The Endophytic Bacteria Bacillus velezensis
Lle-9, Isolated from Lilium leucanthum, Harbors Antifungal Activity and Plant
1Beijing Academy of Agriculture and Forestry Sciences, Beijing, P.R. China
2Genomics and Bioinformatics Division, Institute of Biotechnology and Genetic Engineering (IBGE), The University of Agriculture, Pakistan
3Pharmaceutical Research Laboratory, Biotechnology Research Department, Ministry of Education, Mandalay Division, Myanmar
J. Microbiol. Biotechnol. 2020; 30(5): 668-680
Published May 28, 2020
Copyright © The Korean Society for Microbiology and Biotechnology.
Endophytic bacteria, antifungal activity, secondary metabolites, plant growth promotion
The plant-associated bacteria termed as ‘plant growth-promoting rhizobacteria (PGPRs)’ have long been considered as important players in growth promotion and disease resistance in plants . These PGPRs utilize several mechanisms to exert direct or indirect beneficial effects on associated plants. Synthesis of plant growth hormones, phosphate solubilization, nitrogen fixation, siderophores and ACC deaminase production are some of the key mechanisms through which these PGPRs promote plant growth [2, 3]. The PGPR-mediated disease resistance is conferred through various mechanisms either by blocking the rhizopshere colonization of pathogens or through secretion of antimicrobial compounds that degrade the cell walls of pathogens [4, 5].
Among the isolated PGPRs, several species of the genus
Lily, belonging to family
In the current study, an endophytic bacterial strain of
Materials and Methods
Sample Sterilization and Endophyte Isolation
The bulbs’ preparation, sterilization and isolation of endophyte were carried out using a previously described method . The bulbs were peeled off, and the inner portions were washed with tap water for 5 min followed by treatment with 70% (v ⁄ v) ethanol for 1 min. The samples were then immersed in 10% (concentration of active chlorine) NaClO solution for 20 min and washed with sterile distilled water three times. After surface sterilization, the bulb portions were cut aseptically into approximately 1 cm × 1 cm pieces and placed on LB agar media plates. The plates were incubated at 30°C ± 1°C for 2-3 days until bacterial growth started on the cut bulb portions. The individual bacterial colonies appeared on bulb portions were aseptically inoculated into fresh LB broth and incubated at 30°C ± 1°C until pure cultures were obtained by serial sub-culturing.
Morphological and Molecular Identification of Endophytic Bacteria
The isolated bacterial strain Lle-9 was characterized using colony morphology, growth pattern, Gram staining and scanning electron microscopic (SEM) analysis. The Gram reaction was performed as previously described . Cell morphology of the isolate was determined using an SU8010 Field-Emission Scanning Electron Microscope (SEM, Hitachi, Japan).
For molecular analysis, the endophytic strain Lle-9 was cultured in LB broth for 24 h, incubated at 30°C in a shaker with 220 rpm. The culture was then centrifuged at 4,000 ×
In vitro antifungal assay was conducted to test the antagonistic effects of the isolated endophytic strain Lle-9 against four strains of pathogenic fungi, including
Ethyl Acetate Extraction of Secondary Metabolites
Secondary metabolites of the Lle-9 strain extracted by solvent partition method. The strain was grown in LB broth at 30°C and 150 rpm shaking for 5-6 days. The broth cultures were then centrifuged at 10,000 ×
UHPLC-LTQ-XL-IT-MS/MS Analysis for Secondary Metabolite Profiling
UHPLC-LTQ-IT-MS/MS analysis was performed using the method partially adapted from Lee
Putative Identification of Secondary Metabolites
Putative identification of secondary metabolites was done using molecular networking workflow from the GNPS website (https://gnps.ucsd.edu ) . Raw LC-MS files were converted into mzXML using ProteoWizard 3.0.19140 , and the mzXML file was uploaded to GNPS. A molecular network was created using the default parameters. The spectra in the network were then searched against GNPS' spectral libraries. The library spectra were filtered in the same manner as the input data. All matches kept between network spectra and library spectra were required to have a score above 0.7 and at least 6 matched peaks.
Plant Growth-Promoting (PGP) Assays
For plant growth-promoting assays, the bacterial strain Lle-9 was cultured in 1 ml LB media at 30°C for 48 h with 200 rpm shaking. The culture was then centrifuged at 4,000 ×
ACC Deaminase Detection
The isolated strain Lle-9 was assayed for the production of 1-aminocyclopropane-1-carboxylate (ACC) deaminase according to the method previously developed by Belimov
Organic Acid Production Assay
Organic acids in the isolated strain Lle-9 were detected according to the protocol developed by Cunningham and Kuiack . About 50 μl of the bacterial suspension in MgSO4 (10 mM) was inoculated in 800 μl of Sucrose Tryptone medium (ST) containing 20 g l−1 sucrose and 5 g l−1 tryptone. The ST medium was supplemented with 10 ml of trace element solution. Samples were incubated at 30°C and 200 rpm shaking for 5 days. After incubation, organic acids in samples were detected by adding 100 μl of 0.1% alizarine red S pH indicator. After 15 min, samples with yellow color were considered as positive. Pink-colored samples indicated negative results.
Indole Acetic Acid (IAA) Detection
Indole acetic acid (IAA) production in the isolated strain Lle-9 was assayed according to the method of Gordon and Weber . A bacterial suspension of 150 μl prepared in MgSO4 (10 mM) was inoculated in 3 ml of 1/10 diluted 869-rich medium. The medium was supplemented with various tryptophan concentrations of 0 mg ml-1, 2mg ml-1, 4 mg ml-1, and 6 mg ml-1. Samples were incubated at 30°C for 4 days with 150-180 rpm shaking in the dark. After incubation, the cultures were centrifuged at 4,000 ×
The siderophore production in the strain Lle-9 was evaluated through both qualitative and quantitative tests. The bacterial cells were cultured in liquid 284 medium with chrome azurol S (CAS) shuttle solution, according to the described method . About 50 μl of the bacterial suspension in 10 mM MgSO4 was added to 800 μl of 284 medium prepared with three different iron concentrations. The iron concentrations used were: 0 μM, 0.25 μM, and 3 μM Fe(III) citrate. Samples were incubated at 30°C for 5 days with 150 rpm shaking. After incubation, 100 μl of the blue Chromium Azurol S (CAS) reagent was added to samples followed by incubation for 4 h at room temperature. After incubation, the change of color from blue to orange/yellow was considered as positive. Siderophore concentrations in all samples were further measured at 630 nm. The siderophore quantities were measured as % of siderophore units by the formula: % of siderophore units = Ar − As/Ar * 100 where, “Ar” is the absorbance of reference (CAS reagent); and “As” is the absorbance of sample at 630 nm. The ability of the strain to produce siderophores was further confirmed through qualitative test using CAS agar assay. All assays were carried out in triplicates.
Nitrogen fixing ability of the isolated strain Lle-9 was evaluated on nitrogen-deficient malate medium (NFM).
Phosphate solubilization was evaluated according to the method developed by Mehta and Nautiyal . The
Experimental Design of Greenhouse Test
The growth-promoting effects of the isolated endophytic bacterial strain Lle-9 were evaluated on the Asiatic lilium hybrids ‘Tresor.’ Same-sized bulbs with normal and healthy appearance were selected from the storage house at 4°C. For inoculation, the isolated strain Lle-9 was cultured in 5 ml LB for 10-15 h followed by further inoculation in 50 ml LB for 24 h at 30°C with 220 rpm shaking. After incubation, the culture was re-inoculated in 400 ml LB and was kept to grow at 30°C for 24 h. This culture was then diluted 10 times with normal water and bulbs of Tresor variety were soaked in the diluted culture for 40 min. The non-inoculated bulbs, soaked in simple LB, were used as controls. Soil pots of sizes 20 × 30 cm were prepared with soil mix of peat moss, perlite, and vermiculite in a ratio of 2:1:1. Three lily bulbs, either inoculated or non-inoculated control were sown in each soil pot. Pots were kept in a completely randomized design (CRD). Each treatment contained 5 pots. Pots were kept in plastic trays with holes in the bottom. The plastic trays were watered with equal amounts of normal tap water at regular intervals. Morphological data such as number of flowering shoots, plant height, leaf length, leaf width, bulb size and weight and root length were taken at the peak vegetative and reproductive stage.
The data obtained were subjected to analysis of variance (ANOVA). Means were compared with Student’s
Isolation and Identification of
B. velezensis Strain Lle-9
In our study, several bacterial endophytes were isolated from the bulb samples of
Colony morphology and scanning electron microscope (SEM) analysis of the endophytic bacterial strain ofStrain Lle-9 produced whitish, chalky-colored colonies on LB agar plates ( B. velezensisisolated from L. leucanthum. A). The isolate is of small rod-shaped structures ( B, C).
Molecular analysis indicated that the isolated strain Lle-9 belongs to the genus
Phylogenetic analysis of 16S rRNA gene sequences of the bacterial endophyte Lle-9 isolated fromSequences were aligned through ClustalW using MEGA 7 software. Phylogenetic tree was constructed using Maximum Likelihood method. Bootstrap values are shown as percentages of 1000 replicates; values below 50% are not indicated. Lilium leucanthum. Bacillus cereusMG708176.1, ATCC14579 was used as an outgroup.
Antifungal Activity Analysis
The isolated endophytic strain Lle-9 showed high potential of broad-spectrum antifungal activities against the tested phytopathogens,
In vitro antifungal activities of the isolated endophytic bacterial strain Lle-9 from( Lilium leucanthumagainst four fungal pathogens. A) 5 mm fungus plug was inoculated into the center of PDA medium surrounded by four spots of bacterial inoculum. Plates ( A), ( B), ( C), and ( D) are controls of Botryosphaeria dothidea, Fusarium oxysporum, Fusarium fujikuroiand Botrytis cinerea, respectively. Plates ( E), ( F), ( G), and ( H) contain dual cultures of Lle-9 and the fungal pathogens. ( B) Antifungal activities were measured as size of the zones of inhibition of the pathogenic fungi. Zones of inhibition were expressed as percentages.
Secondary Metabolite Analysis
LC/MS was used to characterize the potential bioactive secondary compounds in ethyl acetate fraction. The compounds identified putatively in the ethyl acetate fraction of
Plant Growth-Promoting (PGP) Assays
The plant growth-promoting effects of Lle-9 were assayed both qualitatively and quantitatively. According to our results, the endophytic bacterial strain Lle-9 showed positive results for all conducted assays.
ACC (Deaminase) and Organic Acid Detection
Qualitative detection of plant growth-promoting traits in the isolated strain.( A) ACC deaminase activity of Lle-9 ( A). Lower well was used as negative control. Organic acid production in the strain Lle-9 ( B). Extreme lower well with pink color was used as negative control. IAA detection in the strain showing change of coloration from yellow to pink ( C). The tubes numbered with 1, 2, 3, and 4 show IAA detection at 0 mg ml-1, 2 mg ml-1, 4 mg ml-1, and 6 mg ml-1, respectively. Siderophore detection was confirmed by a change of color from blue to orange ( D). ( B) Siderophore detection on CAS blue agar plates. Siderophores were detected as a yellow/orange hallow surrounding the bacterial colonies ( A). Closer look of the orange hallow surrounding the colony of Lle-9 ( B). ( C) Nitrogen-fixation by the endophytic strain Lle-9 of B. velezensis. The isolated strain was inoculated on nitrogen-deficient malate medium (NFM) and was assessed for growth in reference to non-nitrogenfixing E. coliDH5α. NFM medium ( A); NFM supplemented with 5 mM NH4Cl ( B). ( D) Phosphate solubilization assay of B. velezensisle-09. Growth of Lle-9 on NBRIP medium ( A). Closer look of the clearing area surrounding bacterial colonies ( B).
The isolated strain Lle-9 was assayed for production of organic acids through a qualitative test. Strain Lle-9 showed moderate to high production of organic acids as revealed by the change of color from pink to yellow (Table 1 and Figs. 4A and 4B).
Indole Acetic Acid
Indole acetic acid (IAA) production in the isolated strain Lle-9 was detected through both qualitative and quantitative tests. Qualitative test confirmed IAA production in Lle-9 as revealed by the change of color of the culture supernatant from yellow to pink (Figs. 4A and 4C). Further, IAA was quantified in the strain at various tryptophan concentrations supplemented in the culture medium. The strain Lle-9 was able to produce IAA at different tryptophan concentrations (Table 1). Different tryptophan concentrations impacted the IAA production in the strain. The IAA content in the isolated strain increased with increasing tryptophan in the culture medium. The strain Lle-9 showed lower IAA content,
Production of siderophores in Lle-9 was assayed both qualitatively and quantitatively at different Fe(III) citrate concentrations added to the culture medium. The endophytic strain Lle-9 was able to produce siderophores as confirmed through a change of color from blue to orange yellow (Figs. 4A and 4D). To further test whether an iron source has any impact on the production of siderophores, the strain was cultured in the liquid 284 medium supplemented with different Fe(III) citrate concentrations (Table 1). The strain Lle-9 showed high siderophore production when cultured in medium without Fe(III) citrate. The total siderophore quantity was reported as 51.3 ± 3.8 (psu) in the culture medium without addition of Fe(III) citrate. However, the siderophore accumulation by the strain declined as the quantities of Fe(III) citrate increased in the culture medium. Significantly greater decrease in siderophores was observed when Fe(III) citrate increased from 0 μM to 0.25 μM. About 33.3 ± 1.5 (psu) siderophores were detected at 0.25 μM Fe(III) citrate in the medium. Further reduction in siderophores was observed when Fe(III) citrate concentration was increased to 3.0 μM. However, this was not significantly different from the quantities obtained at 0.25 μM. At 3.0 μM Fe(III) citrate concentration, the strain Lle-9 accumulated 30.1 ± 1.3 (psu) siderophores. The siderophore production in the endophytic strain Lle-9 was further assayed through a qualitative test using chrome azurol S (CAS) on agar plates. An orange/yellow hallow was observed around the colonies of Lle-9 indicating the production of siderophores. The strain Lle-9 was able to quench the iron from the dye complex that resulted in a change of color from blue to orange/yellow in the form of a hallow surrounding the bacterial colony (Fig. 4B). Further, the diameter of the yellow/orange hallow produced by the strain averaged 15.32 ± 1.3 mm. This test further confirmed the high potential of the isolated endophytic strain Lle-9 to produce siderophores.
Potential for Nitrogen Fixation and Phosphate Solubilization
The nitrogen-fixing potential of the isolated
The phosphate solubilization potential of the endophytic
Plant Growth Promotion
The plant growth-promoting potential of the isolated
Plant growth promotion in Tresor variety upon inoculation of bulbs with the isolated strain Lle-9.Phenotypic differences of Lle-9 inoculated and un-inoculated (control) plants ( A). Differences in bulbs and root elongation between CK and Lle-9 inoculated plants ( B).
In the present study, a new endophytic bacterial strain Lle-9 of
In the present study, the isolated strain Lle-9 showed considerable antagonistic effects against fungal phytopathogens like
The ability of the strain Lle-9 to suppress the growth of phytopathogens could be due to the presence of compounds and metabolites with antimicrobial properties. To provide evidence of this, the ethyl acetate fraction was assessed for potential secondary metabolites with bio-control properties. A number of secondary metabolites were identified and showed close homologies with the already known compounds with bio-control properties. Some of the prominent compounds and group of compounds, putatively identified from the isolated strain were diketopiperazines, cyclo-peptides, linear peptides, latrunculin A, 5alpha-hydroxy-6-ketocholesterol, (R)-S-lactoylglutathione, triamterene, rubiadin, moxifloxacin, 9-hydroxy-5Z,7E,11Z,14Z-eicosatetraenoic acid, D-erythro-C18-sphingosine, citrinin, and 2-arachidonoyllysophosphatidylcholine. Isolation of these secondary metabolites and compounds is evidence that the Lle-9 has a high potential of restricting the growth and proliferation of disease-causing fungal pathogens. Our results are supported by previous identification of these bio-control compounds and metabolites from other
Previous studies have revealed that the plant growth-promoting traits in isolated strains of
Moreover, production of 1-aminocyclopropane-1-carboxylate (ACC) deaminase is one of the important characteristics of plant growth-promoting microbes and endophytes. ACC deaminase cleaves ACC, the immediate precursor of the plant hormone ethylene, to produce α-ketobutyrate and ammonia . Ethylene serves as an important signaling molecule in plants under biotic and abiotic stresses and results in plant growth inhibition . Previous studies have reported that inoculation of plants with ACC deaminase-producing microbes decreased ethylene levels that resulted in decreased inhibition of plant growth under biotic and abiotic stresses [3, 58]. Previous studies showed that improvement of several crops, inoculated with
Indole acitic acid is one of the important auxins that directly support plant growth and productivity. The ability of PGPRs including
Iron is an essential element necessary for growth of plants and microorganisms. However, it is abundantly present in soil in the form of insoluble Fe3+ oxy-hydroxides. Plant-associated microbes reduce Fe3+ to Fe2+ with the help of ferrireductases or solubilize it with extracellular Fe3+ chelators called ‘siderophores’ . These soluble Fe3+-siderophore complexes are then available to both plants and microbes. Species of the genus
Nitrogen is an essential and vital element for the normal growth and developments of plants. Many of the isolated PGPRs including species of
The different strains belonging to the genus
Supplementary data for this paper are available on-line only at http://jmb.or.kr.
This research was funded by the National Key Research and Development Program of China [2017YFD0501005]; Science and Technology Innovation Ability Construction of Beijing Academy of Agricultural and Forestry Sciences (KJCX20170415).
Conflict of Interest
The authors have no financial conflicts of interest to declare.
- Kloepper JW, Leong J, Teintze M, Schroth MN. 1980. Enhancing plant growth by siderophores produced by plant growth-promoting rhizobacteria.
Nature 286: 885-886.
- Rodriguez H, Fraga R. 1999. Phosphate solubilizing bacteria and their role in plant growth promotion.
Biotechnol. Adv. 17: 319-339.
- Glick BR, Cheng Z, Czarny J, Duan J. 2007. Promotion of plant growth by ACC deaminase-producing soil bacteria.
Eur. J. Plant Pathol. 119: 329-339.
- Velkov T, Thompson PE, Nation RL, Li J. 2010. Structure - activity relationships of polymyxin antibiotics.
J. Med. Chem. 53: 18981916.
- Kim YC, Leveau J, McSpadden Gardener BB, Pierson EA, Pierson LS,
et al. 2011. The multifactorial basis for plant health promotion by plant-associated bacteria. Appl. Environ. Microbiol. 77: 1548-1555.
- Tendulkar SR, Saikumari YK, Patel V, Raghotama S, Munshi TK, Balaram P,
et al. 2007. Isolation, purification and characterization of an antifungal molecule produced by Bacillus licheniformisBC98, and its effect on phytopathogen Magnaporthe grisea. Appl. Microbiol. 103: 2331-2339.
- Wang J, Liu J, Chen H, Yao J. 2007. Characterization of Fusarium graminearum inhibitory lipopeptide from
Bacillus subtilisIB. Appl. Microbiol. Biotechnol. 76: 889-894.
- Romero D, Perez-Garcia A, Rivera ME, Cazorla FM, de Vicente A. 2004. Isolation and evaluation of antagonistic bacteria towards the cucurbit powdery mildew fungus Podosphaera fusca.
Appl. Microbiol. Biotechnol. 64: 263-269.
- Borriss R, Chen XH, Rueckert C, Blom J, Becker A, Baumgarth B,
et al. 2011. Relationship of Bacillus amyloliquefaciensclades associated with strains DSM7T and FZB42T: a proposal for Bacillus amyloliquefacienssubsp. amyloliquefacienssubsp. nov. and Bacillus amyloliquefacienssubsp. plantarumsubsp. nov. based on complete genome sequence comparisons. Int. J. Syst. Evol. Microbiol. 61: 1786-1801.
- Perez-Garcia A, Romero D, de Vicente A. 2011. Plant protection and growth stimulation by microorganisms: biotechnological applications of
Bacilliin agriculture. Curr. Opin. Biotechnol. 22: 187-193.
- Wang LT, Lee FL, Tai CJ, Kuo HP. 2008.
Bacillus velezensisis a later heterotypic synonym of Bacillus amyloliquefaciens. Int. J. Syst. Evol. Microbiol. 58: 671-675.
- Dunlap CA, Kim SJ, Kwon SW, Rooney AP. 2015. Phylogenomic analysis shows that Bacillus amyloliquefaciens subsp. plantarum is a later heterotypic synonym of
Bacillus methylotrophicus. Int. J. Syst. Evol. Microbiol. 65: 2104-2109.
- Madhaiyan M, Poonguzhali S, Kwon SW, Sa TM. 2010.
Bacillus methylotrophicussp. nov, a methanol-utilizing, plant-growthpromoting bacterium isolated from rice rhizosphere soil. Int. J. Syst. Evol. Microbiol. 60: 2490-2495.
- Chowdhury SP, Dietel K, Rändler M, Schmid M, Junge H, Borriss R. 2013. Effects of
Bacillus amyloliquefaciensFZB42 on lettuce growth and health under pathogen pressure and its impact on the rhizosphere bacterial community. PLoS One 8: e68818.
- Chowdhury SP, Hartmann A, Gao X, Borriss R. 2015. Biocontrol mechanism by root-associated
Bacillus amyloliquefaciensFZB42- a review. Front. Microbiol. 6: 780.
- Rong LP, Lei JJ, Wang C. 2011. Collection and evaluation of the genus
Liliumresources in Northeast China. Genet. Resour. Crop Evol. 58: 115-123.
- Chau CF, Wu SH. 2006. The development of regulations of Chinese herbal medicines for both medicinal and food uses.
Trends Food Sci. Technol. 17: 313-323.
- You X, Xie C, Liu K, Gu Z. 2010. Isolation of non-starch polysaccharides from bulb of tiger lily (Lilium lancifolium Thunb) with fermentation of
Saccharomyces cerevisiae. Carbohydr. Polym. 81: 35-40.
- Schulz B, Boyle C, Draeger S, Rommert AK, Krohn K. 2002. Endophytic fungi: a source of novel biologically active secondary metabolites.
Mycol. Res. 106: 996-1004.
- Tan RX, Zou WX. 2001. Endophytes: a rich source of functional metabolites.
Nat. Prod. Rep. 18: 448-459.
- Strobel GA, Sears J, Kramer R, Sidhu RS, Hess WM. 1996. Taxol from Pestalotiopsis microspora an endophytic fungus of
Taxus wallachiana. Microbiology 142: 435-440.
- Vincent JM, Humphrey B. 1970. Taxonomically significant group antigens in
Rhizobium. J. Gen. Microbiol. 63: 379-382.
- Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S. 2011. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods.
Mol. Biol. Evol. 28: 2731-2739.
- Khamna S, Yokota A, Lumyong S. 2009. Actinomycetes isolated from medicinal plant rhizospheric soils: diversity and screening of antifungal compounds, indole-3-acetic acid and siderophore production.
World J. Microbiol. Biotechnol. 25: 649-655.
- Lee S, Oh DG, Lee S, Kim G, Lee J, Son Y,
et al. 2015. Chemotaxonomic metabolite profiling of 62 indigenous plant species and its correlation with bioactivities. Molecules 20: 19719-19734.
- Wang M, Carver JJ, Phelan VV, Sanchez LM, Garg N, Peng Y,
et al. 2016. Sharing and community curation of mass spectrometry data with global natural products social molecular networking. Nat. Biotechnol. 34: 828-837.
- Chambers MC, Maclean B, Burke R, Amodei D, Ruderman DL, Neumann S,
et al. 2012. A cross-platform toolkit for mass spectrometry and proteomics. Nat. Biotechnol. 30: 918-920.
- Belimov AA, Hontzeas N, Safronova VI, Demchinskaya SV, Piluzza G, Bullitta S,
et al. 2005. Cadmium-tolerant plant growthpromoting bacteria associated with the roots of Indian mustard ( Brassica junceaL. Czern.). Soil. Biol. Biochem. 37: 241-250.
- Truyens S, Jambon I, Croes S, Janssen J, Weyens N, Mench M,
et al. 2014. The effect of long-term cd and ni exposure on seed endophytes of Agrostis capillarisand their potential application in phytoremediation of metal-contaminated soils. Int. J. Phytorem. 16: 643-659.
- Cunningham JE, Kuiack C. 1992. Production of citric and oxalic acids and solubilization of calcium-phosphate by
Penicillium bilaii. Appl. Environ. Microbiol. 58: 1451-1458.
- Gordon SA, Weber RP. 1951. Colorimetric estimation of indoleacetic acid.
Plant. Physiol. 26: 192-195.
- Schwyn B, Neilands JB. 1987. Universal chemical assay for the detection and determination of siderophores.
Anal. Biochem. 160: 4756.
- Doebereiner J. 1994. Isolation and identification of aerobic nitrogen fixing bacteria, pp. 134-141.
In:Alef K, Nannipieri P (eds), Methods in Applied Soil Microbiology and Biochemistry. Academic, Cambridge, MA, USA.
- Bashan Y, Holguin G, Lifshitz R. 1993. Isolation and characterization of plant growth-promoting rhizobacteria, pp. 331-345.
In:Glick BR, Thompson JE (eds), Methods in Plant Molecular Biology and Biotechnology. CRC Press, BocaRaton, FL, USA.
- Mehta S, Nautiyal CS. 2001. An efficient method for qualitative screening of phosphate-solubilizing bacteria.
Curr. Microbiol. 43: 5156.
- Chen XH, Koumoutsi A, Scholz R, Eisenreich A, Schneider K, Heinemeyer I,
et al. 2007. Comparative analysis of the complete genome sequence of the plant growth-promoting bacterium Bacillus amyloliquefaciensFZB42. Nat. Biotechnol. 25: 1007-1014.
- Rabbee MF, Ali MD, Choi J, Hwang BS, Jeong SC, Baek KH. 2019.
Bacillus velezensis: A valuable member of bioactive molecules within plant microbiomes. Molecules 24: 1046.
- Yao A, Dr HB, Karimov S, Boturov U, Sanginboy S, Sharipov AK. 2006. Effect of FZB 24®
Bacillus subtilisas a biofertilizer on cotton yields in field tests. Arch. Phytopathol. Plant. Protect. 39: 323-328.
- Cai XC, Liua CH, Wang BT, Xuea YR. 2016. Genomic and metabolic traits endow
Bacillus velezensisCC09 with a potential biocontrol agent in control of wheat powdery mildew disease. Microbiol. Res. 196: 89-94.
- Zhang Y, Gao X, Wang S, Zhu C, Li R, Shen Q. 2018. Application of
Bacillus velezensisNJAU-Z9 enhanced plant growth associated with efficient rhizospheric colonization monitored by qpcr with primers designed from the whole genome sequence. Curr. Microbiol. 75: 1574-1583.
- Horst RK. 2013. Field manual of diseases on fruits and vegetables. Springer Science+Business Media Dordrecht.
- Syed-Ab-Rahman SF, Carvalhais LC, Chua E, Xiao Y, Wass TJ, Schenk PM. 2018. Identification of soil bacterial isolates suppressing different
Phytophthoraspp. and promoting plant growth. Front. Plant. Sci. 9: 1502.
- Martínez-Luis S, Ballesteros J, Gutiérrez M. 2011. Antibacterial constituents from the octocoral-associated bacterium Pseudoalteromonas sp.
Revista Latinoamericana Química 39: 75-83.
- Nishanth Kumar S, Mohandas C, Siji J, Rajasekharan K, Nambisan B. 2012. Identification of antimicrobial compound, diketopiperazines, from a
Bacillussp. N strain associated with a rhabditid entomopathogenic nematode against major plant pathogenic fungi. J. Appl. Microbiol. 113: 914-924.
- Yang E, Chang H. 2010. Purification of a new antifungal compound produced by
Lactobacillus plantarumAF1 isolated from kimchi. Int. J. Food. Microbiol. 139: 56-63.
- Wang XM, Bai YJ, Cai YJ, Zheng XH. 2017. Biochemical characteristics of three feruloyl esterases with a broad substrate spectrum from
Bacillus amyloliquefaciensH47. Process. Biochem. 53: 109-115.
- Gill K, Kumar S, Xess I, Dey S. 2015. Novel synthetic anti-fungal tripeptide effective against
Candida krusei. Ind. J. Med. Microbiol. 33: 110-116.
- Kloepper JW, Leong J, Teintze M, Schroth MN. 1980. Enhancing plant growth by siderophores produced by plant growth-promoting rhizobacteria.
Nature 286: 885-886.
- Rao MRK, Philip S, Kumar MH, Saranya Y, Divya D, Prabhu K. 2015. GC-MS analysis, antimicrobial, antioxidant activity of an Ayurvedic medicine,
Salmali Niryasa. J. Chem. Pharma. Res. 7: 131-139.
- Ismail NH, Ali AM, Aimi N, Kitajima M, Takayama H, Lajis NH. 1997. Anthraquinones from
Morinda elliptica. Phytochemistry 45: 1723-1725.
- Ali AM, Ismail NH, Mackeen MM, Yazan LS, Mohamed SM, Ho ASH,
et al. 2000. Antiviral, cyototoxic and antimicrobial activities of anthraquinones isolated from the roots of Morinda elliptica. Pharma. Biol. 38: 298-301.
- Marioni J, da Silva MA, Cabreraa JL, Núñez Montoyaa SC, Paraje MG. 2016. The anthraquinones rubiadin and its 1-methyl ether isolated from
Heterophyllaea pustulatareduces Candida tropicalisbiofilms formation. Phytomedicine 23: 1321-1328.
- Matoba AY. 2012. Fungal keratitis responsive to Moxifloxacin monotherapy.
Cornea 31: 1206-1209.
- Lopes R, Tsui S, Gonçalves PJRO, de Queirozm MV. 2018. A look into a multifunctional toolbox: endophytic
Bacillusspecies provide broad and underexploited benefits for plants. World. J. Microbiol. Biotechnol. 34: 94.
- de Werra P, Péchy-Tarr M, Keel C, Maurhofer M. 2009. Role of gluconic acid production in the regulation of biocontrol traits of
Pseudomonas fluorescensCHA0. Appl. Environ. Microbiol. 75: 4162-4174.
- Todorovic B, Glick BR. 2008. The interconversion of ACC deaminase and D-cysteine desulfhydrase by directed mutagenesis.
Planta 229: 193-205.
- Abeles FB, Morgan PW, Saltveit ME Jr. 1992, pp. 1-13. Ethylene in plant biology, 2nd edn. Academic Press, San Diego.
- Farwell AJ, Vesely S, Nero V, Rodriguez H, McCormack K, Shah S,
et al. 2007. Tolerance of transgenic canola plants ( Brassica napus) amended with plant growth-promoting bacteria to flooding stress at a metal-contaminated field site. Environ. Pollut. 147: 540-545.
- Meng Q, Jiang H, Hao JJ. 2016. Effects of
Bacillus velezensisstrain BAC03 in promoting plant growth. Biol. Cont. 98: 18-26.
- Xu M, Sheng J, Chen L, Men Y, Gan L, Guo S,
et al. 2014. Bacterial community compositions of tomato ( Lycopersicum esculentumMill.) seeds and plant growth promoting activity of ACC deaminase producing Bacillus subtilis(HYT-12-1) on tomato seedlings. World. J. Microbiol. Biotechnol. 30: 835-845.
- Patten CL, Blakney AJC, Coulson TJD. 2013. Activity, distribution and function of indole-3-acetic acid biosynthetic pathways in bacteria.
Crit. Rev. Microbiol. 39: 395-415.
- Idris EE, Iglesias DJ, Talon M, Borriss R. 2007. Tryptophan-dependent production of indole-3-acetic acid (IAA) affects level of plant growth promotion by
Bacillus amyloliquefaciensFZB42. Mol. Plant. Microbe. Interact. 20: 619-626.
- Chagas Junior Af, De Oliveira AG, De Oliveira LA, Dos Santos Gr, Chagas LFB, Lopes da Silva AL, Luz Costa J. 2015. Production of indole-3-acetic acid by bacillus isolated from different soils.
Bulg. J. Agric. Sci. 21: 282-287.
- Raza W, Shen Q. 2010. Growth, Fe3+ reductase activity, and siderophore production by
Paenibacillus polymyxaSQR-21 under differential iron conditions. Curr. Microbiol. 61: 390-5.
- Kesaulya H, Hasinu JV, Tuhumury GNC. 2018. Potential of
Bacillusspp produces siderophores in suppressing the wilt disease of banana plants. IOP Conference Series: Earth Environ. Sci. 102(1): 012016.
- Ferreira CMH, Vilas-Boas A, Sousa CA, Soares HMVM, Soares EV. 2019. Comparison of five bacterial strains producing siderophores with ability to chelate iron under alkaline conditions.
AMB Express 9: 78.
- Tailor AJ, Joshi BH. 2012. Characterization and optimization of siderophore production from
Pseudomonas fluorescensstrain isolated from sugarcane rhizosphere. J. Environ. Res. Dev. 6: 688-694.
- Kumar VS, Menon S, Agarwal H, Gopalakrishnan D. 2017. Characterization and optimization of bacterium isolated from soil samples for the production of siderophores.
Resource-Efficient Technol. 3: 434-439.
- Zhao L, Xu Y, Sun R, Deng Z, Yang W, Wei G. 2011. Identification and characterization of the endophytic plant growth prompter
Bacillus cereusstrain mq23 isolated from Sophora alopecuroidesroot nodules. Braz. J. Microbiol. 42: 567-575.
- Shen FT, Yen JH, Liao CS, Chen WC, Chao YT. 2019. Screening of rice endophytic biofertilizers with fungicide tolerance and plant growth-promoting characteristics.
Sustainability 11: 1133.
- Borriss R. 2011. "Use of plant-associated
Bacillusstrains as biofertilizers and biocontrol agents," in Bacteria in Agrobiology, pp. 41-76. In:Maheshwari DK (ed), Plant Growth Responses. Springer, Heidelberg.
- Compant S, Brader G, Muzammil S, Sessitsch A, Lebrihi A, Mathieu F. 2013. Use of beneficial bacteria and their secondary metabolites to control grapevine pathogen diseases.
Bi°Control 58: 435-455.
- Bach E, Dos Santos Seger GD, De Carvalho Fernandes G, Lisboa BB, Passaglia LMP. 2016. Evaluation of biological control and rhizosphere competence of plant growth promoting bacteria.
Appl. Soil. Ecol. 99: 141-149.
- van Lenteren JC, Bolckmans K, Köhl J, Ravensberg WJ, Urbaneja A. 2018. Biological control using invertebrates and microorganisms: plenty of new opportunities.
Bi°Control 63: 39-59.
- Fan B, Wang C, Song X, Ding X, Wu L, Wu H,
et al. 2018. Bacillus velezensisFZB42 in 2018: the gram-positive model strain for plant growth promotion and biocontrol. Front. Microbiol. 9: 2491.