Assessment of PGPR effects of wheat rhizosphere actinomycetes on tomato seedlings

Introduction

Wheat (Triticum aestivum) is the dominant crop in India and grown all over the world. Wheat belongs to family Gramineae and it is the most important staple food of about two billion people (36% of the world population). Wheat's domestication produced larger grains and a more productive crop, which could not have survived in the wild and required continued intervention of farmers intentionally planting it. It is used for human food and livestock. Wheat (Triticum species) is a crop of global significance and grown in diversified environments. Approximately one-sixth of the total arable land in the world is cultivated with wheat. The rhizosphere is the narrow zone of soil specifically influenced by the root system (Dobbelaere et al. 2003) [13] . This zone is rich in nutrients when compared with the bulk soil due to the accumulation of a variety of plant exudates, such as amino acids and sugars, providing a rich source of energy and nutrients for bacteria (Gray and Smith, 2005) [22] . In the context of increasing international concern for food and environment quality, the use of PGPR for reducing chemical inputs in agriculture is a potentially important issue. PGPR have been applied to various crops to enhance growth, seed emergence and crop yield, and (Dey et al., 2004; Minorsky, 2008) [11,28] . Plant growth promoting rhizobacteria (PGPR) is a group of bacteria that can be found in the rhizosphere (Ahmed et al. 2008) [2] , that colonize the roots of plants that enhance the plant growth. Actinomycetes are filamentous, Gram-positive, a diverse group of heterotrophic prokaryotes forming hyphae at some stage of their growth, hence referred to as filamentous prokaryotes. They are widely distributed in soils, especially in dry soils, slightly acidic soils and rich in organic matter represent a high proportion of biomass (Saadoun et al. 2015) [38] . Among all the actinomycetes, Streptomyces is the predominant genus followed by Actinomadura, Microbiospora, Micromonospora, Nocardia, Nonomurea, Mycobacterium, Frankia, Actinoplanes, Saccharopolyspora and Verrucosispora (Martinez-Hidalgo et al. 2014; Vijayabharathi et al. 2016) [27,44] . Actinobacteria also employ both direct and in-direct mechanisms to influence the plant growth and protection. The direct mechanisms involve the production of vital factors for crop growth such as growth hormones and the assistive actions on nitrogen fixation, phosphate solubilization, and iron acquisition. Plant growth promoting actinobacteria indirectly influence the plant growth by controlling and minimizing the deleterious effects of external stresses of either biotic or abiotic sources through the following modes: competition for nutrients, production of low molecular inhibitory substances such as ammonia, cyanogens, alcohols, aldehydes, sulfides, and ketones, cell-wall degrading enzymes, and secondary metabolites with biocidal properties, in which the latter, two are the key phenomenon deployed by the actinobacterial community (El-Tarabily and Sivasithamparam, 2006; Glick, 2012; Bouizgarne, 2013; Dey et al. 2014) [14,17,7,12] . Actinomycetes are used to develop commercial biocontrol and plant growth-promoting products. It is crucial to recognize the specific challenges they present. To begin with, the interaction between PGPR species and their plant symbionts appears to be specific, even within a crop or cultivar (Chanway et al. 1988; Glick, 1995; Lazarovits and Nowak, 1997) [8,16,26] . Therefore, the aim of my research was to explore the diversity of actinomycetes associated with wheat rhizosphere and their impact on tomato seedlings. This investigation holds significant implications for analyzing plant growth promoting abilities of actinomycetes and their role in enhancing plant growth.

Materials and Methods

Isolation of actinomycetes from the rhizospheric soil of wheat plant 2.1.1 Collection of rhizospheic soil sample

The rhizospheric soil samples were collected from Sri Muktsar Sahib, Punjab, total Five soil samples were collected from five different plant rhizosphere of wheat and mixed, to make it one composite sample. Similarly, four composite samples were taken from two different sites in Sri Muktsar Sahib district, Punjab. The rhizospheric soil sample along with roots pieces was collected around 10 cm apart and upto 1 -2 feet in depth. Composite soil samples were collected and brought to the laboratory in sterile polythene bags and stored at 4ºC for further studies. Before isolation, all the twelve composite samples were dried for 4-5 days at 28°C. Isolation was carried out by using serial dilution method using KenKnight agar and Starch Casein agar plate (Williams and Devies, 1965) [45] . Ten grams of each pretreated soil sample were mixed and shaken in 90 ml sterile distilled water for isolation. Pour plate technique was used for the isolation of actinomycetes, plates were incubated at 28 ± 2°C for 7-10 days. The total actinomycetes count was expressed as colony forming unit per gram (cfu/g soil) of each soil sample. The colonies were streaked for purification and further observations of colony morphological characterization.

Identification of actinomycetes isolates by morphological and biochemical characterization

All indigenous isolates were characterized by polyphasic taxonomy as per Bergy's manual. Isolates were examined for colony morphology (viz., form, elevation, margin etc) colony pigmentation, gram staining and various biochemical reactions as per standard procedures viz., Catalase, oxidase, indole production, MRVP, citrate utilization, starch hydrolysis, lysine decarboxylase, nitrate reduction, hydrogen sulphide production and gelatin liquification tests.

2.2

In vitro screening of actinomycetes isolates for multifarious plant growth promoting (PGP) activities 2.3.1 Phosphate solubilization (Pikovskaya, 1948) Cultivate the actinomycetes culture in nutrient broth and incubated at 28 0 C for 72 h. 100µl of cell free culture supernatant was added to Pikovaskaya's agar wells and plates were incubated at 28 0 C for 24-48 h. Observe yellow zones produced around bit after incubation.

Siderophores Production (Schwyn and Neilands, 1987)

Siderophores production has been done by two ways quantitative and qualitative. Siderophores production was detected qualitatively by chrome azurol-S (CAS) plate assay method. Sterilized blue agar was prepared by mixing chrome azurol-S (CAS) (60.5 mg / 50 ml distilled water) with 10 ml iron solution (1 mM FeCl3.6H2O in 10 mM HCl). This solution was slowly added to Hexadecyl-trimethyl ammonium bromide (HDTMA) (72.9 mg / 40 ml distilled water). Nutrient agar was prepared and autoclaved separately. It was mixed with chrome azurol -S (CAS) (100 ml) (above) and plates were poured. The cell-free supernatant was added to CAS plate wells, which were pre-cut using a sterilized cork borer. The plates were then observed for yellow/orange zones around the wells, measured in mm of diameter. Quantitative estimation of siderophores, chrome azurol-S (CAS) liquid assay method was used as demonstrated by Schwyn and Neilands, 1987, 0.5 ml of 72 h old cell free supernatant of each isolate was mixed with 0.5 ml CAS assay solution in each separate tube.

CAS assay solution Mix 1.5 ml of 1mM FeCl3.6H2O in 10 mM HCl with 7.5 ml of 2mM CAS stock solution (0.121g chrome azurol-S in 100 ml water) and 50 ml of Hexadecyltrimethyl Ammonium Bromide (HDTMA) solution (0.0219 g in 50 ml water). Add 30 ml of piperazine solution (4.307 g in 30 ml water, pH 5.6) and adjust the final volume to 100 ml with distilled water.Add 10 µl of 0.2M 5-sulfosalicylic acid (shuttle solution). The tubes were incubated at room temperature for 10 minutes. Color intensity was measured at 630 nm, with siderophore production indicated by a reduction in blue color, expressed as percent siderophore units (% SU).

Ammonia production

Detection of ammonia production was carried out as described by Lata and Saxena, (2003), 48 h old culture were inoculated in 5 ml of peptone water in each test tube and incubated for 72h at 28 0 C. After incubation 0.5 ml of Nessler's reagent was added in test tubes. Presence of very light brown color (+) indicates small amount of ammonia production and light brown (++) to orange brown color (++++) indicates large amount of ammonia production.

HCN Production

This has been done by following Bakker and Schippers, 1987 method, streak actinomycetes cultures in King's B medium plates amended with glycine (4g/L). Place Whatman Filter paper No.1 already soaked in 2% sodium carbonate and then in 0.5% picric acid inside the top of the plate. Seal plates with parafilm and incubate at 28 °C for 4 days and after incubation observe the change in colour of filter paper

Protease Test

Prepare Skim milk agar plate by adding separately sterilized 1% skim milk to nutrient agar. Add 100µl of 72 h old culture supernatant to the 7mm well already cut on the skim milk agar plates. Incubate the plates at 28°C and observe for production of clear zone around the well (Kaur et al. 1989) [24] .

Chitinase Test

Prepare chitin-nutrient agar plates contain 1% colloidal chitin, place 72 h old culture supernatant in the well already cut on the plate with the help of cork borer and incubate the plates at 28 • C for 2-7 days. After incubation flood the iodine on chitin-nutrient agar plates and observe the plates for production of clear zone around the well (Robert and Selitrennikoff, 1988) [36] .

Auxins production

Auxins production was carried out by inoculate the test organism in nutrient broth and incubate for 72 hours. Prepare supernatant by centrifugation of cultures at 10,000 rpm for 20 min. Now 3 ml of supernatant was pipette into test tube, add 2-3 drops of orthophosphoric acid to 3 ml supernatant followed by 2 ml of salper reagent (1ml of 0.5M Fecl3 in 50 ml of 35% HCIO4). Development of pink colour confirmed the production of IAA (Gorden and paleg, 1957) [19] .

Estimation of PGPR activity screened actinomycetes by pot trial

Pot trials were conducted to assess the impact of PGPR activity from rhizospheric actinomycetes on tomato seedlings. Tomato seeds were inoculated with actinomycete isolates (MKT1, MKT2, MKT3, MKT4, and MKT5). The experiment comprised two sets of treatments: (i) seeds inoculated with actinomycetes (treated), and (ii) seeds without inoculation (control). Seeds were sown in protrays for germination, and seed germination was recorded on the 15th day after sowing. Subsequently, seedlings were transplanted into pots. The experiment followed a completely randomized block design with each treatment replicated four times. Sixty days after sowing, plants from each replication were harvested to evaluate the effects of actinomycetes on leaf number, root and shoot length, root and shoot fresh weight, and root and shoot dry weight (Plate 8).

Effect of liquid formulations on tomato seedlings

Tomato seeds were procurement from local market of Chunni Kalan (Fatehgarh Sahib). Inoculum preparation was done by 48h old culture of different actinomycetes isolates was prepared in SCA broth. The population density that resulted in the formation of 10 8 cfu/ml of actinomycetes isolates was used for preparation of liquid formulation. Subsequently seed sterilization were done by surface sterilization or treated with 70% ethanol for four minutes followed by 2% of HCl for one minute. After that washed several times with sterilized distilled water and blot dry the seeds with bloating paper.

• Seed Bacterization Sterilized tomato seeds were soaked in liquid cultures of different bacterial formulations in flasks and after 2hrs the seeds were sown in pots. Soil was collected from natural habitat and made suitable for growing media preparation after removal of stones and other plant debris. The soil was then mixed with sand and cocopeat thoroughly in ratio (1:1:1).

• Experimental details

The following treatments were applied in pot assay in four replications of each traeetment as T1: Uninoculated control(C), T2: Inoculation with MKT1, T3: Inoculation with MKT2, T4: Inoculation with MKT3, T5: Inoculation with MKT4: T6: Inoculation with MKT5. All the treatments and replications were observed and evaluated using a Completely Randomized Design (CRD).

• Sowing of seeds Seedling production of tomato was completed with cocopeat using portrays, seedling of tomato was raised and transplanted in a pots. Cocopeat was used as a growing media, as it reduces incidence of seedling disease and contains appropriate amount of moisture in it. The seedling tray (protray) was filled with the growing medium (cocopeat). A small dispression (0.5cm) was made with finger tip in the centre of the cell of protray for sowing. One seed per cell was sown and covered with medium. Cocopeat with 300 to 400 percent moisture were used and have no immediate irrigation was required until germination.

• Transplanting

The plants were transplanted in pots containing well prepared growing media. After planting the medium around the stem of the plant was pressed gently with the help of hand. Thorough watering was done immediately after planting. The intercultural operations were carried out including irrigation, hoeing and weeding, spraying against insect pest and disease.

Analysis of Plant Parameter

The plant parameters that have been analyzed and calculated were number of leaves, shoot length and root length after sixty days in centimeters. Shoot or root biomass was done by measuring the fresh weight of shoot or root biomass and excess of water was wiped out by placing it between the folds of filter paper. Then the roots were cut at collar with secateurs and dried in oven at 60°C. Then dry weight was taken and expressed in g/plant. Root: shoot ratio was calculated as Root shoot ratio= Dried root weight/ Dried shoot weight

Results and discussion

Isolation and identification of actinomycetes

In this study, five isolates namely MKT1, MKT2, MKT3, MKT4 and MKT5 were isolated from two sites of Shri Muktsar Sahib district. Similarly, Saadaoui et al. (2024) [37] isolated 14 endophytic actinomycete from wheat roots using Starch casein agar. Gopinath et al. (2018) [18] isolated and screened the effective antibiotic producing actinomycetes from rhizospheric soil of Cipadessa baccifera and Clausena dentate. Sharma et al. (2024) [41] isolated Streptomyces bluencis from the apple rhizosphere and evaluate their effect on plant growth parameters.

Identification of actinomycetes

Morphological characteristics of all five isolates from wheat rhizosphere were found to be circular to irregular, raised (elevation), entire (margin), whitish, greyish and dark greyish in colour on the SCA medium (Table 1 andFig 1). Similar result was found by Dar and Ahmed, (2024) [10] observed that the actinomycetes colonies grow on Starch casein agar in the powdery form and show different shape and colour. Thapa et al. (2024) [43] found that the actinomycetes colonies grown in the form of vegetative or mycelium hyphae, which penetrate into the agar. [39] ; Mondal and Thomas, (2022) [29] ; Somasundaram et al. (2024) [42] and Corral et al. (2022) [9] .

Characterization of actinomycetes isolates for plant growth promoting traits

All the five actinomycetes isolates were screened out for plant growth promoting activities i.e. all the isolates exhibited variation of different plant growth promoting traits. Among all the five isolates, the maximum phosphate solubilization was shown by MKT5 (24 mm) and minimum activity was shown by MKT2 (11 mm) produced yellow zone of solubilization, around the well after two days of incubation on Pikovaskaya's medium. MKT1 did not show phosphate solublization (Table 3 andFig 3). The majority of the phosphorus present in soil in insoluble form which is difficult to intake by plants (Pradhan and Shukla, 2005) [33] . The ability of bacteria to solubilize mineral phosphate has been of interest of agricultural microbiologists as it can increase the availability of phosphorous and iron for plant growth (Panwar et al. 2018) [31] . Endophytic actinomycetes isolated from the roots of eight distinct medicinal plants showed great variation in phosphate solubilization activity on Pikovasaya agar (Hassan et al. 2023) [23] . Out of five isolates, only two isolates i.e. MKT3 (10 mm) and MKT2 (9 mm) were found to be positive for siderophore production in the plate assay. The results (Table 3) were obtained qualitatively in the form of yellowish/ orange coloured zone (mm diameter) produced around the well on chrome azurol-S (CAS) agar plate. In quantitative assay (liquid assay), maximum siderophore activity was shown by MKT2 (33.8%), followed by MKT3 (28.4%). Microorganisms release siderophores, high-affinity ferric iron-chelating compounds, when iron is absent, which transport Fe3+ into microbial cells Ghosh et al. (2020) [15] . Out of six actinomycetes isolates from rhizospheric soil of apple orchards five isolates exhibits siderophore production on chrome azurol-S agar plates in the range of 10-18mm (Sharma et al. 2024) [41] . Auxins production was showcase by three isolates MKT2, MKT4 and MKT5. This test is performed to check the ability of microorganism to produce IAA which is a chief phytohormone. IAA producing bacteria are known to promote root elongation and plant growth (Patten and Glick, 2002) [30] . Indole acetic acid is a main auxin in the plants is known to control many important physiological processes of plants such as cell enlargement, cell division, root initiation, growth rate, and apical dominance, results are show a great agreement with results were found by Anwar et al. (2016) [14] ; Batool et al. (2017) [6] . HCN production was not exhibited by any isolate (Table 5 and Plate 6 A). Actinomycete isolates from groundnut rhizospheric did not exhibits HCN production (Rani et al. 2023) [35] . In the present study all the isolates were found to be positive for ammonia production as all tubes showed colour change in broth from yellow to brown. Ammonia is considered as one of the substances produced by microorganisms that promotes growth and has antimicrobial properties. It helps in disease supression of plants. Zhao et al. (2023) [46] found that all the actinomycetes exhibits ammonia production from different cultures such as manures, humus layer, field soil etc. Isolates were also analyzed for enzyme activity of lytic enzymes such as protease and chitinase test. Enzyme activity was expressed in terms of mm diameter of clear zone produced around the well at 28°C after 48 h. Maximum proteolytic activity was shown by MKT3 (25mm) followed by MKT5 (22mm) and minimum proteolytic activity were shown by remaining isolates i.e. MKT1, MKT2 and MKT4 (20mm). Our findings are in the line with Rahma et al. (2023) [34] ; Sharma et al. (2014) [41] ; MKT3 showed maximum chitinolytic activity (20mm) and MKT1 and MKT4 was represents minimum chitinolytic activity (15mm) whereas MKT5 were found to be negative for chitinase test (Table 3 andFig ) show the maximum production, (+) show the minimum production and (-) for the negativity of the test 3.4 Impact of plant growth promoting rhizospheric actinomycetes on tomato seedlings using pot trials Pot trials were conducted to assess the effect of PGPR activity of actinomycetes on tomato seedlings. Effect of actinomycetes liquid formulations on plant parameters of tomato were accessed under pot trial studies. The effects of PGPR activity of actinomycetes were examined by monitoring the root, shoot length and number of leaves presented, results shown in Table 4. The current study revealed that the maximum root length (8cm) was recorded with treatment (T5) represented by MKT5 isolate, followed by (7.75cm) was observed with treatment (T4) (MKT4) isolate. While, minimum root length (2cm) was recorded in T6 (uninoculated control plants) (Fig. 4). At the same time as in case of shoot length, the maximum 60.50 cm shoot length recorded with treatment T5 (MKT5) isolate followed by (59.25cm) (T4) (MKT4) isolate was observed. Minimum shoot length (51.25cm) was recorded in T6 (uninoculated control) results shown in Fig 5 . Maximum number of leaves (82.25) was recorded by (T5) (MKT5) isolate followed by (T4) (MKT4) isolate i.e. (72.00) and minimum (64.00) was recorded in T6 (uninoculated control) and results shown in Fig 6 . Data appended in Table 5 illustrated that the maximum root and shoot ratio (0.103) observed with T5 inoculated with MKT5 isolate followed by (0.085) T4 inoculated with MKT4 isolate was observed and minimum (0.049) was recorded in T6 that was uninoculated control Fig 7 . Goudjal et al. (2016) [21] confirm the similar results with our pot trial that the actinomycetes show the positive effect on the growth of the tomato plant. Hamada et al.

(2020) proved that the actinomycetes enhance the plant growth as biofertilizer. Statistical analysis using analysis of variance (ANOVA) was performed to determine the significance levels. The results indicated that all parameters were significantly influenced by seed inoculation with actinomycetes

Conclusion

The present study aimed to investigate the plant growthpromoting attributes of actinomycetes from the wheat rhizospheric habitat. Five actinomycete isolates were successfully obtained, each demonstrating excellent plant growth-promoting traits. These isolates were then applied in pot trials to assess their effectiveness. Among them, the isolate MKT5 showed remarkable potential, significantly enhancing root and shoot growth as well as producing a greater number of leaves. This indicates that MKT5 possesses significant plant growth-promoting capabilities compared to other actinomycetes. Therefore, it can be inferred that rhizospheric actinomycetes, such as MKT5, are a viable option for enhancing plant growth and can be utilized as biofertilizers and for other agricultural applications.

Acknowledgment

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