Biological control of root-knot nematode (Meloidogyne javanica) disease by Pseudomonas fluorescens (Chao)

Plant growth-promoting Rhizobacteria is currently developed as an biocontrol agent against many plant pathogens. In this research, biological control of root-knot nematode (Meloidogyne javanica) by Pseudomonas fluorescens was investigated in greenhouse and laboratory experiments. Results showed that 10⁹?(CFU/ml) of P. fluorescens decreased nematode infection and other parameters significantly, compared to the control. P. fluorescens was able to cause destruction of nematode egg mass matrix and significantly decreased nematode egg hatching level. Specific activities of resistance-related enzymes, namely peroxidase (POX) and phenylalanine ammonia lyase (PAL), increased significantly in P. fluorescens-inoculated plants. Maximum activities of POX and PAL were observed at the 5?days after inoculation, respectively. Results suggested that the destruction of eggs and plant defence mechanisms leading to systemic resistance are two main suppression mechanisms used by P. fluorescens against nematode.     

Screening of tomato cultivars in the presence of the root-knot nematode,Meloidogyne javanica

In the present study, 12 varieties of tomato, viz., Arka Vikas, Damayanti, F-hybrid, Hybrid Padmarag, Hybrid Tripti, Marudam, Punjab chhoara, Pusa early dwarf, Punjab kesari, P.K.M.I, Roma and Pusa Ruby were screened for the presence of the root-knot nematode, M. javanica to obtain information on the varying degrees of resistances to tomato cultivars. All the cultivars of tomato tested were found to be infected with the root-knot nematode, M. javanica, however, to a varying extent. Consequently, there was a reduction in the growth parameters of cultivars leading to have an impact on the yield and quality of fruits. The cultivar, Marudam was found resistant while the cultivar the Pusa early dwarf was moderately resistant and rest of the 10 cultivars was highly susceptible.     

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.     

Suppression of fecundity of the root-knot nematode, Meloidogyne javanica, in monoxenic cultures of Arabidopsis thaliana treated with an alkaline extract of Ascophyllum nodosum

Treatment of Arabidopsis thaliana plants with a commercially-available, alkaline extract of the marine brown alga, Ascophyllum nodosum, resulted in a significant decrease in the number of females of the root-knot nematode, Meloidogyne javanica, which developed in the roots compared to those of plants grown in a water control medium. Significant reductions in egg recovery were also achieved from plants treated with the seaweed extract. Similar effects were produced when betaine components of the seaweed extract (γ-aminobutyric acid betaine, δ-aminovaleric acid betaine and glycinebetaine) were used in quantities equivalent to those applied in the seaweed extract treatment. As the experiments were conducted under monoxenic conditions, it can be concluded that the results obtained with the application of either the seaweed extract or betaines are indicative of their effects on the plants and are not dependent on microorganisms associated with the rhizosphere. This revised version was published online in June 2006 with corrections to the Cover Date.     

Effect of amino acids on root-knot nematode (Meloidogyne javanica) infecting tomato plant

Six amino acids viz. DL-methionine, DL-valine, DL-serine, DL-phenylalanine, L-proline and L-histidine were tested against root knot of tomato caused by Meloidogyne javanica. All amino acids showed significant response in plant growth characters with corresponding reduction in the number of galls, adult females, egg masses and juvenile stages within the treated plants. DL-phenylalanine gave significantly higher response in reducing the hatch of egg masses and survival of juveniles in in vitro test compared to control. The highest plant growth and maximum reduction of galling incidence of tomato were recorded in the DL-phenylalanine- treated plants followed by L-proline and L-histidine. All the amino acids gave positive response in suppressing the development of the nematode in the treated plants.     

Effect of root-rot fungus (Macrophomina phaseolina) on the life cycle of root-knot nematode (Meloidogyne javanica) Race-2 on balsam

The penetration of second stage juveniles of Meloidogyne javanica started within 12 hours after inoculation and the rate of penetration gradually increased with the passage of time up to the fifth day in the plants inoculated with root-knot nematode alone and up to the sixth day when plants were infected with root-knot nematode and root-rot fungus. Mostly, the penetration of second stage juveniles of Meloidogyne javanica took place in the meristematic region but in some cases the juveniles also penetrated into the root tips and oriented themselves near the stellar region almost parallel to the longitudinal axis of the roots. The life cycle of Meloidogyne javanica on balsam was completed within 25 days, whereas the duration of the life cycle and fecundity of females was adversely affected in the presence of fungus (Macrophomina phaseolina) and it took about 33 days to complete the life cycle, i.e. the presence of Macrophomina phaseolina delayed the life cycle of the root-knot nematode (Meloidogyne javanica) by eight days.     

Effect of opportunistic fungi on the life cycle of the root-knot nematode ( Meloidogyne javanica) on brinjal

The effect of four opportunistic fungi viz., Paecilomyces lilacinus, Cladosporium oxysporum, Gliocladium virens and Talaromyces flavus on the life cycle of the root-knot nematode, Meloidogyne javanica, on brinjal was evaluated under glasshouse conditions. The results revealed that these fungi affected the penetration and development of M. javanica. The life cycle of M. javanica was delayed by 10, 7, 4 and 2 days in the presence of P. lilacinus, C. oxysporum, G. virens and T. flavus respectively. Fecundity, number of eggs per eggmass and number of larvae was also reduced in the presence of these opportunistic fungi. However, the number of males increased in the presence of opportunistic fungi.     

Evaluation of a Native and Introduced Isolate of Pochonia chlamydosporia against Meloidogyne javanica

Growth chamber and plastic tunnel experiments were conducted to compare the ability of a native and introduced isolate of Pochonia chlamydosporia to colonize the rhizosphere of selected plant species and survive in soil. Effects of the isolates on population density of Meloidogyne javanica and yield of tomato after single or multiple fungal applications were also determined. In growth chamber experiments, both isolates showed a similar ability to colonise the rhizosphere of selected vegetables, except for the introduced isolate, which produced more colony forming units cm^-2 of root surface on tomato and cabbage than the native one. In the tunnel house, both isolates parasitized eggs of M. javanica, and the native but not the introduced isolate increased parasitism after multiple applications. The native isolate was recovered more frequently from soil, and was a better colonizer of tomato roots than the introduced one irrespective of the number of fungal applications. Multiple fungal applications of either isolate reduced the nematode gall rating, and the native isolate also reduced the final egg population in roots. Neither isolates reduced final nematode densities in soil or affected tomato yield when compared to untreated plots.     

Effects of Pseudomonas fluorescens strain UTPF5 on the mobility,mortality and hatching of root-knot nematode Meloidogyne javanica

The root-knot nematode Meloidogyne spp. includes important plant pathogens worldwide. This study has considered nematode Meloidogyne javanica second stage larvae activity in the extracts of Pseudomonas fluorescens strains UTPF5 and cytotoxic effect of the strain on the nematode. The movement of second stage larvae of nematodes in water agar medium at four concentrations of bacterial extracts and second stage larvae mortality rate of hatching nematode and bacterial strains in vitro were affected. Different concentrates of the strain UTPF5 effect nematode larvae movement and disposal of the same. Bacterial extraction kills almost 100% of the larvae hatching after 24?h and a complete ban on egg hatch of biocontrol nematodes and nematode indicated that root-knot nematode larvae movement on the right attract the bacteria P. fluorescens to extract in the first place.     

Histological response of resistant tomato cultivars to infection of virulent Tunisian root-knot nematode (Meloidogyne incognita) populations

The response of a susceptible tomato cultivar (Solanum lycopersicum cv. Rio Grande) to infection by three populations of root-knot nematode (Meloidogyne incognita) was compared histologically with that of Lycopersicon esculentum cv. Monita, L. esculentum cv. VFN8 and Solanum lycopersicum cv. Nemador possessing the Mi-1 resistance gene and accession PI126443 of L. peruvianum possessing the Mi-3 gene. The resistant cultivars showed susceptibility to the Tunisian Meloidogyne populations. Feeding sites were characterised by the development of giant cells that contained granular cytoplasm and several hypertrophied nuclei. The cytoplasm of giant cells was aggregated along their thickened cell walls and consequently the vascular tissues within galls appeared disrupted and disorganised. Feeding site formed on resistant L. esculentum lines and susceptible cultivar Rio Grande are similar according to cell and nucleus number, and the nurse superficies. Resistant accession L. peruvianum PI126443, known to possess heat-stable nematode resistance, also showed susceptible reaction to Tunisian Meloidogyne incognita populations; however, nematode development was reduced in comparison with susceptible plants and less developed feeding cells were observed.     

Bio-control activity of Purpureocillium lilacinum strains in managing root-knot disease of tomato caused by Meloidogyne incognita

The potential of 24 indigenous isolates of Purpureocillium lilacinum (Paecilomyces lilacinus) (Thom) Samson collected from different agro-climatic zones of India was investigated against the root-knot nematode, Meloidogyne incognita. The studies were conducted in vitro (larvicidal, ovicidal and egg-parasitising capacity) and under naturally infested field conditions with selected strains. Repeated field trials were conducted with talc-based preparations of fungal strains at 10 kg ha^?1, which were applied mixed in farm yard manure (FYM) at 1.5 t ha^?1. Results (in vitro) showed that all tested isolates were capable to parasitise eggs, inhibit egg hatching and cause juvenile mortality of M. incognita at various levels. Based on the performance under in vitro studies, eight isolates (NDPL-01, ANDPL-02, SHGPL-03, HYBPL-04, AHDPL-05, PTNPL-06, SNGPL-07 and VNSPL-08) were re-tested to confirm the results. HYBDPL-04 was found causing highest mortality (80%), inhibition of egg hatching (90%) as well as parasitisation of M. incognita eggs (75%). Under field trials also, the best protection of root-knot disease of tomato (Lycopersicon esculentum L.), in terms of reduction of galls (61%) and reproductive factor (P_f/P_i (RF) = 0.2) was achieved through application of HYBDPL-04 + FYM compared to control and other tested isolates. It also enhanced marketable yield of tomato up to 43%. It is concluded that the HYBDPL-04 strain of P. lilacinum is highly effective for management of root-knot disease of tomato under naturally infested field conditions. It is the isolate which produced the maximum number of metabolites which were extracted through high pressure liquid chromatography.     

Evaluation of Pochonia chlamydosporia var. chlamydosporia as a control agent of Meloidogyne javanica on pistachio

The potential of isolates of Pochonia chlamydosporia var. chlamydosporia as biocontrol agents for root-knot nematodes was investigated in vitro and on pistachio plants. On potato dextrose agar, growth of all isolates started at temperatures above 10°C, reached maximum between 25 and 28°C and slowed down at 33°C. On water agar, all isolates parasitized more than 85% of the eggs of Meloidogyne javanica at 18°C after 3 weeks. Filtrates of isolates grown on malt extract broth did not cause more than 5% mortality on second-stage juveniles of M. javanica after 48 h of incubation. A single application of 10×10³ chlamydospores (produced on sand–barley medium) g^–1 soil, was applied to unsterilised soil planted with pistachio cv. Kalehghochi, and plants were inoculated with 3000 nematode eggs. After 120 days in the glasshouse, nematode multiplication and damage were measured. Ability of fungus isolates to survive in the soil and to grow on roots were estimated by counting colony forming units (cfu) on semi-selective medium. Fungal abundance in soil increased nearly 3-fold and 10×10³ and 20×10³ cfu g^–1 root of pistachio were estimated in pots treated with isolates 40 and 50, respectively. Strain 50 was more abundant in soil and on the roots, infected more eggs (40%) on the roots and controlled 56% of total population of M. javanica on pistachio roots, whereas isolate 40 parasitized 15% of the eggs on the roots and controlled ca. 36% of the final nematode population.     

RAPD linkage map of the genomic region encompassing the root-knot nematode (Meloidogyne javanica) resistance locus in carrot

Inheritance studies have indicated that resistance to the root-knot nematode (Meloidogyne javanica) in carrot inbred line ’Brasilia-1252’ is controlled by the action of one or two (duplicated) dominant gene(s) located at a single genomic region (designated the Mj-1 locus). A systematic search for randomly amplified polymorphic DNA (RAPD) markers linked to Mj-1 was carried out using bulked segregant analysis (BSA). Altogether 1000 ten-mer primers were screened with 69.1% displaying scorable amplicons. A total of approximately 2400 RAPD bands were examined. Four reproducible markers (OP-C2₁₇₀₀, OP-Q6₅₀₀, OP-U12₇₀₀, and OP-AL15₅₀₀) were identified, in coupling-phase linkage, flanking the Mj-1 region. The genetic distances between RAPD markers and the Mj-1 locus, estimated using an F₂ progeny of 412 individuals from ’Brasilia 1252’×’B6274’, ranged from 0.8 to 5.7 cM . The two closest flanking markers (OP-Q6₅₀₀ and OP-AL15₅₀₀) encompassed a region of 2.7 cM . The frequency of these RAPD loci was evaluated in 121 accessions of a broad-based carrot germplasm collection. Only five entries (all resistant to M. javanica and genetically related to ’Brasilia 1252’) exhibited the simultaneous presence of all four markers. An advanced line derived from the same cross, susceptible to M. javanica but relatively resistant to another root-knot nematode species (M. incognita), did not share three of the closest markers. These results suggest that at least some genes controlling resistance to M. incognita and M. javanica in ’Brasilia 1252’ reside at distinct loci. The low number of markers suggests a reduced amount of genetic divergence between the parental lines at the region surrounding the target locus. Nevertheless, the low rate of recombination indicated these markers could be useful landmarks for positional cloning of the resistance gene(s). These RAPD markers could also be used to increase the Mj-1 frequency during recurrent selection cycles and in backcrossing programs to minimize ’linkage drag’ in elite lines employed for the development of resistant F₁ hybrids. Received: 22 June 1999 / Accepted: 6 July 1999     

Control of root-knot nematode (Meloidogyne javanica) with combination of Arthrobotrys oligospora and salicylic acid and study of some plant defense responses

Root-knot nematodes (RKN) (Meloidogyne spp.) are economically the most important pathogens of agricultural products. The aim of the present study was to control Meloidogyne javanica by using Arthrobotrys oligospora and salicylic acid (SA) and to analyse the kinetics of enzymes, phenylalanine ammonia lyase (PAL), peroxidase (POX), polyphenol oxidase (PPO) and phenolic compounds accumulation in the root system of tomato after inoculation with M. javanica, A. oligospora and SA. The ability of A. oligospora to produce extracellular proteases was also examined. In greenhouse studies, we used soil drenching of A. oligospora (10⁶ spores/ml) and soil drenching or leaf spraying of SA (5 mM) in six-leaf stage, separately and in combination. Experiments were performed in a completely randomised design. The efficiencies of treatments were appraised by using diameter of galls, number of galls per plant, number of egg masses per plant, number of eggs per egg mass, root and foliage fresh weight. The results showed that the combined application of A. oligospora and SA provided the best nematode control. The activity of the enzymes and phenolic compounds increased in comparison with the control. The nematophagous fungus A. oligospora produced extracellular proteases in the broth culture. Using A. oligospora and SA could be effective in control of M. javanica in tomato.     

Trichoderma harzianum enhances the production of nematicidal compounds in vitro and improves biocontrol of Meloidogyne javanica by Pseudomonas fluorescens in tomato

AIMS: To determine the influence of soil-borne fungus Trichoderma harzianum on the biocontrol performance of Pseudomonas fluorescens strain CHA0 and its 2,4-diacetylphloroglucinol (DAPG) overproducing derivative CHA0/pME3424 against Meloidogyne javanica. METHODS AND RESULTS: Amendment of the culture filtrate (CF) or methanol extract of the CF of a T. harzianum strain Th6 to P. fluorescens growth medium enhanced the production of nematicidal compound(s) by bacterial inoculants in vitro. In addition, bacteria overwhelmingly expressed phl'-'lacZ reporter gene when the medium was amended with CF of T. harzianum. Pseudomonas fluorescens and T. harzianum applied together in unsterilized sandy loam soil caused greater reduction in nematode population densities in tomato roots. CONCLUSIONS: Trichoderma harzianum improves root-knot nematode biocontrol by the antagonistic rhizobacterium P. fluorescens both in vitro and under glasshouse conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: The synergistic effect of T. harzianum on the production of nematicidal compound(s) critical in biocontrol may improve the efficacy of biocontrol bacteria against plant-parasitic nematodes. Considering the inconsistent performance of the biocontrol agents under field conditions, application of a mixture of compatible T. harzianum and P. fluorescens would more closely mimic the natural situation and might broaden the spectrum of biocontrol activity with enhanced efficacy and reliability of control.     

The anti-biofilm potential of pomegranate (Punica granatum L.) extract against human bacterial and fungal pathogens

Infectious diseases caused by bacteria and fungi are the major cause of morbidity and mortality across the globe. Multi-drug resistance in these pathogens augments the complexity and severity of the diseases. Various studies have shown the role of biofilms in multi-drug resistance, where the pathogen resides inside a protective coat made of extracellular polymeric substances. Since biofilms directly influence the virulence and pathogenicity of a pathogen, it is optimal to employ a strategy that effectively inhibits the formation of biofilm. Pomegranate is a common food and is also used traditionally to treat various ailments. This study assessed the anti-biofilm activity of a methanolic extract of pomegranate against bacterial and fungal pathogens. Methanolic extract of pomegranate was shown to inhibit the formation of biofilms by Staphylococcus aureus, methicillin resistant S. aureus, Escherichia coli, and Candida albicans. Apart from inhibiting the formation of biofilm, pomegranate extract disrupted pre-formed biofilms and inhibited germ tube formation, a virulence trait, in C. albicans. Characterization of the methanolic extract of pomegranate revealed the presence of ellagic acid (2,3,7,8-tetrahydroxy-chromeno[5,4,3-cde]chromene-5,10-dione) as the major component. Ellagic acid is a bioactive tannin known for its antioxidant, anticancer, and anti-inflammatory properties. Further studies revealed the ability of ellagic acid to inhibit the growth of all species in suspension at higher concentrations (>75?μg?ml^?1) and biofilm formation at lower concentrations (<40?μg?ml^?1) which warrants further investigation of the potential of ellagic acid or peel powders of pomegranate for the treatment of human ailments.     

Field assessment of efficacy of nitrogen salts to control the root-knot nematode (Meloidogyne javanica) on tomato

Influence of different nitrogen salts at electrical conductivity levels (EC2, 4 and 8?mmhos/cm) on tomato and root-knot nematode (Meloidogyne javanica) and their interactions was evaluated under field conditions. It was found that both diammonium phosphate ((NH₄)₂HPO₄) and ammonium sulphate ((NH₄)₂SO₄) were more effective than ammonium chloride (NH₄Cl) in causing an obvious suppression of M. javanica infection on tomato through reducing root galling and nematode reproduction and improving tomato growth and yield and their suppressive effect was similar to that of oxamyl or ethoprophos. At higher ECs, the tested nitrogen salts did not greatly affect pH, EC and salinity of rhizospheric soil except NH₄Cl at EC8 that caused higher EC and salinity over the untreated control which makes NH₄Cl less suitable candidate. Therefore, the use of (NH₄)₂HPO₄ and (NH₄)₂SO₄ alone or in combination with other control measures could control M. javanica and improve the growth and yield of tomato under field conditions.     

丛枝菌根真菌对不同番茄品种抗根结线虫病的影响

【背景】根结线虫病害严重制约我国设施蔬菜的生产。丛枝菌根真菌(Arbuscular Mycorrhizal Fungi,AMF)作为土壤中最重要的有益真菌之一,可以促进植物生长,提高植物抗病性,减轻土传真菌和线虫病害的发生。在蔬菜保护地栽培中,AMF对于植物线虫病防治作用的研究受到了广泛关注。【目的】针对番茄生产中危害最严重的南方根结线虫(Meliodogyne incognita)病害问题,研究AMF和番茄品种不同组合的抗线虫效应,以期为菌根真菌作为生物防治剂和生物菌肥应用于实际生产提供技术基础。【方法】在灭菌土壤中,人工接种根结线虫,比较不同菌种Rhizophagus intraradices(Ri)、Acaulosporamellea(Am)及菌种组合Rhizophagusintraradices+Acaulosporamellea(Ri+Am)在不同番茄品种(感病品种蒙特卡罗和抗线虫品种仙客1号)上对南方根结线虫侵染和繁殖的影响,研究AMF对根结线虫的拮抗效应。另外,采用南方根结线虫连作发病的土壤,在感病品种蒙特卡洛上接种AMF混合菌种Ri+Am,番茄苗移栽入连作土壤中,测定各生长指标和调查根结和卵块数量,评价接种AMF处理对根结线虫病的防治效果。【结果】在灭菌土壤中,普通番茄品种蒙特卡罗的菌根效应显著优于抗线虫番茄品种仙客1号,表现为前者单位根重的根结和卵块的数量均比对照显著降低,而后者仅降低了卵块数量;蒙特卡罗上接种Ri+Am混合菌种的效果优于接种单一菌种Am和Ri;而仙客1号上接种Ri的效果更好。接种线虫也显著影响了AMF的侵染,而且对抗性品种仙客1号的影响更为明显。但除了接种Am的处理,大多数处理收获时菌根侵染率仍维持较高的水平(70%以上)。在连作土壤中,感病品种蒙特卡罗接种混合菌种Ri+Am具有较好的抗/耐线虫效应,主要表现为促进植株生长,显著降低根结和卵块数量,但菌根侵染率较灭菌土壤低,约为40%。【结论】综合以上结果,表明菌根化苗能够在一定程度上减轻根结线虫病的危害。土壤灭菌条件下,在感病和抗线虫番茄品种上接种AMF能够减轻线虫的侵染和繁殖,而且在感病品种上的防治效果更加显著。在连作土壤中,在番茄感病品种上接种AMF也表现较好的抗线虫效果。     

Influence of organic additives on the incidence of root-knot nematode,Meloidogyne javanica in roots of tomato plants

Glasshouse experiment was conducted to assess the impact of green chopped leaves of four test plants (Lantana camara, Ficus virens, Kigelia pinnata and Ficus bengalensis) and two nematicides (Phorate and Carbofuran) on the plant growth parameters of tomato cv. K25 and on the root-knot development. Results revealed that all the tested treatments significantly (p = 0.05) improved plant growth parameters and reduced root-knot development compared to control. Among the tested organic additives, chopped green leaves of Lantana camara added to soil gave the highest enhancement in plant growth parameters, including plant height, fresh and dry weight, number of fruits and fruit weight with the values of 94.2 cm, 106.8 g, 31.6 g, 7.2 and 153.3 g respectively, as well as a greater reduction of Meloidogyne javanica reproduction and development but exhibiting a lower response compared to nematicides. There was also significant reduction in root-knot development in tomato plants growing in other organic additive amended soil.