Bacterial Community Associated with Autotrophic and Heterotrophic Cultures of Medicinal Plant Smallanthus sonchifolius (Yacon)

Rita M. Moraes, Itamar S. Melo, Joko Sumyanto, Suman Chandra, Vaishali Joshi

1National Center for Natural Products Research, Research Institute for Pharmaceutical Sciences, The University of Mississippi, University, Oxford, USA.
Center of Water and Wetland Resources, The University of Mississippi Field Station, Abbeville, USA.
EMBRAPA/Environment, Rodovia SP 340 - Km 127.5, Caixa Postal 69, CEP, Jaguariúna SP, Brazil.
National Center for Natural Products Research, Research Institute for Pharmaceutical Sciences, The University of Mississippi, University, Oxford, USA.

DOI: 10.4236/ajps.2012.310167                

Abstract

Several unfruitful attempts to grow axenic shoot cultures of Smallanthus sonchifolius, also known as yacon, were made before healthy shoots grew in association with bacteria on half strength Murashigue and Skoog media supplemented with 2.2 μM benzylaminopurine. Twenty-one bacterial isolates were obtained from in vitro S. sonchifolius plantlets, eight of these isolates were identified as Flavimonas oryzihabitans, Curtobacterium pusillum, Sphingomonas paucimobilis, and Microbacterium imperiale. These microorganisms produced indole acetic acid (IAA) at amounts varying between 8.89 to 47.45 μg/mL, reason for being classified as plant growth promoting bacteria (PGPB). The results show that buds associated with bacteria cultured on sucrose free media produced 3.77 new roots measuring 18.33 cm in length after a 30-day growing period. In contrast, buds growing on sucrose supplemented media, the number of roots induced was higher (6.67 to 14 roots/explant) but shorter in length, 4.67 to 5.83 cm. During plant acclimatization to soil, photosynthesis and water efficiency were measured showing that the plants were healthy and vigorous. A slightly higher rate of photosynthesis and water use efficiency was recorded in the plants produced on heterotrophic conditions as compared to plants grown in sucrose free media. Plants adapted well in the soil demonstrating that the PGPB community associated to S. sonchifolius in shoot cultures was not harmful to plant production. The purpose of this study was to demonstrate that the bacteria associated with in vitro S. sonchifolius shoot cultures was not the result of microbial contamination, but rather from symbiotic associations that extended from cultivation in the greenhouse, to culture and back to soil. This is the first report to show that autotrophic cultures may represent a viable alternative to grow healthy plants without eliminating beneficial bacteria associated with the host.

Keywords

Micropropagation; Endophytes; Yacon; IAA; Bacteria

Conflicts of Interest

The authors declare no conflicts of interest.

References

[1]	C.-Y. Ko, A. M. Al-Abdulkarim, S. M. Al-Jowid and A. Al-Baiz, “An Effective Disinfection Protocol for Plant Regeneration from Shoot Tip Cultures of Strawberry,” African Journal of Biotechnology, Vol. 8, No. 11, 2009, pp. 2611-2615.
[2]	E. Bunn and B. Tan, “Microbial Contaminants in Plant Tissue Culture Propagation,” In: K. Sivasithamparan, K. W. Dixon and E. L. Barrett, Eds., Microorganisms in Plant Conservation and Biodiversity, Kluwer Academic Publishers, 2002, pp. 307-335.
[3]	C. Lefiet and A. C. Cassells, “Microbial Harzards in Plant Tissue and Cell Cultures,” In Vitro Cellular and Developmental Biology-Plant, Vol. 37, 2001, pp. 133-138.
[4]	C. Leifert, C. E. Morrisand, W. M. Waites, “Ecology of Microbial Saprophytes and Pathogens in Tissue-Culture and Field-Grown Plants—Reason for Contamination Problems in Vitro,” Critical Review Plant Science, Vol. 13, No. 2, 1994, pp 139-183.
[5]	P. R. Hardoim, L. S. van Overbeek and J. D. van Elsas, “Properties of Bacterial Endophytes and Their Proposed Role in Plant Growth,” Trends in Microbiology, Vol. 16, No. 10, 2008, pp. 463-471. doi:10.1016/j.tim.2008.07.008
[6]	K. I. Miller, C. Qing, D. M. Y. Sze and B. A. Neilan, “Investigation of the Biosynthetic Potential of Endophytes in Traditional Chinese Anticancer Herbs,” PLOS ONE, Vol. 7, No. 5, 2012, e35953. doi:10.1371/journal.pone.0035953
[7]	H. Tamta, N. D. Pugh, P. Balachandran, R. M. Moraes, J. Sumiyanto and D. S. Pasco, “Variability in in Vitro Macro-Phage Activation by Commercially Diverse Bulk Echinacea Plant Material Is Predominantly Due to Bacterial Lipoproteins and Lipopolysaccharides,” Journal of Agricultural and Food Chemistry, Vol. 56, No. 22, 2008, pp. 10552-10556. doi:10.1021/jf8023722
[8]	H. W. Zhang, Y. C. Song and T. X. Tan, “Biology and Chemistry of Endophytes,” Natural Products Reports, Vol. 23, No. 5, 2006, pp. 753-771. doi:10.1039/b609472b
[9]	G. Berg, L. Eberl and A. Hartmann, “The Rhizosphere as a Reservoir for Opportunistic Human Pathogenic Bacteria,” Environmental Microbiology, Vol. 7, No. 11, 2001, pp. 1673-1685. doi:10.1111/j.1462-2920.2005.00891.x
[10]	E. Bent and C. P. Chanway, “The Growth-Promoting Effects of a Bacterial Endophyte on Lodgepole Pine Are Partially Inhibited by the Presence of Other Rhizobacteria,” Canadian Journal of Microbiology, Vol. 44, No. 10, 1998, pp. 980-988. doi:10.1139/w98-097
[11]	G. Strobel, B. Daisy, U. Castillo and J. Harper, “Natural Products from Endophytic Microorganisms,” Journal of Natural Products, Vol. 67, No. 2, 2004, pp. 257-268. doi:10.1021/np030397v
[12]	P. Thomas, S. Kumari, G. K. Swarna, D. P. Prakash and M. R. Dinesh, “Ubiquitous Presence of Fastidious Endophytic Bacteria in Field Shoots and Index-Negative Apparently Clean Shoot-Tip Cultures of Papaya,” Plant Cell Reports, Vol. 26, No. 9, 2007, pp. 1491-1499. doi:10.1007/s00299-007-0363-2
[13]	C. V. De Almeida, F. D. Andreote, R. Yara, F. A. O. Tanaka, J. L. Azevedo and M. De Almeida, “Bacteriosomes in Axenic Plants: Endophytes as Stable Endosymbionts,” World Journal of Microbiology and Biotechnology, Vol. 25, No. 10, 2009, pp. 1757-1764. doi:10.1007/s11274-009-0073-8
[14]	B. Reiter and A. Sessitsch “Bacterial Endophytes of the Wildflower Crocus albiflorus Analyzed by Characterization of Isolates and by a Cultivation-Independent Approach,” Canadian Journal of Microbiology, Vol. 52, No. 2, 2006, pp. 140-149.
[15]	T. Murashige and F. Skoog, “A Revised Medium for Rapid Growth and Bioassays with Tobacco Tissue Cultures”, Physiologia Plantarum, Vol. 15, 1962, pp. 473-497. doi:10.1111/j.1399-3054.1962.tb08052.x
[16]	W. L. Araujo, W. Maccheroni Jr., C. I. Aguilar-Vildoso, P. A. V. Barroso, H. O. L. Saridakis and J. L. Azevedo, “Variability and Interactions between Endophytic Bacteria and Fungi Isolated from Leaf Tissues of Citrus Rootstocks,” Canadian Journal of Microbiology, Vol. 47, No. 3, 2001, pp. 229-236. doi:10.1139/w00-146
[17]	L. Miller and T. Berger, “Bacterial Identification by Gas Chromatography of Whole Cell Fatty Acids,” Avondale, Pa.: Hewlett-Packard, Hewlett-Packard Application Note, 1985, pp. 1-8.
[18]	J. Heyrman, J. Mergaert, R. Denys and J. Swings, “The Use of Fatty Acid Methyl Ester Analysis (FAME) for the Identification of Heterotrophic Bacteria Present on Three Mural Paintings Showing Severe Damage by Microorganisms,” FEMS Microbiology Letters, Vol. 181, No. 1, 1999, pp. 55-62. doi:10.1111/j.1574-6968.1999.tb08826.x
[19]	S. A Gordon and R. P. Weber, “Colorimetric Estimation of Indoleacetic Acid,” Plant Physiology, Vol. 26, No. 1, 1951, pp. 192-195.
[20]	SYSTAT, Software Inc. San Jose, USA.
[21]	S. Lee, M. Flores-Encarnacion, M. Contreras-Zentella, L. Garcia-Flores, J. Escamilla and C. Kennedy, “Indole-3-acetic Acid Biosynthesis Is Deficient in Gluconacetobacter diazotrophicus Strains with Mutations in Cytochrome C Biogenesis Genes,” Journal of Bacteriology, Vol. 186, No. 16, pp. 5384-5391.
[22]	R. Mendes, A. A. Pizzirani-Kleiner, W. L. Araujo and J. M. Raaijmakers, “Diversity of Cultivated Endophytic Bacteria from Sugarcane: Genetic and Biochemical Characterization of Burkholderia cepacia Complex Isolates,” Applied Environmental Microbiology, Vol. 73, 2007, pp 7259-7267. doi:10.1128/AEM.01222-07
[23]	A. C. F. Dias, E. F. C. Costa, F. D. Andreote, P. T. Lacava, M. A. Teixeira, L. C. Assump??o, W. L. Araújo, J. L. Azevedo and I. S. Melo, “Isolation of Micropropagated Strawberry Endophytic Bacteria and Assessment of Their Potential for Plant Growth Promotion,” World Journal of Microbiology and Biotecnology, Vol. 25, No. 2, 2009, pp 189-195. doi:10.1007/s11274-008-9878-0
[24]	H. J. Jones, “Plants and Microclimate: A Quantitative Approach to Environmental Plant Physiology,” 2nd Edition, Cambridge University Press, Cambridge, 1992.
[25]	J. W. Zhang, J. D. Marshall and L. Fins, “Correlated Population Differences in Dry Matter Accumulation, Allocation and Water Use Efficiency in Three Sympatric Conifer Species,” Forest Science, Vol. 42, No. 2, 1996, pp. 242-249.
[26]	I. Zaspel, A. Ulrich, B. Boine and T. Stauber, “Occurrence of Culturable Bacteria Living in Micropropagated Black Locust Cultures (Robinia pseudoacacia L.),” European Journal of Horticultural Science, Vol. 73, No. 5, 2008, pp. 231-235.
[27]	M. F. Abreu-Tarazi, A. A. Navarrete, F. D. Andreote, C. V. Almeida S. M. Tsai and M. Almeida, “Endophytic Bacteria in Long-Term in Vitro Cultivated “Axenic” Pineapple Microplants Revealed by PCR-DGGE,” World Journal of Microbiology and Biotechnology, Vol. 26, No. 3, 2010, pp. 555-560.
[28]	J. W. Kloepper, M. N. Schroth and T. D. Miller, “Effects of Rhizosphere Colonization by Plant Growth-Promoting Rhizobacteria on Potato Plant Development and Yield,” Phytopathology, Vol. 70, 1980, pp. 1078-1082. doi:10.1094/Phyto-70-1078
[29]	H. Asghar, Z. Zahir, M. Arshad and A. Khaliq, “Relationship between in Vitro Production of Auxins by Rhizobacteria and Their Growth-Promoting Activities in Brassica juncea L.,” Biology and Fertility of Soils, Vol. 35, No. 4, 2002, pp. 231-237. doi:10.1007/s00374-002-0462-8
[30]	E. A. Barka, A. Belarbi, C. Hachet, J. Nowak and J.-C. Audran, “Enhancement of in Vitro Growth and Resistance to Gray Mould of Vitis vinifera Co-Cultured with Plant Growth-Promoting Rhizobacteria,” FEMS Microbiology Letters, Vol. 186, No. 1, 2000, pp. 91-95.
[31]	S. Compant, C. Clement and A. Sessitsch, “Plant Growth-Promoting Bacteria in the Rhizo and Endosphere of Plants: Their Role, Colonization, Mechanism Involved and Prospect for Utilization,” Soil Bioogy and Biochemistry, Vol. 42, 2010, pp. 669-678. doi:10.1016/j.soilbio.2009.11.024