| Cucumber Fusarium wilt caused by Fusarium oxysporum f.sp.cucumerinum(FOC)is one of the most important destructive diseases in cucumber production,which seriously affects the yield and quality,and often leads to crop failure and causes great economic losses.At present,using chemical fungicides is a main method to control cucumber Fusarium wilt,but the long-term and large-scale use of chemical fungicides is easy to cause environmental pollution,pesticide residues and drug resistance of pathogenic microorganisms.Therefore,there is an urgent need for a safe and effective control method of cucumber Fusarium wilt.Rapid and accurate identification of pathogens can provide scientific basis for timely and effective disease control.Biological control has become a research hotspot of plant disease control because of its environmental friendly,safety and effectiveness.In this study,a rapid,accurate,specific and sensitive LAMP(loop-mediated isothermal amplification)system for detecting F.oxysorum f.sp.cucumerinum based on its specific marker gene fragment was established,and an antagonistic bacterium was screened from soil.Meanwhile,the biological control mechanism of the bacterium was studied from the aspects of physiology,biochemistry,soil microecology and transcriptome.The main results were as follows:Four LAMP primers were designed based on the sequence of the FOC-specific random amplified polymorphic DNA(RAPD)marker OPZ-12865.LAMP reactions were performed at different temperatures and durations,and the optimal temperature and duration were 63°C and 60 min,respectively.Hence,a LAMP assay for detecting FOC was established.The specificity of the LAMP method was evaluated against 119 isolates of FOC and other pathogens,and only FOC isolates yielded positive results.In sensitivity tests,the lowest concentration of genomic DNA required for the LAMP assay was 10 fg in a 25?L reaction.The LAMP assay was successfully applied to detect FOC in cucumber tissues and soil from infested fields,and the positive ratios of LAMP,PCR,and traditional tissue isolation for detecting FOC from diseased cucumber root samples were 100%,86.6%and 83.3%,respectively.The antagonistic bacterial strains against the pathogen FOC were isolated from healthy cucumber rhizosphere soils by soil dilution and plate confrontation methods.The antagonistic strains were identified based on morphological characteristics,physiological and biochemical indexes,whole cell fatty acids,16S r RNA and gyr B gene sequence homology.The inhibitory effects of antagonistic strains on mycelial growth and spore germination of FOC were also tested,and the control effects of fermentation liquid on cucumber Fusarium wilt were evualuted in greenhouse pot and field experiments.The results showed that 102 strains of bacteria were isolated from healthy ccucumber rhizosphere soil,and one strain FJ17-4 which identified as Bacillus velezensis showed the strongest antimicrobial capability against FOC.FJ17-4 severely disrupted the mycelial growth of the pathogen and caused abnormal phenomena such as mycelial deformity,distortion,swelling,shrinkage and winding.The control effects of 50 fold liquid fermentation liquid,living bacterial cell suspension and sterile filter solution on pot experiment were 70.20%,58.87%and 47.80%,respectively,and in field experiment were 69.53%,58.46%and 36.12%respectively.With the OD600 value of fermentation broth as the optimization standard,the fermentation medium and conditions of B.velezensis strain FJ17-4 were optimized by single factor and orthogonal test.The results showed that the optimum medium is 12.5 g·L-1soybean meal,5.0 g·L-1corn flour and 12.5 g·L-1 potassium dihydrogen phosphate.And the optimum cultural conditions are found to be p H 7.0,fermentation temperatures 30?,liquid loading(medium volume)50 m L in a 250 m L flask,inoculum size 12.5%,speed 180 r·min-1,incubation time 40 h.The OD600 value of the optimized cell growth was 25.62%higher than that before optimization.In order to determine the physiological mechanism of cucumber resistance to Fusarium wilt induced by B.velezensis strain FJ17-4,the activities of SOD,CAT,POD,PAL and PPO were tested by commercial reagent kit.The results showed that the activities of SOD,CAT,POD,PAL and PPO in cucumber roots treated with FJ17-4 were higher than those treated with water(CK).The activity of SOD and POD reached the peak on the 7th day,which was43.65%and 33.63%higher than that of the control.PAL activity showed two peaks on the3rd and 7th day,which were 1.39 and 1.32 times of the control,respectively.The activity of PPO and CAT reached the peak on the 5th day,which were 1.38 and 2.00 times of the control.It indicated that induced resistance is one of the important mechanisms of strain FJ17-4 to control cucumber Fusarium wilt.The colonization capacity and dynamics of strain FJ17-4 in cucumber and soil,and the effect of FJ17-4 on the quantities of culturable microorganisms(bacteria,actinomycetes,fungi and Fusarium oxysporum)in cucumber rhizosphere soil were determined by double antibiotic(Rifampicin,Kanamycin)resistance labeling,plate cultural colony counting methods and scanning electron microscope observation.The results showed that strain FJ17-4R labelled by the resistance of Rifampicin(300?g·m L-1)-Kanamycin(200?g·ml-1)could successfully(stably)colonize in cucumber root,stem,leaf and soil by different inoculation methods.The highest quantity of FJ17-4R in soil,roots and stems of greenhouse pot tests by irrigating method were 4.90×105 cfu·g-1,3.90×105 cfu·g-1 and 3.05×105 cfu·g-1,respectively,while that in field planting soil,roots and stems were 5.50×105 cfu·g-1,4.00×105 cfu·g-1 and3.68×105 cfu·g-1,respectively.And the highest quantity of FJ17-4R in roots of greenhouse pot by leaf spraying and stem daubing methods were 8.0×105 cfu·g-1and 1.5×105 cfu·g-1.Greenhouse pot and field planting tests demonstrated that the quantities of culturable microorganisms(bacteria,fungi and actinomycetes)in cucumber rhizosphere soil treated with strain FJ17-4R by root irrigating was significantly higher(P<0.05)than that in the control,while the quantity of F.oxysporum was significantly decreased(P<0.05).The results indicated that strain FJ17-4R had good colonization ability.Meanwhile,the strain FJ17-4R can change soil microbial structure and inhibit the F.oxysporum.The total DNA from cucumber rhizosphere soils was extracted by Fast DNA SPIN Kit,and the effects of B.velezensis FJ17-4 on composition and diversity of bacterial and fungal communities were investigated by using Illumina Hi Seq high-throughput sequencing technology combined with bioinformatics analysis.The results of high-throughput sequencing showed that 7 729 bacterial OTUs and 3 271 fungal OTUs were obtained from6 cucumber rhizosphere soil,and the coverage of bacterial and fungal libraries was more than 99%.The results of principal component analysis showed that there was higher similarity in the structure of rhizosphere soil microbial community among the three replicates in the treatet group.The microbial community structure and abundance of bacteria and fungi at phylum and genus level were significant different between treatment group and control group.The results of microbial function analysis showed that B.velezensis FJ17-4had no significant effect on the functional composition and relative abundance of bacteria,but had significant effects on the function and relative abundance of fungi.The relative abundance of plant beneficial functional microorganisms(Arbuscular mycorrhiza,Ectomycorrhizal and Endophyte)were increased,and the relative abundance of plant harmful microorganisms(such as plant pathogens)was decreased.B.velezensis FJ17-4could affect the microbial community composition and diversity,and increase the relative abundance of functional microorganisms in rhizosphere soil,thus the rhizosphere soil microecological environment was improved.The transcriptome of cucumber root samples were sequenced by Illumina hiseq TM 2500and analyzed by bioinformatics method.The results showed that 39.24GB of Clean Data was obtained by transcriptome sequencing,the content of GC was more than 42.39%,and the percentage of Q30 bases was above 90.91%.2379 Unigenes were merged and assembled from Clean Data.The clean reads of each sample was compared with the related reference genome,and the comparison efficiency ranged from 91.03%to94.76%.Compared with the control group,there were 1041 differentially expressed genes in the treatment group,of which 477 were up-regulated and 564 were down-regulated.The differentially expressed genes in response to the induction of B.velezensis FJ17-4 were mainly involved in carbon metabolism,starch and sucrose metabolism,phenylpropanoid biosynthesis,biosynthesis of amino acid,glycolysis/gluconeogenesis and photosynthesis.In addition,it was also found that the expression of many genes related to induced resistance,including genes related to salicylic acid(SA),ethylene,and Jasmonic acid(JA)signaling pathways,were significantly up-regulated.Ten differentially expressed genes with annotated functions were selected for q RT-PCR validation analysis,and the results showed that the expression trend of 10 genes was consistent with the results of transcription sequencing.The results indicated that the response of cucumber to the resistance induced by B.velezensis FJ17-4 is a process of multi gene participation and multi metabolism co-regulation.Moreover,the transcriptome sequencing data of this study are reliable and the results cover a wide range,which providing a scientific data for further exploring the mechanism of resistance to Fusarium wilt induced by B.velezensis FJ17-4. |
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