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In Vitro Activation of Seed-Transmitted Cultivation-Recalcitrant Endophytic Bacteria in Tomato and Host–Endophyte Mutualism

Division of Biotechnology, Endophytic and Molecular Microbiology Laboratory, ICAR-Indian Institute of Horticultural Research, Hessaraghatta Lake, Bengaluru 560089, India
*
Author to whom correspondence should be addressed.
Current Address: Thomas Biotech & Cytobacts Centre for Biosciences, 318 Talacauvery Layout, Amruthahalli, Bengaluru 560092, India.
Current Address: Department of Biotechnology, Centre for Post-Graduate Studies, Jain University, Bengaluru 560011, India.
Microorganisms 2019, 7(5), 132; https://doi.org/10.3390/microorganisms7050132
Received: 19 April 2019 / Revised: 9 May 2019 / Accepted: 10 May 2019 / Published: 14 May 2019
(This article belongs to the Special Issue Plant Control of Symbiotic Microbe Behavior and Reproduction)
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Abstract

This study was aimed at exploring seed transmission of endophytic bacteria in tomato utilizing aseptic in vitro conditions. Cultivation-based studies were undertaken on two tomato cultivars “Arka Vikas” and “Arka Abha” employing surface sterilized seeds, aseptically germinated seeds and in vitro grown seedlings at different stages. Bacillus sp. appeared primarily as seed externally-associated bacteria. Tissue homogenate from extensively surface-sterilized seeds, day-3 germinating seeds, or 10-day in vitro seedlings did not show any cultivable bacteria on two bacteriological media. Indexing of 4-week old healthy seedlings with seed-coat removal following seed germination showed bacterial association in 50–75% seedlings yielding 106–107 cfu g−1 tissues. Four endophytic bacteria appeared common to both cultivars (Kosakonia, Ralstonia, Sphingomonas, Sphingobium spp.) with three additional species in “Arka Abha”. The bacterial strains showed a manifold increase in growth with host-tissue-extract supplementation. Seed inoculations with single-isolates stimulated germination or enhanced the seedling growth coupled with the activation of additional endophytic bacteria. In vitro seedlings upon recurrent medium-indexing over eight weeks showed gradual emergence of endophytic bacteria. The study reveals the seed internal colonization by different bacterial endophytes in a cultivation-recalcitrant form, their activation to cultivable state during seedling growth and transmission to seedlings with mutualistic effects. View Full-Text
Keywords: bacterial activation; cultivation recalcitrant endophytic bacteria (CREB); in vitro plant cultures; host–microbe interactions; plant growth promotion; plant tissue culture; seed transmission; Solanum lycopersicum L.; viable but non-cultivable (VBNC) bacteria bacterial activation; cultivation recalcitrant endophytic bacteria (CREB); in vitro plant cultures; host–microbe interactions; plant growth promotion; plant tissue culture; seed transmission; Solanum lycopersicum L.; viable but non-cultivable (VBNC) bacteria
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    Description: Figure S1. Monitoring the tomato seeds for the efficacy of surface sterilization through different steps. (a, b) Seed wash solutions of tomato ‘Arka Vikas’ and ‘Arka Abha’ (direct and after Tween-20 step) showing several bacterial colonies on nutrient agar (NA) comprising of Bacillus spp./ spore formers, and (c, d) very few or no colonies after ethanol wash or NaOCl treatment Figure S2. Tissue homogenate (100 seeds 10 ml-1) from surface-sterilized seeds of tomato ‘Arka Vikas’ and ‘Arka Abha’ and the five decimal serial dilutions applied on nutrient agar showing particulate matter at the original homogenate applied spots (100) mimicking colony growth (with no cfu upon its re-streaking or dilution-plating) Figure S3. Day-10 in vitro grown seedling of tomato ‘Arka Vikas’ and ‘Arka Abha’ on sucrose-minus Murashige and Skoog medium [29] showing clear medium devoid of any microbial association and the seed coat (indicated by arrowhead) detached from the seedling base or carried at the distal end of the cotyledon Figure S4. SP-SDS on the root tissue homogenate from individual bacteria index-positive 3 weeks old ‘Arka Vikas’ (Av) and Arka Abha (Ab) seedlings showing the association of a single organism in most cases Figure S5. A different seed lot of the tomato ‘Arka Vikas’ and ‘Arka Abha’ monitored at surface sterilization through seed wash solution spotting on NA (direct and after Tween-20 step) displaying Bacillus spp./ spore formers (a, b), and inoculum carry over after ethanol wash or NaOCl treatments (c, d) due to difference in the initial bacterial load, or a reduction in the efficacy of the NaOCl
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Shaik, S.P.; Thomas, P. In Vitro Activation of Seed-Transmitted Cultivation-Recalcitrant Endophytic Bacteria in Tomato and Host–Endophyte Mutualism. Microorganisms 2019, 7, 132.

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