Nectar Yeasts Research Articles

Transcriptional Responses to Priority Effects in Nectar Yeast.

Priority effects, where the order and timing of species arrival influence the assembly of ecological communities, have been observed in a variety of taxa and habitats. However, the genetic and molecular basis of priority effects remains unclear, hindering a better understanding of when priority effects will be strong. We sought to gain such an understanding for the nectar yeast Metschnikowia reukaufii commonly found in the nectar of our study plant, the hummingbird-pollinated Diplacus (Mimulus) aurantiacus. In this plant, M. reukaufii can experience strong priority effects when it reaches flowers after other nectar yeasts, such as M. rancensis. After inoculation into two contrasting types of synthetic nectar simulating early arrival of M. rancensis, we conducted whole-transcriptome sequencing of 108 strains of M. reukaufii. We found that several genes were differentially expressed in M. reukaufii strains when the nectar had been conditioned by growth of M. rancensis. Many of these genes were associated with amino acid metabolism, suggesting that M. reukaufii strains responded molecularly to the reduction in amino acid availability caused by M. rancensis. Furthermore, investigation of expression quantitative trait loci (eQTLs) revealed that genes involved in amino acid transport and resistance to antifungal compounds were enriched in some genetic variants of M. reukaufii. We also found that gene expression was associated with population growth rate, particularly when amino acids were limited. These results suggest that intraspecific genetic variation in the ability of nectar yeasts to respond to nutrient limitation and direct fungal competition underpins priority effects in this microbial system.

Endophytic bacteria in Camellia reticulata pedicels: isolation, screening and analysis of antagonistic activity against nectar yeasts.

Camellia reticulata, an ancient plant species endemic to Yunnan Province, China, remains underexplored in terms of its endophytic bacterial communities. The plant tissue pedicel serves as the connection between the flower and the stem, not only delivers nutrients but also transmits metabolic substances from endophytic bacteria to the nectar during long-term microbial colonization and probably improves the antagonistic activity of nectar against yeast. Hence, 138 isolates of endophytic bacteria have been isolated in this study from the pedicels of 12- and 60-year-old C. reticulata. Comparative analysis revealed significantly higher density of endophytic bacteria in older trees. Among these isolates, 29 exhibited inhibitory effects against nectar yeasts. Most of the isolates displayed positive results for Gram staining, catalase reaction, gelatin liquefaction, and motility. Additionally, the isolates demonstrated the ability to utilize diverse substrates, such as glucose, nitrate, and starch. Based on 16S rRNA molecular biology analysis, these isolates were identified to be 11 different species of 6 genera, with the majority belonging to Bacillus genus. Notably, C1 isolate, identified as Bacillus spizizenii, exhibited strongest antagonistic effect against three yeasts, i.e., Metschnikowia reukaufii, Cryptococcus laurentii, and Rhodotorula glutinis, with minimum inhibitory concentration values below 250 μg/mL. Major metabolites of B. spizizenii were aminoglycosides, beta-lactams, and quinolones, which possess antimicrobial activities. Furthermore, KEGG enrichment pathways primarily included the synthesis of plant secondary metabolites, phenylpropanoids, amino acids, alkaloids, flavonoids, neomycin, kanamycin, and gentamicin. Therefore, antagonistic activity of B. spizizenii against yeasts could be attributed to these antibiotics. The findings highlight the diverse endophytic bacteria associated with C. reticulata, indicating their potential as a valuable resource of bioactive metabolites. Additionally, this study provides new insights into the role of endophytic bacteria of pedicels in enhancing nectar resistance against yeasts.

Disruption of pollination by herbivores is rescued by nectar yeasts

Abstract Attracting pollinators to achieve successful reproduction is a key challenge for wild plants that may be disturbed by complex multispecies interactions in nature. Pairwise plant–pollinator interactions have traditionally been studied for decades, while ignoring other ecological players may obscure a comprehensive understanding on how plants recruit partners or combat enemies in the pollination process. Hence, integrated studies considering the inherent complexity of ecological interactions are needed, which may open up new perspectives for deciphering intricate systems and predicting ecological consequences. We examined the presence of nectar yeasts using a combination of high‐throughput sequencing, cultivation and microscopy and quantified floral herbivory by evaluating the incidence of flowers with visible holes in 13 natural populations of Iris bulleyana in the Hengduan Mountains of southwest China during 2017–2022. We combined yeast inoculation and herbivore manipulation treatments to illustrate the isolated and combined impacts of two contrasting nectarivorous organisms, the ascomycetous yeast Metschnikowia reukaufii and adult sawflies, on pollinator visitation and plant reproductive success in two populations. In the lab, we first employed gas chromatography–mass spectrometry to profile the volatile metabolites of yeast‐inoculated nectar relative to control, followed by a behavioural bioassay to test the preference of honeybees for these microbial volatiles. Yeasts commonly inhabited floral nectar and insect herbivores frequently bit holes in the perianth tube to consume nectar and nectaries. Nectar yeasts indirectly facilitated plant reproduction through increased pollinator visits, probably because of microbial metabolism as honeybees preferred nectar volatiles produced by yeasts in behavioural bioassays. Insect herbivores increased total floral visits but reduced legitimate visits by inducing legitimate‐to‐robbing behavioural changes of honeybees, thus leading to lower seed production. The detrimental effect of herbivory was mitigated by the presence of yeasts, which diminished the relative proportion of robbing visits and thereby ‘rescued’ flowers from reproductive failure. Synthesis: Overall, we found contrasting effects of non‐pollinator species, including both micro‐ and macro‐organisms, on plant–pollinator interactions in a biodiversity hotspot, where pollination deficit may be a ubiquitous phenomenon. Our findings suggest that both microbial and herbivory effects are likely to be important in explaining the exact causes of pollen limitation in species‐rich areas, highlighting the biological context dependence of species interactions in natural ecosystems.

Effect of combining ultrasound and UVC treatments for processing orange juice and mango nectar on their microbiological, physicochemical, and sensory characteristics

The present study aims to evaluate the effect of ultrasound (US), UVC radiation, and combined US+UVC treatments on the inactivation of total aerobic mesophilic bacteria (TAMB) and mold and yeast in orange juice and mango nectar. The microbial inactivation after each treatment, physicochemical and sensory properties of treated products, and the microbial response during their storage at 4 °C for 12 days were assessed. Fresh squeezed orange juice and mango nectar were treated by US, UVC, or US+UVC treatments for 20 min at 15 °C; the initial TAMB and mold and yeast counts were determined. The combined treatment US+UVC showed a better inactivation level in TAMB, molds, and yeasts in orange juice and mango nectar, resulting in lower microbial counts at the storage end. Titrable acidity, °Brix, and pH remained unchanged, while ascorbic acid losses and color changes were observed. Additionally, the sedimentation index (SI) was analyzed during the storage of orange juice, observing that US+UVC reduced the SI and stabilized the orange juice for 12 days. For all tested conditions, mango nectar preserved its sensory properties. Industrial relevanceCombining food processing technologies such as ultrasound (US) and UVC radiation can improve the process's effectiveness. The US+UVC treatment enhanced the microbial inactivation (total aerobic mesophilic bacteria and mold and yeast) of orange juice and mango nectar, reduced the orange juice's sedimentation index during storage, and did not modify the sensory properties of mango nectar. Thus, the combined US+UVC treatment is an alternative to sustainably preserving and stabilizing orange juice and mango nectar.

The indirect effect of nectar-inhabiting yeasts on olfactory responses and longevity of two stink bug egg parasitoids

Adult parasitoids are well known to feed on sugar-rich resources such as floral nectar. Recently, an increasing body of evidence has shown that nectar is ubiquitously colonized by microorganisms and, as a consequence, microbial metabolic activity can affect several traits of floral nectar. Yet, how the fermentation of nectar by yeasts impacts the olfactory responses and performance of parasitoids is largely understudied, especially in the case of egg parasitoids. In this study, we investigated whether fermentation by the nectar yeasts Metschnikowia gruessii and M. reukaufii affects the olfactory responses of Trissolcus basalis and Ooencyrtus telenomicida, two egg parasitoid species associated with the southern green stink bug Nezara viridula. We also investigated how yeast fermentation affects the longevity and survival of the egg parasitoids. Results of static four-chamber olfactometer tests showed that nectar fermented by M. gruessii (but not by M. reukaufii) was attractive to both egg parasitoid species, whereas no significant yeast-mediated effects were found in terms of wasp longevity. Gas chromatography coupled with mass spectrometry (GC-MS) showed a clear separation of the volatile profiles among M. gruessii, M. reukaufii and non-fermented control nectar supporting the results of the insect bioassays. The results of our study highlight the need to consider the role of microbes when studying interactions between flower nectar and egg parasitoids and could have implications from a conservation biological control perspective.

Nature's Most Fruitful Threesome: The Relationship between Yeasts, Insects, and Angiosperms.

The importance of insects for angiosperm pollination is widely recognized. In fact, approximately 90% of all plant species benefit from animal-mediated pollination. However, only recently, a third part player in this story has been properly acknowledged. Microorganisms inhabiting floral nectar, among which yeasts have a prominent role, can ferment glucose, fructose, sucrose, and/or other carbon sources in this habitat. As a result of their metabolism, nectar yeasts produce diverse volatile organic compounds (VOCs) and other valuable metabolites. Notably, some VOCs of yeast origin can influence insects’ foraging behavior, e.g., by attracting them to flowers (although repelling effects have also been reported). Moreover, when insects feed on nectar, they also ingest yeast cells, which provide them with nutrients and protect them from pathogenic microorganisms. In return, insects serve yeasts as transportation and a safer habitat during winter when floral nectar is absent. From the plant’s point of view, the result is flowers being pollinated. From humanity’s perspective, this ecological relationship may also be highly profitable. Therefore, prospecting nectar-inhabiting yeasts for VOC production is of major biotechnological interest. Substances such as acetaldehyde, ethyl acetate, ethyl butyrate, and isobutanol have been reported in yeast volatomes, and they account for a global market of approximately USD 15 billion. In this scenario, the present review addresses the ecological, environmental, and biotechnological outlooks of this three-party mutualism, aiming to encourage researchers worldwide to dig into this field.

Highlighted articles for March 2022.

Highlighted articles for March 2022.

Contrasting effects of nectar yeasts on the reproduction of Mediterranean plant species.

Yeasts are often present in floral nectar and can influence plant fitness directly (independently of pollinators) or indirectly by influencing pollinator visitation and behavior. However, few studies have assessed the effect of nectar yeasts on plant reproductive success or compared effects across different plant species, limiting our understanding of the relative impact of direct vs. indirect effects. We inoculated the nectar of six plant species in the field with the cosmopolitan yeast Metschnikowia reukaufii to analyze the direct and indirect effects on female reproductive success over 2 years. The pollinator assemblage for each species was recorded during both flowering years. Direct yeast effects on female fecundity were statistically nonsignificant for all plant species. There were significant indirect, pollinator-mediated effects on fruit production and seed mass for the two species pollinated almost exclusively by bumblebees or hawkmoths, with the direction of the effects differing for the quantity- and quality-related fitness components. There were no consistent effects of the yeast on maternal fecundity for any of the species with diverse pollinator assemblages. Effects of M. reukaufii on plant reproduction ranged from negative to neutral or positive depending on the plant species. The among-species variation in the indirect effects of nectar yeasts on plant pollination could reflect variation in the pollinator community, the specific microbes colonizing the nectar, and the order of microbial infection (priority effects), determining potential species interactions. Elucidating the nature of these multitrophic plant-pollinator-microbe interactions is important to understand complex processes underlying plant pollination.

Nectar Yeast Community of Tropical Flowering Plants and Assessment of Their Osmotolerance and Xylitol-Producing Potential.

Floral nectar is colonised by microbes, especially yeasts which alter the scent, temperature, and chemical composition of nectar, thereby playing an essential role in pollination. The yeast communities inhabiting the nectar of tropical flowers of India are not well explored. We isolated 48 yeast strains from seven different tropical flowering plants. Post MSP-PCR-basedscreening, 23 yeast isolates and two yeast-like fungi were identified, which belonged to 16 species of 12 genera viz. Candida (2 species), Aureobasidium (2 species), Metschnikowia (2 species), Meyerozyma (1 species), Saitozyma (1 species), Wickerhamomyces (1 species), Kodamaea (2 species), Pseudozyma (1 species), Starmerella (1 species), Hanseniaspora (1 species), Rhodosporidiobolus (1 species), Moesziomyces (1 species), and two putative novel species. All yeast strains were assessed for their osmotolerance abilities in high salt and sugar concentration. Among all the isolates, C. nivariensis (SRA2.2, SRA1.1 and SRA2.1), M. caribbica (SRA4.8 and SRA4.6), S. flava SRA4.2, and M. reukaufii SRA3.2 showed significant growth in high concentrations of sugar (40-50% glucose), as well as salt (12-15% NaCl). All 25 strains were also screened for their ability to utilise xylose to produce xylitol. Meyerozyma caribbica was the most efficient xylitol producer, wherein three strains of this species (SRA4.6, SRA4.1, and SRA4.8) generated 18.61 to 21.56gl-1 of xylitol, with 0.465-0.539gg-1 yields. Through this study, we draw attention towards the tropical floral nectar as a potential niche for the isolation of diverse, osmotolerant, and xylitol-producing yeasts. Such osmotolerant yeasts have potential applications in food industries and biofuel production.

The role of plant–pollinator interactions in structuring nectar microbial communities

Abstract Floral nectar harbours a diverse microbiome of yeasts and bacteria that depend predominantly on animal visitors for their dispersal. Since pollinators visit specific sets of flowers and carry their own unique microbiota, we hypothesize that plant species visited by the same set of pollinators may support non‐random nectar microbial communities linked together by the type of pollinator. Here we explore the importance of plant–pollinator interactions in the assembly of nectar microbiome and study the role of plant geographic location as a determinant of microbial community composition. We intensively sampled the nectar of 282 flowers of 48 plant species with beetles, birds, long‐tongued and short‐tongued insects as pollinators in wild populations in South Africa, one of the world's biodiversity hotspots, and using molecular techniques we identified nectar yeast and bacteria taxa. The analyses provided new insights into the richness, geographic structure and phylogenetic characterization of nectar microbiome, and compared patterns of composition of bacteria and yeast communities in relation to plant and pollinator guild. Our results showed that plant–pollinator interactions played a crucial role in shaping nectar microbial communities. Plants visited by different pollinator guilds supported significantly different yeast and bacterial communities. The pollinator guild also contributed to the maintenance of beta diversity and phylogenetic microbial segregation. The results revealed different patterns for yeast and bacteria; whereas plants visited by beetles supported the highest richness and phylogenetic diversity of yeasts, bacteria communities were significantly more diverse in plants visited by other insect groups. We found no clear microbial spatial segregation at different geographical scales for bacteria, and only the phylogenetic similarity of yeast composition was correlated significantly with geography. Synthesis. Interactions of animal vector, plant host traits and microbe physiology contribute to microbial community assemblages in nectar. Our results suggest that plants visited by the same pollinator guild have a characteristic nectar microbiota signature that may transcends the geographic region they are in. Contrasted patterns for yeast and bacteria stress the need for future work aimed at better understanding the causes and consequences of the importance of plants and pollinators in shaping nectar microbial communities in nature.

Nitrogen Assimilation Varies Among Clades of Nectar- and Insect-Associated Acinetobacters.

Floral nectar is commonly colonized by yeasts and bacteria, whose growth largely depends on their capacity to assimilate nutrient resources, withstand high osmotic pressures, and cope with unbalanced carbon-to-nitrogen ratios. Although the basis of the ecological success of these microbes in the harsh environment of nectar is still poorly understood, it is reasonable to assume that they are efficient nitrogen scavengers that can consume a wide range of nitrogen sources in nectar. Furthermore, it can be hypothesized that phylogenetically closely related strains have more similar phenotypic characteristics than distant relatives. We tested these hypotheses by investigating the growth performance on different nitrogen-rich substrates of a collection of 82 acinetobacters isolated from nectar and honeybees, representing members of five species (Acinetobacter nectaris, A. boissieri, A. apis, and the recently described taxa A. bareti and A. pollinis). We also analyzed possible links between growth performance and phylogenetic affiliation of the isolates, while taking into account their geographical origin. Results demonstrated that the studied isolates could utilize a wide variety of nitrogen sources, including common metabolic by-products of yeasts (e.g., ammonium and urea), and that phylogenetic relatedness was associated with the variation in nitrogen assimilation among the studied acinetobacters. Finally, nutrient source and the origin (sample type and country) of isolates also predicted the ability of the acinetobacters to assimilate nitrogen-rich compounds. Overall, these results demonstrate inter-clade variation in the potential of the acinetobacters as nitrogen scavengers and suggest that nutritional dependences might influence interactions between bacteria and yeasts in floral nectar.

Integrating microbes into pollination.

Microbes (fungi, bacteria and viruses) living within flowers are hypothesized to affect pollination. We evaluate current support for this idea at each stage of the pollination process. Evidence to date is convincing that microbes influence pollinator attraction, but data are heavily weighted toward bumblebees and the effects of nectar yeasts. Effects of microbes on the efficacy of pollinator visits is understudied and variable outcomes from field studies suggest quality of pollinator visits, not only quantity, are likely involved. The effect of microbes on pollen performance is underappreciated. Beyond the effect of pathogenic viruses, the impacts of pollen-transmitted endophytic microbes on pollen viability or tube growth are unknown but could affect the outcome of pollen receipt. Future research integrating microbes into pollination should broaden taxonomic diversity of microbes, pollinators and plants and the processes under study.

Towards a better understanding of the role of nectar-inhabiting yeasts in plant\u2013animal interactions

Flowers offer a wide variety of substrates suitable for fungal growth. However, the mycological study of flowers has only recently begun to be systematically addressed from an ecological point of view. Most research on the topic carried out during the last decade has focused on studying the prevalence and diversity of flower-inhabiting yeasts, describing new species retrieved from floral parts and animal pollinators, and the use of select nectar yeasts as model systems to test ecological hypotheses. In this primer article, we summarize the current state of the art in floral nectar mycology and provide an overview of some research areas that, in our view, still require further attention, such as the influence of fungal volatile organic compounds on the foraging behavior of pollinators and other floral visitors, the analysis of the direct and indirect effects of nectar-inhabiting fungi on the fitness of plants and animals, and the nature and consequences of fungal-bacterial interactions taking place within flowers.

The Impact of Yeast Presence in Nectar on Bumble Bee Behavior and Fitness

The presence of yeasts in pollen and floral nectar is rather the norm than the exception. Due to the metabolic activities of yeasts, sugar and amino acid composition of nectar often drastically change and may negatively impact the nutritional value of nectar for pollinators and hence insect fitness. On the other hand, the presence of yeasts in floral nectar may also increase its nutritional value due to yeast's probiotic effect and the release of yeast's metabolites. In this study, we investigated whether the presence of defined flower‐ and insect‐associated yeasts affected individual and colony fitness of the bumble bee pollinator Bombus terrestris. Specifically, we tested whether the presence of yeasts in nectar affected bumble bee foraging behavior and nectar consumption, individual growth and colony development, larval and queen mortality, and mating success. Quantitative analyses of sugar and amino acid profiles showed that nectar yeasts significantly affected the chemical composition of nectar. However, dual‐choice experiments indicated that yeast inoculation did not significantly affect foraging behavior or consumption rates. Nest development, on the other hand, was significantly affected by the presence of yeasts, but effects largely depended on species identity, with Candida bombiphila, Metschnikowia gruessii, and Rhodotorula mucilaginosa having the largest positive impact on colony growth. Interestingly, the effects at the colony level were more pronounced than at the individual level. In vitro growth tests further showed that yeasts have the potential to suppress the growth of the bumble bee gut pathogen Crithidia bombi. Overall, these results demonstrate that nectar‐inhabiting yeasts can have diverse effects on bumble bee fitness and therefore may mediate plant–pollinator mutualisms.

The impact of yeast presence in nectar on bumble bee behavior and fitness

AbstractThe presence of yeasts in pollen and floral nectar is rather the norm than the exception. Due to the metabolic activities of yeasts, sugar and amino acid composition of nectar often drastically change and may negatively impact the nutritional value of nectar for pollinators and hence insect fitness. On the other hand, the presence of yeasts in floral nectar may also increase its nutritional value due to yeast's probiotic effect and the release of yeast's metabolites. In this study, we investigated whether the presence of defined flower‐ and insect‐associated yeasts affected individual and colony fitness of the bumble bee pollinatorBombus terrestris. Specifically, we tested whether the presence of yeasts in nectar affected bumble bee foraging behavior and nectar consumption, individual growth and colony development, larval and queen mortality, and mating success. Quantitative analyses of sugar and amino acid profiles showed that nectar yeasts significantly affected the chemical composition of nectar. However, dual‐choice experiments indicated that yeast inoculation did not significantly affect foraging behavior or consumption rates. Nest development, on the other hand, was significantly affected by the presence of yeasts, but effects largely depended on species identity, withCandida bombiphila,Metschnikowia gruessii, andRhodotorula mucilaginosahaving the largest positive impact on colony growth. Interestingly, the effects at the colony level were more pronounced than at the individual level. In vitro growth tests further showed that yeasts have the potential to suppress the growth of the bumble bee gut pathogenCrithidia bombi. Overall, these results demonstrate that nectar‐inhabiting yeasts can have diverse effects on bumble bee fitness and therefore may mediate plant–pollinator mutualisms.

Draft Genome Sequence of Novel Metschnikowia sp. Strain JCM 33374, a Nectar Yeast Isolated from a Bumblebee.

Here, we report the draft genome sequence of Metschnikowia sp. strain JCM 33374, a nectar yeast isolated from a bumblebee (Bombus diversus). The genome of 20.1 Mb is a naturally heterozygous diploid. Phylogenetic analysis with related taxa demonstrated that the strain is very likely a novel species.

Addition of pollen increases growth of nectar-living yeasts.

Nectar is frequently inhabited by a limited number of microorganisms. Nonetheless, these species can quickly attain relatively high cell densities. This is quite surprising because of the limited availability of nutrients and unbalanced Carbon/Nitrogen ratios. Because nectar yeasts commonly aggregate around pollen and pollen grains are particularly rich in proteins, it has been suggested that the presence of pollen in nectar contributes to enhanced growth of yeasts in nectar, but compelling experimental evidence is still lacking. In this study, we conducted in vitro growth experiments to investigate whether the addition of pollen to sugar water increased growth of yeasts that naturally occur in nectar and honey provisions: Metschnikowia reukaufii, Starmerella orientalis and Torulaspora delbueckii. Our results indicate that yeasts benefit from the addition of pollen to a sugar-dominated medium, but the effects depended on type of pollen used. Overall, these results demonstrate that pollen plays an important role in the population dynamics of nectar-inhabiting yeasts and supports the idea that the chemical composition and the concentration of dehisced pollen may be more important factors determining the population growth of nectar yeasts than the chemistry of the nectar itself.

Corrigendum to: Nectar yeasts of two southern Spanish plants: the roles of immigration and physiological traits in community assembly.

Corrigendum to: Nectar yeasts of two southern Spanish plants: the roles of immigration and physiological traits in community assembly.

Associative learning and memory retention of nectar yeast volatiles in a generalist parasitoid

Understanding how animals learn is crucial to interpreting animal behaviour. Flower-visiting insects, such as bees and parasitoids, are excellent animal models to study visual and olfactory learning, including memory phenomena. The diversity of resources flower-visiting insects exploit predisposes them to learn and remember the colours, shapes and odours associated with rewarding experiences (e.g. flowers), allowing them to focus on the most rewarding resources. Recent research has shown that nectar-living microbes release volatile organic compounds (VOCs) that contribute to overall flower scent. Nevertheless, little is known about the extent to which nectar microbiota mediate insect learning of floral preferences. In this study, we investigated whether VOCs produced by nectar microbes serve as a learning cue to parasitoids and how long any developed preference is maintained. Experiments were performed using the generalist aphid parasitoid Aphidius ervi and three nectar yeasts, including the nectar specialist Metschnikowia reukaufii and the generalist species Hanseniaspora uvarum and Sporobolomyces roseus. Results showed that naive parasitoids had an innate preference for nectar fermented by the nectar specialist M. reukaufii, but not by the other two yeasts which had either a neutral (H. uvarum) or deterrent (S. roseus) effect. When parasitoids were conditioned with yeast-fermented nectar, they were strongly attracted to their odours 2 and 24 h after conditioning, but not after 48 h. Furthermore, when parasitoids were conditioned to one yeast-fermented nectar, they also showed increased attraction to other yeast-fermented nectars. This generalization suggests that their learning ability may have broader ecological consequences. However, this generalized response to other yeast VOCs lasted for only 2 h. We conclude that parasitoids show conditioned responses to the scent of yeast-fermented nectar, and yeasts, therefore, may play an important but understudied role in shaping their foraging behaviour.

Nectar yeasts enhance the interaction between Clematis akebioides and its bumblebee pollinator.

It has been hypothesised that intense metabolism of nectar-inhabiting yeasts (NIY) may change nectar chemistry, including volatile profile, which may affect pollinator foraging behaviours and consequently plant fitness. However, empirical evidence for the plant-microbe-pollinator interactions remains little known. To test this hypothesis, we use a bumblebee-pollinated vine Clematis akebioides endemic to southwest China as an experimental model plant. To quantify the incidence and density of Metschnikowia reukaufii, a cosmopolitan NIY in floral nectar, a combination of yeast cultivation and microscopic cell-counting method was used. To examine the effects of NIY on plant-pollinator interactions, we used real flowers filled with artificial nectar with or without yeast cells. Then the volatile metabolites produced in the yeast-inoculated nectar were analysed with coupled gas chromatography and mass spectrometry (GC-MS). On average 79.3% of the C.akebioides flowers harboured M.reukaufii, and cell density of NIY was high to 7.4×104 cells mm-3 . In the field population, the presence of NIY in flowers of C.akebioides increased bumblebee (Bombus friseanus) pollinator visitation rate and consequently seed set per flower. A variety of fatty acid derivatives produced by M.reukaufii may be responsible for the above beneficial interactions. The volatiles produced by the metabolism of M.reukaufii may serve as an honest signal to attract bumblebee pollinators and indirectly promote the female reproductive fitness of C.akebioides, forming a potentially tripartite plant-microbe-pollinator mutualism.