**Sustainable Agronomic Strategies for Enhancing the Yield and Nutritional Quality of Wild Tomato,** *Solanum Lycopersicum* **(l) Var** *Cerasiforme* **Mill**

**Kanagaraj Muthu-Pandian Chanthini, Vethamonickam Stanley-Raja, Annamalai Thanigaivel, Sengodan Karthi, Radhakrishnan Palanikani, Narayanan Shyam Sundar, Haridoss Sivanesh, Ramaiah Soranam and Sengottayan Senthil-Nathan \***

Sri Paramakalyani Centre for Excellence in Environmental Sciences, Manonmaniam Sundaranar University, Alwarkurichi, Tirunelveli 627412, Tamil-Nadu, India; clairdelune127@gmail.com (K.M.-P.C.); stanleyraja@gmail.com (V.S.-R.); www.thani@gmail.com (A.T.); karthientomology@gmail.com (S.K.); kani.palani5@gmail.com (R.P.); krn.shyamsundar@gmail.com (N.S.S.); Sivanesh2020@gmail.com (H.S.); soranamr@gmail.com (R.S.)

**\*** Correspondence: senthil@msuniv.ac.in; Tel.: +91-463-428-3066

Received: 30 April 2019; Accepted: 28 May 2019; Published: 13 June 2019

**Abstract:** Urbanization and global climate change have constrained plant development and yield. Utilization of wild gene pool, together with the application of sustainable and eco-friendly agronomic crop improvement strategies, is being focused on to tackle mounting food insecurity issues. In this aspect, the green seaweed, *Ulva flexuosa*, was assessed for plant biostimulant potential on cherry tomato, in terms of seed priming effects, nutrition and yield. SEM-EDX analysis of *U. flexuosa* presented the occurrence of cell wall elements (O, Na, Mg, S, Cl, K and Ca). The phytochemical analyses of liquid seaweed extract (EF-LSE) revealed the presence of carbohydrates, protein, phenols, flavonoids, saponins, tannins and coumarins. The EF-LSEs were found to stimulate seed germination in a dose-dependent manner, recording higher seed germination, and biomass and growth parameters. The seedlings of treated seeds altered the biochemical profile of the fruit, in terms of TSS (93%), phenol (92%), lycopene (12%) and ascorbic acid (86.8%). The EF-LSEs positively influenced fruit yield (97%). Henceforth, this investigation brings to light the plant biostimulant potential of the under-utilized seaweed source, *U. flexuosa,* to be useful as a bio fertilizer in agronomic fields for a cumulative enhancement of crop vigour as well as yields to meet the growing food demands.

**Keywords:** seed priming; seaweed extract; biostimulant; germination energy; seedling vigour

#### **1. Introduction**

Agriculture is facing various crises that are worsening with time. Increasing food production to meet or feed the mounting population is a foremost challenge. This can be accomplished by further use of farm lands for an overall hike in food production or technically enhancing the yields from pre-existing lands by application of fertilizers or implementation of novel approaches; for instance, precision farming systems viz., cutting-edge irrigation arrangements, and ecologically accomplishable crop revolutions [1]. Crop diseases decrease yield, resulting in a prominent crisis to food security, creating a global malnutrition spree affecting nearly 815 million people [2]. Henceforward, natural fertilizers are well thought out as probable as well as safe alternatives to chemical fertilizers [3]. Additionally, the presence of several horticultural important traits in the wild gene pool makes them suitable as potential breeding candidates for crop improvement [4]. In this aspect, plant secondary metabolites are being emphasized for their disease regulator competences and combined in more than a few defense control programs [5].

The marine ecosystem serves as a rich source of bioactive compounds, such as sulfated polysaccharides, terpenoids, phenolics, lactones, sterol and fatty acids, possessing pharmacological and plant growth-stimulating properties [6]. Seaweeds form a key portion of these bioactive natural composites, with over 9000 species, known for their biostimulator potentials. Additionally, seaweed products as biostimulants that can enhance crop production are also being focused on. Biostimulants are materials supplementary to fertilizers, which endorse plant growth at lower concentrations [7]. In addition, seaweeds are extensively applied in the fields of agriculture and horticulture to improve quality and quantity, and the results are promising [8]. Innumerable seaweeds are being applied as liquid fertilizers to upsurge crop yields, as they are rich in macro-nutrients, besides trace elements essential for the development and enrichment of plants. Commercial seaweed products are also being marketed successfully [9]. Besides being inexpensive, the seaweed extracts have surplus allelopathic chemicals that promote seed germination as well as emergence rates. Seaweed extracts are known to have a positive impact on prime stages of plant ontogenesis—starting from seed germination to seedling growth [10]. Furthermore, seaweeds are reported with higher amounts of growth hormones, attributed to their plant biostimulant activities [11].

Seed germination is a decisive procedure in plant growth, and the enrichment of germination potentialities of a seed can eventually enable a surge in crop yields, and is dependent on numerous chemical factors (soil moisture salinity, metal, mineral composition) [12]. The emergence of seed is promoted by various methods for enhanced agricultural yields, like exposing them to biostimulants or growth promoting hormones by the process of seed priming. As the very first stage of plant growth, germination is defined as an outcrop of the radicle from the tissues enfolding the seed [13]. As germination rates may vary among species, the analyses of germination rates might be directly proportional to the growth rates and consequently, their yields [14,15].

Cherry tomato, *Solanum lycopersicum* (L.) var. *cerasiforme* Mill. is a widespread, table purpose tomato variety, bearing bright red color, and small fruits. It is a probable ancestor of a cultivated tomato variety with small fruits bright red in color, resembling a cherry and tasting excellent [16]. They are also favorable candidates in breeding programs for their genetic diversity, offering the selection of parental traits along with extensive geographic ranges. [17]. With the debarring effects of crop growth promoting chemicals that alter soil ecology and have hazardous environmental and health impacts, researchers are concentrating on the allegation of naturally benign substitutes to increase yields, while offering effective crop protection. The favorable agronomic traits of cherry tomato (an intermediary genetic admixture flanked by wild currant-type tomatoes and domesticated garden tomatoes), such as higher nutrient composition, offer plans for balanced utilization to unravel indigenous complications encompassing crop adaptation to climatic variations or, to endorse functional food consumption [18].

Seed priming of native seed species can evoke ecological restoration, stimulating the expression of dormant genes responsible for the expression of favorable agronomic traits. Since the sources of natural varieties are collected from the wild and their sources are limited, there is a persistent requirement for novel methods for seed-based restoration technologies. In this aspect, seed priming could be a strategy for sustainable seedling establishment, plant growth, and restoration of native seed. Hence, this research intended to discover the bio stimulator potentials of liquid seaweed extract of green alga, *U. flexuosa*, along with screening of their phytochemical and elemental composition on cherry tomato.

#### **2. Materials and Methods**

#### *2.1. Seed Collection and Preparation*

Cherry tomato seeds, variety ATL-01-19, were purchased from Tamil Nadu Agricultural University, Coimbatore, Tamil Nadu, India. Seeds of undeviating dimensions and hue were carefully chosen for the study, surface sterilized with 0.1% mercuric chloride, washed thrice in sterile distilled water. The experiment was carried out at Sri Paramakalyani Centre for Excellence in Environmental Science, Manonmaniam Sundaranar University, Alwarkurichi, from July to August, 2018.

#### *2.2. Seaweed Collection*

The seaweed, *Ulva flexuosa* (Ulvaceae), was collected from the rocks in coastal areas at Colachel beach (Figure 1), Kanyakumari (8◦14 5168 N and 77◦14 35.209 E) during a low tide period (August 2018). It was washed in seawater several times to remove impurities, sand particles, and epiphytes, and brought to the research laboratory in taped up polythene bags. The seaweed was washed thoroughly in tap water several times, wearied and spread on blotting paper to remove excess water, and shade dried for 2 to 3 hours.

#### *2.3. Preparation of Starter Fertilizer Solution (SFS)*

Ammonium dihydrogen phosphate (Merck) was used as a starter fertilizer solution by mixing 1 mg of NH4H2PO4 in sterile distilled water (10 mL), designated as positive control.

#### *2.4. Preparations of Liquid Seaweed Extract (EF-LSE)*

The washed seaweed was then cut into minor fragments, boiled in distilled water (100 gms/1 L) for one hour in an autoclave (121 ◦C, 15 psi of pressure), and filtered through a cheese cloth (double layered), yielding 890 ml of LSE. The LSE was stored at 4 ◦C in a refrigerator until further use. The test concentrations of EF-LSE were prepared by diluting the extract with distilled water (20%, 40%, 60%, 80% and 100%).

#### *2.5. EF-LSE Analysis*

#### 2.5.1. Physicochemical Analysis

Physicochemical structures of the EF-LSEs—pH, electrical conductivity, and color appearance—was determined. pH was determined using a pH meter (ELICO LI 120, Hyderabad, India), whereas conductivity was done with the help of a conductivity meter (Microprocessor EC Meter 1615, Parwanoo, Himachal Pradesh, India) and expressed in ds/m. The colour of the EF-LSE was visually observed and noted.

#### 2.5.2. Elemental Composition of EF-LSE Using X-ray–Energy Dispersive Spectroscopic (ED) Analysis

The elemental composition of *U. flexuosa* was performed using EDAX (BRUKER) to elucidate the components present in the seaweed cell wall.

#### 2.5.3. Phytochemical and Biochemical Screening

Phytochemical [19] and biochemical screening of *U. flexuosa* was performed to analyse the quantitative amounts of phenol [20], chlorophyll [21] and protein [22] by standard procedures.

#### *2.6. Preparation of Seeds*

Tomato seeds, tomato wild relative, *Solanum lycopersicum* (L.) var. *cerasiforme* Mill, were used for all the tests. Tomato seeds, without any visible signs of infection, of uniform size, shape and colour, were carefully chosen, surface sterilized using 0.1% mercuric chloride, before and after rinsing in sterile distilled water. The seeds were used for further analysis.

To investigate the possible effects of UF-LSEs on tomato plant's vegetative growth and yield, small pot field experiments containing sterilized soil were conducted by sowing the primed seeds in a tray. The seedlings (2-3 true leaf stage) were transplanted into autoclaved pot mixture (red soil: cow dung: vermiculate at 2:1:1, w/w/w) in the surface sterilized (1% mercuric chloride) pots (15 cm diameter, 750 mL volume) at 1 seedling/pot. The pots were labeled based on the treatments. Vegetative growth parameters were analyzed by sampling from seedlings selected from randomized block designs.

#### *2.7. Biostimulant Assays*

#### 2.7.1. Seed Bioassay

#### Seed Germination Test

Seed germination assay was conducted using five surface sterilised seeds per assay, replicated five times. The seeds were exposed to EF-LSE extracts (10 mL), SFS in sealed and labelled conical flasks and kept in a shaker for 12 hours. Seeds in 10 mL sterile distilled water were used as control. The seeds were then removed and spread on a filter paper to blot out the solutions at room temperature for 24 hours. The treated seeds were placed in pre-labelled sterile petri dishes (9 cm) over filter paper (Whatman No. 5) that was moistened (sterile distilled water), instantaneously taped up with parafilm (Merck) to prevent moisture loss, and incubated (25 ± 2 ◦C/alternative 16 h light-8 h dark). The plates were checked for radicle protrusion (>2 mm) on a daily basis (hint of germination). Ten seeds were tested for each concentration of EF-LSE.

Germination was recorded every day by counting the emerging hypocotyls. The mean germination time (MGT) was premeditated [23] by counts made on the time taken for 1%, 10%, 25%, 50%, 75% and 100% of the seeds to germinate and expressed as days.

$$\text{MGT} = \frac{\Sigma \text{ (n T)}}{\Sigma \text{ } n} \tag{1}$$

where,

*n* = number of newly germinated seeds at time T (25 ◦C)

T = hours from the beginning of the germination test

Σ *n* = final germination

\*100% will refer to the total number of seeds germinated after exposure to the highest EF-LSE concentration.

The germination percentage (GP) was calculated using the following formula:

$$\text{GP} = \frac{\text{Number of seconds gemimated}}{\text{Total number of seconds}} \times 100\tag{2}$$

Seed Germination Energy (GE) was calculated according to the following formula:

$$\text{GE} = \frac{\text{Number of germinating seeds}}{\text{No. of total seeds per test post generation for 3 days}} \times 100\tag{3}$$

Seedling vigour index (SVI) SVI was calculated [24] by the following formula:

$$\text{SVI} = \text{Seedling length (cm)} \times \text{generation } \% \tag{4}$$

Seed Imbibition

The biomass (wet and dry weight, mg) of the seeds primed in EF-LSEs and SFS solutions for 24 hours were determined with the help of an electronic balance after oven-drying at 40 ◦C for two days. Seed imbibition was determined by measuring the weight of seeds (100 seeds/treatment) before and during priming with SFS and EF-LSEs at 6, 12, 24, 36 and 48 hours and plotting the water imbibition curve by determining the seed moisture content (MC) and through means of which, the seed imbibition time was calculated [25]. Seeds primed in distilled water served as control. The MC was calculated by the formula:

$$\text{Moisture content} = \frac{\text{Fresh weight} - \text{Dry weight}}{\text{Dry weight}} \times 100\tag{5}$$

#### 2.7.2. Growth Parameter Assay

Growth parameters such as lengths of total plant, plumule and radicle length (cm) and root-shoot lengths and ratio were measured with a Vernier calliper post 5 days and 20 days for petri plate and glasshouse tests, respectively.

#### 2.7.3. Fruit Yield and Quality Parameter assay

The seedlings derived from primed seeds were cultured as per recommended standard procedures [26] were selected on the basis of randomised block design, performing yield and quality parameter assays, repeating each test five times. The effect of EF-LSE on the yield of cherry tomato plants was estimated by fruit weight. The quality parameters of treated cherry tomato plants were calculated in terms of total soluble solids (TSS) [27], ascorbic acid [28], lycopene [29] and phenol [20] contents.

#### *2.8. Statistical Analysis*

All the tests were repeated five times. The effect of EF-LSE on seeds was determined by analysis of variance, one-way (ANOVA), and the treatment means were compared by Tukey-family error test (*p* < 0.05) by using Minitab®17 software package (LEAD Technologies Inc., Charlotte, NC, USA).

#### **3. Results**

#### *3.1. Seaweed Collection and Identification*

The seaweeds collected from Colachel beach (Figure 1) were subjected to microscopical (Nikon Phase Contrast, Japan) and macroscopical analyses and confirmed to be Ulva flexuosa Wulfen (Ulvaceae), based on morphological characteristics and organoleptic features (Table 1). The seaweed belonging to the green alga, phylum Chlorophyta, is tubular and branched (Figure 2). The cells were observed to be arranged in transverse rows.


**Table 1.** Organoleptic features of *Ulva flexuosa*.

**Figure 1.** Sample collection site of seaweed from Colachel beach, Kanyakumari, Tamil Nadu, India.

**Figure 2.** Macroscopic and microscopic images of *U. flexuosa*. ((**A**): Sample collection site, Colachel beach; (**B**): *U. flexuosa*; (**C**,**D**): Macroscopic image; Microscopic image of *U. flexuosa*-(**E**): 10×; (**F**): 40× and (**G**): 100×, showing blue coloured pigments on cell wall).

#### *3.2. EF-LSE Analyses*

#### 3.2.1. Physicochemical Screening

The EF-LSEs appeared as pale greenish yellow in colour. The pH of the EF-LSEs of doses 20%, 40%, 60%, 80% and 100% was measured and found to be 7.160, 7.240, 7.34, 7.4 and 7.58, respectively. The pH of control was 7 and that of SFS was 4.2. The electrical conductivity (EC) of the respective EF-LSE doses was recorded as 0.96 ds m−1, 1.01 ds m−1, 1.08 ds m−1, 1.54 ds m−<sup>1</sup> and 2.8 ds m−1, respectively (Table 2).


**Table 2.** Physico-chemical properties of LSEs, pH and EC (dS m<sup>−</sup>1).

Columns denoted by a different letter are significantly different at *p* ≤ 0.05.

3.2.2. Elemental Composition of EF-LSE Using X-ray–EDS Analysis

The elemental composition of the seaweed elucidated via EDX analysis (Figure 3) revealed the presence of seven compounds on seaweed cell surface—oxygen, Na, Mg, S, Cl, K and Ca. Oxygen was present in higher quantities (56.25%), followed by chlorine (13.4%), sulphur (7.79), and potassium (7.52%). Magnesium (5.3%), calcium (4.89%) and sodium (4.84%) were also recorded.

**Figure 3.** SEM-EDX—Energy Dispersive Spectrum of *U. flexuosa* showing the presence of various elements in their cell wall.

#### 3.2.3. Phytochemical Screening

The phytochemical analysis of EF-LSE revealed the presence of carbohydrates, protein, phenols, flavonoids, saponins, tannins and coumarins (Table 3).


**Table 3.** Phytochemical composition of EF-LSE.

#### 3.2.4. Biochemical Screening

The seaweed examined qualitatively revealed the presence of 1 mg/g of phenol, 6.1% protein, and 0.9 mg/g of total chlorophyll contents.

#### *3.3. Biostimulant Assays*

The cherry tomato seeds purchased were sown in a greenhouse, according to standard horticultural methods (Figure 4).

**Figure 4.** Cherry tomato plants.

#### 3.3.1. Effect of EF-LSEs on Cherry Tomato Seeds

Germination of cherry tomato seeds was initiated on day 2 in seeds treated with 80% and 100% of EF-LSEs. The EF-LSEs were able to initiate germination of the seedlings in a dose-dependent manner (Figure 5). EF-LSE treated seeds emerged early when compared to the control (Figure 6). In addition, the EF-LSE treated seeds in doses 20%, 40%, 60%, 80% and 100% exhibited lower MGT (Figure 7) of 4, 3.4, 3, 2.6 and 2.2 days (*F*6,28 = 3.23; *p* < 0.0001), respectively, compared with seeds treated with SFS, 4 days (*F*6,28 = 3.23; *p* < 0.0001) as well as that of the control, 4.9 days (*F*6,28 = 3.23; *p* < 0.0001).

**Figure 5.** Effect of LSEs on growth of tomato seedlings.

The germination time course for control seeds was longer (Figure 6), taking 86.54, 93.2, 110.64, 116.4 and 125.80 hours (*F*4,20 = 30.33; *p* < 0.0001) for 1%, 10%, 25%, 50%, 75% and 100% of seeds to germinate. Seeds treated with SFS exhibited a time course almost similar to that of control, 85.54, 91.8, 109.34, 114.51 and 125.37 hours (*F*4,20 = 30.33; *p* < 0.0001). The germination time course decreased with increase in EF-LSE concentrations. The time taken for emergence of 1% of seeds decreased from 76.4

(*F*4,20 = 44.77; *p* < 0.0001) to 56.8 (*F*4,20 = 36.81; *p* < 0.0001) and 50.8 (*F*4,20 = 31.39; *p* < 0.0001) hours in seeds treated with 20%, 40% and 60% EF – LSEs, respectively.

**Figure 6.** Effect of EF-LSEs on germination of tomato seeds—germination time course.

Similarly, the rate of emergence of seeds increased, resulting in a decreased germination time course, further to 44.6 (*F*4,20 = 15.97; *p* < 0.0001) and 40 (*F*4,20 = 13.66; *p* < 0.0001) hours in 80% and 100% EF-LSE treatments, respectively. As a series, the time taken for the emergence of 100% of seeds in treatment decreased from 163.2 (*F*4,20 = 30.33; *p* < 0.0001) to 148.8 (*F*4,20 = 38.16; *p* < 0.0001) hours in the respective control and SFS treated seeds. The EF-LSE treated seeds took 132 ((*F*4,20 = 44.77; *p* < 0.0001), 112.8 ((*F*4,20 = 36.81; *p* < 0.0001), 100.8 ((*F*4,20 = 31.39; *p* < 0.0001), 91.1 (*F*4,20 = 15.97; *p* < 0.0001) and 76.8 ((*F*4,20 = 13.66; *p* < 0.0001) hours for 100% emergence at treatment concentrations of 20%, 40%, 60%, 80% and 100%, respectively.

**Figure 7.** Effect of LSEs on germination of tomato seeds—mean germination time.

The seeds in the petri dishes treated with distilled water exhibited 84.8% GP (*F*6,28 = 6.4; *p* < 0.0001) at the end of seven days. SFS treated seeds showed a GP of 86.6% GP (*F*6,28 = 6.4; *p* < 0.0001). Seeds treated with 20%, 40%, 60%, 80% and 100% EF-LSE, displayed 93%, 94%, 95%, 96% and 97% (*F*6,28 = 6.4; *p* < 0.0001) GP, respectively (Figure 8). The number of days taken for all the EF-LSE treated seeds to germinate also decreased to 5.5, 4.7, 4.2, 3.6 and 3.2 days (*F*4,20 = 13.66; *p* < 0.0001).

**Figure 8.** Effect of LSEs on germination percentage of cherry tomato seeds.

The germination energy (Figure 9) of the seeds treated with EF-LSEs were higher, in the range of 55.6, 85.4, 91.6, 94.6 and 97.8 (*F*6,28 = 31.34; *p* < 0.0001) exposed to 20%, 40%, 60%, 80% and 100% EF–LSEs, respectively, which was very high in comparison with the control (10%) and that of SFS-treated (20%) (*F*6,28 = 31.34; *p* < 0.0001) seeds.

**Figure 9.** Effect of LSEs on germination energy of cherry tomato seeds.

The seedling vigour index of the control seeds and SFS treated seeds were 457.18 (*F*6,28 = 32.86; *p* < 0.0001) and 743.75 (*F*6,28 = 32.86; *p* < 0.0001), respectively. However, the SVI of seeds treated with EF-LSEs (Table 4) of dosages 20%, 40%, 60%, 80% and 100% were enhanced to 1003.59, 1047.04, 1060.24, 1236.96 and 1281.31 (*F*6,28 = 32.86; *p* < 0.0001), respectively (Table 4).

The biomass of the cherry tomato seeds was determined after 48 hours of priming. Seeds that were not treated with any extracts unveiled respective dry and wet weights of 0.013 mg (*F*6,28 = 32.08; *p* < 0.0001) and 0.113 mg (*F*6,28 = 19.23; *p* < 0.0001). The dry weights of the tomato plants treated with SFS, EF-LSE extracts – 20%, 40%, 60%, 80% and 100% were found to be 0.0218 mg, 0.0262 mg, 0.0296 mg, 0.0316 mg, 0.042 mg and 0.0528 mg (*F*6,28 = 32.08; *p* < 0.0001) respectively. The wet weights of the tomato plants treated with SFS, EF-LSE extracts—20%, 40%, 60%, 80% and 100% were found to be 0.1396 mg, 0.1434 mg, 0.144 mg, 0.152 mg, 0.2 mg and 0.308 mg (*F*6,28 = 19.23; *p* < 0.0001), respectively (Table 4).


**Table 4.** Seedling vigour index and biomass (wet and dry weight) of tomato seeds treated with LSEs.

Columns denoted by a different letter are significantly different at *p* ≤ 0.05.

The moisture content of the dry seeds was 7.8%, which increased after six hours in all SFS and EF-LSE treatments. The moisture content of control seeds increased to 8%, 9%, 9.4%, 9.7% and 9.9% at 6, 12, 24, 36 and 48 hours. A comparatively higher imbibition occurred in EF-LSE treated seeds, with a minimum imbibition of 8.5% and a maximum of 9.5 after six hours, in 20% and 100% primed seeds (Figure 10).

**Figure 10.** Effect of EF-LSEs on seed imbibition capabilities of tomato seeds.

#### 3.3.2. Effect of EF-LSEs on Growth Parameters of Cherry Tomato

The effect of EF-LSEs on the growth of tomato seedlings tested exhibited significant differences in total seedling length (Figure 11), radicle, and plumule length (Figure 12).

The height of the tomato seedlings at the end of five days was 5.4 cm (*F*6,28 = 30.98; *p* < 0.005), with radicle and plumule lengths of 4 cm (*F*6,28 = 42.16; *p* < 0.005) and 3 cm (*F*6,28 = 42.94; *p* < 0.005), respectively (Figure 12), with 1.26 radicle: plumule ratio (*F*5,24 = 32.20, *p* < 0.005) (Figure 13).

The SFS was able to induce the seedling length to 8.6 cm (*F*6,28 = 30.98; *p* < 0.005) with a corresponding radicle and plumule lengths, ratio of 4.2 (*F*6,28 = 42.16; *p* < 0.005) and 3.72 cm (*F*6,28 = 42.94; *p* < 0.005), 1.36 (*F*5,24 = 32.20, *p* < 0.005), respectively. The EF-LSEs had a positive effect in stimulating the seedling height and their respective radicle and plumule lengths to 10.8 cm (*F*6,28 = 30.98; *p* < 0.005), 5.12 cm (*F*6,28 = 42.16; *p* < 0.005) and 4.12 cm (*F*6,28 = 42.94; *p* < 0.005), respectively, at 20% concentration, exhibiting radicle: plumule ratio of 1.36 (*F*5,24 = 32.20, *p* < 0.005).

**Figure 11.** Effect of EF-LSEs on growth parameters of tomato seedling and plant height.

**Figure 12.** Effect of EF-LSEs on growth parameters of tomato seeds (plumule-radicle length).

The EF-LSEs had a positive effect in stimulating seedling height and their respective radicle and plumule lengths to 10.8 cm (*F*6,28 = 30.98; *p* < 0.005), 5.12 cm (*F*6,28 = 42.16; *p* < 0.005) and 4.12 cm (*F*6,28 = 42.94; *p* < 0.005), respectively, at 20% concentration, exhibiting radicle: plumule ratio of 1.36 cm (*F*5,24 = 32.20, *p* < 0.005). Similarly, the 40% EF-LSE treated seeds exhibited respective seedling height, radicle and plumule lengths, radicle: plumule ratio of 12.07 cm (*F*6,28 = 30.98; *p* < 0.005), 5.72 cm (*F*6,28 = 42.16; *p* < 0.005) and 5 cm (*F*6,28 = 42.94; *p* < 0.005) and 1.59 (*F*5,24 = 32.20, *p* < 0.005). Likewise 60% and 80% EF-LSE treated seeds were observed with seedling lengths of 13.09 cm and 13.84 cm (*F*6,28 = 30.98; *p* < 0.005), with corresponding radicle and plumule lengths of 7.6 cm (*F*6,28 = 42.16; *p* < 0.005) and 5.8 cm (*F*6,28 = 42.94; *p* < 0.005) as well as 8.2 cm (*F*6,28 = 42.16; *p* < 0.005) and 6.4 cm

(*F*6,28 = 42.94; *p* < 0.005) besides the radicle: plumule ratios of 1.76 and 1.9 (*F*5,24 = 32.20, *P* < 0.005), respectively. The seeds treated with 100% EF-LSE exhibited the highest seedling length of 14.1 cm (*F*6,28 = 30.98; *p* < 0.005) with radicle and plumule lengths of 8.2 cm (*F*6,28 = 42.16; *p* < 0.005) and 6.4 cm (*F*6,28 = 42.94; *p* < 0.005), respectively. They revealed a radicle: plumule ratio of 2.04 (*F*5,24 = 32.20, *p* < 0.005).

**Figure 13.** Radicle-plumule as well as root-shoot ratio of tomato seedlings.

The effect of EF-LSEs on the growth of tomato plants tested exhibited significant differences in total plant height (Figure 11), root, and shoot lengths (Figure 14).

**Figure 14.** Effect of EF-LSE on root-shoot lengths of tomato plants.

The height of the tomato plant at the end of 20 days was 16 cm (*F*6,28 = 40.45; *p* < 0.003), with root and shoot lengths of 6 cm (*F*6,28 = 30.87; *p* < 0.005) and 10 cm (*F*5,24 = 32.40; *p* < 0.005), respectively, with 0.68 root: shoot ratio (*F*6,28 = 18.76; *p* < 0.000). The SFS was able to induce plant height to 19 cm (*F*6,28 = 40.45; *p* < 0.003) with a corresponding root, shoot lengths and ratio of 7 cm (*F*6,28 = 30.87; *p* < 0.005) and 12.2 cm (*F*5,24 = 32.40; *p* < 0.005), and 0.82 (*F*6,28 = 18.76; *p* < 0.00001), respectively. The EF-LSEs had a positive effect in stimulating tomato plant height and their respective root and shoot lengths to 23.4 cm (*F*6,28 = 40.45; *p* < 0.003), 9.9 cm (*F*6,28 = 30.87; *p* < 0.005) and 13.22 cm (*F*5,24 = 32.40; *p* < 0.005), respectively, at 20% concentration, exhibiting root: shoot ratio 0.88 (*F*6,28 = 18.76; *p* < 0.0001). Similarly, the 40% EF-LSE treated seeds exhibited respective plant height, root and shoot lengths, root: shoot of 24.6 cm (*F*6,28 = 40.45; *p* < 0.003), 10.1 cm (*F*6,28 = 30.87; *p* < 0.005) and 14.6 cm (*F*5,24 = 32.40; *p* < 0.005) and 1 (*F*6,28 = 18.76; *p* < 0.000). Likewise, 60% and 80% EF-LSE treated seeds were observed with total plant heights of 28.38 cm and 31cm (*F*6,28 = 40.45; *p* < 0.003), with corresponding root and shoot lengths of 12.38 cm (*F*6,28 = 30.87; *p* < 0.005) and 15.8 cm (*F*5,24 = 32.40; *p* < 0.005) as well as 13.82 cm (*F*6,28 = 30.87; *p* < 0.005) and 16 cm (*F*5,24 = 32.40; *p* < 0.005) besides the root: shoot ratios of 1.41 and 1.606 (*F*6,28 = 18.76; *p* < 0.0001), respectively (Figure 13). The seeds treated with 100% EF-LSE exhibited the highest plant height of 32 cm (*F*6,28 = 40.45; *p* < 0.003) with root and shoot lengths of 14.54 cm (*F*6,28 = 30.87; *p* < 0.005) and 17.48 cm (*F*5,24 = 32.40; *p* < 0.005), respectively. They revealed a root: shoot ratio of 1.776 (*F*6,28 = 18.76; *p* < 0.0001).

#### 3.3.3. Effect of EF-LSEs on Fruit Quality Parameters of Cherry Tomato

The treated plants displayed a yield of 2.617 kg/plant compared with that of the control: 1.07 kg/plant (*F*1,8 = 40.92; *p* < 0.0001). Further, the quality parameters analyses revealed the presence of increased amounts of 6.54 (Brix) TSS, 13.2 mg/100 g FM ascorbic acid, 88.45 μg g−<sup>1</sup> FM lycopene and 7.26 mg g−<sup>1</sup> DM phenol contents, compared with that of the control, which recorded 5.2 (Brix) TSS (*F*1,8 = 57.1; *p* < 0.002), 10.48 mg/100 g FM ascorbic acid (*F*1,8 = 53.52; *p* < 0.0001), 55.206 μg g−<sup>1</sup> FM lycopene (*F*1,8 = 61; *p* < 0.003) and 5.72 mg g−<sup>1</sup> DM phenol (*F*1,8 = 47. 8; *p* < 0.0001) contents (Table 5).


**Table 5.** Yield, TSS, Ascorbic acid, lycopene and phenol contents of treated cherry tomato fruits.

Rows denoted by a different letter are significantly different at *p* ≤ 0.05.

#### **4. Discussion**

The indiscriminate application of fertilizers has not only intoxicated the environment, but also lost their efficiency. Alternative naturally benign bases of fertilizers, sourced from biological sources such as plants, animals and micro-organisms, have paved the way for the practice of "organic farming". Many eco-friendly bioactive compounds from seaweeds have been widely used in the agricultural field as plant growth promoters. Seaweeds are reported for their copious amounts of novel as well as assorted range of marine secondary metabolites [30]. Global population growth has seen leaps and bounds in the recent years, posing food insecurity [31]. With the foremost necessity of augmentation of crop production, farmers are in stress to improve yields of agriculturally important crops. As an imperative crop, the germination capability of tomato seed is valued to be around 70%. Seed emergence is mainly prejudiced by the equipoise, flanked by the growth skills of the embryo, in addition to the mechanical resistance of the endosperm, which should be debilitated for germination [32]. Seaweeds are being sought out as potential enhancers of crop growth and yield and are replacing chemical

fertilizers owing to higher efficiencies, broader action range, eco-friendly nature, and cost-effective feature. Seaweeds, reported with outstanding plant growth promoting potentials, increased plant height, root as well as shoot lengths, consequently, are designated as plant growth biostimulants, as reviewed by Khan et al. [7] and Craigie [33] As a crucial and initial plant growth activity, the evaluation of a seed's germination and associated parameters can help in determining the rate of a crop success, in terms of yield and economy [34]. As contemplation, the current investigation was performed to determine the plant growth stimulant activities of green seaweed *U. flexuosa* (Chlorophyceae).

As the germination of a seed counts on various physical aspects, together with nutrient composition [12], preliminary tests of the extracts were performed by analyzing the pH and electrical conductivity (EC) of the extracts. The nutrient content of a solution, in terms of salts and electrolyte concentration can be determined by measuring their EC. The EF-LSE of *U. flexuosa* was found to possess a neutral pH and an optimum EC that indicates the presence of salts, for instance, boron, zinc, magnesium, calcium and other essential plant nutrients in a nutritive solution [35]. Higher rates of EC of nutrient or fertilizer solutions are proven with the stimulation of favourable agronomic traits, such as increase in nutritional quality, colour gradient and quality of tomato fruits [36,37]. However, solutions outside the optimum EC had an inhibitory effect on plant growth activities [38]. A nutrient solution within optimum EC was found optimal for the growth stimulation of lettuce in glasshouse conditions [39]. Henceforth, the EF-LSEs were designated as ideal to be tested for biostimulant potential by means of seed priming.

Additional experiments were carried out to determine the phytochemical as well as the elemental composition of the EF-LSEs. A preliminary phytochemical screening of the EF-LSE was done, which exposed the existence of more than a few compounds, such as carbohydrates, protein, phenols, flavonoids, saponins, tannins, and coumarins. Carbohydrates from different seaweeds were found to act as growth promoters of several crops such as tomato, soybean, duckweed and mung bean [40–42]. Proteins from seaweeds are recorded for their enhanced plant biostimulant activities in mung bean [43], and cherry tomato plants [44]. Proteins help plants to alleviate stress and increase their tolerance levels against abiotic stress like heat, cold, salt and even heavy metals [45].

Seaweeds are rich in phenolic compounds with varied bioactive properties [46,47]. Rajauria et al. [48] identified and characterized eight phenolic compounds from brown Irish seaweed *Himanthalia elongate*, which exhibited strong antioxidant activities. Chanthini et al. [6] correlated the levels of phenolic compound concentration with their antifungal potential. *U. flexuosa* had a considerate amount of phenols (1 mg/g of dry weight). Farasat et al. [49] detected higher phenolics as well as flavonoid levels from *U. flexuosa* and other edible green seaweeds. Besides, *Ascophyllun nodosum* extracts were able to increase the levels of phenols and flavonoids together, post application [50]. Saponins showcase a wide array of biological activities that play a pivotal role in plant growth as well as defense [51]. Coumarins also play a crucial part in plant development. These compounds have been proven with plant growth promotion capabilities alone and also in combination with phytohormones in faba bean [52]. Besides, the coumarin compounds were able to stimulate seed germination and seedling growth of wheat and sorghum seeds at optimum concentrations [53].

Tuhy et al. [54] testified that plant biomass surged by treatment with seaweed-derived micronutrients. The composition and functioning, together with the yield of all the plants, are reliant on their chlorophyll contents [55]. The chlorophyll content of *U. flexuosa* ranged up to 0.9 mg/g, which is relatively high among several other green seaweeds. This was also in agreement with the results published by Rathod [56]. The elemental composition performed revealed the presence of seven elements (O, Na, Mg, S, Cl, K and Ca) present on cell wall surface. The seaweed was 56.25% *w*/*v* of oxygen and along with high phenol content may be regarded as excellent candidates of antioxidizing agents [57]. Plant growth promoting elements such as sodium and potassium present in the cell wall of the seaweed makes them appropriate biofertilizers, besides a broad-spectrum of applications in the agricultural sector [58]. Several other mineral compounds such as chlorine, magnesium and calcium that are critical plant micronutrients are extant in the cell surface.

Tomato seeds treated with EF-LSEs displayed a positive response with respect to early germination, mean germination time, germination percentage, energy as well as better seed vigour index. Seed priming treatments achieved with quite a few plant derivatives such as plant hormones have been operational in the enhancement of seed germination of *Angelica glauca*, a threatened medicinal herb [59] as well as endive and chicory [60]. Seaweed extracts have been proven to show development enhancing properties on plant as well as seeds of various plants [61,62]. Also, priming seeds promoted early emergence of brinjal and tomato seeds compared with un-primed [63].

The seeds primed with EF-LSEs of *U. flexuosa* was analysed in different concentrations, comparing with the standard SFS solution and the control. The EF-LSE treated seeds displayed an increased germination percentage, exhibiting a lower mean germination time (MGT), taking only 2.2 days to emerge. *Codium tomentosum*, a green seaweed extract-treated aubergine seeds displayed lower MGT [64]. The potential of EF-LSEs to stimulate seed germination was reported long back in ornamental plants [65], green Chilies and Turnip [66]. Kavipriya et al. [67] also reported that priming of green gram seed with different seaweed extracts such as *Ulva lactuca* and *Caulerpa scalpelliformis* induced faster seed germination. Rapid seed emergence was recorded by priming the red gram seeds with the extracts of *Sargassum myriocystum* [68]. Furthermore, the positive effects of seaweed on the germination of green [69] and black gram [70] were also noted.

The comparative increase in seed emergence is correlated with seed eminence that is appeared to be augmented by the treatment of EF-LSEs. In addition, Amabika and Sujatha [68] proved that seed quality can be assessed by determining their seedling vigour index. Higher SVI of EF-LSE treated seeds implies an upliftment in seed quality. This was also evident from the results of other parameters of EF-LSE treated seeds, exhibiting higher seedling-plant height, radicle-root, as well as plumule-shoot lengths and dry-wet weight, in comparison with the control. Similar results of increased SVI from seed priming with EF-LSEs of *U. lactuca, U. reticulata, Padina pavonica*, *S. johnstonii* were correlated with that of increased seed germination and growth rates of brinjal and tomato, along with chilli [63].

As the primary developmental plant growth phase, the radicle and plumule are of prominent importance to determine the foundation of a plant. Seeds with an eminent radicle and plumule grow hastily, besides having an amplified competence [71]. Longer radicle lengths are also indicators of greater plant establishment efficiency. Seeds that produce shortened radicle-plumule might have issues in nutrient conduction to the embryo [72]. The EF-LSE primed seeds exhibited higher lengths of radicle and plumule, which improved with higher concentrations. EF-LSEs of seaweeds, *S. wightii* and U. *lactuca*, were demonstrated in their latent seed germination, besides plant growth promotion capabilities [73,74]. Similar increase of radicle-plumule lengths of tomato seeds was observed with the EF-LSEs of *C. sertularioides* and *S. liebmannii* [75].

The growth enhancement displayed by the EF-LSE primed seeds is owed to the occurrence of essential plant macro- and micro-nutrients, in addition to phytohormones. Di Filippo-Herrera et al. [76] reported the biostimulant activity of red seaweeds (*Acanthophora spicifera*, *Gelidium robustum*, and *Gracilaria parvispora*) and brown seaweeds (*Macrocystis pyrifera, Sargassum horridum* and *Ecklonia arborea*) primed on seeds of mung bean, which is primarily due to their nutritional and hormonal constituents. The fact that seaweeds stimulate plant growth has been documented by various researchers worldwide [77,78].

Similarly, EF-LSEs were earmarked for plant growth promotion by amplifying the growth of root and shoot of the tomato seeds, thereby displaying increased heights, compared to the control. Plant height was higher compared with seeds treated with SFS. Seaweed-treated seeds exhibiting higher root shoot lengths and ratio were previously reported in many studies [74,79,80]. Unlike the plant assessment results, comparatively developed shoot lengths remained. This suggests that the distribution of photosynthates and other compounds that aid in plant growth has shifted towards the shoot or increased in the above ground area. This could pave the way for the increase of plant yield [76].

Seed weight is considered an ecologically crucial character in plant progress, by way of influencing the establishment capacity of a seedling, as well as plant height and yield. This was proved by Wuff [81], who found that the *Desmodium paniculatum* seeds of higher biomass produced a high yield. In addition, the EF-LSE treated seeds displayed increased wet and dry weights equated with control, besides improving with an EF-LSE concentration. This was in agreement with the results published by Karthikeyan and Shanmugam [82], who studied the effect of *Kappaphycus alvarezii* extract on peanuts. Vijayakumar et al. [83] also reported the increase in seed weight of *Capsicum annum* by treatment with *Codium decorticatum* EF-LSE. Increased seed weights produced plants with higher height, shoot mass, and yield [84]. Seed weight increase might be due to the production and accumulation of storage oils and several proteins that might promote plant growth abilities [85]. Seeds take in surrounding water, protoplasmic macromolecules by imbibition. The EF-LSEs are actively imbibed in the seed through capillary action, thus increasing biomass. Seeds with higher biomass have been reported to have better seedling growth. Sun et al. [86]. reported that maize seeds with increased biomass had high yields.

The biomass of EF-LSE treated seeds increased with time, thus revealing that the higher imbibing rate occurs at later hours of priming. This is attributed to the active process of enzymatic breakdown and mitosis essential for emergence. The increased imbibing rate of seeds exposed to EF-LSEs is primarily due to the abundance of plant essential nutrients and phytohormone composition of the EF-LSEs. *U. lactuca* and *P. gymnospora* primed tomato seeds exhibited higher imbibition rates during later stages of priming and hence were reported as more successful and better candidates for developing effective biostimulants to improve the growth of tomato plants (Hernández-Herrera [9]).

With respect to the biostimulant potentials of seaweed extract, the yield and quality parameters analyzed presented favorable results. The EF-LSE primed seeds observed an increase in yield, attributed to the presence of phyto-hormones and various plant growth promoting elements, as evident from the EDX analysis of the seaweed. Furthermore, the flavour of the cherry tomato fruit is directly proportional to the amount of TSS [87]. The nutritional value of these tomatoes is based on ascorbic acid content, which was also enhanced by the EF-LSE. Lycopene content, which is the reason for the ripening of fruits at an optimum stage, was also enhanced by EF-LSE treatment. Phenolic compounds, an indication of plant innate defense system, were augmented on treatment with seaweed extracts. These results are in agreement with that of Murtic et al. [88].

Since the cherry tomato gene pool, a wild relative of tomato, provides an opportunity to produce more nutritive and resilient tomato cultivar varieties, an attempt to preserve and conserve this inclusive gene pool in gene banks is critical [89]. Additionally, these wild relative crop types are nutritional repositories, whose cultivation shall be enhanced to meet the increasing global food security targets. The establishment of these species can be further stimulated by seed priming techniques. The application of seaweed extracts as a seed priming agent towards the improvement of agronomic traits of cherry tomato have resulted in positive responses to amplified seed germination capabilities, germination energy, and augmented seedling establishment. In addition, seed priming effects have induced a long-lasting priming effect by altering plant and fruit physiology in a favorable way, by increasing their biochemical constituents and fruit yield. Hence, this study proves the potential of *U. flexuosa* as a potential agricultural biostimulant that is both economic and effective.

**Author Contributions:** K.M.-P.C. and S.S.-N.; methodology, K.M.-P.C.; software, K.M.-P.C. and S.S.-N.; validation, V.S.-R., A.T., S.K., and R.S.; formal analysis, S.S.-N.; investigation, K.M.-P.C., resources, R.P., and A.T.; data curation, S.S.-N.; writing—original draft preparation, K.M.-P.C.; writing—review and editing, K.M.-P.C., and S.S.-N.; visualization, K.M.-P.C.,V.S.-R., N.S.S., H.S. and R.P.; supervision, S.S.-N.; project administration, R.S.; funding acquisition, S.S.-N.

**Funding:** This research was funded by the Department of Biotechnology, Ministry of Science and Technology grant number BT/IN/Indo-US/Foldscope//39/2015 dated 20 April 2018.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

#### **Abbreviations**


#### **References**


© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

## *Article* **Wild** *Vigna* **Legumes: Farmers' Perceptions, Preferences, and Prospective Uses for Human Exploitation**

#### **Difo Voukang Harouna 1,3, Pavithravani B. Venkataramana 2,3, Athanasia O. Matemu 1,3 and Patrick Alois Ndakidemi 2,3,\***


Received: 17 April 2019; Accepted: 24 May 2019; Published: 31 May 2019

**Abstract:** The insufficient food supply due to low agricultural productivity and quality standards is one of the major modern challenges of global agricultural food production. Advances in conventional breeding and crop domestication have begun to mitigate this issue by increasing varieties and generation of stress-resistant traits. Yet, very few species of legumes have been domesticated and perceived as usable food/feed material, while various wild species remain unknown and underexploited despite the critical global food demand. Besides the existence of a few domesticated species, there is a bottleneck challenge of product acceptability by both farmers and consumers. Therefore, this paper explores farmers' perceptions, preferences, and the possible utilization of some wild *Vigna* species of legumes toward their domestication and exploitation. Quantitative and qualitative surveys were conducted in a mid-altitude agro-ecological zone (Arusha region) and a high altitude agro-ecological zone (Kilimanjaro region) in Tanzania to obtain the opinions of 150 farmers regarding wild legumes and their uses. The study showed that very few farmers in the Arusha (28%) and Kilimanjaro (26%) regions were aware of wild legumes and their uses. The study further revealed through binary logistic regression analysis that the prior knowledge of wild legumes depended mainly on farmers' location and not on their gender, age groups, education level, or farming experience. From the experimental plot with 160 accessions of wild *Vigna* legumes planted and grown up to near complete maturity, 74 accessions of wild *Vigna* legumes attracted the interest of farmers who proposed various uses for each wild accession. A X2 test (likelihood ratio test) revealed that the selection of preferred accessions depended on the farmers' gender, location, and farming experience. Based on their morphological characteristics (leaves, pods, seeds, and general appearance), farmers perceived wild *Vigna* legumes as potentially useful resources that need the attention of researchers. Specifically, wild *Vigna* legumes were perceived as human food, animal feed, medicinal plants, soil enrichment material, and soil erosion-preventing materials. Therefore, it is necessary for the scientific community to consider these lines of farmers' suggestions before carrying out further research on agronomic and nutritional characteristics toward the domestication of these alien species for human exploitation and decision settings.

**Keywords:** non-domesticated legumes; *Vigna racemosa*; *Vigna ambacensis*; *Vigna reticulata*; *Vigna vexillata*; Tanzania; wild food legumes

#### **1. Introduction**

Legumes (family: Fabaceae) possess an undeniable vital nutritional value for both humans and animals due to their protein content. They are known to be the second most valuable plant source of nutrients for both humans and animals, and the third largest family among flowering plants, with about 650 genera and 20,000 species [1]. Some of the most commonly domesticated, grown, and commercialized legumes such as soybeans, cowpeas, common beans, and other forms have demonstrated considerable contribution to the global food security [2]. Yet, their production rate remains unsatisfying compared with their consumption rate due to biotic and abiotic challenges [3]. Therefore, there is a need to look for alternatives. A systematic screening of the hitherto wild non-domesticated and wild relatives of the domesticated species within the commonly known and the little-known genera of legumes might be a promising strategy.

The *Phaseolus* and *Vigna* genera comprises the most widely consumed legumes, namely common beans (*Phaseolus vulgaris*) and cowpea (*Vigna unguiculata*) [2,4,5]. Within each genus, there are fewer domesticated edible species as compared with the numerous non-domesticated wild species. Some domesticated or semi-domesticated species have been termed as neglected and underutilized species due to little attention being paid to them or the complete ignorance of their existence by agricultural researchers, plant breeders, and policymakers [6]. This study mainly focuses on the genus *Vigna*.

The genus *Vigna* is a huge and important set of legumes consisting of more than 200 species [7]. It comprises several species of agronomic, economic, and environmental importance. The most common domesticated ones include the mung bean [*V. radiata* (L.) Wilczek], urd bean [*V. mungo* (L.) Hepper], cowpea [*V. unguiculata* (L.) Walp.], azuki bean [*V. angularis* (Willd.) Ohwi & Ohashi], bambara groundnut [*V. subterranea* (L.) Verdc.], moth bean [*V. aconitifolia (Jacq.) Maréchal*], and rice bean [*V. umbellata* (Thunb.) Ohwi & Ohashi]. Many of these species are valued as forage, green manure, and cover crops, besides their value as high protein grains. The genus *Vigna* also comprises more than 100 wild species that do not possess common names apart from their scientific appellation yet [8]. They are given different denotations such as underexploited wild *Vigna* species, non-domesticated *Vigna* species, wild *Vigna*, or alien species, depending on the scientist [2,7,9].

The rapid evolution, distribution, and spreading of improved bred crop varieties due to breeding programs and domestication in order to respond to food security challenges have also impacted positively on the negligence and disappearance of wild crop relatives [10,11]. This is certainly a negative impact vis-à-vis the species' biodiversity conservation. From that perspective, one could imagine and question the awareness, beliefs, and preferences of some generations regarding the origin of the consumed modern crops. This may explain the stigma about the consumption and even the existence of these wild legumes, and therefore their rejection as food while they have been used as such in the past in some cases.

Food acceptability and food choices are usually influenced by many factors in which sensory preferences play an important role [12]. The nutritional composition is also a very essential characteristic to consider in food selection and consumption, as it is directly linked to consumers' health and well-being. Unfortunately, this parameter may only be seriously considered in parts of the world where food accessibility, availability, and affordability are not challenged. Hence, much is needed to be done in this line to investigate the nutritional composition of wild crop foods together with close understanding of their social acceptability.

Investigations on the chemical composition of wild *Vigna* legumes seem to be less attractive to the scientific community for reasons yet to be established. Research in that line has remained silent and undocumented for more than a decade [13]. The latest report shows that some of the wild *Vigna* accessions studied present nutrient levels comparable to those of some domesticated species with exceptionally higher levels of sulfur amino acids [13]. However, it is highly necessary at this point to think about the acceptability of these wild legumes by farmers and consumers before any further research is conducted in order to orient the improvement, adoption, and domestication for a proper exploitation to the benefit of mankind.

This study explores experienced legumes, cultivating farmers' awareness, perception, acceptability, and preferred uses for some accession of wild *Vigna* legumes (*Vigna racemosa*, *Vigna ambacensis*, *Vigna reticulata,* and *Vigna vexillata*). The study has been organized into two parts, considering farmers' awareness in the first part and preferences for wild legumes in the second.

#### **2. Materials and Methods**

#### *2.1. Study I: Explorative Survey*

The aim of this study was to ascertain farmers' awareness about the existence of wild non-domesticated legumes and their uses in addition to challenges and experiences related to the growth and domestication of wild legumes.

The study was conducted among legume farmers in a mid-altitude agro-ecological zone (Arusha Region) and a high altitude agro-ecological zone (Kilimanjaro Region) of Tanzania where legume cultivation is intensified, as shown in Figure 1A [14]. A purposive sampling from a crop-growing population of 0.13% (37,985) from Arusha [15] and 0.17% (56,710) from Kilimanjaro [16] were used to obtain a representative sample size. The total number of farmers involved in legume improvement programs included 50 from the Seliani Agricultural Institute (TARI), Arusha and 100 from the Tanzania Coffee Research Institute (TaCRI), Moshi, Kilimanjaro regions, respectively (Figure 1B). A systematic selection of farmers who had at least two years of trying locally improved legume varieties was performed. An individual face-to-face interview with the help of a semi-structured questionnaire prior to participant experimental plot visit was executed to obtain a broad range of individual opinions and explore their awareness of wild legumes. The questionnaire consisted of 24 items including sociodemographic characteristics. The items were categorized and analyzed to assess the sociodemographic characteristics of participants, their prior knowledge/awareness about wild legumes, and the uses of wild legumes as known by experienced farmers as well as some challenges faced by legume farmers.

#### *2.2. Study II: Farmers' Preferences and Perceptions of Wild Vigna Legumes*

The main aim of this study was to identify farmers' perceptions and prospective uses of preferred accessions of wild legumes based on morphological agronomic characteristics in order to direct the domestication process.



GRC, IITA: Genetic Resource Center, Germplasm Health Unit, International Institute of Tropical Agriculture (IITA), Headquaters, PMB 5320, Oyo Road, Idi-Oshe, Ibadan-Nigeria. AGG: Australian Grain Genebank, Department of Economic Development, Jobs, Transport and Resources, Private Bag 260, Horsham, Victoria 3401.

**Figure 2.** Microphotographs illustrating seed morphology of some wild *Vigna* species; Four (4) seeds per accession were pictured under the same conditions to give an image of the morphology and the relative size. Distances of lines in the background are 1 cm in the vertical and horizontal directions. Source: Images taken and compiled by the authors based on seeds requested from the Australian Grain Genebank (AGG) (**a**–**e**,**q**–**t**) and the Genetic Resources Center, International Institute of Tropical Agriculture, (IITA), Ibadan-Nigeria (**f**–**p**).

#### 2.2.1. Sample Collection

One hundred and sixty (160) accessions of wild *Vigna* species of legume were obtained from gene banks as presented in Table 1 with their details in Appendix A. All the accessions were planted in an experimental plot following the augmented block design arrangement [18] and allowed to grow until near maturity before inviting the farmers to explore their opinions. Since the accessions did not show uniform growth patterns due to their genetic differences, farmers were invited when more than 50% of the accessions reached maturity. An illustration of the seeds of some of the samples is also shown in Figure 2. In addition, three domesticated *Vigna* legumes—that is, cowpea (*V. unguiculata*), rice bean

(*V. umbellata*), and a semi-domesticated landrace (*V. vexillata*)—were used as checks. The checks were also obtained from the Genetic Resource Center (GRC-IITA), Nigeria, the National Bureau of Plant Genetic Resources (NBPGR), India and the Australian Grain Genebank (AGG), Australia respectively.

#### 2.2.2. Experimental Design and Study Site

The study was conducted in two agro-ecological zones located at two research stations in Tanzania during the main cropping season (March-September 2018). One was at the TaCRI, located at Hai district, Moshi, Kilimanjaro region (latitude 3◦13 59.59" S, longitude 37◦14 54" E). The site is at an elevation of 1681 m above sea level, with a mean annual rainfall of 1200 mm and mean maximum and minimum temperatures of 21.7 ◦C and 13.6 ◦C, respectively. The second site was at the Tanzania Agricultural Research Institute (TARI), Selian Arusha in the northern part of Tanzania. TARI-Selian lies at latitude 3◦21 50.08" N and longitude 36◦38 06.29" E at an elevation of 1390 m above sea level (a.s.l.) with mean annual rainfall of 870 mm. The mean maximum and minimum temperatures ranged from 22 ◦C to 28 ◦C and 12 ◦C to 15 ◦C, respectively.

The 160 accessions of wild *Vigna* legumes were planted in an augmented block design field layout following the randomization generated by the statistical tool on the website (http://www.iasri.res.in/ design/Augmented%20Designs/home.htm) [19] for 160 treatments with three checks. The field was monitored and maintained in good conditions from germination to near maturity of 75% of all the accessions before inviting farmers to assess their opinions.

#### 2.2.3. Participants and Data Collection

Participants in the previous study (Study 1) in the Arusha (N1 = 50) and Kilimanjaro (N2 = 100) regions also participated in this study. Field visits were done in groups of five participants. A trained research assistant was recruited to guide the participants around the experimental field from the first to the last block or vice versa. A semi-structured questionnaire was used to collect information on the most preferred accessions (at least 10), and reasons for each selection were given. Every accession was assigned a number to ease participant selection. The number of times each accession was selected was divided by the total number of selections and multiplied by 100 to give the percentage of selection of each accession.

#### 2.2.4. Focus Group Discussion

Participants in their respective regions were further grouped into two groups based on their gender, men and women, giving a total of four group interviews. Each group was invited to participate in an animated video-recorded focus group interview to ascertain their opinion about wild *Vigna* legumes, as obtained in the previous studies. The recorded videos (04) were transcribed verbatim and translated from Swahili language to English. The transcripts were cross-checked with the recordings by the interviewers to align transcripts with notes on non-verbal responses. A coding framework was developed based on the interview objectives and the interview guide. The qualitative data analysis package NVivo 11 (QRS International, 2015) was used to code and organize the data systematically as described by other workers [12]. Key concepts and categories were identified.

#### *2.3. Data Analysis*

For study I, the collected information during the survey was grouped, coded, organized, and analyzed using the statistical package IBM SPSS Statistic 20.0 (New York, NY, USA). Analysis consisted of the descriptive statistics as well as the binary logistic regression to test for the relationship between the prior knowledge about the wild *Vigna* legumes and the farmers' sociodemographic characteristics.

In the case of study II, data were coded and entered in the statistical package IBM SPSS Statistic 20.0 and analyzed. Analysis included descriptive statistics and likelihood ratio test of X2 to determine the relationship between the preferences and the farmers' gender, farming experience, and research location [20].

#### **3. Results**

#### *3.1. Study I*

#### 3.1.1. Sociodemographic Characteristics of Participants

The results from the sociodemographic characteristics showed that 64% and 36% were female and male farmers, respectively (Figure 3a). Most of the participants were above 45 years old, with the highest level of education being primary (Kilimanjaro) and secondary (Arusha). Furthermore, most of the farmers had a reasonable number of years of experience farming legumes, varying from two to more than 35 years of farming (Figure 3d). The intervals of years of farming experience and the percentages of participants with the longest farming experience were 6–10 and 16–20%, respectively (Figure 3d).

#### 3.1.2. Prior Knowledge/Awareness about Wild Legumes

Less than 30% (28% and 26% in both study sites) of the experienced participants involved in the study were aware of the existence of wild legumes (Figure 4). According to the binary logistic regression analysis (Table 2), the model including the farmers' sociodemographic characteristics as explanatory variables and prior knowledge of legumes as a dependent variable is a good fit with the data as *p* = 0.633 > 0.05 (*Hosmer and Lemeshow test*). This explains that the variance in the outcome is significant (X<sup>2</sup> = 40.632, df = 19, p.003) (Omnibus Tests of Model Coefficients). The results show that there is no significant association between the prior knowledge about wild legumes and the overall gender (Wald = 0.495, df = 1, *p* > 0.05) (Table 2). However, there is a slightly effect associated with being a female farmer and prior knowledge (B = 0.303, *p* = 0.482). No significant relationship existed between the overall farmers' age groups and their prior knowledge of wild legumes (Wald = 7.061, df = 6, *p* = 0.315 > 0.05), although there is a slight significance relationship with the youngest age group [15–20] (Wald = 4.113, df = 1, B = 2.982, *p* = 0.043), as shown in Table 2. In the same vein, the test shows that the education level (Wald = 3.962, df = 4, *p* = 0.411) as well as their farming experience (Wald = 5.462, df = 7, *p* = 0.604) do not have any influence to their prior knowledge about wild legumes. On the contrary, the location (research site) has a significant effect on their prior knowledge of wild legumes (Wald = 9.884, df = 1, B = 1.687, *p* = 0.002).

#### 3.1.3. Prior Uses of Wild Legumes

A few participants who had prior knowledge of wild legumes mentioned several uses attributed to the wild legumes they had seen before. Some of the uses mentioned were livestock feed, human food, and soil fertility ingredients as well as botanical pesticides (Table 3).

#### 3.1.4. Challenges Faced by Legume Farmers

Diseases and drought (or reduced rainfall) were the most challenges faced by the farmers in both mid and high altitude agro-ecological zones (Figure 5). Apart from diseases and reduced rainfall issues, other reported challenges were related to market, pest, and storage (Figure 5). Taste and cooking aspects were not of very serious concern to the farmers in the two zones, since most of them seemed to be comfortable with the taste and cooking aspects of their legumes.

**Figure 3.** Sociodemographic characteristics of participants (**A**): participants' gender per study area (%); (**B**): participants' age groups; (**C**): participants' education level; and (**D**): participants' legumes farming experience.

#### *3.2. Study II*

3.2.1. Farmers' Preferred Accessions of Wild *Vigna* Legumes

The study shows that 74 accessions out of the 160 planted and grew to an appreciable level at the screening moment and were selected based on the participants' personal preferences (Figure 6). In the high-altitude zone (Kilimanjaro), only five (5) accessions (TVNu-293, TVNu-758, AGG308107WVIG 2, AGG308101WVIG 1, and TVNu-1546) were selected by the farmers more than half of the time, while in the mid-altitude zone (Arusha), none of the accessions had up to 50% selection (Figure 6). The five most selected accessions in the mid-altitude zone—TVNu-293 (36%), TVNu-758 (36%), AGG51603WVIG 1 (30%), AGG308099WVIG 2 (40%), and AGG53597WVIG 1 (34%)—were different from those selected in the high-altitude zone, except for TVNu-293 and TVNu-758.

The likelihood ratio test revealed that the wild *Vigna* selection (preferences) significantly depended on the farmers' gender (G2 = 130.813, df = 73, *p* < 0.000), farming experience (G2 = 669.196, df = 511, *p* < 0.000), and location (G<sup>2</sup> = 1110.606, df = 73, *p* < 0.000).

**Figure 4.** Participants' prior knowledge of wild legumes. TaCRI: Tanzania Coffee Research Institute.

**Figure 5.** Participants' challenges faced during legumes cultivation in the two study areas: (**a**) Arusha and (**b**) Kilimanjaro.


*Agronomy* **2019**, *9*, 284

a. dependent variables from the independent variables; S.E.: Standard errors associated with coe

Sig.: Significance level (p-value); EXP(B):

Exponentiation

 of the coe

fficients (odd ratios for the predictors); C.I.: Confidence Interval.

fficients; Wald: Wald X2 value; df: Degree of freedom for each of the tests of the coe

 the fficients;



#### 3.2.2. Prospective Uses of Farmers' Preferred Accessions of Wild *Vigna* Legumes

The suggested uses of selected accessions were based on their personal assessment and preferences. Some accessions were selected for more than one use, and the number of selections for every accession is shown on Figure 7a–e. Other uses were proposed by farmers that better suited the accession of their choice. Four main uses (human food, animal feed, forage, and cover crop) were proposed as a result of farmer's preferences and perceptions. Therefore, a total of 31 accessions were preferred as human food (Figure 7a), 49 were preferred as animal feed (Figure 7b), 27 were preferred as forage (Figure 7c), 28 were preferred as cover crop (Figure 7d), and 44 were given specific personal uses (Figure 7e), respectively.

Four accessions were selected at least 30 times or more as human food, while 27 accessions were selected less than 30 times for the same purpose (Figure 7a). The four most selected accessions for this purpose were TVNu-1359 (36), AGG308099WVIG 2 (34), AGG53597WVIG 1 (32), and AGG51603WVIG 1 (30), respectively.

**Figure 7.** *Cont*.

**Figure 7.** *Cont*.

**Figure 7.** (**a**) Wild *Vigna* legumes suggested as a human food; (**b**) Wild *Vigna* legumes suggested as animal feed; (**c**) Wild *Vigna* legumes suggested as forage; (**d**) Wild *Vigna* legumes suggested as cover crop; and (**e**) Wild *Vigna* legumes given specified uses.

Four other accessions were also selected at least 30 times or more by participants as animal feed in the two study sites combined. The selected accessions were TVNu-1546 (18 + 55), TVNu-293 (12 + 34), TVNu-758 (18 + 26), and AGG308101WVIG 1 (35), respectively (Figure 7b).

Only one accession was selected up to 30 times to serve as forage (Figure 7c), while none of the preferred as cover crop accessions were chosen up to 30 times by the participants in both study sites (Figure 7d).

Out of the 44 selected accessions with specified uses, only two accessions—AGG308107WVIG 2 (35) and AGG308100WVIG 3 (36)—were selected more than 30 times (Figure 7e).

All of the non-domesticated wild *Vigna* legumes subjected to this study belonged to four species, *V. racemosa*, *V. reticulata*, *V. vexillata*, and *V. ambacensis*. In summary, it has been shown that the *V. vexillata* accessions were more preferred, followed by *V. reticulata* and *V. racemosa* (Figure 8). Despite the higher number of *V. ambacensis* accessions as compared with *V. racemosa*, it was less selected than *V. racemosa*.

**Figure 8.** Wild *Vigna* legumes selected according to their species.

From their sight and appraisal of the wild *Vigna* legumes, other uses could be organic manure (locally known as '*Mbolea*'—fertilizer), business use, medicinal uses, preventers of soil erosion, and vegetable food for accessions with nice leaves (Figure 9). For personal uses, none of the accessions was selected up to 30 times or more. However, five accessions were selected more than 20 times at least for a specific use. The selected accessions were AGG308100WVIG 3 (24) and TVNu-738 (24) for soil erosion mitigation, and TVNu-1582 (22), TVNu-1546 (26), and AGG308107WVIG 2 (28) for soil fertility as an organic manure agent, respectively (Figure 9).

**Figure 9.** Specified uses of wild *Vigna* legumes as proposed by farmers in the two agro-ecological zones of Tanzania.

3.2.3. Farmers' Perception of Wild *Vigna* Legumes

From the focus group discussion, most farmers perceived some accessions of wild *Vigna* legumes as good material for future promising business in the field of agriculture due to their high seed production, resistance to drought conditions, and high production of leaves, which can benefit both humans and animals as forage. For example, a male farmer from the Arusha region during the group discussion enthusiastically responded when the interviewer asked whether they would be willing to adopt some of the wild *Vigna* legumes presented to them for the first time. He said: "Yes, I like some of these beans because many people don't know about them, they are found in the bush but people don't know that they can be useful, so if we discover their usefulness, this can be a great source of good business because they seem to have a higher productivity as compared with other known beans." To support the view, another voice rose in the hall and said: "Yes, I also like some, because after seeing these crops planted in the farm (referring to the wild legumes of study), I discovered that there are other new varieties of legumes, and this may be another source of food. I also realized that some of them have nice leaves that can be used as vegetables, and some can help us feed our cattle."

A smaller proportion of farmers (represented by 26% and 28% in study I, as shown in Figure 3), who curiously noticed the existence of wild legumes before the study, confirmed having seen some of the planted legumes of the study and having consumed them or used them as medicine for animals and even humans. One of the most interesting views that supported this point was from one of the old female farmers in the Arusha region, who said: "This variety with [a] large number of leaves lying on the ground (referring to one of the varieties of the study with a spreading growth habit), I have consumed them several times when I was a kid. Back then, our mothers used to go to the bush and harvest their leaves, and then go to town and buy maize and come back to cook them together. Myself, I have eaten them and we used to call that meal {*Ngolowo*}, which is very delicious and when we mix it with milk, it looks similar to another meal called {*Rojo*}. So for that one, it is not a poison, because I have eaten it before, it is a food, the leaves are eaten and the seeds are also eaten; it is called {*Ngolowo*}". All her mates in the hall during the group discussion listened to her speech with very attuned ears and clapped at the end. A similar view came from the group of men, which was articulated in these terms: "I have seen these beans before growing in the bush and we were using them as food and feed for animals; then, when I saw it here, I just confirmed that it is edible. Animals enjoy them so much. We used to take them from the bush and consume them and we had no health problems with them, and after I saw it here in the farm, I just realized that it is a normal food. It has never affected our people negatively after consuming them.

However, most of the participants in general proposed that more research and improvements were needed, especially in terms of the toxicity and nutritional benefits, as well as the seed color of the legumes to increase their acceptability for efficient exploitation and utilization. "One of the varieties I saw in the farm numbered 132 looks nice; it looks similar to (Choroko, Swahili word for Mung bean). So, I think that if it can be improved, it will be good for business because it has high productivity and nice leaves, but we don't know if it is not toxic or can negatively affect our health", said a participant who was supported by another one, who said: "similar to this one (participant showing some seeds harvested from the experimental fields), if the color can be improved, it will be very nice, because people in the market don't like buying black-colored beans. Their reason is that the black-colored seeds turn the cooking water black and that is not preferable for them. The black-colored seed beans are only preferred during hunger seasons; that is, seasons where less rainfall has affected the crop yield in the community."

#### **4. Discussion**

The explorative survey above shows that women were more engaged in legume farming in the two zones compared with men. Similarly, the contribution of women in agricultural activities is well-known in Africa [21]. In this study, no statistical significance was found between gender influence and prior knowledge about legumes. This means that being a woman or a man does not influence the probability of being aware of wild legumes.

Legume farming was mainly practiced by the older participants (Figure 3b). This indicates that the younger generations in the areas were not very interested in legume-farming activities or farming other crops. In general, belonging to any age group did not influence the prior knowledge about the legumes, due to the long period of disappearance of the wild genotypes, which led to the ignorance of many generations of people [2,8,11]. However, belonging to the 15 to 20-year-old age group showed a slight influence on the prior knowledge of wild legumes. This may suggest that farmers in this age range may possess some understanding of wild legumes.

The education level of farmers and their farming experience showed no significant influence on their prior knowledge of wild legumes, which meant that being educated or well experienced in farming legumes did not influence the knowledge of wild legumes. This showed that both experienced and non-experienced farmers as well as educated and non-educated farmers might have the same perception and prior background about wild legumes. In addition, it implied that both farming experience and level of education may not be necessary when making policy decisions about the implementation or adoption of a wild legume as a new crop. However, this is in contradiction with other studies carried out using other domesticated crops such as rice and maize [22,23]. Then, it is necessary for further research to try such experiences with other wild crops in other parts of the world to ascertain this fact.

From the results, the location (research site) has a significant effect on the prior knowledge of wild legumes, meaning that being in the Arusha region increased the chance of knowing wild legumes. Decision making regarding the adoption of wild *Vigna* legumes needs to take the location of farmers into consideration. This is in line with earlier reports [24]. This could be explained by the Arusha region being more populated by a certain ethnic group of people (called the Maasai) who are well-known in Tanzania for their indigenous ethno-medical knowledge of plants [25,26]. They are also found in the high-altitude agro-ecological zone (Kilimanjaro), but they are more concentrated in the mid-altitude agro-ecological zone of Arusha [25].

The ignorance of the wild legumes by the majority of participants in the two study sites may be due to the high and long-term distribution of bred, improved, and landrace varieties of legumes that led to the disappearance, rejection, and negligence of the original wild legumes [2]. However, the numerous challenges (biotic, abiotic, and policy) faced by the improved varieties have recently raised scientific concerns [3]. Therefore, it might be important to go back to the wild and investigate other legumes with good characteristics in relation to their acceptability in order to mitigate the global food insecurity challenge, as pointed out by earlier reports [27].

It is noted from this study that despite the high ignorance noted by the majority, the wild legumes are still used for various purposes, including human consumption by a minority. It has also been noted that ignorance or knowledge/awareness of wild legumes significantly depends on the location of the farmers rather than their gender, age group, or farming experience. This could be explained by some ethnic groups of people with significant traditional and indigenous knowledge of plants being concentrated in some parts of the world [25]. Then, it would be wise to carry out more investigation on such legumes in order to domesticate more varieties possessing resistance to the current legumes challenges. From this study, the main challenges experienced by legume farmers in the two study sites were diseases and low rainfall, which might definitely be due to climate change, as it is global challenge [28]. Therefore, alternatives varieties of legumes with resistance to climate variability and diseases would be of great benefit to such similar communities. The study also attempted to screen some accession of choice by the same farmers based on the general appearance, pods, and seeds of some of the wild legumes in order to select varieties for domestication.

Furthermore, it was observed that the prior knowledge about wild legumes is independent of gender, age, education level, and farming experience, but dependent on the farmers' location. However, it is curiously noted that after carefully sighting the wild *Vigna* legumes performing in the field by participants, it is revealed that there is a significant relationship between the farmers' preferences and their gender, farming experience, and location (likelihood ratio test). This could explain that the knowledge of wild legumes increases farmers' attraction and preferences of wild legumes depending on their gender, farming experience, and location. Less than 50% (74 out of 160) of the planted accessions were preferred by farmers in both research sites (Figure 6), showing that several accessions had common preferences depending on the locations. Although this could be

influenced by the number of accessions that reached an appreciable growth level by the selection period, the selection should depend on other parameters such as farmers' gender (G<sup>2</sup> = 130.813, df = 73, *p* < 0.000), farming experience (G<sup>2</sup> = 669.196, df = 511, *p* < 0.000), and location (G2 = 1110.606, df = 73, *p* < 0.000), as confirmed by the X2 test. In a similar study, significant correlations between preferences of male and female farmers in an on-farm trial indicated that both groups have similar criteria for the selection of rice varieties in India [29]. Experiments investigating farmers' knowledge about unknown or wild food crops are lacking or almost non-existent in the literature [12]. The wild *Vigna* species are not well-known legumes, which could be the reason taxonomic characterizations have still been under investigation by scientists until recently [30].

The ignorance of wild legumes by the majority explains the few uses suggested by the farmers as compared with the uses suggested after field visits to farms with wild *Vigna* legumes (Figure 4 and Table 3). Several uses have been suggested by farmers after sighting the wild *Vigna* legumes in farms, showing their interest and motivation to adopt some of the wild crops for human benefit. This is in accordance with findings from earlier research studies carried out with domesticated legumes possessing characteristics that are not well-known [31,32]. It was observed that the farmers were willing to adopt some of the crops for several human exploitation purposes, although some need more improvement. It is also noted that some farmers even had experience consuming some of the wild *Vigna* legumes. Therefore, farmers generally perceived the wild *Vigna* legumes as exploitable resources for a variety of purposes that lack awareness and scientific attention. A recent report also demonstrated participant eagerness to adopt wild vegetables (duckweed) as human food upon first-time observations from a picture [12].

This study also shows that there is a high probability that any sample of farmers taken in Tanzania and any other region of the world would ignore the existence of wild legumes. Therefore, considering food insecurity levels in the developing world, the dependence on a few accessions of legumes, and the challenges faced by farmers and consumers regarding domesticated legumes, there is a need to further study these un-exploited legumes and orient their utilization. Very limited reports approaching the assessment of participants, farmers, or consumers' perception, appreciation, or adoption of wild plants as human food exist.

#### **5. Conclusions**

The existence of non-domesticated wild legumes is highly ignored by many farmers despite the presence of a large existing number in the gene banks and bushes around the world. The ignorance of wild legumes is generally not related to the farmers' gender, age group, or farming experience, while it is significantly related to their location. Besides, preferences in some accessions of wild legumes depend on the gender, farming experience, and location. In addition, the discovery of the wild *Vigna* legumes for the first time motivates the attraction of farmers to prefer them for various purposes. Farmers perceived wild *Vigna* legumes as human food, animal feed, medicinal plants, soil enrichment material, and soil erosion-preventing materials. Therefore, it is necessary for the scientific community to give better attention to these so-called alien species in order to improve their agronomic, nutritional, and physiological characteristics with prior consideration of farmers' and consumers' preferences and perception to orient their domestication, as it is the case here.

**Author Contributions:** P.A.N. conceived and designed the experiments; D.V.H. performed the experiments, collected data, analyzed the data, and wrote the first draft of the manuscript; P.B.V. and A.O.M. supervised the research and internally reviewed the manuscript; and P.A.N. made the final internal review and revised the final draft of the manuscript.

**Funding:** This research was partially funded by the Centre for Research, Agricultural Advancement, Teaching Excellence and Sustainability in Food and Nutrition Security (CREATES-FNS) through the Nelson Mandela African Institution of Science and Technology (NM-AIST) under the reference number P220/CAM.16. The research also received funding support from the International Foundation for Science (IFS) through the grant number I-3-B-6203-1.

**Acknowledgments:** This research was partially funded by the Centre for Research, Agricultural Advancement, Teaching Excellence and Sustainability in Food and Nutrition Security (CREATES-FNS) through the Nelson

Mandela African Institution of Science and Technology (NM-AIST). The authors acknowledge the additional funding support from the International Foundation for Science (IFS) through the grant number I-3-B-6203-1. The authors also acknowledge the Genetic Resources Center, International Institute of Tropical Agriculture (IITA), Ibadan-Nigeria as well as the Australian Grains Genebank (AGG) for providing detailed information on wild *Vigna* and seed materials for this research. The support this study received from the N2 Africa project based at the Nelson Mandela African Institution of Science and Technology (NM-AIST) is also well acknowledged. The authors are thankful to Frank E. Mmbando, Theresia L. Gregory, and Lameck Makoye of the Tanzania Agricultural Research Institute (TARI), Selian-Arusha, Tanzania for their technical support in recruiting farmers and reviewing the questionnaire. The authors also thank Djomo Raoul Fani (Department of Agricultural Economics, Federal University of Agriculture, Nigeria) and Elmugheira Mockarram Ibrahim Mohammed (Department of Forest Management Sciences, Faculty of Forest Sciences & Technology, University of Gezira, Sudan) for their technical advises regarding the data analysis.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **Appendix A**


**Table A1.** Wild *Vigna* legumes accessions used in the study.


**Table A1.** *Cont.*


**Table A1.** *Cont.*


**Table A1.** *Cont.*

#### **References**


© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

#### *Review*
