Department of Employment, Economic Development and Innovation

Cryptic species of Phomopsis from sunflower in Australia revealed by

molecular, morphological and pathogenicity studies

SM Thompson, AJ Young, RG ShivasAgri-Science Qld, DEEDI Australia

2© The State of Queensland, Department of Employment, Economic Development and Innovation, 2010

Australian sunflower industry• Area

30-40,000 ha Qld, NSW 62,000t seed 80% monos, 20% polys, birdseed

• Production25-30,000t oil mono + poly

• Consumption95% human: 92% oil (80% mono 12%poly), 3% confectionary seed, 5% horse, bird

• Demand = 60,000t oil

Australia = net importer of 30,000t oil/annum

S Thompson, Clifton, Qld. 2010

(Source: Nick Goddard, AOF March 2011)

3© The State of Queensland, Department of Employment, Economic Development and Innovation, 2010

Project overview

• Grains Research and Development Corporation (GRDC) funded project (IDM Project DAQ 00154)

Identification of causal agent(s)DistributionBiology Management

• Sue Thompson – PathologyAnthony Young – MolecularRoger Shivas - Taxonomy

4© The State of Queensland, Department of Employment, Economic Development and Innovation, 2010

Zero and Min Till ……but increasing incidence of stubble-borne pathogens

Home for beneficial insects , water retention, compaction BUTProtection for multiple pathogens increased survival

Phomopsis/Diaporthe spp.Phoma spp.Sclerotinia minor, Sclerotinia sclerotiorum, Sclerotium rolfsiiFusarium spp. – wheat, sorghum, cornDiplodia spp. - cornAscochyta rabiei - chickpeas

Stubble assists survival and build-up of multiple pathogens Photo: S Thompson DEEDI 2010 Moree

5© The State of Queensland, Department of Employment, Economic Development and Innovation, 2010

Background - Genus Diaporthe• Common name: Phomopsis Stem Canker

• Phomopsis sp. - name given to the anamorph, asexual state

• Diaporthe sp. - name given to the teleomorph, sexual stateeg. Phomopsis/Diaporthe helianthi

• Wide host range : woody shrubs (roobios), weeds (saffron thistle), crops (soybean, sunflower, grapes), junipers, peach, plum

Perithecia of a Diaporthe sp.on PDA S Thompson 2009Pycnidia of a Phomopsis sp. on seed S Thompson 2010

6© The State of Queensland, Department of Employment, Economic Development and Innovation, 2010

History of Stem Canker on Sunflower

• 1980/81 : Yugoslavia Phomopsis/Diaporthe helianthi (Munt-Cvet.et al)

• 1982 - present : Brazil, France, US, Italy, Iran, Russia, Germany, Argentina Stem Canker on sunflower = Phomopsis/Diaporthe helianthi

• 1981 - present : Australia Intermittent records of unidentified Phomopsis sp.

• 2000/01 : AustraliaPhD study by E. Miric – sunflower, soybean, noogoora burr Conclusion - Phomopsis species in Australia not D. helianthi

7© The State of Queensland, Department of Employment, Economic Development and Innovation, 2010

Phomopsis Stem Canker on Sunflower–the Australian Story• 2009

First significant outbreak of Phomopsis/Diaporthe spp. after prolonged wet weather

• 2009 – present: increasing incidence stem lesions, lodging patches

Lodging and lesions typical of Phomopsis Stem Canker Photos: S Thompson DEEDI

8© The State of Queensland, Department of Employment, Economic Development and Innovation, 2010

Why is the outbreak significant?• No previous epidemics recorded for Aus sunflowers

• Potential for significant yield losses - 40%+ losses recorded US, Russia, Yugoslavia (Croatia, Serbia), France.

Photo: S Thompson DEEDI 2010

9© The State of Queensland, Department of Employment, Economic Development and Innovation, 2010

Symptoms and yield loss

Characteristic brown to brown-black lesions dotted at the nodes

Mid-stem lodging

Pith damage behind the lesion

10© The State of Queensland, Department of Employment, Economic Development and Innovation, 2010

Survival and spread• Pycnidia and perithecia survive in stubble/trash and seed • Role of infected seed in distribution in Australia?

eg. D. adunca (Plantago lanceolata) infects via conidia• Wet conditions, spread in free water, rain splash, wind• Temperatures 21-26°C

Photo: S Thompson

Photos S Thompson DEEDI

Pycnidia on infected floret

Pycnidia on dry stem at node

Pycnidia and conidia on infected seed

11© The State of Queensland, Department of Employment, Economic Development and Innovation, 2010

Disease distribution

Seed productionWild sunflowers

First outbreak Phomopsis spp.

DARWIN

Sydney

Brisbane

Perth

Kununurra

Commercial sunflowers

Disease survey, Dinolite technology Kununurra WA 2010 Photo: S Thompson

12© The State of Queensland, Department of Employment, Economic Development and Innovation, 2010

Identification of causal species• Three-pronged diagnostic approach

Pathogenicity testingMolecular phylogenyMorphological characterisation

Shape and size of pycnidia, conidia

Dinolite – see the magnified image in the field or lab

Highly virulent isolate

13© The State of Queensland, Department of Employment, Economic Development and Innovation, 2010

Step 1: Purification of isolates

Photos: S.Thompson DEEDI

Pure isolates for path testing, molecular testing and taxonomy studies

14© The State of Queensland, Department of Employment, Economic Development and Innovation, 2010

Step 2: Pathogenicity testing 200+ isolates

• V6-8 leaf stage• stem slit - quick screening

(Herr et al 1983, van Rensburg et al 2006)) • direct inoc (Miric et al 2002)• mycelium infused agar plug• 23-25°C

+3

+7

+14

15© The State of Queensland, Department of Employment, Economic Development and Innovation, 2010

Step 3: Molecular analysis

• 21 isolates representing a range of virulences and morphological characteristics selected for sequencing

• ITS rDNA sequencing

Range of morphological characteristics Photos: S Thompson DEEDI

16© The State of Queensland, Department of Employment, Economic Development and Innovation, 2010

Diaporthe phaseolorum isolate ZJ4 gi|...

Diaporthe helianthi strain HUNA gi|51...

Diaporthe helianthi strain mky12 gi|2...

12024 263-264

Diaporthe helianthi A2 gi|14599167|em...

Diaporthe helianthi strain STE-U 5355...

Diaporthe helianthi strain STE-U 5344...

Diaporthe helianthi gi|14599179|emb|A...

Diaporthe helianthi strain HZ02 gi|21...

Diaporthe ambigua isolate Di-C002-9 g...

Diaporthe helianthi (ambigua) gi|1459...

Phomopsis sp. 47-2 gi|26279969|gb|AY1...

T11961B 121-122

Diaporthe angelicae isolate Ph-C168/1...

Diaporthe lusitanicae isolate Ph-C170...

Diaporthe sp. DAR73819 gi|166208795|g...

Diaporthe sp. DAR73811 gi|166208796|g...

T12003B 119-120

Phomopsis longicolla isolate T30 gi|2...

Diaporthe helianthi strain mky13 gi|2...

Phomopsis viticola isolate Pvi3 gi|21...

Diaporthe helianthi isolate 33a-2 gi|...

Diaporthe helianthi strain ATCC 62680...

Diaporthe helianthi strain Su 12/04 g...

Diaporthe helianthi strain USH1 gi|51...

Diaporthe helianthi strain FR001 gi|5...

Diaporthe helianthi strain Su 3/04 gi...

Diaporthe helianthi strain Xa 12 gi|2...

Diaporthe helianthi strain Su 5/0 gi|...

Diaporthe helianthi strain ATCC 52472...

Diaporthe helianthi strain Ar 3 gi|22...

64

64

44

30

99

67

62

17

55

40

48

30

48

43

9

39

7

2

1 Helianthus annuus

Helianthus annuus

Helianthus annuus

A

B

C

Helianthus annuus

Helianthus annuus

Helianthus annuus

Helianthus annuus

Fraxinus excelsior

Bruguiera gymnorhiza

Vitis vinifera

Vitis vinifera

Pinus massoniana

Vitis vinifera

Unidentified

Unidentified

Carthamus lanatusCarthamus lanatus

Foeniculum vulgare

Foeniculum vulgare

Gossipum hirsutum

Foeniculum vulgare

Litchi chinensis

Helianthus annuus

Helianthus annuus

Helianthus annuus

Helianthus annuus

Helianthus annuus

Xanthium strumarium

Helianthus annuus

Helianthus annuus

Arctium lappa

D

Molecular taxonomy

Phylogeny – A Young, DEEDI

17© The State of Queensland, Department of Employment, Economic Development and Innovation, 2010

•i

Step 4: Morphological characterisation

Alpha and beta conidia - presence/absence, dimensions Pycnidia and perithecia - shape, dimensions

Pycnidia, conidia and mycelium Photos: S Thompson DEEDI

18© The State of Queensland, Department of Employment, Economic Development and Innovation, 2010

Results to date :• Isolates grouped into 4 + clades

1+ clade highly virulent - one species to be described 2+ clades less virulent - two species to be described

• NOT Phomopsis/Diaporthe helianthi

• Case-study in support of a revision of Genus Diaporthe?

Bellata NSW Photo: S Thompson DPI 2005

19© The State of Queensland, Department of Employment, Economic Development and Innovation, 2010

Classical Diaporthe Taxonomy

• Traditionally, species ID based on :host morphological characteristics (eg. size, shape of conidia, pycnidia), cultural characteristics (eg. colour, density of mycelium)

Unreliable? Production of alpha and/or beta conidia a distinguishing characteristic but enviromental conditions and age of fruiting body/culture may influence development of spore typeseg. D. adunca on scapes (Linders et al 1996)

Phomopsis sp.perithecia Pycnidia oozing conidiaBeta spores – presence, size, shape

20© The State of Queensland, Department of Employment, Economic Development and Innovation, 2010

The Diaporthe puzzle -a case for revision?• Some single Phomopsis/Diaporthe species now recognised to have

multiple hosts eg. Sunflower Clade C isolatesAll correctly identified ?eg. Grapes: many Phomopsis spp. recorded, including P. helianthi

• Molecular diagnostics -Useful toolUsed in conjunction with other diagnostic techniquesSeparation and more accurate differentiation of species

• 3 + undescribed species associated withPhomopsis Stem Canker on sunflower -a small part of the Diaporthe puzzle

21© The State of Queensland, Department of Employment, Economic Development and Innovation, 2010

Acknowledgements• Tom Gulya, USDA Fargo ND.

• Mal Ryley, Stephen Neate DEEDI, Toowoomba

• Loretta Serafin, NSW I&I, Tamworth

• Pacific Seeds, Nuseed, HSR Seeds

• Australian Sunflower Assoc, Australian Oilseeds Federation

• University of Queensland, Liz Aitken

• GRDC, DEEDI

Photo: S Thompson, Bellata NSW 2005

22© The State of Queensland, Department of Employment, Economic Development and Innovation, 2010

Contact details:

sue.thompson@qld.gov.au

anthony.young@qld.gov.au

roger.shivas@qld.gov.au