Biocontrol Potential Of Metarhizium Isolates Against Aphids And Hydrophobicity-Related Traits Of Beauveria Bassiana, Metarhizium Spp. And Paecilomyces Fumosoroseus For Use In Microbial Control

Entomopathogenic fungi,such as Beauveria,Metarhizium and Paecilomyces,are fungal biocontrol agents against various insect pests.Virulence and hydrophobicity of their conidia are important concerns for choosing fungal candidates for the development of commercial formulations because the virulence determines the biocontrol potential and the hydrophobicity mediates adhesion of conidia to insect cuticle and affects their dispersivity in aqueous dilutions for field spray.In this study,the biocontrol potential of 23 isolates of Metarhizium spp.against green peach aphid Myzus Persicae was evaluated in attempt to select highly aphidicial candidates.Hydrophobicite rates and superficial areas of aerial conidia from 48 isolates of Beauveria bassiana,Metarhizium spp.and Paecilomyces fumosoroseus were measured for correlation analysis.Contributions of conidial wall proteins to the hydrophobicity were examined to find useful hydrophobicity-related traits for the formulation design of fungal candidates.The results are summarized below.Biocontrol potential of Metarhizium isolates against M.persicae.Twenty-three isolates of M.anisopliae(Ma),M.anisopliae var.anisopliae(Maan),M.anisopliae var. acridum(Maac),M.anisopliae var.majus(Mam) and M.flavoviride var.minus(Mfm) derived from different host insects worldwide were evaluated for their aphid biocontrol potential,which is not well known.The apterous adults of M.persicae were exposed to the mean concentrations of 11.5,99 and 1179 conidia/mm² as fungal treatments plus blank control in triplicate leaf-dish bioassays of each isolate.All the tested isolates were proven pathogenic to M.persicae at 21±1℃and 14:10 L:D during 8-day observation and caused corrected mortalities of 0.8-95.3%at the high concentration despite natural aphid mycosis rarely attributed to Metarhizium spp.The bioassay data of the 10 isolates causing＞50% mortalities were well fitted to a time-concentration-mortality model,yielding the estimates of their time-depending LC₅₀s and concentration-depending LT₅₀s.The four isolates Ma 759, Ma 576,Maan 4132 and Maan 2080 were selected as excellent candidates for use in aphid control due to the smaller LC₅₀s of 44-80 conidia/mm² on day 8,the shorter LT₅₀s of 4.9-6.8 days at 100 conidia/mm²,and the high percentages of 91-98%mycotized among killed aphids.These indices highlight their high virulence to M.persicae and desired sporulation potential on cadavers.The biocontrol potential of other isolates with computable LC₅₀s and LT₅₀s is discussed with the percentages of mycotized cadavers.Correlation of conidial hydrophobicity to superficial area per conidium.The hydrophobicity rates(H_r) of aerial conidia from 48 isolates of B.bassiana,Metarhizium spp. and P.fumosoroseus were measured in aqueous-solvent partitioning system.The measured H_r values ranged from 59.7(Pf 6032) to 92.2%(Mam 978) and differed significantly among the tested isolates.Differences were also significant among the H_r values of 69.5-87.2% (mean:78.6%) within B.bassiana,of 77.1-92.2%(83.3%) within Metarhizium spp.and of 59.7-79.2%(72.5%) within P.fumosoroseus.The H_r means differed significantly from one to another fungal species.Superficial areas(S_a) of aerial conidia estimated by measuring widths and lengths of the 48 isolates spanned from 7.9(Pf 6032) to 25.3μm² per conidium (Mam 978).The S_a mean(±SD) of 14.4±3.8μm² from the Metarhizium isolates was significantly larger than those of 10.1±1.4 and 9.0±0.7μm² from the isolates of B.bassiana, and P.fumosoroseus;the latter two means had no significant difference.The S_a estimates were also significantly different within each group of the tested isolates.Interestingly,the arcsin-squared roots of the H_r values from the 48 isolates were significantly correlated to the log₁₀-transformed S_a estimates.The fitted coefficient of determination(r²=0.55) for the linear correlation indicates that 55%of the variation in conidial hydrophobicity is attributed to the superficial areas of the tested isolates based on their conidial sizes.Moreover,the S_a estimates were inversely correlated to the densities of aerial conidia at equal weight (r²=0.95).Thus,conidial size that varies with fungal isolates is a factor influential upon conidial hydrophobicity and useful for formulation design.Correlation of hydrophobicity rates to the contents of TFA-soluble proteins from conidial walls.Cell wall proteins were sequentially extracted with boiling sodium dodecyl sulphate(SDS),iced formic acid(FA) and iced trifluoroacetic acid(TFA) from the aerial conidia of six isolates of B.bassiana,M.anisopliae and P.fumosoroseus selected on a basis of their distinguished H_r and S_a values.The contents of TFA-soluble proteins(P_c) fell in a range of 21.5-91.9μg/mg conidia or 2.7-44.8μg per 10⁷ conidia,differing significantly among the tested isolates.The H_r values of the extracted conidia differed significantly among the treatments of the control,SDS and FA for each isolate or among the tested isolates under each treatment.The FA treatments reduced drastically the H_r values of B. bassiana and P.fumosoroseus isolates whereas no difference was significant between the control and SDS treatments.For the M.anisopliae isolates,however,the H_r values were similar before the TFA treatment.The TFA-soluble proteins in the FA-extracts of two B. bassiana isolates were proven the hydrophobins Hyd1(13.81 kDa) and Hyd2(11.98 kDa) known from the fungal species by Western blotting with their polyclonal antibodies.For all the isolates,the H_r values were significantly correlated to the P_c measured asμg per 10⁷ conidia(r²=0.79) and reduced to only 3.5-9.0%by the FA/TFA treatments,which removed hydrophobin-based rodlet layers from conidial surfaces.However,the correlation was not significant(r²=0.44,P=0.15) when the P_c measured asμg/mg conidia were concerned.This indicates that the hydrophobicity of aerial conidia depends on both TFA-soluble protein content and conidial size.Aqueous dispersancy of emulsifier to the oil formulations of aerial conidia with distinguished hydrophobicity traits.For each of the six isolates selected by distinguished H_r,S_a and P_c values,aerial conidia harvested from rice cultures were suspended in the emulsions consisting of 94.5-98.0%liquid paraffin and 2.9-5.5%emulsifier 'AEO-3' (increased at 0.5%interval),generating eight emulsifiable formulations at the standardized concentration of 1×10¹⁰ conidia/ml.The ratios of the emulsifier were assessed for their dispersancy(D_i) to the formulated conidia in 100-fold aqueous dilution of each formulation. As a result,the dispersivity of the oil-formulated conidia in the aqueous dilution generally increased with the emulsifier ratio at the tested range and,at a given ratio,differed significantly among the tested isolates.The highest emulsifier ratio(5.5%) led to more than 50%dispersivity in the dilutions of the isolates with lower H_r,S_a and P_c values,warranting a normal quality of field spray with conventional sprayers.Emulsifying the oil-formulated conidia in water was easiest for Pf 6032(close to 100%),followed by Bb2 860 and Pf 4205. In contrast,the emulsification was most difficult for Mam 978(only 6.1%),followed by Bb 2864 and Ma 456.The trends of the D_i values over the different emulsifier ratios fit well the logistic equation with high coefficients of determination(0.93≤r²â‰¤0.98).The fitted potential of the emulsifier dispersancy ranged from 26.8(Mam 978) to 91.7%(Pf 6032).For the oil formulations,the fitted increase rates of dispersancy(r_d) over the emulsifier ratios were inversely correlated to the H_r values of the six isolates(r²=0.94,P＜0.001).Conclusively, fungal candidates with higher condidial hydrophobicity need inclusion of more emulsifier in the oil formulation for qualified field spray.In summary,we found for the first time several Metarhizium isolates highly virulent to aphids and thus a necessity to re-evaluate the biocontrol potential of Metarhizium spp. against aphids.A method to quantify conidial hydrophobicity of fungal biocontrol agents was improved and applied to evaluating intra- and inter-specific hydrophobicity variation of 48 fungal isolates.Both TFA-soluble protein contents and superficial areas of aerial conidial were found hydrophobicity-related traits that can be utilized to screen fungal candidates with desired hydrophobicity and to help formulation design.This was confirmed by evaluating aqueous disperancy of a selected emulsifier to oil-formulated conidia of fungal isolates with distinguished traits relating to conidial hydrophobicity.