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Villari C., Tomlinson J. A., Battisti A., Capretti P., Faccoli M.

Detection of blue-stain fungi associated to Ips acuminatus in the Italian Alps using LAMP technology ...aka the use of LAMP technology for non-diagnostic purposes. Villari C., Tomlinson J. A., Battisti A., Capretti P., Faccoli M.

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Villari C., Tomlinson J. A., Battisti A., Capretti P., Faccoli M.

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  1. Detection of blue-stain fungi associated to Ips acuminatus in the Italian Alps using LAMP technology ...aka the use of LAMP technology for non-diagnostic purposes. Villari C., Tomlinson J. A., Battisti A., Capretti P., Faccoli M.

  2. There are several factors that lead bark beetles to rise from a non-outbreak to an outbreak population • Factors interacting with host plant resistance are among of the most important (climate change, atrophic action ecc.) Dendroctonus ponderosae (Photo Richard Waring, Oregon State University)

  3. Bark beetle are associated with blue-stain fungi • Blue-stain fungi role in bark beetle population dynamics?? • Do associated fungi differ in composition and frequency in bark beetle outbreak and non-outbreak conditions ?? • very few information • no verified hypotheses

  4. Study system: Ips acuminatus on Pinus sylvestris • Associated with: • Nutritional fungus: Hyalorhinocladiella macrospora • Blue-stain fungus: ?? • Ophiostoma clavatum • or • Ophiostoma brunneo-ciliatum

  5. Aimsof the study: • to survey which species (O. clavatum or O. brunneo-ciliatum) is actually the main blue-stain fungus associated to I. acuminatus in the southern Alps • to verify if outbreak and non-outbreak populations of I. acuminatus show significant differences in the occurrence of the associated blue-stain fungi

  6. Methods: • Developing of two real-time LAMP assays that respectively detect the presence of O. clavatum or O. brunneo-ciliatum directly from the vector • Developing of a third LAMP assay specific for I. acuminatus as an internal control • Employing of the assays with Italian populations of I. acuminatus

  7. Lamp assay development: • O. clavatum assay and O. brunneo-ciliatum primers were manually designed in the β-tubulin gene • I. acuminatus primers were manually designed in the COX I gene • Setting the optimal conditions to use the LAMP assays with Genie II

  8. Sensibility test Ophiostoma clavatum Ophiostoma brunneo-ciliatum Fungal DNA was diluted in insect DNA, to account for potential inhibitory effects Ips acuminatus

  9. Specificity test

  10. Specificity test

  11. Survey on the Italian I. acuminatus populations • 2 tree per site • 10 insects per tree Outbreak population

  12. DNA was extracted with a protocol for museal specimens (no insect tissues homogenization) • Both the assays for the fungi were carried out separately with all the samples • Each reaction was run in triplicate, and the sample was considered as positively associated to each fungus only if at least two of the replicates had a positive response • Reactions with the I. acuminatus assay were carried out only with those samples for which both the fungal assays had a negative response

  13. Results: Frequency of association of blue-stain fungi with Ips acuminatus

  14. Results: Effects of population characteristics on the frequency of association of O. clavatum with I. acuminatus z = −2.38 Pr = 0.017 GLMM with binomial distribution. Tree within site as random factors

  15. Conclusions: • We developed three LAMP assays which resulted to be specific and sensible • We found that the blue-stain fungus associated to I. acuminatus in the Italian Alps is O. clavatum • We found that the occurrence of O. clavatum is lower in outbreak than non-outbreak • We have shown how LAMP technology can be useful also for ecological and biological studies, rather than only for diagnostic

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