Field studies of control of Anoplophora glabripennis (Coleoptera: Cerambycidae) using fiber bands containing the entomopathogenic fungi Metarhizium anisopliae and Beauveria brongniartii

The Asian longhorned beetle, Anoplophora glabripennis, was first found attacking urban street trees in the United States in 1996 and in Canada in 2003. This tree-killing invasive insect has long been a major pest in China and is difficult to control because immature stages live within wood and long-lived adults are often located high in tree canopies. A microbial control product (Biolisa Kamikiri) consisting of non-woven fiber bands impregnated with cultures of an entomopathogenic fungus, Beauveria brongniartii, is marketed in Japan for control of a congeneric orchard pest. Replicated field trials were conducted in Anhui, China to compare Biolisa Kamikiri with similarly prepared bands containing Metarhizium anisopliae for control of A. glabripennis. One fungal band was placed at 2–2.5 m height, around the stem or major scaffold branch on each of 40 willow trees (Salix spp.) per plot, with five plots for each fungal treatment and five control plots. Adult beetles collected from fungal-treated plots 7–22 days after bands were attached to trees died faster than adults from control plots. Beetles exposed to B. brongniartii bands consistently died faster than controls throughout this period, while results from plots with M. anisopliae bands were not as consistent in differing from controls. Numbers of adult beetles from plots of each fungal species dying in <10 days were greater than controls (16% of beetles) but did not differ between fungal treatments (34–35%). Oviposition in fungal-treated plots was approximately half that in control plots. Locations of adult beetles and oviposition scars within tree canopies were quantified to determine optimal locations for band placement. Most adult beetles were found >3.5-m high in trees, with adults in B. brongniartii-treated plots higher within trees than adults in other plots.     

Virulence of entomopathogenic hypocrealean fungi infecting <Emphasis Type="Italic">Anoplophora glabripennis</Emphasis>

Twenty isolates of four species of entomopathogenic hypocrealean fungi (Beauveria bassiana, Beauveria brongniartii, Isaria farinosa, and Metarhizium anisopliae) were found to be pathogenic to adults of the Asian longhorned beetle, Anoplophora glabripennis. Survival times for 50% of the beetles tested (ST₅₀) ranged from 5.0 (M. anisopliae ARSEF 7234 and B. brongniartii ARSEF 6827) to 24.5 (I. farinosa ARSEF 8411) days. Screening studies initially included strains of B. brongniartii, which is registered as a microbial control agent in Europe, Asia and South America but not in North America. At that time, we could not confirm that this fungal species is native to North America which added uncertainty regarding future registration of this species for pest control in the USA. Therefore, subsequent bioassays documented median survival times for three M. anisopliae isolates (5–6 days to death) and two of these isolates are suggested for further development because they are already registered for pest control in the USA. An erratum to this article can be found at     

Reduction in fitness of female Asian longhorned beetle (Anoplophora glabripennis) infected with Metarhizium anisopliae

Bioassays were conducted to document the effects of Metarhizium anisopliae infection on adult female Asian longhorned beetle (Anoplophora glabripennis) reproduction before death and subsequent survival of offspring. The effect of infection on fecundity was evaluated for females already laying eggs and for newly eclosed females using M. anisopliae isolates ARSEF 7234 and 7711, respectively. Decreased longevity and oviposition compared with controls were observed in females that were already laying eggs when exposed to M. anisopliae ARSEF 7234. Newly eclosed females exposed to M. anisopliae ARSEF 7711 displayed shortened longevity (10.0 ± 0.7 days vs 74.3 ± 6.8 days for controls) and decreased oviposition (1.3 ± 0.7 eggs per ARSEF 7711-exposed female vs 97.2 ± 13.7 eggs per female for controls) compared with controls. Percentages of eggs that did not hatch were greater for both groups of fungal-treated females compared with controls and 60.0% of unhatched eggs contained signs of fungal infection. The percentage of larvae dying within 9 weeks of oviposition was higher for sexually mature females exposed to ARSEF 7234 compared with controls and >40% of dead larvae displayed signs of fungal infection. Thus, for both stages of females and both fungal isolates, fewer surviving larvae were produced after female fungal infection compared with controls. M. anisopliae infection affects female fitness by decreasing female longevity, by decreasing female oviposition before death and through horizontal transmission of M. anisopliae to offspring.     

Evaluation of Beauveria bassiana and B. brongniartii strains and four wild-type fungal species against adults of Bactrocera oleae and Ceratitis capitata

The virulence of two isolates of the hyphomycete fungi, Beauveria bassianaand B. brongniartii, and additional fungal species isolated from diseased Bactrocera oleae pupae and Sesamia nonagrioideslarvae were assessed against adults of the olive fruit fly B. oleae and the Mediterranean fruit fly Ceratitis capitata (Diptera: Tephritidae). Contact and oral bioassays revealed that moderate to high mortality rates for the olive fruit fly occurred when the adults were exposed to conidia of Mucor hiemalis, Penicillium aurantiogriseum, P. chrysogenum and B. bassianaisolates. A strain of M. hiemalis isolated from S. nonagrioides larvae was the most toxic resulting in 85.2% mortality to the olive fruit fly adults. B. brongniartiiand B. bassiana were the most pathogenic to the C. capitataadults causing 97.4 and 85.6% mortality. Metabolites collected from the M. hiemalis and P. chrysogenum isolates were toxic to adults of both species.     

Comparing virulence of North American Beauveria brongniartii and commercial pathogenic fungi against Asian longhorned beetles

In the USA, the development and field application of Beauveria brongniartii (Sacc.) Petch (Hypocreales: Clavicipitaceae) to control the invasive Asian longhorned beetle, Anoplophora glabripennis (Motschulsky) (Coleoptera: Cerambycidae) have been hampered because it was unknown whether this fungal species is native to North America. With the recent confirmation of the occurrence of B. brongniartii in North America there is renewed interest in this species, particularly as it is an effective pathogen of cerambycids in Japan. However, based on partial sequences of the nuclear intergenic BLOC region the commercially available B. brongniartii strain NBL 851 (Idemitsu Kosan, Tokyo, Japan) belongs instead to the species Beauveria asiatica Rehner and Humber. Further, bioassays using two inoculation methods confirmed that commercially available strains of B. asiatica (NBL 851) and Metarhizium brunneum (F52) (Hypocreales: Clavicipitaceae) were significantly more virulent and resulted in lower median survival times (9.5–7.5 d) of A. glabripennis adults than two North American B. brongniartii isolates (ARSEF 6215 and ARSEF 10279) (24–31 d). The virulence of North American B. brongniartii isolates is not well-documented in the literature. To our best knowledge this is the first account of the virulence of native North American B. brongniartii being evaluated for biological control of any invasive insect pest.     

Horizontal transmission of mycotic infection in adult tsetse,Glossina morsitans morsitans

AdultGlossina morsitans morsitans exposed to wet conidia ofBeauveria bassiana andMetarhizium anisopliae suffered high mortalities ranging from 90 to 100% by 2 weeks post-exposure. Infected ♂ ♂ maintained in the same cages with non-infected ♀♀ throughout the experimental period transmitted the fungal infection to the ♀♀ resulting in mortalities of 65% withB. bassiana and 55% withM. anisopliae. Likewise, infected ♀♀ maintained together with non-infected ♂♂ transmitted the infection to the ♂♂ resulting in mortalities of 75% withB. bassiana and 45% withM. anisopliae. Female tsetse flies infected withB. bassiana andM. anisopliae and maintained in the same cages with non-infected ♀♀ also transmitted infection to the non-infected tsetse resulting in mortalities of 62% and 48% withB. bassiana andM. anisopliae respectively. Infected tsetse exposed to non-infected tsetse of the opposite sex for only 30 min were also able to transmit the fungal infection. Pupae produced by female tsetse infected withB. bassiana andM. anisopliae exhibited higher pupal mortality than those produced by non-infected ♀♀. However, pupae exposed directly to dry spores ofB. bassiana andM. anisopliae had no increase in pupal mortality but adults emerging from theB. bassiana-exposed pupae had markedly reduced longevity.      

Dissemination of entomopathogenic fungi using Busseola fusca male as vector

The stem borer, Busseola fusca (Fuller), is an important pest of maize Zea mays L. and sorghum Sorghum bicolor (L.) in eastern and southern Africa. To control this pest, biological control methods including the use of entomopathogenic fungi are being considered. The pathogenicity of one isolate of Metarhizium anisopliae (Metsch.) Sorok. and one isolate of Beauveria bassiana Bals. (Vuill.) were first tested on different developmental stages of B. fusca including eggs, neonate, 2nd and 3rd-instar larvae. Both fungal isolates were pathogenic to all the stages tested. However, differences in mortality were observed among larvae that hatched from treated egg masses. Experiments were conducted thereafter to test whether B. fusca males could serve as a vector for fungal conidia to contaminate B. fusca females and subsequently eggs and larvae. Results demonstrated that B. fusca males successfully transferred inoculum to females during copulation, which in turn transmitted it to the eggs they laid on maize plants, resulting in the decrease of leaf damages.     

Preservation of aerial conidia and biomasses from entomopathogenic fungi Beauveria brongniartii and Metarhizium anisopliae during lyophilization

In this study, we assessed the stability provided by different formulations to aerial conidia or biomasses (conidia, blastospores, and mycelia) of Beauveria brongniartii and Metarhizium anisopliae subjected to lyophilization. First, the impact of the freezing and drying processes on spore survival was evaluated. Whereas unprotected B. brongniartii spores showed high cryosensitivity, those of M. anisopliae were markedly harmed by the drying process. Then, the protective efficiency of 14 excipients was systematically evaluated and optimized regarding required concentrations. Fructose, glucose, and saccharose significantly enhanced viabilities for B. brongniartii and M. anisopliae spores following lyophilization, especially as a result of their cryoprotective effects. In addition, the effect of various bulking agents on spore survival was studied and dextran 4 was selected to enhance the physical properties of the lyophilized products. The combination of fructose and dextran 4 was further applied to prepare lyophilized biomasses of both fungi. In comparison to freshly harvested biomasses, the lyophilized products showed similar growth rates and a comparable production of virulent secondary metabolites such as destruxin A, destruxin B, or oosporein, suggesting their applicability as biological control agents.     

Influence of Temperature on Virulence of Fungal Isolates of <Emphasis Type="Italic">Metarhizium anisopliae</Emphasis> and <Emphasis Type="Italic">Beauveria bassiana</Emphasis> to the Two-Spotted Spider Mite <Emphasis Type="Italic">Tetranychus urticae</Emphasis>

Twenty-three isolates of Metarhizium anisopliae (Metschnikoff) Sokorin and three isolates of Beauveria bassiana (Balsamo) Vuillemin (Ascomycota: Hypocreales: Clavicipitaceae) were assessed for their virulence against the two-spotted spider mite, Tetranychus urticae Koch (Acari: Tetranychidae). Based on the screening results, nine isolates of M. anisopliae and two isolates of B. bassiana were tested for their virulence against young adult (1- to 2-day-old) female T. urticae at constant temperatures of 20, 25, 30 and 35°C. At all temperatures tested, all the fungal isolates were pathogenic to T. urticae but mortality varied with isolates and temperatures. Fungal isolates were more virulent at 25, 30 and 35°C than at 20°C. The lethal time to 50% mortality (LT₅₀) and lethal time to 90% mortality (LT₉₀) values decreased with increased temperature. There were no significant differences in virulence between fungal isolates at 30 and 35°C; however, significant differences were observed at 20 and 25°C.     

Entomogenous Fungi as Promising Biopesticides for Tick Control

When ticks were sealed in nylon tetrapacks and infected with the entomogenous fungi, Beauveria bassiana and Metarizium anisopliae and maintained in potted grass in the field, the fungal oil formulations (10⁹ conidia per ml) induced 100% mortality in larvae of Rhipicephalus appendiculatus and Amblyomma variegatum, whereas mortalities in nymphs varied between 80–100% and in adults 80–90%. The aqueous formulations (10⁹ conidia per ml) induced mortalities of 40–50% and reductions in egg hatchability of 68% (B. bassiana) and 48% (M. anisopliae) when sprayed on Boophilus decoloratus engorging on cattle. The strains of B. bassiana and M. anisopliae isolated from naturally infected ticks were also found to induce high mortalities in both R. appendiculatus and A.variegatum in tetrapacks placed in potted grass. Both aqueous and oil-based formulations were found to be effective, although the latter induced higher mortalities. These fungal strains in aqueous formulation (10⁸ conidia per ml) suppressed on-host populations of adult R. appendiculatus by 80% (B. bassiana) and 92% (M. anisopliae) when sprayed on tick-infested grass once per month for a period of 6 months. The feasibility of using entomogenous fungi for tick control in the field is discussed.