Temperature-responsive sensing regulates biocontrol factor expression in Pseudomonas fluorescens CHA0.

In the plant-beneficial, root-colonizing strain Pseudomonas fluorescens CHA0, the Gac/Rsm signal transduction pathway positively regulates the synthesis of biocontrol factors (mostly antifungal secondary metabolites) and contributes to oxidative stress response via the stress sigma factor RpoS. The backbone of this pathway consists of the GacS/GacA two-component system, which activates the expression of three small regulatory RNAs (RsmX, RsmY, RsmZ) and thereby counters translational repression exerted by the RsmA and RsmE proteins on target mRNAs encoding biocontrol factors. We found that the expression of typical biocontrol factors, that is, antibiotic compounds and hydrogen cyanide (involving the phlA and hcnA genes), was significantly lower at 35 degrees C than at 30 degrees C. The expression of the rpoS gene was affected in parallel. This temperature control depended on RetS, a sensor kinase acting as an antagonist of the GacS/GacA system. An additional sensor kinase, LadS, which activated the GacS/GacA system, apparently did not contribute to thermosensitivity. Mutations in gacS or gacA were epistatic to (that is, they overruled) mutations in retS or ladS for expression of the small RNAs RsmXYZ. These data are consistent with a model according to which RetS-GacS and LadS-GacS interactions shape the output of the Gac/Rsm pathway and the environmental temperature influences the RetS-GacS interaction in P. fluorescens CHA0.

Soil tillage affects the community structure of mycorrhizal fungi in maize roots

In this study we tested whether communities of arbuscular mycorrhizal fungi (AMF) colonizing the roots of maize (Zea mays L.) were affected by soil tillage practices (plowing, chiseling, and no-till) in a long-term field experiment carried out in Tanikon (Switzerland). AMF were identified in the roots using specific polymerase chain reaction (PCR) markers that had been developed for the AMF previously isolated from the soils of the studied site. A nested PCR procedure with primers of increased specificity (eukaryotic, then, fungal, then AMF species or. species-grouop specific) was used. Sequencing of amplified DNA confirmed that the DNA obtained from the maize roots was of AMF origin. Presence of particular AMF species or species-group was scored as a presence of a DNA product after PCR with specific primers. We also used single-strand conformation polymorphism analysis (SSCP), of amplified DNA samples to-check if the amplification of the DNA from maize roots matched the expected profile for a particular AMF isolate with a given specific primer pair. Presence of the genus Scutellospora, in maize roots was strongly reduced in plowed and chiseled soils. Fungi from the suborder Glomineae were more prevalent colonizers of maize roots growing in plowed soils, but were also present in the roots from other tillage treatments. These changes in community of AMF colonizing maize roots might be due to (1), the differences in tolerance to the tillage-induced disruption of the hyphae among the different AMF species, (2) changes in nutrient content of the soil, (3) changes in microbial activity, or (4) changes in weed populations in response to soil tillage. This is the first report on community composition of AMF in the roots of a field-grown crop plant (maize) as affected by soil tillage.

Evolution of uni- and bifactorial sexual compatibility systems in fungi.

Mating systems, that is, whether organisms give rise to progeny by selfing, inbreeding or outcrossing, strongly affect important ecological and evolutionary processes. Large variations in mating systems exist in fungi, allowing the study of their origin and consequences. In fungi, sexual incompatibility is determined by molecular recognition mechanisms, controlled by a single mating-type locus in most unifactorial fungi. In Basidiomycete fungi, however, which include rusts, smuts and mushrooms, a system has evolved in which incompatibility is controlled by two unlinked loci. This bifactorial system probably evolved from a unifactorial system. Multiple independent transitions back to a unifactorial system occurred. It is still unclear what force drove evolution and maintenance of these contrasting inheritance patterns that determine mating compatibility. Here, we give an overview of the evolutionary factors that might have driven the evolution of bifactoriality from a unifactorial system and the transitions back to unifactoriality. Bifactoriality most likely evolved for selfing avoidance. Subsequently, multiallelism at mating-type loci evolved through negative frequency-dependent selection by increasing the chance to find a compatible mate. Unifactoriality then evolved back in some species, possibly because either selfing was favoured or for increasing the chance to find a compatible mate in species with few alleles. Owing to the existence of closely related unifactorial and bifactorial species and the increasing knowledge of the genetic systems of the different mechanisms, Basidiomycetes provide an excellent model for studying the different forces that shape breeding systems.

The effects of arbuscular mycorrhizal fungi inoculation on Euterpe oleracea mart. (açaí) seedlings

With the objective of verifying the response of Euterpe oleracea seedlings to seven arbuscular mycorrhizal fungi species, an experimental trial was carried out under greenhouse conditions. Seeds of E. oleracea were sown in carbonized rice husk. Germinating seeds were initially transferred to plastic cups, containing fumigated Reddish Yellow Quartz Sand and inoculated with arbuscular mycorrhizal fungi. Two months later, seedlings were transferred to 2 kg black plastic bags, containing the same soil without fumigation. Plant growth and mineral nutrients were evaluated nine months after mycorrhizal inoculation. Differential effects were observed among the species tested, with Scutellispora gilmorei being the most effective ones in promoting growth and nutrient content of E. oleracea seedlings. The increment resulted from inoculation with S. gilmorei were 92% in total plant height, 116% in stem diameter, 361% in dry matter production, 191% in N, 664% in P, 46% in K, 562% in Ca, 363% in Mg and 350% in Zn contents, comparing to uninoculated controls. Infected root length was positively correlated to nutrient content and plant growth. It was concluded that growth and nutrient uptake of E. oleracea seedlings could be significantly improved by inoculation of effective arbuscular mycorrhizal fungi.

Fusarium oxysporum strains as biocontrol agents against Fusarium wilt: effects on soil microbial biomass and activity

Before planning the large-scale use of nonpathogenic strains of Fusarium oxysporum as biocontrol agents of Fusarium wilt, their behaviour and potential impact on soil ecosystems should be carefully studied as part of risk assessment. The aim of this work was to evaluate the effects of antagonistic F. oxysporum strains, genetically manipulated (T26/6) or not (233/1), on soil microbial biomass and activity. The effects were evaluated, in North-western Italy, in two soils from different sites at Albenga, one natural and the other previously solarized, and in a third soil obtained from a 10-year-old poplar stand (Popolus sp.), near Carignano. There were no detectable effects on ATP, fluorescein diacetate hydrolysis, and biomass P that could be attributed to the introduction of the antagonists. A transient increase of carbon dioxide evolution and biomass C was observed in response to the added inoculum. Although the results showed only some transient alterations, further studies are required to evaluate effects on specific microorganism populations.

Mycorrhizal colonization and phenolic compounds accumulation on roots of Eucalyptus dunnii maiden inoculated with ectomycorrhizal fungi

Compatibility between Eucalyptus dunnii and the ectomycorrhizal fungi Hysterangium gardneri and Pisolithus sp. - from Eucalyptus spp. -, Rhizopogon nigrescens and Suillus cothurnatus - from Pinus spp.-, was studied in vitro. Pisolithus sp., H. gardneri and S. cothurnatus colonized the roots. Pisolithus sp. mycorrhizas presented mantle and Hartig net, while H. gardneri and S. cothurnatus mycorrhizas presented only mantle. S. cothurnatus increased phenolics level on roots. Pisolithus sp. and R. nigrescens decreased the level of these substances. The isolates from Eucalyptus seem to be more compatible towards E. dunnii than those from Pinus. The mechanisms involved could be related, at least in the cases of Pisolithus and Suillus, to the concentration of phenolics in roots.

Deleterious impact of a virulent bacteriophage on survival and biocontrol activity of Pseudomonas fluorescens strain CHAO in natural soil.

Many biotic and abiotic factors affect the persistence and activity of beneficial pseudomonads introduced into soil to suppress plant diseases. One such factor may be the presence of virulent bacteriophages that decimate the population of the introduced bacteria, thereby reducing their beneficial effect. We have isolated a lytic bacteriophage (phi)GP100) that specifically infects the biocontrol bacterium Pseudomonas fluorescens CHA0 and some closely related Pseudomonas strains. phiGP100 was found to be a double-stranded-DNA phage with an icosahedral head, a stubby tail, and a genome size of approximately 50 kb. Replication of phiGP100 was negatively affected at temperatures higher than 25 degrees C. phiGP100 had a negative impact on the population size and the biocontrol activity of P. fluorescens strain CHA0-Rif (a rifampicin-resistant variant of CHA0) in natural soil microcosms. In the presence of phiGP100, the population size of strain CHA0-Rif in soil and on cucumber roots was reduced more than 100-fold. As a consequence, the bacterium's capacity to protect cucumber against a root disease caused by the pathogenic oomycete Pythium ultimum was entirely abolished. In contrast, the phage affected neither root colonization and nor the disease suppressive effect of a phiDGP100-resistant variant of strain CHA0-Rif. To our knowledge, this study is the first to illustrate the potential of phages to impair biocontrol performance of beneficial bacteria released into the natural soil environment.

ppGpp controlled by the Gac/Rsm regulatory pathway sustains biocontrol activity in Pseudomonas fluorescens CHA0.

In Pseudomonas fluorescens CHA0 and other fluorescent pseudomonads, the Gac/Rsm signal transduction pathway is instrumental for secondary metabolism and biocontrol of root pathogens via the expression of regulatory small RNAs (sRNAs). Furthermore, in strain CHA0, an imbalance in the Krebs cycle can affect the strain's ability to produce extracellular secondary metabolites, including biocontrol factors. Here, we report the metabolome of wild-type CHA0, a gacA-negative mutant, which has lost Gac/Rsm activities, and a retS-negative mutant, which shows strongly enhanced Gac/Rsm-dependent activities. Capillary electrophoresis-based metabolomic profiling revealed that the gacA and retS mutations had opposite effects on the intracellular levels of a number of central metabolites, suggesting that the Gac/Rsm pathway regulates not only secondary metabolism but also primary metabolism in strain CHA0. Among the regulated metabolites identified, the alarmone guanosine tetraphosphate (ppGpp) was characterized in detail by the construction of relA (for ppGpp synthase) and spoT (for ppGpp synthase/hydrolase) deletion mutants. In a relA spoT double mutant, ppGpp synthesis was completely abolished, the expression of Rsm sRNAs was attenuated, and physiological functions such as antibiotic production, root colonization, and plant protection were markedly diminished. Thus, ppGpp appears to be essential for sustaining epiphytic fitness and biocontrol activity of strain CHA0.

Evolution of the P-type II ATPase gene family in the fungi and presence of structural genomic changes among isolates of Glomus intraradices.

BACKGROUND: The P-type II ATPase gene family encodes proteins with an important role in adaptation of the cell to variation in external K+, Ca2+ and Na2+ concentrations. The presence of P-type II gene subfamilies that are specific for certain kingdoms has been reported but was sometimes contradicted by discovery of previously unknown homologous sequences in newly sequenced genomes. Members of this gene family have been sampled in all of the fungal phyla except the arbuscular mycorrhizal fungi (AMF; phylum Glomeromycota), which are known to play a key-role in terrestrial ecosystems and to be genetically highly variable within populations. Here we used highly degenerate primers on AMF genomic DNA to increase the sampling of fungal P-Type II ATPases and to test previous predictions about their evolution. In parallel, homologous sequences of the P-type II ATPases have been used to determine the nature and amount of polymorphism that is present at these loci among isolates of Glomus intraradices harvested from the same field. RESULTS: In this study, four P-type II ATPase sub-families have been isolated from three AMF species. We show that, contrary to previous predictions, P-type IIC ATPases are present in all basal fungal taxa. Additionally, P-Type IIE ATPases should no longer be considered as exclusive to the Ascomycota and the Basidiomycota, since we also demonstrate their presence in the Zygomycota. Finally, a comparison of homologous sequences encoding P-type IID ATPases showed unexpectedly that indel mutations among coding regions, as well as specific gene duplications occur among AMF individuals within the same field. CONCLUSION: On the basis of these results we suggest that the diversification of P-Type IIC and E ATPases followed the diversification of the extant fungal phyla with independent events of gene gains and losses. Consistent with recent findings on the human genome, but at a much smaller geographic scale, we provided evidence that structural genomic changes, such as exonic indel mutations and gene duplications are less rare than previously thought and that these also occur within fungal populations.

Contribution of the Global Regulator Gene gacA to Persistence and Dissemination of Pseudomonas fluorescens Biocontrol Strain CHA0 Introduced into Soil Microcosms.

Structural and regulatory genes involved in the synthesis of antimicrobial metabolites are essential for the biocontrol activity of fluorescent pseudomonads and, in principle, amenable to genetic engineering for strain improvement. An eventual large-scale release of such bacteria raises the question of whether such genes also contribute to the persistence and dissemination of the bacteria in soil ecosystems. Pseudomonas fluorescens wild-type strain CHA0 protects plants against a variety of fungal diseases and produces several antimicrobial metabolites. The regulatory gene gacA globally controls antibiotic production and is crucial for disease suppression in CHA0. This gene also regulates the production of extracellular protease and phospholipase. The contribution of gacA to survival and vertical translocation of CHA0 in soil microcosms of increasing complexity was studied in coinoculation experiments with the wild type and a gacA mutant which lacks antibiotics and some exoenzymes. Both strains were marked with spontaneous resistance to rifampin. In a closed system with sterile soil, strain CHA0 and the gacA mutant multiplied for several weeks, whereas these strains declined exponentially in nonsterile soil of different Swiss origins. The gacA mutant was less persistent in nonrhizosphere raw soil than was the wild type, but no competitive disadvantage when colonizing the rhizosphere and roots of wheat was found in the particular soil type and during the period studied. Vertical translocation was assessed after strains had been applied to undisturbed, long (60-cm) or short (20-cm) soil columns, both planted with wheat. A smaller number of cells of the gacA mutant than of the wild type were detected in the percolated water and in different depths of the soil column. Single-strain inoculation gave similar results in all microcosms tested. We conclude that mutation in a single regulatory gene involved in antibiotic and exoenzyme synthesis can affect the survival of P. fluorescens more profoundly in unplanted soil than in the rhizosphere.

Rapid genotypic change and plasticity in arbuscular mycorrhizal fungi is caused by a host shift and enhanced by segregation.

Arbuscular mycorrhizal fungi (AMF) are among the most abundant symbionts of plants, improving plant productivity and diversity. They are thought to mostly grow vegetatively, a trait assumed to limit adaptability. However, AMF can also harbor genetically different nuclei (nucleotypes). It has been shown that one AMF can produce genotypically novel offspring with proportions of different nucleotypes. We hypothesized that (1) AMF respond rapidly to a change of environment (plant host) through changes in the frequency of nucleotypes; (2) genotypically novel offspring exhibit different genetic responses to environmental change than the parent; and (3) genotypically novel offspring exhibit a wide range of phenotypic plasticity to a change of environment. We subjected AMF parents and offspring to a host shift. We observed rapid and large genotypic changes in all AMF lines that were not random. Genotypic and phenotypic responses were different among offspring and their parents. Even though growing vegetatively, AMF offspring display a broad range of genotypic and phenotypic changes in response to host shift. We conclude that AMF have the ability to rapidly produce variable progeny, increasing their probability to produce offspring with different fitness than their parents and, consequently, their potential adaptability to new environmental conditions. Such genotypic and phenotypic flexibility could be a fast alternative to sexual reproduction and is likely to be a key to the ecological success of AMF.