First Report of Ceratocystis fimbriata Causing Wilt of Loquat in Pakistan

Loquat (Eriobotrya japonica Lindl.) is an important tree fruit crop in Pakistan with a planted area of 1,415 ha and approximately 8,823 metric tons produced annually. During November 2016, in the city of Taxila (33°44′13″N, 72°47′57″E) in Punjab Province, wilt symptoms were observed on several loquat orchards. Initially, symptoms appeared as irregular brown lesions in the center and/or on the margins of leaves. At later stages, all leaves on the tree became yellow and wilted. Irregular streaks and dark brown discoloration in the xylem of the trunk were evident in cross sections. Disease was severe on 64% of trees surveyed. Samples were collected from 20 symptomatic trees. Isolations from transverse sections of discolored xylem tissues were performed on carrot discs that had been surface sterilized in 100-ppm streptomycin solution. After consistent sporulation of a fungus on the carrot discs, the ascospore masses formed at the tip of perithecia were transferred to malt extract agar (MEA). Observed perithecia were black and globose (173.7 to 254.4 × 157.6 to 273.5 µm) with long black necks (203 to 974 µm). Ascospores were hyaline and hat shaped (4.2 to 6.1 × 2.8 to 4.8 µm). Cylindrical endoconidia (11 to 26 × 2.8 to 4.6 µm) and doliform endoconidia (4.2 to 8.5 × 7.0 to 13.2 μm) were arranged in chains. Aleurioconidia that were pyriform and dark brown (13 to 19.5 × 8.5 to 14 μm) appeared on MEA after 45 days. For molecular identification, genomic DNA was extracted from a representative isolate (PD202). The primers ITS1F and ITS4 were used to amplify and sequence the ITS1, 5.8S, and ITS2 rDNA region (Thorpe et al. 2005). BLAST analysis of sequenced isolate (GenBank accession no. MG197748) showed 99 to 100% homology to Ceratocystis fimbriata (isolates KY580869 and KU877208, respectively). Based on morphological and molecular characteristics, the fungus was identified as C. fimbriata (Engelbrecht and Harrington 2005). A pathogenicity test was conducted on 10 1-year-old loquat seedlings (cv. Golden Yellow) in plastic pots containing sandy soil. After a 5-mm-diameter plug from the edge of a 10-day-old fungal culture on MEA was inserted into a slit (5 mm wide × 7.5 mm long) in the bark, the wound was covered with moist cotton and secured with Parafilm to maintain humidity. Controls were 10 seedlings inoculated with sterile MEA plugs. The seedlings were placed in a greenhouse at 25°C with 85% relative humidity. All inoculated plants wilted within 8 to 15 days after inoculation and showed symptoms closely resembling those of naturally infected loquat. Control plants remained symptomless. The fungus was consistently reisolated from symptomatic plant parts, confirming pathogenicity. C. fimbriata has been reported to cause wilt of loquat in China (Li et al. 2014). There are also reports of C. fimbriata causing wilt of pomegranate and eucalyptus in Pakistan (Alam et al. 2017a, b). However, to the best of our knowledge, this is the first report of loquat wilt caused by C. fimbriata in Pakistan. The disease could represent a threat for loquat production owing to its increasing cultivation in Pakistan. Therefore, appropriate management strategies should be carried out to limit the spread of the pathogen.