COMPARATIVE STUDY ON PLANT GROWTH PROMOTION BY ENDOPHYTIC PSEUDOMONAS SPP . AND BACILLUS SPP . OF SOLANUM LYCOPERSICUM

Minimization of deleterious effects of chemical fertilizers on health, ecosystem and economy can only be achieved by finding healthy, ecofriendly and cheap alternatives. Naturally selected symbiotic relationship between the endophytic bacteria and their host plants makes them an ideal candidate as biofertilizer. They can synthesize various plant growth hormones as well as assist their host in uptake of nutrients from soil. The study was designed to compare plant growth promotion of Solanum lycopersicum by Bacillus spp., Pseudomonas spp. and total endophytic community isolated from roots of S. lycopersicum, grown in the soil samples collected from various locations of Kathmandu valley of Nepal. Tomato seeds were inoculated with mixtures of eight endophytic strains of Bacillus spp. and Pseudomonas spp., and crude endophytes obtained from each location separately. Endophytic treatments, except Pseudomonas spp., inhibited seminal root growth during 12-days germination period. However, after plantation, root and shoot biomass was enhanced by the endophytes, with no significant differences among the bacterial treatments. The shoot height was also enhanced, among which Pseudomonas spp. had the strongest effect. In phosphate solubilization assay, out of seventy-two isolates each of Bacillus spp. and Pseudomonas spp. tested, twenty-four isolates of Pseudomonas spp. and sixteen isolates of Bacillus spp. could solubilize phosphate. Higher number of phosphate solubilizing isolates of Pseudomonas spp. might provide a possible explanation for the greater enhancement of shoot height by Pseudomonas spp. as compared to Bacillus spp.


Introduction
Consumption of chemical fertilizers is increasing to keep up with the demands of growing world population.The average application of chemical fertilizers in Nepal was 31 kg/hectare in 1995, which is expected to reach up to 131 kg/hectare by 2017 (Shrestha, 2010).However, the excessive use of chemical fertilizers can result in various environmental problems, such as of deterioration of water quality, harming of beneficial organisms (Hautier et al., 2009), increased emission of greenhouse gases, and in a large scale may also contribute to climate change (Crutzen et al., 2007).
Chemical fertilizers such as Ammonium sulphate and Urea are found to cause acidification of the soil (Chitrakar, 1990).As the pH of the soil decreases, the amount of soluble Phosphorous, which has maximum solubility in the pH range of 6.2-7.0, also decreases.It may be daunting to use Phosphate fertilizers to cope with this problem.However, phosphate fertilizers cannot be the ultimate solution since the soluble phosphates are rapidly immobilized to insoluble forms with aluminum and iron ( Sharma et al., 2013) which results in the loss of 70% of the soluble phosphates from the phosphate fertilizers.Other 10% is lost to run-off and leaching (Sashidhar and Podile, 2010).Hence, only 10-20% of the soluble phosphates added is available for the utilization by plants.On the other hand, phosphorus for the production of phosphate fertilizers is derived from phosphorite, a non-renewable resource which has been estimated to deplete severely within the next 50-100 years (Cordell, 2010).Therefore, it is necessary to find alternative solutions to address the need of soluble phosphorus in agriculture.
Endophytic bacteria can be an environment friendly, sustainable alternative to chemical fertilizers.These bacteria reside inside plant tissues (Hallmann et al., 1997) where they benefit the host plant by making nutrients Research Article available to the plant, production of plant growth hormones and control of phytopathogens (Bloemberg and Lugtenberg, 2001).They are also termed as plant growth promoting bacterial endophytes (PGPBEs) (Hardoim et al., 2008); and are free living rhizospheric bacteria, able to infect the plant through active penetration under favorable conditions.Endophytes are environment friendly and also economical.Compared to large industries required to manufacture chemical fertilizers, microbial fertilizers such as endophytic bacteria can be cultured with the help of small budget and resources.Likewise, as endophytes are component of nature, they are less likely to degrade the soil quality and soil microbiome.Hence it is necessary to develop microbial fertilizers to replace chemical fertilizers in order to ensure healthy growth in crop yields.
Despite of all these advantages, very few works have been conducted regarding the isolation of endophytes in Nepal.National Agricultural Research Council (NARC) soil division has one of the few laboratories in Nepal, actively isolating local and imported strains of Rhizobium and providing them to farmer as biofertilizer packets for legumes (National Agricultural Research Council, 2015).However, research regarding other strains of endophytic bacteria must also be done in order to use them as fertilizers on crops other than legumes.Hence this study was designed to isolate the root endophytes of Solanum lycopersicum (tomato) grown in different soil samples from Kathmandu valley and assess their role in growth promotion of tomato plants.

Sample Preparation
Soil samples were collected from three different locations each of Kathmandu (Thankot, Balaju and Muhan Pokhari), Lalitpur (Lele, Godawari and Bagdol) and Bhaktapur districts (Bodey, Kamal Binayak and Surya Binayak).Tomato seeds of Srijana variety (hybrid) from Gorkha Seeds Co. Pvt.Ltd were germinated on moist filter papers in Petri plates and incubated at 30 o C in dark for 3 days, followed by incubation in 14 hr light/10 hr dark photoperiod conditions.Germinated seedlings were then transferred to plastic pots (Changshu Yangyuan Chemical China) containing approximately 1000 cm 3 soil sample and grown in screen house.

Isolation of Bacterial Endophytes
Bacterial endophytes were isolated from the roots of 7 weeks old tomato plants.Roots were surface disinfected by emerging in 70% ethanol for 30 seconds and then 2% sodium hypochlorite for 5 minutes, followed by three rinses in sterilized distilled water, and then mashed in 1000 µL of sterile saline to prepare crude root extract (Long et al., 2003).To isolate Bacillus spp., the crude root extract was subjected to heat shock at a constant temperature on water bath at 80 o C for 15 min (Marquis and Bender, 1985) and then spread on Nutrient Agar.
The isolation of Pseudomonas spp. was done using King's B medium and Pseudomonas Selective Agar Base (HiMedia Laboratories) supplemented with CFC (Cetrimide, Fucidin, Cephaloridine).Disinfection was verified by plating aliquots of the sterile distilled water used in the final rinse on Nutrient Agar plates and incubating the plates at 37 o C for 48 hours (Mendes et al., 2007).Eight different colonies of Bacillus spp.and Pseudomonas spp.were isolated from each of the nine samples.

Phosphate Solubilization
The ability of isolated Pseudomonas spp.and Bacillus spp. to solubilize phosphate was studied using Pikovskaya agar (HiMedia Laboratories) plates.Isolates were spotted onto Pikovskaya agar and incubated for 10 days at 37 °C.The presence of a clear halo zone around the bacterial colony due to the utilization of tricalcium phosphate present in the medium was considered as an indicator for positive test of phosphate solubilization.

Evaluation of Effect of Endophytes on Plant Growth Promotion
Surface sterilized seeds of tomato (Srijana hybrid) were separately soaked in three different treatments of bacterial broth cultures (O.D. 0.24 A) viz., crude root extract, mixture of eight colonies of Bacillus spp.and mixture of eight colonies of Pseudomonas spp.isolated from each of the nine samples.Seeds soaked in nutrient broth were used as control.Following germination of the seeds (12 days), germination percentage and seminal root length were measured, and the three seedlings were grown in each pots containing sterile soil (~1000 cm 3 ) inside the screen house for the next 4 weeks.Three replications were done for each treatment and the pots were arranged in a randomized complete block design (RCBD) with the help of R programming (R Development Core Team, 2008).Shoot length of seedlings were measured each week after planting to assess any difference in the plant growth.After 4 weeks, one plant from each of the pots was uprooted and the biomass of the shoot and the root were taken using a weighing machine (OHAUS -PRECISION Standard).

Statistical Analysis and Data Presentation
Data were statistically analyzed using IBM SPSS (version 23).Data satisfying for normality test (Kolmogorov-Smirnov) and homogeneity of variance (leven's test), were subjected to One-way ANOVA at P<0.05 level of significance, and the means were separated using the DMRT test at P<0.05.Graphs were prepared in Microsoft Office Excel©.

Results and Discussions
The effect of endophytic bacteria on seed germination was assessed.In this assessment, seed germination remained unaffected by the endophytic treatments (Fig. 1).However, the seminal root lengths of the germinating seeds were inhibited by the crude endophytic bacteria and the Bacillus spp.(Fig. 2).The seminal root lengths of the germinating seeds treated with Pseudomonas spp.however was not significantly different from that of control.Non-significant difference in germination percentage between the bacterial treatments and control might be attributed to insufficient release of gibberellic acid by bacteria to impart any significant difference in seed germination.Likewise, it is also possible that the endophytes could not enter the seeds until its germination.And, only after seeds germinated, endophytes were able to reach the tissues through their seminal roots and could affect the root growth.The inhibition of seminal root growth by the Bacillus spp.treatments can be imputed to auxin induced synthesis of ethylene which inhibits root elongation at high concentrations.Indole acetic acid synthesized by bacteria may be involved in the rise of ethylene level to an excessive concentration as demonstrated by (Romano et al., 1993).IAA at a concentration greater than 1 uM, can stimulate the activity of the enzyme ACC synthase, which is responsible for the production of ethylene (Lieberman, 1979).Various experiments have shown endophytic Bacillus spp. to produce greater concentrations of auxin (Araújo et al., 2005;Murugappan et al., 2013;Talboys et al., 2014).The treatments of the seeds with endophytic Pseudomonas spp.showed significant enhancement of the production of root biomass compared to the control (Fig. 3).However, the dry weight of the plants developed from the seeds treated with Pseudomonas spp. was not significantly greater than those treated with Bacillus spp. or crude endophytes.The shoot biomass production was significantly enhanced by the endophytic treatments compared to the control (Fig. 4).However, there was no significant difference among the bacterial variation.Shoot height growth was significantly enhanced by endophytic treatments compared to the control.Among the endophytes, Pseudomonas spp.exhibited greatest effect on the growth of shoot height (Fig. 5).This result was consistent throughout the four weeks of observation (Fig. 6).The observed promotion of shoot and root biomass production in the plants compared to that of the control can be related to various services of the endophytic bacteria to the plants that they inhabit.For host plants, endophytes can enhance plant nutrient uptake, enhance tolerance to abiotic stresses, inhibit infection by plant pathogens, and produce growth regulators, which consequently, increase biomass yield of the plants (Compant et al., 2005;Hardoim et al., 2008;Muthukumarasamy et al., 2002).It is important to note that the seminal root growth was inhibited during seed germination; however, the root biomass during the active plant growth phase in the soil was promoted.Ethylene, as proposed, might have inhibited the root elongation during the germination phase (without added nutrient); but during the active plant growth phase in the soil, plants had access to the nutrients in the soil.Endophytic bacteria might also have enhanced the nutrient uptake by various mechanisms to convert the nutrients into plant-utilizable forms, counteracting the inhibiting effect on root growth.
Endophytic treatments showed significantly better growth of shoot heights compared to the control.One of the various reasons for such result could be the bacterial production of cytokininone of the few plant growth promoters, that promote cell division (D' Agostino and Kieber, 1999).Both Bacillus spp.and Pseudomonas spp.have been found to synthesize cytokinins (Hussain and Hasnain, 2009).The other reason could be the production of auxins by endophytes (Patel and Patel, 2014) which might have resulted in significantly better growth of plants compared to that of the control.In addition to such hormonal regulation, endophytic bacteria is also found to have phosphate solubilizing activities.
Among, seventy-two isolates each of Bacillus spp.and Pseudomonas spp., sixteen isolates of Bacillus spp.and twenty-four isolates of Pseudomonas spp.showed positive tests for mineral phosphate solubilization.This higher number of phosphate solubilizing isolates of Pseudomonas spp. as compared to that of Bacillus spp., provide a possible link to having stronger shoot height promotion by Pseudomonas spp.The other possible reason could be the activity of ACC deaminase.Inoculation of maize with the PGPR containing ACC-deaminase, resulted in a significant increase in the root growth, shoot length and shoot weight of plants (Arshad et al., 2008).Akhgar et al., (2014), found that fluorescent Pseudomonas group of Canola (Brassica napus), were able to utilize ACC as a sole source of nitrogen.Also, all the strains possessing ACC deaminase tested positive for IAA production.Likewise, Glick, ( 2005), identified that the deleterious effects of high levels of ethylene, produced due to overproduction of IAA, is reduced by ACC deaminase.
Likewise, non-significant difference between the shoot height promotions due to crude endophytes as compared to that due to Bacillus spp., might be ascribed to the production of bacteriocins by Bacillus spp.Endophytic Bacillus spp.have been found to produce bacteriocins as biocontrol compounds (Araújo et al., 2005;Ashwini and Srividya, 2013;Gray et al., 2006;I. Hammami et al., 2009;Ines Hammami et al., 2012;He et al., 2006;Hu et al., 2010;Lee et al., 2009;Mouloud et al., 2013).Hence, when, the crude endophytes were used to inoculate the seeds, Bacillus spp.might have dominated the endophytic population and therefore, the plant growth promotion shown by crude endophytes were similar to that shown by Bacillus spp.

Conclusion
Endophytic bacteria promote plant growth through mechanisms such as phosphate solubilization, phytohormones production and lowering of plant ethylene levels.Endophytes had no effect in germination of tomato seeds, but increased root and shoot biomass, and the shoot length.Among the endophytic treatments, Pseudomonas spp. as compared to Bacillus spp. or the crude endophytes were more effective in tomato shoot height promotion.Hence, the use of Pseudomonas spp.provides promises as an alternative to chemical fertilizer for sustainable tomato cultivation.However, further investigations are needed to assess the efficiency of the endophytes as biofertilizer in field conditions.

Fig. 1 :Fig. 2 :
Fig. 1: Effect of bacterial treatments on the germination percentage of tomato seeds.Germination percentage of untreated seeds represent control.Similar letter above Mean±S.E.(harmonic mean sample size, n=9) represent non-significant difference at P ≤0.05, DMRT

Fig. 3 :
Fig. 3: Effect of bacterial treatments on the dry weight of roots.Dry weight of roots of the untreated plants represent control.Similar letter above Mean ± S.E.(harmonic mean sample size, n=9) represent nonsignificant difference at P ≤0.05, DMRT.

Fig. 4 :
Fig. 4: Effect of bacterial treatments on the dry weight of shoots.Dry weight of shoots of untreated plants represent control.Similar letter above Mean±S.E.(harmonic mean sample size, n=9) represent nonsignificant difference at P ≤0.05, DMRT.

Fig. 5 :
Fig. 5: Effect of bacterial treatments on the shoot height after 4 weeks.Shoot height of untreated plants represent control.Similar letter above Mean±S.E.(harmonic mean sample size, n=19.2) represent nonsignificant difference at P ≤0.05, DMRT.

Fig. 6 :
Fig. 6: The growth trend of the seedlings over 4 weeks as effects of various endophytic treatments as compared to control.Shoot height of untreated plant represent the control.Error bars represent the standard error of means.