Leaf Gas Exchange and Root Nodulation Respond to Planting Density in Soybean [Glycine max (L) Merrill]

Planting density influences structural characteristics and affects mineral nutrient acquisition, irradiance and photosynthesis amongst plants. An experiment was conducted to determine the effect of planting density on leaf gas exchange and nodulation of soybean (Glycine max (L) Merrill). e experiment was conducted as a randomized complete block design (RCBD) in a 5 by 2 factorial treatment arrangement and was replicated three times. Planting density (10, 12, 20, 40, and 80 plants m) and soybean varieties (EAI 3600 and DPSB 19) were first and second factors, respectively. Collected data were subjected to analysis of variance in GENSTAT. Significantly different treatment means were separated using Tukey’s honestly significant difference test at 0.05 significance level. Higher planting density significantly increased (푝 < 0.001) interception of photosynthetically active radiation. Increasing number of plants per unit area significantly (푝 < 0.001) reduced root nodulation, stomata conductance, sub-stomatal CO2 concentration, photosynthetic and transpiration rates. Total chlorophyll content was not responsive to planting density though concentration of chlorophyll “a” content was significantly (푝 < 0.005) higher at lower plant density than at higher plant density. Soil moisture status increased with reduction in plant density. Indeterminate variety DPSB 19 had higher rates of stomata conductance, photosynthesis and sub-stomatal CO2 concentration compared to determinate variety EAI 3600.


Introduction
Planting density affects plant structural characteristics and may help improve disease avoidance, lodging resistance, adaptation to mechanical harvesting and seed yield [1]. A higher plant density has the potential to increase competition for nutrients, light and space while lower plant density may lead to inefficient use of natural resources and inputs [2,3]. Total dry weight of leaves, leaf area index (LAI), crop growth rate (CGR) and nodulation are all dependent on plant density [2]. In soybeans, leaf gas exchange, grain quality such as protein, oil and mineral contents depend on field production conditions with planting population playing a significant role [4].
Previous plant density studies have recommended different plant populations for optimization of soybean plant growth and yield. In United States of America, optimum plant populations for soybean vary from 30 to 50 plants m −2 [5] while in Iran and India, soybean yields were optimized at 60 plants m −2 [6,7]. In Turkey, Mehmet [8] found highest soybean yields at plant density of 12.5 plants m −2 while Rahman and Hossain [1] recommended a planting density of 80-100 plants m −2 in Bangladesh. In Ethiopia, the recommended plant population for soybean is about 40 plants m −2 [3]. e reported results on soybean plant population studies suggest that plant density may vary with type of variety used and that some areas, depending on geographical location, have capacity to support higher planting densities than others due to differences in soil and other environmental conditions [9]. e advent of climate change due to global warming in recent years has also brought with it various biotic and abiotic stresses which make it prudent to adjust agronomic practices in line with prevailing climatic conditions. Considering that leaf gas exchange and root nodulation may vary with planting density, this study was undertaken to determine the effect of plant density on leaf gas exchange and root nodulation of determinate and indeterminate soybean cultivars in Kenya.

Site Description.
e experiment was conducted at Egerton University agronomy teaching and research farm in Njoro, Kenya. Egerton University teaching and research farm (0°22′S; 35°56′E) is at an altitude of 2267 m.a.s.l. e site has mollic andosols and pH of soils ranged from 6.85 to 6.94. e experiment was conducted over two seasons; the first season was during long rains of March to July 2018 while the second season was during short rains of September to November 2018.

Experimental Design and Treatments.
e experiment was conducted in a randomized complete block design (RCBD) with a 5 × 2 factorial treatment arrangement and replicated 3 times. Treatments consisted of two factors: factor 1 being planting density and factor 2 being cultivars. Treatment combinations are presented in Table 1

Chlorophyll Content Determination.
Chlorophyll "a" chlorophyll "b" and total chlorophyll contents were analyzed on a third trifoliate leaf at 50% flowering using a procedure described by Goodwin and Britton [10].

Intercepted Photosynthetically Active Radiation.
Intercepted photosynthetically active radiation (IPAR) was measured at 50% flowering stage using an AccuPar/LAI Ceptometer (LP-80, Decagon Devices, Inc., Pullman, USA). Measurements were done above and below canopy of soybean plants. Calculation of percent IPAR used the following modified Purcell [11] formula as follows.

Measurement of Stomata Conductance.
Stomata conductance was determined on three plants per plot at 50% flowering stage of soybean growth on abaxial side of a middle leaflet of a third trifoliate leaf from top of the plant. It was measured between 12.00 and 14.00 hours on sunny days using a steady state leaf porometer (SC1, Decagon Devices, Inc., Pullman, USA).

Determination of Sub-Stomatal Carbon Dioxide Concentration, Leaf Temperature Photosynthetic and Transpiration Rates.
Sub-stomatal carbon dioxide concentration, leaf temperature, photosynthetic and transpiration rates were determined on two plants per plot at 50% flowering stage of soybean on a middle leaflet of a third trifoliate leaf from top of the plant. Measurements were done between 12.00 and 14.00 hours during sunny days using a TPS-2 portable photosynthesis system (V2.02-PP systems Inc., Amesbury, USA).

Root Nodulation.
Total number of nodules per plant were determined by counting number of nodules formed on five plants randomly selected in a net plot at 50% flowering stage.

Data Analysis
Data were checked for fulfilment of analysis of variance (ANOVA) assumption of normality by using Shapiro-Wilk normality test in Genstat release 18.1. Data were considered normally distributed when -value for Shapiro-Wilk statistic was greater than the threshold -value of 0.05. Data that did not meet the aforesaid ANOVA assumption were subjected to square root transformation before analysis. Data were then subjected to analysis of variance (ANOVA) using linear mixed model for factorial experiment in GENSTAT (REML) and statistically significant treatment means were separated using Tukey's honestly significant difference test at 0.05 significance level. Cultivar EAI 3600 at 12 plants m −2 3 Cultivar EAI 3600 at 20 plants m −2 4 Cultivar EAI 3600 at 40 plants m −2 5 Cultivar EAI 3600 at 80 plants m −2 6 Cultivar DPSB 19 at 10 plants m −2 7 Cultivar DPSB 19 at 12 plants m −2 8 Cultivar DPSB 19 at 20 plants m −2 9 Cultivar DPSB 19 at 40 plants m −2 10 Cultivar DPSB 19 at 80 plants m −2

Soil Moisture Status.
Soil moisture content (Table 2) was responsive to interaction of plant density and cultivar (푝 < 0.05), density and season (푝 < 0.05) and then cultivars and seasons (푝 < 0.001). Lowest soil moisture level was attained at the highest plant density of 80 plants m −2 by cultivar DPSB 19. Plant density and season interaction led to reduced soil moisture content during short rainy season across all plant densities. Season by cultivar interaction led to increased soil moisture during long rains by a determinate cultivar EAI 3600.

Chlorophyll Content.
Total leaf chlorophyll and chlorophyll "b" contents were not responsive to variations in plant density, cultivars and seasons. However, chlorophyll "a" content significantly (푝 < 0.05) changed with plant density (Figure 1). Planting soybean at the lowest plant density of 10 plants m −2 led to increased concentration of chlorophyll "a" which was 25.38% more than the lowest chlorophyll "a" levels obtained at plant density of 20 plants m −2 .

Intercepted Photosynthetically Active Radiation (IPAR).
Interception of photosynthetically active radiation significantly (푝 < 0.001) changed with interaction of plant density and cultivars (

Relationships of Some Leaf Gas Exchange Parameters and Soil Moisture Status.
ere were positive and linear relationships between stomata conductance and leaf substomatal carbon dioxide concentration, soil moisture level and stomatal conductance, leaf sub-stomatal carbon dioxide concentration and photosynthetic rate while stomatal conductance and leaf temperature had negative linear relationship ( Figure 6). Coefficient of determination ( 2 ) for

Discussion
Root nodulation, stomata conductance, sub-stomata carbon dioxide concentration, photosynthetic rate, transpiration and chlorophyll "a" content were higher at lower planting density. Interception of photosynthetically active radiation and leaf temperature increased with increased plant population. Determinate growth habit led to increased interception of photosynthetically active radiation (IPAR) by cultivar EAI 3600 while higher rates of stomata conductance, transpiration and sub-stomatal carbon dioxide concentration were registered with the indeterminate cultivar DPSB 19. ese results are in agreement with reports by Koesmaryono et al. [12], Zhou et al. [13] and Moreira et al. [14] whose studies indicated reductions in stomata conductance, photosynthetic and transpiration rates at lower plant populations of soybean. Similarly, studies with sorghum by Li et al. [15] also reported reductions in stomata conductance and photosynthetic rate at higher planting populations relative to lower planting density. On the other hand, studies with pigeon peas by Wilson et al. [16] showed that varying planting density did not have a significant effect on stomata conductance, photosynthetic and transpiration rates which is at variance with findings of this study.
respective positive relationships imply that 72.67%, 62.49%, and 57.24% variations in sub-stomatal carbon dioxide concentration, stomatal conductance and photosynthetic rate amongst different planting density treatments may be attributed to differences in levels of stomatal conductance, soil moisture and sub-stomatal carbon dioxide concentration, respectively.  Advances in Agriculture 6 significantly higher during long rains compared to short rains. Soil moisture levels during long rains were 74.17% higher than during short rains. Tanaka and Shiraiwa [24] reported that indeterminate soybean varieties have higher numbers of stomata and epidermal cells per unit area compared to determinate soybean varieties. is, in addition to the fact that indeterminate soybean varieties have smaller leaflets with a potential of minimizing leaf overlaps within a plant, explains the prevalence of increased levels of stomata conductance, photosynthetic and transpiration rates by indeterminate variety DPSB 19 compared to a determinate cultivar EAI 3600. Increased leaf temperature at higher plant population could have been a result of stomata closure to limit water loss from plant tissues which meant that there was a reduction in evaporative cooling of plants offered by increased transpiration rates [25]. Higher soil moisture depletion by the determinate variety (EAI 3600) relative to indeterminate cultivar DPSB 19 could have been due to increased evaporation demand that comes with vigorous plant growth and increased leaf area which characterize determinate plant growth habit [26].

Conclusions
High planting density increased interception of photosynthetically active radiation but reduced root nodulation, stomata conductance, sub-stomatal carbon dioxide concentration, photosynthetic and transpiration rates. Indeterminate cultivar DPSB 19 had higher rates of stomata conductance, photosynthesis and sub-stomatal carbon dioxide concentration compared to determinate cultivar EAI 3600.
Higher number of plants per unit area led to reduced soil moisture level which could have contributed to poor root growth and related reduction in nodulation due to penetration resistance of roots from drier soils [17]. Lower soil moisture levels at higher plant density could also have contributed to both increased desiccation and death of rhizobium or reduction in the efficiency of nitrogenase enzymes to facilitate biological nitrogen fixation [18]. Overall, soybean nodulation was optimized during short rains relative to long rains. Long rainy season was characterized by lower temperatures (mean of 18.9°C) and higher amount of rainfall (total 847.9 mm) compared to short rains mean temperature of 22.03°C and total rainfall of 234.7 mm. Excessive soil moisture levels and low temperature suppress root nodulation and biological fixation in soybean [19,20].
Results of this study, which correspond to findings from previous studies by Hailey and Higley [21], Gilbert et al. [22] and Nasaruddin and Ridwan [23], have shown a positive and linear relationship between soil moisture levels and stomata conductance, stomatal conductance and photosynthetic rate and sub-stomatal carbon dioxide concentration and photosynthetic rate. Lower soil moisture status at higher plant density could have led to reductions in stomata conductance. Reduction in stomata conductance meant reduced diffusion of carbon dioxide and water in and out of plant tissues leading to lower photosynthetic and transpiration rates. Stomata conductance, carbon dioxide concentration and transpiration were higher during long rains compared to short rains. Variations in levels stomata conductance, photosynthetic and transpiration rates between long and short rains may also be associated with differences in soil moisture levels between the two seasons considering that soil moisture level was