Seed Biopriming by Endophytes for Enhanced Field Performance of Hybrid Maize (Zea mays L.) COH(M) 8

Maize (Zea mays L.) belongs to the poaceae family and is the third most important cereal crop after wheat and rice and is grown throughout the year in India. Maize is a C4 plant, makes effective use of moisture and sunlight to produce high yields and total dry matter (Bell, 2017). It is a cross-pollinated crop native to Mexico and has wider adaptability under varied agro-climatic conditions with the highest genetic gain among the cereals. The demand of maize production on a global scale is rising as a source of food, oil, forage and biofuel for the growing world’s population.

Endophytic microorganisms are generally known as microbes that colonize the plant parts and that do not harm the plant. They improve plant growth by secreting phytohormones and consequently help in nutrition improvement using bidirectional nutrient transfer and enhancement of the health of plants by protecting them against phytopathogens (Shen et al., 2019). Endophytes are numerous and have been found in many plants; they became important due to their ability to create a wide range of bioactive compounds and essential enzymes (Rajamanikyam et al., 2017). When endophytes are inoculated into a plant, they usually result in significant increase of biomass, as well as aid commercial agriculture (Shen et al., 2019).

Endophytes are found in all or most of the plants and in most cases, endophytes are transmitted through seeds, where they begin to promote plant development and health as soon as the seeds germinate (Kandel et al., 2017). Some other endophytes can be found in the soil and provide benefits to plants (Verma and White, 2018). One of the most important aspects of quality seeds is the production of pest and disease-free seeds which will enhance the vigour, viability and optimum field stand. This can be achieved through seed biopriming with endophytes. During seed priming with endophytes, it enters into the seeds and adapts to the existing conditions. It is capable of fixing nitrogen, solubilization of phosphate, enhancing the uptake of phosphorus and production of siderophores and plant hormones such as auxin, abscisic acid, ethylene, GA₃ and IAA, which are important for plant growth and development (Xu et al., 2014). Besides it offers protection of the host plant against various stress condition. with this view, the present study has been undertaken to get enhanced field performance of Hybrid maize COH(M) 8 through seed biopriming with endophytes.

Collection of experiment materials

The genetically pure and freshly harvested seeds of maize hybrid COH(M) 8 collected from the Department of Millet, Tamil Nadu Agricultural University, Coimbatore used as source material for this study. The endophytic microorganisms Viz., B. bassiana, M. anisopliae and B. subtilis were used for this study. The population dynamics or CFU/ml solution of B. bassiana and M. anisopliae through Potato Dextrose Agar (PDA) medium and B. subtilis through Luria-Bertani (LB) medium was 2 x 10-8.
 
Field experiment
 
The field experiment was carried out in field No 37D, Department of Seed Science and Technology, TNAU, Coimbatore in 2021-2022 and the maximum and minimum temperature of 34.5°C and 20.5°C respectively and an average rainfall of 589 mm were recorded. The experiment was conducted, with the seeds bioprimed with endophytes along with foliar application of fungal and bacterial endophytes to study the morphological, reproductive and seed yield attributes. Endophytic priming solution was prepared using double distilled water and seeds were soaked in priming solution for 12 hrs at the ratio of 1:1 volume/volume and the temperature of 10-15°C was maintained. The seed was removed from the solution after the priming (12 hrs) and rinsed with water and then dried back to their original moisture content. The treatment includes T₀ - Control, T₁ - Hydro priming, T₂ - Seed priming with B. bassiana 5%, T₃ - Seed priming with M. anisopliae 5%, T₄ - Seed priming with B. subtilis 8%, T₅ - Seed priming with B. bassiana 5% and foliar spray of B. bassiana 0.5%, T₆ - Seed priming with M. anisopliae 5% and foliar spray of M. anisopliae 0.5% and T₇ - Seed priming with B. subtilis 8% and foliar spray of B. subtilis 0.5% with three replication in a randomized block design (RBD). The foliar spray was given at 30^th and 50^th DAS. the size of plot was 4.0 x 3.0 m² with the crop spacing of 60 cm x 30 cm. The observations were taken at different crop growth stages.
 
Observations recorded
 
Field emergence (%)
 
Field emergence was recorded by counting the number of hills germinated in each plot at fifteen days after sowing.
 
Plant height (cm)
 
Plant height was measured in ten randomly selected plants from each treatment in each replication, from the base to the tip of the leaf and the mean value was expressed in cm.
 
No. of leaves/ plant
 
The number of leaves on ten randomly chosen plants in each plot was counted and the mean number was computed.
 
Leaf length (cm)
 
The leaf length was observed at the 3rd leaf from the top of the plant using a scale in randomly selected ten plants and the mean length was depicted in centimetre.
 
Leaf breadth (cm)
 
The leaf breadth was taken at the 3rd leaf from the top of the plant using a scale in randomly selected ten plants. Mean values was expressed in centimetre.
 
Chlorophyll content (mg/g)
 
Leaf chlorophyll was measured by the method suggested by Yoshida et al., (1971) and the optical density of extract was measured at 645 nm, 663 nm and 652 nm in a spectrophotometer.
 
Days to 1^st and 50% tasselling (DAS)
 
The number of days taken for the appearance of first tasselling and the number of days taken by 50% of the plants in each plot for tasselling were recorded.
 
Days to 1^st and 50% silking (DAS)
 
The number of days taken for the appearance of first silking and then days taken by 50% of the plants in each plot for silking were observed.
 
Cob length and breadth (cm)
 
In randomly selected ten plants, the length and breadth of the cob were measured using a measuring scale and the mean value was calculated and expressed in centimeters.
 
100 seed weight (g)
 
Hundreds of seeds were counted with eight treatment in each replications and weight was observed and expressed in grams.
 
Seed yield per plot (kg)  
 
Seeds were threshed from each plot separately, dried to 13% moisture content, weighed and expressed in kilogram.

Seed yield/ha (kg/ha)

Computed seed yield was calculated from plot yield and expressed in kg per hectare.
 
Statistical analysis
 
The analysis of variance was carried out and a comparison was done by Duncan’s Multiple Range Test (DMRT). The mean difference is significant at the P-values < 0.05. Statistical analysis was performed using the SPSS 16.0 software (SPSS Inc., Chicago, USA).

Sustainable agriculture is fundamentally concerned with improving and decreasing the adverse effects of agricultural production. Global agriculture must expand food production to meet the needs of a growing population and decrease its dependency on synthetic chemicals. So, utilizing the numerous advantageous interactions that take place between plants and microbes is crucial. It enhances the nitrogen fixation, acquisition and uptake of essential nutrients, the encouragement of shoot and root growth and the control or suppression of disease to boost plant growth and development.

In this regard, the results revealed that seed biopriming with endophytes exhibited significant effects on field emergence. Priming with M. anisopliae @ 5% (T₃) recorded the maximum increase of field emergence (90%) that was 5.88% more over nonprimed seeds (T₀) (85%) (Fig 1). An increase in germination percentage might be due to the combined effect on the production of hydrolytic enzymes and growth hormones like IAA and gibberellins through endophytic seed treatments (Schulz and Boyle, 2005). This is in agreement with other studies showing that priming mainly improves germination as a result of the enhanced water uptake and more favourable water relations and also increases the production of hydrolytic enzymes in primed seeds (Lechowska et al., 2019). Earlier reports corroborate the findings of this study by Yuan et al., (2007) who observed that the germination of rice seeds treated with endophytes were significantly higher than without seed priming.



The maximum plant height at 15 DAS (58.5 cm) was observed in seed priming with M. anisopliae @ 5% (T₃) whereas control (T₀) registered the minimum plant height (53.8 cm). Seed priming compined with foliar spray of M. anisopliae (T₆) recorded higher plant height at 45 DAS (208.4 cm) and 75 DAS (247.8 cm), with an increase of 9.62% and 10.28% respectively over control (Table 1). According to Elena et al., (2011), inoculating M. anisopliae with tomato plants increased plant biomass, particularly plant height, root length and dry biomass from the roots and shoots. Additionally, 10, 15 and 30 days after M. robertsii inoculation, tomato plantlets exhibit an increase in plant height, root length and shoot dry biomass compared to control plants (Siqueira et al., 2020). The finding was in line with M. anisopliae colonization having enhanced growth in soybean (Khan et al., 2012) and maize (Gayathri et al., 2020). The results are supported by Endophytes that have been producing phytohormones like particularly gibberellins (GAs) and indole-3-acetic acid (IAA), which play a role in cell division and elongation thereby increasing plant growth.



In terms of leaf parameters, endophyte treatments do not influence the number of leaves and leaf breadth. In the case of leaf length, seed priming and foliar spraying with endophytes recoded higher leaf length viz., B. bassiana (T₅) (76.1 cm), M. anisopliae (T₆) (76.0) and B. subtilis (T₇)  (75.5 cm) and control (T₀) registered the lowest one (Table 2). A similar result was observed in treated broad bean seeds with M. anisopliae and B. bassiana (Jaber and Enkerli, 2017).



The significant difference was observed in chlorophyll (chl) a and b and total chlorophyll content in endophytic treatments. The result revealed that seed priming and foliar spraying of M. anisopliae (T₆) recorded maximum value of chl a 0.423 mg/g, chl b 0.229 mg/g and total chl 0.652 mg/g. Control (T₀) recorded the lowest valve of chl a 0.367 mg/g, chl b 0.195 mg/g and total chl 0.562 mg/g (Fig 2). M. anisopliae has enhanced chloroplast metabolism due to increased chlorophyll content (Shi et al., 2010). This outcome is consistent with that of Khan et al., (2014), who showed that endophytes inoculated with tomato plants increased the amount of chlorophyll content and additionally, the synthesis of IAA has been shown to increase the production of photosynthetic pigments and metabolites (Duca et al., 2014). The previous research finding was in line with endophytic bacteria had enhanced the chlorophyll content registered in mustard, which could significantly increase the enzymes needed for chlorophyll biosynthesis (Kang et al., 2014).



In phenological characters, days taken to initiation of flowering were reduced in seed priming with foliar spray when compared to other treatments, but there was no significant difference observed in days to 1^st tasselling, 50 percent tasselling and days to 50 percent silking. A significant difference was observed only in days to 1^st silking in both priming and foliar spraying of B. bassiana (T₅), M. anisopliae (T₆) and B. subtilis (T₇) at two stages (Table 3). The duration was higher in control plants, plant growth and development are enhanced by endophytes through secreting phytohormones and improved nutrition uptake through bidirectional nutrient exchange given by Andreozzi et al., (2019).



Seed yield parameters such as cob length and seed yields were significantly influenced by the endophytes, but cob breadth and 100 seed weight were not influenced by the endophytic treatments. The maximum cob length (21.2 cm) was noticed in priming and foliar spraying of M. anisopliae (T₆). While analyzing the seed yield of various treatments, Seed priming and foliar spray of M. anisopliae (T₆) recorded a maximum seed yield of 6.893 kg/plot and the control (T₀) recorded the lowest seed yield 6.400 kg/plot. Similarly seed priming and foliar spray of M. anisopliae (T₆) increased seed yield (kg/ha) 7.71% over control (Table 4) (Fig 3). Endophytes may improve plant growth through their favourable effects on photosynthesis and chlorophyll content. Because of high photosynthetic activity and increase in nutrient exchange thereby accumulation of more dry weight (e.g., increased primary metabolites viz., sugars, proteins and fatty acids), which could have lead to an increased seed yield. The result was in concurrence with Stefan et al., (2013) who observed the higher yield of bean plants inoculated with endophytes associated with higher photosynthetic activities. The present results showed that the seed yield increased significantly in inoculated plants, as that of previous research finding which was in line with the Inoculation of M. anisopliae in maize seeds resulting in a significant increase in seed yield (Kabaluk and Ericsson, 2007). Similarly, inoculation with B. bassiana enhances the plant growth and increases the seeds yield of soybean recorded by Russo et al., (2019).

Based on the results, it can be concluded that the selected endophytes viz., Beauveria bassiana, Metarhizium anisopliae and Bacillus subtilis inoculated in the form of both priming and foliar application had enhanced the maize plant growth and development. It is concluded that among the endophytes M anisopliae seed priming and foliar application had improve the plant growth, photosynthetic pigments and seed yield of maize under field conditions.