Control Of Fusarium Wilt Disease Of Cucumber By Application Of Bio-Organic Fertilizer And Its Working Mechanism

Vascular wilt of cucumber, caused by Fusarium oxysporum f. sp. cucumerinum J. H. Owen (FOC) is an economically important disease throughout the world. The wilt incidence rate can reach 50% in field, leading to important losses of cucumber and limits production in many areas of the world. Chemical control of Fusarim wilt relies to a large extent on the use of fumigant methyl bromide and other fungicides. However, environmental pollution caused by the residues of these fungicides limited their use in many countries. Environmental-friendly biological control represents an alternative for protection of plants against Fusarium wilts. The introduction of antagonistic microorganisms into soil has been proposed for the biocontrol of soi-borne diseases. Currently, it is believed that a combination of antagonists with suitable mature compost may be more efficient in inhibiting disease than using single antagonistic microbes or compost alone. In this study, we screened a new biocontrol agent SQR 9 from cucumber rhizosphere soil. In order to exploit the mechanisms of Fusarim wilt control, we isolated and purified and the antifungal componds produced by SQR 9 and its rhizosphere colonization. The soil bacterial community responses and induced systemic resistance triggered by application of bio-organic fertilizers were also studied.The morphology, biochemical and physiological properties and the sequence of 16S rDNA indicated that SQR 9 was a Bacillus subtilis strain. The strain showed significantly depressed mycelium growth of Fusarium oxysporum f. sp. cucumerinum on PDA by the dual culture technique. A total of 11 gene fragments of the expected size correlated with antibiotic biosynthesis, including surfactin, fengycin, iturin, bacillomycin, subtilosin, subtilisin were efficiently amplified from B. subtilis SQR 9. The methanol-extracts from the culture broth supernant inhibited Fusarium oxysporum growth in in vitro assays. The mycelic growth inhibition rate was 54.9% when 2-fold diluted crude extract was used in PDA. When compared with the control, the spore germination rates were decreased by 70.1% and 83.6% by adding 1-fold diluted and original crude extract into the water-agar medium. The crude extact showed significant inhibitory activities against several soilborne pathogens in cylinder-plate assays. HPLC-ESI-MS analysis suggested that SQR 9 produced two groups of ion peaks with mass difference of 14 Da at m/z (M+H)+1063.6,1017.5, 1031.6,1045.6,1059.6 and 1506.0,1435.0,1449.9,1463.9,1492.9,1477.9. Concerning the literatures and our results, the two compounds might be bacillomycin and fengycin with structural analogs with different side chain length (-CH2-), respectively.The shuttle plasmid pHAPⅡwas used to introduce plasmid-borne gfp genes into SQR 9 by electroporation methods. The gyp-transformant SQR 9-gfp could be easily distinguished by their fluorescence when visualized by fluorescence microscopy. The SQR 9-gfp strain could be colonized on cucumber rhizosphere soil by root-dip inoculation. The strain colonized on cucumber root surfaces most (106-107CFU·g-1 root), followed by rhizosphere soil and internal roots, and the bulk soil. SQR 9-gfp strain was mainly distributed on elongation zone, the root hair zone of cucumber primer roots and the lateral root junctions. Very few cells were found in the root tips.Greenhouse experiments were carried out to evaluate the effect of organic fertilizer inoculated and solid-fermented with Bacillus subtilis SQR 9 and Trichoderma harzium SQR T-037 alone or in combination, hereby defined as bio-organic fertilizers BIO A, BIO B and BIO AB, on the control cucumber Fusarium wilt. The bioorganic fertilizer products were applied into soil during the nursery phase of cucumber seedlings followed by a second application to Fusarium-infested soil when cucumber seedlings were transplanted. Compared with the control treatment, the shoot and root biomass of bio-organic fertilizer-treated plants were promoted by 1.9 to 2.3-fold and 2.1-fold, respectively. Plant height was increased by 72.7%. The organic fertlizer also had a positive effect on plant growth, but the effect was less signifcantly than bio-organic fertilizers. The control treatment had the highest wilt disease incidence rate (73%) 60 d after transplantation. Biocontrol was also achieved by applying bioorganic fertilizers to Fusarium-infested soil. The best biocontrol was obtained by application of BIO AB, which significantly decreased the desease incidence (only 15%). And application of organic fertilizer also reduced the disease incidence (43.2%).Plate counting and Real-time PCR were used to detect the rhizosphere colonization by the pathogen and the two biological agents. Populations of FOC in BOF treated soil declined to 103 CFU·g-1 root after 60 days of inoculation, while those in control treatment remained high (2.48×10S CFU·g-1 root). Application of organic fertilizer alone reduced the FOC population by 84.6%, compared with the control. The treatment BIO AB had the lowest number of FOC (1.2×103 CFU·g-1 root). Real-time PCR analysis with SCAR primer FocF3 (F)/FocR7 (R) earlier designed to be F. oxysporum f. sp. cucumerinum specific was carried out to determine copy number of the target DNA present in cucumber rhizosphere. The DNA copy number varied from 3.4×105 to1.32x107 copy·g-1 root. In agreement with the plate counting, application of bio-organic fertilizers produced a suppressive effect on DNA copy number. The counts of FOC in BIO AB treatment were two orders of magnitude lower than the control. The level of rhizosphere colonization by B. subtilis SQR 9 was monitored in all treatments. Using B. subtilis-selective medium, No background Bacillus-like colonies were found on the non-inoculated plants (CK,OF and BIO B) roots, while the BIO A and BIO AB-treated roots were colonized by 6.03x104 and 1.62x104 CFU·g-1 fresh root B.subtilis 60 d after pathogen challenge.SQR-T037 ITS regions were amplified using primers and probe specific for Trichoderma harzium in Real-time Taqman PCR assay. The non-inoculated treatments had a backgroud growth of T. harzium of 102-103 copy·g-1 root, while in BIO B and BIO AB treatment, the number of T-037 reached 4.9-5.0 log copy·g-1 root, indicating that B. subtilis SQR 9 and T. harzianum SQR-T037 was successfully colonized in cucumber rhizosphere and thus effectively protected the plants from being attacked by pathogens.Before the outbreak of Fusarium wilt, the leaf MDA contents in organic fertilizer and bio-organic fertilizer treated cucumber plants were significantly lower than control 30d after transplantation. The activities of catalase (CAT), peroxidase (POD), superoxidase (SOD) and phenylalanine ammonia-lyase (PAL) were increased by 35.4-59.0%,46.1-86.8%, 33.3% and 71.2-109.3% by bio-organic fertilizer application, respectively, when compared with the control. The activities ofβ-1,3-glucanase and chitinase were also promoted by 58.5-86.5% and 52.1-71.4%, respectively.In order to study the bacterial community responses to application of organic fertilizer and bio-organic fertilizer, we analysed the bacterial richnesses, diversities and community compositons of soil samples in bio-organic fertilizer (BIO AB), organic fertilizer (OF) and the control (CK) treaments using 454-sequencing. The observed OTUs were distributed across three bacterial phyla, Firmicutes, Proteobacteria and Actinobacteria in all samples, suggesting the three samples may have similar bacterial composition. However, the number of OTUs of some groups within one phyla among each sample varied. In BIO AB and OF samples, Bacillaceae, Paenibacillaceae and Planococcaceae were the highest in OTUs in Bacilli class, while Bacillaceae, Paenibacillaceae and Clostridiaceae more abundant in CK treatment. The Bacillaceae accounted for 37.82% and 43.55% of OTUs in BIO AB and OF samples, while only 20.51% of OTUs in the control were assigned as Bacillaceae. On the genus level, Bacillus, Methylocystaceae, Paenibacillus, Actinomadura and Actinoalloteichus were the five most abundant genuses in BIO AB and OF soil samples, while Bacillus, Asticcacaulis, Haliangium and Clostridium were most abundant in the control. It is also noteworthy that Bacillus accounted for 35.24% and 41.21% in BIO AB and OF, respectively, while it only taken up for 13.06% in the control treatment. Moreover, the bacterial diversity of control was significantly different from BIO AB and OF treated soil. Analysis on Thetayc (community structure similarity) and Jclass (community overlap) estimators suggested that there were significant difference in bacterial community structure between BIO AB, OF and the control, while no significant difference was observed between the treatments of BIO AB and OF.