Previously undescribed species of Diaporthe responsible for stem cankers on Australian sunflowers – an ongoing investigation of causal species Sue Thompson1 Yu Pei Tan2, Stephen Neate1, Elizabeth Aitken3 Roger Shivas 2




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Previously undescribed species of Diaporthe responsible for stem cankers on Australian sunflowers – an ongoing investigation of causal species
Sue Thompson1

Yu Pei Tan2, Stephen Neate1, Elizabeth Aitken3 Roger Shivas 2

1Agri-Science Queensland, DEEDI, PO Box 102, Toowoomba, Qld 4350, Australia. sue.thompson@deedi.qld.gov.au; stephen.neate@deedi.qld.gov.au

2Plant Pathology Herbarium, Ecosciences Precinct, DEEDI, 41 Boggo Road, Dutton Park, Qld 4102, Australia. yupei.tan@deedi.qld.gov.au; roger.shivas@deedi.qld.gov.au

3School of Agricultural and Food Sciences, University of Queensland, St Lucia, Brisbane Qld 4072, Australia. e.aitken@uq.edu.au

ABSTRACT

  • Three newly described pathogenic Diaporthe species were associated with the first damaging outbreaks of phomopsis stem canker in Australian sunflowers.

  • Symptoms included light brown to dark brown irregularly shaped lesions centred at the stem nodes, pith deterioration and mid-stem lodging. Pathogenic Diaporthe isolates were also isolated from the harvested seed of infected crops.

  • Although symptoms appeared similar to those caused by Diaporthe (Phomopsis) helianthi which is exotic to Australia, molecular analysis and morphological characterisation of the pathogenic isolates indicated that other Diaporthe species and not D. helianthi are associated with the outbreak.

  • Research to date has included pathogenicity testing, molecular phylogeny analysis, morphological characterisation and taxonomic descriptions of three groups within the isolated populations.

  • This study clearly shows that more than one pathogenic Diaporthe species is associated with sunflower stem cankers in Australia. Furthermore it supports the view that a taxonomic review of Order Diaporthales should be undertaken.


Key words: Diaporthe - new species – pathogenic – Phomopsis - phylogeny – sunflower- taxonomy

INTRODUCTION

In Australia, sunflower (Helianthus annuus L.) has been grown commercially since the introduction of the Russian self-pollinating variety Peredovik in 1969 (Blamey et al., 1987). It is a significant break crop in the wheat growing areas in New South Wales and Queensland, with annual production highly dependent on the comparative price of sunflowers relative to sorghum and maize. Australia’s annual production of 62,000 t seed (25-30,000t oil) from approx. 40,000 ha is unable to meet domestic demand, resulting in the importation of up to 40,000t oil per annum (AOF, Goddard, pers. com. 2011).

In 2009, mid-stem lodging and premature senescence caused significant damage in sunflower crops in New South Wales and Queensland following a prolonged period of wet weather. Pith damage behind elongated brown to brown-black lesions weakened the stems leading to mid-stem lodging as the heads filled (Thompson et al., 2010). Taller hybrids appeared to lodge more often than shorter varieties. Although no formal measurements were taken, field observations did not reveal any differences between hybrids for lesion size or severity.

Although symptoms appeared similar to those caused by the exotic pathogen Diaporthe/Phomopsis helianthi (Muntanola-Cvetkovic et al., 1981), the investigation highlighted the difficulty of accurately identifying Diaporthe species. Host association has often been the basis for species identification of Diaporthe species, as morphological and cultural characteristics have been considered inadequate or unreliable for species differentiation (van Rensburg et al., 2006). However, recent studies have demonstrated that a number of Phomopsis species have wide host ranges (van Niekerk et al.,2005; Ash et al., 2010), and more than one species can occur on a single host (Mostert et al., 2001, Santos & Phillips, 2009). Diaporthe helianthi has also been recorded from hosts other than sunflower in Croatia (Vrandecic et al., 2010) further reinforcing the potential for error when identifying Diaporthe species on sunflower on the basis of host alone.

To accurately identify the species associated with stem cankers on sunflower in Australia, we used incorporated pathogenicity testing, molecular analyses and morphological characterisation.

A number of pathogenic previously undescribed Diaporthe species have been identified from wild, inbred and hybrid sunflowers grown throughout NSW and Queensland. Three new species have recently been described (Thompson et al., 2011) and pathogenic isolates of a number of other previously undescribed species associated with sunflower in Australia are currently being investigated.


MATERIALS AND METHODS

Pathogenicity

Isolates were obtained from stems, leaves and seed of both cultivated and wild sunflower plants exhibiting symptoms of stem canker across NSW and Qld, as well as from hosts other than sunflower.

Small excised stem and leaf pieces were surface-sterilised and incubated (Thompson et al. 2011) to produce pycnidia. Conidia oozing from pycnidia were streaked onto potato dextrose agar (Oxoid) (PDA) amended with 100 µg ml-1 streptomycin sulphate (PDAS). Hyphal tips taken from all isolates were grown on PDAS to establish pure isolates. Cultures incubated for 7 d under ambient light at 23–25 °C were used as inoculum for pathogenicity testing.

Pathogenicity was determined by inoculating five plants of the sunflower hybrid Hyoleic 41 per isolate at the V6–V8 (Schneiter and Miller, 1981) growth stage and grown in a cabinet under a 25 °C 12 h light / 20 °C 12 h dark cycle using the wound inoculation method (adapted from Herr et al., 1983 and van Rensburg et al., 2006). A 5 mm cube of colonised PDAS was inserted into a 5–10 mm long slit made in the sunflower stem at a node, sprayed with distilled water and wrapped with Parafilm™. Control plants were wounded nodes as per the treated plants, then wrapped with permeable film without placing an agar cube in the wound. All plants were sprayed with distilled water, placed in a dew chamber and incubated at 25 °C 12 h light / 20 °C 12 h dark for 48h then returned to a growth cabinet under the light and temperature regime described above. Plants were assessed for lesion development at 14 d after inoculation on a scale of 1 to 5 where: 1 = low level discolouration at site of inoculation, 2 = very small lesion or slight discolouration 1–2 mm diam; 3 = necrotic lesions 2–5 mm, some light stem streaking, some leaf wilting and twisting; 4 = lesions 5–10 mm diam, significant necrosis and dark stem streaking, obvious leaf and plant wilting and stunting, some lodging; 5 = very severe necrosis and lesions, dark streaking, leaf necrosis, twisting and wilting, severe stunting, lodging or plant death..



Morphology

Methods used to describe the morphologies of the anamorph (three species), teleomorph (one species) and type cultures of the three newly described species in Table 1 are in Thompson et al.( 2011).



DNA isolation, amplification and analyses

Mycelia from PDA cultures was macerated with 0.5 mm glass beads (Daintree Scientific) in a Tissue Lyser (QIAGEN). Genomic DNA was then extracted with the Gentra Puregene DNA Extraction kit (QIAGEN) according to the manufacturer’s instructions.

The primers ITS1 and ITS4 (White et al., 1990) were used to amplify the ITS region of the ribosome genes. To further differentiate D. angelicae, D. stewartii, D. gulyae and P. dauci, the primers EF1-728F (Carbone and Kohn, 1999) and EF2 (O’Donnell et al., 1998) were used to amplify part of the translation elongation factor 1-alpha (TEF-1) gene. ITS and TEF loci were amplified with the Phusion High-Fidelity PCR Master Mix (Finnzymes). PCR products were purified with the QIAquick PCR Purification Kit (QIAGEN), and sequenced on the 3730xl DNA Analyzer (Applied Biosystems).

Sequences generated in this study were assembled using Vector NTi Advance 11.0 (Invitrogen) and deposited into GenBank (Table 1). These sequences were aligned with sequences from representative Diaporthe/Phomopsis species from GenBank (Figure 1) in MEGA 5.05 (Tamura et al., 2011). The sequences obtained from GenBank are listed by their taxon names followed by strain numbers in the trees (Figure 1). Nomenclatural novelties were deposited in MycoBank (www.MycoBank.org) (Crous et al., 2004).Further detail of the DNA methods are in Thompson et al. (2011).


RESULTS

Ongoing investigation has revealed that at least eight previously undescribed Diaporthe species are associated with stem cankers on sunflower in Australia (Table 1).

Pathogenicity testing revealed that the species have a range of virulence levels with the most virulent isolates (virulence ratings 4 and 5) causing severe stem lesions, stem streaking and lodging within 7 days of inoculation. Table 1 lists comparative virulence levels of three newly described (Thompson et al. 2011) pathogenic Diaporthe species (D. gulyae, D. kongii, D. kochmanii) and five other species, listed as Diaporthe sp. nov 1-5, which are currently undergoing analyses.

Based on preliminary pathogenicity testing and molecular phylogeny (Table 1, Figure 1), Diaporthe sp. nov 4 and Diaporthe sp. nov. 5 may also be undescribed species highly pathogenic on sunflower. Other Diaporthe species from Australian sunflower in this study are listed in Table 1, Diaporthe ambigua, was not virulent in pathogenicity tests and is considered to be a saprophyte on sunflower.



Figure 1: Maximum likelihood phylogenetic tree based on partial ITS sequence of selected Diaporthe/Phomopsis isolates collected from sunflower and other hosts in Australia during this study. Newly described species are in boldface.

Table 1. Virulence and identification of Diaporthe spp. isolated from Australian sunflower stem cankers.


Genbank No.

BRIP1 No.

Collection Location

Host

Diaporthe species

Virulence Rating4

JF431294

54028

Ryeford, Qld

sunflower

D. gulyae

4

JF431299

54025

Hermitage, Qld

sunflower

D. gulyae

5

JF431300

54032

Childers, Qld

sunflower

D. kongii

3

JF431301

54031

Childers, Qld

sunflower

D. kongii

3

JF431295

54033

Gatton, Qld

sunflower

D. kochmanii

2

JF431296

54034

Gatton, Qld

sunflower

D. kochmanii

3




54136a

Somerton, NSW

lupin

D. sp.nov.1

nt3




54671a

Somerton, NSW

sunflower2

D. sp.nov.1

nt




54256a

Childers, Qld

soybean

D. sp.nov. 2

nt




54118c

Clifton, Qld

sunflower

D. sp.nov. 2

nt




54120a

Gatton, Qld

maize

D. sp.nov. 3

nt




54118b

Clifton, Qld

sunflower

D. sp.nov. 3

nt




54611a

Kingsthorpe, Qld

sunflower

D. sp.nov. 4

4




54737b

Kingsthorpe, Qld

sunflower

D. sp.nov. 4

nt




54736j

Premer, NSW

sunflower

D. sp.nov. 5

nt




54290d

Hay, NSW

sunflower

D. sp.nov. 5

5




54327b

Darlington Pt, NSW

sunflower

D. ambigua

1

1 Isolate number, ex-type cultures in bold. 2 Re-isolated after pathogenicity testing. 3 nt: testing incomplete

4 Plants were assessed for lesion development at 14 d after inoculation on a scale of 1 to 5 where: 1 is a low level of discoloration and 5 is severe necrosis or plant death.
DISCUSSION

Stem canker caused by Diaporthe helianthi, first recorded in Yugoslavia in 1980 (Muntañola-Cvetković et al., 1981), is one of the most serious diseases of sunflower. Yield reductions of up to 50% have been recorded in Europe (Masirevic and Gulya, 1992, Debaeke et al, 2003) and is widespread in the USA (Gulya et al., 1997). D. helianthi has never been recorded in Australia. It has long been suggested that multiple Diaporthe species are associated with cankers on sunflower. In the former Yugoslavia, although only P. helianthi was considered responsible for the serious disease outbreaks it was also suggested that other species were present (Muntanola-Cvetkovic et al., 1985).

Acimovic and Strasser (1982);Yang and Gulya (1984) and Gulya et al. (1997) also suggested that more than one species or biotype of Phomopsis species may be pathogenic on sunflower, while in Australia, studies by Miric et al. (2002) indicated that at least one or perhaps several pathogenic Diaporthe species could be found on sunflower. Australian isolates appeared distinct from non-Australian isolates examined in Miric’s study which included P. helianthi.

Researchers are commonly using molecular technology, in particular phylogenies derived from DNA sequence analyses of the ribosomal internal transcribed spacer (ITS) regions of the nuclear ribosomal RNA to separate and characterise species (Castlebury et al., 2003; van Niekerk et al., 2005; Hyde et al., 2010). Translation elongation factor 1-α (TEF-1α) has also been used to further identify species. These tools have allowed more accurately separation and characterisation of species and are helping to further define species boundaries by providing more specific genetic evidence in support of taxonomic differences (van Rensburg et al., 2006; Rossman et al., 2007; Hyde et al., 2010; Thompson et al., 2011; Udayanga et al., 2011).

To date, three previously undescribed pathogenic Diaporthe species with a range of virulences on sunflower have been identified and described from our study. The symptoms and aggressiveness of the new species Diaporthe gulyae, appear almost identical to those of D. helianthi. The other two newly described species, D. kongii and D. kochmanii, are less aggressive than D. gulyae (Thompson et al. 2011). At least two other virulent and undescribed species, Diaporthe sp. nov 4 and Diaporthe sp. nov. 5 are currently being investigated.

These results support the preliminary findings by Miric et al. (2001) that the Diaporthe species pathogenic on sunflower in Australia are not D. helianthi. It is evident that there is a spectrum of virulent Diaporthe species pathogenic on sunflower in Australia. With the aid of molecular techniques unavailable to earlier researchers such as Wehmeyer (1933), Muntañola-Cvetković et al. (1981) and Herr et al. (1983), we consider it likely that the sunflower disease that was once considered due largely or even exclusively to D. helianthi, will be found to be a complex of species within the Diaporthe genus with the mix of species varying at each locality.

We suggest that as Diaporthe isolates from other sunflower growing regions in Europe and the USA are tested more intensively that a number of these newly described species including D. gulyae, D. kongii and D. kochmanii will also be identified from localities outside Australia. Additionally, it is highly likely that other previously unidentified or misidentified species will be identified from sunflower and other crops worldwide and reclassified as a result.

The results of our investigation of the Diaporthe (Phomopsis) species associated with stem cankers on sunflower present as a significant case study in support of the views of Farr et al., (2002), Hyde et al,. (2010), Santos et al., (2010) and Udayanga et al., (2011) by highlighting the need for the re-evaluation of the identification and classification of Diaporthales.


ACKNOWLEDGEMENTS

The authors would like thank Dr Malcolm Ryley (Department of Employment, Economic Development and Innovation, QLD) and Dr Tom Gulya USDA-ARS Northern Crops Laboratory ND, USA, for their pathology advice; Loretta Serafin (NSW Department of Agriculture) for agronomic expertise, the Australian Oilseeds Federation; Australian Sunflower Association and Grains Research Development Corporation for financial support. We also acknowledge the support of the University of Queensland, Pacific Seeds Pty. Ltd., Nuseed Pty. Ltd., HRS Seeds Pty, Ltd., Ornasun Pty.,Ltd. and Avosun Pty. Ltd.


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