Direct trade-off between cyanogenesis and resistance to a fungal pathogen in lima bean (Phaseolus lunatus L.)

1.  Plants are simultaneously attacked by multiple herbivores and pathogens. While some plant defences act synergistically, others trade-off against each other. Such trade-offs among resistances to herbivores and pathogens are usually explained by the costs of resistance, i.e. resource limitations compromising a plant's overall defence.
2.  Here, we demonstrate that trade-offs can also result from direct negative interactions among defensive traits. We studied cyanogenesis (release of HCN) of lima bean (Fabaceae: Phaseolus lunatus ) and effects of this efficient anti-herbivore defence on resistance to a fungal pathogen (Melanconiaceae: Colletotrichum gloeosporioides ).
3.  Leaf tissue destruction by fungal growth was significantly higher on high cyanogenic (HC) lima bean accessions than on low cyanogenic (LC) plants. The susceptibility of HC accessions to the fungal pathogen was strongly correlated to reduced activity of resistance-associated polyphenol oxidases (PPOs) in leaves of these plants. LC accessions, in contrast, showed high PPO activity, which was correlated with distinct resistance to C. gloeosporioides .
4.  Experimentally applied, gaseous HCN reduced PPO activity and significantly increased the size of lesions caused by C. gloeosporioides in LC leaves.
5.  Field observations of a wild lima bean population in Mexico revealed a higher infection rate of HC compared to LC plant individuals. The types of lesions observed on the different cyanogenic plants in nature were similar to those observed on HC and LC plants in the laboratory.
6.   Synthesis. We suggest that cyanogenesis of lima bean directly trades off with plant defence against fungal pathogens and that the causal mechanism is the inhibition of PPOs by HCN. Our findings provide a functional explanation for the observed phenomenon of the low resistance of HC lima beans in nature.     

Cyanide Production by Rhizobacteria and Potential for Suppression of Weed Seedling Growth

Rhizobacteria strains were characterized for ability to synthesize hydrogen cyanide and for effects on seedling root growth of various plants. Approximately 32% of bacteria from a collection of over 2000 isolates were cyanogenic, evolving HCN from trace concentrations to >30 nmoles/mg cellular protein. Cyanogenesis was predominantly associated with pseudomonads and was enhanced when glycine was provided in the culture medium. Concentrations of HCN produced by rhizobacteria were similar to exogenous concentrations inhibiting seedling growth in bioassays, suggesting that cyanogenesis by rhizobacteria in the rhizosphere can adversely affect plant growth. Growth inhibition of lettuce and barnyardgrass by volatile metabolites of the cyanogenic rhizobacteria confirmed that HCN was the major inhibitory compound produced. Our results suggest that HCN produced in the rhizospheres of seedlings by selected rhizobacteria is a potential and environmentally compatible mechanism for biological control of weeds. Received: 13 December 2000/Accepted: 6 February 2001     

Effects of Proteus vulgaris growth on the establishment of a cheese microbial community and on the production of volatile aroma compounds in a model cheese

Aims:  To investigate the impact of Proteus vulgaris growth on a multispecies ecosystem and on volatile aroma compound production during cheese ripening.
Methods and Results:  The microbial community dynamics and the production of volatile aroma compounds of a nine-species cheese ecosystem were compared with or without the presence of P. vulgaris in the initial inoculum. Proteus vulgaris was able to colonize the cheese surface and it was one of the dominant species, representing 37% of total isolates at the end of ripening with counts of 9·2 log₁₀ CFU g⁻¹. In the presence of P. vulgaris , counts of Arthrobacter arilaitensis , Brevibacterium aurantiacum and Hafnia alvei significantly decreased. Proteus vulgaris influenced the production of total volatile aroma compounds with branched-chain aldehydes and their corresponding alcohols being most abundant.
Conclusions:  Proteus vulgaris was able to successfully implant itself in a complex cheese ecosystem and significantly contributed to the organoleptic properties of cheese during ripening. This bacterium also interacted negatively with other bacteria in the ecosystem studied.
Significance and Impact of the Study:  This is the first time that the impact of a Gram-negative bacterium on cheese microbial ecology and functionality has been described.     

Volatile-mediated killing of Arabidopsis thaliana by bacteria is mainly due to hydrogen cyanide

The volatile-mediated impact of bacteria on plant growth is well documented, and contrasting effects have been reported ranging from 6-fold plant promotion to plant killing. However, very little is known about the identity of the compounds responsible for these effects or the mechanisms involved in plant growth alteration. We hypothesized that hydrogen cyanide (HCN) is a major factor accounting for the observed volatile-mediated toxicity of some strains. Using a collection of environmental and clinical strains differing in cyanogenesis, as well as a defined HCN-negative mutant, we demonstrate that bacterial HCN accounts to a significant extent for the deleterious effects observed when growing Arabidopsis thaliana in the presence of certain bacterial volatiles. The environmental strain Pseudomonas aeruginosa PUPa3 was less cyanogenic and less plant growth inhibiting than the clinical strain P. aeruginosa PAO1. Quorum-sensing deficient mutants of C. violaceum CV0, P. aeruginosa PAO1, and P. aeruginosa PUPa3 showed not only diminished HCN production but also strongly reduced volatile-mediated phytotoxicity. The double treatment of providing plants with reactive oxygen species scavenging compounds and overexpressing the alternative oxidase AOX1a led to a significant reduction of volatile-mediated toxicity. This indicates that oxidative stress is a key process in the physiological changes leading to plant death upon exposure to toxic bacterial volatiles.     

Methylobacterium sp. resides in unculturable state in potato tissues in vitro and becomes culturable after induction by Pseudomonas fluorescens IMGB163

Aims:  To induce growth of endophytic bacteria residing in an unculturable state in tissues of in vitro -grown potato plantlets. To isolate and identify the induced bacteria and to localize the strains in tissues of in vitro -grown potato plantlets.
Methods and Results:  The inoculation of in vitro -grown potato plants with Pseudomonas fluorescens IMBG163 led to induction of another bacterium, a pink-pigmented facultative methylotroph that was identified as Methylobacterium sp . using phylogenetic 16S rDNA approach . Two molecular methods were used for localizing methylobacteria in potato plantlets: PCR and in situ hybridization (ISH/FISH). A PCR product specific for the Methylobacterium genus was found in DNA isolated from the surface-sterilized plantlet leaves. Presence of Methylobacterium rRNA was detected by ISH/FISH in leaves and stems of inoculated as well as axenic potato plantlets although the bacterium cannot be isolated from the axenic plants.
Conclusion:  Methylobacterium sp. resides in unculturable state within tissues of in vitro -grown potato plants and becomes culturable after inoculation with P. fluorescens IMBG163.
Significance and Impact of the Study:  In order to develop endophytic biofertilizers and biocontrol agents, a detailed knowledge of the life-style of endophytes is essential. To our knowledge, this is the first report on increase of the culturability of endophytes in response to inoculation by nonpathogenic bacteria.     

Comparative genomics-guided loop-mediated isothermal amplification for characterization of Pseudomonas syringae pv. phaseolicola

Aims:  To design and evaluate a loop-mediated isothermal amplification (LAMP) protocol by combining comparative genomics and bioinformatics for characterization of Pseudomonas syringae pv. phaseolicola (PSP), the causal agent of halo blight disease of bean ( Phaseolus vulgaris L.).
Methods and Results:  Genomic sequences of Pseudomonas syringae pathovars, P. fluorescens and P. aeruginosa were analysed using multiple sequence alignment. A pathovar-specific region encoding pathogenicity-related secondary metabolites in the PSP genome was targeted for developing a LAMP assay. The final assay targeted a polyketide synthase gene, and readily differentiated PSP strains from other Pseudomonas syringae pathovars and other Pseudomonas species, as well as other plant pathogenic bacteria, e.g. species of Pectobacterium , Erwinia and Pantoea .
Conclusion:  A LAMP assay has been developed for rapid and specific characterization and identification of PSP from other pathovars of P. syringae and other plant-associated bacteria .
Significance and Impact of the Study:  This paper describes an approach combining a bioinformatic data mining strategy and comparative genomics with the LAMP technology for characterization and identification of a plant pathogenic bacterium. The LAMP assay could serve as a rapid protocol for microbial identification and detection with significant applications in agriculture and environmental sciences.     

Adaptation to flavomycin in the ruminal bacterium, Prevotella bryantii

Aims:  The recent EU ban of growth-promoting antibiotics in animal production was based on fears concerning antibiotic resistance being transmitted to human pathogens. This paper explores the adaptation mechanism of a common ruminal bacterium, Prevotella bryantii , to one of the banned compounds, flavomycin (flavophospholipol).
Methods and Results:  Growth in the presence of flavomycin (2 and 20  μ g ml⁻¹) was characterized by a concentration-dependent increase in the length of the lag phase, which decreased after previous flavomycin exposure. From growth patterns on solid medium, decreased sensitivity appeared to be due to a whole-population adaptation. Proteomic analysis indicated upregulation of three native proteins occurred following flavomycin adaptation. Further analysis of two of these proteins resulted in no database matches, suggesting that they may be species-specific. Flavomycin adaptation also resulted in co-adaptation to bacitracin and vancomycin.
Conclusions:  Adaptation of P. bryantii to flavomycin, which also resulted in co-adaptation to bacitracin and vancomycin, may involve an increased availability of undecaprenyl pyrophosphate.
Significance and Impact of the Study:  The use of flavomycin, and similar growth-promoting antibiotics, in animal production may prompt adaptive responses in ruminal bacteria which can significantly change their antibiotic sensitivity.     

Mechanism of insoluble phosphate solubilization by Pseudomonas fluorescens RAF15 isolated from ginseng rhizosphere and its plant growth-promoting activities

Aims:  To investigate the mechanism of insoluble phosphate (P) solubilization and plant growth-promoting activity by Pseudomonas fluorescens RAF15.
Methods and Results:  We investigated the ability of Ps. fluorescens RAF15 to solubilize insoluble P via two possible mechanisms: proton excretion by ammonium assimilation and organic acid production. There were no clear differences in pH and P solubilization between glucose-ammonium and glucose-nitrate media. P solubilization was significantly promoted with glucose compared to fructose. Regardless of nitrogen sources used, Ps. fluorescens RAF15 solubilized little insoluble P with fructose. High performance liquid chromatography analysis showed that Ps. fluorescens RAF15 produced mainly gluconic and tartaric acids with small amounts of 2-ketogluconic, formic and acetic acids. During the culture, the pH was reduced with increase in gluconic acid concentration and was inversely correlated with soluble P concentration. Ps. fluorescens RAF1 showed the properties related to plant growth promotion: pectinase, protease, lipase, siderophore, hydrogen cyanide, and indoleacetic acid.
Conclusion:  This study indicated that the P solubility was directly correlated with the organic acids produced.
Significance and Impact of the Study:  Pseudomonas fluorescens RAF15 possessed different traits related to plant growth promotion. Therefore, Ps. fluorescens RAF15 could be a potential candidate for the development of biofertilizer or biocontrol agent.     

Pseudomonas corrugata: plant pathogen and/or biological resource?

Pseudomonas corrugata is the causal agent of tomato pith necrosis yet it is also used in the biological control of plant pathogenic bacteria and fungi. Potentially it could be used in other fields, such as the production of commercial biomolecules with a wide range of application and including bioremediation. This review reports the multiple characteristics of the bacterium, highlights its known molecular features and speculates on the possible underlying mechanisms of action.
Taxonomy:  Bacteria; Proteobacteria ; γ subdivision; order Pseudomonadales ; family Pseudomonadaceae ; genus Pseudomonas .
Microbiological properties:  Gram-negative, oxidase-positive, non-spore forming rods; non-fluorescent on King's B medium; produces wrinkled and rarely smooth colonies on yeast peptone glucose agar or nutrient dextrose agar; yellow to brown diffusible pigments are frequently produced.
Disease symptoms:  The typical symptom on tomato is necrosis and/or hollowing of the pith of the stem; the syndrome determines loss of turgidity of the plant, hydropic/necrotic areas and long conspicuous adventitious roots on the stem.
Biological control agent: In vitro assessed against plant pathogenic fungi and bacteria, as well as the phytotoxin indicator microorganims Rhodotorula pilimanae and Bacillus megaterium ; in vivo used against pre- and post-harvest plant pathogens.     

Modulation of the bacterial response to spectral solar radiation by algae and limiting nutrients

SUMMARY 1. The response of bacterial production (measured as [³H]TdR incorporation rate) to spectral solar radiation was quantified experimentally in an oligotrophic high-mountain lake over 2 years. Bacterial responses were consistent: ultraviolet-B (UVB) was harmful, whereas ultraviolet-A (UVA) + photosynthetically active radiation (PAR) and PAR enhanced bacterial activity. Full sunlight exerted a net stimulatory effect on bacterial activity in mid-summer but a net inhibitory effect towards the end of the ice-free period.
2. Experiments were undertaken to examine whether the bacterial response pattern depended on the presence of algae and/or was modulated by the availability of a limiting inorganic nutrient (phosphorus, P). In the absence of algae, [³H]TdR incorporation rates were significantly lower than when algae were present under all light treatments, and the consistent bacterial response was lost. This suggests that the bacterial response to spectral solar radiation depends on fresh-C released from algae, which determines the net stimulatory outcome of damage and repair in mid-summer.
3. In the absence of algae, UVB radiation inhibited bacteria when they were strongly P-deficient (mean values of N : P ratio: 46.1), whereas it exerted no direct effect on bacterial activity when they were not P-limited.
4. P-enrichment of lake water markedly altered the response of bacteria to spectral solar radiation at the end of ice-free period, when bacteria were strongly P-deficient. Phosphorus enrichment suppressed the inhibitory effect of full sunlight that was observed in October, both in whole lake water (i.e. including algae) and in the absence of algae. This indicates that the bacterial P-deficiency, measured as the cellular N : P ratio, was partly responsible for the net inhibitory effect of full sunlight, implying a high bacterial vulnerability to UVB.     

Hydrogen cyanide production by Pseudomonas aeruginosa at reduced oxygen levels

Hydrogen cyanide production by Pseudomonas aeruginosa growing in a synthetic medium required aerobosis but operated efficiently at low dissolved oxygen concentration. Half maximum levels of cyanogenesis occurred at 0.015 microM oxygen; maximum cyanogenesis occurred over a wide range, 0.1-180 microM, of oxygen concentrations. These cells lost the ability to produce cyanide upon aerobic incubation in the absence of both the carbon energy source (L-glutamate) and the metabolic precursor of hydrogen cyanide (glycine). This loss of cyanogenesis was dependent on oxygen concentration; 1.0 microM oxygen produced no detectable loss, whereas 180 microM oxygen caused a rapid decline in cyanogenic ability. The endogenous cyanide production rate of cells in the presence of carbon energy source was not significantly influenced by oxygen concentration. During the batch culture cycle, the acquisition of the ability to produce HCN was preceded by oxygen reduction to growth-limiting levels. Cells which had lost the ability to produce hydrogen cyanide by oxygen treatment required protein synthesis before they could again become cyanogenic.     

Quantification of Pseudomonas aeruginosa hydrogen cyanide production by a polarographic approach

Pseudomonas aeruginosa is an opportunistic pathogen responsible for numerous infections acquired in hospital especially in persons whose immune systems are weakened, such as with patient suffering from AIDS or cystic fibrosis. This bacterium produces a great diversity of virulence factors among them hydrogen cyanide (HCN) which is one of the most potent and toxic. A precise quantification of HCN or CN(-) ion is essential to understand the involvement of this toxin in the pathogenesis of P. aeruginosa. In the present study, we present a new technique based on a polarographic approach to measure the production kinetics of HCN/CN(-) by P. aeruginosa strains, in several media commonly used in microbiology labs. The method was validated using mutants (hcnB- and hcnC-) which are unable to produce detectable HCN/CN(-). The kinetics of HCN/CN(-) production by P. aeruginosa in Luria Bertani (LB) medium showed a parabolic shape with a peak observed at 4, 5 and 8h for strains PA14, PAO1 and MPAO1, respectively. When bacteria were grown in ordinary nutrient broth (ONB) 2.5% medium, a less adapted medium for bacterial growth, the general profile of the kinetics was conserved but peak production was delayed (10 and 12h for PAO1 and MPAO1, respectively). When the bacteria were cultured in minimum medium MMC, bacterial growth was particularly slow and HCN/CN(-) production was markedly reduced. Taken together, this new polarographic method appears as a useful technique to detect and quantify HCN/CN(-) in routine media where the bacteria can express and regulate high amounts of toxins. With this method, we demonstrate that HCN/CN(-) production by P. aeruginosa is maximal at the end of the exponential growth phase and depends on the richness of the growth medium used.     

Paenibacillus polymyxa antagonizes oomycete plant pathogens Phytophthora palmivora and Pythium aphanidermatum

Aim:  To find sustainable alternatives to the application of synthetic chemicals for oomycete pathogen suppression.
Methods and Results:  Here, we present experiments on an Arabidopsis thaliana model system in which we studied the antagonistic properties of rhizobacterium Paenibacillus polymyxa strains towards the oomycete plant pathogens Phytophthora palmivora and Pythium aphanidermatum . We carried out studies on agar plates, in liquid media and in soil. Our results indicate that P. polymyxa strains significantly reduced P. aphanidermatum and P. palmivora colonization in liquid assays. Most plants that had been treated with P. polymyxa survived the P. aphanidermatum inoculations in soil assays.
Conclusions:  The antagonistic abilities of both systems correlated well with mycoidal substance production and not with the production of antagonistic substances from the biocontrol bacteria.
Significance and Impact of the Study:  Our experiments highlight the need to take biofilm formation and niche exclusion mechanisms into consideration for biocontrol assays performed under natural conditions.     

Isolation and activity of Xenorhabdus antimicrobial compounds against the plant pathogens Erwinia amylovora and Phytophthora nicotianae

Aims:  Broad-spectrum antibiotics produced by symbiotic bacteria [entomopathogenic bacterium (EPB)] of entomopathogenic nematodes keep monoxenic conditions in insect cadavers in soil. This study evaluated antibiotics produced by EPB for their potential to control plant pathogenic bacteria and oomycetes.
Methods and Results:  Entomopathogenic bacterium produce antibiotics effective against the fire blight bacterium Erwinia amylovora, including streptomycin resistant strains, and were as effective in phytotron experiments as kasugamycin or streptomycin. Xenorhabdus budapestensis and X . szentirmaii antibiotics inhibited colony formation and mycelial growth of Phytophthora nicotianae. From X . budapestensis, an arginine-rich fraction (bicornutin) was adsorbed by Amberlite^® XAD 1180, and eluted with methanol : 1  n HCI (99 : 1). Bicornutin inactivated zoospores, and inhibited germination and colony formation of cystospores at <<25 ppm. An UV-active molecule (bicornutin-A, MW = 826), separated by HPLC and thin-layer chromatography, was identified as a novel hexa-peptide : RLRRRX.
Conclusions:  Xenorhabdus budapestensis produces metabolites with strong antibacterial and cytotoxic activity. Individual compounds can be isolated, identified and patented, but their full antimicrobial potential may be multiplied by synergic interactions.
Significance and Impact of the Study:  Active compounds of two new Xenorhabdus species might control plant diseases caused by pathogens of great importance to agriculture such as Erw. amylovora and P . nicotianae .     

Isolation and characterization of plant growth-promoting strain Pantoea NII-186. From Western Ghat Forest soil, India

Aims:  To isolate plant growth-promoting bacterium from Western Ghat forests in India.
Methods and Results:  A Gram-negative, rod shaped, cream white coloured strain Pantoea NII-186 isolated from Western Ghat soil sample. The taxonomic position of the bacterium was confirmed by sequencing of 16S rRNA and phylogenetic analysis. A strain grew at a wide range of temperature ranging from 5–40°C, but optimum growth was observed at 28–30°C. It showed multiple plant growth-promoting attributes such as phosphate solubilization activity, indole acetic acid (IAA) production, siderophore production and HCN production. It was able to solubilize (28 μg of Ca₃PO₄ ml⁻¹ day⁻¹), and produce IAA (59 μg) at 28°C. The solubilization of insoluble phosphate was associates with a drop in the pH of the culture medium. Pantoea sp. NII-186 tolerate to different environmental stresses like 5–40°C, 0–7% salt concentration and 4–12 pH range.
Conclusions:  The 16S rRNA gene sequence confirmed that the isolate NII-186 was belongs to Pantoea genus and showed considerable differences in physiological properties with previously reported species of this genus. Isolate NII-186 possessed multiple attributes of plant growth-promoting activity.
Significance and Impact of the Study:  Hence in the context it is proposed that Pantoea sp. NII-186, could be deployed as an inoculant to attain the desired plant growth-promoting activity in agricultural environment.     

Monalysin, a novel ß-pore-forming toxin from the Drosophila pathogen Pseudomonas entomophila, contributes to host intestinal damage and lethality

Pseudomonas entomophila is an entomopathogenic bacterium that infects and kills Drosophila. P. entomophila pathogenicity is linked to its ability to cause irreversible damages to the Drosophila gut, preventing epithelium renewal and repair. Here we report the identification of a novel pore-forming toxin (PFT), Monalysin, which contributes to the virulence of P. entomophila against Drosophila. Our data show that Monalysin requires N-terminal cleavage to become fully active, forms oligomers in vitro, and induces pore-formation in artificial lipid membranes. The prediction of the secondary structure of the membrane-spanning domain indicates that Monalysin is a PFT of the ?-type. The expression of Monalysin is regulated by both the GacS/GacA two-component system and the Pvf regulator, two signaling systems that control P. entomophila pathogenicity. In addition, AprA, a metallo-protease secreted by P. entomophila, can induce the rapid cleavage of pro-Monalysin into its active form. Reduced cell death is observed upon infection with a mutant deficient in Monalysin production showing that Monalysin plays a role in P. entomophila ability to induce intestinal cell damages, which is consistent with its activity as a PFT. Our study together with the well-established action of Bacillus thuringiensis Cry toxins suggests that production of PFTs is a common strategy of entomopathogens to disrupt insect gut homeostasis.     

Bioaccumulation of Amylose-Like Glycans by Helicobacter pylori

Background:  Helicobacter pylori cell surface is composed of lipopolysaccharides (LPSs) yielding structures homologous to mammalian Lewis O -chains blood group antigens. These structures are key mediators in the definition of host-microbial interactions and known to change their expression pattern in response to environmental pressure.
Aims:  The present work is focused on the identification of new H. pylori cell-surface glycosides. Special attention is further devoted to provide insights on the impact of in vitro subcultivation on H. pylori cell-surface phenotypes.
Methods:  Cell-surface glycans from H. pylori NCTC 11637 and two clinical isolates were recovered from the aqueous phase resulting from phenol:water extraction of intact bacteria. They were evaluated in relation to their sugars and glycosidic-linkages composition by CG-MS, size-exclusion chromatography, NMR, and Mass Spectrometry. H. pylori glycan profile was also monitored during subcultivation in vitro in agar and F12 liquid medium.
Results:  All three studied strains produce LPS expressing Lewis epitopes and express bioaccumulate amylose-like glycans. Bioaccumulation of amylose was found to be enhanced with the subcultivation of the bacterium on agar medium and accompanied by a decrease in the expression of LPS O -chains. In contrast, during exponential growth in F12 liquid medium, an opposite behavior is observed, that is, there is an increase in the overall amount of LPS and decrease in amylose content.
Conclusions:  This work shows that under specific environmental conditions, H. pylori expresses a phase-variable cell-surface α-(1→4)-glucose moiety.