Resistance analysis of the rice variety Huaidao 5 against root-knot nematode Meloidogyne graminicola

Meloidogyne graminicola has emerged as one of the most destructive plant-parasitic nematodes affecting rice(Oryza sativa)production worldwide.Resistance to M.graminicola in rice could be the most effective option for its management.However,sources of germplasm with resistance to M.graminicola in rice remain limited.Here,we describe the root attraction,gall formation and genetic analysis of the resistance to M.graminicola in the rice variety Huidao 5.A nematode attraction assay showed that second-stage juveniles(J2s)of M.graminicola were attracted at the root tip of Huaidao 5 within 8 h without a significant reduction in attraction compared to the susceptible rice variety Nanjing 9108.Microscopic observation of the infection revealed that the J2s invaded root tissues 12 h after inoculation,but their subsequent movement to the root tip was hindered in Huaidao 5,resulting in decreased nematode number compared to Nanjing 9108.Additionally,we used the soil and hydroponic culture systems to simulate upland and flooding conditions in the paddy fields respectively,and found that gall number was significantly reduced,and nematode development was clearly suppressed in Huaidao 5.To investigate the genetic basis of this resistance,cross breeding was performed between the Huaidao 5 and Nanjing 9108 varieties.There was no reduction in the resistance of the F_(1) offspring to M.graminicola in the greenhouse or field trials,suggesting that a dominant gene could control resistance in Huaidao 5.In summary,this study provides a detailed characterization of a novel source of resistance to M.graminicola in rice,which is of great potential for use in crop breeding.

mgr-mir-9 implicates Meloidogyne graminicola infection in rice by targeting the effector MgPDI

MicroRNAs (miRNAs),a class of small non-coding RNAs,are crucial endogenous gene regulators in a range of animals,including plant-parasitic nematodes.Meloidogyne graminicola is an obligate sedentary endoparasite of rice and causes significant yield losses.A number of studies focused on the roles of M.graminicola effectors during the parasitic process;however,how nematode miRNAs regulate its effectors needs elucidating.In this research,we analyzed a cluster of M.graminicola miRNAs obtained at the second-stage juveniles (J2s) stage that are closely linked to the regulation of M.graminicola effectors.There are 49 767 105 total clean reads obtained from three libraries.A total of 233 known miRNAs and 21 novel miRNAs were identified.Among the known miRNAs,mgr-lin-4,mgr-mir-1,mgr-mir-100,mgrmir-86,mgr-mir-279,mgr-mir-87,mgr-mir-71,mgr-mir-9,mgr-mir-50,mgr-mir-72,and mgr-mir-34 are the most abundant11 miRNAs families.Moreover,the expression levels of selected miRNAs were validated by real-time quantitative PCR.We hypothesized that these miRNAs might regulate the expression of secreted effectors during the J2s stage to facilitate its infection.Consistent with this,we found that mgr-mir-9 targets MgPDI,an important M.graminicola effector mRNA.In addition to that,J2s treated with mgr-mir-9 mimics showed down-regulation of MgPDI expression and reduced reproductive ability,alluding mgr-mir-9 is involved in nematode infection.These results provide novel insight into the regulatory functions of M.graminicola miRNAs during the infection and identify miRNAs and their effector targets as potential key management targets to limit parasite survival during the early stages of infection.

Potential global distribution of the guava root-knot nematode Meloidogyne enterolobii under different climate change scenarios using MaxEnt ecological niche modeling

In recent years,Meloidogyne enterolobii has emerged as a major parasitic nematode infesting many plants in tropical or subtropical areas.However,the regions of potential distribution and the main contributing environmental variables for this nematode are unclear.Under the current climate scenario,we predicted the potential geographic distributions of M.enterolobii worldwide and in China using a Maximum Entropy(MaxEnt)model with the occurrence data of this species.Furthermore,the potential distributions of M.enterolobii were projected under three future climate scenarios(BCC-CSM2-MR,CanESM5 and CNRM-CM6-1)for the periods 2050s and 2090s.Changes in the potential distribution were also predicted under different climate conditions.The results showed that highly suitable regions for M.enterolobii were concentrated in Africa,South America,Asia,and North America between latitudes 30°S to 30°N.Bio16(precipitation of the wettest quarter),bio10(mean temperature of the warmest quarter),and bio11(mean temperature of the coldest quarter)were the variables contributing most in predicting potential distributions of M.enterolobii.In addition,the potential suitable areas for M.enterolobii will shift toward higher latitudes under future climate scenarios.This study provides a theoretical basis for controlling and managing this nematode.

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.

新颖杀线虫剂MCW-2对爪哇根结线虫(Meloidogyne javanica)的防治活性

随着甲基溴等高效土壤熏蒸剂以及非熏蒸杀线虫剂的退出或禁用,当前线虫的防治难度日益加大。由于杀线虫剂市场小于除草剂、杀菌剂和杀虫剂,许多农化企业认为开发新颖杀线虫剂无利可图。此外,对人类与非靶标生物安全的有效物质的开发可行性极低。目前,广泛应用的杀线虫剂有涕灭威、硫线磷、克百威、丙线磷、苯线磷、噻唑磷、

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.

Evaluation of Chinese rice varieties resistant to the root-knot nematode Meloidogyne graminicola

The root-knot nematode Meloidogyne graminicola, which is distributed worldwide, is considered a major constraint on rice production in Asia. The present study used the root gall index and number of nematodes inside the roots to evaluate resistance/susceptibility to M. graminicola in different subpopulations of Oryza sativa （aus, hybrid aus, indica, hybrid indica, temperate japonica, tropical japonica）. Nematode development in highly resistant varieties was also evaluated. Analyses of randomly selected 35 varieties showed the number of M. graminicola nematodes inside the roots correlated very strongly （r=0.87, P≤0.05） with the nematode gall index, and the results from pot and field experiments revealed similar rankings of the varieties for resistance/susceptibility. Among the 136 tested varieties, temperate japonica displayed the highest gall index, followed by tropical japonica, indica, hybrid indica, aus, and hybrid aus. Zhonghua 11 （aus）, Shenliangyou 1 （hybrid aus） and Cliangyou 4418 （hybrid indica） were highly resistant to M. graminicola under both pot and field conditions. Further examination of nematode development suggested that compared to susceptible rice, M. graminicola penetrated less often into highly resistant varieties and more frequently failed to develop into females. The promising varieties found in the present research might be useful for the breeding of hybrid rice in China and for the further development of practical nematode management measures.

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.

Evaluation of the biocontrol potential of Aspergillus welwitschiae against the root-knot nematode Meloidogyne graminicola in rice(Oryza sativa L.)

The root-knot nematode Meloidogyne graminicola is considered one of the most devastating pests in rice-producing areas,and nematicides are neither ecofriendly nor cost effective.More acceptable biological agents are required for controlling this destructive pathogen.In this study,the biocontrol potential of Aspergillus welwitschiae AW2017 was investigated in laboratory and greenhouse experiments.The in vitro ovicidal and larvicidal activities of A.welwitschiae metabolites were tested on M.graminicola in laboratory experiments.The effect of A.welwitschiae on the attraction of M.graminicola to rice and the infection of rice by M.graminicola was evaluated in a greenhouse.The bioagent AW2017 displayed good nematicidal potential via its ovicidal and larvicidal action.The best larvicidal activity was observed at a concentration of 5×AW2017,which caused an 86.2%mortality rate at 48 h post inoculation.The highest ovicidal activity was recorded at a concentration of 5×AW2017,which resulted in an approximately 47.3%reduction in egg hatching after 8 d compared to the control.Under greenhouse conditions,the application of A.welwitschiae significantly reduced the root galls and nematodes in rice roots compared to the control.At a concentration of 5×AW2017,juveniles and root galls in rice roots at 14 d post inoculation(dpi)were reduced by 24.5 and 40.5%,respectively.In addition,the attraction of M.graminicola to rice roots was significantly decreased in the AW2017 treatment,and the development of nematodes in the AW2017-treated plants was slightly delayed compared with that in the PDB-treated control plants.The results indicate that A.welwitschiae is a potential biological control agent against M.graminicola in rice.

Morphological and molecular characterization of the rice root-knot nematode, Meloidogyne graminicola, Golden and Birchfeild, 1965 occurring in Zhejiang, China

The rice root-knot nematode Meloidogyne graminicola is a severe pest of rice. In China, it was first reported from Hainan Province, and later from several other provinces. In the present study, a rice root-knot nematode population found from the rice cultivation areas of Zhejiang Province, China is characterized via molecular analysis using internal transcribed spacer(ITS) and cytochrome c oxidase subunit Ⅱ(coxⅡ)-16 S rRNA genes and scanning electron microscopy(SEM) observations of males and the second-stage juveniles. Morphometric data and molecular sequence comparisons for all M. graminicola populations occurring in China are also provided. The overall morphology of M. graminicola found in Zhejiang match well with the original description, though males have a slightly longer body and stylet, and a shorter tail, while the second-stage juvenile is also slightly longer than in the original description. This is the first report of M. graminicola from Zhejiang. Phylogenetic studies based on coxⅡ suggest that all the Chinese populations belong to Type B. This study expands knowledge of the increasing distribution and phylogenetic relationships of M. graminicola that occur in China.

Potassium sulphate induces resistance of rice against the rootknot nematode Meloidogyne graminicola

Potassium(K),an important nutrient element,can improve the stress resistance/tolerance of crops.The application of K in resisting plant-parasitic nematodes shows that the K treatment can reduce the occurrence of nematode diseases and increase crop yield.However,data on K_(2)SO_(4)induced rice resistance against the root-knot nematode Meloidogyne graminicola are still lacking.In this work,K_(2)SO_(4)treatment reduced galls and nematodes in rice plants and delayed the development of nematodes.Rather than affecting the attractiveness of roots to nematodes and the morphological phenotype of giant cells at feeding sites,such an effect is achieved by rapidly priming hydrogen peroxide(H_(2)O_(2))accumulation and increasing callose deposition.Meanwhile,galls and nematodes in rice roots were more in the potassium channel OsAKT11 and transporter OsHAK5 gene-deficient plants than in wild-type,while the K_(2)SO_(4)-induced resistance showed weaker in the defective plants.In addition,during the process of nematode infection,the expression of jasmonic acid(JA)/ethylene(ET)/brassinolide(BR)signaling pathway-related genes and pathogenesis-related(PR)genes OsPR1 a/OsPR1 b was up-regulated in rice after K_(2)SO_(4)treatment.In conclusion,K_(2)SO_(4)induced rice resistance against M.graminicola.The mechanism of inducing resistance was to prime the basal defense and required the participation of the K^(+)channel and transporter in rice.These laid a foundation for further study on the mechanism of rice defense against nematodes and the rational use of potassium fertilizer on improving rice resistance against nematodes in the field.

温度对南昌市郊区南方根瘿线虫(Meloidogyne incognita)发育的影响

一、前言寄生于植物并导致严重病害的根瘿线虫(Meoloidogyne)是变温动物。了解这类动物二期幼虫侵入寄主植物之后的发育起点温度及达到性成熟而产卵所需的有效积温,不但在理论上而且在生产上都有重要意义。然而有关这方面的文献不多,提出过报告的作者有Tyler(1933)乌斯基诺夫,Milne和Plessis(1964),Starr和mai(1976),Bird和Milln(1979)。南昌市郊侵害多种蔬菜作物的根瘿线虫是M.incognita,值得注意的是,上述作者中只有Bird和Milln在澳大利亚对这个种进行过观察。该作者的观察结果是否适合于南昌市郊这个居群?我们在防治实践中应该认定的数据是什么?问题必须予以回答。

RNAi of <i>MiASB</i>caused high mortality of <i>Meloidogyne incognita</i>juveniles and inhibited the nematode disease

The southern root-knot nematode, Meloidogyne incognita, is one of the most prevalent and damaging plant-parasitic nematodes in the world and causes serious damages to agricultural production. We cloned a mitochondrial ATP synthase b subunit gene fragment of M. incognita (MiASB) based on the nematode genomics prediction. By soaking in the MiASB dsRNA solution, the hatching of RNAi treated eggs was reduced by 60% compared to negative control and by 64% compared to untreated control. Mortality of RNAi treated second stage juvenile (J2) was 8.6 times higher than that of negative control and 26 times higher than the untreated control. Inoculating the RNAi treated egg masses and J2 to tomato seedlings showed the pathogencity was significantly reduced. For the RNAi treated egg masses, the amount of root galls on silence treated seedlings was reduced by 92% compared to that on the negative control seedlings, and reduced by 93% compared to that on untreated control seedlings. For the treated J2, the amount of root galls on silence treated seedlings was reduced by 83% and 86% compared to negative and untreated control seedlings, respectively. The study revealed the MiASB silence had a positive effect on prevention and control of root-knot nematode disease, and also showed that the MiASB may be involved in the pathogenesis of nematode, which provided new ideas and ways to the research of nematode pathology and nematode disease control.

Study on PCR rapid molecular detection technique of Meloidogyne vitis

Meloidogyne vitis is a new root-knot nematode parasitic on grape root in Yunnan Province,China.In order to establish a rapid,reliable and specific molecular detection method for M.vitis,the species-specific primers were designed with rDNA-ITS(ribosomal DNA internal transcribed spacer)gene fragment as the target.The reaction system was optimized and the reliability,specificity and sensitivity of primer were testified,therefore,a rapid PCR detection method for M.vitis was established.The result showed that the optimal annealing temperature of the primers was 53℃,which was suitable for the detection of different life stages of M.vitis.Specificity test showed that the specific fragment size of 174 bp was obtained from M.vitis,but other five non-target nematodes did not have any amplification bands,thus effectively distinguish M.vitis and the other five species,and could specifically detect the M.vitis from mixed populations.Sensitivity test showed that this PCR technique could detect the DNA of a single second-stage juvenile(J_(2))and 10^(-4)female.Futhermore,this PCR technique could be used to detect directly M.vitis from soil samples.The rapid,sensitive and specific PCR molecular detection technique could be used for the direct identification of a single J_(2)of M.vitis and the detection of M.vitis in mixed nematode populations and the detection of two J_(2)s or one male in 0.5 g soil samples,which will provide technical support for the investigation of the occurrence and damage of M.vitis and the formulation of efficient green co ntrol strategies.

Effect of Pleurotus ostreatus on Dynamics of Meloidogyne arenaria Population and Control Effectiveness

The control effects and application methods of Pleurotus ostreatus on the peanut root-knot nematode disease, Meloidogyne arenaria, were systematically studied in the greenhouse. Results of pot experiments showed that P. ostreatus could effectively reduce the 2nd-instar population density in the soil, and the larva reduced by 80% compared with the control. P. ostreatus could markedly lower the infecting number of the peanut root-knot nematodes, the nematode density inside the root knots declined by 40.00% -82.64%, and the peanut root knots also reduced by 86.96% - 94. 03%. The fungus could defer the initial infection of M. arenaria to the roots, the epidemic rate of the peanut root-knot nematode disease was significantly cut down, and the increase of root-knot changed from the way of Logistic curve in the control to the way of linear curve in the treatment. The final control effectiveness was about 80%. The results of pot trial also indicated that the control effectiveness is closely related to the application time and the amount of this fungus. The key factor affecting control effectiveness was the application time of P. ostreatus in the soil and the suitable time was 20 days before sowing.

Interactions between a Root Knot Nematode (<i>Meloidogyne exigua</i>) and Arbuscular Mycorrhizae in Coffee Plant Development (<i>Coffea arabica</i>)

This paper focuses on parasitic root knot nematodes (Meloidogyne exigua) and how to decrease their pathogenic effect on coffee plants (Coffea arabica), by examining the behaviour of and the interactions between nematodes, coffee plant and arbuscular mycorrhizae (AM). The experiment was carried out at the seedling stage, with six (6) treatments (plants with M. exigua, plants with arbuscular mycorrhizae, plants with both organisms, and the same time, first mycorrhizae plants, then nematodes were inoculated and vice versa). After 5 months the measured variables were: dry biomass (roots and shoot), nematode knots caused by M. exigua in root, nematode juvenile (J2) found in 100.0 g of soil, and mycorrhizal percentage. Plant nutrients (P and N) contents were analysed. Significant differences were found in all the variables, but concentration N content in plants. Plants with mycorrhizae and plants with mycorrhizae and then inoculated with nematodes have the same behaviour. Control plants and plants with nematode and then inoculated with mycorrhizae behave similarly. It is thought that arbuscular mycorrhizae are formed before the nematode infestation, allowing coffee plants to regain the energy lost by the parasitic interaction. AM may help coffee plants with lignifications of the plant cell wall cuticle. As the cuticle thickens it is more difficult for nematodes to penetrate and enter into plant roots. Therefore, arbuscular mycorrhizae help coffee plants to uptake and transport nutrients, improving its nutritional status and stabilizing nematode attacks. It is suggested that symbiotic interactions help neutralize parasitic interactions.

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.

Comparative study of <i>Fusarium oxysporum</i>f sp. <i>lycopersici</i>and <i>Meloidogyne incognita</i>race-2 on plant growth parameters of tomato

Many species of soil-inhabiting fungus Fusarium, cause severe yield loss in many crops. Experiments were conducted in net house condition with complete randomized block design to determine the individual effect of different in-oculum levels of root-knot nematode, Meloidogyne incognita, Race-2 and Fusarium oxysporum f sp. lycopersici on plant growth parameters viz., Plant length, fresh and dry weight and number of fruits of tomato var. P21. The experimental results showed that both the pathogens cause significant reduction in plant growth parameters. However, the fungus was not much effective on plant growth parameters in comparison to root-knot nematode. Greatest reduction in plant growth parameters was recorded in plants inoculated with 8000 J2/Kg soil of Meloidogyne in-cognita race 2. The threshold level of root-knot nematode was 1000 J2/kg soil while threshold level of Fusarium was @ 1 g/Kg soil. Inoculum level of Fusarium oxysporum f sp. lycopersici and Meloidogyne in-cognita race-2 was pathogenic and caused significant reduction at and above 1 g/kg soil and 1000 J2/Kg soil respectively.