Ceratocystiopsis quercina sp. nov. Associated with Platypus cylindrus on Declining Quercus suber in Portugal.

Platypus cylindrus is the most common ambrosia beetle in stands of Quercus suber in Portugal. This insect farms specialized fungi in sapwood galleries, using its mycangia to carry and store these organisms. Some ectosymbiotic fungi carried by P. cylindrus are phytopathogenic and cause extensive tree mortality and severe economic losses. To understand the role of P. cylindrus fungal symbionts in stands of Q. suber we examined beetle galleries present in declining and/or dying cork oak trees during field surveys. Logs with active galleries were obtained in situ and from captured emerging beetles. Insects were aseptically dissected, and their mycangia and intestine were retrieved. Morphological and molecular profiles of fungal isolates obtained from cultured insect parts were carried out to accurately characterize and identify isolated fungi. Molecular characterizations were performed with DNA sequence data from four loci, i.e., LSU, SSU, 5.8S-ITS2-28S, and TUB. Morphological results consistently showed a collection of Ophiostoma-like fungal axenic isolates, while phylogenies inferred that this collection constitutes an undescribed taxon reported herein for the first time in association with P. cylindrus in Portuguese cork oak stands. The novel species was erected as Ceratocystiopsis quercina sp. nov. and constitutes a new phytopathogenic fungal species associated with symptoms of vegetative cork oak decline.

Symbiotic Fungi of an Ambrosia Beetle Alter the Volatile Bouquet of Cork Oak Seedlings.

In Portugal, fungal symbionts of the ambrosia beetle Platypus cylindrus affect tree vigor of cork oak (Quercus suber) and are linked with the cork oak decline process. Fungal symbionts play crucial roles in the life history of bark and ambrosia beetles and recent work indicates complex interactions on the fungal and plant metabolic level. Colonized trees may respond with an array of currently unknown volatile metabolites being indicative of such interactions, acting as infochemicals with their environment. In this study, we examined volatile organic compounds (VOCs) of cork oak seedlings wound inoculated with strains of three fungal associates of P. cylindrus (Raffaelea montetyi, R. quercina, and Ceratocystiopsis sp. nov.) over a 45-day period by means of thermodesorption gas chromatography-mass spectrometry techniques. Fungal strains induced largely quantitative but species-specific changes among the 58 VOCs characterized. Overall, monoterpenes-the major volatiles of cork oak foliage-were significantly reduced, possibly a result of fungal biotransformation. Acetophenone, sulcatone, and nonanal-volatiles known for mediating ambrosia beetle behavior-increased in response to fungal inoculation. Qualitative VOC profiles of excised tissue of wood lesions (21 VOCs) and pure fungal cultures (60 VOCs) showed little overlap with seedling VOCs, indicating their plant-derived but fungal-induced origin. This chemoecological study expands on the limited knowledge of VOCs as infochemicals emitted from oak trees threatened by oak decline in relation to beetle-vectored ophiostomatoid fungi. It opens new avenues of research to clarify mutualistic or pathogenic aspects of these complex symbiotic interactions and develop new control strategies for P. cylindrus, including its mycobiota.

Microbiome Associated with the Mycangia of Female and Male Adults of the Ambrosia Beetle Platypus cylindrus Fab. (Coleoptera: Curculionidae).

The ambrosia beetle Platypus cylindrus Fab. (Coleoptera: Curculionidae) is a major cork oak pest in Portugal. Female and male beetles have different roles in host tree colonization and are both equipped with prothoracic mycangia for fungal transportation. Despite a known beneficial role of bacteria in ambrosia beetles, information on bacterial composition associated with prothoracic mycangia structures is scarce. Bacterial community from mycangia of P. cylindrus male and female beetles collected from cork oak galleries was investigated by means of 16S metagenomics. Mycangia anatomical structure was also explored with histological techniques and X-ray computed microtomography to highlight evidence supporting biological sexual dimorphism. A bacterial community with highly diverse bacterial taxa with low abundances at the genus level was revealed. Lactobacillales, Leptotrichia, Neisseria, Rothia, and Sphingomonadaceae were significantly more abundant in males, while Acinetobacter, Chitinophagaceae, Enterobacteriaceae, Erwiniaceae, Microbacteriaceae, and Pseudoclavibacter were more abundant in females. Additionally, a core bacteriome of five genera was shared by both sexes. Histological examination revealed visible connections linking external and internal tissues in females, but none in males. Overall, these results provide the first insights into sexual differentiation for bacteria in a Platypodinae beetle species, identifying key patterns of bacteria distribution in the context of beetle ecology and functional behavior.

Effect of Monochamus galloprovincialis feeding on Pinus pinaster and Pinus pinea, oleoresin and insect volatiles.

In Portugal, the pine black sawyer Monochamus galloprovincialis is the principal vector of the pinewood nematode, Bursaphelenchus xylophilus, the causal agent of pine wilt disease (PWD), a lethal phyopathogen with major ecological and economic consequences to European forestry. The aim of this study was to determine the influence of M. galloprovincialis feeding on the volatiles emitted by pine trees. This study focused on the pine species which are most relevant to Portugal, that is, Pinus pinaster (maritime pine) and Pinus pinea (stone or umbrella pine), assessing to what extent pine chemotypes might influence feeding by the insect vector. Preliminary evaluation of each maritime pine essential oil allowed recognizing the existence of two main chemotypes (C1 and C2) and absence of chemical variability in P. pinea. Emission of volatiles from pine trees was evaluated before and during 24 h of feeding by a mixed-sex pair of newly emerged, unfed M. galloprovincialis. Volatiles were also collected from the oleoresin released from the feeding wounds as well as from the insects after feeding. Pine volatiles were collected by solid phase microextraction (SPME) and insect volatiles extracted with pentane, and all analysed by gas chromatography-mass spectrometry (GC-MS) and by GC for component identification and quantification, respectively. Of the seventeen emitted volatiles detected in SPME analyses of P. pinaster, ß-pinene, α-pinene, ß-caryophyllene, and germacrene D showed the highest average fold increases as a result of M. galloprovincialis feeding. When grouped by P. pinaster chemotype, C1 and C2 groups of trees showed different patterns of responses. ß-Caryophyllene and germacrene D showed the highest fold increase in C1 trees, whereas ß-pinene and α-pinene clearly dominated in C2 trees. Likewise, the oleoresin volatiles from C1 trees were dominated by δ-3-carene and/or ß-pinene, whereas α-pinene and ß-pinene were the main volatile components from oleoresin of C2 trees. Nine components were detected in P. pinea volatiles, of which limonene showed the highest fold increase as a result of insect feeding. The volatiles collected from the insects after they had fed on P. pinaster included α-pinene, ß-pinene, and abietic acid, and by the straight-chain n-alkanes n-C27, n-C29, and n-C25, together with the methyl-branched hydrocarbons 3-meC29, 2-meC28, and 3-meC27. A better understanding of the responses of different P. pinaster chemotypes to feeding by M. galloprovincialis may be helpful in the development of new lures to improve pine sawyer trapping in integrated pest management for control of PWD.

Efficacy of sulfuryl fluoride against the pinewood nematode, Bursaphelenchus xylophilus (Nematoda: Aphelenchidae), in Pinus pinaster boards.

BACKGROUND: The pinewood nematode (PWN) Bursaphelenchus xylophilus is an important conifer disease worldwide. It is the direct cause of the death of millions of pines in south-east Asia (mainly Japan, China and Korea) and has been established in Portugal since 1999. The phasing out of methyl bromide has created an urgent need for alternative treatment of wood packaging materials. The effect of sulfuryl fluoride (SF), a broad-spectrum fumigant used to control insects, was tested in Pinus pinaster boards naturally infested by PWN. RESULTS: Boards were fumigated for 24 h at three different temperatures (15, 20 and 30 °C) with dosage ranges of 3169-4407, 1901-4051 and 1385-2141 gh m(-3) respectively. Treated wood was sampled for nematode identification and counting, before treatment and after 24 h, 72 h and 21 days. No survival was found in the 15 °C and 30 °C treatments, while at 20 °C the mortality ranged from 94.06 to 100%. Some reasons for the survival at 20 °C are presented. CONCLUSION: Results confirm SF to be an effective quarantine treatment for PWN at 15 and 30 °C. Further studies are needed to obtain the most effective dosage at 20 °C, and to determine the toxicity of SF fumigation on B. xylophilus at other temperatures, especially at 25 °C.

Ambrosia fungi in the insect-fungi symbiosis in relation to cork oak decline.

Ambrosia fungi live associated with beetles (Scolytidae and Platypodidae) in host trees and act as a food source for the insects. The symbiotic relation is important to the colonizing strategies of host trees by beetles. Ambrosia fungi are dimorphic: they grow as ambrosial form and as mycelium. The fungi are highly specialized, adapted to a specific beetle and to the biotope where they both live. In addition other fungi have been found such as tree pathogenic fungi that may play a role in insects host colonization success. Saprophytic fungi are also present in insects galleries. These may decompose cellulose and/or be antagonistic to other less beneficial fungi. This paper summarizes the importance of ambrosia fungi and the interaction with insects and hosts. The possibility of the transport of pathogenic fungi by Platypus cylindrus to cork oak thus contributing for its decline is discussed.