In vitro inhibitory effects of rosemary and sage extracts on mycelial growth and sclerotial formation and germination of Sclerotinia sclerotiorum

The anti-fungal efficacy for two Labiate plants, rosemary (Rosmarinus officinalis L.) and Greek sage (Salvia fructicosa Mill.), against Sclerotinia sclerotiorum fungus (Lib.) de Bary has been investigated. The inhibitory effect of these plants as crude leaf ethanolic extract on the radial mycelial growth as well as on sclerotial production and germination was measured in vitro at various concentrations (stock?=?0.5?g dry leaf powder/ml ddH₂O) in the growth medium. In general, rosemary extract revealed a remarkable anti-fungal effect against the fungus, being more inhibitory than Greek sage in this respect. This was evident as total inhibition of radial mycelial growth by rosemary occurred at 10% extract concentration, while sage was half as potent producing such an effect at double the concentration (20%). Both rosemary and sage extracts were more inhibitory to sclerotial formation than to mycelial growth as the fungus ceased to produce any sclerotia at the lower concentrations of 5 and 5–10%, respectively. In addition, rosemary was highly effective in inhibiting sclerotia germination as total inhibition of germination occurred at 20% extract concentration at three?days and onward after incubation. Moreover, at this level, the survival of sclerotia was totally lost when examined after 12?days of incubation. For sage, inhibition of sclerotial germination/death was only 20% at 12th day of incubation. The results of this study indicate that the extracts of rosemary and Greek sage leaves could become natural alternatives to synthetic fungicides to manage diseases of S. sclerotiorum.     

Secretome analysis reveals effector candidates associated with broad host range necrotrophy in the fungal plant pathogen Sclerotinia sclerotiorum

Background

The white mold fungus Sclerotinia sclerotiorum is a devastating necrotrophic plant pathogen with a remarkably broad host range. The interaction of necrotrophs with their hosts is more complex than initially thought, and still poorly understood.

Results

We combined bioinformatics approaches to determine the repertoire of S. sclerotiorum effector candidates and conducted detailed sequence and expression analyses on selected candidates. We identified 486 S. sclerotiorum secreted protein genes expressed in planta, many of which have no predicted enzymatic activity and may be involved in the interaction between the fungus and its hosts. We focused on those showing (i) protein domains and motifs found in known fungal effectors, (ii) signatures of positive selection, (iii) recent gene duplication, or (iv) being S. sclerotiorum-specific. We identified 78 effector candidates based on these properties. We analyzed the expression pattern of 16 representative effector candidate genes on four host plants and revealed diverse expression patterns.

Conclusions

These results reveal diverse predicted functions and expression patterns in the repertoire of S. sclerotiorum effector candidates. They will facilitate the functional analysis of fungal pathogenicity determinants and should prove useful in the search for plant quantitative disease resistance components active against the white mold.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-336) contains supplementary material, which is available to authorized users.     

A robust sampling approach for identification and quantification of methyl jasmonate in leaf tissue of oilseed rape for analysis of early signaling in sclerotinia sclerotiorum resistance

Introduction – Methyl jasmonate (MJA), which is a natrual hormonal regulator, is thought to be essential for the regulation of systemic defence responses. The information about MJA levels in plant tissues is helpful for the study of the disease resistance mechanism and genetically engineered cultivars with increased resistance. Therefore, the quantification of MJA levels in plant tissues by means of a sensitive and reliable method is of interest. Objective – Development of a film extraction method coupled with GC for determination of methyl jasmonate in leaf tisssue of oilseed rape for analysis of early signalling in sclerotinia sclerotiorum resistance. Methodology – A robust polydimethylsiloxane film was prepared and used for extraction of MJA in leaf tissues. By using in‐solution extraction mode, optimum extraction efficiency was achieved with methanol–water (1 : 5, v/v) as extraction medium at 40°C for 60 min. Results – Under the optimal conditions, a detection limit of 0.2 ng/mL was achieved. Excellent reproducibility was found over a linear range of 1–1000 ng/mL. MJA in leaves infected by sclerotinia sclerotiorum was determined, with the results showing that basal levels of MJA (15 ng/g) were present in noninfested controls, but increased to 313 ng/g 10 h after fungal attack. Conclusion – The film extraction method is a simple, rapid and inexpensive sampling technique for determination of endogenous MJA in plant tissues that can be applied to most plants. Copyright © 2009 John Wiley & Sons, Ltd.     

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.     

核盘菌弱毒株Ep-1PN培养性状的有性遗传特性*

观察和测定了核盘菌（Sclerotiniasclerotiorum（Lib．）deBary）弱毒株Ep-1PN单孢分离物的生长、菌落扩展和菌落形态等培养性状。结果发现：来自7个子囊盘的1574个单孢分离物中有1560个分离物的培养性状与正常菌株的没有显著差异,与亲本相似的分离物只有6个,其它8个分离物介于弱毒株Ep-1PN与正常菌株之间。弱毒株Ep-1PN培养性状的有性遗传不遵循孟德尔核遗传规律。     

向日葵菌核病拮抗菌XRK5的筛选与鉴定

从向日葵根际分离了640个细菌分离物,以向日葵菌核病菌(Sclerotinia sclerotiorum)为靶标菌,通过平板对峙法获得了18个具有拮抗性能的细菌,其中XRK5具有较强拮抗能力,且拮抗性能稳定,具有较好的生防应用潜力。经过形态观察、生理生化特征及16SrRNA序列分析,将XRK5鉴定为辣椒溶杆菌(Lysobacter capsici),XRK5的16SrRNA序列在GenBank中的注册号为FJ959348。     

粉红黏帚霉67-1菌株寄生核盘菌菌核的酶学动态研究

对粉红黏帚霉67-1菌株侵染核盘菌菌核过程的多种细胞壁降解酶活性进行了连续测定,以研究几丁质酶等在这一寄生互作体系中的可能作用。结果表明:葡聚糖酶活性变化表现活跃,且随寄生过程呈增加趋势,配对法T检验结果表明,第10d的处理与对照酶活性差异达到最大;几丁质酶、蛋白酶活性变化表现较低,而纤维素酶未检测得到。酶学动态变化与之前石蜡切片显微观察的结果在时间上表现一致;认为葡聚糖酶可能是粉红黏帚霉67-1菌株寄生核盘菌菌核的关键酶。