Mycelial Formulation And Epizootic Potential Of Obligate Aphid Pathogen Pandora Nouryi (Entomophthorales)

Entomophthoralean fungi include over 30 obligate or non-obligate aphid pathogens which are fungal biocontrol agents of global aphids. This study sought to explore epizootic mechanism and biocontrol potential of Pandora nouryi, an obligate pathogen infecting aphids in Asia and Europe. Emphasis was placed upon a mechanism involved in resting spores formation in vivo, mycelium formulation and application for epizootic induction. The results are summarized below.Mechanism involved in resting spore formation in vivo. Among several Pandora species infecting aphids, only P. nouryi is known to produce resting spores (azygospores) in vivo for surviving host absence in situ. To explore a possible mechanism involved in resting spore formation in vivo, the cohorts of 40-60 nymphs of green peach aphid Myzus persicae produced within 24 h on cabbage leaf discs in petri dishes were exposed to spore showers of P. nouryi at the concentrations (C) from a very few to nearly 2000 conidia/mm2 and then reared at the regimes of 10-25℃(T) and 8-16 h daylight (HL) or ambient (17.5±3.1℃, 13:11 L:D). Aphid mortalities observed in 7-11 days from 36-83 cohorts showered at the gradient spore concentrations at each regime showed typical sigmoid trend and fit well a general logistic equation (0.79≤r2≤0.88), yielding similar LC50S of 1.7-6.1 conidia/mm2 with no significant difference from one regime to another. The proportions (P) of cadavers forming resting spores in the cohorts also fit the same equation (0.73≤r2≤0.85) at all tested regimes except at 10℃, a temperature below the low limit of an optimal thermal range for both host and pathogen. This indicates the dependence of resting spore formation in vivo on C. The effects of T and HL on P over C were well elucidated by the fitted complex logistic equations P=0.578/{l+exp[1.710-(0.136-0.00537)C]} and P=0.534/{1+exp[1.639+(0.034-0.0053HL)C]} (both r2=0.79). Our results highlight that the resting spore formation in vivo of P. nouryi is regulated primarily by the concentration of host-infecting conidia discharged from cadavers and facilitated by lower temperature and longer daylight.Sporulation, storage and infectivity of P. nouryi grown on novel granules of broomcorn millet and polymer gel. Pandora species dominates aphid mycosis worldwide but requires fastidious nutrition for growth and saturated humidity for spore discharge and transmission in host cohorts. Twelve preparations of 2-5-mm-long granules (3-4 mg/cm) were made of the mixtures of 80-95% millet powder with 5-20% polymer gel of polyacrylamide, polyacrylate or acrylate-acrylamide copolymer for solid culture of P. nouryi. The preparations were inoculated with mycelia at optimized biomass level of 30 mg/g dry granules plus 87.5% water content, followed by static incubation at 20℃for 4-12 days. The obligate aphid-pathogenic fungus grew well on all preparations but best on 10% copolymer-inclusive one. An 8-day culture of this preparation discharged maximal yield of 58.5x104 conidia/mg granule at 100% RH within 6 days in the temporal discharge pattern of 31.1,30.6,15.8,10.3,7.9 and 4.3% on days 1-6, respectively. Surprisingly, the discharge occurred at non-saturated humidity, forming typical spore halo at 86-97% RH. Stored at 6℃, granular cultures with>85% water content had 2-fold longevity (120 days) and half-decline period (34-36 days) of those observed at ambient temperature. The steadily high water content resulted in a longevity 40 days longer than that decreasing at 6℃despite similar half-decline periods. However, conidia from 70-day-stored granules were less infective to Myzus persicae nymphs than those from fresh ones due to a difference between their LC50s on days 4-6 after inoculation. The novel millet-gel granules were not only suitable for culturing P. nouryi for high sporulation capacity but also capable of reducing humidity requirement for spore discharge, thus increasing their potential for use in aphid control.Epizootic potential of P. nouryi in caged aphid population. To evaluate the epizootic potential of P. nouryi in aphid population, we manufactured small uniform alginate pellets (-1 mg each in dry weight) entrapping-1% P. nouryi mycelia as inoculum source and-5% millet powder as nutritional source to support fungal growth and spore discharge and released them in M. persicae population developing on cabbage plants in meshed field cages (2×2×2 m). The millet-inclusive pellets showed a sporulation capacity (28.4×104 conidia per capita) 8.5-fold higher than that of the millet-free pellets at 20℃and sporulated 3.6-13.6-fold more and much longer than mycotized adult cadavers at 4-28℃under saturated humidity. These pellets, irrespective of drying or not, retained sporulation capability after 120-day storage at 6℃. The releases of 5-20 pellets per plant in four cages resulted in successful colonization of P. nouryi in aphid cohorts during observation from late autumn through spring at weekly interval. Aphid mortalities attributed to P. nouryi mycosis were consistently detectable but very low in all the cages, irrespective of the timing of release and the density of released pellets, until in late spring 1.3-13% mortalities occurred in different cages. Cadavers forming resting spores on sampled plants took high proportions (32-95%) on most sample occasions. Conclusively, resting spore formation in vivo was causative of the low prevalence level of P. nouryi, reducing its biocontrol potential against aphids. The alginate pellet formulation entrapping both mycelia and millet powder is recommended for improving preparations of resting spore-free fungal pathogens, such as P. neoaphidis, for biological control of aphids.In conclusion, our results highlight that the resting spore formation in vivo of P. nouryi is regulated primarily by the concentration of host-infecting conidia discharged from cadavers and facilitated by lower temperature and longer daylight. The millet-gel and millet-alginate technology developed in this study provides novel means to formulating P. nouryi and other entomophthoralean fungi for high sporulation capacity.