Introduction

Rice is the primary and fundamental food source providing 20% of the global calories consumed by over half the world's population and fulfils the dietary requirements of a large proportion of the human population especially in Asia and Africa1. It is cultivated on 165.25 mh with an annual global production of around 787.29 mt 3. India has the largest land area under rice cultivation, but occupies second place in the tonnage globally after China. This is largely due to lower productivity rate of rice in India (4.21 tons/ha) over China (7.11 tons/ha) and the rest of the world 4.76 tons/ha2. In India, rice is grown in all states. West Bengal is the highest rice-producing state (16.72 mt), while Punjab has highest productivity rate (5.10 ton/ha). Uttar Pradesh is the second largest producer of rice, where around 5.60 mha of land is used to grow rice, with a total production of around 15.2 mt4.

Despite of being highest in rice acreage in the world, the annual gross productivity of rice in India is 40% less than China2. The significantly lower rice productivity rate in India is due to regular occurrence of biotic and abiotic stresses in rice cultivation5. Drought or lesser availability of water to paddy crop is a major and challenging abiotic stress to rice crop5. Rice plants require saturated or semi-saturated condition to maintain proper growth and development for better productivity. The crop yield is hampered severely when there is a limitation in the water availability5,6. The moderate drought can potentially reduce rice crop yields by up to 9–10% globally7,8,9.

Trichoderma spp. are a group of filamentous fungi that colonize the rhizosphere in all natural habitates, and may also form symbiotic relationships with plants7,11. They are renowned for their multifunctional capabilities, including suppression of plant pathogens2,12, promotion of plant growth, and modification of plant responses to abiotic stresses13. Several studies have demonstrated the positive impact of Trichoderma spp. in enhancing drought tolerance in rice14,15. Some species of Trichoderma, for example T. harzianum is an efficient mineral solubilizer and pathogen suppressor that can colonize the roots of crop plants and act as an efficient mycoparasite16,17,18. The biopriming with Trichoderma spp. can enhance nutrient absorption and improves water-holding capacity19, leading to gradual improvement in the plant growth and development under water stress conditions20,21,22. Besides the plant breeding for drought tolerance, no other reliable and effective technique has been developed to protect rice from water stress due to low rainfalls. In recent years potential of microorganisms in imparting tolerance against water stress has been evaluated23. Trichoderma spp. have demonstrated promising potential that can be exploited commercially to protect plants under moderate level of droughts24,25. Trichoderma spp. have been reported in reducing the impact of biotic and abiotic stress on plants26,27. The present study was carried out to examine and ascertain the role of Trichoderma spp. in protecting rice plants from the impact of water stress.

Results

Isolation and characterization of Trichoderma species

The DNA of Trichoderma species was sequenced by Macrogen® Incorporation, South Korea using universal primer (ex, 16S V3-V4, ITS, 18S) and Sanger sequencing method. The 16S and 18S rDNA gene sequences showed 99% similarity of the isolates with Trichoderma spp. (Fig. 1). The isolate showed 99 homologies with the ITS region of Trichoderma spp. Thus, the isolates were identified as Trichoderma harzianum, T. viride, T. virens, T. hamatum and T. koningii. The 16S and 18S rDNA gene sequences were deposited in NCBI gene bank under the accession number listed in Table 1. However, to avoid error in the grouping by ITS region, it is recommended to perform sequencing of the conserved region genes such as the TEF gene, Beta-tubulin gene, and Histone H3 gene for species-level identification in fungal isolates, especially, the TEF gene which shows a high level of sequence polymorphism among closely related species, even in comparison to the intron-rich portions of protein-coding genes such as the calmodulin gene, beta-tubulin gene, and histone H3 gene.

Figure 1
figure 1

Dendrogram showing the homology similarity search for Trichoderma harzianum, T. virens, T. viride, T. hamatum and T. koningii isolates.

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Table 1 Trichoderma isolates evaluated in the study.
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Plant growth

The rice cv. Swarna Sub-1 grown under water deficiency condition (75, 50 and 25% water availability of the recommended amount) showed the symptoms of water stress, exhibiting reduced plant growth, with shorter stems and smaller leaves in comparison to plants received the recommended level of irrigation. The symptoms varied with the level of water stress. In general, the rice cv. Swarna Sub-1 was found highly sensitive to drought (water stress). However, the soil application of T. harzianum AMUTHZ82 subsided the water stress symptoms and promoted shoot and root length by 19.7 and 16.8% with 75% watering, 17.4 and 14.1% with 50% watering and 12.3 and 11.2% with 25% watering over respective controls (Tables 2, 3). Next in effectiveness was T. viride AMUTVR73, other isolates did not produce the significant effect. The seed treatment also increased the length of shoot and root i.e., 16.1 and 13.4% (T. harzianum AMUTHZ82) and 10.6 and 8.2% (T. viride AMUTVR73) under 50% drought condition. Next in effectiveness were T. viride AMUTVR72, T. hamatum AMUTHAM_71, T. harzianum AMUTHZ81 and T. viride AMUTVR72 which significantly increased shoot and root length under drought conditions (P ≤ 0.05; Tables 2, 3). However, under normal irrigation (100% watering) highest promotion in the plant growth of rice was recorded, 21.3–23.6% (soil application) and 18–19.2% (seed treatment with T. harzianum AMUTHZ84. Next in effectiveness was T. viride AMUTVR73 (19.8 and 17.5%) over untreated control (Tables 2, 3).

Table 2 Effects of Trichoderma spp. against different water stress levels on the plant growth (shoot length (cm)) of rice cultivar Swarna sub-1.
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Table 3 Effects of Trichoderma spp. against different water stress levels on the plant growth (root length (cm)) of rice cultivar Swarna sub-1.
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Plant biomass production

The biomass production of rice plants grown under water stress conditions (75, 50 and 25% water availability of the recommended amount) was adversely affected. The fresh weight of shoot and root decreased by 21–26%, 36–45% and 52–62% at 75, 50, 25% watering, respectively over 100% watering (control). The soil application of T. harzianum AMUTHZ82 significantly promoted the plant biomass production under 75 and 50% water availability with a maximum increase 20.6 and 17.6% in shoot fresh weight and 18.5 and 15.3% in root fresh weight respectively, over control (P ≤ 0.05). Next in effectiveness was T. viride AMUTVR73, followed by T. harzianum AMUTHZ84 and T. viride AMUTVR71 which also significantly promoted the fresh weight of shoot and root (P ≤ 0.05; Tables 4, 5) over control. Under 25% water availability, T. harzianum AMUTHZ82 scored highest increase in fresh weight of shoot and root (13.8 and 11.3% respectively; Tables 4, 5), followed by T. viride AMUTVR73 (11.4 and 10.4%), and T. hamatum AMUTHAM_71 (8.2 and 8.1%) over control (P ≤ 0.05).

Table 4 Effects of Trichoderma spp. against different water stress levels on the plant biomass (Shoot fresh weight (gm)) of rice cultivar Swarna sub-1.
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Table 5 Effects of Trichoderma spp. against different water stress levels on the plant biomass (root fresh weight (gm)) of rice cultivar Swarna sub-1.
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However, in 100% watered plants, maximum increase in biomass production was recorded with the soil application of T. harzianum AMUTHZ84 (22.6% and 21.4% respectively; Tables 4, 5) followed by seed treatment (22.1 and 20.7%). Next in effectiveness were T. viride AMUTVR73 (20.4 and 20.9%), T. harzianum AMUTHZ83 (19.3 and 19.3%) and T. viride AMUTVR71 (11.8 and 18.4%) over respective control. Other strains viz., T. koningii AMUTKNG_70 and T. virens AMUTVIRENS_71 did not significantly influence the biomass production of rice over respective control (P ≤ 0.05; Tables 4, 5). Overall, the soil application of Trichoderma AMUTHZ82 isolates induced 7–11% greater enhancement in the biomass production of rice in comparison to seed treatment.

Grain yield

The water stress caused significant decline (12–51%) in per plant grain yield production of rice cv. Swarna Sub-1 over normal irrigated control (Table 6). However, soil application of Trichoderma spp. improved the grain yield in both normal and water stress conditions. In normal irrigated condition, maximum increase in production of grain yield was recorded with the treatment of T. harzianum AMUTHZ84 (24.68%), followed by T. viride AMUTVR73 and T. harzianum AMUTHZ82 (22.08 and 20.13%, respectively; Table 6). Under 75 and 50% of water availability, T. harzianum AMUTHZ82 was found superior among all isolates in increasing grain yield by22.79 and 19.63%, respectively, over control (Table 6). Next in effectiveness were T. viride AMUTVR73, T. harzianum AMUTHZ84 and T. hamatum AMUTHM_71 which significantly increased grain yield of rice under drought conditions over control. Under 25% water availability of recommended amount, none of the isolates of Trichoderma were found effective in significantly enhancing the grain yield of rice plants over control. Overall, the soil application of Trichoderma AMUTHZ82 isolates induced 10–22% greater grain yield production of rice in comparison to seed treatment (Table 6).

Table 6 Effects of Trichoderma spp. against different water stress levels on the grain yield (gm/plant) of rice cultivar Swarna sub-1.
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Photosynthesis and transpiration rate

Rice plants grown under drought conditions experienced decline in the photosynthesis rate and transpiration rate (Figs. 2, 3). This resulted in reduced plant growth, and quality biomass production. The photosynthesis rate was found maximum in the rice plants grown under normal condition (100% water availability) with the soil application of T. harzianum AMUTHZ82 (7.12 µmol m−2 s−1), followed by T. viride AMUTVR72, T. harzianum AMUTHZ84, T. harzianum AMUTHZ83 and T. hamatum AMUTHAM_71. Lowest photosynthesis rate was recorded under 25% of water availability in comparison to 100% water availability. Soil application with T. harzianum AMUTHZ82 significantly enhanced the photosynthesis rate of rice plants growing under 75% (37% increase), 50% (19% increase) and 25% water availabilities (9% increase) over respective control. The seed treatment with Trichoderma spp. also minimized the negative effect of water stress on photosynthesis, but less than the soil application.

Figure 2
figure 2

The photosynthesis rate in the leaves of rice plants under different water stress levels. Error bars show standard error.

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Figure 3
figure 3

The transpiration rate in the leaves of rice plants under different water stress levels. Error bars show standard error.

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The transpiration rate was also influenced under low water availability with the treatment of Trichoderma spp. The plants treated with T. harzianum AMUTHZ82 showed minimum transpiration rate (2.36 µmol m−2 s−1), followed by T. viride AMUTVR72, T. hamatum AMUTHAM_71 and T. harzianum AMUTHZ84 under normal irrigated condition. The reduction in transpiration rate was observed lesser under 50% and 25% drought condition with the treatment of Trichoderma spp. over normal irrigated plants. Whereas, maximum transpiration rate was observed in untreated plant receiving recommended level of irrigation (Fig. 2, 3).

Leaf pigments

Total chlorophyll content (Chlorophyll A and B) in the leaves of rice plants under water stress conditions (75, 50 and 25% water availability of recommended amount) was recorded with a 20, 37 and 49% decline, respectively over control (Fig. 4). Under regular irrigation (100% watering), soil application of T. harzianum AMUTHZ84 resulted a maximum increase (21.3%) in chlorophyll content, followed by T. viride AMUTVR73 (17%) in the leaves of rice plants. Whereas, under 75 and 50% watering regime, T. harzianum AMUTHZ82 increased the total chlorophyll content (16.6 and 13.1%) over respective control (Figs. 4, 5). T. harzianum AMUTHZ83 and T. viride AMUTVR73 were found next in increasing chlorophyll content under 25% and 50% water stress conditions. The plants treated through seed treatment of Trichoderma isolates under water stress had significantly greater content of leaf pigment, but less than soil application. The plants receiving 25% water of the recommended amount recorded with 2–7% increase in total chlorophyll content with the soil application or seed treatment with Trichoderma isolates (Figs. 4, 5).

Figure 4
figure 4

Effects of soil application of Trichoderma isolates on the total chlorophyll (chlorophyll A and B) content in the leaves of rice plants under different water stress levels. Error bars show standard error. Where CH = Chlorophyll.

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Figure 5
figure 5

Effects of seed treatment of Trichoderma isolates on the total chlorophyll (chlorophyll A and B) content in the leaves of rice plants under different water stress levels. Error bars show standard error. Where CH = Chlorophyll.

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Discussion

The rice cv. Swarna Sub-1 was found sensitive to drought (water stress), and exhibited significant reduction in the plant growth, biomass production, leaf pigments, photosynthesis etc. grown under water stress conditions (75, 50 and 25% water of the recommended amount) in comparison to plants received normal irrigation (100% water availability of the recommended level). Adequate supply of water to a plant is essential for carrying out various physiological, biological and biochemical activities of the plant28,29. A decrease in the water supply influences the physiological activities especially the photosynthesis30,31, and subsequently the plant growth and development32, which directly affect the biomass production33,34 as observed in the present study.

The soil application of Trichoderma spp. especially T. harzianum AMUTHZ82 and T. viride AMUTVR73 subsided the negative effect of water stress on rice plants, and promoted the shoot and root growth, and biomass production of rice cv. Swarna Sub-1. Trichoderma spp. are universally occurring beneficial microbes in varied kind of soils13, and are generally known to suppress plant pathogenic fungi17,28, by parasitizing them or by producing metabolites, toxins, enzymes, etc. toxic to pathogens28,35. Trichoderma spp. also solubilize minerals present in soil, and produce hormones36,37, which may ultimately lead to plant growth promotion14,38,39. Some strains of Trichoderma spp. form endophytic relationship with roots, and develop symbiotic association with plants13,40. With such association, plants are able to efficiently use the water and nutrients in soil41, and are able to survive under various stress conditions such as nutrition deficiency, water stress, etc.42,43.

Khan et al.44 conducted a study in which the application of T. harzianum significantly enhanced plant height, root length, and shoot biomass of rice cv. Swarna Sub-1, under water-deficient conditions. Similarly, Haque et al.45 demonstrated that T. harzianum treated rice plants exhibited improved water-use efficiency, enhanced photosynthetic activity, and increased grain yield compared to non-treated plants during drought stress. Trichoderma spp. enhance plant health and stimulate the synthesis of plant growth-promoting substances such as auxins and cytokinins28, which play crucial roles in root development, nutrient uptake, and overall plant growth46,47. Additionally, T. harzianum triggers the expression of stress-responsive genes in rice, activating defence mechanisms and antioxidant systems that counteract the detrimental effects of water stress. The present study has demonstrated that soil application of T. harzianum AMUTHZ82 or T. viride AMUTVR73 may efficiently subside the impact of water stress at least up to 50% water availability levels and may enhance the plant growth and biomass production by 17–21%.

Conclusion

The rice cv. Swarna Sub-1 was found sensitive to drought (water stress), and exhibited significant reductions in the plant growth, biomass production, leaf pigments, photosynthesis etc. grown under water stress conditions (75, 50 and 25% water of the recommended amount) in comparison to plants received normal irrigation (100% water availability of the recommended level). The soil application of Trichoderma spp. especially T. harzianum AMUTHZ82 and T. viride AMUTVR73 subsided the negative effect of water stress on rice plants, and promoted the shoot and root growth and biomass production of rice. The present study has demonstrated that soil application of T. harzianum AMUTHZ82 or T. viride AMUTVR73 may efficiently reduce the impact of water stress at least up to 50% water availability levels and may enhance the plant growth and biomass production by 17–21%.

Materials and methods

Seeds of rice cv. Swarna Sub-1 were procured from Regional Centre, International Rice Research Institute, New Delhi, India.

Isolation and characterization of Trichoderma species

The local isolates of different Trichoderma spp. were isolated from rice fields using the serial dilution method on selective medium21. The corresponding GPS locations (latitude and longitude) of sampling sites were given in Table 7. The plates after inoculation were kept in an incubator at 25 ± 2 °C for 10 days. However, the fungus almost fully colonized the medium in 7 days. After incubation the Trichoderma colonies were pure cultured using the same culture medium and conditions. Thereafter, morphological characters of the Trichoderma isolates were studied to identify the species, followed by molecular characterization to confirm the identity48. The DNA of Trichoderma species was sequenced by Macrogen® Incorporation, South Korea, and the homology search was carried out using BLAST on the 18s rRNA-ITS region gene sequences to find and obtain the closest neighbour sequences from the NCBI database49. The MEGA-X software was used to access the Clustal-W alignment tool, and used to align all of the sequences. Simple comparison with the database of known species/isolates of Trichoderma harzianum, T. viride, T. virens, T. hamatum and T. konigii allowed the identification of the species. The UPGMA algorithm included in MEGA-X was used to create the phylogenetic tree (Fig. 1). The sequences have been deposited to the NCBI database and GenBank accession numbers listed in Table 1.

Table 7 List of the sampling sites for isolation of Trichoderma species along with corresponding GPS locations (latitude and longitude) in Aligarh and adjoining districts of Uttar Pradesh.
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Mass multiplication of Trichoderma spp.

Eleven isolates of Trichoderma spp. as enlisted in Table 1 were mass cultured on sorghum grains in conical flasks. The seeds were soaked for 12 h. in distilled water containing sucrose (5%) and chloramphenicol (0.0003%). The seeds were kept in 500 ml capacity conical flasks and sterilized at 15 kg/cm2 steam pressure at 121 °C for 20 min. Next day the flasks were inoculated with the fungus, and incubated in an incubator for 10 days at 25 ± 2 °C. The sorghum seeds colonized by Trichoderma spp. were crushed with distilled water in an electrical grinder. The fungal suspension containing 10 g Trichoderma colonized seeds was applied to the soil (1 kg) in each pot a day before the planting of rice seedlings. For seed treatment, paste of sorghum seeds was applied to the rice seeds (@10 g/kg seeds), 3 h before their sowing in the nursery bed.

Treatments and plant cultures

The seedlings of rice cultivar Swarna Sub-1 were transplanted in the two sets of pots (1 seedling/pot) filled with sterilized soil and FYM (4:1). In one set of pots Trichoderma isolates were applied as soil application (@10 g/kg soil), whereas, in other set rice seedlings raised from Trichoderma treated seeds were planted. Four watering treatments viz., 100, 75, 50 and 25% of recommended irrigation were maintained for each isolate of Trichoderma spp. with five replicate/pots. Under 100% watering, the pots were watered regularly on alternate days. For 75% watering pots were watered every 4th day, and for 50% and 25% watering every 6th and 8th day, respectively. Similar treatments for drought have also been maintained in other researches50. Further, the soil moisture percentage is an important matrix for determining the drought level, but it is very difficult to use it as a treatment because of it changes with the progress of time. Since, there were 11 Trichoderma isolates, a total of 480 Pots were maintained, which were arranged in a complete randomized block design on the roof top receiving uniform sunlight. At maturity, 3 months after transplanting, the plants were harvested to measure plant length (shoot and root) and fresh weight per plant. The treatments for each Trichoderma isolate for soil application or seed treatment were as follows.

T1 = Plant + 100% watering (control)

T2 = Plant + 75% watering

T3 = Plant + 50% watering

T4 = Plant + 25% watering

T5 = Plant + 100% watering + Trichoderma isolate

T6 = Plant + 75% watering + Trichoderma isolate

T7 = Plant + 50% watering + Trichoderma isolate

T8 = Plant + 25% watering + Trichoderma isolate

Statistical analysis

The data obtained from the five replicates were used to calculate the means. However, replicate data were used to analyze least significant difference (LSD at P ≤ 0.05) with the help of Agricolae Package of R software51. Tukey HSD test was employed to mark significantly different treatments in the Tables. Two factor ANOVA was used to evaluate the data on plant growth variables.

Ethical statements

Both the authors have approved the content and authorship of the submitted manuscript, and all prevailing local, national, and international regulations and conventions, and scientific and ethical practices have been respected.

Plant compliance statement

The studies in this research article (plant field studies and experimental research, as well as the collection of plant material) were undertaken following laws and regulations pertinent to the institution.