Summary Bacillus subtilis and other Bacilli have long been used as biological control agents against plant bacterial diseases but the mechanisms by which the bacteria confer protection are not well understood. Our goal in this study was to isolate strains of B. subtilis that exhibit high levels of biocontrol efficacy from natural environments and to investigate the mechanisms by which these strains confer plant protection. We screened a total of sixty isolates collected from various locations across China and obtained six strains that exhibited above 50% biocontrol efficacy on tomato plants against the plant pathogen Ralstonia solanacearum under greenhouse conditions. These wild strains were able to form robust biofilms both in defined medium and on tomato plant roots and exhibited strong antagonistic activities against various plant pathogens in plate assays. We show that plant protection by those strains depended on widely conserved genes required for biofilm formation, including regulatory genes and genes for matrix production. We provide evidence suggesting that matrix production is critical for bacterial colonization on plant root surfaces. Finally, we have established a model system for studies of B. subtilis-tomato plant interactions in protection against a plant pathogen.

Our previous work showed that a consortium of three plant growth-promoting rhizobacterium (PGPR) strains (Bacillus cereus AR156, Bacillus subtilis SM21, and Serratia sp. XY21), termed as BBS for short, was a promising biocontrol agent. The present study investigated its effect on drought tolerance in cucumber plants. After withholding watering for 13 days, BBS-treated cucumber plants had much darker green leaves and substantially lighter wilt symptoms than control plants. Compared to the control, the BBS treatment decreased the leaf monodehydroascorbate (MDA) content and relative electrical conductivity by 40% and 15%, respectively; increased the leaf proline content and the root recovery intension by 3.45-fold and 50%, respectively; and also maintained the leaf chlorophyll content in cucumber plants under drought stress. Besides, in relation to the control, the BBS treatment significantly enhanced the superoxide dismutase (SOD) activity and mitigated the drought-triggered down-regulation of the expression of the genes cAPX, rbcL, and rbcS encoding cytosolic ascorbate peroxidase, and ribulose-1,5-bisphosphate carboxy/oxygenase (Rubisco) large and small subunits, respectively, in cucumber leaves. However, 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity was undetected in none of the culture solutions of three BBS constituent strains. These results indicated that BBS conferred induced systemic tolerance to drought stress in cucumber plants, by protecting plant cells, maintaining photosynthetic efficiency and root vigor and increasing some of antioxidase activities, without involving the action of ACC deaminase to lower plant ethylene levels.

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Wheat sharp eyespot, primarily caused by a soil-borne fungus Rhizoctonia cerealis, has become one of the most serious diseases of wheat in China. In this study, an ethylene response factor (ERF) gene from a wheat relative Thinopyrum intermedium, TiERF1, was characterized further, transgenic wheat lines expressing TiERF1 were developed, and the resistance of the transgenic wheat lines against R. cerealis was investigated. Southern blotting analysis indicated that at least two copies of the TiERF1 gene exist in the T. intermedium genome. Yeast one-hybrid assay indicated that the activation domain of TiERF1 is essential for activating the transcript of the reporter gene with the GCC-box cis-element. The TiERF1 gene was introduced into a Chinese wheat cultivar, Yangmai12, by biolistic bombardment. Results of PCR and Southern blotting analyses indicated that TiERF1 was successfully integrated into the genome of the transgenic wheat, where it can be passed down from the T0 to T4 generations. Quantitative reverse transcription-PCR analysis demonstrated that TiERF1 could be overexpressed in the stable transgenic plants, in which the expression levels of wheat pathogenesis-related (PR) genes primarily in the ethylene-dependent signal pathway, such as a chitinase gene and a β-1,3-glucanase gene, were increased dramatically. Disease tests indicated that the overexpression of TiERF1 conferred enhanced resistance to sharp eyespot in the transgenic wheat lines compared with the wild-type and silenced TiERF1 plants. These results suggested that the overexpression of TiERF1 enhances resistance to sharp eyespot in transgenic wheat lines by activating PR genes primarily in the ethylene-dependent pathway.

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Bipolaris sorokiniana is an economically important phytopathogen of wheat and other cereal species. In this paper, a novel pathogen-induced ethylene-responsive factor (ERF) gene of wheat, TaPIEP1, was isolated and characterized. The transcript of TaPIEP1 was significantly and rapidly induced by treatments with B. sorokiniana, and with ethylene (ET), jasmonate (JA), and abscisic acid. Molecular and biochemical assays demonstrated that TaPIEP1 is a new ERF transcription activator belonging to B-3c subgroup of the ERF family. Transgenic wheat lines overexpressing TaPIEP1 were generated by biolistic bombardment and molecular screening. Compared with the host wheat Yangmai12, six stable transgenic wheat lines overexpressing TaPIEP1 that exhibited significantly increased resistance to B. sorokiniana were identified by molecular detection in the T(0)-T(4) generations and by disease resistance tests. The degree of the enhanced resistance was correlated with an accumulation of the transcript level of TaPIEP1. Furthermore, the transcript levels of certain defense-related genes in the ET/JA pathways were markedly increased in the transgenic wheat plants with enhanced resistance. These results reveal that TaPIEP1 overexpression in wheat could obviously improve resistance to B. sorokiniana via activation of some defense genes, and TaPIEP1 gene may be useful in improving crop resistance to the pathogen.

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A simple and efficient copper-catalyzed method for synthesis of 3,4-disubstituted isoquinolin-1(2H)-one derivatives via cascade reactions of substituted 2-halobenzamides with beta-keto esters under mild conditions has been developed, and the method has economical and practical advantages.