Pathogenicity of Beauveria bassiana (Bals.-Criv.) Vuill. and Metarhizium anisopliae (Metschn.) Sorokin against Galleria mellonella L. and Tenebrio molitor L. in laboratory assays

The pathogenicity of 23 isolates of Beauveria bassiana (Ascomycota, Hypocreales: Cordycipitaceae) and four of Metarhizium anisopliae (Ascomycota, Hypocreales: Clavicipitaceae) was tested against Galleria mellonella (Lepidoptera: Galleriidae) and Tenebrio molitor (Coleoptera: Tenebrionidae) larvae in laboratory assays, using 2•106 conidia mL-1 fungal suspensions. The commercial myco-insecticide Naturalis (Intrachem Bio Italia, Italy), containing the ATCC 74040 B. bassiana strain, was included in the assays for comparison. Mycosed larvae were counted 1, 2, 3, 7, 10, 14 and 17 days after inoculation and the cumulative mortality data were used to calculate mean survival time (MST) and lethal times (LT50 and LT95). No difference between B. bassiana and M. anisopliae were detected in the pathogenicity against the two insect species. However, a wide variability occurred among fungal isolates within species. The two B. bassiana isolates AL1 and ALB55 killed G. mellonella larvae within the shortest time (MST of 2.2 and 2.3 days, respectively), as well as the ALB55 did against T. molitor larvae (MST of 2.8 days). Naturalis was superior to these two B. bassiana isolates, causing a MST of 1.1 day or shorter on the insect larvae. Overall, G. mellonella resulted more sensitive than T. molitor, as showed also by the non-inoculated controls, for which MSTs were 7.7 and 8.4 days, respectively. Due to the rapid and effective insecticide action, the ALB55 B. bassiana isolate can be considered as a new promising candidate for the microbial pest control

Aleurocanthus spiniferus, an alien invasive threat to Europe. AssociatEd bacterial community and natural enemies

Aleurocanthus spiniferus also known as orange spiny whitefly (OSW), is a pest native to tropical Asia that in the last century has spread throughout Asia, reaching Africa, Australia, and Pacific islands. In 2008 the first European OSW population was recorded in Apulia region (South East Italy) and allowed EPPO to add the species as a quarantine threat to Europe now in the A2 list. In the following years OSW spread and invaded new territories of Italy, Croatia and Montenegro. Although OSW polyphagy is already well-known, new associations with autochthonous and allochthonous plants have been reported showing its host-shifting ability. To counteract an upcoming pan-Mediterranean invasion updated bio-ethological information of the pest and the role of possible natural enemies are essential to implement a correct IPM strategy. Field samplings have been aimed at the identification of natural enemies and the evaluation of their efficacy. Furthermore, through insect small-RNA sequencing and by Denaturing Gradient Gel Electrophoresis (DGGE) technique coupled with 16S-rRNA gene sequencing, the primary symbiotic bacteria of OSW have been identified. Sampling on natural enemies highlighted the presence of predatory species belonging to the Coccinellidae family. Besides to the almost ineffective populations of Oenopia conglobata and Clithostetus arcuatus, new findings detected scattered Delphastus sp. populations along the western coast of Italy. Both adult and larvae of this ladybird species preyed OSW developmental stages. The evaluation of the role of Delphastus sp. as biocontrol agent is underway. The first study on OSW microbiota allowed to find symbiotic bacteria commonly associated with the genus Aleurocanthus: Portiera sp., Serratia sp., Wolbachia sp., Rickettsia sp. and, although sporadically, other species. Further studies will target the functional role of these symbionts to develop an effective IPM tailored for Countries at risk

Reduction of post-harvest injuries caused by Drosophila suzukii in some cultivars of sweet cherries using a high carbon dioxide level and cold storage

9openInternationalItalian coauthor/editorEfficient strategies are required in sweet cherry fruits to control the spotted wing drosophila (SWD), Drosophila suzukii, due to its adverse economic effect on farmers. Cold storage (CS) and storage with elevated carbon dioxide (CO2) are environmentally safe approaches for the pest control of stored fresh fruit. These strategies are effective in controlling a wide variety of insect species, without allowing toxic compounds to accumulate. The purpose of this study was to assess the effectiveness of a post-harvest application of CO2 treatment at 50%, cold treatment at 4 °C (CT), and a combination of both (CO2-CT) in controlling the early stages of SWD within four cultivars of freshly harvested cherry fruit, namely “Burlat-Bigarreau”, “Giorgia”, “Ferrovia”, and “Lapins”. In addition, an evaluation of the quality attributes of the cherries (skin firmness, berry firmness, strong soluble material, and titratable acidity) was carried out at harvest and after 10 and 20 days of storage. All treatments significantly reduced the rate of emergence of SWD when compared to the control (untreated cherry at 24 °C), and 100% SWD mortality was obtained in Burlat-Bigarreau (CO2-CT). In addition, over the entire storage time, the quality parameters were preserved in the samples stored at 4 °C and in the samples with combined treatments in comparison with the control.openMostafa, Manal; Ibn Amor, Abir; Admane, Naouel; Anfora, Gianfranco; Bubici, Giovanni; Verrastro, Vincenzo; Scarano, Luciano; El Moujabber, Maroun; Baser, NurayMostafa, M.; Ibn Amor, A.; Admane, N.; Anfora, G.; Bubici, G.; Verrastro, V.; Scarano, L.; El Moujabber, M.; Baser, N

Biological notes on Parahypopta caestrum and first microbiological control assays

We provide notes about the biology and the ethology of Parahypopta caestrum (Hubner) (Lepidoptera Cossidae), based on field observations conducted in asparagus plantations in Apulia region, Italy. Furthermore, the effect of 6 entomopathogenic nematode (EPN) strains (Steinernematidae and Heterorhabditidae) and 3 entomopathogenic fungal (EPF) isolates (Beauveria bassiana) was evaluated in laboratory assays against III instar larvae of the asparagus moth. The results showed that all the nematodes and fungal strains affected the asparagus moth survival, except the Steinernema affine and Heterorhabditis bacteriophora strains. Steinernema feltiae and B. bassiana showed the best performances, killing on average 90% of the P. caestrum larvae. Considering the lack of effective chemical control means, the microbiological control of the asparagus moth by EPNs and EPFs reveals promising perspectives and needs further investigations

Effect of Beauveria bassiana and Metarhizium anisopliae on the Trialeurodes vaporariorum-Encarsia formosa system

The virulence of three strains of Beauveria bassiana and one of Metarhizium anisopliae was tested against Trialeurodes vaporariorum and its parasitoid Encarsia formosa in laboratory assays. These strains were previously selected for their virulence against Galleria mellonella and Tenebrio molitor. The commercial B. bassiana strain ATCC 74040, both as pure fungal culture and formulated myco-insecticide (Naturalis), was included in the assays as positive control. First, the entomopathogenic fungal strains were tested for their virulence against T. vaporariorum nymphs on tomato leaf disks. Then, the E. formosa development was evaluated under treatment with the entomopathogenic fungal strains at five different time points from the parasitization of T. vaporariorum nymphs. The virulence of our entomopathogenic fungal strains was superior to that of ATCC 74040,although not significantly, resulting in a cumulative mortality (CM) of T. vaporariorum nymphs 7 days after inoculation (DAI) greater than 86 %. Our M. anisopliae strain CIST8 was the most effective (96.6 % CM 7 DAI), even superior to the myco-insecticide Naturalis (94.2 %), which was more effective than the ATCC 74040 pure strain (85.6 %). The entomopathogenic fungal strains, and especially Naturalis, negatively affected E. formosa development and its parasitization activity of T. vaporariorum nymphs. This effect was more pronounced when the fungal strains were applied before parasitization. Results suggest that the application of entomopathogenic fungi is incompatible with E. formosa release on crops

Effects of entomopathogenic fungi on Encarsia formosa Gahan. (Hymenoptera: Aphelinidae) activity and behavior

The effects of a previously selected entomopathogenic fungus, Beauveria bassiana AL1 strain, and the B. bassiana-based mycoinsecticide Naturalis were evaluated on the Trialeurodes vaporariorum-Encarsia formosa system. The parasitoid's ability to recognize uninfected and infected hosts was examined by evaluation of E. formosa tropism in 'choice' conditions and its activity (residence time on leaf, searching time, handling hosts time and hosts acceptance) in no-choice conditions. Finally, the role of E. formosa in transmitting the mycoses from infected to uninfected host populations was estimated. E. formosa showed no differential tropism in 'choice' conditions. It was able neither to locate the host at distance nor to discriminate between infected and uninfected hosts. E. formosa was able to vector fungal propagules from contaminated to uncontaminated hosts during its activity. The results of these laboratory experiments have clarified some biological and behavioral aspects of pathogen-host-parasitoid interactions

Interactions between plant‐beneficial microorganisms in a consortium: Streptomyces microflavus and Trichoderma harzianum

The construction of microbial consortia is challenging due to many variables to be controlled, including the cross-compatibility of the selected strains and their additive or synergistic effects on plants. In this work, we investigated the interactions in vitro, in planta, and at the molecular level of two elite biological control agents (BCAs), that is Streptomyces microflavus strain AtB-42 and Trichoderma harzianum strain M10, to understand their attitude to cooperate in a consortium. In vitro, we observed a strong cross-antagonism between AtB-42 and M10 in agar plates due to diffusible metabolites and volatile organic compounds. In liquid co-cultures, M10 hindered the growth of AtB-42 very likely because of secondary metabolites and strong competition for the nutrients. The interaction in the co-culture induced extensive transcriptional reprogramming in both strains, especially in the pathways related to ribosomes, protein synthesis, and oxidoreductase activity, suggesting that each strain recognized the counterpart and activated its defence responses. The metabolome of both strains was also significantly affected. In contrast, in the soil, M10 growth was partially contrasted by AtB-42. The roots of tomato seedlings inoculated with the consortium appeared smaller than the control and single-strain-inoculated plants, indicating that plants diverted some energy from the development to defence activation, as evidenced by the leaf transcriptome. The consortium induced a stronger transcriptional change compared to the single inoculants, as demonstrated by a higher number of differentially expressed genes. Although the cross-antagonism observed in vitro, the two strains exerted a synergistic effect on tomato seedlings by inducing resistance responses stronger than the single inoculants. Our observations pose a question on the usefulness of the sole in vitro assays for selecting BCAs to construct a consortium. In vivo experiments should be preferred, and transcriptomics may greatly help to elucidate the activity of the BCAs beyond the phenotypic effects on the plant

Designing a synthetic microbial community devoted to biological control: The case study of Fusarium wilt of banana

Fusarium oxysporum f. sp. cubense (Foc) tropical race 4 (TR4) is threatening banana production because of its increasing spread. Biological control approaches have been widely studied and constitute interesting complementary measures to integrated disease management strategies. They have been based mainly on the use of single biological control agents (BCAs). In this study, we moved a step forward by designing a synthetic microbial community (SynCom) for the control of Fusarium wilt of banana (FWB). Ninety-six isolates of Pseudomonas spp., Bacillus spp., Streptomyces spp., and Trichoderma spp. were obtained from the banana rhizosphere and selected in vitro for the antagonism against Foc TR4. In pot experiments, a large community such as SynCom 1.0 (44 isolates with moderate to high antagonistic activity) or a small one such as SynCom 1.1 (seven highly effective isolates) provided similar disease control (35% symptom severity reduction). An in vitro study of the interactions among SynCom 1.1 isolates and between them and Foc revealed that beneficial microorganisms not only antagonized the pathogen but also some of the SynCom constituents. Furthermore, Foc defended itself by antagonizing the beneficial microbes. We also demonstrated that fusaric acid, known as one of the secondary metabolites of Fusarium species, might be involved in such an interaction. With this knowledge, SynCom 1.2 was then designed with three isolates: Pseudomonas chlororaphis subsp. piscium PS5, Bacillus velezensis BN8.2, and Trichoderma virens T2C1.4. A non-simultaneous soil application of these isolates (to diminish cross-inhibition) delayed FWB progress over time, with significant reductions in incidence and severity. SynCom 1.2 also performed better than two commercial BCAs, BioPak® and T-Gro. Eventually, SynCom 1.2 isolates were characterized for several biocontrol traits and their genome was sequenced. Our data showed that assembling a SynCom for biocontrol is not an easy task. The mere mixtures of antagonists (e.g., SynCom 1.0 and 1.1) might provide effective biocontrol, but an accurate investigation of the interactions among beneficial microorganisms is needed to improve the results (e.g., SynCom 1.2). SynCom 1.2 is a valuable tool to be further developed for the biological control of FWB.This research was partially funded by the Horizon 2020 project entitled ‘Microbial Uptakes for Sustainable Management of Major Banana Pests and Diseases (MUSA)’ (grant no. 727624) and the project entitled ‘PROdotti, servizi e TEcnologie innovative per il ConTrollo bIOlogico e la difesa ecososteNibile in agricoltura (PROTECTION)’ (call Horizon 2020 PON, Fondo Crescita Sostenibile of the Ministero dello Sviluppo Economico, grant no. F/050421/01-03/X32). In accordance with Article 17, paragraph 2, of the Nagoya Protocol on Access and Benefit-Sharing Clearing-House (ABS-CH), access permit to collect the genetic resources here described (microorganisms) were issued by the ‘Dirección General de Biodiversidad y Calidad Ambiental del Ministerio para la Transición Ecológica,’ Spain, with the Prior Informed Consent (PIC) of the ‘Dirección General de Protección de la Naturaleza del Gobierno de Canarias,’ Spain. Reference number of the permit: ESNC34. ABS-CH Unique Identifier (UID): ABSCH-IRCC-ES-242814-1. All the microorganisms isolated in this study are deposited in the culture collection of CNR-IPSP, Bari, Italy.Peer reviewe