Rapid Detection And Disinfection Treatment Methods Of Main Quarantine Pathogenic Bacteria In Rice Seeds

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.