Multitrait characterization of salt-tolerant plant growth-promoting bacteria under salinity stress

Introduction

Soil salinity ranks among the most pressing threats to global agriculture and food security. Excess soluble salts compromise soil structure and fertility, suppress plant growth, depress crop yields, and disturb soil microbial communities (Tarolli et al., 2024). This salinization arises from a combination of natural drivers arid climates, scarce rainfall, and high evaporation and anthropogenic pressures, including improper irrigation, inadequate drainage, and overuse of fertilizers (Tarolli et al., 2024). Salt stress is a leading constraint on agricultural output, contributing, together with drought, to an estimated 20-50% of global crop losses (Ha-Tran et al., 2021). Secondary salinization has already compromised nearly one-fifth of irrigated land worldwide (Mishra et al., 2023), and the arid and semi-arid regions most susceptible to this damage span roughly 40% of Earth's land surface, sustaining close to two billion people (Hanin et al., 2016). With climate change and intensifying agricultural practices compounding the problem (Mukhopadhyay et al., 2020), developing sustainable mitigation strategies has become essential to securing food production and long-term agricultural resilience (Mukhopadhyay et al., 2020). Among the eco-friendly solutions being explored, plant growth-promoting bacteria (PGPB) have gained recognition as a powerful biological tool for easing salinity stress in crops (Kumar et al., 2020) [1] . These salt-tolerant bacteria help plants cope with saline conditions through several mechanisms producing 1aminocyclopropane-1-carboxylate deaminase, indole-3-acetic acid, antioxidants, extracellular polymeric substances, and volatile organic compounds (Kumar et al., 2020) [1] . Because such microbes enhance plant salt tolerance and help recover yields in saline-prone environments, inoculating crops with them is becoming increasingly valuable in modern agronomy (Ilangumaran et al., 2017) [2] . Halo-tolerant plant growth-promoting rhizobacteria, in particular, serve as effective agents against both biotic and abiotic stresses while boosting productivity in salt-affected farming systems (Kumawat et al., 2023). Deployed as green bioinoculants, they show real promise for reinforcing plant defenses under salt stress and helping meet the world's growing food demand (Ha-Tran et al., 2021). It is within this context that the present study set out to characterize salt-tolerant plant growth-promoting bacteria isolated from rhizosphere soil and root/shoot samples collected from salt-affected rice fields (Multitrait Characterization of Salt-Tolerant Plant Growth-Promoting Bacteria under Salinity Stress, n.d.). Fifteen isolates (IIRRSTB-1 through IIRRSTB-15) were systematically assessed for a range of plant growth-promoting traits hydrogen cyanide production, phosphate and zinc solubilization, ACC deaminase activity, IAA production, and nitrogenase activity under both non-saline and saline conditions (5% and 10% NaCl), using standard protocols (Multitrait Characterization of Salt-Tolerant Plant Growth-Promoting Bacteria under Salinity Stress). This multitrait screening across increasing salinity levels was designed to screen out isolates capable of sustaining their growth-promoting functions under salt stress, laying the groundwork for their potential development as bioinoculants in sustainable crop production across saline environments.

Materials and methods

Sample collection

Rhizosphere soil and root/shoot samples were collected from salt-affected rice fields at Agricultural Research Station (ARS) Machilipatnam, ARS Kunaram, ARS Kampasagar (Nelaguda and adjoining villages), and Krishi Vigyan Kendra (KVK) Gangavathi during the rice-growing season, and transported under refrigeration in sterile, labelled polythene bags to ICAR-Indian Institute of Rice Research, Rajendranagar, Hyderabad.

Isolation and purification

Serial dilutions of rhizosphere soil and surface-sterilized plant tissues were plated on nutrient agar (NA) with 5 % and 10 % NaCl. Morphologically distinct colonies were purified by repeated streaking and maintained on NA slants at 4 °C; 15 isolates (IIRRSTB-1 to IIRRSTB-15) were selected for further study.

Salt-tolerance screening

Overnight cultures were spot-inoculated (6 µl) on NA plates with 0 %, 5 %, and 10 % NaCl, incubated at 28 ± 2 °C for 48 h, and scored visually on a 1-5 scale (++++ = luxuriant growth).

Morphological characterization

Colony form, margin, elevation, texture, and appearance were recorded for all isolates after 48 h on NA medium.

Plant growth-promoting trait assay

Isolates were assessed qualitatively and quantitatively for HCN production, phosphate and zinc solubilization, ACC deaminase activity, and indole-3-acetic acid (IAA) production under non-saline, 5 % and 10 % NaCl, following standard protocols (Bakker & Schippers, 1987; Pikovskaya, 1948; Fasim et al., 2002; Honma & Shimomura, 1978; Gordon & Paleg, 1957); nitrogenase activity was additionally measured via the acetylene reduction assay (ARA).

Results

Morphological characterization of salt-tolerant isolates

The fifteen cultures exhibited wide variation in colony form, margin, elevation, texture, and appearance (as shown in Table 1, Fig. 1). As these traits are descriptive rather than quantitative, no isolate was ranked as high-or low-performing on morphological grounds. Round colonies were the most prevalent, recorded for seven isolates (IIRRSTB-6, 7, 8, 11, 12, 13, and 14), followed by irregular colonies in five isolates (IIRRSTB-1, 3, 4, 5, and 15) (as shown in Table 1). Oval, rhizoid, and punctiform forms were restricted to IIRRSTB-2, IIRRSTB-9, and IIRRSTB-10, respectively (as shown in Table 1). Convex elevation was the dominant type, observed in eleven isolates (IIRRSTB-2, 3, 5, 6, 7, 8, 10, 11, 13, 14, and 15), whereas raised elevation was confined to IIRRSTB-1, 4, 9, and 12 (as shown in Table 1). In terms of appearance, opaque/white colonies were most frequent (IIRRSTB-2, 3, 4, 6, 8, 10, and 12), followed by milky white (IIRRSTB-1 and 5), creamy opaque (IIRRSTB-7 and 13), creamy, white, milky, and opaque (as shown in Table 1).

Comparative salt-tolerance performance

Under both control and 5 % NaCl conditions, all fifteen cultures displayed luxuriant growth (++++) (as shown in Table 2). Differences among isolates became apparent only at 10 % NaCl: the high-performing group (IIRRSTB-1, 2, 4, 7, 9, 11, 12, and 13) sustained luxuriant growth (++++), the intermediate-performing group (IIRRSTB-3, 6, 8, and 14) showed good growth (+++), and the low-performing group (IIRRSTB-5, 10, and 15) exhibited only moderate growth (++) (as shown in Table 2). Notably, no isolate showed poor growth (+) at 10 % NaCl (as shown in Table 2).

Qualitative PGP activity under salinity stress

At 10 % NaCl, HCN production was retained by the high-performing isolates IIRRSTB-1, 2, 4, 7, 9, 12, and 13 (+) (Fig. 7), while the remaining eight isolates were negative (-) (as shown in Table 3). Phosphate-solubilizing activity persisted only in IIRRSTB-1, 2, 4, 7, and 13 (+) (Fig. 2), and zinc-solubilizing activity only in IIRRSTB-2, 6, and 12 (+) (as shown in Table 3, Fig. 4). ACC-deaminase activity was retained by IIRRSTB-1, 2, 4, 7, 9, and 12 (+) (Fig. 5), and IAA production by IIRRSTB-1, 2, 7, and 12 (+) (Fig. 3)(as shown in Table 3). Considering all five qualitative traits collectively at 10 % NaCl, IIRRSTB-2 and IIRRSTB-12 emerged as the most consistently highperforming isolates, retaining activity for HCN, phosphate solubilization, zinc solubilization, ACC deaminase, and IAA (as shown in Table 3). IIRRSTB-1 and IIRRSTB-7 retained four traits, IIRRSTB-4 retained three, IIRRSTB-9 and IIRRSTB-13 retained two, and IIRRSTB-6 retained one; the remaining isolates (IIRRSTB-3, 5, 8, 10, 11, 14, and 15) were uniformly negative for all five traits and were therefore categorized as low-performing for qualitative PGP retention (as shown in Table 3).

Quantitative PGP performance at 10 EC

HCN production was highest in IIRRSTB-2 (35.1) and IIRRSTB-13 (33.7), with intermediate activity in IIRRSTB-12 (27.0), IIRRSTB-7 (15.0), IIRRSTB-1 (14.5), and IIRRSTB-9 (9.6), and the lowest detectable value in IIRRSTB-4 (6.1); all other isolates were non-detectable (as shown in Table 4). Phosphate solubilization was highest and comparable in IIRRSTB-1 (156.0) and IIRRSTB-7 (148.1), followed by IIRRSTB-2 (108.8); lower activity was recorded for IIRRSTB-13 (19.5) and IIRRSTB-4 (7.5), with no detectable activity in the remaining isolates (as shown in Table 4). Zinc solubilization was detected exclusively in IIRRSTB-2 (19.9), IIRRSTB-6 (14.8), and IIRRSTB-12 (10.0) (as shown in Table 4). ACC-deaminase activity was maximal in IIRRSTB-2 (84.5) and IIRRSTB-7 (68.2), intermediate in IIRRSTB-4 (19.1), and comparable at lower values in IIRRSTB-9 (12.1) and IIRRSTB-12 (12.0), with the lowest detectable activity retained by IIRRSTB-1 (6.0); the remaining isolates were non-detectable (as shown in Table 4). Acetylene-reduction activity was retained by only three isolates -IIRRSTB-2, IIRRSTB-4, and IIRRSTB-12 (Fig. 6) while all other cultures were non-detectable across salinity levels (as shown in Table 4). IAA production (µg mg⁻¹ protein) was highest in IIRRSTB-2 (53.4) and IIRRSTB-7 (41.0), with lower detectable activity in IIRRSTB-12 (25.0) and IIRRSTB-1 (8.9); the remaining isolates were non-detectable (as shown in Table 4). Across the high-stress conditions examined, IIRRSTB-2 proved the most consistently high-performing isolate: it retained luxuriant growth (++++) at 10 % NaCl (as shown in Table 2), remained positive for all five qualitative PGP traits (as shown in Table 3), and recorded the highest quantitative values at 10 EC for HCN production (35.1), zinc solubilization (19.9), ACC deaminase (84.5), acetylene-reduction activity, and IAA production (53.4) (as shown in Table 4). IIRRSTB-1 and IIRRSTB-7 performed strongly but in a more traitspecific manner, particularly for phosphate solubilization at 10 EC (156.0 and 148.1, respectively) (as shown in Table 4). IIRRSTB-12 was equally resilient, retaining all five qualitative traits (as shown in Table 3) together with detectable quantitative activity for HCN (27.0), zinc solubilization (10.0), ACC deaminase (12.0), acetylene-reduction activity, and IAA (25.0) at 10 EC (as shown in Table 4). Low-performing status varied with the endpoint considered: at 10 % NaCl, IIRRSTB-5, 10, and 15 were low-performing, showing only moderate growth (as shown in Table 2); for quantitative PGP activity at 10 EC, IIRRSTB-3, 5, 8, 10, 11, 14, and 15 formed the low-performing group, being non-detectable for the measured parameters (as shown in Table 4). IIRRSTB-4, 6, 9, and 13 exhibited intermediate or trait-specific performance rather than uniformly low activity. These classifications reflect laboratory observations at 10 % NaCl or 10 EC and do not establish statistical superiority or field performance.

Discussion

Salinity stress remains a major limiting factor for crop productivity worldwide, and salt-tolerant plant growth-promoting rhizobacteria (PGPR) are increasingly recognized as a promising biological approach for enhancing crop performance in saline agro-ecosystems (Shultana et al., 2022) [3] . Since native strains isolated from saline habitats tend to outperform those from non-saline environments in boosting plant salt resistance (Shultana et al., 2020) [4] , recovering indigenous halotolerant PGPR from salt-affected rice fields is a sensible starting point for developing effective bio-inoculants. In this study, 15 morphologically distinct isolates (IIRRSTB-1 to IIRRSTB-15) were obtained from rhizosphere soil and root/shoot tissue of rice grown in saline fields, using nutrient agar supplemented with 5% and 10% NaCl. This approach to recovering diverse halotolerant bacteria from saline environments echoes previous work: Ramadoss et al. (2013) [5] isolated 84 strains from saline habitats, Albdaiwi et al. (2019) [6] recovered 74 isolates from durum wheat growing near the Dead Sea, and Pallavi et al. (2023) [7] obtained 156 isolates from the Sundarbans mangroves. Similarly, halotolerant riceassociated PGPR including Pseudomonas stutzeri and Klebsiella pneumoniae have been reported to improve seedling growth under saline conditions (Khumairah et al., 2022) [8] . The colony-level diversity observed here (variation in shape, margin, elevation, texture, and surface appearance) reflects the standard use of morphological characterization as a first-pass screening tool in rhizobacterial studies (Damodaran et al., 2013; Pallavi et al., 2023) [9,7] . Growth response to increasing salinity. All 15 isolates grew vigorously at 0% and 5% NaCl, with clear differentiation emerging only at 10% NaCl: eight isolates sustained luxuriant growth, four showed good growth, and three showed only moderate growth. This pattern aligns with earlier reports Ramadoss et al. (2013) [5] found that while all 84 isolates thrived at 5% NaCl, only a quarter tolerated 20% NaCl, and Pallavi et al. (2023) [7] noted that just 20 of 156 isolates survived 15% (2.56 M) NaCl. Similar tolerance thresholds have been documented for multifunctional PGPR tested across 5-9% NaCl (Oo et al., 2020) [10] and for biofilm-forming rhizobacteria capable of surviving 5%, 10%, and 15% NaCl (Haque et al., 2020) [11] ; Sultana et al. (2019) likewise recovered rice-associated isolates tolerant of up to 2.60 mol L -1 NaCl. The ability of most isolates here to grow at 10% NaCl (~1.7 M) therefore confirms genuine halotolerance consistent with their saline paddy origin. Strain-dependent retention of PGP traits. A key outcome of this study is that select isolates preserved multiple plant growth-promoting (PGP) traits despite salt stress. IIRRSTB-2 and IIRRSTB-12 remained positive for all five qualitative traits at 10% NaCl; IIRRSTB-1 and IIRRSTB-7 retained four; IIRRSTB-4 retained three; IIRRSTB-9 and IIRRSTB-13 retained two; IIRRSTB-6 retained one; and the remaining seven isolates (IIRRSTB-3, 5, 8, 10, 11, 14, and 15) lost all activity. Harboring multiple PGP traits within a single strain is widely considered advantageous for biofertilizer development (Gupta et al., 2019) [12] , and multi-trait screening is the accepted strategy for identifying strong halotolerant candidates (Khan et al., 2021) [13] . Salinity is known to modulate trait expression in a strain-specific manner (Oliva et al., 2023) [14] , and nutrient-solubilizing ability generally weakens as NaCl concentration rises (Dragojević et al., 2023) [15] a trend that mirrors the progressive decline in activity observed among the lower-performing isolates in this collection. Hydrogen cyanide production. HCN, a volatile metabolite produced by genera such as Bacillus, Pseudomonas, Alcaligenes, Aeromonas, and Rhizobium (Martínez-Viveros et al., 2010) [16] , contributes to biocontrol of soil-borne pathogens (Elshahat et al., 2016; Shah et al., 2021; Fasusi et al., 2023) [18,17] . Only seven isolates retained HCN activity at 10% NaCl, with IIRRSTB-2 (35.1) and IIRRSTB-13 (33.7) recording the highest values at 10 dS m -1

. Notably, none of the isolates in Ramadoss et al. (2013) [5] produced HCN at all, making the HCN-positive strains identified here a relatively rare and potentially valuable biocontrol resource under saline conditions. Phosphate and zinc solubilization. Phosphate-solubilizing bacteria are particularly important components of salt-tolerant microbial communities, since salinity reduces phosphorus availability in soil (Çelik et al., 2024) [19] . Only five isolates retained qualitative phosphate solubilization at 10% NaCl, with IIRRSTB-1 (156.0) and IIRRSTB-7 (148.1) showing the highest quantitative activity, followed by IIRRSTB-2 (108.8) at 10 dS m -1 . These values are broadly comparable to previous reports of salt-tolerant phosphate solubilizers, such as Enterococcus faecium and Pantoea dispersa, which promoted mungbean growth under salinity (Kumawat et al., 2021) [20] , and Bacillus halotolerans AD9, which solubilized 86 ± 3.06 µg mL -1 phosphate under high-salt conditions (Kapadia et al., 2022) [21] . Zinc solubilization was even more restricted, retained only by IIRRSTB-2 (19.9), IIRRSTB-6 (14.8), and IIRRSTB-12 (10.0). This trait carries agronomic significance because saline, sodic, and calcareous soils frequently induce zinc deficiency (Acharya et al., 2024) [22] ; PGPR can improve zinc bioavailability through organic-acid secretion, proton extrusion, and siderophore-mediated chelation (Otlewska et al., 2020) [23] . The dual retention of phosphate-and zinc-solubilizing activity by strains such as IIRRSTB-2 under salt stress strengthens their potential as multifunctional inoculants. ACC deaminase activity. ACC deaminase degrades the ethylene precursor 1-aminocyclopropane-1-carboxylate into ammonia and α-ketobutyrate, thereby reducing stress-induced ethylene levels and protecting root growth (Nadeem et al., 2010; Kumar et al., 2020; Ilangumaran et al., 2017) [24,1,2] . Bacterial inoculants producing this enzyme reliably alleviate salinity stress by lowering ethylene output (Gupta et al., 2019) [12] , and this capacity persists in at least some halotolerant PGPR from saline habitats (Etesami et al., 2018) [25] . Here, six isolates retained ACC deaminase activity at 10% NaCl, led by IIRRSTB-2 (84.5) and IIRRSTB-7 (68.2) at 10 dS m -1 . The cooccurrence of ACC deaminase and IAA production in strains like IIRRSTB-2 is especially valuable, as these traits work together to stimulate root development (Gupta et al., 2019) [12] . Nitrogenase activity. Nitrogen-fixing capacity, assessed via the acetylene reduction assay (ARA), was retained only by IIRRSTB-2, IIRRSTB-4, and IIRRSTB-12. ARA remains a standard method for estimating nitrogen-fixation potential in PGPR collections (de Souza et al., 2014; Khumairah et al., 2022) [8] . Non-symbiotic nitrogen fixation is well documented in genera such as Azospirillum, Azotobacter, Burkholderia, Herbaspirillum, Bacillus, and Paenibacillus (Otlewska et al., 2020) [23] , and diazotrophic activity has previously been detected in rice tissue using this assay (Rangjaroen et al., 2014). Inoculation with diazotrophic consortia has been shown to boost rice yield while cutting nitrogen fertilizer requirements (Ríos-Ruiz et al., 2020) [26] , highlighting the practical value of the nitrogenase-positive isolates identified in this study. However, ARA is a short-term enzymatic assay and does not, on its own, provide a definitive measure of biological nitrogen fixation (Boddey et al., 1987) [27] . IAA production. Indole-3-acetic acid is the most commonly reported PGP trait among halotolerant bacteria (Pallavi et al., 2023) [7] . It supports lateral root formation, root-hair elongation, and improved water and nutrient uptake under stress (Etesami et [25,28,23] , and IAA-producing halotolerant strains have been shown to enhance wheat root and shoot elongation under salinity (Otlewska et al., 2020) [23] . Only four iso lates in this study retained IAA production at 10% NaCl, with IIRRSTB-2 (53.4 µg mg -1 protein) and IIRRSTB-7 (41.0) recording the highest levels at 10 dS m -1 values comparable to those reported for Bacillus sp. AD32 (42.67 ± 1.20 µg mL -1 ) by Kapadia et al. (2022) [21] and to the top IAA producers characterized by Pallavi et al. (2023) [7] . IIRRSTB-2 as the elite multifunctional isolate. Considering all measured parameters together, IIRRSTB-2 stood out as the most consistently high-performing strain: it sustained luxuriant growth at 10% NaCl, retained all five qualitative PGP traits, and recorded the highest quantitative values for HCN, zinc solubilization, ACC deaminase, nitrogenase activity, and IAA production. Single strains combining multiple PGP functions have proven effective elsewhere-for instance, Curtobacterium albidum SRV4, which produces EPS alongside nitrogen fixation, IAA, and ACC deaminase, helped mitigate rice salinity stress (Kumar et al., 2020) [1] . Multi-trait-selected halotolerant isolates have similarly alleviated salt stress in soybean at concentrations up to 240 mM NaCl (Khan et al., 2021) [13] , while multifunctional consortia (Pseudomonas sp. P8, Peribacillus sp. P10, and Streptomyces sp. X52) showed promise in saline maize soils (Peng et al., 2021) [29] , and native PGPR consortia have outperformed commercial biofertilizers under saline conditions (Karnwal, 2023). Given this precedent, the promising strains identified here particularly IIRRSTB-2 warrant molecular identification through 16S rRNA sequencing, as done in comparable studies (Albdaiwi et [6,7,13,30] , followed by pot and field validation. Study limitations. It should be emphasized that these classifications rest solely on laboratory observations at 10% NaCl or 10 dS m -1 and do not establish statistical superiority or predict field performance. Further work assessing additional traits such as EPS production and biofilm formation, both of which support PGPR survival and Na + /K + homeostasis under salinity (Shultana et al., 2020; Mohammed, 2018) [4, 31] alongside in-planta inoculation trials, will be necessary to fully establish the agronomic potential of the IIRRSTB collection, and of the elite strain IIRRSTB-2 in particular.

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