Grafting is a common practice in perennial crops to combine desirable combinations of rootstock and scion traits. Although grafting has been called a “surgical alternative to breeding” for its utility in creating desirable combinations of traits (Albacete et al., 2015), it is better understood as a synergistic counterpart to breeding. Physical attachment of rootstocks bred for disease resistance and nutrient use efficiency to scions bred for fruit quality and quantity enables divergent sets of traits to be combined without incurring linkage drag and yield penalties. However, breeding programs have not yet optimized both rootstocks and scions in many grafted perennial crops, including Theobroma cacao.

Valued for its pods, which are used in chocolate, cocoa butter, and other economically important commodities, cacao is grown in lowland tropical regions such as West Africa, Latin America, and Southeast Asia. Modern cacao breeding programs have only existed since the 1930s, despite the popularity of this crop, which translates to a limited number of generations given the timeframe required for breeding perennials (DuVal et al., 2017). To date, these programs have focused primarily on generating elite scions, leaving the development of nutrient-efficient, stress-tolerant, disease-resistant rootstocks as a significant research gap. One reason may be that at least two of the three primary breeding objectives (bean quality, yield, and disease resistance) are controlled by scions, and soil-borne pathogens that can be controlled with resistant rootstocks are not as significant as foliar and pod pathogens in cacao (Bailey and Meinhardt, 2016). Another reason for the relative disinterest in rootstocks may be the dominance of seedlings over vegetative propagation methods in West Africa, which produces 70% of the world's cacao (Yin, 2004). Difficulties inherent in phenotyping belowground traits may also contribute. Yet rootstock breeding could play a critical role in the future of cacao. Meeting rising consumer demand without conversion of additional land to cacao production will require increasing resource use efficiency, especially at the root-soil interface.

Studies of nutrient use efficiency in cacao rootstocks are rare, but have shown potential that should be exploited to develop rootstocks that effectively capture soil nutrients, even in depleted soils. A comparison of two cacao rootstocks found differences in nitrogen use efficiency and growth response that were related to root dry weight (Ribeiro et al., 2008). Evidence from other tropical perennials such as mango supports a role for rootstock as well as scion identity in leaf nutrient content (Sarkhosh et al., 2021). This can be traced in some cases to soil resource acquisition. In grapevine, for example, contrasting effects on scion vigor were traced to differences in responsiveness to heterogeneous soil nitrogen and in transcriptomic reprogramming of nitrogen-related genes (Cochetel et al., 2017). Further exploration of homologous root and rhizosphere traits in cacao is warranted.

Root associations with the rhizosphere microbiome, or rhizobiome, may also play an important role in soil resource acquisition and nutrient use efficiency in perennial systems but have not been fully characterized in cacao. Grafting is known to affect rhizobiome composition: Small but significant rootstock effects on rhizosphere bacterial communities have been identified in grafted tomato (Poudel et al., 2019) and grapevine (D'Amico et al., 2018; Marasco et al., 2018). These effects may be small [around 3% of variation in tomato rhizosphere bacterial communities, far lower than soil compartment and site effects (Poudel et al., 2019)] but stronger than scion effects (Liu et al., 2018). Furthermore, these root-rhizobiome interactions can affect nutrient uptake (D'Amico et al., 2018). Research in cacao has shown that management practices impact fungal communities (Arévalo-Gardini et al., 2020) and total microbial communities (Buyer et al., 2017), but the effect of rootstock identity on root-soil-microbe interactions affecting plant nutrition has not been examined.

We grafted seedlings of eight rootstock populations with three scion genotypes under greenhouse conditions to test the individual and interactive effects of rootstock and scion identity on cacao seedling nutrition and rhizobiome composition. Furthermore, we investigated the nature of plant-soil-microbe interactions affecting nutrient uptake using a variety of statistical techniques. We hypothesized that (a) rootstock and scion identity would both affect leaf nutrient content; (b) rootstock would have a stronger influence than scion on rhizobiome composition; and (c) soil physicochemical parameters and microbial community composition would interactively affect plant nutrition. Because nutritionally relevant plant-microbe interactions could occur at many scales, we explored potential impacts of individual taxa, the entire rhizobiome, and subsets of bacterial and fungal communities.

Our findings of dominant rootstock effects on plant nutrition and microbial interactions in cacao suggest that rootstock breeding could be employed to increase nutrient use efficiency. In addition, this strategy would not compromise cacao bean quality, which is under scion control (Prawoto, 1990). Differences in plant nutrition among rootstocks and scions appear to be important primarily for the first few months after grafting. If differences had been observed among rootstocks in nutrient accumulation at both time points, it would have warranted investigation of the genetic basis of differences in nutrient uptake and partitioning as well as rootstock-specific microbial taxa that may have contributed. As the plant matures, plant physiological processes may demand stoichiometric nutrient ratios regardless of plant genotype, an explanation supported by the stronger correlations among nutrients at sixteen as compared to 4 months after grafting (Supplementary Figures 1, 2). Nonetheless, juvenile vigor of rootstocks and young grafts is a relevant trait for field production and a better understanding of the genes responsible for differentiation among rootstocks and scions in this study could help inform cacao breeding programs. A useful next step would be to validate these results by planting the same genotypes in the field under fertile and nutrient-limited conditions.

In accordance with our second hypothesis, rootstock effects outweighed scion effects on root-associated microbes. Bacterial diversity and community composition of both bacteria and fungi followed a similar trend in which rootstocks clustered in two groups: BYNC/EQX3348/IMC14/PA107 and SCA6/T294/T384/T484. Identifying the mechanisms responsible for the higher bacterial diversity in the first group and differences in rhizobiome composition was beyond the scope of this study, but we suggest that genome sequencing and analysis of root exudates from all eight rootstock populations would be useful starting points. The functional implications of reduced bacterial and fungal diversity for nutrient uptake by rootstocks SCA6, T294, T384, and T484 also deserve further investigation. D'Amico et al. (2018) found reduced rhizosphere bacterial diversity in one Vitis vinifera rootstock, including lower abundance of potassium-solubilizing taxa, that was hypothesized to be related to frequent potassium deficiencies in that rootstock.

We used multiple statistical methods to test our hypothesis that soil physicochemical parameters and microbial communities would impact plant nutrition, and to understand the nature of these relationships. The results of stepwise regression show that it may be important to develop new approaches to cacao nutrition that factor in soil biology as well as fertility. However, because this was a greenhouse study, limited variation in soil physicochemical parameters may have led to underestimation of the importance of these variables for plant nutrition. Applying the statistical methods used in this study to field data would be a valuable area for future research. Soil pH, for example, strongly affects nutrient availability and microbial communities in tropical agroecosystems (Juo and Franzluebbers, 2003), but did not show up as particularly important in stepwise regression, likely because of low variability in this study (Figure 4). Interestingly, despite limited variation, soil organic matter (SOM, also a key management concern in tropical soils and particularly in intensive cacao production systems) was the most important variable in multiple plant nutrient models (Figure 4). Increasing SOM in soils under cacao should remain a high priority given its apparent role in plant nutrition as well as significance for microbially-mediated soil health and C sequestration.

We interpret the insignificant nutrient-microbe correlations and Mantel tests and the results of stepwise regression as a call for more complex understanding of plant-soil-microbe interactions at intermediate scales, e.g., between binary associations and entire communities. Identification of functional guilds, statistical methods to identify the most pertinent features of high-dimensionality datasets, and synthetic-community-based studies could prove useful here among other methods (recently reviewed by Gupta et al., 2021). Plant growth promotion or enhanced plant nutrient content mediated by these intermediate-scale groups of microorganisms could result from direct mechanisms (e.g., direct nutrient transfer by symbionts) or indirect mechanisms (e.g., increased bioavailability of soil nutrients), but in either case would be an emergent property of the group rather than attributable to a single organism. Still, we gain insight into potentially beneficial members of the rootstock-responsive taxa identified in this study from single-strain or low-complexity inoculation studies common in the literature.

Many of the rootstock-responsive bacterial taxa identified belonged to orders associated with plant growth promotion, although incomplete taxonomic assignments limit our ability to confirm the presence of plant-growth-promoting rhizobacteria (PGPR) here. For example, the Rhizobiales, Frankiales, and Xanthomonodales, identified as enriched in BYNC, EQX3384, T294, and T384 (Figure 5) include free-living and associative nitrogen-fixing bacteria. Unfortunately, these could be identified to the genus level only in some cases (Pedomicrobium, Devosia, Mycoplana, Hyphomicrobium, Bauldia, Rhodanobacter, Chujaibacter, and Jatrophihabitans), and N fixation is not a conserved trait for all of these genera (Rivas et al., 2002; DeAngelis and Pold, 2020). The Rhizobiales, which include both diazotrophs and pathogens (Carvalho et al., 2010), were the most abundant order across all samples (Supplementary Figure 3). Future metagenomic studies of the cacao rhizobiome could screen for gene clusters involved in N fixation that might become part of biologically-based nutrient management strategies.

The Pseudomonodales and Bacillales, present at low relative abundance in all rhizobiome samples and identified in rootstock-responsive bacteria (Supplementary Figure 3, “Other”), contain taxa capable of increasing plant productivity in many plant hosts by both direct and indirect methods, e.g., producing growth-promoting auxins or suppressing pathogens (Kloepper et al., 2004; Lugtenberg and Kamilova, 2009). Furthermore, strains belonging to the Bacillales have shown promise in cacao. Bacillus subtilis inoculation of germinating seeds increased leaf area by 14%, shoot height by 8%, and graft success rate by 24% (Falcäo et al., 2014), and repeated inoculations of seeds and seedlings increased shoot height by 22% (Leite et al., 2013). Inoculation of 1-month-old cacao seedlings with other Bacillus spp. increased height and biomass by up to 37 and 73%, respectively (Gupta et al., 2011). However, inoculation with Pseudomonas sp. did not promote cacao growth in another study (Guillermo Ramírez et al., 2016).

As with bacteria, incomplete taxonomic assignments limit our ability to clarify the ecological roles of many of the rootstock-responsive fungi. Unfortunately, many of the rootstock-responsive fungi could not be identified by genus or species, the taxonomic levels used for functional guild assignment in fungal trait databases such as Fun^Fun (Zanne et al., 2020), FUNGuild (Nguyen et al., 2016), and FungalTraits (Põlme et al., 2020). Two of the four fungal orders detected, the Glomerellales and the Chaetothyriales, have been found at low abundance (2.0–2.3%) in the cacao phyllosphere, but their roles there were not defined (Wemheuer et al., 2020). The majority of studies investigating fungi as potential plant-growth-promoting agents in cacao have focused on mycorrhizae (Chulan and Ragu, 1986; Cuenca et al., 1990; Chulan and Martin, 1992; Tchameni et al., 2012; Guillermo Ramírez et al., 2016) or Trichoderma sp. (Bae et al., 2009; Tchameni et al., 2011). While 112 ASVs belonging to the genus Trichoderma and 8 belonging to the mycorrhizal order Glomerales were found in fungal rhizobiomes in this study, none were among the rootstock-responsive taxa (Supplementary Figure 4).

The possible presence of pathogenic taxa among rootstock-responsive fungi could have had detrimental impacts on leaf nutrition in diseased plants. The Glomerellales (not to be confused with the Glomerales) contain Colletotrichum sp., cacao pathogens responsible for anthracnose (Nascimento et al., 2019). While two of the rootstock-responsive Glomerellales strains were identified as Musicillium sp., the other two were not identified to the genus level and the possible presence of Colletotrichum sp. could not be ruled out. A member of the Hypocreales enriched in T484 (Figure 5, “Other”) was identified as Fusarium sp. This genus is present at low relative abundance in cacao agroforestry systems (e.g., 1.3–6.0%, Arévalo-Gardini et al., 2020). While it contains pathogens that cause cushion gall, dieback, and other diseases in cacao (Ploetz, 2006; Adu-Acheampong et al., 2012; Rosmana et al., 2013), it also contains strains that may provide biocontrol against Phytophthora palmivora, the causal agent of black pod rot (Hanada et al., 2010).

Despite limited attention to cacao rootstocks and rhizosphere microbial associations to date, our results highlight the potential of coordinated breeding efforts in these areas, combined with informed soil nutrient management, to enhance plant nutrition in cacao. This will be a challenging endeavor for numerous reasons: time and cost required to generate new perennial germplasm, dispersed smallholder production adding logistical difficulties to participatory cacao research and breeding, and remaining knowledge gaps in the genetic basis of cacao-rhizobiome interactions. Nonetheless, significant interest by consumers and industry in sustainable cacao production will help advance the field and translate research into practice.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: NCBI (accession: PRJNA755178).

AD, AP, AT, and JS contributed to the study design and data collection. JS, AD, and TC analyzed the data. JS wrote the manuscript with input from all authors. All authors approved the final version for publication.

JS, AD, AT, and TC are employed by Mars Wrigley. The remaining author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.