Native Strains T. longibrachiatum UCF17-M4 and Trichoderma sp. UCPF2 Reduce Cd Uptake in Cacao CCN51 Under Controlled Conditions

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The article has scientific and practical significance. It considers the possible role of fungi of the genus Trichoderma in reducing the accumulation of the heavy metal cadmium in cocoa. The authors conducted a large number of experiments, modeling and controlling the conditions of interaction of fungi with different concentrations of cadmium. The authors showed the ability of several Trichoderma species to significantly reduce the concentration of cadmium in the environment due to the accumulation of this heavy metal in the fungal biomass. In this case, the differences are manifested at the species level and less at the strain level. The effect of fungi on the accumulation of cadmium in plants turned out to be quite contradictory and ambiguous and manifested itself both in the direction of increasing the accumulation of cadmium by plants and in the direction of decreasing its concentration in cocoa plants. The obtained results allowed the authors to identify promising Trichoderma species and strains for further research aimed at reducing the cadmium content in cocoa plants. The work was carried out at a modern methodological level, the experiments are well described. The processing of the obtained results was carried out using a wide range of statistical methods. Tables and figures supplement the description of the results. In general, the article is quite original, provides valuable information on the role of fungi in cadmium detoxification in agroecosystems where cocoa is grown. It may be of interest to readers of the Journal.

Comments.

The introduction focuses on cadmium pollution of soils and plants in areas in South America where cocoa is grown. Only at the end is one paragraph devoted to previous studies of the role of fungi of the genus Trichoderma. Obviously, this part of the article should have paid more attention to the role of fungi in cadmium detoxification. What types of fungi, in addition to Trichoderma, in this regard were studied. This aspect is mentioned in the discussion of the results, but it should also have been taken into account when justifying the purpose of the work.

The description of the original Trichoderma strains selected for the study looks very schematic. These strains were isolated from local agroecosystems and selected based on previous positive effects on cocoa. It seems that this description is insufficient. This section does not specify the environment from which the strains were isolated. Obviously, we are talking about soil fungi, but there is no mention of this. This is especially true for those strains for which no data have been published. How were the strains stored in collection – on what medium and under what conditions?

It is not clear how the seedlings were inoculated. It is obvious that the inoculum was added to the soil in close proximity to the seedlings or in another way?

The main question for the authors is concerning the role of the soil taken into account in the experiments? Fungi of the genus Trichoderma belong to soil micromycetes. It is obvious that the interaction with the plant in most cases occurred indirectly through the soil. The decrease in the concentration of cadmium entering the plant is also associated with its detoxification in the soil. However, the article does not describe anywhere what soil was used in these experiments. Was the soil sterilized before sowing? Was the natural composition of microbial community in the agricultural soil used in the experiments known? What soil properties were taken into account (pH, type, structure, humidity, temperature, etc.)?

Fig. 1 looks too cumbersome and difficult to perceive. White numbers on a yellow background are not visible. This graph can be considered as an addition to Fig. 1S, where the results are shown quite clearly. Probably, Fig. 1 can be removed or moved to the Supplementary, and the description to it can be left in the text. But keep Fig 1S in the text.

In the discussion, the authors mention the possible influence of fungal and bacterial associations on cadmium detoxification. The issues of the possible use of microorganism associations in the detoxification of heavy metals are very important. When it comes to soil, it is obvious that there cannot be individual microorganisms here, but they are in close interaction, often forming biofilms of complex composition. In such cases, detoxification is associated with the metabolic products of such biofilms, in particular with the production of an extrapolymeric substance. It will be interesting to know what the authors think about the possibility of creating (modeling) such associations that could include different species of the genus Trichoderma? Perhaps this should have been given more attention in the discussion of the results.

Comments on the Quality of English Language

The English could be improved to more clearly express the research.

Author Response

REVIEWER 1

Comments and Suggestions for Authors

Comments 1: The introduction focuses on cadmium pollution of soils and plants in areas in South America where cocoa is grown. Only at the end is one paragraph devoted to previous studies of the role of fungi of the genus Trichoderma. Obviously, this part of the article should have paid more attention to the role of fungi in cadmium detoxification. What types of fungi, in addition to Trichoderma, in this regard were studied. This aspect is mentioned in the discussion of the results, but it should also have been taken into account when justifying the purpose of the work.

Author response 1:

We thank the reviewer for their insightful comments and suggestions. In response, we have revised the introduction and the following paragraph in the introduction has been modified:

"It is well known that several Trichoderma species establish beneficial interactions with various plants, including cacao [24,25]. This saprophytic fungus is commonly found colonizing soil and rhizospheric ecosystems. As described by Abdul-Halim et al. (2023), members of this genus are generally not considered pathogenic or harmful to plant development. Furthermore, some are regarded as strategic partners in agriculture and industry, mainly due to their roles as biocontrol agents, their plant growth-promoting effects, and their physiological capacity to produce industrially important metabolites [26–28; Mukherjee et al., 2013]. In addition, certain Trichoderma strains have also been reported to exhibit heavy metal tolerance and uptake capabilities [29–33]. These properties are attributed to several physiological and biochemical mechanisms, such as adsorption, heavy metal flux across the cellular membrane, and intracellular chelation by metallothionein (MTs) and specific peptides [34]. Although several studies have demonstrated the cadmium removal capacity of Trichoderma strains [58–59], little is known about their effectiveness in association with cacao plants. Therefore, this study aims to determine the Cd-uptake capacity of 12 native Trichoderma strains isolated from cacao-growing soils in Amazonas Region under in vitro conditions and to evaluate their effectiveness in reducing Cd levels in cacao plants.

Comment 2: The description of the original Trichoderma strains selected for the study looks very schematic. These strains were isolated from local agroecosystems and selected based on previous positive effects on cocoa. It seems that this description is insufficient. This section does not specify the environment from which the strains were isolated. Obviously, we are talking about soil fungi, but there is no mention of this. This is especially true for those strains for which no data have been published. How were the strains stored in collection – on what medium and under what conditions?

Author Response 2:

Thank you for your insightful observation. We have revised the paragraph to provide a clearer and more detailed description of the Trichoderma strains used in this study. The text was modified as follows:

“Twelve native Trichoderma strains (Table 1) from cacao agroecosystems in five provinces of Amazonas Region,  were used in this studio. These strains were obtained from rhizospheric soil samples, using the serial dilution method on Potato Dextrose Agar (PDA) medium. The selection was based on previously reported positive effects on cacao [35,36]. All strains are stored in the fungal collection of the Laboratorio de Investigación en Sanidad Vegetal (LABISANV) at the Instituto de Investigación para el Desarrollo Sustentable de Ceja de Selva of the Universidad Nacional Toribio Rodríguez de Mendoza (UNTRM). For each biological assay, the strains were reactivated using PDA by puncture inoculation, and incubated at 28°C for seven days until visible mycelial growth was observed”.

Comment 3: It is not clear how the seedlings were inoculated. It is obvious that the inoculum was added to the soil in close proximity to the seedlings or in another way?

Author’s Response 3:

Thank you for pointing this out. To clarify how the seedlings were inoculated, the following sentences have been incorporated to section 2.3 y 2.4. 

Section 2.3:

“The sediment was then dried at 30°C for 48 hours. The dried Trichoderma spores were used as active compounds for plant inoculation. To this, the spores were resuspended in 250ml of sterile distilled water and the concentration was determined using a Neubauer chamber. Finally all the inocula concentrations were adjusted to a 1×10⁶ spores ml -1”.

Section 2.4:

“The seeds, previously separated from the mucilage, were planted in each bag and inoculated with 250 ml of the Trichoderma spore suspension at a concentration of 1×10⁶ spores ml -1. This was applied directly to the soil around each seed, to ensure close contact between the inoculum and the emerging radicle.”

Comment 4: The main question for the authors is concerning the role of the soil taken into account in the experiments? Fungi of the genus Trichoderma belong to soil micromycetes. It is obvious that the interaction with the plant in most cases occurred indirectly through the soil. The decrease in the concentration of cadmium entering the plant is also associated with its detoxification in the soil. However, the article does not describe anywhere what soil was used in these experiments. Was the soil sterilized before sowing? Was the natural composition of microbial community in the agricultural soil used in the experiments known? What soil properties were taken into account (pH, type, structure, humidity, temperature, etc.)?

Author Response:
Thank you for pointing this out. For the sterilization process, the soil was solarized. To this, the substrate was covered with transparent plastic and exposed to direct sunlight for seven consecutive days, with daily temperatures ranging between 15.5-30.3 °C, according to data from the Servicio Nacional de Meteorología.
The native microbial community of the agricultural soil was not assessed, as proper characterization would require complementary strategies such as next-generation sequencing (NGS) and biodiversity analysis. Although this is indeed an interesting area of study, it falls outside the scope of the present work.
To clarify this point the following sentences has been modified in Section 2.4:

“Polyethylene nursery bags (1Kg capacity) were filled with a prepared substrate composed of Cd-free agricultural soil (Cd<0.0001 ppm) from cacao-growing fields (pH = 7.82 ± 0.34 ; CE =1.20 ± 0.20 ), sand and rice-husk (as an organic matter source) at 2:1:1 rate. The substrate was previously sterilized through solarization by covering it with plastic and exposing it to direct sunlight for seven consecutive days.  During this period, daily temperatures ranged between 15.5 and 30.3 °C, according to data from the Bagua Chica Meteorological Station (Servicio Nacional de Meteorología).

As the main objective of the study was to evaluate the reduction in cadmium uptake by the plant mediated by the Trichoderma inoculum, detailed physicochemical properties of the soil were not assessed at this stage. Referential values for parameters such as pH and electrical conductivity (EC) of the agricultural soil were recovered, however these were not measured at the end of the experiment. 

In addition, this was mentioned in the discussion section:

“Regarding the cadmium-reducing uptake in plants by Trichoderma strains, T. longibrachiatum UCF17-M4 and Trichoderma sp. UCPF2-C1 reduced the Cd concentration in cacao plants. Previous studies have shown that T. longibrachiatum has bioremediation potential for heavy metals. Furthermore, in addition to metal removal [53], strains of this species have been associated with enhanced plant stress tolerance and improved soil quality [54–56]. 

In response to heavy metal toxicity, Trichoderma has been shown to induce the expression  of genes related to reactive oxygen species (ROS) synthesis and detoxification. A recent study reported that T. reesei overexpressed genes associated with the MAPK signaling pathway, thereby enhancing the detoxification of ROS. Additionally, genes related with ABC transporters, viral myocarditis and ErbB signaling pathway were also  upregulated [57].  Beyond the fungal mechanisms involved in the response to cadmium stress, it is also important to consider that environmental conditions play a critical role in the detoxification process, as they influence the bioavailability of the metal. For instance, Cadmium concentrations tend to increase with higher total Cd levels and lower pH values. Moreover, in some cases, Cd concentrations also increase with decreasing organic matter content (%OM) [6]. Although soil physicochemical properties were not measured in this study, their potential influence should be considered in future experiments”.

Comment 5: Fig. 1 looks too cumbersome and difficult to perceive. White numbers on a yellow background are not visible. This graph can be considered as an addition to Fig. 1S, where the results are shown quite clearly. Probably, Fig. 1 can be removed or moved to the Supplementary, and the description to it can be left in the text. But keep Fig 1S in the text.

Author response:

Thank you for your valuable comment. We fully agree with the Reviewer's suggestion. In response, we have moved the original Figure 1 to the Supplementary Material as Supplementary Figure S2. Moreover the color scale was modified. Instead, we have included a revised figure that, more accurately, presents the post hoc results of the contrast analysis for the Strain × Cd concentration interaction for both, Cd-Removal Efficiency and Cd-Biomass accumulation. This figure shows the significant differences among treatments, providing clearer and more interpretable information.
In addition, the following sentences were modified:

Section 3.1.

“Furthermore, post hoc analysis revealed significant differences in 636 out of 1128 interactions (Figure S2). Moreover, as shown in Figure 1, the multiple comparisons showed significant differences in 254 of 630 comparisons, with purple highlights indicating the most significant differences (p < 0.05, indicated by asterisk)”.


“Additionally, post hoc analysis identified significant differences in 828 out of 1128 interactions (Figure S2), and 372 of 630 multiples comparisons (Figure 1), further suggesting that this response depends on both the Trichoderma strain and Cd concentration in the culture medium”.

Comment 6: In the discussion, the authors mention the possible influence of fungal and bacterial associations on cadmium detoxification. The issues of the possible use of microorganism associations in the detoxification of heavy metals are very important. When it comes to soil, it is obvious that there cannot be individual microorganisms here, but they are in close interaction, often forming biofilms of complex composition. In such cases, detoxification is associated with the metabolic products of such biofilms, in particular with the production of an extrapolymeric substance. It will be interesting to know what the authors think about the possibility of creating (modeling) such associations that could include different species of the genus Trichoderma? Perhaps this should have been given more attention in the discussion of the results.

We fully agree with the comment. The scientific literature provides strong support for the critical role that diverse microorganisms play in contaminant detoxification processes, particularly in biologically active zones such as the plant rhizosphere. In this environment, multiple microbial communities converge and establish complex, coordinated interactions. Due to this complexity, there is growing interest in both the basic and applied study of so-called synthetic communities. In this context, and because of our previous experience characterizing other types of Cd-accumulating microorganisms, we consider the evaluation of microbial consortia a highly promising approach.
Moreover, as it is mentioned in the Discussion section, there is evidence of cadmium-reducing effects of Trichoderma (alone or in consortia) in other plant models. To clarify our position, the following paragraph was modified:

Though not extensively studied in cacao, Trichoderma inoculation has demonstrated cadmium-reducing effects in other plant models. For example, in Vigna radiata, inoculation with the hyper-tolerant strain Trichoderma sp. TF-13 reduced lead (Pb) and Cd uptake in root and aerial tissues by 34/39% and 47/38%, respectively [58]. Similar effects were observed in Cicer arietinum plants inoculated with a consortium of Pseudomonas fluorescens PGPR-7 and Trichoderma sp. T-4, where joint inoculation reduced Cd uptake in roots by 38% in plants exposed to 25 μg kg -1 Cd in soil [59]. In addition with reduction of Cd accumulation, the improvement of plant growth and physiological parameters were also reported [58, 60]. Furthermore, a recent metabolomic analysis identified the production of 43 key metabolites, including nicotinic acid, succinic acid, and fumaric acid, involved in Cd detoxification in Nicotiana plants inoculated with T. nigricans T32781 [60]. These findings suggest that the use of Trichoderma, either alone or in consortium, is a promising strategy for reducing cadmium uptake and enhancing plant growth in Cd-contaminated environments. In this context, further experimentation with Trichoderma strains UCF17-M4 and UCPF2-C1 must be performed. Particularly as part of a fungal consortium considering the natural complexity of soil environments and the potential for synergistic interactions among microorganisms.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Manuscript ID: Microbiolres-3611756

Title: Native Strains T. longibrachiatum UCF17-M4 and Trichoderma sp. UCPF2-C1, Reduce Cd-Uptake in CCN51 Cacao Under Controlled Conditions

 

This study investigated that native strains T. longibrachiatum UCF17-M4 and Trichoderma sp. UCPF2-C1 significantly reduced cadmium accumulation in cocoa stems of CCN51. I encourage the authors to improve the overall quality of this manuscript, which is not limited by the points I listed below.

 

Detailed comments:

1. The article dose not explain why it focuses on CCN 51. The specificity of CCN 51 and its representativeness should be explained.
2. There are date deficiencies in table 2. The data in this table needs to be supplemented or the reason for the missing data explained.
3. The yellow color block in Figure 1 does not show the numbers clearly and needs to be changed to a different color.
4. Line 136: The basic physical and chemical properties of agricultural soil should be given.
5. Line 147: It would be more appropriate to change directly to dry to constant weight as final dry weight.

Author Response

REVIEWER 2

Comments 1:  The article does not explain why it focuses on CCN 51. The specificity of CCN 51 and its representativeness should be explained

Author Response:

We appreciate the reviewer’s comment. The focus on CCN 51 is due to its widespread cultivation and economic importance. In addition to its representativeness and agronomic relevance, native varieties have not been fully identified or characterized; they are often grouped under the general term “criollo,” which increases the likelihood of misidentification. 

We have now added a brief explanation in the section 2.4

“Three days before planting, CCN51 cacao seeds were germinated. This clone was selected for its widespread cultivation, economic significance, and genetic uniformity, which make it a suitable model for evaluating plant responses under controlled experimental conditions. To this, the cacao pods were washed with clean water and disinfected using a sodium hypochlorite (NaClO) solution (5%) for 5 minutes”.

Comment 2:

There are date deficiencies in table 2. The data in this table needs to be supplemented or the reason for the missing data explained.

Authors' response:

We appreciate the reviewer’s observation. In some cases, data could not be included in Table 2 due to the spectrophotometer returning unreliable readings, likely due to sample-related interference. These instances have now been clearly indicated in the revised table 2. Moreover this was also included in the text. 

footnote:  

“The results represent the mean of three replicates ± SD, grown at different cadmium concentrations (0, 100, 200, and 300 ppm). When unreliable spectrophotometric readings were obtained, they were represented as “nd” to indicate undetermined values”

Section 3.1.

“On the other hand, in some cases, such as strains CNF20-C1 and UCPF2-C1, efficiency values could not be determined due to unreliable readings, and these cases were indicated as "not determined" (nd) in Table 2.

 

Comment 3: The yellow color block in Figure 1 does not show the numbers clearly and needs to be changed to a different color. 

Authors' response:

Thank you for your valuable comment. We fully agree with the Reviewer's suggestion. In response, we have moved the original Figure 1 to the Supplementary Material as Supplementary Figure S2. Moreover the color scale was modified. Instead, we have included a revised figure that, more accurately, presents the post hoc results of the contrast analysis for the Strain × Cd concentration interaction for both, Cd-Removal Efficiency and Cd-Biomass accumulation. This  figure shows the significant differences among treatments, providing clearer and more interpretable information.

In addition, the following sentences have been modified:

Section 3.1.
"Furthermore, post hoc analysis revealed significant differences in 636 out of 1128 interactions (Figure S2). Moreover, as shown in Figure 1, the multiple comparisons showed significant differences in 254 of 630 comparisons, with purple highlights indicating the most significant differences (p < 0.05, indicated by asterisk)".

"Additionally, post hoc analysis identified significant differences in 828 out of 1128 interactions (Figure S2), and 372 of 630 multiples comparisons (Figure 1), further suggesting that this response depends on both the Trichoderma strain and Cd concentration in the culture medium".

Comment 4: Line 136: The basic physical and chemical properties of agricultural soil should be given.

Authors' Response
As the main objective of the study was to evaluate the reduction in cadmium uptake by the plant mediated by the Trichoderma inoculum, detailed physicochemical properties of the soil were not assessed at this stage. Referential values for parameters such as pH and electrical conductivity (EC) of the agricultural soil were recovered, however these were not measured at the end of the experiment.

To clarify this point the following paragraph has been modified in Section 2.4:

“Polyethylene nursery bags (1Kg capacity) were filled with a prepared substrate composed of Cd-free agricultural soil (Cd<0.0001 ppm) from cacao-growing fields (pH = 7.82 ± 0.34 ; CE =1.20 ± 0.20 ), sand and rice-husk (as an organic matter source) at 2:1:1 rate. The substrate was previously sterilized through solarization by covering it with plastic and exposing it to direct sunlight for seven consecutive days.  During this period, daily temperatures ranged between 15.5 and 30.3 °C, according to data from the Bagua Chica Meteorological Station (Servicio Nacional de Meteorología).

In addition, this was mentioned in the discussion section:

"Regarding the cadmium-reducing uptake in plants by Trichoderma strains, T. longibrachiatum UCF17-M4 and Trichoderma sp. UCPF2-C1 reduced the Cd concentration in cacao plants. Previous studies have shown that T. longibrachiatum has bioremediation potential for heavy metals. Furthermore, in addition to metal removal [53], strains of this species have been associated with enhanced plant stress tolerance and improved soil quality [54–56]. In response to heavy metal toxicity, Trichoderma has been shown to induce the expression  of genes related to reactive oxygen species (ROS) synthesis and detoxification. A recent study reported that T. reesei overexpressed genes associated with the MAPK signaling pathway, thereby enhancing the detoxification of ROS. Additionally, genes related with ABC transporters, viral myocarditis and ErbB signaling pathway were also  upregulated [57].  Beyond the fungal mechanisms involved in the response to cadmium stress, it is also important to consider that environmental conditions play a critical role in the detoxification process, as they influence the bioavailability of the metal. For instance, Cadmium concentrations tend to increase with higher total Cd levels and lower pH values. Moreover, in some cases, Cd concentrations also increase with decreasing organic matter content (%OM) [6]. Although soil physicochemical properties were not measured in this study, their potential influence should be considered in future experiments".

Comment 5: Line 147: It would be more appropriate to change directly to dry to constant weight as final dry weight.

Authors' Response:

Done, the sentence was modified