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.