Soil salinity is a major significant global problem that leads to reduced agricultural production and threats crop yield. Moreover, salt stress in agricultural land is predicted to elevate further as a result of climate change, industrial waste released into soil, excessive use of agro-chemicals, widespread introduction of low-quality water, and intensive farming (Machado and Serralheiro, 2017; Hussain H. A. et al., 2019; Ullah et al., 2021). Salt stress constrains crop yield by activating an osmotic and ionic imbalance inside plant cells. Plant growth triggered by salt stress disturbs morphological, physiological, and molecular attributes, along with increased oxidative stress that subsequently retards plant productivity (Bibi et al., 2019; Hussain S. et al., 2019). Elevated levels of Na⁺ in plant cells disrupt K⁺/Na⁺ balance, hamper uptake of water, and trigger the formation of reactive oxygen species (ROS) affecting metabolism of plants (Parihar et al., 2015; Gul Jan et al., 2019). High saline environment for plants has also shown to increase levels of 1- aminocyclopropane-1-carboxylic acid (ACC), the precursor of ethylene which promotes epinasty (Ali et al., 2014; Müller and Munné-Bosch, 2015). Although several conventional techniques can be employed to reduce soil salinity, such as better drainage management, leaching, surface flushing, irrigation of freshwater, and chemical treatments, yet reclamation of salt-affected land is financially expensive along with scarce availability of acceptable quality of irrigation water (Qadir et al., 2008). Thus, agricultural production in these saline environments requires development of crops with high salt tolerance. Conventional breeding and genetic alteration techniques to develop salinity-resistant plant species have met with limited success, necessitating the development of novel innovative approaches. In the last few decades, scientists across the world have been working to find a sustainable solution to this which is cost-effective, quick to implement, and can mitigate salt-related problems in crops to enhance agricultural productivity and sustainability under various environmental stresses (Mbarki et al., 2018; Moghimi et al., 2018). Plant–microbe interactions, especially the use of endophytes, are an effective approach to enhance agricultural output in both normal and stress conditions (White et al., 2019; Gorai et al., 2020; Mengistu, 2020; Anand et al., 2023). Endophytes are defined as “the microorganisms which spend a whole or part of their life cycle colonizing inter- or intracellularly, healthy living tissues of the host without causing any symptoms” (Petrini, 1991). Endophytic fungi supply secondary metabolites and raise tolerance against abiotic stresses after boosting the defense mechanism (Galeano et al., 2021; Gupta et al., 2021). Endophytic fungi have the capability to produce an array of bioactive components, promote plant growth, enhance accessibility of nutrients, and improve plant fitness by increasing water-use efficiency. Endophytic fungi produce various metabolites such as polyphenols, flavonoids, and phytohormones (auxin, gibberellins, and cytokinin, etc.) that greatly influence metabolism, reproduction, and overall growth of plant in various stresses (Baron and Rigobelo, 2022; Jan et al., 2022). Endophytic fungi also exhibit ACC deaminase activity which subsequently limits ethylene production (Singh et al., 2015). Colonization of plants with endophytic fungi has been reported to mitigate salt effects on plant development by various mechanisms, viz. increasing levels of protective metabolites and osmoprotectants, producing antioxidants to prevent ROS damage, reducing root respiration induced by salt, and improving phytohormone profile (Waqas et al., 2012; Zhang F. et al., 2019; Gupta et al., 2021). The use of endophytic fungi can prove to be a sustainable and cost-effective approach to enhance the salt tolerance of commercially important crops. Therefore, the current study aimed at isolation of endophytic fungi from Catharanthus roseus, collected from fields with high salinity stress and screening them for plant growth promotion and halotolerance abilities. The culture exhibiting good plant growth promotion properties and halotolerance was subjected to in vivo studies for its ability to mitigate salt stress in Vigna radiata. Vigna radiata is one of the most important and highly cultivated summer crops and is considered valuable due to its medicinal and nutritional properties. Its biochemical characteristics such as germination, growth rate, biomass, protein, and carbohydrate are affected by salt stress (Sehrawat et al., 2019). Effect of colonization of selected culture in V. radiata plants was determined on various physiological and biochemical parameters under salt stress. Salinity stress can also result in DNA damage in plant tissue (Alotaibi, 2021; Darwish et al., 2023). High salinity-induced oxidative stress is characterized as generation of ROS and imbalance of antioxidants molecules (AbdElgawad et al., 2016). One of the major outcomes of persistent oxidative stress is DNA damage (Barzilai and Yamamoto, 2004). Antioxidants are the molecules which prevent the formation of ROS by scavenging and converting them to less reactive molecules (Santos-Sánchez et al., 2019). Endophytic fungi have the potential to produce antioxidants that prevent ROS generation and protect DNA damage (Kaur et al., 2020). Genoproptective effect of selected culture in plants exposed to salt stress was also evaluated using comet assay.

In the present study, 15 endophytic cultures were isolated from different parts of C. roseus and screened for salt tolerance and plant growth promotion abilities, viz. IAA production, phosphate solubilization, siderophore, HCN, and ammonium production. An isolate CR7 exhibiting high salt tolerance (up to 15% NaCl) and plant growth promotion activities was selected for further studies. The culture produced high level of IAA and was also positive for phosphate solubilization, HCN, and ammonium production. No siderophore production was detected. Based on preliminary screening, endophytic isolate CR7 was selected and identified according to standard taxonomic key including colony diameter, color, and morphology of hyphae and conidia. The colonies were fast growing reaching a diameter of 4 cm after 5 days on PDA plates when incubated at 30°C, sporulating, cinnamon brown in color, darkening upon age and showed reverse pigmentation (Figure 1A). The conidial heads were compact, biseriate, and columnar. Conidia were small, round, smooth walled, and light brown in color (Figure 1B). Characterization of culture CR7 at molecular level was carried out by amplification of internal transcribed spacer (ITS) regions including 5.8S rDNA and DNA sequencing. After sequencing, a sequence of 484 bp was obtained which has been deposited in GenBank under accession number OQ353072. The sequence on alignment with homologous nucleotide sequences obtained from type strains revealed maximum similarity (100%) with A. terreus (Figure 1C). The culture has been submitted to National Center for Microbial Resource (NCMR) at National Center for Cell Science (NCCS), Pune, Maharashtra, India, under accession number NFCCI5502.

One of the greatest challenges facing agriculture worldwide and affecting food security is salinity of the soil. Plant–microbe interactions are emerging as a vital strategy for alleviating salt stress and restoring crop sustainability. Plant growth-promoting endophytic microbes have the ability to mitigate the effect of salt stress and enhance plant growth and nutrient uptake (Kumar et al., 2020; Gupta et al., 2021; Choudhary et al., 2022). A survey of literature revealed that many endophytic fungi have been reported to possess plant growth promotion potential (Numponsak et al., 2018; Khushdil et al., 2019; Mehmood et al., 2019; Javed et al., 2020). In the present study, endophytic fungi were isolated and evaluated for plant growth promotion ability and salt tolerance. On the basis of preliminary screening, a culture CR7 was selected which showed good plant growth promotion activities and exhibited halotolerance. The culture CR7 was identified as A. terreus on morphological and molecular basis. In the present study, selected isolate A. terreus CR7 produced high amount of IAA, phosphate solubilization potential, and also exhibited HCN and ammonium production. IAA production and phosphate solubilization ability of A. terreus have been also reported by Khushdil et al. (2019) and Khan et al. (2022). In our study, it was found that isolate A. terreus CR7 was able to grow in high salt concentration up to 15% NaCl. Isolates of A. terreus with ability to grow in up to 0.87 and 2.9% NaCl have been reported by Khushdil et al. (2019) and Khan et al. (2022), respectively. In the present study, no siderophore production was observed in the isolated culture, but other workers have documented the production of siderophore by A. terreus (Khan et al., 2022). ACC is precursor of ethylene, which has a significant negative impact on growth and development of plants (Saberi Riseh et al., 2021; Gamalero and Glick, 2022; Shekhawat et al., 2022). ACC deaminase enzyme mitigates the effect of ethylene and metabolizes ACC to ammonia and α-ketobutyrate. In this study, A. terreus CR7 produced high amount of ACC deaminase (86.36 ± 2.70 μmol α -ketobutyrate/h/mg protein). This was confirmed by FTIR analysis which demonstrated that ACC was metabolized by ACC deaminase as peaks corresponding to α- ketobutyrate and ammonia were observed. Numerous studies have reported that endophytic fungi produce ACC deaminase enzyme and mitigate the negative impact of salt stress in plants (Afridi et al., 2019; Zhang S. et al., 2019; Ji et al., 2021; Khan et al., 2022). The effect of A. terreus CR7 in promoting growth and mitigating salt stress was evaluated further in V. radiata plants using pot experiment. Aspergillus terreus CR7-associated plants showed significant enhancement in all the growth parameters inculding root length, shoot length, number of leaves, and fresh and dry biomass. Improvement in growth attributes by A. terreus has also been reported by other workers in wheat, pearl millet, maize, and rice by Khushdil et al. (2019), Khan et al. (2022), and Siddiqui et al. (2022).

A biochemical marker for the behavior of plants under salt stress is decrease in levels of photosynthetic pigments viz. chlorophyll a, b, and total chlorophyll. Under salt stress, a significant amount of Na⁺ ions may accumulate in chloroplast, inhibiting photosystem II and preventing photosynthesis, which ultimately has an impact on plant growth (Jansen et al., 1996; Sudhir and Murthy, 2004). In salt stress, ethylene production also increases, which is responsible for blocking the pathway for biosynthesis of chlorophyll. Carotenoids also play a role in defense mechanisms, by scavenging singlet oxygen or shielding chlorophyll from the damaging effects of photooxidation. Under salt stress, decrease in carotenoid content causes an increase in ROS production, which retards growth of plant by inducing oxidative damage (Behera et al., 2002; Ashraf and Harris, 2013). In our study, it was observed that under salt stress, chlorophyll content was significantly increased in inoculated plants compared to un-inoculated plants. The results shown in this study are in accordance with previous findings, where inoculation with halotolerant endophytic fungi increased the content of chlorophyll in various plants, viz. rice, maize, soyabean under salinity stress etc. (Jogawat et al., 2013; Asaf et al., 2018; Jan et al., 2022). The possible mechanism reported for this protection is inhibition of uptake of Na⁺ ions by fungal inoculated plants, which raises the content of chlorophyll and carotenoid in V. radiata plants under salt stress (Asaf et al., 2018). Salt stress also reduces the ability of plant cells to store water, which shows effect on leaf water content, and results in a build-up of Na⁺ ions inside plant cells (Zhao et al., 2021). The lower relative water content exhibits negative osmotic imbalance ultimately inhibiting plant growth (Hardoim et al., 2008). In this study, A. terreus CR7 inoculated plants had higher relative water content compared with un-inoculated plants. Similar results have also been reported by Gul Jan et al. (2019) that showed increase in relative water content in endophytic fungi inoculated plants under salt stress. Salt stress also modulates the levels of endogenous IAA. The results showed that the amount of endogenous IAA in un-inoculated plants reduced under salt stress, while A. terreus CR7 inoculated plants showed good endogenous IAA production. Increase in production of endogenous IAA in V. radiata could also be due to augmentation with IAA produced by A. terreus CR7.

Endophytic fungi are able to alleviate the negative impacts of salt stress by boosting a variety of physiochemical and biochemical processes in plants, such as production of antioxidant enzymes and secondary metabolites (Verma et al., 2022). In this study, A. terreus CR7 inoculated plants had a substantially higher DPPH radical scavenging activity compared with un-inoculated plants. Moreover, production of secondary metabolites such as phenolic compounds is another essential strategy for avoiding stress-related changes. Phenolic compounds can remove ROS from a stressful environment and prevent membrane deterioration. An increased production of phenolics in fungal inoculated plants under salt stress has also been demonstrated by other researchers (Khushdil et al., 2019; Mehmood et al., 2019). The present study also demonstrated that A. terreus CR7 increased the content of phenol in inoculated plants under salt stress. The increase in antioxidant activity, as revealed in DPPH radical scavenging assay, could also be due to increased phenolic content. Another important osmoprotectant molecule shown to increase under stress is proline. Proline content was also found to be enhanced in salinity-stressed plants, which decreased after inoculation with A. terreus CR7. Similar results showing reduction in proline content after fungal inoculation have been reported by other workers (Abdelaziz et al., 2019; Gul Jan et al., 2019; Badawy et al., 2021). On the contrary, increase in proline content in fungal inoculated plants has also been reported (Jan et al., 2022; Khan et al., 2022). The observed decrease in the production of proline in the present study could be due to antioxidant activity of endophytic fungus, which mitigated the osmotic stress by scavenging ROS.

The generated oxidative stress under high salt conditions is also reflected in biochemical alterations that cause salinity-induced harm to biomolecules and plant cell structures (Ma et al., 2020). Increase in the oxidation of unsaturated fatty acids by ROS leads to various products such as malondialdehyde, which is an indicator of oxidative stress-induced membrane damage (Zhang et al., 2016). In the present study, under salt stress, the amount of MDA increased reflecting ROS production in salt-stressed plants. After colonization with A. terreus CR7, MDA level was decreased which could be due to scavenging of ROS by the endophytic fungus. Increase in MDA content in salt stress and its mitigation by endophytic fungi have also been reported by other workers (Ta¨ıbi et al., 2016; Hnilickova et al., 2021). Salt stress can also raise electrolyte discharge within plant tissue, which can further damage or displace the cellular membrane stability (Demidchik et al., 2014). In the present study, A. terreus CR7 inoculated plants showed lower electrolyte leakage compared with un-inoculated plants, indicating decreased levels of stress. Catalase and superoxide dismutase are antioxidant enzymes that can mitigate the detrimental effect of ROS in stressful conditions. Numerous studies have reported the activation of antioxidant enzyme system mediated by endophytic fungi under salt stress (Radhakrishnan et al., 2015; Li et al., 2017). In our study also, it was observed that the presence of A. terreus CR7 in V. radiata plants under salt stress increased the activity of antioxidant enzymes as compared to the non-inoculated plants.

The cell viability and oxidative stress in roots were qualitatively examined using in vivo histochemical labeling with fluorescent dyes. Propidium iodide (PI), a fluorescent dye, is used to stain DNA molecules and evaluate cell membrane damage. PI enters through damaged cell membranes and intercalates with DNA, resulting in red fluorescence. In the current study, high salt stress affected the viability of cells causing damage, which was visualized as higher intensity of red color. The intensity of red color was reduced in case of fungal colonized roots indicating protective effect. Similar results showing impact of salinity-induced stress on the cell membrane in pea root tips have been reported (Alharbi et al., 2022). Under salt stress, oxidative stress rises which leads to the accumulation of hydrogen peroxide in plants, which can be detected after staining the root tissues with fluorescent dye 2′, 7′-dichlorodihydrofluorescein diacetate (H₂DCFDA) (Liu et al., 2020). H₂DCFDA is most extensively used dye for detecting cellular H₂O₂. It is cell permeable, diffuses into cells, and is deacetylated by cellular esterases to generate 2′, 7′-dichlorodihydrofluorescein (H₂DCF). In the presence of H₂O₂, H₂DCF is quickly oxidized to 2′, 7′-dichlorofluorescein (DCF), resulting in green fluorescence (Galluzzi and Kroemer, 2014). In the present study, green fluorescence was enhanced in salt-stressed plants in contrast to control plants, indicating increase in H₂O₂ levels. The intensity of green color reduced in case of fungal colonized plant roots. Localization of glutathione (GSH) in plant roots was visualized using monochlorobimane (MCB) dye. MCB is a cell permeant dye with high affinity for GSH. Endogenous glutathione-S-transferase (GST) enzymes catalyze the dye's reaction with GSH, resulting in blue fluorescence (Gautam et al., 2021). In this study, amount of glutathione was increased under salt stress as indicated by increased intensity of the blue color in comparison with control. The fungal colonized root showed reduced blue color intensity indicating reduction in level of glutathione.

Salt stress has been shown to increase formulation and accumulation of ROS that results in oxidative stress and cause DNA damage (Nisa et al., 2021). Salinity-induced ROS-dependent DNA damage and alteration in plants has been reported by Alotaibi (2021) and Darwish et al. (2023). Comet assay was used to examine the damage to DNA caused by salt stress and the protective effects of A. terreus CR7. This assay, commonly referred to as a single-cell gel electrophoresis assay, is an efficient way to identify DNA damage in individual cells. The principle behind this is that under the influence of an electric field DNA fragments, which are negatively charged move toward anode resulting in comet-like appearance. The degree of DNA strand breakage is estimated by the extent of migration. Genotoxic damage induced by salt stress can be assessed using various parameters viz., tail length, tail moment, and olive tail moment (Lu et al., 2017). Comet assay has also been employed by other workers to assess salinity-induced DNA damage (Alotaibi, 2021; Darwish et al., 2023). In this study, all the examined DNA damage parameters viz., tail length, tail moment, and olive tail moment were improved in A. terreus CR7 treated plants compared to control plants. This is the first study documenting the alleviation of salt stress in V. radiata by an endophytic A. terreus CR7. In addition to this, the genoprotective effects of endophytic fungi under salt stress are also being reported for the first time.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary material.

PChau: Data curation, Formal analysis, Investigation, Methodology, Writing—original draft. MandS: Data curation, Formal analysis, Methodology, Writing—original draft. AS: Data curation, Formal analysis, Methodology, Writing—original draft. MangS: Formal analysis, Methodology, Writing—original draft. PChad: Supervision, Writing—review & editing. AK: Data curation, Investigation, Methodology, Supervision, Validation, Visualization, Writing—review & editing.