Mechanisms involved in biocontrol by microbial inoculants

Biological control offers alternative environmentally friendly strategies for the control of phytopathogens in agriculture and horticulture. Biocontrol metabolites are designed so that they do not have any adverse effects on host plants or on indigenous microflora and, in addition, resistance to these metabolites does not appear to develop. As promising alternatives to chemical pesticides, some biocontrol agents have been found to produce a variety of antifungal secondary metabolites and lytic enzymes. The 2,4-diacetylphloroglucinol is a secondary metabolite produced by Pseudomonas fluorescens F113, a strain capable of protecting sugar beet against the causal agent of ’damping off’, Pythium ultimum; environmental and genetic factors involved in 2,4-diacetylphloroglucinol production are discussed. Stenotrophomonas maltophilia strain W81 (P) produces chitinase and protease enzymes and is capable of conferring plant protection against the disease-causing activity of Pythium ultimum in vitro; transposon mutagenesis and subsequent in vivo assays have demonstrated that the biocontrol ability of W81 (P) is mediated by lytic enzyme production

Biological control of phytopathogens by phloroglucinol and hydrolytic enzyme producing bacterial inoculants

Envionmental and consumer concerns have focused interest on the development of biological control as an alternative, environmentally-friendly strategy for the protection of agricultural and horticultural crops against phytopathogens. Biological control agents. producing a variety of secondary metabolites and hydrolytic enzymes, have been identified among fungi, actinomyceles and bacteria. Pseudomonas fluorescens FII3 and Stenotrophomonas maltophilia W81 inhibit growth of the phytopathogenic fungus Pythium ultimum in vitro and are capable of protecting sugar beet against the effects of damping-off under soil conditions. Transposon mutagenesis of strains FIl3 and W81 has demonstrated that the biocontrol abilities of these strains are mediated by 2,4-diacetylphloroglucinol (pm...) or lytic enzyme production, respectively. Globodera roslochiensis is a phytopathogenic cyst neml1.tode of major agronomic importance. Purified PHL, lytic enzymes, and chitinolytic or phloroglucinol-producing bacterial inoculants negatively influence hatch of G. rostochiensis eggs and decrease subsequent viability of juvenile cyst nematodes in vitro. Similar results were obtained under soil conditions

The influence of in-egg mortality and spontaneous hatching on the decline of Globodera rostochiensis during crop rotation in the absence of the host potato crop in the field

Summary - The decline of Globodera rostochiensis (Woll.) popUlations in two fields was 57% during the first and 40.3% during the second year of crop rotation in the absence of a potato crop in Co. Cork. Ireland. The decline was found to be due to both spontaneous hatch and in-egg mortality. Spontaneous hatch accounted for between 75.8 and 80.2% of the decline experienced during rotation. In egg mortality was found to be responsible for a reduction of approximately 10% in the total number of viable eggs in both fields, and was positively correlated with soil temperture in the field. The number of culturable bacteria and the composition of the bacterial microflora in the cysts was found to change with increasing soil temperatures. The increase in cellulase-positive isolates reflected the effect of temperature on egg degradation

Quantitative Real Time PCR analysis confirms the influence of PA2206 on gene expression linked to the oxidative stress response.

<p>Bacterial cultures were grown to exponential phase, and aliquots were then either untreated or exposed to a 1 mM concentration of H₂O₂ for 10 mins. Expression of <i>PA3032</i>, <i>PA2821</i>, <i>PA0729.1</i> and <i>PA5129</i> was measured as fold change relative to the <i>proC</i> housekeeping gene. Mean values are represented ± standard error. Consistent with the array data, expression of <i>PA3032</i> and <i>PA2821</i> was increased in the <i>PA2206⁻</i> mutant (white bar) compared to <i>PA2206^C</i> (striped bar) and the wild-type (black bar) strain. Similarly, expression of <i>PA0729.1</i> and <i>PA5129</i> was approximately 3-fold less in the <i>PA2206⁻</i> mutant strain (p-value of <0.05 by student's ttest).</p

<i>PA2214-15</i> is under the direct transcriptional control of the PA2206 LysR regulator.

<p>(<b>A</b>) Comparative genomic analysis of the metabolic-centric <i>PA2206</i> region in <i>P. aeruginosa</i> PAO1, <i>P. fluorescens</i> Pf-5, and <i>P. fluorescens</i> Pf-O1 based on the Pseudomonas Genome Database. Single genes and operons are denoted by colour and pattern fill while homology is denoted by connecting shaded regions. <i>P. fluorescens</i> encodes a <i>PA2206</i> homologue, which is adjacent to truncated genes corresponding to fragments of <i>PA2207</i> and <i>PA2212</i>, both of which are downstream of a conserved <i>PA2214-2216</i> homologous operon. TTT denotes a tripartite tricarboxylate transport system, FAH denotes a putative fumarylacetoacetate hydrolase activity, while Aldedh denotes a putative aldehyde dehydrogenase activity. (<b>B</b>) Promoter-fusion analysis of the <i>PA2214</i> upstream region revealed significantly increased promoter activity in the <i>PA2206^C</i> strain relative to <i>PA2206⁻</i>. The increase in promoter activity was consistently observed in three independent experiments consisting of three biological replicates (** p-value<0.01 by student's ttest). (<b>C</b>) <i>PA2214-lacZ</i> promoter fusion analysis performed in <i>E. coli</i> harbouring a <i>PA2206</i>-pBBR1MCS5 construct compared to vector control revealed direct regulation. Promoter activity was below baseline in the vector control (compared to empty pBBR1MCS5 plasmid), while an average of 6 Miller Units was consistently detected in the overexpressing strain (** p-value of <0.01 by student's ttest).</p

Susceptibility of wild-type, <i>PA2206⁻</i> and <i>PA2206</i>^C strains to oxidative stress.

<p>Mean values are represented ± standard deviation.</p

Bacterial strains and plasmids used in this study.

<p>Bacterial strains and plasmids used in this study.</p

PA2206 binds a LysR-box overlapping the predicted −35 box in the <i>PA2214</i> promoter.

<p>(<b>A</b>) EMSA analysis of the <i>PA2214-15</i>, <i>PA2206</i> and <i>PA2216</i> promoter fragments revealed <i>PA2214-15</i> to be a direct PA2206 target. The protein concentration is marked below each lane while DNA promoter fragments were used at 15 fmoles. Three complexes were observed upon protein interaction with the <i>PA2214-15</i> (1–3) promoter fragment at low nanomolar protein concentrations, with the C2/C3 complexes predominant at higher concentrations of PA2206 protein. The <i>PA2206</i> (4–6) and <i>PA2216</i> (7–9) promoter fragments did not form a complex with the PA2206 protein. (<b>B</b>) Schematic diagram of the location and arrangement of the LysR boxes identified upstream of the predicted transcriptional start site of <i>PA2214</i>. The primer positions for each of the truncated promoter fragments are outlined below and aligned with the LysR boxes contained within each amplicon. (<b>C</b>) Mobility shift analysis of the PA2206 protein interaction with the <i>PA2214</i> promoter region. Protein was added to each reaction at 300 nM and DNA promoter fragments were used at 15 fmoles. The strong shift in lane 4 confirms that PA2206 binds to LysR box 3 overlapping the p<i>PA2214</i> predicted −35 region and that this region is sufficient for the interaction to occur. PA2206 protein did not cause a shift for any of the other truncated promoter fragments.</p

PA2206 is induced in response to H₂O₂ and is required for lethality in a zebrafish embryo model of infection.

<p>(<b>A</b>) Bacterial cultures were grown to exponential phase, and aliquots were then either untreated or exposed to 1 mM, 5 mM and 10 mM concentrations of H₂O₂. <i>PA2206</i> gene expression was measured as fold change relative to the <i>proC</i> housekeeping gene and subsequently calculated as fold change relative to the untreated sample, following 10, 20, 30, and 60 minute periods of exposure to H₂O₂. Mean values are represented ± standard error (** p-value≤0.005, *** p-value≤0.001 calculated using one-way ANOVA). (<b>B</b>) Kaplan-Meier survival curve. Twenty-six hours Zebrafish embryos were injected with ∼300 colony forming units of either mPAO1 wild-type or <i>PA2206⁻</i> mutant into their blood island. Embryos were monitored for survival daily. Mean values of three biological experiments (each 10 embryos) are shown. Statistical analysis showed that at 2 and 3 days post infection, mPAO1 wild-type killed significantly more embryos compared to the <i>PA2206⁻</i> mutant (p-value = 0.0159 using the log-rank test).</p

PA2206 has a global regulatory role in <i>P. aeruginosa</i>.

<p>(<b>A</b>) BioPerl analysis revealed the presence of the putative PA2206 consensus sequence in the promoters of the <i>pvdS</i> and <i>PA4881</i> genes. The arrangement and sequence similarity of the consensus sequence was similar to that of the <i>pcaQ</i> LTTR from <i>S. meliloti</i>. (<b>B</b>) (i) EMSA analysis of the <i>pvdS</i> promoter region with the PA2206 protein revealed an interaction at nanomolar concentrations. Concentrations as low as 800 nM were able to shift the <i>pvdS</i> promoter. (ii) The absence of binding to the <i>PA0982</i> promoter probe indicates the requirement for specificity in the predicted consensus sequence. (iii) No interaction was observed between PA2206 and a truncated <i>pvdS</i> promoter fragment located downstream of the putative LysR box suggesting that this sequence alone is sufficient for the protein-DNA interaction to occur.</p