Microorganisms constitute less than 0.5 per cent (w/w) of the soil mass, yet they have a major impact on soil properties and processes. The seemingly rigid mass of clay, sand and silt is home for a complex microbial community including bacteria, fungi, actinomycetes, algae, protozoa and viruses. The soil bacteria and fungi play pivotal roles in various biogeochemical cycles (BGC) (Wall and Virginia 1999) and are responsible for the cycling of organic compounds. Soil microorganisms also influence above ground ecosystems by contributing to plant growth and soil fertility (Yao et al 2000). Thus the intimate association of microbes and soil particulates is critical for total ecosystem survival. Hence, the integrity of the total ecosystem, above and below ground, rests on the stability of the soil microbial community. The destruction of the soil micro-biota, through mismanagement or environmental interferences results in decline or even death of the above ground plant population.

An understanding of soil microbes, their properties and the nature of the interaction with and within their environment is essential. With this background, the study on assessment of rhizosphere microbial diversity of red sanders was undertaken.

Pterocarpus santalinus popularly known as red sanders (Fabaceae) is a tropical dry deciduous forest species. Red sanders is an endangered timber tree species, endemic to southern India. It grows in approximately 5,160 km² of fragmented forest landscape of southern Andhra Pradesh and in a few sporadic patches in Tamil Nadu and Karnataka states.

The wood is primarily used for making musical instruments and luxury furniture. It also yields santalin dye (Ferreira et al 2004) which finds use in colouring foodstuff (Mulliken and Crofton 2008) and pharmaceutical preparations (Schmidlin et al 2008). Additionally, the extracts obtained from the tree bark and wood are known to have several medicinal properties (Saikia et al 2006). The tree can grow only in dry plains of India like eastern Ghats and eastern plains of Karnataka like Kolar, Tumkur and Chitradurga. It grows in parts of Tirupati hills and Srisailam in Andhra Pradesh (Vedavathy et al 1997). It prefers lateritic and gravelly soil and cannot tolerate water logging. In natural habitat, the tree experiences hot, dry climate with normal rainfall of 88-105 cm received from northeastern and southwestern monsoons.

Red sanders has the prominent importance in the business. Research initiatives are under way to exploit its potential application in soaps, dyes, toys etc. Despite a ban on exploitation and sale, good quality red sanders fetched any price between Rs 3 and 4 lakhs per tonne in the global market, while the inferior type was sold as fuelwood like other timbers (Bhagyaraj and Ramana 2013). Red sanders has fallen back into the ‘endangered’ category in the International Union for Conservation of Nature’s (IUCN) Red List (Jha 2022).

The red sanders wood has different uses in traditional and folklore medicines and is used for the treatment of diabetes, prickly heat, skin diseases, ulcers, eye diseases inducing vomiting, mental aberrations, snake bites, scorpion stings and for various other ailments. The heartwood is known to have anti-hyper glycemic activity; is anti-pyretic, anti-inflammatory, anthelmintic, tonic, hemorrhage, dysentery, aphrodisiac and diaphoretic activities and is a cooling agent (Ramabrahmam and Sujatha 2016).

Red sanders rhizosphere soil (R) samples were collected from different locations viz Rangapuram, Athiyandal, Karumandurai, Balapuram and Attur. The samples were packed in sterile polythene bags and brought to the laboratory for further analysis.

For comparison, non-rhizosphere soil samples (S) adjacent to red sanders plantations were also collected and analysed for microbial population. The samples were collected from each location during March 2023, packed in sterile polythene bags and brought to the laboratory for further analysis. These were stored at 4°C till processed for further biological and physico-chemical properties.

Serial dilution of the samples: One gram fresh soil was transferred to 100 ml sterile distilled water to get 10⁻² dilution. After thorough shaking, 1 ml of the dilution was transferred to 9 ml water blank to get 10⁻³ dilution. Likewise, the samples were diluted serially with 9 ml water blanks until appropriate dilution was obtained.

The bacteria were enumerated by plating one ml of 10⁻⁵ dilution in the sterile Petri dishes using nutrient agar medium. The colonies appearing on the plate after 48 h of incubation at 30°C were counted and expressed as number of CFU per g of soil. Fungi were enumerated by plating one ml of 10⁻⁴ dilution in the sterile Petri dishes using Martin’s rose Bengal agar medium. The colonies appearing on the plate after 2-3 days of incubation were counted and expressed as number of CFU per g of soil. For actinomycetes, one ml of 10⁻⁶ dilution was transferred to sterile Petri dishes and plated in Kenknight’s agar medium and incubated. The colonies of actinomycetes that appeared after 10-14 days were counted and expressed as number of CFU per g of soil.

Isolation of diazotrophic and phosphate solubilizing microorganisms: The dinitrogen utilizing microbes such as Azotobacter, Azospirillium and phosphate solubilizing microorganisms were isolated from rhizosphere soil samples collected from various locations viz Rangapuram, Athiyandhal, Karumandurai, Balapuram and Attur.

Data in Table 1 show that among the locations, Balapuram rhizosphere soils recorded maximum population of bacteria (31.0 × 10⁵ CFU/g soil) and Rangapuram the minimum (16.0 × 10⁵ CFU/g soil). The difference between bacterial population in rhizpsphere and non-rhizosphere soils was also the highest at Balapuram (11.0 × 10⁵ CFU/g soil) and lowest at Rangapuram and Karumandurai (6.0 × 10⁵ CFU/g soil each). The highest fungal population was recorded in the rhizosphere soils at Attur (33.0 × 10⁴ CFU/g soil) and the lowest at Balapuram (21.0 10⁴ CFU/g soil). The difference between fungal population in rhizosphere and non-rhizosphere soils was also highest at Rangapuram (14.0 × 10⁴ CFU/g soil) and the lowest at Balapuram (7.0 × 10⁴ CFU/g soil). Maximum population of actinomycetes was observed in rhizosphere soils at Rangapuram (24.0 x 10⁶ CFU/g soil) and minimum at Attur (10.0 x 10⁶ CFU/g soil). The difference between actinomycetes population in rhizosphere and non-rhizosphere soils was also recorded highest at Rangapuram (8.0 x 10⁶ CFU/g soil) and lowest at Attur (3.0 x 10⁶ CFU/g soil).

The results show that Balapuram rhizosphere and non-rhizosphere soils were most rich in bacterial but poor in fungal population; Rangapuram rhizosphere and non-rhizosphere soils were most rich in actinomycetes but poor in bacterial population and Attur rhizosphere and non-rhizosphere soils were most rich in fungal but poor in actinomycetes population, among the five locations.

Data in Table 2 depict that the bacterial population of Azototobacter, Azospirillum and Phosphobacteria was recoded maximum 31.0, 36.0 and 34.0 × 10⁵ CFU/g soil in the rhizosphere soils at Athiyandal, Balapuram and Attur and minimum 14.0, 18.0 and 17.0 at Rangapuram, Attur and Athiyandal respectively. Thus the Azototobacter population was highest and Phosphobacteria population was lowest in the rhizosphere soils of Athiyandal, whereas, Phosphobacteria population was highest and Azospirillum population was lowest in the rhizsphere soils of Attur, among all the locations.

Data given in Table 3 exhibit that the mean rhizosphere and non-rhizosphere soils ratio of microflora was maximum (1.64) at Rangapuram and minimum (1.44) at Athiyandal. The rhizosphere and non-rhizosphere soils ratio of fungi and bacteria was maximum at Rangapuram (1.82 and 1.60 respectively). The rhizosphere and non-hizosphere soils ratio of fungi was minimum (1.50) at Balapuram and of bacteria at Karumandurai (1.37). The rhizosphere and non-rhizosphere soils ratio of actinomycetes was maximum (1.80) at Karumandurai and minimum at Athiyandal, Balapuram and Attur (1.40 each).

Highest pH of 6.57 each was recorded in rhizosphere at Karumandurai and Attur and lowest 6.41 at Balapuram (Table 4). The difference between pH of rhizosphere and non-rhizosphere soils was highest (0.38) at Rangapuram and lowest (0.08) at Balapuram. Highest EC in rhizosphere soils and difference between EC of rhizosphere and non-rhizosphere soils (1.10 and 0.36 dS/m respectively) were recorded at Athiyandal and lowest (0.79 and 0.06 dS/m respectively) at Karumandurai. Organic carbon in rhizosphere soils was recorded maximum at Athiyandal and Balapuram (0.87% each) and difference between organic carbon of rhizosphere and non-rhizosphere soils was maximum (0.17%) in rhizosphere soils of Athiyandal. The difference between organic carbon of rhizosphere and non-rhizosphere soils was minimum at Karumandurai and Balapuram (0.10% each).

Data in Table 5 indicate that available N in rhizosphere soils was maximum (459.94 kg/ha) at Karumandurai and minimum at Rangapuram (192.33 kg/ha); the difference in available N between rhizosphere and non-rhizosphere soils was maximum at Attur (115.05 kg/ha) and minimum at Athiyandal (27.70 kg/ha). Available P in rhizosphere soils was maximum (16.42 kg/ha) at Attur and minimum at Rangapuram and Balapuram (11.94 kg/ha each); the difference in available P between rhizosphere and non-rhizosphere soils was maximum at Attur (3.00 kg/ha) and minimum at Balapuram (0.37 kg/ha). Available K in rhizosphere soils was maximum (280.00 kg/ha) at Karumandurai and minimum at Attur (238.93 kg/ha); the difference in available K between rhizosphere and non-rhizosphere soils was maximum at Balapuram (33.74 kg/ha) and minimum at Attur (21.47 kg/ha).

The higher number and occurrence of more microflora in the rhizosphere soil compared to non-rhizosphere soil was reported by Olahan et al (2016) in jute and Bopaiah and Shetty (1991) in pepper, cacao, pineapple and coconut. Li et al (2016) reported that all soil nutrient contents and microbial diversity indices were higher in rhizosphere soil compared to non-rhizosphere soil. Aparna et al (2020) also found that the microflora was more in rhizosphere than in the non-rhizosphere.

The photosynthetically fixed carbon that enters the soil ecosystem as plant biomass and root exudates is the prime source of energy for microbes. Hence, maximum microbial activity occurs in an ecosystem encompassed by the soil and root interface that is rhizosphere. Thus the provision of energy to the microbial community by root exudates, dead roots and sloughed cells results in an intense microbial activity and microbial interaction in rhizosphere over non-rhizosphere soils. The quality and quantity of microbial community changes with the growing season of the plants, kind of plants and locations. Hence, microbial habitats are produced continually through root growth. Thus understanding the biological activities of a particular rhizosphere provides a model for successful modification of native microbial community dynamics.

The microbial dynamics in red sanders rhizosphere soils were collected from various locations and compared with non-rhizosphere soils. In general, the density of bacteria, fungi and actinomycetes was higher in rhizosphere soils.

The soils of Attur had the lowest population of actinomycetes owing to their acidic pH (Meiklejohn 1957, Alexander 1977).

The wide variation between bacterial and actinomycetes population among locations and between rhizospher and non-rhizospher soils might be due to varied physico-chemical properties of the soils viz pH, organic carbon and available nutrients. This finding is in accordance with the report of Shail and Dubey (1997) who reported that changes in average number of fungi and bacteria in Kumaun Himalaya soils were positively correlated with soil moisture and negatively with soil pH.

The highest available nitrogen and organic matter content of the Athiyandal and Balapuram soils might be responsible for greater bacterial and actinomycetes observed in the study area. The dinitrogen fixing bacterial isolates viz Azotobacter and Azospirillum were obtained from various locations of red sanders rhizosphere. Among the various diazotrophs, Azospirillum dominated at all the locations except Attur since Azospirillum is a neutral preferring nitrogen fixer (Alexander 1977).

Athiyandal soils harboured greater number of Azotobacter which could be due to higher organic matter content of the Athiyandal soils. Similar to the present study, the occurrence of Azotobacter and Azospirillum in various tropical forests has been reported by many workers (Nagaraj 1989, Paul 1999, Venkatachalam 2003, Vennila et al 2023).

Insoluble inorganic compounds of phosphorus are largely unavailable to plants, but many microbes bring the phosphate into solution. Hence, these microbes dominate in fertile soils that is, soils low in available phosphorus. In the present investigations, maximum phosphate solubilizing microbes were obtained from the soils of Attur whose available phosphorus content was the least among the locations studied.

The rhizosphere effect or the degree of stimulation of an activity of population by the plant root was described by evaluating the ratio of activity per unit weight of rhizosphere soil and the activity per unit weight of non-rhizosphere soil. The values greater than one indicate selective stimulation in the rhizosphere; equivalence of the two activities suggests no rhizosphere effect, whereas, R/S ratio of less than one reveals inhibition of the activity in the rhizosphere. In the current study, the R/S ratio of microbes’ population was more than one at all locations. This might be due to the presence of numerous antagonistic bacteria at this location. Even though the values are more than one, the ratio should be more than 10 for their effective activity (Rao 1997).

It can be concluded that there were differences in bacterial, fungal and actinomycetes population size in rhizosphere and non-rhizosphere soils of red sanders. In general, the dynamics of microflora varied widely among locations as well as between red sanders rhizosphere and non-rhizosphere soils. The density of bacteria was found to be higher in Balapuram rhizosphere and fungal population in Attur rhizosphere soils. Among the red sanders rhizosphere, Athiyandal harboured greater number of microbial population while Rangapuram and Balapuram rhizosphere soils exhibited on bar microbial dynamics. Comparing rhizosphere and non-rhizosphere soils, non-rhizosphere soils were microbially more diverse. The knowledge obtained from the present study would be of help in understanding the natural changes of the microbial population size in rhizosphere soils under red sanders.

R: Rhizosphere soils, S: Non-rhizosphere soils

R: Rhizosphere soils, S: Non-rhizosphere soils

R: Rhizosphere soils, S: Non-rhizosphere soils

References