A fungal cell wall integrity-associated MAP kinase cascade in Coniothyrium minitans is required for conidiation and mycoparasitism

Coniothyrium minitans is an important biocontrol agent against Sclerotinia diseases. Previously, a conidiation-deficient mutant ZS-1T1000 was screened out from a T-DNA insertional library of C. minitans. CmBCK1, encoding MAP kinase kinase kinase and homologous to BCK1 of Saccharomyces cerevisiae, was disrupted by T-DNA insertion in this mutant. Targeted disruption of CmBCK1 led to the mutants undergoing autolysis and displaying hypersensitivity to the cell wall-degrading enzymes. The △CmBCK1 mutants lost the ability to produce pycnidia and conidia compared to the wild-type strain ZS-1. △CmBCK1 mutants could grow on the surface of sclerotia of Sclerotinia sclerotiorum but not form conidia, which resulted in much lower ability to reduce the viability of sclerotia of S. sclerotiorum. Furthermore, CmSlt2, a homolog of Slt2 encoding cell wall integrity-related MAP kinase and up-regulated by BCK1 in S. cerevisiae, was identified and targeted disrupted. The △CmSlt2 mutants had a similar phenotype to the △CmBCK1 mutants. The △CmSlt2 mutants also had autolytic aerial hyphae, hypersensitivity to cell wall-degrading enzymes, lack of conidiation and reduction of sclerotial mycoparasitism. Taken together, our results suggest that CmBCK1 and CmSlt2 are involved in conidiation and the hyperparasitic activities of C. minitans.     

Factors Affecting Biological Control of Sclerotinia sclerotiorum by Fungal Antagonists

Studies were conducted to determine the effects of soil moisture (9, 16 or 24% w/w) and temperature (5, 15, 20 or 25°C) on the control of sclerotia of Sclerotinia sclerotiorum by five fungal agents in sterile and natural field soil. All five biocontrol agents were effective in reducing the survival of sclerotia of S. sclerotiorum in sterile soil under dry (9% moisture) or wet (24% moisture) conditions at 20°C, but only Coniothyrium minitans was effective in natural soil. Coniothyrium minitans was the most effective in reducing sclerotial viability at the temperature range of 15–25°C. Trichoderma virens was effective against sclerotia of S. sclerotiorum to a lesser extent than C. minitans , and in non-autoclaved soil, it performed best at 25°C. Although Epicoccum purpurascens , Talaromyces flavus and Trichothecium roseum were effective against sclerotia of S. sclerotiorum in some instances, they were less effective than C. minitans and T. virens . Sclerotia of S. sclerotiorum conditioned for myceliogenic germination were more vulnerable to attack by the biocontrol agents than dormant sclerotia. The implications are discussed with respect to enhancement of biological control of crop diseases caused by S. sclerotiorum in different geographic regions.     

Elucidation of <Emphasis Type="Italic">veA</Emphasis>-dependent genes associated with aflatoxin and sclerotial production in <Emphasis Type="Italic">Aspergillus flavus</Emphasis> by functional genomics

The aflatoxin-producing fungi, Aspergillus flavus and A. parasiticus, form structures called sclerotia that allow for survival under adverse conditions. Deletion of the veA gene in A. flavus and A. parasiticus blocks production of aflatoxin as well as sclerotial formation. We used microarray technology to identify genes differentially expressed in wild-type veA and veA mutant strains that could be involved in aflatoxin production and sclerotial development in A. flavus. The DNA microarray analysis revealed 684 genes whose expression changed significantly over time; 136 of these were differentially expressed between the two strains including 27 genes that demonstrated a significant difference in expression both between strains and over time. A group of 115 genes showed greater expression in the wild-type than in the veA mutant strain. We identified a subgroup of veA-dependent genes that exhibited time-dependent expression profiles similar to those of known aflatoxin biosynthetic genes or that were candidates for involvement in sclerotial production in the wild type.     

Water potential affects Coniothyrium minitans growth, germination and parasitism of Sclerotinia sclerotiorum sclerotia

Water availability is an important environmental factor which has major effects on fungal activity. The effects of osmotic (KCl amended agar) and matric Polyethylene glycol ((PEG) 8000 amended agar) potentials over the range -0.1 to -5.0MPa on mycelial growth and conidial germination of eight isolates of the sclerotial parasite Coniothyrium minitans was assessed. The influence of soil water potential on the ability of three selected isolates (LU112, LU545, and T5R42i) to parasitise sclerotia of the plant pathogen Sclerotinia sclerotiorum was determined. For all eight C. minitans isolates, decreasing osmotic and matric potentials caused a reduction in mycelial growth and conidial germination. Isolates were more sensitive to decreasing matric potential than osmotic potential. Across the isolates, growth at an osmotic potential of -5.0MPa was 30-70% of the growth seen in the control, whereas less than 20% of the control growth was seen at the corresponding matric potential. Across all isolates no conidial germination was seen at matric potential of -5.0MPa. The C. minitans isolates varied in their sensitivity to decreasing water potentials. Mycelial growth and conidial germination of three isolates (LU112, Conio, and CH1) were more tolerant of low osmotic potential and matric potential with respect to mycelial growth. Isolates T5R42i and LU430 were least tolerant. In contrast, conidial germination of isolates Conio, LU545, and T5R42i were less sensitive to decreasing matric potential. Soil water potential was seen to affect infection and viability of sclerotia by the three C. minitans isolates. Isolate LU545 reduced sclerotial viability over a wider water potential range (-0.01 to -1.5MPa) compared with LU112 (-0.01 to -1.0MPa), with isolate T5R42i being intermediate. Indigenous soil fungi (Trichoderma spp. and Clonostachys rosea) were recovered from sclerotia but did not result in reduction in sclerotial viability. The relevance of these results in relation to biocontrol activity of C. minitans in soil is discussed.     

Biological control of Botrytis gray mould and Sclerotinia drop in lettuce

Research was carried out to evaluate the effectiveness of the biological control of two most important fungal diseases of lettuce (Lactuca sativa L.): 1) Botrytis Gray Mould caused by Botrytis cinerea Pers. ex Fr.; 2) Sclerotinia Drop caused by two pathogenic fungi, Sclerotinia sclerotiorum (Lib.) De Bary and/or Sclerotinia minorJagger. Biological control in lettuce was carried out: 1) using Coniothyrium minitans Campbell, an antagonist fungus that attacks and destroys sclerotia within the soil; 2) identifying lettuce genotypes showing less susceptibility or tolerance. The object of this research was to find control strategies to reduce chemical treatments. The use of resistant varieties is one of the most economical ways to control vegeTable diseases. The lettuce genotypes showing in preliminary trials the best behaviour to the sclerotial diseases were compared in a randomized complete block experiment design and replicated four times. Observations were carried out from February up to April registering the number of diseased plants and yield. The pathogens were isolated on PDA medium and identified. The isolates grown onto PDA plates, after incubation for 6 weeks, allowed obtaining sclerotia that were the target of C. minitans in biological control trials. In laboratory, in controlled conditions, 27 small plots (30 cm in diameter each) with disinfected soil were performed. In 18 plots 9 sclerotia were inoculated (per plot, three of each fungus) and in 9 plots of them a suspension of the antagonist fungus was added. Subsequently, three lettuce varieties were transplanted. For each variety were compared: 1) untreated plots; 2) treated plots with sclerotia only; 3) treated plots with sclerotia and C. minitans suspension. The number of diseased plants was recorded. According to symptom evaluation scale, ranged from 0 (no disease) up to 10 (100% necrotic leaves or dead plants) the plants were grouped into infection classes, calculating the McKinney index. In greenhouse trials, "LM 1307" genotype showed less significant susceptibility to Botrytis Gray Mould (0-2% of affected plants), while "Ninja" and "Charmy" showed 4-11% and 16-26% of diseased plants, respectively. The yields were 69.7, 62.7, 55.3 t/Ha, respectively. In laboratory tests, the McKinney index gave the following results: no disease in all untreated plants; 38.3, 54.2 and 89.2% in "LM 1307", "Ninja" and "Charmy" treated with sclerotia only, respectively; 2.5, 7.5 and 20.8% in "LM 1307", "Ninja" and "Charmy" treated with sclerotia and C. minitans, respectively. In conclusion, the less susceptibility of the genotypes to sclerotial diseases and the use of hyperparasites of sclerotia of phytopathogenic fungi exhibited best results.     

Ciborinia gentianae sp. nov., the causal organism of sclerotial flower blight of cut-flower gentians

A new Ciborinia causing sclerotial flower blight of cut-flower gentians (Gentiana triflora var. japonica and interspecific hybrids between related species or varieties) is described as Ciborinia gentianae on the morphological basis of sclerotia and apothecia. The characteristics of Ciborinia gentianae are (1) an abundant production of spermodochia in the hollow cavity of host stems; (2) flat and thin sclerotia produced beneath the epidermis and the inclusion of host vascular remnants within their medulla; (3) globose cells composed of ectal excipulum of apothecia; (4) elongated cells with a slight apical swelling in ectal excipulum at the apothecial margin; and (5) tetra nucleate ascospores. Asci and ascospores mounted in Melzer's reagent measured 156–208 × 8–12 μm and 11.8–15 × 5.5–7.1 μm, respectively.     

L-arginine is essential for conidiation in the filamentous fungus Coniothyrium minitans

Conidiation is important in the life cycles of mitosporic fungi for survival and transmission. A full-length cDNA of one gene named CMCPS1 encoding L-arginine-specific carbamoyl-phosphate synthase was obtained from Coniothyrium minitans, a sclerotial parasite of the plant pathogenic fungus Sclerotinia sclerotiorum. T-DNA insertional disruption of CMCPS1 resulted in conidiation deficiency of mutant ZS-1T2029, and this was confirmed with the RNAi technique. The phenotype was restored by complementation with L-arginine, and the effect of L-arginine on conidiation may be mediated by nitric oxide, which is catalyzed by nitric oxide synthase (NOS). Conidiation of ZS-1T2029 was restored by sodium nitroprussiate, a NO donor; and conidiation of wild type strain ZS-1 could be suppressed by L-NAME, an inhibitor of NOS. The highest amount of NO in mycelia was detected at an early stage of conidiation (72 hpi) in liquid shake culture medium. Staining with the NO-sensitive fluorescent probe, DAF-FM DA, gave strong fluorescent signals in primordia and young pycnidia. This work presents the first report that L-arginine is involved in conidiation of C. minitans, and the possibility of L-arginine-derived nitric oxide-mediated conidiation among fungi and possible modes of action are discussed.     

Polyamine metabolism during sclerotial development of Sclerotinia sclerotiorum

A study on polyamine metabolism and the consequences of polyamine biosynthesis inhibition on the development of Sclerotinia sclerotiorum sclerotia was conducted. Concentrations of the triamine spermidine and the tetramine spermine, as well as ornithine decarboxylase and S-adenosyl-methionine decarboxylase activities, decreased during sclerotia maturation. In turn, the concentration of the diamine putrescine was reduced at early stages of sclerotial development but it increased later on. This increment was not related to de novo biosynthesis, as demonstrated by the continuous decrease in ornithine decarboxylase activity. Alternatively, it could be explained by the release of putrescine from the conjugated polyamine pool. α-Difluoro-methylornithine and cyclohexylamine, which inhibit putrescine and spermidine biosynthesis, respectively, decreased mycelial growth, but did not reduce the number of sclerotia produced in vitro even though they disrupted polyamine metabolism during sclerotial development. It can be concluded that sclerotial development is less dependent on polyamine biosynthesis than mycelial growth, and that the increase of free putrescine is a typical feature of sclerotial development. The relationship between polyamine metabolism and sclerotial development, as well as the potential of polyamine biosynthesis inhibition as a strategy for the control of plant diseases caused by sclerotial fungi are discussed.     

Sclerotinia nivalis, sp. nov., the pathogen of snow mold of herbaceous dicots in Northern Japan

A newSclerotinia, previously reported asS. intermedia from Japan, is described asSclerotinia nivalis on the morphological basis of the sclerotial anamorph and teleomorph produced in culture. The characters assigning this species to the genusSclerotinia are the tuberoid sclerotia superficially produced on suscepts, the small sclerotia produced on aerial mycelium in culture, the interhyphal spaces in medullary tissue of sclerotia, and the globose cells constructing the ectal excipulum of apothecia. It is distinguishable fromS. sclerotiorum, S. minor, andS. trifoliorum by the intermediate sized sclerotia in culture, binucleate ascospores, the molecular mass of major proteins of sclerotia, and the patterns of esterase isozymes in sclerotial extracts. AlthoughS. nivalis causes snow mold of various dicots, it is a mesophile having an optimum temperature for mycelial growth of around 20°C. It attacks edible burdock(Arctium lappa), Chryhsanthemum morifolium, Ambrosia elatior, carrot(Daucus carota), Angelica acutiloba, Ajuga reptans, andPlantago lanceolata.     

Association of aflatoxin biosynthesis and sclerotialdevelopment in Aspergillus parasiticus

Secondary metabolism in fungi is frequently associated with asexual and sexual development. Aspergillus parasiticus produces aflatoxins known to contaminate a variety of agricultural commodities. This strictly mitotic fungus, besides producing conidia asexually, produces sclerotia, structures resistant to harsh conditions and for propagation. Sclerotia are considered to be derived from the sexual structure, cleistothecia, and may represent a vestige of ascospore production. Introduction of the aflatoxin pathway-specific regulatory gene, aflR, and aflJ, which encoded a putative co-activator, into an O-methylsterigmatocystin (OMST)-accumulating strain,A. parasiticus SRRC 2043, resulted in elevated levels of accumulation of major aflatoxin precursors, including norsolorinic acid (NOR), averantin (AVN), versicolorin A (VERA) and OMST. The total amount of these aflatoxin precursors, NOR, VERA, AVN and OMST, produced by the aflR plus aflJ transformants was two to three-fold that produced by the aflR transformants. This increase indicated a synergisticeffect of aflR and aflJ on the synthesis of aflatoxin precursors. Increased production of the aflatoxin precursors was associated with progressive decrease in sclerotial size, alteration in sclerotial shape and weakening in the sclerotial structure of the transformants. The results showed that sclerotial development and aflatoxin biosynthesis are closely related. We proposed that competition for a common substrate, such as acetate, by the aflatoxin biosynthetic pathway could adversely affect sclerotial development in A. parasiticus. This revised version was published online in June 2006 with corrections to the Cover Date.     

Microscopic observations on the interaction of the mycoparasite Verticillium biguttatum with Rhizoctonia solani and other soil-borne fungi

The mycoparasitic interactions of Verticillium biguttatum with Rhizoctonia solani and with a variety of other soil-borne fungi were investigated in dual cultures. V. biguttatum interacted with various soil fungi by appressed growth along the host hyphae and infrequent penetrations. Intracellular growth and subsequent sporulation, however, only occurred with R. solani, a few binucleate Rhizoctonia and Ceratobasidium spp., and Sclerotinia sclerotiorum. Effective mycoparasitism on sclerotia was restricted to those belonging to R. solani.Electron-microscopic observations revealed that V. biguttatum can penetrate the host cell with infection tubes. This process is probably mediated by enzymatic hydrolysis of the cell wall. Subsequently, trophic hyphae develop within the host cytoplasm, ultimately resulting in death of the host cell.     

Comparison of real-time PCR and microscopy to evaluate sclerotial colonisation by a biocontrol fungus

The biocontrol agent Trichoderma harzianum colonises sclerotia of the plant pathogenic fungus Sclerotinia sclerotiorum. Plating of sclerotia typically has been used to determine the incidence of mycoparasitism, but does not quantify the extent to which individual sclerotia are colonised. We developed a specific PCR primer/probe set for the green fluorescent protein (GFP)-transformant T. harzianum ThzID1-M3, which exhibited high precision and reproducibility. Quantitative real-time PCR was evaluated along with epifluorescence microscopy and image analysis to investigate dynamics of colonisation of sclerotia in non-sterile soil. Amounts of ThzID1-M3 DNA and S. sclerotiorum DNA from entire individual sclerotia were quantified using real-time PCR. Epifluorescence micrographs were captured from sclerotial thin-section samples, and GFP fluorescence from these was quantified using computer image analysis in order to estimate colonisation on a per-sclerotium basis. As determined by either method, ThzID1-M3 colonised sclerotia in soil, and both methods quantified colonisation dynamics over time. In a separate experiment, colonisation of sclerotia on agar plates was observed using confocal laser scanning microscopy to view the GFP-fluorescing hyphae of ThzID1-M3. This method, while highly labour-intensive, provided high spatial resolution of colonisation dynamics. Thus, each method has advantages: microscopy combined with image analysis can provide useful information on the spatial and temporal dynamics of colonisation, while real-time PCR can provide a more precise assessment of the extent of sclerotial colonisation over time and can more easily be used to sample entire sclerotia.     

Non-gramineous hosts ofMyriosclerotinia borealis

On the basis of cultural, anatomical, and electrophoretic studies,Myriosclerotinia borealis (=Sclerotinia borealis) is shown to occur on cultivated non-gramineous plants includingIris ensata var.hortensis (Japanese iris),I. pseudoacorus, I. hollandica (Dutch iris), Perko PVH (a hybrid green manure crop betweenBrassica campestris andB. chinensis), Allium fistulosum, andCampanula portenshlagiana. The fungus did not kill these plants, but produced functional sclerotia, capable of carpogenic germination, on decayed leaves or necrotic lesions of overwintered leaves. The fungus seems to act as a saprophyte colonizing senescent leaves and/or as a weak parasite on plants injured by freezing during winter. In culture, the fungus produces discrete tuberoid sclerotia closely attached to the agar surface; rind differentiation is poor on the under surface of sclerotia. Medullary cells are embedded in a gelatinous matrix showing no distinct intercellular spaces. The ectal excipulum of apothecia produced under artificial conditions is composed of globose cells.Myriosclerotinia borealis is thus shown to be very close toCiborinia on the basis of these sclerotial and apothecial characters.     

Cultivation studies on wild and hybrid strains of Pleurotus tuberregium (Fr.) Sing. on wheat straw substrate

Wild of strains Pleurotus tuberregium from Nigeria and the Australasian-Pacific regions and selected hybrids were studied to determine their growth and sclerotia production capacity on wheat straw substrate. Although the Australasian-Pacific strains showed faster growth rates, the wild strains from Nigeria performed better than the Australasian-Pacific strains in sclerotia yield. Under similar conditions, some hybrids had sclerotial yields that were higher than any wild strain. Our study showed the possibilities of using selected strains and the low cost methods adopted in this experiment, for the cultivation of sclerotia of P. tuberregium.     

The Molecular Phylogeny of a Nematode-Specific Clade of Heterotrimeric G-Protein α-Subunit Genes

In animal olfactory systems, odorant molecules are detected by olfactory receptors (ORs). ORs are part of the G-protein-coupled receptor (GPCR) superfamily. Heterotrimeric guanine nucleotide binding G-proteins (G-proteins) relay signals from GPCRs to intracellular effectors. G-proteins are comprised of three peptides. The G-protein α subunit confers functional specificity to G-proteins. Vertebrate and insect Gα-subunit genes are divided into four subfamilies based on functional and sequence attributes. The nematode Caenorhabditis elegans contains 21 Gα genes, 14 of which are exclusively expressed in sensory neurons. Most individual mammalian cells express multiple distinct GPCR gene products, however, individual mammalian and insect olfactory neurons express only one functional odorant OR. By contrast C. elegans expresses multiple ORs and multiple Gα subunits within each olfactory neuron. Here we show that, in addition to having at least one member of each of the four mammalian Gα gene classes, C. elegans and other nematodes also possess two lineage-specific Gα gene expansions, homologues of which are not found in any other organisms examined. We hypothesize that these novel nematode-specific Gα genes increase the functional complexity of individual chemosensory neurons, enabling them to integrate odor signals from the multiple distinct ORs expressed on their membranes. This neuronal gene expansion most likely occurred in nematodes to enable them to compensate for the small number of chemosensory cells and the limited emphasis on cephalization during nematode evolution. [Reviewing Editor: Dr. John Oakeshott] Damien M. O’Halloran and David A. Fitzpatrick contributed equally to this work.     

一株重寄生枝孢菌的基因组测序及重寄生机制分析

【背景】枝孢菌SYC63是一株具有重寄生作用和抗菌活性的潜在生防菌株,目前尚无研究报道该菌株的全基因组序列,因此限制了其开发与利用。对该菌株进行基因组测序与分析,将进一步了解其重寄生的分子机制,为其在生物防治上的应用奠定研究基础。【目的】解析枝孢菌SYC63基因组序列信息,初步探究该菌的重寄生作用机制。【方法】利用二代高通量测序平台对枝孢菌SYC63进行全基因组测序,运用相关软件对其测序数据进行基因组组装、基因功能注释、预测次级代谢产物合成基因簇并分析重寄生相关的碳水化合物酶类基因等。【结果】基因组组装后共得到17个contigs,总长度为31 912 211 bp,GC含量为52.80%,预测到12 327个编码基因。其中,4 029、949和6 595个基因分别能在KEGG、COG和GO数据库中被注释到,同时还预测到25个次级代谢产物合成基因簇。对重寄生机制相关的碳水化合物酶类进行分析并与重寄生菌株(拟盘多毛孢菌、木霉及盾壳霉)比较,发现该菌具有较多的糖苷水解酶和糖脂酶基因,而且细胞壁降解酶类基因经锈菌孢子壁处理后在转录组测序中显著上调表达,初步分析了该菌与重寄生木霉在分子水平上的...