Genetic variation among Australian isolates of Fusarium oxysporum from strawberry

M. Paynter, E. Czislowski, M. Herrington & E. Aitken

Strawberry production in Australia

Annual planting using bare rooted propagated runners

Growers use same land continuously, pre-plant fumigation

Fragaria x ananassa : coastal regions, Qld & WA (winter), Vic (summer)

Farm gate value: $400 million

Queensland: $200 million (40%)

Granite Belt: $35 million, summer focused

Qld industry: almost all year round

Establishment & growth – $136,524

Important to minimise risk of loss to soil-borne diseases

Fusarium wilt

Fusarium wilt – major disease of strawberry

Phase-out of methyl bromide

Fumigant alternatives and soil treatments less effective

Outbreaks of Fusarium wilt – WA, Qld

Crown isolations – 30-40% Fusarium oxysporum virulence status unknown

Industry expansion in the Granite belt has come with challenges

Increase in replanting into used plastic and ratooning

Predicted (HAL) to constrain economic strawberry production

Emphasis on breeding cultivars with resistance

Spread: via infected planting material, water Severity: greater with warm wet weather Infects: via roots, into vascular tissue, limits water

& nutrient uptake Symptoms: wilting, collapse, death of plant

Fusarium oxysporum f. sp. fragariae (Fof)

Difficult to eradicate, can remain viable for many years No cure for affected plants. Genetic resistance - economically viable control method

Genetic diversity & virulence of Fof isolates in Australia

Cultivar responses

Breeding & selection of resistant genotypes - test new cultivars against a wide range of pathotypes

Genetic variation in Australia - unknown

Assessment of virulence to identify highly virulent or diverse strains

Challenge potential breeding lines

Project objectives

25 isolates of F. oxysporum tested for genetic relatedness

Forma specialis unknown (if pathogenic to strawberry)

Obtained from major strawberry production regions

Fof diversity

• nine isolates

• Kabarla - susceptible

• Root dip method

• 1 x 106 conidia/mL

• Rating (0=healthy to 10=dead)

• Re-isolated & tested

• Nitrate non-utilising mutants (Nit mutant)

• ID by phenotype

• Potassium chlorate amended media

• Pairing into VCGs

• Same VCG -Heterokaryon

• Translation elongation factor 1α (TEF)

• Mitochondrial small subunit ribosomal DNA (mtSSU)

• Relationships among the F. oxysporum isolates

Gene analysis Pathogenicity Vegetative

Compatibility Group (VCGs)

Pathogenicity differences among isolates, based on disease severity ratings, 8 weeks post-inoculation

Isolate Mean visual rating (0-

healthy, 10 = dead)

N13581 9.6 d

N17337 9.2 d

N18462 7.8 cd

N15309 7.2 c

N15457 3.4 b

N18582 2.0 ab

N18419 1.4 a

N16004 1.4 a

N18421 0.6 a

More virulent

Less virulent

Pathogenicity results

Rate and degree of symptom expression varied among isolates

Significant variation among isolates

Ratings >2 were designated as Fof

6 isolates caused Fusarium wilt

Four virulent isolates chosen for cultivar evaluations

Means with same subscript are not significantly different at P=0.05

Vegetative Compatibility Group (VCGs) results

Sectors generated on MM amended with

potassium chlorate

Heterokaryon formation formed by nit

mutants belonging to the same VCG

Nit mutant generated from all isolates

All combinations of isolates were paired on MM (medium)

Belong to the same VCG by prototrophic heterokaryon formation

Most mutants of an individual isolate were compatible with each other. 6 isolates were incompatible

4 isolates from Sunshine coast region – VCGa

All 4 isolates sampled from WA – VCGb

Remaining isolates, only compatible with one another - single-member VCGs

N18842

N16004

N17203

N16818

N15915

N15457

N15309

N13581

N10226

N9103

Fof_Maff744009

N16239

N16240

N17337

N18462

Fof_KJ776745.1

N9551

N17350

N9055

N9054

N10010

N16893

N18582

N18936

N16999

N18437

SA126

Foz_39298

Fov_KF466424.1

54

63

86

48

51

42

36

86

64

0.01

i

ii

iii

iv

v

vi

vii

viii

Phylogenetic tree – TEF dataset, 29 nucleotide sequences

Rooted with F. verticillioides (Fov)

ML method based on GTR model

Bootstrap values from 1,000 reps.

Fof_Maff744009 from Japan

Fof_KJ776745.1 from Turkey

Foz_39298 included as outgroup

Lineages i to vii - Fof

VCGa, Qld isolates – lineage i

VCGb, WA isolates – lineage ii

TEF sequences

ix x

mtSSU sequences

mtSSU phylogenetic tree

25 nucleotide sequences

ML method based on GTR model

Bootstrap values from 1,000 reps.

Foz BRIP39298 included as outgroup

N9551

N18936

N18842

N18582

N18462

N17350

N10010

N10226

N13581

N15309

N15457

N15915

N16004

N16239

N16240

N16818

N16893

N16999

N17337

N17203

N9055

N9054

N18437

SA126

Foz_39298 92

66

0.001

TEF & mtSSU datasets

N17337

N18462

N16240

N16239

N15309

N13581

N10226

N15457

N15915

N16004

N16818

N17203

N18842

N16999

N18582

N18936

N10010

N16893

N9054

N9055

N9551

N17350

N18437

Foz_39298

SA126

87

87

41

41

38

94

61

0.001

Phylogenetic tree – TEF & mtSSU datasets

25 nucleotide sequences

ML method based on Tamura 3-parameter model

Bootstrap values from 1,000 reps.

Foz_39298 included as outgroup

VCGb

VCGa

Isolate variation

Diverse range of pathogenicity and genotypes

Variation in disease response- heterogeneous populations

25 isolates categorised into VCGs – 2 distinct VCGs, correlated with differences in geographic origin

Relationships within Fof were clearly defined by TEF analysis

mtSSU analysis was generally insufficient to resolve interspecific genetic variation

Conclusions

Diverse range of pathogenicity and genotypes

Variation in disease response- heterogeneous populations

Pathogenicity tests identified 4 highly virulent isolates

26 isolates categorised into VCGs – 2 distinct VCGs, correlated with differences in geographic origin

Relationships within Fof were clearly defined by TEF analysis

mtSSU analysis was generally insufficient to resolve interspecific genetic variation

Cultivar responses

Pathogenicity trial

• 4 isolates tested on 8 cultivars

• Root dip method (1 x 106 conidia/mL)

• 9 plants (reps), 6 non-inoculated controls

• Disease severity rating: 0=healthy and 10=dead

• Cultivar x isolate interaction analysed

Varietal differences important aspect in resistance breeding and testing of virulent strains

A range of genotypes tested to measure disease intensity

Cultivar by isolate interactions ?

Glasshouse experiment conducted to verify cultivar responses

Cultivar responses

10 rating times (2 to 14 wks post-inoc)

Significant isolate effect (P<0.05)

Significant cultivar effect

Cultivar x isolate interaction

Cultivar x isolate interaction

Symptom expression in cultivars varied among isolates

Differences in disease expression in cultivars (Festival, Sugarbaby - resistant, Kabarla - susceptible)

N18462 most aggressive isolate (Western Australia)

Camarosa very susceptible to WA strain, high % of strawberry production

WA production – poor soils, no cover cropping, strip fumigation

Infected plant material carrying over from season to season

Risk of increased outbreaks

Utilization of Fof

resistance

Cultivar responses

Significant cultivar differences (P ≤ 0.05)

Significant cultivar differences across isolates

Race structure or cultivar specificity

Festival, Sugarbaby, Rubygem – suitable parents

Some cultivars and breeding lines have been included in current DAF strawberry crossing program at MRF

Continue to screen other genotypes

Conclusions

• Development of new cultivars with disease resistance for the strawberry industry

• Strive to breed multi-pathogen resistance

Maroochy Research Facility (DAF) at Nambour School of Agriculture and Food Sciences, The University of Queensland

This project has been funded by Horticulture Innovation Australia Limited using the Strawberry industry levy and funds from the Australian Government. The Queensland Government has also co-funded the project through the Department of Agriculture and Fisheries

.

Don Hutton Apollo Gomez

Lien Ko Louella Woolcock

Dale McKenna Mary Grace

Tali Grace Warwick Grace

Acknowledgements