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。     

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.     

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.     

莱氏绿僵菌对斜纹夜蛾的致病力及生理效应

【目的】测定莱氏绿僵菌Metarhizium rileyi Nr5772菌株对斜纹夜蛾Spodoptera litura幼虫及蛹的致病能力,研究莱氏绿僵菌侵染后在寄主体内的发育及对寄主的生理效应,探讨莱氏绿僵菌的致病机制。【方法】采用浸渍法测定莱氏绿僵菌孢子对斜纹夜蛾3-6龄幼虫及蛹的致死中浓度(LC_(50))和致死中时(LT_(50))。采用微量注射法接种莱氏绿僵菌虫菌体,在不同时间后采集斜纹夜蛾幼虫血淋巴,在显微镜下检查虫菌体的数量、形态及寄主血细胞数量,并用酶标仪测定寄主血淋巴酚氧化酶(Phenoloxidase,PO)的活性。【结果】M.rileyi孢子对3龄斜纹夜蛾幼虫毒力最强,10 d后LC_(50)=3.12×10~6个孢子/mL,龄期越大,致病力越低;孢子浓度为5×10~9个/mL时,对3龄幼虫的致死速度最快,LT_(50)=4.55 d,致死速度随龄期的增大和浓度的降低逐渐减缓;M.rileyi孢子对蛹的致病力远低于对幼虫的致病力。注射接种虫菌体后,64 h内,虫菌体数量在寄主血腔中以幂函数的形式增长,寄主的血细胞数量没有明显的变化;在侵染初期(接种后44 h内),血淋巴PO活性正常;在侵染后期,虫菌体数量不再增加(55-64 h后),逐渐转化为菌丝体,并快速杀死寄主,PO活性受到抑制。【结论】莱氏绿僵菌Nr5772菌株对斜纹夜蛾幼虫有较强的致病力,应在害虫低龄期应用;莱氏绿僵菌在侵染初期对寄主血细胞和血淋巴PO无影响,后期则完全抑制PO活性。     

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.     

几种虫生真菌附着胞的荧光显微及扫描电镜观察

本研究通过对细脚拟青霉、蝉拟青霉、玫烟色拟青霉、金龟子绿僵菌和莱氏野村菌在疏水表面产生附着胞的荧光显微镜观察和扫描电镜观察,明确五种虫生真菌均可产生附着胞,细脚拟青霉和金龟子绿僵菌产生单附着胞和复合附着胞两种形态,均呈椭圆至长椭圆形,如遇到不适合浸染的部位,则重新产生芽管向前延伸直至找到适合入侵的部位。蝉拟青霉分生孢子多在顶端发芽成人字形,末端形成椭圆形附着胞,该拟青霉再生附着胞能力强。金龟子绿僵菌和莱氏野村菌附着胞表面有较厚的粘液层,而三种拟青霉附着胞表面均未见有粘液层。     

Effect of an entomopathogen on adaptation of Heliothis virescens populations to transgenic host plants

The role of an entomopathogen in evolution of herbivore adaptation to partially resistant host plants was examined using a tritrophic system in the laboratory. We hypothesized that a pathogen should interact with herbivore behavior to accelerate herbivore adaptation to toxic plants: individuals not adapted to toxin tend to move more on toxic plants, and therefore are more likely to encounter a lethal dose of pathogen, further increasing the probability that they will be eliminated by selection. Heliothis virescens (F.) (Noctuidae) was selected for adaptation to transgenic tobacco containing a sublethal concentration of Bacillus thuringiensis Berliner toxin under two treatment regimes: larvae placed on plants treated with infective conidia of the entomopathogenic fungus, Nomuraea rileyi (Farlow) Samson, and larvae placed on plants without fungus.Selection was initiated with a genetically heterogeneous strain created by crossing two laboratory strains of H. virescens, one not adapted to B. thuringiensis toxin, and one highly adapted (>1000-fold) to toxin. This cross was performed four times to create four independent populations. Selection was initiated with F2 offspring from each cross and continued for 8–10 consecutive generations. Adaptation to toxin within each treatment population was quantified every generation by measuring survival and growth of larvae on artificial diet containing a low concentration of B. thuringiensis toxin.In three of four replicates, H. virescens populations exposed to N. rileyi adapted to B. thuringiensis toxin more quickly than populations not exposed. These results supported our hypothesis that the pathogen should accelerate adaptation to toxic plants. However, this hypothesis was contradicted by the result in one replicate, in which the population not exposed to fungus adapted to toxic plants faster. This opposite result could not be explained, but it suggests that there may be substantial variation in herbivore evolution in tritrophic systems.H. virescens populations selected in the presence of fungus and in the absence of fungus did not differ in feeding or in mortality when placed on leaf disks treated with conidia. Thus, populations exposed to N. rileyi on plants for 7–8 generations displayed neither physiological nor behavioral adaptation to N. rileyi.     

Nonspecific Factors Involved in Attachment of Entomopathogenic Deuteromycetes to Host Insect Cuticle

The attachment of the conidia of the insect-pathogenic fungi Nomuraea rileyi, Beauveria bassiana, and Metarrhizium anisopliae to insect cuticle was mediated by strong binding forces. The attachment was passive and nonspecific in that the conidia adhered readily to both host and nonhost cuticle preparations. The hydrophobicity of the conidial wall and the insect epicuticle appeared to mediate the adhesion process. Detergents, solvents, and high-molecular-weight proteins known to neutralize hydrophobicity reduced conidial binding when added to conidium-cuticle preparations. However, these chemicals did not remove the hydrophobic components from the epicuticle or from conidial preparations. The outer surface of the conidium consists of a resilient layer of well-organized fascicles of rodlets. Intact rodlets extracted from B. bassiana conidia bound to insect cuticle and exhibited the hydrophobicity expressed by intact conidia. Both electrostatic charges and various hemagglutinin activities were also present on the conidial surface. However, competitive-inhibition studies indicated that these forces played little, if any, role in the adhesion process.     

Effects of environmental factors on virulence of the entomopathogenic fungus, Nomuraea rileyi, against the corn earworm, Helicoverpa armigera (Lep., Noctuidae)

The effects of environmental factors on infection of the entomopathogenic fungus, Nomuraea rileyi , isolated from the corn earworm, Helicoverpa armigera , in Taiwan, to its host insect were studied in the laboratory. The fungus caused higher larval mortality at 20°C than at 30°C when 5 × 10⁶ conidia/ml were sprayed on the fourth instar. However, mortality of the fifth instar injected with 1 × 10³ conidia/larva was not significantly different when the inoculated larvae were incubated from 15 to 30°C. The fungal development in inoculated larvae was best at 20 and 25°C after shifting from 20°C to either lower or higher temperatures. The germination rate was higher at 20 and 25°C than at 30 or 35°C. Conidial germination was better on the wash-off of insect cuticle than on Sabouraud maltose agar with yeast extract. Sporulation on chill-dried cadavers was maximal at 95 or 100% relative humidity than at lower levels of relative humidity. The time required for sporulation was 2 days less at 100% than at 95% relative humidity. Although photoperiod did not affect fifth instar mortality caused by N. rileyi , the median lethal time (LT₅₀) values were shorter upon incubating under light than in darkness. Incubation of infected cadavers under 12 or 24 h light resulted in 20-fold more conidial production than under full darkness. Therefore, illumination is necessary for development of this isolate on insect cadavers.     

Sem study on the invasion of Nomuraea rileyi (Farlow) on silkworm,shape Bombyx mori Linn. causing green muscardine

The mature conidia of Nomuraea rileyi (Farlow) germinate on the larval integument of Bombyx mori within 24 h and penetrate the cuticle within 36 h after inoculation at 24.0 ± 1.0 °C temperature and 80.0 ± 5.0% relative humidity. The penetrating hyphae multiply by budding and septa formation in the hemocoel, and the larva succumbs to the infection 6–7 days post-treatment. The hyphal bodies elongate and become interwoven with other hyphae forming a mycelial complex across different tissues. The ramification of hyphae along the epidermal tissue results in larval mummification in 7–8 days. Numerous conidiophores emerge, producing a confluent white fungal mat over the entire surface of the host larva by 9–10 days. Pale green conidia develop, making the larval body green. Life cycle of the fungus on B. mori is completed in 10–11 days. This revised version was published online in June 2006 with corrections to the Cover Date.     

Nomuraea rileyi: a plausible fungi selectively controlling lepidopteron, Paraponyx stratiotata L. damaging queen palm (Livistona rotundifolia L.)

Queen Palm (Livistona rotundifolia) is cultivated extensively for both local and foreign ornamental markets with the desired characteristics of even and ever green, pest and disease free healthy palm and quality leaves. The root borer, Paraponyx stratiotata (Lepidoptera) is recently encountered on young palm roots and damaging them within. They emerged as a new pest on L. rotundifolia grown in all the area. Different entomopathogens were evaluated and determined the potential of Nomuroea rileyi selectively due to its greater infective ability. Field collected root borer larvae were reared and N. rileyi was evaluated both in-vitro and in net house conditions. Treatments comprising Beauveria bassiana, Metarhizium anisopliae, Trichoderma sp., N. rileyi, a standard check with 3% Carbofuran and water as a control were evaluated in potted seedlings (3 months old) of queen palms. Among the entomopathogens tested, N. rileyi was found more effective and recorded the best of 61% mortality. Subsequent experiment had been conducted with commercial bio-products alone or in combination with local isolates as treatments; N. rileyi, B. bassiana, M. anisopliae, B. bassiana + M. anisopliae, N. rileyi + M. anisopliae, N. rileyi+ B. bassiana, and water as a control were evaluated. Among these treatments N. rileyi was alone more effective than in combination with or other entomopathogens alone. In vitro study revealed LD50 of N. rileyi as best spore load of 1 x 10(8) spores/ml compare to other treatments. This proves the potential of N. rileyi on P. stratiotata. The application of N. rileyi had claimed not only to reduce the incidence of root damage but also to sustain the growth and vigor of the L. rotundifolia to most fit for exporting.     

重要虫生真菌莱氏野村菌芽生孢子的形态发生

经对比,萨氏麦芽糖-酵母浸粉培养液(SMY)较适合制备莱氏野村菌Nomuraea rileyi芽生孢子。以菌株Nr09接种该培养基,在130r/min、25℃全光照下震荡培养,观察芽生孢子在不同时期发育的形态变化。结果表明,分生孢子萌发产生芽管;24h萌发率为42%,36h时达84%。芽管迅速伸长形成菌丝。在48h前芽生孢子通过菌丝顶端及分枝末端缢缩的方式形成,后期则主要通过芽生孢子继续出芽的方式大量形成,78h芽生孢子数量达到最大值。芽生孢子的形成方式可为一端、两端或多端芽殖。芽生孢子的形成过程可分为5个阶段:(I)分生孢子膨大期;(II)芽管萌发期;(III)芽管延长期;(IV)芽生孢子形成初期;(V)芽生孢子指数生长期。30h后培养液中有少量草酸钙结晶出现并逐渐增多。到84h时有31%的芽生孢子细胞内液泡聚集增大,表明芽生孢子已开始进入衰老阶段。使用指数生长期制备的Nr09芽生孢子进行几丁质酶基因转化,转化效率达79个转化子/μgDNA。     

Effects of natural enemies on relative fitness of Heliothis virescens genotypes adapted and not adapted to resistant host plants

We investigated the potential of two natural enemies of Heliothis virescens (F.) (Noctuidae) to affect its rate of adaptation to tobacco containing Bacillus thuringiensis Berliner toxin. Larval fitness of two laboratory strains of H. virescens, one adapted to B. thuringiensis toxin and one not adapted, was compared on toxic and nontoxic plants, in the presence of the parasitoid Campoletis sonorensis (Cameron) (Ichneumonidae) or the entomopathogenic fungus Nomuraea rileyi (Farlow) Samson. By exposing larvae to plants and enemies for no more than 24 h, we focussed on the behavioral rather than physiological component of their interaction with toxic plants and natural enemies. Parasitism of H. virescens larvae by C. sonorensis during exposure periods of 1–4 h was lower on toxic plants than nontoxic plants and was lower for nonadapted larvae than for toxin-adapted larvae. Decreased larval feeding damage on toxic versus nontoxic plants, and by nonadapted versus adapted larvae, may explain differences in parasitism, because C. sonorensis locates host larvae using cues from damaged plants. Effects of plant toxicity and larval strain on H. virescens survival were numerically consistent with effects on parasitism, but they were not statistically significant. When mean larval survival is used to estimate fitness of the nonadapted genotype relative to the toxin-adapted genotype, we find that C. sonorensis is expected to delay adaptation to toxic plants. Percent infection by N. rileyi of H. virescens larvae exposed to fungus-treated plants for 24 h was greater when plants were toxic, and was greater for nonadapted larvae than toxin-adapted larvae. There were corresponding decreases in larval survival on toxic compared to nontoxic plants, and of nonadapted compared to adapted larvae. Interaction of effects of plant line and larval strain on survival was significant in the presence of fungus, but not in the absence of fungus, which indicates that the effect of toxic plants on the relative fitness of toxin-adapted and nonadapted larvae was mediated by fungus. As in the interaction with C. sonorensis, behavior of larvae on plants may explain differences in susceptibility to N. rileyi. Because nonadapted larvae moved more than toxin-adapted larvae on toxic plants, nonadapted larvae may have been more likely to encounter a lethal dose of conidia. In contrast with C. sonorensis, N. rileyi, which decreased the fitness of the nonadapted genotype relative to the adapted genotype, is expected to accelerate adaptation to toxic plants.     

Phylogenetic relationships among strains of the entomopathogenic fungus, Nomuraea rileyi, as revealed by partial beta-tubulin sequences and inter-simple sequence repeat (ISSR) analysis

AIMS: To elucidate the phyletic relationships among three members of the entomogenous fungal genus, Nomuraea, with an emphasis on N. rileyi. METHODS AND RESULTS: Relationships were evaluated by analysis of the beta-tubulin gene and of inter-simple sequence repeats (ISSR). The amplification product of the partial beta-tubulin gene was larger for N. atypicola than for N. rileyi, and sequencing of this gene fragment confirmed that N. atypicola possesses approximately 25 more nucleotides than N. rileyi and N. anemonoides. Based on neighbor joining and bootstrap analysis of the partial beta-tubulin gene, N. atypicola failed to form a monophyletic grouping with the other two species of Nomuraea. In contrast, the single isolate of N. anemonoides clustered with the N. rileyi isolates, and both taxa grouped with Epichloe typhina (Hypocreales: Clavicipitaceae). Results from this study suggested that N. rileyi and N. anemonoides are closely related to the Clavicipitaceae. In contrast, evidence indicated that N. atypicola is not closely related to this family, and that this taxon is not a Nomuraea. Based on the 83 polymorphic loci of ISSR, it was observed that isolates of N. rileyi from diverse geographical origins were distinctly different from both N. atypicola and N. anemonoides. Considerable heterogeneity was observed in the 18 isolates of N. rileyi tested, and several clusters contained isolates from disparate geographical locations and hosts. However, three isolates from the Philippines (three host species) and three strains isolated from velvetbean caterpillar (Anticarsia gemmatalis) larvae in South America did cluster together. Two other strains from Brazil (isolated from Spodoptera spp.) were distinct from the velvetbean caterpillar isolates from South America. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: The beta-tubulin gene was generally too conserved to resolve intraspecies variability. However, ISSR did identify polymorphisms among the isolates of N. rileyi tested. The results of this study indicate that ISSR may be used as robust molecular markers for studying the population genetics of this entomopathogenic fungus.     

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.     

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).     

Inhibition of phagocytic activity and nodulation in Galleria mellonella by the entomopathogenic fungus Nomuraea rileyi

Changes in phagocytic activity and nodulation in the greater wax moth, Galleria mellonella (L.) (Lepidoptera: Pyralidae), were examined after treatment with the culture fluid of the entomopathogenic fungus Nomuraea rileyi SH1. When isolated hemocytes of G. mellonella were incubated with the conidia of N. rileyi in vitro, the rates of phagocytosis increased at 4 h after incubation but decreased subsequently. On the contrary, the rates of phagocytosis of isolated hemocytes decreased by 80% after 24 h preincubation with the fungal culture fluid (1/200, 1/100 dilutions). Levels of inhibition of phagocytic activity by the culture fluid depended on dilutions used. Galleria mellonella larvae showed a peak of nodulation at 4 h after injection with conidia. The percentage of nodules in hemolymph did not decrease by preinjection with the culture fluid, whereas the percentage of nodule‐containing conidia decreased, depending on the injected fluid. However, phagocytosis and nodulation in G. mellonella did not change after treatment of the culture fluid with proteinase K, indicating that the culture fluid contained proteinaceous immunosuppressive factors. Electrophoretic analysis of the culture fluid and not the fresh medium without culturing the fungus exhibited protein bands. Therefore, N. rileyi possibly secretes toxic proteins that impair cellular immune responses in G. mellonella larvae.     

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。     

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%.