Herbivory‐driven shifts in arbuscular mycorrhizal fungal community assembly: increased fungal competition and plant phosphorus benefits

Abstract

In terrestrial ecosystems, arbuscular mycorrhizal (AM) fungi engage in symbiosis with > 70% of terrestrial plants (Brundrett & Tedersoo, 2018). These fungi occupy the soil where their hyphae grow and forage for resources such as phosphorus (P). Their ability to access such resources is fundamental to their obligate symbiotic relationship with plants, as supply of soil nutrients is exchanged for carbon within the host roots (Smith & Read, 2008). Thus, AM fungi occupy a dual habitat, inhabiting the soil but also plant root systems in which they often form complex and dynamic communities (Öpik et al., 2006). While the symbiosis is often characterised by the transfer of nutrients and carbon, the ecological roles of AM fungi extend beyond the exchanging of resources. They can significantly support plant resilience against various stresses, such as drought, and are important for soil structure, nutrient cycling, and carbon cycling (Powell & Rillig, 2018). Although research into the AM symbiosis advances, key knowledge gaps remain regarding the factors that shape the diversity and community assembly of these fungi within plant roots, especially in the context of other plant biotic interactions. The majority of plants which AM fungi associate with are subject to attack from insect herbivores (Price et al., 2011). For an estimated 350 million years, this relationship has exerted substantial influence on the evolution and diversification of plants (Agrawal et al., 2012). Herbivory is expected to have significant impacts on the AM symbiosis and AM fungi because of the sizable effects on plant carbon budgets along with potential shifts in the needs and allocation of resources of the host plant (Orians et al., 2011). The carbon-limitation hypothesis posits that removal of photosynthetic tissue by insect herbivores would have a negative impact on the AM symbiosis due to diminished carbon availability for the fungi (Gehring & Whitham, 1994). Indeed, this was demonstrated in recent works where aboveground insect herbivory reduced plant carbon allocation to AM fungi within the roots (Charters et al., 2020; Durant et al., 2023). In this way, aboveground herbivory can alter the host plant quality for the fungi as their carbon resources are diminished. It is well-established in ecology that resource availability is a critical determinant of community assembly (Weiher & Keddy, 2001; Tilman, 2004). Decreased availability of a potentially limiting resource is expected to intensify competition between individuals (Johnson, 2010), resulting in shifts in community composition. For AM fungi, their access to carbon from the host has been shown to be related to the nutrient benefit they provide, with potential for adjustments in trade depending on demand (Noë & Kiers, 2018). However, it is important to note these interactions are more nuanced and complex when broader ecological interactions and context are taken into account (Bennett & Groten, 2022). Yet, if the carbon availability for the fungi is decreased, then less competitive taxa are expected to be outcompeted by taxa that can grow and survive with less resources, or those who are more cost-effective for the host when it comes to P delivery (i.e. provide a level of benefit per unit of carbon). Assuming that traits are phylogenetically conserved in AM fungi (Cahill et al., 2008; Powell et al., 2009), competitive exclusion among closely related fungal taxa might be expected to lead to a community in which species are less closely related to each other (i.e. phylogenetic overdispersion; Violle et al., 2011). Should carbon allocation to AM fungi be reduced under herbivory, AM fungal communities would contain taxa that are more distantly related to each other when compared to communities inhabiting herbivore-free plant hosts. That said, increased competition may also result in more closely related communities (phylogenetic clustering) if certain phenotypes, such as those associated with low resource requirements, are associated with competitive dominance (Kraft et al., 2015). In this instance, community assembly may have outcomes analogous to environmental filtering, being based on particular suite of traits that permit community membership (Pausas & Verdú, 2010). Previous research has studied the impact of soil nutrient status on AM fungal community assembly (Liu et al., 2015), yet the role of insect herbivory remains unexplored. While it is expected that aboveground herbivory would significantly affect the community assembly and diversity of AM fungi within plant roots, we have limited data to provide insight on this. Recent work on belowground insect herbivory found root herbivores can significantly reduce species richness and alter community structure of root-colonising AM fungi (Frew, 2022). Studies on other forms of herbivory, such as grazing, have found variable impacts on soil AM fungal communities where intense grazing decreases diversity (Ba et al., 2012) while moderate or light grazing has little effect, or may even increase diversity (Ba et al., 2012; van der Heyde et al., 2017). Other forms of defoliation and damage, such as mechanical mowing in managed systems, are often found to have limited impacts on AM fungal diversity (Zubek et al., 2022), but can still influence interactions between AM fungi and plant communities (Qin et al., 2022). We are aware of only one study which has empirically examined the impacts of aboveground insect herbivory on root AM fungal diversity and composition (Wilkinson et al., 2019) where the authors found no changes in the richness of AM fungi in plant roots, but did observe an increase in community evenness in plants with insect herbivores. The scarcity of studies here highlights a knowledge gap which continues to be overlooked despite its importance, particularly considering the ubiquity and ecological significance of the interactions between insect herbivory and AM fungi. The composition of an AM fungal community within a root system, as a result of assembly processes, is a fundamental determinant of the functional outcome of mycorrhizal symbiosis for the host plant. AM fungal taxa are functionally diverse, with different taxa being more or less associated with particular functions for their host such as P delivery or enhanced plant defence (Hart & Reader, 2002; Sikes et al., 2009; Chagnon et al., 2013). A number of studies have shown how different AM fungal taxa, combinations of taxa or communities can have distinct plant phenotypic outcomes relating to plant growth, nutrient status, and stress tolerance (van der Heijden et al., 1998; Bennett & Bever, 2007; Frew, 2019). Thus, changes in root-dwelling fungal communities will have direct consequences for host plant performance and affect plant productivity and ecosystem functioning (van der Heijden et al., 2008; Bardgett & van der Putten, 2014). We explored how aboveground insect herbivory impacts the taxonomic and phylogenetic diversity and composition of root-colonising AM fungal communities to infer the associated community assembly processes. We hypothesised that herbivory would reduce richness of AM fungi in plant roots, potentially indicating increased competition for carbon resources. By examining the phylogenetic structure of the fungal communities, we expected the herbivory-driven increase in competition for carbon would either (1) increase phylogenetic overdispersion as a result of increased competitive exclusion or (2) increase phylogenetic clustering if competition selects for a particular set of AM fungal traits which confer a competitive advantage (Kraft et al., 2015). We performed a factorial glasshouse study using Sorghum bicolor L. Moench cv ‘MR Taurus’. Our experiment had two treatment factors: herbivory (presence or absence) and AM fungi (presence or absence). Each of the four treatment combinations was replicated 13 times, totalling 52 S. bicolor plants at the experiment initiation. Seeds were surface sterilised with 10% diluted commercial bleach (comprised of 4% sodium hypochlorite), germinated in Petri dishes for 6 d, and then transplanted as individual seedlings into 3.7 l pots. These pots were filled with an autoclave-sterilised 40 : 60 sand/soil mix (Supporting Information Table S1 for soil nutrient data), and plants received an initial dose of the low-P fertiliser Osmocote Native Controlled Release Fertiliser (The Scotts Co. LLC) at the initiation of the experiment. Plants in the ‘with AM fungi’ treatment were inoculated by potting with 150 g of sieved, air-dried field soil inoculum combined with the sterile sand/soil mixture. One week before initiation of the experiment, the inoculum was sourced from the top 20 cm of soil alongside an organically managed arable field in southern Queensland, Australia (−27.4326°, 152.3495°), a site previously verified to harbour a diverse AM fungal community (Ng et al., 2023). Conversely, the ‘no AM fungi’ treatment used 150 g of autoclaved field soil inoculum. All pots were given 300 ml of microbial filtrate, derived from washed field soil passed through a series of sieves (to the smallest aperture of 20 μm to exclude all AM fungal spores; Aguilar-Trigueros et al., 2019), to standardise the non-AM fungal microbial community (Koide & Li, 1989). All plants were grown in the glasshouse and watered ad libitum with tap water, and soil moisture was checked every few weeks with a soil moisture metre to ensure similar moisture status across pots. Plants were grown with c. 13-h day length and day : night temperatures of 28°C : 18°C, while average daylight in the glasshouse for the period was 750 μmol m−2 s−1. Pots were rearranged randomly within the glasshouse chamber every 2 wk. After 8 wk, half of the pots were introduced to five fourth-instar Helicoverpa punctigera larvae each. These larvae had been fed as per the diet medium detailed by Teakle & Jensen (1985) and were sourced from CSIRO Agriculture & Food, Narrabri, Australia. To retain the larvae within their designated pots, all pots (including those without herbivores) were placed into individual enclosures of fine nylon mesh (Bugdorm enclosures; Megaview Science Co. Ltd). At this stage, one replicate plant (with AM fungi, with herbivory) was lost from the experiment. All insects were weighed before being introduced to the plants and again at harvest to obtain a mean insect growth rate per pot. These data represent herbivore performance, a proxy for herbivore treatment intensity across replicates. Plants were harvested 12 wk from germination at which point their roots were separated from the aboveground tissues. After washing the roots, 1 g of fresh roots was collected from each plant by sampling from multiple distinct areas of the outer root system for evaluating mycorrhizal fungal colonisation. The residual plant tissue was dried in an oven at 38°C and subsequently weighed. The aboveground tissue was ground for subsequent chemical analyses, while homogenised dried root samples from the AM fungi plants were reserved for DNA metabarcoding. To measure the phosphorus benefits to the plant from the AM symbiosis, we assessed foliar phosphorus concentration using inductively coupled plasma (ICP) spectroscopy. This was done postdigestion of dried plant material with hydrogen peroxide and nitric acid, as described by Rayment & Lyons (2011). Root carbon concentration was measured using the high-temperature combustion method (LECO elemental analyser) on dried and ground root material. Mycorrhizal fungal colonisation was evaluated in the AM fungal treatment roots, while its absence was confirmed in the ‘No AM fungi’ treatment roots. Freshly harvested root samples were cleared using 10% KOH at 90°C for 15 min and stained with 5% ink-vinegar (Vierheilig et al., 1998). The stained roots were then placed on microscope slides as 5 cm fragments to be examined using the intersect method (McGonigle et al., 1990) across at least 100 intersections per sample at ×200 magnification. DNA was extracted from 70 mg of dried root samples using a DNeasy Powersoil Pro Kit (Qiagen, GmBH) following the manufacturer's instructions with the modification that 0.5 mm fragmented dried roots were added to extraction tubes, rather than soil, which were ground in a FastPrep-24™ (MP Biomedical, Irvine, CA, USA) for 30 s before downstream processing. Sequencing was conducted at Western Sydney University's next-generation sequencing facility, following their protocols. Briefly, the DNA was purified using Agencourt AMPure XP Beads (Beckman Coulter, Brea, CA, USA) and quality-verified using Quant-iT™ PicoGreen fluorescence-based analysis (ThermoFisher Scientific, Waltham, MA, USA). The purified DNA then underwent amplification using polymerase chain reaction (PCR) using the small-subunit (SSU) ribosomal RNA gene with AM fungal-specific primers, WANDA (Dumbrell et al., 2011) and AML2 (Lee et al., 2008). Sequencing was performed on the Illumina MiSeq platform using the Illumina MiSeq reagent kit v3 2 × 300 bp paired-end chemistry as per the manufacturer's instructions. Bioinformatics processing was done using the graphical downstream analysis tool (gDAT; Vasar et al., 2021). From the total 2 × 1952 318 raw reads, cleaning and demultiplexing procedures retained 2 × 1438 308 cleaned reads. This involved checking double barcodes, verifying correct primer sequences, and ensuring an average quality of at least 30. Chimeric sequences (0.1% of cleaned reads) were eliminated using vsearch v.2.15.0. The resulting sequences were identified and assigned to virtual taxa (VT) using the MaarjAM database (Öpik et al., 2010) with a 97% sequence identity and 95% alignment thresholds using Blast+ (v.2.7.1). The top-scoring representative sequences for each VT were selected and aligned using ClustalW. A neighbour-joining phylogenetic tree of these sequences was with based on the method et al., 2021). For all analyses, data was following the described in et To from in sequencing we used the from the et al., & before downstream The of the herbivore treatment on AM fungal VT richness and diversity was by using and then the from the in community composition and structure of the root-colonising AM fungal communities were using analysis & based on To the effects of the herbivore treatment on the changes in community we used using the from the et al., 2013). To potential in the community assembly under herbivory, we the phylogenetic diversity and structure of AM fungal We used (1) phylogenetic diversity which is to the phylogenetic within a (2) the mean which the mean phylogenetic between all VT in a and the mean to measure the between each VT and its all the et al., 2002; et al., 2010). We for these by of the and functions in the et al., 2010). For the two phylogenetic overdispersion due to competitive phylogenetic from environmental (Pausas & Verdú, 2010). We and the from the to the effects of herbivory on these & 2011). To how herbivory plant to AM fungi, we the mycorrhizal growth and the mycorrhizal P These plant mycorrhizal were as with AM plant with AM plant with no AM × where the plant was either the total or tissue P To the effects of herbivory on these plant mycorrhizal as as on AM fungal root plant root : root aboveground P and we using and then from the A using was also to the effects of AM fungi on the mean in of the insect herbivores. which did the of the were to reduce and ensure of residual All and data were using and Our that aboveground insect herbivory diversity and the composition and structure of root-colonising AM fungal communities Herbivory reduced root carbon and increased AM fungal phylogenetic diversity while communities more phylogenetically which competitive exclusion is a of community assembly under herbivory, to the under herbivory, AM fungi a phosphorus benefit to their host plants Plants to insect herbivory AM fungal communities within their roots that were different to herbivore-free plant fungal communities we identified AM fungal VT across all plants, of which were to plants without herbivory, VT were to plants with insect and were in plants from herbivory a in AM fungal VT richness Table and diversity Table by and an outcome may be expected in when competition intensity in this as plants photosynthetic tissue and potentially alter resource in AM fungal diversity have been in studies which have at grazing or belowground insect herbivory (Ba et al., 2012; Frew, 2022). In to shifts in herbivory also AM fungal community structure and composition Table In the roots of herbivore-free plants, the communities of VT from the and of these are all to be that taxa that to hyphae than hyphae et al., 2019). In to with herbivory, the communities also taxa from the et al., 2022), and the are also and are to within the While fungi within this allocation they to have fungal these fungi represent less they may be more competitive when carbon resources from the host are as their limited their carbon needs are than other AM fungal It is for the AM symbiosis to reduce insect herbivore performance, particularly for insects et al., In this instance, we no between the growth of insects on plants with and without AM fungi Although studies have demonstrated AM plant can be significant even between AM fungal of the species et al., 2013). the of research on the effects of AM fungi on plant to insects is limited to a of AM fungal Thus, we have a of the broader of AM fungal diversity on interactions et al., 2022). While AM fungal taxonomic diversity under herbivory, phylogenetic diversity increased and fungal communities more phylogenetically compared with the AM fungal communities inhabiting roots of herbivore-free plants This is to competitive exclusion in community assembly et al., 2002; & Verdú, 2010). In with the carbon concentration of plant roots was reduced in plants by Our support the hypothesis that aboveground herbivory competition between AM fungal taxa, due to carbon the role of competitive exclusion in community characterised by AM fungal communities of more distantly related While in carbon allocation to fungi have been shown (Charters et al., 2020; Durant et al., the consequences of this for AM fungal communities have been unexplored. The scarcity of carbon competition among fungal taxa within the roots to occupy the with an limited this increased from competitive exclusion reduced VT richness and the of these communities were more distantly related to each between AM fungal taxa is to be by phylogenetically conserved traits (Powell et al., While we might that the taxa under herbivory may an ability to grow under low carbon their in their that competition when it comes to resource The traits to may to in their allocation of hyphae to soil or roots et al., 2019), in of the root system, or growth et al., et al., 2022). this the more phylogenetically communities P benefit to their compared with the communities in roots of the herbivore-free plants Thus, while a more might be that competition only selected for more fungal taxa in of their ability to this is less we that competition to functional that in enhanced P benefit to the host et al., 2008). This enhanced P status of the host may also to benefit defence in particularly for plants which more on against herbivory such as plants et al., & In to the mycorrhizal P the mycorrhizal growth was significantly by herbivory However, the root : significantly under herbivory plants with herbivores had root : due to aboveground tissue plants with AM fungi had root : which is an when plants with and without AM fungi et al., as plants with AM fungi are to less in root to forage for resources. While the total colonisation of plant roots by AM fungi was by herbivory arbuscular and colonisation was and in plants increase in the of an increase in the resource between fungi and plants, which may be the as AM fungi P benefit for their under are as and their can be of particular AM fungal taxa (Smith & Read, 2008). While they represent a significant of carbon, their may be of a growth where the fungi carbon to rather than their growth or growth is found in other to low resource or such as plants or in and plants, which more in than tissue to with low resources or resource availability et al., 2008; et al., 2014). Indeed, it is important to be of the and of and that colonisation here was to one the in colonisation by and is to be more nuanced and dynamic than data here are to While of the diversity and community assembly of AM fungi is et al., et al., et al., Vasar et al., 2022), important knowledge gaps Our study for the that aboveground insect herbivory can significantly shape the diversity and phylogenetic structure of root-colonising AM fungal communities and the outcomes for plant growth and P Our that these interactions and how they can AM fungal community The authors the of the on which this work was This work was by an to was by a and are by and The authors would to the and for their access by Western Sydney as of the Western Sydney the of and the which were from with and conducted the experiment and performed the work with from and and the data with and and the of the initial with support from and All authors to the and for that support this study are from the at the following DNA sequencing data are under S1 phylogenetic tree of arbuscular mycorrhizal (AM) fungal virtual of AM fungi on the of the insect of herbivory and AM fungi on root carbon, foliar and and the effects of herbivory on total AM fungal root colonisation. Table S1 nutrient analysis Table at the of herbivory and AM fungi on plant as as fungal and phylogenetic is for the or of Information by the than be to the The is for the or of by the than be to the for the

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