| Fungal biocontrol agents such as Paecilomyces fumosoroseus and Beauveria bassiana in the Hyphomycetes take increasing parts in modern crop protection as more and more formulations of such fungi are incorporated into crop systems for the decreasing dependence of insect pest control on chemical pesticides. Studies on various aspects of the fungal agents have been undertaken in the past few decades and more recent efforts have been made to develop myconisecticides against insect pests because of technical revolution needed for insect control. However, solid fermentation apparatuses suitable for efficient mass production of high-quality aerial conidia as active ingredients of mycoinsecticides are lacking while screening of highly virulent fungal candidates against a specific target pest with peculiar damage behavior is always a routine. To search for alternative strategy and tactics for control of aphids that heavily infest Chrysanthemum morifolium Ramat grown for production of traditional tea 'Hang Bai Ju' in Zhejiang Province, this study sought to select fungal candidates highly virulent to the chrysanthemum aphid, Macrosiphoniella Sanborni, from B. bassiana and P. fumosoroseus strains by optimizing a bioassay protocol in accordance with international standards, and to evaluate interaction of the fungal formulation of a selected B. bassiana strain with sub-lethal application rates of imidacloprid, a common aphidicide. A novel solid fermentation apparatus, namely upright multi-tray conidiation chamber, was developed to fit up bulk production of aerial conidia of the fungal strains as well as other fungal biocontrol agents and tested in four pilot trials with each including 50-kg rice substrate for conidial production. The results are summarized below.Comparative virulence of P. fumosoroseus and B. bassiana isolates to M. Sanborni.Eight isolates of P. fumosoroseus (Pfr116, Pfr612, Pfr2175 and Pfr153) and B. bassiana (Bb2880, Bb2860, Bb2861 and Bb2879) were bioassayed against M. Sanborni under laboratory conditions. Detached leaves of C. morifolium cultivar Xiaobaiju were prepared to have hairy roots grown into nutritional agar in Petri dishes in advance and each were artificially infested with 30-40 newly emerged apterous adults. Aphids on the leaves were separately exposed to conidial suspension spray under a Potter Spray Tower at the concentrations of 21-73 (low), 96-218 (medium) or 508-902 (high) conidia/mm~2 for the P.fumosoroseus isolates, or at 10-47 (low), 76-158 (medium) or 435-646 (high) conidia/mm2 for the B. bassiana isolates. All the assays included black control (sprayed with 0.02% tween 80) and three replicates with each spray. After exposure, the aphids were maintained at the regime of 25°C and 12:12 (L:D) for 8 days and examined daily for mortality records. As a consequence, the low, medium and high concentrations resulted in cumulative mortalities of 17.4-100.0% for P. fumosoroseus and 37.3-100.0% for B. bassiana, respectively. The data resulted from each assay fit very well to a time-concentration-mortality model, generating the estimates of lethal concentrations (LC) variable with days after exposure and lethal times (LT) depending on conidial concentrations. The estimates of LC50 (unit: conidia/mm2) with 95% confidence intervals were 126(107-149) for Pfrll6, 442(329-596) for Pfr612, 213(158-286) for Pfrl53, 234 (168-328) for Pfr2175, 155 (129-186) for Bb2880, 86 (73-101) for Bb2860, 99(73-133) for Bb2861 and 138 (89-214) for Bb2879 on day 5 after exposure, respectively. On day 8, however, the LC50 estimates decreased to 59(49-72), 139 (110-177), 52(34-81), 79(55-115), 63(51-78), 25(20-30), 27(18-40) and 30(16-57) for the concerned isolates, respectively. The estimates of LT50 for the 8 isolates against the pest species decreased with higher conidial concentrations, and the differences among the isolates tended to decline with the increasing concentrations. At 150 conidia/mm2, the LT50 estimates ranged from 4.5 to 7.7 days for the P. fumosoroseus isolates and from 4.5 to 5.1 days for the B. bassiana isolates. However, the LT50 at 500 conidia/mm2 decreased to 2.8-4.9 days for P. fumosoroseus or 3.7-4.0 days for B. bassiana. Based on comparison of both LC50 and LT50 estimates, the isolates Bb2860 and Pfr2175 were desirable candidates with high virulence and fast infection to the aphid pest. Thus, the isolate Bb2860 was chosen for further study as follows.Time and concentration dependent interactions of B. bassiana with sublethal rates of imidacloprid against M. sanborni. A series of laboratory bioassays with each consisting of low, medium and high concentrations of the selected isolate Bb2860 conidia sprayed alone or together with an increasing sublethal rate of imidacloprid (0.01-0.05 a.i. ug/ml) were conducted to quantify the fungal and chemical interactions on apterous adults of M. sanborni. The responses of the aphid species in each of five bioassays fit well to the time-concentration-mortality model, indicating a strong dependence of the fungal andchemical interactions on both concentration and post-spray time. Adding imidacloprid to the fungal sprays at the rates of 0.025-0.05 a.i. (ig /mL significantly, enhanced or accelerated the fungal action on M. sanborni. Based on the LC50 or LC90 estimates and their variances determined by the fitted time-concentration-mortality relationships, the relative potencies of an imidacloprid-inclusive bioassay over those with the fungal spray alone or together with a lower sublethal rate was enhanced significantly and varied over days after spray. These results indicate an alternative tactic for practical control of the aphid pest by a combined formulation or application of B. bassiana and imidacloprid and for management of aphidresistance to the chemical insecticide.New solid-state fermentation chamber for bulk production of aerial conidia of fungal biocontrol agents on rice. A novel solid-state fermentation apparatus, namely an upright multi-tray conidiation chamber, was developed to facilitate the production of aerial conidia of fungal biocontrol agents in the Hypomycetes, such as B. bassiana. To test the performance of the conidiation chamber, four pilot trials were separately carried out for production of fungal conidia of the strain Bb2860 on low-quality rice. The chamber with 25 bottom-meshed metal trays had a capacity of >50 kg rice with each tray holding >2 kg. Overall, the chamber worked very well in all trials, and the high quality of the harvested conidial powder was featured with the purity of 1.7 (1.4-1.9) xlO11 conidia/g, the viability of 94.0% (92.1-95.7%), and the water content of 12.8% (9.9-18.1%). On average, conidial yield reached 14.6 (13.0-17.1) g/kg rice, which was equivalent to 2.4 (1.8-2.7) xlO12 conidia/kg rice after 7-day fermentation in a fully-loaded chamber at the rice consumption rate of 19.9% (16.2-21.8%) only. To monitor temperature and RH inside the chamber during SSF, both variables were hourly recorded by three digital recorders hung in the upper, middle and lower layers of the chamber, respectively. Generally, both variables were well controlled in different seasons despite slight variation within the chamber. The RH was controlled more readily by the mist supply than was the temperature by air conditioner. Thus, the conidiation chamber developed in this study is of high potential for use in large-scale production of aerial conidia of B. bassiana and other fungal biocontrol agents.In summary, a biologically robust bioassay system has been established to quantitatively evaluate separate or interactive effects of fungal biocontrol agents and selected insecticides on insect pests. Our results presented above clearly indicate that B. bassiana Bb2860 was high virulence to M. sanborni and incorporating low rates ofimidacloprid into its formulation enabled to take advantages of both agents for control of the aphid species. With conidial yield, production cost, fermentation controllability, harvest and efficiency in consideration, the novel multi-tray conidiation chamber and associated technology developed in this study has shown a high potential for bulk production of B. bassiana conidia. These progresses would help to facilitate development of the fungal formulation for application to aphid control in the area of chrysanthemum grown for the traditional tea production.
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