Garbelotto, Gonthier, Nicolotti

Transcription

1 Molecular diagnosis of wood rotting fungi in ornamental trees: validation of the method, of a sampling approach, and preliminary ecological notes on decay-associated fungi* Garbelotto, Gonthier, Nicolotti

2

3 wood decay fungi diagnosis and identification prognosis Prediction of severity and evolution of decay process appropriate FRC (Failure Risk Classification) appropriate management practices rapidly progressing root and butt rot agents

4 The best way to detect decay inside trees

5 Resistograph

6 Another way to detect decay inside trees

7 Fruit body type 1999

8 Identification of wood decay fungi in standing trees traditionally based on macro- and micro-morphology of fruiting bodies Overlapping morphological characters Ganoderma resinaceum Perenniporia fraxinea Fruiting bodies rarely visible, often ephemeral advanced stage only

9 Identification of wood decay fungi in standing trees pure culture analysis (i.e. Stalpers 1978) fungal isolation not always feasible identification time-consuming and often complicated

10

11 Decay fungi included in the method Armillaria spp. (Agaricales, Marasmiaceae) Ganoderma spp. (Polyporales, Ganodermataceae) Hericium spp. (Russulales, Hericiaceae) Inonotus/Phellinus spp. (Hymenochaetales, Hymenochaetaceae) Laetiporus spp.(polyporales, Polyporaceae) Perenniporia fraxinea (Polyporales, Polyporaceae) Pleurotus spp. (Agaricales, Pleurotaceae) Schizophyllum spp. (Agaricales, Schizophyllaceae) Stereum spp. (Russulales, Stereaceae) Trametes spp. (Polyporales, Polyporaceae) Ustulina deusta (Xylariales, Xylariaceae) 4 groups 6 groups (Wagner and Fischer 2002)

12 Samples DNA extraction M1

13 M1 Nuc-rDNA 18 S ITS1 ITS2 5.8 S 25 S Fungi Ganoderma spp. Inonotus/ Phellinus spp. ITS1F 228bp Gano2R ITS bp M 1 M F bp Hyme2R 1. Trametes versicolor 2. Phellinus punctatus 3. Ganoderma resinaceum M. DNA ladder 100 bp IT S

14 M2 Nuc-rDNA 5.8 S ITS2 25 S ITS3 Armi2R 185bp Armillaria spp. Laetiporus spp. M 2 M Pleurotus spp. Hericium spp. 25sF LaetR Heri2R Pleu2R 146bp 158bp 200bp Armillaria mellea 2. Hericium flagellum 3. Laetiporus sulphureus 4. Pleurotus ostreatus M. DNA ladder 100 bp

15 M3 Nuc-rDNA 5.8 S ITS2 25 S ITS3 PereR Ste2R Schi2R Ustu2R P. fraxinea 152bp M M Schizophyllum spp. Stereum spp. Ustulina deusta 190bp 240bp 260bp Mt-rDNA ssu Trametes spp. MS1 220bp TraR 1. U. deusta 2. S. hirsutum 3. T. versicolor 4. S. commune 5. P. fraxinea M DNA ladder 100 bp

16 Samples Mhyme Mgano Inonotus/ Phellinus spp. Ganoderma spp. DNA extraction DNA extraction not effective 111 bp 228 bp ~700 bp M1 - No fungi M2 Armillaria spp. Hericium spp. Laetiporus spp. Pleurotus spp. - M3 Fungal taxon not detectable through multiplex method P. fraxinea Schizophyllum spp. Stereum spp. Trametes spp. U. deusta

17 Multi-Gano Nuc-rDNA 18 S ITS1 5.8 S ITS1f GrR GapR G. resinaceum-g. pfeifferi 178bp G. lucidum 193bp GlR GaR Multi-Gano M G. applanatum 200bp G. adspersum- G. australe 211bp G. resinaceum 2. Eur. G. lucidum 3. G. applanatum 4. G. adspersum M 100 bp ladder 200

18 Multi-Hyme Nuc-rDNA ITS2 25 S 25sF PhssR FuscR FomiR InssR IdryaR InocuR Phellinus s.s. 173bp (P.tubercolosus, P.igniarius, P.tremulae) Inonotus s.s. 212bp (I.andersonii, I.hispidus, I.cuticularis) Fuscoporia 223bp (P.gilvus, P.torulosus) I. dryadeus 254bp Fomitiporia 258bp (P.robustus, P.punctatus) Inocutis 265bp (I.dryophilus, I.tamaricis)

19 Fomitiporia Fuscoporia Inonotus dryadeus Inocutis Inonotus s.s. Phellinus s.s. G. resinaceum G. lucidum G. applanatum G. adspersum Samples Mhyme Mgano Inonotus/ Phellinus spp. Ganoderma spp. DNA extraction DNA extraction not effective 111 bp 228 bp ~700 bp M1 - No fungi M2 Armillaria spp. Hericium spp. Laetiporus spp. Pleurotus spp. - M3 Fungal taxon not detectable through multiplex method P. fraxinea Schizophyllum spp. Stereum spp. Trametes spp. U. deusta

20 Aims: 1. to validate the method on wood samples 2. to develop an efficient drilling-based sampling method 3. to infer ecological features of decay agents based on the application of the method in northern Italy

21 Validation I 114 wood samples collected (through a swedish increment borer) from decay-affected trees in central California and northern Italy Wood DNA extraction through QIAmp DNA Stool mini kit (Qiagen) Obtained results From multiplex PCR protocol developed Expected results From analysis of visible fruiting bodies or sequencing Comparison between expected and obtained results

22 Validation II efficiency other non target taxa 9% 8% no fungi expected fungus 83%

23 Validation III specificity 1% 1 99% 107 amplifcazione aspecifica aspecific amplification no amplificazioni aspecifiche no aspecific amplification

24 Degradation ability

25 Root, butt and stem rots

26 Sampling method I testing and optimization 24 trees infected by at least one of the target fungi 40 cm Drilling close to the collar Wood chips

27 Sampling method II testing and optimization comparison of results from single drillings and mixtures of sawdust from 4, 3 and 2 drillings Six transversal drillings 2

28

29 Conclusions on sampling Three options available: 2.1 (large surveys), 4.1 (minimize false negatives, but not best option); 4.2 (best option) Choice depends on type of activity (survey vs. ad hoc diagnosis, and on cost) Tests run on trees less than 120 cm DBH, if larger trees are being analyzed, then consider more than 4 drillings

30 Ecological notes I Unexpected high frequency of Armillaria spp. in urban trees Inonotus/ Phelinus G. resinaceum Armillaria spp. Most frequent fungus at the collar of Platanus sp. in Turin Associated with root failure on Ulmus sp., Celtis australis, Populus nigra

31 Ecological notes II High frequency of Ganoderma spp. in urban trees - G. resinaceum - G. adspersum -nog. applanatum Simultaneous occurrence of Ganoderma resinaceum and P. fraxinea on Celtis australis Occurrence at the tree collar of Phellinus punctatus and Schizophyllum commune First report of U. deusta in the area

32 Ecological notes III Host Species Geographic Origin Wood Decay Fungi Reason for Sending Torrey Pine San Diego Hericium and schizophyllum Tree was flagging on top Valley Oak Livermore Laetiporus Hazard Tree Magnolia Sonoma Armillaria Hazard Tree Tree of Heaven Glendale Pleurotus Hazard Tree Tree of Heaven Glendale Armillaria Hazard Tree Monterey Cypress Soquel Oligoporus balsmeus Conk Grew Inside the Tree Birch Wheatland Ganoderma spp. Dead Tree, Birch Near By Coast Live Oak Palo Alto Ganoderma applanatum Visible Fruiting Body Present Coast Live Oak Cupertino Ganoderma spp. Failed Root Buttress in the Root Crown Area Eucalyptus globulus Ventura Laetiporus gilbertsonii 90% of Large Roots Decayed Coast Live Oak Mountain View Ganoderma applanatum Fruiting Body Present Peach Tree Gridley Phellinus pomaceus Branch Failure 1- Ganoderma applanatum not really associated with most dangerous situations but another G. Sp. is 2- Hazard trees with Armillaria or Laetiporus should be red-flagged. In particular bluegum is very susceptible 3- Phellinus pomaceus responsible for branch failure of fruit trees

33 Sequencing Results Ecological notes III Douglas Fir San Francisco Fomitopsis pinicola Watershed Decay Observed Douglas Fir San Francisco Porodaedalea pini Watershed Decay Observed Douglas Fir Berkeley Porodaedalea pini Decay Observed Madrone Humboldt Co. Dichostereum durum, Phlebia acanthocystis, Dichostereum pallescens Decay Observed Sequoia sempervirens San Martin Coniophora puteana Unusual Fungal Mats on Bark 1- Phellinus pini explains some of the Mortality of DF in SFPUC, but also BRD. 2- Coniophora puteana on standing redwood 3- Fourteen samples were negative, either no target or no fungi were detected. In the second case, most likely because of inhibitors

34 Conclusions: The molecular method is highly specific and efficient Fungal DNA may be successfully extracted from mixtures of sawdust obtained from drillings Optimized sampling Perspectives: target fungi 52% 9% no fungi other fungi 39% Sequencing Bjerkandera sp., (Aesculus sp.) Oxyporus populinus, (Platanus sp.) Gymnopus (=Collybia) fusipes, (Quercus robur) Spongipellis spumeus, (Aesculus sp.) Phellinus cavicola, (Platanus sp., Aesculus sp.) Hyphodontia sp., (Platanus sp.)

35 In the process of developing additional assay for conifers: any samples will be appreciated Inonotus tomentosus Phaeolus schweinitzii Phellinus pini Heterobasidion irregulare/occidentale Fomitopsis pinicola Fomitopsis officinalis Phellinus weirii Echinodontium tinctorium

36

37 SENDING SAMPLES Sample should be sent with next day service within 24 hours of collection Do not send samples in on Fridays Need to let us know you are sending samples with either a phone call or Place samples in paper envelope and fill in one form for each sample

38 Forest Pathology & Mycology Laboratory, UC Berkeley ID Code: Wood Decay Diagnostic Results Collection Date: Submitted by: Received: Tree Species: Done by Technician: Location: Reason For Submission: Wind Throw Hazard Tree Survey Diagnosis: Results Targets Sample Control 1. Fungal DNA 2. Armillaria spp. 3. Fomitiporia (P. punctatus, P. robustus) 4. Fuscoporia (P. contiguous, P. gilvus, P. torulosus) 5. Ganoderma spp. 6. Ganoderma adspersum 7. Ganoderma applanatum 8. Ganoderma lucidum (Eu) 9. Ganoderma resinaceum 10. Hericium spp. 11. Inocutis (I. dryophilus) 12. Kretzschmaria deusta 13. Inonotus dryadeus 14. Inonotus s.s. (I. andersonii, I. hispidus, I. obliquus) 15. Inonotus/Phellinus spp. 16. Laetiporus spp. 17. Perenniporia fraxinea 18. Phellinus s.s. (P. igniarius, P. lundelii, P. tremulae, P. tuberculosus) 19. Pleurotus spp. 20. Schizophyllum spp. 21. Stereum spp. 22. Trametes spp. Diagnosis: Positive For: Negative for all targets but positive for fungal DNA control (decay caused by nontarget fungi.) Assay inconclusive due to excessive decay or inhibition of DNA analysis.

39 RESULTS In about 5-6 weeks unless differently agreed Assay only targets specific fungi not all decay fungi For legal purposes, we stand behind our positives, but it is the collector s responsibility to make sure samples are collected correctly and in the best of ways; so our assay will not stand in trial by itself but it needs to be matched by the professional s diagnosis and correct collecting approach IT ALL DEPENDS ON HOW GOOD YOU ARE AT SAMPLING

40 Thanks Slosson Endowment Ted Swiecki Larry Costello