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Current IssueEditorial BoardOnline First ArticlesArchive

Full Research Article

Enhancing Salinity Tolerance in Triticale via Synergistic Effect of Plant Growth Promoting Bacteria and Arbuscular Mycorrhizal Fungi

T
Taşkın Erol1
0000-0002-4263-3776
F
Fatih Çığ2
0000-0002-4042-0566
İ
İlhan Sabancılar3
0000-0002-0773-2752
K
Kamil Kara1
0000-0002-7729-4532
R
Rojin Özek1
0000-0003-1820-0097
M
Mustafa Ceritoglu2,*
0000-0002-4138-4579
1Department of Organic Agriculture, Kırıkkale University, Kırıkkale Vocational School, Kırıkkale, Türkiye.
2Department of Field Crops, Siirt, Siirt University, Faculty of Agriculture, Türkiye.
3Department of Medical Services and Techniques, Bitlis Eren University Vocational School of Health Services, Bitlis, Türkiye.

  • Submitted23-10-2025|

  • Accepted15-12-2025|

  • First Online 13-01-2026|

  • doi 10.18805/LRF-911

ABSTRACT

Background: Soil salinity is a major abiotic stress factor that severely limits crop productivity worldwide threatening the sustainability of modern agriculture. This study investigated the combined application of plant growth-promoting bacteria (PGPB) and arbuscular mycorrhizal fungi (AMF) in mitigating salt stress in triticale (×Triticosecale).

Methods: Three salinity levels (0, 100 and 200 mM NaCl) and nine microbial treatments including two PGPB strains (KF58B Brevibacterium frigoritolerans and KF63C Paenibacillus xylanilyticus) and two AMF species (Glomus etunicatum and Funneliformis mosseae) were tested using under controlled conditions. Glutathione peroxidase (GSH), catalase (CAT) and malondialdehyde (MDA) alterations were investigated to observe plant stress responses.

Result: Salinity had a significant inhibitory effect on plant growth. Average seedling fresh weight decreased from 0.759 g (control) to 0.393 g under 200 mM NaCl, while root fresh weight dropped from 0.687 g to 0.167 g. Root length declined by 48.2%, from 29.81 cm to 15.44 cm. Enzymatic activities and stress indicators showed notable variations. According to salinity x treatment interaction, the highest CAT (32.77 ng mL-1) and GSH (5.12 ng mL-1) activity were observed in the KF63C + F. mosseae and KF58B + F. mosseae under severe salinity, respectively. MDA reached a peak of 4.81 ng mL-1 under 200 mM NaCl conditions, but, it reduced by 74.6% with KF58B+G. etunicatum combination. Although no statistically significant differences were detected in morphological characteristics at the seedling stage, enzymatic observations indicated stress mitigation in plants. These findings supported the potential of co-inoculation with PGPB (Brevibacterium frigoritolerans and Paenibacillus xylanilyticus) and AMF (G. etunicatum and F. mosseae) on improving salt stress tolerance in triticale during early seedling stage. In paticular, combination of B. frigoritolerans and F. mosseae is recommended to mitigation of salinity stress in triticale during early seedling stage.

INTRODUCTION

Triticale is cultivated worldwide primarily for grain and forage production andmore recently, for bioenergy purposes. A total of 341,000 tons of triticale were produced on 109,605 hectares in Türkiye in 2024. Additionally, the average yield per hectare was calculated to be 3,110 kg (TUIK, 2025). Improving grain yield and quality per unit area in triticale cultivation is of significant importance for both regional and national agriculture.
       
Salinity is one of the critical environmental stress factors that negatively affect agricultural productivity. According to the global report on soil resources, salinity adversely impacts approximately 60 million hectares, or about 20% of irrigated arable land worldwide. Furthermore, it is projected that salinity-affected areas may expand to 50% of total arable land by 2050 (Butcher et al., 2016). Improper irrigation practices and changing environmental conditions have been shown to increase Naz+ and Cl- ion concentrations in soils (Wei et al., 2021). Moreover, salt stress leads to major adverse effects in plants, including ion toxicity, osmotic stress, reduced water uptake, enhanced oxidative stress andoverall inhibition of growth and development (Monisha et al., 2025).
       
One of the mechanisms through which arbuscular mycorrhizal fungi (AMF) promote plant growth is by facilitating nutrient uptake, especially phosphorus (Atakli et al., 2022). This symbiosis is also essential for the acquisition of other elements such as nitrogen, sulfur and zinc (Hodge and Storer, 2015). AMF have been found to reduce or prevent nutrient loss from soil through the production of the glomalin protein, a glycoprotein that significantly improves soil aggregation and prevents nutrient loss via denitrification (He et al., 2020). In general, AMF can mitigate damage caused by soil-borne plant pathogens or parasites, enhance plant resistance or tolerance depending on fungal species andprovide protection against phytopath-ogenic organisms (Song et al., 2015).
       
Beneficial microorganisms are gaining prominence with soil bacteria being the most viable alternative (Akçura and Çakmakçi, 2023). In addition to their direct nutritional contributions to plants, these bacteria can protect plants from multiple environmental stresses through ACC deaminase production and protect against diseases by producing siderophores. AMF can enhance salinity tolerance and mediate plant stress responses (Dastogeer et al., 2020). Evelin et al., (2019) indicated that AMF colonization in roots supports plants to boost salinity stress tolerate. These characteristics make AMF a promising, eco-friendly strategy within sustainable agricultural systems.
       
The central hypothesis of this study is that the combined application of ACC deaminase-active PGPB and AMF strains will synergistically enhance the antioxidant responses of triticale under salinity stress, thereby improving the plant’s physiological tolerance to adverse conditions. Accordingly, the aim of the research is to determine the effects of different PGPB and AMF combinations on the growth performance and molecular defense mechanisms of triticale under salt stress, with a particular focus on alterations in key agronomical and biochemical indicators including CAT, GSH and MDA. The originality of the study lies in the first-time evaluation of two native PGPB isolates with high ACC deaminase and siderophore production capacity in combination with two AMF strains in triticale. Additionally, the systematic assessment of both synergistic and antagonistic interactions among these biological agents highlights another novel aspect of this work. Overall, the study presents an innovative approach that reveals the potential of microbial consortia in managing salinity stress.

MATERIALS AND METHODS

Experimental site and conditions
 
The study was conducted as a pot experiment at 2024 in the climate chamber of Siirt University, Siirt, Türkiye. The climate chamber was maintained at an average temperature of 24±2oC with a photoperiod of 16 hours light and 8 hours dark. Light intensity was set at 400 μmol m-2 s-1 and its distribution was ensured to be homogeneous.
 
Experimental material
 
Tatlicak triticale cultivar was used in the study. The biological materials consisted of two PGPB strains (Brevibacterium frigoritolerans: KF58B and Paenibacillus xylanilyticus: KF63C) and two AMF species (Glomus etunicatum and Funneliformis mosseae). Both strains have high ACC deaminase activity and siderophore production ability. Bacterial materials were colonizated in a nutrient agar medium and transfered into nutrient broth. The density of the nutrient broth medium in which the bacteria were inoculated was adjusted to 108 colony forming unit (cfu) by spectrophotometric method (OD600). Bio-priming process was implemented by the methods of Ceritoglu et al., (2024).
 
Experimental design and lay out
 
The study was designed with a total of 9 treatments under 3 different salinity levels (0, 100 and 200 mM NaCl). Details of the 9 experimental treatments are provided in Table 1.                

Table 1: Experimental treatments including plant growth promoting bacterias and arbuscular mycorrhizal fungis.

       

The experiment was laid out according to completely randomized design with three repplications. A 2:1 (v/v) mixture of sterilized field soil and peat was mixed and filled in two-liter pots. The experimental soil was characterized for physical and chemical properties. Texture was determined using the hydrometer method. The pH and EC were determined via 1:2.5 soil-to-water suspension. Lime content was calculated with calcimeter method that was modified by Elfaki et al., (2015). Organic matter was determined by the method of the Walkley-Black wet oxidation procedure. Macro- and micronutrients were analyzed using inductively coupled plasma optical emission spectrometry (ICP-OES) by protocol of Kacar (2009). The soil was classified as loam and exhibited an slightly alkaline pH. There was no salinity, however, it has low organic matter. The soil contained a moderate amount of lime. Available phosphorus were low, whereas potassium was sufficient. Calcium and magnesium contents were at moderate. Manganese, copper andiron were sufficient, however, zinc content was low (Table 2).

Table 2: Chemical characterization of experimental soil.


       
The experimental soil was classified as loam in texture and exhibited an alkaline pH. It was non-saline and had a low organic matter content. The soil contained a moderate amount of lime. Available phosphorus levels were low, whereas potassium was found to be sufficient. Calcium and magnesium contents were at moderate levels. Manganese, copper andiron were present in adequate amounts; however, zinc content was found to be low (Table 2).
       
Pots were arranged at 80% of field capasity before sowing seeds and sustained through experiment. Seeds were treated for surface sterilization with 70% ethyl alcohol for 1 min and 10% sodium hypochlorite (NaOCl) for 5 min before seed priming process. Seeds were carefully washed with distilled water and then rinsed to remove surface water using sterile filter paper. Seeds were put in bacterial solutions for four hours and dried again (Ceritoglu et al., 2024). After bio-priming process, eight uniform-sized seeds were carefully selected and sown in each pot. Recommended dose of AMF, i.e., 2 g per liter soil, was added into pots under seed bad (Ulukapı et al., 2020). After germination, seedlings were thinned to five plants per pot. The experiment was concluded 40 days after thinning.
 
Data collection
 
Two plant samples were taken from each pot and transferred to a -86oC deep freezer for determination of glutathione peroxidase (GSH), catalase (CAT) and malondialdehyde (MDA). Leaf samples were crushed using liquid nitrogen in a mortar, centrifuged and obtained a homogenate by the methods of Akkemik et al., (2012). CAT, GSH and MDA concentartions were determined by the methods Lakhdar et al., (2011), Paglia and Valentine (1967) and Esterbauer and Cheeseman (1990), respectively. After harvest, plant height (PH), root length (RF), seedling fresh weight (SFW), root fresh weight (RFW), seedling dry weight (SDW) and root dry weight (RDW) was determined to observe morphological changes in the plants.
 
Statistical analysis
 
Two different factor (salinity stress and treatment) were used in the experiment that was laid out under controlled conditions. Normalization of data was controlled by Shapiro and Wilk normalization test. Therefore, completely randomized design with factorial treatment were used in the experiment. The data were subjected to analysis of variance (ANOVA) based on completely randomized design. Mean comparisons were grouped using Tukey’s test via JMP software.

RESULTS AND DISCUSSION

Results
 
 ANOVA results revealed significant differences (p<0.05 and 0.01) in experimental observations. Salinity stress caused statistically significant differences in all measured characteristics where MDA and CAT showed significance at the 5%, while the others were significant at the 1% level. Experiental treatments had no significant differences in growth attributes and biomass, however, caused significant differences (p<0.01) in CAT and GSH, while it was at the 5% in MDA. Similarly, interaction between salinity and treatment (SxT) caused significant differences in antioxidant response observations in which statistical differences was at the 1% in CAT and GSH, but it was 5% in MDA (Table 3).

Table 3: Analysis of variance results for experimental treatments under different salinity conditions.


       
The results indicated that PH, RL, SFW, RFW, SWD and RDW were observed at least under 200 mM NaCl conditions while they were the highest without NaCl treatment. Decreases in PH, RL, SFW, RFW, SWD and RDW under 200 mM NaCl conditions over control were calculated by 19.2%, 48.2%, 48.2%, 75.7%, 11.21% and 69.1%, respectively. The lowest CAT (11.821 ng mL-1), GSH (1.08 ng mL-1) and MDA (1,10 ng mL-1) were observed in control plants, whereas the highest CAT (14.68 ng mL-1), GSH (3.22 ng mL-1) and MDA (2.02 ng mL-1) were determined in 200 mM NaCl exposed plants (Fig 1). CAT, GSH and MDA increased by 83.6%, 24.18% and 198.2% with 200 mM NaCl over control. Experimental treatments were not effective on these growth attributes in which PH, RL, SFW, RFW, SWD and RDW varied between 31.2-38.3 cm, 18.17-22.89 cm, 0.488-0.728 g, 0.292-476 g, 0.0686-0.1038 g and 0.0367-0.0560 g, respectively (Table 4).

Fig 1: Alterations of growth attributes and antioxidant responses in triticale seedlings under different salinity conditions.



Table 4: Alterations in growth attributes with experimental treatments.


       
The lowest GSH (1.25 ng mL-1) was found in KF58B treated plants, while the highest GSH (2.86 ng mL-1) was observed with KF58B+F. mosseae. According to SxT, the lowest GSH (0.37 ng mL-1) was found in control plants under 100 mM NaCl, while the highest GSH (5.12 ng mL-1) was found with KF58B+G. etunicatum under 200 mM NaCl conditions. The lowest CAT (9.531 mL-1) was observed in KF58B+G. etunicatum treated plants, while the highest CAT (20.398 ng mL-1) was found with KF63C+F. mosseae. Overall, SxT interaction indicated that the lowest CAT (6,983 ng mL-1) was found with KF58B+F. mosseae under 200 mM NaCl, whereas the highest CAT (32.770 ng mL-1) was found in KF63C+F. mosseae treated plants under 200 mM NaCl conditions. The lowest MDA (1.07 ng mL-1) was found in KF58B+G. etunicatum treated plants, while the highest one (2.76 ng mL-1) was observed in control plants. According to SxT, the lowest MDA (0.78 ng mL-1) was found in plants treated with KF58B+ G. etunicatum under 100 mM NaCl conditions andthe highest MDA (4.81 ng mL-1) was found in control plants under 200 mM NaCl conditions (Fig 2).

Fig 2: Alterations of antioxidant responses depending on experimental treatments in triticale plants.


       
As a result of the experiment, it was determined that increasing NaCl concentrations in the growth medium adversely affected plant development parameters such as seedling and root length, as well as fresh and dry weights associated with biomass accumulation. It is well known that salt stress has harmful effects on plants at all develop-mental stages, from germination to harvest (Foti et al., 2018). The primary causes of salinity’s inhibitory effects on germination include the accumulation of toxic ions (Na+  and Cl-) and restricted water uptake by seeds. Salinity blocks enzymatic processes that convert endosperm reserves into sugars, thereby preventing proper germination and seedling development (Dash and Panda, 2001). In addition, Kz+ ions that required for osmoregulation, cell growth, membrane polarization, enzyme activity and neutralization of negative ions compete with Naz  accumulation. Moreover, Na+  and Cl-1  enter the cells and cause damage to cell membranes and cytosolic metabolic processes (Zhu et al., 2019). Consequently, increased salinity stress suppressed plant growth and limited dry matter accumulation. These findings are consistent with previous studies that have shown salinity stress restricts growth and biomass production in wheat (El Sabagh et al., 2021), canola (Lone et al., 2022), lentil (Ceritoglu et al., 2023) and triticale (Alagoz et al., 2023).
       
On the other hand, it was observed that antioxidant enzyme activities such as CAT and GSH increased under salt stress along with higher MDA concentrations. Salinity tolerance in plants is closely related to the activities of antioxidant enzymes such as SOD, CAT, GPX, APX and GR, as well as non-enzymatic antioxidant compounds (Gupta et al., 2005). Disruption of electron transport chains causes molecular oxygen to act as an electron acceptor, leading to excessive ROS production under stress conditions. Salt-induced osmotic stress alters plant metabolism and enzymatic activity, resulting in the overproduction of ROS (Menezes-Benavente et al., 2004).
       
Reactive oxygen species such as 1O2, OH-, O2- and H2O2‚  can damage proteins, lipids, carbohydrates and DNA (Groß et al., 2013). Within chloroplasts, photosystems I and II, ubiquinone andmitochondrial ETC complex III are key sites for ROS generation (Gill and Tuteja, 2010). For instance, excessive H2O2 accumulation leads to the collapse of maize leaf veins due to leakage between neighboring cells under salt stress (Menezes-Benavente et al., 2004). Plants respond to salt stress with multigenic mechanisms, including osmotic and ionic homeostasis and antioxidant defense systems involving cellular detoxification (Hasanuzzaman et al., 2021).
       
PGPB and AMF-based treatments did not show significant effects on plant growth and biomass accumulation during the early seedling stage. It is known that the endosperm reserves of seeds play a major role in early seedling development (Açıkbaş et al., 2022). Furthermore, microbial materials generally act more slowly but systematically compared to chemical fertilizers. Thus, it is suggested that biological applications may not have made a noticeable contribution to morphological traits within the short time frame of early seedling development in this experiment. However, molecular analyses revealed significant differences caused by PGPB and AMF applications in triticale seedlings under salt stress. The resistance of PGPB to stress is based on the breakdown of ethylene-which rises sharply under stress-into α-ketobutyrate and ammonia via ACC deaminase activity (del Carmen Orozco-Mosqueda et al., 2020). One of the mechanisms by which AMF promote plant growth is through enhanced nutrient uptake, especially phosphorus. This symbiosis also facilitates the acquisition of nitrogen, sulfur and zinc (Hodge and Storer, 2015). AMF have also been shown to reduce or prevent nutrient loss from soil through the production of glomalin, a glycoprotein that significantly improves soil aggregation and prevents nutrient loss via denitrification (He et al., 2020). Additionally, AMF can reduce the harmful effects of soil-borne pathogens and parasites and enhance plant resistance (Song et al., 2015).
       
Regarding the effects of PGPB and AMF on plant growth, co-inoculation has often been suggested as a better strategy than single inoculation due to enhanced synergistic effects (Nacoon et al., 2020). Bacteria have been found to improve AMF germination by disrupting fungal cell envelopes or releasing volatile compounds (Turrini et al., 2018). Bacillus thuringiensis inoculation in wheat resulted in increases of 38.8% in proline content and 21.4% in soluble sugar content (Huang et al., 2022). Moreira et al., (2022) reported that co-application of PGPB with AMF (Rhizoglomus irregularis) resulted in synergistic effect on stress responses in maize under saline conditions. Sabah et al., (2025) reported that PGPB treatment enhanced AMF root colonization compared to AMF-only treatments. Yadav et al., (2022) indicated that Bacillus sp. and arbuscular mycorrhizal fungi consortia enhance wheat nutrient and yield in the second year field trial in which maximum AMF colonization occurred with PGPB+AMF co-inoculation.

CONCLUSION

While PGPB and AMF applications did not produce significant morphological effects during early seedling development in triticale, they led to notable differences at the molecular level under salt stress. This suggests that these treatments may be effective in stress management during later developmental stages. Another important finding of this study was the emergence of both synergistic and antagonistic interactions among different PGPB and AMF strains. Specifically, bio-priming with Paenibacillus xylanilyticus and soil application of F. mosseae resulted in higher CAT and GSH activation in response to salt stress. Additionally, MDA was reduced by 74.6% in treatments combining Brevibacterium frigoritolerans and G. etunicatum compared to the control under high saline (200 mM) conditions. This outcome is believed to be associated with better utilization of soil moisture and nutrients, as well as the reduction of ethylene synthesis through the ACC deaminase mechanism. It is recommended that dual relationships of PGPB and AMF should be deeply investigated via molecular tests under control and field conditions.

ACKNOWLEDGEMENT

The present study was supported by Kýrýkkale University, Department of Scientific Research Projects under Grant 2024/008.

Disclaimers
 
The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.

CONFLICT OF INTEREST

The authors declare that there are no conflicts of interest regarding the publication of this article. No funding or sponsorship influenced the design of the study, data collection, analysis, decision to publish, or preparation of the manuscript.

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    Enhancing Salinity Tolerance in Triticale via Synergistic Effect of Plant Growth Promoting Bacteria and Arbuscular Mycorrhizal Fungi

    T
    Taşkın Erol1
    0000-0002-4263-3776
    F
    Fatih Çığ2
    0000-0002-4042-0566
    İ
    İlhan Sabancılar3
    0000-0002-0773-2752
    K
    Kamil Kara1
    0000-0002-7729-4532
    R
    Rojin Özek1
    0000-0003-1820-0097
    M
    Mustafa Ceritoglu2,*
    0000-0002-4138-4579
    1Department of Organic Agriculture, Kırıkkale University, Kırıkkale Vocational School, Kırıkkale, Türkiye.
    2Department of Field Crops, Siirt, Siirt University, Faculty of Agriculture, Türkiye.
    3Department of Medical Services and Techniques, Bitlis Eren University Vocational School of Health Services, Bitlis, Türkiye.

    • Submitted23-10-2025|

    • Accepted15-12-2025|

    • First Online 13-01-2026|

    • doi 10.18805/LRF-911

    ABSTRACT

    Background: Soil salinity is a major abiotic stress factor that severely limits crop productivity worldwide threatening the sustainability of modern agriculture. This study investigated the combined application of plant growth-promoting bacteria (PGPB) and arbuscular mycorrhizal fungi (AMF) in mitigating salt stress in triticale (×Triticosecale).

    Methods: Three salinity levels (0, 100 and 200 mM NaCl) and nine microbial treatments including two PGPB strains (KF58B Brevibacterium frigoritolerans and KF63C Paenibacillus xylanilyticus) and two AMF species (Glomus etunicatum and Funneliformis mosseae) were tested using under controlled conditions. Glutathione peroxidase (GSH), catalase (CAT) and malondialdehyde (MDA) alterations were investigated to observe plant stress responses.

    Result: Salinity had a significant inhibitory effect on plant growth. Average seedling fresh weight decreased from 0.759 g (control) to 0.393 g under 200 mM NaCl, while root fresh weight dropped from 0.687 g to 0.167 g. Root length declined by 48.2%, from 29.81 cm to 15.44 cm. Enzymatic activities and stress indicators showed notable variations. According to salinity x treatment interaction, the highest CAT (32.77 ng mL-1) and GSH (5.12 ng mL-1) activity were observed in the KF63C + F. mosseae and KF58B + F. mosseae under severe salinity, respectively. MDA reached a peak of 4.81 ng mL-1 under 200 mM NaCl conditions, but, it reduced by 74.6% with KF58B+G. etunicatum combination. Although no statistically significant differences were detected in morphological characteristics at the seedling stage, enzymatic observations indicated stress mitigation in plants. These findings supported the potential of co-inoculation with PGPB (Brevibacterium frigoritolerans and Paenibacillus xylanilyticus) and AMF (G. etunicatum and F. mosseae) on improving salt stress tolerance in triticale during early seedling stage. In paticular, combination of B. frigoritolerans and F. mosseae is recommended to mitigation of salinity stress in triticale during early seedling stage.

    INTRODUCTION

    Triticale is cultivated worldwide primarily for grain and forage production andmore recently, for bioenergy purposes. A total of 341,000 tons of triticale were produced on 109,605 hectares in Türkiye in 2024. Additionally, the average yield per hectare was calculated to be 3,110 kg (TUIK, 2025). Improving grain yield and quality per unit area in triticale cultivation is of significant importance for both regional and national agriculture.
           
    Salinity is one of the critical environmental stress factors that negatively affect agricultural productivity. According to the global report on soil resources, salinity adversely impacts approximately 60 million hectares, or about 20% of irrigated arable land worldwide. Furthermore, it is projected that salinity-affected areas may expand to 50% of total arable land by 2050 (Butcher et al., 2016). Improper irrigation practices and changing environmental conditions have been shown to increase Naz+ and Cl- ion concentrations in soils (Wei et al., 2021). Moreover, salt stress leads to major adverse effects in plants, including ion toxicity, osmotic stress, reduced water uptake, enhanced oxidative stress andoverall inhibition of growth and development (Monisha et al., 2025).
           
    One of the mechanisms through which arbuscular mycorrhizal fungi (AMF) promote plant growth is by facilitating nutrient uptake, especially phosphorus (Atakli et al., 2022). This symbiosis is also essential for the acquisition of other elements such as nitrogen, sulfur and zinc (Hodge and Storer, 2015). AMF have been found to reduce or prevent nutrient loss from soil through the production of the glomalin protein, a glycoprotein that significantly improves soil aggregation and prevents nutrient loss via denitrification (He et al., 2020). In general, AMF can mitigate damage caused by soil-borne plant pathogens or parasites, enhance plant resistance or tolerance depending on fungal species andprovide protection against phytopath-ogenic organisms (Song et al., 2015).
           
    Beneficial microorganisms are gaining prominence with soil bacteria being the most viable alternative (Akçura and Çakmakçi, 2023). In addition to their direct nutritional contributions to plants, these bacteria can protect plants from multiple environmental stresses through ACC deaminase production and protect against diseases by producing siderophores. AMF can enhance salinity tolerance and mediate plant stress responses (Dastogeer et al., 2020). Evelin et al., (2019) indicated that AMF colonization in roots supports plants to boost salinity stress tolerate. These characteristics make AMF a promising, eco-friendly strategy within sustainable agricultural systems.
           
    The central hypothesis of this study is that the combined application of ACC deaminase-active PGPB and AMF strains will synergistically enhance the antioxidant responses of triticale under salinity stress, thereby improving the plant’s physiological tolerance to adverse conditions. Accordingly, the aim of the research is to determine the effects of different PGPB and AMF combinations on the growth performance and molecular defense mechanisms of triticale under salt stress, with a particular focus on alterations in key agronomical and biochemical indicators including CAT, GSH and MDA. The originality of the study lies in the first-time evaluation of two native PGPB isolates with high ACC deaminase and siderophore production capacity in combination with two AMF strains in triticale. Additionally, the systematic assessment of both synergistic and antagonistic interactions among these biological agents highlights another novel aspect of this work. Overall, the study presents an innovative approach that reveals the potential of microbial consortia in managing salinity stress.

    MATERIALS AND METHODS

    Experimental site and conditions
     
    The study was conducted as a pot experiment at 2024 in the climate chamber of Siirt University, Siirt, Türkiye. The climate chamber was maintained at an average temperature of 24±2oC with a photoperiod of 16 hours light and 8 hours dark. Light intensity was set at 400 μmol m-2 s-1 and its distribution was ensured to be homogeneous.
     
    Experimental material
     
    Tatlicak triticale cultivar was used in the study. The biological materials consisted of two PGPB strains (Brevibacterium frigoritolerans: KF58B and Paenibacillus xylanilyticus: KF63C) and two AMF species (Glomus etunicatum and Funneliformis mosseae). Both strains have high ACC deaminase activity and siderophore production ability. Bacterial materials were colonizated in a nutrient agar medium and transfered into nutrient broth. The density of the nutrient broth medium in which the bacteria were inoculated was adjusted to 108 colony forming unit (cfu) by spectrophotometric method (OD600). Bio-priming process was implemented by the methods of Ceritoglu et al., (2024).
     
    Experimental design and lay out
     
    The study was designed with a total of 9 treatments under 3 different salinity levels (0, 100 and 200 mM NaCl). Details of the 9 experimental treatments are provided in Table 1.                

    Table 1: Experimental treatments including plant growth promoting bacterias and arbuscular mycorrhizal fungis.

           

    The experiment was laid out according to completely randomized design with three repplications. A 2:1 (v/v) mixture of sterilized field soil and peat was mixed and filled in two-liter pots. The experimental soil was characterized for physical and chemical properties. Texture was determined using the hydrometer method. The pH and EC were determined via 1:2.5 soil-to-water suspension. Lime content was calculated with calcimeter method that was modified by Elfaki et al., (2015). Organic matter was determined by the method of the Walkley-Black wet oxidation procedure. Macro- and micronutrients were analyzed using inductively coupled plasma optical emission spectrometry (ICP-OES) by protocol of Kacar (2009). The soil was classified as loam and exhibited an slightly alkaline pH. There was no salinity, however, it has low organic matter. The soil contained a moderate amount of lime. Available phosphorus were low, whereas potassium was sufficient. Calcium and magnesium contents were at moderate. Manganese, copper andiron were sufficient, however, zinc content was low (Table 2).

    Table 2: Chemical characterization of experimental soil.


           
    The experimental soil was classified as loam in texture and exhibited an alkaline pH. It was non-saline and had a low organic matter content. The soil contained a moderate amount of lime. Available phosphorus levels were low, whereas potassium was found to be sufficient. Calcium and magnesium contents were at moderate levels. Manganese, copper andiron were present in adequate amounts; however, zinc content was found to be low (Table 2).
           
    Pots were arranged at 80% of field capasity before sowing seeds and sustained through experiment. Seeds were treated for surface sterilization with 70% ethyl alcohol for 1 min and 10% sodium hypochlorite (NaOCl) for 5 min before seed priming process. Seeds were carefully washed with distilled water and then rinsed to remove surface water using sterile filter paper. Seeds were put in bacterial solutions for four hours and dried again (Ceritoglu et al., 2024). After bio-priming process, eight uniform-sized seeds were carefully selected and sown in each pot. Recommended dose of AMF, i.e., 2 g per liter soil, was added into pots under seed bad (Ulukapı et al., 2020). After germination, seedlings were thinned to five plants per pot. The experiment was concluded 40 days after thinning.
     
    Data collection
     
    Two plant samples were taken from each pot and transferred to a -86oC deep freezer for determination of glutathione peroxidase (GSH), catalase (CAT) and malondialdehyde (MDA). Leaf samples were crushed using liquid nitrogen in a mortar, centrifuged and obtained a homogenate by the methods of Akkemik et al., (2012). CAT, GSH and MDA concentartions were determined by the methods Lakhdar et al., (2011), Paglia and Valentine (1967) and Esterbauer and Cheeseman (1990), respectively. After harvest, plant height (PH), root length (RF), seedling fresh weight (SFW), root fresh weight (RFW), seedling dry weight (SDW) and root dry weight (RDW) was determined to observe morphological changes in the plants.
     
    Statistical analysis
     
    Two different factor (salinity stress and treatment) were used in the experiment that was laid out under controlled conditions. Normalization of data was controlled by Shapiro and Wilk normalization test. Therefore, completely randomized design with factorial treatment were used in the experiment. The data were subjected to analysis of variance (ANOVA) based on completely randomized design. Mean comparisons were grouped using Tukey’s test via JMP software.

    RESULTS AND DISCUSSION

    Results
     
     ANOVA results revealed significant differences (p<0.05 and 0.01) in experimental observations. Salinity stress caused statistically significant differences in all measured characteristics where MDA and CAT showed significance at the 5%, while the others were significant at the 1% level. Experiental treatments had no significant differences in growth attributes and biomass, however, caused significant differences (p<0.01) in CAT and GSH, while it was at the 5% in MDA. Similarly, interaction between salinity and treatment (SxT) caused significant differences in antioxidant response observations in which statistical differences was at the 1% in CAT and GSH, but it was 5% in MDA (Table 3).

    Table 3: Analysis of variance results for experimental treatments under different salinity conditions.


           
    The results indicated that PH, RL, SFW, RFW, SWD and RDW were observed at least under 200 mM NaCl conditions while they were the highest without NaCl treatment. Decreases in PH, RL, SFW, RFW, SWD and RDW under 200 mM NaCl conditions over control were calculated by 19.2%, 48.2%, 48.2%, 75.7%, 11.21% and 69.1%, respectively. The lowest CAT (11.821 ng mL-1), GSH (1.08 ng mL-1) and MDA (1,10 ng mL-1) were observed in control plants, whereas the highest CAT (14.68 ng mL-1), GSH (3.22 ng mL-1) and MDA (2.02 ng mL-1) were determined in 200 mM NaCl exposed plants (Fig 1). CAT, GSH and MDA increased by 83.6%, 24.18% and 198.2% with 200 mM NaCl over control. Experimental treatments were not effective on these growth attributes in which PH, RL, SFW, RFW, SWD and RDW varied between 31.2-38.3 cm, 18.17-22.89 cm, 0.488-0.728 g, 0.292-476 g, 0.0686-0.1038 g and 0.0367-0.0560 g, respectively (Table 4).

    Fig 1: Alterations of growth attributes and antioxidant responses in triticale seedlings under different salinity conditions.



    Table 4: Alterations in growth attributes with experimental treatments.


           
    The lowest GSH (1.25 ng mL-1) was found in KF58B treated plants, while the highest GSH (2.86 ng mL-1) was observed with KF58B+F. mosseae. According to SxT, the lowest GSH (0.37 ng mL-1) was found in control plants under 100 mM NaCl, while the highest GSH (5.12 ng mL-1) was found with KF58B+G. etunicatum under 200 mM NaCl conditions. The lowest CAT (9.531 mL-1) was observed in KF58B+G. etunicatum treated plants, while the highest CAT (20.398 ng mL-1) was found with KF63C+F. mosseae. Overall, SxT interaction indicated that the lowest CAT (6,983 ng mL-1) was found with KF58B+F. mosseae under 200 mM NaCl, whereas the highest CAT (32.770 ng mL-1) was found in KF63C+F. mosseae treated plants under 200 mM NaCl conditions. The lowest MDA (1.07 ng mL-1) was found in KF58B+G. etunicatum treated plants, while the highest one (2.76 ng mL-1) was observed in control plants. According to SxT, the lowest MDA (0.78 ng mL-1) was found in plants treated with KF58B+ G. etunicatum under 100 mM NaCl conditions andthe highest MDA (4.81 ng mL-1) was found in control plants under 200 mM NaCl conditions (Fig 2).

    Fig 2: Alterations of antioxidant responses depending on experimental treatments in triticale plants.


           
    As a result of the experiment, it was determined that increasing NaCl concentrations in the growth medium adversely affected plant development parameters such as seedling and root length, as well as fresh and dry weights associated with biomass accumulation. It is well known that salt stress has harmful effects on plants at all develop-mental stages, from germination to harvest (Foti et al., 2018). The primary causes of salinity’s inhibitory effects on germination include the accumulation of toxic ions (Na+  and Cl-) and restricted water uptake by seeds. Salinity blocks enzymatic processes that convert endosperm reserves into sugars, thereby preventing proper germination and seedling development (Dash and Panda, 2001). In addition, Kz+ ions that required for osmoregulation, cell growth, membrane polarization, enzyme activity and neutralization of negative ions compete with Naz  accumulation. Moreover, Na+  and Cl-1  enter the cells and cause damage to cell membranes and cytosolic metabolic processes (Zhu et al., 2019). Consequently, increased salinity stress suppressed plant growth and limited dry matter accumulation. These findings are consistent with previous studies that have shown salinity stress restricts growth and biomass production in wheat (El Sabagh et al., 2021), canola (Lone et al., 2022), lentil (Ceritoglu et al., 2023) and triticale (Alagoz et al., 2023).
           
    On the other hand, it was observed that antioxidant enzyme activities such as CAT and GSH increased under salt stress along with higher MDA concentrations. Salinity tolerance in plants is closely related to the activities of antioxidant enzymes such as SOD, CAT, GPX, APX and GR, as well as non-enzymatic antioxidant compounds (Gupta et al., 2005). Disruption of electron transport chains causes molecular oxygen to act as an electron acceptor, leading to excessive ROS production under stress conditions. Salt-induced osmotic stress alters plant metabolism and enzymatic activity, resulting in the overproduction of ROS (Menezes-Benavente et al., 2004).
           
    Reactive oxygen species such as 1O2, OH-, O2- and H2O2‚  can damage proteins, lipids, carbohydrates and DNA (Groß et al., 2013). Within chloroplasts, photosystems I and II, ubiquinone andmitochondrial ETC complex III are key sites for ROS generation (Gill and Tuteja, 2010). For instance, excessive H2O2 accumulation leads to the collapse of maize leaf veins due to leakage between neighboring cells under salt stress (Menezes-Benavente et al., 2004). Plants respond to salt stress with multigenic mechanisms, including osmotic and ionic homeostasis and antioxidant defense systems involving cellular detoxification (Hasanuzzaman et al., 2021).
           
    PGPB and AMF-based treatments did not show significant effects on plant growth and biomass accumulation during the early seedling stage. It is known that the endosperm reserves of seeds play a major role in early seedling development (Açıkbaş et al., 2022). Furthermore, microbial materials generally act more slowly but systematically compared to chemical fertilizers. Thus, it is suggested that biological applications may not have made a noticeable contribution to morphological traits within the short time frame of early seedling development in this experiment. However, molecular analyses revealed significant differences caused by PGPB and AMF applications in triticale seedlings under salt stress. The resistance of PGPB to stress is based on the breakdown of ethylene-which rises sharply under stress-into α-ketobutyrate and ammonia via ACC deaminase activity (del Carmen Orozco-Mosqueda et al., 2020). One of the mechanisms by which AMF promote plant growth is through enhanced nutrient uptake, especially phosphorus. This symbiosis also facilitates the acquisition of nitrogen, sulfur and zinc (Hodge and Storer, 2015). AMF have also been shown to reduce or prevent nutrient loss from soil through the production of glomalin, a glycoprotein that significantly improves soil aggregation and prevents nutrient loss via denitrification (He et al., 2020). Additionally, AMF can reduce the harmful effects of soil-borne pathogens and parasites and enhance plant resistance (Song et al., 2015).
           
    Regarding the effects of PGPB and AMF on plant growth, co-inoculation has often been suggested as a better strategy than single inoculation due to enhanced synergistic effects (Nacoon et al., 2020). Bacteria have been found to improve AMF germination by disrupting fungal cell envelopes or releasing volatile compounds (Turrini et al., 2018). Bacillus thuringiensis inoculation in wheat resulted in increases of 38.8% in proline content and 21.4% in soluble sugar content (Huang et al., 2022). Moreira et al., (2022) reported that co-application of PGPB with AMF (Rhizoglomus irregularis) resulted in synergistic effect on stress responses in maize under saline conditions. Sabah et al., (2025) reported that PGPB treatment enhanced AMF root colonization compared to AMF-only treatments. Yadav et al., (2022) indicated that Bacillus sp. and arbuscular mycorrhizal fungi consortia enhance wheat nutrient and yield in the second year field trial in which maximum AMF colonization occurred with PGPB+AMF co-inoculation.

    CONCLUSION

    While PGPB and AMF applications did not produce significant morphological effects during early seedling development in triticale, they led to notable differences at the molecular level under salt stress. This suggests that these treatments may be effective in stress management during later developmental stages. Another important finding of this study was the emergence of both synergistic and antagonistic interactions among different PGPB and AMF strains. Specifically, bio-priming with Paenibacillus xylanilyticus and soil application of F. mosseae resulted in higher CAT and GSH activation in response to salt stress. Additionally, MDA was reduced by 74.6% in treatments combining Brevibacterium frigoritolerans and G. etunicatum compared to the control under high saline (200 mM) conditions. This outcome is believed to be associated with better utilization of soil moisture and nutrients, as well as the reduction of ethylene synthesis through the ACC deaminase mechanism. It is recommended that dual relationships of PGPB and AMF should be deeply investigated via molecular tests under control and field conditions.

    ACKNOWLEDGEMENT

    The present study was supported by Kýrýkkale University, Department of Scientific Research Projects under Grant 2024/008.

    Disclaimers
     
    The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.

    CONFLICT OF INTEREST

    The authors declare that there are no conflicts of interest regarding the publication of this article. No funding or sponsorship influenced the design of the study, data collection, analysis, decision to publish, or preparation of the manuscript.

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