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Article

Effect of Non-Native Endophytic Bacteria on Oat (Avena sativa L.) Growth

by
Krishna Ghimire
1,
Vincent Peta
2,
Heike Bücking
3 and
Melanie Caffe
1,*
1
Department of Agronomy, Horticulture, and Plant Science, South Dakota State University, Brookings, SD 57007, USA
2
Department of Biomedical Engineering, University of South Dakota, Sioux Falls, SD 57107, USA
3
Division of Plant Sciences, University of Missouri, Columbia, MO 65211, USA
*
Author to whom correspondence should be addressed.
Int. J. Plant Biol. 2023, 14(3), 827-844; https://doi.org/10.3390/ijpb14030062
Submission received: 18 August 2023 / Revised: 9 September 2023 / Accepted: 12 September 2023 / Published: 14 September 2023
(This article belongs to the Section Plant–Microorganisms Interactions)
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Abstract

:
Endophytic bacteria are known to influence vital activities of host plants. Endophytes can promote plant growth and provide a defense response against pathogens. The use of endophytes in crop production has the potential to reduce the application of fertilizer and pesticide input and thus improve the sustainability of crop production. In this study, we investigated the effects of seed inoculation with non-native endophytic bacteria, harvested from Brassica carinata, on oat (Avena sativa L.) growth with root vigor assays and greenhouse experiments. For root vigor assay experiments, seeds of two different oat cultivars were treated with 16 endophytic bacteria previously shown to promote growth benefits on multiple crop species. For the greenhouse experiments, the effect of seed inoculation with bacterial isolates was evaluated on ten oat cultivars at two fertilization levels. The root vigor assay showed that multiple isolates, including Bacillus licheniformis, Enterobacter kobei, B. halotolerans, B. cereus, B. aryabhattai, and Lysinibacillus fusiformis, had a positive effect on seedling root growth in one of the two oat cultivars. In the other cultivar, the bacterial isolates had either no effect or a negative effect on root growth. Greenhouse studies showed that the magnitude and direction of the effect of bacterial inoculation on oat growth varied with fertilization levels, bacterial strain, and oat cultivar. However, we identified two cultivars that were more responsive to bacterial inoculation than the others and for which bacterial inoculation of seed resulted in enhanced growth in several traits under both reduced and full nitrogen levels, and this response was observed for the two isolates tested. Our results show that inoculating oat seeds with non-native bacterial endophytes can promote root and shoot growth in oats. Developing biofertilizers that are effective across crop species, crop cultivars, and environmental conditions may be possible if cultivars are selected for their responsiveness across multiple bacterial isolates and in multiple growing environments. Overall, this study indicates that non-native endophytes could be considered for the development of biofertilizers with effectiveness across crop species.

1. Introduction

Oat (Avena sativa L.) is an annual grass widely grown worldwide as a source of forage and animal feed. However, oats are now being increasingly grown as a source of healthy grains for humans [1,2]. Oat consumption provides many health benefits, such as reducing low-density lipoprotein cholesterol and preventing cardiovascular diseases. When consumed regularly, oats exhibit glucose-lowering effects and can reduce the risk of type-2 diabetes [3]. Most health benefits associated with oat consumption are attributed to beta-glucan, a unique soluble fiber present in oats and barley that has been associated with lowering cholesterol and managing blood sugar.
Although synthetic fertilizers have beneficial effects on oat production [4], they are considered environmentally unfriendly [5] and they are a costly input for producers. The excessive and imbalanced use of fertilizers for decades has contributed to greenhouse gas emissions (N2O) and underground water leaching. Thus, exploring alternatives to chemical fertilizers is needed to solve this problem. Endophytes have been proposed as an alternative to replace or reduce the use of fertilizers and pesticides.
Bacterial endophytes are bacteria that invade the tissue of living plants and cause asymptomatic infections within plant tissue [6]. Endophytic bacteria can promote plant growth either directly or indirectly. Directly, they can facilitate the acquisition of resources from the environment, including nitrogen, phosphorus, and iron, and they can modulate plant growth by providing or regulating plant hormones like auxin, cytokinin, or ethylene. Indirect plant growth promotion can occur when endophytic bacteria reduce infection by other pathogenic bacteria, fungi, and nematodes.
Endophytes are known to enhance root growth and root branching, which leads to further plant growth. Endophytes can produce growth regulators and enhance nutrient acquisition, which promotes plant growth [7,8,9,10]. Many phosphate-solubilizing bacteria have the potential to convert insoluble phosphorus to a usable form, which leads to plant crops having a higher yield [11,12].
Many non-leguminous crops like rice, sugarcane, wheat, and maize form an extended niche for various species of nitrogen-fixing bacteria [13]. When non-leguminous plants are inoculated with endophytic bacteria, increased nitrogen accumulation can result from either biological nitrogen fixation or increased nitrogen uptake from soil [13]. Biological nitrogen fixation provided 60% of the nitrogen in some Brazilian sugarcane cultivars and 30 to 60 kg N Ha−1 in rice depending on the cultivar [14]. Bacterial endophytes capable of fixing nitrogen and producing indole acetic acid have also been isolated from oat plants [15,16].
The use of bacterial endophytes as biofertilizers for oat production would reduce the application of chemical fertilizers in conventional systems and would be especially beneficial for oat production under organic management systems. However, for the economical production of biofertilizers, it would be advantageous to identify bacterial isolates that promote growth across crop species. There are multiple reports of non-host plant growth promotion by endophytic bacteria [17]. Mendez-Bravo et al. [18] reported an increase in lateral root, root hair formation, foliar rosette size, and overall biomass in Arabidopsis when inoculated with endophytic bacteria isolated from Avocado. These endophytes were also able to promote growth in Mexican husk tomato. Similarly, bacterial endophytes from Fagonia mollis and Achillea fragrantissima enhanced maize growth [19]. Growth promotion by non-host endophytes has been reported in oats [20,21]. Different Pseudomonas sp. isolated from the rhizosphere of maize plants or field soil showed growth promotion in oats [21]; similarly, Klebsiella sp. isolated from wheat root showed growth promotion in oats [20]. Peta [22] characterized endophytic bacteria isolated from Brassica carinata with growth-promoting effects on wheat, maize, and soybean; however, these isolates were not tested on oats.
Multiple factors can influence the crop response to endophytes. Genotype-specific effects of endophytes on plant growth have been reported. Significant genotype-by-endophyte infection interactions on growth have been observed on ryegrass, wheat, rice, and maize [23,24,25,26]. The level of biological nitrogen fixation has also been reported to be dependent on fertilization levels in maize [27]. Thus, testing potential endophytes on multiple genotypes at various nitrogen levels is necessary for the evaluation of growth promotion abilities of endophytes.
While the isolation and characterization of endophytic bacteria from oats [15,16] were reported previously, the effect on oat growth was not evaluated. Moreover, the effect of inoculation with non-native endophytes was restricted to a few endophytic bacteria and tested on only one oat cultivar [20,21]. More studies are needed to determine the applicability of using non-native endophytes as biofertilizers for crop production.
The objectives of this study were to (1) examine the effects of non-native bacterial endophytes previously isolated from Brassica carinata and shown to have nitrogen-fixing capabilities, to determine any growth enhancement when applied to oats, and (2) evaluate the response of various oat cultivars to endophytic bacterial inoculation under two fertilization levels. For this study, we chose bacterial endophytes isolated from Brassica carinata because they were previously shown to promote growth in other major crops such as wheat, maize, and soybean [22].

2. Materials and Methods

2.1. Root Vigor Assay

Surface-sterilized seeds of oat cultivars Hayden and Gopher were treated with a suspension of 16 endophytic bacterial isolates along with an uninoculated control (treated with nutrient broth without bacteria) (Table 1). The sixteen bacterial endophytes used in this study were isolated from Brassica carinata grown in normal field conditions. The identity of the bacteria was confirmed by sequencing the 16S rRNA gene [22]. Those isolates were previously screened for nitrogen-fixing ability, ACC deaminase production, and indole-3-acetic acid (IAA) production [22]. Six of the isolates (BC03, BC06, BC09, BC12, BC16, and BC17) were able to produce IAA. Three isolates (BC09, BC10, and BC12) were able to solubilize phosphate, and six isolates (BC02, BC07, BC10, BC12, BC13, and BC16) showed the presence of ACC deaminase [22]. Confirmation of nitrogen fixation of the bacterial isolates was achieved using nitrogen-free bacterial media [22]. These bacteria have shown varying levels of growth promotion in wheat, maize, and soybean.
A bacterial suspension was prepared by inoculating Luria Bertani Broth with the bacteria and incubating at 24 °C in a shaker overnight and standardized to an OD of 0.05 at 600 nm with phosphate-buffered saline. Oat seeds were first surface-sterilized with a 5% solution of sodium hypochlorite (NaOCl) for 5 min and air-dried under sterile conditions in a laminar flow hood [20]. The surface-sterilized seeds were placed in 15 mL tubes, and the bacterial suspension was added to the tube and shaken for about a minute to coat the seeds with bacteria. The seeds were treated at the rate of 2 µL of bacterial suspension (0.05 ocular density measured at 600 nm wavelength) per seed.
Inoculated seeds (15 per cultivar) were placed in a line between four sheets of heavy-weight germination paper with 50 mL of distilled water in a sterile square petri plate. The plates were stacked randomly in a growth chamber maintained at 25 °C with a 16-hour photoperiod (16 h of light and 8 h of darkness). The plates were kept in a semi-vertical position to allow for correct root and shoot growth. After 6 days, roots were cut from each seedling and scanned using an Epson flatbed scanner (Epson America, Inc., Los Alamitos, CA, USA). The scanned images of the roots were analyzed with the WhinRhizo software (V5.0, Regent Instrument Inc., Quebec City, QC, Canada) to compute root length, root surface area, and root volume. The experiment was conducted twice using a complete randomized design. Data from the two repetitions were combined for data analysis. The experimental design was a factorial design with 17 × 2 treatments, in which two cultivars were evaluated with 16 bacterial isolates and an uninoculated control. Analysis of variance was conducted with the R statistical program [28]. Least significant difference (LSD) was conducted to test differences between treatments using the agricolae package in R [29].

2.2. Greenhouse Study

Ten oat cultivars, namely Deon, Goliath, Gopher, Hayden, Horsepower, Natty, Saddle, Shelby 427, Sumo, and Warrior, and two endophytic bacterial isolates, namely Bacillus licheniformis (BC02) and Enterobacter kobei (BC06), were used for the greenhouse study. This set of oat cultivars represents cultivars adapted to the main oat grain-producing region of the US with variability in maturity, plant height, seed shape, disease resistance, seed composition, and yield potential. Gopher is an older variety released by the University of Minnesota in 1923. Oat seeds were surface-sterilized by stirring them in a 200 mL of 5% solution of sodium hypochlorite for 10 min and then seeds were rinsed with sterile water and allowed to air dry in a laminar flow hood. The surface-sterilized seeds were inoculated with the bacterial suspension (with 0.05 ocular density measured at wavelength of 600 nm) at a concentration of 2 µL per seed and planted in a sand perlite (60:40) mixture in cone containers. The experiment was conducted in a completely randomized design. Each treatment included 7 plants, each grown individually in a cone. The plants were irrigated with Hoagland solution every other day. One set of plants were irrigated with 50 mL of full-strength Hoagland’s solution to give 100% nitrogen application and another set of plants were irrigated with half-strength Hoagland’s solution that contained only 50% of the nitrogen based on Hoagland’s solution recipe [30]. The experiment consisted of a factorial with ten cultivars, two bacterial treatments and one uninoculated control, and two levels of fertilization. Seven plants were maintained per treatment, resulting in a total of 420 plants (experimental units).
At 42 days after planting, chlorophyll content was measured using a SPAD 502 chlorophyll meter. The roots were cleaned with water and were scanned with an Epson flatbed scanner. The scanned images were processed through the WinRhizo software (V5.0, Regent Instrument Inc., Quebec City, QC, Canada) to determine root length, root surface area, and root volume. The root and shoot material were dried to determine dry root and dry shoot weight. Analysis of variance was conducted with R [28]. Least significant difference (LSD) was conducted to test differences between treatments using the agricolae package in R [29].

3. Results

3.1. Effect of Endophytes on Root Development in Oat Seedlings (Root Vigor Assay)

To screen the growth-promoting ability of a set of 16 endophytic bacteria isolated from Brassica carinata on oats (Table 1), a root vigor assay was performed. The effect of bacterial treatment was evaluated on oat cultivars Hayden and Gopher by measuring root characteristics on 6-day-old seedlings. All factors, including oat cultivars, endophyte isolate, and their interaction, had a significant effect on the total root length, root area, and root volume of oat seedlings.
After 6 days, cultivar Gopher had developed seedlings with significantly larger roots than Hayden whether the seeds were inoculated with endophytes or not. Root length, area, and volume were 28.2 cm, 5.84 cm2, and 0.0979 cm3, respectively, for Hayden and 38.5 cm, 7.34 cm2, and 0.1139 cm3, respectively, for Gopher. The total root length of non-inoculated Gopher seedlings was approximately 37% longer than that of non-inoculated Hayden seedlings. Root area and root volume were also approximately 25% and 16% larger for cultivar Gopher in comparison to Hayden.
Because of the significant interaction between cultivars and endophytic isolates, data analysis was performed for each cultivar separately. For cultivar Gopher, seed inoculation with six of the sixteen endophytic isolates tested (BC02, BC03, BC09, BC15, BC16, and BC17) resulted in seedlings with significantly higher root length and root area in comparison to the non-inoculated control (Figure 1). Three of those isolates (BC02, BC03, and BC09) also enhanced root volume. Endophytic treatment with isolates BC02 and BC03 increased the root length of Gopher seedlings by 34 and 27%, respectively; the root area by 33 and 23%, respectively; and the root volume by 30 and 17%, respectively. Isolate BC12 significantly increased root length but had no effect on root area and root volume. The other nine bacterial isolates (BC04, BC06, BC07, BC8, BC10, BC13, BC14, BC19, and BC20) had no significant effect on root length, root area, or root volume.
For oat cultivar Hayden, however, with a few exceptions, inoculation with endophytic bacteria had no significant effect on root characteristics. Twelve out of sixteen isolates had no effect on root length, while four isolates (BC12, BC13, BC17, and BC20) significantly reduced root length (Figure 1). Only inoculation with isolate BC08 resulted in increased root area and root volume as compared to the non-inoculated Hayden control. Isolates BC16 and BC04 resulted in an increase in root area but had no significant effect on root length and root volume (Figure 1). The response to endophytic inoculation on root growth was more pronounced for oat cultivar Gopher than for Hayden. Only isolate BC16 had some positive effect on root growth across both cultivars. In contrast, isolate BC20 had no effect on the root growth of Gopher seedlings but significantly inhibited root growth for Hayden.

3.2. Effect of Endophytes and Fertilization Level on Root and Shoot Growth of Oat Cultivars in the Greenhouse

For this experiment, a larger set of oat cultivars (ten) with contrasting root and shoot characteristics (Table 2) were considered. The effect on root and plant growth was evaluated at a later stage of plant development (panicle initiation stage with the first spikelet of inflorescence just visible). Two fertilization levels (50% and 100% nitrogen application with Hoagland’s solution) were considered. To keep the number of experimental units to a manageable level, only two endophyte isolates were considered for this experiment, Bacillus licheniformis (BC02) and Enterobacter kobei (BC06). Isolate BC02 was selected because the root vigor assay revealed that seed inoculation with this isolate enhanced seedling root growth in cultivar Gopher. Isolate BC06 did not enhance root growth in the root vigor assay for either cultivar, but it was selected based on prior work [22]. Isolate BC06 was able to produce indole acetic acid in vitro and was able to increase root growth in wheat and soybean [22].
An analysis of variance was conducted to determine the effect of each factor. The effects of the cultivar and fertilization treatments were significant for all traits. Bacterial isolates had a significant effect on shoot dry weight, root dry weight, and chlorophyll content but not on root length, area, or volume. Significant interactions between the three main factors (cultivar, fertilization, and bacterial isolate) were observed for all traits except chlorophyll content. The interaction between cultivar and bacteria was also significant for all traits. The interaction between cultivars and fertilization level was significant for root length and root area. The interaction between bacteria and fertilization level was significant for chlorophyll content. The response to endophyte inoculation varied depending on the trait considered, the oat cultivar, the bacterial isolate, and the nitrogen fertilization level. Due to those complex interactions between factors, the effect of the endophyte treatments on oat root and shoot growth was analyzed at each fertilization level and for each bacterial isolate.

3.2.1. Effect of Fertilization Level on Root and Shoot Growth of Oats

As expected, reduced fertilization resulted in lower average shoot and root dry weight, lower chlorophyll content, and reduced root development (Table 3). The effect of fertilization level on the ten oat cultivars was evaluated by analyzing data collected on the non-inoculated controls. The effects of nitrogen level and cultivars were significant for all six traits evaluated. There was a significant interaction between cultivars and fertilization level for all traits except for chlorophyll content, with cultivars responding differently to reduced fertilization rate (Figure 2). Shoot dry weight was significantly lower under reduced fertilization for all cultivars except for Gopher, Shelby 427, and Hayden (Figure 2). The reduction in root development due to lower fertilization rate was more pronounced for cultivars Deon, Goliath, Saddle, Natty, and Warrior than for Gopher, Horsepower, Shelby 427, and Sumo (Figure 2). Surprisingly, the root system of Hayden was significantly increased under the reduced fertilization rate (Figure 2).

3.2.2. Response of Oat Cultivars to Endophyte Inoculation under Full Fertilization Rate

Under the full nitrogen rate, inoculation with BC02 significantly increased the shoot dry weight in four cultivars (Deon, Hayden, Natty, and Saddle) but significantly decreased the shoot dry weight for cultivar Goliath (Figure 3). Inoculation with BC06 significantly increased shoot dry weight in Deon, Gopher, and Saddle but significantly reduced shoot dry weight for Warrior (Figure 3). Only Deon and Saddle showed an increase in shoot dry weight across bacterial treatments.
Inoculation with bacterial isolate BC02 resulted in an increase in root dry weight for four cultivars (Deon, Natty, Saddle, and Sumo) but significantly decreased root dry weight for two cultivars (Horsepower and Warrior) (Figure 3). Inoculation with BC06 also significantly increased root dry weight in four cultivars (Natty, Saddle, Shelby 427, and Sumo). Three cultivars (Natty, Saddle, and Sumo) out of the ten evaluated showed an increase in root dry weight for both bacterial treatments.
Inoculation with BC02 significantly increased chlorophyll content in 6 out of 10 cultivars (Deon, Gopher, Horsepower, Natty, Sumo, and Warrior). However, inoculation with BC06 significantly increased chlorophyll content in only two cultivars (Gopher and Saddle). Only Gopher showed a consistent increase in chlorophyll content across bacterial treatments (Figure 3).
Inoculation with BC02 significantly increased root length in Hayden and Sumo, but significantly decreased root length in Gopher, Saddle, and Warrior (Figure 3). Inoculation with BC06 significantly increased root length in Hayden and Sumo, and significantly decreased root length in Gopher and Saddle. Interestingly, inoculation of cultivar Saddle with either bacterial isolate led to the development of plants with higher root dry weight but with lower total root length in comparison to the non-inoculated control.
Inoculation with BC02 significantly increased root area in Hayden, Natty, and Sumo, but significantly decreased root area in Goliath, Saddle, and Warrior (Figure 3). Similarly, BC06 inoculation significantly increased root area in Hayden, Horsepower, Shelby 427, and Sumo, but significantly decreased root area in Gopher and Saddle. The same response was observed across isolates for Hayden (increase in root area), Saddle (decrease in root area), and Sumo (increase in root area).
Inoculation with BC02 resulted in an increase in root volume in Deon, Hayden, and Natty, and a decrease in root volume in Goliath. Inoculation with BC06 significantly increased root volume in three cultivars (Hayden, Shelby 427, and Sumo), and decreased root volume in Gopher, Saddle, and Warrior.

3.2.3. Response of Oat Cultivars to Endophyte Inoculation under Half Fertilization Rate

The response of the ten oat cultivars to bacterial inoculation differed between the two fertilization rates. Inoculation with BC02 significantly increased shoot dry weight for Natty, Saddle, Sumo, and Warrior while inoculation with BC06 significantly increased shoot dry weight for Goliath, Horsepower, and Saddle (Figure 4). Consistent with our observations under the full nitrogen rate, seed inoculation for Saddle resulted in an increase in shoot dry weight, irrespective of the bacterial treatment (BC02 or BC06).
Root dry weight significantly increased for four cultivars (Deon, Goliath, Gopher, Sumo, and Warrior) but significantly decreased for Hayden as a result of seed inoculation with BC02 (Figure 4). Inoculation with BC06 resulted in a significant increase in root dry weight for Goliath, Horsepower, and Saddle, but resulted in reduced root dry weight for Hayden and Shelby 427 (Figure 4). The reduction in root dry weight in Hayden was consistent across isolates but was only observed under the half nitrogen rate.
Chlorophyll content was significantly increased for Deon, Gopher, Hayden, and Horsepower from inoculation with either BC02 or BC06. Inoculation with BC02 also increased the chlorophyll content in Sumo, but the opposite was observed for cultivar Natty (Figure 4). Inoculation with BC06 also increased chlorophyll content in Goliath and Saddle) (Figure 4).
Under reduced nitrogen availability, root length in Gopher was inhibited by inoculation with bacterial endophytes irrespective of the isolate (Figure 4). Inoculation with BC06 also reduced the total root length for Hayden (Figure 4). However, inoculation with BC02 significantly increased root length in Deon and Warrior, and inoculation with BC06 resulted in significantly higher total root length for Deon and Natty.
Inoculation with BC02 resulted in an increase in root area for Deon, Goliath, Sumo, and Warrior but a decrease for Gopher and Hayden (Figure 4). Inoculation with BC06 resulted in a significant increase in root area for Deon and Natty but a decrease for Goliath and Gopher (Figure 4). Seed inoculation resulted in a reduction in root area for the oat cultivar Gopher irrespective of the isolate used for inoculation (BC02 or BC06).
Inoculation with BC02 resulted in an increase in root volume for Deon, Goliath, Natty, Sumo, and Warrior; however, the opposite effect (a reduction in root volume) was observed for Hayden. Inoculation with BC06 increased root volume for Deon, Natty, and Saddle, but decreased root volume for Goliath. The root volume of Goliath was impacted in opposite directions (increase or reduction in volume) depending on the bacterial isolate used for inoculation.

3.2.4. Comparison of the 10 Oat Cultivars for Their Growth Response Pattern to Endophyte Inoculation

Across all traits, cultivars, isolates, and fertilization levels, inoculation with bacterial endophytes resulted in enhanced growth characteristics most of the time (86 growth promotion versus 33 inhibitions) (Figure 5). Inoculation of Deon and Sumo seed with bacterial endophytes resulted in overall enhanced plant growth irrespective of the bacterial isolate or nitrogen levels. For those two cultivars, inoculation did not inhibit any trait no matter the isolate or the fertilization level. The enhancement in growth for those two cultivars was more pronounced with isolates BC02 and the growth promotion was stronger under the reduced fertilization level. On the other hand, Shelby 427 was the least responsive to bacterial inoculation, with three positive and one negative response across all traits and treatments. For certain cultivars, the response depended on the nitrogen level. For example, for Hayden, bacterial inoculation enhanced plant growth under the full nitrogen level but inhibited growth under the half nitrogen level. For other cultivars, the response was affected by both the fertilization level and the bacterial isolate. For example, for Warrior, inoculation with BC02 had a growth promotion effect under the half nitrogen level but a negative effect under the full nitrogen rate. Inoculation with BC06 had no effect on Warrior under the half nitrogen level.

4. Discussion

The use of non-native endophytes as biofertilizers has been considered previously. For example, the endophytic rhizobia from clover root were considered for use as biofertilizers in rice [31]. In this study, we examined the potential of bacterial isolates from Brassica carinata as biofertilizers for oat production. Those isolates were previously evaluated for ACC deaminase and indole-3-acetic acid (IAA) production and exhibited plant growth promotion in wheat and soybean [22].
Our first experiment consisted of a root vigor assay to screen multiple bacterial endophyte species for their effect on root development in oat seedlings. The results indicate that inoculation with bacterial endophytes can have positive effects on seedling root growth, but the effect depends on the bacterial isolate and the oat cultivar. Several mechanisms of action may be responsible for the growth promotion; some bacterial isolates that increased root growth in Gopher (BC03, BC09, BC16, and BC17) were shown previously to produce IAA [22]. Isolate BC09, which increased root length in Gopher, can solubilize phosphate [22]. Similarly, BC02 and BC16, which increased the root length in Gopher, were shown previously to exhibit ACC deaminase activity [22].
Bacterial isolates that significantly increased either total root length, root area, or root volume belong to the following species: B. licheniformis, Enterobacter kobei, B. halotolerans, B. cereus, B. aryabhattai. These endophyte species were reported to have positive effects on the growth of ground nut, lentil, wheat, red pepper, soybean, maize, and spinach [32,33,34,35,36,37]. In our study, oat seed inoculation with B. cereus (isolate BC15) enhanced total root length and root area in comparison to the non-inoculated control. Similar results have been reported by Cakmakci et al. [37] where inoculation with B. cereus resulted in enhanced growth in wheat and spinach compared to non-inoculated control. Joo et al. [34] also reported that the growth of red pepper seedlings increased when inoculated with an isolate of B. cereus MJ-1. Gibberellins were produced by B. cereus in culture broth; hence, gibberellins may have been responsible for the growth promotion effect observed on red pepper [34]. B. aryabhattai, which increased root growth in oat seedlings in our study, was also reported to increase root and shoot biomass in soybean and wheat [32]. B. aryabhattai was shown to have zinc-solubilizing capabilities and improve growth, yield, and zinc content in soybean and wheat [32].
The two oat cultivars evaluated in the root vigor assay responded differently to endophytic treatment. Gopher responded with an increase in root growth for nearly half of the bacterial treatments evaluated; however, for Hayden, only three bacterial isolates enhanced root growth and four isolates resulted in a decrease in root length. Gopher is a selection from the cultivar Sixty Day [38] and was released by the University of Minnesota in 1923 (GRIN-Global, NPGS). Sixty Day was introduced from Russia in 1901 [38]. Hayden is a modern oat cultivar released by the South Dakota Experimental Station in 2014 [39]. The genetic backgrounds and breeding procedures are different for these two cultivars. The growth promotion activity of endophytes may be dependent upon the specific combination of oat cultivars and bacteria and how the cultivars can support the growth and colonization by endophytes. In Hayden, BC20 is the only bacteria that inihibited growth for all root traits (length, area, and volume); this endophyte did not produce IAA or ACC deaminase and was not able to solubilize phosphate, so the negative growth response may be due to the metabolic cost of harboring the bacteria. Many researchers have reported cultivar-specific effects of endophytes in wheat, maize, and rice [24,25,26,27].
In the greenhouse study, the magnitude and direction of endophyte effects on oat growth varied with nitrogen levels and bacterial isolates and differed between oat cultivars. This is consistent with previous studies in various crops; the growth response is affected by the inoculant strain, plant species (northern oat grass, wheat, and spinach), plant cultivar, nitrogen availability, and the growth parameters evaluated [37,40,41,42,43].
While the response of oat cultivars to endophyte inoculation was variable, for different traits, similar responses were seen for the traits that were highly correlated such as for root traits. Overall, oat cultivars Deon and Sumo were the most positively affected by bacterial inoculation. Deon is a high-yielding cultivar developed by the University of Minnesota and widely grown in the Northern Great Plains for both conventional and organic oat production. Sumo is a variety developed by South Dakota State University that has been primarily grown under organic management systems. Although the degree of response of the two cultivars was affected by the fertilization level and the bacterial strains and varied based on the trait, bacterial inoculation resulted in an enhanced growth response for those two cultivars. This suggests that it is possible to identify cultivars that would benefit from bacterial seed treatment. Similar observations were made previously. In a study evaluating the effect of endophytes on maize, some cultivars showed more consistent responses than others [44].
For other cultivars, however, such as Goliath, the response was much more complex. While inoculation with BC02 inhibited growth in Goliath under the full fertilization rate, the same strain promoted growth under the limited fertilization level. For other cultivars, the same bacterial isolate would have positive and negative effects depending on the trait. For example, bacterial inoculation (with either bacterial isolate) increased chlorophyll content but inhibited root growth in Gopher at all fertilization levels. A negative effect of a bacterial endophyte on some traits and a positive effect on other traits was reported previously for perennial ryegrass [45]. For Hayden, the root growth response was dependent on the fertilization level; inoculation with either strain enhanced root growth under the full fertilization rate but inhibited root growth under the reduced fertilization rate. The discrepancy in response could be due to a difference among cultivars to support specific bacterial growth and tissue colonization and to a difference in capability to support nitrogen fixation by endophytes with varying nitrogen levels. While plant growth responses to endophytic treatments can be due to enhanced nutrient and water acquisition or to the synthesis of plant hormones, these phenomena are plant–genotype-interaction-specific [46] and influenced by resource availability [47,48].
In the greenhouse study, root growth responses in Hayden and Gopher were not consistent with the results of the root vigor assay. While inoculation with BC02 enhanced root growth for Gopher in the root vigor assay, it was inhibited in the greenhouse study. Similarly, while Hayden was not responsive to BC02 or BC06 in the root vigor assay, inoculation with those two strains had a positive impact on root growth under the full-fertilization rate and a negative impact on root growth under the reduced fertilization rate. The root vigor assay is a quick and relatively easy method to screen many bacteria for plant growth promotion; however, validation of the results in greenhouse or field studies is recommended [22]. It is also likely that other factors such as the stage of the plant and nitrogen level impact the growth response to bacterial endophytes. In the root vigor assay, root traits were measured on seedlings, while in the greenhouse study, the root traits were measured on plants close to heading. In rice, the effect of endophyte inoculation was different at different growth stages, with more cultivars exhibiting enhanced shoot growth at 37 days post-inoculation than at 58 days post-inoculation [49].
Overall, based on the percent change in plant shoot and root traits under both fertilization levels, inoculation with BC02 produced more growth response in oats (65 significant responses with 48 positive and 17 negative responses) compared to BC06 (54 significant responses with 38 positive and 16 negative responses). This is consistent with the greater response to BC02 than BC06 in the root vigor assay. Similar results were obtained in maize where inoculation with an endophyte isolate produced more positive responses compared to the other [43]. Similarly, when oats were inoculated with Pseudomonas strains isolated from the rhizosphere, only some strains significantly increased root and shoot dry weight [21]. BC02 was not able to produce IAA in vitro, but it was able to produce ACC deaminase, which is an inhibitor of ethylene, thus preventing ethylene-induced root growth suppression. Isolate BC06, on the other hand, was able to produce indole acetic acid (IAA) when measured in vitro by a colorimetric assay, which could have helped in growth stimulation. IAA is the most common naturally occurring plant hormone in the auxin class and has a noted impact on the stimulation of rhizogenesis or the production of roots and rhizomes [50]. The differences in root development observed between genotypes when inoculated with BC06 may result from the variation among oat cultivars to support bacterial growth and the amount of IAA produced by the bacteria in different genotypes. The ability of plant roots to exude flavonoids and IAA can impact colonization by endophytes and thus impact a plant’s overall response to endophyte inoculation. Different oat cultivars may have different flavonoid profiles and different capabilities to exude those flavonoids and IAA. Endophytes and their interactions with plant genotypes influence the level of plant hormones, and hormone level is most critical in influencing host growth and physiological outcomes [51]. The root surface area of wheat seedlings decreased by 13–38% when inoculated with an IAA-deficient mutant of salt-tolerant Pseudomomas moraviensis compared to the wild-type strain [52].
Isolate BC06 resulted in similar responses under both full and half fertilization rates; however, isolate BC02 showed more positive responses and less negative responses under the reduced fertilization level. The capacity of endophytic infection to influence the host growth is dependent on nitrogen levels [48,53,54].Ravel et al. [45] and Lewis [55] reported the advantages of endophyte infection at a low nitrogen rate. In sesame, a higher yield was obtained when treated with endophytic bacteria at 25–50% lower fertilization compared to treatment with 100% nitrogen fertilization [56]. However, Cheplick et al. [57] reported a significant increase in biomass with endophyte inoculation at intermediate and high nutrient levels but not with low nutrient application [57].
In certain combinations of oat cultivars, bacterial isolate, and fertilization levels, bacterial inoculation resulted in an inhibition of plant growth (as compared to the control). This inhibition of growth caused by the presence of some endophytes has been observed previously [22,44,57]. There is a metabolic cost to the plant hosting the bacteria, especially under low nutrient availability [42]. In nitrogen-fixing interactions between host and endophyte, the host plant plays an important role by supplying the carbon and energy source for bacterial growth and nitrogen fixation [58]. In the cultivar Warrior, on the other hand, shoot dry weight, root dry weight, root length, and root area were reduced with bacterial inoculation under a high fertilizer rate but enhanced under a low fertilizer level. The inhibition of growth under a high nitrogen level could be associated with differences in alkaloid production by endophytes. Faeth and Fagan [59] suggest that at low soil nitrogen levels, the interaction between host plants and endophytes is potentially mutualistic, and at higher nitrogen levels, there might be net nitrogen loss for the host due to endophytic alkaloid productions; this provides a possible explanation for growth enhancement under low nitrogen but growth suppression at high nitrogen application. Endophytes with a high alkaloid synthesis capacity are thought to consume the majority, if not all, of the nitrogen they stimulate, as well as additional nitrogen from the soil [59].
Nitrogen fertilization can also modify the composition and abundance of root exudates [60]. The root exudates can influence the colonization of the plant by endophytes. This might explain why the response to endophytic inoculations is not consistent for a specific genotype across nitrogen levels. When maize was supplied with increasing amounts of nitrogen, roots secreted more sugars, sugar alcohol, and phenolics, which altered the surrounding soil microbial community. High nitrogen can increase the activity of ammonia-oxidizing and denitrifying bacteria, leading to a decrease in nitrogen use efficiency [60]. Since root exudates can increase rhizospheric bacteria, it may be possible to select cultivars that can secrete reduced root exudates even at high nitrogen application and increase nitrogen use efficiency.
Identifying growth-promoting strains of endophytes is challenging given the variation in direction and magnitude of responses on oats. There are no general and predictable effects of endophyte inoculation on oat growth. Since the response to endophyte inoculation is cultivar-specific and dependent on the growth parameters evaluated, inoculation by multiple endophytes may be considered for enhancing overall plant growth. Several studies have shown that inoculation with multiple endophytes has a greater influence on plant growth promotion than single-strain inoculation [21,61,62,63]. A study in oats reported that the use of two different strains of Pseudomonas sp. (one isolated form maize rhizosphere and another isolated from common reed rhizosphere) was more successful at increasing seedling root length under salt stress than when each strain was used individually [21]. Oliveira et al. [62] used seven different combinations of inoculum using five endophytic species (Gluconacetobacter, diazotrophicus, Herbaspirillum seropedicae, Herbaspirillum rubrisubalbicans, Azospirillum amazonense, and Burkholderia sp.) to evaluate the effect of inoculating endophytic N2-fixing bacteria on sugarcane. The analysis of the biological nitrogen fixation contribution using the 15N-isotope dilution technique showed that inoculation increased the biological nitrogen fixation to the plant tissues and the best treatment was a mixture of all five strains, followed by treatment with a mixture of Herbaspirillum spp. Similarly, Knoth et al. [61] conducted a greenhouse trial with single-strain endophyte and consortia inoculations in poplar clones and they reported that the growth promotion was more pronounced with multi-strain consortia than with single-strain inoculum.
Our results show that endophytic bacteria isolated from B. carinata were able to enhance growth in some oat cultivars. Although we did not evaluate tissue colonization, bacterial endophytes isolated from B. carinata resulted in significant changes in several traits for all cultivars evaluated. Two oat cultivars were identified which were more responsive to inoculation and generally exhibited enhanced growth compared to the non-inoculated check under both fertilization levels. This suggests that the deployment of bacterial endophytes as biofertilizers to enhance oat growth can be successful and can reduce the need for chemical fertilizers in oat production. However, such biofertilizers will need to be deployed for specific plant cultivars that are responsive to bacterial inoculation under various environmental conditions. A better understanding of specific molecular mechanisms responsible for plant cultivar–endophyte interactions would help plant breeders develop improved cultivars with response across environmental conditions and across multiple bacterial strains and species. It would be useful to monitor the colonization of oat tissues following inoculation and elucidate specific mechanisms of growth promotion/inhibition on important oat growth traits. Further studies into the growth-promoting mechanisms employed by these bacteria are necessary in oats and other crops. In addition, field studies are needed to translate and validate the findings of this study to oat production fields.
The endophyte strains evaluated in this study were isolated from a different species than oats (B. carinata). Yet, some of the isolates were effective in promoting growth in oats. Our results indicate that it should be possible to develop biofertilizers that are effective across plant species. This should make the production and deployment of biofertilizers more economical than if developed for each crop species individually. This study opens doors to testing endophytes across crop species and presents valuable findings for the deployment of endophytes as biofertilizers to increase the sustainability of crop production.

Author Contributions

Conceptualization, H.B., M.C., V.P. and K.G.; methodology, K.G., V.P., H.B. and M.C.; software, K.G.; validation, H.B., K.G., M.C. and V.P.; formal analysis, K.G. and M.C.; investigation, K.G. and M.C.; resources, M.C. and H.B.; data curation, K.G.; writing—original draft preparation, K.G. and M.C.; writing—review and editing, M.C., V.P. and H.B.; visualization, K.G. and M.C.; supervision, M.C.; project administration, M.C.; funding acquisition, M.C. All authors have read and agreed to the published version of the manuscript.

Funding

This research was supported by the South Dakota Crop Improvement Association, the South Dakota Agricultural Experiment Station and U.S. Department of Agriculture, National Institute of Food and Agriculture [Hatch SD00H529-14]. The findings and conclusions in this publication have not been formally disseminated by the U.S. Department of Agriculture and should not be construed to represent any agency determination or policy.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data supporting the findings of this study are available from the corresponding author, M.C., upon reasonable request.

Acknowledgments

The authors would like to thank the South Dakota Crop Improvement Association, the South Dakota Agricultural Experiment Station and USDA NIFA for their support.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Percent change in root length (cm), root area (cm2), and root volume (cm3) when inoculated with endophytic bacteria compared to non-inoculated control in Gopher (left column) and Hayden (right column). Data are presented as mean ± standard error. * Asterisks represent statistical differences compared to the control.
Figure 1. Percent change in root length (cm), root area (cm2), and root volume (cm3) when inoculated with endophytic bacteria compared to non-inoculated control in Gopher (left column) and Hayden (right column). Data are presented as mean ± standard error. * Asterisks represent statistical differences compared to the control.
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Figure 2. The percent changes in shoot dry weight, root dry weight, chlorophyll content, root length, root area, and root volume for ten oat cultivars under half nitrogen application compared to full nitrogen application. * Asterisks represent statistical differences compared to the control.
Figure 2. The percent changes in shoot dry weight, root dry weight, chlorophyll content, root length, root area, and root volume for ten oat cultivars under half nitrogen application compared to full nitrogen application. * Asterisks represent statistical differences compared to the control.
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Figure 3. Percent change in shoot dry weight (mg), root dry weight (mg), chlorophyll content, root length (cm), root area (cm2), and root volume (cm3) when inoculated with BC02 and BC06 compared to non-inoculated control under full fertilization rate. Data are presented as mean ± standard error. * Asterisks represent statistical differences compared to the control.
Figure 3. Percent change in shoot dry weight (mg), root dry weight (mg), chlorophyll content, root length (cm), root area (cm2), and root volume (cm3) when inoculated with BC02 and BC06 compared to non-inoculated control under full fertilization rate. Data are presented as mean ± standard error. * Asterisks represent statistical differences compared to the control.
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Figure 4. Percent change in shoot dry weight (mg), root dry weight (mg), chlorophyll content, root length (cm), root area (cm2), and root volume (cm3) when inoculated with BC02 and BC06 compared to non-inoculated control under half fertilization rate. Data are presented as mean ± standard error. * Asterisks represent statistical differences compared to the control.
Figure 4. Percent change in shoot dry weight (mg), root dry weight (mg), chlorophyll content, root length (cm), root area (cm2), and root volume (cm3) when inoculated with BC02 and BC06 compared to non-inoculated control under half fertilization rate. Data are presented as mean ± standard error. * Asterisks represent statistical differences compared to the control.
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Figure 5. Heat map for percent change in root dry weight, root length, root area, root volume, shoot dry weight, and chlorophyll content when inoculated with BC06 and BC02 under full and half nitrogen applications. Color gradient represents the percent change values for each trait. Asterisks represent statistical differences compared to the control.
Figure 5. Heat map for percent change in root dry weight, root length, root area, root volume, shoot dry weight, and chlorophyll content when inoculated with BC06 and BC02 under full and half nitrogen applications. Color gradient represents the percent change values for each trait. Asterisks represent statistical differences compared to the control.
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Table 1. List of endophytic bacteria and 16S rRNA taxonomic identity used in root vigor assay.
Table 1. List of endophytic bacteria and 16S rRNA taxonomic identity used in root vigor assay.
Bacterial ID16S rRNA Taxonomic Identity
BC02Bacillus licheniformis
BC03Enterobacter kobei
BC04Pantoea ananatis
BC06Enterobacter kobei
BC07Bacillus pumilus
BC08Pantoea agglomerans
BC09Brevibacterium halotolerans
BC10Bacillus toyonensis
BC12Bacillus pumilus
BC13Bacillus pumilus
BC14Bacillus thuringiensis
BC15Bacillus cereus
BC16Bacillus aryabhattai
BC17Lysinibacillus fusiformis
BC19Brevibacterium halotolerans
BC20Pseudomonas spp.
Table 2. Shoot and root characteristics for the control treatment (non-inoculated) of ten oat cultivars.
Table 2. Shoot and root characteristics for the control treatment (non-inoculated) of ten oat cultivars.
CultivarsShoot Dry Weight (mg)Root Dry Weight (mg)Chlorophyll Content Root Length (cm)Root Area (cm2)Root Volume (cm3)
Deon681.4 ± 32.9 b188.3 ± 9 de55.5 ± 0.9 ab407.8 ± 16.7 d 104.9 ± 3.8 f 2.2 ± 0.1 de
Goliath761.1 ± 46.0 ab211.4 ± 12.5 cd53.9 ± 1.4 abcd514.5 ± 21.7 b134.9 ± 4.7 b 2.9 ± 0.2 ab
Gopher806.9 ± 34.9 a222.9 ± 8.5 bc50.4 ± 1.2 ef649.4 ± 24.9 a159.6 ± 5.5 a3.2 ± 0.2 a
Hayden769.8 ± 39.4 ab 262.7 ± 9.2 a55.3 ± 1.1 abc486.9 ± 20.7 bc129.0 ± 5.2 bc 2.9 ± 0.2 ab
Horsepower788.1 ± 32.5 ab198.0 ± 9.1 cde49.6 ± 1.2 f518.4 ± 19.7 b125.8 ± 3.2 bcd2.5 ± 0.1 cd
Natty866.9 ± 57.8 a243.4 ± 11.9 ab 52.4 ± 0.9 bcdef483.9 ± 18.6 bc114.5 ± 4.1 def2.2 ± 0.1 de
Saddle770.6 ± 46.7 ab 158.1 ± 9.1 f52.6 ± 1.1 abcde508.5 ± 25.9 bc121.5 ± 5.9 cde 2.7 ± 0.1 cde
Shelby427681.6 ± 35.6 b177.7 ± 11.8 ef51.0 ± 1.2 def450.3 ± 20.9 cd109.5 ± 3.2 ef2.2 ± 0.1 de
Sumo810.0 ± 33.4 a175.5 ± 5.3ef55.9 ± 1.0 a484.9 ± 24.2 bc110.2 ± 2.9ef2.1 ± 0.1 e
Warrior807.7 ± 48.1 a201.5 ± 5.1cde52.4 ± 1.1 cdef466.3 ± 24.2 bcd123.7 ± 2.9 bcd2.7 ± 0.1 bc
Average774.4203.952.9497.1127.22.5
C.V. (%)28.126.111.023.619.2 29.7
Values followed by different letters in a column are significantly different (p < 0.05). Data are presented as mean ± standard error.
Table 3. Mean, range, and standard deviation for biomass and root traits of oat plants grown at two nitrogen levels.
Table 3. Mean, range, and standard deviation for biomass and root traits of oat plants grown at two nitrogen levels.
Half Fertilization RateFull Fertilization Rate
TraitsMeanRangeStandard
Deviation		MeanRangeStandard
Deviation
Shoot dry weight (mg)736.3163–1437208.6906.6293–1608227.7
Root dry weight (mg)200.568–40057.6231.997–45261.8
Chlorophyll content 52.435.1–69.45.756.334–73.45.6
Root length (cm)469.8178.9–836.2116.5517.7156.2–859.1117.7
Root area (cm2)118.353.2–189.224.0130.842.7–217.925.5
Root volume (cm3)2.451.02–5.380.722.720.93–5.810.83
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Ghimire, K.; Peta, V.; Bücking, H.; Caffe, M. Effect of Non-Native Endophytic Bacteria on Oat (Avena sativa L.) Growth. Int. J. Plant Biol. 2023, 14, 827-844. https://doi.org/10.3390/ijpb14030062

AMA Style

Ghimire K, Peta V, Bücking H, Caffe M. Effect of Non-Native Endophytic Bacteria on Oat (Avena sativa L.) Growth. International Journal of Plant Biology. 2023; 14(3):827-844. https://doi.org/10.3390/ijpb14030062

Chicago/Turabian Style

Ghimire, Krishna, Vincent Peta, Heike Bücking, and Melanie Caffe. 2023. "Effect of Non-Native Endophytic Bacteria on Oat (Avena sativa L.) Growth" International Journal of Plant Biology 14, no. 3: 827-844. https://doi.org/10.3390/ijpb14030062

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