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The meadow spittlebug Philaenus spumarius L. is the vector for the bacterium Xylella fastidiosa subspecies pauca, involved in olive quick decline syndrome (OQDS) in Salento (Italy). Control of P. spumarius is key to limiting transmission of the bacterium, and an innovative approach can be based on effective natural compounds and biocontrol agents. Entomopathogenic fungi are an important source of bioactive natural molecules that play a role in the relationship between microorganisms and insects.Pathogenicity bioassays, performed by dipping adults of P. spumarius in either fungal culture suspension (120 mg mLT. chlorosporum GJS 91-150 affected the survival of P. spumarius adults. The lethal effect was not associated with the development of mycelium on the cuticle, but seems due, at least partly, to fungal metabolites released in the culture medium. The fungus tested here has good potential for the development of effective low-environmental impact control strategies for P. spumarius and suppression of X. fastidiosa. © 2022 Society of Chemical Industry.

The meadow spittlebug, Philaenus spumarius, is a highly polyphagous widespread species, playing a major role in the transmission of the bacterium Xylella fastidiosa subspecies pauca, the agent of the “Olive Quick Decline Syndrome”. Essential oils (EOs) are an important source of bio-active volatile compounds that could interfere with basic metabolic, biochemical, physiological, and behavioural functions of insects. Here, we report the electrophysiological and behavioural responses of adult P. spumarius towards some EOs and related plants. Electroantennographic tests demonstrated that the peripheral olfactory system of P. spumarius females and males perceives volatile organic compounds present in the EOs of Pelargonium graveolens, Cymbopogon nardus and Lavandula officinalis in a dose-dependent manner. In behavioral bioassays, evaluating the adult responses towards EOs and related plants, both at close (Y-tube) and long range (wind tunnel), males and females responded differently to the same odorant. Using EOs, a clear attraction was noted only for males towards lavender EO. Conversely, plants elicited responses that varied upon the plant species, testing device and adult sex. Both lavender and geranium repelled females at any distance range. On the contrary, males were attracted by geranium and repelled by citronella. Finally, at close distance, lavender and citronella were repellent for females and males, respectively. Our results contribute to the development of innovative tools and approaches, alternative to the use of synthetic pesticides, for the sustainable control of P. spumarius aiming to contrasting the expansion of X. fastidiosa.

Simple Summary The spotted wing drosophila (SWD) is an invasive fruit fly that causes serious economic damage to many fruit crops. Monitoring is the first step for any management program to determine the characteristics of a pest. For this purpose, there are no efficient baits registered to date. Certain bacteria release chemical compounds that attract SWD. We studied the bacterial impact on the enhanced attractiveness of a commercial bait (Droskidrink®) under field and laboratory conditions. At first, Oenococcus oeni belonging to lactic acid bacteria (LAB) was found to release chemical compounds that were highly attractive for SWD. The attractiveness of Droskidrink® bait was increased by O. oeni culture, resulting in a higher capture rate of SWD in traps. Therefore, our findings suggest the use of the bacterial culture inside the commercial SWD baits. The use of these kinds of baits can minimize the risk of pest outbreaks in fruit orchards in both domestic and wild environments. Our pest management approach is farmer-friendly in all aspects, as well as the food sector. Abstract (1) Monitoring of Drosophila suzukii is based on the use of effective traps and baits. The current baits are insufficient to provide efficient monitoring. The use of bacteria as bio-catalyzers to produce bioactive volatiles may improve flies’ attraction. Thus, we conducted this work to improve Droskidrink® bait’s attractiveness using lactic acid bacteria. (2) Different baits that were based on the use of Droskidrink® were assessed for flies’ attraction in a Droso-Trap® in a vineyard. Oenococcus oeni, Pediococcus spp., and Lactobacillus spp. were used. The performance of the most attractive species, O. oeni, inoculated into Droskidrink® was assessed in laboratory tests. The responses of female flies to volatiles produced by Droskidrink® with O. oeni strains were recorded by electroantennography. (3) Preliminary field assessment of baits recorded O. oeni as the most attractive species. Three strain groups showed adaptation to test conditions. Volatiles extracted by the headspace of baits inoculated with O. oeni, elicited electroantennographic responses from fly antennae. (4) Droskidrink® inoculated with O. oeni is a highly attractive bait for monitoring. These findings will be useful for improving the attractiveness of D. suzukii commercial baits based on the utilization of LAB volatiles in a strain-dependent manner.

Paenibacillus larvae is the causative agent of American foulbrood (AFB), a severe bacterial disease that affects larvae of honeybees. The present study evaluated, in vitro, antimicrobial activity of sixty-one Lactiplantibacillus plantarum strains, against P. larvae ATCC 9545. Five strains (P8, P25, P86, P95 and P100) that showed the greatest antagonism against P. larvae ATCC 9545 were selected for further physiological and biochemical characterizations. In particular, the hydrophobicity, auto-aggregation, exopolysaccharides production, osmotic tolerance, enzymatic activity and carbohydrate assimilation patterns were evaluated. The five L. plantarum selected strains showed suitable physical and biochemical properties for their use as probiotics in the honeybee diet. The selection and availability of new selected bacteria with good functional characteristics and with antagonistic activity against P. larvae opens up interesting perspectives for new biocontrol strategies of diseases such as AFB.

Ascosphaera apis is an entomopathogenic fungus that affects honeybees. In stressful conditions, this fungus (due not only to its presence, but also to the combination of other biotic and abiotic stressors) can cause chalkbrood disease. In recent years, there has been increasing attention paid towards the use of lactic acid bacteria (LAB) in the honeybees’ diets to improve their health, productivity and ability to resist infections by pathogenic microorganisms. The screening of 22 strains of Lactiplantibacillus plantarum, isolated from the gastrointestinal tracts of honeybees and beebread, led to the selection of five strains possessing high antagonistic activity against A. apis. This study focused on the antifungal activity of these five strains against A. apis DSM 3116 and DSM 3117 using different matrices: cell lysate, broth culture, cell-free supernatant and cell pellet. In addition, some functional properties and the antioxidant activity of the five L. plantarum strains were evaluated. All five strains exhibited high antagonistic activity against A. apis, good surface cellular properties (extracellular polysaccharide (EPS) production and biofilm formation) and antioxidant activity. Although preliminary, these results are encouraging, and in future investigations, the effectiveness of these bacteria as probiotics in honeybee nutrition will be tested in vivo in the context of an eco-friendly strategy for the biological control of chalkbrood disease.

Lactic acid bacteria (LAB) are an important group of honeybee gut microbiota. These bacteria are involved in food digestion, stimulate the immune system, and may antagonize undesirable microorganisms in the gastrointestinal tract. Lactobacillus kunkeei is a fructophilic lactic acid bacterium (FLAB) most frequently found in the gastrointestinal tracts of honeybees. Ascosphaera apis is an important pathogenic fungus of honeybee larvae, it can colonize the intestine, especially in conditions of nutritional or environmental stress that cause microbial dysbiosis. In this work, some functional properties of nine selected L. kunkeei strains were evaluated. The study focused on the antifungal activity of these strains against A. apis DSM 3116, using different matrices: cell lysate, broth culture, cell-free supernatant, and cell pellet. The cell lysate showed the highest antifungal activity. Moreover, the strains were shown to possess good cell-surface properties (hydrophobicity, auto-aggregation, and biofilm production) and a good resistance to high sugar concentrations. These L. kunkeei strains were demonstrated to be functional for use in &ldquo, probiotic syrup&rdquo, useful to restore the symbiotic communities of the intestine in case of dysbiosis and to exert a prophylactic action against A. apis.

The meadow spittlebug, Philaenus spumarius L. (Hemiptera, Aphrophoridae) is a commonly found vector of Xylella fastidiosa Wells et al. (1987) strain subspecies pauca associated with the "Olive Quick Decline Syndrome" in Italy. To contribute to the knowledge of the adult P. spumarius chemoreceptivity, electroantennographic (EAG) responses of both sexes to 50 volatile organic compounds (VOCs) including aliphatic aldehydes, alcohols, esters, and ketones, terpenoids, and aromatics were recorded. Measurable EAG responses were elicited by all compounds tested. In both sexes, octanal, 2-octanol, 2-decanone, (E)-2-hexenyl acetate, and vanillin elicited the strongest antennal amplitude within the chemical groups of aliphatic saturated aldehydes, aliphatic alcohols, aliphatic acetates and aromatics, respectively. Male and female EAG responses to sulcatol, (±)linalool, and sulcatone were higher than those to other terpenoinds. In both sexes, the weakest antennal stimulants were phenethyl alcohol and 2-pentanone. Sexual differences in the EAG amplitude were found only for four of test compounds suggesting a general similarity between males and females in antennal sensitivity. The olfactory system of both sexes proved to be sensitive to changes in stimulus concentration, carbon chain length, and compound structure. Compounds with short carbon chain length (C5-C6) elicited lower EAG amplitudes than compounds with higher carbon chain length (C9-C10) in all classes of aliphatic hydrocarbons with different functional groups. The elucidation of the sensitivity profile of P. spumarius to a variety of VOCs provides a basis for future identification of behaviorally-active compounds useful for developing semiochemical-based control strategies of this pest.

In this study we report the effects of fungal metabolites isolated from cultures of the fungus Trichoderma citrinoviride ITEM 4484 on the feeding preference of the aphid Rhopalosiphum padi, a major pest of cereal crops. Different phagodeterrent metabolites were purified by a combination of direct and reverse phase column chromatography and thin-layer chromatography. Chemical investigations, by spectroscopic and chemical methods, led to the identification of different long chain primary alcohols (LCOHs) of the general formula R-OH, wherein R is a long, unbranched, unsubstituted, linear aliphatic group. LCOHs have been reported as components of lepidopteran pheromone blends, but their phagodeterrent effect to aphids is herein reported for the first time. The effects of LCOHs on R. padi were studied by behavioral and electrophysiological bioassays. Feeding preference tests that were carried out with winged and wingless morphs of R. padi showed that LCOHs had high phagodeterrent activity and restrained aphids from settling on treated leaves at a concentration as low as 0.15 mM (0.036 g/l). The results of different electrophysiological analyses indicated that taste receptor neurons located on the aphid tarsomeres were involved in the LCOHs perception. Behavioral assays carried out with some commercial agrochemicals, including azadirachtin A, pyrethrum and a mineral oil-based product, in combination with 1-hexadecanol, the LCOH most abundantly produced by T. citrinoviride ITEM 4484, showed that these different active principles could be applied together, resulting in a useful increase of the phagodeterrent effect. The data shown indicate that these compounds can be profitably utilized for novel applications in biotechnical control of aphid pests. Furthermore, the tested LCOHs have no chiral centers and therefore can be obtained with good yield and at low cost through chemical synthesis, as well as from natural sources.

This study investigated the effects of a strain of the fungus Lecanicillium lecanii (Zimm.) Zare and Gams (Hypocreales: Ascomycota) on the aphid Schizaphis graminum (Rondani) (Hemiptera: Aphididae). The fungus was administered to fourth instar nymphs and to alate and apterous adult morphs as a ground rice-kernel formulation. This study showed that L. lecanii formulation affected the survival of the aphids and interacted differently with the studied morphs, the Lethal Time values being lower for alate compared to apterous morphs and nymphs. The treatment also caused a significant reduction in the fecundity of the three treated aphid groups. Histological analysis revealed that the hyphae invaded the host hemocoel of a limited number of alate and apterous morphs: the fungus only entered through the spiracles. Scanning Electron Microscope observations revealed that L. lecanii adhered to the body surface of both adults and nymphs without differentiation. In conclusion, the present study suggests that this strain might be a good candidate for a programme of biological control of S. graminum and other aphid species.

Chromatin organization in the holocentric chromosomes of the aphid Schizaphis gruminum has been investigated at a cytological level after C-banding, NOR, Giemsa, DAPI and CMA, staining. C-banding technique showed the presence of numerous C bands on the two X chromosomes both in telomeric and intercalary regions, whereas autosomes show a small number of heterochromatic bands. Contrary to the results with other aphid species, in S. gruminum the C-banding pattern is peculiar to each chromosome pair, thus allowing the identification of homologues and the reliable reconstruction of a karyotype. These cytogenetic data could be useful for the identification of chromosomal rearrangement eventually occurred between different S. gruminum biotypes. Moreover, silver staining and fluorescent in situ hybridization (FISH) with a 28s rDNA probe localized rDNA genes on one telomere of each X chromosome; these are the only brightly fluorescent C-positive regions revealed after CMA, staining, whereas all other heterochromatic bands are DAPI positive. Giun Curlo Manicurdi, Dipartimento di Biologiu Animule, Vide Berengario 14, 41 100 Modena, Italy. E-mail: mani

Funghi appartenenti a diverse specie del genere Trichoderma sono stati fino ad ora utilizzati per il controllo di funghi fitopatogeni e il loro impiego ha confermato l'assenza di effetti negativi per la salute umana e l'ambiente. Nostri precedenti studi hanno messo in evidenza per alcune specie del genere Trichoderma una potenziale attività di controllo degli afidi. Tali insetti rappresentano uno dei più importanti gruppi di fitomizi di molte coltivazioni di notevole interesse economico. Il danno provocato alla pianta ospite non è dovuto solo alla sottrazione di elementi nutritivi e ormonali attraverso la suzione della linfa, ma anche e soprattutto, alla capacità di trasmettere virus. Test di preferenza alimentare da noi condotti, hanno evidenziato una significativa attività fagodeterrente di colture dell'isolato di T. citrinoviride ITEM 4484 nei confronti di diverse specie di afidi tra cui il polifago Myzus persicae (Sulzer) e Rhopalosiphum padi un diffuso parassita dei cereali. Ulteriori indagini condotte mediante metodi spettroscopici (essenzialmente NMR e tecniche MS) e volte ad identificare i composti fungini con attività fagodeterrente prodotti dall'isolato di T. citrinoviride hanno permesso di isolare e caratterizzare quattro distinti gruppi di metaboliti tra di loro strutturalmente correlati. I primi due metaboliti, denominati citrantifidiene e citrantifidiol, sono rispettivamente un esa-1,3-dienil estere dell'acido acetico simmetrico ed un cicloesadien-1,3-diolo tetrasostituito. Altri due composti individuati appartengono alla famiglia dei bisorbicillinoidi, e sono il bislongiquinolide e il diidrotricodimerolo. Il quarto gruppo, il quale mostra una spiccata attività fagodeterrente, è composto da diversi alcoli primari a catena lunga: esadecanolo, octadecanolo, cis-9-octadecanolo e trans-9-octadecanolo. Questi composti non hanno centri chirali e oltre ad essere reperibili in natura possono essere facilmente sintetizzati con una buona resa. Per questi alcoli a catena lunga e per altri ad essi chimicamente correlati è stata depositata una domanda di brevetto nazionale (MI2011A000831), con successiva estensione a livello internazionale (PTC IB2012/052383) per la loro attività fagodeterrente che potrà essere utilizzata per il controllo biotecnico degli afidi.

Fungal strains belonging to the genera Fusarium, Paecilomyces and Trichoderma were tested in vitro in order to study their effects against Schizaphis graminum, one of the major pests of cereal crops around the world. Biological assays were performed using a solid formulation that was obtained from fungal cultures grown on rice and then finely ground ( 60%) within 10 minutes after application. As F. larvarum ITEM 2139 did not produce metabolites toxic to mammals, it might be a good candidate as a biocontrol agent of S. graminum in the field.

BackgroundThe activation of autophagy has been extensively described as a pro-survival strategy, which helps to keep cells alive following deprivation of nutrients/growth factors and other stressful cellular conditions. In addition to cytoprotective effects, autophagy can accompany cell death. Autophagic vacuoles can be observed before or during cell death, but the role of autophagy in the death process is still controversial. A complex interplay between autophagy and apoptosis has come to light, taking into account that numerous genes, such as p53 and Bcl-2 family members, are shared between these two pathways.Methodology/principal findingsIn this study we showed a potent and irreversible cytotoxic activity of the stable Curcumin derivative bis-DeHydroxyCurcumin (bDHC) on human colon cancer cells, but not on human normal cells. Autophagy is elicited by bDHC before cell death as demonstrated by increased autophagosome formation -measured by electron microscopy, fluorescent LC3 puncta and LC3 lipidation- and autophagic flux -measured by interfering LC3-II turnover. The accumulation of poly-ubiquitinated proteins and ER-stress occurred upstream of autophagy induction and resulted in cell death. Cell cycle and Western blot analyses highlighted the activation of a mitochondrial-dependent apoptosis, which involves caspase 7, 8, 9 and Cytochrome C release. Using pharmacological inhibitions and RNAi experiments, we showed that ER-stress induced autophagy has a major role in triggering bDHC-cell death.Conclusion/significanceOur findings describe the mechanism through which bDHC promotes tumor selective inhibition of proliferation, providing unequivocal evidence of the role of autophagy in contrasting the proliferation of colon cancer cells.

The effects on animals feeding on resistant plants of widespread use of herbicides were studied. Aphids of the species Sipha (Rungsia) maydis Passerini (Chaitophoridae) and Schizaphis graminum Rondani (Aphididae), which feed on phloem sap of corn (Zea mays L.), were used as test‐organisms. Atrazine, an herbicide that acts via root absorption, is toxic to aphids, and this effect is manifest rather rapidly. In both aphid species was found rather severe tissue damage, which could be responsible for the death of animals. The most striking damage occurred in the digestive apparatus.

We report the effects of some bisorbicillinoids isolated from biomass of the fungus Trichoderma citrinoviride on settling and feeding preference of the aphid Schizaphis graminum. Purification of the fungal metabolites was carried out by a combination of column chromatography and thin-layer chromatography using direct and reverse phases. Chemical identification was performed by spectroscopic methods including nuclear magnetic resonance and mass spectrometry. The identified bisorbicillinoids appeared to be bislongiquinolide, its 16,17-dihydro derivative, trichodimerol, and dihydrotrichodimerol. A feeding preference test with alate morphs of S. graminum was used to identify the active fractions. Among the four bisorbicillinoids, dihydrotrichodimerol and bislongiquinolide influenced aphid feeding preference, restraining specimens from settling on leaves treated with metabolites. Taste neurons sensitive to these compounds, particularly to bislongiquinolide, were located on tarsi of the S. graminum alate morphs.

Two novel metabolites with potential antifeedant activity were isolated from cultures of the fungus Trichoderma citrinoviride strain ITEM 4484 grown in solid-state fermentation on sterile rice kernels. The producing strain was identified at species level by sequence analysis of the internal transcribed spacer regions ITS-1 and ITS-2 of the nuclear rDNA and a fragment of the translation elongation factor gene TEF-1alpha. Fractionation by column chromatography and TLC of the culture organic extract, followed by feeding preference tests on the aphid pest Schizaphis graminum (Rondani), allowed the purification of 5.8 and 8.9 mg/kg of culture of two bioactive metabolites, which were named citrantifidiene and citrantifidiol ( 1 and 2). Citrantifidiene and citrantifidiol, whose structures were determined by spectroscopic methods (NMR and MS) are a symmetrical disubstituted hexa-1,3-dienyl ester of acetic acid and a tetrasubstituted cyclohexane-1,3-diol, respectively. The pure metabolites influenced the feeding preference of S. graminum restraining individuals from feeding on wheat leaves dipped in 5% aqueous methanol solution containing 0.57 mg/mL of citrantifidiene or 0.91 mg/mL of citrantifidiol.

The feeding preferences of alate and apterous morphs of the aphid Schizaphis graminum (Rondani) (Homoptera: Aphididae) were evaluated using leaves treated with powdered rice cultures of four fungal isolates belonging to different species of the genus Trichoderma (Deuteromycotina: Hyphomycetes). All of the fungal isolates restrained alate morphs of S. graminum from visiting treated leaves, but only Trichoderma citrinoviride Bisset also influenced the preference of apterous morphs. Trials carried out with supernatants obtained by centrifuging aqueous suspensions of the fungal cultures showed that the feeding preference of aphids was maintained in the absence of fungal spores and mycelia, supporting the hypothesis that at least part of the fungal metabolites responsible for this effect were water-soluble compounds. Electrophysiological studies showed that the structures involved in the perception of the fungal metabolites are located on the aphid tarsomeres.

Summary Effects of compost products, enriched or not-enriched with a strain of the mycoparasitic fungus Trichoderma atroviride, on the survival of the collembolan Protaphorura armata and the viability of fungal conidia after the transit through the springtail gut were investigated. The effect of compost enriched with Ca-Lignosulphonate (Ca-Ls), a low cost by-product of the acid sulphite pulping process, with lignin-like structure, on P. armata was also evaluated. All compost products enriched or not with the mycoparasitic fungus or Ca-Ls did not affect P. armata survival. No statistical differences were found in animal survival for different types of product or in enriched and not-enriched products. In addition to adults, live juveniles were also observed in all compost products. The gut content of animals, collected at the end of the survival test from compost enriched with T. atroviride, was examined under the light microscope, and in a few cases observations revealed the presence of some T. atroviride conidia. Subsequent tests carried out to study the viability of conidia after the transit through the springtail gut showed that colonies of the fungus developed from all faecal pellets produced by adult and juveniles specimens of P. armata previously fed on conidia of T. atroviride. These results suggest compatibility between Collembola and Trichoderma or Ca-Ls in the composts.

The effects of beauvericin, a toxic fungal metabolite common contaminant of maize and wheat, on aphid fitness were studied in three consecutive generations of females. Aphids were reared on wheat leaves inserted into a sandy substratum wetted with a solution of beauvericin. Ingestion of this solution through leaves did not significantly decrease the lifespan of females of all generations as compared to controls. However, the mean number of offspring from the third generation of treated females was significantly smaller than those in controls. Furthermore, treated second and third generation females produced a greater number of abortive embryos. Histological analysis revealed abundant DAPI and Feulgen positive material in the cytoplasm of some bacteriocytes of treated third generation females. This material was attributed to the endosymbionts of bacteriocytes. Tests by contact were also carried out and revealed a significantly lower survival of treated first instar aphids as compared to controls 18 h after the start of the trial.