ISSN e: 2007-4018 / ISSN print: 2007-4018

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Vol. XXII, issue 2 May - August 2016

ISSN: ppub: 2007-3828 epub: 2007-4018

Scientific article

Trichoderma species from the cacao agroecosystem with biocontrol potential of Moniliophthora roreri

http://dx.doi.org/10.5154/r.rchscfa.2015.08.036

Reyes-Figueroa1, Omar 1 ; Ortiz-García1, Carlos F. 1 ; Torres-de la Cruz2*, Magdiel 2 * ; Lagunes-Espinoza2, Luz del C. 2 ; Valdovinos-Ponce3, Guadalupe 3

  • 1Colegio de Postgraduados, Campus Tabasco. Carretera Cárdenas-Huimanguillo km 3.5. C. P. 86500. Cárdenas, Tabasco, México.
  • 2Universidad Juárez Autónoma de Tabasco. Carretera Villahermosa-Cárdenas km 0.5. C. P. 86039. Villahermosa, Tabasco, México.
  • 3Colegio de Postgraduados, Campus Montecillo. Carretera México-Texcoco km 36.5. C. P. 56230. Texcoco, Estado de México, México.

*Corresponding author. Email: biomag75@hotmail.comTel.: 52 + (914)1192428

Received: August 26, 2015; Accepted: January 28, 2016

This is an open-access article distributed under the terms of the Creative Commons Attribution License view the permissions of this license

Abstract:

Frosty pod rot in cacao (Moniliophthora roreri) is the main limitation on the production of cacao (Theobroma cacao) in Mexico. A sustainable alternative for the control of the disease is the use of the Trichoderma mushroom. The objective of this study was to select isolates that are native to Trichoderma with the best antagonist and physiological in vitro characteristics for the control of M. roreri. For this, 50 isolates of Trichoderma obtained in the cacao agroecosystem were characterized. Mycelial growth and the production of conidia at 25, 30 and 35 °C were considered the physiological variables. Mycoparasitism, antibiosis and potential antagonism were the antagonist variables. Significant differences (P = 0.0001) were found in all evaluated variables. The interval of the optimal temperature for mycelial growth and the production of conidia was 25 to 30 °C. Mycoparasitism varied between 0 and 100 %, and only the isolates of six species showed this characteristic. Antibiosis varied between 6.8 and 55.5 % and potential antagonism varied from 3.4 to 69.0 %. Trichoderma virens (TTC017) and T. harzianum (TTC090, TTC039, TTC073) showed the best potential in vitro biocontrol, so they are promising strains for future investigations on biological control of cacao moniliasis.

Keywords:Frosty pod rot in cacao; biological control; Theobroma cacao; Mycoparasitism.

Introduction

Moniliophthora roreri (Cif. & Par.) Evans et al. is the causal agent of frosty pod rot in cacao (Theobroma cacao L.) (Phillips-Mora & Wilkinson, 2007). In Mexico, M. roreri was reported in 2005, making it the main parasitic limitation on the production of cacao (Phillips-Mora et al., 2006). It has been reported that M. roreri may reduce up to 80 % of the production of cacao, causing negative socio-economic and environmental effects (Torres-de la Cruz, Ortiz-García, Téliz-Ortiz, Mora-Aguilera, & Nava- Díaz, 2011).

Cultural practices have been the most commonly used method for the fight against frosty pod rot in cacao (Soberanis et al., 1999). The use of fungicides has been a scarcely used practice, due to the erratic strategies of fungicide evaluations and to the fluctuating price of cacao (Bateman et al., 2005). Programs for the development of resistant genetic material have also been established (Phillips-Mora, Arciniegas-Leal, Mata-Quiros, & Motomajor-Arias, 2012); however, notable progress has not been obtained in the commercial use of clones with resistance to the disease. Krauss and Soberanis (2001) mention that biocontrol offers some potentials in the sustainable handling of the frosty pod rot through the use of antagonists. The authors reported that the species of the Trichoderma genus showed control on M. roreri.

In the search of biological control agents, one of the basic strategies should be the initial exploration of the natural native enemies (Vázquez, Matienzo, Veitía, & Alfonso, 2008). Based on this reasoning, in the state of Tabasco, Mexico, there are 50 isolates of Trichoderma obtained from the T. cacao rhizosphere which have been grouped into nine species and may be evaluated for the control of M. roreri. Due to this, the objective of this study was to select isolates native to Trichoderma with the best in vitro antagonist and physiological characteristics for the control of M. roreri.

Materials and methods

Trichoderma isolates

Fifty isolates of Trichoderma grouped in nine species were characterized in the present work (Table 1). These isolates were obtained from the T. cacao rhizosphere as a part of a diversity study of Trichoderma in the cacao agroecosystem in the state of Tabasco, Mexico, which were confirmed in kind through morphology and ITS sequences (Torres-de la Cruz et al., 2015). Currently, these isolates are a part of the Trichoderma collection of the División Académica de Ciencias Biológicas, Universidad Juárez Autónoma de Tabasco. The M. roreri isolate was provided by the Laboratorio de Fitopatología of the Colegio de Postgraduados Campus Tabasco, previously identified through morphology and ITS sequences (GenBank access number: GU108605). All isolates were preserved and multiplied in the middle of the Potato Dextrose Agar (PDA) culture.

Table 1. Isolates native to Trichoderma evaluated against Moniliophthora roreri, which causes frosty pod rot in cacao in the state of Tabasco, Mexico. Isolates: T. asperellum (Samuels, Lieckf & Nirenberg), T. pleuroticola (Yu & Park), T. spirale (Bissett), T. harzianum (Rifai), T. virens (Mill, Giddens & Foster) Arx., T. koningiopsis (Samuels, Suárez & Evans), T. longibrachiatum (Rifai), T. brevicompactum (Kraus, Kubicek & Gams) and T. reesei (Simmons).

Municipality / Municipio Locality / Localidad North Latitude / Latitud Norte West Longitude / Longitud Oeste IsolateZ / AislamientoZ Species / Especie Isolate number / Número de aislamiento
Paraíso Ranchería Moctezuma 1 18° 22’ 19.3” 93° 13’ 42.4” TTC001 T. asperellum 4
TTC002 T. pleuroticola
TTC003 T. spirale
TTC004 T. spirale
Ejido Occidente- San Francisco 18° 20’ 37.9” 93° 15’ 24.5” TTC007 T. pleuroticola 2
TTC008 T. pleuroticola
Cardenas Poblado C-28 18° 01’ 46.7” 93° 29’ 42.0” TTC009 T. harzianum 1
TTC014 T. virens
Ranchería Ocampo 18° 01’ 49.3” 93° 21’ 29.7” TTC015 T. virens 3
TTC016 T. virens
Ranchería Rio Seco 1 18° 03’ 47.2” 93° 23’ 04.4” TTC017 T. virens 1
Comalcalco Ranchería Arena 4 18° 13’ 25.4” 93° 23’ 31.1” TTC020 TTC021 T. koningiopsis
TTC023 T. virens
TTC024 T. asperellum 4
T. asperellum
Ranchería Gregorio 18° 14’ 59.9” 93° 17’ 12.7” TTC026 TTC027 TTC028 T. longibrachiatum
Méndez 1 T. longibrachiatum 3
T. longibrachiatum
Cunduacán Ejido José María 18° 08’ 30.1” 93° 17’ 29.1” TTC031 TTC032 T. virens
Pino Suárez T. longibrachiatum 2
Ranchería La Piedra 1 18° 07’ 45.2” 93° 11’ 52.4” TTC035 T. asperellum
TTC037 TTC039 T. harzianum 3
T. longibrachiatum
Huimanguillo Ranchería Villa Flores 2 17° 48’ 06.6” 93° 24’ 16.5” TTC045 TTC047 T. brevicompactum
T. brevicompactum 2
Ranchería Paredón 1 17° 44’ 59.6” 93° 23’ 57.8” TTC050 T. brevicompactum 2
TTC051 T. asperellum
Ejido Rafael Martínez 17° 42’ 56.8” 93° 23’ 45.7” TTC054 T. spirale 1
Jalpa de Méndez Ranchería Hermenegildo 18° 10’ 33.4” 93° 08’ 48.5” TTC056 T. harzianum
Galeana 2 TTC058 TTC059 T. virens 3
T. virens
Ejido Huapacal 18° 11’ 48.1” 93° 10’ 28.5” TTC062 T. harzianum 2
TTC063 T. koningiopsis
Centro Ranchería Plátano y cacao 4 17° 59’ 04.2” 93° 10’ 07.9” TTC077 T. harzianum
TTC081 T. reesei 3
TTC084 T. koningiopsis
Ranchería Buena vista 1 17° 56’ 59.2” 93° 06’ 23.2” TTC085 T. spirale
TTC086 T. brevicompactum 2
Ranchería González 4 17° 57’ 29.6” 93° 05’ 25.7” TTC090 T. harzianum
TTC088 T. longibrachiatum 2
Tacotalpa Ejido Zunuy y patastal 17° 28’ 10.3” 92° 50’ 56.7” TTC093 T. spirale 1
T. harzianum
Ranchería Puyacatengo 1 17° 39’ 37.2” 92° 54’ 20.7” TTC100 T. longibrachiatum 4
TTC101 T. virens
TTC102 TTC104 T. virens
Teapa Ejido Vicente Guerrero 17° 30’ 15.7” 92° 54’ 02.5” TTC115 T. harzianum 2
TTC113 T. reesei
Ranchería Allende 18° 09’ 38.1” 93° 02’ 00.8” TTC073 TTC076 T. harzianum
T. harzianum
Ejido Allende 2 17° 33’ 20.0” 92° 59’ 08.7” TTC109 T. harzianum 1
Total 50
z The isolates were identified with the letters TTC (Trichoderma Tabasco Cacao) followed by the number of isolates.

Mycoparasitism of Trichoderma on M. roreri

The mycoparasitic ability of the Trichoderma isolates on M. roreri was evaluated according to the pre-colonized Petri dish method (Evans, Holmes, & Thomas, 2003). A fragment of 5 mm in diameter was obtained from M. roreri colonies of 10 days of age and was placed near the edge of a Petri dish with 9 cm in diameter with half PDA. The Petri dishes with an inoculated medium were incubated during 37 days at 25 ± 1 °C in the dark. Subsequently, a 0.5 x 2.5 cm fragment of the Trichoderma inoculum, obtained from a 4 day old colony, was planted on the opposite side of the M. roreri inoculum. The dishes pre-colonized by M. roreri and inoculated with Trichoderma were incubated during 15 days under the same conditions indicated for pre-colonization. Five repetitions of the isolate and five of the control treatment were established. The control treatment comprised pre-colonized dishes of M. roreri without the inoculation of Trichoderma.

After the incubation, 10 samples of 0.5 mm in diameter were extracted initiating the inoculum of M. roreri in direction to the inoculum of Trichoderma. The samples were planted in Petri dishes with PDA mediums and were incubated at 25 ± 1 °C in the dark. The samples were observed during 7 days in order to detect the presence of the mycoparasite (Trichoderma) or phytopathogen (M. roreri) and evaluate the percentage of mycoparasitism with the following formula:

PP = (CT x 100) / N

Antibiosis of Trichoderma on M. roreri

The antibiosis of Trichoderma isolates on M. roreri was evaluated on cultures coupled according to Holmes, Schroers, Thomas, Evans, and Samuels (2004). Antibiosis was calculated by the percentage of radial growth reduction of M. roreri. For this, a fragment of 10 day old M. roreri of 5 mm in diameter was placed on a Petri dish with PDA medium. The inoculated dishes were incubated in the dark during 7 days at 25 ± 1 °C in order to establish the colony. Subsequently, the colony of M. roreri was controlled with the mycoparasite, for which a 5 mm fragment of 4 day old Trichoderma was planted on the opposite side of the M. roreri. All dishes were incubated at 25 ± 1 °C in the dark; five repetitions per isolate and control treatment were established. The control treatment comprised colonies of M. roreri without confrontation with Trichoderma. The radial growth was recorded daily until one of the isolates had mycelial contact with M. roreri. The percentage of mycelial growth inhibition was determined with the Abbott formula (1925):

PA = [(CR - CRT) / CR] x 100

Potential antagonism of Trichoderma on M. roreri

The potential antagonism of the Trichoderma isolates on M. roreri was obtained through the formula:

AP = (MP + PA) / 2

Mycelial growth of Trichoderma

Mycelial growth was evaluated according to Dimbi, Maniania, Lux, and Mueke (2004). A fragment of 5 mm in diameter was obtained from the margins of 4 days old Trichoderma colonies and was placed in the middle of a Petri dish with a PDA medium. The boxes were incubated at 25, 30 and 35 °C (the three temperatures with ± 1 °C) with a photoperiod of 12 h in the light and 12 h in the dark; five repetitions per isolate and per temperature were established. The rate of mycelial growth was recorded each 12 h; the test was finalized when one of the colonies filled the Petri dish. The measurements of the last record were used in the statistical analysis. Radial growth (r) was transformed into the growth area (A, cm2) with the formula A = πr2. The corresponding area to each mm of radial growth was expressed in percentages. Thus, the effect of the temperature on the mycelial growth was evaluated through the inhibition percentage and the increase of the area, when increasing the temperature from 25 to 30 °C and from 25 to 35 °C.

Production of Trichoderma conidia

From the 4 days old Trichoderma cultures, a fragment of 5 mm in diameter was planted in Petri dishes with PDA medium. The inoculated fragment was incubated at 25, 30 and 35 °C (the three temperatures with ± 1 °C), with a photoperiod of 12 h in the light and 12 h in the dark during 16 days. Five repetitions per isolate and per temperature were established. The conidia were cultivated from the culture surface and the count was done in a Neubauer chamber (Hausser Scientific, USA). The concentration of conidia·mL-1 was estimated through the following formula:

C = (Cc) (4 x 106) (Fd / 80)

Statistical analysis

The information on mycoparasitism, antibiosis, potential antagonism, mycelial growth and production of conidia were analyzed under a completely random design. Mycoparasitism, antibiosis and potential antagonism consisted of 50 treatments (each one of the evaluated isolates). Mycelial growth and the production of conidia comprised 40 treatments, as the isolates that did not show mycoparasitism and that had the lowest AP values were discarded. Prior to the analysis, the information on mycoparasitism, antibiosis and potential antagonism were transformed to the arc sine of the square root of the proportion, and the information on mycelial growth and production of conidia was transformed to log (x + 1). The information was subjected to an ANOVA and a separation of Tukey means test (P < 0.05) through SAS® (Statistical Analysis System [SAS], 1998).

Results and discussion

Mycoparasitism of Trichoderma on M. roreri

Table 2 presents the percentage of mycoparasitism of the 50 isolates native of Trichoderma evaluated on M. roreri. The isolates showed significant differences (P = 0.0001) and the percentage varied between 0 and 100 %. New isolates reached 100 % mycoparasitism at 15 days of incubation and 10 isolates did not show this characteristic. The isolates with mycoparasitism on M. roreri belong to the T. harzianum (Rifai), T. virens (Mill, Giddens & Foster) Arx, T. spirale (Bissett), T. brevicompactum (Kraus, Kubicek & Gams), T. koningiopsis (Samuels, Suárez & Evans) and T. asperellum (Samuels, Lieckf & Nirenberg) species. It could be hypothesized that these species produce a matrix of enzymes that allow parasitism of M. roreri. In this regard, mycoparasitism of T. harzianum, T virens, T. asperellum and T. koningiopsis isolates on M. roreri has been reported by Evans et al. (2003) and Krauss et al. (2006), while mycoparasitism of T. brevicompactum and T. spirale was documented for the first time. In this study, the parasitism of isolates of one same species varied, which is consistent with Hoyos-Carvajal, Duque, and Orduz (2008), who indicated that isolates of the same species can act in different ways.

Table 2. Mycoparasitism of 50 isolates native to Trichoderma on Moniliophthora roreri.

Isolate Mycoparasitism (%) Isolate Mycoparasitism (%)
TTC015 100 ± 0 a TTC050 54 ± 5.4 gh
TCC023 100 ± 0 a TTC037 54 ± 5.4 gh
TTC017 100 ± 0 a TTC016 54 ± 5.4 gh
TTC100 100 ± 0 a TTC045 52 ± 4.4 gh
TTC104 100 ± 0 a TTC084 42 ± 4.4 hi
TTC058 100 ± 0 a TTC032 42 ± 4.4 hi
TTC059 100 ± 0 a TTC031 34 ± 5.4 ij
TTC014 100 ± 0 a TTC020 30 ± 0 ijk
TTC062 100 ± 0 a TTC093 26 ± 5.4 jkl
TTC004 96 ± 5.4 ab TTC035 24 ± 5.4 jkl
TTC047 94 ± 5.4 b TTC051 22 ± 4.4 jkl
TTC090 92 ± 4.4 b TTC002 20 ± 0 klm
TTC039 84 ± 5.4 c TTC001 16 ± 5.4 lm
TTC085 78 ± 4.4 cd TTC063 16 ± 5.4 lm
TTC115 76 ± 5.4 cde TTC024 10 ± 0 m
TTC073 74 ± 5.4 cdef TTC003 0 n
TTC086 74 ± 5.4 cdef TTC007 0 n
TTC077 66 ± 5.4 defg TTC008 0 n
TTC056 64 ± 5.4 efg TTC026 0 n
TTC054 64 ± 5.4 efg TTC027 0 n
TTC009 62 ± 4.4 fg TTC028 0 n
TTC076 56 ± 5.4 gh TTC088 0 n
TTC102 56 ± 5.4 gh TTC101 0 n
TTC109 56 ± 5.4 gh TTC081 0 n
TTC021 54 ± 5.4 gh TTC113 0 n
± Standard deviation of the mean. Identical letters do not show a significant difference according to the Tukey test (P = 0.0001).

The isolates that did not show parasitism on M. roreri correspond to T. pleuroticola (Yu & Park), T. longibrachiatum (Rifai) and T. reesei (Simmons). The foregoing coincides with Garcia-Simoes, Tauk-Tornisielo, Rocha-Niella, and Tapia-Tapia (2012), who evaluated this species without finding parasitism on M. perniciosa (Stahel) Aime and Phillips-Mora, a species that is related to M. roreri. Trichoderma pleuroticola has also been reported as a pathogen of edible mushrooms (Sobieralski et al., 2012). The absence of mycoparasitism of these species suggests that M. roreri is not in the group of hosts.

Trichoderma antibiosis on M. roreri

Table 3 presents the results of the antibiosis test of Trichoderma on M. roreri. All isolates showed antibiosis with significant differences between them (P = 0.0001). The percentage of antibiosis fluctuated from 6.8 to 55.5 %; the highest values (40 to 55.5%) were obtained with some isolates of T. asperellum (TCC051 and TCC024) T. koningiopsis (TCC084, TCC020 and TCC063), T. pleuroticola (TCC002), T. virens (TCC021) and T. harzianum (TCC056, TCC077 and TCC115). The antibiotic action of the T. harzianum isolates has also been described on the mycelial growth of M. roreri and M. perniciosa (Bailey et al., 2008).

Table 3. Antibiosis of 50 native species to Trichoderma on Moniliophthora roreri.

Isolate Antibiosis (%) / Isolate Antibiosis (%) /
TTC051 55.5 ± 4.0 a TTC016 30.9 ± 3.0 ghijklmno
TTC084 52.8 ± 2.7 ab TTC026 29.7 ± 4.0 hijklmnop
TTC020 48.9 ± 5.0 abc TTC058 28.5 ± 3.7 hijklmnopq
TTC002 46.0 ± 3.7 abcd TTC076 28.5 ± 4.8 Ijklmnopq
TTC021 44.3 ± 3.8 bcde TTC062 27.8 ± 1.5 Ijklmnopq
TTC024 43.2 ± 4.9 bcdef TTC109 27.3 ± 7.2 jklmnopq
TTC056 41.2 ± 2.7 cdefg TTC003 26.6 ± 3.2 klmnopq
TTC077 41.0 ± 4.0 cdefg TTC113 25.8 ± 4.2mnopq
TTC115 40.5 ± 0.6 cdefg TTC037 25.1 ± 4.7 mnopqr
TTC063 40.2 ± 5.6 cdefg TTC054 24.7 ± 3.8 nopqrs
TTC027 38.7 ± 1.2 cdefgh TTC015 23.0 ± 1.5 opqrst
TTC017 37.9 ± 3.7 defghi TTC045 21.1 ± 2.0 pqrstu
TTC102 37.5 ± 6.7 defghi TTC081 20.3 ± 2.6 pqrstuv
TTC085 37.1 ± 2.3 defghij TTC047 19.9 ± 2.6 qrstuvw
TTC039 36.1 ± 4.0 defghijk TTC104 19.8 ± 6.3 qrstuvw
TTC035 35.6 ± 5.0 defghijkl TTC014 16.8 ± 3.7 rstuvw
TTC001 35.3 ± 3.3 efghijkl TTC031 16.2 ± 2.6 stuvw
TTC004 34.9 ± 1.4 efghijklm TTC086 16.0 ± 1.4 stuvw
TTC088 34.7± 3.9 efghijklm TTC093 15.9 ± 4.0 tuvw
TTC073 33.9 ± 3.5 efghijklmn TTC023 15.6 ± 2.9 tuvw
TTC007 33.4 ± 2.9 fghijklmn TTC059 14.8 ± 3.9 tuvw
TTC028 32.4 ± 2.1 ghijklmno TTC101 14.5 ± 3.4 uvw
TTC090 32.4 ± 3.7 ghijklmno TTC008 13.1 ± 4.1 vwx
TTC050 31.7 ± 2.1 TTC032 12.3 ± 1.5 wx
TTC009 31.5 ± 2.6 TTC100 6.8 ± 2.5 x
± Standard deviation of the mean. Identical letters do not show a significant difference according to the Tukey test (P = 0.0001).

The isolates of T. longibrachiatum, T. reesei and T. pleuroticola showed antibiosis on M. roreri (Table 3), even though they did not present mycoparasitism (Table 2). The presence of antibiosis without mycoparasitism suggest that the isolates have metabolites with antifungal activity, or that they do not allow the growth of the pathogen, due to the space and nutrient competition mechanisms (García-Simoes et al., 2012).

Potential antagonism of Trichoderma on M. roreri

Table 4 shows that there were significant differences (P = 0.0001) in the potential antagonism of Trichoderma on M. roreri; the percentage varied between 6.8 and 68.8 %. To evaluate the antagonism potential, such as the sum of mycoparasitism and antibiosis acting synergistically, it was observed that some isolates of T. virens (TTC017, TTC058 and TTC015), T. harzianum (TTC062 and TTC090) and T. spirale (TTC004) showed the highest values, while the lowest percentages were obtained with the isolates that did not show mycoparasitism. According to Monte (2001), the combination of parasitism and antibiosis could result in significant antagonistic levels.

Table 4. Potential antagonism of 50 isolates native to Trichoderma on Moniliophthora roreri.

Isolate Antagonism (%) / Isolate Antagonism (%) /
TTC017 68.8 ± 1.79 a TTC016 42.2 ± 3.03 hijk
TTC004 65.2 ± 2.59 ab TTC076 42.2 ± 3.56 hijk
TTC058 64.2 ± 1.92 abc TTC109 41.6 ± 6.31 hijk
TTC062 64.0 ± 0.71 abc TTC020 39.6 ± 2.19 ijkl
TTC090 62.2 ± 1.79 abc TTC037 39.4 ± 3.21 ijkl
TTC015 61.6 ± 0.89 abcd TTC051 38.6 ± 3.21 jkl
TTC104 60.0 ± 2.74 bcde TTC045 36.6 ± 2.19 klm
TTC039 59.8 ± 3.90 bcde TTC002 33.0 ± 1.87 lmn
TTC014 58.8 ± 1.79 bcde TTC035 29.8 ± 3.96 mno
TTC115 58.2 ± 2.95 bcde TTC063 28.0 ± 5.05 no
TTC085 57.6 ± 2.79 cde TTC032 27.0 ± 1.73 nop
TTC059 57.6 ± 1.82 cde TTC024 26.6 ± 2.50 nop
TCC023 57.6 ± 1.52 cde TTC001 25.6 ± 2.30 opq
TTC047 57.0 ± 3.16 cde TTC031 25.0 ± 3.80 opq
TTC073 54.0 ± 2.35 def TTC093 20.8 ± 3.96 pqr
TTC077 53.4 ± 3.78 ef TTC027 19.6 ± 0.55 qr
TTC100 53.2 ± 1.30 ef TTC088 17.2 ± 1.92 rs
TTC056 52.4 ± 2.19 efg TTC007 16.8 ± 1.48 rs
TTC021 49.2 ± 4.32 fgh TTC028 16.2 ± 1.00 rs
TTC084 47.4 ± 1.40 fgh TTC026 15.0 ± 2.00 rst
TTC009 46.8 ± 2.17 fghi TTC003 13.2 ± 1.64 st
TTC102 46.8 ± 5.17 fghi TTC113 13.0 ± 2.12 st
TTC086 45.0 ± 3.67 ghij TTC081 10.0 ± 1.22 tu
TTC054 44.4 ± 3.21 hij TTC101 7.2 ± 1.48 u
TTC050 42.8 ± 2.17 hijk TTC008 6.8 ± 1.79 u
± Standard deviation of the mean. Identical letters do not show a significant difference according to the Tukey test (P = 0.0001).

Mycelial growth of Trichoderma

The statistical analysis showed significant differences (P = 0.0001) in the mycelial growth of the Trichoderma isolates in each temperature that was assessed. The results are presented in Table 5. At 25 °C, mycelial growth varied between 21 and 40 mm. The TTC063 and TTC084 strains of T. koningiopsis, and TTC021 and TCC017 of T. virens obtained the highest growth at 60 h. At 30 °C, the mycelial growth fluctuated between 18.3 and 40 mm; the TCC021, TCC017, TCC102 and TCC104 strains of T. virens, TCC084 of T. koningiopsis, and TCC100, TCC073 and TCC090 of T. harzianum expressed the highest growth. When going from 25 to 30 °C, 30 out of 40 isolates increased their growth and three maintained it (TTC021, TTC084 and TTC017). Figure 1 shows the graphic comparison of the changes that happened on the mycelial growth of the Trichoderma isolates with temperature changes. At 35 °C, mycelial growth fluctuated from 2.1 to 29 mm and the TCC017 strain (T. virens) presented the highest growth. At this temperature, all the isolates decreased the mycelial growth with respect to the one obtained at 25 and 30 °C; the inhibition fluctuated from 23 to 99.5 % (Figure 1).

Table 5. Mycelial growth of 40 isolates native to Trichoderma at 25, 30 and 35 °C.

Isolate Mycelial growth (%)
25 °C 30 °C 35 °C
TTC063 40.0 ± 0 a 26.1 ± 0.3 efgh 10.1 ± 0.2 hi
TTC021 40.0 ± 0 a 40.0 ± 0 a 7.9 ± 0.1 jk
TTC084 40.0 ± 0 a 40.0 ± 0 a 19.0 ± 0.7 cd
TTC017 40.0 ± 0 a 40.0 ± 0 a 29.0 ± 1.0 a
TTC100 33.3 ± 1.0 b 40.0 ± 0 a 21.9 ± 0.1 bc
TTC090 32.7 ± 1.0 b 34.0 ± 1.2 b 14.7 ± 0.3 ef
TTC102 31.9 ± 1.4 bc 40.0 ± 0 a 21.0 ± 0.7 bc
TTC104 31.8 ± 1.1 bc 40.0 ± 0 a 23.3 ± 0.1 b
TTC056 31.6 ± 2.2 bcd 32 ± 1.7 bc 9.6 ± 0.1 ij
TTC073 30.7 ± 2.8 bcde 40.0 ± 0 a 23.9 ± 0.5 ab
TTC001 30.5 ± 0.5 bcdef 32.2 ± 0.9 bc 19.9 ± 0.8 bcd
TTC076 30.0 ± 0.1 bcdeg 30.8 ± 2.0 bcd 10.4 ± 0.3 hi
TTC020 29.7 ± 0.5 bcdefgh 32.2 ± 1.4 bc 20.9 ± 0.3 bc
TTC085 29.4 ± 0.6 bcdefghi 27.3 ± 0.6 defg 10.0 ± 0.02 hi
TTC002 29.2 ± 1.8 bcdefghij 30.4 ± 0.5 bcde 20.2 ± 0.5 bcd
TTC051 28.8 ± 1.1 bcdefghij 34.2 ± 0.2 b 21.5 ± 0.9 bc
TTC035 28.6 ± 0.9 bcdefghij 31.6 ± 1.2 bcd 21.0 ± 1.0 bc
TTC004 27.6 ± 3.2 cdefghijk 18.3 ± 1.3 k 2.1 ± 1.0 n
TCC023 27.6 ± 2.0 cdefghijk 29.5 ± 0.6 bcdef 14.0 ± 1.1 efg
TTC024 26.9 ± 1.5 defghijkl 32.5 ± 1.2 bc 19.8 ± 0.9 bcd
TTC039 26.9 ± 0.2 efghijkl 33.6 ± 1.7 b 12.3 ± 0.2 fgh
TTC093 26.0 ± 0.6 fghijkl 18.5 ± 0.7 k 3.2 ± 0.2 m
TTC090 25.6 ± 1.0 ghijklm 40.0 ± 0 a 18.7 ± 1.5 cd
TTC062 25.6 ± 0.7 ghijklm 28.1 ± 0.3 cdefg 11.6 ± 0.4 ghi
TTC016 25.5 ± 1.1 hijklmn 29.7 ± 0.6 bcdef 18.7 ± 0.2 cd
TTC115 25.1 ± 0.5 ijklmn 28.0 ± 1.2 cdefg 7.7 ± 0.2 k
TTC059 24.5 ± 0.6 jklmno 24.9 ± 1.1 ghi 11.6 ± 1.1 ghi
TTC050 24.3 ± 2.2 klmno 22.3 ± 0.4 hij 9.7 ± 0.5 ij
TTC032 24.0 ± 1.3 klmnop 30.3 ± 0.7 bcdef 16.8 ± 1.0 de
TTC077 23.6 ± 0.6 klmnop 30.8 ± 1.5 bcd 19.1 ± 0.5 cd
TTC037 23.2 ± 1.2 lmnop 30.6 ± 0.8 bcd 16.6 ± 0.5 de
TTC047 22.2 ± 2.8 mnop 20.0 ± 2.1 jk 10.6 ± 0.2 hi
TTC109 22.1 ± 0.8 mnop 28.3 ± 1.0 cdefg 7.4 ± 0.3 k
TTC058 22.0 ± 0.7 mnop 24.6 ± 0.1 ghi 12.3 ± 0.1 fgh
TTC054 22.0 ± 1.1 mnop 21.1 ± 3.6 jk 5.5 ± 0.08 l
TTC031 21.8 ± 0.5 nop 25.9 ± 0.1 fghi 11.7 ± 0.9 ghi
TTC086 21.1 ± 1.4 opq 22.3 ± 3.2 ij 10.5 ± 0.7 hi
TTC014 20.9 ± 0.3 opq 23.1 ± 1.0 hij 11.7 ± 0.4 ghi
TTC015 20.6 ± 2.0 pq 22.3 ± 1.3 hij 11.6 ± 0.1 ghi
TTC045 18.6 ± 2.6 q 25.3 ± 3.5 ghi 6.7 ± 0.1 kl
± Standard deviation of the mean. Identical letters do not show a significant difference according to the Tukey test (P = 0.0001).

Figure 1. Inhibition changes or increase of mycelial growth of native isolates to Trichoderma when surpassing 25 to 30 °C and 25 to 35 °C.

All isolates grew in the three increased temperatures; however, the range of favorable temperatures for 82 % of the isolates was 25 to 30 °C. In this regard, Jalil, Norero, and Apablaza (1997) reported that the optimal temperature for T. harzianum is 27 °C. The notable growth of all strains in an interval between 25 and 35 °C suggests the adaptability of the high temperatures where cacao is grown.

Production of Trichoderma conidia

The statistical analysis showed significant differences (P = 0.0001) in the production of conidia of the Trichoderma isolates in each evaluated temperature (Table 6). At 25 °C, the production fluctuated from 1.4 x 107 to 3.1 x 109 conidia·mL-1. The strains with the highest production were TTC063 and TTC051 of the T. koningiopsis and T. asperellum species, respectively. At 30 °C, the production of conidia varied between 0.05 x 108 and 2.6 x 109 conidia·mL-1; the TTC102 and TTC017 strains of T. virens, and TTC115 of T. harzianum presented the highest production. At this temperature, 65 % of the isolates increased the production of conidia in relation to the production obtained at 25 °C (Figure 2). At 35 °C, the production of conidia varied between 0 and 3.9 x 108 conidia·mL-1. The TTC084 isolates (T. koningiopsis), TTC104 and TTC016 (T. virens) showed the highest production. At this temperature, 97.5 % of the isolates decreased the production of conidia, with respect to the obtained conidia at 25 and 30 °C; six isolates showed total inhibition (Figure 2). The inhibition fluctuated from 12.3 to 100 %. Only the TTC104 (T. virens) isolate increased the production of conidia, in regards to the values obtained at 25 and 30 °C.

Table 6. Conidia production of 40 isolates native to Trichoderma at 25, 30 and 35 °C.

Isolate Production (conidia·mL-1)
25 °C 30 °C 35 °C
TTC063 3.1 x 109 ± 1.6 x 108 a 1.3 x 109 ± 3.3 x 108 efgh 1.2 x 106 ± 2.1 x 105 lm
TTC051 2.1 x 109 ± 2.4 x 108 b 1.1 x 109 ± 1.6 x 108 fghij 2.7 x 106 ± 1.0 x 105 I
TTC001 1.9 x 109 ± 3.5 x 107 bc 1.8 x 109 ± 5.4 x 107 bc 4.9 x 106 ± 1.5 x 105 h
TTC035 1.8 x 109 ± 1.1 x 108 bc 1.2 x 109 ± 1.0 x 108 efghi 1.3 x 107 ± 2.7 x 106 g
TTC102 1.8 x 109 ± 1.4 x 108 bc 2.6 x 109 ± 2.4 x 108 a 4.2 x 107 ± 3.5 x 106 e
TTC084 1.6 x 109 ± 1.2 x 108 bcd 8.9 x 108 ± 3.9 x 106 ijklm 3.9 x 108 ± 2.6 x 107 a
TTC002 1.6 x 109 ± 2.1 x 108 cd 1.3 x 109 ± 2.2 x 108 efgh 2.1 x 106 ± 5.1 x 105 Ij
TTC020 1.4 x 109 ± 3.6 x 107 de 1.2 x 109 ± 5.2 x 107 efghij 1.7 x 106 ± 1.0 x 105 jk
TTC015 1.3 x 109 ± 2.1 x 108 def 1.2 x 109 ± 1.1 x 108 efghi 2.5 x 105 ± 0 pq
TTC073 1.3 x 109 ± 2.3 x 108 defg 1.1 x 109 ± 1.3 x 108 fghij 6.4 x 106 ± 3.5 x 105 h
TTC024 1.3 x 109 ± 2.8 x 107 defg 1.7 x 109 ± 7.4 x 107 bcd 1.3 x 106 ± 3.0 x 105 kl
TTC037 1.2 x 109 ± 1.3 x 108 efg 1.5 x 109 ± 1.4 x 108 cdef 8.6 x 107 ± 1.7 x 106 cd
TTC045 1.1 x 109 ± 1.2 x 108 efgh 2.8 x 108 ± 2.8 x 107 opqr 6.6 x 107 ± 1.0 x 106 d
TTC021 1.0 x 109 ± 8.7 x 107 fghi 1.7 x 108 ± 1.0 x 107 qr 1.3 x 106 ± 1.7 x 105 klm
TTC016 1.0 x 109 ± 6.8 x 107 fghij 1.4 x 109 ± 1.4 x 108 defg 1.6 x 108 ± 1.5 x 107 b
TTC090 1.0 x 109 ± 8.9 x 107 fghij 1.5 x 109 ± 1.9 x 108 bcde 1.4 x 106 ± 1.9 x 105 kl
TTC115 9.9 x 108 ± 4.1 x 107 ghijk 2.3 x 109 ± 6.8 x 107 a 1.0 x 106 ± 0 m
TTC032 8.4 x 108 ± 3.7 x 107 hijkl 9.7 x 108 ± 8.2 x 107 hijkl 7.4 x 107 ± 4.4 x 106 d
TTC017 8.2 x 108 ± 2.7 x 107 ijklm 1.9 x 109 ± 2.1 x 108 b 2.6 x 107 ± 3.0 x 106 f
TTC109 7.9 x 108 ± 2.3 x 107 jklmn 1.4 x 109 ± 2.7 x 107 defg 1.0 x 108 ± 2.7 x 106 c
TTC058 7.6 x 108 ± 4.4 x 107 klmn 1.0 x 109 ± 1.5 x 108 ghijk 2.8 x 105 ± 3.7 x 104 nop
TCC023 7.2 x 108 ± 7.8 x 106 lmno 1.3 x 109 ± 8.5 x 107 efgh 3.5 x 105 ± 1.7 x 104 n
TTC085 7.1 x 108 ± 1.6 x 107 lmno 8.1 x 108 ± 1.0 x 108 jklm 0 ± 0 r
TTC077 6.6 x 108 ± 1.5 x 107 lmno 1.7 x 109 ± 3.4 x 108 bcd 4.1 x 107 ± 2.7 x 106 e
TTC062 6.3 x 108 ± 3.6 x 107 mno 9.0 x 108 ± 1.0 x 108 ijklm 2.6 x 105 ± 2.3 x 104 nopq
TTC039 6.3 x 108 ± 2.1 x 107 mno 1.5 x 109 ± 8.8 x 107 cdef 1.4 x 107 ± 5.1 x 105 g
TTC031 6.3 x 108 ± 2.6 x 107 mno 7.1 x 108 ± 4.1 x 107 klmn 2.2 x 106 ± 2.1 x 105 Ij
TTC014 6.3 x 108 ± 1.2 x 107 no 1.4 x 109 ± 1.1 x 108 defg 2.5 x 105 ± 1.1 x 104 opq
TTC047 6.2 x 108 ± 2.3 x 107 no 9.0 x 108 ± 5.0 x 107 ijklm 0 r
TTC086 5.8 x 108 ± 2.6 x 107 op 8.2 x 108 ± 8.0 x 107 jklm 2.5 x 105 ± 0 pq
TTC009 4.7 x 108 ± 3.1 x 107 pq 8.0 x 108 ± 8.6 x 107 jklm 1.3 x 107 ± 3.8 x 106 g
TTC100 4.7 x 108 ± 4.1 x 107 pq 1.5 x 109 ± 2.6 x 108 cdef 1.3 x 107 ± 3.4 x 106 g
TTC004 4.3 x 108 ± 1.9 x 106 qr 5.6 x 108 ± 2.7 x 107 mnop 0 r
TTC059 4.3 x 108 ± 2.7 x 107 qr 4.1 x 108 ± 5.6 x 107 nopq 3.4 x 105 ± 2.5 x 104 no
TTC050 3.6 x 10 8 ± 5.0 x 107 r 6.0 x 108 ± 1.1 x 108 lmno 0 r
TTC054 3.4 x 108 ± 2.4 x 107 r 3.8 x 108 ± 2.3 x 107 nopq 0 r
TTC076 3.3 x 108 ± 1.4 x 107 r 2.0 x 108 ± 2.6 x 107 pqr 2.7 x 105 ± 2.9 x 104 nopq
TTC104 1.8 x 107 ± 3.7 x 106 s 0.05 x 108 ±4.8 x 103 r 1.9 x 108 ± 1.7 x 107 b
TTC056 1.5 x 107 ± 3.5 x 106 s 1.1 x 108 ± 2.2 x 107 qr 2.0 x 105 ± 0 q
TTC093 1.4 x 107 ± 2.8 x 106 s 1.1 x 108 ± 1.4 x 107 qr 0 r
± Standard deviation of the mean. Identical letters do not show a significant difference according to the Tukey test (P = 0.0001).

Figure 2. Inhibition changes or increase in the production of isolate conidia native to Trichoderma when surpassing 25 to 30 °C and 25 to 35 °C.

Given that the spores are the active structures of the mushrooms that are the agents for biocontrol, the production of conidia is an important characteristic for the selection of promissory isolates (Vélez-Arango, Estrada-Valencia, González-García, Valderrama- Fonseca, & Bustillo-Pardey, 2001). In this regard, 85 % of the isolates produced conidia in the interval from 25 to 35 °C (Table 6), which could be explained by the tropical origin of the isolates; however, the range of the favorable temperature for production was 25 to 30 °C. The isolates with the most production of conidia in the range of evaluated temperatures were: TTC063 (T. koningiopsis); TTC051, TTC001, TTC035 (T. asperellum); TTC102 (T. virens); and TTC115 (T. harzianum), with a superior production of 1 x 109 conidia·mL-1.

Conclusions

The results show that in the cacao agroecosystem, six out of nine existing species of Trichoderma present antagonistic capacities on M. roreri. According to the data presented in this work, the Trichoderma spp. isolates showed an intraspecific variability in regards to parasitism, antibiosis, mycelial growth and sporulation. The rank of favorable temperature for the native isolates of Trichoderma was 25 to 30 °C. Based on the evaluated characteristics, the promissory isolates for the biological control of M. roreri are: TTC017 of T. virens, and TTC090, TTC039 and TTC073 of T. harzianum. The variability of the isolates in the assessed characteristics demonstrates the importance of in vitro characterization and manifests the potential of the native species of Trichoderma for the development of bio fungicides on M. roreri. Future studies will have to be implemented for the evaluation of the isolates selected under field conditions.

Acknowledgements

  • This work was financed by the Consejo Nacional de Ciencia y Tecnología (CONACYT) and the Programa de Mejoramiento del Profesorado (PROMEP) of the Secretaría de Educación Pública.

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Figures:

Figure 1. Inhibition changes or increase of mycelial growth of native isolates to Trichoderma when surpassing 25 to 30 °C and 25 to 35 °C.
Figure 2. Inhibition changes or increase in the production of isolate conidia native to Trichoderma when surpassing 25 to 30 °C and 25 to 35 °C.

Tables:

Table 1. Isolates native to Trichoderma evaluated against Moniliophthora roreri, which causes frosty pod rot in cacao in the state of Tabasco, Mexico. Isolates: T. asperellum (Samuels, Lieckf & Nirenberg), T. pleuroticola (Yu & Park), T. spirale (Bissett), T. harzianum (Rifai), T. virens (Mill, Giddens & Foster) Arx., T. koningiopsis (Samuels, Suárez & Evans), T. longibrachiatum (Rifai), T. brevicompactum (Kraus, Kubicek & Gams) and T. reesei (Simmons).
Municipality / Municipio Locality / Localidad North Latitude / Latitud Norte West Longitude / Longitud Oeste IsolateZ / AislamientoZ Species / Especie Isolate number / Número de aislamiento
Paraíso Ranchería Moctezuma 1 18° 22’ 19.3” 93° 13’ 42.4” TTC001 T. asperellum 4
TTC002 T. pleuroticola
TTC003 T. spirale
TTC004 T. spirale
Ejido Occidente- San Francisco 18° 20’ 37.9” 93° 15’ 24.5” TTC007 T. pleuroticola 2
TTC008 T. pleuroticola
Cardenas Poblado C-28 18° 01’ 46.7” 93° 29’ 42.0” TTC009 T. harzianum 1
TTC014 T. virens
Ranchería Ocampo 18° 01’ 49.3” 93° 21’ 29.7” TTC015 T. virens 3
TTC016 T. virens
Ranchería Rio Seco 1 18° 03’ 47.2” 93° 23’ 04.4” TTC017 T. virens 1
Comalcalco Ranchería Arena 4 18° 13’ 25.4” 93° 23’ 31.1” TTC020 TTC021 T. koningiopsis
TTC023 T. virens
TTC024 T. asperellum 4
T. asperellum
Ranchería Gregorio 18° 14’ 59.9” 93° 17’ 12.7” TTC026 TTC027 TTC028 T. longibrachiatum
Méndez 1 T. longibrachiatum 3
T. longibrachiatum
Cunduacán Ejido José María 18° 08’ 30.1” 93° 17’ 29.1” TTC031 TTC032 T. virens
Pino Suárez T. longibrachiatum 2
Ranchería La Piedra 1 18° 07’ 45.2” 93° 11’ 52.4” TTC035 T. asperellum
TTC037 TTC039 T. harzianum 3
T. longibrachiatum
Huimanguillo Ranchería Villa Flores 2 17° 48’ 06.6” 93° 24’ 16.5” TTC045 TTC047 T. brevicompactum
T. brevicompactum 2
Ranchería Paredón 1 17° 44’ 59.6” 93° 23’ 57.8” TTC050 T. brevicompactum 2
TTC051 T. asperellum
Ejido Rafael Martínez 17° 42’ 56.8” 93° 23’ 45.7” TTC054 T. spirale 1
Jalpa de Méndez Ranchería Hermenegildo 18° 10’ 33.4” 93° 08’ 48.5” TTC056 T. harzianum
Galeana 2 TTC058 TTC059 T. virens 3
T. virens
Ejido Huapacal 18° 11’ 48.1” 93° 10’ 28.5” TTC062 T. harzianum 2
TTC063 T. koningiopsis
Centro Ranchería Plátano y cacao 4 17° 59’ 04.2” 93° 10’ 07.9” TTC077 T. harzianum
TTC081 T. reesei 3
TTC084 T. koningiopsis
Ranchería Buena vista 1 17° 56’ 59.2” 93° 06’ 23.2” TTC085 T. spirale
TTC086 T. brevicompactum 2
Ranchería González 4 17° 57’ 29.6” 93° 05’ 25.7” TTC090 T. harzianum
TTC088 T. longibrachiatum 2
Tacotalpa Ejido Zunuy y patastal 17° 28’ 10.3” 92° 50’ 56.7” TTC093 T. spirale 1
T. harzianum
Ranchería Puyacatengo 1 17° 39’ 37.2” 92° 54’ 20.7” TTC100 T. longibrachiatum 4
TTC101 T. virens
TTC102 TTC104 T. virens
Teapa Ejido Vicente Guerrero 17° 30’ 15.7” 92° 54’ 02.5” TTC115 T. harzianum 2
TTC113 T. reesei
Ranchería Allende 18° 09’ 38.1” 93° 02’ 00.8” TTC073 TTC076 T. harzianum
T. harzianum
Ejido Allende 2 17° 33’ 20.0” 92° 59’ 08.7” TTC109 T. harzianum 1
Total 50
z The isolates were identified with the letters TTC (Trichoderma Tabasco Cacao) followed by the number of isolates.
Table 2. Mycoparasitism of 50 isolates native to Trichoderma on Moniliophthora roreri.
Isolate Mycoparasitism (%) Isolate Mycoparasitism (%)
TTC015 100 ± 0 a TTC050 54 ± 5.4 gh
TCC023 100 ± 0 a TTC037 54 ± 5.4 gh
TTC017 100 ± 0 a TTC016 54 ± 5.4 gh
TTC100 100 ± 0 a TTC045 52 ± 4.4 gh
TTC104 100 ± 0 a TTC084 42 ± 4.4 hi
TTC058 100 ± 0 a TTC032 42 ± 4.4 hi
TTC059 100 ± 0 a TTC031 34 ± 5.4 ij
TTC014 100 ± 0 a TTC020 30 ± 0 ijk
TTC062 100 ± 0 a TTC093 26 ± 5.4 jkl
TTC004 96 ± 5.4 ab TTC035 24 ± 5.4 jkl
TTC047 94 ± 5.4 b TTC051 22 ± 4.4 jkl
TTC090 92 ± 4.4 b TTC002 20 ± 0 klm
TTC039 84 ± 5.4 c TTC001 16 ± 5.4 lm
TTC085 78 ± 4.4 cd TTC063 16 ± 5.4 lm
TTC115 76 ± 5.4 cde TTC024 10 ± 0 m
TTC073 74 ± 5.4 cdef TTC003 0 n
TTC086 74 ± 5.4 cdef TTC007 0 n
TTC077 66 ± 5.4 defg TTC008 0 n
TTC056 64 ± 5.4 efg TTC026 0 n
TTC054 64 ± 5.4 efg TTC027 0 n
TTC009 62 ± 4.4 fg TTC028 0 n
TTC076 56 ± 5.4 gh TTC088 0 n
TTC102 56 ± 5.4 gh TTC101 0 n
TTC109 56 ± 5.4 gh TTC081 0 n
TTC021 54 ± 5.4 gh TTC113 0 n
± Standard deviation of the mean. Identical letters do not show a significant difference according to the Tukey test (P = 0.0001).
Table 3. Antibiosis of 50 native species to Trichoderma on Moniliophthora roreri.
Isolate Antibiosis (%) / Isolate Antibiosis (%) /
TTC051 55.5 ± 4.0 a TTC016 30.9 ± 3.0 ghijklmno
TTC084 52.8 ± 2.7 ab TTC026 29.7 ± 4.0 hijklmnop
TTC020 48.9 ± 5.0 abc TTC058 28.5 ± 3.7 hijklmnopq
TTC002 46.0 ± 3.7 abcd TTC076 28.5 ± 4.8 Ijklmnopq
TTC021 44.3 ± 3.8 bcde TTC062 27.8 ± 1.5 Ijklmnopq
TTC024 43.2 ± 4.9 bcdef TTC109 27.3 ± 7.2 jklmnopq
TTC056 41.2 ± 2.7 cdefg TTC003 26.6 ± 3.2 klmnopq
TTC077 41.0 ± 4.0 cdefg TTC113 25.8 ± 4.2mnopq
TTC115 40.5 ± 0.6 cdefg TTC037 25.1 ± 4.7 mnopqr
TTC063 40.2 ± 5.6 cdefg TTC054 24.7 ± 3.8 nopqrs
TTC027 38.7 ± 1.2 cdefgh TTC015 23.0 ± 1.5 opqrst
TTC017 37.9 ± 3.7 defghi TTC045 21.1 ± 2.0 pqrstu
TTC102 37.5 ± 6.7 defghi TTC081 20.3 ± 2.6 pqrstuv
TTC085 37.1 ± 2.3 defghij TTC047 19.9 ± 2.6 qrstuvw
TTC039 36.1 ± 4.0 defghijk TTC104 19.8 ± 6.3 qrstuvw
TTC035 35.6 ± 5.0 defghijkl TTC014 16.8 ± 3.7 rstuvw
TTC001 35.3 ± 3.3 efghijkl TTC031 16.2 ± 2.6 stuvw
TTC004 34.9 ± 1.4 efghijklm TTC086 16.0 ± 1.4 stuvw
TTC088 34.7± 3.9 efghijklm TTC093 15.9 ± 4.0 tuvw
TTC073 33.9 ± 3.5 efghijklmn TTC023 15.6 ± 2.9 tuvw
TTC007 33.4 ± 2.9 fghijklmn TTC059 14.8 ± 3.9 tuvw
TTC028 32.4 ± 2.1 ghijklmno TTC101 14.5 ± 3.4 uvw
TTC090 32.4 ± 3.7 ghijklmno TTC008 13.1 ± 4.1 vwx
TTC050 31.7 ± 2.1 TTC032 12.3 ± 1.5 wx
TTC009 31.5 ± 2.6 TTC100 6.8 ± 2.5 x
± Standard deviation of the mean. Identical letters do not show a significant difference according to the Tukey test (P = 0.0001).
Table 4. Potential antagonism of 50 isolates native to Trichoderma on Moniliophthora roreri.
Isolate Antagonism (%) / Isolate Antagonism (%) /
TTC017 68.8 ± 1.79 a TTC016 42.2 ± 3.03 hijk
TTC004 65.2 ± 2.59 ab TTC076 42.2 ± 3.56 hijk
TTC058 64.2 ± 1.92 abc TTC109 41.6 ± 6.31 hijk
TTC062 64.0 ± 0.71 abc TTC020 39.6 ± 2.19 ijkl
TTC090 62.2 ± 1.79 abc TTC037 39.4 ± 3.21 ijkl
TTC015 61.6 ± 0.89 abcd TTC051 38.6 ± 3.21 jkl
TTC104 60.0 ± 2.74 bcde TTC045 36.6 ± 2.19 klm
TTC039 59.8 ± 3.90 bcde TTC002 33.0 ± 1.87 lmn
TTC014 58.8 ± 1.79 bcde TTC035 29.8 ± 3.96 mno
TTC115 58.2 ± 2.95 bcde TTC063 28.0 ± 5.05 no
TTC085 57.6 ± 2.79 cde TTC032 27.0 ± 1.73 nop
TTC059 57.6 ± 1.82 cde TTC024 26.6 ± 2.50 nop
TCC023 57.6 ± 1.52 cde TTC001 25.6 ± 2.30 opq
TTC047 57.0 ± 3.16 cde TTC031 25.0 ± 3.80 opq
TTC073 54.0 ± 2.35 def TTC093 20.8 ± 3.96 pqr
TTC077 53.4 ± 3.78 ef TTC027 19.6 ± 0.55 qr
TTC100 53.2 ± 1.30 ef TTC088 17.2 ± 1.92 rs
TTC056 52.4 ± 2.19 efg TTC007 16.8 ± 1.48 rs
TTC021 49.2 ± 4.32 fgh TTC028 16.2 ± 1.00 rs
TTC084 47.4 ± 1.40 fgh TTC026 15.0 ± 2.00 rst
TTC009 46.8 ± 2.17 fghi TTC003 13.2 ± 1.64 st
TTC102 46.8 ± 5.17 fghi TTC113 13.0 ± 2.12 st
TTC086 45.0 ± 3.67 ghij TTC081 10.0 ± 1.22 tu
TTC054 44.4 ± 3.21 hij TTC101 7.2 ± 1.48 u
TTC050 42.8 ± 2.17 hijk TTC008 6.8 ± 1.79 u
± Standard deviation of the mean. Identical letters do not show a significant difference according to the Tukey test (P = 0.0001).
Table 5. Mycelial growth of 40 isolates native to Trichoderma at 25, 30 and 35 °C.
Isolate Mycelial growth (%)
25 °C 30 °C 35 °C
TTC063 40.0 ± 0 a 26.1 ± 0.3 efgh 10.1 ± 0.2 hi
TTC021 40.0 ± 0 a 40.0 ± 0 a 7.9 ± 0.1 jk
TTC084 40.0 ± 0 a 40.0 ± 0 a 19.0 ± 0.7 cd
TTC017 40.0 ± 0 a 40.0 ± 0 a 29.0 ± 1.0 a
TTC100 33.3 ± 1.0 b 40.0 ± 0 a 21.9 ± 0.1 bc
TTC090 32.7 ± 1.0 b 34.0 ± 1.2 b 14.7 ± 0.3 ef
TTC102 31.9 ± 1.4 bc 40.0 ± 0 a 21.0 ± 0.7 bc
TTC104 31.8 ± 1.1 bc 40.0 ± 0 a 23.3 ± 0.1 b
TTC056 31.6 ± 2.2 bcd 32 ± 1.7 bc 9.6 ± 0.1 ij
TTC073 30.7 ± 2.8 bcde 40.0 ± 0 a 23.9 ± 0.5 ab
TTC001 30.5 ± 0.5 bcdef 32.2 ± 0.9 bc 19.9 ± 0.8 bcd
TTC076 30.0 ± 0.1 bcdeg 30.8 ± 2.0 bcd 10.4 ± 0.3 hi
TTC020 29.7 ± 0.5 bcdefgh 32.2 ± 1.4 bc 20.9 ± 0.3 bc
TTC085 29.4 ± 0.6 bcdefghi 27.3 ± 0.6 defg 10.0 ± 0.02 hi
TTC002 29.2 ± 1.8 bcdefghij 30.4 ± 0.5 bcde 20.2 ± 0.5 bcd
TTC051 28.8 ± 1.1 bcdefghij 34.2 ± 0.2 b 21.5 ± 0.9 bc
TTC035 28.6 ± 0.9 bcdefghij 31.6 ± 1.2 bcd 21.0 ± 1.0 bc
TTC004 27.6 ± 3.2 cdefghijk 18.3 ± 1.3 k 2.1 ± 1.0 n
TCC023 27.6 ± 2.0 cdefghijk 29.5 ± 0.6 bcdef 14.0 ± 1.1 efg
TTC024 26.9 ± 1.5 defghijkl 32.5 ± 1.2 bc 19.8 ± 0.9 bcd
TTC039 26.9 ± 0.2 efghijkl 33.6 ± 1.7 b 12.3 ± 0.2 fgh
TTC093 26.0 ± 0.6 fghijkl 18.5 ± 0.7 k 3.2 ± 0.2 m
TTC090 25.6 ± 1.0 ghijklm 40.0 ± 0 a 18.7 ± 1.5 cd
TTC062 25.6 ± 0.7 ghijklm 28.1 ± 0.3 cdefg 11.6 ± 0.4 ghi
TTC016 25.5 ± 1.1 hijklmn 29.7 ± 0.6 bcdef 18.7 ± 0.2 cd
TTC115 25.1 ± 0.5 ijklmn 28.0 ± 1.2 cdefg 7.7 ± 0.2 k
TTC059 24.5 ± 0.6 jklmno 24.9 ± 1.1 ghi 11.6 ± 1.1 ghi
TTC050 24.3 ± 2.2 klmno 22.3 ± 0.4 hij 9.7 ± 0.5 ij
TTC032 24.0 ± 1.3 klmnop 30.3 ± 0.7 bcdef 16.8 ± 1.0 de
TTC077 23.6 ± 0.6 klmnop 30.8 ± 1.5 bcd 19.1 ± 0.5 cd
TTC037 23.2 ± 1.2 lmnop 30.6 ± 0.8 bcd 16.6 ± 0.5 de
TTC047 22.2 ± 2.8 mnop 20.0 ± 2.1 jk 10.6 ± 0.2 hi
TTC109 22.1 ± 0.8 mnop 28.3 ± 1.0 cdefg 7.4 ± 0.3 k
TTC058 22.0 ± 0.7 mnop 24.6 ± 0.1 ghi 12.3 ± 0.1 fgh
TTC054 22.0 ± 1.1 mnop 21.1 ± 3.6 jk 5.5 ± 0.08 l
TTC031 21.8 ± 0.5 nop 25.9 ± 0.1 fghi 11.7 ± 0.9 ghi
TTC086 21.1 ± 1.4 opq 22.3 ± 3.2 ij 10.5 ± 0.7 hi
TTC014 20.9 ± 0.3 opq 23.1 ± 1.0 hij 11.7 ± 0.4 ghi
TTC015 20.6 ± 2.0 pq 22.3 ± 1.3 hij 11.6 ± 0.1 ghi
TTC045 18.6 ± 2.6 q 25.3 ± 3.5 ghi 6.7 ± 0.1 kl
± Standard deviation of the mean. Identical letters do not show a significant difference according to the Tukey test (P = 0.0001).
Table 6. Conidia production of 40 isolates native to Trichoderma at 25, 30 and 35 °C.
Isolate Production (conidia·mL-1)
25 °C 30 °C 35 °C
TTC063 3.1 x 109 ± 1.6 x 108 a 1.3 x 109 ± 3.3 x 108 efgh 1.2 x 106 ± 2.1 x 105 lm
TTC051 2.1 x 109 ± 2.4 x 108 b 1.1 x 109 ± 1.6 x 108 fghij 2.7 x 106 ± 1.0 x 105 I
TTC001 1.9 x 109 ± 3.5 x 107 bc 1.8 x 109 ± 5.4 x 107 bc 4.9 x 106 ± 1.5 x 105 h
TTC035 1.8 x 109 ± 1.1 x 108 bc 1.2 x 109 ± 1.0 x 108 efghi 1.3 x 107 ± 2.7 x 106 g
TTC102 1.8 x 109 ± 1.4 x 108 bc 2.6 x 109 ± 2.4 x 108 a 4.2 x 107 ± 3.5 x 106 e
TTC084 1.6 x 109 ± 1.2 x 108 bcd 8.9 x 108 ± 3.9 x 106 ijklm 3.9 x 108 ± 2.6 x 107 a
TTC002 1.6 x 109 ± 2.1 x 108 cd 1.3 x 109 ± 2.2 x 108 efgh 2.1 x 106 ± 5.1 x 105 Ij
TTC020 1.4 x 109 ± 3.6 x 107 de 1.2 x 109 ± 5.2 x 107 efghij 1.7 x 106 ± 1.0 x 105 jk
TTC015 1.3 x 109 ± 2.1 x 108 def 1.2 x 109 ± 1.1 x 108 efghi 2.5 x 105 ± 0 pq
TTC073 1.3 x 109 ± 2.3 x 108 defg 1.1 x 109 ± 1.3 x 108 fghij 6.4 x 106 ± 3.5 x 105 h
TTC024 1.3 x 109 ± 2.8 x 107 defg 1.7 x 109 ± 7.4 x 107 bcd 1.3 x 106 ± 3.0 x 105 kl
TTC037 1.2 x 109 ± 1.3 x 108 efg 1.5 x 109 ± 1.4 x 108 cdef 8.6 x 107 ± 1.7 x 106 cd
TTC045 1.1 x 109 ± 1.2 x 108 efgh 2.8 x 108 ± 2.8 x 107 opqr 6.6 x 107 ± 1.0 x 106 d
TTC021 1.0 x 109 ± 8.7 x 107 fghi 1.7 x 108 ± 1.0 x 107 qr 1.3 x 106 ± 1.7 x 105 klm
TTC016 1.0 x 109 ± 6.8 x 107 fghij 1.4 x 109 ± 1.4 x 108 defg 1.6 x 108 ± 1.5 x 107 b
TTC090 1.0 x 109 ± 8.9 x 107 fghij 1.5 x 109 ± 1.9 x 108 bcde 1.4 x 106 ± 1.9 x 105 kl
TTC115 9.9 x 108 ± 4.1 x 107 ghijk 2.3 x 109 ± 6.8 x 107 a 1.0 x 106 ± 0 m
TTC032 8.4 x 108 ± 3.7 x 107 hijkl 9.7 x 108 ± 8.2 x 107 hijkl 7.4 x 107 ± 4.4 x 106 d
TTC017 8.2 x 108 ± 2.7 x 107 ijklm 1.9 x 109 ± 2.1 x 108 b 2.6 x 107 ± 3.0 x 106 f
TTC109 7.9 x 108 ± 2.3 x 107 jklmn 1.4 x 109 ± 2.7 x 107 defg 1.0 x 108 ± 2.7 x 106 c
TTC058 7.6 x 108 ± 4.4 x 107 klmn 1.0 x 109 ± 1.5 x 108 ghijk 2.8 x 105 ± 3.7 x 104 nop
TCC023 7.2 x 108 ± 7.8 x 106 lmno 1.3 x 109 ± 8.5 x 107 efgh 3.5 x 105 ± 1.7 x 104 n
TTC085 7.1 x 108 ± 1.6 x 107 lmno 8.1 x 108 ± 1.0 x 108 jklm 0 ± 0 r
TTC077 6.6 x 108 ± 1.5 x 107 lmno 1.7 x 109 ± 3.4 x 108 bcd 4.1 x 107 ± 2.7 x 106 e
TTC062 6.3 x 108 ± 3.6 x 107 mno 9.0 x 108 ± 1.0 x 108 ijklm 2.6 x 105 ± 2.3 x 104 nopq
TTC039 6.3 x 108 ± 2.1 x 107 mno 1.5 x 109 ± 8.8 x 107 cdef 1.4 x 107 ± 5.1 x 105 g
TTC031 6.3 x 108 ± 2.6 x 107 mno 7.1 x 108 ± 4.1 x 107 klmn 2.2 x 106 ± 2.1 x 105 Ij
TTC014 6.3 x 108 ± 1.2 x 107 no 1.4 x 109 ± 1.1 x 108 defg 2.5 x 105 ± 1.1 x 104 opq
TTC047 6.2 x 108 ± 2.3 x 107 no 9.0 x 108 ± 5.0 x 107 ijklm 0 r
TTC086 5.8 x 108 ± 2.6 x 107 op 8.2 x 108 ± 8.0 x 107 jklm 2.5 x 105 ± 0 pq
TTC009 4.7 x 108 ± 3.1 x 107 pq 8.0 x 108 ± 8.6 x 107 jklm 1.3 x 107 ± 3.8 x 106 g
TTC100 4.7 x 108 ± 4.1 x 107 pq 1.5 x 109 ± 2.6 x 108 cdef 1.3 x 107 ± 3.4 x 106 g
TTC004 4.3 x 108 ± 1.9 x 106 qr 5.6 x 108 ± 2.7 x 107 mnop 0 r
TTC059 4.3 x 108 ± 2.7 x 107 qr 4.1 x 108 ± 5.6 x 107 nopq 3.4 x 105 ± 2.5 x 104 no
TTC050 3.6 x 10 8 ± 5.0 x 107 r 6.0 x 108 ± 1.1 x 108 lmno 0 r
TTC054 3.4 x 108 ± 2.4 x 107 r 3.8 x 108 ± 2.3 x 107 nopq 0 r
TTC076 3.3 x 108 ± 1.4 x 107 r 2.0 x 108 ± 2.6 x 107 pqr 2.7 x 105 ± 2.9 x 104 nopq
TTC104 1.8 x 107 ± 3.7 x 106 s 0.05 x 108 ±4.8 x 103 r 1.9 x 108 ± 1.7 x 107 b
TTC056 1.5 x 107 ± 3.5 x 106 s 1.1 x 108 ± 2.2 x 107 qr 2.0 x 105 ± 0 q
TTC093 1.4 x 107 ± 2.8 x 106 s 1.1 x 108 ± 1.4 x 107 qr 0 r
± Standard deviation of the mean. Identical letters do not show a significant difference according to the Tukey test (P = 0.0001).