Detection of β-tubulin gene from benomyl sensitive isolates of Colletotrichum gloeosporioides causing anthracnose disease in mango

Twenty six isolates of Colletotrichum gloeosporioides from anthracnose infected mango fruits were isolated from different places of Tamil Nadu, India and these isolates were identified as C. gloeosporioides by Internal transcribed spacer (ITS) and species specific (CgInt) primers. The sensitivity of C. gloeosporioides isolates to benomyl fungicide were evaluated at five different concentrations viz., 0.5, 1, 2, 5, 10 mg/l through poisoned food technique and were expressed very less to highly sensitiveness to the fungicide. All the twenty six isolates were able to grow at the lowest concentration of 0.5 mg/l benomyl amended medium and the average mycelial growth was 70.70 mm at even days after inoculation. At the highest concentration of 10 mg/l benomyl, only two isolates viz., MCG 7 and 16 were able to grow with the mycelial diameter of 12.00 and 18.00 mm, respectively and 100% inhibition was found in the remaining isolates. In addition, benzimidazole sensitive and resistance β tubulin gene sequences of TUB 1 and TUB2 were amplified from the benomyl sensitive isolates of C. gloeosporioides. The results indicated the differential resistance or sensitivity to benomyl fungicide against C. gloeosporioides and thereby allowed to identify the variability and diversity of the isolates on regional basis.


INTRODUCTION
Many diseases are affecting mango, of which anthracnose caused by Colletotrichum gloeosporioides (Penz.)Sacc is the most devastating disease and major constraint in production and export of mango.Several fungicides have been used to manage the mango anthracnose as pre-and post-harvest sprays, including benzimidazoles, triazoles, strobilurins, acid amides, dithiocarbamates and heterocyclic compounds (Fei and Wang, 2004).Hot benomyl dips have been reported for effective control of anthracnose in harvested mango fruit (Kim et al., 2007).Benomyl has been used in agriculture for approximately 30 years and numerous cases of *Corresponding author.E-mail: archanas_agri@yahoo.co.in.Tel: +91 9597754699.
Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License resistance have been reported, including Colletotrichum spp. on various crops (Whiteside, 1980;Picinini, 1994;Hewitt, 1998).Benzimidazoles are specific inhibitors of microtubule assembly that act by binding to their heterodimeric subunit (Davidse, 1986).Several studies have demonstrated that benzimidazole resistance is associated with point mutations in codon 198 or 200 of the β-tubulin gene in various fungi, including Venturia inaequalis, Botrytis cinerea, Monilinia fructicola, C. gloeosporioides and Mycosphaerella fijiensis (Ma et al., 2003;Peres et al., 2004;Chung et al., 2006;Kim et al., 2007).Other point mutations in the β-tubulin gene (Jung et al., 1992;Cooley and Caten, 1993) as well as in the putative leucine zipper protein CaBEN1 (Nakaune and Nakano, 2007) have also been associated with resistance to benzimidazoles.Maymon et al. (2006) reported that sequence analyses of the β-tubulin genes, TUB1 and TUB2, of five sensitive and five resistant representative isolates of C. gloeosporioides from Limonium spp.revealed that the benomyl resistant isolates had an alanine substitute instead of a glutamic acid at position 198 in TUB2.Benzimidazole resistant isolates of C. gloeosporioides from different crops have been identified, but the molecular characteristics of these resistant isolates are still not well defined (Pei, 1981;Tsai et al., 2006).The molecular identification and monitoring of benzimidazole resistant fungi has been recognized as a useful tool in the study of fungicide resistance (Ma et al., 2003;Canas-Gutierrez et al., 2006).Housekeeping genes including the nuclear β-tubulin genes, which show higher DNA sequence variability, are more extensively used to develop diagnostics for fungi, (Fraaije et al., 2001;Mostert et al., 2006;Aroca et al., 2008).Partial βtubulin sequences from C. gloeosporioides isolates of mango were amplified (Albertini et al., 1999;Yarden and Katan, 1993) as a molecular tool for the identification of benzimidazole-resistant isolates of C. gloeosporioides.Sanders et al. (2000) evaluated 158 isolates of C. gloeosporioides and reported, 17.7% were resistant to benomyl with 8.5% highly resistant due to mutations in the β -tubulin gene using this information, C. gloeosporioides isolates from mango fruits collected in India were evaluated for benomyl sensitivity and mutations in the β -tubulin gene in the present study.

C. gloeosporioides from mango
Twenty six isolates of C. gloeosporioides collected from different districts of Tamil Nadu, India were evaluated in the study (Table 1).Their pathogenicity, variability in virulence was previously reported by Archana et al. (2014).

Sensitivity of C. gloeosporioides to benzimidazole fungicidebenomyl
Each isolate was cultured on Potato Dextrose Agar (PDA) medium Archana et al. 807 in petri plates at 28±2°C for three days, after that mycelial disc (3 mm diameter) were cut from colony margins and a single disc was transferred to the centre of Petri plate containing PDA medium amended with 0.5, 1, 2, 5 and 10 mg/ml of the active ingredient of benomyl fungicide, which was added to PDA medium after autoclaving.Test cultures were incubated in dark at 28±2°C and then mycelial growth was measured 11 days after inoculation (Chung et al., 2010) and control plate was maintained by growing the isolates in the Petri plate containing PDA medium alone.
PCR amplification using the oligonucleotide primers ITS 4 (TCCTCCGCTTATTGATATGC) and CgInt (GGCCTCCCGCCTCCGGGCGG) were used for species identification.PCR reaction were as described above with amplification as follows: denaturation at 95°C for 5 min, 30 cycles of 30 s. at 95°C, 30 s at 48°C and 90 s at 72°C and a final extension of 10 min at 72°C (Maymon et al., 2006) in an Eppendorf gradient thermal cycler.
Reactions prepared as above and PCR reactions were performed with 60 s of denaturation at 94°C; followed by 35 cycles consisting of 30 sec at 94°C, 30 sec at 50°C and 60 sec at 72°C; and a final extension period of 10 min at 72°C.

Agarose gel electrophoresis
Amplification products for the above 5 evaluations were separated in 1.2% (w/v) agarose gel in1x TAE buffer (0.4 M Tris, 0.2 M acetic acid, 10mM EDTA; pH 8.4) containing 0.5 µg/ml ethidium bromide.Electrophoresis was carried out at 85 V and the gel was documented using an Alpha Imager (Alpha Innotech Corporation, San Leandro, CA, USA).The sizes of the PCR products were determined by comparing with standard 100 bp or 1 kb molecular marker (Bangalore Genei Pvt. Ltd., Bangalore, India).

Statistical analysis
The data were statistically analyzed using the IRRISTAT version 92 developed by the International Rice Research Institute Biometrics unit, Philippines (Gomez and Gomez, 1984).Data were subjected to analysis of variance (ANOVA) at two significant levels (P< 0.05 and P< 0.01) and means were compared by Duncan's Multiple Range Test (DMRT).

Molecular detection of C. gloeosporioides isolates using ITS and CgInt primers
PCR amplification using universal primers of ITS 1 and ITS 4 produced the expected 560 bp fragment, confirming that 26 isolates belonged to the genus Colletotrichum (Figure 1).PCR amplification with CgInt and ITS 4 species specific primers produced the expected 450 bp amplicon confirming the isolates were C. gloeosporioides (Figure 2).

Screening of C. gloeosporioides against benzimidazole fungicidebenomyl
The sensitivity of C. gloeosporioides isolates to the fungicide benomyl was evaluated based on the method of poisoned food technique at five different concentrations viz., 0.5, 1.0, 2.0, 5.0 and 10.0 mg/l (Figure 3).The growth of mycelia on the benomyl amended PDA medium was directly correlated to the sensitiveness of the isolates benomyl.Based on this, twenty six C. gloeosporioides isolates were designated as susceptible, moderately resistant and resistant to the fungicide benomyl.At the lowest concentration of 0.5 mg/l benomyl amended medium, all the twenty six isolates were able to grow and the diameter of the mycelium was measured eleven days after inoculation.The isolate MCG 21 produced the least growth of 27.23 mm followed by MCG 22(33.24mm) and   MCG 13 (33.72 mm) which were significantly different from other isolates.The mycelial growth of 90 mm was observed in the isolates of MCG 3, 16, 23 and 26 at 0.5 mg/l concentration (Table 2).At the concentration of 1 mg/l of benomyl, the percent inhibition over control was higher in the isolate MCG 21 accounting 73.16%, which was on par with the isolates MCG 10 (71.60%) and MCG 22 (71.21%).Least inhibition of 3.06% was observed in

PCR amplification of β-tubulin gene from C. gloeosporioides
In all the twenty six isolates of C. gloeosporioides, partial βtubulin gene sequences of TUB 2 and TUB 1 were amplified by the appropriate primers from genomic DNA (Figure 5).

Nucleotide sequence accession number
The sequence obtained in this study was assigned GenBank accession number of β-tubulin resistance gene isolates MCG 11-KJ470629.1 and MCG 16-KJ462468.1

DISCUSSION
The fungus C. gloeosporioides is known to be highly variable in nature.All twenty six isolates of the present study were identified as C. gloeosporioides based on the symptoms on host, substrate, conidial size and shape, shape of appressoria, growth rate in culture, colour of cultures as reported by von Arx (1970) and Sutton (1980).
The traditional method of relying on cultural and morphological grounds for identification of C. gloeosporioidesis variable rarely standardized and tends to mislead, since it is subjective in nature.Molecular genetic studies have provided useful data for clarifying the systematics of the genus Colletotrichum (Martinez et al., 2002).The polymerase chain reaction (Mullis and Faloona, 1987) offers the opportunity to characterize fungal symbionts by amplification of specific sequences and provide very accurate quantitative data required for control and quarantine decisions.Hence, the present study was resorted into exploitation of ITS and CgInt derived primers for identification.The ITS region contains two variable non-coding regions that are nested within the rDNA repeat between the highly conserved small subunit, 5.8S and large subunit rRNA genes (Gardes and Bruns, 1993).The internal transcribed spacer regions (ITS1 and ITS4) within the nuclear ribosomal gene clusters are attractive loci of PCR-based detection assays since they are readily accessible using universal primers (White et al., 1990).In the present study, ITS 1 and ITS 4 primers amplified a fragment of 560 bp corresponding to the region of 18S rDNA sequence in all the twenty six isolates and further confirmed to the group of Colletotrichum.The results confirmed the findings of Kamle et al. (2013) in amplification of ITS region of C. gloeosporioides from mango yielded 560 bp amplicon.Further, identification of ITS regions of nuclear rDNA has been good targets for differentiation and phylogenetic analysis of fungi (Dunne et al., 2002).In the present research, a sensitive PCR-based species-specific designed primer pair of CgInt and ITS 4 was used with the aim to detect C. gloeosporioides of mango anthracnose pathogen from the infected fruit tissue, which amplified the DNA at 450 bp in all isolates.These results were supported by the earlier findings for the taxonomic identification of Colletotrichum species which have been used to distinguish between Colletotrichum acutatum, C. gloeosporioides and Colletotrichum fragariae (Forster and Adaskaveg, 1999;Urena-Padilla et al., 2002).Similarly, Sakinah et al. (2013) reported that, 38 Colletotrichum isolates from banana were identified as C. gloeosporioides (35) and Colletotrichum musae (3) through the amplification of ITS regions and β-tubulin gene using ITS 4 and 5 and Bt2a and Bt2b primers, respectively.Fungicide resistance is a key factor in limiting the efficacy and disease control strategy.The resistance may also be an important aid to our understanding, at a molecular level, of the fungicidal mechanism of action.In the present study, the sensitivity of C. gloeosporioides isolates to benomyl, a benzimidazole fungicide was determined, based on the mycelial growth at five different concentrations viz., 0.5, 1, 2, 5 and 10 mg/l.The twenty six isolates were  10 MCG 11 MCG 12 MCG 13 MCG 14 MCG 15 MCG 16 MCG 17 MCG 18 MCG 19 MCG 20 MCG 21 MCG 22 MCG 23 MCG 24 MCG 25 MCG 26  categorized as three viz., susceptible, moderately resistant and resistant, since significant variation was observed among the isolates in respect to sensitivity.In aqueous solution benomyl is rapidly hydrolysed to methyl benzimidazole -2carbamate and this is probably the active fungi toxicant against pathogen.At the highest concentration of 10 mg/l benomyl, only isolates MCG 7 and MCG 16 were able to grow eleven days after inoculation, which confirmed their sensitiveness of the remaining isolates to benomyl.This might be due to benomyl does not inhibit the spore germination and conidial production but acts primarily by slowing the mycelial growth before or after infection (Peres et al., 2002).
Based on this, isolate MCG 21 was considered as sensitive MCG 5 as moderately resistant and MCG 16 as resistant to benomyl fungicide.This varied reaction obtained in the present study are similar to the findings of Chung et al. (2010), which demonstrated the sensitivity of thirty one C. gloeosporioides isolates from mango and strawberry to benzimidazole fungicides viz., benomyl, carbendazim and thiabendazole.In that, they reported that seventeen isolates of C. gloeosporioides were grouped as sensitive, two isolates were moderately resistant, nine isolates were resistant and three isolates were classified as highly resistant based on the colony diameter on PDA medium amended with benomyl at 10, 100 and 500 mg/ml concentration.It confirms the earlier reports of Sariah (1989), where the growth of sensitive isolates of Colletotrichum capsici were completely inhibited at 2.5 μg/ml benomyl while the resistant isolates grew on agar containing 1000 μg/ml fungicide.Peres et al. (2004) reported that mycelial growth of sensitive isolates was completely inhibited at 1.0 µg/ml of benomyl, whereas resistant isolates grew even at 10 µg/ml of benomyl.Similarly, Joshi et al. (2013) reported that, out of 30 isolates of C. gloeosporioides, isolate Cg 42 from mandarin orange was found to be insensitive to Benomyl with mean IC50 value of 318.85 μg/ml; while Cg 41 and Cg 43 was highly sensitive to Benomyl.In addition  Nalumpang et al. (2010) reported the sensitivity of C. gloeosporioides isolates from mango to carbendazim fungicide at various concentrations of 0.1, 1, 10, 100, 500 and 1,000 mg/l.They found that, among 59 isolates of C. gloeosporioides, 49 isolates were highly resistant (HR) phenotypes, 4 isolates were sensitive (S) phenotypes, none showed weakly resistance (WR) and moderately resistance (MR) phenotypes to carbendazim.
Benzimidazole fungicides act by inhibition of tubulin biosynthesis (Davidse, 1973), due to the mutations of βtubulin gene which has been related to specific amino acid substitutions at several distinct regions within the ßtubulin molecule (Fujimura et al., 1992).All the twenty six isolates of the current study were used to identify the presence of resistance and susceptible β-tubulin genes.Most often benzimidazole tolerance is due to mutations in the β -tubulin gene which reduce benzimidazole binding (Cooley and Caten, 1993;Reijo et al., 1994).These mutations can be used to rapidly identify tolerant strains with nucleic acid-based methods (Luck and Gillings, 1995).
Further, rapid identification of tolerant strains can aid in determining the fungicide resistance management policies.In the present research, partial β -tubulin gene sequences were amplified by the appropriate primers to the DNA fragment for TUB 2 and TUB 1 gene.Isolates MCG 3,MCG 7,MCG 8,MCG 11,MCG 12,MCG 13,MCG 16,MCG 19,MCG 20,MCG 21 had showed amplification product at 563 bp for benomyl resistant gene while the isolates of MCG 2, MCG 3, MCG 6, MCG 19, MCG 21, MCG 24, MCG 25 produced amplification for the primers of β-tubulin sensitive gene.In addition, alternative gene TUB 1 β-tubulin was amplified from four isolates viz., MCG 19, MCG 20, MCG 24 and MCG 25 at 506 bp. Peres et al. (2004) found that eight isolates of C. gloeosporioides were amplified by the portion of βtubulin gene, with primer TB2R and TB2L.
Resistance to benzimidazole fungicides has been reported in many filamentous fungi, whereby the fungicide is unable to bind to a mutated β-tubulin protein containing an altered amino acid sequence (Davidse and Flach, 1977).Several studies have demonstrated that sequence analysis of the β-tubulin gene of benomylresistant fungi revealed that, resistance almost always is due to point mutations in codon 198 or 200 of the βtubulin gene.Amino acid substitutions at these two codon positions result in different benomyl-resistance phenotypes (Albertini et al., 1999;Yarden and Katan, 1993).

Conclusion
The study reveals that a rapid benomyl tolerance screening could be necessary for examining the potential of different fungicide resistance.Pathogen C. gloeosporioides responsible for anthracnose in mango is Archana et al. 813 composed of both benomyl-resistant and sensitive populations that are genetically distinct, which might be due to a single β-tubulin gene existing within the fungal genome; however, in others, an additional gene may be present that is divergent in sequence.

Figure 3 .
Figure 3. Sensitivity of C. gloeosporioides isolates to benomyl fungicides (Poisoned food method using PDA medium amended with different concentration of benomyl to evaluate the sensitivity of C. gloeosporioides).
Poisoned food method using PDA medium amended with different concentration of benomyl to evaluate the sensitivity of C. gloeosporioides

Figure 4 .
Figure 4. Efficiency of benomyl fungicide against C. gloeosporioides isolates in vitro analysis.

Table 1 .
Characterization of C. gloeosporioides isolates based on cultural characters in PDA medium.
fg Mean of three replications; in a column, means followed by a common letter are not significantly different at 5% level by DMRT.

Table 2 .
Sensitivity of C. gloeosporioides isolates from mango to benomyl fungicide.