| The pine wilt disease caused by the pine wood nematode, Bursaphelenchus xylophylus Nickle has been becoming one of the most serious epidemic diseases of the pine (Pinus spp.) forests in the south of China. The pine sawyer, Monochamus alternatus Hope is the most important vector of the pine wood nematode. Because most of its life time is inside the wood, Monochamus alternatus is difficult to control by spraying chemical insecticides. Biological control, therefore, provides a viable alternative for controlling the vector beetle. The entomopathogenic fungus Beauveria bassiana has been considered to be the most important natural pathogen and the most promising biocontrol agent of the pine sawyer. The application of non-woven fiber bands impregnated with B. bassiana cultures onto infested trees have been demonstrated to be one of the most convenient and effective methods.At the adult stage, the beetle will eclose and look for food and location for oviposition. During this procession, the volatile chemicals play important roles in mediating beetles'behavior. Thus, the attractants have been developed to monitor the population dynamics and suppress the adult populations of pine sawyer. In order to integrate the massive natural epizootic potential of B. bassiana and the merits of synergistic function of attractant, the combined applications of mycoinsecticide and semiochemical attractants were studied for sustainable control of the pine sawyer, Monochanus alternatus. Through the developments of ISSR (Inter-Simple Sequence Repeat) and SSR (Simple Sequence Repeat) molecular markers of Beauvera spp., the studies were conducted to investigate the survival capacity, prevalence and persistence course of the released stain in the field as well as its infective rate on the target insect pest. Based on the theories and methods of community ecology and molecular ecology, further analyses were carried out to evaluate the effects of the released strain on local Beauveria population genetic structures and the nontarget insect pests. From the results of this study, systematical evaluations are made based on the factors of environmental safety of release, ecological effects of the introduced strain, and the mechanisms for sustainable control of the Monochamus alternatus by Beauveria bassiana. Our researches including the following aspects:First, with a scanning electron microscope (SEM), 7 types of sensilla of M. alternatus were observed: Sensilla trichoid, sensilla basiconica, sensilla digit-like, sensilla rod-like, sensilla bottle-like, sensilla bud-like, sensilla chaetica. Overall, Sensilla trichoid and sensilla basiconica are the most abundant sensilla on the antenna surface. The data of electroantennographic (EAG) responses of different parts of the antennae toα-Pinene indicated that in contrast to the basal part, the apical part had the strongest response (2.3951±0.26mV) EAG value was significantly increased at the dose of more than 5 mg/ml. Carene could provoke the most significant EAG response by the male beetles while the females have the strongest response to Rα-Pinene. In general, the findings showed that the mixture of compounds Rα-Pinene and carene elicited the strongest EAG responses by both the male and female beetles.Second, the field trapping in four years showed that different attractant formulae had varied degree of trapping ability to pine sawyer, and some of them could also attract other species of Coleopteran or Hemipteran insects. Among them, the formula Ma02 showed the significantly higher attractancy than other 8 tested attractants with the mean capturing effect was 67.6-77.0 individuals per trap. The percentage of female beetles with pregnant eggs captured by Ma02 was 61.43%, and was significantly lower than those of Ma99-1(100%), Ma03(96.0%) and Ma04(96.67%). The percentage of the beetles carring nematode captured by Ma02 was 25.0% for the male, and 34.8% for the female. The observation of trapping capacity indicated that the effective distance ranged from 20-50m by Ma02 but significantly reduced when beyond 70m. The effectively attractive area could be up to 0.78 ha. The above results showed that the attractant Ma02 could attract not only the ovipositing stage of female adults, but also the nutrition replenishing stage beetles. The attractiveness of three different attractant dispensers to Monochamus alternatus adults was also investigated. The attractant dispenser B (specified control dispensing plastic bottle) had comparatively stronger effect, could be used for up to 38 days. The attractiveness of different dose of attractant showed that the efficiency enhanced with the increase of attractant volume. The effect was not significantly enhanced when the dosage of attractant was increased from 20 ml to 80 ml per bottle, but was significantly improved when the dosage reached up to 120 ml. As for trap, Xuanzhou trap designed by us was superior to the imitated Japanese trap, the trapping efficiency was 36.4 individuals/trap, 9.7 individuals/trap, respectively.Third, combined application of attractants and fungi. The virulence of different entomogenous fungi on adult pine sawyers, Monochamus alternatus was tested. Beauveria bassiana strain Bb202-1 was the highest virulent, with sawyer mortality up to 86.9%, infection rate to 75%, and LT50 at 6.77d. In order to compare the efficacy of different combined applications with control, Five treatments were conducted: (1) control (CK); (2) treated with non-woven fiber bands impregnated with Bb202-1 alone (NFB); (3) fungal bands combined with attractant (NFB+Attractant); (4) fungal bands combined with bait tree (NFB+bait tree); and (5) fungal bands combined with attractant and bait tree (NFB+Attractant+bait tree). Relative population dynamics of Monochamus alternatus captured by attractant-baited trap were monitored for five treatments every four days. The results showed that the treatments of NFB+bait tree and NFB+Attractant+bait tree had obviously increased controlling effects, the relative beetle population densities all declined by nearly 50%. The pine tree mortality in plots using five different treatments against the Monochamus alternatus were investigated in years 2004 and 2005. The results showed that the decrease rates of dead pine trees in the treatment plots of NFB+bait tree and NFB+Attractant+bait tree was 65.66 and 70.56% in 2004, respectively and no dead pine tree was found in 2005. The tree mortality was decreased by 100%, which were significantly higher than the other treatments. But there was no significant difference between the NFB+bait tree and NFB+Attractant+bait tree plots. The efficacy of the NFB and NFB+Attractant was also significantly higher than that of control, the tree mortalities were decreased by 83.33% and 76.67% in 2005, respectively. The cage experiment by importing the adult beetles and rearing inside the treatment plots showed that the mean percentage infection rates of the imported beetles in 4 fungal bands treated plots were significantly different from the control, but the infection declined with time. The infection rates in 4 fungal bands treated plots were ranged at 53.33-63.33% 2 days after bands application, 30-40% for 12th day, 25-35% for 22th day, but the highest infection of untreated control was only 6.67%. The cage experiment by exporting the adult beetle and rearing outside the treatment plots showed that the infections of the exported beetles from NFB+Attractant and NFB+bait tree were significantly higher than those of the control at 2, 12, and 22 days after band application. The mean percentage of infection of the beetles exported from NFB+Attractant and NFB+bait tree was 50% and 60% at 2th day, 25% and 35% at 12th day, 20% and 25% at 22th day, respectively. The anatomization of log section in the cage showed that the number of invading holes and larvae of the treatment of NFB+Attractant and NFB+bait tree were decreased by 50.02-54.41% and 25.9-41.02%, respectively, which were significantly lower than that of the control. The above results showed that the combined applications of fungal bands and semiochemical attractant evidently demonstrated synergistic effects by complementing each other's disadvantages to offer the great potential for controlling Monochamus alternatus.Fourth, the molecular ecology study using molecular markers. ISSR markers were used to investigate genetic diversity among 39 isolates of Beauveria spp. (36 B. bassiana, one B. brongniartii, one B. amorpha, and one B. velata) isolated from different insect hosts and geographical origins. Eighteen of 33 primer pairs harboring different SSR were chosen for their good reproducibility and the generation of high polymorphisms. In total, one hundred and sixty-eight highly reproducible fragments were amplified from all 39 isolates with an average yield of 9.3 markers per primer pair. Among these, 161 (95.8%) were polymorphic among strains. For 36 B. bassiana isolates, 8.9 (1-13) markers per primer were acquired, and the total of 123 fragments were amplified, in which 102 (82.9%) were polymorphic. Among the 168 polymorphic bands, 7 bands were considered to be specific for B. brongniartii isolate Bbr06, 14 bands for B. amorpha isolate Ba08, and 18 bands for B. velata isolate Bv01. Within 36 B. bassiana isolates, genetic similarity ranged from 0.651 to 0.972. However, the genetic similarity values among different Beauveria species ranged from 0.411 to 0.720, suggesting that ISSR technique was successful in differentiating the 3 closely related species from B. bassiana. The results also indicated that there was a positive association between the genotypes of B. bassiana isolates and their geographical origins, but no clear correlation with their insect hosts.Four enriched microsatellite libraries, (CA)n, (CT)n, (AAT)n and (ATG)n were constructed for Beauveria bassiana strain Bb202-1 by using magnetic beads. In total, 429 positive clones were obtained. Of which, 177 clones were for the library constructed by probing with (CA)12 , 108 clones for (CT)12 probe, 72 clones for (AAT)10 probe, and 72 clones for (ATG)10 probe. PCR detection showed that the percentages of positive clones in library (CA)12 (98%) and (CT)12 (92.7%) were higher than those of other two libraries. The percentages of positive clones in library (AAT)10 and (ATG)10 were 29.2% and 47.2%, respectively. Of 165 sequenced positive clones, 65 could successfully provide working primer pairs, accounting for 30% of the total number. Of 65 loci isolated, 54 were perfect (83.07%) and the rest were imperfect (16.93%). Among those, 52 loci could yield amplified products with expected sizes, and of these, 44 amplified SSR products were polymorphic by PAGE analysis, and 27 of them were highly polymorphic, accounting for 41.54%. Investigation of the population dynamics of B. bassiana surviving in soil with a selective dodine-oat medium showed that the inoculum density of B. bassiana in soil of the 4 fungal treatments plots was higher than that of untreated control plot, which suggested that the released strain had higher ability of colonizing in the soil of pine ecosystem. The estimates of community biodiversities of insect hosts and entomopathogenic fungi for different periods showed that B. bassiana had abundant diversity of the insect hosts where the species richness of insect hosts was up to 33, the abundance was 311 and the relative abundance was 60.14%. After fungal releases with an inoculative application strategy, the community diversity index of both insect hosts and entomopathogenic fungi were relatively stable, and did not pose any threat to community diversity of both insect hosts and entomopathogenic fungi. On the contrary, the diversity indexes were increased at the second year after application.Genotyping analysis of B. bassiana strains collected before and after fungal release showed that 2 out of 27 polymorphic SSR markers were specific to the released strain. The detection of recovery showed that the released strain had the higher recovery rates from both the soil and cadaver samples. The recovery rates ranged from 16.67%-66.66% for soil samples and 7.69%-82.14% for dead insects, indicating that the released strain had successfully colonized and dispersed in the pine ecosystem. Analysis of population genetic structure revealed that the artificial releasing strain in an inoculative way also did not impact the genetic diversity of indigenous B. bassiana populations. Analysis of host species infected by the released strain showed that the released strain could also invade 19 species of non-target insect hosts at different seasons. Of those, the species of Dasychira axutha, Monochamus alternatus, Shirahoshizo patruelis, and Lasius niger played crucial roles for the released strain to transfer among different food chains and provided an assurance for maintaining the stability of inoculum density and genetic diversity in the pine ecosystem. In short, the released strain could colonize and persist in the pine ecosystem by surviving as a saprophyte in soil and jumping among different insect hosts to maintain the sustainable control effect against the beetles in the field.
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