| SummaryFungal biocontrol agents,such as Beauveria bassiana and Metarhizium spp.,have been developed into a variety of formulations for use in control of insect pests worldwide. Fungal conidia usually used as active ingredients of the formulations are often exposed to environmental stresses after field application.Of those,solar UV irradiation is detrimental to the exposed conidia and thus an important factor to affect field efficacy and persistence. This study sought to assess conidial tolerances of 60 B.bassiana and Metarhizium strains to simulated UV-B and UV-A irradiations based on a novel system of quantitative stress bioassay in vitro.Two isolates of B.bassiana and M.anisopliae with high biocontrol potential were selected for further in vivo bioassays to evaluate the effects of three UV-B doses on their virulence to aphids sprayed with a concentrated spore suspension.The results are summarized below.In vitro responses of 60 fungal isolates to UV-B irradiation.All 60 isolates[24 B. bassiana(Bb),20 M.anisopliae(Ma),seven M.anisopliae var.acridum(Maac),five M. anisopliae var.anisopliae(Maan),one M.anisopliae var.majus(Mam) and three M. flavoviride var.minus(Mfm) with different host and geographic origins]were separately cultured on the slants of 1/4 SDAY(w/v:1%dextrose,0.25%peptone,0.25%yeast extract and 2%agar) for 7 days at the regime of 25℃and L:D 12:12.Conidia from the slant cultures were suspended in a species-specific germination broth in 0.02%Tween-80.The broth consisted of 0.5%peptone and 2%sucros for B.bassiana and of 0.25%peptone, 0.2%trehalose and 0.25%yeast extract for Metarhizium spp.Three 10-μl aliquots (replicates) of each suspension were dripped onto sterilized glass slides and each was smeared evenly in an area of ca.10 mm diameter.The smeared slides were placed in the sample tray(10×120×260 mm) of a UV irradiation chamber,named Bio-Sun++(Vilber Lourmat,Marne-la-Vallee,France),and then exposed to the irradiation of the weighted UV-B wavelength of 312 nm at the gradient doses of 0.005-1.1 J/cm2(D) After exposure, all slides were immediately transferred to moisture-saturated chambers and incubated for 24 h at 25℃and L:D 12:12.Subsequently,the smeared area of each slide was stained with cotton blue and examined for the counts of germinated and non-germinated conidia from three view fields at 400×magnification(＞100 conidia per field).The ratio of the percent germination of the irradiated conidia over the control germination was defined as the survival index(Is).The Is-D relationship observed from the stress bioassay of each isolate fit well the logistic survival equation Is=1/[1+exp(a+bD)](r2≥0.97).The median lethal doses(LD50) of the UV-B irradiation estimated from the fitted equations for the tested isolates fell in a range of 0.05-0.65 J/cm2.The most UV-B tolerant isolate was Maac 5734, followed by Mfm 3606 and Mfm 3341.However,none of the tested isolates could survive a cumulative UV-B dose of 2.439 J/cm2 measured from the sunlight of a sunny day of summer in Hangzhou.In vitro responses of 24 Metarhizium isolates to UV-A irradiation.The same stress bioassay method was adopted to assess the in vitro responses of 24 Metarhizium isolates to the irradiation of the weighted UV-A wavelength of 365 nm at the doses of 1.0-18.0 J/cm2. Based on the Is-D relationships well fitted to the survival equation(r2≥0.97),the LD50 estimates for the UV-A tolerances of the tested 24 isolates spanned from 2.78 to 10.46 J/cm2. The three UV-B tolerant isolates appeared on the tope five list of the UV-A tolerance. However,the LD50 for the most UV-A tolerant isolate(Mfm 3606) was far below on the UV-A dose of 20 J/cm2 in accumulated in a full day of Beirut under clear-sky conditions.In vivo responses of two selected isolates to three UV-B doses.Two ARSEF isolates (Bb 2860 and Ma 759) with high biocontrol potential against aphids and spider mites were selected to assay the effects of three UV-B doses on the performance of green peach aphid Myzus persicae apterae sprayed with the spore suspension of 1×108 conidia/ml in 0.02% Tween-80.Batches of 30-40 aphids on a cabbage leaf disc were separately sprayed with 1 ml spore suspension of the same concentration in an automatic Potter Spray and then exposed to the UV-B irradiations expected to cause 10%,50%and 90%viability losses, respectively.The three UV-B doses determined by the fitted Is-D relationship for each isolate were 0.13,0.30 and 0.65 J/cm2 for Ma 759,and 0.03,0.45 and 0.86 J/cm2 for Bb 2860,respectively.After irradiation,all treated aphids on leaf discs were reared for 8 days in Petri dishes at 25℃and L:D 12:12 and monitored daily for mortality records.Mycosed cadavers were incubated for 2-3 days in moist chambers for fungal outgrowths.Treatments with the same fungal spray but no irradiation or with neither spray nor irradiation were included as controls for both isolates.All the assays were repeated three times.As a result, the tested UV-B doses significantly reduced the corrected mortalities of the aphids sprayed either with Bb 2860 or with Ma 759 but had no significant influence on the percentages of mycotized cadavers.Compared to the fungal treatment not irradiated,the low,medium and high UV-B doses reduced the aphid mortalities of Bb 2860 by 13.3%,58.9%and 82.3%, and of Ma 759 by 18.1%,54.0%and 88.7%,respectively.Overall mean(±SD) spray-to death periods of the aphids mycosed by Bb 2860(n=280) and Ma 759(n=257) at the low, medium and high UV-B does were estimated as 3.89±0.73 and 4.53±1.41 days.The UV-B effect on the mortality of M.persicae was proven to depend upon the irradiated dose.Conclusively,our results highlight a wide range of inter- and intra-specific variations in conidial tolerance to either UV-B or UV-A irradiation and thus the merits and feasibilities of choosing UV-tolerant candidates for fungal formulations based on their dose-survival responses in vitro,which are best represented by the absolute LD50 estimates.The dose dependence of the UV-B effect on fungal infection in vivo supports photoprotection measures to reducing solar UV-B damages to fungal formulations in the field.These results would increase our knowledge on the stress biology of the fungal biocontrol agents and suggest a strategy for proper application of fungal formulations for improved field efficacy by spraying them at evening time to avoid solar UV irradiation for the first half day.
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