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. 2021 May 24;11(6):293. doi: 10.1007/s13205-021-02818-4

Encapsulation of Pseudomonas libanensis in alginate beads to sustain bacterial viability and inoculation of Vigna unguiculata under drought stress

Pablo Souza-Alonso 1,2,✉, Miguel Rocha 3, Inês Rocha 1, Ying Ma 1, Helena Freitas 1, Rui S Oliveira 1
PMCID: PMC8144263  PMID: 34136330

Abstract

Conventional agricultural practices based on the application of synthetic fertilizers are increasingly considered as unsustainable. Under a forecasted scenario of drought for the next decades, there is a global demand for innovative and sustainable approaches to ameliorate plant performance. Here, encapsulating beneficial microbes (BMs) to promote plant growth is gaining attention. This study evaluates bacterial encapsulation using polymeric beads of alginate, testing the survival of Pseudomonas libanensis TR1 stored up to 90 days. Produced beads were subjected to different treatments (fresh, air-dried and pulverized), which resulted in a variable size range (1200–860 µm). After storage, bacterial viability was maintained, and air-dried beads displayed a higher number of colony-forming units (2 × 107). Then, a glasshouse experiment investigated the drought resistance (plant growth, biomass, and photosynthetic responses) of Vigna unguiculata plants inoculated with these alginate beads. After 10 days of complete water restriction, turgidity and relative water content of V. unguiculata were still high under drought stress (> 80%). Leaf and root growth and biomass did not evidence significant changes after water restriction even after P. libanensis inoculation. Plant photosynthetic parameters (stomatal conductance, net photosynthetic rate, leaf CO2 concentration, or Fv'/Fm') were slightly affected due to inoculation but the level of stress-induced minimal plant responses. In our experiment, water restriction might have been insufficient to downregulate photosynthetic efficiency and reduce plant growth, limiting our understanding of the role of P. libanensis inoculation in alleviating drought stress in V. unguiculata, but highlighting the important relationship between the stress level and agricultural benefits of using encapsulated BMs.

Keywords: Alginate beads, Bacterial encapsulation, Bacterial storage, Drought stress, Cowpea growth, Pseudomonas

Introduction

Within a global climate change scenario, environmental alterations represent an important challenge for future agricultural practices and cropping systems (Porter et al. 2014). Major factors influencing plant development, changes in the water regime and temperature together with the respective plant stresses, are likely to enhance the severity of agricultural problems in the upcoming decades (Zhang et al. 2019). The shifts in rainfall patterns and the rising temperature worldwide are indeed exacerbating the drought, which contributes to land degradation (Mirzabaev et al. 2019) and represents one of the most important limiting factors for crop production over large agricultural areas (Leng et al. 2015; Fahad et al. 2017). In fact, the land percentage affected by drought duplicated in the last 40 years affecting more people worldwide than any other natural hazard (FAO 2020). The frequency, intensity, and duration of heat-related events, including droughts, are projected to increase in the upcoming years, negatively affecting agricultural systems not only in semi-arid environments but also in humid areas (Mirzabaev et al. 2019 and references therein).

Under this uncertain scenario, intensive and unsustainable agricultural practices are increasingly questioned (Foley et al. 2011; Tilman et al. 2001) whereas a more holistic vision that includes better soil and water management and recognizes the value of soil microbes is required (Baulcombe et al. 2009). Here, the inclusion of beneficial microbes (BMs) in agriculture, with their wide range of plant growth-promoting traits, has emerged in the last decades as a feasible strategy to mitigate the negative influence of agrochemicals (Hayat et al. 2010; Glick et al. 2012; Ahemad and Kibret 2014; de Souza et al. 2015; Vejan et al. 2016; Begum et al. 2019). Within the variety of microorganisms that influence plant-soil relationships, plant growth-promoting bacteria (PGPB) are one of the most relevant contributors that benefit agricultural crops. In this sense, the application of PGPB has arisen as a cost-effective and feasible strategy to increase plant performance under drought conditions (Kumar et al. 2016; Oliveira et al. 2017a, b; Chandra et al. 2018a; Shirinbayan et al. 2019; Pereira et al. 2020). Although beneficial traits of PGPB are widely distributed among different bacterial taxa, different species belonging to Azospirillum, Azotobacter, and particularly Bacillus and Pseudomonas are the most studied (Martínez-Viveros et al. 2010). Among them, the ubiquitous genus Pseudomonas is widely used as PGPB in agricultural studies, and it contains a large number of species that can improve plant performance under drought stress (Sandhya et al. 2009; Kumar et al. 2016; Bahmani et al. 2018; Chandra et al. 2018a, b; Niu et al. 2018; Rocha et al. 2019a).

Despite the growing interest and multiple uses of BMs, the selection of appropriate bacterial strains in combination with suitable inoculation methods must be considered for its application in agriculture. Although the use of PGPB can be regarded as a feasible strategy to promote plant growth under drought stress, designing efficient ways to inoculate them is crucial to obtain their beneficial effects (Glick et al. 2012). Moreover, maintaining bacterial viability and effectiveness during field application is challenging (Lugtenberg and Kamilova 2009; Glick et al. 2012). After inoculation in the field, high competition in a hostile environment leads to a rapid decay in the numbers and activities of inoculated PGPB (Martinez-Viveros et al. 2010), thereby reducing the potential for effective associations with host plants. Hence, the development of appropriate delivery systems that can maintain the capacity of BMs for longer periods is crucial (Ma et al. 2019; Rocha et al 2019b). In the last years, encapsulation or microencapsulation (depending on the capsule size) of BMs for agriculture purposes using renewable, affordable, and sustainable materials as carriers has gained attention (Bashan et al. 2002, 2014; Schoebitz et al. 2013; Rocha et al 2019c). Furthermore, the formulation and conservation of BMs maintaining cell viability in the long-term are also highly valuable in terms of commercialization (Timmusk et al. 2017).

Therefore, the objectives of our study were to (i) explore the preparation of different biodegradable and adaptable alginate beads that can encapsulate and maintain the viability of the PGPB Pseudomonas libanensis; and (ii) test the capacity of encapsulated P. libanensis to alleviate drought stress in cowpea (Vigna unguiculata (L.) Walp.). Cowpea is a widely cultivated legume throughout the world, particularly in Asia, Africa, and Latin America, with high environmental (N2 fixation), agronomic, and economic relevance as it produces grain with high protein content and nutrient-rich edible leaves, adequate to fulfilling both human and animal dietary protein requirements (Timko and Singh 2008; Gonçalves et al. 2016). We hypothesize that P. libanensis encapsulated in alginate beads will maintain high viability and its inoculation on V. unguiculata will contribute to ameliorate plant responses under water limitation.

Materials and methods

Selected species, experimental design and preparation of alginate beads

The PGPB strain selected for the experimental design was Pseudomonas libanensis TR1, which was originally isolated from the rhizosphere of Trifolium repens grown in serpentine soils in Bragança, northeast Portugal. This bacterial strain has previously demonstrated beneficial traits, such as nitrogen (N2) fixation, P solubilization, and production of 1-aminocyclopropane-1-carboxylate (ACC) deaminase, siderophores, indole-3-acetic acid (IAA) and ammonia (NH3). Also, P. libanensis TR1 showed resistance to adverse environmental factors such as tolerance to a wide range of temperature (4–38 °C), salinity of up to 8% and severe drought (− 1.5 Mpa) (Ma et al. 2016). The bacterial strain was obtained from the culture collection of the Centre for Functional Ecology, University of Coimbra.

Pseudomonas libanensis was cultivated in 50 ml of Luria–Bertani (LB) medium and incubated at 27 (± 1 °C) under constant agitation (Ma et al. 2019). Optimal growth time was previously obtained by plotting the standard growth curve. After overnight growth, P. libanensis culture was centrifuged at 2817 g for 5 min, the supernatant discarded, and the cells re-suspended in 200 ml of sterile saline solution (0.85% NaCl) at an approximate concentration of 108 colony forming units (CFU) ml−1. All procedures were done under sterile conditions. The elaboration of alginate beads was based on González et al. (2018) with slight modifications. Sodium alginate (2%, Panreac) was dissolved in distilled water (15 min, 90 °C) under constant agitation (600 rpm). After cooling the alginate solution to room temperature to avoid damage to bacterial cells, a saline suspension containing P. libanensis was then mixed with the solution in a ratio of 1:5 (bacterial solution:alginate). Once prepared, the mixture was extruded applying continuous pressure using a syringe (60 ml) equipped with a fine needle (0.2 mm diameter) into a CaCl2 solution (2%) under agitation (300–400 rpm). The syringe was maintained at a constant distance (5 ± 1 cm) to the surface of the CaCl2 solution to create spherical beads. Alginate beads were maintained in the CaCl2 solution for at least 1 h. After that, beads were sieved and washed several times with sterile distilled water to remove the excess of CaCl2.

Based on the type of the drying procedure, 3 treatments were created: fresh beads (no drying process), air-dried beads (drying overnight at room temperature in a laminar flow chamber at 22–23 °C), and pulverized beads (fast drying in a hood chamber and pulverized). Pulverization was gently carried out using mortar and pestle. Hereafter, these treatments will be referred to as BF (fresh beads), BAir (air-dried beads), and BP (pulverized beads). After drying, beads were stored in sterile plastic bags at room temperature. The bacterial viability test lasted 3 months and intermediate measurements to evaluate P. libanensis viability were carried out at 1, 6, 12, and 90 days of storage. Three replicates were performed for each treatment. After bead formation, three small groups of beads (approximately 15 beads each) of each treatment were selected and the beads observed under the microscope (5x, Leica DM4000 B LED) and photographed using a coupled camera (Leica DFC295). To measure their size and identify relevant features, beads were visually analyzed using specialized software (Leica Application Suite, LAS 4.0).

In order to select the appropriate conservation temperature for the experiment, we previously explored the variability of P. libanensis recovery in beads preserved at different temperatures. First of all, we prepared fresh beads and air-dried beads (dried 24 h at room temperature) following the same procedure described above. Beads were then separated and preserved under 2 different storage conditions: refrigerated (4 °C) or room temperature (22–24 °C). The viability of P. libanensis recovered from all treatments were virtually equal (data not shown) and, therefore, the room temperature was selected instead of refrigeration to simplify the experimental procedure.

Exploring the viability of Pseudomonas libanensis in polymeric alginate beads

The maintenance of bacterial viability was one of the main objectives of our experiment. The experimental setup comprised a factorial design using the type of bead formation (3 levels) and storage time (4 levels) as factors. The viability of P. libanensis trapped into fresh, air-dried (BAir), and pulverized beads (BP) was tested immediately after bead formation, at different intermediate times, and also at the end of the storage (1, 6, 12, 90 days). Beads from different treatments were stored separately in sealed Petri dishes (allowing gas exchange) under laboratory conditions, away from direct light and with constant temperature (22–24 °C) and humidity conditions (30–40%). Bacterial viability during storage was assessed for 90 days because loss of viability of P. libanensis during this period would compromise the V. unguiculata inoculation experiment. At each sampling date, bacterial viability was verified by assessing the number of viable colony-forming units (CFU) after dissolving the alginate as detailed in González et al. (2018). Briefly, 0.2 g of beads from each treatment were dissolved for 30 min (at room temperature) by immersion into a citrate buffer solution (20 ml) containing: sodium citrate 55 mM, EDTA anhydride 30 mM, and NaCl 150 mM, (pH = 8). Citrate solution containing beads was shaken for 20 min and then centrifuged (× 4000 g), the supernatant discarded, and the pellet suspended in NaCl (0.85%). The solution containing suspended P. libanensis was serially diluted and plated on LB agar medium. Then, plates were incubated for 24 h at 27 ºC and CFU were counted by direct observation. The process was repeated in the same manner at each sampling date. Based on the viability results obtained, a glasshouse experiment was designed to evaluate the effectiveness of inoculation of alginate beads containing P. libanensis in the mitigation of drought stress in V. unguiculata plants.

Alleviation of drought stress in Vigna unguiculata inoculated with alginate beads

Experimental design

The experimental setup comprised a 4 × 2 factorial design with the factors being microbial inoculation and drought stress, respectively. Regarding microbial inoculation, treatments were arranged in a randomized block design that included (i) non-inoculated control seeds; (ii) seeds bacterized with liquid inoculum of P. libanensis; (iii) seeds + BF beads; and (iv) seeds + BAir beads. Two bead preparations (BF and BAir, Sect. 2.1) with enough material and consistent results (higher cell viability) to satisfy the requirements of a full randomized glasshouse experiment were selected. BP beads were excluded since to some extent it seemed repetitive for the experimental design to consider two treatments (Bp and Bf) that would provide a very similar initial number of CFU. A complementary treatment was included where seeds were bacterized with the liquid inoculum containing P. libanensis as described in Rocha et al. (2020), based on the increase in plant biomass and seed yield observed in previous inoculation studies (Ma et al. 2019; Rocha et al. 2020).

Initially, plastic pots (1 L) were filled with an organic substrate (pH 6.0, electrical conductivity 200 µs cm−1, organic matter 60%, total nitrogen 143.8 mg kg−1, total phosphorus 62.7 mg kg−1, total potassium 415.1 mg kg−1) mixed with zeolite (10:1, v/v) and maintained for 3 days in a glasshouse for stabilization. Before seeding, all V. unguiculata seeds were sterilized in sodium hypochlorite (1%) for 5 min and rinsed with abundant sterile distilled water. For the bacterized treatment (Bact), seeds with were immersed (1 h) in a P. libanensis solution containing 107 CFU ml−1, then air-dried and dressed with biochar (0.25% per seed weight) (Ecochar, IberoMassa Florestal, Portugal) using a sticker solution of 2% gum arabic. After seed preparation, a small hole (2 cm in depth) in the center of the pots was filled with 0.1 g of alginate beads containing P. libanensis, and seeds were carefully placed above (two seeds per pot).

Each treatment was subjected to two levels of water regime: normal water supply (75% of field water capacity) or drought (no water) and replicated five times. Pots were watered every 2–3 days for 40 days at 75% capacity. Water supply was calculated by using control pots to measure field water capacity (measuring the volume of water required to fulfill 100% of field capacity). After 40 days, drought treatment was applied to half of the pots. Following previous experimental designs, drought stress was induced by withholding water supply (Diallo et al. 2001; Fan et al. 2015; Kumar et al. 2016; Chandra et al. 2018a, b), starting on day 40 and extending water restriction for 10 days until the end of the assay. During the experiment, plants were grown in a glasshouse under natural light conditions with an average photoperiod of 12/12 h (day/night cycle). Pots were periodically rotated to different bench positions to minimize differences in light interference. Within this time, the ranges of temperatures and relative humidity were 8–42 °C and 20–85%, respectively.

Plant measurements and harvest conditions

To evaluate the effect of bacterial inoculation on plant performance, photosynthetic parameters were estimated at the end of the drought period, immediately before plant harvest. Plant gas exchange parameters including net photosynthetic rate (μmol CO2 m−2 s−1), stomatal conductance (H2O m−2 s−1), and intercellular CO2 concentration (μmol CO2 mol−1) were measured in healthy and fully expanded leaves using an infrared gas analyzer (IRGA) LI-6400 XT portable photosynthesis system (LI-COR Inc., Lincoln, NE, USA) coupled to a leaf chamber (6400–40 leaf chamber fluorometer, LCF) that also allows estimating in vivo chlorophyll fluorescence. The leaf chamber was placed on a stable surface to ensure stability during the readings and measurements, that were carried out on a sunny day between 10:00 and 12:00 h. To ensure stable CO2 reference concentration, the 6400–01 CO2 mixer containing a CO2 cartridge was installed. Reference CO2 concentration was settled at 400 µmol mol−1 and the flow rate was 300 μmol s−1. Gas exchange measurements were averaged (10 s) and recorded as single point measurements. Photosynthetic parameters were measured in 5 plants per treatment.

At the end of the experiment (day 50 after V. unguiculata seeding), the following growth parameters were measured: shoot length (plant size up to the last internode), number of leaves, root length, fresh and dry weight, the presence of root nodules, specific root length (SRL, root length to dry mass) and specific leaf area (SLA). SLA was measured in living, relatively young, and undamaged leaves. External leaves were sampled to provide the fairest comparison across individuals. Relative water content (RWC) was also measured using the following equation RWC (%) = [(FW − DW)/(TW − DW)] × 100; where FW = fresh weight, DW = dry weight, TW = turgid weight. Shoots and roots of V. unguiculata plants were placed in paper bags and dried in an oven at 50 °C until constant weight. Then, leaf mass fraction (LMF, root biomass to total biomass) and root mass fraction (RMF, root biomass to total biomass) were also calculated based on biometric results. LMF and RMF indices have been recommended as a good approach to estimate biomass allocation patterns (Poorter and Sack 2012).

Statistical analyses

Data normality and the homogeneity of variances were initially checked using the Kolmogorov–Smirnov (K–S) test and Levene's test, respectively. Initially, bead size was compared using one-way analysis of variance (ANOVA). Then, the effect of different treatments and storage time were included as independent variables to explore the viability of P. libanensis using two-way ANOVA with Dunnett´s T3 as the post-hoc test in the comparison of bead size (variances were not homogeneous) and Tukey´s HSD to compare the effect of storage.

For the glasshouse experiment, data obtained from the harvest was also initially explored through two-way ANOVA using microbial inoculation and drought stress as the independent variables of the model. In case interaction was found between independent variables, the analysis of simple main effects was explored through Tukey’s HSD (independent factor treatment, 4 groups) or Student’s t test (independent factor drought, 2 groups). The final length of V. unguiculata plants was compared using covariance analysis (ANCOVA), using the initial length as covariable with Tukey's HSD as the post-hoc tests. Photosynthetic parameters (photosynthetic rate, stomatal conductance, CO2 concentration, and Fv'/Fm') were also analyzed through ANCOVA, using ambient conditions (air temperature, relative humidity, light intensity) as covariables. All statistical analyses were carried out using the IBM SPSS Statistics 21.0 software package (IBM SPSS Inc., Chicago, IL, USA).

Results

Bead size and Pseudomonas libanensis viability test

The initial comparison of alginate beads indicated that bead size was significantly higher in beads with large water capacity, like BF (P < 0.001). Post-hoc results consistently arranged beads in the following order according to their size BF > BAir > BP (Fig. 1). In terms of P. libanensis viability, two-way ANOVA indicated that the two factors date and treatment exerted a significant effect both individually and in combination (P ≤ 0.001, in all cases). Therefore, instead of considering the main effect, single effects between levels of each factor were explored through post-hoc tests. After 90 days, a slight (BP) to moderate (BAir) decrease in the number of CFU was observed (Fig. 2). On the other hand, the comparison between different treatments indicated a large difference of bacterial viability (CFU) favoring BAir at the beginning of the experiment (day 1, P ≤ 0.001) that was maintained at the end of the assay (day 90, P ≤ 0.001). Although total numbers of CFU were proximate, differences in cell viability were maintained between treatments after 90 days of storage: BAir, (2.00 × 107) > BF (1.80 × 106) > BP (1.76 × 106).

Fig. 1.

Fig. 1

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External appearance of alginate beads containing Pseudomonas libanensis TR1 cells with the following order: BF, upper left; BAir, upper right; BP, lower left. BF, fresh beads; BAir, air-dried beads; BP, pulverized beads. Lower right chart indicates bead size comparison between treatments (mean ± SE). Different letters indicate significant ANOVA differences at P ≤ 0.05 significant level, using Dunnett´s T3 as the post hoc test (variances were not homogeneous)

Fig. 2.

Fig. 2

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Survival of Pseudomonas libanensis TR1 during different recovering times: from 1 day (date 1), 3 days (date 2), 12 days (date 3) up to 90 days (date 4). BF, fresh beads; BAir, air dried beads; BP, pulverized beads. Different letters indicate significant differences within the same treatment during the storage and asterisks denote differences between treatments within the same date (BAir > BF = BP) according to Tukey´s at P ≤ 0.05 significance level

Plant growth and photosynthetic parameters

During the first 40 days, V. unguiculata plants were grown with similar irrigation conditions. Results from ANOVA indicated that regardless of the treatment applied, no significant differences were detected in plant shoot growth before the implementation of water restriction (Fig. 3).

Fig. 3.

Fig. 3

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Variation of shoot length during the first 40 days. Data were collected in 10-day intervals (dates 1, 2, 3, 4). No differences between treatments were detected at each date. Bact, seeds directly inoculated with P. libanensis; BF, fresh beads; Bair, air-dried beads

Photosynthetic parameters were measured at the end of the drought period (day 50). Two-way ANOVA detected differences between treatments for CO2 concentration (P = 0.011), stomatal conductance (P ≤ 0.001) and Fv'/Fm' (P ≤ 0.001) (Table 1). No differences were found between treatments due to the application of different irrigation levels. With regular water supply, post-hoc comparisons indicated that bacterial inoculation resulted in a trend of increased intercellular CO2 concentration and Fv'/Fm' with a decrease in stomatal conductance (Fig. 4). Under drought conditions, only stomatal conductance was significantly reduced.

Table 1.

Two-way ANOVA results for glasshouse experiment, including the effect of independent variables (treatment and water level) and their interaction (T*W) for the different parameters measured at plant harvest and photosynthetic parameters

Treatment Water T*W
F P F P F P
Plant parameters Leaf length (cm) 0.912 0.448 0.649 0.427 1.278 0.302
Root length (cm) 1.146 0.348 2.973 0.096 0.414 0.744
Rate of change (%) 1.919 0.147 0.969 0.333 0.639 0.596
True leaves (n) 2.354 0.094 0.210 0.650 0.174 0.913
SLA (m2 kg−1) 0.429 0.733 0.562 0.459 0.353 0.787
RWC (%) 0.954 0.428 47.424  ≤ 0.001 1.781 0.174
Leaf biomass (g DW−1) 1.719 0.186 0.0006 0.979 0.734 0.540
Root biomass (g DW−1) 1.607 0.211 2.585 0.119 2.382 0.091
Total biomass (g DW−1) 1.949 0.145 0.048 0.827 0.744 0.535
SRL (cm g−1) 1.362 0.275 0.763 0.389 1.677 0.195
LMF (leaf DW−1 total DW−1) 0.835 0.486 1.594 0.217 3.104 0.043
RMF (root DW−1 total DW−1) 0.835 0.486 1.594 0.217 3.104 0.043
Net photosynthetic rate (μmol CO2 m−2 s−1) 2.148 0.122 0.634 0.434 0.273 0.844
Photosynthetic parameters CO2 concentration (μmol CO2 mol−1) 4.234 0.011 0.624 0.437 0.291 0.832
Stomatal conductance (H2O m−2 s−1) 25.993  ≤ 0.001 1.099 0.305 1.929 0.153
Fv'/Fm' 9.298  ≤ 0.001 3.793 0.064 2.704 0.069
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Values in bold indicate significant differences at P < 0.05

SLA Specific leaf area; RWC relative water content; SRL specific root length; LMF leaf mass fraction; RMF root mass fraction; DW dry weight

Fig. 4.

Fig. 4

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Mean (± SE) of a photosynthetic rate, b intercellular CO2 concentration, c stomatal conductance, and d Fv'/Fm' of Vigna unguiculata plants after 10 days of drought. Bact, seeds directly inoculated with P. libanensis; BF, fresh beads; Bair, air-dried beads. Different letters indicate significant differences in pairwise comparison using HSD Tukey as the post-hoc test at the P ≤ 0.05 significance level. Capital letters indicate differences between treatments with regular water supply; lower case letters indicate differences with water restriction

Harvest and plant biometrical measurements

Results from two-way ANOVA indicated that the effects of independent variables on different plant parameters were practically absent and only RWC was affected by the application of water restriction (Table 1). Additionally, an interactive effect between treatment and watering level (T * W) was detected in LMF and RMF. No differences were detected between microbial treatment or water restriction in plant length or biomass (Fig. 5). After a detailed root observation, the presence of nodules was not detected. Shoot length of inoculated and non-inoculated plants was compared considering the last 10 days of the assay (days 40 to 50, when the water restriction was applied). Two-way ANOVA also indicated the absence of effect in each of the two independent variables. Responses of RWC were homogeneous within irrigation regimes, but it was significantly reduced in control, BF, and BAir when regular water supply and drought were compared (Fig. 6). No differences were detected between irrigation regimes in seeds directly inoculated with P. libanensis.

Fig. 5.

Fig. 5

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Mean (± SE) of a leaf and root length and b leaf and root biomass (dry weight, DW) of Vigna unguiculata plants grown under different inoculation and water treatments. Bact seeds directly inoculated with P. libanensis; BF, fresh beads; Bair, air-dried beads. No significant differences were detected in two-way ANOVA using Tukey’s as post-hoc test at the P ≤ 0.05 significance level

Fig. 6.

Fig. 6

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Mean (± SE) of a relative water content (RWC) and b specific leaf area (SLA) of plants grown under different inoculation and water treatments. Bact, seeds directly inoculated with P. libanensis; BF, fresh beads; Bair, air-dried beads. Asterisks indicate significant differences between watered and non-watered Vigna unguiculata plants within the same treatment at *(P ≤ 0.05) and **(P ≤ 0.01) significance level

Discussion

Size and viability of alginate beads

Two major features included in our assay (i) preventing the decline of the population of the inoculated bacteria during bead formation, together with (ii) the extension of bacterial shelf life, are considered fundamental aspects of bead formulation (Bashan et al. 2014). Initially used in the 80 s (Bashan 1986), alginate has been widely employed in the formation of different types of beads for agricultural experimentation (Schoebitz et al. 2013; Bashan et al. 2014; Liffourrena and Lucchesi 2018), representing by far the preferential material for BMs encapsulation (Bashan et al. 2014). Naturally originated from brown algae, alginate is a sustainable material, easy to obtain, inexpensive, and degradable under field conditions. Alginate has demonstrated the capacity to maintain cell viability for different periods; after preparation, beads can be directly inoculated or preserved, while maintaining cell viability for 3 years (Trivedi and Pandey 2008) or even after 14 years of storage (Bashan and González 1999). So far, the usefulness of alginate beads to serve as carriers for P. libanensis had not been tested, but natural qualities of alginate -non-toxicity, ease of gelation and handling, progressive degradation- were confirmed along the experiment. During the initial 10 days, alginate beads were still visible firmly adhered to seedling roots (personal observation), but were completely dissolved by the end of the assay, suggesting that alginate bead degradation could lead to a progressive release of the encapsulated bacteria.

The tested methods for P. libanensis encapsulation produced beads of different dimensions, although size variability was not excessive. In this sense, in a comparison between different sizes, Berninger et al. (2016) reported similar viability levels, but the bacterial release from smaller beads was 10 times higher than that observed in larger size beads. Nevertheless, in many cases, the application of beads with different size relies on specific criteria or available equipment. For instance, larger beads (up to several mm) can be produced for direct soil application, whereas beads with the smaller size (50–500 µm) can be used for different inoculation methodologies (Berninger et al. 2016).

As expected, the viability of P. libanensis trapped into the alginate matrix suffered a slight decay at the end of the experiment. After 90 days of storage, cell viability was reduced one order of magnitude (from 108 to 107 CFU g−1), which is in accordance with previous observations. Only considering the process of bead production and desiccation, it was found that the viability of a highly resistant and spore-forming Bacillus tumefaciens decreased from an average of 3.5 × 107 to 1.3 × 106 CFU/100 mg beads (Berninger et al. 2016), corresponding to a survival rate of 4%. Still, this number of CFU represents an adequate value for microbial inoculation, more valuable considering the minimal requirements for preparation and conservation of alginate beads and the rapid decline of PGPB inoculated without encapsulation (Martínez-Viveros et al. 2010). In our case, alginate beads can be theoretically considered as appropriate carriers for P. libanensis storage as these beads matched the 3 main assumptions to be considered a good matrix (Bashan et al. 2014): (i) supported the growth of our bacterial strain, (ii) maintained the necessary number of viable cells for an acceptable time, and (iii) inoculated enough microorganisms to reach a threshold number of bacteria (the last assumption is inferred by the numbers of CFU obtained after 90 days of storage).

Impact of encapsulated Pseudomonas libanensis on plant performance under drought stress

The microcosm study was conducted to assess the extent to which the use of encapsulated P. libanensis may improve the growth and photosynthetic responses of V. unguiculata under drought stress. Contrary to the expected, at the end of the assay plant-related parameters did not evidence a clear trend of amelioration after the inoculation with P. libanensis. Despite that, we further expected a notorious influence in plant parameters in response to water upholding. Within certain limits, the more intense the effect of drought stress suffered by V. unguiculata plants, the larger the benefits obtained by inoculation using alginate beads. Beyond this threshold, association with BM can no longer be effective or the damage becomes irreversible. However, no significant differences were detected.

Due to its important role in tissue metabolic activity, RWC in plant leaves is considered an essential criterion for assessing plant water status. In this regard, an increase in RWC represents important evidence of enhanced plant drought tolerance (Ngumbi and Kloepper 2016). As expected, the RWC of plants subjected to water restriction was significantly reduced regardless of the microbial treatments applied. Contrary to previous studies where the inoculation of PGPB increased plant responses in comparison with untreated plants, the inoculation of P. libanensis did not show any noticeable effect on RWC. A possible explanation could be that after 10 days of water restriction, turgidity and RWC levels of V. unguiculata were still high even under drought stress (> 80%). These high RWC values contrast with the dramatic fall (50–70%) observed when water restriction was applied in other crops, such as maize (Sandhya et al. 2010; Vardharajula et al. 2011; Naseem and Bano 2014; Naveed et al. 2014). Nevertheless, RWC values were in a similar range as those observed in previous studies where V. unguiculata was grown under drought stress (Diallo et al. 2001), confirming some degree of drought tolerance in cowpeas (Shackel and Hall 1983). It has been also suggested that drought responses can be related to the phenological period, since later stages (e.g. flowering) may show more sensitivity to drought stress (Diallo et al. 2001). Values of RWC indicated that water availability did not limit plant development, something that can be inferred by taking a closer look at plant biomass production. Also, the absence of differences in LMF and RMF indices evidences similar biomass allocation patterns in V. unguiculata plants. Among these potential indicators, SRL of fine roots, which is useful to characterize economic aspects of root systems (Ostonen et al. 2007), did not show significant alterations.

Reduction in the RWC of plant leaves is a physiological signal that directly triggers stomatal closure. This is of particular relevance in cowpeas, which have stomata that are very sensitive to soil drying (Bates and Hall 1981). Nevertheless, as stated above, RWC values observed in our study did not reach threshold values to obligate complete stomatal closure neither in watered nor in non-watered plants, or at least to reduce stomatal conductance. On the contrary, photosynthetic parameters indicated that control plants presented increased values of stomatal conductance. Surprisingly, the trend of decrease in stomatal conductance in plants inoculated with P. libanensis was accompanied by a simultaneous increase in the intercellular CO2 concentration and Fv'/Fm'. In C3 plants, such as V. unguiculata, stomatal conductance plays a major role in photosynthesis responses (Medrano et al. 2002) and by extension, plant physiology. Although stomatal conductance values suggest a probable reduction in CO2, gas availability in the mesophyll did not seem to be limited as indicated by values of intracellular CO2 concentration. Also, the increase in the Fv'/Fm' parameter indicated that the efficiency of PSII in the light-adapted state was adequate and even ameliorated in inoculated plants where the absorbed radiation is used to carry out carbon (C) fixation without constrains. Therefore, considered globally, the photosynthetic machinery of V. unguiculata was unaffected due to the level of water stress applied and only slightly influenced due to the application of P. libanensis inoculation treatments.

Although the symbioses with BMs confer important benefits to the host plants, the maintenance of root C fluxes represents an important energetic investment (~ 20% of the net C fixed by photosynthesis) (Nguyen et al. 2003; Jones et al. 2009). Plant dependence on BMs usually relates to the stress level (Ma et al. 2016), and the symbiotic efficiency is expected to increase with the increasing level of the drought stress (Figueiredo et al. 1998). The cost–benefit balance in the mutualist investment depends on resource availability and thus, altering environmental conditions could downregulate allocation pathways involved in symbiosis (Friesen and Friel 2019). In our study, the use of a quality substrate containing elevated organic matter and nutrient content, instead of poor nutrient quality soils could have also contributed to increased water retention and avoided nutrient limitation. Therefore, we argue that the imposed stress level after 10 days of water restraint might have not induced V. unguiculata plants to invest in the association with BMs to alleviate drought effects, probably allocating photoassimilates differently.

The results obtained in this study are limited to fully unfold the role of P. libanensis inoculation in alleviating drought stress in V. unguiculata. Although the reproducibility of the results would benefit from repeating the glasshouse trial, in our study the absence of an evident benefit on the growth of V. unguiculata suggests that growth conditions should be modified. While the stress level could be arguably insufficient, it is important to remark that drought conditions applied in our study were similar to those commonly found in the literature to induce water stress in inoculated plants (Diallo et al. 2001; Fan et al. 2015; Kumar et al. 2016; Chandra et al. 2018a, b). However, the temperatures inside the glasshouse and water restriction were probably insufficient to affect plant responses. Vigna unguiculata plants grew adequately during the timing of the assay and the growth capacity during water restriction (the last 10 days) was maintained. Consequently, no clear differences in terms of growth or photosynthetic inhibition were detected regardless of the treatment applied. Remarkably, V. unguiculata has previously shown a certain degree of tolerance to drought (Shackel and Hall 1983; van Duivenbooden et al. 2002; Hall 2012) and therefore, water restriction as applied in this experiment might have been insufficient to produce some damage in the photosynthetic apparatus and limit plant growth.

Conclusions

Improving the survival rate of selected bacteria and the extension of the viability period are two major aspects of the application of BMs in agriculture. Our study showed that alginate beads containing cells of P. libanensis can be stored for up to 90 days while maintaining adequate cell viability. When applied in the glasshouse trial, alginate beads were progressively dissolved in the plant rhizosphere. Nevertheless, although punctual differences between treatments were observed, our results suggest that other factors, such as nutrient content, water availability, or plant tolerance could have allowed V. unguiculata to avoid drought stress. This avenue of research has important implications for predicting how experimental conditions will influence the necessity and conditions for plant inoculation. These findings contribute to set adequate conditions for using BMs for agricultural purposes, highlighting the importance of the relationship between the stress level and plant benefits obtained by using bacterial inoculants in agriculture.

Acknowledgements

We sincerely thank the comments provided by the editor, handling editor, and two anonymous reviewers that significantly ameliorated the last version of this manuscript. I. Rocha sincerely acknowledges the support of the Portuguese Foundation for Science and Technology (FCT) through the research Grant SFRH/BD/100484/2014, the European Social Fund and Programa Operacional do Capital Humano (POCH). Also, authors thank the support provided by the European Structural and Investment Funds in the FEDER component, through the Operational Competitiveness and Internationalization Programme (COMPETE 2020), (PTDC/AGR-TEC/1140/2014, Funding Reference: POCI-01-0145-FEDER-016801); and finally national funds through the FCT (Project PTDC/AGR-TEC/1140/2014). This work was carried out at the R&D Unit Centre for Functional Ecology—Science for People and the Planet (CFE), with reference UIDB/04004/2020, financed by FCT/MCTES through national funds (PIDDAC).

Author contributions

Conceptualization: IR, PS-A, RSO; methodology: MR, IR, PS-A; formal analysis and investigation: PS-A; writing–original draft preparation: MR, PS-A; writing–review and editing: PS-A, IR, YM, HF, RSO; funding acquisition: HF, RSO; resources: HF, RSO; supervision: RSO.

Funding

I. Rocha acknowledges the support of the Portuguese Foundation for Science and Technology (FCT) through the research Grant SFRH/BD/100484/2014, the European Social Fund, and Programa Operacional do Capital Humano (POCH). This work was supported by the European Structural and Investment Funds in the FEDER component, through the Operational Competitiveness and Internationalization Programme (COMPETE 2020) (Project No. 016801 (PTDC/AGR-TEC/1140/2014); Funding Reference: POCI-01–0145-FEDER-016801); and national funds through the FCT under the Projects PTDC/AGR-TEC/1140/2014. This work was carried out at the R&D Unit Centre for Functional Ecology—Science for People and the Planet (CFE), with reference UIDB/04004/2020, financed by FCT/MCTES through national funds (PIDDAC).

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