Transmissible hypovirulent element in isolate Ep-1PN ofSclerotinia sclerotiorum

The cause of virulence decline of hypovirulent isolate Ep-1PN ofSclerotinia sclerotiorum (Lib.) de Bary has been studied through transmitting test and analysing its offsprings derived from single ascospore. When the colony of a normal strain Ep lPNA 183 intermingled with the colony of Ep-lPN, its margin became abnormal, just like EplPN. Cultures isolated from toptip of the colony of Ep-1PNA 183 showed the hypovirulence and its associated properties of Ep-1PN. Moreover, a series of cultures which have characteristics between EplPN and normal strain could be isolated from stipes of Ep-1PN. More than 200 offsprings derived from single ascospore of Ep-1PN were normal strains. According to those results, there exists a transmissible cytoplasmic hypovirulent element in hypovirulent isolate EplPN of S.sclerotiorum.     

蓝花楹内生拮抗细菌筛选及对茎腐病的防治研究

【目的】筛选蓝花楹茎腐病的内生拮抗细菌,并探讨其生物防治作用。【方法】采用稀释平板法从健康蓝花楹根茎分离内生菌株; 以蓝花楹茎腐病原菌(Fusarium chlamydosporum)作为供测菌,用5点对峙法进行拮抗作用初筛、平板扩散法复筛获得拮抗菌株,经形态学和16SrDNA序列比对,对其进行鉴定。采用温室盆栽和田间试验检测拮抗菌的生防效果。【结果】分离获得24株内生菌株,其中zhu66抑菌率达到97.8%,鉴定为解淀粉芽孢杆菌(Bacillus amyloliquefaciens),GenBank序列登录号为JF899258。盆栽试验结果表明,解淀粉芽孢杆菌能有效预防和治疗蓝花楹茎腐病。在一定浓度下,浓度越高防效越好,以10⁷cfu/mL浓度最佳。灭菌土防效优于自然土; 生防菌先于病原菌施入防效优于生防菌与病原菌同时接种,病原菌先于生防菌接种方式防效较低。菌株能显著促进蓝花楹地径、苗高与冠幅生长,浓度越高,促生效果越好。田间试验进一步验证,蓝花楹茎腐病不同发病程度的生防效果有显著差异,无病区预防作用十分显著,不发生茎腐; 重度发生区几乎没有效果,轻度发生区防效明显,防效达74.1%,中度发生区有一定效果,且随着时间延长,防效增加。【结论】解淀粉芽孢杆菌zhu66对蓝花楹茎腐病有良好的生防潜力。     

青葙根水提物化感活性及抑菌活性的研究

以水稻（Oryza sativaL．）等5种植物和油菜菌核病（Sclerotinia sclerotiovum（Lib）de Bary）等4种植物病原病菌为受试材料,通过生物测定的方法研究了青葙（Celosia argenteas Linn．）根水提物化感活性及抑菌活性。结果表明,与对照相比,在250-4000μg／mL浓度范围内,提取物对稗草（Echinochloa crusgalli（L．）Beauv．）和醴肠（Eclipta prostrateL．）种子的萌发,水稻和稗草幼苗苗高,烟草（Nicotiana tabacum L．）和醴肠幼苗的单株鲜重不产生显著影响,而在相同的浓度范围内,对千金子（Leptochloa chinensis（Linn）Nees）幼苗的根长和单株鲜重产生显著抑制作用。对5种植物相对应的其它生长特性方面,只有在高于250μg／mL浓度时才能受到显著抑制。在25-400μg／mL的浓度范围内,提取物对油菜菌核病、小麦赤霉病（Fusorium graminearum Schw．）和杨树溃疡病（Dothiorella gregaria Sacc．）病菌的生长产生显著抑制作用,当浓度达到50μg／mL时,提取物也显著抑制烟草灰霉病（Botrytis cinerea Pers.）病菌的生长,400μg／mL时,对4种植物病原真菌的抑制率分别为45．90％、46．49％、45．64％和41．66％。上述表明,青葙根水提物具有开发为生物源农药的潜力。     

White-spot disease of Chinese soft-shelled turtles （Trionyx sinens） caused by Paecilomyces iilacinus

Chinese soft-shelled turtles （Trionyx sinens） in culture farms using an artificial warming system in Zhejiang, China, often show typical signs of white-spot disease such as white spots on their bodies, skin lesions, anorexia and eventually death. The sick turtles were mostly 5-80 g in weight. A suspected fungal pathogen was isolated from the sick turtles and verified as Paecilomyces lilacinus by sequence analysis of the internal transcribed spacer （ITS） of its ribosomal DNA （rDNA）. Detailed morphological examinations were also conducted to confirm the white-spot disease.     

青葙根水提物化感活性及抑菌活性的研究

以水稻(Oryza sativa L.)等5种植物和油菜菌核病(Sclerotinia sclerotiovum(Lib)de Bary)等4种植物病原病菌为受试材料,通过生物测定的方法研究了青葙(Celosia argenteas Linn.)根水提物化感活性及抑菌活性。结果表明,与对照相比,在250～4 000μg/mL浓度范围内,提取物对稗草(Echinochloa crusgalli(L.)Beauv.)和醴肠(Eclipta prostrate L.)种子的萌发,水稻和稗草幼苗苗高,烟草(Nicotiana tabacum L.)和醴肠幼苗的单株鲜重不产生显著影响,而在相同的浓度范围内,对千金子(Leptochloa chinensis(Linn.)Nees)幼苗的根长和单株鲜重产生显著抑制作用。对5种植物相对应的其它生长特性方面,只有在高于250μg/mL浓度时才能受到显著抑制。在25～400μg/mL的浓度范围内,提取物对油菜菌核病、小麦赤霉病(Fusarium graminearum Schw.)和杨树溃疡病(Dothiorella gregaria Sacc.)病菌的生长产生显著抑制作用,当浓度达到50μg/mL时,提取物也显著抑制烟草灰霉病(Botrytis cinerea Pers.)病菌的生长,400μg/mL时,对4种植物病原真菌的抑制率分别为45.90%、46.49%、45.64%和41.66%。上述表明,青葙根水提物具有开发为生物源农药的潜力。     

毛霉BFL-5菌株的分离鉴定及染料脱色特性

筛选对染料具有良好脱色能力的真菌.采集当地腐朽树木样品,以染料脱色为目标,筛选、分离真菌菌株;采用真菌26S rDNA D1/D2区域鉴定法对菌种的26S rDNA进行测序.以花生壳等为固定化培养载体,培养得到固定化真菌,用于对含酸性红B,酸性金黄G,直接湖蓝5B,直接耐晒黑G等染料的模拟染料废水脱色实验.初步鉴定其为...     

Functional genomics in the rice blast fungus to unravel the fungal pathogenicity

A rapidly growing number of successful genome sequencing projects in plant pathogenic fungi greatly increase the demands for tools and methodologies to study fungal pathogenicity at genomic scale. Magnaporthe oryzae is an economically important plant pathogenic fungus whose genome is fully sequenced. Recently we have reported the development and application of functional genomics platform technologies in M. oryzae. This model approach would have many practical ramifications in design and implementation of upcoming functional genomics studies of filamentous fungi aimed at understanding fungal pathogenicity.     

甘蔗黑腐病病原菌的鉴定

在云南开远6根发病的甘蔗茎上分离到甘蔗黑腐病病原菌,鉴定了该病害的病原菌,以期为防治和控制该病害提供一定的理论依据.基于致病性测定,ITSI-5.8S-ITS2 rDNA序列的系统发病分析和形态学观察结果,甘蔗黑腐病是由多脂长喙壳菌(Ceratocystis adiposa(Butl.)Moreau)引起的,该病菌导致甘蔗黑腐病在中国属首次报道.     

甘蔗黑腐病病原菌的鉴定

 在云南开远6根发病的甘蔗茎上分离到甘蔗黑腐病病原菌,鉴定了该病害的病原菌,以期为防治和控制该病害提供一定的理论依据.基于致病性测定,ITSI-5.8S-ITS2 rDNA序列的系统发病分析和形态学观察结果,甘蔗黑腐病是由多脂长喙壳菌（Ceratocystis adiposa(Butl.)Moreau）引起的,该病菌导致甘蔗黑腐病在中国属首次报道.     

Agrobacterium tumefaciens-mediated genetic transformation of the phytopathogenic fungus Penicillium digitatum

Agrobacterium tumefaciens-mediated transformation （ATMT） system was assessed for conducting insertional mutagenesis in Penicillium digitatum, a major fungal pathogen infecting post-harvest citrus fruits. A transformation efficiency of up to 60 transformants per 106 conidia was achieved by this system. The integration of the hph gene into the fungal genome was verified by polymerase chain reaction （PCR） amplification and sequencing. These transformants tested were also shown to be mitotically stable. Southern blot analysis of 14 randomly selected transformants showed that the hph gene was randomly integrated as single copy into the fungal genome of P. digitatum. Thus, we conclude that ATMT of P. digitatum could be used as an alternatively practical genetic tool for conducting insertional mutagenesis in P. digitatum to study functional genomics.     

Investigation of the biological roles of autophagy in appressorium morphogenesis in Magnaporthe oryzae

Magnaporthe oryzae has been used as a primary model organism for investigating fungus-plant interaction. Many researches focused on molecular mechanisms of appressorium formation to restrain this fungal pathogen. Autophagy is a very high conserved process in eukaryotic cells. Recently, autophagy has been considered as a key process in development and differentiation in M. oryzae. In this report, we present and discuss the current state of our knowledge on gene expression in appressorium formation and the progress in autophagy of rice blast fungi.     

A wound-induced small polypeptide gene family is upregulated in soybean nodules

Small peptides function as key signals in processes, such as plant cell differentiation, organ development and defenses to biotic stresses. A large number of small peptide precursor genes have been predicted from the analysis of the soybean (Glycine max) whole genome DNA sequence. However, most of these genes have unknown characteristics and functions. In this report, we systemically searched for the gene families of small peptide precursors that are up-regulated in soybean nitrogen-fixing root nodules. We found 212 genes (encoding peptides shorter than 150 amino acids) that were up-regulated, and among them, 79 genes belong to 38 multiple-gene families, but the other 133 genes are unique. Twenty-eight of 38 families are conserved in Arabidopsis, but the other 10 only exist in legumes. We also identified 16 out of the 38 members of the wound-induced polypeptide (WIP) gene family to be upregulated in nitrogen-fixing nodules. We further analyzed homologs of WIP genes in Medicago, Lotus, Arabidopsis and Oryza species and found that a few homologous genes from Medicago truncatula and Lotus japonicus were also upregulated in their nodules and some WIP genes were induced by specific fungal pathogens on soybean and rice. Structure prediction indicated that all WIP prepropeptides contain a conserved DUF3774 domain (including two hydrophobic regions) and most of them have an N-terminal signal sequence. Fluorescence microscopy analysis of two WIP prepropeptides fused to GFP revealed that these proteins are located on the plasma membrane of tobacco leaf cells. Interestingly, 34 soybean WIP genes are clustered onto three soybean chromosomes, different from known peptide gene families (such as CLE). Among them, 11 highly identical genes are aligned on the 6th chromosome, 12 on the 12th, and 11 on the 13th chromosomes. Most of WIP genes from the 12th chromosome share the highest identities with their homologs on the 13th chromosome, suggesting that ancestral WIP genes could have originated from the 13th chromosome, then spread onto the 12th chromosome by chromosome homologous recombination; the new WIP genes could have existed in multiple copies by gene duplication which then spread onto the 6th chromosome. In Arabidopsis and Oryza species, half of the WIP genes are also aligned on one chromosome and showed higher identity with those from the soybean 12th and 13th chromosomes, suggesting that WIP genes originated from one common ancestor.     

Obtaining transgenic rice resistant to rice fungal blast disease by controlled cell death strategy

The strategy of the two-component system,composed of Barnase and Barstar which encode RNase and a specific inhibitor to the RNase respectively, is adopted to obtain transgenic rice resistant to rice fungal blast disease. In this study, two chimeric promoters, induced by rice blast fungus pathogen (Magnaporthe grisea), are fused with Barnase respectively to construct two plant expression vectors, pWBNBS and pPBNBS together with the Barstar driven by CaMV 35S promoter. The resistance of the transgenic rice lines to rice blast fungus disease and rice blight disease are evaluated. The results show that (1) the expression of Barnase is induced in rice leaves when inoculated with the spores of Magnaporthe grisea; (2) the induced expression level of Barnase surpasses the level of Barstar, which elicits a similar hypersensitive response (HR) in the leaves, and the transgenic plant shows high resistance to the rice fungal blast disease; and (3) transgenic rice plants also show obvious resistance to rice bacterial blight disease. Taken together, these results suggest that the transgenic rice plants harboring this two-component system acquire relatively broad spectrum resistance against pathogens, especially high resistance to rice fungal pathogen.     

三七根腐病拮抗菌的筛选及活性产物的初步分离

 以文山州三七根腐病主体病原菌坏损柱孢(Cylindrocaipon destructans)为靶标,对其拮抗菌进行了筛选,获得了抑菌作用明显的3株拮抗细菌:Y-4-70,X-5-15和X-5-32;并对拮抗菌X-5-15代谢产物的抑菌活性进行了初步研究,发现该菌株发酵液石油醚萃取物对病原菌有较好的活性.     

Biological and chemical diversity of cytotoxin-producing symbiotic marine fungi in intertidal zone of Dalian

In this study, the biological and chemical diversity of 8 symbiotic marine fungal strains, with strong cytotoxicity against brine shrimp larvae, were investigated by nucleotide sequencing, morphology and cluster analysis of HPTLC fingerprint. These strains were identified by ITS rDNA sequencing, phylogenic analysis, and morphology to be Hypocrea lixii, Chaetomium globosum, Aspergillus fumigatus, Asp. clavatus and Alternaria sp. Their differences in secondary metabolites were shown by cluster analysis of digitalized colors of HPTLC spots, a newly developed method, which produced a similar dendrogram with that of ITS cluster analysis. Furthermore, this method can fully display intraspecific differences and even the remarkable difference in Aspergillus strains which goes beyond the boundary between genera. Their biological-chemical diversity may be the basis of their potent cytotoxicity and implies their potential in producing diversified antitumor or pesticidal constituents.     

十堰辣椒根腐病病原菌分离鉴定及高氏15号的生物防治作用研究

为明确辣椒根腐病病原,从田间采集分离、纯化、鉴定、柯赫氏法则验证病原菌,并开展高氏15号生物菌剂对辣椒根腐病的防治效果和促生作用研究.结果表明,通过形态学和分子生物学鉴定(ITS、EF-1a、IGS基因序列比对及进化树分析)发现,引起该地区辣椒根腐病的病原物为茄病镰刀菌(Fusarium solani).柯赫氏法则完成了辣椒根腐病的回接鉴定试验.田间防效和促生结果显示,多粘类芽孢杆菌在240 g/667 m2,高氏15号菌剂在50 g/穴和75 g/穴,甲霜·恶霉灵1 500倍液防治辣椒根腐病效果最好,均达到70%以上.经试验处理的株高、根长、整株鲜重、根鲜重和根干重和空白对照相比有显著差异性.     

Construction of Streptomyces lydicus A01 transformant with the chit33 gene from Trichoderma harzianum CECT2413 and its biocontrol effect on Fusaria

Streptomyces lydicus A01 resists many plant pathogens (including Fusarium spp.) by producing the antifungal agent natamycin, which binds to the ergosterol of fungal cell membranes and inhibits the growth of pathogens. Trichoderma harzianum CECT2413 is a widely-distributed soil fungus that antagonizes several plant fungal pathogens (including Fusarium spp.) by producing chi-tinase and degrading chitin, a major component of the fungal cell wall. This study attempted to enhance the biocontrol effect of S. lydicus A01 on Fusarium spp. by transforming the chitinase gene of Trichoderma. Chitinase and natamycin could act synergisti-cally on both the cell walls and cell membranes of pathogens. The 33-kD chitinase-encoding gene (chit33) was cloned and conju-gal-transformed from T. harzianum CECT2413 to S. lydicus A01, and then confirmed via polymerase chain reaction (PCR) assays. Subsequent analyses using the 3,5-dinitrosalicylic acid (DNS) method and ultraviolet spectrophotometry showed that compared with its wild type strain (WT), the S. lydicus A01 conjugal transformant (CT) with chit33 gene exhibited substantially higher chi-tinase activity and natamycin production. The resistance of S. lydicus A01-chit33 CT and WT to four Fusaria in crops and vegetables was tested via the cup-plate method. Compared with the WT, the conjugal transformant of S. lydicus A01 with chit33 gene from T. harzianum CECT2413 showed greatly increased biocontrol effect on fusarium disease. This study would be beneficial to the development of high-quality antifungal bio-agents for agricultural applications via the synergy between the previously non-existent and pre-existing functions achieved through heterogeneous gene transformation.     

基于全基因组序列的尖孢镰刀菌分泌蛋白预测及其特征分析

尖孢镰刀菌是一种以土壤习居为主要特征的植物病原菌,广泛分布于世界各地,且寄主广泛,能引起豆科、茄科、葫芦科等科100多种植物根腐、茎腐、茎基腐等病害,严重时造成植株萎蔫死亡,严重影响着植物的产量和品质.同时,目前生产上对于该菌的防治多使用甲霜灵·锰锌、多菌灵、百菌清等化学药剂,然而,防治效果不佳,急需开发针对该菌新的作用靶标的药剂.前人已经明确植物病原真菌分泌蛋白在侵染、操控植物等过程中发挥着重要功能,然而,学术界尚未见有关该菌中分泌蛋白的报道.本研究以尖孢镰刀菌菌株(Fusarium oxysporumf.sp.lycopersici 4287)的蛋白序列为基础数据,以分泌蛋白所具有的N-端含有信号肽、不含有跨膜结构域、没有GPI锚定位点、将蛋白分泌在胞外4大特征为依据,利用生物信息学在线分析程序明确该菌中含有778个分泌蛋白,并对上述蛋白开展特征分析,明确上述蛋白的氨基酸长度主要集中在101～400aa、氨基酸组成中以G,T,S,A较多,信号肽长度主要集中在16～21aa、信号肽氨基酸组成中以P,S,A,T,G较多,信号肽切割位点为T-X-T类型.为今后进一步开展尖孢镰刀菌的分泌蛋白功能解析和开发新型药剂靶标提供了重要的理论基础.     

Plant pathogens. Mitochondrial control of fungal hybrid virulence

Hybrid species of fungal pathogens that infect wild and cultivated plants are emerging with new virulence and host ranges, posing a threat to agriculture and forestry. Here we show that the virulence of hybrid species of the basidiomycete fungus Heterobasidion annosum (Fr) Bref, a causal agent of root and butt rot in conifers and one of the most economically important forest pathogens, is controlled by their mitochondrial genome. Our results indicate that cooperation between organelles that contain genetic information may influence the phenotype of hybrid phytopathogens.