Plant biostimulants - Determination of Azospirillum spp.

This document provides the methodology for the enumeration and determination of Azospirillum spp. 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 Azospirillum spp.

Dieses Dokument stellt die Methodik für die Zählung und Bestimmung von Azospirillum spp. in Pflanzen-Biostimulanzien in Übereinstimmung mit der Verordnung (EU) 2019/1009 des Europäischen Parlaments und des Rates [1] bereit.

Biostimulants des végétaux - Détermination d'Azospirillum spp.

Le présent document fournit la méthodologie de dénombrement et de détermination d’Azospirillum spp. dans les produits biostimulants des végétaux conformément au Règlement (UE) 2019/1009 du Parlement Européen et du Conseil [1].

Rastlinski biostimulanti - Ugotavljanje prisotnosti in števila Azospirillum spp.

General Information

Status
Not Published
Public Enquiry End Date
02-Jun-2023
Technical Committee
Current Stage
4020 - Public enquire (PE) (Adopted Project)
Start Date
21-Mar-2023
Due Date
08-Aug-2023
Completion Date
02-Aug-2023

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SLOVENSKI STANDARD
oSIST prEN 17713:2023
01-maj-2023
Rastlinski biostimulanti - Ugotavljanje prisotnosti in števila Azospirillum spp.
Plant biostimulants - Determination of Azospirillum spp.
Pflanzen-Biostimulanzien - Bestimmung von Azospirillum spp.
Biostimulants des végétaux - Détermination d'Azospirillum spp.
Ta slovenski standard je istoveten z: prEN 17713
ICS:
65.080 Gnojila Fertilizers
oSIST prEN 17713:2023 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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oSIST prEN 17713:2023

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oSIST prEN 17713:2023


DRAFT
EUROPEAN STANDARD
prEN 17713
NORME EUROPÉENNE

EUROPÄISCHE NORM

April 2023
ICS 65.080 Will supersede CEN/TS 17713:2022
English Version

Plant biostimulants - Determination of Azospirillum spp.
Biostimulants des végétaux - Détermination Pflanzen-Biostimulanzien - Bestimmung von
d'Azospirillum spp. Azospirillum spp.
This draft European Standard is submitted to CEN members for enquiry. It has been drawn up by the Technical Committee
CEN/TC 455.

If this draft becomes a European Standard, CEN members are bound to comply with the CEN/CENELEC Internal Regulations
which stipulate the conditions for giving this European Standard the status of a national standard without any alteration.

This draft European Standard was established by CEN in three official versions (English, French, German). A version in any other
language made by translation under the responsibility of a CEN member into its own language and notified to the CEN-CENELEC
Management Centre has the same status as the official versions.

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, Türkiye 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 European Standard. It is distributed for review and comments. It is subject to change without
notice and shall not be referred to as a European Standard.


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
© 2023 CEN All rights of exploitation in any form and by any means reserved Ref. No. prEN 17713:2023 E
worldwide for CEN national Members.

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Contents Page
1 Scope . 7
2 Normative references . 7
3 Terms and definitions . 7
4 Enumeration of Azospirillum spp. . 7
4.1 General . 7
4.2 Sample preparation . 8
4.2.1 General . 8
4.2.2 Liquid -based water- formulations . 8
4.2.3 Liquid – oil based (emulsifiable concentrate -EC) formulations . 8
4.2.4 Solid - Wettable Powder (WP) formulations . 8
4.2.5 Solid - Water dispersible granules (WDG) formulations . 8
4.2.6 Solid – Pellets, granules, microgranuls (slow release) formulations . 8
4.2.7 Solid - substrate . 8
4.3 Requirements (apparatus) . 8
4.4 Serial dilution . 9
4.5 Preparation of the culture media . 9
4.6 Preparation of serial dilution for MPN count . 9
4.7 Incubation of tubes . 9
4.8 Counting . 9
4.9 Method for estimating MPN count . 10
5 Species Determination of Azospirillum spp. via genetic analysis . 10
5.1 General . 10
5.2 Preparation of the sample for the genomic DNA extraction . 10
5.2.1 Isolation and preparation of the microorganism . 10
5.2.2 Sample concentration . 10
5.2.3 DNA extraction and storage . 11
5.2.4 Partial PCR Amplification of the 16S rRNA Genes . 11
Annex A (normative) Formula of the culture media . 13
A.1 0,1 M Phosphate Buffer Saline (PBS) . 13
A.2 Composition of Nfb semisolid medium . 13
A.2.1 Ingredients . 13
A.2.2 Method to prepare Trace element solution . 13
A.2.3 Method to prepare Vitamin solution. 14
A.2.4 Sterilization and preparation of MPN tubes . 14
A.3 Nutrient agar . 14
A.4 Nutrient broth . 14
Annex B (normative) Table of MPN values [9] . 15
Annex C (informative) Repeatability and reproductibility data . 17
C.1 General . 17
C.2 Microbial analysis-Materials used in the interlaboratory comparison study . 17
C.3 Microbial analysis-Interlaboratory comparison results . 18
C.4 Genetic analysis-Materials used in the interlaboratory comparison study . 19
2

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C.5 Genetic analysis- Interlaboratory comparison results . 20
Annex ZA (informative) Relationship of this European Standard and the essential
requirements of Regulation (EU) 2019/1009 making available on the market of EU
fertilising products aimed to be covered . 21
Bibliography . 22

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European foreword
This document (prEN 17713:2023) 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 CEN Enquiry.
This document will supersede CEN/TS 17713:2022.
In comparison with the previous edition, the following technical modifications have been made:
This document has been prepared under a Standardization Request given to CEN by the European
Commission and the European Free Trade Association, and supports essential requirements of EU
Directive(s) / Regulation(s).
For relationship with EU Directive(s) / Regulation(s), see informative Annex ZA, which is an
integral part of this document.

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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 SR M/564 and M/564 Amd1, 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
Standardization 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
plant 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 plant
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 plant biostimulants, and
will promote and support commercialisation of the European biostimulant industry.
Plant 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 plant biostimulants in agriculture based on live microorganisms
belonging to the genera Azospirillum.
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 plant 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
- 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.
5

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IMPORTANT — It is absolutely essential that tests conducted in accordance with this document
be carried out by suitably trained staff.

6

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1 Scope
This document provides the methodology for the enumeration and determination of Azospirillum
spp. 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.
1
prEN 17702-1:— , Plant biostimulants — Sampling and sample preparation — Part 1: Sampling
3 Terms and definitions
For the purposes of this document, the following terms and definitions 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
azospirillum spp.
gram-negative bacteria that belong to the alphaproteobacterial phylum
Note 1 to entry: Azospirillum is a Gram-negative, microaerophilic, non-fermentative and nitrogen-
fixing bacterial genus. Azospirillum are gram-negative, do not form spores, and have a slightly-twisted
oblong-rod shape. Azospirillum have at least one flagellum and sometimes multiple flagella. The genus has
about 20 species, the relationships between all the species have not been resolved in details, however most
likely they constitute a coherent group.
Note 2 to entry: They are aerobic non-fermentative chemoorganotrophs, vibroid, produce several
hormones, mainly auxins (not described for all species yet), and most of them are diazotrophic (fix
atmospheric nitrogen gas into a more usable form).
1
[SOURCE: prEN 17724:— , 3.2.2.1]
4 Enumeration of Azospirillum spp.
4.1 General
This procedure is meant to determine the number of colony-forming units (CFU) of Azosprillium
spp, per gram or per millilitre. The method, in order to be fast, cheap and repeatable, is based on
serial dilutions and plating [6].

1
Under preparation
7

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4.2 Sample preparation
4.2.1 General
A representative sample of the product to be analysed as per the requirements of prEN 17702-
1
1:— will be prepared according to following procedure, which takes into consideration the
different formulations of Plant 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 of maximum speed [7].
4.2.3 Liquid – oil based (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 of maximum speed [7].
4.2.4 Solid - Wettable Powder (WP) formulations
Dispense 25 gr 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 of maximum speed [7].
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 of maximum speed. If required help
the dispersion of the formulations with other apparatus such as a laboratory paddle blender after
having sieved (100 mesh sieve) the particles and resuspend them in the same suspension [7].
4.2.6 Solid – Pellets, granules, microgranuls (slow release) formulations
Dispense 25 g (or more for low concentrated products) of sample in 225 g of sterile Phosphate
Buffer Solution (PBS) maintained at room temperature, in a sterile bag and disperse them using a
magnetic stirrer for 40 min at half of maximum speed and then sieve in a 100 mesh sieve and if
material remains in the sieve repeat the process for a maximum of three times. Put attention to all
the buffer used to make the exact final calculation [7].
4.2.7 Solid - substrate
Dispense 25 g (or more for low concentrated products) of sample in 225 g 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 of maximum speed [7].
4.3 Requirements (apparatus)
— Graduated pipettes (1 ml and 10 ml);
— Dilution Bottles or flasks;
— Petri Dishes Clear, Uniform, flat-bottomed;
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— Hot Air Oven - Capable of giving uniform and adequate temperature, equipped with a
thermometer calibrated to read up to 250°C and with vents suitably located to ensure prompt
and uniform heating;
— Autoclave/Steam sterilizer;
— Incubator;
— Hand Tally or Mechanical counting Device;
— pH meter.
4.4 Serial dilution
The principle in counting bacteria by dilution is to serially dilute them to reduce the bacterial
density to the level where individual cells can be differentiated.
The diluent is the PBS (see Annex A, A.1).
4.5 Preparation of the culture media
The preparation and the composition of N-free semisolid medium (Nfb) is described in Annex A.
The preparation and performance of culture media is a fundamental step to ensure the integrity
of microbiological examination.
When ready-to-use media are used, the manufacturers of this available media should have a
quality programme that ensure the quality of the media they supply according to
2
EN ISO 11133:2014 [6]. Under this conditions, the user/laboratory not need to run additional
testing on such media, but shall ensure the storage condition according to the ones recommended
by the manufactures.
For diluents and media prepared by the user/laboratory directly from commercially available
dehydrated formulations and/or from basic individual components, the performance of these
2
diluents/media should be evaluated according to EN ISO 11133:2014 [6].
4.6 Preparation of serial dilution for MPN count
−8
Prepare the sample as described in 4.3. Make serial dilutions up to 10 dilution. Pipette out 1 ml
−4 −8
aliquots of 10 to 10 dilution and deliver it to screw cap tubes or test tubes containing N-free
semi solid Nfb media. 5 replicates are necessary.
4.7 Incubation of tubes
Label the tubes and incubate at (36 ± 1) °C for 3 days to 4 days in vertical position in a test tubes
stand. Do not disturb the medium during the entire period of incubation.
4.8 Counting
Count the tubes which have turned blue and have developed typical white sub-surface pellicle.
Count the tubes as +ve (positive) or –ve (negative) for the presence of sub-surface pellicle and
consider for the purpose of calculation.

2
As impacted by EN ISO 11133:2014/A1:2018 and EN ISO 11133:2014/A2:2020).
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4.9 Method for estimating MPN count
To calculate the most probable number of organisms in the original sample, select as P the
1
number of positive tubes in the least concentrated dilution in which all tubes are positive or in
which the greatest number of tubes is +veand let P and P represent the numbers of positive
2 3
tubes in the next two higher dilutions.
Then find the row of numbers in Table B.1 in Annex B in which P and P correspond to
...

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