Rapid Ō’hi’a Death (ROD): The Disease That's Killing Native Hawaiian Trees · 2017-07-09 ·...

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Rapid Ō’hi’a Death (ROD):The Disease That's Killing Native Hawaiian Trees

Lisa Keith, Research Plant PathologistUSDA‐ARS, DKI‐PBARC

Better Border Biosecurity ConferenceWellington, New Zealand

5/9/16

Flint Hughes (USDA Forest Service) & J. B. Friday (UH Mānoa CTAHR) Lionel Sugiyama (USDA ARS), Wade Heller (RCUH) & Blaine Luiz (ARS/UHH)

Travis Sowards, Ric Lopez (USDA IPIF)Chris Fettig, Phil Cannon, Sheri Smith, Leif Mortenson (USDA FS)

Kyson Dunn (HCC) & Nathanael Friday (Dartmouth College)Brian Bushe (UH Mānoa, Ag. Diagnostic Service Center, Hilo, HI)

Rob Hauff (DLNR DOFAW)Wil Guerrero, Amy Takahashi & Lance Sakaino (HDOA)

Christy Martin (CGAPS)Carter Atkinson & Jim Jacobi (USGS)

Tom Harrington (Iowa State)Josh Atwood (HISC)Evelyn Wight (TNC)Springer Kaye (BIISC)

Irene Barnes & Mike Wingfield (FABI, South Africa)Scott Geib & Eva Brill (USDA ARS)

Elizabeth Stacy (University of Hawai‘i at Hilo)Aileen Yeh (HARC)

Gordon Bennett & Curtis Ewing (UH Mānoa, CTAHR)

Teamwork!

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Awesome Lab Crew:Lionel Sugiyama, Wade Heller & Blaine Luiz

Rapid Ō’hi’a Death (ROD)

‘Ōhi‘a, Metrosideros polymorpha

Ceratocystis story in Hawaii

Biology/Genetics

Management Strategies

Outreach

ROD; why now?

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‘Ōhi‘a, Metrosideros polymorpha, Myrtaceae

Dominant tree in 80% of Hawaiian forests

Both primary succession and old‐growth species

Forms almost pure stands

Important for watershed, wildlife, and cultural values

Photo credit Jack Jeffery

29,632 hectares

1,299 hectares

16,238 hectares

9,950 hectares

37,359 hectares

252,591 hectares347,069 total hectares (1 hectare = 2.47 acres)

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2008

There has been a noticeable increase in ‘ōhi‘a mortality in the Puna district of Hawai‘i Island

Pictometry courtesy of USGS

2012

Rapid Ō’hi’a Death (CeratocystisWilt of Ō’hi’a)

Symptoms: physical manifestation of the host’s response

Rapid browning of leaves

Complete foliar death

Leaves remain attached

“2 weeks”

Dead trees

Signs: physical manifestation of the causal agent

Discoloration

OdorPhoto credit Blaine Luiz

© www.doctorasky.com

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Field Sampling: Finding the right samples

Fungal Isolation

10% bleach/lactic acid

Sterile dH2O

Isolations from cookies

Isolationsfromfungal

perithecia

40X

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Identification Growth & conidia morphology

Ceratocystis sp.

Large, diverse complex of species that cause wilt diseases

40X

400X

PCR and Sequencing of ITS

Molecular Analysis

(Molecular ID by Dr. Wade Heller)

Ceratocystis fimbriata isolate CBS115167 18S ribosomal RNA gene, partial sequence; internal transcribed spacer 1, 5.8S ribosomal RNA gene, and internal transcribed spacer 2, complete sequence; and 28S ribosomal RNA gene, partial sequence Sequence ID: gb|KC493164.1|Length: 621Number of Matches: 1

Score Expect Identities Gaps Strand

1048 bits(567) 0.0 583/590(99%) 3/590(0%) Plus/Plus

Query 1 CGGAGGGATCATTACTGAGTTTTTGTACTCTATAAACCATGTGTGAACATACCTATCTTG 60 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 18 CGGAGGGATCATTACTGAGTTTTTGTACTCTATAAACCATGTGTGAACATACCTATCTTG 77 Query 61 TAGTGAGATGAATTGCTGTTTTGGTGGTAGGGCCCTTCTGAAGGGTACCGCTGCCAGCAG 120 |||||||| ||||||||| |||||||||||| ||||||||||||| |||||||||||||| Sbjct 78 TAGTGAGACGAATTGCTGCTTTGGTGGTAGGACCCTTCTGAAGGGCACCGCTGCCAGCAG 137 Query 121 TATATAAGTCTCGCCACTGTAAACTCTTACTATTTTCTTAATTTTCATTGCTGAGTGGCA 180 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 138 TATATAAGTCTCGCCACTGTAAACTCTTACTATTTTCTTAATTTTCATTGCTGAGTGGCA 197 Query 181 TAACTATAAAAAAGTTAAAACTTTCAACAACGGATCTCTTGGCTCTAGCATCGATGAAGA 240 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 198 TAACTATAAAAAAGTTAAAACTTTCAACAACGGATCTCTTGGCTCTAGCATCGATGAAGA 257 Query 241 ACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTG 300 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 258 ACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTG 317 Query 301 AACGCACATTGCGCCTGGCAGTATTCTGCCAGGCATGCCTGTCCGAGCGTCATTTCACCA 360 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 318 AACGCACATTGCGCCTGGCAGTATTCTGCCAGGCATGCCTGTCCGAGCGTCATTTCACCA 377 Query 361 CTCAAGGACTCCTTTGTTCTTGGCGTTGGAGGTCCTGTTCTCCCCTGAACAGGCCGCCGA 420 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 378 CTCAAGGACTCCTTTGTTCTTGGCGTTGGAGGTCCTGTTCTCCCCTGAACAGGCCGCCGA 437 Query 421 AATGTATCGGCTGTTATACTTGCCAACTCCCCTGTGTAGTATAAAATTTCTAATTTTTAC 480 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 438 AATGTATCGGCTGTTATACTTGCCAACTCCCCTGTGTAGTATAAAATTTCTAATTTTTAC 497 Query 481 ACTTTGAAGTTCTTGTGTAACACGCCGCTAAAcccccccccTCAACTTTTGTTGAACTTT 540 |||||||||||||||||||||||||||||||| ||||||||||||||||||||||||| Sbjct 498 ACTTTGAAGTTCTTGTGTAACACGCCGCTAAA---CCCCCCTCAACTTTTGTTGAACTTT 554 Query 541 CACAAGGTTGACCTCGGATCAGGTAGGAATACCCGCTGAACTTAAGCATA 590 |||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 555 CACAAGGTTGACCTCGGATCAGGTAGGAATACCCGCTGAACTTAAGCATA 604

whitni13.blogspot.com

Important Tree Diseases (not found in Hawaii):

Sycamore, Mango, Cacao, Citrus spp., Coffee, Rubber tree, Prunus spp., Fig, Poplar, Acacia spp., and Eucalyptus spp.

Found in Hawaii on: Okinawan sweet potato,

Mountain (Tongan) Yam, Sweet potato, Syngonium, and Taro

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Pathogenicity Tests

Greenhouse80 days

Signs & Symptoms

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Re‐isolation by carrot baiting40X

40X

© www.doctorasky.com

Survey & Distribution:Rate of spread

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Work in Progress Survey

Occurrence and distribution of Ceratocystis sp.

Biology/Phylogenetic Analysis

Rapid, Field‐Friendly Assays

Host Susceptibility

Management Strategies/ Quarantine

Biology: Where is it from?

80+ ‘ōhi‘a isolates

Population genetics: 7 genes have been sequenced; microsatellite markers

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Temperature & Growth

? ?B, Leilani Estates G, Opihikao

Growth & Spore Production

Perithecia containing ascospores

Cross section of wood containing aleurioconidia

PeritheciaAscosporesEndoconidiaAleurioconidia

400X40X

400X

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CBS117355 C. platani (Platanus sp., France)

Cf AF100 C. platani (Platanus sp., Italy)

CBS114719 Ceratocystis sp. (Syngonium, HI)

CBS114723 C. fimbriata (I. batatas, NC)

CBS114720 Ceratocystis sp. (C. esculenta, HI)

CBS115164 Ceratocystis sp. (C. esculenta, HI)

CBS118126 C. polychroma (S. aromaticum, Indonesia)

C1959 C. tiliae (T. americana, IA)

99

64

97

100

100

88

84

98

0.02

Ceratocystis sp. AC (M. polymorpha, HI)

C. fimbriata D (I. batatas, HI)

Ceratocystis sp. B (M. polymorpha, HI)

Phylogenetic analysis of 6 Hawaii Ceratocystis isolates and their most similar published related isolates with accepted species designations. Homology analysis is based on 442 nucleotides of the Ceratoplatanin gene.

Dr. Wade Heller

Evolutionary Relationships of Taxa(Ceratoplatanin)

Evolutionary Relationships of Taxa(rDNA ITS, Ceratoplatanin, β‐tubulin,

TEF1‐α, Actin, and GAPDH)

Phylogenetic analysis of Hawaii Ceratocystis isolates including 3511 nucleotides from 6 genomic (rDNA ITS, Ceratoplatanin, β‐tubulin, TEF1‐α, Actin, and GAPDH)

Dr. Wade Heller

Ceratocystis sp. AC [IT’S A] (M. polymorpha, HI)

Ceratocystis sp. AC [IT’S C] (M. polymorpha, HI)

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Ceratocystis Wilt of Ō’hi’a

Orchidland

Hawaiian Acres

Photo Credit JB Friday

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Wailuku River above Hilo

Photo Credit Flint Hughes

Wailuku River above Hilo


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Native Forest at 3,500 ft, Upper Wailuku River above Hilo


Wao Kele o Puna

Strawberry guavaPhoto Credit Flint Hughes

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Native ‘ōhi‘aforest near Pahoa, 2005


2015


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Kealakekua





Host Susceptibility


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Field Sampling


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Molecular Detection of Ceratocystis,the Causal Agent of ROD

1. Collect Sample 2. Extract DNA 3.qPCR: quantitative real‐time PCR

‐ Specific DNA target in fungal genome

‐ Control DNA target in plant genome

‐ Detection of fluorescence indicates target is present

4. Analyze Amplification Data

Very small amount needed (≤ 100 mg)

Threshold

Ceratocystis targetOhia target

‐ Fluorescence above defined threshold is indicative of a positive sample

‐ Replicate reactions and positive/negative controls are performed to ensure reliability of data

(Developed by Dr. Wade Heller)

* Reduces time from 2 weeks to <24 hours(wood, sawdust, frass, soil)

How does the pathogen move?

SoilVehiclesPhoto Credit JB Friday

Insect frass

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Wind dispersal of infected frass?

www.soest.Hawaii.edu

www.pubs.usgs.gov





Host Susceptibility


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http://en.wikipedia.org/wiki/Ceratocystis_fimbriata

Host Susceptibility Testing

Sweet potato

Syngonium

Taro

Coffee

In search of Ceratocystis fimbriatain Hawaii

Syngonium as a carrier host?Photo Credit Lionel Sugiyama

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Ohia isolate

Syngonium isolate

Ceratocystis fimbriata: Ō’hi’a v. Syngonium

Photo Credit Lionel Sugiyama

Pictures 8 days post inoculation

Cross‐Host Inoculation

(Syngonium/Ō’hi’a)

Ō’hi’a isolate killed Ō’hi’a Syngonium isolate did not kill Ō’hi’a

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Cross‐Host Inoculation

(London Plane)

Resistance Testing:Ō’hi’a varieties and species

Photo Credit Lara Hughes

Metrosideros excelsapōhutukawa

Photo Credit By Bjankuloski06en ‐ Own work, CC BY‐SA 3.0, https://commons.wikimedia.org/w/index.php?curid=7405753

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Host Susceptibility


Management StrategiesDon’t move ‘ōhia wood!

Sanitation Boots, cutting tools used in infected forests

10% chlorine bleach, 70% isopropyl (rubbing) alcohol, 90% ethanol, 4 oz/gal Lysol® concentrate: 15 seconds

NOT Simple Green

Launder clothes used in forest; Power‐wash undersides of vehicles, construction equipment with soap

Heat Treatments & Fungicides

HDOA Quarantine

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Management Strategies

HDOA QuarantineNo movement of ‘ōhi‘a plants or plant parts including flowers, leaves, seeds, stems, twigs, cuttings, “untreated” wood, logs, mulch, green waste, and frass (sawdust from boring beetles) from Hawai‘i Island (Except by permit from HDOA)

After February 2016 no soil

What to do with infected dead trees?

• Cut them down

• Cut them up

• Don’t let sawdust blow around

• Cover with a tarp (3‐6 months)

• Or burn them in place!

• Don’t move the woodPhoto Credit JB Friday

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www.rapidohiadeath.org

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Rapid ‘Ōhi‘a Death; Why Now?

A unique environment on Hawaii Island?

Nursery crops as carrier hosts?

Ō’hi’a: the right tree at the wrong time?

Has Ceratocystis been flying under the radar in Hawaii? (coffee/cacao/eucalyptus/aroids)

A tale of 2 species? (stay tuned)

Thank you

For more information, please contact:

Lisa Keith, Ph.D.Research Plant PathologistUSDA/ARS, DKI‐PBARC

64 Nowelo St.Hilo, HI 96720

USA

phone: 808‐959‐4357email: [email protected]

Rapid Ō’hi’a Death (ROD): The Disease That's Killing Native Hawaiian Trees · 2017-07-09 ·...

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