Bacterial artificial chromosome library construction of root-knot nematode resistant pepper genotype HDA149 and identification of clones linked to Me3 resistant locus

Pepper （Capsicum annuum. L.） is a widely cultivated vegetable crop worldwide and has the second largest planting area and the first largest vegetable output and value in China. Pepper root-knot nematode （Meloidogyne spp.） is one of the most serious pests of pepper, which caused huge losses every year. Previous studies showed that the Me3 gene is resistant to a wide range of Meloidogyne species, including M. arenaria, M. javanica, and M. incognita. HDA149, a double haploid pepper genotype, harboring the root-knot nematode resistance gene Me3, was used to construct bacterial artificial chro- mosome library （BAC） via the vector of CopyControFM pCC1 in this study. The library consists of 210 200 BAC clones and is equivalent to 5.3 pepper genomes. The average insert size is 95 kb, and most of them are 90-120 kb; but the empty clones are less than 3%. In order to screen the BAC library easily, 550 super pools with 384 BAC clones of each pool were further developed in this study. Specific primers from Me3 gene locus were used for BAC library screening, and more than 20 positive BAC clones were obtained. Then the selected positive BAC clones were analyzed by restriction enzyme digestion, BAC-end sequencing, marker development, and new positive BAC clones exploration, respectively. Finally, the contig with total length of about 300 kb linked to the Me3 locus was constructed based on chromosome walking strategy, which made a solid foundation for the cloning of the important root-knot nematode resistance gene Me3.

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.

A fragment of a 70-kDa Heterodera glycines heat shock protein(HgHSP70)interacts with soybean cyst nematode-resistant GmSHMT08

Soybean cyst nematode(SCN)Heterodera glycines is considered as the major constraint to soybean production.Gm SHMT08 at Rhg4 locus on chromosome 08,encoding a serine hydroxylmethyltransferase,is a major gene underlying resistance against H.glycines in Peking-type soybeans.However,the molecular mechanism underpinning this resistance is less well characterized,and whether Gm SHMT08 could interact with proteins in H.glycines remains unclear.In this study,yeast two-hybrid screening was conducted using Gm SHMT08 as a bait protein,and a fragment of a 70-kDa heat shock protein(Hg HSP70)was screened from H.glycines that exhibited interaction with Gm SHMT08.This interaction was verified by both GST pull-down and bimolecular fluorescence complementation assays.Our finding reveals Hg HSP70 could be applied as a potential candidate gene for further exploring the mechanism on Gm SHMT08-mediated resistance against SCN H.glycines.

Genetic Mapping of Root-knot Nematode Resistance Genes in Solanaceous Vegetables

Root-knot nematodes are becoming more and more harmful to solanaceous vegetables. The most economical and effective way to deal with root-knot nematode disease is to breed varieties with disease resistance. This paper reviewed the recent research progress and achievements of tomato, pepper, eggplant and potato on root-knot nematode disease resistance gene characteristics, disease resistance gene mapping and disease resistance gene molecular markers, and prospects for future research directions.

Study on the Activity of Herbal Extract to Kill Root-knot Nematode of Cucumber Seedlings Stage

Present mature plants hydroponic technology was used,combined with some excellent characteristics,such as growth conditions was easy to control and process of root growth was easy to continuously observe,the nematicidal activity of 5 kinds of Chinese herbs extracts and the compound solution of Avermectin,with strong contact toxicity effect indoor,was systematically studied and investigated the affection on the root-knot nematode parasitized on the cucumber seeding stage. It is found that under the premise of no influence on root growth of cucumber,extracts from Picrorhiza scrophulariiflora and Punica granatum showed strong prevention and nematicidal activity,and had the similar efficacy of Avermectin; while the extracts from Cibotium barometz,Aucklandia lappa Decne and Fructus cnidii showed low nematicidal activity and various degrees inhibition effect on plant growth.

Investigation on Pathogenic Factors of Tobacco Root-Knot Nematode Disease in Gengma County,Yunnan Province

Tobacco root-knot nematode disease has caused severe damage in Geng- ma County, Yunnan Province. In order to identify the pathogenic factors of the tobac- co root-knot nematode disease in this county, the pathogenic nematodes, hosts and environment of tobacco fields in Mengsa, Hepai and Sipaishan 3 main tobacco-grow- ing towns in Gengma County were investigated and analyzed based on the local re- lated field survey on tobacco root-knot nematode disease in this county in 2012. The results showed the incidence and severity of the tobacco root-knot nematode disease were all higher than those of previous years. dominant pathogens of the tobacco root-knot The species identification showed the nematode disease were Meloidogyne arenaria and M. javanica in Gengma County. The lacking of disease-resistant culti- vars, poor management and climatic anomaly were the main causes of the tobacco root-knot nematode disease in Gengma. According to the occurrence characteristics of the disease, the agricultural prevention-based control measures were proposed.

Effect of Anti-nematode Preparations on Physiological Traits of Tomato Leaves Affected by Root-knot Nematode

[ Objective ] The paper was to study the effects of anti-nematode preparations with different mechanisms on changes of enzyme systems and membrane permeability of tomato leaves, so as to provide reference basis for effective control of soil root-knot nematode in greenhouse. [ Method] With tomato seedlings af- fected by root-knot nematode as material, changes of superoxide dismutase（SOD）, peroxidase（ POD）, relative conductivity and malondialdehyde （MDA） in toma- toes were tested after the seedling soil was treated by preparations of Wuxianmei, Hailvsu, Duxiandna and Avermectin. [ Result] After treated by different prepara- tions, SOD and POD activity of tomato leaves were higher than control, and that treated by Wuxianmei was the highest. In addition to Duxiandna, the relative con- ductivity and MDA content of other treatments were significantly lower than control. When tomatoes were planted for 70 d, the effect of Avermectin against reot-knot nematode Was the best of 66.3%. [ Conclusion] After tomatoes were infected by root-knot nematode, different preparation treatments all had certain control effect, which made the physical indicators of tomato have obvious change. Integrated control of multiple preparations in greenhouse was beneficial to control soil root-knot nematode.

Study on Controlling Tobacco Root-knot Nematode by Fertilizer and Pesticide Synergy

Synergism between pesticide and fertilizer in controlling root-knot nematode was investigated by field randomized block test. The results showed that the best control efficiency was shared by 0.5% abamectin and 25% mixed pesticide （abamectin and carbosulfan）, 24.1% and 28.0%, respectively. The positive effect was found by synergism between pesticide and fertiUzer in controlling reot-knot nematode. The economic benefit and agricultural benefit were gained by controlling root-knot nematodes. The results suggested that the agricultural and ecological benefit could be gained through the integration of agricultural practice and chemical technology in controlling root-knot nematode.

The synergistic advantage of combining chloropicrin or dazomet with fosthiazate nematicide to control root-knot nematode in cucumber production

The highly-damaging root-knot nematode(Meloidogyne spp.,RKN)cannot be reliably controlled using only a nematicide such as fosthiazate because of increasing pest resistance.In laboratory and greenhouse trials,we showed that chloropicrin(CP)or dazomet(DZ)synergized the efficacy of fosthiazate against RKN.The combination significantly extended the degradation half-life of fosthiazate by an average of about 1.25 times.CP or DZ with fosthiazate reduced the time for fosthiazate to penetrate the RKN cuticle compared to fosthiazate alone.CP or DZ combined with low or medium rate of fosthiazate increased the total cucumber yield,compared to the use of each product alone.A low-dose fosthiazate with DZ improved total yield more than a low dose fosthiazate with CP.Extending the half-life of fosthiazate and reducing the time for fosthiazate or fumigant to penetrate the RKN cuticle were the two features that gave the fumigant-fosthiazate combination its synergistic advantage over these products used singularly.This synergy provides the opportunity for farmers to use a low dose of fosthiazate which lowers the risk of RKN resistance.Farmers could combine DZ at 30 g m^-2 with fosthiazate at a low rate of 0.375 g m^-2 to control RKN and adequately control two major soil-borne diseases in cucumber greenhouses.

Effects of Two Composted Plant Pesticide Residues,Incorporated with Trichoderma viride,on Root-Knot Nematode in Balloonflower

Plant pesticide residues, such as chinaberry （Melia toosendan） residue and sand cypress （Sabina vulgaris） residue, are pesticidal plant materials discarded after the bioactive ingredient has been extracted with organic solvents. The only option for botanical pesticide residue utilization has been as landfill. Chinaberry residue （CBR） and sand cypress residue （SCR） were collected and composted in Yangling, Shaanxi Province, China. We studied the effects of chinaberry residue compost （CBRC）, CBRC incorporated with Trichoderma viride （CBRCT）, sand cypress residue compost （SCRC）, and SCRC incorporated with T. viride （SCRCv） on the root-knot nematode, Meloidogyne incognita, infesting the balloonflower （Platycodon grandiflorum）. Bioassay results indicated that stock solutions of the CBRCT and SCRCT extracts significantly inhibited egg hatching and caused high larval mortality, followed in degree by the CBRC and SCRC extracts. The CBR and SCR extracts caused very low inhibition of eggs and larvae. Supplementing potting mixtures with these four composts reduced the severity of root galling and increased the proportion of marketable roots. The severity of root galling decreased and the average weight of the marketable roots increased with an increase in all the composts when supplemented at rates from 5 to 30%. CBR- and SCR-supplemented pot soils also inhibited the nematodes, but CBR and SCR applied to the soil had a phytotoxic effect and inhibited balloonflower growth. Supplementing field soil with the composts reduced the severity of root galling and the populations of southern root-knot nematodes in the soil. CBRCT and SCRCT clearly enhanced the average weight of the marketable roots by 30.45 and 26.64%, respectively. Continuous supplementation with CBRCT or SCRCT in the same field significantly enhanced the control of the root-knot nematode, and the populations of nematodes continued to decrease with second inoculations. The populations of total Trichoderma spp. were distinctly enhanced and were maintained at high levels for a long time in the supplemented soils.

Chemical mutagenesis and soybean mutants potential for identification of novel genes conferring resistance to soybean cyst nematode

The resistance of soybean(Glycine max(L.) Merr.) to soybean cyst nematode(SCN, Heterodera glycines Ichinohe), which is a devastating pathogen in soybean production and causes a large quantity of annual yield loss worldwide, can shift during the long-term interaction and domestication. It is vital to identify more new resistance genetic sources for identification of novel genes underlying resistance to SCN for management of this pathogen. In the present study, first, two ethane methylsulfonate-mutagenesis soybean M2 populations of PI 437654, which shows a broad resistance to almost all of SCN races, and Zhonghuang 13, which is a soybean cultivar in China conferring strong resistance to lodging, were developed. Many types of morphological phenotypes such as four-and five-leaflet leaves were observed from these two soybean M2 populations. Second, 13 mutants were identified and confirmed to exhibit alteration of resistance to SCN race 4 through the forward genetic screening of 400 mutants of the PI 437654 M2 population, the rate of mutants with alteration of SCNinfection phenotype is 3.25%. Third, these identified mutants were further verified not to show any changes in the genomic sequences of the three known SCN-resistant genes, GmSHMT08, GmSNAP18 and GmSANP11, compared to the wildtype soybean; and all of them were still resistant to SCN race 3 similar to the wild-type soybean. Taken together, we can conclude that the 13 mutants identified in the present study carry the mutations of the new gene(s) which contribute(s) to the resistance to SCN race 4 in PI 437654 and can be potentially used as the genetic soybean sources to further identify the novel SCN-resistant gene(s).

Enhanced Stem Nematode Resistance of Transgenic Sweetpotato Plants Expressing Oryzacystatin-I Gene

Enhanced stem nematode resistance of transgenic sweetpotato （cv. Lizixiang） was achieved using Oryzacystatin-I （OCI） gene with Agrobacterium tumefaciens-mediated transformation. A. tumefaciens strain EHA105 harbors a binary vector pCAMBIA1301 with OCI gene, gusA gene and hptII gene. Selection culture was conducted using 25 mg L-1 hygromycin. A total of 1 715 plants were produced from the inoculated 1 450 cell aggregates of Lizixiang via somatic embryogenesis. GUS assay and PCR analysis of the putative transgenic plants randomly sampled showed that 90.54% of them were transgenic plants. Transgenic plants exhibited significantly enhanced resistance to stem nematodes compared to the untransformed control plants by the field evaluation with stem nematodes. Stable integration of the OCI gene into the genome of resistant transgenic plants was confirmed by Southern blot analysis, and the copy number of integrated OCI gene ranged from 1 to 4. Transgene overexpression in stem nematode-resistant plants was demonstrated by quantitative real-time PCR analysis. This study provides a way for improving stem nematode resistance in sweetpotato.

Hatching and development of maize cyst nematode Heterodera zeae infecting different plant hosts

The occurrence, distribution, and rapid molecular detection technology of Heterodera zeae Koshy et al. 1971, have been reported in China. We explored the biological characteristics of H. zeae sampled in Henan Province, China to understand its interaction with plants. Cysts and second-stage juveniles(J2s) were identified under an optical and scanning electron microscope, internal transcribed spacer(ITS) phylogenetic tree, and sequence characterized amplified region(SCAR)-PCR analyses. The optimum hatching temperatures of H. zeae were 30°C and 28°C, with cumulative hatching rates of 16.5 and 16.1%, respectively, at 30 days post-hatching(dph). The hatching rate of H. zeae eggs was improved by 20-and 50-time maize soil leachate and root juice, and 10-time root exudates. The hatching rate in 10-time root exudates was the highest(25.9%). The 10-time root exudates of maize and millet produced the highest hatching rate at 30 dph(25.9 and 22.9%, respectively), followed by wheat(19.9%), barley(18.3%), and rice(17.6%). Heterodera zeae developed faster in maize than in other crops. Fourth-stage juveniles(J4s) were detected in maize roots 8 days post-inoculation(dpi) at 28°C but not in other crops. Combined with hatching tests, the Huang–Huai–Hai summer maize region and the south and central-southwest mountainous maize areas are highly suitable for H. zeae in China. This is the first systematically study of the hatching and infection characteristics on different plant hosts of corn cyst nematode H. zeae in temperate regions. This study laid a theoretical foundation for the rapid spread and high environmental adaptability of corn cyst nematode.

Preparation and efficacy evaluation of Paenibacillus polymyxa KM2501-1 microbial organic fertilizer against root-knot nematodes

Root-knot nematodes(RKNs) cause huge yield losses to agricultural crops worldwide. Meanwhile, livestock manure is often improperly managed by farmers, which leads to serious environmental pollution. To resolve these two problems, this study developed a procedure for the conversion of chicken manure to organic fertilizer by larvae of Hermetia illucens L. and Bacillus subtilis BSF-CL. Chicken manure organic fertilizer was then mixed thoroughly with Paenibacillus polymyxa KM2501-1 to a final concentration of 1.5×10^(8)CFU g^(-1). The efficacy of KM2501-1 microbial organic fertilizer in controlling root-knot nematodes was evaluated in pot and field experiments. In pot experiments, applying KM2501-1 microbial organic fertilizer either as a base fertilizer or as a fumigant at the dose of 40 g/pot suppressed root-knot disease by 61.76 and 69.05% compared to the corresponding control treatments, respectively. When applied as a fumigant at the dose of 1 kg m;in field experiments, KM2501-1 microbial organic fertilizer enhanced the growth of tomato plants, suppressed root-knot disease by 49.97%, and reduced second stage juveniles of RKN in soil by 88.68%. KM2501-1 microbial organic fertilizer controlled RKNs better than commercial bio-organic fertilizer in both pot and field experiments. These results demonstrate that this co-conversion process efficiently transforms chicken manure into high value-added larvae biomass and KM2501-1 microbial organic fertilizer with potential application as a novel nematode control agent.

Control of Southern Root-knot Nematodes on Tomato and Regulation of Soil Bacterial Community by Biofumigation with Zanthoxylum bungeanum Seed

Biofumigation is an environmentally friendly strategy used to control nematodes and plant diseases.The volatile oil of Zanthoxylum bungeanum has high insecticidal and antibacterial activity.However,it is not known if the seed of Z.bungeanum is a suitable material for biofumigation to control southern root-knot nematodes(SRKN)on tomato,and how it may regulate the soil bacterial community structure.We used pot experiments in the greenhouse to determine the effects of Z.bungeanum seeds on SRKN,plant growth parameters,soil physicochemical and microbial characteristics.A total of 26 volatile components,including nematicidally active substances,were identified from Z.bungeanum seeds.Z.bungeanum seed biofumigation significantly reduced the SRKN population by 88.89%and 81.55%on the 50th and 100th day after transplanting,respectively.Compared to the control,the total soluble sugar,soluble solids,soluble protein,titratable acid,root activity and the fruit yield per tomato plant increased significantly.The content of soil alkali-hydrolyzed nitrogen,available potassium and the soil enzyme activities were also significantly increased.The soil bacterial diversity and the co-occurrence network complexity were increased by Z.bungeanum seed biofumigation.Relativelymore keystone OTUs in biofumigation soil had potential plant growth-promoting capabilities.The function of Z.bungeanum seed increasing tomato production in SRKN-infected soil depends on directly killing SRKN and improving soil properties.These results indicate that Z.bungeanum seed can be used as both a nematicide and a high quality organic fertilizer in tomato production.

Breeding Soybeans for Resistance to Physiological Race 4 of Cyst Nematode

Soybean cyst nematode causes serious damage to soybean production. In 1991, we started breeding studies on the resistance of soybeans to the cyst nematode. We found that near the Beijing area the dominant race of the cyst nematode was race 4. We made more than 50 combinations of cross. The best combination was Dan 8 X PI 437654 which resulted in marked segregation in plant height, pod habit, resistance to cyst nematode and maturity. We obtained many new soybean lines highly resistant to the cyst nematode through the pedigree method of selection, enlarging the number of plants of good combinations, alternative breeding in the North and in the South, and identification at an early generation. We now have released three soybean cultivars, Zhonghuang 12, Zhonghuang 13 and Zhonghuang 17 with moderate resistance to the cyst nematode in Beijing, Anhui, Tianjin and Northern China. In addition, we obtained many lines which were highly resistant to the cyst nematode.

Micropropagation of Pinus densiflora and the evaluation of nematode resistance of regenerated microshoots in vitro

To accelerate the breeding and selection of Pinus densiflora Siebold and Zucc. resistance to pine wilt disease, a micropropagation system was established and nematode resistance evaluated in vitro. Cotyledon-hypocotyl explants from 28-day-old seedlings were first cultured on Gresshoff and Doy medium supplemented with 4.0 mg L^(-1) 6-benzyladenine and 0.2 mg L^(-1) a-naphthaleneacetic acid(NAA) to stimulate the formation of buds. Induced buds were subsequently subcultured on Gupta and Durzan medium supplemented with 0.1%(w/v)activated charcoal for elongation. Stem sections derived from shoots were used as explants for the further multiplication. Roots were formed from shoots transferred to woody plant medium containing 0.2 mg L^(-1) NAA for4 weeks. The nematode resistance test showed that symptoms in micropropagated shoots after infection with pine wood nematode(PWN) were similar to those in plants infected in the field. The wilting rate varied from 20 to100% among different clones 18 days after inoculation.The most susceptible clone was Clone 6-4 with a 100%wilting rate, while Clone 8-4 showed a relatively high resistance with a 20% wilting rate. The number of nematodes recovered from Clone 8-4 shoots was significantly lower(P = 0.05) than from Clones 5-10 and 16-4. This work contributes to the breeding of PWN resistance in P.densiflora.

Soybean cyst nematode-resistance: Gene identification and breeding strategies

Soybean cyst nematode(SCN,Heterodera glycines Ichinohe)is the most economically damaging disease of soybean worldwide,and breeding host plant resistance is the most feasible option for SCN management.In this review,we summarise the progress made so far in identifying nematode-resistance genes,the currently available sources of resistance,possible mechanisms of SCN resistance and strategies for soybean breeding.To date,only two sources of SCN resistance have been widely used,from the accessions PI 88788 and Peking,which has resulted in a shift in SCN resistance and created a narrow genetic base for SCN resistance.These resistant germplasms for SCN are classified into two types according to their copy number variation in a 31-kb genomic region:PI 88788-type resistance requires high copy numbers of a rhg1 resistance allele(rhg1-b)and Peking-type resistance requires both low copy numbers of a different rhg1 resistance allele(rhg1-a)and a resistant allele at another locus,Rhg4.Resistance related to rhg1 primarily involves impairment of vesicle trafficking through disruption of soluble NSF-attachment protein receptor(SNARE)complexes.By contrast,resistance via Rhg4 involves disturbance of folate homeostasis at SCN feeding sites due to alteration of the enzymatic activity of serine hydroxymethyltransferase(SHMT).Other potential mechanisms,including plant defences mediated by salicylic acid(SA)and jasmonic acid(JA)signalling modulation,have also been suggested for SCN resistance.Indeed,genome-wide association studies(GWAS)have identified other candidate SCN resistance genes,such as Gm SNAP11.Although gene functional analysis in a transient expression system could increase the efficiency of candidate gene identification,information on novel genes and mechanisms for SCN resistance remains limited.Any beneficial candidate genes identified might,when fully exploited,be valuable for improving the efficiency of marker-assisted breeding and dissecting the molecular mechanisms underlying SCN resistance.

Toxicity Test of Different Pesticides on Tobacco Root-knot Nematodes

[Objectives]This study was conducted to quickly screen out effective and safe agents for controlling tobacco root knot nematode disease.[Methods]Six pesticides were tested and screened indoors at five different concentrations.[Results]The six pesticides all had toxic effects on the second-instar larvae of tobacco root-knot nematode,and the corrected mortality was positively correlated with the concentration and time of the pesticides,with the correlation coefficients above 0.8.From the perspective of inhibitory activity,the order was fosthiazate>abamectin>emamectin benzoate>cadusafos>ethoprophos>carbosulfan.Four pesticides that can be used in the field were selected.[Conclusions]This study provides a theoretical basis for field pesticide selection.

Determination of Nematicidal Effects of Some Biopesticides against Root-Knot Nematode （Meloidogyne incognita） on Kiwifruit

The aim of this study was to determine the nematicidal effects of some biopesticides againist root-knot nematodes （RKNs） （Meloidogyne incognita） on Bruno type kiwifruit seedlings. Research was conducted in the Atattirk Central Horticultural Research Institute （ACHRI, Yalova, Turkey） greenhouse during 2013-2014. Biopesticides observed in this study were： sesame oil （Devalone EC）, castor oil （Ricinus communis）, Myrothecium verrucaria （M. verrucaria strain AARC-0255, Inferno DF）, Glomus spp. （Endomycorrhizal fungi, Endo Roots Soluble SP） and Paecilomyces lilacinus （Entomopathogenic fungi, P. lilacinus strain PL1, Bionematon SL）. Nematicide Nemacur EC 400 （fenamiphos） was used as treated control （TC） and trials were set up as randomized parcel design （RPD） with seven characters and 10 replicates. Reproduction ratio was determined according to scale values in trial applications and second stage juvenile （J2） populations of M. incognita in soil. The results showed that the best nematicidal effects against RKNs were determined in castor oil （77.92% in 2013 and 73.61% in 2014） and P. lilacinus （61.03% in 2013 and 55.55% in 2014）.