根结线虫拮抗菌的分离与鉴定.docx

Journal of Plant Biology 4Isolation and Identification of Antagonistic bacteria for Meloidogyne incognita in ShangqiuZhifang ZuoSchool of life science, Shangqiu Normal University, Shangqiu 476000, Henan, ChinaAbstract Biological control becomes increasingly important in the prevention of root-knot nematode disease. Screening root-knot nematode pesticides may be necessary for nematode control. We isolated two new antagonistic bacteria Z-2-36 and Z-7-5 from twenty eight soil samples by first and second screenings according to knockdown rates of their culture filtrates against Meloidogyne incognita instar larvae (J2). Knockdown rates were 95% and 97%, respectively. These two bacterial strains also promoted root growth and reduced the number of root knots. Through morphological and molecular characterizations, the isolated antagonistic strains Z-5-54 and Z-7-5 were identified as Arthrobacter pascens and Staphylococcus species.Keywords NematodeIsolationIdentificationAntagonistic bacteriaArthrobacter pascensStaphylococcusZ. Zuo (*)Subtropical Horticulture Research Institute, Jeju National UniversityJeju 690-756, Koreae-mail:IntroductionThe soil-borne root-knot nematode disease greatly affects the world agricultural production (Xiaoning Yan 2003). According to FAOs conservative estimate, nematodes damaged 21 kinds of crops costing $ 7.7 billion economic losses every year (Ning Xie 2005). Long-term use of chemical pesticides against the root-knot nematodes brought a series of side effects such as damage to the ecological balance, pollution to water and soil and the rise in drug resistance (Mankau.R.1980). Development of biological nematode pesticides should be an important direction. Bio-pesticides not only decrease harm to the environment, but also contribute to environmental conservation. Many plant health-promoting rhizobacteria (PHPR) which may produce nematicide substance or change the relationship between root excretion and nematodes to control nematodes have been discovered including Pseudomonas fluorescens, Bacillus sphaericus, Bacillus subtillis, Pseudomonas chlororaphis, but some of the rhizobacteria have not been identified (Mingliang Zhu 2004). We isolated and identified Arthrobacter pascens and Staphylococcus strains in Shangqiu for the first time, and their pesticidal activities will be studied.Materials and methodsFirst and second screening of antagonistic bacteriaBacterial strains were obtained by dilution -separation and purification method. Strains were cultivated in Salt Czapek Dox agar liquid medium for 5 days, then frozen and centrifuged to get the supernatant for the test of killing rates of J2 Plant materialsIdentification of antagonistic bacterial Genomic DNA was isolated with the CTAB procedure. Polymerase chain reactions (PCR) were carried out with Genomic DNA template with the following primers: 8A 5-AGAGTTTGATCCTGGCTCAG-3, 1542P 5-AAGGAGGTGATCCAGCCGCA-3. Sequencing was done by biological company at the end.Mode cultivar Moneymaker were tested forresistant activities of antagonistic bacteria for M.incognita in this protocol.Table 1 First screening of nematode strains death rates after 24h Strains J2 death rate（100%）Correction knockdown rate(100%)Z-1-1591.0%90.7%Z-1-4076.0%75.0%Z-2-183.0%82.5%Z-2-3691.0%90.7%Z-3-3890.0%89.7%Z-4-169.0%68.0%Z- 5-5492.0%91.8%Z-6-2883.0%82.5%-Z-7-593.0%92.8%Z-7-1663.0%61.9%CK3.0%-Table 2 Second screening of nematode strains death rates after 48h Strains J2 death rate（100%）Correction knockdown rate(100%)Z-2-36Z-7-5CK95%97%4%94.8%96.9%-ResultsFirst and second screeningAntagonistic bacterial strains with more than 50% death rates for J2 in 24h were collected in the first screening (Table 1), and ten strains were collected and tested for the second screening in the same way. Z-2-36 and Z-7-5 showed high death rates ( 90% vs. J2) in 48h in the second screening (Table 2).Tomato plants analysesMode cultivar Moneymaker which dressed seed with liquid bacterial suspension were planted in the greenhouse, and after 45 weeks 50 M.incognitaofsecond instar larvae were CKZ-5-54inoculated to test the effect of M.incognita on the seeding plants. The number of tomato root knots was detected one month later (Fig 1). Z-2-36 and Z-7-5 not only promoted root growth but also reduced the number of root knots of M.incognita. Z-7-5Molecular analysesPCR products from Z-2-36 and Z-7-5 with universal primers were analyzed by electrophoresis on agar with Ethidium bromide gels (Fig. 2a), and our bar gene was verified by Colony PCR before sequencing (Fig. 2b). The sequencing results and sequence alignments showed that Z-5-54 was Arthrobacter pascens and Z-7-5 was Staphylococcus. Fig.1 Moneymaker root knots in the greenhouse one month later after inoculated with J22000bp1000bp750bp500bp250bp100bp12M 2000bp 1000bp 750bp 500bp 250bp 100bp21MabFig.2 Results of PCR amplification with the primer 8A,1542P to Z-5-54,Z-7-5 a:Genome DNA amplificationb:Colony PCR amplification to PCR productionDiscussion Root-knot nematodes biocontrol resource refers to all predators in nature, including Nematophagous fungi, Obligate endoparasitic bacteria, Rhizobacteria, Actinomycetes, Protozoa Predacious nematodes and so on. Pasteuria is one of the most studied PHPR (Mingliang Zhu 2004). In our study, two new antagonistic strains Arthrobacter pascens and Staphylococcus that were isolated and identified through screening experiment and bioassay showed a significant level of resistance to M.incognita both in the liquid suspension experiment and in the pot experiment. Identification of