中国稻瘟病菌的致病性和其DNA指纹图谱间关系的研究

通过在稻瘟病菌Pyricularia oryza。基因组文库中的筛选,找到一个散布的并具有基因组特异性的层重复顺序POR6。本文报道用这一株针对6个日本菌株和26个中国北方菌株进行DNA指纹作图的结果。其中22个中国北方菌株按其杂交带型百分相似率被分成8个株系。一些在我们实验室保存的菌株用传统方法鉴定发现在转管过程中会发生致病性变异。当用POR6作探针与这些菌株NNA的EcoRV酶切片段杂交时,检测出它们的无性世代中出现数条EcoRV多态性片段。     

稻瘟病菌株的DNA指纹分析及小种鉴别

基于对稻瘟病菌(Pyricularia oryzae)基因文库的分析,我们找到了一套含重复顺序的克隆。其中POR6和POR7被证实具有高度的多态性并随机散布于稻瘟病菌生理小种的致病性时,可以获得可分辨的基因组特异的杂交带型。我们还分析了致病性与8个稻瘟病菌株DNA指纹图谱之间的关系,结果表明各个小种组合间的百分相似率S_xy,值与该小种组合间共同侵染的鉴别品种数目有正相关性。     

云南稻瘟病菌系谱与致病型的关系

为探究稻瘟病菌无性世代DNA水平的变异,寻找云南稻瘟病菌谱系(genetic lineage,G)和致病型之间的对应关系,根据稻瘟病菌散布的重复序列Pot2(Pyricularia oryzac transposon),对云南水稻主产区稻瘟病菌菌株DNA进行了rep-PCR(repetitive polymerase chain reaction)扩增,获得rep-PCR指纹。聚类分析将134个稻瘟病菌代表菌株划分为G1～G8等8个谱系,揭示云南水稻主产区稻瘟病菌无性系丰富的遗传多样性。进一步接种分析了8个谱系的29个稻瘟病菌菌株对33个云南主产区水稻品种的亲和性,依其毒性谱,采用STATISTICAL5．0软件的UP-GMA程序进行聚类分析,将其划分为P1～P6等6个致病型群(pathotype group)。结果表明同一谱系的稻瘟病菌菌株多数对应2～3个致病型群,少数1个或4个致病型群;但G1～G8等8个谱系中的部分菌株都可对应致病型群P2。因此,云南水稻主产区稻瘟病菌谱系和致病型群之间属于复杂关系类型。此外,33个水稻品种中的合系16和京国92抗全部29个稻瘟病菌株,云粳20和合系30对全部供试菌株表现感病,这对云南水稻主产区品种布局提供了稻瘟病抗性方面的依据。因此,从育种应用和生产实际需要出发,水稻品种抗瘟谱测定仍然必要。     

稻瘟病菌群体的分子遗传学研究I.广东省1998~1999年稻瘟病菌的遗传多样性

从80个随机引物中筛选到带型清晰、多态性及重复性均好的10个引物,对采自广东省1998-1999年四个自然生态稻作区的101个稻瘟病菌菌株进行随机扩增多态性DNA (Random Amplified Polymorphic DNA, RAPD) 指纹分析。10个引物共扩增出113条多态性带,表明广东省稻瘟病菌具有丰富的遗传多样性;RAPD分析可为该菌的遗传多样性分析提供大量的分子标记。对菌株间相似性系数和应用加权算术平均组对法 (Unweighted Pair Group Method using Arithmetic Average, UPGMA) 构建的聚类树状图进行分析,以相似性系数为0.62阀值时,可将101个菌株划分为14个遗传宗谱;其中宗谱1及宗谱2的菌株数占总数的80.2%,为优势宗谱; 其余的20个菌株分别归属于其他12个宗谱,由此说明广东省的稻瘟病病原菌群体既存在很突出的优势宗谱,又存在较多具遗传多样性的小宗谱。分析不同稻作生态区的菌株发现,每个稻作生态区既有共同的宗谱,又有其特异的宗谱;广东省稻瘟病菌群体遗传多样性的组成在不同生态稻作区是相对地比较稳定的。分析不同年份和早晚稻生长季节采集的菌株发现,广东省稻瘟病菌群体遗传多样性在年份和早晚稻生长季节之间也存在一定的特异性。     

稻瘟病菌群体的分子遗传学研究Ⅰ——广东省1998—1999年稻瘟病菌的遗传多样性

从80个随机引物中筛选到带型清晰,多态性及重复性均好的10个引物,对采自广东省1998-1999年四个自然生态稳作区的101个稻瘟病菌菌株进行随机扩增多态性DNA（Random Amplified PolymorphicDNA,RAPD)指纹分析。10个引物共扩增出113条多态性带,表明广东省稻瘟病菌具有丰富的遗传多样性;RAPD分析可为该菌的遗传多样性分析提供大量的分子标记。对菌株间相似性系数和应用加权算术平均组对法（Unweighted Pair Group Method using Arithmetic Average,UPGMA)构建的聚类树状图进行分析。以相似性系数为0.62阀值时,可将101个菌株划分为14个遗传宗谱;其中宗谱1及宗谱2的菌株数占总数的80.2%,为优势宗谱,其余的20个菌株分别归属于其他12个宗谱,收此说明广东省的稻瘟病病原菌群体既存在很突出的优势宗谱,又存在较多具遗传多样性的小宗谱,分析不同稻作生态区的菌株发现,每个稻作生态2区既有共同的宗谱,又有其特异的宗谱;广东省稻瘟病菌群体遗传多样性的组成在不同生态稻作区相对地比较稳定的,分析不同年份和早晚稻生长季节采集的菌株发现,广东省稻瘟病菌群体遗传多样性在年份和早晚稻生长季节之间也存在一定的特异性。     

水稻品种多样性田间稻瘟病菌群体遗传结构分析

利用稻瘟病菌的一段倒位重复序列Pot2设计的一对引物,采用rep—PCR分子指纹技术对来自石屏县净种杂交稻田块、净种糯稻田块以及间种杂交稻糯稻田间的251个稻瘟病单孢分离菌株进行扩增．结果表明,所有供试菌株均分别扩增到9—17条DNA带,大小从400bp到23kb左右,但大多数带主要集中在5—10kb之间．所有菌株共扩增出的DNA指纹带中,约65％的为多态性DNA带,35％的为共同扩增带．将供试菌株扩增带诺进行聚类分析,比较间裁与净载田间病菌群体遗传结构的组成差异结果表明,在不同遗传相似水平,菌株遗传宗群复杂度与栽培方式有一定相关性。间栽田间病菌遗传宗群较净栽田问复杂,为3—5个,且优势宗群群不明显;而在净栽糯稻或净栽杂交稻田间遗传宗群较为简单,只有1—3个,且优势宗群明显．本试验结果证明水稻品种多样性有利于稻瘟病菌稳定化选择。     

稻瘟病菌群体的分子遗传学研究I.*广东省1998~1999年稻瘟病菌的遗传多样性

从80个随机引物中筛选到带型清晰、多态性及重复性均好的10个引物,对采自广东省1998-1999年四个自然生态稻作区的101个稻瘟病菌菌株进行随机扩增多态性DNA (Random Amplified Polymorphic DNA, RAPD) 指纹分析。10个引物共扩增出113条多态性带,表明广东省稻瘟病菌具有丰富的遗传多样性;RAPD分析可为该菌的遗传多样性分析提供大量的分子标记。对菌株间相似性系数和应用加权算术平均组对法 (Unweighted Pair Group Method using Arithmetic Average, UPGMA) 构建的聚类树状图进行分析,以相似性系数为0.62阀值时,可将101个菌株划分为14个遗传宗谱;其中宗谱1及宗谱2的菌株数占总数的80.2%,为优势宗谱; 其余的20个菌株分别归属于其他12个宗谱,由此说明广东省的稻瘟病病原菌群体既存在很突出的优势宗谱,又存在较多具遗传多样性的小宗谱。分析不同稻作生态区的菌株发现,每个稻作生态区既有共同的宗谱,又有其特异的宗谱;广东省稻瘟病菌群体遗传多样性的组成在不同生态稻作区是相对地比较稳定的。分析不同年份和早晚稻生长季节采集的…     

江苏省稻瘟病菌有性态的研究

用标准菌株对1997－1999年在江苏吴江市、宜兴市、通州市、高邮市和赣榆县采集的325个稻瘟病菌单孢分离菌株的可育性和交配型进行了测定,结果表明江苏省稻瘟病菌菌株的育性较低,可交配率为22．77％,可育率仅为7．08％。不同年份、不同地区采集的稻瘟病菌菌株的性亲和力和交配型有较大的差异,三年的交配率分别为26．61％、8．26％和33．64％;通州地区和赣榆地区菌株的交本相对较高,分别为26．15％和25．42％,宜兴地区菌株的交配率较低,只有15．38％。江苏省稻瘟病菌菌株的交配型在不同年份亦出现很大差别,1997年29个可交配菌株中有21个菌株表现为MAT1－2交配型,而1999年36个可交配菌株均为MAT1－1交配型。用江苏省稻瘟病菌的可育菌株进行互交,25个组合中只有6个组合能产生子囊壳和子囊,但均不产生子囊孢子,提示江苏省稻瘟病菌在田间产生健康有性后代的几率不大。对杂交后代的遗传学分析表明,菌株的交配型是受单基因控制的。     

稻瘟病菌微波诱发突变体的分析

通过诱变获得突变体是研究稻瘟病菌变异机制的基础。本文用微波炉对稻瘟病菌分生孢子进行低强度短时间处理获得了一批形态发育和致病性突变体,并对它们进行了分析。突变体1-40-271菌落呈白色,产孢与萌发均正常,但萌发后即便在人工疏水表面上也不能形成附着胞,且丧失了致病性;突变体2-20-6菌落呈黄色,孢子萌发率为1％,萌发的孢子其附着胞形成率仅为0．01％,致病性减弱;突变体2-30-3菌落呈黄色,形成的附着胞大部分不正常,但致病性正常。Rep-PCR指纹分析发现,突变体2-20-6和2-30-3比其相应野生型少1条带,而突变体1-40-271与其野生型比较没有变化,说明微波可能造成稻瘟病菌基因组DNA缺失或点突变而发生变异。继代分析表明微波处理获得的稻瘟病菌形态和致病性突变体是稳定的。     

稻瘟病菌微波诱发突变体的分析*

通过诱变获得突变体是研究稻瘟病菌变异机制的基础。本文用微波炉对稻瘟病菌分生孢子进行低强度短时间处理获得了一批形态发育和致病性突变体,并对它们进行了分析。突变体1-40-271菌落呈白色,产孢与萌发均正常,但萌发后即便在人工疏水表面上也不能形成附着胞,且丧失了致病性;突变体2-20-6菌落呈黄色,孢子萌发率为1%,萌发的孢子其附着胞形成率仅为0.01%,致病性减弱;突变体2-30-3菌落呈黄色,形成的附着胞大部分不正常,但致病性正常。Rep-PCR指纹分析发现,突变体2-20-6和2-30-3比其相应野生型少1条带,而突变体1-40-271与其野生型比较没有变化,说明微波可能造成稻瘟病菌基因组DNA缺失或点突变而发生变异。继代分析表明微波处理获得的稻瘟病菌形态和致病性突变体是稳定的。     

Genome Analysis of Environmental and Clinical P. aeruginosa Isolates from Sequence Type-1146

The genomes of Pseudomonas aeruginosa isolates of the new sequence type ST-1146, three environmental (P37, P47 and P49) and one clinical (SD9) isolates, with differences in their antibiotic susceptibility profiles have been sequenced and analysed. The genomes were mapped against P. aeruginosa PAO1-UW and UCBPP-PA14. The allelic profiles showed that the highest number of differences were in “Related to phage, transposon or plasmid” and “Secreted factors” categories. The clinical isolate showed a number of exclusive alleles greater than that for the environmental isolates. The phage Pf1 region in isolate SD9 accumulated the highest number of nucleotide substitutions. The ORF analysis of the four genomes assembled de novo indicated that the number of isolate-specific genes was higher in isolate SD9 (132 genes) than in isolates P37 (24 genes), P47 (16 genes) and P49 (21 genes). CRISPR elements were found in all isolates and SD9 showed differences in the spacer region. Genes related to bacteriophages F116 and H66 were found only in isolate SD9. Genome comparisons indicated that the isolates of ST-1146 are close related, and most genes implicated in pathogenicity are highly conserved, suggesting a genetic potential for infectivity in the environmental isolates similar to the clinical one. Phage-related genes are responsible of the main differences among the genomes of ST-1146 isolates. The role of bacteriophages has to be considered in the adaptation processes of isolates to the host and in microevolution studies.     

Identification of a variable region of the African swine fever virus genome that has undergone separate DNA rearrangements leading to expansion of minisatellite-like sequences

Nucleotide sequencing identified a tandemly repeated sequence array 22 x 10(3) base-pairs from the right-hand DNA terminus of the African swine fever virus (ASFV) genome. The sequence of the repeat array and sequences closely flanking it were compared in the genomes of four groups of ASFV isolates that had very different restriction enzyme site maps. Arrays present in one group of ASFV isolates from East Zambia/Malawi varied in length and contained between 8 and 38 copies of a 17-nucleotide repeat unit. Repeat arrays in a second group of ASFV isolates from Europe were less variable in length but consisted of different types of repeat unit that were divergent in sequence. A third genetically diverse ASFV isolate. LIV 13 from a South Zambia Game Park, contained repeat unit types that were similar to those of European viruses. MFUE6 isolate from an East Zambia Game Park contained a shorter version of the European repeat unit. An eight-base-pair core sequence was conserved between the East Zambia/Malawi and European and LIV 13 repeat units. These tandemly repeated sequence arrays share a number of properties with chromosomal minisatellite DNA. Similar tandem repeat arrays have not been described in poxviruses.     

New class of leukemogenic ecotropic recombinant murine leukemia virus isolated from radiation-induced thymomas of C57BL/6 mice

We previously reported the establishment of several lymphoid cell lines from X-ray-induced thymomas of C57BL/Ka mice, and all, except one, produce retroviruses (P. Sankar-Mistry and P. Jolicoeur, J. Virol.35:270-275, 1980). Biological characterization of five of these new primary radiation leukemia viruses (RadLVs) indicated that they had a B-tropic, fibrotropic, and ecotropic host range and were leukemogenic when reinjected into C57BL/Ka newborn mice. The leukemogenic potential of one isolate (G(6)T(2)) was further assessed and shown to be retained after prolonged passaging on fibroblasts in vitro. Restriction endonuclease analysis of the DNA of four of our new RadLV isolates (G(6)T(2), Ti-7, Ti-8, and Ti-9) revealed that G(6)T(2) and Ti-7 murine leukemia virus (MuLV) genomes had identical restriction maps, whereas Ti-8 and Ti-9 genomes were different from each other and from the G(6)T(2) and Ti-7 genomes. The physical maps of these genomes were similar to that of known ecotropic MuLV genomes (including the C57BL/Ka endogenous ecotropic MuLV) within their long terminal repeats, env, the right portion of pol, and the left portion of gag. However, a region covering the end of gag and the beginning of pol was different and showed several similarities with xenotropic MuLV genomes of BALB/c, AKR, and C58 mice previously mapped. Our results suggest that these primary RadLV genomes are recombinants between the parental ecotropic MuLV genome and a nonecotropic (xenotropic) sequence. This nonecotropic gag-pol region might be important in conferring the leukemogenic potential to these isolates. Therefore, these RadLVs appear to form a new class of leukemogenic recombinant MuLVs recovered from leukemic tissues of mice. They appear to be distinct from the recombinant AKR mink cell focus-inducing MuLVs which have a dual-tropic host range and harbor xenotropic env sequences. To further study the leukemogenic potential of these RadLVs, the genome of one of them (G(6)T(2)) was cloned in Charon 21A as an infectious molecule.     

Genome re-sequencing analysis uncovers pathogenecity-related genes undergoing positive selection in Magnaporthe oryzae

Rice blast caused by Magnaporthe oryzae(M.oryzae) is one of the most destructive diseases,which causes significant rice yield losses and affects global food security.To better understand genetic variations among different isolates of M.oryzae in nature,we re-sequenced the genomes of two field isolates,CH43 and Zhong-10-8-14,which showed distinct pathogenecity on most of the rice cultivars.Genome-wide genetic variation analysis reveals that ZHONG-10-8-14 exhibits higher sequence variations than CH43.Structural variations(SVs) detection shows that the sequence variations primarily occur in exons and intergenic regions.Bioinformatics analysis for gene variations reveals that many pathogenecity-related pathways are enriched.In addition,193 candidate effectors with various DNA polymorphisms were identified,including two known effectors AVR-Pik and AVR-Pital.Comparative polymorphism analysis of thirteen randomly selected effectors suggests that the genetic variations of effectors are under positive selection.The expression pattern analysis of several pathogenecity-related variant genes indicates that these genes are differentially regulated in two isolates,with much higher expression levels in Zhong-10-8-14 than CH43.Our data demonstrate that the genetic variations of effectors and pathogenecity-related genes are under positive selection,resulting in the distinct pathogenicities of CH43 and Zhong-10-8-14 on rice.     

Evolution of an avirulence gene, AVR1-CO39, concomitant with the evolution and differentiation of Magnaporthe oryzae

The significance of AVR1-CO39, an avirulence gene of the blast fungus corresponding to Pi-CO39(t) in rice cultivars, during the evolution and differentiation of the blast fungus was evaluated by studying its function and distribution in Pyricularia spp. When the presence or absence of AVR1-CO39 was plotted on a dendrogram constructed from ribosomal DNA sequences, a perfect parallelism was observed between its distribution and the phylogeny of Pyricularia isolates. AVR1-CO39 homologs were exclusively present in one species, Pyricularia oryzae, suggesting that AVR1-CO39 appeared during the early stage of evolution of P. oryzae. Transformation assays showed that all the cloned homologs tested are functional as an avirulence gene, indicating that selection has maintained their function. Nevertheless, Oryza isolates (isolates virulent on Oryza spp.) in P. oryzae were exceptionally noncarriers of AVR1-CO39. All Oryza isolates suffered from one of the two types of known rearrangements at the Avr1-CO39 locus (i.e., G type and J type). These types were congruous to the two major lineages of Oryza isolates from Japan determined by MGR586 and MAGGY. These results indicate that AVR1-CO39 was lost during the early stage of evolution of the Oryza-specific subgroup of P. oryzae. Interestingly, its corresponding resistance gene, Pi-CO39(t), is not widely distributed in Oryza spp.     

Characterization of novel <Emphasis Type="Italic">Trichoderma</Emphasis> spp. isolates as a search for effective biocontrollers of fungal diseases of economically important crops in Argentina

Monoconidial cultures of 33 isolates of Trichoderma from Buenos Aires Province, Argentina were characterized on the basis of twenty eight morphological, physiological and biochemical features. All of them were screened for proteinase, endochitinase and β-1,3 glucanase activity. Universally primed PCR (UP-PCR) and inter-simple sequence repeat (ISSR) techniques were used to examine the genetic variability among isolates, which resulted in 127 bands for the total number of isolates. These results were subjected to numerical analysis revealing 20 haplotypes grouped in five clusters. The ability of Trichoderma isolates to antogonize soil-borne fungal plant pathogens using a dual culture assay was done against five fungal species: Alternaria sp., Bipolaris sorokiniana, Fusarium graminearum, F. solani, and Pyricularia oryzae. The highest inhibition values (85% RI) were obtained against B. sorokiniana and P. oryzae. Three isolates of T. harzianum named as FCCT2, FCCT3 and FCCT9 were capable of causing a high growth inhibition on four of the fungal species assayed, which was in agreement with their higher extracellular hydrolytic activity. Our results suggest that these isolates have the potential to be effective agents for biocontrol of cereal and tomato fungal pathogens.     

Structural organization of the termini of the L and S components of the genome of pseudorabies virus.

The sequences of several hundred nucleotides around the junctions between the L and S components in concatemeric DNA and in mature virion DNA were ascertained. The two ends of the mature genome (which are joined in concatemeric DNA) show no sequence homology. Several directly repeated elements are present near both ends of the genome. Furthermore, the last 82 nucleotides at the left end of the L component (and of the genome) are repeated in inverted form (inverted repeat within the L component [IRL]) approximately 350 to 600 nucleotides downstream (depending on the virus isolate) bracketing the UL2 component. A comparison between the sequences at the right and left ends of the L component of the genome showed patchy homology, probably representing a vestigial inverted repeat bracketing the L component (IRL). Furthermore, less than 5% of the genomes have an L component that is in the orientation opposite to that of most of the viral genomes, indicating that the vestigial IRL that brackets the UL sequence may be sufficient to mediate inversion of the L component in some of the genomes. On the other hand, the UL2 component, which is bracketed by a perfect IRL, does not invert to a greater extent than does the L component (if it inverts at all). Analysis of the nucleotide sequence at the concatemeric junction of three different pseudorabies virus isolates showed almost complete sequence conservation. The sequence and organization of the repeated elements in the different isolates were almost identical, despite their different histories and origins. The high degree of conservation of these repeated elements implies that they may fulfill an essential function in the life cycle of the virus.     

Chromosomal heteromorphism linked to the mating type locus of the oomycetePhytophthora infestans

The mating type locus of the oomycete,Phytophthora infestans, is embedded in a region of DNA that displays distorted and non-Mendelian segregation. By using DNA probes linked to the mating type locus to genetically and physically characterize that region, a large zone of chromosomal heteromorphism was detected. LocusS1 was shown to represent a tandemly repeated array of DNA that was typically present in a hemizygous state in A1 isolates while being absent from A2 isolates. The analysis of the parents and progeny of seven crosses indicated that the tandem array was linked in cis to the A1-determining allele of the mating type locus. A worldwide survey of genotypically diverse field isolates ofP. infestans indicated thatS1 was present in each of 48 isolates of the A1 mating type that were tested, but was absent in 46 of 47 A2 strains. Physical analysis ofS1 indicated that the tandemly repeated DNA sequence spanned about 300 kb and had evolved from a 1.35-kb monomer. Internal deletions occurred withinS1 during sexual propagation. This and other mutations apparently contributed to a high degree of polymorphism within theS1 array.