| Bacterial fruit blotch(BFB)is a quarantine seed borne disease caused by Acidovorax citrulli.Due to its wide spread and severe incidence,it has seriously affected the development of watermelon and melon industries in China.Different forms of nitrogen fertilizer have a significant impact on the occurrence of plant diseases,mainly due to differences in host disease resistance and pathogen pathogenicity under different forms of nitrogen fertilizer treatment.In this study,the effects of different forms of nitrogen fertilizer on watermelon disease resistance were systematically studied by measuring the resistance to fruit spot disease,biomass,changes in stem conducting tissue,expression of disease resistance related genes,and the utilization ability of A.citrulli in watermelon stem and leaf juice under different forms of nitrogen fertilizer treatment.By studying the role of nitrogen metabolism regulation system,NtrBC in nitrogen source utilization,stress tolerance,and pathogenicity of A.citrulli,we further explored the impact of different forms of nitrogen fertilizer on the pathogenicity of watermelon acid phagobacteria.The main research results are as follows:1.Applying nitrate nitrogen fertilizer can effectively reduce the incidence BFB of watermelon.It was found that the disease index of watermelon seedlings under single application of ammonium nitrogen fertilizer(79.92)was significantly higher than that under single application of nitrate nitrogen treatment(52.75)and 1:1 mixed application of nitrogen fertilizer treatment(53.96)by inoculating A.citrulli with spray.Further research found that applying nitrate nitrogen was beneficial for watermelon growth and promotes its absorption of calcium elements,conducive to the growth of watermelon stem conducting tissue,It is beneficial to the expression of disease resistance related genes in watermelon.In addition,single application of ammonium nitrogen fertilizer can promote the accumulation of nitrogen in watermelon,which is conducive to the growth of A.citrulli.2.In this paper,the bioinformatics analysis of NtrB and NtrC was carried out to predict their main domains and functions,using the Aac5 as the research object.Single gene deletion mutant strains and double gene deletion mutant strains of ntrB and ntrC as well as their complementary strains were constructed.3.The NtrBC system plays a key role in nitrogen source utilization in A.citrulli.After measuring the nitrogen source utilization ability of each tested strain in MMX medium,it was found that the utilization ability of ΔntrB to ammonium sulfate,urea,and some amino acids was significantly enhanced,while ΔntrC lost its nitrate utilization ability,and its utilization ability to some amino acids was also significantly decreased.The nitrogen source utilization capacity ofΔntrBC is basically consistent with that of ΔntrC.The results of q RT-PCR assay showed that the deletion of ntrB gene significantly upregulated the expression of key genes about nitrate utilization Aave＿2107,nas S,and nas T,while the deletion of the ntrC significantly downregulated nas S,and significantly upregulated the expression of nas T.In addition,the deletion of ntrB and ntrC genes also affected the expression of some amino acid metabolism related genes.4.The NtrBC system plays an important role in the stress tolerance of A.citrulli.The ability to resist oxygen stress,high salt stress,and copper ion stress of each mutant strain was tested,and the results showed that the resistance of each mutant strain to stress was significantly enhanced.5.The NtrBC system plays a key role in the pathogenesis of A.citrulli.By measuring the pathogenicity and other pathogenicity related phenotypes of the tested strains,it was found that the pathogenicity,in vitro growth,in vivo colonization,swimming,twitching,and seed surface adhesion abilities of ΔntrB and ΔntrC decreased significantly,while the biofilm formation ability increased significantly.In addition,the results of q RT-PCR assay showed that the expression of key genes of T3 SS in ΔntrB,such as hrp G,hrp E,hrp X,and hrc J,flagellar related genes flh D,flh C,fli A,fli C,and fli M,and pil A,were significantly downregulated compared to Aac5.The ntrC deletion significantly downregulated hrp E,hrp X,hrc J,and pil A,but significantly upregulated flh D,flh C,fli A,and fli C.6.In the study of the regulatory relationship between ntrB and ntrC genes,it was found that the deletion of the ntrB significantly upregulated the ntrC,while the deletion of the ntrC had a small impact on the expression of the ntrB.After measuring the expression of ntrB and ntrC at different culture times,it was found that the expression of ntrB and ntrC was significantly upregulated with the prolongation of culture time.7.NtrC plays an important role in the colonization of nitrate nitrogen treated watermelon stems in A.citrulli.After measuring the colonization of Aac5 and ΔntrC in the stem of watermelon treated with different forms of nitrogen fertilizer,it was found that the difference in growth between Aac5 and ΔntrC in the watermelon stem under nitrate nitrogen treatment was significantly higher than that under single ammonium nitrogen treatment,indicating that changes in the stem environment after nitrate nitrogen application had a more significant impact on ΔntrC than Aac5. |
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