Identification And Transformation Of Endophytic Bacteria Isolated From Maize For Biological Control

Maize is one of the most important crops in the world. Banded leaf and sheath blight (BLSB) is a traditional soil-borne disease caused by Rhizoctonia solani Kuhn, which is becoming the most important and serious disease of maize and leads to a considerable deduction of the yield and poor quality. Traditional agricultural control is inefficient, although chemical control have an obvious and fast effect on BLSB, it is easy to cause a series of environment problems, resulting in serious environmental pollution, disruption of ecological balance and drug tolerance of pathogens. According to some reports, there is no maize variety that is highly resistant to BLSB yet. Genetic engineering technique is an efficient and general method with an obvious effect in breeding technique for disease control by transferring some resistance genes into maize, but it will lead to the emergence of new race of pathogen and resistance loss of genetically modified maize varieties. Using biocontrol bacteria is an ideal method to control the disease due to its fast proliferation and it is environment-friendly, avoiding the emergence of resistance loss of maize varieties as well.In order to improve the resistance to BLSB, we isolated and identified some endophytic bacterial strains from maize tissues, which are non-pathogenic and confer resistance to BLSB. We also constructed an expression vector plnaQ-PKana-avrRxol-Kana and transformed strain LV-12with it successfully. These assays laid a solid foundation for study of BLSB control in the future. Now we have obtained the research results as follows:1. We obtained70endophytic bacterial strains (24strains from roots,22strains from leaves,13strains from sheathes and11strains from stems) from healthy maize tissues by surface sterilizing and dilution culturing method.2. We obtained4bacterial strains effective in suppressing R. solani Kuhn AG-1-IA through screening assays on PDA plates and on leaves of maize. 3. Based on the characterization of morphological, some physiological/biochemical features and the sequences of their16S rDNA, strain LV-A, strain LV-B and strain LV-8were identified as Bacillus amyloliquefaciens and strain LV-12was identified as Pseudomonas fluorescens.4. We constructed a Pseudomonas-Escherichia shuttle vector pInaQ-Kana by inserting multiple cloning site and Kanamycin-resistant expression cassette. We successfully transformed strain LV-12with the vector pInaQ-Kana.5. We amplified a promoter from Kanamycin-resistant expression cassette and an avirulence gene avrRxol from Xanthomonas oryzae pv. oryzicola and made the gene avrRxol located downstream of the promoter. We constructed an expression vector pInaQ-PKana-avrRxol-Kana and transformed strain LV-12with it successfully.6. Bacterial suspensions of strain LV-12containing vector pInaQ-PKana-avrRxol-Kana were infiltrated into the intercellular spaces of leaves of maize line B73, which contains a resistance gene Rxol corresponding to the avirulence gene avrRxol, with a needleless syringe to test the ability to elicit HR. No obvious HR occurred3d after infiltration compared to bacterial suspensions of X. oryzae pv. oryzicola, which is a positive control.