Detection and Investigation of Soil Biological Activity against Meloidogyne incognita

Greenhouse experiments with two susceptible hosts of Meloidogyne incognita, a dwarf tomato and wheat, led to the identification of a soil in which the root-knot nematode population was reduced 5- to 16-fold compared to identical but pasteurized soil two months after infestation with 280 M. incognita J2/100 cm³ soil. This suppressive soil was subjected to various temperature, fumigation and dilution treatments, planted with tomato, and infested with 1,000 eggs of M. incognita/100 cm³ soil. Eight weeks after nematode infestation, distinct differences in nematode population densities were observed among the soil treatments, suggesting the suppressiveness had a biological nature. A fungal rRNA gene analysis (OFRG) performed on M. incognita egg masses collected at the end of the greenhouse experiments identified 11 fungal phylotypes, several of which exhibited associations with one or more of the nematode population density measurements (egg masses, eggs or J2). The phylotype containing rRNA genes with high sequence identity to Pochonia chlamydosporia exhibited the strongest negative associations. The negative correlation between the densities of the P. chlamydosporia genes and the nematodes was corroborated by an analysis using a P. chlamydosporia-selective qPCR assay.     

In vitro studies of antagonistic fungi against the root-knot nematode,Meloidogyne incognita

The present study was carried out in vitro to determine the efficacy of indigenous fungi isolated from egg masses of root-knot nematode, Meloidogyne incognita on egg parasitism, egg hatching, mobility and mortality against root-knot nematode, M. incognita. The tested fungi were Acremonium strictum, Aspergillus terreus, A. nidulans, A. niger, Chetomium aubense, Chladosporium oxysporum, Fusarium chlamydosporium, F. dimarum, F. oxysporum, F. solani, Paecilomyces lilacinus, Pochonia chlamydosporia, Trichoderma viride and T. harzianum. All tested fungi showed varied effects against the nematodes. Culture filtrates of A. strictum was very effective against the nematode in regards to egg parasitism (53%), egg hatching inhibition (86%) and mortality (68%) compared to controls. A. strictum was found to have an advantage over P. lilacinus, P. chlamydosporia, T. viride and T. harzianum in that it caused greater mortality of the second stage juveniles (J₂). A. terreus did not show egg parasitism but was found to be highly toxic against second stage juveniles (J₂) causing high mortality (around 68%). Thus, A. strictum and A. terreus showed good biocontrol potential against root-knot nematode, M. incognita under in vitro conditions.     

Biocontrol Efficacy Among Strains of Pochonia chlamydosporia Obtained from a Root-Knot Nematode Suppressive Soil

Three Pochonia chlamydosporia var. chlamydosporia strains were isolated from a Meloidogyne incognita-suppressive soil, and then genetically characterized with multiple Pochonia-selective typing methods based on analysis of ß-tubulin, rRNA internal transcribed spacer (ITS), rRNA small subunit (SSU), and enterobacterial repetitive intergenic consensus (ERIC) PCR. All strains exhibited different patterns with the ERIC analysis. Strains 1 and 4 were similar with PCR analysis of ß-tubulin and ITS. The strains'' potential as biological control agents against root-knot nematodes were examined in greenhouse trials. All three P. chlamydosporia strains significantly reduced the numbers of nematode egg masses. When chlamydospores were used as inoculum, strain 4 reduced egg numbers on tomato roots by almost 50%, and showed effects on the numbers of J2 and on nematode-caused root-galling. A newly developed SSU-based PCR analysis differentiated strain 4 from the others, and could therefore potentially be used as a screening tool for identifying other effective biocontrol strains of P. chlamydosporia var. chlamydosporia.     

Root-knot Nematode Problem of Some Winter Ornamental Plants and Its Biomanagement

A microplot study under field conditions was carried out during 2 consecutive years to assess the effect of root-knot nematode infection (2,000 Meloidogyne incognita eggs/kg soil) on three winter ornamental plants: hollyhock (Althea rosea), petunia (Petunia hybrida), and poppy (Papaver rhoeas). Effects of root-dip treatment with the biocontrol agents Pochonia chlamydosporia, Bacillus subtilis, and Pseudomonas fluorescens and the nematicide fenamiphos were tested. The three ornamental species were highly susceptible to M. incognita, developing 397 and 285 (hollyhock), 191 and 149 (petunia), and 155 and 131 (poppy) galls and egg masses per root system, respectively, and exhibited 37% (petunia), 29% (poppy), and 23% (hollyhock) (P = 0.05) decrease in the flower production. Application of fenamiphos, P. chlamydosporia, P. fluorescens, and B. subtilis suppressed nematode pathogenesis (galls + egg masses) by 64%, 37%, 27%, and 24%, respectively, leading to 14% to 29%, 7% to 15%, 14% to 36%, and 7% to 33% increase in the flower production of the ornamental plants, respectively. Treatment with P. fluorescens also increased the flowering of uninfected plants by 11% to 19%. Soil population of M. incognita was decreased (P = 0.05) due to various treatments from 2 months onward, being greatest with fenamiphos, followed by P. chlamydosporia, B. subtilis, and P. fluorescens. Frequency of colonization of eggs, egg masses, and females by the bioagents was greatest by P. chlamydosporia, i.e., 25% to 29%, 47% to 60%, and 36% to 41%, respectively. Colonization of egg masses by B. subtilis and P. fluorescens was 28% to 31% and 11% to 13%, respectively, but the frequency was 0.3% to 1.3% in eggs. Rhizosphere population of the bioagents was increased (P = 0.05) over time, being usually greater in the presence of nematode.     

Nematicidal activity of fervenulin isolated from a nematicidal actinomycete, <Emphasis Type="Italic">Streptomyces</Emphasis> sp. CMU-MH021, on <Emphasis Type="Italic">Meloidogyne incognita</Emphasis>

An isolate of the actinomycete, Streptomyces sp. CMU-MH021 produced secondary metabolites that inhibited egg hatch and increased juvenile mortality of the root-knot nematode Meloidogyne incognita in vitro. 16S rDNA gene sequencing showed that the isolate sequence was 99% identical to Streptomyces roseoverticillatus. The culture filtrates form different culture media were tested for nematocidal activity. The maximal activity against M. incognita was obtained by using modified basal (MB) medium. The nematicidal assay-directed fractionation of the culture broth delivered fervenulin (1) and isocoumarin (2). Fervenulin, a low molecular weight compound, shows a broad range of biological activities. However, nematicidal activity of fervenulin was not previously reported. The nematicidal activity of fervenulin (1) was assessed using the broth microdilution technique. The lowest minimum inhibitory concentrations (MICs) of the compound against egg hatch of M. incognita was 30 μg/ml and juvenile mortality of M. incognita increasing was observed at 120 μg/ml. Moreover, at the concentration of 250 μg/ml fervenulin (1) showed killing effect on second-stage nematode juveniles of M. incognita up to 100% after incubation for 96 h. Isocoumarin (2), another bioactive compound produced by Streptomyces sp. CMU-MH021, showed weak nematicidal activity with M. incognita.     

Measuring abundance,diversity and parasitic ability in two populations of the nematophagous fungus Pochonia chlamydosporia var. chlamydosporia

Abundance, genetic diversity and parasitic ability in the facultative nematode parasite Pochonia chlamydosporia var. chlamydosporia were compared in soils from two sites in Portugal under long-term tomato cultivation where root-knot nematodes (Meloidogyne sp.) were present. Fungal abundance assessed by selective agar or real-time quantitative PCR with specific primers was similar in both soils. PCR fingerprinting of isolates with ERIC primers indicated that the dominant P. c. var. chlamydosporia biotypes (profiles A and B) in both soils were very closely related, although a second biotype (profile C) was detected in one soil. When tomato plants infected with M. incognita were grown in the two soils, only profiles A and B were recovered from eggs. Primers based on polymorphisms in vcp1 demonstrated that isolates with profiles A and B were likely to prefer root-knot nematodes, whereas profile C preferred cyst nematodes. In the soil containing profiles A, B and C, egg parasitism by P. chlamydosporia was estimated at 1% using water agar plates with antibiotics but fewer than 0.2% of M. incognita eggs were shown to be infected with P. c. var. chlamydosporia when using species-specific β-tubulin-PCR primers. In contrast, the soil containing only profile B showed 22% egg parasitism on water agar plates and more than 2.5% of eggs were confirmed as P. c. var. chlamydosporia by species-specific β-tubulin-PCR primers. The results, which reveal limited diversity within the fungus at the two sites, are discussed in relation to biological control of plant-parasitic nematodes.     

Interaction between Meloidogyne incognita and Pochonia chlamydosporia and their effects on the growth of Phaseolus vulgaris

An experiment was conducted to test the effect of different doses of 2, 4 and 8?g/2?kg of soil of Pochonia chlamydosporia against the root-knot nematode (Meloidogyne incognita) on Phaseolus vulgaris. It was observed that inoculation of plant with the nematode alone, and 15?days prior to fungal inoculation, reduced the plant growth when compared with the plant with fungal application followed by the nematode. Plant length, fresh and dry weight, chlorophyll, carotenoid, protein contents and nitrate reductase activity decreased in nematode-infested plants. Application of higher dose of 8?g/2?kg of soil of P. chlamydosporia increased all the plant growth parameters as well as biochemical parameters. Highest number of galls per root system was recorded on the plants infested with nematode but not treated with the fungus. However, application of fungus prior to nematode inoculation improved the plant growth and reduced the number of galls and the number of egg masses per root system.     

Management of root-knot nematode,Meloidogyne incognita and soil borne fungus,Fusarium oxysporum in cucumber using three bioagents under polyhouse conditions

Complex diseases caused by Meloidogyne incognita and Fusarium fungus in cucumber is the most destructive disease under polyhouses. The experiment was conducted in the polyhouse of the Department of Horticulture, CCS HAU, Hisar, Haryana, India during summer season (2015–16) to evaluate the potential of bacterial and fungal biocontrol agents against Meloidogyne incognita and Fusarium oxysporum f. sp. cucumerinum in cucumber. Bioagents - Trichoderma viride (Tv), Pseudomonas fluorescence (Pf), Purpureocillium lilacinum (Pl) were taken 10 and 20 g kg⁻¹ seed and bioagents liquid formulation, 10- and 15-ml kg⁻¹ seed, were mixed with the potted soil. Chemical as well as untreated check were also maintained. All the treatments significantly improved the plant growth parameter, viz., shoot length (SL), root length (RL), fresh shoot weight (FSW), fresh root weight (FRW), dry shoot weight (DSW) and dry root weight (DRW) as compared to untreated check. However, significant reduction in nematode population and maximum improvement in plant growth parameter was recorded with carbofuran followed by higher dose of bioagents liquid formulation. Among the bioagents, bioagents liquid formulation was most effective in suppressing root knot nematode galling (43 / root system) and final population in soil (131 J₂s / 200 cc soil) and fungus wilt incidence (25 %) at 30th day of after germination and significantly improved the plant growth parameters - shoot length (147.3 cm), fresh shoot weight (55.6 g), dry shoot weight (22.51 g) and dry root weight (4.50 g) from other bioagents. Bioagents liquid formulation was effective in suppression of root-knot nematode and fungus complex disease than the powder formulations of bioagents. More studies should be needed in future to evaluate the efficacy of bioagents as seed treatments and soil applications under field conditions.     

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.     

Influence of Glomus intraradices and Soil Phosphorus on Meloidogyne incognita Infecting Cucumis melo

The interaction among Glomus intraradices, Meloidogyne incognita, and cantaloupe was studied at three soil phosphorus (P) levels in a greenhouse. All plants grew poorly in soil not amended with P, regardless of mycorrhizal or nematode status. In soil amended with 50 μg P /g soil, M. incognita suppressed the growth of nonmycorrhizal plants by 84%. In contrast, growth of mycorrhizal plants inoculated with M. incognita was retarded by only 21%. A similar trend occurred in plants grown in soil with 100 μg P /g soil. Mycorrhizal infection had no effect on the degree of root-knot gall formation and did not affect the number of nematode eggs per egg mass. Mineral levels in plant shoots generally declined as soil P levels increased and were not significantly influenced by G. intraradices or M. incognita.     

Relationships Between Tolerance and Resistance to Meloidogyne incognita in Cotton

The southern root-knot nematode, Meloidogyne incognita, is the most damaging pathogen of cotton in the United States, and both resistance and tolerance to M. incognita could be valuable management approaches. Our objectives were to evaluate advanced cotton breeding lines for resistance and tolerance to M. incognita and to determine if a relationship between resistance and tolerance exists. Reproduction of M. incognita was evaluated on 17 breeding lines, a susceptible control (Delta and Pine Land DP5415), and a resistant control (M-120) in two greenhouse trials with six replications in a randomized complete block design. Two-week-old seedlings were inoculated with 8,000 M. incognita eggs and assessed for egg production 8 weeks later. Reproduction on the resistant control was only 10% of that on the susceptible control. Eight breeding lines supported 45% to 57% less (P <= 0.05) nematode reproduction than the susceptible control, and none of them were as resistant as M-120. Yield was determined in 2001 and 2002 in fumigated (1,3-dichloropropene at 56 liters/ha) and nonfumigated plots in a strip-plot design with three replications in a field naturally infested with M. incognita. Yield suppression caused by nematode infection differed among genotypes (P ≤ 0.05 for genotype × fumigation interaction). Six genotypes in 2001 and nine in 2002 were tolerant to M. incognita based on no difference in yield between the fumigated and nonfumigated plots (P ≥ 0.10). However, only three genotypes had no significant yield suppression in both years, of which two also were resistant to M. incognita. Regression analysis indicated that yield suppression decreased linearly as nematode resistance increased.     

Growth of Isolates of Paecilomyces lilacinus and Their Efficacy in Biocontrol of Meloidogyne incognita on Tomato

The potential of 13 Paecilomyces lilacinus isolates from various geographic regions as biocontrol agents against Meloidogyne incognita, the effects of temperature on their growth, and the characterization of the impact of soil temperature on their efficacy for controlling this nematode were investigated. Maximum fungal growth, as determined by dry weight of the mycelium, occurred from 24 to 30 C; least growth was at 12 and 36 C. The best control of M. incognita was provided by an isolate from Peru or a mixture of isolates of P. lilacinus. As soil temperatures increased from 16 to 28 C, both root-knot damage caused by M. incognita and percentage of egg masses infected by P. lilacinus increased. The greatest residual P. lilacinus activity on M. incognita was attained with a mixture of fungal isolates. These isolates effected lower root-galling and necrosis, egg development, and enhanced shoot growth compared with plants inoculated with M. incognita alone.     

Expression of serine proteases in egg-parasitic nematophagous fungi during barley root colonization

Nematophagous fungi Pochonia chlamydosporia and P. rubescens colonize endophytically barley roots. During nematode infection, serine proteases are secreted. We have investigated whether such proteases are also produced during root colonization. Polyclonal antibodies against serine protease P32 of P. rubescens cross-reacted with a related protease (VCP1) of P. chlamydosporia, but not with barley proteases. These antibodies also detected an unknown ca. 65-kDa protein, labeled hyphae and appressoria of P. chlamydosporia and strongly reduced proteolytic activity of extracts from fungus-colonized roots. Mass spectrometry (MS) of 32-kDa protein bands detected peptides homologous to VCP1 only in Pochonia-colonized roots. Peptides homologous to barley serine carboxypeptidases were found in 65 kDa bands of all roots. RT-PCR detected expression of VCP1 and a new P. chlamydosporia serine carboxypeptidase (SCP1) genes only in fungus-colonized roots. SCP1 shared limited sequence homology with VCP1 and P32. Expression in roots of proteases from nematophagous fungi could be greatly relevant for nematode biocontrol.     

Evaluation of Blattisocius dolichus (Acari: Blattisociidae) for biocontrol of root-knot nematode,Meloidogyne incognita (Tylenchida: Heteroderidae)

The root-knot nematode, Meloidogyne incognita Kofoid and White (Tylenchida: Heteroderidae), is one of the most important plant parasitic nematodes attacking many plant roots. In this paper, the predation and biocontrol efficiency of Blattisocius dolichus Ma (Acari: Blattisociidae) on this nematode were studied. The predation rate and the environmental factors affecting predation rate of B. dolichus on second-stage juveniles of M. incognita (Mi-J2) were studied in experimental arenas in plastic dishes. Both female and male mites had greater capability in consuming Mi-J2, and daily predation rate of female and male mites was not less than 35 and 29.6 nematodes within seven days, respectively. Temperature, nematode density and starvation affected B. dolichus predating on Mi-J2, and when offered 300 nematodes per dish, the predation rate of mites starved for 96 h was the highest at 25 °C, with female and male mites consumed 50.5 and 54 nematodes in 24 h, respectively. The biocontrol of B. dolichus against M. incognita was carried out on potted water spinach Ipomoea aquatic in a greenhouse. The water spinach roots were inoculated with 1,000 Mi-J2 ten days after releasing 100, 200, 300, 400 and 500 mites per pot. Compared to the nematodes–alone control, the number of root-knots of all mite treatments and the number of egg masses of the treatments with 300, 400 and 500 mites significantly decreased. Effect of releasing 500 mites on control of the root nematode M. incognita was best among all mite treatments, reduced the root-knots and egg masses 37.1 and 55.1 %, respectively, but no significant difference was observed compared to 400 mites treatment.     

Host-Parasite Relationships of Meloidogyne arenaria and M. incognita on Susceptible Soybean

Pathogenicity and reproduction of single and combined populations of Meloidogyne arenaria and M. incognita on a susceptible soybean (Glycine max cv. Davis) were investigated. Significant galling and egg mass production were observed on roots of greenhouse-grown soybean inoculated with M. arenaria and M. incognita, in combination and individually. M. arenaria produced more galls and egg masses than M. incognita, whereas in combined inoculation with both nematode species, gall and egg production was intermediate. In growth chamber tests, inoculations with M. arenaria and M. incognita, singly or in combination, produced more galls and egg masses at 30 C than at 25 C. At 25 C, M. arenaria alone produced significantly more galls and egg masses than the combined M. arenaria plus M. incognita, while M. incognita produced the fewest. At 30 C, numbers of egg masses produced by M. arenaria did not differ significantly from combined M. arenaria and M. incognita. In temperature tank tests, M. incognita produced more galls and egg masses at 28 C than at 24 C soil temperature. In contrast, numbers of galls, egg masses, and eggs of M. arenaria were slightly higher at 24 C than at 28 C. Combined inoculum of both nematode species produced greater numbers of galls at 24 C than at 28 C.     

Pre-cold acclimation promotes cryoprotectant accumulation and enhances freezing tolerance of Meloidogyne incognita

Strong evidence suggests that cryoprotectant accumulation during pre-cold acclimation protects cells against freezing injuries caused by cellular dehydration. In this study, the concentrations of trehalose and glycerol were measured in Meloidogyne incognita and it was found that both cryoprotectants were significantly accumulated in second-stage juveniles (J2) of M. incognita after acclimation at 4°C. However, compared with non-acclimated samples, only a higher level of trehalose was induced in the egg masses of M. incognita in response to cold treatment. Further characterizations indicated that pre-cold acclimation efficiently accelerated the speed of larvae hatching from egg masses that were subjected to freezing at −1°C. In addition, the survival rate and pathogenicity of M. incognita J2 that had been acclimated prior to freezing were significantly enhanced when compared with non-acclimated J2 individuals. As far as we know, this is the first time that this phenomenon has been reported in M. incognita.     

Pasteuria penetrans for Control of Meloidogyne incognita on Tomato and Cucumber,and M. arenaria on Snapdragon

Meloidogyne incognita and Meloidogyne arenaria are important parasitic nematodes of vegetable and ornamental crops. Microplot and greenhouse experiments were conducted to test commercial formulations of the biocontrol agent Pasteuria penetrans for control of M. incognita on tomato and cucumber and M. arenaria on snapdragon. Three methods of application for P. penetrans were assessed including seed, transplant, and post-plant treatments. Efficacy in controlling galling and reproduction of the two root-knot nematode species was evaluated. Seed treatment application was assessed only for M. incognita on cucumber. Pasteuria treatment rates of a granular transplant formulation ranged from 1.5 × 10⁵ endospores/cm³ to 3 × 10⁵ endospores/cm³ of transplant mix applied at seeding. Additional applications of 1.5 × 10⁵ endospores/cm³ of soil were applied as a liquid formulation to soil post-transplant for both greenhouse and microplot trials. In greenhouse cucumber trials, all Pasteuria treatments were equivalent to steamed soil for reducing M. incognita populations in roots and soil, and reducing nematode reproduction and galling. In cucumber microplot trials there were no differences among treatments for M. incognita populations in roots or soil, eggs/g root, or root condition ratings. Nematode reproduction on cucumber was low with Telone II and with the seed treatment plus post-plant application of Pasteuria, which had the lowest nematode reproduction. However, galling for all Pasteuria treatments was higher than galling with Telone II. Root-knot nematode control with Pasteuria in greenhouse and microplot trials varied on tomato and snapdragon. Positive results were achieved for control of M. incognita with the seed treatment application on cucumber.     

Effects on plant growth and root-knot nematode infection of an endophytic GFP transformant of the nematophagous fungus Pochonia chlamydosporia

Pochonia chlamydosporia (Pc123) is a fungal parasite of nematode eggs which can colonize endophytically barley and tomato roots. In this paper we use culturing as well as quantitative PCR (qPCR) methods and a stable GFP transformant (Pc123gfp) to analyze the endophytic behavior of the fungus in tomato roots. We found no differences between virulence/root colonization of Pc123 and Pc123gfp on root-knot nematode Meloidogyne javanica eggs and tomato seedlings respectively. Confocal microscopy of Pc123gfp infecting M. javanica eggs revealed details of the process such as penetration hyphae in the egg shell or appressoria and associated post infection hyphae previously unseen. Pc123gfp colonization of tomato roots was low close to the root cap, but increased with the distance to form a patchy hyphal network. Pc123gfp colonized epidermal and cortex tomato root cells and induced plant defenses (papillae). qPCR unlike culturing revealed reduction in fungus root colonization (total and endophytic) with plant development. Pc123gfp was found by qPCR less rhizosphere competent than Pc123. Endophytic colonization by Pc123gfp promoted growth of both roots and shoots of tomato plants vs. uninoculated (control) plants. Tomato roots endophytically colonized by Pc123gfp and inoculated with M. javanica juveniles developed galls and egg masses which were colonized by the fungus. Our results suggest that endophytic colonization of tomato roots by P. chlamydosporia may be relevant for promoting plant growth and perhaps affect managing of root-knot nematode infestations.