Endophytic actinomycetes diversity in the Tomato plant was explored in this study. The surface sterilization process is the basic step to isolate and purify the endophytes. The addition nystatin and cycloheximide (at 50 µg/ml supplemented with ISP2 agar plate surface-sterilized sample showed no microbial contamination. In addition, ISP2 agar plates spread with the last washed water also did not have microbes. The results show the surface sterilization protocol is very effective. Similar validation of sterilization was done by Cao et al., (2004)
A total of forty-five plant samples were randomly collected from fifteen different agro sites. The sampling sites are shown in Fig 1. The highest number of actinomycetes were recorded in Madurai (54.16%) followed by Dindugal (16%) and the lowest count was recorded in Tuticorin (8.33%) (Fig 2).
Fig 1: Showing the different locations from where tomato plants were collected for actinomycetes isolation.
Fig 2: A pie chart illustrating the total number of actinomycetes strains isolated from different districts of Tamil Nadu.
Among the two methods tested, the direct streaking method was best with 80% (192) collection while serial dilution yielded 20% (48) (Table 1). Thus, a total of 240 isolates were obtained. Madurai recorded the highest number of isolates in serial dilution and direct streak method 20 and 110 of isolates, while the lowest no of isolates were recorded in Dindugal 4 by serial dilution method) and in Tuticorin 12 by direct streaking method (Fig 3). The results were recorded in Table 2. The microbial colonies were segregated based on different morphological characters. They are separated and purified as separate strains (Fig 4). As for the isolation methods, sample streaking had the best result compared with grinding plant parts and spreading. As we directly streak plant parts and placed plant parts in the ISP2 media, the positive environment around the plant parts might favour the endophytes to come and colonize. While grinding plant parts could be stressful leading to sudden change in environment thus a low number of colonies. The serial dilution plate count method for actinomycetes isolation from crushed plant extract was cultured by serial dilution technique. From this method, only 48 different colonies were isolated and purified. Previously Costa et al., (2012)
isolated 158 endophytic bacteria from the bean in a TSA (Tryptic Soy Agar) medium by serial dilution technique. Similar isolation of endophytic bacteria from the leaves of Anredera cordifolia
CIX1 was reported by Nxumalo et al., (2020).
By placing the cut plant parts, helps enlarged colonies attracting all actinomycetes to come out of plant parts and colonize in the ISP2 medium. This result coincided with Tan et al., (2006)
reported as 619 actinomycetes were isolated from tomato plants by the plant streak method.
Table 1: Showing samples that were collected from different districts, their code and number of strains.
Table 2: Showing the number of endophytic actinomycetes count from serial dilution method and direct streaking method from different districts.
Fig 3: Growth of Actinobacterial colony for isolation by serial dilution method and streak plate method.
Fig 4: Purified endophytic actinomycetes isolate colonies in ISP2 medium.
Based on the morphological analysis was performed for all 240 endophytic isolates, they are categorized as aerial hyphae, colony appearance and pigment producing strains. The total number of isolates and their category is given in Table 3. According to the preliminary morphological identification, the most abundant genus was Streptomyces
sp. a similar finding was reported for different hosts plant wheat by Coombs and Franco (2003)
and neem plant Verma et al., (2009).
Accordingly, there were more aerial hyphae (49.5%) followed filamentous (45%) and less pigment producing strains (6%).
Table 3: Different morphological characteristics of actinomycetes count from different sites of Tamil Nadu.
These isolates belong to the different genera of actinomycetes. Out of 240 isolates, 23.33% (n=56) are small-sized colonies, 22.08% (n=53) are large-sized colonies, 22.08% (n=53) rough-surfaced colonies, 10.83% (n=26) smooth-surfaced colonies, 7.5% (n=18) dry textured colonies, 8.3% (n=20) viscid textured colonies and 5.8% (n=14) pigment-producing colonies appeared in ISP2 agar medium (Table 3).
Three major criteria were analysed in the growth pattern of actinomycetes in ISP2 and results were recorded in Table 3. The first condition for growth is aerial hyphae, which is recorded in all the districts 49.5% (n=119). Among these, the highest aerial hyphae colonies (n=80) were recorded in Madurai (33.3%) while the lowest number (n=8) was recorded in Tirunelveli (3.3%). This study coincides with (Basavaraj et al., 2010)
reports such as morphological and cultural characteristics of the strain actinomycetes A-4 showed cellular and aerial growth as well as soluble pigment formation in various ISP media.
The second condition is filamentous appearance found in 44.58% (n=107) endophytic strains. Madurai recorded highest as 19.1% (n=46) and Tuticorin lowest with n=8. The filamentous appearance was continuous, and connecting producing aerial or substrate mycelium. Similar observation was reported in the diversity of filamentous soil actinomycetes by Chaudhary et al., (2013).
Similar results have been reported by (Karkouri et al., 2019)
from their studies associated with 22 different actinomycetes sp. using ISP2 agar medium. The entire actinomycetes diversity from all the collected plants in different districts sites where a major significant quantity of actinomycetes was reported in MSS (130).
A total of 14 (5.83%) pigment-producing strains were recorded in (Table 3). ISP2 medium supports the growth and pigment production of versatile growth types of actinomycetes (Fig 5). Actinomycetes are capable of producing coloured substances certain actinomycetes produce melanin pigments. This study covers 14 morphologically different actinomycetes producing different pigments (Fig 4, shows different shades). This study coincides with Srinivasan et al., (2016)
in actinomycetes strain Ac 14 with grey color aerial mycelium, dark brown substrate mycelium and produces diffusible dark brown pigment.
Fig 5: Sample plates showing colonies of pigment-producing different actinomycetes.