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    26 July 2021, Volume 37 Issue 7
    Plant Nematode Diseases: Serious Challenges to China’s Food Security
    PENG De-liang
    2021, 37(7):  1-2. 
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    Research Progress on the Genomics of Plant-Parasitic Nematode
    PENG Huan, ZHAO Wei, YAO Ke, JIANG Chen, HUANG Wen-kun, KONG Ling-an, ZHENG Jing-wu, PENG De-liang
    2021, 37(7):  3-13.  doi:10.13560/j.cnki.biotech.bull.1985.2021-0710
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    Plant-parasitic nematodes parasitize various plants,resulting in crop yield reduction and serious economic losses. Genomics study of plant parasitic nematode play an irreplaceable role in revealing the molecular mechanism of interaction between parasitic nematodes and plants,as well as breeding nematode-resistant varieties. With the continuous development of high-throughput sequencing technology,the genome of multiple important plant parasitic nematodes has been revealed and reported. The evolution and origin of plant-parasitic nematodes have been more clearly defined by the development of comparative genomics,and some progress has been made in functional genomics of plant parasitic nematodes. This paper gives a brief review on the major research advances of plant-parasitic nematode genomes,including general features,genome variation,sequences of tandem repeat genes,gene regulation and gene co-expression,horizontal gene transfer,effectors discovery and gene family expansions,aiming at providing a reference for the research on the pathogenic molecular mechanism of plant nematodes and the development of new control strategies.

    Research Advances on the Functional Study of Host Resistance Genes to Heterodera glycines
    HAN Shao-jie, ZHENG Jing-wu
    2021, 37(7):  14-24.  doi:10.13560/j.cnki.biotech.bull.1985.2021-0627
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    Soybean plays an essential role in China’s economy. China is currently the largest soybean consumer and importer in the world,and the number of imported soybeans is increasing year by year. Soybean cyst nematode disease is a significant disease threatening major soybean producing areas globally,causing more than one billion US dollars in economic losses worldwide every year. The control of this pathogen is challenging. The use of resistant varieties is the most economical and effective measures to control soybean cyst nematode disease. At present,with the excessive use of a single resistant variety,the races of soybean cyst nematode continue to evolve,leading to reduced resistance and threatening the safety of the soybean industry. With the development of modern biotechnology,many researchers have elucidated the resistances to soybean cyst nematode by means of genetics,transcriptomics,and protein function studies. This review,summarizes the resistance mechanism of known major resistant sites Rhg1and Rhg4 in soybean,as well as the relationship between vlicular transport,phytohormone pathway and the resistance to soybean cyst nematode,discusses the significance of related functional proteins for soybean resistance and possible issues in the future research direction,and prospects the further reaches in this field. This is conducive to fully exploring the potential resistance genes of soybeans,laying a theoretical foundation for the creation of new soybean germplasms resistant to soybean cyst nematode,and serving the safe development of China’s soybean industry.

    Research Progress on Plants Responses to Parasitic Nematodes Infection
    DENG Miao-miao, GUO Xiao-li
    2021, 37(7):  25-34.  doi:10.13560/j.cnki.biotech.bull.1985.2021-0669
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    Plant-parasitic nematode(PPN)is an important class of plant pathogens. They are widely distributed and highly adapted for parasitism by establishing a long-term and stable relationship with their hosts,which can cause serious damages to food crops,horticultural crops and forestry. Among them,root-knot nematode(RKN)and cyst nematode(CN)are the most economically important and best-studied endoparasites. The differences between RKNs and CNs during their infection,feeding cell formation and reproduction lead to the varied host responses to host plants under nematode infection. Substantial research progress has been achieved in the cloning of nematode disease resistance genes,together with the massive study of effector proteins and host responses. This review summarizes recent progress regarding to the molecular basis of plant-nematode interaction,mainly focusing on three aspects including host disease-resistant genes,nematode secretions,and plant hormone responses,with emphasis on the differences between CN and RKN. The review also discusses the potential application prospect of the knowledge derived from the theoretical research in nematode control.

    Recognition Mechanism of Plant-parasitic Nematodes in Response to Semiochemicals
    LI Chun-jie, WANG Cong-li
    2021, 37(7):  35-44.  doi:10.13560/j.cnki.biotech.bull.1985.2021-0514
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    Semiochemicals secreted by the host root and rhizosphere microorganisms are important cues for host seeking,host location and infection of plant-parasitic nematodes(PPN). Currently,exploring semiochemicals and the associated molecular targets during the pre-parasitic stage of plant-parasitic nematodes to develop plant- or nematode- derived nematicides is an international research hotspot and frontier for nematologists. The focus of this review is on the international and national research progress of PPN on recognition mechanism of semiochemical signals. Firstly,PPN recognition or chemotaxis to semiochemical signals is defined and major semiochemical signal substances reported recently are listed. The chemotaxis is compared between the top two economically important nematodes(root-knot nematodes and soybean cyst nematode). Further,based on the chemosensory function of Caenorhabditis elegans,the molecular regulation of PPN chemotaxis is stated,as well as the potentially molecular transduction mechanism of PPN chemosensory,and its research significance and difficulty are discussed. Finally,the future important research directions of PPN signal recognition mechanism are prospected.

    Research Advances of Biology in Ditylenchus destructor Thorne,1945
    ZHAO Hong-hai, LIANG Chen, ZHANG Yu, DUAN Fang-meng, SONG Wen-wen, SHI Qian-qian, HUANG Wen-kun, PENG De-liang
    2021, 37(7):  45-55.  doi:10.13560/j.cnki.biotech.bull.1985.2021-0600
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    The potato rot nematode,Ditylenchus destructor Thorne,1945,is an important plant-pathogenic nematode in the world and a plant quarantine pest on national agriculture of China. It causes harms to many underground fruit-bearing crops such as sweet potato(Ipomoea batatas),potato(Solanum tuberosum),garlic(Allium sativum),etc.,and can also grow and reproduce on a great number of weeds and fungi. D. destructor is benefited by coolness and high humidity,whereas may be adversely affected by high temperature and desiccation. The nematode is mainly carried by infected vegetative propagation materials of host crops for both long-distance dissemination and local dispersal. There are obvious variations in its persistent survival,infection and damage performances among some different host crops,but the related causes and mechanisms have been still essentially unknown. D. destructor is here reviewed in the geographical distribution,host range,reproduction and development,infection cycle,environmental adaptation,and survival and parasitism related molecular mechanisms,main research topics that need to be further explored are pointed out,aiming to provide novel ideas and serviceable references for the research and practice in forewarning and management of the high risk pest.

    Effects of Plant Hormones on the Establishment and Development of Plant Parasitic Nematodes’ Feeding Sites
    HUANG Wen-kun, YU Jing-wen, JIA Jian-ping, PENG De-liang
    2021, 37(7):  56-64.  doi:10.13560/j.cnki.biotech.bull.1985.2021-0332
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    Root-knot nematode(Meloidogyne spp.)and cyst nematode(Heterodera spp.)are the two most harmful endoparasitic nematodes to the agricultural production. After the nematodes invading the plant,they absorb nutrients and complete their life cycle by forming giant cell or syncytia at the feeding sites. Plants establish a good interaction with invaded nematodes by controlling the levels of auxin,ethylene,cytokinin,gibberellin,abscisic acid and other plant hormones. This paper reviews the establishment and formation of nematode feeding sites,the expression and regulation of plant hormone-related genes in nematode feeding sites,and the effects of plant hormones on the establishment and formation of nematode feeding sites,as well as discusses the issues related to the synergistic effect of plant hormones on the feeding sites of nematodes and the latest research,aiming to reveal the effects of plant hormones on the establishment and development of feeding sites of plant parasitic nematodes and to provide references for the integrated management of crop nematode diseases.

    Advances of Non-coding RNA in Interactions Among Biocontrol Bacteria and Plant Nematodes and Host
    CHEN Li-jie, YANG Fan, FAN Hai-yan, ZHAO Di, WANG Yuan-yuan, ZHU Xiao-feng, LIU Xiao-yu, DUAN Yu-xi
    2021, 37(7):  65-70.  doi:10.13560/j.cnki.biotech.bull.1985.2021-0501
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    Plant nematodes are one of the most harmful and difficult pathogens in world agriculture. They can parasitize many plant species and cause billions of dollars of loss in each year. Biocontrol bacteria may activate the expressions of some disease-resistant genes,thus activate the some immune pathways,therefore inhibiting the infection,growth and development of plant nematodes. This characteristic makes the plant,biocontrol bacteria and nematodes constitute a new bio-regulatory relationship and it becomes a new model of plant immunity research. Non-coding RNA is a hot research topic in the field of biology,and it may be involved in the regulation of plant immunity by plant nematodes and other organisms such as biocontrol microorganisms. In this paper, the roles of miRNA, lncRNA and circRNA in the interactions among microorganisms, plant nematodes and their hosts are reviewed. miRNA, as an emerging post-transcriptional regulator of gene expression, largely affects the development of plants and regulates their immune responses. Pseudomonas can induce changes in miRNA and lncRNA in plants, and then these non-coding RNAs may participate in the process of biocontrol bacteria activating plant immunity. Finally, the future research direction of molecular mechanism of biocontrol bacteria regulating plant immunity against nematodes infection is prospected.

    Potential Function of 4-coumaric Acid-CoA Ligase in Response to Soybean Cyst Nematode Stress
    WANG Hui, ZHANG Shun-bin, JIN He, WANG Han, ZHANG Geng-hua, XIA Shi-ning, CHEN Jing-sheng, DUAN Yu-xi
    2021, 37(7):  71-80.  doi:10.13560/j.cnki.biotech.bull.1985.2021-0378
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    Soybean cyst nematode(SCN,Heterodera glycines)is one of the important targets of pest control strategies in soybean production areas. The prevention and control of SCN has always been one of the hot spots in the field of nematodes. SCN infection not only damage the underground part of soybean plant,but also damage the above ground part and thus affects soybean yield. Therefore,it is necessary to analyze the SCN resistance mechanism in order to achieve the purpose of prevention and control. That SCN successfully parasitizes host plants requires degradation and fusion of its cell wall to form a syncytium that provides the only source of nutrients for its growth and development. The cell wall resistance that hinders the movement of SCN and the establishment of syncytia is the key for soybean to resist SCN,while lignin is an important component of cell wall resistance. The biosynthesis of lignin mainly includes shikimic acid metabolic pathway,phenylpropane metabolic pathways,and specific pathways for lignin synthesis,4-coumarate-coenzyme A ligase(4CL),as an important turning enzyme connecting the phenylpropane metabolic pathway and lignin-specific synthesis pathway,determines the synthesis of lignin,which may be an important regulatory factor in response to SCN stress. This article starts from the nematode invasion that requires cell wall degradation and fusion to establish syncytium,discusses the cell wall resistance caused by lignin,analyzes the response mechanism of 4CL in cell wall resistance,and provides a scientific basis for further exploring the SCN stress mechanism.

    Research Advance of Calreticulin from Plant Parasitic Nematode
    WEI Ying, LUO Meng, DAI Liang-ying, PENG De-liang, LIU Jing
    2021, 37(7):  81-87.  doi:10.13560/j.cnki.biotech.bull.1985.2021-0583
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    Plant parasitic nematodes,as obligate living parasites,affect plant growth and development by infecting roots and cause huge losses in agricultural production in China. Calreticulin are multifunctional proteins which are located in the endoplasmic reticulum(ER)in eukaryotic cells. Calreticulin contains three structural functional domains:N-functional domain,P-functional domain,and C-functional domain. The calreticulin have been found in Meloidogyne incognita,Heterodera avenae,Aphelenchoides besseyi,Radopholus similis,Ditylenchus angustus,and Bursaphelenchus xylophilus. We summarized the structural characteristics,transcriptional sites in nematodes,suppression plant defense response and promoting parasitism of calreticulin from plant parasitic nematode. The insight into the function of calreticulin from plant parasitic nematode in infecting plants provide a theoretical basis for the prevention and controlling of plant parasitic nematode in China.

    Identification of Rice Genes Responding to Both the Nematode Effector MgMO237 and Its Interacting Protein OsCRRSP55
    LI Zhi-wen, LIU Pei-yan, CHEN Jian-song, LIAO Jin-ling, LIN Bo-rong, ZHUO Kan
    2021, 37(7):  88-97.  doi:10.13560/j.cnki.biotech.bull.1985.2021-0508
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    Previous studies showed that the Meloidogyne graminicola effector MgMO237 may interact with the rice protein OsCRRSP55 and inhibit rice defense responses against M. graminicola. Plant protein CRRSPs are involved in plant responses to both biotic and abiotic stresses. Understanding the influence of the interaction between MgMO237 and OsCRRSP55 on the expressions of their downstream genes will provide a theoretical basis for elucidating the mechanism of rice defense responses against root-knot nematodes. Bioinformatics analysis indicated that the OsCRRSP55 protein contained an N-terminal signal peptide and two conserved domains called the domain unknown function 26(DUF26),in which occurred a conserved motif C-X8-C-X2-C,but had no transmembrane domains. RT-qPCR analysis indicated that the OsCRRSP55 expressed in the rice roots and it was significantly up-regulated in the galls at 7 d infection by M. graminicola compared with that in healthy root tip. The expression levels of OsCRRSP55 and its promoter binding transcription factor OsWRKY47 significantly increased in the rice treated with salicylic acid and methyl jasmonate compared with those in wild-type ones. Transcriptome sequencing showed that a jasmonic acid-responsive gene OsERF87 was down-regulated in MgMO237-transgenic rice,but up-regulated in rice overexpressing OsCRRSP55 by RT-qPCR,showing the OsERF87 was co-regulated by OsCRRSP55 and MgMO237. In sum,these results demonstrate that the M. graminicola effector MgMO237 may affect the jasmonic acid signal transmission by interacting with OsCRRSP55,which results in the suppression of plant defense responses to M. graminicola.

    Mitochondrial Genome and Phylogeny of Aphelenchoides medicagus
    XUE Qing, DU Hong-rui, XUE Hui-ying, WANG Yi-hao, WANG Xuan, LI Hong-mei
    2021, 37(7):  98-106.  doi:10.13560/j.cnki.biotech.bull.1985.2021-0628
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    Aphelenchoides medicagus is a facultative plant-parasitic nematode that may complete its life cycles on various fungi,meanwhile it has weak parasitism on soybean and alfalfa. In presented study,Illumina platform was used to sequence A. medicagus genome in low coverage. Assembly was made by extracting mitochondrial reads and subsequently assembled by the seed sequence. For the AT rich non-coding region,amplification was made using PCR and sequenced by Sanger method. The jointed result from the assembled and amplified sequences revealed the mitochondrial genome of A. medicagus consisted of 14 411 bp nucleotides,including 12 protein coding genes,22 tRNA and 2 rRNA genes after gene annotation. These genes are arranged in same way as those in A. besseyi,Bursaphelenchus xylophilus,and B. mucronatus. The phylogeny analysis using amino acid sequences showed that A. medicagus and A. besseyi clustered together as a sister group,which together with B. xylophilus and B. mucronatus to form a highly supported monophyletic Aphelenchoididae clade. This study demonstrates that low-coverage whole-genome sequencing method can be used to assemble the most of mitochondrial genome,and thus support this technique to be a feasible tool in mitochondrial genome sequencing.

    Cloning and Functional Analysis of Dd-mel-26 Gene from Ditylenchus destructor
    GAO Bo, MA Juan, LI Xiu-hua, LI Jiao-sheng, WANG Rong-yan, CHEN Shu-long
    2021, 37(7):  107-117.  doi:10.13560/j.cnki.biotech.bull.1985.2021-0599
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    To further understand the nematode Ditylenchus destructor and seek the target gene resources with RNAi application value,a potential lethal gene substrate specific adaptor gene,Dd-mel-26,was selected from the gene pool of D. destructor. The gene was cloned by RACE,and its function was analyzed by bioinformatics,real-time PCR and dsRNA induced in vitro RNAi of nematode target gene. The results showed that the full-length cDNA of Dd-mel-26 gene was 1 594 bp and contained an open reading frame of 1 158 bp. It was predicted to encode a protein containing 385 amino acids with a molecular weight of 43.52 kD and an isoelectric point of 5.4. Its genome structure consisted of 10 exons and 9 intron sequences. Dd-MEL-26 belonged to the MATH-BTB protein family and did not possess signal peptides. It is predicted that Dd-MEL-26 interacted with related proteins in the nucleus to perform its function. Phylogenetic analysis demonstrated that Dd-MEL-26 was grouped into plant parasitic nematodes,and was closely related to Meloidogyne enterolobii. Silencing the gene via dsRNA significantly increased the nematode fertility and vertical migration. Therefore,the Dd-mel-26 gene can positively regulate the propagation and vertical migration of D. destructor under certain conditions. However,deeper study may be needed if this gene is used as RNAi target for achieving the control of this nematode.

    Comparison of Molecular Characteristics of the Hybrid Progenies from Different Haplotypes of Ditylenchus destructor
    NI Chun-hui, LI Hui-xia, LI Wen-hao, LIU Yong-gang, XU Xue-fen, HAN Bian
    2021, 37(7):  118-126.  doi:10.13560/j.cnki.biotech.bull.1985.2021-0576
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    In order to clarify whether there are reproductive isolation and the variation in the hybrid offspring between different haplotypes,1 of 3rd to 4th stage juveniles and 5 males from different haplotypes were selected to hybridize,and the nematode amounts,ITS-rDNA sequences and secondary structures of F1 generations were analyzed. The results showed that the nematode amount of F1 generations reproduced after hybridized were generally low,and F1 amount of the different cross groups were ≤ 6 per treatment. In some groups,only one nematode was found in one treatment. Due to the low amount of offspring,the ITS-rDNA sequences of 5 F1 generations among 11 groups were successfully sequenced. The ITS-rDNA sequence of F1 generations were different with 0-86 bp from their parents,and all of the secondary structures of ITS were different from those of the parents. The results of ribosomal ITS-RFLP showed diversiform. Phylogenetic analysis showed that some of the F1 generation groups were closer to the male parents,and some were closer to the female parents. No reproductive isolation was found in different haplotypes,but variation appeared mainly in the H9 helix region of ITS1 region in the F1 generation of hybrids,and the degree of variation was directly proportional to the differences between parents. The analysis of differentiation on hybrid generations from different haplotypes of D. destructor were conducted through simulation tests after population diffusions. The results may provide references both to further researches on genetic diversity and control practice of D. destructor.

    Optimization of Fermentation Conditions of Myrothecium verrucaria ZW-2,a Biocontrol Strain Against Heterodera glycines and Analysis of Active Substances
    CHEN Qian, ZHANG Lu-yuan, CHEN Bo-chang, WU Hai-yan
    2021, 37(7):  127-136.  doi:10.13560/j.cnki.biotech.bull.1985.2021-0454
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    Myrothecium verrucaria ZW-2 strain had obvious nematicidal activity against Heterodera glycines. In order to determine the optimal fermentation conditions and metabolites of the strain,the orthogonal experiment with four factors and three levels was used to optimize the fermentation conditions and analyze the metabolomics. The results showed that the strain ZW-2 was cultured on Czapek medium with the optimal culture temperature of 28℃,rotation speed of 180 r/min,bottle volume of 100 mL,medium pH of 8,and the optimal carbon and nitrogen sources were sucrose and sodium nitrate. After metabolomics analysis,20 differential metabolites,including amide compounds and aliphatic compounds,were screened according to the information of the charge ratio of metabolites,the accurate molecular weight and the score of first-order fragment ions. The above experiment results by strain ZW-2 further provide important theoretical basis and application value for the development and utilization of this strain.

    Screening of Protein Interacting with Purpureocillium lilacinum Cyclophilin PlCYP6
    LIU Juan, ZHU Chun-xiao, XIAO Xue-qiong, MO Chen-mi, WANG Gao-feng, XIAO Yan-nong
    2021, 37(7):  137-145.  doi:10.13560/j.cnki.biotech.bull.1985.2021-0010
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    The cyclophilin PlCYP6 that is involved in the responses of Purpureocillium lilacinum to salt stresses was used as the research object,and the putative interaction protein of PlCYP6 in P. lilacinum was screened using immunoprecipitation combined with mass spectrometry(IP-MS),yeast two-hybrid assay and other techniques. Results indicated that 482 proteins was specially fished by PlCYP6,and mainly functioned in cell metabolism. Among them,alcohol dehydrogenase 1(ADH1)directly interacted with PlCYP6,and the domain,WD40 repeat,of PlCYP6 was involved the interaction between them. Meanwhile,both PlCYP6 and ADH1 were induced by NaCl stress. These findings reveal that ADH1 is a putative interaction protein of PlCYP6,which lays a foundation for further uncovering the mechanism of responses of P. lilacinum to salt stresses.

    Cloning,and Functional Analysis of Gene OsRAI1 Resistant to Hirschmanniella mucronate in Rice
    SHAN Cao-mei, YE Lei, ZHANG Lian-hu, KUANG Wei-gang, SUN Xiao-tang, MA Jian, CUI Ru-qiang
    2021, 37(7):  146-155.  doi:10.13560/j.cnki.biotech.bull.1985.2021-0362
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    The plant-parasitic nematode Hirschmanniella mucronata is one of the most serious rice nematodes in Jiangxi province. In the early work of the laboratory,the up-regulated OsRAI1 gene was screened by transcriptome analysis of resistant/susceptible rice root tissues infected by H. mucronata. The full length of the gene was cloned,its protein structure was predicted,and subcellular localization were analyzed. The expression conditions of OsRAI1 protein were optimized by IPTG induction. The results showed that the ORF of OsRAI1 gene was 1 065 bp in length,encoding 354 amino acids,and it belonged to the bHLH transcription factor family. The protein was hydrophilic with molecular weight of 37.90 kD,pI value of 4.86,fat coefficient of 68.33,total average hydrophilicity(GRAVY)of -0.415,and non-transmembrane protein. The results of protein interaction prediction demonstrated that the protein may interact with each other through the HLH region between the two proteins. The results of subcellular localization showed that the protein was expressed in nucleus. The protein expression was the highest when IPTG concentration was 0.6 mmol/L and induced at 16℃ for 18 h. The results lay a foundation for understanding the molecular mechanism of H. mucronate resistance.

    Effects of Biofumigation on Root-knot Nematodes and Soil Nematode Community
    JIN Na, WANG Xue-yan, LIU Qian, PENG De-liang, PENG Huan, JIAN Heng
    2021, 37(7):  156-163.  doi:10.13560/j.cnki.biotech.bull.1985.2021-0670
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    Root-knot nematodes are important pathogens threatening agricultural production. The control effects of cabbage biofumigation on tomato root-knot nematodes in the field were studied,the soil nematodes were isolated by the suspended centrifugation method and the influence of biofumigation on the soil nematode community was evaluated. The results showed that cabbage biofumigation reduced the damage of root-knot nematode to tomato. The control efficacy of biofumigation was 58.6% and 45.8% respectively at 60 and 90 d post transplantation,which was equivalent to that of the chemical pesticide fosthiazate. In addition,biofumigation resulted in the increase of the number of bacterivores nematodes and omnivore predator nematodes,and reduction of the number of fungivores nematodes and parasitic nematodes. Moreover biofumigation increased the maturity index(MI)of soil free-living nematodes,reduced the plant parasite index(PPI)and PPI/MI. It is indicated that cabbage biofumigation presents some inhibitory effect on plant parasitic nematodes,reduces the number and intensity of soil interference,and plays a positive role in the soil ecological environment. The research results show that cabbage biofumigation may effectively prevent and control vegetable root-knot nematodes,which is an environmentally friendly green prevention and control technology.

    Control of Root-knot Nematode Disease by Compounding Biological Agents from Plant and Microorganisms
    SHU Jie, ZHANG Ren-jun, LIANG Ying-chong, CHEN Ya-qiong, ZHANG Juan, GUO Jian, CHEN Sui-yun
    2021, 37(7):  164-174.  doi:10.13560/j.cnki.biotech.bull.1985.2021-0408
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    Tomato root-knot nematode disease seriously affects agricultural production. The compound of two biological agents from plant origin and microbial source are used to control tomato root-knot nematode disease,trying to explore a new feasibility to meet the need of green agricultural production. Three strains(SJ5,SJ17 and SJ19)of killing second-stage juveniles(J2)of root knot nematodes were isolated from the soil by the dilution coating plate method with an inhibitory rate of 89.23%,87.95%,and 88.97%,respectively,and they were identified as Pseudomonas psychrotolerans,Chryseobacterium gleum and Bacillus subtilis by 16S rDNA amplification and sequencing. The plate inhibition zone method proved that there was no antagonism among the three kinds of biocontrol bacteria,and between the biocontrol bacteria and azadirachtin,thus they were suitable for compounding. Preliminary verification by pot experiment showed that the compound preparation treatment group from the filtrates by three kinds of biocontrol bacteria fermented for 48 h(the effective number of viable bacteria in the fermentation broth was more than 300 million/mL)and the azadirachtin diluent(1 000 times)was effective against root-knot nematodes The control effect of the disease reached 67.75%,and there was no significant difference in the control effect compared with the chemical pesticides(fluopyram and thiazophos)treatment group,and the tomato’s plant height,stem circumference,seedling and root biomass were all significantly increased. The preliminary conclusion from the potted experiment reveals that the compound scheme has the application potential to control tomato root-knot nematode disease.

    Screening of Bacillus sp. Against Vegetable Root-knot Nematode and Study on Its Biocontrol Mechanism
    ZHANG Jie, XIA Ming-cong, ZHU Wen-qian, LIANG Juan, SUN Run-hong, XU Wen, WU Chao, YANG Li-rong
    2021, 37(7):  175-182.  doi:10.13560/j.cnki.biotech.bull.1985.2021-0409
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    Bacillus sp. are a group of important biocontrol agents for the management of plant disease. In order to search for excellent Bacillus strain for vegetable root knot nematode,62 Bacillus sp. were isolated from the suppression soil of root knot nematode. The mortality of the second stage juvenile and the inhibition of egg hatch by the fermentation filtrates of the strains were conducted to primarily screen biocontrol strains,and pot experiments were used to determine the control effects of biocontrol strains. Results demonstrated that treatments with bactreial suspensions of strains NB-04 reduced root-knot index by 61.6%. Further based on morphological characteristics and 16S rDNA gene sequence analysis,strain NB-04 was identified as Bacillus methylotrophicus. To preliminary study the biocontrol mechanism of NB-04,the influence of fermention filtrates on M. incognita egg catalase,acetylcholinesterase,carboxylesterase,and the content of egg total sugar and protein were determined. The results showed that the activity of egg catalase,acetylcholinesterase,and carboxylesterase decreased significantly,as well as the content of total sugar and protein significantly declined. This study provides the basis for the research and development of bio-nematicide.

    Duplex-RPA Detection for Bursaphelenchus xylophilus and Bursaphelenchus mucronatus
    FANG Yuan, WU Xun, LIN Yu, WANG Hai-yan, WU Hui-ping, JU Yu-liang
    2021, 37(7):  183-190.  doi:10.13560/j.cnki.biotech.bull.1985.2021-0579
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    The aim of this work is to develop a rapid recombinase polymerase amplification(RPA)method for simultaneously detecting Bursaphelenchus xylophilus and Bursaphelenchus mucronatus. The forward RPA primer Bx-rpa-F,Bm-rpa-F and the common reverse RPA primer Bxm-rpa-R were designed according to the sequence variation of ITS from B. xylophilus and B. mucronatus. The concentration of the three RPA primers in duplex-RPA was optimized,duplex-RPA detection system was established,and its specificity and sensitivity were analyzed. The results showed that duplex-RPA simultaneously amplified DNA extracted from B. xylophilus and B. mucronatus at 37℃ within 30 min. The RPA product of B. xylophilus amplified with Bx-rpa-F/Bxm-rpa-R was 346 bp,and that of B. mucronatus with Bm-rpa-F/Bxm-rpa-R was 189 bp. In addition,duplex-RPA presented the best amplification efficiency when the concentration of Bx-rpa-F/Bm-rpa-F/Bxm-rpa-R was 5∶3∶8. Duplex RPA had high specificity and sensitivity for the detection of B. xylophilus and B. mucronatus. The detection limit of duplex-RPA for B. xylophilus was 10 pg/μL,which was equivalent to 1/100 single nematode;while the detection limit of duplex-RPA for B. mucronatus was 100 pg/μL,which was equivalent to 1/10 single nematode. The sensitivity was lower than that by conventional method,however,it met the requirement for detecting an individual nematode. In conclusion,duplex-RPA may simultaneously detect B. xylophilus and B. mucronatus from infected pine wood samples. Moreover,duplex-RPA has the advantages of simple operation,high detection efficiency,strong specificity,high sensitivity and low requirements for instruments and equipment,which provides a new method for quarantine and identification of B. xylophilus.

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    2021, 37(7):  191-191. 
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    2021, 37(7):  192-192. 
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