Co-Cultivation of Grapevines and Transgenic American Chestnuts: Improving Water Relations through Niche Facilitation

We assessed the growth of transgenic hybrid American chestnuts and growth and productivity of grapevines within a co-cultivation framework. Our results were indicative of asymmetric competition, in that co-cultivation strongly affected the vegetative growth of grapevines in a density-dependent manner, with more limited impact on American chestnut. Although mean chestnut height and mean basal diameter did not vary significantly across the varying vine planting densities, we found a non-significant increase in mean chestnut height for trees growing in the one and three vine planting densities when compared to monoculture chestnuts, and a modest increase in chestnut basal diameter for trees grown with a single vine. Thus, there may be a real impact of density on chestnut performance that this study could not detect because of our relatively modest statistical replication. Observed growth and productivity differences were not informed by our photosynthesis data, as we did not find a density effect on photosynthetic rates, gs, or ψleaf for either crop under either light condition. As expected, we observed a plant effect on photosynthetic rates and gs, such that grapevines performed higher rates of photosynthesis and exhibited greater stomatal conductance than chestnuts under both light conditions. Overall, these findings further our understanding of presently under-examined American chestnut physiology by characterizing it within the context of a well-studied crop–wine grapes. These results also suggest that co-cultivated grapes and chestnuts at the single vine planting density exhibit mutual, albeit asymmetric, facilitation, with a discernible shift toward competition at the three and five vine planting densities for grapes. Collectively, these results are supportive of the viability of their joint cultivation within the Northeast, as premised by traditional agroforestry practices observed in other geographic regions.

The asymmetric facilitation we observed may relate to the varying physiological responses of grape and chestnut in response to resource limitation. Although interspecies competition tends to increase under favorable environmental conditions, such as those present through the duration of this study, (Holmgren et al., 1997; Koffel et al., 2021), chestnut’s pronounced morphological and physiological plasticity may allow it to withstand some of this competition. Under low light conditions, many shade-tolerant plants invest more biomass in aboveground tissues (Holmgren et al., 1997), and this biomass partitioning strategy has been documented for American chestnut (Belair et al., 2018; Evans et al., 2023; Wang et al., 2006). For instance, we observed a modest, albeit statistically insignificant, trend of increased SLA for chestnuts planted with three or five grapevines compared to those planted with a single vine–a common adaptive response in eastern deciduous tree species–which can increase photosynthetic rates under light limitation (Givnish, 1988; Gottschalk, 1994). Likewise, the lack of a density effect on gas exchange rates for chestnut observed here is consistent with the intermediate level of shade tolerance that has previously been proposed for American chestnut, where light response curves demonstrate that photosynthetic capacity remains high for this species even under light limitation (Belair et al., 2018; Evans et al., 2023; Joesting et al., 2007; Joesting et al., 2009; Onwumelu et al., 2022). Accordingly, growth has been shown to remain strong in American chestnut at PPFD values as low as 360 mmol m-2s-1 (32% full sunlight) (Wang et al., 2006). As such, American chestnut is able to tolerate the shade produced by substantial competition, such as what we observed through co-cultivation with varying numbers of grapevines, without facing a growth penalty. Conversely, chestnut growth suffers under substantial water limitation when light is not limiting (Baurle et al., 2006; Brown et al., 2014). Our study did not incorporate or experience drought conditions, limiting our ability to assess how each crop would respond and interact under water limitation. Likewise, we were unable to observe performance under excess soil saturation at this upland study site. Our Central New York site received 113.6 cm of precipitation over the year of this study, which is comparable to the 30-year mean annual precipitation of 101.2 cm (National Oceanic and Atmospheric Administration; 1991–2024; Station USW00014771). Future studies could incorporate irrigation to assess each crop’s response to excess soil saturation.

Concerning grapevine growth and productivity, total vine length increased substantially for grapevines in the single vine density compared to monoculture and other planting densities. Because we observed no statistically significant impact of density on leaf water potential for grapevines, water appeared to not be limiting for grapevines, and differences in access to light or other resources may explain variations in growth across density treatments. Excessive vegetative growth is often a sign that water and nutrient resources are overly abundant, and can reduce vine fruitfulness, decrease fruit quality, and increase fruit disease (Wolf, 2008). As such, it is possible that the reduction in vegetative growth that we observed at the three- and five-vine planting densities is not necessarily negative in the context of producing higher quality fruits, and that mixed-species competition during favorable environmental conditions through co-cultivation could help to achieve a more balanced vine. Accordingly, there was no statistically significant difference between grape cluster count and mass at the three and five planting densities compared to the single-vine density and monocultures. Moreover, while we did not observe a quantitative tradeoff in grapevine vegetative and fruit growth and the single-vine density, we were unable to assess if the increased vegetative growth impacted fruit quality. Grape quality results from a complex balance of sugar and acid levels (Wolf, 2008), and various co-planted partner species can affect these components in different ways (Steiner et al., 2021). Similarly, grape varieties differ in how they respond to such partners (Postles et al., 2013), highlighting the importance of selecting co-planted crop species based on the specific requirements of each grape cultivar (Downey et al., 2006). Additionally, these measurements did not differentiate between primary and secondary shoots, the latter of which tends to exhibit reduced fruiting potential compared to primary shoots (Wolf, 2008).

This research also sought to characterize chestnut photosynthetic and water use physiology within the context of grapevine physiology, a well-studied crop. Grapevines are known to perform exceptionally high rates of photosynthesis with correspondingly high rates of water loss (Moutinho-Pereira et al., 2004). Transgenic American chestnuts performed relatively moderate rates of photosynthesis, even under the low light condition (Table 3), when also compared to documented maximum rates of photosynthesis described in Joesting et al. (2007) for seedlings of co-occurring forest tree species, such Acer saccharum and Betula populifolia, under various shelterwood canopy density treatments (Ellsworth and Reich, 1992; Wayne and Bazzaz, 1993), and when compared to other chestnut types (Knapp et al., 2014). Additionally, we reported increases in net photosynthesis with increased light, supporting previous findings that American chestnuts display plasticity in physiological responses to light (Evans et al., 2023; Joesting et al., 2009; Wang et al., 2006). Given the absence of a density effect on photosynthetic rates in either crop, the observed differences in growth may not reflect recent carbon acquisition but may instead reflect residual effects of physiological performance in previous growing seasons under unfavorable conditions.

Regarding American chestnut water relations, this work supports previous research by Bauerle et al. (2006) and Onwumelu et al. (2022) in characterizing American chestnuts as having relatively high WUE when compared to other tree species, and here, when compared to grapevines. Stomatal regulation of photosynthesis was stronger for chestnut than grape, in agreement with prior research into American chestnut gas exchange (Onwumelu et al., 2022). Further, grape plants showed little to no decline in gₛ as ψleaf became more negative, suggesting a relatively water-permissive strategy. In contrast, chestnut plants under shade exhibited a marginal, albeit statistically insignificant, decrease in gₛ with declining ψleaf, indicative of relatively more conservative stomatal regulation. However we caution that ψleaf values were all moderate, so we did not observe the full space of potential gₛ as ψleaf covariation. These trends suggest that grapevines may prioritize carbon gain under water-limited conditions, while chestnuts exhibit greater stomatal sensitivity to water status.

This study had several limitations. First, it was only modestly replicated with three co- cultivation plots established in one specific location in central New York. The facilitation or competition arising from co-cultivation is often context specific, depending on variables such as climate, topographical position, or soil conditions (Holmgren et al., 1997). A higher degree of replication would increase statistical power while also elucidating some of the context specificity of interspecific interactions. Secondly, we did not explicitly account for the actual light intensities experienced across the experiment, which has previously been found to strongly explain physiological responses to shading (Niinemets, 2007). Future studies should incorporate PAR measurements that capture the actual light conditions experienced by each co-cultivation unit during photosynthetic data collection. Thirdly, this study did not examine differences in growth performance across genetic backgrounds, which have been characterized for American chestnuts in previous work (Clark et al., 2012; Clark et al., 2023, Wegner et al., 2025). Moreover, we assessed height and basal diameter growth for chestnuts, although other aspects of morphology which are important within forest contexts differ within orchard settings. For instance, American chestnuts grown under substantial light limitation tend to exhibit reduced height growth and a broader crown morphology characterized by pronounced lateral branch growth (Wang et al., 2006). While the shorter morphology which may result from co-cultivation at high grapevine planting densities may be beneficial in orchard structural training where access to lower growing branches is prioritized, light limitation generally has a negative impact on nut yield and quality in chestnuts (Araki and Hall, 2000). Because this study was conducted early in the chestnut trees’ development, it is too soon to assess the effects of planting density on nut productivity. Additionally, it is too early to assess the blight tolerance of hybrid transgenic American chestnuts, or how infection may influence American chestnut growth. Long-term monitoring will be necessary to evaluate potential trade-offs between growth and blight tolerance across different co-cultivation densities.

Evaluating the viability of the enforcado vineyard agroforestry system in the northeastern U.S. will require continued analysis of interactions between wine grape and tree species under varying environmental conditions and competitive pressures. In this experiment, conditions were generally favorable, which may have intensified competition between crops, consistent with the stress-gradient response hypothesis. The relative competitiveness of grape alongside the stress tolerance of chestnut allowed each to persist under resource competition–most plausibly resulting from moderate light limitation. This pattern aligns with the complementary trait dynamics outlined in the refined Stress-Gradient Hypothesis (Maestre et al., 2009; Rolhauser et al., 2023). Still, it remains unclear whether grape and chestnut would facilitate each other’s survival under coinciding stressful conditions of light, water, and other resource limitations. Interestingly, prior studies have proposed that the enforcado system tends to experience a lower incidence of pest and disease attack compared to monoculture plantings, due to its enhanced habitat complexity (Oliva Oller et al., 2022; Oliva Oller et al., 2025). Ultimately, vegetative growth is not the sole indicator of overall plant performance in orchard and vineyard contexts, and future analyses of nut and fruit yield and quality will be important for advancing this framework in the Northeast. The joint cultivation of wine grapes and chestnuts not only has the potential to enhance each crop’s performance under increasingly variable climate conditions, but also to advance local biodiversity through tree species restoration. In contrast to forest restoration contexts, agroforestry systems are relatively controlled and managed, such that blight tolerant chestnuts can grow with reduced pressure from pests, pathogens, and competition. These controlled environments may allow for more precise selection of individuals that exhibit both vigorous growth and high nut quality during breeding efforts.