Effect And Mechanism Of Microecological Regulation Against Southern Blight Of Dendrobium Officinale

Dendrobium officinale Kimura et Migo is a rare traditional Chinese medicinal herb and has been favored for its health benefits.At present,due to the lack of wild resources caused by excessive excavation,most of the sources of D.officinale are cultivated artificially.During artificial cultivation,various diseases occur in D.officinale.Southern blight is one of the common diseases,which directly threatens the safety and economic benefits of D.officinale.Based on isolation and identification of southern blight pathogen,functional strains were screened and explored for prevention of the disease,which might provide technical support for biological control and green cultivation of D.officinale.First,the southern blight pathogen of D.officinale was isolated and identified.Combined with the traditional isolation and culture as well as Koch’s rule,the southern blight pathogen DQ was determined.Through morphological observation,it was found that pathogen DQ formed white sericate hyphae,and sclerotia formed 10 days later.ITS sequence analysis showed that the highest similarity between pathogen DQ and Athelia rolfsii was 100%;phylogenetic analysis showed that DQ was clustered with A.rolfsii.Therefore,DQ was identified as A.rolfsii.Secondly,functional strains effectively preventing southern blight of D.officinale were screened.A series of endophytes isolated from D.officinale(endogenous bacteria)and endophytes from other medicinal plants(exogenous bacteria)stored in the laboratory were used as testing strains.The results of plate confrontation antagonism test showed that endophytic bacteria Sphingomonas ZJSH1,had no inhibitory activity against pathogen DQ.The inhibition activity of the endogenous strains Bacillus FHGXJ8,FHJXJ5,XSG14 were between 60.00-74.50%and the inhibition activity of the exogenous strains Bacillus B1-6,WZZ-6,as well as Pseudomonas JX3-1,Y11T3-1 were between 46.00-65.00%.The above single strains and mixed strains were used to analyze the disease resistance of D.officinale in vitro and on tissue culture seedlings.The results indicated that:① When a single strain was used,the endogenous strains had a better control effect(0-100.00%)than exogenous strains(0-75.00%)in vitro,but the control effect of endogenous strains(0-100.00%)on the tissue culture seedlings was worse than exogenous strains(50.00-100.00%);② the control effect of mixed strains was significantly better than that of single strains in vitro or on tissue culture seedlings of D.officinale.Comparatively,the fermentation supernatant had poorer control effect in vitro(0-66.67%)and on tissue culture seedlings(0%)than that of the bacterial cells,which showed 16.67-100.00%and 66.67-83.33%inhibitoty.The fermentation supernatant plus bacterial cells had the best inhibition activity,with 41.67-100.00%in vitro and 83.33-100%on tissue culture plants,respectively.It can be seen that both endogenous and exogenous bacteria have control effect against southern blight of D.officinale.Finally,the possible mechanism of functional strains was preliminary studied.Nine antibiotic-encoding genes were detected by specific PCR amplification among eight strains.It was found that three encoding genes(Iturin A、Surfactin and Phenazine)were detected from 5 strains.Among them,strains FHGXJ8,B1-6,and WZZ-6 contain Iturin A encoding gene;Surfactin encoding gene was amplified in strain FHGXJ8 as well as WZZ-6;and Phenazine encoding gene was successfully amplified in strains JX3-1,Y11T3-1.These antibiotic-encoding genes may play a role in prevention disease,but the products need to be further analyzed.In addition,analysis of siderophore,IAA,and ACC deaminase activity of functional strains indicated that strains JX3-1 and Y11T3-1 could secrete siderophore;all strains produced IAA,with the highest IAA yield for strain ZJSH1(26.20 μg/mL).ACC deaminase activity was present in strains JX3-1,Y11T3-1 and FHJXJ5.In terms of the control effect of single strain and mixed strains,antibiotic,Fe element competition and hormone regulation might be involved in antagonistic effect,which may be an important reason for the enhanced antagonistic activity of the mixed bacteria.In conclusion,southern blight pathogen DQ was used to test the control effect of endogenous and exogenous bacteria against southern blight of D.officinale.It was found that the antagonistic effect of endogenous strains was better than that of exogenous strains,and the mixed strains was more effective than the single one.The control effect of fermentation supernatant plus bacteria cell was the best,and the main control mechanisms of functional strains were antibiotics,competition of Fe elements and hormone regulation.These results provide a good basis for further study of disease prevention and application of D.officinale.