Synergistic Effects Of Bacillus Thuringiensis With Pseudaletia Unipuncta Granulosis Virus-Ps And Its Application

Bacillus thuringiensis (Bt) has been extensively used for four decades as biopesticide due to its safety to environment and human health. The widespread use of Bt is often challenged by the efficacy of controlling pests. Pseudaletia unopuncta granulovirus (PuGV), which contains a special protein called enhancin, can synergize the infection of nucleopolyhedtovirus and enhance the toxicity of Bt to pests. In this study, with Bt and PuGV-Ps propagated in the larvae of P. separata by PuGV as materials, the synergistic effect and characterization of Bt toxicity to lepidoperous species with PuGV-Ps were investigated. The mechanisms of enhancement were analyzed in respect of proteolytic activity of PuGV-Ps, degradation ofδ-endotoxin, influence of midgut enzyme and damage to peritrophic membrane (PM). The enhancin gene of PuGV-Ps was cloned and expressed. A preparation of Bt enhanced by PuGV-Ps was devised and its application effect was also studied. The main results were as fellows:1 Enhancement of B. thuringiensis Toxicity to Lepidopterous Species with P. unipuncta Granulovirus-Ps: Enhancement of toxicity of B. thuringiensis with PuGV-Ps was demonstrated by bio-assays employing larvae of several lepidopterous species. Combinations of Bt and PuGV-Ps were synergistic and enhanced toxicity against Plutella xylostella, Helicoverpa armigera, and Spodoptera exigua. The co-toxicity coefficients (CTC) of Bt combined with PuGV-Ps to different larvae were diverse from 127 to 146. Denatured PuGV-Ps also enhanced toxicity of Bt to larvae of P. xylostella with CTC value of 136, indicating PuGV-Ps contained some synergistic factors. Besides the increase of mortality to larvae, the rate of larval death of P. xylostella was also accelerated by adding PuGV-Ps in Bt, and the median survival time (LT50) reduced 38 percent compared with the treatment of Bt alone at concentration of 250μg/mL. The toxicity of transgenic Bt cotton to H. armigera were also elevated by adding PuGV-Ps. PuGV-Ps also enhanced the effect of Bt on the larval development of S. exigua, with less weight of larvae and pupae, delay of pupating and lower ratio of pupation.2 Purification of Enhancin from P. unipuncta Granulovirus-Ps and Evaluation of Its Synergistic Effect to Bt: PuGV-Ps was propagated in the larvae of P. separata infected by P. unipuncta granulovirus. SDS-PAGE showed the capsules of PuGV-Ps contained a special protein called enhancin with molecular weight of 108 kD. The capsules was dissolved in alkaline solution of 0.02 mol NaOH, and then filtered through on a column of Sephadex G-200 and enhancin proteins were purified from the concentrated crude protein extract. The synergistic effects of PuGV-Ps enhancin on B. thuringiensis were tested by bio-assays employing larvae of several lepidopteral species, such as P. xylostella, H. armigera, and S. exigua. The CTC of Bt combined with PuGV-Ps enhancin to larvae of different species were range from 116 to 155. The results showed that PuGV-Ps enhancin was a synergistic factor, and could enhance the toxicity of Bt to larvae of Lepidoptera.3 Characterization of B. thuringiensis Enhancement by P. unipuncta Granulouvirus-Ps: The synergistic effects of PuGV-Ps on Bt were tested by bio-assays employing larvae of P. xylostella. The CTC of Bt combined with PuGV-Ps in different ratios were diverse from 105.3 to 195.0, showing a positive synergistic effect of PuGV-Ps on Bt. Among the mixtures, the most significant effect was found in the ratio of Bt and PuGV-Ps being 4:1, in which the LC50 was 0.039mg/mL. When environmental temperature was low (16℃and 20℃), the synergistic effects were statistically significant, while there were no differences at temperature of 28℃and 32℃comparing with the treatment of feeding the insects with Bt alone. The synergistic effect was elevated along with increase of pH value. In higher pH value of 8 and 9, PuGV-Ps elevated mortalities of P. xylostella larvae by Bt up to 16.67% and 23.33%, respectively. The co-effects of Bt and PuGV-Ps were also varied along with the larval age. Mortalities of 2nd and 3rd instar larvae increased 50.00% and 30.31% in the treatment of Bt+PuGV-Ps compared with that of Bt respectively, but there were no significant improvement in that of 1st and 4th instar larvae. Much higher synergistic effect was observed when oral inoculation with PuGV-Ps 2 h prior to Bt treatment. Comparing with the treatment of oral inoculation with Bt and PuGV-Ps simultaneously, The mortality at oral inoculation with PuGV-Ps 2 h prior to Bt increased 66.67% 48 h after treatment .4 Effect of P. unipuncta Granulovirus-Ps on the Degradation ofδ-endotoxin from B. thuringiensis: Total proteolysis of PuGV-Ps was measured by using azocasein under different pH condition from value 7.38 to 10.38, and the protease activity was improved with the rising of pH value. All the four kinds of inhibitors tested inhibited the activity of PuGV-Ps proteolysis, among which soybean trypsin inhibitor (STI) made the greatest affect. That indicated the proteolytic activity of PuGV-Ps was due to several proteases, mainly from trypsin-like enzyme. SDS-PAGE analysis showed that large amounts of activated toxin proteins were yielded fromδ-endotoxin of B. thringiensis incubated with PuGV-Ps under alkaline condition. Theδ-endotoxin of 130 kD degraded into toxic protein fragments with molecular weights from 47 kD to 110 kD. PH value influenced the degradation effect greatly, which elevated along with the increase of pH value. In buffer of 0.1mol Na2CO3 pH value 10.7, theδ-endotoxin completely cleaved into 47 kD, 60 kD and 61 kD activated toxins and resisted further degradation. The amount of PuGV-Ps and the time of incubation influenced the degree of degradation. STI also inhibited the degradation ofδ-endotoxin by PuGV-Ps.5 Elucidation of Inhibition Further Degradation ofδ-endotoxin in Midgut Juice from S. exigua by P. unipuncta Granulovirus-Ps: The proteolytic activity of midgut juice from S. exigua was influenced by PuGV-Ps. Under the suitable pH value, the total proteolysis activity of midgut juice reduced for some degree by PuGV-Ps, but there were some difference between in vitro and in vivo test. SDS-PAGE analysis showed that PuGV-Ps also affected the degradation ofδ-endotoxin of B. thuringiensis in midgut juice from S. exigua. The yield of active toxin proteins molecular weight from 60 kD to 87 kD was not influenced obviously by PuGV-Ps, but the further degradation of activated toxin was inhibited. The inhibiting effect was getting more notable with the degradation time and the rising of incubating temperature. Degradations ofδ-endotoxin by midgut juice in different buffers were disagreed, indicating that the saline of Na2CO3 was an important factor to increase the further degradation ofδ-endotoxin.6 Verification of the Damage of P. unipuncta Granulovirus-Ps and B. thuringiensis to Peritrophic Membrane of S. exigua: Using scanning electron microscope and SDS-PAGE gel, impact of B. thuringiensis, P. unipuncta granulovirus-Ps and enhancin from PuGV-Ps on peritrophic membrane of S. exigua was studied. Scanning electron microscope pictures indicated that exterior PM wall of normal S. exigua was smoothness and few rumple, while inner PM wall texture was thick and with some granulation. There were no hole and crack in the membrane. When insect feed PuGV-Ps or enhancin, the exterior PM wall turned to crimple and inner PM wall to thin and smoothness. When treated with Bt alone, the structure of PM was also changed in some degree. No hole or crack was found in all the treatments. SDS-PAGE gel analysis showed that the proteins in PM, which molecular weights was from 80 kD-200 kD, were partly degraded to small proteins under 78 kD, and a 28 kD protein was degraded completely. This 28 kD protein was also dismissed in the treatment of feeding Bt alone. In vitro tests gave more evidence that several different protiens in PM of S. exigua were degraded by enhancin of PuGV-Ps, and the 28 kD small molecular protein also degraded by PuGV-Ps enhancin andδ-endotoxin from Bt.7 Molecular Cloning and Sequence Analysis of Enhancin Gene from P. unipuncta Granulovirus-Ps: Using the DNA from PuGV-Ps as template and the nucleotide sequences of the enhancin genes from H. armigera granulovirus and Trichoplusia ni granulovirus for reference, we designed a primer for PCR and amplified a 2.7 kb specific fragment by PCR reaction. Purified PCR product was cloned into plasmid pEASY-E2 and a recombinant plasmid pEASY-En was constructed. The PCR product was proved to be the full long fragment of the gene encoding enhancin of PuGV-Ps by DNA sequencing. Sequence analysis revealed that enhancin gene from PuGV-Ps had 99.56% identity to enhancing gene from PuGV, which was reported previously. The different nucleotides were mostly concentrated in 5'terminal half and shared 98.60% identity from 1 to 500 nt, while in 3'terminal half only one nucleotide was changed from 1 to 500 nt, indicating that the 3'terminal half had the greater conservation.8 Expression and bioassay of P. unipuncta granulovirus-Ps Enhancin Gene in Escherichia coli: A 2.7kb enhancin gene of PuGV-Ps cloned into pEASY-E2 vector was recombinanted into Escherichia coli BL21(DE3). When induced by IPTG at 37℃, the target protein with molecular weight of 108kD was expressed successfully. The target protein was linked with 6×His tags and purified by Ni2+ column, indicating the target protein was enhancin, which was expressed by the inserted foreign gene. It was in favor of the expressing of enhancin gene by adding 0.2 percent of glucose. The extracted expressing protein showed synergistic activity to Bt. In the treatment of 600μg/g Bt, the mortality of S. exigua was increased 10.00 percent by adding 300μg/g extracted enhancin. When treating larvae of H. armigera, the mortality was elevated to 38.67 percent from 23.67 percent by adding 400μg/g extracted protein into 400μg/g Bt. The synergistic effect was advanced with the increasing of extracted protein.9 Producing and Application of Preparation of B. thuringiensis Enhanced by P. unipuncta Granulovirus-Ps: According to the toxicity of B. thuringiensis to the larvae of Lepidoptera enhanced by PuGV-Ps, a preparation of Bt+PuGV-Ps was prepared. This preparation was a water powder formulation and produced through the process of propagated PuGV-Ps in cultured P. separata, fermented Bt in liquid substrate and spray drying. It was composed by 3.0×109 OB/g PuGV-Ps and 1.0×1010 spores/g Bt, and the CTC was 162.57. PuGV+Bt almost had no virulence to vertebrate and did no harm to fish, bird and bee. Field trial showed, at the concentration of 2000μg/mL, Bt+PuGV-Ps got control effects of 86.74 percent and 61.22 percent to P. xylostella and S. exigua respectively, exceeding effects by Bt remarkablely. When used to control Canphalocrocis medinalis at dosage of 1500g/ha, the efficacy was up to 80 percent, while efficacy of Bt was 71.15 percent. PuGV-Ps+Bt only reduced 3.92 percent of the amounts of spiders, while Abamactin killed spiders up to 34.39 percent.