Fungi of genus (: ) 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 (Badler, 1992). Cladistic stud- (Tainter & Baker, 1996). ies have shown that ambrosia fungi such as In the forest, there are several examples species of 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 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 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.

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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 et al., 1981). Cultures were incubated at 25±1ºC in The molecular analysis of the isolates was darkness. Pure cultures of each were based on the amplification and sequencing of obtained and the isolates were grouped ac- the 18S rDNA region, according to Cassar & cording to their macroscopic characteristics. Blackwell (1996), Jones & Blackwell (1998) In the present work only representative iso- and Rollins et al. (2001). DNA was extracted lates of Raffaelea-like cultures were chosen using the PuregeneDNA® kit. PCR amplifi- to continue the studies. cation and sequencing of the SSU-rDNA was performed as described by Henriques (2007). Morphological characterisation Homologous sequences were obtained from GenBank (NCBI) using Basic Local Align- Fungal identification was based on cultural ment Search Tool (BLAST). Sequences were and morphological features according to El- aligned using BioEdit v. 7.0.5.3. The phylo- lis (1971, 1976), Lanier et al. (1978), Kiffer genetic analysis was performed with MEGA & Morelet (1997) and Barnett & Hunter v.4.0 (Tamura et al., 2007). In the phylogenetic (1998). Conidia biometry of the different tree, downloaded sequences are indicated by isolates was assessed on cultures grown on their GenBank accession numbers. A member Fungi of Raffaelea genus (Ascomycota: Ophiostomatales) associated to Platypus Cylindrus (Coleoptera: Platypodidae) in Portugal 99 of Ophiostomatales was used as an outgroup cording to their macroscopic characteristics ( schenckii Hekt. & Perkins). and representative isolates from each group were chosen to pursue the studies: PC05.005, PC05.006, PC05.007and PC06.001, integrated RESULTS AND DISCUSSION into the Forestry Fungi Collection of the Insti- tuto Nacional de Recursos Biológicos (INRB). One of the most frequent fungi isolated Other isolates from the same work collection from P. cylindrus and its galleries belong to were also used in molecular assays. Raffaelea genus, obtained in particular from the intestinal content and from the mycangia Morphological characterisation (approximately 40% and 30% of all the iso- lated fungi, in both organs, in females and The cultural characterisation of the colo- males, respectively) (Henriques, 2007). Four ny’s surface and reverse is shown in Table 1 apparently different groups were obtained ac- and their observed aspects are in Figure 1.

Table 1 – Macroscopic description of the Raffaelea cultures on PDA

Figure 1 – Typical cultural aspect (surface, reverse) of Raffaelea isolates. 100 REVISTA DE CIÊNCIAS AGRÁRIAS

Isolate PC05.005. Hyphae hyaline and hyphal ends sometimes developing into to- septate that bound together forming compact rulose swellings. Conidiophores unbranched, hyphae ropes. Conidiophores macronematous distinct from hyphae bearing them, hyaline, and mononematous, erect, septate, slender solitary or clustered together to form spo- with a tapered apex, producing simpodulos- rodochia. Conidiae blastosporic, unicellular poric conidiae that leave cicatricial scars in and hyaline, usually solitary but sometimes the conidiogenous cells. Conidiae unicellu- upon germination in situ appear to be in mo- lar, hyaline, with variable forms (triangular, nilioid chains, smooth-walled with variable oval and allantoid) and measuring 5,0-(5,8)- forms (triangular, oval or fusiform), measu- 8,4 x 1,7-(2,1)-3,3 µm. ring 5-(9)-20x2,5-(3,7)-7,5 µm. Isolate PC05.006. Hyphae hyaline and Isolate PC06.001. Hyphae hyaline and septate that bound together forming compact septate, long, ascendant, erect and vigorous, hyphae ropes. Conidiophores macronema- simple or feebly ramified. Conidiophores tous and mononematous, erect, septate, slen- macronematous and mononematous, erect, der with a tapered apex, producing simpodu- septate, slender with a tapered apex, produ- losporic conidiae that leave cicatricial scars cing simpodulosporic. Conidiae adherent in in the conidiogenous cells but not so pronou- a mucilaginous droplet, leaving a discreet nced as in isolate I5.05. Conidiae unicellular cicatricial scars in the conidiogenous cells. and hyaline, with variable forms (triangular, Conidiae unicellular and hyaline, smooth- oval and fusiform) and measuring 3,3-(3,7)- walled, rounded apex and truncated base, 5,0 x 1,7-(1,8)-3,3 µm. with variable forms (pyriform, claviform and Isolate PC05.007. Hyphae hyaline and cuneiform) and measuring 5,0-(7,8)-10,0 x septate repeatedly branched and interlocked, 2,5-(3,7)-5,8 µm.

Figures 2 – Conidiophores and conidia of fungi of Raffaelea genus, a-b) isolate PC05.005: a) conidia allantoid (x600), b) conidia triangular (x600); c-e) isolate PC05.006: c) conidia fusiform to allantoid (x600), d) conidia triangular (x600); e) conidia fusiform (x600); f) isolate PC05.007: conidia pyriforme to globose (x1000); i) isolate PC06.001: conidia pyriform truncated (x1000). Fungi of Raffaelea genus (Ascomycota: Ophiostomatales) associated to Platypus Cylindrus (Coleoptera: Platypodidae) in Portugal 101

Cycloheximide sensitivity However, species of Ophiostoma have a peculiar cell wall (composed by cellulose All the isolates have been grown at the and ramnose) whose structure prevent the same different cycloheximide concen- antibiotic molecule entrance in the cell, trations, occurring a growth diminution thus making these fungi tolerant to cyclo- with the antibiotic concentration increase heximide. Given that all the Raffaelea (Figure 3). The cycloheximide is an anti- isolates have been grown as the O. ulmi biotic that inhibits the protein synthesis in control, it was verified the behaviour simi- the majority of the eukaryotic organisms. larity with this complex.

Figure 3 – Results of the cycloheximide sensitivity test for two Raffaelea isolates in comparison with an Ophiostoma ulmi isolate.

Molecular analysis nus in the Ophiostomatales. The sequen- ces comparison of some isolates suggests The partial sequencing of the rDNA the hypothesis that at least three distinct small subunit (18S) of Raffaelea isolates groups of Raffaelea sp. are associated to allowed, one more time, to locate this ge- P. cylindrus.

Figures 4 – Phylogram obtained from distance analysis using the bootstrap method with Neighbour-joining search (nr. replicates=1000) with Kimura-2-parameter substitution model. rDNA sequence alignment on small subunit region (SSU-rDNA) data of Raffaelea spp. obtained from Platypus cylindrus and their galleries on cork oak. The tree was rooted to (AB374289). 102 REVISTA DE CIÊNCIAS AGRÁRIAS

The phylogram analysis also suggests that Blume (Kubono & Ito, 2002; Kinuura & Ko- isolate PC06.001 is close to R. montetyi and bayashi, 2006). A recently identified Raffae- PC05.007 is related to R. canadensis. Isolates lea species associated with the ambrosia bee- PC05.005 and PC05.006 appear to be very tle Eichhoff was related similar originating a separate group also clo- with a new devastating disease of Lauraceae se to Ophiostoma spp.. Nevertheless, conju- plants in South Carolina (USA) (Fraedrich et gating molecular and morphological analysis al., 2007). is possible to suggest that PC05.005 is next to R. ambrosiae. Twelve species were described within Ra- CONCLUSION ffaelea, the majority being associated with ambrosia insects (Kubono & Ito, 2002; Bis- In the last decade, the insect P. cylindrus by et al., 2006). Concerning P. cylindrus, R. has been considered one of the most impor- ambrosia and R. monteyi had been already tant biotic agents directly involved in cork identified as the main ambrosia fungi (Arx oak decline. Being an ambrosia beetle, it es- & Hennebert, 1965; Morelet, 1998). In bi- tablishes symbioses with fungi that it carries bliographical terms it is also necessary to and inoculates in the host tree to favour its consider Sporothrix sp. described for Baker settlement. (1963) and later classified asR. ambrosiae by The relative importance of the isolated Arx & Hennebert (1965). In the same way, fungi is quite variable: besides those invol- isolates of Cephalosporium sp. made by ved in insect nourishment, some could be po- Baker (1963) and one brownish fungus not tentially pathogenic to cork oak, while others identified by Cassier et al. (1996) but later could have an antagonistic action or be sim- classified as R. montetyi by Morelet (1998) ply saprobes that are involved in a commen- must be considered. salist relation with the host tree. Raffaelea is a mitosporic genus poorly stu- In this work, Raffaelea spp. were the died perhaps for its cryptic nature: although fungi most frequently isolated, especially cosmopolite (Kiffer & Morelet, 1998), living from the mycangia and the intestinal con- in symbiosis with insects they are not com- tent both of female and male insects, thus monly observed. Even if their sexual phase leading to the conclusion that this genus is still unknown, observations of the conidial includes the primary ambrosia fungi asso- development of Raffaelea spp. are concor- ciated with P. cylindrus. All the conduc- dant with the position of this genus within ted essays pointed out that Raffaelea spp. the Ophiostomatales group (Gebhardt & are closely related to Ophiostomatales. Oberwinkler, 2005). Although the relations inside each group Studies on oak decline in Europe show are not satisfactorily clear their morpho- that fungi of the complex Ophiostoma/ Cera- logical characterisation and rDNA sequen- tocystis are frequent pathogens of species of ce comparison corroborate the hypothesis Quercus (Badler, 1992; Degreef, 1992; Dela- that at least three distinct species of Raffa- tour et al., 1992). Santos et al. (1999) regis- elea sp. are associated with P. cylindrus: tered the occurrence of Ophiostoma sp. in Q. R. ambrosiae, R. montetyi and probably R. suber in Portugal. The effect of R. ambrosia canadensis. This last one was never clear- and R. montetyi in Q. suber is still unkno- ly associated to that interaction. wn; however, in Japan the pathogenicity of To fully clarify the taxonomic status of Ra- R. quercivora was proven (Kubono & Ito). ffaelea species associated with P. cylindrus, This primary ambrosia fungus of P. querci- either in Portugal or in the other Mediter- vorus Murayama was associated with a mass ranean countries, additional sequence data mortality of fagaceous, especially Q. serrata need to be generated. Also, many more isola- Thunb., Q. mongolica Fich. and Q. crispula tes must be brought into this study. Fungi of Raffaelea genus (Ascomycota: Ophiostomatales) associated to Platypus Cylindrus (Coleoptera: Platypodidae) in Portugal 103

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