An Improved Assay For Rapid Detection Of Bacterial Fruit Blotch And Disinfection Of Infected Watermelon Seeds

Watermelon and melon fruits are important economic crops in China.Since 2003,China has been the biggest country in the world,in term of the production and consumption of these crops.Bacterial fruit blotch of watermelon(BFB)is a seedborne disease caused by Acidovorax avenae subsp citrulli.BFB led to an economic loss of about 500 million Chinese Yuan every year in China.The research object of this article is to establish an efficient system to detect and eliminate the causal pathogen for BFB.This approach has been one of the research hotspots,as BFB is recently the most popular bacterial disease in both domestic and foreign area.The rapid detection method of the seedborne disease is based on the use of the selective medium of BFB,followed by PCR analysis.However,the screening efficiency of the previously reported selective media was not always high,and therefore the subsequent PCR detection was less effective.On the other hand,the disinfection of infected seeds mainly depends on chemical agents.In the case of successful elimination of BFB by chemicals,the chemical treatment frequently inhibited seed germination.On the basis of previous studies,in this article,the author developed an:improved assay for rapid detection and disinfection of BFB.The main results are as follows:1.In order to improve the specificity and accuracy of BFB selective medium,the author preliminarily examined the existence of antibiotic genes in BFB through two different bioinformatics database.The ARDB database showed that BFB mainly included Baca domain kind of antibiotic-resistance genes,which mainly present the resistance to bacitracin.On the other hand,the NCBI database showed that BFB included the domain of beta-lactamase,suggesting that the bacterium could be tolerated against penicillin and cephalosporin antibiotics.Accordingly,it could be possible to optimize the existing semi-selective mediun through gene prediction.2.The plate coating method was used to assess BFB resistance to antibiotic compounds predicted by bioinformatics.The results showed that the predicted antibiotic combination of ampicillin and cephalothin performed most significant inhibitory effect on mixed bacteria,but showed no significant inhibitory effect on BFB.Obviously,this combination was most effective to screen BFB.According to the results of the present quantitative analysis,the author obtained the most suitable concentration of ampicillin and cephalothin;eventually,the novel selective medium was completed and designated as EXYL medium.After comparing the actual screening results from the different selective media,the author indicated that EXYL medium significantly improved the screening accuracy of BFB;in fact,the detection rate increased from 8(with the pre-existing TWZ medium)to 39%(with EXYL medium).In conclusion,the optimized medium has simple composition and facilitates the screening of BFB.3.In addition to antibiotics,the short wavelength ultraviolet(UV-C)was used as an effective physical antimicrobial method,aiming at increasing a screening selective pressure.In order to explore the genes and proteins related to UV-C resistance in BFB,five species of sequenced typical pathogenic bacteria,including Pseudomonas syringe,A.avenae subsp.avenae,Escherichia coli and P.syringe pv.tomato,were chosen to compare the main UV-related genes and protein structures.As a result,the author found that BFB has highest UV resistance,suggesting that the bacterium carries the UVDE genes.According to the reference data for UV resistant bacteria(Deinococuu sp.and Radiodurans sp.),the best screening time was shown to be 20?J/cm2.This UV irradiation was used as assistant screening factor and combined with optimized EXYL medium.Eventually,the actual screening sensitivity for fruit blotch was improved from 8%to 55%.4.Based on the sterilizing effect and seed germination rate,the author developed a new type of safe bactericide(named CSP)and its sterilization method.The result shows that soaking in CSP not only promoted the seed gerninating vigor,but also accelerated seed germination rate.CSP-treated seeds showed highest germination rate 48 h after the treatment,and the maximum root length was 28.75 mm.This was significant advantage against the seeds treated with other chemical reagents or pesticides.Field culturing assay further confirmed that the CSP-treated seeds generated healthy plants with prevention rate close to 98,8%,which was considerably higher than the previous data(82.6%with bactericide formaldehyde).At last the staining assay showed that dehydrogenase content of the CSP-treated seeds was highest and its conductivity stable,suggesting that the seed vigor was highest.Thus,the CSP treatment was effective to completely kill BFB inside the watermelon seeds without causing any detrimental effect on seed germination.In conclusion,this study developed a new selective medium EXYL and a UV-C-aided screening procedure for BFB,which significantly increased the sensitivity and accuracy of the detection of BFB in water melon seeds.In addition,we developed a new type of safe bactericide CSP and its sterilization method,which could quickly and completely kill BFB.Lastly the author would like to emphasize that this study has an important practical significance for the basic research and actual prevention and control of BFB.