Pathogenicity Differentiation And Genetic Diversity Of Thielaviopsis Basicola Causing Tobacco Black Root Rot

Tobacco black root rot is a worldwide disease, caused by Thielaviopsis basicola (Berk.&Br.) Ferr., the pathogen is associated with the roots of wide range of plants in nature and is recognized as a pathogen that causes black root rot on many important agricultural crops worldwide. However, until now little is known about the extent of morphological variation within T. basicola or about the level of genetic diversity within this fungal species in China.In order to explore the morphological, pathogenicity differentiation and molecular diversity of T. basicola, the following studies were carried out.(1) The pathogen was isolated from diseased tobacco tissues and soil samples of tobacco root rot were collected from5different Chinese geographic regions, including Yunnan, Gansu, Guizhou, Chongqing and Henan provinces. Pathogenicity of the all isolates was tested according to Koch's postulates. Then using traditional morphological classification and rDNA-ITS sequences analysis methods, the T. basicola isolates were further identified.(2) The culture characteristics of three isolates with different colony characteristics that obtained from tobacco black root rot were studied.(3)Tested the pathogenicity differentiation of some T. basicola isolates, and the disease resistances of7main tobacco cultivars against different T. basicola isolates were also evaluated.(4) According to Punja&Sun's grouping identification standard, grouping identification of all isolates was studied.(5)The molecular genetic diversity of all T. basicola isolates including32isolates from carrots was analyzed by the inter-simple sequence repeat (ISSR) technique.(6) The initial interactions between T. basicola and root hairs of tobacco were examined microscopically to make the infection mechanism of T. basicola clear. The results were as followings.1. The specimens of diseased tobacco plants with typical syptoms of black root rot and its soil was collected, the geographic origins of the specimens were Yunnan, Gansu, Guizhou, Chongqing and Henan provinces in China. Using the improved carrot disks methods more than20isolates were collected. After further purification and single spore isolation,45wild types and13white aberrant phenotypes of the pathogen were obtained. The results of experiments accoding Koch's postulates revealed that these wild type isolates were the pathogen of the disease. The morphology and rDNA-ITS molecular identification results show that:all the isolates including two from carrot were Thielaviopsis basicola (Berk.&Br.) Ferr. in classification. The rDNA-ITS sequence analysis suggested that the white aberrant phenotypes were identical to it's wild types in sequences, but completely different from the isolates growing on other host plants.2. Biological characteristics of three T. basicola isolates CPW1-1, CWH-1and HLL-1' with different colony characteristics were studied. The results showed:HLL-1'could not develop conoly on Czapek media, CPW1-1and CWH-1could but conoly is very thin. PSA, PCA and PDA were the most favorable media of three isolates respectively. The temperature has a significant effect on the mycelial growth of the tested isolates, they could grow in5℃～35℃, and the optimum temperature was25℃. The tested strains could grow under the pH=4-12, while under pH=7,10and6were optimum pH values for the mycelial growth of CPW1-1, CWH-1and HLL-1', respectively. CPW1-1and CWH-1could use all of the tested carbon sources except xylose, beef ertract and casein was their favorable nitrogen sources. Under the same culture conditions, the conidia germination rate of CPW1-1was higher than that of CWH-1, however under the organic nutrition, CWH-1was higher than CPW1-1. That showed that nutrition enhanced the conidia germination rate of CWH-1. Dark conditoin was favorable for the germination. The optimum conidia germination temperature of CPW1-1, CWH-1and HLL-1'were20℃,15℃and20℃, respectively.3. Pathogenicity differentiation against black root rot was compared by root cutting inoculation method within15T. basicola isolates, and their pathogenicity showed significant difference. The disease index of15isolates ranged from19.2to62.5, in which4isolates showed high pathogenicity,10isolates were at moderate level, and HLL-1'the albino shouwed weak pathogenicity, and no weak pathogenicity was found in wild type isolates. The disease resistances were significant difference among the7cultivars when different cultivars inoculated by a certain T. basicola isolate, the disease resistance changed from resistant, moderate resistant, susceptible and highly susceptible. Based on the experimental results, it was showed that the resistance of Guiyan4, Qinyan96and Zhongyan100was higher than those of other tested cultivars.4. According to Punja &Sun's Grouping identification standard of T. basicola, the extent of grouping identification was studied in those58T. basicola isolates from tobacco including45wild types and13albino isolates. The results showed that six general morphological groups could be distinguished among the isolates, in which group I, group II and group III were wild types, group Ⅳ and group V were aberrant phenotypes (albino and mycelial). The characteristics of colony and chlamydospores of another aberrant phenotype HLL-1'was completely different from that of the five groups above, colony of it was flat and white, the chlamydospores were borne either in clusters or singly, phialospores could be borne, so it was assigned to be the new group——group Ⅵ. The isolating ratios of groups Ⅰ, Ⅱ, Ⅲ, IV, V and VI were41.4%,32.8%,3.4%,19.0%,1.7%and1.7%respectively. Group I was mainly distributed in Chongqing and Yunnan, group III and V in Guizhou, group IV mainly in Chongqing, Henan and Yunnan, group VI only in Henan, and group II was distributed in all investigating provinces.5. The ISSR amplification system of T. basicola was optimized and established with the parameters, such as the concentrations of Taq DNA polymerase, Mg2+, dNTP and DNA templates, the annealing temperature. A total of6specific ISSR primers were screened from20ISSR primers, and the average percentage of polymorphic loci of populations was70.91%, which suggested all tested isolates of T. basicola had high genetic diversity (the average value of Shannon index I is0.5504). The genetic variation was significant among populations (average Nei's index h was0.3726, and average GST is0.1657). The tested isolates were clustered into7genetic lineages.6. The above results showed that the pathogen virulence differentiation had obvious correlation with its cultural characteristics and morphological grouping, whereas the ISSR clustering groups was insignificant correlation with the virulence and morphological grouping of the isolates, but they were closely related to the different host sources.7. The ultrastructure of initial interactions between T. basicola and tobacco was examined microscopically. It showed that the pathogen invaded the tobacco directly; the culture time of T. basicola affects germination time, germ tube morphology and infectivity of the conidial. The structure that formed is different when the young endoconidia invade into the root hairs from into the epidermic cells. No appressoria were formed when invade into the root hair. The maturity conidial germination time is a little longer and appressoria formed on the top of germtubes. And the chlamydospore could invaded directly, and the top of germtube swollen slightly.