Identification of neuropeptides,flp-1 and flp-12 targeting neuromuscular system of rice root knot nematode (RRKN) Meloidogyne graminicola

Root-knot nematodes (RKNs), Meloidogyne spp, are found in all temperate and tropical areas, and are among the most damaging plant pathogens worldwide. M. graminincola is an economically important root parasite on upland, lowland and deepwater rice. FMRFamide-like peptides (FLPs) play significant role as neurotransmitters or neuromodulators in the nervous system and proposed as one of the important targets for the plant parasitic nematode management. Therefore, for the first time, we have cloned and characterized two neuropeptide genes (flp-1 and flp-12) from the cDNA of preparasitic second stage juveniles of M. graminicola. The flp-12 contains putative 22 residue long signal peptide at N-terminal suggesting function as an extra-cellular protein. We have found highly conserved motif LFRGR in flp-1. These two flp genes could be interesting and potential targets for functional validation to explore their utility for designing management strategies.     

Rice root-knot nematode (Meloidogyne graminicola) infestation in rice

Rice (Oryza sativa) is an important staple food crop for majority of human population in the world in general and in Asia in particular. However, among various pests and diseases which constitute important constraints in the successful crop production, plant parasitic nematodes play an important role and account for yield losses to the extent of 90%. The major nematode pests associated with rice are Ditylenchus angustus, Aphelenchoides besseyi, Hirschmanniella spp., Heterodera oryzicola and Meloidogyne graminicola. However, rice root-knot nematode (M. graminicola) happens to be the most important pest and is prevalent in major rice producing countries of the world. In India, the distribution of M. graminicola in rice growing areas of different states has been documented in nematode distribution atlas prepared by All India Coordinated Research Project (Nematodes) and published by Directorate of Information and Publications of Agriculture, Indian Council of Agricultural Research, New Delhi, India during 2010. M. graminicola affected rice plants show stunting and chlorosis due to the characteristic terminal swellings/galls on the roots which ultimately result in severe reduction in growth and yield. Number of eco-friendly management technologies against M. graminicola have been developed and demonstrated, including the use of bioagents for minimising the losses due to rice root-knot nematode. This review is focused on collating information to understand the current scenario of rice root-knot nematodes with greater emphasis on its ecological requirements, damage symptoms, biology, morphology, host range and management strategies.     

Host response of rice genotypes to the rice root-knot nematode (Meloidogyne graminicola) under aerobic soil conditions

Aerobic rice is a production system where adapted rice varieties are established via direct seeding in non-puddled, non-flooded, non-saturated fields and grown under conditions similar to upland conditions. On land cultivated continuously with aerobic rice, a yield reduction has been observed. The rice root-knot nematode Meloidogyne graminicola is considered one of the possible causes of these yield reductions. Resistance to and tolerance for M. graminicola are essential traits for aerobic rice cultivars in alleviating this problem. In our study, the host response of nineteen aerobic, seven upland and four lowland rice genotypes which are either being used in the International Rice Research Institute’s aerobic rice breeding programme or already cultivated by farmers in Asia was evaluated under aerobic soil conditions in an outdoor raised-bed experiment. Our study showed a large variation in susceptibility and sensitivity to M. graminicola infection among the rice genotypes examined. Resistance comparable to the resistant reference genotypes included in the experiment (CG14, TOG5674, TOG7235) was not found but in terms of susceptibility, the upland genotype Morobereken may be an interesting less-susceptible genotype. Tolerance was found and in terms of sensitivity, the high yielding aerobic genotype IR78877-208-B-1-2 may be an interesting tolerant genotype. Our study also allowed the identification of rice genotypes that are either highly susceptible or sensitive to M. graminicola infection and of which the cultivation should be discouraged. On average, M. graminicola caused an almost 30% reduction in yield. Excluding the two susceptible and three resistant reference genotypes included in the experiment, most affected was dry-shoot biomass (23.6% reduction) followed by root length, which was more affected than fresh-root weight (19.8 vs. 8%) and grain filling (17.3%), while plant height and the number of spikelets/panicle were less affected (10.2 and 8.1%, respectively). Neither tillering nor the number of panicles/plant were affected.     

Mining the secretome of the root-knot nematode Meloidogyne chitwoodi for candidate parasitism genes

Parasite proteins secreted at the interface of nematode and host are believed to play an essential role in parasitism. Here, we present an efficient pipeline of bio-informatic algorithms and laboratory experiments to identify candidate parasitism genes within nematode secretomes, i.e. the repertoire of secreted proteins in an organism. We performed our approach on 12 218 expressed sequence tags (ESTs) originating from three life stages of the plant parasitic nematode Meloidogyne chitwoodi —a molecularly unexplored root-knot nematode species. The ESTs from M. chitwoodi were assembled into 5880 contigs and open reading frames translated from the consensus sequences were searched for features of putative signal peptides for protein secretion and trans-membrane regions, resulting in the identification of 398 secretome members. The products of parasitism genes are secreted by a range of organs, including the oesophageal, amphidial and rectal glands, the intestine, and the hypodermis. To localize the site of expression in M. chitwoodi , we subjected the most abundant secretome members to in situ hybridization microscopy. We found hybridization of one tag in the dorsal oesophageal gland, seven in the two subventral oesophageal glands, two in the intestine and one tag hybridized to the tail tip in the proximity of the phasmids. Four sequences showed similarity to putative parasitism genes from other nematode species, whereas seven represented pioneering sequences. Our approach presents an efficient method to identify candidate parasitism genes, which does not require sophisticated cDNA isolation and selection protocols, and can therefore be used as a powerful starting point for the molecular investigation of parasites.     

Pathogenicity of root knot nematode Meloidogyne incognita on okra and its management through botanicals

An investigation was carried out to study the pathogenicity of root knot nematode Meloidogyne incognita on okra and its management through various organic amendments. The inoculum level of 1000 juveniles per plant showed significant reduction in various plant growth parameters, which reveals that M. incognita is a potential pathogen of okra. With the increase in inoculums level of M. incognita (J₂), there was a progressive decrease in various plant growth parameters. The maximum reduction in plant growth parameters was observed at an inoculum level of 4000 juveniles per plant. The efficacy of five organic amendments viz. groundnut cake, castor cake, sunflower cake, linseed cake and sawdust was tested against root knot nematode M. incognita. Amending the soil with different oil cakes was found to be effective in reducing the nematode soil population, number of females, number of egg masses as well as root gall formation in okra. The highest increase in plant growth (13%) and maximum reduction in number of galls (54%), number of females (57%) and number of egg masses (55%) was recorded on application of groundnut cake.     

Biological control of rice root-knot nematode,Meloidogyne graminicola through mixture of Pseudomonas fluorescens strains

The plant growth promoting rhizobacterium, Pseudomonas fluorescens strains PF1, TDK1, and PY15 were evaluated individually and in combinations for their efficacy against root-knot nematode, Meloidogyne graminicola, in rice plants under in vitro, glass house and field conditions. Culture filtrates of these strains either individually or as mixture inhibited egg hatching and caused mortality of juveniles of M. graminicola in vitro. The efficacy was more pronounced when filtrates of the strain were used as mixtures than as individual strains. Mixtures of P. fluorescens strains signficantly reduced M. graminicola infestation when applied as bacterial suspensions through seed treatment. The higher activity of peroxidase and chitinase enzymes was observed in plants treated with P. fluorescens mixtures than the plants treated with individual strains, two strain mixtures and untreated control. In field trials on rice, talc formulations of the P. fluorescens strains individually as well as mixtures were evaluated as seed treatment, soil treatment and combination of both. A mixture of the three strains was the most effective when applied either as seed + soil treatment or as seed treatment alone. The introduced P. fluorescens strains survived endophytically on rice roots. The application of the P. fluorescens mixture PF1 + TDK1 + PY15 in seed + soil treatment resulted in higher grain yield which provided a 27.3% increase over the control followed by P. fluorescens mixture PF1 + TDK1 + PY15 in seed treatment alone, which increased the grain yield of rice by 24.7% compared to the control.     

Management of root knot nematode, Meloidogyne incognita in cucumber (Cucumis sativus) using silicon

Silicon (Si) has been reported to effectively manage some pests and diseases of plants. This study was conducted to determine the effect of Si concentration, mode, and frequency of application in managing Meloidogyne incognita in cucumber. A susceptible cultivar of cucumber (cv. Cyclone) was planted in pots containing heat-sterilized soil. Three weeks after planting, the plants were inoculated with 1,000 juveniles/ pot. Uninoculated plants were provided to serve as control. Three concentrations of Si in the form of sodium metasilicate was applied on the leaves and roots alone and also on both the leaves and roots. Application was done once during the growing period and weekly until seven days before harvest. Leaf and root application of Si was found to significantly increase (p = 0.0029) the fresh top weight of inoculated and uninoculated plants. On the other hand, inoculation of root-knot nematode significantly increased the fresh root weight of cucumber which could be due to enlargement of roots or formation of galls. Interestingly, the inoculated plants gave significantly higher marketable yield than uninoculated ones. Application of Si at the rate of 200 ppm significantly increased the marketable yield compared to the higher rate of Si (400 ppm). At 200 ppm, one application of Si both on the leaves and roots significantly reduced the number of galls in inoculated plants. This was comparable to the same concentration applied continuously on the roots and at higher concentration (400 ppm) applied continuously on the leaves and on the roots. On the other hand, single root application of Si at the rate of 400 ppm gave the lowest number of eggmasses, however, it was comparable to the same Si concentration applied singly on the leaves and applied continuously both on the leaves and roots. These treatments, however, were at par with continuous application of the lower rate of Si (200 ppm) on the leaves and both leaves and roots.     

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.     

Host response of rice genotypes from crosses of high-yielding and drought-tolerant Oryza sativa advanced breeding lines to the rice root-knot nematode Meloidogyne graminicola

The host response to Meloidogyne graminicola infection of 30 advanced breeding lines developed from crosses between high-yielding and drought-tolerant Oryza sativa genotypes was evaluated in outdoor raised beds. None of the advanced breeding lines showed resistance to M. graminicola comparable to the M. graminicola-resistant African rice (O. glaberrima) genotype TOG5674. However, rice genotype IR86815-23-4-1-2 was identified tolerant which showed no yield reduction even when infected with M. graminicola. IR82635-B-B-143-1, IR85733-19-4-1-1, IR85733-19-4-2-4, IR85,735-42-1-4-1 and IR 85735-42-1-4-4 were less sensitive to M. graminicola infection with less than 20% yield reduction compared to other rice genotypes. These rice genotypes could alleviate yield losses in M. graminicola-infested rice fields in regions with limited resources to control M. graminicola and when resistant cultivars are not available.     

Host response of Oryza glaberrima and O. sativa rice genotypes to the rice root-knot nematode Meloidogyne graminicola in a hydroponic system under growth chamber

The host response of 25 rice genotypes belonging to Oryza glaberrima and Oryza sativa to Meloidogyne graminicola infection was examined in a hydroponic system. The M. graminicola can build up high population densities in a hydroponic system. Resistance to this nematode species was found in O. glaberrima genotypes which supported significantly lower nematode numbers per plant and per unit root than O. sativa genotypes. The M. graminicola-infected O. sativa genotypes showed a higher root galling index than the O. glaberrima genotypes. The hydroponic system is efficient and reliable method to examine the host response of rice genotypes to M. graminicola infection, and can be useful for the fast screening of high numbers of rice genotypes for the selection of M. graminicola-resistant rice germplasm for breeding purposes.     

Identification of functional candidate genes for drought tolerance in rice

Drought tolerance (DT) in rice is known to be controlled by many quantitative trait loci (QTLs) and involved differential expression of large numbers of genes, but linking QTLs with their underlying genes remains the most challenging issue in plant molecular biology. To shed some light on this issue, differential gene expression in response to PEG simulated drought in 3 unique genetic materials (a lowland rice, IR64 and its derived line, PD86 which has 11 introgressed DT QTLs, and a upland rice IRAT109) was investigated using a PCR-based subtractive hybridization strategy. More than 300 unique subtracted cDNA sequences, covering genes of diverse cellular activities and functions, were identified and confirmed by semi-quantitative and quantitative RT-PCR. Detailed bioinformatics analyses of the data revealed two interesting results. First, the levels and mechanisms of DT of the three rice lines were associated with the number and types of differentially expressed genes, suggesting different DT mechanisms in rice are controlled by different sets of genes and different metabolic pathways, and most differentially expressed genes under drought were able to contribute to DT. Second, there appeared a high correspondence in genomic location between DT QTLs and clusters of differentially expressed genes in rice, suggesting some DT QTLs may represent clusters of co-regulated and functionally related genes. Thus, differential gene expression analyses using genetically characterized materials can provide additional insights into the molecular basis of QTLs and convergent evidence to shortlist the candidate genes for target QTLs. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Bin-Ying Fu and Jian-Hua Xiong are contributed to this work equally.     

MicroRNA-target gene responses to root knot nematode (Meloidogyne incognita) infection in cotton (Gossypium hirsutum L.)

MicroRNAs (miRNAs) are a large class of small regulatory RNA molecules, however no study has been performed to elucidate the role of miRNAs in cotton (Gossypium hirsutum) response to the root knot nematode (RKN, Meloidogyne incognita) infection. We selected 28 miRNAs and 8 miRNA target genes to investigate the miRNA-target gene response to M. incognita infection. Our results show that RKN infection significantly affected the expression of several miRNAs and their targeted genes. After 10 days of RKN infection, expression fold changes on miRNA expressions ranged from down-regulated by 33% to upregulated by 406%; meanwhile the expression levels of miRNA target genes were 45.8% to 231%. Three miRNA-target pairs, miR159-MYB, miR319-TCP4 and miR167-ARF8, showed inverse expression patterns between gene targets and their corresponded miRNAs, suggesting miRNA-mediated gene regulation in cotton roots in response to RKN infection.     

Interaction of vesicular-arbuscular mycorrhizal fungi and root knot nematode on cultivars of tomato and white clover susceptible to Meloidogyne hapla

The interactive effects of vesicular-arbuscular mycorrhizal (VAM) fungi and root-knot nematode (Meloidogyne hapla) were studied on nematode-susceptible cultivars of tomato (cv. Scoresby) and white clover (cv. Huia) at four levels of applied phosphate. The relative merits of simultaneous inoculation with mycorrhizal fungi and nematodes and of inoculation with mycorrhizal fungi prior to nematode inoculation were evaluated. Mycorrhizal plants were more resistant than non-mycorrhizal plants to root-knot nematode at all phosphate levels and growth benefits were generally greater in plants preinfected with mycorrhizal fungi. Nematode numbers increased with increasing levels of applied phosphate. In mycorrhizal root systems, nematode numbers increased in the lower phosphate soils; at higher phosphate levels nematode numbers were either unaffected or reduced. The numbers of juveniles and adults per gram of root were always lower in mycorrhizal treatments. Mycorrhizal root length remained unaffected by nematode inoculation. Mycorrhizal inoculation thus increased the plants' resistance to infection by M. hapla. This was probably due to some alteration in the physiology of the root system but was not entirely a result of better host nutrition and improved phosphorus uptake by mycorrhizal plants.