橡胶树胶孢炭疽菌效应蛋白基因CgE35对孢子产量的影响

橡胶树炭疽病是橡胶树的重要真菌病害之一,主要由胶孢炭疽菌引起。效应蛋白是病原菌侵染植物的重要致病因子,在寄主与病原菌的相互识别中发挥重要作用。为了进一步阐明橡胶树炭疽菌的致病机理,课题组前期对橡胶树胶孢炭疽菌的效应蛋白在基因组水平上进行了预测,其中一个编码候选效应蛋白的基因被命名为CgE35。本研究对CgE35基因进行了扩增,并对其所编码的效应蛋白CgE35进行了生物信息学的分析,分析结果表明,该基因编码一条由200个氨基酸组成的多肽,分子质量约为21.7 kD,其氨基段含有一段由24个氨基酸组成的典型信号肽序列,且该蛋白不含任何跨膜结构域和已知的保守结构域。为了进一步研究该基因的功能,我们根据同源重组原理,构建了 CgE35基因的敲除载体pCB1532-CgE35,通过PEG介导的原生质体转化法将其转入胶孢炭疽菌原生质体细胞中进行同源交换,经过对转化子进行抗性筛选和分子鉴定,获得CgE35基因的敲除突变体ACgE35,并对其孢子产量和菌落生长情况进行分析,发现胶孢炭疽菌CgE35基因的敲除突变体ACgE35的孢子产量明显低于野生型菌株,而二者的菌落生长情况没有差异。以上结果表明,橡胶树胶孢炭疽菌CgE35基因编码一个未知功能的分泌蛋白,该基因与胶孢炭疽菌的产孢能力有关,但与其菌丝的生长关系不大。本研究结果为阐明胶孢炭疽菌的致病机理,建立新型橡胶树炭疽病防控策略提供理论依据。     

油菜黑胫病菌（Leptosphaeria biglobosa）生物学特性研究

为了解我国油菜黑胫病病原菌（Leptosphaeria biglobosa）的生物学特性,在实验室里研究了温度、pH值、光照、碳源及氮源等条件对该病菌营养生长及分生孢子产生的影响。结果表明：病原菌在5℃～35℃均可进行营养生长并产孢,最适生长和产孢温度为25℃;在pH4～10的范围内该菌均能生长并产孢,在pH7的条件下生长最快,产孢最多。光暗交替有利于菌丝生长和产孢,而持续光照不利于菌丝生长,尤其不利于产孢。最适的碳源、氮源分别是可溶性淀粉、牛肉膏。分生孢子的致死温度为52℃。     

Attachment and adhesion of conidia of Stagonospora nodorum to natural and artificial surfaces

Attachment and adhesion of conidia of a wheat-isolate of Stagonospora nodorum to leaf and artificial surfaces was studied. Attachment of conidia was a non-viable process, separate from adhesion, that occurred rapidly and irreversibly. Attachment involved conidial-surface carbohydrates and was partially influenced by surface hydrophobicity. The subsequent adhesion, via the secretion of extracellular matrix from conidia, was a viable process that induced the complete cover of conidia in response to wheat leaf surface components containing epi-cuticular wax and to a lesser extent to barley but inducing only partial covering on glass. Results suggest that specific surface components from the compatible host promote rapid attachment and adhesion of S. nodorum conidia.     

The spore coat of the bean anthracnose fungus Colletotrichum lindemuthianum is required for adhesion, appressorium development and pathogenicity

The spores (conidia) of the bean anthracnose fungal pathogen, Colletotrichum lindemuthianum, adhere to the aerial parts of plants to initiate the infection process. In previous studies we have shown that the Colletotrichum spores are surrounded by a fibrillar spore coat, comprising several major glycoproteins. Previous evidence showed that a monoclonal antibody (UB20) that recognised these glycoproteins was able to inhibit adhesion of spores to a hydrophobic surface. In this paper we have further studied the role of the spore coat in adhesion, germination and fungal development by studying the effects of UB20 and protease treatment of spores. The latter treatment has previously been shown to remove the spore coat. Spores germinate on glass, polystyrene and water agar, however, appressoria only develop on glass or polystyrene, showing a requirement for a hard surface. Removal of the spore coat with protease inhibits adhesion at 30 min, before the secretion of ECM glycoproteins. Protease treatment also inhibits the development of appressoria and reduces pathogenicity on leaves. Incubation of spores with the MAb UB20 inhibits adhesion at 30 min, but does not affect appressorium formation or pathogenicity. The results suggest that an intact spore coat has two functions; it is required for adhesion to a hydrophobic surface and for the detection of a hard surface necessary for appressorium formation. We suggest that contact with a hard surface, rather than adhesion, is the key event leading to appressorium formation.     

Components of Liquid Media on the Production of High Spore Concentrations of Lecanicillium lecanii （Zimm.） Gams and Zare

Two Lecanicillium lecanii isolates, ATCC26854 and V3, were evaluated for spore production in liquid media. Both isolates have interesting known properties for the production of high concentrations of chitinases （ATCC26854）, and an outstanding pathogenic activity against the green cabbage aphid, Brevicorine brassiacae （L.）. The isolates were grown on thirteen different liquid media, which had been used to produce other entomopathogenic Hyphomycetes. Experiments were carried out at 27 -4- 1 ℃ with a 12：12 photoperiod using shake flasks. The production of spores was quantified during a seven days period, and the effects of the media were evaluated by determining spore concentration and morphology. Submerged conidia yields were higher with ATCC26854 than with V3 in all thirteen media, while higher concentrations （5.3×10^9, 4.6 ×10^9 and 3.4 ×10^9 conidia/mL） were found with ATCC26854 isolate in the Camaron, Minerales and Jenkins-Prior medium, respectively; lower yields （2.3 ×10^8, 2.2 ×10^8 and 2.3 ×10^8 conidia/mL） were found with the V3 isolate in Catroux, TKI and Camaron media, respectively. Spore production curves were adjusted to different sigmoid models. The process was better explained by the Richards model （r^2 = 0.99）. Concerning conidia morphology, submerged conidia seemed to look like aerial conidia, but they were different in size （ATCC26854 2.73-6.99 μm and V3 5.28-14.29 μm）; however, the dimensions fall within the ranges reported for L. lecanii. The analysis of shake flask cultures with the Richards model allowed selecting two low-cost liquid medium, Camaron and Jenkins-Prior, for scaling up conidia production for use in aphid biological control programs.     

Cytological and biochemical changes induced by chitosan in the pathosystem Alternaria alternata-tomato

The cytological and biochemical response of the fungus Alternaria alternata to chitosan application in tomato fruits was evaluated. The research was developed in the following stages: microscopically to observe the degree of damage that chitosan causes over the conidia and hyphae of the fungus at the structural level and during the infection process in tomato tissue. Biochemically we tried to identify the elicitation of the phytoalexin rhisitin and other compounds involved in resistance. At the microscopic level, mycelium and conidia of chitosan-treated of A. alternata showed cell wall disintegration, plasma membrane retraction, cellular distortion, release of the apical portion of the conidia and lysis of fungal cells. Hyphae and conidia were susceptible to chitosan application. Infection always took place in chitosan treated and inoculated tomatoes and it was difficult to observe ultrastructural alterations due to chitosan application. The phytoalexin rhisitin was not isolated from any of the treatments but other compounds such as alkenes, fatty acids and vitamin E whose antimicrobial effects have been reported were detected. The elicitation of precursor compounds in the pathosystem A. alternata-tomato was more associated with the infection process than with the chitosan application. Further studies are necessary to confirm these findings.     

玫烟色拟青霉孢子萌发和附着胞形成的影响因子研究

用不同种类、不同浓度的培养液离体培养玫烟色拟青霉孢子,观察其萌发过程,结果显示,玫烟色拟青霉孢子萌发可以产生单极芽管和双极芽管,而且芽管顶端膨大可形成附着胞,附着胞形态以球形和棍棒状为主;不同培养液中,孢子萌发和附着胞形成率不同,其中0.1%的菜青虫体壁浸出液(CL)培养液中孢子萌发率和附着胞形成率最高,分别达到79.01%和18.69%。不同基质对玫烟色拟青霉孢子萌发和附着胞形成的影响也不同,蝉翅较适合玫烟色拟青霉孢子萌发和附着胞形成。     

Sources and seasonal dynamics of inoculum for brown spot disease of pear

The dynamics of the production of Stemphylium vesicarium conidia and Pleospora allii ascospores from different inoculum sources on the ground were compared in a model system of a wildflower meadow mainly composed of yellow foxtail, creeping cinquefoil and white clover. The meadow was either inoculated (each October) or not inoculated with a virulent strain of S. vesicarium, and either covered or not covered with a litter of inoculated pear leaves. Spore traps positioned a few centimetres above the ground were exposed for 170 7-day periods between October 2003 and December 2006. Ascospores and conidia were trapped in 46 and 25% of samples, respectively. Ascospore numbers trapped from the pear leaf litter were about five times higher than those from the meadow, while conidial numbers were similar from the different inoculum sources. The ascosporic season was very long, with two main trapping periods: December–April, and August–October; the former was most important for the leaf litter, the latter for the meadow. The conidial season lasted from April to November, with 92% of conidia caught between July and September. The fungus persistently colonized the meadow: the meadow inoculated in early October 2003 produced spores until autumn 2006. The present work demonstrates that orchard ground is an important source of inoculum for brown spot of pear. Thus, it is important to reduce inoculum by managing the orchard ground all year long.     

Laboratory shelf-life of oil-formulated conidia of the locust and grasshopper fungal pathogen Metarhizium flavoviridae Gams & Rozsypal,in mixtures with the pyrethroid insecticide lambda-cyhalothrin

The addition of 20 ppm lambda-cyhalothrin pyrethroid insecticide to Metarhizium flavoviridae (Fungi: Deuteromycetes) dry conidia in oil kept germination above 80% for 2 and 12 months at temperatures of approximately 30 and 8°C respectively. At 30°C, conidia in oil alone maintained viability for only a few weeks, but this was doubled at 8°C. Even dry conidia retained good viability for only 5 months at the higher temperature. The extended viability of conidia given by adding the pyrethroid to the oil formulation is regarded as very valuable for areas where the fungus is to be used against locusts and grasshoppers.     

枸杞炭疽病对成熟果实侵染过程的显微观察

以往研究认为,枸杞炭疽病菌(C.gloeosporioides Penz)仅侵染花、花蕾、青果,一般不会对成熟果实进行侵染.笔者近期研究证明枸杞炭疽病可对成熟果实造成严重侵染,显微观察进一步证实:孢子接种后4 h开始入侵,48 h细胞消融,几无正常细胞,在切片中可清楚地观察到大量繁殖的菌丝体断面和枸杞炭疽病孢子.