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 1

The koa industry in Hawaii ~ $31 million annually 2

3

• Koa is a dominant canopy species in mesic-montane forests in Hawaii • It is a highly valuable timber tree species endemic to the Hawaiian Islands 4

Close canopy koa in rainforest environment 5

Open canopy koa in dry environment 6

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 7

Canopy nodulation in Acacia koa James Leary 8

Major threats to koa • Deforestation • Grazing • Kikuyu grass • Vascular wilt and dyeback disease caused by Fusarium oxysporum f. sp. koae 9

Koa forests have been degraded over the last two centuries by deforestation and pasture conversion 10

Koa in a pastureland 11

A degraded pasture pastureland 12

A koa forest changed to a degraded pastureland 13

Cattle destroy the young koa seedlings 14

Removal of cattle leads to dominant occupation of kikuyu grass ( Pennisetum clandestinum ) 15-45 Mg/ha aboveground biomass 15

The Fusarium wilt of koa Caused by Fusarium oxysporum f. sp. koae Caused by Fusarium oxysporum f. sp. koae seedling mortality 16

One year old sapling with natural infection 17

Stem lesion on koa sapling infected with Fusarium wilt 18

19 Crown dieback on koa sapling

20 Discolored branch of an infected koa

21 Shoot dieback of infected tree

22 Ruptured bark of infected koa

23 Ruptured vascular system with fermented sap

Crown dieback in native forest 24

25 Completely dead koa

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. 26

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 27

Statewide survey for F. oxysporum f. sp. koae (Foxy) Key people Forests and nursery sample sites Nick Dudley Robert James Phil Cannon Miles Foxy was isolated from the roots, stems and branches of diseased koa seedlings and trees 28

29 Stem sample of infected koa

30 Symptomatic koa tree

Stem dissection of symptomatic koa tree 31

Close-up of FOXY mycelium on stem of an infected seedling 32

Positive FOXY root sample: close-up 33

Fusarium spores Macroconidia 34

Fusarium spores Chlamydospores 35

Inoculation Methods Komada agar Cornmeal perlite PDA for producing clamydospores 36 Dibble tubes Peat moss & perlite

Isolate Trials: Identifying strains of Fusarium oxysporum with high virulence on koa • All isolates were identified as Fusarium Nick Dudley oxysporum 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 & Robert James New Isolate Trial 4B 100 Virulent 90 80 Percent of Seedlings Killed 70 60 50 40 30 20 10 0 Isolate Number 38

Nick Dudley & Summary of virulence of Fusarium Robert James oxysporum 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

Screening Families for Resistance to Fusarium oxysporum f. sp. koae Tyler Jones • A mixture of ten virulent isolates are used • % of resistant seedling in the families • A family represents seeds from the same mother tree 40

Healthy Seedling with seedling wilt symptoms Seedling killed by koa wilt Roots of dead seedling 41

T-15 42 A susceptible family

T-24 43 A family showing no symptom of wilt

Koa Wilt Resistance Trial: Distribution of Mortality 360 seedlings Number of seedlings died 13th week 1 7 13 19 23 Assessment week 44

Koa family resistance screening trials • Screened >700 families in total • Retested families to verify results • 153 families have been planted in seed orchards  Highly Resistant families ( >75%)…..……….. .. 11%  Moderately Resistant families (40-75% )……...…38 %  Susceptible families ( <40%)……………… ……..51 %  Survival ranged from 0 – 96%  Average Survival was ~38% 45

Koa wilt resistance selection strategy Collect seeds Resistance testing at Maunauli (HARC) Resistant seedlings from Select mother the best families trees in forest Seed Production (F 1 ) Seed Orchard 46

Koa seed orchard at Maunauli Maunawili Average Survival: 60% Wild population Average Survival: 36% 47

Locations of Wilt Resistant Koa Seed Orchards Nick Dudley 28 34 43 153 families have been planted in seed orchards 48

Molecular screening for Fusarium wilt resistance Isabel Rushanaedy • 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 Chitinase 49 Chitin

Expression of chitinases in resistant and susceptible koa The expression of the chitinase genes is more highly upregulated in the resistant koa. 18S rRNA gene was used as an internal control for normalization of expression levels 50 Rushanaedy I et al. (2012) Tropical Plant Biol. 5:244 – 252.

Expression of disease resistance genes after mechanical treatment Stressed Unstressed Kazue Ishihara Sample collection RNA extraction NanoString or qRT-PCR 51

Microarray and NanoString analyses 10-60 min post mechanical stress 104 NBS-LRR gene sequences in the A. koa trancriptome Fold change Ring-finger Zinc-finger WRKY NBS-LRR protein transcription protein Disease factor Resistance Protein Transcription factors 52

Differential expression of pathogenesis-related (PR) genes 6h post mechanical stress * p < 0.05 ** p < 0.01 Fold change expression S U S U S U  -1,3-glucanase Akchit-1a Akchit-3 Chitinases 53

Determining disease resistance to F. oxysporum Survival recorded after 100 days of inoculation Inoculation with F. oxysporum p < 0.03 % Survival Stressed Unstressed Stressed Unstressed Seedlings were grown from seeds collected from one mother tree. 54

Significance of proanthocyanidins (PAs) in koa Maia Corpuz Kaua’i Hawai’i Maui Oahu 55

LC-MS of procyanidin B2 identified in koa C 30 H 26 O 12 Molecular mass = 578.5 g/mol Intens. Intens. Standard Standard Maia_koa_proB2_042417(1)_Tray02-A1_01_4857.d +MS, 10.0min #985 [M +H] + Procyanidin B2 8 8 x10 x10 579.3 579.3 1.25 1.5 1.00 OH 1.0 0.75 0.50 580.3 0.5 581.3 0.25 575.2 582.3 577.2 585.4 0.0 0.00 7 Plant extract x10 Maia_koa_BIstem_042417(1)_Tray02-A2_01_4859.d Intens. Plant extract +MS, 10.2min #1015 570 575 580 585 590 595 m/z Procyanidin B2 7 x10 [M +H] + 579.4 2.0 2.5 579.4 2.0 1.5 1.5 OH 1.0 575.2 1.0 576.2577.2 580.4 0.5 0.5 581.4 578.2 582.3583.3 585.4 566.6 594.0 0.0 0.0 0 5 10 15 20 25 30 Time [min] 570 575 580 585 590 595 m/z 56

Vegetative propagation of disease resistant koa Tyler Jones Koa clone grown in tissue culture Rooted koa cutting 57

Seedlings obtained from rooted cuttings of resistant lines 58

Summary of disease resistant koa rootability Stock plant rootability varied from 0%-100% 18% of the resistant stocks from Oahu showed high rootability. Only 2% of the resistant stocks from Maui showed high rootability. 59

Resistant koa families propagated from stem cuttings Resistant clone at 1 year Resistant clones of 26 families have been successfully propagated Resistant clone at 2.5 years 60

Resistant koa families propagated from stem cuttings 61 Some resistant clones at Hawaii Agriculture Research Center

Private landowner economic reforestation 3 months 2 years 4 years 62

Acknowledgments • 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 63