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Journal of General Microbiology ( I986), 132, 77 1-777. Printed in Great Britain 77 1 Extracellular Pectinolytic Enzymes of Fungi Elicit Phytoalexin Accumulation in Carrot Suspension Culture By M A G D Y A M I N , F U M I Y A K U R O S A K I * A N D A R A S U K E N I S H I Faculty of Pharmaceutical Sciences, Toyama Medical and Pharmaceutical University, Sugitani, Toyama 930-01, Japan (Received 17 June 1985; revised 7 October 1985) A heat-labile elicitor of phytoalexin accumulation in carrot (Daucus curota L.) was detected in culture filtrate of Chaetomium globosum grown in Czapek-Dox medium. The elicitor activity correlated with the activity of pectinolytic enzymes in the culture filtrates. The culture filtrates induced the release of heat-stable elicitors from carrot cell homogenates, pectic fraction of carrot cell walls and citrus pectin. The coincidence of heat sensitivity and sodium periodate inactivation profiles of elicitor and pectinolytic activity suggested that the activity of pectinolytic enzymes is essential for the release of the heat-stable elicitor. The culture filtrates of Botrytis cinerea, Fusarium rnoniliforrne and Helrninthosporium oryzue showed similar elicitation patterns. INTRODUCTION Phytoalexins are low-M, antimicrobial compounds which have been shown to accumulate in many species of higher plants after infections and appear to be a widespread mechanism by which plants attempt to protect themselves against microbes (Kui., 1972; Grisebach & Ebel, 1978). Molecules of microbial origin which trigger phytoalexin accumulation in plants have been called elicitors (Keen et al., 1972). Many elicitors have been reported, including fungal wall polysaccharides, glycoproteins and lipids (West, 1981). Lee & West ( I98 1 a, b) and Davis et al. (1984) showed that pectinolytic enzymes released into the culture filtrates of Rhizopus stolonifer and Erwinia carotovora elicited phytoalexin accumulation in castor bean and soybean, respectively. Kurosaki & Nishi (1983, 1984) showed that carrot produces 6-methoxymellein as a common phytoalexin after being challenged by various fungi, and that wall fragments released from carrot cells by a commercial pectinase or protease elicit the production of the phytoalexin. These findings suggested that fungal pectinolytic enzymes play a role in the elicitation of phytoalexin production in carrot-fungi interactions. In this work, we present evidence that the extracellular pectinolytic enzymes of fungi serve as elicitors of phytoalexin accumulation in carrot by releasing fragments from pectic polysaccharides of the plant cell wall. METHODS Micro-organisms and cultures. Chaetomium globosum and Botrytis cinerea were kindly provided by Dr Tsubaki, Institute of Biological Sciences, Tsukuba University, Japan. Fusarium monilijbrmt)and Helminthosporium oryzae were obtained from Toyama Agricultural Experimental Station, Japan. All fungi were maintained on agar slants of yeast/malt medium [yeast extract 0.4% (w/v), malt extract 1 % (w/v) and glucose 0.4% (w/v)]. Cultures for testing elicitor activity were obtained by inoculating the stock culture on agar slants of Czapek-Dox medium [sucrose 3% (w/v), N a N 0 3 0.3% (w/v), KzHPOj 0.1% (w/v), MgS0,.7HI0 0.05:/, (w/v), KCl 0.05% (w/v) and FeSO,. 7H,O 0.001 % (wlv)]. After incubation for 7 d at 25 "C, spores were collected in sterile deionized water, and 1 ml samples (approx. lo6 spores) were used to inoculate 100 ml Czapek-Dox medium in 250 ml flasks. After 72 h incubation on a reciprocal shaker (72 strokes min-I) at 25 "C, these cultures were used for testing the presence of substances with elicitor activity. 0001-2766 0 1986 SGM Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Wed, 03 May 2017 22:30:38 772 M . A M I N , F . K U R O S A K I A N D A . NISHI Preparation ojculture3ltrates. mycelial walls arld myceliul extracts. Culture filtrates were separated frotn mycelia by suction filtration. The filtrates were dialysed against several changes of water (4"C), sterilized by membrane filtration (Millipore filter GS 0.22 pm) and kept at - 30 "C until use. Mycelial walls were prepared as described by Yoshikawa et al. (198 1). Cell-free mycelial extract was obtained by centrifugation of the homogenized mycelia. The clear extract was collected and kept at - 30 "C until use. Production of 6-methosymellein in carrot suspensioii culture. Carrot cells derived from a root of carrot (Duucus carota L. cv. Kintoki) were cultured in the synthetic medium of Murashige & Skoog (1962) according to Okamura et ul. (1975). The substance to be tested was added to a 7-d-old culture of carrot cells (late exponential phase). The cells were harvested after 24 h by suction filtration for determination of their 6-methoxymellein content. Production of' 6-mefho.~ymelluinin carrot roofs. Carrot roots were surface sterilized by soaking in ethanol for 15 min and discs (5 mm thick, 20 mm diameter) were prepared using a sterile cork borer. The test substance was placed on the cut surface of the discs. In some experiments, the cut surface was covered by a heat-sterilized glass microfibre filter (Whatman, 25 mm diameter) beforr: inoculation. After 24 h incubation at 27 "C in sterile Petri dishes, the discs were homogenized with sand and their 6-methoxymellein contents were determined. Determination of' 6-metho~xymellein.Procedures f x extraction and purification of 6-methoxymellein from cultured carrot cells and carrot roots were described previously (Kurosaki & Nishi, 1983). Extracts were developed on TLC plates (Merck, silica gel 60 F 254) with benzene/methanol (100:2, v/v), and 6-methoxymellein content was determined as described by a scanning method in which the absorbance of the sample was measured at 265 nm and that of the reference at 400 nm using a ddal-wavelength chromatoscanner (Kurosaki & Nishi, 1983). Assuy of' elicitor activity. Elicitor activity was determined by the production of 6-methoxymellein in cultured carrot cells or carrot root discs. The activity was expressed as pg 6-methoxymellein per 100 ml culture or per root disc. Heat-labile elicitors were sterilized by membrane filtration, and heat-stable elicitors by autoclaving (1 20 "C for 15 min) before addition to cultured carrot or carrot root discs. Pectinolytic activity in fungal culture jiltrates. This was assayed from the rate of production of new reducing termini resulting from the hydrolysis of polygalacturonic acid (Sigma, grade 11) at 30 "C. The reaction mixtures contained 0.5 ml culture filtrate and 9.5 ml 0.251'4 (w/v) polygalacturonic acid sodium salt in 0.2 M-acetate buffer (pH 5.2). The reaction was stopped by mixing 0.2 11-11 reaction mixture with 0.2 ml alkaline copper reagent and reducing ends were measured by the modified Nelson procedure (Somoygi, 1952). Controls were run by adding the alkaline copper reagent to the reaction mixture betore incubation. Carrot pectic polysaccharides. Cell wall materials 'were isolated from suspensions of cultured carrot cells and purified according to Asamizu & Nishi (1979). Pectic polysaccharides were extracted with 0.5% (w/v) ammonium oxalate at 90 "C. The extract was dialysed against distilled water and lyophilized. The lyophilized materials were dissolved in acetate buffer (pH 5.2) to give a final concentration of 0.1% (w/v), then autoclaved and stored at -30 "C until use. RESULTS Elicitation of 6-methoxymellein production by C . globosum in carrot roots The cut surfaces of carrot root discs were inoculated, either directly or after being covered by glass microfibre filters, with 0.2 ml of a suspttnsion of either growing C. globosum mycelia (72 h culture) or purified fungal walls. After 24 h incubation at 27"C, the accumulated 6methoxymellein was determined (Table 1). Carrot roots infected directly or indirectly with the growing mycelia accumulated significant aniounts of 6-methoxymellein. The glass fibre paper prevented direct contact of the mycelia with the root discs: microscopic examination of root surfaces after incubation showed no sign of penetration. The wall preparations did not elicit an appreciable increase in 6-methoxymellein. Elicitor activity of the culture Jiltrates of C . globosum Autoclaved and unautoclaved culture filtrates (1 ml), mycelial extract (1 ml) or purified fungal walls (0.1 g in 10 ml deionized water) was ,3dded to 100 ml carrot cell culture to test elicitor activity. The results (Table 2) indicated that the culture filtrates of C.globosum elicited 6methoxymellein production in carrot cell culture but that the activity was destroyed by autoclaving. Carrot cells treated with mycelial walls or cell extract contained less 6methoxymellein than the control indicating that these fractions did not show elicitor activity. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Wed, 03 May 2017 22:30:38 Elicitation of' phytoalexin production 773 Table 1. Elicitor activity of C.globosum in carrot roots The results are means of three separate experiments, ksu. Elicitor activity Treatment (pg 6-methoxymellein per disc) Uninoculated medium Direct contact with mycelia Indirect contact with mycelia' Direct contact with mycelial wall Indirect contact with mycelial wall* 10.5 0-01 12.3 0.69 9.2 k 0.18 1.5 0.33 11.3 _+ 0.20 * The cut surface of the carrot root disc was covered with glass microfibre filter paper before inoculation. Table 2. Elicitor actiziit)?oj'C. globosum in carrot cell culture The results are the means of three separate experiments, f s u . Elicitor activity (pg b-methoxymellein per 100 ml culture) , Treatment Uninoculated medium Mycelial walls Mycelial extract Culture filtrates Unautoclaved 8.6 6.5 3.8 18.6 k 1.40 k 1.20 f 0.52 f 1.80 1 Autoclaved (1 20 "C for 15 min) 8.8 k 1.31 3.8 f 0-30 4.6 0.40 8.4 f 0.51 Releuse of' heat-stuble elicitor .from carrot cell homogenates Boiled and unboiled culture filtrates of C. globosum were added to carrot cell homogenates and incubated at 30 "C. At intervals, portions of the incubation mixture were withdrawn, autoclaved and added to carrot cell culture to test elicitor activity (Fig. 1). The elicitor activity of the incubation mixture was low at time 0 but it increased with time and two peaks of elicitor activity appeared during the incubation of cell homogenates with the unboiled culture filtrates. Boiled culture filtrates did not enhance the elicitor activity of the homogenates during the incubation. These results suggested that a heat-labile factor in the culture filtrates of C . globosum stimulated the release of heat-stable secondary elicitors from carrot cells. Since we have shown that a commercial pectinase elicits the production of phytoalexin in carrot cells (Kurosaki & Nishi, 1984), we did experiments to determine whether the heat-labile factor derived from the fungus was a pectinase or related enzyme. Releuse oj' heat-stable elicitor-.from carrot pectic polysaccharides and citrus pectin Pectic polysaccharides of carrot and citrus pectin were digested by culture filtrates of C. globosum and the hydrolysates were assayed for heat-stable elicitor (Fig. 2). The pectic fraction of carrot elicited 6-methoxymellein accumulation ; elicitor activity was increased by digestion with unboiled culture filtrates of C. globosum for up to 2 h, but further digestion resulted in complete loss of elicitor activity. Unhydrolysed citrus pectin did not elicit 6-methoxymellein production, while incubation of citrus pectin with the fungal culture filtrates resulted in multiple peaks of heat-stable elicitor activity. Coincidence oj elicitor and pectinolytic acticities in the .fiingal culture filtrates Preliminary experiments to characterize the fungal elicitor showed that the elicitor activity was destroyed by heat or incubation with sodium periodate. This suggested that the elicitor might be a glycoprotein in which both peptide (heat-sensitive) and sugar moieties (periodate-inactivated) are essential for elicitor activity. Teinperature- and periodate-sensitivity were therefore used to Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Wed, 03 May 2017 22:30:38 774 M . A M I N , F . K I J R O S A K I A N D A . NISHI I 0 1 I 2 3 4 5 Incubation time (h) 1 1 6 7 Fig. 1. Release of heat-stable elicitor from carrot cell homogenates. Cultured carrot cells (7 d old, 4 g fresh weight) were suspended in 10 ml acetate buffer (pH 52), killed by autoclaving and homogenized by sonication. A 10 ml portion of the homogenate was mixed with an equal amount of filter-sterilized culture filtrate of C.globosum and incubated at 30 "C. At intervals, 2 ml samples were withdrawn, chilled in an ice bath and centrifuged. The supernatants were autoclaved to destroy the heat-labile elicitor and assayed for heat-stable elicitor activity as described in Methods. Control experiments were run with fungal culture filtrates boiled at 100 "C for 15 min. 0, Boiled fungal filtrates; 0 , unboiled fungal filtrates. Each point represents the mean of two separate experiments, the results of which agreed to within 10%. 0 1 2 3 4 0 1 2 3 4 Incubation time (h) Fig. 2. Release of heat-stable elicitors from carrot pectic polysaccharides and citrus pectin. Experimental procedures were the same as for Fig. 1 except that carrot pectic fraction and citrus pectin (each 0.1%, w/v) were used instead of carrot cell homogenates. (a) Elicitor released from carrot pectic fraction with boiled (0) and unboiled ( 0 )C. globosum culture filtrates and (b) elicitor released from citrus pectin with boiled (0) and unboiled ( 0 )culture filtrates. Each point represents the mean of two separate experiments, the results of which agreed to within 10%. correlate elicitor and pectinolytic activities. The results in Fig. 3(a) show that the elicitor and pectinolytic activities of C. globosum culture filtrates exhibited similar profiles of temperature sensitivity except at 40 "C. Incubation at 613 "C for 15 min caused loss of 7 0 4 0 % of both the activities (Fig. 3 b). When culture filtrates were incubated with sodium periodate (final concentration, 0.014 M) at 30 "C, the elicitor activity decreased progressively in parallel with the pectinolytic activity during the incubat ion (Fig. 4). Elicitor activity in the culture filtrates of other fungi Pectinolytic enzymes have been detected in many fungi and bacteria (Rexova-Benkova, 1976). We tested the culture filtrates of threl? other fungi for the presence of heat-labile elicitor and the ability to release heat-stable elicitor from citrus pectin (Table 3). The results, indicating Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Wed, 03 May 2017 22:30:38 Elicitation o j phytoalexin production 775 120 100 h 8 80 W .-wx 60 2 40 20 n " 0 20 40 60 80 100 120 0 Temperature ( O C) 5 10 15 Time (min) 20 Fig. 3. Effect of temperature on elicitor and pectinolytic activity of C. gfobosum culture filtrates. C. globosum was grown in Czapek-Dox medium for 72 h at 25 "C. The culture filtrate (20 ml) was dialysed, lyophilized and redissolved in 10 mlO.2 M-acetate buffer (pH 5.2). and 1 ml portions were incubated at temperatures of 0 to 120 "C for 15 min (a) or at 60 "C for 0 to 20 min (6).After incubation, the solutions were chilled in an ice bath and a 0.5 ml portion was used for the assay of elicitor activity (a);the other 0.5 ml was assayed for pectinolytic activity (0). The activities are expressed as percentages of the value obtained when the incubation was done at 0°C. Each point represents the mean of two separate experiments, the results of which agreed to within 10%. 100 - 80 h 8 A .-+ 2 60 40 20 0 0 1 2 3 4 5 6 7 Incubation time (h) Fig. 4. Effect of periodate treatment on elicitor and pectinolytic activity of C. globosum culture filtrates. Culture filtrate was prepared as described for Fig. 3, and 1 ml portions were incubated with 20 pl0.7 Msodium periodate at 30°C. At the indicated times, 0.1 ml was withdrawn, mixed with 2p1 ethylene glycol and diluted with 0.9 mlO.2 M-acetate buffer (pH 5.2). A 0.5 ml portion was used for the assay of elicitor (a)and the other 0.5 ml was assayed for pectinolytic activity (A). Controls (open symbols) were run as above, but with ethylene glycol added before the addition of periodate. The activities are expressed as percentages of the value obtained immediately after the addition of periodate (0 time). Each point represents the mean of two separate experiments, the results of which agreed to within 10%. the heat sensitivity of the elicitor in the culture filtrates and the release of heat-stable elicitor from citrus pectin after 30 min digestion, supported the idea that pectinolytic activity of these fungi served as an elicitor of phytoalexin accumulation in carrot. DISCUSSION The results presented in this paper strongly suggest that phytoalexin accumulation in carrot suspension culture is triggered by extracellular pectinolytic enzymes released from various fungi. The results in Table 1 indicate that direct contact of carrot tissue with fungal mycelia is not necessary for elicitation of phytoalexin production and hence that the elicitor activity resulted from substance(s) diffusing either from the fungus or the carrot root. The accumulation of 6methoxymellein in carrot suspension cultures after the addition of C. globosum culture filtrates and the loss of elicitor ability by autoclaving indicated that the elicitor is a heat-labile substance Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Wed, 03 May 2017 22:30:38 776 M . AMIN, F. KUROSAKI A N D A . NISHI Table 3. Elicitor activity in !he culture filtrates of various jungi (a)Culture filtrates of fungi (1 ml) were obtained after cultivation for 72 h in Czapek-Dox medium and added to a carrot cell suspension after being hcated (100 "C, 15 min) or unheated. (b) Culture filtrate (1 ml) was mixed with 1 ml 0-1% citrus pectin in acetate buffer (pH 5-2) and incubated for 30 min at 30 "C. After the treatment, the samples were autoclaved and added to a carrot cell suspension. Control experiments were run with heat-inactivated (100 "C, 15 min) fungal culture filtrate. The results in both ( a )and (b)are means of three experiments, +set. The 6-methoxymellein contents of carrot cell cultures supplied with 0.2 M-acetate buffer alone and with 0.1 % citrus pectin in 0.2 M-acetate buffer were 8.7 .+ 0.5 and 5.2 f 0.7 pg per 100 ml culture, respectively. Elicitor activity (pg 6-methoxymellein per 100 ml culture) ( a ) Culture filtrates of fungi Fungus C. globosum B . cinerea F . monillforme H . oryzae rpL.-- v Heated at 100°C for 15 min Unheated 8.6 k 0.2 11.3 k 0.9 7.5 0.5 16.5 k 0.5 z2.2 35.0 33.8 37.5 k k 2 k 2.2 0.7 3.7 1.5 (b) Citrus pectin incubated with culture filtrates of fungi 7 Heated at 100°C for 15 min 4.8 k 0.6 10.5 h 1.3 9.5 k 0.5 16.7 1.7 Unheated 30.2 48.8 50.0 52.0 k 2.2 k 3.8 k 3.0 k 4.0 released into the culture filtrates of the fungus (Table 2). The coincidence of the heat sensitivity (Fig. 3) and periodate inactivation (Fig. 4) profiles of elicitor and pectinolytic activity in the culture filtrates of C. globosum suggests that pectinolytic activity in the culture filtrates is essential for elicitor activity. Culture filtrate 5 of C. globosum catalysed the release of heat-stable elicitors from carrot cell homogenates, pectic: fraction of carrot cell wall and citrus pectin (Figs 1 and 2). These results suggest that the heat-stable elicitors may be hydrolysis products of the pectic fraction of plant cell wall released by the activity of pectinolytic enzymes in the culture filtrates. We observed multiple peaks of elicitor activity when carrot cell homogenates and citrus pectin were incubated with fungal culture filtrates. Pectinolytic enzymes that catalyse the release of active elicitors may also inactivate them during further incubation (Bruce & West, 1982; Davis et al., 1984). The pectinase-released elicitors are probably not a single molecular species but rather heterogeneous cellular fragments containing oligogalacturonides as essential moieties. Jin & West (1984) have shown that galacturonide oligomers of appropriate size are responsible for the elicitor activity in casbene synthesis; oligomers smaller than the nonamer showed little or no elicitor activity. Nothnagel et al. (1983) reported that several distinct elicitors for glycinol production are released from both cell walls of soybean and citrus pectin by partial acid hydrolysis. The fact that the pectic fraction of carrot had elicitor activity before digestion by the culture filtrates (Fig. 2) may be attributable to the extraction procedure, which involves pronase treatment. Pronase has been shown to release heat-stable elicitor from carrot cell homogenates (Kurosaki & Nishi, 1984). The difference in response of cultured carrot cells to the hydrolysates of carrot pectic fraction and citrus pectin may be partly related to differences in their chemical composition. For instance, the pectin preparation of carrot contained 20 % galacturonic acid (Asamizu & Nishi, 1979) while citrus pectin contains 75% uronic acid. Our present findings are analogous to those obtained by Lee & West (198 1 a, b) and Davis et nl. (1984). They demonstrated that pectin-degrading enzymes from R . stolonifer and E. carotovora are elicitors of phytoalexins in castor bean and soybean, respectively. It has also been shown that these enzymes can release heat-stable elicitor from plant cell wall and citrus pectin. These observations, together with the present results, suggest that phytoalexin production in many plant species is triggered by pectinolytic endcymesof infecting micro-organisms, and the plant cells recognize the partial degradation products of cell wall polymer released by the enzymes as the endogenous elicitor. Unlike soybean, however, carrot cells did not respond to fungal wall preparations, which are a potent elicitor of phytoalexin production in leguminous plants (West, 1981). Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Wed, 03 May 2017 22:30:38 Elicitation of phytoalexin production 777 REFERENCES ASAMIZU,T. & NISHI,A . (1979). Biosynthesis of cell wall polysaccharides in cultured carrot cells. PIanta 146, 49-54. BRUCE, R. J . & WEST, C. A . (1982). Elicitation of casbene synthetase activity in castor bean. Plant Physiology 69, 1181-1 188. DAVIS,K . R., LYON, G. D., DARVILI., A . G. & ALBERSHEIM, P. (1984). Host--pathogen interactions. XXV. Endopolygalacturonic acid lyase from Erwinia carotovoru elicits phytoalexin accumulation by releasing plant cell wall fragments. Plant Physiology 74, 52-60. GRISEBACH,H. & EBEL, J . (1978). Phytoalexins, chemical defense substances of higher plants'? Angewandte Chemie International (Edition in English) 17, 635 647. JIN, D. F. & WEST, C. A . (1984). Characteristics of galacturonic acid oligomers as elicitors of casbene synthetase activity in castor bean seedlings. Plant Physiology 74, 989 -993. KEEN,N . , PARTIDGE, J . & ZAKI,A . (1972). Pathogenproduced elicitor of a chemical defense mechanism in soybean monogenically resistant to Phytophthora megasperma var. sojue. Ph~~topathologjJ 62, 768. KuC, J . (1972). Compounds accumulating in plants after infection. In Microbial Toxins P'ZII, pp. 21 1 247. Edited by K . Solomon. New York: Academic Press. KUROSAKI,F. & NISHI, A. (1983). Isolation and antimicrobial activity of the phytoalexin 6-methoxymellein from cultured carrot cells. Phyrochemnrry 22, 669-672. KUROSAKI,F. & NISHI, A . (1984). Elicitation of phytoalexin production in cultured carrot cells. Physiological Plant Pathology 24, 169 1 76. LEE, S. C. & WEST,C. A . (1981a). Polygalacturonase from Rhizopus stolonijer, an elicitor of casbene synthetase activity in castor bean (Ricinus communis L.) seedlings. Plant Physiology 67, 633-639. LEE, S. C. & WEST, C. A . (1981b). Properties of Rhizopus stolonijer polygalacturonase, an elicitor of casbene synthetase in castor bean (Ricinus communis L.) seedlings. Plant Physiology 67, 640-645. MURASHIGE, T. & SKOOC,F. (1962). A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiologia plantarum 15, 473-497. NOTHNAGEL, E. A., MCNEIL,M., ALBERSHEZM, P. & DELL,A . (1983). Host--pathogen interactions. XXII. A galacturonic acid oligosaccharide from plant cell walls elicits phytoalexins. Plant Physiology 71, 916926. OKAMURA, S., SUEKI,K . & NISHI,A. (1975). Physiological changes of carrot cells in suspension culture, during growth and senescence. Physiologia plantarum 33, 251-255. REXOVA-BENKOVA, L. (1976). Pectic enzymes. Adaances in Carbohjvdmte Chemistrji and Biochemisrrj. 153, 375-380. SOMOGYI,M. (1952). Notes on sugar determination. Journal of Biological Chemistrj. 195, 19-23. WEST,C. A. (1981). Fungal elicitors of the phytoalexin response in higher plants. Naturwissensclrafien 68, 447-457. YOSHIKAWA, M., MATAMA,M . & MASAGO,H . (198 1 ). Release of a soluble phytoalexin elicitor from mycelial walls of Phytophthora megasperma var. sojae by soybean tissues. Plant Physiology 67, 1032-1035. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Wed, 03 May 2017 22:30:38