Colletotrichum asianum is one of the most important pathogens that cause anthracnose on mango plants. To establish a method for rapid detection of C. asianum, a C. asianum-specific sequence was obtained by comparing genomic sequences of different Colletotrichum species, and a pair of specific primers: caf1 (5'-CCGTCAGACGGAATTATCAGC-3') and car1 (5'-CCGATCCTGTCTTTGAAATGG-3') was designed accor-dingly. Specificity test results showed that a 419-bp target amplicon could only be obtained from C. asianum rather than other Colletotrichum species and non-Colletotrichum fungal and bacterial species that we tested in this study using the primer pair caf1/car1, with a detection sensitivity of 1 pg·μL^-1. The 419-bp amplicon could be detected from both mango leaves artificially inoculated with C. asianum strain YN55-1 and from naturally infected mango leaves with anthracnose symptoms. The specific PCR system developed in this study provides a technical support for monitoring and rapid diagnosis of mango anthracnose caused by C. asianum in the field.

China is the largest apple producer in the world. Apple stem grooving virus (ASGV), which impairs the growth and yield of apple trees, is widely distributed in apple producing areas of China. Virus detection is crucial for the prevention and control of apple viral diseases. In this research, we designed primers and probes according to the conserved regions of ASGV coat protein gene. A droplet digital PCR (ddPCR) for ASGV detection was established and assessed. The results showed that the ddPCR could detect ASGV specifically with high repeatability. The sensitivity of ddPCR is 10 times higher than that of qPCR. The ddPCR established in this research could be applied for virus detection in field samples of orchard and virus-free seedlings.

Bacterial leaf streak is an important rice disease caused by Xanthomonas oryzae pv. oryzicola (Xoo). In this paper, we developed a rapid and effective method using PCR combined with propidium monoazide (PMA) to distinguish dead and living Xoo cells. The results showed that PMA at 5 μg·mL^-1 could effectively inhibit the DNA amplification from dead cells after an incubation period of 5 min and light exposuring for 20 min. The minimum detectable concentration of living Xoo cells is 2×10⁴ CFU·mL^-1. The PMA-PCR method established in this study can effectively distinguish the dead and living cells of Xoo and avoid false-positive results.

China is now the world′s biggest sweet cherry producing country by growth area. severe viral diseases curbed cherry production in Yunnan province, China. A survey on the occurrence of cherry viral diseases in Lijiang, Luliang and Xuanwei counties of Yunnan province showed that viral diseases occurred in 3 cultivation areas on 6 varieties, with infection rates of 10%-30%. The major symptoms included dwarfing, malformation, leaf narrowing, leaf shrinking, and mottled, mosaic and brown spots on leaves. Mixed samples from 3 areas were collected for each variety, and the presence of viruses was tested by small RNA deep sequencing combined with RT-PCR. The results showed that 5 viruses were detected from Luliang. They were Cherry virus A (CVA), prunus necrotic ningspot virus (PNRSV), little cherry virus-1 (LChV-1), plum bark necrosis and pitting-associated virus (PBNSPaV) and citrus leaf blotch virus (CLBV). There were also CVA and PNRSV detected in Xuanwei. CVA, PNRSV, PBNSPaV, CLBV and LChV-2 were detected in Lijiang. Sequence analyses showed that the nucleotide sequences of these virus displayed high homology to related viruses in GenBank. PNRSV showed a higher relevance ratio than other viruses. Thus, we speculate that it is a dominant virus in cherry-producing areas of Yunnan.

Pepper anthracnose, caused by Collectotrichum acutatum infection, is the most destructive fungal disease in pepper production, which seriously threatens yield and quality of pepper. The genetic transformation with higher efficiency for the pepper pathogen C. acutatum is still not available. Our research provided optimal conditions to obtain protoplasts with higher efficiency at 2% of the enzyme in 1 mol·L^-1 NH₄Cl solution , 2 h of germination for the germ tube of conidium, and 2-3 h of incubation with application of the protoplasts , the GFP gene was successfully introduced into the wild type C. acutatum strain HHDL02 through modified PEG-mediated transformation. We further evaluated some of the transformants for colony morphology, growth rate, conidium morphology, conidial germination, appressorium formation, conidial production quantity and pathogenicity, which exhibited no obvious differences as compared to the wild type strains. In addition, visualizing the fluorescence signals of their progenies of the transformants suggested that the GFP gene integrated in C. acutatum strain HHDL02 was genetically stable. These results indicated that the protoplasts produced efficiently with the optimal approach from germ tube and the subsequent transformation mediated by PEG was suitable for the genetic operation of C. acutatum, and also helpful for the study on the mechanism of pathogenesis on pepper plants.

The aim of this study was to establish a SYBR Green I quantitative real-time PCR method (qPCR) for detection of melon yellow spot virus (MYSV). The specific primer pair for qPCR was designed based on the conserved sequence of MYSV nucleocapsid protein gene, and the annealing temperature, primer concentration, specificity and sensitivity of the primer were optimized. The results showed that the optimal annealing temperature of the optimized qPCR method was 61.3 ℃, the optimal primer concentration was 0.65 μmol·L^-1, the specificity and repeatability were good and this method was 100 times more sensitive than PCR. The cycle threshold of the qPCR standard curve was linear with the template concentration, and the correlation coefficient was 0.999 7. The results of field samples showed that the established qPCR method can be used for the quantitative detection of MYSV.

The occurrence of Phomopsis stem blight, caused by Phomopsis longicolla (syn. Diaporthe longicolla) and other Diaporthe/Phomopsis genus fungi, is one of the most serious soybean stem diseases in Huang-Huai-Hai and other soybean-production areas of China. Using resistance varieties is the most economical and effective method to control the disease. To establish a technical system for the indoor and rapid identification of soybean resistance to Phomopsis stem blight, and apply it to identify the disease-resistant germplasm resources of soybean, we compared five inoculation methods, including hypocotyl inoculation with wounding, cut-seedling inoculation, etiolated hypocotyl inoculation, mycelium inoculation on seed, and mycelium inoculation on stem. We found that the hypocotyl inoculation with wounding method has the advantages of simple operation, short test period, easy to determine the results, and good stability. In addition, five representative P. longicolla strains with different virulence levels and from different areas were selected and the evaluation criteria of resistance identification results were established, making up a complete set of resistance identification technical system. The system was used to identify 62 popular soybean varieties collected in 2020 from Huang-Huai-Hai and other regions. Among these soybean varieties, 31% and 68% of the varieties showed high resistance to the strong virulence strain DT3-3-1 and the weak virulence strain ZZ1-1, respectively; and 13 (21%) varieties were highly resistant to all five P. longicolla strains. The results revealed that there are soybean germplasm resources resistant to Phomopsis stem blight in China. The technical system established in this study provides a new candidate method for mining the soybean germplasm resources which are resistant to Phomopsis stem blight.

In this study, a rapid fluorescence quantitative PCR (qPCR) detection and the risk early warning system for Plasmodiophora brassicae in soil was established. The minimum detection threshold for P. brassicae DNA with the specific primer PbF/PbR was 1.612×10^-6 ng·μL^-1, which was 1 000 times higher than the sensiti-vity in that of conventional PCR. The minimum detection limit of qPCR for P. brassicae was 10 spores·g^-1 in artificially infested substrate and soil, which was far lower than the threshold for causing clubroot disease, with 100 spores·g^-1 in substrate and 1 000 spores·g^-1 in soil, respectively. The qPCR assay was used to quickly and accurately quantify P. brassicae in 27 field soil samples in Sichuan, Hubei, Jiangsu, Shandong, Liaoning, Shanxi Provinces, and Inner Mongolia and Ningxia Autonomous Regions, which provided a basis for formulating disease prevention and control strategies.

Tomato brown rugose fruit virus (ToBRFV) is an emerging virus and seriously threatens the safety of tomato production. To achieve rapid and simple detection of the virus, we developed a colloidal gold immunochromatographic strip for ToBRFV. Here, we used ToBRFV particles as the immunogen and produced 17 monoclonal antibody lines by hybridoma technology. Two different monoclonal antibodies were individually used for the colloidal gold-labeled antibody and the capture antibody on the detection line of the nitrocellulose membrane and combined to prepare colloidal gold immunochromatographic strips with 272 paired combinations. The immunochromatographic strip prepared with one paired antibodies could specifically recognize ToBRFV within 5 minutes, without reacting with tomato mottle mosaic virus, tomato mosaic virus, tobacco mosaic virus, cucumber mosaic virus, pepper mild mottle virus, potato virus X, potato virus Y,PVY, tomato chlorosis virus, tomato spotted wilt virus, or tomato yellow leaf curl virus. The sensitivity test results showed that the minimum detection threshold was 12 800 times for ToBRFV-infected tomato leaf extracts and 50 ng ToBRFV particles. The resultant colloidal gold immunochromatographic strip, which is sensitive and specific for ToBRFV, easy to use, and competent for large-scale sample detection, can be applied in the precise monitoring and early warning of ToBRFV.

Ditylenchus destructor is an important plant pathogenic nematode of sweet potato and potato, and also an important quarantine pest in China. To achieve accurate, rapid and visual detection of this nematode, the rDNA-ITS sequence of D. destructor was used as the target to construct the recombinase polymerase amplification combined with a lateral flow dipstick (RPA-LFD) visual rapid detection assay. The assay can detect D. destructor specifically within 15 min at 39 ℃, and the detection limit for a single nematode (J4) of type A (sweet potato population) and type B (potato population) is 3 125^-1 nematodes. It can directly detect D. destructor in soil and sweet potato stem tissue, with a sensitivity of one J4 nematode per 10 g soil and one J4 nematode per 2 g plant tissue. In addition, the assay also has the advantages of simple operation, low cost and visual result. The assay will lay a foundation for the early warning and port quarantine of D. destructor.

In this study, we aimed to develop an efficient protoplast transformation system in Alternaria alternata. Several main parameters for isolation of protoplasts, such as the mycelial age, enzyme system, and digesting time, were analyzed and optimized for the strain LI1 of A. alternata with strong pathogenicity. The combined conditions for highest efficiency of protoplast isolation for LI1 strain were as below: the mycelia cultured in CM liquid medium for 20 hours, 0.7 mol·L^-1 NaCl as the osmotic stabilizer, enzyme mixture including 1% Driselase, 1% Lysing enzyme and 1% Snailase for 4 hours of incubation with the mycelia at 28 ℃ at 110 r·min^-1. Moreover, the regeneration of protoplasts was tested with transformation of the plasmid pCT74 DNA containing GFP reporter and the Hyg B resistant genes into LI1 by using PEG/CaCl₂-mediated method. The following confirmation of transformants by PCR and growth phenotypes indicated that the GFP gene was successfully integrated into the LI1 strain genome. In summary, we successfully developed the protoplast transformation system and generated the GFP transgenic strain of A. alternata, which would benefited for understanding of the infection, host colonization, and pathogenic mechanism of this fungus.

Plenodomus lindquistii is a quarantine fungus in the list of quarantine pests in China. A species-specific real-time polymerase chain reaction (PCR) assay was developed for the detection of P. lindquistii. A pair of specific primers and a TaqMan-MGB probe was designed and synthesized according to the difference of ITS sequence between P. lindquistii and related species. A novel real-time fluorescent PCR was established to detect P. lindquistii. The minimal detectable concentration of targeted DNA was 0.1 pg in 20 μL reaction mixture. Optimal primer concentration and probe concentration were 0.6 μmol·L^-1 and 0.3 μmol·L^-1, respectively. The method could be used for the detection and preliminary screening of the samples suspected of carrying P. lindquistii. The method was specific, rapid, sensitive and completed within a single tube, the whole reaction took about one hour, without post-PCR handling, which provided a valuable tool for early rapid detection and identification of P. lindquistii.

Spongospora subterranea f. sp. subterranea (S. subterranea) is the pathogen of powdery scab disease on potatoes. For the detection of S. subterranea in potato tubers and soil, two pairs of specific primers A5/A9, C3/C8 and one pair of specific primers QF/QR were designed from the conserved region of internal transcribed spacer (ITS) regions and mitochondrial DNA to perform the conventional PCR and quantitative PCR, respectively. The results of specificity assay showed that the primers A5/A9 and C3/C8 amplified 264 bp and 367 bp products from S. subterranea DNA but did not amplify DNA from healthy potato or a range of soil-borne microbes including related species. The primers QF/QR had only one absorption peak for S. subterranea. These results indicated that the three pairs of primers have good specificity. The results of sensitivity assay indicated that diluted S. subterranea DNA was detected at a minimum concentration to 13.8 fg·μL^-1 using real-time PCR, which was 1000 times higher than that of the conventional PCR. In addition, the curve relationship between different content of plasmid DNA and corresponding (Ct) value is y=-3.893 9 x+35.228, R²=0.9966, showing a good linear relationship. A total of 18 samples of infected symptomatic potato tubers and 18 soil samples collected from different areas were detected by conventional PCR and real-time PCR. The detection rates of primers A5/A9, C3/C8, and QF/QR to diseased tissues were 100%, and the detection rates to soil samples were 44.44%, 66.67%, and 100%, respectively. In this study, the PCR-based methods were developed for the detection and quantification of the potato pathogen S. subterranea in infected tuber and soil, which will help to provide a scientific basis for early diagnosis and prevention of potato powdery scab.

In order to accurately evaluate the level changes of the Stemphylium solani associated with diseased tomato debris in the soil, a real-time quantitative PCR assay for quantitative detection of Stemphylium DNA in tomato leaf spot was developed. Based on the gene sequence encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in S. solani, a Stem-g7F/Stem-g7R primer pair was designed for which a 150 bp fragment was specifically amplified from the genomic DNA as template. The sensitivity of real-time quantitative PCR established in this study was 10³ times higher than that of conventional PCR. There was a good linear(R²= 0.992)relationship between the threshold cycle and the template concentration. The results showed that the initial copy numbers of DNA in crop residues was 3.69 × 10⁹copies/g. After treatment at 27℃ with humidity 80% for 30 days, the limit of DNA detection from the leaf tissues was decreased to 1.21 × 10⁶ copies/g, in contrast that of 1.29 × 10¹⁰copies/g at the relative humidity of 20%. Therefore, qRT-PCR assay is a highly rapid and reliable method to quantify S. solani in the artificial infected tomato debris. Application of the assay for the fungal detection and quantification may potentially improve the prevention for tomato leaf spot disease at the earlier stage and management on the dynamic of disease spread.

Hirschmanniella oryzae is widely distributed in rice producing areas, and it has been affecting the growth of rice and causing some yield losses worldwide. The specific primers for the loop-mediated isothermal amplification (LAMP) were designed based on rDNA ITS sequences of H. oryzae by comparing with related sequences of Hirschmanniella spp. deposited in GenBank. Two external Primers (F3:5′-ATCTTGTCCTTTGGCACG-3′,B3:5′-CGGTTGAACAAACAACGT-3′) and two inner primers (FIP:5′-CAGCATAGCAACAGAATGAATTCACGGTCGTAAACCTAATACGCG-3′,BIP :5′-TTGTACTACAATGGATTGTTTTCGCCTGATCCATCCACCCATG-3′) were designed. The optimal reaction conditions were screened based on electrophoresis detection and visual observation with SYBR Green I staining method, and the optimal conditions are: LAMP amplification under 57℃ for 45 min, the concentration of dNTPs and MgSO₄ used in the reaction volume is 1.4 mmol·L^-1 and 7 mmol·L^-1, respectively. The results showed that the LAMP method could detect and identify single juvenile, female or male of H. oryzae, and also could directly detect H. oryzae from mixed nematode species and rice root samples, the sensitivity of LAMP assay was 1/1 000 of single nematode DNA.

Citrus leaf blotch virus (CLBV) is the type species of the genus Citrivirus, family Betaflexiviridae. Previous studies have shown that CLBV occurs widely in kiwi plants cultivated in Shaanxi. To further simplify the detection of CLBV and explore its pathogenic mechanism, the coat protein (cp) gene of CLBV isolates from kiwi was obtained by RT-PCR amplification, and inserted into the prokaryotic expression vector pET-30a (+), which was transferred into the E.coli BL21(DE3) cells. A 59 kDa fusion protein was successfully expressed. The purified fusion protein was used for subcutaneous multi-point immunization of rabbits, and the obtained antiserum was purified and concentrated to finally obtain a polyclonal antibody with a concentration of 3.78 mg·mL^-1. After diluting antibody 1∶1 000, 500 pg of antigen can be detected, and Dot-ELISA can directly be used for on-field virus detection in kiwi samples. The preparation of polyclonal antibodies provides convenience for the detection of CLBV, and is also an important tool for the subsequent study of the pathogenic mechanism of CLBV.

Camptotheca acuminata witches′-broom-like disease was founded in Wenshan of Yunnan Province. To determine the pathogen and develop rapidly detection approach, near-full-length phytoplasmal 16S rDNA gene sequences were amplified via conventional PCR and nested-PCR using universal primer pairs P1/P7, R16F2n/R16R2 from DNAs extracted from the symptomatic shoots and leaves of C. acuminata plants. The amp-licons were cloned and sequenced. Phylogenetic analysis indicates that the pathogen is a member of phytoplasma group 16SrXXXII. Then, a set of primers and TaqMan probe specific for the phytoplasma were designed accor-ding to the conserved region of 16S rDNA gene. The standard plasmid DNA of Camptotheca acuminata witches′-broom phytoplasma was prepared, and the concentrations of primers and probe were optimized. The evaluation assay indicated that a good linear correlation was demonstrated in the standard curve with the correlation coefficient (r²) of 0.999. The established real-time fluorescence quantitative PCR detection method could specifically detected the phytoplasma in witches′-broom-like disease in C. acuminata. To our best knowledge, it is the first report of a 16SrII group phytoplasma associated with witches′-broom disease in C. acuminata in Yunnan Province. And it is the first study to determine the pathogen of Camptotheca acuminata witches′-broom disease , and a TaqMan probe-based qPCR method was established for the pathogen detection of this disease. The results of this study can provide reference for rapid detection and identification of Camptotheca acuminata witches′-broom phytoplasma.

Sugarcane mosaic disease wildly exists in sugarcane-cultivating fields and causes huge impact on sugar industry in China. Sugarcane streak mosaic virus (SCSMV), has been noted to become widely spread in recent years. Although serology-based detection techniques for SCSMV are established, rapid, accurate, and high-throughput detection methods are much less developed. Here, the PAb-^SCSMVCP (Polyclonal antibody of ^SCSMVCP) was produced and its specificity was tested. There is no serological cross-reaction observed with other viruses that cause sugarcane mosaic disease too. Based on the PAb-^SCSMVCP, the immune-detection methods, including double antibody sandwich (DAS) ELISA, dot blotting, and western blotting, were successfully deve-loped. Besides, the colloidal gold immunochromatographic strips were well-prepared and successfully used for rapid and accurate SCSMV detection in the wild field for the first time. Our studies provide serology based high-throughput, rapid, sensitive and quick diagnose methods for SCSMV detection in Chinese sugarcane-cultivating regions at first, which might contribute to the sugarcane mosaic disease control in China.

Gray mold, caused by Botrytis cinerea, is one of the main diseases on cherry flowers, leaves, and fruits in the middle and late growth period and the storage period with high incidence rate. Based on the internal transcribed spacer (ITS) sequence, a pair of primers were designed and used to develop recombinase polymerase amplification combined with a lateral flow dipstick (LFD-RPA) assay. The results showed that the optimal reaction temperature and time were 37℃ for 30 min for specific detection of B. cinerea with the sensitivity at 100 fg·μL^-1, which was slightly lower than conventional PCR at 10 fg·μL^-1 and real-time quantitative PCR at 7.43 fg·μL^-1. However, the detection process was relatively simple and rapid, just including RPA reaction in 30 min and LFD detection in 10 min. This method can be of great value for the surveillance of pathogen and early diagnosis of the disease.

The pathogen Didymella glomerata is included in the list of Chinese quarantine pests. The diseased plant materials are the important carrier of organism for the disease transmission. The accurate, sensitive and rapid detection method at ports of entry and land border in comprehensive quarantine system would be the powerful measure for the disease prevention and control. In this study, a pair of primers and a TaqMan-MGB probe based on the actin gene sequence of D. glomerata and its similar species were designed and synthesized for real-time fluorescent PCR assay. The optimal primer concentration and probe concentration were 0.6 μmol·L^-1 and 0.6 μmol·L^-1, respectively. The detection limit of this method was 20 pg of total DNA in 20 μL reaction mixture. The real-time PCR method was rapid, sensitive and completed within a single tube, without post-PCR handling of the amplification products, which provides a valuable tool for early rapid detection and identification of D. glomerata.

Xanthomonas oryzae pv. oryzicola (Xoc) causes bacterial leaf streak (BLS) in host plant rice. Xoc is dependent on the type III secretion system (T3SS) encoded by hrp cluster (T3SS genes) for the secretion of the type III effector proteins into rice cells leading to disease resistance or susceptibility in rice. To analyze the regulating expression of T3SS gene in Xoc, in this study we designed a bridge vector strategy in which a promo-ter segment or complementary fragment of target gene was constructed on a high-copy bridge vector to obtain a fusion element which then was subcloned into a backbone vector to obtain a vector for transcriptional expression or protein expression analysis. In order to verify the feasibility of this strategy, the promoter-probe-vectors and protein expression vectors of selected hrpG, hrpX and hrcC genes were constructed, and these vectors were introduced into the Xoc mutants of the virulence regulator genes trh, lrpX and zur. The results of the GUS activities, quantitative real-time PCR and protein immunohybridization showed that the transcriptional expression le-vels, mRNA levels and protein expression levels of hrpG, hrpX and hrcC genes were consistent in the mutants of trh, lrpX and zur. This indicates that this bridge vector strategy can be effectively used to analyze the T3SS gene transcriptional expression and protein expression. In conclusion, the application of bridge vector can overcome the shortcomings of low-copy vector DNA genetic manipulation efficiency. This strategy is also applicable to study the regulating expression mechanism of virulence-associated genes, which will provide an efficient working system for Xoc-rice interaction research.

Bacterial stem and root rot of sweet potato caused by Dickeya dadantii is a new quarantine plant disease in China. Recent outbreaks of this disease threaten sweet potato production in China. Specific and sensitive detection of D. dadantii is critical for the pathogen identification, distribution monitoring and the disease control. Here, conserved signature genes specific to D. dadantii were found by the whole genome sequences analysis of the strains belonging to the genus Dickeya. Primers targeting to the conserved signature genes were designed and tested by PCR. One set of primers Dad1-F (5′-CATATCAACCAGACCAGCCGTT-3′) and Dad1-R (5′-CGGCCTGCTTTTAAACAACGTATTA-3′), which targets to a conserved signature gene encoding an unknown protein with the accession number WP_077245517, specifically amplified a 167 bp fragment from D. dadantii by conventional PCR and real-time fluorescent quantitative PCR, providing a specific and sensitive method for identification and monitoring of D. dadantii in the field.

Ceratocystis spp. are important soilborne pathogens to many crops. Isolating the pathogen from soil sample has been a challenge due to various technical issues. In this study, carrot was selected as a bait for enriching and isolating Ceratocystis spp. from soil after comparing with potato and sweet potato. A series of experiments were conducted including thickness of carrot slices, relative humidity of soil, temperature of incubation, lighting, and moisture maintenance, which resulted in an optimized protocol as following. Carrot was sliced around 3-5 mm thick, which were placed in soil with water content of 10% to 30% in a container closed. The samples were incubated at 22℃-26℃ for either 24 h with a 12 h light /12 h dark cycle or 24 h in the dark. The pathogen could be recovered from the minimum number of spores 10 per gram soil after 8 days of incubation. This method was successfully applied on 108 soil samples of natural and tillage areas from Xinjiang, Henan, Hunan and Yunnan provinces of China, in which Ceratocystis spp. were isolated for all the 45 samples from Mengzi and Lincang cities of Yunnan. The developed method provide advantages of fast and easy operation, low equipment requirement, and low cost. It can be effectively applied to Ceratocystis isolation from large soil samples.

A SYBR GreenⅠ RT-qPCR method for Grapevine virus B (GVB) was established. An excellent linear correlation(0.999)and amplification efficiency (102.4%) were obtained from standard curve. The detection limit of the method was 10^-4 dilution fold, which was 100 times higher than conventional RT-PCR. The coefficient of variation ranging from 0.00% - 0.65% intra group and 0.02% - 2.00% inter group indicated the excellent stability and reproducibility of the method. The RT-qPCR method could be used to detect a wide range of field sample types. The dormant branches and old petioles were the best materials for GVB detection, the detection rates of which were all 100% and were the same as conventional RT-PCR. However, for samples from other seasons and positions, the detection rates of RT-qPCR (43% to 74%) were generally higher than that of conventional RT-PCR (5% to 71%). Especially for the samples in spring and young leaves in each season, the detection rates of RT-qPCR were 31 and 38 percent higher than conventional RT-PCR, respectively. Six out of 52 samples (belonging to 21 cultivars) from 13 provinces in China were detected to be positive by RT-qPCR (11.5%), which was two times that of conventional RT-PCR (5.8%).

According to the heat shock protein 70 (Hsp70) gene sequence of Tomato chlorosis virus (ToCV), a specific primer pair for ToCV real-time fluorescent quantitative PCR was designed. The recombinant plasmid ToCV-1 is used as a standard to establish a SYBR Green I real-time fluorescence quantitative method and a series of optimization include primers concentration, annealing temperature, specificity, sensitivity, reproducibility and stability were performed. The results showed that, the optimized annealing temperature is 63 ℃, and the primer concentration is 0.3 μmol·L^-1. The melting curve for specific peak proved that this method has good specificity. By comparing with the sensitivity of conventional PCR, we found that the SYBR Green I real-time fluorescence quantitative PCR was 100 times more sensitive. The method has good repeatability and stability. The rapid detection method based on SYBR Green I real-time quantitative PCR has high speed, strong specificity, high sensiti-vity and good repeatability. It can be used for the quantitative detection of ToCV.

Heterodera filipjevi is one of the important pathogenic nematodes that endanger the cereal crops in temperate zone, and it caused serious influence of the yield and quality. H. filipjevi is a newly identified species of cereal cyst nematode in central China. More than three years studies showed that Xuchang population is a new pathotype of H. filipjevi, and its pathogenicity was stronger than other populations of cereal cyst nematode in Huang-Huai Plain. This study aims to establish a rapid and accurate molecular detection system of Xuchang population, lay the foundation for monitoring and controlling Xuchang population of H. filipjevi and provide the basis for breeding and utilization of resistant varieties. This study focused on using RAPD-SCAR technique to analyze 9 CCN populations of 5 different CCN pathotpyes from Huang-Huai Plain. Moreover, 8 extra CCN populations were added to verify the specificity and validity of SCAR markers. 331 random primers were screened and two random primers with RAPD method were found related to Xuchang population, Primer S86, S178 could product polymorphic bands of Xuchang population about 550 bp and 1 200 bp respectively. The two RAPD markers related to Xuchang population of H. filipjevi had been transformed into SCAR markers successfully. The results of the specific detection showed that the two SCAR markers can detect the Xuchang population from other CCN populations in Huang-huai floodplain. The two SCAR markers can be used to detect and identify H. filipjevi Xuchang population.

Analysis and evaluation the pathogenicity of Verticillium dahliae Kleb. is essential for breeding of disease-resistant cotton plants and integrated control against Verticillium wilt. In this paper, we focus on the calibration of disease index in different batch tests and the standard of classification on virulence patterns of Verticillium dahliae based on 167 isolates collected from three major cotton growing regions in China. We found a set of strain and variety, the intermediate virulent strain Vd076 as the reference strain and the Jimian 11 with stable susceptibility as the corrected differential host facilitated the dataset compatibility after correction of the different batch tests data when the disease index of Jimian 11 reaches 50.0±5.0 by infection of Vd076. Based on the phylogenetic analysis, the criteria of classification for pathogenicity of V. dahlia were set up in which the average of the corrected disease indexes for three patterns of strains with high virulence, moderate virulence and low virulence were﹥40.0, 20.1~40.0 and 20.0, respectively. The analysis of different groups of differential hosts supported that Zhongmiansuo 41, Yumian 21, Lumianyan 28, Zhongmiansuo 35, Zhongmiansuo 8 and Jimian 11 were to be the differential hosts. This study provides the technical support for the virulence identification and variation of V. dahliae.

Bakanae is an important disease of rice across all major rice-growing regions in China leading to serious rice yield losses. Here we describe a loop-mediated isothermal amplification (LAMP) assay on direct detection of F. andiyazi in the diseased rice tissues, for rapid diagnoses of rice bakanae caused by this pathogen. Based on the sequence of trichothecene 3-O-acetyltransferase (TAT) gene in F. andiyazi, a set of species-specific primers was designed and screened to generate a positive color (sky blue) in the presence of F. andiyazi with the addition of hydroxynaphthol blue (HNB) indicator prior to amplification, whereas no color change (purple) in the negative reaction tubes of other species isolates under isothermal condition at 64℃ for 80 min. Using the TAT-Fan-LAMP assay with the detection limit at 100 pg·μL^-1 genomic DNA for F.andiyazi, we successfully and rapidly diagnosed the suspected diseased rice plant samples collected from Jiangning, Nanjing and Jurong, Zhenjiang in Jiangsu Province. This method can be used as a new way of diagnosing rice bakanae caused by F. andiyazi.

Bacterial fruit blotch (BFB), caused by Acidovorax citrulli, is one of the most destructive seed-borne diseases of watermelon and other cucurbits. This seed-borne quarantine disease has been reported in most watermelon and melon producing regions worldwide. Since the infected seeds are the most important primary inoculum source of BFB, seed health testing (SHT) and subsequent elimination of the contaminated seeds is one of the most effective methods to control the disease. Bio-PCR is widely used in SHT for the detection of A. citrulli. As A. citrulli has abundant genetic diversity and many other closely related species, specific and sensitive primers are very important for the success of the seed health testing. To screen the best primers for detection of A. citrulli by Bio-PCR, 33 bacterial strains including 17 strains of A. citrulli, 10 strains of other Acidovorax species and 6 strains of other genera of plant pathogenic bacteria were chosen to evaluate 7 pairs of primers reported so far. Results indicated that the primer pair SEQID4^m/SEQID5 had the best specificity that could distinguish all the tested strains of A. citrulli from those of other species and genera. The sensitivity of this primer pair was further tested with the Bio-PCR assay of A. citrulli. Results showed that the detection limit of the assay was 10² CFU·mL^-1 for the pure bacterial culture, while for watermelon seeds the detection limit were 0.01 CFU·g^-1 and 0.1 CFU·g^-1 when seed extracts were cultured on semi-selective media ASCM and EBBA, respectively.