CEN/TS 17707:2022

Plant biostimulants - Determination of the yeast and mould content

This document specifies a horizontal method for the enumeration of yeasts and moulds present in plant biostimulants intended for use in agriculture, by means of the colony count technique after aerobic incubation at 25 °C ± 2,5 °C.
This document allows the enumeration of yeasts and moulds, in technical and formulated plant biostimulant, both in liquid and solid state. The method is applicable to microbial plant biostimulant except those composed of fungi or yeast to verify that the concentration of yeast and moulds does not exceed the respective limits described in the EU Fertilisers Regulation [1].
If necessary, yeast and mould enumerated can be identified using suitable identification tests.

Pflanzen-Biostimulanzien - Bestimmung des Gehalts an Hefen und Schimmelpilzen

Dieses Dokument legt ein horizontales Verfahren zur Zählung von Hefen und Schimmelpilzen in Pflanzen-Biostimulanzien, die zur Verwendung in der Landwirtschaft bestimmt sind, mit Hilfe des Koloniezählverfahrens nach aerober Bebrütung bei (25 ± 2,5) °C fest.
Dieses Dokument hat die Zählung von Hefen und Schimmelpilzen in technischen und formulierten Pflanzen-Biostimulanzien, die im flüssigen oder im festen Zustand vorliegen können, zum Gegenstand. Das Verfahren ist auf mikrobielle Pflanzen-Biostimulanzien anwendbar, ausgenommen jene, die aus Pilzen oder Hefe bestehen, um zu überprüfen, dass die Konzentration von Hefe und Schimmelpilzen die jeweiligen Grenzwerte, die in der EU-Düngeprodukteverordnung [1] angegeben sind, nicht überschreitet.
Gegebenenfalls können die gezählten Hefen und Schimmelpilze durch geeignete Identifizierungsprüfungen identifiziert werden.

Biostimulants des végétaux - Détermination de la teneur en levures et en moisissures

Le présent document spécifie une méthode horizontale pour le dénombrement des levures et des moisissures présentes dans les biostimulants des végétaux destinés à être utilisés en agriculture, par la technique de comptage des colonies après incubation aérobie à 25 °C ± 2,5 °C.
Le présent document permet le dénombrement des levures et des moisissures, dans les biostimulants des végétaux de qualité technique, à la fois à l’état liquide et à l’état solide. La méthode est applicable aux biostimulants microbiens des végétaux, excepté ceux composés de champignons ou de levures, pour vérifier que la concentration des levures et des moisissures ne dépasse pas les limites correspondantes indiquées dans le Règlement UE sur les fertilisants [1].
Si nécessaire, les levures et les moisissures dénombrées peuvent être identifiées par des essais d’identification appropriés.

Rastlinski biostimulanti - Določanje kvasovk in plesni

Ta dokument določa vodoravno metodo za ugotavljanje števila kvasovk in plesni, prisotnih v rastlinskem biostimulantu, namenjenem za uporabo v kmetijstvu, s tehniko štetja kolonij po aerobni inkubaciji pri 25 °C ± 2,5 °C.
Ta dokument dovoljuje štetje kvasovk in plesni v tehničnih ter formuliranih rastlinskih biostimulantih tako v tekočem kot v trdnem stanju. Metoda se uporablja za mikrobne rastlinske biostimulante, razen tistih, ki so sestavljeni iz gliv ali kvasovk za preverjanje, da koncentracija kvasovk in plesni ne presega ustreznih mejnih vrednosti, opisanih v Uredbi EU o sredstvih za gnojenje [1].
Po potrebi je mogoče preštete kvasovke in plesni identificirati z ustreznimi identifikacijskimi preskusi.

General Information

• Status: Withdrawn
• Publication Date: 22-Mar-2022
• Withdrawal Date: 13-Apr-2025
• ICS: 65.080 - Fertilizers
• Technical Committee: CEN/TC 455 - Plant Biostimulants and Agricultural Micro-Organisms
• Drafting Committee: CEN/TC 455/WG 3 - Pathogenic and non-pathogenic microorganisms
• Current Stage: 9960 - Withdrawal effective - Withdrawal
• Start Date: 27-Nov-2024
• Completion Date: 14-Apr-2025
• Directive: 2019/1009 - Regulation (EU) 2019/1009 of the European Parliament and of the Council of 5 June 2019 laying down rules on the making available on the market of EU fertilising products and amending Regulations (EC) No 1069/2009 and (EC) No 1107/2009 and repealing Regulation (EC) No 2003/2003
• Mandate: M/564 - Fertilising products in support of Regulation (EU) 2019/1009
• Ref Project: SIST-TS CEN/TS 17707:2023 - Plant biostimulants - Determination of the yeast and mould content

Relations

• Replaced By: EN 17707:2024 - Plant biostimulants - Determination of the yeast and mould content
• Effective Date: 15-Feb-2023

Standards Content (Sample)

SLOVENSKI STANDARD
01-februar-2023
Rastlinski biostimulanti - Določanje kvasovk in plesni
Plant biostimulants - Determination of the yeast and mould content
Pflanzen-Biostimulanzien - Bestimmung des Gehalts an Hefen und Schimmelpilzen
Biostimulants des végétaux - Détermination de la teneur en levures et en moisissures
Ta slovenski standard je istoveten z: CEN/TS 17707:2022
ICS:
65.080 Gnojila Fertilizers
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

CEN/TS 17707
TECHNICAL SPECIFICATION
SPÉCIFICATION TECHNIQUE
March 2022
TECHNISCHE SPEZIFIKATION
ICS 65.080
English Version
Plant biostimulants - Determination of the yeast and
mould content
Biostimulants des végétaux - Détermination de la Biostimulanzien für die pflanzliche Anwendung -
teneur en levures et en moisissures Bestimmung des Gehalts an Hefen und Schimmelpilzen
This Technical Specification (CEN/TS) was approved by CEN on 3 January 2022 for provisional application.
This Technical Specification was corrected and reissued by the CEN-CENELEC Management Centre on 27 April 2022.

The period of validity of this CEN/TS is limited initially to three years. After two years the members of CEN will be requested to
submit their comments, particularly on the question whether the CEN/TS can be converted into a European Standard.

CEN members are required to announce the existence of this CEN/TS in the same way as for an EN and to make the CEN/TS
available promptly at national level in an appropriate form. It is permissible to keep conflicting national standards in force (in
parallel to the CEN/TS) until the final decision about the possible conversion of the CEN/TS into an EN is reached.

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.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATIO N

EUROPÄISCHES KOMITEE FÜR NORMUN G

CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels
© 2022 CEN All rights of exploitation in any form and by any means reserved Ref. No. CEN/TS 17707:2022 E
worldwide for CEN national Members.

Contents Page
European foreword . 3
Introduction . 4
1 Scope . 6
2 Normative references . 6
3 Terms and definitions . 6
4 Principles . 7
4.1 General . 7
4.2 Plate count method . 7
5 Diluent and culture media . 7
5.1 Diluent . 7
5.2 Culture media . 7
6 Apparatus and glassware . 7
7 Handling of plant biostimulants and sampling . 8
8 Procedure . 8
8.1 General . 8
8.2 Test portion and initial suspension . 8
8.3 Serial dilutions . 9
8.4 Plate-count methods . 9
9 Counting of colonies . 10
10 Expression of results . 10
10.1 Method of calculation . 10
10.2 Interpretation . 11
11 Test report . 12
Annex A (informative) Diluent . 13
Annex B (informative) Culture media . 14
Bibliography . 17

European foreword
This document (CEN/TS 17707:2022) has been prepared by Technical Committee CEN/TC 455
“Plant biostimulants”, the secretariat of which is held by AFNOR.
Attention is drawn to the possibility that some of the elements of this document may be the subject
of patent rights. CEN shall not be held responsible for identifying any or all such patent rights.
This document has been prepared under a Standardization Request given to CEN by the European
Commission and the European Free Trade Association.
Any feedback and questions on this document should be directed to the users’ national standards
body. A complete listing of these bodies can be found on the CEN website.
According to the CEN/CENELEC Internal Regulations, the national standards organisations of the
following countries are bound to announce this Technical Specification: 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 the United Kingdom.
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 fertilizing products (“FPR” or “Fertilising Products Regulation”).
This standardization request, presented as 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 plant, 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 Fertilising
Products Regulation.
This document is applicable to all microbial biostimulants in agriculture.
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. Mycorrhizae
- symbiotic exchanges i.e. Rhizobiaceae/Faba
- secretions mimicking plant hormones (i.e. Trichoderma)
By regulating plant physiological processes
- for e.g. 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.

1 Scope
This document specifies a horizontal method for the enumeration of yeasts and moulds present
in plant biostimulants intended for use in agriculture, by means of the colony count technique
after aerobic incubation at 25 °C ± 2,5 °C.
This document allows the enumeration of yeasts and moulds, in technical and formulated plant
biostimulant, both in liquid and solid state. The method is applicable to microbial plant
biostimulant except those composed of fungi or yeast to verify that the concentration of yeast and
moulds does not exceed the respective limits described in the EU Fertilisers Regulation [1].
If necessary, yeast and mould enumerated can be identified using suitable identification tests.
2 Normative references
There are no normative references in this document.
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
3.1
yeast
mesophilic aerobic microorganism which, using mycological agar medium under the conditions
described in this document, develops matt or shiny round colonies (3.3) on the surface of the
medium, usually having a regular outline and a more or less convex surface
3.2
mould
mesophilic aerobic filamentous microorganism which, on the surface of mycological agar medium
under the conditions described in this document, usually develops flat or fluffy spreading colonies
(3.3) often producing spores or conidia
3.3
colony
localized visible accumulation of microbial mass (such as prokaryotes, bacteria, micromycetes,
yeast and fungi) or organisms (such as Dreissena species) developed on or in a solid nutrient
medium from a viable particle or organism
Note 1 to entry: Frequently, micro colonies from nearby viable particles, before becoming visible, fuse into
one macro colony. The number of visible colonies is, therefore, usually and underestimate of the number of
viable particles.
[ISO 6107-6:2021, [4], modified]
3.4
product
portion of an identified plant biostimulant product received in the laboratory for testing
3.5
sample
portion of the product (3.4) that is used in the test to prepare the initial suspension
3.6
initial suspension
suspension (or solution) of the sample (3.5) in a defined volume of an appropriate diluent
3.7
sample dilution
dilution of the initial suspension (3.6)
4 Principles
4.1 General
This method aims enumeration of colonies on a selective agar medium.
4.2 Plate count method
The plate count consists in the following steps:
— Preparation of poured plates, or spread plates using a specific culture medium. Depending on
the expected number of colonies, a specified quantity of the sample (if the product is liquid),
or of an initial suspension (in the case of other products), or decimal dilutions of the
sample/suspension are inoculated.
— Aerobic incubation of the plates at 25 °C ± 2,5 °C for 3 days to 5 days.
— Calculation of the number of colony-forming units (CFU) of yeasts and moulds per gram or
per millilitre of sample from the number of colonies obtained on plates chosen at dilution
levels producing countable colonies. Moulds and yeasts are counted separately, if necessary.
NOTE An alternative condition for incubation is 22,5 °C ± 2,5 °C, for 5 days to 7 days, using the culture
medium without antibiotic. If necessary, to distinguish yeast colonies from bacterial colonies, the identity
of any doubtful colonies is confirmed by examination with a binocular magnifier or microscope.
5 Diluent and culture media
5.1 General
The following diluents and culture media are suitable for enumeration of yeast and moulds. Other
diluents and culture media may be used if they have been demonstrated to be suitable for use.
Diluent and culture media may be prepared using the descriptions provided or from dehydrated
culture media, according to the instructions from the manufacturer. The instructions provided by
the supplier should be followed.
NOTE Ready-to-use media can be used when their composition and/or growth yields are comparable
to those of the formulae given in the present document.
5.2 Diluent
See Annex A for the recipe of the diluent to be use in the preparation of the initial suspension and
further decimal dilutions.
5.3 Culture media
See Annex B for the list and recipes of the possible media to be use in the inoculation by plating
technique of the initial suspension and the further decimal dilutions.
6 Apparatus and glassware
Use the laboratory equipment, apparatus and glassware typical of microbiological laboratory. See
CEN/TS 17708 for a detailed list.
7 Handling of plant biostimulants and sampling
It is important that the laboratory receives a sample which is truly representative and has not
been damaged or changed during transport or storage.
Sampling is not part of the method specified in this document (CEN/TS 17707): refer to
CEN/TS 17702-1.
If necessary, the product to be tested may be equilibrated at room temperature before starting
the analysis.
8 Procedure
8.1 General
Use sterile material, equipment and aseptic techniques to prepare the sample, initial suspension
and dilutions. In the case of the preparation of the initial suspension, the time that elapse between
the end of the preparation and the moment the inoculum comes into contact with the culture
medium shall not exceed 45 min, unless specifically mentioned in the established protocols or
documents.
Appropriate negative controls (diluent-only) should be run concurrently with the sample serial
dilutions. This step can be performed by incubating an aliquot of the diluent (i.e. 9 ml) at the same
conditions of the test to verify the absence of turbidity to assess the sterility of the diluent. Or,
alternatively, can be spread 1 ml of the diluent over a surface of the same agar medium used in
the analysis. The plate is incubated at the same conditions of the test to verify the absence of
growth to assess the sterility of the diluent.
8.2 Test portion and initial suspension
8.2.1 General
A representative sample of the product is taken to prepare the initial suspension according to
following procedure which takes into consideration the different formulations of biostimulants
based products:
8.2.2 Liquid – water based – formulations
Dispense 25 ml of sample in 225 ml of sterile Phosphate Buffer Solution (PBS) (see Annex A)
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.
8.2.3 Liquid – oil based (emulsifiable concentrate – EC) formulations
Dispense 25 ml or g
...