Differential expression of genes involved in entomopathogenicity of the fungi Metarhizium anisopliae var. anisopliae and M. anisopliae var. acridum (Clavicipitaceae)

Expression analysis of the genes involved in germination, conidiogenisis and pathogenesis of Metarhizium anisopliae during its saprophytic and pathogenic life stages can help plan strategies to increase its efficacy as a biological control agent. We quantified relative expression levels of the nitrogen response regulator gene (nrr1) and a G-protein regulator of genes involved in conidiogenesis (cag8), using an RT-qPCR assay. Comparisons were made between M. anisopliae var. anisopliae and M. anisopliae var. acridum during germination and conidiogenesis and at different stages of pathogenesis. The cag8 gene was repressed during germination and induced during conidial development and the pathogenic phase, and the nrr1 gene was induced during germination, conidiogenesis and the pathogenic phase. Both genes were more expressed in M. anisopliae var. anisopliae, demonstrating that different varieties of M. anisopliae differ in activation of genes linked to virulence for certain environments and hosts. This suggests that differences among these varieties in the ability to adapt could be attributed not only to specific genomic regions and genes, but also to differential gene expression in this fungus, modulating its ability to respond to environmental stimuli.     

Characterization of a small cryptic plasmid from endophytic Pantoea agglomerans and its use in the construction of an expression vector

A circular cryptic plasmid named pPAGA (2,734 bp) was isolated from Pantoea agglomerans strain EGE6 (an endophytic bacterial isolate from eucalyptus). Sequence analysis revealed that the plasmid has a G+C content of 51% and contains four potential ORFs, 238(A), 250(B), 131(C), and 129(D) amino acids in length without homology to known proteins. The shuttle vector pLGM1 was constructed by combining the pPAGA plasmid with pGFPmut3.0 (which harbors a gene encoding green fluorescent protein, GFP), and the resulting construct was used to over-express GFP in E. coli and P. agglomerans cells. GFP production was used to monitor the colonization of strain EGE6gfp in various plant tissues by fluorescence microscopy. Analysis of EGE6gfp colonization showed that 14 days after inoculation, the strain occupied the inner tissue of Eucalyptus grandis roots, preferentially colonizing the xylem vessels of the host plants.     

Differential expression of the pr1A gene in Metarhizium anisopliae and Metarhizium acridum across different culture conditions and during pathogenesis

The entomopathogenic fungi of the genus Metarhizium have several subtilisin-like proteases that are involved in pathogenesis and these have been used to investigate genes that are differentially expressed in response to different growth conditions. The identification and characterization of these proteases can provide insight into how the fungus is capable of infecting a wide variety of insects and adapt to different substrates. In addition, the pr1A gene has been used for the genetic improvement of strains used in pest control. In this study we used quantitative RT-PCR to assess the relative expression levels of the pr1A gene in M. anisopliae and M. acridum during growth in different culture conditions and during infection of the sugar cane borer, Diatraea saccharalis Fabricius. We also carried out a pathogenicity test to assess the virulence of both species against D. saccharalis and correlated the results with the pattern of pr1A gene expression. This analysis revealed that, in both species, the pr1A gene was differentially expressed under the growth conditions studied and during the pathogenic process. M. anisopliae showed higher expression of pr1A in all conditions examined, when compared to M. acridum. Furthermore, M. anisopliae showed a greater potential to control D. saccharalis. Taken together, our results suggest that these species have developed different strategies to adapt to different growing conditions.     

Diversity and identification of methanogenic archaea and sulphate-reducing bacteria in sediments from a pristine tropical mangrove

Mangrove sediments are anaerobic ecosystems rich in organic matter. This environment is optimal for anaerobic microorganisms, such as sulphate-reducing bacteria and methanogenic archaea, which are responsible for nutrient cycling. In this study, the diversity of these two functional guilds was evaluated in a pristine mangrove forest using denaturing gradient gel electrophoresis (DGGE) and clone library sequencing in a 50 cm vertical profile sampled every 5.0 cm. DGGE profiles indicated that both groups presented higher richness in shallow samples (0–30 cm) with a steep decrease in richness beyond that depth. According to redundancy analysis, this alteration significantly correlated with a decrease in the amount of organic matter. Clone library sequencing indicated that depth had a strong effect on the selection of dissimilatory sulphate reductase (dsrB) operational taxonomic units (OTUs), as indicated by the small number of shared OTUs found in shallow (0.0 cm) and deep (40.0 cm) libraries. On the other hand, methyl coenzyme-M reductase (mcrA) libraries indicated that most of the OTUs found in the shallow library were present in the deep library. These results show that these two guilds co-exist in these mangrove sediments and indicate important roles for these organisms in nutrient cycling within this ecosystem.     

Endophytic Colonization of Potato (Solanum tuberosum L.) by a Novel Competent Bacterial Endophyte,Pseudomonas putida Strain P9, and Its Effect on Associated Bacterial Communities

Pseudomonas putida strain P9 is a novel competent endophyte from potato. P9 causes cultivar-dependent suppression of Phytophthora infestans. Colonization of the rhizoplane and endosphere of potato plants by P9 and its rifampin-resistant derivative P9R was studied. The purposes of this work were to follow the fate of P9 inside growing potato plants and to establish its effect on associated microbial communities. The effects of P9 and P9R inoculation were studied in two separate experiments. The roots of transplants of three different cultivars of potato were dipped in suspensions of P9 or P9R cells, and the plants were planted in soil. The fate of both strains was followed by examining colony growth and by performing PCR-denaturing gradient gel electrophoresis (PCR-DGGE). Colonies of both strains were recovered from rhizoplane and endosphere samples of all three cultivars at two growth stages. A conspicuous band, representing P9 and P9R, was found in all Pseudomonas PCR-DGGE fingerprints for treated plants. The numbers of P9R CFU and the P9R-specific band intensities for the different replicate samples were positively correlated, as determined by linear regression analysis. The effects of plant growth stage, genotype, and the presence of P9R on associated microbial communities were examined by multivariate and unweighted-pair group method with arithmetic mean cluster analyses of PCR-DGGE fingerprints. The presence of strain P9R had an effect on bacterial groups identified as Pseudomonas azotoformans, Pseudomonas veronii, and Pseudomonas syringae. In conclusion, strain P9 is an avid colonizer of potato plants, competing with microbial populations indigenous to the potato phytosphere. Bacterization with a biocontrol agent has an important and previously unexplored effect on plant-associated communities.The colonization of plant tissue is of paramount importance for successful application of plant-growth-promoting bacteria. For instance, efficient root colonization was shown to be important for the control of Fusarium oxysporum in tomato by a phenazine-1-carboxamide-producing Pseudomonas chlororaphis strain (6). The migratory response of bacterial inoculants to compounds released by plant roots is often the first step required for establishment of the bacteria in the rhizosphere and rhizoplane (29, 48, 50). Following the initial colonization by introduced bacteria, these organisms may spread further to the aerial parts of the plant (35). The degree of root colonization by bacterial inoculants depends on, in addition to the mode of application (35), factors intrinsic to the organism used, like the presence of flagella (11) and/or the presence of particular outer membrane lipopolysaccharides (13). Such features may differ from strain to strain. Once attached to plant roots, the inoculant bacteria may evoke “protective” responses in the plants, enabling them to resist phytopathogen attack (4).Endophytic colonization, characterized by colonization of internal plant tissues concomitant with growth and systemic spread, can be an important factor for plant growth (23, 24). For instance, cells of Rhizobium etli G12 (used as a biocontrol agent) marked with a green fluorescent protein were visible in root hairs, around epidermal cells, and within the vascular tissue of Arabidopsis thaliana plants (22), and these plants exhibited maximum control of the nematode Meleidogyne incognita. Moreover, cells of the green fluorescent protein-labeled plant-growth-promoting bacterial strain Burkholderia phytofirmans PsJN were present in xylem vessels and different plant organs, including inflorescences, of grape plants (7). Endophytic colonization was also observed for the nitrogen-fixing bacteria Acetobacter diazotrophicus in sugarcane (12) and Serratia marcescens in rice (20). Hence, different types of bacteria apparently have the capacity to colonize the internal compartments of plants and eventually interact with their hosts, thus occupying niches inside plants where they may evoke responses that are important for plant health maintenance and nutrient acquisition.Introduced bacteria can communicate with each other using a range of signaling systems, including quorum sensing, and most likely also with (related) bacteria indigenous to plants and even with their host plant (41). We hypothesize that introduced strains also impact plant-associated indigenous microbial communities by cross talk with members of these communities, by competition for nutrients or space, or by production of antibiotics. Shifts in endophytic bacterial communities are therefore expected to occur after bacterial inoculation. This is a phenomenon that has been observed after inoculation of Madagascar periwinkle (Catharanthus roseus) and Cleveland tobacco (Nicotiana clevelandii) plants with Methylobacterium mesophilicum (1).In situ microscopic detection of endophytes allows determination of the preferred colonization site. However, it is hard to detect cells (e.g., cells marked with gfp) at levels below certain threshold levels in plants, especially when they are grown under nonsterile conditions. For this reason it is not possible to precisely determine in situ interactions between inoculants and indigenous bacterial populations. Molecular fingerprinting techniques, like PCR-denaturing gradient gel electrophoresis (DGGE), are suitable for studying microbial communities in the rhizospheres (14, 26, 44, 51), rhizoplanes (48), and endospheres (18, 38, 42, 51) of different plant species. The use of bacterial group-specific primer systems has been proposed for studies of different taxonomic groups in the plant endosphere (52). The impact of factors like crop history, plant growth stage, and cultivar (genotype) on plant-associated microbial populations can be established by using multivariate statistical analyses (17, 40, 51). The combination of molecular fingerprinting techniques and multivariate analyses enabled us to show that the plant growth stage and cultivar contributed strongly and significantly to the composition of plant-associated microbial communities (37, 51).The aim of this study was to establish the fate and impact of strain P9 during endophytic colonization of potato plants. The polyphasic approach used allowed us to investigate the presence of strain P9 at culture-dependent and -independent levels in plants.