Elsevier

Applied Soil Ecology

Volume 167, November 2021, 104013
Applied Soil Ecology

Co-inoculations with rhizobia and arbuscular mycorrhizal fungi alters mycorrhizal composition and lead to synergistic growth effects in cowpea that are fungal combination-dependent

https://doi.org/10.1016/j.apsoil.2021.104013Get rights and content

Highlights

  • NFB and AMF could be a alternative tool to improve cowpea performance.

  • Positive cowpea response to dual inoculation depends on the AMF composition.

  • NFB affect AMF colonization and composition in cowpea roots.

  • Among the AMF species applied, R. irregularis was the most aggressive colonizer.

Abstract

Cowpea can effectively form tripartite symbiotic associations with nitrogen-fixing bacteria (NFB) and arbuscular mycorrhizal fungi (AMF) although the selection of compatible AMF species and rhizobial strains which are promoting cowpea growth remains a challenge. The aims of the current study were 1) to evaluate the response of cowpea plants to a symbiotic NFB and a multi-AMF inoculum and 2) to explore any interaction between the symbiotic NFB and the different AMF isolates. In a pot gnotobiotic trial, cowpea plants grown under limited N supply, were inoculated with or without a symbiotic nitrogen-fixing bacterium, Sinorhizobium meliloti, and combinations of three different AMF species namely Dominikia disticha, Claroideoglomus etunicatum and Rhizophagus irregularis. Experimental evaluation was determined through the measurement of above ground biomass, nutrient content and AMF root colonization. The presence of AMF species on cowpea roots was also determined with cloning and sequencing. Inoculation with both AMF and S. meliloti led to increased cowpea biomass production compared to inoculation with AMF only, but the presence of a positive effect depended on the specific AMF partners used. Inoculation with AMF alone had a highly positive impact on the growth and P uptake of cowpea, but the NFB inoculation was needed to address N deficiency in planta. The presence of both symbionts generally led to increased AMF colonization of the cowpea roots, however, plant colonization depended on the AMF species, and became even negative, when all three AMF inocula were used together. The AMF composition in plant roots was also altered in the presence of the S. meliloti. Plant nitrogen content of cowpea plants significantly increased under the presence of both symbionts compared to AMF alone, while phosphorus content was hardly affected by dual inoculations. The results show positive synergistic effects of the different AMF species and S.meliloti. Inoculation with all AMF isolates and S. meliloti led to high above ground biomass production and accumulation of N. The presence of S. meliloti increased P content in plants not inoculated with AMF. Finally, the efficiency of synergism depends on the specific AMF partners used and it is not related to their colonization levels.

Introduction

Climate change is the most important threat and challenge that we have to face since it jeopardizes food security globally (Yadav et al., 2015). The design, development and implementation of specific strategies aimed at minimizing external input without reducing the productivity of agricultural ecosystems are critical to face these challenges. In this effort, legumes have dominant position due to their multi-functional characteristics (Voisin et al., 2014). Numerous studies have demonstrated the significant benefits resulting from the cultivation of legumes in reducing greenhouse gas emissions (Jeuffroy et al., 2013; Omirou et al., 2020), in soil fertility (Bichel et al., 2016; Carranca et al., 2015) as well as human and animal nutrition (Tharanathan and Mahadevamma, 2003). Cowpea (Vigna unguiculata (L.) Wale) is one of the most important grain legumes cultivated in arid and semi-arid regions while it is a critical source of income for farmers with a global gross production value of US $1.5 billion (FAO stat).

In Cyprus, cowpea is used as a vegetable via the consumption of green immature pods which are the main products and are harvested several times during the growing season (Omirou et al., 2019). To maintain high quality and quantity of pods farmers are using significant amount of chemical nitrogenous and phosphorus fertilizers, thereby posing a significant risk for the environment. The improvement of cowpea environmental performance is linked to the reduction of using external inputs of nutrient and the development of “tools” to enhance the nutrient use efficiency of the crop. It has been proposed that microbial inocula could be an alternative tool to enhance the productivity of agricultural ecosystems under climate change (Kavadia et al., 2020). Indeed, cowpea's are associated with symbiotic nitrogen fixing bacteria (NFB) and several studies demonstrated that plant inoculation with NFB significantly improves their productivity (Figueiredo et al., 1999; Kebede et al., 2020; Oliveira et al., 2017). Cowpea is also colonized by arbuscular mycorrhizal fungi (AMF) and in combination with NFB form tripartite symbioses.

A main challenge of tripartite symbiosis in legumes is to understand how taxonomic diversity of mycorrhiza interacts with symbiotic nitrogen fixing bacteria and how this interaction affects plant performance. Recent studies suggest that the use of multi-strain AMF inocula could be beneficial for plants (Yang et al., 2017; Crossay et al., 2019) due to functional complementarity (Maherali and Klironomos, 2007; Yang et al., 2017). To obtain maximum performance of AMF and NFB inoculation, the selection of appropriate AMF isolates that are compatible with NFB and vice-verca, is essential to avoid ineffectiveness, incompatibility and inadaptation to the local conditions (Hart et al., 2018). For AMF biofertilizers, colonization ability as well as efficiency (in terms of plant growth and performance) are the main factors that should be taken into account during their development (Giovannini et al., 2020). However, these properties are affected by the presence of NFB. Previous studies have shown that the establishment of AMF species both in roots and nodules of chickpeas roots could be influenced by different NFB strains (Tavasolee et al., 2011). Recently, our group showed that inoculation of cowpea plants with a Bradyrhizobium isolate altered the AMF community structure and colonization in cowpea roots (Omirou et al., 2016).

Numerous studies demonstrated synergistic effects of rhizobial and mycorrhizal symbioses in legumes (Kaschuk et al., 2010; Larimer et al., 2014; van der Heijden et al., 2016). However, the presence of both symbionts in legume roots is not always beneficial for the plant and neutral or negative responses may be observed (Ballhorn et al., 2016; Bethlenfalvay et al., 1982; Püschel et al., 2017). Larimer et al. (2010) revealed synergistic responses in plant growth under the presence of both symbionts while the same authors, in later studies, showed that synergism under co-inoculation may depend on nutrient availability levels (Larimer et al., 2014). Abiotic and biotic conditions may also determine the plant response to dual symbiosis. For example, under moderate drought stress, the performance of P. vulgaris to dual inoculation depended on the plant variety and the symbiont involved (Ide Franzini et al., 2010). It has been shown that both symbionts, as well as the interaction between them and the host plant, affected symbiotic efficiency on legumes (Redecker et al., 1997; Ruiz-Lozano and Azcon, 1993).

Therefore it is evident that the selection and application of NFB and AMF exhibiting synergistic effect on plant performance is essential to enhance crop productivity. The main aim of this study was to assess and characterize the interactions occurring between three different AMF species in the presence or absence of symbiotic N fixing bacterium when colonizing cowpea. In addition, we addressed the impact of t multi-strain inoculation on cowpea in terms of plant biomass, phosphorus and nitrogen content of the above ground biomass. The study was conducted using synthetic communities of AMF assemblages' combinations of AMF isolates belonging to different species Dominikia disticha, Glaroideoglomus etunicatum and Rhizophagus irregularis and Sinorhizobium meliloti. We put down the hypothesis that i) NFB could significantly affect the AMF colonization in cowpea roots and ii) NFB could change the profile of the different AMF species found in cowpea roots.

Section snippets

Preparation of AMF inocula

Three AMF isolates belonging to three different species namely Dominikia disticha (abbreviated with number 3 in the manuscript), Claroideoglomus etunicatum (abbreviated with number 5 in the manuscript) and Rhizophagus irregularis (abbreviated with number 21 in the manuscript) affiliated in two families, Glomeraceae and Claroideoglomeraceae. The AMFs were isolated from sand dunes from several areas in Greece. Spores from the sand derived inocula were isolated and re-propagated in a sterilized

Biomass and nutrient response to inoculations

The above ground biomass of cowpea was significantly affected by AMF (F4,35 = 7.29 p = 0.00022) and NFB (F1,35 = 13.69, p = 0.00073) inoculation. In detail, the inoculation of cowpea plants with S. meliloti caused an eight-fold increase of the above ground biomass of cowpea compared with not inoculated control plants. Similarly, the application of any combination of AMF inocula caused an order of magnitude increase of cowpea biomass compare to plants that didn't receive any inoculation (Table 2

Discussion

In the current study we examined how the combination of different AMF species affected cowpea growth and nutrient acquisition in the presence or the absence of S. meliloti, a symbiotic nitrogen fixing bacterium. Additionally, we examined how the co-inoculation with S. meliloti, affected the AMF colonization as well as the incidence of the various AMF species included in multi-strain AMF combinations. Cowpea AMF root colonization 60 days after the initiation of the experiment ranged from 5.23 to

Conclusions

Plant inoculation with both S. meliloti and AMF could be an alternative tool for farmers to improve cowpea performance through enhanced nutrient acquisition. It was shown that the occurrence of positive synergistic effects under dual inoculations strongly depends on the species composition of the AMF inoculum. Moreover, the presence of symbiotic S. meliloti affected not only the AMF colonization percentage but also the abundance of the AMF inoculum species which eventually colonize plant roots,

Funding

This work was financially supported by European Union within the SUPREME ERANET-MED project ΚΟΙΝΑ/ERANETMED/0316/02.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Informed consent was obtained from all individual participants included in the study.

Availability of data and materials

All data generated or analyzed during this study are included in this article.

CRediT authorship contribution statement

The authors read and approved the final manuscript

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

We acknowledge the technical assistance of Mrs. Louiza Konstantinou and Mrs. Evdokia Neophytou. We thank Dr. Andreas Mavrogenis for his valuable comments.

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