Root-associated bacterial endophytes from Ralstonia solanacearum resistant and susceptible tomato cultivars and their pathogen antagonistic effects.

Reshmi Upreti,Pious Thomas

doi:10.3389/fmicb.2015.00255

Abstract

This study was undertaken to assess if the root-associated native bacterial endophytes in tomato have any bearing in governing the host resistance to the wilt pathogen Ralstonia solanacearum. Internal colonization of roots by bacterial endophytes was confirmed through confocal imaging after SYTO-9 staining. Endophytes were isolated from surface-sterilized roots of 4-weeks-old seedlings of known wilt resistant (R) tomato cultivar Arka Abha and susceptible (S) cv. Arka Vikas on nutrient agar after plating the tissue homogenate. Arka Abha displayed more diversity with nine distinct organisms while Arka Vikas showed five species with two common organisms (Pseudomonas oleovorans and Agrobacterium tumefaciens). Screening for general indicators of biocontrol potential showed more isolates from Arka Abha positive for siderophore, HCN and antibiotic biosynthesis than from Arka Vikas. Direct challenge against the pathogen indicated strong antagonism by three Arka Abha isolates (P. oleovorans, Pantoea ananatis, and Enterobacter cloacae) and moderate activity by three others, while just one isolate from Arka Vikas (P. oleovorans) showed strong antagonism. Validation for the presence of bacterial endophytes on three R cultivars (Arka Alok, Arka Ananya, Arka Samrat) showed 8–9 antagonistic bacteria in them in comparison with four species in the three S cultivars (Arka Ashish, Arka Meghali, Arka Saurabhav). Altogether 34 isolates belonging to five classes, 16 genera and 27 species with 23 of them exhibiting pathogen antagonism were isolated from the four R cultivars against 17 isolates under three classes, seven genera and 13 species from the four S cultivars with eight isolates displaying antagonistic effects. The prevalence of higher endophytic bacterial diversity and more antagonistic organisms associated with the seedling roots of resistant cultivars over susceptible genotypes suggest a possible role by the root-associated endophytes in natural defense against the pathogen.

Highlights

Endophytic microorganisms colonize plants internally without any apparent adverse effects on the host (Hallmann et al, 1997; Gaiero et al, 2013)
Confocal Imaging of Seedling Roots Seedling roots were examined for bacterial colonization through confocal laser scanning microscopy (CLSM) after SYTO-9 staining
Root tissues were examined after surface sterilization which involved a quick dip in 90% ethanol, a rinse in sterile distilled water (SDW) and 1 min sodium hypochlorite (2% available chlorine) treatment followed by six SDW rinses

Summary

Introduction

Endophytic microorganisms colonize plants internally without any apparent adverse effects on the host (Hallmann et al, 1997; Gaiero et al, 2013). Bacterial endophytes are generally known to enter the host from the surrounding soil through wounds in the roots (Hallmann et al, 1997; Compant et al, 2010) or through root hairs (Prieto et al, 2011; Mercado-Blanco and Prieto, 2012) They traverse the root cortex and reach various plant organs through the vascular system (Hallmann et al, 1997; Compant et al, 2010, 2011) while some use the apoplastic route (Sattelmacher, 2001; Reinhold-Hurek et al, 2007). A recent study employing banana shoot tissue has shown abundant endophytic bacteria in the two intracellular niches, namely in the cytoplasm and in the perispace between the cell wall and plasma membrane, and the terms ‘Cytobacts’ and ‘Peribacts’ have been coined to recognize the microorganisms in the respective intracellular niches (Thomas and Reddy, 2013; Thomas and Sekhar, 2014)

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Conclusion