influence of incorporation depth on chloramben activity. Stroller, E. W. 1969. Kinetics of amiben absorption and metabolism as related to species sensi- tivity. Plant Physiol. AA:85A-860. Stroller, E. M.,and L. M. Wax. 1968. Amiben metabolism and selectivity. Weed Sci. 16:283-288. A8. A9. 50. 51. 52. 53. 5A. 55. 56. 57. 58. 71 Swan, D. G.,and F. W. Slife. 1965. The absorption, translocation and fate of amiben in soybeans. Weeds 13:133-138. Swanson, C. R., R. E. Kadunce, R. H. Hodgson, and D. S. Frear. 1966. Amiben metabolism in plants. 1. Isolation and identification of an N-glu- cosyl complex. Weeds lA:3l9-323. Thompson, R.,and A. Johnson. 1951. The inhibitory action of saliva on the diphtheria bacillus: hydrogen peroxide, the inhibitory agent pro- duced by salivary streptococci. J. Infect. Dis. 88:81-85. Toussoun, T. A.,and Z. A. Patrick. 1963. Effect of phytotoxic substances from decomposing plant residues on root rot of bean. Phytopathology 53:265-270. Toussoun, T. A., A. R. Weinhold, R. G. Linderman,and Z. A. Patrick. 1968. Nature of phytotoxic sub- stances produced during plant residue decom- position in soil. Phytopathology 58:Al-A5. Tsao, P. H.,and J. L. Bricker. 1966. Chlamydospores of Thielaviopsis basicola as surviving propa- gules in natural soils. Phytopathology 56: 1012-101A. Tsao, P. H.,and A. C. Canetta. 196A. Comparative study of quantitative methods used for estimating the population of Thielaviopsis basicola in soil. Phytopathology 5A:633-635. Tu, C. M.,and W. B. Bollen. 1968. Effect of paraquat on microbial activities in soil. Weed Research 8:28-37. USDA. 1969. Fats and oils situation. 2A7(A):6. Vasil'ev, D. S.,and R. G. Chanukvadze. 1969. Use of Amiben and Ramrod on soybeans in the piedmont zone of the Krasnodar region. Byull. Nauch. - Tech. Inform. Maslich. Kul't. 1969:56-58. Weindling, R. 193A. Studies ona.1ethal principle effective in the parasitic action of Tricho- derma lignorum on Rhizoctonia solani and other soil fungi. Phytopathology 2A:1153-1l79. 72 59. Wilkinson, V.,and R. L. Lucas. 1969. Influence of herbicides on the competitive ability of fungi to colonize plant tissues. New Phytol. 68:701- 708. 60. Wyse, D. L. 197A. Interaction of herbicides, disease and cultural practices on growth and develop- ment of navy bean. Ph.D. Thesis, Michigan State University, East Lansing. 167 p.