D2.3.0_AGROECOseqC_WP2_10.5281zenodo.14168308

Towards climate-smart sustainable management of agricultural soils AGROECOlogical strategies for an efficient functioning of plant - soil biota interactions to increase SOC sequestration AGROECOseqC Deliverable D2.3.0 Phosphorus-Solubilizing Fungi in Sustainable Agricultural Practices Due date of deliverable: M54 Actual submission date: 15.11.2024 Deliverable D2.3.0 Phosphorus-Solubilizing Fungi in Sustainable Agricultural Practices This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement N° 862695 2 GENERAL DATA Grant Agreement: 862695 Project acronym: EJP SOIL Programme title: Towards climate-smart sustainable management of agricultural soils Programme website: www.ejpsoil.eu Project title: Project website: Start date of the project: February 1st, 2020 Project duration: 60 months Name of lead contractor: INRAE Funding source: H2020-SFS-2018-2020 / H2020-SFS-2019-1 Type of action: European Joint Project COFUND DELIVERABLE NUMBER: D2.3.0 DELIVERABLE TITLE: Phosphorus-Solubilizing Fungi in Sustainable Agricultural Practices DELIVERABLE TYPE: Report WORK PACKAGE N: WP2 WORK PACKAGE TITLE: Plant Communities And Rhizosphere PlantMicrobe Interactions DELIVERABLE LEADER: Akin UN, TAGEM (Turkiye) AUTHOR: DOI: Akin UN, Sahimerdan Turkolmez, Alessandra Trinchera, Elena Testani, Dylan Warren Raffa, Skaidre Suproniene, Sara Sanchez-Moreno, Jim Rasmussen, Simon Sail, Marjoleine Hanegraaf, Sebastien Fontaine 10.5281/zenodo.14168308 LICENSE DISSEMINATION LEVEL: CC BY 4.0 CO Deliverable D2.3.0 Phosphorus-Solubilizing Fungi in Sustainable Agricultural Practices This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement N° 862695 3 ABSTRACT This study explores the potential of phosphorus-solubilizing fungi to enhance sustainable agriculture by supporting the phosphorus cycle in soils across various agroecological sites in Europe and Turkiye. Three treatments were compared in each core site: the first two with increasing levels of agroecological intensification (T1 < T2) and a third one, taken as a control (T3), where no agroecological intensification was introduced (e.g., business as usual). By examining microbial activity under treatments T1, T2, and T3, we evaluated how different levels of agroecological intensification influence fungal communities and their phosphorus-solubilizing capacities. By examining microbial activity under treatments T1, T2, and T3, we evaluated how different levels of agroecological intensification influence fungal communities and their phosphorus-solubilizing capacities. Treatment T2, characterized by the highest agroecological intensification, showed a marked increase in colonyforming units (CFU), reflecting heightened biological activity. However, despite this increase, phosphorus solubilization efficiency was limited, particularly in soils dominated by pathogenic fungi like Fusarium spp., Rhizopus spp., and Alternaria spp., which are known to hinder plant growth. Conversely, non-pathogenic fungi such as Trichoderma spp., Talaromyces spp., and Clonotachys spp. displayed promising phosphorus-solubilizing abilities without compromising plant health. These beneficial fungi not only contribute to phosphorus cycling but also support soil health by reducing reliance on synthetic fertilizers and enhancing nutrient availability. The presence of these fungi underscores their potential as biofertilizers in sustainable agriculture. Our findings emphasize the importance of an integrated soil management strategy that combines nutrient management with pathogen control to maintain long-term soil fertility and crop productivity. This study highlights that while high CFU counts can signal increased biological activity, effective phosphorus solubilization relies on fostering beneficial microbial communities over pathogenic ones. Future research should further investigate the long-term impacts of agroecological intensification on microbial diversity and phosphorus availability, focusing on the role of plant-microbe interactions to promote resilient, sustainable agricultural systems. Deliverable D2.3.0 Phosphorus-Solubilizing Fungi in Sustainable Agricultural Practices This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement N° 862695 4 Table of Contents ABSTRACT ............................................................................................................................................................... 3 Table of Contents ................................................................................................................................................... 4 List of Tables ........................................................................................................................................................... 4 1. Introduction .................................................................................................................................................... 5 2. Materials and Methods .................................................................................................................................. 6 2.1. Description of experimental sites .......................................................................................................... 6 2.2. Sampling ............................................................................................................................................... 7 2.3. Method .................................................................................................................................................. 7 3. Main results .................................................................................................................................................... 8 3.1. Observed Dominated Fungi ................................................................................................................... 8 3.2. CFU Results by Each Core Site ............................................................................................................... 9 4. Conclusions ................................................................................................................................................... 12 5. List of references .......................................................................................................................................... 13 List of Tables Table 1: Description of the AGROECOseqC experimental sites, including the country, country code, coordination, climate and soil properties, treatment codes, management practices, and the amount of fertilization applied in each treatment. .................................................................................................................. 7 Table 2: Commonly observed fungi and separated as beneficial and harmful based on their effect in soils and plants ....................................................................................................................................................................... 8 Table 3:Colony forming units per gram (CFU/g) and average solubilization efficiency (mg/L) of dominated fungi with also indicated alongside their respective phosphate solubilization efficiency in mg/L. across different treatments. ........................................................................................................................................................... 11 List of Figures Figure 1: Position map of n. 8 AGROECOseqC experimental sites .......................................................................... 6 Figure 2: CFU Results For Each Experimental Site ................................................................................................... 9 List of acronyms and abbreviations WP Work Package EU European Union P Phosphorus PS Phosphorus-solubilizing Deliverable D2.3.0 Phosphorus-Solubilizing Fungi in Sustainable Agricultural Practices This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement N° 862695 5 Introduction The rapid population growth after the 1950s triggered a global food security crisis, creating an urgent need to increase agricultural productivity. In response, synthetic fertilizers and chemical pesticides became widely adopted, providing short-term boosts in crop yields. However, the intensive use of these chemicals has led to serious environmental costs, such as degraded soil health, reduced biodiversity, and disrupted ecosystems (Ayala and Rao, 2002; Savci, 2012). Today, sustainable agriculture has become an urgent necessity rather than a choice, especially as rising food demand requires more efficient use of limited resources. Phosphorus (P) is an essential macronutrient for plant growth and energy transfer, yet it is also one of the most limited nutrients in agriculture (Raghothama, 1999). Plants can only absorb P in specific forms, and large reserves of P often accumulate in soils in forms unavailable to plants (Asomaning, 2020). Overuse of synthetic phosphate fertilizers not only leads to economic losses but also causes the accumulation of toxic metals in the soil, further degrading soil health and productivity over time (Wang et al., 2017). This situation underscores the need for more sustainable and environmentally friendly technologies. Phosphorus-solubilizing (PS) microorganisms, particularly fungi, offer a promising solution by making insoluble P in the soil accessible to plants. Fungi can release P ions through the production of organic acids, increasing its availability for plant uptake (Havlin et al., 1999; Sembiring et al., 2017). Genera such as Aspergillus, Penicillium, and Fusarium are well-known for their phosphorus-solubilizing abilities (Karpagam and Nagalaksmi, 2014). Additionally, fungi play a critical role in decomposing organic matter, thereby enhancing soil fertility and structure (Anand et al., 2016). PS fungi species, such as Aspergillus, Penicillium, Trichoderma, and Fusarium, enhance P availability to plants through the production of organic acids, while other species like Acremonium and Chaetomium can reduce the reliance on phosphate fertilizers and help mitigate environmental pollution. These fungi show strong potential as biofertilizers due to their PS capacities, which promote plant growth and soil health (Karpagam and Nagalaksmi, 2014). A common approach to identify and measure the PS ability of these fungi is the Pikovskaya Method, which utilizes a special medium containing insoluble phosphates. Developed by Pikovskaya (1948), this method identifies effective PS fungi by the clear zones they form in the medium, indicating phosphate solubilization. It also enables the comparison of solubilizing capacities across fungal species, aiding in the selection of the most suitable fungi for agricultural applications (Asomaning, 2020; Karpagam and Nagalaksmi, 2014). In this study, soil samples collected from various experimental sites across Europe and Turkey were examined to identify phosphorus-solubilizing fungi, employing the Pikovskaya method for isolation. The fungal species' colonization rates and their capacity to solubilize phosphorus were thoroughly assessed. The phosphorussolubilizing efficiency of these isolated fungi was measured using the P Olsen method, enabling the identification of species with high solubilization potential. Through this analysis, valuable insights were gained into the biodiversity of soil fungi, emphasizing the importance of preserving, enriching, and leveraging the diversity of these microorganisms to support soil health and fertility. Deliverable D2.3.0 Phosphorus-Solubilizing Fungi in Sustainable Agricultural Practices This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement N° 862695 6 Materials and Methods The AGROECOseqC project operated by applying a multifunctional approach, based on a multi-site study, representative of different European cropping systems under different pedoclimate regions. Description of experimental sites Seven European experimental sites, located in Italy (IT, S1), France (FR, S2), Belgium (BE, S3), The Netherland (NL, S4), Lithuania (LT, S5), Spain (ES, S6), Denmark (DK, S7), and an extra-European one in Turkiye (TK, S8,) were considered. In Figure 1, a map of the twelve experimental site location is reported. Figure 1: Position map of n. 8 AGROECOseqC experimental sites Six of the experimental sites (S1, S2, S3, S4, S5, and S6) were part of long-term experimental, whereas the remaining two sites (S7 and S8) were recently established. Notably, only S1 has been managed organically since 2017 (Table 1). At each site, three different treatments were evaluated: T1 and T2 represented increasing levels of agroecological intensification (with T1 < T2), while T3 served as a control with no agroecological intensification applied, reflecting typical conventional practices. The T1 and T2 treatments involved one or more agroecological practices, either individually or in combination, such as no-tillage, cover cropping, crop rotation, soil amendment or farmyard manure application, and crop residue return. These agroecological practices were applied as forms of "treatment" to assess their level of intensification. Table 1 provides a description of each of the eight European experimental sites, detailing key information and the specific agroecological practices used within each cropping system. Deliverable D2.3.0 Phosphorus-Solubilizing Fungi in Sustainable Agricultural Practices This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement N° 862695 7 Sampling In each site, T1, T2 and T3 treatment were tested in four replicates (as randomized blocks), for a total of 3 treatments x 4 blocks = 12 plots/site and n. 96 plots in total. Four soil samples per plot were collected in each experimental site at maximum plant demand and then mixed to form n.1 soil composite sample per plot (four replicates per treatment). Also, roots were collected following the same sampling procedures. All the soil parameters and bioindicators were determined on the composite soil or root samples collected in each block. The case studies approached agroecological intensification differently in their comparative systems, each working within distinct agroecosystems and employing practices tailored to their specific contexts. Table 1 provides a summary of the strategies introduced in relation to the controls. Soil samples were collected from a depth of 0–20 cm for each replicate during the period of maximum plant nutrient uptake, which corresponds to the period of highest fungal activity. Method Soil samples were collected from a depth of 0-20 cm across various regions and analyzed to identify PS microorganisms. The Pikovskaya Agar Medium was sterilized at 121°C and poured into petri dishes, where 1 gram of each soil sample was added. The dishes were incubated at 25°C for one week, and zones of P solubilization were observed. Colonies forming these zones were transferred to PDA medium for purification. Purified isolates were then incubated at 25±1°C with a 12-hour light/dark cycle and classified based on morphological characteristics. Selected representative isolates were further identified by their colony morphology and reproductive structures, and examined microscopically. The colony-forming units (CFU) per gram of soil were calculated following Christensen’s formula, considering colony counts, dilution factor, and soil moisture content (Christensen, 1981). Subsequently, quantitative P solubilization was assessed. Soil samples from each partner country were dissolved in sterile distilled water, inoculated with 5 mg of phosphorus-solubilizing fungi, and incubated at 26°C for 10 days. The solubilized P content was then measured using the Olsen P method. Non-pathogenic fungi with high PS potential were prioritized for further evaluation as candidates for sustainable agricultural applications. Calculated results were evaluated statistically by ANOVA one way method. Table 1: Description of the AGROECOseqC experimental sites, including the country, country code, coordination, climate and soil properties, treatment codes, management practices, and the amount of fertilization applied in each treatment. Deliverable D2.3.0 Phosphorus-Solubilizing Fungi in Sustainable Agricultural Practices This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement N° 862695 8 Main results Observed Dominated Fungi The fungi observed in each treatment from each experimental field were listed. Table 2 presents the fungi commonly found in soil samples, categorizing them into beneficial and harmful groups. Table 2: Commonly observed fungi and separated as beneficial and harmful based on their effect in soils and plants Order Beneficial Fungi Harmful Fungi 1 Trichoderma spp. Aspergillus spp. 2 Talaromyces spp. Mucor spp. 3 Clonotachys spp. Rhizopus spp. 4 Fusarium spp. 5 Bipolaris spp. 6 Cercospora spp. 7 Rhizoctonia spp. 8 Penicillium spp. 9 Alternaria spp. 10 Cladosporium spp. 11 Phytophthora spp. 12 Pythium spp. 13 Verticillium spp Deliverable D2.3.0 Phosphorus-Solubilizing Fungi in Sustainable Agricultural Practices This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement N° 862695 9 CFU Results by Each Core Site AGROECOseqC experimental sites were reported the results of the CFU in six experimental sites based on applied agroecological managements and evaluated statistically. S3 and S4 were not evaluated due to the lack of meaningful data. Results shows in Figure 1 with statistical analysis and Table 3 with CFU, observed dominated fungi and their average solubilization efficiency (mg/L). Upon examination of samples from organically managed S1 site (Italy), it was observed that the soil is characterized by very high fungal activity and richness. Although harmful fungi such as Fusarium were among the dominant species observed, their P solubilization capabilities were also investigated. It was found that these fungi possess significant P solubilization abilities, and the density of the colonies formed was notably high across all treatments. T2 treatment gave the significantly highest average colony count (p<0.05) at 371,029 CFU/g, followed by T1 at 234,296 CFU/g and T3 at 163,925 CFU/g. Additionally, the results of the Phosphate Solubilization Efficiency of the fungi, revealed that the highest P solubilizers are found in the T2 treatment. Identifying these fungi at the species level and categorizing them by genus is anticipated to offer valuable guidance for future research. Analysis of soil samples from S2 site (France) showed the soil is rich in fungal activity. However, it was found that the PS capabilities of the dominant fungi are low. Fungi such as Fusarium spp., Alternaria spp., and Bipolaris spp., which are among the dominant species, are thought to potentially cause diseases and yield losses in crops grown in these soils. Despite a high soil fungal activity observed, these fungi may have pathogenic effects, underscoring the need for an effective plant protection program. The results showed T3 exhibited the highest average colony count at 267,457 CFU/g, followed by T2 with 246,002 CFU/g, and T1 at 155,865 CFU/g. Although T3 showed the highest colony density, statistical analysis indicated that these differences were not significantly different (p <0.05). Analysis of soil samples from LAMMC, Lithuania (S5), indicates that the soil is moderately rich in fungal activity. However, the PS capabilities of the dominant fungi have been found to be low. Even thought, the T2 treatment has the highest average colony count at 169,156 CFU/g, followed by T1 at 132,418 CFU/g, and T3 at 112,623 CFU/g, differences between treatments were not statistically significant. Analysis of soil samples from INIA-CSIC, Spain (S6), indicates that the soil is rich in fungal activity. However, the PS capabilities of the dominant fungi have been found to be low. Among the dominant fungi, while there are beneficial species present, harmful fungi are also identified, with the latter forming more dominant colonies compared to the beneficial ones. These harmful fungi may potentially cause diseases and yield losses in crops grown in the soil. Despite the moderate levels of biological activity concerning beneficial fungi, the presence of potentially pathogenic fungi underscores the need for an effective plant protection program. even the T2 treatment exhibited the highest average colony Figure 2: CFU Results For Each Experimental Site