INTRODUCTION Actinobacteria are a group of prokaryotic microorganisms and are Gram-positive with high G+C content in their DNA (Lo et al., 2002). They are widely distributed in soil, water and plants. They are aerobic organisms and are well known for the production of secondary metabolites. The most important and dominant genus within Actinobacteria is Streptomyces (Ceylan et al., 2008). Streptomyces provides more than half of the naturally occurring antibiotics (Bérdy, 2005) with high commercial value and continue to be routinely screened for interesting bioactive substances (Takahashi, 2004; Meena et al., 2013). The actinomycines represent an important class of natural products that continue to be a focus of many research areas (Kurosawa et al., 2006; Praveen et al., 2008). Actinobacteria isolated from soil samples have the potential to inhibit the growth of plant pathogens and improve the plant health (Jeffrey et al., 2007; Kekuda et al., 2010). Few examples of Actinobacteria used to manage Citrus diseases are Streptomyces chumphonensis against Citrus green mould caused by Penicillium digitatum, Streptomyces sp. against Citrus canker, Citrus foot rot and Citrus nematode etc. Xu et al. (2018) reported the structure and function of the global Citrus microbiome from bulk and rhizosphere soils of Citrus spp. in six continents, accordingly dominant prokaryotic phyla reported were Proteobacteria, Actinobacteria, Acidobacteria, and Bacteroidetes. Li et al. (2021) reported that the microbial therapy using Actinobacteria, Yeast, Rhizobium, along with other beneficial bacteria, and plant growth regulators is promising in managing greening disease in Citrus. In accordance with these reports, the present study was carried out to explore the rhizosphere of healthy acid lime trees for isolation of actinobacteria and to choose the potential candidates among them for plant growth promotion and disease management in acid lime crop. MATERIALS AND METHODS Collection and pretreatment of soil samples. Rhizosphere soil samples from healthy acid lime trees were collected during the survey conducted in various districts of North Tamil Nadu such as Vellore, Tiruppattur, Ranipet, Kanchipuram, Tiruvallur, Dharmapuri, Krishnagiri, Perambalur, Ariyalur and Namakkal. The survey was taken up during March to April, 2021. Collected soil samples were air dried separately for one week to reduce the population of Gram negative bacteria and were sieved separately. Heat treatment of the samples was done by placing them in hot air oven at 121°C for 1 hr to prevent the growth of other bacteria (Gurung et al., 2020). Isolation and characterization of actinobacteria. Isolation of actinobacteria from pretreated soil samples was carried out by the serial dilution and plating technique (Aparanji and Venkata 2013) using Starch Casein Agar (SCA) medium (Küster and Williams, 1964). It was prepared by adding the required components in distilled water and boiled to dissolve the content completely. The prepared medium was sterilized by autoclaving at 15 lbs pressure (121°C) for 20 minutes. From each preprocessed rhizophere soil samples, one gram of soil was taken and added to a test tube containing nine ml sterile water and shaken vigorously using an orbital shaker at 200rpm for 10 min at 25 ± 2°C and these test tubes were considered as stock culture for the soil samples. Then, one ml of aliquot from the stock solution was transferred aseptically to a test tube containing nine ml of sterile water and mixed well. From this test tube, one ml of aliquot was again transferred and mixed with another nine ml of distilled water to make 10-3 dilution. Similarly, serial dilutions up to 10-6 were made for all soil samples (Oskay et al., 2004). One ml of aliquot from the aqueous dilutions of 10-2 and 10-3 was taken and spread onto the Petri plate containing SCA medium. The plates were incubated at 28°C, and observed from 5th day onwards to 20th day. After incubation, colonies with suspected actinobacteria morphology were sub-cultured on SCA medium and incubated at 28°C for 2 to 5 days. Pure cultures were maintained in SCA slants and stored in glycerol broth at 4°C for further studies. Cultural and morphological characteristics of actinobacteria were recorded (Basavaraj et al., 2010). Biochemical characterization of actinobacteria. Reaction of actinobacteria isolates with Gram stain was assessed and biochemical tests such as citrate utilization test, starch hydrolysis test and catalase test (Sapkota et al., 2020) were carried out. For Citrate utilization test, Simmons citrate agar medium was poured on sterile Petri plate and allowed to solidify. The isolates were inoculated and incubated for 5-7 days at 35°C to 37°C. Colour change in the reaction was observed. Starch hydrolysis test was performed by streaking a loop ful of colony at the centre of the Petri plate containing Starch agar medium and incubated upto seven days at 37°C. After incubation 2-3 drops of 10 per cent iodine solution was added directly onto the edge of colonies. The observation was recorded after 10-15 minutes. Similarly, Catalase reaction was tested with a loop full of actinobacteria colony, which was transferred to the surface of a clean, dry glass slide. A drop of H2O2 (3%) was added on the culture and observed for the development of oxygen bubbles. RESULTS AND DISCUSSION Rhizosphere soil samples were collected from matured, healthy acid lime trees belonging to the age group starting from 6 yrs to15 yrs in the orchards located in North Tamilnadu, India. Ten actinobacteria species viz., ACT- VEL-1, ACT- TPT-2, ACT- RAP-3, ACT- TRV-4, ACT- KAP-5, ACT- PER-6, ACT- DAM-7, ACT- KRG-8, ACT- NAM-9 and ACT- ARL-10 were isolated and maintained for further studies in Starch Casein Agar (SCA) medium (Table 1). In agreement with these findings, Xu et al. (2007) isolated various species of actinobacteria and reported that rhizosphere soils were the rich source of beneficial microbes, where prokaryotes dominated and actinobacteria played important role in symbiotic association with crop roots. Tindall et al. (2010) described the polyphasic approach for characterization of actinobacteria that depicted the variety of phenotypic, chemotaxonomic, and genotypic data and most actinobacteria were characterized and classified on the basis of their morphology Since, the morphological characteristics were one of the most basic indexes providing in-depth information on a taxon, cultural characteristics of isolated actinobacteria were documented in this study. The isolated 10 actinobacteria isolates were observed over their growth on SCA medium. Initially, they produced the colonies with smooth surface and on maturation their colony morphology and colour expressed variations. Colony colour of the ten isolates was found to be in different shades of grey and white. After maturation, the colony colour at the reverse side of the Petri plates showed sharp variation. The isolates viz., ACT- VEL-1, ACT-RAP-3, ACT-KAP-5, ACT-PER-6 and ACT-KRG-8 were produced different shades of yellow colour colonies, ACT-TPT-2 and ACT-TRV-4 produced pale pink and cinnamon brown colour colonies respectively. Isolates, ACT-DAM-7 and ACT-ARL-10 produced strikingly different colour colonies with violet and pale violet colour. Isolate ACT-NAM-9 alone had grey colour colonies. Similarly, Van Thanh et al., (2019) isolated 26 endophytic actinobacteria from Horsetail plant and were classified into five color groups as White, Grey, Pink and Brown and Blue based on the color of sporulating aerial mycelium. Among them, Grey group accounted for the biggest portion with 12 strains followed by White group and Brown group. This study also reported that the colony colour of the isolated ten actinobacteria were grouped into two as grey and white. Colony surface showed two different characters such as granular and powdery colonies; out of ten isolates, the following six isolates viz., ACT- VEL-1, ACT-RAP-3, ACT-KAP-5, ACT-PER-6, ACT-DAM-7 and ACT-KRG-8 had granulated colonies and the remaining four isolates, ACT-TPT-2, ACT-TRV-4,ACT-NAM-9 and ACT-ARL-10 produced powdery colonies. Likewise differences were also observed in colony texture. Colonies of the isolates like, ACT-TPT-2, ACT-PER-6 and ACT-DAM-7 were discrete, while ACT- VEL-1, ACT-RAP-3, ACT-KAP-5 and ACT-KRG-8 produced lichenoid colonies. The remaining three isolates such as ACT-TRV-4, ACT-NAM-9 and ACT-ARL-10 were observed to be with butyrous consistency. The observed characters were comparable with the findings of Basavaraj et al. (2010). They documented the antibiotic producing potential of many actinobacteria and studied their cultural characters using crowded technique. Accordingly the isolates exhibited the variations in colony colour from grey to white. Two types of colony texture viz., lichenoid and butyrous were reported among them. William Whitman and Aidan Parte (2012) also documented related findings in Streptomycetales members of actinobacteria and they explained that most of the actinobacteria colonies were discrete and lichenoid and few of them were leathery and butyrous. These colonies initially had a smooth surface, but later develop a weft of aerial mycelium, which appeared floccose and granular. Phan Thi Hong-Thao et al. (2016) also isolated endophytic actinobacteria from orange tissues and reported the similar observations on the colony characteristics. Accordingly the colony surface was powdery and curled. Aerial and substrate mycelia were grey to light brown or light yellow to brownish yellow on medium. Soluble pigments were found to seep into the medium of the isolates viz., ACT-TPT-2, ACT-DAM-7and ACT-ARL-10,but non soluble pigment production was observed with the other seven colonies as they retain their pigments in their colonies itself (Table 2; Plate1). Similarly, William Whitman and Aidan Parte, (2012) explained that Microtetraspora and Streptomyces strains produced diffusible melanoid pigments with diverse molecular structures that typically appear black or brown, red, yellow, orange, pink, brownish, distinct brown, greenish brown, blue, or black, depending on the strain, the medium used, and the age of the culture. Soluble yellow pigment production was observed on media viz., ISP2 and ISP3 by actinobacteria isolatedfrom orange crop (Phan Thi Hong-Thao et al., 2016). Biochemical characteristics of isolated actinobacteria. Biochemical characteristics were analyzed using Gram staining technique. The colonies of the 10 isolates were observed to retain the purple colour after Gram staining under light microscope which indicated that the isolates were Gram positive. It was well established by many studies that Actinobacteria species like Streptomyces, Micromonospora, Rhodococcus, and Salinisporas are Gram-positive bacteria with high G+C DNA content that constitute one of the largest bacterial phyla, and are ubiquitously distributed in both aquatic and terrestrial ecosystems (Muthu et al., 2013; Van Thanh et al., 2019). In Citrate utilisation test, the positive reaction was indicated by the change of colour from green to blue, which was due to the alkylation process by actinobacteria. The isolates viz., ACT- VEL-1, ACT- TPT-2, ACT- TRV-4, ACT- KAP-5, ACT- PER-6, ACT- KRG-8 and ACT- ARL-10 showed positive reaction whereas the isolates, ACT- RAP-3, ACT- DAM-7 and ACT- NAM-9 were negative for this test. Formation of clear zone around the colonies was observed for the positive result of starch hydrolysis test. Among the 10 isolates, seven isolates exhibited the positive reaction .The remaining three isolates like, ACT- TPT-2, ACT- PER-6 and ACT- NAM-9 were found to express negative reaction. When the colonies of all isolates were treated with H2O2 (3%), oxygen bubbles were observed. It indicated the positive reaction for catalase test (Table 3; Plate 2). Agreeing with these findings Sapkota et al. (2020) reported that Streptomyces spp belongs to the family Streptomycetaceae and the order Streptomycetales were aerobic, Gram-stain-positive, non-acid-fast bacteria that form extensively branched substrate and aerial mycelia. They were Catalase-positive and reduced nitrates to nitrites and degraded polymeric substrates such as adenine, gelatin and hypoxanthine. Both the morphological and biochemical characteristics revealed that out of ten isolates, five isolates viz., ACT- VEL-1, ACT-TRV-4, ACT-KAP-5, ACT-KRG-8and ACT-ARL-10 were Streptomyces spp. and further exploration on the plant growth promoters and antibiotics production by these Streptomyces spp. will definitely give a lead for improving the Citrus crop health.