Eggplant (Solanum melongena L.) is a popular vegetable that is grown in both tropical and subtropical regions all year long. The crop is cultivated on small family farms and is a good source of income for resource-limited farmers in Pakistan. In early May 2019, leaf spots on eggplant (cv. Bemisaal) were observed in an experimental field (31°26'14.0"N 73°04'23.4"E) at the University of Agriculture, Faisalabad, Pakistan. Early symptoms were small, circular, brown, necrotic spots uniformly distributed on leaves. The spots gradually enlarged and coalesced into large, nearly circular or irregularly shaped spots that could be up to 3 cm in length. The center of the spots was light tan, surrounded by a dark brown ring, a chlorotic halo, and tended to split in the later developmental stages. Disease incidence was approximately 35% in the infected field. The causal agent of this disease was isolated consistently by plating surface sterilized (1% NaOCl) sections of symptomatic leaf tissue onto potato dextrose agar (PDA). After 6 days incubation at 25°C with a 12-h photoperiod, fungal colonies had round margins and the cottony mycelia were dark olivaceous with a mean diameter of 7.5 cm. For conidial production, the fungus was grown on potato carrot agar (PCA) and V8 agar media under a 16-h/8-h light/ dark photoperiod at 25°C. Conidiophores were septate, light to olive golden brown with a conidial scar, from which conidia were produced. Conidia were borne singly or in short chains and were obpyriform to obclavate, measured 29 ± 4.8 × 13.25 ± 2.78 μm (n=30) with zero to three longitudinal and two to six transversal septa. The morphological characters matched those of Alternaria alternata (Fr.) Keisel (Simmons et al. 2007). DNA was extracted using the DNAzol reagent (Thermo Fisher Scientific MA, USA). For molecular identification, internal transcribed spacer (ITS) region between ITS1 and ITS2, actin gene (β-Actin), translation elongation factor (TEF-1α) gene, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene of two representative isolates (JLUAF1 and JLUAF2) were amplified with primers ITS1/ITS4 (White et al. 1990), β-Actin 512 F/783 R, EF1-728F/-986R (Carbone et al. 1999), and gpd1/gpd2 (Berbee et al. 1999), respectively. The sequences were deposited in GenBank (accession nos. MT228734.1 and MT228735.1 for ITS; MT260151.1 and MT260152.1 for β-Actin, MT260163.1 and MT260164.1, for TEF-1a, and MT260157.1 and MT260158.1 for GAPDH). BLASTn analysis of these sequences showed 100% identity with the sequences of A. alternata for ITS rDNA, β-Actin, TEF-1α, and GAPDH, respectively. Based on the morphological characters and DNA sequences, the leaf spot isolates of eggplant were identified as A. alternata. To confirm the pathogenicity on eggplant, six-week old healthy potted eggplants of cv. Bemisaal were sprayed at the true leaf stage with conidial suspensions of A. alternata (106 conidia/ml; obtained from 1-week-old cultures) amended with 0.1% (vol/vol) of Tween 20 until runoff (1.5 to 2 ml per plant) using an atomizer in the greenhouse. Three plants were inoculated with each of the two isolates (JLUAF1 and JLUAF2), whereas three control plants were sprayed with sterile distilled water amended with 0.1% Tween 20. The plants were incubated at 25 ± 2°C in a greenhouse, and the experiment was conducted twice. After 10 days of inoculation, each isolate induced leaf spots which were similar to typical spots observed in the field, whereas the control plants remained symptomless. The fungus was re-isolated from symptomatic tissues. Re-isolated fungal cultures were morphologically and molecularly identical to A. alternata, thus fulfilling Koch’s postulates. Previously, A. alternata has been reported to cause leaf spots on eggplant in India (Raina et al. 2018). To our knowledge, this is the first report of A. alternata causing leaf spot on eggplant in Pakistan. The disease could represent a threat for eggplant crops due to its increasing cultivation. It is important to develop disease management strategies for Alternaria alternata causing leaf spot of Eggplant in Pakistan.
Citrus reticulata cv. ‘Kinnow’ mandarin is the most popular and widely grown fruit crop in Pakistan. During 2017, a survey was conducted to the local citrus fruit markets of Faisalabad, Pakistan. Citrus fruits (n=50) exhibiting stem end rot and fruit rot were collected with 15% disease incidence. The stem end region showed light to dark brown lesions and white fungal growth was also observed in the severely infected fruit. Infected fruit were excised into 2mm2 segments, surface disinfected with 1% NaClO, rinsed with sterilized water and dried. Later, these tissues were placed on potato dextrose agar (PDA) medium and subsequently incubated at 25 °C. Purified isolates produced white colonies with beige pigmentation. The frequency of fungal isolation was 47%. Microscopic observations revealed that macroconidia (n=50) had 5 to 6 septations, with a prominent dorsiventral curvature, tapered and elongated apical cell, and a foot shape basal cell. The macroconidia were measuring 22 to 45 × 2.9 to 4.3 µm with an average of 31 × 3.6 µm. However, microconidia were not observed. Chlamydospores were globose, intercalary, solitary, or in pairs, appearing in chains (Leslie and Summerell 2006). For molecular identification, DNA was extracted from all isolates. The internal transcribed spacer region (ITS) ITS1/4 (White et al. 1990), translation elongation factor-1 alpha (TEF) EF1/2 (O’Donnell et al. 1998), and RNA polymerase II subunit 1 (RPB1) (O'Donnell et al. 2013) were amplified using PCR and the product was subsequently sequenced. Based on BLAST analysis, the isolate was identified as Fusarium equiseti (FUS-21). The sequences of the representative isolate FUS-21 were deposited in the GenBank with accession numbers (ITS, MH581300), (TEF, MK203749), and (RPB1, MW596599) showing more than 99% similarity with ITS accession GQ505683, TEF accession GQ505594, and 100% to RPB1 accession JX171481. To determine the pathogenicity, 40 healthy surface disinfested citrus fruit were taken. The fruit were inoculated by creating artificial wounds on the surface with a sterilized needle and 10 μL of 105 spores/mL was deposited in the wounds. In case of control fruit were inoculated with 10 μL sterilized distilled water only, and incubated at 25 °C. All fruit inoculated with the putative pathogen, developed symptoms like the original fruit from which they were isolated. The pathogenicity test was repeated twice. Visible white mycelium appeared at the stem end region and the fruits became dried as the infection progressed. However, the control fruit remained asymptomatic. The pathogen was re-isolated from infected fruit and identified based on morphometric and molecular analysis. Previously we have reported F. oxysporum causing citrus fruit rot in Pakistan (Moosa et al. 2020). This is the first report of F. equiseti causing post-harvest rot of citrus fruits in Pakistan. Kinnow is an important fruit crop of Pakistan with huge export value the management of Fusarium rot is quite important to save the loss of fresh produce.
Ear rots occur globally in maize causing reduction in yield and quality of grains. In recent years, Pakistan is facing an increased mycotoxin contamination in maize caused mainly by Aspergillus spp. Information on the occurrence and precise identification of Aspergillus spp. in maize is an important step to mitigate the impact of mycotoxins and enhance food safety in Pakistan. During 2016, harvested maize cobs of fifteen varieties were obtained from the Department of Plant Breeding and Genetics, University of Agriculture, Faisalabad, Pakistan. To isolate the pathogen, 30 cob’s grains from each cultivar were collected, surface sterilized, placed onto potato dextrose agar (PDA) in Petri plates, and the plates were incubated at 26°C. Cultural and morphological characteristics studies showed that 10–15% samples of each genotype were infected by Aspergillus niger. Fungal colonies were white first and then covered with a dense layer of conidiophores and conidia. The conidial heads were radiate, vesicles and covered with irregular metulae and phialides. Conidia were globose or subglobose (3 to 4.8 μm in diameter), dark brown to black, with rough cell walls. The morpho-cultural characters were in compliance with the description of A. niger (1). For molecular identification, genomic DNA of four representative isolates were extracted using DNAzol reagent (Thermo Fisher Scientific MA, USA). PCR amplification and sequencing of the internal transcribed spacer (ITS)-rDNA, β-tubulin (BenA), partial RNA polymerase II largest subunit (RPB2) and translation elongation factor-1 alpha (TEF-1α) gene regions were performed. Primers were ITS1/ITS4 (2), Bt2a/Bt2b (3), RPB2-5F/RPB2-7cR (4), A-EF_F/A-EF_R (5), respectively. The obtained sequences were deposited in GenBank with accession numbers MN922042.1, MN922043.1, MN922044.1 and MN922045.1 for ITS-rDNA; MT117920.1, MT117921.1, MT117922.1, MT117923.1 for the BenA; MT318284.1, MT318285.1, MT318286.1, MT318287.1 for RPB2 and MT318307.1, MT318308.1, MT318309.1, MT318310.1 for TEF-1α. BLASTn analysis showed 99% identity with submitted sequence of A. niger for ITS rDNA, BenA, RBP2 and TEF-1α. To confirm the Koch's postulates Maize cultivars were grown in the research field of the Department of Plant Pathology, Univ. of Agriculture, Faisalabad, Pakistan. For inoculation, a spore suspension was prepared by adding distilled water to Petri plate of A. niger culture to dissolve and harvested the spore. So, the concentration of the spore inoculum used was determined with haemocytometer and adjusted to 1 x 106/spores/ml in sterile 0.01% Triton X-100. Using 10 ml syringe the top ears of maize were inoculated 30-35°C with A. niger at milk growth stage by using 0.5-1 ml of inoculum per ear. After 14 days, disease symptoms similar to those observed in the field appeared in all inoculated but not in uninoculated ears of maize. A. niger had colonized the ears as evidenced by visible sporulation on wounded kernels. These tests were repeated twice in the field, and Koch's postulates were satisfied after re-isolating the A. niger from the inoculated maize. A. niger contamination in maize was previously reported in USA (6) however, to the best of our knowledge this is the first report of A. niger causing pre-harvest ear rot of maize in Pakistan. This finding will help to plan effective disease management strategies against pre-harvest ear rot of maize in Pakistan.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.