捕食线虫丝孢菌的菌寄生性测定

研究了节丛孢Arthrobotrys、单顶孢Monacrosporium和隔指孢Dactylella三个捕食线虫丝孢菌属16个菌株,对水稻立枯丝核菌RhizoctoniasolaniAG1、大豆核盘菌Sclerotiniasclerotiorum、茄科镰刀菌Fusariumsolani和恶疫霉Phytophthoracactorum四种常见土壤植物病原真菌的菌寄生性。结果表明供试菌可以通过弹簧式菌丝圈缠绕、类附着胞结构吸附、简单的菌丝缠绕或者贴附寄主菌丝生长四种方式寄生病原菌。其中,绝大多数菌株对立枯丝核病菌有寄生作用,一些供试真菌对其它三种病原真菌有寄生现象。利用孢子液浸泡法测定了其中5种捕食线虫真菌对核盘菌菌核的寄生能力,显示有较高寄生率。     

Quantification of mycoparasitism by the nematode-trapping fungus Arthrobotrys oligospora on Rhizoctonia solani and the influence of nutrient levels

Abstract The influence of nutrient level, type of carbon source and nitrogen concentration on the parasitism of Arthrobotrys oligospora on Rhizoctonia solani were investigated by quantification of coiling frequency. Changes in coiling frequency were also compared with changes in hyphal density and colony radial growth rate. Increasing concentrations of corn meal agar gave increasing coiling frequency up to a concentration of half the recomended strength. At higher concentrations the coiling frequency was constant, although the hyphal density of both fungi increased over the whole concentration range. Coiling frequency was positively correlated with the probability of hyphal encounter, calculated as the product of the hyphal densities of the two fungi, except at high CMA concentrations. Amongst several carbohydrates tested, glucose resulted in the highest, and sucrose the lowest, coiling frequency. The effect of the different carbohydrates on coiling frequency was not correlated with the hyphal densities of the fungi. Addition of a nitrogen source, NaNO₃, removed the differences in coiling frequency between glucose and sucrose and increased coiling frequencies on both sugars.     

Induced hydrolytic enzymes of ectomycorrhizal fungi against pathogen Rhizoctonia solani

Interactions between ectomycorrhizal fungi (Suillus laricinus, S. tomentosus, Amanita vaginata and Gomphidius viscidus) and the pathogen Rhizoctonia solani in co-culture were studied using both light and scanning electron microscopy. S. laricinus, S. tomentosus and A. vaginata inhibited the growth of the pathogen. Moreover, A. vaginata exhibited coiling around and penetration of the hyphae into R. solani was observed in the interaction zone. Furthermore, the production of chitinases, beta-1,3-glucanases and beta-glucosidases by these ectomycorrhizal fungi on colloidal chitin or cell walls of R. solani was evaluated: chitinases were not induced by colloidal chitin but all three enzymes were induced by R. solani cell walls. No correlation between inhibition rate and production of lytic enzymes was found.     

In vivo study of trichoderma-pathogen-plant interactions, using constitutive and inducible green fluorescent protein reporter systems

Plant tissue colonization by Trichoderma atroviride plays a critical role in the reduction of diseases caused by phytopathogenic fungi, but this process has not been thoroughly studied in situ. We monitored in situ interactions between gfp-tagged biocontrol strains of T. atroviride and soilborne plant pathogens that were grown in cocultures and on cucumber seeds by confocal scanning laser microscopy and fluorescence stereomicroscopy. Spores of T. atroviride adhered to Pythium ultimum mycelia in coculture experiments. In mycoparasitic interactions of T. atroviride with P. ultimum or Rhizoctonia solani, the mycoparasitic hyphae grew alongside the pathogen mycelia, and this was followed by coiling and formation of specialized structures similar to hooks, appressoria, and papillae. The morphological changes observed depended on the pathogen tested. Branching of T. atroviride mycelium appeared to be an active response to the presence of the pathogenic host. Mycoparasitism of P. ultimum by T. atroviride occurred on cucumber seed surfaces while the seeds were germinating. The interaction of these fungi on the cucumber seeds was similar to the interaction observed in coculture experiments. Green fluorescent protein expression under the control of host-inducible promoters was also studied. The induction of specific Trichoderma genes was monitored visually in cocultures, on plant surfaces, and in soil in the presence of colloidal chitin or Rhizoctonia by confocal microscopy and fluorescence stereomicroscopy. These tools allowed initiation of the mycoparasitic gene expression cascade to be monitored in vivo.     

Frozen in time: a new method using cryo-scanning electron microscopy to visualize root-fungal interactions

A new method of sample preparation for cryo-scanning electron microscopy was used to visualize internal infection of wheat (Triticum aestivum) roots by the pathogenic fungus Rhizoctonia solani AG-8. The new method retained fungal hyphae and root cells in situ in disintegrating root tissues, thus avoiding the distortions that can be introduced by conventional preparation by chemical fixation, dehydration and embedding. Infected roots frozen in liquid nitrogen were cryo-planed and etched (sublimed) at -80 degrees C for a critical length of time (up to 9 min) in the microscope column to reveal plant and fungal structures in three dimensions. Root and fungal structures were well preserved irrespective of infection severity. Root and hyphal cell walls were clearly seen and hyphal architecture within and between root cells was preserved. This rapid method permits three-dimensional in situ visualization of fungal invasion within roots and has broad application for examination of diseases caused by other necrotrophic fungi.     

The production of antifungal volatiles by Bacillus subtilis

A strain of Bacillus subtilis which produces an antibiotic metabolite was also found to produce a volatile compound(s) which was antifungal to Rhizoctonia solani and Pythium ultimum.
Growth of the fungi was severely impaired in the presence of the volatiles and physiological abnormalities of the hyphae were observed, including hyphal distortion and vacuolation. A range of media were tested for volatile production and potato dextrose agar (PDA) was found to be the most active. Temperature had a considerable effect on antifungal volatile activity with the greatest inhibition occurring at 30°C. Addition of iron (III) chloride to Sabouraud's glucose agar (SGA) also enhanced the antifungal effect. The volatiles were found to be water soluble and remained active when trapped in SGA.     

Induction of trap formation in nematode-trapping fungi by a bacterium

Three soil bacterial strains were identified as Chryseobacterium sp. TFB on the basis of their 16S rRNA gene sequences. Conidia of Arthrobotrys oligospora produced a few mycelial traps (MT) and conidial traps (CT) when cultured with bacterial cells that they did not produce when cultured with a bacterial cell-free culture filtrate. However, co-culture of A. oligospora with bacterial cells and bacteria-free filtrate simultaneously induced MT and CT in large amounts. With the increased concentration of bacteria-free filtrate, the number of typical CT increased, but conidial germination was progressively inhibited. Scanning electron microscopy of A. oligospora co-cultured with bacteria revealed that bacterial attachment to hyphae was a prerequisite to trap formation and that bacteria-free filtrate facilitated bacterial attachments to hyphae. The results that the addition of nutrients in co-culture medium decreased the number of traps suggest that this type of trap formation may be favoured at a low nutrient status. Eight fungi tested were able to form MT and CT when co-cultured with bacterial cells and bacteria-free culture filtrate, but the abilities varied among species. This study provides novel evidence that under laboratory conditions, soil bacteria attaching to hyphae could induce traps in nematode-trapping fungi.     

Bioactive endophytic streptomycetes from the Malay Peninsula

Three novel endophytic streptomycetes have been isolated and characterized from plants with ethnobotanical uses on the Malay Peninsula including: Thottea grandiflora (family -Aristolochiaceae), Polyalthia spp. (family -Annonaceae), and Mapania sp. (family -Cyperaceae). Each isolate, as studied by scanning electron microscopy, has small hyphae, and produces typical barrel-shaped spores arising by hyphal fragmentation. Interestingly, although none has any detectable antibacterial killing properties, each has demonstrable killing activity against one or more pathogenic fungi including organisms such as Phytophthora erythroseptica, Pythium ultimum, Sclerotinia sclerotiorum, Mycosphaerella fijiensis and Rhizoctonia solani. Molecular biological studies on the rRNA gene sequence of each isolate revealed that it is distinct from all other genetic accessions of streptomyectes in GenBank, and each bears some genetic similarity to other streptomycetes. The bioactivity of each microbe was extractable in various organic solvents.     

The influence of Bacillus subtilis RB14-C on the development of Rhizoctonia solani and indigenous microorganisms in the soil

The effect of soil inoculation with an antagonistic strain Bacillus subtilis RB14-C on the development of Rhizoctonia solani and changes occurring in soil and rhizosphere microbial communities were studied. RB14-C was added to the soil as a water suspension of the cells or as a broth culture. Application of cell suspensions to non-planted soil reduced the number of culturable bacteria. The density of R. solani and the number of filamentous fungi were not significantly affected by RB14-C. A similar effect was observed in the rhizosphere of tomato plants growns in bacterized soil. Broth cultures of RB14-C suppressed R. solani 1 d after inoculation, but after 3 d there was no difference in the pathogen density between soil amended with broth culture and control soil. In microcosm studies, cell suspensions of RB14-C also did not inhibit growth of R. solani on filters buried in soil. However, an inhibitory effect was obtained when a broth culture of the bacterium was used. The effect of RB14-C on fungal biomass was also estimated by measurement of ergosterol concentration in soil. It was found that ergosterol was mostly derived from R. solani and that there were no significant differences in its content between untreated soil and soil treated with RB14-C. The results suggest that suppression of Rhizoctonia damping-off by B. subtilis RB14-C probably is not related to the reduction of the pathogen population in the soil.     

Effect of a preparation from Chaetomium fungi on the growth of phytopathogenic fungi

We studied the fungicidal activity of a biological preparation from the fungi of the genus Chaetomium against soil phytopathogenic fungi Rhizoctonia solani and Fusarium oxysporum. The inhibitory effect of the preparation under study depended on its concentration, duration of storage, and growth characteristics of pure cultures of the phytopathogens. The highest (98.8%) inhibitory activity was observed on day 3 of the interaction with Rhizoctonia solani. After a 2-year storage, this preparation was capable of inhibiting the growth of the phytopathogens only at high doses. The preparation precluded the development of bare patch and increased the productivity of potato plants. The preparation may serve as an alternative to chemical fungicides for plant protection.     

Analysis of the dynamics of fungal communities in soil via fungal-specific PCR of soil DNA followed by denaturing gradient gel electrophoresis

A molecular method for profiling of fungal communities in soil was applied in experiments in soil microcosms, with two objectives, (1) to assess the persistence of two selected fungal species in soil, and (2) to analyze the response of the natural fungal community to a spill of sulphurous petrol in the same soil. To achieve the aims, two soil DNA extraction methods, one originally designed for the direct extraction of bacterial community DNA and the other one aimed to obtain fungal DNA, were tested for their efficiency in recovering DNA of fungal origin from soil. Both methods allowed for the efficient extraction of DNA from introduced Trichoderma harzianum spores as well as Arthrobotrys oligospora mycelial fragments, at comparable rates. Several PCR amplification systems based on primers specific for fungal 18S ribosomal RNA genes were tested to design strategies for the assessment of fungal communities in soil. The PCR systems produced amplicons of expected size with DNA of most fungi studied, which included members of the Ascomycetes, Basidiomycetes, Zygomycetes and Chytridiomycetes. On the other hand, the 18S rRNA genes of Oomycetes (including key plant pathogens) were poorly amplified. Plant (Solanum tuberosum), nematode (Meloidogyne sp.) and bacterial DNA was not amplified. For studies of soil fungal communities, a nested PCR approach was selected, in which the first PCR provided the required specificity for fungi, whereas the second (nested) PCR served to produce amplicons separable on denaturing gradient gels. Denaturing gradient gel electrophoresis (DGGE) allowed the resolution of mixtures of PCR products of several different fungi, as well as products resulting from mixed-template amplifications, into distinct banding patterns. The persistence of fungal species in soil was assessed using T. harzianum spores and A. oligospora hyphal fragments added to silt loam soil microcosms. Using PCR-DGGE, these fungi were detectable for about 14 days and 2 months, respectively. Both singly-inoculated soils and soils that had received mixed inoculants revealed, next to bands resulting from indigenous fungi, the expected bands in the DGGE profiles. The A. oligospora specific amplicon, by virtue of its unique migration in the denaturing gradient, was well detectable, whereas the T. harzianum specific product comigrated with products from indigenous fungi. PCR-DGGE analysis of DNA obtained from the silt loam soil treated with dibenzothiophene-containing petrol showed the progressive selection of specific fungal bands over time, whereas this selection was not observed in untreated soil microcosms. Cloning of individual molecules from the selected bands and analysis of their sequences revealed a complex of targets which clustered with the 18S rDNA sequences of the closely-related species Nectria haematococca, N. ochroleuca and Fusarium solani. Fungal isolates obtained from the treated soil on PDA plates were identified as Trichoderma sp., whereas those on Comada agar fell into the Cylindrocarpon group (anamorph of Nectria spp).     

In vitro attachment of phylloplane yeasts to Botrytis cinerea, Rhizoctonia solani, and Sclerotinia homoeocarpa

The ability of yeasts to attach to hyphae or conidia of phytopathogenic fungi has been speculated to contribute to biocontrol activity on plant surfaces. Attachment of phylloplane yeasts to Botrytis cinerea, Rhizoctonia solani, and Sclerotinia homoeocarpa was determined using in vitro attachment assays. Yeasts were incubated for 2 d on potato dextrose agar (PDA) prior to experimentation. A total of 292 yeasts cultured on PDA were screened for their ability to attach to conidia of B. cinerea; 260 isolates (89.1%) attached to conidia forming large aggregates of cells, and 22 isolates (7.5%) weakly attached to conidia with 1 or 2 yeast cells attached to a few conidia. Ten yeasts (3.4%), including 8 isolates of Cryptococcus laurentii, 1 isolate of Cryptococcus flavescens, and an unidentified species of Cryptococcus, failed to attach to conidia. All non-attaching yeasts produced copious extracellular polysaccharide (EPS) on PDA. Seventeen yeast isolates did not attach to hyphal fragments of B. cinerea, R. solani, and S. homoeocarpa after a 1 h incubation, but attachment was observed after 24 h. Culture medium, but not culture age, significantly affected the attachment of yeast cells to conidia of B. cinerea. The 10 yeast isolates that did not attach to conidia when grown on agar did attach to conidia (20%-57% of conidia with attached yeast cells) when cultured in liquid medium. Attachment of the biocontrol yeast Rhodotorula glutinis PM4 to conidia of B. cinerea was significantly greater at 1 x 10(7) yeast cells x mL(-1) than at lower concentrations of yeast cells. The ability of yeast cells to attach to fungal conidia or hyphae appears to be a common phenotype among phylloplane yeasts.     

An ultrastructural study of cell-cell interactions in capture organs of the nematophagous fungus Arthrobotrys oligospora

Abstract A detailed ultrastructural analysis was made of interactions between individual cells within the same adhesive network (trap) of the nematophagous fungus Arthrobotrys oligospora . These interactions were confined to traps which had captured nematodes, and occurred concurrently with the fungus-nematode interactions. The process was initiated by the anchoring of 2 cells of different loops constituting the trap network, by means of the adhesive coating of these cells. Subsequently, penetration tubes were formed. As in nematode-fungus interactions, the walls of these tubes arose from underneath the original trap cell walls. Two response were observed: either only one of the anchored cells formed a penetration tube, which penetrated the opposite cell and subsequently digested its contents; or both cells formed penetration tubes simultaneously, which were directed against each other. In the latter case, no penetration of other cells was observed, and elongated tubes were occasionally formed. The above mechanism differed from 2 other modes of interaction also observed, namely fusion of intact cells (anastomosis) and development of new hyphae inside dead hyphal cells. In the latter case the newly formed cells developed from the cross-wall of the neighbouring intact cell.     

Laser microdissection of fungal septa as visualised by scanning electron microscopy

Laser microdissection has been proven a successful technique to isolate single cells or groups of cells from animal and plant tissue. Here, we demonstrate that laser microdissection is suitable to isolate subcellular parts of fungal hyphae. Dolipore septa of Rhizoctonia solani containing septal pore caps were cut by laser microdissection from sections of mycelium and collected by laser pressure catapulting. Subsequently, microdissected septa were visualised using a wheat germ agglutinin labelling of cell walls, septa and septal pore caps and scanning electron microscopy. The use of laser microdissection on fungal cells opens new ways to study subcellular fungal structures and the biochemical composition of hyphal cells.     

Arbuscular mycorrhizal structure and fungi associated with mosses

We investigated the colonization and diversity of arbuscular mycorrhizal (AM) fungi associated with 24 moss species belonging to 16 families in China. AM fungal structures, i.e. spores, vesicles, hyphal coils (including intracellular hyphae), or intercellular nonseptate hyphae, were found in 21 moss species. AM fungal structures (vesicles, hyphal coils, and intercellular nonseptate hyphae) were present in tissues of 14 moss species, and spores and nonseptate hyphae on the surface of gametophytes occurred in 15 species. AM fungal structures were present in 11 of the 12 saxicolous moss species and in six of the ten terricolous moss species, but absent in two epixylous moss species. AM fungal structures were only observed in moss stem and leaf tissues, but not in rhizoids. A total of 15 AM fungal taxa were isolated based on trap culture with clover, using 13 moss species as inocula. Of these AM fungi, 11 belonged to Glomus, two to Acaulospora, one to Gigaspora, and one to Paraglomus. Our results suggest that AM fungal structures commonly occur in most mosses and that diverse AM fungi, particularly Glomus species, are associated with mosses.     

The interface between the arbuscular mycorrhizal fungus Glomus intraradices and root cells of Panax quinquefolius: a Paris-type mycorrhizal association

Two major types of arbucular mycorrhizal associations, the Arum-type and the Paris-type, have been identified based on morphological features. Although the Paris-type is the most common, it is the Arum-type that has been most intensively studied in terms of structure/function because of its prevalence in agronomically important plant species. In this study, the interface between the host cell cytoplasm and intracellular hyphae (extensive hyphal coils and arbusculate coils), which typify the Paris-type mycorrhiza, was studied. Using immunofluorescence techniques combined with laser scanning confocal microscopy, dramatic changes in the cytoskeleton in colonized cells were observed. Changes in the positioning of both host cell microtubules and actin filaments occurred in colonized plant cells. Both microtubules and actin filaments were associated with the hyphal coils and the arbusculate coils. An interfacial matrix, of host origin, was demonstrated between hyphal coils and arbusculate coils using various affinity techniques. It formed an apoplastic compartment consisting of cellulose and pectins between the fungus and host cell cytoplasm. There was less labelling adjacent to the fine branches of arbusculate coils compared to the hyphal coils. These observations show some similarities to those seen with Arum-type mycorrhizas.     

Mycoparasitism of Pythium ultimum by Antagonistic Binucleate Rhizoctonia Isolates in Agar Media and on Capsicum Seeds

Hyphal interactions between two antagonistic binucleate Rhizoctonia isolates (BNR) and the seedling dampingoff pathogen, Pythium ultimum var. sporangiiferum, were observed by both light-and scanning electron microscopy (SEM), on agar media and on capsicum seeds in sterilized potting mix. Both BNR isolates displayed similar mycoparasitic behaviour towards P. u. sporangiiferum on agar media. This included parallel growth along the pathogen hyphae, formation of hook-shaped hyphal tips and coils on the surface of P. u. sporangiiferum hyphae and penetration and growth within pathogen structures. Disruption of cytoplasmic streaming and disorganisation of pathogen cytoplasm were also observed. SEM observations revealed alterations in P. u. sporangiiferum cell wall structure and the presence of penetration holes apparently due to digestion by the BNR. P. u. sporangiiferum was also parasitised by both BNR isolates on capsicum seed coats, with parallel growth, hook formation and coils commonly observed. The above observations indicated that mycoparasitism is a possible mode of action of BNR against P. u. sporangiiferum.     

Tobacco leaf spot and root rot caused by Rhizoctonia solani Kühn

Rhizoctonia solani Kühn is a soil-borne fungal pathogen that causes disease in a wide range of plants worldwide. Strains of the fungus are traditionally grouped into genetically isolated anastomosis groups (AGs) based on hyphal anastomosis reactions. This article summarizes aspects related to the infection process, colonization of the host and molecular mechanisms employed by tobacco plants in resistance against R. solani diseases. TAXONOMY: Teleomorph: Thanatephorus cucumeris (Frank) Donk; anamorph: Rhizoctonia solani Kühn; Kingdom Fungi; Phylum Basidiomycota; Class Agaricomycetes; Order Cantharellales; Family Ceratobasidiaceae; genus Thanatephorus. IDENTIFICATION: Somatic hyphae in culture and hyphae colonizing a substrate or host are first hyaline, then buff to dark brown in colour when aging. Hyphae tend to form at right angles at branching points that are usually constricted. Cells lack clamp connections, but possess a complex dolipore septum with continuous parenthesomes and are multinucleate. Hyphae are variable in size, ranging from 3 to 17 μm in diameter. Although the fungus does not produce any conidial structure, ellipsoid to globose, barrel-shaped cells, named monilioid cells, 10-20 μm wide, can be produced in chains and can give rise to sclerotia. Sclerotia are irregularly shaped, up to 8-10 mm in diameter and light to dark brown in colour. DISEASE SYMPTOMS: Symptoms in tobacco depend on AG as well as on the tissue being colonized. Rhizoctonia solani AG-2-2 and AG-3 infect tobacco seedlings and cause damping off and stem rot. Rhizoctonia solani AG-3 causes 'sore shin' and 'target spot' in mature tobacco plants. In general, water-soaked lesions start on leaves and extend up the stem. Stem lesions vary in colour from brown to black. During late stages, diseased leaves are easily separated from the plant because of severe wilting. In seed beds, disease areas are typically in the form of circular to irregular patches of poorly growing, yellowish and/or stunted seedlings. RESISTANCE: Knowledge is scarce regarding the mechanisms associated with resistance to R. solani in tobacco. However, recent evidence suggests a complex response that involves several constitutive factors, as well as induced barriers controlled by multiple defence pathways. MANAGEMENT: This fungus can survive for many years in soil as mycelium, and also by producing sclerotia, which makes the management of the disease using conventional means very difficult. Integrated pest management has been most successful; it includes timely fungicide applications, crop rotation and attention to soil moisture levels. Recent developments in biocontrol may provide other tools to control R. solani in tobacco.