Microbial Ecology

, Volume 58, Issue 4, pp 952–964 | Cite as

Endophytic Bacteria Associated with Growing Shoot Tips of Banana (Musa sp.) cv. Grand Naine and the Affinity of Endophytes to the Host

Plant Microbe Interactions

Abstract

A cultivation-based assessment of endophytic bacteria present in deep-seated shoot tips of banana suckers was made with a view to generate information on the associated organisms, potential endophytic contaminants in tissue-cultured bananas and to assess if the endophytes shared a beneficial relationship with the host. Plating the tissue homogenate from the central core of suckers showed colony growth on nutrient agar from just 75% and 42% of the 12 stocks during May and November, respectively (average 58%; 6 × 103 colony-forming units per gram), yielding diverse organisms belonging to firmicutes (Bacillus, Brevibacillus, Paenibacillus, Virgibacillus, Staphylococcus spp.), actinobacteria (Cellulomonas, Micrococcus, Corynebacterium, Kocuria spp.), α-proteobacteria (Paracoccus sp.), and γ-proteobacteria (Pseudomonas, Acinetobacter spp.). Each shoot tip showed one to three different organisms and no specific organism appeared common to different sucker tips. Tissue homogenate from shoot tips including the ones that did not yield culturable bacteria displayed abundant bacterial cells during microscopic examination suggesting that a high proportion of cells were in viable-but-nonculturable state, or their cultivation requirements were not met. Direct application of cultivation-independent approach to study endophytic bacterial community using bacterial 16S ribosomal RNA universal primers resulted in high interference from chloroplast and mitochondrial genome sequences. Dislodging the bacterial cells from shoot tips that did not show cultivable bacteria and incubating the tissue crush in dilute-nutrient broth led to the activation of four organisms (Klebsiella, Agrobacterium, Pseudacidovorax spp., and an unidentified isolate). The endophytic organisms in general showed better growth at 30–37 °C compared with 25 °C, and the growth of endophytes as well as pathogenic Erwinia carotovora were promoted with the supply of host tissue extract (HTE) while that of the isolates from nonplant sources were inhibited or unaffected by HTE, suggesting an affinity or dependence of the endophytes on the host and the prospect of an HTE-based assay for discriminating the nonendophytes from endophytes.

Notes

Acknowledgements

This study was supported by a grant from the Department of Biotechnology, Govt. of India, New Delhi, India under the project “Identification of covert endophytic microbes in plant tissue cultures and their management and control.” The supply of suckers of different banana cultivars by Dr. L. B. Naik/Dr. Saxena, laboratory assistance by G. K. Swarna, N. Shivrudriah, and B. Hanumanthraju (IIHR, Bangalore, India) are acknowledged. IIHR Contr. No. 55/2008

Supplementary material

248_2009_9559_MOESM1_ESM.rtf (43 kb)
Supplementary Table 1 Growth of selected bacterial isolates from different phylogenetic groups in dilute-nutrient broth or broth supplemented with filter-sterilized host tissue extract (HTE) (RTF 42 kb)
248_2009_9559_MOESM2_ESM.pdf (151 kb)
Supplementary Fig. 1 Phylogenic tree based on 16S rRNA gene sequence of different endophytic bacterial isolates retrieved from banana shoot tips. Phylogenic relationships were estimated according to Jukes–Cantor model and the tree was constructed by the neighbor-joining method with 100–1,000 bootstrap resamplings. Genetic distances are indicated along the branch (PDF 150 kb)
248_2009_9559_Fig3_ESM.gif (132 kb)
Supplementary Fig. 2

Shoot-tip homogenate of banana under phase contrast (1000×) displaying plastids (P) and mitochondria (M) together with bacterial cells in the background (a), and filtered tissue homogenate (2.5 µm) displaying abundant bacterial cells after the sedimentation of larger particles (b) (GIF 132 kb)

248_2009_9559_Fig3_ESM.tif (3.7 mb)
High Resolution Image (TIFF 3.73 mb)

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Copyright information

© Springer Science+Business Media, LLC 2009

Authors and Affiliations

  1. 1.Division of BiotechnologyIndian Institute of Horticultural ResearchBangaloreIndia

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