Fusarium circinatum, F. tucumaniae, F. virguliforme and five formae speciales of F. oxysporum have been included in the “List of Quarantine Pests of Import Plants in the People’s Republic of China” in 2007. This article aims at revising the scientific names of these species and formae speciales in accordance with the current taxonomic system of the genus Fusarium and related genera in Nectriaceae. It is suggested that the Chinese name for the causal agent of pine pitch canker is revised as huan zhuang lian bao (环状镰孢). The Chinese and scientific names of the oil palm wilt pathogen are revised as you zong lian bao (油棕镰孢) and F. elaeidis. It has been clarified that the pathogens responsible for South American soybean sudden death syndrome, F. tucumaniae, and North American soybean sudden death syndrome, F. virguliforme, are the same species in the genus Neocosmospora, and the Chinese name and scientific name are now revised as cai dou xin chi qiao (菜豆新赤壳) and N. phaseoli, respectively. Additionally, this study indicates that the pathogens responsible for the banana wilt (race 4 and non-Chinese race), celery wilt, asparagus wilt, and strawberry wilt are not monophyletic, suggesting the necessity for taxonomic revisions of these formae speciales of F. oxysporum. These updates are references for future revisions and renewal of the quarantine pest lists.
DNA barcoding, as a new technology for species identification, is becoming mature in the identification of species of animals, plants and fungi. It provides a powerful tool for plant quarantine and identification of invasive alien species. This paper summarizes the research progresses on fungal DNA barcoding and analyzes the current situation of DNA barcode collection of quarantine fungal species in five major trading countries/regions including China and the United States. The existing problems in DNA barcode screening and existing database resources of some important quarantine fungi including species in genera Puccinia and Phytophthora are analyzed. The idea of screening DNA barcodes of quarantine fungi at the whole genome level is put forward to improve the accuracy of species identification. The suggestions of strengthening international cooperation and establishing a database of quarantine fungal DNA barcodes, and so on, are also advanced.
Poplars are widely used in wood production, shelterbelt, and garden landscape construction, with significant economic and ecological value. Poplar leaf rust mainly occurs on poplar leaves with a severe impact on seedling growth. In 2023, leaf rust occurred seriously in the poplar nursery in Handan of Hebei Province. In this study, the pathogen of poplar leaf rust from Beijing and Hebei was identified by observing morphological characteristics of urediniospores on leaves and sequence comparison of rDNA ITS regions. The results showed that the pathogen responsible for poplar leaf rust in Beijing and Hebei was Melampsora larici-populina. The quarantine species M. medusae was not detected in these test samples. Eight distinct loci of the ITS sequence were used to differentiate M. medusae from the other closely related Melampsora species, providing some data for monitoring the quarantine species M. medusae.
A fungal strain 8033-2 isolated in bud of rhododendron imported from Belgium was identified by morphological characteristics, sequence analysis of internal transcribed spacer (ITS) region, translation elongation factor (EF-1α) and RNA polymerase Ⅱ second largest subunit (RPB) gene and pathogenicity test. The colony of isolate 8033-2 is white, initially hairy, with more aerial hyphae, and turned into dense, grey, olive green when well developed and mature. Conidia single-celled, smooth, nearly spherical or elliptical, gray brown, with a size of 8.04(6.32-11.49)×5.60(4.38-7.40) µm. The sequence similarities of ITS, EF-1α and RPB genes between isolate 8033-2 and the corresponding sequences of Seifertia azaleae in GenBank were 100%, 100% and 99.90%, respectively. The phylogenetic tree based on ITS, EF-1α and RPB gene sequence showed that isolate 8033-2 clustered with S. azaleae. Artificial wound inoculation caused typical symptom of brown necrosis in healthy buds. The isolate 8033-2 was identified as Seifertia azaleae, which is the first interception of this quarantine pest at border ports of China.
Monilinia fructicola, causal agent of brown rot, is one of the most important fungal pathogens of stone fruit. M. fructicola is a quarantined pathogen in China, causing significant production losses and ecological losses. In this study, the optimized Maxent model and ArcGIS were used to predict the potential geographic distribution of M. fructicola in China under different climate scenarios in the present and future, in order to reducing the risk of the spread of the disease under future climate change. The results showed that the variance of temperature change (bio4), the average temperature of the hottest season (bio10), the precipitation of the driest month (bio14), and the variance of precipitation change (bio15) were the key environmental variables affecting the distribution of M. fructicola. Under the current climate conditions, the total suitable distribution area of M. fructicola in China is 3.64×106 km2, accounting for 38.12% of total land area of China; the highly suitable areas are mainly distributed in Yunnan, central Guizhou, southern Hunan, Zhejiang, southern Anhui, southern Gansu, southern Shaanxi, and Hebei. Under the two future climate scenarios (SSP126 and SSP585), the total suitable area of the pathogen in China will decrease, but the area of low suitable area will increase. In addition, the centroid of the suitable area under both climate scenarios in 2041-2060 tends to migrate from Chongqing to the southwest.
In order to realize high efficiency and high precision quarantine identification and epidemic monitoring of apple quarantine pathogen, the detection method of Neofabraea perennans by droplet digital PCR (ddPCR) was established. This method provides technical services and support for the prevention of pest from ports and the protection of China’s ecological security. N. perennans was used as the research object, and the β-tubulin gene (TUB2) was identified by bioinformatics analysis as a target for specific primer and probe design. The specific detection system of N. perennans by ddPCR was established by optimizing the concentration of primers, probes and annealing temperature. The results showed that the detection limit of ddPCR was 0.16 copies/μL and the detection concentration of genomic DNA was 10.0 pg/μL, being consistent with the result of qPCR. The linear correlation coefficient in the range of quantitative detection was more than 0.999, and the RSD was less than 25%, indicating that the detection system had good repeatability and could be used for rapid and accurate detection of N. perennans in imported fruit samples.
Verticillium alfalfae is an important quarantine pathogen, which can cause serious damage to more than a dozen crops. The sequence characterized amplified region (SCAR) marker of V. alfalfae was chosen as the target gene, and a detection method combining RPA isothermal amplification technique and CRISPR-Cas12a system was established for specific detection of V. alfalfae. Under the optimized experimental conditions, 10.6 pg genomic DNA can be detected by using CRISPR-Cas12a based fluorescence and lateral flow strip method after the genomic DNA was amplified at 39 °C for 30 minutes. The spiked recovery experiment indicated that the established RPA/CRISPR-Cas12a can be used for the rapid detection of V. alfalfae in plant tissues.
Heterobasidion parviporum is a strong devastating pathogen and great harmful to coniferous forests. It is not distributed in our country until now, but there is transmission risk of being introduced through imported wood. In order to establish a KASP detection method for H. parviporum, multiple sequence comparison was performed on 223 genes from Heterobasidion and its similar genus, and then a SNP site was screened on H. parviporum GAPDH gene, which can be used as marker in this study. The primers Hpar-FAM-F1, Hpar-HEX-F2 and Hpar-R were designed based on design rules for H. parviporum KASP molecular detection. Test results show that it could successfully identify H. parviporum from 22 similar species including H. irregulare, H. occidentale and others by genotyping, indicating that the marker has high specificity. DNA concentration of H. parviporum was ten-fold diluted from initial concentration 100 ng/μL in sensitivity testing. The reaction result of gradient 10-4 and 10-5 were close to that of double diluted water, and therefore, the minimum concentration of the marker detected was up to 0.1 ng/μL. Model positive sample tests were performed in two forms, one was basidiocarp powder mixed with wood and another was adding DNA of H. parviporum in wood. Results show that model positive samples and the reference species displayed typical genotyping on axis. The KASP marker could accurately identify H. parviporum on coniferous wood, and the KASP method can be used to detect H. parviporum on imported wood.
Albonectria rigidiuscula is listed as Chinese quarantine pest, and has been intercepted at many ports in China, which may cause invasion risks. A precise enzyme mediated duplex exponential amplification (EmDEA) assay was developed for detection of A. rigidiuscula. Based on translation elongation factor (TEF1-α) gene of A. rigidiuscula and related species, five upstream DNA primers, six downstream DNA primers, and four RNA probes were designed and synthesized. The minimum detection limit is up to 0.5 pg/uL. The detection results of suspected sample indicate this method is useful to detect and screen A. rigidiuscula. The method is rapid, sensitive and completed within a single tube, without post-PCR handling of the amplification products. The entire reaction process takes about 30 minutes. This result provides an important reference for the detection and screening of A. rigidiuscula in the port.
To accurately and rapidly detect the quarantine fungus Setophoma terrestris, a species-specific real-time polymerase chain reaction assay was developed in this study. A TaqMan-MGB probe was designed and synthesized according to the difference of rDNA ITS sequence between S. terrestris type strain and its closely related species. The whole reaction process took only about 1 h. The minimum detection limit was up to 1 pg/μL, and the optimized reaction conditions resulted in a final primer concentration of 0.6 μmol/L and a final probe concentration of 0.6 μmol/L. The method was highly accurate, rapid and sensitive, not only suitable for the detection of pure culture, but also for the detection of S. terrestris in onion tissue samples, providing a new reliable technological method for agroforestry production and customs inspection and quarantine.
