Publications by authors named "Nasir Ahmed Rajput"

10 Publications

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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

Eremophilane Sesquiterpenes and Benzene Derivatives from the Endophyte Microdiplodia sp. WGHS5.

Chem Biodivers 2021 Apr 19;18(4):e2000949. Epub 2021 Mar 19.

College of Plant Protection, State and Local Joint Engineering Research Center of Green Pesticide Invention and Application, Nanjing Agricultural University, Nanjing, 210095, P. R. China.

Three new eremophilane sesquiterpenes phomadecalins G-I (1-3) and two new benzene derivatives microdiplzenes A and B (12 and 13), together with nine known eremophilane sesquiterpenes (4-11 and 14) were isolated from an endophytic fungus, Microdiplodia sp. WGHS5. Their structures were elucidated by the interpretation of HR-ESI-MS and NMR data; meanwhile, the absolute configurations of new compounds were determined on the base of ECD calculations. All compounds were evaluated for the antimicrobial activities and antiproliferative effect on human gastric cancer cell lines (BGC-823).
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http://dx.doi.org/10.1002/cbdv.202000949DOI Listing
April 2021

Species Diversity of and Causing Potato Blackleg Disease in Pakistan.

Plant Dis 2020 May 8;104(5):1492-1499. Epub 2020 Mar 8.

Université Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC), 91198, Gif-sur-Yvette, France.

Potato blackleg is caused by a diverse species of pectinolytic bacteria. In Pakistan, approximately 90% of the pathogens involved belong to Survey (2014 to 2017), sampling, and isolation from different potato growing areas of Punjab, Pakistan depicted an overall disease incidence of approximately 15%. Thirty-six pectinolytic strains confirmed through biochemical and pathogenicity testing were characterized via gene to identify them at the species level. To further validate the identification, one strain from each species SS26 (), SS28 (), SS70 (), SS90 (), SS95 (), and SS96 () were selected for draft genome sequencing and multilocus sequence analysis of 13 housekeeping genes (, , , , , , , , , , , , and ). Phylogenetic analysis revealed considerable genetic diversity in the genus . DNA-DNA hybridization and average nucleotide identity values of the strains selected for genome sequencing were determined with other reference and strains. Moreover, all six representative strains were also phenotypically characterized on the basis of metabolism of different carbon sources. Overall, on the basis of genotypic and phenotypic characteristics, these 36 isolates were grouped into six species: , , , , , and .
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http://dx.doi.org/10.1094/PDIS-08-19-1743-REDOI Listing
May 2020

Nematicidal metabolites from endophytic fungus Chaetomium globosum YSC5.

FEMS Microbiol Lett 2019 07;366(14)

College of Plant Protection, State & Local Joint Engineering Research Center of Green Pesticide Invention and Application, Nanjing Agricultural University, Nanjing 210095, P. R. China.

Management of nematodes is a very hectic job due to a highly diverse group of organisms. To find lead compounds for new nematicide development, five metabolites (1-5) were isolated from the culture broth of Chaetomium globosum YSC5 and tested for nematicidal activities against the second stage juveniles (J2s) of Meloidogyne javanica. The results revealed that chaetoglobosin A (1), chaetoglobosin B (2) and flavipin (3) exhibited strong adverse effects (91.6, 83.8 and 87.4%, respectively) on J2 mortality at 200 μg/mL with LC50 values of 88.4, 107.7 and 99.2 μg/mL after 72 h, respectively, while 3-methoxyepicoccone (4) and 4,5,6-trihydroxy-7-methylphthalide (5) showed moderate effects (78.0 and 75.5%, respectively) with LC50 values of 124.0 and 131.6 μg/mL, respectively. Furthermore, in pot assay compounds 1 and 2 appeared to be promising metabolites at 200 μg/mL that significantly reduced nematode reproduction and showed a positive influence on plant growth. Our findings could be helpful for development of new potential bio-based pesticides for integrated management of plant-parasitic nematode.
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http://dx.doi.org/10.1093/femsle/fnz169DOI Listing
July 2019

Influence of entomopathogenic fungus, Metarhizium anisopliae, alone and in combination with diatomaceous earth and thiamethoxam on mortality, progeny production, mycosis, and sporulation of the stored grain insect pests.

Environ Sci Pollut Res Int 2017 Dec 10;24(36):28165-28174. Epub 2017 Oct 10.

University of Agriculture, Faisalabad, Pakistan.

The stored grain insects cause great damage to grains under storage conditions. Synthetic insecticides and fumigants are considered as key measures to control these stored grain insect pests. However, the major issue with these chemicals is grain contamination with chemical residues and development of resistance by insect pests to these chemicals. Biological control is considered as a potential alternative to chemical control especially with the use of pathogens, alone or in combination with selective insecticides. The present study was conducted to evaluate the synergism of Metarhizium anisopliae with diatomaceous earth (DE) and thiamethoxam against four insect pests on the stored wheat grains. In the first bioassay, the M. anisopliae was applied at 1.4 × 10 and 1.4 × 10conidia/ml alone and in integration with two concentrations (250 and 500 ppm) of tested DE. The tested fungus when combined with DE and thiamethoxam possessed synergistic impact as compared to their individual efficacy. Adult mortality increased with respect to increased exposure interval and doses. In the second bioassay, M. anisopliae was applied at 1.4 × 10 conidia/ml individually and in combination with three concentrations (0.50, 0.75, and 1.00 ppm) of thiamethoxam. Results concluded that M. anisopliae integrated with DE and thiamethoxam provides more effective control of stored grain insect pests.
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http://dx.doi.org/10.1007/s11356-017-0383-6DOI Listing
December 2017

Overexpression of a Phytophthora Cytoplasmic CRN Effector Confers Resistance to Disease, Salinity and Drought in Nicotiana benthamiana.

Plant Cell Physiol 2015 Dec 6;56(12):2423-35. Epub 2015 Nov 6.

Department of Plant Pathology, Nanjing Agricultural University, Nanjing, China

The Crinkler (CRN) effector family is produced by oomycete pathogens and may manipulate host physiological and biochemical events inside host cells. Here, PsCRN161 was identified from Phytophthora sojae based on its broad and strong cell death suppression activities. The effector protein contains two predicted nuclear localization signals and localized to nuclei of plant cells, indicating that it may target plant nuclei to modify host cell physiology and function. The chimeric gene GFP:PsCRN161 driven by the Cauliflower mosaic virus (CaMV) 35S promoter was introduced into Nicotiana benthamiana. The four independent PsCRN161-transgenic lines exhibited increased resistance to two oomycete pathogens (P. parasitica and P. capsici) and showed enhanced tolerance to salinity and drought stresses. Digital gene expression profiling analysis showed that defense-related genes, including ABC transporters, Cyt P450 and receptor-like kinases (RLKs), were significantly up-regulated in PsCRN161-transgenic plants compared with GFP (green fluorescent protein) lines, implying that PsCRN161 expression may protect plants from biotic and abiotic stresses by up-regulation of many defense-related genes. The results reveal previously unknown functions of the oomycete effectors, suggesting that the pathogen effectors could be directly used as functional genes for plant molecular breeding for enhancement of tolerance to biotic and abiotic stresses.
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http://dx.doi.org/10.1093/pcp/pcv164DOI Listing
December 2015

A Phytophthora sojae cytoplasmic effector mediates disease resistance and abiotic stress tolerance in Nicotiana benthamiana.

Sci Rep 2015 Jun 3;5:10837. Epub 2015 Jun 3.

Department of Plant Pathology, Nanjing Agricultural University, Nanjing, China.

Each oomycete pathogen encodes a large number of effectors. Some effectors can be used in crop disease resistance breeding, such as to accelerate R gene cloning and utilisation. Since cytoplasmic effectors may cause acute physiological changes in host cells at very low concentrations, we assume that some of these effectors can serve as functional genes for transgenic plants. Here, we generated transgenic Nicotiana benthamiana plants that express a Phytophthora sojae CRN (crinkling and necrosis) effector, PsCRN115. We showed that its expression did not significantly affect the growth and development of N. benthamiana, but significantly improved disease resistance and tolerance to salt and drought stresses. Furthermore, we found that expression of heat-shock-protein and cytochrome-P450 encoding genes were unregulated in PsCRN115-transgenic N. benthamiana based on digital gene expression profiling analyses, suggesting the increased plant defence may be achieved by upregulation of these stress-related genes in transgenic plants. Thus, PsCRN115 may be used to improve plant tolerance to biotic and abiotic stresses.
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http://dx.doi.org/10.1038/srep10837DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4454142PMC
June 2015

A Virulence Essential CRN Effector of Phytophthora capsici Suppresses Host Defense and Induces Cell Death in Plant Nucleus.

PLoS One 2015 26;10(5):e0127965. Epub 2015 May 26.

Department of Plant Pathology, Nanjing Agricultural University, Nanjing, 210095, China.

Phytophthora capsici is a soil-borne plant pathogen with a wide range of hosts. The pathogen secretes a large array of effectors during infection of host plants, including Crinkler (CRN) effectors. However, it remains largely unknown on the roles of these effectors in virulence especially in P. capsici. In this study, we identified a cell death-inducing CRN effector PcCRN4 using agroinfiltration approach. Transient expression of PcCRN4 gene induced cell death in N. benthamiana, N. tabacum and Solanum lycopersicum. Overexpression of the gene in N. benthamiana enhanced susceptibility to P. capsici. Subcellular localization results showed that PcCRN4 localized to the plant nucleus, and the localization was required for both of its cell death-inducing activity and virulent function. Silencing PcCRN4 gene in P. capsici significantly reduced pathogen virulence. The expression of the pathogenesis-related gene PR1b in N. benthamiana was significantly induced when plants were inoculated with PcCRN4-silenced P. capsici transformant compared to the wilt-type. Callose deposits were also abundant at sites inoculated with PcCRN4-silenced transformant, indicating that silencing of PcCRN4 in P. capsici reduced the ability of the pathogen to suppress plant defenses. Transcriptions of cell death-related genes were affected when PcCRN4-silenced line were inoculated on Arabidopsis thaliana, suggesting that PcCRN4 may induce cell death by manipulating cell death-related genes. Overall, our results demonstrate that PcCRN4 is a virulence essential effector and it needs target to the plant nucleus to suppress plant immune responses.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0127965PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4444017PMC
April 2016

Construction of an immobilised acetylcholinesterase column and its application in screening insecticidal constituents from Magnolia officinalis.

Pest Manag Sci 2015 Apr 20;71(4):607-15. Epub 2014 Oct 20.

College of Plant Protection, Jiangsu Key Laboratory of Pesticide Science, Nanjing Agricultural University, Nanjing, China; Key Laboratory of Integrated Management of Crop Diseases and Pests, Ministry of Education, Nanjing, China.

Background: Application of a matrix-immobilised target enzyme for screening inhibitors is widely used in drug development, but there are few studies in insecticide discovery. In this paper, an economical and effective immobilised acetylcholinesterase (AChE) column was prepared using the sol-gel embedment method, which was further combined with high-performance liquid chromatography for screening the AChE inhibitors and insecticidal compounds from complex natural products.

Results: AChE inhibitory constituents magnolol and honokiol were isolated from the ethanol extract of Magnolia officinalis, with IC50 values of 0.069 and 0.057 mM respectively. In an in vivo bioassay, magnolol and honokiol showed insecticidal activity against Nilaparvata lugens, with LC50 values of 0.324 and 0.137 mM, which are comparable with that of commonly used insecticide chlorpyrifos (0.233 mM). Moreover, molecular docking was carried out against a homology model of N. lugens AChE. The complexes showed that magnolol and honokiol placed themselves nicely into the active site of the enzyme and exhibited an interaction energy that was in accordance with our activity profile data.

Conclusion: These results demonstrate that magnolol and honokiol have great applied potential to be developed as natural insecticides, and an immobilised AChE column is very useful as a rapid screening tool for target enzymes towards potent inhibitors.
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http://dx.doi.org/10.1002/ps.3908DOI Listing
April 2015

Phytophthora sojae effector PsCRN70 suppresses plant defenses in Nicotiana benthamiana.

PLoS One 2014 23;9(5):e98114. Epub 2014 May 23.

Department of Plant Pathology, Nanjing Agricultural University, Nanjing, China.

Phytophthora sojae, an oomycete pathogen, produces a large number of effector proteins that enter into host cells. The Crinklers (Crinkling and Necrosis, CRN) are cytoplasmic effectors that are conserved in oomycete pathogens and their encoding genes are highly expressed at the infective stages in P. sojae. However, their roles in pathogenesis are largely unknown. Here, we functionally characterized an effector PsCRN70 by transiently and stably overexpressing it in Nicotiana benthamiana. We demonstrated that PsCRN70 was localized to the plant cell nucleus and suppressed cell death elicited by all the tested cell death-inducing proteins, including BAX, PsAvh241, PsCRN63, PsojNIP and R3a/Avr3a. Overexpression of the PsCRN70 gene in N. benthamiana enhanced susceptibility to P. parasitica. The H2O2 accumulation in the PsCRN70-transgenic plants was reduced compared to the GFP-lines. The transcriptional levels of the defense-associated genes, including PR1b, PR2b, ERF1 and LOX, were also down-regulated in the PsCRN70-transgenic lines. Our results suggest that PsCRN70 may function as a universal suppressor of the cell death induced by many elicitors, the host H2O2 accumulation and the expression of defense-associated genes, and therefore promotes pathogen infection.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0098114PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4032284PMC
February 2015