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.

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.

Some factors interfering with plant protection from phytoparasitic nematodes are reviewed in the light of changes brought about by the global warming in action. The mechanisms mainly concern changes in temperature and water regimes. The effects of climate changes on the epidemiology and management of the main phytoparasitic species occurring in Mediterranean environments include the alteration of the reproductive cycles due to plants productivity, the geographic dispersion by more northern or higher altitude shifts, the spread of vectors. Other related indirect mechanisms are feedback effects due to the reactions of cultivated species or weeds, and those related to natural enemies. The potential management of some operational tools are briefly discussed, including the development and application of models and monitoring. An exemple of modeling changes induced by increasing temperatures on the carrot cyst nematode Heterodera carotae is briefly discussed.

The bacteria associated to egg masses and second stage juveniles (J2) of root-knot nematodes (Meloidogyne spp.)were studied using three different soils from naturally infested farms in Apulia (Italy). The species were isolatedand identified with 16S rRNA sequencing or characterized with metabolic profiles. They included members ofgenera Pseudomonas and Bacillus, together with Lysinibacillus sphaericus and an unclassified Chryseobacterium sp.To identify potential biocontrol agents, a greenhouse assay was carried out with isolates of B. cereus, B. licheniformis,L. sphaericus, P. fluorescens and P. brassicacearum. The bacteria were introduced in soil on tomatoseedlings before inoculation with juveniles of Meloidogyne incognita, and significantly reduced the nematodedensities after two reproductive cycles. However, plants and root weights were not affected by treatments. In asecond assay with higher bacterial inocula, B. licheniformis and P. fluorescens significantly reduced the J2 gallingon tomato roots, at 21 dpi. The potential of rhizosphere bacteria in management of root knot nematodes isdiscussed.

EU legislation restricted many chemicals for root-knot nematodes (Meloidogyne spp., RKNs) control. Alternative ways of plant protection were hence investigated, based on the use of microbial formulations exploiting plant parasites antagonism. RKNs are severe and widespread pests causing extensive damage to crops in greenhouse and field. Several antagonistic microorganisms are suitable for biocontrol, including the nematophagous hyphomycete Pochonia chlamydosporia that parasitizes eggs to acquire additional nourishment and face competition with other soil microorganisms. A commercial product (POCHAR) was developed by Microspore based on P. chlamydosporia with other microbial inocula that can be applied through irrigation. The aim of this study was to test POCHAR's efficacy against RKNs on potato and its promotion effect on tomato in two field trials. Moreover, the research included trials to evaluate the best method for open-field application in crops not managed through drip irrigation, opening up the possibility to treat large areas without major technological needs. RKNs control with POCHAR represented a viable alternative to chemicals. In conclusion, the organic approach developed through the bioformulated product highlighted effective RKNs management, with a potential to sustain both plant nutrition and the related root protection needs.

The aim of this study was to test the efficiency of industrial bioformulations against RKNs attacking potato and other horticultural crops. Also, the research included trials to evaluate the best method for open-field application.

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.

The use of predatory nematodes as biological control agents of phytoparasitic nematodes has been re-evaluated only recently. To be effective, predatory nematodes should be easy to grow, cheap, and available on a commercial scale.In addition, they must show a reproductive rate sufficient to sustain a high population density and a significant longevity and stability for storage. The predators are not phytopathogenic nematodes, and among the positive outcomes of their introduction are remarkable their environment compatibility and safety for other non-target organisms, as well as the ability to search for prey. These features are present in Diplogasteridae. Their adaptability enables them to withstand changing climatic conditions, as well as the temporal variability between predator and prey. It should also be emphasized the ability to disperse, persist and reproduce in the absence of prey, the spectrum of action, all factors ideal for the management of phytoparasitic species. Adverse factor is cannibalism, due the lack of preys, that can reduce their biological control potential. We herein present preliminary data on occurrence, reproduction and intestine bacterial flora of the predatory nematode, Koerneria sudhausi.

A new species of Hirsutella was isolated from unidentified mites on Petri plates inoculated with soil and root fragments collected from asparagus rhizosphere at Virú, Northern Peru. The fungus differs from other Hirsutella species by an envelope surrounding the conidium, conidia dimension and DNA sequences. In PDA cultures, the mycelium produced aerial hyphae with conidiogenous cells mainly at right angles, occasionally showing a secondary conidiophore. The solitary conidia are cymbiform, slightly apiculate, 5.0-6.0 × 3.0-4.0 ?m. Phylogenetic analyses with partial rRNA and ?-tubulin gene sequences confirmed the fungus as an Hirsutella (Ophiocordycipitaceae). Closest species shown by maximum likelihood and neighbor-joining trees were H. nodulosa and H. aphidis, from which the new species differs for conidium or conidiogenous cells dimensions, lack of synnemata and host type. A recombination event was also detected in the rRNA of the holotype strain, involving Ophiocordyceps sinensis as major parent and O. cochlidiicola as minor parent. A complement, inverted insertion was also found in its rRNA, involving part of the ITS2 and 5.8S regions, flanked by two short nucleotide arrays. Due to conidia dimension and phylogenetic position, the fungus is described as Hirsutella tunicata sp. nov. A review of mononematous Hirsutella species is provided.

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.