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.     

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。     

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.     

Infection Process and Pathogenic Mechanism of <Emphasis Type="Italic">Phomopsis asparagi</Emphasis>, the Asparagus Stem Blight Pathogen

The infection process was explored by light and electron microscopy techniques, as well as bioassays assessing phytotoxins and cell wall-degrading enzymes. We found that germ tubes of asparagus stem blight fungus were produced at 0-24 h after culture on dextrose agarose medium, and mycelia were formed at 24-48 h. Then, mycelia grew and spread continuously, making incursions into host tissues after 4 days. The conidial fructification began to form after 8 days. Subsequently, pycnidia were produced after about 12 days, with conidia released after about 16 days. Interestingly, through culture, extraction and bioassay of phytotoxin culture filtrates, no overt damage of asparagus tissues was found. As for cell wall-degrading enzymes, PG showed the highest activity, followed by Cx and PMG; PGTE and PMTE displayed the lowest activities. Finally, we demonstrated that permeable reducing sugars and relative electric conductivity in the culture increased after incubation in cell wall-degrading enzyme solutions, in an enzyme concentration dependent manner.     

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.     

Pectolytic enzymes produced in vitro and during colonization of melon tissues by Didymella bryoniae

Pectolytic enzymes produced by Didymella bryoniae in a liquid medium containing pectin as sole carbon source and in inoculated etiolated hypocotyls of 10 melon cultivars, as well as those constitutively expressed in spores, were studied by isoelectric focusing, quantitatively and qualitatively. Five constitutive pectin lyase (PNL) isoenzymes differing in isoelectric joint (pI), one acidic (pI 3.9) and four basic (pI 8.4, 8.9, 9.3, 9.9) were expressed in extracts from spores. The same PNL isoenzyme pattern was detected in culture filtrates and in infected tissues of all the melon cultivars tested. Polygalacturonase (PG) activity, represented by a single inducible acidic band (pI 4.6) was detected only in culture filtrates. A single constitutive basic pectin methylesterase (PME) isoenzyme (pI > 10.0) was also found in spores, culture filtrates and inoculated melon tissues. All cultivars were susceptible at the seedling growth stage, but with differences in disease severity; cultivars Amarillo Oro and Juane Canari were, respectively, the least and most susceptible. Pectin lyase activity was highly correlated with disease severity. In rotted tissues and culture filtrates, an increase in pH to values over 7.0 was recorded, values optimal for PNL activity. In this plant–pathogen interaction, PNL activity represents the principal pectolytic component and these isoenzymes were associated with the onset of disease, disease severity and an increase in pH of infected tissue.     

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.     

软腐欧氏杆菌在离体条件下产生胞外酶的比较研究

 3种软腐细菌在21,26和31℃下及6种培养液中的生长量和产生胞外酶的能力各不相同。Ecc在26℃生长最好,Eca为21℃,Ech在三种温度下的生长量差别不大。与其它2种细菌比较,Ech的生长及产生果胶裂解酶(PL)的能力总是最强的。随着温度的升高,各种细菌的PL活性都下降。果胶水解酶(PG)变化不大,但Ech在高温下产PG有减少趋势,且总没有另二种细菌的活性强。Eca以26℃时产PG最适宜。Ech在较高温度下蛋白酶活性大大下降,而Ecc,变化不大,Eca的蛋白酶活性则随温度升高而加强。培养液的无细胞滤液对马铃薯块茎组织的浸离力与其中PL的活性有密切关系,但没有或只有很低果胶酶活性,而蛋白酶活性正常的这种滤液也能使小薯碟浸离。Eca在31℃培养后果胶酶活性很低,蛋白酶活性很高,其无细胞滤液对马铃薯组织也表现较强浸离力。     

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.     

Modulation of host pH during the wheat–Fusarium culmorum interaction and its influence on the production and activity of pectolytic enzymes

The effect on ambient pH of Fusarium culmorum during its growth on mineral medium and in apoplastic fluids from infected wheat seedlings, and the effect on the production and activity of the enzymes pectin lyase (PNL) and polygalacturonase (PG), were investigated. Fungal development on a weakly buffered mineral medium in the pH range 5·0–8·0, with pectin as the sole carbon source, led to pronounced alkalinization, reaching values above 8·0. The increase in ambient pH was accompanied by enhancement of total PNL activity. Pectin lyase secretion was detected at pH 5·0 as a single isoenzyme. An additional isoenzyme was apparent during the increase in medium pH. Polygalacturonase was detected as a single isoenzyme only during early growth on medium buffered at pH 5·0. At this stage, the initial medium pH of 5·0, corresponding to the optimum pH for PG activity, appeared to be the most suitable for the activation of early production of this enzyme. During growth in acidified yeast extract medium the fungus secreted ammonia and increased medium pH. Similarly, in apoplastic fluids from inoculated seedlings the concomitant ammonia accumulation and rise in pH were recorded. This trend was accompanied by an increase in PNL, which could therefore function at close to its optimal pH. The results suggest that during infection of wheat seedlings by F. culmorum , pH modulation can lead to PNL production and activity, thus promoting colonization of host tissue.     

Effect of culture filtrates of seventeen fungicolous fungi on sporulation of cucumber powdery mildew

Culture filtrates of 17 different fungal species thriving upon other fungi were tested for their ability to reduce sporulation of cucumber powdery mildew,Sphaerotheca fuliginea.All culture filtrates reduced the number of healthy conidiophores. However, the differences in activity between the various treatments were not as conspicuous as after application of spore suspensions. The best results were obtained with culture filtrates ofCalcarisporium arbuscula. These reduced the number of healthy conidiophores to ca. 2% of the unsprayed control plants.     

The effect of phenolic compounds on growth,polygalacturonase production and activity of Fusarium oxysporum f. vasinfectum

The effect of catechol, phloroglucinol, gallic acid, phloretin and catechin on the growth ofFusarium oxysporum f.vasinfectum, on the ability of the fungus to produce polygalacturonase (PG) and on the activity of this enzyme has been investigated.Catechol and particularly phloretin inhibited fungal growth and PG formation at a concentration of 0.006 M. Phloroglucinol, although not especially inhibitory to growth, effectively inhibited PG formation. Polygalacturonase activity was slightly inhibited by catechol, phloroglucinol, and gallic acid in all concentrations tested.In all other cases no or only slight inhibitory effects were found.     

大豆毛口壳叶斑病菌产毒素的研究

 针对目前生产上新发现的大豆毛口壳叶斑病菌（Aristastoma sp.）产生的毒素进行了研究。该病菌的Czapek培养液经乙酸乙酯萃取可获得毒素粗提物。通过对该菌产毒条件的研究表明,pH 7的改良Richard培养液产生毒素生物活性最强,培养温度25℃、黑暗条件下持续振荡培养20 d最有利于该病菌毒素的产生。毒素活性测定表明,毒素对大豆叶片产生与病菌作用相同的褪绿症状,对种子胚根生长有抑制作用,对幼苗有致萎作用,说明毒素是大豆毛口壳叶斑病病菌侵染过程中的主要致病因子。     

Production of Indole-3-acetic Acid and Tryptophol by Pythium ultimum and Pythium Group F: Possible Role in Pathogenesis

Pythium group F is a minor pathogen which induces symptomless infections that occur frequently and results in yield losses in tomato soilless cultures. To elucidate the mode of action of this microorganism, the influence of culture filtrates of Pythium group F on tomato growth was investigated and compared to that of the pathogen Pythium ultimum. Depending on metabolite production by the fungus, marked differences were observed in plant response. Pythium group F crude culture filtrates or low molecular weight fractions (< 500) caused swelling behind the root tip and reduced root growth; the cohesion and adherence of cells within the cortical area were also affected. These symptoms were similar to those observed on plants treated with indole-3-acetic acid. By contrast, P. ultimum filtrates caused a marked distortion of cell shape accompanied with folding of host cell walls, particularly in the cortical area. These symptoms were characteristic of the activity of toxic compound(s) on host cells. Chemical analysis of the filtrates demonstrated that indole-3-acetic acid and tryptophol were produced by Pythium group F and P. ultimum. However, Pythium group F took up and metabolized more indole-3-acetic acid precursors, especially tryptophan, a key amino acid in the pathways leading to indole-3-acetic acid synthesis. The fact that Pythium group F and P. ultimum transformed tryptamine and indole-3-acetaldehyde into indole-3-acetic acid and tryptophol confirms the existence of a tryptamine pathway within both fungi. These results support the hypothesis that auxins facilitate Pythium group F infections. On the other hand, toxin(s) and hydrolytic enzymes are likely involved in P. ultimum pathogenesis.     

枯萎病菌毒素培养滤液对唐菖蒲幼苗毒性的初步研究

 对唐菖蒲枯萎病菌进行了产毒培养基的筛选,研究毒素培养滤液对胚根生长的抑制作用、对幼苗根系活力的影响及对幼苗的致萎作用。结果表明：唐菖蒲枯萎病菌在Czapek培养基中培养15 d所产生的培养滤液对唐菖蒲胚根的抑制作用最强,胚根生长抑制率高达99.79%;而菌丝干重在PD培养基中培养13 d达到最高值1 242.27 mg;毒素培养滤液经121℃灭菌20 min后对唐菖蒲胚根仍有很强的抑制作用,表现出较高的热稳定性;与对照相比,毒素培养滤液能够显著降低唐菖蒲幼苗根系活力,随处理时间的延长和处理浓度的增加,萎蔫级数逐渐增加。说明枯萎病菌毒素培养滤液是唐菖蒲枯萎病的致病因子,可以利用其筛选唐菖蒲抗枯萎病品种。     

Production of the phytotoxins, solanapyrones A and C and cytochalasin D among nine isolates of Ascochyta rabiei

Nine isolates of Ascochyta rabiei from Pakistan were grown in still culture on Czapek Dox liquid medium supplemented with aqueous extracts of the seed of one of two cultivars, a desi (6153) or a kabuli (Cypressa). Toxicity of the culture filtrates from both media was assayed with cells isolated from the leaves of both cultivars and concentrations of the phytotoxins. solanapyrone A and solanapyrone C, in the filtrates were determined by high-performance liquid chromatography (HPLC). For eight of the fungal isolates, toxicity and solanapyrone concentration were correlated, r varying from 0·383 to 0·859 according to medium and assay cultivar, but culture filtrates of another isolate, which was the most active by more than an order of magnitude, contained cytochalasin D but neither of the solanapyrones.     

Toxic culture filtrates produced byCalonectria ilicicola, causal agent of red crown rot of soybean

Eleven soybean cultivars with different levels of susceptibility to virulent isolate SG915 ofCalonectria ilicicola were examined for reaction to metabolites produced by the isolate. When the culture filtrate from isolate SG915 was applied to trifoliates from 11 cultivars, cvs. ‘Cajun’ and ‘Asgrow 7986’ exhibited reduced wilting severity. However, there was no correlation between sensitivity to culture filtrate and susceptibility to the fungal isolate. Wilting severity on cv. ‘Riverside 699’ was greatest when trifoliates were treated with culture filtrates from isolates SG915 (highly virulent) and C31 (less virulent). The dilution end-point for culture filtrates of virulent isolate SG915 was determined to be 1:8. Nonautoclaved culture filtrates caused complete wilt of soybean trifoliates after 36 h, but autoclaved culture filtrates demonstrated a reduced ability to wilt leaves. Electrolyte leakage from treated leaf tissues increased over time regardless of the concentrations of culture filtrate tested. The greatest electrolyte losses were observed during the initial 30 min incubation of leaf tissues. The highest concentration of culture filtrate (50%, v/v) induced more electrolyte loss than the low concentration (10%, v/v) or control. These results suggest that toxic metabolites ofC. ilicicola may be involved in disease development with leaf symptom expression.     

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.     

Polygalacturonase S31PG1 from <Emphasis Type="Italic">Geotrichum candidum</Emphasis> citrus race S31 expressed in <Emphasis Type="Italic">Schizosaccharomyces pombe</Emphasis> versus S31PG2 regarding soft rot on lemon fruit

Gene S31pg1, which encodes a polygalacturonase (PG), was previously isolated from citrus race S31 of Geotrichum candidum, the causal agent of citrus sour rot. We have now isolated and sequenced an additional PG gene, S31pg2, with 95% identity to S31pg1 in the mature proteins. To evaluate the contribution of the two PG genes in the development of citrus sour rot, each gene was expressed in the fission yeast Schizosaccharomyces pombe. Both genes conferred PG activity to the yeast. Crude enzyme solutions containing S31PG1 severely degraded the albedo tissue of lemon peel, but those containing S31PG2 did not. Concentrated crude S31PG1 solutions also caused soft rot on lemon fruit, indicating that not S31PG2 but S31PG1 is an important pathogenicity factor in citrus sour rot. Next, the protopectinase (PP) activity of each PG was measured. Although S31PG1 and S31PG2 are highly homologous, S31PG1 had high PP activity, whereas S31PG2 had much lower activity. PG from G. candidum noncitrus race S63 (nonpathogenic to citrus fruits) was also assayed but did not have any PP activity at all. These results suggest that the different PP activities of the PGs are a key to the pathogenicity of G. candidum to lemon fruit.     

草莓灰霉病菌的培养、毒素提取及生物测定

 草莓灰霉病菌(Botrytis cinerea Persoon)在25℃、pH3~4的Peberdy培养基中生长良好,并可产生引起草莓愈伤组织细胞死亡的毒素。产生毒素的高峰期为静置培养20天。灰霉菌发酵液中的毒素可用氯仿进行提取,经提取后的毒素液在269nm处有吸收峰。本研究建立了利用草莓愈伤组织细胞的荧光活性测定毒力的方法。