Microbial degradation of Diospyros melanoxylon biomass by Trichoderma atroviride for plant growth promotion of finger millet

Abstract

Utilisation of microbial inoculants as decomposers of plant biomass could be a sustainable approach for environmental management of biowaste and improving agricultural productivity. Malabar Ebony, locally known as ‘Kendu’ (Diospyros melanoxylon) is one of the dominant plant species in the Eastern Ghats of India having great economic value where the leaves are used to prepare ‘traditional cigarette’ in this region. The production process results in accumulation of leafy wastes causing environmental concerns. To address this issue, a native Trichoderma atroviride WCF2 strain was employed for enhanced degradation of Kendu leaves to further observe its growth promoting effects on finger millet (Eleusine coracana) landrace. The fungus exhibited degradation of Kendu leaves biomass with extracellular production of cellulase, pectinase and amylase enzymes demonstrating enhanced organic matter decomposition. The semi-digested biomass was having higher compost characteristics, with percentage of TC, TN, TH TS and moisture content of 31.96%, 1.03%, 5.05%, 0.863% and 71.31% respectively, C:H ratio of 6:32, C: N ratio of 30:88 and C: H:N ratio of 31.96:1.03. Pot experiments showed T. atroviride WCF2 treated biomass increased 39% plant height and 66% grain weight along with other plant growth parameters in millet landrace. This is the first report of T. atroviride being utilised for Kendu leaf waste management and growth promotion in millet landraces. Thus, this native fungus could be utilised for improvement in the soil nutrients, crop enhancement and agroecosystem of tribal farmers along with conservation of crop landraces in biodiversity rich site like Koraput, designated as Globally Important Agricultural Heritage System (GIAHS).

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-026-35942-3.

Introduction

Millet, an ancient grain known for its resilience, reemerged as a sustainable and nutrient rich alternative to modern cereals. It provides higher levels of iron, zinc, calcium, and vitamin B, which help fight micronutrient deficiencies in tribal communities. With high fiber content, abundant antioxidants, and excellent ability to withstand drought and poor soils, millets not only provide nutritional security but also support sustainable livelihoods for indigenous communities^1–3. Millets ranking sixth among crops after rice, wheat, maize, barley, and sorghum are food crops well-suited for the dryland agriculture of arid and semi-arid tropical areas as their cultivation takes place on marginal, low-fertility soils⁴. Considering their hardiness, millets have been promoted as a solution to combat food insecurity in regions vulnerable to fluctuating temperatures and rainfall, especially in India and sub-Saharan Africa. In 2019-20, the State of Odisha located in the east coast of India alone produced 132.84 thousand tonnes millet, where the crop is cultivated predominantly by the tribal population⁵. These tribal farmers prefer Farm Yard Manure (FYM) prepared from forest biomass generally composed of shredded leaves. These compost-based organic substrates are supposed to improve soil fertility, enhance plant nutrition, and reduce the need for harmful farm chemicals along with mitigating soil degradation caused by intensive agriculture⁶. This semi digested biomass when prepared through beneficial microorganisms (bacteria, fungi, and actinomycetes) have been proved to enhance plant growth, impart biological control, and act as phyto-stimulants or biofertilizers under adverse conditions⁷.

Globally, various species of Trichoderma have been used as biomass degrader in addition to their role as biocontrol agent. Trichoderma spp. are ubiquitous free-living fungi (Hypocreaceae family), found to play a major role in agricultural production, which is capable of producing diverse plant growth-promoting compounds, including enzymes and phytohormones^8–12. In addition to this, these fungal species have globally proved their capability to produce various lytic enzymes like cellulases, amylases, pectinases, etc. for the efficient degradation of leafy biomass and biowastes generated from agricultural and forest activities^13–15. In Odisha State of India, harvesting and processing of Kendu leaves by the tribals is such an activity which generates lots of leafy biowastes. Kendu or the ‘Malabar Ebony’ (Diospyros melanoxylon) is known as the “Green gold of Odisha,” which is widely distributed in the forest areas of Odisha and eastern India where the leaves are used for making traditional cigarette “Bidi.” The Kendu leaf trade in Odisha alone is around Rs.1100 crores (131 million USD) where approximately 3.0 lakh quintals of leaves are produced annually, accounting for about 20% of the total production of India¹⁶. However, approximately 20% of the total collected leaves are rejected during the grading process, leading to challenges in solid waste management. The leaf litter of Diospyros melanoxylon is particularly recalcitrant compared to that of many other species, primarily due to its unique chemical composition. Its leaves are structurally tough and contain high levels of refractory compounds such as lignin, along with various phytochemicals including tannins, flavonoids, and saponins. These constituents collectively strengthen the leaf structure and enhance its resistance to microbial decomposition^17,18.

In this context, the present study reports, for the first time, the isolation of Trichoderma atroviride from crop landraces in Odisha’s Eastern Ghats (GIAHS region), exhibiting cellulase, amylase, and pectinase activities, thereby revealing the unexplored Trichoderma diversity in this region. Under the above-mentioned facts, an attempt was undertaken to utilise these Kendu leaf (Diospyros melanoxylon) biomass through the degradation by a native strain of Trichoderma atroviride and subsequently applying the degraded biomass on a local Finger Millet landrace (Eleusine coracana) for promotion of plant growth and yield. Though various species of Trichoderma have been utilised as plant inoculants, to the best of our knowledge, no study has been carried out on the application of Trichoderma atroviride for the degradation of forest biomass like Kendu leaves and subsequent application on Finger Millet. Previously, there is also no report of isolation of Trichoderma atroviride from maize seed land race in any part of the globe. The results of the study indicated the potential of native microorganisms for utilisation of forest biomass for the preparation of potential compost for the globally important Millet crop and simultaneously improvement of agroecosystem and lower farm inputs of marginal tribal farmers.

Materials and methods

Isolation and identification of Trichoderma atroviride

The Trichoderma atroviride strain was isolated from seeds of a local Maize landrace from Malipadar village, Gupteswar Gram Panchayat Biodiversity Management Committee, Koraput, Odisha, India GPS Location (18° 40’ 57.39” N and 82° 21’ 25.13” E). Standard isolation protocol of Agarwal and Sinclair¹⁹ was followed where the seeds were then surface sterilized by 70% ethanol for 60 s followed by 1% NaOCl solution for 60 s and plated on the potato dextrose agar media after thorough washing with sterilised water. To check the efficacy of surface sterilization, 100 µl water from the last wash was transferred onto potato dextrose agar plates for the appearance of microbial colony, if any²⁰. All the plates were incubated in a BOD incubator for 3–5 days at 28℃ or till the appearance of microbial colonies. Fungal colonies emerging out of the seeds were isolated and preserved in PDA slants at 4 °C.

Identification of the fungal strain was carried out both by characterizing the morphological and microscopic features followed by molecular methods. Color and other features of fungal colony was observed visually whereas and the structure of conidiospores was observed under phase contrast microscope (Leica DM2000 LED)²¹ and identified following Gams and Bissett²². Molecular characterization, identification, and phylogenetic analysis of the fungal isolate were carried out through partial gene sequencing of the Internal Transcribed Spacer (ITS) region, which includes ITS1, the 5.8 S rRNA gene, ITS2, and a partial sequence of the large subunit (LSU) rRNA gene, resulting in a 579 bp sequence. The sequencing was performed at the Microbial Type Culture Collection and Gene Bank (MTCC), Council of Scientific and Industrial Research - Institute of Microbial Technology (CSIR–IMTECH), Chandigarh, India.

Production of cell wall degrading enzymes by Trichoderma atroviride

Capacity of the fungal strain to produce various hydrolytic enzymes was evaluated by growing in specific media and observing the enzyme hydrolyzing zones²³. Production of amylase enzyme was tested by growing the fungal strain on starch agar medium (Himedia) and then development of a clear zone surrounding the fungal colony after flooding with Lugol’s iodine solution (1% iodine in 2% potassium iodide w/v). Production of protease enzyme was evaluated by growing the fungal isolate on skim milk agar plates followed by formation of clear zone of skim hydrolysis²⁴. Cellulase Test was carried out by growing the fungus on CMC agar medium containing carboxymethyl cellulose (5 g/L), Peptone (5 g/L), NaCl (5 g/L), beef extract (3 g/L), and agar (20 g/L) with pH of 7.0. After five days of incubation, the plates were flooded with aqueous solution of Congo red (0.1% w/v) for 15 min followed by flooding with 1 M NaCl for 15 min. Production of cellulase was detected by the formation of transparent zone around the fungal colony in contrast to bright red colour formed on the plate by the Congo red²⁵. Pectinase production was observed on two pectinase screening agar media such as MP5 and MP7 containing polygalacturonase and pectate lyase as the substrates respectively. The fungal isolate was grown on these media followed by flooding the plates with Lugol iodine solution (1% iodine in 2% potassium iodide w/v) solution to detect the clear halo zone around the colony²⁶.

The Enzymatic Index (EI) for all of the aforementioned four enzymes were calculated by using the expression of Florencio et al.²⁷ as follows:

Preparation of Kendu leaves and inoculum containing T. atroviride

Dried Kendu leaves were collected from the forest areas of Nayagarh District of Odisha, India and immediately stored at 4 °C until use. The plant has been identified by Dr. C. Kalidass, Scientist (Taxonomy and Conservation), Regional Plant Resource Centre (RPRC), Bhubaneswar, India. A voucher specimen of this material has been deposited in the herbarium of RPRC with reference no. 12,631. This study complies with relevant institutional, national, and international guidelines and legislation. Appropriate permissions for collection of plant leaves has been accorded from the owner of the plants used in this study.

Leaves were sun-dried for five to six hours to get rid of any excess moisture and insects before proceeding for microbial treatment. Leaves were first surface sterilised with 70% ethanol followed by 0.1% mercuric chloride solution and then rinsed thoroughly with sterilized water. After complete drying, the leaves were cut into small pieces. Ten grams each of dried leaf cuttings were kept in conical flasks (1 L size) and sterilized at 121 °C under 15 lb pressure for 30 min^28,29. A homogenized fungal inoculum containing Trichoderma atroviride was prepared by cultivating the fungus in Potato Dextrose broth at 28 °C for 5 to 6 days. An aliquot of 10 mL of the broth containing T. atroviride (approximately 10⁶ spores/mL) was used as the inoculum for the subsequent experiment, resulting in an inoculation density of about 10⁶ spores per gram of leafy biomass.

Biodegradation of Kendu leaf biomass with Trichoderma atroviride

For the biodegradation of Kendu leaves with T. atroviride, conical flasks were prepared as follows:

Control (C) : Uninoculated sterilised Kendu leaves without water and fungal inoculum.

Treatment-1 (T-1) : Kendu Leaves inoculated with 10 ml of Sterilized Water.

Treatment-2 (T-2) : Kendu Leaves inoculated with 10 ml of T. atroviride broth.

Each flask of T-2 was added with 10 ml of fungal inoculum in seven days interval. Same amount of sterilised water was also added to each flask of T-1 in equal time interval. No inoculum or sterilised water was added to ‘Control (C)’ flask. All the flasks were tightly plugged and incubated at room temperature in laboratory, allowing biodegradation to proceed for up to 100 days.

Analysis of physio-chemical parameters of the partially degraded Kendu leaf biomass

After incubation for 100 days, the partially degraded biomass from each of the experimental flask was examined for its total wet and dry mass of compost. Air-dried leafy biomass was utilized for the estimation of chemical composition. To determine the moisture content and total solids in the biomass, 1 g of air-dried biomass was placed in a hot air oven set at 100 °C for 4 to 5 h or until it reached a constant weight. The final weight of the biomass was recorded and used to calculate the moisture and total solid content as outlined³⁰. Important parameters such as moisture content (MO, %), Total Carbon (TC, %), Total Hydrogen (TH, %), Total Nitrogen (TN, %), Total Sulphur (TS, %), Carbon: Hydrogen ratio (C/H ratio, %) Carbon: Nitrogen ratio (C/N ratio, %) were calculated following standard protocols by a CHNS–O Analyzer (Elementar-UNICUBE)³¹.

Biodegradation of leaf cell wall through fungal enzymatic activities

To determine the extent of structural degradation of various leaf carbohydrates, estimation of cellulose, hemicellulose, lignin and glucose was carried out following the standardized protocol of “National Renewable Energy Laboratory (NREL)”³². In brief, 300 mg dried biomass were treated with 3 ml of 72% H₂SO₄ (W/W) and incubated at 30 °C in a water bath for 1 h followed by secondary hydrolysis at 121 °C for 1 h in autoclave. Prior to secondary hydrolysis, 84 ml water was added to the tubes to make the final concentration of H₂SO₄ to 4%. After the secondary hydrolysis, the 4% H₂SO₄ treated supernatant was used to estimate cellulose by using glucose assay kit (Coral Clinical System) and acid soluble lignin (ASL) was determined by Nano Drop spectrophotometer (ND-100) at 240 nm using the extinction coefficient 60 L g^{− 1}cm^{− 133}.

Effect of partially degraded Kendu leaf biomass on early seedling Vigor of millet

Finger Millet (Eleusine coracana) landrace Variety ‘Sanatara’ was obtained from MS Swaminathan Research Foundation, Jeypore, Koraput, Odisha. Prior to treatment with the degraded biomass, the millet seeds were surface sterilized with 70% ethanol for 60s followed by 0.1% NaOCl solution for 60s and then washed thoroughly with distilled water. Approximately 300 g of sterilised soil (typical forest agricultural soil) was mixed with 0.5 gm of degraded biomass (T-1 and T-2 separately) and kept in plastic cups. For control, the untreated biomass was mixed with soil in separate cups. Surface sterilised Millet seeds were kept on the above soil preparations and allowed germinate for 6 days.

The germinability of treated seeds were calculated as per Swain et al.³⁴ as mentioned below:

Ten healthy seedlings from each control and treatment were randomly chosen on the sixth day of germination in order to measure the lengths of the roots and shoots. Same 10 Seedlings were also used to take fresh weight. Vigor index (VI) was calculated as per Sreekissoon et al.³⁵ as mentioned below:

Observation of growth of treated seedlings in pot culture

Uniformly grown seedlings on previous treatments and control were then transferred to earthen pots (size 8’’ × 8’’) containing sterilised soil mixed with the corresponding degraded biomass in the ratio of 100:1 for observation of further growth and development³⁶. In this was following three types of pots were prepared:

1. Pots containing untreated biomass: Control (C).

2. Pot containing biomass treated with sterilised water: Treatment-1 (T-1).

3. Pot containing biomass treated with Trichoderma atroviride: Treatment-2 (T-2). All the pots containing the corresponding seedlings were kept under field conditions. No external fertilizer or organic manure was added during the crop growth. Observation of various agronomic traits like plant height, leaf number, Finger length, Peduncle length, Ear head length, grain weight, etc. were carried out up to harvesting. Shoots and root weights of each control and treatments were taken after harvesting³⁷. To observe the establishment of Trichoderma atroviride in the millet plant, various plant parts like sections of leaf, stem, seed, and root were surface sterilised and then plated on PDA media which was followed by the appearance of fungal colonies.

Statistical interpretations

The results of the study are expressed as the mean of replicates, with all sampling and analysis experiments conducted in replicates. The treatments were carried out in a completely randomized block design. The experimental data results were computed with one-way analysis of variance (One-way ANOVA) by P < 0.05 significance level using ‘Microsoft excel’ and ‘ggplot2 R package’³⁸.

Results and discussion

Isolation and identification of seed endophytic fungi

Crop landraces have been useful sources of beneficial microorganisms having the potential for multipurpose applications. In this context, an attempt was taken in the current study to isolate biomass degrading fungi from a local maize landrace. Based on morphological and molecular characteristics, the fungal isolate was identified as Trichoderma atroviride WCF 2 (Fig. 1). The phylogenetic analysis confirmed close relationship with the corresponding strain recovered from the NCBI database (Fig. 2). The ITS sequence data of the isolate was deposited in the NCBI GenBank with Accession number PQ035928.

Fig. 1.

Culture characteristics and microscopic features of Trichoderma atroviride WCF 2 isolate; (a) appearance of colonies from maize seeds (b) Trichoderma atroviride strain grown on PDA plates and (c) Microscopic image of conidiophores.

Fig. 2.

Phylogenetic status of Trichoderma atroviride WCF 2 by Maximum parsimony (MP) analysis of a dataset of the ITS region. Bootstrap support values for MP equal to or greater than 50% (black) given above the nodes. The tree was rooted to Protocrea pallida CBS 299.78.

Evaluation of enzyme production by Trichoderma atroviride

Production of three hydrolytic enzymes like cellulases, amylases and pectinases by Trichoderma atroviride WCF2 was evaluated on specific media and the Enzymatic Index (EI) was calculated. In the current investigation, T. atroviride WCF2 showed cellulase activity with an EI of 1.5 (SEM = 0.06) (Fig. 3). These three enzymes aid in breaking down of plant cell walls which is crucial for the successful initiation of compost preparation. In the current study, the T. atroviride showed EI 1.2 (SEM = 0.03) for amylase and EI 1.9 (SEM = 0.07) for pectinase enzymes (Fig. 3). However, no reports were previously available on the ability of Trichoderma atroviride to produce these enzymes altogether.

Fig. 3.

(a) Evaluation of Trichoderma atroviride for the ability to produce biomass degrading enzymes viz. amylase, cellulase and pectinase (b) Enzymatic Indices (EI) of the three hydrolytic enzymes.

Biological degradation of Kendu leaf biomass by Trichoderma atroviride WCF2

Physio-chemical properties of the compost

Trichoderma spp. generally enhances degradation of biomass by releasing extracellular hydrolytic enzymes, while artificial inoculation speeds up decomposition and nutrient return to the soil^39,40. In the current study, leaf biomass of Kendu plant was allowed to be digested with the Trichoderma atroviride isolated from local maize seeds. After 100 days of digestion with a spore concentration of 10⁶ spores/g of biomass, the physico-chemical analysis revealed that the fungal-treated biomass exhibited a higher degree of degradation compared to the untreated control (C) and treatment T1. Overall physical appearance of the fungal treated biomass was darker than the control and T1 (Fig. 4). Trichoderma atroviride treated leafy biomass (T2) was significantly lighter in comparison to uninoculated treatments indicating faster decomposition. The weight of leafy biomass decreased by 2% with uninoculated control (C) and 3% with the sterilized water treatment (T1) whereas with Trichoderma atroviride (T2) treatment, the leafy biomass lost 26% of weight after 100 days of biodegradation. Moisture content of control, T1 and T2 were 6.75% (SEM = 0.27), 60% (SEM = 2.4) and 71.31% (SEM = 0.86) respectively (Table 1). The optimal moisture content for composting typically ranges from 40% to 60%³⁰ and might reach up to 80%⁴¹. The semi-degraded biomass (T2) treated with Trichoderma atroviride showed healthier compost characteristics, with TC% as 31.96% (SEM = 0.44), TN% as 1.03%(SEM = 0.05), TH% as 5.05% (SEM = 0.096), and TS% as 0.863% (SEM = 0.01) (Table 1). This is resulted due to the ammonification of the fungal treated biomass, leading to higher C, H, N, and S concentration which are the key indicators of compost quality, maturity, and suitability for use⁴². The fungal-treated biomass had a C/H ratio of 6.32% (SEM = 0.17), a C/N ratio of 30.88% (SEM = 0.33), and a moisture content of 71.31% (SEM = 0.86), which allowed the fungus to accelerate the breakdown of organic molecules. In this investigation, the C/N ratio for the Trichoderma treated compost was found to be 31.96: 1.03, whereas in the untreated (Control) C/N ratio was 42.99:1.36. The Trichoderma atroviride isolate in the current study helped in degradation of polysaccharides in comparison to the control followed by break down of cellulose, hemicellulose and glucose resulting in stable compost (Fig. 5). This feature presents it as a promising cost-effective option to be employed for producing leafy biomass compost, as well as biofertilizer to enhance crop yields.

Fig. 4.

Degradation of leafy biomass by Trichoderma atroviride for 100 days Untreated control (C) (only Kendu leaves), T1 (Kendu leaves + sterilized water), and T2 (Kendu leaves + Trichoderma atroviride).

Table 1.

Physical and chemical characterization of digested biomass used for composting.

Parameters	C (Control)	T1 (Kendu Leaves + Sterilized Water)	T2 (Kendu Leaves + Trichoderma atroviride WCF 2)
MO %	6.75	60	71.31
TC %	42.99	41.5	31.96
TH%	5.41	5.18	5.05
TN%	1.36	1.46	1.03
TS%	0.11	0.098	0.863

Fig. 5.

Physio-chemical properties of the compost were analysed after 100 days of digestion. (a) Dry wight of digested compost, (b) Moisture content, (c) C/N ratio, (d) C/H ratio, (e) Total Glucose, (f) Total Cellulose, (g) Total Hemicellulose, (h) Total Lignin. Different bars represent the least significant differences among the means of treatments. (P ≤ 0.05).

Germination and early vigor in finger millet

The semi-degraded biomass T1, T2 (T. atroviride) and control (C) were employed as biofertilizer to examine their effect on the growth of millet seedlings. The seed germination rate, vigor index, root & shoot length, and root and shoot fresh weights were measured after 6 days of inoculation (Fig. 6). The germination percentage of seeds sown on Trichoderma-treated biomass was 92.22% which was significantly higher than the control (59.32%) and at par with T1 (95.5%) (Fig. 7a). where an increase in root length (3.5 ± 0.22) and root weight was also observed (0.0051 ± 0.0017) (Fig. 7d and f). Similarly, the vigor Index of seedlings sown on Trichoderma treated biomass was 198.22 whereas VI in control (C) was 102.51 and T1 was 192.61 (Fig. 7b). In our study, Kendu leaf biomass treated with Trichoderma atroviride significantly enhanced millet seedling growth by promoting early vigor and root development.

Fig. 6.

Enhancement of germination and seedling growth in the millet landrace ‘Sanatara’ through the application of Trichoderma atroviride digested Kendu leaf biomass.

Fig. 7.

Various agronomic parameters of Millet seedlings (landrace Sanatara) after 6 days of sowing on biomass treatment with Trichoderma atroviride (T2), sterilised water (T1) and untreated biomass (C) (a) Germination percentage (b) Vigor Index (c) Shoot length, (d) Root length (e) fresh weight of shoot and (f) fresh weight of root. Plotted as box-and-whisker (horizontal lines and whiskers representing median values and total data range, respectively for all treatments. Different bars represent the least significant differences among the means of treatments. (P ≤ 0.05).

Growth promotion of millet seedling under pot culture

The effect of biomass on growth of Millet seedlings further was investigated in earthen pot experiments, where major agronomic parameters showed significant improvement in Trichoderma-treated biomass compared to the control and T1 (Fig. 8). Biomass treated with Trichoderma atroviride (T2) had notable positive impact on plant height, leaf numbers, and number of panicles, while T1 and the control resulted in slower plant growth after being inoculated with their respective digested biomass. Substantial increase in plant height was observed in Trichoderma treated seedlings after 9 weeks of transplantation. Average plant height in fungal-treated biomass was 46.1 cm, followed by T1 (28.18 cm) and the control (17.68 cm) (Fig. 9a). Similarly, in T2, the greatest number of 11.5 leaves and 8 panicles were observed whereas T1 and the control (C) had 5 panicles each where T1 and C had an average of 7.25 and 6.87 leaves, respectively (Fig. 9b and c).

Fig. 8.

Effect of partially digested Kendu leaf biomass by Trichoderma atroviride (T2), sterilised water (T1) and uninoculated Control (C) on the growth of millet seedlings (landrace ‘Sanatara’).

Fig. 9.

Agronomic characters of finger millets observed in Control and treatments T1 & T2 (a) Periodic increase of plant heights (b) Total number of leaves (c) Total No. of Panicle emergence, (d) panicle and finger characters (e) Dry panicle weight (f) Post-harvest dry biomass of millet plants.

Those of other parameters like the average finger length (FL), peduncle length (PL), and earhead length (EL) of millet panicles grown in biomass treated with Trichoderma were 3.8 cm, 4.8 cm, and 3.93 cm, respectively. In contrast, both T1 and the control (C) showed significantly lower values, with T1 having 1.73 cm FL, 1.7 cm PL, and 3.93 cm EL, and control (C) having 1.2 cm FL, 1.4 cm PL, and 1.6 cm EL (Fig. 9d). Similarly, the total grain weight of Millet landrace ‘Sanatara’ was highest in T2, weighing 2.18 g, followed by T1 at 0.74 g and the control (C) at 0.27 g (Fig. 9e). After harvest, plant samples from all three treatments were examined for total shoot and root biomass dry weight. The T. atroviride treatment (T2) in millet plants yielded the highest shoot dry biomass with 3.39 g, followed by T1 with 1.33 g and the control (C) with 0.55 g. However, for root dry biomass, T2 showed a lower value (1.72 g) as compared to T1 (2.31 g), but it was still higher than the control, which had a root dry biomass of 1 g. However, although there was some improvement in root growth, the root dry biomass value of T2 was lower compared to T1. Similar observation was reported by Nieto-Jacobo et al.⁴³, who noted the negative impact of Trichoderma on root length in Arabidopsis, indicating that the effect of Trichoderma on root growth can vary across plant species.

Colonization of T. atroviride in millet plant

The colonisation of the fungus in millet plants was examined by plating sections from various plant parts. It was observed that the Trichoderma atroiviride could colonise in roots only (Fig. 10).

Fig. 10.

Isolation of Trichoderma atroviride from roots of millet plants grown in compost digested by the same fungus.

Discussion

Several species of Trichoderma including Trichoderma atroviride have previously been isolated from soil, crop cultivars and other plant sources^44–46. However, this is the first report of occurrence of Trichoderma atroviride from seeds of a maize landrace which has been traditionally cultivated in this tribal area in the Odisha State of India. This particular region belongs to the “Globally Important Agricultural Heritage System” declared by the United Nations Educational, Scientific and Cultural Organization. Further, this is also a part of the great Eastern Ghats Mountain ranges of India. The floral and faunal significance of this biodiversity rich site have been frequently discussed. However, the presence of beneficial microorganisms in various habitats has been less explored. In this context, utilisation of the current Trichoderma atroviride strain having the ability to produce various hydrolytic enzymes and subsequent crop enhancement adds special significance to this GIAHS.

As far as cellulase activity by fungi is concerned, EI value of 1.5 or above has been considered to be effective for applications like lignocellulosic hydrolysis under solid-state fermentation⁴⁷, enhancing plant growth and antioxidative defence in rice, and increasing straw degradation capacity⁴⁰. In other Trichoderma species, Enzymatic Index as high as 1.74 for cellulase has also been reported^27,47. Apart from that Trichoderma viride and Trichoderma pseudokoningii have proved their ability to produce extracellular α-amylase and glucoamylase on various substrates^48,49 and Trichoderma viride, isolated from agricultural waste and farmyard manure could show pectinolytic activity⁵⁰. The C/N ratio of the initial substrate is a key parameter in composting, as carbon provides structure and energy for microbes, while nitrogen facilitates the synthesis of amino acids, proteins, and nucleic acids essential for microbial growth and reproduction⁵¹. Organic material decomposes quickly at an optimal C: N ratio of 30:1 or 31:1, while ratios that are too high or low can hinder the process or release excess nitrogen as ammonia^52–54. In this study, the C/N ratio of the Trichoderma-treated compost was recorded as 31.96:1.03, while the untreated control exhibited a ratio of 42.99:1.36. Overall, the Trichoderma-treated leafy biomass showed enhanced degradation efficiency, suggesting its potential use as a fertilizer to improve crop productivity.

Similar enhancement of seedling growth, germination rate, seedling vigor and increased physiological performance following application of Trichoderma was also observed earlier^55–57. However, in some cases though the Trichoderma viride significantly improved several agronomic parameters, at the same time, reduction in root length, leaf number, and panicle length was also observed⁵⁸. However, when combined with farmyard manure (FYM), it enhanced both growth and yield, outperforming the use of inorganic fertilizers alone. Biomass treated with T. atroviride showed comparatively better effects than other treatments, demonstrating growth promotion in a millet landrace. Most of the Trichoderma treatments had a positive impact on plant growth and yield parameters^59,60. However, different Trichoderma strains showed varying levels of effectiveness in promoting plant growth. For example, Trichoderma harzianum has been widely studied for its biocontrol properties, particularly against soil-borne pathogens. While it improves plant health and growth, its performance is not always consistent across different crops or environmental conditions⁸.

As observed in previous studies, Trichoderma primarily colonized the roots where the hyphae were extended in the root system, enhancing surface area for nutrient absorption and making water available to plants^61,62 simultaneously contributing to soil aggregation and enhancing porosity and aeration⁶³. Most of the previously described Trichoderma strains have been isolated from soil where few reports described its colonisation in seeds of local landraces. In the present study, we isolated the endophytic Trichoderma atroviride from a local maize landrace from a location which is a part of the “Globally Important Agricultural Heritage System (United Nations Educational, Scientific and Cultural Organization). Various species and strains of Trichoderma have been utilized in various agricultural applications, particularly in biocontrol, where they promote plant growth, act as antagonists against pests and pathogens, aid in biomass degradation, and are also involved in cellulase production through submerged fermentation^8,64. Currently, in India over 250 commercial formulations are available, with the majority being biofertilizers or biopesticides derived from Trichoderma viride³⁶. Various Trichoderma spp. has also accelerated the rate of biomass decomposition by producing cell wall-degrading enzymes, thereby increasing the availability of nutrients in the soil for other organisms to utilize^65,66. Decomposition of empty fruit bunches (EFB) and palm oil mill effluent (POME) were significantly accelerated by Trichoderma spp. reducing the process time from 4 to 6 months to just 21–45 days⁶⁷. In this context, the utilisation of native Trichoderma atroviride having the capability to produce enzymes such as amylase, cellulase, and pectinase collectively could be a novel ecofriendly approach to manage the biowaste or biomass generated from Kendu leaf processing. Further, the biologically degraded Kendu leaf biomass also promoted growth and yield in local millet landrace. This is very encouraging as the tribals mostly cultivate local millet landraces with less or no fertiliser input. Kendu leaf business also serves as an important source of income for the same tribals. Under these circumstances, the application of digested biomass significantly improved millet crop performance by enhancing germination percentage, vigor index, plant height, root length, number of leaves, and number of panicles compared to the untreated control. This treatment also resulted in notable increases in finger length, peduncle length, and earhead length, collectively contributing to higher overall yield. Thus, the utilization of biologically degraded Diospyros melanoxylon (Kendu) leaf biomass would addresses the challenge of waste management and would also enhance soil organic carbon, improve nutrient cycling, and support microbial diversity, all of which are central pillars of agroecology strengthening tribal agroecological resilience. Upscaling and mass production would reduce dependence on chemical inputs, sustain local production systems, promote resource circularity, ecological intensification subsequently support community-based agrodiversity conservation in tribal landscapes.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

SSS-Lead the wet lab and green house experiments, methodology, Formal analysis of result analysis and original drafting of the manuscript; MG-Assisted in wet lab and green house experiments and manuscript proof reading; OPM: Assisted in wet lab and green house experiments and manuscript proof reading; BP: Analysis and manuscript proof reading SN- Conceptualization, Project administration, Supervision, experiment design, Writing - review & editing.

Funding

The authors didn’t receive fundings particularly for the current investigation.

Data availability

1. Details of plant material deposited in the herbarium would be given on request to the communicating author.2. ITS sequence of the Trichoderma atroviride is available in the link: https://www.ncbi.nlm.nih.gov/nuccore/PV094632.1/.

Declarations

Competing interests

The authors declare no competing interests.

Ethical approval and consent to participate

Not applicable.

Consent for publication

All the authors have given their consent for publication.