RESEARCH ARTICLE

RAPD and Nuclear rDNA ITS PolymorphismWithin Macrophomina phaseolina Isolatedfrom Arid Legumes of Western Rajasthan

Ragini Gautam • S. K. Singh • Vinay Sharma

Received: 27 December 2012 / Revised: 11 June 2013 / Accepted: 19 June 2013

� The National Academy of Sciences, India 2013

Abstract Concurrent heat and moisture stress often

favours root diseases caused by Macrophomina phaseolina

in arid legumes. Molecular analysis revealed 92 % varia-

tion within M. phaseolina populations as compared to 8 %

among populations. The first three principle coordinates of

PCA analysis accounted for a 69.61 % of total variance and

Eigen vectors revealed 22.89 % of total variability. The

mean values of all the four populations together for Nei’s

gene diversity (h) was 0.1990 and Shannon’s information

index (i) was 0.3113. The result showed that the genetic

diversity of M. phaseolina isolates of population 2 (cow-

pea) was richest among all the four populations. Analysis

of molecular variance indicated that main proportion of

genetic variation was within the host than among different

hosts. Out of 13 representative isolates seven were

molecularly identified as Rhizoctonia bataticola and six as

M. phaseolina upon sequencing of 5.8S RNA region.

Besides length variation in ITS-1, 5.8S rRNA gene, ITS-2

and total length, the authors report insertion/deletions at a

number of places in 13 isolates sequenced. This study

underlines that M. phaseolina distribution is independent of

host and/or geography and validates the utility of ITS

rDNA region as a reliable indicator of phylogenetic inter-

relationships as an additional approach for identification of

the genus Macrophomina and assessing its genetic

diversity.

Keywords R. bataticola � M. phaseolina �Genetic diversity � RAPD � rDNA analysis

Introduction

The fungus Macrophomina is an anamorphic Ascomycete

which has two asexual sub-phases: (1) a sclerotial phase

Rhizoctonia bataticola and (2) a pycnidial phase Macro-

phomina phaseolina [1, 2]. It is one of the most devastating

seed and soil borne pathogen, infecting over 500 plant

species throughout the world [3, 4]. Under moisture stress

condition, the fungus causes many diseases like seedling

blight, collar rot, stem rot, charcoal rot, root rot and dry

root rot in various economically important crops.

Unfortunately, arid legumes due to their inherent

drought hardy characteristics are mostly grown under rain

fed conditions where land conditions are not suitable for

cultivation of cereals. During kharif, low and erratic rain-

fall, short and long duration of soil moisture stress, low

microbial population of sandy soils along with concurrent

heat stress favours occurrence of dry root rot caused by M.

phaseolina in mild to severe form in all the arid legumes

[5, 6].

The identification of isolates of M. phaseolina is usu-

ally based on morphological criteria, but due to extensive

variation in the phenotypes of the isolates these criteria

are often not reliable. Although only a single species has

been recognized in the genus Macrophomina, high levels

of variations have been found in the degree of pathoge-

nicity [7]. The genetic diversity of Macrophomina could

favour its survival and adaptation to variable environ-

ments because of significant morphological [8], physio-

logical [3] pathogenic [8, 9] and genetic [7, 10–16]

diversity.

R. Gautam � S. K. Singh (&)

Central Arid Zone Research Institute, Jodhpur 342003,

Rajasthan, India

e-mail: sksingh1111@hotmail.com

V. Sharma

Department of Biosciences and Biotechnology, Banasthali

Vidhyapeeth, Banasthali 304022, Rajasthan, India

123

Proc. Natl. Acad. Sci., India, Sect. B Biol. Sci.

DOI 10.1007/s40011-013-0207-5

Molecular markers, especially DNA techniques are

quick and reliable methods to establish the identities of

wild collections and are helpful in revealing genetic

diversity both at inter and -intra specific levels and has

resolved many taxonomic problems. PCR based molecular

approaches have been used to resolve genetic variations

among M. phaseolina isolates [13, 14, 17]. Molecular

characterization of M. phaseolina on the basis of patho-

genicity, host and/or geographical origin have been

reported by using RAPD, simple sequence repeats (SSR)

and URP-PCR [12–14]. Identification and detection of M.

phaseolina by using species specific oligonucleotide

primers and probes have also been carried out by Babu

et al. [18].

The objectives of the present study were: (1) to develop

better understanding of Macrophomina population diver-

sity in arid legumes occurring in western Rajasthan and (2)

to optimize breeding strategies, genetic diversity studies in

M. phaseolina isolates sampled from different arid legumes

using RAPD and nuclear rDNA ITS analysis.

Material and Methods

Isolation of Fungus

Diseased plants of four arid legumes (clusterbean, cowpea,

moth bean, horse gram) were collected from three districts

of Rajasthan viz., Jodhpur, Bikaner and Jaipur during rainy

season of 2011. The infected root bits were surface steril-

ized using sodium hypochlorite for 5 min and washed

thrice in sterilized distilled water. These infected root bits

were then placed in petri plates containing potato dextrose

agar (39 g PDA; Hi Media Company) culture medium and

incubated at 25 ± 2 �C for 5 days. The growing tips of the

mycelia along with the culture medium of root rot patho-

gens were aseptically transferred in PDA test tubes slants

and again incubated at 25 ± 2 �C for 7 days. Pure cultures

were stored at 4 �C until used and/ or sub-cultured. To

perform molecular analysis, the isolates of M. phaseolina

were divided into four populations based on the crop from

which they were isolated.

Morphological Characterization

All the Macrophomina cultures were sub-cultured in petri

plates containing PDA for 5 days. A uniform disc of 5 mm

was cut using Cork Borer and placed in the centre of the

petri plates and kept at 25 ± 2 �C in a BOD incubator for

5 days. The colony growth pattern, colony colour and size

of micro-sclerotia were measured. The sclerotial size was

measured using compound microscope, ocular and stage

micrometers using the following formula.

Calibration Factor ¼Number of divisions of stage micrometers

Number of divisions of ocular micrometers� 10

Genomic DNA Isolation and Quantification

For DNA isolation, 5 mm pieces of growing mycelia were

transferred to malt extract-dextrose broth culture medium

(malt extract 10 g; dextrose 5 g with anti-bacterial agent

streptocyclin 150 mg in one liter of sterilized distilled

water) and incubated in an incubator at 25 ± 2 �C for

7 days. The genomic DNA was extracted from 100 mg of

fresh mycelium of each M. phaseolina isolate, crushed

with micro-pestle in liquid nitrogen. The Hi Pura kit of Hi

Media Company and protocol suggested by Birren and

Lai [19] and Sambrook et al. [20] were followed for the

same. The eluded genomic DNA was quantified with UV/

Vis spectrophotometer by measuring OD260/OD280. The

quantified DNA samples were diluted in TE buffer to

make a final concentration of 50 ng/ll for PCR

amplifications.

RAPD Analysis

A set of 22 decamer random primers of OPA, OPB and

OPP series (Operon Technologies) were used for initial

screening of all the isolates of M. phaseolina. Finally, the

data of 10 RAPD primers exhibiting consistent results was

used for analysis. Each amplification was performed in a

total reaction mixture of 25 ll. Each reaction mixture

contained: decamer primer, 1 ll (50 pmol ll-1); dNTP

mix, 2 ll (2 mM each of dATP, dGTP, dCTP and dTTP

from MBI, Fermentas); MgCl2, 1 ll (25 mM, MBI, Fer-

mentas); Taq DNA polymerase, 0.4 ll (5U ll-1, Sigma

Chem); 109 PCR buffer, 2.5 ll (Sigma Chem), 4 ll of

genomic DNA (50 ng ll-1) in dH2O. PCR amplifications

were performed in a gradient thermal cycler (Corbett

Research, USA) with initial denaturation step of 94 �C for

3 min followed by 38 cycles of 94 �C for 40 s, 50 �C for

40 s and 72 �C for 2 min and final elongation at 72 �C for

10 min.

Amplicons were separated on 1.5 % agarose gel

(Sigma) pre-stained with ethidium bromide solution using

19 TAE buffer. The gel was run for 3 h at 50 V and the

RAPD amplicon profiles were recorded using Syngene

Gel Documentation System with Genesnap software. The

size of amplified fragments was determined using 1 kb

ladder (MBI Fermentas). All RAPD reactions were per-

formed twice to test the reproducibility of the amplicon

profile.

R. Gautam et al.

123

ITS Amplification

Primers namely ITS-1 (50 TCC GTA GGT GAA CCT GCG

G 30) and ITS-4 (50 TCC TCC GCT TAT TGA TAT GC 30)developed by White et al. [21] were used for amplification.

Each PCR amplification was performed in a total volume

of 50 ll containing: 1 U Taq DNA polymerase (Sigma

Chem), 2.5 mM MgCl2, 160 lM dNTP mix (MBI Fer-

mentas), 50 pmol of each of the ITS-1 and ITS-4 primers

(Bangalore Genei), and 50 ng genomic DNA in dH2O. The

reactions were performed in a gradient thermal cycler with

the following conditions: 1 min denaturation at 95 �C, 30 s

annealing at 50 �C, 90 s elongation at 72 �C, for 34 cycles

with a final elongation step of 72 �C for 10 min.

Amplified ITS regions were sequenced employing ABI

Prism DNA sequencer (Applied Biosystems, Carlsbad, CA,

USA) using ITS-1 and ITS-4 primers separately for DNA

labeling by the BigDye terminator method at South Cam-

pus, Delhi University, New Delhi. The sequenced data

obtained from the ITS-4 primer were inversed using Gene

Doc software [22] and clubbed with the sequence data

obtained with the ITS-1 primer, to obtain the complete

sequence of the ITS region. Comparison of nucleotide

sequences was performed using the basic local alignment

search tool (BLAST) available at the National Centre for

Biotechnology Information (NCBI) database (http://www.

ncbi.nlm.nih.gov). Molecular characterization of fungal

isolates was done on the basis of similarity with the best

aligned sequence of BLAST search.

Molecular Analysis of RAPD

The RAPD amplification products were scored as present

(1) and absent (0) of scorable loci for each primer isolate

combination. Molecular data were entered into a binomial

matrix and were used to determine Jaccard’s similarity

coefficient with NTSYS-pc software [23, 24]. Most infor-

mative primers selected were based on high polymorphism

information content (PIC) value of individual primers.

PIC ¼Xn

i¼1

2Fð1� FÞ

where F is frequency of presence of marker band, i is

discrimination rate (DR) which was estimated to test the

efficacy of individual primers in distinguishing the isolates,

employing the following formula.

DR = number of pairs of isolates differentiated/total

number of pairs.

To perform molecular analysis, all the 33 isolates of M.

phaseolina were divided into four populations based on the

arid legume crop from which they were isolated. Principal

coordinate analysis via covariance matrix was calculated

using GenALEx 6 software [25]. On the other hand,

diversity in the frequency of fragment size of RAPD pat-

terns was apportioned within and among M. phaseolina

isolates using Shannon’s information index (i) [26] and

gene diversity index (h) following Nei [27] using PopGen

32 programme.

Sequence Analysis

Nucleotide sequence comparisons were performed by using

the BLAST (NCBI) databases. The multiple sequence

alignment of the ITS region (ITS-1, 5.8S r-RNA gene and

ITS-2) representing the 13 isolates of M. phaseolina was

performed using CLUSTAL X 1.83 software to detect

single nucleotide polymorphism. The phylogenetic rela-

tionship among the isolates was estimated after the con-

struction of a phylogram based on multiple sequence

alignment of rDNA ITS sequences with the Tree View

software [28].

Results

Besides other pathogens associated to the diseased roots of

arid legumes, 33 root samples were found infected with M.

phaseolina. Morphologically, these isolates were grouped

into three colony growth patterns, namely restricted,

feathery and dense type. Most of the isolates were recorded

with feathery colony growth. The colony colour varied

from white, grey, brown to black. Microscopically, the

micro-sclerotial size varied from 38.5 lm (JD-HG2

to128.2 lm, JAI-MB11). The details of the district, host

and fungal characteristics are given in Table 1. The results

indicated that there was no consistency with regard to

colony growth pattern, colony colour and/or micro-scle-

rotial size as the isolates collected from different localities

i.e., Jodhpur, Bikaner and Jaipur of arid legume crops

recorded with different colony growth patterns and colony

colours within locality and/or host crop. The results indi-

cated that the morphological characterization of M.

phaseolina isolates were highly variable, inconsistent and

were insufficient to group them into distinct clusters.

Out of 22 decamer random primers initially tested, 10

primers detected intraspecific variations generating scor-

able amplicons, reproducible patterns that has generated

119 marker bands in the range of 300–4,025 bp. Of which,

94 marker bands were polymorphic amounting to 76.7 %

polymorphism and the polymorphism ranged between 50

and 94.11 %. The number of PCR amplification products

ranged from 9 (OPB-04: OPP-16) to 17 (OPA-02) with an

average of about 12 bands per primer (Table 2). The primer

OPA-02 was the most informative primer which exhibited

94.11 % polymorphism in RAPD banding patterns

RAPD and Nuclear rDNA ITS Polymorphism

123

(Fig. 1A, B). The PIC value varied from 86 to 91 % with

an average of 88.3 %. The primer OPA-02 exhibited the

maximum PIC value of 91 % which was closely followed

by OPA-13 and OPA-16 with 90 % polymorphic content.

It is evident from the dendrogram that all the isolates

were clearly delineated into six main clusters and five

isolates JD-HG8, JD-CP3, BK-MB5, JAI-CB3 and JD-CP2

as distinct isolates (Fig. 2). Cluster I contained three iso-

lates of Jodhpur: JD-HG1, JD-MB2 and JD-MB3. Cluster

II compiled of 10 isolates of Jodhpur: JD-HG2, JD-HG3,

JD-HG7, JD-HG5, JD-HG4, JD-CP1, JD-CP5, JD-HG6,

JD-CP4 and JD-HG8. Cluster III comprised of 10 isolates

of Jaipur and Bikaner: JAI-CB4, BK-MB6, BK-MB7,

JAI-CP6, BK-MB8, JAI-MB9, JAI-MB13, JAI-CP7,

JAI-MB11 and JAI-MB10. Cluster IV, V and VI each

contained two isolates namely: JAI-MB12 and JAI-CP8, JD-

CB1 and JD-CB2 and JD-MB1 and JD-MB4, respectively.

The comparative growth patterns of representative iso-

lates of RAPD clusters and all the distinct isolates can be

seen in Fig. 3. Based on the clustering patterns of different

M. phaseolina, 13 representative isolates from RAPD

cluster and all distinct isolates were selected for nuclear

rDNA ITS region sequencing. All selected isolates were

subjected to PCR amplification of ITS region and com-

passing 5.8S gene region using universal primers ITS-1 and

ITS-4. All the representative isolates generated a single

Table 1 Morphological characterization of Macrophomina phaseolina isolates causing root diseases in arid legumes

S. No. District Host Isolate Colony growth

pattern

Colony colour Micro Sclerotia

size (lm)

Disease

symptoms

1. Jodhpur Horse Gram JD-HG1 Restricted Black 66.8 Root rot

2. Jodhpur Horse Gram JD-HG2 Feathery Grey 38.5 Root rot

3. Jodhpur Horse Gram JD-HG3 Feathery Grey 58.1 Root rot

4. Jodhpur Horse Gram JD-HG4 Restricted White 54.2 Dry root rot

5. Jodhpur Horse Gram JD-HG5 Feathery Grey 79.8 Root rot

6. Jodhpur Horse Gram JD-HG6 Dense Black 50.7 Root rot

7. Jodhpur Horse Gram JD-HG7 Feathery Black 44.4 Root rot

8. Jodhpur Horse Gram JD-HG8 Feathery White 62.1 Seedling rot

9. Jodhpur Cowpea JD-CP1 Dense Brown 75.2 Dry root rot

10. Jodhpur Cowpea JD-CP2 Feathery White 43.8 Dry root rot

11. Jodhpur Cowpea JD-CP3 Dense Grey 62.7 Charcoal rot

12. Jodhpur Cowpea JD-CP4 Feathery Grey 110.0 Root rot

13. Jodhpur Cowpea JD-CP5 Restricted Grey 39.3 Dry root rot

14. Jodhpur Clusterbean JD-CB1 Feathery Grey 83.2 Root rot

15. Jodhpur Clusterbean JD-CB2 Feathery Brown 62.7 Root rot

16. Jodhpur Moth bean JD-MB1 Feathery Grey 74.6 Root rot

17. Jodhpur Moth bean JD-MB2 Feathery Brown 73.5 Dry root rot

18. Jodhpur Moth bean JD-MB3 Restricted White 46.1 Dry root rot

19. Jodhpur Moth bean JD-MB4 Feathery Black 114.0 Dry root rot

20. Bikaner Moth bean BK-MB5 Feathery Grey 120.8 Dry root rot

21. Bikaner Moth bean BK-MB6 Dense Grey 89.4 Root rot

22. Bikaner Moth bean BK-MB7 Feathery Black 114.0 Root rot

23. Bikaner Moth bean BK-MB8 Feathery Black 51.3 Dry root rot

24. Jaipur Clusterbean JAI-CB3 Restricted White 72.3 Seedling rot

25. Jaipur Clusterbean JAI-CB4 Dense Black 85.5 Charcoal rot

26. Jaipur Moth bean JAI-MB9 Dense Black 43.3 Dry root rot

27. Jaipur Moth bean JAI-MB10 Restricted White 103.1 Charcoal rot

28. Jaipur Moth bean JAI-MB11 Feathery Brown 128.2 Charcoal rot

29. Jaipur Moth bean JAI-MB12 Dense Black 84.3 Charcoal rot

30. Jaipur Moth bean JAI-MB13 Dense Black 112.2 Dry root rot

31. Jaipur Cowpea JAI-CP6 Feathery Grey 124.8 Root rot

32. Jaipur Cowpea JAI-CP7 Restricted White 59.8 Charcoal rot

33. Jaipur Cowpea JAI-CP8 Feathery Black 75.2 Charcoal rot

R. Gautam et al.

123

band of *500 bp which included partial sequences of 18S

gene complete sequence of ITS-1, 5.8S gene, ITS-2 and

partial sequences of 28S gene upon direct sequencing

(Fig. 4).

All the nucleotide sequences were subjected to BLAST

search for molecular identification of the isolates up to

species level. Out of 13 isolates, seven were molecularly

identified as R. bataticola (sclerotial phase) and six as M.

phaseolina (pycnidial phase). The molecular identification

of the best aligned reference gene sequence with their error

values and the maximum identities are given in Table 3.

The conserved 5.8S rDNA region was recorded with a

uniform nucleotide length of 158 bp except in JAI-CB3

which was recorded with 157 bp length. Besides length

variations in ITS-1, 5.8S rRNA gene, ITS-2 and total

length, many insertions/deletions at a number of places

among 13 isolates sequenced have been reported (Table 4).

The nucleotide sequences were subjected to multiple

sequence alignment. The phylogram generated using the

tree view software programme further delineated these 13

Fig. 1 A RAPD profiles of

Jodhpur isolates of M.

phaseolina amplified by OPA-

02 primer. B RAPD profiles of

Bikaner and Jaipur isolates of

M. phaseolina amplified by

OPA-02 primer

Table 2 Details of primer code, GC content, per cent polymorphism and PIC values of RAPD primers

S.n. Primer code Primer

Sequence

GC (%) No. of bands No. of polymorphic

bands

Polymorphism

(%)

PIC values

1. OPA-02 TGC CGA GCT G 70 17 16 94.11 91 %

2. OPA-10 GTG ATC GCA G 60 13 11 84.61 88 %

3. OPA-13 CAG CAC CCA C 70 12 9 75.00 90 %

4. OPA-16 AGC CAG CGA A 60 15 14 93.33 90 %

5. OPB-04 GGA CTG GAG T 70 9 5 55.55 86 %

6. OPB-05 TGC GCC CTT C 70 10 5 50.00 89 %

7. OPB-10 CTG CTG GGA C 70 12 10 83.33 89 %

8. OPB-13 TTC CCC CGC T 70 10 7 70.00 86 %

9. OPP-09 GTG GTC CGC A 70 12 10 83.33 88 %

10. OPP-16 CCA AGC TGC C 70 9 7 77.77 86 %

Total 119 94

Average 76.70 88.30

RAPD and Nuclear rDNA ITS Polymorphism

123

Fig. 2 Dendrogram of 33

isolates of M. phaseolina based

on 10 RAPD informative

primers

Fig. 3 Colony growth patterns

of 13 M. phaseolina

representative isolates

Fig. 4 ITS profiles of 13 M.

phaseolina representative

isolates

R. Gautam et al.

123

isolates representing all the six phylogenetic RAPD clus-

ters and distinct genotypes (Fig. 5). An insight of phylo-

gram revealed close lineages of GenBank reference

sequences FJ415067, HM990163, HQ625641 and

HQ392809 with most of the representative isolates having

significant Bootstrap values. All the gene sequences have

been submitted to NCBI database and assigned GenBank

Accession numbers from JQ954868 to JQ954880.

Upon BLAST search, six representative isolates were

identified as M. phaseolina and seven as R. bataticola

(Table 3). Six isolates identified as M. phaseolina exhib-

ited the maximum identities ranging from 95 to 99 % with

three GenBank reference sequences, whereas all seven R.

bataticola isolates exhibited 96–99 % identities with the

reference sequence HQ392809. A high degree of

nucleotide sequence variation allowed separation of all the

13 isolates of M. phaseolina in the present study.

The summary of analysis of molecular variance (AM-

OVA) of RAPD data using GenaLX is shown in Table 5. The

analysis revealed 92 % variations within populations as

compared to 8 % among populations of M. phaseolina. Four

M. phaseolina populations were subjected to principle

coordination analysis (PCA) using GenaLX program

(Fig. 6). The first three principle coordinate accounted for

29.58, 24.76 and 15.27 respectively amounting to a total of

69.61 % of total variance. The Eigen vector analysis indi-

cated that the contributions of the first three factors were

9.73, 8.14 and 5.02, respectively (explaining a total of 22.89

of total variability). Coefficient of gene differentiation

between populations (Gst) was 0.2134. The gene flow (Nm)

Table 3 Molecular

identification of representative

isolates on the basis of ITS

sequences using BLAST

programme with that of

GenBank reference sequences

S. No. Isolate Molecular

identification

GenBank

accession

Error

value

Max. identities

(%)

1. JD-HG1 Macrophomina phaseolina FJ415067 0.0 99

2. JD-HG5 M. phaseolina FJ415067 0.0 99

3. JD-CP2 M. phaseolina FJ415067 0.0 98

4. JD-CP3 M. phaseolina HQ625641 0.0 99

5. JD-CP5 Rhizoctonia bataticola HQ392809 0.0 99

6. JD-CB1 R. bataticola HQ392809 0.0 99

7. JD-CB2 M. phaseolina HM990163 0.0 98

8. JAI-CB4 M. phaseolina HM990163 0.0 95

9. JD-MB3 R. bataticola HQ392809 0.0 99

10. JD-MB4 R. bataticola HQ392809 0.0 99

11. BK-MB5 R. bataticola HQ392809 0.0 99

12. BK-MB8 R. bataticola HQ392809 0.0 96

13. JAI-MB13 R. bataticola HQ392809 0.0 99

Table 4 Nucleotide base pair

lengths of nuclear ribosomal

RNA gene region of 13

representative M. phaseolina

isolates of RAPD sub-clusters

S. No. Genotype GenBank

accession number

ITS-1 (bp) 5.8S (bp) ITS-2 (bp) Total (bp)

1. JD-HG1 JQ954868 181 158 152 491

2. JD-HG5 JQ954869 180 159 152 491

3. JD-CP2 JQ954870 181 158 152 491

4. JD-CP3 JQ954871 181 158 152 491

5. JD-CP5 JQ954872 181 158 152 491

6. JD-CB1 JQ954873 181 158 152 491

7. JD-CB2 JQ954874 182 158 150 490

8. JAI-CB4 JQ954875 181 157 152 490

9. JD-MB3 JQ954876 181 158 152 491

10. JD-MB4 JQ954877 181 158 152 491

11. BK-MB5 JQ954878 181 158 152 491

12. BK-MB8 JQ954879 181 158 152 491

13. JAI-MB13 JQ954880 181 158 152 491

RAPD and Nuclear rDNA ITS Polymorphism

123

varied from 0.4665 to 27.6041 between pair wise populations

and was recorded 1.8427 among all the populations.

To perform genetic analysis of RAPD data, all the 33

isolates of M. phaseolina were divided into four popula-

tions viz. Population 1 (eight isolates of horse gram),

Population 2 (eight isolates of cowpea) Population 3 (four

isolates of clusterbean) and Population 4 (13 isolates of

moth bean). A perusal of distribution of populations across

Principle Coordinates exhibited that the population 2 and

population 4 were widely distributed across all the three

Principle Coordinates as compared to others.

The result revealed significant genetic diversity among

M. phaseolina isolates. A summary of mean genetic vari-

ation statistic of all the four populations and mean of all

loci is presented in Table 6. The mean values of all the four

populations together for Nei’s gene diversity (h) was

0.1990 and Shannon’s Information Index (i) was 0.3113.

The result reveals that the genetic diversity of M. phase-

olina isolates of population 2 (cowpea) was the richest

among all the four populations. Nei’s unbiased measures of

genetic distance were employed to further elucidate the

gene differentiation among populations (Table 7). The

Nei’s genetic distance ranged from 0.0217 to 0.0951 and

the genetic identities ranged from 0.9093 to 0.9551. The

largest distance occurred between population 1 (horse

gram) and 3 (clusterbean) and the least between population

2 and 4 and vice versa for genetic identity.

Discussion

A comparison of morphological vis-a-vis RAPD markers

revealed that there is no consistency in grouping of M.

phaseolina isolates as morphologically similar isolates

Fig. 5 Phylogram generated

using tree view of multiple

sequence aligned rDNA region

of 13 M. phaseolina isolates

Fig. 6 The principal

coordinates analysis (PCA) of

four M. phaseolina populations

using GenALEx software

Table 5 Summary AMOVA

tableSource df SS MS Est. Var. %

Among pops 3 48.383 16.128 0.806 8

Within pops 29 284.587 9.813 9.813 92

Total 32 332.970 10.619 100

R. Gautam et al.

123

were genetically cataloged into different RAPD clusters.

For instance, M. phaseolina isolates belonging to same

colony growth pattern and/or colony colour fall into dif-

ferent phylogenetic clusters i.e., isolates JD-HG2, JD-HG3,

JD-HG5, JD-CP4, JD-CB1, JD-MB1, BK-MB5 and JAI-

CP6 recorded with feathery colony growth pattern and grey

colony colour were delineated into different phylogenetic

clades. Interestingly, phylogenetic clustering was also not

in accordance with the host from which the isolates were

isolated. The isolates belonging to the different hosts can

be seen in almost all the major phylogenetic clusters.

The results indicated that measures of relative genetic

distances among populations did not completely correlate

the geographical distances of places of their origins and/or

hosts suggesting that the fungus Macrophomina is neither

confined to geographical location and/or is host specific in

nature. The present results are in agreement with earlier

molecular studies. Rajkumar et al. [29] suggested that

grouping of isolates was not related to sampling location.

Baird et al. [30] collected M. phaseolina from a wide spread

host and geographic range across the United States and

noticed grouping of isolates independent of host and geog-

raphy. By contrast, a few researchers have claimed to have

distinguished M. phaseolina isolates into area specific

groups [31–33] and/or exhibited host specificity [12, 34–36].

DNA markers, including the random amplified poly-

morphic DNAs (RAPDs) produced by PCR can be used for

the characterization of microorganisms and detection of

microbial diversity [37]. In this study, RAPD markers

system revealed high levels of polymorphism among M.

phaseolina isolates amounting to 76.7 % indicating its

effectiveness in evaluating genetic diversity of M. phase-

olina. The average PIC values of 88.3 % with the maxi-

mum of 91 % by OPA-02 closely followed by OPA-13 and

OPA-16 with 90 % polymorphic content shows the

robustness of the primers used in the present study. The

significance of assessment of wild genetic diversity within

M. phaseolina using DNA based markers like RAPD [12,

29, 32, 34, 38, 39], ISSR [40], SSR [30, 41], AFLP [33,

42], RFLP and rDNA sequencing [36], Universal Rice

Primers [14], RAPD, ITS-AFLP and ITS sequencing and

Oligonucleotides specific probes [31] have also been

reported . The high degree of uniformity in the total length

of 490–491 bp in 13 sequenced isolates of M. phaseolina

validates the conserved nature of nuclear ribosomal RNA

gene region whereas, length polymorphism in the intron

and exon regions of the gene indicate genetic variability

among the isolates, indicating faster rates of evolution.

The clustering of GenBank reference sequences with most

of the test isolates validate that the isolates belong to the genus

Macrophomina and at the same time exhibited substantial

intra-specific genetic diversity due to variations in the

nucleotide sequences by way of SNPs, INDELS and ITS

length polymorphism. Further, two isolates namely JD-HG5

and JD-CP2 of M. phaseolina emerged out to be the most

distinct out groups despite exhibiting 99 and 98 % identities

with the GenBank reference sequence FJ415067 due to

SNP’S and insertions/deletions. The delineation of all the 13

isolates of M. phaseolina with high Boot Strap values strongly

supports wide geographical distributions of the fungus which

is further validated by the fact that out of 13 isolates, nine

isolates exhibited 99 % identities with GenBank reference

sequences from entirely different geographical regions.

High degree of polymorphism in restriction pattern and

variability in nucleotide sequence of nrDNA ITS regions

have also been reported among isolates of Macrophomina

isolates [31, 34]. Saleh et al. [36] based on RFLP and

sequences of rDNA-ITS regions divided 143 isolates into

Table 6 Summary of genetic

variation statistics for all loci

na observed number of alleles,

ne effective number of alleles,

h Nei’s gene diversity,

i Shannon information index

Locus (mean) sample size na ne h i

Pop 1 8 1.2605 1.1504 0.0878 0.1321

Pop 2 8 1.5546 1.3451 0.1962 0.2912

Pop 3 4 1.4454 1.3171 0.1766 0.2581

Pop 4 13 1.5294 1.3235 0.1825 0.2705

Mean of all loci 33 1.7899 1.3247 0.1990 0.3113

Table 7 Matrix of unbiased genetic identity and genetic distance according to Nei [27] among four populations of M. phaseolina based on

RAPD markers

Population Pop 1 Pop 2 Pop 3 Pop 4

Pop1 – 0.9406 0.9093 0.9461

Pop2 0.0612 – 0.9500 0.9785

Pop3 0.0951 0.0513 – 0.9551

Pop4 0.0554 0.0217 0.0459 –

Nei’s genetic identity (above diagonal) and genetic distance (below diagonal)

RAPD and Nuclear rDNA ITS Polymorphism

123

different clusters and reported that most of the isolates

from maize and soybean were clubbed together into the

same cluster indicating that the fungus is not host specific.

The AMOVA analysis revealed greater variations (92 %)

within populations as compared to among populations

(8 %) indicates that the greater proportion of variability

within host as compared to different hosts indicating that

the fungus is not host specific in nature. The results of PCA

analysis clearly indicated that population 2 representing

cowpea and population 4 of moth bean were more diverse

as compared to that of horse gram and clusterbean. Present

study based on RAPD genetic analysis and nrDNA ITS

sequencing revealed significant genetic diversity among

isolates of M. phaseolina collected from different locations

and arid legume hosts underlining non-host specific and

wide distribution of the fungus across geographic locations.

Therefore evaluation of arid legume germplasm may be

carried out against genetically diverse populations of M.

phaseolina developed in a multiple sick plot irrespective of

host and/or geographic location to identify field resistance

against the pathogen.

Acknowledgments The authors are thankful to Dr. M.M. Roy,

Director, Central Arid Zone Research Institute, Jodhpur for providing

necessary laboratory and field facilities. First author is grateful to the

University Grants Commission for providing financial assistance in

the form of fellowship to carry out this study.

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