Introduction

The production of cocoa (Theobroma cacao L.) as part of agroforestry systems is an increasingly growing activity in the Amazon region of La Paz, Bolivia. Cacao is an important economic crop of the Bolivian Amazon, Bolivian producers grow cacao and extract fruits from wild stands in the Beni River region and valleys of the foothills of the Andes, and the traditionally used germplasm group is now called the Bolivian National Cacao (CNB), Alto Beni in the department of La Paz, since the 60s it has developed the cultivation to become the region with the largest cocoa production capacity in Bolivia, home to the largest number of producers and cultivated hectares, as well as the most important cooperative, El Ceibo^1,2.

Cocoa (T. cacao) is native to the jungle regions of tropical America, from Peru to Mexico, its main use being the production of chocolate, followed by other uses such as cosmetics and raw materials³. Its cultivation in tropical regions stands out and its yield is affected by several diseases such as black pod rot, its control could be achieved if integrated management strategies are established, with the combination of biological and chemical methods, genetic control and cultivation methods in an integrated program⁴.

Selection criteria for the CNB were described in Alto Beni-Bolivia, and emphasize that Bolivia is one of the few countries with cocoa in the wild and is positioned among the main holders of fine and aroma cocoa in the world, however, despite its existence being known and cultivated for more than 200 years, there is not enough information available to initiate a program for the selection of outstanding individuals⁵. Studies were carried out on genetic variability of CNB, indicating that wild cocoa is known by several names: "criollo cocoa", "Bolivian national cocoa", "Amazonian cocoa", "wild cocoa" and "cocoa". Wild cocoa has several advantages: it is a non-perishable product that can be harvested, fermented and dried in areas distant from the markets, it has great adaptability to ecoregions, and it can be produced even in long periods of flooding^2,6. However, the development period of seedlings in the nursery is longer than the other types of cocoa, which makes it difficult to scale up production.

To obtain optimal yields, management of this crop is required, such as obtaining quality rootstocks, which can also provide some tolerance to attack by fungi or other pathogens⁷. The use of rootstocks, obtained from botanical seeds, allows to reduce production costs, the characteristics described for CNB and IMC-67 foreign cocoa suggest a wide potential for its production as rootstocks in the region. However, it is necessary to optimize production conditions during the nursery phase, to maximize its development and productivity. Given the relevance of this crop for the country and the growing demand for sustainable agricultural practices², the use of beneficial microorganisms such as Trichoderma is presented as a promising alternative to improve crop productivity and health, reducing dependence on agrochemicals⁸.

Regarding Trichoderma, its characteristics stand out as the mechanism of action as a biocontrol agent, to mitigate the attack of phytopathogens, as an alternative to synthetic pesticides, along with other benefits conferred by the development of crops through its symbiotic interaction with plants and bioremedial effects in contaminated fields⁸. It is also added that ideal biocontrol agents such as Trichoderma have qualities such as the production of antibiotics, lytic enzymes that degrade cell walls of plant pathogens or the induction of a defense response in plants⁹.

Different studies verified the qualities of Trichoderma under nursery conditions for seedling production, such as the evaluation of 15 endophytic strains to characterize their influence on seedling inoculation in the establishment of endophytic growth in cocoa seedlings¹⁰. Effects of endophytic colonization of T. hamatum (DIS 219b) with or without exposure to drought conditions were also verified, obtaining as effects an increase in fresh root weight, root dry weight and root water content in cocoa seedlings¹¹. Other authors evaluated the effect of different agroecological management on the growth of seedlings in the nursery phase, such as T. harzianum combined with leachate from banana rachis, vermicompost, Beauveria, among others¹². The effect of Trichoderma sp. on the agronomic characteristics of T. cacao ecotypes under nursery conditions was also evaluated, comparing treatments based on T. harzianum + CCN51 and T. afroharzianum + TSHS565 with 100 % colonization of root hairs and trichomes in stems¹³. Trichoderma is presented as an alternative for the organic production of cocoa, improving the agronomic characteristics of the crop in nursery conditions.

At present, there is little information regarding alternatives to improve the production of CNB rootstocks, who’s organoleptic and resistance characteristics are desirable for cocoa production⁶. In addition, there is a need to promote the production of T. cacao seedlings with the use of organic inputs, as organic certification is a requirement for many markets, to improve the valuation and price of a product such as cocoa from the Yungas area². Thus, this research aimed to evaluate the effect of the application of T. harzianum with different doses (0, 80, 100 and 120 %), comparing 2 types of rootstocks of cocoa seedlings, CNB and IMC-67.

Materials and methods

Geographical location. The study was carried out in the nursery of the Sapecho Experimental Station (EES), dependent on the Faculty of Agronomy of the Universidad Mayor de San Andrés (UMSA), located in the fourth municipal section of Palos Blancos, Sud Yungas Province of the department of La Paz, Bolivia. The EES is located between the coordinates 15º 32' 54.4" south latitude, 67º 19' 47.8" west longitude, at an average altitude of 450 meters above sea level, average temperature of 26º C and an average rainfall of 1800 mm year^-1 and at a distance of 260 km from the city of La Paz. It is noted that the only collection of CNB that is held so far in the country is safeguarded in the EES⁵. The evaluation period was from May to August 2021, in a nursery covered with 50 % saran semi-shade polyethylene mesh.

Methods. As biological material, CNB seeds and foreign cocoa IMC-67 and T. harzianum obtained from the Phytopathology laboratory of the Faculty of Agronomy of the UMSA were obtained. The seeds were obtained from cobs collected in plots of the EES and later the preparation method used by the EES personnel was used, for the removal of the mucilage, by mechanical action using sawdust. They were then placed in a pre-germinating bed, with a substrate composed of fine sand and sawdust, spaced 2 cm apart. Regular watering was carried out to ensure uniform emergence.

Substrate preparation. The pots were filled in black polyethylene bags (11*27 cm) with a mixture of substrate: 50 % local soil, 30 % ordinary sand, 20 % bocashi fertilizer and 10 % decomposed sawdust obtained by wetting for one week. Likewise, 1 kg of substrate sample was collected for subsequent shipment and analysis at the Soil and Water Laboratory of the Faculty of Agronomy (LAFASA) to know the physicochemical characteristics of the soil. The results obtained corresponded to a silty loam texture, nitrogen content 0.35 %, organic matter 5.77 %, organic carbon 3.35 %, available phosphorus 41.60 ppm, exchangeable potassium 1.90 mEq 100 g^-1, exchangeable magnesium 3.14 mEq 100 g^-1, exchangeable acidity (Al + H) 0.44 mEq 100 g^-1, electrical conductivity in H₂O 1:5 0.67 mmho cm^-1, pH in H₂O ratio 1:5 6.1 slightly acidic.

Inoculation and application of T. harzianum. The preparation of the concentrations was carried out based on the procedure used and recommended by Cadena et al.¹⁴, which, for a 20 L backpack, uses 200 g (fresh weight) or 68 g (dry weight) of the fungus, which represents 100 % concentration. For the present study, a 3.6 L solution for 120 seedlings or pots (30 mL per pot) was prepared for each application. The solution had water and 20 % manure tea as components. In the first application, it was double the calculated amounts of T. harzianum (10.88, 13.6, 16.32 g), 15 days before seed planting, with the aim of contributing to the development of the fungus¹⁴. Subsequently, it was applied every 15 days after sowing to each established treatment, until the stage of grafting cocoa seedlings, which is carried out in the nursery when a recommended diameter of 4 to 5 mm in diameter is reached¹⁵, and 5 mm is the diameter traditionally used in the region for this stage.

Cultural work. Irrigation was carried out according to the demand of the crop and rainfall, together with the "weaning" of the seedlings, a practice that consists of the removal of the seed testa. Likewise, phytosanitary control was carried out through manual control, removing damaged leaves, weed control and cleaning, together with the use of homemade inputs such as pyrethrum (dose of 1 mL for every 2 L of water), to repel insects.

Treatments. 8 treatments were established in a completely randomized design (CRD) with a two-factor arrangement and 3 replicates each, with Factor 1: application dose T. harzianum (0, 80, 100 and 120 %) and Factor 2 type of patterns: CNB and foreign cocoa (IMC-67). Obtaining a total of 24 experimental units (UE) with 10 plants each, making a total of 240 cocoa rootstock seedlings, as detailed in Table 1.

Study variables. Height cm (AP), measured from the base of the plant to the apical bud on the main stem of the plant, stem diameter mm (DT), measured with a digital vernier at the base of the root crown, and number of leaves (NH), quantified by each plant, were evaluated every 15 days. A total of 7 evaluations were recorded in a total of 105 days. Leaf area was determined using statistical models (linear regression equations) to estimate leaf area¹⁶.

Statistical analysis. It was performed using an analysis of variance and Duncan's mean comparison test at 5 % significance, using the InfoStat program to verify the differences between treatments.

Results

Figure 1 Plant height (cm) assessed every 15 days for the eight treatments 

Plant height (cm).Figure 1, the increase, in the different treatments for a period of 105 days. T₄ (120 %, CNB), T₈ (120 %, IMC-67) and T₆ (80 %, IMC-67) reached higher average heights compared to the other treatments. The lowest heights corresponded to the CNB with doses below 120 %.

Table 2, after 30 days of evaluation, highly significant differences were obtained (p˂0.01) for the AP variable for both study factors and their interaction, with a coefficient of determination (R²) equal to 0.97. It is noteworthy that, at 105 days, the highest PA was for T₈ (120 %, IMC-67) with 41.14 cm, which is distinguished from the other treatments. The second PA was for T₄ (120 %, CNB) with 35.68 cm and T₆ (80 %, IMC-67) with 33.70 cm, which do not statistically differ from each other. For the CNB, all treatments obtained lower heights, with the exception of T₄, to which a dose of Trichoderma of 120 % was applied.

Figure 2 Plant diameter (mm) evaluated every 15 days for the eight treatments 

Stem diameter (mm). IMC-67 rootstocks had a greater increase trend compared to CNB rootstocks, with the exception of T₄ (120 %, CNB) which presented an increase in DT similar to T₈ (120 %, IMC-67) (Figure 2).

Table 3, highly significant differences (p˂0.01) were obtained for both factors (type of pattern and dose), attributing the effect of Trichoderma in each stage evaluated, together with the interaction of both factors, with an average of 6.98 mm, in foreign cocoa and CNB with 6.57 mm, corresponding to the dose 120 % at 105 days. T₄ (120 %, CNB), T₅ (0 %, IMC-67), T₆ (80 %, IMC-67) and T₈ (120 %, IMC-67) reached diameters greater than 5 mm at 90 days of evaluation (Table 3), in addition, no differences were observed between T₄ and T₈ at 90 days, while at 105 days, T₈ differed from the other treatments followed by T₄.

As verified in Figure 3, there is a proximity between the NH of the CNB and the IMC-67 for the highest dose of Trichoderma (120 %). On the other hand, the lower doses presented lower averages for NH in both types of rootstocks, verifying that the dose factor incurs significant differences at 105 days (Table 4).

Figure 3 Number of leaves, evaluated every 15 days for the eight treatments 

For NH, the analysis of variance (Table 4) shows statistical differences (p˂0.01) for the dose factor of T. harzianum, but not for the graft bearing factor and interaction (p˃0.05). At 105 days, the dose of 120 % influenced a higher NH per plant for both types of cocoa, with the average being equal to 13.4 for IMC-67 and 12.77 for CNB, they do not differ statistically from each other, but they do differ from the other treatments.

Leaf area (cm ² ). According to the analysis of variance for the leaf area variable (Table 5), there were highly significant differences for the dose factor and the interaction (p˂0.01). The highest values corresponded to the dose of 120 % in both types of cocoa (85.05 cm² IMC-67 and 78.82 cm² CNB).

Discussion

The differences in PA between CNB and IMC-67 can be explained by the differences that distinguish CNB from other genotypes, since it is found in wild conditions and in culture with different degrees of crossing with introduced materials⁶. However, with the application of Trichoderma, the CNB had an increase in height similar to IMC-67 for the dose of 120 %. T₈ had an average development index of 0.34 cm day^-1 that exceeded that obtained by Gamboa Tabares et al.¹⁷ which was 0.2 cm day^-1 for the growth of the same clone (IMC-67), verifying that this fungus is also associated with an increase in the efficiency of photosynthetic and respiratory activities that indirectly help the plant to grow and develop¹⁸. The frequency of application can have a significant effect, since the application of T. harzianum in seedbeds and 15 days after planting stimulates growth in seedlings, such as tomato cultivation¹⁹.

When comparing different organic fertilizers in the production of cocoa seedlings, PA was obtained in a range of 30.7 to 39.1 cm, however, these were obtained up to 138 days²⁰, heights between 28.93 and 33.43 cm were observed for the ICS 6 rootstock at 90 days, which differ from those obtained in this study, and they can be explained by the difference between genotypes²¹. Likewise, an evaluation of several Trichoderma strains had averages of 29.83, 27.73 and 23.60 cm for T. harzianum with the clones CCN 51, TSH 565 and native fine aroma, at 90 days after planting in Peru¹³, confirming that native cocoa may present a slower development compared to other genotypes. However, the effect of Trichoderma could be related to the genotype or species, T. harzianum increased AP, NH and dry biomass of the leaf area in Cedrela odorata seedlings, while in Leucaena leucocephala and Albizia saman it only caused increases in basal diameter²². Because the fungus did not have a significant effect on native cacao, contrary to the results of this study, T. harzianum caused an increase in PA for CNB¹³. T. longibrachiatum can promote a growth of (plant or height ≥13 %)²³.

After 138 days of evaluation, diameters between 6.62 (poultry manure) and 6.86 (soil + bocashi 2-1) mm were obtained, through the application of different organic fertilizers in the production of seedlings of the IMC-67 clone, without obtaining significant statistical differences²⁰. Therefore, it is observed that, with the application of Trichoderma, it is possible to obtain the DT for grafting in shorter periods of time for both genotypes. On the other hand, the ICS 6 rootstock obtained between 5.79 and 6.21 mm during an evaluation period of 3 months, values higher than those obtained in the present study²¹. These differences may be related to the characteristics of the ICS 6 rootstock and the type of substrate used. At 90 days, the DT of 4.4, 3.77 and 4.25 mm, with application of T. harzianum, highlighting that, for a type of cocoa native to Peru, the DT obtained (4.4 mm) exceeded the other rootstocks¹³. Higher DT at 135 days, between 7.22 and 8.16 mm through the application of liquid fertilizers based on T. harzianum, exceeded treatment with the application of a conventional fertilizer (4.59 mm)¹². On the other hand, the determining factor is the genotype of the rootstock used, obtaining a SD of 4.58 mm for IMC-67 at 90 days of culture, while the one obtained in this study was 5.45 mm¹⁷. This difference can be attributed to the use of the Trichoderma fungus in growth, due to its benefits as a defense stimulant and seedling growth promoter^24,25. Its effect as a biocontrol can act by promoting plant development, colonizing the soil and/or part of the plant and preventing the multiplication of pathogens²⁶, considering that there was no presence of diseases during the evaluation period. Many species of this genus can produce the auxin indole-3-acetic acid, which has been suggested to promote root growth, however, the mechanisms and molecules involved in promoting growth are multivariable and are affected by environmental conditions²⁷.

For the NH variable, higher averages were obtained, which in a longer evaluation period obtained an average of 11.5 leaves per plant, which did not obtain significant differences and evaluated an average of 11.4 leaves for a substrate with local soil + bocashi^20,21. At 90 days, higher averages (9.67, 10.0 and 8.33 leaves per plant) were recorded when comparing the values of this study, with the exception of T₄ (120 %, CNB) which reached 8.7 leaves¹³. The same authors conclude that the application of Trichoderma in cocoa ecotypes presented effects with ideal agronomic characteristics, and that the native ecotype with a fine aroma with better results in terms of NH, adding that T. harzianum + CCN 51 obtained 100 % colonization in the root hairs. At 135 days, they also recorded higher averages (19 to 20 leaves per plant) when applying Trichoderma-based products¹². Therefore, the application of Trichoderma with the highest dose does influence a greater number of leaves and, therefore, a greater photosynthetic activity²⁸. As for the use of biostimulants or plants inoculated with Trichoderma, they also generate positive effects on the number of leaves in the cultivation of passion fruit and tomato in a protected environment^29-31.

Also, studies support that Trichoderma sp. significantly improves growth and development, influencing the leaf area^32,33. For the total and aboveground biomass variables, the combination of T. harzianum and compost in pine seedlings allows for higher and homogeneous values when compared to a control³⁴, considering that fertilizer offers better conditions for the development of the fungus.

The effect of Trichoderma spp. as a growth promoter is due to the fact that it increases the total surface area of the root system, enabling greater access to the mineral elements present or that they are capable of solubilizing and increasing the availability of phosphates, iron, copper, manganese and zinc, or other elements, as well as increasing the efficiency of the plant to use some nutrients such as nitrogen³⁵. Likewise, Trichoderma species are widely known as growth promoters, and that this trait is more specific to the isolate than to the species, with individual isolates showing varying degrees of plant specificity²⁵. They also indicate that growth promotion mechanisms include pathogen control, improvements in nutrient absorption, increased carbohydrate metabolism and photosynthesis, in addition to phytohormone synthesis.

The results obtained indicate an increase in AP, DT, NH and leaf area in relation to the controls in the CNB and IMC-67 genotypes with the application of T. harzianum at a dose of 120 %, support as an alternative that can contribute to a greater use of the genetic potential of CNB, improving the conditions of its propagation in nursery conditions. It is also highlighted that the CNB, characterized by presenting a slow growth to obtain seedlings compared to hybrid seedlings, obtained growth characteristics similar to the hybrid IMC-67, with the application of Trichoderma.