Molecular Techniques for Forest Health Monitoring. Rachel Linzer & Matteo Garbelotto UC Berkeley

Transcription

1 Molecular Techniques for Forest Health Monitoring Rachel Linzer & Matteo Garbelotto UC Berkeley November 19, 2008

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3 Questions to address using molecular methods 1. Is a particular pathogen present (yes/no)? 2. Quantity of pathogen present? 3. Which pathogens generally, or which of a predetermined set are present? 4. Genetic fingerprints of pathogen individuals present? 5. Is pathogen alive or dead?

4 Some general benefits of molecular detection methods Fungal fruitbodies/cultures not necessary Can characterize microbes resident on hosts without symptoms or signs Sensitive Fast turnaround Very small amounts of material required Little training required for data interpretation

5 Polymerase Chain Reaction (PCR)

6 Case Study 1: Quantifying Leptographium spores on beetles L. wageneri conidia Bark beetle Threshold cycle (Ct) Real-time PCR Spore count (log 10)

7 Case Study 1: Quantifying Leptographium spores on beetles Highest L. wageneri spore loads on the bark beetle, Hylastes macer, an hypothesized vector

8 Case Study 2: Quantifying airborne Fusarium circinatum spores Monterey pine with pitch canker Filter paper traps airborne spores DNA extraction Real-time PCR Threshold cycle (Ct) Spore count (log 10)

9 Case Study 2: Quantifying airborne Fusarium circinatum spores Spore deposition higher in the rainy season, November to April

10 Case Study 3: Detecting multiple decay fungi in wood Multiplex reaction Common forward primer Reverse primer to detect: M1 ITS1-F All fungi Ganoderma spp. F115 Inonotus/Phellinus group M2 ITS3 Armillaria spp. 25sF Laetiporus spp. Pleurotus spp. Hericium spp. M3 ITS3 P. fraxinea Schizophyllum spp. Stereum spp. MS1 Trametes spp.

11 Case Study 3: Detecting multiple decay fungi in wood Multiplex reaction Common forward primer Reverse primer to detect: Mgano ITS1-F G. adspersum, G.pfefferi, G. applanatum (NA) (after + for G. applanatum (EU) Ganoderma ) G. lucidum (EU) G. resinaceum, G. lucidum (NA) Mhyme 25sF Fomitiporia (P. punctatus, P. robustus ) (after + for Fuscpporia (P. contiguus, P. gilvus, P. torulosus ) Inonotus/Phellinus I. dryadeus group) Inocutis (I. dryophilus ) Inonotus s.s. (I. andersonii, I. hispidus, I. obliquus ) Phellinus s.s. (P. igniarius, P. lundelii, P. tremulae, P. tuberculosus)

12 Case Study 4: Origins of Phytophthora ramorum in CA Microsatellites 3 tandem repeats of C A 5 tandem repeats of C A

13 Case Study 4: Origins of Phytophthora ramorum in CA New infestations (<5 yrs) Intermediate infestations (>5<10 yrs) Old infestations (>10 yrs)

14 Case Study 5: RT-PCR to assess Phytophthora viability PCR (stable DNA) > RT-PCR (unstable mrna) > Culturing

15 Case Study 5: Results of testing environmental samples PCR (stable DNA) RT-PCR (unstable mrna) > > Culturing Phytophthora is viable but not culturable

16 Case Study 5: Results of testing environmental samples PCR (stable DNA) RT-PCR (unstable mrna) > > Culturing Phytophthora is not viable

17 Case Study 6: Comparison of multiple methods for P. ramorum diagnostics From CA bay From other plant spp.

18 What s s on the horizon for molecular methods in forest health? Improvements in: Portability Cost-effectiveness Ease of use Ease of data interpretation High throughput identification: micro and macroarrays Improved integration of molecular and spatial data What is the pathogen doing and how is it doing it? (genomics and proteomics)

19 Acknowledgements THANKS TO THOSE WHOSE RESEARCH I CITED: Garbelotto lab: Sarah Bergemann, Antonio Chimento, Matteo Garbelotto, Fabio Guglielmo, Kevin Maeda, Sylvia Mascheretti, Kabir Peay, Noah Rosenzweig, Wolfgang Schweigkofler, Anna-Maria Vettraino Outside collaborators: Santa-Olga Cacciola, Daniel Cluck, Peter Croucher, Paolo Gonthier, Giovanni Nicolotti, Kerry O Donnell, William Otrosina, Simone Prospero, T. Smith, Sherri Smith, S. Sukno, Andrea Vannini