FUNGI OF RAFFAELEA GENUS (ASCOMYCOTA: OPHiostomATALES) ASSOCIATED to PLATYPUS CYLINDRUS (COLEOPTERA: PLATYPODIDAE) IN PORTUGAL FUNGOS DO GÉNERO RAFFAELEA (ASCOMYCOTA: OPHiostomATALES) ASSOCIADOS A PLATYPUS CYLINDRUS (COLEOPTERA: PLATYPODIDAE) EM PORTUGAL MARIA LURDES INÁCIO1, JOANA HENRIQUES1, ARLINDO LIMA2, EDMUNDO SOUSA1 ABSTRACT Key-words: Ambrosia beetle, ambrosia fun- gi, cork oak, decline. In the study of the fungi associated to Platypus cylindrus, several fungi were isolated from the insect and its galleries in cork oak, RESUMO among which three species of Raffaelea. Mor- phological and cultural characteristics, sensitiv- No estudo dos fungos associados ao insec- ity to cycloheximide and genetic variability had to xilomicetófago Platypus cylindrus foram been evaluated in a set of isolates of this genus. isolados, a partir do insecto e das suas ga- On this basis R. ambrosiae and R. montetyi were lerias no sobreiro, diversos fungos, entre os identified and a third taxon segregated witch quais três espécies de Raffaelea. Avaliaram-se differs in morphological and molecular charac- características morfológicas e culturais, sensibi- teristics from the previous ones. In this work we lidade à ciclohexamida e variabilidade genética present and discuss the parameters that allow num conjunto de isolados do género. Foram the identification of specimens of the threetaxa . identificados R. ambrosiae e R. montetyi e The role that those ambrosia fungi can have in segregou-se um terceiro táxone que difere the cork oak decline is also discussed taking em características morfológicas e molecula- into account that Ophiostomatales fungi are res dos dois anteriores. No presente trabalho pathogens of great importance in trees, namely são apresentados e discutidos os parâmetros in species of the genus Quercus. que permitem identificar espécimes dos três táxones. É ainda discutido o papel que estes fungos ambrósia podem ter no declínio do sobreiro, sabido que fungos Ophiostomata- 1 Instituto Nacional de Recursos Biológicos, I.P. les são patogénios de grande importância em Edifício da ex-Estação Florestal Nacional, Quinta plantas lenhosas, nomeadamente em espécies do Marquês, 2780-159 Oeiras do género Quercus. [email protected] ; [email protected]; [email protected]; Palavras-chave: Declínio, fungo ambrósia, [email protected] insecto ambrósia, sobreiro. 2 Dep.º de Protecção de Plantas e Fitoecologia, Insti- tuto Superior de Agronomia, Universidade Técnica de Lisboa INTRODUCTION Comunicação apresentada no 5º Congresso da Socie- dade Portuguesa de Fitopatologia, Coimbra, 2007 Many insects use vegetal resources, from herbaceous plants to frondose trees. Recepção/Reception: 2008.02.19 Some constitute primary pests for their Aceitação/Acception: 2008.08.09 hosts, attacking vigorous plants and over- FUNGI OF Raffaelea GENUS (Ascomycota: Ophiostomatales) associated TO Platypus CYLINDRUS (COLEOPTERA: Platypodidae) IN Portugal 97 coming its defences, while others do not In addition to fungi directly related to insect have such ability, colonizing only weak- nourishment, others have been found, such as ened plants and carrying allies that break pathogenic fungi that may play an essential these barriers. Fungi, viruses and nema- role in insect selection and tree colonization. todes are frequently involved with insects Those fungi could play both roles, thus con- in those relations, weakening the hosts and tributing to the establishment of insect popu- thus leaving them accessible to the insects. lations. Among those are Botryodiplodia, The microorganisms, in turn, find a way Ceratocystis, Graphium, Leptographium and to overcome distances between the hosts Ophiostoma (Badler, 1992). Cladistic stud- (Tainter & Baker, 1996). ies have shown that ambrosia fungi such as In the forest, there are several examples species of Ambrosiella are closely related to of insects that establish symbioses with Ascomycetes species of either Ophiostoma other organisms causing severe damages or Ceratocystis (Cassar & Blackwell, 1996) in the attacked trees, namely the Dutch elm and species of Raffaelea are related to Ophi- disease caused by Ophiostoma ulmi and O. ostoma genus (Henriques, 2007), based on novo-ulmi (Buisman) Nannf, vectored by rDNA sequences and confirmed by patterns Scolytus spp. bark beetles (Jacobi et al., of cycloheximide sensitivity. According to 2007; Six et al., 2005) or Ophiostoma spp. Harrington et al. (2008), Raffaelea fungi do of maritime pine, carried by Ips sexdenta- not form a sexual state, and thus the rules of tus (Lieutier & Levieux, 1985; Levieux et nomenclature do not allow describing them as al., 1989). species of Ophiostoma. Nevertheless species The insect Platypus cylindrus Fab. is of Raffaelea could be described as a genus of known to attack mainly dead or weakened ambrosia beetle symbionts within the genus trees. However, since the 1980’s, its popu- Ophiostoma. Also, the results of the sequence lation outbreaks seemed to be related to the analysis of 18S-rDNA, if R. hennebertii Scott cork oak decline in Portugal and other Med- & du Toit is excluded, revealed that Raffaelea iterranean countries. This beetle establishes resolves a monophyletic lineage which forms symbioses with fungi that are carried in a group very close to species of Ophiostoma specialized organs – mycangia – as well in (Jones & Blackwell, 1998). the intestine and on the body surface (Sousa Studies of oak decline in Europe showed et al., 1995; Henriques et al., 2006. Such that the complex Ophiostoma/ Ceratocystis fungi are so called ambrosia as they act as is pathogenic to Quercus trees (Badler, 1992; a nourish source for the insect descendants Degreef, 1992; Delatour et al., 1992). In after being inoculated and cultivated in the addition, R. quercivora Kubono & Ito was galleries. The observation of those galleries proven to be pathogenic to fagaceous trees in confirms the existence of a light-coloured, Japan, being associated with mass mortality thin wall cover, constituted by mycelium of adult trees, particularly Q. serrata and Q. of the symbiotic fungi (Inácio et al., 2005; mongolica (Kubono & Ito, 2002). Sousa & Inácio, 2005). The aim of the present study was to de- The taxonomy of ambrosia fungi is termine the correct identity of Raffaelea- somewhat confused and the general papers like isolates occurring in association with on this issue were published a long time P. cylindrus on cork oak and to discuss its ago. Those works placed ambrosia fungi pathogenicity on host trees. To accomplish within four mitosporic genera, Ambro- this goal, fungi isolated both from insects siella, Monacrosporium, Phialosphoropsis and their galleries were morphologically and Raffaelea but is clear that many more characterised and subjected to DNA analyses genera are involved including Acremonium, of their small subunit region of rDNA (SSU- Candida, Fusarium and Graphium (Batra, rDNA). An additional test of cycloheximide 1963; Baker 1963). sensitivity was also performed. 98 REVISTA DE CIÊNCIAS AGRÁRIAS MATERIAL AND METHODS potato-dextrose agar (Difco PDA, USA) af- ter five to ten days, in the darkness at 25±1 Four infested logs of cork oak trees that ºC. Structures were mounted in sterilized exhibit decline symptoms from the regions distilled water, and 40 measurements at x600 Chamusca (Ribatejo province), Montemor magnification were made for each isolate. and Grândola (Alentejo province) were col- The 95% confidence levels were calculated lected and the associated insects captured in and the extremes of spore measurements fabric traps, attached to the log with a sili- were given. Images were taken from slides cone joint. Those samplings were repeated mounted in sterilized distilled water. Macro- during 2005, 2006 and 2007. scopic characters of colonies were described A total of 100 insects per location were after 21 days of growth; colour names are aseptically dissected to obtain their my- from Saccardo (1891). cangia, intestine and parts of the exoskel- eton (elytra). The logs were cut in order to Cycloheximide sensitivity identify the different gallery sections: cork, inner-bark, pre-parental section, larval sec- The effect of different concentrations of tion and gallery end. One complete gallery cycloheximide (0, 5, 10, 100, 500 and 1000 was observed from each log (fragments of ppm) was tested on isolates of each Raffaelea wood with 1 cm2) and six samples (frag- group. The appropriate amount of cyclohex- ments of wood with 1 cm2) of each section imide was added to autoclavated MEA. were collected. All the pieces were surface Media were dispensed into 9 cm diam Petri sterilised with a sodium hypochlorite solu- dishes (20 ml/plate). The center of each plate tion (1%) for 1 min and rinsed with sterilized was inoculated with a 5 mm diam mycelial distilled water. They were plated into 9 cm plug from the advancing margin of a MEA- diameter Petri dishes with malt extract agar grown culture and incubated at 27,5±1ºC in (Difco MEA, USA) added with streptomycin darkness for five days (Harrington, 1981; M. (Sigma-Aldrich, USA) (500 mg/l) and MEA Wingfield, pers. com). One isolate of Ophi- added with cycloheximide (Sigma-Aldrich, ostoma ulmi (GU81158) from the UIPP For- USA) (500 mg/l). The former is a large spec- estry Fungi Collection was used at the same trum antibiotic and the latter has both anti- time as a positive control. bacterial and antifungal action and could be used to distinguish fungi of the Ophiostoma Molecular analysis genus (Harrington, 1981; Hawksworth