Fungicide tolerance of Trichoderma asperelloides and T . harzianum strains

Tolerance in isolations of Trichoderma was developed by exposing two strains of T. harzianum and three of T. asperelloides to increasing concentrations of chemical fungicides. This isolation of Trichoderma was exposed to three fungicides: Captan, Thiabendazol and the mixture Captan-Carboxin. Some selected lines of these strains reached tolerance to Captan and partial tolerance to the mixture Captan-Carboxin. The biological and genetic changes in these tolerant lines were monitored by determining the relative growth rate of the fungus, inhibition of Fusarium and by analyzing the genomic changes through UP-PCR. The results show that the tolerance to fungicides can be developed without affecting the parameters of biological activity in these lines of Trichoderma (growth and parasitism against Fusarium). Chemical tolerance to the fungicide was verified by means of changes at the DNA level (UP-PCR), mainly in the lines tolerant to Captan. This suggests that Trichoderma survives in environments with remnants of fungicide molecules.


INTRODUCTION
A strategy of biological control of plant diseases caused by soil-borne plant pathogen fungi is the use of species of Trichoderma, these includes species of economic importance on industrial purposes for production of antibiotics and enzymes.In agriculture, these fungi, improves plant growth and development, has biological control activity against other fungi and nematodes [1][2][3][4].It has been found that the persistent use of fungicides could weak the natural antagonistic activity [5].However, Trichoderma has the capability of degradading xenobiotic compounds [6][7][8].There are Trichoderma tolerant strains that can survive field concentrations of chemical fungicides.We now have several approaches that can be used to obtain Trichoderma strains resistant to chemical fungicides.Goldman et al. [9] and Mukherjee et al. [10] have sccesfully obtained T. viride and T. pseudokoningii strains tolerant to chemical fungicides.The resistance mechanism of some fungi to chemical fungicides is due to genetic mutations, which reduces the susceptibility to the fungicides and decreases their efficacy [9,[11][12][13].

Strategy for the Selection of Tolerant Trichoderma Lines to Chemical Fungicides
Before the selection experiments were started, the Trichoderma strains were grown in potato dextrose agar (PDA) supplemented with the chemical fungicides at increasing concentrations.The final concentrations used in the field are: Captan 1132.5 ppm, a mix 1:1 Captan-Carboxim 2000 ppm, and Thiabendazole 450 ppm.The objective was to determine the natural fungicide tolerance of the five Trichoderma strains.The selection of tolerant lines to chemical fungicides was performed by successive cultures of the Trichoderma strains in PDA supplemented with the correspondent fungicide at increasing concentrations.Five mm diameter disks from Trichoderma 10 days old cultures were placed on PDA with the chemical fungicides, and mycelial growth was measured on days 1, 2, 3, 4, 5, 7 and 11.Trichoderma lines displaying more than 20 mm of growth were selected to be grown under the following chemical fungicide concentration in subsequent rounds of selection.Strains that did grow 20 or more mm in diameter after 10 days of incubation, continue in the selection media; on the contrary, strains that grew poorly (less than 20 mm of diameter) or did not sporulate, were discarded.Tolerant strains were subjected to further selection experiments with increasing fungicide concentrations until the Trichoderma lines were able to sporulate.
To evaluate the tolerance of the selected Trichoderma lines to the chemical fungicides, they were grown in 30 ml of liquid medium (yeast extract 2.5%, glucose 2.5%, NaNO 3 0.2%) in 125 ml flasks erlenmeyer supplemented with the fungicides, for four days at 28˚C, at 125 rpm.This experiment was performed twice, and in each one 2 replicates were set for each Trichoderma line.

Evaluation of the Antagonistic Activity and Growth of the Tolerant Trichoderma Selected Lines
The antagonistic activity of the selected tolerant Trichoderma strains was compared to the wild type strains by placing a 3 mm diameter disk from a Fusarium oxysporum 5 to 8 day old culture on PDA.After 24 h, a 3 mm diameter disk of the Trichoderma strain was placed 3 mm apart from the plant pathogen.Each treatment was done by triplicate, and incubated at 25  1˚C under lighT.The antagonistic activity of the Trichoderma strains was estimated according to two criteria,: the plant pathogen growth inhibition radius (IR) and the antagonism class system described by Bell et al. [15].Means of growth rate and IR was analyzed by ANOVA and Fisher's least significant difference (LSD) test to determine statistically significant differences.

Identification of Molecular Characteristics of Trichoderma Fungicide Tolerant Selected Lines
The DNA of the Trichoderma fungicides tolerant strains was analyzed through universal primer PCR marker (UP-PCR), a multi-site amplification technique [16,17].The amplifications patterns of these strains were compared to the wild type strain.DNA extraction was performed from 200 mg of lyophilized fungal mycelia according to the method described by [18].PCR amplification mixture was compose of PCR buffer 1X, MgCl 2 3 mM, dNTPs 0.2 mM, primer 1.6 M, Taq DNA polymerase 1 U, 25 ng of DNA distilled water to a final volume of 25 l.The following amplification program was used: initial denaturation at 94˚C during 2.5 min, followed by 30 cycles of 92˚C during 50 s, 53˚C during 90 s and 72˚C during 30 s, with a final extension at 72˚C during 3 min.UP primers used were L-45 (5' GTAAAA CGACGGCCAGT 3') and L-15 (5' GAGGGTGGCGG CTAG 3').All amplification reactions were performed at least by duplicate.Amplification products were separated in 2% agarose, stained with ethidium bromide and visualized on a UV transilluminator.Additionally, a specific DNA fragment of the β-tubulin gene was amplified and used as target to diagnosed resistance to the fungicide Thiabendazole [19].

Selection of Tolerant Trichoderma
Lines to Chemical Fungicides After five rounds of selection, it was noticed that although 10 out of 15 Trichoderma lines used in the experiments accomplished the mycelial growth selection parameter, of at least 20 mm of colony radius in 10 days, the speed of growth in all cases was lower than the wild type strains (Table 1).Natural tolerance to the field dose of the chemical fungicide Captan (1132, 5 ppm) was achieved in all the T. asperelloides and T. harzianum strains evaluated in this study.In general, isolates of T. harzianum were less tolerant to the chemical fungicides than isolates of the T. asperelloides species.At the end of the 9 rounds of selection with the chemical fungicides, tolerance to Captan varied between 176% and 207% of the dose recommended for field application.Isolates of T. harzianum could not develop tolerance to the fungicide Thiabendazole and the mixture Captan-Carboxim.T. asperelloides isolates T-19, T-84, and T-109 were able to grow and to sporulate in the culture medium containing 75% of the dose recommended for field application (2000 ppm).In contrast, none of the evaluated strains were able to develop tolerance to the fungicide Thiabendazole at a concentration below 20 ppm.Selected tolerant strains cultured in liquid medium supplemented with chemical fungicides Captan and Captan-Carboxin (Table 2) do not shown differences from the wild type strains grown without fungicides, after fourdays of culture (data not shown).
Analysis of the growth rate, (mm/hr) of the chemical fungicide tolerant Trichoderma lines compared to the wild type strains, show that this parameter was affected in 8 of the 10 tolerant selected lines.Statistical analysis indicate that the growth rate of six tolerant lines was lower than that of the wild type strains  3).

Antagonism Tests of Tolerant
Trichoderma Lines to Chemical Fungicides The antagonism test was performed with the plant pathogen Fusarium oxysporum and measured as the IR.It was observed that all tolerant Trichoderma strains kept their antagonism class 2 similar to the Trichoderma wild type, but strain T. asperelloides T-19 Thiabendazole shifted to class 3 of antagonism (Table 3).Comparison of the IR mean values displayed by the Trichoderma fungicide tolerant lines indicated that some lines have IR values that are significantly higher than the wild type strain, as in the case of T. harzianum T-7 selected with  Captan and strain T. asperelloides T-19 selected against Captan-Carboxin.While in the other cases, the IR was the same or significantly lower than the wild type strain (Table 3).Taking in account that one of the criteria used in the selection experiments was the ability of the tolerant lines to sporulate, the microscopic study performed indicates that all the selected Trichoderma lines kept this characteristic except for T. asperelloides T-19 exposed to Thiabendazol (data not shown).

Molecular Analysis
PCR analysis of the Captan-Carboxin lines and the wild type Trichoderma strains showed different amplification patterns such as deletion or addition of DNA bands.DNA amplified with primer UP-L45 indicated that the strains T. asperelloides, T-19 and T-84, selected with the fungicide mixture Captan-Carboxin contain the same genetic changes compared to the wild type strains, lost a of 1400 bp DNA band, while the bands of 1150, 500 and 450 bp were new in the fungicide treated lines (Figure 1).
Although the PCR diagnostic test designed to identify Thiabendazole susceptible/resistant genotypes indicated that there were no changes in the β-tubulin gene (Figure 2(A)), a change at the DNA level was observed when primer UP-L45 was used.This change is illustrated by the appearance of a new 400 bp band in both selected Trichoderma lines (Figure 2(B)).Treatment of the Trichoderma strains with the chemical fungicide Captan induced the largest changes at DNA level of the fungicides, primer UP-L45 was used for detection (Figure 3).

DISCUSSION
T. asperelloides and T. harzianum contain strains that could be of importance in biological control of plant pathogens [20][21][22].Trichoderma strains used in this study were isolated from different geographical areas and from different sources.All of them were also naturally tolerant to the recommended concentration of the chemical fungicide Captan, and exposure of Trichoderma strains to increasing concentrations of this fungicide allowed for the selection of tolerant lines.Fungicide resistance is a stable, inheritable adjustment by a fungus to a fungicide, resulting in reduced sensitivity of the fungus to the fungicide.Resistant isolates are less affected or not inhibited at all by application of a fungicide [23].The fungicide can in fact still can control sensitive isolates, causing natural resistant isolates to potentially may become dominant in populations under selection pressure of fungicide.This phenomenos happens in assays, evidencing the fact that Trichoderma has a natural ability to tolerate fungicides, which is called 'natural' or 'inherent resistance'.Resistance is as a response to repeated use of the fungicide, or to the repeated use of another chemically related fungicide and/or by a biochemical mechanism of antifungal action [24].
Ruocco et al. [25] explained that the ability of Trichoderma to withstand relatively high concentrations of a variety of synthetic and natural toxic compounds, including its own antibiotics, depends on efficient cell detoxification mechanisms supported by a complex system of membrane pumps.Now it is well know that the genome of Trichoderma includes ABC transporters (ATPbinding cassette (ABC) transporters), which are members of a protein superfamily that effluxes drugs from cells of target organisms.Thus transporters may provide a mechanism of protection against cytotoxic drugs and xenobiotic agents.The natural function of ABC transporters in plant pathogenic fungi may relate to transport of plant-defense compounds or fungal pathogenicity factors [26].The ABC transporters may explain the natural tolerance of fungicides on Trichoderma, and their ability to successfully to survive in extreme environments.
Growth of T. asperelloides and T. harzianum strains in liquid medium with the fungicides Captan and Captan-Carboxin confirmed that the selected lines have developed a mechanism to tolerate the exposure to homogenous concentrations of the chemical fungicides.Tolerance to the fungicide mixture Captan-Carboxin was obtained in the treated lines of T. asperelloides strains T-19, T-84 and T-109, while some degree of tolerance to Thiabendazole was only obtained with the T. asperelloides strains T-19 and T-84.These data suggested de-toxification mechanisms are restricted to particular strains, and are not present in all the specimens of a taxa.
In some cases, growth rate and IR of the Trichoderma tolerant lines were affected by the exposure to the chemical fungicides.The antagonism capacity under in vitro conditions was only negatively affected in one out of the 10 tolerant lines obtained.A similar phenomenon was found in Penicillium on Imazalil resistance and sensible strains on which was no difference in spore production and radial growth [27].In two cases the antagonistic capacity was superior in tolerant lines (T.asperelloides T109 Captan/Carboxin and T. harzianum T53 Captan).Analogous results were obtained by Mukherjee et al. [10], with mutants of benomyl-tolerant strains of T. pseudokoningii, which were superior to the wild type in biocontrol potential on S. rolfsii.A correlation between fungicide resistance and antagonistic activity is suggested by Marra et al. [28], affirming that the upregulated expression of ABC transporter genes of T. atroviride during the three-way interaction with various plants and fungal pathogens, possibly supports both antagonistic activity and root colonization.
DNA changes were observed in T. asperelloides lines T-19 and T-84 treated with Thiabendazole (benzimidazole group) (Figure 2(B)).The results of the diagnostic test designed by Cañas (2004) indicated that there were not changes in the -tubulin gene level.Nevertheless benzimidazole resistance was conferred by point mutations in the β-tubulin gene in most phytopathogenic fungi.However, exceptions have also been noticed through via site-directed mutagenesis, a mutation that confers benomyl tolerance to other fungi does not impart resistance in T. viride [29].Kawchuk et al. [30] established that the amino acid sequences of the β-tubulin genes from several thiabendazole-resistant and sensitive isolates were identical in Gibberella pulicaris.This analysis confirmed that the β-tubulin gene was not linked to thiabendazole resistance.These results suggest that there must be other genomic regions involved in the resistance to benzimidazoles, but the exact molecular mechanism for this resistance still unknown.
Differences in the number of genetic changes observed in the Trichoderma strains treated with chemical fungicides could be due to their mode of action or to the approach used for tolerance development.It has been described that protectant fungicides such as Captan, induce mutations in several genes, contrary to systemic fungicides in which target a particular gene or gene product [9,[11][12][13].This coincides with the results, since high genetic changes observed in the Captan tolerant Trichoderma lines as compared to the wild type strains.
The results suggest that it is possible to develop Trichoderma tolerant lines to some chemical fungicides.
Most importantly, the changes induced by this tolerance, in most cases, does not negatively affect the antagonistic activity of the biological control strains, and in some other cases, the growth rate and the IR are increased.The molecular study performed permitted us to recognize changes at the genomic level, which in most cases are not related to the loss of biological fitness of the fungal strains.

1
Antagonism class determined according to Bell et al., (1982) determined after 67 hours of culture on PDA; 2 Mean values followed by the same letter within each Trichoderma strain and column are not significative different (LSD,  = 0.05).

Table 1 .
Mean growth value of selected Trichoderma asperelloides and T. harzianum isolates exposed to several concentrations of the chemical fungicides Thiabendazole, Captan-Carboxin, and Captan compared to the wild type strains after 5 rounds of selection.Growth mean value in bold correspond to the Trichoderma isolates selected to continue in the fungicide tolerance selection experiments.

Table 2 .
Maximum concentrations tolerated by Trichoderma asperelloides and T. harzianum strains after multiple increasing exposures to the chemical fungicides Thiabendazole, Captan-Carboxin, and Captan, under laboratory conditions.

Table 3 .
Mean growth rate of Trichoderma strains and antagonism against to Fusarium oxysporum caused by wild-type and selected fungicide tolerant lines of Trichoderma asperelloides and T. harzianum.