Specialized naphthoquinones present in Impatiens glandulifera nectaries inhibit the growth of fungal nectar microbes

Abstract

The invasion success of Impatiens glandulifera (Himalayan balsam) in certain parts of Europe and North America has been partially attributed to its ability to compete for bee pollinators with its rich nectar and due to its capacity to produce and release allelopathic 1,4-naphthoquinones (1,4-NQs) from its roots and leaves. Given that other 1,4-NQs present in the digestive fluids of certain carnivorous plants are proposed to control microbial colonization, we investigated the potential for the 1,4-NQs, 2-methoxy-1,4-naphthoquinone (2-MNQ) and lawsone, to fulfill an analogous role in the nectaries of I. glandulifera. Both 2-MNQ and lawsone were detected in the floral nectaries of I. glandulifera at levels comparable to leaves and roots, but were discovered to be at significantly higher levels in its extra-floral nectaries (EFNs) and to be present in EFN nectar itself. Nectar microbe inhibition assays revealed that the common nectar bacteria Gluconobacter oxydans and Asaia prunellae are not inhibited by 2-MNQ or lawsone, although both compounds were found to inhibit the growth of the common fungal nectar microbes Metschnikowia reukaufii and Aureobasidium pullulans. Taken together, these findings suggest that 2-MNQ and lawsone could serve to protect the rich nectar of I. glandulifera against fungal growth. The high abundance of 2-MNQ and lawsone in I. glandulifera EFNs may also point to an unsuspected mechanism for how allelopathic 1,4-NQs are leached into the soil where they exhibit their known allelopathic effects.

Keywords: Impatiens glandulifera (Himalaya balsam); allelopathy; naphthoquinone; nectar.

Figure 1

Targeted profiling of specialized naphthoquinones in Impatiens glandulifera organs. Pool sizes of 2‐methoxy‐1,4‐naphthoquinone (2‐MNQ) (a) and lawsone (b). Note that open flowers included tissues from petals, spurs, reproductive parts, receptacles, and sepals. Images of unopened (c) and open (d) I. glandulifera flowers with white arrowhead pointing to floral nectaries (spurs). Image of I. glandulifera shoot with white arrow and inset showing an example of a sampled extra‐floral nectary (EFN) (e). All data are means ± SEM (n = ≥3 biological replicates). Different English alphabet letters above bars indicate significant differences via analysis of variance (ANOVA) followed by a post hoc Tukey HSD test (α = 0.05). Due to the greater than 10‐fold difference in 2‐MNQ pool sizes between EFNs versus all other organs, a second statistical test was performed to examine differences in mean 2‐MNQ pool sizes between all organs excluding EFNs. For this planned comparison, the more conservative Bonferroni method was used. Different Greek letters above bars indicate significant differences via ANOVA followed by a post hoc Bonferroni test (α = 0.001) between all samples except EFNs

Figure 2

Antimicrobial activity of lawsone and 2‐MNQ against nectar microbes. Representative images of inhibition zone clearing in an antimicrobial assay using lawsone and 2‐MNQ on the nectar microbe Metschnikowia reukaufii (a). Inhibition zone areas of Gluconobacter oxydans and Asaia prunellae treated with 10 mM of lawsone, 2‐MNQ, or a DMSO control (b). Inhibition zone areas of M. reukaufii treated with various concentrations of lawsone and 2‐MNQ (c). Inhibition zone areas of Aureobasidium pullulans treated with various concentrations of lawsone and 2‐MNQ (d). (*) indicates treatments significantly different from DMSO control (p ≤ 0.05 by a pair wise t test, n = 3)

Figure 3

Detection of lawsone and 2‐methoxy‐1,4‐naphthoquinone (2‐MNQ) in nectar from Impatiens glandulifera extra‐floral nectaries (EFNs). Five EFNs collected from I. glandulifera were dipped in 1 ml HPLC‐grade water to wash off visible nectar droplets (see Figure 1e inset). The resulting wash was concentrated to 100 μl and 25 μl was directly analyzed by HPLC with diode array detection to look for the presence of lawsone and 2‐MNQ. Traces corresponding to 100 pmol of authentic lawsone and 2‐MNQ standards have been offset for clarity. Retention times for lawsone and 2‐MNQ were 22.5 and 24.5 min, respectively