Millet Cultures Of Entomophthoralean Fungi Pandora Neoaphidis And Zoophthora Radicans: Sporulation Capacity, Infectivity And Preservation

Both Pandora and Zoophthora are important genera in the Entomophthorales and are regarded as fungal biocontrol agents of insect pests. For instance, Pandora neoaphidis is an obligated aphid pathogen that causes epizootics in aphid populations worldwide. The fungus Zoophthora radicans is also an obligate insect pathogen capable of infecting a wide range of host insects, particularly Homoptera and Lepidoptera. Technical difficulties with propagation in vitro of Pandora and Zoophthora remain obstacles to utilizing these important fungal pathogens for insect control because of their host specificity and fastidious nutritional requirements. In the present study, cereals such as millets were used as substrate for granular cultures of P. neoaphidis F98028 and Z. radicans ARSEF1100 in order to develop a solid culture technology for easy and cheap propagation of entomophthoralean inocula. Sporulation capacity and timing pattern were relied upon to evaluate the quality of millet cultures and identify potential factors influential on the quality. Infectivity or virulence of the inocula derived from the millet cultures was assayed against the green peach aphid Myzus persicae and the diamondback moth Plutella xylostella, respectively. The effect of the environmental factors on the decline rate of sporulation capacity of the millet cultures during long-term storage were determined by modeling analysis. The results are summarized as follows.Millet cultures of P. neoaphidis. Shelled grains of the broomcorn millet Panicum miliaceum and the foxtail millet Setaria italica were used as substrate to directly culture P. neoaphidis F98028 in vitro. To initiate the culture, steamed millets with water content of 36.5% were inoculated with mashed colonies of P. neoaphidis grown on egg yolk-milk agar and then incubated at 20°C and L:D 12:12. The inoculation was completed by mixing 15-g (dry wt) millets (in a 100-ml flask) with mashed pieces of half a colony in 3-ml modified Sabouraud dextrose broth. During a 21-day period of incubation, 20 millets were sampled at 3-day intervals from day 3 on and individually assessed for sporulation capacity and timing pattern using a self-designed device for spore collection. As a consequence, the broomcorn millet cultures incubated for 6-15 days were capable of producing 16.8-23.4×l0~4 conidia per millet and discharging conidia for 6 days. So cultured millets individually sporulated over twice more or longer than did M. persicae apterae of killed by P. neoaphidis (8.4×10~4 conidia per cadaver). The best foxtail millet cultures attained by 12-day incubationdischarged 5.6x104 conidia per millet, a sporulation capacity lower than that of the broomcorn millets. Based on differences in grain sizes and sporulation capacities between both millets, the cultures grown on the broomcorn millets were superior to those on foxtail millets and also sporulated much better than aphid cadavers killed by the same fungal species.Comparative virulence of P. neoaphidis cultures. Both broomcorn millet cultures achieved by 15-day incubation and mycelial mats produced in liquid culture were bioassayed on M. persicae nymphs by exposing them to conidial showers at 9 concentrations for inoculation. Based on the modeling of the resultant time-dose-mortality data, the estimates of LC50 for the cultured millets were 124.8, 21.4, 7.3, and 4.9 conidia/mm2 on days 4, 5, 6, and 7, respectively;the corresponding estimates for mycelial mats were 175.0, 22.1, 10.6, and 7.7 conidia /mm2. The largest LT50, 6.9 days, corresponded to exposures of 5.0 and 8.0 conidia/mm2 for the cultured millets and mycelial mats. Higher conidial concentrations led to difference-diminishing LT50 estimates for both bioassays. Conclusively, the millet cultures of P. neoaphidis are biologically similar to the mycelial mats and highly virulent to the aphid species tested.Preservation of P. neoaphidis millet cultures Broomcorn millet cultures of P.neoaphidis with initial sporulation capacity (Cs) of 32.0x104 conidia/millet and watercontent (Cw) of 48.7% were prepared by 15-day incubation at 20°C, then kept inpaper-sealed (ambient humidity) or parafilm-sealed (saturated) beakers or in 85 and 98%RH chambers, and monitored for Cw and Cs variations during 200-day storage at 5, 10 and20°C, respectively. The paper-sealed cultures, associated with 21-25% of Cw, were bestpreserved at 5°C, resulting in a 120-day Cs similar to that of fresh cadavers. Constant orvariable high RH at 5°C resulted in significantly higher Cw and lower Cs despite longerpersistence during the storage. The regimes at >10°C preserved the cultures for <40 days.The observations fit well to the model CJ=35.28/{l+exp[-2.36+(-0.003Cw+0.001Cw7>]}(r2=0.95) for all regimes of temperature (7) or C,=35.55/[l+exp(-2.33+O.001Cw0] (r2=0.93)at 5°C only. The C,-decline rate of-0.003Cw+0.001Cwror 0.00\CW over days (t) highlightsthe primary effect of Cw. The daily Cj-decline rates fitted for the best-stored cultures andair-dried cadavers stored at 5°C were surprisingly identical. The results suggest a possiblemean to proper manipulation of the millet cultures through appropriate Cw control.Sporulation capacity, preservation and infectivity of Z. radicans millet cultures to Plutella xylostella. The broomcorn millet culture method was applied to granular culturesof Z radicans ARSEF1100. Steamed millets inoculated with mashed colony pieces in Sabouraud dextrose broth were incubated at 15°C and L:D 12:12 for up to 24 days. The millet cultures incubated for >12 days produced 12.0-14.9xl04 conidia/millet during a 7-day period. The maximal sporulation capacity associated with the 21-day-old culture was about half of that of Z radiccms-killed P. xylostella larvae (28.7x104 conidia/cadaver), which individually were at least three times larger than the millets. Based on the time-dose-mortality responses of second instar larvae to Z radicans in three independent bioassays, the conidia ejected from the millet cultures, the mycelial mats produced in liquid culture, and larval cadavers displayed insignificant variation in infectivity to the host species, and yielded similar LC50 and LT50 estimates. The effects of varying water content (Cw) on the decline of sporulation capacity (Cs) over storage days (t) were clarified by fitting the observations at 5°C to the logistic model Cr=3.34/[l+exp(-3.68+1.14Cw0] (r2=0.99). The G-decline rate of 1.14CW over days (t) showed the primary effect of Cw. Thus, manipulation or formulation of the broomcorn millet cultures for longer storage or better application would be possible if cautious Cw control at the low temperature was taken into account. Conclusively, the broomcorn millet-based technology for production of granular cultures of Z radicans was easy, inexpensive and highly efficient.Based on the results presented above, it was successful using the millets as solid substrate for preparing granular cultures of P. neoaphidis and Z radicans. The millet cultures prepared with this novel method have proven to biologically function as mycotized aphid cadavers regarding either sporulation or infectivity. The millet cultures hence can be regarded as mimic cadavers and preserved for several months at 5°C if their water content control is appropriate. This technology may suit to production of other culturable but nutritionally fastidious entomopathogens in the Entomophthorales and thus would facilitate the study and utilization of such fungi for insect control.