Citrus x aurantiifolia, a new host report of Macrophomina phaseolina in Iran
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Charcoal root rot-like symptoms were observed on Mexican lime (Citrus x aurantiifolia) plants in a nursery located in Hormozgan province, southern Iran. The fungus was identified as Macrophomina phaseolina based on morphological and molecular characteristics. Pathogenicity tests revealed the association of six M. phaseolina isolates with disease. Reisolation from roots of inoculated plants yielded isolates of M. phaseolina with morphological characteristics identical to those of the original isolates used for inoculations, thus fulfilling Koch’s postulates. This is the first record of charcoal rot caused by M. phaseolina on citrus in Iran.
KeywordsCharcoal rot Macrophomina phaseolina Mexican lime Citrus x aurantiifolia
Macrophomina phaseolina is a soil-borne, microsclerote-producing fungus with a worldwide distribution. It causes charcoal rot and ashy stem blight of several important crops including sorghum, sunflower, corn, melon and beans (Frederiksen 1986; Mahdizadeh et al. 2011; Mahmoud and Budak 2011; Olaya et al. 1996; Pearson et al. 1986). M. phaseolina is especially prevalent in subtropical and tropical arid climates. This fungus usually infects plants that are subjected to severe stresses induced by drought and high temperatures (Olaya et al. 1996). In Iran, M. phaseolina causes significant damage in soybean (Raeyatpanah et al. 2002) and sunflower (Razavi and Pahlavani 2004). During the last decade, many other crops including marigold, cantaloupe, cumin, hemp, mung bean, okra, tomato, turnip, watermelon (Mahdizadeh et al. 2011) and strawberry (Sharifi and Mahdavi 2012) were reported as new hosts for M. phaseolina.
Pathogenicity tests, repeated twice, were performed to establish the association of six hyphal-tipped M. phaseolina isolates, including the isolate IRAN 3046C, with disease. Each isolate was used to inoculate 70-day-old healthy Mexican lime plants growing in plastic pots containing autoclaved peat/soil mixture for 70 days. Inocula of M. phaseolina isolates, consisting of microsclerotia, were obtained free of culture medium by aseptically placing a small, colonised agar block from an actively growing culture in a flask containing sterile potato dextrose broth (PDB). The flask was incubated at room temperature for three months until a thick mat composed predominantly of microsclerotia formed on the surface of the broth. The mat was separated from the medium by vacuum filtration, rinsed three times in sterile distilled water, and dried at 35 °C for 72 h. The dried mycelial mats, consisting mostly of microsclerotia, were then ground with a mortar and pestle and passed through a 325 μm mesh to obtain smaller clumps. Prior to the experiment, the germination of microsclerotia on water agar medium was determined to be 80%. Microsclerotia were mixed with 1000 g of sterile air-dried sand and stored at 4 °C until used for inoculations (Goudarzi et al. 2008). The 70-day-old plants were transplanted into the autoclaved peat/soil mix (5000 g in plastic pots) infested with the microsclerotia/sand mix at the rate of 100 viable microsclerotia g−1 soil and maintained in a greenhouse at 30± 2 °C. Ten control plants were transferred to non-infested soil. All plants were watered once a week. After three weeks, all inoculated plants began to show symptoms on leaves and roots, similar to the symptoms of the nursery plants. By five weeks, disease severity ranged from 75 to 100% depending on isolate. Extent of colonisation was rated according to a 1 to 9 scale in which 1 refers to no visible symptoms and no formation of sclerotia, whereas 9 indicates all tissues of the root are colonised and densely covered by sclerotia (Olaya et al. 1996). No symptoms were observed on control plants. Microsclerotia were produced after seven weeks on roots of 85% of the surviving plants. For each isolate tested, M. phaseolina was reisolated only from inoculated plants, fulfilling Koch’s postulates.
To the best of our knowledge, this is the first record of occurrence of charcoal rot on citrus caused by M. phaseolina in Iran. Similarly, charcoal rot of citrus has been reported from Kenya (Kung'u et al. 2002) and India (Chakraborty et al. 2011). Based on the incidence and severity of symptoms, charcoal root rot of citrus, an emerging disease, is considered as a potential threat to citrus industry in southern Iran.
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