Molecular and Morphological Diversity of Rhizoctonia bataticola Causing Dry Root Rot Disease from India

Rhizoctonia bataticola (Taub.)Butl.a omnipresent soil-borne fungus having pycnidial state as Macrophomina phaseolina (Tassi.) Goid., causes dry root rot disease in a wide range of hosts starting from cereal crops to horticultural and medicinal crops. The present study was aimed to find out genetic and morphological variability among the twenty five indigenous isolates of R. bataticola collected from geographically distinct regions (10 states) infecting 19 different crop plants. Sclerotial morphology of the isolates was studied and they were confirmed to species using species-specific primers. All the isolates were grouped into four different types of sclerotia. Genetic diversity of all these isolates was analysed using random amplified polymorphic DNA (RAPD) markers and inter simple sequence repeat (ISSR) markers. Ten random primers for RAPD and six primers for ISSR were tested for amplification of genomic DNA of R. bataticola isolates. Best six primers for RAPD and five primers for ISSR were further analysed. Unweighted pair-group method using arithmetic means (UPGMA) clustering of data showed that isolates did clearly differentiate to the specific group according to the geographical origins. ISSR markers were found more efficient than RAPD markers to correlate the genetic diversity with the grouping of isolates according to the geographical regions.But RAPD markers were found efficient todifferentiate R. bataticola isolates into different clusters according to their sclerotial morphology.It was the first study that investigated genetic relationships with two PCR-markers among different isolates of R. bataticola belongs to different hosts from India.

Dry root rot of different agriculturally important crops viz.Black gram, Chick Pea, Cotton, Cowpea, Jute, Mungbean, Onion, Pea, Rice, Soybean, Spinach and Wheat etc. is caused by Rhizoctonia bataticola (Taub.)Butler in different parts of world including India 36 .The perfect stage of R. bataticola is Thanatephorus cucumeris (Frank.)Donk.It also produces pycnidia and this stage is known as Macrophomina phaseolina (Maubl.)Ashby.R. bataticoa is very diversified and cosmopolitan soil borne fungus, producing different types of symptoms viz., blight, rot, wilt and decay 15 .The hyphae are septate and 1.5 -2.5 µm thick, and produce black and irregular sclerotia up to 100 µm in diam.resembles charcoal powder.
A number of workers have carried out a series of comprehensive studies with reference to its incidence, etiology and genetic variability 41,42,2,27 .They found that isolates of R. bataticola are very diverse in nature with high phenotypic, pathogenic and genetic variation and even differences may occur in the different isolates from a single host.A high level variation in the sclerotial morphology of R. bataticola was reported by no. of workers 32,39 .
PCR based molecular marker techniques were used to determine and evaluate the genetic diversity among different isolates of M. phaseoliona 21 .Genetic variability within the population was varied with the type of the marker 28   .Restriction Fragment Length Polymorphism (RFLP) of rDNA-ITSregions 2 .Random Amplified Polymorphic DNA 1,2,44 , Amplified Fragment Length Polymorphism(AFLP) 9,35 Universal Rice Primer PCR (URP-PCR) 18 Inter simple sequence repeats (ISSR) , 17,30,21 Repetitive Sequence-Based Polymerase Chain Reaction (Rep-PCR) 30 and Simple Sequence Repeats (SSR) 6 were explored extensively to determine the variation of different isolates of R. bataticola mostly from single host plant.Of these techniques, RAPD is simple, low cost and requires small amount of material.Inter simple sequence repeat (ISSR) markers are powerful tools to reveal the variation in genome microsatellite regions.Both RAPD and ISSR markers have been successfully used to differentiate and identify many fungi 44,21 .
The objective of present investigation was to study the morphological and genetic variability among the different isolates of R. bataticola obtained from geographically distinct regions (10 states) over a range of 19 host plant species using RAPD and ISSR markers.

Collection of R. bataticola isolates
Twenty five isolates taken under consideration from different places and sources were collected from Indian Type Culture Collection (ITCC), Division of Plant Pathology, IARI, New Delhi (Table 1, Fig. 1).All the 25 isolates were taken for the morphological characterization and for variability studies using RAPD and ISSR markers.

Morphological Identification of R. bataticola
The phenotypic characteristics of Rhizoctonia isolates were investigated based on sclerotial morphology.

Genomic DNA Extraction
Genomic DNA was extracted from frozen mycelium of R. bataticola based on Cetyltrimethyl ammonium bromide (CTAB) mini extraction method. 12he DNA concentration and purity of the samples was determined with Nano Drop Spectrophotometer (Thermo Scientific).
PCR protocol was standardized varying the cycle number, annealing temperature and the concentration of target DNA.The optimized protocol is described here; PCR amplifications were performed in a total volume of 20µl by mixing 2 µl of 10X Taq buffer, 0.5µldNTPs (100Mm dNTPs Mix), 5 pmol of each primer(0.5µl), 1 U (0.3µl) of Taq DNA polymerase,0.5µl of 50 mMMgCl 2 and 35-50 ng of template DNA(2 µl).The PCR reaction was carried out for 25 cycles of denaturation at 95 °C for 30 s, followed by annealing at 56 °C for 1 min, extension at 72°C for 2 min.and the final extension step of 72°C for 10 min.

Variability among the different isolates of R. bataticola using different RAPD Markers
DNA from 25 isolates was subjected to genetic diversity analysis by the RAPD method using 10 randomly chosen 10-base random primers.Among them six primers were chosen for further studies (Table 3).PCR reactions were carried out in 0.2 ml thin-walled PCR tubes with a total reaction volume of 25 µl containing 2.5¼l of 10X buffer, 2.5mM of each dNTPs( 0.5¼l) ,1¼l of primer, 0.5¼l MgCl2 (25mM) and 1U (0.2¼l) of Taq polymerase (Bangalore, Genei).The PCR amplification conditions were initial denaturation at 94°C for 3 min followed by 40 cycles of denaturation at 94°C for 1 min, primer annealing at 35°C for 1 min, primer extension at 72°C for 2 min, and a final primer extension at 72°C for 5 min.Six primers of RAPD were selected for final analysis based on informative banding patterns, clarity, and repeatability.

Variability among the different isolates of R. bataticola using different ISSR Markers
ISSR analysis was carried out amplifying the genomic DNA using six ISSR primers.Among them five primers were chosen for further studies (Table 3).The PCR amplification conditions were: Initial denaturation at 95 °C for 5 min followed by 40 cycles of denaturation at 94 °C for 1 min, primer annealing at 34 °C for 1 min, primer extension at 72°C for 1 min, and a final primer extension at 72°C for 5 min.A total of six primers were screened initially.Five primers were selected for final analysis based on informative banding patterns, clarity, and repeatability.

Statistical analysis
RAPD and ISSR reproducible fragments were scored as present (1) or absent (0), and bands were entered in a computer file as a binary matrix, one for each molecular marker.The binary matrix was analyzed using the NTSYS-pc (Numerical Taxonomy and Multivariate Analysis System) package, version 2.0 to calculate the similarity values.After obtaining the similarity matrix, clustering was performed by sequential agglomerative hierarchical nested clustering (SAHN).The graphical representation of the cluster was obtained by using the unweighted pair group method of mathematical averages (UPGMA).Bootstrap analysis of the data was performed with 1000 replications using the WINBOOT program to estimate structural stability of clusters.Polymorphic information content (PIC) or average heterozygosity was calculated as per the formula of. 34Average heterozygosity (Hav) is obtained by taking the average of PIC values obtained for all the markers.Multiplex ratio (MR) for each assay was estimated by dividing the total number of bands (monomorphic and polymorphic) amplified by the total number of assays.Marker index (MI) was obtained by multiplying the average heterozygosity (Hav) with MR . 28A principal coordinate analysis (PCO) was performed EIGEN procedure in the NTSYSpc in order to highlight the resolving power of the ordination.To estimate the congruence among dendrograms, cophenetic matrices for each marker type were computed and compared using the Mantel matrix correspondence test.This test was performed using the MXCOMP procedure in the NTSYSpc.

RESULTS
The present research was undertaken to study the sclerotial and genetic variability among the different isolates of R. bataticola obtained from geographically distinct regions (10 states) infecting 19 different crop plants.All the isolates were divided into four different groups based on their high level variation in sclerotial shape, size and arrangement.RAPD and ISSR markers were used to estimate the genetic variability.ISSR markers were found more efficient than RAPD markers to correlate the genetic diversity with the grouping of isolates according to the geographical regions.But RAPD markers were found efficient to differentiate R. bataticola isolates into different clusters according to their sclerotial morphology.

Morphological Identification
Morphological features of 25 isolates of R. bataticola were observed by growing the isolates on PDA medium.Sclerotial morphology was considered to assess the possible variation among different isolates.

Variability in Sclerotial morphology
Among the isolates, the size of sclerotia ranged from 29.4-100 X 21.1-52.9¼m.Based on sclerotial shape, size and arrangements, the isolates were divided into four different groups (Table 2).The observations were made under the compound microscope and pictures were taken for further documentation (Fig. 2).

Molecular identification of R. bataticola based on species specific marker
Molecular characters of 25 isolates of R. bataticola were analyzed in order to resolve their identification based on species specific marker.The primers used yielded single amplified product of 350 bp with all the R. bataticola isolates tested (Fig. 3).The specificity of the primers was tested on representative species of common soil borne microbes like Trichoderma harzianum, Colletotrichum gloeosporioides, Fusarium oxysporum and Chaetomium globosum.The primer pair was found to be specific for R. bataticola as none of the other soil microbes tested could yield any amplification product under identical conditions of amplification.

RAPD Analysis of R. bataticola
A total of 238 bands were produced for Rhizoctonia isolates and all of them were found to be polymorphic.Maximum number (59) bands were obtained with OPA-11, followed by OPE-14 (45 bands), OPO-4 (41), OPN-4 (35), OPN-20 (30), OPS-18 (26) and all of them were polymorphic (Table 3).The size of the fragments varied from 0.4 to 2.5kb.The data obtained from RAPD analysis of 25 isolates of Rhizoctonia species with six primers was subjected to UPGMA analysis.A dendrogram was prepared using the similarity coefficient of The dendrogram obtained separated R. bataticola isolates (No. 2 and 15) completely from all other isolates and revealed more than 75 percent similarity among themselves.These isolates were characterized based on their sclerotial morphology which were large and highly connected.Isolate (Nos.14,16,17,18,20,21,22,23,24,25) were having large, round to irregular and free sclerotia separated from all other isolates and show 72-94% similarity.
Similarly isolate 8 and 13 were characterized as the isolates having small, round and free sclerotia and also clustered together with This dendrogram revealed the presence of the variability among the different isolates by separating based on their sclerotial morphology.

ISSR Analysis of R. bataticola
A total of 250 bands were produced for Rhizoctonia isolates and all of them were found to be polymorphic.Maximum number (53) of bands were obtained with ISSR-1 and ISSR-2, followed by ISSR-3 (49 bands), ISSR-4 (46), ISSR-6 (49) and allof them were polymorphic (Table 3).The size of the fragments varied from 0.7 to 2.5kb.The data obtained from RAPD analysis of 25 isolates of R. bataticola with five primers was subjected to UPGMA analysis.A dendrogram was prepared using the similarity coefficient of ISSR marker.The dendrogram showed the presence of differences among R. bataticola isolates.
Therefore, morphological and molecular variability were assessed in the present study.Twenty five isolates were collected from different host plants growing in different parts of the country.These isolates were characterized and grouped by their sclerotial morphology.All the isolates from Jute, Pea, Soybean, Chick Pea, Cotton, Balsam, Onion, black gram, Cowpea, Spinach, Wheat, Rice, Castor, Anola, Strawberry, Mungbean, burning bush and Sarpgandha were found variable in the shape and size of sclerotia.The size of the sclerotia was 29.4-100 X 21.1-52.9µm in the present study.The biggest sclerotia reported was 101.51µm in Gliri-cidia isolate of R. bataticola while cowpea isolate produced the smallest sclerotia (66.88 µm) 10 whereas 25 reported the size varied from 58.83 µm to 126.63 µm. 29also reported the morphological variability in R. bataticola collected from different pulse crops.Therefore, it is clear that variability exists in sclerotial morphology.
These morphologically characterized 25 isolates of R. bataticola were also assessed at their molecular level.PCR-RAPD analysis is one of the simplest tools available for the analysis of genetic differences at DNA level without demanding any prior information about the genomic sequences.
Molecular markers are useful tools for assessing variation rapidly within and among species 11 In this study, we have compared two different molecular marker systems, RAPD and ISSR, to define genetic relationships among isolates of R. bataticola, and to investigate which marker system can be more effectively used.In RAPD, a total of 238 bands were produced for R. bataticola isolates and all of them were found to be polymorphic.Maximum number (59) of bands were obtained with OPA-11 and all of them were polymorphic.The size of the fragments varied from 0.4 to 2.5kb.The higher level of polymorphism detected by RAPD markers revealed the selective capacity of this marker among R. bataticola isolates. 40In the present study, the variability based on PCR-RAPD was coinciding with the groups made based on sclerotial morphology.
The ISSR markers were successfully used to identify genetic differences in R. bataticola 17 .The results of the present study clearly demonstrated that R. bataticola from different parts of the country were highly variable and ISSR markers are suitable to reflect the genetic diversity among the isolates.A high level of polymorphism in R. bataticola was found in the isolates collected from different hosts and regions using different molecular tools 3,19,23,43 Many number of workers demonstrated the genetic diversity among M. phaseolina isolates . 1,2,5,6,7,17,18,26,30,31,33,35.The high genetic diversity was observed not only in isolates from distinct hosts and geographical origins but also in isolates collected from a single host or single region.
The number of alleles at a locus and their frequency of distribution are responsible for Polymorphism thereby genetic variation in a population 28 .RAPD and ISSR markers target different portions of the genome resulted the difference in discrimination among the isolates. 21The genetic variation usually depends on the number of polymorphic bands obtained with each marker . 8t was the first study that investigated genetic relationships among different isolates of R. bataticola from different host plants from India and also that compared two PCR-markers to define genetic relationships among isolates of R. bataticola.

Fig. 4 .
Fig. 4. Dendrogram constructed with UPGMA clustering method based on Random Amplified Polymorphic DNA (RAPD) -PCR for 25 isolates of R. bataticolafrom different geographical areas of India.
Rhizoctonia bataticola is a non-host specific fungus causing dry root rot disease in a DEVI et al.: GENETIC DIVERSITY OF Rhizoctonia bataticola ISOLATES

Fig. 5 .
Fig. 5. Dendrogram constructed with UPGMA clustering method based on Inter-Simple Sequence Repeat (ISSR) markers for 25 isolates of R. bataticola from different geographical areas of India

Table 1 .
Sources and location of different R. bataticola isolates *Indian Type Culture Collection

Table 2 .
Variability in Sclerotial morphology in different R. bataticolaisolates

Table 3 .
List of Random Amplified Polymorphic DNA (RAPD) and Inter-Simple Sequence Repeat (ISSR) primers and the resulted DNA polymorphisms applied to differentiate isolates of R. bataticola OPA -Operon Series A primer, OPN -Operon Series N primer, OPE -Operon Series E primer, OPO -Operon Series O primer, OPS -Operon Series S primer

Table 4 .
Different groups of R. bataticola isolates based on Inter-