FUNGI ASSOCIATED TO PLATYPUS CYLINDRUS FAB. (COLEOPTERA: PLATYPODIDAE) IN CORK OAK FUNGOS ASSOCIADOS AO INSECTO PLATYPUS CYLINDRUS FAB. (COLEOPTERA: PLATYPODIDAE) EM SOBREIRO JOANA HENRIQUES, MARIA DE LURDES INÁCIO, EDMUNDO SOUSA ABSTRACT Platypus cylindrus é uma praga que desde Platypus cylindrus is a pest that since the os anos 80 do século passado tem sido refe- 80’s of the last century has been considered rida como agente de mortalidade do sobrei- a cork oak mortality agent in Portugal. It ro em Portugal. É um insecto ambrósia que is an ambrosia beetle that establishes com- estabelece simbioses complexas com fungos plex symbioses with fungi whose role in the cujo papel não está completamente esclare- insect-fungus-host interaction has not been cido na interacção insecto-fungo-sobreiro. completely clarified. In order to characterize Com o objectivo de caracterizar a micoflora P. cylindrus associated micoflora in Portugal, associada a P. cylindrus em Portugal foram fungi were isolated from different beetle or- efectuados isolamentos a partir de diferen- gans and from its galleries in cork oak trees. tes órgãos do insecto e suas galerias em so- Fungi of the genera Acremonium, Aspergillus, breiro. Identificaram-se fungos dos géneros Beauveria, Botrytis, Chaetomium, Fusarium, Acremonium, Aspergillus, Beauveria, Bo- Geotrichum, Gliocladium, Nodulisporium, trytis, Chaetomium, Fusarium, Geotrichum, Paecilomyces, Penicillium, Raffaelea, Scyta- Gliocladium, Nodulisporium, Paecilomyces, lidium, Trichoderma and of the order Muco- Penicillium, Raffaelea, Scytalidium, Tricho- rales were identified. An actinomycete of the derma e da ordem Mucorales. Foi igualmen- genus Streptomyces was also identified. Some te identificado um actinomiceta do género of these genera were related for the first time Streptomyces. Alguns destes géneros são to this interaction. In the present work the iso- referidos pela primeira vez nesta interacção. lated fungi are characterized and their contri- No presente trabalho caracterizam-se os fun- bution for beetle population establishment and gos isolados e discute-se a sua contribuição tree weakness is discussed. para o estabelecimento das populações do insecto e enfraquecimento das árvores. Key-words: ambrosia beetle, decline, inte- raction, mycoflora,Quercus suber. Palavras-chave: insecto ambrósia, declínio, interacção, micoflora,Quercus suber. RESUMO INTRODUÇÃO Instituto Nacional de Recursos Biológicos, I.P. Scolytidae and Platypodidae are among the Edifício da ex-Estação Florestal Nacional, Quinta do Marquês, 2780-159 Oeiras most successful wood-inhabiting beetles cau- [email protected]; lurdes.inacio@efn. sing damage of economic significance to tre- com.pt; [email protected] es and timber (Cassier et al., 1996). Platypus cylindrus Fab. is a cork oak (Quercus suber L.) Recepção/Reception: 2009.01.15 pest that has come to assume an increasing im- Aceitação/Acception: 2009.05.14 portance in Portugal and Mediterranean basin FUNGI ASSOCIATED TO PLATYPUS CYLINDRUS FAB. (COLEOPTERA: PLATYPODIDAE) IN CORK OAK 57 countries (Ferreira & Ferreira, 1989; Chakali when excavating tunnels. Auxiliary ambrosia et al., 2002; Riziero et al., 2002; Sousa et al., fungi are transitory, non-specific with respect 2005). Until recently, damages produced by to symbiont insect and may appear after in- this insect were limited to dead or weakened sect development. They may not be present trees. The understanding of recent population in larval cradles or in adult beetles; and their outbreaks, mainly in Portuguese cork oak habitat and distribution range are unrestric- stands, can be based on three assumptions: (i) ted and unrelated to that of the ambrosia be- gradual changes of the cork oak stand dyna- etles. mics, (ii) development of more specific host Several fungi have already been isolated colonization mechanisms and (iii) changes on from P. cylindrus and from galleries in Quer- the insects and their natural enemies’ popula- cus spp. (Baker, 1963; Cassier et al., 1996; tion dynamics (Sousa & Inácio, 2005). Within Sousa et al., 1997; Morelet, 1998; Henriques the host colonization strategies, it’s essential et al., 2006). Their constant presence asso- to consider the fungal symbiosis that may ciated to the insect allows us to presume that contribute to the host weakness and create bet- they play an important role in the symbiosis. ter conditions for the establishment of insects Besides the implication in insect feeding, am- (Henriques et al., 2006). brosia fungi might also be involved in other In deed, P. cylindrus, as almost all members processes such as host weakness, through of the Platypodidae, is denominated an am- pathogenic action; decomposition of ligno- brosia beetle because larvae and adults feed cellulolitic compounds, which helps gallery mainly on fungi (ambrosia fungi) that cover construction and enables fungi colonization; the gallery walls (Batra, 1963). This insect- and/or antagonism that controls the growth fungi relation is expressed in an ectosymbio- of other fungi (Sousa & Inácio, 2005; Henri- sis in which the fungi live outside the insects’ ques et al., 2006). body but are temporarily stored in special ec- The aim of the present work is to charac- todermical organs for dissemination purposes terize the micoflora associated toP. cylindrus (Francke-Grosmann, 1967). Insects carry via- and discuss its contribution for beetle popu- ble inoculum in sac-like structures, called my- lations’ establishment and host weakness. cangia, located in the prothorax; the inoculum is protected from desiccation during the entire life of the beetle and is disseminated into new Material AND METHODS breeding sites at the time of tunnel excavation (Batra, 1963; Sousa & Inácio, 2005). Four infested logs of cork oak trees exhi- Ambrosia fungi definition includes a set biting decline symptoms were collected from of concepts whose interception allows the three of the main Portuguese cork oak produc- classification of several fungi as ambrosia: i) tive regions: Chamusca (Ribatejo province), direct participation in insect feeding; ii) pre- Montemor and Grândola (Alentejo province). sence inside insect galleries in the host; iii) The logs were maintained in the laboratory in dimorphism, meaning ability to grow both order to capture 100 P. cylindrus insects, males as yeast and mycelia; iv) possible specificity and females, as they emerged. Samplings were in the insect-fungi-host relationship (Batra, repeated during 2005, 2006 and 2007. The in- 1963; Beaver, 1989). sects were aseptically dissected in mycangia, Batra (1985) grouped ambrosia fungi as intestine and parts of the exoskeleton (elytra). primary and auxiliary. Primary ambrosia fun- The logs were cut in order to identify the diffe- gi are highly insect species specific and their rent insect gallery sections: cork, inner-bark, distribution correspond to those of insect pre-parental section, larval section and gallery symbionts. They are present and dominant end. One complete gallery was observed from in the tunnels and isolated regularly from the each log and six samples (fragments with 1 mycangia of the beetles in the flight stage or cm2) of each section were collected. 58 REVISTA DE CIÊNCIAS AGRÁRIAS The samples were surface sterilized with Results a sodium hypochlorite solution (1%) for 1 min, rinsed with distilled sterilized water and Fifteen genera were isolated from the di- then plated in malt extract agar (MEA, Dif- fferent insect parts and from all cork oak gal- co, USA) added with streptomycin (500 mg/l) lery sections (Table 1). The identified fungi and MEA added with cycloheximide (500 are classified in very distinct orders including mg/l). The cultures were incubated at 24±1ºC the Ascomycota (Eurotiales, Heliotioales, in darkness. Pure cultures of each fungi isolate Hypocreales, Ophiostomatales, Saccharomy- were obtained and identified as genus based cetales, Sordiales and a genus not assigned on morphological features according to Ellis to any order) and Basidiomycota (Xylariales) (1971, 1976), Lanier et al. (1978), Kiffer & although all the obtained genera, except Cha- Morelet (1997) and Barnett & Hunter (1998). etomium, were isolated in the mitosporic sta- Cultural description of colonies was made on te. Also, an actinomycete was isolated from potato dextrose agar (PDA, Difco, USA). the insect (Streptomycetales). Table 1 – Genera isolated from Platypus cylindrus intestine, exoskeleton and mycangia and from the different sections of insects galleries on cork oak. * Streptomyces (order Actinomycetales) is an actinomycete, the other genera described belong to Fungi. The isolated genera were described ac- isolated, probably belonging to different spe- cording to the morphological features ba- cies. Their microscopic features are: hyphae sed on optical microscopic observations septate and branched, conidiophores macro- and literature guides. In table 2 the main nematous, mononematous, erect, simple, often cultural features for all isolated genera are with a foot cell, with a terminal vesicle bearing described. short branches or phialides radiating from the From the genus Aspergillus several individu- entire surface. When present, the branches are als with different cultural characteristics were in one or several series and the terminal ones FUNGI ASSOCIATED TO PLATYPUS CYLINDRUS FAB. (COLEOPTERA: PLATYPODIDAE) IN CORK OAK 59 Table 2 – Cultural characteristics on PDA of the isolated genera. always bear phialides. Conidiogenic cells mo- Penicillium produces conidiophores