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First Report of Strawberry Leaf Spot Caused by Alternaria alternata in Pakistan

Mehmood, N., Riaz, A., Naz, F., Hassan, I., Jaabeen, N., Anwaar, S., Rosli, H., Gleason, M. L.
Plant disease 2018 v.102 no.4 pp. 820
Alternaria alternata, DNA primers, Fragaria ananassa, agar, conidia, conidiophores, disease incidence, droplets, fungi, genes, growth chambers, hyphae, internal transcribed spacers, leaf area, leaf spot, leaves, pathogenicity, photoperiod, photosynthesis, polygalacturonase, polymerase chain reaction, relative humidity, sodium hypochlorite, strawberries, surveys, Italy, Japan, Korean Peninsula, New Zealand, Pakistan
Strawberry (Fragaria × ananassa Duch.) has recently become an economically important fruit crop in Pakistan and is cultivated on approximately 1,500 ha each year. A survey of 91 farmers’ fields located across Punjab and Khyber Pakhtunkhwa (KPK) provinces and Islamabad (Islamabad Capital Territory), conducted during March and April of 2014–15 and 2015–16, revealed black leaf spot symptoms on strawberry leaves. Leaf spots were irregular in shape, 3 to 6 mm in diameter, and blackish brown. Disease affected yield by reducing active photosynthetic leaf area. Disease incidence was 17 to 55% in visited fields. Portions of symptomatic leaf tissue (3 × 5 mm) were excised from the margins of lesions, surface disinfected with 2% sodium hypochlorite for 2 min, rinsed three times in sterile distilled water (SDW), and blotted dry. Leaf tissue was then placed on Czapek Dox agar (CDA) and incubated at 25 ± 2°C with a 12-h photoperiod for 3 to 6 days. Morphology of isolates was examined; colonies were initially white, then turned light to dark brown and grayish black and produced abundant aerial hyphae. Conidiophores were 22 to 66 µm long × 2 to 4.5 µm wide, straight or flexuous. Conidia were pale to brown, obclavate to pyriform or ellipsoid, with beaks at tips; size ranged from 5 to 60.5 µm long × 6.5 to 26 µm wide. Conidia had 1 to 6 transepta and 0 to 3 longisepta. Based on morphological characteristics of representative group, the fungus was tentatively identified as Alternaria alternata (Simmons 2007). To fulfill Koch’s postulates, pathogenicity tests were conducted by choosing one representative isolate from each regions viz. NAS-AA1, NAS-AA8, and NAS-AA12 on detached trifoliate strawberry leaves collected from healthy plants (cv. Chandler). Each of 10 disinfected leaves was inoculated with a single 10-μl droplet of conidial suspension (10⁶ spores/ml) of representative isolates, and 10 additional leaves (controls) were inoculated with SDW. After inoculation, leaves were incubated in a growth chamber at 25 ± 2°C with a 12-h photoperiod at 70 to 80% relative humidity. Leaf spot symptoms were observed on the inoculated leaves after 4 to 7 days, and morphology of isolates was identical to that of the original isolates, whereas no symptoms were recorded on the control leaves. For molecular identification, PCR amplification of NAS-AA1, NAS-AA8, and NAS-AA12 was carried out by utilizing rDNA-ITS with primer pair ITS1 and ITS4 (White et al. 1990) and endopolygalacturonase gene (EndoPG) using primer pair PG3 and PG2b (Andrew et al. 2009). Sequences were submitted to GenBank with accessions MF966136, MF966143, and KX768145 (ITS) and MF964623, MF974542, and MF176237 (EndoPG). BLAST analysis of sequences from both genes revealed 99 to 100% similarity with previously reported sequences of A. alternata with GenBank entries of ITS (JN107734, KJ957793, and KY609180) and of endoPG (KY923228, KP123996, and KP789546), respectively. A. alternata was previously reported on strawberry foliage in Italy (Wada et al. 1996), New Zealand, Japan (Takahashi et al. 1997), and Korea (Cho and Moon 1980). To our knowledge, this is the first report of A. alternata on strawberry in Pakistan. A. alternata could pose a serious risk to the emerging strawberry industry of Pakistan.