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 amplificationaspecifiche 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   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: Diagnosis:  Positive For: ______

______ Negative for all targets but positive for fungal DNA control (decay caused by non- target fungi.)

 Assay inconclusive due to excessive decay or inhibition of DNA analysis. 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 WWW.MATTEOLAB.ORG Slosson Endowment Ted Swiecki Larry Costello Thanks