| Banana is one of the most important fruits in the world.Banana Fusarium wilt(BFW)is a soil-borne disease caused by Fusarium oxysporum f.sp.cubense(Foc).The chlamydospores of Foc survive in soil for decades,infect suitable host from the rhizosphere,damage the bulbs and vascular bundles of banana plant,cause the whole banana plant to wither.Effective control of BFW has become a worldwide problem.Biological control technology is one of important measures to control BFW.Antimicrobial volatile compounds produced by microorganisms are the hotspot of current research.However,there is no report on the field control of BFW with Streptomyces and its volatile compounds.In this study,the control effect of Streptomyces TF78 and its volatile antifungal compound on BFW and the mechanism of its volatile antifungal compound on Foc were studied.The results are as follows:(1)Thirty-five Fusarium strains were isolated from 38 diseased banana corm samples collected from the main banana producing areas in Guangxi.Based on the result of PCR amplification with specific primers,34 strains of Foc were finally confirmed,of which 9 strains were Fusarium oxysporum f.sp.cubense race 1(Foc1)and 25 strains were Fusarium oxysporum f.sp.cubense race 4(Foc4),and all Foc4 were tropical race 4(FocTR4).The ISSR molecular fingerprint technology was used to analyze the genetic diversity of Foc from Guangxi and other provinces and the clustering results showed that Foc in Guangxi clustered together with other provinces’ and no new separate branches appeared.The result showed that there were two physiological races(Foc1 and Foc4)in Guangxi and all Foc4 strains belonged to FocTR4,FocTR4 was the dominant population of Foc in Guangxi.The pathogenicity of 8 strains of Foc1 and 22 strains of Foc4 were tested by root drench in pot experiment and result showed that pathogenicity of Foc1 and Foc4 has been differentiated and highly pathogenetic strains appeared.Therefore,the highly pathogenic FocTR4 strain(NO.F4)was selected as the target pathogen for subsequent research.(2)A total of 2466 strains of microbes were isolated from the rhizosphere soil of healthy banana plants in diseased banana plantations.After the plate confrontation test,217 strains of fungi,40 strains of bacteria and 76 strains of actinomycetes were found to have antagonistic activity against F4.Fifty-one strains with better antagonistic effect were selected for pot experiment and the result showed that actinomycete TF78 had 82.71% control effect on BFW,which was a highly efficient antagonistic strain.The strain TF78 was identified as S.misionensis by morphological characteristics,culture characteristics,physiological,biochemical test and 16 S r RNA gene sequence comparison analysis.(3)Two banana plantations with 30% incidence rate of BFW(located in Jinling town and Tanluo town of Nanning)were selected.TF78 inoculants were applied to banana tissue culture plants before and 5 months after planting.The control groups were commercial biological control agent Genhushi and the blank control group.Ten months after banana planting,the incidences rate of BFW were investigated by TF78 treatment,Genhushi treatment and blank control.The results showed that the incidence rate of BFW of TF78 inoculantion treatment,Genhushi treatment and blank control was 24.44%,20% and 55.56%respectively in the experimental area of Jinling Town and the control effects of TF78 and Genhushi to BFW were 55.30% and 62.92% respectively.In addition,the incidence rate of BFW of TF78 inoculantion treatment,Genhushi treatment and blank control was 35.56% 、 34.44% and 64.44% respectively in the experimental area of Tanluo Town and the control effects of TF78 and Genhushi to BFW were 45.32% and 47.52% respectively.The results showed that control efficiency of TF78 was close to commercial biological control agent Genhushi,and TF78 had obvious control effect on BFW in pot and field.(4)High throughput sequencing of 16 S and ITS amplified fragments was used to analyze the difference of microbial community between the soil treated with TF78 and the blank contrast soil and the results showed that the relative abundance of F.oxysporum in the contrast soil was 23.22% and 18.03%,respectively,and the relative abundance of F.oxysporum in the soil treated with TF78 decreased to 6.04% and 5.6%,which was significantly different in Jinling experimental site.The microbial abundance index(Chao1 index,ACE index)and microbial diversity index(Shannon index,Simpson index)of the soil treated with TF78 in the two experimental sites were both higher than those of the blank contrast soil,but there was no significant difference between them.It was certifiede that F.oxysporum is one of the most dominant population in the soil with BFW,strain TF78 inhibited the proliferation of Foc,reduced the number of Foc,and realized the decrease of BFW.(5)The antagonistic effect of volatile substances produced by strain TF78 on the growth of FocTR4 mycelia was determined by plate to plate method and results showed that the volatile substances produced by TF78 strain strongly inhibited the mycelial growth of FocTR4.Three volatile compounds were detected and identified by headspace solid phase microextraction and GC-MS,among which 1,4-dichlorobenzene was the dominant chemical substance.The control effect of 1,4-dichlorobenzene on BFW was 85.51% in pot experiment.The results showed that the volatile substances produced by Streptomyces TF78 had good control effect on BFW.(6)The effect of 1,4-dichlorobenzene on the growth of FocTR4 mycelium and conidia germination were determined by plate to plate method.The results showed that 3.56 g / L of 1,4-dichlorobenzene completely inhibited the growth of FocTR4 mycelium and 1.11 g / L of 1,4-dichlorobenzene completely inhibited the conidia germination of FocTR4.The effect of 1,4-dichlorobenzene on the process of FocTR4 conidia infecting banana roots was observed by SEM.The results showed that 1,4-dichlorobenzene significantly weakened the adhesion and infection of FocTR4 conidia to roots.By measuring the effect of1,4-dichlorobenzene on the conductivity of foctr4 cells,it was found that1,4-dichlorobenzene increased the membrane permeability of foctr4 cells and made the cell electrolyte overflow.Moreover,the observations with scanning electron microscopy showed that after 1,4-dichlorobenzene treating FocTR4 mycelium,the surface of the dried mycelium was seriously concave,the mycelium was dried,the mycelium was deformed and the cell wall was perforated.Transmission electron microscopy found that after1,4-dichlorobenzene treatment of FocTR4 mycelium,the cell wall of the mycelium became thin,been partially damaged,and severe separation of the plasm wall appeared;the cell membrane was damaged and blurred,and cytoplasm flowed out;most of organelles were dissolved,only a few rough endoplasmic reticulum was seen.The results showed that the apparent mechanism of 1,4-dichlorobenzene inhibiting FocTR4 was that1,4-dichlorobenzene damaged the cell wall and membrane structure of FocTR4,destroyed integrity of cell wall and cell membrane of FocTR4,caused cell electrolyte and cytoplasm to flow out and dissolution of organelles,which ultimately led to death of hypha cell.(7)The effect of 1,4-dichlorobenzene on the expression of FocTR4 gene was analyzed by high-throughput transcriptome sequencing.Result of transcriptome and Q-PCR verification showed that comparing with the gene expression of FocTR4 in blank control at the corresponding time point,after 1,4-dichlorobenzene fumigation for 12 h,48h and 96 h,the gene expression of FocTR4 changed as follows:(1)the genes expression were significantly down regulated in the follow pathways: cell wall component amino sugar metabolic pathway(gene FOIG07229/ FOIG07604),cell membrane component glycerophospholipid metabolic pathway(gene FOIG05866),autophagy-yeast pathway(gene FOIG00132);(2)the genes expression were significantly up-regulated in the follow pathways: cell membrane components steroid biosynthesis pathway(gene FOIG06054),protein utilization related protein processing in endoplasmic reticulum(gene FOIG00610/FOIG01078),proteasome(gene FOIG07364),protein export(gene FOIG07364),various types of N-glycan biosynthesis(gene FOIG06943),energy metabolism related pentose phosphate pathway(gene FOIG02062/FOIG03869),glycolysis / gluconeogenesis(gene FOIG03966),fatty acid metabolism(gene FOIG03459),citrate cycle(TCA cycle)(gene FOIG08859),detoxification related peroxisome(gene FOIG02066),Longevity regulating pathway(gene FOIG02066),immunity related folate biosynthesis(gene FOIG09195),self-healing ability related valine,leucine and isoleucine degradation(gene FOIG03075).It was proved that the molecular mechanism of 1,4-dichlorobenzene inhibiting FocTR4 was that 1,4-dichlorobenzene affected FocTR4 on the morphogenesis and self-regulation of cell wall and cell membrane of FocTR4;reduced the ability of self-regulation and adaptation to environmental changes,accelerated the speed of protein utilization,interfered the process of cell energy metabolism and function implementation of self repair,immunity and detoxification,and thus interfered with the normal material and energy metabolism process and important physiological activity process of FocTR4.(8)Through plate confrontation method,the antifungal activity of strain TF78 against Pyricularia oryzae,Botrytis cinerea,Fusarium oxysporum f.sp.cucumebrium,Phomopsis sp.,Pestalotiopsis sp.,Botryosphaeria sp.,Colletotrchum sp.,Fusarium solani,Fusarium proliferatum was determined and the result showed that strain TF78 inhibited the growth of mycelium of above 9plant pathogens.The control effect of the volatile substance of strain TF78 on post-harvest mango stalk rot and banana anthracnose was measured in a sealed desiccator and the result showed that the control effect of volatile substances on mango stalk rot and banana anthracnose reached 90.92% and 52.13%,respectively.The control effect of strain TF78 on teak canker and sheath rot of banana was respectively measured by inoculation on the young stem of teak and the sheath of banana with the spores of strain TF78 and pathogen at the same time.The result showed that the control effect of spores of strain TF78 on teak canker and sheath rot of banana was 20.31% and 27.67%.It was certificated that the volatile substances of strain TF78 had a good control effect on the disease control in relatively closed environment,such as root disease and storage disease and the control methods for the disease of the above ground were needed to be explored.In conclusion,a FocTR4 strain with high pathogenicity was obtained as the target pathogen;a strain of S.misionensis TF78 was isolated from banana rhizosphere soil,which has good control effect on BFW in pot and field;TF78reduced the amount of F.oxysporum in the field soil;the volatile substances produced by TF78 strain had good control effect on BFW;1,4-dichlorobenzene is a major chemical produced by TF78 strain;1,4-dichlorobenzene has good pot control effect on BFW;1,4-dichlorobenzene can inhibit the growth of FocTR4 hypha and the germination of conidia,weak the adhesion and infection of FocTR4 conidia to banana roots,increase the membrane permeability of FocTR4 cells and make the cell electrolyte overflow;1,4-dichlorobenzene can damage the cell wall and membrane structure of FocTR4,destroy the integrity of cell wall and membrane of FocTR4;the volatile substances of strain TF78 have good control effect on the disease control of relatively closed environment,such as root disease and storage disease.In addition,according to the results of transcriptome sequencing,it is speculated that 1,4-dichlorobenzene affects the morphogenesis of FocTR4 cell wall and cell membrane and interferes with the normal process of material and energy metabolism and important physiological activity of FocTR4. |
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