Maritime pine (Pinus pinaster Aiton) is a western Mediterranean and north-African typical species that stretches down to the Atlantic coast. Most extensive forests are located in Spain, France and Portugal. In Spain, P. pinaster is an important source of natural goods; it covers naturally the largest surface (600.000 ha) (Del Río et al., 2004) and is the pine species more intensively used in reforestation (800.000 ha) for wood and resin production, with 270.000 ha managed for resin tapping in the old sixties (40.000 ton per year, Serrada, 2004).

During the last years a general decline has appeared in some Maritime pine stands located at the north and centre of Spain (Álvarez et al,. 2008a). Symptoms expressed by declining maritime pines include sparse tree crowns, with unusual crown transparency, and short, yellow-green needles, and death (Fig. 1). Blue-stain was always visible in the wood since the first stages of the disease, suggesting damages caused by ophiostomatoid or other fungi as Diplodia pinea. However, no damages caused by insects are usually found in these P. pinaster stands. The symptoms spread following a gradient of mortality indicating damages caused by a biotic agent. At the final stages of the disease, symptoms appear with extraordinary virulence being visible a sudden and entire necrosis of the crown, and the dead of the tree in some weeks after the more evident symptoms appearance (Alvarez et al., 2008b, 2009). The mortality rate was important reaching 60% of the trees in some heavily affected stands. Apparently, there is no relationship between the decline and resin tapping, as decline can be also found in non-tapped trees.

Ophiostomatoid fungi are commonly associated to blue-stain diseases in conifers (Solheim & Langstrom, 1991; Wingfield et al., 1993; Hausner et al., 2005). Ceratocystis spp., Ophiostoma spp. and Ceratocystiopsis spp. are the main species causing blue-stain in stems, or roots of conifers (Solheim et al., 1993; Grylls & Seifert, 1999), appearing both in their teleomorphic or anamorphics states (Graphium spp., Leptographium spp., Chalara spp.; Jacobs & Wingfield, 2001). Thus, Leptographium wagenerii is causing black-stain in roots of conifers in North America (Otrosina et al., 1999) and other species like L. wingfieldii has been commonly associated to a general decline of P. sylvestris in Poland (Jankowiak et al., 2007). In addition, O. minus has been shown as one of the most virulent ophiostomatoid fungi on pines (Masuya et al., 2003). This fungus was associated with an important decline of Pinus sylvestris in France (Piou & Lieutier, 1989) and recently related with important diseases like these caused by Dendroctonus frontalis (the Southern Pine Beetle) in North America (Six & Klepzig, 2004) or by the pine wood nematode pathogen Bursaphelenchus xylophilus (Maehara et al., 2005).

Although pathogenic and saprophytic fungal taxa have been described associated with different conifers (Ganley & Newcombe, 2006; Hu et al., 2007; Zamora et al., 2008; Botella et al., 2010) the ophiostomatoid fungi associated to Pinus pinaster, particularly in Spain, and its influence on the health status of the tree, are still barely known. The main aim of this study was to identify the ophiostomatoid fungi associated to P. pinaster trees showing decline symptoms.

The intensity of defoliation in the sampled stands ranged from 10 to 100% and the mortality associated reach the 60% of the trees at the Burgos stand, the more heavily affected by the decline.

A total of 21 fungal species were isolated and identified from the eighteen trees (Table 1). Eleven isolates belonged to the ophiostomatoid group being Ophiostoma and Ceratocystis, with three species each, the more represented genera. Ceratocystiopsis only appeared with two species. Three unidentified ophiostomatoids appeared as well with a low frequency in the P. pinaster samples. Ophiostoma minus (Hedgcock) was the most frequently isolated fungus, followed by O. ips (Rumbold). Some other taxa without any phytopathological importance were also identified (i.e. Penicillium sp. (Link), Aspergillus niger (Tiegh). Rhizopus sp. (Ehrenb)).

Canonical Correspondence Analysis (CCA) showed all the independent variables analyzed with significant values of probability: (i) stand (Burgos/Avila) (P = 0.002, data not shown ), (ii) tissue type (xylem/phloem/needles) (P = 0.022, Figure 4), (iii) level of sample in the tree (levels 1, 2, 3, 4, 5, 6, Figure 5) (P = 0.044) and (iiii) healthy status of the tree (healthy, diseased, dead) (P = 0.046, data not shown).

The relative frequency of taxa was higher in Avila (61.3%) than in Burgos (38.6%). O. minus was more abundant in Burgos (13.1%) than in Avila (8.9%), whereas O. ips and C. fasciata were more frequent in this second location with 11.3% and 8.9%, respectively (Table 1). Analyzing the vegetal tissues, Ophiostoma minus appeared three times more frequently in the phloem of Ávila pine trees whereas in Burgos, this appearance was similar for both tissues (Table 2, Fig. 4). For Ophiostoma ips, we found the same pattern for both localities.

Almost all of the ophiostomatoid species appeared more frequently in the phloem (50.3%) than in the xylem (42.5%) (Fig. 4). The axis of this correspondence analysis explained the 38% of the data variability and the three types of tissue showed significant values of probability (P-needles = 0.032, P-phloem = 0.004 and P-xylem = 0.05). The more generalist species showed a clearer trend to appear on the needles such as Penicilium sp. or Phoma sp (Saccardo) whereas the ophiostomatoid species appeared exclusively in the vascular tissues. Thus, O. minus has a frequency of 13.7% in the phloem and 8.3% in the xylem, O. ips, 9.5% and 5.9 % and C. fasciata with 7.1% and 5.3% respectively (Table 2).

The correspondence analysis shows ophiostomatoid especies (excepting Ceratocystis alba) in other tissues that collar (heights 1) and needles (heights 6) (Figure 5). However, the generalist species showed a clear affinity for these tree positions, the closest to the ground and needles and clearly separated from the others. In this figure, variables explained the 4.1% of the total variability of the data and only the sampling positions 1 and 6 showed significant statistical differences (P-values of 0.018 and 0.033, respectively). The two more abundant phytopathogenic fungi, Ophiostoma minus and O. ips, appeared distributed in a fairly homogeneous pattern. The height 5 (terminal guide) hosted almost all the species of fungi identified, contributing almost with a quarter of the total variability (24.4%).

O. minus was mainly found in dead and diseased trees while O. ips appeared almost twice in healthy trees than in died ones (Table 3). Ceratocystiopsis fasciata (Olchow. and Reid) had also a remarkable representation (12.5%) and mainly in dead trees (8.9%). Other ophiostomatoid fungi were found with lower representation (between 0.6% and 1.7%) like Ceratocystis allantospora (Griffin), Ceratocystis angusticollis (Wright and Griffin), Ceratocystis arborea (Olchow and Reid), Ceratocystiopsis alba (DeVay et al.), Ophiostoma piliferum (Fries) and the three unidentified specimens: Ophiostoma type 1, Ophiostoma type 6 and Ophiostoma type 7. Fungal species explained 37.4% of the affinity between plots. Ophiostoma minus and Ophiostoma ips had the highest representation in the sampling units as observed in the correspondence analysis graph. Most of the affinity between plots was explained by the presence of these two species. Ceratocystiopsis fasciata also highlights among the ofiostomatoid group.

In this study we identified eleven ophiostomatoid taxa associated to Pinus pinaster decline and, excluding typical laboratory contaminants (i.e. T. roseum), three of them were remarkable for their prevalence: Ceratocystiopsis fasciata, Ophiostoma ips and O. minus.

Ceratocystiopsis fasciata is an cryptic species not mentioned in any other publication, only one about its morphological description (Grylls & Seifert, 1999). In this study, it has emerged strongly related to the dead trees, but not particularly with the diseased trees suffering the decline. These results seems to indicate that C. fasciata is not the main fungus causing the decline, and their presence on the trees might be subsequent to the decline.

Ophiostoma ips is a pathogenic species and is listed as the most frequent of those registered associated to Ips sexdentatus (Zou et al., 2001; Fernández et al., 2004; Bueno et al., 2010) and Orthotomicus erosus (Romón et al., 2007). Although it can kill trees (Fernández et al., 2004) O. ips could be not so virulent as O. minus (Popp et al., 1995; Zhou et al., 2002). In the present study it has emerged as the second more abundant (15.4% of the total), mainly associated to healthy trees.

Several reasons suggest that O. minus could be the more important ophiostomatoid related to P. pinaster decline in Burgos and Avila stands. : (1) it was the most prevalent, being isolated from the 22.0% of all identified trees, (2) it was the species more associated to diseased (7.7%) and dead trees (9.5%), (3) many references establish the high pathogenicity of this fungus (Langstrom et al., 1993; Popp et al., 1995; Jankowiak, 2006; Jankowiak & Rosa, 2007), even higher than O. ips in all the pathogenicity tests (Lieutier et al., 1989; Popp et al., 1995) and, (4) is a fungus associated in at least other two important pathologies around the world: the Southern Pine Beetle outbreak in Norteamerica (Six & Klepzig, 2004), and the Pine Wood Nematode disease (Togashi, 2004). This fungus is one of the most virulent Ophiostomatoids that have been ever described, since it has the capacity to kill trees when it was inoculated (Masuya et al., 2003). Its strong presence in the samples and the detection on diseased and dying trees (Table 3) indicate that it could play an important role in the decay of the trees sampled. However, pathogenicity proofs should be performed to definitively stablish its specific role in the decline.

However, we should take into account the possibility that the present state of the decline may be due to a complex relationship between fungal (Ophiostoma minus, Heterobasidion annosum, recently detected in some areas close to our sampling plots (Prieto-Recio et al., 2012), or even other pathogens as Phytophthora cinnamomi...), soil, climatic and silvicultural factors. Anyway, further work is necessary to determine the pathogenicity of O. minus and other associated ophiostomatoids fungi on adult trees, with the aim to definitively establish the association of this fungus with the maritime pine decline.

O. minus have also been associated with a decline of Pinus sylvestris in France (Piou & Lieutier, 1989). The relationship between O. minus and the damage observed in this host suggested that this fungus had an important role in the decline. On the other hand, Jankowiak et al. (2007), using two-year-old Scots pine seedlings, demonstrated that inoculation with O. minus produced significantly larger lesions on Picea abies than other ophiostomatoid fungi. . Comparing with other ophiostomatoid fungi, lesions induced by O. minus were significantly larger than lesions induced by O. ips (Popp et al., 1995). These results suggest O. minus is a good candidate to be involved in the P. pinaster decline. However, pathogenicity proofs are needed to definitively confirm its importance on this decline.

O. minus have been associated with Dendroctonus frontalis in North America (Six & Klepzig, 2004). This bark beetle attacks and kills southern pines, introducing fungi into them. Ophiostoma minus may initially aid beetles in killing trees, but later this bluestain fungus becomes an antagonist, competing with larvae for host phloem (Scott et al., 2008). Mite’s abundance was strongly correlated with O. minus and was an important driving force in promoting bluestain prevalence within trees (Lombardero et al., 2003). Spring abundances of mites and the prevalence of O. minus during D. frontalis infestation were strong predictors of beetle population decline (Hofstetter et al., 2006a, 2006b). Further studies on the interactions of O. minus with other insect or fungi associated to Pinus pinaster decline should be performed to establish their influence on the disease.

O. minus have been also associated to Pine Wood Nematode (PWN) pathogen B. xylophilus (Warren et al., 1995). This nematode produced a large population after eating this fungus, (Togashi, 2004). PWN was detected in Portugal three years ago, and recently in the Spanish border (Cáceres and Pontevedra provinces; DOE 2009, DOG 2012, respectively). The symptoms and the analysis performed by the CESANFOR Diagnostic Center (Castilla & León Government, Spain) discarded the presence of Bursaphelenchus xylophilus in the areas affected by Pinus pinaster decline. This fungus has been also associated to PWN vectors. Among ophiostomatoid fungi, O. minus and O. piceae were the most frequently isolated species from M. galloprovincialis adults (Jankowiak & Rossa, 2007). The interaction of O. minus with the PWN and its insect vector should be taken into account if the disease appears in these areas in the future.

In our study of declining P. pinaster stands in Northern Spain we found a total of 21 fungal taxa isolated and identified; eleven of these species belonged to the ophiostomatoid group. Ophiostoma minus, O. ips and C. fasciata were the most frequently isolated fungi, mainly associated to dead and diseased trees. Our results suggest paying more attention to O. minus as a potential agent of decline in P. pinaster stands. However, more studies are needed to stablish the importance of abiotic (drought, resin tapping...) or biotic (Heterobasidion annosum, or Phytophthora cinnamomi...) in the P. pinaster decline.