Abstract：Bacterial pustule is a serious seed-borne disease, which dramatically reduces soybean(Glycine max) yield. The disease becomes more and more serious around the country these years. This research selected house-keeping gene of DNA replication and repair protein (recF) as target gene, and developed a bacterial pustule detection system based on padlock probe combined with dot-blot hybridization. The results indicated that the Padlock probe could detect the bacterial pustule pathogen specifically, and its sensitivity could be up to 100 fg/μL. The system also facilitated the detection of Xanthomonas axonopodis pv. glycines in soybean seedlots with 0.1% artificially infested seed. On the other hand, 45 samples of commercial soybean seeds were detected by using this method, and detected 4 samples positive with the bacterial pustule pathogen. The results indicated that the detecting system based on padlock probe combined with dot-blot hybridization could detect Xanthomonas axonopodis pv. glycines accurately, and provide useful references for prevention and control of bacterial pustule.
袁美丽.1983,吉林省大豆细菌病害种类鉴定结果[J].吉林农业大学学报,4(1):6.(Yuan M Y.1983.identification results of soybean bacterial diseases species in Jilin Province[J]. Journal of Jilin Agricultural University,4(1):6.) 赵海红.2013.大豆细菌性斑疹病的发病原因及防治方法.黑龙江农业科学,(11): 159.(Zhao H H.2013.The pathogenesis and prevention methods of bacterial pustule of soybean. Heilongjiang Agricultural Sciences,11:159.) Antson D O, Isaksson A, Landegren U, et al. 2000.PCR-generated padlock probes detect single nucleotide variation in genomic DNA[J]. Nucleic acids research, 28(12): e58-e58. Groth D E, Braun E J.1989. Survival,seed transmission, and epiphytic development of Xanthomonas campestris pv.glycine in the north-central Uunited States. Plant Dis. 73:326-330 Hardenbol P, Baner J, Jain M, et al. 2003. Multiplexed genotyping with sequence-tagged molecular inversion probes. Nat. Biotechnol.,21, 673–678. Kaewnum S, Prathuangwong S, Burr T J. 2005. Aggressiveness of Xanthomonas axonopodis pv. glycines iolates to soybean and hypersensitivity responses by other plants. Plant Pathology, 54: 409–415. Kanjalkar H P, Lokhande S S. 2013.Detection and classification of plant leaf diseases using ANN[J]. International Journal of Scientific & Engineering Research,, 4(8): 1777-1780. Khaeruni A, Suwanto A, Tjahjono B,et al.2007. Rapid Detection of Bacterial Pustule Disease on Soybean Employing PCR Technique with Specific Primers. HAYATI Journal of Biosciences,14(2):p76-80. Lee Y H, Kim N G, Yoon Y N, et al. 2013.Multiplex PCR Assay for the Simultaneous Detection of Major Pathogenic Bacteria in Soybean[J].58(2):142-148. Nilsson M, Malmgren H, Samiotaki M, et al.1994. Padlock probes: circularizing oligonucleotides for localized DNA detection. Science, 265,2085–2088. Prathuangwong S, Amnuaykit K. 1987. Studies on tolerance and rate reducing bacterial pustule of soybean cultivars/lines. Kasetsart Journal (Natural Science), 21, 408–20. Schaad N W, Song W Y, Hatziloukas E. 2000. PCR primers for detection of plant pathogenic species and subspecies of Acidovorax: U.S. Patent 6,146,834[P]. Shoemaker D D, Lashkari D A, Morris D, et al. 1996. Quantitative phenotypic analysis of yeast deletionmutants using a highly parallel molecular bar-coding strategy. Nature Genet, 14, 450–456. Sikora K,Verstappen E,Mendes O,et al. 2012, A universal microarray detection method for identification of multiple Phytophthora spp. using padlock probes[J]. Phytopathology, 102(6): 635-645. Szemes M, Bonants P, Weerdt M D, et al.2005.Diagnostic application of padlock probes-multiplex detection of plant pathogens using universal microarrays. Nucleic Acids Research, Vol. 33, No. 8. Tian Y L, Zhao Y Q, Bai S, et al. 2013. Reliable and sensitive detection of Acidovorax citrulli in cucurbit seed using a padlock-probe-based assay. Plant Dis.,97:961-966. Tian Y L, Zhao Y Q, Xu R, et al. 2014. Simultaneous Detection of Xanthomonas oryzae pv. oryzae and X. oryzae pv. oryzicola in Rice Seed Using a Padlock Probe-Based Assay,104:10, 1130-1137. Xu W, Bai W, Luo Y, et al. 2008. A novel common single primer multiplex polymerase chain reaction (CSP‐M‐PCR) method for the identification of animal species in minced meat[J]. Journal of the Science of Food and Agriculture, 88(15): 2631-2637. Yaya R, Antonius S, Budi T,et al.2000.Survival and Epiphytic Fitness of a Nonpathogenic Mutant of Xanthomonas campestris pv.glycines[J]. Applied and Environmental Microbiology, 66(3):1183-1189.
