Enhancing Chickpea Growth via Arbuscular Mycorrhizal Fungal Inoculation: Facilitating Nutrient Uptake and Shifting Potential Pathogenic Fungal Communities

doi:10.21203/rs.3.rs-4989901/v1

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Enhancing Chickpea Growth via Arbuscular Mycorrhizal Fungal Inoculation: Facilitating Nutrient Uptake and Shifting Potential Pathogenic Fungal Communities

https://doi.org/10.21203/rs.3.rs-4989901/v1

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The plant mycobiome makes essential contributions to the host life cycle in both healthy and diseased states. Arbuscular mycorrhizal fungi (AMF) are the most widespread plant symbionts associated with plant roots, and they perform numerous functions that contribute to plants’ health and physiology. However, there exist many knowledge gaps in how the interactions between AMF and host plants’ root mycobiomes influence the performance of host plants. To this end, we inoculated a local chickpea cultivar grown in an agricultural soil under semi-controlled conditions with Rhizophagus irregularis. The plants were subjected to low or normal levels of phosphorus (P) fertilization. In addition to examining mycorrhizal colonization, plant biomass, and mineral nutrition, we sequenced the ITS region of the rDNA to assess the chickpea mycobiome and identify key fungal taxa potentially responding to AMF inoculation. Our results showed that AMF inoculation had a stronger effect on chickpea aboveground biomass, in addition to mineral nutrition; whereas P fertilization had a more profound effect on belowground traits. Specifically, AMF promoted shoot (p = 0.06), root (p = 0.001), and total aboveground biomass (p = 0.01), while P fertilization enhanced root biomass (p = 0.02), in addition to root diameter (p = 0.007), root volume (p = 0.01), and root length (p = 0.08). Furthermore, the total P (p = 0.05) and Na contents (p = 0.09) were enhanced in the aboveground biomass by AMF inoculation. ITS metabarcoding revealed Ascomycota as the dominant phylum in both roots and soil biotopes, followed by Basidiomycota, Chytridiomycota, Glomeromycota, Monoblepharomycota, Mucoromycota, and Rozellomycota. Ten ASVs were significantly impacted by AMF inoculation in chickpea roots, including important plant pathogens belonging to Didymella, Fusarium, Neocosmospora, and Stagonosporopsis. Surprisingly, a correlation was established between shoot biomass and some fungal taxa that were differentially abundant in roots. This study confirms the significance of AMF inoculation not for only improving chickpeas’ growth and mineral nutrition in semi-arid conditions but also for shaping plants’ fungal community composition, thereby promoting resilience against both biotic and abiotic stressors.

Arbuscular mycorrhizal symbiosis

Phosphorus fertilization

Fungal community composition and structure

Pathogenic fungi

Biostimulant

Semi-arid climate

Food security

Fungal communities (mycobiomes) inhabiting plant roots and rhizospheres play essential roles in plant physiology and ecology. These plant mycobiomes comprise various groups of fungi with distinct roles and functions, the most significant being beneficial and pathogenic fungi. Beneficial fungi, such as mycorrhizal fungi and endophytic fungi, can directly or indirectly enhance plant growth and induce tolerance to biotic and abiotic stress, leading to improved plant performance. Conversely, pathogenic fungi can harm crop growth, leading to yield losses and posing a major threat to agricultural crop production. Therefore, studying plant mycobiomes is essential for better disease management, improved ecological practices, and the development of eco-friendly crop production methods (Anal et al. 2020). However, little is known about the diversity and functions of the mycobiomes of major food crops, as well as their responses to environmental changes and agricultural practices, especially bioinoculation.

The chickpea (Cicer arietinum L.) is a high-value legume crop that is pivotal for achieving food security, addressing climate change issues, and managing soil degradation due to its low agricultural input demand, enabling the crop to perform well on marginal soils. Two types—Desi (small and pigmented) and Kabuli (large and non-pigmented)—are consumed widely across the globe, owing to their high protein (20–30%) and carbohydrate (40%) contents, in addition to being good dietary sources of minerals, such as calcium, magnesium, phosphorus, magnesium, iron, and zinc, and pro-vitamins, such as beta-carotene, and fiber (Kudapa et al. 2018; Kibar et al. 2023). Thus, it is unsurprising that chickpeas rank second after soybeans among the highest-produced food legumes in the world, with an estimated 18095248 tons produced in 2022 (FAOSTAT 2023). The Kabuli chickpea is the main chickpea type cultivated in Morocco, where it constitutes the second-largest food legume crop grown (after faba beans), with an annual production rate of 75000 tons and 30953 tons in 2021 and 2022, respectively, according to FAOSTAT (FAOSTAT 2023).

Morocco’s chickpea productivity decreased by more than half between 2021 and 2022, with the country’s total production dropping to the fifth position in Africa in 2022, as compared to the fourth position held in 2021 (FAOSTAT 2023). Although chickpeas are widely cultivated in many semi-arid and arid climates, the crop faces many challenges, including climatic factors such as drought and heat, pathogen attacks, and low adoption of certified seeds (Houasli et al. 2020; Mohammed et al. 2017; Arriagada et al. 2022). Like other MENA countries, Morocco’s agriculture is severely affected by drought and excessive heat conditions, leading to lower agricultural productivity. Long-term drought and erratic rainfall decreased Morocco’s agricultural productivity by approximately 17% in 2022 (Tsakok 2023). The recent decline in chickpea productivity can be attributed to sparse rainfall and excessive heat experienced in recent years, given that chickpea cultivation in most fields is rainfed.

Fortunately, methods that improve phosphorus-use efficiency in chickpeas have been demonstrated to enhance chickpea growth under drought stress in Moroccan semi-arid pedoclimatic conditions (Chtouki et al. 2022a; Khadraji et al. 2017). Increased supply of P to legumes can stimulate N₂ fixation by alleviating the negative effect of excessive N on nodule formation (Cabeza et al. 2024). In addition to abiotic stress, fungal pathogens—notably, members of the Didymella and Fusarium genera—are harmful for many chickpea cultivars grown locally (Krimi Bencheqroun et al. 2022). For example, annual yield losses due to pathogen infestations, especially Fusarium wilt/root rot, can reach 90–100% in many fields (Elbouazaoui et al. 2022). Thus, improving chickpeas’ resilience to both abiotic and biotic stresses holds significant promise not only for enhancing food security, but also for promoting economic prosperity in the region.

Arbuscular mycorrhizal fungi (AMF) are widely distributed plant symbionts that colonize the roots of most terrestrial plants, including agriculturally important crops (Basiru et al. 2020). AMF inoculation can assist crops in adapting to various abiotic stresses, especially nutrient limitation and water scarcity (Abdalla et al. 2023; Kavadia et al. 2020), resulting in a substantial yield increase (Hijri 2016). The benefits attributed to AMF inoculation often pertain to nutrient acquisition, which usually relies on the soil’s physicochemical characteristics, especially soil phosphorus levels; however, recent evidence indicates that soil biotic communities, both micro- and macrofauna, can strongly predict the success of AMF inoculation in field conditions (Zhai et al. 2023; Zhang et al. 2019). A large-scale study across 54 fields demonstrated that the abundance of pathogenic species, rather than nutrient availability, was the most reliable predictor of plants’ growth response to AMF inoculation (Lutz et al. 2023). That is, declines in several soil-borne pathogens in maize roots following AMF inoculation were positively correlated with plant growth in fields where the mycorrhizal growth response was positive (Lutz et al. 2023). Hence, it may be inferred that the colonization of roots by AMF could offer advantages in enhancing plants’ performance by suppressing root colonization by potential plant pathogens (Ismail and Hijri 2012; Ismail et al. 2011, 2013), in addition to promoting nutrient acquisition. Nevertheless, the efficacy of this capability remains insufficiently examined across agriculturally significant ecosystems and crops.

Recently, there have been increasing efforts to improve chickpeas’ performance under dry climates through non-diazotroph microbial inoculation, especially with arbuscular mycorrhizal fungi and endophytic fungi (Gomez et al. 2024; Rocha et al. 2019); however, growth responses sometimes vary widely across chickpea genotypes (Kavadia et al. 2020; Bazghaleh et al. 2015; Bazghaleh et al. 2018b). It was demonstrated that two chickpea genotypes responded differently to water limitation due to their contrasting abilities to associate with AMF: the AMF-responsive genotype showed greater tolerance to water limitation compared to the other (Kavadia et al. 2020). Moreover, the successful establishment of foreign AMF strains in a new area outside their local ecological range is often challenged by competition from native communities, leading to inconsistent results(Basiru and Hijri 2022a). After establishment, root colonization by AMF can shape the host crop’s microbiome, reflected in plants’ phenotypic expression; however, the extent of this influence is still contingent on geographic location, soil properties, and plant genotype (Li et al. 2021; Lutz et al. 2023). Moreover, little-to-no information exists on how inoculation with non-native AMF can impact chickpeas’ mycobiome, such as whether it will have a dramatic impact on the host plants’ health and physiology.

Therefore, the objective of this study was to investigate whether inoculation with non-native AMF can enhance chickpeas’ performance in semi-arid regions of Morocco. We selected Rhizophagus irregularis DAOM 197198, originally isolated from Pont-Rouge, Quebec, Canada, as it is the most commonly used AMF isolate in commercial inoculants (Basiru et al. 2020). Our hypotheses were as follows: (1) inoculation with R. irregularis enhances chickpeas’ growth and augments its mineral nutrition, particularly under low-phosphorus conditions in calcareous soil; (2) inoculation with R. irregularis impacts the community structure of the rhizosphere and root mycobiome of chickpeas; and (3) inoculation with R. irregularis stimulates or suppresses certain key fungal taxa that contribute to the health of chickpeas, thus acting as indicators of the AMF inoculation’s success in both chickpea roots and the rhizosphere habitats. To test these hypotheses, we conducted an experiment in a naturally lit glasshouse using locally bred Kabuli chickpea plants (cultivar Moubarak) subjected to two different phosphorus fertilization regimes and inoculated with R. irregularis DAOM 197198, a non-native AMF strain. We assessed mycorrhizal colonization, plant biomass, and mineral nutrition. Additionally, we utilized ITS metabarcoding to examine changes in the chickpea mycobiome, and we conducted differential abundance and regression analyses to identify key taxa in the mycobiome that may influence the chickpeas’ growth response to inoculation.

Setup of the semi-controlled experiment

The experiment was conducted outdoors at UM6P’s experimental farm in Ben Guerir, Morocco. The average daily temperature was 22/15°C (day/night), and the average relative humidity was 65%. The soil used in the study was collected from the experimental farm prior to mixing with river sand at a ratio of 2:1 (v:v). The mixed soil and sand were sieved through a 4 mm mesh before being used to fill 10 L pots. Composite samples of this soil mixture were taken randomly for chemical analysis (Table S1). The AMF strain Rhizophagus irregularis DAOM197198 (originally isolated from Pont Rouge, Quebec, Canada) was used for the inoculation; it was propagated in vitro in Medium M using DNA-transformed chicory roots for 4 months.

Chickpea seeds (cultivar Moubarak) were planted on March 08, 2021, at a rate of five seeds per pot, which was reduced to three seedlings after germination. The experimental design was a two-factor randomized block design comprising two fertilization regimes and two AMF levels replicated seven times. The first variable pertained to phosphorus application, comprising two P levels: one at the recommended dose (i.e., 60 kg/ha P₂O₅), and the other at half the recommended dose (30 kg/ha P₂O₅), as described in Table S2. The second treatment involved AMF, also with two levels (i.e., with or without AMF). To prevent post-irrigation inoculum percolation, a piece of filter paper was placed beneath each seed, as shown in Figure S1. To enhance early establishment of the applied AMF, the inoculum was applied as a seed treatment in order to initiate a priority advantage (Basiru and Hijri 2022b). Fertigation was carried out twice at a four-week interval, with total nutrient contents of N (25 kg/ha), P (60 or 30 kg/ha), and K (25 kg/h) as ammonium phosphate, potassium nitrate, and ammonium nitrate (Table S2). The systemic fungicide Thiophanate Methyl was applied at a dose of 100 g/hL six weeks after sowing, following the observation of lesions suspected to be Anthracnose on the shoots of the chickpea plants.

Experimental harvest and measurement of biomass and yield parameters

The shoot and root dry weight was assessed twice: the first biomass assessment was carried out on May 18, 2021, corresponding to 70 DAS while the second biomass assessment took place on June 30, 2021, corresponding to 113 DAS, hereafter referred as ‘harvest 1’ & ‘harvest 2’ respectively. To assess the shoot, root, and total aboveground biomass (shoot and pods), chickpea plants were harvested and weighed before and after drying in an oven at 70°C for 48 hours. Surprisingly, we did not observe any nodules on the roots at both harvests.

Root imaging and mycorrhizal colonization

The root colonization test was carried out at harvest 1. The method was based on the ink–vinegar staining technique, which is considered to be safer and more easily executable compared to other staining techniques (Vierheilig et al. 1998). Briefly, the roots were cut into small fragments of 1–2 cm and cleared with 10% KOH in a microwave at full power for 2 min. The cleared roots were subsequently rinsed with acidified tap water and stained with 5% ink in vinegar. Following staining, 20 root fragments were positioned atop a slide and observed under an optical microscope to examine both the number of colonized roots and the intensity of colonization (Trouvelot 1986). Furthermore, root samples were scanned to produce images, which were then analyzed using WinRHIZO version 2003b (Regent Instruments Inc., Quebec, Canada) to measure root length, average diameter, and volume.

Mineral Content Analyses

To determine the shoots’ mineral contents, shoot biomass was harvested at harvest 1 and dried at 70°C for 72 h to constant weight. Samples including flowers and emerging pods were ground to a fine powder and sent for chemical (N, P, K, Na, Zn) analysis by UM6P’s soil analysis laboratory.

DNA extraction

Rhizosphere soil and root samples from chickpea plants were collected using sterilized scapulae and stored at -20°C before extraction. Approximately 250 mg rhizosphere soil samples from four replicates were utilized per treatment per plant. The chickpea roots were lyophilized, and 50 mg was ground into a powder using tungsten beads in a TissueLyser II (Qiagen, Global Diagnostic Distribution, Temara, Morocco). DNA extraction was carried out using both DNeasy PowerSoil and DNeasy Plant kits according to the manufacturer’s instructions. Subsequently, DNA was quantified using a Qubit Fluorometer (Thermo Fisher, Rabat, Morocco). Extracted DNA samples were stored at -20°C until use.

PCR amplification and Library Prep

The fungal ITS2 region was amplified using the following primer pair containing adapters CS1_ITS3_KYO2 (5’-ACACTGACGACATGGTTCTACAGATGAAGAACGYAGYRAA-3’) and CS2_ITS4 (5’-TACGGTAGCAGAGACTTGGTCTTCCTCCGCTTATTGATATGC-3’). The PCR reaction was performed in a 25 µL reaction volume consisting of 1X Platinum Direct PCR Universal Master Mix (Thermo Fisher, Rabat, Morocco), 0.25 µM of each primer, 1X Platinum GC Enhancer, and approximately 20 ng of template DNA. Thermal cycling was carried out using an Eppendorf Mastercycler Pro PCR instrument (Eppendorf, Hamburg, Germany) with the following cycling conditions: an initial denaturation step of 2 min at 94°C, followed by 35 cycles of denaturation at 94°C for 45 s, annealing at 50°C for 1 min, and elongation at 72°C for 1 min, with a final elongation step of 10 min at 72°C. Each PCR run included negative PCR controls containing only water, and no visible amplification was observed. Amplified products were quantified and visualized on a 1% agarose gel. Subsequently, amplicons were submitted for library preparation and sequencing using Illumina MiSeq PE 2 × 300 bp (Legeay et al. 2024). The sequences were demultiplexed by the service provider.

Bioinformatics

The DADA2 pipeline implemented in R version 4.3.2 was utilized to analyze raw reads (Callahan et al. 2016). Reads exceeding the expected error rate by more than two were discarded, and forward and reverse reads were trimmed at 50 and 100 bp, respectively, due to the low quality at those termini. Chimera sequences were excluded utilizing the consensus method. The resulting amplicon sequence variants (ASVs) underwent taxonomic assignment using the UNITE database (Nilsson et al. 2019) and the default parameters of the “assignTaxonomy” command.

The reads corresponding to plant DNA, amounting to 30% of reads in the root samples, were discarded. The raw data were transformed into compositional data using the microbiome package.

Alpha diversity indices (Shannon and Simpson) were computed using the microbiomeSeq package (Ssekagiri et al. 2017), employing the plot_anova_diversity function and grouping by AMF and environment, with the p-value cutoff set to 0.05. Ordination plots utilizing the Bray–Curtis ecological distance were generated using the microViz package (version 0.12.1) (Barnett et al. 2021). Between-subject global dissimilarity (beta diversity)—specifically, non-phylogenetic (Bray–Curtis dissimilarity)—was assessed by comparing relative abundance data via principal coordinate plots and a permutational multivariate analysis of variance (PERMANOVA).

Differential abundance analysis was carried out using the R package microbiomeSeq (version 0.1). Briefly, this package employs the DESeq2 procedure, originally developed for differential expression analysis of RNA-Seq data, to calculate differentially abundant taxa (Love et al. 2014). The raw counts were filtered to remove samples containing empty reads and transformed using the log-relative transformation. Two independent analyses were performed on root and soil samples using the differential abundance function, which returns the differentially abundant ASVs between inoculated and non-inoculated samples. The p-value and log fold-change thresholds were set to 0.05 and 0, respectively. The plot showing differentially abundant ASVs, corresponding adjusted p-values, and ranked importance were generated by a random forest classifier implemented by the package (Figures S5-S6).

Furthermore, linear modeling of some important genera, whose ASVs were differentially abundant, was conducted using the microViz package. A two-step transformation (total sum scaling (TSS) was adopted; firstly, the count data were transformed to compositional data using the tax_transform function. The second transformation was executed after calling the taxatree_models function, where the trans option was set to log2, and the zero counts were replaced with half the minimum observed abundance proportion (of any taxon) before transformation (Figure S7).

Statistical analysis

Data from the pot experiment underwent analysis of variance (ANOVA) using the Lme4 and LmerTest packages in R Studio software (Bates et al. 2015; Kuznetsova et al. 2017). Factors such as P fertilization and AMF were treated as fixed factors, while Bloc was considered as a random factor. Prior to analysis, normality tests were conducted using the Shapiro–Wilk statistic for all data, and homogeneity of variance was visually assessed by inspecting the distribution of the residuals through QQ plots in R studio. Pairwise comparisons were performed with the emmeans function using Tukey’s test, with treatments deemed significant at a 5% confidence level (p ≤ 0.05). Graphical representations were generated using the ggplot or ggpubr package.

Effects of treatments on root colonization and growth parameters

Microscopic observation revealed successful colonization of chickpea roots by AMF (Figure S5). However, inoculation did not significantly affect the frequency or intensity of AMF colonization in chickpea roots (p = 0.75 for both) (Fig. 1). In contrast, P fertilization significantly influenced mycorrhizal colonization and intensity in the roots, with decreases of 30% and 43% in these variables, respectively, at higher P levels (p = 0.03 and p = 0.06, respectively) (Fig. 1). The interactive effect of AMF inoculation and P fertilization on root mycorrhizal parameters was not significant.

Despite the absence of a significant response to inoculation in mycorrhizal root colonization, we observed an increase in chickpea root and shoot biomass at harvest 1 & 2, with a notably stronger effect at harvest 2. As anticipated, P fertilization tended to show meaningful effects on shoot biomass only at the later stages, resulting in a 9.8% increase in shoot biomass (p = 0.07). Meanwhile, AMF inoculation enhanced shoot biomass at harvest 1 & 2 by 5% and 16%, respectively (p = 0.06 and p = 0.003) (Fig. 2). Although the interaction effect between fertilization and AMF application on shoot biomass was not significant, both factors significantly impacted the root biomass. Higher P fertilization significantly increased the root biomass (p = 0.02), and AMF inoculation significantly increased the root biomass at harvest 2 (p = 0.01) but not at harvest 1 (Table 1 and Fig. 2). Furthermore, the total aboveground biomass at harvest 2 was positively enhanced by both treatments, with AMF inoculation showing a greater growth response (13%, p = 0.001) compared to P fertilization (5.9%, p = 0.1). Additionally, root image analyses indicated that P fertilization had a more significant impact on chickpea root traits compared to AMF inoculation (Fig. 3). Increased P fertilization led to a notable increase in chickpea root average diameter by 6.5% (p = 0.007), root volume by 20% (p = 0.01), and root length by 12% (p = 0.08) (Fig. 3).

Table 1

Analysis of variance (ANOVA) table showing the effects of P fertilization and inoculation treatments on chickpea growth. The AMF and P treatments (especially at harvest 2) nearly significantly increased the shoot dry weight. Significant differences were also observed for root dry weight between the inoculation and P treatments. The interactions between inoculation and P treatments were significant, but only at harvest 2. AMF also significantly impacted the total aboveground biomass of chickpea at harvest 2, while P treatments had no effects.
		Harvest 1 (70DAS)			Harvest 2 (113 DAS)
	Source of Variation	DF	F value	Pr(> F)	F value	Pr(> F)
SDW	AMF	1	3.491	0.067	5.80	0.074
	P	1	0.039	0.849	3.69	0.062
	AMF:P	1	1.148	0.289	0.35	0.555
RDW	AMF	1	0.654	0.450	7.31	0.010
	P	1	0.765	0.386	5.64	0.022
	AMF:P	1	0.430	0.515	4.48	0.040
AGDW	AMF	1			12.62	0.001
	P	1			2.34	0.133
	AMF:P	1			2.50	0.121
SDW = shoot dry weight; RDW = root dry weight; AGDW = total aboveground biomass

Effects of treatments on mineral contents of chickpeas

AMF inoculation positively influenced the aboveground P content, resulting in a 9.5% increase at harvest 2 (p = 0.05). Conversely, P fertilization did not significantly affect the aboveground P content at harvest 1. However, both AMF inoculation and P application positively impacted the sodium content of the aboveground biomass (Table 2). Additionally, a significant positive correlation was observed between P and the contents of potassium, sodium, and zinc (Figure S6).

Table 2

Analysis of variance (ANOVA) table showing the effects of P fertilization and AMF inoculation on chickpeas’ mineral contents. AMF enhanced the phosphorus and sodium contents of chickpeas, whereas P fertilization only tended to exert an impact on sodium content.
	Phosphorus		Sodium		Potassium		Zinc
Source of Variation	F value	Pr(> F)	F value	Pr(> F)	F value	Pr(> F)	F value	Pr(> F)
AMF	3.88	0.05	2.99	0.09	1.57	0.22	0.05	0.82
P	0.04	0.85	4.02	0.09	0.48	0.51	0.39	0.53
AMF:P	0.63	0.43	0.14	0.71	1.23	0.27	0.20	0.66

Fungal community diversity and composition

ITS metabarcoding of chickpea roots and rhizosphere soils revealed substantial fungal diversity in both the soil and root compartments of chickpeas. As expected, there were more fungal taxa in the soil compartment compared to the root compartment (Fig. 4). Ascomycetes dominated the fungal communities of both the rhizosphere soil and root samples, accounting for more than 90% of all phyla, followed by Basidiomycetes, Chytridiomycetes, Glomeromycetes, Monoblepharomycetes, Mucoromycetes, and Rozellomycetes. At the class level, Sodarimycetes, Dothideomycetes, and Glomeromycetes constituted the major taxa in the roots, while Sordariomyctes, Dothideomycetes, Eurotiomycetes, Pezizomycetes, Agaricomycetes, and Wallemiomycetes were highly abundant in the soil samples (Fig. 4A). At the family level, Nectriaceae, Pleosporaceae, an unknown Pleosporales family, Didymellaceae, and Stachybotryaceae had the most profound abundance in chickpea roots, whereas in the soil compartment, the fungal families were more diverse, including major taxa such as Nectriaceae, Stachybotryaceae, Sporomiaceae, Aspergillaceae, Chaetomiaceae, Didymellaceae, Ascobolaceae, Pleosporaceae, and an unknown Pleosporales family (Fig. 4B). The key Glomeromycota genera identified in chickpea soils included Claroideoglomus, Dominikia, Enterophospora, Funneliformis, Kamienskia, and Rhizophagus, while only three genera (Funneliformis, Claroideoglomus, and Rhizophagus) were observed in the chickpea roots (Fig. 5A). These taxa were identified at the species level except for one Glomeraceae species (Fig. 5B). Thus, at the species level, Funneliformis mosseae, Claroideoglomus durumondii, Entrophospora infrequens, and Rhizophagus irregularis were the major AMF colonizing chickpea roots as detected in this study; in addition to these taxa, Kamienskia bistrata, Dominikia aurea, Dominikia iranica, Dominika disticha, and an unknown Glomeraceae species were also found in the rhizosphere soils. Interestingly, the abundance of Funneliformis mosseae was higher in the inoculated roots compared to the non-inoculated control.

Effects of inoculation on fungal Alpha and Beta Diversities

The alpha diversity indices of the fungal communities did not show significant differences in response to AMF inoculation; however, AMF-inoculated samples seemed to exhibit lower Shannon and Simpson indices (Fig. 6). Principal coordinate analysis revealed a clustering of fungal communities between the chickpea root mycobiome and soil samples (Fig. 7). A subsequent PERMANOVA test using Bray–Curtis ecological distances further affirmed this relationship, with a significant difference in Beta diversity between the soil and roots (p = 0.01) (Table 3). However, no significant effects of AMF inoculation were detected on the overall fungal community structure (p = 0.3), although it appeared that the fungal communities tended to cluster between treatments with or without AMF (Fig. 7).

Table 3

Permutational multivariate analysis of variance (PERMANOVA) on chickpea community diversity between the root and soil compartments, and between inoculated and non-inoculated treatments. Fungal community diversity differs significantly between the root and soil compartments, while the effect of inoculation on fungal community diversity was not significant.
	Df	Sum Of Sqs	R2	F	Pr (> F)
Environment	1	7.41	0.42	43.22	0.01	**
AMF	1	0.20	0.01	1.17	0.31

Differentially abundant taxa

To elucidate the shift in fungal community composition between inoculated and non-inoculated conditions, we conducted a differential abundance analysis on both soil and root biotopes. The number of ASVs showing differential enrichment or depletion (log2 fold-change) was higher in soil compared to roots, as shown in Figures S5-6. Specifically, the abundance of 10 ASVs was differentially expressed in roots, where five taxa were enriched while the other five taxa were depleted under AMF inoculation (Table 4).

Table 4

List of ASVs, the corresponding taxonomic classification, and the level of significance of the differentially abundant taxa in chickpea roots. Five taxa, including an unknown *Fusarium* species, *Sacroladium kiliense*, *Fusarium nyagamai*, *Neocomospora rubicola*, and an unknown *Stagnosporopis* species, were significantly depleted in inoculated chickpea roots, whereas *Didymella arachidicola*, *Fusarium equiseti*, an unknown Phaeosphaeriaceae genus, another *Neocosmospora rubicola* ASV, and an unknown *Scutellinia* species were enriched.
ASVs	Class	Order	Family	Genus	Species	Effect	p-Value
ASV139	Sordariomycetes	Hypocreales	Nectriaceae	Fusarium	Fusarium sp.	Depleted	0.0036
ASV314	Sordariomycetes	Hypocreales	Hypocreales_fam_Incertae_sedis	Sarocladium	Sarocladium kiliense	Depleted	0.0029
ASV109	Sordariomycetes	Hypocreales	Nectriaceae	Fusarium	Fusarium nygamai	Depleted	0.00079
ASV102	Sordariomycetes	Hypocreales	Nectriaceae	Neocosmospora	N. rubicola	Depleted	1.36E-06
ASV15	Dothideomycetes	Pleosporales	Didymellaceae	Stagonosporopsis	Stagonosporopsis sp.	Depleted	0.0029
ASV266	Dothideomycetes	Pleosporales	Didymellaceae	Didymella	Didymella arachidicola	Enriched	0.0225
ASV29	Sordariomycetes	Hypocreales	Nectriaceae	Fusarium	Fusarium equiseti	Enriched	0.022
ASV85	Dothideomycetes	Pleosporales	Phaeosphaeriaceae	Phaeosphaeriaceae genus	Phaeosphaeriaceae species	Enriched	0.0036
ASV44	Sordariomycetes	Hypocreales	Nectriaceae	Neocosmospora	Neocosmospora rubicola	Enriched	1.36E-05
ASV274	Pezizomycetes	Pezizales	Pyronemataceae	Scutellinia	Scutellinia sp.	Enriched	0.0029

Correlation between root fungal communities and chickpea phenotypes

Pearson correlation analyses were carried out to examine the relationship between chickpea growth phenotypes and fungal communities in roots. The results demonstrated both positive and negative relationships between root fungal communities and shoot biomass. For example, ASV15 (r = 0.43, p = 0.01), ASV324 (r = 0.42, p = .01), ASV14 (r = 0.40, p = 0.02), ASV29 (r = 0.038, p = 0.030), ASV266 (r = 0.038, p = 0.030), ASV77 (r = 0.38, p = 0.032), ASV44 (r = 0.37, p = 0.036), and ASV18 (r = .0338, p = .005) were positively correlated with shoot biomass. Surprisingly, many of these ASVs were also responsive to AMF inoculation. Among these, ASV29, -44, and − 266 (Fusarium equiseti, Neocosmospora rubicola, and Didymella arachidicola, respectively) were enriched under the AMF inoculation treatment. ASV85, an unclassified species belonging to the Phaeosphaeriaceae, was also enriched under AMF treatment and had a near-significant positive correlation with shoot biomass (r = 0.30, p = 0,08). On the other hand, ASV15, an unknown species belonging to Stagonosporopsis, although depleted under AMF treatment, was positively correlated with shoot biomass (r = 0.43, p = 0.013). Furthermore, among the ASVs that were depleted under AMF treatment, ASV102 (Neocosmospora rubicola; r = -0.11, p = 0.5); ASV139 (an unknown Fusarium species; r = -0.337, p = 0.059); and ASV314 (Sarocladium kiliense; r = -0.096, p = 0.6) showed negative correlations with shoot biomass.

We further tested the correlation results with linear modeling of the effects of AMF on Fusarium and Didymellaceae species that are considered to be key pathogens of chickpeas. Interestingly, the abundance of many Fusarium species—especially F. nirembergiae (p < 0.05), F. nygami, an unknown Fusarium species (identified as Fusarium solani by blasting in NCBI BLAST with 100% similarity and an e value of 0.0), F. tricinctum, and F. tokinense—was drastically reduced (Figure S7A). Our results also showed a decline in members of the Didymellacea family in AMF-inoculated roots—especially an unknown Stagonoporopsis species (ASV15), although best NCBI BLAST hit was Didymella rabiei, with 100% similarity and an e value of 1e^− 173. Conversely, Didymella aradichola and Didymella spp. were increased in AMF-inoculated roots (Figure S7B).

In this study, we investigated the capability of the non-native AMF R. irregularis inoculant cultivated in vitro to enhance the growth and mineral nutrition of chickpeas cultivated under semi-controlled conditions in a semi-arid climate. Additionally, we examined its effects on both the root and rhizosphere soil fungal communities. Through this experiment, we identified significant fungal taxa responsive to AMF inoculation, and we were able to demonstrate the success of inoculation.

Our findings indicate that AMF inoculation surpassed P fertilization in improving chickpea shoot and total aboveground biomass, whereas P fertilization had more profound impact on root growth parameters such as biomass root length, root diameter and root volume. Specifically, AMF inoculation induced a 16% increase in shoot biomass compared to non-inoculated controls, while P fertilization only led to a 9% increase in shoot biomass. The significance of AMF inoculation in promoting chickpea growth has been established previously; however, the extent of this response may vary between genotypes (Bazghaleh et al. 2018a). The observed increase in shoot biomass and root biomass in our study suggests that a single seed treatment with AMF may be adequate to facilitate root colonization and promote plant growth. This finding is consistent with those of Rocha et al. (2019), who found that coating seeds with AMF inoculants can enhance chickpeas’ shoot biomass and pod weight under both greenhouse and field conditions.

It is believed that seed treatment can provide priority advantage to introduced inoculants, allowing for the quicker establishment of the inoculated strain in roots compared to native species (Basiru and Hijri 2022b; Werner and Kiers 2015). This perhaps explains the preference for seed treatment as a method of AMF application by many commercial inoculants (Basiru et al. 2020). Nevertheless, the root colonization assay conducted in this study did not demonstrate any clear priority advantage between the introduced AMF and indigenous species. There was an absence of significant differences in the root colonization parameters between the inoculated and non-inoculated treatments at harvest 1 after planting. Nevertheless, seed treatment might confer an advantage in the early stages of plant development, following a "first come, first served" principle; this assertion is supported by the increased shoot biomass observed in inoculated plants compared to the non-inoculated controls.

The root staining approach utilized to determine mycorrhizal colonization does not enable the differentiation between indigenous and introduced AMF. Conversely, ITS metabarcoding, although not the preferred sequence barcode for AMF community profiling (Iffis et al. 2017), revealed that Funneliformis mosseae and Claroideoglomus durumondii were the most dominant in chickpea roots, while R. irregularis had a low abundance (Fig. 4B). Surprisingly, the relative abundance of F. mosseae increased in chickpea roots in response to AMF treatment, suggesting a shift in the native AMF communities in response to inoculation. Previous studies have shown suppressive, stimulative, and neutral impacts of AMF inoculation on indigenous communities (Basiru and Hijri 2022b; Renaut et al. 2020; Islam et al. 2021). Here, we observed that the introduction of R. irregularis to the native soil appeared to stimulate the native AMF communities. This was indicated by the increased relative abundance of F. mosseae in chickpea roots after the AMF treatment (Fig. 4B). This finding suggests interspecific competition for niche availability between the inoculated AMF strain and the native strain under the current conditions. Extended-duration studies and sampling at multiple timepoints, in conjunction with the utilization of additional AMF-specific primers targeting the 18S rRNA gene, as well as species-specific quantitative qPCR (Badri et al. 2016), would be necessary to further unravel the nature of this interaction.

The growth-promoting role of AMF is predominantly attributed to the facilitation of nutrient uptake scavenging properties of AMF’s extraradical hyphae. It is documented that up to 90% of plants’ P can be obtained by indirect uptake through the AM symbiotic route (Ahammed and Hajiboland 2024; van der Heijden et al. 2015). Additionally, P is often the most limiting nutrient in legume production. When available, P can enhance nodulation and nitrogen fixation (Chtouki et al. 2022b; Nasr Esfahani et al. 2017). Interestingly, our study revealed a positive effect of inoculation on chickpeas’ nutrient contents; AMF inoculation significantly improved the P (9.5%), Na (8.6%), and K (5.5%) contents compared to the controls. However, it was surprising to find that both the root biomass and total aboveground biomass were significantly increased only at the higher P fertilization rate, i.e., 60 kg/ha (333 g/pot) compared to 30 kg/ha (167mg/pot). This could be attributed to the high P-fixing capacity of the soil, indicated by its high pH (8.4) and high calcium content (8.699 g/Kg). In soils with a pH around 8.0, P tends to precipitate with calcium, potentially necessitating more phosphorus for optimal plant growth (Ducousso-Detrez et al. 2022). Consequently, it could be inferred from our study that AMF inoculation enhanced P mobilization from the soil, resulting in greater P uptake and higher shoot P content compared to the non-inoculated control. The increased growth response observed in the AMF treatment under high P fertilization may imply that higher P fertilization is required under these soil conditions to meet the chickpeas' P demand.

Furthermore, we noted a significant positive correlation among phosphorus, sodium, and potassium, indicating a positive interaction among these elements. A positive correlation between phosphorus uptake and potassium has been documented previously in soybeans (Gaspar et al. 2018) and chickpeas (Fotiadis et al. 2020). This observation is unsurprising, as the availability of one nutrient can influence the crop's use efficiency of other nutrients. Therefore, by enhancing phosphorus-use efficiency, AMF can facilitate the uptake of other essential nutrients. Additionally, AMF plays a significant role in osmolyte homeostasis, enabling host plants to adapt to water and salt stress. One such mechanism involves promoting sodium and potassium accumulation (Wang et al. 2023).

In addition to enhancing plant’s nutrition and nutrient-use efficiency, it appears that the growth benefit observed in the present study under AMF treatment was largely due to the modulation of pathogenic communities in both chickpea roots and rhizosphere soil. This is not surprising, as AMF inoculation has long been recognized as a promising potential biocontrol tool (Ismail and Hijri 2012; Ismail et al. 2011, 2013); however, its biocontrol potential is context-dependent, contingent upon factors such as pathogen type and virulence, host type, AMF isolates, and prevailing environmental conditions (Azcón-Aguilar et al. 2002). Three mechanisms have been hypothesized to underlie AMF’s biocontrol ability: modulation of the root and soil microbial community structure, antagonism, and stimulation of plant defenses (Ismail et al. 2011). The present study observed the potential of AMF to suppress root-borne pathogenic fungal communities, thereby serving as prominent tool to better protect plants against pathogens. It is plausible that early colonization of roots by AMF could inhibit the roots’ infestation by fungal pathogens, leading to low disease severity. This finding corroborates recent efforts to reduce the use of pesticides and mitigate associated environmental issues through the implementation of biocontrol techniques for disease and pest management (El Housni et al. 2024).

Consistent with this, AMF inoculation significantly reduced the relative abundance of putative pathogens in chickpea roots, including Fusarium sp., Sarocladium kiliense, Fusarium nygamai, Neocosmospora rubicola, and Stagonosporopsis sp. Interestingly, Fusarium sp. (ASV 139) exhibited the highest negative correlation with shoot biomass, compared to F. nygamai (ASV 109), N. rubicola (ASV 102), and S. kiliense (ASV 314), among the ASVs whose abundances decreased in chickpea roots under AMF inoculation. Fusarium wilt caused by F. solani is a devastating disease of chickpeas in major growing areas of chickpea production (Younesi et al. 2021). This disease can lead to complete loss of up to 100% in highly infested fields, even under favorable growth conditions (Jendoubi et al. 2017). Several isolates of F. solani species, including those from Morocco, have been isolated from diseased chickpeas and were able to induce disease symptoms and reduce agronomic parameters. El Hazzat et al. (2019) demonstrated that the cultivar Moubarak was the most susceptible to F. solani (FS2 isolate) among other locally bred chickpea cultivars. Our results thus show that inoculating a susceptible chickpea variety (e.g., Moubarak) with AMF can potentially confer induced resistance against many pathogens.

Antagonistic interaction between AMF and Fusarium has been documented in previous studies. For instance, the inoculation of potato with R. irregularis (previously named Glomus intradices) inhibited Fusarium sambucinum growth and modulated the expression of trichothecene mycotoxin genes that were responsible for its virulence (Ismail et al. 2011). Moreover, inoculation of papaya with an AMF complex (MTZ01) comprising four fungi (R. intraradices, F. mosseae, Claroideoglomus eutenicatum, and Gigaspora albida) attenuated Fusarium oxysporum infestation of papaya seedlings (Hernández-Montiel et al. 2013). Interestingly, Sohrabi et al. (2015) demonstrated that inoculation with both R. intraradices and F. mosseae reduced F. solani infection in chickpeas, with F. mosseae being more effective. Given that F. mosseae was most notable in chickpea roots and increased in response to R. irregularis inoculation, it could be suggested that the shift in pathogenic fungal communities observed in chickpea roots was induced by native F. mosseae due to the “priming” effect of R. irregularis inoculation.

A recent 18S rDNA metabarcoding study also reported a negative correlation between AMF colonization and the relative frequency of Fusarium in Ricinus communis roots (Rodríguez-Yzquierdo et al. 2023). However, given that Fusarium is a species complex, it is often difficult to predict which strains are responsive to the inoculation effect. This study shows that AMF might not successfully antagonize all Fusarium spp. For instance, inoculation reduced the relative abundances of F. nygami, Fusarium sp. (identified as Fusarium solani), F. tricinctum, and F. tokinense, while the relative abundances of F. hostae, F. equiseti, and F. burgessi were positively increased (Figure S7A). Thus, our work provides both direct and indirect evidence that AMF inoculation can reduce the relative abundance of F. solani in chickpea roots, especially in chickpeas under semi-arid climates.

Furthermore, ascochyta blight caused by Didymella rabei is considered to be an economic pathogen of chickpeas in Morocco. This fungus displays a wide geographic distribution and varying aggression (Krimi Bencheqroun et al. 2022). Aggressive isolates of D. rabiei can cause up to 97% yield loss in chickpeas. Surprisingly, we found two species belonging to Didymellaceae, i.e., Didymella rabiei (ASV15) and Didymella arachidicola (ASV266), among the differentially abundant taxa in chickpea roots. Whereas Didymella arachidicola was enriched in AMF roots, ASV15 (blast-identified as Didymella rabiei) was depleted in AMF roots (Table 4 and Figure S7B). A few studies have indicated the effectiveness of AMF as effective biocontrol agents for foliar diseases, especially blights caused by Didymella (syn. Ascochyta) rabiei (Kashyap et al. 2024). For example, Moarrefzadeh et al. (2021) conducted a series of inoculation trials on two varieties of chickpea (Saral and Bivanij) and observed a remarkable suppression of Didymella (syn. Ascochyta) rabiei. R. irregularis had the highest disease suppression (46.15%), followed by G. versiforme (42.30%) and G. fasciculatum (40%) (Moarrefzadeh et al., 2022). Surprisingly, our study confirms the suppressive effect of AMF on D. rabiei, whose abundance was reduced in the inoculated roots. However, it is equally surprising that the abundance of D. rabiei was positively correlated with shoot biomass data collected at the flowering stage (70 DAS). This was perhaps because the fungus was still at the early stage of infestation when the samples for the ampliconseq were collected.

We observed that AMF and P fertilization increased the shoot and root biomass as well as the P, K, and Na contents of chickpeas. Similarly, fungal ITS metabarcoding demonstrated that much of the growth effect of AMF could be attributed to the reduction in the relative abundances of potential pathogenic fungal communities, most notably Fusarium solani and Aschochyta rabiei. Our results further indicate the potential of biological solutions to address key challenges facing agricultural productivity. While the root staining method indicated that there was no effect of inoculation on the root parameters, the ITS amplicon sequencing method confirmed the persistence of the inoculated strain, although native AMF taxa were the most abundant in roots, and they were likely responsible for the observed shift in root fungal communities. Future long-term studies coupled with multiple sampling will be essential to track the persistence and dynamics of the inoculated AMF in the soil, along with the legacy effects of inoculation in subsequent crops or cropping seasons. Studies are also needed to investigate the responses of different local chickpea breeds to AMF inoculation in natural soils under field conditions, as well as to unravel the molecular mechanisms responsible for pathogen suppression by AMF inoculation.

Acknowledgements

We thank the SIMLAB of UM6P for providing the computational infrastructure used for the bioinformatics data processing.

Author contribution

SB conducted the experiment, analyzed the data, and wrote the manuscript draft. KA, RE, IK, KE, and JLcontributed to the methodology, data curation, review, and editing. MH conceived and designed the study, secured funding, supervised the work, and contributed to writing, reviewing, and editing.

Funding

This work was supported by funding from the OCP Group (Projects AS-85, awarded to MH), and the University Mohammed VI Polytechnic University (UM6P).

Data availability

Raw reads are available in the NCBI repository under the BioProject: PRJNA1102850 and accession numbers: SAMN41030867-SAMN41030928.

Conflict of interest

The authors declare no competing interests.

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Enhancing Chickpea Growth via Arbuscular Mycorrhizal Fungal Inoculation: Facilitating Nutrient Uptake and Shifting Potential Pathogenic Fungal Communities

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Version 1

Abstract

Figures

Introduction

Materials and Methods

Setup of the semi-controlled experiment

Experimental harvest and measurement of biomass and yield parameters

Root imaging and mycorrhizal colonization

Mineral Content Analyses

DNA extraction

PCR amplification and Library Prep

Bioinformatics

Statistical analysis

Results

Effects of treatments on root colonization and growth parameters

Effects of treatments on mineral contents of chickpeas

Fungal community diversity and composition

Effects of inoculation on fungal Alpha and Beta Diversities

Differentially abundant taxa

Correlation between root fungal communities and chickpea phenotypes

Discussion

Conclusions

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1