Improvement of Pseudomonas fluorescens CHA0 biocontrol activity against root-knot nematode by the addition of ammonium molybdate

AIMS: To improve the efficacy of Pseudomonas fluorescens CHA0 and its genetically modified (GM) derivatives by adding ammonium molybdate to control Meloidogyne javanica, the root-knot nematode in mungbean. METHODS AND RESULTS: Culture filtrate of P. fluorescens CHA0 and its GM derivative (antibiotic overproducing strain CHA0/pME3424 and antibiotic-deficient CHA89) obtained from nutrient broth yeast extract medium amended with 1, 2 or 4 mm of ammonium molybdate (NH4-Mo) caused substantial mortality of M. javanica juveniles in vitro. Pseudomonas fluorescens CHA0 or CHA0/pME3424 applied in conjunction with NH4-Mo caused greater reduction of nematode penetration in mungbean roots compared with the bacterial application alone. Ammonium molybdate at 4 mg kg-1 of soil along with CHA0 also enhanced plant height while shoot weight remained unaffected. Either used alone or in conjunction with NH4-Mo, strain CHA89 did not reduce nematode invasion compared with the controls. Bacterial strains did not differ significantly in their colonization potential in the mungbean rhizosphere. Efficacy of the biocontrol bacteria to control root-knot nematode was accentuated when soil was treated with NH4-Mo and zinc (both at 1 mg kg-1 of soil). CONCLUSION: The addition of ammonium molybdate enhances the production of nematicidal compounds by P. fluorescensin vitro and improves bacterial efficacy against root-knot nematode under glasshouse conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: Application of minerals such as ammonium molybdate is appealing because they are cheap and can easily be applied under field conditions to improve biocontrol potential of the bacterial inoculants. They also significantly reduce the amount of biocontrol inoculant biomass required to achieve root-knot disease control, with a consequent reduction in cost.     

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.     

Role of cyanide production by Pseudomonas fluorescens CHA0 in the suppression of root-knot nematode, Meloidogyne javanica in tomato

Summary Pseudomonas fluorescens strain CHA0 produces hydrogen cyanide (HCN), a secondary metabolite that accounts largely for the biocontrol ability of this strain. In this study, we examined the role of HCN production by CHA0 as an antagonistic factor that contributes to biocontrol of Meloidogyne javanica, the root-knot nematode, in situ. Culture filtrate of CHA0, resulting from 1/10-strength nutrient broth yeast extract medium amended with glycine, inhibited egg hatch and caused mortality of M. javanica juveniles in vitro. The bacterium cultured under high oxygen-tension conditions exhibited better inhibitory effects towards nematodes, compared to its cultivation under excess oxygen situation. Growth medium amended with 0.50 or 1.0 mM FeEDDHA further improved hatch inhibition and nematicidal activity of the strain CHA0. Strain CHA77, an HCN-negative mutant, failed to exert such toxic effects, and in this strain, antinematode activity was not influenced by culture conditions. Exogenous cyanide also inhibited egg hatch and caused mortality of M. javanica juveniles in vitro. Strains CHA0 or CHA77 applied in unsterilized sandy-loam soil as drench, caused marked suppression of root-knot disease development incited by M. javanica in tomato seedlings. However, efficacy of CHA77 was noticeably lower compared to its wild type counterpart CHA0. An increased bioavailability of iron following EDTA application in soil substantially improved nematode biocontrol potential of CHA0 but not that of CHA77. Soil infestation with M. javanica eggs resulted in significantly lower nematode population densities and root-knot disease compared to the juveniles used as root-knot disease-inducing agents. Strain CHA0 significantly suppressed nematode populations and inhibited galling in tomato roots grown in soil inoculated with eggs or juveniles and treated with or without EDTA. Strain CHA0 exhibited greater biocontrol potential in soil inoculated with eggs and treated with EDTA. To demonstrate that HCN synthesis by the strain CHA0 acts as the inducing agent of systemic resistance in tomato, efficacy of the strain CHA0 was compared with CHA77 in a split root trial. The split-root experiment, guaranteeing a spatial separation of the inducing agent and the challenging pathogen, showed that HCN production by CHA0 is not crucial in the induction of systemic resistance in tomato against M. javanica, because the HCN-negative-mutant CHA77 induced the same level of resistance as the wild type but exogenous cyanide in the form of KCN failed to trigger the resistance reaction. In the root section where both nematode and the bacterium were present, strain CHA0 reduced nematode penetration to a greater extent than CHA77, suggesting that for effective control of M. javanica, a direct contact between HCN-producing CHA0 and the nematode is essential.     

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

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

Bacterial mixture from greenhouse soil as a biocontrol agent against root-knot nematode, Meloidogyne incognita, on oriental melon

The biological control efficacy of a greenhouse soil bacterial mixture of Lactobacillus farraginis, Bacillus cereus, and Bacillus thuringiensis strains with antinematode activity was evaluated against the root-knot nematode Meloidogyne incognita. Two control groups planted in soil drenched with sterile distilled water or treated with the broadspectrum carbamate pesticide carbofuran were used for comparison. The results suggest that the bacterial mixture is effective as a biocontrol agent against the root-knot nematode.     

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.     

Differential impact of some Aspergillus species on Meloidogyne javanica biocontrol by Pseudomonas fluorescens strain CHA0

AIMS: The aim was to determine the influence of some Aspergillus species on the production of nematicidal agent(s) in vitro and biocontrol of Meloidogyne javanica in tomato by Pseudomonas fluorescens strains CHA0 and CHA0/pME3424. METHODS AND RESULTS: Six species of Aspergillus, isolated from the rhizosphere of certain crops, produced a variety of secondary metabolites in vitro. Culture filtrate (CF) obtained from Ps. fluorescens strain CHA0 and its2,4-diacetylphloroglucinol overproducing mutant CHA0/pME3424 grown in King's B liquid medium caused significant mortality of M. javanica juveniles in vitro. Bacterial growth medium amended with CF of A. niger enhanced nematicidal and beta-galactosidase activities of fluorescent pseudomonads while A. quadrilineatus repressed such activities. Methanol or ethyl acetate extracts of the CF of A. niger markedly optimized bacterial efficacy to cause nematode deaths while hexane extract of the fungus had no influence on the nematicidal activity of the bacterial strains. A. niger applied alone or in conjunction with the bacterial inoculants inhibited root-knot nematode galling in tomato. On the other hand, A. quadrilineatus used alone or together with CHA0 did not inhibit nematode galling but when used in combination with strain CHA0/pME3424 did reduce galling intensity. CONCLUSIONS: Aspergillus niger enhances the production of nematicidal compounds by Ps. fluorescensin vitro and improves biocontrol potential of the bacterial inoculants in tomato while A. quadrilineatus reduces bacterial performance to suppress root-knot nematodes. SIGNIFICANCE AND IMPACT OF THE STUDY: Rhizosphere harbours a variety of micro-organisms including bacteria, fungi and viruses. Aspergillus species are ubiquitous in most agricultural soils and generally produce a variety of secondary metabolites. Such metabolites synthesized by Aspergillus species may influence the production of nematicidal agents and subsequent biocontrol performance of the bacterial inoculants against plant-parasitic nematodes. This fact needs to be taken into consideration when using biocontrol strains in an agriculture system.     

Extracellular protease and phospholipase C are controlled by the global regulatory gene gacA in the biocontrol strain Pseudomonas fluorescens CHA0

Abstract Pseudomonas fluorescens strain CHA0 protects plants from various root diseases. Antibiotic metabolites synthesized by this strain play an important role in disease suppression; their production is mediated by the g lobal ac tivator gene gacA . Here we show by complementation that the gacA gene is also essential for the expression of two extracellular enzymes in P. fluorescens CHA0: phospholipase C and a 47-kDa metalloprotease. In contrast, the production of another exoenzyme, lipase, is not regulated by the gacA gene. Protease, phospholipase and antibiotics of P. fluorescens are all known to be optimally produced at the end of exponential growth; thus the gacA gene appears to be a general stationary-phase regulator.     

Biocontrol of root-knot nematode Meloidogyne incognita by the isolates of Pseudomonas on tomato

Biocontrol of root-knot nematode Meloidogyne incognita was studied on tomato using 15 isolates of fluorescent Pseudomonads isolated from pathogen suppressive soils. Pseudomonas aeruginosa (isolates Pa8, Pa9 and Pa3) caused greater inhibitory effect on hatching of M. incognita than other isolates. In addition, isolates Pa8, Pa9 and Pa3 caused greater colonisation of tomato roots and also caused a greater increase in the growth of tomato seedlings. These isolates also caused a greater increase in growth of tomato and higher reduction in galling and nematode multiplication in a green house test than is caused by other isolates. Isolates Pf1, Pf5, Pf6 and Pa13 were unable to increase growth of tomato and caused less reduction in galling and nematode multiplication compared to other isolates. Only 10 isolates produced siderophores on chromo-azurol sulfonate (CAS) agar medium and isolate Pa12 showed greater production of siderophore followed by Pa11, Pa9, Pf10, Pa3 and Pf5. Similarly, isolates Pa14, Pa12, Pf10, Pa9, Pa8, Pa7 and Pa6 produced greater amount of HCN than the other isolates tested. Isolates Pa8 and Pa9 showed greater production of IAA than the other 13 isolates tested. This study suggests that P. aeruginosa isolates Pa8 and Pa9 may be used for the biocontrol of M. incognita on tomato.     

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.     

In vitro activity of Bacillus firmus against the burrowing nematode Radopholus similis,the root-knot nematode Meloidogyne incognita and the stem nematode Ditylenchus dipsaci

Antagonistic bacteria have been repeatedly shown to be promising microorganisms for the biological control of sedentary and migratory endoparasitic nematodes. Depending on the bacteria involved, the mechanisms of action include: obligate parasitism, reduction in penetration, growth inhibition due to competition for nutrients and antibiosis associated with bioactive metabolites. In the present studies, the mode of action of the antagonist bacteria Bacillus firmus, isolated from a bionematicide, was evaluated. Significant rates of paralysis and mortality were detected after incubation of three nematode species in low concentrations of the pure culture filtrates following removal of the bacterial cells. The same culture filtrates also significantly reduced hatching of Meloidogyne incognita. Pure bacterial cell suspensions added to sand also reduced survival of R. similis in bioassays by 41% over the controls. The mode-of-action responsible for nematode paralysis and mortality was therefore demonstrated to be closely associated with the production of bioactive compounds secondary metabolites by the bacteria.     

Biological control of rice root-knot nematode,Meloidogyne graminicola through mixture of Pseudomonas fluorescens strains

The plant growth promoting rhizobacterium, Pseudomonas fluorescens strains PF1, TDK1, and PY15 were evaluated individually and in combinations for their efficacy against root-knot nematode, Meloidogyne graminicola, in rice plants under in vitro, glass house and field conditions. Culture filtrates of these strains either individually or as mixture inhibited egg hatching and caused mortality of juveniles of M. graminicola in vitro. The efficacy was more pronounced when filtrates of the strain were used as mixtures than as individual strains. Mixtures of P. fluorescens strains signficantly reduced M. graminicola infestation when applied as bacterial suspensions through seed treatment. The higher activity of peroxidase and chitinase enzymes was observed in plants treated with P. fluorescens mixtures than the plants treated with individual strains, two strain mixtures and untreated control. In field trials on rice, talc formulations of the P. fluorescens strains individually as well as mixtures were evaluated as seed treatment, soil treatment and combination of both. A mixture of the three strains was the most effective when applied either as seed + soil treatment or as seed treatment alone. The introduced P. fluorescens strains survived endophytically on rice roots. The application of the P. fluorescens mixture PF1 + TDK1 + PY15 in seed + soil treatment resulted in higher grain yield which provided a 27.3% increase over the control followed by P. fluorescens mixture PF1 + TDK1 + PY15 in seed treatment alone, which increased the grain yield of rice by 24.7% compared to the control.     

Response of five lettuce cultivars to root-knot nematode, Meloidogyne incognita

The root-knot nematode, Meloidogyne incognito (Kofoid et White) Chitwood is an important pathogen of vegetables. Five commercial cultivars of lettuce (Lactuca sativa L.) were evaluated under greenhouse conditions for resistance to Meloidogyne incognita, Benguet population. Plants were inoculated with 1000 eggs collected from 'Apollo' tomato (Lycopersicon esculentum) roots. The degree of galling and number of egg masses were assessed 4 and 8 weeks after inoculation. Host plant response was classified as immune, highly resistant, resistant, moderately resistant, intermediate, moderately susceptible, and highly susceptible based on the resistance index of Kouamè et at., 1998 [RI = (gall2 + egg2)]. Inoculation of 1000 eggs/plant significantly affected the growth and yield of the five lettuce cultivars 4 and 8 weeks after inoculation. A significant interaction was observed between treatment and cultivar during the two evaluation periods in terms of marketable and non-marketable yield, plant height, root weight, number of galls and number of egg masses. A reduction in growth and yield was observed in the cultivars Ballon, Lollo Rosa and Red Wave. Significant differences were noted in the number of galls and egg masses among the different cultivars tested. The highest average number of galls was obtained from the cultivars Red Wave, Ballon and Lollo Rosa. Cultivar Ballon had the highest average number of recovered nematode while Gilaben had the lowest with 15 and 4 per roots, respectively after 4 weeks inoculation. After 8 weeks, nematode was highest in cultivar Red Wave (615) and lowest in Great Lakes (70). Based on the host response, cultivars Great Lakes and Gilaben were rated highly resistant and resistant, respectively, while Red Wave, Ballon and Lollo Rosa were rated intermediate.     

Thiamine-auxotrophic mutants of Pseudomonas fluorescens CHA0 are defective in cell-cell signaling and biocontrol factor expression

In the biocontrol strain Pseudomonas fluorescens CHA0, the Gac/Rsm signal transduction pathway positively controls the synthesis of antifungal secondary metabolites and exoenzymes. In this way, the GacS/GacA two-component system determines the expression of three small regulatory RNAs (RsmX, RsmY, and RsmZ) in a process activated by the strain's own signal molecules, which are not related to N-acyl-homoserine lactones. Transposon Tn5 was used to isolate P. fluorescens CHA0 insertion mutants that expressed an rsmZ-gfp fusion at reduced levels. Five of these mutants were gacS negative, and in them the gacS mutation could be complemented for exoproduct and signal synthesis by the gacS wild-type allele. Furthermore, two thiamine-auxotrophic (thiC) mutants that exhibited decreased signal synthesis in the presence of 5 x 10(-8) M thiamine were found. Under these conditions, a thiC mutant grew normally but showed reduced expression of the three small RNAs, the exoprotease AprA, and the antibiotic 2,4-diacetylphloroglucinol. In a gnotobiotic system, a thiC mutant was impaired for biological control of Pythium ultimum on cress. Addition of excess exogenous thiamine restored all deficiencies of the mutant. Thus, thiamine appears to be an important factor in the expression of biological control by P. fluorescens.     

Secondary metabolites help biocontrol strain Pseudomonas fluorescens CHA0 to escape protozoan grazing

In soil ecosystems, bacteria must cope with predation activity, which is attributed mainly to protists. The development of antipredation strategies may help bacteria maintain higher populations and persist longer in the soil. We analyzed the interaction between the root-colonizing and biocontrol strain Pseudomonas fluorescens CHA0 and three different protist isolates (an amoeba, a flagellate, and a ciliate). CHA0 produces a set of antibiotics, HCN, and an exoprotease. We observed that protists cannot grow on CHA0 but can multiply on isogenic regulatory mutants that do not produce the extracellular metabolites. The in vitro responses to CHA0 cells and its exoproducts included growth inhibition, encystation, paralysis, and cell lysis. By analyzing the responses of protists to bacterial supernatants obtained from different isogenic mutants whose production of one or more exometabolites was affected and also to culture extracts with antibiotic enrichment, we observed different contributions of the phenolic antifungal compound 2,4-diacetylphloroglucinol (DAPG) and the extracellular protease AprA to CHA0 toxicity for protists and to the encystation-reactivation cycle. The grazing pressure artificially produced by a mixture of the three protists in a microcosm system resulted in reduced colonization of cucumber roots by a regulatory isogenic CHA0 mutant unable to produce toxins. These results suggest that exometabolite production in biocontrol strain CHA0 may contribute to avoidance of protist grazing and help sustain higher populations in the rhizosphere, which may be a desirable and advantageous trait for competition with other bacteria for available resources.     

Plant Species, Host Age and Host Genotype Effects on Meloidogyne incognita Biocontrol by Pseudomonas fluorescens Strain CHA0 and its Genetically-Modified Derivatives

Pseudomonas fluorescens strain CHA0 and its antibiotic overproducing derivative CHA0/pME3424 repeatedly reduced Meloidogyne incognita galling on tomato, brinjal, mungbean and soya bean roots but not in chilli. An antibiotic‐deficient derivative, CHA89, did not reduce nematode invasion in any of the plant species tested. When plant species were compared, bacterial inoculants afforded better protection to tomato, mungbean and soya bean roots against root‐knot nematodes than to brinjal and chilli. Antibiotic overproducing strain CHA0/pME3424 markedly reduced fresh shoot weights of chilli and mungbean while antibiotic‐deficient strain CHA89 enhanced fresh shoot weights of mungbean. While strains CHA0 had no significant impact on fresh root weights of any of the plant species, strain CHA0/pME3424 consistently reduced fresh root weights of brinjal and mungbean. In none of the plant species the bacterial strains had an influence on protein contents of the leaves. Regardless of the plant species, the three bacterial strains did not differ markedly in their rhizosphere colonization pattern. However, colonization was highest in brinjal rhizosphere and lowest in the mungbean rhizosphere. A slight host genotype effect on the biocontrol performance of the bacterial inoculants was also detected at cultivar level. When five soya bean cultivars were compared, biocontrol bacteria exhibited best suppression of the root‐knot nematode in cv. Ajmeri. Antibiotic overproducing strain CHA0/pME3424 substantially reduced fresh shoot weights of the soya bean cultivars Centuray 84 and NARC‐I while strain CHA89 enhanced shoot weights of the cultivars Ajmeri, William‐82 and NARC‐II. Wild type strain CHA0 had no significant impact on fresh shoot weights of any of the soya bean cultivars. Strain CHA0/pME3424 reduced fresh weights of root of Century 84, NARC‐I and NARC‐II while strain CHA89 increased root weights. Bacterial rhizosphere colonization was highest in variety NARC‐I and lowest in variety Ajmeri. Plant age had a significant impact on the biocontrol performance of bacterial inoculants against nematodes. The biocontrol effect of all bacterial strains was more prominent during early growth stage (7 days after nematode inoculation). A strong negative correlation between bacterial rhizosphere colonization and nematode invasion in soya bean roots was observed.