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A survey on entomopathogens was carried out in Arasbaran Biosphere Reserve soils during June 2018 using Galleria mellonella L. (Lepidoptera, Pyralidae) larvae as bait insect with a modified bait insect technique. Three entomopathogen's categories were recorded in 34 out of 36 soil samples (94.4%) collected from different natural habitat; the entomopathogens were identified as nematodes (23.5%), fungi (61%) and bacteria (15.5%) using molecular and morphological techniques.

Steinernema feltiae belongs to the feltiae-kraussei-oregonensis group, clade III, and is an ubiquitarian species of entomopathogenic nematode. It is found in all types of soil and in all types of habitat. Species identification in the entomopathogenic nematodes genera Steinernema is a very complex task, given the broad variability of both morphological and biological traits within populations of a single species. To accomplish this, molecular techniques have been adopted which, however, require additional knowledge. Particularly relevant would be the possibility of testing in a reliable way the variability between different populations of the same species, which might represent different strains with different biological properties. During numerous samplings in Italy, several strains of S. feltiae were isolated. In this paper we analyze the intraspecific variability of the main morphometric and biological data of juveniles and males of 50 Italian populations of S. feltiae. The aim of our work was to determine if morphometric and biological analysis were useful to identify characters having significant diagnostic value, allowing to reliably discriminate among strains. Seven characters routinely computed for morphology (5 morphometrics for infective juveniles, spicula and gubernaculum shapes for males) and 2 biological performances (time to achieve adult stage, reproduction and progeny) were considered. The results showed extreme variability from both morphological and biological points of view

Steinernema feltiae belongs to the feltiae-kraussei-oregonensis group, clade III, and is an ubiquitarian species of entomopathogenic nematode. It is found in all types of soil and in all types of habitat. Species identification in the entomopathogenic nematodes genera Steinernema is a very complex task, given the broad variability of both morphological and biological traits within populations of a single species. To accomplish this, molecular techniques have been adopted which, however, require additional knowledge. Particularly relevant would be the possibility of testing in a reliable way the variability between different populations of the same species, which might represent different strains with different biological properties. During numerous samplings in Italy, several strains of S. feltiae were isolated. In this paper we analyze the intraspecific variability of the main morphometric and biological data of juveniles and males of 50 Italian populations of S. feltiae. The aim of our work was to determine if morphometric and biological analysis were useful to identify characters having significant diagnostic value, allowing to reliably discriminate among strains. Seven characters routinely computed for morphology (5 morphometrics for infective juveniles, spicula and gubernaculum shapes for males) and 2 biological performances (time to achieve adult stage, reproduction and progeny) were considered. The results showed extreme variability from both morphological and biological points of view

The brown marmorated stink bug, Halyomorpha halys (Stål) (Hemiptera: Pentatomidae) (BMSB) is an exotic invasive insect for Italy and Georgia. At present BMSB is very active and makes alarming situation. Pest characterized by the massive increase and formation the large tracts of agrocenosies and urban area (inducing foliage, coniferous, ornamental plants as well). At present, BMSB has become a key pest in many crop and makes grate economical loses. Nowadays, due to lack of specific natural enemies, population density of this insect is not controlled by them. The aim of our study was to established potential entomopathogenic nematodes (EPNs) and determine their effectiveness on H. halys for its control. In experimental trial four entomopathogenic nematodes were used: two of Georgian species Heterorhabditis bacteriophora (HRB, GEO) and Steinernema borjomiensis; two Italian species Heterorhabditis bacteriophora (HRB, IT) and Steinernema apuliae. In laboratory assay (22°C and 80% RH), the mentioned nematodes were used in the following concentrations: 1:1000, 1:500, 1:200 infective juveniles (IJs) per adult of H.halys. The mortality of tested insects was estimated from the third day after treatment. Significant differences were observed between Georgia and Italian species. HRB (GEO) and S. borjomiensis at the high concentration -1:1000, mortality 46.6% -33.3%, in case 1:500 - 33.3%-32% and in 1:200 - 33.3%-13.3% were reached accordingly. More pathogenic were Italian species. HRB(IT) and S. apuliae in high concentration -1:1000, mortality achieved 93.3% -53.3% in case 1:500 - 93.3%-40%, and in 1:200 - 73.3%-33.2% were observed respectively. The emerging IJs were harvested and counted throughout the interval of 11-15 days.

Adults of Rhynchophorus, both ferrugineus (Olivier, 1790; RPW) and palmarum (Linnaeus, 1758; SAPW) (Coleoptera: Curculonidae) usually carry several associate or symbiotic organisms. We present evidences of nematodes regularly found and born by RPW females. In this contribution we consider the presence of paired pouches, which open on both sides of the membranes between the spiculum ventrale and vaginal base. Such structures, never observed previously, can be inflated and everted by injecting and pressuring the weevil with human physiological solution. In vivo and EtOH study by macrophotography, stereoscopy, bright field light microscopy LM, SEM and Cryo-SEM shows microtrichia-decorated ventral membrane and somewhat sclerotized dorsal cuticle. The cuticle is pitted by single or grouped apparent duct openings. Such openings may correspond to gland outlets and are served either by a pore-channeled sieve or by a long and ducted end-apparatus, on the body side of cuticle. Study of tenth of just cached vagrant wild RPW females reports the regular presence of a number of living nematodes aggregated inside the pouches, and the active dispersion of the worms when disturbed during the weevil genitalia dissection. These nematodes have been isolated and molecularly identified as Monochoides macrospiculum, by sequencing the D2-D3 expansion domains of the 28S rRNA gene. This finding confirms previous observations that the nematode reproduce in the body of RPW and the close association with RPW.In the case of R. palmarum, there was a related association with the nematode Bursaphelencus cocophilus. Dauer juveniles can be found in the tracheal sacs and move to the ovipositor of females to be injected into host palm to induce the red ring disease (RRD). In the same way B. cocophilus, Teratorhabditis palmarum and Diplogasteritus sp. (syn. Acrostichus rhynchophori), nematode species that co-participates of the etiology of RRD in coconut palms in Brazil, cohabit into the insect digestive tract (gut) and fecal material, as from studies with LM and SEM. Dauer juveniles of T. palmarum were also recovered from the excised genital capsules of newly emerged female weevils. More recently, the nematode B. fungivorus was found affecting coconut palms in the state of Para in Brazil, co-determining the RRD. In America, dauer juveniles of RPW could spread the RRD in healthy palms, by using the same mechanism of infection of R. palmarum. Evidences suggest a close interrelationship between the weevils and proper species of nematodes, that will be demonstrated in future studies. By the time, authors speculate about the synecological role of the nematodes and the consequence on damages inflicted to infested host palms, in connection with eventual further plant pathogenic partners. The given evidences enforce the presumed weevils vector role and suggests to consider the story of RPW not the introduction and subsequent invasion of a single species, but the biologically coordinated actions of a guild of symbionts.

The pinewood nematode, Bursaphelenchus xylophilus, is the causal agent of the devastating "pine wilt disease" (PWD). It is a quarantine pest originating from North America, and a major invasive pine pest in Eurasia. In Italy and Lebanon, it has never been detected so far, even if its vector Monochamus sp. is present in both countries. To identify the nematofauna associated with the main pine species, surveys in Lebanon (Horsh Beirut and Maten Region) and Southern Italy (Ionic coast and Salenton subregion) have been conducted. Wood samples were taken from pine trees showing declining symptoms; in addition, an evaluation has been carried on the presence of PWN and other nematode species associated with bark and wood boring insects. Nematodes have been extracted from pinewood, and morphological and molecular identification will be performed to identify the species. Considering the wide distribution of Monochamus spp., conifers and climate conditions, Italy and Lebanon show favorable conditions for the development, establishment and spread of PWN. This reinforces the importance of the monitoring system set up which has to contribute to the early detection of the nematode or latent infections.

HSP90 is a highly conserved molecule across all species, from bacteria to humans. It plays critical roles in the development, survival, chaperone function, cell cycle control, oncogenic transformation and also in response to abiotic stresses as heat or cold, or the presence of heavy metals. Little is known about biochemical and molecular responses to cold and heat stresses in entomopathogenic nematodes (EPN). In the present study, the isolation and the molecular characterization of the partial cDNA of the hsp-90 and its corresponding gene from the entomopathogenic nematode Heterorhabditis bacteriophora are reported. Four cDNA variants of the Hb-hsp-90 gene were isolated and sequenced. The unexpected finding of intron presence-absence in the Hb-hsp-90 genes of H. bacteriophora opens some questions about their function and their evolutionary significance.

A portion of the hsp-90 gene was amplified, using degenerate primers, in Steinernema thesami, S. tbilisiensis and Steinernema sp. isolated in Georgia. Two amplified products were obtained for each species and both fragments were cloned and sequenced. Blast search at NCBI revealed that both fragments for each species coded for hsp-90. Sequence analyses revealed that all three Steinernema species contained two different hsp-90 genes and the main difference was the intron presence-absence between hsp-90 genes. The hsp-9o genes containing introns showed conserved intron position between them with high sequence variation. This study showed, for the first time, the presence of two different isoforms for the hsp-90 in nematodes and the conserved position of the intron between the three Steinernema species. Further investigations are ongoing in order to characterize thermotolerant strains.

Pests residing in cryptic habitats, as insects which bore into the plant tissue (woodboring insects) or under the bark (bark beetles)the soil (wireworms) are very difficult to control because chemical pesticides are not able to reach the target. Usually, the only option to reduce infestations is the removal and destruction of infested or injured plants. One potential alternative to chemical insecticides for the control of cryptic insects can be the use of microbial control agents, as entomopathogenic nematodes (EPNs) and fungi (EPFs), because they may be able to penetrate into cryptic habitats and to be horizontally transmitted within the pest populations. Parahypopta caestrum (HUbner) and Cossus cossus (L.) (Lepidoptera, Cossidae) are highly-destructive cryptic pests in Europe. Parahypopta cuestrumcan I be considered the key pest of Asparagus spp. in Italy, due to its high destructiveness and the lack of effective control options available. The soil-borne larvae bore mines into the roots and the shoots, causing the total destruction of plantations after 2-3 I years. The goat moth C. cossus (L.) is a wood-boring pest whose larvae bore large galleries under the bark and even deeply into trunks and branches of fruit and forest trees, reducing plant growth and vigour, and causing limbs and branches to fall. Preliminary assays were performed in laboratory conditions in order to evaluate the infectivity of several EPF and EPN autochthonous strains against P. caestrum and C. cossus larvae. Results revealed the efficacy of these microbial control agents in killing the larvae, although a wide inter- and intra-specific variability in virulence was detected among different microbial strains. Considering the lack of effective chemical control means, the microbial control of the Asparagus moth and the goat moth by EPNs and EPFs reveals promising perspectives and needs further investigations.

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Specimens of nematode belonging to Oscheius genis was isolated through the Galleria bait method from soil collected in a karst cave of Tuscany (Central Italy). Molecular and morphological analyses were performed. Total DNA was extracted from individual nematodes and the mitochondrial COI, the ITS containing region and the 18S rRNA gene were amplified and sequenced. BLAST search at the NCBI discriminate this new taxon, similar to other Oscheius. This species belongs to Dolichura group. Cuticle is finely annulated, stoma is short and cheilorhabdion is simple, not well cuticularized. Female body is almost straight upon fixation, the reproductive system is didelphic and tail is short, conoid with pointed tip. Males are rare and similar to female in general morphology except for smaller size. Male body is straight when heat-killed, testis is single, ventral reflexed. Thei show peloderan bursa , tail short rounded and spicules slender and small. Ingective juveniles are slender withelongate tail and have stoma morphology similar to adult. The nematodes were cultured in Petri dishes on several substrates: Nutrient Agar, Escherichia coli, Botritis cinerea, meat baby food, without satisfactory results. Only Petri dishes method with G. mallonella larvae produced IJs, suggesting the entomopathogenicityof this new taxon.

Insect vectors of plant microorganism are primary targets of insect-borne pathogens control. Evidence about the vector role of Philaenus spumarius (L.) (Hemiptera Aphrophoridae) (Ps) in transmitting Xylella fastidiosa Wells, Raju et al. subsp. pauca OQDS strain (Xf), makes the control of this species of major importance. Data about vector life cycle, behaviour, population dynamics and bionomics are needed to design IPM control strategies (focused on the prevention of plant pathogen transmission) for both Integrated or Organic farming. Ps has an univoltine life cycle, it overwinters as an egg, nymphs develop in spring and female adults undergo a summer ovaric diapause. In OQDS and adjacent areas, the egg hatching occurs during the end of February and March. Juveniles live on herbs and bush twigs in “spittle” made purposely frothing mucus and faeces. Philaenus spumarius adults are active from May to December. Newly-hatched nymphs are gregarious but shift to solitary later in their development. Adults are food search-driven in their mass movements and shift from host to host following sap availability. The population is at maximum as eggs, and it decreases during pre-imaginal life-stages for several, not yet perfectly known mortality factors. Adults are still abundant in fall when they start to lay eggs. Population drops down almost to zero in middle November, but rare individuals are still active in middle December. In OQDS habitats, a mass movement from drying herbs to olive trees occurs around flowering time, and results in Xf acquisition from infected plants and subsequent new infections. We figure a Ps integrated control based on two control actions, namely a) mechanical control of nymphs and b) chemical control of adults. Careful timing both control actions is of paramount importance. We consider nymph control the key control action while adult control either may be a supplementary action in case of high vector population or in case of ineffective control of juveniles. In organic farming, only mechanical control of immatures can be applied. Unfortunately, we have no fully effective insecticides, or further control means options, by the time. Despite pyrethroids or Sweet Orange Essential Oil appear somewhat effective against the adult spittlebugs, their negatively impact on the olive biocenosis because they are broad spectrum. Finally, we are developing our index of impact for vector control actions based on Regione Puglia land use stats and willing to share and discuss the impact of control options with POnTE stakeholders.

The effects of a previously selected entomopathogenic fungus, Beauveria bassiana AL1 strain, and the B. bassiana based mycoinsecticide Naturalis (ATCC 74040 Beauveria bassiana strain) were evaluated on the system Ceratitis capitata - Psyttalia concolor in laboratory assays. First, the entomopathogenic fungal strains were tested for their virulence against 2, 4, and 6-days old puparia of C. capitata. Subsequently, P. concolor emergence from C. capitata puparia treated or not with the fungal strains was evaluated at three different time points (2, 4 and 6 days) from the parasitization and the following pupation. Results showed that the entomopathogenic fungal applications affected the medfly survival. The effect of fungal treatments was higher on 2-day puparia (49.16 and 51.33% of mycosed puparia for ATCC 74040 and AL1 strain respectively) while the rate of mycoses was lower and ranged between 39 and 27.16% when fungal treatments were performed on 4 and 6-day puparia. Furthermore, fungal treatments affected the P. concolor emergence (c.a. 80% in the untreated control) particularly when applied 2 days after the parasitization and the C. capitata pupation (43.16 and 47.83% for the ATCC 74040 and the AL1 strains respectively), while when treatments were performed on older puparia, the P.concolor emergence ranged from 63.33 to 68.66%. Results suggest that the entomopathogenic B. bassiana strains are effective against C. capitata puparia but they may be detrimental against its endoparasitoid P. concolor, particularly when applied in the earlier stages of the parasitization process.