An Assessment of Cosplay of Microbes Treated Seeds on Storability of Rice Seeds

Rice [Oryza sativa (L.) Poaceae] is a staple cereal crop that serves as a crucial source of carbohydrates, proteins, vitamins, minerals and dietary fibre. Its cultivation thrives in humid, tropical and subtropical climates, where optimal conditions promote high yields. However, these same conditions characterized by high temperatures and humidity accelerate seed deterioration during storage, posing challenges for long-term viability and quality maintenance.
 
Physiochemical transformations during storage
 
Rice undergoes notable physicochemical changes during storage, which become evident within approximately three months (Saikrishna et al., 2018). These changes are governed by factors such as:
 
Varietal differences
 
Rice variety determines physical and compositional variations.
 
Storage conditions
 
Temperature and humidity significantly impact the rate of biochemical transformations.
 
Amylose content
 
This influences the texture and retrogradation behaviour of stored rice.
       
Modifications in protein and lipid composition, interactions between proteins and lipid oxidation products and protein-starch interactions collectively influence processing behaviour. Additionally, oxidative reactions in stored rice contribute to decline in nutritional quality and functional properties.
 
Seed ageing and deterioration dynamics
 
Seed ageing is associated with reduced germination rates and vigour, while increasing susceptibility to environmental stresses. Seed degeneration manifesting as loss of quality, viability and metabolic stability is exacerbated in developing regions where ambient storage conditions lack humidity and temperature control.

Mechanisms of accelerated ageing
 
Accelerated ageing methodologies artificially simulate storage-induced deterioration by subjecting seeds to elevated temperatures and high relative humidity (Kapoor et al., 2011). These stress conditions amplify: Membrane permeability defects, increasing solute leakage. Oxidative stress, hastening lipid peroxidation and protein degradation. Enzymatic dysfunction, impacting metabolic pathways essential for germination.
       
Interestingly, studies indicate that primed seeds, particularly onion seeds, exhibit improved performance and extended shelf life under unfavourable storage conditions, as priming mitigates accelerated ageing effects (Yallamalle et al., 2019).

Experiment was carried out in the Department of Seed Science and Technology, College of Agriculture, Vellanikkara (KAU). Freshly harvested seeds were allowed to age naturally for nine months in 700-gauge polythene bags sealed tightly and stored under ambient conditions. The moisture content of seed sample after treatment were set to around 8.1%. The seed quality parameters were assessed as per ISTA guidelines (ISTA, 2019).
 
Seed priming
 
There were eight treatments with seed priming treatments. Seeds were dry dressed with Pseudomonas fluorescens (10 g/kg), Trichoderma viride (4 g/kg), P. fluorescens + T. viride are considered as dry treatments. As for wet treatments seeds were soaked in water (Hydropriming); coconut water (75%); P. fluorescens + coconut water; T. viride + coconut water; P. fluorescens + T. viride + coconut water in rice variety Jyoty, the seeds were soaked in solution for 16 h respectively. Un-primed seeds were used as control.
 
Accelerated aging
 
Artificial aging of seeds was done by considering two parameters temperature and RH as per the method explained by Delouche and Baskin, (1973). The treated seeds were packed in butter paper bags with pin holes to absorb moisture for ageing and placed in the wire gauge / mesh above the water well such that seeds are not in touch with water inside BOD incubator from 0 to 7 days and received a temperature of 40±1oC and a relative humidity of 98%. Accelerated aged samples were taken at daily intervals and received further tests to determine a variety of quality parameters.
 
Germination (G%) and seed vigour (VI)
 
These were estimated as per the procedure prescribed by ISTA, (2019) using between paper method (Roll towel).
 
Moisture content (MC %) and seedling dry weight (g/10 seedlings)
 
Estimated as per the procedure of high constant temperature protocol of ISTA (2010).

Speed of germination (SPG)
 
Estimated using procedure of Maguire (1962) which is known as top of paper method.
 
Mean germination time (MGT)
 
Estimated using the equation formulated by Ellis and Robert (1981).
 
Time taken for 50% germination (T50)
 
Estimated using the procedure given by Coolbear et al. (1984) formulated the 50% germination time equation (T50) as modified by Farooq et al. (2006).
 
Electrical conductivity (ISTA, 2010)
 
The electrical conductivity of the leachate from the grains was measured with a digital conductivity meter.
 
Estimation of ROS scavengers
 
SOD enzyme activity
 
Using Beauchamp and Fridovich’s (1971) method of photochemical inhibition of Nitro Blue Tetrazolium (NBT) reduction, the activity of SOD was assessed. At 560 nm, the absorbance of the reaction mixture was measured.
         
Dehydrogenase enzyme activity (Kittock and Law, 1968)
 
The enzyme dehydrogenase activity is valued at 470 nm using spectrophotometer is the OD value.
       
Analysis of the data on various seed quality parameters was performed following the Completely Randomized Design (CRD) with three replications using OPSTAT, a Statistical software package.

Seed priming is a pre-sowing technique used to improve germination speed and uniformity under adverse environmental conditions. The process involves controlled hydration followed by dehydration, ensuring optimal moisture levels for storage while retaining the physiological benefits of priming (kumar et al., 2015). Primed seeds exhibit enhanced quality traits compared to unprimed seeds (Table 1). Germination rates increased by 2.01% to 23.48%, with the highest germination (83.69%) recorded in seeds treated with a combination of Pseudomonas, Trichoderma and Coconut water, which performed on par with dry treatment using Pseudomonas.


       
This improvement can be attributed by several factors, Microbial enhancement plays a crucial role, as beneficial microbes suppress harmful pathogens and promote overall seed health. Additionally, hormonal stimulation by these microbes contribute to gibberellin production, regulating a-amylase synthesis, which mobilizes starch reserves for early seedling growth. Furthermore, enzymatic activation leads to increased activity of proteases and nucleases, facilitating the digestion of nucleic acids and efficient nutrient translocation to the endosperm, enhancing seed vigour and development (Saudi, 2017).
       
Seedling performance and stress tolerance are significantly enhanced through seed priming treatments. The combined application of Pseudomonas, Trichoderma and Coconut water resulted in the highest germination speed (18.99), closely followed by dry-treated Pseudomonas (18.89) and Pseudomonas combined with Trichoderma (19.02). Additional improvements were observed in seedling length, with the maximum elongation (19.66 cm) recorded in the combined treatment, which was comparable to Coconut water-treated seeds (19.07 cm). The vigour index peaked at 3674, confirming enhanced seedling establishment, while the highest dry weight (0.242 g) was observed in primed seeds, similar to Trichoderma-treated seeds (0.240 g) (Table 2) which are supported by previous studies of Maurya et al. (2024) in Chick pea, Negi et al. (2021) in French bean.


       
Oxidative stress mitigation plays a crucial role in maintaining seed viability. Seeds exposed to oxidative stress accumulate reactive oxygen species (ROS), leading to physiological damage. Protective enzymes such as superoxide dismutase (SOD) and dehydrogenase neutralize these harmful radicals, thereby improving stress resistance (Chatnaparat et al., 2009; Hemsanit et al., 2010). Among treatments, T7 exhibited the highest enzymatic activity, followed by dry-dressed Pseudomonas, reinforcing the role of microbial priming in oxidative stress defense (Table 3). Enzyme activity correlated with previous studies in rice and maize, supporting the beneficial effects of microbial priming (Prathuangwong et al., 2012; Wattanakulpakin et al., 2012).


       
Seed aging is primarily associated with the gradual inactivation of enzymes due to molecular modifications (Radha et al., 2014). Long-term storage leads to reduced SOD and dehydrogenase activity (Fig 1A), consistent with findings in paddy (Vijayan, 2005) and black gram (Manimekalai, 2006). Towards the end of the aging process, all treatments exhibited equal reductions in SOD activity (Fig 1B), indicating uniform oxidative conversion (O‚  to H‚ O) across samples.


       
Adding to, seed priming significantly enhances germination, seedling vigour and enzymatic defense, mitigating the negative effects of storage deterioration. This study provides valuable insights into seed longevity, biochemical stability and storage optimization, contributing to improved agricultural practices.

Seed treatment with root-colonizing organisms such as Pseudomonas fluorescens and Trichoderma viride is an effective strategy for managing rhizosphere-resident microbial antagonists. These biocontrol agents are particularly valued for their strong root colonization ability, their capacity to produce a diverse range of antimicrobial compounds and their role in inducing systemic resistance in plants (Vanitha et al., 2009; Erdogan and Benlioglu, 2010). The application of P. fluorescens, either alone or in combination with other biocontrol agents or organic treatments, has been shown to enhance seed longevity and quality. This effect is largely attributed to the presence of endogenous plant growth regulators, including gibberellins, cytokinins and indole-acetic acid, which contribute to increased mineral availability and improved water uptake. These factors collectively support seed vigour and resilience during storage.
       
Furthermore, studies indicate that seven days of continuous accelerated aging is equivalent to nine months of natural aging, providing a rapid method for assessing seed deterioration and viability under controlled conditions. This correlation between accelerated and natural aging highlights the effectiveness of biological seed treatments in mitigating storage-related stress and preserving seed quality over extended period.

The present study was supported by Department of Seed Science and Technology, Kerala Agricultural University, Dr. Santhoshkumar A.V. (Professor and head, Department of Forest Biology and Breeding, College of Forestry, Vellanikkara) for lab assistance.
 
Disclaimers
 
The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.
 
Informed consent
 
All animal procedures for experiments were approved by the Committee of Experimental Animal care and handling techniques were approved by the University of Animal Care Committee.

The authors declare that there are no conflicts of interest regarding the publication of this article. No funding or sponsorship influenced the design of the study, data collection, analysis, decision to publish or preparation of the manuscript.