| As natural components of the ecosystem,plant endophytic bacteria and host plants have extensive interaction.Recent studies have shown that endophytic bacteria have stable ecological niche in the host plants and establish a synergistic symbiotic relationship with host plants.In addition to helping the host grow and develop,endophytic bacteria are crucial for nitrogen fixation and enhancing the host’s resilience to stress.As the carriers of exogenous genes,endophytic bacteria are able to have an effect on their host plant.They may also make for effective biological fertilizer targets.Therefore,the study of endophytic bacteria of mulberry is of great significance to further understand the growth and development of mulberry.In this study,the hybrid mulberry cultivar Guisangyou 12 and wild mulberry resource(Chuansang)were used as materials.Five samples,namely leaves of tissue culture Guisangyou 12,leaves of outdoor Guisangyou 12,seeds of Guisangyou 12,leaves of tissue culture(Chuansang)and leaves of outdoor(Chuansang),were isolated and identified.Two dominant bacteria genera,Acinetobacter sp.H1 and Bacillus sp.H2,were screened based on vitality and colonization.Acinetobacter sp.was transformed by expression vector transformation,and the colonization ability of Acinetobacter sp.in mulberry and its promotion effect on mulberry were characterized by vector visual labels.The following summarize the primary results of this research:1.Isolation and identification results of culturable endophytic bacteria in mulberry In the present study,a total of 5 samples of 2 kinds of mulberry trees were separated and purified by tissue separation method and plate purification method.It was found that there were differences in the number and species of endophytic bacteria in different growing environments and different mulberry resources.(1)20 strains of culturable endophytic bacteria were isolated from the leaves of Guisangyou 12 which in natural environment.They belong to Acinetobacter sp.,Bacillus sp.,Niallia sp.and Priestia sp.under Proteobacteria and Firmicutes in the bacterial domain,respectively;(2)11 strains of culturable endophytic bacteria were isolated from the leaves of sterile culture Guisangyou12.They belonged to Proteobacteria and Firmicutes in the bacterial domain of Bacillus sp.and Pseudomonas sp.;(3)163 strains of culturable endophytic bacteria were isolated from Guisangyou 12 seeds.All of them belong to Proteobacteria in the bacterial domain of Enterobacter sp.,Atlantibacter sp.,Escherichia sp.,Klebsiella sp.,Kosakonia sp.,Lelliottia sp.,Pseudescherichia sp.,Salmonella sp.,Pantoea sp.;(4)128 strains of culturable endophytic bacteria were isolated from outdoor mulberry leaves.They belonged to Proteobacteria and Firmicutes in the bacterial domain of Bacillus sp.,Pseudomonas sp.,Priestia sp.,Neobacillus sp.,Rossellomorea sp.,Fictibacillus sp.,Lysinibacillus sp.,Peribacillus sp.,Brevibacillus sp.,Paenibacillus sp.;(5)235 strains of culturable endophytic bacteria were isolated from mulberry leaves of tissue culture,both belonging to Proteobacteria,the genus Acinetobacter sp.and Rhizobium sp.These results showed that 557 strains of endophytic bacteria belonged to Proteobacteria or Firmicutes,which classified to 22 genera,among which Acinetobacter spp.was the most abundant(196 strains,accounting for 35.2%),and Bacillus spp.was the second most abundant(87 strains,accounting for 15.6%),therefore,they were identified as the dominant bacteria in mulberry.2.Bacterial colony architectural,physiological and biochemical properties of mulberry dominant bacteriaBy morphologically observing the dominating bacteria,the morphological characteristics of endophytic Acinetobacter and Bacillus colonies in mulberry were identified,and the physiological and biochemical indices of the dominant bacteria were identified.Acinetobacter single colony is milky white round,0.5-0.7mm in size,shiny,opaque,raised and neatly edged;Bacillus single colony is opalescent round,2.2-2.4mm in size,glossy,opaque,raised,and neatly edged.In this study,Gram staining was negative for Acinetobacter and positive for Bacillus.Both of them were aerobic bacteria,and the contact enzyme reactions were positive.Methyl red test was positive for Acinetobacter,but negative for Bacillus.Acinetobacter was negative and Bacillus was positive in starch hydrolysis test.3.Engineering modification of dominant endophytic bacteria of mulberry and its promotion effectAcinetobacter sp.was modified by the method of expression vector transformation.It was determined that the endophytic bacteria showed GUS activity after the i GUS vector was colonized in mulberry,which indicated that the endophytic bacteria had the potential ability to communicate genetic material with mulberry.It was found that fluorescently labeled Acinetobacter-EGFP was mainly concentrated around the root tip and stomata in mulberry,indicating that the endophytic bacteria colonized stably in the roots,leaf tissues,and other organs of mulberry,and could still be detected in the long-term culture of mulberry.These results indicated that the endophytic bacteria carrying the marker gene could colonize the mulberry stably.After 30 days of inoculation,it was detected that the endophytic bacteria carrying egfp reporter genes had an effect on the growth and development of mulberry.The results showed that the endophytic bacteria significantly increased the plant height,root length,fresh weight and dry weight of mulberry,but had little effect on the chlorophyll content of leaves.This study demonstrated the ubiquity and biodiversity of endophytic bacteria in mulberry trees.After infecting mulberry trees,endophytic bacteria will find suitable ecological niches to colonized and exist for a long time.Endophytic Acinetobacter carrying reporter genes can promote the growth and development of mulberry seedlings,proving that endophytic Acinetobacter is an important potential microbial resource for mulberry trees.The data obtained in this study is of great significance for mulberry resources development and germplasm resources cultivation. |
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