Isolation of Tomato Seed-Associated Microbiota through Controlled Germination and Comparative Culturing

Summary

This protocol combines standardized seed surface sterilization with controlled germination to enable the reproducible isolation of tomato seed-associated microbiota during the seed-to-seedling transition. The approach enhances recovery of culturable microorganisms that are underrepresented in dry seeds and supports comparative culturomic studies during early seedling development.

Abstract

Surface sterilization is a critical yet frequently under-optimized step in studies aiming to isolate seed-associated microorganisms, as excessively harsh treatments can reduce or distort endogenous microbial populations. This protocol presents a reproducible approach for isolating culturable tomato seed-associated microbiota using surface-sterilization conditions based on previously validated methodologies, combined with controlled seed germination. Rather than defining universally optimal sterilization treatments, the approach applies standardized conditions that can be adjusted according to seed characteristics while minimizing bias and preserving endogenous microbial populations.

To enhance recovery of culturable microorganisms associated with the seed-to-seedling transition, the protocol incorporates a controlled germination step prior to microbial isolation. In contrast to conventional approaches based on the maceration of dry, non-germinated seeds, controlled germination promotes microbial activation during the seed-to-seedling transition under sterile conditions. Comparative colony-forming unit (CFU)-based analyses of equivalent biological replicates, each consisting of pooled material from 3–5 dry seeds or 3–5 germinated seedlings, revealed a marked shift in the culturable community following germination. Genera that were weakly represented in dry seeds became enriched after germination, whereas other taxa that were more abundant in dry seeds decreased in relative representation, supporting studies of early plant-microbe interactions during seedling establishment.

Across biological replicates, genera including Stutzerimonas, Stenotrophomonas, and Priestia were consistently enriched among isolates recovered from germinated seedlings, whereas Paenibacillus remained abundant across conditions but showed marked shifts in species-level composition. By integrating standardized sterilization conditions with controlled germination and comparative analysis of dry and germinated material, this protocol provides a robust and reproducible framework for the culturomic isolation of seed-associated microbiota with potential functional relevance during early seedling development and supports studies of early plant–microbe interactions during seedling establishment.

Introduction

Seeds harbor diverse microbial communities composed of bacteria and fungi associated with both internal tissues and external surfaces^1,2,3. These microorganisms are increasingly recognized as integral components of plant biology because they can influence plant performance from the earliest developmental stages, including germination, seedling establishment, and responses to biotic and abiotic stress^1,2,3. In ecological terms, seeds function not only as propagules for plant reproduct....

Protocol

1. Preparation of reagents, media, and sterile materials

1. Prepare a 70% (v:v) ethanol solution using sterile distilled water.
2. Prepare a 20% (v:v) working solution from commercial bleach containing 5% available sodium hypochlorite by diluting 20 mL of bleach in 80 mL of sterile distilled water.
3. Prepare sterile distilled water for washing steps.
4. Prepare sterile 0.45% NaCl for homogenization and serial dilution, and use this solution consistently throughout the workflow.
5. Prepare Luria Bertani (LB) agar culture medium (per liter: 10 g of tryptone, 5 g of yeast extract, 10 g of NaCl, and 15 g of agar) and ....

Results

Performance of surface sterilization and control of external contamination
Surface sterilization using standardized conditions based on previously validated methodologies resulted in effective reduction of external microbial contamination while preserving the recovery of culturable microorganisms from seed-derived material. Plating of the final rinse water showed no detectable colony growth, indicating effective removal of surface-associated microorganisms under the conditions used.

Discussion

The results presented here demonstrate that the integration of standardized surface sterilization with controlled seed germination provides a robust framework for the culturomic isolation of seed-associated microbiota. A key outcome of this protocol is the ability to balance effective removal of external contaminants with preservation of endogenous microbial populations. This balance is critical in culture-based studies, where overly stringent sterilization can reduce microbial recovery, while insufficient sterilization .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Agar	Sigma-Aldrich	01916-500G	Component of LB agar medium; 15 g/L.
Autoclave	Lab1st	SV120H	Used to sterilize instruments/materials or validate aseptic treatment.
Bacteria DNA extraction kit	Promocell	PK-MB708-206-100	DNA extraction
Colony counting/recording setup	OCS	B079Y5W57N	Needed to record CFUs, morphotypes, and plate differences; manuscript does not specify counter/model.
Cryostorage vials	Sigma-Aldrich	Z359033	Implied for storage of glycerol stocks at -80 °C; not named explicitly but needed for preservation step.
Digital isolate record system	Not specified in manuscript	Not applicable	Used to record isolate metadata: origin, replicate, condition, sterilization treatment, isolation date, plate medium, morphology, identification status.
Ethanol	VWR	83813440	Used to prepare 70% v/v ethanol solution for seed surface sterilization.
Filter paper	Fisher Scientific	11718772	Optional alternative germination support.
Forceps/Tweezers	Sigma-Aldrich	930229	Used to transfer seeds and collect germinated seedlings.
Genomic DNA extraction kit	PromoCell	PK-MB708-206-100	Used to extract genomic DNA from purified bacterial cultures; exact kit/method not specified.
Glycerol, sterile 80%	VWR	24388364	Mixed 1:1 v/v with liquid culture for cryostock preparation.
Laminar flow hood Bio48	Faster	LAB-CAP-22	Required for aseptic seed handling, sterilization transfers, and preparation of sterile materials.
LB agar medium	VWR	846495000	Standard medium used for comparative culturing; per liter: 10 g of tryptone, 5 g of yeast extract, 10 g of NaCl, 15 g of agar.
Microcentrifuge tubes	Ratiolab	RATI5615000	Used for seed sterilization and sample processing.
MS medium	Sigma	M0404-10L	Mentioned as sterile germination support for axenic germination.
NZYTaq II 2× Master mix	Nzytech	MB35803	PCR mix for amplification
Parafilm	Parafilm/Merck	HS234526A	Used to seal germination plates while reducing contamination.
Petri dishes	Labbox	PDIP-E9N-500	Used for germination setup, plating, and handling sterilized seeds.
Phytoagar	Duchefa Biochemie	9002-18-0	Mentioned as an optional alternative sterile germination support.
Pipettes set and tips	Merck	EP3123000918-1EA	Used for removing sterilization solutions, washing, dilution, and plating.
R2A agar medium	HI-MEDIA	M1743-500G	Optional additional medium for extended culturomic workflows.
Sodium chloride, NaCl	Fisher Chemical	10040460	Used to prepare sterile 0.45% NaCl for homogenization and serial dilutions; also part of LB medium.
Sodium hypochlorite	Carlo Erba	370323	Used to prepare 20% v/v sodium hypochlorite solution for seed surface sterilization. Corrosive; requires PPE.
Sterile disposable pestles	Eppendorf	EP0030120973	Used for homogenization of dry seeds and germinated seedlings.
Vortex mixer	Fisher Scientific	15901137	Used to resuspend microbial cells from homogenates.