Essential oils as soil biofumigants for the control of the root‐knot nematode Meloidogyne incognita on tomato

The effectiveness of soil fumigation with 50, 100 and 200 µL kg^?1 soil of essential oils (EOs) from the plant species Eucalyptus citriodora, Eucalyptus globulus, Mentha piperita, Pelargonium asperum and Ruta graveolens was assessed against the root‐knot nematode Meloidogyne incognita on potted tomato. Plant growth parameters and number of galls, nematode eggs and juveniles on tomato roots were evaluated after two months of maintenance of the treated plants at 25°C in greenhouse. EOs of E. globulus and P. asperum significantly reduced nematode multiplication and gall formation on tomato roots at all the tested rates, whereas the EOs of E. citriodora, M. piperita and R. graveolens were more suppressive at levels greater than 50 µL kg^?1 soil. Biofumigation with EOs of E. globulus and P. asperum resulted also in the largest increase of tomato plant top and root biomass. The five samples of EOs had a different chemical composition as determined by GC and GC‐MS. Structure–activity relationship based on the main constituents of the tested EOs and their nematicidal effect on M. incognita is discussed.     

Mode of action and efficacy of iprodione against the root‐knot nematode Meloidogyne incognita

The ban and restriction on the use of several synthetic chemicals for controlling plant parasitic nematodes, and concern about their side effects necessitate the availability of effective methods of control with low toxicity to humans and non‐target organisms. Therefore, efficacy and mode of action of iprodione, a dicarboximide fungicide, was evaluated against the root‐knot nematode Meloidogyne incognita, in vitro and in vivo conditions, in comparison with the nematicides fenamiphos, fosthiazate and oxamyl at 7.00, 1.66 and 1.66 mL/5 L water, respectively. In vitro, iprodione showed nematostatic rather than nematicidal activity against second‐stage juveniles of M. incognita in contrast to fenamiphos, fosthiazate and oxamyl which were nematicidal. In the in vivo experiment with tomato, iprodione controlled M. incognita less than fenamiphos, fosthiazate and oxamyl. No visual symptoms of phytotoxicity were observed. Therefore, iprodione can be a useful chemical for controlling nematode populations if included in an Integrated Pest Management program.     

Divergent expression of cytokinin biosynthesis,signaling and catabolism genes underlying differences in feeding sites induced by cyst and root‐knot nematodes

Cyst and root‐knot nematodes are obligate parasites of economic importance with a remarkable ability to reprogram root cells into unique metabolically active feeding sites. Previous studies have suggested a role for cytokinin in feeding site formation induced by these two types of nematodes, but the mechanistic details have not yet been described. Using Arabidopsis as a host plant species, we conducted a comparative analysis of cytokinin genes in response to the beet cyst nematode (BCN), Heterodera schachtii, and the root‐knot nematode (RKN), Meloidogyne incognita. We identified distinct differences in the expression of cytokinin biosynthesis, catabolism and signaling genes in response to infection by BCN and RKN, suggesting differential manipulation of the cytokinin pathway by these two nematode species. Furthermore, we evaluated Arabidopsis histidine kinase receptor mutant lines ahk2/3, ahk2/4 and ahk3/4 in response to RKN infection. Similar to our previous studies with BCN, these lines were significantly less susceptible to RKN without compromising nematode penetration, suggesting a requirement of cytokinin signaling in RKN feeding site formation. Moreover, an analysis of ahk double mutants using CycB1;1:GUS/ahk introgressed lines revealed contrasting differences in the cytokinin receptors mediating cell cycle activation in feeding sites induced by BCN and RKN.     

The plant cell inhibitor KRP6 is involved in multinucleation and cytokinesis disruption in giant-feeding cells induced by root-knot nematodes

The plant cell cycle inhibitor gene KRP6 has been investigated in roots infected by plant-parasitic root-knot nematodes (Meloidogyne spp.). Unexpectedly, KRP6 overexpressing lines revealed a distinct role for this specific KRP as an activator of the mitotic cell cycle. This function was confirmed in Arabidopsis thaliana suspension cultures ectopically expressing KRP6. A blockage in the mitotic exit was observed in cell suspensions and in giant cells resulted in the appearance of multi-nucleated cells. KRP6 expression during nematode infection and the similarity in phenotypes among KRP6 overexpressing cell cultures and giant-cell morphology strongly suggest that KRP6 is involved in multinucleation and acytokinesis occurring in giant-cells. Once again nematodes have been shown to manipulate the plant cell cycle machinery in order to promote gall establishment.     

Effects of crop plants on abundance of Pochonia chlamydosporia and other fungal parasites of root‐knot and potato cyst nematodes

The effects of a host plant on reproduction/abundance of fungal populations in relation to soil nutrients released by plants in the rhizosphere were studied. Abundance in the soil and potato rhizosphere of the fungi Paecilomyces lilacinus, Monographella cucumerina (CABI 380408) and Pochonia chlamydosporia var. chlamydosporia (Pc280, potato cyst nematode biotype) and P. chlamydosporia var. catenulata (Pc392, root‐knot nematode biotype) were assessed. The different ability of break crops (oilseed rape, sugarbeet and wheat) in the potato rotation to support Pa. lilacinus, Pochonia isolates Pc280 and Pc392 and abundance of the latter two isolates in soil and rhizosphere of potato plants infected with Meloidogyne incognita were also studied. Potato chits and crop seedlings were planted into boiling tubes containing 5000 chlamydospores or conidia g^?1 in acid washed sand (pH 6) and kept in a growth chamber at 20°C, and 16 h of light for up to 9 weeks. The abundance of the fungi in sand (fallow) differed significantly between fungal species, being in general less abundant in the absence than in the presence of the plant, although there was no interaction between plant species and fungal isolate. There was evidence of a different response to Me. incognita for Pc392 than for Pc280 but there was no significant effect of the presence of the nematode on the rate of increase of the fungus.     

Manipulation of two α‐endo‐β‐1,4‐glucanase genes,AtCel6 and GmCel7, reduces susceptibility to Heterodera glycines in soybean roots

Plant endo‐β‐1,4‐glucanases (EGases) include cell wall‐modifying enzymes that are involved in nematode‐induced growth of syncytia (feeding structures) in nematode‐infected roots. EGases in the α‐ and β‐subfamilies contain signal peptides and are secreted, whereas those in the γ‐subfamily have a membrane‐anchoring domain and are not secreted. The Arabidopsis α‐EGase At1g48930, designated as AtCel6, is known to be down‐regulated by beet cyst nematode (Heterodera schachtii) in Arabidopsis roots, whereas another α‐EGase, AtCel2, is up‐regulated. Here, we report that the ectopic expression of AtCel6 in soybean roots reduces susceptibility to both soybean cyst nematode (SCN; Heterodera glycines) and root knot nematode (Meloidogyne incognita). Suppression of GmCel7, the soybean homologue of AtCel2, in soybean roots also reduces the susceptibility to SCN. In contrast, in studies on two γ‐EGases, both ectopic expression of AtKOR2 in soybean roots and suppression of the soybean homologue of AtKOR3 had no significant effect on SCN parasitism. Our results suggest that secreted α‐EGases are likely to be more useful than membrane‐bound γ‐EGases in the development of an SCN‐resistant soybean through gene manipulation. Furthermore, this study provides evidence that Arabidopsis shares molecular events of cyst nematode parasitism with soybean, and confirms the suitability of the Arabidopsis–H. schachtii interaction as a model for the soybean–H. glycines pathosystem.     

Genetic Diversity of the root‐knot nematode Meloidogyne enterolobii in Mulberry Based on the Mitochondrial COI Gene

This study explores the genetic diversity and structure of Meloidogyne enterolobii in mulberry in China. The COI mitochondrial gene (mtCOI) in M.enterolobii populations in Guangdong, Guangxi, and Hunan Provinces was PCR‐amplified, sequenced, and analyzed for genetic diversity. The total number of variations, haplotypes (Hap), the average number of nucleotide differences (k), haplotype diversity (H), and nucleotide diversity (π) of mtCOI were 25, 11, 4.248, 0.900, and 0.00596, respectively. Insignificant differences in Fst value (0.0169) and a high level of gene flow (7.02) were detected among the 19‐mulberry root‐knot nematode populations, and high genetic variation within each population and a small genetic distance among populations were observed. Both phylogenetic analyses and network mapping of the 11 haplotypes revealed a dispersed distribution pattern of 19 mulberry root‐knot nematode populations and an absence of branches strictly corresponding to the 19 range sampling sites. The neutrality test and mismatch analysis indicated that mulberry root‐knot nematode populations experienced a population expansion in the past. The analysis of molecular variance (AMOVA) revealed that the genetic differentiation of M. enterolobii was mainly contributed by the variation within each group. No significant correlation was found between the genetic distance and geographical distance of M. enterolobii populations. The findings of this study provide a profound understanding of the M. enterolobii population and will inform the development of strategies to combat and manage root‐knot nematodes in mulberry.     

Preferential expression of a plant cystatin at nematode feeding sites confers resistance to Meloidogyne incognita and Globodera pallida

The expression patterns of three promoters preferentially active in the roots of Arabidopsis thaliana have been investigated in transgenic potato plants in response to plant parasitic nematode infection. Promoter regions from the three genes, TUB-1, ARSK1 and RPL16A were linked to the GUS reporter gene and histochemical staining was used to localize expression in potato roots in response to infection with both the potato cyst nematode, Globodera pallida and the root-knot nematode, Meloidogyne incognita. All three promoters directed GUS expression chiefly in root tissue and were strongly up-regulated in the galls induced by feeding M. incognita. Less activity was associated with the syncytial feeding cells of the cyst nematode, although the ARSK1 promoter was highly active in the syncytia of G. pallida infecting soil grown plants. Transgenic potato lines that expressed the cystatin OcIDeltaD86 under the control of the three promoters were evaluated for resistance against Globodera sp. in a field trial and against M. incognita in containment. Resistance to Globodera of 70 +/- 4% was achieved with the best line using the ARSK1 promoter with no associated yield penalty. The highest level of partial resistance achieved against M. incognita was 67 +/- 9% using the TUB-1 promoter. In both cases this was comparable to the level of resistance achieved using the constitutive cauliflower mosaic virus 35S (CaMV35S) promoter. The results establish the potential for limiting transgene expression in crop plants whilst maintaining efficacy of the nematode defence.     

An unconventionally secreted effector from the root knot nematode Meloidogyne incognita,Mi-ISC-1, promotes parasitism by disrupting salicylic acid biosynthesis in host plants

Plant-parasitic nematodes need to deliver effectors that suppress host immunity for successful parasitism. We have characterized a novel isochorismatase effector from the root-knot nematode Meloidogyne incognita, named Mi-ISC-1. The Mi-isc-1 gene is expressed in the subventral oesophageal glands and is up-regulated in parasitic-stage juveniles. Tobacco rattle virus-induced gene silencing targeting Mi-isc-1 attenuated M. incognita parasitism. Enzyme activity assays confirmed that Mi-ISC-1 can catalyse hydrolysis of isochorismate into 2,3-dihydro-2,3-dihydroxybenzoate in vitro. Although Mi-ISC-1 lacks a classical signal peptide for secretion at its N-terminus, a yeast invertase secretion assay showed that this protein can be secreted from eukaryotic cells. However, the subcellular localization and plasmolysis assay revealed that the unconventional secretory signal present on the Mi-ISC-1 is not recognized by the plant secretory pathway and that the effector was localized within the cytoplasm of plant cells, but not apoplast, when transiently expressed in Nicotiana benthamiana leaves by agroinfiltration. Ectopic expression of Mi-ISC-1 in N. benthamiana reduced expression of the PR1 gene and levels of salicylic acid (SA), and promoted infection by Phytophthora capsici. The cytoplasmic localization of Mi-ISC-1 is required for its function. Moreover, Mi-ISC-1 suppresses the production of SA following the reconstitution of the de novo SA biosynthesis via the isochorismate pathway in the cytoplasm of N. benthamiana leaves. These results demonstrate that M. incognita deploys a functional isochorismatase that suppresses SA-mediated plant defences by disrupting the isochorismate synthase pathway for SA biosynthesis to promote parasitism.     

CCS52 and DEL1 genes are key components of the endocycle in nematode‐induced feeding sites

The establishment of galls and syncytia as feeding sites induced by root‐knot and cyst nematodes, respectively, involves a progressive increase in nuclear and cellular size. Here we describe the functional characterization of endocycle activators CCS52A, CCS52B and a repressor of the endocycle, DEL1, during two types of nematode feeding site development in Arabidopsis thaliana. In situ hybridization analysis showed that expression of CCS52A1 and CCS52B was strongly induced in galls and syncytia and DEL1 was stably but weakly expressed throughout feeding site development. Down‐regulation and over‐expression of CCS52 and DEL1 in Arabidopsis drastically affected giant cell and syncytium growth, resulting in restrained nematode development, illustrating the need for mitotic activity and endo‐reduplication for feeding site maturation. Exploiting the mechanism of endo‐reduplication may be envisaged as a strategy to control plant‐parasitic nematodes.     

Biofumigant effect of new defatted seed meals against the southern root‐knot nematode,Meloidogyne incognita

In recent years, Brassica carinata defatted seed meals (DSMs) have been successfully applied as an amendment in the control of pests and soil pathogens in several countries with clear advantages for the environment and soil fertility. The effectiveness of this product is clearly linked to the release of bioactive compounds by the well‐known glucosinolate (GL)–myrosinase (MYR) system. Until now, this commercial know‐how has been limited to allyl‐isothiocyanate, the breakdown product of hydrolysis catalysed by MYR of the GL sinigrin. Brassicaceae germplasm is an extremely wide‐ranging family, considering that more than 3500 species have been classified in this family in nature, which contains around 200 different GLs. Therefore, there is a great potential for the availability of new still unexplored bioactive compounds. This study evaluated, in controlled glasshouse conditions, the effect of biofumigation on the nematode Meloidogyne incognita and that of biostimulation on tomato plants of 13 DSMs obtained from different Brassicaceae species at different levels of soil inoculation. Among the tested DSMs, the best results for all inoculations were achieved by Eruca sativa (rocket), Barbarea verna (land cress) and Brassica nigra (black mustard), whereas the other species gave either alternate results or results not different from untreated or sunflower DSM controls. All the DSMs, including sunflower, determined a clear positive effect on plant vigour. These first results open new perspectives for the application of biofumigation in plant protection and management.     

Expression of tomato salicylic acid (SA)‐responsive pathogenesis‐related genes in Mi‐1‐mediated and SA‐induced resistance to root‐knot nematodes

The expression pattern of pathogenesis‐related genes PR‐1, PR‐2 and PR‐5, considered as markers for salicylic acid (SA)‐dependent systemic acquired resistance (SAR), was examined in the roots and shoots of tomato plants pre‐treated with SA and subsequently infected with root‐knot nematodes (RKNs) (Meloidogyne incognita). PR‐1 was up‐regulated in both roots and shoots of SA‐treated plants, whereas the expression of PR‐5 was enhanced only in roots. The over‐expression of PR‐1 in the whole plant occurred as soon as 1 day after SA treatment. Up‐regulation of the PR‐1 gene was considered to be the main marker of SAR elicitation. One day after treatment, plants were inoculated with active juveniles (J2s) of M. incognita. The number of J2s that entered the roots and started to develop was significantly lower in SA‐treated than in untreated plants at 5 and 15 days after inoculation. The expression pattern of PR‐1, PR‐2 and PR‐5 was also examined in the roots and shoots of susceptible and Mi‐1‐carrying resistant tomato plants infected by RKNs. Nematode infection produced a down‐regulation of PR genes in both roots and shoots of SA‐treated and untreated plants, and in roots of Mi‐carrying resistant plants. Moreover, in resistant infected plants, PR gene expression, in particular PR‐1 gene expression, was highly induced in shoots. Thus, nematode infection was demonstrated to elicit SAR in shoots of resistant plants. The data presented in this study show that the repression of host defence SA signalling is associated with the successful development of RKNs, and that SA exogenously added as a soil drench is able to trigger a SAR‐like response to RKNs in tomato.     

Cell wall breakdown during the formation of syncytia induced in plants by root knot nematodes

Bird Alan F. 1972. Cell wall breakdown during the formation of syncytia induced in plants by root knot nematodes. International Journal for Parasitology, 2: 431–432. Examination under the light muscope of thin (2 μm) serial sections of syncytia embedded in epoxy resin reveals a close association between these structures and adjacent cells. Sometimes these cells protrude into the cytoplasm of a syncytium but at other times their walls and those of syncytia break down and permit cytoplasmic contact. These results support the hypothesis that syncytia are formed partly by incorporation of cells whose walls have dissolved rather than by expansion of a single cell. They also provide an explanation for the observed variability in nucleic acid content of syncytial nuclei.     

Activation of geminivirus V‐sense promoters in roots is restricted to nematode feeding sites

Obligate sedentary endoparasitic nematodes, such as the root‐knot and cyst nematodes, elicit the differentiation of specialized nematode nurse or feeding cells [nematode feeding sites (NFS), giant cells and syncytia, respectively]. During NFS differentiation, marked changes in cell cycle progression occur, partly similar to those induced by some geminiviruses. In this work, we describe the activation of V‐sense promoters from the Maize streak virus (MSV) and Wheat dwarf virus (WDV) in NFS formed by root‐knot and cyst nematodes. Both promoters were transiently active in microinjection experiments. In tobacco and Arabidopsis transgenic lines carrying promoter–β‐glucuronidase fusions, the MSV V‐sense promoter was activated in the vascular tissues of aerial plant parts, primarily leaf and cotyledon phloem tissue and some floral structures. Interestingly, in roots, promoter activation was restricted to syncytia and giant cells tested with four different nematode populations, but undetectable in the rest of the root system. As the activity of the promoter in transgenic rootstocks should be restricted to NFS only, the MSV promoter may have utility in engineering grafted crops for nematode control. Therefore, this study represents a step in the provision of some of the much needed additional data on promoters with restricted activation in NFS useful in biotechnological nematode control strategies.