A valid laboratory bioassay system for evaluating M. anisopliae efficacy under desiccation stress
Water stress tolerance of fungal strains is usually evaluated using various salts to create different water potential scenarios. However, in testing the virulence of the strains, the salt can affect the life cycles of hosts. In this paper, a novel laboratory bioassay system was used to test the efficacy of M. anisopliae under desiccation stress on T. molitor larvae in dry substrate.
Extreme environmental trials of pathogens demand specific hosts. The yellow mealworm, T. molitor, is a freeze-susceptible, stored product pest. When provided with sufficient food supply, T. molitor larvae have low humidity tolerance and can survive under relatively xeric conditions because of their ability to metabolize water from ingested food [12].
Clopton et al. [13] sterilized adult and larval T. molitor by incubation at 36°C to 37°C for 5 d to eliminate the effect of existing gregarine infections on the tests. In the present study, the host insects were cultured and sterilized by generational dilution in sterile wheat bran substrates, and the insects were almost fully sterilized when given enough generation culture. This new method may provide host insects for strict experimental infections.
The efficacy of M. anisopliae under desiccation stress was tested in dry wheat bran substrate with initial moisture content of 8%. At this low moisture level, M. anisopliae was difficult to grow, but the isolate MAX-2 was still active, whereas the other isolates showed very low efficacy. This result suggests that the infection of sterile T. molitor larvae in wheat bran substrates with low moisture content could constitute a valid laboratory bioassay system to study M. anisopliae efficacy under desiccation stress.
Efficacy of M. anisopliae isolate MAX-2
This study demonstrated that M. anisopliae isolate MAX-2 had pathogenicity against T. molitor larvae in all the tested moisture levels, particularly lower moisture levels, and showed relatively high tolerance to desiccation stress.
Daoust et al. [14] indicated that the efficacy of M. anisopliae against insects depends on conidial germination. Conidial germination of all tested isolates in the present study showed a tendency to decrease with the decrease in substrate moisture content within the tested scope (8% to 35%). The mortality of larvae for the isolates in different moisture levels also showed the same tendency, which indicates the correlation between conidial germination and efficacy of M. anisopliae. However, the mortality for MAX-2 decreased much more slowly than those of the other isolates. At the substrate with 8% moisture, which was too low for M. anisopliae to facilitate germination, MAX-2 still showed medium mortality of 41% versus low mortality < 5% for the other isolates against T. molitor larvae. Howard et al.[15] observed that high virulence of M. anisopliae against mosquitoes is not significantly affected by low viability, and they deduced that the difference is possibly due to the different abilities of the fungal conidia to germinate on mosquito cuticles and the agar. Leger [16] also reported the existence of two diverse sets of selection pressures on Metarhizium spp., one for optimum characteristics for soil survival and another for virulence to insects. Although desiccation stress restricted the viability of MAX-2 on the substrate, this isolate might have more excellent ability to germinate and infect after attachment to the host by its own high trehalase activity, some other mechanism, or the induction of the hosts. Further studies on the possible mechanisms of water stress response and high efficacy for MAX-2 are recommended.
Sporulation of entomopathogenic fungi is significantly affected by moisture content, commonly between 1:0.35 and 1:0.60 (wet substrate: water) in mass production, of the solid substrate [17]. The optimum moisture levels of the substrate for M. anisopliae range from 57% to 58% [18]. In the present study, conidial germination and the efficacy of M. anisopliae were tested with a dry substrate at moisture levels from 8% to 35%, at which all isolates caused 100% mortality, except for MAQ-28 (95% mortality). The moisture contents of substrates decreased as water evaporated over time. To avoid contamination, the moisture levels were determined by testing the initial moisture contents of the substrates before inoculation. This study was conducted to test the efficacy of M. anisopliae under desiccation stress. The substrates become drier over the testing course, and the tested efficacies of the isolates might be slightly negative for the tested moisture levels.
Infection characteristics of MAX-2 under desiccation stress
M. anisopliae invades and infects the body of an insect by direct penetration of the cuticle or breathing apertures, ingestion into the digestive tract, or wounds [19]. The infected insects lose their appetite and exhibit somewhat sluggish behavior. Some changes in color might be observed shortly before death. At high humidity, the hyphae emerge through the cuticle and form a hyphal layer on the surface of the insect, and the conidium then emerges after death [20, 21]. The outward signs of infection on T. molitor larvae inflicted with M. anisopliae isolate MAX-2 under desiccation stress differed from those in the wet microhabitat. The treated larvae showed dark black internodes and fungal growth after death in the wet microhabitat. However, local black patches appeared on the cuticles and the cadavers dried, and no fungal growth after death was observed under desiccation stress. This phenomenon was possibly due to the possible production of defense measures by the larvae against a finite number of conidia, which had contact with the larvae in the dry microhabitat. Insects usually activate polyphenol oxidase and melanize their cuticles when wounded or infected with microbial pathogens to heal wounds or prevent microbial intrusion [22]. The local black patches on T. molitor larvae in the dry microhabitat could come from their own polyphenol oxidase activity or resistance to other pathogens. This phenomenon was supported by the few larvae that survived and exuviated, leaving the shell with local black patches (Figure 3i). The wet substrate allowed the production of mass mycelia and conidia, which added to the initial inoculum concentration and increased the penetration efficiency. In addition, the mycelia and its toxin released in the substrate could be ingested by the larvae. The larvae had little chance to protect against invasion, and no local black spots were found. This observation was supported by the high mortality in the wet microhabitat for all isolates. Whether the different symptoms suggest diverse infection mechanisms to T. molitor larvae is worthy of further investigation.
Efficacy of M. anisopliae isolate against pests under desiccation environment
As an alternative to chemical control, the use of fungal insecticides for the biological control of insect pests has attracted significant interest. However, entomopathogenic fungi have not achieved wide-scale use in agriculture in spite of their apparent efficacy in small-scale field trials, mainly because they require high humidity and temperature to grow and disperse. M. anisopliae is a common soil-borne entomopathogenic fungus that is found worldwide, and environmental factors affect its persistence and activity. Moisture level is a major factor that affects the ability of fungi to survive, propagate, and infect and kill their host [23]. The field moisture level usually does not satisfy the requirements for germination and growth of M. anisopliae[24]. Studies on drought tolerance, which is a key part of stress tolerance, are important for the use of fungi in biocontrol [5, 25]. Our results indicate that M. anisopliae isolate MAX-2 maintained high efficacy under desiccation stress, and exhibited great potential for development.
The isolate was obtained from Shangri-la in Yunnan, China. This region is at high altitude with an extensive annual arid period, high UV radiation, and dry and windy weather. The fungi might have developed desiccation tolerance to adapt to the extreme environment, such as low humidity. The tolerance of this fungus to other stressors needs further investigation. The characteristics of MAX-2 provide genetic resources of resistance, and indicate the potential of developing a biopesticide from the fungal isolate for managing pests under desiccation stress.