| 308Verticillium dahliae isolates were recovered from three cotton-planting regions inChina and of which, pathogenicity of167isolates was identified using a method namedvermiculate and sand in bottomless paper pot dipping in quantitative spores. The number ofdefoliating and non-defoliating isolates was also tested based on specific deofliating andnon-defoliating primers. Correlation between fingerprint of ISSR and ISSR and thepathogenicity was analysised. And a mutant library, which initiated from a high virulent V.dahliae strain Vd080, was constructed by agrobacterium tumefaciens-mediatedtransformation (ATMT). Three genes related to biological characteristics and pathogenicitywere cloned and analyzed. The main conclusions showed as fllowing:1. According to cultural characteristics on potato dextrose agar (PDA),308V. dahliaeisolates were divided into five culture types (A-E). Both type A (0.6%) and type B (72.7%)produced massive microsclerotia, moverover, type A produced black mycelia while there wasa white circle at the colony edge of Type B. Type C (23.7%) produced a few microsclerotiadistributed at the edge of colony which formed a black circle. Both type D (1.6%) and type E(0.1%) did not produce microsclerotia under room temperature, in addition, there were raisedaerial mycelium at the colony surface of type E Among them, Type B was the dominant groupaccounted for72.9%. The cultural characteristics of isolates from Yangtze River regionshowed highest variation, followed by those from Yellow River and Xinjiang.2. Based on the results of the pathogenicity tests,167V. dahliae isolates were clusteredinto three groups, which showed strong (average disease index varied53.4~71.5), moderate(average DI varied20.9~51.7%) and weak (average DI varied6.4~27.4) pathogenicity oncotton, respectively. Moderate virulent isolates distributed dominantly in China (57.5%).Differentiation tendency of isolates recovered from Huanghe Basin cotton-planting region andYellow River Basin showed consistently. Isolates exhibited similarly pathogenicity. Amongthem, moderate pathogenic isolates belonged to the dominant group. However, in Xinjiang,the number of weak pathogenic isolates was largest. 3. Among167tested isolates,91%of them were identified as defoliating V. dahliae withthe specific deofliating and non-defoliating primers (D-1/D-2, ND-1/ND-2), which widelydistributed main cotton producing regions. Most of the defoliating isolates belonged to strongand moderate pathogenicity groups. Howerver, The majority of non-defoliating isolatesexhibited weak pathogenicity. The results of greenhouse tests showed that bothnon-defoliating and defoliating isolates could lead to the leaf-defoliation of cotton seedlings.Compared with non-defoliating isolates, the cotton seedlings inoculated with defoliatingisolates showed defoliation3-6days earlier and the defoliating percentage significantly higher.Under the inoculating concentration of1×10~7spores per milliliter, the defoliating percentageof cotton seedlings inoculated with defoliating isolates was significantly higher than thoseinoculated with non-defoliating isolates. With the increasing of inoculating concentration, thedefoliating percentage of cotton seedlings inoculated non-defoliating isolates obviouslyincreased. When the inoculating concentration reached to1×10~8spores per milliliter, there wasnot distinct difference between cotton seedlings inoculated with non-defoliating anddefoliating isolates. The above results showed that non-defoliating isolates needed longerperiod and higher inoculating concentration to cause the defoliation of cotton leaves.4. SSR markers were explored from the genome database of V.dahliae on lettuce. Highspecific primers were designed based on the flanking sequence of SSR.13pairs of SSR withhigher polymorphism were selected for molecular fingerprint analysis. The results showedthat167tested isolates of V. dahliae from China were distinctly classified into two groups.GroupⅠcontained141isolates, of which，strong and moderate pathogenicity isolatesaccounted for89.4%；GroupⅡcontained26isolates, of which，84.6%of the isolates wereweak pathogenicity. Nine pairs of ISSR primer were used to analyze the genetic diversity of167tested isolates of V. dahliae from China. Clustering analysis results showed that167tested isolates were classified into strong, medium and weak three different pathogenicitygroups (strong, moderate and weak). There were17(10.2%),123(73.7%) and27(16.2%)isolates in strong, moderate and weak pathogenicity groups respectively. The study indicatedthat there was a significant correlation between fingerprint of ISSR and ISSR and thepathogenicity. Genetic relationship among isolates with similar pathogenicity was relativelyclose.5. A mutant library containing2000mutants of a high virulent and deofliating V. dahliaestrain Vd080was constructed by the ATMT. Fresh A.tumefaciens cells with OD600≈0.4, freshV. dahliae spore suspension,200μM Acetosyringone and48hours co-cultivation period at25℃were optimizing conditions. Mutant ratio could reach150~540transformants per106spores under the optimizing conditions.The results of PCR and Southern blot indicated thatT-DNA inserted into the genome of Vd080successfully, and T-DNA in mutants selected randomly were mostly single copy. All of the tested transformants maintained their resistanceto hygromycin B.6. Three mutants, with significant variation in biological characteristics andpathogenicity compared with wild strain Vd080, were screened from the mutantlibrary.VdT286showed significantly lower spore yield, growth rate and non-microsclerotia.In addition, its pathogenicity decreased slightly. Both VdT1023and VdT1053exhibitedsignificantly lower pathogenicity. Vd1053did not produce microsclerotia and the secretioncrude toxin of VdT1023increased significantly. Using TAIL-PCR, three genes related tobiological characteristics and pathogenicity of V. dahliae were cloned by amplying flankingsequence of inserted T-DNA. In VdT286, T-DNA was inserted into CYC8(glucose repressionmediator protein)，which was a key gene in the control of spore yield, growth rate of myceliaand microsclerotia producing of V. dahliae. It was located in the fifth chromosome with3201bp in full length and contained7exons and6introns. The length of its cDNA was2679bpcoding892aa. The function of CYC8was still not reported in V. dahliae yet. In VdT1023,T-DNA was inserted upside promoter region of VDAG00467, which was a new unknown genewithout annotation. It was located in the second chromosome with1152bp in full lengthwithout intron, Coding protein contained383aa. In VdT1053, T-DNA disrupted VDAG00607,which was located in the second chromosome with1947bp in full length and contained1intron. Its coding product was phosophoglycerate mutase containing648aa. |
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