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.     

Effects of Fluctuating Moisture and Temperature Regimes on Sporulation of Beauveria bassiana on Cadavers of Rhodnius prolixus

The effects of both moisture and temperature on the sporulation of an isolate of Beauveria bassiana , highly virulent to the Chagas' disease vector, Rhodnius prolixus , were studied in the laboratory under fluctuating regimes. Quantitative assays for conidial production from funguskilled cadavers from 10-day-old tests showed that recycling was highly dependent on the duration of exposure to high relative humidity (RH). On first-instar larval cadavers, sporulation reached more than 10 5 conidia/nymph under most regimes with a favourable phase combining high humidity (97% RH) and temperatures of 20 or 25 C for at least 12 h/day. Sporulation on third- and fifth-instar nymphs required a longer period (16 h/day or more) of favourable conditions (97% RH and 25 C) per day. The intensity of the conidial production from Rhodnius cadavers was enhanced when diurnal temperatures were high (28 and 35 C). From a vector control standpoint, daily high humidity requirements appear to be a crucial constraint. The recycling ability of B. bassiana on fungus-killed R. prolixus is most likely to contribute to the regulation of this triatomine vector if applications are made during the rainy seasons and in the most favourable habitats.     

Infection of the Coffee Berry Borer Hypothenemus hampei (Coleoptera: Scolytidae) by Brazilian Isolates of the Entomopathogenic Fungi Beauveria bassiana and Metarhizium anisopliae (Deuteromycotina: Hyphomycetes)

The virulence of four fungal isolates (three Beauveria bassiana and one Metarhizium anisopliae ) against adult female coffee berry borers (CBB) was investigated. The most virulent isolate from initial bioassays, B. bassiana LPP1, with a LT 50 of 3.4 days, was further investigated by application to berries prior to infestation and to berries already infested with CBB. At the highest concentration applied to berries (1 ×10 7 conidia mL -1 ), CBB mortality was 83% (berries inoculated prior to infestation) and 62% (berries inoculated after infestation).     

Laboratory Evaluation of the Pathogenicity of Three Isolates of the Entomopathogenic Fungus Beauveria bassiana (Bals.) Vuillemin on the American Cockroach (Periplaneta americana)

Mycopesticides can be ideal for the biocontrol of cockroaches because the habitat of these insects promotes initial fungal infection and its subsequent spread. The pathogenicity of three isolates of Beauveria bassiana to the American cockroach was tested. The insects were treated in three different ways, by direct contact with spore mass, a spore-wheat flour mixture and a spray of an aqueous spore suspension. A mortality of 100% in the first treatment, 67-100% in the second treatment and 17-75% in the third treatment was observed. These results suggest that B. bassiana spore formulations in food baits can be developed for cockroaches.     

Generation of Benomyl Resistant Beauveria bassiana Strains and Their Infectivity Against Helicoverpa armigera

Beauveria bassiana transformants were obtained by conventional protoplasting and transformed by eletroporation and polyethylene glycol (PEG) treatment. These displayed mitotic stability in Beauveria bassiana . Strains transformed with pSV50 harbouring the β-tubulin gene of Neurospora crassa grew well on benomyl concentrations of 10 μg ml -1 unlike the recipient strain. The transformants were mitotically stable on either selective or non-selective medium. The efficiency of transformation by linear and circular pSV50 cosmid was 8 and 10 transformants per mug DNA per ml viable protoplast by electroporation, respectively, and 4 and 6 by the protoplast PEG method, respectively. Southern blot and hybridization of undigested fungal DNA of wild type and four transformants, probed with β-tubulin sequence of pSV50, showed hybridization at high M r region of genomic DNA in four transformants, whereas in wild type genomic DNA, no homology of the sequence was observed. Digested genomic DNA, of four transformants gave a complex hybridization pattern. Virulence tests of the transformants showed that there was no significant loss in the pathogenicity toward Helicoverpa armigera third instar larvae. This method of transformation should prove useful with entomopathogenic fungal species in which a genetic transformation system has not yet been established.     

Effects of Temperature, Humidity and Inoculation Method on Susceptibility of Schistocerca gregaria to Metarhizium flavoviride

The effects of temperature on conidial germination and susceptibility of adults of the desert locust , Schistocerca gregaria, to four isolates of Metarhizium flavoviride were determined . In addition , the effects of inoculation method (topical or spray) , spore carrier (oil or water) and ambient relative humidity (RH) on susceptibility of the locust to the most thermotolerant isolate (Mf324) were investigated . There were differences among the isolates in the effects of temperature on germination of conidia after a 24 - h incubation period . Over 90% of conidia of all isolates germinated after 24 h at 30 o C . In contrast , at 40 o C , none of the isolates germinated for up to 72 h . However , there were differences in germination between the isolates at 35 o C . Locust mortality and disease progression were significantly affected by temperature . At both 25 and 30 o C , all isolates induced 98 - 100% mortality within 8 days; however , there were differences between isolates at 35 o C . None of the isolates caused significant mortality at 40 o C . Humidity and inoculation method had no influence on levels of mortaility in fungus treated locusts . In contrast , carrier type significantly affected cumulative mortality . Topical oil treatment resulted in higher overall mycosis than the three other treatments . Control mortality on the other hand was strongly affected by inoculation method and to a lesser extent by humidity and carrier . In these conditions , application of oil by spray was generally toxic at all humidities whereas topical application of water was most toxic at near saturation . The results of these laboratory studies demonstrate the importance of strain selection , formulation and application method in the development of a microbial control agent against the desert locust . Low RH should not impede use of this fungus under dry conditions .     

Sporulation of Metarhizium anisopliae and Beauveria bassiana on Coptotermes formosanus and in vitro

The sporulation of 22 total isolates of Metarhizium anisopliae and Beauveria bassiana was quantified on cadavers of the Formosan subterranean termite, Coptotermes formosanus. Conidial production increased significantly over 11 days post-death. Effects of isolates of M. anisopliae and B. bassiana on in vivo sporulation were significant. Although the overall effects of fungal species on in vivo sporulation were not significant, the interactions between fungal species and certain times post-death were significant, indicating different sporulation patterns between the two fungal species. B. bassiana isolates could be categorized into a group with high total sporulation (day 11) and low quick sporulation (on days 2 and 3), while M. anisopliae isolates fell into another group with high quick sporulation and low total sporulation. This could give M. anisopliae an advantage over B. bassiana in termite microbial control due to termite defensive social behaviors. Conidial production was significantly higher in vitro than in vivo. In vitro and in vivo sporulation differed by as much as 89x and 232x among the selected isolates of B. bassiana and M. anisopliae, respectively. Correlation between in vivo and in vitro conidial production was positive and significant. This may allow preliminary in vitro screening of a large number of isolates for high in vivo sporulation.     

白僵菌、绿僵菌对雷公藤丽长角巢蛾的侵染力测定

经室内侵染力测定,球孢白僵菌Beauveria bassiana和金龟子绿僵菌Metarhizium anisopliae皆能侵染雷公藤丽长角巢蛾Xyrosaria lichneuta幼虫,不同菌株的侵染力存在显著差异。3株白僵菌中,Bb27菌株毒力最强,菌液为1×10^5～1×10^9个·mL^-1五个不同浓度梯度的孢子悬浮液时,害虫15d校正死亡率在58．3％-100％之间;LT50分别为9．36d,6．26d,3．89d、2．54d．2．10d;LC50为4．87×10^4个·ML^-1。3株绿僵菌中,MaZPTP01菌株毒力最强,5种孢子浓度15d校正死亡率分别为52．4％、69．0％、76．2、92．9％、95．2％;LT50分别为11．47d、6．89d、4．36d、2．68d、2．13d;LC50为7．39×10^4个·mL^-1。Bb27和MaZFIT01菌株可应用于林间作进一步防治试验。     

Diversity of indigenous Beauveria and Metarhizium spp. in a commercial banana field and their virulence toward Cosmopolites sordidus (Coleoptera: Curculionidae)

Metarhizium anisopliae and Beauveria bassiana sensu lato were isolated, from 7 and 41 % of soil samples from a commercial banana field, with average fungal density of 4.3 × 10³ and 8.2 × 10³ CFU g^?1 soil, respectively. Twenty-one morphologically distinct B. bassiana and four M. anisopliae sensu lato isolates from different plots within the field were further characterized. ISSR fingerprinting revealed six different clusters for B. bassiana, whereas gene sequencing revealed three M. anisopliae sensu stricto and one unclassified Metarhizium sp. Bioassays with one or more representative isolates from each Metarhizium species and B. bassiana cluster showed that all indigenous isolates had lower virulence and significantly greater ST₅₀s than reference (exotic) isolates. The data suggest that the low virulence of most indigenous isolates toward Cosmopolites sordidus adults and their relatively low density in soil samples, may help explain the low occurrence of epizootics caused by entomopathogenic fungi in populations of this pest, also known to burrow under the soil surface in banana plantations.     

Laboratory and Field Trials of Bait Formulations of the Fungal Pathogen, Metarhizium flavoviride, Against a Tropical Grasshopper and Locust

Extrusion technology was used to produce maize-starch 'contact bait' carriers, and vegetable-oil suspensions of Metarhizium flavoviride were incorporated into the baits after extrusion. Laboratory bioassays using the locust, Schistocerca gregaria, demonstrated mortality and reduction of 8 1 feeding after exposure to the baits, and gave an LC 50 of 1.5 10 spores l slope 0.63 for fifth instar nymphs. In laboratory trials, maize-starch contact baits showed improved efficacy relative to wheat-bran edible bait formulations of M. flavoviride. Field cage experiments, undertaken in Mali, using fourth and fifth instar nymphs of the grasshopper, Hieroglyphus dagenensis, demonstrated significant mortality with both fresh and weathered maize-starch contact bait formulations of M. flavoviride, relative to untreated controls.     

Comparative impact of artificial selection for fungicide resistance on Beauveria bassiana and Metarhizium brunneum

Hypocreales fungi such as Beauveria bassiana (Balsamo) Vuillemin and Metarhizium brunneum Petch can be negatively affected by fungicides thereby reducing their biocontrol potential. In a previous study, we demonstrated enhanced fungicide resistance in B. bassiana through artificial selection. However, it is not clear if the enhanced resistance was because of improved germination, vegetative growth, or both. Additionally, the enhanced fungicide resistance has only been demonstrated in B. bassiana, and therefore it is of interest to investigate the potential to enhance resistance in other fungi. Thus, the objectives in this study were to determine the potential to enhance fungicide resistance in M. brunneum through artificial selection, and investigate if selection is based on germination, vegetative growth, or both in B. bassiana and M. brunneum. Selection for resistance to fenbuconazole, and triphenyltin hydroxide was assessed through inhibition evaluations on solid media, and germination and mycelial growth in liquid media. Increased resistance after selection was observed for all fungicide-fungus combinations on solid and or liquid media. Selection resulted in increased resistance to fenbuconazole in both fungi in solid and liquid media; in liquid culture fungicide resistance in B. bassiana was manifested by increased germination and mycelial growth, whereas in M. brunneum fungicide resistance concerned only mycelial growth. Selection for resistance to triphenyltin hydroxide varied in the different media. For B. bassiana, triphenyltin hydroxide resistance was enhanced on solid media but not in liquid, whereas enhanced resistance of M. brunneum was detected in both media. Fungicide sensitivity and selection potential differs based on the medium and fungal species. Selection for fungicide resistance, had negative effects on other beneficial traits when fungicide pressure was removed, for example, some selected populations showed decreased germination or growth, relative to their nonselected control populations. Additionally, reduced virulence to the greater wax moth, Galleria mellonella (L.), was observed in all fungal populations that were exposed to fungicide resistance regimes. We conclude that it is possible to use genetic selection to enhance fungicide resistance in B. bassiana and M. brunneum, but before use the resulting populations should be screened for inadvertent negative impacts on beneficial traits.     

寄主和分布区系不同的球孢白僵菌菌株近缘关系的研究

观察、测定了来源不同的球孢白僵菌 1 9个菌株的培养性状、产孢量、萌发中时、水分活性、紫外照射活率、水浴活率等指标 ,将培养性状编码后 ,由所得的 8个指标对供试菌株求相关系数矩阵 .分析表明 ,不同菌株近缘关系的远近主要由菌株原始分布区系距离远近所决定 ,受寄主不同的影响次之 .来源于相同 (或相近 )地区的菌株相关系数可达极显著水平 ,而从相同寄主角度分析 ,得到菌株间的相关系数有高有低 ,多表现为相关不显著     

Effect of temperature on virulence of Beauveria bassiana and Metarhizium anisopliae isolates to Tetranychus evansi

The virulence of three isolates of Beauveria bassiana (Bals.) Vuill. and 23 isolates of Metarhizium anisopliae (Metschnik.) Sorok. (Ascomycota: Hypocreales) against the tomato spider mite, Tetranychus evansi Baker and Pritchard (Acari: Tetranychidae), was assessed in the laboratory. The effect of temperature on germination, radial growth and virulence of selected isolates (two isolates of B. bassiana and nine of M. anisopliae) on T. evansi was also investigated in the laboratory. All the fungal isolates tested were pathogenic to the adult females of T. evansi, and there were significant differences in mortality between fungal isolates. The lethal time to 50% mortality (LT(50)) values ranged from 4.2 to 8.1 days and the LT(90) values from 5.6 to 15.1 days. Temperature had significant effects on germination, radial growth and virulence of the various isolates. The best fungal germination was observed at 25 and 30 degrees C, while for the fungal radial growth it was 30 degrees C. All the isolates germinated and grew at all temperatures, but germination and radial growth varied with isolate and temperature. The selected isolates were all virulent to T. evansi, but virulence varied also with isolate and temperature.     

Characterization of Beauveria bassiana and Metarhizium anisopliae isolates for management of tarnished plant bug,Lygus lineolaris (Hemiptera: Miridae)

Selected morphological and physiological characteristics of four Beauveria bassiana (Balsamo) Vuillemin isolates and one Metarhizium anisopliae (Metschnikoff) Sorokin isolate, which are highly pathogenic to Lygus lineolaris (Palisot de Beauvois) (Hemiptera: Miridae), were determined. There were significant differences in conidial size, viability, spore production, speed of germination, relative hyphal growth, and temperature sensitivity. Spore viability after incubation for 24h at 20 degrees C ranged from 91.4 to 98.6% for the five isolates tested. Spore production on quarter-strength Sabouraud dextrose agar plus 0.25% (w/v) yeast extract after 10 days incubation at 20 degrees C ranged from 1.6x10(6) to 15.5x10(6)conidia/cm(2). One B. bassiana isolate (ARSEF 1394) produced significantly more conidia than the others. Spore germination was temperature-dependant for both B. bassiana and M. anisopliae. The time required for 50% germination (TG(50)) ranged from 25.0 to 30.9, 14.0 to 16.6, and 14.8 to 18.0h at 15, 22, and 28 degrees C, respectively. Only the M. anisopliae isolate (ARSEF 3540) had significant spore germination at 35 degrees C with a TG(50) of 11.8h. A destructive sampling method was used to measure the relative hyphal growth rate among isolates. Exposure to high temperature (40-50 degrees C) for 10min had a negative effect on conidial viability. The importance of these characteristics in selecting fungal isolates for management of L. lineolaris is discussed.     

Mortality in Three African Tephritid Fruit Fly Puparia and Adults Caused by the Entomopathogenic Fungi, Metarhizium anisopliae and Beauveria bassiana

The pathogenicity of 13 isolates of Metarhizium anisopliae and two isolates of Beauveria bassiana to Ceratitis capitata and Ceratitis var. rosa fasciventris exposed as late third instar larvae in sand was evaluated in the laboratory. All isolates caused a significant reduction in adult emergence and a corresponding large mortality on puparia of both species. All isolates also induced large deferred mortality in emerging adults following treatment as late third instar larvae. On C. capitata , seven isolates ( M. anisopliae ICIPE 18, 20, 32, 60 and 69 and B. bassiana ICIPE 44 and 82) caused significantly higher mortality on puparia than other isolates. With the exception of ICIPE 32, the other four isolates of M. anisopliae above were the most pathogenic against C. r. fasciventris . Dose-response study carried out with these isolates of M. anisopliae on the two species of flies above plus another species, Ceratitis cosyra showed that the dose-mortality regression lines of ICIPE 18 and 20 were steeper with lower LC 50 values when compared with ICIPE 60 and 69 on the three species. When these two isolates were evaluated with regard to their pathogenicity to different pupal age, adult emergence was found to increase with increasing pupal age with a corresponding decrease in mortality in puparia and emerging adults in the three species of fruit flies. M. anisopliae ICIPE 18 and 20 were equally pathogenic to all pupal ages tested in C. capitata and C. cosyra but ICIPE 18 was more pathogenic to older puparia of C. r. fasciventris than ICIPE 20. Our results suggest that soil inoculation with M. anisopliae under mango trees might form an important component of integrated pest management strategies in areas where these three species of fruit fly coexist.     

Laboratory evaluation of entomopathogenic fungi Beauveria bassiana and Metarhizium anisopliae against puparia and adults of Ceratitis capitata (Diptera: Tephritidae)

Laboratory experiments were done to measure the pathogenicity of 10 autochthonous isolates of Beauveria bassiana (Balsamo) Vuill. and of five Metarhizium anisopliae (Metsch.) Sorok. toward puparia and adults of the Mediterranean fruit fly, Ceratitis capitata (Wiedemann) (Diptera: Tephritidae). Although all isolates applied via inoculation of the fungal suspensions on the ventral surface of the abdomen were pathogenic to adults, with mortality rates ranging from 30 to 100% and average survival times (ASTs) from 6.5 to 8.6 d, when C. capitata puparia were immersed in the conidial suspensions, only B. bassiana Bb-1333 and EABb 01/103-Su and M. anisopliae EAMa 01/58-Su isolates caused >50% mortality of puparia. In a second series of bioassays conducted on five selected isolates, adults were sprayed with four 10-fold concentrations ranging from 1.0 x 10(5) to 1.0 x 10(8) colony-forming units (cfu)/ml. The median lethal concentrations (LC50) of the four most virulent isolates ranged from 4.9 x 10(5) to 2.0 x 10(6) cfu/ml with estimated time to kill 50% of the insects ranging from 4.6 to 5.3 d. The effect of a sublethal dose (ca. LD50) of either B. bassiana EABb 01/103-Su or M. anisopliae EAMa 01/58-Su isolate was studied by reciprocal crossing. Treatment with B. bassiana reduced fecundity and fertility at 6, 8, and 10 d after treatment, with fecundity and fertility reductions ranging from 20.0 to 71.2% and from 33.6 to 60.0%, respectively. These reductions occurred in pairing combinations of treated females with either treated or nontreated males. M. anisopliae was more effective in reducing fecundity and fertility at 6 d after treatment, with the reduction varying from 58.4 to 72.1% and from 28.6 to 45.9%, respectively. In addition, the first oviposition was significantly delayed for 1 d in females treated by either fungal species. The above-mentioned five selected isolates were assayed against C. capitata puparia treated as late third instars in sterilized soil at 25'C under three moisture conditions (-0.1, -0.01, and -0.0055 MPa). At -0.01 MPa, all isolates were low pathogenic to C. capitata puparia, whereas significant differences in the puparia mortality occurred between isolates at -0.1 and -0.0055 MPa. The highest pupal mortalities ranged from 52.5 to 70.0%, as a function of soil moisture and were caused by EAMa 01/58-Su and Bb-1333 isolates.     

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.     

Effects of Beauveria bassiana and Metarhizium anisopliae on mortality, fecundity and egg fertility of Tetranychus evansi

Abstract:  The susceptibility of various developmental stages of Tetranychus evansi Baker & Pritchard (eggs, larvae, protonymphs, deutonymphs and adults) to the entomopathogenic fungi Metarhizium anisopliae (Metschnikoff) Sorokin and Beauveria bassiana (Balsamo) Vuillemin was evaluated under laboratory conditions. Three concentrations (3.0 × 10⁶, 1.0 × 10⁷ and 1.0 × 10⁸ conidia/ml) of both fungi were used for each stage. The effect of fungal infection on fecundity and egg fertility was also investigated using both fungal species. Deutonymphs that survived the infection and developed into adult females were allowed to oviposit. Adults and deutonymphs were more susceptible to fungal infection than larval and protonymphal stages at all the concentrations. Nevertheless, the concentration level influenced the mortality of the different mite stages. Eggs were also susceptible to fungal infection and mortality was dose-dependent. Fungus-treated female mites laid fewer eggs than the controls but there was no significant difference in egg hatchability between the treatments.     

Evaluation of Beauveria brongniartii and its Metabolite Oosporein Regarding Phytotoxicity on Seed Potatoes

The phytotoxic potential of Beauveria brongniartii and its main secondary metabolite oosporein were evaluated against seed potatoes ( Solanum tuberosum ) both in vitro and in situ . In a tray test, potatoes were inoculated using conidiospores and grown either in garden soil or in B. brongniartii -enriched soil. The only significant effect of the treatment was the fact that the weight of the roots was different compared with the controls. The weight of haulm and tubers was unaffected by the treatment with B. brongniartii . No oosporein was detected in the potatoes, although the density of B. brongniartii in the soil was forty times higher than recommended for the usage of this biological control agent (BCA). This indicates that oosporein does not enter the food chain via potato tubers. The effect of BCA on seed potatoes was also tested in situ . No B. brongniartii was detected in the potato field before the application of BCA. Three months after application, an average of 3 ×10 4 colony forming units per gram soil (dry weight) were recorded. Although Melolontha melolontha larvae damaged 60% of the potatoes, only 3% of the tissue samples were infected with B. brongniartii . The results obtained in this study suggest that B. brongniartii and its secondary metabolite oosporein pose no risks to potato plants, tubers, or to their consumers.