朱红毛斑蛾实时荧光定量PCR内参基因的筛选

筛选朱红毛斑蛾Phauda flammans(Walker)在不同成虫组织、性别及发育阶段处理条件下稳定表达的内参基因,为进一步开展朱红毛斑蛾相关基因的定量研究提供参考.本研究以不同成虫组织(头、胸、腹、足、翅和触角)、不同成虫性别和不同发育阶段(卵、幼虫、蛹和成虫)为实验材料,对10个候选内参基因进行实时荧光定量PC...     

朱红毛斑蛾嗜食性的研究

通过野外调查和室内选择实验,研究了朱红毛斑蛾的食性。自然条件下,朱红毛斑蛾为害榕属植物中的小叶榕和垂叶榕,为害率分别为67.39%和47.61%。在T50时,朱红毛斑蛾对印度榕、对叶榕、无花果、黄葛树、高山榕、小叶榕和垂叶榕的平均取食量分别为0、0、0、0、0、8.62±1.6 cm2和3.79±1.8 cm2;平均取食选择指数分别为0、0、0、0、0、0.69和0.31,说明朱红毛斑蛾仅为害小叶榕和垂叶榕,且对小叶榕有明显偏好性。研究结果证明朱红毛斑蛾为寡食性害虫,提示在园林绿化过程中应尽可能避免选择易受害的单一树种大面积栽种。     

Growth and insect assays of Beauveria bassiana with neem to test their compatibility and synergism

Beauveria bassiana is being used as a biopesticide for many insect pests. Neem oil (azadirachtin) is an eco-safe popular botanical pesticide. A biopesticde with a neem compatible isolate of B. bassiana will enable their simultaneous use in pest management. A sample of 30 isolates of B. bassiana from culture collections was screened for compatibility with a commercial formulation of neem oil (Margoside®) at the field recommended dose (0.3%, v/v). Compatibility was tested in vitro through germination and growth assays. In all isolates, conidial germination was delayed but not significantly decreased by neem. In the growth assays, 23 isolates were found compatible with neem. In the neem sensitive isolates, growth was decreased but not totally inhibited. The effect of combined treatment with B. bassiana and neem in comparison to single treatments with either of them on Spodoptera litura Fabricius was tested in laboratory bioassays. The combined treatment was found to have synergistic effect on insect mortality when a B. bassiana isolate compatible with neem was used, while, with an isolate sensitive to neem, an antagonistic effect was observed.     

Effect of combination treatment with entomopathogenic fungi Beauveria bassiana and Nomuraea rileyi (Hypocreales) on Spodoptera litura (Lepidoptera: Noctuidaeae)

In biocontrol of insect pests, efficacy of treatment with multiple pathogens has not been frequently investigated but may have potential for effective management. The possible advantage of a combination treatment with two entomopathogenic fungi - Beauveria bassiana and Nomuraea rileyi - was assessed in laboratory bioassays on second instar Spodoptera litura. From among the fungal isolates of an epizootic population, two isolates of each fungus differing in virulence to S. litura were chosen, one highly virulent and the other with low virulence. The bioassays were carried out at either a continuous temperature of 25±1°C or at a temperature cycle of 32±2°C 8 h/21±2°C 16 h to mimic the field temperatures during the epizootic. Treatments with the two fungi were done both simultaneously and sequentially. In combination treatments at 25±1°C, in all isolate combinations, a majority of the larvae showed N. rileyi induced mycosis; the percentage mortality and speed of kill of insects in these treatments was similar to the N. rileyi isolate used in the combination treatments. At the temperature cycle of 32±2°C 8 h/21±2°C 16 h, in all combination treatments, all the dead insects exhibited B. bassiana mycosis; the mortality pattern was similar to the B. bassiana isolate used in the combination treatments. The adverse effect of high temperature on virulence of N. rileyi was however, not evident in in vitro growth assays. Combination treatment with both fungi did not have a synergistic effect on insect mortality.     

Germination, Radial Growth, and Sporulation of Beauveria bassiana and Metarhizium anisopliae Isolates and Their Virulence to Chilo partellus (Lepidoptera: Pyralidae) at Different Temperatures

The effect of temperature regimes on conidia germination, radial growth and virulence of Beauveriabassiana and Metarhizium anisopliae against the spotted stalk borer Chilo partellus was investigated with Ethiopian isolates. Conidia germination, radial growth and sporulation of all isolates were retarded at 15 and 35°C. A suitable temperature range for the isolates was between 20 to 30°C. Conidia germination was less tolerant of low temperature (15°C) than radial growth. Radial growth and sporulation reacted differently to temperature. At both 25 and 30°C, all isolates induced 100% mortality to C. partellus larvae in six days. The LT₅₀ decreased for the isolates with increasing temperature.     

Effect of exposure method to Beauveria bassiana and conidia concentration on mortality, mycosis, and sporulation in cadavers of Chilo partellus (Lepidoptera: Pyralidae)

The effects of exposure methods, conidial concentrations, and temperature on mortality, mycosis and sporulation in second instar Chilo partellus cadavers infected by Beauveria bassiana was investigated in laboratory studies. Larvae directly sprayed with conidia, exposed to conidia-treated leaves, and dipped into conidial suspension resulted in high mortality (98-100%). The longest LT50 (3.5 days) and days to mortality (2.6 days) were observed in the treated-leaves exposure method. The shortest LT50 (1 day) and days to mortality (1 day) were recorded for the dipping method. With increasing conidial concentrations, there were decreasing LT50 and days to mortality. Larvae exposed to treated leaves and larvae directly sprayed with conidial suspensions produced high mycoses in cadavers. Exposure of larvae to treated-leaves resulted in high sporulation. At lower concentrations of conidia, both mycosis and sporulation in cadavers were high. The optimum temperature for mycosis was 20 and 15 degrees C for sporulation.     

Evaluation of Beauveria bassiana and Metarhizium anisopliae for Controlling Chilo partellus (Lepidoptera: Crambidae) in Maize

Maize (variety Katumani) was planted in a greenhouse and plants were infested with 20 Chilo partellus second instar larvae 3 and 4 weeks after plant emergence. One isolate of Beauveria bassiana (BB-01) and four isolates of Metarhizium anisopliae (PPRC-4, PPRC-19, PPRC-61 and EE-01) were sprayed onto the leaf whorl at 2×10⁸ conidia/mL 24 h after infestation. Leaf damage by the larvae was greatly reduced by the treatments. The mean daily temperature and relative humidity in the greenhouse ranged from 10 to 35°C and 30 to 90%, respectively. The growth of infested unsprayed plants was less than that of fungi treated plants. Stem tunneling (1-5%), deadheart (0-33%), number of attacked nodes (0.3-2.5) and holes (0.2-3.3) were also reduced in plants sprayed with conidial suspensions. Isolates PPRC-4, PPRC-19 and PPRC-61 seemed to be the best candidates for further development.     

美国白蛾高毒力球孢白僵菌菌株筛选

【目的】美国白蛾Hyphantria cunea(Drury)是世界性检疫害虫,目前已经入侵到我国长江下游地区(从辽宁丹东至安徽芜湖)。作者在前期研究中,在芜湖地区成功获得了被球孢白僵菌Beauveria bassiana侵染的美国白蛾僵虫。本研究拟在前期已获取菌株的基础上,筛选生物学性状良好且对美国白蛾幼虫具有高致病力的球孢白僵菌菌株,以期为我国长江下游地区利用昆虫病原真菌防治美国白蛾奠定基础。【方法】对采集自美国白蛾僵虫的7株球孢白僵菌菌株(Bb01-1,Bb08-5,Bb08-6,Bb08-11,Bb22-2,Bb22-3和Bb22-5)进行生物学特性研究,以候选菌株菌落形态、生长速率、产孢量、萌发率和抗紫外能力等参数确定优良菌株;并将生物学性状良好的球孢白僵菌菌株配制成5个浓度(5×10~6,1×10~7,5×10~7,1×10~8和5×10~8孢子/m L)的孢悬液,采用浸渍法处理美国白蛾4龄幼虫,以幼虫死亡率、侵染率等指标评价菌株的致病力。【结果】供试菌株Bb22-2,Bb22-3和Bb22-5为粉状菌落,其菌落直径大,产孢早且孢子层厚;同时,这3个菌株在菌落生长速率和产孢量以及孢子萌发率、紫外照射处理后的萌发率也均显著优于其他4个菌株。毒力测定结果表明,这3个菌株对美国白蛾4龄幼虫表现出很强的致病力,各菌株处理11 d后美国白蛾幼虫校正死亡率为80%~90%。这3个菌株对美国白蛾幼虫的侵染率均高(侵染率53%~87%),并且呈现随浓度升高侵染率上升的趋势。其中,菌株Bb22-2侵染率均要高于其他2个菌株。【结论】本研究成功筛选获得了生物学性状良好且对美国白蛾高毒力的球孢白僵菌菌株。鉴于中国长江下游地区气候特点与美国白蛾生物学习性,可以预期采用昆虫病原真菌防治美国白蛾在该地区将具有很好的应用前景,但其实际应用价值尚需进一步的田间试验进行确认。     

Laboratory and field evaluations of the susceptibility of immature Choristoneura rosaceana (Lepidoptera: Tortricidae) to Beauveria bassiana (Hypocreales: Cordycipitaceae)

Abstract

The susceptibility of immature Choristoneura rosaceana (Harris) to Beauveria bassiana-GHA (BotaniGard® 22WP) was evaluated under laboratory and field conditions. Although egg masses ≤24 h old were susceptible to infection by topically sprayed B. bassiana spores in the laboratory and resulting mycosis significantly reduced the percent of neonates able to emerge, no significant egg mass infections resulted from orchard treatment of ≤24–48 h eggs. Exposure to high levels of the B. bassiana product on apple leaf surfaces in the laboratory caused significant dose-related mortality in first to fourth instar C. rosaceana. First instar C. rosaceana were the most susceptible of the larval stages assayed in the laboratory although only 36% of neonates introduced into the orchard prior to Beauveria treatment and 27% of the neonates emerging from orchard treated egg masses became infected. Fourth instar mortalities were similar but moderate when treated with 1×10⁸ spores mL^?1 in the orchard and on leaf surfaces in the laboratory. Beauveria bassiana-induced larval mortalities were significantly higher when the spores were applied directly to the larval integument as opposed to the leaf surface where the insect would encounter the pathogen in a treated orchard. Feeding of C. rosaceana larvae on antibiotic containing meridic diet prior to their use in trials did not impact the susceptibility of the larvae to B. bassiana. Antibiotic containing meridic diet significantly reduced larval C. rosaceana mortality when treated larvae or the spores were placed directly on the diet as opposed to leaf tissue.     

In vivo passages of heterologous Beauveria bassiana isolates improve conidial surface properties and pathogenicity to Nilaparvata lugens (Homoptera: Delphacidae)

In entomopathogenic hyphomycetes, desired candidates against the brown planthopper, Nilaparvata lugens (a sap-sucking rice pest in Asia), are lacking. In this study, 21 Beauveria bassiana isolates from heterologous host insects showed low pathogenicity to third-instar nymphs sprayed at the high concentration of ∼1000 conidia/mm², causing only 2-23% mortalities. Of those, three isolates killed significantly more nymphs (up to 45-62%) after two in vivo passages but no more after further passage. Conidial hydrophobicity rates (H_r), zeta potentials (P_z), and subtilisin-like protease (Pr1) activities (A_p) of these isolates showed the same trends in the three host passages (N: 0-3). In multivariate correlation, the variables N, H_r and P_z were found contributing 89% to the mortality variation (r² = 0.89). Significant positive correlations were also found between H_r and N (r² = 0.64), P_z and N (r² = 0.52), A_p and N (r² = 0.51), H_r and A_p (r² = 0.45), and P_z and A_p (r² = 0.57), respectively. However, irregular changes of H_r and P_z occurred in four other isolates, whose pathogenicity to N. lugens was not enhanced by repeated host passages, resulting in no correlation between the variables. Our data indicate that the conidial surface properties H_r and P_z associated with cuticle adhesion reflect the heterologous host-induced adaptation and help to select fungal candidates against N. lugens from repeated in vivo passages.     

Susceptibility of apple clearwing moth larvae,Synanthedon myopaeformis (Lepidoptera: Sesiidae) to Beauveria bassiana and Metarhizium brunneum

Apple clearwing moth larvae, Synanthedon myopaeformis (Lepidoptera: Sesiidae) were found to be susceptible to infection by two entomopathogenic fungi: an indigenous fungus isolated from S. myopaeformis cadavers and identified as Metarhizium brunneum (Petch); and Beauveria bassiana isolate GHA. In laboratory bioassays, larvae exhibited dose related mortality after exposure to both the M. brunneum and Beauveria bassiana with 7 day LC₅₀'s of 2.9×10⁵ and 3.4×10⁵ spores/mL, respectively. Larval mortalities caused by the two isolates at 1×10⁶ spores/mL were not significantly different and 73% of the M. brunneum-treated, and 76% of the B. bassiana-treated larvae were dead 7 days post treatment, with LT₅₀'s of 5.5 and 5.1 days, respectively.     

Efficacy of entomopathogenic fungi Beauveria bassiana and Metarhizium anisopliae against Thaumetopoea pityocampa Shiff. (Lepidoptera: Thaumatopoeidae)

Abstract

In this study, Metarhizium anisopliae TR 106 and Beauveria bassiana TR 217 was tested against fourth instar larvae of Thaumetopoea pityocampa. The LT₅₀ and LT₉₀ of 1?×?10⁶ concentration of M. anisopliae against T. pityocampa were 3.60 and 4.11 for direct application, while these were 2.87 and 3.60?days, respectively in leaves application. The LT₅₀ and LT₉₀ of the 1?×?10⁸ concentration of the same isolate were 2.50 and 2.95?days for direct application, and 2.98 and 3.74?days for leaves application. The LT₅₀ of insect and leaves application for 1?×?10⁶ of B. bassiana were 3.75 and 3.49?days, respectively. The LT₉₀ of same concentration for insect application was 4.48?days, while LT₉₀ for leaves application was 4.63?days. Similarly, LT₅₀ of insect and leaves application for 1?×?10⁸ of B. bassiana were 3.03 and 3.31?days, while LT₉₀ were 3.68 and 4.29?days, respectively. Approximate 100% mycosis was observed in all treatments.     

球孢白僵菌对象虫金小蜂安全性评价和亚致死效应研究

为明确球孢白僵菌Beauveria bassiana对烟草甲Lasioderma serricorne(Fabricius)幼虫、成虫的致病力以及对象虫金小蜂Anisopteromalus calandrae(Howard)的安全性,本研究采用浸渍法测定球孢白僵菌对烟草甲和象虫金小蜂的LC₁₀、LC₂₅、LC₅₀,以球孢白僵菌亚致死浓度LC₁₀和LC₂₅胁迫象虫金小蜂成虫后,分析其F₁代实验种群生存、发育和繁殖的变化。结果表明：(1)在1×10⁹孢子/mL的球孢白僵菌浓度下,烟草甲幼虫的致死率可达到92.62%,烟草甲成虫的致死率为100.00%,象虫金小蜂成虫的致死率可达98.73%,亚致死浓度LC₁₀和LC₂₅分别为2.125×10⁵和7.114×10⁵孢子/mL。(2)受LC₁₀和LC₂₅胁迫处理象...     

Interaction of HaNPVs with two novel insecticides against Helicoverpa armigera Hubner (Noctuidae: Lepidoptera)

Nucleopolyhedrosis viruses can be utilized for effective management of agriculture pests. Their efficacy can be increased if they are mixed with certain insecticides. In the current study, HaNPV was mixed with two insecticides: spinetoram and emamectin benzoate in various combinations and applied to larvae of H. armigera in laboratory conditions. There were a total of 15 combinations of HaNPV with each of the two insecticides in addition to five doses of HaNPV and three doses of insecticides alone. The synergistic and antagonistic effects of combinations were explored. The results revealed that there was synergistic effect of HaNPV @ 0.5 × 10⁹ PIB/ml × Spinetoram @ 40, 20, 10 ml/100 L of water. In case of emamectin benzoate, synergistic effects were recorded at 1 × 10⁹ PIB/ml HaNPV × emamectin benzoate @ 100 ml/100 L of water. However, 0.5 × 10⁹ PIB/ml HaNPV has synergistic effects with all three doses of emamectin benzoate. The results suggested that HaNPV can be used in combination with spinetoram and emamectin benzoate for the management of resistant population of H. armigera.     

Effects of Bacillus thuringiensis toxin Cry1Ac and Beauveria bassiana on Asiatic corn borer (Lepidoptera: Crambidae)

In this study, interactions between Cry1Ac, a toxic crystal protein produced by Bacillus thuringiensis (Berliner), and Beauveria bassiana on the mortality and survival of Ostrinia furnacalis was evaluated in the laboratory. The results showed that Cry1Ac is toxic to O. furnacalis. Not only were larval growth and development delayed, but pupation, pupal weight and adult emergency also decreased when larvae were fed on artificial diet containing purified Cry1Ac toxin. When third instars O. furnacalis were exposed to combination of B. bassiana (1.8 × 10⁵, 1.8 × 10⁶ or 1.8 × 10⁷ conidia ml⁻¹) and Cry1Ac, (0.2 or 0.8 μg g⁻¹), the effect on mortality was additive, however, the combinations of sublethal concentrations showed antagonism between Cry1Ac (3.2 or 13 μg g⁻¹) and B. bassiana (1.8 × 10⁵ or 1.8 × 10⁶ conidia ml⁻¹). When neonates were reared on sublethal concentrations of Cry1AC until the third instar, and survivors exposed B. bassiana conidial suspension, such treatments showed additive effect on mortality of O. furnacalis except for the combination of Cry1Ac (0.2 μg g⁻¹) and B. bassiana (1.8 × 10⁶ conidia ml⁻¹) that showed antagonism.     

Selection of isolates of entomopathogenic fungi for controlling Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae) and their compatibility with insecticides used in tomato crop

The activity of Metarhizium anisopliae and Beauveria bassiana towards eggs and larvae of Tuta absoluta (Meyrick) was evaluated. Our data showed that the isolates were pathogenic to both developmental stages tested and the eggs were more susceptible than the 1st instars. The isolates URPE-6 and URPE-19 of M. anisopliae were more pathogenic to eggs and larvae, respectively. The compatibility of these two isolates with the insecticides chlorfenapyr, spinosad, indoxacarb, abamectin, and neem were evaluated. Spinosad and indoxacarb were compatible with the two M. anisopliae isolates in all tested concentrations. At the average recommended concentration, chlorfenapyr was compatible to URPE-6 and abamectin to UFPE-19. The use of entomopathogenic fungi associated with compatible insecticides may be a useful alternative to control T. absoluta.     

玫烟色棒束孢几丁质酶的转基因球孢白僵菌菌株的获得及其对马尾松毛虫的毒力增效作用

本文根据GenBank中已登录的蜡蚧蚧霉Lecanicillium lecanii,粉棒束孢Isaria farinosa,球孢白僵菌Beauveria bassiana和金龟子绿僵菌Metarhizium anisopliae的几丁质酶基因序列的同源性比较, 以它们高度保守的核苷酸序列设计一对引物,采用RT-PCR和3′/5′-RACE相结合的方法,首次从玫烟色棒束孢Isaria fumosorosea中克隆出几丁质酶Ifuchi1全长cDNA基因。其全序列长为1 542 bp,编码阅读框由1 275个核苷酸组成, 5′非翻译区（5′-UTR）与3′非翻译区（3′-UTR）分别为33个核苷酸和234个核苷酸。基因编码424个氨基酸, 信号肽长度为24个氨基酸, 成熟蛋白理论分子量为47.6 kDa,理论等电点为4.89。该蛋白可归于几丁质酶18族V类。运用制备芽生孢子转化法将Ifuchi1基因导入球孢白僵菌。最适产酶时间36 h条件下, 挑选的转基因菌株的几丁质酶活性相对于野生型菌株提高了86.2%;在对马尾松毛虫的生物测定中,在孢子浓度为1×10⁷个孢子/mL时,与野生型菌株Bb13相比,转基因菌株的致死中时间LT₅₀平均缩短了29%,同时死亡率提高了52.9%;野生型菌株和转基因菌株致死中浓度LC₅₀分别为4.71×10⁶ 个孢子/ mL和1.74×10⁶ 个孢子/ mL,降低了约1.7倍的剂量,故通过基因工程方法获得的转基因工程菌株显著地提高了球孢白僵菌的杀虫效率。     

Pine processionary moth (Thaumetopoea pityocampa,Lepidoptera: Thaumetopoeidae) larvae are highly susceptible to the entomopathogenic fungi Metarhizium brunneum and Beauveria bassiana

The larvae of the pine processionary moth (PPM), Thaumetopoea pityocampa, feed on the needles of pine and cedar. The urticating hairs of older instars pose a threat to human and animal health. Strains of the entomopathogenic fungi, Metarhizium brunneum (V275, ARSEF 4556) and Beauveria bassiana (KTU-24), were assayed against first to fourth instar T. pityocampa using doses ranging from 1?×?10⁵ to 1?×?10⁸ conidia mL^?1. The three strains differed slightly in their virulence but caused 100% mortality of all instars at the highest dose. The newly emerged or first instar larvae were extremely susceptible with 100% mortality being achieved 2–4 days post inoculation with V275 at all but the lowest dose. The fourth instar larvae appeared to be less susceptible than earlier instars. There was good horizontal transmission of conidia from treated to un-inoculated larvae. However, mortality was higher in third and fourth instars and where the ratio of inoculated versus untreated larvae was high. This we presume is due to spores being more readily trapped by the urticating hairs found on third and older instar larvae. Injection of the nests offers a simple and environmentally friendly way of controlling the pest with reduced risk to operators.     

Laboratory and field evaluation of Beauveria bassiana against sugarcane stalkborers (Lepidoptera: Pyralidae) in the lower Rio Grande Valley of Texas

Of approximately 18,200 ha planted to sugarcane in south Texas, only approximately 80 ha (<0.5%) are treated with insecticides because this type of control is widely regarded as ineffective against stalkboring pyralids, the key pests of sugarcane. Therefore, nonchemical control measures, such as resistant varieties and biological controls, must be evaluated to mitigate the losses caused by stalkborers. We performed laboratory and field evaluations on the use of Beauveria bassiana (Balsamo) Vuillemin (Deuteromycotina: Hyphomycetes) (strain GHA) against the Mexican rice borer, Eoreuma loftini (Dyar) (Lepidoptera: Pyralidae), which causes damage in south Texas estimated at between 10 and 20 million dollars annually. We also performed bioassays against the sugarcane borer, Diatraea saccharalis (F.) (Pyralidae), the key pest in other sugarcane growing areas. In the bioassays, E. loftini was substantially more susceptible to B. bassiana than D. saccharalis, based on both 5-d LD50 values and survival times. A commercial oil-based formulation of B. bassiana was evaluated in the field using the following treatments: oil alone (control), B. bassiana + oil, and B. bassiana + Silwet L-77 carrier at an application rate of 5 x 10(13) spores per hectare. Neither numbers of E. loftini per stalk, nor stalk damage (approximately 20% bored internodes) were significantly affected by treatment. The application of B. bassiana + Silwet significantly affected the numbers of internodes showing high damage, but not those with low or medium damage. Analysis of yield data and juice quality showed no significant treatment effects. We conclude that the application of Beauveria + Silwet offers the best chances for reducing damage caused by E. loftini of those treatments tested. However, reductions in insect incidence or damage did not result in measurable increases in yield or sugar quality, probably because of insufficient coverage. Effective control of stalkboring pyralids in sugarcane using B. bassiana will likely require improvements in delivery technology.