Comparison of Water,Oils and Emulsifiable Adjuvant Oils as Formulating Agents for <Emphasis Type="Italic">Metarhizium Anisopliae</Emphasis> for Use in Control of <Emphasis Type="Italic">Boophilus Microplus</Emphasis>

Studies were conducted to identify oil-based formulating agents (paraffinic oil, palm oil and emulsifiable adjuvant oils (EAOs)) for Metarhizium anisopliae that were superior to water with simple surfactants using a germination test and a bioassay against Boophilus microplus. Germination of conidia in all formulations, except 10% coconut EAO, produced more than 68% germination at 24 h and nearly 100% at 48 h. Coconut oil (average survival time (AST)=4.6±0.28 days) and 10% liquid paraffin EAO (AST=4.4±0.15 days) enhanced the pathogenicity of M. anisopliae to B. microplus relative to water (AST=8.4±0.42 days). M. anisopliae in 10% liquid paraffin EAO was the most effective formulation having a moderately high germination after 24 h and a low AST as well as a high AST in the control. In the second experiment, germination of conidia in 2% liquid paraffin EAO and 2% Cropspray was higher than in 2% Codacide oil at 24 h, however, all treatments reached 100% germination after 48 h. The ASTs of the EAO based M. anisopliae formulations (Average AST=6.4±0.54 days) were similar but lower that the ASTs of the controls (Average AST=9.6±0.28 days).     

Field observations of the effects of fenitrothion and Metarhizium anisopliae var. acridum on non-target ground dwelling arthropods in the Sahel

The effect of the chemical insecticide, fenitrothion, and a mycoinsecticide based on Metarhizium anisopliae var. acridum on the activity of non-target epigeal arthropod scavengers was investigated in areas of open savannah in southeast Niger Republic, West Africa. Both insecticides were applied as full cover sprays to unreplicated 800 ha plots to assess their season-long control of Sahelian grasshoppers. Compared with control plots, fenitrothion caused an immediate but temporary reduction in grasshopper numbers, whereas M. anisopliae var. acridum provided delayed but prolonged control. Scavenging rates of pyrethroid-killed grasshoppers placed along transects in unsprayed plots and those treated with fenitrothion and M. anisopliae var. acridum at various intervals after spraying were assessed. In the fenitrothion plot, an immediate reduction in scavenging activity occurred that was still apparent after 40 days at the plot center, although recovery at the plot edges was more rapid. By contrast scavenging rates remained high over equivalent areas in the M. anisopliae var. acridum and two untreated plots. Concurrent to the scavenging study, counts of grasshopper cadavers resulting from the spray treatments were conducted. These counts revealed that the density of grasshopper cadavers remained low throughout the M. anisopliae var. acridum plot and explained <1% of the reduction in live grasshoppers resulting from treatment, compared with >20% in the fenitrothion plot. This shortfall in grasshopper cadavers resulting from the spray treatment in the M. anisopliae var. acridum plot was unexpected because in a monitoring study, fungus-killed (unlike pyrethroid-killed) grasshoppers were unattractive to scavengers and readily persisted in this plot, and thus should have become apparent. Given we did not observe significant grasshopper dispersal, the scarcity of cadavers generated in the M. anisopliae var. acridum plot, together with unquantified visual observations, suggests that predation of infected but living grasshoppers was high. Our data provide circumstantial evidence that the different effects of chemical and biological grasshopper control on grasshopper natural enemies may influence the efficacy of large-scale treatments.     

Expression of scorpion toxin LqhIT2 increases the virulence of Metarhizium acridum towards Locusta migratoria manilensis

LqhIT2 is an insect-specific neurotoxin from the venom of scorpion. In this study, the LqhIT2 gene was introduced into the entomopathogenic fungus, Metarhizium acridum. The virulence of the genetically modified strain MaLqhIT2 was then evaluated against locusts (Locusta migratoria manilensis). Compared with the wild-type strain, the median lethal cell density (LC₅₀) for MaLqhIT2 was a 22.6-fold lower, and the median times to death (LT₅₀) for MaLqhIT2 were reduced by 30.3 and 29.6 %, respectively, after topical inoculation and injection. MaLqhIT2 also grew significantly faster in the hemolymph than wild-type strain. There were no significant differences in germination, appressorium formation and sporulation in locust carcasses between the MaLqhIT2 and wild-type strain. These results indicate that LqhIT2 increased the virulence of M. acridum towards locusts by shortening the in vivo infection period, without affecting cuticle penetration or conidia formation in the carcasses. LqhIT2 thus shows considerable potential for increasing fungal virulence against locusts.     

Molecular detection of the airborne entomopathogen fungus Metarhizium acridum using specific oligonucleotides

We describe a technique to detect the presence of airborne conidia from the fungus M. acridum (formerly Metarhizium anisopliae var. acridum) (Hypocreales: Clavicipitaceae) with great accuracy. Airborne conidia were collected using Hirst-type spore traps. DNA extractions were optimized to achieve the best possible recovery. DNA was examined using polymerase chain reaction (PCR) with specific oligonucleotides to enable the detection of a single conidium. Experiments using a mini-wind tunnel were conducted to validate the method. Subsequently, this technique was applied to an agricultural region of Mexico, where M. acridum was sprayed to control the grasshopper, Sphenarium purpurascens, population (Orthoptera: Pyrgomorphidae). M. acridum conidia were detected 2 days after spraying in San Mateo Coatepec (Puebla, site of grasshopper study).     

Effect of formulation on the virulence of Metarhizium anisopliae conidia against mosquito larvae

Metarhizium anisopliae conidia were formulated with three granular carriers and nine dust diluents and stored over an 8- to 12-month period at 4° or 20°C. The virulence of formulations, with the exception of two dust preparations, was reduced significantly compared to unformulated conidia against Culex pipiens pipiens larvae. The formulation components most detrimental to conidial virulence were corn cob granules, diatomaceous earth, and two Kaolinite diluents. This was exampled by a decline in virulence from ca. 100% for unformulated conidia to 36% or below for these formulations. LT₅₀ values also increased from 2.4–2.6 days for unformulated conidia to above 6 days. In contrast, a diluent derived from dried castor oil (Thixcin R) significantly enhanced conidial virulence at several doses above that of unformulated conidia against C. pipiens larvae. Enhancement occurred whether conidia were formulated prior to storage or stored separate from the diluent and mixed prior to application. The Thixcin R formulation was more effective against Anopheles stephensi larvae, but virulence was reduced against Aedes aegypti larvae. A bentonite formulation (Bentone-38) also maintained conidial virulence effectively, but Thixcin R was a superior diluent. It was shown that conidial virulence of formulations was not correlated with differences in conidial viability. The preparations that were applied dry by a surface method were more virulent than when an aqueous suspension containing a surfactant was used. The results demonstrate the need to assess efficacy of mycoinsecticidal formulations in a virulence bioassay prior to field testing.     

Behavioural changes in Schistocerca gregaria following infection with a fungal pathogen: implications for susceptibility to predation

1. Field observations have indicated that infection of locusts and grasshoppers by the fungal entomopathogen Metarhizium anisopliae var. acridum may result in a substantial increase in the host's susceptibility to predation, before death is caused directly by the disease. 2. Laboratory experiments were conducted to examine how the behaviour of the desert locust Schistocerca gregaria Forskål changes following infection by M. anisopliae var. acridum to explore some potential mechanisms underlying this phenomenon. 3. In the first experiment, which involved monitoring general locust activity in small cages throughout the disease incubation period, infected locusts were observed to increase locomotion and bodily movement from 3 days after infection until death (average survival time of 11 days). There was some evidence of reduced feeding and mating behaviour following infection. 4. In a second experiment, locusts were exposed individually to a simulated predator attack and the initiation and strength of any escape responses were measured. Infected locusts were observed to have a reduced escape capability (both the propensity to escape and the strength of the response). In contrast to the relatively early changes in general activity observed in the first experiment, this was only apparent at the late stages of infection shortly before death. 5. Both an increase in movement and general apparency early in the infection process, and reduced escape capability late on, suggest mechanisms whereby the susceptibility of locusts and grasshoppers to predation might be enhanced following infection with M. anisopliae var. acridum.     

Effect of formulated Metarhizium anisopliae on eggs and eclosing nymphs of Triatoma infestans

Little is known about the ovicidal effects of fungi that attack nymphs and adults of triatomine vectors. A combined formulation of Metarhizium anisopliae IP 46 conidia prepared with diatomaceous earth (DE) and vegetable oil was tested against eggs of Triatoma infestans. Eggs were highly susceptible to fungal infection at relative humidity close to saturation [>98% relative humidity (RH)] but not at 75% RH regardless of the formulation applied. Susceptibility of eggs decreased with longer post‐ovipositional embryonation periods before treatments. The eventual eclosion of nymphs was best suppressed by application of conidia prepared with DE + oil and at a >98% RH incubation. Moreover, nymphs were less affected by the fungus when exposed for only a 24‐h period after eclosion to a treated surface than individuals that were in constant contact with the conidia. These findings contribute to a better understanding of the potential of M. anisopliae as an agent against all developmental stages of T. infestans.     

Infection of Blissus antillus (Hemiptera: Lygaeidae) Eggs by the Entomopathogenic Fungi Metarhizium anisopliae and Beauveria bassiana

This study determined the pathogenicity and virulence of Beauveria bassiana and Metarhizium anisopliae to eggs of the chinch bug Blissus antillus (Hemiptera: Lygaeidae). Eggs were inoculated under laboratory conditions by immersion in concentrations of 1 × 10⁴ and 5 × 10⁶ conidia/ml. Inoculated eggs were kept under controlled conditions. Evaluations were carried out daily for 20 days. M. anisopliae isolates were highly virulent to eggs, even at 1 × 10⁴ conidia/ml. All B. bassiana isolates tested were considered to be of low virulence or avirulent. The most virulent isolate tested was ESALQ 818 (M. anisopliae), which caused 96.7% infection, when eggs were immersed in suspensions of 1 × 10⁴ conidia/ml. Conidial production on infected eggs was observed to be highest for M. anisopliae isolate CG144, with a mean value of 11.6 × 10⁵ conidia/ml/egg. Infection of Blissus eggs oviposited on plant stems was greater when M. anisopliae isolate CG144 was formulated in mineral oil (63.5% mortality) than when formulated in Tween 80 (27.1% mortality).     

Invasion of the pathogenic fungus Metarhizium anisopliae through the guts of germfree desert locusts,Schistocerca gregaria

Less than 1% of an ingested inoculum of the pathogenic fungus Metarhizium anisopliae was retained for long enough (ca. 24 h) in the gut of the desert locust, Schistocerca gregaria, for germination and penetration to have occurred. The residual inoculum did not initiate an infection in guts of fed conventional or axenic locusts. However, symptoms of mycosis (hyphal bodies in the haemolymph, fungal penetration of the hindgut intima and epithelium, tetanic paralysis) were consistently observed in axenic but not conventional locusts which were starved post-inoculation.It is concluded that the antifungal toxin produced by the gut bacteria defends the desert locust against gut invasion by Metarhizium anisopliae during periods of starvation when the physical defences, prominent in fed insects, are less apparent.     

Thermal ecology of Zonocerus variegatus and its effects on biocontrol using pathogens

1 Thermal behaviour of the variegated grasshopper, Zonocerus variegatus, was investigated in the humid tropical zone of southern Benin, west Africa, in the dry seasons of 1996 and 1998. In 1998, investigations included studies of a population of grasshoppers sprayed with an oil‐based formulation of the entomopathogenic fungus Metarhizium anisopliae var acridum. 2 Body temperature measurements and observations of thermal behaviour both in the field and on thermal gradients in the laboratory, suggest that Z. variegatus was not an active behavioural thermoregulator. Although it did show shade‐seeking behaviour at high temperatures, no overt behavioural postures or microhabitat selection associated with heat gain and elevation of body temperatures was observed. Moreover, no alterations to thermal behaviour were found in response to infection by Metarhizium. 3 Body temperatures exhibited by Z. variegatus in the field will lengthen disease incubation of M. anisopliae var acridum compared with laboratory maintained, constant temperature conditions and may have a significant impact on pathogens with a lower thermal tolerance. 4 Habitat structure appeared to be an important factor determining the extent of body temperature elevation. The effect of habitat differences on infection and growth of M. anisopliae var acridum and other entomopathogenic fungi is discussed.     

Morphological Alterations of Metarhizium anisopliae During Penetration of Boophilus microplus Ticks

Chronological histological alterations of Metarhizium anisopliae during interaction with the cattle tick Boophilus microplus were investigated by light and scanning electron microscopy. M. anisopliae invades B. microplus by a process which involves adhesion of conidia to the cuticle, conidia germination, formation of appressoria and penetration through the cuticle. Twenty-four hours post-infection conidia are adhered and germination starts on the surface of the tick. At this time, the conidia differentiate to form appressoria exerting mechanical pressure and trigger hydrolytic enzyme secretion leading to penetration. Massive penetration is observed 72 h post-inoculation, and after 96 h, the hyphae start to emerge from the cuticle surface to form conidia. The intense invasion of adjacent tissues by hyphae was observed by light microscopy, confirming the ability of M. anisopliae to produce significant morphological alterations in the cuticle, and its infective effectiveness in B. microplus.     

Effects of Metarhizium anisopliae on Meccus pallidipennis (Hemiptera: Reduviidae) over different types of wall surfaces

The entomopathogenic fungus Metarhizium anisopliae is virulent for the insect triatomine Meccus pallidipennis. To evaluate the functionality of a fungal formulation (vegetable oil and emulsifiers) of this fungus, virulence was assayed by insect mortality on the pronotum of third instar nymphs (N3) M. pallidipennis in laboratory conditions and ST₅₀ was calculated. Mortality was evaluated directly: 100%, 97.33% and 98.66% mortalities were caused by formulation, emulsified formulation and fungal conidia, respectively, at day 8 of insect infection. Another bioassay was carried out in simulated external conditions (peridomicility) using red and gray brick walls, a stone fence and mountain soil (experimental units). These simulated conditions were infected with 10?ml of a 1?×?10⁹?conidia/ml emulsified formulation by means of a manual sprinkler prior to the placement of the nymphs. Ten N3 M. pallidipennis were deposited in each experimental unit and insect mortality was monitored every 12?h for 22 days. Each treatment was replicated four times. With the red brick wall, a mortality of 90% at day 22 and a ST₅₀ of 15 days were obtained on N3 M. pallidipennis; with the gray brick wall, 100% mortality and a ST₅₀ of 13 days; and with the stone fence, 88.33% mortality and a ST₅₀ of 21 days. The results obtained in this research work indicate that the formulation with conidia of the M. anisopliae strain EH-473/4 may be auxiliary in the development of strategies for the control of Chagas disease insect transmitters such as M. pallidipennis.     

Influence of Unrefined Corn Oil and Surface-active Agents on the Germination and Infectivity of Alternaria helianthi

A dew immediately after inoculation is normally required for the mycoherbicidal activity of Alternaria helianthi on common cocklebur. The formulation of A. helianthi conidia in an emulsion of unrefined corn oil enabled the pathogen to infect the weed, regardless of whether dew was immediate or delayed for 24 h. Corn oil emulsion and the surface-active agents Tween 20, 40, 60 and 80 maintained the germination ability of 14-day-old conidia to some degree for up to 4 days in suspension. Both the corn oil emulsion and Silwet stimulated the germination of A. helianthi on common cocklebur leaves, but Silwet did not enhance infectivity when dew was delayed. Unrefined corn oil enhanced mycoherbicide efficacy by protecting the conidia during a dew-free period and by stimulating germination when a dew occurred. Corn oil emulsion, Silwet and Tween 20, 40, 60 and 80 reduced the dew period required for disease activity. Unrefined corn oil emulsion has potential as a formulation for the application of this mycoherbicide in the field because it maintained the germination ability and virulence of conidia on the weed during a dew-free period.     

Microcycle conidiation and the conidial properties in the entomopathogenic fungus Metarhizium acridum on agar medium

Conidiation of the entomopathogenic fungus Metarhizium acridum on agar media was investigated. M. acridum CQMa102 exhibits two different conidiation patterns on agar media: normal conidiation in which conidia are formed on extended hyphae and microcycle conidiation in which conidiation occurs directly after conidia germination. Microcycle conidiation resulted in a mass of conidia produced via budding by accelerated development at the inoculation site. The mean total conidial yield (conidiation at day 10) was 4–5-fold greater after microcycle conidiation than during normal conidiation. Insect pathology assays indicated that microcycle conidia produced on SYA agar were as effective as normal aerial conidia against the locust. Ultraviolet (UV)-resistance tests showed no significant differences between the two types of cell propagules. However, microcycle conidia were more heat resistant than normal aerial conidia, and accumulated higher levels of trehalose in response to heat induction compared to normal aerial conidia.     

The genome sequence of the biocontrol fungus Metarhizium anisopliae and comparative genomics of Metarhizium species

Background

Metarhizium anisopliae is an important fungal biocontrol agent of insect pests of agricultural crops. Genomics can aid the successful commercialization of biopesticides by identification of key genes differentiating closely related species, selection of virulent microbial isolates which are amenable to industrial scale production and formulation and through the reduction of phenotypic variability. The genome of Metarhizium isolate ARSEF23 was recently published as a model for M. anisopliae, however phylogenetic analysis has since re-classified this isolate as M. robertsii. We present a new annotated genome sequence of M. anisopliae (isolate Ma69) and whole genome comparison to M. robertsii (ARSEF23) and M. acridum (CQMa 102).

Results

Whole genome analysis of M. anisopliae indicates significant macrosynteny with M. robertsii but with some large genomic inversions. In comparison to M. acridum, the genome of M. anisopliae shares lower sequence homology. While alignments overall are co-linear, the genome of M. acridum is not contiguous enough to conclusively observe macrosynteny. Mating type gene analysis revealed both MAT1-1 and MAT1-2 genes present in M. anisopliae suggesting putative homothallism, despite having no known teleomorph, in contrast with the putatively heterothallic M. acridum isolate CQMa 102 (MAT1-2) and M. robertsii isolate ARSEF23 (altered MAT1-1). Repetitive DNA and RIP analysis revealed M. acridum to have twice the repetitive content of the other two species and M. anisopliae to be five times more RIP affected than M. robertsii. We also present an initial bioinformatic survey of candidate pathogenicity genes in M. anisopliae.

Conclusions

The annotated genome of M. anisopliae is an important resource for the identification of virulence genes specific to M. anisopliae and development of species- and strain- specific assays. New insight into the possibility of homothallism and RIP affectedness has important implications for the development of M. anisopliae as a biopesticide as it may indicate the potential for greater inherent diversity in this species than the other species. This could present opportunities to select isolates with unique combinations of pathogenicity factors, or it may point to instability in the species, a negative attribute in a biopesticide.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-660) contains supplementary material, which is available to authorized users.     

Effectiveness of Metarhizium anisopliae formulations against dengue vectors under laboratory and field conditions

The oviposition behaviour of Aedes aegypti and the effectiveness of Metarhizium anisopliae conidia formulated in water or oil-in-water against A. aegypti adults and eggs were tested in multi-choice and no-choice tests in oviposition devices under laboratory conditions. Both females and males rested in the devices, regardless of the formulation, and were not repelled by the presence of conidia (up to 10⁶?conidia/cm²) without oil or formulated with oil on treated filter paper arranged in the device. However, at higher oil concentrations (≥0.1?μl/cm²), regardless of the presence of conidia, the number of eggs laid by gravid females on the filter paper dropped. The susceptibility of adults, especially of males, to fungal infection increased up to a 15-day incubation. An elevated number of larvae (≥41%) eclosed from eggs laid on the moistened filter paper in the device even without submersion of eggs in water, and these larvae subsequently died. In the laboratory, 1?μl/cm² oil combined with 10⁶?conidia/cm² clearly reduced eclosion to 1.8% after submersion of eggs in water compared to ≥13% eclosion in the control. In field tests in Goiânia, Brazil, eclosion of aedine larvae from eggs laid on filter paper previously treated with oil-in-water formulated conidia dropped to between 0% and 36% compared to 22–50% in the control. Promising results of laboratory and field tests with M. anisopliae formulated in water or oil-in-water and tested in a device emphasised the effectiveness of a fungus-based formulation for aedine mosquitoes in peridomestic areas.     

Field observations of the effect of Metarhizium anisopliae var. acridum on the saxaul locust,Dericorys albidula Serville (Orthoptera: Dericorythidae)

Dericorys albidula Serville (Orthoptera: Dericorythidae) is a major pest of Haloxylon ammodendron and other saxaul plant species in the Qom province, Iran. Using fungal insecticides can be an alternative method for chemical insecticides. Effect of insecticide fungi, Metarhizium anisopliae var. acridum, on the various nymphs of D. albidula was studied in the field through 2005 and 2006. Fixed concentrations of fungi (10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰ and 10¹³ spore mL^?1) were prepared as gasoline formulation and were sprayed on the locusts on H. ammodendron trees, and mortality percentage was recorded 15 days after treatment. The results showed that various concentrations significantly affected on the second, third, fourth and fifth nymphal instars of D. albidula compared to control in 2006, although this effect was lower in 2005 on nymphs. Mortality of the highest concentration (10¹³ spore mL^?1) was higher (17.6–24%) than other concentrations in all tests, but these values were not notable. The results of this study showed that using M. anisopliae var. acridum diluted in gasoline can be effective on nymphal instars of locust, D. albidula, in two continuous years.     

Differential susceptibility of Amblyomma maculatum and Amblyomma americanum (Acari:Ixodidea) to the entomopathogenic fungi Beauveria bassiana and Metarhizium anisopliae

Nymph and adult ticks from Ambylomma americanum and Ambylomma maculatum were treated with conidia and blastospores of the entomopathogenic fungi Beauveria bassiana (90517) and Metarhizium anisopliae (20500). Fungal suspensions of conidia harvested from potato dextrose plates containing 10⁸ conidia/ml caused greater than 90% mortality in adult A. maculatum but less than 10% mortality in adult A. americanum over a 28 day time course. Similarly, infection with M. anisopliae (10⁸ conidia/ml) resulted in 60 and 15% mortality in A. maculatum and A. americanum, respectively. Nymphs of both tick species were more susceptible to fungal infection reaching mortality rates of almost 100% for A. maculatum and over 35% for A. americanum. Scanning electron microscopy of infected ticks showed rapid attachment, germination, and proliferation of fungal spores on A. maculatum cuticles, and to a much lesser extent on A. americanum cuticles. Pentane extracts of A. americanum cuticle hydrocarbons inhibited germination and hyphal growth of B. bassiana conidia, whereas no inhibition was observed using A. maculatum extracts. Significant mortality towards A. americanum was observed (>60%, 28 days) only when the ticks were treated with B. bassiana directly from the growth medium (10⁷ blastospores/ml, grown for 3–4 days in Sabouraud dextrose + 0.5% yeast extract liquid media). These results indicate tick species display differential susceptibility to the entomopathogenic fungi B. bassiana and M. anisopliae, and that the ability to overcome fungistatic compounds present in the tick epicuticle may determine the likelihood of successful infection and virulence.     

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.     

Discovery of a North American genetic variant of the entomopathogenic fungus <Emphasis Type="Italic">Metarhizium anisopliae</Emphasis> var. <Emphasis Type="Italic">anisopliae</Emphasis> pathogenic to grasshoppers

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% (LT₅₀ = 6.7 days, LT₉₀ = 9.6 days) at 12 days post-inoculation compared to 100% (LT₅₀ = 4.1 days, LT₉₀ = 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% (LT₅₀ = 4.4 days, LT₉₀ = 5.4 days) mortality attained within 7 days compared to 100% (LT₅₀ = 4.7 days, LT₉₀ = 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. GenBank Accession Nos. DQ342236, DQ342237.