Study Of The Pdftr1 Gene Mediated Copper Ion Regulation Of Growth,Development And Pathogenicity Of Penicillium Digitatum In Citrus

As an important fruit in China,citrus is susceptible to green mold disease caused by Penicillium digitatum after harvest,resulting in almost 90%of postharvest losses.Copper preparations have been widely used in the prevention and control of various diseases,and previous studies have found that copper sulfate can also be used in the prevention and control of P.digitatum.In this study,we expanded and screened the P.digitatum mutant library,resequenced and found the plasma membrane iron permease gene Pdftr1,knocked out and complemented the gene Pdftr1 by homologous recombination,we explored the mechanism of Pdftr1 in P.digitatum through in vitro experiments and inoculated fruits.The main results were as follows:1.The gene Pdftr1 that affects the sensitivity of P.digitatum to copper ions was cloned.By optimizing the agrobacterium-mediated transformation system of P.digitatum,a total of 1200 transformants were obtained.The sensitive and resistant transformants were screened from 2600 transformants using PDA medium containing 0.08 g/L and 0.5 g/L copper sulfate,respectively.Finally,two sensitive transformants P7-64 and P11-52 were screened and re-sequenced,the gene Pdftr1 was found at the T-DNA insertion site of the transformant P7-64.Through homologous recombination,the gene Pdftr1 of P.digitatum was knocked out and complemented,and 60 knockout transformants and 50 complement transformants were obtained,respectively.2.The sensitivity of P.digitatum to copper ions mediated by gene Pdftr1 was studied.The wild strain P44,the knockout strain△Pdftr1 and the complementary strain△Pdftr1-f were cultured under different concentrations of copper sulfate.The results showed that the growth ability of the Pdftr1 deletion mutant was significantly reduced under copper sulfate culture,indicated that the gene Pdftr1 mediated the sensitivity of P.digitatum to copper ions.Further experiments showed that the Pdftr1 deletion strain was also susceptible to the four inorganic copper preparations commonly used in production,but not to organic copper preparations.3.The growth development and pathogenicity of P.digitatum mediated by gene Pdftr1were studied.The wild strain P44,the knockout strain△Pdftr1 and the complementary strain△Pdftr1-f were cultured under different in vitro conditions,the results showed that the gene Pdftr1 was not associated with colony morphology,growth rate,oxidative stress,cell wall integrity and Na⁺stress of P.digitatum,as well as the sensitivity to Mg²⁺,Fe²⁺,Fe³⁺and Zn²⁺,while the gene may be associated with K⁺,prochloraz and propiconazole resistance,and with Al³⁺susceptibility.After 4 days of inoculation with different fungal solution on citrus fruit,the pathogenicity analysis showed that the diameter of the diseased spot of the fruit inoculated with△Pdftr1 was not significantly different from that of P44and△Pdftr1-f,showed that the gene did not affect the pathogenicity of P.digitatum.After the fruit was inoculated with different fungal solution and then with 3 g/L copper sulfate for 4 days,the diameter of△Pdftr1 lesion（2.13±0.49 cm）,significantly lower than P44（5.6±0.89 cm）and△Pdftr1-f（4.55±0.40 cm）,and still showed significantly higher copper sensitivity than P44 and△Pdftr1-f on citrus fruit.In summary,the study of the action targets of copper ions on P.digitatum,provided a theoretical basis and a new direction for the subsequent research and development of green control agent.