Identification And Pathogenicity Of Bacteria Associated With Kiwifruit Canker

Kiwifruit canker (caused by Pseudomonas syringae pv. actinidiae) is a worldwidebacterial disease which could cause destructive effects on kiwifruit orchards and thusresult in serious economic loss. It has been found in China, Japan, Korea, Italy, NewZealand and other main kiwifruit production countries. Currently, the disease occurs withincreasing frequency in main kiwifruit production areas in China, even broken out inindividual years. Due to the fact that there is no systematicresearch on the pathogen of kiwi canker, the study collected and isolated samples fromtissues of kiwifruit canker which is wildly occurred in Anhui, Shaanxi and Chongqing.The related bacteria will be further identified with phenotypic identification（Morphological, physiological and biochemical）, molecular characteristics analysis,and pathogenicity test will also be carried out in the research. The main results are thefollowing:1. Separation and phenotypic identification of bacteria related to kiwifruit cankerdisease.Three culture medium was used to separate the samples by grinding diluteseparation method, and328high frequency and representative bacterial isolates werepurified, these bacterial in BPA medium culture has three types as following:311strains with a white colonies, circular bulge, smooth and wavy edge, including282gramnegative, active, aerobic, LOPAT test (++-++), positive for seven leaf glycosideshydrolysis strains, gelatin liquefaction test, glucose, sucrose, trehalose, cellobiose,lactose, maltose, and it can use glucose to produce acid and produce fluorescent pigmenton KB medium, these bacterial were preliminarily identified as Pseudomonas fluorescens.Other29strains were gram negative, facultative anaerobic, movable, LOPAT test (+---+), negative for fluorescent pigment production and gelatin liquefaction, positive forcatalase reaction and seven leaf glycosides hydrolysis. The second category is yellow, smooth circular, entire margin. The third is milk-white, producing yellow pigment,growing fast.2. Analysis of the molecular characteristics for the relation bacteria with kiwifruitcanker disease.By use of the specific primers PsaF1/R2and PsaF3/R4of P. s. pv. actinidiaePCR amplification has been done for the whole genome DNA of125bacterial strains.The results showed that only two fragments amplified for the three strains of P.s. pv. actinidiae from Italy(F285, Fc84, F28c) can be detected in the positive around175bp and280bp, and the two fragment were not detected fromthe separated bacteria strains.Approximately1500bp amplified DNA fragments from125bacterial strains wasobtained by the bacteria16S rRNA fragment universal primers P1F/P2R. Enzymedigestion was done separately for the16S rRNA-PCR amplification fragment byrestriction enzymes HaeIII and MSPI, the results of PCR-RLFP showed that the strainsare divided into four times with enzyme HaeIII, another are divided into five times withMSPI. These strains’ phenotypic characterization results are in accord with the enzymedigestion results.14representative bacterial strains were selected, and16S rRNAfragment of these bacteria were amplified, cloned, and sequenced, separately. Theobtained sequences was further analyzed in the NCBI database by BLAST software,the results showed that the16S rRNA sequences of three P. s. pv. actinidiae (F285,Fc84, F28c) had the highest similar (99%) to P. s. pv. actinidiae deposited in GenBank,the strains AHK-1, AHK-2, A1-3, A2-1, A2-2had the highest similar(99%) to P.fluorescens, the strains A17, F-7had the highest similar (99%) to Rahnella spp., thestrain C101, AL-3had the highest homology (99%) to Xanthomonas spp., and the strainsB-2, C-3had the highest similar (99%) to Pantoea agglomerans.The PCR amplification was done pseudomonas ITS universal primers D21/D22forthe genome DNA of14representative bacteria strains, the results showed that the bands of P. s. pv. actinidiae and P. fluorescens was inspected in the location of600bp, which isagreed with the expected fagement. However, the targeted fragment was not detected byPCR amplification for the strains of Rahnella spp., Xanthomonas spp. and Pantoeaagglomerans. ITS fragment of these bacteria was amplified, cloned, and sequenced,separately. BLAST searches with the DNA sequences obtained showed that the rRNA-ITSsequences of Pseudomonas isolates (AHK-1, AHK-5A1-3，A2-1，A2-2) had the highesthomology (97to99%) to that of P. fluorescens strains A506,62e, and2313, while therRNA-ITS sequence of strain F285from Italy had the highest homology (99%) to that of P.syringae pv. actinidiae strains PA835and Kw-11. For sequences with the ITS sequencesof known bacteria phylogenetic analysis, it is showed that P. fluorescens, P. syringae and P.avellanae belong to different branches (89%bootstrap support), and this experimentseparated from Pseudomonas strains are closely adjacent to P. fluorescens (95%bootstrapsupport), the strains P. s. pv. actinidiae from Italy are close to P. s. pv. actinidiae (63%bootstrap level).3. Pathogenicity assays of the relation bacteria with kiwifruit canker disease.The tobacco leaves, twigs and1-2year branches on vitro health kiwifruit plant,twigs and leaves on living kiwifruit plant were inoculated with bacterial suspension of14representative bacterial strains（107~108CFU/mL）by wounded and non-woundedmethods. Three days later, it is observed allergic reaction on tobacco leaves by thesestrains including Rahnella spp., Xanthomonas spp., P.fluorescens and P.s. pv. actinidiae,but causing weak allergic yellow lesion by Pantoea agglomerans after ten days. Only P.fluorescens and P. s. pv. actinidiae strains can cause symptom on the branches, twigs andleaves, no symptoms was found with other strains. There is no symptom on the tissue bysterile water. According to the Koch’s rule, P. fluorescens and P. s. pv. actinidiae strainswere successfully isolated from the infected tissue. Therefore, we can conclude that P.fluorescens is the causal agent of kiwifruit bacterial canker disease. So far, it is the first time of P. fluorescens as a new causal agent of bacterial cankerdisease of kiwifruit in China.