Planting environment affects effectiveness of genotype diversity in increasing biomass and yield of a spring wheat mixture

INTRODUCTION

The relationship between biodiversity and ecosystem functioning has been a major focus of research in ecology. Previous studies have shown that increasing plant diversity has positive effects on ecosystem functioning, such as increasing primary productivity (Grange et al. 2021^; Tilman et al. 2001), increasing ecosystem stability (Craven et al. 2018^; Hautier et al. 2015), promoting nutrient cycling (Cong et al. 2014^; Steinbeiss et al. 2008^; Zhu et al. 2023a) and improving resistance to diseases and pests (Brooker et al. 2021^; Hahn and Cammarano 2023^; Mitchell et al. 2002^; Peacock et al. 2001). In agriculture, increasing crop diversity, i.e. intercropping, aims to exploit this biodiversity effect to improve the efficiency of resource utilization and production (Gaba et al. 2014^; Gardarin et al. 2022^; Lithourgidis et al. 2011). However, due to the differences in phenology, plant type, grain size and harvest logistics, mixed planting of multiple crops often brings extra burden to farmers during sowing, maintenance and harvest. However, this would be well solved, if mixtures of varietal genotypes with similar growth and development can bring the same effects as species mixtures. In other words, genotype mixtures would lead to biodiversity benefits.

As early as the 1980s and 1990s, research has demonstrated that mixing several varieties of a crop could increase grain yield and reduce disease and pest pressure (Smithson and Lenne 1996^; Wolfe 1985). Like species diversity, genotype diversity can also affect the structure and functioning of ecosystems (Hughes et al. 2008). Some studies further verified the important role of genotype diversity in increasing plant biomass or productivity (Crawford and Whitney 2010^; Crutsinger et al. 2006^; Drummond and Vellend 2012^; Hughes et al. 2016^; Johnson et al. 2006^; Tomimatsu et al. 2014) including crops (Fang et al. 2014^; Huang et al. 2024^; Juskiw et al. 2001^; Kathju et al. 2003^; Kong et al. 2023^; Mengistu et al. 2010^; Schöb et al. 2015^; Zhu et al. 2000). However, the large variation in magnitude of genotype diversity effects questions whether genotype diversity can play a stable positive effect (Borg et al. 2018^; Kiær et al. 2009^; Reiss and Drinkwater 2018). Besides, in crop systems, agronomists usually evaluate the success of crop mixture cultivation from agronomically relevant variables, such as biomass, yield and disease resistance, while ecologists assess the potential ecological factors and mechanisms underlying such effects. Here we aim to understand the ecological mechanisms supporting over-yielding and whether crop mixtures can be used to make agriculture more sustainable.

The sampling effect (SE) and complementarity effect (CE) are two components of the net genotype diversity effect (Crawford and Rudgers 2013^; Hughes et al. 2008^, 2016^; Johnson et al. 2006). In other words, the net genotype diversity effect might be dominated by one of the two, or the combination of both effects. A previous study found that with the increase of genotypic richness of Oenothera biennis, the diversity of arthropods also increased and the additive (i.e. SE) and non-additive (i.e. CE) effects among genotypes jointly played a role (Johnson et al. 2006). In contrast, other studies found CEs play a greater role in aboveground net primary productivity than SEs (Crutsinger et al. 2006). Therefore, it remains unclear to what extent sampling and CEs contribute to genotype mixture effects on yield. Another way to study mechanisms underlying biodiversity effects is the quantification of plant–plant interaction intensities, which are supposed to be less negative with increasing complementarity among plants in a community (Schöb et al. 2018). Modern crop cultivars tend to represent weak competitors to reduce the mutual competition between neighboring plants, thus improving the population-level yield (Denison 2015^; Denison et al. 2003). Furthermore, studies indicated that increasing genotype richness might further reduce negative plant–neighbor interactions through complementarity, and therefore further increase the population-level yield (Bossdorf et al. 2009^; Fridley et al. 2007^; Genung et al. 2012). However, the ecological mechanisms of genotype diversity in crops have been little studied, and whether a mixture of multiple genotypes could reduce intraspecific competition intensity in wheat is still uncertain.

Recent studies have verified the importance of the environment on the effects of crop species diversity or genotype diversity on productivity (Chen et al. 2021^; Engbersen et al. 2022^; Gallandt et al. 2001^; Reiss and Drinkwater 2018). However, the contrasting results limit our ability to resolve differences between studies. Some studies showed that genotype or species diversity often has more obvious effects under the condition of stress or limited resources (Hughes and Stachowicz 2009^; Reusch et al. 2005), but Weigelt et al. (2009) suggested the slopes of the biodiversity–productivity relationships increased with increasing levels of management intensity. Therefore, we need to understand how the over-yielding effects of genotype diversity vary under different environmental conditions.

Pot and field experiments are the two main methods in agricultural research along with their own advantages and disadvantages, such as pot experiments being easy to control and manage and field experiments being closer to the actual situation of crop production. Moreover, both pot and field experiments have often been used interchangeably to test hypotheses about biodiversity–ecosystem functioning relationships and their underlying mechanisms, but the results were not always completely consistent due to the objective, differential environmental conditions of pots compared with fields (Passioura 2006^; Poorter et al. 2012). For example, researchers often found that plants growing in the glasshouse had higher specific leaf area or leaf biomass than in the field (Lóopez-Castañeda et al. 1995^; Poorter and De Jong 1999^; Rebetzke et al. 2004). Thus, it is important to assess the genotype diversity effects in the situations of both pot and field experiments, and to judge how well pot experiments can represent field experiments.

In this study, we selected nine different genotypes of spring wheat (Triticum aestivum L.) and designed two planting patterns, a monoculture of one genotype and a mixture of nine genotypes, to simulate the possible effects of genotype richness. The experiment was conducted in both pot and field conditions for one growing season and we collected relevant trait data of spring wheat to verify our hypotheses: (i) genotypic diversity can promote productivity, such as biomass and yield; (ii) planting environment (i.e. pot and field) can affect character and strength of the diversity effect and (iii) CEs might contribute more to the net diversity effect than SEs.

MATERIALS AND METHODS

Experimental design

In this study, we selected two commonly used experimental methods to conduct an experiment: artificial pot conditions and real field conditions. The pot experiment was carried out from March to July 2016 in a rainout shelter near the field. The shelter was normally kept open but was closed manually when raining. We selected circular plastic pots (diameter 20 cm and height 26 cm) as the containers for planting. Soil in pots was taken from the top layer of the field and mixed with vermiculite at a uniform 1:3 ratio. We ensured each pot had the same soil weight (14.0 kg) and was watered with nutrient solution (1 g N, 0.27 g P and 0.35 g K per pot) before sowing. We conducted three planting treatments: monoculture, mixture and single plant (Fig. 1a–d). In mixture, one of the nine genotypes was used as the center plant, and the other eight genotypes were placed around it in random order and equal distance; while in monoculture, all the nine positions were of the same genotype. To ensure uniformity of mixture, each of the nine genotypes was used separately as a center plant. In addition, we had single plants in pots to observe the maximum potential of each genotype as a single plant without neighbors (Fig. 1d). Monocultures had three replications, mixtures and single treatments had four replicates, respectively. Therefore, there are a total of 99 pots in this pot experiment. All pots were distributed by using a completely randomized design. Pots were watered by the weighing method to 70% of the soil field capacity every day. Weeds were pulled by hand, and pests and diseases were controlled by spraying insecticide and fungicide once at each of the jointing stage, heading stage and grain filling stage, according to the local farmer’s management practices.

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The field experiment was conducted simultaneously with the pot experiment to test the realistic field production environment. Seeds were sown by hand at a depth of 3 cm with a planting density of 484 plants m⁻² and the plot size was 1 m × 1 m. To ensure the same genotype layout as potted plants and to facilitate sampling of wheat individuals, we selected the central sampling plot as the sampling area (Fig. 1e–g). Genotypes outside the sampling plot were planted randomly, but they were not recorded but used for total aboveground biomass and yield data collection. The monocultures had three replicates, and to ensure the survival of central sampling plants in the mixture, we performed six replicates, and selected all the surviving five replicates for data analysis. In order to measure each genotype’s performance in the field, a single plant was sown in the center of the sample area (1 m × 1 m) with four replicates (Fig. 1h). Therefore, there are a total of 108 plots in this field experiment, 27 plots for monoculture, 45 plots for mixture and 36 plots for single plants. All plots in the field experiment were distributed using a completely randomized design. 4 mm rainfall sprinkler irrigation was carried out with micro-spray equipment to ensure seed germination. The fertilizer of N, P and K was 31.8, 8.6 and 11.1 g m⁻², respectively, in the field according to the fertilization practices of local farmers. Weeds were pulled by hand, and pests and diseases were controlled by spraying insecticide and fungicide once at each of the jointing stage, heading stage and grain filling stage, following the local farmer’s management practices.

RESULTS

Effect of genotype diversity on spring wheat traits under different environments

Phenotypic characteristics at the sampling plot level were all significantly affected by the planting environment, with higher values observed in pots compared with those in the field (Fig. 2; Supplementary Table S2). The aboveground biomass, grain yield, stem weight, leaf weight, spike weight and grain number were affected by the interaction of planting environment and planting pattern: mixtures increased these variables under pot conditions but not under field conditions (Fig. 2; Supplementary Table S2).

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Compared with monocultures, genotype mixtures significantly enhanced the aboveground biomass and grain yield by 14.5% and 8.2%, respectively, in pots (Fig. 2a and b). However, no significant effects of genotype diversity on aboveground biomass and seed yield were found in the field, neither at the sampling plot level (Fig. 2a and b) nor at the actual production level (Supplementary Fig. S1). Plant height and stem thickness in mixtures were slightly, but not significantly higher than in monocultures under the two conditions (Fig. 2c and d). The stem weight, leaf weight, spike weight and grain number showed the same characteristics, that was, they were significantly increased in mixtures compared with monocultures only under pot conditions (Fig. 2e–h).

SE, CE and net effect

The CE, rather than the SE, was more likely to influence the diversity effect; the values of CE were higher than those of SE both in pot and field conditions (Fig. 3). In the field, the CE of aboveground biomass was significantly positive (95% confidence interval [CI]: 0.19–2.73), while the SE was negative. But both CE and SE of grain yield were negative, which indicated that monocultures and mixtures did not produce differences in grain yield under field conditions. In the pot, the SEs of aboveground biomass and yield were negative, while the CEs were significantly positive (95% CIs: 9.88–12.86 for aboveground biomass and 2.68–5.68 for grain yield), and were higher than the SE, resulting in a positive net diversity effect (95% CIs: 7.23–10.83 for aboveground biomass and 0.65–4.25 for grain yield).

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Plant–plant interactions in communities

We found that the effects of the neighbor plant’s biomass on the central plant’s grain yield and harvest index were significantly dependent on the planting pattern and planting environment (Fig. 4; Supplementary Table S3). In monocultures, the relationship between the neighboring biomass and grain yield of the central plant was negative (P = 0.30 and 0.37, respectively, in pot and field), and the relationship with the harvest index was significantly negative (P = 0.003 and 0.03 in pot and field, respectively). However, in mixtures, the relationship between the neighboring biomass and the central plant’s grain yield or harvest index changed to be a non-significant positive correlation. Furthermore, compared with monocultures, mixtures significantly changed these relationships in the pot environment, but not in the field environment (Supplementary Table S3). This indicated that mixtures reduced the negative impact of neighbors on the focal plant, especially in the pot environment. Additionally, the RII further demonstrated that mixtures significantly (P < 0.01, Welch’s t-test) reduced the intensity of plant–plant interactions compared with monocultures under pot conditions (Fig. 5).

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DISCUSSION

Previous studies on the impact of species diversity or genotypic diversity on productivity have shown that the environment plays a crucial role in the realization of diversity effects (Chen et al. 2021^; Engbersen et al. 2022^; Gallandt et al. 2001^; Reiss and Drinkwater 2018). Our results are consistent with them, and we found that the positive effect of genotypic diversity was not expressed in the rainfed field environment but was observed in the artificial pot environment, which received more water supply. Although some studies indicated that with more effective use and sharing of resources, genotype diversity or species diversity often exhibit more positive effects under stressful conditions characterized by limited resources (Hughes and Stachowicz 2009^; Reusch et al. 2005^; Zhu et al. 2023b), it is speculated that our opposite result can be explained by the varying effects of different planting environments on the ecological mechanisms underlying diversity effects.

From the perspective of diversity effect composition, we found that the SE exhibited a weak negative effect under both pot and field conditions (Fig. 3). This suggests that no specific spring wheat genotype dominated or suppressed others in mixtures, regardless of whether they were under field or pot conditions. However, the CE revealed distinct results between different environments, with no significant effect in the field but significant positive effects in the pot (Fig. 3). This suggests that the manifestation of the positive net effects of mixtures in pots is mainly driven by complementarities between genotypes. This is consistent with the findings of recent studies, which demonstrated that the yield increase of crop mixtures was primarily due to CEs (Cai et al. 2019^; Fang et al. 2014^; Li et al. 2020^; Su et al. 2023^; Wuest et al. 2021). Previous studies have demonstrated that the effectiveness of genotype diversity depends on the size of the potential genetic variation; the greater the difference in traits among individuals, the stronger the potential ecological CE (Drummond and Vellend 2012^; Hughes et al. 2008). Environmental conditions are important factors affecting the expression of this genetic variation (Hoffmann and Merilä 1999), and the full potential of genotypic differences is more easily expressed under relatively favorable conditions (Charmantier and Garant 2005).

In our study, the nine spring wheat genotypes, all of which are dryland wheat cultivars, have been adapted to the local water-scarce growing environment through a long-term drought domestication process. In the field environment, individual trait differences between genotypes were small. However, in the pot environment, greater water availability (2.8 times that of the field) promoted the expression of potential genetic variation, resulting in significant differences in individual traits among the spring wheat genotypes. We observed that the individual traits of spring wheat under pot conditions were much greater than under field conditions (Supplementary Figs S2 and S4) and showed higher ratios of coefficients of variance (Supplementary Fig. S3). This implies that mixed populations of spring wheat in pots exhibit greater ecological niche differentiation compared with those in fields, which is an important factor in promoting the emergence of CEs in mixtures.

Based on the ecological niche differentiation among spring wheat genotypes in pots, genotype mixtures can more fully utilize resources. For example, in pots we observed that the mixtures exhibited a higher specific leaf area and lower root biomass compared with the monocultures (Supplementary Fig. S5), showcasing the benefits of collective production in utilizing aboveground light and belowground resources. This is consistent with previous studies (Kong and Zhao 2023^; Li et al. 2023), which have demonstrated that genotype mixtures can optimize canopy structure and enhance the efficiency of capturing light, thereby increasing grain yield. In addition, compared with monocultures, mixtures of spring wheat reduced intraspecific competition. The negative correlations in monocultures between neighbor biomass and the focal plant’s grain yield or harvest index turned to positive or unrelated in mixtures (Fig. 4) and the RII was increased (Fig. 5). These results are consistent with the results of Parachnowitsch et al. (2014) and Cook-Patton et al. (2011), who found that competition intensity is highest in monocultures, but decreased with higher genotype diversity, while the productivity also increased with genotypic richness. This reflects the important relationship between grain yield benefits driven by spring wheat genotype diversity and niche partitioning reducing plant interaction intensity and increasing complementarity.

Capitalizing on the positive effects of genotypic diversity to increase crop yields is currently a hot topic among ecologists and agronomists. By analyzing the underlying ecological mechanisms, mixtures designed using a trait-based approach prove to be more effective in generating positive effects (Barot et al. 2017^; Montazeaud et al. 2020). It is interesting to note from this study that the influence of the planting environment on diversity effects is reflected in the realization of the unique genotypic potential. In the arid and semi-arid Loess Plateau region, where rainfed agriculture is prevalent, limited rainfall suppressed the genetic potential of the genotypes, leading to similar individual characteristics across all genotypes. This, in turn, limited the ecological niche differentiation within the mixtures, thereby restricting the emergence of effective positive complementarities. Therefore, we hypothesize that the over-yielding effect of genotype diversity may also be observed under water-stressed field conditions when appropriate water replenishment strategies are implemented. In the future, it will be necessary to investigate the positive effects of diversity by identifying varieties or management methods that are suitable for local conditions.

CONCLUSIONS

Our results showed that the positive diversity effect of genotype mixtures of spring wheat was only evident under pot conditions, not under field conditions. In pots, compared with the field rainfed environment, higher water availability promoted the expression of the genotypic potential of spring wheat genotypes, resulting in larger plant differences and ecological niche differentiation, and reduced competition between neighboring plants. This further led to a significantly positive CE and a net diversity effect on both biomass and grain yield. In the present study, the positive effects of genotype diversity occurred under the artificial pot condition but not under the real field conditions due to the different characteristics of the growing environment. This suggests that positive effects found under artificial (pot) conditions are not necessarily retained under more realistic field conditions.