Possible phytotoxic metabolites in culture filtrates of Diaporthe phaseolorum var. sojae

Filter-sterile culture filtrates of an isolate of Diaporthe phaseolorum var. sojae (Phomopsis sojae), causal agent of pod and stem blight and seed decay of soybeans (Glycine max), grown on Czapeks-dox broth significantly (P = 0.05) inhibited germination of cabbage (Brassica oleracea), cantaloupe (Cucumis melo), onion (Allium cepa), soybean, and wheat (Triticum vulgare) seeds and wilted soybean seedling cuttings within 24 hr. Inhibition of seed germination and severity of soybean seedling wilt increased with increased concentrations of culture filtrates and increased incubation time of the cultures.     

Occurrence of Phomopsis longicolla β Conidia in Naturally Infected Soybean

Although Phomopsis longicolla is primarily known as a seedborne pathogen, it can be isolated from all parts of the plant. The disease lesions observed on the basal parts of soybean stems were slightly sunken with irregular shapes and sizes, bordered by a thin black margin. Within the lesions themselves, large and diffusely distributed pycnidia with α and β conidia, typical of the genus Phomopsis, were observed. The percentages of the two types of conidia varied considerably, but β conidia were predominant in most of the pycnidia. The presence of these reproductive organs indicated that the symptoms could have been caused by Phomopsis sojae. However, after isolation on a nutritive medium, all cultural and morphological characteristics clearly indicated that the isolated fungus was P. longicolla, whose identification was subsequently confirmed by sequencing three genomic regions. Monosporic isolates, with different ratios of α and β conidia, exhibited a high level of pathogenicity on soybean, after artificial inoculation. Both types of conidia were observed on the stems of the inoculated soybean plants. Beta conidia also formed quickly on medium made of soybean seeds and mature stems after exposure to low temperatures (?10°C). This study suggests that P. longicolla is capable of a massive production of β conidia, not only in old fungal cultures as it had until now been believed, but also in infected soybean plants in the field.     

Reaction of Diaporthe longicolla to a strain of Sarocladium kiliense

Phomopsis seed decay of soybean [Glycine max (L.) Merr.] is a seedborne fungal disease caused by Diaporthe longicolla that causes yield losses and reduced seed quality. Biocontrol of this pathogen by a strain of Sarocladium kiliense isolated from a culture of D. longicolla was investigated in vitro and in vivo. A zone of inhibition formed between the two fungi in vitro, but was poorly sustained, and inhibition of conidial germination of D. longicolla by a culture filtrate of S. kiliense was not significant, though D. longicolla mycelial growth was inhibited by a 25% culture filtrate. Co-inoculation of both fungi failed to reduce seed rot or increase seed germination in greenhouse and growth chamber experiments, respectively. Co-inoculation of both fungi also failed to reduce pycnidial development in colonies of D. longicolla growing on leaf pieces, but soaking of the leaf pieces with S. kiliense conidia for one or three days prior to inoculation with D. longicolla eliminated pycnidial development completely. Although it was not possible to reproduce the parasitism of S. kiliense on D. longicolla under the experimental and environmental conditions used, the potential to use S. kiliense as a protectant biocontrol for soybean fungal pathogens should be further investigated.     

Development of Heterodera glycines on Soybean Damaged by Soybean Looper and Stem Canker

Short-term greenhouse studies with soybean (Glycine max cv. Bragg) were used to examine interactions between the soybean cyst nematode (Heterodera glycines) and two other common pests of soybean, the stem canker fungus (Diaporthe phaseolorum var. caulivora) and the soybean looper (Pseudoplusia includens), a lepidopterous defoliator. Numbers of cyst nematode juveniles in roots and numbers of cysts in soil and roots were reduced on plants with stem cankers. Defoliation by soybean looper larvae had the opposite effect; defoliation levels of 22 and 64% caused stepwise increases in numbers of juveniles and cysts in both roots and soil, whereas numbers of females in roots decreased. In two experiments, stem canker length was reduced 40 and 45% when root systems were colonized by the soybean cyst nematode. The absence of significant interactions among these pests indicates that the effects of soybean cyst nematode, stem canker, and soybean looper on plant growth and each other primarily were additive.     

MoNSTR‐seq,a restriction site‐associated DNA sequencing technique to characterize Agrobacterium‐mediated transfer‐DNA insertions in Phomopsis longicolla

Phomopsis longicolla (Hobbs) causes Phomopsis seed decay and stem lesions in soybean (Glycine max). In this study, a novel, high‐throughput adaptation of RAD‐seq termed MoNSTR‐seq (Mutation analysis via Next‐generation DNA Sequencing of T‐DNA Regions) was developed to determine the genomic location of T‐DNA insertions in P. longicolla mutants. Insertional mutants were created via Agrobacterium tumefaciens‐mediated transformation, and one mutant, strain PL343, was further investigated due to impaired stem lesion formation. Mutation analysis via Next‐generation DNA Sequencing of T‐DNA Regions, in which DNA libraries are created with two distinct restriction enzymes and customized adapters to simultaneously enrich both T‐DNA insertion borders, was developed to characterize the genomic lesion in strain PL343. MoNSTR‐seq successfully identified a T‐DNA insertion in the predicted promoter region of a gene encoding a cellobiose dehydrogenase (CDH1), and the position of the T‐DNA insertion in strain PL343 was confirmed by Sanger sequencing. Thus, MoNSTR‐seq represents an effective tool for molecular genetics in P. longicolla, and is readily adaptable for use in diverse fungal species.

Significance and Impact of the Study

This study describes MoNSTR‐seq (Mutation analysis via Next‐generation DNA Sequencing of T‐DNA Regions), an adaptation of restriction site‐associated DNA sequencing (RAD‐seq) to identify the position of transfer‐DNA (T‐DNA) insertions in the genome of Phomopsis longicolla, an important pathogen of soybean. The technique enables high‐throughput characterization of mutants generated via Agrobacterium tumefaciens‐mediated transformation (ATMT), thus accelerating gene discovery via forward genetics. This technique represents a significant advancement over existing approaches to characterize T‐DNA insertions in fungal genomes. With minor modifications, this technique could be easily adapted to taxonomically diverse fungal pathogens and additional mutagenesis cassettes.     

Dermatophytes isolated from domestic animals in Barcelona,Spain

A comparative study was conducted with soybean material presenting symptoms of the Diaporthe/Phomopsis complex that was collected in two distant geographical regions of the world: Beltsville, MA, USA, and Vojvodina, Yugoslavia. Contrasting with earlier findings, great variability in the disease symptoms was observed, and one or more Phomopsis species could be isolated from lesions presenting similar characteristics. Among the thirty-three isolates obtained from the lesions the following species were identified: D. phaseolorum var. caulivora, P. phaseoli (teleomorph D. phaseolorum var. sojae, rare), P. longicolla (found for the first time on the soybean fields of Yugoslavia), Phomopsis sp., and one culture showing intermediate characters of D. phaseolorum var. caulivora and D. phaseolorum var. sojae. Much diversity was also found in the cultural characters of the the isolates from both localities, presumably indicating evolutionary and adaptation processes. This revised version was published online in June 2006 with corrections to the Cover Date.     

Cultural Growth Patterns and Incompatibility Reactions in Diaporthe and Phomopsis Populations

Comparative studies were carried out with cultures isolated from Phomopsis condiomata and/or Diaporthe ascomata found on naturally-infected living stems or on overwintered stem fragments of Arctium lappa L., Helianthus annuus L., Glycine max (L.) Merr., and Xanthium italicum Moretti, growing in or nearby the same fields in Vojvodina. Results underscored the heterogeneity of the Phomopsis anamorphs in the D. arctii (Lasch) Trav, complex, Intra-specific vegetative incompatibility groups were distinguished among Phomopsis and/or Diaporthe populations from A. lappa. More than one phytopathogenic Phomopsis and/or Diaporthe species were identified on X. italicum. P. longicolla Hobbs was isolated for the first time in Yugoslavia from soybean stem debris. The holomorph Diaporthe (Phomopsis) helianthi Muntanola- Cvetkovic, Mihaljcevic et Petrov showed distinguishing characteristics with respect to other congeneric fungi.     

Growth and development of Arabidopsis thaliana seedlings in interaction with fungi isolated from stem-end rot of avocado fruits

Abstract

The influence of Phomopsis viticola, Diaporthe phaseolorum and Pseudofusicoccum stromaticum isolated from avocado stem-end rot on growth of Arabidopsis seedlings was investigated. We found responses that are specific for each fungus, whereas P. viticola and D. phaseolorum strongly repressed root growth, P. stromaticum induced growth and branching, and enhanced shoot biomass production. Detailed microscopy and structural analyses revealed that the fungi did not affect cell division in root meristems but rather influenced cell elongation and differentiation, and these responses were related to changes in auxin responsiveness. P. stromaticum strongly acidifies the growth medium and this correlated with induction of the jasmonic acid responsive gene construct LOX2:GUS in leaves, whereas P. viticola and D. phaseolorum showed much reduced acidification properties. Taken together, our results show that fungal isolates from the stem-end of avocado fruits interact with Arabidopsis plants in highly diverse and contrasting manners influencing growth, patterning and defence.     

Replacement Series: A Tool for Characterizing Competition between Phytoparasitic Nematodes

The replacement series approach was used to detect and define competition between Meloidogyne incognita (Mi) and Rotylenchulus reniformis (Rr) on soybean. In three greenhouse tests, soybean cv. Davis seedlings were inoculated with 1,000 vermiform nematodes in the following Mi:Rr ratios: 0:0, 100:0, 75:25, 50:50, 25:75, and 0:100. After 86 days, relative nematode-yield values (number of each species in mixed culture divided by number in nonmixed culture) were calculated based on nematodes in soil per gram of dry root tissue. Calculated values were plotted and the resulting line compared with a reference line representing equal inter- and intraspecific competition predicted by the replacement series. Relative yields for Mi were higher than predicted at all ratios where Mi and Rr occurred together (lack-of-fit regression, F= 5.9401, P = 0.0008), indicating increased reproduction in the presence of Rr. Relative yields for Rr did not differ from predicted yields (lack-of-fit regression, F= 0.7565, P = 0.5203), indicating no effect of Mi on Rr. These relationships were not detected using analysis of variance. The relationship between Mi and Rr was independent of host colonization by Diaporthe phaseolorum var. caulivora, the stem canker fungus.     

Bestimmung pektolytischer Enzyme bei mehreren Schadpilzen von Citrusfrüchten

Characterization of pectolytic enzymes From several fungi causing post-harvest decay of citrus fruits From culture filtrates of Penicillium digitatum Sacc., Trichoderma viride Penz., Phomopsis citri (Faw.) cf. Diaporthe citri (Faw.) Wolf, and Colletotrichum gloeosporioides Penz. endo-polygalacturonase and endo-polymethylgalacturonase have been isolated. Both enzymes were active under acid conditions only. The last named fungus additionally produced pectic acid-transeliminase, the activity was found under alkaline conditions.     

Phomopsis (Diaporthe) Species as the Cause of Soybean Seed Decay in Korea

Soybeans are an important crop known to harbour a complex of Diaporthe and Phomopsis species. This complex has been reported to be involved in several soybean diseases, including Phomopsis seed decay. In this study, two species of Diaporthe/Phomopsis fungi from soybean plants were identified by morphological and molecular characterizations. Koch's postulates were confirmed by pathogenicity tests on hypocotyls of soybean seedlings. Phomopsis longicolla was found to be the most common and virulent pathogen to soybeans in Korea. Phomopsis sp., which was considered as a new soybean pathogen, might have been introduced from other plants given that similar strains of Phomopsis sp. have infected fruit trees in China, Japan and Portugal and vegetable plants in the United States.     

Viability staining of soybean suspension-cultured cells and a seedling stem cutting assay to evaluate phytotoxicity of Fusarium solani f. sp. glycines culture filtrates

The phytotoxicity of culture filtrates of Fusarium solani f. sp. glycines, the fungus causing sudden death syndrome (SDS) of soybean (Glycine max), was tested with a viability stain of soybean suspension-cultured cells and a stem cutting assay of soybean seedlings. Suspension-cultured cells from a SDS-susceptible soybean cultivar were exposed to cell-free culture filtrates of F. solani f. sp. glycines or other F. solani isolates for 2, 4, 6, and 8 days and then stained with 0.1% phenosafranin. The percentage of dead soybean suspension-cultured cells was greater (P<0.001) with filtrates prepared from F. solani f. sp. glycines than from other F. solani isolates, and dead cells increased over time and with higher concentrations of culture filtrate. Cuttings of soybean seedlings with their stems immersed in culture filtrates of F. solani f. sp. glycines isolates developed SDS-like foliar symptoms, but not when immersed in filtrates of other isolates. There was a positive correlation (r=0.94, P<0.001) between soybean foliar symptom severity and percentage of stained soybean suspension-cultured cells. Both methods were used to determine the phytotoxicity of fungal culture filtrates. Received: 9 December 1997 / Revision received: 10 August 1998 / Accepted: 28 August 1998     

Fusarium-produced vitamin B6 promotes the evasion of soybean resistance by Phytophthora sojae

Plants can be infected by multiple pathogens concurrently in natural systems. However,pathogen–pathogen interactions have rarely been studied. In addition to the oomycete Phytophthora sojae, fungi such as Fusarium spp. also cause soybean root rot. In a 3-year field investigation, we discovered that P. sojae and Fusarium spp. frequently coexisted in diseased soybean roots. Out of 336 P. sojae–soybean–Fusarium combinations,more than 80% aggravated disease. Different Fusarium species all enhanced P...     

Interaction between pathogenic and saprobic fungi isolated from soybean roots and seeds

A variety of interactions was recorded in culture between 11 saprobic fungi isolated from soybean (Glycine max) roots and seeds and the soybean pathogens Cercospora sojina, Colletotrichum truncatum, Macrophomina phaseolina, Phomopsis sojae, and Septoria glycines. The most active saprobes were Aspergillus terreus, Chaetomium cupreum, Epicoccum nigrum, Gliocladium roseum, Myrothecium roridum, Penicillium thomii, and Trichothecium roseum. Hyphal lysis of several fungal pathogens by Acremonium sp., C. cupreum and P. thomii was recorded perhaps because of parasitism by G. roseum and T. roseum. In greenhouse studies, seeds coated with G. roseum, P. thomii, and T. harzianum emerged significantly (P=0.05) more than those coated with A. terreus and the control. In field studies, seeds coated with a conidial suspension of A. terreus, G. roseum, P. thomii or Trichoderma harzianum produced a significantly greater stand than the control. The area of cotyledons covered with lesions caused by C. truncatum was significantly less on seeds coated with G. roseum, P. thomii and T. harzianum than the control.     

Chaetomium globosum for reducing primary inoculum of Diaporthe phaseolorum f. sp. meridionalis in soil-surface soybean stubble in field conditions

The potential of an antibiotic-producing isolate of Chaetomium globosum (CgA-1) to suppress Diaporthe phaseolorum f. sp. meridionalis (Dpm) in soybean stubble was studied in field microplots of no-tillage, minimum-tillage, and shallow plowing. Mature soybean stems colonized in vitro with Dpm were spread on the soil surface and C. globosum ascospore suspension, without nutrient supply, was sprayed over the entire plot prior to any tillage operation. Perithecial formation and survival of Dpm in soybean stems, concomitantly with colonization by C. globosum, were monitored for a 180-day period (mid-autumn through winter and mid-spring), which is the normal interval between soybean harvest and sowing. The proportion of soybean stem segments occupied by Dpm and number of perithecia formed decreased linearly with time and showed a strong negative correlation with increase in the occupation by C. globosum. At the end of the study, which coincided with the soybean sowing season, the soybean stubble was free from viable Dpm and was colonized by C. globosum. The effectiveness of C. globosum in eliminating the pathogen from surface-borne residue or harrowed-in residue was similar but much slower than in the shallow-plowed microplots. C. globosum successfully competed with major interfering fungi such as, Trichoderma, Nigrospora, and Fusarium in colonizing the soybean stems above and under the soil surface. The data provide strong evidence for use of the antibiotic-producing isolate of C. globosum to control soybean stem canker disease.     

Roles of small RNAs in soybean defense against Phytophthora sojae infection

The genus Phytophthora consists of many notorious pathogens of crops and forestry trees. At present, battling Phytophthora diseases is challenging due to a lack of understanding of their pathogenesis. We investigated the role of small RNAs in regulating soybean defense in response to infection by Phytophthora sojae, the second most destructive pathogen of soybean. Small RNAs, including microRNAs (miRNAs) and small interfering RNAs (siRNAs), are universal regulators that repress target gene expression in eukaryotes. We identified known and novel small RNAs that differentially accumulated during P. sojae infection in soybean roots. Among them, miR393 and miR166 were induced by heat‐inactivated P. sojae hyphae, indicating that they may be involved in soybean basal defense. Indeed, knocking down the level of mature miR393 led to enhanced susceptibility of soybean to P. sojae; furthermore, the expression of isoflavonoid biosynthetic genes was drastically reduced in miR393 knockdown roots. These data suggest that miR393 promotes soybean defense against P. sojae. In addition to miRNAs, P. sojae infection also resulted in increased accumulation of phased siRNAs (phasiRNAs) that are predominantly generated from canonical resistance genes encoding nucleotide binding‐leucine rich repeat proteins and genes encoding pentatricopeptide repeat‐containing proteins. This work identifies specific miRNAs and phasiRNAs that regulate defense‐associated genes in soybean during Phytophthora infection.     

Host-Pathogen Interactions: XII. Response of Suspension-cultured Soybean Cells to the Elicitor Isolated from Phytophthora megasperma var. sojae, a Fungal Pathogen of Soybeans

The glucan elicitor isolated from the mycelial walls of Phytophthora megasperma var. sojae, the fungus which causes stem and root rot in soybeans, stimulates the activity of phenylalanine ammonia-lyase and the accumulation of glyceollin in suspension-cultured soybean cells. Nigeran, a commercially available fungal wall glucan, was the only other compound tested which has any activity in this system. Glyceollin is a phenylpropanoid-derived phytoalexin which is toxic to P. megasperma var. sojae. Evidence is presented to support the hypothesis that the action of elicitors in stimulating phytoalexin synthesis is not species or variety specific but, rather, is part of a general defensive response of plants.     

Reaction of soybean callus to culture filtrates of Phialophora gregata

Soybean [Glycine max (L.) Merr.] calli derived from susceptible and resistant soybean genotypes were exposed to the culture filtrates of pathogenic and non-pathogenic isolates of Phialophora gregata (Allington and Chamberlain) W. Gams. The rate of browning, growth and viability (measured by 2,3,5-triphenyltetrazolium chloride reduction) of the callus were determined after various exposure times to the fungus culture filtrates. Callus from susceptible Century, Cumberland, Corsoy 79, Harosoy and Clark 63 were sensitive to the culture filtrates of pathogenic isolates of P. gregata. Callus from Plant Introductions 437833 and 84946-2, when treated with fungal culture filtrates, did not develop browning and callus growth and cell viability were not decreased compared to untreated controls. Culture filtrates from non-pathogenic isolates of the fungus did not affect the growth of susceptible and resistant callus. Tobacco (Nicotiana tabacum L.) callus was not sensitive to the culture filtrate of a P. gregata isolate pathogenic to soybean. The fungal culture filtrate, based on limited evaluation, appears to be selective towards soybean callus. Based on this initial work, it appears that soybean callus bioassays have utility for evaluating soybean for resistance to P. gregata as well as assessing pathogenicity of fungus isolates.