Effect of Pythium spp. and glyphosate on phytoalexin production and exudation by bean (Phaseolus vulgaris L.) roots grown in different media

Kievitone, phaseollinisoflavan and phaseollin were detected in roots of bean seedlings (Phaseolus vulgaris L.) grown in natural soil. Comparison of phytoalexin production by roots grown in different media indicated that these phytoalexins were probably induced by microorganisms in soil. The influence of common root rot pathogens of bean, Pythium spp., on phytoalexin production was determined. Pythium ultimum elicited kievitone, phaseollinisoflavan and phaseollin in roots grown in sterilized silica sand. P. sylvaticum induced only kievitone and phaseollin in the same growth medium. Glyphosate did not significantly affect the accumulation of phytoalexins within 3 days. However, by day 5, significantly more phaseollin was detected in the roots of Pythium inoculated plants treated with glyphosate than in Pythium inoculated plants not treated with glyphosate. In a hydroponic system, both Pythium spp. elicited accumulation of kievitone and phaseollin in root tissue, and both phytoalexins were exuded into the bathing solution. Glyphosate application did not significantly affect accumulation or exudation of phytoalexins by bean roots in the hydroponic system. The results from this study illustrate the nature and extent of phytoalexin production by bean roots in the absence and presence of microbes.     

Formation of phytoalexins within and outside lesions of Botrytis cinerea in French bean leaves

Rapidly spreading lesions and lesions restricted in size developed in primary leaves of French bean (Phaseolus vulgaris) in response to infection byBotrytis cinera isolates BC-1 and BC-5, respectively. These isolates caused similar differential lesions in leaves of cucumber, flax, lettuce and tomato. To determine whether phytoalexin accumulation was correlated with the resistant reaction in bean leaves, accumulation of phytoalexins was examined in necrotic areas of both types of lesions and in their surrounding green tissues. Phaseollin was the predominant phytoalexin, both inside and outside lesions, whereas phaseollidin and sometimes also phaseollinisoflavan were always present in lower concentrations. Phaseollin accumulated earlier and to higher levels within and around lesions of isolate BC-5 than of isolate BC-1. Relatively low concentrations of phaseollin were detected in the more remote green areas, including the petiole, of leaves bearing a spreading lesion. The phaseollin metabolite, 6a-hydroxyphaseollin, was found only inside lesions and in a narrow zone around lesions of both types. The authors consider the possibility that the differing concentrations of phytoalexins in the infected tissues are not a determining factor for the differential interactions betweenB. cinerea and bean leaves, but are rather the result of it.     

Induction of phytoalexins in adzuki bean after inoculation with <Emphasis Type="Italic">Phytophthora vignae</Emphasis> f. sp. <Emphasis Type="Italic">adzukicola</Emphasis>

To identify phytoalexins of adzuki bean elicited in response to attempted infection of Phytophthora vignae f. sp. adzukicola, we isolated compounds from adzuki bean and evaluated their antifungal activity. Seven flavonoids (daidzein, genistein, 2′-hydroxygenistein, coumestrol, dalbergioidin, kievitone, and phaseol) were identified from epicotyls wound-inoculated with a mycelial suspension of an avirulent race of P. vignae f. sp. adzukicola. Of those compounds, kievitone and dalbergioidin accumulated to higher levels in incompatible interactions compared to compatible interactions 48 h after inoculation. Kievitone strongly inhibited the germination of encysted zoospores, and dalbergioidin were slightly suppressive. From these results, we concluded that kievitone and dalbergioidin are phytoalexins in adzuki bean.     

Temperature-induced susceptibility of soybeans to Phytophthora megasperma f.sp. glycinea: phenylalanine ammonia-lyase and glyceollin in the host; growth and glyceollin I sensitivity of the pathogen

In unwounded soybean hypocotyls, pulse labelled with [¹⁴C]phenylalanine and inoculated with Phytophthora megasperma f.sp. glycinea, rates of [¹⁴C]-incorporation and glyceollin I accumulation were higher in resistant than in susceptible responses throughout the time-course of the experiment. This distinction was masked in hypocotyls that were wounded and inoculated. In such hypocotyls, high rates of [¹⁴C]-incorporation developed that were similar for the first 11 h in resistant and susceptible responses, although much more glyceollin I accumulated in the former. High rates of [¹⁴C]-incorporation also developed in uninoculated wounded hypocotyls but only small amounts of glyceollin I of high specific radioactivity were detected. Estimates of phenylalanine ammonia-lyase activity indicated that the metabolic flux through phenylalanine was limited in wounded controls but potentially very high in resistant responses. Differences in rates of [¹⁴C]-incorporation and in specific radioactivity of accumulated glyceollin I presumably indicate differences in the relative contributions of mobile internal pools and externally applied phenylalanine, in addition to rates of biosynthesis. Rapid decline in [¹⁴C]-glyceollin I was demonstrated in wounded controls in pulse-ch0ase experiments with phenylalanine as chase, but not in inoculated hypocotyls, due to continued [¹⁴C]-incorporation during the chase period. Rapid metabolism was demonstrated in all interactions and in wounds when cinnamic acid was used as the chase, but there was no evidence that differences in glyceollin I accumulation were due to differential rates of metabolism. Additional evidence for metabolic activity was provided by pulse feeding with [¹⁴C]glyceollin I. It is concluded that the stimulus of wounding or infection induces a metabolic pathway in which glyceollin I is not an end product. The accumulation of higher levels of glyceollin I in resistant than in susceptible responses appears to be due to earlier initiation and subsequently higher rates of biosynthesis in the former.     

Changes in biochemistry and cellular ultrastructure support different resistance mechanisms to Phytophthora sojae in nonhost common bean and host soybean

Phytophthora root and stem rot of soybean caused by Phytophthora sojae is a destructive disease affecting soybean production worldwide. In nature, soybean is the only economically important cultivated host of P. sojae. The aim of this study was to explain different resistance mechanisms to P. sojae in nonhost common bean and host soybean as a basis for the control of Phytophthora root and stem rot of soybean via nonhost resistance. Observations and measurements of disease resistance-related variables showed slight differences in structural and biochemical resistance mechanisms between common bean and soybean. P. sojae infection induced a stronger hypersensitive response in nonhost common bean than in host resistant soybean. Moreover, phytoalexin phaseollidin synthesis-related vestitone reductase gene was extremely highly up-regulated, and phytoalexin glyceollin synthesis-related isoflavone reductase gene was slightly less up-regulated in common bean than in soybean, which resulted in a higher level of phaseollidin and a lower level of glyceollin in common bean. Phaseollidin had stronger inhibitory effects on mycelial growth and oospore formation of P. sojae than glyceollin, and more cell wall depositions and callose accumulated in common bean, which are probably related to the stronger resistance of nonhost common bean to P. sojae.     

Polygalacturonase isoenzymes and oxalic acid produced by Sclerotinia sclerotiorum in soybean hypocotyls as elicitors of glyceollin

The polygalacturonases (PG) and oxalic acid produced by Sclerotinia sclerotiorum in infected soybean hypocotyls were investigated as elicitors of the phytoalexin glyceollin I.Purification to homogeneity through isoelectrofocusing and ion-exchange fast protein liquid chromatography revealed three endo-PG isoenzymes (PG-I, PG-II and PG-IV) and one exo-PG (PG-III) in 6-day-old etiolated soybean hypocotyls infected with the B-24 isolate of S. sclerotiorum.PG-I and PG-III, in the range of concentrations tested (0·15–1·2 reducing units ml⁻¹), did not act as elicitors of glyceollin I synthesis. Some elicitor activity was shown by PG-II at 0·6–1·2 reducing units ml⁻¹. PG-IV, at lower doses (0·038–0·30 reducing units ml⁻¹), was even more effective in inducing phytoalexin synthesis. However higher concentrations of PG-IV induced tissue softening and decreased phytoalexin accumulation.PG-II and PG-IV released heat-stable elicitors from purified soybean cell walls supporting the evidence that uronides are intermediate inducers in elicitation by endo-PGs. Oxalic acid was an active elicitor of glyceollin I over the range of concentrations tested (0·31–20 m ) with the maximum at a concentration of 5 m . The inability of oxalic acid to release uronides from purified cell walls makes it unlikely that uronide intermediate elicitors are involved in elicitation by oxalic acid.     

Bacterial induction of the accumulation of phaseollin,pisatin and rishitin and their antibacterial activity

Bean hypocotyls, pea pods and tomato fruits were tested for phaseollin, pisatin and rishitin production when challenged with the phytopathogenic bacteriaErwinia carotovora, Pseudomonas phaseolicola, P. pisi andP. solanacearum, and their isolated extracellular polysaccharides. All bacteria induced phytoalexin accumulation, whereas only phaseollin and pisatin, but not rishitin, were elicited by EPS. The inhibitory effect of these three phytoalexins on bacterial growth was studied in liquid medium; whereas phaseollin and pisatin strongly inhibited growth, only a slight inhibitory effect resulted from the presence of rishitin in the medium.Samenvatting Bonehypocotylen, erwtepeulen en tomatevruchten werden onderzocht op hun vermogen tot vorming van respectievelijk faseolline, pisatine en rishitine, na inoculatie met de fytopathogene bacteriënErwinia carotovora, Pseudomonas phaseolicola, P. pisi enP. solanacearum en na behandeling met oplossingen van hun extracellulaire polysacchariden (EPS). Alle bacteriesoorten induceerden fytoalexinevorming, terwijl hun EPS wel faseolline- en pisatine-, maar geen rishitinevorming induceerden. Faseolline en pisatine remden de groei van de bacteriën in vloeibaar medium sterk; rishitine daarentegen had slechts een geringe groeiremming tengevolge.     

Suppression of sclerotinia stem rot of soybean by lactofen herbicide treatment

ABSTRACT Severity of Sclerotinia stem rot of soybean after treatment with lactofen (Cobra) and other herbicides was assessed in field experiments conducted in Michigan from 1995 to 1997. At sites where disease pressure was high, disease severity was reduced 40 to 60% compared with controls when lactofen was applied at the V3 (1995 and 1996) or R1 (1997) growth stages. Corresponding seed yields were unchanged or up to 20% greater when lactofen was applied at the R1 stage in 1997. Disease severity was not reduced by lactofen treatments in years and at sites where disease pressure was low to medium, and corresponding yields often were reduced by 10%. High levels of glyceollin accumulated in lactofen-injured leaves collected from field plots in 1996 and 1997. High glyceollin content in lactofen-treated leaves was associated with significant reductions in lesion size when leaves were challenge-inoculated with Sclerotinia sclerotiorum.     

Sensitivity to,and metabolism of,phaseollin in relation to the pathogenicity of different isolates of Botrytis cinerea to bean (Phaseolus vulgaris)

The pathogenicity of five isolates ofBotrytis cinerea to bean (Phaseolus vulgaris) was compared with their sensitivity to the phytoalexin phaseollin and their ability to metabolize phaseollin. Three of the five isolates were pathogenic to bean, as they formed spreading lesions on bean leaves; the other two isolates were considered nonpathogenic to bean, since they produced lesions limited in size or no lesions at all. The isolates were about equally sensitive to phaseollin, except for one nonpathogenic strain which was more sensitive than the other ones to higher concentrations of phaseollin. The three pathogenic isolates metabolized phaseollin to 6a-hydroxyphaseollin readily in shake cultures containing 9 μg phaseollin/ml, while nonpathogenic isolates were not able, or less able, to do so.     

Phaseollidin stored in vacuoles and the phytoalexin response in bean

In time-course experiments, the amounts of phytoalexin in hypocotyls of the resistant bean cultivar Flor de Mayo were determined after infection with Colletotrichum lindemuthianum . Phaseollin and phaseollidin accumulated as a defence response. However, phaseollidin accumulated earlier than phaseollin and at greater concentrations (155  +  14 compared with 120  +  9  μ g g⁻¹ fresh weight). Phaseollidin was found conjugated as a glucoside in the tissue. Fungal treatment apparently led to a decline of approximately 50% in phaseollidin conjugate after only 7 h after infection. Isolation of vacuoles confirmed the presence of phaseollidin glucosides in this organelle. Treatment of the tissue or isolated protoplasts with a fungal elicitor also produced a decrease, by half, of phaseollidin conjugate concentrations from vacuoles isolated from both sources. The contribution of pre-existing pools of phaseollidin glycoside to the accumulation of this phytoalexin is discussed.     

The possibility of factors other than phytoalexin accumulation preventing fungal growth in the incompatible interactions of soybean with Phytophthora megasperma f. sp. glycinea

Soybean ( Glycine max ) cv. Harosoy 63 is resistant to race 1 and susceptible to race 9 of Phytophthora megasperma f. sp. glycinea (Pmg). In detached primary leaves inoculated with zoospores, growth of race 1 was completely suppressed 16 h after inoculation, while race 9 was unaffected. The amount of the phytoalexin glyceollin that accumulated, however, was not significantly different in either the incompatible or compatible interaction 16 h after inoculation. At the circumference of the inoculated area, a slight accumulation of phytoalexin was observed only in the incompatible interaction 20 h or more after inoculation. Tolerance of race 9 to the phytoalexin was significantly higher than that of race 1 when the phytoalexin was added to agar. Moreover, race 9 degraded glyceollin faster than race 1. On leaves inoculated at separate points with either race, the lesion associated with race 9 never colonized areas inoculated with race 1. These results suggest that factor(s) other than the accumulation of phytoalexin in soybean tissue might cause cessation of growth of Pmg.     

Studies on pathogenesis of Rhizoctonia solani in beans: an evaluation of the possible roles of phenylacetic acid and its hydroxy derivatives as phytotoxins

All isolates of Rhizoctonia solani used in this study, when grown in Weinhold medium supplemented with phenylalanine, accumulated phenylacetic acid (PAA) and its ortho-, meta- and para-hydroxy derivatives in culture. Major metabolites which accumulated were PAA and m-hydroxyphenylacetic acid (m-OH-PAA). However, the accumulation in vitro of total phenylacetic acid derivatives by isolates of R. solani was not related to their pathogenicity and/or virulence. On the contrary, the non-pathogenic and weakly virulent isolates of R. solani produced the highest quantities of PAA-derivatives per unit weight of mycelium in comparison to the highly virulent isolates.The phenylacetic acid derivatives used in this investigation, when bioassayed at high concentrations, caused inhibition of growth of tomato and bean seedlings. The most active compounds were PAA and -α-methoxy-phenylacetic acid (MeO-PAA). However, when m-OH-PAA was assayed at low concentrations there was a stimulation of seedling growth. In addition, when m-OH-PAA solutions were injected into bean hypocotyls or into tobacco leaves they never caused necrotic lesions or symptoms resembling those caused by R. solani on beans.These results indicate that PAA-derivatives are not directly involved in pathogenesis of R. solani in beans.     

Phytoalexin production in French bean leaves infected by botrytis cinerea

French bean (Phaseolus vulgaris) leaves were inoculated with three pathogenic and two nonpathogenic isolates ofBotrytis cinerea and the infected tissues, containing either spreading lesions or lesions limited in size, were investigated for the presence of phytoalexins. In most cases phaseollin, phaseollidin, phaseollinisoflavan, the phaseollin metabolite, 6a-hydroxyphaseollin, and a few unidentified antifungal compounds were found; phaseollin was predominant. The concentration of phaseollin accumulating in leaves infected by the nonpathogenic isolate BC-5 was about twice as high as that in infections produced by pathogenic isolates. In contrast, leaves infected by the nonpathogenic isolate BC-6 only contained low concentrations of phaseollin.Our data do not provide strong evidence that phaseollin is the principal factor that limits lesion development.Samenvatting Bladeren van boon (Phaseolus vulgaris) werden geïnoculeerd met drie pathogene en twee niet-pathogene isolaten vanBotrytis cinerea, als gevolg waarvan zich uitbreidende lesies, resp lesies van beperkte omvang ontstonden. Deze lesies werden samen met een omringend groen gedeelte onderzocht op de aanwezigheid van fytoalexinen. Hiertoe werden deze stoffen geëxtraheerd uit drooggevroren bladmateriaal, en gezuiverd m.b.v. kolomchromatografie over Sephadex LH-20 (Tabel 1) en dunnelaag-chromatografie (Tabel 2). Kwantitatieve analyse vond plaats d.m.v. in situdensitometrie.In de meeste gevallen werden phaseolline, phaseollidine, phaseollineïsoflavaan, een omzettingsprodukt van phaseolline, nl. 6a-hydroxyphaseolline, en enkele onbekende fungitoxische stoffen aangetoond (Fig. 1). Phaseolline was het meest voorkomende fytoalexine (Fig. 2 en 3). De concentratie van phaseolline in bladeren geinfecteerd met het niet-pathogene isolaat BC-5 was ongeveer tweemaal zo hoog als in bladeren geïnfecteerd met een pathogeen isolaat (isolaat BC-1, BC-3 en BC-4). Daarentegen bevatten bladeren die met het niet-pathogene isolaat BC-6 waren geinfecteerd slechts lage concentraties phaseolline.De hoeveelheden phaseolline en 6a-hydroxyphaseolline, omgerekend per lesie, namen snel toe in zich uitbreidende lesies van isolaat BC-1, terwijl de toenamesnelheid van deze stoffen afnam in lesies van isolaat BC-5, die beperkt van omvang bleven (Fig. 4).De resultaten geven geen duidelijke aanwijzingen dat phaseolline de belangrijkste remmende factor zou zijn bij het beperkt blijven van lesies.     

谷胱苷肽及其类似物诱导大豆活性氧和植保素的积累

 谷胱苷肽(Glutathione, GSH)是一种重要的抗氧化剂, 但它能诱导一些豆科等植物的防卫反应。本文采用大豆悬浮细胞体系分析了GSH及其衍生物诱导大豆活性氧和大豆植保素大豆素(glyceollin)的积累。GSH诱导大豆素和黄苷元的积累, 并有一定的剂量关系。N-乙酰半胱氨酸、巯基乙醇、二硫苏糖醇没有激发子活性, 但氧化型谷胱苷肽能激发大豆的防卫反应。在巯基被修饰的化合物中, S-对叠氮苯甲基谷胱苷肽和S-对氯苯甲基谷胱苷肽能够诱导大豆素和H₂O₂的产生, S-己基谷胱苷肽也能诱导一定量的H₂O₂积累。水杨酸和蛋白酶抑制剂DFP能增强芫菁素或者酵母细胞壁激发子诱导的大豆素积累, 但对GSH没有相似的增强效果。这些结果表明GSH中的巯基并不是激发所必需的, 大疏水基团能弥补巯基的作用。另外, GSH诱导大豆素积累信号传导途径可能与芫菁素的不同。     

In vitro Effect of the Phytoalexin Glyceollin I on Polygalacturonase and Oxalic Acid Production ofSclerotinia sclerotiorum

The polygalacturonase (PG) activity in culture filtrates fromSclerotinia sclerotiorum is reduced when glyceollin I, the major soybean phytoalexin, is present in the culture medium. When the enzyme activities in the culture filtrates are expressed per unit of fungal growth, PG activity decreases with increasing concentration of glyceollin I in the culture medium. The phytoalexin does not influence the isoenzyme pattern. This suggests that glyceollin I may inhibit quantitative but not qualitative enzyme production. Only the highest glyceollin I concentration tested inhibits oxalic acid production. The inhibitory effect on mycelial growth is confirmed. The data suggest a further hypothesis about the role of phytoalexin during pathogenesis.     

Accumulation of isoflavonoid phytoalexins in leaves of Arachis hypogaea differing in reaction to rust (Puccinia arachidis) and early leafspot (Cercospora arachidicola)

Groundnut leaves, naturally infected in the field with Cercospora arachidicola or artificially infected in the greenhouse with C. arachidicola or Puccinia arachidis , accumulated isoflavonoid phytoalexins. Major compounds included medicarpin, formononetin, demethylmedicarpin, three isoflavanones and the isoflavone, daidzein. In both greenhouse and field experiments cv. Egret was less susceptible than cv. P84/5/244 to both pathogens, and accumulated phytoalexin (medicarpin) concentrations which were up to three times greater. Higher concentrations and greater variation of phytoalexins were detected when both cultivars were infected with C. arachidicola than when they were infected with P. arachidis.     

非寄主和寄主种子分泌物对大豆疫霉活性的影响

为探寻非寄主和寄主种子分泌物中抗病信号分子,通过显微观察,采用菌丝生长速率法和离体接种法对不同种子分泌物处理后大豆疫霉Phytophthora sojae的游动孢子数、孢子囊数、游动孢子释放后残留的空囊数、成囊和未成囊的游动孢子数、萌发和未萌发的胞囊数、菌落直径、卵孢子数进行测量,并计算抑制率,明确非寄主菜豆和寄主大豆抗病品种、感病品种种子分泌物对大豆疫霉游动孢子趋化性、生长发育和侵袭力的影响。结果显示,非寄主菜豆种子分泌物不吸引大豆疫霉游动孢子,显著抑制大豆疫霉孢子囊形成、胞囊萌发和卵孢子产生,抑制率依次为97.3%、73.0%和17.5%,然后溶解胞囊,最终导致游动孢子对下胚轴侵袭力降低,抑制率为67.1%。寄主大豆种子分泌物能吸引大豆疫霉游动孢子,感病品种种子分泌物吸引力高于抗病品种。感病品种种子分泌物对大豆疫霉生长发育无显著影响,但促进大豆疫霉游动孢子侵袭力;抗病品种种子分泌物显著抑制大豆疫霉孢子囊形成、胞囊萌发和卵孢子产生,抑制率依次为86.6%、34.3%和12.8%,然后溶解胞囊,但作用强度小于非寄主菜豆种子分泌物,最终导致游动孢子对下胚轴的侵袭力降低,抑制率为24.2%。表明非寄主菜豆和寄主大豆抗病品种的种子分泌物对大豆疫霉有抑菌活性,大豆疫霉的非寄主和寄主抗病性与种子分泌物有关。     

Cultivar Resistance to Anthracnose Disease of Cowpea (Vigna unguiculata (L.) Walp.) caused by Colletotrichum destructivum O'Gara

The infection process of Colletotrichum destructivum, a hemibiotrophic anthracnose fungus, was studied by light microscopy in two cowpea (Vigna unguiculata) cultivars which differ in disease reaction type. Large, multilobed, intracellular infection vesicles, followed by necrotrophic, radiating, secondary hyphae were produced in tissues of the susceptible cv. IT82E-60. In the resistant cv. TVx3236, both the production of appressoria and their melanisation were impaired, resulting in reduced penetration. Where penetration occurred, the initially-infected epidermal cells underwent a hypersensitive response, restricting the growth of multilobed vesicles and thereby blocking the destructive necrotrophic phase of disease development. The phytoalexins kievitone and phaseollidin accumulated earlier and more rapidly in stem tissues of the resistant cultivar, associated with the appearance of delimited, necrotic spots on inoculated surfaces. In contrast, delayed and slower accumulation of these compounds occurred in the compatible interaction, together with the development of typical spreading, water-soaked, anthracnose lesions.     

Chitosan oligomers and copper sulfate induce grapevine defense reactions and resistance to gray mold and downy mildew

ABSTRACT Chitosan (CHN), a deacetylated derivative of chitin, was shown to be efficient in promoting plant defense reactions. CHN oligomers of different molecular weight (MW) and degree of acetylation (DA) triggered an accumulation of phytoalexins, trans- and cis-resveratrol and their derivatives epsilon-viniferin and piceid, in grapevine leaves. Highest phytoalexin production was achieved within 48 h of incubation with CHN at 200 mug/ml with an MW of 1,500 and a DA of 20% (CHN1.5/20), while oligomers with greater MW were less efficient, indicating that a specific MW threshold could be required for phytoalexin response. Treatment of grapevine leaves by highly active CHN1.5/20 also led to marked induction of chitinase and beta-1,3-glucanase activities. CHN1.5/20 applied together with copper sulfate (CuSO(4)) strongly induced phytoalexin accumulation. CuSO(4) alone, especially at low concentrations also elicited a substantial production of phytoalexins in grapevine leaves. Evidence is also provided that CHN1.5/20 significantly reduced the infection of grapevine leaves by Botrytis cinerea and Plasmopara viticola, and in combination with CuSO(4) conferred protection against both pathogens.