Arbuscular Mycorrhizal Fungi in Common Bean Roots: Agricultural Impact and Environmental Influence

Abstract

Although many plant families are predominantly mycorrhizal, few symbiotic relationships between plants and arbuscular mycorrhizal fungi (AMF) have been thoroughly studied. Mycorrhized plants tend to exhibit greater tolerance to soil-borne pathogens and enhanced plant defence. Legumes, including common bean (Phaseolus vulgaris L.), are essential sources of protein globally. To improve common bean productivity, identifying efficient native microsymbionts is crucial. This study aimed to identify native AMF associated with common bean roots that could act as biostimulants and protect against soil diseases under varying environmental conditions. Agronomic trials were conducted at MBG-CSIC (Pontevedra, Spain) in 2021 and 2022, testing combinations of nitrogen fertilization, Burkholderia alba, Trichoderma harzianum, and a control. Traits such as nodulation, biomass, plant vigor, disease severity, nutrient content, and yield were evaluated. Four AMF species across three genera were identified. No consistent pattern was observed in AMF influence on agronomic traits. However, reduced mycorrhization in 2022 was associated with decreased nodulation, likely due to higher temperatures. Surprisingly, yields were higher in 2022 despite lower colonization. These findings suggest that intelligent use of AMF could reduce pesticide use, enhance sustainability, and promote healthier food systems. Continued research and conservation efforts are essential to optimize their benefits in legume production.

1. Introduction

Soil is a non-renewable resource and acts as one of the vital life-supporting components of the biosphere. Beneficial microorganisms play a fundamental role, especially the arbuscular mycorrhizal fungi (AMF), nitrogen-fixing microorganisms, and plant growth-promoting rhizobacteria (PGPR) [1,2]. These fungi not only promote plant development but also enhance resistance to soil-borne diseases [3]. Mycorrhizal fungi form a mutualistic relationship with the roots of many plant species. AMF is formed primarily by fungi belonging to the phylum Glomeromycota, which penetrate the root cells of most vascular plants, forming branching structures called arbuscules. These fungi enhance plant nutrient uptake, particularly phosphorus, and contribute to soil aggregation and stability. Relatively few of the mycorrhizal relationships between plant species and fungi have been examined to date, but 95% of the plant families are predominantly mycorrhizal, either in the sense that most of their species associate beneficially with mycorrhizae or are absolutely dependent on mycorrhizae. Mycorrhized plants are often tolerant to diseases caused by microbial soil-borne pathogens and can assist in plant defence both above and belowground, since mycorrhizae have been found to excrete enzymes that are toxic to soil pathogens of crops. By facilitating nutrient uptake, enhancing plant resilience, and influencing ecosystem dynamics, mycorrhizae contribute significantly to plant health and agrosystems’ functions. The resident mycorrhizae benefit from a share of the sugars and carbon produced during photosynthesis, while the plant effectively accesses water and other nutrients, such as nitrogen and phosphorus, which are crucial to promote the plant’s growth under different agrosystems [4].

Today, as in the past, common global problems are arising, such as feeding a rapidly growing human population. Malthus [5] was among the first to highlight the problem of population growth outpacing the global capacity to produce enough food. His predictions were not fulfilled due to the technological advances of the 19th and 20th centuries and the discovery of new resources. In agriculture, the ongoing challenge is to ensure safe and nutritious food for a growing population, pushing scientists to develop sustainable, low-impact solutions. To meet rising food demand, the use of synthetic fertilizers has increased; over a quarter of the global population relies on them to boost crop yields [6].

Legumes provide a rich source of protein for human nutrition. The common bean (Phaseolus vulgaris L.) is among the three most important legumes worldwide, along with soybean (Glycine max) and peanut (Arachis hypogaea) [7]. In Europe, a large area of snap and dry beans is cultivated, producing 1,500,000 mg in 2020, with 166,000 mg produced in Spain [8,9].

An increase in common bean production is crucial to extend the identification of new competitive and efficient bean microsymbionts (µ-symbionts). Cultivation of legumes can save huge amounts of environment-polluting nitrogen fertilizers, decreasing N₂O emissions as well as protecting ground and surface water resources from toxicity while improving human health and soil fertility, contributing globally to the welfare of mankind as a whole. In this context, up to 20 rhizobial species, including alpha- and beta-Proteobacteria, have been described as bean µ-symbionts. The use of N-fixing bacteria is currently in use, especially in soybeans [10,11], with Bradyrhizobium being the most common genus [12]. The use of other nitrogen-fixing bacteria is limited due to their host specificity, making it essential to develop versatile strains that can nodulate various legume species.

Biostimulants are gaining prominence in agriculture for their ability to improve crop productivity. They stimulate natural plant processes, promoting growth and stress tolerance with lower reliance on chemical fertilizers and pesticides [13,14,15,16]. There is also a trend towards the use of growth-promoting microbiota, including Trichoderma, mycorrhizae, and even their combinations [17], but not including N-fixing bacteria. Mycorrhizal fungi, nitrogen-fixing bacteria, and phosphorus solubilizers act synergistically to improve crop yield, plant health, and soil organic matter. These microorganisms basically work on the supply of nitrogen and phosphorus to the plant. Other important functions are more abundant root development and a protective effect against fungal root diseases. Previous studies have mostly focused on single microorganisms or factors, with few reports on the effects of microbial interactions.

The initial purpose of this research was to study the effect of three factors (nitrogen fertilization, inoculation with Trichoderma harzianum, and inoculation with Burkholderia alba) on the agricultural performance of two common bean genotypes (Galaica and Matterhorn) in different years. There is insufficient research on the local adaptation of native AMF to improve yield and stress tolerance in leguminous crops under different environmental conditions. During the development of the experiment, the opportunity arose to study the native AMF of the MBG-CSIC soils to assess their effect on the agricultural performance of bean plants and the possible interaction with the factors studied.

This study aimed to identify native AMF associated with common bean roots that act as biostimulants and offer protection against soil pathogens, promoting sustainable production. Additionally, the environmental factors that affect root colonization were studied.

2. Materials and Methods

2.1. Bean Genotypes

The Galaica and Matterhorn varieties of common bean, from the germplasm collection of the Biology of Agrosystems Group at the MBG-CSIC, were used in the field trials. Galaica, released in 2018 (plant variety 20170135, registered at the Spanish Office for Plant Varieties), belongs to the international market class ‘Favada’ [18] of the Andean genetic pool. It has indeterminate climbing habit (type IV, [19]), very large white seeds, and very late vegetative cycle. Matterhorn [20] is derived from the cross Alpine x 90012. It belongs to the ‘Great Northern’ international market class of the Mesoamerican genetic pool and was released in 1998. It has an upright type II growth habit [19] and is also disease-resistant with high-yield characteristics, along with early to mid-season maturity and medium-sized seeds (Figure 1).

2.2. Field Trials and Traits Evaluated

The agronomic trials were conducted in 2021 and 2022 at the experimental field of the MBG-CSIC (42°4′ N, 8°34′ W, 85 masl). The texture of the soil is sandy loam (62.0% sand), 26.6% silt and 11.4% clay, with a pH of 5.7 (H₂O 1:2:5), organic matter content of 4.1%, available P of 140.8 ppm (Olsen method), assimilable K of 156.4 ppm (ClNH displacement), and exchangeable mg of 51.1 ppm (ClNH displacement). The seeds of Galaica and Matterhorn genotypes were inoculated with Burkholderia alba sp. nov [21], a symbiotic nitrogen-fixing bacterium, and the beneficial fungus for the plants Trichoderma harzianum Rifai.

The experimental plot had 0.75 m² area. The distance between rows was 0.50 m, and between bean plants, it was 0.10 m. Fifteen plants were grown in each plot, with two replications, and five plants were measured from each plot. The experimental design chosen is the split-plot design, a special type of complete block design that is often used in factorial experiments (Figure 2).

A control check and three single factors, along with their combinations, were included in the experiments:

0: Control check.

Single factors:

N: nitrogen fertilization (40 kg ha⁻¹);

T: Trichoderma harzianum;

B: Burkholderia alba.

Combined factors:

T-N;

N-B;

T-B;

T-N-B.

The proportion of mycorrhization (MP, %) was determined in the plots that presented mycorrhized plants.

The following agricultural traits were determined based on a plot average:

Number of nodules (NN);

Fresh weight of nodules (FWN) (g);

Dry weight of aerial part (DWA) (g);

Dry weight of root (DWR) (g);

Plant vigor (PV) (1–5);

Plant with fungi (PF) (%);

Disease severity at flowering (DSF) (%);

Disease severity in pod (DSP) (%);

Leaf nitrogen content (LNC) (mg N/g leaf);

Leaf phosphorus content (LPC) (mg P/g leaf);

Pods/plant (PP);

Seeds/pod (SP);

Plant yield (PY) (g);

Ten seed weight (SW) (g);

Seed water absorption (WA) (%);

Seed tegument proportion (TP) (%);

Yield (Y) (kg/ha).

Daily environmental conditions were recorded by the meteorological station at the MBG-CSIC (connected to the AEMET—Spanish National Meteorological Agency):

Rainfall (R);

Maximum temperature (MT);

Growing Degree Days (GDD).

GDD or heat units were calculated as follows: GGD = ∑(MT-BT), where BT is the base temperature; in the case of the common bean, it is 10 °C. GDD is frequently used to describe the timing of biological processes and to estimate the growth and development of crops during the growing season. Data collected from May to September also allow retrospective estimation of the crop growth stage [22].

Samples of fine roots from five bean plants were collected from each plot at the MBG-CSIC, placed in bags, preserved in 70% ethanol, and sent to the EFA-DEPO for the identification of native AMF and the assessment of the percentage of colonized roots.

2.3. Statistical Analysis

A split-plot design was used (Badii et al., 2007) [23]. Measurements were repeated in each of the plots in years 1 and 2. Since some of the response variables are count variables and do not meet the assumptions required to adjust a random-effects analysis of variance (ANOVA), the models were estimated using mixed models (Jiang, 2007) [24]. The interpretation of these models is similar to that of ANOVA but allows for greater flexibility, as it does not have such rigid assumptions. All analyses were performed using the free software R 4.2.3. (R Core Team, 2021) [25].

2.4. Molecular Methodology

Identification of natural AMF species was made by amplification and sequencing of the partial small subunit (SSU) ribosomal RNA gene. All PCR experiments were performed using ADN preparations consisting of pooled roots of each plot. DNA extractions from 32 root samples were carried out in 2021 and 2022. For each sample, fresh root was placed in a PCR tube and total DNA was extracted using the E.Z.N.A^® Fungal DNA Mini Kit (Omega Bio-Tek, Inc. Norcross, GA, USA). All samples were analyzed in triplicate.

The partial small subunit (SSU) ribosomal RNA gene was amplified using nested PCR [26] with the universal primers NS1 and NS4 in the first round and AML1 and AML2 in the second.

In the first reaction, 1 µL of the DNA template was introduced into a microcentrifuge tube containing one PuReTaqM Ready-To-Go™ PCR Bead (GE Healthcare. Chicago, IL, USA) and 0.5 µL of each primer (10 µM), and sterile water was added until a final volume of 25 µL was reached. SSU amplification reactions were performed in a PCR thermocycler (Biometra. Jena, Germany) under the following conditions: one cycle at 94 °C for 3 min, followed by 30 cycles at 94 °C for 30 s, 40 °C for 1 min, and 72 °C for 1 min and a final cycle at 72 °C for 10 min. The first PCR product was used in a second PCR reaction using the primers AML1 and AML2 with the following conditions: initial denaturation at 3 min for 94 °C, 30 cycles of 1 min at 94 °C, 1 min at 50 °C, and 1 min at 72 °C, followed by a final extension of 10 min at 72 °C (Lee et al., 2008) [27]. The PCR products were electrophoresed in a 2% agarose gel (w/v) in 0.5 X TBE at 120 volts. In each assay, a 100 bp standard ladder (marker XIV, Roche Diagnostics. Barcelona, Spain) was also run. The gels were stained with GelRed™ (Biotium Inc., Fremont, CA, USA) and examined under a UV transilluminator.

Products of PCR obtained by AML1 and AML2 primers were purified by using High-Purity PCR Product Purification Kit (Roche Applied Science. Penzberg, Germany). Sequencing reactions were conducted using the BigDye^® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems. Foster City, CA, USA) in a thermocycler under the following conditions: 96 °C for 1 min, 25 cycles at 96 °C for 10 sec, 50 °C for 5 s, and 60 °C for 4 min. The products from sequencing reactions were precipitated in absolute ethanol, then denaturized by using the Template Suppression Reagent (Applied Biosystems) at 94 °C for 3 min and loaded in an ABIPrism^TM 3500 Genetic Analyzer (Applied Biosystems). The sequences were analyzed with the program Sequencing Analysis 5.1.

The sequences generated in this study were compared with related sequences from NCBI’s GenBank sequence database using the megablast algorithm in the BLASTn program (Maryland, USA) [28]. For phylogenetic analysis, highly similar sequences (percentage of sequence identity of 99–100%) were added to the alignments. All sequences were assembled, trimmed to ensure that all had the same start and end points, added to the corresponding outgroup, carefully checked visually using BioEdit v. 7.0.9 software [29], and aligned using the multisequence alignment ClustalW program (with manual adjustment. Dublin, Ireland) [30] included in the BioEdit software.

2.5. Percentage of Colonization by Mycorrhiza-Forming Fungi

To estimate colonization by mycorrhiza-forming fungi, the clarification and staining method proposed by Phillips and Hayman [31] was used, but without phenol. Roots were clarified in 10% KOH in water (w/v) and kept in a water bath at 90 °C for about 1 h. Clarification was completed by treating the roots with 10 volume hydrogen peroxide for about two hours. After removal of the hydrogen peroxide, the roots were immersed in 0.1M HCl for a few minutes at room temperature. Finally, they were stained with 0.05% trypan blue in lactoglycerol in a water bath at 90 °C for 5 min.

To quantify the percentage of root colonized by mycorrhiza-forming fungi, the root/reticulum intersection method established by Ambler and Young, 1977 [32] was used. Each stained root sample was placed on a plate with a grid of 1.23 × 1.23 cm cells. The plate was placed under a stereomicroscope, and points of intersection between the root and the lattice were observed, recording the presence or absence of any of the characteristic structures of the fungus (arbuscules, hyphae, and vesicles) at each point. The percentage of intersections showing mycorrhization with respect to the total number of intersections counted is considered the percentage of colonized roots. A minimum of 100 intersection points per sample was observed to achieve an accuracy of ±3% [33].

3. Results

The native AMF identified in the plant roots in the years of the experiments (2021 and 2022) are displayed in

Table 1. Summary of the native AMF identified in the plant roots in 2021 and 2022 in the bean genotypes Matterhorn and Galaica.

Genus/Species	Phylum/Family	Year
		2021	2022
	Bean genotype: Matterhorn
Acaulospora sp.	Glomeromycota/Acaulosporaceae	x
Funneliformis mosseae	Glomeromycota/Glomeraceae	x
Gigaspora margarita	Glomeromycota/Gigasporaceae	x	x
Gigaspora sp.	Glomeromycota/Gigasporaceae	x	x
Glomus sp.	Glomeromycota/Glomeraceae	x	x
Paraglomus occultum	Glomeromycota/Paraglomeraceae	x	x
Rhizophagus sp.	Glomeromycota/Glomeraceae		x
Rhizophagus clarus	Glomeromycota/Glomeraceae	x	x
Scuttelospora sp.	Glomeromycota/Gigasporaceae		x
	Bean genotype: Galaica
Acaulospora sp.	Glomeromycota/Acaulosporaceae	x
Acaulospora spinosa	Glomeromycota/Acaulosporaceae	x
Gigaspora margarita	Glomeromycota/Gigasporaceae		x
Glomus sp.	Glomeromycota/Glomeraceae	x	x
Rhizophagus sp.	Glomeromycota/Glomeraceae		x
Rhizophagus clarus	Glomeromycota/Glomeraceae	x	x
Rhizophagus irregularis	Glomeromycota/Glomeraceae		x
Scuttelospora sp.	Glomeromycota/Gigasporaceae		x

.

The genetic differences between Galaica and Matterhorn genotypes are considerable, since they belong to different gene pools, as already mentioned. Galaica, as a germplasm belonging to the Andean gene pool, has a type IV indeterminate climbing and very aggressive growth habit; therefore, its biological cycle is very long. On the other hand, Matterhorn, from the Mesoamerican gene pool, has a type II determinate erect growth habit, and its biological cycle is short. In the experiment of 2021, the beginning of flowering was at 48 days after sowing on average in Matterhorn and 65 days in Galaica. In 2022, the beginning of the flowering of the Matterhorn was 45 days and 71 days in Galaica.

Table 2. The experimental design of this study, including the different factors in 2021, and the agricultural performance of the bean genotypes.

Plot	Rep	Factors	Bean Genotypes	Mycorrhizal Fungi	MP (%)	NN	FWN	DWA	DWR	PV	PF	DSF	DSP
2021:3E01	1	T	MATTERHORN	Glomus sp.	76	59	1.97	24.1	2.7	3	26.9	16.7	26.2
2021:3E02	2	T	MATTERHORN	Glomus sp.	77	60	1.65	10.2	1.3	3	10.4	4.8	35.7
2021:3E03	1	T	GALAICA	Acaulospora sp.	76	12	0.22	29.6	1.9	3	23.1	14.3	23.8
2021:3E04	2	T	GALAICA	Glomus sp.	60	9	0.51	25.0	1.4	3	32.1	9.5	21.4
2021:3E05	1	T-N	MATTERHORN	Glomus sp.	39	24	0.14	16.8	2.1	4	47.1	21.4	35.7
2021:3E06	2	T-N	MATTERHORN	Rhizophagus clarus	46	140	5.15	21.1	4.5	4	33.3	33.3	42.9
2021:3E07	1	T-N	GALAICA	Rhizophagus clarus	38	111	5.36	39.6	3.2	3	22.2	28.6	31.0
2021:3E08	2	T-N	GALAICA	Rhizophagus clarus	24	18	0.71	26.2	2.6	3	23.1	21.4	31.0
2021:3E09	1	T-B	MATTERHORN	Glomus sp.	58	127	4.26	13.8	1.8	3	29.2	11.9	19.1
2021:3E10	2	T-B	MATTERHORN	Gigaspora margarita	44	121	2.43	14.5	1.9	3	14.3	4.8	9.5
2021:3E11	1	T-B	GALAICA	Rhizophagus clarus	63	92	2.08	38.2	1.5	3	17.2	14.3	23.8
2021: 3E12	2	T-B	GALAICA	Glomus sp.	68	99	3.73	28.6	2.4	3	3.7	14.3	16.7
2021:3E13	1	T-N-B	MATTERHORN	Rhizophagus clarus	56	91	1.70	17.8	1.5	5	42.9	7.1	47.6
2021:3E14	2	T-N-B	MATTERHORN	Paraglomus occultum	30	113	3.13	37.0	4.2	5	33.3	7.1	47.6
2021:3E15	1	T-N-B	GALAICA	Rhizophagus clarus	64	17	0.67	35.5	4.6	5	12.5	14.3	21.4
2021:3E16	2	T-N-B	GALAICA	Rhizophagus clarus	50	4	0.08	45.4	2.4	5	10.7	14.3	11.9
2021:3E17	1	0	MATTERHORN	Acaulospora sp.	70	9	0.09	27.5	2.7	2	44.0	9.5	88.1
2021:3E18	2	0	MATTERHORN	Glomus sp.	74	104	3.86	23.1	2.4	3	18.5	4.8	92.9
2021:3E19	1	0	GALAICA	Rhizophagus clarus	72	64	1.30	29.2	0.8	3	34.6	21.4	40.5
2021:3E20	2	0	GALAICA	Acaulospora spinosa	64	29	1.13	24.9	0.2	3	19.2	23.8	40.5
2021:3E21	1	N	MATTERHORN	Rhizophagus clarus	57	146	5.42	33.7	3.9	4	32.0	9.5	100.0
2021:3E22	2	N	MATTERHORN	Rhizophagus clarus	50	15	0.79	38.2	4.8	4	37.5	14.3	100.0
2021:3E23	1	N	GALAICA	Rhizophagus clarus	61	10	0.15	30.3	2.2	3	22.2	35.7	54.8
2021:3e24	2	N	GALAICA	Rhizophagus clarus	50	12	0.24	30.0	3.2	3	20.0	28.6	47.6
2021:3E25	1	B	MATTERHORN	Glomus sp.	28	106	1.90	12.0	1.9	2	14.8	35.7	73.8
2021:3E26	2	B	MATTERHORN	Gigaspora sp.	22	66	2.47	9.1	2.0	2	25.0	16.7	81.0
2021:3e27	1	B	GALAICA	Glomus sp.	56	62	1.08	33.1	1.6	3	6.7	31.0	47.6
2021:3E28	2	B	GALAICA	Glomus sp.	42	218	4.72	25.5	2.3	3	10.0	21.4	42.9
2021:3e29	1	N-B	MATTERHORN	Funneliformis mosseae	36	276	7.38	58.4	7.5	4	25.0	11.9	97.6
2021:3E30	2	N-B	MATTERHORN	Glomus sp.	28	65	1.19	18.5	1.6	4	41.4	14.3	97.6
2021:3e31	1	N-B	GALAICA	Glomus sp.	69	357	4.88	21.4	1.3	5	10.0	14.3	40.5
2021:3E32	2	N-B	GALAICA	Rhizophagus clarus	65	77	3.46	52.8	2.8	5	6.9	14.3	42.9
				Mean	53.5	84.8	2.308	27.84	2.54	3.5	23.43	17.04	47.92
				Minimum	22	4	0.08	9.1	0.2	2	3.7	4.8	9.5
				Maximum	77	357	7.38	58.4	7.5	5	47.1	35.7	100.0
				Standard deviation	16.5	80.1	1.971	11.62	1.41	0.9	11.93	8.95	28.14
Plot	Rep	Factors	Bean Genotypes	Mycorrhizal Fungi	LNC	LPC	PP	SP	PY	SW a	WA	TP	Y
2021:3E01	1	T	MATTERHORN	Glomus sp.	54.9	3.62	20.3	4.4	23.8	2.97	122.5	13.9	1189
2021:3E02	2	T	MATTERHORN	Glomus sp.	56.6	4.04	13.8	4.8	21.8	2.74	91.0	14.1	1091
2021:3E03	1	T	GALAICA	Acaulospora sp.	41.0	2.79	9.5	3.2	19.1	9.78	124.3	19.8	954
2021:3E04	2	T	GALAICA	Glomus sp.	36.3	2.59	16.3	3.6	35.9	8.51	94.2	19.0	1794
2021:3E05	1	T-N	MATTERHORN	Glomus sp.	61.2	3.08	28.9	4.8	33.7	2.89	140.7	13.0	1683
2021:3E06	2	T-N	MATTERHORN	Rhizophagus clarus	59.7	2.24	38.3	4.6	48.3	3.07	126.0	13.8	2414
2021:3E07	1	T-N	GALAICA	Rhizophagus clarus	50.6	3.62	17.2	3.6	32.1	7.76	133.4	20.8	1606
2021:3E08	2	T-N	GALAICA	Rhizophagus clarus	28.4	2.65	15.1	3.4	28.9	8.02	126.7	19.2	1445
2021:3E09	1	T-B	MATTERHORN	Glomus sp.	53.4	3.52	20.3	5.6	26.8	3.44	87.3	13.5	1339
2021:3E10	2	T-B	MATTERHORN	Gigaspora margarita	47.9	3.22	19.6	5.4	30.3	3.86	90.4	14.8	1516
2021:3E11	1	T-B	GALAICA	Rhizophagus clarus	31.8	2.80	9.9	3.2	18.8	8.02	123.6	21.3	939
2021:3E12	2	T-B	GALAICA	Glomus sp.	45.7	3.06	13.0	3.0	26.1	8.65	128.4	21.2	1303
2021:3E13	1	T-N-B	MATTERHORN	Rhizophagus clarus	51.3	3.43	27.0	5.2	34.8	2.68	145.4	13.9	1740
2021:3E14	2	T-N-B	MATTERHORN	Paraglomus occultum	46.5	2.80	25.2	5.6	31.5	2.89	139.0	13.8	1576
2021:3E15	1	T-N-B	GALAICA	Rhizophagus clarus	36.2	2.94	15.9	3.2	26.4	7.80	132.8	20.0	1322
2021:3E16	2	T-N-B	GALAICA	Rhizophagus clarus	27.8	2.89	19.6	3.2	50.4	8.64	130.6	18.9	2521
2021:3E17	1	0	MATTERHORN	Acaulospora sp.	27.6	1.93	12.2	4.0	14.1	2.62	124.5	14.8	706
2021:3E18	2	0	MATTERHORN	Glomus sp.	51.5	3.47	14.1	5.2	18.6	2.58	124.9	15.9	928
2021:3E19	1	0	GALAICA	Rhizophagus clarus	29.5	2.33	10.6	2.6	14.5	7.80	132.2	20.6	724
2021:3E20	2	0	GALAICA	Acaulospora spinosa	35.7	2.25	10.7	2.6	18.3	8.32	131.9	21.2	915
2021:3E21	1	N	MATTERHORN	Rhizophagus clarus	41.3	2.20	16.5	4.4	16.8	2.53	125.5	16.4	839
2021:3E22	2	N	MATTERHORN	Rhizophagus clarus	47.7	3.12	22.2	5.2	21.2	2.43	60.3	30.2	1061
2021:3E23	1	N	GALAICA	Rhizophagus clarus	34.3	2.44	5.3	2.6	5.7	7.25	141.5	22.4	286
2021:3E24	2	N	GALAICA	Rhizophagus clarus	38.1	2.49	6.3	2.2	9.4	8.17	136.1	21.5	472
2021:3E25	1	B	MATTERHORN	Glomus sp.	44.9	3.74	6.9	5.2	7.6	2.56	109.2	15.7	378
2021:3E26	2	B	MATTERHORN	Gigaspora sp.	54.4	4.50	13.0	5.0	17.0	2.87	106.3	22.8	851
2021:3E27	1	B	GALAICA	Glomus sp.	34.3	2.78	4.9	2.2	5.7	7.05	142.7	22.3	283
2021:3E28	2	B	GALAICA	Glomus sp.	31.5	1.86	8.9	3.0	16.7	8.09	129.7	21.2	835
2021:3E29	1	N-B	MATTERHORN	Funneliformis mosseae	42.7	2.87	17.9	5.4	17.0	2.29	145.7	15.9	851
2021:3E30	2	N-B	MATTERHORN	Glomus sp.	53.1	2.96	18.8	5.4	20.8	2.18	161.2	15.8	1038
2021:3E31	1	N-B	GALAICA	Glomus sp.	36.0	3.05	9.8	2.8	13.0	7.69	131.6	22.3	650
2021:3E32	2	N-B	GALAICA	Rhizophagus clarus	29.0	2.20	14.4	2.8	20.3	7.71	134.9	19.2	1013
				Mean	42.52	2.922	15.70	3.98	22.66		124.20	18.41	1133.2
				Minimum	27.6	1.86	4.9	2.2	5.7		60.29	13	283
				Maximum	61.2	4.50	38.3	5.6	50.4		161.2	30.2	2521
				Standard deviation	10.22	0.611	7.34	1.14	10.78		20.66	3.90	538.9

a: The mean, maximum, minimum, and standard deviation for seed weight are not displayed since Galaica and Matterhorn belong to different genetic pools, and their seed sizes are very different. MP: proportion of mycorrhization; NN: number of nodules; FWN: fresh weight of nodules; DWA: dry weight of aerial part; DWR: dry weight of root; PV: plant vigor; PF: plant with fungi; DSF: disease severity at flowering; DSP: disease severity in pod; LNC: leaf nitrogen content; LPC: leaf phosphorous content; PP: pods/plant; SP: seeds/pod; PY: plant yield; SW: 10 seed weight; WA: water absorption; TP: seed tegument proportion; Y: yield

displays the experimental design of this study, including the different factors, the mycorrhizal fungi identified in each experimental plot, and the agricultural performance of the two bean genotypes studied in 2021. The percentage of root colonized by mycorrhiza-forming fungi reached a high value of 53.5% on average. Glomus sp. displayed three values over 70% in the bean genotype Matterhorn, Acaulospora sp. showed two scores over 70% in Matterhorn and Galaica, and R. clarus scored more than 70% in Galaica. The percentage of mycorrhization refers to native mycorrhizae in general, not only to the identified species, since there may be other species that we have not detected.

There was a notable difference in grain size between the bean genotypes studied due to their wide genetic differences. The Andean pool is characterized by large or extra-large grain. In the case of Galaica, as displayed in Table 2, the mean grain weight was 80.8 g 100 seeds⁻¹, ranging from 70.5 to 97.8 g 100 seeds⁻¹. In contrast, Matterhorn showed the characteristic medium grain size of the Mesoamerican pool, with a mean of 27.9 g 100 seeds⁻¹, and a range of variation from 21.8 to 38.6 g 100 seeds⁻¹.

The range of variation of the number of nodules was 4–357 per plant, with a mean of 84.8. In general, Matterhorn had more nodules per plant than Galaica; the plots inoculated with B. alba alone or combined with other factors displayed a high number of nodules, highlighting the combination of N-B factors in Galaica with 357 nodules in a plot with the native mycorrhizae Glomus. However, the highest value of dry weight of nodules, 1.58, corresponded to Matterhorn, with N-B treatment and presence of F. mosseae.

Regarding the dry weight of the vegetative parts of the plants, aerial and root, the highest values, respectively, were 58.4 g and 7.5 g, both in Matterhorn, N-B treatment, and F. mosseae. Plant vigor, visually estimated, generally presents the highest values in Galaica, with several mycorrhizae being present, especially R. clarus, and the N-B and also N-T-B factors.

The incidence of fungal diseases in the bean crop was estimated visually using the proportion of plants with fungi, the disease severity at flowering, and the disease severity in pods. Phytopathological analyses were carried out at the EFA-DEPO, identifying Fusarium solani, F. oxysporum, F. proliferatum, Alternaria sp., and Albifimbria verrucaria. The least affected plot was the one with the Matterhorn genotype with T-B treatment and G. margarita, displaying 14.3% of plants with fungi and the minimum severity values in flowering (4.8%) and in pod (9.5%).

The nitrogen and phosphorus content in plant leaves are important variables from the point of view of plant physiology and crop growth. The highest values of both elements were found in the Matterhorn genotype. The maximum values of nitrogen content were 61.2 (mgN/gleaf) (N-T treatment and Glomus), 59.7 (mgN/gleaf) (N-T and R. clarus, and 56.6 (mgN/gleaf) (T and Glomus). Regarding the phosphorus content, the highest values were 4.50 (mgP/gleaf) (B treatment and Gigaspora), 4.04 (mgP/gleaf) (T and Glomus), and 3.74 (mgP/gleaf) (B and Glomus).

The quality of the bean grain was assessed on two traits: water absorption (%) and tegument proportion (%). The more water the grain absorbs, the better the cooking process will go, and the more product will be obtained for consumption. Both bean genotypes showed good results in several experimental plots. Matterhorn has presented values of 161.2% (with Glomus sp. and N-B treatment), 145.4% (with R. clarus and N-T-B), 140.7% (with Glomus sp. and N-T), and 139.0% (with P. occultum and N-T-B). Galaica had maximum water absorption values of 142.7% (with Glomus sp. and B) and 141.5% (with R. clarus and N). The proportion of grain tegument after soaking and before cooking is an important aspect for its consumption because it indicates the palatability of the product, and reduced values of the proportion of tegument are preferred. Matterhorn has presented the best values of proportion of tegument in grains from several plots: 13.0% (with Glomus sp. and N-T treatment), 13.5% (with Glomus sp. and T-B), 13.8% (with R. clarus and N-T and also with P. occultum and N-T-B), and 13.9% (with Glomus sp. and T, and also with R. clarus and N-T-B). In the case of Galaica, the best value was 18.9% (with R. clarus and N-T-B treatment).

The productivity of the two bean genotypes studied was estimated using three traits: pods per plant, production per plant, and crop yield. The highest number of pods per plant corresponded to Matterhorn with 38.3 (N-T treatment and R. clarus). The average for Matterhorn was 19.7 pods/plant, and for Galaica, it was 11.7 pods/plant, with the late genotype having the highest score of 19.6 pods/plant with N-T-B treatment and R. clarus. The productivity of grain per plant reached the value of 50.4 g in Galaica (N-T-B treatment and R. clarus), while in the case of Matterhorn, it reached a maximum of 48.3 g/plant with an N-T treatment and R. clarus. This is a very variable character, with a minimum of 5.7 g/plant in Galaica, with N treatment and R. clarus. Crop yield is one of the most important agricultural traits, as it is related, on the one hand, to the fitness of the plants, and on the other hand, to the economic relevance of the crop. The average yield was 1133.2 kg/ha, with a small difference between Matterhorn (1199.9 kg/ha) and Galaica (1066.5 kg/ha). Relevant yield values were obtained in Galaica with 2521 kg/ha (N-T-B treatment and R. clarus) and Matterhorn scoring 2414 kg/ha (N-T treatment and R. clarus).

Table 3. The experimental design of this study, including the different factors in 2022, and the agricultural performance of the bean genotypes.

Plot	Rep	Factors	Bean Genotypes	Mycorrhizal Fungi	MP (%)	NN	FWN	DWA	DWR	PP	SP	PY	SW a	WA	TP	Y
2022:2E01	1	T	MATTERHORN	Paraglomus occultum	24	8	0.01	20.1	0.47	19.0	5.2	12.1	3.67	126.4	8.9	606
2022:2E02	2	T	MATTERHORN	Paraglomus occultum	45	16	0.75	19.6	0.80	26.0	5.6	13.5	3.15	120.3	11.9	677
2022:2E03	1	T	GALAICA	Rhizophagus clarus	22	29	0.52	55.7	1.91	11.0	1.2	20.9	8.25	126.4	9.6	1043
2022:2E04	2	T	GALAICA	Glomus sp.	24	57	1.37	23.5	0.84	15.0	1.4	33.6	9.09	119.3	9.3	1680
2022:2E05	1	B-T	MATTERHORN	Rhizophagus clarus/Glomus sp.	30	0		15.5	1.03	36.0	4.4	17.8	3.15	115.9	10.2	889
2022:2E06	2	B-T	MATTERHORN	Glomus sp.	25	133	0.13	9.5	1.48	24.0	4.2	5.0	2.45	158.4		252
2022:2E07	1	B-T	GALAICA
2022:2E08	2	B-T	GALAICA	Glomus sp.	15	64	1.08	9.1	0.87	58.0	2.2		8.55	116.5	8.1
2022:2E09	1	N-T	MATTERHORN	Rhizophagus sp.	20	13	0.02	27.6	2.24	24.0	4	16.2	4.00	102.8	9.7	811
2022:2E10	2	N-T	MATTERHORN	Glomus sp.	16	2	0.01	21.0	1.57	32.0	5.2	21.7	3.89	104.6		1084
2022:2E11	1	N-T	GALAICA	Glomus sp.	20	6	0.19	28.4	2.70	15.0	2.4		9.56	116.8	10.5
2022:2E12	2	N-T	GALAICA	Glomus sp.	21	68	1.31	106.1	2.53	14.0	2.2	50.7	7.60	117.2	8.7	2534
2022:2E13	1	N-B-T	MATTERHORN	Gigaspora margarita/Glomus sp.	15	16	0.02	13.2	0.39	28.0	5.6	18.0	3.45	102.0	8.6	901
2022:2E14	2	N-B-T	MATTERHORN	Rhizophagus sp.	25	20	0.53	37.1	3.19	33.0	4.6	20.9	3.62	105.0	10.0	1047
2022:2E15	1	N-B-T	GALAICA	Rhizophagus clarus/Glomus sp.	18	11	0.02	40.2	1.64	28.0	2	26.9	8.43	116.4	8.5	1344
2022:2E16	2	N-B-T	GALAICA	Rhizophagus clarus/Scuttelospora sp.	22	7	0.02	64.1	1.93	18.0	2.8	73.0	6.98	128.4	10.8	3650
2022:2E17	1	0	MATTERHORN	Glomus sp.	20	14	0.00	17.7	0.68	30.0	4.2	10.9	2.94	109.9	9.7	546
2022:2E18	2	0	MATTERHORN	Glomus sp.	22	11	0.03	24.1	1.09	23.0	4.2	6.6	1.73	160.1		332
2022:2E19	1	0	GALAICA	Rhizophagus irregularis	30	0		30.2		5.0	1.6	36.2	9.68	114.0	8.3	1811
2022:2E20	2	0	GALAICA		0	4	0.00	7.7		16.0	2.6	42.1	9.66	121.6	8.4	2105
2022:2E21	1	B	MATTERHORN	Rhizophagus clarus/Gigaspora margarita	22	13	0.02	19.8	1.26	29.0	4.2
2022:2E22	2	B	MATTERHORN		0	0		14.3	1.25	25.0	3.8
2022:2E23	1	B	GALAICA	Glomus sp.	22	6	0.02	17.8	0.91	8.0	1.4		9.90	11.4	10.4
2022:2E24	2	B	GALAICA
2022:2E25	1	N	MATTERHORN	Rhizophagus clarus	25	10	0.03	51.3	2.23	28.0	4.6	5.6	3.71	119.1	7.7	282
2022:2E26	2	N	MATTERHORN	Scutellospora sp./Gigaspora sp.	22	7		47.7	2.37	50.0	3.8	23.1	3.56	109.0	9.7	1154
2022:2E27	1	N	GALAICA	Gigaspora margarita	20	20	0.10	19.2	0.99	16.0	2	43.4	8.48	115.4	8.2	2169
2022:2E28	2	N	GALAICA	Glomus sp.	25	6	0.05	29.6	2.19	33.0	2.4	69.0	9.00	122.8	8.7	3452
2022:2E29	1	N-B	MATTERHORN	Gigaspora sp.	30	14	0.01	21.6	1.83	21.0	5.4	8.7	3.17	122.4	10.1	434
2022:2E30	2	N-B	MATTERHORN	Rhizophagus clarus/Glomus sp.	28	25	0.02	35.6	1.94	24.0	4.4	8.6	3.08	114.3	8.4	430
2022:2E31	1	N-B	GALAICA	Rhizophagus sp.	26	5	0.04	24.0	0.98	20.0	2.4	17.2	7.83	113.3	9.5	861
2022:2E32	2	N-B	GALAICA	Rhizophagus clarus/Glomus sp.	26	175	2.67	23.8	1.18	30.0	2.8	68.4	8.12	121.6	9.7	3419
				Mean	22.0	25.3	0.345	29.17	1.516	24.63	3.43	26.81		115.40	9.35	1340.4
				Minimum	0	0	0.00	7.7	0.39	5.0	1.2	5.0		11.4	7.7	252
				Maximum	45	175	2.67	106.1	3.19	58.0	5.6	73.0		160.1	11.9	3650
				Standard deviation	8.2	39.5	0.631	20.07	0.723	11.15	1.38	20.38		24.31	1.01	1018.8

a: The mean, maximum, minimum, and standard deviation for seed weight are not displayed since Galaica and Matterhorn belong to different genetic pools, and their seed sizes are very different. MP: proportion of mycorrhization; NN: number of nodules; FWN: fresh weight of nodules; DWA: dry weight of aerial part; DWR: dry weight of root; PP: pods/plant; SP: seeds/pod; PY: plant yield; SW: 10 seed weight; WA: water absorption; TP: seed tegument proportion; Y: yield.

shows the experimental design of the experiment, including the different factors, the mycorrhizal fungi identified in each experimental plot, and the agricultural performance of the two bean genotypes studied in 2022. The percentage of root colonized by AMF showed low values, compared to the year 2021, with an average of 22.0% and a maximum percentage of 45% in P. ocultum with Matterhorn. There were three values of 30% in R. clarus/Glomus sp. with Matterhorn, R. irregularis with Galaica, and Gigaspora sp. with Matterhorn, and close values were scored by R. clarus/Glomus sp. in Matterhorn and Galaica and Rhizophagus sp. in Galaica. As in 2021, there was a notable difference in grain size between the bean genotypes studied; in the case of Galaica, the mean grain weight was 86.5 g 100 seeds⁻¹, ranging from 69.8 to 99.0 g 100 seeds⁻¹. Matterhorn has a mean seed weight of 32.6 g 100 seeds⁻¹ and a range of variation from 17.3 to 40.0 g 100 seeds⁻¹.

The range of variation of the number of nodules per plant was 0–175, with a mean of 25.3. Galaica had more nodules per plant (average 32.7) than Matterhorn (average 18.9). Two plots with Glomus sp. and Rhizophagus clarus/Glomus sp. inoculated with B. alba had 133 and 175 nodules per plant, respectively. It is noteworthy that two plots with R. clarus/Glomus sp. and R. irregularis, both inoculated with B. alba, did not have any nodules. Regarding the dry weight of the vegetative parts of the plants, aerial and root, the highest values, respectively, were 106.1 g (Galaica, Glomus sp., N-T treatment) and 3.19 g (Matterhorn, Rhizophagus sp., N-T-B).

In 2022, the quality of the bean grain was assessed again upon water absorption (%) and tegument proportion (%). Matterhorn has presented values of 160.1% (with Glomus sp. and 0 treatment), 158.4% (with Glomus sp. and T-B), and 126.4% (with R. clarus and T). Maximum values of Galaica were 128.4% (with S. clarus/Scuttelospora sp. and N-T-B) and 126.4% (with R. clarus and T). Matterhorn presented good values of proportion of tegument in grains from several plots: 7.7% (with R. clarus and N) and 8.4% (with R. clarus/Glomus sp. and N-B). Galaica also had good scores of 8.1 (with Glomus sp. and T-B), 8.2% (with G. margarita and N), 8.3% (with R. irregularis and 0 treatment), and 8.4% (no mycorrhizae and 0).

The productivity of the two bean genotypes studied was estimated by means of pods per plant, production per plant, and crop yield. The highest number of pods per plant corresponded to Galaica with 58.0 (with Glomus sp. and T-B), while Matterhorn had a maximum of 50.0 (with Scutellospora sp./Gigaspora sp. and N treatment). Productivity of grain per plant reached the highest values in Galaica: 73.0 4 g (with R. clarus and N-T-B treatment), 69.0 g (with Glomus sp. and N), and 68.4 g (with R. clarus and N-B). The productivity of Matterhorn was very low, with a maximum of 23.1 g (with Scutellospora sp./Gigaspora sp. and N). It was again a very variable character, with low values under 10 g/plant in five plots of Matterhorn, four of them with Glomus sp. The average crop yield was 1384.6 kg/ha, higher than the 2021 yield, with relevant values obtained in Galaica: 3650 kg/ha (with R. clarus and N-T-B treatment), 3452 kg/ha (with Glomus sp. and N), and 3419 kg/ha (with R. clarus and N-B).

Table 4. Combined analysis of variance of the quantitative variables studied in the bean genotypes in 2021 and 2022 growing seasons.

Source of Variation	df	MS	df	MS	df	MS	df	MS	df	MS	df	MS
	NN		FWN		DWA		DWR		PP		SP
T	62	8111.15 ***	56	2.13	62	331.22 ***	62	2.69 ***	60	191.37 ***	60	0.48 ***
G		2995.60		1.03		449.92 *		1.91		386.66 ***		53.56 ***
Y		38937.60 ***		37.70 ***		45.93		18.12 ***		504.19 ***		2.30 ***
TxG		2185.30		1.15		94.56		1.02		110.60 ***		0.47
GxY		0.13		0.05		7.26		0.61		91.58 **		0.00
TxGxY		2163.56		2.13		231.32 *		0.34		84.31 ***		0.11
	PY		SW		WA		TP		Y
T	55	669.18 *	60	0.68 ***	60	1536.07 ***	55	6.68 **	55	1672937.90 *
G		1899.02 *		269.10 ***		7.13		22.41 ***		4747548.12 **
Y		14857.21 ***		4.34 ***		2073.43 ***		767.94 ***		37143034.22 ***
TxG		98.37		0.45 ***		460.03 ***		5.85 *		245937.07
GxY		1842.30 **		1.76 ***		298.89		19.75 **		4605753.29 **
TxGxY		166.71		0.48 ***		398.20 ***		10.28 ***		416780.63

T: treatments. G: genotypes. Y: years. df: degrees of freedom. MS: mean squares. p < 0.05 *, p < 0.01 **, and p < 0.001 ***.

displays the combined analysis of variance of the quantitative variables studied in the bean genotypes in the 2021 and 2022 growing seasons. In nodulation traits, there were significant differences between treatments for NN, but not for FWN. There are also differences between years, but no interactions. Regarding plant growth, expressed as DWA and DWR, there were differences between treatments, differences between genotypes also DWA, due to the different growth of the two genotypes, there were differences between years in DWR and a triple interaction in DWA. In the productivity traits studied, PP, SP, PY, and Y, there were differences between treatments, between genotypes, and between years. All the interactions were significant for PP; for SP, the TxG was significant; for PY, the GxY interaction was significant; and for Y, GxY was significant. Finally, regarding quality, expressed as SW, WA, and TP, there were differences in all three traits between treatments; no differences between genotypes in WA; differences between the three traits across years; and the interactions were all present, except GxY in WA.

According to meteorological data, seasonal rainfall between May and September showed no significant differences (2021: 314.1 mm; 2022: 271.4 mm). Additionally, all plots were drip irrigated at a constant rate of 37.5 mm per week. However, seasonal temperatures during this period differed considerably between the two years (

Table 5. Temperatures in May-September in 2021 and 2022 during the bean crop growing period.

Month	Growing Degree Days		Days with TMAX ≥ 30 °C
	2021	2022	2021	2022
May	69.5	69.0	0	2
June	232.5	228.5	5	2
July	260.5	363.5	4	13
August	293.0	356.5	4	14
September	273.5	273.0	1	2
TOTAL	1129.0	1290.5	14	33

). Bean plant flowering occurred in July and August, and the higher temperatures recorded in 2022 compared to 2021 may have influenced both plant growth and the mycorrhizal colonization of the roots.

4. Discussion

Bio-based agronomic practices promote ecological functions and economic sustainability, aligning with the UN Sustainable Development Goals by conserving natural resources, enhancing ecosystem services, and reducing crop management costs and environmental impacts. This approach replaces intensive agriculture reliant on synthetic inputs with agroecological technologies that emphasize biodiversity management. Key to this model are soil microorganisms—bacteria and fungi—that form mutualistic relationships with plants, facilitating nutrient cycling, growth, and stress resilience, thereby improving resource-use efficiency and reducing environmental harm [34,35].

The AMF constitutes a promising microbiological input for the development of sustainable agriculture; their role in the functioning of ecosystems and their potential as biological fertilizers are perhaps sufficient reasons to consider them as one of the important components in modern agroecology. Every day, there is more of a need to adopt agricultural development strategies to ensure stable food production that is consistent with environmental quality. The objectives pursued are food security, eradicating poverty, and conserving and protecting the environment and natural resources. The use of AMF fits very well into the multiple objectives pursued by sustainable agriculture. At an environmental level, they contribute to increased crop productivity, regeneration of degraded plant communities, and maintenance of the ecosystem balance. At an economic level, they contribute to the efficient use of fertilizers, and at a social level, they contribute to integrated rural development, with the use of natural resources (development of native inoculum) at a local scale, thus favouring the establishment of agroecosystems of sustained production.

The Glomeromycota phylum is the most commonly used arbuscular mycorrhizal fungi (AMF) in agricultural practices, functioning as a photosynthetic enhancer [36]. In the experiments, four AMF families were found: Acaulosporaceae, Gigasporaceae, Glomeraceae, and Paraglomeraceae. Most evidence suggests that Glomeromycota rely on land plants for carbon and energy. However, recent circumstantial evidence indicates that some species may be capable of independent existence. Arbuscular mycorrhizal species are terrestrial and widely distributed in soils worldwide, where they form symbiotic relationships with the roots of most plant species [37].

The AMF application has shown promising results in alleviating drought stress and has also positively influenced the nutritional value and chemical composition of pods and seeds of common bean. However, these benefits vary depending on the irrigation regime and the timing of pod and seed harvest [16]. In the experiments, the effect of AMF on two genotypes of common bean, Galaica and Matterhorn, was studied in combination with three agricultural factors: nitrogen fertilization, inoculation with T. harzianum, and inoculation with B. alba, together with their combinations and a check control.

The variability of native mycorrhizae found in the trials can be considered remarkable, with four species and three genera in each of the two years of evaluation. The proportion of mycorrhization in 2021 showed very high values, which is considered positive for crop growth. It is noteworthy that the genus Glomus showed some values higher than 70% that year, as did Acaulospora and R. clarus. Razakatiana et al. [35] reported scores of mycorrhization of 62.5% with Acaulospora, which increases up to 80.0% when co-inoculated with a cocktail of 10 Rhizobium spp. strains, while for Glomus, they reported 30.0% of mycorrhization that grows up to 95% in association with the Rhizobium cocktail.

A decrease in the proportion of mycorrhization was observed in the second year of experimentation, which did not correspond to the productivity of the bean plants, since yields were higher in 2022 than in 2021. The unexpected increase in yield despite reduced AMF colonization in 2022 can be explained by physiological and ecological factors. During the reproductive stage, plants often limit AMF colonization to prioritize energy for flowering and seed development. Additionally, higher temperatures may have accelerated phenology and nutrient mineralization, improving nutrient uptake independently of AMF [38]. Ecologically, reduced colonization might be offset by increased microbial activity or better water-use efficiency. These factors together may account for the higher yields observed. In 2022, the maximum score of mycorrhization was achieved by Paraglomus occultum (45%), which, in this case, was not associated with the rhizobacteria B. alba. The AMF Acaulospora sp. was not detected in roots this year. The reasons for the reduction in the quantitative reduction of mycorrhization in 2022 compared to 2021 could be of an environmental nature, particularly due to the temperature differences between the two years.

Temperature variations can markedly affect AMF colonization in legumes. A meta-analysis revealed that increased temperatures generally enhance AM fungal colonization and plant biomass, while decreased temperatures can limit these benefits. However, the response can vary depending on the plant species and environmental conditions [39].

According to Mather et al. [38], high temperature stress decreased root colonization in plants. In studies involving non-leguminous plants, such as maize, high-temperature stress led to a significant reduction in AMF colonization, dropping from approximately 75–80% under normal conditions to about 40–45% under heat stress. While this study focused on maize, it suggests that elevated temperatures can adversely affect AMF colonization, which may have implications for legumes as well. Table 4 shows that the maximum temperatures in 2022 were higher than in 2021, which could produce some heat stress in the bean plants. Soudzilovskaia et al. [40] found a relationship between the mean temperature of the warmest month at a site and percent AMF colonization, with an optimum occurring at a mean monthly temperature of 19.5 °C. In the experiments reported here, the mean monthly temperature in 2021 was 18.4 °C in July and 19.5 °C in August, while in 2022, it was 22.3 °C in July and 21.5 °C in August during the flowering period of the bean plants, which means that temperatures in 2021 were more adequate for the mycorrhization process.

Gao et al. [41] found that elevated temperature significantly decreased G. mosseae colonization rate in the roots by 49.5% (under Cd exposure), and Nazari et al. [42] reported that when the temperature is above 30 °C, fungal activity decreases due to plant growth disturbances. In our experiments, there were 27 days over 30 °C in 22 during the flowering time (July–August) and only 8 in the same period in 2021. The above-mentioned could explain why the proportion of mycorrhization in 2022 was lower than in 2021, since temperature plays a pivotal role in the establishment and efficiency of AMF colonization in legume crops. Both excessively high and low temperatures can disrupt the symbiotic relationships, potentially leading to reduced plant growth. Understanding these temperature effects is crucial for optimizing legume cultivation, especially in the context of climate change and its impact on agricultural systems [43].

The experimental results were grouped into several areas: nodulation, plant growth and vigor, presence of diseases, physiological nitrogen and phosphorus content, and crop productivity. The influence of the different mycorrhizae identified on the agronomic behaviour of the bean genotypes does not seem to follow a pattern in the experiments.

In nodulation, the effect of Glomus has been notable, in terms of the number of nodules and their fresh weight, and in the most positive cases, combined with the presence of B. alba. F. mosseae, also with B. alba, has offered good results. In the nodulation, a relationship has been detected between low mycorrhization in 2022 and the reduction in the number of nodules per plant and their weight in this year. Species within the genus Glomus are typically generalist symbionts found across diverse habitats, indicating a tolerance to a range of environmental stresses; they also exhibit significant functional diversity, with different isolates contributing to substantial variations in plant growth responses [44]. The dual inoculation is likely to produce synergistic effects in severely nutrient-depleted soils. This hypothesis is supported by the fact that the main mechanism by which inoculation with AM fungi acts on plant functions seems to be improved phosphatase activity, particularly that of acid phosphatases responsible for organic P hydrolysis and involved in supplying the high P requirements of N₂-fixing nodules [35].

Nodule formation in the 2021 and 2022 trials showed very similar values in the two bean genotypes studied, and an increase in the number of nodules was detected in plants inoculated with B. alba, which was more pronounced in the case of co-inoculation with T. harzianum. Leaf nitrogen content also responded positively to inoculation with B. alba, especially phosphorus, which reached its highest value. However, inoculation with this symbiotic bacterium had no effect on plant productivity. Regarding N fertilization, it reduced the number of nodules due to the availability of inorganic nitrogen in the soil for plant growth [36,37].

Razakatiana et al. [35] studied the effect of Acaulospora sp. and Glomus sp., along with a cocktail of 10 Rhizobium spp. strains, in low-fertility tropical soil. They identified a positive effect of this co-infection on plant growth and on the total N content of the plants, along with a synergistic effect on the total P content, the number of nodules, and the mycorrhizal rate of the plants. In a controlled co-inoculation experiment, Tajini and Drevon [45,46] found that low phosphorus content improves mycorrhizal colonization. In this case, colonization by native mycorrhizae is important despite the high phosphorus content in the soil, which represents one of the significant contributions of this research

In the study of aerial and subaerial growth of plants and vigor, the effect of R. clarus has been positive, which is associated with the presence of the combination of nitrogen and B. alba. The species F. mosseae, combined with B. alba and nitrogen, has also provided good results.

Regarding the incidence of diseases, there are several combinations that could be the most suitable for crop protection. Glomus is associated with T. harzianum; G. margarita is associated with T. harzianum and B. alba; and R. clarus is associated with nitrogen, T. harzianum, and B. alba. According to these results, the presence of T. harzianum seems relevant for bean crop health, and in some cases, it is in a consortium with B. alba. AMF are capable of promoting plant growth and effectively suppressing several plant diseases. The interaction of these microorganisms in the plant rhizosphere affects plant growth and microbial community composition [47,48].

With regard to leaf nitrogen content, Glomus seems to play a relevant role, along with T. harzianum, and there are also positive results regarding the effect of R. clarus with T. harzianum. In terms of phosphorus content, Glomus also stands out, both with B. alba and with T. harzianum, while Gigaspora also plays a positive role with B. alba. The results agree with Razakatiana et al. [35] in their experiment of co-inoculation with Rhizobium spp., Glomus sp., and Acaulospora sp.

According to Fueyo [49] and Puerta Romero [50], grain quality will depend, at least in different Spanish regions, on grain size, although this criterion does not show an international trend [51]. In the 2021 and 2022 trials, the grain weight ranged from 1.7 (Matterhorn) to 9.9 (Galaica) (g 10 grains-1). Seed size is determined by the size of the pod, since it is maternal tissue and is primarily determined by the genotype of the mother plant. In addition to genetic effects, environmental factors are relevant to grain size at harvest [49].

Other relevant traits for the commercial quality of bean grain for consumption are water absorption and the proportion of seed coat [52,53]. In the 2021 and 2022 trials, water absorption ranged from 108 to 136%. Water absorption is a very important characteristic for cooking [53], since low water absorption by soaked beans leads to the “hard-to-cook” phenomenon, whereby a significant proportion of the beans remain hard during cooking and reduce their commercial value. The seed coat ratio is of great importance for the final quality of the product for consumption. Sanz and Atienza [52] developed an overall quality index for bean grains based on tasting tests, which considers the proportion of seed coat and its perception during tasting. The seed coat ratio of the Galaica variety in 2021 and 2022 showed high values between 14.4 and 18.0%, which is higher than the usual values in its production area in the northwest of Spain.

Crop productivity was estimated on the basis of grain weight, number of pods per plant, productivity per plant, and crop yield. Glomus had an effect on grain weight and nitrogen, with there being an association with T. harzianum and B. alba, as did R. clarus with T. harzianum. Acaulospora and G. margarita, both with T. harzianum, also had an effect on increasing grain weight. The number of pods per plant increased due to the effect of R. clarus, with the presence of T. harzianum and nitrogen fertilization. Glomus also stands out as being associated with the same factors. Productivity per plant was favored by R. clarus, which is associated with T. harzianum and nitrogen. Glomus, which is associated with T. harzianum, was also highlighted. The crop yields in both bean genotypes had outstanding values with respect to productivity in the humid temperate zone in which the experiment was carried out. R. clarus, combined with T. harzianum and nitrogen, has led to yield values above 2000 kg/ha. Glomus, combined with T. harzianum, has also given an adequate yield.

The interaction of rhizosphere microorganisms, such as AMF, fungi of the genus Trichoderma, and bacteria of the genus Rhizobium and similar (such as B. alba), is usually classified as a component of a biological control agent (BCA). Nitrogen fixers and plant growth-promoting microorganisms (PGPM) depend on different factors to express their potential beneficial effects. Interactions between microbial combinations like AMF, nitrogen-fixing bacteria, and T. harzianum can lead to both synergistic and antagonistic effects. Synergies may enhance nutrient uptake and plant growth [35], while competition for colonization or plant resources can cause negative outcomes [54]. Understanding these mechanisms is key to developing effective microbial consortia for sustainable and productive legume cultivation.

It is difficult to predict the outcome of interactions between plants and beneficial soil microorganisms and, even more so, between species of microorganisms. However, the microbial communities associated with the root plant system are considered to play a key role in the development of sustainable agricultural practices. There is information regarding the positive synergistic effect of AMF together with Rhizobium spp. [35], but there are no references available regarding the role that B. alba may play, either individually or in association with AMF. The response of plants to inoculation depends on the functional compatibilities in the physiology and biochemistry of the interaction between the microbial components; thus, it gives different answers, depending on the combination of microorganisms [55]. Arbuscular mycorrhizal fungi (AMF) and nitrogen-fixing bacteria form complex interactions not only with plants but also with each other, leading to a “tripartite symbiosis”. This three-way association is particularly important in legumes, where AMF enhances phosphorus uptake, while nitrogen-fixing bacteria supply biologically available nitrogen through root nodules. The synergy between these microorganisms supports improved nutrient acquisition, plant growth, and stress tolerance. Focusing on AM symbiosis specifically within Fabaceae is crucial because these plants rely heavily on such mutualistic relationships to thrive, making this tripartite interaction a key driver of sustainable legume production.

In conclusion, effective AMF use could reduce pesticide reliance in legume farming, leading to more affordable, sustainable, and healthier food production, although environmental factors may affect colonization rates. Through research and conservation, the potential of AMF can be harnessed to boost agricultural productivity, reduce environmental damage, and support global sustainability. This study demonstrates the potential of native AMF as biostimulants in common bean production and highlights the importance of microbial synergy for sustainable agriculture. Future research should focus on validating AMF effects across different locations and varieties and use high-throughput omics approaches to better understand the mechanisms involved.