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.

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.

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.

Effects of interacting conditions of sub-lethal levels of antioxidants, water potential ( ) and temperaturewere evaluated on growth, sclerotial characteristics, aflatoxin B1 (AFB1) production and aflD (=nor-1) geneexpression by Aspergillus flavus strain RCP08108. These studies were carried out on peanut meal extractagar osmotically modified to-2.8,-7.1,-9.9 and-16.0 MPa and incubated at 28 and 20 oC. The foodgrade antioxidants added were butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) at(1 + 1 mM-M1) and (5 + 5 mM-M2). To relate the aflD expression after toxigenic A. flavus grew underinteracting stress conditions, real-time PCR was used. Antioxidant mixtures caused a higher and significant(p < 0.001) reduction in growth rate. The major impact on size and volume sclerotia was producedby  ; followed by antioxidant mixtures. High AFB1 levels were observed in response to the M1 appliedat-7.1 MPa. Induction of the aflD gene was observed in response to the M1 treatment at-2.8,-7.1 and-9.9 MPa; but significant decreases of AFB1 production and aflD transcripts were observed; when thefungus grew in the presence of the M2 treatment.These results showed that it is necessary to apply food-grade antioxidants into the peanut storagesystem at levels higher than 5 mM. This is an important tool to avoid sub-lethal antioxidant doses thatcan lead to fungal growth, increase resistance structures, and stimulate aflD gene expression and AFB1accumulation in this substrate.

The objective of this study was to isolate andidentify native entomopathogenic fungi from differentcomponents of maize agroecosystem, and evaluate theirvirulence against Tribolium confusum, Rhyzopertha dominica,and Sitophilus zeamais, three insect pest vectors ofaflatoxigenic fungi. Paecilomyces and Metarhizium werethe most abundant genera isolated from the soil. Identificationof fungal cultures by DNA extraction, amplification,and sequencing showed that all isolates macro- andmicromorphological identified as P. lilacinus were Purpureocilliumlilacinum. The isolate JQ926223 showed thelowest LT50 for T. confusum (4, 66 days) and R. dominica(9, 38 days), and the isolate JQ926212 demonstrated similarLT50 for the three insects evaluated between the rangeof 11, 7 to 14, 95 days. Maximum mortality rate wasobserved for the isolate JQ926223. The isolates of Purpureocilliumlilacinum JQ926223 and JQ926212 may beconsidered good candidates for biologic control in theecosystem of stored maize.

Root knot nematodes (Meloidogyne spp.) are important pests of a wide range of crops, including tomato. Resistance of tomato to root-knot nematodes is conferred by the single dominant gene Mi-1, which currently is present in all commercially available resistant tomato cultivars. However, several resistance-breaking populations are being collected worldwide. Two isolates coming fromthe same standard population of Meloidogyne incognita, one selected for virulence against Mi-1 (SM1) and the other left avirulent (avr1), have been used in this study. qRT-PCR was used to detect transcript levels of the manganese superoxide dismutase (Mn-SOD) gene from (a)virulent pre-parasitic second-stage juveniles (J2). Over-expression of the Mn-SOD gene was found in the virulent isolate compared with the avirulent counterpart. The enzyme activity of membrane-bound mitochondrial Mn-SOD was assayed in J2 and adult females as the fraction of total SOD activity insensitive to hydrogen peroxide (H2O2). J2 from SM1 showed about a two-fold higher enzyme activity than J2 from avr1; conversely, no difference was found when adult females were tested. Proteins of J2 extracts were separated by n-PAGE on special mini-gels and stained for SOD. One slow migrating and three fast migrating bands were stained. SOD activity of the slow migrating band was H2O2-insensitive and enriched by treatment with the detergent Triton X-100. J2 survival was monitored in suspensions provided or not provided with the cell oxygen radical generator paraquat, at high concentration. Virulent J2responded to paraquat treatment by increasing life extension with respect to control conditions; by contrast, avirulent J2 suffered major mortality in the presence of paraquat. Mn-SOD gene expression is discussed in relation to nematode fitness in oxidative stresses and vir phenotype.

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.

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.