Studies on characterization of insect - plant circulating bacteria and application toward control of insect pests by these bacteria

摘要

Many bacterial strains were isolated from sevral species of insects (silkworm, mulberry pyralid and rice brown planthopper) and their diet plants. Several bacterial species, such as Erwinia herbicola - group bacteria (Er. ananas and Er. herbicola) and Enterobacter cloacae, were commonly isolated from both insects and plants. particularly, Er. herbicola - group bacteria formed a dominant flora in all samples. The growing ability of Er. herbicola - group bacteria and Ent. cloacae in the silkworm guts were examined. Plant- origin strains of Er. herbicola - group bacteria and Ent. Cloacae grew in the guts as well as insect - origin strains of them. Furthermore, a large number of these bacteria were isolated from fecal samples of silkworm ingesting them. These results suggest that Er. herbicola- group bacteria and Ent. cloacae on plant leaves are ingested by insects, grow in the insect guts, and transmit again to plant leaves through feces of insects. We named the this type of bacteria as 250L癷nsect- plant circulating bacteria”.Three characteristics of insect- plant circulating bacteria (ice nucleation active (INA) abilities, multiple drugs - resistance, and conjugative ability in insect guts) were investigated. All the 20 insect and plant-origin INA strains of Er. Herbicola - group bacteria, including “Er. herbicola” M1 (IceE) and Er. ananas IN10 (ina A) previously reported, were identified as Er. herbicola. Furthermore, the structure of ice nucleation (IN) genes was compared among 20 strains by PCP-RFLP method. PCR products from the whole IN gene containing R domain were markedly varied in their sizes within 3.8 kb to 4.4 kb, and RFLP analysis of the IN genes revealed that the R-domains were varied in size within the region from PvuII site to DraI site, classifying into 12 groups. Nine plant-origin strains of Ent. cloacae were resistant to 5 antibiotics (streptomycin, kanamycin, tetracycline, ampicillin, and chloramphenicol), and harbored a large plasmid designed as pMUL1, whereas insect- origin strains were resistant to onlyampicillin. The plasmid pMUL1 was hybridyzed with all the probe of these antibiotic- resistant genes and transfer to epiphytic Er. herbicola - group bacteria together with 5 drugs -resistance, indicating that pMUL1 is a conjugative and multiple drug - resistant plasmid. In addition, the gene transfer among epiphytic Er. herbicola - group bacteria, or between Er. herbicola -group bacteria and Ent. cloacae in silkworm guts were investigated. Both plasmids pBPW1 and RSF1010 transferred frequently to recipient strains. These results suggest that such a in insecta conjugation among epiphytic bacteria and insect- resident bacteria may play an important role in the evolution of plant- and insect - associated bacteria.To evaluate the suitability of these INA bacteria as a biocontrol agent against the insect pests, the cold hardiness of the larvae of mulberry pyralid and silkworm ingesting INA strains of Er. ananas and Pseudomonas syringae was monitored by supercooling point (SCP) and freezing mortality by cold incubation (-5 deg C). Cold-hardiness of mulberry pyralid and silkworm larvae ingesting Er. ananas decreased markedly (SCP and mortarity increase to -4.7 deg C and 80 percent, respectively) and a large number of INA bacteria was isolated from these larvae, showing good “gut-colonization” by these bacteria. In contrast, the cold-hardiness of mulberry pylarid larvae ingesting InA strain of Pseudomonas syringae was the almost similar level as that in the control treatments. Furethermore, the cold-hardiness of mulberry pyralid larvae ingesting the suspension of Ent. cloacae WBM (INA), which was constructed by transformation of IN gene of Er. ananas IN10 into Ent. cloacae WBMH-3, were examined. Remarkable decrease of cold- hardiness of mulberry pyralid larvae ingesting WBM(INA) was detected and kept much stably until at least 9 days after treatment, which is caused by better “gut-colonization” of Ent. cloacae than