【作者】 莫瑾；

【导师】 朱水芳； 廖晓兰；

【作者基本信息】 湖南农业大学 ， 生物安全与检疫， 2020， 博士

【摘要】 我国是水稻种子出口大国,特别是杂交稻种为我国种业发展和世界粮食安全做出了重要贡献。但随着各进口国提出的严格的检疫要求,因水稻种传病菌快速检测和除害处理技术的缺乏常导致货物滞港、退运和销毁等问题,严重阻碍我国水稻种子出口。因此,建立快速检测技术和有效的除害处理技术对杂交水稻国际化推广至关重要。本研究主要针对稻种进口国提出的检疫要求,对水稻细菌性谷枯病菌(Burkholderia glumae)、水稻细菌性褐斑病菌(Pseudomonas syringae pv.syringae)、水稻细菌性叶鞘腐病菌(Pseudomonas fuscovaginae)、水稻白叶枯病菌(Xanthomonas oryzae pv.oryzae)、水稻细菌性条斑病菌(Xanthomonas oryzae pv.oryzicola)等检疫性病菌进行快速检测技术研究,建立水稻种子快速有效的检疫方案,以期规范检疫方法,缩短检疫时间,提高口岸检疫效率。同时针对稻种出口前除害处理要求,建立适用于水稻种子调运中方便有效的除害处理方法,为保障我国水稻种子顺利出口提供技术支撑。主要研究结果如下:1.水稻种子重要种传病菌快速检测技术的研究1.1根据SyrB基因和PfsI/R基因设计特异性PCR引物,结合脂肪酸鉴定法,建立P.syringae pv.syringae和P.fuscovaginae的快速检测方法;根据含铁细胞接受因子基因设计特异性PCR引物,结合生化差异性指标,建立能够区分X.oryzae pv.oryzae和X.oryzae pv.oryzicola的方法,该方法的应用较现行国家标准中所使用的生化鉴定方法快3-5 d。1.2根据ITS基因(genebank D87080)和gryB基因(genebank AB207074)设计的特异性检测引物和探针,建立了双重PCR和实时荧光PCR检测B.glumae的方法,检测灵敏度可达到4.0×10~2cfu/mL。该引物已应用于现行的国家标准,在国家标准的基础上进一步建立了双重PCR检测体系,检测成本减少了二分之一,操作更为简便,且通过增加一个基因位点的检测和增加实时荧光PCR检测方法,结果更为可靠。1.3采用三对检测引物对B.glumae、P.fuscovaginae以及X.oryzae pv.oryzae或X.oryzae pv.oryzicola进行多重PCR分析,建立了多重PCR反应体系,方法特异性强,检测灵敏度可达到10~3cfu/mL。通过一次三重PCR检测能完成四种病菌的筛选,较原来三个独立实验的检测时间缩短了三分之二,提高了检测效率。2.水稻细菌性谷枯病菌鉴定图谱库的建立2.1变性高效液相色谱(DHPLC)分子标本图谱库:通过对B.glumae常规PCR扩增产物进行DHPLC同源性分析,获得16个吸收峰参比分子标本图谱。将待鉴定的PCR产物与分子标本进行DHPLC同源性比对,即可快速地对其进行验证。建立的用于鉴定B.glumae的分子标本图谱库,较目前的验证PCR产物的测序和分子杂交等方法,只需要具备常规PCR操作基础即可完成,操作简便,鉴定速度由2-4 d缩短至1 d。2.2基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS)蛋白质指纹图谱数据库:采用MALDI-TOF-MS对B.glumae标准菌株进行分析,获得了B.glumae蛋白质指纹图谱数据库,采用8株B.glumae阳性菌株对数据库进行验证,证明该方法准确可靠,B.glumae指纹数据库的建立填补了MALDI-TOF-MS蛋白质指纹数据库的空白,鉴定速度较常规生化鉴定(4-6 d)、脂肪酸鉴定(3-4 d)和Biolog鉴定(3-4 d)缩短至1-2 d,可应用于水稻种子中病原菌的初步筛选和鉴定。3.水稻种子快速检测技术方案的建立及应用3.1增效培养基:在半选择性培养基和富集培养基中添加终浓度为2.5%(v/v)的增效因子D+H(2:1),B.glumae生长速度可加快5-6 h,检出率提高了30%-40%。3.2水稻种子快速检疫方案:采用0.001%吐温-磷酸盐缓冲液25℃±1℃浸泡水稻种子4 h后,浸泡液涂布于上述3.1增效培养基上,B.glumae的检测效果显著性增加(P<0.01),B.glumae检测灵敏度至少可达到2.5×10~2cfu/g。建立了吐温-磷酸盐缓冲液处理水稻种子、选择性增效培养基培养病菌结合PCR-DHPLC分子标本图谱库和MALDI-TOF-MS蛋白质指纹图谱数据库等鉴定手段的快速检疫方案。规范了检测流程,较现行的国家标准增加了MALDI-TOF-MS和PCR-DHPLC等技术鉴定手段,较B.glumae检疫鉴定国家标准检测时间由原来的4-9 d缩短至1-5 d,较X.oryzae pv.oryzae和X.oryzae pv.oryzicola检疫鉴定国家标准检测时间由原来的5-15 d缩短至1-5 d。该方案对83批次水稻种子进行检测,8批次检出X.oryzae pv.oryzae,7批次检出X.oryzae pv.oryzicola,1批次检出P.syringae pv.syringae,阳性对照组均检出阳性病菌,检出率100%。该方案快速准确,能够满足口岸“快检快放”的要求,具有在口岸检疫部门推广应用的价值。4.水稻种子除害处理方法的研究研究得到了有害生物及其在种子中的最佳致死温度和时间:X.oryzae pv.oryzicola、X.oryzae pv.oryzae53℃处理20 min;B.glumae55℃处理15 min;P.syringae pv.syringae、P.fuscovaginae51℃处理15 min。种子中有害生物在实验室条件下55℃干热处理20 min以及应用种子烘干机55℃处理25 min,可对批量水稻种子进行除害处理,发芽率无显著影响,B.glumae等病原细菌灭活率>99%。建立的种子干热除害处理方法,保证了种子活性同时达到除害的目的,为口岸仓储的种子出口前除害处理方法提供了技术支撑。更多还原

【Abstract】 China is a major exporter of rice seeds,especially hybrid rice seeds that have made important contributions to the development of the seed industry and world food security.But with the strict quarantine requirements by importing countries,the lack of rapid detection and treatment technology of rice seed-borne diseases often leads to problems such as detention,return and destruction,and severely hinders the export of rice seeds.Therefore,the establishment of rapid detection technology and effective pests treatment technology is very important for the international promotion of hybrid rice.Based on the quarantine requirements raised by several importing countries for the hybrid rice seed exported from China,this study mainly focused on developing rapid quarantine and identification technology research on five major transmitted quarantine pathogens in rice species,including Burkholderia glumae,Pseudomonas syringae pv.Syringae,Pseudomonas fuscovaginae,Xanthomonas oryzae pv.oryzae,Xanthomonas oryzae pv.oryzicola.The quarantine system for rice seeds developed in the study can standardize the quarantine method,shorten the quarantine identification time,as well as improve the quarantine rate.The study provided the optimal pests treatment conditions and established an efficient and effective treatment method for the transportation of rice seeds,which provide important technical support to accelerate the process of customs clearance and meet requirements of the pre-export detoxification treatment of hybrid rice seeds during the export trade.The main findings of this study are summarized as follows:1.Research on rapid detection technology for important seed-borne pathogens of rice seeds1.1 I found that PCR specific primers combined with fatty acid identification methods can quickly and efficiently identify P.syringae pv.syringae and P.fuscovaginae.For the PCR specific primers used in the test,P.syringae pv.syringae identification primers were designed based on the SyrB gene:SyrB-F,SyrB-R and P.fuscovaginae identification primers were designed based on the PfsI/R quorum sensing locus:PfsI/R-F,PfsI/R-R.In addition,this study provided evidence that the specific primer(designed based on the Putative siderophore receptor gene cds)and indicators of biochemical diversity can accurately identify the seed-borne pathogens of rice seeds:X.oryzae pv.oryzae,X.oryzae pv.oryzicola.1.2 Built upon the specific detection primers designed for ITS gene(genebank D87080)and the specific detection primers and probes designed for the gryB gene(genebank AB207074),I developed double PCR and real-time fluorescence PCR methods for detecting B.glumae and the sensitivity of such test reaches 4.0×10~2cfu/mL.The primer has been applied to the national standard of China,and a double PCR detection system has been established.The detection cost is reduced by one-half,and the operation is more convenient.The result is more reliable after adding a gene locus in the PCR test and using the real-time Fluorescence PCR method.1.3 Three pairs of detection primer pairs B.glumae,P.fuscovaginae and X.oryzae pv.Oryzae or X.oryzae pv.oryzicola were used for multiplex PCR analysis,and a multiplex PCR reaction system was established.The results show that the method has strong specificity and the detection sensitivity can reach 10~3cfu/mL.The screening of four pathogens can be completed through one triple PCR detection,which shortens the detection time by two-thirds compared with the original three independent experiments,and improves the detection efficiency.2.Establishment of identification fingerprint library of B.glumae2.1 Denaturing high-performance liquid chromatography(DHPLC)molecular sample library:Through the DHPLC homology analysis of B.glumae conventional PCR amplification products,16 reference molecular specimen maps of absorption peaks were obtained.DHPLC homology comparison of the PCR product to be identified with the molecular specimen can quickly verify it.A molecular sample library for identification of B.glumae was established.Compared with the sequencing and molecular hybridization methods used to verify PCR products,the operation is simple and efficient.That is,it only requires to use the basic and conventional PCR operations,but the identification speed is shortened from 2-4 d to 1 d.2.2 Matrix-assisted laser desorption ionization time-of-flight mass spectrometry(MALDI-TOF-MS)method used to establish a protein fingerprint database for identification:In the study,MALDI-TOF-MS analysis of 8 strains of B.glumae standard strains were performed to obtain a fingerprint profile of the protein and then the MALDI-TOF-MS protein fingerprint database was used to identify the positive strains.The findings suggest that the method is accurate and reliable,and the identification speed is about 1-2 days,which is faster than those using conventional biochemical identification(4-6 d),fatty acid identification(3-4 d)and Biologic identification(3-4 d).The establishment of B.glumae fingerprint map filled the gap of MALDI-TOF-MS protein fingerprint database,which can be applied to the preliminary screening and identification of pathogenic bacteria in rice seeds.3.Establishment and application of technical program for rapid detection of rice seeds3.1 Synergistic medium:Adding synergistic factor D+H(2:1)to a final concentration of2.5%(v/v)in semi-selective medium and enriched medium can accelerate the growth rate of B.glumae by 5-6 h and increase the detection rate by 30%-40%.3.2 Rapid quarantine program for rice seed:By soaking rice seeds in a 0.001%Tween-phosphate buffer solution at 25°C±1°C for 4 h and then applying the synergistic selective medium to the 3.1 study,the detection effect of B.glumae in long-term preserved rice seeds was significantly increased(P<0.01).After fresh rice treated by this method,the B.glumae detection sensitivity is at least 2.5×10~2cfu/g.A rapid quarantine program for rice seeds was established,including Tween-phosphate buffer treatment of rice seeds,selective synergistic medium cultivation of pathogens,PCR-DHPLC molecular specimen library and MALDI-TOF-MS protein fingerprint database and other identification methods.This program standardized the detection process,and added MALDI-TOF-MS,PCR-DHPLC and other technical identification methods to the current national standards.Specifically,the testing time for B.glumae quarantine and identification is shortened to 1-5 d,compared with4-9 d under the national standard.Also,the testing time for X.oryzae pv.oryzae and X.oryzae pv.oryzicola quarantine and identification is shortened from 5-15 d to 1-5 d.In the study,eighty-three batches of rice seeds were tested for pathogens using the system.X.oryzae pv.oryzae was detected in 8 batches of rice seeds,X.oryzae pv.oryzicola was detected in 7 batches of rice seeds and P.syringae pv.syringae was detected in a batch of rice seeds.Since the system developed in the study is rapid and accurate,it meets the requirements of the“quick inspection and quick clearance”proposed by the customs and is a valuable technical process to be applied widely in the port quarantine departments.4.Research of disinfection treatment method in rice seedBy studying the heat resistance of pests and their heat resistance in seeds,the best lethal temperature and time for pests were obtained:After treatment with X.oryzae pv.oryzicola,X.oryzae pv.oryzae at the temperature of 53°C for 20 min;B.glumae after heat treatment at55°C for 15 min;P.syringae pv.syringae and P.fuscovaginae after treatment at 51°C for 15min.The dry heat treatment in the seeds at 55°C for 20 min and the continuous temperature control at 55°C for 25 min with the seed dryer control can kill the bulk rice seeds,and there is no significant effect on the seed germination rate after treatment.It has significant inactivation effects on bacterial diseases such as B.glumae(>99%inactivation rate).The established dry-heat treatment method for seeds has ensured the rice seed activity while achieving the purpose of pest treatment,provides technical support for the pre-export treatment methods of seeds stored in ports.更多还原