Ophiostomatoid Fungi Transported by Ips sexdentatus ( Coleoptera ; Scolytidae ) in Pinus pinaster in NW Spain

Ips sexdentatus (Coleoptera; Scolytidae) is one of the main vectors of ophiostomatoid blue stain fungi that can cause mortality of healthy conifers. For this reason, our objective was to identify the fungal species carried by this bark beetle in Maritime pine (Pinus pinaster) in north-western Spain. We collected insects from naturally infected pines placed them on malt extract agar (MEA) and left to walk freely on culture plates. Plant tissues (phloem and xylem) from adult pines were cultivated in moist chambers and also on MEA. At the same time, we inoculated pine logs with living insects in the laboratory. Four ophiostomatoid fungi appeared: Ophiostoma ips, Ophiostoma brunneo-ciliatum, Ceratocystiopsis minuta and Ophiostoma sp., as well as Graphium and Sporothrix imperfect stages. Moreover there were seven saprophytic species: Penicillium sp., Trichoderma sp., Verticillium sp., Mucor sp., Aspergillus niger, Gliocladium viride and Scopulariopsis brevicaulis, and the pathogenic Ophiostoma ips. The fructification percentage of the ophiostomatoid species was low, however; its imperfect stage Sporothrix/Hyalorhinocladiella produced high quantity of conidiophores.

Bark beetles damage trees directly but greater problems arise from their symbiotic phytopathogenic blue stain or saprophytic fungi.Furthermore, the commensal mites can also vector blue stain fungal spores (Doberski 1978, Bridges and Moser 1983, Moser 1985).
Ophiostomatoid fungi are often associated with bark beetles and regarded responsible for many tree diseases, such as blue-stain in conifers (teleomorphs: Ceratocystis, Ophiostoma and Ceratocystiopsis), black-stain in roots of conifers (Leptographium wagenerii), or Dutch elm disease caused by Ophiostoma novo-ulmi (Seifert 1993, Harrington 1993).In addition, some of their associated fungi are non-pathogenic and present a specific association with bark beetles.
The association between blue-stain fungi and bark beetles was first postulated by Hartig (1878) and by Von Schrenk (1903).Many other authors have discussed this association (Francke-Grosmann 1967, Harrington 1988) and most believe that it is a symbiotic relationship.Fungi benefit from dispersal by the insects, and probably helping it later with the establishment of the population by contributing to exhausting tree resistance (Christiansen et al. 1987, Lieutier 1993, 2002, Solheim et al. 2001).However the advantages for the insect are mixed.Lombardero et al. (2003) postulated that Ophiostoma minus is an antagonistic fungus transported on the exoskeleton of Dendroctonus frontalis.Their larvae do not survive in the fungal presence, but yet otherwise they feed on Ceratocystiopsis ranaculosus and Entomocorticium sp.(Barras and Perry 1972).Recently Scott et al. (2008) found that the successful relationship between Dendroctonus frontalis and Entomocorticium sp. is likely mediated by antibiotic producing actinomycetous bacteria, which selectively inhibit O. minus.
Few fungus-bark beetle studies have been done in the Iberian Peninsula (Fernández et al. 2004, Romón et al. 2007).The former study describes the threshold level of pathogenecity of Ophiostoma ips in Pinus sylvestris and the latter relates fungi to bark beetles without indicating insect body parts, where spores are found.
The aim of our study was to demonstrate the effectiveness of Ips sexdentatus as a vector of Ophiostomatoid fungi in Pinus pinaster in NW Spain.We identified 1) fungal species associated with Ips sexdentatus colonizing trees in the forest, 2) insect body parts, in which fungi are transported, and 3) fungal species that the beetle can vector under laboratory conditions.The sex vector effect was also considered for all three objectives.

Insects and Pine Tissues Collection
In 2006, Ips sexdentatus adults and colonized plant material were collected in Quintana del Castillo forest (León province, NW Spain, UTM coordinates 29T, X-738530, Y-4729138, altitude 1100 m.a.s.l.).Three hundred bark beetles were collected from a 40-years old P. pinaster stand (mean DBH: 28 cm, mean height 12 m) affected by fire.Trees were dead standing but with greenyellow needles still remaining in the crown.Ips sexdentatus specimens were collected at the end of September, when the population was at an endemic level and when the last generation have their galleries already built.Each collected beetle was individually stored in one Ependorff tube at 6ºC and subsequently sexed.
Colonized tissues (sapwood and phloem) were obtained from four trees naturally infested by I. sexdentatus.70 cm long logs with 32 cm diameter were stored at the laboratory at 6 ºC until the extraction of the tissues.
Four non-colonized healthy P. pinaster logs collected in the same area (12.5-18 cm in diameter and 60-73 cm in length) were inoculated in the laboratory according to the methodology described by Furniss et al. (1990).One hundred insects (50 males and 50 females) were inoculated into four logs.For each log, a 5 mm diameter cork borer was used to create twenty five holes leaving 10 cm gaps from the edges to avoid desiccation.Thereafter, one insect was placed on each wound and crushed with the removed bark disk.Inoculated logs were then stored in the lab for one month at 25ºC.

Fungal Isolation and Identification
Different methodologies were employed for fungal isolations and identification from naturally infected pine tree tissues, logs inoculated in the laboratory, and from insects.60 samples were taken from naturally infested logs, 30 phloem samples and 30 sapwood ones, all of them from 3 different pine logs.In each log, 10 different samples of 3 cm 2 of phloem were taken and subdivided in 5 parts of 1 cm length × 0, 5 cm wide.From the xylem, we took Pressler´s samples of 5 cm length and 0.5 cm in diameter that we divided in 5 parts of 1 cm length.All the samples were kept in a dark moist chamber at 25ºC during 30 days to promote fungal fructification.
Tissue samples were cultured on MEA+ antibiotic medium (33 g of malt extracts, 16 g of agar and 250 mg of tetracycline per litre of distilled water).Petri dishes were stored for 7 days in the dark at 25ºC and then carefully examined using NIKON SMZ-2T binoculars.Fruiting bodies from the samples were identified with a microscope Nikon Eclipse E-400 model.
Insects associated fungi were cultured by two methods: 1) leaving the insect freely move on Petri dishes with MEA+antibiotic for 2 hours and, 2) placing different insect body parts (mandibles, legs, pronotum and elytra) directly onto.After two hours, the walking insects were placed in 1.5 ml Ependorff test tubes and washed in 400 μl sterile water with 1% Tween-80 and vortexed at 40 Hz for 30 seconds (Lieutier et al. 1989).The rinsing water (400 μl) was plated on MEA using a sterile pipette, incubated for 2 weeks in the dark at 25ºC, and monitored for fungal growth.
The insect body parts (mandibles, pronotum, legs and elytra) were cultured on MEA+antibiotic.Petri dishes were stored in the dark at 25ºC for one week.Fungal colonies were subcultured to sterile MEA+tetracycline, after 30 days identified based on microscopic features and classified according to biometric characteristics.
Sapwood and phloem samples from artificially infested logs were cultured in a moist chamber and on the MEA+tetracycline media at 25ºC in the dark, subcultured, and identified under microscope.
Seven different keys were used for fungal identification: Sutton 1980, Fassatiová 1986, Wolf-aardt et al. 1992, Wingfield et al. 1993, Muñoz et al. 1996, Kiffer and Morelet 1997, Jacobs and Wingfield 2001.Fungal species abundance data were analyzed by a generalized lineal model using binomial distribution and the Link Logit function.Fungal diversity was analyzed by Poisson distribution with Link Log function.Data from both experiments were analyzed using STATISTICA 5.5 program (p < 0.05).

Fungal Identification
Twenty-five taxa, including twelve Sporothrix states were identified after analysis.All identified fungal isolates belong to the Eumycotina group.
Ophiostoma ips, Ophiostoma brunneo-ciliatum, Ceratocystiopsis minuta and Ophiostoma sp. were isolated in their sexual state from the moist chamber experiment.Only Ophiostoma ips was isolated simultaneously from moist chamber and culture media.Different asexual states of Ophiostoma genus, such as Graphium (acquired from logs stored in moist chambers), or Sporothrix and Hyalorhinocladiella (obtained by both methodologies) were also isolated.The existing similarity between these genera recommends the use of Sporothrix "sensu lato", including the Hyalorhinocladiella form under that genus (Lieutier et al., 1989).Consequently, 11 conidial types were distinguished and each was categorized within Sporothrix 2, through Sporothrix 12. Isolates without conidia but with Sporothrix biometric mycelial characteristics were grouped to Sporothrix 1 (Lieutier et al. 1989).One fungal taxon was classified as Deuteromycete 1 attending to the characteristics of the asexual fruiting body (no spores were found).
Isolates belonging to Trichoderma, Penicillium, Aspergilus niger, Verticillium, Gliocladium viride, Mucor and Scopulariopsis brevicaulis were cultured from insect bodies and plant material on MEA medium.

Fungi Isolated from the Logs Naturally Colonized by Insects
The following six Ophiostomatoid species, including the imperfect states were identified from the sapwood and the phloem of the logs: Ophiostoma ips, Ophiostoma brunneo-ciliatum, Ceratocystiopsis minuta, a species identified only at the genus level (Ophiostoma sp.), Sporothrix 12 and Graphium sp.Also one unidentified fungus Deuteromycete 1 was detected (Table 1).Only O. brunneo-ciliatum and Graphium sp. were present on the totality of the logs, but O. brunneociliatum appeared on both tissues (log × tissue) and occurred more frequently.The number of isolates was higher in phloem than sapwood.O. brunneo-ciliatum was most abundant fungus in the phloem (p = 0.003), and O. ips was the second most frequent, seldom found in the sapwood (p = 0.000).The anamorphic states of Ophiostoma sp., Sporothrix 12 and Graphium sp. were detected more frequently in phloem than sapwood, although the differences were significant only for Sporothrix 12 (p = 0.004).Ceratocystiopsis minuta and Ophiostoma sp. were found in phloem, and Deuteromycete 1 from sapwood was detected with low frequency.
The fungal diversity detected in the phloem (1.5 fungal species per sample) was much higher than diversity in sapwood (0.4 fungi per sample) (data not shown).

Walking Insect and Dilution Method
Six fungal species were isolated using the two techniques and from both sexes of Ips sexdentatus (Table 2): Trichoderma sp., Penicillium sp., Aspergillus niger, Verticillium sp., Gliocladium viride and Sporothrix 1.With the exception of Sporothrix 1, all fungi were isolated from walking males; in addition Trichoderma sp., Penicillium sp. and Verticillium sp. were isolated from females.Dilution method yielded Trichoderma sp., Penicillium sp. and occasionally Sporothrix 1 on females.
Highest records frequency for both methodologies were found for Trichoderma sp.(40%  walking, 92% dilution) with significant differences between methods (p = 0.000) and relation methodology × sex interaction (p = 0.005).The highest rate of recurrence was found in males isolated by dilution method with 96% success rate in contrast to walking technique, which resulted in 12% recovery.In contrast to the dilution method results, walking method generated higher incidence of Trichoderma sp.isolated from females than males.Percentages of abundance were much lower for the remaining fungal species; 20% of samples yielded Penicillium sp., 10% Verticillium sp. and 4% Aspergillus niger and Gliocladium viride.Sporothrix 1 was occasionally recovered from females by dilution method.With reference to methodology, only significant differences were noticed for Trichoderma sp. and Penicillium sp.
(p = 0.026 and p = 0.000).Trichoderma spp.are very fast growing molds and may easily overgrow slower growing ophiostomatoid fungi.Fungal diversity was not significantly different despite the slightly higher average number of fungi per insect in females than in males (1.0 and 0.8) (Table 3).4).O. ips was isolated only from the male pronotum and elytra.Sporothrix 1 and Sporothrix 10 were isolated from every body part of both sexes.The recovery rates were highest for Penicillium sp., Sporothrix 10, Sporothrix 1 and Sporothrix 2 (the last, isolated only from the males), whereas the isolation frequency of the remaining fungi was low.Sex differences depend on the body part (sex × body part interaction: p < 0.05; Table 5).Penicillium sp. was recovered in highest frequency from the elytra of both sexes and Sporothrix 1 was found primarily on the mandibles.Sporothrix 10 was often isolated from male elytra and pronotum and from female pronotum.With the exception of Sporothrix 3 and Sporothrix 2, differences in abundance percentage for sex × body part interaction was not significant for any of the isolated fungi.
The fungal diversity analysis showed statistically significant differences between sexes for the body part cultures.On average, 1.9 fungi per male and 1.2 fungi per female were isolated (p = 0.012) (Table 3).

Fungi Isolated from Inoculated Logs
Eight fungal taxa were isolated from tissues (sapwood or/and phloem) of I. sexdentatus inoculated logs.Penicillium sp. and Trichoderma sp. were more commonly isolated from both tissues inoculated by males and females (Table 5).
Logs inoculated with females showed highest fungal abundance in the sapwood (Trichoderma sp.followed by Penicillium sp).The Ophiostoma  anamorphs Sporothrix 1, 9 and 10 were most frequently isolated from the phloem.The logs inoculated with males exhibited high recovery rate of Penicillium sp.followed by Trichoderma sp. in the sapwood, whereas the remaining taxa were only found in the phloem.Fungal diversity recovered from phloem was greater than sapwood (1.6 and 1.1 respectively; p = 0.172).Furthermore, female inoculated logs had a greater fungal diversity in comparison with male inoculated logs (1.5 and 1.3; p value no statistically significant; data not shown).
In our study, O. brunneo-cilliatum represented the most frequently recovered species from the phloem (60%) followed by O. ips, also isolated from phloem (33.3%).These results concur with Jankowiak's (2005) findings, where he states that fungal occurrence associated with Ips typographus was higher in the phloem galleries (99.1%) than sapwood (52.1%) of Picea abies.Fernández et al. ( 2004) first described the presence of O. ips in Spain and found the highest degree of association of this fungus with I. sexdentatus in Pinus sylvestris.Romón et al. (2007) isolated O. ips from I. sexdentatus and other bark beetles species colonizing Pinus radi ata.In other European countries, O. ips was more frequently isolated from Ips species such as I. acuminatus and different secondary bark beetles (Lieutier et al. 1991, Mathiesen-Käärik 1953, Kirschner 1998).
Some authors found that Ceratocystiopsis minuta, is an unspecific fungus commonly found in many bark beetles in Europe, such as Ips species (I.acuminatus, I. sexdentatus) (Kirschner 1998, Solheim 1986), or I. typographus found in Picea abies with more degree of association (Viiri and Lieutier 2004).
In our study we also found two anamorphic states: Sporothrix 12 and Graphium sp.Lieutier et al. (1989) isolated Sporothrix with 100% success rate from blue stain areas close to I. sexden tatus galleries on Pinus sylvestris; finding 10% sexual forms, most of them corresponding to Ophiostoma ips.After sampling deep-xylem and non-stained areas they recorded 90% of Sporo thrix anamorph, of which 5% corresponded to O. brunneocilliatum and 21% to O. ips.In the non-stained galleries, only 30% of our isolates were Sporothrix and no sexual fruiting bodies were found.In Lieutier's et al. (1989) survey of sexual fruiting state, the highest levels were detected in O. ips whereas in our study, O. brunneo-cilliatum produced the most fruiting bodies.Lieutier et al. failed to find blue stain fungi in the sapwood, or saprophytic fruiting bodies, whereas Jankowiak (2006) isolated many non-ophiostomatoid fungi, mainly Penicillium sp. and Trichoderma sp. from sapwood in quantities similar to results in our study.

Insect Body Parts Utilized for Fungal Transport
Walking and dilution methods, as well as insect body part culturing technique were used to answer the following objectives of our study: 1) detection of fungal species vectored by insects, 2) identification of sexual or asexual stages of vectored fungi, and 3) identification of fungal species successfully inoculated into logs.Seven fungi from colonized trees were identified and only O. ips was also detected in the lab by insect body part culturing technique.
The number of ophiostomatoid fungi isolated by the walking and dilution techniques was low (only Sporothrix was detected), however, many other saprophytic fungi, such as Trichoderma sp. and Penicillium sp. were present (Table 2).Disregarding the likelihood of contamination in the laboratory, we considered the possibility that insect mycangia, which are used for transport of ophiostomatoid spores (Lévieux et al. 1991), failed to produce effective inoculations because of the limited contact with the culture media, thus preventing spores to germinate.Conversely, saprophytic fungi that are commonly transported on exoskeleton grew very well (Six 2003a).Lieutier et al. (1989) found saprophytes as Penicillium, Trichoderma, Aspergillus, or Cladosporium on Ips sexdentatus, and, Peverieri et al. (2006) recovered these fungi from Tomicus destruens.Considering the low economical importance in the forest, saprophytes are not often referenced in publications; however, some of them are very important biological control agents, for example Trichoderma spp.Whitney and Blauel (1972) reported ascosporic masses of several species of Ceratocystis, Ceratocystiopsis and Ophiostoma dispersed in conifer resin but not in water.These findings substantiate the lack of ophiostomatal isolations from insect washing dilutions.
From the insect body part cultures, O. ips and different conidia of 8 Sporothrix forms were identified (Table 4).Sporothrix conidia were frequently isolated from pronotum and mandibles whereas O. ips was mainly cultured from pronotum.Moreover, 3 non-ophiostomatoid fungi were isolated from insects, most often from elytra and legs.Therefore, these findings confirm that mandibles and pronotum of I. sexdentatus are involved in ophiostomatoid spore dispersal, whereas elytra, pronotum and legs could be implicated in transporting non-ophiostomatoid spores.Lévieux et al. (1989) noticed spore masses of various shapes situated on pronotal sides, with limited quantities in rounded hollows of the strial punctures of elytra and its declivity, and occasionally located under the abdominal sternites, or in the punctures of the external side of mandibles.

Which Fungal Species Can Be
Inoculated by the Beetle into the Trees?
Regarding the insect's ability to inoculate fungi into the pine trees, five different conidia from ophiostomatoid fungi were isolated in the sapwood and phloem (Sporothrix 1, 2, 8, 9 and 10) and three saprophytic fungi: Trichoderma sp., Penicillium sp., and Mucor sp.(Table 5).Sporothrix spp. was rarely occurred; Sporothrix 1 and 10 were more frequently found in the phloem, however, none occurred in sapwood.
Out of seven identified fungi, only O. ips was detected also in the lab.The perithecium of this fungus was frequently recovered from cultures containing Sporothrix after placing wood chips in the plates.
All the ophiostomatoid fungi were the anamorphs whereas if we compared with the fungi isolated from naturally colonized tissues trees, we can see that 6 ophiostomatoid species were found, 4 of them, were teleomorphs.Several explanations could be given for these differences: the different climatic conditions for the fungi growth between the lab and the field (anaerobic conditions could stop fungi fructification).Secondly, the technique used at the lab that consisted in crashing the beetle inside the hole made with the cork-borer.That also could be augmenting the saprophytic fungi presence due to the intestinal content of the insects.The last explanation for this difference in the frequency of the ophiostomatoid fungi could be the high presence of these two ubiquitous saprophytes (Trichoderma and Penicillium species) and their antagonistic mycoparasitic nature, that may interact in significant, but still unknown, the ways with bark beetle-fungal associates (Six 2003b).It is interesting to note that in the whole study, 0% of sexual fructification was obtained in MEA Petri dishes where these two saprophytic fungi were present; that could support the inhibition hypothesis.We have also to take account that no controls were used in this experiment and this lack didn't leave us to determine if the high presence of the saprophytic fungi could be due to contaminations in the lab inoculation procedure, but the naturally presence in logs of the mentioned fungi is well know and noticed from several authors and studies, as we mentioned before.
Using the same methodology as in our study, Viiri and Lieutier (2004) isolated 11 ophiostomatal fungi in Picea abies but the logs were disinfected with 70 % alcohol before inoculations with Ips typographus.Lieutier et al. (1989) also disinfected logs prior to inoculation with Ips sexdentatus and used sterilized distilled water with Tween 80, recovering Sporothrix with 68% success rate.

Conclusion
We isolated twenty-five fungal taxa associated with Ips sexdentatus: 4 Ascomycetes, 1 Zygomycetes, and 20 Deuteromycetes, including 13 anamorphs (12 Sporothrix spp.and 1 Graphium sp).The isolates were obtained by different methodologies; 6 taxa from the walking+dilution method and 10 taxa more grew on insect body parts.Only Penicillium and Sporothrix 1 were obtained by both techniques.From the seven fungal taxa isolated from naturally infested pine logs, only O. ips was associated with insects using the body part culturing technique.
The highest frequency of fungal spores carried by insects corresponded to Penicillium sp. on the elytra.Ophiostomatoid fungi had the highest diversity on pronotum followed by mandibles, legs and elytra.However, only the values for Sporothrix 1 on mandibles and Sporothrix 11 on pronotum were significant (Table 4).
The total fungal diversity (16 fungal taxa isolated from insects) was higher in males than females.Moreover, males carried more than twice as many ophiostomatoid fungal species, (9) than females (4) but differences were not statistically significant.Only the value for Sporothrix 3 sex × body part interaction was statistically significant.(p = 0.047).In general, higher numbers of ophiostomatoid fungi appeared in phloem tissue and were associated with males.
The inoculated log results revealed 8 taxa, including 5 ophiostomatoid (Table 5), and highest abundance in females associated with Sporothrix 1 and 10, although the values were no significant.The value for Sporothrix 9, (fungus transported only by females) was statistically significant.
Further studies using molecular identification will be necessary to provide more information about the fungal diversity associated with I. sexdentatus, the identification of the Sporothrix anamorphs and their teleomorphs.

Table 1 .
Percentages of different fungi isolated from sapwood and phloem naturally colonized by Ips sexdentatus the log and the interaction tissue * : significant differences (p > 95%).

Table 4 .
Percentages of presence of the different fungi taxa isolated from Ips sexdentatus according to the sex, body parts, and interaction sex

Table 5 .
Percentages of presence depending on the sex, the tissue and the interaction sex × tissue of the different fungi isolated from the sapwood and phloem of the inoculated logs at the laboratory with Ips sexdentatus insects.(-) 1 : number of Petri dishes., * : significant differences (p > 95%).