Tomato plant growth promotion potential of Janibactersp strain RC18

Actinomycetes are indispensable in the search for novel, potent and eco friendly fertilizers. The aim of this study was to investigate the ability of Janibacter sp. RC18 to enhance tomato plant growth. Janibacter sp. RC18 isolated from turmeric rhizosphere was screened for in vitro and in vivo plant growth promoting traits. The in vitro plant growth promotion traits exhibited by the strain were phosphate solubilization and ammonia production. The strain RC18 inoculated tomato plant recorded significant (P< 0.05) increase in plant height (45.67cm ± 1.5), number of leaves (69.67 ± 3.5), weight (19.40 g ± 0.53), and high soil phosphate solubilizing bacterial count (7.2x10 5 CFU/g) compared to the uninoculated control. High endogenous plant nutrient content N, P, K, (4.4%, 0.8 % and 1.1%) was also obtained with inoculation of Janibacter. Significant (p< 0.01) positive correlation was recorded with endogenous plant nutrient content and all the plant growth parameters except with the number of flowers. Findings from this study indicate that Janibactersp RC18 is a good candidate for bio-inoculant formulation.


Introduction
The goal number 2 of the sustainable development goals of United Nations General Assembly 2015 is to achieve zero hunger by the year 2030 which involves: promoting sustainable agricultural practices (United Nations, 2015).Therefore, it is expected that by 2030 agricultural productivity should be doubled, also that eco-friendly agricultural practices that help preserve ecosystems and progressively improve soil health should be adopted.
Actinomycetes due to their filamentous nature are considered as transitional groups between bacteria and fungi, they are Gram-positive bacteria that are saprophytic in nature (Barka et al., 2016;Olarenwaju & Babalola, 2019).
They are widely distributed in terrestrial ecosystems and constitute a large component of soil microbial population, especially plant rhizosphere soil.Actinomycetes have the attention of agroindustries as a source of novel agro agents, as plant growth promoters and as biocontrol tools (Anwar et al., 2016;Arumugam et al., 2017).Actinomycetes are unlimited producers of various agro active compounds such as antifungals, antibacterial, indole acetic acids, siderophores, chitinases and phytohormones and have phosphate and potassium solubilizing abilities (Anwar et al., 2016;Olarenwaju et al., 2017;Chitraselvi, 2018).
Actinomycetes play a major role in plant growth, therefore have been frequently screened for potential agroactive agents for sustainable agriculture (Xue, et al., 2013;Wang, et al., 2015).

Inoculation of plants with actinomycetes by
several studies have demonstrated it to be a promising agricultural approach that enhances soil health, increases crop production and controls plant disease (Gopalakrishnan et al., 2015;Jog et al., 2016;Radha, 2016;Bhatti et al., 2017;Htwe, et al., 2019).Significant increase in agronomic properties of agricultural important crops in response to inoculation with actinomycetes have been repeatedly reported; Gopalakrishnan et al. (2015) on cowpea, Robles-Hernández et al. (2015) on pepper and Toumatia et al. ( 2016) on wheat.
Over the years there has been an enormous research interest towards actinomycetes, but its agricultural application has been poorly investigated in Nigeria.Hence the objective of this study was to evaluate the effect of actinomycetes inoculation on tomato plants.

Experimental actinomycete
In a previous study by Osaro-Matthew et al. (2020) (Pikovskaya, 1948) containing tri-calcium phosphate (TCP), was used for evaluating the selected actinomycetes isolates for phosphate solubilisation ability.Selected strain was inoculated onto a Pikovskaya's agar plate and incubated for 5 days at 28 o C, solubilization of phosphate was observed by a clear zone around the isolate.

Ammonia Production
The selected Isolate was inoculated in triplicates into 5 ml of peptone water and incubated at 28 • C for 10 days in a rotary incubator at 120 rpm.After incubation, 0.5 ml of prepared Nessler's reagent was added to each culture.Ammonia production was indicated by the appearance of yellow that changed to brown (Cappuccino & Sherman, 2002).

Hydrogen Cyanide Production
Isolate was streaked on an actinomycetes isolation agar modified with 4.4 glycine / l to assay its hydrogen cyanide production ability.A whatman filter paper No. 1 dipped in 2% sodium carbonate in 0.5% picric acid for a minute was placed beneath the petri dish lids.The lids were sticked to the plates with paraffin and incubated at 28 • C for 10 days.Orange to red color change of filter paper was HCN production positive (Lorck, 1948).

Soil and Seedling Preparation
Plastic pots and bags were surface sterilized with 1% sodium hypochlorite and filled with 1kg of garden soil sterilized at 121 o C for 20 min.Tomato (Roma VF) seeds sterilized in 70% ethanol were planted in sterile soil using a broadcast method for 15 days.

Preparation of Janibacter sp strain RC18 Inoculum and Organic Fertilizers
The optimized starch soybean broth was used in the cultivation of Janibacter sp strain RC18 which involved a 7-day fermentation process carried out on a rotary-incubator at 30 °C and 150 rpm.Janibacter sp strain RC18 cells were harvested after incubation by centrifugation at 10,000 rpm for 20 min.Thereafter, pellet cells were washed twice with distilled water and re-suspended into 0.01M phosphate buffer (pH 7).The optical density (OD) of the prepared inoculum was adjusted to 0.1 at 600 nm using a spectrophotometer to obtain cell concentration of 10 8 CFU/ml (Aka & Babalola, 2016).The positive control used was Nutriplant liquid organic fertilizer at a ratio of 0.01:1 (1ml of liquid organic fertilizer to 100ml of water), whereas sterile water was used as negative control.

Pot Experiment
On the 15 th day, the tomato plants were transplanted into sterilized plastic bags.The treatments were applied to the plant in triplicates after one week of transplant which constitutes 10ml each of both Janibacter and the organic fertilizer preparations.Subsequently the liquid organic fertilizer was added weekly till the 42 nd day, then the plants were uprooted and measurements of the length (height), number of leaves, branches and flowers were taken, and plants were weighed (Gopalakrishnan et al., 2015;Nnabude et al., 2015).

Plant Mineral Content Analysis
The plant roots were dried and ground to allow it to pass 0.5 mm sieve.The calcium and magnesium contents were determined using an atomic absorption spectrophotometer.Micro-kjeldahl was used for N content analysis, colorimetric method was adopted for the estimation of the phosphorus content and the flame photometer method was adopted for the determination of the potassium content (Carpenter & Hendricks, 2003).

Total phosphate solubilizing Bacterial count
The after-planting soil samples from the pot experiments were evaluated for the presence of phosphate solubilizing bacteria.One gram of each sample was prepared and inoculated into Pikovskaya's agar, incubated at 28 o C for 24 hours after which the total viable counts of the phosphate solubilizers were taken.

Statistical analysis
All experiments were carried out in triplicates and statistically analyzed using SPSS (statistical package for social science) version 27.Data obtained were compared using analysis of variance (ANOVA), the means of the various groups were compared with the Duncan multiple range test and correlation analysis was carried out on appropriate data.

In -vivo and in vitro Screening for Plant Growth Promotion Activities
Strain R 1 03 was screened for in vivo plant growth promotion traits as shown in Table 1.The result obtained showed that strain was a phosphate solubilizer and an ammonia producer.
As represented in Table 2, soil treatment with Janibacter sp.Strain RC18 significantly (P<0.05)enhanced the growth of tomato (Roma VF) based on the following results obtained from the plant: height (45.67 ± 1.5 cm), fresh plant weight (19.40 ± 0.53 g), number of leaves (69.67 ± 3.5) and number of branches (9.00 ± 1), compared to the data obtained from positive control (fertilized) and negative control (unfertilized-uninoculated).There was no significant (p>0.05)difference in the number of flowers observed on the tomato plants treated with Janibacter, organic fertilizer and the untreated control (UFUI).However, tomato plants in pots treated with Janibacter numerically had a higher number of flowers (1 ± 1) compared to the fertilized and negative control plants (0.00 ± 0).

Total Phosphate Solubilising Bacteria (PSB) Count
The after-harvest phosphate solubilizing bacterial count from soil samples Fig. 1, indicates that the highest PSB count was obtained from soil treated with Janibacter sp.Strain RC18 (7.2x10 5 CFU/g) and the lowest PSB count was found in UFUI (unfertilizeduninoculated) soil (4.1x10 5 CFU/g).The Effect of Janibacter sp.RC 18 inoculation on plant mineral content As presented in Fig. 2, significantly (p<0.05)higher content of primary nutrient N, P, K, (4.4%, 0.8 % and 1.1%) respectively, and secondary nutrient Ca (2.53%) and Mg (1.66%) were observed in the inoculated tomato plants.This was followed by the fertilized plants and the least nutrient uptake was observed in the negative control plant (UFUI)

Relationship between Plant Growth and Plant Nutrient Uptake
The result of correlation relationships between plant nutrient uptake and plant growth parameters as represented in Table 3 revealed that plant N, P, K uptake correlated positively and significantly (p<0.01) with plant height, number of leaves, number of branches and fresh weight.

In vitro Plant Growth Promotion Activities
The agronomic potential of Janibacter sp.strain RC18 was revealed by the result obtained from In vitro screening for selected plant growth promotion traits viz phosphate solubilization, ammonia production and hydrogen cyanide production.Strain RC18 was a phosphate solubilizing actinomycetes, an ammonia producer but a non-producer of hydrogen cyanide.This result is in accordance with the report of Radha (2016) who reported that 41 actinomycetes exhibited some plant growth promoting potential but were all negative for hydrogen cyanide production.Olarenwaju & Babalola (2019) attributed the increase in available soil phosphate to the presence of phosphate solubilizing actinomycetes.Rodriguez et al. (2016) stated that the ability of microorganisms to produce ammonia is an indication that it can increase the soil available nitrogen.The exhibition of these plant growth promoting traits makes strain RC18 a good biofertilizer candidate.

In -vivo Plant Growth Promotion Activities
Janibacter sp RC18 being an actinomycetes of rhizosphere origin was believed to have ability to colonize and enhance plant growth as expected from most rhizobacteria.Janibacter sp strain RC18 showed that it can promote tomato plant growth In vivo in a pot experiment.
Using organic fertilizer as a positive control, strain RC18 significantly (P<0.05)increased plant height (25.6%), number of leaves (46.42%), number of branches (25.9%) and fresh weight (34.54%).This result agrees with some studies: Gopalakrishnan et al. (2015) inoculated chickpea with Streptomyces sp and obtained a significant increase in growth and yield.In 2016 actinomycetes were inoculated into wheat plants and increase in growth and yield were recorded (Anwar et al., 2016).Htwe et al. (2019)

Plant Mineral content
There was an increase in nutrient content in plants treated with strain RC18 compared with un-inoculated positive and negative control.Several functionalities associated with actinomycetes, especially production of indole acetic acid (IAA) might explain the increase in the nutrient content of the tomato plant.Although strain RC18 was not assayed for production of IAA which enhances uptake of nutrient by increasing the size and surface area of the root (Aeron et al., 2011), some researchers have shown that most actinomycetes can produce IAA (Anwar et al., 2016;Bhai et al., 2016).
In agreement with this result are the observations of Htwe et al. (2018)  The correlation analysis of plant growth parameters, and nutrient content, showed positive correlation between plant growth parameters (plant height, number of flowers, number of leaves.and fresh weight) and uptake of nutrients (N, Ca, P, K and Mg).The negative correlation between uptake of N and number of flowers might be linked to nitrogen toxicity to flowering stage (Tilley, 2020).This implies that plant growth and development largely depend on adequate concentration of mineral nutrients in the soil which was achieved with the inoculation of RC18.Rosenani et al. (2016) has shown that there is a correlation between nutrient uptake and plant growth.

Conclusion
Sustainable agriculture assures food security for the growing population.The In vitro and In vivo plant growth promotion traits of Janibacter sp.RC18 viz phosphate solubilization, ammonia production and increase in tomato plant agronomic properties is evidence that it is a promising microbial resource for green and sustainable agriculture.

Table 1 :
Preliminary screening of Janibacter sp RC18for plant growth promotion trait

Table 2 :
Effect of Janibacter Sp Strain RC 18 on Tomato (Roma VF) Growth At 8 Weeks After Planting Values show means of triplicates ±SD.Values with different superscript on the same column are significantly different at P≤0.05.

Table 3 : Correlation coefficient between plant growth and plant nutrient uptake Growth parameters Endogenous plant mineral content
*Means significant at p<0.05, ** means significant at p<0.01