Research Article


Ophiostomatoid fungi associated with declined Pinus pinaster stands in Spain


Gonzalo Álvarez

Instituto Universitario de Investigación y Gestión Forestal Sostenible. Universidad de Valladolid-INIA. Avenida de Madrid 44, 34004, Palencia, España

Mercedes Fernández

Instituto Universitario de Investigación y Gestión Forestal Sostenible. Universidad de Valladolid-INIA. Avenida de Madrid 44, 34004, Palencia, España

Julio J. Diez

Instituto Universitario de Investigación y Gestión Forestal Sostenible. Universidad de Valladolid-INIA. Avenida de Madrid 44, 34004, Palencia, España



Aim of study: We studied the presence of fungi and distribution patterns in relation to the health status of declining Pinus pinaster trees.

Area of study: Trees in two declining stands in Central Spain were allotted to three declining classes.

Material and Methods: Trees in two declining stands in Central Spain were allotted to three declining classes (healthy, declining and recently dead) and 3 trees of each class were felled in each stand. Wood slides (phloem and xylem) were taken at six positions along the trees and samples collected from fungal identification.

Main results: A total of 21 fungal taxa were isolated and identified; eleven of these species belonged to the Ophiostomatoid group. Ophiostoma minus was the most frequently isolated fungus and was identified in 22% of the samples, mainly associated to dead and diseased trees.

Research highlights: Together these results suggest a putative association of O. minor with the decline in this area, and thus we suggest paying more attention to this fungus as a potential agent of decline in P. pinaster stands.

Keywords: Ophiostomatoid fungi; forest pathology; bluestain fungi; multivariate analyses.

Abbreviations: UTM: Universal Transverse Mercator coordinate system; MEA: Malt Extract Agar; DNA: Deoxyribonucleid acid; ITS: Internal Transcribed Spacer; BLAST: Basic Local Alignment Search Tool; CCA: Canonical Correspondence Analyses; PWN: Pine Wood Nematode.

Citation: Alvarez, G., Fernandez, M., Diez, J.J. (2015). Ophiostomatoid fungi associated with declined Pinus pinaster stands in Spain, Volume 24, Issue 1, e-006, 9 pages.

Received: 5 Feb 2014. Accepted: 15 Dec 2014

Copyright © 2015 INIA. This is an open access article distributed under the Creative Commons Attribution License (CC by 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Funding: The autor(s) received no specific funding for this work.

Competing interests: The authors have declared that no competing interests exist.

Correspondence should be addressed to Julio J. Diez:





Materials and methods







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.

Figure 1. Pinus pinaster stand affected by decline in Burgos province.

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 Dendroctonusfrontalis (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.

Materials and MethodsTop


Pinus pinaster samples were collected from two plots located in two pine stands with decline symptoms in Castilla & León region (Spain). These plots were selected because of the severity of the damages. One is sited at Burgos province (La Horra, UTM coordinates 37T, X-722292, Y-331796, soil type Inceptisol, suborder Xerochrepts) and the other at Ávila province (San Esteban del Valle, UTM coordinates 29T, X-738530, Y-4729138, soil type Inceptisol, suborder Xerochrepts and Xerumbrepts). The Burgos stand is 820 m.a.s.l., with dried gypsum soils and 500 millimetres rain per year. The maximum annual temperature is 29 °C and the minimum –2 °C. The Avila stand is about 1250 m.a.s.l., with dried soils and 1600 mm rain per year. Maximum annual temperature is 30 °C and minimum 0 °C.

In October 2007, nine trees were randomly selected per stand according to their health status (i) three healthy, (ii) three with decline symptoms and (iii) three recently dead (Fig. 2). The trunk was cut with a chainsaw and samples were collected from six different levels (Fig. 3): (1) the collar, (2) the middle of the trunk, (3) the first alive branch, (4) a branch cross-section, (5) the terminal guide and (6) some needles from the top of the crown. Fungal identification was performed using phloem and xylem pieces from these slides. A total of 792 moist chambers were performed ([18 trees x 5 levels x 2 tissues (xylem and phloem) x 4 replicates each]+ [18 trees x 1 level x 1 tissue (needles) x 4 replicates each]).

Figure 2. Health status of Pinus pinaster trees selected for the study: (i, left) three healthy, (ii, center) threes with different degrees of decline symptoms and (iii, right) three recently dead.

Figure 3. Different levels where samples were taken from Pinus pinaster trees, 1: collar, 2: middle of the trunk 3: fist alive branch, 4: branch cross section, 5: terminal guide, 6: needles.

Fungal isolation and identification

In order to identify the fungal taxa, xylem and phloem samples (1 x 2 cm2) as well as needles (2 cm in length) were sampled from each tree, and processed using moist chamber and culture media methodologies, as it has been outlined in previous fungal studies (Santamaría & Diez, 2005; Zamora et al., 2008; Botella et al., 2010; Botella & Diez, 2011). The moist chamber is based in finding fruiting bodies on plant tissues (xylem, phloem and needles) after incubation in Petri dishes at room temperature (25°C ± 2°C) (Zhou et al., 2001) in diffused daylight containing wet paper. A total of 792 moist chambers were performed ([18 trees x 5 levels x 2 tissues (xylem and phloem) x 4 replicates each]+ [18 trees x 1 level x 1 tissue (needles) x 4 replicates each]).

The culture media method included growing mycelia on culture media. Tissue samples were cultured on MEA+tetracycline (33 g of malt extracts, 16 g of agar and 250 mg of tetracycline per litre of distilled water) and were processed into a laminar flow hood to avoid contaminations. Only the surface of needles was sterilized due to the fact that sodium hypochlorite used in sterilization can affect the growth of the ophiostomatoid fungi in the wood. Samples were washed in running tap water for one minute, soaked in 70% alcohol for two minutes, and soaked twice in 3% sodium hypochlorite solution, for two minutes each time. Finally, the samples were immersed twice in sterile distilled water, for two minutes each time, to remove any possible remains of the hypochlorite. Petri dishes were stored for 7 days in the dark at 25ºC and then carefully examined using binoculars. Fruiting bodies from the samples were identified with a microscope Nikon Eclipse E-400 model. A total of 792 Petri dishes were performed ([18 trees x 5 levels x 2 tissues (xylem and phloem) x 4 replicates each]+ [18 trees x 1 level x 1 tissue (needles) x 4 replicates each]).

Fungi in Petri dishes were identified according to morphological characteristics of spores and other reproductive structures, such as size, shape and colour. Different taxonomic keys were used for fungal identification (Barnett & Hunter, 1998; Hanlin, 1998; Goidanich, 1990; Lanier et al., 1978; Sutton, 1980; Kiffer & Morelet, 1999). For those structures belonging to the ophiostomatoid fungi Wolfaardt et al., (1992) and Grylls & Seifert (1999) keys were used. Some recalcitrant species like Ophiostoma ips (Rumb.) Nannf. were determined by DNA sequencing (primers ITS1 and ITS4) and BLAST match (Kuekam et al., 2013).

Statistical analysis

In order to assess the influence of the main explanatory variables on the fungal occurrence, a Canonical Correspondence Analysis (CCA) was carried out. The presence or absence of fungal species found for each sample was considered as the dependent variable. The independent variables considered were (i) stand (Burgos/Avila), (ii) tissue type (xylem/phloem/needles), (iii) level of sample in the tree (levels 1, 2, 3, 4, 5, 6) and (iiii) healthy status of the tree (healthy, diseased, dead). A forward selection procedure using the Monte Carlo’s test was then applied to test the degree of significance, with 499 permutations for exploratory analysis and 999 for final results (Legendre & Legendre, 1998). The constrained ordination was performed by using default settings and untransformed species data by means of CANOCO for Windows version 4.5 (Ter Braak & Smilauer, 2002).


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)).

Table 1. Absolute and relative prevalence of the fungal species . At each location, nine trees and six sampling position along each individual tree were studied.

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).

Figure 4. Correspondence analysis considering sampled tissues and identified species. X: xylem, P: phloem, N: needles. Alt: Alternaria sp., Aspnig: Aspergillus niger, Ceralb: Ceratocystiopsis alba, Cerfas: Ceratocystiopsis fasciata, Cerall: Ceratocystis allantospora, Cerang: Ceratocystis angusticollis, Cerarb: Ceratocystis arborea, Did: Didymella sp., Epi: Epicoccum sp., Ophips: Ophiostoma ips, Ophmin: Ophiostoma minus, Ophpil: Ophiostoma piliferum, Ophtil: Ophiostomatal with perithecia type 1, Ophti6: Ophiostomatal with perithecia type 6, Ophti7: Ophiostomatal with perithecia type 7, Pen: Penicillium sp., Pho: Phoma sp., Pitcha: Pithomyces chartarum, Rhi: Rhizopus sp., Sep: Septonema sp, Triros: Trichotecium roseum.

Figure 5. Correspondence analysis considering sampled tree levels (1-6) and identified species. Alt: Alternaria sp., Aspnig: Aspergillus niger, Ceralb: Ceratocystiopsis alba, Cerfas: Ceratocystiopsis fasciata, Cerall: Ceratocystis allantospora, Cerang: Ceratocystis angusticollis, Cerarb: Ceratocystis arborea, Did: Didymella sp., Epi: Epicoccum sp., Ophips: Ophiostoma ips, Ophmin: Ophiostoma minus, Ophpil: Ophiostoma piliferum, Ophtil: Ophiostomatal with perithecia type 1, Ophti6: Ophiostomatal with perithecia type 6, Ophti7: Ophiostomatal with perithecia type 7, Pen: Penicillium sp., Pho: Phoma sp., Pitchithomyces chartarum, Rhi: Rhizopus sp., Sep: Septonema sp, Triros: Trichotecium roseum.

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.

Table 2. Prevalence of fungi (%) per location and vegetal tissues.

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 with7.1% and 5.3% respectively (Table 2).

The correspondence analysis shows ophiostomatoid especies (excepting Ceratocystisalba) 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, Ophiostomaminus 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). Ceratocystiopsisfasciata (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. Ophiostomaminus and Ophiostomaips 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. Ceratocystiopsisfasciata also highlights among the ofiostomatoid group.

Table 3. Absolute and relative prevalence of the fungal species related to the healthy state of the sampled trees.


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.

Ceratocystiopsisfasciata 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.

Ophiostomaips 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 Orthotomicuserosus (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 Dendroctonusfrontalis 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.


The authors wish to thank to Gemma Pérez from Centro de Sanidad Forestal de Calabazanos (Junta de Castilla & León) for their useful help during the sampling and also to Dr. M. Wingfield and Dr. T. Zhou for the molecular identification of recalcitrant isolates.


Alvarez G, Fernández M, Diez JJ, 2008a. Hongos asociados al decaimiento del pino pinaster. Master Thesis, University of Valladolid. Spain.
Alvarez G, Fernández M, Diez JJ, 2008b. Hongos asociados al decaimiento de Pinus pinaster en España. Proc. XIV Congreso Sociedad Española de Fitopatología. Lugo (Spain). September 15-19.
Alvarez G, Fernández M, Diez JJ, 2009. Ophiostoma minus asociado a masas de Pinus pinaster (Aiton) con síntomas de decaimiento en Castilla y León. Proc. V Congreso Forestal Español. Ávila (Spain). September 21-25.
Barnett HL, Hunter BB, 1998. Illustrated genera of imperfect fungi. The American Phytopathological Society. St. Paul, Minnesota. USA. 218 pp.
Botella L, Diez, JJ, 2011. Phylogenic diversity of fungal enddophytes in Spanish stands of Pinus halepensis. Fungal Divers 47(1), 9–18.
Botella L, Santamaría O, Díez JJ, 2010. Fungi associated with the decline of Pinus halepensis in Spain. Fungal Divers 40, 1–11.
Bueno A, Diez JJ, Fernández MM, 2010. Ophiostomatoid fungi transported by Ips sexdentatus (Coleoptera; Scolytidae) in Pinus pinaster in NW Spain. Silva Fennica 44 (3), 387-397.
Del Río M, Bravo F, Pando V, Sanz G, Sierra de Grado R, 2004. Influence of individual tree and stand attributes in stem straightness in Pinus pinaster Ait. stands. Ann For Sci 61, 141-148.
DOE, 2009. Decreto 268/2009, de 28 de diciembre, por el que se adoptan medidas fitosanitarias para la erradicación y control del nematodo de la madera del pino Bursaphelenchus xylophilus (Steiner et Buhrer) Nickle et al., se regulan las indemnizaciones derivadas de la adopción de estas medidas y se dictan sus normas de aplicación para el año 2009 en la Comunidad Autónoma de Extremadura (Spain). Diario Oficial de Extremadura 248, pp 35397-35415.
DOG, 2012. Resolución do 16 de abril do 2012, da Secretaría Xeral de Medio Rural e Montes, polo que se establecen as restricións ás autorizacións de cortas de especies sensibles ao nematodo do piñeiro (Bursaphelenchus xylophilus) na zona demarcada das Neves. Diario Oficial de Galicia (Spain). 77. pp 14879-14880.
Fernández MM, García AE, Lieutier F, 2004. Effects of various densities of Ophiostoma ips inoculations on Pinus sylvestris in north-western Spain. For. Path 34(4), 213-223.
Ganley RJ, Newcombe G, 2006. Fungal endophytes in seeds and needles of Pinus monticola. Mycol Res 110, 318–327.
Goidanich G, 1990. Manuale di patologia vegetale. Edizioni Agricole della Calderini, Bologna, Italy. 1283 pp.
Grylls B, Seifert KA, 1999. A synoptic key to species of Ophiostoma, Ceratocystis and Ceratocystiopsis. In: Ceratocystis and Ophiostoma. Taxonomy, Ecology and Pathogenicity (Wingfield MJ, Seifert KA, Webber JF. eds). pp 261-268.
Hanlin RT, 1998. Illustrated Genera of Ascomycetes. The American Phytopathological Society. St. Paul, Minnesota, USA. 164 pp.
Hausner G, Iranpour M, Kim JJ, Breuil C, Davis CN, Gibb EA, Reid J, Loewen PC, Hopkin AA, 2005. Fungi vectored by the introduced bark beetle Tomicus piniperda in Ontario, Canada, and comments on the taxonomy of Leptographium lundbergii, Leptographium terebrantis, Leptographium truncatum and Leptographium wingfieldii. Can J Bot 83, 1222.
Hofstetter RW, Cronin JT, Klepzig KD, Moser JC, Ayres MP, 2006a. Antagonisms, mutualisms and commensalisms affect outbreak dynamics of the southern pine beetle. Oecologia 147, 679-691.
Hofstetter RW, Klepzig KD, Moser JC, Ayres MP, 2006b. Seasonal dynamics of mites and fungi and their interaction with southern pine beetle. Environmental Entomology 35, 22-30.
Hu HL, Jeewon R, Zhou DQ, Zhou TX, Hyde HD, 2007. Phylogenetic diversity of endophytic Pestalotiopsis species in Pinus armandii and Ribes spp.: evidence from rDNA and β- tubulin gene phylogenies. Fungal Divers 24, 1–22.
Jacobs K, Wingfield MJ, 2001. Leptographium species: tree pathogens, insect asociates, and agents of blue-stain. American Phytopathological Society, St. Paul, Minnesota, USA. 207 pp.
Jankowiak, R, 2006. Fungi associated with Tomicus piniperda in Poland and assessment of their virulence using Scots pine seedlings. Ann Forest Sci 63, 801-808.
Jankowiak R, Rossa R, Bilanski P, 2007. Contribution to pathogenicity of three blue-stain fungi associated with the pine sawyer beetle (Monochamus galloprovincialis) (Coleoptera: Cerambycidae) to Scots pine in Poland. Phytopathol Polon 46, 37-46.
Jankowiak R, Rossa R, 2007. Filamentous fungi associated with Monochamus galloprovincialis and Acanthocinusaedilis (Coleoptera: Cerambycidae) in Scots pine. Pol Bot J 52, 143-149.
Kiffer E, Morelet M, 1999. Les deuteromycetes, classification et clés d’identification generique. INRA. París. France. 306 pp.
Kuekam GN, Wingfield MJ, Roux J, 2013. Ceratocystis species, including two new taxa, from Eucalyptus trees in South Africa. Aus Plant Pathol 42(3), 283-311.
Langstrom B, Solheim H, Hellqvist C, Gref R, 1993. Effects of pruning young Scots pines on host vigour and susceptibility to Leptographium wingfieldii and Ophiostoma minus, two blue-stain fungi associated with Tomicus piniperda. Eur J Forest Pathol 23, 400-415.
Lanier L, Joly P, Bondoux P, Bellemère A, 1978. Mycologie et Patologie Forestières. Masson, Paris. 487 pp.
Legendre P, Legendre L, 1998. Numerical Ecology. Elsevier, Amsterdam, Holland. 852 pp.
Lieutier F, Yart A, Garcia J, Ham MC, Morelet M, Levieux J, 1989. Blue-stain fungi associated with two bark beetle pests of Scots pine (Pinus sylvestris): a preliminary study of fungal aggressiveness to the host tree. Ann Sci Forest 46, 210-216.
Lombardero MJ, Ayres MP, Hofstetter RW, Moser JC, Lepzig KD, 2003. Strong indirect interactions of Tarsonemus mites (Acarina: Tarsonemidae) and Dendroctonus frontalis (Coleoptera: Scolytidae). Oikos 102, 243-252.
López SR, Romón P, Iturrondobeitia JC, Goldazarena A, 2007. Los escolítidos del País Vasco. Guía práctica para su identificación y control. Servicio Central de Publicaciones del Gobierno Vasco. Vitoria-Gasteiz, Spain. 198 pp.
Maehara N, Hata K, Futai K, 2005. Effect of blue-stain fungi on the number of Bursaphelenchus xylophilus (Nematoda: Aphelenchoididae) carried by Monochamus alternatus (Coleoptera: Cerambycidae). Nematology 7, 161-167.
Martínez P, Alves F, Diez, J, 2012. In vitro and in vivo interactions between Trichoderma viride and Fusarium circinatum. Silva Fennica 46(3), 303–316.
Masuya H, Kaneko S, Yamaoka Y, 2003. Comparative virulence of blue-stain fungi isolated from Japanese red pine. J. For. Res 8, 83-88.
Otrosina WJ, Bannwart D, Roncadori, RW, 1999. Root-infecting fungi associated with decline of longleaf pine in the southeastern United States. Plant and Soil 217, 145-150.
Piou D, Lieutier F, 1989. Symptomatological observations and possible roles of Ophiostoma minus and Tomicus piniperda in the decline of Scots pine in the forest of Orleans. Ann Sci Forest 46, 39-53.
Popp MP, Johnson JD, Lesney MS, 1995. Characterization of the induced response of slash pine to inoculation with bark beetle vectored fungi. Tree Physiology 15, 619-623.
Prieto-Recio C, Romeralo C, Bezos D, Martin-Garcia J, Martinez-Alvarez, P, Botella L, Diez JJ, 2012. First report of Heterobasidion annosum on Pinus pinaster in Spain. Plant Dis. 96, 770.
Romón P, Zhou XD, Iturrondobeitia JC, Wingfield MJ, Goldazarena A, 2007. Ophiostoma species (Ascomycetes: Ophiostomatales) associated with bark beetles (Coleoptera: Scolytinae) colonizing Pinus radiata in northern Spain. Can J Microbiol 53, 756-767.
Santamaria O, Diez JJ, 2005. Fungi in leaves, twigs and stem bark of Populus tremula from northern Spain. Forest Pathol 35, 95–104.
Scott JJ, Dong-Chan O, Yuceer C, Klepzig M, Clardy KD, Curie CR, 2008. Bacterial protection of beetle-fungus mutualism. Science 322, 63.
Serrada, R, 2004. Apuntes de Selvicultura. Fundación Conde del Valle Salazar. Escuela de Ingeniería Técnica Forestal. Madrid.
Six DL, Klepzig KD, 2004. Dendroctonus bark beetles as model systems for studies on symbiosis. Symbiosis 37, 207-232.
Solheim H, Langstrom B, Hellqvist C, 1993. Pathogenicity of the blue-stain fungi Leptographium wingfieldii and Ophiostoma minus to Scots pine: effect of tree pruning and inoculum density. Can. J. For. Res 23, 1438-1443.
Solheim H, Langstrom B, 1991. Blue-stain fungi associated with Tomicus piniperda in Sweden and preliminary observations on their pathogenicity. Ann Sci Forest 48, 149-156.
Sutton BC, 1980. The coelomycetes. Fungi imperfecti with pycnidia, acervuli and stromata. Commonwealth Mycological Institute, England, UK. 696 pp.
Ter Braak CJF, Šmilauer P, 2002. CANOCO Reference Manual and CanoDraw for Windows User’s Guide: Software for Canonical Community Ordination (version 4.5). Microcomputer Power (Ithaca NY). USA.
Togashi K, 2004. A new method for loading Bursaphelenchus xylophilus (Nematoda: Aphelenchoididae) on adult Monochamus alternatus (Coleoptera: Cerambycidae). J Econ Entomol 97, 941-945.
Warren JE, Edwards OR, Linit MJ, 1995. Influence of bluestain fungi on laboratory rearing of pinewood nematode infested beetles. Fund Appl Nematol 18.
Wingfield MJ, Seifert KA, Webber JF, 1993. Ceratocystis and Ophiostoma. Taxonomy, Ecology and Pathogenicity. American Phytopathological Press, St. Paul, Minnesota, U.S.A. 293 pp.
Wolfaardt JF, Wingfield MJ, Kendrick WB, 1992. Synoptic key and computer database for identification of species of Ceratocystis sensu lato. S. Afr. J. Bot. 58, 277-285.
Zamora P, Martínez-Ruiz C, Diez JJ. 2008. Fungi in needles and twigs of pine plantations from northern Spain. Fungal Divers 30, 171–184.
Zhou XD, De Beer ZW, Wingfield BD, Wingfield MJ, 2001. Ophiostomatoid fungi associated with three pine-infesting bark beetles in South Africa. Sydowia 53, 290-300.
Zhou XD, De Beer ZW, Wingfield BD, Wingfield MJ, 2002. Infection sequence and pathogenicity of Ophiostoma ips, Leptographium serpens and L. lundbergii to pines in South Africa. Fungal Diversity 10, 229-240.