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Full Research Article

Tolerance Level of Stylosanthes guianensis Cultivar Cook against Salinity Stress

A.Z. Fauzan1,*, P.D.M.H. Karti2, I. Prihantoro2, L. Abdullah2
  • 0009-0009-1939-2693
1Graduate School of Nutrition and Feed Science, Faculty of Animal Science, IPB University (Bogor Agriculture University), Bogor 16680, Indonesia.
2Department of Animal Nutrition and Feed Technology, Faculty of Animal Science, IPB University (Bogor Agriculture University), 16680, Bogor, Indonesia.

ABSTRACT

Background: Stylosanthes guienensis Cook cultivar has the potential to be cultivated in saline areas, especially in the tropics. This study aims to evaluate the tolerance level of Stylosanthes guianesis cultivar Cook to different levels of salinity stress.

Methods: The method used was single randomized complete block design (CRD) with 5 treatments and 20 replicates. The experimental design consists of P0 (NaCl 0 ppm), P1 (NaCl 2000 ppm), P2 (NaCl 3000 ppm), P3 (NaCl 4000 ppm) and P4 (NaCl 5000 ppm). Parameters measured include plant viability, vertical height of plants, number of trifoliate leaves, number of leaflets, plant weight and leaf color. Data were analyzed using ANOVA and if the data were significantly different, further tested using Duncan with SPSS software version 25.

Result: The results showed that Stylosanthes guianensis Cook in NaCl treatment had a significant effect (P<0.05) on plant vertical height, number of leaflets, plant length and plant weight and NaCl treatment up to the level of 5000 ppm decreased plant viability and leaf color became darker. The conclusion of the study showed that Stylosanthes guianensis Cook can survive in saline conditions up to 2000 ppm and at moderate stress (3000 ppm) experienced a significant decrease.

INTRODUCTION

Stylosanthes guianensis is a legume native to Central and South America and can grow in tropical to subtropical regions, including Suriname, Venezuela, Brazil and Indonesia (Palsaniya et al., 2017). Stylosanthes guianensis as a feed source has a fairly high nutrient content with a crude protein content of 10.5%, crude fiber 25% and crude fat 3% (Schultze-Kraft et al., 2023). The International Center for Tropical Agriculture reported that there are ≥500 Stylosanthes cultivars in various countries since the 1960s (Santos-Garcia et al., 2012). One cultivar that is widely cultivated in Indonesia is S. guianensis cv. Cook. This plant has the advantage of improving soil fertility. According to Latief et al., (2020) S. guianensis has the ability to fix nitrogen from the air through a symbiotic process with Rhizobium bacteria so that it can improve the quality of tropical grazing land.
       
Efforts to develop and spread S. guianensis cv. Cook need to be done especially in marginal areas such as saline soils. Indonesia has 81,290 km of coastline (Tome et al., 2024) that can be utilized as land for S. guianensis cultivation. Currently, S. guianensis has been cultivated as a forage that grows in soils that tend to be acidic with a pH ranging from 3-5 (Putra et al., 2021). The Agricultural Research and Development Agency, (2020) reported that 6.2% of Indonesia’s total land area is saline soils ranging from 0.5 - 32 dS  m-1. Saline soils have low availability of P, N, Cu, Mn, Fe and Zn nutrients, high osmotic pressure, low activity of soil microorganisms and weak water and air movement (Liu et al., 2017). The use of saline soils causes lethargy and death in non-halophytic plants (Wang et al., 2022). The level of tolerance of plants to salinity is divided into four categories: halophytes (>4000 ppm), salt-tolerant  plants (2000-4000 ppm), moderate salt-tolerant (1000-2000 ppm), salt-sensitive plants (<1000 ppm) (Moussa et al., 2022).
       
Most cultivars of Stylosanthes guianensis have sensitivity to salinity as in the study (Liu et al., 2022) S. guianensis variety FM05-2 claimed to be sensitive to 100 mM salinity. Studies on S. guianensis cv. Cook on saline soils has not been reported in Indonesia, so research on the growth of S. guianensis cv. Cook on saline soils as a basis for effective development of S. guianensis. This study aims to evaluate the tolerance level and obtain information on the adaptation limit of S. guianensis cv. Cook to different levels of salinity stress.

MATERIALS AND METHODS

Study period and location
 
This research was conducted from January to May 2024 at the Feed Plant Tissue Culture Laboratory, Division of Feed and Pasture Plant Science and Technology, Faculty of Animal Science, Bogor Agricultural University.
 
Work procedure
 
Seeds of Stylosanthes guanensis cultivar Cook were obtained from the Agrostology Field Laboratory of IPB University. Stylo seeds were given seed treatment by soaking the seeds in NaClO solution for 10 minutes, after which they were immersed in warm water at 60oC for 3 minutes, then soaked with distilled water for 24 hours. Seeds were sterilised in a laminar air flow using clorox solution and distilled water. Seeds that have been sterilised can be planted on control media as many as 20 seeds and 80 seeds in NaCl treatment. Each seed was put into one bottle measuring 10.4 × 4.9 cm with a volume of 100 ml. The time of planting seeds into the media shows the age of 0 Days After Planting (DAP). Observations of this study were carried out by observing morphological characteristics (vertical height, number of trifoliate leaves and leaf blade) which were carried out every three days, leaf color was observed and crown weight was weighed on the 30th day. Observations and measurements started from the first day after planting until 30 DAP.
 
Statistical analysis
 
This study was conducted using a single randomised complete block design (CRD) consisting of 5 treatments and 20 replicates. Data from the study were analysed by analysis of variance (ANOVA), using the method of Steel and Torrie, (1995) if significantly different (P<0.05) Duncan’s further test was conducted with IBM SPSS Statistic version 25. While the results of leaf colour analysis at the age of 30 days using Munshell Color Chart 1.0.11. The results of the study using the stress index formula based on Fernandez, (1992):


 
Keterangan :
IC = Stress Index.
Hc = Average yield of all replicates per treatment under stressed conditions.
Hp =  Average yield of all replicates under no stress (control) condition.

RESULTS AND DISCUSSION

Viability of Stylosanthes guianensis under different salinity stresses
 
Plant viability illustrates the level of tolerance of S. guianensis to salinity stress, which is very important specially to evaluate the quality and growth potential of S. guianensis. The results of S. guianensis viability with different NaCl levels (30 DAT) are presented in Fig 1. Viability of S. guianensis  cv. Cook decreased as the NaCl level increased. Media treated with NaCl at the highest level had lower growth by 40% than without NaCl. The decrease in viability occurs due to increased osmotic stress, impaired nutrient absorption, cell death and even excessive Na+ and Cl- ion poisoning, thus disrupting cell ionic balance and physiological processes such as respiration and photosynthesis. In accordance with the research of Veraplakorn et al., (2013) excess Na+ and Cl- ions damage the cell membrane of S. guianensis so that the metabolic process is disrupted, severe cell membrane damage causes plant cell death.

Fig 1: Viability of S. guianensis with different NaCl levels (30 DAT).


       
Growth stages can show different sensitivity to salinity stress. (Tolib et al., 2017) reported that legumes are still tolerant in moderate salinity soils at 2500 ppm and are not resistant to high salinity. NaCl stress causes plant cell death and reduced growth (up to 40%) either by inhibiting water uptake or toxic effects of ions in the cell. According to (Farooq et al., 2017) ion toxicity in plant tissues damages cell structure and enzyme function thereby inhibiting growth and causing plant development to be inhibited up to 70%.
 
Vertical height of S. guianensis under different salinity stresses
 
The results of vertical height of S. guianensis at different NaCl levels are presented in Table 1. Giving the highest level of NaCl had a very significant effect at 15 DAP. Media without NaCl (control) showed the best vertical height compared to S. guianensis with NaCl media. The decrease in vertical height of S. guianensis was influenced by the level of NaCl which caused differences in the expression of plant height in each treatment. This results in cell damage in S. guianensis inhibiting growth.

Table 1: Vertical height of S. guianensis at different NaCl levels.


       
The concentration of NaCl in the media affects osmosis and causes water to move out of S. guianensis cells. Based on Veraplakorn et al., (2012) the height growth of S. guianensis CIAT184 began to be significantly inhibited at a salinity of 20-25 mM NaCl (about 1100-1400 ppm). According to Chen et al., (2023), an increase in osmotic pressure causes cell turgor pressure to decrease so that stem growth is inhibited and as a result, plants become short. Excess Na+ and Cl- ions in the media interfere with the activity of gibberellin hormone in S. guianensis. Gibberellin hormone plays a role in cell elongation, excess Na+ and Cl- ions in gibberellin activity causes inhibited stem growth (Ramadhani and Ulpah, 2022).
 
Number of trifoliate leaves under different salinity stresses
 
Stylosanthes guianensis has trifoliate leaves or consists of three leaflets attached to one stalk. The results of the number of trifoliate leaves at different NaCl levels are presented in Fig 2. Trifoliate leaves began to form on day 12 with the highest number in media without NaCl. The provision of more NaCl caused a decrease in the number of trifoliate leaves reaching 72.7% of the media without NaCl (control). The ability of NaCl can reduce the absorption of water by the roots, the potential water in the media becomes lower than the potential water in the root cells.

Fig 2: Number of Trifoliate Leaves of S. guianensis with Different NaCl Levels (30 DAP).


       
Media supplemented with NaCl can increase osmotic pressure, so that plants are dehydrated and interfere with the division of meristematic cells responsible for the formation of primordial leaves of S. guianensis. According to Rizki et al., (2022) the number of Stylosanthes trifoliate leaves at two weeks after planting in the range of 3-6 leaves. Zhao et al., (2021) reported that salinity stress affects the slow formation of primordial leaves, so it is necessary to have genetic mutations that can protect plants from this stress. Salinity stress inhibits respiratory enzymes and increases free radicals, thus impairing leaf respiration of S. guianensis. In line with the statement of Das et al., (2019) salinity stress inhibits the activity of enzymes such as pyruvate dehydrogenase and isocitrate dehydrogenase that play a role in cellular respiration.
 
Number of leaves of s. guianensis under different salinity stresses
 
The results of the number of leaves of S. guianensis against different NaCl levels have been done presented in Table 2. The higher the NaCl level in the media, the fewer leaf blades produced. The decrease in the number of leaves shows a significant inhibitory effect on the number of leaves due to high NaCl levels. The impact will be increasingly visible as the level and duration of NaCl exposure increase in the media. High NaCl levels in the media interfere with nutrient absorption by means of Na+ ion concentrations competing with K+, Ca2+ and Mg2+  ions in S. guianensis cells and can interfere with cell metabolism. In line with the research of Feng et al., (2020) which states that Na+ ions have a high affinity which causes the absorption of nutrient ions to be inhibited, so that photosynthesis is disrupted and leaf growth is inhibited. High NaCl concentrations increase reactive oxygen species in plant cells, inhibit axillary branching and reduce leaf blade production Chauhan et al., (2024).

Table 2: Number of leaves of S. guianensis under different salinity stresses.


 
Leaf length of s. guianensis under different salinity stresses
 
The difference in leaf surface area significantly affects the rate of photosynthesis that takes place in S. guianensis. The results of leaf length of S. guianensis with different NaCl levels are presented in Table 3. Long-term exposure to NaCl and high NaCl levels can cause the growth of S. guianensis leaf length to be inhibited. The provision of higher levels of NaCl causes water stress, as a result the water content transported for photosynthesis is reduced.

Table 3: Leaf length of S. guianensis at different NaCl levels.


       
High NaCl levels can be toxic to S. guianensis by damaging cells and inhibiting metabolic processes. This process disrupts the process of cell division at the leaf growth point, so that the leaves cannot grow optimally long. Research by Feng et al., (2020) showed that higher NaCl causes water stress, thus reducing the availability of water for photosynthesis and inhibiting the activity of photosynthetic enzymes, such as RuBisCO (ribulose-1,5-bisphosphate carboxylase/oxygenase) which is important in CO2 fixation. Disruption of photosynthesis causes leaf damage and leaf length growth is not optimal. In accordance with Zhao et al., (2021)  If the plant lacks water, the photosynthetic rate will drop, so that cells will be regulated to perform limited growth.
 
Fresh weight of S. guianensis under different salinity stresses
 
The measurement results of S. guianensis weight with different NaCl levels are presented in Fig 3. The highest NaCl level (P4) showed the most significant decrease in S. guianensis weight (25.97%) compared to media without NaCl (control). NaCl application to S. guianensis causes dehydration due to NaCl ions pulling water out of the cells, reducing turgor and inhibiting growth. In accordance with Liu et al., (2017) high salinity causes damage to cell structures resulting in loss of cell turgor and cell death which results in the growth of S. guianensis being inhibited. Prolonged exposure to NaCl and high levels in tissue culture media can result in necrosis (cell death) of apical buds and young leaf tissue. Tissue death in S. guianensis directly reduces mass, so the weight of S. guianensis is reduced. Hossain et al., (2024) stated that the provision of higher NaCl levels in legumes [Vigna mungo Hepper] resulted in a decrease in fresh and dry weights due to a decrease in photosynthetic capacity and osmotic imbalance in plant tissues.

Fig 3: Fresh weight of S. guianensis under different salinity stresses (30 HST).



Stress index of S. guianensis cv. cook
 
Salinity stress index is an important method to measure crop tolerance to salinity stress. This method can be used to develop plant varieties that are more tolerant to salinity. The results of the calculation of the stress index in S. guianensis cv. Cook at different NaCl levels are presented in Table 4. Moderate stress began to appear at 3000 ppm salinity, which indicates S. guianensis cv. Cook is not tolerant to moderate salinity. The increase in index value is due to the high NaCl level damaging the cell membrane, thus disrupting the metabolism and physiology of S. guianensis cv. Cook.

Table 4: Stress index of S. guianensis at different NaCl levels.


       
Exposure to NaCl in the media also triggered changes in gene expression in S. guianensis cv. Cook and has an impact on the stress index value. This indicates that S. guianensis cv. Cook does not have the ability to adapt to medium-high salinity because it experiences a significant reduction in growth. According to Fuentes et al., (2010) S. guianensis cv. CIAT-184 shows moderate tolerance to salinity conditions, growth begins to be suboptimal in moderate to high salinity soils. Salinity stress in S. guianensis cv. Cook increased peroxidase and decreased root nodulation. Salinity stress inhibits root nodulation by suppressing the growth of rhizobium bacteria that are sensitive to saline areas (Yunus et al., 2024). Based on Ghosh et al., (2025) reported that plants exposed to salinity stree need calsium and potassium supplementation to increase protein fraction.
 
Leaf color of S. guianensis under different salinity stresses
 
Leaf color is an important parameter to provide information on stress, health and nutrient content in plants. The observation results of S. guianensis leaf color at different NaCl levels are presented in Table 5. The number after GY/Y indicates the brightness of the leaf, where a higher number indicates a lighter color. The value after the slash represents chroma, with high numbers indicating a more saturated and intense color, while low numbers indicate a more muted color. The leaf color of S. guianensis (Table 5) in NaCl supplemented media showed green-yellow and dark yellow colors, while without NaCl was dominated by 5GY 3/4 at 36%, meaning that the leaf color has two shades, namely GY (Green-Yellow) with 3/4 which tends to be higher. The more chlorophyll in the leaves, the more green the color response is expressed (Prihantoro et al., 2023). Exposure to NaCl in the media affects leaf color due to excess NaCl uptake, which inhibits water absorption and stimulates abscisic acid and anthocyanin production, causing leaf wilting.

Table 5. Leaf Color of S. guianensis under Different Salinity Stresses.

CONCLUSION

Stylosanthes guianensis cv. Cook can survive in saline conditions up to 2000 ppm and at moderate stress (3000 ppm) has decreased significantly. Increasing NaCl in media causes a decrease in the growth of S. guianensis cv. Cook and changes in leaf color to yellow and darker. Further research can be carried out by providing genetic mutations with gamma rays and increasing salinity tolerance in S. guianensis cv. Cook.

ACKNOWLEDGEMENT

The present study was supported by the Ministry of Education, Culture, Research and Technology of the Republic of Indonesia and the Directorate General of Higher Education, Research and Technology through the Master Program of Education Leading to a Doctoral Degree for Excellent Graduates (PMDSU). Research Implementation Assignment Agreement Number: 006/C3/DT.05.00/PL/2025. Additionally, the authors express their sincere appreciation to Thabed Tholib Baladraf for his valuable assistance in reviewing and improving the writing of this manuscript.
 
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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