家蚕病原球孢白僵菌的原生质体再生回复及核型分析

家蚕病原球孢白僵菌（Beauveriabassiana）原生质体的分离制备、性状及再生回复,并用脉冲凝胶电泳（PFGE）技术分析了其核型。以6mg／mLDriselase为酶解液,0.7mol／LNaCl液（pH5.8）为渗透压稳定剂,在30℃下轻轻振荡处理幼嫩菌丝1．5h,是原生质体分离的适宜条件。原生质体的无核率为26．5％,有核率为73.5％,其中单核率为53.5％。再生回复的形式可观察到三种,其培养基的渗透压稳定剂以0.7mol／L葡萄糖较为适当。球孢白僵菌至少具有6条染色体,估算大小为2.5～6.6Mb,核型大小为26.5Mb。     

Selection of a Highly Virulent Fungal Isolate,Metarhizium anisopliae Ma TB 101, for Control of Taro Beetle,Papuana uninodis (Coleoptera: Scarabaeidae)

Fungal isolates (31 Metarhizium anisopliae var. anisopliae , five M. anisopliae var majus , three Beauveria bassiana and four B. brongniartii ) originating from a wide range of geographical locations, insect species and soil types were tested against Papuana uninodis (Coleoptera: Scarabaeidae). In a first test series, spores were applied topically to third-instar larvae and adults. The most effective strain against P. uninodis larvae and adults was Ma TB 101, a M. anisopliae isolate from adult P. woodlarkiana . For adults, strain Ma FI 384, a M. anisopliae from Popillia japonica , was almost equally effective. The 11 most effective isolates (nine M. anisopliae var. anisopliae , one B. brongniartii and one B. bassiana ) with LT values of less than 6 weeks in 50 adults and/or less than 4 weeks in larvae were tested for their efficacy against adults and larvae of P. uninodis by application of spores to soil (107 spores/g). Ma TB 101 was significantly more effective against both adults and larvae (LT ca. 10 days) than all other isolates (LT > 50 50 3 weeks). Two other M. anisopliae strains, Ma F 248 from soil and Ma FI 384 from P.japonica , were more effective than most isolates in adults. The latter three M. anisopliae isolates were tested in a concentration series against third-instar larvae and adults. Mortality was concentration related. Isolates Ma F 248 and Ma FI 384 did not achieve 50% mortality within the test period at concentrations lower than 107 spores/g of soil or feed. For concentrations of Ma TB 101 from 1 107 to 2 105 spores/g the LT ranged from 13 to 30 days in adults and 12 to 24 50 days in third-instar larvae. Accordingly, concentrations causing 50% mortality (LC ) for Ma 50 TB 101 were significantly lower than for the two other M. anisopliae isolates tested.     

绿僵菌大孢变种的生物学特征及其对蛴螬的毒力研究

以报道较多的蛴螬病原真菌 布氏白僵菌、绿僵菌小孢变种作参照菌,系统研究了新分离鉴定的绿僵菌大孢变种的生物学特性,测定了其在偏低的自然气温下对蛴螬的致病能力.结果表明,绿僵菌大孢变种与参照菌的培养要求接近,最适宜的温度为25 ℃.3种供试的培养基中,PPDA为最适宜的培养基,其次为SDAY.在偏低的温度环境下绿僵菌大孢变种对蛴螬的毒杀能力强于布氏白僵菌,土壤和喷雾处理后累计僵虫率分别达88.23%和76.47%,有很好的田间应用前景.     

酿酒酵母染色体DNA样品制备和CHEF分析

等高锁状均匀电场（contolllsclampedhomogeneouselectncfield,CHED电泳技术与分子杂交、酵母人工染色体（yeastartificialchromosome,YAC）等分子生物学技术的有机结合,推动了真菌电泳核型、染色体作图和染色体DNA长度多型性（chromosomalDNAlellgthpolymorphism,CLP...     

The cryopreservation of some large ciliate entodiniomorphid protozoa taken from the rumen

M.H.P. FUNGARO, M.L.C. VIEIRA, A.A. PIZZIRANI-KLEINER AND J.L. DE AZEVEDO. 1996. Random amplified polymorphic DNA (RAPD) was used in order to analyse the relationships among 13 isolates of Metarhizium anisopliae var. anisopliae . Six of them were isolated from Deois flavopicta (Stal) (Hemiptera—Homoptera: Cercopidae) in different regions of Brazil. The other seven were isolated from soil in Paraná State in Southern Brazil. The isolates were grouped by cluster analysis using Dice similarity index. The results show that isolates of M. anisopliae var. anisopliae are extremely diverse (47% similarity) but those isolated from D. flavopicta present only a moderate degree of variation (82% similarity) when compared with the wide diversity (31% similarity) found in the group isolated from soil. These results suggest that M. anisopliae var. anisopliae has developed host specificity.     

由部分核糖体RNA序列确定的绿僵菌属种系统发育关系

本研究应用特异寡聚核苷酸引物和双脱氧核苷酸终止法测定绿僵菌Metarhizium不同种的部分rRNA序列。这些序列分别选自小亚基(18S)和大亚基(25S)rRNA上的不同区域。校排的不同序列用于计算核苷酸的差异。绿僵菌的系统发育关系采用最大可能性(Maximum-Likelihood)方法分析。所有的分类单元聚类成二个分支。在产生圆柱状瓶形小梗的种中,M.anisopliae var.anisopliae,M.anisopliae var.majus,M.brunneum(NRRL1944),M.guizhouense,M.pingshaense和未知种M.sp.2组成一个密切相关的群组,而M.anisopliae(ACCC 30104)位于这群组之外。在产生棍棒状瓶形小梗的种中,M.album,M.cylindrosporae和M.flavoviride互相独立成支。实验结果不支持Metarhizium只能区分为M.anisopliae和M.flavoviride二个种的分类观点。文中还讨论了经典分类标准的系统发育价值。     

Characterization of Metarhizium species and varieties based on molecular analysis, heat tolerance and cold activity

Aims:  The genetic relationships and conidial tolerances to high and low temperatures were determined for isolates of several Metarhizium species and varieties.
Methods and Results:  Molecular-based techniques [AFLP and rDNA (ITS1, ITS2 and 5·8S) gene sequencing] were used to characterize morphologically identified Metarhizium spp. isolates from a wide range of sources. Conidial suspensions of isolates were exposed to wet heat (45 ± 0·2°C) and plated on potato dextrose agar plus yeast extract (PDAY) medium. After 8-h exposure, the isolates divided clearly into two groups: (i) all isolates of Metarhizium anisopliae var. anisopliae ( Ma-an ) and Metarhizium from the flavoviride complex ( Mf ) had virtually zero conidial relative germination (RG), (ii) Metarhizium anisopliae var. acridum ( Ma-ac ) isolates demonstrated high heat tolerance ( c . 70–100% RG). Conidial suspensions also were plated on PDAY and incubated at 5°C for 15 days, during which time RGs for Ma-an and Ma-ac isolates were virtually zero, whereas the two Mf were highly cold active (100% RG).
Conclusions:  Heat and cold exposures can be used as rapid tools to tentatively identify some important Metarhizium species and varieties.
Significance and Impact of the Study:  Identification of Metarhizium spp. currently relies primarily on DNA-based methods; we suggest a simple temperature-based screen to quickly obtain tentative identification of isolates as to species or species complexes.     

两株椰心叶甲分离物的鉴定及其系统发育分析

采用形态学方法对2株从自然罹病死亡的椰心叶甲虫尸上分离到的致病菌株Dz01和Ma4进行了鉴定,发现2个菌株在菌丝、瓶梗和分生孢子等形态特征上与金龟子绿僵菌小孢变种基本一致,可将2个菌株鉴定为金龟子绿僵菌小孢变种。基于Dz01和Ma4菌株和其它31个代表绿僵菌主要种或变种菌株rDNA上ITS1-5.8S-ITS2区序列构建的最大简约树显示,Dz01和Ma4菌株均聚在金龟子绿僵菌小孢变种所构成的分支中,这为2个菌株形态学鉴定结果提供了分子依据。     

A chromosome 1-specific DNA library from the domestic pig (Sus scrofa domestica).

Chromosomes were prepared from lymphocytes of a male domestic pig and flow-sorted on a dual-laser FACS. Twenty spots were observed, corresponding to the known pig karyotype of 18 pairs of autosomes plus the X and Y. DNA was isolated from 10,000 copies of the presumed chromosome 1 spot, restricted with Sau3A, ligated into the vector pGEM4z, and PCR amplified using universal primers; the products were then re-ligated into pUC18. After transformation into Escherichia coli, 210,000 independent colonies were obtained, 5% of which contained only vector DNA. The average insert size of the library was 405 bp. Southern blotting revealed that 36% of the clones contained single-copy DNA and that the remainder contained moderately or highly repetitive DNA. Screening with a (CA)n probe revealed that roughly 1% of the clones contained microsatellite sequences. A bulk insert of the library was biotinylated by PCR and used as a probe for chromosomal in situ suppression hybridization to pig chromosomes, which confirmed that the library is specific for chromosome 1. However, sequences from the centromeric and telomeric regions seem to be underrepresented in the library.     

Division frequency of pea protoplasts in relation to starch accumulation

Division frequency of alginate-embedded pea (Pisum sativum var. Belman) protoplasts derived from embryonic shoot tips was studied quantitatively by image analysis in relation to starch accumulation and protoplast size. Protoplast divisions were observed from day 4 on and the number of protoplasts undergoing division increased in a stepwise manner to 70% the following days. The starch content increased rapidly during the first 3 days of culture prior to the onset of division and resulted a 4.2-fold increase in the intracellular starch area and a 3.0-fold increase (from 27% to 80%) in the number of protoplasts containing starch. Subsequent periods with rapid increases the number of dividing protoplasts were preceded by further starch accumulation. Dividing protoplasts were 33–60% smaller and contained 8–42% less starch than non-dividing protoplasts. However, calculations showed that, in the dividing protoplasts, the relative area covered by starch was 6–12% higher than in non-dividing protoplasts. These data suggest that starch accumulation precedes division of pea protoplasts.     

A quantitative analysis of shotgun cloning in Bacillus subtilis protoplasts

Summary We used the Escherichia coli-Bacillus subtilis shuttle vector pHP13, which carries the replication functions of the cryptic B. subtilis plasmid pTA1060, to study the effects of BsuM restriction, plasmid size and DNA concentration on the efficiency of shotgun cloning of heterologous E. coli DNA in B. subtilis protoplasts. In a restriction-deficient strain, clones were obtained with low frequency (19% of the transformants contained a recombinant plasmid) and large inserts (>6 kb) were relatively rare (12% of the clones contained inserts in the range of 6–9 kb). The efficiency of shotgun cloning was severely reduced in restricting protoplasts: the class of large inserts (>6 kb) was under-represented in the clone bank (4% of the clones contained inserts in the range of 6–6.1 kb). Furthermore, BsuM restriction caused structural instability of some recombinant plasmids. Transformation of protoplasts with individual recombinant plasmids showed that plasmid size and transforming activity were negatively correlated. The size effect was most extreme with cut and religated plasmid DNA. The yield of clones was independent of the DNA concentration during transformation. It is therefore unlikely that clones were not detected because of simultaneous uptake of more than one plasmid. It is concluded that shotgun cloning in B. subtilis protoplasts is inferior to that in competent cells.     

Genetic polymorphisms in three subtilisin-like protease isoforms (Pr1A, Pr1B, and Pr1C) from Metarhizium strains

Restriction fragment length polymorphisms (RFLP) were examined in three isoforms of a gene family encoding subtilisin-like proteases (Pr1A, Pr1B, and Pr1C) in several isolates of the entomopathogenic fungus Metarhizium anisopliae. RFLP variation was not observed in any of the Pr1 genes from isolates within the same genetically related group. Between genetically related groups and between isolates from disparate geographical areas, the greatest variation in RFLP patterns was observed for Pr1A. When variation does occur at Pr1B and Pr1C, it was generally observed at an EcoRI site. Metarhizium anisopliae var. majus strain 473 and a M. flavoviride isolate were most dissimilar in RFLP patterns at all Pr1 genes when compared to the M. anisopliae strains. We suggest that Pr1 genes represent a gene family of subtilisin-like proteases and that the Pr1A gene encodes for the ancestral subtilisin-like protease which has subsequently duplicated and rearranged within the genome.     

Mortality of Delia floralis, Galleria mellonella and Mamestra brassicae treated with insect pathogenic hyphomycetous fungi

Abstract:  The susceptibility of Delia floralis eggs, neonates and larvae and the susceptibility of Galleria mellonella and Mamestra brassicae larvae to seven different Norwegian isolates of the insect pathogenic, hyphomycetous fungi Tolypocladium cylindrosporum , Metarhizium anisopliae and Beauveria bassiana , were investigated. Metarhizium anisopliae isolate ARSEF 5520 was highly virulent to G. mellonella larvae and caused 100% mortality when tested at a concentration of 3.6 × 10⁶ conidia/ml. The same M. anisopliae isolate was not virulent to D. floralis larvae. Isolates of T.cylindrosporum , were equally virulent to G. mellonella and D. floralis causing up to 36.0% mortality of larvae. It is suspected, however, that the use of grated rutabaga as a food source in the D. floralis bioassay reduced the fungal virulence of both M. anisopliae and T. cylindrosporum to D. floralis . Among three T. cylindrosporum isolates tested at a concentration of 1.0 × 10⁷ conidia/ml against eggs of D. floralis none of them reduced the hatching percentage. One isolate, ARSEF 5525 did, however, significantly reduce the longevity of neonates. Beauveria bassiana isolates ARSEF 5510 and ARSEF 5370 tested at a concentration of 1.0 × 10⁷ conidia/ml resulted in M. brassicae mortality levels of 70.0 and 55.0%, respectively. The B. bassiana isolate ARSEF 5557, however, was not virulent to M. brassicae . Among the three isolates tested against M. brassicae the two virulent isolates produced a red pigment, probably oosporein, when cultured in Sabouraud dextrose agar.     

Characterization of a newly discovered China variety of Metarhizium anisopliae (M. anisopliae var. dcjhyium) for virulence to termites, isoenzyme, and phylogenic analysis

The efficacy of a new virulent Metarhizium anisopliae variety (M. anisopliae var. dcjhyium, DQ288247) obtained from Odontotermes formosanus in China was evaluated against the subterranean termite, O. formosanus, in the laboratory. The new variety was compared with four other virulent M. anisopliae isolates and was found to be highly infectious and virulent against termites. M. anisopliae var. dcjhyium could cause approximately 100% mortality of termites 3 days post-inoculation in the concentration of 3x10(8) conidia/ml. There were also differences in relative hyhal growth and isoenzymes. M. anisopliae var. dcjhyium showed a different isoenzyme band pattern from the four isolates of M. anisopliae (AB027337, AB099510, AB099941 and AF280631). The phylogenetic tree of the 18S rDNA sequences revealed the taxonomic and evolutionary position of M. anisopliae var. dcjhyium. M. anisopliae var. dcjhyium and four isolates of M. anisopliae formed a monophyletic group, supported by a 99% bootstrap value. M. anisopliae var. dcjhyium formed a distinct variety, which had a special characterization of unique bands of isoenzyme, high virulence and low repellency against termites when compared with four other isolates of M. anisopliae.     

金龟子绿僵菌原生质体的制备和再生及其羟化酶活性研究

对金龟子绿僵菌(Metarhizium anisopliae)原生质体的制备和再生的影响因素进行实验,并在此基础上考察了甾体底物对原生质体羟化酶的诱导作用。结果表明,原生质体制备的合适条件是:42 h的菌丝体用纤维素酶(10 mg/mL)和蜗牛酶(5 mg/mL)的混合酶在含有0.8 mol/L甘露醇的pH 5.8磷酸缓冲液中,28℃震荡(80 r/min)酶解3 h,原生质体产量可达到6.12×10~7/mL,在含有0.6 mol/L KCl的双层马铃薯培养基上再生率达到7.79%。经过6 h底物诱导的菌丝体制备的原生质体细胞色素P450的表达量比没经过诱导的菌丝体制备的原生质体高约40%,证明该菌羟化酶系统的可诱导性。由于没有细胞壁的阻碍经过底物诱导的原生质体能够高效的将底物转化为产物,且副产物相对较少。     

克鲁斯假丝酵母及其近似种的脉冲电泳核型分析

用钳位均匀电场脉冲电泳(CHEF)系统分析了克鲁斯假丝酵母(Candida krusei),郎比可假丝酵母(C. lambica)和粗状假丝酵母(C. valiad)的模式菌株的电泳核型,发现这三种表型相似的假丝酵母却具有互不相同的染色体DNA分子带型,为其分类学研究提供了可靠的鉴别依据。在常规分类学研究的基础上,测定了AS 2.75(原定种名为(C. incospicua),AS2.1182(原定种名为 C. lambica)和AS 2.1772(未定种)等三株假丝酵母的G+C含量和脉冲电泳核型。通过对已报道的C. inconspicu的G+C含量及上述三种假丝酵母模式菌株的脉冲电泳核型的比较分析证明,AS 2.75和AS 2.1772为粗状假丝酵母(C. valida),AS 2.1182为克鲁斯假丝酵母(C. krusei)。     

Electrophoretic Karyotypes of Fusarium spp.

Migheli, Q., Berio, T., and Gullino, M. L. 1993. Electrophoretic karyotypes of Fusarium spp. Experimental Mycology 17, 329-337. The electrophoretic karyotype of 17 antagonistic and pathogenic strains of Fusarium spp. has been established by using contour-clamped homogeneous electric field gel electrophoresis. Intact chromosomal DNA was prepared from fungal protoplasts with standard procedures. Up to 11 distinct chromosomal bands were resolved after 184 h of migration at 50 V. Polymorphic karyotypes were observed in different species of Fusarium, formae speciales of F. oxysporum , and races of F. oxysporum f.sp. dianthi. Using the Schizosaccharomyces pombe and Saccharomyces cerevisiae chromosomes as size standards, the size of the Fusarium genome was estimated to range from approximately 18.1 to 51.5 Mb. The suitability of electrophoretic karyotyping as a tool for strain characterization, as well as some applications in hybridization analysis of Fusarium spp., is discussed.     

Electrophoretic Karyotype of the Obligate Biotrophic Parasite Plasmodiophora brassicae Wor.

Classical genetic analysis is not possible with the protist Plasmodiophora brassicae due to the intracellular life of this obligate biotrophic parasite . An electrophoretic karyotype has been obtained using contour-clamped homogeneous electric field gel electrophoresis to facilitate gene mapping of P. brassicae. Using two different separation conditions 16 chromosomal bands of P. brassicae were distinguished ranging in approximate size from 2.2 Mb to 680 kb. According to this determination of chromosome number and size, the total genome size of P. brassicae was estimated to be 20.3 Mb. The chromosomal bands were further designated by their hybridization pattern with repetitive elements of P. brassicae . The repetitive element H4 (1800 bp) hybridized with 14 chromosomal bands, but the sequence of H4 showed no homology to known centromere or telomere structures and revealed no repetitive motifs.     

Thermal biology of the meadow grasshopper, Chorthippus parallelus, and the implications for resistance to disease

Abstract.  1. The thermal biology of the meadow grasshopper, Chorthippus parallelus , a common, habitat generalist acridid species found in the U.K., was characterised and the influence of thermoregulatory behaviour for resistance against a temperate ( Beauveria bassiana ) and tropical ( Metarhizium anisopliae var. acridum ) fungal pathogen was determined.
2.  Chorthippus parallelus was found to be an active behavioural thermoregulator, with a preferred temperature range of 32–35 °C.
3. Both pathogens proved lethal to fifth instar and adult grasshoppers. No evidence of behavioural fever in response to infection by either pathogen was found, but normal thermoregulation was found to reduce virulence and spore production of B. bassiana. Normal thermoregulation did not appear to affect M. anisopliae var. acridum .
4. These results suggest that the effects of temperature on host resistance depend on the thermal sensitivity of the pathogen and, in this case, derive from direct effects of temperature on pathogen growth rather than indirect effects mediated by host immune response.
5. The implications for possible risks of exotic pathogens and influence of climate change are discussed.     

Cytogenetic studies of four South American species of Paullinia L. (Sapindaceae)

Four South American species of Paullinia ( P. elegans , P. meliaefolia , P. pinnata , and P. rhomboidea ) were compared using conventional chromosome staining, C-Giemsa and C-chromomycin A₃/4',6-diamidino-2-phenylindole (C-CMA₃/DAPI) banding, and fluorescence in situ hybridization (FISH) with a 45S ribosomal DNA (rDNA) probe. All species showed a somatic complement of 2 n  = 24 chromosomes, agreeing with earlier records in some cases, and showing a tendency for the chromosome number to be conserved in this genus. The chromosome number of P. rhomboidea is a new report. The karyotypes differed in chromosome size and degree of karyotype asymmetry. The chromosomal band patterns and location of the 45S rDNA sites are reported for the first time in the genus. Terminal C-CMA₃ bands were associated with the 45S rDNA sites, but varied in number and size between the species. The occurrence of other C-Giemsa bands that were not revealed by CMA₃ suggests that more than one family of repetitive DNA may be involved in karyotype differentiation. The systematic implications of these results on the infrageneric relationships are discussed.   © 2007 The Linnean Society of London . Botanical Journal of the Linnean Society , 2007, 154 , 313–320.