Colletotrichum gloeosporiodes causing anthracnose on pomegranate in Turkey
During spring 2017, an anthracnose-like foliar disease was observed in the pomegranate orchards in the region. Disease incidence (%) ranged from 15 to 22. The objectives of this study were to characterise this fungal pathogen, based on morphology, molecular characteristics and pathogenicity. Foliar symptoms progressed light to dark brown, concentric, circular, distinct spots with yellowish halos. Also, salmon coloured spore masses were observed on pomegranate cv. Hicaz leaves in Kozan district. Cultural and morphological examinations and DNA sequence data revealed that the fungus associated with symptomatic leaves was Colletotrichum gloeosporioides. Pathogenicity testing was performed by inoculation of healthy leaves with a spore suspension of C. gloeosporioides. This confirmed C. gloeosporioides as causal agent of anthracnose symptoms on pomegranate in Turkey.
KeywordsC. gloeosporioides Pomegranate Pathogenicity PCR
Pomegranate (Punica granatum) is a commercially important fruit both for domestic consumption and as an export commodity in tropical and subtropical countries. In Turkey, total pomegranate production (tonnes) between 2011 and 2016 increased by 114% (TUIK 2018). The Mediterranean Region, including provinces Hatay, Adana, Mersin and Antalya, the main pomegranate growing areas of the region, accounts for approximately 52.7% of the total pomegranate production in Turkey. Pomegranate production is also popular in other parts of the country. At present, the known fungal disease agents of pomegranate are Alternaria spp., Botrytis cinerea, Aspergillus niger, Colletotrichum gloeosporioides, Coniella spp., Nematospora spp., Pilidiella granati, Pestalotiopsis versicolor, Syncephalastrum racemosum, Penicillium spp. and Rhizopus spp. (Palou et al. 2013; Kanetis et al. 2015; Munhuweyi et al. 2016). Among the various fungal diseases, anthracnose caused by Colletotrichum gloeosporioides is one of the most serious diseases of pomegranate worldwide (Munhuweyi et al. 2016). All stages of pomegranate tree development are susceptible to the disease. Latent infection may occur invisibly in healthy plants causing extensive crop losses due to anthracnose disease related symptoms (Munoz et al. 2009). Primary and secondary sources of inoculum are from infected leaves and windborne conidia, respectively (Munhuweyi et al. 2016).
For fungal isolation, the epidermal tissues (approximately 5 mm) of leaves affected were collected and cut into pieces, surface-sterilised by dipping in a 1% sodium hypochlorite (NaOCl) solution for 2 min, rinsed twice in sterile distilled water, and blotted dry on sterile filter papers. Small pieces of disinfested tissues were plated five pieces/plate on the surface of potato dextrose agar (PDA) amended with 100 μg ml−1 streptomycin sulphate (Sigma Aldrich, St. Louis, MO) to inhibit bacterial growth in 90 mm-diam petri dishes. The plates were incubated for 7–10 days at 25 °C in the dark. The morphological characteristics of the fungal structures such as acervuli and conidia originating from the leaves were carefully scraped using a sterile needle and examined under an upright microscope (Nikon Eclipse Ni-U, Japan) equipped with a digital camera (Nikon DS-Ri2, Japan) for measurements and photographs.
A pathogenicity test was carried out by spraying on surface-sterilised and wounded pomegranate (cv. Hicaz) leaves each with 10 μl of a suspension of conidia (4 × 106 conidia ml−1) obtained from a single spore culture. Leaves sprayed with sterile distilled water served as controls. Both inoculated and control leaves were covered with polythene bags. Fifteen seedlings of twenty-month-old pomegranate were kept separately in a growth chamber at 25 °C, 85% relative humidity, and 12-h photoperiod. No symptoms developed on the leaves sprayed with sterile water. Dark-grey spot symptoms reproduced after 9–12 days were similar to those initially observed on the trees. The fungus was successfully reisolated from symptomatic leaves on PDA and exhibited the same morphological characteristics that were observed upon the initial isolation, thus fulfilling Koch’s postulates.
Morphological characters, pathogenicity and the BLAST analysis of the nuclear gene region sequences against the NCBI database as described above revealed that the isolate we obtained in this study could be identified as Colletotrichum gloeosporioides. This pathogen has been reported on Citrus reticulata, Ficus carica, Hedera helix, Persea americana, Persicaria perfoliata and Rhododendron ponticum in Turkey (Farr and Rossman 2018). Although C. gloeosporioides on pomegranate has been previously reported from China, USA, Greece, India, Myanmar, Puerto Rico, and Virgin Islands (Farr and Rossman 2018), to the best of our knowledge, this is the first report of anthracnose on pomegranate in the Mediterranean Region of Turkey. Further studies are required to reliably assess the potential threat posed by this pathogen for commercial pomegranate production and to better define the host range of C. gloeosporioides in Turkey. The present study provides basic information for epidemiologic studies and developing disease management strategies. Also, it should search for effective bio-control agents and fungicides with acceptable residue levels for use in commercial pomegranate production in Turkey.
The authors gratefully acknowledge Mustafa Kemal University and TUBITAK for the financial support. This research was supported by a Grant (No. 16556) from the Coordinatorship of the Scientific Research Projects of Mustafa Kemal University, Hatay, and by project number 117O688 from the Scientific and Technical Research Council of Turkey (TUBITAK), Ankara.
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