Diversity and biotransformative potential of endophytic fungi associated with the medicinal plant Kadsura angustifolia.

This study investigated the diversity and host component-transforming activity of endophytic fungi in medicinal plant Kadsura angustifolia. A total of 426 isolates obtained were grouped into 42 taxa belonging to Fungi Imperfecti (65.96%), Ascomycota (27.00%), Zygomycota (1.64%), Basidiomycota (0.47%) and Mycelia Sterilia (4.93%). The abundance, richness, and species composition of endophytic assemblages were significantly dependent on the tissue and the sampling site. Many phytopathogenic species associated with healthy K. angustifolia were found prevalent. Among them, Verticillium dahliae was dominant with 16.43% abundance. From 134 morphospecies selected, 39 showed remarkable biocatalytic activity and were further identified as species belonging to the genera Colletotrichum, Eupenicillium, Fusarium, Hypoxylon, Penicillium, Phomopsis, Trametes, Trichoderma, Umbelopsis, Verticillium and Xylaria on the basis of the sequence analysis of the internal transcribed spacer (ITS1-5.8S-ITS2). The results obtained in this work show that K. angustifolia is an interesting reservoir of pathogenic fungal species, and could be a community model for further ecological and evolutionary studies. Additionally, the converting potency screening of some endophytic fungi from this specific medicinal plant may provide an interesting niche on the search for novel biocatalysts.

Incidence and impact of Verticillium dahliae in soil associated with certified potato seed lots.

Verticillium dahliae causes Verticillium wilt of potato and can be found in soil associated with potato seed tubers. The purpose of this research was to quantify V. dahliae in soil associated with certified seed tubers and determine if this potential inoculum source is related to disease development in the field. Approximately 68% of seed lots assayed contained V. dahliae-infested soil on seed tuber surfaces (seed tuber soil). Over 82% of seed lots contained V. dahliae in loose seed lot soil obtained from bags and trucks used to transport seed tubers. Most samples contained ≤50 CFU/g but some contained >500 CFU/g. Most isolates (93%) were vegetative compatibility group 4A. Populations of V. dahliae in stem sap increased with increasing inoculum densities in field soils only when V. dahliae concentrations in seed tuber soil were low. High concentrations of V. dahliae in seed tuber soil resulted in greater stem sap colonization when V. dahliae inoculum densities in field soil were low (P < 0.01) and resulted in greater pathogen inoculum densities in postharvest field soils (P = 0.04). Seed tubers contaminated with V. dahliae-infested soils may introduce the pathogen into fields not previously cropped to potato or recontaminate those which have received preplant management practices. Long-term management of V. dahliae requires reducing propagules in soil associated with seed lots.

Verticillium dahliae populations from mint and potato are genetically divergent with predominant haplotypes.

In total, 286 Verticillium dahliae isolates from mint, potato, and other hosts and substrates were characterized for mating type, vegetative compatibility group (VCG), and multilocus microsatellite haplotype to determine population genetic structure among populations infecting mint and potato. Populations from mint and potato fit a clonal reproductive model, with all isolates a single mating type (MAT1-2) and multiple occurrences of the same haplotypes. Haplotype H02 represented 88% of mint isolates and was primarily VCG2B, while haplotype H04 represented 70% of potato isolates and was primarily VCG4A. Haplotypes H02 and H04 typically caused severe disease on mint and potato, respectively, in greenhouse assays regardless of host origin. Principal coordinate analysis and analysis of molecular variance indicated that mint and potato populations were significantly genetically diverged (P = 0.02), and identification of private alleles and estimation of migration rates suggested restricted gene flow. Migration was detected between infected potato plants and seed tubers, infested tare soil, and field soils. Genetic differentiation of V. dahliae from mint and potato may be due to the occurrence of a single mating type and differences in VCG. Populations of V. dahliae in potato and mint were characterized by the presence of aggressive, clonally reproducing haplotypes which are widely distributed in commercial mint and potato production.

Proteomic analysis of the phytopathogenic soilborne fungus Verticillium dahliae reveals differential protein expression in isolates that differ in aggressiveness.

Verticillium dahliae is a soilborne fungus that causes a vascular wilt disease of plants and losses in a broad range of economically important crops worldwide. In this study, we compared the proteomes of highly (Vd1396-9) and weakly (Vs06-14) aggressive isolates of V. dahliae to identify protein factors that may contribute to pathogenicity. Twenty-five protein spots were consistently observed as differential in the proteome profiles of the two isolates. The protein sequences in the spots were identified by LC-ESI-MS/MS and MASCOT database searches. Some of the identified sequences shared homology with fungal proteins that have roles in stress response, colonization, melanin biosynthesis, microsclerotia formation, antibiotic resistance, and fungal penetration. These are important functions for infection of the host and survival of the pathogen in soil. One protein found only in the highly aggressive isolate was identified as isochorismatase hydrolase, a potential plant-defense suppressor. This enzyme may inhibit the production of salicylic acid, which is important for plant defense response signaling. Other sequences corresponding to potential pathogenicity factors were identified in the highly aggressive isolate. This work indicates that, in combination with functional genomics, proteomics-based analyses can provide additional insights into pathogenesis and potential management strategies for this disease.

[Inhibitory effects of natural plant extracts on Verticillium albo-atrum].

This paper studied the inhibitory effects of 54 kinds of ethanol-extracted plant solutions on Verticillium albo-atrum. The results showed that 15 kinds of these extracts could inhibit the growth of verticillium albo-atrum mycelium, with an inhibitory rate more than 50%, among which, the inhibitory rate of Asarum sieboldii, Coptis chinensis, Magnolia officinalis, Acacia catechu, Sophora flavescens, Glycyrrhiza uralensis, Cnidium monnieri, Platycodon grandiflorum and Allium. cepa. extracts was higher than 65%, and that of Cnidium monnieri extract reached 86.84%. A total of 16 kinds of plant extracts decreased the spore germination of V. albo-atrum, with the inhibitory rate higher than 70%, and there were 7 kinds of plant extracts whose inhibitory rate reached 95%. Almost no spore bourgeon was found after treated with Asarum sieboldii, Coptis chinensis, and Magnolia officinalis extracts. The extract of Acacia catechu did not inhibit the growth of mycelium, but restrained the spore germination by 100%. Cnidium monnieri extract could strongly inhibit the growth of mycelium, but had a less effect on spore germination rate (only by 11.3%). The active substances found in natural plant extracts which had the inhibitory effects on pathogen brought us a new and promising method to deal with V. albo-atrum and other vegetable diseases.

Preliminary evaluation of the effectiveness of a Verticillium lecanii isolate in the control of Thrips tabaci (Thysanoptera: Thripidae).

Thrips tabaci is a polyphagous pest that attacks many different crop and ornamental plant species. Recently, the need to reduce the use of insecticides in the control of thrips is more and more increasing. On that account, the possibility to make use of the entomopathogenic fungus Verticillium lecanii (DAOM198499) was evaluated in laboratory conditions. Suspension of 10(3) to 10(7) conidia/ml grown in liquid culture medium with a control (distilled water) treated on second larval stage of T. tabaci in controlled condition (16L: 8D photoperiod, 25 +/- 1 degrees C temperature, 98% RH in first 48h and 70% RH in next days). Preliminary results showed that mortality in lethal suspension (10(7) conidia/ml) of V. lecanii after 24 hours and 2-7 days in lower suspension appears. This suggests that strine DAOM 198499 of V. lecanii has the ability to control T. tabaci in greenhouse conditions.

Quantification in soil and the rhizosphere of the nematophagous fungus Verticillium chlamydosporium by competitive PCR and comparison with selective plating.

A competitive PCR (cPCR) assay was developed to quantify the nematophagous fungus Verticillium chlamydosporium in soil. A gamma-irradiated soil was seeded with different numbers of chlamydospores from V. chlamydosporium isolate 10, and samples were obtained at time intervals of up to 8 weeks. Samples were analyzed by cPCR and by plating onto a semiselective medium. The results suggested that saprophytic V. chlamydosporium growth did occur in soil and that the two methods detected different phases of growth. The first stage of growth, DNA replication, was demonstrated by the rapid increase in cPCR estimates, and the presumed carrying capacity (PCC) of the soil was reached after only 1 week of incubation. The second stage, an increase in fungal propagules presumably due to cell division, sporulation, and hyphal fragmentation, was indicated by a less rapid increase in CFU, and 3 weeks was required to reach the PCC. Experiments with field soil revealed that saprophytic fungal growth was limited, presumably due to competition from the indigenous soil microflora, and that the PCR results were less variable than the equivalent plate count results. In addition, the limit of detection of V. chlamydosporium in field soil was lower than that in gamma-irradiated soil, suggesting that there was a background population of the fungus in the field, although the level was below the limit of detection. Tomatoes were infected with the root knot nematode (RKN) or the potato cyst nematode (PCN) along with a PCN-derived isolate of the fungus (V. chlamydosporium isolate Jersey). Increases in fungal growth were observed in the rhizosphere of PCN-infested plants but not in the rhizosphere of RKN-infested plants after 14 weeks using cPCR. In this paper we describe for the first time PCR-based quantification of a fungal biological control agent for nematodes in soil and the rhizosphere, and we provide evidence for nematode host specificity that is highly relevant to the biological control efficacy of this fungus.