Hypocrea / Trichoderma viridescens ITV 43 with potential for biocontrol of Moniliophthora roreri Cif & Par , Phytophthora megasperma and Phytophthora capsici

Universidad Autónoma del Estado de Hidalgo, Hidalgo, México. Carretera Pachuca Tulancingo km 4.5, Mineral de la Reforma. Hidalgo. México. C.P. 42090. Universidad del Papaloapan. Instituto de Biotecnología. Campus Tuxtepec. Circuito central # 200. Colonia Parque Industrial Tuxtepec, Oaxaca, México C.P. 68301. Instituto Tecnológico de Veracruz. Unidad de Investigación y Desarrollo en Alimentos. Av. M. A. de Quevedo No. 2779 Col. Formando Hogar C. P. 91897 Veracruz, Ver., México.


INTRODUCTION
Cacao (Theobroma cacao L.) is one of the most important tropical crops since the seeds of its fruits are the raw material in the production of chocolate.However, diseases caused by fungi that attack cacao plantation have seriously affected cacao production.Among these, the witches broom caused by Moniliophthora perniciosa and the frosty pod rot caused by Moniliophthora roreri, are the most devastating.Losses are been estimated between 40 to 90% in several countries of America (Phillips-Mora and Wilkinson, 2007;Sánchez-Mora and Garces-Fiallos, 2012).On the other hand, black pod disease caused by species of Phytophthora, which are widely distributed in several countries of America, Asia and Africa cause losses in cacao plantation between 45 and 100% (Ploetz, 2007;Djocgoue et al., 2010).The control of these diseases is carried out by the application of fungicides, plant health practices and use of resistant cocoa varieties (Tondje et al., 2007).However, these conventional methods are not enough to completely stop the development of diseases in cocoa producing regions, being still necessary new tools to integrate into a comprehensive management program of cocoa diseases.An alternative strategy is the use of microorganisms isolated from the cacao tree or from its environment.For example, Trichoderma ovalisporum isolated from Theobroma grandiflorum and from Banisteriopsis caapi in Brazil and Ecuador, respectively demonstrated its ability to antagonize and parasitize M. roreri (Holmes et al., 2004).On the other hand, Trichoderma theobromicola and Trichoderma paucisporum isolated from healthy Theobroma cacao in Peru and Ecuador, respectively, showed an antibiotic effect against M. roreri on cocoa fruit in situ and in vitro experiments (Samuels et al., 2006).Trichoderma martiale isolated in Brazil from Theobroma cacao reduced the severity of symptoms caused by P. palmivora on cocoa fruit in the field (Hanada et al., 2008(Hanada et al., , 2009) ) and Trichoderma stromaticum isolated from Theobroma grandiflorum, was mycoparasitic on Phytophthora palmivora (Hanada et al., 2010) and is the active ingredient of 'Tricovab', a product that is being applied in the field to control black pod disease in Brazil (De Souza et al., 2008).Moreover, Trichoderma asperellum isolated from soil in Cameroon was mycoparasitic on P. capsici, P. citrophthora and P. palmivora, other causal agents of black pod worldwide (Tondje et al., 2007).
In a previous study, 128 fungal isolates were isolated from aerial plant tissues of cacao including tree trunks, stems and fruits with visual symptoms of frosty pod rot and black pod disease (Cuervo-Parra et al., 2011a).One of these isolates named ITV43 was identified to the genus level as Hypocrea/Trichoderma.The objectives of this study were to determine (i) the species of Hypocrea/Trichoderma strain ITV43 and (ii) its antagonistic activity in vitro against the plant pathogens M. roreri, P. megasperma and P. capsici.

Morphological characterization of Hypocrea/Trichoderma ITV43
The Hypocrea/Trichoderma strain ITV43 was cultured on cornmeal agar with 2% (w/v) dextrose (CMD: cornmeal agar 17 g, dextrose 20 g, 1000 ml distilled water) and on synthetic low-nutrient agar (SNA: KH 2 PO 4 1.0 g, KNO 3 1.0 g, MgSO 4 •7H 2 O 0.5 g, KCl 0.5 g, glucose 0.2 g, sucrose 0.2 g, 1000 ml distilled water, agar 20.0 g).Petri dishes (9 cm-diam) were incubated under ambient laboratory conditions of light and dark at 25-28°C for 10 days.The morphology and color of the colony, formation and shape of pustules, diffusing pigment in the agar and odor were recorded.To analyze conidiophores, phialides and conidia, samples of pustules were placed in a drop (3% w/v KOH) and observed with a compound microscope (Leica DM 3000, Leica Microsystems GmbH Wetzlar, Germany) using bright field (BF) and phase contrast (PC).Chlamydospores were observed by examining the reverse of the colony grown on CMD with 20x objective.Thirty measurements for each of the following structures were made: conidium length, width, and length/width ratio (L/W), phialide length, width, length/width ratio (L/W), base of phialide and cells supporting the phialides, chlamydospore length, width, and length/width ratio (L/W).Measurements of the characters were taken from images using the software LAS 3.2.0version Leica Microsystems (GmbH Wetzlar, Germany) and were reported as extremes in brackets separated by the means plus and minus the standard deviation.Also, the 95% confidence interval (CI) was calculated for each parameter.The ITV43 strain was identified using the key to species of Hypocrea with warted conidia (Jaklitsch et al., 2006).

Molecular characterization of Hypocrea/Trichoderma strain ITV43, P. megasperma, P. capsici strains and phylogram tree
Hypocrea/Trichoderma strain ITV43, P. megasperma (HT-ITV08, HT-ITV15, HT-ITV37) and P. capsici (HT-ITV14, HT-ITV16, HT-ITV33) strains were grown on potato broth medium (PD) at room temperature and 250 rpm.PD medium composition (expressed in g per liter in 1000 ml of distilled water) was as follows: glycerol, 20; polypeptone, 10 yeast extract, 10, glucose 5, and 100 ml of potato extract.The mycelium was grown for three days recovering and washing twice with TE buffer (TrisHCl 10 mM, pH 8.0, EDTA 1 mM, 2% SDS).The mycelium was ground and DNA was extracted by the method described by Kurzatkowski et al. (1996).A nuclear DNA region, containing the ITS I/5.8s/ITS II sequence, was amplified by PCR using the ITS1 (5tccgtaggtgaacctgcgg3') and ITS4 (5tcctccgcttattgatatgc3') primers (White et al., 1990).PCR amplification was performed in a final volume of 50 µl of reaction that contained: 10 μL of stamp 5x Colorless, 200 μMdNTPSs, 0.2 μM of each primer, 2,5 units of TaqDNA polimerase and 10-50 ng of template DNA.PCR reaction was placed in a thermocycler (Bio-Rad Gene CyclerTM series model No. 11453 Hercules, California, EE.UU.) under the following parameters: 5 min of initial denaturalization at 95°C, followed by 30 cycles of denaturalization of 1 min at 95°C, 1 min of alignment at 57°C, 1 min of extension at 72°C and a final extension period of 12 min at 72°C.The resulting product was purified with the kit Geneclean® II (Hercules, California, EE.UU.) according to the manufacturer's protocol and DNA was sequenced by the Biotechnology Institute, Cuernavaca, Morelos, México.Fungal DNA sequences were obtained with the program Chromas 1.45 (School of Health Science, Griffith University, Gold Coast Campus, Southport, Queensland, Australia) and a search was conducted with the BLAST (Zhang et al., 2000), for comparison of the homologies with the fungi sequences deposited in the GenBank (www.ncbi.nlm.nih.gov).The DNA sequence was aligned with other sequences through ClustalX (Thompson et al., 1997;Larkin et al., 2007) to identify the variable regions or base sequences.The distance matrixes between all pairs of sequence of multiple alignments were calculated and a tree was generated by the Neighbor-Joining method developed by Saitou and Nei (1987).Two bootstrap analyses were performed with 1000 interactions with the program ClustalX (Thompson et al., 1997).Two phylogram trees were generated and rooted by importing the calculated values with the PhyloDraw program version.0.8 (Graphics Application Lab, 1999).

Confrontation experiment of Hypocrea/Trichoderma against phytopathogenic fungi
The confrontation experiments of Hypocrea/Trichoderma strain ITV43 against M. roreri, P. megasperma and P. capsici strains were performed using the technique described by Szekeres et al. (2006).Petri dishes with PDA medium were seeded at equidistant points and were incubated at 25°C in the dark for 31 days.The antagonistic activity was recorded at 7 and 31 days of incubation.Digital images were taken with a camera Cyber-shot DSC-P72, using the same vertical distance from the camera to the Petri dish (18 cm).The percentage growth inhibition of the pathogenic fungi was calculated using the biocontrol index (BCI) according to the formula reported by Szekeres et al. (2006) Where A is the area of the colony of Hypocrea/Trichoderma and B is the total area occupied by colonies of Hypocrea/Trichoderma and each pathogen.The software ImageJ (on line: http://rsbweb.nih.gov/) was used to calculate the area of the fungi colonies.From the digital images taken with the digital camera, the area was measured and its outline was drawn using the free tool of the program ImageJ.During the analysis, the scale was set to 28.346 pixels per centimeter and the corresponding unit area was calculated in cm 2 .The experiments were made by triplicate and analysis of variance (ANOVA) was performed with the software Statistix9.Means were compared with Tukey test (P ≤ 0.05).Samples obtained from the interaction area between Hypocrea/TrichodermaITV-43 and the phytopathogenic fungi were observed with an optical and scanning electron microscope (JEOL, JSM-5600 LV).

Identification of fungal strain by ITS1-5.8s-ITS2 sequencing and phylogeny
Using ITS1 and ITS4 primers, the ribosomal DNA (rDNA) region containing the ITS1-5.8s-ITS2sequence was amplified from each fungal strain.The amplified region of H./T. viridesens ITV43 had a size of 679 bp and was deposited in the NCBI GenBank (JX144329).The ITS1-5.8s-ITS2 sequence amplified showed the highest similarity of 100 and 94% with DNA H. viridescens sequence (GenBank EF534007) and H. viridescens (GenBank EU280104, EU280137), respectively.The first sequence was obtained from a strain isolated in the United States of America by Liu et al. (2008) and the other two from strains isolated in Colombia and Peru by Hoyos-Carvajal et al. (2009).Phylogenetic analysis of H./T. viridescens ITV43 and 11 representative ITS1-5.8s-ITS2sequences of isolates of Trichoderma/Hypocrea generated a tree by the Neighbor Joining method and formed three majors groups (Figure 2).H./T. viridescens ITV43 grouped with H. viridescens (GU566274).This clade is affiliated with H. viridescens (EU280104 and EU280137) and close to H. viridescens (HQ833353).
According to Tukey´s tests, the highest BCI values were obtained against M. roreri HT-ITV20 (81.63),P. megasperma HT-ITV15 (69.95) and P. capsici ).After 7 days, a progressive inhibition zone produced by H./T. viridescens ITV43 against all phytopathogenic fungi strains was observed and at 31 days of incubation, 100%     P. megasperma by scanning electron and optical microscopy.The effect of H./T. viridescens ITV43 on morphological deformations and disorganization in the structure of the M. roreri cell walls was observed by scanning electron microscope (Figure 5a) and by optical microscopy for M. roreri (Figure 5b), P. capsici (Figure 5c) and P. megasperma (Figure 5d).

DISCUSSION
Fungi of the genus Trichoderma (Hypocrea) have been widely studied due to their antagonistic activity on phytopathogenic fungi in agriculture and are an alternative to synthetic chemical products (Mukherjee et al., 2013).Most Trichoderma species has been isolated from soil, however there are reports of Trichoderma isolates from other sources such as bark, decorticated wood, roots, decaying wood, mushroom compost, leaf litter, as an endophyte from trunks and aerial parts of plants (Harman et al., 2004).In the biological control of cocoa (T.cacao) pod diseases, mycoparasites isolated from cocoa fruits, aerial plant parts and rhizospheric soil from plot planted with cocoa trees have shown great promise in the control of M. roreri (Villamil et al., 2012), causal agent of black pod rot and Phytophthora spp.(Hanada et al., 2009), causal agent of black pod.Only a small number of the vast microbial diversity associated with cacao has been studied and endophytic and epiphytic microbes are candidates for biocontrol of cacao diseases.In this study, we have isolated a Hypocrea/Trichoderma strain from cacao pod with typical symptoms of frosty pod rot and our results show that the strain has strong antagonism against six Phytophthora and three M. roreri strains.Phytophthora and M. roreri strains were also isolated from cocoa pod with typical symptoms of frosty pod rot (Cuervo-Parra et al., 2011a) According to their morphological characteristics, the strain ITV43 was identified as H./T. viridescens that match the descriptions reported by Jaklitsch et al. (2006) and we suggest that this identification is reliable.H./T. viridescens belongs to Viride clade that includes Trichoderma viride, Trichoderma atroviride, Trichoderma koningiopsis and others (Samuels and Ismaiel, 2009).The characteristic of coconut aroma attributed to 6-PAP (6-pentyl-a-pyrone) was detected in H./T. viridescens ITV43 and the antifungal compound 6-PAP (Collins and Halim, 1972;Cutler et al., 1986) is a distinctive feature of many members of the Viride clade (Samuels et al., 2006).Analysis by BLAST of ITS1-5.8s-ITS2DNA sequence of H./T. viridescens ITV43 showed high degree similarity (99-100%) with H. viridescens strains (EU280104, EU280137) and phylogenetic tree (Figure 2) locate H./T. viridescens ITV43 close to H. viridescens (GU441579, GU566274) and T. asperellum (AY380912) strains and located away from T. theobromicola (DQ109525) and T. paucisporum (DQ109527, DQ109526) strains.Although ITS1-5.8s-ITS2DNA sequences are useful for identifying and studying the evolution of microorganisms, do not appear to be useful in identifying some Trichoderma/Hypocrea species (Jaklitsch et al., 2006).Druzhinina et al. (2005) developed the Trich OKEY program for the quick molecular identification of Hypocrea/Trichoderma at the genus and species levels based on an oligonucleotide barcode for the internal transcribed spacers 1 and 2 ((www.isth.info).To identify H./T. viridescens ITV43, we used the ITS1-5.8s-ITS2DNA sequence and the program was not able to identify the strain.Similar results were obtained by Torres-Palacios (2010) who used the program to identify 16 Trichoderma strains isolated near the Nevado de Toluca volcano, Mexico.In that study, 19% of the strains were identify entirely, 19% were identify as new species and 62% were ambiguous mainly viridescens-konongii.Other cases reported by Druzhinina et al. (2005) in which the software initiate ambiguities in Trichoderma/Hypocrea strains are: cerinum=tomentosum, crassum=longipile and orientalis=cerebriformis=longibrachiatum.
In conclusion, the strain ITV43 was identified as H./T.viridescens and was mycoparasitic on M. roreri, P. megasperma and P. capsici.

Figure 2 .Figure 3 .
Figure 2. Phylogenetic tree for ITS1-5.8s-ITS2region of rDNA, of H. viridescens ITV43 and the sequences from specimens related, obtained from GenBank (NCBI).The Kimura distance and the Neighbor-Joining method was used.The numbers adjacent to the branch points of ramification are bootstrap values of 1000 iterations.

Figure 4 .
Figure 4. Digital images taken at: (a) 7 and (b) 31 days of confrontation of H. viridescens ITV43 against T. cacao phytopathogenic fungi (M.roreri, P. capsici and P. megasperma) in PDA medium.Areas of H. viridescens ITV43 colonies (Area T, line blue dots) and the total area occupied by colonies of H. viridescens ITV43 and the phytopathogenic fungi (Area T + P, yellow line).

Figure 5 .
Figure 5. Images showing the effect of H. viridescens ITV43 on phytopathogenic fungi with the scanning electron microscope (a) and light microscope (b, c, d) in PDA medium: (a) H. viridescens (H.v) parasitizing mycelium of M. roreri (M.r) and inducing the lysis and deformation of the pathogen hyphae, (b) mycoparasitism by wrapping the hyphae of M. roreri by H. viridescens, (c) mycoparasitism of hyphae of P. capsici (P.c) by hyphae of H. viridescens, (d) parallel hyphal growth of H. viridescens and P. megasperma (P.m).

Table 1 .
Measurements for each structure of H./T. viridescens strain ITV43.
*Standard deviation; Different letter within the column indicate significant differences (P  0.05, ANOVA and Tukey´s tests).