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    植物病理合辑Plant Protection—Plant Pathology

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    Mapping subgenomic promoter of coat protein gene of Cucumber green mottle mosaic virus
    LIU Mei, LIU Li-ming, WU Hui-jie, KANG Bao-shan, GU Qin-sheng
    2020, 19 (1): 153-163.   DOI: 10.1016/S2095-3119(19)62647-X
    Abstract90)      PDF in ScienceDirect      
    Many plant viruses utilize subgenomic RNA as gene expression strategy, therefore mapping subgenomic promoter (SGP) is extremely important for constructing viral vectors.  Although Cucumber green mottle mosaic virus (CGMMV)-based virus vectors have been constructed, SGP of the coat protein (CP) has not yet mapped.  To this end, we firstly presumed 13 nucleotides upstream of the start codon as the transcription starting site (TSS) as previous study identified by random amplification of cDNA ends (RACE).  Secondly, the region from nucleotides –110 to +175 is the putative CP SGP, as predicted, a long stem loop structure by the secondary structure of RNA covering movement protein (MP) and CP.  To map the CGMMV CP SGP, we further constructed a series of deletion mutants according to RNA secondary structure prediction.  The deletion of TSS upstream significantly enhanced CP transcription when 105 nucleotides were retained before the CP TSS.  For the downstream of CP TSS, we analyzed the expression of enhanced green fluorescent protein (EGFP) in a series of vectors with partial deletion of the CGMMV CP and found that the nucleotides from +71 to +91 played a key role in the EGFP expression at the transcription level, while EGFP showed the highest expression level when 160 nucleotides were retained downstream of the CP TSS.  To confirm these results, we applied online software MEME to predict the motifs and cis-acting elements in the 466 nucleotides covering the sequences of deletion analysis.  Conserved motifs and relative acting elements were in regions in which transcription levels were the highest or enhanced.  To our best knowledge, this is the first mapping of CGMMV SGP.
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    Evaluation of soil flame disinfestation (SFD) for controlling weeds, nematodes and fungi
    WANG Xiao-ning, CAO Ao-cheng, YAN Dong-dong, WANG Qian, HUANG Bin, ZHU Jia-hong, WANG Qiu-xia, LI Yuan, OUYANG Can-bin, GUO Mei-xia, WANG Qian
    2020, 19 (1): 164-172.   DOI: 10.1016/S2095-3119(19)62809-1
    Abstract88)      PDF in ScienceDirect      
    Soil flame disinfestation (SFD) is a form of physical disinfestation that can be used both in greenhouses and on field crops.  Its use for soil disinfestation in different crop growing conditions makes it increasingly attractive for controlling soil-borne pathogens and weeds.  But little is known about the effect on weeds and soilbrone diseases.  This study reports on greenhouses and field crops in China that determined the efficacy of SFD to control weeds, nematodes and fungi.  It also determined the impact of SFD on the soil physical and chemical properties (water content, bulk density, NO3-N content, NH4+-N content, conductivity and organic matter) in three field trials.  A second generation SFD machine was used in these trials.  SFD treatment significantly reduced weeds (>87.8%) and root-knot nematodes (Meloidogyne incognita) (>98.1%).  Plant height and crop yield was significantly increased with SFD treatment.  NO3-N and NH4+-N increased after the SFD treatment, and there was also an increase in soil conductivity.  Water content, bulk density and organic matter decreased significantly in the soil after the SFD treatment compared to the control.  Soil flame disinfestation is a potential technique for controlling weeds and diseases in greenhouses or in fields.  SFD is a non-chemical, safe, environmentally-friendly soil disinfection method. 
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    Molecular and biological characterization of melon-infecting squash leaf curl China virus in China 
    WU Hui-jie, LI Meng, HONG Ni, PENG Bin, GU Qin-sheng
    2020, 19 (2): 570-577.   DOI: 10.1016/S2095-3119(19)62642-0
    Abstract107)      PDF in ScienceDirect      
    It has been reported that squash leaf curl China virus (SLCCNV) infects some Cucurbitaceae crops except for melon (Cucumis melo L.).  A new disease of melon exhibiting severe leaf curl and dwarfing was observed in Hainan Province of China.  In this study, the pathogen was identified as SLCCNV through biological and molecular characterization.  The isolate (SLCCNV-HN) possess a bipartite genome, DNA-A (HM566112.1) with the highest nucleotide identity (99%) to SLCCNV-Hn (MF062251.1) pumpkin and SLCCNV-Hn61 (AM260205.1) squash isolates from China, whereas DNA-B (HM566113.1) with the highest nucleotide identity (99%) to SLCCNV-Hn (MF062252.1).  Phylogenetic analyses based on the full-length SLCCNV-HN DNA-A and -B sequences indicated that SLCCNV-HN melon isolate is clustered with SLCCNV-Hn pumpkin, SLCCNV-Hn61 and SLCCNV-SY squash isolates from southern China, forming an independent cluster.  Infectious clone of SLCCNV-HN was constructed and the melon plants were inoculated and the infection rate is 100%, the systemic symptoms in melon showed identical to those of melon plants infected in fields.  Additionally, melon plants transmission of this virus by Bemisia tabaci with a transmission rate of 95% (19/20) showed leaf curl and dwarf symptoms 15 days post transmission, thereby fulfilling Koch’s postulates.  Analysis of genomic organization and phylogenetic trees indicated that SLCCNV-HN melon isolate belongs to the Begomovirus genus.  To the best of our knowledge, this is the first characterization of melon-infecting SLCCNV through its genome, infectious clone and transmission.
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    Functional analysis and screening small molecules to RpfF protein in Xanthomonas oryzae involved in rice bacterial blight disease
    Mundla SRILATHA, Naina PATYAL, Madhu Sudhana SADDALA
    2020, 19 (3): 735-747.   DOI: 10.1016/S2095-3119(19)62813-3
    Abstract91)      PDF in ScienceDirect      
    Xanthomonas oryzae pv. oryzae (Xoo) is an important rice pathogen.  This is a vascular pathogen entering the plant via the hydathodes causing rice bacterial blight.  It has been known that most regulation of pathogenicity factor F (RpfF) genes in Xanthomonas regulates virulence in response to the diffusible signal factor (DSF).  The RpfF recognized as an attractive drug target in bacterial rice blight disease.  In this study, we performed the gene-gene interaction of RpfF and pathway functional analysis.  3D structure of RpfF protein was predicted using a homology modelling tool Swiss-Model and refined by molecular dynamics (MD) simulation.  The refined model protein was predicted structural assessment using various tools such as PROCHECK, ERRAT, and VERIFY-3D.  We have collected 2 500 rifampicin analogues from Zinc Database by virtual screening.  The screened compounds were docked into the active site of the RpfF protein using AutoDock Vina in PyRx Virtual Screening Tool.  Furthermore, docking result and in silico ADMET analysis described that the compounds ZINC03056414, ZINC03205310, ZINC08673779, ZINC09100848, ZINC09729566, ZINC11415953, ZINC12810788, ZINC24989313, ZINC27441787 and ZINC32739565 have best binding energies and less toxicity than reference compound.  This study revealed that the active site residues such as HIS-118, HIS-147, THR-148, ARG-179, ASP-207, ARG-240 and THR-244 are key roles in the pathogenicity.  It could be beneficial in the design of small molecule therapeutics or the treatment of rice bacterial blight disease.
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    Prevalence and genetic diversity of grapevine fabavirus isolates from different grapevine cultivars and regions in China
    FAN Xu-dong, ZHANG meng-yan, ZHANG Zun-ping, REN Fang, HU Guo-jun, DONG Ya-feng
    2020, 19 (3): 768-774.   DOI: 10.1016/S2095-3119(19)62677-8
    Abstract89)      PDF in ScienceDirect      
    A total of 288 grapevine samples of 61 different grapevine cultivars, collected from 22 provinces and regions, were analyzed by reverse transcription-PCR (RT-PCR) for the presence of grapevine fabavirus (GFabV).  PCR detection results showed the incidences of GFabV were 12.8% (30/235) and 48.1% (25/52) in the asymptomatic and symptomatic vines, respectively.  The genetic diversity of GFabV isolates was analyzed based on partial nucleotide and encoded amino acid sequences of the RNA1 and RNA2 polyprotein genes.  Phylogenetic analyses of the RNA1 and RNA2 gene sequences divided the GFabV isolates into five well-defined groups.  Groups 1, 2, and 4 comprised only Chinese isolates.  This article represents the first report for the prevalence and genetic diversity of GFabV in grapevines grown in China.
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    Monoclonal antibody-based serological detection of potato virus M in potato plants and tubers
    ZHANG Yu, GAO Yan-ling, HE Wan-qin, WANG Ya-qin, QIAN Ya-juan, ZHOU Xue-ping, WU Jian-xiang
    2020, 19 (5): 1283-1291.   DOI: 10.1016/S2095-3119(19)62755-3
    Abstract74)      PDF in ScienceDirect      
    Potato virus M (PVM) is one of the common and economically important potato viruses in potato-growing regions worldwide.  To investigate and control this viral disease, efficient and specific detection techniques are needed.  In this study, PVM virions were purified from infected potato plants and used as the immunogen to produce hybridomas secreting PVM-specific monoclonal antibodies (MAbs).  Four highly specific and sensitive murine MAbs, i.e., 1E1, 2A5, 8A1 and 17G8 were prepared through a conventional hybridoma technology.  Using these four MAbs, we have developed an antigen-coated plate (ACP)-ELISA, a dot-ELISA and a Tissue print-ELISA for detecting PVM infection in potato plants and tubers.  PVM could be detected in infected potato plant tissue crude extracts diluted at 1:10 240 (w/v, g mL–1) by the dot-ELISA or at 1:163 840 (w/v, g mL–1) by the ACP-ELISA.  The Tissue print-ELISA is the quickest and easiest assay among the three established serological assays and is more suitable for onsite large-scale sample detection.  Detection results of the field-collected samples showed that PVM is currently widespread in the Yunnan and the Heilongjiang provinces in China.  The field sample test results of the developed serological assays were supported by the results from RT-PCR and DNA sequencing.  We consider that the newly established ACP-ELISA, dot-ELISA and Tissue print-ELISA can benefit PVM detection in potato plant and tuber samples and field epidemiological studies of PVM.  These assays can also facilitate the production of virus-free seed potatoes and breeding for PVM-resistant potato cultivars, leading to the successful prevention of this potato viral disease.
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    MmNet: Identifying Mikania micrantha Kunth in the wild via a deep Convolutional Neural Network
    QIAO Xi, LI Yan-zhou, SU Guang-yuan, TIAN Hong-kun, ZHANG Shuo, SUN Zhong-yu, YANG Long, WAN Fang-hao, QIAN Wan-qiang
    2020, 19 (5): 1292-1300.   DOI: 10.1016/S2095-3119(19)62829-7
    Abstract102)      PDF in ScienceDirect      
    Mikania micrantha Kunth is an invasive alien weed and known as a plant killer around the world.  Accurately and rapidly identifying M. micrantha in the wild is important for monitoring its growth status, as this helps management officials to take the necessary steps to devise a comprehensive strategy to control the invasive weed in the identified area.  However, this approach still mainly depends on satellite remote sensing and manual inspection.  The cost is high and the accuracy rate and efficiency are low.  We acquired color images of the monitoring area in the wild environment using an Unmanned Aerial Vehicle (UAV) and proposed a novel network -MmNet- based on a deep Convolutional Neural Network (CNN) to identify M. micrantha in the images.  The network consists of AlexNet Local Response Normalization (LRN), along with the GoogLeNet and continuous convolution of VGG inception models.  After training and testing, the identification of 400 testing samples by MmNet is very good, with accuracy of 94.50% and time cost of 10.369 s.  Moreover, in quantitative comparative analysis, the proposed MmNet not only has high accuracy and efficiency but also simple construction and outstanding repeatability.  Compared with recently popular CNNs, MmNet is more suitable for the identification of M. micrantha in the wild.  However, to meet the challenge of wild environments, more M. micrantha images need to be acquired for MmNet training.  In addition, the classification labels need to be sorted in more detail.  Altogether, this research provides some theoretical and scientific basis for the development of intelligent monitoring and early warning systems for M. micrantha and other invasive species. 
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    Biofumigation: An alternative strategy for the control of plant parasitic nematodes
    Rebecca Jean Barnes BRENNAM, Samantha GLAZE-CORCORAN, Robert WICK, Masoud HASHEMI
    2020, 19 (7): 1680-1690.   DOI: 10.1016/S2095-3119(19)62817-0
    Abstract98)      PDF in ScienceDirect      
    Plant-parasitic nematodes wreak havoc on the yield and quality of crops worldwide.  Damage from these pests is estimated to exceed US$100 billion annually but is likely higher due to misdiagnosis.  Nematode damage may be catastrophic, but historically the solution has been damaging as well.  Use of the synthetic nematicide methyl bromide (MBr) poses risks to the environment and to human health.  Biofumigation, the use of plant material and naturally produced compounds to control pests, is an increasingly feasible method of pest management.  The process acts through the growth or incorporation of plant material into the soil, that, over the course of its degradation, releases glucosinolates that break down into nematotoxic isothiocyanates.  These secondary plant metabolites exist naturally in commonly grown plants, most of which belong to the Brassicaceae family.  Research endeavors have increasingly explored the potential of biofumigation.  The reaction of target pests, the selection of biofumigant, and ideal environments for efficacy continue to be evaluated.  This review seeks to provide a cost and benefit assessment of the status of biofumigation for the control of plant-parasitic nematodes as an alternative to conventional methyl bromide usage. 
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    FgHAT2 is involved in regulating vegetative growth, conidiation, DNA damage repair, DON production and virulence in Fusarium graminearum
    Lü Wu-yun, YANG Nan, XU Zhe, DAI Han, TANG Shuai, WANG Zheng-yi
    2020, 19 (7): 1813-1824.   DOI: 10.1016/S2095-3119(19)62819-4
    Abstract126)      PDF in ScienceDirect      
    Histone lysine acetylation is catalyzed by acetyltransferases (HATs), which is important in regulating gene expression and physiological function in eukaryotic cells.  HATs can be classified into two main types: A- and B-type HATs.  Recently, in Fusarium graminearum, it has been reported that A-type HATs are involved in hyphal development, conidiation, sexual reproduction and virulence.  However, the biological roles of B-type HATs are unknown.  Here we report the identification and characterization of two B-type HATs (FgHat1 and FgHat2) in F. graminearum.  Targeted deletion of FgHAT1 did not result in any detectable phenotypes.  However, ΔFghat2 mutants were severely defective in vegetative growth, conidia production and morphogenesis, deoxynivalenol (DON) biosynthesis and virulence.  Interestingly, deletion of FgHAT2 resulted in significantly increased sensitivity to the DNA-damaging agent methyl methanesulfonate (MMS).  Furthermore, double deletion mutants (ΔFghat1ΔFghat2) displayed similar phenotypes to the ΔFghat2 mutants.  Taken together, we conclude that FgHat2 but not FgHat1 plays essential roles in regulating morphogenesis, DNA damage repair, DON production and virulence in F. graminearum.
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    Virome identification in wheat in the Czech Republic using small RNA deep sequencing
    Khushwant SINGH, Jana JAROŠOVÁ, Jan FOUSEK, CHEN Huan, Jiban Kumar KUNDU
    2020, 19 (7): 1825-1833.   DOI: 10.1016/S2095-3119(19)62805-4
    Abstract103)      PDF in ScienceDirect      
    High-throughput deep-sequencing technology and bioinformatics analysis of the small RNA (sRNA) population isolated from plants allows universal virus detection and complete virome reconstruction for a given sample.  In the present sRNA deep-sequencing analysis of virus-infected wheat samples in the Czech Republic, samples were firstly tested for barley yellow dwarf viruses (BYDVs), wheat streak mosaic virus (WSMV) and wheat dwarf virus (WDV) using ELISA, RT-PCR and PCR.  Subsequent sRNA sequencing of these samples yielded more than ~60 million single-end 50-bp reads with high confidence for nine field samples of wheat.  Overall, 16.5% of reads were virus-specific and 83.5% were mapped to the host.  More 21-nt reads (~7.7E+06 reads) were found than 24-nt (~6.20E+06 reads) or 22-nt (~4.30E+06 reads) reads.  De novo assembly of the high-quality contigs revealed the presence of three earlier reported viruses in the Czech Republic: BYDVs (31.48%), WSMV (24.23%) and WDV (26.66%).  We also showed the presence of cereal yellow dwarf virus (14.33%; two species CYDV-RPS and CYDV-RPV (family Luteoviridae/Polerovirus) and wheat yellow dwarf virus (WYDV, 3.30%; Luteoviridae).  Phylogenetic analysis showed CYDV and WYDV grouped separately from BYDVs.  Furthermore, several recombination breakpoints were found among the groups of yellow dwarf viruses (BYDVs, CYDV, and WYDV).  Using RNA deep sequencing, we confirmed the presence of the three known viruses (BYDVs, WSMV, and WDV) and the first record of two species of CYDV and WYDV in wheat in the Czech Republic.
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    A sensitive SYBR Green RT-qPCR method for grapevine virus E and its application for virus detection in different grapevine sample types
    REN Fang, ZHANG Zun-ping, FAN Xu-dong, HU Guo-jun, ZHANG Meng-yan, DONG Ya-feng
    2020, 19 (7): 1834-1841.   DOI: 10.1016/S2095-3119(19)62784-X
    Abstract77)      PDF in ScienceDirect      
    To develop a rapid and high-sensitivity method for detection of grapevine virus E (GVE), a SYBR Green based real-time fluorescence quantitative RT-PCR method (RT-qPCR) was established.  This method could be used to detect GVE specifically, and the sensitivity was about 100 times greater than conventional RT-PCR.  An excellent linear correlation (R2=0.997) and a high amplification efficiency (E=97.5%) were obtained from the standard curve of this method.  Reproducibility tests revealed that the coefficients of variation in the intra- and inter-assay results were 0.31–1.03% and 0.82–2.62%, respectively, indicating a good reproducibility.  The RT-qPCR method could be used to detect GVE in a wide range of grapevine sample types.  The detection rates of RT-qPCR for nearly all sample types from different positions and seasons were higher than conventional RT-PCR.  The detection rates in spring, summer, autumn and winter increased gradually.  Samples in autumn and winter were best for detection, and the detection rates of most samples were 80–100%, which were 10 to 40% higher than conventional RT-PCR.  In general, old petioles and branches were the best tissues for GVE detection.  The detection rates of these samples in each season were all 100%, which were 20 to 40% higher than conventional RT-PCR.  The second highest rates were in the old leaf, with detection rates for RT-qPCR of 80–100% in all seasons, which were 20 to 40% higher than conventional RT-PCR.  GVE could be difficultly detected in young leaves by conventional RT-PCR, and the detection rates were only 0–50%, while by RT-qPCR the rates could increase to 0–80%.  A total of 33 out of 363 samples (belonging to 68 cultivars) from 20 regions in China were detected to be positive by RT-qPCR (9.1%), which was more than twice the rate of the conventional RT-PCR (3.9%). 
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    Identification of a novel emaravirus infecting lilac through next-generation sequencing
    WANG Ya-qin, SONG Yu, CAO Meng-ji, CHENG Qi, WU Jian-xiang, HU Tao
    2020, 19 (8): 2064-2071.   DOI: 10.1016/S2095-3119(19)62806-6
    Abstract85)      PDF in ScienceDirect      
    Emaraviruses are multipartite negative-sense single-stranded viruses that usually infect flowering plants.  Using unbiased next-generation sequencing (NGS) approach, a novel virus from lilac leaves with yellow mottle symptom was identified.  Five viral genomic RNA segments were detected and completely sequenced.  The molecular characteristics of the viral RNAs and proteins resemble those of members of the genus Emaravirus.  The virus was found to be mechanically transmissible to Nicotiana benthamiana. Sequence alignment and phylogenetic analyses of the virus support the proposal of creating a new species in the genus Emaravirus for which the name lilac chlorotic ringspot-associated virus (LiCRaV) is proposed. 
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    Functional characterization of the catalytic and bromodomain of FgGCN5 in development, DON production and virulence of Fusarium graminearum
    WANG Qian-nan, HUANG Pan-pan, ZHOU Shan-yue
    2020, 19 (10): 2477-2487.   DOI: 10.1016/S2095-3119(20)63219-1
    Abstract88)      PDF in ScienceDirect      
    FgGCN5, a GCN5 homolog in Fusarium graminearum, plays a critical role in hyphal vegetative growth, asexual and sexual reproduction, deoxynivalenol (DON) biosynthesis and plant infection.  For nuclear localized GCN5, four conserved sequence motifs (I–IV) are presented in the catalytic domain and a bromodomain in the carboxy-terminus.  As a lysine acetyltransferase, conserved negatively charged residues are present to neutralize the protons from lysine substrates.  However, the role of conserved motifs/domains and residues in FgGCN5 are unclear.  Here, we generated deletion mutant strains for each the conserved motifs/domains and a glutamate residue 130 (E130) replacement mutant.  Deletion of each conserved motif in the catalytic domain and replacement of E130 site resulted in manifold defects in hyphae growth, asexual and sexual development, DON biosynthesis, and plant infection.  Phenotypic defects in the mutant strains were similar to deletion mutants.  The deletion of the bromodomain led a significant reduction in DON production and virulence, with no effects on hyphae growth, asexual or sexual reproduction.  FgGCN5 was further found to localize to the nucleus in conidia and hyphae cells.  In conclusion, FgGCN5 encodes a nuclear localized acetyltransferase.  The conserved motifs in the catalytic domain and E130 are essential for correct functions of the gene.  The conserved bromodomain is important for DON production and pathogen virulence.  This was the first report to identify the functions of conserved motifs/domains in FgGCN5, which will contribute to our understanding of the mechanism(s) by which FgGCN5 regulates F. graminearum
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    Decreasing detection frequency of MITE (MCLas-A) in the population of ‘Candidatus Liberibacter asiaticus’ recently collected in southern China
    CUI Xue-jin, ZENG Chun-hua, LIU Ke-hong, TENG Cai-ling, ZHOU Chang-yong, WANG Xue-feng
    2020, 19 (10): 2597-2601.   DOI: 10.1016/S2095-3119(20)63217-8
    Abstract72)      PDF in ScienceDirect      
    An active miniature inverted-repeat transposable element (MITE), MCLas-A, was previously identified from ‘Candidatus Liberibacter asiaticus’ known to be associated with citrus Huanglongbing (HLB, yellow shoot disease).  To explore the recent transposition status of MCLas-A, 389 ‘Ca. L. asiaticus’ strains collected from nine regions in China were amplified using a specific primer set and three representative ‘Ca. L. asiaticus’ strains were analyzed by next-generation sequencing (NGS) approach.  PCR and genomic analysis showed that the entire MCLas-A was only present in 1.80% (7/389) and the jumping-out type of the MITE was predominant (81.23%) in samples tested, suggesting high frequency transposition occurred in ‘Ca. L. asiaticus’ strains recently collected from China.  Biological roles of transposition of the active MITE remain to be determined.
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    Molecular diversity of barley yellow dwarf virus-PAV from China and the Czech Republic
    May Oo kHINE, brozenká MICHAELA, LIU Yan, Jiban kumar kUNDU, WANG Xi-feng
    2020, 19 (11): 2736-2745.   DOI: 10.1016/S2095-3119(20)63241-5
    Abstract79)      PDF in ScienceDirect      
    Wheat yellow dwarf disease (BYD), caused by different species of barley/cereal yellow dwarf viruses (B/CYDVs), is one of the most serious cereal diseases in China and the Czech Republic.  Because genetic diversity of the virus directly influences disease epidemiology, the molecular diversity and population structure of 24 Chinese isolates and 16 the Czech Republic isolates of BYDV-PAV from different regions in two countries were analyzed by sequencing their coat protein (CP) and readthrough protein (RTP) domain (RTD) genes and comparing the sequences with six CP and 16 RTP sequences of BYDV-PAV isolates from the NCBI database based on nucleotide identity position, phylogenetic analysis and nucleotide diversity.  Nucleotide identities between the Chinese and the Czech Republic isolates for the CP were 76.6–99.4%, 73.9–89.1% for RTD (ORF5), respectively.  The Chinese and the other country isolates showed 74.7–99.2% nucleotide identity for RTP (ORF3+ORF5).  Phylogenetic analysis of CP sequences showed that 20 Chinese isolates clustered in the same clade, but the other four Chinese isolates clustered in another clade with the isolates from the Czech Republic and other counties.  The population of BYDV-PAV in China had greater nucleotide variability and was more divergent than that in the Czech Republic.  Geographical and ecological factors but not hosts might contribute to the population differences in the two countries.
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    StKU80, a component in the NHEJ repair pathway, is involved in mycelial morphogenesis, conidiation, appressorium development, and oxidative stress reactions in Exserohilum turcicum
    GONG Xiao-dong, LIU Yu-wei, BI Huan-huan, YANG Xiao-rong, HAN Jian-min, DONG Jin-gao, GU Shou-qin
    2021, 20 (1): 147-158.   DOI: 10.1016/S2095-3119(20)63233-6
    Abstract75)      PDF in ScienceDirect      
    Homologous recombination (HR) and nonhomologous end joining (NHEJ) are considered the two main double-strand break (DSB) repair approaches in eukaryotes.  Inhibiting the activities of the key component in NHEJ commonly enhances the efficiency of targeted gene knockouts or affects growth and development in higher eukaryotes.  However, little is known about the roles of the NHEJ pathway in foliar pathogens.  Here we identified a gene designated StKU80, which encodes a putative DNA end-binding protein homologous to yeast Ku80, in the foliar pathogen Exserohilum turcicum.  Conserved domain analysis showed that the typical domains VWA, Ku78 and Ku-PK-bind are usually present in Ku70/80 proteins in eukaryotes and are also present in StKu80.  Phylogenetic analysis indicated that StKu80 is most closely related to Ku80 (XP_001802136.1) from Parastagonospora nodorum, followed by Ku80 (AGF90044.1) from Monascus ruber.  Furthermore, the gene knockout mutants ΔStKU80-1 and ΔStKU80-2 were obtained.  These mutants displayed longer septas, thinner cell walls, smaller amounts of substances on cell wall surfaces, and more mitochondria per cell than the wild-type (WT) strain but similar HT-toxin activity.  The mutants did not produce conidia and mature appressoria.  On the other hand, the mutants were highly sensitive to H2O2, but not to ultraviolet radiation.  In summary, the StKU80 plays devious roles in regulating the development of E.?turcicum.
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    Identification and resistant characterization of legumes sources against Meloidogyne incognita #br#
    Pornthip RUANPANUN, Prakit SOMTA
    2021, 20 (1): 168-177.   DOI: 10.1016/S2095-3119(20)63414-1
    Abstract86)      PDF in ScienceDirect      
    Root-knot nematodes (RKNs; Meloidogyne spp.) are becoming a serious problem in legume production.  This study identified Vigna genotypes exhibiting resistance to M. incognita (RKN) and characterized the modes of the resistance to M. incognita.  In total, 279 accessions from 21 Vigna species were screened for resistance based on a galling index (GI) and an egg mass index (EI).  Seven accessions were highly resistant to RKN with GI≤25, namely JP74716 (V. mungo var. mungo; cultivated black gram), JP107881 (V. nepalensis), JP229392 (V. radiata var. sublobata; wild mungbean), AusTRCF118141 (V. unguiculata subsp. unguiculata; cultivated cowpea), AusTRCF306385 (V. unguiculata subsp. unguiculata), AusTRCF322090 (V. vexillata var. vexillata; wild zombi pea) and JP235929 (V. vexillata var. vexillata).  JP229392 and AusTRCF322090 were the most resistant accessions having EI values of 18.74 and 1.88, respectively.  Continuous culture of M. incognita on both JP229392 and AusTRCF322090 resulted in a weakness in pathogenic ability for this RKN.  The resistance in JP229392 and AusTRCF322090 to RKN appeared to be antibiosis that was associated with reduced nematode penetration, retardation of nematode development and impeding giant cell formation.  The Vigna germplasm resistance to RKN identified in this study could be utilized as gene sources for the development of RKN-resistant Vigna cultivars.
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    Beneficial rhizobacterium provides positive plant–soil feedback effects to Ageratina adenophora
    SUN Yuan-yuan, ZHANG Qiu-xin, ZHAO Yun-peng, DIAO Yue-hui, GUI Fu-rong, YANG Guo-qing
    2021, 20 (5): 1327-1335.   DOI: 10.1016/S2095-3119(20)63234-8
    Abstract70)      PDF in ScienceDirect      
    Rhizosphere microbial communities play important roles in facilitating or inhibiting the establishment of exotic species.  Since some invasive plants interact with soil microbial communities such as rhizosphere bacteria, changes triggered by rhizosphere bacteria may alter competitive interactions between exotic and native plants.  This study compared the Bacillus cereus content in soils with different degrees of Ageratina adenophora invasion, and investigated the effects of A. adenophora allelochemicals on B. cereus growth and soil characteristics and the feedback effects of B. cereus on A. adenophora growth.  Bacillus cereus content in the rhizosphere of A. adenophora increased with intensification of the invasion process, and newly invaded soil contained almost twice as much bacteria as noninvaded soil.  When rhizosphere soil was added to the root exudates of A. adenophora, the contents of B. cereus were twice as much as the control, except on the first day.  Certain soil parameters increased significantly, such as ammonium nitrogen (NH4+-N) and available phosphorus (AP), which were increased by 41 and 27%, respectively.  Soil treatment with B. cereus promoted the degradation of two allelochemicals from the rhizosphere of A. adenophora, amorpha-4,7(11)-dien-8-one and 6-hydroxy-5-isopropy1-3,8-dimethyl-4a,5,6,7,8,8a-hexahydraphthalen-2(1H)-one, to varying degrees; and increased the germination rate by 50%, root length by 117%, shoot length by 48% and fresh weight by 81% for A. adenophora compared to those of untreated soil.  Our results confirmed that the invasion of A. adenophora will promote an increase of B. cereus, a beneficial rhizosphere bacterium, which in turn induces a positive feedback effect on A. adenophora.
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    Biological and molecular characterization of tomato brown rugose fruit virus and development of quadruplex RT-PCR detection
    YAN Zhi-yong, ZHAO Mei-sheng, MA Hua-yu, LIU Ling-zhi, YANG Guang-ling, GENG Chao, TIAN Yan-ping, LI Xiang-dong
    2021, 20 (7): 1871-1879.   DOI: 10.1016/S2095-3119(20)63275-0
    Abstract101)      PDF in ScienceDirect      
    Tomato brown rugose fruit virus (ToBRFV) is a novel tobamovirus firstly reported in 2015 and poses a severe threat to the tomato industry.  So far, it has spread to 10 countries in America, Asia, and Europe.  In 2019, ToBRFV was identified in Shandong Province (ToBRFV-SD), China.  In this study, it was shown that ToBRFV-SD induced mild to severe mosaic and blistering on leaves, necrosis on sepals and pedicles, and deformation, yellow spots, and brown rugose necrotic lesions on fruits.  ToBRFV-SD induced distinct symptoms on plants of tomato, Capsicum annumm, and Nicotiana benthamiana, and caused latent infection on plants of Solanum tuberosum, Solanum melongena, and N. tabacum cv. Zhongyan 102.  All the 50 tomato cultivars tested were highly sensitive to ToBRFV-SD.  The complete genomic sequence of ToBRFV-SD shared the highest nucleotide and amino acid identities with isolate IL from Israel.  In the phylogenetic tree constructed with the complete genomic sequence, all the ToBRFV isolates were clustered together and formed a sister branch with tobacco mosaic virus (TMV).  Furthermore, a quadruplex RT-PCR system was developed that could differentiate ToBRFV from other economically important viruses affecting tomatoes, such as TMV, tomato mosaic virus, and tomato spotted wilt virus.  The findings of this study enhance our understanding of the biological and molecular characteristics of ToBRFV and provide an efficient and effective detection method for multiple infections, which is helpful in the management of ToBRFV.
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    Natural nematicidal active compounds: Recent research progress and outlook
    CHEN Ji-xiang, SONG Bao-an
    2021, 20 (8): 2015-2031.   DOI: 10.1016/S2095-3119(21)63617-1
    Abstract108)      PDF in ScienceDirect      

    Plant-parasitic nematodes cause substantial economic losses to global agriculture yearly.  The use of nematicides is an effective way of controlling plant-parasitic nematodes.  However, the long-term use of traditional organophosphorus and carbamate chemical nematicides can lead to increased nematode resistance.  With the increasing awareness of the necessity for the protection of the environment and human health, highly toxic nematicides no longer meet the developmental requirements of modern agriculture.  Recently, many studies have been undertaken on the isolation and nematicidal activity of natural products against plant-parasitic nematodes and Caenorhabditis elegans.  As an important model nematode, C. elegans plays a vital reference role in studying plant-parasitic nematodes regarding nematicidal activity, metabolic mechanism, and modes of action and target.  We reviewed the latest research progress of natural nematicidal active compounds against plant-parasitic nematodes and C. elegans over the past ten years, discussed the structure-activity relationship and mechanism of action, and examined the development and application of natural nematicidal active compounds.

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    The transcription factor FgNsf1 regulates fungal development, virulence and stress responses in Fusarium graminearum
    SHI Dong-ya, REN Wei-chao, WANG Jin, ZHANG Jie, Jane Ifunanya MBADIANYA, MAO Xue-wei, CHEN Chang-jun
    2021, 20 (8): 2156-2169.   DOI: 10.1016/S2095-3119(20)63339-1
    Abstract68)      PDF in ScienceDirect      
    Nutrient and stress factor 1 (Nsf1), a transcription factor containing the classical Cys2-His2 (C2H2) zinc finger motif, is expressed under non-fermentable carbon conditions and in response to salt stress in Saccharomyces cerevisiae.  However, the role of Nsf1 in filamentous fungi is not well understood.  In this study, the orthologue of Nsf1 was investigated in Fusarium graminearum (named FgNsf1), a causal agent of Fusarium head blight (FHB).  The functions of FgNsf1 were evaluated by constructing a FgNSF1 deletion mutant, designated as ΔFgNsf1, and its functional complementation mutant ΔFgNsf1-C.  Gene deletion experiments showed that the mycelial growth rate, asexual and sexual reproduction of ΔFgNsf1 were significantly reduced, but the pigment production of ΔFgNsf1 was remarkably increased compared with the PH-1 and ΔFgNsf1-C.  In addition, the tolerance of ΔFgNsf1 to osmotic pressures, cell wall-damaging agents and oxidative stress increased significantly.  Sensitivity tests to different fungicides revealed that ΔFgNsf1 exhibited increased sensitivity to carbendazim (MBC) and tebuconazole, and enhanced tolerance to fludioxonil and iprodione than PH-1 and ΔFgNsf1-C.  The virulence of ΔFgNsf1 to wheat coleoptiles and flowering wheat heads were dramatically decreased, which was consistent with the decrease in the yield of deoxynivalenol (DON).  All of these defects were restored by target gene complementation.  These results indicated that FgNsf1 plays a crucial role in vegetative growth, asexual and sexual reproduction, stress responses, fungicide sensitivity, and full virulence in F. graminearum.
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    Robust molecular detection of the new Tomato brown rugose fruit virus in infected tomato and pepper plants from Turkey
    Hakan FIDAN, Pelin SARIKAYA, Kubra YILDIZ, Bengi TOPKAYA, Gozde ERKIS, Ozer CALIS
    2021, 20 (8): 2170-2179.   DOI: 10.1016/S2095-3119(20)63335-4
    Abstract89)      PDF in ScienceDirect      
    Tomato brown rugose fruit virus (ToBRFV) causes severe fruit loss in tomato (Solanum lycopersicum) and pepper (Capsicum annuum) plants.  It is an emerging Tobamovirus that is spreading globally.  The major challenge is to develop a reliable method for the detection of the virus, and to better characterize the symptoms it causes.  The aims of this study, therefore, were to characterize the symptom development on tomato and pepper plants, and to establish a reliable detection method for the virus.  Following infection of the tomato and pepper plants with ToBRFV, the leaves turned chlorotic, mosaic or mottled, while the fruit became rugose, necrotic and marbled, and showed discoloration with yellow or brown spots.  Transmission electron microscopy (TEM) revealed single rod-like virus particles characteristic of the Tobamoviruses.  Classical reverse transcription PCR (RT-PCR) and quantitative PCR (qPCR) with specific primers and probes confirmed that the virus is ToBRFV.  We found that the resistance genes from tomato, Tm-22, and pepper, L1, L2, L3 and L4, did not confer resistance to ToBRFV.  Here, we present a PCR-based method as a diagnostic test for detecting ToBRFV in infected seeds.  This method will help to prevent further spread of the virus in commercial seeds.
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    Transcriptomic analysis reveals the transcription factors involved in regulating the expression of EPSPS gene, which confers glyphosate resistance of goosegrass (Eleusine indica)
    ZHANG Chun, YU Chao-jie, ZHANG Tai-jie, GUO Wen-lei, TIAN Xing-shan
    2021, 20 (8): 2180-2194.   DOI: 10.1016/S2095-3119(21)63682-1
    Abstract99)      PDF in ScienceDirect      
    Glyphosate inhibits the enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) and overexpression of the EPSPS gene is one of the molecular mechanisms conferring glyphosate resistance in weeds.  A regulatory sequence of EPSPS gene was isolated previously, and an alteration in its 5´-untranslated region (UTR) pyrimidine (Py)-rich stretch element is involved in the regulation of EPSPS expression in glyphosate-resistant (GR) Eleusine indica.  However, the transcription factors involved in this regulatory sequence remain to be elucidated.  In this study, we investigated the regulatory network of EPSPS overexpression associated genes in a GR E. indica population by RNA-seq.  The differentially expressed transcript analyses revealed that glyphosate treatment caused an increase in the expression of 2 752 unigenes and a decrease in the expression of 4 025 unigenes in the GR E. indica, compared to the glyphosate-susceptible (GS) E. indica.  Among them, 1 373 unigenes were identified to be co-expressed with the EPSPS gene in GR E. indica.  GO and KEGG pathway analyses showed that the up-regulated unigenes were mainly enriched in chloroplasts and associated with the shikimate biosynthesis pathway, chlorophy II and peroxisome metabolism processes.  Notably, the expression of a Shikimate kinase which catalyzed the conversion of Shikimate to Shikimate 3-phosphate (S3P, a substrate of EPSPS), was also up-regulated.  Eight transcription factors were identified as likely to be involved in the regulation of the EPSPS expression, and three of them (ARF2, ARF8 and BPC6) showed more binding sites because of a (CT)n insertion of the 5´-UTR Py-rich stretch element in GR.  However, the yeast one-hybrid assay illustrated that ARF8 and BPC6 could bind to the 5´-UTR Py-rich stretch element of wild type EPSPS, but could not bind to the mutated form.  Our data suggests that the transcriptional regulation of EPSPS expression is complex and was significantly altered in GR E. indica.  These discoveries provide new references for further study of the EPSPS overexpression mechanism that endows glyphosate resistance. 
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    Wheat straw biochar amendment suppresses tomato bacterial wilt caused by Ralstonia solanacearum: Potential effects of rhizosphere organic acids and amino acids
    TIAN Ji-hui, RAO Shuang, GAO Yang, LU Yang, CAI Kun-zheng
    2021, 20 (9): 2450-2462.   DOI: 10.1016/S2095-3119(20)63455-4
    Abstract84)      PDF in ScienceDirect      
    Complex interactions based on host plant, rhizosphere microorganisms and soil microenvironment are presumed to be responsible for the suppressive properties of biochar against soil-borne diseases, although the underlying mechanisms are not well understood.  This study is designed to evaluate the efficacy of biochar amendment for controlling tomato bacterial wilt caused by Ralstonia solanacearum, and to explore the interactions between biochar-induced changes in rhizosphere compound composition, the pathogen and tomato growth.  The results showed that biochar amendment decreased disease incidence by 61–78% and simultaneously improved plant growth.  The positive ‘biochar effect’ could be associated with enhanced microbial activity and alterations in the rhizosphere organic acid and amino acid composition.  Specifically, elevated rhizosphere citric acid and lysine, but reduced salicylic acid, were induced by biochar which improved microbial activity and rendered the rhizosphere unsuitable for the development of R. solanacearum.  In addition, nutrients which were either made more available by the stimulated microbial activity or supplied by the biochar could improve plant vigor and potentially enhance tomato resistance to diseases.  Our findings highlight that biochar’s ability to control tomato bacterial wilt could be associated with the alteration of the rhizosphere organic acid and amino acid composition, however, further research is required to verify these ‘biochar effects’ in field conditions.
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    Elimination of grapevine fleck virus and grapevine rupestris stem pitting-associated virus from Vitis vinifera 87-1 by ribavirin combined with thermotherapy
    HU Guo-jun, DONG Ya-feng, ZHANG Zun-ping, FAN Xu-dong, REN Fan
    2021, 20 (9): 2463-2470.   DOI: 10.1016/S2095-3119(20)63336-6
    Abstract85)      PDF in ScienceDirect      
    Vitis vinifera 87-1 plants infected by grapevine fleck virus (GFkV) and grapevine rupestris stem pitting-associated virus (GRSPaV) were used as the plant materials for virus elimination treatment.  This study evaluated the effects of ribavirin at different concentrations (15 and 25 μg mL–1; R15 and R25, respectively), thermotherapy (37°C; T), and the combination of ribavirin and thermotherapy (R15+T and R25+T) on eliminating viruses from grapevine plants in vitro.  Both R15 and R25 had phytotoxic effects and weakened plant growth.  Thermotherapy positively affected the growth of grapevine plants.  Plant height was significantly greater in T, R15+T, and R25+T than in CK, R15 and R25.  The proportion of dead plants after T, R15+T, and R25+T was 51.4, 11.4, and 8.6%, respectively.  The survival rates of regenerated plants after all treatments were >68.0%.  Ribavirin concentration and treatment time were related to the regeneration of shoot tips and elimination efficiencies of the two viruses.  The survival rates of plants after R15+T for 30, 40, and 50 days were 97.3, 90.7, and 74.4%, respectively.  The elimination rates of GRSPaV from plants in the three time quantum were 55.6, 84.6, and 93.8%, respectively.  The elimination rate of GFkV was 23.9% higher in R25 (35/44) than in R15 (25/45), and that of GRSPaV was 7.0% higher in R25 than in R15.  The combination of thermotherapy and chemotherapy was found to have a positive effect on the eradication of GFkV and GRSPaV, and R25+T for 50 days was able to completely eliminate the two viruses from in vitro grapevines.  
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    Combining simplified DNA extraction technology and recombinase polymerase amplification assay for rapid and equipment-free detection of citrus pathogen Phytophthora parasitica
    CHEN Wei-yu, YU Jia, XU Heng, LU Xin-yu, DAI Ting-ting, TIAN Yue-e, SHEN Dan-yu, DOU Dao-long
    2021, 20 (10): 2696-2705.   DOI: 10.1016/S2095-3119(20)63459-1
    Abstract75)      PDF in ScienceDirect      
    Foot and root rot caused by Phytophthora parasitica is a substantial threat to citrus cultivation, affecting both yield and quality.  Thus, rapid and accurate detection of P. parasitica plays an important role in disease management.  The aim of this study was to develop a simple diagnostic method to detect P. parasitica infection by combining recombinase polymerase amplification and lateral flow strips (LF-RPA).  To establish the LF-RPA assay of P. parasitica, the primers and probe designed based on the Ypt1 gene were tested for specificity to P. parasitica, which showed no cross-reactivity with DNAs of other related oomycete species.  The LF-RPA assay detected the amount of genomic DNA of P. parasitica which was as low as 1 pg.  To make the LF-RPA assay useful in low-resource settings, four simplified DNA extraction methods were compared, after which the LF-RPA assay was applied, with no specialized equipment, to analyze a diverse range of citrus tissues by using a simplified PEG-NaOH method for DNA extraction.  This method was successful in detecting P. parasitica in infected plant samples within 30 min.  Combining the LF-RPA assay and a simplified DNA extraction method could be a potential  detection test for P. parasitica, especially in areas with limited resources.
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    Isolation and molecular characterization of entomopathogenic nematode, Heterorhabditis sp. from an arable land in Nigeria
    Fisayo Y. DARAMOLA, Osarenkhoe O. OSEMWEGIE, Stephen O. OWA, Samuel B. ORISAJO, Evbuomwan IKPONMWOSA, Elizabeth T. ALORI
    2021, 20 (10): 2706-2715.   DOI: 10.1016/S2095-3119(21)63609-2
    Abstract73)      PDF in ScienceDirect      
    The occurrence of entomopathogenic nematodes (EPNs) in arable soil samples from Nigeria was investigated using Baermann extraction tray and insect-bait (White’s trap) techniques.  Isolates were tested for infectivity using the larvae of Galleria mellonella (greater moth) and Tenebrio molitor (mealworm).  The study revealed a new species of Heterorhabditis (MT371593) in soil samples that were randomly collected from an arable farmland cultivated with cassava TMS-30572 at the Teaching and Research Farm of Landmark University, Nigeria.   Amplification of the internal transcribed spacer region (ITS) of the ribosomal DNA produced a nucleotide sequence of 933 base pairs (bp).  A BLASTN search of GenBank showed that the sequence of the Nigerian isolate is identical at 99% similarity to that of Heterorhabditis sp. from Thailand.   Infectivity test of the isolate showed 100% mortality against T. molitor larvae within 48 h of exposure while only 80% mortality was recorded for G. mellonella after 1 week of exposure.  This is the first account of Heterorhabditis sp. in Nigeria.  The varying degrees of infectivity against mealworm and greater moth observed in this study proved that the Nigerian isolate of Heterorhabditis sp. could potentially be an attractive option in the management of insect pests of cash crops.
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    The putative elongator complex protein Elp3 is involved in asexual development and pathogenicity by regulating autophagy in the rice blast fungus
    ZHANG Li-mei, CHEN Shu-ting, QI Min, CAO Xue-qi, LIANG Nan, LI Qian, TANG Wei, LU Guo-dong, ZHOU Jie, YU Wen-ying, WANG Zong-hua, ZHENG Hua-kun
    2021, 20 (11): 2944-2956.   DOI: 10.1016/S2095-3119(20)63493-1
    Abstract53)      PDF in ScienceDirect      
    Autophagy is responsible for maintaining fundamental cellular homeostasis and is, therefore, essential for diverse development processes.  This study reported that PoElp3, the putative catalytic subunit of Elongator complex, is involved in the maintenance of autophagy homeostasis to facilitate asexual development and pathogenicity in the rice blast fungus Pyricularia oryzae.  It was found that the ΔPoelp3 strains were defective in vegetative growth, conidiation, stress response, and pathogenicity.  The mutants exhibited hyper-activated autophagy in the vegetative hyphae under both nutrient-rich and nutrient-deficient conditions.  The hyper-activation of autophagy possibly suppressed the production of vegetative hyphae in the ΔPoelp3 strains.  Moreover, the ΔPoelp3 strains were found to be more sensitive to rapamycin during vegetative- and invasive-hyphal growth but have no effect on Target-of-Rapamycin (TOR) signaling inhibition.  Taken together, these results demonstrated that PoElp3 is involved in asexual development and pathogenicity by regulating autophagy in the rice blast fungus. 
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    Three Sclerotinia species as the cause of white mold on pea in Chongqing and Sichuan of China
    DENG Dong, SUN Su-li, DU Chen-zhang, XIANG Chao, LONG Jue-chen, CHEN Wei-dong, ZHU Zhen-dong
    2021, 20 (11): 2957-2965.   DOI: 10.1016/S2095-3119(21)63629-8
    Abstract84)      PDF in ScienceDirect      
    White mold of pea caused by Sclerotinia sclerotiorum is a common disease in China.  However, we discovered that the diverse Sclerotinia species could cause white mold on pea plants in Chongqing and Sichuan of China during recent disease surveys.  Thus, the objective of this study was to confirm the causal agents from diseased pea plants.  The obtained isolates of white mold from Chongqing and Sichuan were identified by morphological characters and molecular characterization to determine the pathogen species, and their pathogenicity was confirmed on pea through completing Koch’s postulates.  Fungal isolates of Sclerotinia-like were obtained from diseased plants or sclerotia.  Based on morphological characteristics and molecular characterization, 30 isolates were identified to three species, six isolates as S. minor, seven as S. sclerotiorum, and 17 as S. trifoliorum.  In pathogenicity tests on pea cultivars Zhongwan 4 and Longwan 1, all 30 isolates caused typical symptoms of white mold on the inoculated plants, and the inoculated pathogens were re-isolated from the diseased plants.  This study confirmed that white mold of pea was caused by three Sclerotinia species, S. sclerotiorum, S. minor and S. trifoliorum in Chongqing and Sichuan. It is the first report that S. minor and S. trifoliorum cause white mold of pea in Southwest China.
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    Three sensitive and reliable serological assays for detection of potato virus A in potato plants
    WU Jia-yu, ZHANG Yu, ZHOU Xue-ping, QIAN Ya-juan
    2021, 20 (11): 2966-2975.   DOI: 10.1016/S2095-3119(20)63492-X
    Abstract99)      PDF in ScienceDirect      
    Vegetative propagation of seed potato often allows passaging of viruses to seed tubers, resulting in significant yield losses and reduction of potato tuber quality.  Thus, virus detection approach is crucial for effective virus management programs and the production of virus-free seed potatoes.  Among the reported potato-infecting viruses, potato virus A (PVA) is considered as one of the most important viruses in potato-growing regions worldwide.  This study prepared four hybridoma lines secreting PVA-specific monoclonal antibodies (MAbs) (2D4, 8E11, 14A6 and 16H10) using purified PVA virions as an immunogen.  Western blotting results indicated that all the four MAbs reacted strongly and specifically with the putative capsid protein of PVA.  Using these four MAbs, this study developed antigen-coated plate enzyme-linked immunosorbent assay (ACP-ELISA), Dot-ELISA and Tissue print-ELISA for detection of PVA infection in potato plants.  The results indicated that PVA can be detected in crude tissue extracts from infected potato plants diluted up to 1:327 680 (w/v, g mL–1) by ACP-ELISA or up to 1:10 240 by Dot-ELISA.  The Tissue print-ELISA is the quickest and easiest approach among the three serological assays, and is more suitable for onsite large-scale potato screening programs.  Further analyses of field-collected potato samples showed that the sensitivities and specificities of the three serological approaches were similar to those of RT-PCR in PVA detection and confirmed that PVA is currently widespread in Yunnan and Zhejiang provinces of China.  Hence, the results strongly suggest that these highly sensitive serological approaches based on PVA-specific MAbs are useful and powerful for PVA-free seed potato production programs and PVA field surveys. 
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