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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Main Authors: 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
Format: Article
Language:English
Published: Frontiers Media S.A. 2020-07-01
Series:Frontiers in Plant Science
Subjects:
Online Access:https://www.frontiersin.org/article/10.3389/fpls.2020.00903/full
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spelling 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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