Production and regulation of extracellular proteases from the entomopathogenic fungus Isaria fumosoroseus (Cordycipitaceae; Hypocreales) in the presence of diamondback moth,Plutella xylostella cuticle

Extracellular proteases of entomopathogenic fungi have been implicated as components of the insect infection process. To elucidate their role in the infection process, it is essential to characterize these enzymes. During the current studies synthesis and regulation of extracellular proteases (Pr1 and Pr2) by Isaria fumosoroseus (Cordycipitaceae; Hypocreales) isolate IF28.2 were investigated as a function of carbon source (with special reference to diamondback moth cuticle), temperature and pH. The highest level of Pr1 and Pr2 activity were found in the supernatants from 1% glucose plus 1% diamondback moth cuticle (18.83±1.25 and 12.44±1.36 U/mg per h for Pr1 and Pr2, respectively). Maximum Pr1 production by the depressed mycelia was observed from the supernatants having diamondback moth cuticle as a nutrient source, whereas depressed mycelia showed maximum Pr2 activity from the cultures having chitin as the basic nutrient. The optimum pH for Pr1 and Pr2 activity was 8 while 35°C was the best temperature for protease production.     

Effect of Ifchit1 gene of Isaria fumosorosea on mortality,oviposition and oxidase activities of Bemisia tabaci

Isaria fumosorosea is one of important entomopathogenic fungi showed a good potential in controlling Bemisia tabaci. The effects of I. fumosorosea ⊿Ifchit1 mutant (Ifchit1 gene deletion mutant) on the mortality, oviposition, and host immunological response of B. tabaci, on Brassica campestris L. plant, were evaluated under laboratory conditions. The wild-type fungal strain infection significantly increased insect mortality and reduced the oviposition effeciency of B. tabaci, whereas the ⊿Ifchit1 mutant was much less effective, resulting in higher survival and ovipositing of B. tabaci. The activities of four insect enzymes were examined during a time course of fungal infection. Insect phenoloxidase, perioxidase, and catalase activities were decreased in whiteflies treated with the wild type and mutant I. fumosorosea strain at 12–36?h post treatment. However, these enzyme activities increased in fungal-treated whiteflies as compared to controls between 36 and 60?h post-infection, reaching peak values. Superoxide dismutase activity in fungal-treated whiteflies was higher than that in controls during the entire experimental time course examined. The overall enzyme activity profiles in ⊿Ifchit1 mutant-treated whiteflies were significantly different from wild-type strain treatments. Our results showed that loss of the Ifchit1 gene in I. fumosorosea affects whitefly mortality, ovipositioning and various antioxidant enzyme activities, providing new insights into the role of chitinases in I. fumosorosea-insect host–pathogen interactions.     

球孢白僵菌对四种十字花科蔬菜害虫的兼控潜力评价

研究了分离自小猿叶甲的一株球孢白僵菌(Beauveria bassiana(SCAU-BB01D))对十字花科蔬菜4种非目标害虫烟粉虱Bemisia tabaci、小菜蛾Plutella xylostella、黄曲条跳甲Phyllotreta striolata和菜缢管蚜Lipaphis erysimi的致病能力。结果表明,在实验室条件下,该菌株对以上4种害虫均有不同程度的侵染力。1×108孢子/mL的白僵菌悬浮液对1,2,3,4龄烟粉虱若虫的侵染率分别为84.88,86.81,55.94和38.78%;对小菜蛾2,3和4龄幼虫的侵染率为67,59和44%;对黄曲条跳甲幼虫和成虫分别为63%和60%;对菜缢管蚜成蚜和若蚜的侵染率分别为44%和35%。通过机率值分析,得出该菌株对烟粉虱1~4龄若虫的LT50分别为4.14,3.78,6.24和7.59 d;对小菜蛾2~4龄幼虫的LT50分别为7.29,8.16和9.82d;对黄曲条跳甲成虫和幼虫的LT50分别为11.22和8.11 d;对菜缢管蚜成蚜和若蚜的LT50分别为11.01和12.15 d。说明该球孢白僵菌菌株对烟粉虱、小菜蛾和黄曲条跳甲3种非目标害虫具有一定的兼控作用,在蔬菜害虫的生物防治中具一定的应用潜力。     

Host plants influence susceptibility of whitefly Bemisia tabaci (Hemiptera: Aleyrodidae) to the entomopathogenic fungus Isaria fumosorosea (Hypocreales: Cordycipitaceae)

We quantified the tritrophic effect of host plant on the susceptibility of the sweetpotato whitefly Bemisia tabaci (Genn.) to a fungal pathogen in the laboratory. Second-instar whiteflies reared on cucumber, eggplant, tomato and bean plants for six generations were exposed to conidial suspensions of Isaria fumosorosea isolate IF-1106. Our results did not detect differences in response (proportional survival or median lethal time, LT₅₀ days) among insect populations derived from different plants that were treated with 10⁷ conidia/ml. However, at concentrations ≤ 5×10⁶ conidia/ml, whiteflies reared on bean and tomato died significantly more quickly (i.e. LT₅₀ of 4–5 days) compared with cucumber and eggplant reared populations (5–7 days). Bean and tomato-reared populations were also more susceptible to mycosis (LC₅₀ ≈ 6 × 10⁵ conidia/ml) compared with those reared on cucumber (1.9 × 10⁶ conidia/ml) and eggplant (1.5 × 10⁶ conidia/ml). A separate study confirmed that this differential response of whitefly populations to I. fumosorosea was not explained by differences in deposition rate of conidia on leaf surfaces (i.e. a dosage effect). Our findings show that host plants affect the pathogenicity and virulence of a herbivore pathogen, but depend on the rate of exposure (inoculum) applied.     

Agroecological basis for the design of biotechnological traps based on Isaria fumosorosea for the biological control of Bemisia tabaci in strawberry crops

Isaria fumosorosea is an entomopathogenic fungus that is used as a control alternative for nymphs and adults of Bemisia tabaci. Currently there are some commercial products, however, in greenhouse or field, these do not reach the levels of control as in the laboratory because the viability of the spores decreases as a result of the conditions of application of these products in situ. The objective of this work is to implement, through agroecological data, a system of biotechnological traps based on I. fumosorosea to increase the control efficiency mainly of adults of B. tabaci in strawberry greenhouses. One way to quantify the degree of infestation of a crop is the use of yellow traps, likewise to determine the spatial distribution of adults. The Taylor method [(1984). Assessing and interpreting the spatial distributions of insect populations. Annual Reviews of Entomology, 29, 321–357) was used in five different strawberry cultivation models, finding aggregate and regular distributions. Finally, once the crop model with the highest degree of infestation was selected, the designed traps were tested and mortalities were obtained between 50% and 90% in both the laboratory and the greenhouse. The biotechnological traps based on I. fumosorosea both in the laboratory and in the greenhouse had statistically the same effect as those used under the traditional method used in the field that is aspersion; therefore, this alternative method of application can be a tool important for the biological control of this pest.     

玫烟色拟青霉对小菜蛾致病力的时间-剂量-死亡率模型模拟

小菜蛾Plutella xylostella是我国南方十字花科蔬菜上的重要害虫,已对田间常用的化学杀虫剂产生了严重的抗性。为寻找有效的小菜蛾生物防治措施,本实验研究了一株分离自家白蚁的玫烟色拟青霉Paecilomyces fumosoroseus (SCAU-PFCF01)对小菜蛾2～4龄幼虫的致病力。实验采用浸液法,供试浓度为1×10³、1×10⁴、1×10⁵、1×10⁶和1×10⁷个孢子/mL。结果表明：随玫烟色拟青霉孢子浓度的升高,小菜蛾的感病死亡率增加,在浓度为1×10⁷ /mL时,小菜蛾2、3和4龄幼虫的累计死亡率分别为96%、85%和80%。玫烟色拟青霉对小菜蛾各龄幼虫的致病力与供试龄期有关,其感病的敏感顺序为2龄、3龄和4龄。用时间 剂量 死亡率模型（time-dose-mortality model,TDM）对各龄幼虫的致病力数据进行模拟,所建模型均顺利通过Hosmer-Lemeshow拟合异质性检验,表明模型拟合良好,并由模型估计出了该菌株对小菜蛾各龄幼虫的致死剂量与致死时间。2龄幼虫接种后第7天、3龄幼虫接种后第5天、4龄幼虫接种后第4天的LC₅₀估计值分别为1.17×10⁴、1.44×10⁴和5.21×10⁴ /mL,LC₉₀估计值分别为1.98×10⁶、3.82×10⁷和1.29×10⁸ /mL。玫烟色拟青霉对小菜蛾幼虫的致死时间与浓度相关,供试各龄幼虫的LT₅₀值随着孢子悬浮液浓度的增加而递减,在1×10⁵～1×10⁷ /mL的范围内,2龄幼虫的LT₅₀值从3.16天降低到1.72天,3龄幼虫的LT₅₀从3.21天降低到1.83天,4龄幼虫的LT₅₀从3.69天降低到2.04天。即2龄幼虫致死所需的时间最短,其次为3龄幼虫,4龄幼虫致死所需的时间最长。结果显示了该株玫烟色拟青霉在小菜蛾的生物防治中具较强的应用潜力。     

玫烟色棒束孢相容性农药筛选及混用对小菜蛾的防效

为了明确玫烟色棒束孢Isaria fumosoroseus与常用农药的相容性,本研究测定了7种常用农药制剂对玫烟色棒束孢菌丝生长、孢子萌发以及产孢量的影响,测试了相容性较好杀虫剂与玫烟色棒束孢混用对小菜蛾Plutella xylostella Linnaeus的防治效果。结果表明,田间使用浓度下,氯虫苯甲酰胺、双丙环虫酯和四氯虫酰胺对菌丝抑制作用相对较小,田间浓度和亚致死浓度的阿维菌素对菌丝生长具有促进作用。所有供试农药在田间使用浓度下对孢子萌发均具有明显抑制作用,抑制作用随农药浓度降低而减弱,其中噻虫啉和四氯虫酰胺对孢子萌发抑制作用相对较小。所有测试药剂对产孢量具有显著抑制作用,抑制作用随农药浓度降低而减弱,其中噻虫啉和氯虫苯甲酰胺抑制作用较小。玫烟色棒束孢3种浓度（9×107、9×106、9×105个孢子/mL）分别与次亚致死剂量四氯虫酰胺混用对小菜蛾的LT50分别为2.19 d、2.94 d、5.16 d,杀虫速度高于单用相应浓度真菌处理（2.34 d、3.29 d、5.33 d）和四氯虫酰胺单独处理（10.60 d）。综合分析结果表明,低剂量的四氯虫酰胺、噻虫啉和氯虫苯甲酰胺与玫烟色棒束孢相容性相对较好,是田间菌药混用可供选择对象;次亚致死剂量的四氯虫酰胺与真菌混用防控小菜蛾具有协同增效作用。     

玫烟色棒束孢侵染对小菜蛾幼虫体内不同酶活的影响

在实验室条件下,测定了昆虫病原真菌玫烟色棒束孢Isaria fumosorosea的侵染对小菜蛾Plutella xylostella3龄幼虫体内酚氧化酶(PO)和不同抗氧化酶类,包括超氧化物歧化酶(SOD)、过氧化物酶(POD)、过氧化氢(CAT)及谷胱甘肽S-转移酶(GSTs)活力的变化。结果表明,玫烟色棒束孢侵染小菜蛾3龄幼虫后,体内PO、SOD、POD、CAT和GSTs活力均受到明显的影响。其中感病小菜蛾幼虫体内的PO活力始终高于同期未感染的小菜蛾幼虫,当接菌40h时,酚氧化酶活力达到最大37.4U/g,为对照的2.6倍,而SOD、POD、CAT和GSTs活力在感病前期明显高于对照,接菌40–48h各种酶活力均达到最大,当接菌56h酶活力开始下降,64h时酶活力均明显低于对照。可见玫烟色棒束孢的侵染严重干扰了小菜蛾幼虫体内正常的生理活动。     

A new host plant species for the cassava biotype of Bemisia tabaci (Gennadius) (Hom., Aleyrodidae)

Abstract:  The cassava biotype of Bemisia tabaci (Gennadius) is known to colonize cassava ( Manihot esculenta Crantz) whereas the sweet potato biotype colonizes various plant and weed species but not cassava. Both biotypes have Solanum melongena L. and Solanum aethiopicum L. as common host plant species. This study shows for the first time that both biotypes can successfully feed, survive and reproduce on a new host plant species, Nicotiana debneyi Domin. These findings have important implications for better understanding the disease epidemics, related to whitefly transmissible geminiviruses.     

Development and reproduction of Bemisia tabaci （Homoptera： Aleyrodidae） on three bean species

Abstract The development, survivorship and reproduction of Bemisia tabaci B-biotype on three bean species were studied at 26±1°C in the laboratory. The developmental periods from egg to adult varied from 27.80 days on garden beans to 18.20 days on soybeans. The survivorship from egg to adult on soybeans, cowpeas and garden beans was 77.14, 70.14 and 64.28%, respectively. The average longevity of female adults ranged from 12.30 days on soybeans to 9.80 days on garden beans, and the oviposition of B. tabaci varied from 160.85 eggs on soybeans to 98.00 eggs on garden beans. Life table parameters were calculated as biological attributes for Bemisia tabaci populations fed on three bean species. The results indicated that the intrinsic rate of increase (r_m), the finite rate of increase (λ) and net reproductive rate (R₀) were high for populations fed on soybeans, with values of 0.1857, 1.2041 and 82.1576, respectively. The corresponding values were less for populations fed on garden beans, with values of 0.1097, 1.1159 and 31.2661, respectively. The parametric values for cowpeas were intermediate between soybeans and garden beans but no significant difference were observed for the r_m values for soybeans and cowpeas. Experimental evidence in our investigation indicated that Bemisia tabaci is best adapted and shows the greatest preference for soybean of the three bean species tested in this study.     

Development and reproduction of ‘B＇ biotype Bemisia tabaci （Gennadius） （Homoptera： Aleyrodidae） on four ornamentals

Abstract Effects of four commercial ornamentals on the development, survivorship and reproduction of the whitefly B biotype Bemisia tabaci (Gennadius) were studied in the laboratory (temperature 26 ± 1 °C; relative humidity 75%–90%; L: D 14:10). The total survivorship from egg to adult on hibiscus ( Hibiscus rosa-sinensis L.), poinsettia ( Euphorbia pulcherrima Will), cottonrose hibiscus ( Hibiscus mutabilis L.) and variegated leafcroton ( Codiaeum variegatum'Aucubaefolium' ) were 33.69%, 40.55%, 79.11%, and 29.39%, respectively. The developmental periods from egg to adult varied from 23.12 days on cottonrose hibiscus to 32.13 days on hibiscus. The average longevity of adult females ranged from 6.87 days on variegated leafcroton to 21.07 days on poinsettia. The average numbers of eggs laid per female were 9.20, 25.13, 54.45, and 26.79 on the above respective hosts. The intrinsic rates of natural increase ( r_m ) for B biotype B. tabaci on cottonrose hibiscus was the highest. Based on life table analyses of whitefly populations, cottonrose hibiscus was the most suitable host for B biotype B. tabaci in this study.     

寄主转换对B型烟粉虱生长发育和繁殖的影响

在实验室恒温条件下,对B型烟粉虱Bemisiatabaci(Gennadius)寄主转换后的生长发育和繁殖进行初步研究。结果表明:B型烟粉虱从番茄转移到国抗22棉花上后,第1代转寄主烟粉虱的发育历期比第2代长19.2%,存活率低16.4%,第2代和第3代之间没有明显差异。受体寄主对B型烟粉虱发育速率的恢复有一定的影响。     

Effect of male and female multiple mating on the fecundity, fertility, and longevity of diamondback moth, Plutella xylostella (L.)

Abstract:  The effect of diamondback moth (DBM), Plutella xylostella (Lep., Plutellidae) male and female multiple mating on fecundity, fertility, and longevity was studied. Males could mate for five times with virgin females during scotophase. The successful copulation rates, fecundity of female, and longevity of both females and males decreased when male mating times increased, whereas copulation duration increased. Correlation coefficient between copulation duration and male mating times was significant ( r  = 0.7358, P = 0.0001, spearman rank-order correlation). There were linear relationships between mating history of males and longevities of males and females, and regression relationships between them were significant. Mated females had similar daily reproductive pattern, which laid the most eggs on the first day after mating in spite of their mates' mating history. Virgin females laid some infertile eggs before they died. Most of the females mated once during their lifespan but 19.9% of females mated twice when one female kept with one male during scotophase. There were no significant differences in the fecundity, fertility and longevity between the single- and twice-mated females. Correlation coefficient between copulation duration and female mating times was not significant ( r  = 0.0860, P = 0.8575). Results suggested that DBM females may be monandrous. Multiple mating did not increase male or female mating fitness.     

绿僵菌素对爪哇棒孢霉SP053菌株的影响及其混用对小菜蛾的联合毒力

为了寻找更有效的利用虫生真菌爪哇棒孢霉Isaria javanicus防治小菜蛾Plutella xylostella的新方法, 测定了绿僵菌素（destruxin）对SP053菌株生长发育与产孢量与萌发率的影响, 利用协同毒力指数法和毒力回归分析法评价了两者混用对小菜蛾2龄幼虫的联合毒力。结果表明: 绿僵菌素对SP053菌株菌丝生长、孢子萌发率和孢子产量均无显著影响（P>0.05）。而一定浓度的绿僵菌素与SP053菌株分生孢子混用具有增效作用, S50-CD100（指爪哇棒孢霉SP053菌株的分生孢子浓度为50×10⁵个/mL和绿僵菌素粗毒素的浓度为100 mg/L的组合; 依此类推）、S25-CD100、S25-CD50及S12.5-CD100等混配组合都有显著的增效效果, 尤其以S25-CD100组合增效效果最好, 其48 h和72 h的协同毒力指数（c.f.）分别达到52.31和31.07。毒力回归分析结果也显示, 添加绿僵菌素粗毒素25~100 mg/L的混剂的LC50值明显降低, 比如添加100 mg/L绿僵菌素粗毒素的混剂（SP053-CD100）, 其48和72 h的LC50分别为17.45和10.55 （×10⁵个孢子/mL）, 而SP053菌株分生孢子单剂的LC50相应值为>50和35.85（×10⁵个孢子/mL）。本研究证明绿僵菌素与爪哇棒孢霉SP053菌株混用有增效作用, 对改进小菜蛾的生物防治方法有一定指导意义。     

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。     

外源基因导入对棉花烟粉虱种群的影响

以转基因棉花和对应的常规棉花亲本为材料,探讨外源基因导入对棉花烟粉虱Bemisia tabaci(Gennadius)种群的影响。结果表明,在转基因棉花GK12、33B、SGK上,烟粉虱从卵发育到成虫羽化的历期分别为15.56、15.35和15.25 d,较对应的常规棉亲本SM3(19.38 d)、33(20.81 d)、SY321(18.76 d)分别短24.55%、26.23%、18.71%;GK12和33B棉花上烟粉虱的存活率(69.16%)分别较对应的常规棉亲本SM3(54.76%)和33(64.91%)高26.29%和12.81%,而SGK(63.21%)上烟粉虱的存活率与对应的常规棉亲本SY321(62.61%)之间差异不显著;烟粉虱在GK12(84.00)和33B(77.25)上的产卵量分别较SM3(62.25)、33(70.00)高34.93%和10.35%,但SGK和SY321之间差异不显著;在转基因棉花GK12、33B、SGK上,烟粉虱的雌雄性比分别比对应的常规棉亲本高26.71%、46.23%和19.17%。结果表明,外源基因导入后,有助于烟粉虱的发育,提高了烟粉虱的产卵量和雌雄性比,从而促进了烟粉虱种群的上升。     

4种寄主植物对烟粉虱发育、存活及繁殖的影响

在实验室条件下[温度(26±1)℃,相对湿度(75±5)%,光照L∶D=14∶10],对烟粉虱[Bemisia tabaci(Gennadius)]在烟草、假连翘、一品红、扶桑等4种寄主植物上的发育、存活、繁殖力及若虫个体形态进行了研究。结果表明:烟粉虱在上述4种寄主植物上从卵至成虫的发育历期分别为:17.63、25.70、23.15、20.32 d,存活率为:62.63%、58.48%、43.69%、52.45%。烟粉虱在烟草、假连翘、一品红、扶桑上的单雌产卵量分别为337.80、307.40、157.00、57.20粒,种群增长指数(I)分别为117.95、107.86、37.86、16.24。     

C_V型烟粉虱在三种变叶木上的发育和繁殖

CV型烟粉虱Bemisia tabaci(Gennadius)是园林观赏植物上的重要害虫。本文在室内(26±1)℃下比较了CV型烟粉虱在撒金变叶木(Codiaeum variegatun‘Aucubaefolium’)、美丽变叶木(C.variegatumnov.)和彩叶变叶木(C.variegaum‘Mons-florin’)3个变叶木品种上的发育和繁殖情况,结果表明在撒金变叶木上的发育历期最长,为32.8 d;在美丽变叶木和彩叶变叶木上分别为29.7 d和29.5 d,后两者差异不显著。彩叶变叶木上烟粉虱的世代存活率最高,达58.8%,其次为美丽变叶木为46.5%,在撒金变叶木上的存活率最低,为40.5%,三者间差异显著。CV型烟粉虱在撒金变叶木上的产卵前期最长,为0.8 d。烟粉虱成虫彩叶变叶木上的平均寿命最长,为14.9 d。CV型烟粉虱在3个品种上的平均单雌产卵56～81粒,相互间差异不显著。烟粉虱在撒金变叶木、美丽变叶木和彩叶变叶木3个品种上的rm分别为0.0397、0.0625和0.0860。综合比较,这3种变叶木均为CV型烟粉虱较合适的寄主。     

温度对烟粉虱发育和繁殖的影响

研究了7个不同温度梯度对烟粉虱(Beimisia tabaci )B生物型发育、存活和繁殖的影响。在17℃时,烟粉虱从卵到成虫羽化需要48．7天,而在29℃则仅需要13．9天。烟粉虱发育的最佳温度是26℃,低温(＜20℃)和过高的温度(＞32℃)对烟粉虱发育有抑制作用。经线性回归分析,烟粉虱的发育起点温度约为13℃,有效积温为263．8℃。烟粉虱在20℃时,其成虫寿命和产卵量都是32℃时2倍多,单雌最高产卵量达到208粒。烟粉虱的存活率以26℃时最高,低温和高温对烟粉虱的存活率有负面影响。烟粉虱的内禀增殖率在26℃和29℃时较高,分别为0．1456和0．1470。所有数据显示26℃是烟粉虱种群发育和繁殖的最佳温度。