Leaf growth, gas exchange and assimilation performance of cowpea varieties in response to Bradyrhizobium inoculation.

Supplying nitrogen to crops through selecting high N fixing legumes and effective inoculant is one of the key strategies to improve crop productivity. However, studies related to the effect of Bradyrhizobial inoculation on leaf growth, its functioning in relation to photosynthesis, and transpiration efficiency (WUE) of cowpea [Vigna unguiculata (L.) Walp] varieties in the tropics were inadequate. A two-year field experiment was conducted at three sites to evaluate the effect of inoculation on leaf growth, gas exchanges and photosynthetic efficiency of cowpea varieties. The study treatments were composed of four varieties, Keti (IT99K-1122), TVU, Black eye bean, and White wonderer trailing and three levels of inoculation (non-inoculated or inoculated with Bradyrhizobium strains CP-24 or CP-37). Gas exchange was measured on live plants at 67-77 days after sowing, between 8:00 to 11:00 a.m. and 14:00 to 16:00 p.m. Leaf growth parameters (leaf number and leaf area) were measured by destructive sampling, and the yield data was determined by harvesting plants in the three central rows at physiological maturity. Variety TVU performed best in terms of leaf number, photosynthesis rate, and WUE. Whereas, Black eye bean revealed superior performances for leaf area, leaf area index, and stomatal conductance compared with the rest two varieties. The effect of inoculation was significant with 14.0, 23.8, 13.7, and 11.0% advantage in leaf area, leaf area index, net photosynthesis, and WUE, respectively. Moreover, the performance of cowpea of the 2018 cropping season showed a relative advantage over 2019 in terms of leaf number, leaf area, leaf area index, net photosynthesis, and stomatal conductance. Therefore, inoculating cowpea varieties with effective Bradyrhizobium strain can be a viable alternative to enhance growth, gas exchange, photosynthetic efficiency, and grain yield.

Introduction

Cowpea [Vigna unguiculata (L.) Walp] is one of the most popular grain legumes grown in Africa and some regions of America and Asia (Boukar et al., 2011). It has multiple purposes serving as an affordable source of quality protein for both rural and urban dwellers in high undernourished areas (Antova et al., 2014). Its leaves and green pods are used as vegetables, and dried grains are used in many different dishes (Kyei-Boahen et al., 2017). The protein concentrations of cowpea leaves and dry grains range from 27 – 43% and 21–33%, respectively (Abudulai et al., 2016). In Ethiopia, cowpea is cultivated mainly for food in dry grain forms since very long time, but utilization of its leaf as a green vegetable has been recently gotten attention (Mulugeta et al., 2016). Cowpea has many non-food desirable benefits in the farming system including supporting sustainability due to its excellent N2-fixing ability (Appiah et al., 2015; Bittenbender 1990). Moreover, its tolerance to drought and adaptability to stressful environments make it an ideal crop under changing climate conditions (Bisikiwa et al., 2014).

The growing impact of climate change on crop production calls for the need of improved agronomic practices, including the use of inoculants and improved crop varieties (Olivares et al., 2013). Moreover, low N contents of most tropical soils are among the various factors that limit the growth and photosynthesis rates of crops (Belane and Dakora 2010; Hailu et al., 2015). Legume inoculation with effective inoculants can be an alternative for the sustainable improvement of soil fertility to enhance crop yields (Samago et al., 2018). Thus, supplying N to crops and cropping systems through either inoculation or utilization of legumes is expected to increase photosynthetic processes, leaf area production, leaf area duration as well as net photosynthesis rate (Ahmad et al., 2009). In this regard, N2-fixing legumes, including cowpea, have an advantage over non-legumes in their ability to meet their photosynthetic N requirements from symbiotic N2-fixation and thereby fulfill the N requirement for morpho-physiological functions of plants (Hikosaka and Terashima 1995). Among the legumes, cowpea, which is widely grown in sub-Saharan Africa, including Ethiopia, is a major player in sustaining the fertility of soils under smallholder farms. It is estimated that cowpea can fix up to 200 kg ha−1 of N (Dakora et al., 1987) and can contribute up to 92 kg ha−1 of N to the soil (Chikowo et al., 2004).

Leaf functioning regulates leaf transpiration rates, metabolite flux, long-distance signaling, and the influx of CO2 and water (Lawson 2009). Thus, selecting crop varieties having a high carbon yield in stressing environments would require a prior understanding of leaf growth, stomatal functioning in relation to photosynthetic gas exchange, and leaf water relations (Lawson 2009; Wang et al., 2005). On the other hand, the threat of climate change has affected important natural resources, including water (Chatterjee et al., 2012). In this regard, crop varieties having higher water use efficiency (WUE) are preferred which in turn could be improved via the N2 – fixation (Xu and Hsiao 2004). Evidently, Batterman et al. (2013) have reported that N2-fixing plants alleviate nitrogen shortage, thereby responding to climate change by increasing the capacity to fix CO2. Similarly, N2-fixing plants demonstrated higher WUE compared with the non-fixing plants (Adams et al., 2016). N2 - fixation influences WUE because leaf transpiration and carbon fixation are related to leaf nitrogen (Osnas et al., 2013). Thus, increasing leaf nitrogen can increase consumption of intercellular CO2, because a strengthened CO2 diffusion gradient helps to maintain the supply of CO2 to Rubisco (Adams et al., 2016). Therefore, leaf nitrogen and photosynthetic rates have strong positive relationships (Evans, 1989). Collectively, these pieces of evidence highlight the demands for the establishment of a link between N2 - fixation driven plant N nutrition improvement with leaf growth and photosynthetic gas exchange and assimilation in leaves of cowpea varieties. The question was which varieties of cowpea are the best candidate to improve the aforementioned biological traits in response to the inoculation with elite Bradyrhizobium at three locations in the tropics for two consecutive experiment periods: 2018 and 2019.

Materials and methods

Description of the experimental sites

The experiment was conducted during the main rainy season of the years 2018 and 2019 at three sites in southern Ethiopia. The three sites are situated at: Hawassa (7o 3′ N; 38o 30′ E, 1700 m a.s.l.), Boricha (6°17′N; 38°04′E, 1866 m a.s.l.) and Dore (7°3′N; 38°28′E, 1694 m a.s.l.). They are selected as they are among the vulnerable areas in southern Ethiopia to climate change. In addition, the population is prone to food insecurity (Atara et al., 2019). It is worth to noting that the plots used in the 2018 cropping season were changed during 2019 in order to avoid the residual effects of inoculants.

Soil and climate characteristics

Soil and climatic data are summarized in Tables 1 and 2, respectively. The soil texture of the study sites was clay loam for Boricha and Hawassa, and loam for Dore sites. The pH of the experimental soil was found suitable for cowpea production as it is characterized by a pH of about neutral (Onyibe et al., 2006). As per the limit set by Havlin et al. (1999) and (Herrera (2005)), the total N ranges from low to moderate and the organic carbon very low to low, respectively. The soils were not out of range for legume production in terms of the available P, cation exchange capacity, and exchangeable cations content (FAO 2006) (Table 1). During the 2018 experimental season Boricha, Dore, and Hawassa sites received a mean monthly rainfall of 116, 115 and 107 mm, respectively. However, the sites were characterized with 169, 166, and 141 mean monthly rainfall, respectively, during the second season. The mean atmospheric temperature were within the range of 19.0–20.3 °C for both years (Table 2). A detailed description has been given in Ayalew et al. (2021).

Table 1

Soil physico-chemical properties at 30 cm of soil depth at Boricha, Dore and Hawassa experimental sites during the cropping seasons of 2018 and 2019. Adapted from Ayalew et al., (2021).

Locations	Texture	pH	OC	TN	Avail. P	CEC	K	Ca	Mg
			%		mg kg−1	cmol/kg
2018 cropping season
Boricha	Clay loam	5.7	2.1	0.2	21.4	9.5	1.7	8.3	1.2
Dore	Loam	6.2	0.8	0.1	9.5	7.8	1.6	8.9	1.2
Hawassa	Clay loam	7.1	1.5	0.1	67.4	20.8	3.8	14.8	2.6
2019 cropping season
Boricha	Clay loam	6.5	2.0	0.2	3.2	17.9	1.5	6.7	1.3
Dore	Loam	6.9	1.2	0.1	3.0	7.5	2.1	10.3	2.5
Hawassa	Clay loam	7.2	1.7	0.2	51.7	22.8	3.5	11.1	2.0
Methods	Bouyoucos Hydrometer	pH meter	Walkley and Black	Kjeldahl	Olsen	Ammonium acetate	Mehlich-3

CEC = Cation exchange capacity; OC = Organic carbon; TN = Total nitrogen; Avail. P = Plant-available phosphorus.

Table 2

Monthly rainfall and temperature records at Hawassa, Boricha and Dore sites during cropping seasons of 2018 and 2019. Adapted from Ayalew et al., (2021).

Month	Precipitation (mm)						Average temperature (°C)
	Boricha		Dore		Hawassa		Boricha		Dore		Hawassa
	2018	2019	2018	2019	2018	2019	2018	2019	2018	2019	2018	2019
June	133	125	154	141	142	128	18.5	18.9	19.9	20.1	19.5	19.8
July	50	168	68	178	67	158	18.7	18.5	19.4	19.4	19.2	19.3
Aug.	135	161	145	175	150	160	18.4	19.2	19.1	20.9	19.1	19.9
Sept.	122	128	107	139	91	128	20.1	20.5	20.2	20.7	19.8	20.4
Oct.	140	263	102	196	83	133	19.4	19.8	20.3	20.6	20.0	20.5
MA	116	169	115	166	107	141	19.0	19.4	19.8	20.3	19.5	20.0
GMT	580	844	575	829	533	706

MA = monthly average, GMT = total for the growing months.

Experimental material sources and experimental design

The seeds of four cowpea varieties, Keti (IT99K-1122), TVU, Black eye bean, and White wonder trailing, were provided by Melkassa Agricultural Research Center, Ethiopia. These cowpea varieties are under production in Ethiopia, which were released for commercial production between the years 1970–2012 (MoA 2018). They differ regarding the time required to reach maturity at this experiment locations with the range of 121–129 days (Ayalew et al., 2021). CP-24 and CP-37 Bradyrhizobium strains were provided by Hawassa University, Agriculture College. The strains, after isolation, were subjected to authentication and characterization (Degefu et al., 2018). Furthermore, the symbiotic effectiveness was confirmed using sand culture, before use in field conductions (Ayalew and Yoseph 2020).

The treatments consisted of four cowpea varieties and three inoculation doses in Randomized Complete Block Design with a factorial arrangement, replicated four times. A spacing of 0.5, 0.2, 0.5, and 1 m were used for row - row, plant - plant, plot – plot, and block – block arrangements, respectively, with 3.5 ∗ 3.4 m individual plot size. The seeds were inoculated with 10 g of peat-based inoculant per kg of seeds (Rice et al., 2001) containing 6.5×108 viable bacterial cells g−1 peat of Bradyrhizobium strain CP-24 and CP-37. The seeds were inoculated under shade and air dry for some minutes prior to planting to maintain cells viability and avoid fungal growth, respectively (Rice et al., 2001). The plots used in 2018 were changed in 2019 to avoid the residual effects of inoculants). The detailed design and procedures are described in Ayalew et al. (2021).

Photosynthetic rate and gas exchanges

Net photosynthesis rate (Pn) [μmol (CO2) m−2 s−1], transpiration rate (E) [mmol (H2O) m−2 s−1], internal CO2 concentration (Ci) and stomatal conductance (gs) were measured using a portable infrared gas analyzer (IRGA) (ADC BioScientific LCi-SD System Ltd., UK, 2011) at 70–72 and 75–77 days after sowing, at flowering, during 2018 and 2019 crop seasons, respectively. Measurements were made between 8:00 to 11:00 a.m. and between 14:00 to 16:00 p.m. During the measurement, the system was calibrated with the ambient carbon dioxide concentration (Cref) 377-μmol mol−1. The temperature of leaf chamber (Tch) varied between 27 °C and 36 °C, and ambient atmospheric pressure (p) was 833 mBar and PAR (Q) at leaf surface reached a maximum of 2100 μ mol m−2 s−1. The average leaf temperature varied between 22 °C and 37 °C. Instantaneous WUE [μmol (CO2) mmol (H2O)−1] was computed as photosynthetic assimilate produced per unit of water molecule transpired. The measurements were made on the two upper fully expanded central trifoliate leaves per plant and three plants per plot, which made the total measurements of six per plot.

Growth assessment

At about mid flowering (70 and 78 DAS for 2018 and 2019, respectively), five plants per plot were randomly taken from the second inner row at both sides of the plot, uprooted and the shoot part cut off from the root, and subjected to leaf growth parameter measurement, i.e. leaf number plant−1 and leaf area (LA) (cm2). The leaf area was measured using an automatic leaf area meter (model LI 31000A Li-Cor, Lincoln, USA). Then, leaf area index (LAI) and the leaf area ratio (LAR) were calculated using Eq. (1)and Eq. (2), respectively.

Plant height was measured from the ground level to the tip of the plants from those plants uprooted for nodulation data determination.

Nitrogen content and grain yield

The yield data was determined by harvesting plants in the three central rows at physiological maturity as described in detail by Ayalew et al. (2021), a paper published from the same experiment with this, and in this paper the yield data was used to show the relation between leaf growth, gas exchange and photosynthesis with that of yield performance of cowpea.

Statistical analysis

The dataset was subjected to a generalized linear model procedure of SAS software version 9.4 (SAS Institute, 2012). Inoculants and cowpea varieties were considered as fixed factors and growing season and location as random variables. The two seasons were combined in the analysis when the test for variances was homogeneous, and separate analysis was done when the variances between seasons were heterogeneous. Variety responses and inoculation effects on leaf number, leaf area, leaf area index, plant height, net photosynthesis, stomatal conductance, internal CO2 concentration and shoot nitrogen content were analyzed for the 2018 and 2019 growing seasons separately. Since the data for those parameters were heterogeneous upon testing homogeneity of variance over seasons, it helped to compare inter-annual variation. Again, since the data for these parameters were homogeneous over seasons, the analysis of interaction effect was conducted for the varieties response and inoculation effect on leaf area ratio, leaf transpiration rate, water use efficiency and grain yield. The data from the three sites were combined as the variances among locations were homogenous. Treatments were compared by the least significant difference (LSD) method for a significance level of P ≤ 0.05. Correlation analysis was done using Pearson's simple correlation coefficients to test the relationships between leaf development, gas exchanges and yield.

Results

Varietal effects on growth traits

Leaf number plant−1 was significantly influenced by cowpea variety (Table 3). Variety TVU produced the greatest number of leaves plant−1 followed by White wonderer trailing and Keti (IT99K-1122), while the lowest number of leaves plant−1 were recorded in the Black eye bean variety (Table 3). The relatively broad-leaved Black eye bean variety produced the largest LA, while White wonderer trailing variety presented the lowest LA in both years (Table 3). However, Black eye bean and TVU in 2018, and Keti and White wanderer trailing in 2019, did not significantly (P ≥ 0.05) differ for LA. Similarly, Black eye bean recorded the highest LAI, followed by TVU and Keti (IT99K-1122) followed by White wonderer trailing. The LAR was numerically higher for Black eye bean followed by TVU with the lowest from White wonderer trailing (Table 4). Plant height was significantly influenced by the cowpea varieties, being highest for variety of Black eye bean, and lowest for Keti (Table 3).

Table 3

Effects of cowpea varieties and Bradyrhizobium strains inoculation on leaf number (LN), leaf area (LA), leaf area index (LAI) and plant height (PH) of cowpea grown at three sites during the cropping seasons of 2018 and 2019.

Treatments	2018				2019
	LN	LA [cm−2]	LAI	PH [cm]	LN	LA [cm−2]	LAI	PH [cm]
Variety
Keti	100b	1919b	1.9b	15.8c	83.5c	1291c	1.3c	30.7b
TVU	124a	1947ab	1.9b	16.8bc	132.5a	1484b	1.5b	30.9b
Black eye bean	91b	2066a	2.1a	28.0a	74.3c	1721a	1.7a	47.2a
White wonderer trailing	117a	1600c	1.6c	22.4ab	117.3b	1247c	1.2c	35.5ab
Bradyrhizobium
Non-inoculated	91b	1729b	1.6b	20.2	90.5b	1290	1.3	35.7
CP-24	122a	2005a	2.1a	21.5	110.8a	1573	1.6	38.7
CP-37	111ab	1920a	2.0a	20.5	104.4a	1446	1.4	35.4
F-statistics
Variety	8107∗∗∗	1425494∗∗	1.4∗∗	1145∗∗	27341∗∗	9445749∗	9.5∗	4345∗∗∗
Bradyrhizobium	11944∗∗∗	954749∗	3.4∗∗∗	20ns	5145ns	5126226ns	5.1ns	309ns
Variety x Bradyrhizobium	267ns	428621ns	0.5ns	14ns	1405ns	2446716ns	2.5ns	174ns

Means followed by different letters in a column are significantly different at ∗: P ≤ 0.05; ∗∗: P ≤ 0.01; ∗∗∗: P ≤ 0.001 by the LSD test.

Table 4

Effect of cowpea varieties and Bradyrhizobium strains inoculation on leaf area ratio (LAR), leaf transpiration rate (E) and water use efficiency (WUE) of cowpea grown at three sites for the pooled results of the cropping seasons 2018 and 2019.

Treatments	LAR [cm2 g−1]	E [mmol (H2O) m−2 s−1]	WUE [μmol (CO2) mmol (H2O)−1]
Variety
Keti (IT99K-1122)	91.1	6.1ab	3.6ab
TVU	94.8	5.8b	4.0a
Black eye bean	100.0	6.4a	3.6b
White wonderer trailing	82.0	6.1ab	3.6b
Bradyrhizobium
Non-inoculated	85.73b	6.2	3.5b
CP-24	104.2a	6.1	3.9a
CP-37	86.1b	5.9	3.8a
F-statistics
Variety	4143ns	1.6∗	3.4∗∗
Bradyrhizobium	10724∗∗	1.9ns	4.4∗∗
Variety x Bradyrhizobium	456ns	1.8ns	1.1ns

Means followed by different letters in a column are significantly different at ∗: P ≤ 0.05; ∗∗: P ≤ 0.01; ∗∗∗: P ≤ 0.001 by the LSD test.

Inoculation effects on growth traits

Plants inoculated with either of the Bradyrhizobium strains produced more leaves plant−1 compared with the non-inoculated control (Table 3). However, the two strains did not significantly differ for their effects on the number of leaves plant−1. Inoculation of cowpea with Bradyrhizobium strains showed a significant effect on LA and LAI in the 2018 cropping season compared with the control, however, the effect was not significant in 2019. The two strains did not differ for LA and LAI in the 2018 cropping season (Table 3). LAR was also significantly influenced by inoculation with Bradyrhizobium strains, with the higher LAR from CP-24 inoculated plants (Table 4). However, the strain CP-37 did not differ from the control for LAR.

Varietal effect on physiological gas exchanges

There was no difference in net photosynthesis among varieties (Table 5). However, there was a significant difference among cowpea varieties for stomatal conductance (gs) during 2018 (Table 5). Among the varieties, the White wonderer trailer and Black eye bean exhibited the highest gs in 2018. However, the varieties Black eye bean and White wonderer trailing did not significantly differ for leaf gs. The variety Keti recorded the lowest value for stomatal gs compared with the other three varieties in 2018 but not 2019. Similarly, the varieties differed significantly for internal carbon dioxide concentration (Ci) in 2018 cropping season. As indicated in Table 5, the variety Black eye bean (274.2) and TVU (274.0) exhibited higher Ci followed by Keti (269.0 μmol mol−1). The lowest C was recorded for the White wonderer trailing variety. The effect of varieties on Ci was not significant in 2019, however, the relative performance among the varieties followed the same trend for Ci in 2018 (Table 4). The highest E was recorded in the variety Black eye bean which significantly differed from TVU. Regarding the WUE generally, TVU variety recorded the highest values for WUE, while the other varieties did not differ one another (Table 4).

Table 5

Effects of cowpea varieties and Bradyrhizobium inoculation on net photosynthesis (Pn), stomatal conductance (gs) and internal carbon dioxide concentration (Ci) of cowpea grown at three sites during the cropping seasons of 2018 and 2019.

Treatments	2018			2019
	Pn [μmol (CO2) m−2 s−1]	gs [mmol m−2 s−1]	Ci (μmol.mol-1)	Pn [μmol (CO2) m−2 s−1]	gs [mmol m−2 s−1]	Ci [μmol.mol-1]
Variety
Keti (IT99K-1122)	21.3	1.3b	269.0ab	19.8	1.3	246
TVU	22.1	1.5ab	274.0a	20.7	1.0	247
Black eye bean	21.1	1.9a	274.2a	20.2	1.2	248
White wonderer trailing	20.9	1.9a	259.9b	20.6	2.0	236
Bradyrhizobium
Non-inoculated	19.5b	1.3b	265.7	19.4b	1.3	246
CP-24	22.6a	1.8a	271.8	21.1a	1.5	244
CP-37	22.0a	1.8a	270.4	20.5ab	1.3	243
F-statistics
Variety	9.7ns	3.12∗∗∗	1609.8∗∗	6.5ns	6.61ns	1080ns
Bradyrhizobium	128.9∗∗∗	4.34∗∗∗	487.0ns	32.0∗∗	0.79ns	160ns
Variety x Bradyrhizobium	8.7ns	0.30ns	280.4ns	5.6ns	0.74ns	735ns

Means followed by different letters in a column are significantly different at ∗: P ≤ 0.05; ∗∗: P ≤ 0.01; ∗∗∗: P ≤ 0.001 by the LSD test.

Effects of inoculation on physiological gas exchanges

Inoculation with Bradyrhizobium significantly increased net photosynthesis during both 2018 and 2019 cropping seasons compared with the control but without significant differences between the Bradyrhizobium strains (Table 5). Stomatal conductance gs, also markedly affected with Bradyrhizobium inoculation in 2018 with both strains (Table 5) and resulted in increased transpiration efficiency, WUE, than non-inoculated plants (Table 4). However, the influence of the two strains on WUE did not differ significantly.

Correlation analysis

The Pearson's correlation coefficients for comparison of the degree of association between leaf growth, gas exchange and/or seed yield are presented in Table 6. There were significant positive correlation between yield and its components and all the leaf development parameters. For example, significant positive correlations occurred between number of pods and leaf number (0.83∗∗), leaf area (0.69∗∗), and LAI (0.65∗∗). Similarly, the biological yield (the aboveground biomass including the straw and pods yields) was significantly positively correlated with leaf number (0.66∗∗), leaf area (0.62∗∗) and LAI (0.58∗∗) indicating the contribution of leaf growth performance for better biomass accumulation. More importantly, grain yield showed significant positive correlation with leaf number (0.90∗∗∗), leaf area (0.74∗∗) and LAI (0.72∗∗), indicating the importance of leaf growth performance for grain yield improvement. Grain yield and WUE were positively correlated although not significantly (0.55ns), whereas, grain yield correlated negatively with leaf transpiration (−0.57∗). Biological yield had highly significant positive correlation with number of pods (0.91∗∗∗), and grain yield also demonstrated significant positive correlation with pod numbers per plant (0.93∗∗∗) and biological yield (0.86∗∗∗).

Table 6

Simple Pearson's correlation on leaf growth, gas exchanges and yield of cowpea. Data on yield and its components are given by Ayalew et al. (2021), the yield data was published from the same experiment with this one.

	LN	LA	LAI	LAR	PH	Pn	gs	Ci	E	WUE	PNP	BY	GY
LN	1
LA	0.80∗∗	1
LAI	0.81∗∗	0.99∗∗∗	1
LAR	-0.01ns	0.06ns	0.07ns	1
PH	-0.41ns	-0.08ns	-0.07ns	0.35ns	1
Pn	0.60∗∗	0.54ns	0.62∗∗	0.10ns	-0.10ns	1
gs	0.17ns	0.26ns	0.28ns	-0.17ns	0.49ns	0.07ns	1
Ci	-0.26ns	-0.29ns	-0.27ns	-0.20ns	0.38ns	-0.53ns	0.59∗	1
E	-0.53ns	-0.41	-0.44ns	0.28ns	0.55ns	-0.25ns	0.18ns	0.21ns	1
WUE	0.61∗	0.64∗∗	0.70∗∗	-0.06ns	-0.41ns	0.62∗∗	-0.27ns	-0.41ns	-0.75∗∗	1
PNP	0.83∗∗∗	0.69∗∗	0.65∗∗	-0.29ns	-0.29ns	0.41ns	0.13ns	-0.16ns	-0.53ns	0.52ns	1
BY	0.66∗∗	0.62∗	0.58∗	-0.45ns	-0.05ns	0.30ns	0.43ns	0.07ns	-0.27ns	0.28ns	0.91∗∗∗	1
GY	0.90∗∗∗	0.74∗∗	0.72∗∗	-0.37ns	-0.44ns	0.43ns	0.24ns	-0.15ns	-0.57∗	0.55ns	0.93∗∗∗	0.86∗∗∗	1

LN = leaf number; LAR = Leaf area ratio, PH = Plant height; Pn = Net photosynthesis; gs = stomatal conductance; Ci = internal CO2 concentration; E = leaf transpiration; PNP = pod number per plant; BY = Biological yield; Grain yield.

There was significant positive correlation between net photosynthesis and leaf number (0.60∗∗), and LAI (0.62∗∗), while positive non-significant correlation with leaf area. WUE exhibited a significant positive correlation with leaf number (0.61∗), leaf area (0.64∗∗) and leaf area index (0.70∗∗). Furthermore, WUE showed a positive significant (0.62∗∗) and a negative significant (0.75∗∗) correlation with net photosynthesis and leaf transpiration, respectively.

Discussion

Effects of inoculation on plant development and photosynthesis

The significant improvement in leaf number per plant at flowering due to Bradyrhizobium inoculation of the 2018 observation, can be associated with the improved vegetative growth because of enhanced nitrogen supply by biological nitrogen fixation. In this experiment, inoculation with the Bradyrhizobium demonstrated an advantage of about 41% N contribution compared with the non-inoculated control (Ayalew et al., 2021). The subsequent improvement in LAI with inoculation is associated with the advantage in LA development due to inoculation. In agreement with these observations, a higher leaf growth in inoculated cowpea plants was also reported by Agba et al. (2013). Furthermore, Pereira et al. (2019) reported the LA improvement for inoculated cowpea varieties. Agba et al. (2013) and Kaweski (1994) and Okon (1989) have concluded that N2-fixation by Rhizobia inoculated legumes enhances leaf expansion. Plant nutrition management practices like inoculation with elite strains aim to optimize photosynthesis rate and yield, mainly by optimizing LAI (Mitchell and Hardy 2000), due to the fact that leaf area growth determines the light interception capacity and thereby the resource use efficiency of crops (Sarathi et al., 2015).

It has been indicated that total leaf photosynthesis increases with an increase in LAI (Mitchell and Hardy 2000). Connected to this, the correlation analysis found a strong and significant relationship between LAI and net photosynthesis, and grain yield (Table 6), indicating a link between LAI with capacity of assimilation and yield (Mitchell and Hardy 2000). Higher yield recorded from this experiment (Ayalew et al., 2021) was attributed to the inoculation driven improvement in leaf characteristics. The advantage for LAR due to inoculation is associated with a corresponding leaf growth improvement, as inoculation enhanced leaf area. In agreement, Pereira et al. (2019) also reported significant increase in leaf area ratio due to inoculation of faba bean (Vicia faba L.) plants. However, during the cropping season of 2019, inoculation did not lead to a significant effect on most of the leaf growth characteristics. Rainfall was higher at all sites during 2019, with 141–168 mm monthly range, what may have led to lower grain yield response of cowpea to inoculation (Ayalew et al., 2021). Higher rainfall during the 2019-cropping season, might have affected the survival, establishment and symbiotic properties of Bradyrhizobium strains (Hamdi 1999). Climatic condition are among the most important factors affecting inoculant performance (Kunert et al., 2016) and the high rainfall in 2019 (Table 2) could explain the non-significant effect of inoculation on leaf development. High precipitation can affect microbial respiration, biomass nutrition and enzyme activities (Odette et al., 2016), and thus impair the performance of the inoculated Rhizobia.

The improvement in net photosynthesis with inoculation (Table 5) might be due to the improved supply of N nutrition from symbiosis (41% more fixed N was achieved compared with the non-inoculated control) and thereby improved leaf metabolic processes. Nitrogen is the major nutrient required for the synthesis of the macromolecules responsible for CO2 assimilation and radiation interception (ribulose-1, 5-bisphosphate carboxylase-oxygenase -Rubisco), thereby improving the rate of net photosynthesis when enough N is available for plants. N2 fixation and net photosynthesis are, therefore, metabolically interlinked in nodulating legumes. In agreement with the current result, Belane and Dakora (2015) have reported improvement of net photosynthesis in cowpea due to inoculation.

The net photosynthesis response was relatively higher in 2018 compared with 2019, what might be related with the poor crop-inoculant performance in 2019 due to high rainfall. Supporting the current result, Pule-Meulenberg et al. (2010) reported differences in net photosynthesis due to environmental effects on the host plant performance. A significant improvement with Bradyrhizobium inoculation in leaf gs was recorded. The increased gs might be associated with the increased N concentration in the leaf due to N fixation that reached 104.9 kg ha−1 (Ayalew et al., 2021, submitted). In accordance with the findings of this experiment, Augé et al. (2015) reported improvement in stomatal functioning due to symbioses. Furthermore, the higher LAI due to inoculation might play a role in the modification of the stomatal conductance (Blatt et al., 2017), because symbiosis modifies stomatal behavior (Augé et al., 2015). A change in leaf gs and E in cowpea due to rhizobial inoculation was also reported elsewhere (Matiru and Dakora 2005) where stomatal conductance increased. Similarly, the improvement in WUE of cowpea due to inoculation highlights the importance of the cowpea-Bradyrhizobium symbiosis for enhanced water relationships. The improvement in WUE with inoculation could be related to the improved nitrogen availability; supporting the optimal physiological functions. The symbiosis-triggered enhancement in cowpea WUE was also reported by Tankari et al. (2019) and Yahaya et al. (2019) supporting the current findings. Generally, the current study is innovative, because crop production has becoming vulnerable to climate changes and the rural community is under food insecurity pressure (Atara et al., 2019). Thus, the legume and its interaction with Rhizobia brings a potential alternative to face the climate changes due to biological nitrogen fixation capacity (Ayalew et al., 2021).

Varietal effect on plant development and photosynthesis

Significant genotypic variations and inoculation effect on leaf growth and photosynthetic gas exchanges were observed, which could give indications of superiority when comparing cowpea genotypes and Bradyrhizobium strains for agronomic fitness during the era of climate changes (Anyia and Herzog 2004). For example, a large number of leaves, as observed in variety TVU, could be an important attribute from the viewpoint of yield and microclimate improvement. Corroborating this statement, variety TVU having higher leaf number results with higher grain yield (Ayalew et al., 2021). The significant positive correlation between leaf number and grain yield confirmed this relationship (Table 6). Moreover, it will serve for a more effective light interception, thereby improving net photosynthesis (Mitchell and Hardy 2000). This is also confirmed by the significant positive correlation (r = 0.6, p = 0.001) between net photosynthesis and leaf number (Table 6). Increase in leaf area heads to improved photosynthesis (Wenshi et al., 2020). According to Liu et al. (2014), net photosynthesis rate is directly proportional to the leaf light intercepting capacity. This is supported by the significant positive correlation between LAI and net photosynthesis in this study. Furthermore, the higher yield observed for the variety TVU over the years in three sites (Ayalew et al., 2021) validates the link between leaf development and photoassimilates production leading to higher yield. Simultaneous increases in leaf development and photosynthetic rate with enhancing crop yield were also observed by Wenshi et al. (2020). Similarly, Malone et al. (2002) reported that yield increases with the increase in LAI, which is in agreement with our study manifested by the significant positive relationship between LAI, and grain yield.

Black eye bean variety performed best for LA and LAI over the years and seasons and this was associated with its broad leaf nature compared with the other three varieties. For instance, calculating the leaf size as LA over leaf number, Black eye bean leaf size exceeded that of the small leaf producing variety-White wonderer trailing by 39%. In agreement with this result, Pereira et al. (2019), Peksen et al. (2005) and Bisikiwa et al. (2014) reported a difference in leaf growth among cowpea varieties including leaf number and area. The improved LAI observed for the Black eye bean variety could be associated with its higher LA and the strong positive (r = 0.99, p = 0.001) correlation between LA and LAI (Table 5). Agba et al. (2013) and Toker et al. (2002) also observed differences in LAI among cowpea varieties. The higher plant height from the Black eye bean variety is attributed to the growth behavior of the variety. Kyei-Boahen et al. (2017) found the differences in shoot performance among cowpea varieties. In agreement with the current result, significant difference in plant height among cowpea varieties were reported also by Bisikiwa et al. (2014) and El Naim and Jabereldar (2010).

The relatively higher photosynthesis rate in 2018 might explain the poor performance of the varieties caused by higher precipitation in 2019. The subtleties in host plant performance could account for the differences in net photosynthesis rate between genotypes in time and space (Pule-Meulenberg et al., 2010). Martí nez-Acosta et al., 2020 reported negative effect of excess water supply on stomatal conductance, photosynthesis and transpiration. Varieties of White wonderer trailing and Black eye bean exhibited the highest leaf gs compared with the other three varieties. The variability for stomatal gs among varieties could be attributed to the genetic variation among the tested varieties. The higher leaf gs for White wonderer trailing might also be associated with the relatively low LAI demonstrated by this variety, which could increase the conductance per unit of leaf area. Processes such as canopy interception, evapotranspiration and photosynthesis are affected by the LAI (Liu et al., 2014). In agreement with the current result, Augé et al. (2015) reported a variation in leaf stomatal conductance among cowpea varieties.

The highest and the lowest transpiration rate exhibited by Black eye bean and TVU, respectively, might be related to the leaf growth characteristics of each variety. The higher LA and resulting LAI for the Black eye bean might be the cause for higher transpiration rate observed in this variety, which exposes the leaf to more water loss at the expense of CO2 diffusion in the process of photosynthesis. Leaf area contributed for variation in leaf water loss (Wang et al., 2019). The negative correlation between transpiration with leaf number and leaf area in this study (Table 6), indicates the association amongst leaf growth characteristics and leaf transpiration rate. The current result also agreed with the previous findings of Driss and Bill (2001). Similarly, the variation in WUE among the varieties might be caused due to the variation in genetic characteristics of the varieties and their interactions with environment. For instance, the higher WUE for TVU is linked with the lower stomatal gs and leaf E (Tables 4 and 5). The significant negative correlation between WUE and leaf transpiration rate, and the negative correlation between the WUE and leaf gs, confirms the inverse association between higher leaf transpiration rate and stomatal gs with WUE (Tables 4 and 6). In confirmation, the variety Black eye bean with the highest leaf E and stomatal gs, recorded the lowest WUE. High WUE can be achieved either through lower stomatal conductance or higher photosynthetic capacity or a combination of both (Condon et al., 2002). In line with the current findings, Damba et al. (2019) presented a variation among cowpea varieties for WUE. In good agreement with the current findings, a previous study by Tomás et al. (2012) confirmed the existence of variation in WUE among cowpea varieties. The internal CO2 concentration was lower for the variety White wonderer trailing compared with the other three varieties in 2018, which might be attributed to the relatively lower net photosynthetic rate during the same year. In accordance with this, Janoudi et al. (1993) reported a reduction in net photosynthesis due to a limited CO2 concentration.

Conclusion

Inoculation with Bradyrhizobium strains CP-24 and CP-37 significantly increased leaf growth performance of cowpea in terms of leaf number, leaf area, leaf area index and leaf area ratio. The tested varieties also showed a significant difference regarding to leaf growth characteristics except leaf area ratio, whereby variety TVU exhibits higher performance for number of leaf per plant. Whereas, the variety Black eye bean performed best for LA, LAI and plant height. Inoculation improved net photosynthesis rate, stomatal conductance, and WUE of cowpea varieties. The varieties also differed for physiological parameters with a high performance of TVU for WUE, and White wonderer trailing for stomatal conductance. Furthermore, yield showed significant positive correlation with leaf development traits in terms of leaf number, leaf area and leaf area index. Therefore, the observed improvement in leaf growth and physiological traits of inoculated cowpea varieties confirms the importance of Bradyrhizobium inoculation to enhance the morpho-physiological performance and associated yield advantage of cowpea varieties.

Declarations

Author contribution statement

Tewodros Ayalew: Conceived and designed the experiments; Performed the experiments; Analyzed and interpreted the data; Wrote the paper.

Tarekegn Yoseph; Petra Högy; Georg Cadisch: Conceived and designed the experiments; Analyzed and interpreted the data; Wrote the paper.

Funding statement

This work was supported by a PhD scholarship at the Hawassa Universtiy, in the framework of the German-Ethiopian SDG Graduate School “Climate Change Effects on Food Security (CLIFOOD)” between the Food Security Center, University of Hohenheim (Germany) and the Hawassa University (Ethiopia), supported by the DAAD with funds from the Federal Ministry for Economic Cooperation and Development (BMZ).

Data availability statement

Data included in article/supplementary material/referenced in article.

Declaration of interests statement

The authors declare no conflict of interest.

Additional information

No additional information is available for this paper.