To study the effect of cropping and plant cover on soil microbial diversity three adiacent sites characterized by the same soil, but differing for land use and cover, were sampled at Carovigno (Brindisi). Soil samples were collected from a traditional greenhouse producing horticultural crops, a close olive grove and an adiacent mediterranean (Quercus ilex) forest spot. The soil bacterial communities from replicated samples were identified with a metagenomic Next Generation Sequencing (NGS) approach. Total RNAs were extracted from 2 g soil subsamples and the V3-V4 hypervariable regions of the 16S rRNAs were sequenced with the Illumina MiSeq technology. A total of 2.46 x 10^6 reads was produced from 13 samples, of which 85% passed the quality threshold, yielding an average of 97 - 239 x 10^3 reads per sample. Almost all (99%) sequences belonged to the Kingdom Bacteria, and 30% were informative up to the species level. Using the Greengenes classification system, the average number of species per sample was around 10^3. Most represented phyla in all samples were Proteobacteria, Actinobacteria, Firmicutes, Planctomycetes and Verrucomicrobia, with Alpha-, Beta- and Gamma-Proteobacteria as most represented classes. NGS data showed that samples from cultivated soils (olive and vegetables) had higher frequencies (5-10%) of Bacillales, which were under-represented in the mediterranean forest. Data analysis at the species level is under course to identify changes in the bacterial composition at deeper taxonomic levels, as related to agricultural practices.

Olive anthracnose, a fungal disease caused by species of the genus Colletotrichum, is responsible for severe yield losses and poor oil quality. Typical symptoms appear in autumn or early winter, when the drupes begin to ripen. Under favorable conditions, symptoms on branches and leaves can also occur, leading to chlorosis, severe defoliation, and death of woody organs. Symptomless infection of flowers and blights have also been reported. Latent fruit infections could play an important role as the inoculum source for the autumn-winter epidemics. Application of systemic fungicides has proved effective in field trials, and pre-flowering sprays contribute to reduce latent infection and the inoculum density for autumn infection. However, public concerns about potential risks on the environment and human health promoted the search for alternative and sustainable means. Therefore, the activity of a new sulfur-based product and biocontrol agents (Bacillus subtilis, and endophytic isolates of Aureobasidium pullulans) in reducing the incidence of olive anthracnose was evaluated under field conditions. The sulfur-based product and B. subtilis applied at the pre-flowering stage were as effective as the chemical fungicides in reducing the incidence of latent infections on drupes. Moreover, some endophytic strains of A. pullulans provided high protection levels against Colletotrichum spp. when applied at the pre-flowering and veraison stages. Overall, data indicated that olive anthracnose can be controlled by using biological means and new products could be considered for introduction in the list of the organic product specification.

Biocontrol effects of arbuscular mycorrhizal fungi against nematodes have been reported in various plants. Literature data suggest that mycorrhizal symbiosis affects plant-water relationships as well. Moreover, it is well established that water deficit and infection with plant parasitic nematodes represent two environmental stresses with interacting effects under field conditions. Few data are available on the effect of combined mycorrhizae and water stress on the development of nematode feeding sites. We studied the impact of Rizophagus intraradices symbiosis on Meloidogyne incognita and tomato (cv San Marzano nano) interaction, with or without water stress. Plants inoculated or not with R. intraradices, maintained in growth chamber at 25°C, were exposed to mild water stress and subsequently infected with J2s of M. incognita. Galls hand-dissected at 7 and 14 days were processed for light microscopy observations. The analysis performed on cross sections of galls i) with or without mycorrhizae, ii) with water stress and iii) with mycorrhizae and water stress, showed changes in the morphology of galls and nematode feeding sites, affecting density and dimensions of nuclei. The symbiosis with R. intraradices and water stress hampered development and structure of giant cells, showing an effect on the modulation of host plant metabolism. NGS-based analysis of galls transcriptome is under study, to unravel the molecular pathways involved in this multiple interaction. Research partially funded by CNR, Progetto Premiale Aqua.

Small RNAs play a key role in the plant-parasite interaction, regulating critical effector genes needed for infection. However, little is known about the effects of endophytes on non coding (nc)-RNAs expression in plant. To elucidate micro(mi)RNAs and other ncRNAs regulatory participation in plant-endophyte interactions, we used Illumina's NGS technology to sequence small RNAs (sRNAs) in tomato roots inoculated and not inoculated with the fungus Pochonia chlamydosporia. In both treatments, Sly-miR166a/b was the most abundant tomato miRNA, followed by sly-miR166c-3p. The two miRNAs together accounted for 81% and 74.1% of the annotated tomato miRNAs in P. chlamydosporia not inoculated or inoculated roots. Such highly expressed miRNAs are likely to have important roles in roots, considering that in epigeal parts of tomato and other plants the most abundant miRNA reported is miRNA-156. Endophytism by P. chlamydosporia affected miRNAs and other nc-sRNAs expression, with 26 miRNAs differentially expressed between the two treatments (up regulated with fold changes 2 to 9). Their 154 potential target genes involve apoptosis, primary metabolism and binding functions i.e. Squamosa promoter binding-like protein. Comparative analysis showed that 48 out of 5055 P. chlamydosporia down-regulated tomato genes, from a previous RNAseq experiment, are miRNA targets (with fold changes 2 to 16). Furthermore, five miRNAs (sly-miR9473-5p, sly-miR169c, sly-miR169a, sly-miR9476-5p and sly-miR1918) were found only in presence of the fungus. We also identified many other classes of sRNAs, including transfer RNA (tRNA)-derived sRNAs, some of which were also differentially expressed between the two treatments. Data provide valuable clues to understand the properties of sRNAs with a new insight on the role of miRNAs and other sRNAs in the host-endophyte interaction. A better understanding of ncRNA-mediated plant-endophyte interaction may sustain management of pests and diseases, and promote growth. MiRNA-based manipulations as gene suppressors, i.e. artificial miRNAs, may emerge as a new alternative approach for the improvement of crops and control of nematode pests.

The molecular mechanisms active during the endophytic phase of the fungus Pochonia chlamydosporia are still poorly understood. In particular, few data are available on the links between the endophyte and the root response, as modulated by noncoding small RNAs. In this study, we describe the microRNAs (miRNAs) that are differentially expressed (DE) in the roots of tomato, colonized by P. chlamydosporia. A genome-wide NGS expression profiling of small RNAs in roots, either colonized or not by the fungus, showed 26 miRNAs upregulated in inoculated roots. Their predicted target genes are involved in the plant information processing system, which recognizes, percepts, and transmits signals, with higher representations in processes such as apoptosis and plant defense regulation. RNAseq data showed that predicted miRNA target genes were downregulated in tomato roots after 4, 7, 10, and 21 days post P. chlamydosporia inoculation. The differential expression of four miRNAs was further validated using qPCR analysis. The P. chlamydosporia endophytic lifestyle in tomato roots included an intricate network of miRNAs and targets. Data provide a first platform of DE tomato miRNAs after P. chlamydosporia colonization. They indicated that several miRNAs are involved in the host response to the fungus, playing important roles for its recognition as a symbiotic microorganism, allowing endophytism by modulating the host defense reaction. Data also indicated that endophytism affects tRNA fragmentation. This is the first study on miRNAs induced by P. chlamydosporia endophytism and related development regulation effects in Solanum lycopersicum.

MicroRNAs (miRNAs) are endogenous small-RNAs transcribed from non-coding DNA, matching a target messenger RNA to repress translation or induce cleavage. They act in almost every biological plant activity e.g. development, abiotic stress tolerance, signal transduction, and in defense from pathogens or parasites. To elucidate miRNAs role in plant-endophyte interactions, we constructed libraries from roots of Solanum lycopersicum endophytically colonized (Pmi) or not (Pm) by the hyphomycete Pochonia chlamydosporia. This fungus shows endophytic behaviour with growth promotion or nematode biocontrol effects. No data are available on tomato miRNAs role and targets in the endophytic interaction. Illumina(TM) NGS of small-RNAs yielded 9 o 106 (Pmi) and 12 o 106 (Pm) reads per library. CLC Genomics Workbench was used for trimming, counting, annotation and data analysis. Non-redundant, unique small-RNAs (869178 in Pmi, 958026 in Pm), were produced. MiRNAs expression was affected by endophytism. Analyses of tomato miRNAs (miRBase, rel.21), revealed miR156 and miR168 (conserved across higher plants), as most abundant in roots. Four further miRNAs (miR169a, miR169c, miR9473 and miR9476), out of 75 known in tomato, were expressed only in Pmi, with seven further (miR169d, miR1917, miR169e, miR394, miR167a, miR5300 and miR9475) over-expressed and 27 down-regulated (fold change range: 1.2-4.8). 37 remaining miRNAs were equally expressed in both conditions. A Pmi comparative analysis showed that 1732 out of 5055 Pmi down-regulated genes were miRNA targets, involved in structural protein, metabolism, transcription factor, growth and development, stress-related, signaling pathways, storage and other processes.

Biological control relying on soil microorganisms may offer feasible and sustainable perspectives in many agrosystems for management of soil pests such as root-knot nematodes (RKNs, Meloidogyne spp.). The rhizosphere environment shows a complex of soil microbial communities, including beneficial organisms such as specialized bacterial pathogens and/or of rhizosphere fungi. New advanced technologies like Next Generation Sequencing (NGS) may enlarge our knowledge about the biodiversity and role of these rhizosphere communities. The numbers of microbial species usually range around 10(3) - 10(4) taxonomic units . g(-1) of soil. This dimension suggests that the nematode antagonists known today represent only a fraction of the total number potentially available. The soil microbiome activity may lead to soil suppressivity, a stable nematode control effect related to species undetected or undescribed. Known parasitic microorganisms like the bacterium Pasteuria penetrans show specific and density-dependent links with the host. Other species such as the nematophagous fungus Pochonia chlamydosporia can also induce plant growth promotion effects. NGS transcriptomic data indicate that it can elicit a plant response to many biotic and abiotic stresses. In conclusion, the biodiversity of antagonists and the mechanisms they exert in nematode regulation have yet to be fully explored. However, new high-throughput analytical technologies may fill this gap. Experimental assays for RKN management and/or plant growth promotionindicate that biological management of nematodes is a promising alternative to chemicals. However, this approach requires detailed knowledge about the composition, role and effects of the microbial community present in soil and/or about the right and accurate aggregate of biological entities to apply.

Soil microbiome has a significant impact on phytoparasitic nematodes. However, given the number of species present in soil, its role is difficult to study with traditional approaches. Advanced technologies, i.e. Next Generation Sequencing (NGS), allow the identification and quantitative determination of almost all species in a sample, enlarging our view about their rhizosphere effects. Metagenomic studies showed that microbial species may reach 104 or more taxonomic units in a few g of soil. Comparing these numbers with the nematode bacterial antagonists known we can infer that biocontrol studies have yet a large space to explore. The activity of soil microbiome on nematodes can show suppressivity, but active species may remain undetected or unknown. To measure suppressive potential, a study was carried out with soil from a carnation crop with patchy Meloidogyne spp. infestations. After 4-years continuous croppings on tomato, 40% of pots showed nematode extinction, suggesting suppression or biological containment on a long time scale. In vitro NGS studies are needed to identify the role of a specific microbial component. The endophytic and nematode parasitic fungus Pochonia chlamydosporia showed differential expression of resistance and defensive genes in colonized tomato roots. Depending on the experimental approach, NGS studies provide a wide basis to understand the impact of soil microbiome and how phytonematode attacks may be balanced through management.