Semiochemical management of pine sawyer beetles monochamus galloprovincialis (olivier) and m. Sutor (linnaeus)

Laburpena

Pine sawyer beetles Monochamus galloprovincialis and M. sutor, are secondary pests of pines in Europe and North Africa that have become important since the former was identified as the vector in Europe of the Pine wood nematode (PWN) Bursaphelenchus xylophilus. The latter species has been hypothesized as capable of transmitting B. xylophilus if the nematode ever became associated with it. PWN is the causal agent of pine wilt disease (PWD), a wilting disease that has caused dramatical economic and environmental losses in pine stands of several countries in Eastern Asia. The disease has been introduced and spread through Portugal in Europe, and it eventually have crossed into Spain four times since 2008 . Since PWN introduction in Asian countries, management of PWD through the insect vectors has been recommended as one of the most promising strategies. Traps baited with specific lures have showed effective and preferable to insecticides due to the hazard they represents to non-target organisms and human health. Significant progress on the chemical ecology of M. galloprovincialis and M. sutor has been performed in recent years, resulting on highly attractive lures with practical applications in PWD management. The present thesis provides advances in the development of lures and traps especific for the semiochemical management of Monochamus galloprovincialis and M. sutor. Advances in Monochamus antenal physiology as a basis for further lure improvement are also presented. Finally, application of these tools to biological control by entomopathogenic fungi, by means of auto dissemination tactics, has been also researched. An effective trapping system is needed, not only for monitoring the insect vector but also for direct control of its population. Trapping may also provide key information on the nematode load carried by the beetles, allowing early detection of infections, provided that captured beetles remain alive within the trap. Effective attractants have been developed in recent years that are commonly used in combination with diverse standard trap designs. In our experiments, several trap designs were developed and compared to commercial standard models in order to determine which designs maximized the number of attracted insects actually caught and the proportion of them remaining alive. In total, 12 trap designs were evaluated in five field experiments carried out in France, Spain and Portugal. Teflon coating applied to the whole trap and extended, ventilated collecting cups resulted in a significant improvement of trap performance. These modifications led to significant increases of pine sawyer catches, up to 275%, when applied to multiple-funnel or black cross-vane traps, compared to standard designs. Furthermore, a significant proportion of the captured beetles remained alive within the trap. These findings have been used to develop new commercial traps (Econex Multifunnel-12® and Crosstrap®; Econex, Murcia, Spain) available to forest managers. A model for insect survival within the trap was also fitted. Elapsed time between consecutive samplings, mean relative humidity and maximum radiation were the three most significant variables. Thus, traps should provide a suitable sample of live insects if sun exposure of the trap is minimized and a reasonable sampling schedule is implemented. Studying M. galloprovincialis antennal physiology will provide clues for lure further improvement. The response of its olfactory receptor neurons (ORNs) to several odorants was tested using single sensillum electrophysiology. Behaviourally active pheromone and kairomone (host and sympatric bark beetle pheromones) odors were tested alongside smoke compounds released by burnt wood that are potentially attractive to the insect. The antennae bore several types of sensillae. Two areas in the proximal and distal ends of each antennal segment were covered with basiconic sensillae that responded to the odor stimuli. Sensillae basiconica contained one or two cells of different spike amplitude. The 32 male and 38 female ORNs tested responded with excitations or inhibitions to the different plant odors. In general the response of male and female receptors was very similar so they were pooled to perform a cluster analysis of ORN responses. Six ORNs were clearly specialized for pheromone reception. Responses to kairomone and smoke odors were less specific than those of pheromone, but a group of 9 cells was clearly excited by smoke compounds (mainly eugenol and 4-methyl-2-methoxyphenol), a group of 8 cells was very responsive to ¿-pinene, ß-pinene and cis-verbenol, and a group of 14 cells responded to a wider range of compounds. The rest of the cells (47%) were either non-responsive or slightly inhibited by smoke compounds. Dose-response curves were also obtained for several compounds. Among field effective attractants identified in recent years are the specific M. galloprovincialis aggregation pheromone, host pine kairomones such as ¿-pinene and bark beetle kairomones like ipsenol and 2-methyl-3-buten-2-ol. The main objective of our study was to optimize the combination of these volatiles to improve lure attractiveness and specificity. Based on ten complementary field experiments, we found a pheromone dose-response of trap catches and that the best combination of attractants associated the aggregation pheromone and two bark beetles kairomonal compounds, ipsenol and 2-methyl-3-buten-2-ol. By contrast the addition of pine terpenes, such as ¿-pinene, did not significantly improved M. galloprovincialis trap capture while increasing the capture of non target species, including natural enemies. The use of pine terpenes would be advisable only if priorizing to maximize removal of vectors. While this research has lead to the development a new, highly attractive commercial lure for mature pine sawyers, none of the tested blends were successful in attracting immature pine sawyer adults. Further investigation is needed to develop attractants for these beetles. Recent reporting of 2-undecyloxy-1-ethanol as the M. sutor male-produced aggregation pheromone opened the possibility of developing an efficient lure for this species. It has also been reported that European Ips bark beetle pheromone compounds, ipsenol, ipsdienol, cis-verbenol and 2-methyl-3-buten-2-ol, alone or supplemented by host volatiles ¿-pinene and 3-carene, kairomonally attracted this species. Besides, smoke volatiles from burnt trees might play a role in M. sutor host location. Field trapping experiments during three years in three countries (Spain, Sweden and Austria), aimed to develop an efficient pheromone-kairomone lure operative for M. sutor management were carried out. Electroantennographic responses by M. sutor to Ips pheromones and to the Pityogenes chalcographus pheromone chalcogran were also studied. GC-EAG recording showed that M. sutor males and females clearly responded to ipsenol and ipsdienol, and females also to 2-methyl-3-buten-2-ol. Chalcogran elicited a neat response to M. sutor female antennae. In field tests, ipsenol was the most attractive kairomone to both sexes of M. sutor, whereas ipsdienol, cis-verbenol and 2-methyl-3-buten-2-ol resulted very weakly attractive and chalcogran was unattractive. When combined with the pheromone, most Ips kairomones increased catches of both sexes, but it was significant only for ipsenol. On the contrary, chalcogran resulted completely ineffective. Thus, ipsenol not only was shown the strongest individual kairomone for M. sutor but it resulted also the best single kairomone to be combined with the pheromone. Results, on the other hand, of a blend of smoke volatiles from burnt pines, tested in Spain and Austria, were negative, pointing to that smoke cues are not likely involved in host finding by this species. Semiochemical management of M. galloprovincialis through biological control has been also approached. Three entomopathogenic fungal strains, Isaria farinosa (Holmsk.) Fr., Lecanicillium attenuatum (Zare & W. Games) and Beauveria pseudobassiana (Bals.) Vuill. were isolated in Spain from naturally infected Monochamus galloprovincialis. This is the first time that these entomopathogenic fungi have been isolated from M. galloprovincialis beetles. Assays showed the B. pseudobassiana EABps 11/01-Mg strain to be highly virulent against the pine sawyer. Horizontal and vertical transmission were assessed for both aqueous (1×108 conidia/ml) and dry (4.25×109 conidia/g) conidial formulations. Evidence of horizontal or vertical transmission was not found when insects were inoculated with the aqueous conidial suspension. However, when dry conidia were applied, 100% of the horizontally-infected insects died and their average survival times (AST) were significantly reduced (from 21.10 and 25.00 days in controls to 10.40 and 10.00 days in infected males and females, respectively). Compared to control females, numbers of egg-laying wounds, eggs laid, live larvae after 5 days and larvae entering the xylem after 6 months were significantly reduced in both inoculated females and clean females that had mated with inoculated males, pointing to horizontally-induced reduction of progeny. These results validate the potential of the isolated B. pseudobassiana strain as an important natural population regulator. Through auto-dissemination techniques (i.e ¿lure and infect¿), this biological agent could be used for the integrated control of pine wood nematode vectors and constitutes a new tool for Pine Wilt Disease management.