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.     

Protozoan growth rates on secondary-metabolite-producing Pseudomonas spp. correlate with high-level protozoan taxonomy

Different features can protect bacteria against protozoan grazing, for example large size, rapid movement, and production of secondary metabolites. Most papers dealing with these matters focus on bacteria. Here, we describe protozoan features that affect their ability to grow on secondary-metabolite-producing bacteria, and examine whether different bacterial secondary metabolites affect protozoa similarly. We investigated the growth of nine different soil protozoa on six different Pseudomonas strains, including the four secondary-metabolite-producing Pseudomonas fluorescens DR54 and CHA0, Pseudomonas chlororaphis MA342 and Pseudomonas sp. DSS73, as well as the two nonproducers P. fluorescens DSM50090(T) and P. chlororaphis ATCC43928. Secondary metabolite producers affected protozoan growth differently. In particular, bacteria with extracellular secondary metabolites seemed more inhibiting than bacteria with membrane-bound metabolites. Interestingly, protozoan response seemed to correlate with high-level protozoan taxonomy, and amoeboid taxa tolerated a broader range of Pseudomonas strains than did the non-amoeboid taxa. This stresses the importance of studying both protozoan and bacterial characteristics in order to understand bacterial defence mechanisms and potentially improve survival of bacteria introduced into the environment, for example for biocontrol purposes.     

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.     

Influence of Host Plant Genotype, Presence of a Pathogen, and Coinoculation with Pseudomonas fluorescens Strains on the Rhizosphere Expression of Hydrogen Cyanide- and 2,4-Diacetylphloroglucinol Biosynthetic Genes in P. fluorescens Biocontrol Strain CHA0

The production of hydrogen cyanide (HCN) and 2,4-diacetylphloroglucinol (DAPG) is a major factor in the control of soil-borne diseases by Pseudomonas fluorescens CHA0. We investigated the impact of different biotic factors on the expression of HCN–in comparison to DAPG biosynthetic genes in the rhizosphere. To this end, the influence of plant cultivar, pathogen infection, and coinoculation with other biocontrol strains on the expression of hcnA-lacZ and phlA-lacZ fusion in strain CHA0 was monitored on the roots of bean. Interestingly, all the tested factors influenced the expression of the two biocontrol traits in a similar way. For both genes, we observed a several-fold higher expression in the rhizosphere of cv. Derakhshan compared with cvs. Goli and Naz, although bacterial rhizosphere colonization levels were similar on all cultivars tested. Root infection by Rhizoctonia solani stimulated total phlA and hcnA gene expression in the bean rhizosphere. Coinoculation of strain CHA0 with DAPG-producing P. fluorescens biocontrol strains Pf-68 and Pf-100 did neither result in a substantial alteration of hcnA nor of phlA expression in CHA0 on bean roots. To our best knowledge, this is the first study investigating the impact of biotic factors on HCN production by a bacterial biocontrol strain in the rhizosphere.     

Systemic Resistance in Tomato Induced by Biocontrol Bacteria Against the Root-Knot Nematode, Meloidogyne javanica is Independent of Salicylic Acid Production

Salicylic acid (SA)‐mediated induction of systemic resistance by Pseudomonas aeruginosa strain 7NSK2 and P. fluorescens strain CHA0 against soil‐borne fungi and viruses have been reported. The role of SA biosynthesis in the enhancement of defence mechanism against plant‐parasitic nematodes by these bacterial strains in tomato is not known. To better understand the importance of SA in rhizobacteria‐mediated suppression of root‐knot nematodes, biocontrol potential of SA‐negative or SA‐overproducing mutants against Meloidogyne javanica was evaluated with their respective wild type counter parts. Culture supernatant of 7NSK2, CHA0 and their respective mutants caused significant mortality of M. javanica juveniles in vitro. SA deletion in 7NSK2 and SA overproduction in CHA0 did not influence bacterial efficacy to cause nematode deaths. Similarly, culture supernatants resulting from King's B liquid medium amended with FeCl₃ did not influence nematicidal activity of the bacterial strains. Strain CHA0 induced juvenile deaths more than 7NSK2 did. In pot experiments, the bacterial strains applied in unsterilized sandy loam soil markedly reduced final nematode population densities in roots and subsequent root‐knot infection in tomato seedlings. SA‐negative or overproducing derivatives prevented tomato roots in kinetics similar to those with their respective wild types. When soil iron concentration was lowered by the addition of ethylenediamine di(o‐hydroxyphenylacetic acid), nematode biocontrol by the bacterial strains (both wild type and mutants) remained unaltered. To understand the mechanism involved in rhizobacteria‐mediated suppression of root‐knot nematode in tomato, bacterial performance was assessed in a split root trial in which one‐half of the root system was treated with bacterium while the other inoculated with nematode. Compared with the controls, application of the bacterial cell suspension to one‐half of the root system lowered the populations of root‐knot nematode in non‐bacterized nematode‐treated sections indicating enhanced defence in the non‐bacterized half. With respect to nematode infection, mutants induced systemic resistance to a similar extent as that caused by the wild types in both wild type tomato and NahG tomato plants. It is concluded that fluorescent pseudomonads induce systemic resistance against root‐knot nematode via a signal transduction pathway, which is independent of SA accumulation in roots.     

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.     

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.     

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.     

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.     

An insight into the feeding ecology of deep-sea canyon nematodes — Results from field observations and the first in-situC feeding experiment in the Nazaré Canyon

Submarine canyon systems provide a heterogeneous habitat for deep-sea benthos in terms of topography, hydrography, and the quality and quantity of organic matter present. Enhanced meiofauna densities as found in organically enriched canyon sediments suggest that nematodes, as the dominant metazoan meiobenthic taxon, may play an important role in the benthic food web of these sediments. Very little is known about the natural diets and trophic biology of deep-sea nematodes, but enrichment experiments can shed light on nematode feeding selectivity and trophic position. An in-situ pulse-chase experiment (Feedex) was performed in the Nazaré Canyon on the Portuguese margin in summer 2007 to study nematode feeding behaviour. ¹³C-labelled diatoms and bacteria were added to sediment cores which were then sampled over a 14-day period. There was differential uptake by the nematode community of the food sources provided, indicating selective feeding processes. ¹³C isotope results revealed that selective feeding was less pronounced at the surface, compared to the sediment subsurface. This was supported by a higher trophic diversity in surface sediments (Θ⁻¹ = 3.50 ± 0.2) compared to the subsurface (2.78 ± 0.6), implying that more food items may be used by the nematode community at the sediment surface. Predatory and scavenging nematodes contributed relatively more to biomass than other feeding types and can be seen as key contributors to the nematode food web at the canyon site. Non-selective deposit feeding nematodes were the dominant trophic group in terms of abundance and contributed substantially to total nematode biomass. The high levels of ‘fresh’ (bioavailable) organic matter input and moderate hydrodynamic disturbance of the canyon environment lead to a more complex trophic structure in canyon nematode communities than that found on the open continental slope, and favours predator/scavengers and non-selective deposit feeders.     

Feeding of the Nematode Acrobeloides nanus on Bacteria

Information on the effect of bacteria-feeding nematodes on bacterial populations in the soil is sparse. We have isolated, cultured, and microscopically examined bacteria and nematodes coexisting within an agricultural soil and have studied their feeding relationship. The bacterium Pseudomonas corrugata isolate 2140R is a biocontrol agent against the pathogenic fungus Gaeumannomyces graminis var. tritici. The nematode Acrobeloides nanus is a cosmopolitan, bacteria-feeding organism widespread in agricultural and arid soils throughout Australia. Using light and electron microscopy, we observed the ingestion and breakdown of P. corrugata in the pharynx of A. nanus and bacterial passage through the nematode intestine as well as the accumulation of fluorescent compounds from ingested and broken P. fluorescens in the lumen of the nematode''s intestine. We also observed A. nanus feeding, growing, and reproducing on the Gram-positive bacterium Clavibacter toxicus, the causative agent of the disease annual ryegrass toxicity, and detected crushed bacteria in the nematode''s intestine.     

Secondary Metabolites of <Emphasis Type="Italic">Pseudomonas fluorescens</Emphasis> CHA0 Drive Complex Non-Trophic Interactions with Bacterivorous Nematodes

Non-trophic interactions are increasingly recognised as a key parameter of predator–prey interactions. In soil, predation by bacterivorous nematodes is a major selective pressure shaping soil bacterial communities, and many bacteria have evolved defence mechanisms such as toxicity. In this study, we show that extracellular secondary metabolites produced by the model soil bacterium Pseudomonas fluorescens CHA0 function as a complex defence strategy against bacterivorous nematodes. Using a collection of functional mutants lacking genes for the biosynthesis of one or several extracellular metabolites, we evaluated the impact of bacterial secondary metabolites on the survival and chemotactic behaviour of the nematode Caenorhabditis elegans. Additionally, we followed up the stress status of the nematodes by measuring the activation of the abnormal DAuer Formation (DAF) stress cascade. All studied secondary metabolites contributed to the toxicity of the bacteria, with hydrogen cyanide efficiently repelling the nematodes, and both hydrogen cyanide and 2,4-DAPG functioning as nematicides. Moreover, these metabolites elicited the DAF stress response cascade of C. elegans, showing that they affect nematode physiology already at sublethal concentrations. The results suggest that bacterial secondary metabolites responsible for the suppression of plant pathogens strongly inhibit bacterivorous nematodes and thus likely contribute to the resistance of bacteria against predators in soil.     

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.     

Relation between nematode communities and trophic state in southern Swedish lakes

The aim of this study was to examine whether littoral nematode community patterns are shaped by lake trophic state. It was hypothesized that trophic level is associated negatively with the proportion of omnivores and positively with the percentages of bacterial feeders, but not at all with the diversity, abundance, and biomass of freshwater nematodes. Sediment samples were taken at littoral sites of eight southern Swedish lakes of different trophy in spring and autumn 2007. Trophic level was found to strongly influence species richness, as oligotrophic and mesotrophic lakes supported the greatest species numbers, whereas nematode abundance, biomass, and Shannon index were unaffected. Furthermore, our results indicated effects on the nematode community’s trophic structure, with a larger proportion of predatory nematodes in oligotrophic and mesotrophic lakes but no differences in the other feeding types (bacteria, algae and suction feeders, omnivorous species). Multivariate analysis indicated a shift in species compositions along the threshold from mesotrophic to eutrophic conditions, with the presence of Tobrilus gracilis, Monhystera paludicola, Brevitobrilus stefanskii, and Ethmolaimus pratensis related to the latter. Nematode communities in oligotrophic and mesotrophic lakes were characterized by a similar species composition, with pronounced occurrences of Eumonhystera longicaudatula, Semitobrilus cf. pellucidus, Prodesmodora circulata, and Rhabdolaimus terrestris. Overall, the results suggested that lake trophic state is a major factor structuring littoral nematode communities, although intra-lake variations might be of importance as well.     

Potential Role of Pathogen Signaling in Multitrophic Plant-Microbe Interactions Involved in Disease Protection

Multitrophic interactions mediate the ability of fungal pathogens to cause plant disease and the ability of bacterial antagonists to suppress disease. Antibiotic production by antagonists, which contributes to disease suppression, is known to be modulated by abiotic and host plant environmental conditions. Here, we demonstrate that a pathogen metabolite functions as a negative signal for bacterial antibiotic biosynthesis, which can determine the relative importance of biological control mechanisms available to antagonists and which may also influence fungus-bacterium ecological interactions. We found that production of the polyketide antibiotic 2,4-diacetylphloroglucinol (DAPG) was the primary biocontrol mechanism of Pseudomonas fluorescens strain Q2-87 against Fusarium oxysporum f. sp. radicis-lycopersici on the tomato as determined with mutational analysis. In contrast, DAPG was not important for the less-disease-suppressive strain CHA0. This was explained by differential sensitivity of the bacteria to fusaric acid, a pathogen phyto- and mycotoxin that specifically blocked DAPG biosynthesis in strain CHA0 but not in strain Q2-87. In CHA0, hydrogen cyanide, a biocide not repressed by fusaric acid, played a more important role in disease suppression.     

Impact of Biocontrol Pseudomonas fluorescens CHA0 and a Genetically Modified Derivative on the Diversity of Culturable Fungi in the Cucumber Rhizosphere

Little is known about the effects of Pseudomonas biocontrol inoculants on nontarget rhizosphere fungi. This issue was addressed using the biocontrol agent Pseudomonas fluorescens CHA0-Rif, which produces the antimicrobial polyketides 2,4-diacetylphloroglucinol (Phl) and pyoluteorin (Plt) and protects cucumber from several fungal pathogens, including Pythium spp., as well as the genetically modified derivative CHA0-Rif(pME3424). Strain CHA0-Rif(pME3424) overproduces Phl and Plt and displays improved biocontrol efficacy compared with CHA0-Rif. Cucumber was grown repeatedly in the same soil, which was left uninoculated, was inoculated with CHA0-Rif or CHA0-Rif(pME3424), or was treated with the fungicide metalaxyl (Ridomil). Treatments were applied to soil at the start of each 32-day-long cucumber growth cycle, and their effects on the diversity of the rhizosphere populations of culturable fungi were assessed at the end of the first and fifth cycles. Over 11,000 colonies were studied and assigned to 105 fungal species (plus several sterile morphotypes). The most frequently isolated fungal species (mainly belonging to the genera Paecilomyces, Phialocephala, Fusarium, Gliocladium, Penicillium, Mortierella, Verticillium, Trichoderma, Staphylotrichum, Coniothyrium, Cylindrocarpon, Myrothecium, and Monocillium) were common in the four treatments, and no fungal species was totally suppressed or found exclusively following one particular treatment. However, in each of the two growth cycles studied, significant differences were found between treatments (e.g., between the control and the other treatments and/or between the two inoculation treatments) using discriminant analysis. Despite these differences in the composition and/or relative abundance of species in the fungal community, treatments had no effect on species diversity indices, and species abundance distributions fit the truncated lognormal function in most cases. In addition, the impact of treatments at the 32-day mark of either growth cycle was smaller than the effect of growing cucumber repeatedly in the same soil.     

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.     

Non-pathogenic Fusarium solani represses the biosynthesis of nematicidal compounds in vitro and reduces the biocontrol of Meloidogyne javanica by Pseudomonas fluorescens in tomato

AIMS: The aim of the present investigation was to determine the influence of various Fusarium solani strains 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: Culture filtrates (CF) of P. fluorescens strain CHA0 and its diacetylphloroglucinol-overproducing derivative CHA0/pME3424 caused substantial mortality of M. javanica juveniles in vitro. Bacterial growth medium amended with the growth medium of F. solani repressed the nematicidal activity of the bacteria. Methanol extract of F. solani CF resulting from Czapek's Dox liquid (CDL) medium without zinc amendment repressed the nematicidal activity of the bacteria while the CF obtained from CDL medium amended with zinc did not. Conidial suspension of F. solani strain Fs5 (repressor strain for the biosynthesis of nematicidal compounds in P. fluorescens) reduced biocontrol potential of the bacterial inoculants against M. javanica in tomato while strain Fs3 (non-repressor) did not. CONCLUSIONS: Fusarium solani strains with increased nematicidal activity repress the biosynthesis of nematicidal compounds by P. fluorescens strains in vitro and greatly alter its biocontrol efficacy against root-knot nematode under natural conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: Fusarium solani strains are distributed worldwide and found in almost all the agricultural fields which suggest that some mycotoxin-producing strains will also be found in almost any soil sample taken. Besides the suppressive effect of these metabolite-producing strains on the production of nematicidal compound(s) critical in biocontrol, F. solani strains may also affect the performance of mycotoxin-sensitive biocontrol bacteria effective against plant-parasitic nematodes.     

Zinc and glycerol enhance the production of nematicidal compounds in vitro and improve the biocontrol of Meloidogyne javanica in tomato by fluorescent pseudomonads

AIMS: To assess the effects of various carbon and mineral sources on the nematicidal potential of biocontrol inoculants of Pseudomonas aeruginosa IE-6S+ and Ps. fluorescens CHA0 under laboratory and glasshouse conditions. METHODS AND RESULTS: Culture filtrates of strains IE-6S+ and CHA0, cultured in nutrient yeast extract broth, caused substantial mortality of the juveniles of Meloidogyne javanica. The nematicidal activities of the culture filtrates were altered after amendment with various carbon and mineral sources. Soil amendment with zinc alone or in combination with glycerol improved the biocontrol efficacy against root-knot nematode, promoted tomato plant growth and enhanced bacterial rhizosphere and endophytic colonization. CONCLUSIONS: Appropriate quantities of glycerol and zinc alone or in combination enhance the nematicidal activity of Ps. aeruginosa and Ps. fluorescens. Glucose reduces the activity of these bacteria against nematodes. SIGNIFICANCE AND IMPACT OF THE STUDY: Minerals and carbon sources are appealing because they are easy and economical to provide during liquid fermentation of inoculants or as fertilizer amendments to improve the biocontrol activity of indigenous and introduced bacteria.     

Decoupled responses of soil bacteria and their invertebrate consumer to warming,but not freeze–thaw cycles,in the Antarctic Dry Valleys

Altered temperature profiles resulting in increased warming and freeze–thaw cycle (FTC) frequency pose great ecological challenges to organisms in alpine and polar ecosystems. We performed a laboratory microcosm experiment to investigate how temperature variability affects soil bacterial cell numbers, and abundance and traits of soil microfauna (the microbivorous nematode Scottnema lindsayae) from McMurdo Dry Valleys, Antarctica. FTCs and constant freezing shifted nematode body size distribution towards large individuals, driven by higher mortality among smaller individuals. FTCs reduced both bacterial and nematode abundance, but bacterial cell numbers also declined under warming, demonstrating decoupled consumer–prey responses. We predict that higher occurrence of FTCs in cold ecosystems will select for large body size within soil microinvertebrates and overall reduce their abundance. In contrast, warm temperatures without FTCs could lead to divergent responses in soil bacteria and their microinvertebrate consumers, potentially affecting energy and nutrient transfer rates in soil food webs of cold ecosystems.