Studies on the co-inoculation effect of phosphobacteria and arbuscular mycorrhizae fungi on the growth and yield of finger millet

Introduction

Finger millet or ragi (Eleusine coracana L.) is a nutrient-dense small grain crop, and it is mostly cultivated in arid and semiarid regions of Asia and Africa. It is a short, profusely tillering plant with finger-like spikes that produce small reddish-brown seeds. Ragi grains are highly rich in calcium, protein, essential amino acids (e.g., methionine, cystine, tryptophan), complex carbohydrates, iron, phosphorus, potassium, B-complex vitamins, and other minerals. They also contain high levels of dietary fibre, polyphenols, and antioxidants. India is the largest producer of finger millet in the world, cultivated over an area of 1.19 million ha, with a total production of 1.98 million tonnes and a productivity of 16.61 q/ha. Karnataka is the leading producer, accounting for 56.21% of the total cultivated area and 59.52% of the country's production. Some of the major finger millet-producing states include Tamil Nadu (9.94% and 18.27%), Uttarakhand (9.40% and 7.76), and Maharashtra (10.56% and 7.16) (Sakamma et al., 2017) [7] . In India, farmers use a significant amount of chemical fertilizers for crop production, especially for vegetables; this pollutes the soil, which leads to soil pollution and groundwater contamination, ultimately causing health hazards (Thakur and Paika, 2024). To avoid environmental pollution, especially soil pollution, most scientists recommend using biofertilizers alongside inorganic fertilizers in an appropriate manner to maintain soil health and productivity (Singh et al., 2024) [9] . The essential nutrients are required for plant growth. Phosphorus is second to nitrogen, as it contributes 0.2% of plant dry weight. Phosphatesolubilizing microorganisms (PSMs) can convert soil P into a form that plants can absorb and utilize, and the application of PSMs is currently an important measure for increasing the available P content in soil (Yadav et al., 2017; Yadav, 2020) [16,17] . Arbuscular mycorrhiza (AM) forms a symbiotic relationship with 83% of dicots and 79% of monocots; only a few major cultures are unable to accept mycorrhizal symbiosis (Slimani et al., 2024) [11] . Mycorrhizal fungi are also useful for improving soil structure, water-holding capacity, biological control of root pathogens, and resistance to biotic and abiotic stresses, helping to improve plant growth and increase the production of fruits (Delaeter et al., 2024) [2] . They also improve the absorption of minor elements, produce plant hormones, and increase the activity of nitrogen-fixing organisms in the root zone (Selim S. et al., 2024) [8] . AM fungi and Phosphobacterial inoculation enhance plant growth and development by producing growth-promoting substances and play an important role in P solubilization and mobilization, and are involved in the uptake of macro and micronutrients and protecting plants from stresses and pathogens.

Materials and Methods

Location of the study

All laboratory experiments were conducted in the Department of Agricultural Microbiology, Faculty of Agriculture, Annamalai University, Annamalai Nagar, Tamil Nadu. Field experiments were conducted in Pochampalli village, Dharmapuri district, Tamil Nadu.

Cultivar

Ragi seeds (Paiyur 2) were obtained from the Vegetable Research Station, Palur, Panruti.

Quantification of Phosphobacteria

Ragi rhizosphere soil samples were collected from different locations in the Dharmapuri district for the isolation of phosphobacteria. Pikovskaya's medium was used to isolate phosphobacteria. Phosphobacteria were enumerated from the rhizosphere soils of different ragi-grown fields using the serial dilution plate technique (Sperber et al., 1958) [12] . A 10 -4 dilution was achieved by diluting the soil samples. One mL of samples from the final dilution was plated, and the plates were incubated for up to two weeks at 28±2 °C. Bacterial colonies showing a clear zone were identified. Then the efficient isolates were selected and showed the highest phosphate solubilization efficiency.

Collection of biofertilizer

The AM Fungi (Glomus fasiculatum) biofertilizer is collected from the Department of Microbiology, TNAU, Coimbatore.

Molecular characterization of the efficient bacterial strain

Molecular identification was carried out by 16s rRNA gene sequencing. Amplification of 16s rRNA gene was carried out for the samples using universal primers 27F: AGAGTTTGATCMTGGCTCAG and 1492R: TACGGYTACCTTGTTACGACTT were used for amplification. Expected band was amplified in all the samples PCR-generated amplicon was confirmed and purified using GeneJET PCR purification kit (Thermo Scientific, EU-Lithuania) to remove the primer dimer and other carryover contaminations. The quality of the product was assessed using 1.8% agarose gel along with 100bp DNA ladder assize standard and the product was found to be good for sequencing. PCR products were purified and prepared for Cycle sequencing using the Big Dye® Terminator 3.1 sequence kit (Applied Biosystems, Foster City, California, USA). After cycle sequencing, the products were purified using Ethanol-EDTA purification protocol to remove the un-incorporated dntp's, ddntp's and primer dimer. The purified cycle sequencing products were dissolved in 12µl Hi-Di formammide and the samples were subjected for denaturation at 95ºC for 5mins. Denatured products were subjected for sequencing in forward and reverse direction using Genetic Analyzer 3500 (Life Technologies Corporation, Applied Biosystems® California 94404, USA) as per manufacture's instruction. Sequences were aligned and edited using Mega software version 11 (Tamura et al., 2020) to confirm the species.

Experimental design

The experimental design was laid out in Randomized Block Design (RBD). The treatment details are given below: T 1: Control T 2: 50% P (RDF) T 3: 75% P (RDF) T 4: 100% P (RDF) T 5: 75% P + Bacillus megaterium T 6: 75% P + Glomus fasiculatum T 7: 75% P + Bacillus megaterium + Glomus fasiculatum T 8: 50% P + Bacillus megaterium T 9: 50% P + Glomus fasiculatum T 10: 50% P + Bacillus megaterium + Glomus fasiculatum

Biometric observation

The plant growth parameters such as plant height, dry matter production (kg ha -1 ), number of tillers per plant, number of ear heads hill -1 , 1000 grain weight (g), finger length (cm), and grain yield (t ha -1 ) of ragi at different graded levels of phosphorus were recorded.

Results

The field experiments were conducted to investigate the effect of co-inoculation of Bacillus megaterium and Glomus fasiculatum at different graded levels of P 2 O 5 (50%, 75%, 100%) in ragi var. Paiyur 2.

The effect of co-inoculation of Bacillus megaterium and Glomus fasiculatum on the plant height of ragi at different graded levels of phosphorus.

In the experimental study, among all the treatments, the plant height of ragi was recorded as maximum at harvest for treatment T 7 (107.5 cm), followed by treatment T 4 (102.8 cm). The minimum plant height was recorded in the treatment T 1 control (88.2 cm). The data pertaining to plant height were presented in Table 1.

The effect of co-inoculation of Bacillus megaterium

and Glomus fasiculatum on the dry matter production of ragi at different graded levels of phosphorus.

In the experimental study, among all the treatments, the dry matter production of ragi was recorded as maximum at harvest for the treatment T 7 (7008 kg ha⁻¹) followed by T 4 (6642 kg ha⁻¹). The minimum dry matter production was recorded in the treatment T 1 control (4679 kg ha⁻¹). The data were presented in Table 2.

The effect of co-inoculation of Bacillus megaterium and Glomus fasiculatum on the number of tillers of ragi at different graded levels of phosphorus.

In the experimental study, among all the treatments, the maximum number of tillers was recorded at harvest in the treatment T 7 (7.0), followed by T 4 (6.4). The minimum number of tillers was recorded in the treatment T 1 control (4.4). The data were presented in Table 3.

The effect of co-inoculation of Bacillus megaterium and Glomus fasiculatum on the number of ear heads hill-1 of ragi at different graded levels of phosphorus.

In the experimental study, among all the treatments, the maximum number of ear heads hill -1 was recorded at maximum at harvest in the treatment T 7 (9.00), followed by T 4 (8.78). The minimum number of ear heads hill-1 was recorded in the treatment T 1 control (7.46). The data were presented in Table 4.

The effect of co-inoculation of Bacillus megaterium

and Glomus fasiculatum on the 1000-grain weight (g) of ragi at different graded levels of phosphorus.

In the experimental study, among all the treatments, the maximum 1000-grain weight was recorded at harvest in the treatment T 7 (3.13 g), followed by T 4 (3.10 g). The minimum 1000 grain weight was recorded in the treatment T 1 control (2.42 g). The data were presented in Table 5. The result clearly established that co-inoculation of Bacillus megaterium and Glomus fasiculatum significantly increased the yield of ragi by increasing the 1000-grain weight.

Table 5: The effect of co-inoculation of Bacillus megaterium and Glomus fasiculatum on the 1000-grain weight (g) of ragi at different levels of phosphorus.

S. No.

Treatments 1000 grain weight (g) 1.

T5: 75% P + Bacillus megaterium 3.07 6.

T6: 75% P + Glomus fasiculatum 3.04 7.

T7: 75% P + Bacillus megaterium + Glomus fasiculatum 3.13 8.

T8: 50%P + Bacillus megaterium 2.83 9.

T9: 50% P + Glomus fasiculatum 2.71 10.

T10: 50% P + Bacillus megaterium + Glomus fasiculatum 2.91 S.Ed 0.08 CD(p = 0.05) 0.17

The effect of co-inoculation of Bacillus megaterium

and Glomus fasiculatum on the finger length (cm) of ragi at different graded levels of phosphorus. Finger length of ragi in response to inoculation of Bacillus megaterium and Glomus fasiculatum was observed at harvest, and the results are presented in Table 6. An increase in finger length was observed both in inoculated and uninoculated controls. The co-inoculated ragi at all the phosphorus levels recorded higher values than the single inoculation. The maximum finger length was recorded in the treatment T 7 (9.8 cm), followed by T 4 (9.4 cm), and the minimum finger length was recorded in the treatment T 1 control (6.9 cm). In the experimental study, among all the treatments, the grain yield was recorded as maximum at harvest for treatment T 7 (3.08 t ha -1 ), followed by treatment T 4 (3.04 t ha -1 ). The minimum grain yield was recorded in the treatment T 1 control (2.31 t ha -1 ). The data pertaining to grain yield were presented in Table 7.

Molecular characterization of the efficient bacterial strain

Molecular identification of the one best isolate of Bacillus was subjected to 16s rRNA sequencing. The 16s rRNA analysis suggested that strains found to be Priestia megaterium (B-1) (Accession No. PV611841).

Discussion

In the present study, Phosphobacteria populations, Percentage colonization, Spore number were significantly higher in co-inoculated ragi plants compared to single inoculation. Similar findings were reported by Begum and Priya (2004) [1] . Vazquez et al., (2000) [14] reported that Bacillus amyloliquefaciens, Vibrio proteolyticus and Enterobacter asburiae from the roots of black and white mangrove produced succinic, lactic, isovaleric, isobutyric and acetic acids. In the present study, Bacillus megaterium produced tartaric, malice, glyoxalic, gluconic and lactic acids. Lakshmanan et al., (2000) [5] reported that synergistic interaction of Glomus sp. and Bacillus polymyxa significantly increased the plant height and plant dry matter production of Clitoria ternatea L. In the present study, all the graded levels of phosphorus with co-inoculation significantly increased the plant height and dry matter production. Interestingly, the dual inoculation recorded higher increase in the above growth parameters compared to single inoculation. Kalavathi et al., (2000) [4] reported that the co-inoculation effect of Glomus sp. and Pseudomonas striata or Bacillus polymyxa on neem (Azadirachta induce) increased the seedling growth. Jancy Sathiyavathi (2002) [3] found that the dual inoculation with Glomus sp. and B. megaterium significantly increased the leaf length, number of tillers than the single inoculation at each of three phosphorus level.

Summary and Conclusion

The present study emphasized that the applying of 75% of the phosphate, along with co-inoculation of Bacillus megaterium and Glomus fasiculatum, increased ragi yield, thereby reducing chemical fertilizer use by up to 25% and minimizing cultivation costs in an eco-friendly manner.

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