Genotypic analysis of Nomuraea rileyi collected from various noctuid hosts

The genetic diversity of 79 Nomuraea rileyi isolates from various lepidopteran hosts in Asia, North America, and South America was evaluated using amplified fragment length polymorphism (AFLP) analysis. Cluster analysis separated the N. rileyi isolates into two major groups and seven subgroups. The resulting dendrogram generally classified the N. rileyi isolates based on insect host and geographical region. The haplotypic diversity index of N. rileyi subpopulations from each location and host origin was ranging from 0.8788 to 1.000. However, analysis of molecular variance (AMOVA) demonstrated no significant differences (p =0.3421) among N. rileyi isolates from different continents. Whereas the genetic variation among the N. rileyi populations from the different host insects within each continent was significantly different (p <0.0001).     

Use of a colorimetric system to detect enzymes expressed by germinating conidia of entomopathogenic fungi

An apiZYM system, with 19 substrates, was used to detect enzymes expressed by germinating conidia of Nomuraea rileyi (5 isolates), Nomuraea atypicola, Nomuraea anemonoides, Beauveria bassiana and Metarhizium anisopliae. Similar enzyme profiles were obtained for two of the N. rileyi isolates (Mississippi, Ecuador) regardless of whether culture medium (Sabouraud-maltose-yeast) or cuticle (from larvae of Trichoplusia ni, Heliothis zea or Heliothis virescens) were used as substrates. Centroid-clustering analysis revealed three distinct enzyme profiles.Mention of a proprietary product in this paper does not consitute a recommendation for use by USDA.     

引起蜘蛛流行病的紫色野村菌种群异质性

应用ISSR分子标记对安徽琅琊山自然保护区和牯牛降自然风景区引起蜘蛛流行病的紫色野村菌Nomuraea atypicola遗传多样性进行了研究。筛出的9个ISSR引物共扩增出117条带,多态性比率高达94%。ISSR扩增结果显示:22株紫色野村菌之间的遗传分化较大,Jaccard遗传相似系数为0.48-0.88,平均为0.71。UPGMA方法构建的系统树表明,在遗传相似系数0.60处,22株紫色野村菌按照两个自然保护区明显分为2大类群。第一大类是分离自琅琊山不同蜘蛛的10株菌株,其种群内平均遗传相似系数为0.74。第二大类是采自牯牛降不同蜘蛛的12株菌株,其种群内平均遗传相似系数为0.76。种群内的遗传相似性虽高于种群间的遗传相似性,但未见两株菌株的ISSR指纹图谱相同或高度相似。这表明,不管是在琅琊山还是在牯牛降,紫色野村菌的两个种群均无优势的流行菌株。两地的蜘蛛流行病都是由高度异质的紫色野村菌种群造成的。紫色野村菌的菌株遗传相似性和地理来源相关而和寄主无关。     

Identification of Virulent Isolates of the Entomopathogenic Fungus Nomuraea Rileyi (F) Samson for the Management of Helicoverpa Armigera and Spodoptera Litura

Eleven geographical isolates of the entomopathogenic fungus Nomuraea rileyi (Farlow) Samson of Helicoverpa armigera (Hubner)/Spodoptera litura (Fabricius) origin were studied for efficacy against the two host insects. Laboratory bioassays at a concentration of 2 x 10(8) conidia ml(-1) indicated that N. rileyi isolates of S. litura origin were better in terms of time taken for mycosis and mortality in both the test larvae: S. litura (77-80% mortality in 7 days) and H. armigera (79-85% in 8 days). Among the isolates of S. litura origin, geographical isolates from Hyderabad and Karimnagar were superior in terms of high percent kill as well as 100% germination of conidia within 48 h. Fastest germination was observed with Karimnagar isolate followed by Hyderabad isolate. Conidial yield was highest on barley-carrot extract-yeast extract medium. However in terms of material cost, barley-yeast extract medium was the lowest. The Karimnagar isolate of S. litura origin gave the highest conidial yield on barley-yeast extract medium. Chitinolytic enzyme profiles of different isolates revealed polymorphism in all the isolates from S. litura origin. Overall among the parameters studied the best traits were found in the Karimnagar isolate of S. litura origin.     

Phylogenetic and structural analyses of the mating-type loci in Clavicipitaceae

Entomopathogens and other econutritional fungi belonging to Clavicipitaceae were phylogenetically analyzed on the basis of the 18S rRNA gene and mating-type genes (MAT1-1-1 and MAT1-2-1). The phylogenies of the mating-type genes yielded better resolutions than that of 18S rRNA gene. Entomopathogens (Cordyceps bassiana, Cordyceps brongniartii, Cordyceps militaris, Cordyceps sinclairii, Cordyceps takaomontana, Isaria cateniannulata, Isaria farinosa, Isaria fumosorosea, Isaria javanica, Lecanicillium muscarium and Torrubiella flava) were considered as a phylogenetically defined group, and were closely related to mycopathogens (Lecanicillium psalliotae and Verticillium fungicola). They located at more descendant positions in the mating-type trees than other fungi, and lacked the mating-type gene MAT1-1-3. The deletion of MAT1-1-3 was supposed to have occurred once in Clavicipitaceae, and a good indication for the evolution of Clavicipitaceae. Other entomopathogens (Cordyceps cylindrica, Cordyceps subsessilis, Metarhizium anisopliae and Nomuraea rileyi) and pathogens of plants, nematodes and slime molds, were relatively related to each other, and possessed MAT1-1-3, but were supposed to be heterogeneous. Root-associated fungi did not form any clade with other species.     

Identification and analysis of genes differentially expressed in the Spodoptera litura fat body in response to the biocontrol fungus, Nomuraea rileyi

Nomuraea rileyi is an important pathogenic fungus that can successfully control Spodoptera litura. However, little is known on how S. litura responds to N. rileyi infection. A forward suppression subtractive hybridization (SSH) cDNA library was constructed from the S. litura fat body and the up-regulated genes were identified to isolate differentially expressed genes in response to N. rileyi. A total of 345/1175 random clones screened by cDNA array dot blotting were sequenced, resulting in 117 uniquely expressed sequence tags (ESTs). Potential functional genes were identified by BLAST searches and were categorized into seven groups associated with different biological processes based on the literature and gene ontologies. Among 117 genes, 74 had matches in the non-redundant (NR) protein database and were found to be involved in different biological processes, while 43 of the screened genes were classified to the "unknown function" gene group. Notably, only two genes had previously been reported in S. litura and most of the screened genes showed less similarity to known sequences based on BLASTn results, suggesting that 115 genes were found for the first time in S. litura. Semi-quantitative RT-PCR analysis of seven randomly selected genes revealed that most were differentially expressed after N. rileyi infection. qRT-PCR analysis confirmed that four genes (Hsp70, Hsp90, gallerimycin, and cysteine proteinase) were significantly up-regulated after N. rileyi infection. Taken together, the present study identified up-regulated S. litura genes in response to N. rileyi infection. Further investigations are needed to unravel the molecular mechanisms of the genes or proteins potentially involved in the S. litura innate immune defense against N. rileyi infection.     

莱氏野村菌Cq菌株几丁质酶基因的克隆与表达分析

【目的】为揭示昆虫病原真菌分泌的几丁质酶对宿主感染致病时的作用,对莱氏野村菌Cq菌株几丁质酶基因进行了克隆与表达,并检测了表达产物的活性。【方法】采用CTAB法提取菌体DNA,设计特异性引物,多次PCR扩增克隆莱氏野村菌Cq菌株几丁质酶基因全序列,并克隆基因的ORF片段chit1,与载体pPIC9K相连接,构建表达载体pPIC9K-Chit1,转入毕赤酵母感受态细胞中,然后通过1.5mg/L浓度的G418筛选及PCR验证,将阳性转化子进行诱导培养,对发酵液分别进行酶活性测定试验、几丁质酶透明圈验证试验和SDS-PAGE电泳检测。【结果】莱氏野村菌Cq菌株几丁质酶基因全长序列为2756bp(NCBI登录号:EU795711),PCR扩增得到开放阅读框ORF片段chit1为1827bp,其中包含3个内含子,5′端非编码区长76bp,3′端非编码区240bp,编码424个氨基酸的几丁质酶前体,理论信号肽剪切位点在Gly(20)与Leu(21)之间;毕赤酵母重组细胞发酵液中几丁质酶活性随着发酵时间的延长而增加,72h达到最大值482.5U/100μL,透明圈活性验证试验显示,在含1%的几丁质平板上可出现明显的透明圈,表达产物SDS-PAGE电泳检测其分子量为41.0kDa。【结论】本研究克隆到莱氏野村菌Cq菌株几丁质酶基因,其ORF成功重组到毕赤酵母中并表达出有活性的几丁质酶。基因表达产物的利用对进一步研究病原真菌染病昆虫的机制等具有重要意义。     

莱氏野村菌疏水蛋白基因Nrhyd的克隆及其表达特征分析

运用SMART RACE RT-PCR技术,从虫生真菌莱氏野村菌中克隆出完整的疏水蛋白基因Nrhyd编码区序列,以RT-qPCR技术,对莱氏野村菌Nrhyd基因在不同生长条件下的表达特征进行分析,从mRNA转录水平上探讨了不同培养条件对Nrhyd基因表达的影响。结果表明：Nrhyd基因全长733bp,5′非翻译区132bp,3′非翻译区262bp,开放阅读框（ORF）339bp,编码111个氨基酸,前体蛋白理论分子量10.6kDa,理论等电点为6.19。液体摇瓶培养条件下,Nrhyd基因的表达受抑制呈逐渐降低的趋势。固体培养条件下,Nrhyd基因表达量随着分生孢子的产生而升高,到产孢量达到最大的第8天时,表达量最高,以后随着产孢量的下降,基因的表达量降低。因此推测,Nrhyd基因在莱氏野村菌分生孢子形成过程中起重要作用。与不同真菌疏水蛋白基因的系统进化分析表明,Nrhyd基因与绿僵菌来源的同源基因关系最近。     

柱孢绿僵菌和绿色野村菌分类地位的研究

通过测定柱孢绿僵菌 Metarhizium cylindrospora、绿色野村菌 Nomuraea viridula及莱氏野村菌N. rileyi的ITS1-5.8S-ITS2 rDNA区域序列,为柱孢绿僵菌和绿色野村菌正确归属提供了新的分子依据。依据形态学和分子生物学的证据将绿色野村菌组合到绿僵菌属中,并对野村菌属的存在提出疑议。     

Characterization of an Indonesian Isolate of Paecilomyces reniformis

An entomopathogenic fungus (IndGH 96), identified as Paecilomyces reniformis, was isolated from long-horned grasshoppers (Orthoptera: Tettigoniidae) in Sulawesi, Indonesia. The phenotypic and molecular data identified the IndGH 96 as a P. reniformis. We present the first comprehensive characterization of this species using morphological features, sequencing of the ITS1-5.8s-ITS2 region, D1/D2 region of 28S of rDNA, and a portion of the tubulin gene, and laboratory bioassays. Distinguishing features include a hyphal body stage during vegetative growth and the production of distinctly curved, light-green conidia. High dosage bioassays showed that IndGH 96 was infectious to both long-horned and short-horned grasshoppers but not to the house cricket, Acheta domestica, or to the lepidopterans velvetbean caterpillar, Anticarsia gemmatalis or fall armyworm, Spodoptera frugiperda. Phenotypic and genetic analyses suggest that IndGH 96 and other isolates of P. reniformis are more closely related to Nomuraea rileyi than to other species of Paecilomyces.     

Molecular phylogeny of asexual entomopathogenic fungi with special reference to Beauveria bassiana and Nomuraea rileyi

The phylogenetic lineage, taxonomic affiliation and interrelationships of important asexual entomopathogenic fungal genera were studied using the sequences of partial regions of β-tubulin and rRNA genes. The species structures of Beauveria bassiana and Nomuraea rileyi were also investigated. A total of 147 fungal entries covering 94 species were analysed. Phylogenetic analysis placed all the asexual entomopathogenic fungal species analysed, in the family Clavicipitaceae of the order Hypocreales of Ascomycota. Deep phylogenetic lineages were observed in B. bassiana iterating the complex nature of this species. Some of the isolates assigned to this species separated out more distinctly than morphologically distinguishable genera. Cryptic speciation was also evident in N. rileyi. It is concluded that the asexual fungi with entomopathogenic habit arose from a single lineage in sexual Clavicipitaceae.     

Genomic characterization of Saccharomyces cerevisiae strains isolated from wine-producing areas in South America

AIMS: The wide use of yeast inoculum for wine fermentations permit the spreading of commercial Saccharomyces strains in wine areas all over the world. To study the impact of this practice on the autochthonous yeast populations it is necessary to have tools that permit the evaluation of the geographical origin of native isolates and differentiate them from commercial strains. METHODS AND RESULTS: Electrophoretic karyotyping and mitochondrial DNA restriction analysis were used to characterize the genome of native S. cerevisiae isolates associated to wine from three countries in South America. Both methods revealed differences in the genomic structure between these populations, in addition to differences between sub-populations collected in wine-producing areas in Chile. CONCLUSIONS: Our data support that molecular polymorphism analysis may be useful to evaluate the geographical origin of native isolates of yeast strains for industrial use. Furthermore, these findings are in agreement with the idea of a clonal mode of reproduction of wine yeasts in natural environments. SIGNIFICANCE AND IMPACT OF THE STUDY: This study permits the characterization of native yeast isolates in relation to their geographical origin. This procedure could be used as a tool for evaluating if a native isolate derives from the region were it was collected or if it is a strain derived from a commercial strain by microevolution.     

Soil Treatment with Nomuraea rileyi: A Promising Technique for the Control of Spodoptera litura on Groundnut

Soil treatment with Nomuraea rileyi conidia; together with its substrate, crushed sorghum, resulted in high mortality of Spodoptera litura larvae due to mycosis. The persistence of N. rileyi conidia, similarly applied to the soil in a groundnut field, was high, even at 2 weeks after application.     

Cellular immune responses and FAD-glucose dehydrogenase activity of Mamestra brassicae (Lepidoptera: Noctuidae) challenged with three species of entomopathogenic fungi

Abstract.  Cellular immune responses and glucose dehydrogenase activity are examined in the larvae of Mamestra brassicae (Lepidoptera: Noctuidae) injected with three entomopathogenic fungi, Beauveria bassiana , Nomuraea rileyi and Paecilomyces tenuipes . Total haemocyte count and the number of spherulocytes increase significantly over 2 h in larvae infected with N. rileyi , the most virulent fungal pathogen of M. brassicae . However, glucose dehydrogenase is not activated in the haemolymph of larvae inoculated with N. rileyi . By contrast, P. tenuipes , the least virulent fungal species, but with the highest proteolytic activity, activates glucose dehydrogenase and the number of nodules formed is significantly larger in larvae inoculated with P. tenuipes . It is suggested that the different virulence of each fungal species is caused by specific immune responses in the larvae. Haemopoiesis is affected by the fungal conidia and this is investigated by culture of the haemopoietic organs of M. brassicae in vitro . The proportion of spherulocytes discharged from the organs is typically high after all of the fungal treatments. In addition, the anterior and posterior haemopoietic organs, according to the histological locations in larva, show different patterns of haemopoiesis in response to fungi.     

A semi-defined medium for culturing Nomuraea rileyi

A semi-defined medium was successfully used to culture several natural and mutant isolates of the entomopathogenic fungus, Nomuraea rileyi. The medium contains only two complex ingredients at very low levels, yet permits germination and mycelial growth of N. rileyi. The medium also facilitates recovery and detection of hydrolytic enzymes. Use of the formulation could simplify nutritional, biochemical and physiological studies with N. rileyi and also might be used to culture other entomopathogenic fungi.This article reports the results of research only. Mention of a proprietary product does not constitute an endorsement or a recommendation for its use by USDA.     

Field evaluation of soybean engineered with a synthetic cry1Ac transgene for resistance to corn earworm, soybean looper, velvetbean caterpillar (Lepidoptera: Noctuidae), and lesser cornstalk borer (Lepidoptera: Pyralidae)

A transgenic line of the soybean 'Jack', Glycine max (L.) Merrill, expressing a synthetic cry1Ac gene from Bacillus thuringiensis variety kurstaki (Jack-Bt), was evaluated for resistance to four lepidopteran pests in the field. Jack-Bt and genotypes serving as susceptible and resistant controls were planted in field cages and artificially infested with larvae of corn earworm, Helicoverpa zea (Boddie), and velvetbean caterpillar, Anticarsia gemmatalis (Hübner), in 1996, 1997, and 1998, and also with soybean looper, Pseudoplusia includens (Walker), in 1996. Susceptible controls included Jack (1996-1998), 'Cobb' (1996), and Jack-HPH (1996). GatIR 81-296 was used as the resistant control in all 3 yr. Compared with untransformed Jack, Jack-Bt showed three to five times less defoliation from corn earworm and eight to nine times less damage from velvetbean caterpillar. Defoliation of GatIR 81-296 was intermediate between that of Jack and Jack-Bt for corn earworm, and similar to that of Jack for velveltbean caterpillar. Jack-Bt exhibited significant, but lower resistance to soybean looper. Jack-Bt also showed four times greater resistance than Jack to natural infestations of lesser cornstalk borer, Elasmopalpus lignosellus (Zeller), in conventional field plots at two locations in 1998. Data from these experiments suggest that expression of this cry1Ac construct in soybean should provide adequate levels of resistance to several lepidopteran pests under field conditions.     

Potential application of the entomopathogenic fungus, shape Nomuraea rileyi, for control of the corn earworm, shape Helicoverpa armigera

The entomopathogenic fungus, Nomuraea rileyi, caused 90.5–100% mortality in fourth-instar larvae of the corn earworm, Helicoverpa armigera, when applied at 10⁷ conidia/ml to corn silks, and leaves of soybean, tomato and chrysanthemum. The LT₅₀ was 5.9–6.7 days. The fifth-instar larvae showed a mortality of 94.6% on soil with 20% water content, and 41.7% on 10% water content when the soil surface was sprayed with 10⁸ conidia/ml suspension. Five fungicides, eight insecticides and nine herbicides, which are commonly used in corn fields, were evaluated for inhibition to conidial germination by a paper disk test. Among them, only two fungicides, viz., maneb and propineb, were highly inhibitory, while insecticides and herbicides examined were not inhibitory to the fungus. Field applications of N. rileyi conidial suspension to neonate larvae were found to be as effective as 40.46% carbofuran (EC) at 800-fold dilution in controlling corn earworm based on marketable ears. It is thus suggested that N. rileyi has potential to be a microbial control agent for this insect.     

Entomogenous fungus Nomuraea rileyi inhibits host insect molting by C22-oxidizing inactivation of hemolymph ecdysteroids

The entomogenous fungus Nomuraea rileyi reportedly secretes a proteinaceous substance inhibiting larval molt and metamorphosis in the silkworm Bombyx mori. We studied the possibility that N. rileyi controls B. mori development by inactivating hemolymph molting hormone, ecdysteroids. Incubation of ecdysone (E) and 20-hydroxyecdysone (20E) in fungal-conditioned medium resulted in their rapid modification into products with longer retention times in reverse-phase HPLC. Each modified product from E and 20E was purified by HPLC, and identified by NMR as 22-dehydroecdysone and 22-dehydro-20-hydroxyecdysone. Some other ecdysteroids with a hydroxyl group at position C22 were also modified. Injection of the fungal-conditioned medium into Bombyx mori larvae in the mid-4th instar inhibited larval molt but induced precocious pupal metamorphosis, and its injection into 5th instar larvae just after gut purge blocked pupal metamorphosis. In hemolymph of injected larvae, E and 20E disappeared and, in turn, 22-dehydroecdysone and 22-dehydro-20-hydroxyecdysone accumulated. These results indicate that N. rileyi secretes a specific enzyme that oxidizes the hydroxyl group at position C22 of hemolymph ecdysteroids and prevents molting in B. mori larvae.     

几种虫生真菌对斜纹夜蛾的致病性

用布氏白僵菌、球孢白僵菌、玫烟色拟青霉、绿僵菌和莱氏野村菌5种真菌的固体培养物,对斜纹夜蛾2、3龄幼虫进行了毒力测定.结果表明：布氏白僵菌和莱氏野村菌两种菌株对斜纹夜蛾幼虫有明显的致病效果,对2龄幼虫的致死中时(LT₅₀)分别为2.95 d和4.10 d,累计校正死亡率分别为100%和95.2%;对3龄幼虫的致病力低于2龄,致死中时(LT₅₀)分别为19.67 d和19.63 d,累计校正死亡率分别为56.6%和52.2%.玫烟色拟青霉、球孢白僵菌两菌株也有一定的致病力,对2龄幼虫的致死中时(LT₅₀)分别为4.89 d 和6.34 d,累计校正死亡率分别为85.7%和71.4%.