Daniel J. Ballhorn

Airborne Induction and Priming of Plant Defenses against a Bacterial Pathogen

Herbivore-induced plant volatiles affect the systemic response of plants to local damage and hence represent potential plant hormones. These signals can also lead to “plant-plant communication,” a defense induction in yet undamaged plants growing close to damaged neighbors. We observed this phenomenon in the context of disease resistance. Lima bean (Phaseolus lunatus) plants in a natural population became more resistant against a bacterial pathogen, Pseudomonas syringae pv syringae, when located close to conspecific neighbors in which systemic acquired resistance to pathogens had been chemically induced with benzothiadiazole (BTH). Airborne disease resistance induction could also be triggered biologically by infection with avirulent P. syringae. Challenge inoculation after exposure to induced and noninduced plants revealed that the air coming from induced plants mainly primed resistance, since expression of PATHOGENESIS-RELATED PROTEIN2 (PR-2) was significantly stronger in exposed than in nonexposed individuals when the plants were subsequently challenged by P. syringae. Among others, the plant-derived volatile nonanal was present in the headspace of BTH-treated plants and significantly enhanced PR-2 expression in the exposed plants, resulting in reduced symptom appearance. Negative effects on growth of BTH-treated plants, which usually occur as a consequence of the high costs of direct resistance induction, were not observed in volatile organic compound-exposed plants. Volatile-mediated priming appears to be a highly attractive means for the tailoring of systemic acquired resistance against plant pathogens.

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

A broad range of chemical plant defenses against herbivores has been studied extensively under laboratory conditions. In many of these cases there is still little understanding of their relevance in nature. In natural systems, functional analyses of plant traits are often complicated by an extreme variability, which affects the interaction with higher trophic levels. Successful analyses require consideration of the numerous sources of variation that potentially affect the plant trait of interest. In our recent study on wild lima bean (Phaseolus lunatus L.) in South Mexico, we applied an integrative approach combining analyses for quantitative correlations of cyanogenic potential (HCNp; the maximum amount of cyanide that can be released from a given tissue) and herbivory in the field with subsequent feeding trials under controlled conditions. This approach allowed us to causally explain the consequences of quantitative variation of HCNp on herbivore-plant interactions in nature and highlights the importance of combining data obtained in natural systems with analyses under controlled conditions.

Comparing responses of generalist and specialist herbivores to various cyanogenic plant features

Plants are obliged to defend themselves against multiple generalist and specialist herbivores. Whereas plant cyanogenesis is considered an efficient defence against generalists, it is thought to affect specialists less. In the present study, we analysed the function of various cyanogenic features of lima bean [Phaseolus lunatus L. (Fabaceae)] during interaction with different herbivores. Three cyanogenic features were analysed, i.e., cyanogenic potential (HCNp; concentration of cyanogenic precursors), β‐glucosidase activity, and cyanogenic capacity (HCNc; release of cyanide per unit time). In no‐choice and free‐choice feeding trials, five lima bean accessions were offered to generalist desert locust [Schistocerca gregaria Forskål (Orthoptera: Acrididae)] and specialist Mexican bean beetle [Epilachna varivestis Mulsant (Coleoptera: Coccinellidae)]. The HCNc was the most important parameter determining host plant selection by generalists, whereas choice behaviour of specialists was strongly affected by HCNp. Although locusts were effectively repelled by high HCNc, this cue was misleading for the detection of suitable host plants, as extensive consumption of low HCNc plant material resulted in strong intoxication of locusts. Balancing cyanide in consumed leaf area, the quantitative release of gaseous cyanide during feeding, and cyanide in faeces suggested that specialists metabolized significantly lower rates of cyanide per consumed leaf material than generalists. We hypothesize that specialists are able to avoid toxic concentrations of cyanide by using HCNp rather than HCNc as a cue for host plant quality, and that they exhibit mechanisms that reduce incorporation of host plant cyanide.

Dual benefit from a belowground symbiosis: nitrogen fixing rhizobia promote growth and defense against a specialist herbivore in a cyanogenic plant

Legume-associated nitrogen-fixing bacteria play a key role for plant performance and productivity in natural and agricultural ecosystems. Although this plant-microbe mutualism has been known for decades, studies on effects of rhizobia colonisation on legume-herbivore interactions are scarce. We hypothesized that additional nitrogen provided by rhizobia may increase plant resistance by nitrogen-based defense mechanisms. We studied this below-aboveground interaction using a system consisting of lima bean (Phaseolus lunatus L.), rhizobia, and the Mexican bean beetle (Epilachna varivestis Muls.) as an insect herbivore. We showed that the rhizobial symbiosis not only promotes plant growth but also improves plant defense and resistance against herbivores. Results of our study lead to the suggestion that nitrogen provided by rhizobia is allocated to the production of nitrogen-containing cyanogenic defense compounds, and thereby crucially determines the outcome of plant-herbivore interactions. Our study supports the view that the fitness benefit of root symbioses includes defence mechanisms and thus extends beyond the promotion of plant growth. Since the associations between legumes and nitrogen-fixing rhizobia are ubiquitous in terrestrial ecosystems, improved knowledge on rhizobia-mediated effects on plant traits―and the resulting effects on higher trophic levels―is important for better understanding of the role of these microbes for ecosystem functioning.

Quantitative effects of cyanogenesis on an adapted herbivore.

Plant cyanogenesis means the release of gaseous hydrogen cyanide (HCN) in response to cell damage and is considered as an effective defense against generalist herbivores. In contrast, specialists are generally believed not to be affected negatively by this trait. However, quantitative data on long-term effects of cyanogenesis on specialists are rare. In this study, we used lima bean accessions (Fabaceae: Phaseolus lunatus L.) with high quantitative variability of cyanogenic features comprising cyanogenic potential (HCNp; concentration of cyanogenic precursors) and cyanogenic capacities (HCNc; release of gaseous HCN per unit time). In feeding trials, we analyzed performance of herbivorous Mexican bean beetle (Coleoptera: Coccinellidae: Epilachna varivestis Mulsant) on selected lines characterized by high (HC-plants) and low HCNp (LC-plants). Larval and adult stages of this herbivore feed on a narrow range of legumes and prefer cyanogenic lima bean as host plant. Nevertheless, we found that performance of beetles (larval weight gain per time and body mass of adult beetles) was significantly affected by lima bean HCNp: Body weight decreased and developmental period of larvae and pupae increased on HC-plants during the first generation of beetles and then remained constant for four consecutive generations. In addition, we found continuously decreasing numbers of eggs and larval hatching as inter-generational effects on HC-plants. In contrast to HC-plants, constantly high performance was observed among four generations on LC-plants. Our results demonstrate that Mexican bean beetle, although preferentially feeding on lima bean, is quantitatively affected by the HCNp of its host plant. Effects can only be detected when considering more than one generation. Thus, cyanide-containing precursors can have negative effects even on herbivores adapted to feed on cyanogenic plants.

Induced plant defense via volatile production is dependent on rhizobial symbiosis

Nitrogen-fixing rhizobia can substantially influence plant–herbivore interactions by altering plant chemical composition and food quality. However, the effects of rhizobia on plant volatiles, which serve as indirect and direct defenses against arthropod herbivores and as signals in defense-associated plant–plant and within-plant signaling, are still unstudied. We measured the release of jasmonic acid (JA)-induced volatiles of rhizobia-colonized and rhizobia-free lima bean plants (Fabaceae: Phaseolus lunatus L.) and tested effects of their respective bouquets of volatile organic compounds (VOCs) on a specialist insect herbivore (Mexican bean beetle; Coccinellidae: Epilachna varivestis Mulsant) in olfactometer choice trials. In a further experiment, we showed that VOC induction by JA reflects the plant responses to mechanical wounding and insect herbivory. Following induction with JA, rhizobia-colonized plants released significantly higher amounts of the shikimic acid-derived compounds, whereas the emission of compounds produced via the octadecanoid, mevalonate and non-mevalonate pathways was reduced. These changes affected the choice behavior of beetles as the preference of non-induced plants was much more pronounced for plants that were colonized by rhizobia. We showed that indole likely represents the causing agent for the observed repellent effects of jasmonic acid-induced VOCs of rhizobia-colonized lima bean plants. Our study demonstrates a rhizobia-triggered efficacy of induced plant defense via volatiles. Due to these findings, we interpret rhizobia as an integral part of legume defenses against herbivores.

Local adaptation of aboveground herbivores towards plant phenotypes induced by soil biota.

Background Soil biota may trigger strong physiological responses in plants and consequently induce distinct phenotypes. Plant phenotype, in turn, has a strong impact on herbivore performance. Here, we tested the hypothesis that aboveground herbivores are able to adapt to plant phenotypes induced by soil biota. Methodology and Principal Findings We bred spider mites for 15 generations on snap beans with three different belowground biotic interactions: (i) no biota (to serve as control), (ii) arbuscular mycorrhizal fungi and (ii) root-feeding nematodes. Subsequently, we conducted a reciprocal selection experiment using these spider mites, which had been kept on the differently treated plants. Belowground treatments induced changes in plant biomass, nutrient composition and water content. No direct chemical defence through cyanogenesis was detected in any of the plant groups. Growth rates of spider mites were higher on the ecotypes on which they were bred for 15 generations, although the statistical significance disappeared for mites from the nematode treatment when corrected for all multiple comparisons. Conclusion/Significance These results demonstrate that belowground biota may indeed impose selection on the aboveground insect herbivores mediated by the host plant. The observed adaptation was driven by variable quantitative changes of the different separately studied life history traits (i.e. fecundity, longevity, sex-ratio, time to maturity).

Quantitative variability of cyanogenesis in Cathariostachys madagascariensis—the main food plant of bamboo lemurs in Southeastern Madagascar

Giant bamboo (Cathariostachys madagascariensis) is a major food plant for three sympatric species of bamboo‐eating lemurs (Hapalemur aureus, H. griseus, and Prolemur simus) in the rain forests of southeastern Madagascar. This plant species is strongly cyanogenic. However, quantitative data on cyanide concentration in C. madagascariensis are scarce. Previous studies reported 15 mg cyanide per 100 g fresh shoot material (corresponding to approx. 57 µmol cyanide per gram dry weight). However, we found mean concentrations (±SE) ranging from 139.3±19.32 in ground shoots to 217.7±16.80 µmol cyanide per gram dry weight in branch shoots. Thus, cyanogenesis of C. madagascariensis was up to four times higher than reported before. In contrast to the strongly cyanogenic shoots no cyanide could be detected in differently aged leaves of C. madagascariensis confirming earlier studies. Within individual shoots fine‐scaled analysis revealed a characteristic ontogenetic pattern of cyanide accumulation. Highest concentrations were found in youngest parts near the apical meristem, whereas concentrations decreased in older shoot parts. Beyond the general intra‐individual variability of cyanogenic features analyses indicated site‐specific variability of both, the ontogenetic pattern of cyanide concentration as well as the total amount of cyanide accumulated in shoots. Additionally, analyses of soluble proteins—one important nutritive measure affecting food plant quality—demonstrated a converse quantitative relation of protein concentrations in leaves to cyanide concentration in shoots at the site‐specific level. We, thus, suggest integrative analyses on quantitative variation of cyanogenesis together with nutritive plant parameters in future studies. This approach would allow obtaining more detailed insights into spatial variability of giant bamboos overall browse quality and its impact on lemur herbivores. Am. J. Primatol. 71:305–315, 2009.

Constraints of Simultaneous Resistance to a Fungal Pathogen and an Insect Herbivore in Lima Bean ( Phaseolus lunatus L.)

The existence of tradeoffs among plant defenses is commonly accepted, however, actual evidence for these tradeoffs is scarce. In this study, I analyzed effects of different direct defenses of wild lima bean plants (Phaseolus lunatus) that were simultaneously exposed to a fungal pathogen (Colletotrichum lindemuthianum) and an insect herbivore, the Mexican bean beetle (Epilachna varivestis). Although plants were derived from spatially widely separated populations, I observed a common tradeoff between resistance to pathogens and herbivores. Plants with high levels of anti-herbivore defense (cyanogenesis) showed low levels of resistance to pathogens (polyphenol oxidase activity and phenolic compounds), and vice versa. Competition for resources generally is considered to be the basis for tradeoffs. However, I report direct inhibition of polyphenol oxidase by cyanide, making simultaneous expression of both defenses at high levels impossible. I argue that populations composed of individuals investing in one type of defense have an advantage in environments that periodically favor either pathogen or herbivore plant antagonists.

Quantitative Variability of Direct Chemical Defense in Primary and Secondary Leaves of Lima Bean (Phaseolus lunatus) and Consequences for a Natural Herbivore

Ontogenetic variability in chemical plant defenses against herbivores is a common phenomenon, but the effects of this variability on herbivore–plant interactions are little understood. In a previous study on lima bean (Phaseolus lunatus), we found a trade-off between cyanogenesis, a direct defense, and the release of herbivore-induced volatile organic compounds (VOCs; mainly functioning as an indirect defense). Moreover, the expression of these two defenses could change during plant ontogeny. The present study aimed at elucidating whether such ontogenetic changes in plant defense can affect herbivore–plant interactions. We quantified feeding rates of a natural insect herbivore, the Mexican bean beetle (Epilachna varivestis), on primary and secondary leaves of individual lima bean plants. These insects strongly preferred low cyanogenic primary leaves over high cyanogenic secondary leaves. Although weakly defended by cyanogenesis, lima beans’ primary leaves showed protein concentrations and photosynthetic activities that did not differ significantly from secondary leaves at the time of analysis. Based on our findings, we suggest that lima beans’ long-lived primary leaves function as efficient source organs, even beyond the stage of seedlings. This hypothesis may explain why primary leaves express a strong indirect defense by the release of herbivore induced VOCs.