Aclees sp. cf. foveatus (Coleoptera: Curculionidae) is spreading in Central Italy, causing severe infestation on fig trees. There are very few information for this pest and no natural enemy is reported. Here, we report the first recovery of a natural enemy associated with this invasive weevil, a strain of the entomopathogenic fungus Beauveria bassiana. The potential use of entomopathogenic fungi and nematodes as biocontrol agents was tested against adults in laboratory trials. In agree with the detection in nature, only treatments with B. bassiana were able to control the insects. This result opens new frontiers for the environmental friendly control strategies against this weevil

GARGANI, ELISABETTA

MAZZA, GIUSEPPE

PENNACCHIO, FABRIZIO

ROVERSI, PIO FEDERICO

STRANGI, AGOSTINO

TORRINI, GIULIA

English

CREA Journals (Consiglio per la ricerca in agricoltura e l’analisi dell’economia agraria)

REDIA, XCIX, 2016: 29-33(*) Consiglio per la ricerca in agricoltura e l’analisi dell’economia agraria (CREA-ABP) – Research Center forAgrobiology and Pedology, Via Lanciola 12⁄A, 50125, Florence, Italy;e-mail: elisabetta.gargani@crea.gov.itGargani E., Mazza G., Benvenuti C., Torrini G., Strangi A., Pennacchio F., Roversi P.F. – Biological control of Acleessp. cf. foveatus and first recovery of an associate Beauveria bassiana strain.Aclees sp. cf. foveatus (Coleoptera: Curculionidae) is spreading in Central Italy, causing severe infestation on figtrees. There are very few information for this pest and no natural enemy is reported. Here, we report the first recoveryof a natural enemy associated with this invasive weevil, a strain of the entomopathogenic fungus Beauveria bassiana.The potential use of entomopathogenic fungi and nematodes as biocontrol agents was tested against adults in laboratorytrials. In agree with the detection in nature, only treatments with B. bassiana were able to control the insects. This resultopens new frontiers for the environmental friendly control strategies against this weevil. KEY WORDS: alien invasive pest, biocontrol, entomopathogenic fungi, fig weevil, natural enemies.ELISABETTA GARGANI (*) - GIUSEPPE MAZZA (*) - CLAUDIA BENVENUTI (*) - GIULIA TORRINI (*)AGOSTINO STRANGI (*) - FABRIZIO PENNACCHIO (*) - PIO FEDERICO ROVERSI (*)BIOLOGICAL CONTROL OF ACLEES SP. CF. FOVEATUS AND FIRST RECOVERYOF AN ASSOCIATE BEAUVERIA BASSIANA STRAININTRODUCTIONFew severe enemies are reported for Ficus carica L. (e.g.POLLINI, 1998; MAZZA et al., 2014a), but in 2005 the intro-duction of the invasive Aclees sp.cf. foveatus Voss (Coleoptera:Curculionidae) in Central Italy, in particular in Tuscany, causedserious symptoms of decay on fig trees (CIAMPOLINI et al.,2005; BENELLI et al., 2014). At now, the systematic of this weevil remains unclear, mainlydue to the absence of a recent revision of the entire genusAclees (MORIMOTO, 1982) and, as expected, information onbiology, and management of this weevil are scanty (CIAMPOLINIet al., 2007, 2008; BENELLI et al., 2014). Since the situation in Tuscany is already compromised andthe species spread in five on ten provinces (PhytosanitaryService of Tuscany, pers. com.), the aim of this paper is toimprove the knowledge on the potential biological controlagainst this invasive weevil. In addition, with this aim, wereport the finding of the first natural enemy associated to thisinsect: a strain of the entomopathogenic fungus Beauveriabassiana Balsamo (Vuillemin), directly isolated from a deadadult found in Prato province, an area of recent introduction.MATERIAL AND METHODSINSECT COLLECTION AND LABORATORY MAINTENANCEAdults with a balanced sex ratio, were collected duringfield surveys from July to October 2015, in fig orchardslocated in three sites between Prato and Lucca provinces(Fig. I.1). The specimens were transferred to the facilitiesof CREA-ABP in Florence (Italy), sexed following thecriteria used in THU et al. (2010) and maintained at 26-28°C with 16:8 (L:D) photoperiod, in cages with chipboardand wood portions of fig, with the addition of apple slices. ISOLATION AND IDENTIFICATION OF THE FUNGAL STRAINThe fungal strain was isolated from a dead adult (Fig. I.2)collected during field survey in Carmignano (Prato), Tuscany(N 43.81.43, E 11.01.94., altitude ca. 186 m a.s.l.). The adultwas stored in sterile 50 mL tube until fungal isolation. Theisolate was subcultured in Petri dishes on Sabouraud DextroseAgar (SDA) plus 0.25% (w/v) yeast extract (SDAY) and main-tained in a climatic chamber at 25 ± 1 °C. Fungal strain was subcultured and an aliquot of myceliumand conidia (about 10 mg maximum) was collected, frozenat -140 °C and pulverized with Precellys 24 (Precellys).Genomic DNA was extracted using the DNeasy Plant MiniKit (QIAGEN) according to the manufacturer’s instructions.Amplification and sequencing of ITS locus and β-Tubulinwas performed according to WHITE et al. (1990) and GLASS& DONALDSON (1995) using the universal primers ITS1 - ITS4and Bt2a-Bt2b.PCR product was sequenced at the Centro di Servizi per leBiotecnologie di Interesse Agrario Chimico ed Industriale(CIBIACI), Università degli Studi di Firenze, Italy. Thesequence was submitted to GenBank: accession n. KU726564and KU867831.Maximum Likelihood (ML) phylogenetic reconstructionmethod was used to identify fungal strain at species level. Thealignment obtained from homologous ITS sequences wasused for the subsequent analyses. ML tree was built withMEGA 6.0 (TAMURA et al., 2013) using TN93 as substitutionmatrix assuming a gamma-distributed substitution rate amongsites and performing 2000 bootstrap replicates.Phylogenetic tree based on Bayesian Inference (BI) wasconstructed using BEAST v. 2.3.2 (DRUMMOND & RAMBAUT,2007) assuming the same substitution matrix and the coales-cent constant population model. Analyses were run for 10million generations, sampling every 1000 generations (first10% discarded as burn-in). Five independent Markov chainMonte Carlo (MCMC) analyses were performed starting froma randomly chosen tree. Maximum clade credibility tree wassummarized.PATHOGENICITY ASSAYSEntomopathogenic nematodes and fungi were selectedto perform lab control trials on the adults. Bioassays were– Received 4 April 2016     Accepted 19 May 2016http://dx.doi.org/10.19263/REDIA-99.16.05accomplished using formulated insecticide products; inparticular the biological agents are all commercialproducts in Italy, with the exception of one of thenematodes, belonging to a laboratory collection.Pathogenicity assays with entomopathogenic nematodesCapsanem® containing Steinernema carpocapsae (Weiser)nematodes (Koppert B.V., The Netherlands), andHeterorhabditis bacteriophora Poinar ItH-LU1 (collectionof the Section of Entomology and Zoology - DISSPA,University of Bari) were used in this study. In particular, H.bacteriophora was reared at 24 °C on the greater wax mothGalleria mellonella L. larvae and the Infective Juveniles thatemerged from cadavers were recovered using modifiedWhite traps (KAYA & STOCK, 1997). After storage at 12 °Cfor 2 weeks, they were kept at 20 °C for 24 h prior to testing.Experimental unit (n=15 for each of the two treatments)consisted in a plastic cup (4.5 cm diameter, 7 cm deep)filled with 60 cc of sterile peat and water (1:1). A dose of50 IJs/cm2 (800 nematodes/insect) was applied onto thesoil surface of each cup in 0.5 ml of water. In the controlcup (n=15), we added only sterile water. An adult wasinserted in each cup and fed ad libitum with apple. Cupswere closed with perforated plugs and then, they wereincubated at 24 ± 2 °C, 60-80% RH, under a naturallight:dark (L:D) cycle regime (about 11:13) for 28 days.Insect mortality was checked daily.Pathogenicity assays with entomopathogenic fungiMetarhizium anisopliae var. anisopliae (Metschnikoff)Sorokin strain F52 (Met52®, Novozymes A/S - 9 x 108CFU/g) and B. bassiana strain ATCC74040 (Naturalis®,Biogard/CBC - 2.3x107 conidia/ml) were used in thepresent study.Experimental unit (n=15 for each treatment) consisted inthe same plastic cup described above. For both fungi(Met52® and Naturalis®) the label dose was applied ontothe soil surface of each cup. In the control cup (n=15),only sterile water was added. A single adult was insertedin each cup and the setting of the experimental units wasmade as for the test described above.An additional biossay was performed with Naturalis®,treating single adults (n=15) by direct application of 1 µLsuspension of the product. The adults were kept in thesame experimental units used above. Control adults (n=15)received only 1 µL of distilled water.Insect mortality was checked daily. Dead weevils weresurface sterilized with 1% sodium hypochlorite, rinsedthree times with sterile distilled water, and placed onmoistened filter paper in Petri dishes at 25 ± 1 °C todetermine the presence of external sporulation accordingto CITO et al. (2014).DATA ANALYSISLethal time to 50% (LT50) was estimated. Data from thepathogenicity bioassays were compared using WilcoxonGehan test (statistic: χ2). All tests were performed withSPSS v. 15.0 software.RESULTSISOLATION AND IDENTIFICATION OF THE FUNGAL STRAINOn the basis of morphological and molecular data, thefungal strain found in association with the dead weevilwas identified as Beauveria bassiana. Consensus treeobtained by ML and BI algorithm support thisidentification (Fig. II), however some differences could befound between the tree reconstructions, in particular BIalgorithm distinguish Beauveria lii and Beauveria varroaefrom B. bassiana while ML bootstrap does not. A possibleexplanation for the low ML resolution could be the30 E. GARGANI ET AL. REDIA, Vol. XCIX, 2016Fig. I – (1) Sites of collection of Aclees sp. cf. foveatus in Tuscany (Italy). (2) Dead specimen of Aclees sp. cf.foveatus found in Carmignano (Prato) and infected by Beauveria bassiana strain Bb 15/I13.1 2BIOLOGICAL CONTROL OF ACLEES SP. CF. FOVEATUS AND FIRST RECOVERY OF AN ASSOCIATE BEAUVERIA BASSIANA… 31Fig. II – Species identification of fungal isolate based on two phylogenetic reconstructions using Maximum Likelyhood(ML) and Bayesian Inference (BI) methods. (1) ML tree include isolate 15/I13 in Beauveria bassiana clade (-lnL = -1537.1707); bootstrap values are reported near respective nodes. (2) BI tree identify isolate 15/I13 as B. bassiana (-lnL =-1779.38); each node was annotated with its posterior probability.1 2closeness of these species (in particular B. lii) from B.bassiana (ZHANG et al., 2013).The strain (labeled as Bb 15/I13) was isolated andstored into the Ult-Freezer-Haier at -80 °C as frozen driedcultures at the entomopathogenic micro-organismscollection of the CREA-ABP of Florence. PATHOGENICITY ASSAYSNo mortality was observed using entomopathogenicnematodes, nor by using the commercial productCapsanem® neither with the lab strain ItH-LU1 and,likewise, no effect was obtained by the treatments with thecommercial product Met52®, based on M. anisopliae. On the contrary, noteworthy are the results obtainedwith both the bioassays using B. bassiana Naturalis®. Thepercentage of mortality for both treatments was 100% and90% and the percentage of sporulation was 90% and 80%,respectively. In particular, the LT50 for the first assay is 8.5days and for the second one is 7 days. There was nodifference in the mortality caused by the two differenttreatments (χ2 = 0.38, d.f. = 1, P = 0.538), while significantdifferences were found between both treatments and thecontrol (χ2 = 13.91, d.f. = 1, P < 0.0001; χ2 = 11.76, d.f. =1, P < 0.001, respectively; Fig. III).DISCUSSIONThis is the first finding of an entomopathogenic fungusassociated to Aclees sp.cf. foveatus. This record agreeswith the promising results obtained from the pathogenicityassays with Beauveria bassiana performed in labcondition. As highlighted also for another invasive weevil,Rhynchophorus ferrugineus (Olivier) (MAZZA et al.,2014b) and as indicated in the results, Aclees showed adifferent susceptibility towards different entomo -pathogenic fungi, while no effect was obtained using ourentomopathogenic nematodes. The use of B. bassiana,moreover found as the only natural enemy in ourenvironments, should be seriously considered forbiological control and for inclusion in integrated pestmanagement programs. Further studies are necessary tobetter understand the species behaviour in order to addressmanagement techniques with special reference to thetemporal placement of the treatments against adults. In thecase of Aclees, most of the life cycle is completed withinthe fig trunk, making the preimaginal stages inaccessibleto direct-contact treatment, thus the adults are the onlyexposed stage that can be infected upon the emergence.Moreover it is important to continue the research of newbiocontrol agents for an environmental friendly approach. ACKNOWLEDGMENTSWe would like to thank E. Tarasco (DISSPA) to provideHeterorhabditis bacteriophora strain, G.P. Barzanti(CREA-ABP) for the help in the isolation andidentification of the fungal strain. Thanks to Siro Petracchi(Associazione produttori fichi secchi di Carmignano), IvoPoli and Claudio Lorenzi (fig nurseries owners). Thanks toNancy Ricceri for the english revision.RIASSUNTOPROVE DI CONTROLLO BIOLOGICO DI ACLEES SP.CF. FOVEATUS E PRIMO RITROVAMENTOIN NATURA DI UN CEPPO DI BEAUVERIA BASSIANAASSOCIATO AD ESSOAclees sp. cf. foveatus (Coleoptera: Curculionidae) si starapidamente diffondendo in Italia centrale, causando graviinfestazioni su alberi di fico. Pochissime sono le informazionisu questo fitofago e  non risultano segnalati nemici naturali.In questo articolo, si riporta il primo ritrovamento di un nemiconaturale associato a questo curculionide invasivo, un ceppodel fungo entomopatogeno Beauveria bassiana. Prove di pato-genicità, su adulti, sono state effettuate in laboratorio per valu-tare la potenziale efficacia di funghi entomopatogeni e nematodicome agenti di biocontrollo. In sintonia con il ritrovamentoin natura, solo i trattamenti con B. bassiana sono stati in gradodi controllare gli insetti. Questo risultato apre nuove frontiereper strategie di controllo del curculionide con prodotti ad ele-vata sostenibilità ambientale.REFERENCESBENELLI G., MEREGALLI M., CANALE A., 2014 – Fieldobservations on the mating behavior of Aclees sp. cf.foveatus Voss (Coleoptera: Curculionidae), an exoticpest noxious to fig orchards. - Journal of InsectBehavior, 27: 419-427.CIAMPOLINI M., PERRIN H., REGALIN R., 2005 – Acleescribratus, nuovo per l’Italia nocivo al fico allevato invivaio. - Informatore Agrario, 61: 69-71.CIAMPOLINI M., REGALIN R., FARNESI I., LORENZI C., 2007– Prime osservazioni sulla bio-etologia di Acleessp.(Curculionidae, Molytinae) esiziale a Ficus carica L.in Italia. - Bollettino di Zoologia Agraria e diBachicoltura, 39: 51-60.CIAMPOLINI M., FARNESI I., SCARSELLI F., LORENZI C.,2008 – Contro il curculionide del fico decisiva la lottaagli adulti. - Informatore Agrario, 64: 57.CITO A., MAZZA G., STRANGI A., BENVENUTI C., BARZANTIG.P., DREASSI E., TURCHETTI T., FRANCARDI V., ROVERSIP.F., 2014 – Characterization and comparison ofMetarhizium strains isolated from Rhynchophorusferrugineus. - FEMS Microbiology Letters, 355: 108-115.DRUMMOND A., RAMBAUT A., 2007 - BEAST: Bayesianevolutionary analysis by sampling trees. BMC Evol Biol7: 214.32 E. GARGANI ET AL. REDIA, Vol. XCIX, 2016Fig. III – Survival analysis of Aclees sp. cf. foveatus adults treated with Naturalis®. Lines with the same let-ter are not significantly different.GLASS N.L., DONALDSON G.C., 1995 – Development ofprimer sets designed for use with the PCR to amplifyconserved genes from filamentous Ascomycetes. -Applied and Environmental Microbiology, 61: 1323-1330.KAYA H.K., STOCK S.P., 1997 – Techniques in InsectNematology, In: L.A. Lacey Ed , Manual of Techniquesof Insect Pathogens Academic Press, London, UK. pp.281-324.MAZZA G., CHOUAIA B., LOZZIA G.C., MONTAGNA M., 2014a– The bacterial community associated to an Italianpopulation of Psacothea hilaris: a preliminary study. -Bulletin of Insectology, 67: 281-285.MAZZA G., FRANCARDI V., SIMONI S., BENVENUTI C., CERVOR., FALEIRO J.R., LLACER E., LONGO S., NANNELLI R.,TARASCO E., ROVERSI P.F., 2014b – An overview on thenatural enemies of Rhynchophorus palm weevils, withfocus on R. ferrugineus. - Biological Control, 77: 83-92. MORIMOTO K., 1982 – The family curculionidae of Japan. I.Subfamily Hylobiinae. - Esakia: 51-121.POLLINI A., 1998 – Manuale di Entomologia Applicata.Edagricole, Bologna, Italy.TAMURA K., STECHER G., PETERSON D., FILIPSKI A., KUMARS., 2013 _ Mega6: Molecular evolutionary geneticsanalysis version 6.0. - Molecular Biology and Evolution,30: 2725-2729.THU P.Q., QUANG D.N., DELL B., 2010 – Threat to cedar,Cedrela odorata, plantations in Vietnam by the weevil,Aclees sp. - Journal of Insect Science, 10: 192. availableonline: insectscience.org/10.192.WHITE T.J., BRUNS T., LEE S., TAYLOR J., 1990 –Amplification and direct sequencing of fungal ribosomalRNA genes for phylogenetics. In: M. Innis, D. Gelfand, J.Sninsky, T.J. White Eds., Pcr protocols: A guide tomethods and applications . - Academic Press, New York,USA. pp. 315-322.ZHANG S.L., HE L.M., CHEN X., HUANG B., 2013 –Beauveria lii sp. nov. isolated from Henosepilachnavigintioctopunctata. - Mycotaxon, 121: 199-206.BIOLOGICAL CONTROL OF ACLEES SP. CF. FOVEATUS AND FIRST RECOVERY OF AN ASSOCIATE BEAUVERIA BASSIANA… 33

BIOLOGICAL CONTROL OF ACLEES SP. CF. FOVEATUS AND FIRST RECOVERY OF AN ASSOCIATE BEAUVERIA BASSIANA STRAIN

https://journals-crea.4science.it/index.php/redia/article/download/1321/984

BIOLOGICAL CONTROL OF ACLEES SP. CF. FOVEATUS AND FIRST RECOVERY OF AN ASSOCIATE BEAUVERIA BASSIANA STRAIN

Abstract

Similar works

Full text

Available Versions

CREA Journals (Consiglio per la ricerca in agricoltura e l’analisi dell’economia agraria)