The biological materials used for the realization of this work consisted of the seeds of two (02) varieties of bean and (04) biostimulant products. Red bean mainly cultivar DOR-701 (small dark red bean commonly called Meringue) and white bean mainly cultivar MEX-142 (small white bean) collected at the Agricultural Research Institute for Development (IRAD) of the station multipurpose school in Foumbot, a town located in the Noun division, West region of Cameroon.

Biostimulant products in particular BS1, BS2, BS3 and BS4 based on natural products were provided by the BioTid Research Team of the Laboratory of Plant Physiology and Biochemistry (LPBV) of the Higher Teachers’ Training College of the University of Yaoundé 1.

NPK chemical fertilizer (20-10-10) was used as an amendment for the positive control treatment (T+) and was purchased at the Mokolo market in Yaoundé 2^nd district.

The plots were cleared manually and loosened using a pickaxe, then 12 blocks were formed using a hoe, the blocks were separated from each other by an interval of 60 cm. The experimental device was that of randomized blocks with 6 treatments and 3 repetitions, in non-sterile conditions. The treatments were:

T− = plot treated with water (negative control).

T+ = plot treated with NPK 20-10-10 chemical fertilizer (positive control).

F1 = plot treated with BS1 (based on Tithonia diversifolia + Emilia Coccinea).

F2 = plot treated with BS2 (based on Tithonia diversifolia + Clams).

F3 = plot treated with BS3 (based on Sonchus Oleraceus).

F4 = plot treated with BS4 (based on Urtica sp.).

The replicates were separated from each other by an interval of 25 cm.

A good quantity of seeds of the two varieties of beans were soaked in water 24 hours before sowing, in order to accelerate their germination. Seeds were sown manually using a dibbler and 5 seeds were sown in holes of 3 cm deep, at 25 cm intervals on the lines and between the lines. Treatments (biostimulants and chemical fertiliser) were carried out once a week and watering every two days. When the plants reached 15 to 20 cm in height, they were hoeed and the soil ridden up to the first two leaves; then every 30 cm, the oars were planted and angled inwards then were tied at the top. Thinning took place 14 days after sowing, and weeding took place 30 days after sowing using a hoe.

The effect of the various treatments on the development of the plants was evaluated by taking agronomic parameters every 7 days from the end of the second week after sowing and extended over the following 12 weeks until at plant maturity. The agromorphological measurements taken were leaf number, plant size, stem diameter, branching number, pH, sunshine, soil humidity and temperature. After flowering, the number of flowers and pods was counted every two days until maturation.

The maturation of the pods began on the 60^th day and on bean plants having reached maturity, a good quality of leaves harvested in the different modalities were sent to the Laboratory of Plant Physiology and Biochemistry (LPBV) of the Higher Teachers’ Training College of the University of Yaoundé 1 for the purpose of analyzes. While the harvested seeds were later sent to the Laboratory of Food Sciences and Metabolism (LabSAM) of the University of Yaoundé 1 for analysis purposes as well. For each variety, two types of samples were used, namely: treated raw beans and control raw beans. All analyses on samples were carried out in triplicate.

For each treatment 0.5 g of fresh bean leaves was used for sample’s analyses. The extraction and quantification of samples were carried out according to the method reported by [15] - [22] modified respectively for total chlorophylls (652 nm), total phenols content (760 nm), total proteins (595 nm), phenylalanine ammonia-lyase (290 nm), peroxidase (470 nm), polyphenol oxidases (330 nm). The total phenols were measured in mg per g of fresh weight while that of the total proteins concentration was expressed in mg equivalent (Eq) of bovine serum albumin (BSA) per g of fresh weight (FW). The phenylalanine ammonia-lyase, peroxidase and polyphenol oxidase specific activity were expressed in μmol/min/mL.

For each treatment a mass of 5 g fresh bean was used for sample’s analyses of the determination of water and dry matter content carried out according to the method reported by [23] , while a mass of 0.1 g of dry bean powder was used for sample’s analyses of the total nitrogen content that was determined after mineralization of the samples according to the Kjeldahl method [24] and monitoring of the assay by the method of [25] . The total amount of nitrogen was determined by the method [26] . The extraction and quantification of samples were carried out according to the method reported by [27] - [29] respectively for total lipids, total sugars, iron (Fe) and zinc (Zn) minerals. The dry matter and water content were expressed as percentage of fresh matter (g/100g FW), while the total protein content in the samples were expressed in g/100g dry matter (DM), with the conventional coefficient of conversion of nitrogen into protein used, i.e. 6.25 according to [26] . The total lipids and the total sugars were expressed in g/100g of DM, while the mineral content was expressed in mg/100g of dry matter.

Bean powder was used to determined the oxalates content (1 g), phytates content (2 g), tannins content (1 g) and saponins content (5 g) according to the method reported by [30] - [34] . The oxalates, phytates, tannins and saponins content were expressed in g/100g of dry matter.

The analysis of variance (ANOVA) was performed using Tukey’s test at the 5% probability threshold using XLSTAT software (version 21.0 for Windows) to compare the different modalities and make the graphic representations. A four-factor ANOVA (modality, variety, day and repetition) was carried out for the analysis of agromorphological parameters (diameter and height of the stem, number of leaves, branching, flowers, pods) and physicochemical characterization of substrates during cultivation, and to two factors (modality and variety) for the biochemical analysis of seeds (total proteins, total sugars, total lipids, iron, zinc, oxalates, phytates, tannins, saponins) and leaf extracts of bean (total chlorophyll, total phenols, total proteins, peroxidases, polyphenol oxidases and phenylalanine ammonia-lyases). The Principal Components Analyzes (PCA) were carried out using the Pearson correlation test.

Analysis of variance of agromorphological parameters of bean plants throughout this experiment revealed that the variable “modality”, and “day” (of taking the measurements) each had highly significant effects (P < 0.0001) on the plant responses (the height of the plant, the diameter of the stem, the number of leaves, the branching, the number of flowers, and the number of pods). The variable “variety” and the interaction “modality*variety” showed highly significant effects (P < 0.0001) on height, number of leaves, branching, number of flowers and number of pods; and the interaction “modality*variety*day” also showed a highly significant effect (P < 0.0001) on these parameters except for branching. The coefficient of determination (R²) for all the vegetative variables is close to 100% indicating that the model used is reliable and reproducible ( Table 1(A) and Table 1(B) ). Amongst these variables, the one that most influences plant growth in terms of height, diameter, number of leaves, branching, number of flowers is the variable “day”, while the variable “variety” is the most influential for the number of pods.

Values in bold correspond to tests so the null hypothesis is rejected with a significant level alpha = 0.05. DF is the degree of freedom; F is the F test value and P is the probability.

Plants treated with biostimulants (BS1, BS2, BS3 and BS4) showed higher growth in agromorphological parameters (height, stem diameter, number of leaves, branching, number of flowers, number of pods) compared to the negative control ( Figure 1 and Figure 2 ). However, the modalities that showed the most efficiency in terms of rapid increase in height, number of leaves, number of branches, number of flowers and pod were the BS2 and BS4 modalities compared with the positive control (T+) ( Figure 1 and Figure 2 ). The number of pods was three (03) times greater in the BS2 and BS4 modalities compared to the negative control (T−). A similarity was noted in the increase in stem diameter between the treated plants and the positive control ( Figure 1(B) ).

Regardless the modality, the number of leaves, branches, flowers and the number of pods were greater in red bean plants compared to white bean plants. However, the white bean plants were more important in terms of height compared to the red bean plants except for the negative control. The leaves and pods of plants treated with biostimulants (BS1, BS2, BS3 and BS4) were larger compared to the negative control ( Figure 2 ).

The analysis of variance (ANOVA) of the physicochemical characteristics of the substrates (pH, temperature, insolation and relative humidity) in the field during the growth of bean plants revealed that the variables “modality” and “day”, the interaction “modality*variety” each had a significant (P < 0.001) effect on soil pH and a highly significant (P < 0.0001) effect on field soil temperature ( Table 2 ). The coefficient of determination R² for all the variables is close to 100% indicating that the model is reliable and reproducible. Among these variables, the one that most influences soil pH was the interaction “modality*variety”, while the variable “day” has the most influence on soil temperature ( Table 2 ).

Low insolation (Low^-) and high humidity (Wet⁺) relative to the soil in the field were recorded throughout the experiment. There exists a significant difference variation in soil temperature and pH between the treated modalities and the control modalities ( Figure 3 ). Overall, the modalities (BS1, BS2, BS3 and BS4) improve the soil temperature and pH more than the controls.

Table 2. Analysis of the variance of the evolution of the average pH and average temperature of the bean growth substrate during the experiment.

Values in bold correspond to tests so the null hypothesis is rejected with a significant level alpha = 0.05. DF is the degree of freedom; F is the F test value and P is the probability.

Analysis of variance of biomarkers accumulation in bean leaves revealed that overall, the variables “modality” and/or “variety” each had a highly significant effect (P < 0.0001) on the content of total chlorophylls, total phenols, total proteins, polyphenol oxidases, peroxidases and phenylalanine ammonia-lyases. The coefficient of determination R² for all the variables was between 89% and 100%, indicating thus that the model used was reliable and reproducible ( Table 3 ). There was a highly significant effect of the interaction “modality*variety” (P < 0.0001) on the accumulation of total chlorophylls, total phenols and defence-related enzymes. Amongst the two variables (“modality” and “variety”), the one that most influences the accumulation of total chlorophylls, peroxidases and phenylalanine ammonia-lyases was the “modality”, while the variable “variety” has the most influence on the accumulation of total phenols, total proteins and polyphenol oxidases.

Values in bold correspond to tests so the null hypothesis is rejected with a significant level alpha = 0.05. DF is the degree of freedom; F is the F test value and P is the probability.

The biostimulant modalities (BS1, BS2, BS3 and BS4) improve very significantly overall the accumulation of total chlorophylls, total phenols, total proteins, polyphenol oxidases (PPO), peroxidases (POX) and phenylalanine ammonia-lyase (PAL) in plants compared to controls. The positive control modality (T+) improves the accumulation in these biomarkers compared to the negative control modality (T−) ( Figure 4 and Figure 5 ). The accumulation of these biomarkers varies from one variety to another. The best accumulation in these biomarkers was obtained with the BS4 modality for the white variety and the BS3 modality for the red variety. The total phenol content is about 5 times higher in the BS4 and BS1 modalities compared to the negative control (T−) of the white variety and 2 times higher in this modality compared to the positive control (T+) of the white variety ( Figure 4(B) ).

Overall, the PPO, POX and PAL contents were 2 to 3 times higher in the BS3 modality of the red variety compared to the controls (T+ and T−) and 3 to 4 times higher in the BS4 modality of the white variety compared to the controls (T+ and T−) ( Figure 5(A)-(C) ). In the white bean, we can observe a better accumulation in total phenols, total proteins, PPO, POX and PAL compared to the red bean.

The effect of biostimulants on plant growth and seed filling was found to be very significant compared to controls. Indeed, the appearance of the seeds (size, shape and color) was different depending on the method of treatment applied during growth. The biostimulant BS2 and BS4 were those which presented the best seeds respectively for the white and red bean. The yield was also improved at the end of the culture with a better yield for the reds treated with BS4, while in the whites it was BS2 ( Table 4 ).

The analysis of variance (ANOVA) of macronutrients, minerals, and antinutrients accumulation in bean seeds showed that the variables “modality” and “variety” each had a highly significant effect (P < 0.0001) on the content of dry matter, water, proteins, lipids, total sugars, iron, zinc, oxalates, phytates, tannins, and saponins. The coefficient of determination R² for all the variables was between 96% and 100%, indicating thus that the model used is reliable and reproducible ( Table 5 ). Amongst these variables (“modality” and “variety”), the one that most influences the accumulation of macromolecules (proteins, lipids and total sugars), minerals (iron and zinc), phytates and saponins was the variable “modality”, while that which most influences the accumulation of dry matter, water, oxalates and tannins was the variable “variety”.

Values in bold correspond to tests so the null hypothesis is rejected with a significant level alpha = 0.05. DF is the degree of freedom; F is the F test value and P is the probability.

The dry matter content in red bean seeds treated with BS3 and BS4 was higher compared to the controls (T+ and T−) and to the BS1 and BS2 treatments. However, regardless of the modality, the dry matter and water content were the same in white bean seeds. There was no significant difference between the positive control modality (T+) and the negative control modality (T−) ( Table 6 ). Red bean presents a better dry matter accumulation compared to white bean, while water accumulation was higher in white bean compared to red bean regardless the modality.

The superscript values of different letters in the same line are significantly different (P < 0.05).

The accumulation of total proteins, lipids and sugars in bean seeds treated with biostimulants (BS1, BS2, BS3 and BS4) was greater compared to controls. The positive control modality (T+) improves the accumulation of these macromolecules more than the negative control modality (T−) ( Table 7 ). Proteins content nearly doubled in BS3 and BS4 treated bean seeds compared to controls. While the lipids content was higher in bean seeds treated with BS1 compared to controls. The accumulation of total sugars was twice as high in the bean seeds treated with BS3 and BS4 compared to the controls ( Table 7 ). The white bean has a higher content of total proteins and lipids in the seeds compared to the red bean. On the other hand, the total sugars content was higher in red beans compared to white beans ( Table 7 ). Overall, BS3 and BS4 were the modalities that accumulated more macromolecules in bean seeds compared to BS1 and BS2 modalities and control modalities.

The superscript values of different letters in the same line are significantly different (P < 0.05).

Bean plants treated with biostimulants (BS1, BS2, BS3 and BS4) significantly improve minerals accumulation (iron and zinc) in bean seeds. However, the BS3 modality increases the accumulation of these minerals much more compared to the controls. The iron content in bean seeds treated with BS3 was approximately twice that of the negative control. The positive control modality (T+) improves the accumulation of these minerals more than the negative control modality (T−) ( Table 8 ). The mineral content (iron and zinc) also varies from variety to variety. It was higher in white beans compared to red beans.

Table 8. Effect of biostimulants (BS1, BS2, BS3 and BS4) on the accumulation of Iron (A) and Zn (B) in bean seeds of three months old white bean (White) and red bean (Red) compare to the negative (T−) and positive (T+) controls modalities.

The superscript values of different letters in the same line are significantly different (P < 0.05).

Globally, the positive control modality (T+) increases highly significantly accumulation of antinutrients (oxalates, phytates, tannins and saponins) in bean seeds compared to biostimulant modalities (BS1, BS2, BS3 and BS1). The accumulation of these antinutrients differs from variety to variety. The phytates content was approximately twice as high in the seeds of the positive control compared to the seeds of BS2, while the tannin content was three times higher in the positive control compared to BS4 and twice as high compared to BS3. The saponin content was half as high in the bean seeds treated with BS2 compared to the positive control. The positive control modality (T+) also increases highly significantly the accumulation in these antinutrients compared to the negative control modality (T−) ( Table 9 ). The content of oxalates, tannins, and saponins was higher in red beans compared to white beans, while the phytates content was higher in white beans compared to red beans ( Table 9 ). However, regardless the modality, the seeds of the positive control (T+) are those which present more accumulation in these antinutrients (oxalates, phytates, tannins and saponins).

The superscript values of different letters in the same line are significantly different (P< 0.05).

The white bean Principal component analysis (PCA) shows that the first two principal components explain respectively 51.42% and 29.00% of the total variance and define a plan allowing more than 80.43% of the variabilities to be taken into account ( Figure 6(A) ). Macromolecules (proteins, lipids and total sugars) are strongly positively correlated with each other as well as with BS1 and BS2. Iron is strongly positively correlated with BS1 and moderately with zinc and dry matter. Tannins and oxalates are strongly negatively correlated with the positive control (T+) and inversely negatively correlated with phytates and saponins. The water content is strongly negatively correlated with the negative control (T−). The positive control modality (T+) accumulates more antinutrients and the modality (BS4 and negative control T−) accumulates them less. The dissimilarity of nutrients and antinutrients of white beans in the different modalities shows that nutrients are accumulated more in BS2 and BS3 modalities, followed by BS1 ( Figure 6(B) ). The red beans PCA shows that the first two principal components explain respectively 53.26% and 26.09% of the total variance and define a plan allowing more than 79.34% of the variabilities to be taken into account ( Figure 7(A) ). There is a strong positive correlation between the contents of lipids, dry matter, proteins and total sugars as well as the BS1 and BS2 modalities. Iron is moderately positively correlated with macromolecules (lipids, proteins, total sugars) and dry matter. Tannins and oxalates are strongly negatively correlated with each other and with the positive control (T+). The water content is strongly negatively correlated with the negative control and inversely negatively correlated with the saponins, phytates and zinc contents. The control modalities (T+ and T−) accumulate them less compared to BS3 and BS4. The accumulation of antinutrients such as tannins and saponins is greater in the positive control modality. The dissimilarity of nutrients and antinutrients of red beans in the different modalities shows that nutrients are accumulated more in BS1 and BS2 modalities ( Figure 7(B) ).

Principal component analysis (PCA) of all white and red bean agromorphological responses (plant height, stem diameter, number of leaves, branches, number of flowers, number of pods, total chlorophylls, total phenols, total proteins, PPO, POX, PAL) as well as the physicochemical parameters of the substrates during plant growth (pH and temperature) and the content of macromolecules, minerals and antinutrients in the seeds of these plants revealed that there is a strong negative correlation between growth biomarkers (total chlorophylls, total phenols, total proteins) and defense biomarkers (POX, PAL, PPO) in white bean leaves ( Figure 8(A) ). Moreover, they are strongly negatively correlated with plant height, plant diameters and the content of minerals (iron and zinc), proteins, lipids, water in seeds and modalities BS1, BS2 and BS3. A strong positive correlation was observed between the contents of antinutrients (saponins, oxalates and tannins) and between agromorphological parameters (number of leaves, pods, branches, flowers). The total sugars content of the seeds is weakly positively correlated with the dry matter content and the BS2 and BS3 modalities of the white variety. Physicochemical parameters (pH and soil temperature) are weakly positively correlated with biomarkers of white bean growth and defense.

The dissimilarity of parameters (agromorphological and physicochemical), growth and defense biomarkers, as well as dry matter, water, minerals and antinutrient contents of white and red beans shows that in red beans, BS1 and BS2 are the best modalities followed by BS3, BS4 and finally the positive control (T+) which accumulates more antinutrients ( Figure 8(B) ). Whereas for white beans, BS2 and BS3 are the best modalities followed by BS1 and BS4 and finally the positive control (T+).