During the spring of 2015, fruit rot symptoms were observed on pomegranate fruit in an orchard located in Oaxaca, Mexico. Characteristic lesions were collected and a fungus was isolated from these lesions. Based on morphological characteristics, analysis of rDNA-ITS sequences, and pathogenicity tests on pomegranate fruits, the causal agent was identified as Pilidiella granati. This is the first report of P. granati causing pomegranate fruit rot in Mexico.
Pomegranate (Punica granatum) is an important shrub of the tropical and subtropical regions of the world, which is valued for its delicious edible fruit (Badizadegan and Khabbazian 1977). The edible part of the fruit is called arils, which are eaten fresh and can be preserved as syrup or used for making juice and jam. In addition, the tree is also valued for its pharmaceutical properties. Pomegranates can help prevent or treat various disease risk factors including high blood pressure, high cholesterol, oxidative stress, hyperglycemia, and inflammatory activities (Zarfeshany et al. 2014). The fruit peel, stem, root bark and leaves are a good source of secondary products such as tannins, dyes and alkaloids (Mars and Marrakchi 1999).
In Mexico, pomegranate production is mainly concentrated in seven states (Oaxaca, Hidalgo, Guanajuato, Morelos, Sonora, Estado de México, and Puebla). In 2014, the production was 4362 ton distributed in 716 ha (SIAP 2015).
During the spring of 2015, severe symptoms of a fruit rot disease were observed on 60 to 85% of pomegranate fruits in an orchard located in Oaxaca, Mexico. The initial symptoms consisted of dark brown lesions that later increased in size and resulted in fruit rot.
To isolate the fungus, pieces of 5 × 5 mm were taken from the margins of diseased tissue, surface disinfested by immersion in a 1% sodium hypochlorite solution (NaOCl) for 2 min, rinsed three times with sterile distilled water and dried on sterilized paper. The pieces were placed in Petri plates containing potato dextrose agar (PDA) (Difco®, USA) and plates were incubated at 25 °C for 6 days in darkness. Subsequently, mycelial plugs of 5 mm in diameter from the edge of any fungal hyphae that developed from the fruit tissues were transferred aseptically to fresh PDA. To obtain single spore cultures, a single germinated conidium was removed and transferred to the new Petri plate containing fresh PDA. Cultures were maintained as mycelial plugs in 15% glycerol at −80 °C.
For the morphological characterization of the fungus, slide preparations were made in lactic acid using longitudinal sections of pycnidia present on the fruit and examined with a compound microscope (Olympus BX41®, Japan). The qualitative and quantitative morphological characteristics of 20 pycnidia and 100 conidia were recorded. The identification of the fungal species was done with the use of species description and a key reported by Van Niekerk et al. (2004).
Fungal colonies with white aerial mycelia and concentric rings of black pycnidia were observed after 9 days of incubation (Fig. 1a, b). Pycnidia were solitary, globose and black with thin and membranous walls, 90 to 140 μm in diameter. Conidia were hyaline, one-celled, ellipsoid to fusiform, and 12 to 16 × 3.2 to 5.1 μm. These morphological characteristics were consistent with Pilidiella granati (syn. Coniella granati).
To confirm the identity of the fungus, genomic DNA was extracted from mycelia using a DNeasy Plant Mini Kit (Qiagen®, CA, USA) according to the manufacturer protocol. The quality of the DNA was verified by electrophoresis in a 1.0% agarose gel. DNA concentrations were quantified using a NanoDrop Lite Spectrophotometer (Thermo Scientific®, USA) and the samples were diluted to 50 ng μL−1 for PCR reaction.
The rDNA internal transcribed spacer region (ITS1–5.8S–ITS2) of a representative isolate was amplified using primers ITS5/ITS4 (White et al. 1990). The amplified PCR product was purified using the Wizard SV Gel and PCR Clean-Up System (Promega®, USA) and sequenced directly using ITS5/ITS4 primers with the Big Dye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems®, USA) and analyzed with an ABI PRISM 3130xl Genetic Analyzer (Applied Biosystems®, USA). The resulting 579-bp sequence was deposited in GenBank (Accession number KX369239) and was compared with other ITS sequences. BLASTn analysis of the sequence showed a 99% nt identity with those of P. granati from pomegranate from South Africa (KT279814) and China (HQ166057). The isolate used in this study was deposited in the Culture Collection of Phytopathogenic Fungi of the Department of Agricultural Parasitology at the Chapingo Autonomous University (Texcoco, Estado de México, Mexico) under the accession number “UACH-T156”.
Pathogenicity tests were carried out on 10 ripe pomegranate fruits. Fruit were previously disinfested by immersing them in 1% sodium hypochlorite solution for 2 min, washed three times with sterile distilled water and dried on sterilized paper towels. Fruit were wounded in four areas with a sterile toothpick (3 mm of depth) and 10 μL of a conidial suspension (1 × 106 conidia mL−1) were pipetted into each wound, on each of ten fruit. Five fruit were mock inoculated with distilled water as a control. All fruit were placed in plastic containers and maintained at 25 °C for 10 days. The entire experiment was conducted twice. All inoculated fruits developed fruit rot within 6 to 9 days after inoculation (Fig. 1c), whereas no symptoms were observed on the control fruits (Fig. 1d). Furthermore, the pathogen was re-isolated from all inoculated fruit and was identified to be P. granati as described above, fulfilling Koch”s postulates.
To the best of our knowledge, this is the first report of P. granati causing pomegranate fruit rot in Mexico. This pathogen has been previously reported causing cankers on shoots, fruit rot and crown rot of pomegranate in Brazil (Mendes et al. 1998), Greece (Tziros and Tzavella-Klonari 2008), Spain (Palou et al. 2010), Israel (Levy et al. 2011), Iran (Mirabolfathy et al. 2012), Turkey (Çeliker et al. 2012), China (Chen et al. 2014), Italy (Pollastro et al. 2016), and USA (Michailides et al. 2010; KC and Vallad 2016).
Pomegranate is highly susceptible to P. granati, and the disease caused by this plant pathogenic fungus has resulted in substantial losses and is threatening the expansion of pomegranate cultivation in Greece (Thomidis 2015) and other countries (Palou et al. 2010). According to Sharma and Tegta (2011), the incidence of the disease is higher in regions where infected fruit are often seen in the form of dark brown to black fruit mummies scattered on the orchard floor. Besides, the use of fungicides may be the best method to protect the fruit and control this disease. However, there is limited information about the chemical control of P. granati (Thomidis 2015).
Badizadegan M, Khabbazian GH (1977) Study of pomegranate cultivation in Fars Provinces, Publication No. 7. Shiraz University Res. Cen., 69 p
Çeliker NC, Uysal A, Çetinel B, Poyraz D (2012) Crown rot on pomegranate caused by Coniella granati in Turkey. Aust Plant Dis Notes 7:161–162
Chen Y, Shao DD, Zhang AF, Yang X, Zhou MG, Xu YL (2014) First report of a fruit rot and twig blight on pomegranate (Punica granatum) caused by Pilidiella granati in Anhui Province of China. Plant Dis 98(5):695
KC AN, Vallad GE (2016) First report of Pilidiella granati causing fruit rot and leaf spots on pomegranate in Florida. Plant Dis 100(6):1238
Levy E, Elkind G, Ben-Arie R, Ben-Ze’ev IS (2011) First report of Coniella granati causing pomegranate fruit rot in Israel. Phytoparasitica 39:403–405
Mars M, Marrakchi M (1999) Diversity of pomegranate (Punica granatum L.) germplasm in Tunisia. Genet Resour Crop Evol 46:461–467
Mendes MAS, Silva VL, Dianese JC, Ferreira MASV, Santos CEN, Gomes Neto E, Urben AF, Castro C (1998) Fungos em plantas no Brasil. EMBRAPA-SPI/EMBRAPA-CENARGEN, Brasil, Brasilia
Michailides TJ, Puckett R, Morgan D (2010) Pomegranate decay caused by Pilidiella granati in California. Phytopathology 100:S83
Mirabolfathy M, Groenewald JZ, Crous PW (2012) First report of Pilidiella granati causing dieback and fruit rot of pomegranate (Punica granatum) in Iran. Plant Dis 96(3):461
Palou L, Guardado A, Montesinos-Herrero C (2010) First report of Penicillium spp. and Pilidiella granati causing postharvest fruit rot of pomegranate in Spain. New Disease Report 22: 21.
Pollastro S, Dongiovanni C, Gerin D, Pollastro P, Fumarola G, De Miccolis A, Faretra F (2016) First report of Coniella granati (Sacc.) Petr. & Syd. as a causal agent of pomegranate crown rot in Southern Italy. Plant Disease 100(7):–1498
Sharma RL, Tegta RK (2011) Incidence of dry rot of pomegranate in Himachal Pradesh and its management. Acta Hortic 890:491–499
SIAP (2015) Servicio de Información Agrícola y Pecuaria Accessed 1 July 2016Available at: http://www.siap.sagarpa.gob.mx
Thomidis T (2015) Pathogenicity and characterization of Pilidiella granati causing pomegranate diseases in Greece. Eur J Plant Pathol 141:45–50
Tziros GT, Tzavella-Klonari K (2008) Pomegranate fruit rot caused by Coniella granati confirmed in Greece. Plant Pathol 57(4):783
Van Niekerk JM, Groenewald JZ, Verkley GJM, Fourie PH, Wingfield MJ, Crous PW (2004) Systematic reappraisal of Coniella and Pilidiella, with specific reference to species occurring on Eucalyptus and Vitis in South Africa. Mycol Res 108:283–303
White TJ, Bruns T, Lee S, Taylor JW (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ (eds) PCR protocols: a guide to methods and applications. Academic Press, California, pp 315–322
Zarfeshany A, Asgary S, Javanmard SH (2014) Potent health effects of pomegranate. Advanced Biomedical Research 3:100
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Cintora-Martínez, E.A., Leyva-Mir, S.G., Ayala-Escobar, V. et al. Pomegranate fruit rot caused by Pilidiella granati in Mexico. Australasian Plant Dis. Notes 12, 4 (2017). https://doi.org/10.1007/s13314-017-0230-0
- Punica granatum
- Pilidiella granati
- Fruit rot