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Construction of a hypervirulent and specific mycoinsecticide for locust control.

Fang W, Lu HL, King GF, St Leger RJ - Sci Rep (2014)

Bottom Line: We found that expression of four insect specific neurotoxins improved the efficacy of M. acridum against acridids by reducing lethal dose, time to kill and food consumption.Coinoculating recombinant strains expressing AaIT1(a sodium channel blocker) and hybrid-toxin (a blocker of both potassium and calcium channels), produced synergistic effects, including an 11.5-fold reduction in LC50, 43% reduction in LT50 and a 78% reduction in food consumption.However, specificity was retained as the recombinant strains did not cause disease in non-acridids.

View Article: PubMed Central - PubMed

Affiliation: Institute of Microbiology, College of Life Sciences, Zhejiang University, Hangzhou. 310058, Zhejiang, China.

ABSTRACT
Locusts and grasshoppers (acridids) are among the worst pests of crops and grasslands worldwide. Metarhizium acridum, a fungal pathogen that specifically infects acridids, has been developed as a control agent but its utility is limited by slow kill time and greater expense than chemical insecticides. We found that expression of four insect specific neurotoxins improved the efficacy of M. acridum against acridids by reducing lethal dose, time to kill and food consumption. Coinoculating recombinant strains expressing AaIT1(a sodium channel blocker) and hybrid-toxin (a blocker of both potassium and calcium channels), produced synergistic effects, including an 11.5-fold reduction in LC50, 43% reduction in LT50 and a 78% reduction in food consumption. However, specificity was retained as the recombinant strains did not cause disease in non-acridids. Our results identify a repertoire of toxins with different modes of action that improve the utility of fungi as specific control agents of insects.

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LT50 values of the wild-type strain versus transformants at four inoculation doses (100, 500, 1,000 and 10,000 conidia per insect).WT: the wild-type strain; AaIT1 and Hybrid are transformants expressing AaIT1 and hybrid-toxin, respectively. AaIT1/Hybrid: a 1:1 combination of transformants AaIT1 and hybrid-toxin. At inoculation dose of 100 conidia per insect, the LT50 of WT could not be calculated because of its failure to kill.
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f2: LT50 values of the wild-type strain versus transformants at four inoculation doses (100, 500, 1,000 and 10,000 conidia per insect).WT: the wild-type strain; AaIT1 and Hybrid are transformants expressing AaIT1 and hybrid-toxin, respectively. AaIT1/Hybrid: a 1:1 combination of transformants AaIT1 and hybrid-toxin. At inoculation dose of 100 conidia per insect, the LT50 of WT could not be calculated because of its failure to kill.

Mentions: The virulence of each transformant was initially surveyed by calculating the median lethal time (LT50, time taken to kill 50% of insects) using 1,000 conidia per grasshopper (M.femur-rubrum) (Fig. 2). Strains expressing each of the toxins killed significantly faster than the wild-type fungus (P < 0.001). Expressing AaIT1, Hv1a, Hv1c or hybrid-toxin reduced LT50 values by 16%, 18.5%, 17% and 31%, respectively. Expression of hybrid-toxin killed grasshoppers significantly faster than expression of AaIT1, Hv1a or Hv1c (P < 0.001), while AaIT1, Hv1a and Hv1c killed at similar speeds (P > 0.25). Because AaIT1 blocks NaV channels and hybrid-toxin targets both CaV and KCa channels, we next looked for additive or synergistic effects between AaIT1 and hybrid-toxin by co-inoculating grasshoppers with conidia of 324-AaIT1 (transgenic M. acridum ARSEF324 expressing AaIT1. The naming system is also used for other transgenic strains) and 324-HYBRID. The wild-type fungus was lethal at 500 conidia per grasshopper (LT50 9.5 days) but 100 conidia failed to kill, whereas grasshoppers were killed by 100 conidia carrying genes for hybrid toxin (LT50 8.6 days) or AaIT1 (LT50 12.8 days), with hybrid-toxin killing significantly faster (P < 0.001). Grasshoppers co-inoculated with 50 conidia of both 324-AaIT1 and 324-HYBRID died significantly faster (LT50 7.5 days) than insects exposed to single toxins (P < 0.01). At 500 conidia per insect, LT50 values were reduced by 20% (expressing AaIT1), 33% (expressing hybrid-toxin) and 43% (combining 324-AaIT1 and 324-HYBRID), compared to wild-type fungus (LT50 9.5 days). At higher inoculums (1,000 or 10,000 conidia per insect), the results show trends that are similar but toxin expression has comparatively less affect than with lower inoculums. Strains expressing either hybrid-toxin or AaIT1 killed significantly faster than the wild-type fungus (P < 0.001), and a combination of 324-HYBRID and 324-AaIT1 killed significantly more rapidly than the 324-HYBRID alone (P < 0.001). However, even though 10,000 conidia of a 1:1 combination of 324-HYBRID and 324-AaIT1 achieved an LT50 value of only 3.9 days, the 11.4% reduction compared with 324-HYBRID alone is less than that seen with smaller inoculums (Fig. 2). This data indicates that 324-HYBRID and 324-AaIT1 have synergistic or at least additive interactions. A binomial test confirmed synergistic interactions 4–8 days post-inoculation with 100, 500 and 1,000 conidia per insect (χ2 > 16.8). With 10,000 conidia, synergistic effects were detected 4 and 5 days post-inoculation (χ2 > 10.2); however, only additive effects were observed at 6 and 7 days post-inoculation (χ2 < 2.2).


Construction of a hypervirulent and specific mycoinsecticide for locust control.

Fang W, Lu HL, King GF, St Leger RJ - Sci Rep (2014)

LT50 values of the wild-type strain versus transformants at four inoculation doses (100, 500, 1,000 and 10,000 conidia per insect).WT: the wild-type strain; AaIT1 and Hybrid are transformants expressing AaIT1 and hybrid-toxin, respectively. AaIT1/Hybrid: a 1:1 combination of transformants AaIT1 and hybrid-toxin. At inoculation dose of 100 conidia per insect, the LT50 of WT could not be calculated because of its failure to kill.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4256560&req=5

f2: LT50 values of the wild-type strain versus transformants at four inoculation doses (100, 500, 1,000 and 10,000 conidia per insect).WT: the wild-type strain; AaIT1 and Hybrid are transformants expressing AaIT1 and hybrid-toxin, respectively. AaIT1/Hybrid: a 1:1 combination of transformants AaIT1 and hybrid-toxin. At inoculation dose of 100 conidia per insect, the LT50 of WT could not be calculated because of its failure to kill.
Mentions: The virulence of each transformant was initially surveyed by calculating the median lethal time (LT50, time taken to kill 50% of insects) using 1,000 conidia per grasshopper (M.femur-rubrum) (Fig. 2). Strains expressing each of the toxins killed significantly faster than the wild-type fungus (P < 0.001). Expressing AaIT1, Hv1a, Hv1c or hybrid-toxin reduced LT50 values by 16%, 18.5%, 17% and 31%, respectively. Expression of hybrid-toxin killed grasshoppers significantly faster than expression of AaIT1, Hv1a or Hv1c (P < 0.001), while AaIT1, Hv1a and Hv1c killed at similar speeds (P > 0.25). Because AaIT1 blocks NaV channels and hybrid-toxin targets both CaV and KCa channels, we next looked for additive or synergistic effects between AaIT1 and hybrid-toxin by co-inoculating grasshoppers with conidia of 324-AaIT1 (transgenic M. acridum ARSEF324 expressing AaIT1. The naming system is also used for other transgenic strains) and 324-HYBRID. The wild-type fungus was lethal at 500 conidia per grasshopper (LT50 9.5 days) but 100 conidia failed to kill, whereas grasshoppers were killed by 100 conidia carrying genes for hybrid toxin (LT50 8.6 days) or AaIT1 (LT50 12.8 days), with hybrid-toxin killing significantly faster (P < 0.001). Grasshoppers co-inoculated with 50 conidia of both 324-AaIT1 and 324-HYBRID died significantly faster (LT50 7.5 days) than insects exposed to single toxins (P < 0.01). At 500 conidia per insect, LT50 values were reduced by 20% (expressing AaIT1), 33% (expressing hybrid-toxin) and 43% (combining 324-AaIT1 and 324-HYBRID), compared to wild-type fungus (LT50 9.5 days). At higher inoculums (1,000 or 10,000 conidia per insect), the results show trends that are similar but toxin expression has comparatively less affect than with lower inoculums. Strains expressing either hybrid-toxin or AaIT1 killed significantly faster than the wild-type fungus (P < 0.001), and a combination of 324-HYBRID and 324-AaIT1 killed significantly more rapidly than the 324-HYBRID alone (P < 0.001). However, even though 10,000 conidia of a 1:1 combination of 324-HYBRID and 324-AaIT1 achieved an LT50 value of only 3.9 days, the 11.4% reduction compared with 324-HYBRID alone is less than that seen with smaller inoculums (Fig. 2). This data indicates that 324-HYBRID and 324-AaIT1 have synergistic or at least additive interactions. A binomial test confirmed synergistic interactions 4–8 days post-inoculation with 100, 500 and 1,000 conidia per insect (χ2 > 16.8). With 10,000 conidia, synergistic effects were detected 4 and 5 days post-inoculation (χ2 > 10.2); however, only additive effects were observed at 6 and 7 days post-inoculation (χ2 < 2.2).

Bottom Line: We found that expression of four insect specific neurotoxins improved the efficacy of M. acridum against acridids by reducing lethal dose, time to kill and food consumption.Coinoculating recombinant strains expressing AaIT1(a sodium channel blocker) and hybrid-toxin (a blocker of both potassium and calcium channels), produced synergistic effects, including an 11.5-fold reduction in LC50, 43% reduction in LT50 and a 78% reduction in food consumption.However, specificity was retained as the recombinant strains did not cause disease in non-acridids.

View Article: PubMed Central - PubMed

Affiliation: Institute of Microbiology, College of Life Sciences, Zhejiang University, Hangzhou. 310058, Zhejiang, China.

ABSTRACT
Locusts and grasshoppers (acridids) are among the worst pests of crops and grasslands worldwide. Metarhizium acridum, a fungal pathogen that specifically infects acridids, has been developed as a control agent but its utility is limited by slow kill time and greater expense than chemical insecticides. We found that expression of four insect specific neurotoxins improved the efficacy of M. acridum against acridids by reducing lethal dose, time to kill and food consumption. Coinoculating recombinant strains expressing AaIT1(a sodium channel blocker) and hybrid-toxin (a blocker of both potassium and calcium channels), produced synergistic effects, including an 11.5-fold reduction in LC50, 43% reduction in LT50 and a 78% reduction in food consumption. However, specificity was retained as the recombinant strains did not cause disease in non-acridids. Our results identify a repertoire of toxins with different modes of action that improve the utility of fungi as specific control agents of insects.

Show MeSH
Related in: MedlinePlus