| Zanthoxylum is an important eco-economic plant and has a long history of cultivation in China.Z.armatum not only can strengthen the ecological protection but also can increase the economic income and the employment in the mountainous region of the southwest.With the strong support of the government,they have been widely planted in poor mountainous areas in southwestern China,and have become an important economic source for farmers in low-altitude mountainous areas.Since 2018,black rot of fruit and leaf spot on Zanthoxylum armatum has been observed in Yongshan County,Yunnan Province,which caused serious economic loss to local growers.The disease occurs suddenly and without good control measures.In order to scientifically prevent and control the disease,this study conducted a detailed study of the disease,including disease investigation,pathogen identification and screening of control agents,established a rapid detection system for pathogenic bacteria,and performed transcriptome sequencing analysis and resistance of pathogenic bacteria under pesticide stress.It is expected to provide theoretical basis and practical technology for the prevention and control of the disease,and lay the foundation for the further research of the disease.The main results as follows.(1)Fruit black rot and leaf spot of Zanthoxylum armatum were mainly distributed in Yongshan County.The disease mainly harms fruits and leaves,and it is only observed in Z.armatum Yongqing No.1.In the high-temperature weather in early June,brown to black near-round spots appeared on the surface of the fruit and leaves without signs,and the lesions were about 0.5-3 mm in diameter,and gradually expanded to the entire fruit and leaves.It can complete a large area of diffusion.After about three weeks,the affected fruit turns black,dry and rots and falls off with the wind.The investigation found that the disease almost only harmed Z.armatum Yongqing No.1,the onset of Ludian,Qiaojia and Yong Among the three counties,Ludian County has the most serious diseases.It has reached a devastating disaster in 2019,with a highest incidence of 96.7%and a disease index of 51.8.(2)Pathogeny studies confirmed that Pseudomonas oryzihabitans and P.fulva were the pathogens of fruit black rot,and P.fulva was also the pathogen of leaf spot disease and pathogenicity test shows that only Z.armatum Yongqing No.1 is the susceptible variety.Approximately 400 samples were collected,and 497 strains were isolated.According to Kochs postulate and identification,385 pathogenic isolates were identified as Pseudomonas oryzihabitans or P.fulva.P.fulva can cause fruit and leaf diseases,while P.oryzihabitans can only cause fruit diseases.The inoculation results showed that P.oryzihabitans can only cause black rot of peppercorn fruits and the lesions are moist and spread rapidly,while P.fulva can cause black rot and leaf spot of fruit.The lesions are dry and spread slowly.However,P.oryzihabitans had stronger pathogenicity,severity and incidence of fruit than P.fulva.The pathogenicity test results show that Z.armatum is a susceptible disease variety with an incidence rate of up to 95%,and Z.armatum var.novemfolius has a certain resistance and the incidence rate is less than 14%.Z bungeanum and Z.schinifolium are completely resistant varieties with an incidence rate of 0.This result is consistent with the field survey results.Through disease investigation,pathogen identification and host range testing,disease occurrence areas,pathogen types,and susceptible hosts were determined,which will provide a basis for future control of the disease,selection of control methods,and selection of Zanthoxylum cultivation.(3)The rapid detection system of pathogens was constructed by designing p4033-F/gp4033-R(P.oryzihabitans)and gp8055-F/gp8055-R(P.fulva)specifically for two pathogens.A relatively complete rapid disease detection system has been established to achieve rapid quarantine and early diagnosis of the disease.The rapid detection of plant bacterial diseases is an important means for quarantine,diagnosis and control of diseases.The specific primers gp4033-F/gp4033-R(P.oryzihabitans)and gp8055-F/gp8055-R(P.fulva)were designed based on the differences in the gap1 gene.Primer specificity and sensitivity were verified by three methods:ordinary PCR,fast PCR and fluorescent quantitative PCR.The sensitivities of gp4033-F/gp4033-R to P.oryzihabitans are 100 pg/μL for DNA,104 CFU/mL for bacterial concentration,and 10-1 pg/μL for DNA concentration,respectively.The sensitivities of gp8055-F/gp8055-R to P.fulva are 101 pg/μL for DNA,104 CFU/mL for bacterial concentration,and 100 pg/μL for DNA concentration,respectively.Field samples can also be tested accordingly.The target primers were designed using the gap1 gene differences,and three types of PCR were used for verification and sensitivity testing.The results showed that the primers had better specificity and higher sensitivity.(4)The 37 fungicides were screened by the bacteriostatic circle method and the turbidimetric method,and the agent with the best bacteriostatic effect was tetramycin,followed by methomycin.In LB medium,0.3%tetramycin has the best antibacterial effect.The diameter of the zone of inhibition is 58.3±1.2 mm(P.oryzihabitans)and 47.3 ± 1.9 mm(P.fulva)When 50 μL of the active ingredient 2 mg/mL is added.In LB liquid medium,0.3%tetramycin still had the best antibacterial effect.EC50=267 ng/L,EC90=23165 ng/L(P.oryzihabitans)and EC50=10296 ng/L,EC90=1.4952 × 107 ng/L(P.fulva).The drug screening results showed that antibiotics had better bacteriostatic effects than copper and P.fulva was significantly more resistant than P.oryzihabitans.The screening of fungicides provides the basis for field control and specific fungicide production.(5)The transcriptome of the main pathogens(P.oryzihabitans)under tetramycin stress,we can initially understand the resistance mechanism of pathogens to tetramycin.The drug-resistant of P.oryzihabitans to tetramycin was studied by transcriptome sequencing under tetramycin stress.Through co-cultivation of P.oryzihabitans and tetramycin,RNA extraction,library construction,sequencing and sequence analysis,a total of 1446 significantly differently expressed genes were obtained,622 were up-regulated and 824 were down-regulated.The 19 differential genes selected to verify transcriptome sequencing by fluorescence quantitative PCR.GO enrichment analysis showed that the biological process,BP type had the most differential genes,with a total of 605 annotations.KEGG enrichment analysis showed that the global and overview maps had the most metabolic pathways.The most significant scatter plot of the KEGG pathway was Ribosome,followed by Quorum sensing and Peptidoglycan biosynthesis and degradation proteins pathways.Through transcriptome sequencing analysis,the metabolic differences of pathogenic bacteria under drug stress were clarified,and the physiological mechanism of drug resistance was basically clarified,providing a basis for the subsequent development of specific drugs and resistance breeding.(6)Through gene screening,cloning,expression verification and functional test,it is preliminarily speculated that rpsJ and K07 may be involved in the response mechanism of P.oryzihabitans to tetramycin.The K07 and rpsJ genes were stably cloned and transformed from 15 candidate genes.The original DH5α,unloaded DH5α,K07-DH5α,and rpsJ-DH5α strains were obtained.The diameters of bacteriostatic circles in LB solid medium were 32.6±1.0 mm(original DH5α),32.7±1.4 mm(unloaded DH5α),31.3±1.6 mm(K07-DH5α),and 27.8±2.5 mm(rpsJ-DH5α).EC50 in LB liquid medium were 491882 ng/L,493909 ng/L,524465 ng/L,and 611456 ng/L.The K07-DH5α strain showed almost no drug resistance,while the rpsJ-DH5α strain showed significant drug resistance.In order to further verify the resistance of two genes in P.oryzihabitans.Tetramycin co-cultivation methods at concentrations of 8.57 ng/L,10.00 ng/L,12.00 ng/L,15.00 ng/L,20.07 ng/L(EC50),30.00 ng/L,and 60.00 ng/L.The relative expression of each gene was determined by fluorescent quantitative PCR.They reached relative expression peaks at 30.00 ng/L(K07)and 60.00 ng/L(rpsJ),indicating that the two genes are related to the tetramycin resistance mechanism of P.oryzihabitans strains.Exploring resistance-related genes can further understand the drug-resistant preparations of pathogenic bacteria,and can provide a basis for subsequent selection of disease-resistant breeding,improvement of drugs,and formulation of prevention and control programs. |
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