白僵菌的致病力与菌株生长特性及几丁质酶活性的关系

将辽宁、河南、山东等地采集到的70个寄生于桃小食心虫的球孢白僵菌菌株以3龄桃小食心虫进行筛选,得到了46-1,46-2和37-3三个致病率分别为96.7％、92.8％和93.3％的优良菌株。以四个代表高,中、低致病率的菌株46-1、46-2、7-1(43.3％)和66(33.3％),测定其菌丝的生长量,产孢量和几丁质酶活性的结果显示菌株的致病力与菌丝生长量、几丁质酶活性、菌株产孢量呈显著的正相关。     

球孢白僵菌生物学特性与其对小菜蛾致病力相关性分析

为明确球孢白僵菌的各项生物学指标与其对小菜蛾致病力间的相关性,对一株球孢白僵菌菌株GXU-Bb进行单孢分离,测定了菌株GXU-Bb及其分离子菌株GXU-Bb16、GXU-Bb10、GXU-Bb13和GXU-Bb05这5个菌株的生长速率、产孢量、孢子萌发率、胞外蛋白酶水平和几丁质酶水平等多项生物学指标。结果表明,各菌株间生长速率和孢子萌发率无显著差异;产孢量变化范围1.54×10^7-2.98×10^7个/ml;胞外蛋白酶水平变化范围1.04-1.19;几丁质酶水平变化范围1.05-1.14;各菌株间对小菜蛾二龄幼虫的致病力差异较大,校正死亡率变化范围为39.81%-83.79%。将各生物学指标与菌株对小菜蛾二龄幼虫的校正死亡率进行回归分析得出,球孢白僵菌对小菜蛾的致病力与菌株生长速率、孢子萌发率无显著相关关系;与产孢量、胞外蛋白酶水平呈极显著正相关关系;与几丁质酶水平呈显著正相关关系。     

球孢白僵菌BB-T02生物学特性及对两种检疫性粉蚧的侵染活性

南洋臀纹粉蚧和石蒜绵粉蚧是近年我国新记录的两种检疫性有害生物,目前分别在福建省的番荔枝果园和多肉植物种植基地发生为害严重。为明确球孢白僵菌BB-T02对这2种检疫性粉蚧的生防潜力,本研究在该菌株生物学特性的基础上,测定该菌株对两种检疫性粉蚧的致病力和胞外酶活性。结果表明,球孢白僵菌BB-T02培养最适的温度为28℃、光周期8L:16D、碳源为麦芽糖、氮源为蛋白胨,在此条件下培养10 d菌落直径和产孢量分别可达5.60 cm和4.22×10⁸孢子/cm²。菌株BB-T02侵染南洋臀纹粉蚧和石蒜绵粉蚧10 d后的LC₅₀分别为5.00×10⁵孢子/mL和2.17×10⁵孢子/mL,1.00×10⁸孢子/mL处理的累计致死率分别为85.39%和88.76%、LT₅₀分别为5.78 d和5.19 d。菌株BB-T02蛋白酶活性的变化幅度较大,在第5 d达峰值22.68 U/mL;几丁质酶和脂肪酶活性的变化幅度相对平稳,均在第6 d分别达到峰值13.19 U/mL和9.77 U/mL。综上,球孢白僵菌BB-T02生长速度快、产孢量高,对南洋臀纹粉蚧和石蒜绵粉蚧的侵染活性强,可用于这两种检疫性粉蚧的生物防治。     

桃小食心虫高致病力白僵菌菌株筛选

对来源于不同地点或寄主的14株球孢白僵菌进行僵虫率、在虫体上菌丝生长时间、产孢时间及单虫产孢量测定,综合评价各菌株对桃小食心虫幼虫的致病力,选择较好的菌株进行毒力测定。结果表明：基于僵虫率、菌丝生长时间、产孢时间及单虫产孢量4个指标,筛选出 3 株具有较强致病力的优良菌株Bb7、Bb9和Bb14;在孢子浓度为1.0×107 孢子/mL时,对桃小食心虫幼虫的致死率分别为73.33%、77.78%和80.00%, LT50分别为8.45、7.49、8.42 d;LC50分别为1.76×105、3.03×106、2.10×105孢子/mL。     

松阿扁叶蜂越冬期感病幼虫病原菌的分离鉴定及林间应用研究

实地调查了徂徕山林场松阿扁叶蜂越冬期幼虫感病死亡情况,并对引起幼虫感病死亡的致病菌分离纯化,进行了形态鉴定和分子鉴定。结果表明,松阿扁叶蜂越冬感病幼虫在自然状态下感病致死率可达8.9%～21.3%,其病原分离物可分为真菌菌株和细菌菌株两大类。其中,真菌感染致死率为67.8%,细菌感染致死率为32.2%。从松阿扁叶蜂死亡幼虫虫体中分离到5株细菌[嗜虫沙雷氏菌(Serratia entomophila)、河生肠杆菌(Enterobacter amnigenus)、鲍氏不动杆菌(Acinetobcter baumannii)、黏质沙雷菌(Serratia marcescens)、枯草芽孢杆菌(Bacillus subtilis)和2株真菌(粉质棒束孢(Isaria farinosa)、环链棒束孢(Isaria cateinannulata)]。其中,2株细菌菌株(黏质沙雷菌、枯草芽孢杆菌)和2株真菌菌株(粉质棒束孢、环链棒束孢)对松阿扁叶蜂幼虫的室内试验校正致死率为24.2%～68.9%;林间试验校正致死率为16.7%～62.4%。其中,环链棒束孢的校正死亡率为62.4%。     

球孢白僵菌SJ-05几丁质酶基因的克隆与序列分析

几丁质酶在昆虫真菌侵染寄主过程中起着重要的作用。本文对采自内蒙古准格尔旗沙棘木蠹蛾的球孢白僵菌菌株SJ-05进行了几丁质酶的基因分析,从中克隆了几丁质酶Bbchit基因,得到了重组质粒pMD19-T/chit。测序结果表明,Bbchit基因核苷酸序列阅读框架全长为1047 bp,编码348个氨基酸,推测分子量为36.924 ku,等电点为5.94。氨基酸序列比对结果表明,与球孢白僵菌Bb0062菌株（AAN41259.1）的氨基酸序列相似性最高,为99.71%。     

防治假眼小绿叶蝉的虫生真菌高毒菌株的筛选及田间防治效果

茶小绿叶蝉Empoasca spp.是我国最重要的茶树害虫,几乎对所有化学农药都产生了很高的抗药性,亟需开发经济有效的生物农药。本研究通过对从茶小绿叶蝉上分离的1株爪哇棒束孢Isaria javanicus和4株自其它叶蝉上分离的球孢白僵菌Beauveria bassiana的胞外蛋白酶活性测定,初选出活性较高的爪哇棒束孢和两株球孢白僵菌;对假眼小绿叶蝉低龄若虫的生物测定结果显示,爪哇棒束孢毒力最强,分别比两株球孢白僵菌高2.33～7.26倍和6.08～27.26倍,在1×108孢子mL 1浓度条件下第13 d的致死率达100%,LT50为5.9 d。通过随机区组设计进行的田间防治试验结果表明,使用爪哇棒束孢菌株配制的IJOD-1悬乳剂以7.5×1013孢子hm 2的剂量常量喷施,对假眼小绿叶蝉虫口的防治效果在第11 d时为50.32%;第25 d时追加1次放菌,在第55 d时的防治效果达68.40%。     

寄生沙棘木蠹蛾的白僵菌菌株特性及其胞外蛋白酶、几丁质酶的活性

［目的］ 明确寄生沙棘木蠹蛾的白僵菌不同菌株的生物学特性,以筛选优良菌种加以利用。［方法］ 测定温湿度对分离自沙棘木蠹蛾幼虫和蛹的5株白僵菌菌株分生孢子萌发的影响,并采用平板测定法和Lowry及DNS测定法对白僵菌各菌株胞外蛋白酶和几丁质酶活性进行测定。［结果］ 白僵菌SJ 01、SJ 02、SJ 03、SJ 04、SJ 05菌株在15～35 ℃范围内均可萌发,15、20 ℃和35 ℃时孢子萌发率相对较低,25 ℃时5株菌株萌发率均高,为90％～98％。相对湿度为41％、50％和61％时,各菌株均不萌发;76％和80％时,SJ 05的萌发率明显高于其他菌株,分别达60％和80％。50 ℃高温胁迫24 min和30 min后,各菌株萌发率均显著降低,几乎不萌发。胁迫12 min和18 min后,SJ 05的萌发率较其他菌株高,表明其抗高温性强。5株菌株均可产生胞外蛋白酶和几丁质酶,其中SJ 05菌株胞外蛋白酶产量为55.3 U/mL,几丁质酶产量为137.9 U/mL,其蛋白酶和几丁质酶活性均显著高于其他菌株。［结论］ SJ 05菌株具有较强的耐热性,一定的抗低湿能力,较高的蛋白酶和几丁质酶活性,为最优菌株。     

白僵菌菌株的紫外线诱变选育

从自然罹病的桃小食心虫和蛴螬僵尸上分离到产孢量较高的球孢白僵菌菌株46-1和卵孢白僵菌菌株4号,经紫外线照射处理,获得2个球孢白僵菌菌株23号和53号、1个卵孢白僵菌菌株45号,它们的产孢量和毒力稳定高于亲本,而且生产性状和田间防治应用效果良好。     

茶园昆虫病原真菌群落组成及生态位

本文系统调查了安徽省石台县仙寓山粗放经营茶园昆虫病原真菌物种多样性和生态位特征。结果表明,在植被单一、干扰较多的茶园,昆虫病原真菌种群结构较为单一。共采集到116株标本,其中优势种群为环链棒束孢、球孢白僵菌和粉棒束孢,分别占总数的32.8%、27.6%和19.8%。分析了3种优势种昆虫病原真菌的时间和营养生态位。结果表明,环链棒束孢的时间生态位最大,粉棒束孢最小;球孢白僵菌营养生态位宽度最大,粉棒束孢营养生态位宽度最小。昆虫病原真菌之间以环链棒束孢和球孢白僵菌的时间生态位重叠度最大,为0.776,环链棒束孢和粉棒束孢的营养生态位重叠度最大,为0.785。可见,粗放经营茶园中环链棒束孢发生时间长,分布范围广,生存能力强。     

玫烟色棒束孢的生物学特性与其对小菜蛾致病力的相关性

测定了玫烟色棒束孢原始亲本菌株PFCF-O、退化亲本菌株PFCF-D和单孢分离子菌株PF-CF-D58、PFCF-D20、PFCF-D50的菌落生长速率、产孢量、孢子萌发率和胞外蛋白酶水平等生物学指标以及各菌株对小菜蛾2龄幼虫的校正死亡率。结果表明,菌株PFCF-O的生长速率呈先快后慢的趋势,产孢量为2.7×107个/mL,孢子萌发率为62.59%,胞外蛋白酶水平为1.16,对小菜蛾2龄幼虫校正死亡率达97.74%。而菌株PFCF-D、PFCF-D20、PFCF-D50和PFCF-D58间平均生长速率和孢子萌发率无显著差异,产孢量变化范围为1.05×107~2.34×107个/mL,胞外蛋白酶水平变化范围为1.02~1.07,各生物学指标及其对小菜蛾2龄幼虫的校正死亡率(19.86%~51.44%)都显著低于菌株PFCF-O。相关性分析结果显示,玫烟色棒束孢对小菜蛾的致病力与菌株生长初期的生长速率、产孢量、孢子萌发率和胞外蛋白酶水平呈极显著的正相关关系,与菌株生长后期的生长速率呈极显著负相关关系。     

Physiological effect of chitinase purified from Bacillus subtilis against the tobacco cutworm Spodoptera litura Fab.

An extracellular chitinase was purified from Bacillus subtilis. The lethal concentration (LC₅₀) was determined by using chitinase in first, second, and third instars of Spodoptera litura Fab. Chitinase showed the highest insecticidal activity at 6 μM concentration within 48 h. The nutritional indices were also significantly affected by the 6 μM concentration (P < 0.05). Food consumption, efficiency of conversion of ingested and digested food, relative growth rate, and consumption values declined significantly while approximate digestibility was increased. Our study indicates that treatment of host plant leaves with the chitinase can regulate (reduce) larval growth and weight, and enhance the mortality. This may serve as an effective biocide and alternative to Bt toxin.     

寄主植物对甜菜夜蛾生长发育和消化酶活性的影响

通过室内饲养和生化测定,研究了甜菜、玉米和黄瓜3种寄主植物的营养物质及其对甜菜夜蛾幼虫发育、成虫产卵及5龄幼虫体内蛋白质和总氨基酸含量、淀粉酶和脂肪酶活力的影响.结果表明:与取食玉米和黄瓜的个体相比,取食甜菜的甜菜夜蛾,其幼虫期和蛹期较短,分别为11.4天和5.6天;蛹期存活率最高,达100%;成虫寿命较长,为26.3天;单雌产卵量最高,为301.8粒.取食3种寄主植物,甜菜夜蛾5龄幼虫体内的蛋白质含量无差异,但总氨基酸含量差异显著,此外,幼虫体内淀粉酶和脂肪酶活力也存在显著差异,取食甜菜的个体>取食玉米的个体>取食黄瓜的个体.取食不同寄主植物,甜菜夜蛾幼虫的消化酶和脂肪酶活力与其发育适合度呈正相关.     

Isolation and partial characterization of an antifungal protein from the endophytic Bacillus subtilis strain EDR4

An antifungal protein E2, from the culture filtrate of the endophytic Bacillus subtilis strain EDR4 of wheat with a high activity against numerous fungal species in vitro and take-all in wheat caused by Gaeumannomyces graminis var. tritici in vivo, was purified by (NH₄)₂SO₄ precipitation, hydrophobic-interaction chromatography, anion-exchange chromatography and polyacrylamide gel electrophoresis (PAGE). The molecular mass of the protein was about 377.0 kDa determined by gel permeation chromatography (GPC) using a Superdex 200 10/300 GL pre-packed column and the pI value of the protein detected by isoelectric focusing PAGE was 6.59. Following sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) the antifungal protein showed a band with a molecular mass of 39.1 kDa, which suggest that the native protein consists of multi-subunits. The amino acid sequences of three peptides from the antifungal protein were obtained by using a nano-ESI-MS/MS (Q-TOF2) System. The protein isolated may be regarded as a new protein according to amino acid sequences of three peptides. The purified protein exhibited inhibitory activity on mycelium growth of e.g. Fusarium graminearum, Macrophoma kuwatsukai, Rhizoctonia cerealis, Fusarium oxysporum f.sp. vasinfectum, Botrytis cinerea and G. graminis var. tritici (Ggt). Scanning electron microscopy showed that hyphae of Ggt treated with the antifungal protein were severely deformed. The antifungal protein E2 exhibited ribonuclease and hemagglutinating activities as well as a trifle protease activity. However, no β-1,3-glucanase, β-1,4-glucanase, chitinase or protease inhibitory activities were detected.     

多杀菌素亚致死浓度对小菜蛾解毒酶系活力的影响

采用多杀菌素亚致死浓度,以浸叶法分别处理小菜蛾Plutella xylostella (L.)敏感种群(SS)和亚致死选育种群 的3龄幼虫,分别测定饲喂处理6、12、24、48和72 h后小菜蛾体内羧酸酯酶(CarE)、谷胱甘肽S-转移酶(GST) 和多功能氧化酶(MFOs)的活性,分析了酶活性的变化动态。结果表明,SS种群小菜蛾CarE的活性在不同时间段波动较大,经多杀菌素处理后,开始时段比活力增加,随着处理时间的延长,比活力逐渐被抑制,Sub-SS种群的GarE活力高于SS种群;多杀菌素对GST具有明显的诱导作用,亚致死浓度处理后GSTs比活力呈上升趋势,且具有一定的时间效应;对细胞色素P450酶系的O-脱甲基酶活性具有明显的抑制作用,多杀菌素亚致死浓度连续处理5代后,该酶活性更低。     

水稻纹枯病菌拮抗细菌的筛选及鉴定

为获得对水稻纹枯病有生防效果的拮抗细菌,从江苏南京、徐州和常州等地采集的土样中分离细菌分离物1914株,采用平板对峙法筛选获得70株对水稻纹枯病菌有较强抑菌活性的分离物,其中11株对5种水稻病害病原菌均有抑制作用;对11株拮抗菌进行田间防效和室内促生试验,测定菌株分泌的抑菌物质和促生物质,并进行种属鉴定.结果表明,拮抗菌对水稻纹枯病的盆栽和田间小区防效在48.41％和43.03％以上;均可产生蛋白酶与嗜铁素,而不产生几丁质酶,除XF-174外其余10个菌株均可产生纤维素酶;对水稻苗株高和鲜重具有促生作用,并均可产生赤霉素(GA3);除ZF-273和XF-174外的9个菌株可产生吲哚乙酸(IAA),且细菌发酵液中IAA和GA3含量与水稻株高和鲜重的增长率呈正相关.结合各菌株形态特征、生理生化特性和16S rDNA与gyr-B序列分析结果,鉴定SF-181为枯草芽胞杆菌Bacillus subtilis,XF-174为荧光假单胞菌Pseudomonas fluorescens,其余9个菌株为解淀粉芽胞杆菌B.amyloliquefaciens.     

Biochemical studies on sheep body louse Bovicola ovis (Schrank) (Phthiraptera: Trichodectidae): comparison of pyrethroid and organophosphate resistant and susceptible strains

Strains of sheep louse Bovicola ovis (Schrank) with various levels of resistance to pyrethroid and one strain with high degree of resistance to organophosphate (OP) insecticides were used to investigate the biochemical mechanisms of insecticide resistance, i.e., enhanced levels of general esterases, specific acetylcholinesterases (AChE), glutathione S-transferase (GST), and mixed function oxidases. Native gel electrophoresis combined with quantitative enzyme assays showed analogous expression profiles of several esterase isozymes in all the strains tested. The determination of the sensitivity of each esterase isozyme to five inhibitors (acetylthiocholine iodide, butyrylthiocholine iodide, paraoxon eserine sulfate, and pCMB) led to the identification of nine esterases in the B. ovis strain. Gel electrophoresis results are supported by enzyme assay studies where, except for the OP resistant strain, no differences in esterase activities were detected in all the pyrethroid resistant and susceptible strains assayed. Statistical analyses demonstrated that some strains have elevated GST activities compared to the susceptible reference strain.     

不同培养基继代培养蜡蚧轮枝菌对产孢量和烟粉虱毒力的影响

为了探究不同培养基继代培养的蜡蚧轮枝菌对烟粉虱的作用效果,通过继代培养方法测定JMC-01、JMC-02菌株的产孢量、对烟粉虱的毒力及其几丁质酶活性。结果表明,JMC-01、JMC-02菌株在添加蝉蜕和烟粉虱的PDA培养基中连续培养5代的产孢量、对烟粉虱的毒力、几丁质酶活性均高于无添加的PDA培养基,且JMC-01菌株的各项参数均高于JMC-02菌株。JMC-01菌株在添加蝉蜕的PDA培养基中T_1和T_3代的产孢量最大,均为1.279×10~7个/mL,T_3代对烟粉虱的毒力最大,为88.33%,T_5代的几丁质酶活最高,为0.268 3 U/mL;在添加烟粉虱的PDA培养基中,JMC-01菌株的最大产孢量、对烟粉虱的最高毒力和最高几丁质酶活分别出现在S_2、S_4和S_5代,分别为1.270×10~7个/mL、88.33%和0.325 4 U/mL。回归分析结果显示,JMC-01、JMC-02菌株几丁质酶活性与烟粉虱校正死亡率呈显著的正相关,且拟合优度较好。本试验表明JMC-01、JMC-02菌株均对烟粉虱具有较强的毒力,添加蝉蜕和烟粉虱的培养基可以维持菌株生长特性、增加产孢量、延缓菌株退化、增强菌株的几丁质酶活性,为烟粉虱的生物防治提供了新的菌种资源。     

Pyrethroid resistance and esterase activity in three strains of the cotton bollworm, Helicoverpa armigera (Hübner)

Microplate assay technique for estimation of esterase activity in a single insect was used in combination with dose mortality bioassays to detect pyrethroid resistance in three strains of Helicoverpa armigera and to study the frequency of pyrethroid resistant individuals within the population of the same strain at two larval stages, third and fifth instar. The third and fifth instar larvae of the field strains i.e., Nagpur strain and Delhi strain that displayed high degree of resistance towards deltamethrin, had higher esterase activity compared to a susceptible laboratory strain. The frequency distribution of individuals with elevated esterase activity above 1.00 absorbance unit was correlated with the resistance level of the strains. The frequency of resistant individuals in the third instar larvae of Nagpur strain and Delhi strain were 29% and 23%, respectively compared to 4% in the susceptible strain. The resistant individuals in the resistant strains have markedly increased in the fifth instar larvae with a frequency distribution of 63% and 90% in Delhi strain and Nagpur strain, respectively, while only 14% of individuals was found to have elevated esterase activity in the susceptible strain. The results demonstrated the role of esterase in pyrethroid resistance in H. armigera. Microplate assay proved to be a rapid and reliable biochemical technique for monitoring of pyrethroid resistance in H. armigera.     

Evaluation of the effect of chitin-rich residues on the chitinolytic activity of Trichoderma harzianum: In vitro and greenhouse nursery experiments

The chitin-rich residues obtained from several industries have numerous applications in diverse fields such as biotechnology, pharmacology, medicine and agriculture. In this study, three different chitin-rich residues were evaluated with regard to enhancement of the chitinolytic activity of the mycoparasitic fungus Trichoderma harzianum, used for biological control of Fusarium oxysporum in greenhouse nurseries. Chitosan (CHIT), shrimp shell powder (SSP) and mushroom wastes (MW) activated the expression of the T. harzianum genes encoding the NAGase and chitinase activities (exc1, exc2, chit42, chit33 and chit37), the highest in vitro activities of T. harzianum being seen with MW. Under greenhouse nursery conditions, the treatments involving amendment with these residues and with T. harzianum increased the NAGase activity (between 5- and 20-times higher than peat) and the chitinase activity (between 3- and 6-times higher than peat) of the growing media and maintained the shoot dry weight of plants infected with F. oxysporum. Moreover, the incorporation of these residues into the growing media enhanced the growth (first leaf length, stem length and shoot dry weight) of muskmelon seedlings. The use of these residues with T. harzianum as amendments of growing media enhanced the growth of muskmelon seedlings and decreased the weight loss due to the pathogen, giving an added-value to these residues and enhancing their management.