Effect of chitinase expression in Pseudomonas putida on plant disease control
Date Issued
2004
Date
2004
Author(s)
Yang, I-Hua
DOI
zh-TW
Abstract
土壤中存有大量根圈細菌，這些細菌能夠群聚並長期存活於植物根圈，分泌物質提供植物養份，並促進植物生長，被稱為促進植物生長之根圈細菌(plant growth-promoting rhizobacteria, PGPR)。許多PGPR菌株為螢光假單胞菌(fluorescent pseudomonads)，已知多種Pseudomonas fluorescens及Pseudomonas putida菌株能防治植物之葉部及根部病害。以生化方法收集P. putida拮抗菌株LY95及其產生幾丁質分解酵素之遺傳工程菌株的周質、細胞質、內膜和外膜分段液，比較幾丁質分解酵素的活性，在P. putida LY95(pMMST2000)外膜及內膜可偵測到幾丁質分解酵素，利用西方免疫法估計其分子量約為94 kDa。 P. putida LY95(pMMST2000)經免疫螢光染色，以螢光顯微鏡及雷射共軛焦掃描顯微鏡觀察，均可在其一細胞表面觀察到螢光。螢光基質分析顯示P. putida LY95(pMMST2000)之培養上清液也可偵測到幾丁質分解酵素活性；以P. putida LY95(pMMST2000)培養18小時之上清液處理百合及番茄之萎凋病菌及灰黴病菌，皆有抑制孢子發芽的作用。於溫室中，P. putida LY95(pMMST2000)能有效地降低百合及番茄萎凋病及灰黴病的罹病度，顯示表現幾丁質分解酵素分佈於細胞表面的方式，可增加P. putida之抑菌活性及生物防治活性。
Rhizosphere bacteria are present in large number in the soil where nutrients are provided by plant root exudates. Their application can stimulate plant growth, therefore being named plant growth-promoting rhizobacteria (PGPR). Fluorescent Pseudomonads such as Pseudomonas fluorescens and Pseudomonas putida are able to suppress plant diseases. In this study, the periplasmic, cytoplasmic, inner membrane and outer membrane fractions of a Pseudomonas putida wild type strain LY95, and its genetic engineering strains were isolated by biochemical method. The inner and outer membrane locations of chitinase in P. putida LY95(pMMST2000) were indicated by Western blot analysis with ChiA1 antibody. Molecular mass of detected protein was estimated to be 94 kDa. Fluorescence was observed on bacteria cell surface of FITC-labelled P. putida LY95(pMMST2000) by fluorescence and confocal laser scanning microscopy. By fluorometric assay, chitinase activity was detected in the culture supernatant of P. putida LY95(pMMST2000). The culture supernatant of P. putida LY95(pMMST2000) was capable of inhibiting conidial germination of Botrytis spp. and Fusarium spp. Application of P. putida LY95(pMMST2000) effectively decrease disease severity caused by Botrytis spp. and Fusarium spp. on lily and tomato, suggesting that expression of chitinase in P. putida transformant and translocation of chitinase to bacterial suface could increase its antifungal and biocontrol activity.
Subjects
生物防治
抑菌活性
促進植物生長之根圈細菌
螢光假單胞菌
細菌幾丁質分解酵素
chitinase
biocontrol
antifungal activity
plant growth-promoting rhizobacteria
fluorescent Pseudomonads
Type
other
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