Identification and Control of Latent Bacteria in in vitro Cultures of Sweetpotato [Ipomoea batatas (L.) Lam]
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 ba...
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doaj-712789cc86f04860b02df8631f5dfc412020-11-25T03:08:03ZengFrontiers Media S.A.Frontiers in Plant Science1664-462X2020-07-011110.3389/fpls.2020.00903550873Identification and Control of Latent Bacteria in in vitro Cultures of Sweetpotato [Ipomoea batatas (L.) Lam]Myriam Lorena IzarraAna Luz PantaCarmen Rosa MazaBrenda Carina ZeaJuan CruzadoLiliam Rosario GutarraCristina R. RiveraDavid EllisJan Frederik KreuzeBacterial 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® (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.https://www.frontiersin.org/article/10.3389/fpls.2020.00903/full16S rRNAendogenous microbestissue culturecontaminationgenetic resources |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Myriam Lorena Izarra Ana Luz Panta Carmen Rosa Maza Brenda Carina Zea Juan Cruzado Liliam Rosario Gutarra Cristina R. Rivera David Ellis Jan Frederik Kreuze |
spellingShingle |
Myriam Lorena Izarra Ana Luz Panta Carmen Rosa Maza Brenda Carina Zea Juan Cruzado Liliam Rosario Gutarra Cristina R. Rivera David Ellis Jan Frederik Kreuze Identification and Control of Latent Bacteria in in vitro Cultures of Sweetpotato [Ipomoea batatas (L.) Lam] Frontiers in Plant Science 16S rRNA endogenous microbes tissue culture contamination genetic resources |
author_facet |
Myriam Lorena Izarra Ana Luz Panta Carmen Rosa Maza Brenda Carina Zea Juan Cruzado Liliam Rosario Gutarra Cristina R. Rivera David Ellis Jan Frederik Kreuze |
author_sort |
Myriam Lorena Izarra |
title |
Identification and Control of Latent Bacteria in in vitro Cultures of Sweetpotato [Ipomoea batatas (L.) Lam] |
title_short |
Identification and Control of Latent Bacteria in in vitro Cultures of Sweetpotato [Ipomoea batatas (L.) Lam] |
title_full |
Identification and Control of Latent Bacteria in in vitro Cultures of Sweetpotato [Ipomoea batatas (L.) Lam] |
title_fullStr |
Identification and Control of Latent Bacteria in in vitro Cultures of Sweetpotato [Ipomoea batatas (L.) Lam] |
title_full_unstemmed |
Identification and Control of Latent Bacteria in in vitro Cultures of Sweetpotato [Ipomoea batatas (L.) Lam] |
title_sort |
identification and control of latent bacteria in in vitro cultures of sweetpotato [ipomoea batatas (l.) lam] |
publisher |
Frontiers Media S.A. |
series |
Frontiers in Plant Science |
issn |
1664-462X |
publishDate |
2020-07-01 |
description |
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® (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. |
topic |
16S rRNA endogenous microbes tissue culture contamination genetic resources |
url |
https://www.frontiersin.org/article/10.3389/fpls.2020.00903/full |
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