| Vernicia fordii（Tung oil tree）is one of the four major woody oil plants in China.Tung oil is one of the best drying oils in the world,traditionally used as coatings and paints,and now used as impregnating materials for large-scale integrated circuit boards.In addition,it has indispensable uses in aerospace science and industry,electronic technology,and medical hygiene.As the primarily and origin country,China has a long history of cultivation.However,Fusarium wilt disease,caused by Fusarium oxysporum f.sp.fordiis（Fof-1）,has caused devastating damage to the industry of tung oil trees and resulted in huge economic losses,which occurs in more than 90 counties and cities of 8 provinces nationwide.It becomes a serious threat to nearly one million hectares of tung oil forests all over the country.Plant Fusarium wilt is one of the top ten fungal diseases of plants,which has harmed more than 120 species of plants in the world and has no effective control measures at present,especially the lack of effective biological control strains.Fusarium wilt desease is a soil-borne disease,and its control is very difficult.Research on hybrid breeding and molecular breeding of tung oil tree is time-consuming and difficult;chemical control produces pesticide residues and pollutes the environment.The utilization of microbial control has the advantages of ecology,sustainability and efficiency.In the area of Fusarium wilt disease,we found that most of tung trees are infected by Fof-1,but very few tung trees grow normally.To provide effective biocontrol strains against Fusarium wilt disease,we took wilt-susceptible and wilt-resistant tung trees in the study to analyze the community structures of microorganisms in root rhizosphere.The antagonistic microorganisms of Fusarium wilt disease were isolated and identified from roots of tung trees.Burkholderia arboris Ba1 was isolated from roots of tung trees and the antagonistic ability was investigated.Furthermore,antagonistic compounds of Ba1 were identified.The main results are as follows:（1）Microbial community structure in the rhizosphere of susceptible and resistant tung trees was significantly different,and Burkholderia sp.was one of the major antagonistic strains enriched in the rhizosphere of wilt-resistant tung trees.Roots of tung trees that were susceptible and resistant to Fof-1 were collected in the area of Fusarium wilt disease,and microorganisms of rhizosphere soil collected from tung trees were sequenced by high-throughput sequencing technology.It’s found that pathogenic fungi Fusarium sp.,antifungal bacteria Burkholderia sp.could distinguish the wilt-susceptible and wilt-resistant groups.At the same time,Poplar（Populus deltoides×P.euramericana）and tomato（Solanum lycopersicum）are susceptible to Fusarium wilt disease;rice（Oryza sativa）and maize（Zea mays）are resistant to Fusarium wilt disease.The plants were divided into two groups,the poplar-tomato wilt-susceptibile group（PDESL）and rice-corn wilt-resistant group（OSZM）.Both were planted in the soil of the area of Fusarium wilt disease.The rhizosphere microbial community structure of the two groups were further analyzed.It’s found that pathogenic fungi Fusarium sp.and antifungal bacteria Burkholderiaceae could distinguish two groups.（2）Isolation and identification of Burkholderia arboris Ba1 against Fof-1.Roots and rhizosphere of tung oil trees were cultured in specific medium-MEA medium（containing two antibiotics）used to isolate and screen antagonistic microorganisms.It’s found that the number of isolated Burkholderia sp far exceeded that of other antagonistic micoorganisms.Besides,its antagonistic effect was superior to other antagonistic bacteria.Antagonistic bacteria were PCR amplified and identified by specific 16S r RNA primers,which were identified as Burkholderia arboris and named as Ba1.A phylogenetic tree within the genus Burkholderia was constructed,and it’s found that Ba1 belonged to Burkholderia cepacia complex（Bcc）.Bcc bacteria are closely related species in the genus Burkholderia,which are studied intensively for their biotechnological applications in plant growth promotion,biological control of plant pests and bioremediation.A rapid and efficient method to isolate Burkholderia sp.from rhizosphere soil and roots of tung trees were established.Burkholderia arboris Ba1 isolated and identified in this experiment was deposited for patent purpose,and the deposit number is CGMCC No.16905.（3）Burkholderia arboris Ba1 had antagonistic effect on various plant pathogens including Fusarium sp.We determined antagonism of B.arboris Ba1 found in this experiment.It’s found that Ba1 had obviously antagonistic effect against plant pathogenic fungi including Fusarium oxysporum f.sp.fordiis,F.oxysporum f.sp.cucumerium,Monilinia fructigena,Magnaporthe oryzae,F.graminearum through confrontation experiments.Furthermore,microscopic observations revealed that Ba1 confrontation culture with Fof-1 resulted in abnormality of Fof-1 hyphae and spores.According to the single factor experiment of Ba1,King A and King A+Fe3+media were the best antagonistic formulation of Ba1.Furthermore,germination test of tung trees’seeds soaked by antimicrobial agents revealed that King A solution could better promote seed germination.（4）Ba1 could produce antagonistic secondary metabolites,diketopiperazines and pyrrolopyrazinone.Firstly,it’s determined by the clapboard experiment that Burkholderia arboris Ba1 didn’t have antagonistic effect by producing volatile substances.Secondly,the non-volatile crude extract of Ba1 had antagonistic effect against Fof-1.Thirdly,analysis of crude extract of Ba1 by ultra performance liquid chromatography-tandem quadrupole mass spectrometry（UHPLC-MS-MS）and detection of crude extract compounds and corresponding standards by liquid chromatography-mass spectrometry（LC-MS）,it’s found that Ba1 produced9 compounds,belonging to diketopiperazines and pyrrolopyrazinone.It’s reported that diketopiperazines have antibacterial,antifungal,antiviral,antitumor,antimalarial and other biological activities,and pyrrolopyrazinone have anti-inflammatory and anti-tumor effects.After applying purified DKPs,it was found that DKPs had a significant antifungal effect against pathogen Fof-1.Two types of antagonistic compounds were produced mainly through non-ribosomal peptide synthases（NRPSs）pathways.Finally,gene clusters related to the two types of compounds synthesis wre found by sequencing and analyzing of Ba1 entire genome,and there were 44 genes and 39 genes in genome 1 and 50 genes in genome 2. |
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