Colonisation of sclerotia of Sclerotinia sclerotiorum by a fungivorous nematode

Aphelenchoides saprophilus nematodes fed on sclerotia, mycelium, and alginate-formulated pellets of Sclerotinia sclerotiorum, mycelium of Trichoderma harzianum, and mixed fungal cultures. As many as 500 nematodes were found inside individual sclerotia. Results suggest potential impacts of fungivory on S. sclerotiorum and its ecological interactions with plant hosts and biocontrol fungi.     

Effects of colonisation by different strains of Coniothyrium minitans on the viability of sclerotia of Sclerotinia sclerotiorum

White mold is a major disease in commercial soybean production. An effective measure to reduce the negative effects of Sclerotinia sclerotiorum is the use of bio-fungicides. Strains of Coniothyrium minitans were isolated and efficacy tests against S. sclerotiorum was studied. The efficacy of pycnidiospores sprays of strain N09 (GenBank Accession No HQ908274) from Iowa, USA and strain CON/M/91-08 of Contans® WG were compared in a series of experiments. Sclerotia viability was significantly (P < 0.05) lower in both sclerotia-infested-sterilized-soils (SISS) and sclerotia-infested-unsterilized-soils (SIUS) sprayed with N09 compared with CON/M/91-08 and control at 3°C for 75d and 90d sampling. Similarly, sclerotia viability was significantly (P < 0.05) lower at 23°C for 45, 60 and 75d sampling in SISS and 45, 75 and 90 d sampling in SIUS compared with CON/M/91-08 and control. In contrast, viability of N09 colonies were significantly (P < 0.05) higher than that of CON/M/91-08 both at 3°C and 23°C in SISS across sampling periods. While in SIUS, N09 colonies were significantly higher at 3°C for 15, 30, 45, 75 and 90 d sampling, and at 23°C for 30, 60 and 75 d sampling. Also, (1) N09 had a faster growth rate and produced 1.5 times more pycnidiospores than CON/M/91-08; (2) mycoparasitism by N09 was faster than CON/M/91-08; and (3) co-inoculation of sclerotia and the strains, N09 showed lower sclerotia reproduction than CON/M/91-08. Our data suggest that the new strain N09 has a greater efficiency than CON/M/91-08 in killing sclerotia.     

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.     

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.     

Effect of volume and concentration of conidial suspensions of Coniothyrium minitans on infection of Sclerotinia sclerotiorum sclerotia

White mould, caused by Sclerotinia sclerotiorum, is a serious disease affecting a wide range of agricultural and horticultural crops. Biological control is one option available to limit its damage. Field experiments to evaluate various concentrations and volumes of Coniothyrium minitans spore suspensions applied to S. sclerotiorum-infected bean crops were conducted in 1997 and 1998. Percentage sclerotia infected by C. minitans were scored. Three replicate experiments were performed in time in 1997 with 21 combinations of isolates, volumes and concentrations, including two controls. In 1998, 22 combinations of isolates, volumes and concentrations plus two controls were used, combined with the absence or presence of a maize buffer, with two replicates for each. Isolates as well as concentration and volume had no effect on infection by C. minitans, but there was a significant effect of total dose (volume×concentration) of inoculum applied over the full range from 100 L ha^-1 at 10⁴ conidia mL^-1 to 1000 L ha^-1 at 10⁷ conidia mL^-1. Percentage infected sclerotia increased linearly with log (dose) as well as from 1 to 4 weeks after application of C. minitans, and reached a level of about 100% at high doses under the humid conditions of 1998. Apothecia of S. sclerotiorum developing from sclerotia in collected soil samples from the 1997 experiment showed no significant effect of C. minitans inoculum dose, but there was a significant effect of the replicate experiments. The influence of weather conditions is highlighted, and the implications of the results for cost-effective biocontrol of S. sclerotiorum are discussed.     

菌核病防治研究进展

菌核病是一种寄主种类广泛的重大植物病害,可侵染450多种重要作物和草类,在我国每年给油菜、大豆以及多种蔬菜带来10～30亿元的损失.介绍了菌核病的症状、危害以及致病机理等,概述了主要的防治措施,并报道了国内外在关于菌核病生物防治、转基因育种、分子机理等方面的研究进展.     

Suppressive soil against Sclerotinia sclerotiorum as a source of potential biocontrol agents: selection and evaluation of Clonostachys rosea BAFC1646

The fungal diversity structures of soils that are suppressive and non-suppressive to Sclerotinia sclerotiorum were characterised and screened for fungal strains antagonistic to the S. sclerotiorum pathogen. Soil suppressiveness was associated with a particular fungal diversity structure. Principal component analysis showed that antagonism by fungal species in suppressive soils was associated with the occurrence of Fusarium oxysporum, Fusarium solani, Talaromyces flavus var. flavus and Clonostachys rosea f. rosea. In particular, C. rosea f. rosea occurred exclusively in suppressive soil samples, suggesting that this morpho-species plays an important role in suppression of S. sclerotiorum diseases. One strain of C. rosea f. rosea (BAFC1646) was selected for further experiments. Dual-culture assays confirmed the antagonistic behaviour of C. rosea f. rosea BAFC1646 against three different S. sclerotiorum strains. Antifungal activity was corroborated by diffusion assays with metabolite extracts. Greenhouse assays with soybean plants showed that the selected C. rosea f. rosea strain reduced the percentage of dead plants when co-inoculated with S. sclerotiorum. In addition, inclusion of C. rosea f. rosea alone increased shoot lengths significantly. In this work, we established the involvement of fungal species in soil suppressiveness and in further assays confirmed that C. rosea f. rosea BAFC1646 exhibits a bioprotective effect against S. sclerotiorum in soybean plants.     

黄绿木霉菌代谢产物对大豆菌核病核盘菌的抑菌能力研究

利用室内实验的方法,研究了黄绿木霉对大豆菌核病菌的抑菌能力。黄绿木霉在与核盘菌（Sclerotinia sderotiorum）对峙培养过程中,5-7d即可将菌核病菌完全覆盖,在形成的菌核上会有黄绿木霉孢子出现,扫描电镜中观察到核盘菌的菌丝体发生变形;其代谢产物在经121℃处理25min后,抑菌作用仍可高达100％;在对黄绿木霉发酵过程中,利用PD培养基即可达到有效的控制大豆菌核病菌菌丝生长的目的,而且利用PD培养基发酵3-4d的黄绿木霉代谢产物抑菌效果最好,抑菌中浓度最低,发酵时间过长与过短,均不利于对病原菌的抑制。经黄绿木霉代谢产物处理的菌核菌电导率与呼吸强度均发生变化,在光学显微镜下观察到菌核菌菌体发生变化,且处理后的菌核菌致病力明显降低。     

Trichoderma harzianum T39 Preparation for Biocontrol of Plant Diseases-Control of Botrytis cinerea , Sclerotinia sclerotiorum and Cladosporium fulvum

Isolate T39 of Trichoderma harzianum (TRICHODEX) is a commercial biocontrol agent. It controls Botrytis cinerea (grey mould) in greenhouse crops and in vineyards, Sclerotinia sclerotiorum (white mould) in various greenhouse and field crops, Cladosporium fulvum (leaf mould) in tomato, and the powdery mildews Sphaerotheca fusca in cucurbits and Leveillula taurica in pepper. T. harzianum T39 was applied in vineyards and greenhouses as part of grey mould management programmes in alternation with chemical fungicides. In the present study, the effect of T39 on diseases of greenhouse crops was demonstrated. The biocontrol agent was applied in formulations containing two concentrations of the active ingredient, or in the presence of oil in cucumber and tomato greenhouses. Suppression of B. cinerea , C. fulvum and S. sclerotiorum was similar when T39 was applied at final active ingredient rates of 0.2 or 0.4 g l -1 , except for one sampling date in one experiment. The addition of JMS Stylet-Oil did not contribute to the control of the above mentioned diseases achieved by T39.     

Trichoderma harzianum enhances the production of nematicidal compounds in vitro and improves biocontrol of Meloidogyne javanica by Pseudomonas fluorescens in tomato

AIMS: To determine the influence of soil-borne fungus Trichoderma harzianum on the biocontrol performance of Pseudomonas fluorescens strain CHA0 and its 2,4-diacetylphloroglucinol (DAPG) overproducing derivative CHA0/pME3424 against Meloidogyne javanica. METHODS AND RESULTS: Amendment of the culture filtrate (CF) or methanol extract of the CF of a T. harzianum strain Th6 to P. fluorescens growth medium enhanced the production of nematicidal compound(s) by bacterial inoculants in vitro. In addition, bacteria overwhelmingly expressed phl'-'lacZ reporter gene when the medium was amended with CF of T. harzianum. Pseudomonas fluorescens and T. harzianum applied together in unsterilized sandy loam soil caused greater reduction in nematode population densities in tomato roots. CONCLUSIONS: Trichoderma harzianum improves root-knot nematode biocontrol by the antagonistic rhizobacterium P. fluorescens both in vitro and under glasshouse conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: The synergistic effect of T. harzianum on the production of nematicidal compound(s) critical in biocontrol may improve the efficacy of biocontrol bacteria against plant-parasitic nematodes. Considering the inconsistent performance of the biocontrol agents under field conditions, application of a mixture of compatible T. harzianum and P. fluorescens would more closely mimic the natural situation and might broaden the spectrum of biocontrol activity with enhanced efficacy and reliability of control.     

Potential biocontrol activity of Arthrobotrys oligospora and Trichoderma harzianum BI against Meloidogyne javanica on tomato in the greenhouse and laboratory studies

In this investigation, the biological control activity of Arthrobotrys oligospora and Trichoderma harzinum BI against the root-knot nematode, Meloidogyne javanica, infecting tomato, was assessed both in in vitro and in in vivo experiments. In greenhouse experiments, tomato seedlings at six-leaf stage were inoculated with 10⁶?spores/ml of A. oligospora and T. harzianum BI and number of 2000 nematode eggs per individual seedling. In in vitro assays, the per cent inhibition of nematode eggs hatching, the death per cent of second-stage juvenile (J2) and proteolytic activity on casein hydrolysis was evaluated. Results showed that A. oligospora and T. harzianum BI decreased the mean numbers of galls, eggmasses and egg per eggmass significantly (p?<?0.05) compared with control. Percentage hatching inhibition of M. javanica treated with A. oligospora and T. harzianum BI was 25 and 52%, respectively. Moreover, A. oligospora and T. harzianum BI significantly increased (p?<?0.05) the mortality rate of M. javanica (J2) after two and four days (74, 85 and 53, 63%, respectively). A. oligospora and T. harzianum BI had a proteolytic activity of 3.9 (U/min per ml) and 2.4 (U/min per ml) at pH 5.0, respectively. Our data suggest that the application of these two fungi in tomato rhizosphere infected with root-knot nematode M. javanica had antagonistic effects on the infection and reproduction of this nematode and the ability to control its population.     

Biological control of Sclerotinia disease by Aspergillus sp. on oilseed rape in the field

Sclerotinia sclerotiorum causes serious yield losses to many crops worldwide. Aspergillus sp. Asp-4, previously shown to inhibit germination of sclerotia of S. sclerotiorum in vitro and in the field, was evaluated in field trials for suppression of this pathogen on oilseed rape. Spray application of Asp-4 to the soil prior to sowing rice in a rice–oilseed rape rotation resulted in a significant reduction in incidence of Sclerotinia stem rot on oilseed rape compared with the non-treated control in two field trials. This application of Asp-4 also resulted in a significant reduction in germination of sclerotia relative to the non-treated control in these field trials, suggesting that this reduction in sclerotial germination led to disease control. Microscopic examination demonstrated that Asp-4 could effectively colonise external and internal portions of sclerotia of S. sclerotiorum in vitro. Incubation of Asp-4 with sterile sclerotial material induced production of β-glucanase and chitinase activities by this isolate; β-glucanase and chitinase being potentially capable of degrading the glucan and chitin polymeric components of sclerotia. Incubation of Asp-4 with sterile sclerotial material also resulted in a significant reduction in dry weight of this sclerotial material relative to the non-treated control in 96?h in vitro experiments. Experiments reported here indicate that Aspergillus sp. Asp-4 has promise as a biological control agent for S. sclerotiorum on oilseed rape. Experiments reported here suggest that disease control results from inhibition of germination of sclerotial resting structures due to mycoparasitic colonisation by Asp-4.     

Hydrocarbons,free fatty acids,and amino acids of sclerotia of Sclerotinia sclerotiorum

Summary Sclerotia of Sclerotinia sclerotiorum (Lib.) D By. were obtained from commercial pea-and bean-cleaning operations or grown on potato-dextrose agar and synthetic glucose-and sucrose-salts agar media. The crude fat (ether extract) content of sclerotia varied from 0.8 to 1.5%. Extraction and fractionation of the lipids followed by gas chromatographic analysis showed that sclerotia from pea cleanings contained one predominant hydrocarbon which was absent from sclerotia produced in the laboratory. Sclerotia from natural sources and grown in the laboratory contained a similar distribution of C₁₈ unsaturated free fatty acids, however, quantitative differences were noted. Palmitic, oleic and linoleic were the major free fatty acids of the laboratory-grown sclerotia while a high proportion of linoleic acid was also found in sclerotia from natural sources. Sclerotia were fractionated into water-soluble and water-insoluble fractions. After acid hydrolysis of the waterinsoluble fraction, both fractions were analyzed for amino acids. Twenty-one compounds, including 2 unknowns, were detected in the soluble fraction. The hydrolyzates contained 19 amino acids, including the same 2 unknowns. Two compounds tentatively identified as ornithine and -aminobutyric acid were found only in the water-soluble fraction. The relative amino acid composition of the water-insoluble fraction of sclerotia from various sources was fairly constant but the arginine content decreased on the synthetic media.     

The importance of water potential range tolerance as a limiting factor on Trichoderma spp. biocontrol of Sclerotinia sclerotiorum

Biological control of fungal phytopathogens is often more variable in efficacy compared with disease suppression achieved by conventional pesticide use. Matching the environmental range of a potential biocontrol agent with that of the target phytopathogen is necessary if consistent disease suppression is to be achieved under field conditions. Strains of Trichoderma that could parasitise sclerotia of Sclerotinia sclerotiorum had their spore germination and mycelial growth (five strains) and ability to parasitise sclerotia (two strains) tested under a range of water potentials under laboratory conditions. Relative mycelial growth and germination of all strains decreased with decreasing osmotic and matric potentials, with matric potential having a greater impact on growth and germination over the range examined. Trichoderma harzianum LU698 mycelial growth was the least affected by decreasing osmotic potential than the other isolates, and Trichoderma atroviride LU141 growth least affected by decreasing matric potential. The germination of LU698 and LU144 was also generally less affected by decreasing osmotic potential, although generally decreasing matric potential had the greatest affect on the germination of LU698 along with T. atroviride LU132. Soil treatments of LU698 and Trichoderma asperellum LU697 reduced sclerotial viability in all but the lowest soil water potential tested, with LU698 being most effective at ?0.1 and ?0.3 MPa after 28 days and LU697 most effective at ?0.01 and ?1.5 MPa after 28 days. We conclude that differences in the tolerance of potential biocontrol agents to changing water potential is an important experimental factor to consider when assaying biocontrol or making predictions of biocontrol efficacy in the field.     

Biological control of Sclerotinia sclerotiorum attacking soybean plants. Degradation of the cell walls of this pathogen by Trichoderma harzianum (BAFC 742)

Two experiments of biological control of Sclerotinia sclerotiorum, one in the greenhouse and the other in the field, were carried out with soybean and Trichoderma harzianum as host and antagonist, respectively. Significant control of disease was achieved in both experiments, but there were no significant differences in plant growths. In the greenhouse, the application of T. harzianum as alginate capsules, increased the survival of soybean plants more than 100% with respect to the disease treatment. In the field, T. harzianum treated plants survived 40% more than those from the disease treatment, showing a similar survival level to control plants. Besides, a significant reduction (62.5%) in the number of germinated sclerotia was observed in the Trichoderma treated plot. Chitinase and 1,3-β- glucanase activities were detected when T. harzianum was grown in a medium containing Sclerotinia sclerotiorum cell walls as sole carbon source. In addition, electrophoretic profiles of proteins induced in T. harzianum showed quantitative differences between major bands obtained in the media induced by S. sclerotiorum cell walls and that containing glucose as a sole carbon source. This revised version was published online in June 2006 with corrections to the Cover Date.     

Mycoparasitism studies of Trichoderma harzianum against Sclerotinia sclerotiorum: evaluation of antagonism and expression of cell wall-degrading enzymes genes

Trichoderma spp. are known for their biocontrol activity against several plant pathogens. A specific isolate of Trichoderma harzianum, 303/02, has the potential to inhibit the growth of Sclerotinia sclerotiorum, an important agent involved in several crop diseases. In this study, the interaction between T. harzianum 303/02 and mycelia, sclerotia and apothecia of S. sclerotiorum was studied by scanning electron microscopy. RT-qPCR was used to examine the expression of 11 genes potentially involved in biocontrol. T. harzianum 303/02 parasitizes S. sclerotiorum by forming branches that coil around the hyphae. The fungus multiplied abundantly at the sclerotia and apothecia surface, forming a dense mycelium that penetrated the inner surface of these structures. The levels of gene expression varied according to the type of structure with which T. harzianum was interacting. The data also showed the presence of synergistic action between the cell-wall degrading enzymes.     

Effect of culture conditions on spore shelf life of the biocontrol agent Trichoderma harzianum

The influence of pH, carbon:nitrogen (C:N) ratio, carbon content and harvesting time on spore attributes of the biocontrol agent Trichoderma harzianum was evaluated. The effect of these culture parameters on viability, shelf-life and ultrastucture was also assessed. pH was a key parameter to manipulate for both growth and sporulation, while carbon concentration and C:N ratio strongly affected spore production time. At fixed pH, the C:N ratio had a limited influence on production yield, but was critical for spore shelf-life. The highest spore longevity was found in a medium with a C:N ratio of 14 and a pH of 7.0, when most resulting spores were still alive after 45 d storage. These spores also remained viable during storage under a broad range of relative humidities, indicating that they would be more sustainable in the field.     

Colony Age of Trichoderma azevedoi Alters the Profile of Volatile Organic Compounds and Ability to Suppress Sclerotinia sclerotiorum in Bean Plants

Common bean (Phaseolus vulgaris L.) is one of the most important crops in human food production. The occurrence of diseases, such as white mold, caused by Sclerotinia sclerotiorum can limit the production of this legume. The use of Trichoderma has become an important strategy in the suppression of this disease. The aim of the present study was to evaluate the effect of volatile organic compounds (VOCs) emitted by Trichoderma azevedoi CEN1241 in five different growth periods on the severity of white mold in common bean. The in vitro assays were carried out in double-plate and split-plate, and the in vivo assays, through the exposure of the mycelia of S. sclerotiorum to the VOCs of T. azevedoi CEN1241 and subsequent inoculation in bean plants. Chemical analysis by gas chromatography coupled to mass spectrometry detected 37 VOCs produced by T. azevedoi CEN1241, covering six major chemical classes. The profile of VOCs produced by T. azevedoi CEN1241 varied according to colony age and was shown to be related to the ability of the biocontrol agent to suppress S. sclerotiorum. T. azevedoi CEN1241 VOCs reduced the size of S. sclerotiorum lesions on bean fragments in vitro and reduced disease severity in a greenhouse. This study demonstrated in a more applied way that the mechanism of antibiosis through the production of volatile compounds exerted by Trichoderma can complement other mechanisms, such as parasitism and competition, thus contributing to a better efficiency in the control of white mold in bean plants.