The virulence of seventeen strains of entomopathogenic nematodes, belonging to four species of Steinernema and two of Heterorhabditis, against the chestnut insect parasites Curculio elephas, C. glandium, Pammene castanicola, Cydia splendana and C. fagiglandana, was tested in laboratory. The bioassays were conducted on the insect larvae collected in three different years (2010-2012) in chestnut groves of Etna. Most of the strains tested resulted good biocontrol agents for all the treated insect pests. The strains S. feltiae ESA and S. feltiae EPP were the most effective strains autochthonous of Etna when all the species of insect pests are considered as a whole

Clausi, Mirella

Leone, Diego

Rappazzo, Giancarlo

Tarasco, Eustachio

Vinciguerra, Maria Teresa

English

Vinciguerra, MARIA TERESA

Rappezzo, Giancarlo

TARASCO, Eustachio

Archivio istituzionale della ricerca - Università di Bari

REDIA, XCVII, 2014: 133-136(*) Dipartimento di Biologia Animale “M. La Greca”, Università di Catania, via Androne 81, 95124, Catania, Italy; e-mail:mclausi@unict.it(**) Dipartimento di Scienze del Suolo, della Pianta e degli Alimenti (DiSSPA), Università degli Studi di Bari “A. Moro”, viaAmendola 165/A, 70125, Bari, ItalyClausi M., Leone D., Vinciguerra M.T., Rappazzo G., Tarasco E. – Laboratory tests on the biocontrol of chestnutinsect pests on Etna (Sicily, Italy) by means of entomopathogenic nematodes.The virulence of seventeen strains of entomopathogenic nematodes, belonging to four species of Steinernemaand two of Heterorhabditis, against the chestnut insect parasites Curculio elephas, C. glandium, Pammene castanicola,Cydia splendana and C. fagiglandana, was tested in laboratory. The bioassays were conducted on the insect larvae col-lected in three different years (2010-2012) in chestnut groves of Etna. Most of the strains tested resulted good bio-control agents for all the treated insect pests. The strains S. feltiae ESA and S. feltiae EPP were the most effectivestrains autochthonous of Etna when all the species of insect pests are considered as a whole.KEY WORDS: Curculio, Pammene, Cydia, Steinernema, Heterorhabditis.MIRELLA CLAUSI (*) - DIEGO LEONE (*) - MARIA TERESA VINCIGUERRA (*)GIANCARLO RAPPAZZO (*) - EUSTACHIO TARASCO (**)LABORATORY TESTS ON THE BIOCONTROL OF CHESTNUT INSECT PESTSON ETNA (SICILY, ITALY) BY MEANS OF ENTOMOPATHOGENIC NEMATODESINTRODUCTIONAmong the insects there are numerous parasites that cancause damage to chestnuts, reducing their quality andcommercial value. A research on the chestnut pests ofEtna was considered useful because recently in Sicily therehas been a revival of this crop, which has traditionally beenused only for timber, and there are some attempts toconvert coppice stands into orchards and to produce goodquality nuts for the alimentary market. Moreover, becauseof the poor commercial use made of the nuts so far, thereis no literature regarding the nut parasite situation in Sicily.From studies conducted in Sicily (CLAUSI andVINCIGUERRA, 2005; VINCIGUERRA and CLAUSI, 2006;RAPPAZZO et al., 2006) chestnut pests in Etna Vulcan areabelong to two species of Coleoptera Curculionidae,namely Curculio elephas Gyll. and Curculio glandiumMarsh., and to three Lepidoptera Tortricidae, Cydiasplendana (Hb.), Cydia fagiglandana (Zel) and Pammenecastanicola Trematerra, 2009, the last one previouslyidentified as P. fasciana (L.) and recently described as anew species (TREMATERRA and CLAUSI, 2009) and untilnow reported only from Etna. These species represent areal menace for chestnut and for this reason it would bevery useful to adjust suitable tools of biological controlagainst them. In the previous work (VINCIGUERRA andCLAUSI, 2006) some preliminary tests on the ability ofdifferent species and strains of EPN to infect these insectpests were conducted but a more punctual study wasneeded. The present investigation is based on severallaboratory tests conducted on the biocontrol of late instarlarvae of C. elephas, C. glandium, C. splendana, C. fagi -glandana and P. castanicola, extracted from chestnuts andfrom acorns collected in seminatural mixed groves onEtna, by means of strains and species of ento -mopathogenic nematodes (EPNs) of the genera Hete -rorhabditis Poinar and Steinernema Travassos. Therestriction on the use of many pesticides, particularly inprotected areas as the natural Park of Etna, requires toresort to alternative products such as EPNs as biocontrolagents. Aim of this study was to determine which chestnutpests are sensitive or resistant to EPN + bacteriacomplexes and which species or strain of nematodes arethe most suitable to such control strategies, alsocomparing commercial to autochthonous strains, most ofwhich collected in the same sites where pests are present. MATERIALS AND METHODSCOLLECTION OF INSECTS AND SELECTION OF EPNThe efficacy of 17 EPN strains (Table 1) was tested inlaboratory; these strains belonged to the species Steinernemafeltiae (Filipjev, 1934) Wouts, Mrácek, Gerdin & Bedding,1982, S. ichnusae Tarasco, Mrácek, Nguyen, Triggiani, 2008,S. kraussei (Steiner, 1923) Travassos, 1927, S. carpocapsae(Weiser, 1955) Wouts, Mrácek, Gerdin & Bedding, 1982, S.vulcanicum Clausi, Longo, Rappazzo, Vinciguerra, 2011,Heterorhabditis bacteriophora Poinar, 1976 and H. megidisPoinar, Jackson & Klein, 1987; 13 of them were from Italy(12 from Sicily and 1 from Apulia) and 4 were commercialproducts. The commercial strains were S. feltiae, H. megidisand H. bacteriophora, furnished by IntrachemBio, and S.feltiae furnished by B.T. Biotecnologie; the native strainswere 5 of S. feltiae (ESA, EPP, EMM1, EPC, ETA), 4 of S.kraussei (EBL, EPL, ESC3, EMM2), 1 of S. vulcanicum(ESC1), 1 of S. ichnusae (EMA), all collected in various sitesof Etna, 1 of H. bacteriophora (BAL1) from the island ofPantelleria (Sicily) and 1 of S. carpocapsae (MR7) fromApulia (Tarasco et al., 2014). The native strains come fromthe EPN collections of the Department of Biological,Geological and Envi ronmental Sciences, University of– Received 26 August 2014     Accepted 24 September 2014      Published 10 December 2014SHORT NOTECatania, and from DiSSPA, University of Bari “A. Moro”.The experimental bioassays were performed in laboratoryagainst the chestnut pests Curculio elephas, C. glandium, P.castanicola, C. splendana and C. fagiglandana. Theexperiments were performed in a span of 3 years, 2010-2012, and following the trend of infestations of 5 chestnutgroves of Etna in the three years. All insect pests used fortesting were obtained from chestnuts collected on Etna; thelarvae were extracted putting the chestnuts in a stove at atemperature of 40° C and collecting the larvae which cameout from the nuts. Before the experiments, all larvae werepreviously checked to make sure there were no infections ofany kind. In all bioassays a dose of 750 IJS x ml and 10insect larvae in a 5x5 Petri dish for each EPN were used.The Petri dishes were kept at the temperature of 10° C,more or less corresponding to the mean soil temperature inEtna chestnut groves in Autumn. Each treatment wasreplicated three times. The insect larvae that died showingthe typical characteristics caused by the infestation (colour,odour, consistence) were transferred in White traps wherethe nematodes completed their life cycle and abandonedthe insect body.LABORATORY BIOASSAYS2010. Efficacy of different EPNs strains against curcu lionidlarvae. In this bioassay all the already cited EPN strains wereused but only to test the susceptibility of the larvae of eachCurculio species.2011. Efficacy of different EPNs strains against the mainchestnut insect pests.In 2011 only three species of chestnut pests werecollected: C. elephas, C. glandium and P. castanicola. Nocommercial strain was used; three the selected nativestrains, S. feltiae EPP, S. kraussei EMM2 and H.bacteriophora BAL1, used against the chestnut pests. Inthis experiment the larvae of Curculio were tested withoutdistinguishing the species. The selected EPNs were testedalso on larvae of Galleria mellonella (L.) as control. 2012. Efficacy of selected EPNs against all chestnut pestsIn 2012 all five species of chestnut pests were collected.No commercial strain was used; five the selectedautochthonous strains, S. feltiae ESA, EPP, EMM1, S.kraussei EMM2 and H. bacteriophora BAL1, used against134 M. CLAUSI ET AL. REDIA, Vol. XCVII, 2014Table 1 – EPNs tested in the experimentsStrain/species Locality/CompanyS. feltiae ESA S.Alfio (CT, Sicily)S. feltiae EMM1 Mt. Monaco (CT, Sicily)S. feltiae EPP Piano Porcheria (CT, Sicily)S. feltiae EPC Pietracannone (CT, Sicily)S. feltiae ETA Tarderia (CT, Sicily)S. feltiae IntrachemBioS. feltiae B.T. BiotecnologieS. ichnusae EMA Triciala (CT, Sicily)S. carpocapsae MR7 Manfredonia (FG, Apulia)S. vulcanicum ESC1 Salto del cane (CT, Sicily)S. kraussei ESC3 Salto del cane (CT, Sicily)S. kraussei EBL Piano Balilla (CT, Sicily)S. kraussei EPL Piano Lepre (CT, Sicily)S. kraussei EMM2 Mt. Monaco (CT, Sicily)H. bacteriophora BAL1 Pantelleria (TP, Sicily)H. bacteriophora IntrachemBioH. megidis IntrachemBiothe 5 chestnut pests. Also in this case the larvae of bothCurculio species and those of both Cydia species have beenused without distinguishing the species of the same genus.The selected EPNs were tested also on larvae of G.mellonella as control. DATA ANALYSISThe mortality data of larvae were assessed using analysisof variance (ANOVA); Tukey’s (HSD) test was used tocompare means. Before conducting ANOVA, allpercentages were transformed using the arcsine squareroot transformation. All data were processed utilizingStatistic 9.0. A p value of 0.05 was used in all analyses.RESULTSFigure I, concerning the bioassays of 2010, shows thepercentages of larvae of the two species of Curculioinfected by each EPN strain. Both species were infectedby almost all EPN strains used in the test. The highestmortality was obtained with both commercial strains ofHeterorhabditis: H. bacteriophora caused 90% of mortalityin C. glandium and 80% in C. elephas. H. megidis caused60% of mortality in C. glandium and 100% in C. elephas.All the native strains showed to be less efficacious of thecommercial strains of Heterorhabditis. Among the nativestrains the most virulent against C. glandium were H.bacteriophora BAL1, found in Pantelleria, and S. krausseiEMM2 and S. feltiae EMM1, both found in chestnut soilof Etna, at “Monte Monaco”. On the contrary, the twoindustrial strains of S. feltiae, BT and Intrachem, and thenative strain S. feltiae ETA did not show any virulenceagainst the weevils. Both species of Curculio were alsosusceptible to S. feltiae EMM1 and, to a lesser extent, to S.feltiae EPC and to S. vulcanicum ESC1, both from Etnachestnut groves, and to S. carpocapsae, from Apulia.Figure II, concerning the bioassays of 2011, shows thatthe three selected EPN strains had the same efficacyagainst the larvae of P. castanicola (about 60%), whileagainst the weevils the highest mortality was obtained withS. feltiae EPP (60% of infected animals); S. krausseiEMM2, differently from the previous test, resultedinexplicably less virulent (only 20% of larvae infected)while H. bacteriophora BAL1 showed the same virulenceon C. elephas (about 42%) as in the previous test. All thethree strains, however, were very effective with the larvaeof G. mellonella.Figure III concerns the bioassays of 2012. In this test allthe EPN strains used were very active against pests larvae.As regards the larvae of Curculio, the mortality rangedfrom 30 to 45 %; S. feltiae EMM1 was less effective thanin 2010 while S. feltiae EPP was more effective and S.feltiae ESA maintained the same virulence degree. Theleast virulent strain towards almost all insects was H.bacteriophora BAL1. Cydia species were mainly infectedby S. feltiae ESA and S. feltiae EMM1, with a mortality ofabout 80%. Against P. castanicola the most effective strainswere S. feltiae ESA and S. feltiae EPP, with a mortality ofabout 70%. In many cases the mortality of the moths washigher than that of G. mellonella. DISCUSSION AND CONCLUSIONThe tests showed that the virulence of the same infectiousstrain of EPN is often different in respect of the targetLABORATORY TESTS ON THE BIOCONTROL OF CHESTNUT INSECT PESTS ON ETNA (SICILY, ITALY).… 135Fig. I – Mortality of Curculio glandium and C. elephas larvae 10 days after the treatment with 17 species andstrains of EPNs. Columns marked with the same letter are not statistically different at p < 0.05, according tothe Turkey’s HSD test.Fig. II – Mortality of chestnut pests’ larvae 10 days after the treatment with 3 species and strains of EPNs. Columns markedwith the same letter are not statistically different at p < 0.05, according to the Turkey’s HSD test..136 M. CLAUSI ET AL. REDIA, Vol. XCVII, 2014Fig. III – Mortality of chestnut pests’ larvae infested 10 days after the treatment with 5 species and strains of EPNs.Columns marked with the same letter are not statistically different at p < 0.05, according to the Turkey’s HSD test.species and also that different strains of the same nematodespecies can have a different ability to infect the same insectspecies; moreover, even the virulence of a single strainagainst the same pest has resulted different in different tests.Notwithstanding all these variations, still these tests assessedthe virulence of many EPN strains, most of whichautochthonous of Etna, against all chestnut pests. To obtain useful results in the biological control of suchparasites, preliminary laboratory tests of the relativeefficacy of the strains to be used are needed to select themost suitable ones, but the success of such a tool in thefield, however, largely depends also on the correctmethodology of application of the EPN suspension and onthe ability of the nematodes to survive in the soil for thetime needed to localize and infest the larvae. In the specificcase of the chestnuts of Etna, it is important to evaluatethe EPN ability to persist for long after insemination in thechestnut soil. The results of the bioassays show that thestrains S. feltiae ESA and S. feltiae EPP were the mosteffective strains autochthonous of Etna when all thespecies of insect pests are considered as a whole. Thecommercial strains of H. bacteriophora showed to be moreeffective than H. bacteriophora BAL1, native of Pantelleria,but a comparison with strains of the same speciesautochthonous of Etna was not possible, since no speciesof Heterorhabditis has ever been found so far in chestnutsoil. Some autochthonous strains of Steinernema, however,showed good applicative potentialities against all chestnutinsect pests and probably they would be more able topersist in Etna soil than the species of Heterorhabditis,more susceptible to the low temperatures present at thehigh altitudes where chestnut are present, so much so thatno species of this genus have ever been found during thethorough screening for autochthonous EPN strains andspecies made by the authors in Etna chestnut soil.REFERENCESCLAUSI M., VINCIGUERRA M.T., 2005 – I nematodientomopatogeni in un progetto per lo sviluppo sostenibiledei castagneti.- Nematol. Medit., 33: 91-94.RAPPAZZO G., CLAUSI M., CARDILLO S., VINCIGUERRAM.T., 2006 – Molecular identification of Curculioglandium Marsham, 1802 reported as chestnut insect pestin Sicily (Italy) for the first time.- Redia, 89: 11-13.TARASCO E., CLAUSI M., RAPPAZZO G., PANZAVOLTA T.,CURTO G., SORINO R., ORESTE M., LONGO A., LEONED., TIBERI R., VINCIGUERRA M. T., TRIGGIANI O., 2014 –Biodiversity of entomopathogenic nematodes in Italy. -Journal of Helminthology, 1-8. DOI:10.1017/S0022149X14000194.TREMATERRA P., CLAUSI M., 2009 – Pammene castanicolasp.n. a new Lepidoptera Tortricidae from Sicily. Boll.Zool. agr. Bachic., 41 (2): 59-64.VINCIGUERRA M.T., CLAUSI M., 2006 – Biological control ofchestnut insect pests by means of entomopathogenicnematodes. - Adv. Hort. Sci., 20:40-44

Laboratory tests on the biocontrol of chestnut insect pests on Etna (Sicily, Italy) by means of entomopathogenic nematodes

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Laboratory tests on the biocontrol of chestnut insect pests on Etna (Sicily, Italy) by means of entomopathogenic nematodes

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