Transition to agroecology promotes mutualistic and reduces antagonistic fungi in lettuce roots

doi:10.21203/rs.3.rs-3093424/v1

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Transition to agroecology promotes mutualistic and reduces antagonistic fungi in lettuce roots

https://doi.org/10.21203/rs.3.rs-3093424/v1

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Efficient and sustainable food production constitutes a significant challenge. Conventional agriculture involves the regular application of pesticides and fertilizers, with a wide range of negative externalities including the compromise of ecosystems stability, pernicious effects on human health, and social problems. These practices may negatively affect soil microorganisms including two groups of widely distributed plant root mutualists: arbuscular mycorrhizal fungi (AMF) and dark septate endophytic fungi (DSEF). These fungi improve host plant nutrition and growth, and protect them against stress. Agroecology has emerged as a demand for a paradigm shift in productive systems and involve the reduced or null inputs of agrochemicals that are replaced by ecological services provided by biodiversity and biotic interactions. Notwithstanding, the knowledge on how agroecological practices affect crops interactions with belowground mutualists and antagonists is limited. In this study, we evaluated through direct observation and molecular methods, the abundance and diversity of mutualistic -AMF and DSEF- and the abundance of antagonistic fungi Olpidiaster brassicae (ex Olpidium) in lettuce roots cultivated in transition to agroecology (TA) and conventional horticulture (CH) fields located in central Argentina. We found that TA, mainly through avoiding agrochemical applications, increased root colonization levels of AMF and DSEF, molecular sequences abundance of AMF, and diversity of both mutualists while decreased percentage of colonization of the pathogen O. brassicae in lettuce. Therefore, through their effects on soil fungi, agroecological managements could benefit plant growth and health.

horticulture

symbiosis

arbuscular mycorrhizal fungi

dark septate endophytic fungi

sustainable agriculture

Efficient and sustainable food production is one of the greatest challenges facing humanity in the context of global change (Godfray et al., 2010; Pawlak & Kołodziejczak, 2020). Nowadays, the agricultural practices based on the regular application of pesticides and fertilizers dominate in most world’s arable lands (Wilkes & Wilkes, 1972; Ramankutty et al., 2018). These practices have many negative consequences such as compromising ecosystem stability through environmental degradation and biodiversity loss (Foley et al., 2005, 2011), as well as adverse effects on human health and increment of social conflicts (El Bilali et al., 2019; Kalyabina et al., 2021). These agricultural practices (hereafter called ‘conventional’) also affect the soil physicochemical properties and microbiomes (Bulluck et al., 2002; Dubey et al., 2019). Even worst, there is evidence that only a small proportion of agrochemicals applied to crops reach the target organisms, while the rest accumulate in soils and water, affecting the soil biota (Riah et al., 2014; Meena et al., 2020).

Among soil biota, there is a wide array of fungi that inhabit plant roots, some of which are harmful while others beneficial (Zeilinger et al., 2016). Among the latter, arbuscular mycorrhizal fungi (AMF), Glomeromycotina (Spatafora et al., 2016), are one of the most widespread plant root mutualists (Smith & Read, 2008). They provide plants access to soil nutrients, such as phosphorous and nitrogen, and protection against harmful organisms and abiotic stress, among other benefits (Dowarah et al., 2021; Jajoo & Mathur, 2021; Marro et al., 2022). Another group of widespread plant root symbionts are the "dark septate endophytic fungi" (DSEF), a polyphyletic group within the phylum Ascomycota (Jumpponen & Trappe, 1998; Jumpponen, 2001; Ruotsalainen et al., 2021). Although direct nutrient exchange between plants and DSEF has not been clearly established, they participate in mineralizing nitrogen and phosphorus, improving the acquisition of these nutrients and plant performance (Newsham, 2011; Andrade-Linares et al., 2011; Prema-Sundara-Valli et al., 2018; Vergara et al., 2017; 2019). In addition, DSEF have also been reported to protect plants against both abiotic and biotic stress (Narisawa et al., 2004, 2018; Andrade-Linares et al., 2011; Prema et al., 2018; Santos et al., 2021; Akhtar et al., 2022).

Plants, including most cultivated species, are usually co-colonized by both types of mutualistic fungi, AMF and DSEF (Das et al., 2010; Priyadharsini et al., 2012; Andrade-Linares et al., 2011; Massenssini et al., 2014; Surendirakumar & Pandey, 2016; Gao et al., 2016). It has been documented that the use of pesticides negatively affect root fungal symbionts and their benefits to cultivated plants (Druille et al. 2013 a,b, Schreiner & Bethlenfalvay, 1997; Kjøller & Rosendahl, 2000; Calonne et al., 2012, Spagnoletti & Chiocchio, 2020; Caccia et al. 2023). In turn, while pathogens could be also negatively affected by pesticide application, some of them are resistant such as fungal species belonging to the genus Olpidiaster (ex Olpidium, Chytridiomycota), which are vectors of two lettuce viruses: "Mirafiori lettuce virus," causing big vein disease, and "Lettuce big-vein associated virus," suspected to cause ring necrosis (Maccarone, 2013; Verbeek et al., 2013). These fungi are capable of remaining dormant in the soil and on plant debris for several years in the form of chlamydospores (Blancard et al., 2003 in Barrière et al. 2014).

Given the negative effects of conventional agriculture, it is claimed that food production should shift to agroecological management (Garibaldi et al., 2017; Seppelt et al., 2020). In a broad sense, the agroecological management seeks to increase the biodiversity and biotic interactions and take advantage of the ecosystem services they provide. In this way, agroecology aims to replace external inputs (such as synthetic fertilizers and pesticides) and rely more on beneficial interactions with soil microorganisms (Altieri, 2018). However, few studies have evaluated the effects of agroecological management on soil fungi and their interactions with plants. Recently, we found that transitioning to agroecology resulted in a higher accumulation of soil fungal richness compared to conventional horticulture at the regional scale, with no significant changes in average local richness and composition (Grilli et al., 2023). Despite there may be no major shifts in fungal communities in the soil, there may be changes in their functioning and/or the interactions they establish with plants because many sequences may be due to relict DNA or strains inactivated by agrochemical use (Carini et al., 2016, Edlinger et al., 2022); though, this issue has been poorly studied.

In this study we aimed to evaluate the intensity of the interaction and the composition of mutualistic and antagonistic fungi in lettuce roots cultivated in crop fields transitioning to agroecology (TA) and with conventional horticulture (CH) located in the green belt of Córdoba city (central Argentina). To this end, we compared a) the percentage of colonization by AMF, DSEF (mutualists) and Olpidaster brassicae (antagonist) in lettuce roots cultivated in TA and CH managements; b) the composition (i.e. richness and abundance) of fungal symbionts in the plant roots using metagenomic tools.

Study area

The study was carried out in an area devoted to fruit and vegetable production, called “green belt”, in the surroundings of Córdoba city, central Argentina. Mean annual temperature in this area is 17 ºC, with thermal amplitude of 14 ºC. Mean annual precipitation is 750 mm mainly concentrated in the warm season of the southern hemisphere (October to April). (Ghuida-Daza & Sanchez 2009).

Experimental design

Seven sites in the green belt of Córdoba were selected, namely: Guiñazú, Horacio, Malvinas, Rosa, Rossi, San Carlos 1, and San Carlos 2. In each site, paired farms including lettuce among their cultivated plants, one with CH and the other in TA managements were selected, with the exception of San Carlos 2 that only presented a CH farm (see Fig. 1). Sites were separated from each other by a minimum of 1.56 km and fields within each site varied between 0.4 and 15.6 hectares. The TA involves reduced or no use of synthetic inputs while the CH involves the application of agrochemicals. All the TA farms use chicken or goat manure and/or ‘Bocashi’ as fertilizers. Bocashi is a Japanese term that means “fermented organic matter”. Most of the CH uses urea and NPK fertilizers. For pests, TA farms apply extracts of Melia azederach and/or a combination of extracts of Allium sativun and Capsicum spp. or even nothing while CH apply different synthetic pesticides (See Grilli et al. 2023 for more details).

In order to encompass different stages of the plant life cycle, we conducted the sampling in two distinct periods (October 4 and 31, 2019) at the beginning and at the end of the crop cycle, in 4 and 8 week old lettuce roots. In each sampling date, roots of 4 lettuce plants were collected with a shovel for each crop field, with the exception of the conventional management field in Rossi site during the first collection, from which only one individual of lettuce could be recovered.

Quantification of root fungal colonization percentages

Roots were taken to the laboratory where they were washed and dried at 60°C for 72 h for their conservation until the determination of percentages of root fungal colonization. In the case of the roots from the second sampling, a part of each one was separated before drying for molecular analyzes. Roots were rehydrated and stained following the hot staining methodology proposed by Brundrett et al. (1996). Subsequently the colonization percentages of AMF (i.e. aseptate hyphae, vesicles and arbuscules -the nutrient exchange structures-), DSEF (i.e. brown septate hyphae and sclerotia) and O. brassicae (i.e. sporangia) were quantified by observation under an optical microscope (Nikon Eclipse E200) at 2000x magnification following the interception method proposed by McGonigle et al. (1990). For each individual, 100 interceptions were covered.

Molecular analysis

The environmental DNA was extracted from 1 g of pooled sample soil per farm with the DNeasy PowerSoil DNA Isolation Kit (QIAGEN) following the manufacturer instructions. Fungal sequences were amplified using the nuclear ITS2 rRNA gene with the forward primer ITS3-kyo2 (5′-GATGAAGAACGYAGYRAA-3′) and the reverse primer ITS4 (5′-TCCTCCGCTTATTGATATGC-3′) designed by Toju et al. (2012). A 10 µl PCR master mix was prepared for each sample and contained 1 µl 10x buffer mix without MgCl2 (RocheHigh Fidelity Fast Start Kit), 1.8 µl 25 mM MgCl2, 0.5 µl DMSO, 0.5 µl 20xAA Loading Reagent (Fluidigm Corpor-ation, South San Francisco, USA), 0.2 µl 10 mM dNTPs each (Roche High Fidelity Fast Start Kit), 0.1 µl Fast Start High Fidelity Taq Polymerase (5 U/µl) (Roche High Fidelity Fast Start Kit) and 1.9 µl PCR grade water. 6 µl PCR master mix, 2 µl DNA and 2 µl barcode primers. Thermal profile was 50°C for 2 min, 70°C for 20 min, 95°C for 10 min, followed by 32 cycles at 95°C for 25 s, 60°C for 30 s and 72°C for 90 s, and a finishing step at 72°C for 5 min (Lange et al., 2014). In order to identify sequences from different soil samples we used Illumina indexes. This approach uses tagmentation to enzymatically fragment and tag the sample DNA with adapters in random positions. This step is followed by a 32 cycle PCR (60° C annealing temperature) in order to add the sequencing indices, PCR cleanup and normalisation. Amplicon libraries were sequenced using 2 x 250 bp paired – end (PE) reads on an Illumina MiSeq sequencing platform (Illumina Inc., San Diego, CA, USA). Sequencing was done at the Unidad de Genómica (INTA – Hurlingham, BA, Argentina).

Bioinformatics

Bioinformatic analysis were performed as in Grilli et al. (2023). In short, the Illumina MiSeq paired-end reads (2 x 250 bp) sequences were cleaned, filtered and analyzed by DADA2 package (Callahan et al., 2016). The 13 samples in this study were amplified successfully. Forward and reverse primers together with their complements were removed from raw sequences with Cutadapt v2.8 (Martin, 2011). Reads with expected errors higher than 2 were discarded. Cleaned and filtered sequences were dereplicated. The amplicon sequence variants (ASVs) were inferred in this study as the standard unit of gene-marker instead of coarser and less accurate denovo OTU clustering approach (Callahan et al., 2017). Single forward reads (R1) were used because they give better results than assembled reads (Pauvert et al., 2019). Sequence variants identified as bimeras were removed by “consensus” decision across samples. Singleton ASVs were considered as artifactual and removed. Taxonomic identity of ASVs was inferred using the AssignTaxonomy() function against the fungal UNITE + INSD taxonomic database version 9.0 (Abarenkov et al., 2022; Nilsson et al., 2019). Representative ASV sequences were submitted to GenBank under BioProject ID: *********.

Statistical analyses

Generalized linear mixed models with Tweedie distribution were used to compare the variables total AMF colonization and percentage of arbuscules, DSEF colonization, and O. brassicae colonization. Tweedie distribution adjusts well to non-negative, right-skewed, zero inflated, and continuous data (Bonat & Kokonendji 2017) such as these response variables. For molecular data, we included AVSs of Glomeromycota, Olpidiaceae and the DSEF species classified into the orders Helotiales, Pleosporaes, Chaetothyriales, Capnodiales, Chaetosphaeriales, Eurotiales, Hypocreales, Microascales, Sordariales and Xylariales (Addy et al., 2005; Jumpponen, 2001; Knapp et al., 2015). The variation of ASV richness and abundance between farming systems was assessed using a generalized linear model with a poisson and a negative binomial family distribution in the overdispersed models. Management variable was used as a fixed factor with two levels: TA and CH, sampling time was used as a fixed factor with two levels: first and second sampling, we include the interaction management × sampling time, the site variable was included as a random factor (only for root colonization data). Analyzes were carried out with the glmmTMB package of R (Magnusson et al., 2017; R Development Core Team, 2021). Graphics were performed with R package ‘ggplot2’ (Wickham 2016; Wickham et al. 2021). R version 1.4.1717 was used.

Root fungal colonization

The percentage of total AMF colonization and colonization by arbuscules was higher in the lettuce roots from fields with TA than in those collected from CH fields. Sampling time was also significant because roots collected in the second sampling were more colonized by AMF than roots collected in the first sampling. We also found a significant interaction term between management × sampling time on AMF colonization because the difference between TA and CH was higher in the second sampling time (Table 1, Fig. 2). The percentage of DSEF colonization was also higher in roots from TA. There was also a significant interaction term between management × sampling time but no effect of sampling time (Table 1, Fig. 2). In turn, the colonization by the pathogenic O. brassicae was higher in plants from CH than from TA, though was not affected neither by the sampling time nor the interaction management x sampling time. (Table 1, Fig. 2).

Table 1

Generalized lineal mixed models outputs for the root colonization percentages by arbuscular mycorrhizal fungi (AMF), arbuscules of AMF, dark septate endophytic fungi (DSEF), and *Olpidiaster brassicae* between lettuce plants from fields with transition to agroecology (TA) and lettuce plants collected in fields with conventional horticulture (CH). Z and p values are shown. Bold values indicate significant differences.
	AMF			arbuscules AMF			DSEF			Olpidiaster brassicae
	Z	±	p	Z	±	p	Z	±	p	Z	±	p
Management	-3.162	±	0.00157	-2.821	±	0.004792	-3.836	±	0.000125	2.734	±	0.00625
Sampling time	4.286	±	1.82e-05	3.453	±	0.000555	1.202	±	0.229282	-1.373	±	0.16966
Management x Sampling time	-3.009	±	0.00262	-0.378	±	0.705741	2.112	±	0.034659	-0.476	±	0.63394

Molecular analyzes

Consistently with the root fungal colonization, AMF sequences abundance was higher in roots from TA fields than in those from CH (Table 2, Fig. 3a). Similar pattern was observed for richness of both AMF and DSEF mutualist fungi (Table 2, Fig. 3b). Instead, DSEF sequences abundance was also higher in TA but did not significantly differed from CH (Table 2, Fig. 3a). In turn, the abundance of O. brassicae sequences was almost three times higher in CH than TA management, although this difference was also not significant (Table 2, Fig. 3a).

Table 2

Molecular analyses outputs for arbuscular mycorrhizal fungi (AMF), dark septate endophytic fungi (DSEF), and *Olpidiaster brassicae* root sequences abundance and richness between lettuce plants from fields with transition to agroecology (TA) and lettuce plants collected in fields with conventional horticulture (CH). Z and p values are shown. Bold values indicate significant differences.
		Management
Abundance		Z	p
	AMF	-5.96	< 0.0001
	DSEF	-1.16	0.25
	Olpidiaster brassicae	1.26	0.234
Richness	AMF	-2.70	0.007
	DSEF	-2.35	0.02
	Olpidiaster brassicae	-0.04	0.97

Global food production occurs at a crossroads. While there is a need to intensify production to meet the demands of a growing global population, it is also necessary to reduce the negative impacts of this intensification; otherwise, production becomes unsustainable for the environment and detrimental to human health (Foley et al., 2005; 2011; Kalyabina et al., 2021). There is a wealth amount of studies on how agroecology increase biodiversity, ecological processes and the associated ecosystem services, although they mostly focused on aboveground organisms (Garibaldi et al., 2017). The positive effects of agroecological managements on root mutualists richness and their interactions with a cultivated plant, accompanied with a negative effect on the fungal pathogen O. brassicae, is novel evidence that such agroecological aboverground benefits can be also enhanced belowground.

Recently, it has been observed that the richness and abundance of soil fungi did not differ at a local scale between TA and CH fields (Grilli et al., 2023). The strong differences in root colonization by mutualistic fungi observed here in the same fields, suggests that while CH may not have major observable negative effects on soil fungal communities, it may have profound effects on their interactions with cultivated plants. These results suggest that CH, possibly through the application of agrochemicals, has no major effects on the composition of soil fungi because their propagules persist in soils, but on their functioning and the interactions that they establish with plants. Though, these effects on the interactions with plants might have effects on soil fungal community composition over longer terms.

Some of the findings of this study are in line with previous results showing that reduction of chemical input-based agriculture enhanced AMF colonization (e.g., Galván et al., 2009; Banerjee et al., 2019), albeit Galvan et al. (2009) did not observe increased AMF richness. The higher percentage of root colonization by fungal mutualists may have nutritional consequences on plants (Treseder, 2013) but also affect the aboveground plant interactions, such as herbivore-plant interactions (Hartley et al., 2009). The higher AMF colonization and colonization by arbuscules of AMF in the second sampling time may be explained by plants stage of development, the 8 week plants possibly can provide more carbon fixed by photosynthesis to AMF symbionts than the 4 week lettuce plants. Additionally, there was an effect of interaction management × sampling time for both mutualist fungi -AMF and DSEF- suggesting that agrochemical negative effects in the CA fields may accumulate over time.

It has been recently shown that the higher mycorrhizal colonization promoted by AMF from TA management did not enhance lettuce grow, neither nutrient content, but plants were less attacked by leaf miners (Caccia et al., 2023). In that study instead, the inoculation with soil microorganisms, irrespective of the presence of AMF, promoted increases in leaf phosphorus content compared to sterile soil, suggesting that DSEF, but not AMF, could play an important role in the nutrition of this plant. Accordingly, it has been observed that DSEF can improve growth and nutrition of tomato plants under regular, biotic stress, and abiotic stress conditions (Andrade-Linares et al., 2011; Vergara et al., 2017; Prema et al., 2018). Prema et al. (2018) also found that Cadophora orchidicola (Heliotales) doubled the biomass of tomatoes and these effects depended on root colonization. Also, it has been observed that DSEF improved nitrogen content and micronutrient levels in rice plants (Vergara et al., 2019).

It has been proposed that the higher root colonization by mycorrhizal fungi also protect plants against root fungal pathogens (Newsham et al. 1995, Chagnon et al. 2013). Then, the decreased in O. brassicae colonization in TA management observed here, could be the consequence of the increases in AMF and DSEF colonization. Conversely, the decrease in the colonization of AMF and DSEF under CH could explain the increase in the colonization of O. brassicae, together with its resistance to agrochemicals.

Altogether, these findings suggest that AMF and DSEF root colonization may have complementary benefits on cultivated plants. Indeed, Gao et al. (2016) found that the combined inoculation with AMF and DSEF improved cucumber plant growth, yield, and protection against nematode Meloidogyne incognita compared to simple inoculation treatments with AMF or DSEF.

Fungal molecular sequences abundance in lettuce roots showed comparable patterns to percentage of root fungal colonization for AMF and was accompanied with higher fungal richness in both groups of symbionts (i.e. AMF and DSEF) in TA. The lower richness of AMF and DSEF in roots from CH fields is consistent with previous evidence showing that conventional intensification reduces interaction network complexity in wheat root microbiome (Banerjee et al. 2019). The higher richness of AMF in TA is relevant because it has been shown that distinct AMF taxa differ in their effects on plant nutrition and stress alleviation (Marro et al. 2022) and there could exist functional complementarity in the benefits provided by them (Maherali & Klironomos 2007). Similarly, genomic and molecular data on DSEF supports their functional heterogeneity given by distinctive metabolic machineries among different taxa (Knapp & Kovács 2016; Knapp et al. 2018). Anyway, whether a generalized functional complementarity between AMF and DSEF taxa exists will require further insights to be proved. What has been proven is that more complex fungal communities tend to increase the services that mycobiomes provide to agroecosystems, such as increases in crop productivity and health (Frąc et al., 2022).

All in all, our findings show that TA management promote higher fungal mutualist richness and stronger interactions with lettuce roots accompanied with less intense fungal pathogen infection. This management may lead to more resistant and resilient agroecosystems in comparison to CH crop lands that are poorer in biodiversity and ecological interactions, and thus more sensitive to biotic and abiotic stressors (Cappelli et al., 2022).

Overall, our results show that transition to agroecology (TA) management improves colonization percentage levels and sequences abundance of mutualist arbuscular mycorrhizal fungi (AMF), and diversity of both mutualistic fungi: AMF and dark septate endophytic fungi (DSEF); while diminish colonization percentage and has no effect in sequences abundance of the pathogenic fungi Olpidiaster brassicae in lettuce roots. This suggests that a) the application of agrochemicals in conventional management negatively affects most of the fungi in lettuce roots, including important symbiotic organisms, and b) the pathogen O. brassicae is not affected by agrochemicals. Therefore, through its effects on soil fungi, agroecological management can benefit plant health.

Acknowledgements

We thank the farmers of the sampled sites from Córdoba green belt (Argentina) for kindly letting us extract soil samples from their fields. The authors also wish to acknowledge the assistance of the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) and the Universidad Nacional de Córdoba (U.N.C), both of which have provided facilities used for this study. We also thank Magalí Burni who collaborated in the root staining process.

Funding

This research program is funded by Secretaría de Ciencia y Tecnología de la Universidad Nacional de Córdoba (Secyt), Ministerio de Ciencia de la Provincia de Córdoba and Fondo para la Investigación Científica y Tecnológica (FONCYT).

Authors contribution

All authors contributed to the study conception, design and samplings. Staining of roots and quantification of fungal colonization percentages was performed by VB. DNA extraction and bioinformatic work was performed by GG. Statistical analyses were performed by GG and VB. Graphics were performed by GG and VB. The first draft of the manuscript was written by VB and CU, and all authors revised and commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Competing Interests

The authors have no relevant financial or non-financial interests to disclose.

Data availability

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

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Transition to agroecology promotes mutualistic and reduces antagonistic fungi in lettuce roots

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Experimental design

Quantification of root fungal colonization percentages

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Root fungal colonization

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