Effect of Plant Growth-Promoting Rhizobacteria at Various Nitrogen Rates on Corn Growth

Plant growth-promoting rhizobacteria (PGPR) colonize plant roots and promote plant growth by producing and secreting various chemical regulators in the rhizosphere. With the recent interest in sustainable agriculture, an increasing number of researchers are investigating ways to improve the efficiency of PGPR use to reduce chemical fertilizer inputs needed for crop production. Accordingly, greenhouse studies were conducted to evaluate the impact of PGPR inoculants on biomass production and nitrogen (N) content of corn (Zea mays L.) under different N levels. Treatments included three PGPR inoculants (two mixtures of PGPR strains and one control without PGPR) and five N application levels (0%, 25%, 50%, 75%, and 100% of the recommended N rate of 135 kg N ha). Results showed that inoculation of PGPR significantly increased plant height, stem diameter, leaf area, and root morphology of corn compared to no PGPR application under the same N levels at the V6 growth stage, but few differences were observed at the V4 stage. PGPR with 50% of the full N rate produced corn biomass and N concentrations equivalent to or greater than that of the full N rate without inoculants at the VT stage. In conclusion, mixtures of PGPR can potentially reduce inorganic N fertilization without affecting corn plant growth parameters. Future research is needed under field conditions to determine if these PGPR inoculants can be integrated as a bio-fertilizer in crop production nutrient management strategies.


Introduction
Commercial fertilizers, especially N sources, are essential for maintaining global crop production and fulfilling food requirements for a rapidly growing world population with limited land resources [1] [2] [3] [4]. In 2014, over 11.7 million tonnes of N fertilizer were applied to US agricultural crops [5]. This number is expected to increase in the coming years because inorganic N is an indispensable input in crop production. For example, Stewart et al. [1] evaluated several longterm studies to determine the effect of eliminating N fertilizer, and predicted that corn and cotton (Gossypium hirsutum L.) yields would decline by 41% and 37%, respectively, without N fertilizer. Optimal crop yields also depend upon the nitrogen use efficiency (NUE) of crops. Generally, NUE is very low (~33%; [6]) due to various soil processes and environmental factors [7] [8]. For example, over half of the N applied can be lost from agricultural systems as gaseous loss (N 2 , nitrous oxide, NH 3 etc.), runoff (NO 3 ), or leaching (NO 3 ) into groundwater [9] [10]. Changing this poor NUE requires more effective management practices.
Microorganisms that promote plant growth may be worth evaluating as a prospective tool to improve fertilizer use efficiency [11] [12] [13] [14] [15]. Plant growth-promoting rhizobacteria (PGPR) are free-living microbes that live on or around the roots [16] and that stimulate plant growth and enhance root development and architecture [17] [18] [19] [20]. Kumar et al. [21] reported that applying Pseudomonas aeruginosa LES4 at half the recommended fertilizer rate resulted in growth of sesame (Sesamum indicum L.) that was equivalent to treatments at the full fertilizer rate, and that the oil yield increased 33.3%, and protein yield increased 47.5% compared to the full fertilizer rate. Adesemoye et al. [19] found that on tomato (Solanum lycopersicum) supplementing 75% of the recommended fertilizer with a mixture of Bacillus spp. and arbuscular mycorrhiza fungus (AMF) resulted in growth, yield, and uptake of N and P equivalent to the full fertilizer rate without inoculants. Similar results also showed that inoculating P. thivervalensis and Serratia marcesens to soil with 75% of the recommended chemical fertilizer rate for corn [22] and inoculating Rhodopseudomonas palustris to soil with 50% of recommended chemical fertilizer rate for Chinese cabbage (Brassica rapachinesis; [23]) resulted in the same plant biomass and yield as with the full rate.
Among the genera of PGPR, Bacillus is the most widely used to enhance plant growth and suppress plant diseases [24] due to their capacity to form stable endospores that can be inoculated onto crop seeds. Also, their wide metabolic capabilities allows them to play important roles in soil ecosystem functions and processes, such as soil carbon, nitrogen, and sulfur cycling, and transformation of other soil nutrients [25]. Huang et al. [26] isolated four Bacillus strains from rainforest soils that increased plant height and shoot biomass of Arabidopsis, corn, and tomato under greenhouse conditions. In another study, Wani and Khan [27] reported that Bacillus strains enhanced plant height and plant fresh However, the range of enhancement was much lower when sufficient N was supplied. Inoculating plants with Bacillus strain PSB10 also resulted in enhanced nodulation, chlorophyll, leghemoglobin, seed yield, and grain protein of chickpea (Cicer arietinum L.) in chromium-stressed soils [27]. In another study, Meng et al. [28] inoculated nine types of plants under greenhouse conditions with the B. velezensis strain and found that some of the plants increased growth at various levels in different plant parts. Growth promotion by Bacillus has also been observed with canola (Brassica napus L., [29], corn [30], soybean (Glycine max, [31], sugar beet (Beta vulgaris, [32]), and wheat (Triticuma estivum L., [33]).
Numerous studies and reviews have reported plant growth promotion, increased yield, phytohormone production, soil P solubilization, and enhanced N uptake through inoculation with Bacillus spp. However, most of these studies were conducted using single-strain inoculations and the positive effects were only shown under specific conditions, and hence, growth promotion was limited when using single-strain inoculations [34]. For example, B. velezensis inoculation increased dry leaf weight, but not root weight for several vegetative crops [28]. In a study on canola, de Freitas et al. [29] reported that Bacillus spp. had no effect on plant growth when rock phosphate was applied; while seed yield was increased, there was no effect on P uptake with triple superphosphate. Similarly, de Freitas [33] reported in a pot study that B. polymyxa tended to enhance wheat grain yield, but no differences in total-N or shoot dry matter yield were observed as compared to the uninoculated control.
A few studies have reported that mixtures of PGPR strains generally cause more consistent positive effects on plant growth than do individual strains [35] [36] [37]. In addition, some studies have suggested that PGPR are more effective under limited nutrient conditions [26] [38] [39]. For example, a greenhouse study showed that B. polymyxa had a better stimulatory effect on corn plant growth and N, P, and K uptake in nutrient-deficient soils than in nutrient-rich soils [39]. However, limited information exists concerning the effects of Bacillus spp. mixtures on corn growth with reduced levels of N fertilizers. Therefore, the objectives of this study were to: 1) evaluate the impact of PGPR mixtures on corn root growth and biomass production under different N levels; 2) investigate the potential of PGPR mixtures to allow a reduction in the amount of inorganic N fertilizer needed by resulting in corn plant growth and nutrient uptake levels equivalent to those at the recommended N fertilizer rate; and 3) determine the optimal N rate for stimulating PGPR growth-promoting effects on corn.

Greenhouse Experiment
A greenhouse container study was conducted at Auburn University's Horticulture Paterson Greenhouse (HP) in Auburn, AL, USA. This study consisted of two separate experiments conducted with the same treatments. The first experi-

Data Collection
Corn plants were harvested at the V4, V6, and VT vegetative growth stages.

Data Analysis
An analysis of variance (ANOVA), using a general linear model (GLM) of SAS 9.4 [44], was used to analyze each response variable in this experiment. The least significant difference test (LSD) at a 0.05 probability level was used to identify significant differences among treatments. Significant interactions (P ≤ 0.05) were observed between the two experiments and the N rates. Thus, treatment means for each N rate were analyzed separately by experiment. Significant interactions between N rate and PGPR treatments (P ≤ 0.05) were observed for some response variables, thus, the LSD test was conducted to identify significant difference among PGPR treatments at each N level for these response variables.
Also, comparisons were made to determine the effects of each PGPR inoculant at each N level to the non-inoculated full N rate treatment (standard application rate) using the LSD test.

Plant Growth Parameters
Plant height is often correlated with the number of leaves per plant and can potentially affect corn yield [45]. Nitrogen levels significantly affected the corn vegetative growth parameters evaluated (plant height, stem diameter, and leaf area) in this study from the V4 to VT stages ( Table 2 and Table 3). There were no significant differences in either experiment or clear tendencies observed among the N levels evaluated for plant height at the V4 and VT stages in either experiment. Plants receiving 75% (P = 0.0052) and 100% (P = 0.0327) of the recommended N rate were significantly taller than those with no N application at the V6 stage in the first experiment (HP1  (Table 3). Moreover, inoculation of the PGPR mixture 2 significantly increased plant height compared to the N100P0 treatment (P = 0.0260) at the V4 stage in HP1 (Table 2). Stem diameter was significantly affected by N levels, especially during the latter vegetative growth stages ( Table 2 and Table 3). Plants grown with 50% (P = 0.0042), 75% (P = 0.0050), and 100% (P = 0.0002) of the recommended N rate had significantly greater stem diameter than the no N control at the VT stage during HP1, with an increase of 15.2%, 14.9%, and 18.4%, respectively. Nitrogen rate also significantly affected corn stem diameter at the VT stage in HP2, and plants with 100% of the recommended N rate had the largest stems. Although, there were no significant differences among N treatments for stem diameter at the V4 and V6 stages in HP2, there was a tendency for greater stem diameter with increasing N rates. Fancelli and DouradoNeto [49] reported that stronger stems were directly related to increased productivity since it is involved in the storage of soluble solids, which may subsequently be used in the formation of seeds. PGPR inoculations (averaged across N rates) had minimal impact on stem diameter of corn, no significant difference was observed at the V4 and VT stages and a significant decrease in stem diameter was observed at the V6 stage in HP1 ( Table 2 and Table 3). However, when conducting direct comparisons between each PGPR mixture at each N level, PGPR mixture 1 at the V4 stage in HP1 tended to increased stem diameter for the no N fertilizer (N0P1) treatment, and was even significantly greater than that of the N100P0 (recommended N rate without PGPR) treatment (P = 0.0467).
There were no significant differences among N levels on leaf area at the V4 and VT stages for both experimental times ( Table 2 and Table 3). However, average leaf area at the recommended N rate was significantly larger than that of the no N fertilizer (P = 0.0013) or 25% of recommended N rate treatment (P = 0.0005) at the V6 stage in HP1. The leaf area was not influenced by PGPR applications for both experiments ( Table 2 and Table 3), while PGPR inoculations showed an increasing tendency at some N levels.
Leaf greenness (SPAD readings) was significantly affected by N levels at V6 in HP1 and at the VT stage during both experiments ( Table 2 and Table 3). SPAD readings increased with increasing N rates throughout the plant growth stages; therefore, higher chlorophyll content was observed when relatively high N fertilizer rates were applied. The effects of microbial inoculations on leaf greenness varied depending on growth stage and N level for both experiments (Table 2 and Table 3).
Significant differences were observed between PGPR inoculants at the V6 and VT stages in HP1. An interaction of N level and PGPR inoculation was observed for SPAD readings at the V6 stage in HP1. A significant increase in chlorophyll content was observed after inoculation of PGPR mixtures 1 & 2 when 50% of the recommended N rate (P = 0.0322) was compared to the no-PGPR control at the same N rate. When conducting direct comparisons between each PGPR mixture at each N level, PGPR mixture 1 at the V6 stage (P = 0.0398) and PGPR mixture 2 at the VT stage (P < 0.0001) had significantly greater SPAD readings than the non-inoculated control, with an increase of 4.5% and 12.3%, respectively. Moreover, PGPR mixture 1 with no N application (N0P1; direct comparison between each PGPR at each N level) had the greatest leaf area compared to other treatments, and was significantly greater than that of the N100P0 treatment (P = 0.0183) at the V6 stage in HP2.
The uptake of N by corn is low during early development and increases as it nears tasseling [50], which means that N generally has minimal effects on plant growth during the seedling stages. This likely is why minimal differences were observed for the plant growth parameters at the V4 stage. Moreover, another important factor that may affect seedling growth is the emergence day. Earlier emergence can lead to taller plants, greater stem diameter, and leaf area. Since temperature and sunlight duration increased from March to June; therefore, greater plant growth parameters (plant height, stem diameter, and leaf area) were found in HP2, rather than in HP1.
Overall, applying PGPR had positive effects on plant growth during the vegetative stages, especially at the V6 stage when corn plants' need for N from soil begins to escalate. In this study, PGPR mixture 1 showed positive effects on plant height, stem diameter, and leaf greenness of corn, while PGPR mixture 2 tended to only increase leaf area and leaf greenness of corn. The difference between these two microbial inoculated mixtures may be due to the capacity of the different Bacillus spp. responses to the soil N conditions. Our results are consistent with previous studies which indicated that PGPR can increase plant height [37] [51] [52], increase plant stem diameter [53] [54], and enhance the number of leaves and leaf area of corn [55] [56]. Most of these improvements in plant growth were observed prior to the corn reaching the tasseling stage. In this study, PGPR showed more positive effects under low N soils than the soil with high rates of N fertilization. Consistently, several studies have demonstrated that when nutrient levels are high in soil, PGPR's efficacy to improve plant growth is low [12] [57] [58]. One possible reason is that the production of ethylene under low levels of nutrients could be catabolized by 1-aminocyclopropane-1-carboxylate (ACC) deaminase, produced by PGPR, to NH 3 and α-ketobutyrate [59]. In addition, in nutrient rich soil, plants could obtain enough N from soil by their own root absorption, thus, plants will not need rhizobacteria-enhanced N uptake.

Root Morphology
The influence of PGPR inoculants on root morphological parameters varied with N level and growth stage (Table 4 and Table 5). A significant N and PGPR interaction was observed for total root length at the V6 and VT stages in HP2 (  (Table 4). The PGPR mixture 2 significantly increased total root length by 13.3% (P = 0.0494) and 31.3% (P = 0.0160) with 75% and 100% of the recommended N application rate at the V6 stage and up to 13.9% (P = 0.0024) and 15.6% (P = 0.0418) with 50% and 75% of recommended N rate at the VT stage, respectively. An increase in total root length of 16.1% (P = 0.0013) was observed with the inoculation of PGPR mixture 1 at the VT stage for half the recommended N rate in HP2 (Table 5).
These results indicated that the selected PGPR strains in this experiment could potentially promote root growth even under N-limited conditions. Our results are consistent with those observed in several studies which have indicated that PGPR inoculations effectively increased the root length and surface area [18] [60], suggesting this resulted from PGPR synthesis of phytohormones and other secondary metabolites [61]. It is also worth mentioning that the corn hybrid used in this experiment has a high root strength (8/10) which means it has an innate capacity to grow a strong root system, which may have masked some of the potential positive effects of PGPR on root growth.
Root morphological parameters, especially total root length and root surface area, play an important role in the capture of belowground nutrient resources for plant development [62] [63] and root morphological parameters may exhibit higher water retention [64]. Several studies have reported that root structure and morphology are influenced by soil microorganisms such as rhizobacteria [52] [64] [65] [66]. El Zemrany et al. [64] investigated the root characteristics of corn where seeds were inoculated with PGPR Azospirillumlipoferum CRT1 during the early growth stages (for 35 days after planting, DAP) and demonstrated that plants inoculated with PGPR significantly increased root biomass, total root length, and root surface area at 26, 30, and 35 DAP. Calvo et al. [52] reported that Bacillus spp. mixtures could increase total root length, root surface area, root volume, and total length of fine roots of corn compared to the non-inoculated control when urea ammonium nitrate (UAN) was present at the V2 stage, while positive effects resulted when calcium ammonium nitrate (CAN) was applied at the V4 stage.

Biomass Accumulation and N Uptake
Significant differences were observed among N levels for biomass of roots, stems, and leaves. Plant aboveground biomass tended to increase with increasing N rate at the V6 and VT stages, no significant differences were observed at the V4 stage in both experiments ( Table 6, Figure 1 and Figure 2). At the V4 stage, the no N treatment had the greatest plant biomass when compared with other N rates with the same PGPR treatment, especially in HP1. The no N control had the greatest root biomass on average (Figure 1(a)). At the V6 and VT stages, the relative high N rates (N75 and N100) had the greatest plant biomass regardless    Figure 1 and Figure 2). The lack of PGPR effects on plants evaluated at the V4 stage may be due to the low rate of biomass accumulation and nutrient uptake during the early corn growth.
In contrast, no significant difference between non-PGPR and PGPR treatments on biomass accumulation evaluated at the VT stage may be due to the small amount of nutrients provided by PGPR could not satisfy the high nutrient requirements during the late vegetative growth stage. Nitrogen and PGPR interactions were observed for plant biomass accumulation at the V6 stage in HP1 ( showed lower stem and leaf biomass than the no-PGPR control at the V6 stage. These results indicated that PGPR inoculation induced an increase of plant biomass that was slightly greater than the non-PGPR treatment at the different N levels, especially with low or half-rate N application. Plant tissue N concentrations were significantly different among N treatments, with N concentrations tending to increase with increasing N rate regardless of whether the PGPR inoculants were added or not at the VT stage for both experimental times (Table 6 and Figure 3). Plants receiving 75% and the full N rate had significantly greater root, stem, and leaf N concentration compared to 25% of recommended N rate and unfertilized control, while the half N rate treatments also significantly increased plant tissue N concentrations compared to the unfertilized control ( Figure 3).  Figure 3. These results indicated that under a properly managed greenhouse condition that prevented nutrient loss through leaching, 50% or 75% of the recommended N rate could satisfy plant N requirements during the vegetative growth stages, which may mask the positive effects of inoculated PGPR strains [67]. No significant differences were observed for the response of corn N concentrations to PGPR inoculation ( Figure 3). Also, there was no N and PGPR interaction observed for plant tissue N concentrations (  [73]. Biari et al. [73] indicated that inoculation of PGPR strains can increase corn growth parameters, such as plant height and shoot dry weight and also enhance grain dry weight and seed quality (100-seed weight and nutrients content). Therefore, PGPR treatments in our experiment that enhanced plant growth parameters and biomass accumulation could lead to a potential increase in corn yield. In addition, these positive effects of PGPR are mainly attributed to its capacity to promote better absorption of essential nutrients that are responsible for the high rate of photosynthesis [52] [73]. Consistently, a stronger root system, greater SPAD reading and dry weight biomass were observed with PGPR application in our experiment.

Conclusion
Overall the selected PGPR mixtures applied with half the recommended N rate promoted corn growth and produced corn biomass and tissue N concentrations equal to or greater than that of the full N fertilization rate under greenhouse conditions. The high amounts of N fertilization may have masked the potential fore, PGPR inoculants should be considered as tools that will complement nutrient efficiency practices by increasing the plant's nutrient uptake efficiency, thereby reducing N losses and reducing the amount of applied N. Further studies are needed in order to determine the threshold of N fertilization reduction that could be achieve when PGPR inoculants are applied to different crops and with different types of nitrogen fertilizers, as well as investigate the optimal field management practices for simulating the efficacy of PGPR under field conditions.