J. J. Diez

Attractive responses by Monochamus galloprovincialis (Col., Cerambycidae) to host and bark beetle semiochemicals

Abstract:  The pine sawyer Monochamus galloprovincialis is the European vector of the recently introduced pine wood nematode. This nematode is the causal organism of pine wilt disease, a serious tree killer in East Asia. Efficacious baits and traps to monitor and control this beetle are now required. The effect of bark beetle (Ips spp.) pheromone components, released individually (ipsenol) or in blends (ipsenol, ipsdienol, cis‐verbenol and methyl‐butenol), together with host volatiles (turpentine or α‐pinene and ethanol) on M. galloprovincialis trap catches has been studied in Spain. A kairomonal response by male and female of M. galloprovincialis to Ips semiochemicals was found. Beetles were more attracted to host blends supplemented with bark beetle pheromones than to host volatiles alone. Ipsenol alone was attractive to pine sawyers, and was synergistic with α‐pinene and ethanol. The full blend of the four Ips semiochemicals and the host compounds was highly attractive. Multiple‐funnel traps were as effective as black cross‐vane traps in capturing this insect when the escape of trapped beetles was prevented. Trapping of non‐target bark beetle predators was also evaluated. The trogossitid Temnochila coerulea and clerid Thanasimus formicarius were kairomonally attracted to and killed in traps baited with bark beetle pheromones. These results suggest that effective monitoring of M. galloprovincialis would be possible by baiting any of these traps with host volatiles and Ips semiochemicals, but reduction of the lure components and trap modification to minimize impact on predators should be considered.

Phylogenic diversity of fungal endophytes in Spanish stands of Pinus halepensis

Fungal endophytes were collected from 168 Pinus halepensis trees sampled in 55 Spanish stands. In total, 229 endophytic isolates were grouped into 92 morphotypes according to their mycelium features. Thus, twigs enclosed 63.76% of the total endophyte isolates and needles 36.24%. Likewise, twig samples also yielded a higher endophyte species richness. Analysis of ITS rDNA region generated 38 different Ascomycota taxa and confirmed the endophytic stage of several pathogens previously associated with the Pinus halepensis decline in Spain. Naemacyclus minor, Brunchorstia pinea, Lophodermium pinastri, Phomopsis sp., Diplodia pinea, Pestalotiopsis besseyi and Truncatella angustata were isolated. Sequencing of LSU rDNA region verified the 38 taxa and contributed to infer their phylogenic relatedness using the Neighbour-Joining method. In the bootstrap consensus tree five classes were observed. Dothideomycetes resulted to be the dominant class because of its high isolation frequency (52.4%) and richest species composition (39.5%). On the contrary, class Eurotiomycetes was the least abundant (3.5%) and along with class Pezizomycetes harboured the lowest species richness (7.9%). Class Sordariomycetes and Leotiomycetes had intermediate abundance and species composition percentages. This study represents the first work concerning the taxonomy of the fungal endophytic community of the Mediterranean host species Pinus halepensis using molecular tools. The data provided here may help to establish the cause of Pinus halepensis decline.

An operative kairomonal lure for managing pine sawyer beetle Monochamus galloprovincialis (Coleoptera: Cerymbycidae)

Abstract:  Monochamus galloprovincialis Olivier (Col., Cerambycidae) is a vector of the pine wood nematode, Bursaphelenchus xylophilus, causing the destructive pine wilt disease. An effective lure for monitoring and/or mass‐trapping would be of great interest in the management of this pine sawyer. Males and females of this species show an attractive kairomonal response to blends composed of four pheromone compounds used by Ips spp. bark beetles and two host volatiles from pines. This six‐component lure is highly attractive but may to be too complex and costly for practical use as each component is released from a separate lure. The role of each component, ipsdienol, ipsenol, cis‐verbenol, methyl butenol, α‐pinene and ethanol as attractants for M. galloprovincialis was field tested in Spain to obtain a simpler but equally effective bait. Ipsenol was confirmed as the strongest kairomonal signal to M. galloprovincialis synergizing response to α‐pinene by 95 times. The addition of methyl butenol to this blend doubled the number of males and females trapped. On the other hand, neither ipsdienol, cis‐verbenol nor ethanol improved the results when incorporated into the above three‐component blend. A lure consisting of ipsenol, methyl butenol and α‐pinene may be very cost‐efficient in operational monitoring or mass trapping of M. galloprovincialis. Three potentially repellent candidates, (−)verbenone, methyl cyclohexenone and trans‐conophthorin, were also tested against the attractive three‐component bait. trans‐Conophthorin significantly reduced male catches of M. galloprovincialis; methyl cyclohexenone had no effect. Verbenone significantly enhanced the response of females to the attractive combination of α‐pinene, ipsenol and methyl butenol.

Fungi associated with the decline of Pinus halepensis in Spain

Fungal species richness and composition within needles and twigs in 55 stands of Pinus halepensis, spread out over the whole Iberian Peninsula, were determined. The aim was to evaluate the relationships of fungal communities with local environmental variables, in order to analyze the potential causes of the current decline of this pine species in Spain. A total of 35 fungal taxa were isolated from 1980 moist chambers analysed (990 per vegetal tissue). A taxon within the Alternaria alternata complex was most frequent, followed by Leptostroma pinastri, Aspergillus niger, Diplodia pinea and Phomopsis sp. At the tree level, tissue was a significant response variable and a higher species richness was found in needles as compared to twigs. On the other hand, the multivariate analysis showed the environmental variables ‘age’, ‘shadow’, ‘elevation’, ‘mean temperature’, ‘illumination’ and ‘availability of water’ significantly influenced fungal species composition. In particular, ‘mean temperature’, was an important variable implicated in the general weakening of this thermophilic pine species, and appeared to be inversely correlated with the occurrence of several conifer pathogens such as Brunchorstia pinea, Cytospora sp., Diplodia pinea, Naemacyclus niveus, Pestalotiopsis stevensonii and Sclerophoma pythiophila. This study shows a possible combined effect of abiotic and biotic stresses in causing the general decline of Allepo pine in Spain.

Olfactory Sex Attraction and Mating Behaviour in the Pine Sawyer Monochamus galloprovincialis (Coleoptera: Cerambycidae)

The pine sawyer Monochamus galloprovincialis Olivier (Coleoptera: Cerambycidae) is a vector of the pine wood nematode Bursaphelenchus xylophilus (Steiner and Buhrer) Nickle. Male and females of this species have a kairomonal attraction to host volatiles and Ips semiochemicals. Once on the host tree, males and females copulate and oviposition occurs. Bioassays using Y-tube olfactometry revealed that females were attracted to volatile compounds produced by males, but not to volatiles produced by females. However, immature males did not seem to release attractants for mature females and immature females did not show any attraction for mature males. A experiment designed to know about mating behaviour in this specie revealed that most of the males encountered females while they were walking. In all pairs both sexes contacted with antennae before copulation, however, in some occasions copulation did not proceed just after antennal contact. On the contrary, in all pairs observed, the male licked the elytra of the female with his mouth palpi just before copulation (licking); immediately afterwards, the male grabbed the female with his forelegs and mounted her suggesting a chemically mediated mate recognition by males.

Kairomonal responses of Tomicus destruens (Col., Scolytidae) to host volatiles α‐pinene and ethanol

The Mediterranean pine shoot beetle Tomicus destruens is one of the most damaging bark beetles attacking Mediterranean pine forests in southern Europe and north Africa. We studied the attractiveness of the host volatiles α‐pinene and ethanol at a range of release rates, alone or in combination, to T. destruens, in order to develop an attractive lure for the management of this beetle. T. destruens was attracted slightly to the host volatile α‐pinene, but a strong synergistic effect was found in the attraction towards monoterpene when ethanol was added to the bait. The highest catches of T. destruens were obtained by the optimal blend releasing 300 mg/day of α‐pinene and 900 mg/day of ethanol. In contrast to data reported for the related species T. piniperda, Mediterranean pine shoot beetles were clearly attracted to baits releasing ethanol alone (1350 mg/day). trans‐Verbenol, which was also added to host volatiles in some tests, did not affect the response. The use of the attractive blend proposed would have a low impact on the natural enemy population of Thanasimus formicarius because of asynchronies in flight periods. Other non‐target insects, such as the facultative predator or competitor Oxipleurus nodieri, were also significantly attracted. These results allow the development of an operative lure for the effective monitoring of T. destruens, although improvements by the addition of other host volatiles should be studied.

Female recognition and sexual dimorphism of cuticular hydrocarbons in Monochamus galloprovincialis (Coleoptera: Cerambycidae).

ABSTRACT Monochamus galloprovincialis Olivier (Coleoptera: Cerambycidae) is the European vector of the pine wilt nematode, Bursaphelenchus xylophilus (Steiner and Buhrer) Nickle, which causes the pine wilt disease. A series of experiments were carried out to determine the existence of a contact pheromone on the females cuticle. All males tested tried to copulate with freshly killed females, but none tried to copulate with the same females after they had been washed with hexane. When the hexane extract was reapplied to the extracted females, 50% of the males tried to copulate with them. This shows that females have a contact pheromone on their cuticle that stimulates male copulatory behavior. Cuticular compounds (mainly hydrocarbons) were sampled with solid phase microextraction and solvent extraction. No marked differences between sampling methods were observed. Individual peak analysis revealed that 23 compounds were significantly more abundant in females and eight were more abundant in males. Correspondence analysis found significant differences between the global hydrocarbon profile of male and female M. galloprovincialis. Individually, these compounds represented a small percentage of the total blend. This suggest that males of M. galloprovincialis may distinguish females by a blend of several, not very abundant, compounds that are present in both sexes but in different proportions.

Spanish population of Gremmeniella abietina is genetically unique but related to type A in Europe.

Genetic structure of the European Gremmeniella abietina var. abietina was analyzed in this study. Ninety-two Spanish isolates, six Swiss isolates of Alpine biotype, 76 Finnish isolates of biotype A and 54 Finnish and seven Russian isolates of biotype B were collected. Genetic variation of different populations was analyzed using sequence analysis of specifically amplified markers GAAA1000, GAAA800 and ACA900. Variation in the GAAA1000 marker was significant, and composed of 33 alleles divided into the following four studied populations: five alleles in the Alpine type, 12 in biotype B, 16 in biotype A and two in the Spanish population. Based on variation in GAAA1000 marker, a subset of isolates were further analyzed using GAAA800 and ACA900 sequences, which showed lower overall genetic variability, and no variation among the Spanish population. Genetic differentiation analysis revealed a high genetic differentiation among populations. Finally, clustering analysis of GAAA1000 sequences showed that the Spanish isolates clearly separated from the rest of the biotypes, whereas the Alpine type was closely related to the B type. However, one of the A-type isolates had an identical GAAA1000 allele with the prevailing allele among Spanish isolates. Altogether, our data suggest that the Spanish population is genetically highly differentiated from any other G. abietina population in Europe with a probable A-type origin.

Influence of the ectomycorrhizal fungus Laccaria laccata on pre-emergence, post-emergence and late damping-off by Fusarium oxysporum and F. verticillioides on Stone pine seedlings.

In greenhouse experiments, the ectomycorrhizal fungus Laccaria laccata was evaluated for biological control of preemergence, post-emergence and late damping-off of Pinus pinea caused by Fusarium verticillioides and F. oxysporum. In pre-emergence damping-off assays, preinoculation with Laccaria laccata did not significantly improve germination of seeds and no statistical significant differences were found in Fusarium treatments when compared with controls. At 18 weeks after sowing, inoculation with L. laccata reduced the incidence of post-emergence damping-off but differences were significant only in F. oxysporum treatments. Pinus pinea transplanted plants were used in late damping off assays, and only F. oxysporum produced significant damage. Inoculation with L. laccata did not attenuate significantly the virulence of F. oxysporum. However, the percentage of mycorrhization did not reached significant level, so the amount of mycorrhizal fungus was insufficient for effective protection. Although very low percentages of mycorrhization were recorded in all mycorrhized treatments, and Fusarium occurrence significantly reduced mycorrhization, those levels have been efficient to reduce damage in F. oxysporum post-emergence damping-off assays. In short, pre-emergence damping-off was not found; only F. oxysporum produced significant damage on P. pinea seedlings and L. laccata reduced damage when the percentage of mycorrhization reached a significant level. These results have been compared with previous work on P. sylvestris inoculated with the same mycorrhizae isolate and Fusarium pathogens.

Towards standardised crown condition assessment in poplar plantations

Abstract• This work aims at developing new tools for rapid assessment of forest health indicators in poplar plantations.• Crown transparency and discoloration were visually evaluated in all trees of four 15 m-radius sub-plots in 32 poplar clonal plantations, which were chosen according to a factorial scheme with three factors: tree age, site quality and understorey vegetation management. A subset of trees was assessed using digital photos processed with a semi-automatic image analysis system (the CROCO software) in order to compare visual and digital crown transparency estimates.• Poplar crown conditions were better in young stands and rich sites. Harrowing understorey vegetation improved tree health in poor sites. Samples of 20 trees per stand provided the same information about crown transparency and discoloration as 60 trees. Calibration curves of digital crown transparency estimates were successfully fitted against visual crown transparency estimates. The same effects of stand age and site quality could be detected with digital crown transparency as response variable.• The use of digital photos processed with CROCO in ca. twenty trees per stand is therefore recommended to accurately and objectively monitor crown condition in clonal poplar plantations.Résumé• Une étude a été réalisée pour développer des méthodes rapides et standardisées de suivi de l’état sanitaire des houppiers en plantation de peuplier.• La transparence et la décoloration des houppiers ont été estimées visuellement sur tous les arbres de quatre placettes d’un rayon fixe de 15 m, dans 32 plantations monoclonales de peuplier choisies selon un plan factoriel complet croisant trois facteurs : l’âge des plantations, la fertilité du site et la gestion du sous-bois. La moyenne des notes de transparence et de décoloration des houppiers a été calculée sur un nombre décroissant de placettes par plantation afin de tester l’influence de la taille de l’échantillon sur la qualité de l’estimation. Des photographies numériques du houppier d’un souséchantillon de peupliers ont été également prises puis traitées à l’aide du logiciel CROCO afin de comparer les estimations visuelles et numériques de la transparence du houppier.• L’état sanitaire des houppiers était meilleur dans les jeunes plantations de peupliers et les sites les plus fertiles. Le hersage de la végétation du sous-bois a permis d’améliorer l’état sanitaire des peupliers dans les sites les plus pauvres. Les valeurs moyenne de transparence et de décoloration du houppier par plantation, calculées à partir des données d’une seule placette de 15 m de rayon — soit une vingtaine d’arbres — ne différaient pas significativement de celles calculées à partir des données de trois placettes et permettaient de détecter les mêmes effets des caractéristiques du peuplement et du site. Des courbes de calibration de la transparence du houppier estimée à partir de photos numériques et du logiciel CROCO ont pu être ajustées aux observations de transparence. Les effets de l’âge des plantations, de la fertilité du site et de la gestion du sous-bois ont pu également être détectés avec ces valeurs numériques calculées.• Il peut donc être recommandé d’utiliser des photos numériques traitées à l’aide du logiciel CROCO pour estimer la transparence du houppier d’une vingtaine d’arbres afin d’évaluer rapidement, précisément et objectivement l’état sanitaire des plantations monoclonales de peuplier.