20 February 2024, Volume 54 Issue 1
    

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    REVIEW
  • LI Yanzhong, YANG Bo
    Acta Phytopathologica Sinica. 2024, 54(1): 1-14. https://doi.org/10.13926/j.cnki.apps.001601
    Abstract ( ) Download PDF ( )   Knowledge map   Save
    Alfalfa (Medicago sativa L.) is the most important pasture crop in China in terms of its planting area and status in animal husbandry. Verticillium wilt is a devastating disease worldwide, and China has listed it as an import plant quarantine object. The causal agent was previously known as Verticillium albo-atrum and renamed Verticillium alfalfae in 2011. However, on the list of quarantine diseases for the plants imported to China, the causal organism of alfalfa Verticillium wilt was still designated as V. albo-atrum. This undoubtedly brings much confusion to the quarantine work. In China, the disease was ever found in Zhangye City, Gansu Province, in 2016, and has now been widely distributed in the Hexi Corridor, the most important commercial alfalfa production area in China. It is vital to encourage relevant researchers to pay more attention to this dangerous disease so as to strengthen integrated control measures of the disease. This paper reviews research progress on Verticillium wilt of alfalfa, including the causal pathogen species, distribution, infection cycle and management measures, and suggests future research directions.
  • XU Ming, XU Jing, LIU Huiquan
    Acta Phytopathologica Sinica. 2024, 54(1): 15-25. https://doi.org/10.13926/j.cnki.apps.000870
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    Chitin is an important component of fungal cell wall. It imparts strength and rigidity to fungal cell wall due to its crystalline nature. Chitin plays an important role in infection-related morphogenesis of phytopathogenic fungi, such as the infection structure and the infection hyphae. During the interaction between plants and plant pathogenic fungi, chitinases secreted by plants can degrade chitin to chitin oligomers. As a pathogen-associated molecular pattern (PAMP), chitin oligomer can be recognized by the membrane-located chitin receptors to stimulate plant immune responses. To successfully infect plants, plant pathogenic fungi have evolved a variety of strategies to inhibit the chitin-triggered plant immunity responses. Specifically, plant pathogenic fungi secrete many proteins such as effector proteins, polysaccharides or chitin deacetylases, chitinases and proteases to inhibit the plant defense responses. These proteins are secreted into the apoplast or inside the cell to destroy the plant immune system, which promotes the nutrient acquisition, colonization, growth, and development of the pathogenic fungi. This review summarizes the molecular mechanism of chitin recognition in plants and the main strategies to inhibit chitin-triggered plant immune responses of plant pathogenic fungi. We also discuss future research trends in this field.
  • ETIOLOGY
  • WANG Xuewen, YU Cun
    Acta Phytopathologica Sinica. 2024, 54(1): 26-35. https://doi.org/10.13926/j.cnki.apps.001616
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    To investigate the causal agent of samara brown spot occurred on Acer truncatum Bunge in Guiyang City, Guizhou Province, we collected diseased samara for pathogen isolation. YB26, a representative fungal strain causing the disease through pathogenicity test, was identified as Alternaria alternata based on combined results of morphological characteristics and multigene (rDNA-ITS, Alt a1 and GAPDH) phylogenetic analysis. Then the biological characteristics of A. alternata strain YB26 were determined by testing the mycelial growth rate under different carbon and nitrogen sources, temperatures, pH values, media and light conditions. The results showed that the suitable conditions for vegetative growth of YB26 were mannitol as carbon source, beef extract as nitrogen source, Sabouraud glucose agar (SDA) as culture medium, temperature of 28 ℃, pH value of 7, and incubation under darkness. Furthermore, the inhibitory effects of 7 fungicides against strain YB26 were tested, among which 25% pyrisoxazole had the best antifungal activity, with the EC50 value of 0.8323 mg·L-1. This is the first report of samara brown spot on A. truncatum caused by A. alternata. The results provide a basis for the diagnosis and control of the disease on A. truncatum.
  • CHEN Chen, SHA Pengfei, JIANG Xue, JIANG Shan, YANG Feng, SHEN Yingchun, CHEN Xijun
    Acta Phytopathologica Sinica. 2024, 54(1): 36-48. https://doi.org/10.13926/j.cnki.apps.001024
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    Ligustrum japonicum leaf spot occurs universally in Wenhuilu campus of Yangzhou university, Yangzhou, Jiangsu, China every spring. The aim of this study was to identify the pathogen species, determine the biological characteristics, and screen out effective pesticides for the prevention and control of the disease.The multiple assays were conducted for the pathogen identification including tissue separation and single-spore purification, pathogenicity test both in vitro and vivo to fulfill Koch’s postulates, and morphological and phylogenetic analysis based on a combined ITS, GAPDH, RPB2 and TEF1 sequence dataset, the biological characterization,and the sensitivity test to the five fungicides. We obtained the purified fungal culture which was further identified as Alternaria alternata, a new pathogen causing leaf spot on L. japonicum. The effect of various carbon and nitrogen sources for growing of the representative isolate was investigated and A. alternata isolate was suitable for growing on the optimized medium supplied with sucrose, fructose, and potassium nitrate at 25 ℃, pH 9.0. In addition, 5 fungicides were assessed on the inhibitory effect against mycelial growth of A. alternata isolate in which azoxystrobin displayed the strongest inhibitory activity with EC50 value of 0.080 6 μg·mL-1, followed byprochloraz, pyraclostrobin and tebuconazole with EC50 values of 2.272 2 μg·mL-1, 3.934 9 μg·mL-1 and 6.400 0 μg·mL-1, respectively, while difenoconazole exhibited the least sensitivity at EC50 value of 15.486 0 μg·mL-1. These results indicated that these fungicides could be used for the prevention and control of L. japonicum leaf spot disease.
  • WANG Fei, YANG Jin, LI Xuemeng, ZHAO Ying, LIU Yuxia, QIN Yanhong, GAO Suxia, WEN Yi, LU Chuantao
    Acta Phytopathologica Sinica. 2024, 54(1): 49-58. https://doi.org/10.13926/j.cnki.apps.001602
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    The diseased plants of Achyranthes bidentata showing typical symptoms of Fusarium wilt were collected from Jiaozuo, Henan Province, China. Tissue separation method was used to obtain potential pathogenic fungal isolates, and the pathogenicity of these isolates was determined by root-dipping inoculation method. Fusarium proliferatum was identified as the causal agent of the disease based on morphological characteristics, ITS sequence and polygenic analysis (EF1-α, Tub, RPB2 and PRO1/2). F. proliferatum isolates showed maximum radial growth at 28 ℃ and pH 7.0 on oatmeal agar medium under dark conditions. The pathogen could utilize multiple carbon and nitrogen sources, with the best carbon and nitrogen sources of sucrose and peptone, respectively. To our knowledge, this is the first report of F. proliferatum causing Fusarium wilt on A. bidentata. The results provide a scientific basis for diagnosis and control of Fusarium wilt of A. bidentata. Toxicity test of the six fungicides on F. proliferatum showed that these fungicides had certain inhibitory effects on the pathogen, and tebuconazole and fludioxonil exhibited relative higher inhibitory effects, with EC50 values of 3.03 and 2.36 mg·L-1, respectively.
  • TANG Yafei, LI Zhenggang, SHE Xiaoman, YU Lin, LAN Guobing, DING Shanwen, HE Zifu
    Acta Phytopathologica Sinica. 2024, 54(1): 59-68. https://doi.org/10.13926/j.cnki.apps.001322
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    Three Broussonetia papyifera samples suspected to be infected by Begomovirus, with yellow mosaic leaves, were collected from Lianping county, Heyuan city, Guangdong province. Total DNA was extracted from suspected samples individually, and was used as template for PCR detection with degenerate Begomovirus pri-mers AV494/CoPR. The PCR detection result showed that three suspected samples were infected by Begomovirus. The full genome sequence of virus isolated from Broussonetia papyifera in Guangdong (GS-2021) was obtained by RCA amplification, followed by enzyme digestion, cloning and sequencing. GS-2021 was a bipartite virus, including DNA-A and DNA-B components. The full sequence of DNA-A (GS-2021-A) was 2 777 nt in size, and encoded seven ORFs. The full sequence of DNA-B (GS-2021-B) was 2 742 nt in size, and encoded two ORFs. GS-2021 shared the higher similarity with all isolates of clerodendrum golden mosaic China virus (ClGMCNV). GS-2021-A shared a 93.0%-93.9% identity with DNA-A of all isolates of ClGMCNV, and the highest identity (93.9%) is with the Fujian Fz7 isolate (GenBank accession number: FJ011668). GS-2021-B shared an 86.3%-89.6% identity with DNA-B of all isolates of ClGMCNV, and the highest identify (89.6%) is with the Fujian Fz7 isolate (GenBank accession number: FJ011669). GS-2021 was closely related to five isolates of ClGMCNV from Fujian, Zhejiang, Jiangsu and the United States, which belonged to the same clade. In addition, GS-2021 clustered with Fz7 isolate from Fujian in a small clade, and had the closest relationship with it. Recombination analysis showed that there was no obvious gene recombination event in GS-2021. Based on the latest demarcation threshold for Begomovirus, GS-2021 was a new strain of ClGMCNV. In this study, Begomovirus was detected on Broussonetia papyifera for the first time. The full viral genome sequence of this virus was obtained and identified as a new strain of ClGMCNV. This result shows that Broussonetia papyifera is a newly discovered natural host for Begomovirus.
  • DU Jiang, MA Zhennan, WANG Chenyan, CUI Liyan, WANG Defu, NIU Yanbing
    Acta Phytopathologica Sinica. 2024, 54(1): 69-81. https://doi.org/10.13926/j.cnki.apps.001317
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    Citrullus lanatus is an important horticultural plant. Viral diseases on C. lanatus have become more and more serious in recent years. In order to identify the viruses that infect watermelons, the small RNA deep sequencing was used to analyze samples with mosaic and shrinking symptoms from Taigu District, Shanxi Province. RT-PCR and bioinformatics methods were used to analyze the pathogens. The results showed that the watermelon samples exhibiting mosaic and shrinking symptoms were infected by five viruses including cucurbit aphid-borne yellows virus (CABYV), cucurbit melo cryptic virus (CmCV), watermelon virus A (WVA), watermelon crinkle leaf-associated virus 2 (WCLaV2) and cucumber green mottle mosaic virus (CGMMV). The coat protein (CP) sequences of five viruses were amplified by RT-PCR, and further analyzed by sequence identity analysis and phylogenetic analysis. It was found that the nucleotide sequence of CABYV-SXJZ (GenBank No : OP957280) obtained in this study had the highest identity with that of CABYV-Inner Mongolia (GenBank No : EU262627), reaching 100%. The nucleotide sequence of watermelon WVA isolate WVA-SXJZ (GenBank No : OP957281) has the highest identity with that of watermelon WVA isolate WVA-Huizhou (GenBank No : MK292710) and watermelon WVA isolate WVA-KF15 (GenBank No : KY363796), which are also from China, reaching 93.6% and 99.9%, respectively. The nucleotide sequence of WCLaV2 isolate WCLaV2-SXJZ (GenBank No : OP957282) has the highest identity with that of WCLaV2 Brazilian watermelon isolate Ju-01 (GenBank No : LC636075), reaching 99.6%. The nucleotide sequence of CmCV isolate CmCV-SXJZ (GenBank No : OP957283) obtained from watermelon for the first time in this study has 99.9% identity to that of Chinese melon isolate CmCV-HLJ (GenBank No : MH479773). The nucleotide sequence of CGMMV isolate CGMMV-SXJZ (GenBank No : OP957284) obtained in this study has the highest identity with CGMMV isolate GDLZ (GenBank No : MK933286), CG038 (GenBank No : MH271443), CGMMV-pXT1 (GenBank No : KY753929), eWT (GenBank No : KY753928), C284R (GenBank No : KY753927), CGMMV-XG (GenBank No : KP868654), JD2 (GenBank No : KM873785) and Anhui (GenBank No: KT236095), reaching 99.8%.
  • CELL BIOLOGY, PHYSIOLOGY, BIOCHEMISTRY, AND MOLECULAR BIOLOGY
  • TENG Hui, YANG Jishuo, LI Siyu, ZHOU Dongmei, WANG Nan, SUN ShuYan, LIU Huaqi, WEI Lihui, WANG Yunpeng, DENG Sheng
    Acta Phytopathologica Sinica. 2024, 54(1): 82-91. https://doi.org/10.13926/j.cnki.apps.001023
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    Lotus is the most important aquatic vegetable in China and has the largest cultivation area. With the expansion of cultivation and the simplification of agricultural practices, various lotus diseases have been becoming more serious, among which lotus rhizome rot disease is the most harmful and the most difficult to control. At present, there are few research reports on the pathogenesis of Fusarium spp. causing lotus rhizome rot disease around the world. From the microscopic and ultra-microscopic levels, this study revealed the changes to the rhizome and its vascular tissues during Fusarium infection. The observation of paraffin-embedded tissue sections showed that with the development of the disease, necrotic cells stained by toluidine blue appeared around the vascular bundles, and the closer to the bundles, the more numerous necrotic cells and the more densely distributed. In order to further reveal the influence of pathogen infection on vascular bundles, brightfield optical and scanning electron microscopy were used to observe the lotus fibers in the diseased lotus rhizomes. The results showed that the smooth and compact lotus fibers became rough and dispersed with the disease tending to be more severe, and a large number of galls-like protrusions and other possible metabolic attachments were attached to the surface. These changes to the vascular bundles are likely to reduce the water and nutrient transportation, and finally the aboveground parts of the plant exhibited the symptoms of water loss and wilting. The results of this study revealed the cellular biological mechanism of the disease symptom development, and provided a basis of histology for the breeding of resistant varieties in the future.
  • LI Yueyue, ZHANG Shuiying, ZHOU Wenpeng, ZHANG Tianyi, LAN Pingxiu, TAN Guanlin, LI Fan
    Acta Phytopathologica Sinica. 2024, 54(1): 92-101. https://doi.org/10.13926/j.cnki.apps.001324
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    Tomato mottle mosaic virus (ToMMV), a member of the genus Tobamovirus, showed stronger pathogenicity than tobacco mosaic virus (TMV) and tomato mosaic virus (ToMV) in most solanaceae crops. To figure out the cytopathological changes of tobacco plants that were infected by ToMMV, we constructed a full-length infectious cDNA clone of ToMMV, and carried out transmission electron microscopy observation. The results showed that the rod-shaped virions of ToMMV were 220-310 nm in length and 16-18 nm in diameter. ToMMV virions were also observed in the seeds of ToMMV-infected Nicotiana tabacum var. Xanthi nc. ToMMV virions were dispersed or angularly-layered in cytoplasm or vacuoles. Multivesicular bodies, small vesicles, myeloid bodies, mitochondrial cristae were observed to be reduced or disappeared in both N. tabacum var. Xanthi nc and N. rustica infected with ToMMV. In addition, broken chloroplast membranes and large amount of small peripheral vesicles protruding into the disintegrated chloroplasts were observed in the infected N. tabacum var. Xanthi nc showing mosaic and distortion symptoms, while large amount of small vesicles were observed in the cells of infected N. tabacum var. Xanthi nc showing leaf narrowing. However, chloroplasts changes were less frequent in N. rustica with systemic necrosis symptoms. In this study, the construction of ToMMV infectious clone and the observation of cytopathological changes in tobacco plants infected with ToMMV, provided important bases for the further study of the pathogenesis of ToMMV and its interaction with the host.
  • XING Qikai, WANG Xinfang, PENG Junbo, ZHANG Wei, YAN Jiye, LI Yonghua
    Acta Phytopathologica Sinica. 2024, 54(1): 102-115. https://doi.org/10.13926/j.cnki.apps.001625
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    Lasiodiplodia theobromae is an important plant pathogenic fungus, infecting more than 500 species of woody plants and causing serious damages worldwide. The secreted proteins of pathogenic fungi play important roles in pathogen invasion, expansion, and colonization in plants. Although there are more reports on the involvement of classical secreted proteins in fungal pathogenicity, the roles of non-classical secreted proteins in the pathogenesis of plant pathogenic fungi have rarely been investigated. In this study, 238 candidate non-classical secreted protein-coding genes were obtained by bioinformatic prediction based on the whole genome sequence of L. theobromae. Gene functional prediction and GO enrichment analysis showed that these genes are enriched in the carbon-sulfur lyase activity pathway. The annotation results based on PHI-base showed that 15 non-classical protein-coding genes might be related to the pathogenicity of L. theobromae. Predication of subcellular location using ApoplastP showed that 4 of them were located in plant apoplast and 11 were located in plant proplastids. Reverse transcription quantitative PCR (RT-qPCR) analysis results further verified that the transcriptional expression levels of the above candidate non-classical secreted protein-coding genes were significantly changed after treatment with grapevine branch tissues, indicating that these non-classical secreted proteins may play important roles in grapevine-L. theobromae interaction. The results lay a foundation for further understanding of the pathogenic mechanism of non-classical secreted proteins of L. theobromae.
  • JIANG Jia, TIAN Mengjun, NIE Tingbin, YANG Zhihui, ZHU Jiehua
    Acta Phytopathologica Sinica. 2024, 54(1): 116-126. https://doi.org/10.13926/j.cnki.apps.001631
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    The potato early blight caused by Alternaria solani is an important disease in potato production, which seriously affects the yield and quality of potatoes. To reveal the interaction mechanism between A. solani and its host plant potato, we screened the effector candidates produced by A. solani during its interaction with potato plant using RNA transcriptome sequencing technology and biological software (signalp-4.1, tmhmm-2.0c, phobius101 and ProtComp v3) combined with characteristics analysis of effector proteins. As a result, 137 differentially expressed candidate proteins were obtained, and four (Gene03396, Gene06670, Gene02858 and Gene06508)of them were selected for further analysis. Based on the result of RNA-seq analysis, Gene03396, Gene06670 and Gene02858 had an up-regulated expression pattern at three time points (3, 4 and 5 dpi) compared with that at 0 dpi, whereas Gene06508 had a downregulated expression pattern. The four proteins do not have a common motif according to the MEME analysis result. Transient overexpression of the target effector candidate genes in tobacco showed that Gene03396 and Gene02858 directly induced tobacco cell necrosis, and Gene06670 inhibited programmed cell death (PCD) induced by BCL2-associated X, indicating that effector candidates function by stimulating plant immunity or inhibiting host defense response. The subcellular localization results revealed that Gene03396 was located on the cell membrane, while Gene06670 and Gene02858 may be located in the intercellular space. These results provide a basis for further identifying the effector proteins of A. solani and revealing the underlying pathogenic mechanism of the pathogen.
  • GENETICS OF DISEASERESISTANCE AND PATHOGENICITY
  • ZHANG Ce, GUO Qingchen, PANG Huihui, ZHANG Siyuan, YIN Wen, MA Rui, YU Dan, ZHAO Jing
    Acta Phytopathologica Sinica. 2024, 54(1): 127-136. https://doi.org/10.13926/j.cnki.apps.000868
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    Poplar leaf rust is serious harm to the tree health and planting resistance cultivar is an effective way to control the disease. The breakdown of resistance owing to the variation of pathogen, it is urgent for exploring new resistance resources. Plant nonhost resistance, characterized by broad-spectrum and durability, provides a new resolution for resistance breeding. In the present study, the histology observation and the expression pattern of defense-related genes of wheat leaves inoculated with Melampsora larici-populina (Mlp), the casual agent of popular leaf rust, were analyzed. The results demonstrated that although Mlp urediniospores normally germinated on wheat leaves, only 3.7% recognized the stomata, and a very few of them were able to develop sub-stomatal vesicle-like (SSVL) structure and infection hypha. The immediately induction of H2O2 and callose at the infection site arrested the further infection of the rust fungi. In addition, no cell death was observed, suggested that the defense reaction of wheat against Mlp belongs to type I nonhost resistance. Two genes involved in jasmonic acid synthesis, LOX1 and AOS1, as well as PR1a, the downstream pathogenesis related gene of JA pathway, were all induced by Mlp infection, highlighted an important role of JA pathway in wheat nonhost resistance to Mlp. This study provided groundwork for further elucidation of the molecular mechanism of wheat nonhost resis-tance to poplar leaf rust.
  • GOU Jinyu, SUN Dan, SHANG Jiawei, TONG Zhaoyang, XU Xiaowei, WANG Fengtao, FENG Jing, HOU Lu, LIN Ruiming
    Acta Phytopathologica Sinica. 2024, 54(1): 137-146. https://doi.org/10.13926/j.cnki.apps.000869
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    Barley spot blotch, caused by the facultative B. sorokiniana, is an important foliar disease on barley, being widely prevalent in most barley-growing regions in the world. It is most destructive especially in the regions with warm and moist climates, consequently bringing about serious yield losses.Presently, spot blotch is the first important fungal epidemic disease in the spring barley-growing regions in northeastern China.The disease usually occurs in mid- and late-growth stages of barley. Growing resistant varietiesis, therefore,the most cost-effective measure for the disease control. In this study, two highly virulent dominant B.sorokiniana isolates Z12028 and Z15525 derived from northeastern China were selected to identify spot blotch resistance in barley germplasm accessions at the seedling and adult plant stages. Only one accession 2013F61903 was found to be highly resistant to Z12028 at the seedling stage, and no immune one accession to B. sorokiniana was detected. Nine accessions, such as ZDM00009,ZDM00013,ZDM00094,ZDM08888,ZDM01414,ND14049,ND B112,Newdale, and kenpimai 9 among the tested germplasm accessions, were found to be of allstage resistance to the both highly virulent isolates; fourteen accessions (4.3%)like ZDM00074,Bowman and Stander were resistant only at the seedling stage but susceptible at the adult plant stage, and another 72 accessions(28.3%) of adult plant resistance to spot blotch. Barley accessions of adult plant resistance accounted for 28.1% and 29.5% to Z12028 and Z15525, respectively. As a result, the percentage of adult plant resistant accessions to spot blotch was significantly higher than that of those with all stage resistance. The results of this study provided valuable resistant resources for exploiting new spot blotch resistance genes.
  • PLANT DISEASE AND CONTROL
  • WANG Yi, WANG Yaoyao, ZHANG Mingxin, PENG Na, WANG Xiu, GAO Weiwei
    Acta Phytopathologica Sinica. 2024, 54(1): 147-159. https://doi.org/10.13926/j.cnki.apps.000864
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    The root rot caused by Fusarium and Ilyonectria is the most serious disease in American ginseng. The prevention of this disease mainly relies on the utilization of fungicides. However, it is unknown whether it could be controlled by calcium fertilizer. In order to reduce the use of pesticides and find possible physiological and ecological mechanisms of calcium in root rot disease control, a pot experiment was conducted to investigate the effects of calcium application and inoculation. During the cultivation of ginseng, different amounts of calcium oxide (0、0.5、1.5、4.5 g·kg-1 ) was applied to the soil. The root biomass, disease incidence, root nutrient content, soil physicochemical properties and soil microbial community were measured. The results showed that low calcium additions increased root rot disease index, however, high calcium additions decreased root rot disease index. Compared to the control, adding 4.5 g·kg-1 calcium oxide decreased root rot disease index of I. mors-panacis by 87.69% (P < 0.05), meanwhile, the root rot disease index of F. solani was reduced by 66.67% (P>0.05). The calcium content in American ginseng roots from the two inoculation groups were increased by 101.28% and 61.50% (P<0.05), respectively. Nitrogen and phosphorus content in the roots were positively correlated with the root rot disease index of F. solani. On the other hand, the structure and composition of soil microbial communities inoculated with two pathogens differed significantly. The calcium treatment mainly affected the diversity and composition of fungal community, while having little effect on the bacterial commun-ity. Additionally, treatment with 4.5 g·kg-1 calcium oxide inhibited the relative abundance of Didymella and Phoma. Overall, the potential mechanism for the reduction of root rot disease in American ginseng by more than 65% through the application of high-dose calcium is related to increased calcium content in roots, reduced nitrogen and phosphorus content in roots, and decreased the relative abundance of potential pathogens in soil. This study provides a theoretical basis for reducing root rot disease of American ginseng through rational application of calcium fertilizer.
  • ZHANG Xiaoyan, XIE Zhao, WANG Xiuhua, LU Baohui, GAO Jie
    Acta Phytopathologica Sinica. 2024, 54(1): 160-169. https://doi.org/10.13926/j.cnki.apps.001025
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    In order to obtain high-quality and sufficient protoplasts of Botrytis cinerea that can be used for genetic transformation, effects of multiple parameters including mycelial age, combinations of lytic enzymes, types of osmotic stabilizers, enzymatic hydrolysis temperature, and time of enzyme digestion on protoplasts preparation were studied. The optimal lytic enzyme was determined to be the combination of driselase, snailase, and lysing enzyme at an active ingredient of 1%, 0.1% and 1%, respectively, and the mycelial age, composition and concentration of osmotic stabilizer, enzyme digestion temperature, and enzyme digestion time were as follows: mycelia of JA-6 was cultivated on PDA at 25 °C for 36 h, osmotic stabilizer contained 0.6 mol·L-1 KCl and 50 mmol·L-1 CaCl2, and enzyme digestion time was 3 h at 120 r·min-1 at 28 °C. Enzymatic hydrolysis of 5 g·mL-1 of B. cinerea mycelium can yield 1.06×107 protoplasts·mL-1 under above optimal protoplast preparation conditions. No significant differences of the colony morphology, growth rate, conidial production and pathogenicity were observed between the regenerated strain and the wild-type strain. The GFP (green fluorescent protein) gene was subsequently transformed into B. cinerea JA-6 by PEG mediated transformation. The fluorescence signal of the transformants can be stably inherited. The established protoplast preparation method in the present research would meet the requirements of genetic transformation of B. cinerea for further study.
  • LI Xinlei, ZHU Mengkun, LI Wei, CAO Shulin, DENG Qingchao, FANG Shouguo, CHEN Huaigu, SUN Haiyan
    Acta Phytopathologica Sinica. 2024, 54(1): 170-179. https://doi.org/10.13926/j.cnki.apps.000865
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    In the researches of chemical control of plant diseases, the mechanism of action of fungicides and pathogen resistance to fungicides are the focus of attention, while the mechanisms of how pathogenic bacteria response to mitigate chemical stress is often ignored. The transcriptome sequencing technology was used to study the stress response mechanism of Fusarium graminearum to succinate dehydrogenase inhibitor (SDHIs) fungicides in this study. The results showed that 1 180 up-regulated and 937 down-regulated differentially expressed genes were identified in the pathogen treated with pydiflumetofen and benzovindiflupyr compared to the control. Complex II on mitochondrial electron transfer chain is the target of SDHIs fungicides. Therefore, the genes rela-ted to mitochondrial electron transfer chain were focused in this study. The results showed that F. graminearum responded to the stress of SDHIs fungicides by upregulating the expression of genes such as Succinate dehydrogenase, Ubiquinol-cytochrome C reductase, Fumarylacetoacetate hydrolase (FAH), ABC and MFS transpor-ters, and downregulating the expression of genes in the SWI / SNF complex and other genes. FgFAH gene was induced to express after being treated with SDHIs fungicides. The sensitivity of FgFAH gene-knockout mutant to SDHIs fungicide was increased compared to wild type, indicating that this gene may mediated the stress response of F. graminearum to SDHIs fungicide. The results of this study will provide a research basis for studying stress response mechanism of pathogen to chemical agents and research ideas for developing sensitizers of fungicides in the future.
  • WANG Shuang, ZHAO Fumei, TIAN Yuting, QIAO Qi, ZHANG Desheng, WANG Yongjiang, ZHANG Zhenchen
    Acta Phytopathologica Sinica. 2024, 54(1): 180-187. https://doi.org/10.13926/j.cnki.apps.001314
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    Virus disease is a main constraint that influences sweet potato yield and quality. The most important viruses for sweet potato are the sweet potato chlorotic stunt virus (SPCSV) of the genus Crinivirus and sweet potato feathery mottle virus (SPFMV) of the genus Potyvirus. SPCSV-infected sweet potato storage roots incline to develop severe viral disease at the seedling stage. However, the effect of whitefly (Bemisia tabaci) in sweet potato fields on the proportion of SPCSV-viruliferous storage roots and viral disease occurrence remains largely unknown. Here, we report that the amount of whitefly and the rate of SPCSV-viruliferous whiteflies in sweet potato fields were closely related to the viruliferous rate of storage roots. When there was a high number of viruliferous whiteflies in sweet potato fields, a high rate of viruliferous storage roots were triggered, even though virus-free sweet potato cuttings that would not induce severe symptoms in above ground plants had been planted. The SPCSV infection rate and double infection of storage roots with SPCSV and SPFMV presented significant positive correlations with the virus-like symptom rate in sprouts generated from the storage roots. The symptom rate in sprouts can be predicted by the viruliferous rate of storage roots.
  • DU Mengyuan, LIU Di, CHEN Li, HOU Yanhong, CHEN Qi, FAN Zhiye, LI Leilei, SHEN Hailong, WANG Wenhao, SHI Xingkai, LI Shimin
    Acta Phytopathologica Sinica. 2024, 54(1): 188-194. https://doi.org/10.13926/j.cnki.apps.001315
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    In order to clarify the variation of virus content of wheat yellow mosaic virus (WYMV) in various disease grade and tissue parts, wheat samples with typical incidence levels of 0, 1, 2 and 3 were collected from fields with uniform incidence of wheat yellow mosaic disease. Healthy wheat samples from virus-free fields were used as blank control. The absolute quantification of the content of wheat yellow mosaic virus in roots, stems and leaves was carried out by real-time fluorescent quantitative PCR (RT-qPCR). The results showed that the content of WYMV in wheat roots was significantly higher than that in leaves (P<0.05), and the virus content in wheat roots of grade 0 was 13.97 times higher than that in leaves. In wheat tissues of different disease grade, the virus content in roots, stems and leaves increased significantly with the aggravation of disease grade (P<0.05). The virus content in roots increased rapidly and then slowed down. From grade 0 to grade 1, the virus content in roots increased by 3.95×106 copies·μL-1L. However, from grade 1 to grade 3, the virus increase was only 1.45×106copies·μL-1. The virus content in stems and leaves showed a trend of uniform increase, and there was a strong positive correlation between the virus content in stems and the disease grade (R2=0.996 7). This study confirmed that the root is the key tissue site of WYMV virus infection in wheat. At the same time, it was found that the content of WYMV in the roots of diseased grade 0 wheat was much higher than that in the control group. The content of WYMV in the stem correlates closely with viral symptoms. The higher the content of stem virus, the more obvious the symptom of wheat.
  • RESEARCH NOTES
  • LI Xin, XIE Xuewen, ZHANG Zhuqing, SHI Yanxia, LI Lei, FAN Tengfei, LI Baoju, CHAI Ali
    Acta Phytopathologica Sinica. 2024, 54(1): 195-198. https://doi.org/10.13926/j.cnki.apps.001605
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    Pepper (Capsicum annuum L.), belonging to the Solanaceae family, is an economically important vegetable crop in China. In April 2021, severe downy mildew-like symptoms were observed on pepper in Ningxiang County, Changsha City, Hunan Province. The diseased pepper plants exhibited large, interveinal chlorotic lesions on the adaxial surface of the leaves, which coalesced and turned necrotic as disease progressed. Meanwhile, white to gray mycelia could be seen on the abaxial side. Based on morphological characteristics, rDNA-ITS and cox2 sequence analysis, and the result of pathogenicity test, the pathogen was identified as Peronospora capsici. This is the first report of downy mildew caused by P. capsici on pepper in Hunan Province, China, and the sequence information of P. capsici was for the first time deposited in the NCBI nucleotide database.
  • CAI Gaolei, ZHOU Yang, ZHANG Zezhi, SONG Wei, ZHANG Han, SUN Liangxue, KE Zunwei, LIU Xingang
    Acta Phytopathologica Sinica. 2024, 54(1): 199-203. https://doi.org/10.13926/j.cnki.apps.000877
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    In May of 2021, a leaf spot disease of the herbal medicine Dysosma versipellis was observed,and it occured seriously in Shiyan of Hubei Province. Diseased leaves of D. versipellis were collected, pathogen was isolated by tissue cultural-method, and its pathogenicity was confirmed according to the Koch's postulates. Based on the morphological characteristics and phylogenetic analysis by combination of ITS region and EF1-α gene sequences, pathogen was identified as Alternaria alternata. This is the first report leaf spot of Dysosma versipellis caused by A. alternata in China.
  • SONG Wanduo, YAN Liying, YU Dongyang, KANG Yanping, LEI Yong, CHEN Yuning, HUAI Dongxin, WANG Xin, WANG Zhihui, LUO Huaiyong, JIANG Huifang, LIAO Boshou
    Acta Phytopathologica Sinica. 2024, 54(1): 204-208. https://doi.org/10.13926/j.cnki.apps.001028
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    In order to improve the efficiency and reliability of bacterial wilt resistance screening in peanut, a novel water culture inoculation system was developed. Resistant peanut variety ‘Zhonghua21’ and susceptible ‘Zhonghua12’ were inoculated with mixed suspension of three Ralstonia solanacearum isolates under (28 ± 2) ℃, and 16 h light/8 h dark conditions. The minimum concentration for distinguishing the resistant and susceptible peanut lines was 1×107 CFU·mL-1. The optimal time for scoring the resistance was 12 d after inoculation. 36 peanut accessions were inoculated by the mixed bacterial suspension in water culture, in which 25 ones were resistant and 11 ones were susceptible. The same set of peanut accessions was also tested for their resistance to bacterial wilt in natural disease nursery, from which 27 ones were resistant and nine were susceptible. The consistency of these accessions for resistance to bacterial wilt both in water culture and in natural disease nursery was more than 80%. Therefore, the established method was quick, simple and high throughput for identifying resistance to bacterial wilt in peanut.
  • WANG Zhiyuan, FENG Ruying, LOU Yinian, LIANG Kaili, LIU Jingying, XU Xiongbiao
    Acta Phytopathologica Sinica. 2024, 54(1): 209-214. https://doi.org/10.13926/j.cnki.apps.001313
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    Small RNA (sRNA) triggered RNA silencing plays significant roles in viral resistance response by plants. Analysis of virus-derived sRNA profiles in plants is an efficient method for virus identification and de novo assembly of viral genomes.In this study,suspected virus-infected Hibiscus tiliaceus samples collected from Nanning were used for sRNA library construction and subsequent deep sequencing.After the assembly of total sRNAs,H. tiliaceus leaves were found to be infected by hibiscus chlorotic mottle virus (HCMV).The library gene-rated about 11.77 million sRNA reads,of which 15 093 can be mapped onto viral genomes.Using de novo assembly and GenBank Virus RefSeq database blast,a candidate virus covers 18.8% of the full length genome nucleotide sequence of a badnavirus, which has the highest similarity to hibiscus bacilliform virus isolate GD-1 (HBV-GD1).To confirm the existence of HCMV in the samples,a fragment of about 1 400 nucleotides encoding Reverse transcriptase /RNase H was obtained by PCR,and confirmed by Sanger sequencing.This is the first report about using sRNA deep sequencing technology to identify the Hibiscus tiliaceus infecting virus, and this provides important information for further study on the molecular characteristics of HCMV and virus-host interactions.
  • QIU Yanhong, ZHANG Haijun, WANG Dexin, WANG Hongyang, WEN Changlong, XU Xiulan
    Acta Phytopathologica Sinica. 2024, 54(1): 215-219. https://doi.org/10.13926/j.cnki.apps.001318
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    During a survey in 2022, cucumber displayed yellowing and chlorotic symptoms on leaves in Qingzhou and Shouguang, Shandong Province. In order to investigate the virus species, eight samples were collec-ted, and total RNA was extracted from each sample. Three high quality RNA samples were mixed together for the next-generation sequencing (NGS). The NGS clean data were assembled into large contigs and then compared with the GenBank Virus RefSeq database using BLASTn software. The BLAST results were verified with reverse transcription-polymerase chain reaction (RT-PCR), confirming that the cucumber samples were infected by five species of virus, including watermelon silver mottle virus, cucumber mosaic virus, cucurbit aphid-borne yellows virus, zucchini yellow mosaic virus and watermelon mosaic virus. After cloning the full-length of S RNA (GenBank No. OQ184866), we found that the N gene of WSMoV was 828 nt in length,encoding 275 aa, sharing 92%-98% sequence identity with other WSMoV isolates. Further phylogenetic analysis showed that the N gene formed an independent cluster and was mostly related to the cucumber isolate. This is the first report that watermelon silver mottle virus infects cucumber in Shangdong Province.
  • CHEN Yahan, WU Chonggao, FANG Jianhui, ZHU Yaling, XU Honggang, CHENG Xiaoyue, OUYANG Jingzheng
    Acta Phytopathologica Sinica. 2024, 54(1): 220-224. https://doi.org/10.13926/j.cnki.apps.000904
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    To clarify the taxonomic status of the phytoplasma causing willow witches’-broom in Gansu Pro-vince, this study used nested PCR technique to clone the 16S rDNA gene using total DNA collected from different regions and species of willow witches’-broom samples as templates, and nine 1 250 bp target fragments were obtained using P1/P7 and R16F2n/R16R2; the sequences of the above genes were compared by homology, The results showed that the 16S rDNA gene of willow witches’-broom phytoplasma strain had the highest homology with Willow proliferation phytoplasma (Willow proliferation) in Inner Mongolia, both above 99.1%; the phylogenetic tree was constructed and found that the 16S rDNA gene of willow witches’-broom clustered with members of 16SrVI-A subgroup. The phylogenetic tree was constructed, and the 16S rDNA gene of willow witches’-broom clustered with members of the 16SrVI-A subgroup; and the virtual RFLP analysis reported that the highest sequence similarity with the one obtained in this study was the reference pattern of the 16SrVI-A subgroup, with a similarity coefficient around 0.98, so the willow witches’-broom phytoplasma in Gansu Province could be classified into the 16SrVI-A subgroup. Meanwhile, the phytoplasma resources were preserved by grafting and identified as vector insects based on amplification of the 16S rDNA gene in the willow shoulder reticulata.