球孢白僵菌单孢子分离株在继代培养过程中菌落局变的遗传分析

研究了球孢白僵菌Beauveria bassiana单孢株继代过程中的菌落局变现象,并通过AFLP分析了角变子与原菌株间在分子水平上的差异。结果表明,野生型出发菌株Bb13的单孢分离株13S5和13S8在继代培养过程中均发生生长速率加快和产孢量下降现象。13S5在前5代,13S8在前6代产孢量有所下降但不显著,至第10代时产孢量比第1代分别下降了81.7%和69.0%。菌株角变现象在继代培养5-6代后表现明显,而13S8角变子出现的频率更高。AFLP指纹图谱分析表明,用20个引物组扩增出的98个位点中,两个角变子与野生型菌株间有12条差异条带,变异率达12.2%。由此证明单孢子分离株在继代培养中发生菌落局变后遗传物质已产生了变异。     

禾谷镰孢菌高产毒菌株的构建*

为研究禾谷镰孢菌Fusarium graminearum Schw.单端孢霉烯族毒素生物合成基因（产毒基因）在寄主体内的表达,作者构建了带报告基因GUS（(-葡糖苷酸酶基因）的质粒pGUSTRI6P5,并通过对野生型菌株的转化获得禾谷镰孢高产毒菌株。该质粒含有由TRI5（禾谷镰孢单端孢霉二烯合酶基因）启动子（TRI5 Prom）驱动的GUS基因编码区、潮霉素B抗性基因和拟枝孢镰孢F. sporotrichioides的产毒调控基因TRI6（FSTRI6）。用pGUSTRI6P5转化野生型菌株GZ3639后,在含潮霉素 B的培养基上选取抗性菌落,单孢分离获单孢菌株（转化子）。在GYEP（葡萄糖-酵母粉-蛋白胨）液体培养基上,转化子B4-1和B16-1的GUS比活力强,15-AcDON（15-乙酰脱氧雪腐镰刀菌烯醇）产量高,且两者呈正相关（相关系数（r）分别为0.9839和0.9523）。B4-1和B16-1两个转化子可作为研究禾谷镰孢与其寄主相互作用的工具菌株。     

球孢白僵菌野生及转基因菌株的退化

球孢白僵菌在人工培养基上继代培养容易发生退化变异,表现为菌丝徒长以及产孢量减少和毒力的逐渐下降,给真菌杀虫剂的生产和使用造成较大的损失.没有丢失被插入的外源毒力基因的工程株同样出现退化.培养基成分和含水量以及培养温度和光照等环境因子皆可不同程度地诱发退化的发生,但退化主要由其遗传机制决定,包括线粒体基因突变、异核现象、准性生殖、核基因突变以及有性重组等;菌落局变实质上也是菌种老化的征兆.真菌杀虫剂生产中防止退化的对策除减少传代次数外,还要根据具体菌株确定适宜的环境控制细则.     

胶孢镰刀菌产生串珠镰刀菌素的不稳定性

从陕北克山病病区分离到的两株串珠镰刀菌素产生菌株——胶孢镰刀菌(Fusarium subglutinans Wollenw.et Reinking)陕-6号和2-17号进行单孢分离,分别得到23和19个单孢分离株。这些单孢菌株可分为两种培养型:一种形态上与原始菌株相似,产生串珠镰刀菌素,产色素,具有大、小分生孢子,转管八次产毒量有下降;另一种则不产串珠镰刀菌素和孢子,无色素,后者在二株菌的单孢分离菌中所占比例分别为60.9％和15.8％。由此可见,胶孢镰刀菌产毒稳定性受异核体和该菌单核变异性共同影响。     

蛹虫草单孢菌株的生物学特性及稳定性

蛹虫草菌株在继代培养过程中极易退化。本研究选取5株蛹虫草菌株作为出发菌株,基于单孢分离和显微技术从每株出发菌株中获得50株单孢菌株。通过PCR方法对单孢菌株进行交配型类型鉴定,全部为单一交配型单孢菌株,且不同出发菌株分离得到的MAT1-1和MAT1-2交配型单孢菌株比例差异较大,分别为27:23、34:16、42:8、28:22和7:43。从不同出发菌株获得的单孢菌株中随机选择MAT1-1和MAT1-2交配型单孢菌株各5株(共计50株),进行菌落直径、产孢量和虫草素含量测定。与出发菌株相比较,14株单孢菌株菌落直径具有显著差异(其中10株显著减小),24株产孢量具有显著差异且全部下降,29株单孢菌株虫草素含量具有显著差异(其中21株显著下降)。进一步,将50株单孢菌株连续继代培养,测定菌株菌落直径、产孢量和虫草素含量,计算第七代与第一代比值评估菌株性状稳定性。结果表明,与出发菌株相比较,14株单孢菌株菌落直径比值具有显著差异且全部增加,41株单孢菌株产孢量比值具有显著差异(其中40株显著下降),44株虫草素含量比值具有显著差异且全部下降。本研究表明同一菌株中的不同单孢菌株个体之间,在生...     

胶孢镰刀菌产生串珠镰刀菌素的不稳定性

从陕北克山病病区分离到的两株串珠镰刀菌素产生菌株－胶孢镰刀菌陕－６号和２－１７号进行单孢分离,分别得到２３和１９个单孢分离株。这些单孢菌株可为两种培养型：一种形态上与原始菌株相似,产生串珠镰刀菌素,产色素,具有大、小分生孢子,转管八次产毒量有下降,另一种则不产串珠镰刀菌素和孢子,无色素,后者在二株菌的单孢分离菌中所占比例分别为６０．９％和１５．８％。由此可见,胶孢镰刀菌产毒稳定性受异核体和该菌单     

链格孢霉醇和链格孢霉醇单甲醚产生菌株的筛选

本文对分离自小麦、马铃薯、番茄和茄子上链格孢霉属(Alternaria)2个种(链格孢和茄链格孢)的96个菌株,用枯草杆菌生长抑制试验筛选链格孢霉醇(AOH)和链格孢霉醇单甲醚(AME)的产生菌株,有48株产生毒性作用(占所测菌株的50％)。18株产强、中毒性菌用高效液相色谱分析,有13株产AOH和AME(占所测菌株的72.2％)。链格孢的产毒素菌株率比茄链格孢低。但产毒素含量却是前者明显高于后者。其中产AOH和AME的最高含量,链格孢菌株XA-8分别为280和5140mg/kg,而茄链格孢菌株SA-10分别为95.9和94.3mg/kg。     

不同寄主来源的串珠镰孢生物学性状及其在无性后代的遗传与变异

自安徽、山东、湖北、江苏等地多点采集棉花红腐病、水稻恶苗病、玉米穗腐病的病组织,经分离、鉴定和纯化,获得107个串珠镰孢（Fusarium monilifoFine）菌株。对上述来源于棉花、水稻、玉米的串珠镰孢的菌落形态、生长速率和产孢量等生物学性状及其在无性后代的遗传与变异进行了研究。结果表明,不同寄主来源的串珠镰孢菌株的菌丝生长温度范围和最适温度大致相同,但在菌落形态特别是色素方面存在明显差异,生长速率和产孢量也存在显著差异。棉花菌株的平均生长速率最大,玉米菌株生长速率最小,水稻菌株生长速率居中,相同群体的不同菌株间生长速率有极显著差异;玉米菌株产孢量最大,棉花菌株产孢量最小,水稻菌株产孢量居中。方差分析显示,不同寄主菌株群体间产孢量存在显著差异,而同一寄主群体的不同菌株间产孢量均无显著差异,说明菌株产孢量大小主要与其寄主种类有关,而与地区来源关系不大。遗传测定结果表明,分离自棉花、玉米和水稻的串珠镰孢的菌落形态和生长速率在单分生孢子后代均可稳定遗传;产孢量性状遗传有两种情况：分离自棉花和水稻的串珠镰孢菌株Fm1和Fm31的产孢量性状在单分生孢子后代均可稳定遗传,而分离白玉米的串珠镰孢菌株Fm19的产孢量性状在单分生孢子第一代（CG1）发生变异。     

河北省马铃薯早疫病菌群体遗传结构的研究

通过对2009－2010年河北省145株及外省30株马铃薯早疫病菌对代森锰锌的敏感性、产孢量两个表型性状和AFLP基因型的测定,揭示了河北省早疫病菌群体的遗传结构。毒力测定表明所有供试菌株对代森锰锌均表现敏感,其EC50范围介于1.04－4.86μg/mL之间,平均为2.93μg/mL。被测河北省47株早疫病菌产孢量平均为85个孢子/mm2,大大高于30个对照菌株的产孢量,对照菌株的平均产孢量为50个孢子/mm2,菌株间产孢量存在明显差异。AFLP聚类分析揭示出了马铃薯早疫病菌丰富的遗传多样性,每个菌株都具有独特的AFLP基因型。早疫病菌AFLP基因型与地理来源存在着密切的相关性,与菌株产孢量有一定的相关性,但与代森锰锌敏感性无相关性。     

禾谷镰孢菌高产毒菌株的构建

为研究禾谷镰孢菌Fusarium graminearum Schw.单端孢霉烯族毒素生物合成基因（产毒基因）在寄主体内的表达,作者构建了带报告基因GUS（β-葡糖苷酸酶基因）的质粒pGUSTRI6P5,并通过对野生型菌株的转化获得禾谷镰孢高产毒菌株,该质粒含有由TRI5（禾谷镰隐单端孢霉的二烯合酶基因）启动子（TRI5 Prom)驱动的GUS基因编码区、潮霉素B抗性基因和拟枝孢镰孢F.sporotrichioides的产毒调控基因TRI6（FSTR16）。用pGUSTRI6P5转化野生型菌株GZ3639后,在含潮霉素B的培养基上选取抗性菌落。单孢分离获单孢菌株（转化子）。在GYEP（葡萄糖-酵母粉-蛋白胨）液体培养基上,转化子B4-1和B16-1的GUS比活力强,15-AcDON(15-乙酰脱氧雪腐镰鼠菌烯醇）产量高,且两者呈正相关（相关系数（r)分别为0.9839和0.9523)。B41-1和B16-1两个转化子可作为研究禾谷镰孢与其寄主相互作用的工具菌株。     

脐腹小蠹高致病力虫生真菌菌株的筛选

【目的】脐腹小蠹Scolytus schevyrewi Semenov是白榆的钻蛀性害虫之一,为找到对脐腹小蠹的高致病力虫生真菌菌株。【方法】通过室内喷雾法研究了5株虫生真菌菌株对脐腹小蠹幼虫的致病力,结合各菌株菌落直径、孢子萌发率、产孢量和菌落生长速率4个培养特征,筛选到Bb773和Pc546 2株优良的菌株,在此基础上,进一步对这2株菌株的几丁质酶、蛋白质酶和荧光素二乙酸酯(FDA)水解酶活性进行了测定。【结果】菌株Bb773和菌株Pc546在浓度为1×107孢子/mL悬浮液处理后,10 d的后脐腹小蠹幼虫的校正死亡率分别达96.67%和90.00%,致死中时分别为3.61 d和4.18 d。菌株Bb773的几丁质酶、FDA水解酶和蛋白质酶活性在接种后第2~10天均高于菌株Pc546,2株菌株蛋白质酶活性差异均达到了显著水平(P<0.05)。【结论】确定了菌株Bb773在脐腹小蠹害虫生物防治中具有潜在的应用价值。     

Repeated in vitro subculturing alters spore surface properties and virulence of Metarhizium anisopliae

Repeated subculturing caused rapid changes in the spore surface properties and virulence of Metarhizium anisopliae. Of the two strains evaluated, M. anisopliae V245 attenuated more rapidly than V275. Electrophoretic mobility and Radial Flow Chamber assays were used for the first time to generate qualitative and quantitative information on the adhesive forces of M. anisopliae conidia. Independent of strain, adhesion, hydrophobicity and spore-bound Pr1 declined after the first subculture; however, spore surface charge decline was erratic. Adhesion and hydrophobicity stabilized after the third subculture, whereas spore-bound Pr1 continues to decline following repeated subculturing. Decline in spore bound Pr1 was directly correlated with decline in virulence, however, such correlation with adhesion, hydrophobicity or surface charge could not be established. Because spore-bound Pr1 activities were directly correlated with M. anisopliae virulence; it could be used as a quality-control marker to monitor changes in virulence.     

Hypovirulence-associated traits induced by a mycovirus of Cryphonectria parasitica are mimicked by targeted inactivation of a host gene.

Expression of the Vir2 gene of Cryphonectria parasitica is down-regulated in strains of the fungus containing a double-stranded RNA genetic element that reduces fungal virulence (W. A. Powell and N. K. Van Alfen, Mol. Cell. Biol. 7:3688-3693, 1987). We have sequenced the Vir2 gene and characterized its structure; the mRNA contains a short open reading frame whose product has structural similarities to several fungal pheromones. A null mutant was constructed by homologous recombination to determine the function of the Vir2 gene and whether its disruption resulted in any of the altered phenotypes exhibited by many hypovirulent strains, such as reductions in virulence, pigmentation, and sporulation. The Vir2 null mutant (18dm) exhibited a wild-type phenotype with respect to gross colony morphology, growth rate, pigmentation, asexual spore viability, and virulence in apple fruit and chestnut trees. However, numbers of asexual fruiting bodies (pycnidia) and conidia were reduced significantly in comparison with the wild-type strain EP155/2. In sexual crosses of 18dm with a wild-type strain of the opposite mating type, perithecia (sexual fruiting bodies) developed but were barren. Deletion of the Vir2 gene results in a phenotype that mimics that of many double-stranded-RNA-containing hypovirulent strains; i.e., the null mutant exhibits significant reductions in asexual sporulation and pycinidum production as well as impaired sexual crossing ability. To our knowledge, this is the first report of the partial reproduction of a virus-induced phenotype by deletion of a virus-perturbed host gene.     

Molecular studies of co-formulated strains of the entomopathogenic fungus,Beauveria bassiana

A 28S rDNA intron was used as a molecular marker to distinguish between two single spore strains of Beauveria bassiana, Bb123 and Bb151. When co-formulated and assayed against larvae of Galleria mellonella these strains exhibited no synergistic increase in virulence, rather Bb123 usually dominated. This study shows that the success of any strain to infect Galleria is dependent on the dose and method of inoculation (injection versus immersion). The result of co-formulated strains grown on solid culture also showed that usually one strain dominated, i.e., strain displacement could happen both in vivo and in vitro. The speed by which one strain was displaced following successive sub-culturing on PDA partly depended on the ratio of Bb151 and Bb123. The co-formulated inoculum could widen the window over which parent strains would be active on different water activity media. Co-infection did result in heterokaryosis within the Galleria host. Molecular studies also showed that the heterokaryon was not stable and could revert back to the parent strain.     

Effects of major spore-specific DNA binding proteins on Bacillus subtilis sporulation and spore properties

Sporulation of a Bacillus subtilis strain (termed alpha(-) beta(-)) lacking the majority of the alpha/beta-type small, acid-soluble spore proteins (SASP) that are synthesized in the developing forespore and saturate spore DNA exhibited a number of differences from that of the wild-type strain, including delayed forespore accumulation of dipicolinic acid, overexpression of forespore-specific genes, and delayed expression of at least one mother cell-specific gene turned on late in sporulation, although genes turned on earlier in the mother cell were expressed normally in alpha(-) beta(-) strains. The sporulation defects in alpha(-) beta(-) strains were corrected by synthesis of chromosome-saturating levels of either of two wild-type, alpha/beta-type SASP but not by a mutant SASP that binds DNA poorly. Spores from alpha(-) beta(-) strains also exhibited less glutaraldehyde resistance and slower outgrowth than did wild-type spores, but at least some of these defects in alpha(-) beta(-) spores were abolished by the synthesis of normal levels of alpha/beta-type SASP. These results indicate that alpha/beta-type SASP may well have global effects on gene expression during sporulation and spore outgrowth.     

Variation in virulence of Coxsackie virus B3 in the hearts of mice. I. Comparison of mortality and virus growth in the heart and other organs

Virulence of Coxsackie virus B3 (CB3) was compared in mice between strain SK-74 isolated from a patient and strain T-70 isolated from a healthy child as well as between prototype strain Nancy and its mouse-passaged derivative strain PMH. Strain SK-74 showed a high mortality (40-80%), while strain T-70 did not induce deaths in mice (mortality: 0%). Strain PMH showed a high mortality (65-85%), but strain Nancy did not cause deaths in the mice. In agreement with the mortality trend, virus titers in the heart and other organs of mice inoculated with strain SK-74 and PMH were higher than those in mice inoculated with T-70 and Nancy strains. Since virus titers in the heart remained higher than those in other organs, the heart was regarded as the main organ in which CB3 could replicate and induce cell degeneration. The present results suggest that a marked variation in virulence in the hearts of mice among CB3 strains occurred and that passage through the hearts of mice enhances the virulence of CB3 in the hearts of mice.     

Natural isolates of Saccharomyces cerevisiae display complex genetic variation in sporulation efficiency

Sporulation is a well-studied process executed with varying efficiency by diverse yeast strains. We developed a high-throughput method to quantify yeast sporulation efficiency and used this technique to analyze a line cross between a high-efficiency oak tree isolate and a low-efficiency wine strain. We find that natural variation in sporulation efficiency mirrors natural variation in higher eukaryotes: it shows divergence between isolated populations, arises from loci of major effect, and exhibits epistasis. We show that the lower sporulation efficiency of the wine strain results from a failure to initiate sporulation, rather than from slower kinetics of meiosis and spore formation. The two strains differentially regulate many genes involved in aerobic respiration, an essential pathway for sporulation, such that the oak tree strain appears better poised to generate energy from this pathway. We also report that a polymorphism in RME1 that affects sporulation efficiency in laboratory strains also cosegregates with significant phenotypic differences in our cross of natural isolates. These results lay the groundwork for the study of variation in sporulation efficiency among natural isolates of yeast.