Genetic variation among isolates of <em>Bipolaris maydis</em> using RAPD_PCR
10.6092/issn.2531-7342/6195
Il seccume fogliare del mais è una malattia diffusa in molte aree dell’India con clima caldo-umido. Il patogeno responsabile è Bipolaris maydis (Y. Nisik. &amp; C. Miyake) Shoemaker. Dieci isolati del fungo sono stati selezionati per il presente studio da diverse aree geografiche dell’India. Uno screening preliminare è stato effettuato per l'analisi della variazione molecolare usando 32 primer delle serie OPM, OPB e OPN (Operon Technologies, Inc., USA). Dei 32 primer utilizzati per l'amplificazione del DNA da tutti gli isolati di B. maydis, due hanno dato prodotti di amplificazione sub-ottimali o non distinti. Il dendrogramma ottenuto dai coefficienti di similarità con il metodo UPGMA-SAHN è risultato in due cluster principali, uno con Bm-3 e Bm-2 ed un secondo con il resto degli isolati raggruppati in due sottogruppi. Il primo sottogruppo ha incluso Bm-4 e BM-7, mentre Bm-1, Bm-5 e BM-9 sono stati raggruppati nel secondo sottogruppo. Gli isolati Bm-8 e Bm-10 sono risultati diversi dal resto degli isolati, andando a costituire un gruppo a parte. Nel presente studio non è stato possibile stabilire una correlazione significativa tra la similarità genetica e le origini geografiche degli isolati.
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Ramesh Chaudary Sharma
Italian Journal of Mycology
molecular markers
Genetic variation among isolates of <em>Bipolaris maydis</em> using RAPD_PCR
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Articles
2016-06-23
2016-06-23
2016-04-21
2018-08-20
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Ital J Mycol
Ramesh Chaudary Sharma
2016/06/23
10.6092/issn.2531-7342/6195
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Plant Pathology, Indian Agricultural Research Institute, New Delhi 110012
2531-7342
molecular markers
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Genetic variation among isolates of <em>Bipolaris maydis</em> using RAPD_PCR
https://italianmycology.unibo.it/article/download/6195/5969
45
M. R. Karimishahri, R. C. Sharma                             Italian Journal of Mycology vol. 45 (2016) ISSN 2531-7342
DOI: 10.6092/issn.2531-7342/6195




 Genetic variation among isolates of Bipolaris
 maydis using RAPD_PCR
 ___________________________________________
                 Mahmoud Reza Karimishahri, Ramesh Chaudery Sharma

 Division of Plant Pathology, Indian Agricultural Research Institute, New Delhi 110012 India

 Correspondig Author e-mail: mrkarimi@iripp.ir


 Abstract

    Maydis Leaf Blight prevalent in many parts of India is a major threat to maize cultivation when grown in warm
    and humid climates. The pathogen responsible is Bipolaris maydis (Y. Nisik. & C. Miyake) Shoemaker. Ten
    isolates of the fungus were selected from different geographical regions in India for the present study.
    Preliminary primer screening was carried out using 32 primers from OPM, OPB and OPN series (Operon
    Technologies, Inc., USA) for molecular variation analysis. Out of 32 primers used for amplification of DNA of
    all isolates of B. maydis, two resulted in either sub-optimal or non-distinct amplification products. The
    similarity coefficients subjected to UPGMA-SAHN to produce a dendrogram, resulted in two major clusters,
    which separated Bm-6 from rest of the isolates, which were grouped in two major cluster, one having Bm-3 and
    Bm-2 and the second one having the rest of the isolates in two sub clusters. The first sub cluster included Bm-4
    and Bm-7 while Bm-1, Bm-5 and Bm-9 were grouped in second sub cluster. Isolates Bm-8 and Bm-10 were
    different from rest of the isolates in the second sub cluster thus remained one grouped. In present investigations
    though significant correlation between the genetic similarity and geographical origins could not be established.

    Keywords: maydis leaf blight; India; molecular markers


 Riassunto

    Il seccume fogliare del mais è una malattia diffusa in molte aree dell’India con clima caldo-umido. Il patogeno
    responsabile è Bipolaris maydis (Y. Nisik. & C. Miyake) Shoemaker. Dieci isolati del fungo sono stati
    selezionati per il presente studio da diverse aree geografiche dell’India. Uno screening preliminare è stato
    effettuato per l'analisi della variazione molecolare usando 32 primer delle serie OPM, OPB e OPN (Operon
    Technologies, Inc., USA). Dei 32 primer utilizzati per l'amplificazione del DNA da tutti gli isolati di B. maydis,
    due hanno dato prodotti di amplificazione sub-ottimali o non distinti. Il dendrogramma ottenuto dai coefficienti
    di similarità con il metodo UPGMA-SAHN è risultato in due cluster principali, uno con Bm-3 e Bm-2 ed un
    secondo con il resto degli isolati raggruppati in due sottogruppi. Il primo sottogruppo ha incluso Bm-4 e BM-7,
    mentre Bm-1, Bm-5 e BM-9 sono stati raggruppati nel secondo sottogruppo. Gli isolati Bm-8 e Bm-10 sono
    risultati diversi dal resto degli isolati, andando a costituire un gruppo a parte. Nel presente studio non è stato
    possibile stabilire una correlazione significativa tra la similarità genetica e le origini geografiche degli isolati.

    Parole chiave: seccume fogliare del mais; India; marker molecolari



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M. R. Karimishahri, R. C. Sharma                         Italian Journal of Mycology vol. 45 (2016) ISSN 2531-7342
DOI: 10.6092/issn.2531-7342/6195



 Introduction

 Maize has occupied an important place not only in India but elsewhere as well, due to its great significance
 as food, feed and industrial utilization. In India 61 diseases have been reported on maize (PAYAK and
 SHARMA, 1985). Among them Maydis Leaf Blight (MLB) prevalent in many parts of India, is a major
 threat to maize cultivation when grown in warm and humid climates. The disease has its presence in Jammu
 and Kashmir, Himachal Pradesh, Sikkim, West Bengal, Meghalaya, Punjab, Haryana, Rajastan, Delhi, Uttar
 Pradesh, Bihar, Gujarat, Maharashtra and Andhra Pradesh. Maydis Leaf Blight is incited by a fungal
 pathogen earlier referred to as Drechslera maydis (Y. Nisik. & C. Miyake) Subram. & B.L. Jain, but during
 the course of time the international scientific community has changed the generic name of the pathogen to
 Bipolaris maydis (Y. Nisik. & C. Miyake) Shoemaker. Thus here after the name of the pathogen is cited as
 Bipolaris maydis.
 In recent years, plant pathologists interested in genetic variation in pathogen populations have adopted the
 use of molecular markers as population genetics tools. Motivating this shift has been the availability of a
 myriad of molecular techniques which makes the quantification of genetic variation a relatively
 straightforward endeavor (MICHELMORE and HULBERT, 1987; BROWN, 1996). Molecular markers such as
 allozymes (LEUNG and WILLIAMS, 1986; GOODWIN et al., 1993), restriction fragment length
 polymorphisms (RFLP) (MCDONALD et al, 1990; KOHLI et al, 1992, MILGROOM et al, 1992) and random
 amplified polymorphic DNA (RAPD) (HAMELIN et al, 1994; PEEVER and MILGROOM, 1994) have been
 extensively used to characterize pathogen populations. More recently, amplified fragment length
 polymorphisms (AFLP) (MAJER et al, 1996; MUELLER et al, 1996) have proven to be highly polymorphic
 and robust markers and will likely be used extensively with plant pathogenic fungi in the future. In contrast
 to virulence and fungicide resistance markers, molecular markers are presumed to be selectively neutral and
 therefore may be used to study evolutionary processes in addition to selection (MILGROOM and FRY, 1997).
 The RAPD method was shown to be reproducible and to yield more objective results than symptom
 observation. In addition to morphological characteristics it can be rapid and reliable tool to identify a fungal
 isolate. RAPD patterns were extremely useful to identify false classified isolate. Clearly, new approaches are
 needed to improve the taxonomy of Bipolaris and changes should reflect their genetic relations. Recently, the
 phylogenetic relationships reflected in similarities at the level of DNA sequences have been adopted for
 comparison among many phenotypically uncertain (BAUN, 1992).To evaluate such similarities, DNA base
 composition, DNA homologies have been studied and compared (MULLANEY and KLICH, 1990;
 METZENBERY, 1991). Keeping these concerns in view, the present study was initiated to study genetic
 variation in isolates of B. maydis by PCR-RAPD.

 Materials and Methods

 Ten isolates of Bipolaris maydis (Y. Nisik. & C. Miyake) Shoemaker were selected for the present study.
 These isolates were collected from different geographical regions in India. The isolates with their designation
 (Bm) are listed in Tab. 2 and Fig. 1.




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M. R. Karimishahri, R. C. Sharma                              Italian Journal of Mycology vol. 45 (2016) ISSN 2531-7342
DOI: 10.6092/issn.2531-7342/6195


 Tab.1 Place of collection and designation given to individual isolates of Bipolaris maydis (Bm)
 Tab.1. Elenco, luoghi di provenienza e sigle degli isolati di Bipolaris maydis (Bm) utilizzati nel lavoro



                                                                                     Designation to
                    Place of collection        Location (State)
                                                                                     isolates

                    Changla                    Udaipur (Rajasthan)                    Bm-1
                    Jashipur                   Orissa (East) (Orissa)                 Bm-2
                    Assam                      Assam (East) (Assam)                   Bm-3
                    Almora                     Almora (Uttarakhand)                   Bm-4
                    Nia-Village                Udaipur (Rajasthan)                    Bm-5
                    IARI                       Delhi (New Delhi)                      Bm-6
                    Karnal                     Karnal (Haryana)                       Bm-7
                    Gorang                     Udaipur (Rajasthan)                    Bm-8
                    Pantnagar                  Pantnagar (Uttarakhand)                Bm-9
                    Undithal                   Udaipur (Rajasthan)                    Bm-10




 Fig. 1 Map of India with places where isolates were collected
 Fig.1 Mappa dell’India con i siti di provenienza degli isolati




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M. R. Karimishahri, R. C. Sharma                        Italian Journal of Mycology vol. 45 (2016) ISSN 2531-7342
DOI: 10.6092/issn.2531-7342/6195



 Fungal multiplication
 Potato dextrose broth was used to get mycelial growth of fungus for extraction of DNA. One hundred and
 fifty ml of medium was dispensed in 500 ml conical flasks and sterilized at 15 p.s.i. for 20 min. Each flask
 was inoculated with 6 mm diam. mycelial disc of the fungus, taken from the actively growing single spore
 cultures of different isolates growing on PDA. The inoculated flasks were incubated for 10 days at 28±1° C.
 At the end of incubation period, the mycelial mats were harvested by filtering through Whatman paper n.1
 filter paper, washed with sterilized water thrice, blot dried and stored in aluminum foils at –20° C.


 Genomic DNA isolation
 DNA extraction was done by procedures given by MURRAY and THOMPSON (1980) and ZOLAN and
 PUKILLA (1985) with slight modifications. Mycelial mats were weighed (1 g), ground in liquid N2 in pre-
 chilled pestle and mortar, taking care not to let the samples to thaw. Powdered mycelium was transferred into
 centrifuge tubes carrying 25 ml of pre-heated (65° C) 2 % CTAB extraction buffer to make a slurry. The
 tubes were incubated at 65° C for an hour and stirred occasionally with the help of sterile glass rod. Equal
 volume of chloroform isoamyl alcohol (24:1) was added to each tube and mixed gently. Samples were
 centrifuged at 10,000 rpm for 10 min. at room temperature. Upper aqueous phase was separated and
 precipitated with 0.6 vol. of ice-cold isopropanol and 0.1 vol. of 3M-sodium acetate (pH 5.2) and centrifuged
 at 15,000 rpm for 15 minutes. The pellets obtained were washed with 70% ethanol and kept for drying at
 room temperature (25° C). Total nucleic acid obtained was dissolved in sterile distilled water and stored at –
 20° C in small aliquots.


 Purification and quantification
 RNase treatment was given to remove RNA from the total nucleic acid. 2 µl of RNase from stock solution
 was added to nucleic acid extracted and incubated at 37° C for an hour. DNA concentration of samples and
 purity were determined by taking ultra-violet absorbance at 260 nm and 280 nm in a NanoDrop™ 8000
 spectrophotometer and rechecked by running samples on 1% agarose along with 1kb molecular weight
 marker (MBI, Fermentas).


 Random Amplification of Polymorphic DNA
 Optimization of Polymerase Chain Reaction (PCR)
 Composition of PCR reaction was optimized by varying the concentration of template DNA (25, 50, 75 and
 100 ng), Taq DNA Polymerase (0.5, 1.0 and 1.5 units) and MgCl2 concentration (3, 5, and 7.5 mM). The
 standardized amplification assay was as follows: Template DNA 25 ng; Taq DNA Polymerase (Banglor
 geni) 0.5 units; MgCl2 5 mM; dNTP (Genei) 100 µM each of dATP, dGTP, dCTP, dTTP, primer (Operon
 Technology) 1µM, Buffer (Genei)-1x in a reaction volume of 25 µl. Different PCR protocols given by
 PASCUAL et al., (2000) and LEE and TAYLOR (1990) were tested for obtaining best amplification of nucleic
 acid of the isolates under investigation. The Polymerase chain reaction was performed using Bio-Rad Gen
 cyclerTM, with the following temperature profile: the initial denaturation at 94° C for 2 min followed by 45
 cycles of three steps of denaturation at 92° C for 1 min. annealing at 37° C for 1 min. and extension at 72° C
 for 2 min. followed by final one elongation cycle of 72° C for 5 min.


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M. R. Karimishahri, R. C. Sharma                         Italian Journal of Mycology vol. 45 (2016) ISSN 2531-7342
DOI: 10.6092/issn.2531-7342/6195



 Primer screening and selection
 Preliminary primer screening was carried out using 32 primers from OPM, OPB and OPN series (Operon
 Technologies, Inc. USA) for molecular variation analysis. Out of 32 primers used for amplification of DNA
 of all isolates of B. maydis, 2 resulted in either sub-optimal or non-distinct amplification products. Therefore,
 these were discarded and remaining 30 were used for PCR amplification, which were found to give
 reproducible and scorable bands with high percentage of polymorphism. PCR amplification with each of the
 primers was done twice before scoring for presence and absence of bands. Each amplification product was
 considered as RAPD marker and recorded across all samples. The RAPD pattern of each isolate was
 evaluated assigning character state 1 to all the bands that could be reproducibly detected in the gel and 0 for
 the absence of band at same locus. Data was entered using a matrix in which all observed bands or characters
 were listed.


 Analysis of genetic diversity based on RAPD markers
 Estimation of genetic distance
 The data matrix thus generated was used to calculate Jaccard’s similarity coefficient for pair wise
 comparisons. For this purpose, NTSYS-pc Version 2.0 (ROHLF, 1998) software was used.
 Cluster analysis
 The RAPD data were analyzed using the Statistical Package for the Social Sciences (SPSS) software, 2nd
 edition (WILKINSON et al., 1992), through which similarity coefficients were calculated and a dendrogram for
 genetic distances constructed. In the present study, cluster analysis was done using commonly adopted
 clustering algorithm in genetic diversity analysis, namely UPGMA (Unweighted Pair Group Method using
 Arithmetic averages) (CRISCI and ARMENGOL, 1983). The resultant similarity matrices based on Jaccard’s
 measure were further analysed by performing sequential, agglomerative, hierarchical and nested clustering
 (ROHLF, 1998) using the UPGMA method. The results of clustering were plotted in the form of
 dendrograms.


 Results and Discussion

 Primer survey was carried out using 32 primers from OPM, OPB & OPN series of Operon Technologies,
 USA. It is indicated that out of 32 primers screened for amplification of DNA of isolates of B. maydis 30
 were found to give reproducible and scorable bands with high percentage of polymorphism (94 %). All the
 bands scored were polymophic in the amplification products maximum polymorphism was observed in
 OPN1, OPN3, OPN6, OPN8, OPN14, OPN15, OPN17, OPN 18 and OPN20 with 100 percent polymorphism
 as total number of bands obtained were polymorphic for these primers, while primers OPM19, OPM11,
 OPM2, OPM8, OPM12, OPM13 and also OPB6 gave a good indication of the variability, as the also resulted
 in more than percentage polymorphism (Fig. 2).
 Scoring and analysis of RAPD-PCR amplification
 The total number of bands obtained was 346 out of which 328 were polymorphic. Genetic variation observed
 in the study was maximum between isolates from Bm-6 and Bm-5 (66.2 per cent), closely followed by
 isolates Bm-6 and Bm-3 (64.4 per cent). Isolates from Bm-1 and Bm-5 were genetically closer than any other
 isolates with 60 percent, similarly.

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DOI: 10.6092/issn.2531-7342/6195




 Fig. 2 Genetic polymorphism between various isolates of Bipolarsis maydis by RAPD_PCR using different random
 primers
 Fig. 2 Polimorfismo genetico fra gli isolati di Bipolaris maydis determinato con RAPD_PCR usando differenti primers
 random

 Genetic similarity of 58.7 per cent between Bm-1and Bm-9 isolates were closely behind them. Bm-2 isolate
 was genetically closely related with Bm-3 isolate (50.9 per cent). This isolate exhibited maximum
 divergence at genetic level from Bm-6 with dissimilarity of 64.4 per cent. It was clearly observed that Bm-1
 and Bm-5 which had a common geographical origin were genetically closer. The isolates Bm-8 and Bm-10
 from Udaipur exhibited higher similarity among them. Similarly the East Indian isolates, Bm-3 and Bm-2
 had high genetic similarity. However, isolate Bm-6 was equidistant from rest of the isolates used in the
 present study (Tab. 1).
 The similarity coefficients subjected to UPGMA-SAHN to produce a dendrogram, resulted in two major
 clusters (Fig. 3) which separated Bm-6 from rest of the isolates, which were grouped in two major cluster,
 one having Bm-3 and Bm-2 and the second one having the rest of the isolates in two sub clusters. The first
 sub cluster included Bm-4 and Bm-7 while Bm-1, Bm-5 and Bm-9 were grouped in second sub cluster.
 Isolates Bm-8 and Bm-10 were different from rest of the isolates in the second sub cluster thus remained one
 grouped.
 It could be noted that there are two major clusters formed (Fig. 3). The prominent outcome of this clustering
 is that the East Indian isolates Bm-2 and Bm-3 were clustered in a separated sub group while Udaipur isolate
 including Bm-1 and Bm-5 were clustered in one sub group, the second cluster branched out into one cluster.
 The Bm-6 isolate was separate from rest of this isolates.
 Several primers were observed to be good markers for some isolates of B. maydis in the present study. The
 banding pattern by OPM14 primer distinguished the isolate Bm-6 and isolate Bm-7 and Bm-1 isolate from
 rest of the isolates (Fig. 2).




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DOI: 10.6092/issn.2531-7342/6195




 Fig. 3 Dendrogram showing relationship between different isolates of Bipolaris maydis based on RAPD analysis using
 UPGMA
 Fig. 3 Dendrogramma degli isolati di Bipolaris maydis basato su analisi RAPD usando il metodo UPGMA



 Conclusions

 Random amplified polymorphic DNA (RAPD) has increasingly been used for the determination of
 polygenetic relationships and for the estimation of genetic diversity within fungal population. More recently
 the use of the molecular markers has given a boost to the analysis of accurate variation among various
 isolates of the pathogen.
 BAKONYI et al. (1994) reported that species of Bipolaris formed a compact group, while Drechslera showed
 a strong dissimilarity; species of Exserohilum were scattered within the latter. SILVAKUMAR et al. (1995)
 reported that strains of Corynespora could be distinguished from a member of the closely related genus
 Helminthosporum on the basis of amplified ITS fragment size, but could not be typed individually as the ITS
 regions of all isolates exhibited identical size and restriction endonuclease digestion pattern.
 ABADI et al. (1996) estimated DNA variability between thirteen isolates of Exserohilum turcicum races 0, 1,
 2, 23 and N by RAPD and suggested that race 0, 1, and N may be grouped separately from race 23. BAKONYI
 et al. (1995) reported that Drechslera formed a large heterologus group, while species of Bipolaris and
 Exserohilum were more closely related. PELTONEN et al. (1996) reported that all Drechslera teres isolates
 could not be according to geographic origin, aggressiveness, growth rate or morphological features,
 indicating that the primers used in this study were natural markers for these characters. The primers were
 however, able to differentiate between isolates of Helminthosporium species. NICHOLSON et al. (1993)
 compared isolates of B. maydis from China (Races O, C, and T) using two techniques RAPD analysis
 indicated that race O and C isolates were more similar to each other than that of the race T isolates.
 The selection of primers has great significance in pathotype identification and characterization and molecular
 mapping of the genome. These primers can also serve as a reference for integral comparison in future
 studies. In the presents study, characterization of B. maydis isolates by RAPD has proved useful in separating
 all the isolates from each other. It has also provided with primer marker that can be used to separate and
 distinguish each isolate. Unique band of 500 kb size by Bm-6 obtained by amplification with OPM 14 primer
 can be used further for development of SCAR marker. This possibility of distinguishing different isolates by
 rather simpler technique of genomic fingerprinting based on PCR-RAPD, could be of great important for use
 in epidemiological , taxonomical and evolutionary aspects.
 In present investigations though no correlation could be found between morphological and genetical
 attributes, significant correlation between the genetic similarity and geographical origins could be

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M. R. Karimishahri, R. C. Sharma                       Italian Journal of Mycology vol. 45 (2016) ISSN 2531-7342
DOI: 10.6092/issn.2531-7342/6195



 established. These findings are similar to those obtained by FABRE et al. (1995) in a study on Colletotrichum
 lindemuthianum from different countries, they investigated grouping by DNA polymorphism in relation to
 the geographical origins of the isolates were formed two groups with isolates from Latin America occurring
 in both groups.
 RAPD analysis offers the possibility of creating polymorphism without any prior knowledge of the DNA
 sequences of test organism. The patterns produced are highly polymorphic, allowing discrimination between
 isolates of a species, if sufficient numbers of primers are screened. The method is also an economic
 technique for screening large number of samples. Although, some researchers are critical of the poor
 reproducibility of RAPD pattern, in present investigation, it was found that once the optimal RAPD
 conditions for a given species are established, the method works well for fungal samples even on crude DNA
 extracts.

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M. R. Karimishahri, R. C. Sharma                           Italian Journal of Mycology vol. 45 (2016) ISSN 2531-7342
DOI: 10.6092/issn.2531-7342/6195




 Fig. 4. Symptoms of maydis leaf blight caused by Bipolaris maydis
 Fig. 4 Sintomi del seccume fogliare causato da Bipolaris maydis




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