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Article

Phenology, Nitrogen Status, and Yield of Red Clover (Trifolium pretense L.) Affected by Application of Vitamin B12, Humic Acid, and Enriched Biochar

by
Dorna Saadat
,
Arthur Siller
and
Masoud Hashemi
*
Stockbridge School of Agriculture, University of Massachusetts, Amherst, MA 01003, USA
*
Author to whom correspondence should be addressed.
Agronomy 2023, 13(12), 2885; https://doi.org/10.3390/agronomy13122885
Submission received: 3 November 2023 / Revised: 20 November 2023 / Accepted: 22 November 2023 / Published: 24 November 2023
(This article belongs to the Special Issue Effects of Cover Crops, Crop Rotation, and Intercropping on Natural Soil Fertility)
Browse Figures
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Abstract

:
Studies on vitamin B12’s influence on the flowering and yield parameters of red clover (Trifolium pretense L.) are not well documented. A greenhouse experiment investigated the effect of the solo and combined application of vitamin B12 with humic acid and enriched biochar on the morphology, nitrogen status, and biomass yield of the shoots and roots of red clovers. Two levels of vitamin B12 (0, 20 mg pot−1) mixed with four growing media were laid out as a randomized complete block design. The results indicated that vitamin B12 markedly led to (A) a decrease in the flowering time by 5 days (100.2 to 95.9 days), while the co-application of B12 and humic acid resulted in further reduction (84.5 days); (B) an increased total number of stems (73.0 to 78.6 plant−1); (C) a boost in the root dry weight by 60% (3.8 to 6.4 g) while having no significant influence on the shoot dry weight; (D) a decrease in the leaf trichome density by 30% (49.0 to 35.0 plant−1); (E) a rise in the N content of the roots (107.8 to 173.3 mg plant−1), while having no influence on shoot N content. biochar’s influence on the phenology and productivity of red clovers was minimal. The results highlighted the importance of the application of manure, which is naturally rich in vitamin B12 and humic acid, to forage crops, including red clover.

1. Introduction

Soil is a cornerstone of food, feed, and fiber production, and ninety-five percent of our food comes from soil [1,2]. The concept of soil health is defined as the continued capacity of soil to function as a vital living ecosystem that sustains plants, animals, and humans (https://www.nrcs.usda.gov/wps/portal/nrcs/main/soils/health/ accessed on 2 November 2023). Non-sustainable agricultural practices, including the use of synthetic fertilizers [3,4], lead to a loss of soil organic matter (SOM), subvert the soil ecology, and negatively impact soil health [5,6,7]. Rhizosphere management strategies, including nutrient-rich rhizosphere (i.e., amending soil with organic matter or biostimulants), can directly contribute to the soil nutrient pool and minimize the reliance on excessive chemical input, especially in intensive livestock farming systems [8,9].
Red clover (Trifolium pretense L.) is a short-lived perennial forage crop and has been widely cultivated as a monoculture or more commonly mixed with grasses as hay and pasture in cool climates, including Europe and North America [10,11]. T. pretense is native to Europe, but currently, it acclimatizes to various edaphic and climate conditions. It is now considered the second-most important forage legume after alfalfa in terms of cultivation area in the world [12,13]. Abiotic factors, such as soil amendment and bio-stimulants, can play a key role in fostering symbiotic N2 fixation and improving crop productivity [14,15]. Generally, the quality of forages (grasses and legumes) declines as plants reach the flowering stage. Therefore, accelerating flowering time is considered positive and may provide an opportunity for increasing the frequency of harvesting.
Vitamin B12, cyanocobalamin, is a member of the corrinoids and is considered a comparatively large molecule (1355.38 g mol−1). In a natural habitat, field soil is a rich source of vitamin B12 (estimated concentration of 15 g kg−1), where it is synthesized by specific bacteria and archaeon [16,17,18] and absorbed by plant roots [19,20]. Reports have revealed that the application of cyanocobalamin as a biostimulant may result in promoting plant yield and indirectly plant metabolism by enhancing rhizobia activity and developing plant growth, including leaf area, shoot, and root development [21,22,23].
Humic substances, including humic acids, emanating from the biochemical degradation of living organisms impose a significant positive influence on plant growth and development [24,25,26], especially root proliferation and the formation of lateral roots [27,28]. Moreover, humic acid could facilitate the absorption of micro- and macro-nutrients by buffering pH, chelating unavailable nutrients, and increasing plant biomass [29,30,31]. Analogous research has declared that the application of humic compounds to the aerial part of plants stimulated early flowering [32], increased leaf area [33], and photosynthetic pigments by enhancing nitrogen absorption or light acquisition [24,34,35].
Biochar, as a soil amendment, can improve all aspects of soil health, including physical, chemical, and biological characteristics [36,37,38]. Some studies reported a neutral or even counter-productive influence of biochar on plant growth (i.e., it acts as a nutrient competitor, adsorbs nitrogen, or disturbs the chemical communication between plants and soil biota) [39,40,41,42]. However, generally, research has revealed a positive effect of biochar on plant growth by promoting root systems [43], net photosynthesis rates [44], and shoot growth [45], thus enhancing overall plant productivity [46,47]. Specifically, in leguminous families, biochar has altered plant yield [48], nodulation rates [46,49], nitrogen fixation [50], and the population of N-fixing bacteria [51].
Since high crop productivity with “high input, high output” contributes to the farming system for higher yields, the aim of this research was first to direct attention towards soil health and evaluate the effect of vitamin B12 as a biostimulant on the yield parameters of red clover, especially when it is integrated with different growing media. Forage crops are of optimum quality when they are managed to be in a shorter reproductive stage. For example, in the case of legumes, the time of cutting should be as soon as they start to flower [52]. Therefore, the current study aimed to evaluate the time of flowering as an important agronomic factor. Although the effect of vitamin B12 on some plant species has been reported, the earlier focus has been mainly on the vegetative stage of plants [21,22,23]. There is a paucity of studies addressing the effect of the exogenous solo or combined application of vitamin B12 on the reproductive stage of plants and whole-plant biomass yield.

2. Materials and Methods

A greenhouse experiment was conducted in 2022 at the University of Massachusetts, Amherst, College of Natural Science Greenhouse to investigate the effect of biostimulants (humic acid and vitamin B12 and biochar enriched with compost) on the morphology, physiology, and root and shoot biomass of red clover. The greenhouse conditions were set at a 12:12 (L:D) photoperiod; a day temperature of 21 ± 2 °C; a night temperature of 18 ± 2 °C; and a 55 ± 5% relative humidity.
Growing media and treatments: Marathon red clover (T. pretense L.) was grown for two weeks before being transplanted into the main experimental pots. In this experiment, we used transparent plastic pots to observe the rooting system expansion. The dimensions of the pots are illustrated in Figure 1. The two growing media used in this experiment were potting soil (Promix BX) fortified with mycorrhizae (1 kg pot−1 total weight) and field soil (6 kg pot−1 total weight), collected from the University of Massachusetts, research farm. The selected physical and chemical characteristics of the field soil used in treatments 7 and 8 are presented in Table 1. The potting soil was mixed thoroughly with each treatment, including humic acid and sugar maple hardwood biochar enriched with compost, using a blending machine for 10 min. A schematic drawing of the experimental treatments is presented in Figure 1. The treatments consisted of the following:
  • Potting soil + Biochar-compost + (Plus) Vitamin B12
  • Potting soil + Biochar-compost − (Without)Vitamin B12
  • Potting soil + Humid acid + Vitamin B12
  • Potting soil + Humic acid − Vitamin B12
  • Potting soil + Vitamin B12
  • Potting soil − Vitamin B12
  • Field soil + Vitamin B12
  • Field soil − Vitamin B12
The biochar treatment consisted of 8% of the total weight of the growing media. The biochar in this study was made of hardwood sugar maple. Commercially purchased humic acid was a granular sodium salt made by Sigma-Aldrich (CAS-No 68131-04-4, Saint Louis, MO, USA). It was dissolved in water at a rate of 300 mg lit−1 and applied to the growing media. The commercially purchased vitamin B12 (95.0%) was a crystal powdered formula (Catalog number: C04491G, TCI America, Portland, OR, USA) and applied at a rate of 20 mg per potting soil container (based on 1 kg pot−1 total weight) and 120 mg pot−1 of field soil container (based on 6 kg pot−1 total weight). Vitamin B12 was dissolved in distilled water and applied at three time frames, including post-transplant, three, and six weeks after transplanting. This study was performed under unsterilized conditions.
Measurements: Morphological parameters: The total number of stems, flowers, trichome thickness and density, and flowering time were recorded just prior to the clover harvest. The non-glandular trichome density on the leaf margin images was captured using a stereo microscope (Nikon SMZ800N, Tokyo, Japan). A cohort of leaves was selected by choosing the middle leaf of the trifoliate (7-week-old leaves) of the second node from the apex for all three plants in a pot. Trichome numbers were counted in each specific area of the virtual ruler on images under the microscope to keep uniformity. The flowering time was recorded considering the time from planting date to full bloom.
Nitrogen status and yield parameters: The nitrogen status of plants was measured seven weeks after transplanting using an SPAD meter (The Soil Plant Analysis Development chlorophyll meter by SPAD-502 Plus, Konica Minolta, Tokyo, Japan). For SPAD readings, a cohort of leaves was chosen by considering the terminal leaves (7-week-old leaves) of the second node from the apex for all three plants in a pot. Clovers were harvested 100 days after transplanting for yield determination. The harvested time was selected based on the early flowering stage, which is a trade-off between forage quality and biomass. Harvested plants were divided into roots and above-ground parts, including stems, leaves, and flowers. Plant parts were oven-dried at 60 °C individually to a constant weight and ground into a fine powder (1 mm screen) by Foss Cyclotec 1093, Hilleroed, Denmark, and analyzed for the total N content using the Kjeldahl procedure [53].
Statistical analyses: All quantitative data were compared by an analysis of variance (ANOVA) and proc mixed using SAS studio (SAS Institute Inc., Cary, NC, USA). The main effects consisted of the growing media (four treatments) and vitamin B12 (two levels). Each treatment consisted of three biological observations and four replications (12 plants in total). The experiment was laid out as a randomized complete design. The test of significance for the mean separation was analyzed using Tukey’s test at p  ≤  0.05 and sliced for growing media and vitamin B12 application.

3. Results

3.1. The Effect of Vitamin B12 and Soil Amendments on the Phenological Parameters

3.1.1. Flowering Time and Number

The vitamin B12 application markedly impacted the flowering time. In the absence of vitamin B12, it took the red clover plants approximately 100 days to reach the flowering stage, while in the presence of vitamin B12, the flowering time was reduced by an average of 5 days (p < 0.001) (Table 2).
The clovers grown in the field soil did not produce any flowers by the harvesting time (110 days after planting) (p < 0.001) (Table 3). While the application of enriched biochar to the potting soil had no significant effect on flowering time (96.9 days), the addition of humic acid to the potting soil reduced the flowering time by approximately six days (90.6 days). Interestingly, vitamin B12 and humic acid demonstrated a synergistic effect in the presence of both; the flowering time was reduced by an additional five days (84.5 days) (p < 0.001) (Figure 2). As mentioned above, the clovers grown in the field soil (treatments 7 and 8), despite producing buds, did not reach the blooming stage by the harvesting time (Figure 2).

3.1.2. Total Number of Flowers and Buds

The application of vitamin B12 resulted in approximately 30% more flowers and buds (albeit non-significant) (Table 2). Similarly, amending soil with either humic acid or enriched biochar had no significant effect on the total number of flowers and buds at the harvest time (Table 3). The clovers in the potting soil produced on average of 9.9 flowers per plant. Although this was not statistically significant, the plants that received humic acid produced 11.2 flowers, whereas those that were amended with enriched biochar produced only 6.2 flowers per plant. As mentioned above, the plants grown in the field soil, whether they received vitamin B12 or not, did not flower by the time of harvest. Unlike the flowering time, no significant effect (p > 0.05) was detected for the interactive effect of vitamin B12 and the growing media on the flower number.

3.1.3. Trichome Density and Flower Cluster

An interesting result obtained in this study was the observation of the influence of vitamin B12 on the non-glandular trichome density on the leaf margin of the red clover and also the appearance of flower clusters. In general, the application of vitamin B12 reduced the density of the trichomes (Figure 3). Based on our measurement, in the presence of vitamin B12, the clover leaves had approximately 30% fewer trichomes compared with those that did not receive vitamin B12 (Table 2).
Vitamin B12 also influenced the cluster and the pigmentation (color) of the clover flowers grown in the potting soil + humic acid growing media so that the flowers bloomed with less-dense clusters and a pale pink color (Figure 4).

3.1.4. Total Number of Stems

The application of vitamin B12 promoted tillering; thus, the clovers produced more stems (78.6) compared to the clovers grown without vitamin B12 (73.0) (Table 2) (p < 0.05). Also, the number of stems per plant was influenced significantly by the growing media. Across the four growing media, the clovers grown in field soil produced the minimum stem number (45.0) (p < 0.05), whereas the maximum stem number was produced in the clovers grown in potting soil alone (94.1) (Table 3). The interactive effect of vitamin B12 and the growing media was not statistically significant (p > 0.05).

3.2. The Effect of Vitamin B12 and Growing Media on the Yield Parameters of Red Clover

3.2.1. Buds and Flowers Dry Matter

Despite the considerable difference in the flowers’ and buds’ dry weights between the plants that received vitamin B12 (0.96 g plant−1) and those that did not (0.55 g plant−1), this difference was not statistically significant (Table 2). The interactive effects of vitamin B12 and the growing media on the biomass yield of the flowers and buds differed significantly (p < 0.05) (Figure 5). With the co-application of humic acid and vitamin B12, the flower dry mass increased dramatically (Figure 5). In contrast, amending the potting soil with enriched biochar considerably reduced the flower dry weight, mainly due to the production of fewer flowers, as reported above (Table 3). The highest flower dry matter was obtained from the pots amended with humic acid (1.57 g), and the lowest dry matter pertained to the clovers grown on enriched biochar (1.30 g). As mentioned earlier, no flower growth was initiated in the field soil by the time of harvest.

3.2.2. Root Dry Matter

The addition of vitamin B12 to the growing media resulted in approximately 60% more root biomass (Table 2). Similarly, the growing media influenced the root dry matter significantly (p < 0.05). The plants in potting soil + humic acid produced the highest root dry matter (6.2 g plant−1) compared with potting soil alone or the field soil (4.1 and 4.2 g plant−1, respectively) (Table 3). Figure 6 demonstrates the root condition at the harvesting time when grown in different treatments. The interaction of between the growing medium and vitamin B12 was not statistically significant.
Although not statistically significant, the plants that received humic acid produced 11.2 flowers, whereas those that were amended with enriched biochar produced only 6.2 flowers per plant. As mentioned above, the plants grown in the field soil, whether they received vitamin B12 or not, flowered by the time of harvest. Unlike the flowering time, no significant effect (p > 0.05) was detected for the interactive effect of vitamin B12 and the growing media on the flower number.

3.2.3. Shoot Dry Matter

The application of vitamin B12 to the red clovers resulted in no significant influence on the shoot dry weight (p < 0.05) (Table 2). However, the growing media treatment affected the shoot dry weight significantly (Table 3). The shoot dry weight of the three potting soil treatments ranged between 24.0 g plant−1 (no amendment) and 27.2 g plant−1 in the potting soil amended with humic acid.
As mentioned earlier, the root dry weight of the plants that received vitamin B12 was considerably higher than that of those without vitamin B12 (Table 2). As a result, the root: shoot ratio in the presence and absence of vitamin B12 varied significantly and was 30% and 18%, respectively. Our data indicated that the influence of vitamin B12 on clover biomass is primarily focused on the roots rather than the shoots. Similar to vitamin B12, adding humic acid and enriched biochar enhanced the root dry weight rather than the shoot dry weight (Table 3). Therefore, while the root: shoot ratio in the amended potting soil (either with humic acid or enriched biochar) was 23%, the ratio in the potting soil alone was only 17%. In the current study, the plants grown in the field soil performed poorly. These plants not only did not produce flowers at the harvesting time, but their shoot and root dry weights were also considerably lower than those of the plants grown in potting soil.

3.3. The Effect of Soil Amendment and Vitamin B12 on the N-Status of Red Clover

3.3.1. SPAD Value

The application of vitamin B12 did not influence the N status of the leaves (Table 2). The measured SPAD values, as an indicator of the N status of the leaves with and without vitamin B12, were almost similar. However, amending the growing media with humic acid and enriched biochar to some degree increased the SPAD values (Table 3). The highest SPAD values were recorded for the clover leaves that received enriched biochar (45.2), followed by those that received humic acid (44.9) (albeit with no significant differences). The readings for the potting soil alone were approximately 10% lower than those for the amended treatments. Figure 7 exhibits the interactive effect of the vitamin B12 application and the growing media. The interaction was significant only with potting soil alone (no amendment) and the field soil treatment. In other words, amending the potting soil with either humic acid or enriched biochar did not boost the influence of vitamin B12 on the SPAD values.

3.3.2. Nitrogen Content

In addition to the SPAD readings, the nitrogen concentration of the clovers was also evaluated by measuring N in the roots and shoot. The nitrogen concentration in the roots and shoots of the red clovers was not affected by either the application of vitamin B12 or the growing medium (Table 2 and Table 3). The interactive effects of vitamin B12 and the soil amendments were also insignificant (p < 0.05).
Overall, the nitrogen concentration of the roots was approximately 18% lower than the nitrogen concentration of the shoots. Although the differences between the nitrogen concentrations in the roots and shoots of the different treatments were not statistically significant, the nitrogen yields (N concentration × dry matter) of the roots and shoots and the total (root + shoot) nitrogen yield were considerably different. The clovers amended by humic acid had considerably higher root, shoot, and total nitrogen yields (1145.9 mg plant−1, followed by the plants amended by enriched biochar (949.8 mg plant−1). The difference in the nitrogen yields of the clovers grown in potting soil alone compared with the humic acid and enriched biochar treatments was 21% and 5%, respectively (Table 3).

4. Discussion

The enhancement effect of various soil amendments on soil health and crop productivity has been extensively reported [23]. However, there is still a gap in information about the influence of vitamin B12, humic acid, and biochar on forage crops, more specifically, the morphology, nitrogen status, and productivity of red clover.
Phenological parameters: The results of the current study denoted that the growing medium may induce a remarkable effect on the flowering time. Indeed, de novo flowering can be critical for domestication, geographical expansion, genetic research [54], and yield improvement [55]. In many crops, delaying or accelerating the flowering time can improve the crop yield by protecting the plant’s specific susceptible growth stage from being exposed to certain biotic and abiotic stresses such as frost, temperature fluctuations, diseases, and drought [56,57,58]. In some vegetable crops, the avoidance of flowering can be an imperative goal [59]. In grass and legume forages, however, the appearance of flowers coincides with a decline in their nutritional values, including an increase in their fiber content and a decrease in their protein level [60]. Floral initiation can be altered by both internal cues (i.e., genetics and nutrition, hormonal induction) and external factors such as photoperiod, temperature, and vernalization [61,62,63] to regulate flowering locus T (FT), a vital signal sent from leaves to the shoot apical meristem to change a floral transition [64,65].
The results of the current experiment indicated that when humic acid was added to growing media, the flowering time was reduced by approximately 4 days. This reduction may be attributed to a humic-substance-promoting effect on root growth [32,66], which can lead to the uptake of more nutrients [67] and subsequently accelerate the growth period, thus shortening the transition from the vegetative to the reproductive stage. Additionally, humic acid can increase the nitrogen content, which, in turn, can enhance the photosynthesis rate [68], therefore reducing the vegetative period [66,69]. Also, humic compounds may improve cell membrane permeability [69] or induce hormone-like activity (i.e., auxins and gibberline), which can affect and regulate FT transcription and modulate flowering [61,68].
Although in all growing media, humic acid could initiate the flowering time 4 days earlier (90.6 days), the co-application of vitamin B12 with humic acid shortened the flowering time by an extra six days (84.5 days). While there is a paucity of studies reporting the effect of vitamin B12 on flowering time, the results of the current study demonstrated a synergistic effect of vitamin B12 and humic acid. Perhaps one of the contributing factors to the vitamin B12 effect could be attributed to the role of cobalt. Cobalt is the vital core element of vitamin B12 and an indispensable part of many enzymes and coenzymes, and its activity can promote atmospheric nitrogen fixation by promoting nodulation in legumes [70]. Consequently, cobalt can raise the N concentration in roots; thus, plants reach their optimum N level through the upregulation of miR172c levels. An increase in or overexpression of mir172c levels in leaves encourages the degradation of the transcripts of GmTOE4a, which can result in floral initiation [71,72].
In a multiple-cutting forage system, earlier flowering may provide an opportunity to have an extra harvest, which enhances growers’ income, especially in short-growing-season areas. The longest flowering time was observed in the potting soil amended with enriched biochar. The results confirm an earlier study that concluded that biota from biochar-amended soil was less beneficial to the growth of legumes [42]. On the contrary, some evidence proved that biochar’s effect on plant growth depended on the rate of its application. While biochar can simulate flowering at low doses, it may inhibit flower formation at higher doses [73,74,75]. In the current study, we used 8% biochar in potting soil, which should be considered a high application rate. Research has also indicated that an unknown effect of biochar on flowering could be ascribed to the type of biochar feedstock or pyrolysis temperatures [76,77].
In all the growing media, excluding the field soil (with no flowers at harvesting time), the application of vitamin B12 did not induce any significant effect on the number and dry weight of flowers. Surprisingly, the interaction of vitamin B12 and the growing media was not significant for the number of flowers but was significant for the flowers’ and buds’ dry weights. Perhaps these differences are a result of the individual weights (sizes) of the flowers, which were greater.
As a part of the vegetative stage, the clover developmental stage starts as a rosette consisting of many branches. The significant increase in the stem number of the clovers that received vitamin B12, without any significant difference in shoot dry weight, may be attributed to many factors, such as the stem length, leaf area, number of leaves, and weight per unit area [52].
The function of non-glandular trichomes in plants is primarily referred to as a structure for reducing water loss and deterring predators [78,79]. One notable result of this research, which is being reported for the first time, was that vitamin B12 altered the trichome density. In the presence of vitamin B12, the trichome density was abated, while in the absence of vitamin B12, the trichome density was increased. Previous evidence has suggested that a higher trichome density was the result of insufficient nitrogen [80,81]. Probably, in our clover study, a lack of vitamin B12 led to a lower N input into roots and thus formed a greater number of trichomes. Another explanation could be related to the interplay between the trichome density and the leaves’ maturity stage. The trichome density is higher in younger leaves and progressively diminishes as a leaf matures [79]. Since the leaves in the current study were a cohort, the formation of higher trichome numbers in the clovers without vitamin B12 could be partly due to a delay in reaching the maturity stage compared to the clovers that received vitamin B12 and were able to reach the maturity stage sooner.
N-status: In the current study, we evaluated the nitrogen status of clovers in two ways. Although vitamin B12, enriched biochar, and humic acid did not significantly change the SPAD value, the interaction between vitamin B12 and the growing media was significant. Yun and Collogues (2023) denoted that when all plants flowered simultaneously, they showed similar SPAD values [71]. Possibly, in our study, the SPAD value showed differences because the plants flowered at different times. The measured nitrogen concentration in the roots and shoots confirmed the measured SPAD values. The total N yield (nitrogen concentration × dry matter) of both the shoots and roots improved with the application of vitamin B12. The effect of vitamin B12 was more pronounced on the total N yield of the roots than that of the shoots. For example, the nitrogen yields of the clover roots in the presence and absence of vitamin B12 were 173.3 and 107.8 mg plant−1, respectively. The impact of the soil amendment on the nitrogen yield of the roots and shoots was even greater than that of the application of vitamin B12. Humic acid had the highest influence on the root and shoot nitrogen yields (173.0 and 972.9 mg plant−1, respectively), followed by enriched biochar, with 152.0 and 949.8 mg plant−1. The influence of the soil additives and vitamin B12 on the root and shoot biomass was primarily due to their effect on biomass rather than the N contents in these two organs.
Yield and growth-related factors: Overall, the clovers in the amended potting soil produced more shoot biomass than those in the field soil. The field soil used in this study was acidic and deficient in most macro- and micronutrients. We were interested to see if the application of vitamin B12 could compensate for low soil fertility, which, as it turned out, was not the case. The application of vitamin B12 to the potting soil increased the root biomass by approximately 60% but did not influence the yield of the aerial parts. Although amending the soil with humic acid and enriched biochar improved the root biomass, the differences between the treatments were not statistically significant. The results partially agree with earlier studies [14,68], which found that the positive effect of humic acid was more pronounced on roots than shoots. Liu and colleagues (1998) concluded that amending soil with a humic compound stimulated proliferation, branching, and the initiation of root hair growth [82]. Thus, the root dry weight and nutrient uptake were improved [83,84]. Further studies may shed light on the exogenous and endogenous factors that can accentuate the effect of humic acid on root growth and development via auxins as a primary dominant signal in promoting the mitotic activity of pericyclic cells in the process of primordia initiation [85,86,87]. An earlier report discovered that humic acid could trigger lateral root formation faster than auxins in 24 h [88]. On the contrary, amending the potting soil with enriched biochar had no influence on the root dry matter. There is considerable heterogeneity regarding the influence of biochar on plants’ productivity. While some research proclaimed that biochar application resulted in yield improvement [45,89], other reports propounded a null or negative response of plants’ biomass when amended with biochar [90,91,92]. Nitrogen immobilization [93] or species-specific phytotoxicity released from fresh biochar [94] have been mentioned to justify the null or negative growth responses to biochar amendment. Moreover, biochar application may alter soil microbiota [42,51], resulting in a negative plant response. Interestingly, the co-application of vitamin B12 with enriched biochar did not compensate for biochar’s null or negative effect on both the root and shoot growth. However, the co-application of vitamin B12 and humic acid enhanced the root growth.
This study was focused on the effect of vitamin B12 on some phenological, morphological, and biomass traits. Some further parameters and aspects, such as the rhizosphere–root chemical interaction, must be considered for future research. Additionally, the timing of application pre/post-planting could produce additional results.

5. Conclusions

Strategies to improve soil organic matter and the application of biostimulants enhance soil health through rhizosphere modification and contribute to the soil nutrient pool and nutrient accessibility. The results of the current study support the hypothesis that the presence of vitamin B12 can positively change the phenology, nitrogen yield, and biomass of roots and shoots. More specifically, the results of the current study demonstrated the importance of the application of manure, which is naturally rich in vitamin B12 and humic acid, to forage crops, including red clovers. Additionally, the presence of humic acid in manure or its application as a biostimulant can improve multiple characteristics of clovers, including the earlier flower initiation and total nitrogen yield of both roots and shoots. The influence of biochar amendments on the phonology and productivity of the clovers was minimal, partly caused by the short duration of the study.

Author Contributions

Conceptualization, D.S.; data curation, D.S. and A.S.; formal analysis, D.S.; funding acquisition, M.H.; investigation, D.S.; methodology, D.S.; project administration, M.H.; resources, M.H.; software, D.S.; supervision, M.H.; validation, A.S.; visualization, D.S.; writing—original draft, D.S.; writing—review and editing, M.H. and A.S. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Data Availability Statement

The data supporting the findings of this study are available from the authors upon request.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Pozza, L.E.; Field, D.J. The science of soil security and food security. Soil Secur. 2020, 1, 100002. [Google Scholar] [CrossRef]
  2. Handelsman, J.; Cohen, K. A World without Soil: The Past, Present, and Precarious Future of the Earth Beneath our Feet. In A World without Soil: The Past, Present, and Precarious Future of the Earth Beneath Our Feet; Yale University Press: New Haven, CT, USA, 2021. [Google Scholar]
  3. Wu, L.; Jiang, Y.; Zhao, F.; He, X.; Liu, H.; Yu, K. Increased organic fertilizer application and reduced chemical fertilizer application affect the soil properties and bacterial communities of grape rhizosphere soil. Sci. Rep. 2020, 10, 9568. [Google Scholar] [CrossRef] [PubMed]
  4. Bai, Y.-C.; Chang, Y.-Y.; Hussain, M.; Lu, B.; Zhang, J.-P.; Song, X.-B.; Lei, X.-S.; Pei, D. Soil chemical and microbiological properties are changed by long-term chemical fertilizers that limit ecosystem functioning. Microorganisms 2020, 8, 694. [Google Scholar] [CrossRef] [PubMed]
  5. Wang, L.; Qi, S.; Gao, W.; Luo, Y.; Hou, Y.; Liang, Y.; Zheng, H.; Zhang, S.; Li, R.; Wang, M.; et al. Eight-year tillage in black soil, effects on soil aggregates, and carbon and nitrogen stock. Sci. Rep. 2023, 13, 8332. [Google Scholar] [CrossRef] [PubMed]
  6. Beillouin, D.; Corbeels, M.; Demenois, J.; Berre, D.; Boyer, A.; Fallot, A.; Feder, F.; Cardinael, R. A global meta-analysis of soil organic carbon in the Anthropocene. Nat. Commun. 2023, 14, 3700. [Google Scholar] [CrossRef] [PubMed]
  7. Frouz, J. Soil biodiversity conservation for mitigating climate change. In Climate Change and Soil Interactions; Elsevier: Amsterdam, The Netherlands, 2020; pp. 1–19. [Google Scholar] [CrossRef]
  8. Hakim, S.; Naqqash, T.; Nawaz, M.S.; Laraib, I.; Siddique, M.J.; Zia, R.; Mirza, M.S.; Imran, A. Rhizosphere engineering with plant growth-promoting microorganisms for agriculture and ecological sustainability. Front. Sustain. Food Syst. 2021, 5, 617157. [Google Scholar] [CrossRef]
  9. Shen, J.; Li, C.; Mi, G.; Li, L.; Yuan, L.; Jiang, R.; Zhang, F. Maximizing root/rhizosphere efficiency to improve crop productivity and nutrient use efficiency in intensive agriculture of China. J. Exp. Bot. 2013, 64, 1181–1192. [Google Scholar] [CrossRef]
  10. Putnam, D.H.; Orloff, S.B. Forage Crops. Encycl. Agric. Food Systems. 2014, 381–405. [Google Scholar] [CrossRef]
  11. Taylor, N.L. Clovers around the world. Clover Sci. Technol. 1985, 25, 1–6. [Google Scholar] [CrossRef]
  12. Taylor, N.L.; Quesenberry, K.H. Red Clover Science; Springer Science & Business Media: Dordrecht, The Netherlands, 1996; Volume 28. [Google Scholar] [CrossRef]
  13. Annicchiarico, P.; Barrett, B.; Brummer, E.C.; Julier, B.; Marshall, A.H. Achievements and challenges in improving temperate perennial forage legumes. Crit. Rev. Plant Sci. 2015, 34, 327–380. [Google Scholar] [CrossRef]
  14. Tan, K.H.; Tantiwiramanond, D. Effect of humic acids on nodulation and dry matter production of soybean, peanut, and clover. Soil Sci. Soc. Am. J. 1983, 47, 1121–1124. [Google Scholar] [CrossRef]
  15. Quilliam, R.S.; DeLuca, T.H.; Jones, D.L. Biochar application reduces nodulation but increases nitrogenase activity in clover. Plant Soil 2013, 366, 83–92. [Google Scholar] [CrossRef]
  16. Smith, E.L. Vitamin B12. Soil Sci. 1960, 90, 77. [Google Scholar] [CrossRef]
  17. Kononova, M.M. Soil Organic Matter; Pergamon Press, Inc.: New York, NY, USA, 1966. [Google Scholar]
  18. Lochhead, A.G.; Thexton, R.H. Vitamin B12 as a growth factor for soil bacteria. Nature 1951, 167, 1034. [Google Scholar] [CrossRef] [PubMed]
  19. Lilly, V.G.; Leonian, L.H. Vitamin B1 in soil. Science 1939, 89, 292. [Google Scholar] [CrossRef] [PubMed]
  20. Robbins, W.J.; Hervey, A.; Stebbins, M.E. Studies on Euglena and vitamin B12. Bull. Torrey Bot. Club 1950, 77, 423–441. [Google Scholar] [CrossRef]
  21. Soppelsa, S.; Kelderer, M.; Casera, C.; Bassi, M.; Robatscher, P.; Matteazzi, A.; Andreotti, C. Foliar applications of biostimulants promote growth, yield and fruit quality of strawberry plants grown under nutrient limitation. Agronomy 2019, 9, 483. [Google Scholar] [CrossRef]
  22. Mozafar, A. Enrichment of some B-vitamins in plants with application of organic fertilizers. Plant Soil 1994, 167, 305–311. [Google Scholar] [CrossRef]
  23. Rehim, A.; Amjad Bashir, M.; Raza, Q.U.A.; Gallagher, K.; Berlyn, G.P. Yield enhancement of biostimulants, Vitamin B12, and CoQ10 compared to inorganic fertilizer in radish. Agronomy 2021, 11, 697. [Google Scholar] [CrossRef]
  24. Rasouli, F.; Nasiri, Y.; Asadi, M.; Hassanpouraghdam, M.B.; Golestaneh, S.; Pirsarandib, Y. Fertilizer type and humic acid improve the growth responses, nutrient uptake, and essential oil content on Coriandrum sativum L. Sci. Rep. 2022, 12, 7437. [Google Scholar] [CrossRef]
  25. Elshamly, A.M.; Nassar, S.M. Stimulating growth, root quality, and yield of carrots cultivated under full and limited irrigation levels by humic and potassium applications. Sci. Rep. 2023, 13, 14260. [Google Scholar] [CrossRef] [PubMed]
  26. Abdellatif, I.M.Y.; Abdel-Ati, Y.Y.; Abdel-Mageed, Y.T.; Hassan, M.A.M.M. Effect of humic acid on growth and productivity of tomato plants under heat stress. J. Hortic. Res. 2017, 25, 59–66. [Google Scholar] [CrossRef]
  27. Nardi, S.; Schiavon, M.; Muscolo, A.; Pizzeghello, D.; Ertani, A.; Canellas, L.P.; Garcia-Mina, J.M. Molecular characterization of humic substances and regulatory processes activated in plants. Front. Plant Sci. 2022, 13, 851451. [Google Scholar] [CrossRef] [PubMed]
  28. Chen, X.; Kou, M.; Tang, Z.; Zhang, A.; Li, H.; Wei, M. Responses of root physiological characteristics and yield of sweet potato to humic acid urea fertilizer. PLoS ONE 2017, 12, e0189715. [Google Scholar] [CrossRef]
  29. Mackowiak, C.L.; Grossl, P.R.; Bugbee, B.G. Beneficial Effects of Humic Acid on Micronutrient Availability to Wheat. Soil Sci. Soc. Am. J. 2001, 65, 1744–1750. [Google Scholar] [CrossRef]
  30. Tahir, M.M.; Khurshid, M.; Khan, M.Z.; Abbasi, M.K.; Kazmi, M.H. Lignite-derived humic acid effect on growth of wheat plants in different soils. Pedosphere 2011, 21, 124–131. [Google Scholar] [CrossRef]
  31. Jing, J.; Zhang, S.; Yuan, L.; Li, Y.; Chen, C.; Zhao, B. Humic acid modified by being incorporated into phosphate fertilizer increases its potency in stimulating maize growth and nutrient absorption. Front. Plant Sci. 2022, 13, 885156. [Google Scholar] [CrossRef]
  32. Baldotto, M.A.; Baldotto, L.E.B. Gladiolus development in response to bulb treatment with different concentrations of humic acids. Rev. Ceres 2013, 60, 138–142. [Google Scholar] [CrossRef]
  33. Wang, Y.; Lu, Y.; Wang, L.; Song, G.; Ni, L.; Xu, M.; Nie, C.; Li, B.; Bai, Y. Analysis of the molecular composition of humic substances and their effects on physiological metabolism in maize based on untargeted metabolomics. Front. Plant Sci. 2023, 14, 1122621. [Google Scholar] [CrossRef]
  34. Khorasaninejad, S.; ََAlizadeh Ahmadabadi, A.; Hemmati, K. The effect of humic acid on leaf morphophysiological and phytochemical properties of Echinacea purpurea L. under water deficit stress. Sci. Hortic. 2018, 239, 314–323. [Google Scholar] [CrossRef]
  35. Melero, S.; Vanderlinden, K.; Ruiz, J.C.; Madejon, E. Long-term effect on soil biochemical status of a Vertisol under conservation tillage system in semi-arid Mediterranean conditions. Eur. J. Soil Biol. 2008, 44, 437–442. [Google Scholar] [CrossRef]
  36. Jutakanoke, R.; Intaravicha, N.; Charoensuksai, P.; Mhuantong, W.; Boonnorat, J.; Sichaem, J.; Phongsopitanun, W.; Chakritbudsabong, W.; Rungarunlert, S. Alleviation of soil acidification and modification of soil bacterial community by biochar derived from water hyacinth Eichhornia crassipes. Sci. Rep. 2023, 13, 397. [Google Scholar] [CrossRef] [PubMed]
  37. Chen, Y.; Qiu, Y.; Hao, X.; Tong, L.; Li, S.; Kang, S. Does biochar addition improve soil physicochemical properties, bacterial community and alfalfa growth for saline soils? Land Degrad. Dev. 2023, 34, 3314–3328. [Google Scholar] [CrossRef]
  38. Khosravi, A.; Zheng, H.; Liu, Q.; Hashemi, M.; Tang, Y.; Xing, B. Production and characterization of hydrochars and their application in soil improvement and environmental remediation. Chem. Eng. J. 2022, 430, 133142. [Google Scholar] [CrossRef]
  39. Kavitha, B.; Reddy, P.V.L.; Kim, B.; Lee, S.S.; Pandey, S.K.; Kim, K.H. Benefits and limitations of biochar amendment in agricultural soils: A review. J. Environ. Manag. 2018, 227, 146–154. [Google Scholar] [CrossRef] [PubMed]
  40. Regmi, A.; Singh, S.; Moustaid-Moussa, N.; Coldren, C.; Simpson, C. The negative effects of high rates of biochar on violas can be counteracted with fertilizer. Plants 2022, 11, 491. [Google Scholar] [CrossRef] [PubMed]
  41. Joseph, S.; Kammann, C.I.; Shepherd, J.G.; Conte, P.; Schmidt, H.-P.; Hagemann, N.; Rich, A.M.; Marjo, C.E.; Allen, J.; Munroe, P.; et al. Microstructural and associated chemical changes during the composting of a high temperature biochar: Mechanisms for nitrate, phosphate and other nutrient retention and release. Sci. Total Environ. 2018, 618, 1210–1223. Available online: https://www.sciencedirect.com/science/article/pii/S0048969717325512 (accessed on 23 November 2023). [CrossRef]
  42. Hol, W.G.; Vestergård, M.; ten Hooven, F.; Duyts, H.; van de Voorde, T.F.; Bezemer, T.M. Transient negative biochar effects on plant growth are strongest after microbial species loss. Soil Biol. Biochem. 2017, 115, 442–451. [Google Scholar] [CrossRef]
  43. Feng, L.; Xu, W.; Tang, G.; Gu, M.; Geng, Z. Biochar induced improvement in root system architecture enhances nutrient assimilation by cotton plant seedlings. BMC Plant Biol. 2021, 21, 269. [Google Scholar] [CrossRef]
  44. Ren, T.; Wang, H.; Yuan, Y.; Feng, H.; Wang, B.; Kuang, G.; Wei, Y.; Gao, W.; Shi, H.; Liu, G. Biochar increases tobacco yield by promoting root growth based on a three-year field application. Sci. Rep. 2021, 11, 21991. [Google Scholar] [CrossRef]
  45. Wan, H.; Liu, X.; Shi, Q.; Chen, Y.; Jiang, M.; Zhang, J.; Cui, B.; Hou, J.; Wei, Z.; Hossain, M.A.; et al. Biochar amendment alters root morphology of maize plant: Its implications in enhancing nutrient uptake and shoot growth under reduced irrigation regimes. Front. Plant Sci. 2023, 14, 1122742. [Google Scholar] [CrossRef] [PubMed]
  46. Haddad, S.A.; Mowrer, J.; Thapa, B. Biochar and compost from cotton residues inconsistently affect water use efficiency, nodulation, and growth of legumes under arid conditions. J. Environ. Manag. 2022, 307, 114558. [Google Scholar] [CrossRef]
  47. Ren, H.; Guo, H.; Islam, M.S.; Zaki, H.E.M.; Wang, Z.; Wang, H.; Qi, X.; Guo, J.; Sun, L.; Wang, Q.; et al. Improvement effect of biochar on soil microbial community structure and metabolites of decline disease bayberry. Front. Microbiol. 2023, 14, 1154886. [Google Scholar] [CrossRef] [PubMed]
  48. Zhang, L.; Wu, Z.; Zhou, J.; Zhou, L.; Lu, Y.; Xiang, Y.; Zhang, R.; Deng, Q.; Wu, W. Meta-Analysis of the Response of the Productivity of Different Crops to Parameters and Processes in Soil Nitrogen Cycle under Biochar Addition. Agronomy 2022, 12, 1857. [Google Scholar] [CrossRef]
  49. Martínez-Gómez, Á.; Poveda, J.; Escobar, C. Overview of the use of biochar from main cereals to stimulate plant growth. Front. Plant Sci. 2022, 13, 912264. [Google Scholar] [CrossRef] [PubMed]
  50. Xiu, L.; Zhang, W.; Wu, D.; Sun, Y.; Zhang, H.; Gu, W.; Wang, Y.; Meng, J.; Chen, W. Biochar can improve biological nitrogen fixation by altering the root growth strategy of soybean in Albic soil. Sci. Total Environ. 2021, 773, 144564. [Google Scholar] [CrossRef] [PubMed]
  51. Cole, E.J.; Zandvakili, O.R.; Blanchard, J.; Xing, B.; Hashemi, M.; Etemadi, F. Investigating responses of soil bacterial community composition to hardwood biochar amendment using high-throughput PCR sequencing. Appl. Soil Ecol. 2019, 136, 80–85. [Google Scholar] [CrossRef]
  52. Nelson, C.J.; Moser, L.E. Plant factors affecting forage quality. In Forage Quality, Evaluation, and Utilization; Cover Crops and Soil Ecosystem Services; American Society of Agronomy, Crop Science Society of America, & Soil Science Society of America: Madison, WI, USA, 1994; pp. 115–154. [Google Scholar] [CrossRef]
  53. Bremner, J.M. Determination of nitrogen in soil by the Kjeldahl method. J. Agric. Sci. 1960, 55, 11–33. [Google Scholar] [CrossRef]
  54. Li, C.; Li, Y.-H.; Li, Y.; Lu, H.; Hong, H.; Tian, Y.; Li, H.; Zhao, T.; Zhou, X.; Liu, J.; et al. A domestication-associated gene GmPRR3b regulates the circadian clock and flowering time in soybean. Mol. Plant 2020, 13, 745–759. [Google Scholar] [CrossRef]
  55. Borghi, M.; Perez de Souza, L.; Yoshida, T.; Fernie, A.R. Flowers and climate change: A metabolic perspective. New Phytol. 2019, 224, 1425–1441. [Google Scholar] [CrossRef]
  56. Chandler, J.; Corbesier, L.; Spielmann, P.; Dettendorfer, J.; Stahl, D.; Apel, K.; Melzer, S. Modulating flowering time and prevention of pod shatter in oilseed rape. Mol. Breed. 2005, 15, 87–94. [Google Scholar] [CrossRef]
  57. Patrick, J.W.; Stoddard, F.L. Physiology of flowering and grain filling in faba bean. Field Crops Res. 2010, 115, 234–242. [Google Scholar] [CrossRef]
  58. Schiessl, S. Regulation and subfunctionalization of flowering time genes in the allotetraploid oil crop Brassica napus. Front. Plant Sci. 2020, 11, 605155. [Google Scholar] [CrossRef]
  59. Jung, C. Flowering time regulation: Agrochemical control of flowering. Nat. Plants 2017, 3, 17045. [Google Scholar] [CrossRef] [PubMed]
  60. Lorenzo, C.D.; García-Gagliardi, P.; Antonietti, M.S.; Sánchez-Lamas, M.; Mancini, E.; Dezar, C.A.; Vazquez, M.; Watson, G.; Yanovsky, M.J.; Cerdán, P.D. Improvement of alfalfa forage quality and management through the down-regulation of MsFTa1. Plant Biotechnol. J. 2020, 18, 944–954. [Google Scholar] [CrossRef] [PubMed]
  61. Chen, W.; Zhao, L.; Liu, L.; Li, X.; Li, Y.; Liang, G.; Wang, H.; Yu, D. Iron deficiency-induced transcription factors bHLH38/100/101 negatively modulate flowering time in Arabidopsis thaliana. Plant Sci. 2021, 308, 110929. [Google Scholar] [CrossRef]
  62. Sheldon, C.C.; Rouse, D.T.; Finnegan, E.J.; Peacock, W.J.; Dennis, E.S. The molecular basis of vernalization: The central role of FLOWERING LOCUS C (FLC). Proc. Natl. Acad. Sci. USA 2000, 97, 3753–3758. [Google Scholar] [CrossRef]
  63. Jaeger, K.E.; Graf, A.; Wigge, P.A. The control of flowering in time and space. J. Exp. Bot. 2006, 57, 3415–3418. Available online: https://academic.oup.com/jxb/article-abstract/57/13/3415/480219 (accessed on 2 November 2023). [CrossRef]
  64. Corbesier, L.; Vincent, C.; Jang, S.; Fornara, F.; Fan, Q.; Searle, I.; Giakountis, A.; Farrona, S.; Gissot, L.; Turnbull, C.; et al. FT protein movement contributes to long-distance signaling in floral induction of Arabidopsis. Science 2007, 316, 1030–1033. [Google Scholar] [CrossRef]
  65. Taoka, K.I.; Ohki, I.; Tsuji, H.; Kojima, C.; Shimamoto, K. Structure and function of florigen and the receptor complex. Trends Plant Sci. 2013, 18, 287–294. [Google Scholar] [CrossRef]
  66. Markadeh, M.S.; Chamani, E. Effects of various concentrations and time of humic acid application on quantitative and qualitative characteristics of cut stock flower (Matthiola incana’Hanza’). J. Sci. Technol. Greenh. Cult. 2014, 5, Pe157–Pe170. [Google Scholar]
  67. Cimrin, K.M.; Yilmaz, I. Humic acid applications to lettuce do not improve yield but do improve phosphorus availability. Acta Agric. Scand. Sect. B-Soil Plant Sci. 2005, 55, 58–63. [Google Scholar] [CrossRef]
  68. Nardi, S.; Pizzeghello, D.; Muscolo, A.; Vianello, A. Physiological effects of humic substances on higher plants. Soil Biol. Biochem. 2002, 34, 1527–1536. [Google Scholar] [CrossRef]
  69. Chen, Y.; Aviad, T. Effects of humic substances on plant growth. Humic substances in soil and crop sciences: Selected readings. Madison Soil Sci. Soc. Am. 1990, 161–186. [Google Scholar] [CrossRef]
  70. Akeel, A.; Jahan, A. Role of cobalt in plants: Its stress and alleviation. In Contaminants in Agriculture; Springer: Cham, Switzerland, 2020; pp. 339–357. [Google Scholar] [CrossRef]
  71. Yun, J.; Wang, C.; Zhang, F.; Chen, L.; Sun, Z.; Cai, Y.; Luo, Y.; Liao, J.; Wang, Y.; Cha, Y.; et al. A nitrogen fixing symbiosis-specific pathway required for legume flowering. Sci. Adv. 2023, 9, eade1150. [Google Scholar] [CrossRef] [PubMed]
  72. Su, C.; Wang, L.; Kong, F. miR172: A messenger between nodulation and flowering. Trends Plant Sci. 2023, 28, 623–625. [Google Scholar] [CrossRef] [PubMed]
  73. Joseph, S.; Cowie, A.L.; Van Zwieten, L.; Bolan, N.; Budai, A.; Buss, W.; Cayuela, M.L.; Graber, E.R.; Ippolito, J.A.; Kuzyakov, Y.; et al. How biochar works, and when it doesn’t: A review of mechanisms controlling soil and plant responses to biochar. Gcb Bioenergy 2021, 13, 1731–1764. [Google Scholar] [CrossRef]
  74. Keshavarz Afshar, R.; Hashemi, M.; DaCosta, M.; Spargo, J.; Sadeghpour, A. Biochar application and drought stress effects on physiological characteristics of Silybum marianum. Commun. Soil Sci. Plant Anal. 2016, 47, 743–752. [Google Scholar] [CrossRef]
  75. Cole, E.J.; Zandvakili, O.R.; Xing, B.; Hashemi, M.; Barker, A.V.; Herbert, S.J. Effects of hardwood biochar on soil acidity, nutrient dynamics, and sweet corn productivity. Commun. Soil Sci. Plant Anal. 2019, 50, 1732–1742. [Google Scholar] [CrossRef]
  76. Liang, X.; Meng, J.; Yang, X.; Yang, D.; Yuan, J. Organic Compounds in Biochar Stimulate Arabidopsis Flowering. Russ. J. Plant Physiol. 2023, 70, 24. [Google Scholar] [CrossRef]
  77. Guo, Y.; Niu, G.; Starman, T.; Gu, M. Growth and development of Easter lily in response to container substrate with biochar. J. Hortic. Sci. Biotechnol. 2019, 94, 80–86. [Google Scholar] [CrossRef]
  78. Bowley, S.R.; Lackie, S.M. Genetics of nonglandular stem trichomes in red clover (Trifolium pratense L.). J. Hered. 1989, 80, 472–474. [Google Scholar] [CrossRef]
  79. Karabourniotis, G.; Liakopoulos, G.; Nikolopoulos, D.; Bresta, P. Protective and defensive roles of non-glandular trichomes against multiple stresses: Structure–function coordination. J. For. Res. 2020, 31, 1–12. [Google Scholar] [CrossRef]
  80. Bilkova, I.; Kjaer, A.; Van Der Kooy, F.; Lommen, W.J.M. Effects of N fertilization on trichome density, leaf size and artemisinin production in Artemisia annua leaves. In XXIX International Horticultural Congress on Horticulture: Sustaining Lives, Livelihoods and Landscapes (IHC2014): V World 1125; International Society for Horticultural Science: Leuven, Belgium, 2014; pp. 369–376. [Google Scholar] [CrossRef]
  81. Li, Q.; Mao, H.; Dong, X.; Zuo, Z.; Zhou, J. The effects of nitrogen on micro-structure of tomato leaf. In 2014 Montreal, QC, Canada, 13–16 July 2014; American Society of Agricultural and Biological Engineers: St. Joseph, MI, USA, 2014; p. 1. [Google Scholar] [CrossRef]
  82. Liu, C.; Cooper, R.J.; Bowman, D.C. Humic acid application affects photosynthesis, root development, and nutrient content of creeping bentgrass. HortScience 1998, 33, 1023–1025. [Google Scholar] [CrossRef]
  83. Atiyeh, R.M.; Lee, S.; Edwards, C.A.; Arancon, N.Q.; Metzger, J.D. The influence of humic acids derived from earthworm-processed organic wastes on plant growth. Bioresour. Technol. 2002, 84, 7–14. [Google Scholar] [CrossRef]
  84. Pettit, R.E. Organic matter, humus, humate, humic acid, fulvic acid and humin: Their importance in soil fertility and plant health. CTI Res. 2004, 10, 1–17. [Google Scholar]
  85. Zandonadi, D.B.; Canellas, L.P.; Façanha, A.R. Indolacetic and humic acids induce lateral root development through a concerted plasmalemma and tonoplast H+ pumps activation. Planta 2007, 225, 1583–1595. [Google Scholar] [CrossRef]
  86. Casimiro, I.; Marchant, A.; Bhalerao, R.P.; Beeckman, T.; Dhooge, S.; Swarup, R.; Graham, N.; Inzé, D.; Sandberg, G.; Casero, P.J.; et al. Auxin transport promotes Arabidopsis lateral root initiation. Plant Cell 2001, 13, 843–852. [Google Scholar] [CrossRef]
  87. De Smet, I.; Vanneste, S.; Inzé, D.; Beeckman, T. Lateral root initiation or the birth of a new meristem. Plant Mol. Biol. 2006, 60, 871–887. [Google Scholar] [CrossRef]
  88. Trevisan, S.; Pizzeghello, D.; Ruperti, B.; Francioso, O.; Sassi, A.; Palme, K.; Quaggiotti, S.; Nardi, S. Humic substances induce lateral root formation and expression of the early auxin-responsive IAA19 gene and DR5 synthetic element in Arabidopsis. Plant Biol. 2010, 12, 604–614. [Google Scholar] [CrossRef]
  89. Jeffery, S.; Verheijen, F.G.; van der Velde, M.; Bastos, A.C. A quantitative review of the effects of biochar application to soils on crop productivity using meta-analysis. Agric. Ecosyst. Environ. 2011, 144, 175–187. Available online: https://www.sciencedirect.com/science/article/pii/S0167880911003197 (accessed on 2 November 2023). [CrossRef]
  90. Cheng, J.; Lee, X.; Tang, Y.; Zhang, Q. Long-term effects of biochar amendment on rhizosphere and bulk soil microbial communities in a karst region, southwest China. Appl. Soil Ecol. 2019, 140, 126–134. [Google Scholar] [CrossRef]
  91. Manasa, M.R.K.; Katukuri, N.R.; Nair, S.D.; Yang, H.J.; Yang, Z.M.; Rong, B.G. Role of biochar and organic substrates in enhancing the functional characteristics and microbial community in a saline soil. J. Environ. Manag. 2020, 269, 110737. [Google Scholar] [CrossRef] [PubMed]
  92. Sarfraz, R.; Yang, W.; Wang, S.; Zhou, B.; Xing, S. Short term effects of biochar with different particle sizes on phosphorous availability and microbial communities. Chemosphere 2020, 256, 126862. [Google Scholar] [CrossRef]
  93. Kloss, S.; Zehetner, F.; Wimmer, B.; Buecker, J.; Rempt, F.; Soja, G. Biochar application to temperate soils: Effects on soil fertility and crop growth under greenhouse conditions. J. Plant Nutr. Soil Sci. 2014, 177, 3–15. [Google Scholar] [CrossRef]
  94. Anyanwu, I.N.; Semple, K.T. Phytotoxicity of phenanthrene and its nitrogen polycyclic aromatic hydrocarbon analogues in ageing soil. Water Air Soil Pollut. 2015, 226, 347. [Google Scholar] [CrossRef]
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Figure 1. Schematic drawing of the experiment setup. The upper row shows pots that received vitamin B12 and the lower row demonstrates pots with no vitamin B12. The amount of enriched biochar and humic acid is the same in both.
Figure 1. Schematic drawing of the experiment setup. The upper row shows pots that received vitamin B12 and the lower row demonstrates pots with no vitamin B12. The amount of enriched biochar and humic acid is the same in both.
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Figure 2. Interactive effect of vitamin B12 and growing media on flowering time. Clovers were grown in potting soil alone or amended (by either humic acid or enriched biochar and field soil). All pots were grown either with or without vitamin B12. The presented values are an average of 4 replications and three biological observations. ** indicates highly significant differences (p ≤ 0.01), ns indicates non-significant differences, and error bars indicate standard deviations.
Figure 2. Interactive effect of vitamin B12 and growing media on flowering time. Clovers were grown in potting soil alone or amended (by either humic acid or enriched biochar and field soil). All pots were grown either with or without vitamin B12. The presented values are an average of 4 replications and three biological observations. ** indicates highly significant differences (p ≤ 0.01), ns indicates non-significant differences, and error bars indicate standard deviations.
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Figure 3. Stereomicroscopic image of trichome density and thickness in the leaf margin of red clover that received two levels of vitamin B12: +Vit = vitamin B12; −VitB12 = without Vitamin B12 in different growing media; P: potting soil; P + Humic acid: potting soil + humic acid; P + Bio + comp: potting soil + biochar enriched with compost and field soil.
Figure 3. Stereomicroscopic image of trichome density and thickness in the leaf margin of red clover that received two levels of vitamin B12: +Vit = vitamin B12; −VitB12 = without Vitamin B12 in different growing media; P: potting soil; P + Humic acid: potting soil + humic acid; P + Bio + comp: potting soil + biochar enriched with compost and field soil.
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Figure 4. Different shapes and pigmentations of the clusters in flowers that received two levels of vitamin B12: +Vit = vitamin B12; −VitB12 = without Vitamin B12 in different growing media: P: potting soil; P + Humic acid: potting soil + humic acid; P + Bio + comp: potting soil + biochar enriched with compost and field soil.
Figure 4. Different shapes and pigmentations of the clusters in flowers that received two levels of vitamin B12: +Vit = vitamin B12; −VitB12 = without Vitamin B12 in different growing media: P: potting soil; P + Humic acid: potting soil + humic acid; P + Bio + comp: potting soil + biochar enriched with compost and field soil.
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Figure 5. Interactive effect of vitamin B12 and growing media on bud and flower dry matter. Clovers were grown in potting soil alone or amended (by either humic acid or enriched biochar) and field soil. All pots were grown either with or without vitamin B12. The presented values are an average of four replications and three biological observations. ** Indicates highly significant differences (p ≤ 0.05); ns indicates a non-significant difference; and error bars indicate standard deviations.
Figure 5. Interactive effect of vitamin B12 and growing media on bud and flower dry matter. Clovers were grown in potting soil alone or amended (by either humic acid or enriched biochar) and field soil. All pots were grown either with or without vitamin B12. The presented values are an average of four replications and three biological observations. ** Indicates highly significant differences (p ≤ 0.05); ns indicates a non-significant difference; and error bars indicate standard deviations.
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Figure 6. Root appearance in plants that received two levels of vitamin B12: +Vit = vitamin B12; −VitB12 = without Vitamin B12 in different growing media: P: potting soil; P + Humic acid: potting soil + humic acid; P + Bio-comp: potting soil + biochar + compost and field soil.
Figure 6. Root appearance in plants that received two levels of vitamin B12: +Vit = vitamin B12; −VitB12 = without Vitamin B12 in different growing media: P: potting soil; P + Humic acid: potting soil + humic acid; P + Bio-comp: potting soil + biochar + compost and field soil.
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Figure 7. Interactive effect of vitamin B12 and growing media on SPAD readings of red clover leaves. Clovers were grown in potting soil alone or amended (by either humic acid or enriched biochar and field soil). All pots were grown either with or without vitamin B12. The presented values are an average of four replications and three biological observations. ** indicates highly significant differences (p ≤ 0.01), ns indicates non-significant differences, and error bars indicate standard deviations.
Figure 7. Interactive effect of vitamin B12 and growing media on SPAD readings of red clover leaves. Clovers were grown in potting soil alone or amended (by either humic acid or enriched biochar and field soil). All pots were grown either with or without vitamin B12. The presented values are an average of four replications and three biological observations. ** indicates highly significant differences (p ≤ 0.01), ns indicates non-significant differences, and error bars indicate standard deviations.
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Table 1. Chemical properties of the selected field soil.
Table 1. Chemical properties of the selected field soil.
pHMacronutrients (mg kg−1)Micronutrients (mg kg−1)
5.5PKCaMgSBMnZnCuFe
9.21236178737.4029.50.90.64.3
Table 2. Main effect of application of vitamin B12 on phenological parameters, N-status, and dry matter yield of red clover.
Table 2. Main effect of application of vitamin B12 on phenological parameters, N-status, and dry matter yield of red clover.
Phenological and Yield Parameters of Red Clover+Vitamin B12−Vitamin B12
Phenological parametersFlowering time (days)95.9 ± 9.4 a 1100.2 ± 6.4 b
Total number of flowers/buds (per plant)7.7 ± 1.7 a5.9 ± 0.6 a
Total number of stems (per plant)78.6 ± 5.3 a73.0 ± 6.0 b
Trichome density (per plant)35.0 ± 4.1 a49.0 ± 5.0 b
N-statusSPAD value 43.6 ± 5.5 a44.0 ± 7.2 a
Shoot N content (mg g−1)35.4 ± 14.1 a30.8 ± 6.3 a
Root N content (mg g−1)27.2 ± 3.2 a27.4 ± 3.5 a
N yield shoot (mg plant−1)671.3 ± 73.7 a647.7 ± 85.0 a
N yield root (mg plant−1)173.3 ± 11.2 a107.8 ± 1.6 b
Total N yield (mg plant−1)844.6 ± 71.0 a755.5 ± 92.7 a
YieldFlowers/buds dry mass (Per plant) (g)0.96 ± 0.27 a0.55 ± 0.20 a
Shoot dry mass (per plant) (g)21.2 ± 2.3 a20.8 ± 2.8 a
Root dry mass (per plant) (g)6.4 ± 0.4 a3.8 ± 0.5 b
1: The values in each row followed by the same letter are not significantly different at the p ≤ 0.05 level. The presented values are an average of 4 replications and three biological observations.
Table 3. Main effect of growing media on phenological parameters, N-status, and yield of red clover.
Table 3. Main effect of growing media on phenological parameters, N-status, and yield of red clover.
Measured ParametersGrowing Media
Potting SoilPotting Soil + Humic AcidPotting Soil + Biochar-CompostField Soil
Phenological parametersFlowering time (days)94.6 ± 1.9 b 190.6 ± 6.5 a96.9 ± 1.2 b110 ± 2.1 c
Total number of flowers/buds (Per plant)9.8 ± 2.3 a11.1 ± 0.7 a6.2 ± 0.2 ab0.1 ± 0.1 b
Total number of stems (per plant−1)94.1 ± 7.2 a89.1 ± 2.0 a87.1 ± 1.6 a45.0 ± 3.8 b
N-statusSPAD values 40.9 ± 10.1 b44.9 ± 3.3 a45.2 ± 4.3 a44.1 ± 4.7 a
Shoot N content (mg g−1)33.0 ± 5.3 a35.8 ± 20.1 a32.3 ± 3.4 a31.2 ± 8.7 a
Root N content (mg g−1)28.5 ± 2.6 a27.0 ± 10.1 a26.2 ± 2.7 a27.3 ± 4.9 a
N yield shoot (mg plant−1)765.4 ± 142.6 a790.4 ± 43.8 a802.4 ± 36.2 a280.0 ± 56.5 b
N yield root (mg plant−1)125.5 ± 22.9 a166.9 ± 14.6 a151.6 ± 16.0 a118.5 ± 22.6 a
Total N yield (mg plant−1)891.0 ± 138.8 a957.2 ± 44.8 a953.6 ± 41.6 a398.5 ± 74.1 b
Biomass YieldFlowers/buds dry matter (g plant−1)1.0 ± 0.2 ab1.5 ± 0.5 a0.44 ± 0.2 a0.0 ± 0.0 b
Shoot dry weight (g plant−1)23.9 ± 4.4 a27.1 ± 1.3 a24.7 ± 0.4 a8.25 ± 1.5 b
Root dry matter (g plant−1)4.1 ± 0.6 a6.2 ± 0.6 b5.8 ± 0.6 ab4.2 ± 0.7 a
1: The values in each row followed by the same letter are not significantly different at the p ≤ 0.05 level. The presented values are an average of four replications and three biological observations.
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Saadat, D.; Siller, A.; Hashemi, M. Phenology, Nitrogen Status, and Yield of Red Clover (Trifolium pretense L.) Affected by Application of Vitamin B12, Humic Acid, and Enriched Biochar. Agronomy 2023, 13, 2885. https://doi.org/10.3390/agronomy13122885

AMA Style

Saadat D, Siller A, Hashemi M. Phenology, Nitrogen Status, and Yield of Red Clover (Trifolium pretense L.) Affected by Application of Vitamin B12, Humic Acid, and Enriched Biochar. Agronomy. 2023; 13(12):2885. https://doi.org/10.3390/agronomy13122885

Chicago/Turabian Style

Saadat, Dorna, Arthur Siller, and Masoud Hashemi. 2023. "Phenology, Nitrogen Status, and Yield of Red Clover (Trifolium pretense L.) Affected by Application of Vitamin B12, Humic Acid, and Enriched Biochar" Agronomy 13, no. 12: 2885. https://doi.org/10.3390/agronomy13122885

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