| Notoriously slow kill action of fungal cell-based insecticides often depresses commercial interest and genetically engineered mycoinsecticides can accelerate the kill action but associated ecological risk is of public concern. Since little effort has been made to assess control efficacy and ecological safety of transgenic mycoinsecticides under real field conditions, this study sought to fill the gap with the data of two35-day field trials performed in Jinhua, Zhejiang Province. Aerial conidia of a transgenic Beauveria bassiana strain (BbHV8) over-expressing a Bacillus thuringiensis vegetative insecticidal protein (Vip3Aal) for acquisition of fast per os virulence to caterpillars and its parental wild-type strain Bb2860(WT) were formulated for repeated sprays against cabbage pest complex, which was dominated by the cabbage butterfly (CBF) Pieris rapae, followed by the diamond-back moth (DBM) Plutella xylostella and the green peach aphid (GPA) Myzus persicae. Their control efficacies were compared with that of emarnectin benzoate (EB), a chemical insecticide commercially recommended for cabbage pest control. Their field fitness and effects on field spider community and native soil fungi were also evaluated during the cabbage growing season and several post-trial months respectively. An effort was made to establish a technical system that is potential for fast quantification of BbHV8conidia in cabbage foliage and soil samples by means of single-copy specificity of the target gene primers in quantitative real-time PCR (qRT-PCR). The results are summarized below.High field efficacy of BbHV8formulation competed with the EB efficacy in full-seson cabbage pest control. The two fields surrounded by rice and maize crops were transplanted with cabbage seedlings on April2and12respectively, attacking lepidopteran insects, such as CBF and DBM, for egg deposition. The mean CBF density (±SEM) reached0.94(±0.06) and0.93(±0.05) larvae per plant in the two fields on April22and May5, exceeding an economic injury level of0.5larvae per plant for initiatiing Trials1and2respectively. Each field trial included four treatments of four plots (6x11m), i.e.,100-fold aqueous dilution of emulasifiable BbHV8or WT formulation (1010conidia/ml) sprayed at the rate of1013conidia/ha, aqueous EB dilution sprayed at the labeled rate, and100-fold aqueous dilution of emulsion vector (for fungal formulation) sprayed as blank control (BC). All treatments and plots were arranged in terms of a randomized block design and monitored every5days for pest and spider densities until cabbage heads were ready for harvest. Repeated spray was carried out in Trial1at10-day internval, resulting in the respective CBF densities of16.1±1.01,4.310.3,8.2±0.8and1.3±0.2larvae per plant in the BC, BbHV8, WT and EB treatments on day25. The four treatments were infested with the DBM densities of1.50±0.15,0.40±0.08,0.45±0.06and0.75±0.06larvae per plant on day20and of0.13±0.05,0.15±0.05,0.28±0.09and1.95±0.64larvae per plant on day35respectively. GPA density in Trial1began from12.7±1.1aphids per plant and was consistently suppressed to a very low level by BbHV8throughout the tiral but increased to68.6±3.2aphids per plant in EB plots at the end of the trial. Cabbage plants in Trial2suffered from similar damages by main pest populations as seen in Trial1but the CBF density in BC was up to27.7±2.0larvae per plant on day10. For this reason, repeated sprays in Trial2were scheduled on days5,15and35respectively, resulting in somewhat better best control than that in Trial1.Since CBF overwhelmed foliage damage in both field trials and ageing larvae could transfer to adjacent plants from severely damage plants, pest population densities estimated by sampling could not reflect the true damage levels in the fields. Thus, damage of each sampled plant was scaled to five grades in terms of percent foliage loss due to ingestion by CBF larvae and the damge grades were used to compute control efficacy (%) relative to BC. As a result, the control efficacies of BbHV8, WT and EB fell in the respective ranges of65～75%,15～58%and42～85%during Trial1and were averaged as70.5%(±1.2),30.9%(±6.1) and69.7%(±6.9). The efficacies of the three treatments in Trial2spanned from49%to80%,41%to70%and51%to90%, resulting in the overall means of74.5%(±4.3),48.4%(±4.2) and83.5%(±5.5) respectively. In both trials, BbHV8provided a full-season protection from the damage of cabbage pest complex with an overall efficacy similar or close to that achieved by EB application. In contrast, WT showed some degree of control efficacy against the pest complex but the control was insufficient to protect the cabbage from the damage by large CBF population.A supplementary experiment was conducted to bioassay the virulence of BbHV8and WT to fourth-instar CBF larvae sprayed at the same rate as the two field trials during Trial2. Under the spray, LT50, LT95and associated95%confidence intervals were estimated as1.6(1.2～1.9) and4.5(4.3～4.8) days for BbHV8and5.7(5.4～6.0) and11.5(10.6～12.8) days for WT. These estimates indicate that BbHV8killed50%and95%of the tested larvae4-and7-day faster than WT. Ecological safety of transgenic BbHV8released in the field. During Trials1and2, spiders as nontarget predators of pests appeared on sampled plants were counted in situ and the counts on all sampling occasions revealed no negative effect of both BbHV8and WT on the spider population, which was even augmented by the BbHV8treatment but reduced by the chemical control.Surface soil samples were collected back to laboratory from the plots of BbHV8, WT and BC the day after the last spray of Trial1, every10day within the first month post-spray and every month from then on. Colony forming Units (CFUs) of B. bassiana and saprophytic fungi were isolated from the soil samples of each batch using two selective media respectively. As a result, CFU counts per gram of soil sample (dry weight) reached3.1(±0.4)×104and2.1(±0.4)×104in the soi samples taken respectively from WT and BbHV8plots the day after the last spray and declined to4.3(±0.2)×103and3.2(±0.2)×103on day10and to3.8(±0.7)×102and4.1(±0.8)×102on day20. From then on, CFU counts fluctuated at low levels and decreased to only145±59and36±36on day120but became undetectable afterwards. Interestingly, the counts69±40and34±34CFUs/g were found in the BC soil samples taken20and30days after the last spray respectively. Moreover, CFU counts of saprophytic fungi from different batches of soil samples showed seasonal fluctuation but did not differ significantly from one treatment to another within each batch.Seventy-two B. bassiana isolates recovered from the soil samples collected in the BbHV8and WT plots30,60and90days after the last spray were detected for the presence or absence of the target gene vip3Aal in their DNA samples. The target gene was present in33of the36isolates recovered from BbHV8plots but only in two of the36isolates recovered from Bb2860. Interestingly, the occasionally crossing presence of the target gene occurred only in the earlier soil samples. Since B. bassiana also occasionally recovered from the BC plots, our data indicated that the cabbage field lacked native B. bassiana strains and that the fungal sprays did cause a low frequency of crossing between treatments perhaps due to frequent light rains during the trial.Moreover, conidial yields of rHV8and rWT, two isolates recovered from the soil samples of BbHV8and WT treatments90days after the last spray, were26.5%and32.4%lower than BbHV8and WT, respectively, after8-day incubation on SDAY plates while rHV8grew32.2%slower than BbHV8. In stress assays, BbHV8conidia were19.2%and12.5%less tolerant to UV-B irradiation and wet-heat at45℃than the WT conidia while rHV8and rWT showed the reductions of38.9%and39.7%in conidial UV-B resistance and of32.5%and31.0%in conidial thermotolerance compared to BbHV8and WT respectively. Apparently, post-release ecological fitness was low for both BbHV8and WT and even lower for BbHV8.Rapid assessment of transgenic BbHV8conidia in cabbage foliage and soil samples by quantitative real-time PCR (qRT-PCR). High specificity of paired primers Vip3Aal-F/R to the target gene vip3Aal was determined with a melt curve in qRT-PCR, followed by establishing a standard curve for the amplication of vip3Aal from total DNA samples of10-105BbHV8conidia/ml. Since the target gene is signle-copied in each each transgenic cell, the number of transgenic conidia in each sample unit would equal to the number of DNA copies in the total DNA of each sumple unit. Thus, the established standard curve was used to assess conidial denisity in foliage and soil samples to which BbHV8conidia were quantitatively added. As a result, an accuracy of qRT-PCR assessment for the conidial density was2conidia/mm cabbage leave or1～2conidia/mg soil (dry weight). However, percent deviations between assessed and quantified conidial densities in soil samples were larger than those in foliage samples perhaps due to the complexity of soil components, which might affect efficiencies of both target DNA extraction and qRT-PCR amplification.Conlusively, the transgenic mycoinsecticide BbHV8not only competed with the chemical insecticide recommended for the control of cobbage insect pests dominated by P. rapae in two independent field trials but also had no adverse effect on nontarget spider population and native soil fungi. Taken together with a rapid decline rate of its residue in the soil of released field, BbHV8could be of little risk for environmental safety if it was commercially released. |
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