Publications by authors named "Muhammad Waqar Alam"

5 Publications

  • Page 1 of 1

First Record of Chaetomium globosum Causing Leaf Spot of Pomegranate in Pakistan.

Plant Dis 2021 Mar 31. Epub 2021 Mar 31.

Institute of Molecular Biology and Biotechnology, Lahore, Pakistan;

Pomegranate (Punica granatum L.) is a non-climacteric and a favorite fruit of tropical, sub-tropical and arid regions of the world. During a survey in autumn 2019, leaf lesions were observed on plants (cv. Kandhari) in different orchards of Muzaffargarh (30°4'27.7572″ N, 71°11'4.7544″ E), a major pomegranate-producing region in Punjab Province. Disease incidence ranged from 17 to 20%. Leaf lesions were initially small (1 to 3 mm in diameter), round, purple or reddish-brown, scattered spots. At later stages, spots increased in size and the centers of mature lesions became dark red or black with fungal sporulation. To isolate the pathogen, samples of leaf (5 × 5 mm) were cut from the junction of diseased and healthy tissue, surface disinfected in 75% alcohol for 30 s, sterilized with 6% sodium hypochlorite for 3 min, washed with sterile distilled water three times, air dried in laminar flow hood, and cultured on potato dextrose agar (PDA). After one week of incubation at 25 ± 2°C with a 12-h photoperiod, fungal colonies developed, which were initially white and became pale yellow with olivaceous green mycelium after 20 days. On PDA, ascomata were olivaceous green, with a papillate ostiole, globose or ovoidal to obovoidal (155 to 220 × 120 to 240 µm, n=50). Terminal and lateral setae were abundant, brown, and tapering toward the tips (4 to 6 µm, n=50). Asci were greenish and lemon-shaped (6 to 8 × 9 to 13.5 µm, n=50). Ascospores were limoniform and olivaceous gray-brown (10 to 11.5 × 7 to 9 µm, n=50). These morphological characteristics were consistent with the morphology of Chaetomium globosum (Lan et al. 2011; Wang et al. 2016). Genomic DNA was extracted from two isolates and identification of the pathogen was confirmed by amplification and sequencing of the internal transcribed spacer region (ITS) and the partial translation elongation factor 1-α (TEF1) gene using ITS1/ITS4 (White et al. 1999) and EF1-983F/EF1-2218R primers (Wang et al. 2016), respectively. The sequences of the PCR products were deposited in GenBank with accession numbers MW522514, MW522352 (ITS), and MW530423, MW530424 (TEF1). BLAST results of the obtained sequences of the ITS and TEF1 genes revealed 100% (513/513 bp) and 99.78% (927/929 bp) similarity with those of C. globosum in GenBank (ITS: KX834823 and KT898637, and TEF1: MG812564 and KC485028). To confirm pathogenicity, inoculum was prepared by harvesting conidia from 10-day-old culture grown in PDA. The surface-disinfected (70% ethyl alcohol, 30 s) leaves of ten 1-year-old seedlings (cv. Kandhari) were sprayed with a spore suspension (1×106 conidia/ml). Leaves of ten seedlings sprayed with sterile distilled water served as controls. All seedlings were covered with plastic bags and placed in a greenhouse at 26°C with 12 h photoperiod. After eight days, symptoms on inoculated leaves were similar to those observed in the orchards; no symptoms were observed on controls. The fungus was reisolated from all symptomatic tissues. C. globosum has been reported on Punica granatum (Guo et al. 2015), Cannabis sativa (Chaffin et al. 2020) and Brassica oleracea (Zhu et al. 2020). This is the first report of C. globosum causing leaf spot on pomegranate in Pakistan. This finding suggests a potential threat to pomegranate production in Pakistan and further studies should focus on effective prevention and control practices of this disease.
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http://dx.doi.org/10.1094/PDIS-01-21-0200-PDNDOI Listing
March 2021

First Record of Colletotrichum gloeosporioides Causing Anthracnose of Banana in Pakistan.

Plant Dis 2021 Mar 17. Epub 2021 Mar 17.

Institute of Molecular Biology and Biotechnology, Lahore, Pakistan;

Banana (Musa spp.) is one of the most widely grown and consumed fruits in Pakistan and all around the world due to their distinct aroma and taste. In 2018, anthracnose symptoms were observed on banana fruit harvested from different plantations of Sindh- a major banana producing Province of Pakistan. Approximately, 25% of banana fruit collected from different plantations were infected. The symptoms consisted of small brown to reddish-brown spots on the fruit surface and then became sunken lesions as the disease progressed. To identify the pathogen, infected tissues (5 mm in diameter) from the margin of the lesions were surface sterilized by dipping in 1% sodium hypochlorite (NaOCl) for 2 min, 70% ethanol for 30 s, and then rinsed twice with sterile distilled water, plated onto potato dextrose agar (PDA), and incubated at 27°C for 5 days with 12 h light and darkness cycle. Colonies with a similar pattern were consistently isolated and all colonies were sub-cultured using the single-spore method. Colonies first appeared with white colored mycelium and later turned to dark gray. Conidia produced in acervuli were cylindric, hyaline, straight, and aseptate, with both ends rounded. Conidia measured 14.0 ± 0.5 × 3.4 ± 0.6 μm. Conidiomata were dark brown and spherical. On the basis of morphological characterization, the pathogen was identified as Colletotrichum gloeosporioides (Penz.) Penz. & Sacc. (Weir et al. 2012). Two independent isolates (PDL2031 and PDL2032) were used for further genetic analysis. The internal transcribed spacer (ITS) region and chitin synthase 1 (CHS-1) gene were amplified from genomic DNA using primer pairs of ITS1/ITS4 and CHS-79F/CHS-345R, respectively (White et al. 1990; Damm et al. 2012). The GenBank accession numbers (MW493198, MW504711 for ITS and MW530421, MW530422 for CHS-1) of the sequences exhibited 99% to 100% identity to multiple sequences of C. gloeosporioides. To conduct a pathogenicity test, 10 healthy fruits were selected and surface sterilized with 70% ethanol followed by a wash of sterilized water. The fruits were stabbed with a sterile needle and a drop of 20 µl of spore suspension (106 spores/ml) was placed on each wound independently. Meanwhile 10 fruits inoculated with sterile water were treated as controls. The fruits were incubated at 27°C with 90% relative humidity for 10 days. Inoculated fruits exhibited symptoms similar to the original infection. No visible lesions appeared on control fruit. C. gloeosporioides was successfully reisolated from the inoculated fruit, confirming Koch's postulates. Anthracnose of banana is known to be caused by C. musae, C. gloeosporioides, C. siamense, C. tropicale, C. chrysophilum, C. theobromicola, and C. scovillei (Kumar et al. 2017; Peres et al. 2001; Vieira et al. 2017; Zakaria et al. 2009; Zhou et al. 2017). To our knowledge, this is first report of anthracnose of banana caused by C. gloeosporioides in Pakistan. The new disease primarily reduces the quality and yield of Banana. Effective measures should be taken to manage this disease.
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http://dx.doi.org/10.1094/PDIS-01-21-0215-PDNDOI Listing
March 2021

First Report of Lasiodiplodia pseudotheobromae Causing Stem End Rot of Mango Fruit in Pakistan.

Plant Dis 2021 Mar 3. Epub 2021 Mar 3.

Plant Pathology, University of Agriculture, Faisalabad, Punjab, Pakistan;

Mango (Mangifera indica L.) is considered a desirable fruit in international markets and is grown throughout tropical and sub-tropical countries around the world (Alemu, 2014). Stem end rot is the most damaging and complex postharvest disease of mango, resulting in losses of up to 40% in Pakistan, which is the leading producer and exporter (Alam et al. 2017). A field survey was conducted in June of 2017 and 2018 in the Rahim Yar Khan and Multan- major mango producing regions of Punjab Province. After mature but unripe mango fruit (cv. Samar Bahisht Chaunsa) were stored at 12°C for 2 weeks to permit ripening, water-soaked, dark brown to purplish black decay began to appear around the stem end portion. The decay gradually enlarged and covered the whole fruit after 7 days. Disease incidence was estimated at 30%. Small pieces (3 to 4 mm2) from the periphery of 15 diseased fruit were surface disinfected with 1% sodium hypochlorite for 2 min, rinsed three times in sterilized distilled water, air dried, and then placed aseptically onto potato dextrose agar (PDA) medium and incubated at 25°C under a 12-h light/dark photoperiod for 7 days. Twelve single-spore isolates with similar morphology were isolated from the infected tissues. Initially the fungus produced thick, fluffy and greyish-white aerial mycelium, that later turned into dark gray colonies. Conidia were unicellular, ellipsoidal, and initially hyaline, but with age became dark brown and developed a central septum. Conidia measured 24.5 to 31.5 × 11.4 to 15.7 µm (n = 60). Conidiophores were inflated at their base with one diaphragm which reduced to conidiogenous cells. Conidiogenous cells were hyaline and cylindrical. On the basis of morphological characteristics, the fungus was tentatively identified as Lasiodiplodia sp., a member of the family Botryosphaeriaceae (Alves et al. 2008). For molecular identification, genomic DNA was extracted from mycelium following the CTAB method. The internal transcribed spacer (ITS) region of rDNA and translation elongation factor 1-alpha (TEF1-α) gene were amplified using ITS1/ITS4 (White et al. 1990) and EF1-728F/EF1-986R primer sets (Carbone and Kohn 1999), respectively. BLASTn searches of sequences revealed 99% to 100% identity with the reference sequences of various Lasiodiplodia pseudotheobromae isolates (GenBank accession nos. MH057189 for ITS; MN638768 for TEF-1a). The sequences were deposited in GenBank (accession nos. MW439318, MW433883 for ITS; and MW463346, MW463347 for TEF-1a). To fulfill Koch's postulates, a suspension of 105 conidia/ml from a 7-day-old culture of L. pseudotheobromae was used to inoculate fully mature but unripe mango fruit (cv. Samar Bahisht Chaunsa). Fruit were pricked with a sterilized needle to a depth of 4 mm at the stem end portion, injected with 50 μl of the prepared spore suspension (Awa et al. 2012), and stored at 12°C for 3 weeks under 70 to 80% RH. Twenty mango fruit were inoculated, and 10 were inoculated with sterile water only. After 15 days, most fruit showed typical symptoms at the stem end. Reisolations from symptomatic fruit following the procedures described above for isolating and identifying the fungal cultures from infected field samples, consistently yielded a fungus identical to L. pseudotheobromae. Control fruit remained disease-free. Although L. pseudotheobromae was previously reported on several forest and fruit trees (Alves et al. 2008; Awan et al. 2016), this is the first report of the pathogen causing stem end rot disease of mango in Pakistan. This report is important for the new studies aiming at management of stem end rot disease of mango caused by L. pseudotheobromae in Pakistan.
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http://dx.doi.org/10.1094/PDIS-01-21-0099-PDNDOI Listing
March 2021

First report of Alternaria alternata causing fruit rot on Fig (Ficus carica) in Pakistan.

Plant Dis 2021 Feb 22. Epub 2021 Feb 22.

University of Agriculture, Faisalabad, Plant Breeding and Genetics, Faisalabad, Pakistan;

Fig (Ficus carica L.) is among the earliest and widely cultivated fruit trees in the world due to its easy adaptation to diverse climates (Solomons et al. 2006). In July 2020, a rot disease was observed on multiple orchards located in Faisalabad- a region of Punjab Province. The symptoms appeared as light brown, circular to oval, and water-soaked lesions (4-8 mm in diameter). In more advanced stages of the disease, the lesions enlarged in size and leading to rot of the entire fruit. Disease incidence on fruit across the fields ranged from 23 to 29%. To isolate the causal agent, segments (5 mm2) were excised from 15 symptomatic fruit, surface disinfested with 70% ethanol for 1 min, washed in three changes of sterilized water, air dried, transferred aseptically to plates containing potato dextrose agar (PDA), and incubated at 25°C for 7 days with a 12-h photoperiod. Nine single spore isolates with similar morphology were isolated from the infected tissues. The cultured isolates consistently yielded dark brown to black colonies on PDA. Conidia were in chains (average conidial dimension 20 to 28 × 8 to 10 μm), olivaceous to dark brown, with a short conical beak with both transversal (two to five) and longitudinal (one to three) septa. Conidiophores were short, septate, hyaline to olivaceous brown, either branched or unbranched, 20 to 52 μm long, and 1 to 3 μm wide. These cultural and morphological characteristics were consistent with the descriptions of Alternaria alternata (Simmons 2007). The genomic DNA from three isolates was extracted using a PrepMan Ultra kit according to the manufacturer's protocol and amplified using universal primers ITS1/4 (White et al. 1990) and the endopolygalacturonase gene using primers PG3/PG2b (Andrew et al. 2009), and sequenced. The amplified PCR products were deposited in GenBank (accession nos. MW261786, MW433689, MW439319 for ITS and MW249057, MW463344, MW463345 for PG3/PG2b). Blast searches against GenBank showed 99%-100% nucleotide identity with the reference sequences of various A. alternata isolates. The pathogenicity of the representative isolate (PDL 2021) was tested on Fig fruit cv. "Black Mission". For that, 20 asymptomatic and mature fruit were surface-disinfected with 75% ethanol solution for 30 s. The fruit were inoculated by spraying a spore suspension (106 spores/ml) of A. alternata and stored at 25°C and 80% relative humidity. An equal number of fruit inoculated with sterile water were used as negative controls. Symptoms similar to those on the naturally infected fruits began after 4-5 days of inoculation. The negative controls remained healthy. Koch's postulates were fulfilled by reisolating (100%) A. alternata from only the inoculated fruit. Previously, the pathogen has been reported to cause fruit rot of Lychee, Peach and Pomegranate in Pakistan (Alam et al 2017a; 2019b; 2019c). The pathogen has been reported to cause fig fruit rot in California (Michailides et al. 1994). Keeping in view the extent of disease on many fruits, further studies are needed on management options to combat the disease in Punjab Province of Pakistan.
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http://dx.doi.org/10.1094/PDIS-01-21-0090-PDNDOI Listing
February 2021

Antifungal exploitation of fungicides against Fusarium oxysporum f. sp. capsici causing Fusarium wilt of chilli pepper in Pakistan.

Environ Sci Pollut Res Int 2018 Mar 20;25(7):6797-6801. Epub 2017 Dec 20.

Department of Plant Pathology, College of Agriculture, BZU Bahadur Sub Campus Layyah, Layyah, Pakistan.

The research was conducted to evaluate in-vitro efficacy of numerous fungicides against Fusarium oxysporum f. sp. capsici. In present research, six treatments (T) viz. Carbendazim, Benomyl, Topsin-M, Difenoconazole, Nativo, and Alliete along with control, various concentrations (C), days (D), and their interactions, i.e., (T × C), (T × D), (C × D), and (T × C × D) were exploited in a laboratory through food poison technique. Alliete expressed maximum colony growth (1.93 cm) as compared to all other fungicides with respect to control. Interaction between treatments and concentration (T × C) exhibited maximum colony growth of all treatments (Carbendazim, Benomyl, Topsin-M, Difenoconazole, Nativo, and Alliete), i.e., 0.87, 1.23, 1.73, 2.20, 2.53, and 2.93 cm at 300 ppm as compared to 500 and 700 ppm concentrations, respectively. Similar trend was also observed concerning interaction between (fungicides × days) and (tested concentrations × days). Results of the present study revealed that among tested fungicides, Carbendazim at 700 ppm expressed significant reduction in fungal growth.
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http://dx.doi.org/10.1007/s11356-017-1032-9DOI Listing
March 2018