Studies On The Control Effect And Mechanism Of Earthworms And Its Intestinal Bacteria Bacillus Velezensis YFB3-1 To Lily Fusarium Oxysporum Wilt

Lily Fusarium wilt is caused by soil-borne pathogenic fungi such as Fusarium oxysporum,which is the most serious fungal disease and causes the largest economic loss in continuous cropping.To reduce the yield loss of continuous cropping lily,through three consecutive years of field cultivation experiment,we used quicklime（QL）,chemical fungicide thiram（CF）,inoculation with earthworms（IE）and biocontrol agent（BA）approach to deal with the soil of continuous cropping lily.Results showed that the earthworm activities can optimize the rhizosphere soil microbial community,and then significantly reduce the disease index of Fusarium wilt,decreasing the yield loss of continuous cropping lily.The antagonistic bacterium YFB3-1 was isolated and screened from the intestinal tract of earthworm that had strong antibacterial effect on Fusarium oxysporum,and the synergistic effect with earthworm could obvious increase the abundance of beneficial microorganisms in rhizosphere soil of continuous cropping and improve the control effect of lily Fusarium wilt.The main results are as follows:（1）The incidence and disease index of Fusarium wilt increased year by year with the time increase of continuous cropping lily,but compared with CK（no preventive measures were applied）,the above treatments could alleviate the occurrence of lily Fusarium wilt in continuous cropping.Among them,IE treatment had the best effect on reducing the incidence and disease index of lily Fusarium wilt in continuous cropping.Additionally,the disease index of three years were significantly lower than other treatments（p<0.05）,which could better alleviate the decline of lily yield in continuous cropping.Compared with CK,the control effect of lily Fusarium wilt were 43.75%,44.44%and 40.48%in three years continuous cropping,and the yield increased by 17.9%,18.54%and 15.69%,respectively.Compared to the original investment of 18 m^-2earthworms,the number of earthworm in IE treatment increased 22.41 times,The increasing number of earthworms were more conducive to the interaction of earthworms in different soil layers,which promoted the diffusion of organic matter from surface to deep soil and increased the content of organic matter in rhizosphere soil,alleviated soil acidification,and significantly increased the contents of alkali hydrolyzed nitrogen（AN）,available phosphorus（AP）and available potassium（AK）.Earthworm activity optimized the bacterial community structure of rhizosphere soil of continuous cropping lily（Chao1 index and Simpson index are the largest）,improved the species abundance of Rhizobiales,Myxococcales,Streptomycetales,and Pseudomonadales in rhizosphere soil,promoted the growth of lily,and reduced the occurrence of Fusarium wilt.The p H（r=0.8007,p<0.01）and AP（r=0.5804,p<0.01）were the key factors which affected the bacterial community structure of rhizosphere soil in continuous cropping lily.（2）A total of 33 strains were isolated and screened from the intestinal tract of earthworm with good inhibitory effect on F.oxysporum,including 18 bacteria,three fungi,and 12 actinomycetes.The YFB3-1 strain had the best antibacterial effect against F.oxysporum,and the inhibition rate was 80.03%.The YFB3-1 had high broad-spectrum antifungal ability against soil-borne pathogenic fungi,showed good inhibition effect to F.trilobium,F.solani,Alternaria alternata,Sclerotium rolfsii,Phytophthora parasitica,Penicillium cyclopium,and Aspergillus niger,the inhibition rate of the above pathogenic fungi was over 75%.YFB3-1 was identified as Bacillus velezensis by the morphological characteristics of bacteria,colonies and molecular biology.The pot experiment results showed that the prevention effect of YFB3-1 on lily Fusarium wilt was 64.18%,compared with commercial B.subtilis biocontrol agent,increased by 34.38%.（3）The liquid medium and fermentation conditions of YFB3-1 were optimized by single factor and orthogonal experiments.The optimal medium and fermentation conditions were yeast powder 8 g/L,complex amino acid 10 g/L,Na Cl 1 g/L,Mg SO₄·7H₂O 1 g/L,initial p H 7.0,inoculation amount 2.5%,fermentation temperature 30℃,rotation speed 180r/min,culture time 48 h.Under the above conditions,the concentration of YFB3-1 was2.83×10¹⁰CFU/m L,which was 4.93 times higher than the 4.77×10⁹CFU/m L of beef extract peptone liquid medium（NB）before optimization.It provides theoretical basis and technical parameters for the industrialization of YFB3-1.（4）Compared with single inoculation with earthworm or spraying YFB3-1 bacteria liquid,both interactions（T3）optimized the rhizosphere bacterial community structure（Ace index,Chao1 index and Shannon index were the largest）.Compared with CK（without inoculation with earthworm and spraying YFB3-1 bacteria liquid）,the control effect of T3 on lily Fusarium wilt was 37.66%in continuous cropping,and the yield increased by 14.78%.（5）YFB3-1 has the ability to synthesize a variety of antibacterial substances.The whole genome of YFB3-1 was sequenced and assembly by Illumina and Nanopore high-throughput sequencing technology.The results show that YFB3-1 contains only a circular chromosomal genome with a size of 3.93 Mb.Anti SMASH analysis revealed 12 secondary metabolite synthesis gene clusters,including the secondary metabolite synthesis gene clusters of surfactin,fengycin,bacillibactin and bacilysin,which can effectively inhibit pathogenic fungi.LC-MS was used to identify surfactin and fengycin from differences up-regulated metabolites between the fermentation liquid of YFB3-1 different fermentation times.YFB3-1 fermentation liquid could cause the hyphae of the pathogenic fungi F.oxysporum and S.rolfsii to enlargement and rupture,resulting in extravasation of intracellular substances,similar to the reported antifungal phenomena of surfactin and fengycin.It was speculated that surfactin and fengycin were the main active products of YFB3-1 against lily soil-borne pathogenic fungi.