Identification Of Resistant Germplasm And Defence Mechanism Study In Common Bestn(Phaseolus Vulgaris L.) To Fusarium Wilt

Common bean is one of the main legumes for human consumption with high nutritional value andlarge-scale planting area. However, Fusarium wilt (FW) was a serious disease in the agricultrualproduction of common bean and usually brought greater losses to common bean production in thedisease happened year. Therefore, it was significant to ensure the food security and improve theeconomic value of common bean production by screening for stable resistance germplasm resources ofcommon bean, revealing resistance mechanism of the common beans to FW, discovering and takingadvantage of excellent resistance gene so as to improve the resistance of different common beanvarieties. In this study, a transcription factor gene (ftf1genes) from Fusarium oxysporum f. sp. phaseoliwhich is closely related to its virulence was used to design a pair of specific primers. The primers werecombined with real time quantitative PCR to differentiate the resistance level of different common beancultivars accurately. It revealed differentially expressed genes in interaction between common bean andpathogen by cDNA-AFLP analysis, then we cloned two defence-response genes named PvCaM1encoding calmodulin and PvPOX1encoding secretory peroxidase, separately. The exogenous planthormone was used to induce the host resistance and reveal the resistance signalling pathway andresistance mechanism of the common bean to FW so as to supply the gene resources and theoreticalbasis for resistance genetic improvement. The results as followed:1.59Fusarium oxysporum isolates collected from8locations in6provinces were identifiedusing pathogenicity test, morphological and molecular biological methods. The results indicated59F.oxysporum contained37pathogenic isolates. A transcription factor gene (ftf1genes) from Fusariumoxysporum f. sp. phaseoli which is closely related to its virulence was used to design a pair of specificprimers QFopA/QFopB, and149bp amplified product was obtained only from F. oxysporum f. sp.phaseoli. Therefore, the primers was used to identify F. oxysporum f. sp. phaseoli and screening for theresistant resource quantitatively.2.362accessions of common bean germplasm resources were screened for the resistant resourceinoculated with FOP-DM01isolate by the corn meal inoculum after comparation with the hypocotylsdouble holes injection and the root dipped inoculum, and we obtained16resistant cultivars, and2ofthem named with260205and Heiyundou showed stable resistance level, their accession numbers wereF0005035and F0004851, respectively. Using the specific primers QFopA/QFopB, the minimum of1pgpathogen DNA was detected by real time-PCR. The result indicated that FOP-DM01DNAquantifications in the roots and stems of susceptible BRB-130and A0640-1were significantly higherthan those in resistant260205and Heiyundou, which absolutely matched with the phenotypicidentification.3. The differentially expressed genes in interaction between common bean and pathogen wereanalyzed by cDNA-AFLP. Totally443differentially expressed fragments were obtained, and172ofthem were sequenced. The result indicated that a large number of plant resistance-related genes wasobtained, such as the GH3auxin-regulated protein, calmodulin, peroxidase and the expression level wasdeterminated by real time quantitative PCR; The results of scanning electron micrographs (SEM) and transmission electron micrographs (TEM) showed that the pathogens caused serious damage to theorganizational and celluar structure of roots and stems in BRB-130, however, the260205root and steminfected by pathogen were not obviously damaged; Phenylalanine ammonia lyase (PAL) and peroxidase(POX) activity was determinated, the result indicated that the activities of the two enzymes in the rootand stem of260205were significantly higher than BRB-130, which showed that these two plantdefense-related enzymes played an important role in the resistance of common bean to FW. In addition,H₂O₂and O₂^-content in the root and stem of260205and BRB-130were measured, it showed H₂O₂andO₂^-of260205were higher than BRB-130both in root and stem. Therefore, reactive oxygen species(ROS) were testified as an important signalling molecular in the resistant response of common bean.4. A full-length cDNA sequence coding for CaM in common bean was cloned based onexpressed sequence tags from common bean, designated PvCaM1(GenBank accession numberJN418801). Phylogenetic analysis based on the amino acids sequence of PvCaM1showed that theprotein encoded by this gene had the closest relationship with the CaM in Lotus japonicus andwatermelon, the homology were77%and76%, respectively. Real time-PCR analysis indicated that theexpression level of PvCaM1in the interactions between common bean and FOP-DM01isolateincreased significantly. The expression level of PvCaM1in leaves was higher than in roots.Transcriptional level of PvCaM1was up-regulated by exogenous abscisic acid(ABA), methyljasmonate(MeJA) and ethephon(ETH). PvCaM1expressed differentially in the leaves, stems and roots,and the expression level in leaves was higher than that in roots and stems.5. A full-length cDNA sequence coding for POX in common bean was cloned based onexpressed sequence tags from common bean, designated PvPOX1(GenBank accession numberJQ627838). Phylogenetic analysis based on the amino acids sequence of PvPOX1showed that theprotein encoded by this gene had the closest relationship with the POX in soybean and purple medic, thehomology were90%and85%, respectively. Real time-PCR analysis indicated that the enhancedexpression level of PvPOX1significantly correlated with the change of POX activity and H₂O₂content.PvPOX1expressed differentially in the roots, stems, leaves, flowers and pods, the expression level ofleaves was the lowest, and that of pods was the highest. Transcriptional level of PvPOX1wasup-regulated by exogenous salicylic acid(SA), ABA and other abiotic stress such as mechanicalwounding, salt and drought.6. SA, MeJA, ETH, paclobutrazol and nordihydroguaiaretic acid(NDGA) were used to treatBRB-130plants. The results indicated that plants treated by SA on leaves induced SA level in rootsincreased, and significantly enhanced the plant resistance to FOP-DM01isolate. In addition, SAinhibited growth of the pathogen in roots by inducing SAR in plants significantly, and SA had beentested to have no direct antifungal activity to FOP-DM01isolate in vitro; SA enhanced the activities ofPAL and POX, and increased H₂O₂and O₂^-content in common bean root significantly. These enhancedthe resistance to FOP-DM01isolate through induction of HR and SAR of common bean.