Study On Diversity Of Culturable Endophytic Actinobacteria From Moss Of Different Habitats In Yunnan Province And Their Antibacterial Activity Against Ralstonia Solanacearum

Most of the studies on endophytic actinobacteria in the literature at home and abroad have focused on vascular plants,but in view of the moss endophytic actinomyces is not a lot.Therefore,through the separation of endophytic actinobacteria in mosses,understanding the diversity and population structure of endophytic actinobacteria will bring ne w opportunities for the discovery of new species of actinobacteria and secondary metabolites of new structures.Tomato bacterial wilt is called"plant cancer"because of its high incidence and difficulty in preventing.Once infected,tomato yield will be severely reduced or even lost.At present,the prevention and control methods of tomato bacterial wilt mainly focus on chemical control.However,long-term application of chemical pesticides will make the disease more difficult to control,so the urgent tas k of modern agriculture is to find effective biological control methods to control tomato bacterial wilt.In this study,the endophytic actinomycetes of six moss samples were isolated,sorted and merged according to the source,and their diversity was anal yzed.The 16S r RNA gene sequencing for strains with different morphologies,identification of polyphasic taxonomy for the potential new species.All isolated strains were tested for their activity against bacterial wilt in vitro,in vivo activity test were carried out on the strains with excellent activity.The specific contents are as follows:?1?Six media?AAG,CPA,DPA,GS,HV and SSA?were used to isolate 118 strains of actinobacteria from six mosses,including 27 strains from Hypnum plumaeforme,21 strains from Physcomitrella patens,19 strains from each of Chandonanthus hirtellus and Plagiomnium acutum?Lindb.?T.Kop.,17 strains from Meteoriella soluta and 15 strains from Sphagnum palustre.Further through morphology and classifies the culture charac teristics,into 62 different forms of actinobacteria,16S r RNA sequencing of these strains belonging to 12 genera,the main species is Streptomyces?66.1%?,other genera are Micromonospora?11.3%?,Promicromonospora?3.2%?,Rhodococcus?3.2%?,Nocardia?3.2%?,Actinomadura?3.2%?,Microbispora?1.6%?,Mycocibacterium?1.6%?,Kitasatospora?1.6%?,Isoptericola?1.6%?,Herbidospora?1.6%?and Microbacterium?1.6%?,respectively.?2?Two strains LD120^T and LD22^T were identified as new species of Streptomyces and Actinomadura through morphological characteristics,culture characteristics,physiological and biochemical characteristics,chemotaxonomic characteristics and molecular biological characteristics,and named as Actinomadura physcomitrii sp.nov.and Streptomyces physcomitrii sp.nov.,respectively.?3?Agar block method was used to test the activity of 62 strains against bacterial wilt,and obtained 11 strains with antibacterial activity,among which DD18 6 had the strongest inhibitory activity,and the inhibitory diameter was up to 33.00 mm.Selection activity stronger 4 strains?DC6,DD186,LD126 and XG33?and the new species LD120^T potted biological control activity test,the results show that strain DD186 control effect is best,when the spore concentration is 1×10⁷ CFU·g^-1 control effect is best could reach to 72.02%,LD120^T also showed a better control effect,the concentration of spores for the 1×10⁷ CFU·g^-1,control effect could reach to 63.64%.The rest control effect of the three strains is low.?4?Four fermentation media were selected to optimize the fermentation conditions of the above five strains,strain DD186 had the strongest antibacterial activity in PTM medium,and the antibacterial diameter up to 26.70 mm.The HPLC analysis of DC6,LD126 and XG33 and active material tracking showed that they had the same kind of active product,and then selected sterile fermentation liquor of DD186,LD120^T and LD126 filtrate activity in vivo tests.LD126 10³-fold dilution solution control effect is best,could reach 84.07%,followed by DD186 10³-fold dilution solution control effect of 67.48%,LD120^T 10³-fold dilution solution control effect of 49.17%.?5?For DD186,LD120^T and LD126 active products,isolated a known compound from DD186 is identified as bis?2-ethylhexyl?phthalate,three known compounds from LD120^T are identified as bis?2-ethylhexyl?phthalate,N-acetyltyramine and N-acetyltyramine analogue,a compound from LD126 is actinomycin D,which could against Ralstonia solanacearum.This study provides basic data for further research on endophytic diversity,resource utilization,and new species research by analyzing community structure of endophytic actinomycete isolated from moss;it also provides biocontrol bacteria and antibiotics for the control of tomato bacterial wilt,in order to lay the foundation for the biological control of tomato bacterial wilt,and provide a certain experimental reference for the discovery of many types of novel natural products.