Sohail Yousaf

Soil type affects plant colonization, activity and catabolic gene expression of inoculated bacterial strains during phytoremediation of diesel.

The combined use of plants and associated microorganisms has great potential for cleaning up soils contaminated with petroleum hydrocarbons. Apart from environmental conditions the physicochemical properties of the soil are the main factors influencing the survival and activity of an inoculated strain as well as the growth of plants. This study examined the effect of different soil types (sandy, loamy sand and loam) on the survival, gene abundance and catabolic gene expression of two inoculated strains (Pseudomonas sp. strain ITRI53 and Pantoea sp. strain BTRH79) in the rhizosphere and shoot interior of Italian ryegrass vegetated in diesel contaminated soils. High colonization, gene abundance and expression in loamy soils were observed. By contrast, low colonization, gene abundance and absence of gene expression in sandy soil were found. The highest levels of genes expression and hydrocarbon degradation were seen in loamy soil that had been inoculated with BTRH79 and were significantly higher compared to those in other soils. A positive correlation was observed between gene expression and hydrocarbon degradation indicating that catabolic gene expression is necessary for contaminant degradation. These results suggest that soil type influences the bacterial colonization and microbial activities and subsequently the efficiency of contaminant degradation.

Hydrocarbon degradation, plant colonization and gene expression of alkane degradation genes by endophytic Enterobacter ludwigii strains

The genus Enterobacter comprises a range of beneficial plant-associated bacteria showing plant growth promotion. Enterobacter ludwigii belongs to the Enterobacter cloacae complex and has been reported to include human pathogens but also plant-associated strains with plant beneficial capacities. To assess the role of Enterobacter endophytes in hydrocarbon degradation, plant colonization, abundance and expression of CYP153 genes in different plant compartments, three plant species (Italian ryegrass, birdsfoot trefoil and alfalfa) were grown in sterile soil spiked with 1% diesel and inoculated with three endophytic E. ludwigii strains. Results showed that all strains were capable of hydrocarbon degradation and efficiently colonized the rhizosphere and plant interior. Two strains, ISI10-3 and BRI10-9, showed highest degradation rates of diesel fuel up to 68% and performed best in combination with Italian ryegrass and alfalfa. All strains expressed the CYP153 gene in all plant compartments, indicating an active role in degradation of diesel in association with plants.

Phylogenetic and functional diversity of alkane degrading bacteria associated with Italian ryegrass (Lolium multiflorum) and Birdsfoot trefoil (Lotus corniculatus) in a petroleum oil-contaminated environment

Twenty-six different plant species were analyzed regarding their performance in soil contaminated with petroleum oil. Two well-performing species, Italian ryegrass (Lolium multiflorum var. Taurus), Birdsfoot trefoil (Lotus corniculatus var. Leo) and the combination of these two plants were selected to study the ecology of plant-associated, culturable alkane-degrading bacteria. Hydrocarbon degrading bacteria were isolated from the rhizosphere, root interior and shoot interior and subjected to the analysis of 16S rRNA gene, the 16S and 23S rRNA intergenic spacer region and alkane hydroxylase genes. Furthermore, we investigated whether alkane hydroxylase genes are plasmid located. Higher numbers of culturable, alkane-degrading bacteria were associated with Italian ryegrass, which were also characterized by a higher diversity, particularly in the plant interior. Only half of the isolated bacteria hosted known alkane hydroxylase genes (alkB and cytochrome P153-like). Degradation genes were found both on plasmids as well as in the chromosome. In regard to application of plants for rhizodegradation, where support of numerous degrading bacteria is essential for efficient break-down of pollutants, Italian ryegrass seems to be more appropriate than Birdsfoot trefoil.

The Inoculation Method Affects Colonization and Performance of Bacterial Inoculant Strains in the Phytoremediation of Soil Contaminated with Diesel Oil

Plants in combination with microorganisms can remediate soils, which are contaminated with organic pollutants such as petroleum hydrocarbons. Inoculation of plants with degrading bacteria is one approach to improve remediation processes, but is often not successful due to the competition with resident microorganisms. It is therefore of high importance to address the persistence and colonization behavior of inoculant strains. The objective of this study was to determine whether the inoculation method (seed imbibement and soil inoculation) influences bacterial colonization, plant growth promotion and hydrocarbon degradation. Italian ryegrass was grown in non-sterilized soil polluted with diesel and inoculated with different alkane-degrading strains Pantoea sp. ITSI10, Pantoea sp. BTRH79 and Pseudomonas sp. MixRI75 individually as well as in combination. Inoculation generally had a beneficial effect on plant biomass production and hydrocarbon degradation, however, strains inoculated in soil performed better than applied by seed imbibement. Performance correlated with the colonization efficiency of the inoculated strains. The highest hydrocarbon degradation was observed in the treatment, in which all three strains were inoculated in combination into soil. Our study revealed that besides the degradation potential and competitive ability of inoculant strains the inoculation method plays an important role in determining the success of microbial inoculation.

Fungal Endophytic Communities in Grapevines (Vitis vinifera L.) Respond to Crop Management

ABSTRACT We studied the distribution of fungal endophytes of grapevine (Vitis vinifera L.) plants in a subalpine area of northern Italy, where viticulture is of high economic relevance. We adopted both cultivation-based and cultivation-independent approaches to address how various anthropic and nonanthropic factors shape microbial communities. Grapevine stems were harvested from several locations considering organic and integrated pest management (IPM) and from the cultivars Merlot and Chardonnay. Cultivable fungi were isolated and identified by internal-transcribed-spacer sequence analysis, using a novel colony-PCR method, to amplify DNA from fungal specimens. The composition of fungal communities was assessed using a cultivation-independent approach, automated ribosomal intergenic spacer analysis (ARISA). Multivariate statistical analysis of both culture-dependent and culture-independent data sets was convergent and indicated that fungal endophytic communities in grapevines from organically managed farms were different from those from farms utilizing IPM. Fungal communities in plants of cv. Merlot and cv. Chardonnay overlapped when analyzed using culture-dependent approaches but could be partially resolved using ARISA fingerprinting.

Hydrocarbon degradation and plant colonization by selected bacterial strains isolated from Italian ryegrass and birdsfoot trefoil

Aims:  To assess the degradation potential and plant colonization capacity of four alkane‐degrading strains (ITSI10, ITRI15, ITRH76 and BTRH79) in combination with birdsfoot trefoil and Italian ryegrass and to evaluate the diversity of indigenous alkane‐degrading soil bacteria in the rhizo‐ and endosphere.

Bacterial endophytic communities in the grapevine depend on pest management.

Microbial plant endophytes are receiving ever-increasing attention as a result of compelling evidence regarding functional interaction with the host plant. Microbial communities in plants were recently reported to be influenced by numerous environmental and anthropogenic factors, including soil and pest management. In this study we used automated ribosomal intergenic spacer analysis (ARISA) fingerprinting and pyrosequencing of 16S rDNA to assess the effect of organic production and integrated pest management (IPM) on bacterial endophytic communities in two widespread grapevines cultivars (Merlot and Chardonnay). High levels of the dominant Ralstonia, Burkholderia and Pseudomonas genera were detected in all the samples We found differences in the composition of endophytic communities in grapevines cultivated using organic production and IPM. Operational taxonomic units (OTUs) assigned to the Mesorhizobium, Caulobacter and Staphylococcus genera were relatively more abundant in plants from organic vineyards, while Ralstonia, Burkholderia and Stenotrophomonas were more abundant in grapevines from IPM vineyards. Minor differences in bacterial endophytic communities were also found in the grapevines of the two cultivars.

Effect of mixing ratio of food waste and rice husk co-digestion and substrate to inoculum ratio on biogas production.

Aim of this study was to find out suitable mixing ratio of food waste and rice husk for their co-digestion in order to overcome VFA accumulation in digestion of food waste alone. Four mixing ratios of food waste and rice husk with C/N ratios of 20, 25, 30 and 35 were subjected to a lab scale anaerobic batch experiment under mesophilic conditions. Highest specific biogas yield of 584L/kgVS was obtained from feedstock with C/N ratio of 20. Biogas yield decreased with decrease in food waste proportion. Further, fresh cow dung was used as inoculum to investigate optimum S/I ratio with the selected feedstock. In experiment 2, feedstock with C/N ratio 20 was subjected to anaerobic digestion at five S/I ratios of 0.25, 0.5, 1.0, 1.5 and 2.0. Specific biogas yield of 557L/kgVS was obtained at S/I ratio of 0.25. However, VFA accumulation occurred at higher S/I ratios due to higher organic loadings.

Interkingdom transfer of the acne causing agent, Propionibacterium acnes, from human to grapevine

Here, we report the surprising and, to our knowledge, unique example of horizontal interkingdom transfer of a human opportunistic pathogen (Propionibacterium acnes) to a crop plant (the domesticated grapevine Vitis vinifera L.). Humans, like most organisms, have established a long-lasting cohabitation with a variety of microbes, including pathogens and gut-associated bacteria. Studies which have investigated the dynamics of such associations revealed numerous cases of bacterial host switches from domestic animals to humans. Much less is, however, known about the exchange of microbial symbionts between humans and plants. Fluorescent in situ hybridization localized P. acnes in the bark, in xylem fibers, and, more interestingly, inside pith tissues. Phylogenetic and population genetic analyses suggest that the establishment of the grapevine-associated P. acnes as obligate endophyte is compatible with a recent transfer event, likely during the Neolithic, when grapevine was domesticated.

Diversity in Endophyte Populations Reveals Functional and Taxonomic Diversity between Wild and Domesticated Grapevines

Endophytes hold great potential for implementing a more sustainable, bio-based agriculture. Here we investigated the differences among populations of endophytic bacteria isolated from wild and domesticated grapevines to improve our understanding of how agriculture and agro-environment affect endophytic communities. Bacterial isolates from 88 wild and domesticated grapevines that shared the same climate for at least four years were identified and a representative set of 155 strains was characterized for 30 features, including quorum sensing-related, enzyme production, antibiotic resistance, plant growth promotion (PGP), and biocontrol (BiCo) traits. Microbial diversity was greater in wild grapevines (25 genera) than in domesticated grapevines (six genera). Molecular fingerprinting by automated ribosomal intergenic spacer analysis also indicated a greater diversity in wild grapevines. Multivariate analysis of phenotypic traits indicated that strains isolated from wild and cultivated plants often formed distinct clusters, even when they belonged to the same genus. Interestingly, endophytes from domesticated grapevines were more likely to perform well in PGP and BiCo tests than endophytes from wild grapevines, suggesting that the decreased taxonomic diversity in domesticated grapevine microbiota did not correspond to a loss of agriculturally relevant traits.