Analysis of Genetic Diversity Amongst Fusarium spp . Associated with Root Rot of Apple

The Genus Fusarium is associated with crop diseases of horticultural and agricultural commodities and is considered to be a destructive pathogen. Genetic diversity among ten different isolates of Fusarium species isolated from apple rhizosphere from different locations of Himachal Pradesh was analysed using Random Amplified Polymorphic DNA (RAPD-PCR). Molecular characterization using ITS sequencing revealed that out of 10 isolates of Fusarium, three belongs to F. oxysporum, two belongs to F. solani, one belongs to F. equiseti and four were Fusarium species. Seven random primers were used for their genetic characterization using RAPD analysis. The dendrogram obtained from the UPGMA study characterized ten isolates into one main cluster and nine independent branches on the basis of similarity value 0.65.

Fusarium is one of the devastating phytopathogenic fungi belongs to Division Ascomycota, Class: Sordariomycetes, Order Hypocreales, Family: Nectriaceae.This filamentous fungi, can smite any crop since having a broad host range including rice, wheat, horticultural crops, ornamentals and in almost all agricultural commodities (Supyani and Widadi, 2015).Species of Fusarium serving as pathogens to many diseases in crops such as vascular wilt, root rot, corm rot, damping-off, yellows, and others.Mostly Fusarium species are regarded as soilborne fungi because of their abundance in soil and their frequent association with plant roots, as parasites and saprophytes.However, many have active or passive means of dispersal in the atmosphere and are common in colonizers of aerial plant parts, where they may result in diseases of substantial economic importance.Fusarium rot on apples by Fusarium species (Riad and Zeiden, 2015), Sugarcane wilt by Fusarium sacchari (Lin et al., 2014), Pokkah Boeng in sugarcane by Fusarium moniliforme (Vishwakarma et al., 2013), Bakanae in rice by Fusarium fujikuroi (Jain et al., 2014), Oil palm wilt by Fusarium oxysporum f.sp.elaidis (Rusli et al., 2015), Panama disease by Fusarium oxysporum f.sp.cubensis (Zhang et al., 2013), are some important diseases caused by Fusarium spp.
Fusarium taxonomy is very complex and over the last 100 years has persistently been rationalized.The traditional diagnostic method based only on morphological characteristics observed on selective media under specific incubation conditions for detection and identification of Fusarium species.Considerable expertise is required for morphological identification while differentiating closely related species of Fusarium as their morphological features may overlap.Molecular biology has brought many powerful tools to fungal taxonomists including method for identification of isolates, means to illuminate the relationships among fungal species.To define fungal populations at species, intraspecific, race and strain levels, RAPD assays have been extensively used (Miller, 1996;Ingle et al., 2009) and for detecting genetic variability RAPD-PCR technique is also used (Edwards et al., 2002;Sabir, 2006).
The present study was aimed to estimate genetic relatedness among the Fusarium isolates, isolated from apple rhizosphere from different locations of Himachal Pradesh using RAPD markers.

DNA isolation
Thirteen isolates of Fusarium species collected from different locations of Shimla, Kullu and Mandi were cultured on potato dextrose agar (PDA) plates containing 1 µg/ml streptomycin, incubated at 27°C for 3-5 days.The mycelia grown were harvested and total DNA was extracted using protocol described by Sambrook et al., 1989.Crushing of harvested mycelia was done in 1-2 ml of extraction buffer (100mM Tris HCl, 50mM EDTA, 500mM NaCl, 0.01% βmercaptoethanol).130 µl of 10% sodium dodecyl sulphate (SDS) was added to the mixture per ml of extraction buffer and incubated at 65°C for 15 minutes.The sample was centrifuged at 8,000 rpm for 10 min.To the supernatant, equal volume of phenol/chloroform (1:1) was added, mixed thoroughly and centrifuged at 10,000 rpm for 10 min.Aqueous phase containing nucleic acid was collected and 2.5 volume of absolute ethanol was added to precipitate the nucleic acid.Sample was centrifuged at 12,000 rpm for 20 min to pellet down the precipitates.The pellet was then washed with 70% ethanol, air dried and resuspended in 50 µl of 10X TE buffer (10mM Tris HCl, 50mM EDTA).Bands corresponding to genomic DNA were observed by performing electrophoresis in 1% agarose gel.

PCR Purification of Amplified DNA Product
PCR amplified DNA products were purified from gel using gel purification kit (DNA gel/PCR purification miniprep kit, XcelGen) as per the instructions provided in the instruction manual.

Sequencing and Sequence analysis
The eluted DNA was sent to Xcelris™ lab Pvt Ltd (Ahmedabad) for sequencing.Sequence data obtained was analysed using BLAST software.Sequences obtained were submitted to European Nucleotide Archive and accession numbers were obtained.

RAPD product scoring and data analysis
Data was compiled as binary 0-1 matrix, (1) represented the presence of a band and (0) the absence of a band at a particular position.All RAPD bands were considered in statistical analysis.Dendrogram was produced from the distance matrix by Unweighted Pair-Grouped Method by Arithmetic average, contained in the software package NTsys 2.2 version.

DNA isolation
To study the diversity among the Fusarium species, samples were collected from apple rhizosphere in orchards of Himachal Pradesh, India.
All the strains have been purified by growing the single spores on PDA plate and DNA of thirteen isolates was extracted manually using phenol/chloroform method in a good quantity.

Molecular Characterization
Isolated Fusarium strains were confirmed by molecular characteristics i.e. by PCR amplification of ITS region and transcription elongation factor -alpha, recommended as a universal DNA barcode marker for fungi (Schoch et al., 2012).For a single-locus identification in Fusarium, TEF markers has become a choice (Geiser et al., 2004).Three primers sets: ITS FuF-FuR, ITS Fu1F-Fu1R corresponding to ITS region and TEF Fu3F-Fu3R corresponding to transcription elongation factor -alpha region showed amplification at 400 bp which confirmed that ten of the thirteen isolates under study were Fusarium (Table 1).Arif et al., (2012) designed the primers for ITS, TEF for rapid detection of genus Fusarium and F. solani.The primers had shown accurate identification and discrimination of genus Fusarium and F. solani, which have number of applications in screening of infected plants, disease diagnosing and breeding programs.Purified PCR product of primers ITS FuF-FuR and TEF Fu3F-Fu3R of ten fungal isolates were sent to Xcelris™ lab Pvt. Ltd. for sequencing.Sequence data obtained was analysed using BLAST software and found that out of ten Fusarium isolates three isolates (isolate 4, isolate 5 and isolate 12) belongs to F. oxysporum, two isolates (isolate 2 and isolate 13) belongs to F. solani , one isolate (isolate 9) to be F. equiseti and other four belongs to strains of Fusarium species.The sequences were assigned accession numbers by submitting to European

Table 2 .
Sequence of primers, number and size of fragments of Fusarium species amplified by RAPD primers

Table 1 .
PCR amplification of fungal isolates with ITS and TEF primers