Rapid detection of Meloidogyne spp. by LAMP assay in soil and roots

Loop-mediated isothermal amplification (LAMP), a novel DNA amplification technique, has been used to detect a variety of pathogens including viruses, fungi, bacteria and parasites. However, diagnosis of sedentary plant-parasitic nematode (PPN) species has not yet been attempted. In this study, we developed a universal LAMP set (RKN-LAMP) for the diagnosis of four common Meloidogyne species (Meloidogyne incognita, M. arenaria, M. javanica and M. hapla), and M. incognita-specific LAMP set (Mi-LAMP). In both assays, a typical ladder-like pattern on gel electrophoresis was observed in all positive samples but not in the negative controls. Amplification products were further confirmed using restriction analysis of the Hpa II enzyme, detection by visual inspection using SYBR Green I and the lateral flow dipstick (LFD) assay. The two LAMP sets were specifically able to detect four common Meloidogyne species and M. incognita populations having several different geographical origins and pathotypes. No cross reaction with DNA of other PPNs was observed. Sensitivity of the RKN- and Mi-LAMP was 10 and 100 fg of pure genome DNA respectively. Both LAMP sets could also amplified crude DNA isolated from the galled root tissue and from soil containing juveniles of M. incognita. The RKN- and Mi-LAMP sets offer the advantages of simplicity, rapidity and cost effectiveness. Both LAMP sets will be instrumental for the diagnosis of Meloidogyne spp. by local extension and regulatory personnel.     

苏云金杆菌00-50-5发酵上清液对南方根结线虫杀虫活性研究

室内条件下,分别测定了苏云金杆菌Bt 00-50-5菌株液体和固体发酵物对南方根结线虫的毒力效果。结果显示,牛肉膏蛋白胨液体发酵法在72h所得上清液的毒性最高,南方根结线虫死亡率达到99.0%,而固体麸皮发酵物对线虫的最高毒性为发酵8h,南方根结线虫死亡率为89.0%。液体发酵上清液经葡聚糖凝胶G-75柱层析分离,柱下物在280nm处的吸收值呈现出4个峰I、II、III和IV,其值0.601,1.475,1.641和0.392,其中峰II、III、IV处收集的蛋白对南方根结线虫有毒杀作用,分子量最小的IV蛋白杀线虫活性最高。上清液天然聚丙烯酰胺凝胶电泳分离杀虫蛋白结果显示6条清晰蛋白带,其中37kDa蛋白含量最多。     

Greenhouse and field evaluations of commonly occurring weed species for their host suitability to Meloidogyne species

Since weeds serve as hosts for nematode pests, the host status of 20 weed species (commonly occurring in fields of developing farmers) to Meloidogyne incognita and M. javanica, respectively, were investigated. Greenhouse studies showed that seven weed species had Rf values >1 for both nematode species indicating susceptibility, while 13 had Rf values ≤ 1 indicating resistance. Greenhouse results showed that Hibiscus trionum and Amaranthus tricolor were identified as the most susceptible and Chenopodium carinatum and Datura ferox the poorest hosts for M. incognita and M. javanica. For field experiments at Kuruman, Solanum retroflexum was the most susceptible weed to a M. javanica population, while the same was evident for H. trionum at Nelspruit, where a mixed population of M. incognita and M. javanica occurred and at Potchefstroom, where a population of M. incognita was present. Results from this study indicated that certain weed species are highly susceptible to root-knot nematodes and should be removed timely and effectively to prevent population level increases of root-knot nematode pests in the fields of farmers.     

In vitro and in planta nematicidal activity of Fumaria parviflora (Fumariaceae) against the southern root‐knot nematode Meloidogyne incognita

Root and stem extracts of Fumaria parviflora showed strong nematicidal activity against Meloidogyne incognita in in vitro and in planta experiments. Phytochemical screening of F. parviflora revealed the presence of seven classes of bioactive compounds (alkaloids, flavonoids, glycosides, tannins, saponins, steroids and phenols). Quantitative determination of the plant extracts showed the highest percentages of alkaloids (0·9 ± 0·04) and saponins (1·3 ± 0·07) in the roots and total phenolic contents in the stem (16·75 ± 0·07 μg dry g^?1). The n‐hexane, chloroform, ethyl acetate and methanol extracts of roots and stems at concentrations of 3·12, 6·24, 12·5, 25·0 and 50·0 mg mL^?1, significantly inhibited hatching and increased mortality of second‐stage juveniles (J2s) compared with water controls. Percentage J2 mortality and hatch inhibition were directly related to exposure time. In pot trials with tomato cv. Rio Grande, root and stem extracts at concentrations of 1000, 2000 and 3000 ppm, applied as soil drenches, significantly reduced the number of galls, galling index, eggs masses, eggs and reproduction factor compared with the water control. Regardless of concentration, all the extracts significantly increased the host plant growth parameters studied. The n‐hexane extracts from the roots and stem were the most active, followed by the methanol ones, at all concentrations. The in vitro and in planta results suggest that extracts from the roots and stem of F. parviflora may be potential novel nematicides.     

茶枯饼乙醇抽提物对爪哇根结线虫的毒杀活性

【目的】研究了茶枯饼乙醇抽提物对爪哇根结线虫(Meloidogyne javanica)的毒杀活性,以及茶枯饼粉末拌土处理对空心菜和落葵爪哇根结线虫病的盆栽防治效果,为线虫病的生物防治提供理论依据。【方法】以茶枯饼为材料,采用药液直接浸泡法和粉末拌土盆栽法,分别研究了茶枯饼乙醇抽提物对爪哇根结线虫2龄幼虫的毒杀活性、对卵的孵化抑制性,以及茶枯饼粉末拌土处理对空心菜和落葵爪哇根结线虫病的防效。【结果】茶枯饼乙醇抽提物对爪哇根结线虫2龄幼虫有较强的毒杀活性,以50,100,150mg/mL茶枯饼乙醇抽提物处理72h,经清水复苏24h后,校正死亡率分别为39.94%,58.80%,70.68%;对爪哇根结线虫卵的孵化表现出较强抑制活性,以50,100,150mg/mL茶枯饼乙醇抽提物处理5d后,抑制孵化率均达到58%以上。茶枯饼粉末拌土处理对空心菜(9g/L)及落葵(11g/L)爪哇根结线虫均有较好的防治效果,茶枯饼粉末拌土能显著降低根结数及雌虫产卵量,并使植株地上部鲜质量显著增加。【结论】茶枯饼中存在杀线虫成分,可作为潜在的防治根结线虫病资源用于线虫病的防治。     

Nematicidal Activity of Chrysanthemum coronarium

Organic amendments and green manure are potential alternatives to the harmful chemical control means currently used against plant-parasitic nematodes. In this work, Chrysanthemum coronarium was applied to the soil as a green manure to control the root-knot nematodes Meloidogyne incognita and M. javanica. Chrysanthemum coronarium significantly reduced nematode infection of tomato roots and improved plant-top fresh weight, both in the greenhouse and in microplots. Other green manures, derived from Anthemis pseudocotula, wild chickpea (Cicer pinnatifidum), Geranium spp. and wheat, were not as effective as C. coronarium. Chrysanthemum coronarium, retained its nematicidal activity even when applied as a dried material. Only mature C. coronarium plants, in their flowering stage, exhibited nematode control activity, but the green plant parts were more effective than the flowers. An aqueous extract of C. coronarium exhibited in vitro, nematostatic activity towards M. incognita and M. javanica second-stage juveniles and inhibited their hatching from eggs and egg-masses; its nematostatic activity was expressed also against other phytonematode species such as Heterodera avenae and Pratylenchus mediterraneus, but did not affect the beneficial entomopathogenic nematode Steinernema feltiae.     

臭氧水对茄子根结线虫的防治效果评价

根结线虫是具有毁灭性危害的植物内生寄生虫,臭氧水漫灌土壤是较为理想的替代化学药剂的防治方法,但还未见在茄子上使用的报道。为了探究高浓度臭氧水漫灌处理土壤对茄子根结线虫的田间防治效果及对茄子生长的影响,本研究在茄子定植前对土壤进行连续2次臭氧水(9 mg/L)漫灌处理,并对漫灌处理前后和茄子整个生长期的根结线虫病害发生情况及茄子生长情况进行跟踪调查。结果表明,与对照相比,结果期和生长后期根结线虫数量分别下降91.67%和95.97%;幼苗期、结果期和生长后期的防治效果分别为100%、55.36%和65.52%;处理样本的株高显著提高。因此,高浓度臭氧水漫灌处理土壤对茄子根结线虫的防治效果较好且可减少农药使用量,对茄子生长安全,值得推广应用。     

Global status of nematode problems of groundnut, pigeonpea, chickpea, sorghum and pearl millet, and suggestions for future work

Analysis of responses received from 40 cooperators in 20 countries to a questionnaire on nematode problems of groundnut, pigeonpea, chickpea, sorghum and pearl millet suggested that Meloidogyne spp. are internationally important nematode pests of groundnut, chickpea and pigeonpea. Pratylenchus spp. are important on all the five crops. In India, Heterodera cajani and Rotylenchulus reniformis are important pathogens of pigeonpea. Over the last 10–15 years, extensive nematode disease surveys have been undertaken for these crops in Australia, Egypt, India, Jamaica, Senegal, Sudan, Thailand and Zimbabwe; however, < 10% of the total crop areas were covered by these surveys. Except for Brazil, Egypt, USA and Zimbabwe, growers do not use nematicides to control the nematodes. Cultural practices, especially crop rotations, are the most commonly used control measures. Species of Meloidogyne, Pratylenchus and Rotylenchulus on the legumes, and species of Hoplolaimus, Pratylenchus, Quinisulcius and Xiphinema on the cereals, are strongly suspected of increasing the severity of fungal diseases. Work aimed at finding host resistance is being done in Brazil, Fiji, India and the USA, and some sources of resistance have been identified against Meloidogyne arenaria, M. incognita, M. javanica and R. reniformis. Facilities for resistance screening work now exist in many countries. Information on damage thresholds of important pest species are available only from Brazil, Fiji, India and the USA.     

Synergistic interactions betweenRhizoctonia solani Kühn,Verticillium dahliae Kleb.,Meloidogyne spp. andPratylenchus neglectus (Rensch) Chitwood & Oteifa,in potato

Summary Samples of a sandy soil and a marine clay soil sterilized by steam were put in 55-1 containers insulated with polystyrene and placed outdoors on a brick pavement. Sandy soil was infested singly or in all possible combinations with root-knot nematodes (Meloidogyne spp.) and the fungiRhizoctonia solani andVerticillium dahliae, and the marine clay soil was infested with the root-lesion nematodePratylenchus neglectus and the same fungi to evaluate the effects of these organisms on the yield of potato. The experiments were carried out from 1983 to 1986. Tuber yield was reduced by single infestations of theMeloidogyne spp. andV. dahliae but not significantly byR. solani orP. neglectus. A three-factor interaction: nematode ×R. solani × V. dahliae was found in both experiments.R. solani andV. dahliae showed significant synergistic effects when soil was infested with theMeloidogyne spp. orP. neglectus.     

根结线虫污染的温室番茄土壤修复——土壤修复剂稀释倍数筛选

为明确笔者实验室研制的不同稀释倍数的土壤修复剂对被南方根结线虫（Meloidogyn incognita）污染的温室番茄土壤的修复效果,该文用浅盘法分离土壤中线虫,鉴定并记录各处理及清水对照的线虫数量、根系根结率。结果显示：在5种稀释倍数中,稀释125倍的土壤修复剂每株、每次施用500ml最能有效地抑制根结线虫,种群数量可控制在30 individuals·100ml-1 soil以下,并使根系的根结率减少至40%以下,同时能促进有益的小杆线虫(Rhabditis (Oscheius) spp.)种群扩增（388 indi.·100ml-1 soil）。研究结果还显示每25d施用一次土壤修复剂可以较有效地修复被根结线虫污染的土壤。