| Muskmelon (Cucumis melo. L) is one of the ten most popular fruits in the world and has been grown over temperate and tropical lands. Muskmelon planting in China accounts for 48.5% of the total world muskmelon cropping area and 56.3% of its production. Muskmelon fusarium wilt, a vascular wilt, which is caused by soil-borne pathogen Fusarium oxysporum f.sp melonis that infects the crop in the whole growth periods, is always a disaster for farmers all over the world. Thus control of the disease is an urgent need worldwidely. In recent twenty years, the severity of the disease in China becomes heavier than ever due to wide replanting problems from intensive farming. While chemical control is challenged by environment and human safety, farming control such as crop rotation and field management is usually time-and labor-consuming. Therefore, biological control has gathered much attention across many research fields to replace chemical inputs with environment-friedly biotechnological products. However, few reports have been found on the biocontrol mechanisms. Biocontrol products are also rare in market.In the present study, we developed a new bio-organic fertilizer to control muskmelon fusarium wilt disease and then investigated its biocontrol mechanisms on muskmelon by pot experiments and lab tests. Furthermore, we tagged the biological agent of Bacillus subtilis Y-IVI by green fluorescent protein technique to facilitate enumeration of its real population from complex environments. Pot experiment and series of lab tests were carried out to investigate Y-IVI’s promotion effects on muskmelon growth and its colonization ability in the rhizosphere and the interior of plant tissues.The main results obtained were listed as follows:1. Isolation of Fusarium wilt pathogen is the fundamental prerequisite step for biological control of the wilt disease. The pathogen was isolated from tissue of a diseased muskmelon plant (collected from Hexian County, Anhui Province, China) using Fusarium-selective medium. Based on the mycelium, conidiophores and hlamydospore characteristics, the isolated fungus was identified as Fusarium spp. Furthermore, the isolated Fusarium spp was confirmed as the responsible pathogen by procedures described in Koch’s postulation. The wilt disease incidence was 80% after inoculation of the conidia suspension 20 days. Five bacteria (Y-6, Y-8, Y-10, Y-12 and Y-IVI) and two fungi (Al and Pl) with strong antagonistic activities against Fusarium oxysporum f.sp melonis (FOM) were isolated from healthy muskmelon rhizosphere soil by using in vitro antagonism tests. The filtrate suspensions of 5 antagonistic bacteria have no negative effect on muskmelon seed germination. The germination rate of seeds treated with filtrate suspension of Y-10 and Y-IVI were increased by 10% and 5% compared with control, respectively, in addition, the radical length in these two treatments was significantly higher compared with others. The culture filtrates of antagonistic bacteria can highly inhibit pathogen growth in vitro antagonistic tests. The pathogen inhibition rate was 54%,67%,75.3%,68% and 72.4% corresponding to 10% filtrate suspension concentration of microbes Y-6, Y-8, Y-10, Y-12 and Y-IVI, respectively. These results showed that microbes of Y-10 and Y-IVI have stronger inhibition effect on FOM and could be used as potential biocontrol agents. Laboratory tests showed that B. subtilis Y-IVI can produce indole acetic acid, siderophores and ammonia. Based on morphological and biochemical characteristics and 16S rDNA technology, Y-10 and Y-IVI were respectively identified as Paenibacillus polymyxa (Genbank accession number GQ849013) and Bacillus subtilis (Genbank accession number GQ475486). The two antagonistic fungi microbes Al and PI were tentatively identified as Aspergilis spp and penicillum spp, respectively.2. Pot experiments were performed to investigate the effects of different bio-organic fertilizers (BIOs) made from organic fertilizer and different antagonistic microbes(Y-IVI, Y-10, Al and PI). BIOs decreased the incidence of fusarium wilt disease and increased melon yield. The disease incidence of treatments with double application (BIOs applied both in the nursery and the pot soil) was 20%, much lower than control (80%). Application of BIOs strongly reduced the number of pathogen colony-forming units (CFU) in stems and roots of melon. Pathogen populations were 2.27×103 and 6.67×103 CFU g"1 FW (fresh weight) on BIOII-treated stems and roots, respectively, and 8.17×104 and 3.67 x 104 CFU g-1 FW on control stems and roots, respectively; i.e., CFUs were reduced by 97% and 82%, respectively. Microbial community structure was ameliorated by all BIOs. The number of bacteria and actinomycota in rhizosphere soil increased markedly under all BIO applications compared to control. In contrast, pathogen and fungal density was dramatically higher in the rhizosphere of control plants. The activities of defense enzymes in the leaves of melons receiving double application of BIOII were lower than those of control plants. In conclusion, the most effective treatment was double application of BIOII, which minimized the incidence of wilt disease, maximized biomass production, and altered microbial community structure. In addition, greenhouse experiments were carried out to investigate the abilities of Bacillus subtilis Y-IVI to promote plant growth and to colonize the rhizosphere and interior tissues of muskmelon. The inoculation of soil with green fluorescent protein-tagged Y-IVI (GY-IVI) significantly increased plant shoot and root dry weights as compared with the noninoculated soils. The inoculation of soil with B. subtilis GY-IVI maintained approximately 108 colony-forming-units (CFU) of GY-IVI per gram of dry rhizosphere soil for one month. The GY-IVI recovered from the interior of crowns and roots in the inoculated soil were 106 and 107 CFU g-1 dry weight, respectively, suggesting that GY-IVI acted as an endophyte.3. The culture filtrate of B. subtilis Y-IVI has antifungal activity even after diluted to 10% of its suspension. The compounds in extracts were aciduric by adjusting pH to 2 for 24 h and thermostable by heating at 80℃for 2 h. They were also stable to digestion by pepsin and Proteinase K. The crude lipopeptides from culture filtrate were further extracted by HCl precipitation and purified by high-performance liquid chromatography (HPLC). Two peaks that were detected by HPLC had antifungal activities. Liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) analysis showed that the mass spectra of the two peaks were characterized by two series of homologous ion peaks, one with molecular weights of 1028.7,1042.7 and 1056.7 and the other with molecular weights of 1463,1477 and 1491. The two series of compounds were ascribed to iturin A and fengycin, respectively. The maximum production of iturin by strain Y-IVI inoculated in Landy medium was 89.75 mg L-1. Fengycin production was not measured due to lack of its standard reagent. In conclusion, we provided biochemical evidence that strain Y-IVI was able to producing antifungal compounds and hence had great potential to be used in biological control of plant diseases.4. A bio-organic fertilizer (BIO) secondarily fermented with antagonistic strain Bacillus subtilis Y-IVI was used to control this disease. Pot experiments were carried out to investigate the efficacy and elucidate the biocontrol mechanisms of the disease. Application of BIO reduced the incidence of muskmelon wilt disease by 91% and significantly increased plant dry weight by 3.1 times compared with the control amended with nothing. The BIO treatment significantly decreased FOM densities in plant shoots, rhizosphere soil and bulk soil. The colony-forming-unit (CFU) of FOM in rhizosphere soil of the BIO treatment was 1000-fold lower than that in the control. The previously lab-screened bacterial strain, Y-IVI, could effectively colonize rhizosphere soil and plant shoots. The logarithmic CFU of strain Y-IVI maintained between 7.6 and 6.7 in rhizosphere soil sampled from 10 to 60 days after transplanting into the BIO treatment. The average concentration of antifungal lipopeptide Iturin A in the BIO treatment was 78.1μg·g-1 of fresh rhizosphere samples. Ten days after transplanting, the content of salicylic acid in BIO treated plant leaves was 17.5μg·g-1 fresh weight, which was significantly higher than that in the control which showed that the BIO can induce plant systemic resistance. In conclusion, BIO can effectively control muskmelon Fusarium wilt, possibly because the antagonistic microbes in BIO effectively colonized the rhizosphere and plant shoots to preclude pathogen invasion. Furthermore, the antagonistic microbes in BIO produce antifungal lipopeptides in the rhizosphere and induce plant systemic resistance at an early stage of attack by pathogens. We first checked and quantified rhizosphere production of iturin and surfactin by biocontrol agents under interactions of plant-pathogen-biocontrol agents. |
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