A Meloidogyne incognita effector Minc03329 suppresses plant immunity and promotes parasitism

Meloidogyne incognita is a devastating plant-parasitic nematode.Effectors play important roles during the stages of nematodes infection and parasitism,but their molecular functions remain largely unknown.In this study,we characterized a new effector,Minc03329,which contains signal peptide for secretion and a C-type lectin domain.The yeast signal sequence trap experiments indicated that the signal peptide of Minc03329 is functional.In situ hybridization showed that Minc03329 was specifically expressed in the subventral esophageal gland.Real-time qPCR confirmed that the expression level of Minc03329 transcript was significantly increased in pre-parasitic and parasitic second-stage juveniles(pre-J2s and par-J2s).Tobacco rattle virus(TRV)-mediated gene silencing of Minc03329 in host plants largely reduced the pathogenicity of nematodes.On the contrary,ectopic expression of Minc03329 in Arabidopsis thaliana significantly increased plant susceptibility to nematodes.Transient expression of Minc03329 in Nicotiana benthamiana leaves suppressed the programmed cell death triggered by the pro-apoptotic protein BAX.Moreover,the transcriptome analysis of Minc03329-transgenic Arabidopsis and wild type revealed that many defense-related genes were significantly down-regulated.Interestingly,some different expressed genes were involved in the formation of nematode feeding sites.These results revealed that Minc03329 is an important effector for M.incognita,suppressing host defense response and promoting pathogenicity.

Comparative transcriptome analysis provides insights into the molecular mechanism of the anti-nematode role of Arachis hypogaea(Fabales:Fabaceae)against Meloidogyne incognita(Tylenchida:Heteroderidae)

Background:Plant root-knot nematode(RKN)disease is a serious threat to agricultural production across the world.Meloidogyne incognita is the most prominent pathogen to the vegetables and cash crops cultivated.Arachis hypogaea can effectively inhibit M.incognita,but the underlying defense mechanism is still unclear.Methods:In our study,the chemotaxis and infestation of the second-stage juveniles(J2s)of M.incognita to A.hypogaea root tips were observed by the Pluronic F-127 system and stained with sodium hypochlorite acid fuchsin,respectively.The transcriptome data of A.hypogaea roots with non-infected or infected by J2s were analyzed.Results:The J2s could approach and infect inside of A.hypogaea root tips,and the chemotactic migration rate and infestation rate were 20.72%and 22.50%,respectively.Differential gene expression and pathway enrichment analyses revealed ubiquinone and other terpenoid-quinone biosynthesis pathway,plant hormone signal transduction pathway,and phenylpropanoid biosynthesis pathway in A.hypogaea roots responded to the infestation of M.incognita.Furthermore,the AhHPT gene,encoding homogentisate phytyltransferase,was considered to be an ideal candidate gene due to its higher expression based on the transcriptome data and quantitative real-time PCR analysis.Conclusion:Therefore,the key gene AhHPT might be involved in the A.hypogaea against M.incognita.These findings lay a foundation for revealing the molecular mechanism of A.hypogaea resistance to M.incognita and also provide a prerequisite for further gene function verification,aiming at RKN-resistant molecular breeding.

Field evaluation of Streptomyces rubrogriseus HDZ-9-47 for biocontrol of Meloidogyne incognita on tomato

Streptomyces rubrogriseus HDZ-9-47,isolated from eggs of Meloidogyne spp.,was evaluated as a potential biocontrol agent of Meloidogyne incognita under in vitro and protective field.Microscopic observations showed that HDZ-9-47 parasitized eggs of M.incognita within 7 days.In vitro,the culture filtrate of HDZ-9-47 caused 97.0%mortality of second-stage juveniles（J2s）of M.incognita and inhibited more than 50%egg hatching.In the field,compared with the control,the rootknot index and J2s density in the treatment of drench the broth contained 1012 HDZ-9-47 spores were respectively reduced by 51.1 and 80.7%at 90 days post transplantation,which were better than that in other application doses and methods.In addition,reduction rates of root-knot index and J2s density of the treatment of combined application of HDZ-9-47 with biofumigation was 87.1 and 91.0%,respectively,better than either of HDZ-9-47 or biofumigation used alone or fosthiazate treatment.And tomato yield also increased by 16.1%.Together,our results suggest that HDZ-9-47 could be an effective biocontrol agent of M.incognita,and that application of HDZ-9-47 combined with cabbage residue biofumigation was a promising and sustainable option for M.incognita control.

Sources for Heat-Stable Resistance to Southern Root-Knot Nematode (Meloidogyne incognita) in Solanum lycopersicum

Southern root-knot nematode （Meloidogyne incognita） is a major problem in vegetable production in China due to the expansion of plastic tunnel and solar greenhouse. Using resistant cultivars is an effective approach to control the disease. Nine genes, Mi-1 to Mi-9, have been reported and only Mi-1 has been successfully used in tomato breeding. However, Mi-1 is inactive at a temperature above 28~C. In order to identify sources for heat-stable resistance to southern root-knot nematode, 53 genotypes of tomato （Solarium spp.） were inoculated with an isolate of M. incognita in the growth chamber at 28 or 32℃ for initial screening. 28 lines had less than 25 galls and were considered as resistant candidates. The top 60% （16 in total） of resistant candidates obtained from each temperature were subject to re-evaluation at 32~C using the same nematode isolate. Three lines ZN17, ZN 48, and LA0385 showed heat-stable resistance with an average of 10 galls or less per plant. LA0385 is a wild species, while ZNI7 and ZN48 are elite breeding lines. These lines were grown in a greenhouse for two seasons, and also showed high resistance with less than 10 galls per plant. Thus they were considered as good sources for breeding resistance to southern root-knot nematode in tomato.

Identification and Evaluation of Resistance to Meloidogyne incognita in Bitter Gourd Germplasms

[ Objective] This study aimed to identify and evaluate the resistance of bitter gourd germplasms to Meloidogyne incognita. [ Method] A total of 71 bit-ter gourd germplasms were artificially inoculated with M. incognita at seedling stage to investigate the effects of M. incognita on resistance indexes of bitter gourd seedlings. Cluster analysis and subordinate function analysis were performed to identify and evaluate the resistance of experimental materials. [ Result] The infec-tion of M. incognita increased the variation coefficients of resistance indexes. The resistance to M. incognita varied significantly among different bitter gourd germ- plasms. Based on gall index, 71 bitter gourd germplasms were divided into five types by cluster analysis, including resistant materials, moderately resistant materi-als ,moderately susceptible materials, susceptible materials and highly susceptible materials. The subordinate function analysis showed that total subordinate func-tion values of Y107, Y109, Y108, Y51, Y105, Y81, Y89, Y140 and Y94 exceeded 1.95 , indicating high resistance to M. incognita. [ Conclusion] This study could provide resistant materials for breeding root-knot nematode resistant varieties of bitter gourd.

Potential use of cucumber (Cucumis sativus L.) endophytic fungi as seed treatment agents against root-knot nematode Meloidogyne incognita

Seed treatment with endophytic fungi has been regarded as an effective method for plant parasitic nematode control.Endophytic fungi from cucumber seedlings were isolated and screened for their potential to be used as seed treatment agents against Meloidogyne incognita.Among the 294 isolates screened,23 significantly reduced galls formed by M.incognita in greenhouse test.The 10 most effective isolates were Fusarium(5),Trichoderma(1),Chaetomium(1),Acremonium(1),Paecilomyces(1),and Phyllosticta(1) .Their control efficacies were repeatedly tested and their colonizations as well as in vitro activity against M.incognita were studied.They reduced the number of galls by 24.0%-58.4%in the first screening and 15.6%-44.3%in the repeated test,respectively.Phyllosticta Ph511 and Chaetomium Ch1001 had high colonizations on both the roots and the aboveground parts of cucumber seedlings.Fusarium isolates had colonization preference on the roots,their root colonizations ranging from 20.1%to 47.3%of the total root area.Trichoderma Tr882,Paecilomyces Pa972,and Acremonium Ac985 had low colonizations on both the roots and the aboveground parts.Acremonium Ac985,Chaetomium Ch1001,Paecilomyces Pa972,and Phyllosticta Ph511 produced compounds affecting motility of the second stage juveniles of M.incognita.Based on these results,Chae-tomium Ch1001 was considered to have the highest potential as a seed treatment agent for M.incognita biocontrol.

Molecular cloning and functional analysis of the pepper resistance gene Me3 to root-knot nematode

Root-knot nematodes(RKNs)cause severe diseases in peppers annually around the world.In pepper,the Me3 gene provides a heat-stable and broad-spectrum resistance to RKNs.In this study,several simple sequence repeat(SSR)markers and insertion/deletion(In Del)markers were developed to fine map the Me3 gene.Analysis of 2272 individuals(F2progenies)revealed that Me3 was located in a 45-kb DNA region between markers SSR784 and SSR339,in which there were three candidate genes.Among them,as a novel nucleotide binding site and leucine rich repeat(NBS-LRR)family gene,the DNA sequence of Capana09g000163 of pepper line‘HDA149’was 6348 bp in length,with a 2802-bp open reading frame encoding 933 amino acids,including NB-ARC and LRR domains.Tobacco transient expression assays demonstrated that expression of Capana09g000163 triggered a hypersensitive response(HR)in Nicotiana benthamiana leaves.Subcellular localization results showed that the Capana09g000163 protein was localized in the cell nucleus.Ectopic expression of Capana09g000163 in Arabidopsis significantly increased resistance against Meloidogyne incognita compared with the wild-type(WT)Arabidopsis.Furthermore,M.incognita was almost unable to develop in transgenic Arabidopsis expressing Capana09g000163.Taken together,we cloned the Me3 gene and verified that it induced resistance against M.incognita with the methods of map-based cloning and transgenic technology,which may be of great significance to pepper breeding for resistance against RKNs.

八种杀线剂对南方根结线虫的毒杀活性测试

为筛选出高效低毒的烟草根结线虫病防治药剂,采用浸虫法测试了8种低毒杀线(虫)剂对南方根结线虫(Meloidogyne incognita)J_(2)幼虫的毒杀活性。结果表明,8种供试药剂以阿维菌素B_(2)、噻唑膦和氟吡菌酰胺的毒杀效果较好,以50μg/mL处理24 h时线虫的校正死亡率均为100.00%。毒力测试结果表明,阿维菌素B_(2)、噻唑膦和氟吡菌酰胺处理J_(2)幼虫24 h的LC_(50)分别为0.56、8.30、6.24μg/mL,以阿维菌素B_(2)的毒杀效果最佳。

Improvement of Bio-Efficacy to Reduce Bacterial Wilt Complex Disease in Tomatoes through Bacillus amyloliquefaciens and Pseudomonas fluorescens under Field Conditions

Bacterial wilt complex disease of tomato(Solanum lycopersicum L.)was incited jointly by bacterial wilt pathogen Ralstonia solanacearum and Meloidogyne incognita worldwide.Bio-efficacy of bacterial antagonists i.e.B.amyloliquefaciens DSBA-11 and P.fluorescens DTPF-3 was studied against the wilt disease complex in tomato at National Phytotran facility Indian Agricultural Research Institute(IARI),New Delhi,at 26±2°C.Minimum wilt disease incidence(26.00%)with the highest bio-control efficacy(64.15%),less juvenile population(19.33 J2/g of soil)of M.incognita was recorded in the combined application of DTPF-3+DSBA-11 after 30 d of inoculation under glasshouse conditions.In a field study,minimum bacterial wilt disease incidences 19.0%and 20.4%were recorded in the bleaching powder treatment followed by mixed application of DSBA-11+DTPF-3,19.6%and 21.2%wilt incidence in 2014 and 2015 respectively.However,a reduction of root-knot gall index was recorded a maximum of 59.76%and 69.62%in DSBA-11+DTPF-3 treated plants followed by 54.88%and 60.13%over control in DTPF-3 treatment in 2014 and 2015 respectively.The yield of tomato fruit was increased over control by 17.48%and 16.97%in 2014 and 2015 respectively under field conditions.A combination of P.fluorescens DTPF-3+B.amyloliquefaciens DSBA-11 suppressed bacterial wilt and root-knot diseases and also increased the yield of the tomato fruit significantly(p<0.05)under field conditions.

Synthesis, nematicidal activity and docking study of novel chromone derivatives containing substituted pyrazole

A series of chromone derivatives containing substituted pyrazole were designed and synthesized.Preliminary bioassays showed that most of the synthesized compounds exhibited good nematicidal activity in vivo against Meloidogyne incognita at 10 mg/L. Among the tested compounds, A10 and A11 exhibited 100% inhibition rates. In addition, the molecular docking results indicated that both compound A10 and A11 interacts with amino acid residue Tyr121, Trp279, Tyr70, Trp84 and Phe330 of ACh E via hydrogen bond and p–p stacking. This investigation suggested that the chromone containing substituted pyrazole scaffold could be further optimized to explore novel, high-bioactivity nematicidal leads.

Synthesis of 1,2,3-benzotriazin-4-one derivatives containing spirocyclic indoline-2-one moieties and their nematicidal evaluation

To discover new chemotypes of nematicides with proper toxicological profiles, a series of novel 1,2,3-benzotriazin-4-one derivatives were synthesized and further bioevaluated. The bioassay results showed that most of the synthesized compounds were endowed with moderate to good control efficacy against Meloidogyne incognita at 10.0 mg/L in vivo. Among them, compounds 6k and 6p displayed 100%inhibitory activities at this concentration, which implied that they could be used as lead compounds for promising nematicides.