Phenazine-1-carboxamide production in the biocontrol strain Pseudomonas chlororaphis PCL1391 is regulated by multiple factors secreted into the growth medium

Pseudomonas chlororaphis PCL1391 controls tomato foot and root rot caused by Fusarium oxysporum f. sp. radicis-lycopersici. The production of phenazine-1-carboxamide (PCN) is crucial for this biocontrol activity. In vitro production of PCN is observed only at high-population densities, suggesting that production is under the regulation of quorum sensing. The main autoinducer molecule produced by PCL1391 was identified structurally as N-hexanoyl-L-homoserine lactone (C6-HSL). The two other autoinducers that were produced comigrate with N-butanoyl-L-homoserine lactone (C4-HSL) and N-octanoyl-L-homoserine lactone (C8-HSL). Two PCL1391 mutants lacking production of PCN were defective in the genes phzI and phzR, respectively, the nucleotide sequences of which were determined completely. Production of PCN by the phzI mutant could be complemented by the addition of exogenous synthetic C6-HSL, but not by C4-HSL, C8-HSL, or any other HSL tested. Expression analyses of Tn5luxAB reporter strains of phzI, phzR, and the phz biosynthetic operon clearly showed that phzI expression and PCN production is regulated by C6-HSL in a population density-dependent manner. The introduction of multiple copies of the regulatory genes phzI and phzR on various plasmids resulted in an increase of the production of HSLs, expression of the PCN biosynthetic operon, and consequently, PCN production, up to a sixfold increase in a copy-dependent manner. Surprisingly, our expression studies show that an additional, yet unidentified factor(s), which are neither PCN nor C4-HSL or C8-HSL, secreted into the growth medium of the overnight cultures, is involved in the positive regulation of phzI, and is able to induce PCN biosynthesis at low cell densities in a growing culture, resulting in an increase of PCN production.     

Production of the antifungal compounds phenazine and pyrrolnitrin from Pseudomonas chlororaphis O6 is differentially regulated by glucose

Aims: To determine whether glucose in growth medium affects secondary metabolite production and biocontrol efficacy of Pseudomonas chlororaphis O6. Methods and Results: The secondary metabolites pyrrolnitrin and phenazines antagonize phytopathogenic fungi. The expression of the prnA gene encoding tryptophan halogenase, the first step in pyrrolnitrin biosynthesis, required the stationary‐phase sigma factor, RpoS. Mutations in rpoS and prnA in Ps. chlororaphis O6 eliminated antifungal activity against Rhizoctonia solani and Fusarium graminearum. Pyrrolnitrin production was reduced by glucose in growth media, whereas phenazine levels were increased. The efficacy of Ps. chlororaphis O6 in the biocontrol of tomato late blight was reduced by addition of glucose to the growth medium. Conclusions: Regulation by glucose of pyrrolnitrin production influenced the efficacy of the biocontrol of tomato leaf blight. Significance and Impact of the Study: The nutritional regulation of secondary metabolite production from a soil pseudomonad may account, at least in part, for the variability of biocontrol under field conditions.     

Interactions in the tomato rhizosphere of two Pseudomonas biocontrol strains with the phytopathogenic fungus Fusarium oxysporum f. sp. radicis-lycopersici

The fungus Fusarium oxysporum f. sp. radicis-lycopersici causes foot and root rot of tomato plants, which can be controlled by the bacteria Pseudomonas fluorescens WCS365 and P. chlororaphis PCL1391. Induced systemic resistance is thought to be involved in biocontrol by P. fluorescens WCS365. The antifungal metabolite phenazine-1-carboxamide (PCN), as well as efficient root colonization, are essential in the mechanism of biocontrol by P. chlororaphis PCL1391. To understand the effects of bacterial strains WCS365 and PCL1391 on the fungus in the tomato rhizosphere, microscopic analyses were performed using different autofluorescent proteins as markers. Tomato seedlings were inoculated with biocontrol bacteria and planted in an F. oxysporum f. sp. radicis-lycopersici-infested gnotobiotic sand system. Confocal laser scanning microscope analyses of the interactions in the tomato rhizosphere revealed that i) the microbes effectively compete for the same niche, and presumably also for root exudate nutrients; ii) the presence of either of the two bacteria negatively affects infection of the tomato root by the fungus; iii) both biocontrol bacteria colonize the hyphae extensively, which may represent a new mechanism in biocontrol by these pseudomonads; and iv) the production of PCN by P. chlororaphis PCL1391 negatively affects hyphal growth and branching, which presumably affects the colonization and infecting ability of the fungus.     

Intracellular ATP levels affect secondary metabolite production in Streptomyces spp

The addition of extracellular ATP (exATP) to four Streptomyces strains had similar effects: low exATP levels stimulated antibiotic production and high levels reduced it. Compared with antibiotic production, the concentrations of intracellular ATP (inATP) in the tested strains were opposite, which suggests a role of inATP in regulating secondary metabolite production. Under inactivation of the polyphosphate kinase gene (ppk) in Streptomyces lividans, we observed the same results: when the inATP level in the mutant strain was lower than in the parent strain, more antibiotic was produced. Combining all the results, a strong inverse relationship between [inATP] and the secondary metabolite production is suggested by this study.     

Intracellular ATP Levels Affect Secondary Metabolite Production in Streptomyces spp.

The addition of extracellular ATP (exATP) to four Streptomyces strains had similar effects: low exATP levels stimulated antibiotic production and high levels reduced it. Compared with antibiotic production, the concentrations of intracellular ATP (inATP) in the tested strains were opposite, which suggests a role of inATP in regulating secondary metabolite production. Under inactivation of the polyphosphate kinase gene (ppk) in Streptomyces lividans, we observed the same results: when the inATP level in the mutant strain was lower than in the parent strain, more antibiotic was produced. Combining all the results, a strong inverse relationship between [inATP] and the secondary metabolite production is suggested by this study.     

THE TOXIC CYANOBACTERIUM NOSTOC SP. STRAIN 152 PRODUCES HIGHEST AMOUNTS OF MICROCYSTIN AND NOSTOPHYCIN UNDER STRESS CONDITIONS

The understanding of how environmental factors regulate toxic secondary metabolite production in cyanobacteria is important to guarantee water quality. Very little is known on the regulation of toxic secondary metabolite production in benthic cyanobacteria. In this study the physiological regulation of the production of the toxic heptapeptide microcystin (MC) and the non-toxic related peptide nostophycin (NP) in the benthic cyanobacterium Nostoc sp. strain 152 was studied under contrasting environmental conditions. I used a 2(k) levels factorial design, where k is the number of four factors that have been tested: Reduction in temperature (20 vs. 12°C), irradiance (50 vs. 1 μmol · m(-2) · s(-1)), P-PO(4) (144 vs. 0.14 μM P-PO(4)), N-NO(3) (5.88 mM vs. N-NO(3) free). While the growth rate was reduced more than hundred fold under most severe conditions of temperature, irradiance, and phosphate reduction the production of MC and NP never ceased. The MC and NP contents per cell varied at maximum 5- and 10.6-fold each, however the physiological variation did not outweigh the highly significant linear relationship between the daily cell division rate and the MC and NP net production rates. Surprisingly the MC and NP contents per cell showed a maximum under P-PO(4) reduced and irradiance reduced conditions. Both intra- and extracellular MC and NP concentrations were negatively related to P-PO(4) and irradiance. It is concluded that the proximate factor behind maximal cellular MC and NP contents is physiological stress.     

Influence of abiotic stress signals on secondary metabolites in plants

Plant secondary metabolites are unique sources for pharmaceuticals, food additives, flavors, and industrially important biochemicals. Accumulation of such metabolites often occurs in plants subjected to stresses including various elicitors or signal molecules. Secondary metabolites play a major role in the adaptation of plants to the environment and in overcoming stress conditions. Environmental factors viz. temperature, humidity, light intensity, the supply of water, minerals, and CO₂ influence the growth of a plant and secondary metabolite production. Drought, high salinity, and freezing temperatures are environmental conditions that cause adverse effects on the growth of plants and the productivity of crops. Plant cell culture technologies have been effective tools for both studying and producing plant secondary metabolites under in vitro conditions and for plant improvement. This brief review summarizes the influence of different abiotic factors include salt, drought, light, heavy metals, frost etc. on secondary metabolites in plants. The focus of the present review is the influence of abiotic factors on secondary metabolite production and some of important plant pharmaceuticals. Also, we describe the results of in vitro cultures and production of some important secondary metabolites obtained in our laboratory.     

Introduction of the phzH gene of Pseudomonas chlororaphis PCL1391 extends the range of biocontrol ability of phenazine-1-carboxylic acid-producing Pseudomonas spp. strains

Pseudomonas chlororaphis PCL1391 controls tomato foot and root rot caused by Fusarium oxysporum f. sp. radicis-lycopersici. Its biocontrol activity is mediated by the production of phenazine-1-carboxamide (PCN). In contrast, the take-all biocontrol strains P. fluorescens 2-79 and P. aureofaciens 30-84, which produce phenazine-1-carboxylic acid (PCA), do not control this disease. To determine the role of the amide group in biocontrol, the PCN biosynthetic genes of strain PCL1391 were identified and characterized. Downstream of phzA through phzG, the novel phenazine biosynthetic gene phzH was identified and shown to be required for the presence of the 1-carboxamide group of PCN because a phzH mutant of strain PCL1391 accumulated PCA. The deduced PhzH protein shows homology with asparagine synthetases that belong to the class II glutamine amidotransferases, indicating that the conversion of PCA to PCN occurs via a transamidase reaction catalyzed by PhzH. Mutation of phzH caused loss of biocontrol activity, showing that the 1-carboxamide group of PCN is crucial for control of tomato foot and root rot. PCN production and biocontrol activity of the mutant were restored by complementing the phzH gene in trans. Moreover, transfer of phzH under control of the tac promoter to the PCA-producing biocontrol strains P. fluorescens 2-79 and P. aureofaciens 30-84 enabled these strains to produce PCN instead of PCA and suppress tomato foot and root rot. Thus, we have shown, for what we believe is the first time, that the introduction of a single gene can efficiently extend the range of the biocontrol ability of bacterial strains.     

Effects of simulated browsing on growth and leaf chemical properties in Colophospermum mopane saplings

Browsing intensity influences a plant's response to herbivory. Plants face a trade-off between investment in the production of secondary compounds and investment in growth. To elucidate this trade-off, we simulated four browsing intensities (0%, 50%, 75% and 100%) on mopane saplings, Colophospermum mopane (J.Kirk ex Benth.) J.Léonard, in a greenhouse experiment. This showed that, with increasing defoliation intensity, plants change their investment strategy. At intermediate levels of defoliation (50%), mopane saplings increased the synthesis of condensed tannins, so that tannin concentrations followed a hump-shaped relation with defoliation intensity, with significantly higher tannin concentration at intermediate defoliation levels. When defoliated heavily (75% and 100%), tannin concentrations dropped, and plants were carbon stressed as indicated by a reduced growth rate of the stem diameter, and leaf production and mean individual leaf mass were reduced. This suggests that, at intermediate defoliation intensity, the strategy of the plants is towards induced chemical defences. With increasing defoliation, the relative costs of the secondary metabolite synthesis become too high, and therefore, the plants change their growing strategy. Hence, browsers should be able to benefit from earlier browsing by either adopting a low or a relatively high browsing pressure.     

Production of Kluyveromyces spp. and environmental tolerance induction against Aspergillus flavus

The viability and biomass production of three isolates of Kluyveromyces spp. in six different growth media were studied. All yeast isolates showed good growth in all of the media tested, but nutrient yeast dextrose broth (NYDB 75 %) and molasses soy meal (MSB) media were selected for further analyses. The adaptive response of the yeasts to heat shock and water stress was studied, revealing that 60 min of incubation at 45 °C and a water activity value of 0.95 a_w were the appropriate conditions to adapt these yeasts for subsequent analyses. The physiological adaptation did not affect the ecological similarity between biocontrol agents and pathogen. The adapted yeasts also had a negative influence on the growth of Aspergillus flavus RCM89 mycelia and the accumulation of aflatoxin B₁ levels in vitro. These results have important implications for optimizing the formulation process of proven biocontrol agents against A. flavus. In addition, the applications of physiological methods are necessary for increasing the performance of biocontrol agents. Moreover, the physiological methods could enhance survival under environmental stress conditions of biological control agents.     

Evaluation of the plasmid copy number in B. cereus spores, during germination, bacterial growth and sporulation using real-time PCR

Only a small number of studies have measured the plasmid copy number (PCN) variation during bacterial growth. Besides, information about the PCN in spores is still rare. In this work, we utilized a real-time PCR assay to evaluate the PCN of four different plasmids in Bacillus cereus. The PCN was measured in spores as well as during germination, active bacterial growth, and sporulation. Plasmid stability was also evaluated to ensure that plasmid loss does not affect the accuracy of the PCN measurement. We demonstrated that the PCN of low and high copy number plasmids varies with growth phase as well as culture media over B. cereus life cycle. The PCN was minimum during the germination and maximum during the stationary growth phase for all plasmids tested. We also demonstrated that the use of antibiotic in the culture media is not enough to ensure stable inheritance in spores of plasmids carrying antibiotic resistance genes. Moreover, we revealed that the PCN in spores is related to the PCN during endospores formation. Therefore, the plasmid partitioning during sporulation is not influenced by the unequal-size of the forespores and the mother cells, even for a plasmid distributed randomly.     

The importance of water potential range tolerance as a limiting factor on Trichoderma spp. biocontrol of Sclerotinia sclerotiorum

Biological control of fungal phytopathogens is often more variable in efficacy compared with disease suppression achieved by conventional pesticide use. Matching the environmental range of a potential biocontrol agent with that of the target phytopathogen is necessary if consistent disease suppression is to be achieved under field conditions. Strains of Trichoderma that could parasitise sclerotia of Sclerotinia sclerotiorum had their spore germination and mycelial growth (five strains) and ability to parasitise sclerotia (two strains) tested under a range of water potentials under laboratory conditions. Relative mycelial growth and germination of all strains decreased with decreasing osmotic and matric potentials, with matric potential having a greater impact on growth and germination over the range examined. Trichoderma harzianum LU698 mycelial growth was the least affected by decreasing osmotic potential than the other isolates, and Trichoderma atroviride LU141 growth least affected by decreasing matric potential. The germination of LU698 and LU144 was also generally less affected by decreasing osmotic potential, although generally decreasing matric potential had the greatest affect on the germination of LU698 along with T. atroviride LU132. Soil treatments of LU698 and Trichoderma asperellum LU697 reduced sclerotial viability in all but the lowest soil water potential tested, with LU698 being most effective at ?0.1 and ?0.3 MPa after 28 days and LU697 most effective at ?0.01 and ?1.5 MPa after 28 days. We conclude that differences in the tolerance of potential biocontrol agents to changing water potential is an important experimental factor to consider when assaying biocontrol or making predictions of biocontrol efficacy in the field.     

Influence of environmental conditions on putisolvins I and II production in Pseudomonas putida strain PCL1445

Pseudomonas putida strain PCL1445 produces the cyclic lipopeptides putisolvin I and II, which are biosurfactants affecting environmentally important functions including swarming motility, solubility of nutrients and biofilm formation and maintenance. In this study, the effect of relevant nutritional and environmental factors on putisolvin production by PCL1445 was analyzed. Growth and levels of putisolvins I and II biosynthesis were significantly influenced by various carbon sources and by the nature of amino acid supplemented to the culture medium. An increase in the salt concentration of BM medium as well as growth at 1% oxygen significantly increased putisolvin production. In addition, we recently demonstrated that growth at a low temperature has a positive effect on putisolvin production. Analysis of the expression of gacA and gacS in Tn5luxAB mutant strains at a low temperature or under a high salt concentration suggests that biosynthesis of putisolvin is modulated through the GacA/GacS two-component regulatory system in PCL1445 under the tested conditions.     

Medium composition and methyl jasmonate influence the amount and spectrum of secondary metabolites in callus cultures of Zanthoxylum stenophyllum Hemsl.

Callus cultures of Zanthoxylum stenophyllum were initiated in vitro and the effect of growth regulators and elicitors was tested both upon callus growth and secondary metabolite production. On a medium containing naphthaleneacetic acid, kinetin, and 2,4-dichlorophenoxyacetic acid, a yellowish and friable callus was obtained from 90% of cotyledon explants. Callus growth and secondary metabolite accumulation was followed after sub-culturing the established callus culture on different media containing various hormonal combinations. Results indicate that medium containing naphthaleneacetic acid and a higher concentration of 2,4-dichlorophenoxyacetic acid gave the highest stimulation of growth. Addition of an organic nitrogen source also had a positive effect on growth. Rapid HPLC screening of methanol extractable secondary metabolites from calluses showed that phytohormones and nutrients were able to modify the chromatographic pattern of compounds. Calluses grown on the medium giving the highest stimulation of growth show a reduced accumulation of some secondary products, but not all. In response to elicitation by methyl jasmonate, metabolite production was different for the different classes of compounds, and hormonal composition of the culture medium influenced the response. Thus, results confirm the importance of the reciprocal interactions between hormones, nutrients, and elicitors when attempts are made to enhance secondary metabolite accumulation in in vitro cultures.     

Ecophysiological factors affecting growth,sporulation and survival of the biocontrol agent Penicillium oxalicum

The effect of temperature, pH, water potential and sources of nitrogen and carbon on the biocontrol agent Penicillium oxalicum were studied in vitro. The fungus is xerotolerant, mesophillic and has a wide pH tolerance. The parameters evaluated (germination, germ tube length, growth rate and sporulation) showed different sensitivities to the environmental factors. Peptone and free amino acids gave the highest growth rates and high levels of sporulation. Xylose, mannose and fructose gave the highest growth rates and mannose induced strong sporulation. The effect of nutrients (mannose + arginine) and water potential was also studied in vivo. The xerotolerant character of the fungus was confirmed. From this study we consider Penicillium oxalicum ecologically competent to perform effectively as a biocontrol agent in the soil environment. This revised version was published online in June 2006 with corrections to the Cover Date.     

Effect of aposymbiotic conditions on colony growth and secondary metabolite production in the lichen-forming fungus Ramalina dilacerata

The production of secondary metabolites by aposymbiotic lichen-forming fungi in culture is thought to be influenced by environmental conditions. The effects of the environment may be studied by culturing fungi under defined growing parameters to provide a better understanding of the role of the large number of polyketide synthase (PKS) gene paralogs detected in the genomes of many fungi. The objectives of this study were to examine the effects of culture conditions (media composition and pH level) on the colony growth, the numbers of secondary products, and the expression of two PKS genes by the lichen-forming fungus Ramalina dilacerata. Four types of growth media at four different pH levels were prepared to culture spore isolates of R. dilacerata. Colony diameter and texture were recorded. The number of secondary compounds were determined by thin layer chromatography (TLC) and high performance liquid chromatography (HPLC). Expression of two PKS genes (non-reducing (NR) and 6-MSAS-type PKS) were compared with expression of an internal control mitochondrial small subunit gene (mtSSU). The results showed that media containing yeast extracts produced the largest colony diameters and the fewest number of secondary metabolites. Colony growth rates also varied with different media conditions, and a significant negative relationship occurred between colony diameter and number of secondary metabolites. Expression of the NR PKS gene was significantly higher at pH 6.5 on the glucose malt agar than any other media, and expression of the 6-MSAS-type (partially-reducing) PKS gene was significantly higher at pH 8.5 on (malt agar) malt agar than on the other types of agar. Gene expression was correlated with the pH level and media conditions that induced the production of the larger number of secondary substances. This is the first study to examine secondary metabolite production in R. dilacerata by comparing the number of polyketides detected with quantitative polymerase chain reaction (qPCR) of two PKS genes under different culture conditions.     

Influence of soil reaction on diversity and antifungal activity of fluorescent pseudomonads in crop rhizospheres

The diversity and antifungal activity of fluorescent pseudomonads isolated from rhizospheres of tea, gladiolus, carnation and black gram grown in acidic soils with similar texture and climatic conditions were studied. Biochemical characterisation including antibiotic resistance assay, RAPD and PCR-RFLP studies revealed a largely homogenous population. At soil pH (5.2), the isolates exhibited growth with varying levels of siderophore production, irrespective of crop rhizospheres. Two isolates with maximum chitinase production showed antagonism. The bacterial populations in general lacked the ability to produce deleterious traits such as cellulase, pectinase and hydrogen cyanide. However, increased pH levels beyond 5.2 caused reduction in metabolite production with reduced antifungal activity. The homogeneity of the bacterial population irrespective of crop rhizospheres together with decreased secondary metabolite production at higher pH levels reinstated the importance of soil over host plant in influencing rhizosphere populations. The studies also yielded acid tolerant chitinase producing antagonistic fluorescent pseudomonads.     

The effects of temperature on growth and production of the antibiotic granaticin by a thermotolerant streptomycete

The synthesis of granaticin, a polyketide-derived antibiotic synthesized as a secondary metabolite by Streptomyces thermoviolaceus strain NCIB 10076, was studied at different growth temperatures. Quantitative measurements of the antibiotic made during batch fermentations showed that the yield was greatest at 45 degrees C, whereas the rate of synthesis was most rapid at 37 degrees C. The timing of the appearance of granaticin in culture could not be assigned to any particular phase of growth or to de-repression due to depletion of any particular nutrient. However, at all temperatures, appearance of the antibiotic coincided with a rise in ammoniacal nitrogen presumably due to deamination of glutamate, the carbon source for growth. We have previously shown that production of the antibiotic is pH sensitive and that some carbon sources result in higher titres than others. This paper examines the effect of temperature on the physiology of growth and on antibiotic production in more detail under conditions that also allow an exact measurement of granaticin yield.