Host-Pathogen Interactions XV. FUNGAL GLUCANS WHICH ELICIT PHYTOALEXIN ACCUMULATION IN SOYBEAN ALSO ELICIT

A 8jglucan isolated from the mycelial wails of Phytophthora megasperma var. sojae and a glucan purified from yeast extract stimulate the accumulation of phytoalexins in red kidney bean, Phaseolus vulgads, and stimulate the accumulation of the phytoalexin, rishitin, In potato tubers, Solanum tuberosum. These glucans have previously been shown to be potent elicitors of glyceollin accumulation In soybean, Glycine max. Treatment of kidney bean cotyledons with the glucan elicitors resulted in the accuulation of at least five fungistatic compounds. These compounds migrate durig thin layer chromatography identically to the fungistatic compounds wbhch accumulate in kIdney beans which have been inoculated with CoBletotrkhui Hndemuthianum, a fungal pathogen of kidney beans. Potatoes accumulate as much as 29 micrograms of rishitin per gram fresh weight following exposure to the glucan from Phytophthora megasperma var. sojae and as much as 19.5 micrograms of rishitin per gram fresh weight following exposure to yeast glucan. Potatoes accumulated 28 micrograms of rishitin per gram fresh weight following inoculation with live Phytophtbora megasperma var. soJae. Many plants respond to invasion by both pathogenic and nonpathogenic microorganisms by producing phytoalexins (12, 17, 19, 20). Phytoalexins are low mol wt antimicrobial compounds which plants can accumulate in quantities sufficient to inhibit the growth in culture of potential pathogens (6, 8, 11, 25, 28). It is thought that phytoalexin accumulation may be a mechanism by which plants resist the majority of microbes which they encounter (1, 12, 17). Molecules that stimulate phytoalexin production have been isolated from various fungi. These molecules have been termed elicitors (18). An elicitor of glyceollin accumulation in soybeans has been purified from the soybean fungal pathogen Pms3 (4). This Pms elicitor is a /l-glucan and is a component of the mycelial wall (5). Recently, an elicitor of glyceollin has been purified from commercial brewers' yeast extract. The purified elicitor is a glucan which appears to be structurally similar to the Pms elicitor (16). This suggests that soybeans can detect the presence of other fungi by reCognition of a portion of their glucans. Such glucans are present in the cell walls of many different fungi (9). The purpose of this work was to determine whether other higher plants have the ability to respond to Pms and yeast by recognition 1 Research was supported by the Rockefeller Foundation RFGAA57714 and the Department of Energy EY-76-S-02-1426. 2To whom correspondence should be addressed. 3Abbreviation: Pms: Phytophthora megasperma var. sojae. oftheir glucan eicitors. Dark red kidney beans, Phaseolus vulgaris, and potatoes, Solanum tuberosum, have been chosen as models to test whether the Pms elicitor and the yeast elicitor will stimulate the accumulation of phytoalexins in other higher plants. Dark red kidney beans respond to invasion by a variety of microorganisms including Colletotrichum lindemuthianum, the causal agent of anthracnose in beans, by turning brown at the site of inoculation and by accumulating a number of phytoalexins including phaseollin, phaseollidin, phaseollin isoflavin, and kievitone (6-8, 11). This response is also stimulated by exposing the cut surfaces of kidney bean plants to as little as 100 ng of a polysaccharide elicitor which was isolated from the mycelia wails of C. lindemuthianum by Anderson-Prouty and Albersheim (3). Potatoes respond to inoculation with a variety of microorganisms by accumulating a number of phytoalexins including rishitin, lubimin, and phytuberin (21, 22, 25, 28). A polysaccharide has been isolated from the mycelial wails of Phytophthora infestans, the causal organism of potato blight. Exposure of potatoes to this polysaccharide results in the accumulation of phytoalexins (10). MATERIALS AND METHODS An authentic sample of phaseollin was the generous gift of J. A. Bailey. An authentic sample of rishitin was kindly donated by G. D. Lyon. The Pms glucan elicitor was obtained from B. Valent of this laboratory and was the fraction I elicitor purified by the procedure of Ayers et al. (4). The yeast elicitor was obtained from M. Hahn and was purified from yeast extract by the procedure ofHahn and Albersheim (16). A crude C. lindemuthianum elicitor, the concentrated water extract of autoclaved mycelial walls, was prepared by the procedure of Anderson-Prouty and Albersheim (3). Elicitor carbohydrate was quantitated by the anthrone method (13) using glucose as a standard and is reported as itg of glucose equivalents. Dark red kidney beans var. Charlevoix were obtained from the Idaho Seed Bean Co., Twin Falls, Idaho. The beans were soaked in sodium hypochlorite (0.75%) for 5 min, were then rinsed with distilled H20, and were planted in Vermiculite. Bean plants were grown under a photoregime of 14 hr of 2,500 ft-c illumination at 20 C and 10 hr of darkness at 18.5 C. Idaho russet potatoes were obtained from a local grocery and were surface-sterilized for 2 min with 80% ethanol before use. Brewers' yeast was obtained from a local brewer's supply store and was washed with 10 mm potassium phosphate (pH 7.2) prior to use. The Pms was grown in still culture and was prepared for inoculation as previously described (4). Race a of C. lindemuthianum was maintained on agar slants containing the medium described by Mathur et aL (23). Spores of C. lindemuthianum were prepared for inoculation by adding 2 ml of sterile H20 to an agar slant of the culture and by scraping spores from the surface of the agar with a spatula. Inoculation of Bean Tissue and Extraction of Phytoalexins. Red 918 www.plantphysiol.org on July 14, 2017 Published by Downloaded from Copyright © 1978 American Society of Plant Biologists. All rights reserved. HOST-PATHOGEN INTERACTIONS. XV kidney bean cotyledons were excised from 6-day-old seedlings, were soaked in sodium hypochlorite (0.75%) for 5 min, and then rinsed with distilled H20. A thin epidermal strip was cut from the convex side of each cotyledon with the aid of a razor blade. Cotyledons were placed on moist filter paper in sterile Petri plates with their cut surfaces up. Solutions to be tested for elicitor activity were made 10 mm with respect to potassium phosphate (pH 7.2). Test solutions (50 pl) were applied to each cotyledon. Sterile phosphate buffer was applied to cotyledons as a control. The cotyledons were incubated in the dark at 24 C for 22 to 24 hr. Phytoalexins were extracted by soaking the treated cotyledons in 95% ethanol (2.5 ml/cotyledon) for 1 hr with swirling every 10 min. The ethanol extract was decanted and the cotyledons were reextracted with another volume of ethanol for 30 min. The ethanol extracts were combined and centrifuged at 12,000g for 10 min. The supernatant solution was evaporated to dryness under reduced pressure at 38 C and the residue was extracted with ethyl acetate (1 ml for each cotyledon originally extracted) for 1 hr. The ethyl acetate extract was blown dry under a stream of N2 at room temperature. The residue was redissolved in ethyl acetate (10 pl for each cotyledon originally extracted). This fraction is called the "concentrated phytoalexin extract." Detection of Phytoalexins in Kidney Bean Cotyledon Extracts. Portions of the concentrated phytoalexin extracts were spotted onto 0.25-mm thin layer silica plates and the plates were developed with HCCl3-methyl alcohol (100:4, v/v). The plates were dried and the presence of phytoalexins was detected by the use of a bioassay as previously described (3). In this bioassay, spores of Cladosporium cucumerinum are mixed with potato dextrose agar and the mixture is sprayed uniformly onto the thin layer plate. The plate is placed in a covered box and is incubated at 100%1o RH at 24 C for 48 to 72 hr. The spores of this fungus are dark. Regions where phytoalexins are present lack fungal growth and appear as white spots on a dark background. Inoculation of Potatoes and Extraction of Rishitin. Ten cavities, each 2 cm deep x 1.1 cm in diameter, were formed in each potato with a No. 6 cork borer. Solutions to be tested were supplemented with 500 ig/ml penicillin. One ml of a test solution was pipetted into each cavity. The inoculated potatoes were covered with plastic wrap and were incubated at 19 C and 100%1o RH. After 48 hr, the tissue surrounding the treated cavities was removed with a No. 8 cork borer, diameter 1.8 cm, to a depth of 2.4 cm. Rishitin was extracted from the excised tissue by homogenizing the tissue in 70% ethanol (2 ml for every cavity) at 4 C in a Sorvall OmniMixer for 5 min. The homogenized tissue was filtered through cheesecloth and the liquid which passed through the cheesecloth was centrifuged at 16,000g for 20 min. The supernatant solution was evaporated to dryness under reduced pressure at 30 C. The residue was shaken with a mixture of H20 and ethyl ether (40:60, v/v), 10 ml/original cavity. The ether layer was collected and the aqueous phase was reextracted two more times with ether. The ether extracts were combined and evaporated to dryness under reduced pressure at 30 C. The residue was redissolved in methanol (10 pl for each cavity assayed). This fraction is called the "concentrated rishitin extract." Identification and Quantitation of Rishitin. Portions of the concentrated rishitin extract were spotted on a 0.25-mm thin layer silica plate. The thin layer plate was developed with ethyl ether. Rishitin was visualized on the TLC plate as a pink spot when the plates were sprayed with a saturated solution of antimony trichloride in chloroform followed by heating at 60 C for 3 min. Rishitin was also detected as a fungistatic spot on plates which were bioassayed with C. cucumerinum. Rishitin was identified on thin layer plates by comparison of its migration to the migration of a rishitin standard. Rishitin in the potato extracts was quantitated by a modification of the method of Sato and Tomiyama (26) as follows. The silica was scraped from areas corresponding to a rishitin standard from thin layer plates on which potato extracts had been chromatographed. The silica was extracted with 20 ml of ethyl ether. The ether extract was evaporated to dryness under a stream of N2 and the residue was dissolved in 1 ml of hexane. One ml of concentrated H2SO4 was added to the rishitin-containing hexane solution followed by vigorous mixing on a Vari-Whirl mixer. The A of the H2SO layer was measured at 500 nm and the amount of rishitin was calculated by comparison to a standard curve obtained using authentic rishitin. The identity of rishitin in the hexane solution was confirmed by gas chromatography and MS on a HewlettPackard model 5980A GC/MS using a glass column (2 mm x 1.83 m) containing 1% O.V. 17 on Gas-chrom Q.