The study of nonpathogenic Fusarium oxysporum on controlling Fusarium wilt of cucumber

Autor: Chao-Jen Wang, 王照仁
Rok vydání: 2013
Druh dokumentu: 學位論文 ; thesis
Popis: 101
Previous investigations demonstrated that nonpathogenic Fusarium oxysporum (NPFo) isolates, which have biological control potential for managing Fusarium wilt of crops, has not been possible to separate from pathogens through morphological characteristics. Although these two populations can be distinguished from one another using a bioassay, the work is laborious and time-consuming. In order to establish an effective method for screening and differentiating the NPFo isolates from pathogens, a fragment of the intergenic spacer (IGS) region of ribosomal DNA from an NPFo isolate, with biological control ability, was PCR-amplified with published general primers, FIGS11/FIGS12 and sequenced for comparing the nucleotides polymorphism with the Fusarium wilt pathogens. A new primer, NPIGS-R, which was designed based on the IGS sequence, was paired with the FIGS11 primer. These primers were then evaluated for their specificity to amplify DNA of NPFo isolate from other 122 Fusarium wilt isolates of fourteen different formae speciales. In addition, the modified primer pair showed positive amplification from four of the fourteen tested Fo isolates, and these four Fo isolates showed the ability in delaying symptom development of cucumber Fusarium wilt in greenhouse bioassay tests. Seventy-seven Fo isolates were obtained from the soil and plant tissues and then subjected to amplification using the modified primer pair; six samples showed positive amplification. These six isolates did not cause symptoms on cucumber seedlings when grown in peat moss infested with the isolates and delayed disease development when the same plants were subsequently inoculated with a virulent isolate of Fusarium wilt pathogen on cucumber. Moreover, the isolates of nonpathogenic Fo with biological control ability were monophylogeny and could been differentiated from other reference isolates of human and plant pathogens by phylogenic analysis based on the sequences of IGS and EF-1α. Previous study indicated that the colonization and distribution of NPFo isolates could be affected by different inoculation methods. In order to clarify the correlation between the colonization ability and the inoculation methods of NPFo in cucumber, two kinds of analysis methods, such as fluorescent staining and molecular detection, were used to observe the distribution and colonization of the NPFo isolate in plant tissues. Results of the distribution study indicated that the NPFo isolate Fo276 was restricted in root and peg tissues and mainly colonized in epidermis or cortex cell by seed coating, substrate irrigation and root soaking whereas Fo276 could colonize in hypocotyl tissues, stem tissues and vascular tissues by hypocotyl cutting inoculation method. Furthermore, the interaction between pathogenic isolate of Foc0812 and NPFo isolate of Fo276 in plant tissues were also evaluated and analyzed by molecular detection method in this study. The results indicated that the Fo276 still be restricted in root and peg tissues whereas the Foc0812 could reach and colonize in hypocotyl, cotyledonary node and stem tissues at 4 weeks after co-inoculated by root soaking method. However, the Fo276 could been observed in hypocotyl, cotyledonary node and stem tissues whereas the Foc0812 was been restricted in the 1-3 cm of hypocotyl tissues at 4 weeks after co-inoculation with hypocotyl cutting method. Previous study indicated that nonpathogenic Fusarium oxysporum (NPFo) isolates Fo276, Fo95022, Fo95024 and Fo95026 showed the ability in delaying the disease development on Fusarium wilt of cucumber in greenhouse bioassay tests. Moreover, the satisfactory biological control efficacy of NPFo was achieved when the isolate was introduced into plant by hypocotyl cutting inoculation method. The biocontrol efficacy of NPFo isolates in controlling Fusarium wilt of cucumber had been evaluated in field conditions by substrate irrigation and hypocotyl cutting inoculation methods. The preliminary test was achieved at Luzhu in 2010, the isolates Fo276, Fo95024 and Fo95026 were introduced into cucumber plants by substrate irrigation inoculation method, and the result indicated that the disease severity rating of NPFo isolates of Fo276 and Fo95026 provided after 9 weeks were 65.1 and 70.3%, respectively whereas the control treatment was 62.3%. However, the disease severity of the Fo95024 treatment was only 43.1%. In the next year, the NPFo isolates were introduced into cucumber plants by hypocotyl cutting inoculation method and then transplanted in field for evaluating the biocontrol efficacy of Fusarium wilt of cucumber at Yuchi. Disease severity was suppressed by the NPFo isolates Fo95022, Fo95024, Fo95026 and combination (33.3-38.0%) throughout 8 weeks period following the transplantation of seedlings into field. But isolate Fo276 did not suppressed the disease severity of 42.9%, as compared with 56.1% rating for the control.
Databáze: Networked Digital Library of Theses & Dissertations
Externí odkaz: