The use of endophytic microorganisms to minimize the impact of abiotic stress is widely accepted as a successful strategy for restoring agricultural productivity in challenging environments. Plant growth-promoting fungi (PGPF) benefit their hosts in many ways, including increased growth, biomass accumulation, and nutrient uptake and provide resistance to various stresses (Chitnis et al. 2020). Hence, plants like rice and other crops associated with them may have shown better growth and higher yields despite the presence of several biotic and abiotic stresses, which reduces the strain on the economy and the environment by producing more food at a lower cost and reducing the need for breeding and agrochemicals. PGPF can be endophytic, whereby they live inside plant cells and exchange metabolites directly and can be epiphytic, whereby they live freely outside plant cells on the root surface i.e., in the rhizosphere. Since dwelling inside plants’ tissues, endophytes get a chance to communicate with their plant hosts in a very effective manner, which may make them advantageous over rhizospheric fungi (Santoyo et al. 2016). Halophytes have diverse physiological and biochemical strategies to survive and develop in saline environments (Khan et al. 2009). Apart from developing defense strategies against salinity stress, plants establish symbiotic relationships with fungi that aid in their growth and development (Yasmeen et al. 2019). The main property of these halotolerant fungal isolates is their survival capacity in high salinity. They must have some unique mechanisms to survive in a high salt-stressed condition which might help the plants they are associated with. The objective of the study was to discover endophytic fungal strains from the halophyte O. coarctata with diverse PGP attributes, such as phosphate and zinc solubilization, phytohormone production, nitrogen fixation, bio-control, and abiotic stress tolerance properties etc., crucial to create a bio-inoculant that can be utilized in different ecological regions for improving rice yield under saline stress condition (Jorquera et al. 2008).
Taking this into account, three endophytic fungi were isolated from Oryza coarctata and identified, AwOcstreb1 by whole genome sequencing (manuscript under preparation) and phylogenetic analysis based on average nucleotide identity (ANI), as well as Talaromyces adpressus and Talaromyces argentinensis based on morphological features, sequence of their ITS regions and phylogenic analysis. All three showed significant nitrogen fixation ability, solubilization of phosphates and zinc even under high salt (900 mm) stress which is indicative of their effect on the improvement of soil fertility and plant growth and restoration of plant growth under salt stress.
Endophytic fungi increase plant growth, yield, and stress resistance through a variety of strategies. By improving the absorption of nutrients and water, altering the structure of roots, boosting photosynthesis, and accumulating osmo-protectant, fungi assist plants in surviving under high-salinity environments (Sheng et al. 2008; Hajiboland et al. 2010). In our work, AwOcstreb1 visibly improved the structure of the rice plants, even under salt stress. In nutrient-poor soils, fungi play a special role in plant colonization ecology by enhancing soil nutrient cycling, especially for low-mobile ions (P, N, Cu, Mn, and Fe).
Phosphorus and zinc are key nutrients required for higher and sustained plant growth and crop yield (Khan 2015; Kumar et al. 2019). Phosphorus is a crucial component of many important structures in plant cells such as the phospholipids that form the membranes and the sugar-phosphate molecules involved in respiration and photosynthesis, that is necessary for the growth of plants (Abdel Latef and Miransari 2014). But plant-edible phosphorus is very limited in salinity affected areas. The capacity of PGPF strains to solubilize insoluble P and Zn and convert them to form that is accessible to plants is an important key trait for plant improvement. In agreement with our results, other strains of A. welwitschiae have been previously reported as phosphate solubilizers as well as growth promoters of Glycine max L. (Hussain et al. 2021). The two other endophytes belonging to the Talaromyces genus have also been identified as phosphate solubilizers in previous research (Silitonga et al. 2019; Doilom et al. 2020) and reported to be plant growth promoters (Nicoletti et al. 2023) though little is known about their potential as zinc solubilizers or nitrogen fixers.
Another primary limiting element for plant growth and productivity is nitrogen. The correlation between chlorophyll content and grain weight is linked to the plant's ability to acquire nitrogen from the soil. Furthermore, the more chlorophyll a plant has, the greater its growth potential in aerobic rice cultivation (Xiong et al. 2015). Various studies found significant effect of nitrogen level on rice plant height, maximum and productive tiller number, panicle length, spikelet number, filled grain number, paddy yield as nitrogen favors many metabolic processes within the plant system (Awan et al. 2011; Abou-Khalifa 2012; Dinesh et al. 2012; Ehsanullah et al. 2012; Hasanuzzaman et al. 2012; Yoseftabar 2013). A sustainable method for boosting the growth and yields of crop plants is the use of N2-fixing microbes as biofertilizers (Ahemad and Kibret 2014). Fungi can supplement nitrogen by biological nitrogen fixation (BNF) process to the plants as it converts elemental nitrogen into a plant-edible form. This is in agreement with other studies that have found strains from the Aspergillus genus to possess the ability to fix nitrogen (Chuang et al. 2007; Mittal et al. 2008; Yin et al. 2015; Khuna et al. 2021; Sharma and Kumawat 2021) and treating rice plants with atmospheric nitrogen-fixing bacteria results in the improvement of rice growth and yield through a considerable increase in the chlorophyll content of the leaves (Abdullahi et al. 2012) which correlates with our results.
Iron is another vital component involved in chlorophyll synthesis and nitrogen fixation, photosynthetic electron transport, nitrate reduction (Fillat et al. 1995). Siderophores help plants in iron acquisition and prevent phytopathogen colonization by withdrawing iron from the environment (Ahemad and Kibret 2014). Some fungi (Aspergillus niger, Aspergillus ochraceous, Penicillium chrysogenum, Penicillium citrinum) are reported to biosynthesize siderophores for iron transport under iron-deficient conditions which can then be taken up by plants through an iron siderophore transporter (Baakza et al. 2004). This study showed all fungal isolates can produce siderophore, which can be a great boon for their host to uptake iron under deficient soil conditions even under salinity stress. These outcomes demonstrate the potential of this strain to serve as an inoculant for enhancing rice production.
One of the primary processes underlying the direct PGP action on plants is phytohormone production. IAA (auxin) is regarded as the most significant phytohormone as at a low concentration IAA promotes primary root elongation, whereas high concentrations promote lateral and adventitious root development (Duca et al. 2014). A study showed that IAA producing endophytic fungus A. welwitschiae significantly increased the endogenous IAA content of soybean plants (Hussain et al. 2021). One study demonstrated that IAA producing microbes played a vital role in rice roots by regulating endogenous IAA levels and altering root architecture parameters such as the maximum number of roots, lateral roots, root thickness, area, root volume, and bushiness, promoting plant growth, and maintaining biomass during stress (Usha et al. 2005; Ambreetha et al. 2018). In the current study, the fungal isolates were identified as low levels of IAA-producers that may be responsible for root elongation of their host. Among them, AwOcstreb1 produced IAA in comparatively greater amounts and that seemed to stimulate a remarkable overproduction of lateral roots and root hairs in the treated rice plants which resulted in increased root weight and root length. Again, plants exposed to saline soil conditions experience various physiological and morphological changes that can decrease plant productivity (Allakhverdiev et al. 2000). Salinity disrupts the cellular function of plants and affects the branching of their root systems, ultimately resulting in hindering root growth (Yasmeen et al. 2019). IAA can indirectly manage ROS (reactive oxygen species) homeostasis by altering the stability of the stress-induced protein or can directly regulate oxidative stress by prompting ROS detoxification enzymes (Paponov et al. 2008). Combining IAA signaling pathways and ROS mechanisms provides insight into the mechanisms enabling plants to adapt and survive under stress. The manipulation of plant growth hormones could be a valuable technique for shielding plants from the negative effects of environmental stressors (Egamberdieva et al. 2018).
This study also found AweOcstreb1 to produce ACC-deaminase, which is also very important for plant growth-promotion under salt stress condition as it hydrolyzes ACC (1-aminocyclopropane 1-carboxylic acid) into α-ketobutyrate and ammonia, which can be used as a nitrogen source and thus prevent ethylene production (Honma and Shimomura 1978). The substantial increase in ethylene production from ACC induced by salinity stress harms root growth, ultimately impacting the growth of the entire plant (Arshad et al. 2007). Several studies showed that, PGP microbes that produce ACC deaminase have been found to enhance plant growth, especially under stressful conditions (Mayak et al. 2004; Pandey et al. 2005; Bal et al. 2013). Furthermore, by using ACC-deaminase deficient mutant of Pseudomonas fluorescens YsS6 and Pseudomonas migulae 8R6, Ali et al. (2014) showed that ACC deaminase activity is directly responsible for the salt tolerance of P. fluorescens YsS6 and P. migulae 8R6 treated tomato plants. Activity of ACC deaminase is also related with enhanced root growth (Arshad et al. 2007). Sun et al. (2009) demonstrated that, endophytic bacteria Burkholderia phytofirmans PsJN lost their ability to promote the elongation of the roots of canola seedlings upon a deletion mutation in the structural gene of 1-aminocyclopropane-1-carboxylate (ACC) deaminase (acdS). In accordance with the results of previous study, our findings indicated that ACC deaminase producing AwOcstreb1 treatment resulted in enhanced root growth and salinity stress resistance in rice plants. Again, recent research has indicated that PGPR with ACC deaminase activity can promote symbiosis and nitrogen fixation in plants (Okazaki et al. 2004). So, plants inoculated with PGPEF producing IAA and ACC deaminase not only protect plants from stress but also improve root growth facilitating nutrient and mineral uptake such as P, K, N, Zn, Fe etc.
Saline conditions result in changes in the net photosynthesis and stomatal conductance as a result of damage to the photosynthetic machinery (Yasmeen et al. 2019). Salt stress induces an increase in the production ROS, such as H2O2 in plants, resulting in oxidative damage, disrupts the balance within cells and leads to membrane leakage (Tuna et al. 2008). In rice cultivated in conditions with high salt content, membrane deterioration due to ROS is reported, leading to cellular toxicity (Kim et al. 2005). The synthesis of flavonoids and phenolics, which are important for antioxidant mechanisms, reduction of ROS in plant cells, and protection of plants from stress, is heavily reliant on chlorophyll molecules (Ghasemzadeh et al. 2010). The results of this study demonstrated a noteworthy rise in the levels of phenolic and flavonoid compounds, as well as a considerable reduction in H2O2 content and electrolyte leakage in rice seedlings that were inoculated with PGPEF. This study also showed increased sugar content in PGPEF-inoculated plants, which has important involvement in regulating gene related to photosynthesis, metabolism and osmotic stress (Rosa et al. 2009).
Salinity-induced elevation in Na+ concentrations leads to an increase in the Na+/K+ ratio, which can inhibit cytosolic activities, including photosynthesis and respiration (Bhat et al. 2020). Root and shoot Na+/K+ ratio significantly decreased in AwOcstreb1 inoculated plants under salinity stress in our study. In agreement with our result, Kord et al. (2019) and Abdelaziz et al. (2019) reported that the application of P. indica reduced the concentration of Na+ and increased the level of K+ in rice, maize, tomato and barley plants experiencing salt stress (Alikhani et al. 2013; Kord et al. 2019; Ali et al. 2022). The current study showed OsSOS1 gene expression was significantly higher in fungus-inoculated plants compared to uninoculated control under salt stress condition. It has been proposed that SOS1 facilitates cellular signaling to maintain ion (Na+) homeostasis under conditions of salt stress and its increased expression has already been reported to enhance salt tolerance in Arabidopsis, tomato and rice plants (Shi et al. 2003; Olias et al. 2009; Yasmin et al. 2015; Jaemsaeng et al. 2018) which correlate with our result. Piriformospora indica also reported to increase SOS1 expression in tomato roots compared to control to confer salinity tolerance (Ghorbani et al. 2019). After association with PGPEF, rice plants showed improvements in their levels of chlorophyll, flavonoid, phenolic, and sugar content, which contributed to enhanced growth and yield under salt stress. So, the results of the study suggest that utilizing halotolerant PGPEF from O. coarctata as bio-fertilizers can be a viable approach to mitigate yield loss due to salt stress in coastal regions.