Plant biostimulants - Determination of Rhizobium spp.

This document provides the methodology for the enumeration and determination of Rhizobium sp., Mesorhizobium sp., Ensifer sp., or Bradyrhizobium sp. in plant biostimulant products in accordance with the Regulation (EU) 2019/1009 of the European Parliament and of the Council [1].

Pflanzen-Biostimulanzien - Bestimmung von Rhizobium spp.

Dieses Dokument wurde entwickelt, um die Methodik für die Zählung und Bestimmung von Rhizobium sp., Mesorhizobium sp., Ensifer sp. oder Bradyrhizobium sp. in Pflanzen-Biostimulanzienprodukten in Übereinstimmung mit der Verordnung (EU) 2019/1009 des Europäischen Parlaments und des Rates [1] bereitzustellen.

Biostimulants des végétaux - Détermination de Rhizobium spp.

Le présent document fournit la méthodologie de dénombrement et de détermination de Rhizobium sp., Mésorhizobium sp., Ensifer sp. ou Bradyrhizobium sp. dans les produits biostimulants des végétaux conformément au Règlement (UE) 2019/1009 du Parlement européen et du Conseil [1].

Rastlinski biostimulanti - Določanje Rhizobium spp.

General Information

Status
Published
Current Stage
6060 - Definitive text made available (DAV) - Publishing
Due Date
23-Mar-2022
Completion Date
23-Mar-2022

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SLOVENSKI STANDARD
kSIST-TS FprCEN/TS 17718:2021
01-november-2021
[Not translated]
Plant biostimulants - Determination of Rhizobium spp
Biostimulanzien für die pflanzliche Anwendung - Bestimmung von Rhizobium spp
Biostimulants des végétaux - Détermination de Rhizobium spp.
Ta slovenski standard je istoveten z: FprCEN/TS 17718
ICS:
65.080 Gnojila Fertilizers
kSIST-TS FprCEN/TS 17718:2021 en,fr,de

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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kSIST-TS FprCEN/TS 17718:2021
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kSIST-TS FprCEN/TS 17718:2021
FINAL DRAFT
TECHNICAL SPECIFICATION
FprCEN/TS 17718
SPÉCIFICATION TECHNIQUE
TECHNISCHE SPEZIFIKATION
September 2021
ICS 65.080
English Version
Plant biostimulants - Determination of Rhizobium spp
Biostimulanzien für die pflanzliche Anwendung -
Bestimmung von Rhizobium spp

This draft Technical Specification is submitted to CEN members for Vote. It has been drawn up by the Technical Committee

CEN/TC 455.

CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,

Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway,

Poland, Portugal, Republic of North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and

United Kingdom.

Recipients of this draft are invited to submit, with their comments, notification of any relevant patent rights of which they are

aware and to provide supporting documentation.

Warning : This document is not a Technical Specification. It is distributed for review and comments. It is subject to change

without notice and shall not be referred to as a Technical Specification.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels

© 2021 CEN All rights of exploitation in any form and by any means reserved Ref. No. FprCEN/TS 17718:2021 E

worldwide for CEN national Members.
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kSIST-TS FprCEN/TS 17718:2021
prEN 17718:2021 (E)
Contents Page

European foreword ............................................................................................................................... 3

Introduction ............................................................................................................................................ 4

1 Scope ............................................................................................................................................ 5

2 Normative references ............................................................................................................ 5

3 Terms and definitions ........................................................................................................... 5

4 Enumeration of Rhizobium sp., Mesorhizobium sp., Ensifer sp.,

Bradyrhizobium sp.) .............................................................................................................. 6

4.1 General ........................................................................................................................................ 6

4.2 Sample preparation ................................................................................................................ 6

4.2.1 General ........................................................................................................................................ 6

4.2.2 Liquid (based water) formulations ................................................................................... 6

4.2.3 Liquid - based oil, emulsifiable concentrate (EC) formulations ............................. 6

4.2.4 Solid - Wettable Powder (WP) formulations ................................................................. 6

4.2.5 Solid - Water dispersible granules (WDG) formulations .......................................... 6

4.2.6 Solid – Pellets, granules, microgranules - slow release - formulations ............... 6

4.2.7 Solid - substrate ....................................................................................................................... 7

4.3 Serial dilution ........................................................................................................................... 7

4.4 Plate counts of rhizobia in sterile diluent ...................................................................... 7

4.5 Spread-plate counting ........................................................................................................... 8

4.6 Growth Media ........................................................................................................................... 8

4.7 Calculation ................................................................................................................................. 8

5 Species Determination of Rhizobium sp., Mesorhizobium sp., Ensifer sp.,

Bradyrhizobium sp.) via genetic analysis ...................................................................... 9

5.1 Preparation of the sample for the genomic DNA extraction .................................... 9

5.1.1 Isolation and preparation of the microorganism ........................................................ 9

5.1.2 Sample concentration ............................................................................................................ 9

5.1.3 DNA extraction and storage ................................................................................................. 9

5.1.4 Partial PCR Amplification of the 16S rRNA Genes ..................................................... 10

Annex A (informative) Formula of colture media................................................................... 11

Bibliography .......................................................................................................................................... 15

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kSIST-TS FprCEN/TS 17718:2021
prEN 17718:2021 (E)
European foreword
This document (FprCEN/TS 17718:2021) has been prepared by Technical Committee
CEN/TC 455 “Plant Biostimulants”, the secretariat of which is held by AFNOR.
This document is currently submitted to the Vote on TS.

This document has been prepared under a Standardization Request given to CEN by the European

Commission and the European Free Trade Association.
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kSIST-TS FprCEN/TS 17718:2021
prEN 17718:2021 (E)
Introduction

This document was prepared by the experts of CEN/TC 455 ‘Plant Biostimulants’. The European

Committee for Standardization (CEN) was requested by the European Commission (EC) to draft

European standards or European standardization deliverables to support the implementation of

Regulation (EU) 2019/1009 of 5 June 2019 laying down rules on the making available on the

market of EU fertilising products (“FPR” or “Fertilising Products Regulation”). This request,

presented as SR M/564, also contributes to the Communication on “Innovating for Sustainable

Growth: A Bio economy for Europe”. The Working Group 5 “Labelling and denominations”, was

created to develop a work program as part of this request.

The technical committee CEN/TC 455 ‘Plant Biostimulants’ was established to carry out the work

program that will prepare a series of standards. The interest in biostimulants has increased

significantly in Europe as a valuable tool to use in agriculture. Standardization was identified as

having an important role in order to promote the use of biostimulants. The work of CEN/TC 455

seeks to improve the reliability of the supply chain, thereby improving the confidence of farmers,

industry, and consumers in biostimulants, and will promote and support commercialisation of the

European biostimulant industry.

Biostimulants used in agriculture can be applied in multiple ways: on soil, on plants, as seed

treatment, etc. A microbial plant biostimulant consists of a microorganism or a consortium of

microorganisms, as referred to in Component Material Category 7 of Annex II of the EU Fertilizing

Products Regulation.

This document is applicable to all biostimulants in agriculture based on live microorganisms

belonging to the group Rhizobia.

Table 1 summarizes many of the agro-ecological principles and the role played by biostimulants.

Table 1. — Agro-ecological principles and the role played by biostimulants
Increase biodiversity
By improving soil microorganism quality/quantity
Reinforce biological regulation and interactions
By reinforcing plant-microorganism interactions
— symbiotic exchanges i.e. mycorrhize
— symbiotic exchanges i.e. rhizobiaciae/fava
— secretions mimicking plant hormones (i.e. trichoderma)
By regulating plant physiological processes
— for ex growth, metabolism, plant development
Improve biogeochemical cycles
— improve absorption of nutritional elements
— improve bioavailability of nutritional elements in the soil
— stimulate degradation of organic matter

WARNING — Persons using this document should be familiar with normal laboratory practice.

This document does not purport to address all of the safety problems, if any, associated with its

use. It is the responsibility of the user to establish appropriate safety and health practices and to

ensure compliance with any national regulatory conditions.

IMPORTANT — It is absolutely essential that tests conducted in accordance with this document

be carried out by suitably trained staff.
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kSIST-TS FprCEN/TS 17718:2021
prEN 17718:2021 (E)
1 Scope

This document provides the methodology for the enumeration and determination of Rhizobium

sp., Mesorhizobium sp., Ensifer sp., or Bradyrhizobium sp.) in plant biostimulant products in

accordance with the Regulation (EU) 2019/1009 of the European Parliament and of the Council

[1].
2 Normative references

The following documents are referred to in the text in such a way that some or all of their content

constitutes requirements of this document. For dated references, only the edition cited applies.

For undated references, the latest edition of the referenced document (including any

amendments) applies.
FprCEN/TS 17724, Plant Biostimulants – Terminology

FprCEN/TS 17702-1, Plant Biostimulants - Sampling and sample preparation – Part 1: Sampling

3 Terms and definitions

For the purposes of this document, the terms and definitions given in FprCEN/TS 17724 and the

following apply.

ISO and IEC maintain terminological databases for use in standardization at the following

addresses:
• IEC Electropedia: available at https://www.electropedia.org/
• ISO Online browsing platform: available at https://www.iso.org/obp
3.1
rhizobium

beneficial bacteria belonging to the group named Rhizobia, where the most relevant genera are

Rhizobium, Mesorhizobium, Ensifer and Bradyrhizobium

Note to 1 entry: Rhizobium belonging to this group are rhizobium sp., mesorhizobium sp., ensifer sp.,

bradyrhizobium sp..

Note to entry 2: Legumes (Leguminosae or Fabaceae) are considered the second most cultivated crop,

covering 14 % of the total cultivated land worldwide and providing an important source of food for human

beings via direct consumption or indirect consumption via animal feed. Leguminosae can ensure high

quality protein-rich food and feed due to a special symbiosis they have with specific microorganisms

present in the soil that can fix in the rhizosphere, atmospheric nitrogen. Those microorganisms can account

for a 65 % of the total fixed nitrogen. Those microorganisms have originally been called Rhizobium. The

word ‘rhizobium’ is actually derived from two Greek words ‘rhizo’ meaning root and ‘bium’ meaning home.

Since the late nineteenth century, all legume root-nodule bacteria were placed in the genus ‘Rhizobium’.

Gradually it was realized that they were rather diverse. A few slow-growing rhizobia were split off into a

new genus ‘Bradyrhizobium’. In the 1984 edition of Bergey’s Manual of Systematic Bacteriology, all rhizobia

were placed in the family Rhizobiaceae which included Bradyrhizobium and Rhizobium. Since then, the

number of bacterial genera representing rhizobia has increased rapidly; Rhizobia are plant root nodule

inhabiting, associative symbiotic, nitrogen fixing bacteria. Today the classification of the different Rhizobia

species is based the sequence of the 16S rDNA sequence comparison and physiological and biochemical

properties. Considering that taxonomy and phylogeny of bacteria is in continuous evolution and considering

that any current classification scheme is subject to future revision and considering moreover that most of

the Rhizobial species in the alpha-proteobacteria class of phylum proteobacteria in Rhizobiaceae family are

in either the Rhizobium, Mesorhizobium, Ensifer, or Bradyrhizobium genera, for the purpose of this

document we will consider the above mentioned genera as referring to the Rhizobium sp. group.

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kSIST-TS FprCEN/TS 17718:2021
prEN 17718:2021 (E)

Note 3 to entry: Other nodule-forming bacteria belong to the genus Frankia and interact with

non-leguminous species, including woody species of the families Betulaceae and Casuarinaceae. Such

bacteria should be included in the general wording of “Rhizobium” according to the terms of this document.

[SOURCE: FprCEN/TS 17724, 3.2.2.7]
4 Enumeration of Rhizobium sp., Mesorhizobium sp., Ensifer sp.,
Bradyrhizobium sp.)
4.1 General

This procedure is meant to determine the number of colony-forming units (CFU) of the above

mentioned bacteria «Pathogen», per gram or per millilitre. The method, in order to be fast, cheap

and repeatable, is based on serial dilutions and plating.
4.2 Sample preparation
4.2.1 General
A representative sample of the product to be analysed as per the requirements of

FprCEN/TS 17702-1 will be prepared according to following procedure, which takes into

consideration the different formulations of biostimulants based products.
4.2.2 Liquid (based water) formulations

Dispense 25 ml of sample (or more for low concentrated products) in 225 ml of sterile Phosphate

Buffer Solution (PBS) maintained at room temperature, in a flask and shake for 10 min or more

until the distribution is optimal, with a magnetic stirrer at half speed [9].
4.2.3 Liquid - based oil, emulsifiable concentrate (EC) formulations

Dispense 25 ml of sample (or more for low concentrated products) in 225 ml of sterile Phosphate

Buffer Solution (PBS) maintained at room temperature, in a flask and shake for 10 min or more

until the distribution is optimal, with a magnetic stirrer at half speed [9].
4.2.4 Solid - Wettable Powder (WP) formulations

Dispense 25 g of sample (or more for low concentrated products) in 225 ml of sterile Phosphate

Buffer Solution (PBS) maintained at room temperature, in a flask and shake for 20 min or more

until the distribution is optimal, with a magnetic stirrer at half speed [9].
4.2.5 Solid - Water dispersible granules (WDG) formulations

Dispense 25 g (or more for low concentrated products) of sample in 275 g of sterile Phosphate

Buffer Solution (PBS) maintained at room temperature in a flask and shake for 40 min or more

until the distribution is optimal, with a magnetic stirrer at half speed. If required help the

dispersion of the formulations with other apparatus such as a stomacher after having sieved (100

mesh sieve) the particles and resuspend them in the same suspe
...

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