Determination of cellular carbohydrates in peanut fungal pathogens and baker's yeast by capillary electrophoresis and electrochromatography.

In this work, the quantitation of cellular carbohydrates, namely chitin and glucan, in peanut fungal pathogens and baker's yeast was carried out by capillary electrophoresis (CE) and capillary electrochromatography (CEC). The chitin and glucan of the fungi were hydrolyzed by the enzymes chitinase and glucanase, respectively, to their corresponding sugar monomers N-acetylglucosamine (GlcNAc) and glucose (Glc). These two monosaccharides were then tagged with 6-aminoquinoline (6-AQ) to allow their separation and detection in CE and CEC. The 6-AQ derivatives of GlcNAc and Glc formed the basis for the determination by CE and CEC of chitin and glucan in peanut fungi and baker's yeast. Several parameters affecting the separation of the 6-AQ derivatives of GlcNAc and Glc, including the separation voltage and the composition of the running electrolyte, were investigated. Under the optimized separation conditions, the contents of cellular carbohydrates including N-acetylglucosamine, chitin, glucose, and glucan in some fungi, such as Sclerotinia minor, Sclerotium rolfsii, and baker's yeast, were successfully determined. The method described here allowed the assessment of genetic differences in Sclerotium rolfsii isolates from various locations.     

Proteome changes in leaves of Brassica napus L. as a result of Sclerotinia sclerotiorum challenge

Sclerotinia stem rot, caused by the necrotrophic fungal pathogen Sclerotinia sclerotiorum, is a serious disease of canola (Brassica napus L.). To increase the understanding of the B. napus- S. sclerotiorum interaction, proteins potentially involved in mediating this interaction were identified and characterized. Upon infection of canola leaves by S. sclerotiorum, necrosis of host leaves was observed by 12 h and rapidly progressed during the later time points. These morphological observations were supported by microscopic study performed at different time points after pathogen challenge. Leaf proteins were extracted and analyzed by 2-DE, which revealed the modulation of 32 proteins (12 down- and 20 up-regulated). The identities of these proteins were established by ESI-q-TOF MS/MS and included proteins involved in photosynthesis and metabolic pathways, protein folding and modifications, hormone signaling, and antioxidant defense. Gene expression analysis of selected genes was performed by qRT-PCR, whereas the elevated levels of the antioxidant enzymes peroxidase and superoxide dismutase were validated by enzyme assays. To the authors' best knowledge, this is the first proteomics-based investigation of B. napus-S. sclerotiorum interaction, and the roles of many of the proteins identified are discussed within the context of this pathosystem.     

Loss of nutrient-independence for germination by fungal propagules incubated on soils or on a model system imposing diffusive stress

Nutrient independent conidia of Cochliobolus victoriae and sclerotia of Sclerotium cepivorum, Macrophomina phaseolina, and Verticillium dahliae were incubated aseptically on sand through which a dilute salts solution percolated at a flow rate sufficient to inhibit germination. Propagules were then transferred to a static salts solution to assess germination. Conidia of C. victoriae and sclerotia of S cepivorum became nutrient-dependent (6% germination in salts solution) after 9 and 15 days on sand, respectively. Thirty-five days of diffusive stress were required to attenuate the nutrient-independence of M. phaseolina sclerotia. Sclerotia of V. dahliae lost little of their nutrient-independence even after 45 days of diffusive stress. Viability of C. victoriae and S. cepivorum was reduced after 45 days of diffusive stress, but viability of V. dahliae and M. phaseolina was not. Conidia of C. victoriae gradually became nutrient-dependent when incubated for several weeks on each of five soils. A loam and two sandy loam soils were more effective in decreasing the nutrient-independence of conidia than were two clay loams. Sclerotia of M. phaseolina also lost nutrient-independence when incubated on four of the five soils.Interruption of artificially imposed diffusive stress resulted in increased [¹⁴C]exudation from conidia of C. victoriae and sclerotia of M. phaseolina. Germinability on salts solution of C. victoriae conidia previously made nutrient-dependent was significantly increased, when the conidia were kept at 4°C for 3.5 days before germination assay. Conidia of C. victoriae made nutrient-dependent and then incubated on soils labeled with [¹⁴C]glucose, absorbed twice as much ¹⁴C from a loam and two sandy loam soils as from two clay loam soils. Following incubation on four of the five ¹⁴C-labeled soils, the germinability of the conidia in the absence of nutrients was significantly increased over that of conidia not incubated on these soils.The results suggest that a continued minimal stress may be needed to maintain the nutrient-dependence of some fungal propagules in soil. Interruption of nutrient stress appears to allow nutrient-dependent propagules an opportunity to recoup nutrients from the soil solution or to reorganize endogenous energy reserves whereby the potential for germination is increased.     

Partitioning the spatial and environmental variation of Sclerotinia stem rot on soybean

The variance in survival of Sclerotinia sclerotiorum's sclerotia, carpogenic germination (apothecia) as well as Sclerotinia stem rot (SSR) severity (Disease Severity Index (DSI)) on soybean was partitioned among canopy, soil physico-chemistry and microbiology, cultural practices (2 or 3-y-corn rotation/soybean monoculture and mineral fertilization/urban compost), and spatial matrices in two soils. Partial multiple regression was used to partition the individual SSR variables variance while partial canonical redundancy analysis partitioned the DSI-apothecia and apothecia-survival variance. In clay loam, the sclerotial survival and apothecia variance were mainly explained by the spatial structure of soil physico-chemistry while the DSI did not share this spatial structure and was largely explained by the effects of 3-y-corn rotation on canopy and soil, i.e. lower weed biomass, enhanced soybean yield and fewer apothecia were correlated with disease suppressiveness. In sandy loam, the DSI variance was mostly explained by the spatial structure of canopy and physico-chemistry. Disease suppressiveness, by the interaction of 3-y-corn rotation with urban compost, was largely explained by the enhancement of soil properties, i.e. higher aggregate stability, microbial activity and soil solution concentration in exchangeable ions correlated negatively with carpogenic germination. Partitioning the SSR variance among four matrices of spatial and environmental factors allowed for the first time to interpret and quantify the variance of disease development explained by cultural practices in interaction with the main characteristics of this agroecosystem.     

菊花菌核病病原菌鉴定、抗性筛选与防治

菌核病是菊花生产中的一种重要病害.为明确菊花菌核病病原菌的种类、品种抗性差异和有效的防治药剂,本研究采用组织分离法对病原菌进行分离培养,结合形态学特征、生物学特征和分子生物学研究进行鉴定,并筛选了33个切花菊品种以及7种杀菌剂.结果 表明,从感病植株上分离得到的病原菌能够重新感染菊花,并且再次分离得到的菌株与原菌株相同...     

Suppression of Sclerotinia sclerotiorum apothecial formation by the soil bacterium Serratia plymuthica: identification of a chlorinated macrolide as one of the causal agents

A selection of soil bacteria was screened for their ability to interfere with carpogenic germination of Sclerotinia sclerotiorum. Nine out of 300 bacterial isolates were found to significantly suppress apothecial formation. One of these isolates, identified as a strain of Serratia plymuthica, was highly effective in inducing complete suppression of apothecial formation at high concentrations, and also strongly inhibited the germination of ascospores as well as hyphal growth of S. sclerotiorum. A bioassay-guided purification procedure starting with the cell-free supernatant of the bacterial culture led to the identification of a chlorinated macrolide as an active compound able to induce the observed inhibition. Spectroscopic data showed the compound to be identical to haterumalide A. The data presented show the ability of this compound to inhibit apothecial formation and ascospore germination. Other possible mechanisms involved in inhibition of apothecial formation and mycelial and hyphal growth of S. sclerotiorum by the same isolate are discussed. The relevance of our observations to natural systems will be the subject of further research.     

Detection of Sclerotinia sclerotiorum using a monomeric and dimeric single-chain fragment variable (scFv) antibody

Sclerotinia sclerotiorum (Lib.) de Bary is a phytopathogenic fungus capable of causing significant yield losses in numerous crops, including canola, in which the fungus causes sclerotinia stem rot. Immunological detection methods to rapidly determine the presence of S. sclerotiorum on plants may provide growers with a viable diagnostic tool to aid with fungicide use decisions. This paper discusses the generation of a monomeric and dimeric single-chain, variable fragment (scFv) antibody with affinity for S. sclerotiorum using phage display technology. The bacterially expressed and purified scFv is shown to bind S. sclerotiorum with some cross-reactivity with the closely related phytopathogen Botrytis cinerea (Pers.:Fr.). The dimeric scFv displayed improved binding to the fungus as compared to the monomer and could detect the presence of mycelia in inoculated canola petals. To the authors' knowledge, this is the first report of a scFv dimer with affinity for S. sclerotiorum that has the potential for use in the development of a new diagnostic test.     

Reduction of sclerotial inoculum of Sclerotinia sclerotiorum with Coniothyrium minitans

Aspects of the biology of C. minitans and its potential for control of S. sclerotiorum were investigated.Temperatures below 7°C resulted in comparatively slow rates of germination and infection of sclerotia by C. minitans. The optimum temperature for germination, growth, infection of sclerotia, and destructive parasitism by C. minitans was 20°C. The optimum relative humidity for germination, growth and infection by C. minitans was above 95%.Autumn inoculations with suspensions of conidia, pycnidia and mycelium of C. minitans in the field resulted in negligible numbers of sclerotia remaining viable after 1 month. With culture-grown sclerotia 2 months were required for a similar reduction of sclerotial viability. In the absence of C. minitans mulching had no significant effect on sclerotial viability. In the presence of C. minitans mulching did, however, influence the viability and infection by C. minitans of culture-grown sclerotia. Populations of field sclerotia also differed from culture-grown sclerotia in that they harboured an internal population of microorganisms, which included C. minitans, and had a lower level of viability at the commencement of the treatments.A winter application of C. minitans did not result in significant infection of sclerotia nor in a reduction in viability of sclerotia. This failure is believed to have resulted from low temperatures and dry conditions.     

Soil and compost humic fractions regulate the response of Sclerotinia sclerotiorum to exogenously added allelochemical compounds

Allelochemical compounds released by plants to signal their presence and needs interact in soils with very important macromolecules, such as humic acids (HAs), which are able to modulate the ultimate effects on target organisms. Most of the available studies on plants and microorganisms report the effects of allelochemicals or those of humic fractions, separately. In this study, we investigated the combined activity of these two types of compounds on the soil-resident fungus Sclerotinia sclerotiorum. Thus, ferulic acid (FA), caffeic acid (CA), benzoic acid (BA), salicylic acid (SA), gallic acid (GA) and phthalic acid (PA), exogenously applied to the fungal growth medium, were tested both alone and in combination with a soil HA (SHA) and a compost HA (CHA). The two HAs were also tested alone on the fungus. When the allelochemicals were applied alone, only FA, BA and SA evidenced a significant inhibition of mycelial growth, whereas FA, BA and CA increased the number of sclerotia formed. The two HAs alone reduced the early growth of the fungus and markedly stimulated sclerotia formation. A significant attenuation or, in some cases, suppression of the allelochemical effect on mycelial growth was caused by the coexistence in the medium of the allelochemical and each HA, especially CHA. Moreover, in general, the combinations of HA-allelochemical significantly stimulated sclerotia formation, with respect to the sole allelochemical, but decreased it with respect to HA alone. Thus, investigations on the response of fungi to plant-released allelochemicals should not exclude interactive aspects of these compounds with ubiquitous coexisting humic macromolecules.     

Allium white rot and its control

Abstract. White rot ( Sclerotium cepivorum ) causes serious losses in Allium crops throughout the world. The pathogen produces sclerotia which survive for long periods and are the main source of inoculum. Sclerotial germination is stimulated by the host and new sclerotia are produced on the host near the soil surface. Allium crops are cultivated in various systems and environments and no one method of control is effective. There is increasing interest in control strategies based on combinations of treatments which decrease the populations of sclerotia in the soil, thereby improving the effectiveness of present methods of control. Materials and methods being tested for inclusion in programmes of integrated control include germination stimulants, soil fumigants, solar heating, roguing, aerobic composting, microbial control and combined chemical/microbial control with fungicide-resistant micro-organisms.     

Diallyl-disulphide to reduce the numbers of sclerotia of Sclerotium cepivorum in soil

A single injection of 0.2 ml diallyl disulphide (DADS) at 0.156% (v/v) into soil containing naturally-produced sclerotia of Sclerotium cepivorum and maintained in the laboratory at 15°C stimulated sclerotial germination and reduced sclerotial numbers by 67%; ungerminated sclerotia remained viable. Higher concentrations of DADS had no additional effect except that at 20% (v/v), germination was slightly inhibited. A similar reduction in sclerotial numbers was obtained when the mixture of soil and sclerotia was exposed to DADS vapour. Four, monthly applications of DADS at 0.2 ml 0.15% (v/v) per application did not give a further reduction.The effect of DADS was temperature dependant, with a reduction in sclerotial numbers of 65 and 9% at 15 and 5°C respectively.     

Antifungal activity of 4-methyl-6-alkyl-2H-pyran-2-ones

A number of 4-methyl-6-alkyl-alpha-pyrones were synthesized and characterized on the basis of 1H NMR and mass spectroscopy. These compounds were tested in vitro against pathogenic fungi, namely, Sclerotium rolfsii Saccardo, Rhizoctonia bataticola (Taub.) Butler, Pythium aphanidermatum (Edson) Fitz., Macrophomina phaseolina (Tassi), Pythium debaryanum (Hesse), and Rhizoctonia solani Nees. Lower homologues were less effective, whereas compounds such as 4-methyl-6-butyl-alpha-pyrone, 4-methyl-6-pentyl-alpha-pyrone, 4-methyl-6-hexyl-alpha-pyrone, and 4-methyl-6-heptyl-alpha-pyrone were found effective against all of the test fungi. They inhibited mycelial growth by approximately 50% (ED50) at 15-50 microg/mL. 4-Methyl-6-hexyl-alpha-pyrone, which was found most effective, was tested against S. rolfsii in a greenhouse at 1, 5, and 10% concentrations. The 10% aqueous emulsion of 4-methyl-6-hexyl-alpha-pyrone suppressed disease development in tomato by 90-93% as compared with the untreated infested soil in the greenhouse after 35 days of treatment.     

Multivariate effects of plant canopy, soil physico-chemistry and microbiology on Sclerotinia stem rot of soybean in relation to crop rotation and urban compost amendment

The effects of canopy, soil physico-chemical and microbiological variables on Sclerotinia stem rot (SSR) on soybean were assessed in two soils (clay loam and sandy loam) using multiple regression and canonical redundancy analysis (RDA) and their partial form to control for the rotation (2 or 3-y-corn/soybean monoculture) and fertilization (mineral/urban compost) or spatial variables effects. The models revealed the minimal sets of variables that best explain the variance of the survival of Sclerotinia sclerotiorum’s sclerotia, carpogenic germination, disease severity and their associations. In clay loam, the 3-y-corn rotation reduced disease severity mainly through the reduction of weed biomass that favoured carpogenic germination. Urban compost has a conducive effect explained by a better soil surface drainage. Additionally, total N was found suppressive to sclerotial survival. In sandy loam, the carpogenic germination was negatively correlated with high C mineralization quotient and aggregate stability but correlated positively with Ca. Sclerotial survival was negatively correlated with pH and Ca, and positively correlated with biological fertility index. Aggregate stability, Ca and pH were associated with the urban compost. The regression and RDA analyses allowed to identify key variables that drived SSR development and explain their relationship with the cultural practices, soil health, as well as the spatial variation of disease variables.     

Effects of solarization of soil on nematode and fungal pathogens at two sites in victoria

The effect of covering soil with transparent polyethylene sheets, known as soil solarization, on the viability of plant pathogens was determined. The treatment was tested in mid-summer on sandy loams in N.W. and S. Victoria. Columns of moist soil were inoculated with one of a variety of pathogens, viz. Fusarium oxysporum, Pythium irregulare, Plasmodiophora brassicae, Sclerotium cepivorum, S. rolfsii, Sclerotinia minor, Verticillium dahliae and the nematodes Macroposthania xenoplax, Meloidogyne javanica, Pratylenchus penetrans and Tylenchulus semipenetrans. Columns were placed vertically in soil, and then treated either for 4 weeks in N.W. Victoria, or 6 weeks in S. Victoria.Preliminary laboratory tests showed that pathogens were killed by temperatures within the range 38–55°C. The relative sensitivities of pathogens to fluctuating soil temperatures were similar at both sites. The most sensitive were the nematodes, and the fungi V. dahliae, S. cepivorum and S. minor, while F. oxysporum, P. irregulare and P. brassicae were the least sensitive. In N.W. Victoria treatment effects were apparent to 26 cm and most pathogens were not recovered from 0 to 11 cm. In S. Victoria treatment effects were apparent to a depth of 16cm and most pathogens were not recovered from 0 to 6cm.     

Viridepyronone, a new antifungal 6-substituted 2H-pyran-2-one produced by Trichoderma viride

A new antifungal 6-substituted 2H-pyran-2-one, named viridepyronone, has been isolated from a cultural filtrate of a strain of Trichoderma viride showing antagonistic activity in vitro toward Sclerotium rolfsii, which is the causal agent of crown and stem rot of artichoke. Viridepyronone was characterized as 6-(4-oxopentyl)-2H-pyran-2-one 2 with spectroscopic methods. Bioassays showed that viridepyronone had a good antifungal activity against S. rolfsii, and its minimum inhibitory concentration (over 90% inhibition) was found to be 196 microg/mL. This is the first report of viridepyronone produced by any species of fungi.     

河套灌区向日葵菌核病发生程度预测预报

根据2000-2009年河套灌区向日葵菌核病中心病株出现期、始盛期和发生程度资料与同期气象资料进行相关性分析,研究其与气象条件的关系并建立相应的预测方程。结果表明,降水量是影响向日葵菌核病发生的关键因子,温度、湿度、蒸发量、大风日数和日照时数也对菌核病发生时间和发生程度有重要影响。通过逐步回归,建立了分别于11月和翌年5月进行预报的6个模型,均通过了0.05水平的显著性检验,对历史资料的拟合效果较好;通过2010、2011年试报,预报值与实际值相差较小,可投入实际运行。     

Cynodontin: a fungal metabolite with antifungal properties

A red pigment that accumulates in cultures of a Drechslera avenae pathotype with specificity for Avena sterilis was isolated and identified as the anthraquinone cynodontin (3-methyl-1,4,5,8-tetrahydroxyanthraquinone). Satisfactory yield of the compound was obtained with 20-60 day incubations at temperatures between 20 and 27 degrees C. Cynodontin was tested in vitro for fungitoxicity and was found to be a potent inhibitor of the growth of Sclerotinia minor, Sclerotinia sclerotiorum, and Botrytis cinerea and, to a lesser extent, of Verticillium dahliae. The ED(50) values obtained with these fungi were of the same order of magnitude as those of the commercial fungicides dicloran and carbendazim, which were used as reference chemicals. In contrast, the growth of a number of other fungi was not significantly inhibited by cynodontin. Anthraquinone and two other anthraquinone derivatives, emodin and chrysophanol, which were also included in the tests, did not affect the growth of the cynodontin-sensitive fungi. It thus appears that the type and position of the substitutions at the C-ring play a role in the expression of antifungal activity.     

Identification and use of potential bacterial organic antifungal volatiles in biocontrol

Bacteria, isolated from canola and soybean plants, produced antifungal organic volatile compounds. These compounds inhibited sclerotia and ascospore germination, and mycelial growth of Sclerotinia sclerotiorum, in vitro and in soil tests. Ascospore germination in cavity slides was inhibited 54-90% by the volatile producers. When mycelial plugs or the sclerotia, exposed to these volatiles, were transferred to fresh agar plates, the pathogen could not grow, indicating the fungicidal nature of the volatiles. Head space volatiles, produced by bacteria, were trapped with activated charcoal, by passing nitrogen continuously over shake cultures for 48 h. The compounds were eluted from the charcoal with methylene chloride and identified using Gas Chromatography-Mass Spectrometry (GC-MS). The volatile compounds included aldehydes, alcohols, ketones and sulfides. Of the 23 compounds assayed for antifungal activity in divided Petri plates, with filter-disks soaked with these compounds (100 and 150 μl), only six compounds completely inhibited mycelial growth or sclerotia formation, suggesting their potential role in biological control. The compounds are benzothiazole, cyclohexanol, n-decanal, dimethyl trisulfide, 2-ethyl 1-hexanol, and nonanal. Volatiles may play an important role in the inhibition of sclerotial activity, limiting ascospore production, and reducing disease levels. Studies are under way to understand this phenomenon under field conditions. This is the first report on the identification and use of bacterial antifungal organic volatiles in biocontrol.     

Evaluation of Paecilomyces lilacinus,chitin, and cellulose amendments in the biological control of Aspergillus flavus fungi

This study was undertaken to investigate the feasibility of using Paecilomyces lilacinus NRRL 13 866, chitin, and cellulose amendments, in order to reduce survival and prevent germination of sclerotia from Aspergillus flavus NRRL 6556, NRRL 13 048, and A. parasiticus NRRL 13 005, NRRL 13 539 buried in sandy soil in Georgia and Illinois (April–October, 1990). The number of sclerotia that germinated sporogenically in moist chambers following burial in Illinois was twice that of sclerotia buried in Georgia and varied among the four sclerotium-producing Aspergillus spp. strains. Sclerotium viability, as measured by Aspergillus spp. colony formation on potato dextrose agar, was high (>84%) for all strains and treatments (e.g. amendment, location). Even so, A. parasiticus NRRL 13 539 sclerotia were apparently more susceptible to P. lilacinus colonization than sclerotia from other strains. Treatment with Paecilomyces sp. caused a small but significant reduction in germination among sclerotia buried in Georgia.     

核盘菌侵染拟南芥的过程及其芥子油苷的变化

研究了核盘茵侵染拟南芥哥伦比亚生态型(Col-O)过程中,拟南芥的发病症状、活性氧和芥子油苷组分和含量的变化,及其抗性相关基因和芥子油苷生物合成相关基因表达的变化.结果表明,在侵染过程中,拟南芥的受侵染部位出现黄色水渍状病斑,随发病时间的延长病斑逐渐扩大到整株,其相对含水量逐渐下降,相对电导率不断上升,活性氧(过氧化氢...