Meloidogyne Incognita Eggs Research Articles

Activity of Hydroxamic Acids from Secale cereale Against the Plant-Parasitic Nematodes Meloidogyne incognita and Xiphinema americanum

ABSTRACT Cyclic hydroxamic acids are secondary metabolites found in the family Poaceae and have been implicated in the allelopathy of rye (Secale cereale). The toxicity of these compounds against plant-parasitic nematodes is unknown. DIBOA (2,4-dihydroxy-(2H)-1,4-benzoxazin-3(4H)-one), DIMBOA (2,4-hydroxy-7-methoxy-(2H)-1,4-benzoxazin-3(4H)-one), and their degradation products BOA (benzoxazolin-2(3H)-one) and MBOA (6-methoxy-benzoxazolin-2(3H)-one) were screened in vitro against Meloidogyne incognita second-stage juveniles (J2) and eggs and mixed-stages of Xiphinema americanum. Xiphinema americanum was more sensitive to DIBOA and DIMBOA than M. incognita J2, with a maximum apparent mortality of 96 and 92% compared to 73 and 72% at 90 mug/ml. Eggs of M. incognita were less sensitive to the hydroxamic acids than J2; only DIBOA resulted in a 50% reduction in egg hatch, with a lethal concentration (LC(50)) of 74 mug/ml compared to 21 mug/ml for J2. When M. incognita J2 were exposed to DIBOA for 48 h and the compound was removed and replaced with water, the LC(50) value increased from 21.0 to 40.7 mug/ml. MBOA was not toxic to X. americanum or M. incognita eggs, but was toxic to M. incognita J2, with LC(50) values of 44 and 20 mug/ml before and after the compound was removed and replaced with water. BOA was the least toxic hydroxamic acid tested; it did not reduce M. incognita egg hatch after 1 week of exposure or increase X. americanum mortality after 24 h of exposure. While in vitro studies provide a valuable starting point in determining the toxicity of the chemical component of rye, the relevance of the data to soil remains to be determined.

Broad spectrum action of phenazine against active and dormant structures of fungal pathogens and root knot nematode

Antifungal antibiotic from Pseudomonas chlororaphis isolate PA23 was identified as Phenazine using TLC and HPLC. Phenazine recorded the highest inhibition zone of 21 mm with 35.55% percent inhibition of mycelial growth of Pythium aphanidermatum over control. It had a significant effect on the hyphal morphology of P. aphanidermatum and on spore germination of Botryodiplodia theobromae and Alternaria solani. Disorganization of hyphal morphology of P. aphanidermatum includes vacuolization, cell content degeneration and hyphal lysis. Similarly interaction of phenazine with Rhizoctonia solani resulted in abnormal swelling of hyphal tips was noticed in the hyphal tips. Similarly the germination of sclerotia of Macrophomina phaseolina, R. solani and Sclerotium rolfsii were completely inhibited by phenazine at a concentration 50 μl. Incubation of the eggs of the root knot nematode Meloidogyne incognita in 30 μl concentration of phenazine, completely suppressed the hatching of juveniles.

Isolation and characterization of PRA1, a trypsin-like protease from the biocontrol agent Trichoderma harzianum CECT 2413 displaying nematicidal activity

Mycoparasitic Trichoderma strains secrete a complex set of hydrolytic enzymes under conditions related to antagonism. Several proteins with proteolytic activity were detected in culture filtrates from T. harzianum CECT 2413 grown in fungal cell walls or chitin and the protein responsible for the main activity (PRA1) was purified to homogeneity. The enzyme was monomeric, its estimated molecular mass was 28 kDa (SDS-PAGE), and its isoelectric point 4.7-4.9. The substrate specificity and inhibition profile of the enzyme correspond to a serine-protease with trypsin activity. Synthetic oligonucleotide primers based on N-terminal and internal sequences of the protein were designed to clone a full cDNA corresponding to PRA1. The protein sequence showed <43% identity to mammal trypsins and 47-57% to other fungal trypsin-like proteins described thus far. Northern analysis indicated that PRA1 is induced by conditions simulating antagonism, is subject to nitrogen and carbon derepression, and is affected by pH in the culture media. The number of hatched eggs of the root-knot nematode Meloidogyne incognita was significantly reduced after incubation with pure PRA1 preparations. This nematicidal effect was improved using fungal culture filtrates, suggesting that PRA1 has additive or synergistic effects with other proteins produced during the antagonistic activity of T. harzianum CECT 2413. A role for PRA1 in the protection of plants against pests and pathogens provided by T. harzianum CECT 2413 is proposed.

Suppression of Meloidogyne incognita and Meloidogyne hapla with entomopathogenic nematodes on greenhouse peanuts and tomatoes

Abstract We measured the effect of entomopathogenic nematodes on root-knot nematodes (Meloidogyne spp.) infecting tomato and peanut. In growth chamber experiments, peanut seedlings were inoculated with 300 Meloidogyne hapla eggs and 25 infective juveniles (IJs)/cm2 of Steinernema feltiae, Steinernema riobrave, or Heterorhabditis bacteriophora. With the exception of seedling roots treated with H. bacteriophora, M. hapla inside roots and egg recovery from seedlings treated with entomopathogenic nematodes were significantly less than those in the controls. Peanut plants in the greenhouse were infested with 5000 M. hapla eggs and treated 2 weeks before, 1 week before, at the same time, 1 week after, or 2 weeks after with 25 or 125 IJs/cm2 of S. feltiae, S. riobrave, or H. bacteriophora. Pre- and post-infestation applications of S. feltiae suppressed M. hapla penetration but not egg production. Only pre-infestation applications of S. riobrave suppressed M. hapla. Only the high application rate of H. bacteriophora applied at the time of M. hapla infestation was suppressive. Tomato plants in the greenhouse were infested with 5000 Meloidogyne incognita eggs and treated with 25 or 125 IJs/cm2 of Steinernema glaseri or Heterorhabditis megidis applied at the same times as the tomato treatments. The low rate of S. glaseri suppressed M. incognita penetration into tomato roots and the high rate of S. glaseri reduced egg production.

Activity of fungal culture filtrates against soybean cyst nematode and root-knot nematode egg hatch and juvenile motility

Abstract Fungi were isolated from soybean cyst nematode (SCN, Heterodera glycines) eggs collected in China, and 253 fungal isolates were assayed for production of compounds active against SCN and root-knot nematode (RKN, Meloidogyne incognita). Fungal isolates were grown for 3 and 7 days in potato dextrose broth (PDB), the culture broths were sterile-filtered to remove fungal biomass, and the filtrates were placed into 24-well plates to test for effects on egg hatch and juvenile motility. Meloidogyne incognita egg hatch ranged from 2 to 121% of hatch in PDB controls and H. glycines hatch from 15 to 224%. Activities of filtrates harvested after 3 and 7 days were significantly correlated. Only four isolates produced filtrates that significantly inhibited juvenile motility of SCN, RKN or both nematodes. This study identified fungal isolates capable of producing compounds active against these nematodes, and demonstrated that there was a low correlation in activity against SCN and RKN. The active fungal isolates are candidates for studies on identification of potential nematicides.

Isolation of a novel collagen gene (Mj-col-5) in Meloidogyne javanica and analysis of its expression pattern.

Mj-col-5, isolated from the plant parasitic nematode Meloidogyne javanica, has a longer carboxy-terminus than other members of the Caenorhabditis elegans COL-6 subfamily of cuticle collagen, including an extra tyrosine residue, and may form altered nonreducible cross-linkages. By semiquantitative determination at different life stages, Mj-col-5 transcript was shown to be more abundant in eggs than in juveniles/young females and adult females. To characterize further this gene's contribution to the changing cuticle of the nematode, we expressed a fusion protein containing a nonconserved 58-amino-acid sequence from the putative Mj-col-5 gene product and raised rabbit antiserum against the fusion protein. The antiserum detected a strongly reacting band (36 kDa, designated MJE36) on western blots of M. javanica eggs extracted with beta-mercaptoethanol. MJE36 was sensitive to collagenase and was not detected on western blots of extracts from M. javanica second-stage juveniles or adult females. A band of the same molecular size was detected in Meloidogyne incognita egg extracts but not in those of Heterodera avenae. Immunoblot indicated that MJE36 is not present in egg shells of M. javanica.

Pathogenicity of Selected Antagonistic Soil Fungi on Meloidogyne incognita (Kofoid & White) Eggs and Egg Masses under in Vitro and in Vivo Conditions

Four indigenous antagonistic fungi belonging to 3 species, Gliocladium virens Miller Giddens & Foster, Paecilomyces lilacinus (Thon) Samson, Trichoderina harzianum Rifai and T. viride Pers. Ex S. F. Gray evaluated for their pathogenicity to eggs and egg masses of Meloidogyne incognita , in vitro through the Petri-dish assay and in vivo under glasshouse conditions were found pathogenic. Under both the conditions, maximum egg mass and egg parasitization was observed to be by P. lilacinus followed by T. viride , G. virens and T. harizianum . There was a general decline in parasitization by these fungi, under in vivo conditions compared to that under in vitro conditions. Fungal colonization in galled roots was higher than in healthy roots. Comparatively T. viride and G. virens recorded higher root colonization closely followed by P. lilacinus . Among the four fungi , P. lilacinus recorded consistently higher pathogenicity against root-knot nematodes both in vitro and in vivo conditions, indicating that P. lilacinus was more efficient against root-knot nematodes compared to other species.

The Viability of Meldidogyne Incognita Eggs Released From Egg Masses of Different Ages Using Different Concentrations of Sodium Hypochlorite

The proportion of eggs of Meloidogyne incognita which hatched following extraction from egg masses with sodium hypochlorite solution (NaOCl) was shown to be related to the proportion that were embryonated at the time of extraction. Following NaOCl extraction, young egg masses (4-5 weeks after inoculation), which contained proportionally fewer embryonated eggs, yielded proportionally fewer juveniles than did old egg-masses (8-9 weeks). Using eggs extracted with NaOCl as inoculum a similar difference was observed in the numbers of juveniles which invaded tomato roots. Differences between two experiments in the rate at which eggs were released by the same nominal concentration of NaOCl were subsequently attributed to differences in chlorine (Cl) content. A further experiment where the Cl content was assayed showed that the numbers of eggs extracted increased, but that their viability decreased, with increasing concentration and duration of exposure to NaOCl. Overall, NaOCl was most effective when used on older egg masses with a high proportion of embryonated eggs.

The role of the proteinase VCP1 produced by the nematophagous Verticillium chlamydosporium in the infection process of nematode eggs

The infection process of the nematophagous fungus Verticillium chlamydosporium (isolate Vc1O) was studied in two nematode species that differed in susceptibility according to standardized in vitro assays. After 14 d, V. chlamydosporium infected 9 and 80% of Globodera rostochiensis and Meloidogyne incognita eggs, respectively. The purified proteinase VCPl, from V. chlamydosporium, removed the protein coat from eggs of M. incognita but not of G. rostochiensis. VCPl was more effective at hydrolysing proteins from M. incognita eggs than the related enzymes Prl and proteinase K, whereas the latter two were slightly more active against G. rostochiensis eggs than VCPl. Pretreatment of eggs with VCPl increased susceptibility to V. chlamydosporium from 80–100% in M. incognita eggs and from 9 to 29% in G. rostochiensis eggs. These observations suggest that VCPl is a host specificity-determining factor and also confirm it to be a pathogenicity determinant which works best in concert with other enzymes secreted by the actively growing fungus. Fungal growth on the surface of eggs of both species was commensurate with the infection frequencies observed; growth was limited and fewer appressoria were observed on eggs of G. rostochiensis compared with those of M. incognita. Eggs of M. incognita denatured by heat or killed with sodium azide, like eggs treated with VCPl, were more susceptible than untreated, healthy eggs. In contrast heat and azide treatment of G. rostochiensis eggs did not increase the number of infections. Appressoria were observed on the eggs of both nematode species in all treatments, but the numbers and types varied greatly. On M. incognita, the dominant type was swollen and branched laterally from hyphae, while on G. rostochiensis lateral inconspicuous appressoria prevailed. Differentiation of appressoria on live and dead nematode eggs showed that their production was not limited to the parasitic state of the fungus.

Total sterols in root-knot nematode Meloidogyne incognita infected cotton Gossypium hirsutum (L.) plant roots

Two near-isogenic breeding lines of Gossypium hirsutum (L.) (upland cotton) were inoculated with eggs of Meloidogyne incognita (Kofoid and White) Chitwood (RKN) in greenhouse pots. One line was susceptible (ST-213) and the other was resistant (81–249) to RKN. Eight days after inoculation, roots of plants and their controls were analyzed for total sterols by GLC and GLC-MS. The total content in roots of uninoculated roots of susceptible plants was slightly higher than in the roots of uninoculated resistant plants, but significantly lower after inoculation. By comparison, the sterols in roots of the resistant line were significantly higher at 8 days after inoculation. The major sterols in both susceptible and resistant lines were stigmasterol, sitosterol and campesterol, with a lesser amount of cholesterol and a trace of stigmastanol. Because the amounts and distribution of the sterols were similar in susceptible and resistant lines, they do not appear to be a factor associated with RKN growth and development. Sitosterol was the most abundant sterol in RKN eggs which also contained lesser amounts of cholesterol and stigmasterol. However, no stanols were found in RKN eggs.

Alginate films for delivery of root-knot nematode inoculum and evaluation of microbial interactions

A method was developed for utilizing alginate films to deliver inoculum into soil and evaluate microbial antagonistic activity against nematode eggs. Eggs of Meloidogyne incognita were harvested from galled tomato roots (Lycopersicon esculentum), surface disinfested, suspended in 2% (w/v) aqueous sodium alginate, and applied to 2.5 × 5.0 cm polyvinyl chloride coated fiberglass screens (1.5 mm2 mesh size) at a uniform thickness of 0.5 mm. The alginate solution was gelled by dipping in 0.25 M CaCl2. Films containing eggs were observed in vitro and egg development was evaluated. The number of immature eggs and eggs with first stage juveniles declined linearly over time while the number of empty eggs shells, and hatched juveniles increased over time, indicating that the alginate gel did not inhibit development and motility of M. incognita juveniles. In a greenhouse experiment using cucumber (Cucumis sativus) the number of galls g-1 root was correlated with the number of eggs in alginate films placed in each pot at planting. Films containing M. incognita eggs were buried in field soil containing organic amendments, incubated, removed from soil, rinsed with water, and observed. The number of immature eggs in grids from soil amended with chitin or flax seed meal were lower than in untreated soil; percent parasitized eggs was also greater in films from amended soil than from untreated soil.

Tolerance of cotton to Meloidogyne incognita as influenced by phosphorus fertiliser and a vesicular arbuscular mycorrhizal fungus

SummaryCotton was grown in soils of differing phosphorus levels with and without a mycorrhizal fungus and with a range (0 to 3200 eggs/ 100 c‐3 soil) of Meloidogyne incognita eggs added. Tolerance of cotton to M. incognita did not differ between plants grown in different soils with or without the mycorrhizal fungus, though plant size could differ markedly. Plant tolerance was increased by a delay in adding nematodes.

Effect ofAzotobacter chroococcum on the hatching of egg masses and eggs ofMeloidogyne incognita

Investigations were carried out to study the effect ofAzotobacter chroococcum VP-5 on the hatching of egg masses and eggs ofMeloidogyne incognita in vitro. Eggs were more susceptible and required lesser time for antagonistic action of Azotobacter than egg masses.

Fusarium oxysporum f. sp. nicotianae Enzyme Production on Cell Walls Isolated from Meloidogyne incognita-Infected and Healthy Tobacco Roots

SUMMARYFusarium oxysporum f. sp. nicotianae was grown on cell walls from the roots of the flue-cured tobacco cultivars ‘Coker 298’ (resistant to Fusarium) and ‘Virginia Gold’ (susceptible to Fusarium). All plants were grown in a split-root system and Meloidogyne incognita eggs were applied to one-half of the root system of certain ‘Coker 298’ plants. The amounts of polygalacturonase, cellulase, polygalacturonate transeliminase, pectinmethyl transeliminase, polymethyl galacturonase and amylase enzyme activities and soluble protein concentrations produced by the fungus on cell wall preparations from these roots did not differ statistically. However, there was a strong tendency for greater enzyme activity production on cell walls from the nematode-infected roots than on walls from nematode-free roots.

Effect of Temperature On Parasitism of Meloidogyne Incognita Eggs By Dactylella Oviparasitica

Dactylella oviparasitica had an optimum temperature for radial growth on agar of 24-27° C, but it parasitized Meloidogyne incognita eggs on agar and in soil at temperatures of 12-27°. Although egg parasitism took longer at low temperatures, the fungus became a slightly more efficient parasite as temperatures decreased below the optimum for egg production, egg development and hatch.

Effect of Soil Amendments on Hatching of Meloidogyne incognita Eggs

Effect of Soil Amendments on Hatching of Meloidogyne incognita Eggs

Lipid and fatty acid composition of females and eggs of Meloidogyne incognita and M. arenaria

1. 1. Adult females of Meloidogyne incognita and M. arenaria contained 46 and 40 per cent lipid whereas eggs contained 64 and 67 per cent lipit, respectively, on a dry weight basis. 2. 2. In nematodes of both species polar lipid accounted for around 9–11 per cent of the total lipid in females and 6–8 per cent in eggs. 3. 3. Qualitatively the same fatty acids occurred in nematodes of both species, although differences in relative quantities of certain fatty acids appeared in females and eggs between the two species. Relative quantities of certain fatty acids appeared to differ between females and eggs within a nematode species also. 4. 4. The major fatty acid in females and eggs of both M. incognita and M. arenaria was vaccenic acid (18:1 ω7) comprising 96–99 + per cent of the total 18:1 and account for 25–30 per cent of the dry weight of females and over 40 per cent of the dry weight of eggs. 5. 5. The polar lipid from both females and eggs of the two nematode species was consistently higher in iso-18:0 and polyunsaturated fatty acids and much lower in vaccenic acid than the neutral lipid.