Rebuilding the Arbuscular Mycorrhizal Inoculant Pipeline: Integrating Quality-by-Design and Trait–Environment Matching

Abstract

Arbuscular mycorrhizal (AM) fungi offer opportunities for low-input agriculture, but commercial inoculant performance remains variable. This narrative and conceptual review examines the evidence for this variability and proposes a framework for more reliable development. Three findings emerge. Independent assessments reveal recurrent composition and infectivity deficiencies among tested products, without establishing their market prevalence. Root colonisation does not guarantee host benefit, nor host benefit agronomic value. Provenance effects are context-dependent, so local origin can guide sourcing but confers no universal advantage. We distinguish six performance endpoints and five development bottlenecks, separating documented constraints from hypothesised cultivation-associated trait change. A hypothesis-generating trait–environment matching framework relates establishment and host benefit separately to environmental constraints and resident AM fungal community function. A complementary quality-by-design framework begins by diagnosing whether inoculation is needed and which function is required, then links selection, production, storage validation and batch release to targeted deployment. Practical priorities are to select isolates and consortia on demonstrated function, verify retention through production and storage, and test added value against management alternatives. These frameworks require experimental validation. Verified product quality is necessary but insufficient: the selected function must be expressed where its benefit justifies intervention.

1. Introduction

Arbuscular mycorrhizal (AM) fungi associate with most terrestrial plant species, exchanging host carbon for mineral nutrients acquired beyond root nutrient-depletion zones [1,2,3]. Colonisation can also influence stomatal regulation, osmotic adjustment and antioxidant metabolism, improving performance under water deficit or salinity [4,5]. These properties underpin the worldwide marketing of AM fungi as biofertilisers and biostimulants [6,7]. Their agronomic use nevertheless faces two distinct problems that are often conflated.

The first problem is ecological. Because inoculation introduces a living symbiont into an existing ecological system, agronomic responses range from positive to negative according to soil fertility, host genotype, fungal identity and management [8,9,10,11]. Across 54 Swiss maize fields, for example, growth responses ranged from approximately −12% to +40%, with much of the variation explained by soil and microbiome indicators [12]. Even a fully functional inoculant may therefore provide no benefit where resident fungi already supply the required function or where conditions prevent its expression.

The second problem concerns the product itself. Independent assessments report little or no colonisation, inadequate propagule abundance or infectivity, label discrepancies and inconsistent plant responses [13,14,15,16]. Apparent growth benefits sometimes disappear when formulation-borne nutrients are controlled, and unintended organisms, including potential pathogens, have also been detected [15]. Such products fail before context-dependent symbiotic benefits can arise. Because both problems produce the same symptom, an absent growth response, they can only be distinguished by following a common diagnostic sequence: identity, viability, infectivity, establishment, effectiveness and agronomic value. Table 1 defines these endpoints and distinguishes provenance terms (native, local, sympatric and non-native) from experimentally demonstrated local adaptation; these distinctions underpin the review.

Existing contributions have addressed parts of this problem: quality management [17,18,19], regulation [20], inoculant selection [21], field prediction [12], local adaptation [22] and applied research needs [23]. What remains missing is a framework that connects these elements, linking product measurements to the biological need for inoculation, to the selection of fungi for a specified function, and to verification that this function is retained through production and expressed after deployment. Without such a link, a product may conform to its specification yet be deployed where it cannot help, and fungi may be selected for their provenance or production yield rather than for the function required.

Table 1. Working definitions of the principal terms used in this review and the sources from which each definition is derived.

Term	Working Definition as Used in This Review	Key Sources
Performance endpoints (Figure 1)
Identity	Correspondence between the AM fungal taxa declared for an inoculant and those verifiably present, assessed by morphological and molecular methods; a criterion of conformity and traceability	[14,24]
Viability	Proportion of propagules that are alive and physiologically functional; necessary but not sufficient for infectivity	[17,25]
Infectivity	Capacity of an inoculum to establish root colonisation, measured as colonisation frequency and intensity in a defined bioassay; a property of the inoculum, not of the fungal species	[13,17]
Establishment	Colonisation of the target crop by the introduced fungus, distinguishable from total root colonisation only with strain-specific methods. Distinguished here from maintenance during the period in which the function is needed and from persistence after harvest or between seasons, which is required for some uses only	[26,27,28]
Effectiveness	Magnitude of the host response (biomass, nutrient uptake, stress tolerance or yield) attributable to colonisation; high infectivity does not imply high effectiveness	[11,13]
Agronomic value	Benefit that is sufficiently large, reproducible and relevant under practical production conditions to justify inoculation	[10,12]
Provenance and adaptation
Native (indigenous)	Occurring naturally in the region or ecosystem considered, without deliberate introduction. The term describes biogeographic origin only, and the spatial scale to which it refers should be stated. Autochthonous is used as a synonym	[22,29]
Non-native (exotic, introduced)	Originating outside the region or ecosystem of use and introduced there, as is the case for most commercial strains applied away from their site of isolation. Allochthonous is used as a synonym	[29]
Local	Obtained at or close to the site of intended use (the same field, farm or landscape). The term describes proximity; it implies neither association with the target host nor adaptation	This review
Sympatric and allopatric	Sympatric: plant, fungus and soil, or any two of them, originate from the same site. Allopatric: they originate from different sites. The terms describe the shared origin of the partners, not their performance	[22]
Locally adapted	Showing higher performance at home than genotypes of foreign origin (local versus foreign criterion) or than the same genotype elsewhere (home versus away criterion). The performance concerned must be specified: that of the fungus, of the plant or of the association. Local adaptation must be demonstrated experimentally and cannot be inferred from origin	[22,30,31]
Home-field advantage	Greater symbiotic benefit when plant, fungus and soil share a common geographic origin than in allopatric combinations	[22,31]
Other concepts
Propagule	Any fungal structure able to initiate a new colonisation event: spore, colonised root fragment or extraradical mycelium. Propagule abundance per unit mass or volume (the dose) is a product attribute distinct from viability and infectivity	[20,24]
Mycorrhizal inoculum potential	Colonising capacity of a whole soil, integrating all propagule types present, assessed with a standardised trap bioassay	[32,33]
Mycorrhizal growth response	Relative difference in host biomass between colonised and non-colonised plants; ranges from strongly positive to negative and is context-dependent	[11,34]
Monoxenic culture	In vitro co-culture of an AM fungus with a root organ, usually genetically transformed, under aseptic conditions; composition is controlled, but freedom from contaminants must be verified; not host-free	[35,36,37]
Asymbiotic (axenic) culture	Growth and sporulation of an AM fungus in the complete absence of a host, achieved so far with exogenous fatty acids and phytohormones for a small number of isolates	[38,39,40]
Priority effect	Influence of the order of arrival on community assembly; here, suppression of an introduced fungus by a resident community that occupied the root and soil niche first	[27,28]
Trait drift under culture	Hypothesised directional change in fungal functional traits imposed by repeated subculture under artificial conditions; current evidence comes from comparisons among isolates of one species	[41,42,43]
Trait–environment matching	Selection of fungi whose functional traits are compatible with the host genotype, soil, resident microbiome and dominant environmental constraint of the target system	This review; [22,31]
Quality-by-design	Development approach in which the need for inoculation is diagnosed and the desired fungal functional phenotype is defined first, and production, formulation, release and deployment are designed and verified to preserve and express it	This review
Consortium	Defined combination of two or more AM fungal isolates, each verified individually, formulated as one product	This review; [44]
Release criterion and threshold	Criterion: a measurement with a defined method, unit and decision rule that a production lot must satisfy before release. Threshold: an agreed numerical limit for that measurement	This review; [17]

Table 1. Working definitions of the principal terms used in this review and the sources from which each definition is derived.

Term	Working Definition as Used in This Review	Key Sources
Performance endpoints (Figure 1)
Identity	Correspondence between the AM fungal taxa declared for an inoculant and those verifiably present, assessed by morphological and molecular methods; a criterion of conformity and traceability	[14,24]
Viability	Proportion of propagules that are alive and physiologically functional; necessary but not sufficient for infectivity	[17,25]
Infectivity	Capacity of an inoculum to establish root colonisation, measured as colonisation frequency and intensity in a defined bioassay; a property of the inoculum, not of the fungal species	[13,17]
Establishment	Colonisation of the target crop by the introduced fungus, distinguishable from total root colonisation only with strain-specific methods. Distinguished here from maintenance during the period in which the function is needed and from persistence after harvest or between seasons, which is required for some uses only	[26,27,28]
Effectiveness	Magnitude of the host response (biomass, nutrient uptake, stress tolerance or yield) attributable to colonisation; high infectivity does not imply high effectiveness	[11,13]
Agronomic value	Benefit that is sufficiently large, reproducible and relevant under practical production conditions to justify inoculation	[10,12]
Provenance and adaptation
Native (indigenous)	Occurring naturally in the region or ecosystem considered, without deliberate introduction. The term describes biogeographic origin only, and the spatial scale to which it refers should be stated. Autochthonous is used as a synonym	[22,29]
Non-native (exotic, introduced)	Originating outside the region or ecosystem of use and introduced there, as is the case for most commercial strains applied away from their site of isolation. Allochthonous is used as a synonym	[29]
Local	Obtained at or close to the site of intended use (the same field, farm or landscape). The term describes proximity; it implies neither association with the target host nor adaptation	This review
Sympatric and allopatric	Sympatric: plant, fungus and soil, or any two of them, originate from the same site. Allopatric: they originate from different sites. The terms describe the shared origin of the partners, not their performance	[22]
Locally adapted	Showing higher performance at home than genotypes of foreign origin (local versus foreign criterion) or than the same genotype elsewhere (home versus away criterion). The performance concerned must be specified: that of the fungus, of the plant or of the association. Local adaptation must be demonstrated experimentally and cannot be inferred from origin	[22,30,31]
Home-field advantage	Greater symbiotic benefit when plant, fungus and soil share a common geographic origin than in allopatric combinations	[22,31]
Other concepts
Propagule	Any fungal structure able to initiate a new colonisation event: spore, colonised root fragment or extraradical mycelium. Propagule abundance per unit mass or volume (the dose) is a product attribute distinct from viability and infectivity	[20,24]
Mycorrhizal inoculum potential	Colonising capacity of a whole soil, integrating all propagule types present, assessed with a standardised trap bioassay	[32,33]
Mycorrhizal growth response	Relative difference in host biomass between colonised and non-colonised plants; ranges from strongly positive to negative and is context-dependent	[11,34]
Monoxenic culture	In vitro co-culture of an AM fungus with a root organ, usually genetically transformed, under aseptic conditions; composition is controlled, but freedom from contaminants must be verified; not host-free	[35,36,37]
Asymbiotic (axenic) culture	Growth and sporulation of an AM fungus in the complete absence of a host, achieved so far with exogenous fatty acids and phytohormones for a small number of isolates	[38,39,40]
Priority effect	Influence of the order of arrival on community assembly; here, suppression of an introduced fungus by a resident community that occupied the root and soil niche first	[27,28]
Trait drift under culture	Hypothesised directional change in fungal functional traits imposed by repeated subculture under artificial conditions; current evidence comes from comparisons among isolates of one species	[41,42,43]
Trait–environment matching	Selection of fungi whose functional traits are compatible with the host genotype, soil, resident microbiome and dominant environmental constraint of the target system	This review; [22,31]
Quality-by-design	Development approach in which the need for inoculation is diagnosed and the desired fungal functional phenotype is defined first, and production, formulation, release and deployment are designed and verified to preserve and express it	This review
Consortium	Defined combination of two or more AM fungal isolates, each verified individually, formulated as one product	This review; [44]
Release criterion and threshold	Criterion: a measurement with a defined method, unit and decision rule that a production lot must satisfy before release. Threshold: an agreed numerical limit for that measurement	This review; [17]

The present review builds on Salomon et al. [17] and independent product assessments [13,14,15,16,45]. Salomon et al. already address application-oriented selection, storage, labelling and minimum bioassay quality, and identify field prediction and production stability as research needs. Our contribution is to integrate these concerns into an explicit, testable development sequence. Quality-by-design begins with a diagnosis of whether inoculation is justified and which function is required (stage 0), then links selection to verification during production and storage. Trait–environment matching separates predictions for fungal establishment from those for host benefit and specifies potentially inconsistent results (Section 5.3). The six-endpoint diagnostic sequence (Figure 1) and the distinction between strain qualification, formulation validation, lot release and claim validation (Section 6.6) connect product measurements to targeted deployment and net return (Section 6.7). Section 6.8 compares the emphasis of these complementary contributions; the novelty lies in their operational integration rather than in claiming that each component is new.

Our central hypothesis is that reliable inoculants require identification of the functions needed by a target host–environment system, selection of fungi supplying them, and retention of the relevant properties through production and formulation. Propagule yield matters only insofar as the resulting inoculum establishes and expresses the required function, and provenance guides sourcing rather than substituting for demonstrated performance. To develop this argument, we first examine the limitations documented in commercial products (Section 3), the bottlenecks of the development pipeline (Section 4) and the evidence on provenance effects (Section 5). We then propose the trait–environment matching (Section 5.3) and quality-by-design (Section 6) frameworks; the latter begins with the justification for inoculation and connects selection and verification to ecological suitability and economic value. Research priorities (Section 7) distinguish established findings from relationships requiring validation.

Figure 1. A diagnostic sequence for AM fungal inoculant performance. For each of six endpoints, the figure gives the diagnostic question, an appropriate test and a documented failure or gap. Identity is a criterion of conformity and traceability; viability, infectivity and establishment are biological prerequisites for effectiveness; agronomic value depends in addition on consistency and cost. Endpoints 1–3 can be verified before release, whereas endpoints 4–6 also depend on host, soil, resident microbiome and management. Propagule abundance (the dose) is a cross-cutting product attribute, not an endpoint. A poor plant response alone cannot identify where performance was lost. Sources: endpoint 1 [14,46]; 2 [13,15]; 3 [13,16]; 4 [27,28]; 5 [47,48]; 6 [12]; dose [15].

Figure 1. A diagnostic sequence for AM fungal inoculant performance. For each of six endpoints, the figure gives the diagnostic question, an appropriate test and a documented failure or gap. Identity is a criterion of conformity and traceability; viability, infectivity and establishment are biological prerequisites for effectiveness; agronomic value depends in addition on consistency and cost. Endpoints 1–3 can be verified before release, whereas endpoints 4–6 also depend on host, soil, resident microbiome and management. Propagule abundance (the dose) is a cross-cutting product attribute, not an endpoint. A poor plant response alone cannot identify where performance was lost. Sources: endpoint 1 [14,46]; 2 [13,15]; 3 [13,16]; 4 [27,28]; 5 [47,48]; 6 [12]; dose [15].

2. Literature Search and Scope

This narrative and conceptual review does not undertake a quantitative synthesis. Searches of Web of Science, Scopus, PubMed and Google Scholar were conducted without a lower date limit and completed in September 2026. Search terms combined “arbuscular mycorrhiza*” or “AM fungi” with terms related to inoculant quality, production, storage, provenance and performance, including “inoculant”, “biofertili*”, “quality control”, “infectivity”, “root organ culture”, “monoxenic”, “shelf life”, “native”, “local adaptation”, “establishment” and “field inoculation”. In database syntax supporting truncation, the asterisk (*) retrieves variants of a word stem; for example, biofertili* covers biofertiliser and biofertilizer. Syntax and wildcard behaviour differ among databases.

Exact executed search strings and complete screening logs were not retained during preparation of this narrative review. For transparency, the following approximate queries have been reconstructed retrospectively from its search concepts; they illustrate the strategy and are not archived original queries. In Web of Science topic-field syntax, they are:

Product quality: TS = ((“arbuscular mycorrhiza*” OR “AM fungi” OR AMF) AND (inoculant* OR “commercial product*” OR biofertili* OR biostimulant*) AND (quality OR infectivity OR viability OR coloni* OR label* OR composition OR contamina*)).

Production and storage: TS = ((“arbuscular mycorrhiza*” OR “AM fungi”) AND (“root organ culture” OR monoxenic OR “in vitro” OR aeroponic* OR “on-farm” OR asymbiotic) AND (production OR propagation OR formulation OR storage OR “shelf life”)).

Provenance and field performance: TS = ((“arbuscular mycorrhiza*” OR “AM fungi”) AND (native OR indigenous OR “local adaptation” OR “home-field” OR sympatric) AND (inoculation OR establishment OR yield)).

The numbers of records screened by title and abstract and of full texts assessed cannot be reconstructed reliably and are therefore not reported. The reference list contains 99 publications; this is a citation count, not a screening count. Reference lists of relevant publications were also screened, and only English-language publications were considered.

Study selection prioritised evidence relevant to the six endpoints examined in this review, with particular attention to independently conducted product-quality assessments, meta-analyses, multi-site field trials, studies distinguishing introduced from resident AM fungi, and provenance comparisons conducted under common host and soil conditions. Table 2 summarises the product assessments and field studies informing the analysis. Because study identification and selection were non-systematic, the evidence base is neither exhaustive nor intended to be representative of the commercial inoculant market; consequently, the frequency of quality deficiencies or performance failures cannot be estimated. The conceptual frameworks proposed in this review should therefore be regarded as hypotheses and decision-support approaches requiring further experimental validation.

Table 2 is a purposive summary rather than the output of prospectively specified eligibility criteria. It brings together multi-product assessments of declared or detected composition, propagule abundance, infectivity, root colonisation or plant response, and syntheses of those assessments. Its two field datasets comprise 231 potato trials [49] and 54 maize fields [12], illustrating variation in field outcomes at larger scales; no prospectively defined minimum number of fields was used. Individual-product efficacy studies are discussed elsewhere and are not comprehensively represented in Table 2. Favourable outcomes are included: mean potato yield increased by 9.5%, with profitability in almost 79% of trials [49]; some products increased colonisation or biomass [13,16,46]; and maize growth responses reached +40% [12]. A separate targeted search specifically for favourable product assessments cannot be documented. The table may therefore be imbalanced, because independent product audits often address quality concerns and positive single-product trials are incompletely represented. Meta-analyses reporting positive average responses [9,10] are considered separately in Section 1. These limitations preclude inference about the market-wide frequency of deficiencies or benefits.

Table 2. Published assessments of commercial AM inoculant quality and of field-scale inoculation outcomes (2004–2025).

Study	Products or Scope	Approach	Principal Quantitative Outcome
Assessments of commercial product quality
Corkidi et al., 2004 [50]	10 commercial products; nursery conditions	Maize bioassay in three growing media	Colonisation ranged from 0 to 50% and depended on the growing medium; colonisation did not enhance plant growth
Wiseman et al., 2009 [51]	Products marketed for woody landscape plants	Greenhouse bioassays with a laboratory culture as positive control	Colonisation rarely exceeded 5% in maize and sorghum, against 38–61% for the laboratory culture and 74% in seed-grown magnolia
Faye et al., 2013 [46]	12 commercial products	Trap-culture propagation followed by a maize bioassay in soil	Five undeclared species were recovered and four declared species produced no spores; three products increased colonisation above an indigenous soil inoculum
Basiru et al., 2021 [24]	68 products from 28 manufacturers (Europe, America, Asia)	Documentary analysis of declared composition and claims	A few species dominated: Rhizophagus irregularis in 39% of products, Funneliformis mosseae in 21% and Claroideoglomus etunicatum (current name Entrophospora etunicata) in 16%; one third single-species; all species assigned to the Glomeraceae by the authors
Salomon et al., 2022 [13]	28 products; Australia, Europe, North America	Greenhouse bioassays of 25 products in sterilised and non-sterilised soil; field trial of three products	Sterilised soil: 84% produced no colonisation and biomass increased in five of 25 treatments; non-sterilised soil: no increase in colonisation, biomass increased in one of 25; laboratory R. irregularis reached 48% and 79% colonisation
Vahter et al., 2023 [14]	11 products from four producers	DNA metabarcoding compared with label declarations	Two products contained none of the declared species; 10 of 11 contained undeclared species
Koziol et al., 2024 [15]	23 inoculants (commercial products, laboratory-grown fungi, field soil)	Spore enumeration, infectivity and crop response with product-borne fertiliser controlled	Propagule counts differed from declared values by up to 100%; insufficient colonisation by commercial products; contamination by Olpidium; growth benefits often absent once fertiliser was controlled
Koziol et al., 2025 [45]	302 inoculant trials (250 with commercial products) from 28 experiments in six publications	Meta-analysis of studies testing five or more commercial products	Commercial products averaged <9% hyphal colonisation, against 41% for laboratory-grown inoculants; crop growth increased by 19% versus 38%; only 12% of commercial trials combined sufficient colonisation with a growth increase
Boussageon et al., 2025 [16]	16 commercial products (nine agricultural, seven home gardening) and seven research inocula	Controlled greenhouse bioassay	Three of 16 commercial products colonised roots (three of nine agricultural, zero of seven home gardening) and one promoted growth; research inocula colonised consistently; pooling with three earlier surveys, the authors report that 85% of 64 products were of poor quality and did not colonise roots
Field-scale inoculation outcome
Hijri 2016 [49]	231 field trials on potato over four years, North America and Europe; one inoculant (Rhizophagus irregularis DAOM 197198)	Inoculated versus non-inoculated fields; liquid spore suspension applied to seed pieces	Marketable yield 42.2 versus 38.3 t ha−1 (+3.9 t ha−1, 9.5%); inoculation profitable above +0.67 t ha−1, a threshold reached in almost 79% of trials
Lutz et al., 2023 [12]	54 on-farm maize fields, Switzerland	Field inoculation with soil and microbiome predictors	Growth response ranged from −12% to +40%; within this dataset, 86% of the variation was explained, mainly by soil microbiome indicators, with the abundance of pathogenic fungi the best single predictor

Studies are listed chronologically within each group. Percentages and counts are reported as given by the original authors. The studies were identified by the non-systematic search described in Section 2 and do not constitute a representative sample of the market. The pooled estimate of Boussageon et al. [16] and the meta-analysis of Koziol et al. [45] partly re-use data from other studies listed here. Section 2 explains the descriptive selection of studies, the inclusion of favourable outcomes and the limitations of this purposive summary.

Table 2. Published assessments of commercial AM inoculant quality and of field-scale inoculation outcomes (2004–2025).

Study	Products or Scope	Approach	Principal Quantitative Outcome
Assessments of commercial product quality
Corkidi et al., 2004 [50]	10 commercial products; nursery conditions	Maize bioassay in three growing media	Colonisation ranged from 0 to 50% and depended on the growing medium; colonisation did not enhance plant growth
Wiseman et al., 2009 [51]	Products marketed for woody landscape plants	Greenhouse bioassays with a laboratory culture as positive control	Colonisation rarely exceeded 5% in maize and sorghum, against 38–61% for the laboratory culture and 74% in seed-grown magnolia
Faye et al., 2013 [46]	12 commercial products	Trap-culture propagation followed by a maize bioassay in soil	Five undeclared species were recovered and four declared species produced no spores; three products increased colonisation above an indigenous soil inoculum
Basiru et al., 2021 [24]	68 products from 28 manufacturers (Europe, America, Asia)	Documentary analysis of declared composition and claims	A few species dominated: Rhizophagus irregularis in 39% of products, Funneliformis mosseae in 21% and Claroideoglomus etunicatum (current name Entrophospora etunicata) in 16%; one third single-species; all species assigned to the Glomeraceae by the authors
Salomon et al., 2022 [13]	28 products; Australia, Europe, North America	Greenhouse bioassays of 25 products in sterilised and non-sterilised soil; field trial of three products	Sterilised soil: 84% produced no colonisation and biomass increased in five of 25 treatments; non-sterilised soil: no increase in colonisation, biomass increased in one of 25; laboratory R. irregularis reached 48% and 79% colonisation
Vahter et al., 2023 [14]	11 products from four producers	DNA metabarcoding compared with label declarations	Two products contained none of the declared species; 10 of 11 contained undeclared species
Koziol et al., 2024 [15]	23 inoculants (commercial products, laboratory-grown fungi, field soil)	Spore enumeration, infectivity and crop response with product-borne fertiliser controlled	Propagule counts differed from declared values by up to 100%; insufficient colonisation by commercial products; contamination by Olpidium; growth benefits often absent once fertiliser was controlled
Koziol et al., 2025 [45]	302 inoculant trials (250 with commercial products) from 28 experiments in six publications	Meta-analysis of studies testing five or more commercial products	Commercial products averaged <9% hyphal colonisation, against 41% for laboratory-grown inoculants; crop growth increased by 19% versus 38%; only 12% of commercial trials combined sufficient colonisation with a growth increase
Boussageon et al., 2025 [16]	16 commercial products (nine agricultural, seven home gardening) and seven research inocula	Controlled greenhouse bioassay	Three of 16 commercial products colonised roots (three of nine agricultural, zero of seven home gardening) and one promoted growth; research inocula colonised consistently; pooling with three earlier surveys, the authors report that 85% of 64 products were of poor quality and did not colonise roots
Field-scale inoculation outcome
Hijri 2016 [49]	231 field trials on potato over four years, North America and Europe; one inoculant (Rhizophagus irregularis DAOM 197198)	Inoculated versus non-inoculated fields; liquid spore suspension applied to seed pieces	Marketable yield 42.2 versus 38.3 t ha−1 (+3.9 t ha−1, 9.5%); inoculation profitable above +0.67 t ha−1, a threshold reached in almost 79% of trials
Lutz et al., 2023 [12]	54 on-farm maize fields, Switzerland	Field inoculation with soil and microbiome predictors	Growth response ranged from −12% to +40%; within this dataset, 86% of the variation was explained, mainly by soil microbiome indicators, with the abundance of pathogenic fungi the best single predictor

Studies are listed chronologically within each group. Percentages and counts are reported as given by the original authors. The studies were identified by the non-systematic search described in Section 2 and do not constitute a representative sample of the market. The pooled estimate of Boussageon et al. [16] and the meta-analysis of Koziol et al. [45] partly re-use data from other studies listed here. Section 2 explains the descriptive selection of studies, the inclusion of favourable outcomes and the limitations of this purposive summary.

3. Evidence for Limitations in Commercial AM Fungal Inoculants

3.1. Viability, Infectivity and Root Colonisation

Infectivity is tested by root colonisation at a defined dose under conducive conditions. Salomon et al. [13] assessed 28 products on three continents, including 25 in greenhouse bioassays with reference Rhizophagus irregularis cultures. In sterilised soil, 84% produced no detectable colonisation; reference cultures reached 48% and 79% in the two bioassays.

Boussageon et al. [16] compared 16 commercial products (nine agricultural and seven gardening) with seven research inocula. Three commercial products colonised roots and one promoted growth. Research inocula colonised consistently and often improved growth, possibly reflecting higher propagule density. Pooling this survey with three earlier ones [13,15,46], the authors reported that 85% of the 64 commercial products tested were of poor quality and did not colonise roots; this pooled figure overlaps with the individual surveys and is not an additional independent dataset.

Earlier nursery tests of ten products by Corkidi et al. [50] found 0–50% colonisation, which did not enhance plant growth; in the evaluation of Faye et al. [46], only three of twelve products increased colonisation above that obtained with an indigenous soil inoculum. Neither an absent growth benefit nor unchanged total colonisation demonstrates absence of infective propagules.

A negative bioassay may reflect insufficient abundance, reduced viability, dormancy, host incompatibility or delayed colonisation, rather than universally dead propagules. Reference cultures demonstrate that the assay supports symbiosis, as also shown by Wiseman et al. [51] and Koziol et al. [15] (Table 2), but not equal suitability for every taxon or formulation.

Thus, many of the tested products showed insufficient infectivity at the tested dose and assay duration. Non-random sampling, heterogeneous assays and possible publication bias prevent market-wide extrapolation. Some products succeeded [13,16]; recurrent tested deficiencies do not establish general commercial ineffectiveness.

3.2. Product Identity, Composition and Label Accuracy

Vahter et al. [14] used DNA metabarcoding on 11 inoculants from four producers: two lacked detectable declared taxa and ten contained undeclared taxa. These discrepancies concern detected composition, not viability; non-detection depends on sampling, marker coverage and taxonomic resolution.

After trap-culture propagation of twelve products, Faye et al. [46] recovered five undeclared species, while four declared species produced no detectable spores. Trap cultures favour compatible taxa, so failed recovery does not establish absence from the original product.

Among 23 commercial, laboratory and field-soil inoculants, Koziol et al. [15] reported discrepancies of up to 100% between declared propagule counts and observed spore concentrations. These are not equivalent measures: propagules include colonised roots and hyphae. Defined units and infectivity assays are therefore needed. Olpidium, a genus including plant pathogens, was also detected.

Declared diversity is narrow. In the analysis of Basiru et al. [24], across 68 products from 28 manufacturers, R. irregularis occurred in 39%, Funneliformis mosseae in 21% and Claroideoglomus etunicatum in 16%; one third listed a single species. Claroideoglomus etunicatum is now Entrophospora etunicata (Entrophosporaceae) [52], contrary to the authors’ assignment of all declared species to the Glomeraceae. Restricted commercial diversity raises questions about functional breadth, but does not itself demonstrate poor quality.

Product evaluation therefore requires complementary identity, abundance, infectivity and contaminant checks. Labels should distinguish declared composition, molecular detection, spore counts and functional capacity (Table 2).

3.3. From Product Quality to Agronomic Value: A Diagnostic Sequence

Figure 1 organises the six endpoints defined in Table 1 into a diagnostic sequence separating conformity, biological performance and practical benefit.

These endpoints are related but serve different diagnostic purposes. Viability is necessary for infectivity, while root colonisation demonstrates that infectivity has been expressed under the conditions of the assay. Establishment concerns colonisation of the target crop by the introduced fungus and must be assessed separately with strain-specific methods: initial colonisation does not demonstrate maintenance during the period of need, although transient colonisation may still affect the host. Identity, by contrast, concerns conformity and traceability. An undeclared fungus may colonise roots and benefit the plant, but an unresolved discrepancy between declared and actual composition limits attribution of the response and reproducibility across products or batches.

Propagule concentration, product amount and placement determine the delivered dose, a cross-cutting attribute rather than an endpoint. Counts establish neither viability nor infectivity; failed colonisation cannot distinguish dead from dormant propagules, insufficient dose or assay incompatibility (Section 3.1).

Failure at a later endpoint does not, by itself, identify the underlying cause. An absence of plant growth response may reflect insufficient infective dose, loss of viability, poor host compatibility, failure to establish, competition with resident fungi or environmental conditions that limit the benefit of the symbiosis. Conversely, a measurable biological benefit may be too small or inconsistent to provide agronomic value once application costs and management requirements are considered. Plant biomass is therefore a useful response variable, but cannot alone diagnose inoculant quality; root colonisation demonstrates infectivity under the test conditions, but does not establish effectiveness or agronomic value. Each endpoint requires an appropriate measurement, with complementary observations used to locate the limitation within the sequence.

3.4. Persistent Gaps in Quality Assurance

Gianinazzi and Vosátka [19] called for accepted quality standards and registration encouraging compliance more than two decades ago. Vosátka et al. [18] subsequently highlighted the need for inexpensive, reproducible purity tests and strain traceability. Salomon et al. [17] proposed composition criteria and standardised bioassays, but recurring deficiencies show that recommendations have not ensured consistency.

Jurisdictions classify products as biostimulants, biofertilisers or plant-protection products according to use and claims [20]. Some require biological criteria or evidence of effects, but requirements remain heterogeneous; registration cannot signify equivalent identity, infectivity or value across jurisdictions.

Specifications and claims therefore need complementary measurements, acceptance rules and batch-consistency checks. Section 6.6 distinguishes release criteria from evidence of deployment performance.

4. Five Bottlenecks in the AM Fungal Inoculant Pipeline

Manufacturing defects occur within a longer pipeline connecting natural communities to deployment. Successive stages may restrict diversity, modify traits or reduce infectivity, but may also maintain or amplify useful fungi. The question is whether retained properties meet application requirements.

Five interacting bottlenecks are distinguished (Figure 2): obligate biotrophy, cultivation of field-derived fungi, scalable production, formulation and storage, and field establishment. Biotrophy constrains cultivation and production across stages; the others describe transitions. Regulation, cost, application and host genotype are cross-cutting influences (Section 3.4 and Section 6.7). This literature-derived classification is neither exhaustive nor systematic.

Figure 2. The five-bottleneck pipeline in AM fungal inoculant development. Attrition may occur at five bottlenecks (B1–B5) between natural AM fungal diversity and agronomic outcome. The tapering band is schematic: coloured dots denote taxa or functional types (colours have no fixed taxonomic meaning), open dots denote propagules that are no longer viable or infective, and dot numbers are not quantitative. For each bottleneck the principal constraint, its consequence and the level of supporting evidence are indicated (Section 4.1, Section 4.2, Section 4.3, Section 4.4 and Section 4.5). The bottlenecks are interconnected and may require iterative optimisation.

The evidence supporting these bottlenecks varies in strength and scope. Host dependence, differences among production systems, storage-associated losses of infectivity and variable field establishment have experimental support. However, evidence for these individual constraints does not establish that diversity or function declines cumulatively throughout the pipeline. Selective recovery during cultivation provides indirect support for a cultivation filter, while directional changes in fungal traits during prolonged culture, and their consequences for field performance, remain hypotheses to be tested (Section 4.2). Figure 2 distinguishes these levels of evidence and identifies where further validation is needed.

4.1. Bottleneck 1: Obligate Biotrophy

Under natural conditions, AM fungi require living hosts for sustained growth and reproduction. Examined genomes lack conventional multidomain cytosolic fatty-acid synthase and have reduced plant-cell-wall-degrading repertoires [53,54,55,56,57,58]. Host lipids are transferred to fungi; disruption of plant lipid biosynthesis or export impairs arbuscules and function [59,60,61]. Multiplication therefore requires compatible hosts and conditions for both partners.

Defined nutrition and signals can partially bypass this dependence. Myristate supports asymbiotic R. irregularis growth [38], and specific branched-chain or unsaturated fatty acids stimulate secondary spores [39]. Strigolactone, jasmonate, myristate and organic nitrogen yielded infective Rhizophagus clarus HR1 spores without a host [40], opening production possibilities [62].

However, successful growth or sporulation in selected fungi does not establish a broadly applicable production platform. Generalisation across diverse taxa and field-derived isolates, reproducibility over successive production cycles, and the quality and performance of the resulting propagules remain to be established [38,40]. Differences between asymbiotically and symbiotically produced spores also require evaluation rather than being assumed to indicate equivalent or inferior quality. Asymbiotic culture should therefore be viewed as a promising route for reducing host dependence, while host-associated cultivation remains the established basis of AM fungal multiplication.

4.2. Bottleneck 2: Cultivation Filters and Potential Trait Changes

Only a restricted subset of AM taxa is routinely propagated monoxenically; initiating field-derived cultures can be harder than maintaining them [63,64]. Isolation determines the candidates available for selection.

Cultivation may favour readily germinating fungi that colonise and reproduce on the selected host. The commercial dominance of a few species [24] is consistent with this filter, but production yield, formulation and commercial choices provide alternative explanations.

Cultivation-associated trait change is suggested by two R. irregularis studies. Across 14 isolates with zero to >80 in vitro generations, spore dormancy was strongly associated with culture history, whereas the high variation in other asymbiotic traits was unrelated to time in vitro [41]. Among 11 isolates, longer history correlated with greater sporulation and lower host shoot phosphorus [43].

However, both studies compared different isolates rather than following the same lineages through successive generations, and neither assessed field performance. They therefore cannot separate effects of cultivation from pre-existing differences among isolates, demonstrate heritable change within a lineage or establish a general trade-off between production yield and agronomic benefit. Selection during repeated propagation, physiological responses to culture conditions and persistent differences among isolates remain alternative explanations. Systematic domestication towards a ruderal phenotype is consequently a hypothesis rather than a demonstrated outcome.

The potential functional consequences can be considered using the competitor–stress tolerator–ruderal framework adapted to AM fungi [42]. Broad differences in colonisation and biomass allocation have been described, with Glomeraceae often showing rapid intraradical colonisation and Gigasporaceae greater investment in extraradical mycelium [42]. Communities with contrasting strategies can differ in their effects on host nutrient acquisition [65]. Nevertheless, these classifications describe tendencies and cannot substitute for measurements of individual isolates under relevant conditions [66,67]. The key question is therefore which traits are retained, excluded or modified during isolation and propagation, and whether those traits support performance in the intended recipient system.

4.3. Bottleneck 3: Scalable Production

Amplification must provide sufficient infective inoculum at acceptable cost while maintaining identity and quality. Systems differ in hosts, microbial control, yield, labour, infrastructure and isolate compatibility (Table 3); scalability concerns reproducible usable inoculum, not spore yield alone.

Pot and trap cultures accommodate field-derived fungi difficult to culture in vitro [32,68], but may co-propagate organisms from starting material, roots or substrate. Characterising this microbiota is generally harder than characterising that of aseptic root-organ systems.

On-farm systems reduce infrastructure and support decentralised access. Substrates and crop residues require validation; Senegalese work identified substrate sterilisation and irrigation-water salinity as quality determinants [69]. Affordability does not remove verification requirements.

Aeroponics facilitates access to roots and propagule recovery without solid substrate [25]. Suitability depends on fungus–host compatibility, environmental control and operational resources; greater control does not guarantee purity or infectivity.

Monoxenic root-organ culture propagates defined fungi with excised, usually transformed roots under aseptic conditions [35,36,70]. It improves compositional control, subject to contaminant checks, but many field fungi remain difficult to initiate and maintain.

Table 3. Comparison of arbuscular mycorrhizal fungal production systems against the criteria that determine whether a field isolate can be converted into a defined inoculant.

Production System	Freedom from Contaminants	Propagule Yield	Scalability and Cost	Tractability for Newly Isolated Field Strains	Evidence of Culture-Induced Trait Change
Pot and trap culture on a living host [19,32,68]	Low; other soil and root organisms are co-propagated	Moderate	Moderate cost; demanding in space and time	High; the reference route for strains that do not enter in vitro culture	Not quantified
On-farm substrate systems using crop residues [69]	Low	Moderate to high	Low cost; minimal infrastructure; decentralised	Moderate; sensitive to substrate sterilisation and irrigation-water salinity	Not quantified
Aeroponic and hydroponic culture [25,71]	Intermediate	High spore density per unit root mass	Moderate; requires controlled facilities	Low to moderate	Not quantified
Monoxenic root-organ culture [35,36,37,63,70]	High; composition controlled, to be verified for each culture	High per plate; low per unit floor area	Limited by throughput and labour	Low initially; improved by root-fragment initiation and successive subculture	In one species, time in culture was associated with spore dormancy [41] and with higher spore production and lower host shoot phosphorus [43]; drift within a lineage not demonstrated
Asymbiotic culture with fatty acids and phytohormones [38,39,40]	Potentially high under aseptic conditions; absence of contaminants must be verified	Currently limited	Not yet scalable	Very low; demonstrated for few isolates	Unknown; asymbiotic propagules may differ from symbiotic ones

Numbers in brackets after each production system are the key sources. Qualitative ratings summarise the cited literature and are intended for comparison among systems rather than as absolute values.

Table 3. Comparison of arbuscular mycorrhizal fungal production systems against the criteria that determine whether a field isolate can be converted into a defined inoculant.

Production System	Freedom from Contaminants	Propagule Yield	Scalability and Cost	Tractability for Newly Isolated Field Strains	Evidence of Culture-Induced Trait Change
Pot and trap culture on a living host [19,32,68]	Low; other soil and root organisms are co-propagated	Moderate	Moderate cost; demanding in space and time	High; the reference route for strains that do not enter in vitro culture	Not quantified
On-farm substrate systems using crop residues [69]	Low	Moderate to high	Low cost; minimal infrastructure; decentralised	Moderate; sensitive to substrate sterilisation and irrigation-water salinity	Not quantified
Aeroponic and hydroponic culture [25,71]	Intermediate	High spore density per unit root mass	Moderate; requires controlled facilities	Low to moderate	Not quantified
Monoxenic root-organ culture [35,36,37,63,70]	High; composition controlled, to be verified for each culture	High per plate; low per unit floor area	Limited by throughput and labour	Low initially; improved by root-fragment initiation and successive subculture	In one species, time in culture was associated with spore dormancy [41] and with higher spore production and lower host shoot phosphorus [43]; drift within a lineage not demonstrated
Asymbiotic culture with fatty acids and phytohormones [38,39,40]	Potentially high under aseptic conditions; absence of contaminants must be verified	Currently limited	Not yet scalable	Very low; demonstrated for few isolates	Unknown; asymbiotic propagules may differ from symbiotic ones

Numbers in brackets after each production system are the key sources. Qualitative ratings summarise the cited literature and are intended for comparison among systems rather than as absolute values.

For an R. irregularis isolate from the date-palm rhizosphere, cultures initiated from colonised root fragments yielded 1414 ± 65 spores per plate at 16 weeks and outperformed spore-initiated cultures in propagule viability, root colonisation and root growth [37]. The in vitro-produced spores also colonised a host plant more extensively than trap-culture spores in the tested assay. Standardised comparisons of roots and isolates are available [63].

Such results support root-organ culture as an option for producing defined inocula from compatible, functionally selected isolates. They do not establish equivalent performance across fungi or demonstrate industrial scalability: throughput, labour, costs and batch consistency require assessment at the intended production scale. A diversity-oriented pipeline should therefore retain multiple production routes and select among them according to isolate compatibility and product requirements. Requiring successful monoxenic cultivation for every candidate would risk excluding useful fungi before their application potential has been evaluated.

4.4. Bottleneck 4: Formulation, Storage and Delivery

Formulation, storage and delivery must retain biological quality until propagules reach recipient roots in a suitable form and dose.

Spores, colonised roots and hyphae can be carried in solid or liquid formulations [71]. Suitability depends on isolate, propagule, carrier and storage conditions. Refrigeration prolonged infectivity in appropriate carriers [25]; formulations preserving monoxenic propagules have been described [72]. These are validated combinations, not universal formulations.

Shelf life is the period of compliance with biological criteria under declared conditions. Recognisable propagules may no longer be functional. Validation requires measurements through expiry; direct viability tests complement, but cannot replace, infectivity testing at the intended dose.

Preparation, placement and application compatibility must translate packaged quality into an adequate infective dose at the roots.

4.5. Bottleneck 5: Establishment in the Field

Field establishment depends on host, soil, management and resident microorganisms; successful colonisation under controlled conditions guarantees neither crop establishment nor maintenance for the required period.

In a five-year vineyard study, inoculum failed to establish despite a priority advantage; high phosphorus was a possible contributor [27]. Reviewed introductions persisted for months to years, with declining abundance in approximately 60% and apparent exclusion in 30% of assessed cases [28]. These are case-series proportions, not general probabilities.

Colonisation after two vineyard seasons yielded no biomass gain [48]; wheat colonisation increased without additional biomass or mycorrhiza-acquired nutrients [47]. Introduced strains may also replace residents without changing total colonisation [26]. Strain-discriminating methods, baselines and repeated sampling are necessary.

Phosphorus affects both establishment and benefit. High availability can suppress host symbiosis programmes [73]; temperate cereals benefited more under reduced fertilisation and nutrient limitation [74]. Yet in Swiss maize fields, establishment of the introduced strain increased with soil phosphorus, which was associated with fewer native fungi, while benefits declined with phosphorus fertilisation [26]. Phosphorus alone predicts neither endpoint.

Residents may compete with, coexist with or complement introduced fungi. Introductions can suppress, stimulate or minimally alter resident AM fungal communities [28,29,75]. In a growth-chamber experiment with grassland soil, some commercial inocula reduced the growth of native species and increased that of invasive species [75], a context-specific ecological concern.

Field establishment should consequently be assessed as an endpoint in its own right. Three temporal outcomes should be distinguished: colonisation of the target crop by the introduced fungus, maintenance during the period when its function is required, and persistence after harvest or between seasons. Activity during a critical growth period may suffice for an annual crop, whereas longer maintenance may be desirable in perennial crops or restoration. Persistence within the treated system and spread beyond it require separate assessment, particularly where introduction into non-target habitats is a concern (Section 5.4).

5. Beyond “Native Versus Exotic”: Towards Trait–Environment Matching

Local sourcing offers additional candidates, but proximity demonstrates neither adaptation nor superiority. Suitability concerns fungal traits matched to the recipient host, environment and function.

Native/non-native denote regional biogeographic status; local denotes proximity; sympatric denotes shared locality. Local adaptation requires comparative performance evidence (Table 1). Fungal growth or sporulation advantages need not benefit hosts.

Local-versus-foreign comparisons test origins in one environment; home-versus-away comparisons test a genotype across environments [30]. Home advantage may reflect environmental quality; reciprocal comparisons help separate effects.

5.1. Evidence for Home-Field Advantage

A meta-analysis of 1170 comparisons from 139 publications linked plant responses to plant, fungal and soil origins [22]. Fully sympatric combinations tended to outperform fully allopatric ones, but local inocula were not consistently favoured and few experiments manipulated all partners. Provenance effects were context-dependent.

A reciprocal inoculation experiment combined Andropogon gerardii ecotypes, soils and AM fungal communities from phosphorus- and nitrogen-limited grasslands [31]. All-home combinations formed more arbuscules and nutrient responses matched source limitations; associations were more mutualistic in phosphorus-limited soils. Greater fungal hyphal production at home represents a separate performance endpoint.

Sympatric semi-arid grass–soil-biota combinations formed more mycorrhizal structures and improved drought performance, particularly under extreme drying [76]. Associated soil organisms prevent attribution solely to AM fungi.

Native inocula from late-successional grasslands improved prairie establishment and diversity over three to four growing seasons [77,78]. Conspecific-host desert communities improved biomass, photosynthesis and foliar nutrients in one of two shrubs [79]. Benefits alone establish neither local adaptation nor superiority over foreign inocula.

Taken together, the evidence supports home-field advantage in some plant–fungus–soil combinations, with outcomes depending on resource availability, host identity and inoculum composition. Demonstrating its fungal basis requires comparisons that distinguish provenance effects from environmental quality and contributions of other soil organisms. Identified isolates, or characterised AM fungal communities with appropriate microbial controls, can support such tests. Local origin is therefore a useful basis for candidate sourcing, while suitability must be demonstrated for the target environment and the intended performance endpoint.

5.2. Evidence Against a Universal Native Advantage

A foreign-isolate mixture outperformed a 14-species local consortium in alfalfa forage yield, nitrogen and phosphorus over two years; introduced strains persisted in roots [80]. In date palm under salinity, a non-native AM fungal complex and an isolate derived from it also outperformed their native counterparts [81]. Both comparisons confound provenance with fungal identity and composition.

Barley yield responses to an indigenous consortium varied with cultivar and year, and developmental stage affected root fungal community structure [82]. Local origin did not ensure a uniform response.

Arable-field communities promoted less growth and greater below-ground carbon transfer than neighbouring grassland communities, but increased phosphorus accumulation and nitrogen transfer [83]. Management history thus affects costs and nutrient delivery, rather than producing uniformly inferior function.

Provenance should guide sourcing, not stand alone as a functional-quality criterion.

5.3. A Hypothesis-Generating Framework for Trait–Environment Matching

Three processes reconcile contrasting outcomes. Adaptation depends on source–recipient correspondence, but chronic exposure alone demonstrates neither adaptation nor host benefit [31,76]. Management shapes local community function [42,83]. Ecological opportunity depends on residents and priority effects [27,28]; depletion may reduce competition while the conditions causing it still inhibit inoculation.

Figure 3 combines abiotic/resource-constraint intensity with resident-community condition, from intact to depleted or functionally disrupted. Tillage, fertilisation, fallow and cropping history drive that condition rather than measure it. The framework generates hypotheses, not calibrated predictions.

Figure 3. A hypothesis-generating framework for trait–environment matching. The figure is not a predictive model fitted to data; it organises four hypotheses (H1–H4; Table 4). The potential value of inoculation (background shading) is hypothesised to vary with the intensity of abiotic or resource constraint (horizontal axis) and with the depletion or functional disruption of the resident AM fungal community (vertical axis), of which tillage, fertilisation, fallow and cropping history are drivers. The requirement for trait–environment matching is expected to increase with constraint, and the opportunity for establishment with depletion of the resident community, provided that soil conditions and host are compatible. Biotic constraints such as pathogen pressure are not represented. Host genotype and the resident microbiome modulate outcomes in all quadrants.

These dimensions describe the potential need for an additional fungal function and the ecological context in which an inoculant must operate. Both require qualification. The type of environmental constraint matters as well as its intensity, because traits supporting performance under drought may differ from those relevant to phosphorus limitation or salinity. Similarly, resident abundance, infective potential, composition and contribution to plant performance need not vary together. An abundant community may provide limited benefit under the target conditions, while a less abundant community may remain functionally effective. Measurements of resident inoculum potential and composition should therefore be complemented, where feasible, by assessments of the functions relevant to the intended application.

Ecological opportunity is conditional on a compatible host and environment. Depletion of resident fungi may reduce competition, but high phosphorus availability or unsuitable host conditions may still prevent effective symbiosis. Phosphorus also illustrates the interaction between the two dimensions: its availability affects plant nutrient limitation, host regulation of colonisation and resident AM fungal communities [26,73]. Host genotype and responsiveness, together with the wider soil microbiome, further modify outcomes throughout the framework [8,12]. The axes should consequently be interpreted as interacting dimensions rather than independent determinants.

Two limitations define the scope of this framework. First, it represents abiotic and resource constraints more directly than biotic constraints. Aphid herbivory can also alter carbon–nutrient exchange within the symbiosis [34]. In Swiss maize fields, pathogenic fungal abundance predicted inoculation responses better than nutrient availability [12]. A site with weak abiotic limitation but high pathogen pressure can be located on the proposed axes, yet its potential response would remain incompletely represented. Second, neither axis has a common quantitative scale across studies, and thresholds separating the quadrants have not been established. The quadrants therefore identify contrasting scenarios for testing candidate traits and inoculation outcomes, rather than fixed categories or validated recommendations.

Table 4 separates establishment from benefit hypotheses. H1 predicts little added benefit in intact, weakly constrained communities already supplying relevant functions. H2 predicts greater benefit from constraint-matched fungi if established in intact, strongly constrained systems, with competition restricting establishment. H3 predicts that independently measured colonisation traits explain establishment better than provenance in depleted, weakly constrained systems. H4 predicts that trait matching explains benefit better than origin in depleted, strongly constrained systems. All require host–soil compatibility.

Table 4. Predictions derived from the trait–environment matching framework (Figure 3), given separately for establishment of the introduced fungus and for host benefit, with the experimental design and the results that would be inconsistent with them.

Quadrant (Figure 3)	Prediction for Establishment of the Introduced Fungus	Prediction for Host Benefit	Design and Variables	Results Inconsistent with the Prediction, Provided That the Experiment Has Adequate Precision
H1. Weak constraint, intact resident community	Lower than in soils with a depleted resident community	Smaller than in constrained or depleted soils	Inoculated versus non-inoculated plots in fields ranked by inoculum potential and constraint; abundance of the introduced strain (strain-specific markers); yield and nutrient uptake	Effect on yield as large as in constrained or depleted fields
H2. Strong constraint, intact resident community	Restricted by the resident community: lower in non-sterilised than in sterilised soil	Where they establish, fungi with independently verified constraint-matched traits give a larger benefit than unmatched fungi; the advantage is larger with the constraint than without it	Multiple matched and unmatched isolates from local and non-local origins, with and without the constraint, crossed with resident-community treatments; control sterilisation and non-AM microbial effects; measure host benefit and introduced-strain abundance	Matching advantage absent, or no larger under the constraint; establishment comparable between resident-community treatments after accounting for sterilisation effects
H3. Weak constraint, depleted resident community	Colonisation rate explains more of the variation in establishment than provenance	Not predicted by colonisation rate alone; depends on the effectiveness of the established fungus on the host	Fast and slow colonisers of local and non-local origin in soil of low inoculum potential; colonisation rate, abundance of the introduced strain, crop response	Provenance explains as much of the variation in establishment as colonisation rate, or more
H4. Strong constraint, depleted resident community	Greater than under H2, provided that soil conditions and host are compatible with the symbiosis	Trait-matched fungi give a larger benefit than unmatched fungi, whether native or not; the match explains more of the variation in benefit than origin	Factorial comparison of trait-matched and unmatched isolates of native and non-native origin on the target host under the constraint; constraint-specific traits (Table 5)	Similar benefit from matched and unmatched isolates; origin explains more of the variation than the measured traits
All quadrants. Modifiers	Modified by host genotype and by the resident soil microbiome	Modified by the same factors and by biotic constraints such as pathogen pressure, which Figure 3 does not represent	The comparisons above repeated across cultivars and soils; microbiome indicators measured before inoculation	Responses consistent across cultivars and soils

The framework is hypothesis-generating; none of the predictions has been tested in the full design described. Predictions are relative and should be evaluated from effect sizes and their confidence intervals; the absence of a significant difference is not evidence of equivalence. A residual effect of provenance after measured traits are accounted for may reflect traits that were not measured.

Testing these predictions requires comparisons that distinguish provenance from fungal identity, inoculum composition and infective dose. Effects should be assessed using their magnitude and confidence intervals, with predefined equivalence bounds where equivalence is the question: a non-significant difference alone does not demonstrate comparable performance. The date-palm study of Outamamat et al. [81] illustrates the importance of this distinction. Under salinity, the tested native inocula were outperformed by non-native alternatives, but the comparison does not isolate provenance from differences in fungal identity and composition. The appropriate question is therefore not simply whether native AM fungi are better, but which fungal traits improve performance under specified conditions, and whether local sourcing helps identify and retain those traits.

A practical example illustrates how the two axes could be assessed, without reporting a new experiment. Consider date palm grown in saline oasis soils. Quantify the constraint using soil electrical conductivity and irrigation-water salinity, then grow the same cultivar under controlled salinity levels to assess effects on biomass and leaf Na⁺/K⁺ ratio while holding other conditions comparable. Characterise resident abundance by spore density and field-root colonisation, and infective potential with a standardised trap bioassay [33]. To investigate resident function, compare plants in sterilised site soil receiving a characterised resident AM fungal inoculum with plants receiving an equivalent sterilised inoculum; balance carrier and nutrient inputs and add the same AM-propagule-free microbial filtrate to both treatments. Retain intact site soil as an ecological reference. Sterilisation alters soil chemistry, and microbial filtrates do not reproduce the whole non-AM microbiome, so these contrasts provide qualified evidence rather than isolating an AM fungal effect perfectly. Strong salinity effects combined with little resident functional benefit would motivate testing H4, even if spore density were high. Compare multiple salt-tolerant and salt-sensitive isolates from both native and non-native origins, selected using independently measured germination and hyphal-growth responses to NaCl (Table 5) [84], at comparable infective doses. Measure establishment with discriminatory markers and benefit as biomass, ion status and, in subsequent field trials, yield. Comparable designs can address phosphorus limitation or water deficit.

5.4. Ecological Risk and the Rationale for Local Sourcing

Local sourcing also addresses ecological uncertainty. Establishing inocula may persist, colonise non-target hosts or spread off-site [29,75]. Broad host ranges preclude assuming crop confinement, but compatibility alone demonstrates neither dispersal nor off-site establishment.

Outcomes range from failure to persistence with little change or altered resident composition and plant–microbe interactions [29]. Establishment, persistence, spread and ecological effects are separate risk dimensions.

Suitable, documented local isolates may reduce uncertainty from interregional transfers, independently of agronomic superiority. Local origin nevertheless does not guarantee a negligible impact: multiplication, selection and high doses can alter resident interactions. Identity, provenance, suitability and ecological assessment remain necessary [29].

6. A Quality-by-Design Framework for AM Fungal Inoculants

Development should start with intended use, a demonstrated need and the required fungal function. Cultivation, production, formulation and evaluation criteria follow from this objective.

Figure 4 specifies six stages: diagnose biological and practical need (0); source and characterise (1); select on functional performance (2); amplify and verify retained properties (3); validate formulation/storage and release batches (4); and deploy with field evaluation of benefit and costs (5).

Figure 4. A quality-by-design framework for AM fungal inoculants. A diagnostic step (stage 0; Section 6.1) asks whether inoculation is biologically justified. Stages 1–4 (Section 6.2, Section 6.3, Section 6.4, Section 6.5 and Section 6.6) lead from sourcing to release, and stage 5 (Section 6.7) targets deployment to sites where the selected function can be expressed and the expected benefit exceeds the cost. Key actions and the expected output are shown for each stage, with the bottleneck(s) it addresses (Figure 2). Dashed arrows indicate feedback from re-testing of production strains and from field monitoring. The lower panel lists the quality criteria detailed in Table 6.

The framework applies to both single isolates and designed consortia (Section 6.4). It is iterative: evidence from production, storage or field evaluation can prompt revision of candidate selection, product specifications or the intended use. Quality is therefore built into development through explicit objectives and verification at successive stages, rather than assessed only in the finished product.

6.1. Stage 0: Is Inoculation Biologically Justified?

Better products do not establish a need for inoculation where residents supply the required function (H1). Whether AM abundance/diversity should guide crop management is disputed [85,86]. Stage 0 diagnoses additional functional need rather than maximising colonisation.

Diagnosis asks: what resident infectivity is there; what constraint could fungi alleviate; and can the system support benefit? Bioassays estimate soil inoculum potential, not target-crop functional adequacy [33]. Crop responsiveness [8,82], nutrients [73,74], endpoint, habitat capacity and priority effects inform assessment [87].

In a model fitted to 54 Swiss maize fields, soil and microbiome indicators explained 86% of the variation in growth response [12]. Internal cross-validation supports potential but not transferability to other regions, crops or inocula. Diagnosis can combine indicators, bioassays and targeted field comparisons.

Three decisions follow: avoid unsupported inoculation while maintaining residents; promote resident recovery; or consider inoculation. Across 54 field studies, reduced tillage and winter cover cropping each increased colonisation of the following cash crop by approximately 30% (relative increase) [88], without proving recovered function or equivalent yield benefit.

Inoculation becomes a candidate intervention where an identifiable functional deficit could be addressed by selected fungi and the recipient conditions permit their activity. The rationale may be particularly strong where management alone is unlikely to provide the required function within the available time, although inoculation and management changes may also be combined. Stage 0 should therefore specify the intended function, the evidence supporting intervention and the criteria for judging success. Diagnostic costs and the feasibility of alternative interventions enter the economic assessment (Section 6.7).

Evidence of a functional deficit differs from the evidence used to choose an inoculant in its object, its comparison and its timing. Deficit evidence concerns the recipient system: it asks whether the target crop, in its soil and with its resident community, lacks a function that AM fungi could supply. Potential indicators, which do not individually demonstrate a functional deficit, include low inoculum potential of the site soil, low colonisation of the target crop, nutrient limitation, and a host response in site soil to a well-characterised reference inoculum, compared with non-inoculated controls and, where relevant, with management alternatives. Selection evidence concerns candidate fungi: it compares isolates or consortia with one another on the target host under the identified constraint (stage 2; Table 5), and is interpretable only once the deficit and the required function have been specified. A response to a reference inoculum in site soil therefore indicates scope for inoculation but not which fungus to use; conversely, superior performance of a candidate in a screening assay does not show that the target system needs inoculation.

6.2. Stage 1: Source and Characterise

Sample target or comparable hosts and environments exposed to relevant drought, salinity or nutrient limitation. This guides sourcing; adaptation and host benefit require stage-2 testing.

Combine morphology and molecular identification, as applied to Moroccan date-palm isolates [44,68,89,90]. Document resolution; tracking an isolate among conspecific residents may require discriminatory markers beyond taxonomic identification.

Link unique identifiers to reference cultures and collection, isolation and propagation histories. Record locality, host, soils, management and relevant constraints. Source-soil inoculum potential adds community context where feasible, not isolate infectivity [33].

Stage 1 delivers traceable, characterised candidates for functional comparison, production verification and field monitoring.

6.3. Stage 2: Select on Functional Traits Rather than Provenance or Taxonomy Alone

Selection specifies properties, methods and measurement levels: fungus, association, community or product (Table 5). Host drought growth is an association response, not an intrinsic fungal trait. Minimum tests address standardised infectivity, target-host effectiveness under constraint and compatible production of infective propagules.

Document doses and control carriers, nutrients and microorganisms. Tests with and without the constraint distinguish stress-specific from general benefits. Keep production yield separate from host benefit.

Traits vary across and within species. Families differ in colonisation and biomass allocation [91]. Among 24 isolates of four species, mycelial extension and growth responses varied within species, while phosphorus uptake per unit hyphal length was comparatively constant within species but differed among species [92]. Isolates differ in germination and hyphal growth under NaCl [84]; autochthonous isolates of Glomus intraradices and G. mosseae (names as in the original study) supported greater growth of a drought-stressed host than similar allochthonous strains [93], without proving that tolerance derives from origin.

The predictive value of screening measurements must be established rather than assumed. Table 5 therefore identifies candidate selection criteria whose relationships with field establishment and benefit require validation using the same isolates under relevant recipient conditions. A standardised trait framework for AM fungi provides a basis for comparing measurements among laboratories [67], while tests across representative hosts, soils and management conditions can assess the robustness of candidate performance. The geographic origins and relevant environmental histories of the plant, fungus and soil should also be reported, because their combinations can influence the observed response [22].

Retain candidates with defined functional profiles and feasible production, without substituting ease of multiplication for host benefit.

Table 5. Candidate functional traits for inoculant selection, with the level at which each is measured, the target situation and a measurable endpoint.

Candidate Trait	Level of Measurement	Target Situation	Measurable Endpoint	Evidence That the Trait Varies Among Fungi
Rate of root colonisation	Fungus–host association	Depleted resident community; short-cycle crops	Percentage of root length colonised at successive early harvests in a standardised bioassay	Differs among families and isolates [91]
Extraradical hyphal development	Fungus (in symbiosis)	Soils low in available phosphorus	Hyphal length density in soil	Varies within species [92]
Phosphorus transfer to the host	Fungus–host association	Soils low in available phosphorus	Uptake of labelled phosphorus (32P or 33P) from a root-free compartment; plant phosphorus content	Uptake per unit hyphal length differs among species [92]
Tolerance of salinity	Fungus	Saline soils and irrigation water	Spore germination and hyphal growth along a NaCl gradient	Differs among isolates [84]
Function under salinity or water deficit	Fungus–host association	Saline and drought-prone systems	Root colonisation, host biomass and host water or ion status under the controlled constraint	Reported for autochthonous versus allochthonous strains [93], for different inocula under salinity [81] and for sympatric combinations [76]
Carbon cost relative to nutritional return	Fungus–host association	All systems; decisive where growth depressions occur	Indirect indicator: host growth and nutrient response per unit of colonisation. Direct measure: carbon transferred to the fungus, by isotopic labelling	Differs among communities of different management history [83]; reviewed in [11]
Host range and compatibility	Fungus–host association	Rotations and mixed cropping; choice of cultivar	Root colonisation and growth response across the target crop species and cultivars	Depends on host genotype and on the combination of fungus and host [82,94]
Establishment and maintenance in the presence of resident AM fungi	Community	Field use	Abundance of the introduced strain in the target crop during the period of need, with strain-specific markers, in non-sterilised field soil; persistence between seasons recorded separately where relevant to the use	Persistence ranges from months to years among introductions [26,27,28,80]
Propagule production in the intended system	Fungus (in the production system)	Production capacity	Spores or infective propagules per plate or per unit of substrate, and their viability	Depends on culture initiation [37]; associated with time in culture [43]
Tolerance of storage	Product	Formulation and distribution	Infectivity retained after a defined time in the intended carrier and at the intended temperature	Depends on carrier and temperature [25,72]

The correspondences between traits and target situations are candidate selection criteria that remain to be validated against field performance; they are not established predictive relationships. The growth response per unit of colonisation does not measure carbon cost directly; a measurement of carbon transfer is a different level of evidence. Standardised definitions and measurement methods for AM fungal traits are proposed in [67].

Table 5. Candidate functional traits for inoculant selection, with the level at which each is measured, the target situation and a measurable endpoint.

Candidate Trait	Level of Measurement	Target Situation	Measurable Endpoint	Evidence That the Trait Varies Among Fungi
Rate of root colonisation	Fungus–host association	Depleted resident community; short-cycle crops	Percentage of root length colonised at successive early harvests in a standardised bioassay	Differs among families and isolates [91]
Extraradical hyphal development	Fungus (in symbiosis)	Soils low in available phosphorus	Hyphal length density in soil	Varies within species [92]
Phosphorus transfer to the host	Fungus–host association	Soils low in available phosphorus	Uptake of labelled phosphorus (32P or 33P) from a root-free compartment; plant phosphorus content	Uptake per unit hyphal length differs among species [92]
Tolerance of salinity	Fungus	Saline soils and irrigation water	Spore germination and hyphal growth along a NaCl gradient	Differs among isolates [84]
Function under salinity or water deficit	Fungus–host association	Saline and drought-prone systems	Root colonisation, host biomass and host water or ion status under the controlled constraint	Reported for autochthonous versus allochthonous strains [93], for different inocula under salinity [81] and for sympatric combinations [76]
Carbon cost relative to nutritional return	Fungus–host association	All systems; decisive where growth depressions occur	Indirect indicator: host growth and nutrient response per unit of colonisation. Direct measure: carbon transferred to the fungus, by isotopic labelling	Differs among communities of different management history [83]; reviewed in [11]
Host range and compatibility	Fungus–host association	Rotations and mixed cropping; choice of cultivar	Root colonisation and growth response across the target crop species and cultivars	Depends on host genotype and on the combination of fungus and host [82,94]
Establishment and maintenance in the presence of resident AM fungi	Community	Field use	Abundance of the introduced strain in the target crop during the period of need, with strain-specific markers, in non-sterilised field soil; persistence between seasons recorded separately where relevant to the use	Persistence ranges from months to years among introductions [26,27,28,80]
Propagule production in the intended system	Fungus (in the production system)	Production capacity	Spores or infective propagules per plate or per unit of substrate, and their viability	Depends on culture initiation [37]; associated with time in culture [43]
Tolerance of storage	Product	Formulation and distribution	Infectivity retained after a defined time in the intended carrier and at the intended temperature	Depends on carrier and temperature [25,72]

The correspondences between traits and target situations are candidate selection criteria that remain to be validated against field performance; they are not established predictive relationships. The growth response per unit of colonisation does not measure carbon cost directly; a measurement of carbon transfer is a different level of evidence. Standardised definitions and measurement methods for AM fungal traits are proposed in [67].

6.4. Single Strains Versus Designed Consortia

Selection can concern single isolates or defined consortia. Mixtures require additional evidence of compatibility, composition and performance.

Complementarity, not richness alone, motivates consortia. Families differ in biomass allocation [91]; cross-family mixtures improved ultramafic-soil host growth, nutrition and metal tolerance over the single-species inoculations tested [95]. Among three isolates and six hosts, dominance did not necessarily indicate benefit [96].

Yet a meta-analysis of 435 comparisons from controlled experiments found larger biomass responses to single-species than to multi-species inocula [94], without demonstrating competition as the cause. A ten-species trait-diverse community gave weak synergy evidence [65]. Complementarity requires combination-, host- and environment-specific testing.

Table 6. Proposed biological quality criteria for AM fungal inoculants: measurement, decision rule, decision concerned and current status of evidence and standardisation.

Criterion (Endpoint in Figure 1)	Measurement	Decision Rule	Decision Concerned	Current Evidence and Standardisation Needed
Identity and declared composition (1)	Spore morphology and ribosomal DNA sequence of each production strain; strain-specific markers or metabarcoding for multi-strain products	Every declared taxon detected; no undeclared AM fungal taxon above the reporting limit of the assay	Strain qualification; lot release	Undeclared and missing taxa are documented [14,46]. Needed: reference sequences for production strains and an agreed reporting limit
Propagule abundance (dose; a product attribute, not an endpoint)	Direct count of spores and colonised root fragments; most probable number of infective propagules	Measured value not lower than the label claim	Lot release	Counts deviated from label values by up to 100% [15]. The available framework sets no minimum density [17]; methods are reviewed in [97]. Needed: a common definition of propagule and a common counting protocol
Viability (2)	Vital staining or germination assay, where applicable to the propagule type	Proportion of viable propagules reported and used to adjust the recommended dose	Formulation validation; lot release	Rarely measured directly in product audits. No agreed threshold: to be derived for each product from the dose required to pass the infectivity assay
Infectivity (3)	Standardised bioassay on a responsive host in a defined substrate, with non-inoculated and reference-culture controls; colonised root length. For consortia, each member tested separately before blending, or detected in roots with member-specific markers	Colonisation above the non-inoculated control and above a minimum fixed in advance; 20% colonised root length is the value proposed in one reference protocol [17], not a universal standard	Strain qualification; lot release	Most products tested independently produced little or no colonisation [13,16]. Needed: agreed host, substrate, dose, duration and reference culture
Contaminants	Culture-based and molecular screening for a specified list of plant and human pathogens, with a stated limit of detection	Each specified organism below the limit of detection of the stated method, or within the applicable regulatory limit	Lot release	Olpidium was detected in commercial products [15]. In the European Union, the microbiological limits in Annex I, PFC 6(A), concern human-health and hygiene organisms, not plant pathogens [98]. Needed: an agreed, product-relevant list of plant pathogens
Stability and shelf life (2–3)	Viability and infectivity measured over time under the labelled storage conditions	Stability study: both criteria still met at the declared expiry date. Lot control: retained samples of each lot checked against the same criteria	Formulation validation (stability study); lot release (retained samples)	Infectivity declines with time and temperature [25,72]. Expiry to be verified for each isolate and production method [17]
Effectiveness under the target constraint (5)	Host biomass, nutrient uptake or stress indicators relative to non-inoculated and carrier-only controls, in replicated experiments	Effect size relevant to the intended use, reported with its confidence interval; not a significant difference alone	Strain qualification; claim validation	Apparent benefits can disappear when product-borne nutrients are controlled [15]. Specific to host and constraint; a universal threshold is not expected

A criterion is a measurement with a defined method and decision rule; a threshold is an agreed numerical limit. Four decisions are distinguished: qualification of the strain, validation of the formulation, release of the lot and validation of the claim. Numbers in parentheses refer to the performance endpoints in Figure 1. For consortia, identity and infectivity must be shown for each declared member; total colonisation by the mixture is not sufficient.

Table 6. Proposed biological quality criteria for AM fungal inoculants: measurement, decision rule, decision concerned and current status of evidence and standardisation.

Criterion (Endpoint in Figure 1)	Measurement	Decision Rule	Decision Concerned	Current Evidence and Standardisation Needed
Identity and declared composition (1)	Spore morphology and ribosomal DNA sequence of each production strain; strain-specific markers or metabarcoding for multi-strain products	Every declared taxon detected; no undeclared AM fungal taxon above the reporting limit of the assay	Strain qualification; lot release	Undeclared and missing taxa are documented [14,46]. Needed: reference sequences for production strains and an agreed reporting limit
Propagule abundance (dose; a product attribute, not an endpoint)	Direct count of spores and colonised root fragments; most probable number of infective propagules	Measured value not lower than the label claim	Lot release	Counts deviated from label values by up to 100% [15]. The available framework sets no minimum density [17]; methods are reviewed in [97]. Needed: a common definition of propagule and a common counting protocol
Viability (2)	Vital staining or germination assay, where applicable to the propagule type	Proportion of viable propagules reported and used to adjust the recommended dose	Formulation validation; lot release	Rarely measured directly in product audits. No agreed threshold: to be derived for each product from the dose required to pass the infectivity assay
Infectivity (3)	Standardised bioassay on a responsive host in a defined substrate, with non-inoculated and reference-culture controls; colonised root length. For consortia, each member tested separately before blending, or detected in roots with member-specific markers	Colonisation above the non-inoculated control and above a minimum fixed in advance; 20% colonised root length is the value proposed in one reference protocol [17], not a universal standard	Strain qualification; lot release	Most products tested independently produced little or no colonisation [13,16]. Needed: agreed host, substrate, dose, duration and reference culture
Contaminants	Culture-based and molecular screening for a specified list of plant and human pathogens, with a stated limit of detection	Each specified organism below the limit of detection of the stated method, or within the applicable regulatory limit	Lot release	Olpidium was detected in commercial products [15]. In the European Union, the microbiological limits in Annex I, PFC 6(A), concern human-health and hygiene organisms, not plant pathogens [98]. Needed: an agreed, product-relevant list of plant pathogens
Stability and shelf life (2–3)	Viability and infectivity measured over time under the labelled storage conditions	Stability study: both criteria still met at the declared expiry date. Lot control: retained samples of each lot checked against the same criteria	Formulation validation (stability study); lot release (retained samples)	Infectivity declines with time and temperature [25,72]. Expiry to be verified for each isolate and production method [17]
Effectiveness under the target constraint (5)	Host biomass, nutrient uptake or stress indicators relative to non-inoculated and carrier-only controls, in replicated experiments	Effect size relevant to the intended use, reported with its confidence interval; not a significant difference alone	Strain qualification; claim validation	Apparent benefits can disappear when product-borne nutrients are controlled [15]. Specific to host and constraint; a universal threshold is not expected

A criterion is a measurement with a defined method and decision rule; a threshold is an agreed numerical limit. Four decisions are distinguished: qualification of the strain, validation of the formulation, release of the lot and validation of the claim. Numbers in parentheses refer to the performance endpoints in Figure 1. For consortia, identity and infectivity must be shown for each declared member; total colonisation by the mixture is not sufficient.

Co-propagation may change abundances [96]. Separate production and blending control initial composition, not storage survival or root contributions. Specify abundance measures and acceptable variation; product and root proportions are distinct.

Total colonisation does not prove a contribution by every member. Individual assays establish pre-blend infectivity; root-specific detection supports post-application monitoring. Neither alone proves function in mixtures. Verify intended membership given commercial discrepancies [14].

A claim of added benefit should be tested against the best-performing constituent isolate at a comparable total infective dose, with appropriate carrier and nutrient controls. Superior mixture performance supports a consortium advantage under the tested conditions; a stronger claim of synergy additionally requires a defined expectation against which the observed response is compared. Selection should therefore favour the combination that provides a reproducible benefit and can be produced and formulated consistently, rather than the mixture with the greatest number of members.

6.5. Stage 3: Amplify and Verify Retention of Selected Properties

Choose an amplification system that balances production with retained identity, infectivity and selected function (Table 3). Limited drift evidence warrants monitoring without presuming deterioration (Section 4.2). Record subcultures, duration and conditions; retest periodically and after substantial process changes.

Verification should compare the amplified material with the reference performance established during selection, using standardised hosts, inoculum doses and assay conditions. This allows changes in infectivity or host benefit to be distinguished from variation attributable to the test environment. Acceptance criteria should focus on properties relevant to the intended use rather than requiring every measured trait to remain unchanged. For consortia, amplification must also address retention of the intended members and acceptable variation in composition (Section 6.4).

Compatible isolates benefit from monoxenic control with contaminant checks. Root fragments can improve initiation [37]; comparison protocols exist [63]. These findings are candidate-specific.

Monoxenic culture is optional. Pot/trap systems serve recalcitrant fungi [32,68]; on-farm multiplication may suit decentralised needs [69]. Every route requires identity, infectivity, consistency and unintended-organism checks. Stage 3 supplies sufficient qualified inoculum.

6.6. Stage 4: Formulate, Store and Release Against Explicit Biological Criteria

DNA or propagule detection alone cannot justify release. Table 6 separates qualification, formulation/storage validation, batch release and claim substantiation, linking each to measurements and acceptance rules.

Qualification establishes identity, infectivity and function. Formulation validation verifies their retention in carrier, packaging and storage through shelf life [25,72]. Release checks batch specifications; claims require intended-use evidence. Routine release need not repeat full functional/field validation but remains linked to it.

Criteria require method, unit, sampling and acceptance rules. Limits may be regulatory, protocol-based or product-specific. For their reference bioassay, Salomon et al. [17] propose a positive growth response and at least 20% colonised root length; this is not a universal quality or field threshold, and the framework sets no minimum propagule density and leaves expiry dates to be verified by producers.

Counts, infection units, most-probable-number estimates and molecular/lipid quantification measure different properties [97]. Dose–response, application rate and storage losses inform infectivity specifications. Release criteria should support compliance until expiry under validated conditions.

Contaminant criteria must specify the organisms sought, the sampling procedure, the analytical method and the detection limit. A non-detection result cannot establish the absence of all unintended organisms. Under Regulation (EU) 2019/1009, mycorrhizal fungi are permitted under component material category CMC 7. Annex I, PFC 6(A), specifies microbiological limits for microbial plant biostimulants, including Salmonella spp., Escherichia coli, Listeria monocytogenes, Vibrio spp., Shigella spp., Staphylococcus aureus and Enterococcaceae, and applicable plate-count requirements [98]. These are human-health and hygiene requirements; that microbiological table does not specify plant-pathogen limits. Screening for plant pathogens, such as Olpidium [15], and other organisms relevant to the source material, production system and intended use should therefore be defined separately in the product specification.

Effectiveness claims need defined hosts, constraints and management, with practically relevant effects and adequate precision rather than significance alone. Release connects quality, storage, dose and substantiated use.

6.7. Stage 5: Deploy Where Function Can Be Expressed and Benefit Exceeds Cost

Deployment combines verified products with suitable sites. Lutz et al. [12] illustrate targeting through soil/microbiome indicators, subject to validation for the crop, inoculant and region.

Agronomic value is distinct from biological effectiveness. From the grower’s perspective, the incremental net return is the additional revenue and any input-cost savings attributable to inoculation, minus the additional costs relative to the reference practice. These costs include the product, application, any diagnosis or monitoring required in routine use, and associated management changes. Costs incurred during research and product validation should be distinguished from those borne directly by the user. Benefits that cannot be reliably monetised should be reported separately rather than assigned an unsupported financial value.

Potato inoculation at approximately 71 spores per seed piece broke even at +0.67 t ha⁻¹, attained in almost 79% of 231 trials [49]. These historical prices/costs do not transfer universally. Swiss maize responses [12] likewise require marketable output, price and cost data for profitability.

The dose must deliver benefit, not merely colonisation, and it determines the cost per hectare. Seed coating or other root-proximal delivery may reduce the quantities required, if validated [18,24,99]. Fertilisation/tillage affect compatibility and response [74,88]; refrigeration adds logistics [25]. Control storage losses through specifications rather than routinely increasing dose.

Economic assessment must also account for variation among sites, years and cultivars [12,82]. Mean net return alone does not describe the probability or magnitude of a loss in a particular field and season. Trials should therefore report application dose and method, relevant costs, and the distribution of biological and economic responses. Sensitivity analyses can show how conclusions change with product price, crop value and management costs. Comparisons should include feasible alternatives, such as management of resident fungi and on-farm multiplication [69], with their respective quality-control requirements included. Stage 5 thereby defines where deployment is justified and provides feedback for revising product specifications and recommendations.

6.8. Relation to Existing Quality Frameworks

Table 7 compares the emphasis of the proposed frameworks with Salomon et al. [17] and recent independent assessments [15,16,45]. Existing quality requirements remain part of strain qualification and lot release (Table 6). The proposed integration links diagnosis of need, explicit establishment and benefit predictions, verification across stages, and evaluation of deployment and net return.

Table 7. Comparison of the elements addressed by an existing quality management framework, by recent independent product assessments and by the frameworks proposed in this review.

Element	Salomon et al., 2022 [17]	Koziol et al., 2024, 2025 [15,45]; Boussageon et al., 2025 [16]	This Review
Primary purpose	Minimum product quality and a reference bioassay	Independent quantification of the quality and performance of tested products	Integrated diagnostic and development framework from assessment of need to deployment
Starting point	Commercial inoculant quality	Finished product	Recipient need and required function before candidate selection (stage 0)
Justification of inoculation	Management alternatives discussed	Not a focus	Explicit decision among no inoculation, resident recovery or inoculation (Section 6.1)
Performance endpoints	Composition, colonisation and growth	Root colonisation and plant growth; composition, propagule counts and growth with product-borne fertiliser controlled [15]; pooled or meta-analytic summaries [16,45]	Six endpoints separating conformity, biological function and value (Figure 1)
Selection of fungi	Generalist or application-specific fungi	Commercial versus research inocula	Separate establishment and benefit predictions; measured traits distinguished from provenance (Table 4)
Production and storage	Storage, packaging and expiry guidance	Product quality measured at testing; production history not experimentally isolated	Linked verification of selected function after amplification and storage (stages 3–4)
Release decisions	Reference bioassay criteria	Not applicable	Distinct strain, formulation, lot and claim decisions; criteria versus thresholds (Table 6)
Consortia	Declared composition	Mixtures assessed as products; member contributions not resolved	Member verification and comparison with best constituent (Section 6.4)
Deployment and economic value	Field prediction and viability identified as research needs	Mainly controlled product assessments, not full deployment economics	Site targeting and distribution of net returns versus alternatives (Section 6.7)
Nature of the output	Quality recommendations and research priorities	Empirical evidence of deficiencies among tested products	Integrated hypotheses and development decisions requiring validation

This comparison describes the main emphasis of each contribution, not an exhaustive inventory. Elements listed as extensions may be discussed or identified as research needs in earlier work. The earlier product assessments in Table 2 share the empirical assessment focus shown in the third column.

7. Conclusions

Reliable arbuscular mycorrhizal fungal inoculants require both verifiable product quality and a recipient system in which the introduced fungi can provide a useful function. Independent assessments reveal recurrent composition and infectivity deficiencies among tested products, but the available evidence cannot establish their prevalence across the commercial market. At the same time, successful root colonisation does not necessarily translate into host benefit or agronomic value. These findings distinguish product deficiencies from ecological mismatch and show why neither propagule abundance nor geographic origin alone is sufficient to guide inoculant development.

The proposed quality-by-design and trait–environment matching frameworks connect these challenges by starting with the need for inoculation and the function required, then linking candidate selection to verification during production, storage and deployment. Their value lies in making the underlying assumptions testable. Inoculation is expected to provide little additional benefit when resident fungi already supply the required function, whereas fungi with traits matched to a strong environmental constraint should outperform unmatched candidates if they establish. In depleted communities under weak constraint, colonisation traits are expected to explain establishment better than provenance. Following the same isolates through production and storage can further test whether retained infectivity and host benefit predict subsequent performance under the intended conditions. These relationships remain hypotheses requiring experimental validation.

Future research should therefore combine reproducible quality measurements with comparisons that distinguish fungal identity, functional traits, provenance and infective dose. Traceable isolates and defined consortia should be followed from screening to field deployment, with mixtures evaluated against their best-performing constituents and inoculation compared with feasible management alternatives. Trials across representative soils, hosts and seasons should measure establishment separately from plant benefit and assess whether the resulting gains justify application costs and ecological exposure. Such evidence would support decisions about whether to inoculate, which fungi to use and where their functions can add value, while revealing when support for resident communities is the more appropriate intervention.