Isolation,Fermentation And Formulation Of The Entomopathogenic Fungi Highly Toxic To Diaphorina Citri

The Asian citrus psyllid(ACP),Diaphorina citri(Kuwayama),one of the most destructive pests of citrus crop,causes substantial loss to most citrus species worldwide.The most concerning threat caused by D.citri is the transmission of an?-proteobacteria Candidatus Liberibacter asiaticus(CLas),which is known as the pathogen of Huanglongbing(HLB)disease or citrus greening disease,causing heavy economic losses annually in the major citrus-producing regions of China.Chemical pesticides have been employed as the principal strategy for the management of D.citri.However,injudicious pesticide usage has many adverse effects,such as insecticide resistance in insects and pesticide residues.This study was designed to develop the mycopesticides to partially replace the chemical pesticides.The native fungal strains were collected from the cadavers of D.citri from the major citrus-producing regions of China,and screened the isolates with the highest virulence and best growth characteristics.Then the conditions of biphasic(liquid-solid)fermentation and oil-based conidial formulation were optimized for developing the potential mycopesticides.Meanwhile,the secondary metabolites toxic to D.citri were screened and identified from the virulent fungal isolate,and the effect of light stimuli(varied light/dark patterns and wavelengths)on the sporulation of this isolate was analyzed.These findings will facilitate in the development of potential novel mycopesticides and secondary metabolites for the biological control of Asian citrus psyllid.1.Isolation,morpho-molecular characterization and virulence of fungal strains toxic to D.citriThe first part of this study identified potential Chinese native fungal isolates from the cadavers collected from 5 different geographical areas in the south of China.First the 13fungal strains were isolated from varied geographical cadavers,including F-HY002-1A,F-HY002-7A,F-HY002-4A,F-HY002-ACPHali,F-HY010,F-HY006,F-HY015,and F-HY016 from Ganzhou in Jiangxi,F-HY009 and F-HY014 from Zhaoqing in Guangdong,F-HY007 and F-HY013 from Nanning in Guangxi,and F-HY008 from Guilin in Guangxi.The molecular identification was performed on basis of the molecular markers(ITS1-5.8S r DNA-ITS4 and translation elongation factor 1 alpha(tef1)).The phylogenetic analysis classified 13 strains as Beauveria bassiana(3 isolates),Fusarium fujikuroi(5 isolates),and Cordyceps javanica(5 isolates),which was further confirmed by morphological clarification.Among 13 isolates,C.javanica F-HY002-ACPHali and B.F-HY006possessed the highest corrected mortality of 86.52±2.35%and 85.01±1.39%,respectively,with a conidial concentration of 1×10⁸ conidia/ml for 10 days of exposure.Furthermore,these two strains possessed the best growth characteristics(57.67±9.76×10⁵ conidia/cm²and 2.89±0.28×10⁵ conidia/cm²,respectively).Based on these findings,C.javanica F-HY002-ACPHali and B.F-HY006 were used in the followed experiments.2.Optimizing the fermentation and formulation of B.bassiana F-HY006Based on its initial high virulence,B.bassiana F-HY006 was further subjected to adult females of D.citri to calculate the corrected mortality and LT₅₀ values after application of a suspension containing 1×10⁷ conidia/ml(400?l/citrus seedling)for 7days.The results showed corrected mortality of 76±7.08%and LT₅₀ of 4.09(3.64–4.57)d.As its high corrected mortality and low LT₅₀ values,which is its potential for developing mycopesticide,biphasic(liquid-solid)fermentation for conidia production was optimized using several nutritional and environmental factors.The optimal conditions of liquid-state fermentation was 50 g/L of maltose,15 g/L of casein,and 6 of initial p H value at 30?,which resulted in the maximum blastospores concentration(2.2±8.28×10⁹ blastospores/L).The optimal combination for solid-state fermentation was soybean+cottonseed flour(1:1)as solid substrate,24 h:0 h of the light:dark cycle,70%of initial substrate moisture,6.5 of the initial p H,and 10%(v/w)of inoculation size at30? for eight days of growth,which resulted in the highest conidial production(5.7±0.49×10⁹/g dry substrate).To enhance the conidial shelf life and virulence,the oil-based conidial formulation was further optimized using various adjuvants.Sesame oil was proved an effective carrier,which resulted in the highest germination ability(90.33±1.20%)after storage of 30 d.Among other tested adjuvants,the silwet L-77(0.05%)as the best surfactant and carboxymethyl cellulose(CMC,0.05%)as the best stabilizer showed the significantly highest germination abilities(with 87.66±1.20%,89.33±1.45%,respectively)after storage of 30 d.Thus the optimal formulation combination was determined to be sesame oil+silwet L-77(0.05%)+CMC(0.05%).The oil formulation helps in significant enhancement of the conidial shelf life up to 16 weeks at 26?(80.74±0.77%of germination rate),tolerance to thermal stress at 50? for 3 h(80.6±0.92%of germination rate),and virulence against D.citri with increased corrected mortality(100%after 6 d)and decreased LT₅₀(2.50(1.28–2.98)d)compared to unformulated conidia.Therefore,a potential mycopesticide was prepared for the biological control of D.citri based on the optimized biphasic culture growth and formulation of an isolated virulent fungus,B.bassiana F-HY006.3.Optimizing the fermentation and formulation of C.javanica F-HY002-ACPHaliBased on its initial highest virulence,C.javanica F-HY002-ACPHali was further subjected to adults of D.citri to calculate the corrected mortality and LT₅₀ values after application of a suspension containing 1×10⁷ conidia/ml(400?l/citrus seedling)for 7days.The corrected mortality was 71.25±4.72%and LT₅₀ was 5.71(3.99–6.60)d.As its high corrected mortality and low LT₅₀ values,which is potential for developing mycopesticide,the biphasic liquid-solid fermentation for its conidia production was optimized using several nutritional and environmental factors.For liquid fermentation,the optimal condition was 20 g/L of glucose,10 g/L of yeast extract,and 6 of initial p H value at 26°C,which yielded the highest blastospore concentration(8.82±0.14×10⁸blastospores/L).In addition,the overall optimal combinations for solid-state fermentation were soybean+cottonseed flour(1:1)as solid substrate,24 h:0 h of the light:dark cycle,70%of initial substrate moisture,6.5 of the initial p H,and 10%(v/w)of inoculation size at 30? for eight days of growth,which achieved the highest conidial production(7.85±0.26×10⁹/g dry substrate).Furthermore,to enhance conidial shelf-life and virulence,the oil-based conidial formulation was optimized employing several critical adjuvants.Among all carriers,the sesame oil has significant germination ability(94.33±1.20%).For other adjuvants,Tween 80(0.05%),CMC(0.05%)and ascorbic acid(0.3%)was proved optimal with the highest germination abilities(91.33±1.20%,90.66±0.66%and79.11±1.53%,respectively).Thus,the optimal formulation parameters were:Sesame oil+Tween 80(0.05%)+CMC(0.05%)+ascorbic acid(0.3%).The oil-dispersion formulation performed significant enhancements of the conidial persistence(73.26±1.61%)up to 16 weeks at 26?,the tolerances to thermo-stress(89.20±2.43%at 50°C for 3 h)and UV-B stress(79.11±1.53%for 45 min),and the virulence against D.citri with increased corrected mortality(100%after 6 d)and decreased LT₅₀(3.58(2.67–4.01)d)in comparison to that of unformulated conidia.Our results not only provide a potentially valuable native strain for bio-control of D.citri,but also imply the soybean meal+cottonseed flour as valuable solid substrate and full-light exposure as novel environmental stimuli for solid-state fermentation for conidial production,and the sesame oil as an effective carrier for the formulation of the conidia from C.javanica or even other entomopathogenic fungi.4.D.citri-toxic secondary metabolites from C.javanica F-HY002-ACPHaliTo identify the active metabolites to develop the safe agent for bio control of D.citri,the toxicities of the secondary metabolites of C.javanica F-HY002-ACPHali against D.citri were further explored.Six fractions of secondary metabolites eluted by six organic solvents(100:0,80:20,70:30,60:40,30:70,and 0:100 of CHCl3/Me OH(ml/ml))were first separated.The subsequent bioassay with the adults of D.citri indicated the 4^thfraction with the highest corrected mortality(100%)after exposure period of 48 h.Then the compounds from the 4^th fraction were identified by Ultra-Performance Liquid Chromatography/mass spectroscopy(UPLC/MS)analysis,which indicated cinchonine,alantrypinone,component X,and dimethirimol with high abundances.The D.citri-toxic activities of these four components were further determined,which presented the component X with the highest virulence against D.citri(86.67%of corrected mortality,4.93(3.86-5.49)mg/L of LC₅₀)after 72 h of exposure period.These findings provide a new potential metabolite that can be employed as a safe bio control agent for the management of D.citri.5.Effect of light condition on growth and conidial production of C.javanica F-HY002-ACPHaliIn this study,C.javanica F-HY002-ACPHali was selected to check the effect of varied light/dark patterns and wavelengths on conidial production.Five different white light/dark pattern(24 h:0 h,18 h:6 h,12 h:12 h,6 h:18 h and 0 h:24 h)were subjected to C.javanica F-HY002-ACPHali cultured on PDA plates for 5 d,10 d,15 d,20 d and 25 d.From initial findings,two main results are:(1)the maximum conidial production(8.81±16.04×10⁵conidia/cm²)was observed after 15 d under 24 h:0 h light/dark pattern,(2)conidial production starts after 5 days of incubation in 24 h:0 h,18 h:6 h,12 h:12 h,6h:18 h of light/dark pattern,whereas no conidia were produced under 0 h:24 h.Furthermore,blue(454 nm),green(520 nm)and red(660 nm)light was subject to C.javanica at 24 h:0 h of light/dark pattern,which indicated the blue light stimulated the highest level of conidial production(9.14±0.79×10⁵ conidia/cm² at 15 d).Our results stated that the light/dark pattern and wavelength can significantly influence the conidial production of EPFs,and full light pattern with blue wavelength(454 nm)is optimal light condition for C.javanica F-HY002-ACPHali.In conclusion,this study provides a new insight in the development of potential stress-tolerant bio-pesticide based on EPFs.Based on obtaining two fungal isolates(B.bassiana F-HY006 and C.javanica F-HY002-ACPHali)which were high virulent against D.citri,the optimized biphasic(liquid-solid)fermentation and formulation techniques was developed for mycopesticides.For the first time,soybean meal+cotton seed powder were proved that can play a vital role in enhancing conidial production of EPFs and also full light pattern(24 h:0 h)can be employed as a new optimal environmental stimulus during solid-state fermentation.In addition,the oil-dispersion formulation based on sesame oil was proved effective that can help the conidia of F-HY006 and F-HY002-ACPHali in elevating the shelf-life,thermo-/UV-B-tolerance and virulence as compared to unformulated conidia.Meanwhile,among the secondary metabolites of C.javanica F-HY002-ACPHali,a novel active metabolite was identified,component X,which is toxic towards adults of D.citri.Finally,the full light pattern(24 h:0 h)under blue wavelength(454 nm)was found to be best for the sporulation of C.javanica F-HY002-ACPHali.These findings have provided not only two potential mycopesticides,but also a potentially novel functional secondary metabolite for the bio control of D.citri,which is critical for the integrated management of this harmful pest.