Identification and taxonomic classification of microorganism(s) represented for use as supplements under the Fertilizers Act

Introduction

The purpose of this document is to provide guidance to applicants/product proponents on acceptable scientific methodologies and techniques for the identification of microorganisms intended for use as active ingredients in microbial supplements*.

This document focuses on the taxonomic identification of pure cultures of microorganisms belonging to the kingdoms of:

  1. bacteria, of;
  2. archaea, and of
  3. fungi.

It also lists standard resources and reference materials on taxonomic classification and nomenclature of the above groups of microorganisms. The document does not, however, include guidance on identification methods for microbial consortia which contain multiple genera of microorganisms that function as a community.

*Under the federal Fertilizers Act, a "supplement" is defined as any substance or mixture of substances, other than a fertilizer, that is manufactured, sold or represented for use in the improvement of the physical condition of soils or to aid plant growth or crop yields.

I. Microbial Identification

Knowledge of the phenotypic, genotypic and biological characteristics of a microorganism is imperative in differentiating it from its pathogenic and/or toxigenic relatives or other microorganisms that are detrimental to the health of plants, animals, humans and the environment. As such, accurate identification of the active microorganism(s) is a fundamental component of all safety assessments of microbial supplements regulated under the Fertilizers Act. Furthermore, the conclusions pertaining to product safety or its impacts on human health or the environment are valid only if the active microorganism(s) are correctly identified.

Polyphasic approach to microbial identification

The selection of method(s) used for microbial identification depends on the type and nature of the microorganism. The method(s) chosen should be well-described in scientific literature and consistent with those currently used in the field of microbial identification and taxonomic classification and they must enable identification of the organisms to the genus and species and, if possible, strain level. The robustness, precision and validity of the methodologies used to identify the microorganism are critical elements in the assessment of the safety of the product.

The choice of methods for microbial identification is at the discretion of the product proponent. However, the Fertilizer Safety Office recommends that applicants adopt an integrated polyphasic approach that includes classical microbiological and phenotypic analysis along with molecular tools, to accurately identify the active microorganism(s). The strengths and weaknesses of the various identification methods should be taken into consideration, such that the methods chosen complement each other to result in a conclusive and definitive identification of the microorganism, and allow for clear differentiation of the organism from any closely related pathogenic and/or toxigenic species and strains. The methods commonly used in identification and substantiation of taxonomic classification of microorganisms are summarized below.

1. Phenotypic analysis

Preliminary analysis in microbial identification often involves one or more phenotypic methods. Phenotypic methods are suitable for microorganisms that are culturable (i.e., can grow as pure culture on artificial media), have well-established growth parameters, and physiological and biochemical profiles.

a. Analysis of morphological traits

These methods utilize colony and cell morphology to obtain an initial identification of a microorganism. This is accomplished through simple isolation and culturing of the microorganism and subsequent visual observation using microscopy. The morphological properties include:

  1. shape,
  2. size,
  3. surface characteristics and pigmentation,
  4. cell wall characteristics (Gram-staining),
  5. sporulation characteristics,
  6. mechanisms of motility, and
  7. other cellular inclusions and ultrastructural characteristics.

b. Analysis of biochemical, physiological and metabolic characteristics

Phenotypic identification methods include the study of the biochemical profile and metabolic properties of a microorganism by testing its growth requirements, enzymatic activities and cellular fatty acid composition.

The biochemical tests use specific growth media, nutrients, chemicals or growth conditions to elicit an observable or measurable biochemical response from the microorganism, thereby enabling its identification and characterization. These tests include: utilization of carbon and nitrogen sources, growth requirements (anaerobic or aerobic; temperature-optimum and range, pH optimum and range), preferred osmotic conditions, generation of fermentation products, production of enzymes, production of antimicrobial compounds, as well as sensitivity to metabolic inhibitors and antibiotics. Examples of recognized tests include: phenol red carbohydrate, catalase and oxidase tests, oxidation-fermentation tests, methyl red tests, Voges-Proskauer tests, nitrate reduction, starch hydrolysis, tryptophan hydrolysis, hydrogen sulfide production, citrate utilization, litmus milk reactions, etc. Several miniaturized and automated commercial systems are currently available with well-defined quality control procedures that allow for rapid identification of microorganisms.

c. Analysis of Fatty Acid Methyl Ester composition (FAME analysis)

Microorganisms can be identified by analysing the fatty acid profiles of whole cells or cell membranes using gas-liquid chromatography or mass spectrometry. The data on the type, content, proportion and variation in the fatty acid profile are used to identify and characterize the genus and species by comparing it against the fatty acid profiles of known organisms.

The expression of microbial phenotypes is highly dependent on environmental variables (e.g., culture pH, temperature, selective vs non-selective media, depletion of nutrients, presence of stressors etc.), and thus, may introduce inconsistencies in the identification process. The phenotypic methods are only acceptable if the response criteria are sufficient to identify the microorganism with a high level of confidence and distinguish it from phylogenetically close relatives that potentially pose safety concerns. Also, the applicability of the method is based on the robustness of information in reference databases. As such, results from phenotypic methods may require supporting data from other methods to accurately identify a microorganism.

2. Molecular Methods

Development of molecular methods has greatly improved the ability to rapidly detect, identify and classify microorganisms and also establish the taxonomic relationship among closely related genera and species. Identification, using molecular methods, relies on the comparison of the nucleic acid sequences (DNA, RNA) or protein profiles of a microorganism with documented data on known organisms. The molecular methods are considered sensitive enough to allow detection of low concentrations of viable or non-viable microorganisms in both pure cultures and complex samples (e.g., soil, peat, water etc.).

a. Genotypic methods

These include methods such as nucleic acid hybridization (Southern blot analysis or Solution-phase hybridization) and amplification-based or polymerase chain reaction (PCR) technologies. The latter consist of sequence comparisons of conserved genomic regions such as 16S or 18S rRNA, or comparisons of restriction fragment length polymorphisms (RFLP), amplified fragment length polymorphisms (AFLP) or G+C % content in the genomic DNA with corresponding data on known organisms.

Reliable genotypic identification requires databases with accurate and complete sequence information from a large number of taxa. The commonly used gene sequence databases include:

  • GenBank®1;
  • Ribosomal Database Project (RDP)2;
  • Europe's collection of nucleotide sequence data (EMBL)3; and
  • Universal Protein Resource (UniProt)4.

Product proponents are not limited to using the reference materials listed above; these are intended as guidance only.

Some of the limitations associated with genotypic methods also include: difficulties in differentiating between species that share identical and/or similar conserved region sequences, limited information on the quality of sequence data available in public databases and the complexity of taxonomic nomenclature overall. Considering the above, it is important to validate the results of genotypic microbial identification methods with data from other sources (e.g., morphological and/or phenotypic analysis).

b. Protein based methods

Serological methods such as Western blotting, Immuno-precipitation and Enzyme Linked Immuno-sorbant Assay (ELISA) use antibodies to detect specific proteins that are unique and/or characteristic of a microorganism. The applicability of serological methods is dependent on the availability, sensitivity and specificity of the antibodies used. There are commercial kits available for immuno-detection of several microorganisms. Protein based methods also include gel electrophoresis (SDS-PAGE, 2D-gels, etc.) that can separate cellular proteins on a defined matrix and identify microbial proteins of interest by comparing with microorganisms with known protein profiles.

3. Genomics

More recently, complete profiling of the transcriptome, genome, proteome or metabolome have been used to identify and characterise organisms. Several modern technologies such as DNA and protein microarray analyses, mass spectral protein profiling, nuclear magnetic resonance (NMR) spectral analysis, in-silico microbial metabolome platforms are increasingly used in identification and characterization of microorganisms.

The knowledge of the sensitivity and specificity of genomic tools and their application in microbial identification is rapidly evolving. However, challenges related to standardization of genomics methodologies (including optimization of protocols and bioinformatics tools for reliable data annotation, interpretation etc.) continue to hinder their applicability in safety (risk) assessment and regulatory decision making. The Fertilizer Safety Office will consider data generated by genomics-methods on a case-by-case basis. However, validation of genomics data using alternate methods is currently required to substantiate the identification and taxonomic classification of an active microorganism(s) in a supplement product.

II. Taxonomic Classification and Nomenclature:

The taxonomic identification of the microorganism(s) should be based on the currently used and internationally accepted taxonomic classification system. The description of the microorganism(s) in the product and its characteristics must correspond to the characteristics described in standard resources and/or references that are commonly used by the scientific community to validate taxonomic classification. These can include but are not limited to:

  • textbooks such as the Bergey's Manual of Systematic Bacteriology5;
  • The Prokaryotes6;
  • Applied Microbial Systematics7;
  • Principles of fungal taxonomy8 etc.;
  • online resources such as the Catalogue of Life9;
  • PubMed Taxonomy10 and UniProt Taxonomy11 etc.; and
  • peer reviewed journals.

The taxonomic name should follow the nomenclature code officially recognized by the International Committee on Systematics of Prokaryotes (ICSP). Applicants should verify the "Approved List of Bacterial Names" to ensure that the nomenclature is in accordance with the latest Validation List developed and updated by the International Journal of Systematic and Evolutionary Microbiology (IJSEM).

Please note that microbial taxonomic classification and nomenclature, particularly for bacteria, is in a constant state of flux as methodologies evolve to generate more reliable information to identify/classify and/or reclassify the current taxonomic scheme. Cross referencing more than one resource/reference will help in validating the current taxonomic designation and classification of a microorganism.

III. Streamlining the information requirements for taxonomic identification of microorganisms

The following is general guidance to streamline the information requirements for identification of microorganisms represented as active ingredients in fertilizers and/or supplements regulated under the Fertilizers Act. The Fertilizer Safety Office will consider the information for each application on a case-by-case basis.

i. Microorganisms that are exempt from full safety data requirement (Refer to Appendix A)

The Fertilizer Safety Office has conducted a comprehensive safety assessment of microorganisms belonging to the Rhizobial group (Rhizobium spp., Bradyrhizobium spp., Mesorhizobium spp., Sinorhizobium spp.), Bacillus subtilis and Vascular Arbuscular Mycorrhizae (VAM). Based on the currently available scientific literature and well documented history of safe use of these microorganisms in Canada, the Fertilizer Safety Office has exempted these microorganisms from full safety data requirements. However, submission of core safety data (refer to Appendix A) is required for this category of microorganisms.

If the Rhizobia, VAM or Bacillus subtilis strains used as active ingredient in the inoculant product, are obtained from a recognized culture collection, substantiation of taxonomic identification as described under section (ii) below is required. If the microorganism was isolated from a natural environment, applicants are expected to adopt a polyphasic approach (e.g., morphological and biochemical methods) to support the identification of microorganisms as described below under section (iii).

For the Rhizobial group and VAM, identification is required up to the species and, if possible, to strain level. Microorganisms belonging to genus Bradhyrhizobium also require identification up to the species level, and, if possible, to strain level, in order to differentiate the active ingredient from closely related toxigenic species like B. elkanii.

All microorganisms belonging to the genus Bacillus, including Bacillus subtilis must be identified to the strain level, as described in section (iii).

ii. Microorganisms obtained from culture collections

If the microorganism is obtained or purchased from a recognized International Culture Collection such as the American Type Culture Collection (ATCC)12, Germany's Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ)13, USDA-ARS National Rhizobium Germplasm Collection (BRCC)14, or Canadian Collection of Fungal Cultures (CCFC)15 etc., the following information will be required to substantiate the taxonomic identification of the microorganism.

  • a certificate of analysis from the culture collection;
  • description of methods used to identify and substantiate the taxonomic designation, and
  • results of analysis to demonstrate the purity and true-type of the microorganism and its relatedness or similarities to microorganisms that are detrimental to the health of plants, animals and humans and the environment.

iii. Environmental isolates

For all environmental isolates, taxonomic designation should be based on a complementary, polyphasic approach that enables identification and substantiation of the microorganism up to species level, and if possible, up to strain level, and allows differentiation from closely related pathogenic and/or toxigenic species and/or strains. This is particularly important for microorganisms belonging to genera where pathogenic and/or toxigenic species and strains are known to exist. For instance, strain level identification is required for microorganisms belonging to the genera Bacillus, in order to differentiate the microorganism from related phylogenetic kins that include pathogenic and/or toxigenic species (e.g., Bacillus anthracis, Bacillus thuringiensis, Bacillus cereus etc.), and from potentially pathogenic and/or toxigenic strains within a particular species (e.g., Bacillus licheniformis etc.).

Appendix A: Safety data requirements for products regulated under the Fertilizers Act and Regulations

1. Types of Data

Core safety Data:

  • Complete list of ingredients including their sources and proportions. Please note that for some product types the source information should include:
    1. country of origin;
    2. method of manufacture (i.e. by-product of another process, mining of materials, etc.); and
    3. name of the distributor. If the above information is not directly available to the applicant, the distributor may submit it to the CFIA directly, and it will be protected as confidential business information;
  • Method of manufacture;
  • Material Safety Data Sheet (MSDS) for ingredients and/or product (if available);
  • Data support taxonomic identification of any guaranteed microorganism(s)

Results of analysis including any or all of the following:

  • Four sets of analyses for fecal coliform and Salmonella;
  • Four sets of analyses for Arsenic (As), Cadmium (Cd), Cobalt (Co), Copper (Cu), Chromium (Cr), Mercury (Hg), Molybdenum (Mb), Nickel (Ni), Lead (Pb), Selenium (Se), Zinc (Zn);
  • One set of analysis for dioxins and furans

Full safety data package:

Depending on the product type, guidance on the safety standards and type of information required in a full safety data package can be found in one or more of the following documents:

T-4-93 - Standards for metals in fertilizers and supplements

T-4-107 - Registration of supplements under the Fertilizers Act

T-4-112 - Information required for the assessment of by-products and other "waste" materials sold as fertilizers or supplements

T-4-113 - Guidelines to safety assessments of fertilizers and supplements and to information to be submitted in demonstrating product safety

T-4-113 (Supplement 1) - Data requirements for product safety evaluations: explanatory notes

T-4-114 - Sampling procedures for fertilizers

T-4-116 - Safety data requirements for the registration of polyacrylamide, acrylamide-acrylate copolymer and polyacrylate soil supplements as active ingredients under the Fertilizers Act

T-4-118 - Guidelines to safety assessments of microorganisms in fertilizers and supplements regulated under the Fertilizers Act

Registration Guide: Micronutrient Fertilizers

2. Products/ingredients that require the submission of core safety data to support approval or registration

Fertilizers:

  • NPK fertilizers without micronutrients
  • Lesser plant nutrients: Calcium (Ca), Magnesium (Mg), and Sulphur (S)
  • Fertilizer-Pesticide combinations as found in the Fertilizer Pesticide Compendium
  • Low analysis farm fertilizers

Supplements:

  • Indole-3-butyric acid (IBA) at a concentration not exceeding 1% of the product
  • Products used to correct soil acidity or alkalinity (e.g. liming materials)
  • Soil amendments represented to improve the condition of the soil by increasing organic matter (OM)
  • VAMs (Vesicular Arbuscular Myccorhizae) provided the species is substantially equivalent/representative of VAM group
  • Rhizobia (species of the genera Rhizobium, Bradyrhizobium, Mesorhizobium and Sinorhizobium), provided the species is substantially equivalent/representative of the rhizobia group
  • Bacillus subtilis provided the strain is representative of the species and does not produce any toxins

Please note that the presence of other active or inert ingredients in the product may trigger additional safety requirements. If any of the products listed above are made from or contain waste materials, they fall under the next section.

3. Products/ingredients that require the submission of core safety data AND results of analyses to support approval or registration

  • Non-chelated micronutrients
  • Micronutrients chelated with the following chelators: ethylenediamine-tetra-acetic acid disodium salt (EDTA), diethylenetriamine penta-acetic acid (DTPA), ethylenediamine-di-o-hydroxyphenylacetic acid (EDDHA), N-(2-hydroxyethyl) ethylenediamine-N, N', and N'-tri-acetic acid trisodium salt (HEDTA)
  • NPK fertilizers with micronutrients
  • All fertilizers and supplements containing waste derived materials (e.g. composts, processed sewage, pulp and paper sludges, etc.)

4. Products/ingredients that require the submission of a full safety data package AND may require results of analysis to support approval or registration

Fertilizers:

  • Fertilizers containing any of the supplements listed below
  • Micronutrients for foliar use
  • Micronutrients chelated with other chelators than those indicated above
  • NPK fertilizers (with or without micronutrients) containing/coated with polymers
  • Nano-materials and Nano-encapsulated fertilizers
  • Digestates and other products of microbial fermentation

Supplements:

  • Plant growth regulators other than IBA as described above (e.g., NAA, Kinetin)
  • Plant signalling compounds (e.g., LCO's, Hesperetin, Naringenin)
  • Wetting agents and surfactants
  • Water holding polymers and soil flocculants/stabilizers
  • Inoculant extenders (products represented to improve inoculant on-seed survival)
  • Any product represented as a "Bio-stimulant" (e.g., vitamins, enzymes)
  • Inoculants containing viable microorganisms other than VAM and Rhizobia
  • Microbial consortia products

NOTE: All fertilizers and supplements sold and imported into Canada must be safe with respect to human, animal, plant health and the environment. The product safety assessments performed by the Fertilizer Safety Office (FSO) are conducted on case-by-case basis and the information requirements may vary depending on the ingredients in the product (both active and inert), their sources, degradation by-products, manufacturing process, use pattern and application rate. To that effect, the FSO reserves the right to require additional safety information, data, rationale or results of analysis to support the registration/approval of any product regulated under the Fertilizers Act and Regulations.

References:

1 GenBank® of National Centre for Biotechnogy Information (NCBI)

2 Ribosomal Database Project (RDP)

3 EMBL Nucleotide DB (European Molecular Biology Laboratory)

4 Universal Protein Resource Knowledgebase (UniProtKB)

5 Bergey's Manual of Systematic Bacteriology, Second edition, volume 1 to 5, The Williams and Wilkins Company, Baltimore, 2001-2009

6 The Prokaryotes Third edition, 2007, 7000 page 7-volume-set, Springer publication

7 Applied microbial systematics. Priest, F. G.; Goodfellow, M. (Edition) 2000, 500 page, Softcover. ISBN: 978-0-7923-6518-1

8 Principles of fungal taxonomy. P. H. B. Talbot. 3-274 page 1971. ISBN 10: 0333115619

9 Catalogue of life 2010 annual check list

10 PubMed Taxonomy

11 Universal Protein Resource Knowledgebase (UniProtKB)

12 ATCC-The Global Biosource Centre

13 DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH

14 USDA-ARS Rhizobium Germplasm Resource Collection

15 The Canadian Collection of Fungal Cultures, Agriculture and Agri-Food Canada

Additional References

European Union
Guideline developed within the Standing Committee on the Food Chain and Animal Health on the taxonomic level of microorganisms to be included in Annex I to Directive 91/414/EEC. EUROPEAN COMMISSION HEALTH AND CONSUMER PROTECTION DIRECTORATE-GENERAL. Sanco/10754/2005 revision 5, April 2005.

Environment Canada
Guideline for the Notification and Testing of New Substances: Organisms (August 2010)