Cooperation among phosphate-solubilizing bacteria, humic acids and arbuscular mycorrhizal fungi induces soil microbiome shifts and enhances plant nutrient uptake

Abstract

Background: Increasing the presence of beneficial soil microorganisms is a promising sustainable alternative to support conventional and organic fertilization and may help to improve crop health and productivity. If the application of single bioeffectors has shown satisfactory results, further improvements may arise by combining multiple beneficial soil microorganisms with natural bioactive molecules. Methods: In the present work, we investigated in a pot experiment under greenhouse conditions whether inoculation of two phosphate-solubilizing bacteria, Pseudomonas spp. (B2) and Bacillus amyloliquefaciens (B3), alone or in combination with a humic acids (HA) extracted from green compost and/or a commercial inoculum (M) of arbuscular mycorrhizal fungi (AMF), may affect maize growth and soil microbial community. Phospholipid fatty acid (PLFA) and denaturing gradient gel electrophoresis (DGGE) fingerprinting analysis were performed to detect changes in the microbial community composition. Results: Plant growth, N and P uptake, and mycorrhizal root colonization were found to be larger in all inoculated treatments than in the uninoculated control. The greatest P uptake was found when B. amyloliquefaciens was applied in combination with both HA and arbuscular mycorrhizal fungi (B3HAM), and when Pseudomonas was combined with HA (B2HA). The PLFA-based community profile revealed that inoculation changed the microbial community composition. Gram+/Gram− bacteria, AMF/saprotrophic fungi and bacteria/fungi ratios increased in all inoculated treatments. The greatest values for the AMF PLFA marker (C16:1ω5) and AMF/saprotrophic fungi ratio were found for the B3HAM treatment. Permutation test based on DGGE data confirmed a similar trend, with most significant variations in both bacterial and fungal community structures induced by inoculation of B2 or B3 in combination with HA and M, especially in B3HAM. Conclusions: The two community-based datasets indicated changes in the soil microbiome of maize induced by inoculation of B2 or B3 alone or when combined with humic acids and mycorrhizal inoculum, leading to positive effects on plant growth and improved nutrient uptake. Our study implies that appropriate and innovative agricultural © The Author(s) 2021. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Open Access *Correspondence: [email protected]; [email protected] 1 Interdepartmental Research Centre of Nuclear Magnetic Resonance for the Environment, Agro-Food and New Materials (CERMANU), University of Naples Federico II, Via Università 100, 80055 Portici, NA, Italy Full list of author information is available at the end of the article Page 2 of 18 Cozzolino et al. Chem. Biol. Technol. Agric. (2021) 8:31 Background The intensive use of mineral fertilizers and chemical pesticides has been driving the agricultural productivity of the past century. Future challenges for agriculture require innovative cropping technologies for a more efficient management of the limited natural resources to preserve soil fertility and minimize the adverse environmental impact of current agricultural production [1]. In this regard, beneficial plant–microbe interactions can be manipulated to improve crop production in agriculture. Focusing on microbiome engineering is an emerging biotechnological strategy of a sustainable intensification in agriculture [2]. In soil–plant systems, prebiotics are products as organic amendments, usually manure, compost, biochar or humic substances, promoting the growth of probiotics, soil microorganisms already present within the soil– plant system or favoring the establishment of inoculated microorganisms. In the same field, probiotics are specifically considered as beneficial microorganisms, which improve plant fitness and nutrient availability. Use of soil amendments as prebiotics and inoculation with specific probiotics microorganisms represent promising tools to develop and establish a beneficial plant microbiome [2]. Organic fertilizers, such as compost, have the advantage of helping to recycle parts of nutrients, that are already available in the agroecosystem, enriching soil with organic matter that contains nutrients in organic forms less readily accessible to plant uptake and less susceptible to leaching [3, 4]. Thus, their application may increase the nutrient use efficiency, that is one of the final goals of a sustainable intensification of agriculture. In this context, bioeffectors (BEs), including various plant growth-promoting microorganisms (PGPM) and/ or bioactive natural compounds support important functions in sustainable agriculture as supplements to conventional fertilizers; they appear as important boosters of soil fertility enhancing the efficiency of nutrients recycling in the soil [5]. Among microbial BEs, Bacillus and Pseudomonas species are common and abundant bacterial populations in the rhizosphere of various crops [64], suggesting a high competence to colonize plant surfaces and tissues. Strains belonging to these genera provide numerous beneficial traits, such as root growth promotion, solubilization of sparingly soluble nutrients and stimulation of root colonization by mycorrhizal fungi [6, 7]. This last trait plays a key functional role by mediating the transfer of carbon from roots to soil as a source of energy for microbial life and contribute to plant uptake of mineral nutrients and water [8]. However, none of the known PGPM per se has the potential to fulfill the requested requirements of providing a viable alternative to mineral fertilizers [9]. In fact, it has been recently recognized that the interactive and synergistic effects expected from PGPM cannot be achieved with a single application [10, 11]. Mycorrhizal fungi are described as crucial in soil systems since they establish a mutualistic association with the majority of plants [12]. AMF diversity in agricultural soils has been strongly reduced by conventional agricultural practices [13], including overuse of fertilizers, agrochemicals, tillage, long fallow periods. AMF diversity and abundance in agricultural soils could be improved by changing management practices which promote indigenous AMF population, or by reintroducing them through inoculation [14]. The multiple services provided by AMF are the result of the synergistic activities of the bacterial communities living in the mycorrhizosphere, including N fixation, P solubilization, and the production of phytohormones, siderophores, and antibacterial metabolites [15]. Cooperation among beneficial microorganisms has been confirmed for different species of phosphate-solubilising bacteria (e.g., Bacillus spp., Pseudomonas spp.) in diverse plant species [15–20]. Besides synergistic interactions among microbial bioeffectors, a combination with natural bioactive compounds holding plant growth-promoting effects can also provide additional benefits for plant growth. Humic substances (HS) have attracted rising attention by farmers and scientific community owing to their capacity to regulate many ecological and environmental processes and can be used as soil conditioning agents. In fact, HS sustain plant growth and microbial life controlling soil C and N cycling, growth of plants and microorganisms, plant root initiation and architecture, and stabilization of soil structure [21–28]. Several studies reported the synergistic effects of the integrated use of HS plus biofertilizers on plant yield and nutrient uptake [29]. The anatomical and physiological changes in roots induced by HS may favor the fitness of the mutualistic interaction by increasing rhizosphere population and chemotaxis, bacteria attachment and survival on plant surface as well as endophytic management, enhancing the potential contribution of beneficial soil microorganisms as AMF, may result in an improved nutrient use efficiency in plants.