Molecular detection of a bacterial contaminant Bacillus pumilus in symptomless potato plant tissue cultures

Isenegger, Daniel; Taylor, Peter W.; Mullins, Kathleen; McGregor, Graeme; Barlass, M.; Hutchinson, James F.

doi:10.1007/s00299-003-0583-z

Cited by 14 publications

(4 citation statements)

References 25 publications

(17 reference statements)

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“…Covert or inconspicuous bacteria in vitro, unless introduced intentionally, is highly undesirable as they can interfere with research conclusions and act as a potential threat to micropropagation industry (Leifert and Woodward 1998), in vitro gene banks (Van den Houwe and Swennen 2000) and safe exchange of germplasm (Salih et al 2001). Endophytic B. pumilus in symptomless potato tissue cultures was found to be the cause for aberrant RAPD patterns (Isenegger et al 2003). B. pumilus spores could survive even autoclaving temperatures (Leifert et al 1989).…”

Section: Discussionmentioning

confidence: 99%

Isolation of Bacillus pumilus from in vitro grapes as a long-term alcohol-surviving and rhizogenesis inducing covert endophyte

Thomas

2004

J Appl Microbiol

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P . T H O M A S . 2004.Aims: To isolate and characterize the alcohol-surviving covert bacterium associated with grape tissue culture. Methods and Results: Single colony was isolated by plating the spent rectified spirit used during grape culturing and the organism was identified as Bacillus pumilus using partial 16S rDNA sequence data. Spotting tests (1 ll) using 3-day-old broth culture having a spore content of 20-30% showed similar bacterial survival in 25-90% (v/v) aqueous ethanol for 14 days. Survival in 90% ethanol and 90% rectified spirit appeared affected thereafter with no colony growth from 1 ll samples after 4 months. Plating the samples at this stage gave similar CFU ml )1 for 25, 50 and 70% ethanol, a significant reduction in 80% ethanol and very few colonies in 90% ethanol and rectified spirit. B. pumilus-inoculated grape microcuttings showed substantial endophytic colonization of original cuttings (7AE4 · 10 6 CFU g )1 ) followed by the sprout (5AE9 · 10 5 ) and roots (2AE0 · 10 4 ). The bacterium although a poor root colonizer, induced early rooting and more roots in vitro. Inoculation at ex vitro planting resulted in significantly more roots, root weight and shoot growth. Conclusions: Bacillus pumilus could remain as a covert endophyte in grape tissue cultures and survive in aqueous ethanol for extended periods. 90% ethanol was the most effective bactericidal concentration. The bacterium showed endophytic colonization and root and shoot growth promotion. Significance and Impact of the Study: The revelation that general recommendation of 70-80% ethanol may not be the most effective bactericidal concentration for all bacteria, elucidation of the possibility of covert bacterial survival in vitro plant cultures and isolation of a potential plant growth promoting endophyte in grape.

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Section: Discussionmentioning

confidence: 99%

Isolation of Bacillus pumilus from in vitro grapes as a long-term alcohol-surviving and rhizogenesis inducing covert endophyte

Thomas

2004

J Appl Microbiol

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“…and Bacillus spp.1 (Table 5), in mixes with plant material. The detection limit of primers for Bacillus spp.3 (closely related to B. pumilus) primers and for Bacillus spp.1 (closely related to B. cereus) were 20 and almost 5 × 10 3 fold more sensitive, respectively, than those previously reported (Isenegger et al, 2003;Kałużna et al, 2014). Only primers for Janibacter spp.1 were not sensitive enough to detect the bacteria either directly from culture (without DNA extraction), or from contaminated plants (with DNA extraction).…”

Section: Discussionmentioning

confidence: 69%

“…Bacillus spp. and Paenibacillus spp., often found in the soil ( Supplementary Table S3 ), represented the majority of bacteria identified in this research ( Table 4 ), possibly because they can live endogenously within the plant or root as a result of colonization through physical damage, during growth or as an escape of initial sterilization during tissue culture isolation ( Isenegger et al, 2003 ). Indeed, there are reports of autoclave and alcohol resistant, spore-forming bacteria ( Thomas and Soly, 2009 ; Thomas et al, 2009 ; Thomas and Aswath, 2014 ), and thus this cannot be ruled out.…”

Section: Discussionmentioning

confidence: 98%

Identification and Control of Latent Bacteria in in vitro Cultures of Sweetpotato [Ipomoea batatas (L.) Lam]

et al. 2020

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Bacterial microorganisms which are latent in in vitro cultures can limit the efficiency of in vitro methods for the conservation of genetic resources. In this study we screened 2,373 accessions from the in vitro sweetpotato germplasm collection of the International Potato Center in Lima, Peru for bacteria associated with plantlets in tissue culture through a combination of morphological methods and partial 16S rDNA sequencing. Bacteria were detected in 240 accessions (10% of the accessions screened) and we were able to isolate 184 different bacterial isolates from 177 different accessions. These corresponded to at least nineteen Operational Taxonomic Units (OTUs) of bacteria, belonging to the genera Sphingomonas, Bacillus, Paenibacillus, Methylobacterium, Brevibacterium, Acinetobacter, Microbacterium, Streptomyces, Staphylococcus, and Janibacter. Specific primers were developed for PCR based diagnostic tests that were able to rapidly detect these bacteria directly from tissue culture plants, without the need of microbial sub-culturing. Based on PCR screening the largest bacterial OTUs corresponded to a Paenibacillus sp. closely related to Paenibacillus taichungensis (41.67%), and Bacillus sp. closely related to Bacillus cereus (22.22%), and Bacillus pumilus (16.67%). Since in vitro plant genetic resources must be microbe-free for international distribution and use, any microbial presence is considered a contamination and therefore it is critical to clean all cultures of these latent-appearing bacteria. To accomplish this, plantlets from in vitro were transferred to soil, watered with Dimanin R (2 ml/l) weekly and then reintroduced into in vitro. Of the 191 accessions processed for bacterial elimination, 100% tested bacteria-free after treatment. It is suspected that these bacteria may be endosymbionts and some may be beneficial for the plants.

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“…However, because the growth medium employed in tissue culture may not suit bacteria or inhibit their growth, pathogens may be undetected for lengthy periods (Isenegger et al 2003).…”

Section: Resultsmentioning

confidence: 99%

Effect of antibiotics in eliminating bacterial wilt (Ralstonia solanacearum) from in vitro propagated ginger

MARKOS

Feyissa

2020

Cell Biol Dev

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Abstract. Markos T, Feyissa T. 2020. Effect of antibiotics in eliminating bacterial wilt (Ralstonia solanacearum) from in vitro propagated ginger. Cell Biol Dev 4: 46-52. Ginger (Zingiber officinale Roscoe) is a herbaceous perennial. It is cultivated commercially in most tropical regions of the world and is a member of the Zingiberaceae family. In conventional ginger cultivation, underground rhizomes are used as planting material. However, it is afflicted by diseases that result in the senescence and degeneration of tissues. Due to Ralstonia solanacearum infection, substantial rhizome losses have been recorded. Therefore, disease-free planting material is required to cultivate ginger successfully. Plant tissue culture technology has been successfully used to commercialize pathogen-free plants and conserves the germplasm of rare and endangered species. In in-vitro, culture techniques offer an alternative method of plant multiplication and a method for crop enhancement. Frequently, ginger multiplication media are contaminated with R. solanacearum, which can survive endophytically in plantlets, rendering them useless for in vitro propagation. Therefore, an experiment was undertaken to determine the efficiency of antibiotics against the in vitro development of R. solanacearum. Four antibiotics, gentamicin, tetracycline, ampicillin, and streptomycin, at four concentrations, 130 mg/L, 160 mg/L, 200 mg/L, and 250 mg/L, were evaluated for their ability to eradicate bacteria from in-vitro propagated ginger. Gentamicin was proven superior to other therapies, with the maximum inhibition (22 mm) at 250 mg/L, followed by tetracycline at 200 mg/L (18 mm), streptomycin at 130 mg/L (13 mm), and ampicillin at 130 mg/L (12 mm). The R. solanacearum can be eradicated from in vitro propagated ginger by applying antibiotics in the micropropagation media.

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Molecular detection of a bacterial contaminant Bacillus pumilus in symptomless potato plant tissue cultures

Cited by 14 publications

References 25 publications

Isolation of Bacillus pumilus from in vitro grapes as a long-term alcohol-surviving and rhizogenesis inducing covert endophyte

Isolation of Bacillus pumilus from in vitro grapes as a long-term alcohol-surviving and rhizogenesis inducing covert endophyte

Identification and Control of Latent Bacteria in in vitro Cultures of Sweetpotato [Ipomoea batatas (L.) Lam]

Effect of antibiotics in eliminating bacterial wilt (Ralstonia solanacearum) from in vitro propagated ginger

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