Genetic diversity and morphological variability of S clerotinia sclerotiorum isolates of oilseed Brassica in India

Sclerotinia stem rot caused by Sclerotinia sclerotiorum (Lib.) de Bary is one of the most serious disease and causes damage to oilseed Brassica. Seventeen geographical isolates of S. sclerotiorum from different geographical locations of India were compared for their growth under artificial culture conditions and the result was validated through random amplified polymorphic DNA (RAPD) markers. Based on the morphological studies, the isolates were grouped into fast growing (SR-01, SR-02, SR-05, SR-16) and intermediate (SR-03, SR-04, SR-07, SR-10 to SR-13, SR-18), though the rest of the isolates showed comparatively a slow mycelial growth rate. In RAPD analysis for their genetic diversity, fifty decamer primers of arbitrary sequences produced 692 scorable amplicons ranging from 180 to 3900 bp in size. Out of these, 385 fractionated fragments were reported polymorphic. As an average, 13-14 unambiguous and reproducible amplified products were generated by single primer (minimum of 4 and maximum of 23 fragments). Hence, the present investigation confirmed the diversity among the different geographical isolates from India.


INTRODUCTION
Sclerotinia rot, caused by Sclerotinia sclerotiorum (Lib.) de Bery, is one of the most devastating and cosmopolitan plant pathogen belonging to ascomycetes having a wide host range of more than 408 described plant species from 278 genera in 75 families including canola (Boland and Hall, 1994;Ziqin et al., 2008).Sclerotinia stem rot a devastating disease of rapeseed-mustard mainly since 1999 in India, caused up to 40% yield losses (Chattopadhyay et al., 2003).Sclerotinia rot is a threat to cultivation of oilseed Brassica in Rajasthan, Haryana, Madhya Pradesh, Uttar Pradesh, Bihar and Punjab states of India (Agarwal et al., 1997).The initial infection occurs in the late winter or /and early spring and the fungal mycelia grow within and between the plants.Host crops and their cultivars have no resistance to this broad spectrum pathogen (Zhao and Meng, 2003).Genetic diversity among the S. sclerotiorum population was demonstrated by few workers (Harlton et al., 1995;Okabe et al., 1998).Genetic variability within the isolates in different geographical regions is important for documenting the changes occurring in the population.On the basis of morphological differences in mycelial growth, sclerotia and ascospores formation, S. sclerotiorum isolates have been differentiated (Kohn et al., 1990).S. sclerotiorum is a homothallic fungus and able to produce clonal ascospores from self fertilized meiotic reproduction.Genetic analysis shows that isolates of S. sclerotiorum are homogenous with limited variability in 18 and 28 s rDNA regions (Kohli et al., 1995).Lack of variations in virulence among isolates from defined geographical areas has been reported by many workers (Alvarez and Molina, 2000;Atallah et al., 2004;Sexton and Howlett, 2004).For better management of the disease, it is important to understand the epidemiology and genetic diversity in the pathogen population at regional level.Differences in virulence may be detected only when isolates from widely separate geographical region are compared.There has been conclusive evidence to suggest host specialization among isolate of S. sclerotiorum (Kull et al., 2004).
Random amplified polymorphic DNA (RAPD) is a powerful tool for diversity analysis.On the basis of earlier studies, there is a range of markers including a multicopy repetitive element for DNA fingerprinting and nuclear gene polymorphisms (Yu et al., 2006).The aim of the present investigation is to compare different geographical isolates of S. sclerotiorum which originated from completely different agro-climatic conditions infecting oilseed Brassica in India, based on morphological variability and genetic diversity through RAPD marker.

MATERIALS AND METHODS
Seventeen isolates of S. sclerotiorum were originally collected from major oilseed Brassica growing States of India viz., Jammu & Kashmir, Rajasthan, Madhya Pradesh, West Bengal, Punjab, Uttar Pradesh, Uttarakhand and Himachal Pradesh.The isolates were designated as SR-01 to SR-18.A single sclerotium of different geographical isolate was surface sterilized separately in 1% (v/v) sodium hypochlorite and 70% ethanol for 2-3 min and thoroughly washed in sterile distilled water.Individual sclerotium was cut half and placed on potato dextrose agar (PDA) under laminar air flow (Clarkson et al., 2003).Isolates of S. sclerotiorum were subcultured and maintained at 22 ± 2°C on potato dextrose agar (PDA) under 12 h alternate fluorescent lights.

Morphological variability
Fresh cultures of isolates were subjected to detailed morphological and cultural characteristics viz., radial colony growth (mm), number of sclerotia developed in 90 mm Petri plates, size of sclerotia (mm) and weight of single sclerotia (mg).Data of radial colony growth were taken at 72 and 96 h after inoculation while number of sclerotia and size of sclerotia of each isolates were recorded 10 to 15 days after inoculation.Fresh and dry mycelial weight was taken after 10 days growth of isolates grown at 22 ± 2°C on potato dextrose broth (PDB) medium.

Genetic diversity
Mycelial mats grown in PDB and DNA was extracted using the cetyl trimethyl ammonium bromide (CTAB) method of Simpson et al. ( 2004) with some modifications.Equal volume of tris-saturated phenol was added in the tube and mixed for 5 min then centrifuged at 11000 rpm for 20 min at 20°C.After centrifugation, aqueous layer was collected and transferred in to fresh tube.Equal volume of chloroform: iso-amyl alcohol (24:1) was added, mixed and centrifuged.The supernatant was drained out and pellet was washed with 70% alcohol.For removal of RNA and residual protein, 5 µl of RNAse (20 mg/ml) and 20 µl of protinase-K (20 mg/ml) were added in nucleic acid solution (Sambrook et al., 1989).Extracted DNA was quantified using spectrophotometer.In addition, agrose gel electrophoresis was also performed for DNA quantification.

Statistical analysis
A binary matrix was compiled using numerical system of multivariate analysis.The dendogram was constructed by the unweighted paired group method of arithmetic average (UPGMA) based on Jaccard's similarity coefficient with SHAN program of NTsys.The morphological data were statistically analysed using analysis of variance (ANOVA).

Morphological variability among isolates
Results revealed that the mycelial growth rate among the isolates differed considerably.Differences in all the morphological characters of S. sclerotiorum were observed.Based on radial growth, the isolates were classified into three groups: Very fast, intermediate and slow growing.Data after 72 h incubations revealed that the isolates SR-01, SR-02, SR-05 and SR-16 represented significantly fast growing, isolates SR-03, SR-04, SR-07, SR-10 to SR-13 and SR-18 were intermediate, while SR-05, SR-08, SR-09 and SR-14 to SR-17 showed slow radial colony growth.However after 96 h incubation majority of isolates except SR-05, SR-12, SR-14, and SR-15 became fast growing and filled the Petri plate with mycelial growth.Maximum isolates were having whitish mycelial growth with smooth texture.
Isolates SR-09 and SR-11 were with off-white, SR-12 was gray white and SR-01 was dirty white in colour.Fresh and dry mycelial weight of different isolates  1 and Figure 1).

Molecular diversity
DNA of S. sclerotiorum was amplified with 50 decamer primers of arbitrary sequences.Primers were selected that produced a reasonably good number of countable and reproducible PCR products.The RAPD analysis is prone to  reproducibility problems across the laboratories but the results obtained in the investigation were consistent and repeatable.50 primers produced 692 scorable amplicons ranging in size from 190 to 3600 bp.Out of these, 385 fractionated fragments were reported polymorphic.The Polymorphism was detected for 1.3 to 2.7 kb amplified product as in primer OPS-11 (5ˈ-AGTCGGGTGG-3ˈ) (Figure 2).On an average, 13-14 unambiguous and reproducible amplified products were generated by single primer (minimum of 4 and maximum of 23 fragments).
The present investigation confirmed the diversity in S. sclerotiorum isolates determined on the basis of morphological criteria.The segregation of RAPD marker among the isolates is provided (Table 2).The RAPD bands segregation was estimated by theoretical ratio of segregation using χ 2 (qui square) analysis.
Based on Jaccard's similarity coefficient, dendogram for different isolates was prepared using 50 primers (Figure 3).The genetic relationships among the isolates based on the results of RAPD analysis, were also examined.All the 17 isolates of S. sclerotiorum were placed into four groups based on the presence and absence of the amplified products.These major clusters were further divided into sub-cluster.RAPD-PCR analysis proved to be useful in determining the extent of genetic diversity among the S. sclerotiorum isolates under investigation.Values of similarity coefficient of RAPD data ranged from 0.18 to 0.87 (Table 3).Isolates SR-02, SR-05, SR-13, SR-14 and SR-15 formed first clear group with 70% diversity.Second cluster framed the isolates SR-01, SR-07, SR-08, SR-09, SR-10 and SR-11.SR-04 and SR-12 isolates formed the third cluster.Fourth cluster was framed by SR-03, SR-16, SR-17 and SR-18 isolates.This is the first report indicating the existence of three genetically different population of S. sclerotiorum in India collected from oilseed Brassica.

DISCUSSION
Diversity among the Indian geographical isolates of S. sclerotiorum based on their growth behavior with validation using RAPD markers was confirmed.Variability among the isolates has already been reported by Carpenter et al. (1999).All isolates were darkly pigmented.However, intensity of melanisation varied among the pigmented isolates.Primarily, the dark colour of the sclerotia results from the production of melanin.
The main role of melanin in pathogen is to protect the sclerotia from adverse biological and environmental conditions (Butler and Day, 1998).Punja and Damiani (1996) and Zarani and Christensin (1997) recorded differences in growth rates among different isolates obtained from various host species.Based on sclerotial diameter, several workers recorded variation in size of sclerotia among different isolates of the fungus (Dhingra and Sinclair, 1973;Mirza et al., 1985).
During the study, bigger sizes of sclerotia were observed in the same morphological group and genetic cluster.Sun et al. (2005) compared S. sclerotiorum  Bowden and Leslie (1999).They reported that sexual reproduction is considered as an important assumption for molecular divergence among the population from different geographical regions.Isolates of S. sclerotiorum (Lib) de Bary from various agro-climatic conditions has been compared by morphological and genetic analysis.Morphological variations observed in the study in general were strongly correlated to genotypic variations.The pathogen is widely spread and well established in the different geographical regions.Therefore it dealt with the relatedness among 17 S. sclerotiorum isolates as representative samples of the pathogen.

Figure 2 .
Figure 2. RAPD profile of different geographical isolates of S. sclerotiorum.

Figure 3 .
Figure 3. Cluster diagram showing grouping of Sclerotinia sclerotiorum based on RAPD.

Table 1 .
Variability in mycelial growth and sclerotia formation (on PDA) in different geographical isolate of Sclerotinia sclerotiorum.

Table 2 .
Qui squire analysis for allele frequency.
*Number of bands.

Table 3 .
Similarity matrix generated through Jacquard coefficient.The results confirmed our presumption about link of different morphological characters and genetic variations within the isolates.The very fast growing group of isolates was also within the same genetic cluster except SR-16.The molecular biology techniques have added to the analysis of variability in that organism where stable morphological markers are absent.Our finding is also supported byYu et al.