Rice Root-knot Nematode Meloidogyne Research Articles

Optimization of DNA extraction methods for genomic analysis of rice root-knot nematode (Meloidogyne graminicola) using PCR (polymerase chain reaction) and sanger sequencing

Optimization of DNA extraction methods for genomic analysis of rice root-knot nematode (Meloidogyne graminicola) using PCR (polymerase chain reaction) and sanger sequencing

Evaluation of root-gall associated fungal endophytes for the control of Meloidogyne graminicola infecting rice

In the rice-producing regions of India, the infection of the rice root-knot nematode Meloidogyne graminicola represents a significant biotic constraint that leads to substantial yield reduction. Traditional nematode management practices are neither cost-effective nor environmentally friendly. Consequently, there is an urgent need to identify potential biocontrol agents for effectively managing this destructive pest. M. graminicola infestation in rice incites the formation of terminal root galls, which serve as multinucleated food sinks inhabited by nematodes and other endophytic microorganisms. In our study, we isolated and screened fungal endophytes associated with healthy and galled rice roots to identify potential biocontrol agents against the nematode. Among the isolated fungi, a gall-associated endophytic fungus identified as Aspergillus niger isolate F4 exhibited 100% juvenile (J2) mortality under in vitro conditions at 50% culture filtrate concentration, within 24 h of inoculation, with an LC50 of 10.85%. Additionally, it demonstrated ovicidal properties by inhibiting egg hatching by 96.96% compared to the control. Furthermore, the attraction assay revealed a significantly lower number of M. graminicola J2s attracted to rice roots treated with F4 culture filtrate compared to untreated roots. In an in vivo pot experiment, root dipping with F4 culture filtrate reduced the galling index, the number of J2s penetrating the root, and the reproduction factor. Besides the direct effects, inoculation of the fungus also triggered plant defence responses, potentially providing indirect protection against nematode infection. Overall, our study, to the best of our knowledge, is the first to demonstrate that M. graminicola root galls harbour specific endophytic fungi with potent biocontrol properties. The available evidence clearly establishes the potential of Aspergillus niger F4 as an important biological control agent against M. graminicola, offering a novel management approach for this significant rice pest.

MG1 interacts with a protease inhibitor and confers resistance to rice root-knot nematode

The rice root-knot nematode (Meloidogyne graminicola) is one of the most destructive pests threatening rice (Oryza sativa L.) production in Asia; however, no rice resistance genes have been cloned. Here, we demonstrate that M. GRAMINICOLA-RESISTANCE GENE 1 (MG1), an R gene highly expressed at the site of nematode invasion, determines resistance against the nematode in several rice varieties. Introgressing MG1 into susceptible varieties increases resistance comparable to resistant varieties, for which the leucine-rich repeat domain is critical for recognizing root-knot nematode invasion. We also report transcriptome and cytological changes that are correlated with a rapid and robust response during the incompatible interaction that occurs in resistant rice upon nematode invasion. Furthermore, we identified a putative protease inhibitor that directly interacts with MG1 during MG1-mediated resistance. Our findings provide insight into the molecular basis of nematode resistance as well as valuable resources for developing rice varieties with improved nematode resistance.

CRISPR/Cas9-mediated mutagenesis of the susceptibility gene OsHPP04 in rice confers enhanced resistance to rice root-knot nematode.

CRISPR crops carrying a mutation in susceptibility (S) genes provide an effective strategy for controlling plant disease, because they could be 'transgene-free' and commonly have more broad-spectrum and durable type of resistance. Despite their importance, CRISPR/Cas9-mediated editing of S genes for engineering resistance to plant-parasitic nematode (PPN) disease has not been reported. In this study, we employed the CRISPR/Cas9 system to specifically induce targeted mutagenesis of the S gene rice copper metallochaperone heavy metal-associated plant protein 04 (OsHPP04), and successfully obtained genetically stable homozygous rice mutants with or without transgenic elements. These mutants confer enhanced resistance to the rice root-knot nematode (Meloidogyne graminicola), a major plant pathogenic nematode in rice agriculture. Moreover, the plant immune responses triggered by flg22, including reactive oxygen species burst, defence-related genes expression and callose deposition, were enhanced in the 'transgene-free' homozygous mutants. Analysis of rice growth and agronomic traits of two independent mutants showed that there are no obvious differences between wild-type plants and mutants. These findings suggest that OsHPP04 may be an S gene as a negative regulator of host immunity and genetic modification of S genes through the CRISPR/Cas9 technology can be used as a powerful tool to generate PPN resistant plant varieties.

Population dynamics of Meloidogyne graminicola in soil in different types of direct-seeded rice agroecosystems in Hunan Province, China.

The rice root-knot nematode Meloidogyne graminicola is increasingly widely distributed in China and has had a severe incidence in Hunan Province. It is thus necessary to investigate its population dynamics in paddy fields. This study was conducted to ascertain the effect of direct-seeded rice agroecosystems on the population dynamics of M. graminicola and root gall development in rice. The results indicated that the population density of M. graminicola in soil was markedly influenced by the agroecosystem, rainfall and temperature. The population density of M. graminicola J2, and eggs in the soil and root galls, were significantly larger in the dry aerobic rice agroecosystem and in the rain-fed upland agroecosystem than in the lowland double-rice cropping sequence agroecosystem. As it can affect soil moisture rainfall was the key factor affecting the density of nematodes in both the rain-fed upland agroecosystem and the dry aerobic rice agroecosystem. Field flooding was still an effective way to reduce the population density of M. graminicola. In addition, we observed that M. graminicola can lay eggs outside rice roots under laboratory conditions. Therefore, we propose a hypothesis that M. graminicola lays egg masses within roots when the soil moisture is high, but lays eggs outside when the soil moisture is suitable. By clarifying the population dynamics of M. graminicola in different types of direct-seeded rice agroecosystems, this study is conducive to controlling rice root-knot nematodes.

Evaluation of Total Phenol Content of some Common Paddy Cultivars of Odisha with the Application of Biofertilizer in Assessing Resistance to Meloidogyne graminicola

The total phenol content of four common paddy cultivars of Odisha namely Abhisek (R), Manik (MR), Bas-12 (S) and Lalat (HS) were studied under pot culture condition in the net house following CRD design under different treatments using biofertilizer Azospirillum. brasilense alone or in combinations against rice root-knot nematode Meloidogyne graminicola. The objective of the work was to study the quantitative changes in phenolic compounds content and the effect of A.brasilense in the induction of phenolic compounds on the test nematode. It was observed that there was a significant increase in total phenol content in both resistant and moderately resistant paddy cultivars Abhisek and Manik in treatment T2 (Azospirillum brasilense @ 12kg/ha at 15DAT + M. graminicola @ 1000J2 after 7days of Azospirillum inoculation) were seen higher than the susceptible variety Bas-12 and highly susceptible variety Lalat as compared to control T5 with increase percentage of 38.77%, 36.58%, 35.02% and 39.28% respectively in shoots. Similarly the trend was continued in roots containing total phenol content of 0.25mg/g in Abhisek, 0.18mg/g in Manik, 0.21mg/g in Bas-12 and 0.19mg/g in Lalat as compared to other treatments. There were increase in percentage of change (p≤0.05) in roots under treatment T3 (A alone) in all the four varieties followed by T2 (A→N), and T1 (N→A) with respect to control T5. Basically, shoots of control plants in all four cultivars were observed more content of phenolic compounds than their roots. But after M. graminicola invasion and the action of A. brasilense the amount increased in both the roots and shoots of all the cultivars. The T4 (N alone) treatment in all the R, MR, S and HS varieties were recorded higher as compare to T5 but less than the treatments T3, T2, and T1 in both roots and shoots against the test nematode, which could exacerbating for eco-friendly RKN management approach.

Bio-management of rice root-knot nematode, M. graminicola by using various organic amendments and bio-control agents on rice nursery

A screen house experiment was conducted during kharif season, 2020-21 to know the effect of organic amendments and bio-agents (alone and in combination) in comparison with chemical on the population of rice root-knot nematode (Meloidogyne graminicola) in rice. Nursery was grown in 5 kg soil capacity earthen pan filled with infested soil having initial nematode population 285 J2/200 cc soil. Seeds of the rice (Variety- Pusa 1121) were soaked in tap water for 24 h and the sprouted seeds were sown in pots. Hundred seeds of rice were sown in each pot and each treatment was replicated four times. Organic amendments (neem cake and FYM) and bio-agents (Pseudomonas fluorescens, Purpureocillium lilacinum and Trichoderma viride) were added 10 days before sowing. Weighted amount of bio-agents was mixed in neem cake and FYM for enrichment with 7-10 days waiting period. Carbofuran (Furadan 3G) was added at time of sowing. The earthen pots without organic amendments, bio-agents and chemical were treated as control. Observations on plant growth parameters (seedling length in cm fresh and dry seedling weight in g) and nematode multiplication (number of galls/20 seedling, number of eggs/20 seedling and final nematode population in the soil/200 cc soil) were made at transplanting time. The plant parameters were maximum and significantly highest in treated nursery (neem cake @ 50 g/pot + Pseudomonas fluorescens @ 50 g/pot) under screen house conditions. Nematode reproduction and multiplication parameters such as number of galls/seedling, number of eggs/seedling and final nematode population were significantly reduced in neem cake @ 50 g/pot + Pseudomonas fluorescens @ 50 g/pot.

Bioefficacy evaluation of chalcone derivatives against Meloidogyne graminicola infecting rice, Oryza sativa L.

A series of chalcones (1–14) were synthesized, characterized (using IR and 1H NMR techniques), and evaluated with an objective to manage rice root-knot nematode (RRKN) (Meloidogyne graminicola) both under pluronic gel and field conditions. Out of these fourteen compounds, 1-(4-fluoro-phenyl)-3-phenyl-propenone (13) and 1,3-diphenyl-propenone (14) showed promising and dose dependent activity at 10, 20, and 40 mg L−1. A significant reduction in penetration of second stage juveniles (J2s) in rice roots was observed in compounds 13 (9.5–12.0 J2s/plant) and 14 (10.5–13.4 J2s/plant) compared to control plants (PB1121) (13.5–23.6 J2s/plant) in pluronic gel study. The results of field trials indicated that 14, showed significantly (P ≤ 0.05) better plant growth on 28 days after sowing (DAS) compared to 13. Both 13 and 14 reduced gall formation significantly than carbofuran 3 G @1 kg a.i./ha. However, lower concentrations were less effective in field in reducing the gall formation. Also, a significant reduction in the number of galls was observed when soil was drenched with 14 @ 40 mg L−1. However, root dipping was not as effective as soil drenching. The study revealed that both the chalcones have the potential for effective management of RRKN in fields, and can be a better alternative to carbofuran.

Management of rice root-knot nematode (Meloidogyne graminicola Golden and Birchfield)–An integrated approach

An experiment was conducted to know the efficacy of bio agents viz., Purpureocillium lilacinum, Trichoderma harzianum, Bacillus subtilis,Bacillus megaterium, Vermicompost and Consortium of bio-agents (Purpureocillium lilacinum + Trichoderma harzianum + Bacillus subtilis +Bacillusmegaterium +Vermicompost) and Carbofuran3G @ 0.3 i.e., alone for management of rice root-knot nematode Meloidogyne graminicola for one season at Chikadadakatte village of Honnali taluk, Davanagere district during Kharif- 2018. The results revealed that all the treatments were significantly superior over check with respect to growthparameters and nematode population. However, carbofuran 3G significantly reduced the nematode population (275.11 /200 cc soil)which was found to be the best treatment as it recorded highest plant height (114.06 cm), root length (23.13 cm), maximum grain yield (44.60 q/ha) with least RKI (1.20) followed byConsortium of bio-agents (Purpureocilliumlilacinum + Trichoderma harzianum + Bacillus subtilis +Bacillusmegaterium +Vermicompost).

An improved draft genome assembly of Meloidogyne graminicola IARI strain using long-read sequencing.

The rice root-knot nematode Meloidogyne graminicola is a major biotic stress for the rice crop under upland, rain-fed lowland and irrigated cultivation conditions. Here, we present an improved draft genome assembly of M. graminicola IARI strain using the long-read sequencing approach (PacBio Sequel platform). The assembled genome size was 36.86Mb with 514 contigs and N50 value of 105kb. BUSCO estimated the genome to be 88.6% complete. Meloidogyne graminicola genome contained 17.83% repeat elements and showed 14,062 protein-coding gene models, 4,974 conserved orthologous genes, 561 putative secreted proteins, 49 RNAi pathway genes, 1,853 proteins involved in pathogen-host interactions, 1,575 carbohydrate-active enzymes, and 32,138 microsatellites. Five of the carbohydrate-active enzymes were found only in M. graminicola genome and were not present in any other analysed root-knot nematode genome. Together with the previous two genome assemblies, this improved genome assembly would facilitate comparative and functional genomics for M. graminicola.

Identification of multi-facial microbial isolates from the rice rhizosphere and their biocontrol activity against Rhizoctonia solani AG1-IA

Identification of multi-facial biocontrol agents that could act against different pathogens of rice crop shall be an interesting approach to develop effective strategies of biocontrol in practice. To identify the promising multi-facial microbial isolates (MIs) from the rice rhizosphere, twelve native MIs which had already been tested against rice root-knot nematode (Meloidogyne graminicola) were screened in vitro against sheath blight pathogen of rice, Rhizoctonia solani AG1-IA. The MIs exhibited substantial variability in their ability to inhibit the mycelial growth of R. solani. Out of twelve, six most promising MIs were tested against R. solani under pot trials on rice cv. PusaSugandh-5. We observed 30–52% loss in plant growth with R. solani. However, soil treated with MIs significantly attenuated the adverse effect of sheath blight pathogen. Among the MIs, Pseudomonas putida AMUPP-1 was found to be most effective and suppressed the disease severity by 69% leading to a 21–49% improvement in plant growth. Furthermore, we observed that Trichoderma harzianum AMUTH-1, P. fluorescens AMUPF-1 and other MIs had lesser effectiveness compared to P. putida AMUPP-1. Interestingly, P. putida AMUPP-1 was also found to detoxify oxalic acid and able to produce ammonia, hydrogen cyanide, indole acetic acid, siderophore, and solubilized phosphorus. Moreover, validation trials under naturally infested field verified the effectiveness of P. putida AMUPP-1 with 61% improvement in rice yield. In addition to these, we also observed the effect of P. putida AMUPP-1 better than fungicide, propiconazole. The present study has identified the multi-facial nature of P. putida AMUPP-1 which effectively suppressed different group of rice pathogens, R. solani (in the present study) and M. graminicola (in a previous study).

Application of biochar and vermicompost against the rice root-knot nematode (Meloidogyne graminicola): an eco-friendly approach in nematode management

The rice root-knot nematode (Meloidogyne graminicola) is one of the key pests of rice (Oryza sativa L.) in irrigated and upland rice ecosystems inflicting about 20–80% yield loss. While chemical nematicides are widely used to combat this pest, they have a negative impact on the environment as well as on animal and human health. The present study evaluated the efficacy of organic amendments like biochar and vermicompost in managing the rice root-knot nematode (RRKN) at 0.6, 1.2, 2.5, and 5% (w/v) concentrations. Our result showed that biochar and vermicompost exudate neither exhibit any direct killing ability nor altered the infectivity of RRKN second-stage juveniles (J2). Second-stage juvenile hatching was, however, reduced in two higher doses (2.5% and 5%) of vermicompost exudates. When final nematode population was normalized with root weights at 21 days’ post inoculation, a reduction in population build-up was observed with increased doses of vermicompost. A reverse trend was observed for biochar treatments. Overall, our study showed that application of 1.2% biochar and 5% vermicompost could be helpful in mitigating the RRKN stress in rice. Hence, application of biochar and vermicompost could be an effective alternative to toxic chemical nematicides and recommended as eco-friendly management options against M. graminicola in rice.

Synthesis and transfer of RNAi construct for potential knock-down of gene expression of root-knot nematode (Meloidogyne graminicola) to rice (Oryza sativa L.)

Effectors play key roles in the parasitism of the plant-parasitic nematode. Silencing the effector-coding genes was applied to study the function and role of nematode effectors. In this study, the Mgra16281 gene (ID: MK322955.1) encoding an effector with the unknown function was cloned from the rice root-knot nematode Meloidogyne graminicola isolated in Long An province. To knock-down the expression of this gene, an artificial microRNA was synthesized based on the Osa-MIR528 precursor and inserted into an expression vector. This microRNA can be expressed in rice to investigate the function of MGRA16281 of root-knot nematode via host-induced gene silencing approach (HIGS).

Crop rotation sequencing to minimize yield losses of summer-irrigated lowland rice in Myanmar caused by the rice root-knot nematode Meloidogyne graminicola

The effect of different rice-based crop rotation sequences on the population densities of Meloidogyne graminicola and on the yield of rice was evaluated in a microplot experiment under lowland rice field conditions. Ten treatments of cropping sequences were grown in four successive growing seasons: continuous growing of the susceptible rice cultivar Thihtatyin (rice-rice-rice-rice), four treatments of 1- season crop rotation sequences (rice-rice-chickpea-rice, rice-rice-black gram-rice, rice-rice-soybean-rice, rice-rice-cowpea-rice), and five treatments of 2-season crop rotation sequences (groundnut-rice-chickpea-rice, green gram-rice-black gram-rice, cowpea-rice-soybean-rice, sesame-rice-cowpea-rice, sunflower-rice-sesame-rice). Population densities of M. graminicola recovered from the rice plants and the nematode multiplication factors (Mf) in the soil in the continuous rice cropping sequence (rice-rice-rice-rice) was the highest among the ten cropping sequences. Lowest nematode Mf in the soil was observed in the 2-season crop rotation sequence sunflower-rice-sesame-rice and the highest was observed in the 1-season crop rotation sequence rice-rice-cowpea-rice among the nine crop rotation sequences. Highest rice yield was obtained in the 2-season crop rotation sequences green gram-rice-black gram-rice, sesame-rice-cowpea-rice and sunflower-rice-sesame-rice, which were about 2 times higher compared with the 1-season crop rotation sequences and about 3 times higher compared with the continuous rice cropping sequence.

Beneficial bacterial volatile compounds for the control of root-knot nematode and bacterial leaf blight on rice

Microbial volatiles can promote plant growth and suppress diseases, nematodes and insects. Our knowledge of the effects of microbial volatiles on monocots such as rice and its pathogens and pests is still incomplete. As part of a screening initiative for beneficial bacterial species, we identified three strains (Bacillus sp., Paenibacillus sp. and Xanthomonas sp.) lethal to the rice root-knot nematode (Meloidogyne graminicola). Using both in vitro and in planta dual-chamber pot experiments, we found that volatiles from the beneficial bacteria were lethal to M. graminicola J2s and significantly reduced infection of susceptible rice. We conducted similar in vitro and in planta experiments against rice bacterial leaf blight Xanthomonas oryzae pv. oryzae. Exposure to bacterial volatiles inhibited the growth of X. oryzae pv. oryzae by 50–60% in vitro, but did not impact infection in planta. Finally, we found that bacterial volatiles from Bacillus sp. and Xanthomonas sp. inhibited rice germination and seedling development in vitro but did not affect plant growth in planta. These findings suggest that volatiles from beneficial bacteria have the potential to control M. graminicola, but that high volatile concentrations may inhibit plant growth.

Effect of Agrochemicals used Commonly in Rice Ecosystem on Meloidogyne graminicola

The effect of 14 insecticides, 6 fungicides and 6 herbicides used commonly in a rice ecosystem on mortality of infective stage juveniles (J2) of the rice root-knot nematode Meloidogyne graminicola, is reported in-vitro bioassays. Among the insecticides studied more than 50 per cent mortality was recorded with cartap hydrochloride, monocrotophos, chlorpyriphos and azadirachtin with a mean juvenile mortality of 100.00, 84.50, 70.50 and 80.00 per cent, respectively at 96 h post-exposure. Among the fungicides, more than 50 per cent mortality of J2 was observed with propiconazole (81.43%) and mancozeb (57.14%) at 96 h after exposure. Among herbicides, bispyribac sodium and pendimethalin resulted in 95.72 and 67.86 per cent mortality of J2 at 96 h of incubation. The insecticide cartap hydrochloride (50% SP) caused 100 per cent mortality of J2 within 24 h of exposure.

Morphological and molecular characterization of the rice root-knot nematode, Meloidogyne graminicola, Golden and Birchfeild, 1965 occurring in Zhejiang, China

The rice root-knot nematode Meloidogyne graminicola is a severe pest of rice. In China, it was first reported from Hainan Province, and later from several other provinces. In the present study, a rice root-knot nematode population found from the rice cultivation areas of Zhejiang Province, China is characterized via molecular analysis using internal transcribed spacer (ITS) and cytochrome c oxidase subunit II (coxII)-16S rRNA genes and scanning electron microscopy (SEM) observations of males and the second-stage juveniles. Morphometric data and molecular sequence comparisons for all M. graminicola populations occurring in China are also provided. The overall morphology of M. graminicola found in Zhejiang match well with the original description, though males have a slightly longer body and stylet, and a shorter tail, while the second-stage juvenile is also slightly longer than in the original description. This is the first report of M. graminicola from Zhejiang. Phylogenetic studies based on coxII suggest that all the Chinese populations belong to Type B. This study expands knowledge of the increasing distribution and phylogenetic relationships of M. graminicola that occur in China.

Chemical and biological control of root knot nematode (Meloidogyne graminicola) in Paddy Crop

The present field experiment was conducted to evaluate the efficacy of bio agents and chemical nematicides viz. Trichoderma harzianum, Pseudomonas fluorescens, Bacillus subtilis, Carbofuran 3G and Phorate 10G for management of rice root-knot nematode Meloidogyne graminicola during kharif-2014, 2015 and 2016. The present findings revealed that all the treatments were significantly superior over control with respect to plant growth parameters and nematode population. In control, terminal and spiral galls developed on rice roots, and plants suffered 32-41% decrease in the plant growth parameters and resulted upto 27% yield loss. At harvest greater decrease in soil population was recorded after soil application of T. harzianum (51.11%) followed by carbofuran 3G (46.82%). T. harzianum and carbofuran 3G were at par in suppression of root gall and egg mass suppression and T. harzianum caused 61.11 and 58.28% reduction in root gall and egg mass respectively, whereas, carbofuran 3G caused 59.52% and 62.50% reduction in root gall and egg mass respectively.

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.

Nematode Genome Announcement: A Draft Genome for Rice Root-Knot Nematode, Meloidogyne graminicola.

The rice root-knot nematode Meloidogyne graminicola has emerged as a devastating pest of rice in South-East Asian countries. Here we present a draft genome sequence for M. graminicola , assembled using data from short and long insert libraries sequenced on Illumina GAIIx sequencing platform.