Purpose To screen endophytic Bacillus producing volatile organic compounds (VOCs) with antifungal activity, and to explore their biocontrol properties toward the growth and pathogenicity of Curvularia lunata . Methods Two-sealed-base-plate assays were used to estimate the antifungal activities of Bacillus strains against C. lunata . Conjoint analysis of solid-phase microextraction gas chromatography-mass spectrometry and antagonistic experiments were used to identify the VOCs responsible for the antifungal activity. Effects of individual synthetic VOCs were analyzed along with reactive oxygen species (ROS) accumulation in C. lunata conidia. After exposure to individual VOCs, conidia were also sprayed onto maize leaves to evaluate their pathogenicity. Expression levels of virulence-related genes in C. lunata mycelium following exposure to VOCs were analyzed using quantitative real-time PCR. Results Among the ten endophytic Bacillus strains and two plant growth-promoting rhizobacterial (PGPR) strains, only B. subtilis strain DZSY21 strongly inhibited the growth of C. lunata by producing VOCs. 2-Methylbutyric acid, 2-heptanone, and isopentyl acetate produced by strain DZSY21 showed inhibitory effects on the mycelia growth and conidial sporulation of C. lunata . 2-Heptanone and isopentyl acetate also repressed the germination of conidia and the expression levels of virulence-related genes in C. lunata mycelium. Moreover, isopentyl acetate strongly enhanced the accumulation of intracellular ROS in conidia. The disease indexes of maize leaves sprayed with VOC-treated C. lunata conidia were reduced from 60.52 to 26.64%. Conclusion Endophytic B. subtilis strain DZSY21 displayed the potential to control C. lunata by producing VOCs, especially 2-heptanone and isopentyl acetate.

DNA甲基化作为表观遗传的一种表现形式,在植物的生长发育、抗逆和抗病过程中发挥重要作用.前期研究中从杜仲体内分离筛选到一株拮抗内生芽孢杆菌DZSY21,其可在玉米中定殖,定殖后明显增强玉米抗病性.为明确内生菌DZSY21在玉米中定殖与其DNA甲基化水平的关系,该试验利用甲基敏感扩增多态性分析技术(MSAP)和全基因组DNA甲基化(WGBS)分别对内生菌DZSY21处理的玉米基因组进行DNA甲基化水平的定性和定量研究.定性和定量结果均表明拮抗菌DZSY21引入玉米植株,可引起玉米基因组DNA总甲基化水平降低;全基因组DNA甲基化(WGBS)定量测序结果显示玉米基因组DNA总甲基化水平降低了0.89％,DNA甲基化差异性区域数据库中共有5820个差异性甲基化区域(DMRs),且GO聚类分析和Pathway功能显著性富集分析结果显示DMR相关的基因主要涉及糖代谢、氨基酸合成以及蛋白激酶活性等与抗病相关的主要功能.该结果为深入研究DNA甲基化在内生菌DZSY21定殖增强玉米抗病性中的调控机制提供理论基础.

杜仲内生拮抗细菌DZSY21可在玉米中稳定定殖并增强玉米植株的抗病能力.试验从基因水平上对内生拮抗细菌DZSY21诱导玉米产生抗病性的分子机制进行预测,利用转录组测序技术对内生拮抗细菌DZSY21处理玉米后不同生长时间段叶片的总mRNA进行差异表达分析,以Fold change≥2且FDR<0.01为标准,挑选差异表达基因(DEGs).结果显示:以处理0 h作为对照,处理12 h时有2413个差异表达基因,其中上调基因1278个,下调基因1135个;处理24 h时有737个差异表达基因,其中上调基因538个,下调基因199个.在此基础上,根据DEGs功能注释分类,在差异表达基因中筛选与抗病相关的基因,最终获得267个抗病基因.处理12 h筛选得到218个基因,主要涉及脂质转移蛋白、MATE转运蛋白及LysM受体蛋白激酶等26个抗病途径;处理24 h获得71个基因,主要涉及异黄酮还原酶、纤维素合成酶、苯丙氨酸解氨酶、L-抗坏血酸过氧化物酶、GLK转录因子及ACC氧化酶等30个抗病途径,其中不同处理时间段内重复基因23个.上述结果表明,将杜仲内生拮抗细菌DZSY21引入玉米,可调节玉米叶片中抗病相关基因的表达,为进一步寻找抗病基因及其功能鉴定奠定了基础.

Bacillus subtilis DZSY21 isolated from the leaves of Eucommia ulmoides oliv. was labeled by antibiotic marker and found to effectively colonize the leaves of maize plant. Agar diffusion assays and biocontrol effect experiments showed that strain DZSY21 and its lipopeptides had antagonistic activity against Bipolaris maydis, as well as high biocontrol effects on southern corn leaf blight caused by B. maydis. Using MALDI-TOF-MS analysis, we detected the presence of antimicrobial surfactin A, surfactin B, and fengycin in the strain DZSY21. Signaling pathways mediated by DZSY21 were analyzed by testing the expression of key plant genes involved in regulation of salicylic acid (SA) or JA/ET pathways, the defense-related genes PR1 and LOX were concurrently expressed in the leaves of DZSY21-treated plants; this corresponded to slight increase in the expression level of PDF1.2 and decreases in ERF gene transcription levels. The results indicated an induced systemic response that is dependent on the SA and jasmonic acid (JA) pathways. Thus, we hypothesized that the strain DZSY21 inhibits B. maydis by producing antifungal lipopeptides and activating an induced systemic response through SA- and JA-dependent signaling pathways. This work describes a mechanism behind reduced disease severity in plants inoculated with the endophytic bacteria DZSY21.

Colletotrichum sp. DZJ07, a biological control strain isolated from the Eucommia ulmoides, shows po-tential for control of wheat sharp eyespot . To elucidate the ecological adaptability of DZJ07 of wheat, the green fluo-rescent protein gene( gfp) was inserted into the DZJ07 genome by Agrobacterium-mediated transformation method, and a GFP-tagged DZJ07-6 strain was constructed. Colony shape, growth rate, antagonistic effect and efficacy for biologi-cal control of wheat sharp eyespot were analyzed with DZJ07 and the GFP-tagged strain DZJ07-6. No significant differ-ences were obtained for any of the characteristics tested between the two strains. Then colonization ability of the GFP-tagged strain DZJ07-6 on wheat was assessed under greenhouse conditions. And observing by fluorescence microscope showed that the marked strain was able to colonize in the root and stem of wheat, and the population of DZJ07-6 in wheat rhizosphere, root and stem after 34 days were 2. 72 × 106 , 0. 62 × 104 and 2. 26 × 103 cfu/g respectively. The results afforded scientific basis for its colonization and the further research on bio-controlling mechanism.