Introduction
The aerial habitat colonized by microbes is known as the phyllosphere, and the inhabitants are called epiphytes. There are several studies on the inhabitants of flowers and buds [1]. Microbes can be found in different segments of plants as epiphytes on the surface and as endophytes inside plant tissues [2-4]. Most of the research on phyllosphere microbiology has focused on leaves. The phyllosphere environment is commonly occupied by diverse microbes, such as bacteria, filamentous fungi, and yeast, where bacteria are the dominant microorganisms. Bacteria are the most abundant microorganisms on leaves, ranging from 106 to 107 cells/cm2[2,5]. The phyllospheremicrobes facilitate carbon dioxide fixation and the release of molecular oxygen and thus facilitate primary productivity. They can also fix N, absorb minerals,and decrease disease severity [6]. Phyllosphere microbial communities are significantly different from airborne microbial communities, although they are in direct contact with the atmosphere surrounding plants. Bacterial endophytes normally complete their life cycle within host plants without causing damage to the plant, but their multiplication might be limited by the innate immune system of host plants [7]. The phyllosphere can be further subdivided into caulosphere (stems), phylloplane (leaves), anthosphere (flowers), and carposphere (fruits). Various types of microorganisms are found on plant surfaces and in the surrounding environment. Some are beneficial, while others are harmful to plants as plant pathogens [8]. Each leaf creates a specific environment in which only some microorganisms can thrive. This selective effect of leaves on microbial communities can be termed the phyllosphere effect. Phyllosphere interactions depend on environmental factors, host genotypes, and microbial communities. Factors such as water availability, leaf surface topology, nutrient availability, leaf age, plant species, and leaf physiology. On the surface of the leaves, different morphological parts, such as trichomes, veins, cell wall junctions of the epidermis and stomata, are abundant in the microbial community. Therefore, these regions are protected by phyllosphere bacteria from UV radiation as well as other environmental extremes [8-10].
Most phyllospheric bacteria act as commensals without any known effect on their plant host, but multiple bacteria establish a mutualistic relationship with plants. Host plants supply nutrients and provide shelter to bacteria, and bacteria can promote the growth of host plants and confer resistance against insects, pests, and pathogens. The phyllosphere environment is commonly occupied by diverse microbes, such as bacteria, filamentous fungi, and yeast, where bacteria are the dominant microorganisms. Plant growth is promoted by phyllosphere bacteria. Phyllosphere bacteria can suppress and stimulate the colonization and infection of tissues by plant pathogens [4,11]. The global surface area of the phyllosphere has been estimated to be more than 4 x 108 km2, with bacterial populations in this region of 1026 cells [12]. Different bacteria have been isolated from different plant sources, such as carrots, tomatoes, soybeans, cabbage, citrus, pineapple, clover, maize, and wheat [13].Epiphytic bacterial populations differ significantly in size within the plants of the same species [14].These variations are caused by fluctuations in the leaf environment and nutritional conditions.The leaf bacteria differs from the root bacteria. Previous studies showed that pigmented bacteria which are rarely found in the rhizosphere are observed to dominate the leaf surfaces [15].
Materials and methods
Sample collection was performed in two seasons: summer (August) and rainy (January). Three healthy leaves of maize (Zea mays), tomato (Solanum lycopersicum),and cabbage (Brassica oleracea var. capitata) were collected in sterile UV bags from separate fields at the Thirunelvely Agricultural Research Station and Thinnai Organic Farm using a random sampling method. Some parameters of the field, such as temperature, humidity, longitude, and latitude, were measured. The collected samples were brought to the laboratory in ice bags.
Isolation of phyllosphere bacteria
The collected leaves were cut into equal sizes by using a sterile scalpel and imprinted on nutrient agar (NA) media. Replicates were also prepared for each sample. Then, the plates were labeled and incubated at 37°C for 24-48 hours to observe bacterial growth.
Subculturing of phyllosphere bacteria
Surface and hand sterilization were performed using a spirit homogenizer. Spirit lamps wereused.The individual bacteria that grew from the edges of the leaf segments were removed by using a sterile inoculating loop and streaked on fresh NA media. Then, the plates were incubated at 37°C for 24-48 hours. Single colonies of isolates were purified by subculturing on nutrient agar media, and the process was repeated until pure bacterial isolates were obtained, as confirmed by microscopic examination.
Storage of bacterial cultures
After the growth of pure bacterial isolates on plated nutrient agar media, they were streaked on nutrient agar slants and stored in a refrigerator at 4°C-5°C. These stored bacterial cultures were sub-cultured in NA media and used whenever they were required for the assay.
Identification of phyllosphere bacteria by colony morphological characteristics and biochemical tests
Colony Morphological study was done on the basis of shape, elevation, texture, margin, colour, size and pigmentation of colonies. The phyllosphere bacteria were grown on NA medium and incubated at 37o C for 24-48 hours.
Preliminary tests were done, such as Gram staining, endospore staining, capsule staining, motility test, catalase test, and oxidase test.
Biochemical tests such as starch hydrolysis, sugar fermentation tests, citrate utilization tests, gelatin hydrolysis, nitrate reduction test, indole test, and MR-VP test were carried out.
Results and Discussion
The selection of representative colonies was based on their growth on NA media, staining, and microscopic examination. Phyllosphere bacteria were identified through culture and morphological tests (Fig. 1, Table 1, and Table 2), and their biochemical properties were also studied (Table 3).
B1 was isolated from Zeamays and Solanum lycopersicum. B2 was isolated from Zea mays. B3 was isolated from Solanum lycopersicum and Brassica oleracea var. capitata. B4 and B5 were isolated from Brassica oleracea var. capitata. P1 was isolated from Zea mays. S1 was isolated from Zea mays. S2 was isolated from Solanum lycopersicum. L1 was isolated from Brassica oleracea var.capitata, and M1 was isolated from Brassica oleracea var. capitata.
The results from the biochemical characterization are presented in Table 3. The microbial communities of the phyllosphere are diverse, supporting numerous genera of bacteria, filamentous fungi, yeast, algae, and, in some situations, protozoans and nematodes [4]. Bacteria are the most numerous and diverse colonists of leaves, with culturable counts ranging between 102and 1012 cells/g leaf. Ten isolates from the phyllosphere were identified based on biochemical tests and colony morphology as described in Bergey’s Manual of Determinative Bacteriology.
The identified phyllosphere microorganisms are shown in table 1, table 2, and table 3.
B1- Bacillus cereus
B2-Bacillus pumilus
P1-Pseudomonas putida
S1-Staphylococcus equorum
B3-Bacillus subtilis
S1-Staphylococcus epidermidis
L1-Lactobacillus brevis
B4-Bacillus mycoides
M1-Micrococcus luteus
B5-Bacillus licheniformis
UV bags were used to collect the leaf samples to prevent contamination. An ice box was used to prevent dryness of the samples. Healthy leaves were selected for the isolation of beneficial phyllosphere bacteria. A random sampling method was used to collect samples to confirm the uniform distribution of particular isolates. To assess seasonal variation, samples were collected in the summer and rainy seasons. Organic fields were selected to obtain the natural microbial population.
The leaf imprint technique was used to isolate phyllosphere bacteria. This approach is a favorable method for isolating epiphytic microorganisms. Bacillus cereus, Bacillus pumilus, Pseudomonas putida,and Staphylococcus equorum were isolated from Zea mays. Bacillus cereus, Bacillus subtilis, and Staphylococcus epidermidis were isolated from Solanum lycopersicum. Bacillus subtilis, Lactobacillus brevis, Bacillus mycoides, Micrococcus luteus and Bacillus licheniformis were isolated from Brassica oleracea var. capitata.
These ten isolates can be confirmed at the species level by molecular identification in future studies.
The phyllosphere represents a group with immense agricultural and environmental significance. Many studies have investigated the important interactions of phyllosphere microorganisms that may affect the fitness of plant populations and the productivity of crops. Phyllosphere bacteria can promote plant growth and may suppress the colonization of tissues by plant pathogens. Further studies should be performed to confirm whether phyllosphere bacteria protect plants from herbivores and whether they can promote drought tolerance in plants. If this study is performed, it would be beneficial to tropical countries that have long-term dry climates.
Acknowledgments
Not applicable.
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