Bacterial detoxification of plant defence secondary metabolites mediates the interaction between a shrub and frugivorous birds

Trabelcy, Beny; Shteindel, Nimrod; Lalzar, Maya; Izhaki, Ido; Gerchman, Yoram

doi:10.1038/s41467-023-37525-6

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Published: 31 March 2023

Bacterial detoxification of plant defence secondary metabolites mediates the interaction between a shrub and frugivorous birds

Nature Communications volume 14, Article number: 1821 (2023) Cite this article

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Abstract

Many plants produce fleshy fruits, attracting fruit-eating animals that disperse the seeds in their droppings. Such seed dispersal results in a conflict between the plant and the animal, as digestion of seeds can be highly beneficial to the animal but reduces plant fitness. The plant Ochradenus baccatus uses the myrosinase-glucosinolates system to protect its seeds. We show that hydrolysis of the O. baccatus fruit glucosinolates by the myrosinase enzyme inhibited digestive enzymes and hampered digestion in naïve individuals of the bird Pycnonotus xanthopygos. However, digestion in birds regularly feeding on O. baccatus fruits was unaffected. We find that Pantoea bacteria, dominating the gut of these experienced birds as well as the fruits, thrive on glucosinolates hydrolysis products in culture. Augmentation of Pantoea protects both naïve birds and plant seedlings from the effects of glucosinolates hydrolysis products. Our findings demonstrate a tripartite interaction, where the plant-bird mutually beneficial interactions are mediated by a communal bacterial tenant.

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Introduction

Seed dispersal is a fundamental life cycle stage in plants. Seed dispersal functions in the reduction of competition for resources between parent and offspring and among offspring¹, and enables recruitment into new habitats². Endozoochory is a common seed dispersal strategy involving the production of fruits to attract frugivores that feed on the fruits and disperse seeds in their droppings³. Beside dispersal, endozoochory enhances germination by separating seeds from the pulp (often containing germination inhibitors), fertilizing the ground with fecal material, and scarification of the seed coat^4,5,6,7. Interaction between plants and frugivores is assumed mutualistic, with the consumer benefiting from nutrients in fruits and the plant benefiting from seed dispersal^8,9. Nevertheless, this relationship can be complex. Seeds are rich in proteins generally lacking in the fruit pulp^10,11, making seed digestion beneficial¹² and thus hampering plant fitness^13,14, resulting an evolutionary arms race between plants and frugivores¹⁵. Hence, plants face a delicate balance—attracting seed dispersers while evading seed predation and digestion.

Secondary metabolites (SMs) were suggested to play a role in this trade-off^16,17. Glucosinolates (GSLs) are common SMs in plants of the Brassicales order. Intact GSLs are harmless, but when mixed with the myrosinase enzyme (MYR; EC 3.2.1.147), GSLs go through rapid hydrolysis, releasing toxic products (aka the “mustard oil bomb”¹⁸), which protect Brassicales plants from herbivory^18,19 and reduce intake of plant tissue by herbivores²⁰. To prevent self-inflicted damage to the plant, the GSLs and MYR are separately compartmentalized, mixed only upon damage to the tissue^21,22.

The desert plant Ochradenus baccatus (Delile 1813 [Resedaceae]) produces sweet fleshy fruits²³, attracting a variety of animals²⁴. The fruit pulp mainly contains water and sugar, whereas the seeds are rich in protein, fat, and starch²⁵, making them a desired food. Previously, we showed that O. baccatus uses the MYR-GSL mechanism to reduce seed predation by the spiny mouse Acomys russatus (an effective seed predator)²³, by compartmentalizing the GSLs in its pulp and the MYR in its seeds²³. Animals that consume and digest the seeds with the pulp mix the two and thus activate the MYR-GSL mechanism. As a result, the animals are exposed to the GSLs hydrolysis product.

In the desert, O. baccatus fruits are frequently eaten by Pycnonotus xanthopygos (Hemprich and Ehrenberg 1833; white-spectacled bulbuls)^24,26, a songbird common throughout Israel. Pycnonotus xanthopygos swallow the whole fruits and excrete seeds²⁶ and are considered a major seed disperser of O. baccatus. Although passage through the digestive system of P. xanthopygos significantly increases the germination of seeds, the birds digest up to 80% of the consumed seeds, making the interaction multi-faceted¹⁵. Consumption of O. baccatus fruits also results in the reduced digestive ability of the birds¹⁵, suggesting a plant defense mechanism against seed predation by the P. xanthopygos.

Here we report that the MYR-GSL hydrolysis products of O. baccatus fruits inhibit digestive enzymes, hamper P. xanthopygos food digestibility, and have allelopathic effects on seedling growth of O. baccatus. Birds regularly feeding on O. baccatus fruits acquire fruit-associated bacteria that detoxify the MYR-GSL hydrolysis products, recovering digestibility. Finally, the same bacteria passed through the bird’s digestive system improved seedling growth and prevented the MYR-GSL hydrolysis products effect. These results demonstrate a tripartite, multi-kingdom interaction mediated by bacteria and affecting both plant and bird phenotypes.

Results

Ochraceous baccatus fruits hamper digestion in naïve P. xanthopygos

To test the effect of O. baccatus fruits on bird digestion, we captured birds from two locations 95 km apart (Fig. 1a). Birds were captured on O. baccatus plants (hereafter experienced birds) in one (Southern) location, and another group of birds was captured at a (Northern) location far from the edge of O. baccatus distribution (hereafter naïve birds) and acclimated in captivity. We assume that birds collected outside O. baccatus distribution region (naïve birds) had no previous encounter with that plant. Naïve birds acclimated to banana (Fig. 1b, W4–6, yellow) showed reduced digestive ability when fed banana + O. baccatus pulp + seeds extract (Fig. 1c, B+P+S) as compared to experienced birds acclimated to O. baccatus fruits (Fig. 1b, W4–6, blue and Fig. 1c, B+P+S). Feeding on banana or banana mixed with either O. baccatus pulp or seed extract did not have such an effect, regardless of the acclimation diet (Fig. 1c: B, B+P and B+S). These results demonstrate that consumption and thus mixing of O. baccatus fruit pulp and seeds hinders digestion in vivo and that birds experienced with O. baccatus may acquire resistance to the digestibility-reducing effects of MYR-GSL hydrolysis products.

Fig. 1: *Ochradenus baccatus* distribution map and *Pycnonotus xanthopygos* collection sites; experimental timeline; mixture of *O. baccatus* seed extract and fruit pulp inhibits digestion in naïve birds.

Characterization of the MYR-GSL defense mechanism in O. baccatus fruits and its effect on bird digestion

HPLC analysis of the O. baccatus pulp identified the presence of BenzylGSL (Glucotropaeolin) as the main glucosinolate in the fruit pulp (Fig. 2a). Mixing O. baccatus fruit pulp and seeds resulted in the appearance of Benzyl isothiocyanate (BITC), as identified by GC-MS (Fig. 2b). BITC formation is due to the activity of the MYR enzyme found in the seeds that hydrolizes intact BenzylGSL found in the fruit pulp (Fig. 2c). To further explore this interplay in an in vivo context, we used HPLC to quantify the BenzylGSL concentration after the passage of whole fruits through the bird digestive tract (Fig. 2d). BenzylGSL decomposed rapidly in the bird digestive system—starting from 14.38 ± 1.37 µg/g fresh weight (FW) in the O. baccatus fruits to 1.54 ± 0.16 µg/g FW in naïve birds and 1.77 ± 0.2 in experienced bird droppings (Fig. 2d). The 88–89% decrease in concentration is likely due to the hydrolysis of the BenzylGSL in the bird digestive system. No BITC was detected in the droppings of birds feeding on O. baccatus fruits, probably because BITC readily binds to proteins²⁷, such as in the digestive system of the birds, creating conjugates that will be undetectable in the GC/MS.

**Fig. 2: Mixing *Ochradenus baccatus* seeds and fruit pulp activates the MYR-GSL mechanism and inhibits digestive enzymes.**

To test if the reduction in the digestibility of birds feeding on seed extract mixed with fruit pulp juice is mediated by the BenzylGSL hydrolysis products, we tested the effect of BITC on the activity of two major digestive enzymes, amylase and lipase, both likely to take part in seed digestion¹⁵ (commercial enzymes were used; see Methods section). Reduced activity of both enzymes was evident (Fig. 2e, f), suggesting that the BenzylGSL hydrolysis products inhibit digestive enzymes and hamper digestion, highlighting the biochemical mechanism of BITC for seed protection. It should be noted that to avoid the slaughter of many birds, commercial (porcine pancreas) enzymes were used.

Both bird diet and natural history determine the prevalence of fruit-associated Pantoea bacteria

The ability of birds acclimated in captivity to O. baccatus to digest food containing O. baccatus pulp and seeds (Fig. 1c) suggests an “acquired tolerance” mechanism to BenzylGSL hydrolysis products. Such tolerance can be the result of specialized digestive enzymes (as was shown for monoterpenes²⁸) but is unlikely since the distance between the two capture sites is only 95 km that is continuously inhabited by P. xanthopus populations, making genetic mixing of the bird population highly likely. Thus, hypothesizing that this tolerance is based on the activity of gut bacteria, we examined the effect of O. baccatus diet on the overall composition of and specific taxa abundance in bird gut microbiota. We used next-generation 16S rRNA gene sequencing of DNA extracted from the droppings of birds feeding on O. baccatus fruits or banana diets (Fig. 1b, W1–3). For comparison, we also extracted and sequenced bacterial DNA from the O. baccatus and banana feeds. Overall, we detected 1091 amplicon sequence variants (ASVs) in the bird droppings and 233 ASVs in the fruits. We found a significant effect of capture location (naïve vs. experienced; Fig. 3a, triangle vs. circle, respectively) and diet (banana vs. O. baccatus fruits; Fig. 3a, yellow vs. blue, respectively) on birds microbiota composition, as demonstrated by non-metric multidimensional scaling analysis (NMDS; Fig. 3a). Interestingly, samples from experienced birds that were switched to O. baccatus fruit diet clustered together with samples from experienced birds feeding on O. baccatus fruits (Fig. 3a) and were separate from banana-fed naïve birds, demonstrating a strong diet effect.

**Fig. 3: The bird gut microbiota and relative abundance of *Pantoea* are strongly affected by the consumption of *Ochradenus baccatus* fruits.**

Looking at the genus level community composition, Pantoea were the most abundant bacteria in birds feeding on O. baccatus fruits, regardless of their geographical origin (Fig. 3b). Among Pantoea-annotated ASVs, ASV0003 was by far the most abundant, corresponding to >93% of total reads assigned to that genus. In sharp contrast, in birds feeding on the banana diet, the Pantoea genus as a whole was only 0.67% of the bacterial population (Fig. 3c), with ASV0003 representing 58% of Pantoea ASVs (0.4% of total bacteria). Highly consistent with this finding, the Pantoea were also the most abundant bacteria in the O. baccatus fruit microbiota (47.97% of ASVs; Fig. 3c), with AVS0003 comprising 91.4% of the total Pantoea ASVs.

Time course analysis of the effect of diet (O. baccatus fruits or banana) and its shift on the prevalence of Pantoea in the bird droppings supports the notion that Pantoea in the bird gut is closely associated with feeding on O. baccatus fruits (Fig. 3d). At the beginning of the experiment, Pantoea ASV0003 was found only in experienced birds and was completely absent from the naïve bird microbiomes (Fig. 3d). Switching diets resulted in a dramatic, rapid change in the abundances of these bacteria, increasing in birds that were switched from banana to O. baccatus and decreasing in birds that were switched from O. baccatus to banana within 3 days (Fig. 3d). These results demonstrated that feeding on O. baccatus fruits promotes Pantoea in the bird gut microbiome and that these bacteria are transient rather than core members of the bird gut microbiome, originating from the O. baccatus fruits.

Pantoea bacteria growth is improved in the presence of BITC

The elevated level of Pantoea bacteria in the O. baccatus fruits (Fig. 3c) and in droppings of birds feeding on these fruits (Fig. 3c, d) hints that this genus might be selected for by the presence of BITC, the breakdown product of BenzylGSL by the MYR enzyme. To test this, we cultured bacteria from fresh droppings of experienced birds on the day of their capture (Fig. 1a: cultivatable bacteria isolation) and found that some isolates were able to grow in Luria Broth (LB) supplemented with 0.1% BITC. Of the 50 morphologically representative isolates tested, most showed reduced growth as compared to those grown on plain LB. However, a few, and especially isolate #45, showed increased growth in the presence of BITC (Fig. 4a). After DNA extraction, amplification, and sequencing of the 16S rRNA gene of isolate #45, we found it to be 99.78% identical to Pantoea agglomerans and 100% identical to ASV0003 (Supplementary Figs. S1 and S2, respectively; hereafter Pa45), the most dominant taxa in O. baccatus fruit and in the gut microbiome of birds fed on O. baccatus fruit (Fig. 3b, c). These results suggest that the Pantoea not only originates from the O. baccatus fruits but is also selected for by GSL-MYR breakdown products in the O. baccatus fruit.

Fig. 4: *Pantoea* (isolate 45, 100% sequence similarity to ASV0003, see Supplementary Fig. S2) grow and remove BITC and can remediate inhibition of digestive enzymes and improve digestibility in birds feeding on a mixture of *O. baccatus* seeds and pulp.

Pa45 can detoxify BITC and improves digestion in birds

We examined the ability of Pa45 (isolate #45 identified as P. agglomerans) to detoxify BITC. A GC-MS analysis demonstrated that the addition of Pa45 bacterial isolate to LB medium supplemented with 0.1% BITC led to a significant reduction in BITC concentration as compared to the medium without the bacteria (Fig. 4b, c). In agreement, the addition of Pa45 significantly neutralized the inhibition effect of BITC on amylase and lipase activity (Fig. 4d, e, respectively). To test the effect of this bacterium in vivo, we enriched the diets of birds with Pa45 bacteria. Consistent with our in vitro finding, feeding banana-acclimated birds on banana + pulp + seeds resulted in reduced digestibility (as compared to O. baccatus-acclimated birds), while enrichment of the diet with Pa45 improved digestibility, making it comparable to the O. baccatus-acclimated birds (Figs. 1c and 4f). Taken together, we identified a fruit-associated bacterium that neutralizes the inhibitory digestive effect of seed-pulp mixing by removing the GLSs hydrolysis products of the MYR-GSL system.

BITC inhibits O. baccatus seedling development and Pa45 can remediate the BITC effect

Ochradenus baccatus pulp was previously shown to have detrimental effects on the germination of O. baccatus seeds¹⁵. Here we show that supplementing irrigation water with BITC, the main hydrolysis product of the pulp GSLs (Fig. 2b), had deleterious effects on O. baccatus seed development (Fig. 5; +BITC) and that the addition of Pa45 can remediate this effect (Fig. 5; +BITC+Pa45). In the absence of BITC, the addition of Pa45 had no effect on seed development (Fig. 5; +Pa45), suggesting the bacterial effect was due to the neutralization of the BITC.

**Fig. 5: Benzyl isothiocyanate inhibits *Ochradenus baccatus* seedling growth and *Pa45* neutralizes inhibition.**

Discussion

Previous research exploring the effect of microbiota on fruit-frugivore interaction has suggested that, at least in some cases, microorganisms can enhance the attractiveness of fruits to vertebrate herbivores²⁹. Here we explored this interaction for Ochradenus baccatus, a desert shrub that produces fleshy fruits, having overcome seed digestion by frugivores. Besides sugar content, the fruit pulp contains glucosinolates and the seeds contain the myrosinase enzyme. When the seeds and pulp are mixed; for example, in an animal’s digestive tract, toxic hydrolysis products are released by the MYR-GSL mechanism. Previously, this formation of hydrolysis products was shown to encourage rodents to shift from seed predation to seed dispersion²³. Pycnonotus xanthopygos birds (bulbuls) are considered important seed dispersers of O. baccatus, consuming the whole fruit and secreting the seeds²⁶. Nevertheless, our previous study demonstrated a complex interaction between the birds and the plant where passage through the bird’s digestive system dramatically reduced the number of viable seeds (and thus reduced plant fitness). However, the digestive passage also increased the germination of the surviving seeds¹⁵ but hampered the bird’s digestive ability by inhibiting digestive enzyme activity, demonstrating the existence of conflicting interests and an ecological arm race¹⁵.

In the current study, we decipher the mechanism behind these effects, showing that activation of the “O. baccatus-oil bomb“ (i.e., the GSL-MYR system) produces BITC that inhibits bird digestion and digestion enzymes and O. baccatus seed growth. We also show that a bacterium, Pantoea agglomerans³⁰, found to be a common resident in O. baccatus fruits, and acquired from fruit intake by P. xanthopygos, could reduce concentrations of BITC. Furthermore, P. xanthopygos can remediate the physiological consequences of BITC exposure and fully restore bird digestion and seedling growth. These results fit well with the previously suggested role of the gut microbiome in detoxification of “pollutants” in food³¹ for insects³², rodents³³ and sunbirds³⁴, but here we demonstrate that a specific bacterial species can mediate a multitrophic interaction positively affecting both bird digestive physiology and plant demographics (Fig. 6). We also show that P. agglomerans is a transient member of the P. xanthopygos gut microbiome, established and stabilized in this environment by the consumption of O. baccatus by the birds. Switching the birds’ diet from O. baccatus fruits to bananas resulted in a dramatic and rapid decrease of the Pantoea population in the bird gut, and vice versa when switching from bananas to the O. baccatus diet (similar to refs. ^35,36). As an isolate from the bird droppings, the same P. agglomerans showed not only resistance but also improved growth in the presence of GSL hydrolysis products, when most other isolates from the same droppings showed reduced growth as such breakdown products are known to harm many bacteria³⁷ (but see refs. ^38,39 for previous studies on resistant bacteria).

**Fig. 6: The effect of the “*Ochradenus baccatus*-oil-bomb” on the bird and on seedling growth in the presence of *Pantoea* 45 bacterial isolate.**

In summary, the data presented here offer an explanation for the effect of glucosinolates breakdown products on the digestive ability of vertebrates and the detoxification of these products by a specific bacterial species, P. agglomerans, acquired from consumed fruits, and selected for in the presence of glucosinolates hydrolysis products. The bacteria can benefit from these interactions by gaining a unique, SM-protected habitat in both the bird intestine and in the O. baccatus fruits, extending the reach of the latter. Given that Pantoea bacteria are common in many environments, and that glucosinolates are common secondary metabolites, these bacteria may be involved in many other plant–animal interactions (e.g., ref. ⁴⁰). Finally, our results highlight the complexity of conflicting interests between different tripartite kingdom members: a plant, a fruit consumer, and a specific bacterial species.

Methods

This study received and was conducted according to the University of Haifa ethics committee approval number 750/21.

Study species

Ochraceous baccatus (Delile 1813; Resedaceae) is a perennial wild shrub with wide Saharo-Sindian distribution and is found in desert habitats from Pakistan to central Israel⁴¹. The data for O. baccatus range in Israel (Fig. 1a) were taken from Danin and Fragman-Sapir (2016) Flora of Israel Online https://flora.org.il/en/plants/OCHBAC/#moreinfo). All O. baccatus fruit used in the experiments were harvested near the northern coast of the Dead Sea, Israel (31°48’N, 35°27’E) from various shrubs. Fresh fruits were collected once every 3 days, mixed and stored at 4 °C until use. Where required, O. baccatus seeds and pulp were manually separated. The pulp was crushed and squeezed through filter paper for juice. Where designated, seeds were ground in short pulses in a coffee grinder, and the seed powder was suspended in water at 20% wt/vol (e.g., 2 g ground seeds in a total water volume of 10 mL). The pulp juice and seed extract were centrifuged (8000 × g, 5 min) to remove large solids and sterilized by filtration through 0.2 µm syringe filters.

Musa acuminata Colla 1798 (banana) is an evergreen perennial plant belonging to the family Musaceae. Its fruits are often used as a main component in artificial diets of frugivorous birds⁴². Banana pulp contains high amounts of soluble carbohydrates (70% of dry weight) but low levels of protein (1.2%) and lipids (0.2%)⁴³. In our experiments, we used the Grand Nain variety, the most common cultivar grown in Israel.

Pycnonotus xanthopygos (Hemprich and Ehrenberg 1833; white-spectacled bulbuls) is a common Middle East resident⁴⁴. P. xanthopygos were captured from two habitats: one overlapping with the O. baccatus range (31°33’ N, 35°24’ E), and thus assumed to be unexperienced in foraging and consuming O. baccatus fruit^24,26 (i.e., experienced birds; n = 6) and the second outside the O. baccatus distribution area (32°27’ N, 35°33’ E), and thus assumed to be naïve to O. baccatus fruit (i.e., naïve birds; n = 4). Birds were captured using mist nets (Monofilament 9S; mesh size 15 × 15 mm, net size 2.5 × 12 m) on May 2018 at the Jordan Valley, Israel (bird ringing permit #297, Israel Nature & Park Authority). The birds’ ages were 6 months to 1 year. Sex determination in Pycnonotus xanthopygos is very complex without surgery, which we did not do. All birds were released unharmed at the end of the experiment.

Bird capture, housing and diet

After capture, each bird was kept in a separate cage (100 × 80 × 40 cm) in an outdoor enclosure at the Oranim College Campus, Kiryat Tivon, Israel. Upon capture, feeding commenced. In the first week, naïve birds were fed on banana fruit, while experienced birds were fed on O. baccatus fruit (Fig. 1b, W1). At the beginning of the second week, and for 2 weeks (W2–3), naïve birds were fed O. baccatus fruit while experienced birds were fed on banana fruit. During these 3 weeks (Fig. 1b, W1–3), droppings and fruit samples were collected daily and placed in sterile tubes, and two volumes of ethanol were added. Tubes were frozen at −20 °C. Next, at the beginning of week 4 (Fig. 1b, W4), diets were switched back, and until the end of the experiment, naïve birds were fed on bananas, while experienced birds were fed on O. baccatus fruit. At specified intervals (Fig. 1b), one meal of the birds’ diet was supplemented with either water, O. baccatus fruit pulp juice (containing glucosinolates), O. baccatus ground seed extract (containing the myrosinase enzyme) or both pulp juice and seed extract (activating the O. baccatus GSL-Myr system and resulting in GLS breakdown products).

Quantification of digestion ability

Digestibility was quantified as in ref. ¹⁶. Briefly, on the morning of each test (07:00 am), water and food containers were removed from all cages. At 11:00 am, baking paper sheets were placed at the bottom of each cage to collect droppings and a container holding 50 g (fresh weight) of feed mixture (see below) was presented to the birds. The birds were allowed to feed ad libitum for 30 min, after which the dishes were removed. The food remaining in the containers was weighed and the dry weight of the food consumed (gram dry weight) was calculated according to Eq. 1. In all cases, dry weight was determined by drying known amounts of feed at 60 °C to stable weight and calculating the dry mass percentage. In addition, fresh food weight loss due to evaporation was quantified by placing five containers (similar to the containers presented to the birds, containing 50 g of each food) out of the cages during the feeding period and weighing the food after the feeding period (evaporation loss was less than 6% in all cases). Change in food weight was corrected for food weight loss due to evaporation. The 50-g feed was supplemented with 2 mL water (control); 1 mL O. baccatus pulp juice + 1 mL water (pulp, P); 1 mL O. baccatus ground seed extract + 1 mL water (seeds, S); or 1 mL O. baccatus pulp juice + 1 mL of O. baccatus ground seed extract (pulp + seeds, P+S).

The maximal gut passage time of O. baccatus fruits in P. xanthopygos is 91.5 ± 7.3 min²⁶. Therefore, 2 h after the end of feeding (i.e., 13:30 pm), the linings on the cage floors were collected and the droppings scraped into pre-weighed pre-labeled glass Petri dishes (the glass used as “plastic” tended to lose weight do to evaporation of plasticizers). The droppings were dried to a constant weight at 60 °C and food digestibility was calculated according to Eq. 2. After each experiment, the naïve birds were returned to feed on banana and the experienced birds were returned to feed on O. baccatus fruit ad libitum. All birds were seen to be in good health during the experiments and willingly consumed all feeds.

$$\mathop{{{{{{{\rm{Food}}}}}}}\,{{{{{{\rm{consumption}}}}}}}}\limits_{({{{{{\rm{g}}}}}}\,{{{{{\rm{dry}}}}}}\,{{{{{\rm{wt}}}}}})}=\left[\mathop{{{{{{\rm{food}}}}}}\,{{{{{{\rm{supplied}}}}}}}}\limits_{{{{{{\rm{g}}}}}}\,{{{{{\rm{fresh}}}}}}\,{{{{{\rm{wt}}}}}}}-\frac{{{{{{\rm{remaining}}}}}}\,{{{{{\rm{food}}}}}}({{{{{\rm{g}}}}}}\,{{{{{\rm{fresh}}}}}}\,{{{{{\rm{wt}}}}}})}{1\!-\!{{{{{\rm{percentage}}}}}}\,{{{{{\rm{food}}}}}}\,{{{{{\rm{loss}}}}}}\,{{{{{\rm{due}}}}}}\,{{{{{\rm{to}}}}}}\,{{{{{\rm{evaporation}}}}}}}\right]\times\%\,{{{{{\rm{dry}}}}}}\,{{{{{\rm{wt}}}}}}$$

(1)

$${{{{{\rm{Digestability}}}}}}(\%)=\frac{({{{{{\rm{food}}}}}}\,{{{{{\rm{consumption}}}}}}\,({{{{{\rm{g}}}}}}\,{{{{{\rm{dry}}}}}}\,{{{{{\rm{wt}}}}}})-{{{{{\rm{droppings}}}}}}({{{{{\rm{g}}}}}}\,{{{{{\rm{dry}}}}}}\,{{{{{\rm{wt}}}}}}))}{{{{{{\rm{food}}}}}}\,{{{{{\rm{consumption}}}}}}({{{{{\rm{g}}}}}}\,{{{{{\rm{dry}}}}}}\,{{{{{\rm{wt}}}}}})}\times 100$$

(2)

Identification of O. baccatus fruit glucosinolates

Pulp and seeds of O. baccatus fruit stored at 4 °C were separated manually. Three hundred mg of pulp were transferred to an Eppendorf tube containing 1200 µL methanol and 100 μM sinigrin (as internal standard; no sinigrin was found in O. baccatus⁴⁵). The GSLs were extracted on a DEAE column, converted to desulfoGSLs, and identified by HPLC by comparing the retention time and the absorbance spectrum (all according to ref. ⁴⁵).

Quantification of BenzylGSL in fresh O. baccatus fruits and the droppings of birds fed on O. baccatus fruit

Three hundred mg of fresh O. baccatus pulp were placed in an Eppendorf tube containing 1200 µL methanol and 100 μM sinigrin (as internal standard). Simultaneously, 300 μL of droppings from birds fed on O. baccatus fruit (experienced birds in the first day capture and naïve birds after 1 week when the diets were switched) were also transferred to an Eppendorf tube containing 1200 µL methanol and 100 μM sinigrin (as internal standard). The GSLs were extracted, converted to desulfoGSLs, identified, and quantified by HPLC according to ref. ⁴⁵.

Identification of O. baccatus glucosinolates-myrosinase hydrolysis products

To identify GSL hydrolysis products, we used gas chromatography combined with mass spectrometry (GC-MS). Forty mL of ground O. baccatus fruit (pulp and seeds) were placed in a 100 mL Erlenmeyer flask and overlayered with 10 mL of n-Hexane for 2 h. Then, the liquids were transferred to 50 mL tubes, mixed by vortex for 1 min, and centrifuged at 4000 × g for 5 min to remove solids. Next, 5 mL of the upper organic phase was transferred to a glass tube and concentrated to 100 µL under a nitrogen stream. The concentrated solution was transferred to a glass GC vial and 2 µL were injected into an Agilent 7890A gas chromatograph (Agilent Technologies), equipped with a Rxi-5Sil MS capillary column (30 m length; 0.25 mm ID; 0.25 µm coating; Restek) and coupled to a single-quad mass spectrometer detector (Agilent 5975C). The initial oven temperature was 70 °C. It was raised at a rate of 20 °C/min to 250 °C and then at 10 °C/min from 250 °C to 320 °C and held for 10 min. The injector temperature was set at 250 °C and the carrier gas (helium) flow was 1 mL/min. Splitless injection was used with a purge time of 1 min. The transfer line was set at 320 °C. Mass spectrometer conditions were as follows: ion source temperature at 230 °C, quadrupole temperature of 150 °C, and ionization voltage of 70 eV. Mass analysis was performed with an electron impact source under scan mode from 40 to 550 m/z. Identification of GSL hydrolysis products was based on retention time and comparison of fragmentation patterns with the National Institute of Standards and Technology (NIST) library eleven and validated against a commercial standard (Sigma Cat# 622-78-6).

Effect of BITC on digestive enzymes activity

To test if BITC could inhibit digestive enzymes activity, commercial digestive enzymes amylase (Sigma Cat# A3176) and lipase (Sigma Cat# L0382) were tested after being combined with aqueous commercial BITC (0.2%; Sigma Cat# 622-78-6). Deionized water was used as a control. Amylase and lipase assays were performed according to ref. ¹⁵.

DNA extraction from bird droppings and plant material, and 16S rRNA gene amplification and sequencing

To study the effect of the birds’ origin and diet on bacterial community composition, we analyzed the microbiota from droppings of the birds from two habitats, fed as described above (also see Fig. 1b). Bird droppings (~500 μL) were collected daily into a sterile tube, and two volumes of absolute ethanol were added. In addition, bananas and O. baccatus placed outside the cages were collected daily at the time of feeding to analyze fruit microbiota. All samples were stored at −20 °C until DNA extraction. Sample tubes were centrifuged for 10 min at 15,000 × g and the ethanol was removed by pipetting, followed by evaporation for 20 min in a 100 °C dry block. DNA was extracted from the solids using PureLink™ Microbiome DNA Purification Kits (Thermo Fisher Scientific, UK) following the manufacturer’s instructions. DNA was also extracted from three empty tubes that went through the same treatment as negative controls. The extracted DNA was kept at −20 °C until further analyses. For PCR amplification, 10–100 ng DNA was amplified for the V4 region of 16S rRNA gene using the primer pair CS1_515F (ACACTGACGACATGGTTCTACAGTGCCAGCMGC CGCGGTAA) and C1_806R (TACGGTAGCAGAGACTTGGTCTGGACTACHVG GGTWTCTAAT) (Integrated DNA Technologies, USA) as previously published⁴⁶. Amplification was performed in a volume of 25 µL with the 2× Bio-Ready-Mix (Bio-Lab, Israel). Primer concentrations were 0.5 ng/µL. PCR conditions were: 95 °C for 5 min, followed by 28 cycles of 30 s at 95 °C; 45 s at 55 °C and 30 s at 68 °C, and a final elongation step of 7 min at 68 °C. The amplification products were verified by agarose gel electrophoresis and then stored at −20 °C. No amplification (contamination) was observed in the blank samples.

Library preparation and sequencing were performed at the Genome Research Core, Research Resources Center, University of Illinois, Chicago, USA. Briefly, a second PCR amplification was performed for each sample to introduce a unique 10-base barcode obtained from the Access Array Barcode Library for Illumina (Fluidigm, CA, USA; Item# 100-4876). Samples were pooled in equal volume using and purified using an AMPure XP cleanup protocol (0.6×, vol/vol; Agencourt, Beckmann-Coulter). The pooled libraries, with a 15% PhiX spike-in, were loaded onto an Illumina MiniSeq mid-output flow cell (2 × 150 paired-end reads) to generate sequence data. Raw reads were recovered as FASTQ files. Raw sequence data were deposited in the NCBI SRA database under BioProject accession PRJNA869874.

Sequence data processing and analysis

Sequence analysis—the DADA2 pipeline (version 1.18.0)⁴⁷ using R was used for sequence data analyses. Sequences were filtered and trimmed for quality using the “filterAndTrim” command with the parameters maxN = 0, maxEE = 2, trimLeft = 20 bp and truncLen = 153 bp. A sequence error estimation model was carried out using the “learnErrors” option using default parameters. Following these steps, the DADA2 algorithm was implemented for error correction, and a count table containing the ASVs and counts per sample was produced. Sequences were merged using the “mergePairs” command with minimum overlap set at 8 bp. Suspected chimeras were detected and removed using the command “removeBimeraDenovo”. Count tables with ASV sequences and the number of reads per sample were extracted. Taxonomic classification of ASVs was assigned using the SILVA small subunit rRNA database (version 138; https://zenodo.org/record/3731176#.Y9ZyjnZBxaQ), using the command “assignTaxonomy” algorithm with a minimum similarity threshold of 80% and the Phyloseq package (version 1.34.0) used to create the ASVs table. Sequences that were assigned as non-bacterial or unclassified (e.g., Eukaryotes, Chloroplast, and Mitochondria) were removed. Then, we excluded bird fecal samples with total read counts below 3500. Due to the high rate of chloroplast-originating sequences in plant samples, we analyzed all plant samples with bacterial read counts above 1000.

Count data were normalized by the cumulative sum square method (CSS⁴⁸). In order to examine the effect of bird origin (naïve vs. experienced) and diet (banana or O.b.) on the microbial community composition, the permutational analysis of variance (PERMANOVA) test was applied. PERMANOVA was performed using the command “adonis2” in R package “vegan” (version 2.6-2)⁴⁹. In addition, non-metric multidimensional scaling (NMDS) analysis was done in “vegan” based on Bray–Curtis dissimilarities calculated from CSS normalized counts. NMDS was done using the command “metaMDS” in “vegan” with parameters k = 2, try = 1000. Relative abundances for ASVs and genera were calculated based on CSS normalized counts. Linear discriminant effect size (LEfSe) analysis was chosen to calculate differential abundance and identify biomarkers for bird diet. This method is effective in determining which features, in this case ASVs, are most likely to explain observed differences among factor levels⁵⁰. LEfSe was performed using the online Galaxy module (http://huttenhower.sph.harvard.edu/galaxy).

Isolation of cultivatable bacteria from experienced bird droppings and their identification

On the first day of captivity, 500 µL of droppings were collected from each experienced bird and placed into a sterile tube as described above but without the addition of ethanol. The droppings were immediately plated on Luria Broth (LB) agar and incubated at 28 °C for 24–48 h. Representative colonies were selected by morphology and subcultured five times to achieve purity for each isolate. Cultures of each isolate, grown overnight in LB were used for DNA extraction and PCR, as well as biochemical testing. Genomic DNA was purified from cultures using the DNA Fungal/Bacterial Microprep Kit (Zymo Research, USA) according to the manufacturer’s instructions. The near full-length 16S rRNA gene was amplified by PCR using primers 11F (5′-GGATCCAGACTTTGATYMTGGCTCAG-3′) and 1512R (5′-GTGAAGCTTACGG(C/T)TAGCTTGTTACGACTT-3′), modified from ref. ⁵¹. The thermal cycling conditions were 94 °C for 4 min; 30 cycles at 94 °C for 30 s, at 54 °C for 40 s, and at 72 °C for 70 s; and then a final step at 72 °C for 20 min. The PCR products were sequenced by MCLAB (San Francisco, CA) and the sequences were compared to those available in the NCBI nucleotide database, using the standard nucleotide–nucleotide BLAST tool (BLASTN; http://www.ncbi.nlm.nih.gov).

The effect of BITC on isolate growth

Tubes with 3 mL of liquid LB medium supplemented with BITC (0.1% v/v) or water (as a control) were inoculated with the different bacterial isolates (see above) to O.D._600nm of 0.05, measured in a 1 cm cuvette using a spectrophotometer (UV-1650PC, Shimadzu). The cultures were incubated at 28 °C for 24 h. Then, the O.D._600nm of each culture was measured, and the ratio between growth LB medium supplemented with BITC (0.1%) relative to water was calculated.

The effect of isolate Pa45 on BITC amount

A 100-mL Erlenmeyer flask with 40 mL of LB was supplemented with BITC (0.1% v/v) and inoculated with Pa45 (added to a final O.D._600nm of 0.05). Ten mL of n-Hexane (Merck, 110-54-3) was overlayered and the Erlenmeyer was sealed with Parafilm. A sterile medium was used as a negative control. Following 24 h of incubation at 28 °C with shaking at 200 rpm, the liquids were transferred to 50 mL tubes, mixed by vortex for 1 min, and centrifuged at 4000 × g for 5 min for phase separation. Next, 5 mL of the upper organic phase was transferred to a glass tube and concentrated to 100 µL under nitrogen flow. The concentrated solution was analyzed with GC-MS as above and BITC quantification against a calibration curve was performed on BITC dissolved in n-Hexane (1, 3 and 5 µg/mL).

The effects of BITC on O. baccatus seedling growth

To assess the effect of BITC on O. baccatus seedling development, O. baccatus seeds were separated manually immediately after the collection of fruits, and they were germinated as in ref. ¹⁵. Soaking liquids were water, water inoculated with Pa45, water supplemented with BITC (0.1% v/v), and water supplemented with BITC (0.1%) and inoculated with Pa45. The seedlings were left for 2 weeks. Seedling plumule and radicle were measured with a caliber.

Statistical analysis

Statistical analyses were performed using SPSS statistical software version 21.0 (IBM, SPSS Inc., Chicago, IL, USA). Normality was examined using the Shapiro–Wilk test, and data were considered statistically significant for a p value <0.05. Two-tailed t-tests for two independent samples were applied to the effect of food provided for digestion efficiency and for the amount of BITC in the presence or absence of Pa45. A one-way ANOVA followed by Bonferroni multiple comparisons test was done on the quantity of the GSLs in O. baccatus fruits before or after passage through the bird digestive tract and on the raw enzymatic activity for each enzyme.

Data availability

The raw amplicon sequence data generated in this study have been deposited in the NCBI SRA database under accession code PRJNA869874. Raw results of feeding experiments, digestibility, enzyme activity, BenzylGSL and BITC quantification, abundance of bacterial genera, number of OTUs, and seedlings growth are given in the “Supplementary Dataset” file. Source Data are provided with this paper.

References

Howe, H. E. & Smallwood, J. Ecology of seed dispersal. Annu. Rev. Ecol. Syst. 13, 201–228 (1982).
Article Google Scholar
Traveset, A., Heleno, R. & Nogales, M. The ecology of seed dispersal. in Seeds: The Ecology of Regeneration in Plant Communities 3rd edn (ed. Gallagher, R. S.) Ch. 3 (CABI International) https://doi.org/10.1079/9781780641836.0062 (2014).
van der Pijl, L. Principles of Dispersal in Higher Plants (Springer, Berlin) https://doi.org/10.1007/978-3-642-87925-8 (1982).
Díaz Vélez, M. C., Sérsic, A. N., Traveset, A. & Paiaro, V. The role of frugivorous birds in fruit removal and seed germination of the invasive alien Cotoneaster franchetii in central Argentina. Austral. Ecol. 43, 558–566 (2018).
Article Google Scholar
Egerer, M. H., Fricke, E. C. & Rogers, H. S. Seed dispersal as an ecosystem service: frugivore loss leads to decline of a socially valued plant, Capsicum frutescens. Ecol. Appl. 28, 655–667 (2018).
Article PubMed PubMed Central Google Scholar
Shikang, S., Fuqin, W. & Yuehua, W. Does the passage of seeds through frugivore gut affect their storage: a case study on the endangered plant Euryodendron excelsum. Sci. Rep. 5, 1–8 (2015).
Article Google Scholar
Pegman, A. P. M. K., Perry, G. L. W. & Clout, M. N. Size-based fruit selection by a keystone avian frugivore and effects on seed viability. N.Z. J. Bot. 55, 118–133 (2017).
Article Google Scholar
Herrera, C. M. Seed dispersal by vertebrates. in Plant-Animal Interactions, An Evolutionary Approach (eds. Herrera, C. M. & Pellmyr, O.) Ch. 7 (Blackwell Science, 2002).
Schleuning, M. et al. Specialization and interaction strength in a tropical plant-frugivore network differ among forest strata. Ecology 92, 26–36 (2011).
Article PubMed Google Scholar
Izhaki, I. Essential amino acid composition of fleshy fruits versus maintenance requirements of passerine birds. J. Chem. Ecol. 24, 1333–1345 (1998).
Article CAS Google Scholar
Witmer, M. C. & Van Soest, P. J. Contrasting digestive strategies of fruit-eating birds. Fun. Ecol. 12, 728–741 (1998).
Article Google Scholar
Norconk, M. A., Grafton, B. W. & Conklin-Brittain, N. L. Seed dispersal by neotropical seed predators. Am. J. Primatol. 45, 103–126 (1998).
Article CAS PubMed Google Scholar
Hulme, P. Seed-eaters: seed dispersal, destruction, and demography. in Seed Dispersal and Frugivory: Ecology, Evolution and Conservation (eds Levey, D., Silva, W. & Galetti, M.) Ch. 17 (CABI Publishing, 2002).
Janzen, D. H. Seed predation by animals. Annu. Rev. Ecol. Evol. Syst. 2, 465–492 (1971).
Article Google Scholar
Trabelcy, B., Izhaki, I. & Gerchman, Y. The survival contest of endozoochory: conflicting interests in a frugivorous avian plant mutualism. J. Ecol. 110, 526–539 (2022).
Article Google Scholar
Cipollini, M. L. Secondary metabolites of vertebrate-dispersed fruits: evidence for adaptive functions. Rev. Chil. Hist. Nat. 73, 421–440 (2000).
Article Google Scholar
Whitehead, S. R. & Bowers, M. D. Evidence for the adaptive significance of secondary compounds in vertebrate-dispersed fruits. Am. Nat. 182, 563–577. https://doi.org/10.1086/673258 (2013).
Halkier, B. A. & Gershenzon, J. Biology and biochemistry of glucosinolates. Annu. Rev. Plant Biol. 57, 303–333 (2006).
Article CAS PubMed Google Scholar
Kliebenstein, D. J., Kroymann, J. & Mitchell-Olds, T. The glucosinolate–myrosinase system in an ecological and evolutionary context. Curr. Opin. Plant Biol. 8, 264–271 (2005).
Article CAS PubMed Google Scholar
Tripathi, M. K. & Mishra, A. S. Glucosinolates in animal nutrition: a review. Anim. Feed Sci. Technol. 132, 1–27 (2007).
Article CAS Google Scholar
Rask, L. et al. Myrosinase: gene family evolution and herbivore defense in Brassicaceae. Plant Mol. Biol. 42, 93–113 (2000).
Article CAS PubMed Google Scholar
Hopkins, R. J., van Dam, N. M. & van Loon, J. J. Role of glucosinolates in insect–plant relationships and multitrophic interactions. Annu. Rev. Entomol. 54, 57–83 (2009).
Article CAS PubMed Google Scholar
Samuni-Blank, M. et al. Intraspecific directed deterrence by the mustard oil bomb in a desert plant. Curr. Biol. 22, 1218–1220 (2012).
Article CAS PubMed Google Scholar
Bronstein, J. L. et al. Fleshy-fruited plants and frugivores in desert ecosystems. in Seed Dispersal: Theory and its Application in a Changing World (eds Dennis, A. J., Schupp, E. W., Green, R. J. & Westcott, D.W.) Ch. 7 (CABI International) https://doi.org/10.1079/9781845931650.0148 (2007).
Lotan, A. & Izhaki, I. Could abiotic environment shape fleshy fruit traits? A field study of the desert shrub Ochradenus baccatus. J. Arid. Environ. 92, 34–41 (2013).
Article ADS Google Scholar
Spiegel, O. & Nathan, R. Incorporating dispersal distance into the disperser effectiveness framework: frugivorous birds provide complementary dispersal to plants in a patchy environment. Ecol. Lett. 10, 718–728 (2007).
Article PubMed Google Scholar
Petri, L. et al. Cysteine specific bioconjugation with benzyl isothiocyanates. RSC Adv. 10, 14928–14936 (2020).
Article ADS CAS PubMed PubMed Central Google Scholar
Kohl, K. D. et al. Monoterpenes as inhibitors of digestive enzymes and counter-adaptations in a specialist avian herbivore. J. Comp. Physiol. B. 185, 425–434 (2015).
Article CAS PubMed Google Scholar
Peris, J. E., Rodriguez, A., Peña, L. & Fedriani, J. M. Fungal infestation boosts fruit aroma and fruit removal by mammals and birds. Sci. Rep. 7, 5646 (2017).
Article ADS PubMed PubMed Central Google Scholar
Gavini, F. et al. Transfer of Enterobacter agglomerans (Beijerinck 1888) Ewing and Fife 1972 to Pantoea gen. nov. as Pantoea agglomerans comb. nov. and description of Pantoea dispersa sp. nov. Int. J. Syst. Evol. Microbiol. 39, 337–345 (1989).
Google Scholar
Claus, S. P., Guillou, H. & Ellero-Simatos, S. The gut microbiota: a major player in the toxicity of environmental pollutants? NPJ Biofilms Microbiomes. 2, 16003 (2016). (2016).
Article PubMed PubMed Central Google Scholar
Jing, T. Z., Qi, F. H. & Wang, Z. Y. Most dominant roles of insect gut bacteria: digestion, detoxification, or essential nutrient provision? Microbiome 8, 38 (2020).
Article CAS PubMed PubMed Central Google Scholar
Kohl, K. D., Weiss, R. B., Cox, J., Dale, C. & Dearing, M. D. Gut microbes of mammalian herbivores facilitate intake of plant toxins. Ecol. Lett. 17, 1238–1246 (2014).
Article PubMed Google Scholar
Gunasekaran, M., Trabelcy, B., Izhaki, I. & Halpern, M. Direct evidence that sunbirds’ gut microbiota degrades floral nectar’s toxic alkaloids. Front. Microbiol. 12, 639808 (2021).
Article PubMed PubMed Central Google Scholar
Hanschen, F. S., Yim, B., Winkelmann, T., Smalla, K. & Schreiner, M. Degradation of biofumigant isothiocyanates and allyl glucosinolate in soil and their effects on the microbial community composition. PLoS One. 10, 1–18 (2015).
Article CAS Google Scholar
Kellingray, L. et al. Consumption of a diet rich in Brassica vegetables is associated with a reduced abundance of sulphate-reducing bacteria: a randomised crossover study. Mol. Nutr. Food Res. 61, 1600992 (2017).
Article PubMed PubMed Central Google Scholar
Aires, A., Mota, V. R., Saavedra, M. J., Rosa, E. A. S. & Bennett, R. N. The antimicrobial effects of glucosinolates and their respective enzymatic hydrolysis products on bacteria isolated from the human intestinal tract. Appl. Microbiol. 106, 2086–2095 (2009).
Article CAS Google Scholar
Szűcs, Z. et al. Endophytic fungi from the roots of horseradish (Armoracia rusticana) and their interactions with the defensive metabolites of the glucosinolate—myrosinase—isothiocyanate system. BMC Plant Biol. 18, 85. https://doi.org/10.1186/s12870-018-1295-4 (2018).
Sellam, A., Dongo, A., Guillemette, T., Hudhomme, P. & Simoneau, P. Transcriptional responses to exposure to the brassicaceous defense metabolites amalexin and allyl-isothiocyanate in the necrotrophic fungus Alternaria brassicicola. Mol. Plant Pathol. 8, 195–208 (2007).
Article CAS PubMed Google Scholar
Shukla, S. P. & Beran, F. Gut microbiota degrades toxic isothiocyanates in a flea beetle pest. Mol. Ecol. 29, 4692–4705 (2020).
Article CAS PubMed Google Scholar
Miller, A. G. A revision of Ochradenus. Notes Roy. Bot. Gard. Edinb. 41, 491–504 (1984).
Google Scholar
Denslow, J. S., Levey, D. J., Moermond, T. C. & Bernard, C. A synthetic diet for 631 fruit–eating birds. Wilson Bull. 99, 131–135 (1987).
Google Scholar
Laska, M. Food transit times and carbohydrate use in three Phyllostomid bat species. Z. Säugetierkd 55, 49–54 (1990).
Google Scholar
Shirihai, H. The Birds of Israel (Academic Press, UK, 1996).
Trabelcy, B. et al. Investigation of glucosinolates in the desert plant Ochradenus baccatus (Brassicales: Resedaceae). Unveiling glucoochradenin, a new arabinosylated glucosinolate. Phytochem 187, 112760 (2021).
Article CAS Google Scholar
Gunasekaran, M., Lalzar, M., Sharaby, Y., Izhaki, I. & Halpern, M. The effect of toxic pyridine-alkaloid secondary metabolites on the sunbird gut microbiome. NPJ Biofilms Microbiomes. 6, 53 (2020).
Article CAS PubMed PubMed Central Google Scholar
Callahan, B. J. et al. DADA2: high-resolution sample inference from Illumina amplicon data. Nat. Methods 13, 581–583 (2016).
Article CAS PubMed PubMed Central Google Scholar
Segata, N. et al. Metagenomic biomarker discovery and explanation. Genome Biol. 12, R60 (2011).
Article PubMed PubMed Central Google Scholar
Simpson, G. L. et. al. Vegan community ecology package version 2.6-2 (2022).
Paulson, J. N., Stine, O. C., Bravo, H. C. & Pop, M. Differential abundance analysis for microbial marker-gene surveys. Nat. Methods 10, 1200–1202 (2013).
Article CAS PubMed PubMed Central Google Scholar
Felske, A., Rheims, H., Wolterink, A., Stackebrandt, E. & Akkermans, A. D. Ribosome analysis reveals prominent activity of an uncultured member of the class Actinobacteria in grassland soils. Microbiology 143, 2983–2989 (1997).
Article CAS PubMed Google Scholar

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Acknowledgements

We thank the Israeli Science Foundation (ISF grant No. 296/16 to I.I.) and the Middle East Regional Cooperation Program (project TA-MOU-08-M28-013 to Y.G. and I.I.) for their generous funding. We are grateful to R. Haran, who helped capture the birds, and to N. Dainov of the Oranim College animal house staff for her help with animal maintenance.

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Authors and Affiliations

Department of Evolutionary and Environmental Biology, Faculty of Natural Sciences, University of Haifa, Haifa, 3498838, Israel
Beny Trabelcy, Nimrod Shteindel, Ido Izhaki & Yoram Gerchman
Bioinformatic Unit, University of Haifa, Haifa, Israel
Maya Lalzar
Oranim College, Kiryat Tivon, 3600600, Israel
Yoram Gerchman

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Beny Trabelcy
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Nimrod Shteindel
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Maya Lalzar
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Ido Izhaki
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Contributions

Conceptualization: B.T., I.I., and Y.G. Methodology: B.T., I.I., and Y.G. Investigation: B.T. Formal analysis: B.T., M.L., and I.I. Writing—original draft: B.T., M.L., N.S., I.I., and Y.G. Funding acquisition: I.I. and Y.G. Supervision: I.I. and Y.G.

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Correspondence to Beny Trabelcy or Yoram Gerchman.

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Trabelcy, B., Shteindel, N., Lalzar, M. et al. Bacterial detoxification of plant defence secondary metabolites mediates the interaction between a shrub and frugivorous birds. Nat Commun 14, 1821 (2023). https://doi.org/10.1038/s41467-023-37525-6

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Received: 09 September 2022
Accepted: 14 March 2023
Published: 31 March 2023
DOI: https://doi.org/10.1038/s41467-023-37525-6

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