Development and evaluation of compatible microbial consortia for accelerated degradation of heterogeneous crop residues and leaf litter

Introduction

Agricultural residues are an important source of organic carbon and plant nutrients, and their efficient decomposition plays a significant role in nutrient recycling and sustainable residue management. Large quantities of rice, wheat and other crop residues are generated from intensive agricultural production, while their improper disposal, particularly open field burning, results in the loss of valuable organic resources and creates environmental concerns. Biological decomposition using efficient microorganisms offers an alternative approach for converting these residues into useful organic matter and facilitating their incorporation into soil (Choudhary et al., 2016) [1] . The recalcitrant nature of crop residues is mainly associated with their complex lignocellulosic structure comprising cellulose, hemicellulose and lignin. Microorganisms capable of producing hydrolytic and oxidative enzymes can utilize these structural components and contribute to progressive residue breakdown. However, degradation of lignocellulose is a complex process involving several enzymatic activities and different microorganisms may possess different capacities for degrading particular components. Studies on wheat straw have demonstrated that microbial communities with different compositions can exhibit similar overall degradation ability while possessing distinct enzymatic profiles, indicating the importance of functional complementarity among microorganisms (Cortes-Tolalpa et al., 2016) [2] . The use of microbial consortia can therefore provide an advantage over individual microbial inoculants by combining microorganisms with complementary degradative properties. However, the successful development of a consortium requires microorganisms that can coexist and maintain their functional activities without strong antagonistic interactions. This makes compatibility assessment an important preliminary step in consortium formulation. Choudhary et al. (2016) [1] identified efficient fungal isolates from rice and wheat fields and reported that compatible combinations of lignocellulolytic fungi showed potential for accelerating crop-residue degradation.

Similarly, research on synthetic microbial consortia has shown that the composition and functional performance of a consortium can be influenced by the microorganisms selected and their interactions during lignocellulose degradation (Cortes-Tolalpa et al., 2016; Puentes-Tellez et al., 2018) [2,3] . The efficiency of a microbial consortium may further depend on the type and chemical composition of the residue used as substrate. Wheat straw, maize straw, rice straw and leaf litter differ in their relative proportions of cellulose, hemicellulose, lignin and other organic constituents, and consequently may support different patterns of microbial degradation. Thus, evaluation of a consortium against multiple residues is necessary to determine whether its decomposing ability is broad or substrate-specific. Previous studies have primarily evaluated selected microorganisms or consortia against individual residues, particularly rice or wheat straw, highlighting the need for comparative assessment across diverse plant materials (Choudhary et al., 2016; Cortes-Tolalpa et al., 2016) [1,2] . Although lignocellulolytic microorganisms and microbial consortia have been investigated extensively, systematic utilization of already available efficient isolates through compatibility testing, consortium development and comparative evaluation on diverse residues under pot conditions remains important. Therefore, the present study was undertaken (i) To assess the compatibility of selected microbial isolates, (ii) To formulate compatible microbial consortia and (iii) To evaluate their decomposition potential against wheat straw, maize straw, rice straw and leaf litter under pot culture conditions. The study is aimed at identifying effective microbial combinations with broad residue-decomposition potential and establishing their suitability for biological management of agricultural residues.

Materials and Methods

Selection of microbial isolates and compatibility testing

Eight fungal isolates (P2, P3, P15, P18, P19, P24, P28 and P29) and three bacterial isolates (B2, B5 and B10) were taken from Department of Plant Pathology, VNMKV, Parbhani. Compatibility was assessed using cross streak and dual culture assays as described by Dantroliya et al., 2021 [4] . Bacterial combinations were evaluated by cross streaking on nutrient agar and incubation at 30-35 °C for 24 hrs. Fungal combinations were assessed on PDA using 8 mm discs from 7 days old cultures and incubated at approximately 27 ± 1 °C for 5 days. Fungal-bacterial interactions were assessed on modified PDA using a 5 mm fungal disc and bacterial streak, followed by incubation at 28 ± 2 °C. Absence of visible inhibition or antagonism was considered compatibility. All compatibility assays were performed in triplicate.

Consortium formulation

Six pairwise compatible microbial combinations were identified and used to formulate six consortia: three two member fungal consortia, one three-member fungal consortium, one fungal-bacterial consortium and one bacterial consortium. For two member fungal consortia, equal volumes of compatible fungal inoculum were combined in a 1:1 (v/v) ratio and adjusted to approximately 1 × 10 6

CFU ml -1 . For the three member fungal consortium T 4 , comprising P2, P3 and P24, the compatible fungal inoculum were combined for consortium preparation. For the bacterial consortium, compatible bacterial suspensions were combined in a 1:1 ratio and standardized to approximately 1 × 10 7 CFU ml -1 . For the fungus-bacterium consortium, fungal and bacterial suspensions were combined with sterile 4% (w/v) jaggery solution in a 1:1:2 (v/v/v) ratio and acclimatized for 4-6 hrs at 28 ± 2 °C. All preparations were carried out aseptically.

Treatment Consortium

Pot culture experiment

The decomposition experiment was conducted separately for wheat straw, soybean straw, maize straw and tree leaf litter in a completely randomized design (CRD) with three replications. Each substrate had seven treatments, giving 21 pots per substrate and 84 pots in total.

Substrates were chopped into 2-3 cm pieces, soaked in 1% NaOH at a substrate-to-solution ratio of 1:10 (w/v) for 24 hrs, washed with distilled water until neutral, dried at 80 °C for 6 hrs and autoclaved at 121 °C for 20 min. Five hundred grams of substrate were placed in 1 kg capacity pots. Microbial consortia were applied at 10% (v/w), mixed thoroughly and incubated under aseptic conditions. Moisture was maintained at approximately 60% using sterile water. An uninoculated control received the same substrate pretreatment but no microbial consortium.

Observations and analysis

Colour, odour and texture were recorded at fixed intervals, including the initial stage and 30, 60 and 90 days. Total nitrogen was estimated by Kjeldahl's method (Piper, 1966) [5] , total phosphorus by the vanadomolybdate yellow colour method (Kitson et al., 1944) [6] , potassium using a flame photometer (Piper, 1966) [5] and organic carbon by the dry-combustion procedure (Nelson et al., 1982) [7] . C:N ratio was calculated from organic carbon and nitrogen concentrations. The decomposition endpoint was recorded based on visible complete decomposition and stabilization of colour, odour, texture and nutrient parameters. Substrate degradation rate after 90 days was calculated as; initial substrate weight-residual substrate weight initial substrate weight × 100

Statistical analysis

Data were analyzed using analysis of variance as outlined by Gomez et al. (1984) [8] and OPSTAT software. When the F-test indicated significant treatment effects, treatment means were compared using Critical Difference (CD) @ 1% level. SE(m) ± and CD values with the corresponding results.

Initial substrate characterization

Results and Discussion

Compatibility and consortium selection

Among 28 fungal-fungal combinations tested, four were found compatible; P2 × P3, P2 × P24, P3 × P24 and P3 × P28. The remaining 24 fungal combinations showed inhibition, overgrowth or contact-point inhibition. Among 24 bacteria-fungi interactions, only B5 × P2 was compatible. B2 and B10 were incompatible with all tested fungal isolates, while B5 was incompatible with fungal isolates other than P2.

Among the three bacterial pairs, B2 × B5 was compatible, whereas B2 × B10 and B5 × B10 were incompatible. These compatibility results provided the basis for constructing the consortia evaluated in the pot experiment. Compatibility-based selection is consistent with the principle that microbial members of a consortium should be able to coexist without strong antagonistic interactions (Chen et al., 2024) [9] .

Physical transformation during decomposition

Across all four substrates, microbial treatments produced faster visible transformation than the uninoculated, pretreated control. Residues progressively changed in colour, odour and texture during the 90-day period. T 4 showed particularly rapid transformation, with advanced darkening, reduced characteristic odour and development of crumbly or humus-like texture in several substrates. These observations were qualitative and were not treated as an independent quantitative maturity score. Similar changes in physical characteristics and accelerated organic matter transformation following microbial inoculation have been reported during consortium-based decomposition studies (Awasthi et al., 2014; Beary et al., 2002) [10,11] . Final nitrogen concentration was higher than the initial concentration in all four substrates, with the magnitude varying among treatments. T 4 recorded the highest numerical final nitrogen concentration in each substrate: 1.53% in wheat straw, 1.67% in soybean straw, 1.23% in maize straw and 1.71% in leaf litter. The increase in percentage concentration should be interpreted primarily as a concentration effect associated with decomposition and loss of carbon-rich material, rather than as proof of an increase in total nitrogen mass. Microbial inoculation has similarly been reported to influence nitrogen retention and nitrogen concentration during straw composting (Zhou et al., 2023) [12] .

Nitrogen transformation

Phosphorus transformation

Final phosphorus concentration was higher than the initial value in all four substrates. T 4 recorded the highest final phosphorus concentrations: 0.22% in wheat straw, 0.49% in soybean straw, 0.35% in maize straw and 0.45% in leaf litter. These values represent concentrations in the remaining decomposed material and should not be interpreted as a net increase in total phosphorus mass without a nutrient mass balance. Phosphorus release during residue decomposition is known to vary with residue composition and decomposition conditions (Lupwayi et al., 2007) [13] , while recent consortium studies have also demonstrated changes in straw degradation and phosphorus transformation following microbial inoculation (Che et al., 2024) [14] . [15] . Field studies have also shown that residue type and decomposition environment influence nutrient release during residue decomposition (Mishra et al., 2024) [16] . [17] . [12,10] . The recorded the time required to reach its decomposition endpoint based on visible complete decomposition and stabilization of colour, odour, texture and nutrient parameters. Under this endpoint criterion, T 4 required 63 days for wheat straw, 54 days for soybean straw, 57 days for maize straw and 42 days for leaf litter. Because these end-point times were based on visible criteria and not on direct repeated gravimetric measurements of mass loss, they should not be interpreted as direct measurements of 80% gravimetric degradation. [18,14,9] .

Treatment

Organic carbon transformation

C:N ratio

Time required to reach the decomposition endpoint

Conclusion

Compatibility testing demonstrated that microbial interactions were highly combination dependent. Six compatible combinations were selected to construct microbial consortia. Under controlled pot conditions, the three-member fungal consortium T 4 (Corynascus sp. P2 + Myrothecium sp. P3 + Aspergillus sp. P24) consistently produced the highest 90 days substrate degradation rates, rapid physical transformation and the highest final N, P and K concentrations across the four substrates. Treatment T 5 showed the greatest reduction in final organic carbon in several substrates, whereas T 4 recorded the lowest final C:N ratio across all four substrates. The time to endpoint values reported were shortest under T 4 , but these endpoint times were based on visible criteria. The study identifies T 4 as the leading consortium under the controlled experimental conditions and supports its further evaluation under field conditions.

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