Plant Pathol. J. 1-11 (2017) https://doi.org/10.5423/PPJ.OA.04.2017.0081 The Plant Pathology Journal pISSN 1598-2254 eISSN 2093-9280 ©The Korean Society of Plant Pathology Research Article Open Access The Incidence of Alternaria Species Associated with Infected Sesamum indicum L. Seeds from Fields of the Punjab, Pakistan Brian Gagosh Nayyar1*, Steve Woodward2, Luis A. J. Mur3, Abida Akram1, Muhammad Arshad1, S. M. Saqlan Naqvi4, and Shaista Akhund1 1Department of Botany, Pir Mehr Ali Shah Arid Agriculture University, Rawalpindi 46300, Pakistan 2Institute of Biological and Environmental Sciences, University of Aberdeen, Cruikshank Building, St. Machar Drive, Aberdeen AB24 3UU, Scotland, UK 3Institute of Biological, Rural and Environmental Sciences, Aberystwyth University, Edward Llwyd Building, Penglais Campus, Aberystwyth SY23 3DA, Wales, UK 4Department of Biochemistry, Pir Mehr Ali Shah Arid Agriculture University, Rawalpindi 46300, Pakistan (Received on April 10, 2017; Revised on July 9, 2017; Accepted on July 23, 2017) Sesame (Sesamum indicum) is an important oil seed (KP123850.1) in GenBank accessions. The pathogenic- crop of Asia. Yields can be negatively impacted by vari- ity and virulence of these isolates of Alternaria alternata ous factors, including disease, particularly those caused was confirmed in inoculations of sesame plants result- by fungi which create problems in both production and ing in typical symptoms of leaf blight disease. This storage. Foliar diseases of sesame such as Alternaria leaf work confirms the identity of a major source of sesame blight may cause significant yield losses, with reductions leaf blight in Pakistan which will aid in formulating ef- in plant health and seed quality. The work reported fective disease management strategies. here determined the incidence of Alternaria species in- fecting sesame seeds grown in the Punjab, Pakistan. A Keywords : Alternaria, Alt a 1 gene, ITS, Punjab, sesame total of 428 Alternaria isolates were obtained from 105 seeds seed samples and grouped into 36 distinct taxonomic groups based on growth pattern and morphological Handling Associate Editor : Lee, Jungkwan characters. Isolation frequency and relative density of surface sterilized and non-surface sterilized seeds Sesame (Sesamum indicum L.) is one of the oldest and showed that three isolates (A13, A47 and A215) were most important oilseed crops, mainly grown for its oil and the most common morphological groups present. These protein content (Johnson et al., 1979). This crop is an im- isolates were further identified using sequencing of portant source of fats, proteins, vitamins and minerals in the Internal Transcribed Spacer (ITS) region of ribo- the food of rural people, particularly children, throughout somal DNA (rDNA) and the Alternaria major allergen the world (Shahidi et al., 2006). Sesame seed contains oil gene (Alt a 1). Whilst ITS of rDNA did not resolve the (48 to 60%), protein (18 to 23.5%), carbohydrate (13.5%), isolates into Alternaria species, the Alt a 1 sequences and ash (5.3%), with a moisture content of approximately exhibited > 99% homology with Alternaria alternata 5.2% (Obiajunwa et al., 2005; Kahyaoglu and Kaya, 2006). Sesame oil contains the antioxidants sesamoline, sesamin *Corresponding author. and sesamol (Pastorello et al., 2001). Sesame oil is used Phone) +92-334-809-8095, FAX) +92-51-9290160 in cuisine for salad dressings and the manufacturing of E-mail) [email protected] margarine, and is a raw ingredient in industry for making This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// paints, varnishes, soaps, perfumes, insecticides, and phar- creativecommons.org/licenses/by-nc/4.0) which permits unrestricted maceuticals as a vehicle for drug delivery (Grubben and noncommercial use, distribution, and reproduction in any medium, Denton, 2004). provided the original work is properly cited. Cultivation of sesame probably originated in the Harappa Articles can be freely viewed online at www.ppjonline.org. Valley region of the Indian subcontinent as long as ago as 2 Nayyar et al. pre-3000 BC (Ashri, 2007). Currently, sesame is grown The aim of the work described in this paper was to de- commercially in 76 countries in the world but production is termine the Alternaria species associated with sesame in mostly dominated by Asian and African countries (Ashri, the Punjab, Pakistan, as an example of the source of yield 1998). India, Sudan and China are the leading producers of losses affecting production of this important crop in South sesame, followed by Myanmar, Nigeria, Ethiopia, Uganda, Asia. To the best of our knowledge, no such studies have Mexico, Pakistan, Bangladesh, Thailand, Turkey (FAO, been carried out to date in the Punjab. This basic work on 2014). In Pakistan, sesame is grown in all four provinces, testing seeds for the presence of seed-borne pathogens is an but 89% of production is in the Punjab, which is considered important step in the development of disease management a major sesame producing area in South Asia (The Finan- strategies for this crop. cial Daily, 2017). Numerous microorganisms, especially fungi, pose a chal- Materials and Methods lenge to both sesame production and seed storage. Sesame agriculture faces numerous disease problems, including Plant Material and Fungal Isolation. One hundred and vascular wilt, root rot, leaf blight, leaf spot and damping off five seed samples of sesame were collected from the ma- in young seedlings (Farhan et al., 2010). Previous reports jor sesame growing areas of the Punjab, Pakistan (Fig. have shown the presence of Aspergillus and Fusarium 1). Seeds for isolating fungi were selected randomly from in sesame seeds (Mbah and Akueshi, 2000, 2001). How- within a batch and 50% of seeds were surface sterilized in ever, sesame is also susceptible to leaf blight caused by 5% NaOCl for 2 mins. Surface sterilized and unsterilized Alternaria sesami (Kolte, 1985), which is also seed-borne seeds were placed at the rate of 25 seeds into 90 mm diam- (Richardson, 1979). Leaf diseases of sesame, such as blight eter Petri dishes with three layers of well-moistened filter diseases caused by Alternaria spp. result in substantial loss paper discs (Whatman™ 1001-090 Grade 1). Petri dishes in yield and deterioration in quality and vigor of seed (Eni- were incubated at 22 ± 2ºC for seven days with an alternate kuomehin et al., 2002). Of particular economic importance cycle of light and darkness (12 h each) in a Versatile Envi- is leaf blight caused by Alternaria sesami and A. alternata ronmental Test Chamber (Sanyo, Japan); illumination was (Chohan, 1978), which also causes seed rot and pre- and provided by 55W fluorescent lights (Sylvania, Germany) post-emergence damping-off, as well as infecting all aerial giving a light intensity of 125-130 µmol m-2 s-1. The experi- plant parts, resulting in considerable losses in yield, both ment was performed in triplicate. After incubation, emerg- qualitative and quantitative (Naik et al., 2004). Alternaria ing fungal colonies were counted and isolated onto potato leaf spot of sesame has been recognized as a major biotic dextrose agar (PDA, Oxoid, UK) amended with 100 mg L-1 pressure of single origin limiting yields (Lubaina and streptomycin (Singleton et al., 1993). Isolates were main- Murugan, 2013). Symptoms of Alternaria leaf blight in- tained on PDA and identified on the basis of morphological clude the formation of round to irregular spots of up to 10 characters (Ellis, 1971, 1976; Simmons, 2007). mm diameter. Individual spots may coalesce to form large Isolation frequency (Fr) and relative density (RD) of necrotic patches, resulting in premature abscission (Rao fungi in and on seeds were calculated as follows: and Vijayalakshmi, 2000). Alternaria species are dispersed from region to region through various pathways which in- clude air-borne conidia and adherence of soil to seedlings, farm equipment, or animals (Ojiambo, 1997). Where, ns is the number of samples on which a fungus Alternaria species produce non-host specific (e.g., tenu- occurred; N is the total number of seeds sampled; ni is the azonic acid (TeA), alternariol (AOH), alternariol mono- number of isolates of a fungal genus/species, and Ni is the methyl ether (AME), brefeldin A, tentoxin, zinniol) (Saha total number of fungal isolates obtained. et al., 2012) as well as host-specific toxins (Thomma, 2003) which contaminate the product. Consumption of foodstuff Fungal DNA Extraction, PCR amplification, and Se- contaminated with Alternaria toxins has been implicated quencing. Genomic DNA of representative isolates of the in elevated incidence of esophageal carcinoma in humans three most frequent groups of Alternaria (A13, A47, and (Schrader et al., 2001). The wide distribution, high variabil- A215) was extracted by scraping the surface of 14 days ity and influence of Alternaria spp. on crops and humans old cultures grown on PDA. DNA was extracted from 50 warrants accurate identification of the causal agents to for- mg mycelium per isolate using “Plant DNeasy Mini Kit” mulate effective control and management strategies (Hong (Qiagen, UK) by homogenizing in liquid nitrogen and et al., 2005b). following the manufacturer’s protocol. DNA concentra- Alternaria spp. Infecting Sesame Seeds 3 Fig. 1. (A) Sampling sites in the Punjab, Pakistan. (B) Typical leaf blight symptoms on sesame leaves in the field and (C) pods (arrowed) during the sampling in the Punjab,