Gene for gene system in plant fungus interactionVinod Upadhyay
MOLECULAR CHARACTERIZATION OF GENE FOR GENE SYSTEMS IN PLANT- FUNGUS INTERACTION AND THE APPLICATIONS OF AVIRULENCE GENES IN CONTROL OF PLANT PATHOGENS
Systemic Acquired Resistance (SAR) and it’s Significance in Plant Disease Ma...Ankit Chaudhari
Systemic Acquired Resistance (SAR) is a mechanism of induced defense that confers long-lasting protection against a broad spectrum of microorganisms and pests. Presently disease control is largely based on the use of hazardous chemicals viz., fungicides, bactericides and insecticides for either direct or indirect disease management. The hazardous natures of the products on the environment, human and animal health strongly necessitates the search for new safer means of disease control. SAR have high potential to diminish the use of toxic chemicals in the agriculture and has emerged as an alternative, non-conventional, non-biocidal and eco-friendly approach for plant protection and hence for sustainable agriculture. SAR requires the signal molecule salicylic acid (SA) and is associated with accumulation of pathogenesis-related proteins, which are thought to contribute to resistance.
Gene for gene system in plant fungus interactionVinod Upadhyay
MOLECULAR CHARACTERIZATION OF GENE FOR GENE SYSTEMS IN PLANT- FUNGUS INTERACTION AND THE APPLICATIONS OF AVIRULENCE GENES IN CONTROL OF PLANT PATHOGENS
Systemic Acquired Resistance (SAR) and it’s Significance in Plant Disease Ma...Ankit Chaudhari
Systemic Acquired Resistance (SAR) is a mechanism of induced defense that confers long-lasting protection against a broad spectrum of microorganisms and pests. Presently disease control is largely based on the use of hazardous chemicals viz., fungicides, bactericides and insecticides for either direct or indirect disease management. The hazardous natures of the products on the environment, human and animal health strongly necessitates the search for new safer means of disease control. SAR have high potential to diminish the use of toxic chemicals in the agriculture and has emerged as an alternative, non-conventional, non-biocidal and eco-friendly approach for plant protection and hence for sustainable agriculture. SAR requires the signal molecule salicylic acid (SA) and is associated with accumulation of pathogenesis-related proteins, which are thought to contribute to resistance.
An introduction to the concept of Signal transduction mechanism prevalent in lower organisms, particularly bacteria. Also forms a part in many eukaryotic systems of signal transduction, particularly in the plant world.
plant pathogen interaction
different types of pathogens
gene for gene hypothesis
direct receptor model
Elicitor receptor model
suppersor repressor model
gaurd hypothesis
In potato, causes mild mosaic on leaves,Crinkling and necrosis etc. TGB3 (Triple gene block proteins) is expressed by leaky scanning of the TGB2 subgenomic mRNA. TGBp1 with the presence of TGBp2 and TGBp3 can modify the PD size exclusion limit and move between cells.
CaMV Genome organization & their replication, Cauliflower Mosaic Virus belong to Group VII (ds-DNA-RT), Open circular double stranded DNA of 80kb and CaMV replicates by reverse transcription
An introduction to the concept of Signal transduction mechanism prevalent in lower organisms, particularly bacteria. Also forms a part in many eukaryotic systems of signal transduction, particularly in the plant world.
plant pathogen interaction
different types of pathogens
gene for gene hypothesis
direct receptor model
Elicitor receptor model
suppersor repressor model
gaurd hypothesis
In potato, causes mild mosaic on leaves,Crinkling and necrosis etc. TGB3 (Triple gene block proteins) is expressed by leaky scanning of the TGB2 subgenomic mRNA. TGBp1 with the presence of TGBp2 and TGBp3 can modify the PD size exclusion limit and move between cells.
CaMV Genome organization & their replication, Cauliflower Mosaic Virus belong to Group VII (ds-DNA-RT), Open circular double stranded DNA of 80kb and CaMV replicates by reverse transcription
Phytochemical, cytotoxic, in-vitro antioxidant and anti-microbial investigati...IOSR Journals
Zizyphus rugosa Lam. (Family: Rhamnaceae), locally known as “Bon Boroi” or as “Jongli Boroi” in Bangladesh generally found as a herb on the hills in bunches on thorny branches of the Zizyphus rugosa trees. Its bark and wood are used medicinally for dysentery in China, India, Laos, Burma, Sri Lanka, Thailand and Vietnam. Phytochemical screening of the Leaf extract of Zizyphus rugosa Lam showed different phytoconstituents including carbohydrates (monosaccharides, reducing and mixed-reducing sugars), alkaloid, glycosides, steroids, tannins and saponin. No flavonoid was detected. In DPPH and NO radical scavenging methods, IC50 was moderately satisfactory. IC50 was found 179.713μg/ml and 769.909μg/ml respectively compare with the reference ascorbic acid (15.707μg/ml and 82.642μg/ml respectively). In LPO (Lipid peroxidation) assay the Leaf fraction extract showed moderate inhibition potentiality (IC50 402.835μg/ml) in comparison to standard drug BHT (IC50 32.94μg/ml). In CUPRAC assays, the fraction was found to possess low Total antioxidant content, good flavonoid, and moderate amounts of phenolics, tannin and alkaloid content. The Leaf fraction extract was found to show good toxicity to Brine Shrimp nauplii, (LC50 212.402μg/ml & LC90 10715.91μg/ml) compare with the reference anticancer drug vincristine sulphate (LC50 2.47μg/ml & LC90 42μg/ml). In the antimicrobial study the fraction showed moderate activity against only one bacterium (Shiggla sonni) while the standard drug Chloramphenicol showed very good zone of inhibition against all five types (Salmonella typhi, Staphylococcus aureus, Shiggla sonni, Salmonella paratyphi, Salmonella grb) of bacteria. These findings provide scientific basis for the use of Zizyphus rugosa Lam. leaf ethanolic extract in traditional medicine in the treatment of aforementioned diseases. The plant also possesses moderate antimicrobial activity, good cytotoxic and good to moderate antioxidant activity.
Detection of Genetic variation in tissue culture clones of date palm using IS...IJSRD
Date palm is a plant having high nutritional value and long life (yielding up to 100 years). Phoenix dactylifera requires 2-5 males for pollination of 100 females’ plant depending up on genetic and environment factors. Therefore paternity variation expected to very low according to PCR based techniques, Even though we have tried to find out genetic variation among tissue culture cloned plant. Tissue culture technique can be used for genetic improvement of date palm. The main purpose of this study was to evaluate the genetic variation in the tissue culture clones of date palm by using ISSR primers among mother and it’s two clones. The plant DNA was extracted and subjected to detection of genetic variation in two groups of date palm using ISSR primers. In this study ISSR primers produced monomorphic bands within group-1 and group-2. Genetic variation in tissue culture clones of date palm was not detecte by UBC primer series.
Molecular characterization of pea (Pisum sativum L.) using microsatellite mar...IOSR Journals
Nineteen pea (Pisum sativum L.) accessions have been characterized using Simple Sequence Repeats (SSRs). The mains objectives of this study were to examine SSR polymorphism among cultivars and to assess genetic diversity among them. Eight microsatellites, from the Pisum microsatellite consortium (Agrogene ®, France) have been used. Five of the eight SSRs studied gave good electrophoretic profiles and helped us to amplify a number of alleles per locus varying from 3 (PSMPA5 and PSMPA6) to 13 (PSMPSAD126) with a total of 34 and an average number of 6.8 alleles per locus. The Polymorphism Information Content (PIC) varied from 0.18 for PSMPSAD134 to 0.85 for PSMPSAD126, with an average value of 0.62. The five microsatellites analyzed allowed us to separate 18 out of the 19 genotypes studied, and only the two most polymorphic markers (PSMPSAA205 and PSMPSAD126), permit to discriminate among the same genotypes (18) separated using the 5 SSRs. Genetic distances computed have been used to draw the corresponding dendrogram and to distribute genotypes according to their genetic relationship. The genotypes classified within the same group share several agro-morphological characters. Finally, the present study attests that SSR microsatellites are good tools for identifying genotypes and for the assessment of genetic diversity in pea.
Genotyping and subgenotyping of Trichophyton rubrum isolated from dermatophyt...iosrjce
IOSR Journal of Pharmacy and Biological Sciences(IOSR-JPBS) is a double blind peer reviewed International Journal that provides rapid publication (within a month) of articles in all areas of Pharmacy and Biological Science. The journal welcomes publications of high quality papers on theoretical developments and practical applications in Pharmacy and Biological Science. Original research papers, state-of-the-art reviews, and high quality technical notes are invited for publications.
Phylotype Analysis of Ralstonia Solanacearum Causing Bacterial wilt in Eggpla...ijtsrd
Eggplant is prone to attack by several pests including bacteria, fungi, nematodes and insects. In this study, we have analyzed phylotype of bacterial wilt Ralstonia solanacearum infection in eggplant plants collected from Bhubaneswar Orissa in India. Bacterial wilt symptomatic five plant samples were collected from brinjal field in Bhubaneswar in 2016. The samples were macerated in sterile distilled water and grown on Kelman's triphenyltetrazolium chloride TZC agar media. Total genomic DNA of the bacterium were extracted and subjected to PCR amplification using the R. solanacearum specific universal primer pair 759 760. An expected single 280 bp fragment amplified in all the samples confirmed the identity of these as Ralstonia. To reconfirmed isolate of bacterium, the amplicon was sequenced in sequencer. In NCBI blast, the nucleotide sequence was 100 similar with Ralstonia solanacearum strain RS lpxC DOB 1 AB910593 and the sequence was submitted in NCBI database under Acc. No. KY393266. To determined phylotype of strain used specific multiplex PCR with phylotype specific primers Nmult 21F1 2, Nmult 22InF, Nmult 23AF, Nmult 22RR revealed that all the five infected samples belonged to phylotype I as a 144 bp amplicon were observed in agarose gel. On the basis of above finding concluded that the bacterial wilt infected eggplant collected from Bhubaneswar was Ralostonia solanacearum, Phylotype I. Rakesh Kumar | Ramachandran, E. | Koteshwar Yadav "Phylotype Analysis of Ralstonia Solanacearum Causing Bacterial wilt in Eggplants in Orissa in India" Published in International Journal of Trend in Scientific Research and Development (ijtsrd), ISSN: 2456-6470, Volume-3 | Issue-3 , April 2019, URL: https://www.ijtsrd.com/papers/ijtsrd21580.pdf
Rapid identification of dermatophyte species by 28S rDNA Polymerase Chain Rea...iosrjce
IOSR Journal of Dental and Medical Sciences is one of the speciality Journal in Dental Science and Medical Science published by International Organization of Scientific Research (IOSR). The Journal publishes papers of the highest scientific merit and widest possible scope work in all areas related to medical and dental science. The Journal welcome review articles, leading medical and clinical research articles, technical notes, case reports and others.
To study of the genetic variations among the Azospirillum lipoferu isolates u...ijsrd.com
Among free-living microorganisms, which can be practically used in agriculture, bacteria from the Azospirillum genus as well as other endophytes are nowadays thought of as the most active component of associative dinitrogen fixation. The investigation was carried out to study the characterization of Azospirillum lipoferu found in the soils of the ten agro-climatic zones which Karnataka, is classified. By using RAPD markers, 75 bands were scored out of which 78.6 % were found to be polymorphic. Statistical analysis of RAPD data enabled the classification of 10 Azospirillum isolates into two major groups. . In this, the cluster analysis based on 75 RAPD bands revealed that the ten A. lipoferu isolates examined clustered at a linkage distance of about 40 units on the dendrogram. There was no correlation between RAPD and geographical origin of isolates.
Industrial Training at Shahjalal Fertilizer Company Limited (SFCL)MdTanvirMahtab2
This presentation is about the working procedure of Shahjalal Fertilizer Company Limited (SFCL). A Govt. owned Company of Bangladesh Chemical Industries Corporation under Ministry of Industries.
Hybrid optimization of pumped hydro system and solar- Engr. Abdul-Azeez.pdffxintegritypublishin
Advancements in technology unveil a myriad of electrical and electronic breakthroughs geared towards efficiently harnessing limited resources to meet human energy demands. The optimization of hybrid solar PV panels and pumped hydro energy supply systems plays a pivotal role in utilizing natural resources effectively. This initiative not only benefits humanity but also fosters environmental sustainability. The study investigated the design optimization of these hybrid systems, focusing on understanding solar radiation patterns, identifying geographical influences on solar radiation, formulating a mathematical model for system optimization, and determining the optimal configuration of PV panels and pumped hydro storage. Through a comparative analysis approach and eight weeks of data collection, the study addressed key research questions related to solar radiation patterns and optimal system design. The findings highlighted regions with heightened solar radiation levels, showcasing substantial potential for power generation and emphasizing the system's efficiency. Optimizing system design significantly boosted power generation, promoted renewable energy utilization, and enhanced energy storage capacity. The study underscored the benefits of optimizing hybrid solar PV panels and pumped hydro energy supply systems for sustainable energy usage. Optimizing the design of solar PV panels and pumped hydro energy supply systems as examined across diverse climatic conditions in a developing country, not only enhances power generation but also improves the integration of renewable energy sources and boosts energy storage capacities, particularly beneficial for less economically prosperous regions. Additionally, the study provides valuable insights for advancing energy research in economically viable areas. Recommendations included conducting site-specific assessments, utilizing advanced modeling tools, implementing regular maintenance protocols, and enhancing communication among system components.
Immunizing Image Classifiers Against Localized Adversary Attacksgerogepatton
This paper addresses the vulnerability of deep learning models, particularly convolutional neural networks
(CNN)s, to adversarial attacks and presents a proactive training technique designed to counter them. We
introduce a novel volumization algorithm, which transforms 2D images into 3D volumetric representations.
When combined with 3D convolution and deep curriculum learning optimization (CLO), itsignificantly improves
the immunity of models against localized universal attacks by up to 40%. We evaluate our proposed approach
using contemporary CNN architectures and the modified Canadian Institute for Advanced Research (CIFAR-10
and CIFAR-100) and ImageNet Large Scale Visual Recognition Challenge (ILSVRC12) datasets, showcasing
accuracy improvements over previous techniques. The results indicate that the combination of the volumetric
input and curriculum learning holds significant promise for mitigating adversarial attacks without necessitating
adversary training.
Courier management system project report.pdfKamal Acharya
It is now-a-days very important for the people to send or receive articles like imported furniture, electronic items, gifts, business goods and the like. People depend vastly on different transport systems which mostly use the manual way of receiving and delivering the articles. There is no way to track the articles till they are received and there is no way to let the customer know what happened in transit, once he booked some articles. In such a situation, we need a system which completely computerizes the cargo activities including time to time tracking of the articles sent. This need is fulfilled by Courier Management System software which is online software for the cargo management people that enables them to receive the goods from a source and send them to a required destination and track their status from time to time.
Quality defects in TMT Bars, Possible causes and Potential Solutions.PrashantGoswami42
Maintaining high-quality standards in the production of TMT bars is crucial for ensuring structural integrity in construction. Addressing common defects through careful monitoring, standardized processes, and advanced technology can significantly improve the quality of TMT bars. Continuous training and adherence to quality control measures will also play a pivotal role in minimizing these defects.
Vaccine management system project report documentation..pdfKamal Acharya
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Fungal Identification method by RDNA sequence analysis: Molecular approach to revel the role of microbial community in vermicomposting
1. IOSR Journal of Computer Engineering (IOSR-JCE)
e-ISSN: 2278-0661,p-ISSN: 2278-8727, Volume 17, Issue 2, Ver. 1 (Mar – Apr. 2015), PP 103-106
www.iosrjournals.org
DOI: 10.9790/0661-1721103106 www.iosrjournals.org 103 | Page
Fungal Identification method by RDNA sequence analysis:
Molecular approach to revel the role of microbial community in
vermicomposting
Shobha Shouche1*
, Zeemi Nema2
and Praveesh Bhati3
1: Assistant Professor, Govt. Madhav Science College, Ujjain (M.P.) 456010,
India:shobha.shouche@gmail.com
2: research scholar, Govt. Madhav Science College, Ujjain (M.P.) 456010, India: zeeminema15@gmail.com
3: Lecturer, Advance College of Commerce and Science, Ujjain (M.P.) 456010,India:bhati_p212@yahoo.co.in
Abstract: Internal transcribed spacer is a special sequence that present in between sequence of ribosomal
DNA. It is present in multiple copies and act as a conservative sequence. This sequence is unique for particular
living being there for it is used as tool for identification.Present study carried out to identification of floral
degrading fungi by using of PCR method. This process was done with the help of specific primer. The length of
primer was different for different group of fungi. There were four fungi which identified were Aspergillus flavus,
A. fumigatus, A. terrus and Alternaria alternate.
Key words: ITS, PCR, vermicomposting, rDNA, electrophoresis.
I. Introduction
Fungi are the group of heterotrophic filamentous organisms that found everywhere on the earth. Their
diverse nature affects the activity of ecosystem. Among these fungi, genus Aspergillus and Alternaria are the
most common fungi that have a great impact on living being. The Aspergilli are a ubiquitous group of
filamentous fungi spanning over 200 million years of evolution. Among over the 185 Aspergilli are several that
have an impact on human health and society, including 20 human pathogens as well as beneficial species used to
produce foodstuffs, composting process and industrial enzymes (Timberlake and Marshall, 1989). Several fungi
have been pointed for their key role in vermicomposting of different organic wastes. (Taiwo & Oso, 2004;
Peters et al., 2000). The group of fungi mostly characterized on the basis of morphology, physiology and
genomic sequencing. The 18SrRNA sequence is generally used to identify the eukaryotic organism because of
less change occurred in this sequence. The ribosomal DNA (r RNA genes) acquires characteristics which
are appropriate for the recognition of microorganisms at the species level. This rDNA is highly stable and show
a variety of conserved and diverse regions within the genome (Hibbett, 1992). They also occur in multiple
copies in per haploid genome (Bruns, et al., 1991; Yao et al., 1992) arranged in tandem repeats , each repeat
consisting of the 18S small subunit (SSU), the 5.8S, and the 28S large subunit (LSU) genes. Internal transcribed
spacer (ITS) regions have been used successfully to generate specific primers capable of differentiating closely
related fungal species (Bryan et al., 1995).
Therefore we focused on the ITS regions of ribosomal genes (Figure 1) for the identification of floral
waste degrading fungi. In the broader context, taxon-selective amplification of ITS regions is likely to become a
common approach in molecular identification strategies. Taxon-selective ITS amplification has already been
used for detection of the fungal pathogens such as Fusarium (O´Donnell, 1992) and Verticillium spp. (Nazar et
al., 1991). Moller et al. (1999) also developed polymerase chain reaction (PCR) to identify Giberrella fujikuroi
anamorphs in maize kernels using primer pairs based on sequences of RAPD fragments, and were specific for
Fusarium moniliforme and Fusarium subglutinans. Amplification of target DNA through PCR with sequence
specific primers is potentially more sensitive and rapid than microbiological techniques, as a number of
constraints are removed. Unlike culture, PCR does not require the presence of viable organisms for success and
may be performed even when sample volumes are small. The objective of our investigation was to develop a
reliable and sensitive PCR assay for the selective detection of composting fungal species and also find out their
evolutionary correlation between them.
II. Materials And Methods
Fungal isolates
In the present study, following four isolates were used i.e. Aspergillus flavus, A.fumigatus , Alternaria
alternata and A.terreus. The isolates originated from floral waste vermicomposting process done in Govt. MVM
Ujjain (M.P.), India.
2. Fungal Identification method by RDNA sequence analysis: Molecular approach to revel the role ….
DOI: 10.9790/0661-1721103106 www.iosrjournals.org 104 | Page
DNA isolation
Total genomic DNA was isolated from fresh mycelium according to a miniprep protocol described by
Cenis (1992) and Abd-Elsalam (2003). In this method potato dextrose broth medium (Hi-media) was inoculated
with fungal mycelium and left at room temperature for three days. After that, tube allowed to centrifuge at
10,000 rpm for 5 min, so that mycelial mat was pelleted. Now Pellet was washed with 500 μl Tris-EDTA buffer.
The mat was then homogenized by hand in 300 μl of extraction buffer for 5 min. One hundred and fifty micro
liters of 3 M sodium acetate (pH 5.2) was added, and the mixture was cooled to 20°C for 10 min. Fungal debris
was pelleted by centrifugation at 10,000 rpm for 5 min, the supernatant was transferred to a fresh tube, and an
equal volume of isopropanol was added. DNA was then pelleted by centrifugation at 10,000 rpm for 10 min.
Excess salt was removed by washing with 70% ethanol, and DNA was resuspended in Tris-EDTA.
PCR amplification of ribosomal DNA regions
The ITS and the inverting 5.8S coding rDNA were amplified by PCR using the primers ITS described
by White et al. (1990). Each PCR reaction mixture contained 10 ng of genomic DNA, 1μM each of the primers
ITS, reaction buffer (50 mM KCl, 50 mM Tris-HCl; [pH 8.3] 0.1 mg/ml bovine serum albumin), 3 mM MgCl2
,
200μM each of dNTP and 2.5 U of Taq DNA polymerase (Promega, Mannheim, Germany) in a total volume of
50 μl. The PCR profile was enetration at 95°C for 2 min, followed by 30 cycles of 94°C for 1 min, 54°C for 30
s, and 72°C for 1 min. ITS bands of interest were excised from agarose gels and re-amplified by PCR using the
same primer pair that was used for generating the ITS bands (Fig.1).
Fig 1. PCR amplified Gel Picture
Fungal rDNA sequencing:
PCR products were purified to remove excess primer using microconcentrators and after obtaining the
ITS band, it was taken out from the agarose gel was directly sequenced with the Di- Deoxy Terminator
sequencer. Obtained sequences are as follow.
1) Aspergillus fumigatus:
>ATATGAGGAGGCTCGCCGAAGAATGCGAAGGATTCAGCGGTGCGGATTTGGGAAGTTTGCTACG
CCGTGCCGGTTATTCTGCAATCAAAAGACGCGATCAGATCAGCTTTGAGGACTTCGTTGCCGCTAA
GGCTTTCATTCGGCCTAGTGTTACCGATCTCAAAAAGTATGAGAAGCTCAGGAGAGAGTGGAGCG
GCGGCGTGTTGTAGATATCCGACCAGTGTGGCATACAAAGTCAACGAGAGCATTGTGTACGTACG
GGATAATGGTCATATACCAAAAGCTTTGCATGTGCGGATGCTGACAATCTCCTCGCTTGCTTGGAT
GTTTGTACTCAATGAGTCTACCAATCGTTCGTTCTTCCACATAATACCTCCAAGAATTAGACTTGAT
ATCGGGATGCCAGGTTGCTTGGACCATCAGACGAGGGCTTATTTTTAAGGAAACAGTTTCTATAAG
GACTATAACTTTTATAAAGCGGATATATCTCTTGACGAAAACATTGATGCACACCAAGACGCCTCT
CATGTAGACATGACCTGATAAATTAACAGTTGGAAATCCCAATATGTACGCTTCGAATCGACGTGT
GATTACATCCAGAGACGGCAAGTT
2) Aspergillus flavus:
>ATTCCTGAATTCCTTCCTCACCTCCACGATGGTTGACCATATCTCCCCCCGGGCATCTCCCGGACC
GATCCGTTCCTCCCAGACTCGCCGCGCCCGAAAGCTCCGGGATAGCTGTACGAGTTGTGCCAGCTC
AAAAGTGCGATGCACCAAGGAGAAACCGGCCTGTGCTCGGTGTATCGAACGTGGTCTTGCCTGTC
AATACATGGTCTCCAAGCGGATGGGCCGCAATCCGCGCGCTCCCAGTCCCCTTGATTCAACTCGGC
3. Fungal Identification method by RDNA sequence analysis: Molecular approach to revel the role ….
DOI: 10.9790/0661-1721103106 www.iosrjournals.org 105 | Page
GACCATCAGAGAGTCTTCCTTCAGCCAGGTCGGAACAGGGACTTCCGGCGCATAACACGTACTCA
ACGCCTCATGCTCATACGCAGGCCCACACTCATGCTCATTCTCATCCGCAACCGCATCCACAATCT
CATCCTCAATCGAATCAACCACCACACGCTCTGCCCACCCCCAATGGTAGCAGTAGCGTCTCCGCC
ATCTTTTCTCATCAGAGTCCGCCGCCACCCGTGGAGACCCAGGGCCTTGGAGGAGATCTGGCTGGT
CAGGAGCAAAGCACCCTGTCTTCCCTAACAGTCGATTCGGAATTCGGGGGCTCTTTGCAGTCAATG
GAACACGGAAACCATGCCGATTTCTTGGCCGAGTCGACGGGGAGTCTTTTCGACGCGTTTTTGGAA
GTAGGGACCCCCATGATCGACCCGTTCCTCGAGTCGGCCCCACTACCACCGTTTCAGGCGCGCTAT
TGCTGCTTTTCGCTAGCACTACAAACACTGACCCACCTCTTCCCCCACGCCCCGCTGGGCTGTCAAC
TACGGCTGACGGACGGTGAGGACAGTTCGTGCAACCTGATGACGACTGATATGGTCATCTCGGGG
AACAAGAGGGCTACCGATGCGGTCCGGAAGATCCTCGGGTGTTCGTGCGCGCAGGATGGCTACTT
GCTGAGCATGGTCGTCCTTATCGTTCTCAAGGTGCTGGCATGGTATGCTGCGGCAGCAGGCACCCA
GTGTACCTCAACGGCGGCGGGTGGAGAAACCAACAGTGGCAGCTGTAGCAACAGTCCCGCCACCG
TGTCCAGTGGCTGTCTGACGGAAGAGCGCGTGCTGCACCTCCCTAGTATGATGGGCGAGGATTGTG
TGGATGAGGAAGACCAGCCGCGAGTGGCGGCACAGCTTGTTCTGAGTGAACTGCACCGAGTCCAG
TCGCTGGTGAACCTATTGGCCAAGCGCCT GCAA.
3) Alternaria alternata
>GCGTCAGTAACAAATTAATAATTACAACTTTCAACAACGGATCTCTTGGTTCTGGCATCGATGAA
GAACGCAGCGAAATGCGATAAGTAGTGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAAC
GCACATTGCGCCCTTTGGTATTCCAAAGGGCATGCCTGTTCGAGCGTCATTTGTACCCTCAAGCTTT
GCTTGGTGTTGGGCGTCTTGTCTCTAGCTTTGCTGGAGACTCGCCTTAAAGTAATTGGCAGCCGGC
CTACTGGTTTCGGAGCGCAGCACAAGTCGCACTCTCTATCAGCAAAGGTCTAGCATCCATTAAGCC
TTTTTTCAACTTTTGACCTCGGATCAGGTAGGGATACCCGCTGAACTTAAGCATATCAATAAGCGG
AGG.
4) Aspergillus terreus
>ATCTTTATGGCCACCTCCCACCCGTGACTATTGTACCTTGTTGCTTCGGCGGGCCCGCCAGCGTTG
CTGGCCGGCGGGGGGCGACTCGCCCCCGGGCCCGTGCCCGCCGGAGACCCCAACATGAACCCTGT
TCTGAAAGCTTGCAGTCTGAGTGTGATTCTTTGCAATCAGTTAAAACTTTCAACAATGGATCTCTTG
GTTCCGGCATCGATGAAGAACGCAGCGAAATGCGATAACTAATGTGAATTGCAGAATCCAGTGAA
TCATCGAGTCTTTGAACGCACATTGCGCCCCCTGGTATTCCGGGGGGCATGCCTGTCCGAGCGTCA
TTGCTGCCCTCAAGCCCGGCTTGTGTGTTGGGCCCTCGTCCCCCGGCTCCCGGGGGACGGGCCCGA
AAGGCAGCGGCGGCACCGCGTCCGGTCCTCGAGCGTATGGGGCTTCGTCTTCCGCTCCGTAGGCCC
GGCCGGCGCCCGCCGACGCATTTATTTGCAACTTATTTATTCCA
GGTGACCTCGGATCAGTAGGGAA
III. Results
In this present study, special types of oligonucleotide probes were used for identification of ITS
sequence present in fungal rDNA. This process performed by PCR amplification method. After amplification,
PCR products were separated by agarose gel electrophoresis (Fig. 1). On the gel, four different bands were
obtained. Using the universal fungal primers (ITS), PCR products were generated from all of the four different
fungal species. The base pair of these sequences were determined with the help of control bands. Figure 1
illustrates the different sizes obtained for the full ITS region amplified from a selection of different Aspergillus
and Alternaria species. Oligonucleotide probes were designed from ITS sequences available in this study; the
full ITS region was sequenced from each species. One sequencing reaction was performed on each strand.
Sequence result showed that the length of base pairs of ITS were in between 398 - 1216 bp. The shortest length
of bp was 398 of Alternaria alternata while largest length of bp was 1216 of Aspergillus flavus. In this method
known primer were used that was species specific. The primers showed good specificity for ITS sequence of
fungal species. Due to this specificity, fungal species were identified.
IV. Discussion and Conclusion
The rRNA genes, commonly used in identification and taxonomic studies, were confirmed in the
present study to be particularly appropriate for the purpose of providing target sequences for molecular
detection. Differences in the nucleotide composition of the variable ITS region have been successfully employed
to design specific primer sets that amplify DNA selectively among and within species of plant pathogens (Nazar
et al., 1991; Moukhamedov et al., 1994; Schilling et al., 1996; Moricca et al., 1998). O`Donnell (1992) found a
surprising level of divergence for ITS sequences within the species of F. sambucinum. We used ITS primers to
amplify the entire 18S rDNA gene. The amplified DNA was sequenced to develop a genus-specific PCR assay
4. Fungal Identification method by RDNA sequence analysis: Molecular approach to revel the role ….
DOI: 10.9790/0661-1721103106 www.iosrjournals.org 106 | Page
for the rapid identification of Aspergillus and Alternaria genera, isolated from floral waste vermicomposting
mixture.
Nevertheless, the current approach of testing known related species is justified primarily by the use of
rDNA as the basis for specificity. The nucleotide sequence analysis of rDNA region has been widely accepted to
have phylogenetic significance, and is therefore useful in taxonomy and the study of phylogenetic relationships
(Hibbett, 1992). This approach, designing primers from the rDNA region has far superior reliability compared to
the use of random non-defined probes or primers.
Acknowledgement
A) The authors are very thankful to MP- CST, Bhopal (India) to provide initial grants for genetically
identification of floral waste degrading fungi.
B) We are also thankful to our principal Dr. Usha Shrivastava for giving her help and support.
C) We are also thankful to BioAxis DNA Research Centre Private Limited. for their technical assistance.
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