Sources of inoculum and reappearance of spot blotch of wheat in rice–wheat cropping

A study was undertaken to examine the main source of inoculum of Bipolaris sorokiniana responsible for its reappearance in rice–wheat cropping regions of eastern India. Soil samples were collected at monthly intervals during April–October in the years 2000 and 2001 from fields having rice–wheat cropping. Bipolaris sorokiniana conidia were isolated and their viability was found to decline sharply with the onset of flooding in the month of August. In contrast to 82% in April, viability was 4% and <1% in August and September, respectively. Viable conidia were multiplied in the laboratory and inoculated on to susceptible cv. Sonalika under controlled conditions for test of pathogenicity. Appearance of symptoms typical to spot blotch were recorded after 7 days. Twenty-two different species (weeds and grasses) normally found to be associated with rice–wheat fields were tested for the presence of B. sorokiniana to evaluate their possible role as alternative hosts. Only three species, i.e. Setaria glauca, Echinochloa colonum and Pennisetum typhoids, were found to naturally harbour B. sorokiniana. Isolates from these hosts were tested for pathogenicity and also for their possible spread to wheat. When reisolated from these hosts, the pathogen did not infect wheat. Seeds of 25 different wheat genotypes were tested for B. sorokiniana infection. All genotypes were infected and the incidence of infection varied from 26% to 86%. Five isolates of wheat and one isolate from each of the three species (S. glauca, E. colonum and P. typhoids) were subjected to RAPD analysis. Two broad clusters were formed, suggesting that the wheat isolates were different from the isolates originating from other hosts. The results indicate that seeds are the most important source of inoculum for the reappearance of spot blotch of wheat in rice-wheat cropping systems in eastern India.     

呼和浩特市苜蓿根腐病病原菌鉴定及致病性测定

为确定引起呼和浩特市苜蓿根腐病的病原菌种类,采用常规组织分离法对采集的疑似苜蓿根腐病病样进行病原菌分离与培养,利用形态学观察结合分子生物学方法对分离物代表菌株进行鉴定,并采用土壤接种法对代表菌株的致病性进行测定。结果表明,共分离获得6类形态学特征不同的分离物,各随机选择1株代表菌株进行鉴定,结合分子生物学鉴定结果确定呼和浩特市苜蓿根腐病病原菌有6种,分别是麦根腐平脐蠕孢菌Bipolaris sorokiniana、立枯丝核菌Rhizoctonia solani、木贼镰刀菌Fusarium equiseti、变红镰刀菌F. incarnatum、锐顶镰刀菌F. acuminatum和织球壳枯萎菌Plectosphaerella cucumerina,分别分离到1、7、14、26、7和14株菌株,占总分离菌株数的1.45%、10.14%、20.29%、37.68%、10.14%和20.29%。其中,立枯丝核菌的致病力最强,接种苜蓿幼苗发病的病情指数达82.67,其次为木贼镰刀菌、变红镰刀菌、锐顶镰刀菌、麦根腐平脐蠕孢菌和织球壳枯萎菌,病情指数分别为72.67、62.67、58.67、52....     

Are field populations of arbuscular mycorrhizal fungi able to suppress the transmission of seed-borne <Emphasis Type="Italic">Bipolaris sorokiniana</Emphasis> to aerial plant parts?

The development of seed-borne Bipolaris sorokiniana in barley in the presence of arbuscular mycorrhizal fungi was studied. To exploit natural variation in their ability to control disease development, arbuscular mycorrhizal fungi from various Swedish arable soils were multiplied in trap cultures using a mixture of plant species. Six out of eight trap culture soil inocula were able to reduce transmission of B. sorokiniana from seeds to stem bases when grown together with infected barley seed. Based on this result two soil inocula, of different origin, from semi-natural grassland and barley respectively, were chosen for further greenhouse studies. Both soil inocula gave significant reductions in pathogen transmission from seeds to seedlings compared to the untreated control. In addition, treatment with spore inocula, collected from the different trap culture soils, showed disease suppression. Treatment with spores from the pure culture Glomus intraradices gave significant reduction in leaf lesion development. A treatment with the commercial inoculum Vaminoc^® was included and gave some suppression of the pathogen. In conclusion this study has shown that AM soil inocula from trap cultures suppressed the transmission of seed-borne B. sorokiniana in the aerial parts of barley plants.     

The use of ergosterol in the pathogenic fungusBipolaris sorokiniana for resistance rating of barley cultivars

Ergosterol content in the plant pathogenic fungusBipolaris sorokiniana was determined in different matrices including mycelium, spores, culture filtrate and infected barley leaves. Ergosterol was extracted with methanol, hydrolysed with KOH and quantified by reverse phase high performance liquid chromatography (HPLC). Our procedure was used to study how the ergosterol concentration ofB. sorokiniana varied due to fungal age and nutrient availability when growing in liquid medium. It was found that the ergosterol content decreased with fungal age. The decrease was not due to leakage. It was also found that a change to a less nutrient-rich medium caused an increase in ergosterol content whereas a change to a rich medium led to a decrease. The procedure was also used for quantification of fungal infections in complex matrices (e.g. leaves). The development of fungal infection in barley leaves was followed during 10 days. Visual grading of leaf spots was also compared to ergosterol content in three varieties of barley. The ergosterol content in the leaves increased exponentially until day 7, and the grading of the leaf spots was correlated to the ergosterol content. Our results show that, despite a great variation, ergosterol may be used as a biomarker to detect and quantify fungal infections in a given matrix.     

Genetic Analysis of the Role of Trichothecene and Fumonisin Mycotoxins in the Virulence of Fusarium

The phytotoxicity of the Fusarium trichothecene and fumonisin mycotoxins has led to speculation that both toxins are involved in plant pathogenesis. This subject has been addressed by examining virulence of trichothecene and fumonisin-nonproducing mutants of Fusarium in field tests. Mutants were generated by transformation-mediated disruption of genes encoding enzymes that catalyze early steps in the biosynthesis of each toxin. Two economically important species of Fusarium were selected for these studies: the trichothecene-producing species Fusarium graminearum, which causes wheat head blight and maize ear rot, and the fumonisin-producing species F. verticillioides, which causes maize ear rot. Trichothecene-non-producing mutants of F. graminearum caused less disease than the wild-type strain from which they were derived on both wheat and maize, although differences in virulence on maize were not observed under hot and dry environmental conditions. Genetic analyses of the mutants demonstrated that the reduced virulence on wheat was caused by the loss of trichothecene production rather than by a non-target mutation induced by the gene disruption procedure. Although the analyses of virulence of fumonisin-non-producing mutants of F. verticillioides are not complete, to date, the mutants have been as virulent on maize ears as their wild-type progenitor strains. The finding that trichothecene production contributes to the virulence of F. graminearum suggests that it may be possible to generate plants that are resistant to this fungus by increasing their resistance to trichothecenes. As a result, several researchers are trying to identify trichothecene resistance genes and transfer them to crop species.     

拮抗性链霉菌对大丽轮枝菌微菌核形成与萌发的影响

为探索拮抗性链霉菌对棉花黄萎病病原大丽轮枝菌Verticillium dahliae Kleb.的抑菌机制,采用菌丝生长速率法、微菌核萌发法研究了6株拮抗链霉菌无菌发酵滤液对大丽轮枝菌生长、微菌核形成与萌发的影响。链霉菌无菌发酵滤液对大丽轮枝菌菌落生长、菌核形成和微菌核萌发有明显抑制作用。其中菌株B49的抑菌效果最好,5倍稀释发酵液培养14天时对菌落生长的抑菌率达69.7%;菌株B49、D184和Act12的5倍稀释发酵液对微菌核形成的抑制率达100%;将经B49、D184和Act12发酵液处理后丧失形成微菌核能力的大丽轮枝菌菌株转接至不含发酵液的PDA培养基,连续传代至第5代,其仍然不能恢复形成微菌核的能力;微菌核在含有菌株D184 5倍稀释发酵液的培养基上培养168 h时,萌发率仅为38.3%。     

<Emphasis Type="Italic">Leptosphaeria maculans</Emphasis>, a fungal pathogen of <Emphasis Type="Italic">Brassica napus</Emphasis>, secretes a subtilisin-like serine protease

Leptosphaeria maculans,a fungal pathogen of Brassica napus, secretes large amounts of a 28kDa protein (SP2) in liquid culture. This protein shows high sequence similarity to secreted serine proteases from other ascomycetes and is the major component of culture filtrate with protease activity, as analysed on casein zymogels. The sp2 gene is expressed during infection of B.napuscotyledons when L. maculans hyphae are growing between mesophyll cells, as well as at later stages when the fungus invades the vascular tissue.     

Production of phytotoxic metabolites by five species of Botryosphaeriaceae causing decline on grapevines, with special interest in the species Neofusicoccum luteum and N. parvum

In recent years an increasing number of species of Botryosphaeriaceae have been associated with grapevine decline worldwide. Five species isolated from declining grapevines in Spain (Botryosphaeria dothidea, Diplodia seriata, Dothiorella viticola, Neofusicoccum luteum and N. parvum) were checked for toxin production in liquid cultures. Cultural conditions for all fungi were adjusted to obtain optimal production of phytotoxic culture filtrates, by growing the fungi in steady liquid cultures of Czapek–Dox broth for different time intervals. Phytotoxicity of D. seriata and N. parvum reached a maximum after 14 days while the remaining species showed the highest phytotoxicity levels after 21 days in culture. All fungi produced hydrophilic high-molecular weight compounds with phytotoxic properties. In addition, N. luteum and N. parvum produced lipophilic low-molecular weight phytotoxins, not detected consistently among the remaining species. This led to a more exhaustive study on the phytotoxicity of N. luteum and N. parvum. Culture filtrates and corresponding extracts of both species were consistently highly phytotoxic in different assays. The gas-chromatography analysis of the acetylated O-methyl glycosides of the phytotoxic exopolysaccharides produced by N. parvum showed these substances to be composed mainly of glucose, mannose and galactose. Results suggest that phytotoxic metabolites could be involved in the virulence of both species in planta.     

Toxigenic Fusarium Species of Liseola Section in Pre-harvest Maize Ear Rot, and Associated Mycotoxins in Slovakia

The occurrence of Fusarium species of Liseola section and related toxins was investigated for two years (1996 and 1998) on maize ear rot samples collected in the most important areas for maize growing in Slovakia. The species most frequently isolated was F. verticillioides, followed by F. proliferatum in 1996 and F. subglutinans in 1998. Most of the strains belonged to mating populations A, D, and E of the teleomorph Gibberella fujikuroi. Fusarium graminearum was also frequently recovered in both the years of investigations. Toxin analysis of maize ears showed that most of the samples (21 out of 22) were contaminated with at least one toxin. In particular, the concentration of fumonisin B₁, and fumonisin ₂ was up to 26.9 and 5.1gg^-1, respectively in 1996, and up to 12.1 and 6.3gg^-1, respectively in 1998. Beauvericin was detected only in one sample in 1996. Seven samples in 1996 were contaminated by fusaproliferin up to 8.2gg^-1, but just traces of the toxin were found in one sample in 1998. All 29 strains of F. verticillioides, two of three strains of F. proliferatum and none of eight F. subglutinans strains isolated from samples produced fumonisin B₁ in culture on whole maize kernels (0.1–5646 and 940–1200ugg^-1, respectively). Two strains of F. subglutinans and two of F. proliferatum produced beauvericin (up to 65 and 70gg^-1, respectively). Ten strains of F. verticillioides produced beauvericin: 9 strains produced a low amount (up to 3gg^-1), while only one of them produced a high level of toxin (375gg^-1). Fusaproliferin was produced by two F. proliferatum strains (220 and 370gg^-1), by seven F. subglutinans (20–1335gg^-1) and by three F. verticillioides (10–35gg^-1). This is the first report on fusaproliferin production by F. verticillioides, although at low level.     

海洋生境贝莱斯芽孢杆菌TCS001的鉴定及抑真菌活性

为研究海洋生境芽孢杆菌TCS001的分类地位和抑菌活性,通过形态和生理生化特征观察,并结合gyrA序列同源性分析对菌株进行了鉴定;通过平板对峙培养法测定了菌株TCS001对多种植物病原真菌的抑菌谱;采用菌丝生长速率法和凹玻片法,测定了不同浓度TCS001菌株发酵滤液对靶标菌黄瓜灰霉病菌Botrytis cinerea菌丝生长和孢子萌发的影响。结果显示:该菌株为贝莱斯芽孢杆菌Bacillus velezensis,其对6种供试病原菌均有一定的抑制效果,其中对黄瓜灰霉病菌的抑制率最高,达87.66%。不同稀释倍数下,TCS001发酵滤液对黄瓜灰霉病菌均有一定的抑制作用,其中,稀释5倍时对菌丝生长和孢子萌发的抑制率最高,分别为96.24%和98.05%,稀释20倍时抑制率也均达90%以上。形态学观察发现,TCS001发酵滤液可导致黄瓜灰霉病菌孢子萌发芽管中间或顶端膨大畸形。研究表明,海洋生境贝莱斯芽孢杆菌TCS001极具开发为微生物农药的潜能。     

Fungi on roots and stem bases of asparagus in the Netherlands: species and pathogenicity

A survey was made to identify the most important soilborne fungal pathogens of asparagus crops in the Netherlands. Ten plants were selected from each of five fields with a young (1–4 y) first planting, five fields with an old (6–13 y) first planting and five fields with a young replanting. The analysis included fungi present in the stem base and the roots of plants with symptoms of foot and root rot or showing growth decline without specific disease symptoms. Isolates of each species were tested for pathogenicity to asparagus on aseptically grown plantlets on Knop's agar. Symptoms were caused byFusarium oxysporum, F. culmorum, Botrytis cinerea, Penicillium verrucosum var.cyclopium, Cylindrocarpon didymum, Phialophora malorum, Phoma terrestris andAcremonium strictum. F. oxysporum was by far the most common species and was isolated from 80% of the plants. Not all of its isolates were pathogenic to asparagus. Symptoms were caused by 67%, 78% and 93% of the isolates obtained from young first plantings, old first plantings and replantings, respectively.F. culmorum was isolated from 31% of the plants. Two other notorious pathogens of asparagus,F. moniliforme andF. proliferatum, did not occur in our samples.Species causing symptoms in the vitro test that were found on more than 5% of the plants were additionally tested for their pathogenicity in pot experiments.F. oxysporum f.sp.asparagi caused severe foot and root rot, significantly reduced root weights and killed most of the plants.F. culmorum caused lesions on the stem base often resulting in death of the plant.P. terrestris, a fungus only once reported as a pathogen of asparagus, caused an extensive root rot, mainly of secondary roots that became reddish. The fungus was isolated in only a few samples and is not to be regarded as an important pathogen in Dutch asparagus crops.P. malorum caused many small brown lesions on the stem base and incidentally also on the upper part of small main roots. This is the first report of its pathogenicity to asparagus. The fungus is one of the organisms inciting spear rust and it reduced crop quality rather than crop yield.P. verrucosum var.cyclopium andC. didymum did not cause symptoms in pot experiments.Because of its predominance on plants with foot and root rot and its high virulence,F. oxysporum f.sp.asparagi was considered to be the main soilborne pathogen of asparagus in the Netherlands.     

Pathogenic variation and molecular characterization of <Emphasis Type="Italic">Fusarium</Emphasis> species isolated from wilted Welsh onion in Japan

Thirty-two isolates of Fusarium species were obtained from wilted Welsh onion (Allium fistulosum) grown on nine farms from six regions in Japan and identified as F. oxysporum (18 isolates), F. verticillioides (7 isolates), and F. solani (7 isolates). The pathogenicity of 32 isolates was tested on five commercial cultivars of Welsh onion and two cultivars of bulb onion in a seedling assay in a greenhouse. The Fusarium isolates varied in the degree of disease severity on the cultivars. Five F. oxysporum isolates (08, 15, 17, 22, and 30) had a higher virulence on the cultivars than the other isolates. The host range of these five isolates was limited to Allium species. Molecular characterization of Fusarium isolates was performed using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of the internal transcribed spacer (ITS) regions of ribosomal DNA. The 32 isolates were grouped into eight types (four types for F. oxysporum, one for F. verticillioides, and three for F. solani). Restriction patterns of the ITS region were not related to pathogenicity. However, the haplotypes obtained with five enzymes (RsaI, HinfI, HaeIII, ScrFI, and MspI) and the phylogenetic analysis permitted the discernment of the three Fusarium species. The PCR-RFLP analysis should provide a rapid, simple method for differentiating Fusaruim species isolated from wilted Welsh onion in Japan.     

中国小麦条锈病菌CYR32和CYR33的毒性及基因型多样性

为明确我国小麦条锈病菌当前主要流行生理小种CYR32和CYR33的毒性及基因型特征,从全国11个省(区)随机选取29个CYR32菌株和39个CYR33菌株,利用近等基因系及辅助鉴别寄主对其进行毒性鉴定,利用SSR分子标记技术对其进行基因型分析,并对其进行聚类分析。结果显示,CYR32和CYR33菌系各有17种毒性表型,而且在抗病基因Yr2、Yr17、Yr27、Yr32、Yr43、Yr Sp、Yr Exp2、Yr28、Yr V23上都发生了毒性分化,CYR32和CYR33菌系的多样性指数分别为0.089、0.097;CYR32和CYR33菌系的香农信息指数均值分别为0.44和0.45;当相似性系数为0.93时,CYR32和CYR33菌系分别被聚为5个和8个毒性类群;当相似性系数为0.84时,CYR32和CYR33菌系分别被聚为10个和16个基因型类群。表明在中国鉴别寄主上具有相同毒性谱特征的CYR32和CYR33菌系在近等基因和SSR分子标记中发生了不同程度的毒性和基因型分化。     

Differentiation of Phoma foveata from P. exigua using a RAPD Generated PCR-RFLP Marker

Phoma foveata and P. exigua variety exigua both infect potatoes and are morphologically very similar. P. foveata produces a pigment which allows differentiation from P. exigua in culture. Discrimination of the two species based on the production of a secondary metabolite, which is dependent on the growth conditions, is not reliable. Therefore, there is a need to develop nucleic acid based identification markers. A 482bp random amplified polymorphic DNA (RAPD) fragment from P. foveata was isolated and sequenced. Polymerase chain reaction (PCR) primers, developed from the sequence of the RAPD product, amplified a 474bp fragment for P. foveata and P. exigua varieties exigua, diversispora, inoxydibilis and sambuci-nigrae. The similarity of the PCR fragments was demonstrated by sequence analysis and by using the restriction enzymes DdeI and DpnII. P. foveata was distinguished from the four varieties of P. exigua on the basis of the RFLP patterns of the PCR fragment. Ten isolates of P. foveata and nine of P. exigua var. exigua from different geographic locations were tested and all isolates but one showed the restriction digest pattern of the PCR fragment (PCR-RFLP) specific to each species. One isolate of P. foveata demonstrated a PCR-RFLP pattern similar to P. exigua var. exigua leading to the conclusion that the isolate had been previously misidentified as a strain of P. foveata lacking the ability to produce pigment.     

Pathogenicity variation and DNA polymorphism of Bipolaris sorokiniana infecting winter wheat in the Huanghuai floodplain of China

Wheat root rot, caused by Bipolaris sorokiniana, has led to severe losses of wheat products worldwide. To evaluate the pathogenicity and genetic variation of B. sorokiniana, diseased wheat samples were collected from 97 locations in the Huanghuai floodplain of China in 2014 and 2015 for analysis. A total of 673 isolates were obtained, 262 of which were identified as B. sorokiniana. Pathogenicity analysis of the isolates revealed variation in pathogenicity, which was not directly correlated with geographic region. Large variations in pathogenicity were also found within geographic groups. To determine the genetic structure of the populations, PCR was performed with universal rice primers (URP). Cluster analysis based on amplification patterns showed that the classified groups were correlated with geographical regions. Thus, analysis of the genetic diversity of the population indicated a negative correlation with geographic origin, that is, the greater the distance between sites, the lower the genetic variation similarity coefficient. Identification of wheat germplasm resistance showed that resistant cultivars accounted for a low percentage, while susceptible and highly susceptible cultivars were in the majority. Overall, these results are meaningful for developing strategies to prevent and control wheat root rot.     

白僵菌及其伴生菌发酵液对线虫的毒力研究

采用发酵液浸没法,以4种线虫作为靶标,对6株白僵菌、7株伴生菌菌株进行了杀线虫活性筛选,以期获得新的、高效的杀线虫生防菌资源。其中:处理24 h对南方根结线虫Meloidogyne incognita 2龄幼虫及大豆胞囊线虫Heterodera glycines 2龄幼虫校正死亡率均达80%以上的菌株仅有白僵菌Snef2598及伴生菌Snef5;供试菌株对水稻干尖线虫Aphelenchoides besseyi的毒力均较低,其中仅有1株白僵菌校正死亡率达到40%以上。处理48 h对南方根结线虫2龄幼虫校正死亡率达90%以上的菌株有白僵菌4株和伴生菌5株;对大豆胞囊线虫2龄幼虫校正死亡率超过90%的菌株有白僵菌3株和伴生菌1株;供试菌株对水稻干尖线虫毒力均较低,校正死亡率达60%以上的菌株有白僵菌3株和伴生菌2株;几乎所有供试菌株,对小杆线虫Caenorhabditis sp.的毒力均很高,处理24 h及48 h的校正死亡率均达97%以上,且被杀死的小杆线虫体壁以及虫体内部物质均被不同程度地消解。研究表明,球孢白僵菌菌株Snef2598及其伴生菌菌株Snef5对4种靶标线虫的毒力均较好,具有进一步研究的价值。     

Effects of Host and Microbial Factors on Development of Clonostachys rosea and Control of Botrytis cinerea in Rose

Development of Clonostachys rosea in rose leaves and petals and control of Botrytis cinerea by the agent were investigated. C. rosea germinated, established endophytic growth, and sporulated abundantly whether the tissues were mature, senescent or dead when inoculated. Germination incidence was moderate on mature and senescent leaves (47% and 35%) and petals (31% and 43%), and high (>98%) on dead tissues. Sporulation of C. rosea in tissues inoculated when mature, senescent or dead averaged 41%, 61%, and 75% in leaves, and 48%, 87% and 53% in petals. When leaves were wounded with needles before inoculation, germination of C. rosea increased from 45–56% to 90–92%, but sporulation became high (>75%) regardless of wounds. When leaves were inoculated with C. rosea at 0–24h after wounding and subsequently with B. cinerea, germination of the pathogen was reduced by 25–41% and sporulation by 99%. A humid period prior to inoculation of senescent or dead leaves promoted communities of indigenous fungi, reduced sporulation of C. rosea and B. cinerea, and, in dead leaves, increased control of the pathogen associated with C. rosea. Applied at high density, isolates of indigenous Penicillium sp. and Alternaria alternata from rose interacted with C. rosea and reduced control of the pathogen by 16% and 21%, respectively. In conclusion, C. rosea markedly suppressed sporulation of B. cinerea in rose leaves and petals regardless of developmental stage, minor wounds, and natural densities of microflora. This versatility should allow C. rosea to effectively control inoculum production of B. cinerea in rose production systems.     

Genomic Variation within Monilinia laxa, M. fructigena and M. fructicola, and Application to Species Identification by PCR

Brown rot and twig canker of fruit trees are caused by Monilinia laxa, M. fructigena and M. fructicola. The Internal Transcribed Spacer (ITS) between the 18S and the 28S rRNA genes of four M. laxa and four M. fructigena isolates collected in France was amplified by Polymerase Chain Reaction (PCR) using universal primers and sequenced. Multiple alignment of the ITS sequences and comparison with published sequences revealed very little intraspecific variation and a low interspecific polymorphism clustered in two regions. Species-specific PCR primers were designed to amplify a 356bp fragment for each of the three species. The specificity of the three primer pairs was successfully tested with a collection of 17 M. laxa, 18 M. fructigena and 6 M. fructicola isolates collected from different hosts and different countries, unequivocally confirming the identification of each isolate based on morphological and cultural traits. Using stringent PCR conditions, no cross-reaction was observed with any of the isolates tested. The specificity of the PCR assays was also successfully confirmed with DNA extracted from different fungal species, either phylogenetically close to the genus Monilinia or commonly found on diseased fruits. Using this new reliable technique, doubtful isolates can be directly identified in a single PCR run. Moreover, detection and identification of the Monilinia species were successfully achieved directly on diseased fruits. This simple and rapid method can be particularly useful to detect M. fructicola which is a listed quarantine fungus in all European countries.     

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.