Discovery of a North American genetic variant of the entomopathogenic fungus Metarhizium anisopliae var. anisopliae pathogenic to grasshoppers

Abstract

A genetic variant of the entomopathogenic fungus Metarhizium anisopliae var. anisopliae, isolated from a soil in Alberta, Canada, from a location with a history of severe grasshopper infestations, was evaluated for pathogenicity in bioassays of living grasshoppers. Mortality in treated individuals drawn from a laboratory colony was 99% (LT 50 = 6.7 days, LT 90 = 9.6 days) at 12 days postinoculation compared to 100% (LT 50 = 4.1 days, LT 90 = 5.8 days) mortality at 8 days in insects exposed to a commercial isolate of M. anisopliae var. acridum (IMI 330189). Experimental infection of field-collected grasshoppers under laboratory conditions with the native isolate of M. anisopliae var. anisopliae resulted in 100% (LT 50 = 4.4 days, LT 90 = 5.4 days) mortality attained within 7 days compared to 100% (LT 50 = 4.7 days, LT 90 = 6.3 days) mortality in 9 days in insects treated with M. anisopliae var. acridum. Amplification of fungal genomic DNA from the indigenous isolate with primers for the specific detection of M. anisopliae var. anisopliae produced a product almost 300 bp larger than expected based on previously known isolates. This is the first demonstration of a highly virulent, indigenous non-chemical control agent of grasshoppers in North America.

Mechanisms Relevant to the Enhanced Virulence of a Dihydroxynaphthalene-Melanin Metabolically Engineered Entomopathogen

“…In Fig. 8C , the specific IGS band can only be visualized in wild type and transformant M. anisopliae (lanes “S5” and “S9”), indicating the specificity of the two-step nested PCR method for detecting M. anisopliae [37] . The specificity of the PKS primers for detecting the transformant was also confirmed, as a single PKS band of approximately 700 bp was only observed in the transformant ( Fig.…”

“…Whereas DNA extracted from the 0.25 g of loam soil samples containing 1 ml of a spore suspension (1×10 6 ) of either M. anisopliae BCRC35505 or MA05-169 were used as a positive check. The PCR universal primers (8F 5′-GTTTGATCCTGGCTCAG-3′ , 1512R 5′-GGYTACCTTGTTACGACTT3′ ), (ITS4 5′-TCCTCCGCTTATTGATATGC-3′ , ITS5 5′-GGAAGTAAAAGTCGTAACAAGG-3′ ) or specific primers (PKS-TE(a), PKS-TE(b)) were adopted from previously described methods [5] ; the two-step nested PCR procedure described by Entz et al (2008) [37] was followed to detect the M. anisopliae in the soil. In brief, in the 1st step of the nested PCR procedure, the DNA segment between the large subunit rDNA and the intergenic spacer (IGS) region was amplified using primers Ma-28S4 ( 5′-CCTTGTTGTTACGATCTGCTGAGGG-3′ ) and Ma-IGS1 ( 5′-CGTCACTTGTATTGGCAC-3′ ).…”

Differential pathogenicity of Metarhizium anisopliae and the control of the sugarcane root spittlebug Mahanarva fimbriolata

“…Additional benefits of using this particular pathogen are that it is also active against the borer Diatraea saccharalis (Fabricius) but does not affect some of the natural enemies of the spittlebug such as the wasp Salpingogaster nigra (Schiner) and predator ant Pheidole genalis (Borgmeier) (Mendonça and Mendonça, 2005). Several studies have been conducted that focus on the isolation and screening of entomopathogenic fungi which may be effective in controlling the pest insects (Entz et al, 2008;Anand et al, 2009). For example, the pathogenicity of isolates of M. anisopliae from different hosts and regions of Brazil have been tested against the spittlebug M. fimbriolata (Loureiro et al, 2005;Macedo et al, 2006).…”

Effect of Metarhizium anisopliae (Clavicipitaceae) on Rhagoletis mendax (Diptera: Tephritidae) pupae and adults

“…Metarhizium anisopliae var. anisopliae strain S54 cultures were originally obtained from soil collected in southern Alberta, Canada (Entz et al 2005(Entz et al , 2008. Isolates were obtained from soil via washing, dilutions, and plating on dodine-based selective media (Chase et al 1986) during exploration in [2004][2005][2006].…”

“…Conidia of the S54 strain were produced at the University of Lethbridge (Lethbridge, Alberta, Canada) by growth on potato dextrose agar, followed by inoculation and fermentation of sterile, standard flaked barley (Alberta Barley Commission, Calgary, Alberta, Canada) in 1-kg sterile, aerated mushroom spawn bags (SacO2, Microsac, Nevele, Belgium) from liquid culture or conidia subsampled from colonies grown on potato dextrose agar plates. A total of 800 g of dry conidia was produced for field testing and verification of insect infectivity in experiments in Canada (Entz et al 2008). A portion (approximately 10 g) of this product was provided for the blueberry maggot tests conducted in Nova Scotia.…”