Meloidogyne Incognita Eggs Research Articles

Purification and Characterization of Chitinase from the Nematode – Fungus Paecilomyces sp. P1

Purification and Characterization of Chitinase from the Nematode – Fungus Paecilomyces sp. P1

Performance of volatiles emitted from different plant species against juveniles and eggs of Meloidogyne incognita

Several plant species are known to control plant-parasitic nematodes in soil by producing nematicidal volatile organic compounds (VOCs). However, the VOCs activity of many plant species against root-knot nematodes is still unknown. Here, we investigated VOCs emitted from leaves of citronella (Cymbopogon nardus L.) or black pepper (Piper nigrum L.), broccoli shoots (Brassica oleracea L.) and Brazil nuts (Bertholletia excelsa Bonpl.) against second-stage juveniles (J2) and eggs of Meloidogyne incognita. In vitro assays revealed that VOCs emitted from dry plant macerates decreased J2 motility to almost 0% and reduced egg hatching by 47% in comparison with the control. The water exposed to VOCs from citronella, black paper and Brazil nuts decreased the motility of the J2 by 42%, whereas broccoli reduced it to almost 0%. Furthermore, VOCs from Brazil nuts and broccoli shoots killed J2 and reduced the number of galls and eggs in tomato roots under greenhouse conditions. Purified dimethyl sulfate (DMS), dimethyl disulfide (DMDS) and 3-pentanol, previously identified in the broccoli volatilome, were tested on eggs of M. incognita. DMDS and 3-pentanol were shown to reduce egg hatching by 96.8% at 176 mg/L and by 88.4% at 918 mg/L, respectively, whereas DMS increased egg hatching by 13% at 500 and 1000 mg/L. All plant species produced VOCs toxic to eggs and J2, which are the most important developmental stages in terms of nematode management and should be more exploited to reduce root-knot nematode populations in soils prior to host infection or used for prospection of novel nematicidal products.

Cloning of the defense gene PlchiIII and its potential role in the biocontrol of Pratylenchus coffeae nematodes and Meloidogyne incognita eggs in Musa

Abstract A wide range of crops such as bananas and plantains are devastated by plant parasitic nematodes that affect the root system, leading to impaired plant growth and production. In this scenario, the chitinase gene was isolated from the nematode-resistant cultivar of Pisang lilin. The amplified target gene was cloned into pET28a encoding 323 amino acid residues, which shares a high degree of homology with acidic class III chitinase in Musa acuminata, and the product was transformed into E. coli BL21 strain for protein expression. Using affinity chromatography, the recombinant chitinase was purified as a protein of 29 kDa with an optimal activity at pH 7.0. The purified chitinase was capable of degrading the chitin present in the exoskeleton layer of root lesion nematodes (Pratylenchus coffeae) and in the eggshells of root-knot nematodes (Meloidogyne incognita), as well as significantly influencing its development and mortality. Bioassay was performed under in vitro conditions. In root lesion nematodes, 100% mortality was recorded at concentrations of 0.2 µg/mL and 0.15 µg/mL on the 3rd and 5th days, respectively, whereas the egg-hatching rate was inhibited by 100% at concentrations of 0.2 µg/mL and 0.01 µg/mL on the 1st and 5th days, respectively. The results of this study suggest that the PlchiIII gene can be used as a plant-based microbial agent in the biocontrol of nematodes would help in reducing nematode population and increase the yield of banana.

Nematicidal activity of silver nanoparticles of botanical products against root-knot nematode, Meloidogyne incognita

Current study investigated the nematicidal activity of leaf extracts of Conyza dioscoridis, Melia azedarach, and Moringa oleifera that were prepared as silver nanoparticles (Ag-NP). The characterisation and size confirmation of the Ag-NP were done by UV–vis spectrophotometry and the scanning electron microscopy (SEM). The phytochemical contents of crude extracts and the nano formulations were analysed using gas chromatography-mass spectroscopy (GC-MS). Results revealed that silver nanoparticles of C. dioscoridis extractives had great nematicidal activity against the 2nd stage juvenile (J2) and eggs of Meloidogyne incognita. Also, the Ag-NP showed similar nematicidal effect to the reference nematicide; rugby. The GC-MS analysis revealed the increase of certain metabolites due to the formulation of the Ag-NPs. Aromadendrene, 1-hydroxy-1,7-dimethyl-4-isopropyl-2,7-cyclodecdiene, 6-epi-shyobunol, 4-hexylacetophenone, β-isocomene, caryophyllene, β- and α-selinene, α-cadinol, berkheyaradulen, and bis-(2-ethylhexyl)phthalate were increased more than 2.5-folds in the Ag-NP compared the extract. Therefore, the green synthesis of metal nanoparticles might be a safe, effective and affordable nematicide alternatives.

Mutagenic effects of low-energy N+ ion implantation on the propamocarb-tolerance of nematophagous fungus Lecanicillium attenuatum

Lecanicillium attenuatum is an important nematophagous fungus with potential as a biopesticide for control of plant-pathogenic nematodes. However, relatively low fungicide-tolerance limits its application in the field. To improve the propamocarb-tolerance of L. attenuatum, a low-energy N+ ion implantation-based mutagenesis system was established. In the first round of N+ ion implantation, a proper conidiallethality rate (87.2–88.8%) and the highest positive mutation rate (11.2–11.4%) were achieved, and the highest propamocarb-tolerant mutant LA-I-R1-T7-M1 and LA-I-R1-T7-M2 were isolated in the experiment 1 and experiment 2 respectively, having a median effective concentration (EC50) value of 1223.6 and 1173.6μg/ml respectively. Multiple rounds of N+ ion implantation were further employed, but fungal propamocarb-tolerance had not been significantly enhanced until the fifth-round of ion implantation. The LA-I-R5-M1 was the highest tolerant to propamocarb among all the mutants, whose EC50 value was increased 2.54-fold compared to wild-type strain, reaching to the 1298.7μg/ml. Its EC50 value was significantly increased with continuous passaging, which was stable from the fifth- to fifteenth-generation, and then declined. However the EC50 value of the twentieth-generation strain was still higher than the EC50 (1298.7μg/ml) of the first-generation strain. The colony growth, conidia yield and conidial germination of mutants on plates, and their parasitism in the nematode eggs of Meloidogyne incognita and Heterodera glycines were not significantly changed in both single- and multiple-round of N+ ion implantation. In conclusion, we had succeeded in improving the propamocarb-tolerance of L. attenuatum through this low-energy N+ ion implantation-based mutagenesis system. The improved strain LA-I-R5-M1 could be potentially applied in combination with propamocarb in the field.

Screening of cassava varieties for resistance to root-knot nematodes

Twenty cassava cultivars were screened in micro-plot experiments in the green house of Institute of Agricultural Research and Training, Obafemi Awolowo University, Moor Plantation, Ibadan for root-knot nematode resistance. Four weeks after planting, each plant was inoculated with 5000 eggs of Meloidogyne incognita. There were three replications per cultivar. The plants were uprooted and assessed for resistance after 60 days of inoculation. Assessment of plant damaged was determined by gall index and reproductive factor. Based on host status rating, 3 cultivars were found to be resistant, 4 cultivars were tolerant, 10 cultivars ere susceptible while .3 cultivars were hypersusceptible. All the screened cultivars are resistant to cassava mosaic disease and other major pests of cassava like bacterial disease, anthracnose disease, cassava green mite and cassava mealy bug. They are all high yielding, suitable for food, industry and livestock feed.

Sensitivities of nematode-inoculated tomato plants to Nemarioc-AL phytonematicide from fresh Cucumis myriocarpus fruit

Costs related to temperature-dependent drying and grinding equipment of Cucumis myriocarpus fruit for preparation of Nemarioc-AL phytonematicide could inherently restrict on-farm preparation of this product, particularly in resource-poor smallholder farming systems. The objective of this study was to determine the overall sensitivities of tomato (Solanum lycopersicon L.) plants and nematodes to Nemarioc-AL phytonematicide produced using fermented crude extracts of C. myriocarpus fresh fruit thereby establishing the feasibility of producing this product on-farm. Uniform five-week-old tomato cv. ‘Floradade’ seedlings were transplanted into plastic pots containing a growing mixture, with treatments 0, 2, 4, 8, 16, 32 and 64% Nemarioc-AL phytonematicide applied weekly on plants inoculated with 3000 Meloidogyne incognita eggs and second-stage juveniles (J2). At 56 days after initiating the treatments, plant and nematode variables were subjected to analysis of variance. Nematode variables over increasing log2 2-transformed concentrations were subjected to lines of the best fit. Nematodes versus increasing concentrations of phytonematicide exhibited negative quadratic relations, with the model for eggs and J2 in roots and soil being explained by 90 and 95% relations, respectively. Plant variables were subjected to the Curve-fitting Allelochemical Response Data (CARD) computer-based model to generate biological indices. The mean concentration stimulation point (MCSP = Dm + Rh/2) was computed 2.79%, with the overall sensitivity (Σk) of four units. In conclusion, Nemarioc-AL phytonematicide from fermented fresh fruit of C. myriocarpus could be used in managing population densities of Meloidogyne species in tomato production without inducing phytotoxity.

Effects of Tomato Root Exudates on Meloidogyne incognita.

Plant root exudates affect root-knot nematodes egg hatch. Chemicals in root exudates can attract nematodes to the roots or result in repellence, motility inhibition or even death. However, until recently little was known about the relationship between tomato root exudates chemicals and root-knot nematodes. In this study, root exudates were extracted from three tomato rootstocks with varying levels of nematode resistance: Baliya (highly resistant, HR), RS2 (moderately resistant, MR) and L-402 (highly susceptible, T). The effects of the root exudates on Meloidogyne incognita (M. incognita) egg hatch, survival and chemotaxis of second-stage juveniles (J2) were explored. The composition of the root exudates was analysed by gas chromatography/mass spectrometry (GC/MS) prior to and following M. incognita inoculation. Four compounds in root exudates were selected for further analysis and their allopathic effect on M. incognita were investigated. Root exudates from each tomato rootstocks (HR, MR and T strains) suppressed M. incognita egg hatch and increased J2 mortality, with the highest rate being observed in the exudates from the HR plants. Exudate from HR variety also repelled M. incognita J2 while that of the susceptible plant, T, was demonstrated to be attractive. The relative amount of esters and phenol compounds in root exudates from HR and MR tomato rootstocks increased notably after inoculation. Four compounds, 2,6-Di-tert-butyl-p-cresol, L-ascorbyl 2,6-dipalmitate, dibutyl phthalate and dimethyl phthalate increased significantly after inoculation. The egg hatch of M. incognita was suppressed by each of the compound. L-ascorbyl 2,6-dipalmitate showed the most notable effect in a concentration-dependent manner. All four compounds were associated with increased J2 mortality. The greatest effect was observed with dimethyl phthalate at 2 mmol·L-1. Dibutyl phthalate was the only compound observed to repel M. incognita J2 with no effect being detected in the other compounds. Each of the four compounds were correlated with a reduction in disease index in the susceptible cultivar, T, and tomato seedlings irrigated with L-ascorbyl 2,6-dipalmitate at 2 mmol·L-1 showed the best resistance to M. incognita. Taken together, this study provided a valuable contribution to understanding the underlying mechanism of nematode resistance in tomato cultivars.

Isolation and identification of nematode-antagonistic compounds from the fungus Aspergillus candidus.

Culture medium from an isolate of the fungus Aspergillus candidus was extracted, fractionated and examined to discover compounds antagonistic to plant-parasitic nematodes that are important pathogens of agricultural crops. Column, thin layer and preparative chromatographies and spectral and elemental analyses, were used to isolate and identify two major constituents of an active fraction (Fraction F) obtained from the medium. Compound 1 was identified as 2-hydroxypropane-1, 2, 3-tricarboxylic acid (citric acid). Compound 2 was identified as 3-hydroxy-5-methoxy-3-(methoxycarbonyl)-5-oxopentanoic acid, an isomer of 1, 2-dimethyl citrate. Compound 1 and a citric acid standard, each tested at 50 mg mL(-1) in water, decreased hatch from eggs of the plant-parasitic nematode Meloidogyne incognita by more than 94%, and completely immobilized second-stage juveniles after 4-6 days exposure. Fraction F and Compounds 1 and 2 decreased the mobility of adults of the plant-parasitic nematode Ditylenchus destructor in vitro. Fraction F (25 mg mL(-1)) inhibited mobility >99% at 72 hrs. Compounds 1 and 2 (50 mg mL(-1)) each inhibited mobility more than 25% at 24 hr and more than 50% at 72 hr. This is the first assignment of nematode-antagonistic properties to specifically identified A. candidus metabolites.

The role of a phospholipase (PLD) in virulence of Purpureocillium lilacinum (Paecilomyces lilacinum)

Phospholipases are key enzymes in pathogenic fungi that cleave host phospholipids, resulting in membrane destabilization and host cell penetration. However, understanding the role of phospholipases on the virulence of the filamentous fungus Purpureocillium lilacinum has been still rather limited. In this study, pld gene was characterized. It encodes the protein phospholipase D (PLD) in P. lilacinum. This gene, 3303 bp open reading frame fragment (ORF), encodes a protein of 1100 amino acids with high similarity to the same gene from Penicillium oxalicum and Aspergillus fumigatus. Secondary structure prediction showed two PLD phosphodiesterase domains (437–464 bp and 885–912 bp). The pld gene was significantly regulated during infection of Meloidogyne incognita eggs by P. lilacinum. The expression of pld gene using RT-PCR was the highest at 36 and 48 h, which introduce evidence that the presence of M. incognita may induce the expression of the pld gene in P. lilacinum. In addition, maltose and l-alanine were found to increase the expression of pld gene. An acidic environment (pH 3.0–4.0) and moderate temperatures (27–29 °C) are favorable for pld expression in P. lilacinum.

Statistical optimization of medium components for chitinase production by Pseudomonas fluorescens strain HN1205: role of chitinase on egg hatching inhibition of root-knot nematode

In the present research, statistical Plackett–Burman design (PBD) and central composite design (CCD) were used to optimize the medium's components in order to enhance the chitinase activity of Pseudomonas fluorescens strain HN1205. Among the seven nutritional elements that were studied, crab shell powder, CaCl2·2H2O and yeast extract were proved using PBD to have significant effect on chitinase activity. An optimal medium was obtained by applying a three-factor central composite design, which consisted of crab shell powder (1.0 g/L), CaCl2 (0.5 g/L) and yeast extract (0.5 g/L), with the highest chitinase activity of 1.03 U/mL. This value was 2.87-fold higher than the activity obtained in the lowest productive medium in the design matrix. The chitinase produced by the HN1205 strain was partially purified with 80% ammonium sulphate and anion-exchange chromatography and assessed for inhibition of hatching of nematode eggs. The partially purified chitinase significantly reduced the hatch of Meloidogyne incognita eggs (8.1%), as compared to the effect of 10 mmol/L Tris-HCl buffer (pH 8.0) (49.4%) and boiled chitinase (54.7%). This study demonstrated the role of chitinolytic enzymes produced by P. fluorescens strain HN1205. The obtained optimal activity proved that the enzymes could be potential biological control agents.

Toxicity and Biochemical Effect of Organophosphates and Bio-pesticides against Root-knot Nematode, Meloidogyne incognita

This study was carried out to investigate the toxicity and Biochemical effect of two (biopesticides) biofly, abamectin and two organophosphates pesticides cadusafos and fenitrothion against root-knot nematode, Meloidogyne incognita egg filtrates and second stage juveniles (J2) as well as on laboratory experiment, also the inhibitory effect of the tested pesticides to acetylcholenesterase (AChE) and adenosine triphosphatase (ATPase) were determined. Results indicated that the tested pesticides have toxic action against Meloidogyne incognita second-stage juveniles (J2) and egg filtrates after 24 hrs from application to 72 hrs and the toxicity increased with the time. Abamectin was most toxic followed by cadusafos , fenitrothion and biofly was least in its toxicity to M. incognita second stage juveniles (J2 )and egg filtrates while the toxicity are depending on dose. The tested pesticides have inhibitory effect on AChE and ATPase activity and the inhibitory effect increased with AChE in case of abamectin and cadusafos while the potency of the organophosphate pesticides to inhibit ATPase were limited refers to its mode of action.

Management of Meloidogyne incognita in yam-based cropping systems with cover crops

The effect of intercropping cover crops was with yam was assessed for nematode management both in pot and field conditions in Nigeria. The cover crops were sown with yams in pots containing sterile soil and inoculated with 5000 eggs of Meloidogyne incognita. In the field, each cover crop was separately intercropped with yams inoculated with 10,000 nematodes. From both pot and field experiments, no nematode damage was observed on yam tubers that were intercropped with Aeschynomene histrix, Crotolaria juncea, and Tagetes erecta. Damage in intercropped tubers was reduced by 72.7% with Mucuna pruriens, Centrosema pubescens, and Pueraria phaseoloides and by 58.3% with Stylosanthes guianensis compared to yams planted without cover crops; they also had lower (p ≤ 0.05) nematode populations. Cajanus cajan, Lablab purpureus and Vigna unguiculata however, supported high nematode populations and led to nematode damage in intercropped yams. Tubers from M. incognita-inoculated plants were more damaged and lost 42% more weight (p ≤ 0.05) following three months of storage than tubers from uninoculated plants. Intercropping yams with selected cover crops can be useful in managing M. incognita without reducing yam yields.

Nematicidal and quantitative phytochemical analysis of the chromatographic fractions from the leaf and stem of <i>Allamanda cathartica (L)</i>

The environmental pollution of synthetic nematicides is enormous, due to their residual effects and nondegradability. This study was conducted to investigate the possibility of a green alternative that will eventually replace the pollution problems of synthetic nematicides. The activity of plant secondary metabolites against plant parasitic nematodes was tested in-vitro using chromatographic isolates from the stem and leaves of Allamanda cathartica. Isolates from the extracted plant materials were tested at various concentrations (0 mg/mL, 0.06 mg/mL, 0.1 mg/mL and 0.2mg/mL) against Meloidogyne incognita juveniles and eggs. The effect was determined through testing the inhibition of egg hatch and larval mortality. The result showed that egg hatching was totally inhibited among all the isolates at different concentrations except in the control which had 48.39% on day 5. The isolates showed strong nematicidal effect, with percentage mortality of 16.87% at the first hour of exposure which compared well with the commercial nematicide (oxamyl). However the leaves of A. cathartica proved more effective than the stem. Preliminaryphytochemical screening revealed that the quantities of secondary metabolites are more in the leaves of A. carthatica than in the stems. Infra-red spectroscopic data of three of the isolates revealed the presence of alkanes (2930; 2923 and 1460 cm-1) aldehydes (2855; 2851 and 1733cm-1) and phenols at (3650; 1115cm-1). Aliphatic amines (3200; 1460 and 1600 cm-1) and non-cyclic ethers (1310; 1300; 1240 and 1000cm-1) are present in isolate four. This study showed that the leaves of A. cathartica could serve as a bio-pesticide to reduce environmental pollutionKey words: Extraction, n-hexane, Isolation, Oxamyl, Nematodes, Chromatography.

Observations on the effect of trophic conditions onPochonia chlamydosporiagene expression

In vitro assays were performed under varying nutritional conditions to investigate the expression of four genes of the fungus Pochonia chlamydosporia, a nematode parasite and a plant growth promoter. The expression levels of a basic leucine zipper (bZIP) transcription factor, a putative phytase, a phospholipase D (PLD) and a monoxygenase of isolate DSM 26985 were, after 8 h incubation in the presence of Meloidogyne incognita eggs, 3, 14, 900 and 67-fold higher than the fungus alone, respectively. M. incognita uninoculated tomato plants alone induced, at 4 h incubation, a phytase transcription sixfold higher than control. Changes in transcript amounts were not significant when the fungus was in the presence of nematode parasitized plants. Further assays performed at different pHs or in the presence of glucose and NH4+ showed that the bZIP and phytase transcripts reflect early changes in the fungus metabolism. Barley plants treated with P. chlamydosporia isolates IMI 331547 and DSM 26985 showed a pronounced plant growth promotion effect of the former isolate. Phytic acid affected growth of isolates at 1 × 10−4 ppm concentration, however, the colony diameter increased at 10 ppm. Gene expression data showed that P. chlamydosporia metabolism reflects key biochemical signals proceeding from roots and host nematode eggs.

Nematicidal and ovicidal activities of Dryobalanops aromatica and Mentha haplocalyx var. piperascens-derived materials and their formulations against Meloidogyne incognita second-stage juveniles and eggs

Nematicidal and hatching inhibition activities (HIA) of the methanol extracts from Dryobalanops aromatica (DA) and Mentha haplocalyx var. piperascens (MH), their constituents, and emulsified concentrate and granule formulations containing the methanol extracts as active ingredients, were compared with those of commercial nematicides, cadusafos and fosthiazate, against Meloidogyne incognita second-stage juveniles (J2) and eggs using well plate, container and pot assays. In a well plate assay with J2, DA applied at 500, 250 and 125 ppm resulted in 100, 95 and 55% mortality, respectively, while MH showed 80% mortality even at the lower concentration of 63 ppm. MH gave stronger HIA than DA at 1000 and 500 ppm. In addition, α-pinene, camphor, borneol and p-menthole, which are the main components of DA and MH extracts, showed strong nematicidal properties and HIA. In particular, the nematicidal activity of p-menthole among them was the highest and its HIA was also stronger than that of cadusafos and fosthiazate. In a container test using emulsifiable concentrate (EC) and granule (G) formulations containing 50 and 30% DA and MH, respectively, EC of D. aromatica showed a stronger LC50 value (0.203 μl (g soil)−1) than G formulation (0.935 mg (g soil)−1). However, their effects were fewer than MH formulations. The lethality of EC and G formulations containing MH was comparable to that of fosthiazate soluble concentrate and granule formulations. In another test using pots containing soils naturally infected by M. incognita, these plant formulations significantly inhibited the nematode-population density judging from the number of galls that formed on tested tomato roots after 8 weeks. These results suggest that DA and MH methanol extracts, as well as their constituents, might be useful as management agents against M. incognita.

Nematicidal isochromane glycoside from Kigelia pinnata leaves

Synthetic nematicides such as oxamyl and carbofuran play significant roles in the management of plant-parasitic nematodes. However, their negative environmental impacts have it imperative to search for safer alternatives. As part of our contribution in the search for bio-nematicides, compounds from plant extract were screened for possible potent nematicidal agent. A new isochromane carboxylic acid glycoside, isolated from the leaves of Kigelia pinnata (Lam.) Benth (Bignoniaceae) was evaluated for its nematicidal activity. The structure of the proposed compound was characterized by various spectroscopic methods, which included UV, FTIR, 1D-, and 2D-NMR, FAB-MS, TOF-ESI-MS and TOF-ESI-MS/MS (TANDEM). The in vitro experiment conducted on the glycoside against Meloidogyne incognita juveniles and eggs indicated an induced mortality. Its activity can be compared favourably with oxamyl, when tested at 0.1 mg/mL concentration. At four hours of observation, no significant difference (P < 0.05) between oxamyl and the glycoside was observed. The present data sustains that natural glycoside is a promising oxamyl alternate for controlling nematode-induced plant root knots and may contribute to integrated pest management.

A combined cryo-scanning electron microscopy/cryoplaning approach to study the infection of Meloidogyne incognita eggs by Pochonia chlamydosporia

The fungus Pochonia chlamydosporia is a saprophytic soil-dwelling fungus and is also a parasite of the eggs of the root-knot plant-parasitic nematodes (Meloidogyne spp.). Studies on its molecular characterisation, diversity, tritrophic interactions and ecology have been carried out. However, to elucidate the role in soil and rhizosphere ecology of this and other nematophagous fungi used in biological control, and to enhance their exploitation, it is necessary to improve the understanding of the biology and fungus-nematode infection process using different approaches, including microscopy. Low-temperature Scanning Electron Microscopy (cryo-SEM) techniques allow the examination of frozen, fully hydrated samples that can reveal important ultrastructural features occurring through fungus-nematode interactions. A method that combined cryo-SEM with cryoplaning was developed to examine samples of eggs, gelatinous matrix and females of Meloidogyne incognita colonised by P. chlamydosporia. The fungal samples were produced in potato dextrose agar to which different nematode stages were added and processed for cryo-SEM and cryoplaning within a period of 0-72 h post inoculation. The method was found to be rapid and economical, provided clear and detailed external images of the infection process and allowed viewing of sections through structures with minimal processing in comparison to other cryo-SEM techniques and it could be adapted to study other fungus-nematode interactions.

Identification and molecular characterisation of new Mexican isolates of Pochonia chlamydosporia for the management of Meloidogyne spp.

New Mexican isolates of the nematophagous fungus Pochonia chlamydosporia were obtained from nematode infested fields in the vegetable growing area of Tepeaca Valley, Puebla State, Mexico. Based on macro and microscopic morphology, seven ‘putative’ P. chlamydosporia isolates were selected and the DNA extracted for polymerase chain reaction (PCR). Three new isolates of P. chlamydosporia were identified: Pcp2, Pcp21 and Pcp31. The amplification reaction of the internal transcribed spacer (ITS) region revealed a 650 bp amplicon which was used in a maximum likelihood phylogenetic inference analysis. Three groups were recovered in the tree topology, supported by a > 90% bootstrap value. Nucleotide identity values were > 83.6% between the test sequences and the reference sequence. In addition, using specific primers for two existing varieties of P. chlamydosporia, restriction fragment length polymorphism on the ITS products in conjunction with the phylogenetic inferences and the molecular test for detection of P. chlamydosporia vcp1 gene, it was found that all three isolates belong to a new variety which we have named P. chlamydosporia var. mexicana. We compared the chlamydospore production rate, rhizosphere colonisation and egg parasitism percentages of the three native isolates in Meloidogyne spp. with a reference isolate (Pc10). Native isolates produced > 1×106 chlamydospores/50 g of substrate (of which more than 80% were viable), colonised > 80% of the rhizosphere, and parasitised > 60% of Meloidogyne incognita and Meloidogyne arenaria eggs. Meloidogyne hapla egg parasitism was < 60%. Isolates Pcp2 and Pcp21 were identified as potential biological control agents of Meloidogyne spp. to be tested further in greenhouse and field tests.

English

The impact of three levels of&nbsp;Meloidogyne incognita&nbsp;eggs, that is, 1000, 2000 and 3000 eggs/ plant on plant growth parameters of sugarbeet cv. Nejma and its development and reproduction under greenhouse conditions at 17 &plusmn; 5&deg;C was studied. Results indicated that reduction percentage of sugarbeet growth parameters were greatly affected, where the marginal effect was more pronounced on roots than shoots. Moreover, as the inoculation level increased from 1000 up to 3000 eggs of&nbsp;M. incognita&nbsp;/plant, the percentage reduction of plant growth parameters increased, where the highest values were recorded to be 48.97 and 24.18% by 3000 eggs/ plant for shoot dry and whole plant fresh weights, whereas, their lowest values of 30.93 and 4.05% resulted by inoculation level of 1000 eggs/ plant for the same plant growth criteria, respectively. Moreover, the highest rate of nematode reproduction on sugarbeet plants cv. Nejma was recorded by the level of 2000 eggs per plant with value of 1.05 whereas the lowest ones of the same criterion resulted by the levels of 3000 or 1000 eggs per plant that were 0.85 or 0.84, respectively.&nbsp;M. incognita&nbsp;infection to sugar beet plants cv. Nejma at the three levels of egg inoculation obviously reduced N, P, K and total chlorophyll content on leaves of sugarbeet with values of 9.9% (N), 18.5% (P), 8.8% (K), 1.58% (chlorophyll) and 20.0% (N), 27.4% (P), 18.4% (K) and 5.9% (chlorophyll) by 1000 and 3000 eggs/ plant, respectively compared to the uninoculated plant. &nbsp; Key words:&nbsp;Meloidogyne incognita, sugarbeet, inoculum levels.