write a hypothesis about the worms and lima bean plants

Microbes promote lima bean growth

Lima beans are packed with nutrients. They are an excellent source of protein and fiber. They are rich in vitamins and minerals.

Lima beans are also good for the environment and farmers. They are effective as cover crops and as green manure.

The benefit of lima beans stretches down even into their roots. There, they house microbes that transfer – or ‘fix’ – atmospheric nitrogen into the soil. Plants can access this fixed nitrogen, which helps them grow.

Researchers in Brazil have identified which microbes work well with lima beans in the drier, northeastern parts of the country.  The study  was published in Soil Science Society of America Journal.

“We think our findings can be an important component of sustainable agriculture in the region,” says Fatima Maria de Souza Moreira, co-author of the new study.

Microbes are often added to seeds or to the soil when preparing for planting bean crops. These additions – called inoculants – can jumpstart the partnership between the plants and microbes.

“Sustainable agriculture management aims to improve natural, helpful processes like nitrogen fixation by microbes,” says Moreira. “Knowing which inoculants work well with lima beans in northeastern Brazil will provide economic and environmental benefits.”

Lima beans – also called butter beans – are an important crop in many parts of the world. Often, these beans are a vital source of food and income in poorer areas.

In Brazil, lima bean is mainly cultivated by small farmers in semi-arid regions.

“Knowing which microbes are naturally present in the root nodules of lima beans in these areas is important,” says Moreira.

“We can then test which species of microbes work best as inoculants to promote lima bean growth,” she says. “Also, we can determine which of these microbes don’t harm animals and humans.”

In addition, climate change makes it even more crucial to get more information about these microbes.

“Knowing more increases our chances of finding microbes that perform well in different soils and different climatic conditions,” says Moreira.

The researchers collected lima bean plants from farms in the northeast Brazilian state of Piauí. They grew microbes isolated from root nodules. Then they extracted DNA from these microbes and analyzed DNA sequences to identify them.

Most of the microbes the researchers identified belong to a group called  Bradyrhizobium . These microbes are not harmful to humans or animals and have many advantages.

For example,  Bradyrhizobium  microbes are genetically stable. That means they tend to not accumulate many mutations in their DNA, and the same strains can be used in agriculture for many years, even decades.

“In fact,  Bradyrhizobium  strains used in soybean farming in Brazil have been the same since the 1960s,” says Moreira.

Also, the microbes identified in the study were obtained from a semi-arid region with very high temperatures. “They are a great resource for potential use in a warming world,” says Moreira.

The researchers also tested which strains of  Bradyrhizobium  worked best as inoculants for growing lima beans in the lab. They found several strains that helped lima bean plants grow well under both optimal and adverse soil conditions.

“Microbes are versatile lifeforms,” says Moreira. “Very often they work in different soil types and climate regions.”

But that’s not always the case.

That’s why it’s important to have a large number of microbes with diverse genetic characteristics that can be used as inoculants. That will allow farmers and researchers to choose microbes best adapted to specific soils and conditions.

Researchers are now testing the microbes identified in the study under different field conditions.

“We are also studying them to explore other diverse biotechnological applications,” says Moreira. “For sure, the lima bean/ Bradyrhizobium  partnership can be an important component of sustainable agroecosystems.”

Fatima Maria de Souza Moreira  is a researcher at the Universidade Federal de Lavras in Brazil. This work was supported by CAPES, the Brazilian National Council for Scientific and Technological Development, and the Minas Gerais Research and Support Foundation.

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Early damage enhances compensatory responses to herbivory in wild lima bean

Associated data.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

Damage by herbivores can induce various defensive responses. Induced resistance comprises traits that can reduced the damage, while compensatory responses reduce the negative effects of damage on plant fitness. Timing of damage may be essential in determining the patterns of induced defenses. Here, we tested how timing and frequency of leaf damage affect compensatory responses in wild lima bean plants in terms of growth and seed output, as well as their effects on induced resistance to seed beetles. To this end, we applied mechanical damage to plants at different ontogenetical stages, at one time point (juvenile stage only) or two time points (seedling and juvenile stage or juvenile and reproductive stage). We found that plants damaged at the seedling/juvenile stage showed higher compensatory growth, and seed output compared to plants damaged only at the juvenile stage or juvenile/reproductive stage. Seeds from plants damaged at the juvenile and juvenile/reproductive stages had fewer beetles than seeds from undamaged plants, however this was driven by a density dependent effect of seed abundance rather than a direct effect of damage treatments. We did not find differences in parasitism rate by parasitoid wasps on seed beetles among plant treatments. Our results show that damage at the seedling stage triggers compensatory responses which implies that tolerance to herbivory is enhanced or primed by early damage. Herbivory often occurs at several time points throughout plant development and this study illustrates that, for a full understanding of the factors associated with plant induced responses in a dynamic biotic environment, it is important to determine the multitrophic consequences of damage at more than one ontogenetical stage.

Introduction

The nature and magnitude of plant induced responses depend on several factors, such as the type, frequency and timing of damage and herbivore identity ( Karban, 2011 ; Schuman and Baldwin, 2016 ). Herbivore damage can occur at several times throughout a plant’s life, during the same ontogenetical stage or at different stages. However, it is still not fully understood how the timing and frequency of herbivore damage affect the plant’s induced defense responses. Induced plant responses to damage comprise plastic traits that help plants increasing their fitness in the presence of natural enemies ( Karban and Baldwin, 1997 ; Baldwin and Preston, 1999 ). Such responses can be classified as two different strategies, induced resistance and tolerance. Induced resistance includes inducible traits that protect plants from future attacks ( Karban, 2011 ), such as direct defenses, both chemical (e.g. secondary metabolites) and physical (trichomes) defenses, and indirect defenses (volatile compounds and extrafloral nectar), that aid in the recruitment of natural enemies of herbivores ( Gatehouse, 2002 ; Heil and Baldwin, 2002 ; Turlings and Erb, 2018 ).

Tolerance to herbivory refers to the plant's response after herbivory to reduce the negative effects of damage on fitness ( Strauss and Agrawal, 1999 ; Núñez-Farfán et al., 2007 ) and it is the result of compensatory responses which can often be measured as compensatory growth and/or seed production (the ability of a plant to increase its biomass or reproduction after suffering damage). As tolerance involves changes in resource allocation that occur as a response to damage, it is also considered an induced defense ( Karban and Myers, 1989 ). Because resistance and tolerance can be induced by herbivore damage, the time at which damage occurs determines the expression of both strategies. Yet it is still unclear the extent to which the timing of multiple damage events influences the expression of each strategy.

Plants present ontogenetic trajectories consisting of changes in the production and investment of defenses that occur across the developmental stages of the plant ( Boege and Marquis, 2005 ; Barton and Boege, 2017 ). They are influenced by abiotic factors, type of defense, and/or amount and timing of herbivory. For example, in Casearia nitida it was shown that plants damaged at the sapling or reproductive stage compensate for defoliation by growing more new leaves than undamaged plants ( Boege, 2005 ). However, this response varied with the amount of defoliation, since saplings compensated better than reproductive plants at high defoliation levels. The interactions between plants and a dynamic community of antagonists (herbivores) and mutualists (pollinators and natural enemies of herbivores) will be fundamental in shaping the ontogenetical patterns of defensive strategies. In this context, plants can use different strategies depending on the stage at which they interact with other species ( Barton and Boege, 2017 ). For example, plants that suffer a single damage event would favor investment in compensatory responses, while plants that experience repeated damage could favor a higher investment in induced resistance ( Karban et al., 1999 ). Ontogenetic trajectories for tolerance can also be species-specific. Barton (2013) found that tolerance mechanisms were different between two Plantago species depending on their ontogeny. While tolerance in P. lanceolata was associated with flowering and shoot biomass in plants damaged at a mature stage, in P. major it was associated more with biomass and photosynthetic parameters when damaged at seedling and juveniles stages, respectively. Finally, plant resistance and tolerance may have different trajectories. This could also indicate trade-offs between both strategies if the expression of one strategy over the other switches according to the ontogenetical stage. For example, it has been shown that in Raphanus sativus juvenile plants show higher resistance but less tolerance than reproductive plants ( Boege et al., 2007 ), but in Arabidopsis thaliana the cost of tolerance at different stages was not associated with a trade-off with resistance ( Kornelsen and Avila-Sakar, 2015 ).

Plant resistance and tolerance responses to herbivore damage can cascade to other interactions that occur later during the plant’s life. Rusman et al. (2020) studied the interaction between the timing of herbivore damage on flower traits and the pollinator community in Brasica nigra . They found that plants attacked at vegetative stages by some herbivore species had lower number of flowers, fewer flower visitations by pollinators and lower seed output than control undamaged plants, while damage at the budding stage had positive effects on flower visitation. Similarly, previous work with wild lima bean, showed that damage by leaf beetles reduced subsequent attack by seed beetles at a later stage of the plant’s life ( Abdala-Roberts et al., 2016 ; Hernández-Cumplido et al., 2016 ; Bustos-Segura et al., 2020 ). However, this type of induction depends on herbivore species and on the plant’s stage when the first damage occurs (in leaves or bean pods) ( Hernández-Cumplido et al., 2016 ). In another study, Cuny et al. (2018) found that when lima bean plants were exposed to a single herbivory event by leaf caterpillars, plants produced more leaves compared to undamaged plants, indicating leaf overcompensation. The results from the studies above provide strong evidence that induced responses in wild lima bean are dependent on several biotic factors, however the extent to which the frequency and time of damage influence these responses has not been tested. Particularly, it remains unknown whether early leaf damage events can modify or even improve tolerance to herbivory or affect seed quality and preferences of seed feeders and their predators or parasitoids. Analyses of ontogenetic interactions, could provide insights into how ontogenetical trajectories influence defense induction for future interactions in a multitrophic context.

Here we used wild lima bean to test the effects of frequency of damage and damage at different ontogenetic stages on compensatory responses and subsequent interactions with seed insects. To represent better the frequency and timing of herbivory as it occurs in the wild, we applied mechanical damage at two time points representing early damage (at seedling + juvenile stages) or late damage (at the juvenile + reproductive stages). In addition, to analyze the influence of one vs. two damage events at different ontogenetical stages on tolerance and induced resistance, another group of plants was damaged only at the juvenile stage. This design allowed us to test differences in timing (seedling vs. reproductive) and the synergistic effects of different events of damage (one vs two events of damage) that comprise the span of the plant’s development. Once seeds were matured, we examined the effects of leaf damage on seed infestation by exposing seeds to natural populations of seed beetles and their parasitoids. We addressed the following questions: 1) How does the timing and frequency of leaf damage influence plant growth and reproductive output? 2) How does timing and frequency of leaf damage alter the subsequent interactions between seeds and their associated insects? In addition, because we expected that a damage event early in the plant’s life could determine the investment in tolerance to damage at future stages, we tested the hypothesis that damage at the seedling stage will enhance plant tolerance, compared to damage at the juvenile stage or repeated damage at the juvenile and adult stages.

Material and methods

Study system.

Wild lima bean ( Phaseolus lunatus ) is distributed along the Pacific coast from Mexico to South America ( Freytag and Debouck, 2002 ). In the south pacific Mexican coast this annual legume germinates in June–July, produces flowers in October–November and seeds in December–January ( Heil and Silva Bueno, 2007 ; Moreira et al., 2015 ; Hernández-Cumplido et al., 2016 ). During the growing stage, lima bean plants are attacked by a number of leaf-chewing insects, including the velvet armyworm Spodoptera latifascia , Lepidoptera: Noctuidae ( Cuny et al., 2018 ). This polyphagous caterpillar attacks several crops including maize, beans, tomato and chili pepper ( Habib et al., 1982 ; Chabaane et al., 2022 ). During seed production, which lasts around two months, seed beetle species such as Zabrotes subfasciatus (Coleoptera: Chrysomelidae) and Acanthoscelides obtectus (Coleoptera: Chrysomelidae) enter dry pods and lay eggs on lima bean seeds, the larvae develop and pupate inside the seed ( Benrey et al., 1998 ; Alvarez et al., 2005 ; Šešlija et al., 2009 ). They are both considered insect pests that also attack other species of stored cultivated beans ( Birch et al., 1985 ; Paul et al., 2009 ). Both species are parasitized by several solitary parasitoid species, among them, the ectoparasitoid Stenocorse bruchivora ( Aebi et al., 2008 ; Moreira et al., 2015 ; Hernández-Cumplido et al., 2016 ).

To explore the effects of timing of herbivory on plant responses, we conducted an experiment at the field station of the Universidad del Mar, Campus Puerto Escondido during the field season 2019-2020. We planted three seeds of wild lima bean in each of 80 pots (5 l volume) by November 15th, 2019. Seeds were collected in the same site the year before. After germination, we thinned the seedlings to leave only one per pot. Pots were kept in a large tent (Lumite ® 3.66 m × 1.83 m × 1.83 m, Bioquip, CA, USA) to avoid unwanted insects attacking the plants. After two weeks of germination, we selected 64 seedlings (with only one trifolia developed) and distributed them randomly in 16 mesh tents (four plants per tent; Lumite ® 1.83 m × 1.83 m × 1.83 m, Bioquip, CA, USA). Then, we applied damage treatments on these plants with the objective of studying induced plant responses to damage produced at different times throughout plant development, at seedling, juvenile or reproductive stage.

Experimental procedure

Four treatments were randomly assigned to the four plants in each of the 16 tents. The treatments to one plant per tent were as follows: C: control plants with no artificial damage. S-J: Plants damaged at the seedling and juvenile stages (at two and six weeks after germination). J: Plants damaged only at the juvenile stage (at six weeks after germination). J-R: plants damaged at the juvenile and reproductive stages (at six and ten weeks after germination; see Figure 1 ). So that all tents contained one plant of each treatment. For each damaged plant, we cut half of each leaflet with scissors. In addition, we also removed the apical meristem of growing tendrils. This was based on the damage that common herbivores, such as Spodoptera latifascia , produce on wild plants, as they would readily eat both the meristems and leaves (Bustos-Segura, personal observation).

Schematic representation of the experimental design. Damage treatments on lima bean plants consisted of artificial damage on leaves at two different ontogenetical stages (S-J and J-R) or at one (J).

Plant measurements and collection of seed insects

We counted the number of leaves and branches per plant three times throughout plant development (5, 9 and 14 weeks after germination). As the mesh tent was not 100% effective at keeping out naturally occurring insect, we observed some damage on the plants (mainly by Spodoptera spp. and Diabrotica baleata ). Thus, throughout the experiment we recorded the proportion of damaged leaves. We also recorded the date when each plant produced the first flower to calculate the time to flowering (days from germination to the production of the first flower).

When pods were mature, they were removed from the experimental plants and placed inside a single green mesh bag per plant, next to each plant following the methods from Bustos-Segura et al. (2020) . This mesh was wide enough to allow the entrance of seed beetles and their parasitoids, but small enough to hold seeds inside the bags. Twelve weeks after germination, when most of the seeds were collected in mesh bags, the tent was removed to allow natural infestation by seed insects (including seed beetles and their parasitoids). After two weeks of exposure, the seeds were placed in plastic bags for two weeks to allow the emergence of insects developing inside the seeds. Then seeds were placed in a freezer at -20°C for one day. Insects found in each bag were counted and identified. We also counted the number of seeds and weighed 10 healthy seeds per plant.

Statistical methods

All analyses were performed with R system (version 4.2.0; R Core Team, 2022 ). We used generalized linear mixed models (GLMM) to analyze differences among damage treatments on plant and insect variables using package lme4 ( Bates et al., 2015 ). For all models we included damage treatment as a fixed effect and tent as a random effect. For the number of leaves and natural damage we included time point of measurement and the interaction with treatment as another fixed effect, and plant ID as a random effect. For analyzing number of leaves, number of branches, number of seeds, and number of insects, a Poisson error distribution was used. For analyzing the number of seed insects we included the number of seeds (log-transformed) as a covariate to account for the density-dependent relationship between insects and seeds. As seed beetles are attacked by solitary parasitoids, the number of total seed insects (beetles plus parasitoids) indicates the initial infestation by seed beetles. Whenever the dispersion ration of Poisson models was higher than 2, we included an observation level factor as a random factor to control for overdispersion. For comparing natural damage among treatments we used a binomial error distribution (as the proportion of damage leaflets). Time to first flowering was analyzed with a Cox proportional hazards mixed model, with package coxme . Seed mass in seeds was analyzed with a normal error distribution. Differences in parasitism rate among treatments were analyzed with a GLMM and a binomial distribution. We performed a structural equation model (SEM) for testing the causal pathway among Treatment groups, number of trifolia (at the last measurement), number of seeds and number of insects. Parasitism rate was not included in the SEM, given it could only be estimated for 30 plants. We used the piecewiseSEM package in R system that allows to include GLMs and mixed models in a SEM ( Lefcheck, 2016 ).

Overall, there was a significant effect of damage treatment on the number of trifolia across the season ( χ ( 3 ) 2 =10.63; P=0.014, Figure 2A ), with only plants from the J treatment having significantly fewer leaves than control plants ( Table S1 ). The effect sizes of the different contrasts ranged in the lower values (Cohen’s d from -0.04 to 0.11, Table S1 ). Time significantly explained the change in number of trifolia ( χ ( 2 ) 2 =2452; P<0.0001), meanwhile, the interaction between treatment and time was not statistically significant ( χ ( 6 ) 2 =3.15; P=0.79).The number of branches increased with time ( χ ( 2 ) 2 =170; P<0.0001; Figure S1A ), but damage treatment and its interaction with time had a non-significant effect ( χ ( 3 ) 2 =6.26; P=0.1; χ ( 6 ) 2 =1.72; P=0.94, respectively). Despite the plants being inside tents, some insects entered and caused damage. For this minor but uncontrolled damage, there were differences across time ( χ ( 2 ) 2 =1115; P<0.0001), with higher damage at the beginning of the season ( Figure 2B ), but treatment as a main factor did not explain much of the variation ( χ ( 3 ) 2 =5.72; P=0.13). There was however, an effect of the interaction between treatment and time ( χ ( 6 ) 2 =29.38; P<0.0001), where in the middle of the season undamaged control plants received more natural damage than mechanically damaged plants (regardless of the timing of damage).This effect was small compared to the treatment damage and was not detected at the beginning, nor at the end of the season ( Figure 2B ).

Effects of timing and frequency of damage on lima bean leaves. (A) number of leaves, (B) proportion of leaflets with uncontrolled natural herbivory. Treatment groups were: mechanically undamaged control plants (C); plants damaged at the seedling and juvenile stage (S-J), only at the juvenile stage or at the juvenile stage and reproductive stage (J-R). Different letters indicate significant differences among damage treatments within the same time point. ns indicate non-significant difference among damage treatments.

The time from germination to first flowering was affected by the damage treatment ( χ ( 3 ) 2 =7.81; P=0.05). Plants from damage treatments S-J showed the longest time to production of the first flower and control plants the shortest time ( Figure S1B ). However, a multiple comparison posthoc test did not reveal specific differences between treatment pairs.

Fifty-two plants out of 64 produced seeds, but this was not influenced by the damage treatment ( χ ( 3 ) 2 =1.17; P=0.76). There was a significant effect of damage treatment on seed production ( χ ( 3 ) 2 =217; P<0.0001; Figure 3A ). Control plants produced the most seeds, followed by plants damaged at the seedling stage (S-J), and then J and J-R plants. J and J-R plants produced similar number of seeds ( Table S1 ). The effect sizes for comparisons between controls and damage treatments were relatively high ( Table S1 ), while the effect sizes among damage treatments were lower ( Table S1 ). Seed mass was not significantly different among treatments ( χ ( 3 ) 2 =5.55; P=0.14).

Effects of timing and frequency of damage on seed production and interactions with seed insects. (A) number of seeds as a measure of reproductive output and (B) number of seed insects per 100 seeds per plant. The number of insects includes seed beetles and parasitoids, thus indicating the initial infestation by seed beetles. Treatment groups were: mechanically undamaged control plants (C); plants damaged at the seedling and juvenile stage (S-J), only at the juvenile stage or at the juvenile stage and reproductive stage (J-R). Different letters indicate significant differences among damage treatments.

The total number of beetles and parasitoids collected was 87 and 21, respectively. Beetles emerging from the seeds were mostly of the species Acanthoscelides obtectus , with Zabrotes subfasciatus only present in two plants, so both species were pooled for analyses. We found that damage treatment had an effect on the abundance of seed beetles ( χ ( 3 ) 2 =22.83; P<0.0001; Figure 3B ). The number of seed insects in seeds from control plants was not significantly different from plants from the S-J treatment (Tukey’s post-hoc test: P=0.27), but was higher than in J and J-R treatment (Tukey’s post-hoc test: P=0.003 and P<0.001, respectively). When the number of seeds per plant was used as a covariate to explain insect abundance, its effect was highly significant ( χ ( 1 ) 2 =25.41; P<0.0001), but the effect of damage treatment was no longer important ( χ ( 3 ) 2 =0.79; P=0.85), with more insects emerging from plants that produced more seeds. The parasitism rate on beetles was in average 0.122 ± 0.038 and was not different among damage treatments ( χ ( 3 ) 2 =0.62; P=0.89). The structural equation model confirmed the association among variables and showed an association between number of leaves and number of seeds ( Figure 4 ). Damage treatment affected the number of trifolia, however this path does not show an influence on number of seeds. Thus, the effect of treatment on number of seeds was independent from the number of trifolia. There is an indirect effect of treatment on number of seed beetles, mediated by number of seeds, with no direct effect of treatment on number of beetles.

Diagram of the structural equation model including pathways between measured variables. Solid arrows indicate statistically significant positive pathways and dashed arrows indicate no significant pathways. The differences between treatments are given next to pathways from the Damage treatment variable and different letters indicate significant differences between groups according to a Tukey’s post-hoc test. R square values given for each individual endogenous variables are conditional r squares (excluding random effects). All pathways were analyzed with GLMs and a Poisson error distribution. The estimates are given in the log scale. Model goodness-of-fit: Fisher’s C=0.25, P=0.88.

The ability of a plant to recover from herbivore damage is crucial for its success in a natural environment. Plant ontogeny is known to play an important role in determining the compensation and defense responses against herbivory. While most studies on the effects of plant ontogeny on induced responses focus on isolated developmental stages, in nature, herbivores can attack throughout plant development at different points in time. Here we aimed to study the consequences of herbivory occurring at more than one ontogenetic stage with repeated or single damage events, on plant compensatory responses. This approach can give us information about the influence of early damage on the responses to subsequent damage events. We found that early damage (at the seedling and juvenile stages) results in better compensatory growth and seed output than late damage (at the juvenile stage alone, or together with damage at the reproductive stage). But early and repeated damage is still positive in terms of leaf and seed production when compared to a single damage event occurring at the juvenile stage. This indicates that damage at the seedling stage can enhance plant tolerance to future damage. Conversely, we did not find evidence that leaf damage induced defenses on seeds against attack by seed beetles. Infestation by beetles in seeds of damaged plants was not so different from infestation in seeds from undamaged control plants, regardless of the timing and number of damage events.

Plants damaged at the seedling stage compensated by producing more leaves than plants from the other damage treatments, while maintaining a similar leaf production compared to control plants. This suggests that early damage stimulated growth to compensate for the loss of photosynthetic area, as compared to plants damaged at later stages. A larger photosynthetic area as compared to plants damaged later in their development could provide enough resources to produce more seeds ( Tiffin, 2000 ; Wise and Abrahamson, 2005 ). However, the SEM in the present study showed no association between number of trifolia and seed set, but a direct effect of damage treatment on the number of leaves, which indicates a change in resource allocation. A plausible explanation may be that when the apical meristem of seedlings was cut, it stimulated branching and this increased leaf and flower production ( Tiffin, 2000 ). Interestingly, the number of branches was not affected by early damage, so that compensation was only for leaf production with no evidence of change in plant morphology in response to early herbivory. A meta-analysis on defense ontogeny ( Barton and Koricheva, 2010 ) showed that tolerance does not generally change with ontogenetic stage. This could be an indicator that the ontogenetic patterns in tolerance are not consistent and depend on the plant system. If tolerance is an adaptation to the specific community and its interactions, then it could be expected that each plant species evolves compensatory responses well suited for their biotic environment ( Pearse et al., 2017 ). However, this meta-analysis did not include studies that analyze tolerance to seedling damage. As seedlings have limited resources available from photosynthetic area or root reserves, they are expected to invest more in growth than in resistance strategies ( Barton and Boege, 2017 ). Damage at the seedling stage can be particularly relevant, given the lack of resources in this early developmental stage ( Barton and Hanley, 2013 ; Quintero and Bowers, 2013 ), any stressor could drastically alter resource allocation with consequences for the whole plant development. Thus, interactions at the seedling stage deserve more attention in studies of tolerance ontogeny.

Induced responses to damage can also be influenced by the plant’s exposure to previous events. For example, plants can be primed with stimuli such as egg oviposition, early herbivore damage or volatiles from damaged neighbors that prepare them for future attacks and increase the induction of defenses ( Conrath et al., 2006 ; Heil and Kost, 2006 ; Hilker et al., 2016 ; Martinez-Medina et al., 2016 ). However, priming effects are not commonly linked to the responses in different ontogenetical stages. It is possible that when a stimulus occurs in an early ontogenetic stage of the plant, it could prime it to induce responses for later damage events. Thus, priming and ontogenetic trajectories can be linked by these early damage events. Yet, very few studies have examined the effect of an initial herbivory event at early ontogenetic stages on future plant compensatory responses. In wild radish, mechanical damage at the juvenile stage reduced seed output, while damage at a reproductive stage did not ( Boege et al., 2007 ). However, when both types of damage were applied, seed output was similar to that in plants with only juvenile damage. As the specific physiological changes that take place to induce compensation need an external stimulus, it is possible that an early stimulus such as seedling damage, could prime the plant’s compensatory responses to future herbivory. While priming for future attacks is a phenomenon expected to occur in most plant species, it has been mainly tested for resistance and not for tolerance traits ( Conrath et al., 2006 ; Hilker et al., 2016 ). In our study we show that seed output was higher for plants damaged as seedlings after repeated damage at the juvenile stage compared to plants damaged at only the juvenile stage or at the juvenile and reproductive stage, indicating that plants damaged at the seedling stage tolerated damage better than plants damaged at later stages. This, supports the existence of priming for tolerance in lima bean. Importantly, this is not an effect of only repeated damage, since plants damaged at the juvenile and reproductive stage did not compensate better than plants with one damage event at the juvenile stage. Alternatively, it is possible that damage at the seedling stage alters the plant’s resource allocation with an increased investment on growth and seed output, regardless of future damage.

Other types of priming stimuli for tolerance that are independent of damage such as application of oral secretion from herbivores or exposure to plant volatiles, can be tested. Induction of enhanced growth by volatiles has been shown for some plant species, including Arabidopsis thaliana ( Shimola and Bidart, 2019 ), Brassica sp. ( Pashalidou et al., 2020 ), lima bean ( Freundlich et al., 2021 ) and Medicago trunculata ( Maurya et al., 2022 ). In Brassica , volatiles emitted by plants infested with eggs of Pieris brassicae induce a higher reproductive output in undamaged exposed plants than in plants not exposed to volatiles. But when exposed plants were damaged by P. brassicae caterpillars, the reproductive output was similar to that of damaged plants not exposed to plant volatiles ( Pashalidou et al., 2020 ). Thus, in this case tolerance to herbivory was not affected by exposure to eggs-infested plant volatiles. Tolerance can increase plant fitness in environments where herbivory is intense ( Fornoni, 2011 ). Since it allows plants to reproduce despite herbivore damage, it is especially useful when herbivores are adapted to resistance traits ( Best et al., 2008 ; Fornoni, 2011 ). Thus, compensatory responses associated with tolerance to herbivory play an important role in the establishment and maintenance of plant individuals and populations in communities with intense antagonistic interactions. Future studies should examine with more detail different tolerance priming stimuli and their outcome at varying herbivore pressure.

Induced resistance to herbivory at different ontogenetic stages is also an important component of plant defense. Here, our results show that plants damaged at the juvenile and reproductive stages had fewer seed insects than control plants, while plants damaged at the seedling stage had intermediate numbers of seed insects. However, this was driven by a density dependent effect of the number of seeds, with more seed insects present in plants with more seeds. Therefore, we did not detect an induction of seed defenses, which may suggest that this mechanism can vary with environmental conditions and the type of damage. This result could be also influenced by the nature of the damage (herbivores vs. mechanical damage). Induction of plant defenses have been shown to be influenced by direct cues from herbivores such as elicitors or microbes ( Shikano et al., 2017 ), but also the type and amount of damage may play a role. For example, a single mechanical damage event did not induce herbivore associated plant volatiles in lima bean plants, but continuous mechanical damage can replicate the whole spectrum of volatiles as compared to plants damaged by herbivores ( Mithöfer et al., 2005 ). Thus, it seems likely that the mechanical damage used in our study was not enough to induce defenses in the seeds as has been shown in studies using damage by herbivores in lima bean ( Hernández-Cumplido et al., 2016 ; Bustos-Segura et al., 2020 ). Mechanical damage allowed us to control for the amount of damage among plants and between different time points which would not have been possible with herbivores in field conditions. Moreover, the amount of damage is particularly important for analyzing tolerance to herbivory ( Muola et al., 2010 ). Previous studies on indirect defenses, plant volatiles ( Heil and Silva Bueno, 2007 ) and extrafloral nectar ( Blue et al., 2015 ), and on plant-plant communication ( Moreira et al., 2016 ) have shown that mechanical damage induces plant responses in lima bean. However, the difference between the effects of mechanical and natural damage on lima bean seed defenses and tolerance has not been examined.

In synthesis, we provide evidence for the role of ontogeny in plant resource allocation which results in differential growth compensation and tolerance. We also show that frequency together with timing of damage will affect these responses, as early damage influenced the plant’s tolerance to future damage. Given that in nature, interactions with herbivores occur throughout the plant’s life, we emphasize the importance of analyzing the consequences of plant herbivory at multiple ontogenetic stages. Such an approach will increase our understanding of the factors associated with plant adaptations to a dynamic biotic environment.

Data availability statement

Author contributions.

CB-S and BB: Conceptualization. CB-S and RG-S: Investigation. CB-S: Data curation and formal analysis. CB-S: Methodology. BB: Funding acquisition. CB-S: original draft preparation. CB-S and BB: review and editing. All authors contributed to the article and approved the submitted version.

This research was financially supported by the Swiss National Science Foundation (Project No. 31003A_162860) awarded to BB.

Acknowledgments

We thank the Universidad del Mar, campus Puerto Escondido for logistical support, particularly Dr. Jose Arcos and Alfredo Lopez-Rojas. We also thank Lucas Malacari and Yosra Chabaane for help in the field and Philippine Surer for her assistance with the seed analyses. The authors declare no conflict of interest.

Conflict of interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Publisher’s note

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.

Supplementary material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpls.2022.1037047/full#supplementary-material

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Peer-reviewed

Research Article

Cyanogenesis of Wild Lima Bean ( Phaseolus lunatus L.) Is an Efficient Direct Defence in Nature

* E-mail: [email protected]

Affiliation Department of General Botany – Plant Ecology, Universität Duisburg-Essen, Essen, Germany

Affiliations Department of General Botany – Plant Ecology, Universität Duisburg-Essen, Essen, Germany, Departamento de Ingeniería Genética, CINVESTAV – Irapuato, Guanajuato, México

Affiliation Department of Horticultural Science, University of Minnesota, St. Paul, Minnesota, United States of America

• Daniel J. Ballhorn, 
• Stefanie Kautz, 
• Martin Heil, 
• Adrian D. Hegeman

• Published: May 8, 2009
• https://doi.org/10.1371/journal.pone.0005450
• Reader Comments

In natural systems plants face a plethora of antagonists and thus have evolved multiple defence strategies. Lima bean ( Phaseolus lunatus L.) is a model plant for studies of inducible indirect anti-herbivore defences including the production of volatile organic compounds (VOCs) and extrafloral nectar (EFN). In contrast, studies on direct chemical defence mechanisms as crucial components of lima beans' defence syndrome under natural conditions are nonexistent. In this study, we focus on the cyanogenic potential (HCNp; concentration of cyanogenic glycosides) as a crucial parameter determining lima beans' cyanogenesis, i.e. the release of toxic hydrogen cyanide from preformed precursors. Quantitative variability of cyanogenesis in a natural population of wild lima bean in Mexico was significantly correlated with missing leaf area. Since existing correlations do not by necessity mean causal associations, the function of cyanogenesis as efficient plant defence was subsequently analysed in feeding trials. We used natural chrysomelid herbivores and clonal lima beans with known cyanogenic features produced from field-grown mother plants. We show that in addition to extensively investigated indirect defences, cyanogenesis has to be considered as an important direct defensive trait affecting lima beans' overall defence in nature. Our results indicate the general importance of analysing ‘multiple defence syndromes’ rather than single defence mechanisms in future functional analyses of plant defences.

Citation: Ballhorn DJ, Kautz S, Heil M, Hegeman AD (2009) Cyanogenesis of Wild Lima Bean ( Phaseolus lunatus L.) Is an Efficient Direct Defence in Nature. PLoS ONE 4(5): e5450. https://doi.org/10.1371/journal.pone.0005450

Editor: Juergen Kroymann, CNRS UMR 8079/Université Paris-Sud, France

Received: February 15, 2009; Accepted: April 5, 2009; Published: May 8, 2009

Copyright: © 2009 Ballhorn et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: This work was financially supported by the DFG (grant He 3169/4-2) and the Universitaet Duisburg-Essen. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Lima bean (Fabaceae: Phaseolus lunatus L.) represents a prominent experimental model plant for studies of inducible indirect plant defences against herbivores [1] . These indirect (carnivore attracting) defences include the release of herbivore-induced volatile organic compounds (VOCs) and secretion of extrafloral nectar (EFN). Both types of indirect defences have been investigated extensively for the last decade under laboratory [2] – [8] and, more recently, natural field conditions [9] – [14] . In addition to directly attracting carnivores, VOCs of lima bean play a role in defence-associated signalling between plants [6] , and as external signal for priming of induced indirect defences within a single plant [15] .

In contrast to indirect defences, the efficiency of lima beans' direct chemical defences under natural conditions has not been studied until now. A characteristic direct chemical defence of lima bean is cyanogenesis, i.e. the release of toxic hydrogen cyanide (HCN) from preformed cyanide-containing compounds in response to cell damage [16] – [20] . Plants do not rely on a single defence mechanism, but rather express multiple defences comprising the constitutive and induced synthesis of many chemical compounds as well as the production of structural traits [e.g. 21] – [24] . The combination of different traits often leads to the evolution of multiple defence syndromes, since the association with specific ecological interactions results in co-variation of defensive traits [25] , [26] . In a recent series of conceptual and experimental studies, the evolution of defence syndromes has been compellingly illustrated for milkweeds ( Asclepias sp.) [27] , [28] . Nevertheless, the functional interplay of different plant traits involved in a plant's overall defences under natural conditions is poorly understood in many cases. To better understand this functional interplay of different defensive traits, multiple contributing components must be analysed thoroughly.

In natural systems functional analyses of specific plant traits are generally complicated by the variability of internal and external factors affecting the trait of interest. Physiology of plants and plants' attractiveness to herbivores is known to vary widely depending on plant and leaf age [17] , [29] . In addition, microclimatic conditions have a strong impact on plant characteristics and consequently on the interaction of plants with higher trophic levels [30] . Thus, to investigate the efficiency of lima beans' cyanogenesis as a direct defence in nature, we applied an integrative approach combining analyses for quantitative correlations of cyanogenic potential (HCNp; the maximum amount of cyanide that can be released by a given tissue [31] ) and herbivory in the field with feeding trials under controlled conditions. In some plant species — as for example in Sorghum bicolor — intermediates formed in the process of cyanogenesis can be metabolized and correspondingly the amount of cyanide, which can be released from a given tissue, is lower than the amount that is accumulated in cyanogenic precursors [32] . However, in lima beans with high β -glucosidase activity as is often found in wildtypes, the HCNp is a good measure for cyanogenesis because the amount of cyanide contained in cyanogenic precursors corresponds closely to the total amount that is released in response to cell disintegration [17] .

Negative correlations of cyanogenesis and resistance to different types of herbivores have been demonstrated convincingly for several plant species on both extensive [33] and local scales [34] , [35] . Confirming these results in field studies on natural lima bean populations, we found a distinct negative correlation between leaf damage and HCNp. As existing correlations observed in nature do not necessarily mean causal relations, we conducted additional feeding trials under out-door conditions in South Mexico with two natural insect herbivores of lima bean (Chrysomelidae: Cerotoma ruficornis and Gynandrobrotica guerreroensis ). Before natural variability of cyanogenesis may be used for functional analysis of its defensive efficiency in herbivore-plant interactions, the quantitative variation among cyanogenic features must be known. In the present study, we produced clonal plants by stem cuttings that were derived from mother plants growing at a natural site characterized by distinct differences in HCNp. Our experimental design thus allowed us to measure quantitative effects of HCNp on herbivores. This integrative approach considered both the efficiency of cyanogenesis as a direct defence in nature and its quantitative effects in controlled feeding trials.

Results and Discussion

Cyanogenic potential of lima bean in nature.

The cyanogenic potential (HCNp) of 46 individual field-grown lima bean plants was quantitatively analysed. At the natural field site, we considered leaves of a defined developmental stage (young, fully unfolded leaves inserting three positions down the apex) to avoid ontogenetic variability of plant traits including ‘Plant Size’ and ‘Light Exposure’ ( Fig. 1 ) as random factors in an multivariate general linear model (GLM) for assessing potential effects of these factors on HCNp and consumed leaf area ( Table 1 ). Among plants growing in nature, HCNp varied substantially ranging from 10.32 to 43.18 µmol HCN g −1 fwt ( n  = 46 leaves; Fig. 2 ). Lima beans growing in full sunlight showed no significant differences in HCNp when compared to plants growing under shaded conditions (according Mann-Whitney-U test: Z  = −0.770, P  = 0.441; n  = 24 plants in full sun, and n  = 22 growing under shaded conditions; two-tailed P -values are reported; Fig. 1 ). In addition, we found no significant differences in HCNp depending on plant size ( Z  = −0.569, P  = 0.570; n  = 19 plants with less than 20 leaves, and n  = 27 plants with more than 20 leaves; Fig. 1 ). Consequently, the GLM predicted no effects of ‘Plant Size’ or ‘Light Exposure’ on HCNp ( Table 1 ). These findings indicate that microclimatic conditions as well plant age or size have limited impact on leaf HCNp in lima bean plants at natural sites. Alternatively, the results of this field study suggest that the observed quantitative variability of cyanogenic plant features is under genetic control as has been observed for many other cyanogenic plant species [e.g. 30] , [36] – [38] . This high degree of genetic control of cyanogenesis in wild type lima bean was confirmed in our cloning experiments where plant material with defined cyanogenic features was generated for feeding trials.

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• PNG larger image
• TIFF original image

Positions of individual plants at the natural site were generated with GPS-data and combined with schematic illustration of vegetation and surrounding land use pattern. Plants of different size and number of leaves were included in the analysis. Individuals in boxes (HC and LC) were selected for the production of clones for consecutive feeding trials.

https://doi.org/10.1371/journal.pone.0005450.g001

Individual leaves of field-grown lima bean plants ( n  = 1 leaf of a defined ontogenetic stage per plant; n  = 46 plants) were quantitatively analysed for removed leaf area and concentration of cyanogenic precursors (HCNp). Data were analysed using Pearson's correlation ( P <0.01). Individuals in boxes (HC and LC) were selected for the production of clones for consecutive feeding trials.

https://doi.org/10.1371/journal.pone.0005450.g002

https://doi.org/10.1371/journal.pone.0005450.t001

Herbivory at Natural Sites

Among lima bean plants in the field, herbivore damage measured as missing leaf area (of defined leaf stages) showed high quantitative variability (0–741 mm 2 corresponding to 0–33% leaf area removed; Fig. 2 ). None of the leaves considered in this study was missing completely (i.e. damaged on the 100% level). Analysis of individual leaf cyanogenic features revealed a significant negative correlation of leaf damage and HCNp (according to Pearson's correlation: r  = −0.567, P <0.001; n  = 46; Fig. 2 ). Plants with high cyanogenic young leaves showed less damage even of older leaf stages than plants with lower cyanogenic young leaves (pers. observ.). Thus, the correlation of HCNp and missing leaf area in individual leaves was a good predictor for herbivore damage of total plants. This is in accordance with own previous laboratory analyses of HCNp ontogenetic variability in lima beans, since plants characterized by high HCNp in their young leaves showed consistently higher HCNp in intermediate and mature leaf developmental stages than plants with low HCNp in young leaves [17] .

In contrast to significant effects of HCNp on leaf damage, neither plant size ( Z  = −0.312, P  = 0.755) nor plant exposure to light ( Z  = −0.341, P  = 0.733) significantly affected herbivores' preferences (according to Mann-Whitney-U test, two-tailed P -values are reported). Consequently, GLM predicted no significant effects of ‘Plant Size’ or ‘Light Exposure’ on the degree of herbivory observed ( Table 1 ). Critical evaluation of small-scale site-specific variability in growth conditions and plant size (as we conducted here) is crucial to predict causal associations between a defensive plant trait and its efficiency in herbivore deterrence under natural conditions. Although not considered in many field studies, the high impact of positional effects (such as light exposure) as well as effects of plant size or age on the degree of herbivory observed in natural systems is well documented [39] – [42] . Thus, variability of site-specific conditions and plant morphological parameters must be included in meaningful analyses of natural plant-herbivore systems.

Feeding Trials

Field-observations of negatively correlated herbivore damage (missing leaf area) and HCNp suggest that cyanogenesis is important in anti-herbivore defence of lima bean at natural sites, whereas plant size and light exposure of the respective plant did not significantly affect damage of defined leaves by herbivores. Despite these observations, however, a broad array of external factors including microclimatic conditions [43] and plant morphological parameters [44] , biotic factors, such as aggregation phenomena of herbivores [45] , sporadic appearance of carnivores [46] , [47] as well as patch heterogeneity of neighbouring plants [48] , [49] may strongly determine the outcome of damage to individual plants by herbivores.

In order to confirm our field observations on the efficiency of lima beans' cyanide production as plant defence against herbivores, we conducted feeding experiments with two chrysomelid herbivores ( Gynandrobrotica guerreroensis and Cerotoma ruficornis ). These insects commonly occurred on lima bean plants and were collected in natural sex-ratios in the field. Leaf material for the feeding trials was derived from lima bean cuttings prepared from three high (HC) and three low (LC) cyanide containing mother plants growing at the natural field site ( Figs. 1 and 2 ). HC- and LC-cuttings from these plants were cultivated under herbivore (and carnivore) free outdoor-conditions to reduce variation of external factors [17] – [19] , [50] . Quantitative analyses of HCNp in leaves of defined developmental stages using clonal plants (according to plants at natural site three insertion positions down the apex) revealed similar cyanide concentrations as compared to the mother plants and showed low variation in HCNp within HC- and LC-groups. Cyanogenic potential in leaves of clones belonging to the HC-group ranged from 26.19 to 37.24 µmol HCN g −1 leaf fwt whereas HCNp in plants of the LC-group was lower and ranged from 5.25 to 14.51 µmol HCN g −1 leaf fwt. To exclude unknown variability of HCNp from the experimental setup, we measured cyanide in remaining individual leaflets after they had been used in feeding trials. Both lateral leaflets of trifoliate leaves were used in feeding trials according to Heil [9] . Comparative analyses of both lateral leaflets of individual trifoliate leaves revealed a variance of 4.90±4.01% in HCNp (mean±s.e.m.; n  = 84 leaves) when considering the highest cyanogenic leaflet of each pair as 100%. These leaflets with defined cyanogenic features therefore allowed for utilization of naturally occurring HCNp variability for functional analyses of its efficiency in anti-herbivore defence under experimental feeding trial conditions.

Choice Behaviour of Beetles

Cuttings produced from different mother plants were grouped (HC and LC) according to their HCNp. These groups were pooled to avoid potential effects on herbivores resulting from individually different attractiveness of plant material depending on other factors than cyanogenesis. Pairs of leaflets with defined HCNp from different individual plants were used in trials ( Fig. 3 ). Depending on the experimental set-up, we compared high vs. low (1), high vs. high (2) and low vs. low (3) cyanogenic leaflets ( Fig. 3 ). Both insect herbivores investigated showed significant preferences for low over high cyanogenic leaf material in feeding trials ( n  = 14 trials per setup), as measured by consumed leaf area per time [trial 1: Z  = −3.296, P  = 0.001 ( C. ruficornis ); Z  = −3.296, P  = 0.001 ( G. guerreroensis ) according to Wilcoxon signed rank test]. When given the choice between leaflets of similar HCNp, no significant preference was observed [trial 2: Z  = −0.973, P  = 0.331 ( C. ruficornis ); Z  = −0.220, P  = 0.826 ( G. guerreroensis ); trial 3: Z  = −0.596, P  = 0.551 ( C. ruficornis ); Z  = −0.031, P  = 0.975 ( G. guerreroensis ) according to Wilcoxon signed rank test].

Choice behaviour of both beetle species ( C. ruficornis and G. guerreroensis ) was tested in feeding trials, in which leaf material of different cyanogenic quality was offered. In trial 1 high (HC) and low cyanogenic (LC) leaf material was presented to the beetles, whereas trials 2 and 3 served as controls and the beetles were given the choice to select between leaves of similar quality (HC leaves in trial 2 and LC leaves in trial 3), respectively. Data on removed leaf area are means (±s.e.m.) of n  = 14 replications per setup.

https://doi.org/10.1371/journal.pone.0005450.g003

Our results obtained under feeding trial conditions (i) demonstrate a central role of HCNp for feeding choice behaviour of both beetle species and (ii) provide a causal quantitative explanation for variability of feeding damage observed on individual lima bean plants in nature.

Leaf Consumption and Incorporated Cyanide

Beetles' feeding behaviour showed that the concentration of cyanide quantitatively affects repellent activity of leaves and thus represents an important measure of plant defence ( Fig. 4 ). To address the question of whether a quantitative toxic threshold exists, we balanced the total amount of cyanide incorporated by each beetle in a respective 24 h feeding trial using missing leaf area and HCNp of leaflets offered. Individual beetles of both species consumed significantly more leaf material in trials 1 and 3 than in trial 2 (according to LSD post hoc analysis after one-way ANOVA: F  = 23.046, df  = 41, P <0.001, ( C. ruficornis ) and F  = 14.002, df  = 41, P <0.001, ( G. guerreroensis ) ( Fig. 5A ). Thus, total leaf consumption was increased when LC leaf material was available compared to setup 2, in which exclusively HC leaves were offered ( Fig. 5A ). After correcting for lower body weight of C. ruficornis [18.1±4.2 mg ( n  = 42)] as compared to G. guerreroensis [22.3±5.1 mg ( n  = 42)] total ingested leaf material was slightly higher for G. guerreroensis in all feeding trials ( Fig. 5A ).

Consumption of leaf area from leaves of different quality (HC = high cyanogenic, LC = low cyanogenic) by both beetle species under different experimental setups (trials 1, 2 and 3; n  = 14 trials per setup) was correlated to the HCNp of the individual leaves (Pearson's correlation; P <0.01).

https://doi.org/10.1371/journal.pone.0005450.g004

Included were leaves of different cyanogenic quality (HC and LC) in trial 1 or leaves of similar HCNp (HC leaves in trial 2 and LC leaves in trial 3). Data are means (±s.e.m.). Among treatments and species means of consumed leaf area marked with different letters in the upper panel are significantly different [according to post-hoc analysis after one-way ANOVA (LSD; P <0.05)]. A. Total leaf area consumed in feeding trials by both beetle species. B. Total amount of cyanide in ingested leaf material by both beetle species.

https://doi.org/10.1371/journal.pone.0005450.g005

Extensive consumption of LC leaf material in trails 1 and 3 resulted an higher total incorporation of cyanide as compared to amounts incorporated in HC∶HC trials (2) ( Fig. 5B ). ‘Setup’ was a significant source of variance [according to LSD post hoc analysis after one-way ANOVA: F  = 3.253, df  = 41, P <0.049, ( C. ruficornis ) and F  = 3.039, df  = 41, P <0.059, ( G. guerreroensis )]. These results are in accordance with findings in an earlier study [17] and indicate that HC plants are significantly better defended than LC plants showing a non-linear dose-response relationship. The impact of toxic compounds generally depends on the intake per body weight and time (dose), but varies depending on the dilution with non toxic material and on the quantitative availability of plant compounds required for detoxification processes [51] – [54] . In insects – and plants – β -cyanoalanine synthase is an enzyme that catalyzes the conversion of cysteine and cyanide to produce β -cyanoalanine and sulfide. β -cyanoalanine can be readily metabolized into asparagine [55] , [56] . Beetles can tolerate a higher dose of cyanogenic compounds when feeding on LC leaf material than HC material either because of the larger extent of dilution in LC material and/or potentially higher availability of cysteine.

While feeding trials provide striking evidence supporting the role of cyanogenesis as a causal agent moderating herbivore feeding damage in lima beans, it is interesting to see whether the results obtained in feeding trials can be corroborated in the natural system. In addition to livestock vertebrate herbivores, such as cows, goats, horses and donkeys, lima bean in South Mexico is threatened by a diverse range of natural arthropod herbivores including locusts (Pyrgomorphoidea: Pyrgomorphidae: Sphenarium borrei Bol. and one undetermined species belonging to the superfamily Acridoidea), caterpillars (Hesperidae: Proteus urbanus L., Saturniidae: Automeris io Fabricius and one species presumably belonging to the family Geometridae) and a species-complex of at least five sympatric chrysomelid beetles (pers. observ.). One must question whether the herbivores used in the study are of relevance in the natural system.

Chrysomelids used here represented the most prominent insect herbivores found on wild lima bean in coastal area of Oaxaca (pers. observ.). The two species ( Gynandrobrotica guerreroensis and Cerotoma ruficornis ), which we selected for feeding trials, were the most abundant herbivores among Chrysomelids at the study site in August 2007. Feeding damage observed in wild lima bean population could be widely assigned to G. guerreroensis and C. ruficornis by direct observation of feeding beetles and under consideration of typical feeding patterns found on leaves. Observations in nature supported the use of G. guerreroensis and C. ruficornis as representative herbivores in feeding trials and strengthen the significance of the results with relevance to the situation at field sites.

In our present study, we demonstrated that cyanogenesis of lima bean represents an efficient defence against insect herbivores in nature. Quantitative correlations of herbivore damage and cyanogenesis in leaves observed in a natural population of lima bean in Mexico were confirmed under experimental conditions. Balancing leaf consumption and cyanide intake by two natural herbivores revealed a non linear dose-response relationship further indicating substantial repellent activity of high cyanogenic plant individuals or plant parts. Results of the present study, together with recent findings on distinct trade-offs between direct (cyanogenesis) and indirect defences (VOCs) of lima bean, strongly suggest implementation of cyanogenic features in further studies on lima beans' overall defence in nature. In contrast to previous studies, which have focussed exclusively on indirect defences, our findings indicate the general need for consideration of multiple defence syndromes on plant defences rather than continuing to restrict analyses to single defence mechanisms. While the subject of indirect defence efficiency appears well documented, subsequent investigations need to apply an integrative approach and revisit the topic considering quantitative variability of cyanogenesis in natural lima bean populations.

Materials and Methods

Analyses were conducted in August 2007 in the coastal area of Oaxaca, Mexico. We used plant material from a wild lima bean population ca. 10 km west of Puerto Escondido (15°55′N and 097°09′W, elevation 15 m). Plants were collected along a path, which was surrounded by extensively used agricultural areas ( Fig. 1 ). Lima bean plants at the field site were in part shaded by trees and shrubs, while other plants were in full sun. All plants had developed 11–42 leaves at the time of sampling.

Sampling of Plant Material

Leaves of a defined ontogenetic stage were assessed for leaf damage by herbivores. These leaves inserted three positions down the apex and were fully unfolded. Use of defined developmental stages reduced variability of leaf texture due to ontogenetic characteristics. Thus, all leaves analysed here showed the same soft leaf texture. One leaf per plant individual was used ( n  = 46 plants). Leaves were cut off with a razor blade and immediately placed into Ziploc® bags (Toppits, Minden, Germany) containing moist filter paper. In the field, bags were stored in an insulation box, cooled with ice during sampling, and transported to the field laboratory for analyses.

Quantification of Leaf Damage

In the laboratory, leaves were spread on a scaled paper and then digitally photographed (Camedia C-4000 Zoom, Olympus, Hamburg, Germany). Missing leaf area of individual leaves was quantified using the AnalySIS® software (Olympus Soft Imaging Solutions GmbH, Münster, Germany) back in Germany.

Cyanogenic Precursor Concentration

We analysed concentration of cyanogenic precursors in leaf material, i.e. the cyanogenic potential (HCNp) according Ballhorn et al. [17] . The method is based on complete enzymatic degradation of cyanogenic glycosides in closed Thunberg vessels and subsequent spectrophotometric measurement (585 nm) of HCN released from the cyanide-containing compounds using the Spectroquant® cyanide test (Merck KGaA, Darmstadt, Germany). For enzymatic degradation, we used specific β -glucosidase isolated from rubber tree (Euphorbiaceae: Hevea brasiliensis ). This plant species possesses the same cyanogenic glycosides as lima bean, i.e. linamarin and lotaustralin. We added external β -glucosidase in excess to leaf extracts to guarantee total conversion of cyanogenic glycosides into free cyanide and to accelerate the enzymatic reaction.

Cultivation of Plants

Clonal plants used in feeding trials were propagated from stem cuttings of field-grown mother plants each containing one leaf ( n  = 18 cuttings per plant). We selected three mother plants each for propagation of high (HC; more than 25 µmol HCN g −1 fwt) and low (LC; less than 15 µmol HCN g −1 fwt) HCNp to reduce variability of cyanogenesis in the set of experimental plants. The HCNp of clonal plants quantitatively resembled the HCNp of mother plants. Cuttings were rooted in continuously moist soil from the natural site mixed 1∶1 with sand and were transferred in pure natural soil after the first roots had started to grow and then were cultivated in 250 ml plastic pots. Plants were exposed to natural conditions, watered twice a day and fertilized once a week with 50 ml of nitrogen-phosphate fertilizer (Blaukorn®-Nitrophoska®-Perfekt, Compo GmbH & Co. KG, Münster, Germany) in a concentration of 0.25 mg L −1 . Plants were checked three to four times a day for the infestation by herbivores. Herbivores rarely appeared under controlled outdoor conditions, and those that did appear were removed immediately by hand.

In feeding trials, we used phyllophagous beetles Cerotoma ruficornis Olivier and Gynandrobrotica guerreroensis Jacoby (Chrysomelidae: Galerucinae: Luperini: Subtribe Diabroticina). Chysomelids were determined by Astrid Eben (Instituto de Ecología, Veracruz, Mexico). Both beetle species have repeatedly been used in earlier field studies addressing lima bean indirect defences in nature [9] , [10] , [12] , [15] . Beetles were collected in August 2007 on lima beans at the same study site, which was selected for assessing herbivory and parallel quantification of accumulated cyanide in leaves. Beetles were present all day long exhibiting two peaks of feeding and moving activity in the first hours after dawn (8:00 AM–10:00 AM) and dusk (7:00 PM–11:00 PM), respectively (pers. observ.). Beetles appearing on lima bean were collected representing natural ratios of sexes and ages that might display different choice behaviour [e.g. 57] . Insects were kept in transparent 250 ml plastic cups with water supplied on cotton and were deprived of food for 1 d prior to the experiment.

Feeding experiments were carried out under controlled outdoor conditions. At the time of the experiments, potted plants were 60–80 cm tall and had developed 10–15 leaves. As for leaves quantitatively analysed for HCNp and herbivory in nature, leaves that inserted three positions down the apex were analysed for HCNp and used in feeding trials. Leaves (terminal leaflets) were quantitatively analysed for their HCNp prior to the experiment to confirm quantitative stability of cyanogenesis. All three leaflets showed high homogeneity in HCNp per trifoliate leaf excluding any position effects. In accordance to mother plants, leaves with an HCNp of more than 25.0 µmol HCN per g leaf fresh weight were classified as high cyanogenic, while leaves with less than 15.0 HCN per g leaf fresh weight were considered as low cyanogenic. In the feeding experiments, two lateral leaflets derived from leaves of different plant individuals were tested against each other. We used three different experimental setups: (1) high vs. low, (2) high vs. high, and (3) low vs. low cyanogenic leaflets. After the feeding trials, remaining leaf material of individual leaflets was weighted and analysed for HCNp.

For feeding trials, single beetles were placed in 250 ml plastic cups sealed with fabric (anti-aphid net). Experiments were run over 24 hrs and then leaflets were digitally photographed on a scale for quantification of missing leaf area using AnalySIS® software back in Germany. The two leaflets exposed to the same beetle were used as a pair for data evaluation.

Acknowledgments

We thank Julia Göbel (Essen) for help with the quantification of missing leaf area.

Author Contributions

Conceived and designed the experiments: DJB. Performed the experiments: DJB SK. Analyzed the data: DJB SK. Wrote the paper: DJB SK MH ADH.

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