"Role of Arbuscular Mycorrhizal Fungi in Nutrient Uptake and Growth of Durum Wheat"

Soil microbiome is involved at different levels in the food web, in bio-geochemical nutrient cycles and in several interactions with plants. Based on its key role in the agro-ecosystem processes, the soil microbiome has been identified as one of the principal factors in an agriculture addressed to the ecological intensification. Among the several relationships established between plants and soil microorganisms, arbuscular mycorrhizal (AM) symbiosis is the most widespread. Two out of three of all plant taxa (among others the main crops) are involved in the AM symbiosis which takes place between the plant root system and arbuscular mycorrhizal fungi (AMF), a monophyletic group of fungi belonging to the subphylum of Glomeromycotina. Although AM symbiosis can provide several positive services in the agroecosystem, the main benefit has always been highlighted in the increment of plant nutrition. However, the outcome of the AM symbiosis is context dependent. The greatest benefits ascribed to AMF has been observed on plant P acquisition under conditions of soil P-deficiency, whereas their contribution on plant N nutrition is still debate, since positive, neutral and negative effect has been observed. The reason of such contradictory results seems to rely on the soil N availability, since AMF have a notable N demand for their own metabolism and can even compete with the host plant for the soil N under soil N-deficiency. Additionally, although AMF can transfer N from organic source, no information is available on whether or not this ability change varying the organic source composition. Given the role of AMF in nutrient cycling, uptake and transfer to the host plant, increasing our knowledge about their role on plant nutrition is crucial in an agricultural addressed to the environment and economic sustainability. With the view to the agriculture sustainability, the reduction or the absence of tillage can provide several environmental and agronomical benefits. At the same time, different tillage system determines various pedo-climatic micro-environments which can profoundly influence the soil microbial community composition. Different pedo-climatic micro-environments are also observed along the soil profile with the consequence of drastic modifications in the community composition of bacteria and fungi (including AMF) along the soil depth. Modification in the community composition can differ in symbiotic efficiency and therefore drive the outcome of the interaction between crop and soil microorganisms. However, the potential effect of the different communities deriving from the above reported microenvironments on plant growth has not yet been investigated. In order to contribute to fill the above reported gap of knowledge a set of 4 experiments (described in chapter 2) was carried out. Two experiments (paragraph 2.1 and 2.2) aimed to evaluate the effect AM symbiosis outcome varying soil N and P availabilities, and the effect of AMF on plant growth, N uptake and N recovery from the applied fertilizer when N in soil was applied as mineral or organic source. The third experiment (paragraph 2.3) aimed to characterize the AMF community along the soil profile and to evaluate if the observed differences were able to affect the plant growth and nutrient uptake under adequate water availability or under drought stress. Finally, the fourth experiment (paragraph 2.4) focused on the effect of the soil microbial community deriving from different long-term tillage management and depth on plant growth an N uptake. In all the experiments durum wheat was used as focal plant. Results have shown that under soil N-deficiency AMF compete with the host plant determining a decrement of plant growth and N uptake. A negative effect of AMF on plant growth was also observed under very high soil N availability, in absence of other limiting factors. Whereas, a positive AMF effect was observed at intermediate soil N availability, when the host plant is still under N-limiting conditions and the fungal component has satisfied its own demand. In the latter case, AMF have shown the ability to transfer a substantial amount of N derived from mineral fertilizer and organic matter. However, the organic matter composition has strongly affected the effect of AMF on plant performance. In fact, while AMF increased the plant N recovery from the organic patch with a low C:N ratio, a detrimental effect of plant growth and N recovery was observed in presence of an organic source with a high C:N ratio. Results have also shown that the AM symbiosis outcome in presence of different soil N availability conditions may change in relation to the availability of other elements. In fact, while under conditions of high P availability, the mycorrhizal outcome shifted along the entire spectrum of the ecological relationships (mutualism, commensalism or parasitism) depending on the availability of N, under soil P-deficiency, AMF have always provided a benefit to the host plant, regardless the soil N availability. Results of the third experiment have highlighted a significant shift of AM fungal communities with depth and the existence of subsoil specific AM fungal phylotypes. The inoculation with living soil deriving from different depths resulted in variations in root colonization consistent with those detected by molecular analysis, but have had little or no effect on plant performance both with adequate water availability and in presence of drought. On the contrary, significant differences on root colonization, aboveground biomass production and N uptake were observed when plants were inoculated with living soil deriving from different tillage systems and soil depth (paragraph 2.4)

Microplastic incorporation into soil in agroecosystems

Background: We live in a plastic age (Thompson et al., 2009), with microplastic (typically defined as plastic particles < 5 mm) becoming an increasingly appreciated aspect of environmental pollution. Research has been overwhelmingly focused on aquatic systems, especially the oceans, but there is a current shift to more strongly consider terrestrial ecosystems (Rillig, 2012; Horton et al., 2017). In particular agroecosystems are coming into focus as a major entry point for microplastics in continental systems (Nizzetto et al., 2016b), where contamination might occur via different sources as sludge amendment or plastic mulching (Steinmetz et al., 2016). Given the central role of agroecosystems, including their soil biodiversity (Rillig et al., 2016), in food production, such numbers are potential cause for concern. Field data on measured microplastic presence in agricultural soils are still not widely available, but nevertheless this material is certain to arrive at the soil surface. The fate of material deposited at the soil surface is not clear: particles may be removed by wind or water erosion, becoming airborne, or may be lost by surface runoff (Nizzetto et al., 2016a). Nevertheless, a substantial part of the microplastic (or nanoplastic following further disintegration) is expected to enter the soil. The degree of hazard represented by microplastic to various soil biota is not clear. Direct evidence comes from experimental work on earthworms, on which microbeads had negative effects (Huerta Lwanga et al., 2016; also reviewed in Horton et al., 2017). Data on impacts on other soil biota groups are not available. However, Kiyama et al. (2012) have shown that polystyrene beads can be taken up by the nematode Caenorhabditis elegans; this means the material could also accumulate in the soil food web (Rillig, 2012). Movement into soil is an important aspect of assessing risk: will soil biota be exposed to microplastics? Here, we sketch what is known about movement of such particles in soil, which players and factors could influence this, and we chart avenues for research aimed at the movement and distribution of microplastic in agricultural soils

Impacts of arbuscular mycorrhizal fungi on nutrient uptake, N2 fixation, N transfer, and growth in a wheat/faba bean intercropping system

Arbuscular mycorrhizal fungi (AMF) can play a key role in natural and agricultural ecosystems affecting plant nutrition, soil biological activity and modifying the availability of nutrients by plants. This research aimed at expanding the knowledge of the role played by AMF in the uptake of macro- and micronutrients and N transfer (using a 15 N stem-labelling method) in a faba bean/wheat intercropping system. It also investigates the role of AMF in biological N fixation (using the natural isotopic abundance method) in faba bean grown in pure stand and in mixture. Finally, it examines the role of AMF in driving competition and facilitation between faba bean and wheat. Durum wheat and faba bean were grown in pots (five pots per treatment) as sole crops or in mixture in the presence or absence of AMF. Root colonisation by AMF was greater in faba bean than in wheat and increased when species were mixed compared to pure stand (particularly for faba bean). Mycorrhizal symbiosis positively influenced root biomass, specific root length, and root density and increased the uptake of P, Fe, and Zn in wheat (both in pure stand and in mixture) but not in faba bean. Furthermore, AMF symbiosis increased the percentage of N derived from the atmosphere in the total N biomass of faba bean grown in mixture (+20%) but not in pure stand. Nitrogen transfer from faba bean to wheat was low (2.5–3.0 mg pot -1 ); inoculation with AMF increased N transfer by 20%. Overall, in terms of above- and belowground growth and uptake of nutrients, mycorrhization favoured the stronger competitor in the mixture (wheat) without negatively affecting the companion species (faba bean). Results of this study confirm the role of AMF in driving biological interactions among neighbouring plants. and analysis, decision to publish, or preparation of the manuscript

a solution right under our feet?

With growing populations and climate change, assuring food and nutrition security is an increasingly challenging task. Climate smart and sustainable agriculture, that is, conceiving agriculture to be resistant and resilient to a changing climate while keeping it viable in the long term, is probably the best solution. The role of soil biota and particularly arbuscular mycorrhizal (AM) fungi in this new agriculture is believed to be of paramount importance. However, the large nutrient pools and the microbiota of subsoils are rarely considered in the equation. Here we explore the potential contributions of subsoil AM fungi to this agriculture and suggest future research goals that would allow us to maximize their benefits

Subsoil Arbuscular Mycorrhizal Fungi for Sustainability and Climate-Smart Agriculture: A Solution Right Under Our Feet?

With growing populations and climate change, assuring food and nutrition security is an increasingly challenging task. Climate-smart and sustainable agriculture, that is, conceiving agriculture to be resistant and resilient to a changing climate while keeping it viable in the long term, is probably the best solution. The role of soil biota and particularly arbuscular mycorrhizal (AM) fungi in this new agriculture is believed to be of paramount importance. However, the large nutrient pools and the microbiota of subsoils are rarely considered in the equation. Here we explore the potential contributions of subsoil AM fungi to a reduced and more efficient fertilization, carbon sequestration, and reduction of greenhouse gas emissions in agriculture. We discuss the use of crop rotations and cover cropping with deep rooting mycorrhizal plants, and low-disturbance management, as means of fostering subsoil AM communities. Finally, we suggest future research goals that would allow us to maximize these benefits

Transcriptome changes induced by Arbuscular mycorrhizal symbiosis in leaves of durum wheat (Triticum durum Desf.) promote higher salt tolerance

The salinity of soil is a relevant environmental problem around the world, with climate change raising its relevance, particularly in arid and semiarid areas. Arbuscular Mycorrhizal Fungi (AMF) positively affect plant growth and health by mitigating biotic and abiotic stresses, including salt stress. The mechanisms through which these benefits manifest are, however, still unclear. This work aimed to identify key genes involved in the response to salt stress induced by AMF using RNA-Seq analysis on durum wheat (Triticum turgidum L. subsp. durum Desf. Husn.). Five hundred sixty-three differentially expressed genes (DEGs), many of which involved in pathways related to plant stress responses, were identified. The expression of genes involved in trehalose metabolism, RNA processing, vesicle trafficking, cell wall organization, and signal transduction was significantly enhanced by the AMF symbiosis. A downregulation of genes involved in both enzymatic and non-enzymatic oxidative stress responses as well as amino acids, lipids, and carbohydrates metabolisms was also detected, suggesting a lower oxidative stress condition in the AMF inoculated plants. Interestingly, many transcription factor families, including WRKY, NAC, and MYB, already known for their key role in plant abiotic stress response, were found differentially expressed between treatments. This study provides valuable insights on AMF-induced gene expression modulation and the beneficial effects of plant-AMF interaction in durum wheat under salt stress

Exploring the genetic landscape of nitrogen uptake in durum wheat: genome-wide characterization and expression profiling of NPF and NRT2 gene families

Nitrate uptake by plants primarily relies on two gene families: Nitrate transporter 1/peptide transporter (NPF) and Nitrate transporter 2 (NRT2). Here, we extensively characterized the NPF and NRT2 families in the durum wheat genome, revealing 211 NPF and 20 NRT2 genes. The two families share many Cis Regulatory Elements (CREs) and Transcription Factor binding sites, highlighting a partially overlapping regulatory system and suggesting a coordinated response for nitrate transport and utilization. Analyzing RNA-seq data from 9 tissues and 20 cultivars, we explored expression profiles and co-expression relationships of both gene families. We observed a strong correlation between nucleotide variation and gene expression within the NRT2 gene family, implicating a shared selection mechanism operating on both coding and regulatory regions. Furthermore, NPF genes showed highly tissue-specific expression profiles, while NRT2s were mainly divided in two co-expression modules, one expressed in roots (NAR2/NRT3 dependent) and the other induced in anthers and/ovaries during maturation. Our evidences confirmed that the majority of these genes were retained after small-scale duplication events, suggesting a neo- or sub-functionalization of many NPFs and NRT2s. Altogether, these findings indicate that the expansion of these gene families in durum wheat could provide valuable genetic variability useful to identify NUE-related and candidate genes for future breeding programs in the context of low-impact and sustainable agriculture

Subsoil Arbuscular Mycorrhizal Fungi for Sustainability and Climate-Smart Agriculture: A Solution Right Under Our Feet?

With growing populations and climate change, assuring food and nutrition security is an increasingly challenging task. Climate-smart and sustainable agriculture, that is, conceiving agriculture to be resistant and resilient to a changing climate while keeping it viable in the long term, is probably the best solution. The role of soil biota and particularly arbuscular mycorrhizal (AM) fungi in this new agriculture is believed to be of paramount importance. However, the large nutrient pools and the microbiota of subsoils are rarely considered in the equation. Here we explore the potential contributions of subsoil AM fungi to a reduced and more efficient fertilization, carbon sequestration, and reduction of greenhouse gas emissions in agriculture. We discuss the use of crop rotations and cover cropping with deep rooting mycorrhizal plants, and low-disturbance management, as means of fostering subsoil AM communities. Finally, we suggest future research goals that would allow us to maximize these benefits

Soil Nitrogen Form and Availability affect the role of Arbuscular Mycorrhizal Fungi on Nitrogen Uptake and Nitrogen Recovery in Durum Wheat

The term Arbuscular Mycorrhizal (AM) is referred to the symbiosis between arbuscular mycorrhizal fungi (AMF) and plant roots. Such symbiosis is the most widespread among plants (two out of three of the all plant taxa) including the majority of crops. AMF belong to the monophyletic subphylum Glomeromycotina which include obligate biotrophs that entirely relay on the host plant for the carbon source. In exchange, AMF provide their host several benefits which have been recognise in mitigation of biotic and abiotic stress, even though the main benefit ascribe to AMF is improving P uptake. However, although the AM symbiosis is considered as a classic example of a mutualistic symbiosis, many factors influence the outcome of this interaction (e.g. fungal and plant species involved in the symbiosis, nutrient availability, etc.), and under certain condition the presence of AMF have been shown to do not influence or depress the plant growth. Soil N availability is one of the main factors affecting the plant production, and AMF have been shown to be able to transfer N to the host plant. However, the conditions that ruling the uptake and the following transfer of N via AMF to the host plant are still unclear. Here, durum wheat has been grown with or without AMF inoculum in presence of different N form and availability to study if the amount and the form of soil N affect the outcome of the AM symbiosis. The experiment was conducted in pot under natural climatic condition. The soil moisture was monitored twice a week with gravimetric methods and additional water was added when the soil moisture reached the 70% threshold of the holding capacity. Three treatments have been compared: 1) native substrate N content (soil Navailability= 0.45 ‰; control); 2) mineral N in form of (NH4)2SO4 applied in equivalent quantity to 75 kg of N ha-1 (N-min); 3) organic N in from of organic matter of Lolium multiflorum applied in equivalent quantity to 6.5 Mg ha-1(N-org). The organic matter was applied before the sowing phase, distributing the fertilizer homogenously in the substrate 5 to 7 cm below the sowing bed. The mineral treatment was applied in two steps: the first application was done 10 days after the emergence (DAE) distributing 2/3 of the total amount, the second was done 50 DAE applying the rest. In each case the fertilizer had a known amount of 15N to monitoring the N fate. Each treatment was replicated 5 times and the experiment was conducted in a completely randomize design. The experiment lasted until the crop reach the flowering phase (75 DAE). At the end of the experiment the biomass was harvested and split in the two parts below and above; the fresh and dry weights were recorded and the total N and 15N content were assayed. The below biomass was used to assay the total root length and the AMF infection. The data were used to determine the plant biomass production, N uptake and N recovery in the plant biomass. The data obtained were analysed in R using a two-way ANOVA. When statistical significance occurred, means were compared using the Tukey’s test. The results, as expected, have shown a marked effect of the fertilizer treatments. In fact, the treatment N-min determined an average increment respect to the control for both above and below ground biomass; while the treatment N-org determined a decrement for both the biomass portion compare to the control. Such trend was consistent for the others variables investigated. As concern the AMF inoculum, on the biomass, the effect was confined to a decrement of the above ground biomass in the N-org treatment, while no significant effect was detected for the same parameter on the others treatments and on the below ground biomass. A strong interaction has been shown between Fertilization and AMF inoculum in N uptake and N recovery. In particular, the presence of AMF determined a decrement of both parameter in the control and N-org, while a marked increase has been detected for both the parameters in the N-min. In conclusion, the effect of AMF inoculum on total plant biomass was very contained. By contrast, on the others parameters a competitive behaviour for the N source has been shown in a poor-N-environment, while a cooperative behaviour was observed in a reach-N-environment

Early Sowing Allows To Reduce Weed Pressure In No-Till Organic Durum Wheat Production

In organic farming, the adoption of the conventional tillage (CT) technique is considered by many farmers to be necessary to control weeds. Such tillage system, in fact, permits to bury weed seeds deep in the soil by means of soil inversion with moldboard plowing and to eliminate the weed plants that gradually emerge by means of the secondary tillage operations. However, it is also true that intensive tillage progressively reduces the soil organic matter content and the stability of soil aggregates, thus increasing the risk of soil erosion (Six et al. 2000). This is in contrast with one of the basic principles of organic agriculture, which is the conservation of soil fertility. Alternatively to CT, the no tillage (NT) technique can maintain or even enhance soil fertility by increasing C storage, soil biological activity, and soil aggregate stability, but, as a matter of fact, its application relies on herbicide use as the primary weed control mechanism (Gattinger et al. 2011). In the light of these considerations, efforts must be made to revisit the NT technique to make it applicable in organic farming. Without prejudice to the fact that this challenge should be addressed through a systemic approach (Peigné et al. 2007), one possible option could be to take advantage of the possibility given by the NT technique to sow the crop in an earlier period than what usually the farmer does when adopts the CT technique. Anticipating the sowing time would allow operating when most of the weed plants are still poorly developed, so that the sowing operation itself can kill many of them. Moreover, sowing early, when temperatures are still relatively mild, could accelerate the initial growth, thus reducing the period during which the crop is particularly vulnerable to weed competition. Usually, early sowing in the CT systems is not possible since a proper seedbed preparation needs time so that clods formed as a result of plowing could be broken down by natural weathering processes and by one or more secondary tillage operations. Therefore, an experiment was performed under organic management to study the effects of NT compared to CT on durum wheat (Triticum durum Desf.) grain yield, and to verify whether early sowing under NT conditions, compared to sowing at the ordinary time for the study area, can provide an advantage to the crop by increasing its competitiveness against weeds. Furthermore, the above effects were investigated on two durum wheat genotypes highly different for pheno-morphological and agronomic characteristics, assuming for them different competitiveness against weeds