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Garbelotto, Gonthier, Nicolotti 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 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 The best way to detect decay inside trees Resistograph Another way to detect decay inside trees Fruit body type 1999 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 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 2007 www.wooddecay.org 2008 Decay fungi included in the method •Armillaria spp. (Agaricales, Marasmiaceae) •Ganoderma spp. (Polyporales, Ganodermataceae) 4 groups •Hericium spp. (Russulales, Hericiaceae) •Inonotus/Phellinus spp. (Hymenochaetales, Hymenochaetaceae) 6 groups (Wagner and Fischer •Laetiporus spp.(Polyporales, Polyporaceae) 2002) •Perenniporia fraxinea (Polyporales, Polyporaceae) •Pleurotus spp. (Agaricales, Pleurotaceae) •Schizophyllum spp. (Agaricales, Schizophyllaceae) •Stereum spp. (Russulales, Stereaceae) •Trametes spp. (Polyporales, Polyporaceae) •Ustulina deusta (Xylariales, Xylariaceae) Samples DNA extraction M1 M1 Nuc-rDNA 18 S ITS15.8 S ITS2 25 S ITS1F Gano2R ITS4 F115 Hyme2R Fungi 600-800bp Ganoderma spp. 228bp M1 M 1 2 3 Inonotus/ 111 bp Phellinus spp. 1000 1. Trametes versicolor IT S 2. Phellinus punctatus 500 3. Ganoderma resinaceum M. DNA ladder 100 bp 228 200 1 1 1 M2 Nuc-rDNA ITS2 25 S 5.8 S ITS3 Armi2R 25sF LaetR Heri2R 185bp Pleu2R Armillaria spp. Laetiporus spp. 146bp Pleurotus spp. 158bp M2 M 1 2 3 4 Hericium spp. 200bp 1000 1. Armillaria mellea 500 2. Hericium flagellum 3. Laetiporus sulphureus 2 0 0 200 1 8 5 4. Pleurotus ostreatus 1 5 8 1 4 6 M. DNA ladder 100 bp 100 M3 Nuc-rDNA 5.8 S ITS2 25 S ITS3 PereR Ste2R Schi2R Ustu2R M3 P. fraxinea 152bp 1 2 3 4 5 M Schizophyllum spp. 190bp Stereum spp. 240bp 1000 Ustulina deusta 260bp 500 Mt-rDNA 200 ssu 100 MS1 TraR 1. U. deusta 2. S. hirsutum Trametes spp. 220bp 3. T. versicolor 4. S. commune 5. P. fraxinea M DNA ladder 100 bp Samples Mhyme Mgano DNA extraction •DNA extraction not Inonotus/ Ganoderma spp. effective Phellinus spp. 111 bp 228 bp ~700 bp - •No fungi M1 M3 P. fraxinea M2 Schizophyllum spp. - Stereum spp. Trametes spp. Armillaria spp. U. deusta Hericium spp. Fungal taxon Laetiporus spp. not detectable through Pleurotus spp. multiplex method Multi-Gano Nuc-rDNA 18 S ITS1 5.8 S ITS1f GrR GapR GlR GaR Multi-Gano G. resinaceum-G. 178bp M12 34 pfeifferi G. lucidum 193bp G. applanatum 200bp G. adspersum- 500 G. australe 211bp 300 1. G. resinaceum 200 2. Eur. G. lucidum 3. G. applanatum 4. G. adspersum M 100 bp ladder 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) Fomitiporia Fuscoporia G. resinaceum Samples Inonotus dryadeus G. lucidum Inocutis G. applanatum Inonotus s.s. G. adspersum Phellinus s.s. Mhyme Mgano DNA extraction •DNA extraction not Inonotus/ Ganoderma spp. effective Phellinus spp. 111 bp 228 bp ~700 bp - •No fungi M1 M3 P. fraxinea M2 Schizophyllum spp. - Stereum spp. Trametes spp. Armillaria spp. U. deusta Hericium spp. Fungal taxon Laetiporus spp. not detectable through Pleurotus spp. multiplex method 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 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 Validation II efficiency no fungi 8% other non 9% target taxa 83% expected fungus Validation III specificity 1%1 99%107 amplifaspecific cazione amplification aspecifica no amplifno aspecific icazioni amplification aspecifiche Degradation ability Root, butt and stem rots 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 Sampling method II 3 testing and optimization 2 4 6 comparison of results from single drillings 5 and mixtures of sawdust from 4, 3 and 2 1 drillings Six transversal drillings 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 Ecological notes I Unexpected high frequency of Armillaria spp. in urban trees G. resinaceum Inonotus/ Phelinus Armillaria spp. Most frequent fungus at the collar of Associated with root failure on Platanus sp. in Turin Ulmus sp., Celtis australis, Populus nigra 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 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 Ecological notes III Sequencing Results 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 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 other fungi 39% Sequencing 52% Bjerkandera sp., (Aesculus sp.) 9% no fungi Oxyporus populinus, (Platanus sp.) Gymnopus (=Collybia) fusipes, (Quercus robur) Spongipellis spumeus, (Aesculus sp.) Phellinus cavicola, (Platanus sp., Aesculus sp.) Hyphodontia sp., (Platanus sp.) 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 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 email • Place samples in paper envelope and fill in one form for each sample Forest Pathology & Mycology Laboratory, UC Berkeley Wood Decay Diagnostic Results ID Code: Collection Date: ______________ ________________________________________ Submitted by: Received: ______________ ____________________________________ Tree Species: Done by Technician: _____________________________________ ______________ Location: ____________________________________________________________________________ Reason For Submission: Wind Throw Hazard Tree Survey 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
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