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Article

Effects of Inoculation with Different Plant Growth-Promoting Rhizobacteria on the Eco-Physiological and Stomatal Characteristics of Walnut Seedlings under Drought Stress

by
Dawei Jing
1,†,
Binghua Liu
2,†,
Hailin Ma
2,
Fangchun Liu
2,*,
Xinghong Liu
2 and
Liying Ren
3
1
College of Ecology, Resources and Environment, Dezhou University, Dezhou 253023, China
2
Institute of Resource and Environment, Shandong Academy of Forestry, Jinan 250014, China
3
School of Resources and Environment, Linyi University, Linyi 276000, China
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Agronomy 2023, 13(6), 1486; https://doi.org/10.3390/agronomy13061486
Submission received: 4 May 2023 / Revised: 25 May 2023 / Accepted: 25 May 2023 / Published: 28 May 2023
(This article belongs to the Special Issue Rhizosphere Microorganisms)
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Abstract

:
Plant growth-promoting rhizobacteria (PGPR) can promote plant growth and induce systemic resistance to biological and abiotic stresses. However, do all PGPR have significant effects in arid environments, and which PGPR have the most optimal effects? This study used a pot experiment to investigate the effects of inoculation with two different PGPR on the physiological and ecological characteristics of walnut (Juglans regia) seedlings under drought stress: Bacillus subtilis GE1, which secretes protease only, and Pseudomonas brassicacearum X123, which secretes protease and indoleacetic acid (IAA). The leaves inoculated with X123 under drought stress had higher net photosynthetic rate (Pn), transpiration rate (Tr), and stomatal conductance (gs), especially stomatal length and stomatal width, compared to GE1 inoculation under drought stress. Moreover, inoculation with X123 significantly increased the leaf superoxide dismutase (SOD) and ascorbate peroxidase (APX) activities by 20.84% and 12.41%, respectively, and the gibberellin (GA) and zeatin (ZT) contents by 72.07% and 19.17%, respectively, whereas the leaf soluble sugar and soluble protein contents significantly decreased compared with GE1 inoculation. These results indicated that the effects of GE1 inoculation on the physiological and ecological characteristics of walnut seedling leaves were significantly weaker in comparison with X123 inoculation as a result of its functional characteristics. The application of different PGPR on the drought tolerance of J. regia showed significant differences. Therefore, the selection of appropriate PGPR is key to achieving positive treatment effects under drought conditions.

1. Introduction

With the gradual intensification of global warming, more areas are experiencing drought conditions, both in terms of drought intensity and how long the drought lasts for [1], with serious negative effects on the sustainable development of agriculture and ecological environments [2]. In arid and semi-arid regions, most fruit crops are often in a state of water shortage, which negatively affects the growth of fruit trees, fruit yield, and quality [3]. However, traditional drought-resistant measures, such as irrigation and selection of drought-tolerant varieties, are time-consuming and labor-intensive. Therefore, there is an urgent need to develop ways of enhancing the drought-adaptive ability of plants in arid and semi-arid regions.
Walnut is not only an important nut and woody oil fruit tree, but it is also an important timber and greening tree species [4], which has significant economic, ecological, and social benefits [5]. If walnuts experience drought stress while growing, their growth and commerciality both decrease [6]. Wang et al. [7] reported that the relative water content and superoxide dismutase (SOD) activity in leaves of the four tree species in the walnut family showed a decreasing trend, while the soluble sugar content of leaves increased first and then decreased, with the extension of drought stress. Jiang et al. [8] found that with increased and prolonged drought stress, the MDA content of pecan Carya illinoensis leaves increased, as did the membrane lipid peroxidation of leaves and damage to the membrane system. Liao et al. [9] investigated changes in endogenous hormones in the leaves of five different provenances of C. illinoensis under drought stress. Moreover, pecan seedlings grew best under mild drought stress, and their chloroplast structure was severely damaged under moderate drought stress [6]. Therefore, research is required to determine appropriate ways to further enhance the drought adaptability of such species, including walnut, to enable their successful and economically viable cultivation.
Plant growth-promoting rhizobacteria (PGPR) are rhizosphere inhabitants that can promote plant growth and inhibit disease [10,11,12]. Recent research has focused on improving the adaptability of plants in abiotic stress environments by inoculating them with PGPR [13,14]. Arkhipova et al. [15] reported that Lactuca sativa lettuce seedlings inoculated with cytokinin (CTK)-secreting bacteria under moderate drought conditions showed increased levels of CTK, benefiting plant growth. Our previous studies suggested that inoculation with Bacillus cereus L90, which produces high levels of CTK and IAA, improved the photosynthetic characteristics and plant height and ground diameter of walnut seedlings [16]. Gutiérrez-Mañero et al. [17] reported that Bacillus pumilus and Bacillus licheniformis promote plant growth and increase yield by secreting gibberellin (GA). Xi and Ye founded that Bacillus aryabhattai SK1-7, which secretes IAA and protease, increased the plant height, fresh weight, and dry weight of potted tomato seedlings [18]. Other research showed that inoculation of pea (Pisum sativum) with PGPR containing ACC deaminase improved its drought resistance [19], while inoculation of Bacillus tequilensis U36, which secretes IAA, promoted the formation of root hairs and root growth of seedlings, increasing the uptake of water and nutrients, and helping plants to cope with water shortages [20,21]. Thus, various PGPR species have been used to investigate whether they can improve the drought tolerance of plants by PGPR. However, do all PGPR have significant effects in arid environments, and which PGPR have the most optimal effects? We hypothesize that the effects of PGPR would be related to their functional characteristics; however, little information is available on the effects of different PGPRs on the physiological and ecological characteristics of crop plants, including walnut, under drought conditions.
Thus, the current study screened and identified two PGPR strains from rhizosphere soil and investigated their effects on the physiological and ecological characteristics of walnut seedlings under drought stress. It was hypothesized that PGPR inoculation would be beneficial to walnut seedlings by improving their physiological and ecological characteristics under drought stress conditions. These results provide new ideas and a scientific basis for the optimal application of PGPR for improving plant drought resistance and the development and utilization of PGPR resources.

2. Materials and Methods

2.1. Bacterial Screening and Identification

The bacterial strains GE1 and X123 were isolated and purified from the walnut rhizosphere. The GE1 only secreted protease, whereas the X123 not only secreted protease but also produced indoleacetic acid (IAA). The strains were then preserved in the China General Microbiological Culture Collection Center (CGMCC Nos. 11964 and 7668, respectively). The protease activity by the bacterial strain GE1 was 33.6 μg/mL, and the protease activity and IAA production by the bacterial strain X123 were 46.37 μg/mL and 217.56 μg/mL, respectively, using the determination method of ultra-performance liquid chromatography–electrospray ionization tandem mass spectrometry [22]. According to the 16S rRNA sequencing data, the bacterial isolate GE1 indicated 100% similarity with Bacillus subtillus (AJ276351.1), and the X123 showed 99% similarity with Pseudomonas brassicacearum (AF100321). The bacterial strains were indeed identified as B. subtillus and Pseudomonas sp., respectively, based on a comparison of their biochemical characteristics (Table 1) with those described in Bergey’s Manual of Determinative Bacteriology [23]. The 16S RNA gene sequences of these two strains are provided in Supplementary Material File S1.

2.2. Plant Materials and Growth Conditions

The experiment was conducted in the nursery of Shandong Academy of Forestry, Taian (36°23′ N, 117°27′ E), Shandong Province, China. One-year-old seedlings of J. regia L. cv. Jizhaomian, a unique wild walnut resource in China, were used as experimental materials.
On 20 May 2020, plastic pots, each with a height of 36 cm and diameter of 20 cm, were each filled with a 3.75 kg mixture composed of local topsoil, sand, and grass peat in a volume ratio of 5:1:1. Then, walnut container seedlings with a consistent growth were selected and transplanted into the pots, with one plant per pot. The seedlings were grown without additional lighting at a temperature of 20–25 °C and relative humidity of 65–75% during the day and night. All seedlings were irrigated every 2 days prior to the beginning of our experiment.

2.3. Experimental Design

Drought stress treatments were implemented on 2 June 2020. Each pot was well watered before treatment to maintain a consistent initial soil moisture content. Four drought treatments were used in the trial, including a control with normal watering conditions (CK, field moisture capacity = 70–80%) and drought stress without inoculation treatment (DR, 40–50% field capacity), inoculation with GE1 under drought stress (DR + P1, 40–50% field capacity), and inoculation with X123 under drought stress (DR + P2, 40–50% field capacity).
The culture, centrifugation, and dilution of the isolated PGPR strains GE1 and X123, and then inoculation of walnut seedlings with them was conducted according to the method described by Liu et al. [16]. The treatments of DR + P1 and DR + P2 were treated with the corresponding suspensions of bacterial strains GE1 and X123, respectively, and the treatments of CK and DR were irrigated with the same volume of water.
The soil moisture content in the pots was determined at 18:00 h with a digital moisture recorder. The drought stress experiment lasted about for two months, after which the aboveground and underground parts of the seedlings were harvested separately. Twelve fully expanded leaflets (4–5 leaflets from the middle of each compound leaf) from each seedling of three replicates per treatment were collected, frozen with liquid nitrogen, and then stored at −70 °C for the measurement of antioxidant system and other ecophysiological indicators.

2.4. Data Collection and Determinations

Photosynthetic characteristics, including the net photosynthetic rate (Pn), transpiration rate (Tr), stomatal conductance (gs), and intercellular CO2 concentration (Ci) were determined on 13 July 2020 using a portable gas exchange system (LI-6400, LI-COR, USA) on a sunny day from 09:00 to 11:00 h in the morning.
Stomatal length and stomatal width were observed by scanning electron microscopy and measured using the software of image-pro plus 6.0 (Media Cybernetics, Inc., Rockville, MD, USA).
Superoxide dismutase (SOD) activity was assayed by measuring its ability to inhibit the photochemical reduction of nitro blue tetrazolium [24]. Catalase (CAT) activity was measured by monitoring the decomposition of H2O2 [25]. The activities of monodehydroascorbate reductase (MDHAR) and ascorbate peroxidase (APX) were assayed by the method of Nakano and Asada [26]. The contents of AsA and total AsA were calculated according to the standard curve of AsA and expressed as μg∙g−1 FW [27]. Dehydroascorbate (DHA) content was estimated by subtraction of AsA concentration from the total AsA. The contents of reduced glutathione (GSH) and oxidized glutathione (GSSG) were measured according to the method described by Rahman et al. [28]. The contents of GSSG and total GSH were calculated by the standard curve of GSH and expressed as ng∙g−1 FW. GSH was estimated by subtraction of GSSG concentration from total GSH.
The contents of soluble protein and soluble sugar in leaves were determined by the methods of Bradford [29] and Li [30], respectively. To determine the endogenous hormone levels in leaves, the extraction and purification methods of cytokinin zeatin (ZT) and gibberellin (GA) were adopted in this study, modified from Bollmark et al. [31] and He [32].

2.5. Statistical Analyses

The experiments were performed using a completely randomized design. All the measurements were carried out on at least three replicates. The data were compared statistically among four different treatments using analysis of variance (ANOVA) in SPSS software (version 20.0) and LSD (least significant difference) as post hoc tests to differentiate the treatment groups. All data are expressed herein as means ± the standard deviation (SD) of three replicates.

3. Results

3.1. Photosynthetic Characteristics

The photosynthetic characteristics of walnut seedling leaves differed among the different treatments (Table 2). Compared with CK, Pn in the DR group was significantly decreased, whereas it was significantly higher in the DR + P1 and DR + P2 groups. Tr in the DR group was significantly lower than in CK; there was a slight increase in the DR + P1 group compared with the DR group, but this was not significant. Tr in the DR + P2 group significantly increased compared with the DR group. gs in the DR group was significantly lower than in the CK group. There was no significant difference in gs between the DR + P1 and DR groups, whereas gs in the DR + P2 group was significantly higher than in the DR group. Ci was highest in the CK group and lowest in the DR group, although there were no significant differences among the four different treatments. Thus, inoculation with two different PGPR under drought stress improved the photosynthetic capacity of walnut seedling leaves to a certain extent, thereby reducing the damage to walnut seedlings caused by drought stress.

3.2. Stomatal Characteristics

The stomatal length of the DR group was significantly reduced by 16.71% compared with the CK group (Figure 1 and Figure 2). The level in the DR + P2 group was significantly higher than in the DR and DR + P1 groups. Moreover, the stomatal width in the DR group was significantly decreased by 40.51% in comparison with CK. The level in the DR + P2 group was significantly higher than in the DR + P1 group, and although the DR + P2 group was higher than the DR group, the difference was not significant. These results showed that the stomatal length and stomatal width in leaves of walnut seedlings decreased significantly under drought stress, and the decreasing amplitude of stomatal width was greater than that of stomatal length, whereas inoculation with X123 under drought stress could increase the stomatal length and width, but inoculation with GE1 under drought stress had no effect.

3.3. Antioxidant Enzyme Activities

The SOD and CAT activities in walnut seedling leaves increased significantly (by 6.61% and 9.81%, respectively) in the DR group compared with the CK group (Table 3); CAT activity in the DR + P1 group was significantly higher than in the DR group, whereas SOD and CAT activities in the DR + P2 group were significantly increased compared with the DR group, showing 10.96%, 4.08%, and 20.84% increases in SOD activity, respectively, compared to the groups of CK, DR, and DR + P1.
The order of MDHAR activity in leaves was as follows: CK ≈ DR + P2 > DR + P1 > DR. There were no significant differences between the DR + P2 and CK groups, but MDHAR activity was significantly higher than in the DR + P1 and DR groups, with that in the DR + P1 group also being significantly higher than in the DR group. APX activity was highest in the DR + P2 group, being 44.23%, 17.03%, and 12.41% higher compared to the CK, DR, and DR + P1 groups, respectively (Table 2). There was no significant difference between APX activity in the DR + P1 and DR groups, although both were significantly higher than in the CK group (increases of 28.31% and 23.24%, respectively). Thus, SOD, CAT, and APX activities in walnut seedling leaves increased significantly under drought stress and were enhanced to different degrees by inoculation with PGPR, being higher in the DR + P2 group than in the DR + P1 group.

3.4. Non-Enzymatic Antioxidants

In terms of non-enzymatic antioxidants, AsA, GSH, and GSSG were highest in the DR group and significantly higher compared with the CK group; by contrast, their levels in the DR + P1 and DR + P2 groups were significantly lower than in the DR group (Table 4). The DHA content of each group was as follows: DR + P1 ≈ CK > DR + P2 > DR, with the DR group being significantly lower than in the CK group (by 34.32%), whereas levels were significantly higher in the DR + P1 and DR + P2 groups compared with the DR group (by 56.69% and 42.35%, respectively). The level in the DR + P1 group was significantly higher than in the DR + P2 group. Thus, inoculation with PGPR under drought stress reduced the levels of non-enzymatic antioxidant substances (except DHA) in walnut seedling leaves.

3.5. Soluble Protein and Sugar Levels

The amount of osmotic adjustment substances (soluble sugar and soluble protein) in walnut seedling leaves differed substantial among the treatment groups (Figure 3). The soluble sugar content of leaves of the DR group significantly increased (by 6.02%) compared with the CK group. The levels in the DR + P1 and DR + P2 groups were significantly lower than in the DR group, being lowest in the DR + P2 group. The order of soluble protein content of leaves of the different groups was DR > CK > DR + P1 > DR + P2, with significant differences between all groups. The level in the DR group increased significantly by 8.66% compared with the CK group, whereas those in the DR + P1 and DR + P2 groups significantly decreased (by 13.46% and 19.23%, respectively) compared with the DR group. Thus, drought stress promoted the accumulation of soluble sugar and soluble protein in walnut seedling leaves, whereas these levels were significantly reduced by inoculation with either PGPR under drought stress conditions, with the reduction rate of inoculation with X123 being greater than with GE1.

3.6. Endogenous Hormones

The GA content of walnut seedling leaves in different treatment groups increased in the order DR + P2 > CK > DR > DR + P1 (Figure 4). The differences among all treatments were significant, with the GA content decreasing by 10.12% in the DR group compared with the CK group and increasing by 36.02%, 51.33%, and 72.07% in the DR + P2 group compared with the CK, DR, and DR + P1 groups, respectively. Thus, inoculation with X123 under drought stress condition could significantly increase the GA content of walnut seedling leaves. The zeatin (ZT) content in the DR group was significantly reduced by 26.93% compared with the CK group, whereas in the DR + P1 and DR + P2 groups, it was significantly increased compared with the DR group (by 16.45% and 38.77%, respectively), with no significant difference observed between the DR + P2 and CK groups, and the ZT content in the DR + P2 group increased by 19.17% compared to the DR + P1 group (Figure 4). This suggests that the GA and ZT contents of walnut seedling leaves were significantly reduced under drought stress conditions, whereas X123 inoculation significantly increased both GA and ZT contents in leaves, and GE1 inoculation only increased the ZT content and at a lower rate than with X123 inoculation. Thus, there were significant differences in the effects of inoculating with two different PGPRs on the endogenous hormone content of walnut seedling leaves.

4. Discussion

4.1. Photosynthetic Characteristics

Under drought stress, the accumulation of stressed ethylene in plant cells activates the gene encoding chlorophyllase, leading to chlorophyll decomposition, reducing photosynthesis and affecting plant growth [33]. To reduce the transpiration rate, the stomata partially or completely close, which not only reduces water loss but also reduces the entry of CO2, resulting in a decrease in photosynthetic rate [34]. Research has shown that PGPR has a significant effect on promoting plant growth [3,35,36]. Xu et al. [37] concluded that PGPR inoculation under drought stress improved the stomatal conductance and net photosynthetic rate in apple seedling leaves. In the present study, gs and Pn in walnut seedling leaves significantly decreased under drought stress, whereas inoculation with X123 significantly increased these factors, and the increase amplitude was higher than that of inoculation with GE1; this might be because X123 secretes IAA itself, which could increase both root length and root area [38,39], enhancing the ability of walnut seedling roots to absorb soil nutrients and water and helping to alleviate drought stress, which would then result in stomata opening to increase the net photosynthetic rate (Figure 2). Our results indicated that inoculation with X123 improved the photosynthetic capacity of walnut seedlings under drought stress and stimulated the accumulation of photosynthetic products, probably through reduced damage to chloroplasts, resulting in the enhancement of the drought resistance of the seedlings [37]. These results are similar to those reported for Zizyphus jujuba [3].
The stoma is a channel through which water and CO2 in plants are exchanged with the external environment. The size, quantity, and regulating function of stomata are closely related to physiological processes such as photosynthesis and transpiration of leaves [40]. Yu et al. [41] found that stomatal length and width of maize leaves decreased under moderate drought conditions, and the variation amplitude of stomatal width was greater than that of stomatal length. This was basically consistent with the results of this study. Moreover, the stomatal length and stomatal width of walnut seedlings leaves were increased by X123 inoculation under drought stress, and there was a significant effect on stomatal length, this might be related to the increase of ZT content in leaves after inoculation with X123, which could regulate the opening and closing of stomata. Additionally, the stomatal length and stomatal width of X123 inoculation were significantly higher than those of GE1 inoculation. The reason might be related to IAA secreted by X123, which also indicated that stomatal regulation was one of the mechanisms to resist drought stress and adapt to the environment [42].

4.2. Antioxidant System

Drought stress leads to the production of numerous reactive oxygen species (ROS) in plant cells, destroying the balance between the production and scavenging of free radicals and resulting in the accumulation of free radicals; plants are able to increase or activate their antioxidant enzyme systems to prevent ROS damage to cells [43,44,45]. Drought stress significantly increases the activities of SOD, CAT, POD, GR, and APX in leaves, and CAT activity is significantly positively correlated with SOD, POD, and APX activities [46]. In the current study, SOD and CAT activities in walnut seedling leaves significantly increased under drought stress compared with normal watering, which would remove ROS, alleviating oxidative damage of walnut seedlings to a certain extent, and indicating that the walnut seedlings themselves had strong drought resistance. Under drought stress conditions, the SOD and CAT activities in leaves inoculated with X123 were significantly higher than in those of non-inoculated treatments, indicating that inoculating with X123 under drought stress might accelerate the activation of the ROS scavenging system in walnut seedlings and remove ROS more quickly, effectively reducing not only ROS accumulation but also damage to the membrane structure. This was similar to the findings in Lolium perenne by Zhang et al. [36]. Furthermore, the activities of SOD, MDHAR, and APX in leaves inoculated with X123 significantly increased in comparison to GE1 inoculation. The reason might be related to the difference of functional characteristics between X123 and GE1.
Under stress conditions, enzyme activity in the AsA-GSH cycle increases significantly, and the normal metabolism of cells is maintained by clearing numerous ROS [47]. High concentrations of endogenous AsA and GSH maintain a high antioxidant capacity in leaves and reduce the degree of oxidative damage to leaves caused by ROS [48]. In the current study, compared with control plants, the APX activity in walnut seedling leaves without inoculation under drought stress was significantly increased, indicating that these seedlings were able to rapidly remove excess H2O2 and reduce the level of ROS in leaves by consuming AsA under drought stress. However, the APX activity in plants inoculated with X123 under drought stress was significantly higher than in those without inoculation under drought stress, indicating that X123 might promote AsA to remove H2O2 more quickly, resulting in high AsA consumption. This might by why the AsA content of leaves inoculated with X123 was lower than in leaves without inoculation under drought stress. Compared with no inoculation under drought stress, the DHA content of leaves inoculated with X123 was significantly increased, whereas the contents of GSH and GSSG of leaves were significantly decreased, which was not entirely consistent with the results of previous studies [49]. This might be related to differences in the adaptive metabolic regulation of different plants in response to drought stress on the one hand, and differences in the test period and drought stress intensity and duration on the other hand. Thus, the specific mechanisms involved require further study.

4.3. Osmoregulatory Substances and Endogenous Hormones

Soluble sugar and soluble protein are important osmotic adjustment substances in plants under environmental stress conditions and help to maintain osmotic balance and cell membrane stability, remove ROS, and improve plant adaptability [50]. When plants are under drought stress, soluble sugar, soluble protein, and proline accumulate to reduce the osmotic potential and maintain a certain turgor pressure, supporting normal physiological activities and enabling adaptation an arid environment [51,52]. The accumulation of osmoregulatory substances has been demonstrated in many plants under drought stress [35,37,53]. This was further verified by the current study. Compared with normal watering, the soluble sugar and soluble protein contents of leaves without inoculation under drought stress were significantly increased, whereas those in leaves inoculated with X123 were significantly lower than those in all other treatment groups. Thus, the impact of drought stress on walnut seedlings was reduced after inoculation with X123, with a lower concentration of osmotic regulatory substances being able to maintain the activity of protective enzymes, thus improving the drought tolerance of the seedlings. Thus, inoculation with X123 had a significant protective effect on the leaves of walnut seedlings under drought stress. By reducing the soluble sugar and soluble protein contents of leaves, the activities of antioxidant enzymes were maintained, and the drought-resistant ability of walnut seedlings was enhanced. However, according to Wang et al. [54], the soluble sugar and proline contents of maize seedling leaves significantly increased after inoculation with PGPR BBS under drought stress, which was not fully consistent with the results of the current study; this difference might because the drought tolerance of maize itself is weaker than that of walnut seedlings. There might also be differences in the functional characteristics of different PGPR, the intensity and duration of drought stress, and other experimental factors.
Plant endogenous hormones are important regulators of plant growth and development, which are sensitive physiologically active substances for plants to cope with drought stress [55]. In this study, the ZT and GA contents in leaves inoculated with X123 were significantly higher than those inoculated with GE1. This might be because the root tip meristem is the main synthesis site of ZT as well as a synthesis site for GA; in addition, X123 secretes IAA, which promotes the formation, length, and dry weight of plant adventitious roots [56], which then enable transport of root-synthesized ZT and GA to the aboveground plant tissue through the root system. Alternatively, X123 might also stimulate the plants to produce higher concentrations of ZT and GA.

4.4. The Function of Bacteria and Plant Drought Resistance

In the present study, Pn, Tr, gs, stomatal length, and stomatal width in leaves of walnut seedlings inoculated with X123 under drought stress were higher than those inoculated with GE1. The SOD, MDHAR, and APX activities in, as well as ZT and GA contents of, leaves inoculated with X123 were significantly higher than in those inoculated with GE1. However, the soluble sugar and soluble protein contents of leaves significantly decreased following inoculation with X123 compared with inoculation with GE1. Thus, X123 inoculation has significantly more positive effects on the physiological and ecological characteristics of walnut seedlings compared with GE1 inoculation. This might be because of the functional characteristics of the two strains: GE1 can only secrete protease, whereas X123 can secrete both protease and IAA. Given such differences, it is vital that the appropriate PGPR is selected for inoculation.

5. Conclusions

The results of this study indicate that inoculation with Pseudomonas brassicacearum X123 under drought stress improved the photosynthetic characteristics, antioxidant enzyme activities, and hormone contents in leaves of walnut seedlings, and the contents of soluble sugar and soluble protein in leaves significantly decreased. However, the effect of Bacillus subtilis GE1 inoculation on physiological and ecological characteristics of walnut seedlings was significantly weaker than that of X123 inoculation, which might be related to their functional characteristics. These results show that there are significant differences in the application effects of two different PGPR on improving drought tolerance of walnut seedlings. Inoculation with X123 could enhance the drought adaptability of walnut seedlings under drought stress conditions by ameliorating the photosynthetic characteristics, antioxidant enzyme activity, and endogenous hormone content, and reducing the accumulation of osmotic regulatory substances of leaves, which suggests that the selection of PGPR type is the vital factor of the application effect.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/agronomy13061486/s1, Supplementary File S1: The 16S RNA gene sequences of B. subtilis GE1 and Pseudomonas sp. X123 used in our study.

Author Contributions

D.J., F.L. and H.M. conceived and designed the research; D.J., B.L., F.L. and X.L. conducted the experiments; F.L. conducted bacterial screening and identification; D.J., B.L. and F.L. carried out data analysis, original draft preparation, draft submission, and revision; H.M. acquired funding for publication; L.R. contributed materials and analysis tools. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by Forestry Science and Technology Innovation Project of Shandong Province (2019LY009).

Data Availability Statement

Not applicable.

Acknowledgments

We are grateful to the anonymous reviewers for helpful comments on and corrections to the English of the manuscript.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Effects of different treatments on the stomatal length and width in walnut seedling leaves. Bars are means, and error bars are standard deviations (n = 4). CK: normal watering; DR: drought stress without inoculation; DR + P1: inoculation with GE1 under drought stress; DR + P2: inoculation with X123 under drought stress. Different letters indicate significant differences among treatments at p < 0.05 by LSD.
Figure 1. Effects of different treatments on the stomatal length and width in walnut seedling leaves. Bars are means, and error bars are standard deviations (n = 4). CK: normal watering; DR: drought stress without inoculation; DR + P1: inoculation with GE1 under drought stress; DR + P2: inoculation with X123 under drought stress. Different letters indicate significant differences among treatments at p < 0.05 by LSD.
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Figure 2. Leaf stomatal morphology of walnut seedling under different treatments. CK: normal watering; DR: drought stress without inoculation; DR + P1: inoculation with GE1 under drought stress; DR + P2: inoculation with X123 under drought stress.
Figure 2. Leaf stomatal morphology of walnut seedling under different treatments. CK: normal watering; DR: drought stress without inoculation; DR + P1: inoculation with GE1 under drought stress; DR + P2: inoculation with X123 under drought stress.
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Figure 3. Effects of different treatments on the soluble sugar and soluble protein contents in walnut seedling leaves. Bars are means, and error bars are standard deviations (n = 3). CK: normal watering; DR: drought stress without inoculation; DR + P1: inoculation with GE1 under drought stress; DR + P2: inoculation with X123 under drought stress. Different letters indicate significant differences among treatments at p < 0.05 by LSD.
Figure 3. Effects of different treatments on the soluble sugar and soluble protein contents in walnut seedling leaves. Bars are means, and error bars are standard deviations (n = 3). CK: normal watering; DR: drought stress without inoculation; DR + P1: inoculation with GE1 under drought stress; DR + P2: inoculation with X123 under drought stress. Different letters indicate significant differences among treatments at p < 0.05 by LSD.
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Figure 4. Effects of different treatments on the content of endogenous hormones in walnut seedling leaves. Bars are means, and error bars are standard deviations (n = 3). CK: normal watering; DR: drought stress without inoculation; DR + P1: inoculation with GE1 under drought stress; DR + P2: inoculation with X123 under drought stress; GA: gibberellin; ZT: zeatin. Different letters indicate significant differences among treatments at p < 0.05 by LSD.
Figure 4. Effects of different treatments on the content of endogenous hormones in walnut seedling leaves. Bars are means, and error bars are standard deviations (n = 3). CK: normal watering; DR: drought stress without inoculation; DR + P1: inoculation with GE1 under drought stress; DR + P2: inoculation with X123 under drought stress; GA: gibberellin; ZT: zeatin. Different letters indicate significant differences among treatments at p < 0.05 by LSD.
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Table
Table 1. Biochemical characteristics of the bacterial strains of GE1 and X123.
Table 1. Biochemical characteristics of the bacterial strains of GE1 and X123.
GE1Starch hydrolysisIndole testV-P testLecithinase testGas production of glucosePeroxidase testAcid production of glucose
+ a−−−−++
X123Starch hydrolysisIndole testV-P testNitrate reductionFructose testPeroxidase testH2S test
+−++++−
a +: positive; −: negative.
Table
Table 2. Effects of different treatments on the photosynthetic characteristics of walnut seedling leaves.
Table 2. Effects of different treatments on the photosynthetic characteristics of walnut seedling leaves.
TreatmentPn (μmol CO2 m−2 s−1)Tr (mmol H2O m−2 s−1)gs (mol H2O m−2 s−1)Ci (μmol CO2 mol−1)
CK7.96 ± 0.52 a2.58 ± 0.12 a0.34 ± 0.04 a311.11 ± 73.67 a
DR2.17 ± 0.17 b0.74 ± 0.12 c0.18 ± 0.03 b166.00 ± 31.06 a
DR + P16.48 ± 2.37 a1.50 ± 0.90 bc0.24 ± 0.10 ab289.09 ± 148.63 a
DR + P27.45 ± 0.45 a1.94 ± 0.11 ab0.32 ± 0.01 a255.62 ± 36.47 a
Note: Data are mean ± SD. CK: normal watering; DR: drought stress without inoculation; DR + P1: inoculation with GE1 under drought stress; DR + P2: inoculation with X123 under drought stress; Pn: net photosynthetic rate; Tr: transpiration rate; gs: stomatal conductance; Ci: intercellular CO2 concentration. Different letters indicate significant differences among treatments at p < 0.05 by LSD.
Table
Table 3. Effects of different treatments on antioxidant enzyme activities in walnut seedling leaves.
Table 3. Effects of different treatments on antioxidant enzyme activities in walnut seedling leaves.
TreatmentSOD/(U∙g−1 FW)CAT/(U∙g−1 FW)MDHAR/(U∙g−1 FW)APX/(U∙g−1 FW)
CK2840.17 ± 11.30 c905.47 ± 17.00 c11.98 ± 0.15 a964.03 ± 48.46 c
DR3027.87 ± 79.24 b994.30 ± 10.54 b10.55 ± 0.07 c1188.10 ± 14.71 b
DR + P12608.00 ± 82.50 d1179.93 ± 24.92 a11.17 ± 0.23 b1236.90 ± 29.67 b
DR + P23151.40 ± 42.78 a1149.97 ± 9.59 a11.72 ± 0.24 a1390.43 ± 20.27 a
Note: Data are mean ± SD. CK: normal watering; DR: drought stress without inoculation; DR + P1: inoculation with GE1 under drought stress; DR + P2: inoculation with X123 under drought stress; SOD: superoxide dismutase; CAT: catalase; MDHAR: monodehydroascorbate reductase; APX: ascorbate peroxidase. Different letters indicate significant differences among treatments at p < 0.05 by LSD.
Table
Table 4. Effects of different treatments on antioxidant levels in walnut seedling leaves.
Table 4. Effects of different treatments on antioxidant levels in walnut seedling leaves.
TreatmentAsA/(μg∙g−1 FW)DHA/(μg∙g−1 FW)GSH/(ng∙g−1 FW)GSSG/(ng∙g−1 FW)
CK148.30 ± 5.07 c385.80 ± 11.09 a703.50 ± 40.40 c401.52 ± 8.33 b
DR171.04 ± 5.14 a253.39 ± 2.25 c1038.17 ± 18.92 a439.84 ± 9.60 a
DR + P1163.79 ± 1.18 b397.04 ± 13.33 a795.00 ± 22.97 b267.03 ± 11.51 d
DR + P2159.28 ± 2.07 b360.70 ± 4.56 b698.07 ± 18.65 c380.02 ± 9.16 c
Note: Data are mean ± SD. CK: normal watering; DR: drought stress without inoculation; DR + P1: inoculation with GE1 under drought stress; DR + P2: inoculation with X123 under drought stress; AsA: reduced ascorbic acid; DHA: dehydroascorbate; GSH: reduced glutathione; GSSG: oxidized glutathione. Different letters indicate significant differences among treatments at p < 0.05 by LSD.
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Jing, D.; Liu, B.; Ma, H.; Liu, F.; Liu, X.; Ren, L. Effects of Inoculation with Different Plant Growth-Promoting Rhizobacteria on the Eco-Physiological and Stomatal Characteristics of Walnut Seedlings under Drought Stress. Agronomy 2023, 13, 1486. https://doi.org/10.3390/agronomy13061486

AMA Style

Jing D, Liu B, Ma H, Liu F, Liu X, Ren L. Effects of Inoculation with Different Plant Growth-Promoting Rhizobacteria on the Eco-Physiological and Stomatal Characteristics of Walnut Seedlings under Drought Stress. Agronomy. 2023; 13(6):1486. https://doi.org/10.3390/agronomy13061486

Chicago/Turabian Style

Jing, Dawei, Binghua Liu, Hailin Ma, Fangchun Liu, Xinghong Liu, and Liying Ren. 2023. "Effects of Inoculation with Different Plant Growth-Promoting Rhizobacteria on the Eco-Physiological and Stomatal Characteristics of Walnut Seedlings under Drought Stress" Agronomy 13, no. 6: 1486. https://doi.org/10.3390/agronomy13061486

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