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European Journal of Applied Sciences – Vol. 10, No. 2
Publication Date: April 25, 2022
DOI:10.14738/aivp.102.11786. Ritte, I. P., Egnin, M., Idehen, O., Mortley, D., Bernard, G. C., Binagwa, P. H., Brown, A. P., & Bonsi, C. K. (2022). Evaluation of Cowpea
Morpho-physiological and Yield Responses to Vegetative and Pre-Anthesis Water-Deficit Stress Tolerance under Greenhouse
Conditions. European Journal of Applied Sciences, 10(2). 391-411.
Services for Science and Education – United Kingdom
Evaluation of Cowpea Morpho-physiological and Yield Responses
to Vegetative and Pre-Anthesis Water-Deficit Stress Tolerance
under Greenhouse Conditions
Inocent P. Ritte
Department of Agriculture and Environmental Sciences
Plant Biotech and Genomics Research Laboratory
College of Agriculture, Environment and Nutrition Sciences
Tuskegee University, Tuskegee, AL 36088, USA
Marceline Egnin
Department of Agriculture and Environmental Sciences
Plant Biotech and Genomics Research Laboratory
College of Agriculture, Environment and Nutrition Sciences
Tuskegee University, Tuskegee, AL 36088, USA
Osagie Idehen
Department of Agriculture and Environmental Sciences
Plant Biotech and Genomics Research Laboratory
College of Agriculture, Environment and Nutrition Sciences
Tuskegee University, Tuskegee, AL 36088, USA
Desmond Mortley
Department of Agriculture and Environmental Sciences
Plant Biotech and Genomics Research Laboratory
College of Agriculture, Environment and Nutrition Sciences
Tuskegee University, Tuskegee, AL 36088, USA
Gregory C. Bernard
Department of Agriculture and Environmental Sciences
Plant Biotech and Genomics Research Laboratory
College of Agriculture, Environment and Nutrition Sciences
Tuskegee University, Tuskegee, AL 36088, USA
Papias H. Binagwa
Department of Agriculture and Environmental Sciences
Plant Biotech and Genomics Research Laboratory
College of Agriculture, Environment and Nutrition Sciences
Tuskegee University, Tuskegee, AL 36088, USA
Adrianne P. Brown
Department of Agriculture and Environmental Sciences
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European Journal of Applied Sciences (EJAS) Vol. 10, Issue 2, April-2022
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Plant Biotech and Genomics Research Laboratory
College of Agriculture, Environment and Nutrition Sciences
Tuskegee University, Tuskegee, AL 36088, USA
Conrad K. Bonsi
Department of Agriculture and Environmental Sciences
Plant Biotech and Genomics Research Laboratory
College of Agriculture, Environment and Nutrition Sciences
Tuskegee University, Tuskegee, AL 36088, USA
ABSTRACT
Cowpea production is severely hindered by water scarcity; thus, understanding
morpho-physiological response mechanisms of known drought-tolerant cultivars
under water-deficit stress is critical to identify and establish representative yield- related traits of climate-hardy cowpeas. To determine cowpea genotypic variability
to drought-tolerance, seventeen days-post sowing (DPS) greenhouse plants were
subjected to 14-days drought stress without watering, then watered every 10-days
at 25%, 50%, and 75% field capacity (FC) until maturity in two-trial experiment.
Controls were well-watered at 100% FC every 3-days. Drought stress data were
collected on plant height, stem diameter, chlorophyll content and terminal leaflet
expansion rate. At maturity, 83 to 119 DPS, pod number, shoot and root biomass,
and seed yield per plant were recorded. Data were combined and analyzed using
analysis of variance. Drought tolerance was evaluated by percent change in
performance and stress tolerance indexes. Drought stress in both trials impacted
phenotypic expression. Plant height declined by 74%, stem diameter 18.2%,
chlorophyll content, 47.6% terminal leaf length 83.2%, and width 85.2%. Pods per
plant were reduced by 73% and seed yield by 98.8%. The estimated correlation
between morpho-physiological and other yield-related traits of drought-tolerance
indices verified that TVu 11987, LOBIA-I-SEFADE, and TVu 7362 were drought
tolerant along with confirmed tolerant commercial cultivars California Blackeye
No.5, Big Boy, and Lady. These cultivars exhibited different stress-coping strategies
of low water requirements and growth performance to yield reduction. Overall, the
genotypic performance recorded as drought-tolerant characteristics may be
recommended as potential screening factors for donor cultivar traits in cowpea
breeding programs.
Keywords: Cowpea (Vigna unguiculata), Water Stress, Field Capacity, Morpho- physiological Traits, Growth and Grain Yield Response.
INTRODUCTION
Cowpea [Vigna unguiculata (L.) Walp.], Fabaceae, (2n = 2x = 22) is an important legume and
inexpensive source of protein, vitamins, minerals and fiber for millions of low-income
households where it is consumed as dry grain and leaves as vegetables [1], [2]. According to
Food and Agriculture Organization (FAO), 14.5 million ha of land are devoted to cowpea
production, mainly in the African countries of Niger, Nigeria, Burkina Faso, Mali, and Sudan,
accounting for 75% of global grain production and 78% of cultivated area. However, the
average production in most African countries is below 1t ha-1 compared to the potential yield
of up to 3t ha-1 [3], [4] due to abiotic factors such as environmental fluctuations and soil and
water limiting conditions. These factors are important plant growth and development
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Ritte, I. P., Egnin, M., Idehen, O., Mortley, D., Bernard, G. C., Binagwa, P. H., Brown, A. P., & Bonsi, C. K. (2022). Evaluation of Cowpea Morpho- physiological and Yield Responses to Vegetative and Pre-Anthesis Water-Deficit Stress Tolerance under Greenhouse Conditions. European Journal
of Applied Sciences, 10(2). 391-411.
URL: http://dx.doi.org/10.14738/aivp.102.11786
determinants, particularly drought, which can severely limit the productivity and quality of
cowpeas. In the United States (U.S.), cowpea is an introduced field, and horticultural crop with
much of the production predominantly practiced in the southern states where cowpea remains
a staple food [4], [5], [6]. Cowpea production in the U.S. is practiced on about 5,220 ha in which
11,750 tons of cowpeas are harvested [3], [7] as dried seed commonly known as black-eyed
peas or southern peas, and often cooked, canned or frozen. Furthermore, some cowpeas are
harvested while the seeds are high in moisture, and sold fresh [4], [8], [9]. The U.S dry cowpea
production has been continuously declining due to changing cropping systems, environmental
fluctuations and overall food consumption trends as opposed to increased land devoted for
cowpea production in other regions [4], [9]. Other sources suggests that, the collapse of cowpea
acreage is associated with increased acreage of soybean due to ease of mechanization and
reduced pest problems [10]. However, cowpea is considered more drought-tolerant than
soybeans and well adapted to sandy soil types [11].Due to the importance of cowpea
worldwide, versatile end uses and unique grain characteristics, there is a dire need to
understand its tolerance performance under soil water deficit and extreme temperatures to
help develop hardy crops with higher grain yield stability and better stress adaptation to
changes in the global climate.
As global climate change and related problems increase, water shortage is becoming
increasingly alarming [12]. These environmental constraints not only impact plant growth,
yield, and water relations, but also membrane integrity, pigment content, and photosynthesis
[13], [14], especially during pre-anthesis. Cowpea production, especially in Africa, is primarily
grown under rainfed conditions thus, its productivity is essentially hindered by erratic rainfall
patterns which either come late, at the beginning of the season or stops earlier than usual,
leading to severe drought conditions during the growing season [15], [16]. Although cowpea
species are naturally well-adapted to growing in drier regions where other legumes do not
perform well, many varieties are potentially affected by various environmental factors
resulting in a serious reduction of crop yield and quality [17], [18]. Hence, the adaptability of
some cultivars to withstand both heat and drought is of significant interest for gaining insight
into their resiliency in extreme environmental conditions.
Cowpea exhibits inherent genotypic variations in response to drought stress. Some cultivars
are comprised of discrete physiological and morphological traits that enhance their ability to
adapt to different environmental conditions [19]. Purushothaman et al., [20] indicated that root
traits like thick cortex aid cowpea to effectively absorb re-introduced soil moisture after
drought stress which enhances recovery. The application of efficient screening techniques
would facilitate the identification of key traits for breeding to improve drought stress
adaptation, yield, and quality. Phenotypic evaluation is the first step in the screening of
desirable and promising cowpea genotypes with drought adaptive characteristics [21], [22].
Two methods employed include the empirical or performance approach that uses grain yield
and its components as the main criteria, since yield is the integrated expression of the entire
array of traits related to productivity under stress. The second method is the physiological
approach that identifies a specific physiological or morphological trait that significantly
contributes to growth and yield in the event of drought [23]. Physiological parameters like
water potential, relative turgidity, diffusion pressure deficit, chlorophyll stability index and
carbon isotope determination are technically difficult and are associated with a high cost of
time and investment, especially when large numbers of breeding populations are involved [24],
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[25]. Field screening is difficult due to uncertainties of rainfall differences in photoperiod and
temperatures especially in the dry seasons [26]. Pot screening method under greenhouse
conditions is an effective and controlled method for evaluating cowpea agronomic performance
under water-deficit stress and identification of drought-tolerant characteristics of resistant
genotypes [27]. We present in this study a systematic greenhouse evaluation of 15 selected
cowpea cultivars under pre- and post-anthesis water deficit to gain an understanding of
genotypic variation in stress adaptation to explore the possible associations among studied
traits for their potential in developing enhanced phenotypic pools that can be used in breeding.
We investigated the impact of no-watering, different types of soil moisture stresses on the
physiological, morphological, and yield response of cowpea to identify high-performing
germplasm.
MATERIALS AND METHODS
Plant Materials, Soil, and Conditioning
Fifteen cowpea cultivars with known and unknown responses to drought stress were selected
for this work (Table 1). They were comprised of six plant introductions (PIs) requested from
the United States (U.S.) Department of Agriculture (USDA) National Plant Germplasm System
(NPGS) and nine commercial cultivars grown by U.S. farmers. The soil was collected from
Tuskegee University’s George Washington Carver Agricultural Experiment Station situated in
Tuskegee, Alabama (32° 26’ 15.59” N 85° 44’ 0.8.70’ W) in October 2019. The taxonomic
classification of the soil is fine loamy, kaolinitic, thermic Typic Kanhapludults. The soil belongs
to the Marvyn soil series, consisting of very deep, well-drained dark greyish brown loamy sand
at 0-7 inches, a yellowish-brown sandy loam at 7-15 inches, and a sandy clay at a depth below
15 inches. This geographic location has a dominant slope range of 2-5%, mean annual
temperature of 65oF, and annual precipitation of about 54 inches. The soil series are non-saline
(0.5-2.0 mohms/cm) with strong to moderate acid reaction.
The collected topsoil (0-15cm) was sorted to remove large debris (plant residue and pebbles)
manually, then passed through a 0.5cm mesh sieve. Tap water was added to the soil to evenly
moisten, mixed thoroughly and then steam sterilized (Pro-Grow electric soil sterilizer, Model
SS-60R, Brookfield, WI) at 82.2oC for 48 hours. The sterilized soil was air dried for four days
followed by thorough mixing with potting soil (SUNGRO:#52 2.8 CUFT 42/PLT; Sun Gro
Horticulture, Agawam, MA, USA) in a ratio of 2:1, respectively. The potted mixed soil moisture
was determined gravimetrically as described in [28], [29]. Briefly, a 1000 ml graduated cylinder
was drilled in the bottom to allow air to escape when water was added. The cylinder was filled
with a random subsample of air-dried soil, which was tamped to a similar consistency as used
in the pots. The surface of the soil was covered with a paper towel, and 100 ml of water was
poured slowly onto the surface to obtain an even distribution through the column. The cylinder
was covered by aluminum foil to avoid evaporation and allowed to equilibrate for 24 hours. Soil
samples were collected from the cylinder in triplicate about 5 cm above the wetting front and
dried at 105°C to a constant weight, after which dry weight was recorded and gravimetric
moisture fraction was determined using the fresh to dry weight ratio. This information was
used to determine the amount of oven-dry soil per pot and watering to field capacity. Watering
treatments were calculated based on soil moisture equivalent to field capacity (FC) in
percentages; that is 5.5kg of the air-dried mixed soil was transferred to each pot (22cm height
x 22cm diameter) and watered with 935ml tap water to achieve 100% FC. Three soil water- stress treatments were established and classified as 25% FC, 50% FC, 75% FC, and the control
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Ritte, I. P., Egnin, M., Idehen, O., Mortley, D., Bernard, G. C., Binagwa, P. H., Brown, A. P., & Bonsi, C. K. (2022). Evaluation of Cowpea Morpho- physiological and Yield Responses to Vegetative and Pre-Anthesis Water-Deficit Stress Tolerance under Greenhouse Conditions. European Journal
of Applied Sciences, 10(2). 391-411.
URL: http://dx.doi.org/10.14738/aivp.102.11786
or optimum conditions at 100% FC. These FC treatment levels were applied to the plants after
a fourteen-day non-watering period as severe drought.
Table 1. The list of fifteen cowpea cultivars evaluated for drought stress tolerance at Tuskegee
University Agricultural Experiment Station, AL, USA (2019/2020)
SN Plant name Av. 1 Seed
size (mg) Status Origin Drought
sensitivity Other traits
1. Mississippi
Silver 163.33 Comm. Var United States Susceptible Fusarium wilt (R), Root
knot nematodes (R)
2. Top Pick Brown
Crowder 186.67 Comm. Var United States Tolerant Resistant to diseases
3. Top Pick Cream 133.33 Comm. Var United States Susceptible Unknown
4. Big Boy 340.00 Comm. Var United States Tolerant Unknown
5. California
Blackeye No.5 186.67 Comm. Var United States Tolerant Fusarium wilt (R),
Nematodes (R)
6. Lady 90.00 Comm. Var United States Tolerant Unknown
7. Pinkeye Purple
Hull BVR 176.67 Comm. Var United States Tolerant Blackeye Cowpea
Mosaic Virus (R)
8. Black Crowder 196.67 Comm. Var United States Tolerant Unknown
9. TVu 7362 120.00 PI Nigeria Unknown Unknown
10. TVu 11987 143.33 PI Sudan Unknown Unknown
11. LOBIA-I-SEFADE 243.33 PI Afghanistan Unknown Unknown
12. UCR 242 160.00 PI Tanzania Unknown Unknown
13. TVnu 113 30.00 PI Tanzania Unknown Unknown
14. K929 146.67 PI Iraq Unknown Unknown
15. White Acre 130.00 Comm. Var United States Tolerant Early maturity
Av, average; Comm var, commercial variety; PI, plant introduction; R, Resistant. Phenotype
information were obtained from [30], [31]
Experimental Design and Drought Stress Treatment
Experiments were conducted in 2019 (October 2019 – January 2020) at Tuskegee University,
Agricultural Experiment Station (TU-AES) greenhouse. The experimental layout was a
Randomized Complete Block Design (RCRD) as a split-plot with a 4 x 15 x 3 factorial treatments
arrangement. The drought stress treatment levels (25% FC, 50% FC, 75% FC) and the 100% FC
control were the main plots while sub-plots consisted of cowpea cultivars (Table 1) in both
experiments. After filling each pot with mixed soil described above, all pots were watered to
field capacity (100%) by adding 935 ml of tap water and left to sit for 24 hours. Five seeds of
each cultivar were sown in each pot according to experimental design. Following seedlings
emergence, thinning was performed to two fairly vigorous growing seedlings per pot, and soil
moisture was maintained at field capacity until the first trifoliate leaves were fully expanded at
17 days after sowing. These 17 days post sowing (DPS) plants, except for the controls, were
then subjected to a drought stress treatment by suspending watering for 14 days to mimic
severe drought conditions. On the 15th day following the drought stress, watering resumed at
25% FC, 50% FC, 75% FC every 10-days per experimental layout. The controls were well- watered at 100% FC every three days without pre-drought treatment. These treatments were
maintained throughout the remainder of the experimental period until maturity. Plants were
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protected from insects by a weekly spray of pyrethrin concentrate (15cc/gallon of water) from
flowering to maturity, and no fertilizers were applied.
Greenhouse Conditions
Weather parameters such as temperature and relative humidity were recorded using a HOBO
U12-012 data logger (Onset Computer Corporation, MA, USA) throughout the experimental
period. The average maximum and minimum temperature and relative humidity of the
greenhouse are shown in Table 2. The average maximum temperature was either below or
within the optimal range for cowpea (22 oC and 35 oC) as described by Singh, [9].
Table 2. Mean monthly greenhouse weather data during the experimental period
Year Month Tmax (oC) Tmin (oC) RHmax (%) RHmin (%)
2019/2020 October 30.81 18.74 65.00 34.09
November 36.87 13.23 87.32 42.07
December 28.79 8.27 87.05 35.11
January 32.15 13.42 84.43 43.24
Tmax, average maximum temperature; Tmin, average minimum temperature; RHmax, average
maximum relative humidity; RHmin, average minimum relative humidity.
Data Collection and Statistical Analysis
Data on growth parameters were assessed at weekly intervals from drought treatments
initiation at 18 DPS through the water-stress levels for four weeks except for terminal leaflet
expansion rate, which was assessed at 3-day intervals on a tagged leaf from the time the leaf
opened until when the leaf attained full expansion. Traits measured included plant height (PH)
from 1 cm above soil level to the apex of the plant, stem diameter (St. D) measured 1.5cm above
the soil level, chlorophyll content (Ch. C) by using chlorophyll concentration meter, Model MC- 100 (Apogee instruments Logan, UT, USA), and expansion rate:which was determined by
measuring the length (Tl. L) and width (Tl. W) of terminal leaflet of the second trifoliate leaf. At
maturity, plants were harvested to determine yield components and seed yield. These were
number of pods per plant (NP/P), seed yield per plant (SY/P) after shelling the dried pods and
the seeds were weighed and weight recorded. Shoot (SDW) and root (RDW) dry biomass were
determined by separating the shoots and roots that were carefully cleaned and then dried at
70°C until constant weight was attained, and dry weight recorded. All data were recorded as
the average of two plants for each pot in Microsoft Office Excel and utilized for statistical
analysis. The impact of drought stress on the performance of the cowpea cultivars was assessed
based on the percent reduction for each of the studied traits across treatment levels [32], [33].
This assessment enables the estimation of the extent of reduction in performance for a given
trait using the following formula:
% Reduction (% Red)= performance without stress – performance with stress x 100%
performance without stress
Performance was also assessed under drought stress and normal conditions using a stress
tolerance index (STI) to enable relative comparison of the cultivars under drought stress
conditions. STI of the fifteen cowpea cultivars was calculated based on cultivars means for
morpho-physiological and yield-related traits in 25%, 50% and 75% FC by using the formula
demonstrated by Fernandez, [34];
Stress tolerance index; STI = (Ys)(Yp)
(Yp)2
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LOBIA-I-SEFADE displayed a stay-green phenotype. Mississippi Silver (susceptible check)
displayed more chlorotic leaves in 25% FC compared to other cultivars.
Figure 1. Picture of cowpea cultivars after (a) 7 days without watering in drought treated pots,
(b) 14th day without watering, (c) Scorching and chlorosis of trifoliate leaves as a result of
drought stress on Big Boy in 50% FC, (d) Increasing drought stress impacted plant height and
other traits, (e) Yield components of Mississippi Silver were impacted by increasing drought
stress, (f) Cultivar TVu 11987 (left) displayed stay green phenotype whereas Mississippi Silver
displayed more chlorotic leaves in 25% FC (right).
Table 3. Mean squares and significance tests for analysis of variance for nine morpho- physiological and yield traits of the 15 cowpea cultivars evaluated for drought stress
tolerance under three drought treatment levels and well-watered conditions.
SOV DF PH (cm) St. D (mm) Ch. C (μmolm-2) Tl. L (cm)
Cultivars (Cult.) 14 2,605.2*** 2.522*** 7773.6*** 16.078***
Drought treatments (DT) 3 7,271.2*** 0.951*** 5526.4*** 19.789***
Cult*DT 42 114.1ns 0.0861ns 504.9** 0.844ns
Residual 118 126.3 0.0881 296.7 1.177
Grand mean 65.52 3.60 237.86 8.78
SOV DF Tl. W (cm) SDW (g) RDW (g) NP/P SY/P (g/p)
Cultivars (Cult.) 14 9.733*** 8.367*** 0.219*** 2.868*** 2.585***
Drought treatments (DT) 3 9.493*** 20.494*** 2.051*** 20.586*** 23.749***
Cult*DT 42 0.5698ns 1.1612** 0.0843ns 1.3445* 0.8724*
Residual 118 0.6217 0.6354 0.0677 0.9070 0.5611
Grand mean 5.76 2.26 0.43 2.20 1.98
SOV, source of variations; DF, degrees of freedom; PH, plant height; St. D, stem diameter; Ch.
C, chlorophyll content; Tl. L, terminal leaflet length; Tl. W, terminal leaflet width; NP/P,
number of pods per plant; SY/P, seed yield per plant; SDW, shoot dry weight; RDW, root dry
weight; *P < 0.05; **P < 0.01; ***P < 0.001; ns, non-significant.
a