CEN/TC 455/WG 3 - Pathogenic and non-pathogenic microorganisms

This document provides the methodology for the enumeration and determination of Rhizobiaceae (Rhizobium spp., Mesorhizobium spp., Ensifer spp., Bradyrhizobium spp.).
This document is applicable to the blends of fertilizing products where a blend is a mix of at least two of the following component EU fertilising products categories: Fertilizers, Liming Materials, Soil Improvers, Growing Media, Plant Biostimulants and where the following category Plant Biostimulants is the highest percentage in the blend by mass or volume, or in the case of liquid form by dry mass. If Plant Biostimulants is not the highest percentage in the blend, the European Standard for the highest percentage of the blend applies. In case a blend of fertilizing products is composed of components in equal quantity or in case the component EU fertilising products used for the blend have identical formulations , the user decides which standard to apply.

This document specifies general rules for the aerobic preparation of the initial suspension and of dilutions for microbiological examinations of microbial plant biostimulants.
This horizontal method might not be applicable in very detail for certain products. In this case, different methods which are specific to these products can be used if necessary, for justified technical reasons.
This document is applicable to the blends of fertilizing products where a blend is a mix of at least two of the following component EU fertilising products categories: Fertilizers, Liming Materials, Soil Improvers, Growing Media, Plant Biostimulant and where the following category Plant Biostimulants is the highest % in the blend by mass or volume, or in the case of liquid form by dry mass. If Plant Biostimulants is not the highest % in the blend, the European Standard for the highest % of the blend applies. In case a blend of fertilizing products is composed of components in equal quantity or in case the component EU fertilising products used for the blend have identical formulations , the user decides which standard to apply.

This document specifies a method for the detection of Salmonella spp. in biostimulants.
This document is applicable to all microbial biostimulants in agriculture.
This document is applicable to the blends of fertilizing products where a blend is a mix of at least two of the following component EU fertilising products categories: Fertilizers, Liming Materials, Soil Improvers, Growing Media, Plant Biostimulants, and where the following category Plant Biostimulants is the highest % in the blend by mass or volume, or in the case of liquid form by dry mass. If Plant Biostimulants is not the highest % in the blend, the European Standard for the highest % of the blend applies. In case a blend of fertilizing products is composed of components in equal quantity or in case the component EU fertilising products used for the blend have identical formulations , the user decides which standard to apply.

This document gives guidance for the detection and identification of the specified microorganism Escherichia coli in technical and formulated plant biostimulants, both in liquid and solid states, and also the horizontal method for the enumeration of ß-glucuronidase-positive E. coli in plant biostimulants (both in liquid and solid states).
The qualitative method described in this document is based on the detection of E. coli in a non-selective liquid medium (enrichment broth), followed by isolation on a selective agar. Other methods can be appropriate, depending on the level of detection required.
NOTE 1   For the detection of E. coli, subcultures can be performed on non-selective culture media followed by suitable identification steps (e.g. using identification kits).
The quantitative method described in this document uses a colony-count technique at 44 °C ± 1 °C on a solid medium containing a chromogenic ingredient for detection of the enzyme ß-glucuronidase.
NOTE 2   Strains of E. coli which do not grow at 44 °C ± 1 °C and, in particular, those that are ß-glucuronidase negative, such as E. coli O157, will not be detected.
This document is applicable to the blends of fertilizing products where a blend is a mix of at least two of the following component EU fertilising products categories: Fertilizers, Liming Materials, Soil Improvers, Growing Media, Plant Biostimulants, and where the following category Plant Biostimulants is the highest % in the blend by mass or volume, or in the case of liquid form by dry mass. If Plant Biostimulants is not the highest % in the blend, the European Standard for the highest % of the blend applies. In case a blend of fertilizing products is composed of components in equal quantity or in case the component EU fertilising products used for the blend have identical formulations , the user decides which standard to apply.

This document specifies the general rules to determine the concentration of microorganisms present in plant biostimulants expressed as the number of active units per volume or weight, or in any other manner that is relevant to the microorganism, e.g. colony forming units per gram (cfu/g).
This document is applicable to the blends of fertilizing products where a blend is a mix of at least two of the following component EU fertilising products categories: Fertilizers, Liming Materials, Soil Improvers, Growing Media, Plant Biostimulants, and where the following category Plant Biostimulants is the highest % in the blend by mass or volume, or in the case of liquid form by dry mass. If Plant Biostimulants is not the highest % in the blend, the European Standard for the highest % of the blend applies. In case a blend of fertilizing products is composed of components in equal quantity or in case the component EU fertilising products used for the blend have identical formulations , the user decides which standard to apply.

This document specifies a horizontal method for the enumeration and genus/species determination [2], [3], [4] of mycorrhizal fungi in microbial plant biostimulants.
This document is applicable to the blends of fertilizing products where a blend is a mix of at least two of the following component EU fertilising products categories: Fertilizers, Liming Materials, Soil Improvers, Growing Media, Plant Biostimulants and where the following category Plant Biostimulants is the highest percentage in the blend by mass or volume, or in the case of liquid form by dry mass. If Plant Biostimulants is not the highest percentage in the blend, the European Standard for the highest percentage of the blend applies. In case a blend of fertilizing products is composed of components in equal quantity or in case the component EU fertilising products used for the blend have identical formulations , the user decides which standard to apply.

This document was developed to provide the methodology for the enumeration and determination of Azotobacter spp. [2] [3] in microbial plant biostimulants in accordance with the Regulation (EU) 2019/1009 of the European Parliament and of the Council [1].
This document is applicable to the blends of fertilizing products where a blend is a mix of at least two of the following component EU fertilising products categories: Fertilizers, Liming Materials, Soil Improvers, Growing Media, Plant Biostimulants and where the following category Plant Biostimulants is the highest % in the blend by mass or volume, or in the case of liquid form by dry mass. If Plant Biostimulants is not the highest % in the blend, the European Standard for the highest % of the blend applies. In case a blend of fertilizing products is composed of components in equal quantity or in case the component EU fertilising products used for the blend have identical formulations , the user decides which standard to apply.

This document provides a method for verifying that the pathogen Shigella spp. is not present in microbial plant biostimulants.
The detection method for Shigella pathogens is not sensitive and quantification is rarely performed. Detection is usually performed using an enrichment medium followed by subculturing onto a variety of selective media.
This document is applicable to the blends of fertilizing products where a blend is a mix of at least two of the following component EU fertilising products categories: Fertilizers, Liming Materials, Soil Improvers, Growing Media, Plant Biostimulants and where the following category Plant Biostimulants is the highest percentage in the blend by mass or volume, or in the case of liquid form by dry mass. If Plant Biostimulants is not the highest percentage in the blend, the European Standard for the highest percentage of the blend applies. In case a blend of fertilizing products is composed of components in equal quantity or in case the component EU fertilising products used for the blend have identical formulations , the user decides which standard to apply.

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 biostimulants, both in liquid and solid states. The method is applicable to microbial plant biostimulants except those composed of fungi or yeasts.
If necessary, yeasts and moulds enumerated can be identified using suitable identification tests.
This document is applicable to the blends of fertilizing products where a blend is a mix of at least two of the following component EU fertilising products categories: Fertilizers, Liming Materials, Soil Improvers, Growing Media, Plant Biostimulants, and where the following category Plant Biostimulants is the highest % in the blend by mass or volume, or in the case of liquid form by dry mass. If Plant Biostimulants is not the highest % in the blend, the European Standard for the highest % of the blend applies. In case a blend of fertilizing products is composed of components in equal quantity or in case the component EU fertilising products used for the blend have identical formulations , the user decides which standard to apply.

This document specifies a horizontal method for the detection of enteropathogenic Vibrio species (spp.), which causes human illness in or via the intestinal tract. The species detectable by the methods specified include Vibrio parahaemolyticus, Vibrio cholerae and Vibrio vulnificus.
It is applicable to the microbial plant biostimulants.
NOTE 1   The World Health Organization (WHO) has identified that V. parahaemolyticus, V. cholerae and V. vulnificus are the major contaminants of Vibrio spp. [2].
NOTE 2   For confirmation, it is possible to use PCR (Polymerase Chain Reaction) tests; in this case a validation is carried out by the laboratory for the procedure and data generated.
This document is applicable to the blends of fertilizing products where a blend is a mix of at least two of the following component EU fertilising products categories: Fertilizers, Liming Materials, Soil Improvers, Growing Media, Plant Biostimulants, and where the following category Plant Biostimulants is the highest % in the blend by mass or volume, or in the case of liquid form by dry mass. If Plant Biostimulants is not the highest % in the blend, the European Standard for the highest % of the blend applies. In case a blend of fertilizing products is composed of components in equal quantity or in case the component EU fertilising products used for the blend have identical formulations , the user decides which standard to apply.

This document specifies a method for laboratory measurement of the pH value in liquid microbial plant biostimulants, using pH electrodes with a glass membrane.
This document does not apply to plant biostimulants other than microbial plant biostimulants.
This document is applicable to the blends of fertilizing products where a blend is a mix of at least two of the following component EU fertilising products categories: Fertilizers, Liming Materials, Soil Improvers, Growing Media, Plant Biostimulants and where the following category Plant Biostimulants is the highest percentage in the blend by mass or volume, or in the case of liquid form by dry mass. If Plant Biostimulants is not the highest percentage in the blend, the European Standard for the highest percentage of the blend applies. In case a blend of fertilizing products is composed of components in equal quantity or in case the component EU fertilising products used for the blend have identical formulations , the user decides which standard to apply.

This document provides the methodology for the enumeration and determination of Azospirillum spp. in microbial plant biostimulants.
This document is applicable to the blends of fertilizing products where a blend is a mix of at least two of the following component EU fertilising products categories: Fertilizers, Liming Materials, Soil Improvers, Growing Media, Plant Biostimulants and where the following category Plant Biostimulants is the highest percentage in the blend by mass or volume, or in the case of liquid form by dry mass. If Plant Biostimulants is not the highest percentage in the blend, the European Standard for the highest percentage of the blend applies. In case a blend of fertilizing products is composed of components in equal quantity or in case the component EU fertilising products used for the blend have identical formulations , the user decides which standard to apply.

This document specifies a method to verify that the pathogen Staphylococcus aureus is absent from microbial plant biostimulants. The method is based on the enumeration of coagulase-positive staphylococci in a sample by counting of colonies obtained on a solid medium (Baird-Parker medium) after aerobic incubation at 36 °C ± 2 °C.
This document is applicable to all formulations of microbial plant biostimulants in liquid or solid form. This document is not applicable to other fertilizing products.
This document is applicable to the blends of fertilizing products where a blend is a mix of at least two of the following component EU fertilising products categories: Fertilizers, Liming Materials, Soil Improvers, Growing Media, Plant Biostimulants and where the following category Plant Biostimulants is the highest % in the blend by mass or volume, or in the case of liquid form by dry mass. If Plant Biostimulants is not the highest % in the blend, the European Standard for the highest % of the blend applies. In case a blend of fertilizing products is composed of components in equal quantity or in case the component EU fertilising products used for the blend have identical formulations , the user decides which standard to apply.

This document specifies a method for the enumeration of enterococci in microbial and non-microbial plant biostimulants. This document specifies a colony-count technique on a selective medium (Slanetz-Bartley agar) with confirmation on Bile Esculin Azide agar.
This document is applicable to all formulations of microbial and non-microbial plant biostimulants in liquid or solid form. This document is not applicable to other fertilizing products.
This document is applicable to the blends of fertilizing products where a blend is a mix of at least two of the following component EU fertilising products categories: Fertilizers, Liming Materials, Soil Improvers, Growing Media, Plant Biostimulants, and where the following category Plant Biostimulants is the highest % in the blend by mass or volume, or in the case of liquid form by dry mass. If Plant Biostimulants is not the highest % in the blend, the European Standard for the highest % of the blend applies. In case a blend of fertilizing products is composed of components in equal quantity or in case the component EU fertilising products used for the blend have identical formulations , the user decides which standard to apply.

This document specifies a horizontal method for the enumeration of microorganisms that are able to grow and form colonies in a solid medium after anaerobic incubation at 30 °C.
The method applies to microbial plant biostimulants, except those composed of aerobic bacteria.
This method does not apply to the microbiological monitoring of the environment in which microbial plant biostimulants are manufactured.
No information about potential human pathogens can be inferred from anaerobic plate counts.
This document is applicable to the blends of fertilizing products where a blend is a mix of at least two of the following component EU fertilising products categories: Fertilizers, Liming Materials, Soil Improvers, Growing Media, Plant Biostimulants and where the following category Plant Biostimulants is the highest percentage in the blend by mass or volume, or in the case of liquid form by dry mass. If Plant Biostimulants is not the highest percentage in the blend, the European Standard for the highest percentage of the blend applies. In case a blend of fertilizing products is composed of components in equal quantity or in case the component EU fertilising products used for the blend have identical formulations , the user decides which standard to apply.

This document specifies a method for the detection of Listeria monocytogenes in microbial plant biostimulants.
This document is applicable to the blends of fertilizing products where a blend is a mix of at least two of the following component EU fertilising products categories: Fertilizers, Liming Materials, Soil Improvers, Growing Media, Plant Biostimulants and where the following category Plant Biostimulants is the highest percentage in the blend by mass or volume, or in the case of liquid form by dry mass. If Plant Biostimulants is not the highest percentage in the blend, the European Standard for the highest percentage of the blend applies. In case a blend of fertilizing products is composed of components in equal quantity or in case the component EU fertilising products used for the blend have identical formulations , the user decides which standard to apply.

This document specifies a horizontal method for the detection of enteropathogenic Vibrio spp., which causes human illness in or via the intestinal tract [1]. The species detectable by the methods specified include Vibrio parahaemolyticus, Vibrio cholerae and Vibrio vulnificus.
It is applicable to the following:
—   microbial plant biostimulants.
NOTE 1   The World Health Organization (WHO) has identified that V. parahaemolyticus, V. cholerae and V. vulnificus are the major contaminants of Vibrio spp. [1].
NOTE 2   For confirmation, it is possible to use PCR tests; in this case the laboratory validates the procedure and data generated.

This document was developed to provide the methodology for the enumeration and determination of Azotobacter sp. in plant biostimulant products in accordance with the Regulation (EU) 2019/1009 of the European Parliament and of the Council [1].

This document specifies a method for laboratory measurement of the pH value in liquid microbial plant biostimulants, using pH electrodes with a glass membrane.
From the scope of this document plant biostimulants other than microbial plant biostimulants are excluded because there is no essential requirement in the Regulation (EU) 2019/1009 [1] for measuring the pH of non-microbial plant biostimulants.

This document defines general rules for the aerobic preparation of the initial suspension and of dilutions for microbiological examinations of microbial plant biostimulants.
This horizontal method might not be appropriate in very detail for certain products. In this case, different methods which are specific to these products can be used if necessary, for justified technical reasons.

This document provides a horizontal method for enumeration of microorganisms that are able to grow and form colonies in a solid medium after anaerobic incubation at 30 °C.
The method is applicable to microbial plant biostimulants for verifying that the concentration of anaerobes does not exceed the respective limits outlined in the EU Regulation on Fertilising Products [1].
This method does not apply to the microbiological monitoring of the environment in which microbial plant biostimulants are manufactured.
No information about potential human pathogens can be inferred from anaerobic plate counts.

This document specifies general rules to determine the concentration of microorganisms present in plant biostimulant products.
The method is applicable to microbial plant biostimulants for verifying that the concentration of microorganisms does not exceed the respective limits outlined in the EU Regulation on Fertilising Products [1].
This horizontal method might not be appropriate in every detail for certain products. In this case, it is necessary to refer to the methodology of specific determination and quantification of the microorganisms.

This document gives general guidelines for the detection and identification of the specified microorganism Escherichia coli in technical and formulated biostimulant products, both in liquid and solid state, and also the horizontal method for the enumeration of ß-glucuronidase-positive Escherichia coli in plant biostimulants products (both in liquid and solid state).
The qualitative method described in this document is based on the detection of Escherichia coli in a non-selective liquid medium (enrichment broth), followed by isolation on a selective agar medium. Other methods can be appropriate, depending on the level of detection required.
NOTE   For the detection of Escherichia coli, subcultures can be performed on non-selective culture media followed by suitable identification steps (e.g. using identification kits).
The quantitative method described in this document uses a colony-count technique at 44 °C on a solid medium containing a chromogenic ingredient for detection of the enzyme ß-glucuronidase.
WARNING — Strains of Escherichia coli which do not grow at 44 °C and, in particular, those that are ß glucuronidase negative, such as Escherichia coli O157, will not be detected.

This document provides a method for verifying that the pathogen Shigella spp. is not present in microbial plant biostimulants in a concentration that exceeds the respective limits outlined in the EU Regulation on Fertilising Products.
The detection method for Shigella pathogens is not sensitive and quantification is rarely performed. Detection is usually performed using an enrichment medium followed by subculturing onto a variety of selective media.

This document provides the methodology for the enumeration and determination of Azospirillum spp. in plant biostimulant products in accordance to the Regulation of EU fertilising products [1].

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].

This document was developed to provide a horizontal method for enumeration and genera/species determination [1], [2], [3] of mycorrhizal fungi in plant biostimulants products in accordance with the EU Fertilising Products Regulation.

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].

This document describes a method for the detection of Salmonella spp. in biostimulants of the following Product Function Categories (PFCs) and Component Material Category (CMC) of EU fertilizing products, as described in the Regulation (EU) 2019/1009 of the European Parliament and of the Council [1]:
—   PFC 6(A): Microbial plant biostimulant;
—   PFC 6(B): Non-microbial plant biostimulant;
—   CMC 7: Microorganisms.
It requires three successive steps: a selective enrichment, an isolation on a chromogenic agar, and if positive a confirmation with a serological test (and if required, a selective media).

This document provides a method for the detection of Listeria monocytogenes in microbial plant biostimulants for verifying that the concentration of this human pathogen does not exceed the respective limits outlined in the EU Regulation on Fertilising Products [1].

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.

This methodology has been developed to determine enterococci in biostimulants as a single microorganism component or in a mixture with other microorganisms. This document is not applicable to mineral fertilizers that are defined as complementary feeding stuffs composed mainly of minerals and containing at least 40 % crude ash (Regulation (EC) No 767/2009) [3].