不同寄主来源的根虫瘟霉菌株对小菜蛾幼虫的毒力比较

在不同寄主来源的4株根虫瘟霉Zoophthora radicans对小菜蛾 Plutella xylostella 2龄幼虫的生物测定中,发现源于小菜蛾的菌株ARSEF1100毒力最强,在0.53～319.32/mm²的孢子剂量下,接种后第8 天累计死亡率为2.38%～97.44%,虫尸全部表现典型的虫瘟霉症状;源于叶蝉的ARSEF2699和F99101菌株的同日累计死亡率分别为2.38%～50.00% (剂量为1.56～314.84/mm²孢子)和2.38%～57.89% (剂量为1.84～484.08/mm²的孢子);而源于菜粉蝶的ARSEF1342菌株在3.54～633.0/mm²的孢子剂量下只引发6.52%～13.63%的累计死亡率,后3个菌株致死的小菜蛾幼虫仅部分表现典型症状。所获数据经时间剂量-死亡率模型模拟分析,剂量效应参数依次为ARSEF1100 (1.89) > F99101 (1.48) > ARSEF2699 (1.23) > ARSEF1342 (0.37),相互间差异均达极显著水平。接种后4～8 天内,ARSEF1100的LD₅₀值分别为231.68、113.08、71.41、40.87和35.30/mm²的孢子,其毒力远高于其余3个菌株;ARSEF2699的相应LD₅₀值为1344.43、922.39、555.58、410.06和397.07/mm²的孢子;F99101的LD₅₀值为666.86、451.64、413.82、350.65和332.57/mm²的孢子,而ARSEF1342的毒力太弱难以估计。这些结果表明,ARSEF1100菌株最有希望用于小菜蛾的微生物防治。     

大菜粉蝶根虫瘟霉菌株转染小菜蛾后侵染力变化及其与寄主血淋巴酚氧化酶活性的关系

源于大菜粉蝶Pieris brassicae的根虫瘟霉Zoophthora radicans菌株R₀通过反复转染小菜蛾Plutella xylostella而分别获得转染菌株R₁、R₃和R₅。用这些菌株对小菜蛾2龄幼虫进行生物测定,发现菌株对寄主的侵染力有随转染次数增加而增强的趋势。接种后第1～6 天,R₀的LC₂₀（孢子数/mm²）分别为14.7、14.5、9.0、7.1、6.0和5.5;R₁的LC₂₀分别为9.6、5.0、4.2、3.6、3.1和3.0;R₃的LC₂₀分别为4.6、2.9、2.8、2.5、2.4和2.2; R₅的LC₂₀分别为5.2、3.7、3.2、2.8、2.6和2.6,接种后同一天菌株 R₃的LC₂₀值最小即侵染力最强。各菌株感染小菜蛾幼虫后可显著激活寄主血淋巴中的酚氧化酶活性,但R₁、R₃和R₅对酚氧化酶的激活程度显著低于原始菌株R₀。各菌株对小菜蛾的侵染力强弱指标log₁₀ (LC₂₀)与其侵染后寄主血淋巴酚氧化酶活性呈明显正相关（0.852<0.95）,表明R0在对新寄主转染过程中逐渐获得了逃避或克服新寄主免疫防御的能力,从而增强对新寄主的侵染力。     

小菜蛾幼虫血淋巴中β-1,3-葡聚糖结合蛋白的分离及其对根虫瘟霉侵染的免疫活性

用亲和沉淀法从小菜蛾(Plutella xylostella)幼虫血淋巴中分离获得2种β-1,3-葡聚糖结合蛋白,分子质量分别为75.9 ku和83.2 ku,主要存在于幼虫血浆中,但血细胞中未检出.研究表明,β-1,3-葡聚糖结合蛋白能特异性地识别β-1,3-葡聚糖,并显著激活幼虫血淋巴中的酚氧化酶原（ProPO）,与昆布多糖共存时所激活的酚氧化酶（PO）活性显著高于两者单独存在时的PO活性.与4株根虫瘟霉(Zoophthora radicans)菌丝裂解液共存时,β-1,3-葡聚糖结合蛋白能激活幼虫血淋巴中的ProPO,使PO活力显著高于该菌原生质体裂解液所激活的PO活性.显然,β-1,3-葡聚糖结合蛋白只有特异性地识别根虫瘟霉细胞壁中的β-1,3-葡聚糖后才能激活幼虫血淋巴中的ProPO,表明虫霉原生质体可逃避寄主免疫反应.此外,β-1,3-葡聚糖结合蛋白对不同菌株所激活的PO活性存在差异,各菌株所激活的PO活性由高到低依次为：ARSEF1342＞ARSEF2699＞F99101＞ARSEF1100,这与各菌株对小菜蛾的毒力强弱相一致,即菌株逃避寄主免疫识别的能力与其毒力相关.     

昆虫酚氧化酶原激活酶研究

昆虫酚氧化酶原激活酶(Prophenoloxidase activating proteinase,PAP)是酚氧化酶原激活过程中的一个关键酶,是昆虫先天性体液免疫体系的重要组成部分。外界的免疫刺激能够诱导级联反应上游的蛋白对酚氧化酶原激活酶的前体进行剪切激活,而激活后的酚氧化酶原激活酶能够将无活性的酚氧化酶原剪切激活为有活性的酚氧化酶并最终生成细胞毒素物质来消灭外源物。本文综述了昆虫酚氧化酶原激活酶的结构与特性及其在酚氧化酶原级联激活系统中的作用机制,并探讨了寄生蜂对寄主昆虫酚氧化酶原激活酶的调控。     

昆虫酚氧化酶原活化及其在免疫中的作用

1　前言酚氧化酶 (phenoloxidase,PO)以无活性的酶原形式——酚氧化酶原 (prophenoloxi-dase,PPO)存在于昆虫血淋巴、中肠和表皮等组织 ,当病原生物入侵时 ,通过特异性丝氨酸蛋白酶的级联反应 (PPO级联 )被活化 ,参与机体的免疫防御反应 [1] 。本文拟就近年来有关昆虫PPO级联及其在免疫中的作用等研究方面的新进展做一简述。2　PPO的特性及其分子生物学PPO是 PPO级联中一个很重要的成分 ,近年来 ,随着分子生物学技术和其他相关学科的快速发展及向昆虫学领域的日渐渗透 ,有关PPO特性及其分子生物学方面的研究取得了很大进展。目前…     

棉铃虫血淋巴酚氧化酶活性的微量测定

昆虫血淋巴黑化的形成由激活酚氧化酶原的级联系统所引发 ,酚氧化酶在昆虫体液免疫中起着重要作用。用抗凝剂从棉铃虫血淋巴中分离获得了血浆及完整的血细胞 ,以L DOPA为底物 ,牛胰蛋白酶为激活剂 ,测定了血浆及血细胞裂解液中酚氧化酶及酚氧化酶原的活性。结果表明 ,血浆及血细胞中两者都有一定量的分布。这一昆虫血淋巴酚氧化酶的微量测定方法 ,所需样品量少 ,耗时短 ,简便易行。     

褐飞虱和桃蚜粗提液中酚氧化酶原激活系统的激活特点

采用研磨法制备褐飞虱Nilaparvata lugens (Stål)和桃蚜Myzus persicae (Sulzer)粗提液,用于研究几种化学因子对粗提液中酚氧化酶原激活系统(proPO-AS)的激活特点。结果表明, 在0.1～100 mmol/L的Ca²⁺浓度范围内,虱液和蚜液的酚氧化酶(PO)活性随Ca²⁺浓度升高而增强,并在30 mmol/L处PO活性达到最高,在此限之上PO活性反而下降。当昆布多糖浓度从10^-4 mg/mL升至10 mg/mL时,虱液和蚜液的PO活性也随之升高,但再提高昆布多糖浓度却未见PO活性的峰顶出现。在6种葡聚糖对PO的激活作用中,昆布多糖和酵母聚糖能显著增强PO活性,但curdlan对虱液和蚜液的PO无明显激活作用,而甘露聚糖、右旋糖苷和纤维素则降低PO活性。这些结果显示β-1,3-葡聚糖能有效激活proPO-AS。Ca²⁺和丝氨酸蛋白酶抑制剂PMSF加样顺序的不同也会影响PO活性。     

稻纵卷叶螟根虫瘟霉的分离鉴定及其流行病研究

在广东省发现一种新的侵染稻纵卷叶螟幼虫的昆虫病原真菌,经鉴定该病原真菌为根虫瘟霉Zoophthora radicans,这是中国首次报道该菌侵染稻纵卷叶螟。在室内进行的温度和光周期对该菌生长的影响实验表明,该菌在15－25℃均可生长,最适生长温度为25℃;光周期对菌落生长影响不显著。根虫瘟霉主要侵染稻纵卷叶螟3－5龄幼虫,对3－5龄幼虫的侵染率分别为2.71%、24.32%和72.97%。在10－11月,根虫瘟霉可持续引发稻纵卷叶螟幼虫高强度流行病,侵染率高达95%。该菌以休眠孢子越冬,成为次年稻纵卷叶螟幼虫流行病的初侵染源。因此,根虫瘟霉是稻纵卷叶螟幼虫一种有效的自然控制因子。     

环境因素对安徽虫瘟霉生长发育的影响

安徽虫瘟霉是目前仅发现于我国的重要蚜虫病原真菌,对桃蚜具有明显的自然控制作用。本研究了温度和紫外光对安徽虫瘟等固体培养性状及不同温湿度组合对安徽虫瘟霉分生孢子萌发的影响。     

根虫瘟霉原始菌株及转寄主菌株在不同培养条件下胞外蛋白酶的诱导表达特性

通过比较根虫瘟霉Zoophthora radicans 3个菌株（原始菌株R₀、转寄主菌株R₁和R₅）在不同培养条件下诱导胞外蛋白酶的差异,发现在含昆虫表皮和明胶的培养基中,均能诱导表达高水平的胞外蛋白酶,而在营养缺乏的MS培养基和营养丰富的萨氏培养基中则胞外蛋白酶的表达水平均很低。37 kD的丝氨酸蛋白酶在任何条件下都能被诱导表达,是各菌株中的保守序列所编码的胞外蛋白酶。46 kD的金属蛋白酶仅能在含明胶的培养基中被诱导表达,而葡萄糖可抑制其表达。在萨氏培养基中,67 kD 的蛋白酶条带在转寄主过程中消失了,而46 kD的金属蛋白酶条带和117 kD的条带随着转寄主传代数增加而明显,表明菌株在转染过程中选择表达了对新寄主具有较高基质特异性的胞外蛋白酶。     

A pheromone trap for dispersal of the pathogen Zoophthora radicans Brefeld. (zygomycetes: Entomophthorales) amongst populations of the diamondback moth,Plutella xylostella L. (lepidoptera: Yponomeutidae)

A sex pheromone trap designed as a novel pathogen delivery system is described. The strategy envisaged for its use is that male Plutella xylostella moths attracted to the pheromone enter the trap and are dosed with the fungus Zoophthora radicans. After habituation to the pheromone, the moths leave the trap and disperse to the crop carrying the fungal inoculum to susceptible larvae, thus initiating or enhancing an epizootic. A fluorescent marker in the trap was used to demonstrate that moths entered and exited from the trap and carried particles to crop plants at least 5 m away. As a preliminary study, this work shows the potential of this strategy for disseminating Z. radicans in populations of P. xylostella In addition, it could be considered as a model system with implications for other pest species where a pheromone and virulent pathogen are known.     

小菜蛾幼虫对不同寄主的取食嗜好性及其适宜性

对不同寄主种类、不同寄主形态和不同寄主饲喂的小菜蛾(Plutella xylostella)幼虫之间的取食嗜好性比较试验表明,小菜蛾幼虫优先取食大白菜、萝卜或菜心幼苗,其次为油菜和甘蓝幼苗,在大白菜与油菜幼苗之间的取食选择比例是93.33%和6.67％;在甘蓝与菜心幼苗之间的取食选择比例是16.67%和83.33%.小菜蛾幼虫的取食嗜好性受饲喂寄主种类的影响,偏食大白菜或菜心幼苗.小菜蛾幼虫选择寄主取食的次序与寄主体内可溶性糖或淀粉含量没有明显关系,但与两者的相对量呈一定的负相关.取食大白菜或菜心幼苗的小菜蛾生长良好,单头取食达0.583～0.637 cm², 单头体重达2.07～2.18 mg, 与取食甘蓝或油菜幼苗的幼虫在取食量、个体发育方面有明显差异.小菜蛾幼虫也喜好取食已经被虫危害过的幼苗.     

Formulation and application of the entomopathogenic fungus: Zoophthora radicans (Brefeld) Batko (Zygomycetes: Entomophthorales)

Aims: To isolate and formulate a native strain of Zoophthora radicans naturally infecting larvae of diamondback moth, Plutella xylostella, existing in South Australia and to provide evidence that formulation of the fungus is effective against P. xylostella larvae, and therefore, it could be used as a tool in pest management of this insect. Methods and Results: Dose–response bioassays using formulated and unformulated forms of the fungus strain were carried out against third instar larvae of P. xylostella. Results obtained have indicated a significant increase in the larval mortality when higher concentrations of a formulated form of the fungus strain were applied compared to the treatments with the unformulated form (85·0 vs 57·5% of larval mortality, respectively, at the top concentration of 10⁷ conidia/ml). The median lethal concentration (LC50) for a formulated form was 100 times less than that of the unformulated form when they were applied against the third instar larvae of P. xylostella. In addition, the formulation used in the present bioassays has preserved the viability of introduced fungus conidia for longer time in comparison with the unformulated conidia. Conclusions: The effective application of a formulated fungus strain against P. xylostella larvae constitutes the first step towards its use in pest management of this insect. Significance and Impact of the Study: The formulated fungus in inverted emulsion could be used as an alternative tool to insecticides in pest management of P. xylostella larvae because of the development of resistance to insecticides in the treated larvae.     

小菜蛾血淋巴对玫烟色棒束孢入侵的生理防御反应

为了探讨小菜蛾Plutella xylostella血淋巴对玫烟色棒束孢Isaria fumosorosea的防御机制, 利用吉姆萨染色法在光镜下观察了小菜蛾4龄幼虫血细胞感染不同致病力玫烟色棒束孢后的免疫反应。结果表明： 玫烟色棒束孢的入侵可导致小菜蛾血细胞数量发生改变, 表现为入侵初期血细胞总数增加, 不同类型血细胞比例改变等。体表接种后8-45 h, 高致病力菌株PFCF-001处理的幼虫血细胞总数在24 h出现最高值6 250个/mm³, 而低致病力菌株PFCF-D58处理在36 h达到最高值3 000个/mm³, 比高致病力菌株处理滞后12 h。不同菌株处理下虫体参与防御反应的主要血细胞类型为浆血细胞和粒血细胞。小菜蛾幼虫血细胞在感菌初期能够产生粘附、吞噬、包被及形成结节等一系列防卫反应, 但最终无法抵挡高致病力菌株PFCF-001的侵染。结果说明小菜蛾幼虫血淋巴对玫烟色棒束孢的防御反应只有短暂的抑制作用, 不能从根本上清除、 消灭玫烟色棒束孢。     

Insecticidal effects of selected biological control agents on the larvae of Plutella xylostella (Lepidoptera: Plutellidae)

To identify a more effective and safe biological control agent against a common cabbage pest, Plutella xylostella (L.) (Lepidoptera: Plutellidae), the insecticidal effects of selected biological agents were evaluated. The highest insecticidal effects determined were 100, 73.5, 45.5, 47 and 55.3% using toxin HD‐1 (isolated from the Harry Dumagae strain of Bacillus thuringiensis), toxin BTS‐1 (isolated from the tenebrionis strain of B. thuringiensis), B. thuringiensis Berliner, B. thuringiensis israelensis and B. thuringiensis kurstaki, respectively.     

Management of diamondback moth,Plutella xylostella (Lepidoptera: Plutellidae) by mating disruption

Abstract Field trials were conducted in China in 2008 and 2009 to evaluate the efficacy of mating disruption (MD) on diamondback moth, Plutella xylostella, in cabbage, Brassica oleracea var. capitata. Effectiveness was positively correlated with the MD dispenser density in the field. A density of 167 MD dispensers per ha produced an average population decrease of about 50% compared to the conventional‐practice field. Significant fewer males were captured in pheromone‐treated and conventional‐practice fields than in the blank control field, but the difference was not significant between the pheromone‐treated and conventional‐practice fields. In addition, fewer eggs and larvae were observed in pheromone‐treated fields. Our results suggest mating disruption coupled with minimal insecticidal supplements is a promising solution for resistance management and control of diamondback moth infestation.     

Isolation and characterization of Pseudomonas cedrina infecting Plutella xylostella (Lepidoptera: Plutellidae)

The diamondback moth, Plutella xylostella, is one of the most destructive pests worldwide and its management relies exclusively on frequent application of chemical insecticides. Resistance to common insecticides is now widespread, and novel classes of insecticides are needed. Entomopathogenic bacteria and their related products play an important role in the management of this pest. In the present work, one bacterial strain was separated from infected pupae of P. xylostella collected from field and its pathogenicity was evaluated. On the basis of the 16S ribosomal RNA sequencing, BLASTN, and phylogenetic analysis, this bacterial isolate was identified as Pseudomonas cedrina. Oral administration of P. cedrina at levels above 10,000 CFU/ml gave significant mortality to P. xylostella larvae. The pathogenicity was also observed by reduced longevity and fecundity in adult females. However, when live bacterial cells were removed, the cultured broth lost any pathogenicity. In response to the bacterial infection, P. xylostella expressed antimicrobial and stress‐associated genes. A mixture treatment of P. cedrina and Bacillus thuringiensis showed an additive effect on larval mortality of P. xylostella. These results indicated that P. cedrina is an opportunistic entomopathogen without secretion of toxins. Furthermore, the additive effect of P. cedrina and B. thuringiensis provide a new insight to develop new strategy for controlling P. xylostella.     

小菜蛾不同地理种群遗传多样性的ISSR标记研究

为阐明小菜蛾Plutella xylostella不同地理种群的遗传多样性, 应用ISSR技术对我国小菜蛾8个地理种群的遗传多样性进行了研究分析。15条引物扩增出395条ISSR条带, 其中多态性条带占89.11%, 全部个体显示了各自独特的ISSR图谱。ISSR标记的遗传多样性分析结果表明： 小菜蛾无论在物种水平上（P=89.11%, H=0.2706, I=0.4286）, 还是在种群水平上（P=88.80%, H=0.2759, I=0.4349）都表现出较高的遗传多样性。其中, 北京南口种群内遗传变异最大, 海南海口和甘肃兰州种群内遗传变异最小, 南方地区（云南、 湖北）小菜蛾种群遗传多样性明显高于北方地区（北京、 天津、 山东、 黑龙江、 甘肃）种群。据种群变异来源分析, 有5.66%的遗传变异来自种群间, 94.34%的变异来源于种群内（N_m=8.3399）, 不同地理种群间没有明显的遗传分化。本文有关小菜蛾不同地理种群基因流动和遗传变异的研究为小菜蛾抗药性的控制及田间种群的综合防治提供了有价值的分子生物学依据。