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Neoascochyta species cause leaf scorch on wheat in Australia

  • H. GolzarEmail author
  • G. Thomas
  • K. W. Jayasena
  • D. Wright
  • C. Wang
  • M. Kehoe
Article
  • 185 Downloads

Abstract

A conspicuous leaf scorch of wheat associated with Didymellaceae family was observed on commercial wheat varieties in Western Australia during recent years. Two fungal species isolated from leaf lesions were identified based on morphological characteristics and DNA sequencing. Neoascochyta europaea was isolated from symptomatic leaf lesions on the wheat varieties Trojan and Wyalkatchem also Neoascochyta graminicola was detected on Calingiri variety. Pathogenicity tests were conducted and Koch’s postulates were fulfilled by reisolating of these fungal species. This is a first report of Neoascochyta europaea and Neoascochyta graminicola causing leaf scorch on wheat in Australia.

Keywords

Didymellaceae Didymella Neoascochayta Ascochyta Leaf scorch Wheat 

The Didymellaceae family consist of a wide range of fungal species, including Ascochyta spp. and Neoascochyta spp., causing serious economic loses on various crop species. Plant species from the family Poaceae are known to be infected by Didymellaceae species (Punithalingam 1979; Mel'nik 2000). The economic importance of these fungal pathogens is not clear however damaging disease symptoms on wheat and barley have been reported (Cormey et al. 1994; Perelló and Moreno 2003; Bockus et al. 2010; Kosiada 2012).

Didymella exitialia is a common pathogen on wheat and barley in Europe (Punithalingam 1979) and has been reported on wheat causing various leaf scorch severities on different wheat varieties in New Zealand (Cromey et al. 1994; Braithwaite et al. 1998).

In addition the occurrence of A. tritici on wheat, A. sorghi on brome grass and wheat, A. hordei on barley and A. avenae on oat has been reported in New Zealand (Braithwaite et al. 1998; Kosiada 2012). A. tritici, A. graminicola and A. sorghi are considered the cause of ascochyta leaf spot in wheat (Bockus et al. 2010).

The occurrence of A. hordei var. europaea on wheat has been reported to be significant in Argentina and under artificial inoculation up to 45% of leaf area became necrotic (Perelló and Moreno 2003).

The taxonomy of Didymellaceae family was revised based on its phylogenetic analysis because of lacking telemorphic stage and morphological differentiation. Therefore Neoascochyta was identified to be the correct genus for some of the Ascochyta species that have previously been reported (Chen et al. 2017).

Leaf necrosis was observed on the commercial wheat varieties confused with the septoria leaf blotch symptom in recent years (Fig. 1a). Leaf samples from wheat varieties Wyalkatchem and Calingiri grown in wheat belt regions in 2014 and Trojan in Albany 2017 were tested to clarify this confusion. Isolation was made using sections of leaf tissue surrounding the lesions and surface-sterilised by immersion in a 1.25% aqueous solution of sodium hypochlorite for 2 min, rinsed in sterile water and dried in a laminar flow cabinet. The sterilised leaf pieces were then either placed on potato dextrose agar (PDA) and incubated at 22 ± 3 °C, then fungal colonies sub-cultured onto oat meal agar (OMA) and then single-spored to obtain pure cultures; or placed on moist filter paper and incubated at 25 °C with a 12 h dark and light for inducing growth and sporulation.
Fig. 1

Neoascochyta europaea. Leaf scorch bearing pycnidia and pseudothecia structures embedded in the naturally infected leaf tissues of Trojan variety (a), asci and ascospores (b) and pycniospores (c). Bars = 5 mm (a) and 10 μm (b and c)

Morphological characteristics of the isolated fungi were determined and N. europaea and N. graminicola were identified from symptomatic leaf lesions (Figs. 1, 2, 3 and 4).
Fig. 2

Neoascochyta europaea. Responses to artificial inoculation on Trojan variety, 48 h postinoculation (a), and 3 weeks after inoculation in comparison to control pink sign (b). Bars = 5 mm

Fig. 3

Neoascochyta graminicola. Pycnidial structures on the naturally infected of Calingiri variety leaf tissues (a) and pycniospores (b). Bars = 50 μm and 10 μm respectively

Fig. 4

Neoascochyta graminicola. Responses to artificial inoculation on Calingiri variety, 2 weeks post-inoculation (a), in comparison to control (b). Bars = 5 mm

Colonies of N. europaea on OMA were olivaceous grey, 30 to 40 mm in diameter after 7 d. Pycnidia ostiolate, semi-immersed in the media, globose, 85215 μm, conidia hyaline fusoid to cylindrical, guttulets and one septate 12.5–19.5 × 3–5 μm. Pseudothecia dark brown, round, ostiolate, solitary or in-group immersed in the surface of infected leaf tissues 120250 μm in diameter. Asci bitunicate, cylindrical to clavate, ascospores fusoid, hyaline and guttulets, one septate, constricted at the septum 16.5–22.5 × 3.5–5 μm in diameter.

Colonies of N. graminicola on OMA were olivaceous grey 18–24 mm in diameter after 7 d. Pycnidia dark brown 135–240 μm, conidia ellipsoidal to cylindrical, hyaline to light brown and one septate 1420 × 35.5 μm in diameter. Teleomorph of N. graminicola was not detected.

The morphological characteristics of these species are identical to similar species described in previous references (Punithalingam 1969, 1979).

The representative isolates of morphologically identified fungi were grown on PDA for 10 days at 22 ± 3 °C. Genomic DNA was extracted from fungal mycelium with a DNeasy Plant Mini Kit (Qiagen) according to the manufacturer’s instructions. The ITS region was amplified with V9G (De Hoog and Gerrits van den Ende 1998) and ITS4 (White et al. 1990), the LSU region with LR0R (Rehner and Samuels 1994) and LR7 (Vilgalys and Hester 1990), the tub2 region with Bt2a and Bt2b (Glass and Donaldwon 1995) and the rpb2 region with fRPB2-5F and fRPB2-7cR (Liu et al. 1999). PCR products were purified using the QIAquick PCR purification kit (Qiagen) and sent to the Australian Genome Research Facility for direct sequencing. Sequences were analysed and subjected to analysis by BLAST against the available sequences in Genbank using Geneious 9.1.7 (Biomatters). WAC13728 and WAC14075 were confirmed to be a 100% match for N. europaea across the ITS, LSU, rpb2 and tub2 regions. WAC13710 was unable to be determined by the ITS and LSU regions, however gave a 100% match to N. graminicola for the rpb2 and tub2 regions.

A replicated trial was conducted for individual fungal isolates using Wyalkatchem, Calingiri and Trojan varieties. These pathogenicity tests were performed at three leaf stage of seedlings under glasshouse condition using representative isolates of N. europaea and N. graminicola. Conidial suspension (106 spores/mL) was prepared in sterile water using a 21-day-old culture of each of the isolates grown on OMA. The conidial suspension was sprayed on the leaf surface to run-off. Controls were sprayed using sterilised water. The plants were misted for 48 h and then moved to a glasshouse at 22 ± 3 °C. The expression of disease symptoms were evaluated two and 3 weeks post-inoculation. Control plants remained asymptomatic (Figs. 2 and 4). The preliminary infection was evident 48 h post inoculation (Fig. 2a) however there was slow development of disease expression and infection did not develop on the upper leaves in the glasshouse condition (Fig. 2b). The wheat samples received for disease diagnostic and in a survey conducted in the southern regions of WA, the leaf scorching of mature plants was evident (Fig. 1). It seems that environmental factors and monoculture of susceptible wheat varieties are important in disease development. This hypothesis has been supported by the previous research (Perelló and Moreno 2003; Kosiada 2012).

Koch’s Postulates were fulfilled by re-isolation and confirmation by morphological characteristics and molecular identification of N. europaea and N. graminicola. Cultures of N. europaea (WAC14075 and WAC13728) that were isolated on the wheat varieties Trojan and Wyalkatchem and N. graminicola (WAC13710) from Calingiri variety deposited in the Western Australia Plant Pathogen Collection. To our knowledge, there are no previous reports of N. europaea and N. graminicola causing leaf scorch on wheat in Australia.

Notes

Acknowledgements

The authors would like to thank Ms. Yu Pei Tan and Dr. Roger Shivas for their comments on molecular identification of the isolated fungi.

We also would like to acknowledge the support and funding by the Grains Research Development Corporation through the project DAW00229.

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Copyright information

© Australasian Plant Pathology Society Inc. 2019

Authors and Affiliations

  • H. Golzar
    • 1
    Email author
  • G. Thomas
    • 1
  • K. W. Jayasena
    • 2
  • D. Wright
    • 1
  • C. Wang
    • 1
  • M. Kehoe
    • 1
  1. 1.Department of Primary Industries and Regional DevelopmentSouth PerthWestern Australia
  2. 2.AlbanyWestern Australia

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