Variability among the potato sclerotial isolates of Rhizoctonia solani of Mountainous Region , Gilgit-Baltistan , Pakistan

An experiment was carried out to find the variability among twenty sclerotial isolates of Rhizoctonia solani collected from potato growing areas of Gilgit-Baltistan. These isolates were grown in different culture media, mycelial compatibility and fungus protein profile were investigated. Culture media were used to study radial colony growth and sclerotial production of isolates. Radial colony growth (RCG) and sclerotial production (SP) of isolates against culture media ranged from 12.31-21.55; 3.66-22.66 in potato dextrose agar, 12.67-18.56; 4.66-10.66 in czpedox agar, 12.02-20.42; 2.00-8.66 in corn meal agar and 10.54-14.16; 0.00-3.00 in water agar, respectively. These isolates were further classified into three categories on the basis of RCG and SP. Result revealed that out of total isolates, 60% showed medium RCG and 40% fast growth, while 10, 60 and 30% isolates showed low, medium and high SP. Furthermore, sclerotial characteristic such as size, shape and distribution pattern were also recorded. Mycelial compatibility and incompatibility among the R. solani isolates was also studied. The results indicated that out of 190 combinations, 72.10% were compatible, whereas 27.90% were incompatible. Fungus protein profile of twenty isolates of R. solani by sodium dodecyl suplphate gel electrophoresis (SDS-PAGE) recovered sixty one bands with different frequencies among the isolates. Cluster analysis of twenty isolates divided them into two major lineage groups, A and B. Lineage A contained 65% isolates whereas lineage B contained 35% isolates. These lineages were further divided into thirteen clusters (C1-C13); A was comprised of eight and B five clusters, respectively.


INTRODUCTION
Potato (Solanum tuberosum L.) is one of the important vegetable crops in the world.In Pakistan, its production increases year by year.According to Agricultural statistics, potato is grown on an area of 145000 ha during *Corresponding author.E-mail: azharkiu@gmail.com.Author(s)

Morphological and sclerotial variability of R. solani isolates
Morphological and sclerotial variability of R. solani isolates were studied on four solid media viz, PDA, CZPA, CMA and WA.Twenty millilitres of each medium was poured into sterilized Petri-dishes and 5 mm actively growing mycelial plug were inoculated.Experiment was repeated twice and maintained three replicates in each isolate.Radial colony growth was recorded every 24 h during 4 days at 25 ± 2°C.Mean radial colony growth rate per day was determined by dividing colony growth recorded at 96 h by 4 days.Radial colony growth rate (mmd -1 ) was calculated by using following formula as described by Guleria et al. (2007).

Mycelial compatibility
Mycelial discs (5 mm in diameter) taken from the edge of an actively growing colony (4 th day old) of each isolate were placed 40 mm apart on opposite sides of Petri dishes (90 mm in diameter) and incubated at 25 ± 2°C.Two isolates were paired on one Petri dish and the test was repeated twice.The pairings were examined macroscopically for presence of an antagonistic (barrage or aversion) zone in the region of mycelial contact as described by Punja and Grogan (1983).

Extraction of proteins from R. solani isolates
Protein profile of R. solani isolates was carried out using SDS-PAGE as described by Laemmli (1970).Fungus protein was extracted from the mycelium mat of each isolate grown on potato dextrose agar plate for 6 days at 27± 2°C.Mycelium of each isolate was harvested then dried.A dry mycelium of each isolate was ground to make a fine powder with mortar and pestle.
Mycelium flour 0.01 g was added to 400 μl extraction buffer (0.5 M Tris-HCL (pH 6.8), 2.5 SDS, 10% glycerol and 5% 2mercaptoethanol) mixed 400 μl in Eppendorf tube and vortexed (Automatic lab Mixer DH-10).Then, the samples were centrifuged at 15,000 rpm at least ten minutes at ambient temperature.The clear supernatant was transferred into 1.5 ml Eppendorf tubes and stored at 2°C until they were run on the polyacrylamide gel.

Electrophoretic procedure
Fungus protein was analyzed through slab type SDS-PAGE using 12.25% polyacrylamide gel PAGE (Model: AE-6530M, Japan), resolving gel (3.0MTris-HCl) pH9, 0.4% SDS and 4.5% stacking gel (0.4M Tris-HCl pH 7.0, 0.4% SDS).Electrode buffer (0.025 M Tris, 129 M Glycine, 0.125 % SDS) was loaded top pool of apparatus.A 15 μl of the supernatant of isolates along with marker were loaded into the wells of the gel.Apparatus was connected with uninterrupted electric supply (100 V) until the bromophenol blue (BPB) reached the bottom of the gel plate.

Gel staining
Gel were placed in staining solution (0.2% Commassie Brilliant Blue dissolved in 10% glacial acetic acid, 40% methanol and water in the ratio of 10:40:50) for one hour then placed in destaining solution (5% acetic acid and 20% methanol).Destained gels were analyzed directly using photographic method or drying gel by gel-drying processor for about 2-4 h.

Statistical analysis
The design of in vitro experiment was a randomized complete block and analysis of variance (ANOVA) of the data was performed using the statistical package STATISTICA 8.1 and SPSS Version 16.0 for Windows 2007.

Effect of different culture media on colony growth rate and sclerotium production
In this study, different culture media and twenty isolates of R. solani were used to determine suitable medium and variability of radial colony growth, sclerotium production, size, shape and distribution of sclerotia.Four culture media viz., PDA, CZPA, CMA and WA were used.During the studies, it was observed that the colony growth rate and sclerotium production in the PDA ranged from 12. ; 0.00-3.00).Mean radial colony growth and sclerotial production were recorded in PDA (18.29, 9.94), CZPA (16.76,6.83),CMA (15.80,4.79)and WA (12.70, 1.33).The result observed that potato dextrose agar were suitable medium for culture of R. solani (Table 1 and Figure 5).Table 2 showed that the R. solani isolates grown in potato dextrose agar were further categorized on the basis of slow, medium and high.Result indicated that 60% of isolates had medium and 40% had fast growth on the basis of radial colony growth (Figure 2) while on the basis of sclerotium production, 10% isolate had low, 60% medium and 30% high (Figure 3).Besides these, significant variations among sclerotial size, shape and distribution pattern of R. solani isolates were observed (Table 2, Figure 4 and Figure 6).

Mycelial compatibility group
There were one hundred and ninety combinations of the twenty isolates of R. solani.Amongst only 53 showed incompatibility reaction and 137 showed compatibility (Table 3).For combinations which showed antagonistic reactions with each other, a thin band of living or dead mycelia was formed (Figure 7).Based on mycelial compatibility, 72.10% mycelial compatibility and 27.90% non compatible among the tested isolates were shown.

SDS-PAG gel electrophoresis
Twenty isolates of R. solani were subjected to SDS-PAGE gel electrophoresis producing sixty one fungal protein bands.On the basis of presence or absence of protein bands in individual isolates, RS 17 exhibited highest number of protein bands followed by RS 6 , RS 8 , RS 10 , RS 13 , RS 19 and RS 20 whereas on the basis of molecular weight, highest number of bands were produced at 18 kDa and lowest were observed in 45 kDa (Figures 8 and 9).Cluster analysis divided twenty isolates into two major lineage groups, groups A and B. Lineage A contains 65% of isolates whereas lineage B contains 35% isolates (Figure 10).These lineages were further divided into thirteen clusters (C 1 -C 13 ).Lineage A consists of eight cluster while lineage B comprises five clusters.Among the lineage A, C 1 contains three isolates, C 3 , C 4 , and C 6 has two isolates, while C 2 , C 5 , C 7 and C 8 comprises single isolate each.Similarly, in the lineage B, C 9 consists of three isolates while C 10 , C 11 , C 12 and C 13 contain single isolate each (Table 4).

DISCUSSION
Different culture media, mycelial compatibility and protein profile through SDS-PAGE were used to study variability of R. solani isolates.All isolates of R. solani showed variation in radial colony growth and sclerotial production against culture media.Maximum radial colony growth and sclerotial production was found in PDA which was followed by CZPA and CMA, while least radial colony growth and sclerotial production was recorded in WA.The results were in agreement with that of Lalan et al. (2013) who studied colony diameter, growth, colour and sclerotia formation of six isolates of R. solani of soybean and concluded that PDA was best for growth and development among the tested culture media.It has been further observed that PDA is a frequently used medium, due to its simple formulation and supportive nature of different plant pathogenic fungi (Maheshwari et al., 1999;Saha et al., 2008).Numerous researchers have confirmed that PDA is most excellent for colony growth of different fungi (Xu et al., 1984;Meera et al., 2012;   .Rhizoctonia solani isolates classified into three groups on the basis of sclerotium production cm 2 on PDA medium.Saha et al., 2008).R. solani is a complex pathogen with wide host range.Due to their ill-define taxonomy and poor understanding of natural history, its identification and study is always a challenging tasks (Cubeta and Vilgalys, 1997).Variability within the isolates of R. solani has been reported by many researchers (Sherwood, 1969;Neeraja et al., 2002;Linde et al., 2005;Guleria et al., 2007;Thind and Aggarwal, 2008).In our current study, different isolates of R. solani showed considerable variation in terms of redial colony growth, number, size, shape, and distribution pattern of sclerotia which is in agreement with report of Thind and Aggarwal (2008) who studied morphological and sclerotial characteristics of potato R. solani isolate.Yadav and Anamika (2005) studied morphological and culture variation of different isolates of R. solani that caused damping off fenugreek vegetable and concluded that all isolates differ in colony growth, colour, width and sclerotial production.
The finding of the current study revealed that majority of the isolates showed compatibility reaction by pairing.Mycelial compatibility and incompatibility reactions are a useful way of categorizing intraspecific heterogenecity.It is a self and non-self-recognition system controlled by multiple loci, but knowledge of the underlying genetic mechanisms is limited in most filamentous fungi (Glass and Kaneko, 2003).In contrast, isolates that are different at one or some or more of these loci will not anastomose.Rosa et al. (2012) studied compatibility/incompatibility of 433 R. solani isolates and found that about 91% isolates were incompatible, while 9% of the pairing were compatible.Majority of isolates showed compatibility in the same county except few isolates.
In the current study, different fungus protein band were recovered from mountaneous isolates of R. solani.The results also agree with previous finding of Monica et al. (1983) who studied protein pattern of different anastomosis group of R. solani and showed that there was sufficient variability among the isolates particularly AG3 having distinct protein band regardless of isolates sources.Zuber and Manibushanrao (1982) also reported that polyacrylamide gel electrophoresis showed marked variation among five virulent isolates of R. solani.Similarly, El-Akkad (1997) also recorded fungus protein band heterogeneity among the R. solani isolates of AG-4.Hussein et al. (2000) reported that cluster analysis of protein band recoverd by SDS-PAGE form seventeen isolates of multinucleate and binucleate R. solani showed clear-cut differentiating features between Rhizoctonia spp.The results of the current study showed variation in fungus protein banding.However, some isolates gave similar binding pattern, they could be differentiated by some components.Iqbal et al. (2005) reported that similarity might be due to comigration of different peptides on the SDS-PAGE and closely related ancestry.
The grouping on the basis of morphological characters was quite different from that which was on the basis of

Low <5
Medium 5-10 High > 10         SDS-PAGE.This is because of the fact that all the genes are not expressed in a particular environment.However, variability among the isolates was observed.The result of the current study indicates that R. solani of mountainous region Gilgit-Baltistan is composed of pathotype and morphological variability and genetic diversity exists.More studies are needed by using different molecular techniques for understanding of diversity among the isolates.

Figure 2 .
Figure 2. Rhizoctonia solani isolates classified into three groups on the basis of colony growth rate mmd -1 on PDA medium.

Figure 3
Figure 3. Rhizoctonia solani isolates classified into three groups on the basis of sclerotium production cm 2 on PDA medium.

Figure
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Table 1 .
Colony mycelial growth, rate/day and sclerotium production of twenty isolates of R. solani on different media culture at 25±2°C.