This study has provided the first data to show that root aphid populations are affected by the soil moisture status of their host plants. The interactions between host plants and insect herbivores depend on the quality of the host plant (Leather 2017). If a plant offers quality attributes (e.g., chemical, physical), herbivorous insects can utilize these to their advantage. Insect performance and populations are therefore governed by host plant availability, quality, and environmental conditions. This data confirms our hypothesis that root aphid numbers are higher in drought-stressed perennial ryegrass plants in comparison to their well-watered counterparts (Fig. 2). For example, drought increased the root aphid population by 8-fold in endophyte-free plants compared to those that were well-watered, respectively 512 aphids and 62 aphids per g of root (Fig. 2). Contrasting observations have been made linking drought with fitness in other aphid species, reporting reduced aphid fitness (Huberty et al. 2004; Pineda et al. 2016), no effect (Banfield-Zanin et al. 2015; Mewis et al. 2012), or positive effects (Oswald et al. 1997; Tariq et al. 2012). An increase in sap-feeding insects has previously been linked with drought-stressed plants (Khan et al. 2010; White 1969). However, a recent meta-analysis encompassing 55 published studies on the effect of drought on sap-feeding insects, showed that, for Poaceae (n = 24 studies), aphid fitness is generally reduced under low soil moisture conditions (Leybourne et al. 2021). For example, the fitness of the foliage aphid Rhopalosiphum padi L. was reduced when feeding on drought-stressed wheat (Triticum aestivum L.) (Kansman et al. 2020). Multiple factors could explain the variation between these studies including aphid and/or host biology. Specific aphid-plant combinations may influence the response to drought. Various aphid species showed contrasting responses to drought on Arabidopsis plants (Mewis et al. 2012), while a single aphid species can display contrasting responses when feeding on closely related host plants (Hale et al. 2003). Furthermore, aphid species can influence the relationship between drought and aphid fitness and aphid-plant systems. Leybourne et al. (2021) analysed above-ground living aphids largely from the Aphidini and Macrosiphini tribes. However, A. lentisci lives below ground, leading to the conclusion that aphid biology may explain the contrasting response. Other below-ground living aphids can reproduce rapidly under drought conditions (Hein et al. 2005; Kindler et al. 2004; Pretorius et al. 2016). Low soil moisture conditions produce dry, friable soil with numerous airspaces, likely creating favourable conditions for root aphids. In these airspaces A. lentisci surround themselves with copious amounts of flocculent wax, protecting them from harsh environmental conditions (Popay et al. 2021).
In general, aphids feed on plant sap that contains predominantly four non-essential amino acids: glutamine, glutamic acid/glutamate, asparagine/aspartic acid, and serine (Douglas 1993). These serve as a primary nitrogen source for the aphids. Phloem sap varies in composition throughout the developmental stage of the plant, diurnal cycle, season, as well as abiotic factors such as temperature and water availability (Karley et al. 2002; Stallmann et al. 2022). The composition and concentration of such amino acids in the phloem is therefore an important indicator of aphid performance and hence populations (Douglas 1993; Karley et al. 2002). During water deficit, the plant changes its protein metabolism and amino acid synthesis (Sun et al. 2020) which causes the existing proteins to hydrolyse, resulting in a higher concentration of free amino acids (Brodbeck et al. 1987). Although we did not measure amino acids in the phloem directly, this study shows the total amino acid concentration was unsurprisingly higher in drought-stressed plants (Fig. 7b). The PCA analysis indicates a clear separation of the moisture status of the plants, indicating a shift of amino acid concentration in drought-exposed plants. Drought increased amino acid concentration, such as aspartic acid, glutamic acid as well as proline, three non-essential amino acids the most, accounting for 35% of the total amino acid content. These amino acids are well known to be responsive to abiotic stress showing increased concentration in drought stressed plants (Hayat et al. 2012; Nachappa et al. 2016). High levels of glutamic acid have been linked with reduced nutritional quality of phloem sap for aphids (Douglas 1993) and reduced aphid performance (Chen et al. 1997; Weibull 1988). However, all phloem-feeding hemipterans are nutritionally ‘buffered’ from phloem sap fluctuations because they obtain supplementary amino acids from their symbiotic bacteria Buchnera sp. (Shigenobu et al. 2000). These symbiotic micro-organisms are passed from mother to offspring and are essential for survival on a phloem diet (Douglas 2006). Overall, drought-exposed plants offer relatively enriched essential amino acids, nutrients that insects cannot synthesis de novo, as well as non-essential amino acids. Thus, the phloem of drought-exposed plants could be considered more nutritious than that of well-watered plants. Aphids likely utilise the higher availability of amino acids to their advantage, promoting higher root aphid populations in drought-stressed plants. This is consistent with our observations that root aphid feeding reduced amino acid concentration in drought-exposed plants by 10% (Fig. 7b), indicating that aphids deplete the plant of sap. However, our findings contrast with that of Leybourne et al. (2021), who reported that aphid fitness is linked with plant vigour and plant-derived defence compounds, rather than drought-induced altered amino acid concentration. Some aphids, such as Diuraphis noxia, can remobilize plant nutrients when the host is suffering drought stress, causing them to be less affected (Sandström et al. 2000). Similarly, some aphids, such as the cabbage aphid (Brevicoryne brassicae L.), can sequester plant-defence compounds, that improve their tolerance to plant-derived stress compounds (Kazana et al. 2007). However, it is unknown if A. lentisci has similar attributes.
This study also showed that aphid numbers were increased in Epichloë-infected plants, with drought-exposed NZCT and AR37-infected plants harbouring 4-fold and 8-fold more aphids than well-watered plants (Fig. 2). Epichloë endophytes alter the quality of host plants by changing the chemistry (Rasmussen et al. 2008a; Rasmussen et al. 2008b). Insects can respond to such changes in three ways i) negative- alkaloid presence impairs insect performance ii) neutral- insects are not affected iii) positive- herbivorous insects' fitness improves when feeding on plants (Bultman et al. 2003; Saikkonen et al. 1999). Many studies have demonstrated that fungal Epichloë endophytes can reduce insect populations and hence improve plant performance (Caradus et al. 2020). Our results are consistent with previous studies that infection with certain strains of endophyte significantly reduces root aphid populations (Popay et al. 2016; Popay et al. 2021), even in drought conditions. In this study, drought-exposed plants infected with AR37 almost completely suppressed root aphid populations with a mean infestation of 9 aphids/ g of root compared to 36 on NZCT and 512 on endophyte-free. Similar results were found by Popay et al. (2016), who reported strong aphid suppression in AR37 infected plants as well as a toxic effect. These differences in root aphid populations on plants infected with different endophyte strains are likely attributed to the fungal chemistry. It has been hypothesised that ergovaline produced by the NZCT endophyte strain has deterrent effects on root aphids (Popay et al. 2007). It is likely that higher alkaloid concentrations lead to increased exposure to defence compounds, causing reduced aphid fitness because of feeding deterrence and reduced sap ingestion, especially since ergovaline is a lipophilic compound occurring in the roots (Lane et al. 1997a). Ergovaline concentrations in planta are influenced by environmental conditions (Ball et al. 1995; Lane et al. 1997b). Although not significant, ergovaline concentrations in this study were highest in aphid-infested well-watered plants which generally had lower root aphid numbers than drought-exposed plants. In comparison with plants with the NZCT endophyte those with the AR37 endophyte have a greater deterrent effect on root aphids despite not producing ergovaline. The chemical compound produced by the AR37 endophyte responsible for this effect is unknown.
The plant defence theory predicts that plants under abiotic stress, such as drought, will increase their alkaloid concentration (Arachevaleta et al. 1989; Hahn et al. 2008). Miranda et al. (2011) reported that endophyte infection in Italian ryegrass (L. multiflorum) reduced foliage aphids only in drought-stressed plants indicating that alkaloid concentrations may have been highest in plants suffering from the combined stress of insect feeding and drought. Similarly, endophyte infection in tall fescue (Festuca arundinacea) reduced the growth and development of fall armyworm (Spodoptera frugiperda Smith) in only drought-stressed herbage (Bultman et al. 2003). The reason for the effect between root aphid populations and alkaloid concentration in this study is unknown. It is likely that an unknown metabolite responsible for root aphid deterrence may mask these effects since to date, the fungal secondary metabolite responsible for aphid deterrence/toxicity by AR37 has not been determined.
Plant performance is linked to the intensity of aphid pressure on the root system. The highest population of root aphids were found in the 0-10cm section (data not shown). However, when considering the root mass, aphids did not show a preference for a particular soil depth. Young nymphs are highly mobile in the soil (Rasmussen et al. 2008b). Large colonies were often found in areas with greater pore spaces in the soil structure as well as between the soil and planting container where there was prolific root growth. These colonies spread throughout the entire root system significantly reducing the above-ground shoot dry matter by up to 42% in endophyte-free plants (Fig. 5a), confirming previous research in which aphid feeding reduced foliar growth by up to 23% (Popay et al. 2016). While drought did not significantly change the total root biomass, the combined effect of root aphid feeding and drought caused a significant reduction in root growth. This study has provided the first data to show the detrimental effects of root aphids on the total root biomass, reducing root growth in endophyte-free plants by 49% (Fig. 5g) in comparison to plants treated with insecticide. However, some aphids were found on insecticide-treated plants. The applied insecticide is classified as systemic which means that it gets taken up by the plant and circulated in the phloem system to achieve superior efficiency in all parts of the plant. It is believed that ongoing drought conditions may have decreased the plant's ability to fully take up the insecticide, which allowed a minor infestation of root aphids on insecticide-treated control plants. The reduced root growth compromises the plant's ability to access water under drought stress, and yet in the field, plants are often exposed to simultaneous drought and pest pressure. Root aphids prefer to feed on young roots (Popay et al. 2016), suggesting that root morphology plays a significant role in population dynamics. Although not measured in this experiment, it may be that drought-exposed plants had more young roots, that are more efficient in water and nutrient uptake than old roots (Bouma et al. 2001; Eissenstat et al. 1997). Young roots contain less lignin making it easier for the aphid to insert its stylet into the root phloem (Whitham 1978). Plants in this experiment were kept at 1% above the permanent wilting point, which may have caused the development of new roots to maximise water uptake to secure plant survival, hence increasing root aphid populations.
Root aphids are phloem feeders requiring a positive plant turgor pressure to extract the available nitrogen-containing sap (Archer et al. 1995). To see an insect population increase, it is necessary that turgor pressure recovers periodically for the aphids to benefit from the increased nitrogen content (pulsed stress hypothesis by Huberty et al. (2004)). Therefore, the severity of drought plays an important role in predicting herbivore damage (He et al. 2014). In this study, the highest root aphid population was found in plants that were exposed to drought for 4-weeks, and the lowest amount in plants that had just reached the permanent wilting point (Fig. 3). In a similar study, above-ground aphid populations increased when plants were intermittently water-stressed, but not when plants were suffering from prolonged drought periods (Huberty et al. 2004) or were highly drought-stressed (Kansman et al. 2020). In this study, plants were exposed to drought for up to 8 weeks, during which intermittent drought was inflicted as often seen in natural situations. It may be that intermediate recovery of plant turgor pressure was sufficient for the aphids to take advantage of the stress-induced increase in nitrogen/amino acid content in the sap. Hence, if the turgor pressure falls below a certain threshold, it interferes with the aphid’s ability to utilise the available nitrogen in the phloem (Huberty et al. 2004), possibly resulting in lower populations as seen in plants that reached a permanent wilting point.
Pastoral farming in a changing environment
Climate change and extreme weather events, such as drought, are major drivers of pest populations as well as crop production (Skendžić et al. 2021). Despite the importance of the pastoral industry to New Zealand’s economic well-being, few studies have investigated and predicted the impacts of climate change on pasture insects in New Zealand’s farm systems (Dynes et al. 2010; Mansfield et al. 2021). The wide distribution of root aphids in New Zealand and the increasing severity and frequency of droughts makes root aphids an increasingly important group of herbivorous insects. The population increases observed in this study will significantly challenge modern pasture systems and since root aphids are present year-round they can constantly drain the plant resources resulting in reduced plant performance and vigour. With an increase in drought severity, aphids may be able to acclimatise to different environmental conditions utilising the stress-induced increase in nutrient content in the sap. With these changes, pasture systems will need to adapt to maintain production in a more variable and often drier climate. Root aphids are difficult to control due to their small size and below-ground habitat. In the field, the application of synthetic insecticide to prevent population build-up is not feasible. Therefore, an integrated pest management approach is necessary to minimise herbivore pressure. This study provides evidence that climate change-mediated impacts of root aphids can reduce pasture production. Even though Epichloë infections are expected to impose costs on the host grass in the absence of other stress factors (Bronstein 2001; Rodriguez et al. 2009), field observations indicate that endophyte infection is most beneficial to plants during simultaneous biotic and abiotic stresses (Popay et al. 2011). In New Zealand, such combined pressure occurs most often during late summer and autumn (Hume et al. 2007), when plants are the most vulnerable and fungal alkaloid concentrations are typically the highest (Fletcher et al. 2001). Our results have clearly demonstrated that the impact of simultaneous root aphid feeding and drought on plants can be ameliorated by the use of appropriate endophyte strains. Choosing the right endophyte strain is crucial to maximise pasture growth and is dependent on existing pest pressure and location (Caradus et al. 2021; Hewitt et al. 2021). Therefore, fungal Epichloë endophytes continue to be a critical constituent of intensively managed pastoral systems. However, their full potential under resource limitation and herbivorous pressure remains poorly understood, even though it is the combined pressure that can be terminal for pasture grasses. Further field trials are necessary to determine how climate change-mediated impacts of herbivorous insects affect pasture production.