低毒病毒及板栗疫病菌低毒力机制

低毒病毒是一类存在于板栗疫病菌细胞质中自主复制的无衣壳正链RNA病毒.感染低毒病毒后,板栗疫病菌的致病力显著降低,色素分泌减少,菌丝体由感染病毒前的桔黄色变为白色,产孢量降低或不产孢,漆酶表达水平明显下降.低毒病毒侵染性克隆的获得以及高效转化和转染体系的建立,使得低毒病毒成为目前唯一可以进行全面遗传操作的真菌病毒.利用低毒病毒作为探针来探测板栗疫病菌的致病力组成和毒力调节机制,已获得了一些很有意义的发现.本文介绍近几年低毒病毒及其与真菌相互作用的研究进展,包括低毒病毒的基因组和功能基因研究、低毒病毒和线粒体损害引起的板栗疫病菌低毒力机制、板栗疫病菌的RNA沉默系统以及低毒病毒抗RNA沉默的机制.低毒病毒/板栗疫病菌系统已经成为研究病毒与宿主相互作用以及病原真菌致病机理的很好的模式系统.     

三种袋蛾的观察

<正> 笔者于1965—1966年及1977—1979年间,对广西袋蛾作了初步研究,先后调查了桂林、柳州、南宁、钦州、梧州、玉林、百色、河池等40多个县、市,采集到标本13种,有7种得到雄蛾,另6种仅有护囊及其他虫态。寄主有柑桔、黄檀、荔枝、板栗、梨,茶等40多科100余科植物。现将其中3种袋蛾简述如下。     

板栗疫病菌cpfluG基因的功能研究

fluG基因在许多丝状真菌中对无性孢子的发育起调控作用,然而在树木病原菌板栗疫病菌(Cryphonectria parasitica)中,无性孢子的发育调控机制尚未完全明确。本研究克隆了板栗疫病菌的fluG同源基因cpfluG,采用同源重组的方法构建了fluG基因缺失菌株ΔcpfluG。相对于野生型菌株,ΔcpfluG突变株几乎完全丧失产孢能力,色素分泌显著减少,但致病性不受影响。cpfluG基因的缺失导致已知的产孢相关基因cls-31表达量显著下调,但不影响pks基因和cls-32的表达。本研究结果揭示,fluG是板栗疫病菌孢子发育调控的一个关键成员,但板栗疫病菌分生孢子发育的调控细节可能有别于已报道的丝状真菌。     

在板栗疫病菌线粒体中检出低毒病毒编码蛋白p29

p29蛋白是低毒病毒基因组编码的一个木瓜蛋白酶样蛋白。前人研究发现,在宿主板栗疫病菌(Cryphonectria parasitica)体内表达p29,会引起真菌毒力降低,色素产生减少,丧失产生无性孢子的能力。除已知p29与源于高尔基体的膜结构共分离之外,p29在细胞内的其它分布形式未明。本研究在成功制备p29特异抗体和高效分离板栗疫病菌线粒体的基础上,尝试用p29抗体检测线粒体中是否存在p29蛋白。Western印迹结果表明,受CHV1-EP713感染的EP713菌株线粒体中存在与p29抗体特异作用的病毒蛋白。本研究结果暗示,低毒病毒蛋白p29可能参与调控宿主线粒体功能。     

栗疫病菌不同毒力菌株胞外酶活性和草酸产量的比较

对栗疫病菌不同毒力菌株产生胞外酶的种类、活性和草酸产量以及草酸对多聚半乳糖醛酸酶水解聚果胶酸钙的影响进行了研究。所有供试菌株均未能检测到淀粉酶活性。栗疫病菌在培养中可分泌漆酶,多聚半乳糖醛酸酶、蛋白酶、纤维素酶和脂酶,但不同毒力菌株产生这些酶的能力不同。总的来说,强毒力菌株均可分泌这些酶,且活性强,但弱毒力菌株的酶活性较弱或不分泌这些酶。菌丝产量和草酸产量分析表明,强毒力菌株的草酸产量明显高于弱毒力菌株。菌丝产量与草酸产量没有相关性。在没有草酸盐存在的条件下,多聚半乳糖醛酸酶不能降解聚果胶酸钙。     

板栗疫病菌cpnat基因的功能分析

赖氨酸乙酰化是一种重要的蛋白质翻译后修饰,广泛参与多种生命过程的调节.目前,赖氨酸乙酰化修饰在植物病原真菌——板栗疫病菌中的功能和调节机制尚无报道.本研究克隆了板栗疫病菌的编码乙酰转移酶的cpnat基因,成功构建了cpnat基因的缺失突变体Δcpnat,与野生型菌株相比,板栗疫病菌cpnat基因缺失株生长速率不变,气生菌丝变浓密,菌丝末端分支变多,色素明显减少,产孢能力显著降低,致病力也降低.cpnat基因的缺失并不影响参与信号传导途径的cpga1、cpgb1、cpmk1、cpmk2和ste12基因的转录水平,表明其对色素、产孢和致病力的调控是通过尚未被发现的途径实现的.本研究结果为揭示乙酰化修饰在病原真菌中的功能提供了新的知识.     

中国东部栗疫病菌dsRNA群体的多样性

采用生物学测定和凝胶电泳的方法,对中国东部栗疫病菌(Cryphonectria parasitica)的dsRNA病毒进行了研究。根据电泳图谱,中国东部栗疫病菌的dsRNA病毒可以分为5种类型,其中类型1只具有一个12．7kh的条带,占大多数;类型Ⅱ有两条带,一条12．7kh,另一条5．2kb左右,只有一个菌株354;类型Ⅲ则除了12．7kb的条带外,还有3条小于2kb的小片段,有238和250两个菌株;dsRNA属于类型Ⅳ的为269和344两个菌株,它只含有两个1．8—3．1kb的小片段;dsRNA类型Ⅴ只有一个菌株280,除了一条12．7kb分子外,还有4条2．6—3．3kb的小片段。含有dsRNA的菌株培养性状多样,根据菌落特征．栗疫病菌可以划分为5种培养类型,但菌株的培养类型与dsRNA的类型间没有对应的关系。毒力测定表明含有dsRNA病毒的供试菌株大多属于低毒力类型;     

几种植物精油及单体对栗疫病病菌的抑制作用

栗疫病 ( Cryphonectria parasitica)是一种世界范围的林木病害 ,2 0世纪上半叶不到50年的时间里栗疫病将美洲栗几乎毁灭 [1 ] ,欧洲栗也曾遭受巨大损害[2 ] ,中国板栗一向被认为是最抗栗疫病的 [3] ,但由于栽培制度的改变、种质资源的频繁交流、单一化品种的应用等原因 ,原有相对平衡的寄主——病原物系统被迅速打破 ,我国部分地区的栗疫病已经造成了较大损失[4] 。由于欧洲栗的栗疫病菌群体遗传结构较为单一 ,欧洲人利用含有ds RNA的低毒力栗疫病菌菌株对欧洲栗的栗疫病进行生物防治取得了成功 [5] ,但因为这种 ds RNA一般只在具有营养…     

栗疫菌低毒力菌株dsRNA的分离及转移

自云南、广西、江苏、浙江、河北、京郊六个省市的板栗树(Castanea mollissima)溃疡斑上分离了栗疫菌(Endothia parasitica)160株,进行了dsRNA的提取及其毒力测定,获得两株含dsRNA的低毒力菌株(H),其毒力与带有起源于欧洲的dsRNA的低毒力菌株比较相近。比较国内菌株与欧洲低毒力菌株的dsRNA电泳谱带,示有共同分子量为5x106或6．2 x 108的电泳带。与正常毒力菌株(V)混合接种能显著降低致病力。这是欧美大陆发现低毒力菌株后,亚洲地区的首次报告。以5株具有欧洲dsRNA的低毒力菌株作为供体,与云南、江苏、浙江的10株毒力菌株配对,16％(8,50)的毒力菌株发生形态变化,dsRNA转入后毒力明显降低。结果表明,国内毒力菌株可以接受欧洲低毒力因子——dsRNA并转变为低毒力菌株。     

广西翼手类调查

广西地处祖国南方,濒临北部湾,北回归线横贯广西中部,太阳辐射强烈,热量充足,雨水丰沛,不少地方仍保持着高山密林的自然景观。特别是分布有众多的石灰岩山地,明暗石洞比比皆是,这种独特的自然景观,为翼手类的生存和繁衍提供了良好的场所。中国科学院动物研究所汪松等于1962年曾报道过广西西南部翼手类5科,17种(亚种)。 作者从1982-1985年对桂林、柳州、河池、百色、南宁、玉林、梧州等30个县市进行了调查,共得标本800余号,经鉴定隶属6科,15属,35种(包括亚种)(表1)。     

Thyreophagus corticalis as a vector of hypovirulence in Cryphonectria parasitica in chestnut stands

The natural spread of hypovirulence in Cryphonectria parasitica (Murr.) Barr. occurs in chestnut (Castanea sativa Mill) stands and orchards in Italy and other European countries, leading to spontaneous recovery of the diseased trees. Little is known about how hypovirulence spreads in chestnut stands but various corticolous mite species frequently detected on chestnut cankers could be one of the many factors playing a role in the spread. Artificial virulent cankers created in inoculation field tests and treated with Thyreophagus corticalis (Acari, Sarcoptiformes, Acaridae) raised on hypovirulent cultures showed similar growth to those treated with mycelia of the hypovirulent strain over 18 months of inoculation. Cultures re-isolated from virulent cankers treated with mites were found to contain hypovirus like those derived from pairings of virulent and hypovirulent strains. Viral dsRNA could be carried externally and/or ingested by mites from the hypovirulent mycelia and then transmitted to the mycelia of virulent strains, causing their conversion. In a laboratory study, all fecal pellets collected from mites reared on hypovirulent and virulent strains grown on semi-selective media gave rise to colonies of C. parasitica with similar morphological characters and virulence to the original cultures. Field inoculation of stump sprouts with the resulting colonies revealed that mite digestive tract passage did not alter the virulence of the studied strains. These results are of interest for the biological control of chestnut blight.     

Extending chestnut blight hypovirus host range within diaporthales by biolistic delivery of viral cDNA

Biolistic bombardment was used to successfully transform three phytopathogenic fungal species with an infectious cDNA clone of the prototypic hypovirus, CHV1-EP713, a genetic element responsible for the virulence attenuation (hypovirulence) of the chestnut blight fungus, Cryphonectria parasitica. The fungal species included two strains each of C. parasitica and Valsa ceratosperma, as well as one strain of Phomopsis G-type (teleomorph Diaporthe Nitschke); all are members of the order Diaporthales but classified into three different genera. A subset of transformants for each of the fungal species contained CHV1-EP713 dsRNA derived from chromosomally integrated viral cDNA. As has been reported for CHV1-EP713 infection of the natural host C parasitica, biolistic introduction of CHV1-EP713 into the new fungal hosts V ceratosperma and Phomopsis G-type resulted in altered colony morphology and, more importantly, reduced virulence. These results suggest a potential for hypoviruses as biological control agents in plant-infecting fungal pathogens other than the chestnut blight fungus and closely related species. In addition, the particle delivery technique offers a convenient means of transmitting hypoviruses to potential host fungi that provides new avenues for fundamental mycovirus research and may have practical applications for conferring hypovirulence directly on infected plants in the field.     

Papain-like protease p29 as a symptom determinant encoded by a hypovirulence-associated virus of the chestnut blight fungus.

Viral double-stranded RNAs (dsRNAs) responsible for virulence attenuation (hypovirulence) of the chestnut blight fungus, Cryphonectria parasitica, profoundly influence a range of host functions in addition to virulence. The 5'-proximal open reading frame, A, of the prototypical hypovirulence-associated viral dsRNA, L-dsRNA, present in hypovirulent strain EP713, was recently shown by DNA-mediated transformation analysis to suppress fungal sporulation, pigmentation, and accumulation of the enzyme laccase (G. H. Choi and D. L. Nuss, EMBO J. 11:473-477, 1992). We mapped this suppressive activity to the autocatalytic papain-like protease, p29, present within the amino-terminal portion of open reading frame A-encoded polyprotein p69. Mutational analysis revealed that the ability of p29 to alter fungal phenotype is dependent upon release from the polyprotein precursor but is independent of intrinsic proteolytic activity. Deletion of the p29-coding domain within the context of an infectious L-dsRNA cDNA clone resulted in a replication-competent viral dsRNA that exhibited intermediate suppressive activity while retaining the ability to confer hypovirulence. Thus, p29 is necessary but not sufficient for the level of virus-mediated suppression of fungal pigmentation, sporulation, and laccase accumulation observed for wild-type hypovirulent strain EP713 and is nonessential for viral RNA replication and virulence attenuation. These results also illustrate the feasibility of engineering infectious viral cDNA for construction of hypovirulent fungal strains with specific phenotypic traits.     

Transmission of Fusarium boothii mycovirus via protoplast fusion causes hypovirulence in other phytopathogenic fungi

There is increasing concern regarding the use of fungicides to control plant diseases, whereby interest has increased in the biological control of phytopathogenic fungi by the application of hypovirulent mycoviruses as a possible alternative to fungicides. Transmission of hypovirulence-associated double-stranded RNA (dsRNA) viruses between mycelia, however, is prevented by the vegetative incompatibility barrier that often exists between different species or strains of filamentous fungi. We determined whether protoplast fusion could be used to transmit FgV1-DK21 virus, which is associated with hypovirulence on F. boothii (formerly F. graminearum strain DK21), to F. graminearum, F. asiaticum, F. oxysporum f. sp. lycopersici, and Cryphonectria parasitica. Relative to virus-free strains, the FgV1-DK21 recipient strains had reduced growth rates, altered pigmentation, and reduced virulence. These results indicate that protoplast fusion can be used to introduce FgV1-DK21 dsRNA into other Fusarium species and into C. parasitica and that FgV1-DK21 can be used as a hypovirulence factor and thus as a biological control agent.     

Mycoviruses and virocontrol

Viruses are widespread in all major groups of fungi. The transmission of fungal viruses occurs intracellularly during cell division, sporogenesis, and cell fusion. They apparently lack an extracellular route for infection. Recent searches of the collections of field fungal isolates have detected an increasing number of novel viruses and lead to discoveries of novel genome organizations, expression strategies and virion structures. Those findings enhanced our understanding of virus diversity and evolution. The majority of fungal viruses have dsRNA genomes packaged in spherical particles, while ssRNA mycoviruses, possessing or lacking the ability to form particles, have increasingly been reported. This review article discusses the current status of mycovirus studies and virocontrol (biocontrol) of phytopathogenic fungi using viruses that infect them and reduce their virulence. Selected examples of virocontrol-associated systems include the chestnut/chestnut blight/hypovirus and fruit trees/white root rot fungus/mycoviruses. Natural dissemination and artificial introduction of hypovirulent fungal strains efficiently contributed to virocontrol of chestnut blight in European forests. Attempts to control white root rot with hypovirulence-conferring mycoviruses are now being made in Japan.     

A viral gene confers hypovirulence-associated traits to the chestnut blight fungus.

A viral double-stranded (ds)RNA associated with reduced virulence (hypovirulence) and the accompanying biological control of the chestnut blight fungus, Cryphonectria parasitica, was shown recently to contain two contiguous coding domains designated ORF A and ORF B. We report here that transformation of an isogenic virulent, dsRNA-free C. parasitica strain with a cDNA copy of ORF A conferred traits similar to those exhibited by the dsRNA-containing hypovirulent strain: characteristics included reduced pigmentation, reduced laccase accumulation and suppressed conidiation. However virulence was not reduced, indicating an apparent uncoupling of associated traits from hypovirulence. These results establish a direct cause and effect relationship between a viral dsRNA genetic element present in a hypovirulent C. parasitica strain and specific phenotypic traits. They demonstrate further that these traits are not the result of a general reaction of the fungus to the presence of the replicating viral RNA, but are caused by a specific viral coding domain.     

Double-stranded RNA mycovirus from Fusarium graminearum

Double-stranded RNA (dsRNA) viruses in some fungi are associated with hypovirulence and have been used or proposed as biological control agents. We isolated 7.5-kb dsRNAs from 13 of 286 field strains of Fusarium graminearum isolated from maize in Korea. One of these strains, DK21, was examined in more detail. This strain had pronounced morphological changes, including reduction in mycelial growth, increased pigmentation, reduced virulence towards wheat, and decreased (60-fold) production of trichothecene mycotoxins. The presence or absence of the 7.5-kb dsRNA was correlated with the changes in pathogenicity and morphology. The dsRNA could be transferred to virus-free strains by hyphal fusion, and the recipient strain acquired the virus-associated phenotype of the donor strain. The dsRNA was transmitted to approximately 50% of the conidia, and only colonies resulting from conidia carrying the mycovirus had the virus-associated phenotype. Partial nucleotide sequences of the purified dsRNA identify an RNA-dependent RNA polymerase sequence and an ATP-dependent helicase that are closely related to those of Cryphonectria hypovirus and Barley yellow mosaic virus. Collectively, these results suggest that this dsRNA isolated from F. graminearum encodes traits for hypovirulence.     

Cryphonectria radicalis: rediscovery of a lost fungus

In an attempt to isolate the ascomycete Cryphonectria parasitica (Diaporthales, Valsaceae) from dead chestnut stems, we obtained three C. radicalis strains. All three strains were isolated in areas of Switzerland with high chestnut blight incidence. To confirm our species designation, we compared the three C. radicalis strains to hypovirus (hv)-free and hv-infected C. parasitica strains. The comparison revealed several distinctive characteristics. On potato dextrose agar in the dark, the C. radicalis strains produced a fluffy mycelium and small droplets of a purple exudate giving the mycelium a light pinkish appearance. On corn meal medium in the dark, the C. radicalis strains caused a color change of the medium to purple, whereas the C. parasitica strains did not cause any color change. Ascospores from C. radicalis were significantly smaller than C. parasitica ascospores and their dimensions fit within other published size ranges. Southern hybridization analysis of the two species using nuclear and mitochondrial probes support their taxonomic separation. This separation is further supported by the lack of successful interspecific crosses. In virulence tests on chestnut trees, the C. radicalis strains exhibited very low virulence, comparable to highly hypovirulent hv-infected C. parasitica strains. Our results suggest that C. radicalis still coexists with C. parasitica although at a low frequency.     

Double-Stranded RNA Mycovirus from Fusarium graminearum

Double-stranded RNA (dsRNA) viruses in some fungi are associated with hypovirulence and have been used or proposed as biological control agents. We isolated 7.5-kb dsRNAs from 13 of 286 field strains of Fusarium graminearum isolated from maize in Korea. One of these strains, DK21, was examined in more detail. This strain had pronounced morphological changes, including reduction in mycelial growth, increased pigmentation, reduced virulence towards wheat, and decreased (60-fold) production of trichothecene mycotoxins. The presence or absence of the 7.5-kb dsRNA was correlated with the changes in pathogenicity and morphology. The dsRNA could be transferred to virus-free strains by hyphal fusion, and the recipient strain acquired the virus-associated phenotype of the donor strain. The dsRNA was transmitted to approximately 50% of the conidia, and only colonies resulting from conidia carrying the mycovirus had the virus-associated phenotype. Partial nucleotide sequences of the purified dsRNA identify an RNA-dependent RNA polymerase sequence and an ATP-dependent helicase that are closely related to those of Cryphonectria hypovirus and Barley yellow mosaic virus. Collectively, these results suggest that this dsRNA isolated from F. graminearum encodes traits for hypovirulence.