Volatiles from the fungus Fusarium oxysporum affect interactions of Brassica rapa plants with root herbivores

1. Soil is a diverse and heterogeneous environment where chemicals mediate numerous interactions between soil organisms and plants. To date, studies have extensively addressed volatile‐mediated interactions between soil microorganisms and the effects of microbial volatiles on plant growth. Yet, to our knowledge, it remains to be explored whether volatiles from soil‐borne fungi can influence plant interactions with root herbivores, facilitating or hampering performance of competitors that share the same host plant.


Introduction
Soil is the most diverse ecosystem on Earth and harbours dense and complex communities of macro-and microorganisms, including earthworms, arthropods, protozoa, nematodes, fungi, oomycetes, bacteria, and archaea. These soil organisms interact directly or indirectly with each other and constitute dynamic soil food webs with several trophic levels (Susilo et al. 2004;Morriën 2016). In this crowded environment, soil organisms have to compete for resources and escape from predators and parasites, while still interacting with mutualists. Schulz-Bohm et al. 2017). Additionally, microbial volatiles can mediate inter-kingdom interactions with plants and animals (Wenke et al. 2010;Rasmann et al. 2012b;van Dam & Bouwmeester 2016;Schenkel et al. 2018). For example, volatiles emitted by saprophytic Fusarium oxysporum strains can negatively affect egg hatching of the root-knot nematode Meloidogyne incognita (Terra et al. 2018) or reduce nematode population size (Hallmann & Sikora 1994). Also, volatiles from pathogenic and non-pathogenic soil microorganisms can promote plant growth and accelerate plant development (Casarrubia et al. 2016;Cordovez et al. 2017;Piechulla et al. 2017;Moisan et al. 2019). Reciprocally, plant volatiles can affect soil microorganisms. For example, root volatiles can attract beneficial bacteria to the rhizosphere (Rasmann & Turlings 2016;Schulz-Bohm et al. 2018). Therefore, behaviour and performance of one soil organism can be directly affected by volatiles emitted by con-and heterospecific organisms in the soil.
Interestingly, microbial volatiles can indirectly mediate interactions between different organisms. For instance, microbial volatiles can enhance plant resistance to subsequent attackers, by providing chemical protection of the plant, hence preventing further microbial colonisation (Vorholt 2012;Junker & Tholl 2013), and by promoting plant growth and eliciting local and systemic resistance (Bitas et al. 2013;Sharifi & Ryu 2016;Cordovez et al. 2017). Recently, we showed that volatiles from different soil-borne fungi influence compensatory plant growth and resistance against a caterpillar and a root insect herbivore (Moisan et al. 2020). Together, these data indicate that microbial volatiles can indirectly mediate ecological interactions of plants with other organisms.
Yet, it remains largely unknown whether volatiles from a soil-borne fungus, directly and indirectly, impact plant interactions with root herbivores by facilitating or hampering the performance of the attacker. In this study, we investigated the effects of volatiles emitted by saprophytic F. oxysporum on the performance of the cyst nematode Heterodera schachtii and the insect root herbivore Delia radicum on Brassica rapa roots. We hypothesised that F. oxysporum volatiles would negatively impact the performance of the two root herbivores as they both represent competitors that share the same host plant.

Plant, herbivore, and fungal materials
Seeds of the wild turnip, B. rapa (accession Maarssen, The Netherlands), were sterilised as previously described (Moisan et al. 2019). Briefly, all B. rapa seeds were surface sterilised by exposure to chlorine gas for 4 h in a desiccator, and stratified at 4 ∘ C in the dark for 3-4 days. Before the start of the experiments, F. oxysporum f. sp. raphani was cultured on 1/5th PDA medium, either in ø 9 cm Petri dishes for 7 days or in ø 3 cm Petri dishes for 3 days. The plant parasitic nematode, H. schachtii, was reared on roots of sugar beet plants (Beta vulgaris L.), and larvae of the cabbage root fly, D. radicum, were reared on rutabaga (Brassica napus subsp. napobrassica) roots.

Plant exposure to Fusarium oxysporum volatiles
Brassica rapa roots were exposed to volatiles from F. oxysporum using two-compartment pots (Fig. 1). Plants were grown in the top compartment, while the fungus was grown in the bottom compartment. Both compartments were separated by a nylon membrane of 1 μm mesh width (Plastok Associates Ltd., Birkenhead Wirral) that allowed air exchange between the two compartments, while preventing physical contact between the roots and the fungus. Size of the pots differed between the infestations of the two root herbivores as they naturally infest plants at different stages: H. schachtii nematodes perform better on plants in the pre-emergence seedling stage (Fedorko 1962;Griffin 1981), whereas larvae of D. radicum require sufficient development of the root system for feeding, thus adult flies preferably oviposit on larger plants (Ellis et al. 1979;Dosdall et al. 1996). In both experiments, one sterile B. rapa seed was sown in the top compartment filled with a sterile (γ-irradiated) soil mixture (1:1 v/v, ø 2 mm sieved Horticoop potting soil: sand). The bottom compartment contained either a Petri dish with F. oxysporum growing on 1/5th PDA medium or a control Petri dish with 1/5th PDA medium only. In the pots used for infestation with D. radicum larvae, both compartments were connected to each other by a connector (h = 12.5 cm, ø = 12.7 cm), which allowed the weekly replacement of Petri dishes containing the fungus or control Petri dishes with fresh 7-day-old fungus or medium. Volatile exposure was initiated as soon as B. rapa seeds were sown, and was maintained in a greenhouse compartment (21 ± 2 ∘ C; L16:D8; 70 ± 5% RH) throughout the experiment until the harvest of the plants. Volatile exposures (F. oxysporum and control) were replicated 14-15 times for each herbivore infestation.

Plant infestation with Heterodera schachtii cyst nematodes
Two-week-old B. rapa plants were inoculated with 1 ml of inoculum containing 550 ± 20 H. schachtii J2 juveniles, while roots were still exposed to fungal volatiles (see previous section; Fig. 1a). For this, cysts of H. schachtii were incubated in water at 25 ∘ C, 4 days before inoculation. Newly hatched juveniles were sieved, and the concentration of the inoculum was adjusted by counting the juveniles under the microscope and making dilutions. To prevent flushing of the juveniles, no water was added to the plants during the first 2 days after inoculation. Aboveground parts of the plants were harvested 3 weeks after inoculation, and the cysts were given another 2 weeks to ripen. Roots were gently harvested and rinsed with water through two sieves (ø = 0.850 mm and ø = 0.212 mm). Sieves were rinsed with water, and cysts in suspension in this water were collected and counted under the microscope. Roots were also checked under the microscope for remaining cysts. Collected cysts were stored in Petri dishes at 4 ∘ C until measurement. To assess the degree of cyst maturation, cyst colouration was scored based on visual evaluation under the microscope: white to light yellow-coloured cysts were considered as unripe, and orange to dark orange-coloured cysts were considered as ripe (Gardner & Caswell-Chen 1997). To assess cyst size, cysts were photographed (Leica DFC450, Leica Microsystems B.V., Son, The Netherlands) under a microscope (Leica M205C), and pictures (400 dpi resolution) were processed with ImageJ software to measure individual cyst size. In addition, roots and leaves were dried at 70 ∘ C for 3 days and weighed. Effects of F. oxysporum volatiles on the number of collected cysts per g of root dry weight was tested with a Student's t-test (α = 0.05) while the total count of cysts was analysed with a generalised linear model with a quasi-Poisson distribution. Differences of percentages of unripe and ripe cysts collected from control and volatile-exposed plants were tested using a quasi-binomial (to handle binomial overdispersion) generalised linear model with fungal volatile exposure as a fixed factor (α = 0.05). Differences in cyst size were analysed using a linear mixed model with plant replicate as a random factor. Differences of root and leaf dry weight were tested with Student's t-tests, and correlations with the number and size of cysts were assessed with Pearson correlation tests (α = 0.05).

Plant infestation with Delia radicum larvae
Four-week-old B. rapa plants were infested with 10 newly hatched D. radicum larvae, while roots were still exposed to fungal volatiles (see section 'Plant exposure to Fusarium oxysporum volatiles'; Fig. 1b). During the first 2 days after infestation, no water was added to the plant to prevent flushing of the larvae. Two weeks after infestation, plants were harvested and D. radicum individuals were recollected from the soil and roots. Larvae and pupae were counted and individually weighed. The fraction of recollected D. radicum (out of 10 larvae infested per plant) and the fraction of D. radicum pupae (out of the insects recollected) were analysed with a quasi-binomial (to handle binomial overdispersion) generalised linear model and logit link function, using fungal volatiles as a fixed factor. Additionally, a linear mixed model, using plant replicate as a random factor, was used to analyse fresh weight of D. radicum larvae and pupae. Correlations between insect fresh weight, number of insects and root dry weight were assessed with Pearson correlation tests (α = 0.05).

Plant infestation with Heterodera schachtii cyst nematodes
The total count of cysts collected in fungal volatile-exposed plants did not differ with that of control plants ( Fig. 1a; GLM; P = 0.403). However, fewer H. schachtii cysts were retrieved per milligram of dry roots from B. rapa plants exposed to F. oxysporum volatiles than from non-exposed control plants ( Fig. 2a; t = 2.5; P = 0.018). On average, 25% of the cysts collected from control plants were unripe, whereas this number reached 48% in plants exposed to the fungal volatiles ( Fig. 2b; GLM; P = 0.029). Moreover, cysts retrieved from plants exposed to the fungal volatiles were larger than those collected from control plants ( Fig. 2c; F = 5.8; P = 0.026). Exposure to the fungal volatiles increased root and leaf weight of H. schachtii-infested plants ( Fig. 3a; t root = −2.3; P root = 0.034; t leaf = −2.9; P leaf = 0.009). Overall, cyst size was positively correlated with root dry weight ( Fig. 3b; r = 0.493; P = 0.023) and with the number of cysts retrieved ( Fig. 3c; r = 0.449; P = 0.041).

Discussion
Our results show that interactions of B. rapa plants with root herbivores can be affected by volatiles emitted by the soil-borne fungus F. oxysporum. We did not find an effect of fungal volatiles on the performance of the insect root herbivore D. radicum, whereas the development and reproduction of the nematode H. schachtii were negatively and positively affected, respectively. Besides potential direct effects of fungal volatiles on the attackers, plant growth promotion upon fungal volatile exposure suggests possible indirect effects mediated by the plant as well. Together, our data show that fungal volatiles can specifically affect the performance of root herbivores and plant growth upon root herbivory.
Interaction between B. rapa roots and H. schachtii nematodes was hindered upon root exposure to F. oxysporum volatiles. Fungal volatiles could have directly affected the nematodes themselves in the soil. Nematodes are indeed endowed with high sensitivity to volatiles for their navigation in soil (Rasmann et al. 2012a;Rengarajan & Hallem 2016). Previous studies have shown that volatiles from soil or root-associated microorganisms can affect nematode development and behaviour (Wuyts et al. 2006;Turlings et al. 2012;Cheng et al. 2017;Sharma et al. 2019;Wolfgang et al. 2019). For example, volatiles emitted by some F. oxysporum strains negatively affect egg hatching of the root-knot nematode Meloidogyne incognita (Terra et al. 2018). Additionally, some bacterial volatiles can act as nematicides and chemotactic agents to M. incognita (Cheng et al. 2017). Although we expected higher colonisation in fungal volatile-exposed plants that had more roots to colonise, we recorded similar number of cysts in control plants and in fungal volatile-exposed plants. This result suggests that volatiles from F. oxysporum did not impact root colonisation by the nematode.
Interestingly, F. oxysporum volatiles did slow down nematode development and potentially increased their reproduction rate. We hypothesise that these effects result from a modulation of plant defences by F. oxysporum volatiles. We previously demonstrated that pre-exposure of plant roots to volatiles from fungal pathogens, using same experimental set-ups, can alter plant growth and resistance to insect herbivores, despite the fact that the herbivores were not directly exposed to the fungal volatiles themselves (Cordovez et al. 2017;Moisan et al. 2019Moisan et al. , 2020. Furthermore, we show that root pre-exposure to fungal volatiles can affect plant compensatory growth upon herbivory, suggesting an effect on plant primary metabolism (Moisan et al. 2020).
Here, we postulate that upon root penetration and colonisation, juveniles encountered more structural (e.g. cuticle, wax layer) or chemical plant defences (e.g. reallocation of primary metabolites, accumulation of secondary metabolites), resulting in lower colonisation rate (Miroslaw et al. 2005). For instance, exposure of Arabidopsis thaliana plants to volatiles from Bacillus amyloliquefaciens resulted in the elevation of glucosinolates in leaves, which negatively affected the performance of an insect herbivore (Aziz et al. 2016). Survival and development of the nematodes depends on the establishment of the feeding site, that is the syncytium. For this, cyst nematodes manipulate plant machinery by inhibiting plant resistance responses (Gheysen & Mitchum 2011;Bohlmann & Sobczak 2014). Root exposure to fungal volatiles may have modified plant resistance responses to cell damage caused by the nematodes, thus influencing establishment of the syncytium. Additionally, biochemical and physiological changes in the host plant can affect the quantity and quality of nutrients available in the syncytium, thus influencing the cyst content (Betka et al. 1991;Gaur et al. 1995). Cysts, which are the dead body of the female, contain the new progeny in the form of eggs, and larger cysts may support higher numbers of eggs (Atkinson et al. 2001). Thus, plant growth promotion by fungal volatiles could have enabled higher food intake by female nematodes, thus increasing cyst size as a result of increased number of eggs. Further research, involving transcriptomic and metabolomic analyses of the plant responses to the fungal volatiles, will be needed to address the underlying mechanisms. Interestingly, unlike H. schachtii nematodes, larvae of D. radicum performed equally on control plants and plants exposed to fungal volatiles, and plant growth was unaffected. These differences may be explained by the size of the pots used. In the setup which is used for D. radicum, the top compartment was larger than that for H. schachtii, hence increasing the distance between the roots plus the insect herbivores and the source of the fungal volatiles. Thus, plant roots and insect larvae were less likely to be directly exposed to fungal volatiles. However, we recently showed, using the same large pots, that development of D. radicum can be slowed down in B. rapa roots when these plants are pre-exposed to F. oxysporum volatiles (Moisan et al. 2020). Thus, the setup used is effective. The difference in the results of the current study suggests that the direct effects (when the herbivore itself is also exposed to the volatiles, our current study) and the indirect effects (via plant-mediated changes when roots are exposed to the fungal volatiles prior to herbivore attack, Moisan et al. (2020)) of fungal volatiles on the performance of D. radicum can differ. Moreover, fungal volatiles may also elicit biochemical and physiological changes in the roots that can differentially affect plant resistance to specific attackers. For instance, colonisation of A. thaliana roots by the rhizobacteria Pseudomonas simiae led to reduced performance of the caterpillar Mamestra brassicae (Pangesti et al. 2016), whereas it enhanced the performance of the aphid Myzus persicae (Pineda et al. 2013). As D. radicum larvae and H. schachtii nematodes cause different types of root damage, one may expect differences in fungal volatile-induced plant responses as well. Taken together, our findings show that volatiles from a soil-borne fungus can modulate belowground interactions of plants with root attackers.
In conclusion, we show that fungal volatiles can diffuse through the soil matrix and induce plant phenotypic responses that, in turn, can affect the performance of root herbivores. The next step will be to investigate these interactions in more complex and natural ecosystems with highly competitive soil microbiomes and with more dynamic and complex blends of volatiles. Additionally, as F. oxysporum can parasite nematode eggs and can colonise plant roots as an endophyte, it would be interesting to test more soil-borne fungi to determine if such effects on plant interactions with root herbivores can result from a manipulation of the fungus for its own benefit.