Novel mutations in β-tubulin gene in Trichoderma harzianum mutants resistant to methyl benzimidazol-2-YL carbamate

Abstract

Twelve low resistant (LR) mutants of Trichoderma harzianum with the capability of grow fast at 0.8 μg/mL methyl benzimidazol 2 yl carbamate (MBC) were obtained using UV mutagenesis. MR and HR mutants which could grow fast at 10 and 100 μg/mL MBC, respectively, were isolated by step up selection protocols in which UV treated mutants were induced and mycelial sector screening was made in plates with growth medium. Subsequently, β tubulin genes of 14 mutants were cloned to describe the molecular lesion likely to be responsible for MBC resistance. Comparison of the β tubulin sequences of the mutant and sen sitive strains of T. harzianum revealed 2 new MBC binding sites differed from those in other plant pathogens. A single mutation at amino acid 168, having Phe (TTC) instead of Ser (TCC), was demonstrated for the HR mutant; a double mutation in amino acid 13 resulting in the substitution of Gly (GGC) by Val (GTG) was observed in β tubulin gene of MR mutant. On the other hand, no substitutions were identified in the β tubu lin gene and its 5' flanking regions in 12 LR mutants of T. harzianum. 165Ala to Val A. nidulans [ 23] 167 Phe to Tyr Cochliobolus heterostrophus [24], Neurospora crassa [25], Penicillium expansum [26], Saccharomyces cerevisiae [27] 198 Glu to Ala Botrytis cinera [28], M. fructicola [19], Penicillium aurantiogriseum [29], P. expansum [26], Tapesia yallundae [30], Venturia inaequalis [29] Glu to Asp A. nidulans [18] Glu to Gln A. nidulans [18], T. yallundae [30] Glu to Gly N. crassa [31], T. yallundae [30] Glu to Lys A. nidulans [18], Colletotrichum.gloeosporioides [32], M. fructicola [19], P. aurantiogri seum [29], P. expansum [26], Sclerotinia homoeocarpa [29], T. yallundae [30], V. inaequalis [29] Glu to Val P. expansum [26] 200 Glu to Lys B. cinera [33] Phe to Tyr P. aurantiogriseum [29], T. yallundae [30], V. inaequalis [29] 240 Leu to Phe M. laxa [34], T. yallundae [30] 241 Arg to His S. cerevisiae [35]

Predicting Resistance by Mutagenesis: Lessons from 45 Years of MBC Resistance

“…Two substitutions with high resistance factors but not reported in the field are G13V and S168F, both found in Trichoderma harzianum (Li et al, 2013). The S168F mutant had the highest resistance factor with a 1572-fold increase in EC 50 compared to the wild-type, and the G13V mutant had a 96-fold increase.…”

Genetic enhancement of Trichoderma asperellum biocontrol potentials and carbendazim tolerance for chickpea dry root rot disease management

“…Biological control with improved Trichoderma strains has proven to be a potential alternative to chemicals of several soil-borne plant pathogens [ 27 ]. In many cases, individual use of either biocontrol agents or chemicals could not provide satisfactory levels of control over soil-borne [ 8 ]. Attempts have been made to obtain resilient fungicide resistant strains for usage in integrated disease management [ 8 ].…”

“…There have been several successful reports regarding the development of novel Trichoderma strains by using mutagenesis which has led to enhanced fungicide tolerance and biocontrol potential [ 1 ]. Pesticide tolerance in Trichoderma would be a prerequisite to reduce the fungicide resistance in pathogens and to promote the plant health [ 8 ]. Pesticide-resistant Trichoderma in combination with reduced fungicide application would help in reducing the detrimental effects of the latter on soil along with ensuring satisfactory levels of crop protection [ 9 ].…”

“…Among fungicides, carbendazim is one of the most extensively used benzimidazole (methyl benzimadazol-2-yl carbamate) for control of soil-borne diseases through seed treatment and foliar application [ 8 ]. Carbendazim resistance in pathogen populations have been detected in the field shortly after an intensive and exclusive exposure [ 11 ].…”

“…The benzimidazole tolerance in Trichoderma strains are yet to be ascertained for enhanced fungicide tolerance and large-scale field applications. There have been reports to confirm benzimidazole tolerance in Trichoderma with substitution of amino acids as a result of single and double mutation in β-tubulin tub2 region [ 8 , 15 ].…”