Biological control of root-knot nematode,Meloidogyne incognita infesting tomato

Talc based formulations of two antagonistic fungi, Acremonium strictum W. Gams and Aspergillus terreus Thom were tested separately and together for their ability to suppress the development of root-knot disease of tomato caused by the root-knot nematode, Meloidogyne incognita Kofoid & White in two consecutive trials (2007–08). Tomato seedlings were each inoculated with M. incognita at 2 infective second stage juveniles /g of soil. M. incognita caused up to 48% reduction in plant growth parameters compared to un-inoculated control. Control efficacy achieved by combined soil application of both fungi, in terms of galls/root system and soil population/50 ml of soil, was 66 and 69% respectively at 60 days of inoculation compared to control. Soil application by individual fungus did not achieve as much effectiveness as the biocontrol agents applied together. The combined treatment was found to have antagonistic effect on M. incognita development and increased plant vigor. Incorporation of fine powder of chickpea pod waste with talc powder was beneficial in providing additional nutrients to both plant and biocontrol agents and increased the activity of the nematophagous fungi in soil. A. strictum and A. terreus were successfully established in the rhizosphere of tomato plants up to the termination of the experiment.     

Bio-activity of fungal culture filtrates against root-knot nematode egg hatch and juvenile motility and their effects on growth of mung bean (Vigna radiata L. Wilczek) infected with the root-knot nematode,Meloidogyne incognita

Culture filtrates of selected soil fungi, namely Aspergillus flavus, Aspergillus fumigatus, Aspergillus niger, Fusarium oxysporum, Penicillium vermiculatum and Rhizopus nigricans exhibited variable response to egg hatching and mortality of the root-knot nematode, Meloidogyne incognita. Higher concentrations of the culture filtrates of all the fungi inhibited egg hatching and proved to be toxic to the juveniles of M. incognita. In addition, development of the gall and multiplication of M. incognita were also found adversely affected in varying degrees on all the plants of Vigna radiata treated with the filtrates. The culture filtrate of A. niger showed highest toxicity to the nematode than those of any other fungus tested. Soil drench application of the culture filtrates gave better seedling growth and least nematode multiplication in comparison to seed soaking treatment.     

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.     

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.     

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.     

In-vitro Assays of Meloidogyne incognita and Heterodera glycines for Detection of Nematode-antagonistic Fungal Compounds

In-vitro methods were developed to test fungi for production of metabolites affecting nematode egg hatch and mobility of second-stage juveniles. Separate assays were developed for two nematodes: root-knot nematode (Meloidogyne incognita) and soybean cyst nematode (Heterodera glycines). For egg hatch to be successfully assayed, eggs must first be surface-disinfested to avoid the confounding effects of incidental microbial growth facilitated by the fungal culture medium. Sodium hypochlorite was more effective than chlorhexidine diacetate or formaldehyde solutions at surface-disinfesting soybean cyst nematode eggs from greenhouse cultures. Subsequent rinsing with sodium thiosulfate to remove residual chlorine from disinfested eggs did not improve either soybean cyst nematode hatch or juvenile mobility. Soybean cyst nematode hatch in all culture media was lower than in water. Sodium hypochlorite was also used to surface-disinfest root-knot nematode eggs. In contrast to soybean cyst nematode hatch, root-knot nematode hatch was higher in potato dextrose broth medium than in water. Broth of the fungus Fusarium equiseti inhibited root-knot nematode egg hatch and was investigated in more detail. Broth extract and its chemical fractions not only inhibited egg hatch but also immobilized second-stage juveniles that did hatch, confirming that the fungus secretes nematode-antagonistic metabolites.     

The effects of exotoxin (Thuringiensin) from Bacillus thuringiensis on Meloidogyne incognita and Caenorhabditis elegans

A partially purified preparation as well as two formulations of exotoxin from Bacillus thuringiensis (thuringiensin) were evaluated for nematicidal activity. The methods used in our evaluations included direct contact nematicidal assays, hatching tests, infection tests in seed pouches using the cucumber/root-knot nematode (Meloidogyne incognita) system, and greenhouse test using the root-knot nematode. While contact nematicidal activity was not observed against juveniles of M. incognita, 100% mortality occurred when the free-living nematode, Caenorhabditis elegans, was used as the test organism. Nematode infection evaluations in the seed pouch assay showed reduced root galling at relatively high concentrations (>10 mg kg^-1). Greenhouse assays indicated significant reduction in the soil population. However, the degree of control in relation to the amount of material applied, as measured by the gall numbers, larvae from soil/roots, and plant growth parameters, was not considered adequate. Data on the plant response in relation to treatment with different formulations of the toxin are presented.     

Detection and biocontrol potential of <Emphasis Type="Italic">Verticillium leptobactrum</Emphasis> parasitizing <Emphasis Type="Italic">Meloidogyne</Emphasis> spp.

Three isolates of Verticillium leptobactrum proceeding from egg masses of root-knot nematodes (RKN) Meloidogyne spp. and soil samples collected in Tunisia were evaluated against second-stage juveniles (J2) and eggs of M. incognita, to determine the fungus biocontrol potential. In vitro tests showed that V. leptobactrum is an efficient nematode parasite. The fungus also colonized egg masses and parasitized hatching J2. In a greenhouse assay with tomato plants parasitized by M. incognita and M. javanica, V. leptobactrum was compared with isolates of Pochonia chlamydosporia and Monacrosporium sp., introducing the propagules into nematode-free or naturally infested soils. The V. leptobactrum isolates were active in RKN biocontrol, improving plants growth with a significant increase of tomato roots length, lower J2 numbers in soil or egg masses, as well as higher egg mortalities. In a second assay with M. javanica, treatments with three V. leptobactrum isolates reduced egg masses on roots as well as the density of J2 and the number of galls. To evaluate the fungus capability to colonize egg masses a nested Real-time polymerase chain reaction (PCR) assay, based on a molecular beacon probe was used to assess its presence. The probe was designed on a V. leptobactrum ITS region, previously sequenced. This method allowed detection of V. leptobactrum from egg masses, allowing quantitative DNA and fungal biomass estimations.     

Biological control of Meloidogyne incognita and Fusarium solani in dry common bean in the field

Meloidoyne incognita (root-knot nematode) and Fusarium solani (root-rot pathogen) were the common soil-borne pathogens and cause severe damage to bean plants in newly reclaimed sandy soil in Nubaryia district, Behera Governorate, Egypt. The antagonistic effects of Trichoderma album and Trichoderma viride as well as three commercial products namely Rhizo-N^® (Bacillus subtilis), Bio-Arc^® 6% (Bacillus megaterium) and Bio-Zeid^® 2.5% (T. album) were tested against M. incognita and F. solani under naturally infected field conditions. T. album and T. viride highly reduced the frequency (%) population of pathogenic fungi such as Fusarium spp., F. solani and Rhizoctonia spp., than the commercial products. Results indicated that all the tested bio-control agents reduced, significantly, the nematode criteria as evidenced by the number of juvenile (J₂) in soil and number of galls and egg masses on roots of common bean and Fusarium root-rot incidence (%). Rhizo-N^® highly reduced the number of J₂ in soil, while T. album was the best in reducing the number of galls and egg masses in roots. The bio-control agents also increased the plant growth parameters of common bean plants i.e. plant height, plant weight, branch no./plant, pods no./plant, pod weight/plant, pod weight, seeds no./plant, fresh seeds weight/pod, dry seeds weight/pod and dry weight of 100 seeds.     

Isolation of nematophagous fungi from eggs and females of Meloidogyne spp. and evaluation of their biological control potential

Fungi were isolated from Meloidogyne spp. eggs and females on 102 field-collected root samples in China. Of the 235 fungi isolated (representing 18 genera and 26 species), the predominant fungi were Fusarium spp. (42.1% of the isolates collected), Fusarium oxysporum (13.2%), Paecilomyces lilacinus (12.8%), and Pochonia chlamydosporia (8.5%). The isolates were screened for their ability to parasitise Meloidogyne incognita eggs in 24-well tissue culture plates in two different tests. The percentage of eggs parasitised by the fungi, the numbers of unhatched eggs and alive and dead juveniles were counted at 4 and 7 days after inoculation. The most promising fungi included five Paecilomyces isolates, 10 Fusarium isolates, 10 Pochonia isolates and one Acremonium isolate in test 1 or test 2. Paecilomyces lilacinus YES-2 and P. chlamydosporia HDZ-9 selected from the in vitro tests were formulated in alginate pellets and evaluated for M. incognita control on tomato in a greenhouse by adding them into a soil with sand mixture at rates of 0.2, 0.4, 0.8 and 1.6% (w/w). P. lilacinus pellets at the highest rate (1.6%) reduced root galling by 66.7%. P. chlamydosporia pellets at the highest rate reduced the final nematode density by 90%. The results indicate that P. lilacinus and P. chlamydosporia as pellet formulation can effectively control root-knot nematodes.     

Extracellular Protease of Pseudomonas fluorescens CHA0, a Biocontrol Factor with Activity against the Root-Knot Nematode Meloidogyne incognita

In Pseudomonas fluorescens CHA0, mutation of the GacA-controlled aprA gene (encoding the major extracellular protease) or the gacA regulatory gene resulted in reduced biocontrol activity against the root-knot nematode Meloidogyne incognita during tomato and soybean infection. Culture supernatants of strain CHA0 inhibited egg hatching and induced mortality of M. incognita juveniles more strongly than did supernatants of aprA and gacA mutants, suggesting that AprA protease contributes to biocontrol.     

Bio-efficacy of some leaf extracts on the inhibition of egg hatching and mortality of Meloidogyne incognita

Nematicidal activities of extracts from plants were assayed against Meloidogyne incognita in vitro. Leaves of six different plants were collected in and around Aligarh Muslim University Campus. Aqueous extracts of six plants were screened for egg hatchability and nematicidal activity against second stage juveniles of M. incognita in the plant pathology and nematology laboratory, AMU Aligarh. The nematode egg and juveniles were exposed 12, 24 and 48 h in (S, S/2, S/10, S/100) concentrations of plant extracts. The plant extracts of leaves of six plants species viz. Jatropha pandurifolia, Polyalthia longifolia, Wedelia chinensis, Nerium indicum, Duranta repens and Cassia fistula exhibited highly promising mortality of 99.00–72.00% after 48 h of exposure. Aqueous extracts of leaves of J. pandurifolia, P. longifolia, W. chinensis were recorded to be highly effective for inhibition of egg hatching and increasing juvenile mortality of M. incognita. There was a gradual decrease in egg hatching and increase in mortality rate of juveniles of M. incognita with increase in the concentration of leaf extract and exposure time.     

Effects of culture filtrates from the nematophagous fungus Verticillium leptobactrum on viability of the root-knot nematode Meloidogyne incognita

Filtrates of three isolates of the nematophagous fungus Verticillium leptobactrum were evaluated for their nematicidal activity against the root-knot nematode Meloidogyne incognita. The filtrates inhibited egg hatching, with maximum toxicity observed for isolate HR21 at 50% (v:v) dilution, after 7 days exposure. Filtrates also inactivated second-stage juveniles (J2) at 10-50% dilutions. A scanning electron microscopy study of treated eggs showed severe alterations caused by the filtrate of isolate HR43 on M. incognita eggs, which appeared collapsed and not viable, suggesting the production of chitin-degrading enzymes or other active compounds.     

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.     

Potential biocontrol activity of Arthrobotrys oligospora and Trichoderma harzianum BI against Meloidogyne javanica on tomato in the greenhouse and laboratory studies

In this investigation, the biological control activity of Arthrobotrys oligospora and Trichoderma harzinum BI against the root-knot nematode, Meloidogyne javanica, infecting tomato, was assessed both in in vitro and in in vivo experiments. In greenhouse experiments, tomato seedlings at six-leaf stage were inoculated with 10⁶?spores/ml of A. oligospora and T. harzianum BI and number of 2000 nematode eggs per individual seedling. In in vitro assays, the per cent inhibition of nematode eggs hatching, the death per cent of second-stage juvenile (J2) and proteolytic activity on casein hydrolysis was evaluated. Results showed that A. oligospora and T. harzianum BI decreased the mean numbers of galls, eggmasses and egg per eggmass significantly (p?<?0.05) compared with control. Percentage hatching inhibition of M. javanica treated with A. oligospora and T. harzianum BI was 25 and 52%, respectively. Moreover, A. oligospora and T. harzianum BI significantly increased (p?<?0.05) the mortality rate of M. javanica (J2) after two and four days (74, 85 and 53, 63%, respectively). A. oligospora and T. harzianum BI had a proteolytic activity of 3.9 (U/min per ml) and 2.4 (U/min per ml) at pH 5.0, respectively. Our data suggest that the application of these two fungi in tomato rhizosphere infected with root-knot nematode M. javanica had antagonistic effects on the infection and reproduction of this nematode and the ability to control its population.     

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.     

Studies on the disease complex incidence by Meloidogyne incognita and Fusarium solani on Poi,Basella rubra

A survey of Poi crop in Ghaziabad (UP) exhibited a disease complex incidence by Meloidogyne incognita and Fusarium solani causing synergistic effect on the host. Paecilomyces lilacinus was found from the egg masses of M. incognita and Aspergillus niger and Aspergillus terreus from the rhizosphere of root-knot infected Poi crop. Paecilomyces lilacinus parasitised the eggs to a greater extent. The level of parasitism was highest (65%) by P. lilacinus while Aspergillus spp. did not colonise the eggs. Fusarium solani which in the present investigation has been established to be pathogenic to Poi plant.     

Isolation and screening of fungi associated with eggs and females of root-knot nematodes and their biocontrol potential against Meloidogyne incognita in Bangladesh

A total of 297 fungal isolates belonging to 20 genera and 33 species were isolated and identified from eggs and females of Meloidogyne spp. in Bangladesh. The predominant genera were Fusarium, Aspergillus and Penicillium; and the significant ones were Purpureocillium, Trichoderma and Pochonia. The 24 well tissue culture plate screening technique was applied for pathogenicity tests against Meloidogyne incognita in vitro. The average percentages of egg parasitism, egg hatch inhibition and juvenile mortality varied significantly and were ranged from 8.2 to 64.9% (p = 0.05), 24.8 to 72.4% (p = 0.05), and 2.3 to 33.1% (p = 0.05), respectively. Two isolates of Purpureocillium lilacinum (PLSAU 1 and PLSAU 2) and one isolate of Pochonia chlamydosporia (PCSAU 1) reduced more than 60% average root galls of tomato, eggplant and cucumber in greenhouse experiments. This is the first investigation of fungi associated with nematodes in the country and their biological control potential against M. incognita.     

Biological Control of Root-Knot Nematode <i>Meloidogyne incognita</i> in <i>Psoralea corylifolia</i> Plant by Enhancing the Biocontrol Efficacy of <i>Trichoderma harzianum</i> Using Press Mud

Meloidogyne incognita is a plant pathogen causing root-knot disease and loss of crop yield. The present study aimed to use Trichoderma harzianum as a biocontrol agent against plant-parasitic nematodes and used press mud, which is a solid waste by-product of sugarcane, as a biocontrol agent and biofertilizer. Therefore, the combined application of T. harzianum and press mud may enhance nematode control and plant growth. Elemental analysis of press mud using scanning electron microscopy (SEM) integrated with an Energy Dispersive X-ray (EDX) analyzer revealed the presence of different elements such as C, O, Mg, Si, P, K, Ca, Cu and Zn. In addition, a greenhouse study was conducted to investigate the combined effects of press mud and T. harzianum on M. incognita reproduction and growth and the biochemical features of Psoralea corylifolia. The results showed that plant length, dry biomass, leaf area, the number of seeds per plant, chlorophyll a, chl b, carotenoid content, nitrate reductase, carbonic anhydrase, and nitrogen content were significantly increased (P ≤ 0.05) in the T2 plants (plants were treated with 100 g of press mud + 50 mL T. harzianum before one week of M. incognita inoculation), over inoculated plants (IC). Antioxidant enzyme activity of ascorbate peroxidase (APX), catalase (CAT), peroxidase (POD), and superoxide dismutase (SOD) in the foliage of P. corylifolia was significantly increased when plants were treated with press mud + T. harzianum. A significant reduction in the number of egg masses, nematode population, and root-knot index (RKI) was found in plants with T2 plants. These results suggest that the combined application of T. harzianum and press mud has the potential to control the M. incognita infection and can be used as an environmentally safe alternative to chemical nematicides and also help in the removal of sugarcane waste that causes environmental pollution.     

Evaluation of oil dispersion formulation of nematophagus fungus,Pochonia chlamydosporia against root-knot nematode,Meloidogyne incognita in cucumber

Root-knot nematode, Meloidogyne incognita is considered as one of the major non-insect pests of crops. The management of these root feeders becomes highly challenging due to a strong host-parasitic relationship. Pochonia chlamydosporia is a nematophagus fungus that colonizes eggs of nematodes. This study aimed to test the efficacy of P. chlamydosporia (NAIMCC-SF0039) against M. incognita. An oil dispersion formulation of P. chlamydosporia was prepared using emulsifiers and vegetable oil. This formulation had a shelf-life of 90 days (3.3 × 10⁸ CFU/mL) at room temperature (28 ± 1 °C). The inhibitory effect of oil formulation was tested against M. incognita by inoculating it on the egg mass. We found that colonization of the gelatinous matrix occurred on the third day of inoculation followed by complete egg parasitization on the seventh day. A greenhouse trial was laid out to evaluate the biocontrol potential of P. chlamydosporia in cucumber (Cucumis sativus). The results showed that the application of talc formulation of P. chlamydosporia at the rate of 1 kg per acre during planting, followed by delivery of 1 L of oil dispersion formulation through drip lines at 30-day intervals caused the highest reduction of nematode infestation. This treatment recorded 67.9 and 57.5% reduction in egg masses and soil nematode population respectively than that of control.