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Intense association of non-culturable endophytic bacteria with antibiotic-cleansed in vitro watermelon and their activation in degenerating cultures

Abstract

The study was undertaken with a view to unravel the source of bacterial colony growth observed in a section of micropropagated triploid watermelon cultures that were supposedly cleansed of the associated endophytic bacteria through antibiotic treatment, and thereafter maintained under stringent sterility checks to prevent lateral intrusion of contaminants. Five different bacteria were retrieved from colony growth-displaying watermelon cultures that were previously treated with gentamycin and five isolates from cefazolin-treated stocks with the organisms showing tolerance to the respective antibiotic. These watermelon cultures were in degeneration phase (over 6 months after the previous sub-culturing), while the actively maintained counterpart stocks appeared healthy with no colony growth on different bacteriological media during tissue-screenings. The latter cultures, however, revealed abundant motile, tetrazolium-stained bacterial cells in microscopy, suggesting tissue colonization by non-culturable endophytes. PCR screening on healthy cultures endorsed tissue colonization by different bacterial phylogenic groups. A few organisms could be activated to cultivation from healthy watermelon stocks through host tissue extract supplementation, which also enhanced the growth of all the organisms. The study indicated that a fraction of antibiotic-tolerant bacteria survived intra-tissue in non-culturable form during the preceding cleansing activity, multiplied to substantial numbers thereafter, and turned cultivable in degenerating cultures contributed by tissue breakdown products. This study brings out the existence of a deep endophyte association in tissue cultures which is not easily dissociable. It also signifies the utility of in vitro system for investigations into plant–endophyte association and to bring normally non-culturable novel organisms to cultivation facilitating their future exploitation.

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Abbreviations

HTE:

Host tissue extract

NA:

Nutrient agar

TTC:

2,3,5-Triphenyl tetrazolium chloride

TSA:

Trypticasein soy agar

VBNC:

Viable but non-culturable

WMM:

Watermelon multiplication medium

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Acknowledgments

The help rendered by the research fellows Aparna, Reshmi, Lokesh and Mujawar during the conduct of some of the trials is gratefully acknowledged. This study was initiated under the project “Identification of covert endophytic microbes in plant tissue cultures and their management and control” funded by the Department of Biotechnology, Govt. of India, and now completed with funding support from the Indian Council of Agricultural Research under the AMAAS project “Basic and applied investigations on endophytic microorganisms in horticultural crops”. This publication bears IIHR Contribution No. 46/11. Supply of live bacterial cultures for research purpose is subject to their revival from glycerol stocks and the requestor obtaining permission from the Director General, Indian Council for Agricultural Research, New Delhi-110001.

Author information

Correspondence to Pious Thomas.

Additional information

Communicated by P. Kumar.

Electronic supplementary material

Below is the link to the electronic supplementary material. Please use Windows Media Player to open the following supplementary materials.

Supplementary material 1. Tissue squeeze from visibly clean index-negative watermelon cultures showing abundant motile bacteria including cocci, oblong and rod shaped cells under bright field microscopy (100× objective) (MPG 1.26 mb)

Supplementary material 2. Pooled growth from pure cultures of different bacteria isolated from watermelon culture under bright field seen in different planes displaying motility similar to that observed in tissue squeeze (100× objective) (MPG 936 kb)

Supplementary material 3. Tissue squeeze from visibly clean index-negative watermelon cultures showing abundant motile bacteria including cocci, oblong and rod shaped cells under phase contrast in different planes (100× objective) (MPG 2.23 mb)

Supplementary material 4. Pooled growth from pure cultures of different bacteria isolated from watermelon culture under phase seen in different planes with motility as observed in tissue squeeze (100× objective) (MPG 1.26 mb)

Supplementary material 5. Free-hand tissue section from surface-sterilized index-negative watermelon shoots under bright field showing motile bacteria (100× objective) (MPG 1.18 mb)

Supplementary material 6. Free-hand tissue section from surface-sterilized index-positive watermelon culture displaying abundant motile bacteria in different vertical planes (100× objective) (MPG 3.00 mb)

Supplementary material 7. Free-hand sections from surface-sterilized index-positive watermelon culture incubated in TTC solution for 24 h displaying light pink-stained and non-stained motile bacteria in different planes (100× objective) (MPG 3.05 mb)

Supplementary material 8. Surface sterilized tissue from index-negative watermelon culture incubated in TTC solution for 7 days showing abundant pink-stained bacterial cells including motile and non-motile cells in different planes (100× objective) (MPG 1.98 mb)

Supplementary material 9. Pure culture of bacterial isolate GM 03 incubated in TTC solution for 24 h showing pink stained cells with similar motility as seen in tissue sections (100× objective) (MPG 1.09 mb)

Supplementary material 10. Pure culture of bacterial isolate GM 04 incubated in TTC solution for 24 h showing partly pink stained and unstained cells with similar motility as observed in tissue sections (100× objective) (MPG 1.52 mb)

Supplementary material 1. Tissue squeeze from visibly clean index-negative watermelon cultures showing abundant motile bacteria including cocci, oblong and rod shaped cells under bright field microscopy (100× objective) (MPG 1.26 mb)

Supplementary material 2. Pooled growth from pure cultures of different bacteria isolated from watermelon culture under bright field seen in different planes displaying motility similar to that observed in tissue squeeze (100× objective) (MPG 936 kb)

Supplementary material 3. Tissue squeeze from visibly clean index-negative watermelon cultures showing abundant motile bacteria including cocci, oblong and rod shaped cells under phase contrast in different planes (100× objective) (MPG 2.23 mb)

Supplementary material 4. Pooled growth from pure cultures of different bacteria isolated from watermelon culture under phase seen in different planes with motility as observed in tissue squeeze (100× objective) (MPG 1.26 mb)

Supplementary material 5. Free-hand tissue section from surface-sterilized index-negative watermelon shoots under bright field showing motile bacteria (100× objective) (MPG 1.18 mb)

Supplementary material 6. Free-hand tissue section from surface-sterilized index-positive watermelon culture displaying abundant motile bacteria in different vertical planes (100× objective) (MPG 3.00 mb)

Supplementary material 7. Free-hand sections from surface-sterilized index-positive watermelon culture incubated in TTC solution for 24 h displaying light pink-stained and non-stained motile bacteria in different planes (100× objective) (MPG 3.05 mb)

Supplementary material 8. Surface sterilized tissue from index-negative watermelon culture incubated in TTC solution for 7 days showing abundant pink-stained bacterial cells including motile and non-motile cells in different planes (100× objective) (MPG 1.98 mb)

Supplementary material 9. Pure culture of bacterial isolate GM 03 incubated in TTC solution for 24 h showing pink stained cells with similar motility as seen in tissue sections (100× objective) (MPG 1.09 mb)

Supplementary material 10. Pure culture of bacterial isolate GM 04 incubated in TTC solution for 24 h showing partly pink stained and unstained cells with similar motility as observed in tissue sections (100× objective) (MPG 1.52 mb)

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Thomas, P. Intense association of non-culturable endophytic bacteria with antibiotic-cleansed in vitro watermelon and their activation in degenerating cultures. Plant Cell Rep 30, 2313–2325 (2011). https://doi.org/10.1007/s00299-011-1158-z

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Keywords

  • Bacterial endophytes
  • Citrullus lanatus
  • Endophytic microorganisms
  • Microbial contamination
  • Plant tissue culture
  • Tetrazolium staining
  • VBNC
  • Viable but non-culturable bacteria
  • Videography