Variation in Resistance to Benzimidizole in Different Biocontrol Agents Based on Protein Sequence Homology

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Variation in Resistance to Benzimidizole in Different Biocontrol Agents Based on Protein Sequence Homology

B.M.S. Jarullah*, R.B. Subramanian and M.J. Jummanah

¹

BRD School of Biosciences, Sardar Patel University, Vallabh Vidyanagar- 388120, Gujarat, India; ¹King Fahad Medi- cal Research Centre, Faculty of Medicine, King Abdulaziz University, Jeddah, Saudi Arabia

Abstract: Benzimidazole resistance in thirty isolates of the biocontrol agents was studied with reference to specific mutations in the -tubulin genes. Our results suggest that apart from the correlation between specific mutations in the -tubulin gene and variation in resistance, the overall ratio of polar and non-polar amino-acids may also play a vital role in confer- ring benzamidazole resistance to these fungi.

Keywords: Biocontrol agents, benzamidazole, non-polar amino acids, polar amino acids.

1. INTRODUCTION

Integrated Pest Management (IPM) is a sustainable approach to manage pests by combining biological, cultural, physical and chemical tools in a way that minimizes eco- nomic, health and environmental risks. One of the important biological tools used in this approach are the fungal biocontrol agents. An important criterion for the performance of these fungi under field conditions is their ability to resist the chemical compounds present in fungicides, nematicides, anthelmintics, pesticides etc used in the pest management strategies. In this study we have focused on four species of such biocontrol agents. Although a number of studies have been carried out by several investigators on the adverse effect of fungicides on individual species of fungi [1-4], no study has as yet been carried out on the comparative effects of these chemical compounds on the various species of fungal biocontrol agents.

Compounds that inhibit growth of biocontrol fungi in the laboratory tests include benomyl, zineb, captan, methyl para- thion and phenthoate. Benomyl, the active ingredient of the benzamidazole anthelmintics and fungicides, has been reported to be the most common inhibitor of all compounds tested against various fungi [5-6].

Earlier reports on benzimidazole resistance in fungi have suggested its correlation with specific mutations in the – tubulin gene [7-10]. In the present study an attempt has been made to correlate the beta tubulin gene sequences of various biocontrol fungi with their response to carbendazim, a benzimidazole derivative. Twenty five different isolates of nematophagous fungi including the Arthobotrys sps, Dud- dingtonia sps and Paecilomyces sps were used. Five ento- mopathogenic fungi, Beauveria sps was also added in order to compare the variation in resistance amongst these two diverse groups of fungi. The data obtained was then correlated with the –tubulin sequences of these fungi and a detailed study of the sequences of these –tubulin genes in terms of their amino acid composition was done.

*Address correspondence to this author at the BRD School of Biosciences, Sardar Patel University, Vallabh Vidyanagar- 388120, Gujarat, India; Tel:

+91-9824084524; E-mail: [email protected]

2. MATERIALS AND METHODS 2.1. Fungal Culture

One week old pure cultures of fungi maintained on 2%

CMA (Hi Media, Mumbai) or PDA (Hi Media, Mumbai) plates, containing 35g/ml Tetracycline and 100g/ml Am- picilin, depending on the culture requirements, maintained at 27 ºC were used for the assay.

2.2. Benzimidazole Derivative Used

Carbendazim (Bavistin, 50% WP, BASF India Ltd., Mumbai) was used in a range of concentration from 0.1 to 500Kg of active ingredient/ml of nutrient media.

2.3. Assay for Effect of Benzimidazole

Five petri dishes containing the appropriate media amended with carbendazim at 0, 0.1, 1, 100 and 500 g of active ingredient (a.i.)/ml of media were used. Replicates of each Carbendazim concentration were used for each isolate.

Nine millimeter diameter mycelial plugs taken from the edge of a one week old colony of each isolate were placed onto the center of the plates amended with each concentration of the above benzimidazole derivative.

The plates were incubated at 27°C and the radial growth (colony diameter) of each isolate was measured, with the original mycelial plug subtracted from each measurement, at the interval of 24 hours for 5 days. Two measurements, each perpendicular to the other, were taken for each plate and the mean calculated.

From the above data the rate of growth and the percent inhibition in rate of growth for each fungus was calculated and the results compared.

2.4. Sequencing of –Tubulin Gene

2.4.1. Synthesis of -Tubulin Primer and PCR Amplifica- tion

-tubulin gene specific primers were obtained from Ge- nei, Bangalore. These were used to amplify -tubulin gene of

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462 Protein & Peptide Letters, 2007, Vol. 14, No. 5 Jarullah et al.

A. musifomis. PCR reaction was set up under sterile conditions in 200 l capacity PCR tubes. The PCR mixture con- tained 200 ng of template DNA, 33 ng of oligonucleotide primer (5’aac atg cgt gag att gta agt 3’, 5’ tct gga tgt tgt tgg gaa tcc 3’), 1X PCR reaction buffer with 1.5 mM MgCl₂, 250M of each dNTP and 2 unit of Taq DNA polymerase in final reaction volume of 50 l. PCR reaction was conducted using Eppendorf thermal cycler. The thermal profile used was as follows: initial denaturation at 95°C for 2 minutes, followed by 40 cycles of 95°C for 30 seconds, primer an- nealing at 51°C for 1 minute, extension at 72°C for 1 minute and final extension at 72°C for 5 minutes. After completion of the PCR reaction, amplification product was electrophore- sed at 100 volts in 2% Agarose gel, stained with ethidium bromide, viewed under UV light and photographed. DNA ladder (100 bp) was used as molecular size marker.

2.4.2. Cloning of PCR Product into pGEM-T-Easy Vector Amplified genomic DNA of A. musiformis (450 bp) was cloned into pGEM-T-Easy vector. Ligation reaction was set up in a final volume of 10 l containing 1:1 ratio of vector (50 ng pGEM-T-Easy): insert, 1X ligation buffer and 1 unit of T4-DNA ligase. The resultant reaction mixture was incubated at 14-16 ºC for 16 hours.

2.4.3. Transformation

Competent cells were prepared as per the methods of Sambrook et al., 1989.[11] Two microlitres of 0.5 M - mer- captoethanol was mixed in 50 l of E. coli competent cells (Lac Z repressor) by gentle stirring. To this 2 l of well mixed ligation reaction was added, mixed and incubated on ice for 30 minutes. Heat shock treatment was given at 42ºC in water bath exactly for 120 seconds followed by incubation on ice for 5 minutes. To this, 250 l of SOC medium was added and incubated at 37 ºC for 1 hour at 225 rpm.

2.4.4. Analysis of Transformants

Fifty microliters and 200 l of the transformation product obtained was spread on LB / X- Gal / IPTG plates containing 100 g/ml of ampicillin followed by incubation at 37ºC for 18 hours. These plates were shifted to 4°C for 2-3 hours in order to allow proper development of color. Insert containing white colony were used for plasmid isolation and restriction analysis.

2.4.5. Sequencing of -Tubulin Gene

Sequencing of purified plasmid DNA was carried out using an automated DNA sequencer with flourescent dye terminator using ABI 377 PRISM sequencer (Applied Bio- systems, California) at Genei, Bangalore. Similarity search against the databases was done using the Basic Local Align- ment Search Tool in the BLAST network service (National Centre for Biotechnology Information, Bethesda).

2.5. Analysis of –Tubulin Sequences

The sequenced -tubulin gene from Arthrobotrys musi- formis was submitted to the National Center for Biotechnol- ogy Information (NCBI) Database (AY963560).

The –tubulin sequences for the rest of the fungi used in our study were obtained from the database, aligned and a cladogram was generated using ClustalW program (random seed number, 111; bootstrap value, 1000).

This data was then compared with the results obtained from the assay for the carbendazim resistance. A detailed study of the sequences of these –tubulin genes in terms of their amino acid composition was also done using Genedoc, a sequence editing program [12].

3. RESULTS AND DISCUSSION

Based on the study of benzamidazole resistance, these fungi were seen to fall into three different categories: Sensi- tive, Moderately Resistant and Resistant. (Table 1) The sensitive isolates showed a reduction in the rate of growth above 5mm/day (>2.8%), the moderately resistant isolates between 2.5-5mm/day (2-2.8%) and the resistant isolates below 2.5mm/day (<2%). It was interesting to note that all the isolates belonging to one species fell into any one of the three categories.

Table I. Inhibition in the Rate of Growth with Increasing Concentration of Carbendazim

Organisms Percentage Inhibition (Mean ± SD)

Paecilomyces sps 1.67 ± 0.32

Arthrobotrys musiformis 1.90 ± 0.20 Arthrobotrys oligospora 2.34 ± 0.02

Duddingtonia sps 2.72 ± 0.21

Beauveria sps 2.90 ± 0.30

Earlier reports on benzamidazole resistance in fungi had suggested its correlation with specific mutations in the – tubulin gene [7-10]. To confirm this relationship, we further constructed an unrooted phylogenetic tree from the multiple sequence alignment data, of the -tubulin gene sequences of the fungi used in our study, using the CLUSTAL W program (random seed number, 111; bootstrap value, 1000).

When the cladogram generated was compared with our data for resistance, distinct correlation was observed. The comparatively resistant isolates i.e.; Arthrobotrys musiformis and Paecilomyces sps were in close proximity to each other.

The moderately resistant isolates including Arthobotrys oli- gospora and Duddingtonia flagrans were found in one node and the sensitive isolates i.e.; Beauveria sps were placed in an entirely separate node apart from the rest of the species Fig. (1).

We further analyzed the amino acid composition, which is known to be one of the important parameters in determin- ing protein thermostability. It has been reported that thermo- stable proteins contain an increased proportion of charged residues at the expense of uncharged polar amino acids, con- ferring them rigidity and stability by minimizing deamidation

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and backbone cleavages [13]. A detailed study of the sequences of these –tubulin genes, in terms of their amino acid composition suggested a significant relation between resistance and the percentage of polar and non-polar amino- acids in the –tubulin genes Fig. (2).

With the increase in the percentage of non-polar amino- acids in the –tubulin gene of the fungi, there is a significant increase in resistance. Probably the stability of the –tubulin against the benzamidazoles is due to this increase in the percentage of non-polar amino acids in its sequence. The sensitive strains were found to have lower percentage of non- polar amino acids where as the resistant ones showed higher percentage of non-polar amino acids in the aligned region.

It is a well known fact that protein structures are stabi- lized by several factors, such as van der Waals interactions, hydrogen bonds, hydrophobic interactions, effect of hydra- tion on non-polar groups and salt bridges. The hydrophobic forces play a crucial role in the folding of the linear polypep- tide into a compact molecule with a tight hydrophobic core and a hydrophilic surface by excluding solvated water, and thus giving a large entropic gain. The hydrophobic interac-

tions give a gain of 0.0-2.5 (1.5) kcal mol^-1 per methylene group by removing approximately one-third of the ordered solvation water on formation of the secondary structure ele- ments. On further folding into a tertiary structure another one-third of the ordered solvation water is lost.

Although charge–charge interactions increase the number of salt bridges and are associated with enhanced stability of proteins, one must take into consideration that these interactions are also important for the functionality of a protein.

Thus, it can be expected that in cases in which the increase in electrostatic interactions interferes with the protein activity, other mechanisms for resistance development might be used.

Interestingly, the analysis of the kind of mutations that have been reported to confer resistance to benzimidazoles in fungi further supported our hypothesis. Most benzimidazole resistant isolates have been known to exhibit codon changes either at the positions 198 or 200 in beta tubulin gees. The substitution of polar amino acid glutamic acid (GAG) at codon 198 by non polar amino acid alanine (GCG) results in high resistance to benzimidazoles [9, 10, 14].

Figure 1. Cladogram generated by the alignment of –tubulin sequences for the fungi used in the study.

Figure 2. Percentage inhibition in rate of growth compared with difference in percentage of polar and non-polar amino acids in -tubulin sequence.

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464 Protein & Peptide Letters, 2007, Vol. 14, No. 5 Jarullah et al.

Since the principle of protein stability is not only of aca- demic interest but also has practical and technical implica- tions [15], there would definitely be a need to envisage the status of polar amino acid residues, in addition to optimiza- tion of charge–charge interactions on the surface of a protein in designing stable proteins [16].

4. CONCLUSIONS

Our results suggest that not only can the presence of specific mutations in the –tubulin gene be correlated with the variation in resistance, but the overall sequence of the gene with its three dimensional structure and the percentage of polar and non-polar amino-acids also play a vital role in con- ferring benzamidazole resistance to these fungi.

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Received: February 02, 2007 Revised: March 21, 2007 Accepted: March 30, 2007

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