Variability in Fusarium oxysporum f.sp. ciceris causing wilt in chickpea

Fusarium wilt caused by Fusarium oxysporum f.sp. ciceris (Padwick) Matuo and K. Sato is a major constraint in successful cultivation of chickpea. Therefore, in the present study, 24 isolates of F. oxysporum f.sp. ciceris collected from different chickpea growing areas of Punjab and adjoining states were assessed for morphological variations. Among 24 isolates, the maximum (8.78 mm/days) and minimum (5.00 mm/day) growth rate were exhibited by Foc-21 and Foc-15, respectively. The isolates showed growth pattern from appressed, fluffy to less fluffy and mycelial colour varied from different shades of white to purplish white. A significant variation with respect to size of micro (8.9-16.9 x 3.1-6.3 μm) and macro (21.7-64.9 x 2.7-10.0 μm) conidia was also observed. At pathogenic level, twenty isolates were studied where Foc-3, Foc-7 and Foc-22 showed virulence pattern similar to existing races 1, 2, 3 and 4 on three standard differentials viz. JG 62, WR 315 and L 550, whereas the rest of the isolates did not match with any of the existing race reaction. Further, the six selected genotypes could differentiate the isolates into four pathotypes based on their aggressiveness and Foc-8 was found more aggressive (98.48% wilt incidence) whereas Foc-24 was found to be least aggressive (7.22% wilt incidence). At molecular level, the sequences of internal transcribed spacers (ITS) genomic regions of isolates were studied and they showed 99% similarity with Foc sequences by basic local alignment search tool (BLAST) analysis.


INTRODUCTION
Chickpea cultivation is often subjected to several biotic stresses of which diseases like Ascochyta blight, Botrytis grey mould, Verticillium wilt, Sclerotinia stem rot, dry root rot and Fusarium wilt are important.Among them, Fusarium wilt, caused by Fusarium oxysporum f.sp.ciceris (Padwick) Matuo and K. Sato has assumed serious proportions in the recent years.Throughout the world, annual chickpea yield losses due to this disease vary from 10 to 15% (Trapero-Casas and Jimenez-Diaz, 1985), but can reach even 100% under favourable Primer Sequence ITS-Fu-f 5`-CAACTCCCAAACCCCTGTGA-3` ITS-Fu-r 5`-GCGACGATTACCAGTAACGA-3` conditions (Navas-Cortes et al., 2000).In India, yield losses estimates range from 10 -90% every year in different regions in different cultivars (Singh and Dahiya, 1973;Jalali and Chand, 1992).
The fungus is both seed-and soil-borne and may survive in soil for up to six years even in the absence of the host (Haware et al., 1996).Considering the nature of damage and survival ability of the fungus, management of the disease is difficult either through crop rotation or application of fungicides.The most practical and costefficient method for management of chickpea wilt is the use of resistant varieties (Nene and Haware, 1980;Nene and Reddy, 1987;Bakhsh et al., 2007).However, evolution of new races poses a serious threat to deployment of wilt resistance in chickpea.
Therefore, regular monitoring of variation in new isolates collected from different cultivars and geographically distinct regions over the years is critical for successful resistance breeding programme.The conventional approaches to assess variation among the fungal isolates are morphological and virulence analysis.However, with the advent of DNA based molecular techniques it is now possible to assess genetic variation among the isolates.Genotyping of F. oxysporum f.sp.ciceris isolates along with virulence analysis may yield some information relevant to breeding.Keeping this in view, the present study was carried out with the objective of assessment of variability in isolates of F. oxysporum f.sp.ciceris (Foc) collected from chickpea growing areas of Punjab and adjoining states at morphological, pathogenic and molecular level.

Collection, isolation and maintenance of isolates
A large number of isolations were made from stem and root portions of diseased chickpea plants collected from different chickpea cultivars grown in different locations in Punjab and adjoining states.The infected portion was cut into small bits, was surface sterilized in 0.1% mercuric chloride for 40-60 s followed by rinsing twice in sterilized distilled water.Later, these bits were transferred on PDA in Petri plates under complete and aseptic conditions.Plates were incubated at 25°C in BOD incubator to obtainfungal growth.Finally, a total of 24 isolates were purified and maintained on PDA slants at 4°C for further studies and designated as Foc-1, Foc-2 and so on (Table 1).

Cultural and morphological characterization
In cultural characterization, the fresh cultures were grown from 7 days old culture of each isolate separately and incubated at 25 ± 2°C.Each isolate was replicated thrice.The observations on colony diameter, colony colour, rate and pattern of growth were recorded up to 9 days at regular intervals.For morphological characterization, cultures obtained on PDA slants were examined under compound microscope (Leica DM 3000) using image analyzer software at 40x.The size and shape as well as septation of micro and macro conidia of each isolate were recorded.

Pathogenic characterization
For pathogenic characterization, only twenty isolates of F. oxysporum f.sp.ciceris were included in the study since the remaining four isolates seems to be morphologically similar to one or other isolates.All the isolates were subjected to the pathogenicity tests on susceptible and resistant chickpea germplasm lines (JG-62, WR-315, L-550, L-552, GL-26054 and GLK-24092).Each isolate was cultured on potato dextrose broth (PDB) for 15 days at 25 ± 2°C.Ten surface sterilized seeds of 6 differential varieties were sown in three replications in 4 x 9" poly bags containing autoclaved soil and inoculum of respective isolate.In the control, plants were sown in autoclaved soil only without inoculum.Data on disease incidence (% infected plants) were recorded at regular intervals.

Molecular characterization
All the isolates were characterized using internal transcribed spacers (ITS) primers for Fusarium genus and xylanase 3 gene specific markers (Table 2).The cultures from 24 Foc isolates were grown on 100 mL potato dextrose broth in 250 mL Borosil flasks and incubated at 25± 2°C in BOD incubator for fifteen days.DNA from 15 days old broth cultures of each isolate was extracted using modified CTAB extraction method (Murray and Thompson, 1980).DNA was quantified using Thermo Scientific NanoDrop™ 1000 Spectrophotometer and working dilutions were made with DNA concentration of 60 ng/ µl in each isolate.
The amplified ITS genomic region from each isolate was further sequenced by outsourcing from Xcelris Labs Ltd., Ahmedabad India and compared with other Foc sequences submitted at National Center for Boitechnology Information (NCBI).

Cultural and morphological characterization
All the isolates produced well developed colonies on PDA medium with colony diameter ranging from 64.5 to 85.0 mm after 9 days of incubation.Maximum colony diameter, that is, 85.0 mm was observed in isolates Foc-3, Foc-4, Foc-10, Foc-21 and Foc-23, whereas minimum colony diameter, that is, 64.5 mm was observed in Foc-15.However, when growth rate was calculated, Foc-21 exhibited maximum growth rate of 8.78 mm/day and Foc-15 exhibited minimum growth rate of 5.0 mm/day (Table 3).
The six selected genotypes could differentiate the level of aggressiveness of all the isolates, thus giving them the status of pathotypes.Therefore, on the basis of mean aggressiveness, the 20 isolates could be converted into four pathotypes as given in Table 6.The isolates were also differentiated on the basis of their virulence reaction on each chickpea genotype, using DARwin software.The isolates were converted into two groups (Table 7).The first group consisted of two sub groups, that is, 1a (Foc-1, Foc-9, .Similarly, the second group also consisted of two sub groups, that is, 2a (Foc-2, Foc-13 and Foc-18) and 2b  and Foc-10) (Figure 1).The same standard differential was followed by several workers and existence of four races viz; race 1, 2, 3 and 4 was reported (Bayraktar and Dolar, 2012;Mandhare et al., 2011;Barhate and Dake, 2006).Recently, a new race (race 6) was reported to occur in India by Sharma et al. (2014).

Molecular characterization
Fusarium genus specific 18 S ribosomal DNA based ITS markers were used for true identification and study of genetic variation among Foc isolates.The ITS primers amplified a region of ~400 bp size from genomic DNA of all isolates (Figures 2 and 3).Further, amplicons were eluted using gel extraction kit, purified and sequenced by outsourcing from Xcelris Labs Ltd., Ahmedabad India.The sequences were aligned using DNA baser software and compared with other Foc sequences from standard database GenBank.Nineteen isolates were further subjected to Basic Local Alignment Search Tool (BLAST) software to compare the sequences of Foc, previously submitted at National Center for Biotechnology Information (NCBI) data base.All sequences resembled 99% similarity with Foc sequences.Dendrogram on the basis of sequence homologies among all the isolates was also generated by using dendroscope and it was found that 20 isolates were further grouped into three major groups (Figure 6).First major group consisted of 17 isolates .The other two groups consisted of Foc-9 and Foc-10.The ITS region sequence of Foc-15 have been deposited at NCBI, with Accession no.KM253762.
In the present study, presence of 700 bp fragment amplified by xylanase 3 gene (Figures 4 and 5) in all the isolates confirmed absence of race 4 in any of the isolates.Foc race 4 was distinguished with xylanase 3 gene by absence of amplification product only in this    race (Gurjar et al., 2009).Any correlation between geographical region and virulence variation with genetic diversity was not observed in the case of both markers (Singh et al., 2006;Sharma et al., 2009;Mandhare et al., 2011;Sivaramakrishnan et al., 2002).In a similar study by Kelly et al. (1994), it was observed that RAPD marker analysis converged the Foc isolates into two groups, yellowing type and wilt syndrome type.RAPDs markers have also been developed into SCAR markers specific to Foc (Durai et al., 2012).Dubey et al. (2010) observed high level of genetic diversity using ITS-RFLP analysis of Foc isolates, which converged the isolates into six groups.Recently, Sharma et al. (2014) reported occurrence of race 6 in India, which is of Mediterranean and USA region.They also reported application of DArT markers in assessment of genetic diversity in Foc pathogen, with few race specific unique alleles.

Figure 6 .
Figure 6.Maximum-likelihood phylogenetic tree based on the alignment of the partial sequences.

Table 1 .
Location and source of host variety of different isolates of F. oxysporum f.sp.ciceris.

Table 2 .
Sequence of ITS primer.

Table 3 .
Cultural characteristics of different isolates of F. oxysporum f. sp.Ciceris.
Fungal DNA extraction from Foc isolates

Table 4 .
Morphological characterization of different isolates of F. oxysporum f.sp.ciceris.

Table 5 .
Percent wilt incidence induced by different Foc isolates on selected chickpea genotypes.

Table 6 .
Grouping of different isolates as different pathotypes.