​Diversity of Actinomycetes in Tomato Plants

 

Tomato (Lycopersicon esculentum) belongs to the genus Lycopersicon under the Solanaceae family. Tomato is one of the most important “protective foods” because of its special nutritive value. It is one of the most versatile vegetables with wide usage in Indian culinary tradition (Helaly, 2021). Tomato is an important economic crop grown worldwide that is of commercial value and ecological importance. Tomato is the world’s largest vegetable crop after potato and sweet potato, and it tops the list of canned vegetables. The total global area under tomato was 46.16 lakh ha with global production of 1279.93 lakh tonnes. All India production estimate of Tomato in 2021 was estimated at 21 Metric Tonnes, estimated to have amounted to 852 thousand hectares. The cultivation area increased from the previous fiscal year. India ranked second on the list of nations producing tomatoes during the measured time period (Statista.com). 

Endophytic microbes are a diverse group of bacteria, fungus, and actinomycetes that live inside plant tissues (Arini et al., 2021). Among these endophytes, actinomycetes are that associated with plants have played a crucial role in protecting the host from phytopathogenic invasions (Crawford et al., 1993). Several endophytic actinomycetes act as a plant growth promoter by producing plant growth hormones like Indole3-Acetic Acid (IAA) or iron-chelating molecules (Rungin et al., 2012). Endophytic microorganism has been in association for millions of years with their eukaryotic hosts, from lower crops to higher plants, representing an important increasing resource of new secondary metabolites (Firdous et al., 2019). In the last decade, approximately half of the newly discovered (5000 compounds) metabolites were isolated from endophytic strains. Antibiotic and vitamins-producing actinomycetes are beneficial for the plants physiological processes (Trajkovic et al., 2018). Plant endophytic microbes, often exhibit plant identities and plant second genes (Michelmore et al., 2017). These endophytes can live within cells, vascular system, or the capillary cells of the plant (Maggini et al., 2017). They are beneficial to the host plant, e.g., biological control of plant diseases (Liu et al., 2017). Therefore, it is important to have a good understanding of the endophytic actinobacterial communities in the tomato plant. Endophytic Streptomyces sp. also provide advantages to the host plant by enhancing the physiological activity of the plant or through other modes of action and thus may serve as a source of agro-active compounds, biocontrol agents, or plant growth promoters (Fadiji and Babalola, 2020; Vurukonda et al., 2018). The possibilities of using these endophytes as biological control agents against tomato speck, wilt, fusarium crown, root rot are very high (Nandhakumar et al., 2020). Endophytic actinobacteria have attracted attention in recent years, with increasing reports of isolates from a range of plant types, including crop plants (cereals, such as wheat and rice, as well as potatoes, carrots, tomatoes and citrus (Singh and Dubey, 2018) and medicinal plants (Gos et al., 2017). Therefore, the present study aims to screen the diversity of endophytic actinomycetes in tomato plants of southern Tamil Nadu.

Tomato plant sample collection
 
Healthy tomato plants (Lycopersicon esculetum) were collected from five different districts (Tirunelveli - 8.7815° N, 77.3942°E; Tuticorin 9.1727°N, 77.8715°E; Madurai - 9.9420°N, 77.9724°E; Dindukal - 10.4489°N, 77.9360°E and Theni - 10.0015°N, 77.6164°E) of southern Tamil Nadu in India. Plants were collected from three different places for each district. And three samples from each farm at the fruiting stage. Three tomato plants were randomly collected from the corner and centre of the field. Thus, a total of fifteen plants were collected in a sterile polypropylene bag and brought directly to the laboratory for microbiological processing. Thus, 45 samples (3 from each site) were collected from different areas/locations of Tamil Nadu.
 
Sample preparation
 
The collected plants were washed in running water to remove all adherents. Each isolation procedure was done in triplicate for each plant sample. The plant samples were cut to 4-5 cm length using the sterile surgical cutter. The plant parts were shoot tip, stem (upper and lower regions), root-(upper and lower regions). The disinfection and isolation of actinomycetes were performed according to Araújo et al.,  (2000) with minor modifications. The plant parts were disinfected superficially disinfected as 70% alcohol for 1 min, 90% ethanol 1 min, sodium hypochlorite (0.9%) for 4 min, 70 ethanol for 30 seconds, 10% NaHCO3 for 5 min and finally rinsed in sterile, distilled water. Sterile distilled water wash was done for 5 minutes for every change in the solution. To ensure the disinfection protocol, an aliquots of the sterile water used in the final rinse were plated in an ISP2 medium (Yeast malt extract agar- yeast extract - 4.0 g malt extract - 10.0 g dextrose - 4.0 g agar 20.0 g).
 
Actinomycetes isolation
 
Two different methods were followed to evaluate and to get the maximum number of actinomycetes from tomato plants.
 
Serial dilution method
 
Small pieces of shoot tip, stem, root were separately ground separatively with 6 mL of aqueous solution (0.9% NaCl) in a sterile mortar and pestle. The tissue extract was subsequently incubated at 28°C for 3 hours to allow the complete release of endophytic microorganisms from the host tissue. For the isolation of endophytic bacteria, the tissue extract was diluted in an aqueous solution and plated on five ISP2 agar plates supplemented with nystatin and cycloheximide (at 50 µg/ml) for each dilution (from 10-1 to -10-5). The plates were incubated for 15 days at 30°C. Colonies were selected on 7^th, 10^th, 12^th and 15^th days of incubation. The isolated colonies are pure cultures of actinomycetes that were separated on the following methodology, time of appearance, growth rate, and morphology (color, shape, and size). All of the colonies were counted and expressed as CFU (Colony Forming Unit) per one gram of fresh tissue.
 
Direct streaking method
 
The washed and surface-sterilized stems were cut in a cross direction and streaked on the ISP2 agar supplemented with nystatin and cycloheximide (at 50 µg/ml) incubated at 30°C for 7^th, 10^th, 12^th and 15^th days. Thus, actinomycetes alone were allowed to grow and separated as specified in the previous serial dilution method.
 
Identification
 
Each petri dish was evaluated; the colonies were selected according to their time of growth and morphology (color, size and shape). Different strains were identified based on the morphological characteristics of colonies on the plate, areal hyphae on the agar plate, the morphology of spores, and pigment production. Isolates were labelled serially as VITGV. These separated strains were purified and maintained in glycerol suspensions (30% v/v) at -80°C.

Actinomycetes isolation
 
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) for Streptomyces sp.

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).



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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.









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%).



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.

Tomato plants collected from selected locations of Tamil Nadu contains a total of 240 different species of endophytic actinomycetes. Out of which some 5 to 10% are expected to be new species. On an average, a tomato plant harbours 25 to 30 different species of actinomycetes. As actinomycetes are well-known organisms for bioactive compounds, there could be a large symbiotic association ensuring plant protection. The presence of certain pigment-producing species further opens the way to explore their genetic relations.

This work is funded by VIT SEED GRANT of Vellore Institute of Technology, India.

This article does not contain any studies with human participants or animals performed by any of the authors.