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.

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.

Three isolates of Verticillium leptobactrum proceeding from egg masses of root-knot nematodes(RKN) Meloidogyne spp. and soil samples collected in Tunisia were evaluated against second-stage juveniles (J2)and eggs of M. incognita, to determine the fungus biocontrol potential. In vitro tests showed that V. leptobactrum is an efficient nematode parasite. The fungus also colonized egg masses and parasitized hatching J2. In a greenhouse assay with tomato plants parasitized by M. incognita andM. javanica, V. leptobactrum was compared with isolates of Pochonia chlamydosporia and Monacrosporium sp.,introducing the propagules into nematode-free or naturally infested soils. The V. leptobactrum isolates were active in RKN biocontrol, improving plants growth with a significantincrease of tomato roots length, lower J2 numbers in soil or egg masses, as well as higher egg mortalities. In a second assay with M. javanica, treatments with three V. leptobactrum isolates reduced egg masses on roots as well as the density of J2 and the number of galls. To evaluate the fungus capability to colonize egg masses a nested Real-time polymerase chain reaction (PCR) assay,based on a molecular beacon probe was used to assess its presence. The probe was designed on a V. leptobactrum ITS region, previously sequenced. This method allowed detection of V. leptobactrum from egg masses, allowing quantitative DNA and fungal biomass estimations.

Aspergillus flavus and A. parasiticus are the main species from section Flavi responsible for aflatoxinaccumulation in stored peanuts. A real-time PCR (RT-PCR) system directed against the nor-1 gene of theaflatoxin biosynthetic pathway as target sequence was applied to monitor and quantify Aspergillus sectionFlavi population in peanuts. Kernels were conditioned at four water activity (aW) levels and stored duringa 4-month period. The quantification of fungal genomic DNA in naturally contaminated peanut samples wasperformed using TaqMan fluorescent probe technology. Sensitivity tests demonstrated that DNA amountsaccounting for a single conidium of A. parasiticus RCP08300 can be detected. A standard curve relating nor-1copy numbers to colony forming units (cfu) was constructed. Counts of species of Aspergillus sectionFlavi from unknown samples obtained by molecular and conventional count (CC) methodologies werecompared. A correlation between cfu data obtained by RT-PCR and CC methods was observed (r=0.613;p<0.0001); and the former always showed values higher by 0.5-1 log units. A decrease of fungal densitywas observed throughout the storage period, regardless of the quantification methodology applied. Totalaflatoxin levels ranging from 1.1 to 200.4 ng/g were registered in peanuts conditioned at the higher aWvalues (0.94-0.84 aW).The RT-PCR assay developed appears to be a promising tool in the prediction of potential aflatoxigenic risk instored peanuts, even in case of low-level infections, and suitable for rapid, automated and high throughputanalysis.

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.

Filtrates of three isolates of the nematophagous fungus Verticillium leptobactrum were evaluated for their nematicidal activity against the root-knot nematode Meloidogyne incognita. The filtrates inhibited egg hatching, with maximum toxicity observed for isolate HR21 at 50% (v:v) dilution, after 7 days exposure. Filtrates also inactivated second-stage juveniles (J2) at 10-50% dilutions. A scanning electron microscopy study of treated eggs showed severe alterations caused by the filtrate of isolate HR43 on M. incognita eggs, which appeared collapsed and not viable, suggesting the production of chitin-degrading enzymes or other active compounds.

The aim of the work was to examine the effects of sub-lethal doses of butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) (1+1 mmol/l and 5+5 mmol/l), water activities (0.98, 0.95, 0.93, 0.89 aW) and temperatures (28, 20 °C) on growth, sclerotial characteristics, aflatoxin accumulation and aflD (=nor-1) transcript level by Aspergillus parasiticus RCP08300 on peanut based medium. Growth rate and aflatoxin production were inhibited by BHA-BHT mixture (1+1 mmol/l), regardless of environmental factor assayed. Although sclerotia number and aflD expression were stimulated by this treatment, sclerotia dry weight and volume were reduced by 62.3 and 31.2%, respectively. In contrast, when the fungus grew in presence of the higher dose of BHA-BHT mixture none or very low aflatoxin accumulation and aflD expression occurred. Similarly, A. parasiticus growth has been highly influenced by BHA-BHT (5+5 mmol/l) and interacting stress factors. Data show that sub-lethal antioxidant doses significantly reduced growth and aflatoxin accumulation by A. parasiticus but these treatments were not able to repress the expression of the early expression gene (aflD) involved in aflatoxin biosynthesis. Thus, this represents a high potential risk of stored peanuts contamination with aflatoxins

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.

Microorganisms play a key role in preserving soil fertility in forest agro-ecosystems.Arbuscular mycorrhizal (AM) fungi, which form symbioses with the roots of the most importantcrop species, are usually considered biofertilizers. AM fungi improve plant mineral nutrientacquisition, in exchange for C compounds derived from the photosynthetic process, which canresult in a positive host growth response and in an improved response to stress. Previous works,mainly performed in pots, suggest that the effects of the AM symbiosis on plant-water relationshipsare often subtle, transient, and probably circumstance- and symbiont-specific. Additionally, themechanisms involved in the mitigation to water stress effects on plant growth by AM fungi are stillto be elucidated. Within the framework of a project supported by CNR (AQUA), we studied theimpact of the AM symbiosis on tomato (var. San Marzano nano) tolerance to water stress. The firststep consisted in developing a protocol for water stress in presence/absence of an AM fungus. TwoAM species have been selected to be inoculated separately, i.e. Funneliformis mosseae andRizophagus intraradices, while non-mycorrhizal plants acted as controls. Leaf water potential(?leaf) and gas exchanges have been monitored until the plants reached a water stress state. Byvarying the duration of the drought, two levels of water stress were achieved, as confirmed by themeasurement of leaf water potential: moderate stress (water potential approximately -0.8 MPa) andsevere water stress (leaf water potential below -1.2 MPa), no stress (controls, leaf water potential at-0.3 MPa). Water Use Efficiency (WUE) has been calculated, showing a difference between the twoconsidered AM fungi. At the end of the experiment (when plants have reached a severe stress state),AM colonization degree has been calculated, and morphometric parameters have been considered(e.g., shoot length and diameter, internodal distance, stomatal density). For the followingbiochemical and molecular analyses, leaves and roots from all the set of plants have been collected.The levels of ABA, proline, H2O2 have been assessed, and the analysis of CAT and SOD activity isbeing determined. Gene expression analyses (RT-qPCR experiments) will be performed consideringgenes potentially involved in water stress response (e.g., a tomato gene coding for a dehydrin andLeNCED1) and in mycorrhizal symbiosis (e.g., fungal and plant PT genes), on leaves and rootsamples respectively. On the basis of the results so far obtained, R. intraradices has been selectedfor further experiments devoted to verify the impact of the AM symbiosis on the plant responses tobiotic stresses (i.e., aphids and nematodes) during drought conditions.

The role of nematology in food security is discussed. Nematology began with the classification of species. The onset of monocultures required effective nematode management tools. Recently, more complex approaches to management include ecology, natural sciences and genomics, with important fallouts in crop protection. The food security goal is to satisfy any dietary need for the healthy life of all but this message has not spread globally. Benefits achievable through crop technologies are not equally distributed and food security is not yet assured for several million people. Many social, political or economic factors are involved, and nematology may only partially contribute to achieve this goal. However, the scientific knowledge produced can contribute to solving some food security issues. Fundamental conditions are free access to education and science, as well as support for independent production of knowledge, improving regional and international cooperation and research.

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.

La investigación nematologica se ha intensificado cuando los monocultivos necesitaron herramientas eficaces para la gestión de los nematodos. La reducción de la disponibilidad de productos químicos por los riesgos para el medio ambiente y la salud humana ha exigido nuevos enfoques eco-sostenibles en la gestión de las especies màs importantes. Las investigaciones actuales incluyen estudios multidisciplinares, que van desde la biología molecular y la genómica hasta la ecología, en una visiòn holistica de la explotación del microbioma del suelo. El impacto de los microbios del suelo sobre los nematodos se puede observar como "suppressividad". Las especies microbianas activas a menudo son desconocidas. Pero la mayorìa de los microorganismos pueden ser identificados con análisis moleculares o estudios de metagenómica. Se espera que, junto a las aplicaciones de analisis genómicas y ecológia, ellos puedan ofrecer los conocimientos necesarios para métodos alternativos de control. Estos datos también son importantes en las regiones más pobres y marginales. La seguridad alimenticia implica el compromiso ético para una dieta suficiente de todas las personas. En las regiones menos industrializadas los nematodos afectan los ingresos de los agricultores y la disponibilidad de alimentos. El problema fitopatologico causado por los nematodos a menudo no es percibido y su gestión no es adecuada. Las tecnologías aplicadas son elementares con un suceso limitado, sumandose a desafíos como la pobreza, la demografía o los cambios climáticos. Herramientas sostenibles de gestión son demandadas, incluyendose el control biológico de nematodos, plantas mejoradas y tecnologías innovadoras de cultivo y diagnostica. Los problemas son desafiantes en términos de investigación y transferencia de tecnología. Aunque los avances son significativos y han enmejorado la comprensión del problema, su transferencia en el campo no es inmediata. La investigación en nematología, sin embargo, puede contribuir a alcanzar hitos importantes a través de programas de educación e investigación científica.

Nematology originated as a specific monophyletic science stemming from more general disciplines like zoology and natural history, and early studies concerned descriptions and taxonomy. Research work was intensified in the 50's, when monocultures of industrial cropping systems required effective nematode management tools. Actual research is part of advanced endeavours, largely multidisciplinar, integrating approaches ranging from ecology to molecular biology and genome studies. As defined by FAO, food security means that all people have the right to sufficient and safe food. A fundamental ethic committment of research in agriculture is then how to satisfy, in space and time, any dietary need and preference for a healthy life. A key issue concerns the transfer and application of main achievements and knowledge in other regions of the world, with different needs. Challenges include facing the consequences of demography and climate changes or other threats present. We have to recognize that the magnitude of the problems to afford is much more demanding than ever in the history of agriculture, either in terms of research and technology transfer. Given the social, political or economic roots of many food security threats, nematology may only partially contribute to reach this goal. Actual advances of scientific knowledge, spanning from genomes to plant biology and ecosystem services are, however, very significant. To solve or at least alleviate some of the food security problems affecting many rural systems and communities, nematology may contribute to reach specific milestones through the international cooperation. Fundamental are the freedom and right for access to education and scientific information for everyone, and the support of global efforts aiming at an independent production of knowledge.

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.

A review is provided about the most important microbial antagonists of plant parasitic nematodes and their use as biological control agents.

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.

The relationships between primary organic matter, soil biota and soil organic matter are still insufficiently understood, even though knowledge thereof is crucial for both understanding and optimizing sustainable management systems. Long-term field experiments, such as the 50 year V140/00 experiment on sandy soil in Müncheberg, provide the opportunity to analyse soil biota and long-term carbon dynamics in variants subjected to different management. The aim of this study was to relate soil carbon dynamics to soil biodiversity using a combined monitoring and modelling approach. Selected taxa from different functional groups (ecosystem engineers, litter transformers, microfoodwebs) were studied between 2010 and 2013 in treatments with different input of straw, manure and N-fertilizer. In addition, some integrated measures of soil quality were taken. We, inter alia, hypothesized that increased carbon sequestration would coincide with increased soil biodiversity, especially with respect to soil fauna. The results revealed that no simple relationship exists between soil biodiversity and carbon dynamics in the studied soil. The results are discussed in the framework of the ecosystem services concept.

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.

The emergence of virulent root-knot nematode populations, able to overcome the resistance conferred by some of the resistance genes (R-genes) in Solanaceous crops, i.e., Mi(s) in tomato, Me(s) in pepper, may constitute a severe limitation to their use in the field. Research has been conducted to evaluate the durability of these R-genes, by comparing the reproduction of several laboratory-selected and wild virulent Meloidogyne incognita isolates, on both susceptible and resistant tomatoes and peppers. We first show that the Me1 R-gene in pepper behaves as a robust R-gene controlling avirulent and virulent Me3, Me7 or Mi-1 isolates. Although the reproductive potential of the virulent isolates was highly variable on susceptible and resistant plants, we also confirm that virulence is highly specific to a determined Rgene on which selection has occurred. Another significant experimental result is the observation that a reproductive fitness cost is associated with nematode virulence against Mi-1 in tomato and Me3 and Me7 in pepper. The adaptative significance of trade-offs between selected characters and fitness-related traits, suggests that, although the resistance can be broken, it may be preserved in some conditions if the virulent nematodes are counter-selected in susceptible plants. All these results have important consequences for the management of plant resistance in the field.

Expression profiles were identified in the fungusPochonia chlamydosporia, a biological control agent ofplant parasitic nematodes, through a cDNA-amplifiedfragment length polymorphism approach. Two isolates withdifferent host ranges, IMI 380407 and IMI 331547, wereassayed in conditions of saprotrophic-to-parasitic transition,through in vitro assays. Gene expression profiles from threedifferent nutritional conditions and four sampling timeswere generated, with eggs of host nematodes Globoderapallida and Meloidogyne incognita. Expression of transcriptschanged in RNA fingerprints obtained underdifferent nutritional stresses (starvation in presence/absenceof eggs, or rich growth media). Transcript derived fragments(TDFs) obtained from the expression profilescorresponded to 6,800 products. A subset was sequencedand their expression profile confirmed through RT PCR. Atotal of 57 TDFs were selected for further analysis, basedon similarities to translated or annotated sequences. Genesexpressed during egg parasitism for both IMI 380407 andIMI 331547 were involved in metabolic functions, cellularsignal regulation, cellular transport, regulation of geneexpression, DNA repair, and other unknown functions.Multivariate analysis of TDF expression showed threegroups for IMI 380407 and one for IMI 331547, eachcharacterized by expression of genes related to eggsparasitism. Common amplification profiles among TDFclusters from both isolates also reflected a pool ofconstitutive genes, not affected by the nutritional conditionsand nematode associations, related to general metabolicfunctions. The differential expression of parasitism relatedgenes suggest a network of induced/repressed products,playing a role in fungal signaling and infection, with partialoverlaps in host infection and parasitism traits.