Next Article in Journal
Oral Iron Supplementation—Gastrointestinal Side Effects and the Impact on the Gut Microbiota
Previous Article in Journal
Lysinibacillus sphaericus III(3)7 and Plasmid Vector pMK4: New Challenges in Cloning Platforms
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Microbiology Research
Volume 12
Issue 2
10.3390/microbiolres12020032
microbiolres-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Effect of Co-Inoculation of Bacillus sp. Strain with Bacterial Endophytes on Plant Growth and Colonization in Tomato Plant (Solanum lycopersicum)

by
Ahsanul Salehin
1,
Ramesh Raj Puri
2,
Md Hafizur Rahman Hafiz
3,4 and
Kazuhito Itoh
1,3,*
1
The United Graduate School of Agricultural Sciences, Tottori University, 4-101 Koyama-Minami, Tottori 680-8553, Japan
2
Agriculture Victoria Research, Department of Jobs, Precincts and Regions, 110 Natimuk Road, Horsham, VIC 3400, Australia
3
Faculty of Life and Environmental Sciences, Shimane University, 1060 Nishikawatsu, Matsue 690-8504, Japan
4
Department of Crop Physiology and Ecology, Hajee Mohammad Danesh Science and Technology University, Dinajpur 5200, Bangladesh
*
Author to whom correspondence should be addressed.
Microbiol. Res. 2021, 12(2), 480-490; https://doi.org/10.3390/microbiolres12020032
Submission received: 30 March 2021 / Revised: 28 April 2021 / Accepted: 29 April 2021 / Published: 6 May 2021
Browse Figures
Review Reports Versions Notes

Abstract

:
Colonization of a biofertilizer Bacillus sp. OYK strain, which was isolated from a soil, was compared with three rhizospheric and endophytic Bacillus sp. strains to evaluate the colonization potential of the Bacillus sp. strains with a different origin. Surface-sterilized seeds of tomato (Solanum lycopersicum L. cv. Chika) were sown in the sterilized vermiculite, and four Bacillus sp. strains were each inoculated onto the seed zone. After cultivation in a phytotron, plant growth parameters and populations of the inoculants in the root, shoot, and rhizosphere were determined. In addition, effects of co-inoculation and time interval inoculation of Bacillus sp. F-33 with the other endophytes were examined. All Bacillus sp. strains promoted plant growth except for Bacillus sp. RF-37, and populations of the rhizospheric and endophytic Bacillus sp. strains were 1.4–2.8 orders higher in the tomato plant than that of Bacillus sp. OYK. The plant growth promotion by Bacillus sp. F-33 was reduced by co-inoculation with the other endophytic strains: Klebsiella sp. Sal 1, Enterobacter sp. Sal 3, and Herbaspirillum sp. Sal 6., though the population of Bacillus sp. F-33 maintained or slightly decreased. When Klebsiella sp. Sal 1 was inoculated after Bacillus sp. F-33, the plant growth-promoting effects by Bacillus sp. F-33 were reduced without a reduction of its population, while when Bacillus sp. F-33 was inoculated after Klebsiella sp. Sal 1, the effects were increased in spite of the reduction of its population. Klebsiella sp. Sal 1 colonized dominantly under both conditions. The higher population of rhizospheric and endophytic Bacillus sp. in the plant suggests the importance of the origin of the strains for their colonization. The plant growth promotion and colonization potentials were independently affected by the co-existing microorganisms.

1. Introduction

Plant growth-promoting rhizobacteria (PGPR) are becoming more widely accepted in intensive agriculture to enhance sustainable agricultural production in various parts of the world [1]. PGPR contain a diverse range of bacteria and several mechanisms have been proposed though they are not fully understood [2]. In sustainable agricultural practices using PGPR, inoculation techniques for their colonization at the rhizosphere is critical [3]; therefore, a further understanding of the interactions of PGPR with plant and indigenous rhizobacteria is essential.
Bacillus spp. have been recognized as one of the most important PGPR and widely used for sustainable agriculture as biofertilizers and/or antagonists against plant diseases [4,5,6,7,8]. Bacillus spp. have also received considerable attention because of their benefits over other PGPR in producing stable formulations [6,9] and stability in rhizosphere soil in semi-arid deserts [10]. In addition, Bacillus spp. exhibit a significant reduction in disease incidence on various crops by inducing systemic resistance [11,12] and by forming biofilm on root surfaces [13].
In our previous study, when the commercial biofertilizer OYK consisting of the Bacillus sp. strain was applied to sweet potato, no significant plant growth-promoting effect was observed, and the inoculated Bacillus sp. strain was not detected in the plant tubers. The possible reasons were due to competition of the inoculant against indigenous rhizobacteria and endophytes, and a lack of endophytic potential of the inoculant, which was originally isolated from soil [14]. As many endophytic Bacillus strains have been reported in several plants [15,16,17,18,19,20,21,22], it is assumed that endophytic bacteria have some colonization strategies in interaction with plants.
In addition to the individual colonizing ability of PGPR, interactions with other co-existing bacteria would be important to determine the colonization and plant growth-promoting potential. Synergetic effects of the inoculation with the other PGPR have been reported in maize [23], cotton [24], ryegrass [25], strawberry [26], and cucumber [27]. On the other hand, negative interactions with co-existing bacteria should also be considered. They inhibited the colonization of inoculants in sugarcane [28] and reduced the plant growth-promoting effects in tomato plant [29,30].
For efficient and practical use of PGPR, it is essential to understand its colonizing behavior and abilities to compete with co-existing bacteria. Though several studies have been reported on the effects of co-inoculation with multiple bacteria on plant growth, their effects on colonization have not been extensively studied yet. The aim of this study was to evaluate the colonization properties of Bacillus sp. OYK, which was isolated from a soil, in relation to its origin by comparing it with those of the other Bacillus sp. strains isolated from plant endosphere and rhizosphere, and then to elucidate the effects of co-inoculation of the endophytic Bacillus sp. strain with the other endophytes on their colonization and plant growth-promoting activities.

2. Materials and Methods

2.1. Bacterial Strains

In addition to Bacillus sp. OYK, three strains of Bacillus sp.: two strains (Bacillus sp. RF-12 and RF-37) isolated from the rhizosphere of sweet potato and another one (Bacillus sp. F-33) as an endophyte of the same plant cultivated in Japan [16], and three strains of endophytes: Herbaspirillum sp. Sal 6, Klebsiella sp. Sal 1, and Enterobacter sp. Sal 3, isolated from Nepalese sweet potato [15], were used in this study (Table 1).

2.2. Plant Growth Promotion and Colonization of Bacillus sp. Strains in Tomato Plant

To prepare the bacterial inoculum, each Bacillus sp. strain was cultivated in Modified Rennie (MR) [31] liquid medium with shaking at 150 rpm at 26 °C for 3 days. The culture was washed twice with sterilized distilled water by centrifugation at 10000× g at 4 °C for 10 min, and the cell pellet was resuspended with sterilized distilled water at 108 colony forming units (CFU)/mL to prepare an inoculum based on OD–CFU/mL correlated linear equations prepared for each strain.
In this study, we used tomato as a test plant due to the difficulty in preparing bacteria-free plants in sweet potato. Tomato seeds (Solanum lycopersicum L. cv. Chika F1 hybrid, Takii & Co., Ltd., Kyoto, Japan) were surface sterilized with 70% ethanol for 1 min followed by 1% sodium hypochlorite with 3–4 drops of Tween-20 for 13 min and washed 7–8 times with sterilized distilled water. The seeds were sown in the sterilized vermiculite in a Leonard jar [32] supplied with the sterilized Hoagland solution [33], and 1 mL of the inoculum was added onto the seed zone. The jar was put in a ventilated (<0.2 mm pore size) transparent plastic bag (Sun bag, Sigma-Aldrich, Tokyo, Japan), and after thinning out to one plant per jar, the tomato plant was aseptically cultivated in a phytotron (Model- LH 220S, Nippon Medical & Chemical Instruments Co., Ltd., Osaka, Japan) at 28/25 °C (16h/8h, day/night) for 24 days. An autoclaved culture was used as a control, and the experiment was conducted twice, using three plants for each treatment.
After cultivation, the tomato crop was harvested, and the fresh weight and length of the root and shoot were measured. Then, the population of the inoculated strains in the root, shoot, and rhizosphere was determined using two plants for each treatment. A rhizosphere sample was prepared by dipping and gently shaking the roots in sterilized distilled water. After washing the plant surface 6–7 times with sterilized distilled water, the root and shoot samples were separated and macerated with sterilized distilled water using a sterilized mortar and pestle, and the samples were subjected to dilution plating for the determination of CFU/g. At the same time, an aliquot of the final washing solution was directly plated, and no colony was observed. The inoculation experiment was conducted twice.

2.3. Effect of Co-Inoculation on Plant Growth Promotion and Colonization of Bacillus sp. F-33 with the Other Endophytic Strains in Tomato Plant

Bacillus sp. F-33 was used as a representative of the Bacillus sp. strains with the other endophytic strains, Klebsiella sp. Sal 1, Enterobacter sp. Sal 3, and Herbaspirillum sp. Sal 6, to examine the effect of co-inoculation on their plant growth promotion and colonization in the tomato plant.
Each bacterial strain was cultivated under the same conditions as described in Section 2.2 to prepare the inoculum at ca. 108 CFU/mL. In case of co-inoculation, the same volume of individual cell suspension was mixed. The sterilized seeds were sown in the sterilized vermiculite in a capped glass tube (12 cm × 3 cm) supplied with the sterilized Hoagland solution, and 1 mL of the inoculum was added onto the seed zone. The other procedures were the same as those described in Section 2.2 except that the cultivation period was 14 days, and that the plant samples were macerated using a BioMasher (Nippi, Tokyo, Japan). The morphologies of the colonies of the co-inoculated strains were clearly different for counting separately. The inoculation experiment was conducted twice.

2.4. Effect of Time Interval Inoculation on Plant Growth Promotion and Colonization of Bacillus sp. F-33 and Klebsiella sp. Sal 1 in Tomato Plant

Bacillus sp. F-33 and Klebsiella sp. Sal 1 were used as representatives of the Bacillus sp. and the endophytic strains, respectively, to examine the effect of time interval of inoculation on their plant growth promotion and colonization in the tomato plant. The experimental procedures were the same as those described in Section 2.3 except that Bacillus sp. F-33 was inoculated first, and then Klebsiella sp. Sal 1 was separately inoculated 7 days after the first inoculation. The tomato plants were harvested at 14 days after the first inoculation. An experiment with a different order of inoculation, Klebsiella sp. Sal 1 first and Bacillus sp. F-33 s, was also conducted in the same way. The inoculation experiment was conducted twice, but one experiment was done using two plants and one of the plants was used to determine the population.

2.5. Statistical Analysis

Statistical analysis of the data on the plant growth and population of the inoculant obtained in each twice-repeated experiment was performed using the MSTAT-C 6.1.4 [34] software package. Data were subjected to Tukey’s test after one-way ANOVA.

3. Results

3.1. Plant Growth Promotion and Colonization of Bacillus sp. Strains in Tomato Plant

The effects of inoculation of the Bacillus sp. strains on the growth of the tomato plant are presented in Figure 1. All Bacillus sp. strains except for Bacillus sp. RF-37 showed plant growth promotion. The root and shoot weights, and the shoot lengths of the inoculated tomato plant were significantly larger than the control while the root lengths were not affected. More lateral root development was observed in the inoculated tomato plant compared with the control.
The populations of the inoculated Bacillus sp. strains in the rhizosphere, root, and shoot of the tomato plants are presented in Figure 2. All Bacillus sp. strains were detected in the rhizosphere, root, and shoot, and the populations of Bacillus sp. RF-12 and RF-37, which were originally isolated from the rhizosphere of sweet potato, and that of Bacillus sp. F-33, which was originally isolated as an endophyte of sweet potato, were higher than that of Bacillus sp. OYK, which was originally isolated from soil. The populations of the three Bacillus sp. strains were 0.9–2.2, 2.1–2.8, and 1.4–2.2 orders higher than those of Bacillus sp. OYK in the rhizosphere, root, and shoot, respectively. The populations were 2.4–4.0 and 3.1–5.2 orders higher in the rhizosphere than those in the root and shoot, respectively. No colony appeared in the control samples.

3.2. Effect of Co-Inoculation on Plant Growth Promotion and Colonization of Bacillus sp. F-33 with the Other Endophytic Strains in Tomato Plant

The effects of co-inoculation of Bacillus sp. F-33 with the other endophytic strains are presented in Figure 3. The plant growth tended to be promoted by Bacillus sp. F-33 but not significantly. The reduction tendencies of the effects were observed by co-inoculation of Enterobacter sp. Sal 3 and Herbaspirillum sp. Sal 6. In shoot weight and root length, the effects of the co-inoculation seemed to be negative in most cases.
All strains colonized tomato plants, resulting in a large population, in which those of the endophytic strains were 1.5–1.7, 1.7–2.6, and 1.2–2.3 orders higher than those of Bacillus sp. F-33 in the rhizosphere, root, and shoot, respectively (Figure 4). Among the endophytic strains, the populations were not different in the rhizosphere, but the populations of Herbaspirillum sp. Sal 6 were about one order of magnitude higher than Klebsiella sp. Sal 1 in the plant parts. The populations were 1.8–2.7 and 2.3–3.3 orders higher at the rhizosphere than those in the root and shoot, respectively. No colony appeared in the control samples.
In case of the co-inoculation, no apparent change in the population was observed in most cases. In co-inoculation of Bacillus sp. F-33 and Herbaspirillum sp. Sal 6, however, the population in the shoot tended to decrease by 0.8 and 1.8 orders in Bacillus sp. F-33 and Herbaspirillum sp. Sal 6, respectively. In addition, one example of a positive tendency in the co-inoculation was observed in the population of Klebsiella sp. Sal 1 in the shoot, in which a 1.4-order increase was observed.

3.3. Effect of Time Interval Inoculation on Plant Growth Promotion and Colonization of Bacillus sp. F-33 and Klebsiella sp. Sal 1 in Tomato Plant

The effects of the time interval of inoculation of Bacillus sp. F-33 and Klebsiella sp. Sal 1 are presented in Figure 5. The plant growth seemed to be promoted by Bacillus sp. F-33 but not by Klebsiella sp. Sal 1. When Klebsiella sp. Sal 1 was inoculated after Bacillus sp. F-33, the plant growth-promoting effects tended to be reduced in root weight. On the other hand, when Bacillus sp. F-33 was inoculated after Klebsiella sp. Sal 1, the effects seemed to be increased compared with the single inoculation of Klebsiella sp. Sal 1.
In individual inoculation, populations of Klebsiella sp. Sal 1 were 1.9, 1.7, and 3.0 orders higher than those of Bacillus sp. F-33 in the rhizosphere, root, and shoot, respectively, and the populations were 2.7–2.8 and 2.5–3.7 orders higher in the rhizosphere than those in the root and shoot, respectively (Figure 6). When Klebsiella sp. Sal 1 was inoculated after Bacillus sp. F-33, the populations of Bacillus sp. F-33 were similar to those in the individual inoculation. When Bacillus sp. F-33 was inoculated after Klebsiella sp. Sal 1, those were 1.3–2.4 orders lower than those in individual inoculation. The populations of Klebsiella sp. Sal 1 showed similar levels under any conditions. No colony appeared in the control samples.

4. Discussion

Significant plant growth-promoting properties were observed in the Bacillus sp. strains except for Bacillus sp. RF-37 (Figure 1). Similar PGPR properties in Bacillus spp. have been previously reported [35,36,37,38]. In this study, the inoculants stimulated lateral root growth, resulting in greater root weight, which could explain the inconsistent results on root weight and root length in the inoculated plants. As indole-3-acetic acid (IAA) is known to have similar effects on plants [39], the plant growth promotion might be caused by IAA production by the inoculants. In another experiment, Bacillus sp. RF-12 and F-33 showed an IAA-producing ability while Bacillus sp. RF-37 did not (data not shown). However, since Bacillus sp. OYK also showed no activity, the reason for the plant growth promotion is unclear.
In addition to the IAA production, other tomato plant growth-promoting mechanisms by Bacillus spp. strains have been reported as follows: gibberellic acid (GA3) as well as IAA production [37,40,41], organic acid production and phosphate-solubilizing abilities [37,40,41], siderophores production [37,42], nitrogen fixation [37], and 1-aminocyclopropane-1-carboxylate (ACC) deaminase production [37,42].
In our previous study, the inoculated Bacillus sp. OYK strain could not establish its population as an endophyte in sweet potato [14], although Bacillus spp. strains have been reported as indigenous endophytes in sweet potato [15,16], tomato [19], banana [20], and switchgrass [21]. We attributed it to the competition with indigenous rhizobacteria and endophytes, as well as the endophytic ability of the inoculant.
In this study, all Bacillus strains colonized in the rhizosphere and endosphere of the tomato plants cultivated using sterilized vermiculite (Figure 2), suggesting that Bacillus sp. OYK has endophytic potential, and that the presence of indigenous microorganisms inhibited its colonization. However, the 1.4–2.8-orders lower populations of Bacillus sp. OYK in the plants compared with the other Bacillus sp. strains, which were isolated from the rhizosphere or as an endophyte (Figure 2), suggests decreased competitiveness of Bacillus sp. OYK against indigenous plant-associated microbes. Some genes and functions may be involved in the plant colonization ability, and PGPR strains from different habitats may have different interactions with plants. The use of originally plant associated PGPR could establish their populations at the rhizosphere and/or endosphere of plants.
The plant growth-promoting effects of Bacillus sp. F-33 were reduced in the presence of the other endophytes, though the population of Bacillus sp. F-33 was maintained (Klebsiella sp. Sal 1 and Enterobacter sp. Sal 3) or slightly decreased (Herbaspirillum sp. Sal 6) (Figure 3 and Figure 4), suggesting that its phyto-stimulating ability was neutralized by the other strains. As the three co-inoculated strains have IAA-degrading ability [30], they might degrade IAA produced by Bacillus sp. F-33 below the effective level.
Synergetic effects of co-inoculation have been reported [23,24,25,26] while cancelation of the positive effects [43,44,45], and negative effects of co-inoculation have also been reported [29,30]. The effects of the co-inoculation seemed to be dependent on the combination of the strains. In most studies that examined the effects of co-inoculation of PGPR, changes in populations of the PGPR by co-inoculation were not measured. In the limited examples of the study using Azospirillum brasilense Sp245 and Bacillus subtilis 101 [29], and Klebsiella sp. Sal 1 and Herbaspirillum sp. Sal 6 [30], their plant growth promotions were reduced even though the populations of the PGPR were maintained, as observed in this study. In our previous study, diverse endophytic bacterial communities were observed in sweet potato, and some components of the communities disappeared by inoculation of Bacillus sp. OYK [14]. It is crucial to elucidate the mechanisms of the microbial interactions; however, it might be complex given the actual environment.
After the establishment of Bacillus sp. F-33 in the rhizosphere and in the tomato plant, Klebsiella sp. Sal 1 could colonize the same population as the strain was individually inoculated (Figure 6) and inhibited the plant growth-promoting ability of Bacillus sp. F-33 without reducing its population (Figure 5), as in the co-inoculation experiment. The high colonizing potential of Klebsiella sp. Sal 1 seemed not to be affected by the about 2-orders lower population of the previously established Bacillus sp. F-33.
On the other hand, after the establishment of Klebsiella sp. Sal 1, the colonization of Bacillus sp. F-33 was reduced by 1.3–2.4 orders than those in the individual inoculation (Figure 6). The relatively lower potential for colonization of Bacillus sp. F-33 might be the reason. The microbial community structure might be a crucial factor to determine the fate of allochthonous microorganisms, such as a PGPR inoculant. Pre-inoculation of PGPR prior to transplantation could be one practical method to enhance higher colonization in plants.
In spite of the reduced population of Bacillus sp. F-33, the plant growth promotion was increased when the strain was inoculated after Klebsiella sp. Sal 1 (Figure 5). It was suggested that the level of the population is not a determinant of the potential of the strain. Although the population of Bacillus sp. F-33 was maintained both in the co-inoculation and in the inoculation of Klebsiella sp. Sal 1 after Bacillus sp. F-33, the PGPR potential of Bacillus sp. F-33 was reduced in the presence of Klebsiella sp. Sal 1, so unknown factors might be involved in plant growth promotion. In addition, the ratio between the populations might not be constant when plants developed, and the kinetic of the different bacterial populations might not be reflected by one sampling time. Time course analysis after inoculation could reveal the progress of colonization in the plant. The results of this study also indicate that there are different niches for the different strains and the colonization of these niches may not have the same impact on plant growth. It may mean that bacteria are competing for some niche colonization.
In addition to plant growth-promoting properties, the colonization potential should be considered as important criteria when assessing their suitability for commercial development. The lower population of Bacillus sp. OYK, which was isolated from soil, than the other Bacillus sp. strains, which were isolated from either the rhizosphere or endosphere of plant samples, suggests the importance of the origin of the strains for their colonization. The plant growth promotion and colonization potentials were independently affected by the co-existing microorganisms. Further studies are necessary to evaluate the colonization potential of PGPR under field conditions where diverse microorganisms exist.

5. Conclusions

In this study, the higher population of rhizospheric and endophytic Bacillus sp. in the plant suggest the importance of the origin of the strains for their colonization. The plant growth promotion and colonization potentials were independently affected by the co-existing microorganisms.

Author Contributions

A.S. and K.I. conceptualized the study and designed the experiments; A.S. performed the experiments; R.R.P. isolated rhizospheric and endophytic strains; M.H.R.H. assisted data analysis; A.S. wrote the article, with a substantial contribution from K.I. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Kaymak, H.C. Potential of PGPR in agricultural innovations. In Plant Growth and Health Promoting Bacteria; Maheshwari, D., Ed.; Springer: Berlin/Heidelberg, Germany, 2010; Volume 18, pp. 45–79. [Google Scholar]
  2. Glick, B.R. The enhancement of plant growth by free living bacteria. Can. J. Microbiol. 1995, 41, 109–117. [Google Scholar] [CrossRef]
  3. Zakria, M.; Udonishi, K.; Ogawa, T.; Yamamoto, A.; Saeki, Y.; Akao, S. Influence of inoculation technique on the endophytic colonization of rice by Pantoea sp. isolated from sweet potato and by Enterobacter sp. isolated from sugarcane. Soil Sci. Plant Nutr. 2008, 54, 224–236. [Google Scholar] [CrossRef]
  4. Vessey, J.K. Plant growth promoting rhizobacteria as biofertilizers. Plant Soil 2003, 255, 571–586. [Google Scholar] [CrossRef]
  5. Miljaković, D.; Marinković, J.; Balešević-Tubić, S. The significance of Bacillus spp. in disease suppression and growth promotion of field and vegetable crops. Microorganisms 2020, 8, 1037. [Google Scholar] [CrossRef]
  6. Kumar, A.; Prakash, A.; Johri, B.N. Bacillus as PGPR in crop ecosystem. In Bacteria in Agrobiology: Crop Ecosystems; Maheshwari, D.K., Ed.; Springer-Verlag: Berlin/Heidelberg, Germany, 2011; pp. 37–59. [Google Scholar]
  7. Govindasamy, V.; Senthilkumar, M.; Magheshwaran, V.; Kumar, U.; Bose, P.; Sharma, V.; Annapurna, K. Bacillus and Paenibacillus spp.: Potential PGPR for Sustainable Agriculture. In Plant Growth and Health Promoting Bacteria; Maheshwari, D., Ed.; Springer-Verlag: Berlin/Heidelberg, Germany, 2010; Volume 18, pp. 333–364. [Google Scholar]
  8. Govindasamy, V.; Senthilkumar, M.; Kumar, U.; Annapurna, K. PGPR-biotechnology for management of abiotic and biotic stresses in crop plants. In Potential Microorganisms for Sustainable Agriculture; Maheshwari, D.K., Dubey, R.C., Eds.; IK International Publishing: New Delhi, India, 2008; pp. 26–48. [Google Scholar]
  9. Emmert, E.A.B.; Handelsman, J. Biocontrol of plant disease: A (Gram-) positive perspective. FEMS Microbiol. Lett. 1999, 171, 1–9. [Google Scholar] [CrossRef]
  10. Nain, L.; Yadav, R.C.; Saxena, J. Characterization of multifaceted Bacillus sp. RM-2 for its use as plant growth promoting bioinoculant for crops grown in semi-arid deserts. Appl. Soil Ecol. 2012, 59, 124–135. [Google Scholar]
  11. Kloepper, J.W.; Ryu, C.-M.; Zhang, S. Induced systemic resistance and promotion of plant growth by Bacillus spp. Phytopathology 2004, 94, 1259–1266. [Google Scholar] [CrossRef] [Green Version]
  12. Choudhary, D.K.; Johri, B.N. Interactions of Bacillus spp. and plants—with special reference to induced systemic resistance (ISR). Microbiol. Res. 2009, 164, 493–513. [Google Scholar] [CrossRef]
  13. Chen, Y.; Yan, F.; Chai, Y.; Liu, H.; Kolter, R.; Losick, R.; Guo, J. Biocontrol of tomato wilt disease by Bacillus subtilis isolates from natural environments depends on conserved genes mediating biofilm formation. Environ. Microbiol. 2013, 15, 916–927. [Google Scholar] [CrossRef] [Green Version]
  14. Salehin, A.; Hafiz, M.H.R.; Hayashi, S.; Adachi, F.; Itoh, K. Effects of the biofertilizer OYK (Bacillus sp.) inoculation on endophytic microbial community in sweet potato. Horticulturae 2020, 6, 81. [Google Scholar] [CrossRef]
  15. Puri, R.R.; Dangi, S.; Dhungana, S.A.; Itoh, K. Diversity and plant growth promoting ability of culturable endophytic bacteria in Nepalese sweet potato. Adv. Microbiol. 2018, 8, 734–761. [Google Scholar] [CrossRef] [Green Version]
  16. Puri, R.R.; Adachi, F.; Omichi, M.; Saeki, Y.; Yamamoto, A.; Hayashi, S.; Itoh, K. Culture-dependent analysis of endophytic bacterial community of sweet potato (Ipomoea batatas) in different soils and climates. J. Adv. Microbiol. 2018, 13, 1–12. [Google Scholar] [CrossRef]
  17. Marques, J.M.; Da Silva, T.F.; Vollu, R.E.; Blank, A.F.; Ding, G.-C.; Seldin, L.; Smalla, K. Plant age and genotype affect the bacterial community composition in the tuber rhizosphere of field-grown sweet potato plants. FEMS Microbiol. Ecol. 2014, 88, 424–435. [Google Scholar] [CrossRef]
  18. Germida, J.J.; Siciliano, S.D.; Renato, D.F.J.; Seib, A.M. Diversity of root-associated bacteria associated with field-grown canola (Brassica napus L.) and wheat (Triticum aestivum L.). FEMS Microbiol. Ecol. 1998, 26, 43–50. [Google Scholar] [CrossRef]
  19. Tian, B.; Zhang, C.; Ye, Y.; Wen, J.; Wu, Y.; Wang, H.; Li, H.; Cai, S.; Cai, W.; Cheng, Z.; et al. Beneficial traits of bacterial endophytes belonging to the core communities of the tomato root microbiome. Agric. Ecosyst. Environ. 2017, 247, 149–156. [Google Scholar] [CrossRef]
  20. Souza, S.A.; Xavier, A.A.; Costa, M.R.; Cardoso, A.M.S.; Pereira, M.C.T.; Nietsche, S. Endophytic bacterial diversity in banana “Prata Anã” (Musa spp.) roots. Genet. Mol. Biol. 2013, 36, 252–264. [Google Scholar] [CrossRef]
  21. Xia, Y.; Greissworth, E.; Mucci, C.; Williams, M.A.; Bolt, S.D. Characterization of culturable bacterial endophytes of switchgrass (Panicum virgatum L.) and their capacity to influence plant growth. GCB Bioenergy 2013, 5, 674–682. [Google Scholar] [CrossRef]
  22. Liu, B.; Qiao, H.; Huang, L.; Buchenauer, H.; Han, Q.; Kang, Z.; Gong, Y. Biological control of take-all in wheat by endophytic Bacillus subtilis E1R-j and potential mode of action. Biol. Control 2009, 49, 277–285. [Google Scholar] [CrossRef]
  23. Molina-Romero, D.; Baez, A.; Quintero-Hernández, V.; Castañeda-Lucio, M.; Fuentes-Ramírez, L.E.; del Rocio Bustillos-Cristales, M.; Rodríguez-Andrade, O.; Morales-García, Y.E.; Munive, A.; Muñoz-Rojas, J. Compatible bacterial mixture, tolerant to desiccation, improves maize plant growth. PLoS ONE 2017, 12, 1–21. [Google Scholar] [CrossRef]
  24. Marimuthu, S.; Subbian, P.; Ramamoorthy, V.; Samiyappan, R. Synergistic effect of combined application of Azospirillum and Pseudomonas fluorescens with inorganic fertilizers on root rot incidence and yield of cotton. J. Plant Dis. Prot. 2002, 109, 569–577. [Google Scholar]
  25. Castanheira, N.L.; Dourado, A.C.; Pais, I.; Semedo, J.; Scotti-Campos, P.; Borges, N.; Carvalho, G.; Barreto Crespo, M.T.; Fareleira, P. Colonization and beneficial effects on annual ryegrass by mixed inoculation with plant growth promoting bacteria. Microbiol. Res. 2017, 198, 47–55. [Google Scholar] [CrossRef] [PubMed]
  26. Vestberg, M.; Kukkonen, S.; Saari, K.; Prikka, P.; Huttunen, J.; Tainio, L.; Devos, N.; Weekers, F.; Kevers, C.; Thonart, P.; et al. Microbial inoculation for improving the growth and health of micropropagated strawberry. Appl. Soil Ecol. 2004, 27, 243–258. [Google Scholar] [CrossRef]
  27. Raupach, G.S.; Kloepper, J.W. Mixtures of plant growth-promoting rhizobacteria enhance biological control of multiple cucumber pathogens. Phytopathology 1998, 88, 1158–1164. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  28. Oliveira, A.L.M.; Stoffels, M.; Schmid, M.; Reis, V.M.; Baldani, J.I.; Hartmann, A. Colonization of sugarcane plantlets by mixed inoculations with diazotrophic bacteria. Eur. J. Soil Biol. 2008, 45, 106–113. [Google Scholar] [CrossRef]
  29. Felici, C.; Vettori, L.; Giraldi, E.; Forino, L.M.C.; Toffanin, A.; Tagliasacchi, A.M.; Nuti, M. Single and co-inoculation of Bacillus subtilis and Azospirillum brasilense on Lycopersicon esculentum: Effects on plant growth and rhizosphere microbial community. Appl. Soil Ecol. 2008, 40, 260–270. [Google Scholar] [CrossRef]
  30. Dhungana, S.A.; Itoh, K. Effects of co-inoculation of indole-3-acetic acid-producing and -degrading bacterial endophytes on plant growth. Horticulturae 2019, 5, 17. [Google Scholar] [CrossRef]
  31. Elbeltagy, A.; Nishioka, K.; Sato, T.; Suzuki, H.; Ye, B.; Hamada, T.; Isawa, T.; Mitsui, H.; Minamisawa, K. Endophytic colonization and in planta nitrogen fixation by a Herbaspirillum sp. isolated from wild rice species. Appl. Environ. Microbiol. 2001, 67, 5285–5293. [Google Scholar] [CrossRef] [Green Version]
  32. Leonard, L.T. A simple assembly for use in the testing of cultures of rhizobia. J. Bacteriol. 1943, 45, 523–527. [Google Scholar] [CrossRef] [Green Version]
  33. Hoagland, D.R.; Arnon, D.I. The water-culture method for growing plants without soil. Calif. Agric. Exp. Stn. Circ. 1950, 347, 1–32. [Google Scholar]
  34. Freed, R. MSTAT: A software program for plant breeder. In Principles of Plant Genetics and Breeding, 2nd ed.; Acquaah, G., Ed.; Blackwell Publishing: Malden, MA, USA, 2007; Volume 1, pp. 426–431. [Google Scholar]
  35. Saharan, B.S.; Nehra, V. Plant growth promoting rhizobacteria: A critical review. Life Sci. Med. Res. 2011, 21, 1–30. [Google Scholar]
  36. Shen, M.; Kang, Y.J.; Wang, H.L.; Zhang, X.S.; Zhao, Q.X. Effect of plant growth-promoting rhizobacteria (PGPRs) on plant growth, yield, and quality of tomato (Lycopersicon esculentum Mill.) under simulated seawater irrigation. J. Gen. Appl. Microbiol. 2012, 58, 253–262. [Google Scholar] [CrossRef] [Green Version]
  37. Xu, M.; Sheng, J.; Chen, L.; Men, Y.; Gan, L.; Guo, S.; Shen, L. Bacterial community compositions of tomato (Lycopersicum esculentum Mill.) seeds and plant growth promoting activity of ACC deaminase producing Bacillus subtilis (HYT-12-1) on tomato seedlings. World J. Microbiol. Biotechnol. 2014, 30, 835–845. [Google Scholar] [CrossRef]
  38. Batista, B.D.; Lacava, P.T.; Ferrari, A.; Teixeira-Silva, N.S.; Bonatelli, M.L.; Tsui, S.; Mondin, M.; Kitajima, E.W.; Pereira, J.O.; Azevedo, J.L. Screening of tropically derived, multi-trait plant growth-promoting rhizobacteria and evaluation of corn and soybean colonization ability. Microbiol. Res. 2018, 206, 33–42. [Google Scholar] [CrossRef]
  39. Egamberdieva, D. Indole-acetic acid production by root associated bacteria and its role in plant growth and development. In Auxins: Structure, Biosynthesis and Functions; Keller, A.H., Fallon, M.D., Eds.; Nova Science Publishers: Hauppauge, NY, USA, 2012; pp. 103–122. [Google Scholar]
  40. Chowdappa, P.; Kumar, S.M.; Lakshmi, M.J.; Mohan, S.P.; Upreti, K.K. Growth stimulation and induction of systemic resistance in tomato against early and late blight by Bacillus subtilis OTPB1 or Trichoderma harzianum OTPB3. Biol. Control 2013, 65, 109–117. [Google Scholar] [CrossRef]
  41. Bahadir, P.S.; Liaqat, F.; Eltem, R. Plant growth promoting properties of phosphate solubilizing Bacillus species isolated from the Aegean Region of Turkey. Turk. J. Bot. 2018, 42, 1–14. [Google Scholar] [CrossRef]
  42. Abbamondi, G.R.; Tommonaro, G.; Weyens, N.; Thijs, S.; Sillen, W.; Gkorezis, P.; Iodice, C.; de Melo Rangel, W.; Nicolaus, B.; Vangronsveld, J. Plant growth-promoting effects of rhizospheric and endophytic bacteria associated with different tomato cultivars and new tomato hybrids. Chem. Biol. Technol. Agric. 2016, 3, 1. [Google Scholar] [CrossRef] [Green Version]
  43. Schmidt, C.S.; Agostini, F.; Simon, A.M.; Whyte, J.; Townend, J.; Lifert, C.; Killham, K.; Mullins, C. Influence of soil type and pH on the colonization of sugar beet seedlings by antagonistic Pseudomonas and Bacillus strains, and on their control of Pythium damping-off. Eur. J. Plant Pathol. 2004, 110, 1025–1046. [Google Scholar] [CrossRef]
  44. Dandurand, L.; Knudsen, G. Influence of Pseudomonas fluorescens on hyphal growth and biocontrol activity of Trichoderma harzianum in the spermosphere and rhizosphere of pea. Phytopathology 1993, 83, 265–270. [Google Scholar] [CrossRef]
  45. García, L.J.A.; Probanza, A.; Ramos, B.; Barriuso, J.; Gutierrez Mañero, F.J. Effects of inoculation with plant growth promoting rhizobacteria (PGPRs) and Sinorhizobium fredii on biological nitrogen fixation, nodulation and growth of Glycine max cv. Osumi. Plant Soil 2004, 267, 143–153. [Google Scholar] [CrossRef]
Microbiolres 12 00032 g001 550
Figure 1. The effects of inoculation of Bacillus sp. strains on the growth of the tomato plant. The tomato plant was cultivated using sterilized vermiculite, and the parameters were measured at 24 days after seed inoculation. CTL represents the control samples inoculated with autoclaved cultures. The bars represent the standard deviation (n = 6), and different letters indicate significant differences at p < 0.05 by Tukey’s test.
Figure 1. The effects of inoculation of Bacillus sp. strains on the growth of the tomato plant. The tomato plant was cultivated using sterilized vermiculite, and the parameters were measured at 24 days after seed inoculation. CTL represents the control samples inoculated with autoclaved cultures. The bars represent the standard deviation (n = 6), and different letters indicate significant differences at p < 0.05 by Tukey’s test.
Microbiolres 12 00032 g001
Microbiolres 12 00032 g002 550
Figure 2. Colonization of seed-inoculated Bacillus sp. strains in the rhizosphere (a), root (b), and shoot (c) of the tomato plant. The tomato plant was cultivated using sterilized vermiculite, and colonization was examined at 24 days after seed inoculation. No colony appeared in the control samples. The bars represent the standard deviation (n = 4), and different letters indicate significant differences at p < 0.05 by Tukey’s test.
Figure 2. Colonization of seed-inoculated Bacillus sp. strains in the rhizosphere (a), root (b), and shoot (c) of the tomato plant. The tomato plant was cultivated using sterilized vermiculite, and colonization was examined at 24 days after seed inoculation. No colony appeared in the control samples. The bars represent the standard deviation (n = 4), and different letters indicate significant differences at p < 0.05 by Tukey’s test.
Microbiolres 12 00032 g002
Microbiolres 12 00032 g003 550
Figure 3. The effects of co-inoculation of Bacillus sp. F-33 with the other endophytic strains, Klebsiella sp. Sal 1, Enterobacter sp. Sal 3, and Herbaspirillum sp. Sal 6, on the growth of the tomato plant. The tomato plant was cultivated using sterilized vermiculite, and the parameters were measured at 14 days after seed inoculation. CTL represents the control samples inoculated with autoclaved cultures. The bars represent the standard deviation (n = 6), and different letters indicate significant differences at p < 0.05 by Tukey’s test.
Figure 3. The effects of co-inoculation of Bacillus sp. F-33 with the other endophytic strains, Klebsiella sp. Sal 1, Enterobacter sp. Sal 3, and Herbaspirillum sp. Sal 6, on the growth of the tomato plant. The tomato plant was cultivated using sterilized vermiculite, and the parameters were measured at 14 days after seed inoculation. CTL represents the control samples inoculated with autoclaved cultures. The bars represent the standard deviation (n = 6), and different letters indicate significant differences at p < 0.05 by Tukey’s test.
Microbiolres 12 00032 g003
Microbiolres 12 00032 g004 550
Figure 4. The effect of the seed-co-inoculated Bacillus sp. F-33 with the other endophytic strains, Klebsiella sp. Sal 1, Enterobacter sp. Sal 3, and Herbaspirillum sp. Sal 6, on colonization in the rhizosphere (a), root (b), and shoot (c) of the tomato plant. The tomato plant was cultivated using sterilized vermiculite, and colonization was examined at 14 days after seed-co-inoculation. The bracket on the x-axis indicates each population in co-inoculation, and no bracket indicates single inoculation. No colony appeared in the control samples. The bars represent the standard deviation (n = 4), and different letters indicate significant differences at p < 0.05 by Tukey’s test.
Figure 4. The effect of the seed-co-inoculated Bacillus sp. F-33 with the other endophytic strains, Klebsiella sp. Sal 1, Enterobacter sp. Sal 3, and Herbaspirillum sp. Sal 6, on colonization in the rhizosphere (a), root (b), and shoot (c) of the tomato plant. The tomato plant was cultivated using sterilized vermiculite, and colonization was examined at 14 days after seed-co-inoculation. The bracket on the x-axis indicates each population in co-inoculation, and no bracket indicates single inoculation. No colony appeared in the control samples. The bars represent the standard deviation (n = 4), and different letters indicate significant differences at p < 0.05 by Tukey’s test.
Microbiolres 12 00032 g004
Microbiolres 12 00032 g005 550
Figure 5. The effects of the time interval of inoculation on plant growth promotion and colonization of Bacillus sp. F-33 and Klebsiella sp. Sal 1 in the tomato plant. The tomato plant was cultivated using sterilized vermiculite, and the parameters were measured at 14 days after seed inoculation. In the time interval of inoculation, F-33 + Sal 1 and Sal 1 + F-33, the second inoculation was conducted 7 days after the first inoculation and analyzed 7 days after the second inoculation. CTL represents the control samples inoculated with autoclaved cultures. The bars represent the standard deviation (n = 5), and different letters indicate significant differences at p < 0.05 by Tukey’s test.
Figure 5. The effects of the time interval of inoculation on plant growth promotion and colonization of Bacillus sp. F-33 and Klebsiella sp. Sal 1 in the tomato plant. The tomato plant was cultivated using sterilized vermiculite, and the parameters were measured at 14 days after seed inoculation. In the time interval of inoculation, F-33 + Sal 1 and Sal 1 + F-33, the second inoculation was conducted 7 days after the first inoculation and analyzed 7 days after the second inoculation. CTL represents the control samples inoculated with autoclaved cultures. The bars represent the standard deviation (n = 5), and different letters indicate significant differences at p < 0.05 by Tukey’s test.
Microbiolres 12 00032 g005
Microbiolres 12 00032 g006 550
Figure 6. The effect of the time interval of inoculation of Bacillus sp. F-33 and Klebsiella sp. Sal 1 on colonization in the rhizosphere (a), root (b), and shoot (c) of the tomato plant. The tomato plant was cultivated using sterilized vermiculite, and colonization was examined at 14 days after seed inoculation. In the time interval of inoculation, F-33 + Sal 1 and Sal 1 + F-33, the second inoculation was conducted 7 days after the first inoculation and analyzed 7 days after the second inoculation. The bracket on the x-axis indicates each population in the time interval of inoculation, and the arrows on the bracket indicate the order of inoculation. No bracket indicates a single inoculation. No colony appeared in the control samples. The bars represent the standard deviation (n = 3), and different letters indicate significant differences at p < 0.05 by Tukey’s test.
Figure 6. The effect of the time interval of inoculation of Bacillus sp. F-33 and Klebsiella sp. Sal 1 on colonization in the rhizosphere (a), root (b), and shoot (c) of the tomato plant. The tomato plant was cultivated using sterilized vermiculite, and colonization was examined at 14 days after seed inoculation. In the time interval of inoculation, F-33 + Sal 1 and Sal 1 + F-33, the second inoculation was conducted 7 days after the first inoculation and analyzed 7 days after the second inoculation. The bracket on the x-axis indicates each population in the time interval of inoculation, and the arrows on the bracket indicate the order of inoculation. No bracket indicates a single inoculation. No colony appeared in the control samples. The bars represent the standard deviation (n = 3), and different letters indicate significant differences at p < 0.05 by Tukey’s test.
Microbiolres 12 00032 g006
Table
Table 1. Bacterial isolates used in this study [5,6].
Table 1. Bacterial isolates used in this study [5,6].
StrainMost Similar Genus aClassOriginAccession Number
OYKBacillus sp.BacilliSoilLC590219
RF-12Bacillus sp.BacilliRhizosphereLC593252
RF-37Bacillus sp.BacilliRhizosphereLC593253
F-33Bacillus sp.BacilliEndophyticLC430058
Sal 1Klebsiella sp.γ-ProteobacteriaEndophyticLC389410
Sal 3Enterobacter sp.γ-ProteobacteriaEndophyticLC389433
Sal 6Herbaspirillum sp.β-ProteobacteriaEndophyticLC389442
a Based on the 16S rRNA gene sequence in the database.
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Share and Cite

MDPI and ACS Style

Salehin, A.; Puri, R.R.; Hafiz, M.H.R.; Itoh, K. Effect of Co-Inoculation of Bacillus sp. Strain with Bacterial Endophytes on Plant Growth and Colonization in Tomato Plant (Solanum lycopersicum). Microbiol. Res. 2021, 12, 480-490. https://doi.org/10.3390/microbiolres12020032

AMA Style

Salehin A, Puri RR, Hafiz MHR, Itoh K. Effect of Co-Inoculation of Bacillus sp. Strain with Bacterial Endophytes on Plant Growth and Colonization in Tomato Plant (Solanum lycopersicum). Microbiology Research. 2021; 12(2):480-490. https://doi.org/10.3390/microbiolres12020032

Chicago/Turabian Style

Salehin, Ahsanul, Ramesh Raj Puri, Md Hafizur Rahman Hafiz, and Kazuhito Itoh. 2021. "Effect of Co-Inoculation of Bacillus sp. Strain with Bacterial Endophytes on Plant Growth and Colonization in Tomato Plant (Solanum lycopersicum)" Microbiology Research 12, no. 2: 480-490. https://doi.org/10.3390/microbiolres12020032

Article Metrics

Back to TopTop