Publications by authors named "Sudharsan Sadhasivam"

10 Publications

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Host Factors Modulating Ochratoxin A Biosynthesis during Fruit Colonization by .

J Fungi (Basel) 2020 Dec 28;7(1). Epub 2020 Dec 28.

Institute of Postharvest and Food Sciences, The Volcani Center, Agricultural Research Organization, Rishon LeZion 7528809, Israel.

is a strong and consistent ochratoxin A (OTA) producer and considered to be the main source of this toxic metabolite in grapes and grape products such as wine, grape juice and dried vine fruit. OTA is produced under certain growth conditions and its accumulation is affected by several environmental factors, such as growth phase, substrate, temperature, water activity and pH. In this study, we examined the impact of fruit host factors on regulation and accumulation of OTA in colonized grape berries, and assessed in vitro the impact of those factors on the transcriptional levels of the key genes and global regulators contributing to fungal colonization and mycotoxin synthesis. We found that limited sugar content, low pH levels and high malic acid concentrations activated OTA biosynthesis by , both in synthetic media and during fruit colonization, through modulation of global regulator of secondary metabolism, and OTA gene cluster expression. These findings indicate that fruit host factors may have a significant impact on the capability of to produce and accumulate OTA in grapes.
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http://dx.doi.org/10.3390/jof7010010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7823970PMC
December 2020

Functional roles of LaeA, polyketide synthase, and glucose oxidase in the regulation of ochratoxin A biosynthesis and virulence in Aspergillus carbonarius.

Mol Plant Pathol 2021 01 10;22(1):117-129. Epub 2020 Nov 10.

Institute of Postharvest and Food Sciences, The Volcani Center, Agricultural Research Organization, Rishon LeZion, Israel.

Aspergillus carbonarius is the major producer of ochratoxin A (OTA) among Aspergillus species, but the contribution of this secondary metabolite to fungal virulence has not been assessed. We characterized the functions and addressed the roles of three factors in the regulation of OTA synthesis and pathogenicity in A. carbonarius: LaeA, a transcriptional factor regulating the production of secondary metabolites; polyketide synthase, required for OTA biosynthesis; and glucose oxidase (GOX), regulating gluconic acid (GLA) accumulation and acidification of the host tissue during fungal growth. Deletion of laeA in A. carbonarius resulted in significantly reduced OTA production in colonized nectarines and grapes. The ∆laeA mutant was unable to efficiently acidify the colonized tissue, as a direct result of diminished GLA production, leading to attenuated virulence in infected fruit compared to the wild type (WT). The designed Acpks-knockout mutant resulted in complete inhibition of OTA production in vitro and in colonized fruit. Interestingly, physiological analysis revealed that the colonization pattern of the ∆Acpks mutant was similar to that of the WT strain, with high production of GLA in the colonized tissue, suggesting that OTA accumulation does not contribute to A. carbonarius pathogenicity. Disruption of the Acgox gene inactivated GLA production in A. carbonarius, and this mutant showed attenuated virulence in infected fruit compared to the WT strain. These data identify the global regulator LaeA and GOX as critical factors modulating A. carbonarius pathogenicity by controlling transcription of genes important for fungal secondary metabolism and infection.
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http://dx.doi.org/10.1111/mpp.13013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7749749PMC
January 2021

The pH-Responsive Transcription Factor PacC Governs Pathogenicity and Ochratoxin A Biosynthesis in .

Front Microbiol 2020 13;11:210. Epub 2020 Feb 13.

Institute of Postharvest and Food Sciences, Agricultural Research Organization, Volcani Center, Rishon LeZion, Israel.

Pathogenic fungi must respond effectively to changes in environmental pH for successful host colonization, virulence and toxin production. is a mycotoxigenic pathogen with the ability to colonize many plant hosts and secrete ochratoxin A (OTA). In this study, we characterized the functions and addressed the role of PacC-mediated pH signaling in pathogenicity using designed gene knockout mutant. Δ mutant displayed an acidity-mimicking phenotype, which resulted in impaired fungal growth at neutral/alkaline pH, accompanied by reduced sporulation and conidial germination compared to the wild type (WT) strain. The Δ mutant was unable to efficiently acidify the growth media as a direct result of diminished gluconic and citric acid production. Furthermore, loss of resulted in a complete inhibition of OTA production at pH 7.0. Additionally, Δ mutant exhibited attenuated virulence compared to the WT toward grapes and nectarine fruits. Reintroduction of gene into Δ mutant restored the WT phenotype. Our results demonstrate important roles of PacC of in OTA biosynthesis and in pathogenicity by controlling transcription of genes important for fungal secondary metabolism and infection.
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http://dx.doi.org/10.3389/fmicb.2020.00210DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7031272PMC
February 2020

Synergistic Inhibition of Mycotoxigenic Fungi and Mycotoxin Production by Combination of Pomegranate Peel Extract and Azole Fungicide.

Front Microbiol 2019 20;10:1919. Epub 2019 Aug 20.

Department of Food Quality and Safety, Institute for Postharvest and Food Sciences, Agricultural Research Organization, The Volcani Center, Rishon LeZion, Israel.

Fungal plant pathogens cause considerable losses in yield and quality of field crops worldwide. In addition, under specific environmental conditions, many fungi, including such as some and spp., are further able to produce mycotoxins while colonizing their host, which accumulate in human and animal tissues, posing a serious threat to consumer health. Extensive use of azole fungicides in crop protection stimulated the emergence of acquired azole resistance in some plant and human fungal pathogens. Combination treatments, which become popular in clinical practice, offer an alternative strategy for managing potentially resistant toxigenic fungi and reducing the required dosage of specific drugs. In the current study we tested the effect of pomegranate peel extract (PPE) on the growth and toxin production of the mycotoxigenic fungi and , both alone and in combination with the azole fungicide prochloraz (PRZ). Using time-lapse microscopy and quantitative image analysis we demonstrate significant delay of conidial germination and hyphal elongation rate in both fungi following PPE treatment in combination with PRZ. Moreover, PPE treatment reduced aflatoxin production by up to 97%, while a combined treatment with sub-inhibitory doses of PPE and PRZ resulted in complete inhibition of toxin production over a 72 h treatment. These findings were supported by qRT-PCR analysis, showing down-regulation of key genes involved in the aflatoxin biosynthetic pathway under combined PPE/PRZ treatment al low concentrations. Our results provide first evidence for synergistic effects between the commercial drug PRZ and natural compound PPE. Future application of these findings may allow to reduce the required dosage of PRZ, and possibly additional azole drugs, to inhibit mycotoxigenic fungi, ultimately reducing potential concerns over exposure to high doses of these potentially harmful fungicides.
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http://dx.doi.org/10.3389/fmicb.2019.01919DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6710344PMC
August 2019

Identification and Toxigenic Potential of Fungi Isolated from Peppers.

Microorganisms 2019 Aug 30;7(9). Epub 2019 Aug 30.

Department of Food Quality and Safety, Institute for Postharvest and Food Sciences, The Volcani Center, Agricultural Research Organization, Rishon LeZion 7528809, Israel.

peppers are among the most popular horticultural crops produced and consumed worldwide. This study aimed to assess the occurrence of spoilage fungi responsible for post-harvest losses in the most common varieties of peppers collected from retail markets in Nigeria and Ghana. Forty fungal isolates belonging to 7 families, 8 genera, and 17 species were identified on the basis of morphology, culture characteristics, and DNA sequencing of the internal transcribed spacer (ITS) region. spp. (42.5%), spp. (22.5%), and spp. (15%) were found to be the predominant fungal pathogens. Furthermore, potential ability of the isolated mycotoxigenic fungi to produce some major mycotoxins was analyzed using high-performance liquid chromatography (HPLC). Among the 22 isolates analyzed, 11 strains belonging to the genera of , and were found to be able to produce mycotoxins, such as aflatoxin B1, gliotoxin, deoxynivalenol, and citrinin. A better understanding of the role of fungal contaminants in pepper fruits, especially the prevalence of mycotoxigenic fungi and their associated mycotoxigenic potential, will assist in the development of management strategies to control mycotoxin contamination and to reduce toxicological risks related to pepper consumption by humans and animals.
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http://dx.doi.org/10.3390/microorganisms7090303DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6780498PMC
August 2019

Isolation and chemical characteristics of rhamnose enriched polysaccharide from Grateloupia lithophila.

Carbohydr Polym 2018 Sep 2;195:486-494. Epub 2018 May 2.

Centre of Advanced Study in Marine Biology, Faculty of Marine Sciences, Annamalai University, Parangipettai, 608 502, Tamil Nadu, India.

The crude polysaccharide was extracted from Grateloupia lithophila through hot-water extraction and deproteinization. Further, fractionated by anion-exchange column using Q-Sepharose and purified by gel-permeation chromatography using Sepharose 4-LB column. The crude and purified polysaccharide contains high carbohydrate (75.7 and 89.7%), ash (18.2 and 3.2%) and moisture (14.8 and 1.3%); the protein and uronic acid were absent. The molecular weight of crude, fractionated and purified polysaccharide was found to be 37 kDa, 29 kDa and 24 kDa. The monosaccharide composition of the crude polysaccharide was found to be having rhamnose (79.82%), fructose (8.38%), galactose (3.95%), xylose (3.31%) and glucose (1.48%); whereas the purified polysaccharide reported higher amount of rhamnose (95.88%), 1.13% of xylose and 2.21% of fructose. The structural elucidation of the purified polysaccharide was conformed as α-l-rhamnose through polarimetry, FT-IR and H NMR spectroscopy.
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http://dx.doi.org/10.1016/j.carbpol.2018.05.002DOI Listing
September 2018

Isolation, characterization and bioactive potential of sulfated galactans from Spyridia hypnoides (Bory) Papenfuss.

Int J Biol Macromol 2018 Apr 19;109:589-597. Epub 2017 Dec 19.

Centre of Advanced Study in Marine Biology, Faculty of Marine Sciences, Annamalai University, Parangipettai 608 502, Tamil Nadu, India.

The sulfated galactans (SG) of mass 16 kDa was purified from S.hypnoides through anion exchange and gel permeation chromatography. The biochemical properties of SG including carbohydrate, 3,6 anhydrogalactose, sulfate, uronic acid, moisture, ash, carbon, hydrogen, nitrogen contents were estimated. In the purified SG, the presence of major sugars such as galactose and glucose were identified through HPLC and it was further structurally characterised through FT-IR and NMR spectroscopy. Anticoagulant activity of SG was estimated as 25.36 & 2.46 IU at 25 μg/ml (aPTT & PT). SG also showed potential dose dependent antioxidant activity against free radicals such as DPPH (56.41% at 2 mg/ml), hydroxyl radicals (65.58% at 3 mg/ml) and superoxide radicals (73.12% at 0.6 mg/ml). The maximum metal chelating and total antioxidant property (76.42%, 66.81%) was exhibited at 1 mg/ml. The results indicate that the SG from red seaweed represents a good source of polysaccharide with significant anticoagulant and antioxidant properties.
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http://dx.doi.org/10.1016/j.ijbiomac.2017.12.097DOI Listing
April 2018

Rapid Detection and Identification of Mycotoxigenic Fungi and Mycotoxins in Stored Wheat Grain.

Toxins (Basel) 2017 09 25;9(10). Epub 2017 Sep 25.

Department of Food Quality and Safety, Institute for Postharvest and Food Sciences, Agricultural Research Organization, The Volcani Center, Rishon LeZion 7528809, Israel.

This study aimed to assess the occurrence of toxigenic fungi and mycotoxin contamination in stored wheat grains by using advanced molecular and analytical techniques. A multiplex polymerase chain reaction (PCR) strategy was established for rapid identification of mycotoxigenic fungi, and an improved analytical method was developed for simultaneous multi-mycotoxin determination in wheat grains by liquid chromatography-tandem mass spectrometry (LC/MS/MS) without the need for any clean-up. The optimized multiplex PCR method was highly specific in detecting fungal species containing species-specific and mycotoxin metabolic pathway genes. The method was applied for evaluation of 34 wheat grain samples collected from storage warehouses for the presence of mycotoxin-producing fungi, and a few samples were found positive for and species. Further chemical analysis revealed that 17 samples contained mycotoxins above the level of detection, but only six samples were found to be contaminated over the EU regulatory limits with at least one mycotoxin. Aflatoxin B₁, fumonisins, and deoxynivalenol were the most common toxins found in these samples. The results showed a strong correlation between the presence of mycotoxin biosynthesis genes as analyzed by multiplex PCR and mycotoxin detection by LC/MS/MS. The present findings indicate that a combined approach might provide rapid, accurate, and sensitive detection of mycotoxigenic species and mycotoxins in wheat grains.
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http://dx.doi.org/10.3390/toxins9100302DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5666349PMC
September 2017

Antioxidant and anticoagulant activity of sulfated polysaccharide from Gracilaria debilis (Forsskal).

Int J Biol Macromol 2015 Nov 28;81:1031-8. Epub 2015 Sep 28.

Department of Biophysics, All India Institute of Medical Sciences, New Delhi 110 029, India.

Sulfated polysaccharide was isolated from Gracilaria debilis and purified through gel chromatography and their molecular weight was determined through AGE and PAGE. The total sugars in the crude, fractionated and purified polysaccharide were estimated as 52.65%, 59.70% and 67.60%, respectively. The ash and moisture content of crude and purified polysaccharide was found to be 14.2% and 23.5% and the polysaccharide was free from protein contamination. The sulfate and uronic acid contents in the crude, fractionated and purified were estimated as 14.08%, 15.33% and 16.01% and 10.12%, 13.56%, 16.70%. The elemental composition including carbon (crude - 23.12%, purified - 21.05%), hydrogen (crude - 3.4%, purified - 4.13%) and nitrogen (crude - 1.22%, purified - 0.56%) were also analyzed. The anticoagulant activity of the sulfated polysaccharide through APTT and PT was estimated at 14.11 and 8.23IU/mg. The purified polysaccharide with the molecular mass of 20kDa showed highest antioxidant activity (38.57%, 43.48% and 38.88%) in all the assays tested such as DPPH hydroxyl radical, superoxide radical, hydroxyl radical scavenging activities and the structural property was analyzed through FT-IR and (1)H NMR spectrum. The results together suggest that the isolated low molecular weight sulfated polysaccharide will demonstrate as a enormously available alternative natural source of antioxidant for industrial uses.
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http://dx.doi.org/10.1016/j.ijbiomac.2015.09.046DOI Listing
November 2015

Structural characterization and bioactivities of sulfated polysaccharide from Monostroma oxyspermum.

Int J Biol Macromol 2015 Jan 30;72:1459-65. Epub 2014 Oct 30.

Centre of Advanced Study in Marine Biology, Faculty of Marine Sciences, Annamalai University, Parangipettai 608 502, Tamil Nadu, India.

Sulfated polysaccharide was isolated from Monostroma oxyspermum through hot water extraction, anion-exchange and gel permeation column chromatography. The sulfated polysaccharide contained 92% of carbohydrate, 0% of protein, 7.8% of uronic acid, 22% of ash and 33% of moisture respectively. The elemental composition was analyzed using CHNS/O analyzer. The molecular weight of sulfated polysaccharide determined through PAGE was found to be as 55 kDa. Monosaccharides analysis revealed that sulfated polysaccharide was composed of rhamnose, fructose, galactose, xylose, and glucose. The structural features of sulfated polysaccharide were analyzed by NMR spectroscopy. Further the sulfated polysaccharide showed total antioxidant and DPPH free radical scavenging activity were as 66.29% at 250 μg/ml and 66.83% at 160 μg/ml respectively. The sulfated polysaccharide also showed ABTS scavenging ability and reducing power were as 83.88% at 125 μg/ml and 15.81% at 400 μg/ml respectively. The anticoagulant activity was determined for human plasma with respect to Activated Partial Thromboplastin Time (APTT) and Prothrombin Time (PT) was 20.09 IU and 1.79 IU at 25 μg/ml respectively. These results indicated that the sulfated polysaccharide from M. oxyspermum had potent antioxidant and anticoagulant activities.
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http://dx.doi.org/10.1016/j.ijbiomac.2014.09.062DOI Listing
January 2015