Xinqi Huang

Biocontrol of Rhizoctonia solani damping-off disease in cucumber with Bacillus pumilus SQR-N43.

Biological control is an efficient and environmentally friendly way to prevent damping-off disease. Micrographs were used to investigate the ability of Bacillus pumilus (B. pumilus) SQR-N43 to control Rhizoctonia solani (R. solani) Q1 in cucumbers. The root colonization ability of B. pumilus SQR-N43 was analyzed in vivo with a green fluorescent protein (GFP) tag. A pot experiment was performed to assess the in vivo disease-control efficiency of B. pumilus SQR-N43 and its bio-organic fertilizer. Results indicate that B. pumilus SQR-N43 induced hyphal deformation, enlargement of cytoplasmic vacuoles and cytoplasmic leakage in R. solani Q1 mycelia. A biofilm on the root surface was formed when the roots were inoculated with 10(7)-10(8)cells g(-1) of soil of GFP-tagged B. pumilus SQR-N43. In the pot experiment, the biocontrol reduced the concentration of R. solani. In contrast to applications of only B. pumilus SQR-N43 (N treatment), which produced control efficiencies of 23%, control efficiencies of 68% were obtained with applications of a fermented organic fertilizer inoculated with B. pumilus SQR-N43 (BIO treatment). After twenty days of incubation, significant differences in the number of CFUs and the percentage of spores of B. pumilus SQR-N43 were recorded between the N treatment (2.20×10(7)CFU g(-1) of soil and 79%, respectively) and the BIO treatment (1.67×10(8)CFU g(-1) of soil and 52%, respectively). The results indicate that B. pumilus SQR-N43 is a potent antagonist against R. solani Q1. The BIO treatment was more effective than the N treatment because it stabilized the population and increased the active form of the antagonist.

Application of Trichoderma harzianum SQR-T037 bio-organic fertiliser significantly controls Fusarium wilt and affects the microbial communities of continuously cropped soil of cucumber

BACKGROUND The reduction in diversity of the soil microbial community causes the disorder of continuous cropping. The aim of this study was to determine the effects of applying Trichoderma harzianum SQR-T037 bio-organic fertiliser (BIO) on the microbial community in continuously cropped cucumber soil. Four treatments were set: (1) control, where neither seedling nursery soil (N) nor transplanted soil (T) was amended with BIO; (2) N treatment, where nursery soil was amended with BIO (1% w/w) but transplanted soil was not; (3) N + T treatment, where BIO was added to both nursery soil (1% w/w) and transplanted soil (0.5% w/w); (4) uncropped soil, where soil was left uncropped consistently. RESULTS A disease index of 72.2% was found for the control treatment, while the N and N + T treatments had disease indices of only 25 and 15% respectively. Analysis of the denaturing gradient gel electrophoresis (DGGE) profiles showed that the bacterial communities of the N and N + T treatments were similar to those of the uncropped soil but distinct from those of the control soil. The fungal communities of the N and N + T treatments differed from those of both the uncropped soil and the control. CONCLUSION Addition of BIO to both the nursery soil and the transplanted soil can diversify the microbial community in continuously cropped cucumber soil and thus effectively control Fusarium wilt of cucumber plants.

Illumina MiSeq investigations on the changes of microbial community in the Fusarium oxysporum f.sp. cubense infected soil during and after reductive soil disinfestation

Although reductive soil disinfestation (RSD) is increasingly used for the control of soil-borne diseases, its impact on the soil microbial community during and after RSD remains poorly understood. MiSeq pyrosequencing, real-time PCR and denaturing gradient gel electrophoresis were performed to investigate the changes of microbial community in the Fusarium oxysporum f. sp. cubense (FOC) infected soil during RSD and at the simulative banana cultivation after RSD. The results showed that RSD significantly increased soil microbial populations and a different microbial community with the pathogenic soil was established after RSD. Specifically, the number of Firmicutes mainly containing Ruminococcus and Coprococcus followed by a small part of Clostridium which were the dominant bacterial genera significantly increased during RSD. In contrast, Symbiobacterium and Flavisolibacter were the dominant genera in the flooding soil. When the soils were recovered under aerobic condition, the relative abundances of the bacteria belonging to the phylum Bacteroidetes, Acidobacteria, Planctomycetes increased as alternatives to the reducing Firmicutes. For fungi, the population of F. oxysporum significantly decreased during RSD accompanied with the pH decline, which resulted in the significant decrease of relative abundance in the phylum Ascomycota. Alternatively, the relative abundances of some other fungal species increased, such as Chaetomium spp. and Penicillium spp. belonging to Ascomycota and the family Clavulinaceae belonging to Basidiomycota. Then, the relative abundance of Ascomycota re-increased after RSD with Podospora and Zopfiella as dominant genera, whereas the relative abundance of Fusarium further decreased. Overall, the microbial populations and community re-established by RSD made the soil more disease-suppressive and beneficial to the soil nutrient cycling and plant growth compared with the previous pathogenic soil.

Toxic organic acids produced in biological soil disinfestation mainly caused the suppression of Fusarium oxysporum f. sp. cubense

Biological soil disinfestation (BSD) is an effective and environmentally friendly way to suppress soil-borne pathogens. Although it is increasingly used in USA, the Netherlands and Japan, its precise mechanism has not been well quantified so far. Quantitative real-time PCR, denaturing gradient gel electrophoresis and high performance liquid chromatography were used for investigating the role of organic acids in the mechanisms of BSD. The results showed that BSD significantly reduced the population of Fusarium oxysporum in soil. Simultaneously, in BSD treatments, the soil pH significantly decreased and some organic acid producers, such as Clostridia sp., were observed. Four kinds of toxic organic acids to F. oxysporum were detected in soil solutions of BSD treatments. Acetic acid and butyric acid were the primary organic acids, followed by small amounts of isovaleric acid and propionic acid. The verification test directly demonstrated that the toxic organic acids with the maximal doses detected in BSD significantly suppressed F. oxysporum, Rhizoctonia solani and Ralstonia solanacearum.

Changes in the soil microbial community after reductive soil disinfestation and cucumber seedling cultivation.

Reductive soil disinfestation (RSD) has been proven to be an effective and environmentally friendly way to control many soilborne pathogens and diseases. In this study, the RSDs using ethanol (Et-RSD) and alfalfa (Al-RSD) as organic carbons were performed in a Rhizoctonia solani-infected soil, and the dissimilarities of microbial communities during the RSDs and after planting two seasons of cucumber seedlings in the RSDs-treated soil were respectively investigated by MiSeq pyrosequencing. The results showed that, as for bacteria, Coprococcus, Flavisolibacter, Rhodanobacter, Symbiobacterium, and UC-Ruminococcaceae became the dominant bacterial genera at the end of Al-RSD. In contrast, Et-RSD soil involved more bacteria belonging to Firmicutes, such as Sedimentibacter, UC-Gracilibacteraceae, and Desulfosporosinus. For fungi, Chaetomium significantly increased at the end of RSDs, while Rhizoctonia and Aspergillus significantly decreased. After planting two seasons of cucumber seedlings, those bacteria belonging to Firmicutes significantly decreased, but Lysobacter and Rhodanobacter belonging to the phylum Proteobacteria as well as UC-Sordariales and Humicola belonging to Ascomycota alternatively increased in Al- and Et-RSD-treated soils. Besides, some nitrification, denitrification, and nitrogen fixation genes were apparently increased in the RSD-treated soils, but the effect was more profound in Al-RSD than Et-RSD. Overall, Et-RSD could induced more antagonists belonging to Firmicutes under anaerobic condition, whereas Al-RSD could continuously stimulate some functional microorganisms (Lysobacter and Rhodanobacter) and further improve nitrogen transformation activities in the soil at the coming cropping season.

Effects of water regime, crop residues, and application rates on control of Fusarium oxysporum f. sp. cubense

Biological soil disinfestation is an effective method to control soil-borne disease by flooding and incorporating with organic amendments, but field conditions and resources sometimes limited its practical application. A laboratory experiment was conducted to develop practice guidelines on controlling Fusarium wilt, a widespread banana disease caused by Fusarium oxysporum f. sp. cubense (FOC). FOC infested soil incorporated with rice or maize straw at rates of 1.5 tons/ha and 3.0 tons/ha was incubated under flooded or water-saturated (100% water holding capacity) conditions at 30°C for 30 days. Results showed that FOC populations in the soils incorporated with either rice or maize straw rapidly reduced more than 90% in the first 15 days and then fluctuated till the end of incubation, while flooding alone without organic amendment reduced FOC populations slightly. The rapid and dramatic decrease of redox potential (down to -350 mV) in straw-amended treatments implied that both anaerobic condition and strongly reductive soil condition would contribute to pathogen inactivation. Water-saturation combined with straw amendments had the comparable effects on reduction of FOC, indicating that flooding was not indispensable for inactivating FOC. There was no significant difference in the reduction of FOC observed in the straw amendments at between 1.5 and 3 tons/ha. Therefore, incorporating soil with straw (rice or maize straw) at a rate of 3.0 tons/ha under 100% water holding capacity or 1.5 tons/ha under flooding, would effectively alleviate banana Fusarium wilt caused by FOC after 15-day treating under 30°C.

Comparison of the spores of Paenibacillus polymyxa prepared at different temperatures

Paenibacillus polymyxa SQR-21, which is antagonistic against Fusarium oxysporum, is used as a biocontrol agent and, when mixed with organic substances for solid fermentation, produces a bioorganic fertilizer. The spores of P. polymyxa prepared at different temperatures were characterized with respect to the dipicolinic acid content, heat resistance, fatty acid composition and germination. Spores prepared at 37°C showed higher heat resistance than those prepared at 25 and 30°C. However, the germination rate was negatively correlated with the sporulation temperature. The maximum germination rate of the spores prepared at 25°C was 1.3-times higher than the spores prepared at 30°C. The sporulation temperature thus affects the resistance and germination properties of P. polymyxa spores. These results are useful for the production of improved bio-organic fertilizer.

Development of a specific real-time PCR assay targeting the poly-γ-glutamic acid synthesis gene, pgsB, for the quantification of Bacillus amyloliquefaciens in solid-state fermentation

A TaqMan real-time PCR procedure was developed for specific detection and quantification of strains belonging to Bacillus amyloliquefaciens group. The primer pair pgsB726-f/pgsB791-r and the pgsB-probe were designed from one of the poly-γ-glutamic acid synthesis gene (pgsB) of B. amyloliquefaciens. The detection limit was approximately between 10(2)-10(3) cells/mL. A linear correlation between the log10 input pMD-pgsB plasmid DNA copies and the threshold cycle values were observed with a magnitude of linearity in the range of 9.415×10(3)-10(7) copies/mL for the standard curve, which exhibited a slope of -3.35 and an R2 value of 99.8%. Results of validation of this method with artificially contaminated and natural solid-state fermentation samples showed that it was suitable for specific and sensitive detection and quantification for the target strains in solid-state fermentation samples. This could be more useful to understand the fermentation starting strain and the final microbiological properties of fermentation products.

The supernatant of Bacillus pumilus SQR-N43 has antifungal activity towards Rhizoctonia solani

For clarification of the antagonistic mechanism of Bacillus pumilus SQR‐N43 (N43) against Rhizoctonia solani Q1, production of antibiotics by N43 was determined, and the effect of the antibiotics on the pathogen mycelium was microscopically observed. Further more, the control efficiencies of the antifungal compounds on damping‐off disease were investigated. The results obtained are listed as follows: N43 produced antibiotic substances towards R. solani Q1 at logarithmic growth phase. The antibiotics caused hyphal deformation and enlargement of cytoplasmic vacuoles in R. solani Q1 mycelia. 70% saturation of ammonium sulfate made a complete precipitation of the antibiotics in culture broth. When treated with protease K and trypsase, the activities of antibiotics were decreased by 79% and 53%, respectively, compared with control. The antibiotics were sensitive to high temperature and were alkaline stable. The molecular weights of the substances were about 500–1000 Da. The bio‐control efficiencies of the antibiotics had no significant difference with that of N43 cell suspension. It is a first report that B. pumilus strain produced oligopeptides which had inhibitory effect on R. solani Q1 at logarithmic growth phase.