Interaction Between the Entomopathogens Beauveria bassiana, Metarhizium anisopliae and Paecilomyces fumosoroseus and the Mycoparasites Clonostachys spp., Trichoderma harzianum and Lecanicillium lecanii

Biocontrol agents of numerous insect pests and fungal pathogens exist but virtually nothing is known about their interaction if used simultaneously. Our objective was to investigate the compatibility of the entomopathogens Beauveria bassiana, Metarhizium anisopliae and Paecilomyces fumosoroseus, and the broad host-range mycoparasites Clonostachys spp., Trichoderma harzianum and Lecanicillium lecanii. In vitro host-range tests revealed that M. anisopliae was highly susceptible to all mycoparasites tested. B. bassiana was attacked by Clonostachys rosea, and P. fumosoroseus. was resistant to mycoparasites. M. anisopliae but not P. fumosoroseus killed nymphs of Bemisia tabaci in bioassays. B. bassiana and M. anisopliae proved lethal to Cosmopolites sordidus, Diatraea saccharalis and Sitophilus oryzae. Coapplication of mycoparasites with entomopathogens did not affect their biocontrol efficacy in vivo, although the reisolation success of entomopathogens could be significantly reduced, especially from smaller insect species. Trichoderma spp. were reisolated from mycoparasite-treated insects more frequently than C. rosea. The coapplication of the highly susceptible M. anisopliae generally enhanced mycoparasite recovery. Mycofungicide preparations caused some insect mortality but less than a copper hydroxide fungicide which is still permissible in organic agriculture. We concluded that the tested entomopathogens and mycoparasites are compatible elements of integrated pest management.     

Integrated Management of Two-spotted Spider Mite Tetranychus urticae on Hops using Hop ß-acids as an Antifeedant Together with the Predatory Mite Phytoseiulus persimilis

The effects on population development of two-spotted spider mite Tetranychus urticae by sprays of hop β-acids, a repellent and antifeedant of T. urticae, together with an inoculative release of the predatory mite Phytoseiulus persimilis were compared on tall and dwarf hops. The combination of the semiochemical with the predator was more effective than either factor singly on both hop types. The highest numbers of spider mites and their eggs were recorded on untreated plots. The sprays of β-acids slowed the rate of increase of the pest and contributed more to the effect of the combined treatment early in the season when the predator was establishing itself on the crop. By harvest time, the predator was the more influential of the two factors.     

Laboratory investigations on the potential of entomopathogenic fungi for biocontrol of Helicoverpa armigera (Lepidoptera: Noctuidae) larvae and pupae

The susceptibility of third instar Helicoverpa armigera to seven strains of three entomopathogenic fungal species, i.e. Metarhizium anisopliae, Beauveria bassiana and Paecilomyces fumosoroseus, was tested under laboratory conditions using the larval immersion method. High efficacies ranging from 68 to 100% corrected mortality were recorded with more profound effects in treatments with B. bassiana and P. fumosoroseus strains. The median lethal concentration (LC₅₀) for L3 was 6.0×10⁵ in M. anisopliae 79, 1.5×10⁵ in B. bassiana 124 and 4.2×10⁴ in P. fumosoroseus 14. These three strains were further used to characterize the age-dependent mortality of different larval stages (L2-L5) and the effect against pupae of H. armigera. Larval stages did not differ in their mortality but differed i in median lethal time, with shorter values recorded in the second instar. Tested fungi also caused a high reduction between 74.4 and 100% in the emergence of pupae using the soil inoculation method and the pupal immersion technique. All three fungal species, especially P. fumosoroseus, have a high potential for biocontrol of H. armigera larvae and also as a soil treatment targeting the pupae.     

Autodissemination of Beauveria bassiana by Sap Beetles (Coleoptera: Nitidulidae) to Overwintering Sites

An autoinoculative device was used to test the ability of sap beetles (Coleoptera: Nitidulidae) to carry a specific strain of Beauveria bassiana (Balsamo) Vuillemin to overwintering sites in a multiyear field study. The device was baited with the pheromone and coattractants for the dusky sap beetle (Carpophilus lugubris Murray) and placed in the field in the fall of each year. The introduced strain occurred at high frequency among the B. bassiana isolated in the fall of all four years tested (100% of all isolates from 21 of 22 collection dates). The introduced strain of B. bassiana was isolated at high frequency from all the B. bassiana-contaminated sap beetles recovered from the overwintering traps (100% of all isolates from 13 of 23 trap/beetle species combinations) and was highest after the longest fall exposure. The introduced strain was primarily isolated from C. lugubris and C. antiquus, but species distribution was also dependent on the overwintering trap design used. Few non-sap beetles species of insects were recovered from the artificial overwintering sites. Although B. bassiana was isolated from free flying sap beetles caught in traps in the spring of each year, none were infected with the introduced strain. The autoinoculating device provides selective contamination of sap beetles in overwintering sites when used in the fall. It may be useful in providing some control of sap beetles or other insects where limited numbers of mass overwintering sites (such as tree holes) occur.     

Do aphids infected with entomopathogenic fungi continue to produce and respond to alarm pheromone?

The response of pea aphids, Acyrthosiphon pisum, to aphid alarm pheromone was not modified by infection with Beauveria bassiana. Approximately 50% of uninfected and infected aphids responded to synthetic alarm pheromone. The simulated attack of aphids infected with B. bassiana did not elicit a response in uninfected aphids. Preliminary air entrainment experiments of both uninfected aphids and aphids at different stages of B. bassiana (generalist pathogen) or P. neoaphidis (obligate pathogen of aphids) demonstrated that B. bassiana infected aphids produced less alarm pheromone than uninfected aphids and, conversely, P. neoaphidis infected aphids produced more alarm pheromone than uninfected aphids. These results are discussed with particular emphasis on the different life history strategies of these two pathogens. We hypothesise that the obligate, specialist pathogen, P. neoaphidis, is under greater selection pressure to increase pathogen transmission and survival resulting in modified host behaviour, than the generalist pathogen, B. bassiana.     

Whey for mass production of Beauveria bassiana and Metarhizium anisopliae

Spore production of Beauveria bassiana and Metarhizium anisopliae was studied in a novel whey-based culture media. Spore yield and viability were determined for two B. bassiana (GHA-726 and CA-603) and two M. anisopliae (CA-1 and IMI 330189) isolates following production in three whey-based systems: solid, liquid, and a diphasic production system. Our study indicated that whey permeate can be used effectively for production of spores of entomopathogenic fungi. However, spore yield and viability were significantly influenced by fungal isolate, whey concentration, and the type of production process used. Under the conditions defined in the present study, spore yields ranging from 1.3 × 10⁹–10 × 10¹¹ spores l⁻¹ of whey medium could be obtained depending on the strain and production process used. Our study revealed that spores produced by all strains in whey-based solid and liquid media showed between 73–99 % viability; germination rates were comparable with those obtained using the standard SDA medium. In the two-stage production process, the viabilities of conidia produced by GHA-726, CA-603, and CA-1 were 35–86, 32–98, and 6–29 %, respectively; viability was correlated with whey concentration and isolates. Whey permeate can be used as a growth substrate for mass production of biocontrol fungi. We hypothesize that spore yield and viability could be improved by careful selection of whey content in the medium, incorporation of critical additives and optimization of culture conditions.     

Biochemical and molecular characteristics of indigenous strains of the entomopathogenic fungus Beauveria bassiana of Central India

Forty-eight isolates of indigenous strains of Beauveria bassiana from various insect hosts collected from Central India were characterised by employing protease zymography and RAPD analysis. Results of protease zymographic profiles were reproducible and significant enough to contribute to the biochemical diversity of this species. RAPD analysis revealed the presence of high level of genetic diversity and indicated that 0-66% significant differences has evolved between these isolates. The sets of amplified bands showing identical pattern to others were grouped at 100% similarity level. A wide range in the value (0.25-1.00) of Jaccard similarity coefficient was observed among all the isolates. The grouping of the indigenous strains, obtained from numerical analysis of these data, appears to be related to the host specificity in B. bassiana. Clear groups were seen for strains isolated from Lepidopteran and Coleopteran insect hosts.     

Horizontal Transmission of the Entomopathogenic Fungus Beauveria bassiana among the Spruce Bark Beetle, Ips typographus (Col., Scolytidae) in the Laboratory and under Field Conditions

In laboratory and field cage experiments, the horizontal transmission of the entomopathogenic fungus Beauveria bassiana (Boverol®) between adults of the spruce bark beetle Ips typographus was examined. In a laboratory experiment, conidia transfer between treated and untreated beetles at different ratios resulted in 96% mortality (MST 4.3 days) at a ratio of 1:1 and 90% mortality (MST 4.5 days) at a ratio of 1:2. At ratios of 1:5, 1:10 and 1:20, the mortalities at 7 days were 83, 77 and 75%, respectively. In a second laboratory experiment, the efficacy of B. bassiana was investigated after transmission from contaminated to healthy beetles over a period of 5 days. Two weeks later, the mortality of treated and untreated beetles was 99% (control mortality was 44%), while the rate of mycosis was 79% in the treatment compared to 10% in the control beetles. A single contact between one treated male (2.0×10⁵ conidia/beetle) and an untreated female was found to be sufficient to transmit a lethal dose of B. bassiana of about 1.2×10⁴ conidia. Two experiments in field cages in a spruce stand demonstrated horizontal transmission of B. bassiana (Boverol®) between beetles. In the first experiment the transmission was investigated following the introduction of B. bassiana-inoculated beetles into a population of untreated beetles. Significant reductions were observed in the length of maternal galleries, and the number of larvae and pupae. In the second field cage experiment, natural populations of beetles were lured into a flight cage using a pheromone trap, and treated with B. bassiana or left untreated on alternate days. In this experiment, significant reductions were observed in the number of bore holes and the length of maternal galleries, while no larvae, pupae or juveniles were found under the bark of spruce trunks in the Boverol® transmission treatment. It is concluded that B. bassiana has potential for control of I. typographus, and further field experiments are warranted to investigate its efficacy under more practical conditions.     

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.     

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.     

Development of Beauveria bassiana dry formulation for vectoring by honey bees Apis mellifera (Hymenoptera: Apidae) to the flowers of crops for pest control

Using bee pollinators as a means for the dissemination of microbial control agents, such as Beauveria bassiana, against insect pests of agricultural crops is a novel and interesting approach to biological control. In four laboratory trials, one in Canada and three in Jordan, factors affecting the acquisition of B. bassiana by honey bees were evaluated using hive-mounted inoculum dispensers. The numbers of conidia carried by bees emerging from the dispensers differed according to the type of carrier used. Bees that passed through corn flour acquired more inoculum than did those that walked through wheat flour, durum semolina, corn meal, potato starch, potato flakes, oat flour or barley flour. The numbers of conidia acquired by the bees increased with decreasing particle size and moisture content of the carrier, and with increasing density of B. bassiana conidia in the formulation. Time required for a bee to pass through the dispenser did not significantly affect the acquisition of conidia. This study indicated that honeybees (Apis mellifera carnica) have a great potential for vectoring B. bassiana in crop systems. It also opens more avenues for studies on bee delivery of other microbial biological control agents.     

Microbial metabolism of 1-aminoanthracene by Beauveria bassiana

The carcinogen and mutagen, 1-aminoanthracene, was efficiently metabolized by the fungal strain Beauveria bassiana ATCC 7159 to yield three new metabolites identified as 1-acetamido-5-[(4′-O-methyl-β-d-glucopyranosyl)oxy]anthracene, 1-acetamido-8-[(4′-O-methyl-β-d-glucopyranosyl)oxy]anthraquinone, and 1-acetamido-6-[(4′-O-methyl-β-d-glucopyranosyl)oxy]anthraquinone, together with 1-acetamidoanthracene and 1-acetamidoanthraquinone. Formation of these metabolites suggests that the metabolic pathways of 1-aminoanthracene in B. bassiana ATCC 7159 involve acetylation, oxidation, hydroxylation, and O-methylglucosylation.     

4种虫草相关真菌菌丝体粗多糖的生物活性评价

本研究以细脚棒束孢、蛹虫草、蝉棒束孢和球孢白僵菌的菌丝体粗多糖为对象,分析4种虫草相关真菌菌丝体粗多糖含量与生物量的相关性,并进一步对其抗氧化能力和抗肿瘤活力进行评价。液体发酵结果表明,蝉棒束孢MF12、MF13和蛹虫草MF27、MF1的菌丝体粗多糖含量（>40mg/g）显著高于其他菌株,蝉棒束孢MF11、MF13和蛹虫草MF27菌丝体生物量（>12g/L）显著高于其他菌株,但相关性分析表明,4种虫草相关真菌10个菌株菌丝体的多糖含量与生物量之间没有显著相关性;抗氧化活性表明,蛹虫草MF27、MF1和球孢白僵菌MF10菌丝体粗多糖具有良好的体外抗氧化活性,其EC₅₀均小于0.9mg/mL;抗肿瘤活性表明,蛹虫草MF1、MF28、MF27和球孢白僵菌MF10菌丝体粗多糖在体外能有效抑制HepG-2细胞增殖,其IC₅₀均小于1.5mg/mL。综上,蛹虫草MF27、MF1和球孢白僵菌MF10虫草菌株具有良好的开发和应用潜力。     

小菜蛾应对球孢白僵菌感染的免疫应答基因分析

通过室内生物测定,筛选对小菜蛾Plutella xylostella具有高毒力的球孢白僵菌Beauveria bassiana菌株,分析小菜蛾应对球孢白僵菌浸染的免疫应答基因。采用新一代Illumina高通量测序技术对接种/感染48h与未接种的小菜蛾样品分别进行转录组测序,筛选差异表达基因;结合生物信息学分析差异表达基因的功能、分类及涉及的信号通路等。结果表明接种小菜蛾48h后诱导2 434个基因发生差异表达,包括1 080个上调基因和1 354个下调基因。GO分类结果表明,这些差异表达基因（DEGs）被注释到45个GO term中,包括23个生物学过程,12个细胞组分和10个分子功能。KEGG分析表明,1 308个DEGs被富集到25个功能途径,这些DEGs包括497个上调基因和811个下调基因。分析发现,DEGs编码蛋白包含肽聚糖识别蛋白、丝氨酸蛋白酶55、丝氨酸蛋白酶抑制剂以及细胞色素P450 6K1类的免疫相关蛋白。另外,组蛋白、磷酸泛酰半胱氨酸脱羧酶、双链RNA结合蛋白、黑芥子酶等基因在球孢白僵菌侵染过程中也高表达,推测这些基因可能参与小菜蛾应对球孢白僵菌侵染的免疫反应。研究结果为进一步研究小菜蛾免疫相关基因的功能奠定了基础。     

Toxicity of bifenazate and its principal active metabolite, diazene, to Tetranychus urticae and Panonychus citri and their relative toxicity to the predaceous mites, Phytoseiulus persimilis and Neoseiulus californicus

Bifenazate is a novel carbazate acaricide discovered by Uniroyal Chemical (now Chemtura Corporation) for the control of phytophagous mites infesting agricultural and ornamental crops. Its acaricidal activity and that of its principal active metabolite, diazene, were characterized. Bifenazate and diazene had high toxicity and specificity both orally and topically to all life stages of Tetranychus urticae and Panonychus citri. Acute poisoning was observed with no temperature dependency. No cross-resistance was found to mites resistant to several other classes of acaricides, such as tebufenpyrad, etoxazole, fenbutatin oxide and dicofol. Bifenazate remained effective for a long time with only about a 10% loss of efficacy on T. urticae after 1 month of application in the field. All stages of development of the predatory mites, Phytoseiulus persimilis and Neoseiulus californicus, survived treatment by both bifenazate and diazene. When adult females of the two predatory mite species were treated with either bifenazate or diazene, they showed a normal level of fecundity and predatory activity in the laboratory, effectively suppressing spider mite population growth. Even when the predators were fed spider mite eggs that had been treated previously with bifenazate, they survived. These findings indicate that bifenazate is a very useful acaricide giving high efficacy, long-lasting activity and excellent selectivity for spider mites. It is, therefore, concluded that bifenazate is an ideal compound for controlling these pest mites.     

Effect of Plant Type on the Persistence of Beauveria bassiana

Field evaluation of isolate MK 2001 of the entomopathogenic fungus Beauveria bassiana (Balsamo) Vuillemin, revealed conidia persistence and infectivity up to 26 days on foliage of lettuce and celery, two crops with substantial plant differences. Plants were treated at a rate of 100 ml/cage at a concentration of 1×10⁸ conidia/ml. The number of colony-forming units (CFU) recovered immediately after treatments from discs sampled on lettuce and celery leaves were not significantly different, due to similar foliar coverage of conidia by application. Significant differences were observed between both host plants during the 26 days of the field trial. The number of CFUs recovered on lettuce was significantly higher than that on celery leaves. However, for each host plant, there were no significant differences either between lettuce external leaves and internal leaves or between celery canopy and bottom leaves. An in vitro pathogenicity test carried out on Lygus lineolaris adults fed with leaf discs harvested from treated cages resulted in a high pathogenicity of B. bassiana isolate MK 2001. The mortality immediately after treatment did not differ from the death counts taken every other day for 26 days. However, efficacy was significantly different between both plants. Mortality was 91% on lettuce and 78% on celery, 7 days post-treatment. This study highlighted that plant type must be taken into account in foliar application of B. bassiana.     

Biocontrol of Acanthoscelides obtectus and Sitophilus oryzae with diatomaceous earth and Beauveria bassiana on stored grains

Combined treatment with Beauveria bassiana, and diatomaceous earth (DE) was evaluated against the bean weevil Acanthoscelides obtectus and rice weevil Sitophilus oryzae. DE from Argentina was screened both alone or in combination with water or dry fungal formulations. DE killed 100% of A. obtectus and 68% of S. oryzae showing a significantly higher insecticidal effect than the fungal dust. For A. obtectus, median lethal time (MLT) with the DE-dry fungus was significantly lower than with a fungal aqueous-suspension. In S. oryzae, powder formulations with either of B. bassiana or DE showed a MLT significantly higher than the wet treatments.     

印楝素农药与虫生真菌混用防治红树林鳞翅目害虫

采用2种印楝素农药1%印楝素苦参碱乳油和0.3%印楝素乳油,以及2个虫生真菌绿僵菌菌株和白僵菌菌株对桐花长卷蛾、棉古毒蛾和广州小斑螟等3种红树林叶面和种子害虫的室内毒力测定结果表明,两种印楝素农药的单剂(500～1000倍)和与虫生真菌的复配剂(700倍),均对害虫有较强的毒性,一周内害虫死亡率达78.6～100%.两种印楝素农药与虫生真菌混合使用均比各自单独使用的杀虫效果好,其中与虫生真菌的复配剂700倍液的杀虫最快,效果最好,第3天柑橘长卷蛾和广州小斑螟的死亡率达100%,棉古毒蛾的死亡率达93.3%.应用1%印楝素苦参碱乳油和0.3%印楝素乳油与绿僵菌和白僵菌的复配剂500倍液进行林间防治危害桐花树的柑橘长卷蛾的试验,防治效果达65.4%～100%,该复配剂具有内吸传导作用、高效、低毒和安全等优点,值得大力推广应用.     

基于SSR标记的琅琊山球孢白僵菌寄主专化性

球孢白僵菌Beauveria bassiana是一类常见的昆虫病原真菌,其自然侵染的寄主昆虫众多,达15目149科750种。为了解球孢白僵菌自然种群的遗传多样性,探讨种群异质性和寄主来源之间的关系,分析其寄主专化性的强弱,本研究利用SSR分子标记技术,比较了安徽琅琊山国家森林公园的85株球孢白僵菌（寄主种类涉及7目24种）群体遗传多样性差异,通过构建聚类树分析菌株基因型和寄主关联性。结果表明琅琊山球孢白僵菌群体Nei’s基因多样性指数h=0.2906,Shannon信息指数I_s=0.4510,多态位点百分率P为100%。不同寄主目球孢白僵菌遗传多样性水平由高至低为鞘翅目>膜翅目>同翅目>双翅目>鳞翅目>直翅目>半翅目,其中菌株数量较多的鞘翅目、膜翅目和同翅目3个亚种群的遗传多样性较高且水平接近。聚类分析发现8对SSR引物将85株球孢白僵菌分成29个基因型,并在遗传相似系数0.70处分别聚为3个分支。分析寄主类型发现相同基因型的株系可侵染不同目的寄主,而同一类型寄主也可被不同基因型的菌株侵染。球孢白僵菌种群的总体遗传多样性较高,遗传谱系与寄主来源无明显相关性,菌株的寄主专化性弱。     

Potential for using Lecanicillium lecanii for suppression of strawberry powdery mildew

Augmenting native populations of the hyperparasite Lecanicillium lecanii suppressed powdery mildew of strawberry, caused by Sphaerotheca macularis f. sp. fragariae in California field trials. Repeated sprays significantly reduced disease compared to the untreated controls for periods of the fruit production season, suggesting possible use as a partial, but not total disease control strategy.