Virulence of the insect-pathogenic fungi Metarhizium spp. to Mormon crickets, Anabrus simplex (Orthoptera: Tettigoniidae).

The Mormon cricket (MC), Anabrus simplex Haldeman, 1852 (Orthoptera: Tettigoniidae), has a long and negative history with agriculture in Utah and other western states of the USA. Most A. simplex populations migrate in large groups, and their feeding can cause significant damage to forage plants and cultivated crops. Chemical pesticides are often applied, but some settings (e.g. habitats of threatened and endangered species) call for non-chemical control measures. Studies in Africa, South America, and Australia have assessed certain isolates of Metarhizium acridum as very promising pathogens for Orthoptera: Acrididae (locust) biocontrol. In the current study, two isolates of Metarhizium robertsii, one isolate of Metarhizium brunneum, one isolate of Metarhizium guizhouense, and three isolates of M. acridum were tested for infectivity to MC nymphs and adults of either sex. Based on the speed of mortality, M. robertsii (ARSEF 23 and ARSEF 2575) and M. brunneum (ARSEF 7711) were the most virulent to instars 2 to 5 MC nymphs. M. guizhouense (ARSEF 7847) from Arizona was intermediate and the M. acridum isolates (ARSEF 324, 3341, and 3609) were the slowest killers. ARSEF 2575 was also the most virulent to instar 6 and 7 nymphs and adults of MC. All of the isolates at the conidial concentration of 1 × 107 conidia ml-1 induced approximately 100% mortality by 6 days post application of fungal conidia. In conclusion, isolates ARSEF 23, ARSEF 2575, and ARSEF 7711 acted most rapidly to kill MC under laboratory conditions. The M. acridum isolates, however, have much higher tolerance to heat and UV-B radiation, which may be critical to their successful use in field application.

Dual effects of Metarhizium spp. and Clonostachys rosea against an insect and a seed-borne pathogen in wheat.

BACKGROUND: Crops are often prone to both insect herbivory and disease, which necessitate multiple control measures. Ideally, an efficacious biological control agent must adequately control the target organism and not be inhibited by other biological control agents when applied simultaneously. Wheat seeds infected with the plant pathogen Fusarium culmorum were treated with Metarhizium brunneum or M. flavoviride and Clonostachys rosea individually and in combination, with the expectation to control both root-feeding insects and the pathogen. Emerging roots were evaluated for disease and then placed with Tenebrio molitor larvae, which were monitored for infection. RESULTS: Plant disease symptoms were nearly absent for seeds treated with C. rosea, both individually and in combination with Metarhizium spp. Furthermore, roots grown from seeds treated with Metarhizium spp. caused significant levels of fungal infection in larvae when used individually or combined with C. rosea. However, cotreated seeds showed reduced virulence towards T. molitor when compared with treatments using Metarhizium spp. only. CONCLUSIONS: This study clearly shows that seed treatments with both the entomopathogenic fungus M. brunneum and the mycoparasitic fungus C. rosea can protect plant roots from insects and disease. The dual-treatment approach to biological control presented here is consistent with the ideals of IPM strategies.

Diversity within the entomopathogenic fungal species Metarhizium flavoviride associated with agricultural crops in Denmark.

BACKGROUND: Knowledge of the natural occurrence and community structure of entomopathogenic fungi is important to understand their ecological role. Species of the genus Metarhizium are widespread in soils and have recently been reported to associate with plant roots, but the species M. flavoviride has so far received little attention and intra-specific diversity among isolate collections has never been assessed. In the present study M. flavoviride was found to be abundant among Metarhizium spp. isolates obtained from roots and root-associated soil of winter wheat, winter oilseed rape and neighboring uncultivated pastures at three geographically separated locations in Denmark. The objective was therefore to evaluate molecular diversity and resolve the potential population structure of M. flavoviride. RESULTS: Of the 132 Metarhizium isolates obtained, morphological data and DNA sequencing revealed that 118 belonged to M. flavoviride, 13 to M. brunneum and one to M. majus. Further characterization of intraspecific variability within M. flavoviride was done by using amplified fragment length polymorphisms (AFLP) to evaluate diversity and potential crop and/or locality associations. A high level of diversity among the M. flavoviride isolates was observed, indicating that the isolates were not of the same clonal origin, and that certain haplotypes were shared with M. flavoviride isolates from other countries. However, no population structure in the form of significant haplotype groupings or habitat associations could be determined among the 118 analyzed M. flavoviride isolates. CONCLUSIONS: This study represents the first in-depth analysis of the molecular diversity within a large isolate collection of the species M. flavoviride. The AFLP analysis confirmed a high level of intra-specific diversity within the species and lack of apparent association patterns with crop or location indicates limited ecological specialization. The relatively infrequent isolation of M. flavoviride directly from crop roots suggests low dependence of root association for the species.

Stress tolerance and virulence of insect-pathogenic fungi are determined by environmental conditions during conidial formation.

The virulence to insects and tolerance to heat and UV-B radiation of conidia of entomopathogenic fungi are greatly influenced by physical, chemical, and nutritional conditions during mycelial growth. This is evidenced, for example, by the stress phenotypes of Metarhizium robertsii produced on various substrates. Conidia from minimal medium (Czapek's medium without sucrose), complex medium, and insect (Lepidoptera and Coleoptera) cadavers had high, moderate, and poor tolerance to UV-B radiation, respectively. Furthermore, conidia from minimal medium germinated faster and had increased heat tolerance and were more virulent to insects than those from complex medium. Low water-activity or alkaline culture conditions also resulted in production of conidia with high tolerance to heat or UV-B radiation. Conidia produced on complex media exhibited lower stress tolerance, whereas those from complex media supplemented with NaCl or KCl (to reduce water activity) were more tolerant to heat and UV-B than those from the unmodified complex medium. Osmotic and nutritive stresses resulted in production of conidia with a robust stress phenotype, but also were associated with low conidial yield. Physical conditions such as growth under illumination, hypoxic conditions, and heat shock before conidial production also induced both higher UV-B and heat tolerance; but conidial production was not decreased. In conclusion, physical and chemical parameters, as well as nutrition source, can induce great variability in conidial tolerance to stress for entomopathogenic fungi. Implications are discussed in relation to the ecology of entomopathogenic fungi in the field, and to their use for biological control. This review will cover recent technologies on improving stress tolerance of entomopathogenic fungi for biological control of insects.

Heat-induced post-stress growth delay: a biological trait of many Metarhizium isolates reducing biocontrol efficacy?

The habitats of many pest insects have fluctuating climatic conditions. To function effectively, the pathogens of these pests must be capable of infecting and developing disease at a wide range of temperatures. The current study examines ten Metarhizium spp. isolates as to their ability to recover normal metabolic activity after exposure to high temperature for several hours daily; and whether such recovery, with at least some isolates, requires a temporary repair ("retooling") period. Fungal colonies were exposed to 40°C for 4h or 8h followed by 20h or 16h at 28°C, respectively, for three consecutive days. Growth rates during treatments were compared to control plates (constant 28°C) and to plates with growth stoppage by cold treatment (4h or 8h at 5°C per day). All ten isolates survived 3days of cycled heat treatment and resumed normal growth afterward; some isolates however, were considerably more negatively affected by heat-cycling than others. In fact, some isolates underwent greatly reduced growth not only during 8h heating, but also some hours after cessation of heat treatment. This phenomenon is labeled in the current study as "post-stress growth delay" (PSGD). In contrast, all isolates stopped growing during 8h cold treatments, but immediately recommenced growing on return to 28°C. The delay in recommencing growth of some isolates after heat treatment amplifies the effect of this stress. In addition to the studies on the effects of heat cycling on fungal cultures, the effects of imposing such temperature cycling on fungal infection of insects was documented in the laboratory. Three Metarhizium isolates were bioassayed using Galleria mellonella larvae. Treated insects were placed at daily temperature regimes matching those used for the in vitro fungus rate-of-growth study, and insect mortality recorded daily. For all three isolates the levels of insect mortality at the highest-heat dose (40°C at 8h daily) significantly reduced infection. Fluctuating temperatures are likely to be a factor in most pest-insect habitats; therefore, the presence and level of PSGD of each isolate should be a primary consideration in selecting field-appropriate fungal isolates.