Threats to and efforts to protect Acacia koa (koa) in Hawaii Dulal - - PowerPoint PPT Presentation

Threats to and efforts to protect Acacia koa (koa) in Hawaii

Dulal Borthakur University of Hawaii at Manoa Presentation at NASEM on December 1, 2017

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~ $31 million annually

The koa industry in Hawaii

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• It is a highly valuable

timber tree species endemic to the Hawaiian Islands

• Koa is a dominant

canopy species in mesic-montane forests in Hawaii

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Close canopy koa in rainforest environment

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Open canopy koa in dry environment

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Hawaii State koa concentrations

• The natural koa tree populations

are broadly distributed across the Hawaiian Islands.

• Koa is absent in the islands of

Niihau and Kahoolawe

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Canopy nodulation in Acacia koa

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James Leary

Major threats to koa

• Deforestation
• Grazing
• Kikuyu grass
• Vascular wilt and dyeback disease caused by

Fusarium oxysporum f. sp. koae

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Koa forests have been degraded over the last two centuries by deforestation and pasture conversion

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Koa in a pastureland

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A degraded pasture pastureland

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A koa forest changed to a degraded pastureland

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Cattle destroy the young koa seedlings

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Removal of cattle leads to dominant occupation of kikuyu grass (Pennisetum clandestinum)

15-45 Mg/ha aboveground biomass

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The Fusarium wilt of koa

Caused by Fusarium oxysporum f. sp. koae

seedling mortality

Caused by Fusarium oxysporum f. sp. koae

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One year old sapling with natural infection

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Stem lesion on koa sapling infected with Fusarium wilt

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Crown dieback on koa sapling

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Discolored branch of an infected koa

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Shoot dieback of infected tree

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Ruptured bark of infected koa

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Ruptured vascular system with fermented sap

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Crown dieback in native forest

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Completely dead koa

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Important considerations for finding a solution

• Koa is a cross-pollinated heterozygous plant
• Highly self-incompatible and insect pollinated
• Not ‘true-breeding’
• There is no rapid screening method for selection of

resistant koa seedlings.

• Identifying and developing wilt resistant koa

populations are the keys to successful koa restoration and reforestation.

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Steps towards selecting koa for resistance to Fusarium wilt disease

① Virulent isolates of Fusarium oxysporum ② Developing a method of screening koa families ③ Operational koa family screening trials ④ Determining the expression levels of disease resistance

genes in resistant and susceptible koa families

⑤ Vegetative propagation of resistant koa

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Statewide survey for F. oxysporum f. sp. koae (Foxy)

Miles

Forests and nursery sample sites

Foxy was isolated from the roots, stems and branches

• f diseased koa seedlings

and trees

28 Key people

Nick Dudley Robert James Phil Cannon

Stem sample of infected koa

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Symptomatic koa tree

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Stem dissection of symptomatic koa tree

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Close-up of FOXY mycelium on stem of an infected seedling

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Positive FOXY root sample: close-up

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Fusarium spores Macroconidia

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Fusarium spores Chlamydospores

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Inoculation Methods

Komada agar for producing clamydospores Peat moss & perlite Dibble tubes Cornmeal perlite PDA

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Isolate Trials: Identifying strains of Fusarium

• xysporum with high virulence on koa
• All isolates were identified as Fusarium
• xysporum based on morphology
• 25 seedlings were tested with each isolate
• All seeds were from the same family
• Trial was run for 90 days
• Virulent isolates are then used in resistance

screening

37 Nick Dudley

10 20 30 40 50 60 70 80 90 100

New Isolate Trial 4B

Isolate Number

Percent of Seedlings Killed

Virulent

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Summary of virulence of Fusarium

• xysporum isolates

NUMBER OF ISOLATES TESTED: 157 Highly virulent : 10 [6%]

Moderately virulent: 9 [6%] Low: 72 [46%] Non-pathogenic: 66 [42%]

Many isolates remain to be tested

39 Nick Dudley & Robert James

• A mixture of ten

virulent isolates are used

• % of resistant seedling

in the families

• A family represents

seeds from the same mother tree

Screening Families for Resistance to Fusarium oxysporum f. sp. koae

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Healthy seedling Seedling with wilt symptoms Seedling killed by koa wilt Roots of dead seedling

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T-15

A susceptible family

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T-24

A family showing no symptom of wilt

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Koa Wilt Resistance Trial: Distribution of Mortality

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13th week 360 seedlings Number of seedlings died Assessment week

1 7 13 19 23

Koa family resistance screening trials

• Screened >700 families in total
• Retested families to verify results
• 153 families have been planted in seed
• rchards

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 Highly Resistant families (>75%)…..……….... 11%  Moderately Resistant families (40-75%)……...…38%  Susceptible families (<40%)……………… ……..51%

• Survival ranged from 0–96%
• Average Survival was ~38%

Koa wilt resistance selection strategy

Resistance testing at Maunauli (HARC) Select mother trees in forest Seed Orchard Collect seeds Seed Production (F1) Resistant seedlings from the best families

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Koa seed

• rchard at

Maunauli

Maunawili Average Survival: 60% Wild population Average Survival: 36%

Locations of Wilt Resistant Koa Seed Orchards

Nick Dudley

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153 families have been planted in seed orchards

Molecular screening for Fusarium wilt resistance

• Fungal cell wall contains a polymer called chitin.
• Chitin is composed of β-1,4-linked N-acetylglucosamine

units.

• Resistant plants produce enzymes called chitinases that

hydrolyze β-1,4-glycoside bonds in chitin

Chitin Chitinase

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Isabel Rushanaedy

Expression of chitinases in resistant and susceptible koa

Rushanaedy I et al. (2012) Tropical Plant Biol. 5:244–252.

The expression of the chitinase genes is more highly upregulated in the resistant koa.

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18S rRNA gene was used as an internal control for normalization of expression levels

Expression of disease resistance genes after mechanical treatment

Unstressed Stressed

Sample collection RNA extraction NanoString

• r qRT-PCR

Kazue Ishihara 51

Microarray and NanoString analyses

Fold change

Ring-finger protein WRKY transcription factor Zinc-finger protein NBS-LRR Disease Resistance Protein

10-60 min post mechanical stress Transcription factors

104 NBS-LRR gene sequences in the A. koa trancriptome 52

Differential expression of pathogenesis-related (PR) genes

* p < 0.05 ** p < 0.01

6h post mechanical stress

-1,3-glucanase Chitinases

S U S U S U

Akchit-1a Akchit-3 Fold change expression

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Unstressed Stressed

Survival recorded after 100 days of inoculation

Inoculation with F. oxysporum

Determining disease resistance to F. oxysporum

Seedlings were grown from seeds collected from one mother tree.

% Survival

Stressed Unstressed p < 0.03

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Significance of proanthocyanidins (PAs) in koa Kaua’i Maui Hawai’i Oahu

Maia Corpuz

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LC-MS of procyanidin B2 identified in koa

C30H26O12 Molecular mass = 578.5 g/mol

Maia_koa_proB2_042417(1)_Tray02-A1_01_4857.d Maia_koa_BIstem_042417(1)_Tray02-A2_01_4859.d 0.0 0.5 1.0 1.5 8 x10 Intens. 0.0 0.5 1.0 1.5 2.0 2.5 7 x10 5 10 15 20 25 30 Time [min] 575.2 577.2 579.3 580.3 581.3 582.3 585.4 +MS, 10.0min #985 0.00 0.25 0.50 0.75 1.00 1.25 8 x10 Intens. 570 575 580 585 590 595 m/z 566.6 575.2 576.2577.2 578.2 579.4 580.4 581.4 582.3583.3 585.4 594.0 +MS, 10.2min #1015 0.0 0.5 1.0 1.5 2.0 7 x10 Intens. 570 575 580 585 590 595 m/z

Procyanidin B2 Procyanidin B2

OH OH

Standard Plant extract 579.3 579.4 [M +H]+ [M +H]+ Standard Plant extract

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Vegetative propagation of disease resistant koa

Rooted koa cutting Koa clone grown in tissue culture

57 Tyler Jones

Seedlings obtained from rooted cuttings of resistant lines

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Stock plant rootability varied from 0%-100%

Summary of disease resistant koa rootability

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18% of the resistant stocks from Oahu showed high rootability. Only 2% of the resistant stocks from Maui showed high rootability.

Resistant clone at 1 year Resistant clone at 2.5 years

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Resistant clones of 26 families have been successfully propagated

Resistant koa families propagated from stem cuttings

Resistant koa families propagated from stem cuttings

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Some resistant clones at Hawaii Agriculture Research Center

3 months 2 years 4 years

Private landowner economic reforestation

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• Nick Dudley, Hawaii Agriculture Research Center
• Tyler Jones, Hawaii Agriculture Research Center
• Robert James, Phil Canon, and Richard Sneizko from

USDA

• James Leary, Daniel Adamski, Isabel Rushanaedy, Kazue

Ishihara, and Maia Corpuz

• JB Friday and Travis Idol
• USDA-TSTAR Award No. 2009-04862
• NIFA McIntire-Stennis Project Award No. HAW00597-M
• Hawaii State Department of Agriculture

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Acknowledgments