Isothermal DNA amplification facilitates the identification of a broad spectrum of bacteria, fungi and protozoa in Eleutherococcus sp. plant tissue cultures

Abstract

In vitro cultures of Eleutherococcus sieboldianus originating from surface sterilized leaf explants were found to be associated with several microorganisms. The associations included bacteria, fungi and protozoa within the rhizosphere and inside root hairs. To determine if this phenomenon is unique to this species, plant tissue cultures of E. gracilistylus and E. senticosus were included in our studies for comparison. A methodology consisting of isothermal amplification, cloning and sequencing was established for analysing 16S ribosomal DNA of cultivated and non-cultivated bacteria from different tissue types. The same methodology was used to obtain internal transcribed spacer regions and 18S regions of fungal and protozoan rDNA. Comparative analyses of sequencing data resulted in the identification of various genera within the Firmicutes and c-proteobacteria kingdoms and a broad spectrum of fungal genera related to several uncultured fungi. In addition, amoebal and chrysophyte species were detected. Most of the species were identified in different plant organs and in in vitro culture cell types indicating the microorganisms are systemically distributed. The presence of identical microorganisms in different plant species argues for an evolutionary long-lasting and stable association between the plant genus and the microinhabitants.

Development of a loop-mediated isothermal amplification method for the rapid diagnosis of Ascochyta rabiei L. in chickpeas

“…Furthermore, after quality testing with a NanoDrop 2000 and 1% agarose gel electrophoresis, the samples were dyed with EB and examined under UV light (280 nm) and subsequently stored at −20 °C. The DNA solutions from the artificial infected soil samples were extracted via the CTAB method 40 with slight modifications. Briefly, each 0.1 g soil sample was carefully ground with 1 mL lysis buffer (Tris-HCl 100 mM, pH = 9.0, EDTA 100 mM, NaCl 400 mM, 2% SDS) and 0.5 g of quartz sand in a mortar.…”

Functional roles of microbial symbionts in plant cold tolerance

“…In order to further knowledge, future surveys should focus on yeasts, since few articles have reported yeast endophytes in cold environments (Loque et al 2010; Godinho et al 2013; Santiago et al 2016), with none having tested the effects of these fungi on host plant performance. Studies should also focus on unicellular algae and protists, which have been shown to be present in the tissues of temperate and tropical plant species (Trémouillaux‐Guiller et al 2002; Müller & Döring 2009), but the effects of which on plants at low temperatures remain poorly defined. We also recommend studies into the effects of microbial symbionts on plant cold tolerance mediated by the synthesis of secondary metabolites, such as the antibacterial compounds produced by the fungal endophyte Phoma against the ice‐nucleating bacterium Pseudomonas syringae (Moghaddam & Soltani 2014).…”

Comunidade bacteriana endofítica em microplantas de abacaxizeiro: estrutura, diversidade e sua influência na morfofisiologia após antibioticoterapia

“…A ocorrência de espécies de Stenotrophomonas já foi verificada em microplantas de equinácea (LATA et al, 2006), comigo-ninguém-pode (JAN et al, 2004), lavanda-do-mar (LIU; HSU; WU,. 2005) e ginseng siberiano (MULLER;DÖRING, 2009). Ao mesmo tempo estudos com espécies de Burkholderia em abacaxizeiro comprovaram a capacidade dessas bactérias em promover o crescimento vegetal durante a fase de aclimatização de microplantas (WEBER et al, 2003a, b) e aumentar a produtividade de frutos (WEBER et al, 2004), indicando que os benefícios causados pela interação entre plantas e as bactérias dos…”

“…Visando eliminar essa competição sugere-se o uso dos primers 799f (CHELIUS;TRIPLETT, 2001) e 1492R(HEUER et al, 1997) para aumentar a concentração do DNA bacteriano antes da amplificação com grampo GC(ANDREOTE;AZEVEDO;ARAÚJO, 2009). O primer 799f vem sendo utilizado com freqüência, tendo sido relatado em vários estudos recentes em comunidades bacterianas endofíticas(IDRIS et al, 2004; MÜLLER;DÖRING, 2009;.A ARISA, desenvolvida por Fisher e Tripplett (1999) é uma técnica baseada no polimorfismo do comprimento do espaço intergênico ribossomal 16S-23S rRNA; consiste no uso de primer fluorescente na amplificação por PCR do espaço intergênico entre a menor e a maior subunidade do gene rRNA (16S-23S em procarioto). Esta região pode codificar vários tRNAs dependendo da espécie e apresenta heterogeneidade em termos do comprimento e da composição da sequência(GONZÁLEZ et al 2003;RANJARD;POLY;NAZARET, 2000).…”