Research On Population Diversity And Antagonistic Strains Screening Of Endophytic Bacteria In Chinese Jujube

Traditionally microbiological isolating culture，Physiological and biochemical methodand molecular biology technologies have been used to study diversity of genetic, diversityof ecological distribution of the endophytic bacteria and the dynamic distribution indifferent varieties of jujube. The diversity and distribution state of endophytic bacteria wassystemic studied at first time, and endophytic bacteria were compared between health anddisease plants. The objective of this research is to isolate and screen endophytic bacteriafrom jujube to control “Suoguo” disease, and discover the mechanism of the endophyticbacteria against pathogens. The below are main results of the study:1. The conditions for best surface sterilization were: tissues washed with75%ethanolfor3min；leaves immersed in fresh sodium hypochlorite solution (3%available Cl-) for3min, branches immersed in fresh sodium hypochlorite solution (2%available Cl-) for4min, roots immersed in fresh sodium hypochlorite solution (2.5%available Cl-) for4min,fruits immersed in fresh sodium hypochlorite solution (2.5%available Cl-) for3min; thenrinsed with75%ethanol for30s; and finally washed with sterile distilled water.The most kinds of endophytic bacteria were found on TSA、KMB and NA mediathrough the screening of10kinds of culture medium.2. The types and quantities of endophytic bacteria in different tissues of some jujubevarieties planted in different regions of Hebei Province have been analyzed, the result sho-wed that Bacillus sp. and Pseudomonas sp. were distributed in different jujube varieties and different organs in the plants, and their quantities occupied the dominant position. The ty-pes and quantities of endophytic bacteria were different in different varieties, tissues,growth period and cultivated regions of jujube. Basically, the quantity in roots was thelargest, and then was the leaves and fruits, the minimum number of endophytic bacteriawas in branch. Besides, the differences of the endophytic bacteria quantities were also existin different varieties of jujube, the most was in the roots, and then was the leaves andbranch, the endophytic bacteria in fruits was minimum. The isolated bacteria belong to13genera,16species, including Bacillus sp., Pseudomonas sp., Arthrobacter sp., etc. Tengenera were separated from Chinese jujube at first time, such as Nesterenkonia,Stenotrophomonas, Pedobacter，Gamma proteobacterium etc.3. The identified results indicated that “Pozao” jujube plants have plenty of endophytic bacterium in density and population, and the density is higher in root andbranch than in leave. At fruiting stage, the isolated bacteria belong to11genera, includingBacillus sp., Pseudomonas sp., Arthrobacter sp., Staphylococcus sp., etc. The Bacillus sp.is the dominant strain in all jujube tissues. There was distinguishing between the numberand variety of endophytic bacteria in different growth period. The number and variety ofendophytic bacteria was increasing from dormant to full bloom, and was decreasing fromfull bloom to fruit maturation. The isolated probability of endophytic bacteria is not thesame in different tissues, it showed that roots＞branches＞leave＞fruits.4. The variety of endophytic bacteria from the healthy branch、leaves were higher thanthe ones from the diseased plant, but the number was fewer than the diseased tissues. Thedisease incidence and index of fruit were negatively correlated to percentage of Bacillus sp.in jujube. The number and variety of endophytic bacteria from the diseased fruit werefewer than the ones from the healthy fruit. The numbers of endophytic bacteria in thedifferent parts of jujube plants were significant different. Little differences were foundamong upper parts, middle parts and lower parts．Based on the analysis diversity indexes ofShannon, Simpson and Pielou, it is concluded that the maximum of SHANNON index wasin the branches, and the minimum was in the fruits, however, the maximum ofSIMPSON(D) index was in the branches, and the minimum was in the fruits and themaximum of Pielou index was in the leaves while the minimum was in the branches．5. Of the19isolates,7strains are known to express antibiosis activities against plantpathogen,4strains were determined in vitro antagonism to all of the tested plant pathogens.According to morphological observation, physiological characteristics tests, and16SrDNAsequence analysis,4strains were identified as strain of Bacillus sp., Arthrobacter,Pseudomonas, Nesterenkonia.6. Antagonistic characteristics displayed that strain PZ-4was able to obviously inhibitgrowth of eight pathogens, such as Alternaria alternata Allantophomoides carotae,Fusarium graminearum, Bipolaris maydis, Alternaria alternate f.sp.mali, Cytospora sp.,Botrytis cinerea, Botryosphaeria berengeriana f. sp. piricola, the inhibition rate of itsfiltration ranged from41.2%to69.3%on8kinds of pathogens, and the zone of inhibitionwas obviously, it was proved that PZ-4strain has a wider Antibacterial spectrum.According to morphological observation, physiological characteristics tests, and16S rDNAsequence analysis, PZ-4strains was identified as strain of Bacillus subtilis.