Plant biostimulants - Detection of Staphylococcus aureus

This document provides a method for verifying that the pathogen Staphylococcus aureus is present in microbial plant biostimulants according to the limits outlined in the EU Regulation on Fertilising Products [2].

Pflanzen-Biostimulanzien - Nachweis von Staphylococcus aureus

Dieses Dokument stellt ein Verfahren zur Verfügung, mit dem verifiziert werden kann, dass das Pathogen Staphylococcus aureus in Pflanzen-Biostimulanzien die Grenzwerte der EU Dünge-produkteverordnung [2] nicht überschreitet.

Biostimulants des végétaux - Détection de Staphylococcus aureus

Le présent document fournit une méthode permettant de vérifier que le pathogène Staphylococcus aureus est présent dans les biostimulants microbiens des végétaux dans le respect des limites énoncées dans le Règlement UE sur les fertilisants [2].

Rastlinski biostimulanti - Ugotavljanje prisotnosti Staphylococcus aureus

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 17712:2021
01-november-2021
[Not translated]
Plant biostimulants - Detection of Staphylococcus aureus

Biostimulanzien für die pflanzliche Anwendung - Nachweis von Staphylococcus aureus

Ta slovenski standard je istoveten z: FprCEN/TS 17712
ICS:
65.080 Gnojila Fertilizers
kSIST-TS FprCEN/TS 17712:2021 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 17712:2021
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kSIST-TS FprCEN/TS 17712:2021
FINAL DRAFT
TECHNICAL SPECIFICATION
FprCEN/TS 17712
SPÉCIFICATION TECHNIQUE
TECHNISCHE SPEZIFIKATION
September 2021
ICS 65.080
English Version
Plant biostimulants - Detection of Staphylococcus aureus
Biostimulanzien für die pflanzliche Anwendung -
Nachweis von Staphylococcus aureus

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 17712:2021 E

worldwide for CEN national Members.
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kSIST-TS FprCEN/TS 17712:2021
FprCEN/TS 17712:2021 (E)
Contents Page

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

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

1 Scope ............................................................................................................................................ 6

2 Normative references ............................................................................................................ 6

3 Terms and definitions ........................................................................................................... 6

4 Principle ..................................................................................................................................... 7

5 Sampling ..................................................................................................................................... 7

6 Preparation of test sample ................................................................................................... 7

7 Procedure ................................................................................................................................... 7

7.1 Diluent ......................................................................................................................................... 7

7.2 Modified Giolitti-Cantoni broth .......................................................................................... 7

7.3 Baird-Parker agar medium .................................................................................................. 8

8 Apparatus and glassware ..................................................................................................... 8

9 Procedure ................................................................................................................................... 9

9.1 Test portion, initial suspension and dilutions .............................................................. 9

9.1.1 Solid formulations ................................................................................................................... 9

9.1.2 Liquid formulations ................................................................................................................ 9

9.2 Enrichment and isolation ..................................................................................................... 9

10 Precision ................................................................................................................................... 10

11 Test report ............................................................................................................................... 10

12 Performance characteristics of the method ................................................................ 10

12.1 Interlaboratory studies ....................................................................................................... 10

12.2 Sensitivity ................................................................................................................................. 11

12.3 Specificity ................................................................................................................................. 11

Annex A (normative) (Media) .......................................................................................................... 12

A.1 Media composition and preparation .............................................................................. 12

A.1.1 Baird Parker Rabbit Plasma Fibrinogen (RFP) agar [4], [5], [6] .......................... 12

A.1.2 Buffered peptone water (BPW) ........................................................................................ 12

A.1.3 Modified Giolitti-Cantoni broth ........................................................................................ 12

Bibliography .......................................................................................................................................... 14

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kSIST-TS FprCEN/TS 17712:2021
FprCEN/TS 17712:2021 (E)
European foreword

This document (FprCEN/TS 17712:2021) 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 mandate given to CEN by the European Commission

and the European Free Trade Association.
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kSIST-TS FprCEN/TS 17712:2021
FprCEN/TS 17712:2021 (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 to the soil, to 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 microbial biostimulants in agriculture.

The Table 1 below summarizes many of the agro-ecological principles and the role played by

biostimulants.
Table 1 — Agro-ecological principles and the role played by biostimulants [1]
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
- such as growth, metabolism or 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.
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kSIST-TS FprCEN/TS 17712:2021
FprCEN/TS 17712:2021 (E)

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 17712:2021
FprCEN/TS 17712:2021 (E)
1 Scope

This document provides a method for verifying that the pathogen Staphylococcus aureus is present

in microbial plant biostimulants according to the limits outlined in the EU Regulation on

Fertilising Products [2].
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.

EN ISO 7218:2007, Microbiology of food and animal feeding stuffs — General requirements and

guidance for microbiological examinations (ISO 7218:2007)
FprCEN/TS 17724, Plant biostimulants — Terminology
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
staphylococcus aureus

bacterium which forms colonies fitting the description of the species on the specified selective

medium after incubation of 24 h at a temperature of 37 °C under aerobic conditions

Note 1 to entry: S.aureus colony description:
— circular;
— convex;
— entire margin;
— grey to black (due to the reduction of potassium tellurite to telluride).
Note 2 to entry: Colony size varies between 1 mm and 2 mm in diameter.

Note 3 to entry: S. aureus is a facultatively anaerobic, Gram-positive coccus, which appears as grape-like

clusters when visualized under a microscope, and has a round, usually golden-yellow colonies, often with

haemolysis, when grown on selective blood agar plates.

Note 4 to entry: The term ‘Coagulase-positive staphylococci’ refers to bacteria that form typical and/or

atypical colonies on the surface of a selective culture medium and show a positive coagulase reaction when

the test is performed following the method specified in this document.
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kSIST-TS FprCEN/TS 17712:2021
FprCEN/TS 17712:2021 (E)
3.2
detection of the coagulase-positive staphylococci

determination of the detection or non detection of Staphylococcus aureus (3.2), in 25 g or 25 ml of

product, when tests are carried out in accordance with this document
4 Principle

After sample preparation that is specific to microbial biostimulants, for detection of

Staphylococcus aureus, we refer to the sections of EN ISO 6888-3:2003 that are pertinent to the

physical/chemical nature of the test sample for the detection of Staphylococcus aureus. If the

product is liquid, a specified volume of the test sample will be inoculated onto a liquid selective

culture medium. .For products formulated differently, a specified volume of an initial suspension

will be inoculated onto the selective medium. Incubation under anaerobic conditions at 37 °C for

24 and 48 h.

Pour plate and spread plate techniques could be used. Spread plate is considered enough for

detecting the presence of this species. In this case, the inoculation could be done with a sterile

loop (containing around 10 μl of product, dilution o
...

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