*2.2. Experimental Site and Pot Experiment*

The experiment was conducted during the 2020–2021 *Rabi* season (the Indian cropping season starting from the onset of winter from October-November until spring in March– April) in a controlled net house condition at Microbiology, the ICAR-National Rice Research Institute (NRRI), Cuttack, Odisha (latitude—20◦25- N, longitude—85◦55- E with an altitude of 24 m above mean sea level). The pot (5 kg) experiment was conducted with five different species of AMF and six rice varieties with three replications. The treatment details are as follows, T0: Control, T1: *Funneliformis* sp., T2: *Rhizophagus* sp., T3: *Glomus* sp., T4: *Acaulospora* sp. and T5: *Claroideoglomus* sp. In this experiment, four aerobic rice varieties *viz*. V1: CR Dhan 201, V2: CR Dhan 204, V3: CR Dhan 205, V4: CR Dhan 207 (CR Dhan 201, 204, 205, and 207 developed by ICAR-NRRI, Cuttack), and two check varieties *viz*. V5: IR 36 (P-susceptible) and V6: Kasalath IC459373 (P-tolerant) were used, and were collected from the Crop Improvement Division, ICAR-NRRI, Cuttack, India. After germination, three plants per pot were maintained. Soil (completely homogenized and transported to the laboratory in a cool pack) and all the plant samples from each pot were collected from all treatment after 60 days in order to estimate the AMF colonization, growth parameters (root length, shoot length, leaf area, chlorophyll, fresh and dry biomass), P uptake, soil chemical, microbial and enzymatic activities analysis [30].

#### *2.3. Assessment of AMF Colonization and Spore Count*

The method developed by Phillip and Hayman [31] was used to evaluate the rice root colonization of AMF [32]. Freshly collected root samples were gently washed to remove soil that was attached to the root surfaces, submerged in 10% potassium hydroxide (KOH) solution, and autoclaved for 15 min at 121 ◦C. The KOH solution was decanted, and the treated roots were rinsed with tap water three times until no brown colour appeared in the rinsed water. The treated root samples were further immersed in 2% hydrochloric acid (HCl) solution for 5 min. Without being rinsed with water, HCl was decanted, and the root samples were stained with 0.05% trypan blue (HiMedia, Maharashtra, India) in lacto-glycerol (400 mL lactic acid + 400 mL glycerol + 100 mL water) and autoclaved for 15 min at 121 ◦C. After autoclaving, the stained solution was decanted, and the roots were de-stained with lacto-glycerol solution to remove the excess stains and used for microscopic observations. The slide was prepared by keeping 10 segments of the stained root on a clean glass slide and observed under a compound microscope (Zeiss Stemi 508, Oberkochen, Germany). The method described by McGonigle et al. [33] was used to calculate the percentage of root colonization.

AMF root colonization was calculated using the formula:

*% of colonization* = *no. of root segments colonized* ÷ *total no. of root segments* × 100

#### *2.4. Phosphorus Estimation in Plant Sample*

Collected plant samples were dried in a hot air oven maintained at 60 ◦C for up to 5 days in order to attain a constant weight. The determination of P concentration in the plant sample was carried out using the vanadomolybdophosphoric acid method with a spectrophotometer [30,34]. A quantity of 1 gm of the dried plant sample and 10 mL of the concentrate HNO3 were added and kept overnight, following which 10 mL of triacid (HNO3, H2SO4, HCLO4 in a ratio of 9:4:1), was added and mixed properly. The mixture was kept in a hot plate at 100 ◦C for 1 h under a temperature rise up to 200 ◦C until the content reduced to 2–3 mL and turned colourless. The content was cooled and 10 mL of diluted HCL was added and filtered through Whatman No. 42. The filtrate volume was made up to 100 mL with distilled water. A quantity of 5 mL of the digested sample was taken and 10 mL of vanadomolybdate reagent was added (Merck, Darmstadt, Germany) and kept for 30 min. The absorption of the sample was measured at 420 nm with a spectrophotometer (Analytikjena specord-200, Jena, Germany). A standard curve was prepared with a phosphate solution (0.2195 gm of KH2PO4 in 500 mL of distilled water + 25 mL of 7N H2SO4 and made up to 1 L) and the P content of the plant sample was calculated from the standard curve.

#### *2.5. Estimation of Soil Chemical, Enzymatic and Microbial Properties*

The activity of the acid (AcP) and alkaline (AkP) phosphatase of soil samples was estimated by the method of Tabatabai and Bremner [35], using p-nitrophenyl as a substrate and expressed in l g of p-nitrophenyl phosphate (*p*NP) released per gram of soil per hour. Soil fluorescein diacetate activity (FDA) measurement was carried out by using Scherer and Ross [36] as modified by Adam and Duncan [37]. The concentration of fluorescein released during the assay was calculated using the calibrating graph produced from the 0–5 μg fluorescein mL−<sup>1</sup> standard and expressed as μg fluorescein h−1g−<sup>1</sup> soil [27]. Dehydrogenase activity (DHA) was estimated by the method of Casida et al. [38]), using triphenyltetrazolium chloride (TTC) as a substrate. Microbial biomass carbon (MBC) was determined using the chloroform fumigation extraction (CFE) method [39].

#### *2.6. Statistical Analysis*

The R version 4.2.2 [40] was used for statistical computing. For the identification of important variables related to AMF colonization in plants, a stepwise regression model was constructed using the "stepAIC" function available in the MASS package [41]. The Pearson correlation was constructed using the "ggpairs" function available in the GGally package [42].

#### **3. Results and Discussion**

Rice crops are very sensitive to water stress and reduction in water inputs with a consequent decline in yield [43]. Approximately 75% of the rice is produced by a conventional flooding method, and 3000–5000 L of water is needed to produce 1 kg of grains [4,44]. Researchers have developed several technologies to reduce water inputs in rice such as alternate wetting and drying, raised bed rice cultivation, saturated soil culture, a system of rice intensification, ground cover systems, and raised bed systems [45]. Some of the modern technologies additionally require puddling and ponded water during crop growth. In rice cultivation, the aerobic rice has been introduced to minimize the use of water, which is one of the promising water-saving technologies in rice production [46,47]. Aerobic rice reduces water use by 27–51% by limiting water loss due to seepage, percolation, and evaporation and increases water productivity by 32–88% [48]. It has been well documented that microorganisms enhance plant growth under abiotic stress [49].

#### *3.1. Seed Germination of Rice Varieties*

The seed germination percentages of four different aerobic rice varieties (CR Dhan 201, CR Dhan 204, CR Dhan 205, and CR Dhan 207), as well as another P-susceptible variety (IR 36) and P-tolerant variety (Kasalath IC459373) are given in Figure 2. CR Dhan 204 and 207 rice varieties showed the highest germination percentages. However, all the rice types had germination rates of >90%.

**Figure 2.** Percentage of seed germination of six rice varieties.

#### *3.2. AMF Root Colonization in Different Aerobic Rice Varieties*

AMF symbiosis increases nutrient and water uptake in plants by external hyphae, regulation of stomatal conductance and the increased activity of antioxidant enzymes. Under aerobic conditions, rice plants readily form mycorrhizal associations as compared to submerged conditions where the anoxic environment limits the mycorrhizal infection process [50,51]. Rice can also be grown with alternate irrigation to reduce the water input and to create aerobic conditions for better AMF fungi colonization in rice roots. Therefore, an investigation was undertaken to understand the benefits of AMF association for rice plant growth and development under aerobic conditions. Narwal et al. [44] found a 20% increase in the plant biomass and 58% higher colonization of *Glomus intraradices* and *G. mosseae* (currently *Funneliformis mosseae*) in upland rice varieties (Pyari, Satyabhama, CR Dhan 205 and CR Dhan 202) compared to lowland rice varieties (Pusa Basmati (PB) 1509, PB 1121, Pusa Sugandha 5 and PB 1612) in pot experiments with sterile soil. The AM plants enhanced the activities of glutamine synthetase and nitrate reductase; the rice genotypes with higher nitrate reductase and glutamine synthetase (Pyari and Satyabhama) also exhibited more (20%) biomass production and plant N content by 36% [44]. In our study, the results of the different AMF-inoculated rice varieties and its root colonization, presented in Figure 3, indicated that *Funneliformis* sp., *Rhizophagus* sp. and *Glomus* sp. showed higher colonization in CR Dhan 207 (91.75, 91.72 and 87.97%, respectively) and CR Dhan 204 (85.43, 83.19, and 75.37%, respectively), while the other genera of AMF recorded a root colonization in the range of 54.38–74.98%.

#### *3.3. Effect of AMF Inoculation on Physiological and Agronomic Properties*

Inoculation of AMF played an important role in the improvement of the biomass chlorophyll contents and physiological and agronomic parameters of the plant. It is widely believed that the inoculation of AMF provides the highest efficiency to host plants for plant growth. As shown in Figure 4, our results demonstrated that AMF inoculation in different rice varieties significantly increased the agronomic parameters, including root length (cm), shoot length (cm), leaf area (m2), chlorophyll (SPAD), fresh biomass (gm), and dry biomass (gm) compared to the control. The highest shoot and root lengths were found in IR36 (53.40 cm) and CR Dhan 207 (23.973 cm) with the treatment of *Rhizophagus* sp. (Figure 4a,b). In the rice variety CR Dhan 207 (34.127 m2), treatment with *Glomus* sp.

showed the best improvements for the leaf area (Figure 4c). Chlorophyll (SPAD) levels were highest in CR Dhan 204 (32.73) with *Rhizophagus* sp.; CR Dhan 204 (32.53) with *Claroideoglomus* sp.; Kasalath IC459373 (32.43) and CR Dhan 207 (32.40) with *Acaulospora* sp. treatment (Figure 4d). The *Funneliformis* sp. treated with CR Dhan 207 (4.32 gm) and *Rhizophagus* sp. treated with Kasalath IC459373 (2.466 gm) had the maximum performance in terms of plant fresh biomass and dried biomass, respectively (Figure 4e,f). However, the plant growth parameters viz. root length, leaf area, chlorophyll and plant biomass showed themselves to be significantly higher in CR Dhan 207 and CR Dhan 204 inoculated with *Rhizophagus* sp., *Glomus* sp., *Funneliformis* sp., and *Acaulospora* sp.

**Figure 3.** Percentage of AMF colonization (AMFc) in different rice varieties. Abbreviation: percentage of AMF colonization (AMFc).
