Study On Tolerance Against Ultraviolet Radiation And Oxidative Stress As Well As Activity And Types Of Superoxidase Dismutase Of Beauveria Bassiana

The entomopathogenic fungus,Beauveria bassiana,has been developed into numerous formulations for wide application to biological control of insect pests in agriculture and forestry.It is well known that the shelf life and field persistence of a fungal formulation are affected by environmental factors,such as temperature,humidity and solar radiation.For formulation improvement,therefore,it is necessary to understand the tolerance of B. bassian towards main stressful factors so that exploring fungal biocontrol candidates and technical means to improving fungal fitness to variable environments is more effective.This study sought to evaluate quantitatively the conidial tolerances of different B.bassiana strains to ultraviolet(UV-B)radiation and oxidative stress for better candidates.The effects of different medium components and cultural conditions on the tolerance of conidia to oxidative stress were investigated.The activities and types of superoxide dismutases(SOD) in B.bassiana strains were also demonstrated.The results are summarized as follows.UV-B tolerance of B.bassiana strains.Twenty strains of B.bassiana(Bb)with different host and geographic origins were assayed for their tolerance to UV-B(290-320 nm) radiation.For each of the strains,conidia produced on 1/4-SDAY slants at 25℃were suspended in a germination broth(0.5%peptone and 2%sucrose plus 0.02%Tween-80). Each of three 10-μl aliquots(replicates)of the suspension(1×10⁶ conidia ml^-1)was evenly smeared onto the concave round area(10 mm diameter)of sterilized glass slides.The smeared slides were then exposed to the UV-B(312 nm)irradiations of 0-1.6 J cm^-2in a UV-B generator 'Bio-Sun'.After exposure,the slides were incubated for 24 h at the regime of 25℃and L:D 12:12 with saturated humidity.Subsequently,the concave area of each slide was stained with methyl blue and then observed for the counts of germinated and ungerminated conidia under a microscope(400×magnification).As a result,the proportions of germinated conidia(G_p)at the UV-B doses fit well the logistic survival model G_p =1/(1+exp(a+bD)for all the tested strains(r²â‰¥0.93).The median lethal doses(LD₅₀)of the UV-B irradiation estimated from the fitted equations of the tested strains fell in a range of 0.10-0.92 J cm^-2.The LD₅₀of the most UV-b tolerant strain Bb2861 was about the cumulative UV(200-320 nm)radiation(0.63 J cm^-2)on a fine summer day(6 August 2007) in Hangzhou.Oxidative stress tolerance or B.bassiana strains and influential factors in cultures. The conidia of 17 B.bassiana strains produced on SDAY under normal condition and those of the strain Bb2861 under different culture conditions were assayed for their tolerances to oxidative stress.For conidial production of Bb2861,SDAY plates were adjusted with ion concentrations(50,100,150,200,250 and 300μg ml^-1Fe³⁺,Mn²⁺,Cu²⁺or Zn²⁺),pH(4.0, 5.0,6.0,7.0 and 8.0),carbon sources or water activity(a_w)or incubated at different temperatures(15,20,25 and 30℃).For each of the strains or the concerned factors,three 50-μl aliquots(replicates)of conidial suspension(1000 conidia ml^-10.02%Tween 80)were evenly smeared on the plates(9 cm diameter)of SDAY containing 10μg ml^-1methylene blue(as a standard oxidative stress)and then incubated at the regime of 25℃and L:D 12:12 for 4 days.The ratio of the resultant colony diameter of(cross-measured)over that of blank control(not stressed)was defined as relative growth rate(RGR)to depict the tolerance of each strain or factor.The resultant RGR values differed significantly among the 17 strains tested(F_16,64= 28.61,P＜0.01).On average,the RGR ranged from 14.1±5.8%(Bb2881)to 77.11±8.51% (Bb2988),yielding an overall mean of 53.74%for all tested strains.Inclusion of 300μg ml^-1 Cu²⁺or of 50μg ml^-1Zn²⁺in SDAY led to production of Bb2861 conidia most tolerant to the oxidative tolerance(RGR:71.90±7.43%and 74.38±8.14%).Conidia produced at 30℃showed better oxidative tolerance than those produced at lower temperatures(F_3,12= 28.12, P＜0.01),colony growth with no stress at 30℃was slower(2.06±0.09 mm per day)than that at 20℃(3.49±0.09 mm per day).The conidia produced on plates with starch as a mere carbon source had the RGR values of 30.3-42.5%.In contrast,the RGR values were 73.9-89.4%when glucose was used as carbon source for conidial production.The optimized conditions for producing the most stress-tolerant conidia were a medium containing 4% glucose as carbon source and 200μg ml^-1Mn²⁺as additive(with adjusted pH 4.0-6.0),and 25℃for incubation.The activities and types of superoxide dismutases(SOD)in B.bassiana strains.To determine the activities and types of superoxide dismutases(SOD)in B.basstana,cellular extracts were produced from 4-day cellophane-plate colonies of 17 strains grown on 1/4 SDAY at 25℃.The SOD activities of the extracts were assayed based on their inhibitory effect on spontaneous autoxidation of pyrogallol.As a result,total SOD activities fell in a range of 0.13-1.46 U mg^-1proteins for the tested strains.Two SOD types were identified from all the cellular extracts in terms of their sensitivity to KCN or H₂O₂ in native polyacrylamide gel electrophoresis.Fourteen strains produced both MnSOD and FeSOD while the rest yielded only MnSOD or FeSOD,which was found for the first time in B. bassiana.Three strains showing distinctly low,medium and high SOD activities at 25℃differed significantly in relative SOD activity after their cultures were exposed to the thermal stress of 35℃for 3-18 h but had no change in SOD type.The variation of the SOD activities expressed either in normal cultures or in response to the thermal stress could be exploited for selection of stress-tolerant fungal candidates against insect pestsIn summary,quantitative assay methods were developed for successful evaluation of the tolerances of B.bassiana strains to UV-B radiation and oxidative stress.Factors including metal ions,pH,temperature,carbon source and water activity in conidial production were found affecting the tolerance of the resultant conidia to the oxidative stress. The overall activities of two SOD types in B.bassiana were generally at low levels despite significant variation among the tested strains.FeSOD was found for the first time in fungi. The results would increase our knowledge on the stress biology of B.bassiana and suggest a promising approach to improving fungal formulations for better field persistence and performance.