Distribution and genetic diversity of root‐knot nematodes (Meloidogyne spp.) in potatoes from South Africa

A molecular‐based assay was employed to analyse and accurately identify various root‐knot nematodes (Meloidogyne spp.) parasitizing potatoes (Solanum tuberosum) in South Africa. Using the intergenic region (IGS) and the 28S D2–D3 expansion segments within the ribosomal DNA (rDNA), together with the region between the cytochrome oxidase subunit II (COII) and the 16S rRNA gene of the mtDNA, 78 composite potato tubers collected from seven major potato growing provinces were analysed and all Meloidogyne species present were identified. During this study, M. incognita, M. arenaria, M. javanica, M. hapla, M. chitwoodi and M. enterolobii were identified. The three tropical species M. javanica, M. incognita and M. arenaria were identified as the most prevalent species, occurring in almost every region sampled. Meloidogyne hapla and M. enterolobii occurred in Mpumalanga and KwaZulu‐Natal, respectively, while M. chitwoodi was isolated from two growers located within the Free State. Results presented here form part of the first comprehensive surveillance study of root‐knot nematodes to be carried out on potatoes in South Africa using a molecular‐based approach. The three genes were able to distinguish various Meloidogyne populations from one another, providing a reliable and robust method for future use in diagnostics within the potato industry for these phytoparasites.     

Nematicidal activity of Ochradenus baccatus against the root‐knot nematode Meloidogyne javanica

Ochradenus baccatus is a widely distributed shrub in desert regions of the Middle East and North Africa. This plant's nematicidal activity against the root‐knot nematode Meloidogyne javanica was evaluated because it has been found to contain exceptionally high levels of glucosinolates. In in vitro assays with aqueous extracts of the plant, 100% of second‐stage juveniles were immobilized after exposure to 4% root‐core extract for 48 h; 8% root‐core extract suppressed their hatching by 87%, whereas stem, flower and root bark showed lower activity. Incorporation of root core or bark into the soil, as fresh or dry powder at 1 and 0·5% (w/w), respectively, reduced the number of nematodes recovered from the soil by 95–100%, whereas the flower and stem were much less effective. Results from further pot experiments indicated that only the root bark consistently contains nematicidal compounds which are effective in soil, whereas the nematicidal activity of the root core in soil was inconsistent. The presence of non‐volatile lipophilic and lipophobic nematicidal compounds in the root bark was suggested by extraction with different polar solvents, but these compounds do not seem to be isothiocyanates – glucosinolate‐hydrolysed compounds with nematicidal activity. Very poor host status of Ochradenus baccatus to M. javanica, M. incognita and M. hapla, but with root‐penetration rates of juveniles similar to those in tomato roots, suggest that this plant may be used as a cover plant or trap plant to reduce nematode populations in the soil.     

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.     

Host genetic resistance to root‐knot nematodes,Meloidogyne spp., in Solanaceae: from genes to the field

Root‐knot nematodes (RKNs) heavily damage most solanaceous crops worldwide. Fortunately, major resistance genes are available in a number of plant species, and their use provides a safe and economically relevant strategy for RKN control. From a structural point of view, these genes often harbour NBS–LRR motifs (i.e. a nucleotide binding site and a leucine rich repeat region near the carboxy terminus) and are organised in syntenic clusters in solanaceous genomes. Their introgression from wild to cultivated plants remains a challenge for breeders, although facilitated by marker‐assisted selection. As shown with other pathosystems, the genetic background into which the resistance genes are introgressed is of prime importance to both the expression of the resistance and its durability, as exemplified by the recent discovery of quantitative trait loci conferring quantitative resistance to RKNs in pepper. The deployment of resistance genes at a large scale may result in the emergence and spread of virulent nematode populations able to overcome them, as already reported in tomato and pepper. Therefore, careful management of the resistance genes available in solanaceous crops is crucial to avoid significant reduction in the duration of RKN genetic control in the field. From that perspective, only rational management combining breeding and cultivation practices will allow the design and implementation of innovative, sustainable crop production systems that protect the resistance genes and maintain their durability. © 2015 Society of Chemical Industry     

Genetic diversity of the root‐knot nematode Meloidogyne ethiopica and development of a species‐specific SCAR marker for its diagnosis

Meloidogyne ethiopica is an important nematode pathogen causing serious economic damage to grapevine in Chile. In Brazil, M. ethiopica has been detected with low frequency in kiwifruit and other crops. The objectives of this study were to evaluate the intraspecific genetic variability of M. ethiopica isolates from Brazil and Chile using AFLP and RAPD markers and to develop a species‐specific SCAR‐PCR assay for its diagnosis. Fourteen isolates were obtained from different geographic regions or host plants. Three isolates of an undescribed Meloidogyne species and one isolate of M. ethiopica from Kenya were included in the analysis. The results showed a low level of diversity among the M. ethiopica isolates, regardless of their geographical distribution or host plant origin. The three isolates of Meloidogyne sp. showed a high homogeneity and clustered separately from M. ethiopica (100% bootstrap). RAPD screenings of M. ethiopica allowed the identification of a differential DNA fragment that was converted into a SCAR marker. Using genomic DNA from pooled nematodes as a template, PCR amplification with primers designed from this species‐specific SCAR produced a fragment of 350 bp in all 14 isolates of M. ethiopica tested, in contrast with other species tested. This primer pair also allowed successful amplification of DNA from single nematodes, either juveniles or females and when used in multiplex PCR reactions containing mixtures of other root‐knot nematode species, thus showing the sensitivity of the assay. Therefore, the method developed here has potential for application in routine diagnostic procedures.     

Host suitability of Vitis rootstocks to root‐knot nematodes (Meloidogyne spp.) and the dagger nematode Xiphinema index,and plant damage caused by infections

The host suitability of commercial Vitis rootstocks commonly used in Spain (161‐49C, 41B, 1103P, 110R, 140Ru and SO4) to root‐knot nematodes (Meloidogyne arenaria, M. incognita, M. javanica) and Xiphinema index, and damage caused by nematode infection were determined under controlled conditions. The three root‐knot nematodes reproduced with a rate higher than one in all rootstocks, indicating that they are suitable hosts for these nematodes. Growth of rootstocks infected with the root‐knot nematodes was less vigorous than that of nematode‐uninfected controls in the majority of the rootstocks studied. Root infection resulted in moderate to severe root galling in all rootstocks. The shoot and main stem diameters appeared to be the most sensitive variables of damage caused by infection by Meloidogyne spp., with reduction rates from 36% and 53% in 161‐49C to 57% and 66% in 140Ru, respectively. The shoot height was not significantly affected by the root‐knot nematodes and the root fresh weight generally increased as a consequence of intensive galling. The nematode X. index caused significant root damage with a reproduction factor higher than one in all rootstocks. However, reproduction factor was significantly influenced by the rootstock and significantly decreased by about 12‐fold (5·7 to 18·1‐fold) with the increase in inoculum density from 100 to 1000 nematodes per plant. The root dry weight was reduced by X. index infections, and was the plant growth variable most affected by the nematode infection in all rootstocks at both inoculum densities. Meloidogyne arenaria, M. incognita, M. javanica and X. index, prevalent in many world vineyards, are all shown to have a damaging effect on the six tested rootstocks.     

Thermal requirements and population dynamics of root‐knot nematodes on cucumber and yield losses under protected cultivation

Several studies were carried out to determine (i) thermal requirements for development, egg production and emergence of juveniles, and completion of the life cycle of Meloidogyne incognita and Meloidogyne javanica on cucumber, (ii) the maximum multiplication rate and the equilibrium density of root‐knot nematodes on cucumber and yield losses in pot and plastic greenhouse experiments, and (iii) the relationships between relative leaf chlorophyll content (RLCC) and relative cucumber dry top weight biomass (RDTWB) in relation to increasing nematode densities at planting (P_i) in pot experiments. Thermal requirements of M. incognita and M. javanica on cucumber did not differ, irrespective of the biological stage. In the pot experiments, M. javanica completed one generation. The maximum multiplication rate (a) was 833, and the equilibrium density (E) varied according to the effective inoculum densities. The relationship between RDTWB and P_i fitted the Seinhorst damage function model. The RLCC value at 40 or 50 days post‐inoculation also fitted the damage model and was related to RDTWB. In greenhouse experiments, conducted from 2009 to 2012, M. incognita completed three generations. The values for a and E were 1147 and 625 second stage juveniles (J2) per 250 cm³ soil, respectively. The tolerance limit was below zero, and the minimum relative yield ranged from 0·12 to 0·34.     

Cucumis metuliferus is resistant to root‐knot nematode Mi1.2 gene (a)virulent isolates and a promising melon rootstock

Pot experiments were carried out to characterize the response of two Cucumis metuliferus accessions (BGV11135 and BGV10762) against Mi1.2 gene (a)virulent Meloidogyne arenaria, M. incognita and M. javanica isolates and to determine the compatibility and the effect on physicochemical properties of fruit melons. In addition, histopathological studies were conducted. One week after transplanting, plants were inoculated with one J2 cm^?3 of sterilized sand (200 cm³ pots) and maintained in a growth chamber at 25 °C for 40 days. The susceptible cucumber cv. Dasher II or melon cv. Paloma were included for comparison. The number of egg masses and number of eggs per plant were assessed, and the reproduction index (RI) was calculated as the percentage of eggs produced on the C. metuliferus accessions compared to those produced on the susceptible cultivars. The compatibility and fruit quality were assessed by grafting three scions, two of Charentais type and one of type piel de sapo, under commercial greenhouse conditions. The resistance level of both C. metuliferus accessions ranged from highly resistant (RI < 1%) to resistant (1% ≤ RI ≤ 10%) irrespective of Meloidogyne isolates. Melon plants grafted onto C. metuliferus accession BGV11135 grew as self‐grafted plants without negatively impacting fruit quality traits. Giant cells induced by Meloidogyne spp. on C. metuliferus were in general poorly developed compared to those on cucumber. Furthermore, necrotic areas surrounding the nematode were observed. Cucumis metuliferus accession BGV11135 could be a promising melon rootstock to manage Meloidogyne spp., irrespective of their Mi1.2 (a)virulence, without melon fruit quality reduction.     

Root‐knot nematodes (Meloidogyne spp.), a growing problem in French vegetable crops

Root‐knot nematode infestations have become an increasing source of concern in French vegetable production due to recent restrictions on the use of chemical nematicides and the banning of some of the most active compounds. This problem is likely to become particularly severe in the next few years. A survey, based on questionnaires to farmers, was carried out by INRA Sophia Antipolis (PACA centre) between 2007 and 2010 in collaboration with more than 30 research institutes and technical centres, groups of producers and chambers of agriculture. It showed that in the Provence–Alpes–Côte d’Azur (PACA) region, more than 40% of horticultural holdings are infested by Meloidogyne species. It is no longer possible to produce cucurbits on certain plots. Other regions are also affected. Other genera of nematodes that are more difficult for farmers to diagnose (Globodera, Heterodera, Pratylenchus, Ditylenchus) were sometimes detected. Alternatives to chemical control have been tested on several farms and have been found to be highly variable in efficiency. There is therefore a need for researchers and professionals (producers, technical advisers, etc.) to combine forces to monitor this plant health problem and to develop and test new, economically viable and sustainable control methods.     

Meloidogyne luci,a new root‐knot nematode parasitizing potato in Portugal

In 2013, during a field survey conducted in Portugal on potato, Solanum tuberosum, an unusual esterase (EST) phenotype was detected in a root‐knot nematode (RKN) from potato roots collected in Coimbra. This Portuguese isolate was purified and maintained on tomato, S. lycopersicum, and morphological, biochemical and molecular characteristics were studied. Perineal pattern morphology was highly variable, similar to Meloidogyne ethiopica and not useful for identification. The EST phenotype, from young egg‐laying females, displayed three bands similar to the Brazilian M. luci (L3) and distinct from M. ethiopica (E3). Phylogenetic analyses of mitochondrial cytochrome oxidase subunit I and the mitochondrial DNA region between COII and 16S rRNA genes revealed that the Portuguese isolate grouped with M. luci isolates close to M. ethiopica isolates. However, considering the ITS1‐5.8S‐ITS2 region, the Portuguese isolate grouped with isolates of M. luci, M. ethiopica and M. hispanica, which limits the confidence of this region for M. luci diagnosis, and its differentiation from other species with morphological similarities. The M. luci pathogenicity to potato was also assessed in 16 commercial cultivars and compared with M. chitwoodi, considered to be a quarantine RKN species by EPPO. All potato cultivars were susceptible to both Meloidogyne species with gall indices of 5 and higher reproduction factor values ranging from 12.5 to 122.3, which suggests that M. luci may constitute a potential threat to potato production. In the present study, M. luci is reported for the first time attacking potato in Portugal.     

Root‐knot nematodes do not affect black root rot disease severity on watermelon and bottle gourd

The effects of root‐knot nematodes on black root rot of watermelon and bottle gourd were studied using field surveys and co‐inoculation tests with Meloidogyne incognita (southern root‐knot nematode) and Diaporthe sclerotioides. The results of the field survey suggested that root‐knot nematodes had little effect on either the severity of black root rot or infection with D. sclerotioides. None of the three fields showed a significant positive correlation between disease severity and nematode gall index, with low correlation coefficients. Co‐inoculation experiments under controlled conditions found no significant effect of root‐knot nematodes on black root rot of watermelon and bottle gourd based on area under disease progress curves (AUDPC). These results were supported by the quantities of DNA of the two agents in root tissues because no significant difference was found between dual‐ and single‐inoculation treatments with M. incognita and/or D. sclerotioides. These findings suggest that root‐knot nematodes probably do not affect the infection of watermelon or bottle gourd roots by D. sclerotioides or the incidence of black root rot in these crops caused by this fungus.     

Pathogenicity and host‐parasite relationships of the root‐knot nematode Meloidogyne incognita on celery

Pathogenicity and host‐parasite relationships in root‐knot disease of celery (Apium graveolens ) caused by Meloidogyne incognita race 1 were studied under glasshouse conditions. Naturally and artificially infected celery cv. D’elne plants showed severe yellowing and stunting, with heavily deformed and damaged root systems. Nematode‐induced mature galls were spherical and/or ellipsoidal and commonly contained more than one female, males and egg masses with eggs. Feeding sites were characterized by the development of giant cells that contained granular cytoplasm and many hypertrophied nuclei. The cytoplasm of giant cells was aggregated along their thickened cell walls and consequently the vascular tissues within galls appeared disrupted and disorganized. The relationship between initial nematode population density (Pi) and growth of celery plants was tested in glasshouse experiments with inoculum levels that varied from 0 to 512 eggs and second‐stage juveniles (J₂) mL^?1 soil. Seinhorst's model y = m + (1 – m)z^P–T was fitted to height and top fresh weight data of the inoculated and control plants. The tolerance limit with respect to plant height and fresh top weight of celery to M. incognita race 1 was estimated as 0·15 eggs and J₂ mL^?1 soil. The minimum relative values (m) for plant height and top fresh weight were 0·37 and 0·35, respectively, at Pi ≥ 16 eggs and J₂ mL^?1 soil. The maximum nematode reproduction rate (Pf/Pi) was 407·6 at an initial population density (Pi) of 4 eggs and J₂ mL^?1 soil.     

Genetic diversity of the root‐knot nematode Meloidogyne enterolobii and development of a SCAR marker for this guava‐damaging species

The objectives of this work were to evaluate the genetic variability of Meloidogyne enterolobii by molecular markers, and develop species‐specific molecular markers for application in detection. Sixteen M. enterolobii isolates from different geographical regions (Brazil and other countries) and hosts were used in this study. The identification and purification of the populations were carried out based on isoenzyme phenotype. The DNA amplification of the intergenic region (IGS) of the rDNA and of the region between the cytochrome oxidase subunit II (COII) and 16S rRNA genes (mtDNA) produced specific fragments of the expected size for this nematode, i.e. 780 and 705 bp, respectively. Intraspecific variability among the isolates was evaluated with three different neutral molecular markers: AFLP, ISSR and RAPD. The results showed a low level of diversity among the isolates tested, indicating that M. enterolobii is a genetically homogeneous root‐knot nematode species. The RAPD method allowed the identification of a species‐specific RAPD fragment for M. enterolobii. This fragment was cloned and sequenced, and from the sequence obtained, a set of primers was designed and tested. The amplification of a 520‐bp‐long fragment occurred only for the 16 isolates of M. enterolobii and not for the 10 other Meloidogyne species tested. In addition, positive detection was achieved in a single individual female, egg‐mass and second stage juvenile of this nematode. This SCAR species‐specific marker for M. enterolobii represents a new molecular tool to be used in the detection of this nematode from field samples and as a routine diagnostic test for quarantine devices .     

Germination rates of weedy radish populations (Raphanus spp.) altered by crop‐wild hybridisation,not human‐mediated changes to soil moisture

Cultivated plants are known to readily hybridise with their wild relatives, sometimes forming populations with weedier life‐history strategies than their progenitors. Due to altered precipitation patterns from human‐induced global climate change, crop‐wild hybrid populations may have new and unpredictable environmental tolerances relative to parental populations, which would further challenge farming and land‐management weed control strategies. To recognise the role of seed dormancy variation in weed invasion, we compared seedbank dynamics of two cross‐type populations (wild radish, Raphanus raphanistrum, and crop‐wild hybrid radish, R. raphanistrum × R. sativus) across a soil moisture gradient. In a seed‐burial experiment, we assessed relative rates of seed germination, dormancy and seed mortality over two years across cross types (crop‐wild hybrid or wild) and watering treatments (where water was withheld, equal to annual rainfall, or double annual rainfall). Weekly population censuses in 2012 and 2013 assessed the frequency and timing of seedling emergence within a growing season. Generally, germination rates were two times higher and seed dormancy was 58% lower in hybrid versus wild populations. Surprisingly, experimental soil moisture conditions did not determine seedbank dynamics over time. Yet, seed bank dynamics changed between years, potentially related to different amounts of annual rainfall. Thus, variation in seedbank dynamics may be driven by crop‐wild hybridisation rates and, potentially, annual variation in soil moisture conditions.     

植物根结线虫基因组学研究进展

根结线虫是世界农业生产中危害最大的植物病原之一,目前仍缺乏安全有效的防治措施。深入揭示寄生线虫与植物之间互作的分子机制,利用生物技术进行抗性育种被认为是最有前景的抗线虫策略。在根结线虫基因组学研究方面,目前已经构建了北方根结线虫AFLP遗传连锁图谱,南方根结线虫和北方根结线虫基因组测序也已完成;基因组的注释和比较基因组学分析,较全面地描述了根结线虫的遗传组成;以差异表达分析和比较基因组学为主的方法鉴定了大量的重要基因;以RNA干扰、植物转化和蛋白互作为主的根结线虫基因功能研究也取得了一些进展。本文就根结线虫基因组学研究予以综述,并进一步探讨其研究方向和可持续抗线虫新策略的发展前景。