Biodiversity and Bioprospecting of Fungal Endophytes from the Antarctic Plant Colobanthus quitensis.

Microorganisms from extreme environments are considered as a new and valuable reservoir of bioactive molecules of biotechnological interest and are also utilized as tools for enhancing tolerance to (a)biotic stresses in crops. In this study, the fungal endophytic community associated with the leaves of the Antarctic angiosperm Colobanthus quitensis was investigated as a new source of bioactive molecules. We isolated 132 fungal strains and taxonomically annotated 26 representative isolates, which mainly belonged to the Basidiomycota division. Selected isolates of Trametes sp., Lenzites sp., Sistotrema sp., and Peniophora sp. displayed broad extracellular enzymatic profiles; fungal extracts from some of them showed dose-dependent antitumor activity and inhibited the formation of amyloid fibrils of α-synuclein and its pathological mutant E46K. Selected fungal isolates were also able to promote secondary root development and fresh weight increase in Arabidopsis and tomato and antagonize the growth of pathogenic fungi harmful to crops. This study emphasizes the ecological and biotechnological relevance of fungi from the Antarctic ecosystem and provides clues to the bioprospecting of Antarctic Basidiomycetes fungi for industrial, agricultural, and medical applications.

Plant organ and sampling time point determine the taxonomic structure of microbial communities associated to apple plants in the orchard environment.

Plant-associated microbial communities interact with their host and are important components of the biodiversity of natural and agro-ecosystems. Scarce knowledge is available on the establishment of plant microbiota in perennial woody plants. In this work the variability in bacterial and fungal communities in aboveground organs was analysed in leaves, bark, flowers and fruits on three apple cultivars (Gala, Fuji and Golden), in order to understand changes of the microbiota community structure from orchard planting to the first year of fruit production. Our results indicate that Proteobacteria, Bacterioidetes, Actinobacteria and Firmiculites were the dominant bacterial phyla across all samples. The majority of fungal sequences were assigned to Ascomycota and Basidiomycota. The bacterial genera Pseudomonas and Sphingomonas, and the fungal genera Aureobasidium and Filobasidium, represented a major component of the aboveground microbiota. Different parts of the apple plant harboured a specific microbiota and the effect of plant organ on the bacterial and fungal taxonomic structure exceeded the influence of sampling time and plant genotype. This work highlights the specificity of the microbiota associated with aboveground apple organs, changes of the microbiota composition during the plant development from orchard planting to the first year of fruit production, and the negligible effects of apple cultivar.

Tissue age, orchard location and disease management influence the composition of fungal and bacterial communities present on the bark of apple trees.

Plants host microbial communities that can be affected by environmental conditions and agronomic practices. Despite the role of bark as a reservoir of plant pathogens and beneficial microorganisms, no information is available on the effects of disease management on the taxonomic composition of the bark-associated communities of apple trees. We assessed the impact of disease management strategies on fungal and bacterial communities on the bark of a scab-resistant apple cultivar in two orchard locations and for two consecutive seasons. The amplicon sequencing revealed that bark age and orchard location strongly affected fungal and bacterial diversity. Microbiota dissimilarity between orchards evolved during the growing season and showed specific temporal series for fungal and bacterial populations in old and young bark. Disease management did not induce global changes in the microbial populations across locations and seasons, but specifically affected the abundance of some taxa according to bark age, orchard location and sampling time. Therefore, the disease management applied to scab-resistant cultivars, which is based on a limited use of fungicides, partially changed the taxonomic composition of bark-associated fungal and bacterial communities, suggesting the need for a more accurate risk assessment regarding possible pathogen outbreaks.

Fungal biodiversity in the periglacial soil of Dosdè Glacier (Valtellina, Northern Italy).

Periglacial areas are one of the least studied habitats on Earth, especially in terms of their fungal communities. In this work, both molecular and culture-dependent methods have been used to analyse the microfungi in soils sampled on the front of the East Dosdè Glacier (Valtellina, Northern Italy). Although this survey revealed a community that was rich in fungal species, a distinct group of psychrophilic microfungi has not been detected. Most of the isolated microfungi were mesophiles, which are well adapted to the sensitive climatic changes that occur in this alpine environment. A discrepancy in the results that were obtained by means of the two diagnostic approaches suggests that the used molecular methods cannot entirely replace traditional culture-dependent methods, and vice versa.

Fungal endophytic communities in grapevines (Vitis vinifera L.) respond to crop management.

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.

Ecophysiological requirements and survival of a Trichoderma atroviride isolate with biocontrol potential.

Trichoderma atroviride SC1, isolated from decayed hazelnut wood in northern Italy in 2000, is a promising fungal agent for biological control of soil-borne plant pathogens. The objective of this research was to characterize the biology and ecology of this fungus, in order to determine its environmental parameter tolerance levels and its behavior in the phylloplane and soil systems. To better characterize T. atroviride SC1, the influences of pH, temperature, water activity and different nitrogen and carbon sources on its in vitro growth were evaluated. T. atroviride SC1 survival was assessed on strawberry leaves under controlled conditions in a greenhouse and in sterilized and non-sterilized soil samples kept at room temperature. Results showed that isolate SC1 is mesophilic and grows best at 25 degrees C. The fungus tolerates a wide range of pH levels, but growth was reduced on alkaline media (pH >or= 8). The nitrogen and carbon sources peptone, tryptone, nitrate, mannose, galactose and sucrose were associated with the highest mycelial biomass production, as compared with other potential sources of nitrogen and carbon. The fungus survived on strawberry leaves under greenhouse conditions (25 +/- 2 degrees C, RH = 60 +/- 10%) and grew in sterilized soils at room temperature (23 +/- 2 degrees C) for 45 d. However, no increase in mycelial dry weight was observed in non-sterilized soils. T. atroviride SC1 survived under the test conditions, showing a good potential for use in soil and foliar biocontrol applications.

Real-time PCR for detection and quantification of the biocontrol agent Trichoderma atroviride strain SC1 in soil.

Trichoderma (Hypocreales, Ascomycota) is a widespread genus in nature and several Trichoderma species are used in industrial processes and as biocontrol agents against crop diseases. It is very important that the persistence and spread of microorganisms released on purpose into the environment are accurately monitored. Real-time PCR methods for genus/species/strain identification of microorganisms are currently being developed to overcome the difficulties of classical microbiological and enzymatic methods for monitoring these populations. The aim of the present study was to develop and validate a specific real-time PCR-based method for detecting Trichoderma atroviride SC1 in soil. We developed a primer and TaqMan probe set constructed on base mutations in an endochitinase gene. This tool is highly specific for the detection and quantification of the SC1 strain. The limits of detection and quantification calculated from the relative standard deviation were 6000 and 20,000 haploid genome copies per gram of soil. Together with the low throughput time associated with this procedure, which allows the evaluation of many soil samples within a short time period, these results suggest that this method could be successfully used to trace the fate of T. atroviride SC1 applied as an open-field biocontrol agent.