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Article

Pseudomonas sp. UW4 Enhances Drought Resistance in Garlic by Modulating Growth and Physiological Parameters

by
Yiwei Yan
1,
Chunqian Guo
1,
Bernard R. Glick
2 and
Jie Tian
1,3,*
1
Qinghai Key Laboratory of Vegetable Genetics and Physiology, Academy of Agriculture and Forestry Sciences of Qinghai University, Xining 810016, China
2
Department of Biology, University of Waterloo, Waterloo, ON N2L 3G1, Canada
3
Laboratory for Research and Utilization of Germplasm Resources in Qinghai Tibet Plateau, Qinghai Academy of Agricultural and Forestry Sciences, Xining 810016, China
*
Author to whom correspondence should be addressed.
Horticulturae 2025, 11(10), 1170; https://doi.org/10.3390/horticulturae11101170
Submission received: 2 August 2025 / Revised: 17 September 2025 / Accepted: 27 September 2025 / Published: 1 October 2025
(This article belongs to the Section Biotic and Abiotic Stress)
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Abstract

Drought stress is one of the primary abiotic factors negatively affecting garlic growth, development, and yield formation. The application of plant growth-promoting bacteria (PGPB) could enhance plant tolerance to drought stress. The aim of this study was to explore the regulatory effect of the PGPB Pseudomonas sp. UW4 on growth and physiological indexes of garlic under drought stress. The results revealed that drought stresses significantly reduced total root length, total root surface area, root projection area and total root volume, chlorophyll content, antioxidant enzyme activity and osmolyte content (proline and soluble proteins), and increased relative electrical conductivity and malondialdehyde (MDA) content, all of which could be significantly improved by inoculating the roots with strain UW4. Under drought stress, an increase in total surface area of roots of 87.06% and an increase in root projected area of 40.71% were observed upon inoculation with strain UW4. The a, b, and total content of chlorophyll were increased significantly by 83.63%, 217.33% and 100.02%, respectively. The osmolyte content in leaves significantly increased, and decreased significantly in roots. The content of antioxidants also significantly increased. Moreover, the relative electrical conductivity in leaves and roots was decreased by 23.18% and 41.20%, respectively, upon strain UW4 inoculation. The content of malondialdehyde (MDA) was decreased by 25.23% and 54.08%, respectively, in the presence of strain UW4. The result of principal component analysis (PCA) revealed that the key factors influencing drought tolerance in garlic inoculated with Pseudomonas sp. UW4 could be summarized into two categories: photosynthetic pigments and root growth-related factors, and leaf osmotic adjustment and root antioxidant enzyme-related factors. Based on the result of the Mantel test, it can be inferred that there was a connection between the osmoregulation and antioxidant enzyme systems in the roots and leaves. Based on the D values, the comprehensive evaluation result of drought resistance was that the drought resistance of the garlic inoculated with strain UW4 under drought stress was lower than that of the garlic inoculated with UW4 under normal treatment and higher than that of the garlic under normal treatment. Therefore, Pseudomonas sp. UW4 enhanced the drought resistance of garlic seedlings by improving root phenotype and antioxidant enzyme activity, and increasing the content of shoot chlorophyll.

1. Introduction

Garlic (Allium sativum L.) is an aromatic herbaceous annual spice that can be used for cooking purposes to flavor foods. It is also one of the oldest known herbs which has been used as a traditional medicine since ancient times [1,2]. The active components of Allium species are reported to prevent and reduce the risk of cardiovascular and diabetes diseases, and protect against infections by activating the immune system, which has been confirmed by human clinical studies [3]. However, the garlic industry is also significantly affected by drought stress, particularly in the northwest plateau regions of China.
Many crops have been subjected to drought stress globally in the last decade. Agricultural drought is always associated with decreased soil water levels and consequent crop failures, which severely affect the production of food throughout the world [4]. Moreover, serious plant growth problems caused by drought are expected for more than 50% of the arable lands by 2050 [5,6,7]. The annual precipitation in Ledu District, Haidong City, Qinghai Province, China is below 370 mm, with high levels of evaporation and uneven precipitation distribution, which has a significant negative impact on the yield and quality of one of the main cultivars, ‘Ledu purple garlic’.
Plant growth-promoting bacteria (PGPB) can promote the growth of plants even under drought conditions [8]. PGPB also have the ability to increase metabolic versatility, fast growth rate, and biocontrol activities, which could make them survive in a variety of soils and interact with plant hosts directly. That is why PGPB have the ability to influence the growth and morphology of plants [9,10]. PGPB not only help to promote plant growth, but they can also protect plants from being damaged by environmental stresses directly and indirectly, including drought stress [11,12,13]. The PGPB-mediated stress tolerance of plants has already been documented in diverse bacterium–plant interactions. Drought stress commonly enhances ethylene biosynthesis, which in turn reduces root and shoot growth. A large number of strains of plant growth-promoting bacteria (PGPB) possess the enzyme 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase (EC4.1.99.4), a pyridoxal 5′-phosphate (PLP)-dependent enzyme, which converts ACCs, the immediate precursor of ethylene in all higher plants, to α-ketobutyrate (α-KB) and ammonia to lower the level of plant ethylene [14]. Therefore, ACC deaminase-producing bacteria can protect plants from the deleterious effects of environmental stresses, thereby exerting beneficial functions [15,16] Pseudomonas chlororaphis O6 can alter the expression of drought signaling response genes to reduce the effects of drought stress on Arabidopsis thaliana L. [17]. Achromobacter piechaudii ARV8 significantly enhanced the resistance of tomato and pepper plants to drought stress through ACC deaminase-mediated ethylene regulation [18].
Pseudomonas sp. UW4, a representative AcdS-producing plant growth-promoting bacteria (PGPB), can demonstrate robust chemotactic responses toward 1-aminocyclopropane-1-carboxylate (ACC) [19,20]. Through its enhanced ACC deaminase activity, Pseudomonas sp. UW4 confers strengthened chemotaxis and rhizosphere colonization, enhances stress tolerance, and promotes growth and ornamental quality in crops, demonstrating great potential for sustainable horticulture [21,22,23]. The study showed that the efficacy of Pseudomonas sp. UW4’s ACC deaminase (ACDS) also experimentally demonstrated that it can increase the biomass and lipid content of Chlamydomonas under nitrogen-deficient conditions [24]. It has also been confirmed that Pseudomonas sp. UW4 can promote the growth and development of mushrooms by degrading ACC to reduce ethylene levels, alleviating the inhibition of mycelial growth and primordium formation [25]. Moreover, inoculating cadmium-stressed lettuce with Pseudomonas sp. UW4 reduced ethylene synthesis via ACC deaminase, promoting growth in a colonization-dependent manner [26]. Besides, Chandra, D. et al. demonstrated that Pseudomonas sp. UW4 enhanced stress tolerance and improved biochemical and antioxidant properties in wheat through its ACC deaminase activity, thereby increasing yield [27]. However, the effect of Pseudomonas sp. UW4 on garlic plants under drought stress has not been investigated to date. Therefore, this study aimed to reveal the alterations in the physiological and biochemical mechanisms of ‘Ledu purple garlic’ inoculated with Pseudomonas sp. UW4 under drought stress.

2. Material and Methods

2.1. Plant and Biological Materials

The garlic variety used in this study was ‘Ledu purple garlic’, which is the main cultivar in the Qinghai Province of China.
The Pseudomonas sp. UW4 was previously isolated from the rhizosphere of common reeds on the campus of the University of Waterloo, Canada. Pseudomonas sp. UW4 was inoculated in LB (Luria–Bertani) liquid medium for 24 h (30 °C, 160 rpm) and then adjusted to A600 = 0.6–0.8 (the number of colonies in the solution at this concentration is ~107 cfu/mL), and then the cells were collected by centrifugation (4000 rpm, 6 min). The collected bacteria were washed three times with sterile double-distilled H2O and then resuspended with the same amount of sterile water and stored at 4 °C for later use.

2.2. Plant Growth Conditions and Treatments

The cloves of ‘Ledu purple garlic’ were planted in pots containing the same amount of substrate (250 g) and placed in an intelligent artificial climate chamber (Model: HYR-6wE-3) with cultivating conditions of 14 h light/ 10 h dark with a temperature of 25 °C in the light and 15 °C in the dark. The relative humidity of the air in the incubator was 70% [28].
The experiment comprised four treatments, when the garlic seedings reached 10–12 cm. (1) CK group: normal irrigation with 300 mL distilled water every 5 d; (2) PGPB group: normal irrigation and inoculated with strain UW4 (1.0 × 106 colony forming units (CFU) per mL) on the first day; (3) DT group: normal irrigation with 300 mL distilled water on the first day and then drought stress with 30% relative humidity in the light incubator until the soil was moderately arid with 45% relative moisture; and (4) DT + PGPB group: carried out the drought stress with 30% relative humidity of the air in the light incubator after inoculating with UW4 (1.0 × 106 colony forming units (CFU) per mL) on the first day. The soil was moderately arid with 45% relative moisture at the end of the treatment. The test material was taken after 15 days from the start of the treatments.
Each of the experiments described below included three independent biological replicates.

2.3. Determination of Plant Growth Parameters

Ten garlic plants with consistent growth within each treatment were randomly selected for measurements. Plant height, leaf length, and total root length were recorded with a ruler. Root diameter was measured with a vernier scale. Root surface area was measured with a root scanner (Zhejiang Top Cloud-Agri Technology Co., Ltd., Hangzhou, China, Rhizome Analysis System GXY-A). Root tip number and dry weight content of the aboveground part and roots were recorded.

2.4. Determination of the Chlorophyll a, b, and Total Contents

Chlorophyll content was determined according to Li [29]. Some 0.5 g of the test material was weighed into a test tube, the leaves were cut into 2 mm pieces, and extracted with 95% ethanol. After waiting for the leaves to turn white, the absorbance of the liquid was measured after about 14 h. Using 95% ethanol as a reference at room temperature, the absorbance was measured at 645 nm and 663 nm, respectively, and the content of chlorophyll a and chlorophyll b was calculated by Arnon’s formula [30].
Chlorophyll a (chla) content (mg/g) = (12.7 × A663 − 2.69 × A645) ×V/(1000 W); chlorophyll b (chb) content (mg/g) = (22.7 × A645 − 4.68 × A663) × V/(1000 W); total chlorophyll content (mg/g) = chla content + chlb content.
In the formula, A663 and A645 are the absorbance values at the corresponding wavelengths, V is the volume of the extract, and W is the mass of the leaves taken.

2.5. Determination of the Osmolyte Contents

The proline content of garlic leaves and roots was determined as follows [29]. A mixture of 0.3 g fresh samples and 5 mL sulfosalicylic acid was homogenized and then centrifuged at 3000 rpm for 20 min. The supernatant was mixed with 2 mL glacial acetic acid and 2 mL acidic ninhydrin, and the resulting mixture was boiled at 100 °C for 25 min in a water bath. After cooling to room temperature, 4 mL of toluene was added to the liquid. The absorbance of the extracts was evaluated at 520 nm. Anthrone colorimetry was used to measure the soluble sugar content [29] Coomassie Brilliant Blue staining was used to determine the soluble protein content, with bovine serum albumin (BSA) as a protein standard.

2.6. Determination of Malondialdehyde (MDA) and Relative Electric Conductivity

A mixture of 0.5 g fresh plant material and 5 mL of 5% TCA was centrifuged at 12,000 g for 25 min. The supernatant was mixed with 2 mL 0.67% thiobarbituric acid solution and heated for 30 min at 100 °C in a water bath. The samples’ absorbances were evaluated at 450, 532, and 600 nm using a blank containing all reagents. The MDA content of the sample was calculated using the formula:
C (μmol/g) = 6.45 (A532 − A600) − 0.56 A450.
The letter C represents the concentration of the MDA, and the letter A represents the absorbance of the MDA. The malondialdehyde (MDA) content was measured according to the method of Heath et al. [31].
The method of measuring the relative electric conductivity of the garlic seedling was as follows [31]: 1.0 g of plant leaves was placed into test tubes (18 mm × 180 mm), 10 mL of distilled water was added, and the tubes were sealed with plastic caps and shaken by hand for 30 s to let the plant be immersed in distilled water. The tubes were uncapped and placed under vacuum for 10 min, and then kept for 1 h at room temperature before measuring the electrical conductance value (S1) with a conductivity meter. The tubes were then placed in boiling water for 20 min, then cooled to room temperature and the electrical conductance value was measured (S2). The electrical conductance value of distilled water was also measured (S3). The relative electric conductivity of the leaves was calculated using the formula:
Relative electric conductivity (%) = (S1 − S3)/(S2 − S3) × 100%.

2.7. Determination of Antioxidant Enzyme Activity

The method of measuring superoxide dismutase (SOD) activity was as follows [31]. The assay mixture comprised 3 mL reaction mixture containing 50 μL of enzyme extract with 50 mM phosphate buffer (pH 7.8), 200 mM methionine, 75 μM nitroblue tetrazolium (NBT), 3 mM EDTA, and 60 μM riboflavin. Tubes were mixed well and kept for 10 min at 25 °C in an intelligent growth chamber (HYR-6ωE-3). The absorbance was measured at 560 nm with the bovine erythrocyte SOD as an SOD standard.
The method of measuring peroxidase (POD) activity was as follows [32,33]. The mixture contained 28 μL guaiacol, 2 mM H2O2, 0.1 M phosphate buffer (pH 6.0), and 0.1 mL enzyme. The absorbance was recorded at 470 nm with the horseradish peroxidase as a POD standard.
The method of measuring catalase (CAT) activity was as follows [34]. The mixture contained 0.1 mM phosphate buffer (pH 7.0), 10 mM H2O2, and 0.2 mL enzyme extracts. The decrease in absorbance at 240 nm was used to calculate CAT activity. Bovine liver catalase was used as the CAT standard.
Ascorbate peroxidase (APX) activity was assayed according to Nakano [35]. Accordingly, 2 mL reaction mixture (50 mM phosphate buffer, 5 mM ascorbic acid, 25 mM H2O2, 0.1 mM EDTA, and 100 μL of the plant extract) was prepared, and the solution pH was adjusted to 7.0. The oxidation of ascorbate for 40 s was measured at 290 nm.
The method of measuring ascorbate peroxidase (GR) activity was as follows [36]. Reaction buffer was prepared accordingly: 0.2 M phosphate buffer (pH 7.5), 10 mM EDTA (pH 7.5), 10 mM NADPH, and 12 mM 5.5-dithiobis(2-nitrobenzoic acid) (DTNB, Ellman’s reagent); pH 7.5. An aliquot of 0.5 g fresh sample was ground in 2 mL extraction buffer and centrifuged at 10,000× g for 10 min. One mL of the supernatant was mixed with 1 mL of the reaction buffer, and 0.2 U of glutathione reductase was added. Finally, the absorbance was measured at 340 nm. Yeast glutathione reductase was used as the GR standard.
All experiments were performed with three independent biological replicates to ensure statistical robustness.

2.8. Statistical Analysis

Subordinate function values were calculated using the formula:
u(Xj) = (Xj − Xmin)/(Xmax − Xmin) (j = 1, 2, …, n).
Xj indicates the score of the jth comprehensive indicator, Xmin indicates the minimum score of the jth comprehensive indicator, and Xmax indicates the maximum score of the jth comprehensive indicator.
The comprehensive evaluation values (D) of the garlic under different treatments were calculated using the formula:
D = ∑(Uj × Wj) (j = 1, 2, …, n).
D indicates the comprehensive evaluation value for drought tolerance in the garlic exposed to different treatments.
SPSS 26 (https://www.ibm.com/products/spss) (accessed on 3 December 2022) and Origin 2018 (http://www.originlab.com) (accessed on 7 December 2022) were used for statistical analysis of the data.

3. Results

3.1. Effects of UW4 Inoculation on Garlic Plant Growth Parameters Under Drought Stress

Compared to normal irrigation conditions, drought stress significantly reduced the growth of garlic shoots and roots, except for the mean root diameter and total root volume. However, under both water conditions, inoculation with strain UW4 significantly improved the plant height, leaf width, shoot dry weight, total root length, total root surface area, total root volume, number of root tips, root dry weight, and root projection area. Compared to the garlic under drought stress, the increases of total root length, total root surface area, root projection area, and total root volume were 68.16%, 87.06%, 87.65%, and 34.06%, respectively, in plants inoculated with strain UW4 under drought stress. The result showed that strain UW4 could effectively alleviate the inhibitory effect of drought stress on the growth of garlic seedlings, especially on total root length, total root surface area, root projection area, and total root volume (Table 1).

3.2. Effect of Strain UW4 Inoculation on Chlorophyll Content of Garlic Under Drought Stress

Compared with the garlic under normal conditions, the chlorophyll a, b, and total chlorophyll content in garlic under drought stress decreased by 34.29%, 37.50%, and 36.40%, respectively. However, the chlorophyll a, b, and total chlorophyll content in garlic inoculated with strain UW4 under drought stress increased by 83.63%, 217.33%, and 100.02%, respectively, compared with the content in garlic under drought stress without being inoculated with strain UW4. These results indicate that inoculation with strain UW4 effectively alleviates the decrease in chlorophyll content in garlic plants under drought stress (Figure 1).

3.3. Effects of Strain UW4 Inoculation on Osmolyte Content in Garlic Plants Under Drought Stress

Drought stress significantly increased the osmolyte content (proline, soluble total sugars, and soluble proteins) of garlic plants (Figure 2). The contents of soluble proteins in garlic under drought stress were significantly higher than those under normal conditions, which had an increase of 1.57% and 87.33% in leaves and roots, respectively. Proline content increased by 15.33% and 76.41% in leaf and root, respectively. Furthermore, soluble sugar content increased by 17.57% in leaves and 912.33% in roots. The osmolyte contents were significantly increased in leaves but not in roots when the garlic was inoculated with strain UW4 under drought stress. The increase in the contents of leaf proline and soluble total sugars was 44.48% and 59.48%, respectively. On the other hand, the contents of proline and soluble total sugars decreased by 32.36% and 72.79%, respectively, in roots when the garlic was inoculated with strain UW4 under drought stress. The results indicated that UW4 inoculation had different effects on the regulation of the osmolyte content in leaves and roots of garlic under drought stress. Strain UW4 promoted the accumulation of soluble proteins, soluble total sugars, and proline in leaves, but inhibited these values in garlic roots under drought stress.

3.4. Effects of Strain UW4 Inoculation on the Relative Electrical Conductivity and MDA Content in Garlic Plants Under Drought Stress

Under drought stress conditions, the relative electrical conductivity was significantly higher than in the garlic under normal conditions, which was 40.03% and 40.88% in leaves and roots, respectively. However, the relative electrical conductivity was changed significantly in garlic inoculated with strain UW4 under drought stress, which exhibited a decrease of 23.18% and 41.20% in leaves and roots, respectively. Thus, strain UW4 played a significant role in decreasing the relatively high electrical conductivity, which, in turn, reduced membrane permeability.
Moreover, the change of malondialdehyde (MDA) content was similar to that of relative electrical conductivity. MDA content was significantly higher in both leaves and roots in garlic under drought stress compared with the garlic under normal conditions, which had an increase of 49.26% and 117.99%, respectively (Figure 3). In contrast, in the garlic plants inoculated with strain UW4, it decreased by 25.19% and 41.58% in leaves and roots, demonstrating the significant effect of strain UW4 in alleviating membrane damage caused by drought stress.

3.5. Effects of Strain UW4 Inoculation on Garlic Antioxidant Enzyme Activity Under Drought Stress

Under drought stress conditions, the activity of SOD, POD, CAT, APX, and GR significantly increased in leaves compared to normal irrigation (Figure 4). There was an increase of 4.38%, 390.17%, 114.68%, 81.05%, and 100% in SOD, POD, CAT, APX, and GR activity, respectively. In addition, these activities changed in garlic inoculated with strain UW4 under drought stress compared to the garlic in drought stress without the added bacterium. The SOD activity increased by 3.65%, the POD activity increased by 11.11%, the CAT activity increased by 117.95%, the APX activity increased by 14.53%, and the GR activity increased by 81.11%.
However, the changes in these activities in the roots were not similar to those observed in leaves. Compared to normal irrigation, the activity of POD and APX increased by 41.95% and 37.08%, respectively, while the activity of CAT and GR decreased by 56.74% and 86.20%, respectively. Moreover, the activity changed in another way when the garlic was inoculated with strain UW4 under drought stress. Specifically, the activity of CAT, APX, and GR significantly increased by 161.89%, 53.21%, and 451.22%, respectively, compared to garlic under drought stress in the absence of the added bacterium. In addition, the activity of SOD and POD decreased by 63.51% and 30.36%, respectively. These results indicate that strain UW4 inoculation could effectively enhance the antioxidant enzyme activity in leaves and some antioxidant enzyme activity in roots to mitigate oxidative damage caused by drought stress and strengthen garlic’s drought resistance capability.

3.6. Principal Component Analysis (PCA)

Principal components analysis (PCA) was performed on 33 indices of garlic roots and leaves under the four treatments employed (Figure 5). The variance contribution rates of the first principal component (PC1), the second principal component (PC2), and the third principal component (PC3) of garlic plants were 54.98, 32.226, and 12.794, respectively. The eigenvalues of PC1, PC2, and PC3 were all greater than 1, and the cumulative contribution rate was 100%. This indicated that the three principal components could completely reflect the different information of drought tolerance of garlic inoculated with strain UW4. The PC1 coefficient of garlic was positive, and the indices with high load value were chlorophyll a content, chlorophyll b content, total chlorophyll content, root length, root surface area, root projection area, and root volume, indicating that the indices of PC1 could be summarized as photosynthetic pigments and root growth-related factors. The indices with positive PC2 coefficients and high load value included leaf soluble total sugar content and root APX enzyme activity, indicating that PC2 indices could be summarized as leaf osmotic adjustment and root antioxidant enzyme-related factors.
1. Plant height; 2. Leaf width; 3. Dry weight of ground part; 4. Chlorophyll a content; 5. Chlorophyll b content; 6. Total chlorophyll content; 7. SOD enzyme activity in leaves; 8. POD enzyme activity in leaves; 9. CAT enzyme activity in leaves; 10. APX enzyme activity in leaves; 11. GR enzyme activity in leaves; 12. Soluble protein content in leaves; 13. Total soluble sugar content in leaves; 14. MDA content in leaves; 15. Root length; 16, Root volume; 17. Root surface area; 18. Root projection area; 19. SOD enzyme activity in roots; 20. POD enzyme activity in roots; 21. GR enzyme activity in roots; 22. APX enzyme activity in roots; 23. CAT enzyme activity in roots; 24. The number of roots; 25. Root dry weight; 26. Soluble protein content in roots; 27. Soluble sugar content in roots; 28. MDA content in roots; 29. Average diameter in roots; 30. Blade relative conductivity; 31. Relative electrical conductivity of root system; 32. Proline content in leaves; 33. Proline content in roots.
The results of the Mantel test analysis on the indicators selected by the first principal component (PC1) and the second principal component (PC2) compared to the remaining indicators show that PC1 was significantly positively correlated with root soluble protein content, root soluble sugar content, root GR enzyme activity, root MDA content, and root relative electrical conductivity. This indicated that PC1 (representing leaf photosynthetic pigments and root morphological traits) is closely related to the root osmotic regulation system and the root antioxidant enzyme system. Meanwhile, PC2 was significantly positively correlated with leaf SOD, CAT, APX, and GR enzyme activities, suggesting that PC2 (representing the leaf osmotic regulation system and root antioxidant enzyme system) was strongly associated with the leaf antioxidant enzyme system. Based on the results, it can be hypothesized that the osmotic regulation systems and antioxidant enzyme systems in roots and leaves could mutually regulate each other (Figure 6).

3.7. Comprehensive Evaluation of Drought Resistance of Garlic Under Different Treatments

The comprehensive score of principal components was used to evaluate the effect of strain UW4 on drought tolerance factors of garlic under drought stress (Figure 5). The weight of each comprehensive index was calculated according to the eigenvalues of each principal component (F1, F2, F3), and the weights were 0.550, 0.322, and 0.128, respectively. According to the principal component and its contribution rate, a comprehensive evaluation model was constructed as follows: Y = 0.550Y1 + 0.322Y2 + 0.128Y3. Using this function, the comprehensive scores of four treatments were obtained, and each treatment was sorted according to the score level (Table 2). The results showed that the order of drought resistance ability of garlic under four treatments was the PGPB group, DT + PGPB group, CK group, and DT group, which indicated that the drought resistance of garlic inoculated with strain UW4 was greater than that of plants without strain UW4, and the principal component comprehensive score of garlic plants under drought stress treated by strain UW4 was 0.56 times and 1.98 times higher than that of the CK group and the DT group, respectively.

3.8. Analysis of Heat Map Clustering

In order to further clarify the changes of garlic leaf and root indices under drought stress, the measured indices were analyzed by a cluster heat map (Figure 7). The CK group, the PGPB group, and the DT + PGPB group were clustered into one group, while the DT group was clustered into another group, which indicated that the growth and physiological indicators in garlic were significantly regulated when the garlic plants were inoculated with strain UW4. Compared with garlic under drought stress, the proline content, the antioxidant enzyme POD activity, and the soluble protein content in roots and the relative electrical conductivity and MDA content in both leaves and roots were significantly decreased in the garlic plants inoculated with strain UW4 under drought stress. At the same time, the antioxidant enzyme APX activity in roots, the total root length, the root projection area, and the root surface area were significantly increased. The content of chlorophyll a and b, the total chlorophyll content, the antioxidant enzyme activity of CAT and GR, and the content of soluble sugar in garlic plant leaves were also significantly increased. The changes in the above physiological indicators can be visually observed in the pattern diagram (Figure 8).

4. Discussion

Over the past two decades, climate change has become one of the most important concerns affecting the environment. The impacts of abiotic stress factors such as drought, flooding, and salinity on plant growth have increased as a direct consequence of global warming [37]. Climate change plays a significant role in influencing the Earth’s water cycles and in reducing the water availability, which has a negative effect on plant agriculture [38]. Drought stress has become the main impediment to plant crop growth and productivity [39]. In addition, drought stress also restricts plant membrane integrity, pigment content, and cellular element biosynthesis [40,41]. Lack of sufficient water negatively affects agricultural production and causes food shortages; it is therefore necessary to develop a means to reduce this problem [39].
The methods of helping plants respond and adapt to drought stress have been studied for several years. However, most of these technologies are costly and time-consuming [42]. In recent years, PGPB inoculations have been a sustainable alternative strategy for enhancing the ability of plants to adapt to drought stress [43]. In the past two decades, the use of inoculating plants with PGPB to facilitate plant growth has increased dramatically. And the bacteria that have been utilized to facilitate plant growth worked well indeed [44]. Although the exact mechanisms of PGPB in regulating drought stress tolerance are largely unknown, there are still several ways that could be explained. Many studies have indicated that PGPB could promote plant growth directly by improving the acquisition of certain nutrients. In addition, antioxidative enzyme activities, including catalase (CAT), peroxidase (POD), and superoxide dismutase (SOD), were confirmed to increase significantly in plants inoculated with the PGPB UW4 [45,46]. Moreover, a part of newly synthesized ACC could be taken up by the PGPB, which directly leads to a decrease in the amount of ethylene in the plant. And the ACC can be converted by the enzyme ACC deaminase to ammonia and α-ketobutyrate, which could prevent plant ethylene levels from being growth inhibitory [47]. PGPB could also significantly improve plant tolerance to drought stress by regulating ROS (reactive oxygen species) levels [48]. These substances play significant roles in producing antioxidant enzymes to prevent the accumulation of reactive oxygen species (ROS) [49].
PGPB can boost root length and density to promote crop yield. They could also maintain the osmotic balance by maintaining the relative water content [42]. The exudates produced by roots increase microbial populations around the roots. The root exudates could also act as signaling molecules for the microbes associated with the roots, which could attract beneficial microbes to the roots [50]. PGPB colonize plant roots, which can directly or indirectly promote the growth of plants and enhance the ability to respond to drought stress. Plants adapt their root development to cope with drought stress through increasing the root: shoot biomass ratio to help absorb more water, which means the root system architecture is an adaptive feature of drought conditions [39]. Naveed et al. showed that pepper plants inoculated with PGPB had an increase of 40% in root system size to enhance water absorption and resistance to drought stress [51]. In the present study, the addition of strain UW4 led to a significant increase in total root length, total root surface area, root projection area, and total root volume in garlic under drought stress, all consistent with strain UW4 reducing the impact of drought stress and promoting the growth of roots. It has the same results as the pepper inoculated with PGPB [51].
Photosynthesis is the basis for dry matter accumulation and yield formation of crops. Drought stress negatively affects plant metabolism and physiological processes, resulting in a decrease in chlorophyll content in plants. PGPB can ameliorate the damage to plants by promoting the accumulation of photosynthetic pigments. In Yang’s report, there is an increase in chlorophyll content in wheat inoculated with Streptomyces pactum Act12 [52]. And in Mun’s report, inoculating plants with PGPB led to an increase in chlorophyll content [53]. In this study, the contents of chlorophyll a, chlorophyll b, and total chlorophyll in garlic were significantly decreased under drought stress; however, the contents of these compounds were significantly increased in plants treated with the PGPB, indicating that PGPB treatment could maintain the photosynthetic pigment content to ensure the relatively normal photosynthesis of garlic plants.
A plant’s ability to respond to drought stress is also regulated by the accumulation of various organic and inorganic secondary metabolites, a process that is known as osmotic adjustment in the cytosol, which is one of the most significant strategies of plant drought tolerance [54]. Therefore, these osmo-protectants act as plant stress protectants in responding to drought stress, which include sucrose, amino acids, proline, organic acids, and some other compounds [55]. However, several studies indicate that PGPB plays an important role in synthesizing osmolytes, which can act together with plant-produced osmolytes to regulate plant growth synergistically. One of the most investigated solutes is proline, for its important relevance to stress tolerance [49]; the content of proline typically increases in plants under water deficit [56]. The proline content generally significantly increases when plants are inoculated with PGPB. For example, compared to non-treated plants, an accumulation of proline and soluble sugars was observed in maize plants inoculated with Pseudomonas putida GAP-P45 under drought stress; this increased the relative water content, leaf water potential, and plant biomass, all of which improved the physiological and biochemical properties [57]. Similarly, an increase of proline content was found in tomato plants inoculated with phosphate solubilizing bacteria (PSB) (i.e., Bacillus polymyxa) under drought conditions compared with tomato plants under normal conditions [58]. In this study, the osmotic adjustment substances in garlic seedlings, including soluble sugar, soluble protein, and proline, showed different trends under drought stress. The soluble protein in leaves decreased significantly under drought stress, while the soluble sugar and proline in leaves and the soluble sugar, soluble protein, and proline in roots increased significantly under drought stress. The osmotic adjustment substances in the leaves of garlic seedlings treated with strain UW4 under drought stress increased significantly, while the osmotic adjustment substances in the roots decreased significantly. The results showed that strain UW4 could induce the synthesis of sugar, protein, and free proline in garlic leaves, resist adverse environmental factors, and protect tissues from drought stress. Roots could secrete extracellular polysaccharides and amino acids and other substances after treatment with strain UW4. By affecting the plant root environment, regulating the osmotic pressure inside and outside the root system, the tissue was protected from drought stress.
Reactive oxygen species (ROS) are produced by plants under drought stress, which could cause oxidative damage, impairing the normal functions of plant cells, proteins, lipids, and deoxyribonucleic acid [42,59]. In order to limit these effects, plants have developed antioxidant defense systems, including superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), and glutathione reductase (GR) [60,61]. In maize plants inoculated with Bacillus sp. under drought stress, there was a decrease in the activity of the antioxidant enzymes APX and glutathione peroxidase (GPX) [62]. And in basil plants (Ocimum basilicum L.) inoculated with Pseudomonas sp. under drought stress, there was a significant increase in CAT enzyme activity [63]. Moreover, following inoculation with PGPB, there was an increase in the enzyme activity of SOD, CAT, POD, and APX in rice under drought stress compared to normal conditions. Under the same drought conditions in rice, the content of H2O2 and malondialdehyde (MDA) was decreased [64]. There are several different ways to regulate plant growth using different antioxidant defenses. In the present study, the enzyme activity of SOD, POD, CAT, APX, and GR in garlic plant leaves and the enzyme activity of POD and APX in garlic plant roots were significantly higher than those in garlic plants under drought stress. After inoculating the garlic plants with strain UW4, the level of SOD enzyme activity in leaves was increased. The activities of antioxidant enzymes in leaves and roots increased after inoculating plants with UW4 in order to eliminate reactive oxygen species more effectively and avoid oxidative damage to garlic seedlings. Following treatment with strain UW4, the content of MDA and the relative electrical conductivity were both decreased, further demonstrating the effect of inoculating plants with strain UW4 and thereby reducing the damage caused by oxidation.
Drought tolerance of plants is a consequence of many factors, and simplified indicators cannot accurately reflect its regulation ability. Therefore, it is necessary to comprehensively evaluate the drought tolerance of plants by inoculating them with strain UW4. Three principal components with a total contribution rate of 100% were extracted from garlic by principal component analysis (PCA). The visual analysis of the three principal components showed that the strain UW4 inoculation could improve the drought tolerance of garlic plants by enhancing common defense mechanisms, namely, by improving the root phenotype (root surface area and root projection area), increasing photosynthetic pigments and leaf antioxidant enzyme activities. In addition, the principal component comprehensive score of the drought tolerance of garlic by strain UW4 further indicated that it had a good regulatory effect on drought stress. The above experimental results showed that the strain UW4 inoculation endowed garlic with the physiological mechanisms to adapt to drought.

5. Conclusions

UW4 inoculation could effectively promote the accumulation of photosynthetic pigments in garlic leaf and root growth under drought stress, up-regulate the activity of antioxidant enzymes, enhance the scavenging ability of reactive oxygen species, promote the biosynthesis of soluble sugar and soluble protein, and maintain root osmotic pressure, thereby alleviating the damage of drought stress on garlic plants. Principal component analysis showed that UW4 had an obvious regulation effect on root surface area, projection area, photosynthetic pigment content, and CAT and GR enzyme activities in leaves of garlic under drought stress. This study provided a theoretical basis for the drought tolerance mechanism of garlic and had important guiding significance for drought resistance cultivation of garlic.
Based on previous studies, the molecular regulatory mechanism of UW4 has been basically clarified. Therefore, future research should aim to clarify the signal transduction pathways and gene expression changes induced by UW4 inoculation in garlic.

Author Contributions

Y.Y. and C.G. designed and conducted the experiments, calculated the data, and prepared the figures; Y.Y. wrote the manuscript; B.R.G. and J.T. revised the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Key Laboratory Project of Qinghai Science & Technology Department (Grant No. 2023-1_4).

Data Availability Statement

The raw data supporting the conclusions of this article will be made available by the authors on request.

Acknowledgments

The authors gratefully acknowledge the assistance of the staff of the School of Agriculture and Forestry Sciences of Qinghai University and the University of Waterloo for their support.

Conflicts of Interest

The authors declare that they have no known competing financial interests or personal relationships that could have influenced the work reported in this paper.

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Figure 1. Effects of UW4 on the contents of chlorophyll a (A), chlorophyll b (B), and total chlorophyll (C) in garlic seedlings under drought stress. Note—CK: Control group of garlic plants under normal irrigation; PGPB: Treatment group of garlic plants inoculated solely with Pseudomonas sp. UW4; DT: Experimental group of garlic plants subjected to drought stress treatment; DT + PGPB: Experimental group of garlic plants exposed to drought stress and simultaneously inoculated with Pseudomonas sp. UW4. Means with different lowercase letters are significantly different according to Tukey’s HSD test (p ≤ 0.05). Values sharing a common letter are not significantly different. (Same below).
Figure 1. Effects of UW4 on the contents of chlorophyll a (A), chlorophyll b (B), and total chlorophyll (C) in garlic seedlings under drought stress. Note—CK: Control group of garlic plants under normal irrigation; PGPB: Treatment group of garlic plants inoculated solely with Pseudomonas sp. UW4; DT: Experimental group of garlic plants subjected to drought stress treatment; DT + PGPB: Experimental group of garlic plants exposed to drought stress and simultaneously inoculated with Pseudomonas sp. UW4. Means with different lowercase letters are significantly different according to Tukey’s HSD test (p ≤ 0.05). Values sharing a common letter are not significantly different. (Same below).
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Figure 2. Effects of PGPB on contents of soluble protein (A,D), proline (B,E), and soluble sugar (C,F) in garlic seedlings under drought stress.
Figure 2. Effects of PGPB on contents of soluble protein (A,D), proline (B,E), and soluble sugar (C,F) in garlic seedlings under drought stress.
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Figure 3. Effects of strain UW4 on relative electrical conductivity (A,C) and MDA content (B,D) of garlic seedlings under drought stress.
Figure 3. Effects of strain UW4 on relative electrical conductivity (A,C) and MDA content (B,D) of garlic seedlings under drought stress.
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Figure 4. Effects of strain UW4 on activities of SOD (superoxide dismutase) (A,F), POD (peroxidase) (B,G), CAT (catalase) (C,H), APX (ascorbate peroxidase) (D,I), and GR (glutathione reductase) (E,J) in leaf and root of garlic seedlings under drought stress.
Figure 4. Effects of strain UW4 on activities of SOD (superoxide dismutase) (A,F), POD (peroxidase) (B,G), CAT (catalase) (C,H), APX (ascorbate peroxidase) (D,I), and GR (glutathione reductase) (E,J) in leaf and root of garlic seedlings under drought stress.
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Figure 5. Effects of PGPB treatment on the characteristic vectors of growth and physiological indices in roots and leaves of garlic under drought stress. Note: The red circle represents principal component 1 main factor, the basket circle represents principal component 2 main factor, and the green circle represents principal component 3 main factor.
Figure 5. Effects of PGPB treatment on the characteristic vectors of growth and physiological indices in roots and leaves of garlic under drought stress. Note: The red circle represents principal component 1 main factor, the basket circle represents principal component 2 main factor, and the green circle represents principal component 3 main factor.
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Figure 6. Mantel tests between the indicators selected by the first principal component (PC1), the second principal component (PC2), and the remaining variables. Note: PC1 comprised chlorophyll a content, chlorophyll b content, total chlorophyll content, root length, root surface area, root projection area, and root volume. PC2 comprised leaf soluble total sugar content and root APX enzyme activity. X1: Root Soluble Protein Content; X2: Root soluble sugar content; X3: Leaf soluble protein content; X4: Leaf SOD enzyme activity; X5: Leaf POD enzyme activity; X6: Leaf CAT enzyme activity; X7: Leaf APX enzyme activity; X8: Leaf GR enzyme activity; X9: Root SOD enzyme activity; X10: Root POD enzyme activity; X11: Root CAT enzyme activity; X12: Root GR enzyme activity; X13: Root diameter; X14: Number of root tip; X15: Plant height; X16: leaf width; X17: Aboveground dry weight; X18: Root dry weight; X19: MDA content in roots; X20: MDA content in leaves; X21: Leaf Relative Conductivity; X22: Root relative conductivity; X23: Leaf proline content; X24: Root proline content. Statistical significance is indicated as follows: * p < 0.05, ** p < 0.01, *** p < 0.001.
Figure 6. Mantel tests between the indicators selected by the first principal component (PC1), the second principal component (PC2), and the remaining variables. Note: PC1 comprised chlorophyll a content, chlorophyll b content, total chlorophyll content, root length, root surface area, root projection area, and root volume. PC2 comprised leaf soluble total sugar content and root APX enzyme activity. X1: Root Soluble Protein Content; X2: Root soluble sugar content; X3: Leaf soluble protein content; X4: Leaf SOD enzyme activity; X5: Leaf POD enzyme activity; X6: Leaf CAT enzyme activity; X7: Leaf APX enzyme activity; X8: Leaf GR enzyme activity; X9: Root SOD enzyme activity; X10: Root POD enzyme activity; X11: Root CAT enzyme activity; X12: Root GR enzyme activity; X13: Root diameter; X14: Number of root tip; X15: Plant height; X16: leaf width; X17: Aboveground dry weight; X18: Root dry weight; X19: MDA content in roots; X20: MDA content in leaves; X21: Leaf Relative Conductivity; X22: Root relative conductivity; X23: Leaf proline content; X24: Root proline content. Statistical significance is indicated as follows: * p < 0.05, ** p < 0.01, *** p < 0.001.
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Figure 7. Cluster heat map analysis of root and leaf indicators of garlic plants treated with bacterial strain UW4 under drought stress.
Figure 7. Cluster heat map analysis of root and leaf indicators of garlic plants treated with bacterial strain UW4 under drought stress.
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Figure 8. Changes in physiological indicators of garlic after inoculating plants with strain UW4 under drought stress.
Figure 8. Changes in physiological indicators of garlic after inoculating plants with strain UW4 under drought stress.
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Table 1. Effect of strain UW4 on the growth indicators of garlic seedlings under drought stress.
Table 1. Effect of strain UW4 on the growth indicators of garlic seedlings under drought stress.
GroupPlant Height (cm)Leaf Width
(cm)		Shoot Dry Weight
(g)		Total Root Length
(cm)		Total Root Surface Area (cm2)Total Root Volume
(cm3)		Average Root Diameter
(mm)		Number of Root TipsRoot Dry Weight
(g)		Root Projection Area
(cm2)
CK55.00 ± 0.42 b1.97 ± 0.24 a6.24 ± 1.0 a634.24 ± 35.01 c185.17 ± 3.96 b6.91 ± 0.01 d0.84 ± 0.03 b121.00 ± 15.56 ab0.53 ± 0.02 a58.94 ± 1.26 b
PGPB62.00 ± 1.27 a1.83 ± 0.08 ab6.31 ± 2.0 a794.84 ± 18.71 a293.70 ± 2.22 a13.94 ± 2.03 a1.17 ± 0.09 a146.00 ± 5.35 a0.61 ± 0.03 a93.49 ± 7.97 a
DT50.30 ± 2.12 c1.57 ± 0.12 c4.68 ± 0.56 b433.64 ± 16.51 d145.16 ± 10.83 c8.28 ± 1.91 c1.14 ± 0.05 a83.00 ± 22.63 c0.30 ± 0.01 c46.06 ± 3.79 c
DT + PGPB54.23 ± 2.41 b1.73 ± 0.12 b6.31 ± 0.43 a729.19 ± 14.80 b271.53 ± 25.06 a11.10 ± 1.57 b1.25 ± 0.03 a101.33 ± 4.11 b0.40 ± 0.02 b86.43 ± 3.45 a
Note: Means with different lowercase letters are significantly different according to Tukey’s HSD test (p ≤ 0.05). Values sharing a common letter are not significantly different.
Table 2. Comprehensive evaluation of different treatments.
Table 2. Comprehensive evaluation of different treatments.
TreatmentsCI1CI2CI3U2U3D Value
PGPB0.939−0.1551.1600.4450.9130.726
DT + PGPB0.0811.403−0.5251.0000.3130.642
CK0.382−0.963−1.0850.1570.1130.406
DT−1.402−0.2850.4500.3980.6600.226
Note: Values represent composite scores derived from the first three principal components (CI1, CI2, CI3) and two additional indices (U2, U3). The final comprehensive evaluation score (D value) was calculated as the weighted sum of the cumulative variance contribution rates of each principal component. Treatments are listed in descending order of D value.
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MDPI and ACS Style

Yan, Y.; Guo, C.; Glick, B.R.; Tian, J. Pseudomonas sp. UW4 Enhances Drought Resistance in Garlic by Modulating Growth and Physiological Parameters. Horticulturae 2025, 11, 1170. https://doi.org/10.3390/horticulturae11101170

AMA Style

Yan Y, Guo C, Glick BR, Tian J. Pseudomonas sp. UW4 Enhances Drought Resistance in Garlic by Modulating Growth and Physiological Parameters. Horticulturae. 2025; 11(10):1170. https://doi.org/10.3390/horticulturae11101170

Chicago/Turabian Style

Yan, Yiwei, Chunqian Guo, Bernard R. Glick, and Jie Tian. 2025. "Pseudomonas sp. UW4 Enhances Drought Resistance in Garlic by Modulating Growth and Physiological Parameters" Horticulturae 11, no. 10: 1170. https://doi.org/10.3390/horticulturae11101170

APA Style

Yan, Y., Guo, C., Glick, B. R., & Tian, J. (2025). Pseudomonas sp. UW4 Enhances Drought Resistance in Garlic by Modulating Growth and Physiological Parameters. Horticulturae, 11(10), 1170. https://doi.org/10.3390/horticulturae11101170

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