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Alternaria alternata Causing Leaf and Fruit Spot of Olive in Pakistan

Alam, T., Munis, M. F. H.
Plant disease 2019 v.103 no.4 pp. 762
Alternaria alternata, Olea europaea, Sphagnum, agar, cetyltrimethylammonium bromide, color, conidia, conidiophores, cultivars, disease incidence, food safety, fruits, fungi, genes, glucose, growth chambers, internal transcribed spacers, leaves, mycelium, mycotoxins, olive oil, olives, orchards, oxytetracycline, pathogenicity, pathogens, plastic bags, polymerase chain reaction, relative humidity, ribosomal DNA, sodium hypochlorite, tubulin, Pakistan
Olive (Olea europaea) belongs to family Oleaceae, and its plantation is increasing in Pakistan owing to high demand of olive oil. Several cultivated olive varieties have been invaded by different pathogens. Olive fruit infection may lead to the accumulation of deleterious mycotoxin in its oil and pose a significant threat to food safety and security (Visconti et al. 1986). Leaf and fruit spot was observed in Carolea cultivar of olive in two orchards at National Agriculture Research Center, Islamabad (33°40′17.6″ N, 73°07′27.4″ E) during August to September 2017. The leaf symptoms initiated as small spots on the upper surface of the leaves, which proliferated and covered the whole plant. The infected spots were predominantly located at the edges of the leaves and brown V-shaped chevron was observed in the infected leaves. Disease symptoms were also observed on fruit. The fruit infection commenced as small brown spots that later enlarged and became necrotic. The fruit spots were light to dark brown in color and densely sporulated with fungus growth. The estimated average disease incidence in the orchards was 32%. Infected leaves and fruit samples were collected from the olive orchards. The samples were sliced into fine pieces and surface disinfected with 0.5% sodium hypochlorite for 1 min. These disinfected samples were washed with deionized water, dried, and transferred into Petri dishes containing Sabouraud dextrose agar amended with oxytetracycline (50 mg/liter). The Petri dishes were incubated in the dark at 25°C for a week. The morphology of the cultured fungi was observed to be green-greyish with whitish mycelium borders. Conidiophores and conidia were observed in five replicates. Conidiophores were dark green with 27 ± 5 to 99.6 ± 7 µm length and 15 ± 2 to 36 ± 4 µm width. Conidia were 33.6 ± 3 to 52.7 ± 6 µm long and 7 ± 2 to 17 ± 2 µm wide, obclavate with two to four longitudinal septa and one to four transverse septa, and these were light brown to green in color. The morphology was similar to Alternaria alternata (Simmons 2007). To confirm the identity, the genomic DNA was extracted from a single-spore culture of six isolates employing the cetyltrimethylammonium bromide method. Primers ITS1/ITS4 were used to amplify the internal transcribed spacer (ITS) region (White et al. 1990), and primers Bt1a/Bt1b were used to amplify the β-tubulin (TUB) gene of rDNA (Glass and Donaldson 1995). The amplified polymerase chain reaction product was sequenced and deposited in GenBank as ITS sequences (accession nos. MH553296, MH553297, MH716003, and MH716004) and TUB sequences (MH766891 and MH766892). BLAST analysis of ITS and TUB gene revealed 99% similarity (accession no. MG198619) and 100% similarity (accession no. KM396706) with A. alternata, respectively. Olive cultivar Carolea was grown in peat moss, and distilled water was applied twice a week. The pathogenicity tests were performed on fruits and leaves of 2-month-old plants. Five randomly selected plants were spray inoculated with known spore suspensions (10⁵ conidia/ml) and covered with plastic bags. Control plants were sprayed with sterile deionized water. Inoculated and control plants were kept in separate growth chambers at 25°C and 90% relative humidity. The inoculation experiment was performed three times. The symptoms appeared on fruits after 5 days and commenced as pinpoint, globose, and light to dark brown necrotic spots. Similar symptoms could be observed after 8 days of inoculation on leaves. The symptoms were identical to those observed in the orchards. The control fruits and leaves showed no symptoms. Koch’s postulates were confirmed by the reisolation of the pathogen from inoculated fruits and leaves 10 days postinoculation. By using the abovementioned morphological and microscopic analyses, fungus morphology was confirmed to be similar with inoculated A. alternata isolates. Owing to rapid spread and lack of awareness, this disease can become a disaster to actively increasing olive oil production in Pakistan. To our knowledge, this is the first report of olive leaf and fruit spot caused by A. alternata in Pakistan.