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| Rapid Screening of the Wild Coronatine-producing Stains Based on PCR and HPLC |
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Abstract Coronatine (COR) is a chlorosis-inducing non-host-specific phytotoxin that is produced by several pathovars of Pseudomonas syringae including P. syringae pv. tomato, P. syringae pv. glycinea, P. syringae pv. maculicola and P. syringae pv. atropurpurea. COR is a structural and functional mimic of jasmonates but it is more active than jasmonates. COR is involved in a wide array of effects on plant development and defence responses including inhibition of root elongation, hypertrophy, senescence, accumulation of defense-related protease inhibitors, secondary metabolite production, ethylene emission and resistance to abiotic stresses, and can function as a novel plant growth regulator. The objective of this study is to establish a rapid method for screening coronatine producing strains, which plays an important role in enriching its strain resources and promoting the research and application of COR. According to the key genes in COR biosynthesis gene clusters, corS and corR, two sets of PCR primers were designed. Polymerase chain reaction (PCR) and agarose gel electrophoresis gel electrophoresis were conducted for initial screening of wild COR-producing strains. The yield of COR for wild strains was determined by high performance liquid chromatography (HPLC). As a result, the target bands corS (850 bp) and corR (450 bp) were amplified when we used the genomic DNA of a typical COR-producing strain P. syringae pv. glycinea MW123 as a template. And it was consistent with the result of colony PCR of P. syringae pv. glycinea MW123. The PCR method was verified with the typical COR-producing strains and non-COR-producing strains. The results showed that the corS (850 bp) and corR (450 bp) bands were amplified only from the typical COR-producing strains, including P. syringae pv. glycinea MW123, P. syringae pv. glycinea MFB1 and P. syringae pv. tomato DC3000. The 2 target bands were not be obtained when using the non-COR-producing strains as templates. This showed that the designed primers had a fairly good specificity for COR-producing strains. The above PCR method was applied to preliminary screening of 32 wild stains isolated from diseased plants in the field. The specific PCR assay displayed that a 850 bp (corS) band could be obtained from the strains numbered 7, 18, 19 and a 450 bp (corR) band could be obtained only from the strains of 7 and 18. The 2 target bands, corS (850 bp) and corR (450 bp), weren't be observed in the PCR products of the rest wild strains. Among 32 wild strains, 3 wild strains (7, 18 and 19) were picked out as potential COR-producing strains. The selected strains were inoculated and cultivated in HSC culture medium for COR fermentation. Subsequent HPLC analysis showed that the strains of 7 and 18 could synthesize COR and the yields of COR were 6.5 mg/L and 2.1 mg/L, respectively. Two novel producing-COR strains were successfully screened out from 32 wild strains by specific PCR and HPLC. In comparison, the specific PCR is more efficient for COR-producing strains and it is suitable for screening rapidly COR-producing strains which could help to found higher yield of COR-producing strain and provide support for the industrial production and large-scale agricultural applications of COR.
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Received: 30 March 2017
Published: 01 November 2017
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