Application Of Bark Beetle Semiochemicals In Timber Quarantine And The Preliminary Establishment Of Related Bark Beetle Identification Database

Bark beetle is one of approximately 220 genera with 6,000 species of beetles in the family Scolytidae(Coleoptera),which is the most common and important pest infesting forest.As the bark beetle damages in the trunk or the inner bark of trees,it can be easily transmitted during international trade in timber and wooden packing material,so the quarantine of bark beetle is very important.At present,restricted by customs clearance time and quarantine facility,the main method of bark beetle quarantine is to check the cargo with eyes and hand.It is hard to avoid oversight with the backward technology.So it is time to apply new technology to quarantine.On the other hand,the number of bark beetle species is very large and the identification of intercepted and captured bark beetle during quarantine is very difficult.Further more,at present,there are tess systemic professional references about intercepted and captured bark beetle during quarantine in China,so it's time to establish a database of morphologic characteristics of bark beetle for quarantine in order to help quarantine inspection officer correctly identify them.Together with DNA bar-coding technique,the database will greatly promote the ability in identifying intercepted and captured bark beetle during quarantine.In order to solve above-mentioned practical problems during timber quarantine,we carried out research as following.First,we investigated the effects of semiochemicals in trapping the bark beetles in import timber and wooden packing material,and some factors that affected the effects in fields and the laboratory.Second,we set the database of morphological characteristics of intercepted and captured bark beetle during quarantine,and the last,we studied the molecular identification of 15 species.The results were showed as below.1.Application of bark beetle semiochemicals in timber quarantine1.1 EAG and walking responses of bark beetle to different semiochemicalsSemiochemicals which can induce the olfactory response and aggregation behavior of bark beetle include host volatile components(kairomones),pheromones secreted by bark beetles itself or synthesized with host compounds by bark beetles,and the compounds released by tissue of decaying wood under the pressure of microorganism after bark beetle colonization.In the experiment,EAG technology was used to study the olfactory response of three typical bark beetle species to each one of the semiochemicals at different concentration,and the walking responses of the three species to each one and the different combinations of the semiochemicals were observed by using Y-tube olfactometer.The research showed that:(1) Dendroctonus micans and Ips nitidus responded to pheromone of low concentration,which demonstrated that the olfactory response threshold of these two species to pheromone was very low,and the response value increased with the concentration.(2) These three species responded to host volatile components of different concentration,but change patterns of EAG response values with the host volatile component concentration.were different among the three species.The EAG response values of D.micans and I.nitidus were maximal when host volatile components concentrations were low or medium,and the EAG values decreased as the concentration increased On the contrary,the EAG response values of X.perforans increased with the concentration increase,and the EAG values were maximal at high concentration of host volatile components.(3) For the two isomeric compounds ofα-pinene,these three tested bark beetle species were sensitive to(1S)-(-)-α-pinene of low concentration(0.0001μL/mL) and R-(+)-α-pinene of high concentration(10μL/mL).(4) The walking responses of the three bark beetle species to different semiochemicals and.their combinations observed by using Y-tube olfactometer showed that ethanol and Monoterpenoids were synergic in attracting bark beetle,and the host volatile components could enhance the responses of bark beetle to pheromone.1.2 Application of attractant in timber quarantine and the influence of environmental factors to trap effectTrapping experiments were carried out on the import timber to evaluate the trapping effect of seven kinds of bark beetle's attractant.The attractants contain the components of host volatile,bark beetle's pheromone and the synthetic components of the host pathogen.The result showed:(1) the attractants could trap many species of bark beetle in the main imported timber of China,so far,insects of four families(Scolytidae,Platypodidae, Bostychidae and Cleridae) have been trapped from the imported timber of Southeast Asia, Africa,Oceania and Russia,and there are nine genus in Scolytidae,total 18 species,four species in Platypodidae and one specie in Bostychidae.(2) The attractant of BB06 and BB07,which contain many effective components,have good Trapping effect,and the differences with the Canadian attractant were not significant,but the cost is only 1/5 to 1/8 of Canadian attractant,so they are worth being generalized.(3) Trapping effect was influenced by the weather condition(temperature,wind and rain).The trapping effect under the condition of no wind was much better than that under the condition of wind.The trapping effect under the temperature of 29-36℃was much better than that under the temperature of 23-30℃.Furthermore,the rainfall promoted the trapping effect.by increasing the moisture of timber.(4) In order to get good trapping result,the trap should be established on the top of wood pile during timber quarantine.Establishing time of trap should be 24 hours,to get more accurate result,48 hours is recommended.1.3 Development of controlling released attractants(CRA) and the trial of controlling release functionFour CRA were developed according to different quarantine object.Through trapping trial,it can be concluded that the comparison of effective components releasing speed of four CRA is:porous starch CRA＞sustained release capsule CRA≥aqueous microcapsule CRA＞non-woven fabric CRA.On the early stage,the trapping effect of porous starch CRA is the best.But due to the high speed of effective components releasing,the trapping effect of porous starch CRA decreased conspicuously with the time prolonged,and its function of controlling release is not distinct.This kind of CRA can be used for the quarantine of imported timber with short customs clearance time,such as imported container timber.The persistence of porous starch CRA can be greatly prolonged by adding septa of polyvinyl alcohol(PVA).Many kind of CRA fit for different needs can be developed by adjusting the thickness of PVA septa, which have great prospect of application.Effective components releasing of non-woven fabric CRA is gently and slowly,and its function of controlling release is distinct.The trapping effect of non-woven fabric CRA is good on every stage,and it is very convenient to apply in quarantine.This kind of CRA can be used in quarantining imported timber with long customs clearance time,such as imported ship timber and the monitoring of bark beetle.Effective components releasing of sustained release capsule CRA and aqueous microcapsule CRA are moderate,but the persistence is very short.The persistence of aqueous microcapsule CRA can not be greatly prolonged after adding low temperature binders,and its usage is very complex,so it is not the perfect CRA for application. 2.Preliminary establishment of quarantine bark beetle morphological characteristics databaseOne hundred and thirty two species of quarantine bark beetle were selected from the specimens collected in the plant quarantine laboratory of Jiangsu Entry-Exit Inspection and Quarantine Bureau and the insect specimen's museum of Institute of Zoology,Chinese Academy of Sciences.The morphological description and photos of these species have been taken.The morphological identification database contains the familiar species intercepted and captured by quarantine department of China,and many species are first described in Chinese.Distinct identification characters are exhibited through accurate morphological description and plenty of photos with primitive colors in the database,which overcome the shortcoming of the previous database.At the same time,the database includes the host and the distribution of related bark beetle species,which provides useful information to quarantine department and insect research.Identification illustrated handbook of bark beetle from imported timber has been published by Shanghai Scientific and Technical Publishers in 2007 according to above-mentioned data.3.Feasibility study on application of DNA bar-coding technology(based on mtDNA CO I gene) in molecular identification of ScolytideaCO I gene of 15 bark beetle species collected from different regions and host is studied to discuss the feasibility of application of DNA bar-coding technology in Scolytidea molecular identification.The result showed that all fragments of the target gene from 15 samples could be amplified with the common primer and the variation was obviously among species.The DNA data analysis showed that the inter-species genetic distance was no more than 0.019,and there were no significant differences among different geography groups or host groups of the same species.While,the intra-species genetic distance was no less than 0.040,and the average intra-species genetic distance was 0.213.The result of neighbor-joining tree clustered basing on the CO I sequence of 15 samples was consistent with that of morphological analysis.The bootstrap values of the original divergence among different geography groups and host groups of the same species amounted to 99%,which illustrated that the neighbor-joining cluster-analysis based on Kimura-2-Parameter could be utilized in identifying the species of Scolytidea.The study demonstrate that using DNA bar-coding technology(based on mtDNA CO I gene) to identify species of Scolytidea is feasible.The research theoretically founded the strategy of establishing DNA bar-coding database of bark beetle from imported timber,so as to improve the level of bark beetle species identification.Generally,to solve the practical problem of bark beetle quarantine in imported timber and wooden packing material,we carried out the research of semiochemicals-trap application,morphological and molecular identification of bark beetle in this project,and the results would contribute to improving the whole level of bark beetle quarantine.