Broomcorn Millet Culture Of Obligate Aphid Pathogen Zoophthora Anhuiensis And Its Biological Feature And Function

The fungal species, Zoophthora anhuiensis (Zygomycotina: Entomophthorales), is an obligate aphid pathogen found in China only. As an important fungal biocontrol agent, Z anhuiensis frequently causes epizootics in aphid populations in middle-lower reaches of Changjiang River in autumn and winter. However, technical difficulties with propagation in vitro of Z anhuiensis and other obligate insect pathogens remain obstacles to utilization of entomophthoralean fungi in pest control because of their host specificity and fastidious requirements for nutrients. The present study sought to develop a solid-culture-based technology for easy and cheap propagation of Z. anhuiensis inoculum using small grains as substrate for fungal growth. Sporulation capacity and timing pattern were evaluated for the quality of the millet cultures. The Infectivity or virulence of the inocula derived from the cultures was assayed against the green peach aphid, Myzus persicae. The results are summarized below.Broomcorn millet cultures. Broomcom millets (yielded by the crop Panicum miliaceum) from market were used as substrate for solid culture of Z anhuiensis ZU1102, an aphid-derived isolate. To initiate the culture, properly steamed millets with water content of 45% in flasks were mixed with pieces of the fungal colonies grown on egg yolk-milk agar and then maintained for stationary incubation at the regime of 20℃ and 12:12 L:D. During a 15-day period of incubation, 12 millets were sampled from each flask at 2-day intervals from day 3 onwards and individually assessed for their sporulation capacity and timing pattern using a self-designed device for spore collection. As a consequence, the 5-day-old millet cultures had best sporulation capacity, discharging on average 13.0×10⁴ conidia per millet and sporulating for up to 6 days. The millets cultures incubated for 5-11 days discharged conidia 4.2-5.3 times more and twice longer than aphid cadavers killed by Z anhuiensis (2.5×10⁴ conidia per cadaver during ≤60-h sporulation period). The results indicate that the millet cultures of Z. anhuiensis are not only biologically similar to mycosed aphid cadavers but superior to them in sporulation capacity and persistence.Infectivity of the millet cultures to aphids. Nymphs of M. persicae on detached cabbage leaves were exposed to the shower of primary conidia discharged from the 7-day-old millet cultures, generating nine dose treatments ranging from 7.9 to 134.9 conidia per mm². The data resulted from daily observations of the inoculated aphids fit very well to the time-dose-mortality model. Based on the fitted time- and dose-effect parameters for the model, the LC50 and LC₉₀ of the fungus against M. persicae were 59.8 and 354 conidia per mm on day 5 after exposure, 39.5 and 234 conidia per mm² on day 6, and 33.5 and 198conidia per mm2 on day 7, respectively. The LT50 decreased to 4.3 days at the dose of 134.9 conidia per mm2 from 5.1 dasy at 57.7 conidia per mm2. The results indicate that the millet cultures of Z. anhuiensis were highly infective and virulent to M. persicae.Preservation of the millet cultures. To search for a proper method for preservation, the 7-day-old millet cultures of Z. anhuiensis were enclosed in beakers sealed by newspaper or by parafilm excluding or including silica gel at the beaker bottom and then stored at 4-20°C in dark. Their sporulation capacity and water content wee monitored by sampling at 20-day intervals during 80-day storage. As a result, the millet cultures in the three treatments at 20°C retained only 9.2, 8.5 and 7.7% of their sporulcation capacity (12.3xlO4 conidia per millet at the initial time after 20-day storage, respectively. Those stored at 10°C for 20 days lost 76.1, 86.3 and 86.4% of their sporulation capacity, respectively. In contrast, the millet cultures stored at 4°C lost their sporulation capacity by 50.3, 68.8 and 73.8% on day 20 after storage and were capable of discharging 4700-8600 conidia per millet on day 80. The results indicate that the millet cultures in the paper-sealed beaker at 4°C were better preserved than those in other treatments.Based on the results presented above, it was successful using the broomcorn millets as substrate for preparing granular cultures of Z. anhuiensis. The millet cultures made with this novel method have proven biologically similar to mycosed aphid cadavers regarding sporulation and infectivity. Thus, the millet cultures are cadaver-mimic materials and can be preserved at the low temperature for up to 80 days. The method developed in this study would facilitate study and utilization of the entomophthoralean fungi for insect control.