Mycoparasite Coniothyrium Minitans Research Articles

Sclerotinia sclerotiorum Agglutinin Modulates Sclerotial Development, Pathogenicity and Response to Abiotic and Biotic Stresses in Different Manners.

Sclerotinia sclerotiorum is an important plant pathogenic fungus of many crops. Our previous study identified the S. sclerotiorum agglutinin (SSA) that can be partially degraded by the serine protease CmSp1 from the mycoparasite Coniothyrium minitans. However, the biological functions of SSA in the pathogenicity of S. sclerotiorum and in its response to infection by C. minitans, as well as to environmental stresses, remain unknown. In this study, SSA disruption and complementary mutants were generated for characterization of its biological functions. Both the wild-type (WT) of S. sclerotiorum and the mutants were compared for growth and sclerotial formation on potato dextrose agar (PDA) and autoclaved carrot slices (ACS), for pathogenicity on oilseed rape, as well as for susceptibility to chemical stresses (NaCl, KCl, CaCl2, sorbitol, mannitol, sucrose, sodium dodecyl sulfate, H2O2) and to the mycoparasitism of C. minitans. The disruption mutants (ΔSSA-175, ΔSSA-178, ΔSSA-225) did not differ from the WT and the complementary mutant ΔSSA-178C in mycelial growth. However, compared to the WT and ΔSSA-178C, the disruption mutants formed immature sclerotia on PDA, and produced less but larger sclerotia on ACS; they became less sensitive to the eight investigated chemical stresses, but more aggressive in infecting leaves of oilseed rape, and more susceptible to mycoparasitism by C. minitans. These results suggest that SSA positively regulates sclerotial development and resistance to C. minitans mycoparasitism, but negatively regulates pathogenicity and resistance to chemical stresses.

Cloning and Molecular Characterization of CmOxdc3 Coding for Oxalate Decarboxylase in the Mycoparasite Coniothyrium minitans.

Coniothyrium minitans (Cm) is a mycoparasitic fungus of Sclerotinia sclerotiorum (Ss), the causal agent of Sclerotinia stem rot of oilseed rape. Ss can produce oxalic acid (OA) as a phytotoxin, whereas Cm can degrade OA, thereby nullifying the toxic effect of OA. Two oxalate decarboxylase (OxDC)-coding genes, CmOxdc1 and CmOxdc2, were cloned, and only CmOxdc1 was found to be partially responsible for OA degradation, implying that other OA-degrading genes may exist in Cm. This study cloned a novel OxDC gene (CmOxdc3) in Cm and its OA-degrading function was characterized by disruption and complementation of CmOxdc3. Sequence analysis indicated that, unlike CmOxdc1, CmOxdc3 does not have the signal peptide sequence, implying that CmOxDC3 may have no secretory capability. Quantitative RT-PCR showed that CmOxdc3 was up-regulated in the presence of OA, malonic acid and hydrochloric acid. Deletion of CmOxdc3 resulted in reduced capability to parasitize sclerotia of Ss. The polypeptide (CmOxDC3) encoded by CmOxdc3 was localized in cytoplasm and gathered in vacuoles in response to the extracellular OA. Taken together, our results demonstrated that CmOxdc3 is a novel gene responsible for OA degradation, which may work in a synergistic manner with CmOxdc1.

Host Transcriptional Response of Sclerotinia sclerotiorum Induced by the Mycoparasite Coniothyrium minitans.

Mycoparasite Coniothyrium minitans parasitizes specifically the mycelia or sclerotia of Sclerotinia sclerotiorum, a worldwidely spread plant fungal pathogen causing serious diseases on crops. The interaction of C. minitans with S. sclerotiorum remains reciprocal and complex and little is known, especially on the side of the host (S. sclerotiorum). In this study, the early transcriptional response of S. sclerotiorum to the mycoparasitism by C. minitans was explored and the differentially expressed genes (DEGs) were analyzed. Based on GO ontology, KEGG pathway and fungal categories database, 887 up-regulated DEGs were enriched in the growth related function (i.e., rRNA processing, ribosome biogenesis, binding and transport), while the 546 down-regulated DEGs were enriched in the stress-related functions (i.e., oxidoreductase, response to stress and heat and the chorismate biosynthetic process). The expression of shikimate pathway and the biosynthesis of phenylalanine involving genes was significantly suppressed. Furthermore, 581 unenriched DEGs were explored in the parasitizing process and were mapped on the Pfam domains of redox enzymes, Alpha/Beta hydrolase, haloacid dehalogenase, and other universal conserved domain containing proteins. Thirty-two DEGs encoding candidate effectors, with 16 up-regulated and 16 down-regulated, were observed with diverse function. SS1G_11912 (encoding SsNEP2) was significantly up-regulated and may function in the parasitism. The involving of the shikimate pathway of phenylalanine biosynthesis and effector candidates were discussed. The results provide a basal understand on the interaction of S. sclerotiorum and C. minitans.

Nox Complex signal and MAPK cascade pathway are cross-linked and essential for pathogenicity and conidiation of mycoparasite Coniothyrium minitans.

The NADPH oxidase complex of a sclerotial mycoparasite Coniothyrium minitans, an important biocontrol agent against crop diseases caused by Sclerotinia sclerotiorum, was identified and its functions involved in conidiation and mycoparasitism were studied. Gene knock-out and complementary experiments indicated that CmNox1, but not CmNox2, is necessary for conidiation and parasitism, and its expression could be significantly induced by its host fungus. CmNox1 is regulated by CmRac1-CmNoxR and interacts with CmSlt2, a homolog of Saccharomyces cerevisiae Slt2 encoding cell wall integrity-related MAP kinase. In ΔCmNox1, CmSlt2-GFP fusion protein lost the ability to localize to the cell nucleus accurately. The defect of conidiation in ΔCmRac1 could be partially restored by over-expressing CmSlt2, indicating that CmSlt2 was a downstream regulatory factor of CmNox1 and was involved in conidiation and parasitism. The expressions of mycoparasitism-related genes CmPks1, Cmg1 and CH1 were suppressed in the knock-out mutants of the genes in CmNox1-CmSlt2 signal pathway when cultivated either on PDA. Therefore, our study infers that CmRac1-CmNoxR regulates CmNox1-CmSlt2 pathway in regulating conidiation and pathogenicity of C. minitans.

Phosphoribosylamidotransferase, the first enzyme for purine de novo synthesis, is required for conidiation in the sclerotial mycoparasite Coniothyrium minitans

Coniothyrium minitans is an important sclerotial parasite of the fungal phytopathogen, Sclerotinia sclerotiorum. Previously, we constructed a T-DNA insertional library, and screened for many conidiation-deficient mutants from this library. Here, we report a T-DNA insertional mutant ZS-1T21882 that completely lost conidiation. In mutant ZS-1T21882, the T-DNA was integrated into a gene (CmPrat-1) which encodes phosphoribosylamidotransferase (PRAT, EC 2.4.2.14), an enzyme catalyzing the first committed step in de novo purine nucleotide synthesis. Gene replacement and complementation experiments confirmed that phosphoribosylamidotransferase is essential for conidiation of C. minitans. Mutant ZS-1T21882 did not grow on modified Czapek-Dox broth (MCD), but it grew well on MCD amended with IMP or AMP. The conidial production of this mutant was dependent on the dosage of IMP amended. At low concentrations, such as 0.1mM and 0.25mM, the mutant produced very few pycnidia, while up to 0.75mM or higher, the conidiation of this mutant was restored completely. cAMP could not restore the conidiation of mutant ZS-1T21882 when amended into MCD, but could when amended into PDA. Neither GMP nor cGMP could restore the conidiation in MCD or in PDA. Our findings suggest that phosphoribosylamidotransferase is essential for conidiation of C. minitans via adenosine related molecules. Furthermore, when dual cultured with its host, this mutant produced conidia in the host mycelium and on the sclerotia of S. sclerotiorum, but not in dead mycelium or on dead sclerotia, suggesting that C. minitans is likely to able to obtain adenosine or related components from its host during parasitization.

Susceptibility of Sclerotinia sclerotiorum strains different in oxalate production to infection by the mycoparasite Coniothyrium minitans

Three strains of Sclerotinia sclerotiorum, namely Ep-1PB (PB), Ep-1PK (PK) and Ep-1PNA5 (A5), were compared for the production of oxalic acid (OA) on potato dextrose agar (PDA) and Maxwell agar medium (MAM) and for mycelial susceptibility to infection by the mycoparasite Coniothyrium minitans on PDA. Results showed that strain PB produced negligible oxalate, whereas strain PK was detected to produce oxalate, but much less than that produced by strain A5. The three investigated strains differed slightly in mycelial growth rates and mycelial biomass on PDA. However, colonies of strains PB and PK formed on PDA were more susceptible to invasion by C. minitans than colonies of strain A5. Meanwhile, amendment of synthetic oxalate in PDA at 0.25–2.00 mg g−1 medium suppressed aggressiveness of C. minitans in invasion of colonies of S. sclerotiorum strain PB developed on this medium. These results suggest that infection of hyphae of S. sclerotiorum is negatively affected by the presence of oxalate. The importance of oxalate degradation by C. minitans in its mycoparasitism on hyphae of S. sclerotiorum provides a clue for improvement of the biocontrol efficacy of C. minitans in the future.

Some environmental factors affect growth and antibiotic production by the mycoparasite Coniothyrium minitans

The effects of temperature and pH on growth and antibiotic production by three isolates of Coniothyrium minitans (Conio, Contans and IVT1), known to produce the macrolide antibiotic macrosphelide A, were examined in modified Czapek Dox broth (MCD). Antibiotic production was determined by incorporating heated (60°C for 5 min) C. minitans spent culture filtrates of MCD (10%, v/v) into potato dextrose broth and assessing the ability of the filtrates to inhibit growth of S. sclerotiorum. All isolates grew over the temperature range of 10–30°C, with the optimum at approximately 15–20°C. Antibiotics were produced by all isolates at 10–30°C. Culture filtrates of MCD from all isolates incorporated into PDB inhibited growth of S. sclerotiorum by >50%, whereas there was a reduction in inhibition at 30°C for Conio and IVT1 but not Contans. All three isolates grew over the pH range of 3–7, with greater biomass production in buffered pH 3–5 than the unbuffered control (pH 4.8) media. Antibiotics were produced by all isolates at pH 3–5. Culture filtrates of MCD from all three isolates grown at pH 3–5 inhibited growth of S. sclerotiorum, with the greatest effect on inhibition observed at pH 3. There were no differences in growth inhibition between isolates at pH 3 and 4, but culture filtrates from Conio grown at pH 5 inhibited S. sclerotiorum more than those of IVT1 grown at the same pH. The significance of these results for biocontrol and optimizing antibiotic production by C. minitans is discussed.

Chemical and biological control of Sclerotinia sclerotiorum in witloof chicory culture.

Sclerotinia sclerotiorum (Lib.) de Bary is a major pathogen of witloof chicory. For lack of authorised field treatment, post-harvest sprays with dicarboximide fungicides have been standard practice since the 1970s to prevent root rot and chicory heart decay during the forcing phase. However, the registration of procymidone and vinclozolin has been withdrawn in Europe. The development of organic agriculture and the necessity to reduce fungicide applications in conventional agriculture prompted an assessment of the efficacy of new fungicides and the use of the mycoparasite Coniothyrium minitans (Campbell). A mixture of the fungicides fludioxonil and cyprodinil (Switch(®) ) applied on chicory roots achieved a very good control of S. sclerotiorum (up to 95%). The use of C. minitans limited root infection, both when applied in the field (50-65% efficacy) and before the forcing period (post-harvest treatment up to 80%). In organic agriculture, two treatments with C. minitans (in field and later at the forcing period) could improve protection against S. sclerotiorum. In conventional agriculture, after the field biological treatment, a post-harvest chemical treatment could be applied. The addition of other prophylactic methods could lead to a high level of performance in practice against decay caused by S. sclerotiorum.

Effects of soil temperature and moisture on survival of Coniothyrium minitans conidia in central China

A study was conducted to investigate effects of soil temperature and moisture on survival of the mycoparasite Coniothyrium minitans in soils free of its host fungus Sclerotinia sclerotiorum in central China. Survival of C. minitans conidia was monitored in non-irrigated soil (yellow-brown clay soil) and in submerged soil under the influence of seasonal fluctuations of soil temperature from −0.1 to 37 °C and precipitation from 1.2 to 10.4 mm d −1. Results showed that in non-irrigated soil, C. minitans survived for 750 days with the concentration decreasing from 7.2 × 10 5 to 3.9 × 10 4 cfu g −1 soil. In submerged soil, C. minitans survived for 150 days over the summer-to-autumn period and the winter-to-spring period with the concentration decreasing more rapidly than that in non-irrigated soil. Studies on the effects of constant temperature and moisture on survival of C. minitans in soil showed that at 4, 10, 20 and 28 °C, C. minitans survived for 360 days in soil containing 6.3%, 18.5% or 45.4% of water (w w −1). At 30, 35, 37 and 40 °C, survival of C. minitans became moisture dependent. Good survival in soil containing 6.3% of water and poor survival in soil containing 18.5% or 45.4% of water were observed. C. minitans survived very poorly (⩽1 day) at 45 and 50 °C irrespective of soil moisture. These results suggest that C. minitans can adapt to soil temperature and moisture conditions in central China and can be a promising agent used to treat soil for control of S. sclerotiorum.

Molecular cloning, characterization and expression analysis of a pacC homolog in the mycoparasite Coniothyrium minitans

Coniothyrium minitans is a mycoparasite and a promising biocontrol agent of Sclerotinia sclerotiorum, an omniovorous plant pathogenic fungus. Our previous studies indicate that mycoparasitism and antibiosis of C. minitans are greatly affected by ambient pH, implying that a pH-regulatory system may exist in C. minitans. In this study, we cloned a putative pH-responsive transcription factor gene, cmpacC, from strain Chy-1 of C. minitans. The full-length DNA sequence of cmpacC (GenBank Acc. No. FJ176399) has one open reading frame (ORF) and two introns. cmpacC putatively encodes a polypeptide with 596 amino acid residues (GenBank Acc. No. ACN43303). The sequence of this polypeptide has three conserved zinc finger domains. Meanwhile, a 2,740 bp-long 5′ flanking sequence of cmpacC was obtained. It contains five copies of PacC binding sequences (5′-GCCAAG-3′). Real-time RT-PCR analysis indicated that with the increase of ambient pH from 3 to 8, expression of cmpacC, as well as the chitinase gene (cmch1) and the β-1,3-glucanase gene (cmg1), increased consistently in C. minitans. Cloning of cmpacC from C. minitans suggests that the conserved pH-dependent signaling pathway might exist in C. minitans. It provides a clue for further deciphering of the pH-regulatory mechanism responsible for the pH-associated interaction between C. minitans and S. sclerotiorum.

Water-assisted dissemination of conidia of the mycoparasite Coniothyrium minitans in soil

A study was conducted to determine water-assisted dissemination of conidia of Coniothyrium minitans (Cm), a mycoparasite of Sclerotinia sclerotiorum (Ss), in four soils (yellow–brown soil, red-clay soil, fluvo-aquic soil and black soil) and one sand. Conidial suspensions (1×107 conidia mL−1) of Cm were applied to sieved (2 mm screen) soil or sand in glass tubes to test vertical dissemination (VD) and in aluminum boxes to test horizontal dissemination (HD) of conidia. Results showed that conidia of Cm could be disseminated with water and spread in soil or sand for 16–20 cm vertically and for 5–10 cm horizontally. The conidial concentration of Cm was logarithmically reduced with the increase in depth of VD or the distance of HD. Dissemination of Cm conidia in sand was better than that in four soils. Potting experiments were done to further understand the potential of water-assisted dissemination of Cm conidia in suppression of Ss carpogenic germination. Results showed that more apothecia were produced by Ss sclerotia located at the soil surface than those at 5 and 10 cm in depth. The minimum Cm concentration for suppression of Ss carpogenic germination was 1000 conidia g−1 soil. Two-season field trials indicated that water-assisted application of Cm was an effective strategy used at the time for transplanting oilseed rape seedlings to suppress Ss carpogenic germination, thereby reducing the primary infection source for sclerotinia diseases of oilseed rape.

Characterization of some culture factors affecting oxalate degradation by the mycoparasite Coniothyrium minitans

To find possible approaches to utilize the mechanism of oxalate degradation by Coniothyrium minitans (Cm) in controlling the plant pathogen Sclerotinia sclerotiorum (Ss). Differences in oxalate degradation by different Cm strains and effects of the initial oxalate concentration, ambient pH and nutrient factors on mycelial growth and oxalate degradation by Cm were studied in shaken cultures. Results showed that two wild-type Cm strains, Chy-1 and ZS-1, did not differ in oxalate degradation in modified potato dextrose broth (mPDB) amended with oxalic acid (OA). Cm could grow in mPDB amended with sodium oxalate (SO-mPDB) at pH 6.5 or with ammonium oxalate (AO-PDB) at pH 6.2, but oxalate degradation was very low; oxalate degradation was greatly enhanced in SO- or AO-mPDB with pH being lowered to 2.8-2.9. Similarly, oxalate degradation was higher than 90% in OA-amended mPDB at pH 4.4 but was reduced to be <22% at pH 7.0. Five carbon sources and three nitrogen sources investigated and nutrients from mycelia and sclerotia of Ss were favorable for the growth of Cm and OA degradation by Cm. Cm can degrade oxalate under acidic pH. Exudates from mycelia or sclerotia of Ss may serve as nutrients for Cm mycelial growth and degradation of oxalate secreted by Ss. The finding of oxalate degradation laid a foundation for mining-related genes in Cm for engineering plant resistance against Ss. Elucidation of the importance of acidic pH and nutrients from Ss in oxalate degradation by Cm will help to understand the interaction between Cm and Ss.

Antimicrobial activity ofConiothyrium minitansand its macrolide antibiotic macrosphelide A

Assessment of antimicrobial activity of the mycoparasite Coniothyrium minitans and its macrolide antibiotic macrosphelide A. Thirteen isolates of C. minitans were tested for ability to inhibit a number of filamentous fungi, yeasts, oomycetes and bacteria in agar based tests. Activity was found against some ascomycetes, basidiomycetes, oomycetes and Gram-positive bacteria, but not against zygomycetes, yeasts or Gram-negative bacteria tested. Six C. minitans isolates (Conio, Contans, IVT1, CM/AP/3118, B279/1, A1/327/1) were found to produce macrosphelide A in liquid culture and no other antibiotics were detected. On agar, macrosphelide A inhibited growth of some ascomycetes, basidiomycetes, oomycetes and all four Gram-positive bacteria tested, including the medically important Staphylococcus aureus with a minimum inhibitory concentration of < or =500 microg ml(-1). There was no inhibition observed against the yeasts and Gram-negative bacteria when macrosphelide A was tested at 700 microg ml(-1). The spectrum and level of activity of macrosphelide A produced by C. minitans against micro-organisms are extended markedly compared to previous reports. Macrosphelide A was effective against Staph. aureus. Further study on the control of this bacterium is merited in view of the development of antibiotic resistance.

Use of Coniothyrium minitans as a biocontrol agent and some molecular aspects of sclerotial mycoparasitism

The use of the sclerotial mycoparasite Coniothyrium minitans as a biological control agent of diseases caused by sclerotium-forming pathogens especially Sclerotinia sclerotiorum is briefly reviewed. A number of studies have examined production and application methods, integrated control, ecology, and modes of action in order to understand the biology of the mycoparasite and enhance activity and reproducibility of use. Recently, development of a number of molecular-based techniques has begun to allow the examination of genes involved in mycoparasitism. Some of these procedures have been applied to identify pathogenicity genes involved in the infection of sclerotia of S. sclerotiorum by C. minitans and this work is discussed.

Effects of ambient pH and nutritional factors on antifungal activity of the mycoparasite Coniothyrium minitans

Antifungal activity (AA) of the mycoparasite Coniothyrium minitans (CM) against Sclerotinia sclerotiorum (SS), designated as AA CM–SS, was detected by measuring the effect of cell-free cultural filtrates of CM on inhibition of growth (IG) of SS on potato dextrose agar. Using this bioassay method, effects of ambient pH and nutritional factors on AA CM–SS in CM cultures grown in various liquid media were investigated. Results showed that production of AA CM–SS by CM was closely related to ambient pH. After incubation for 9–15 days, AA CM–SS was high in modified Czapek–Dox broth (MCD) with IG values being 78–90%, but low in potato dextrose broth (PDB) with IG values being 9–18%. In contrast, pH value was as low as 3.3–3.6 in the MCD cultures, whereas as high as 6.0–7.2 in the PDB cultures. In the pH-buffered experiment, AA CM–SS was detected in MCD cultures of CM buffered at pH 2–8 with the optimum pH being 3–5, under which AA CM–SS in PDB cultures of CM was greatly enhanced with the IG values reaching up to 46–67%. Results also indicated that AA CM–SS in CM cultures can be affected by nutritional factors and their effects were also related to ambient pH. In the basal medium Czapek mineral salts, most carbon and nitrogen sources investigated were favourable to CM to produce AA CM–SS with the IG values being 73–91% and the final pH values being 2.7–4.1. In the other basal medium potato extracts, a few carbon and nitrogen sources were favourable for CM to produce AA CM–SS with the IG values being 40–60% and the final pH values being 2.7–5.5. In the pH-buffered experiments with selected carbon and nitrogen sources, or with mycelial or sclerotial extracts of SS, AA CM–SS was much higher in CM cultures buffered at pH 3 than that buffered at pH 6. Additional experiments showed that AA CM–SS could be induced by the phytotoxin oxalic acid (OA), but not by sodium oxalate, further suggesting the importance of acidic pH in production of AA CM–SS by CM. Meanwhile, leaf blight incidence on Chinese cabbage caused by SS was greatly reduced in the treatment of CM conidia pre-germinated in PDB plus OA (16 mM) than that in the treatment of CM conidia pre-germinated in PDB alone. This study suggests that ambient pH plays an important role in regulation of the antifungal activity of CM against SS.

Suppression of Sclerotinia sclerotiorum by antifungal substances produced by the mycoparasite Coniothyrium minitans

A study was conducted to investigate production of antifungal substances (AFS) by Coniothyrium minitans (Cm), a mycoparasite of Sclerotinia sclerotiorum (Ss), in modified Czapek-Dox (MCD) broth and potato dextrose broth (PDB), and effects of AFS of Cm on mycelial growth and germination of sclerotia and ascospores of Ss and incidence of leaf blight of oilseed rape caused by Ss. Results showed that mycelial growth of Ss was reduced by 41.6 and 84.5% on 3 day-old cultures grown on potato dextrose agar (PDA) amended with 10% (v v−1) of cultural filtrates of Cm grown in MCD (MCDcm) after incubation for 6 and 15 days, respectively, and by 2.7 and 15.7% on PDA amended with 10% (v v−1) of cultural filtrates of Cm grown in PDB for 6 and 15 days, respectively. In addition to retardation of mycelial growth, morphological abnormality of Ss such as hyphal swellings and cytoplasm granulation were also observed in colonies grown on PDA amended with cultural filtrates of MCDcm. Sclerotia of Ss soaked in the filtrates of MCDcm for 24 h remained viable, but their ability to undergo myceliogenic germination on PDA was delayed, compared to sclerotia treated with MCD. Germination of ascospores of Ss was unaffected on PDA amended with 10% of the filtrates of MCDcm. However, germ tubes of Ss were shortened and deformed by the formation of hyphal swellings in the treatment of MCDcm. Treatment of leaves of oilseed rape with cultural filtrates of MCDcm reduced incidence of leaf blight caused by Ss, compared to the controls (water or MCD). This study suggests that AFS produced by Cm plays an important role in the suppression of mycelial growth and germ-tube development of ascospores of Ss and that there is potential for using AFS of Cm to control leaf blight of oilseed rape caused by ascospores of Ss.

Biocontrol and epidemiology of lettuce drop caused bySclerotinia minorat Bacchus Marsh, Victoria

The mycoparasite Coniothyrium minitans, but not a commercial preparation of Trichoderma or a local isolate of Trichoderma virens, significantly increased the percentage of sclerotia infected and reduced the percentage viability of sclerotia when applied to lettuce plants showing the earliest symptoms of lettuce drop caused by Sclerotinia minor in commercial crops. C. minitans always infected untreated controls in these experiments and was evidently a natural resident in the soils; it is likely that it effects a degree of natural biocontrol of sclerotia of S. minor in these soils. In spring and summer, the viability of untreated sclerotia among plant debris on the surface of the soil declined greatly, providing evidence that prolonging the exposure of sclerotia on the soil surface before cultivation for the next crop can greatly reduce sclerotial inoculum. Far fewer sclerotia were formed on infected, untreated plants in late spring to summer than in autumn and early spring. Epidemics of lettuce drop caused by S. minor on salad lettuces developed rapidly in the last 1 or 2 weeks before harvest. This was as a result of infection mainly via the lower leaves drooping onto the surface of the soil or touching adjacent infected plants. Clumping of diseased plants was evident only late in the epidemic. Cutting of infected mature plants at an early stage of symptom development and leaving them in the field did not reduce the number of sclerotia formed on them compared to plants left in the ground, indicating that cut plants should be removed from the field. Possibilities for disease control are discussed.

Survival of the mycoparasite Coniothyrium minitans on flower petals of oilseed rape under field conditions in central China

Abstract The mycoparasite Coniothyrium minitans is an effective biocontrol agent of stem rot of oilseed rape (Brassica napus) caused by Sclerotinia sclerotiorum. This study was to investigate survival of C. minitans on flower petals of oilseed rape under field conditions in central China using the mutant strain SV-5-2 which is tolerant to the fungicide vinclozolin, and the two bactericides benzylpenicillin sodium (BPS) and streptomycin sulfate (SS). Results showed that potato dextrose agar (PDA) amended with vinclozolin (500 μg ml−1), BPS (500 μg ml−1), and SS (500 μg ml−1) was suitable for detection of conidial germination and mycelial growth of C. minitans strain SV-5-2. Results of the five field experiments (A–E) conducted in 2004 and 2005 showed that C. minitans strain SV-5-2 applied to flower racemes of oilseed rape could be detected for five days in four experiments and three days in one experiment. The rate of survival of C. minitans decreased with time and at the last sampling date, the survival rate was 17.3, 14.3, 35.0, 62.6, and 1.9% for experiments A, B, C, D, and E, respectively. Moreover, survival of C. minitans was affected by weather conditions. Relatively, high rates of survival of C. minitans were observed under sunny weather in the experiments C and D, implying that conidia of C. minitans are tolerant to sunlight irradiation for a period of at least five days in the experiment C and three days in the experiment D. Low survival rates of C. minitans were observed under light rain weather in the experiment A, and heavy rain weather in the experiments B and E. The low recovery rate of C. minitans under rainy conditions suggests a possibility of rain wash of the inoculum applied to the florets. Methods for improving efficacy of C. minitans in the field are discussed.

Biological Control of Sclerotinia Diseases of Rapeseed by Aerial Applications of the Mycoparasite Coniothyrium minitans

Indoor and field experiments were conducted to evaluate the efficacy of applying the mycoparasite Coniothyrium minitans to the aerial parts of rapeseed plants at the flowering stage to control sclerotinia diseases caused by Sclerotinia sclerotiorum. Under controlled conditions, a petal inoculation technique was used to determine the effect of conidial suspensions of C. minitans on suppression of sclerotinia leaf blight. Results showed that C. minitans was effective in inhibiting infection initiated by ascospores of S. sclerotiorum on flower petals by restricting mycelial growth of the pathogen. Suppression of lesion development was related to the conidial concentration of C. minitans, with larger lesions at low concentration (5×103conidia ml−1), but smaller lesions at high concentration (5×104 conidia ml−1 or higher). When C. minitans-treated rapeseed leaves were inoculated with mycelia of S. sclerotiorum, C. minitans failed to prevent infection of leaves, but caused a significant reduction in number of sclerotia produced on the diseased leaves. No significant difference in efficacy was detected between the two isolates of C. minitans, LRC 2137 and Chy-1, on the two rapeseed cultivars, Westar (spring type) and Zhongyou 821 (winter type). Results of field trials showed a significant reduction of stem rot of rapeseed in four (1997, 1999, 2003 and 2004) out of five years by aerial application of C. minitans, compared with controls. No significant difference in suppressive efficacy was observed between the treatments of C. minitans (106 conidia ml−1), C. minitans (106 conidia ml−1) + benomyl (50 μg ml−1) and benomyl (100 μg ml−1) in 2003, and between the treatments of C. minitans (106 conidia ml−1), C. minitans (106 conidia ml−1) + vinclozolin (100 μg ml−1) and vinclozolin (500 μg ml−1) in 2004. Sclerotia of S. sclerotiorum collected from diseased plants in plots treated with C. minitans in 1999, 2000 and 2003, or with C. minitans + benomyl in 2003 were infected by C. minitans at frequencies ranging from 21.3 to 54.5%. This study concludes that aerial spraying of C. minitans is an effective method for controlling sclerotinia diseases of rapeseed.

Effect of Combined Treatment of Pasteurisation and Coniothyrium minitans on Sclerotia of Sclerotinia sclerotiorum in Soil

Integrated control of soil-borne plant pathogens such as Sclerotinia sclerotiorum is becoming more important as the soil fumigant methyl bromide is being phased out of use. Two alternative methods of control that have been found to reduce viability of sclerotia are steam sterilisation (pasteurisation) of soil or the application of the mycoparasite Coniothyrium minitans. This work investigated the possibility of integrating these two control measures. Soil was pasteurised in an autoclave, using a temperature of 80 °C for 3 min to simulate the possible temperatures reached by soil steaming machines for field use. Coniothyrium minitans was subsequently applied to the pasteurised soil to assess the effects of the combination of control measures in reducing sclerotial viability of S. sclerotiorum. Similar results were found in two soil types. Either method used individually was effective in decreasing the number of viable sclerotia, but no further reduction in sclerotial viability was seen when the two methods were combined. Coniothyrium minitans was found to colonise pasteurised sclerotia significantly quicker than untreated sclerotia, and it was seen that there was an increase in number of C. minitans in pasteurised soil in the presence of sclerotia. Experiments were also conducted to investigate the effect of application timing of the biocontrol agent to soil following pasteurisation, in relation to sclerotial infection. Here, two different isolates of S. sclerotiorum were used, with similar results. Application of C. minitans to soil immediately following pasteurisation resulted in sclerotial infection by the mycoparasite, but application 7 days or more after soil pasteurisation resulted in low recovery of the biocontrol agent from sclerotia, possibly due to the mycoparasite being masked by the presence of other fungi which colonised the sclerotia first.