AM fungal Spores and their molecular characterization
Rhizophagus intraradices and Funneliformis mosseae identified based on morphological characterization, previously by the authors from millet ecosystem. They showed superior root colonization potential and secreted efficient root metabolites when inoculated in maize (Mythili and Ramalakshmi 2022). These two spores were selected for the present study. The commercial AM fungal inoculum (Rhizophagus intraradices) and pink pigmented facultative methylotrophic bacteria (PPFM) were obtained from the Department of Agricultural Microbiology, Tamil Nadu Agricultural University, and Coimbatore for comparison studies.
The spores were also identified by molecular methods by extracting spore DNA and PCR amplification of ITS region carried out as per the method described by Manian et al., 2001. Briefly, the AM fungal spores were thoroughly washed in sterile distilled water and centrifuged at 11,000 g for one minute. Then the samples were frozen in liquid nitrogen and crushed using micro pestle. The DNA was extracted using lysis buffer and eluted in 50 µl of 1mMTrisHCL as per the procedure given by. Primer pairs viz., ITS1 and ITS4 were used for identification of AM fungi. Polymerase Chain Reaction (PCR) was carried out in Bio-Rad Thermal cycler T100. The 50 µl PCR mixture contained 20 ng of DNA, 1µM of each dNTP, 1 µM each primer, 1X buffer and 1 U of Taq polymerase. PCR conditions were as follows; initial denaturation for 2 min at 95°C; 35 cycles of 1 min at 94°C, 2 min at 55°C and 2 min at 72°C and final extension of 5 min at 72°C. The PCR product was purified using purification kit as per the method described by the manufacturers. The purified PCR product was sent for automated sequencing (Bio serve, Hyderabad). Sequencing was carried out using ITS1 primer and then the homology analysis of generated sequence was performed by BLAST (Basic Local Alignment Search Tool) of NCBI (http://www.ncbi.nlm.nih.gov/Blast.cgi). The sequencing and homology analysis was repeated three times.
Mass multiplication of AM fungi
The AM fungal inoculum was mass multiplied with maize as host and vermiculite as substrate for 60 days. The inoculum along with root bits was sieved through different sieves viz., 75, 180, 250 and 500micron. The presence of spores in sieved inoculum was estimated along with control (without sieved inoculum) by wet sieving and decantation method as outlined by (Gerdemann and Nicolson 1963).
Standardization of polymers for AM fungi seed coating
An experiment was carried out to evaluate the efficiency of AM fungal spores as seed inoculants using Roll towel method (Phaneendranath 1980). The purpose of this study is to reduce the inoculum load and improve the infection potential of AM fungi. In order to do this several polymers were tested as coating agents. Finger millet seeds (variety: Paiyur2) were surface sterilized with 70% ethanol for 1 minute followed by 5% sodium hypochlorite for 5 minutes and washed several times with sterile water. The surface sterilized seeds were coated with AM fungal inoculum at the rate of 10g per kg of seeds using different polymers at the rate of 0.1 per cent (Carboxy Methyl Cellulose (CMC), Polyvinylpyrrolidone (PVP), Biosticker, rice gruel). The CMC and PVP were purchased from Sigma-Aldrich® and the biosticker was obtained from Tropical Agro Systems Pvt (Ltd.), Coimbatore. The following parameters were recorded after 15 days of the experiment viz., germination percentage, vigour index, shoot length and root length as per method described by Hoagland and Arnon, 1950. Vigour index was expressed in number and was calculated using the formula, Vigour index = Germination percentage x (Root length + Shoot length). AM colonization percentage in roots was assessed according to the method of Phillips and Hayman, 1970.
SEM analysis
The root samples were carefully removed from the treatment and control conditions after 10 days for arbuscules and after 30 days for vesicles and then were cut into small pieces of about 0.5 cm and rinsed with phosphate buffer saline. To check AM colonization, several hand-cut transverse sections were taken. Then the root samples were placed in a tiny plastic tube containing 2.5% glutaraldehyde (v/v) for fixation at room temperature for 1 hour. The root samples were again fixed in 1% osmium tetroxide at 4°C for 40 minutes and then dehydrated using 10% ethanol for 5 minutes. The samples were sputter coated with gold nanoparticles to improve the visibility of AM fungal colonization under SEM. The samples were then metal coated to improve resolution and to prevent the structural damage. The SC7620 Mini Sputter Coater/Glow Discharge System (Quorum Technologies Ltd, United Kingdom) was used to sputter coat the samples. The sputter coated root samples were examined under SEM (Quanta 250, FEI company, United States) and the AM colonization in finger millet seedlings were observed and documented.
Experimental design and description
A pot experiment was performed during two Rabi season (2021 and 2022) at green house, Department of Agricultural Microbiology, to evaluate the efficiency of new AM fungi viz., Rhizophagus irregularis and Funneliformis sp in finger millet crop grown under 50% deficit irrigation. Finger millet variety “ATL 1” was used for the experiment. Finger millet seeds were raised in trays containing mixture of soil and sand (1:1). The soil was collected from the Department of Millets, TNAU, Coimbatore. The physico-chemical chemical properties of soil were pH – 8.3 and EC – 0.35 dS m− 1, OC – 0.72%, N − 228.96 kg ha− 1, P – 39.12 kg ha− 1 and K − 289.93kg ha− 1. The 5 kg soil was filled in a pot measuring inner diameter, length and volume of 15 cm, 30 cm and 5.3 liter. The water holding capacity of soil was determined. 3 healthy seedlings were transplanted to each pot. Plants were grown under drought stressed condition. All the plants were watered regularly with tap water till it reach 50% of its water holding capacity (WHC). The 50% WHC was maintained based on gravimetric method on alternate days. The experiment was completely randomized with ten treatments and three replications per treatment.
Treatment details are T1 – Uninoculated control;T2 – Rhizophagus irregularis (soil application);T3 – Rhizophagus irregularis (seed inoculation);T4 – Funneliformis sp (soil application);T5 – Funneliformis sp (seed inoculation);T6 – Rhizophagus irregularis + Funneliformis sp (soil application);T7 – Rhizophagus irregularis + Funneliformis sp (seed inoculation);T8 – Commercial AM inoculum (soil application); T9 – Commercial AM inoculum (seed inoculation); T10–1% PPFM spray
Seed coating of finger millet seeds were done using 500 micron sieved AM fungal inoculum (10g/kg of seed) coated with 0.8% carboxymethyl cellulose and 10g per plant was inoculated for soil application. PPFM was used as a positive control for drought mitigation. The growth, yield, soil biochemical and antioxidant parameters were taken at 30 and 45days after sowing under 50% deficit irrigation.
Plant growth and yield parameters
Three randomly selected seedlings from each treatment were used to measure the shoot length, root length at 30 and 45 days and expressed in centimeters. The yield parameters were recorded at the time of harvest and expressed as per cent increase over control.
Soil bio-chemical parameters
Root colonization potential of AM fungi in roots was assessed (Phillips and Hayman, 1970). The glomalin content in soil samples was measured using the method of Wright and Upadhyaya, 1998. The blue colour developed was read in a microplate reader (Spectramax i3X) using bovine serum albumin as standard at 595 nm and expressed as mg g− 1 of soil.
The acid and alkaline phosphatase activity (pH – 6.5 for acid and pH – 11 for alkaline) was determined using the method of Tabatabai and Bremner, (1969). The intensity of yellow colour developed was measured at 420 nm in spectrophotometer (Systronics ® Spectrophotometer 166). The phosphatase activity was expressed in µg of p-nitrophenol released per g of soil per hour.
Antioxidant properties
The proline content in the finger millet leaf samples were determined by the method of Bates et al., (1973). The toluene layer was removed and absorbance was measured in spectrophotometer (Systronics ® Spectrophotometer 166) at 520 nm.
The catalase activity was estimated using the method (Chaparro-Giraldo et al. 2000) and absorbance was recorded at 240 nm using spectrophotometer (Systronics ® Spectrophotometer 166). The enzyme activity was expressed in g− 1 min− 1 tissue.
The protein concentration in finger millet leaf samples was determined by the method(Lowry et al. 1951). The colour intensity was measured in spectrophotometer (Systronics ® Spectrophotometer 166) at 660 nm using Bovine serum albumin as standard.
Metabolic profiling of finger millet roots
The 40 days old finger millet seedlings were used for profiling of metabolomes as described by Meena et al., 2017. One seedling from each treatment was removed and the roots were thoroughly washed with water. Then the roots were placed in a beaker containing 50 ml milli Q water. The solution was extracted with equal quantity of ethyl acetate and the ethyl acetate extract was dried and then processed for GC-MS analysis (PerkinElmer®, USA).
Data analysis
The Phylogenetic analysis of Rhizophagus irregularis and Funneliformis sp was performed using distance tree results of NCBI blast (https://blast.ncbi.nlm.nih.gov/Blast.cgi). Pooled analysis was performed for two season experiments. The results of all experiments were statistically analyzed as per the method of Gomez and Gomez (1984). The mean values with standard errors were performed in triplicates. The results of all Enrichment analysis, Pathway analysis and Hierarchical Clustering heatmaps of AM fungi mediated root metabolites was performed using Metaboanalyst 5.0 (Pang et al. 2021).