Exploration of microbial biodiversity for their functionality in different fodder crops in custardapple based Horti-pastoral system

Introduction

Agricultural crops and tree species are ubiquitously associated with a variety of microorganisms. The communities of bacteria, archaea, fungi, and protists that colonize plant tissues and inhabit outer plant surfaces collectively make up the plant microbiota. The plant microbiome, or phytobiome, is defined as the complete set of microbial genomes associated with a host plant (Guttman et al., 2014) [9] . The importance of plant-associated microbes in enhancing host plant health, nutrition, and stress resistance is widely recognized (Vandenkoornhuyse et al., 2015) [19] . However, this relationship is influenced by numerous factors, including host and microbial genotypes, interactions within the microbiota, and various abiotic factors (Hardoim et al., 2015) [10] .

Forests which cover 30% of the total global land area are found to be highly productive components of terrestrial ecosystems (Llado et al., 2017) [12] . Furthermore, trees aid in soil structure maintenance, organic matter decomposition, pollutant degradation, and so on (Thakur et al., 2018) [16] . Some of bacterial taxa previously shown to dominate forest soil ecosystems and play such key roles include members of the genera Pedobacter and Chitinophaga (Bacteroidetes); Pseudomonas, Variovorax, Ewingella, and Stenotrophomonas (Proteobacteria) (Brabcova et al.,2016) [5] ; Burkholderia, Phenylobacterium and Methylovirgula (Pseudomonadota), members of the Rhizobiales and Nitrosopumilus. Soil microorganisms are an important component of the forest ecosystem (Uroz et al., 2016) [18] as they play fundamental roles in most nutrient transformations upon which the stability and sustainable development of forest ecosystems rely. The distribution and diversity of soil microbiomes is influenced by numerous aspects such as soil type, physicochemical characteristics, microclimate, vegetation and land-use (Gupta et al., 2017) [8] . The interaction of plants with microbes in the rhizospheric region helps in maintaining the soil fertility and health of the plant (Kumar et al., 2019) [11] . Bacteria in forest soils play key roles in organic matter decomposition (López-Mondéjar et al., 2016) [13] , nitrogen fixation, and mineral weathering. Their interactions with mycorrhizal fungi, which enhance nutrient cycling and soil connectivity, are supported by mycorrhiza helper bacteria, influencing overall forest soil health and function (Bonito et al., 2014) [4] . Fungi are the primary decomposers in temperate and boreal forest soils, breaking down plant material through extracellular enzymes, and facilitating the cycling of nitrogen, phosphorus, and carbon (Eichlerová et al., 2015) [6] . While fungi have been the main focus of forest soil studies, there is a growing recognition of the significant role bacteria play in these ecosystems. Actinomycetes, a dominant group in soil, contribute to organic matter recycling and are crucial in producing antibiotics, enzymes, and pharmaceuticals. Since the discovery of streptomycin, about two-thirds of known antibiotics have been derived from actinomycetes, highlighting their medical and industrial importance (Arifuzzaman et al., 2010) [1] . Fodder crops exhibit a dynamic microbial status that influences their growth and nutrient content. Microorganisms, such as nitrogen-fixing rhizobia in legumes, aid in nitrogen fixation and enhance plant fertility. Mycorrhizal associations improve nutrient uptake, especially phosphorus. Beneficial soil microbes contribute to pathogen suppression, reducing the need for chemical pesticides. Microorganisms play a role in silage fermentation, preserving fodder quality. Managing microbial interactions is vital for optimizing the nutritional value and sustainability of fodder crops in livestock agriculture. Microbial diversity associated with crops, fruit trees, shrubs, forest tree species is considered important for maintaining the sustainability of agriculture/forest/agroforestry production systems. The microbiome of plants could be classified into three categories, e.g., rhizospheric, phyllospheric and endophytic the rhizosphere is the zone of soil influenced by plant roots through the release of different substrates that affect microbial function and its stability (Suman et al., 2016) [15] . Though lot of studies were conducted across the globe on various ecosystems, the microbial diversity in hortipastroal systems in Deccan plateau is less studied. Hence, an experiment is being proposed to study the microbial diversity and effect on productivity in custard apple based hortipastroal system involving different fodders.

Materials and Methods

Soil Sample Collection

Soil samples were collected from Agroforestry research block-II, PJTASU, Rajendranagar, Hyderabad. The samples were collected near the Custardapple intercropped with fodder crops (Anjan and Guinea grasses) rhizosphere region at 15 cm of depth and kept in sterilized polyethene covers and brought to the laboratory.

Isolation of rhizosphere bacteria, fungi and actinomycetes Bacteria, Fungi and Actinomycetes were isolated by using the serial dilution and plating technique (Arifuzzaman et al., 2010) [1] . For the isolation of bacteria, fungi and actinomycetes, fresh soil samples have to be taken. From which 1 gm of each soil sample was suspended in 9 ml of sterile saline containing 0.85% NaCl and mixed with vortex. The suspension was left for 30 min in the Laminar Air Flow hood. Consequently, 1 ml of suspension was taken and diluted 10-fold in 9 ml of sterile normal saline (0.85%). The suspensions were agitated with vortex and 0.1 ml of suspension was taken from dilutions. Dilutions of 10 -5 and 10 -6 were used for isolation of bacteria while dilutions of 10 -3 and 10 -4 were used for isolation of fungi and actinomycetes. 0.1ml of respective dilutions were spread onto Nutrient agar medium (NA), potato dextrose agar medium (PDA) or Martin 's Rose Bengal agar medium (RBA), Actinomycetes agar medium for isolation of bacteria, fungi and actinomycetes present in rhizosphere soil. These media plates were incubated at 28±2 °C for 24-48 hours, 25±2 °C for 3-4 days, 28±2 °C for 10-15 days for isolation of bacteria, fungus and actinomycetes respectively. The isolates were differentiated based on colony morphology.

Enumeration of microbes in soil samples

Enumeration of bacteria, fungi and actinomycetes was done on Nutrient agar medium, Rose Bengal agar medium and Actinomycetes agar medium respectively, using serial dilution spread plate technique. The media were prepared and sterilized in an autoclave at 15 psi pressure and 121 °C temperature for 20 minutes. Serial dilutions (up to 10%) were made by pipetting 1 ml of the soil suspension into 9 ml of sterile water blank. Finally. 0.1 ml aliquot of the diluted soil suspension was uniformly spread with the help of sterilized spreader on solidified petri plates having respective medium. The petri plates were incubated for 1 to 3 days at 28±2 °C in an inverted position. After incubation, the number of colonies appeared on medium plates were counted, averaged and multiplied by the dilution factor to find the number of cells per gram of soil sample CFU/gm =

Morphological characterization of microbial isolates

All the isolates were checked for their purity and then studied for colony morphology and pigmentation. The cell shape and gram reaction were also recorded as per the standard procedures given by Barthalomew and Mittewar (1950) [3] . Morphological characteristics of the colony of each isolate was examined by growth of the isolates on their respective medium and incubated them for 5 to 7 days. Cultural characterization of isolates were observed by different characteristics of colonies such as shape, colour, margin and elevation were recorded.

Results and Discussion

Isolation and Purification of microbial isolates Microbial isolates were isolated by using nutrient agar for bacteria, potato dextrose agar or rose Bengal agar medium for fungi, Actinomycetes agar medium for actinomycetes and depending on the colony morphology and gram staining the Unique and individual isolates were selected. The isolated colonies were purified by quadrant streak plate method. Moreover, pure cultures were preserved in 20% glycerol stocks to study biochemical characters. Among the 46 bacterial, fungal and actinomycete isolates studied, 16 bacteria included 10 gram-positive and 5 gramnegative strains, while all 16 actinomycete isolates were grampositive. The bacterial colonies were typically round or circular with raised, gummy textures, producing exopolysaccharides under optimal conditions. Colony sizes ranged from 1.5 to 4.4 mm, with colours from watery or milky translucent to creamy or opaque white. Actinomycete isolates were cocci and rod-shaped, predominantly rod-shaped and cream-colored. Pigmentation in actinomycetes varied from cream, dark/light brown, grey, green, to white and ash. Their colony surfaces were described as dry, smooth, rough, and powdery, with various penetrating characteristics. The colony shapes included filamentous, circular, and irregular forms. Fungal colonies exhibited irregular, circular, or filamentous morphology, with colours ranging from green to brownish black and pink. Results was depicted in table 7, 8 and 9. Our results are similar with Azmi et al. (2021) [2] and EI-Tarabily et al., (2021) [7] described various fungal and actinomycetes strains. Similarly, Upadhyay et al. (2015) [17] reported various bacterial strains.

Enumeration of microbes in fodder crops Rhizospheric soil samples

The Enumeration of Total bacteria, fungi and actinomycetes from the rhizosphere soil of fodder crops at three different intervals. The results showed that among the different treatments treated to the two grasses (Anjan and Guinea grass).

Bacteria

At 30 DAT, the highest bacterial population in Anjan grass (M1) was recorded in T3 (PGLM) with 120 × 10⁶ CFU g⁻¹, followed by T4 (NGLM) at 100 × 10⁶, T5 (SGLM) at 83 × 10⁶, and T7 (FYM) at 80 × 10⁶. The lowest was in T1 (control) with 56 × 10⁶ of soil. In Guinea grass (M2), T3 (PGLM) had the highest population of 140 × 10⁶, while the lowest was in T1 at 50 × 10⁶ of soil. At 60 DAT, T3 (PGLM) remained the highest for Anjan grass with 90 × 10⁶ CFU g⁻¹, followed by T4 at 80 × 10⁶, and the lowest in T1 at 40 × 10⁶ of soil. In Guinea grass, T3 had the highest population of 80 × 10⁶, with T1 again the lowest at 40 × 10⁶ of soil. At 90 DAT, T7 (FYM) had the highest population for Anjan grass at 70 × 10⁶, followed by T3 at 60 × 10⁶, while T1 showed 30 × 10⁶ of soil. For Guinea grass, T7 was highest at 65 × 10⁶, with T1 being the lowest at 25 × 10⁶ of soil. Results was presented in table 1.

Fungi

At 30 DAT, the highest fungal count for Anjan grass (M1) was observed in T3 (PGLM) with 35 × 10³ CFU g⁻¹ of soil, followed by T6 (Poultry manure) at 23 × 10³ and T4 (NGLM) at 20 × 10³ of soil. The lowest count was in T1 (control) at 9 × 10³ of soil. In Guinea grass (M2), T3 also had the highest fungal count with 30 × 10³, while T1 showed the lowest at 9 × 10³ of soil. At 60 DAT, the highest fungal count for Anjan grass was in T3

(PGLM) at 20 × 10³ of soil, followed by T4 at 16 × 10³, while T1 had the lowest at 7 × 10³ of soil. For Guinea grass, T3 and T5 both recorded 15 × 10³, while T1 had the lowest at 6 × 10³ of soil. At 90 DAT, the highest fungal count for Anjan grass was in T3 (PGLM) at 13 × 10³, while T1 had the lowest at 5 × 10³ of soil. For Guinea grass, T3 again had the highest count with 13 × 10³, and T1 the lowest at 4 × 10³ of soil. Results was presented in table 2.

Actinomycetes

At 30 DAT, the highest actinomycete count for Anjan grass (M1) was in T3 (PGLM) with 70 × 10³ CFU g⁻¹, followed by T4 (NGLM) at 55 × 10³. The lowest count was in T1 (control) with 24 × 10³ of soil. For Guinea grass (M2), T3 had the highest count of 80 × 10³, and T1 had the lowest at 19 × 10³ of soil. At 60 DAT, the highest count for Anjan grass was in T4 (NGLM) at 35 × 10³, while T1 had the lowest at 12 × 10³ of soil. For Guinea grass, T4 recorded the highest count at 40 × 10³, while T1 had the lowest at 10 × 10³ of soil. At 90 DAT, T4 (NGLM) had the highest count for Anjan grass with 30 × 10³, and T1 had the lowest at 10 × 10³ of soil. For Guinea grass, T4 again had the highest count at 25 × 10³, while T1 recorded the lowest at 4 × 10³ of soil. Results was presented in table 3.

Comparison Between Grasses

Guinea grass (M2) supported higher microbial populations compared to Anjan grass (M1) across most treatments. For instance, in the first interval, (M2) Guinea grass showed higher bacterial, fungal, and actinomycetes counts under Pongamia (T3) and other organic treatments, showed that Guinea grass fasters more favorable conditions for microbial growth, possibly due to root exudates or soil interactions.

Comparison Between Treatments

Treatment T3 (Pongamia green leaf manuring) consistently had the highest microbial populations across both grasses and all intervals, while T2 (100% RDF) and T1 (Control) had the lowest. This demonstrates the superior effect of organic manures, particularly green leaf manuring, in promoting a rich soil microbial environment compared to chemical fertilizers. Among the treatments, T3 (Pongamia green leaf manuring) consistently resulted in the highest bacterial, fungal, and actinomycetes populations across all intervals for both Anjan grass (M1) and Guinea grass (M2). This can be attributed to the rich organic matter, nitrogen content, and slow-release nutrients provided by Pongamia leaves, which faster sustained microbial activity. Other treatments like T4 (Neem green leaf manuring) and T5 (Subabul green leaf manuring) also contributed significantly to microbial populations, though their effects were slightly less than Pongamia. T6 (Poultry manure) and T7 (FYM) showed moderate microbial counts, while T2 (100% RDF) and T1 (Control) consistently had the lowest microbial counts across all intervals, demonstrating that organic manures, especially green leaf manures, promote better microbial activity than inorganic fertilizers. Similar results were obtained by Singh et al. (2021) [14] , enumerated microbial populations in the soil of Custard apple cv. Balanagar plants, showing increased bacterial (26.67 cfug⁻¹), fungal (15.00 cfug⁻¹), and actinomycetes (10.00 cfug⁻¹) populations.

Enumeration of microbes in Custardapple Rhizosphere soil samples:

The Enumeration of Total bacteria, fungi and actinomycetes from the rhizosphere soil of Custardapple at three different intervals.

Bacteria

At the 1st interval (30 DAT), the highest bacterial count was recorded in T7 (75% RDN + 25% N through FYM) with 80 × 10⁶ CFU g⁻¹, followed by T3 (Pongamia green leaf manuring) at 60 × 10⁶, T5 (Subabul green leaf manuring) at 50 × 10⁶, and T2 (100% RDF) and T6 (Poultry manure) both at 40 × 10⁶ of soil. Lower counts were in T4 (Neem green leaf manuring) at 35 × 10⁶, with the lowest in T1 (Control) at 25 × 10⁶ of soil. At the 2nd interval (60 DAT), T7 (FYM) maintained the highest bacterial count at 70 × 10⁶, followed by T3 (55 × 10⁶), T5 (45 × 10⁶), T2 (36 × 10⁶), T6 (33 × 10⁶), and T4 (30 × 10⁶). The lowest was again in T1 (Control) at 20 × 10⁶ of soil. At the 3rd interval (90 DAT), T7 (FYM) had the highest count at 73 × 10⁶, followed by T3 (50 × 10⁶), T5 (44 × 10⁶), T2 (35 × 10⁶), T6 (32 × 10⁶), and T4 (23 × 10⁶) of soil. The lowest bacterial count was in T1 (Control) at 15 × 10⁶ of soil. Results was presented in table 4.

Fungi: At the 1st interval (30 DAT), the highest fungal count was observed in T7 (75% RDN + 25% N through FYM) with 33 × 10³ CFU g⁻¹, followed by T3 (Pongamia green leaf manuring) at 20 × 10³, T5 (Subabul green leaf manuring) with 17 × 10³, and T6 (Poultry manure) at 15 × 10³. T2 (100% RDF) recorded 14 × 10³, T4 (Neem green leaf manuring) had 12 × 10³, while T1 (Control) had the lowest at 9 × 10³. In the 2nd interval (60 DAT), T7 (FYM) again showed the highest fungal count at 25 × 10³, followed by T3 (15 × 10³), T5 (14 × 10³), and T6 (12 × 10³). T2 recorded 10 × 10³, T4 had 9 × 10³, and T1 (Control) had the lowest count at 6 × 10³. By the 3rd interval (90 DAT), T7 (FYM) maintained the highest fungal count at 18 × 10³, followed by T3 (14 × 10³), T5 (12 × 10³), and T6 (9 × 10³). T2 recorded 8 × 10³, T4 had 7 × 10³, and the lowest count was again in T1 (Control) at 5 × 10³. Results was presented in table 5.

Actinomycetes

In the 1st interval (30 DAT), T7 (75% RDN + 25% N through FYM) showed the highest actinomycetes count with 80 × 10³ CFU g⁻¹, followed by T3 (Pongamia green leaf manuring) at 60 × 10³, and T5 (Subabul green leaf manuring) with 50 × 10³. T6 (Poultry manure) recorded 35 × 10³, T2 (100% RDF) had 33 × 10³, T4 (Neem green leaf manuring) recorded 25 × 10³, and T1 (Control) had 19 × 10³. In the 2nd interval (60 DAT), T7 remained highest at 70 × 10³, followed by T3 (55 × 10³), T5 (45 × 10³), T6 (32 × 10³), T2 (30.1 × 10³), T4 (20 × 10³), and T1 (15 × 10³) of soil. By the 3rd interval (90 DAT), T7 and T3 both had 50 × 10³, T5 recorded 40 × 10³, T6 had 30 × 10³, T2 showed 25.083 × 10³, T4 had 17 × 10³, and T1 recorded the lowest at 14 × 10³ of soil. Results was presented in table 6.

Comparison Between Treatments

T7 (75% RDN + 25% N through FYM) was the most effective treatment across all microbial categories-bacteria, fungi, and actinomycetes-showed the highest microbial counts in all three intervals. T3 (Pongamia green leaf manuring) consistently ranked second, supporting significant microbial activity, though lower than T7. T5 (Subabul green leaf manuring) demonstrated moderate effectiveness, while T2 (100% RDF), T6 (Poultry manure), and T4 (Neem green leaf manuring) showed varying levels of microbial populations, generally lower than the top two treatments (T2 and T6). T1 (Control), which had no fertilization or manuring, consistently recorded the lowest microbial populations across all categories. This indicates that organic amendments, particularly FYM and Pongamia leaf manuring, significantly enhance microbial activity in soil compared to chemical fertilizers and no fertilization treatments. In custard apple rhizosphere, the highest microbial populations and fruit yields were associated with treatment T7, while the control and 100% RDF treatments showed the lowest counts and yields. This study highlights a clear correlation between increased microbial diversity and improved yields in both custard apple and fodder crops. The results emphasize the advantages of using organic amendments to enhance soil health, boost crop productivity, and promote the sustainability of custard apple-based horticultural-pastoral systems.

Acknowledgement

AcknowledgementThe author wants to thank chairman Dr.S. Triveni Professor and University Head PJTSAU, Rajendranagar, Hyderabad for his valuable support through research work and to all the lab staff.

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