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Alcoholic nectar and fermenting yeasts: Grevillea 'Robyn Gordon' and Eucalyptus Sideroxylon.

Introduction

Nectar composition varies considerably between species. This may be with respect to sugar composition such as the proportions of xylose, sucrose, fructose and glucose (Ecroyd et al. 1995), sugar concentration (Chalcoff et al. 2006) and viscosity (Chalcoff et. al. 2006) as well as other constituents that are present, such as amino acid content. Not all compounds present in nectar are harmless and nectar toxicity is well known. For example, several heavy metals such as copper, zinc and arsenic have been found in nectar and are believed to be transported from roots to flowers by translocation (Ernst and Bast-Cammer 1980). Toxins also can accumulate due to deposition of airborne particles (Ernst and Bast-Cammer 1980). Other chemicals are believed to be synthesised from nectar constituents inside the flower and comprise an array of alcohols, including several benzyl alcohols and ethyl alcohol (ethanol) (Ernst and Bast-Cammer 1980).

Ethanol has been recorded in floral nectar within a variety of studies, e.g. Birtchnell and Gibson (2008); Herzberg (2004); Jackson and Nicolson (2001). It is hypothesised that yeasts colonise the floral cup and ferment the sugars in the nectar to produce the ethanol (Birtchnell and Gibson 2008). Boutroux (1884) produced the first paper on the occurrence of yeasts in nectar and hypothesised that it was not coincidence that they occurred frequently; he concluded that pollinators were potential vectors in the distribution of yeasts in flowers.

Nectar chemistry and plant distribution can influence whether or not yeasts occur in nectar and which species of yeast occur. Several plant species have active resistance to yeasts because of their production of compounds such as benzyl alcohol, cinnamyl alcohol and phenethyl alcohol, known for their anti-fungal properties (Lawton et al. 1993). Ascomycetous yeasts frequently occur in flowers with sucrose-dominant nectars but rarely occur in flowers with nectar high in monosaccharides (Mittlebach et al. 2015); basidiomycetous yeasts colonise flowers with hexose-dominated nectar (Mittlebach et al. 2015). It must be remembered, however, that not all species of yeast found in nectar are capable of fermentation but the number that do occur provide considerable potential for fermentation of nectar of many species (Fleet 2001). For example, Candida tolerans occurs abundantly in the nectar of Hibiscus spp. and is capable of fermentation (Lachance et al. 1999). Melliferous (honey producing) flora can host several different species of yeast, e.g. Candida spp., in ethanol laden nectar (Birtchnell and Gibson 2008). Jackson and Nicolson (2001) have shown that xylose, a common nectar sugar, is metabolised to produce ethanol in the Proteaceae.

The observed physiological effects of alcohol on pollinators can be cause for concern. Birtchnell et al. (2005) reported hundreds of bees littering the ground after feeding on fermented nectar, the bees showing preference for plants containing the fermented nectar. This also is known for other members of the Hymenoptera, including local species such as native Flower Wasps (Tiphiidae) and European Wasps Vespula germanica (Hassan 1992; Birtchnell et al. 2005). Intoxication of avian pollinators also is widely known.

Ethanol in nectar may have a number of ecological and economic ramifications. Honey production is a multimillion dollar industry for Australia, and potentially could be threatened by this phenomenon through ethanol-induced bee mortality. Fermentation of nectar in the wild appears to be more common in recent years (Birtchnell and Gibson 2008) so it is important to know which plant species produce alcoholic nectar and why. The research presented here is a very small and opportunistic study but contributes to the knowledge gap concerning the occurrence of fermenting yeasts and alcohol content of two common Australian plants. The project aimed to determine:

* whether ethanol occurred in the nectar of the two plants; and

* whether yeasts capable of fermentation occurred in the nectar.

Methods

The study focused on two plant species commonly planted in the Victorian urban environment, thus easily accessible, and well known for their nectar production: Grevillea 'Robyn Gordon' and Eucalyptus sideroxylon. Both species secrete high volumes of nectar ensuring a plentiful and, therefore, comparatively easy-to-obtain supply.

Nectar was obtained from 30 plants of G. 'Robyn Gordon' and 10 plants of E. sideroxylon. Selection of sample sites was arbitrary: there was an abundance of the target species in the areas chosen. Ten plants of G. 'Robyn Gordon' were sampled at each of:

1. The gardens within Deakin University Burwood;

2. The area defined by the junction of The Boulevard and George Street to Ovens Road and Victoria Street in Doncaster, Victoria; and

3. Along the median strip on Dorset Road near the corner of Mt Dandenong Road in Croydon, Victoria.

Five plants of E. sideroxylon were sampled at each of:

1. Grounds surrounding the Montmorency Football Club, Montmorency, Victoria; and

2. The Eastern Golf Club, Doncaster, Victoria.

Nectar Collection

Nectar was collected using a modified glass pipette; the tip was heated over a Bunsen burner and pulled to a fine tip. It also was bent to allow easier access to the nectar in the flower. Pipettes were sterilised in sterilisation envelopes using an autoclave. Envelopes were opened immediately prior to collection to minimise the chance of contamination.

Nectar was procured by inserting the sterile pipette into the flower and then sucking the nectar into the pipette using a pipette filler. The nectar was then placed into a sterilised 1.7 mL Eppendorf tube. This was wrapped in Parafilm and placed inside a jar and immediately stored on ice inside a cool box to avoid the potential for fermentation to occur after the sample had been procured. The samples then were refrigerated at 4[degrees]C until ethanol and microbial analyses were undertaken, usually the next day.

Grevillea flowers occur in racemes and there are around 40 individual flowers on each raceme. Around 10 of these racemes were sampled on each plant to obtain an adequate amount of nectar from each plant. Racemes were located on separate branches of the plant. Despite sampling 10 racemes, 40 flowers were not necessarily sampled from each raceme; sampling was terminated when an adequate volume of nectar was collected, thus the number of flowers used varied from shrub to shrub.

The Eucalyptus nectar was extremely difficult to obtain. Attempts to collect nectar were unsuccessful on several trips, and it was found that the time of day was crucial for success. Subsequently, samples were collected between 0400 and 1200 hours, as this was found to be the time when nectar was most abundant. Between 100 and 200 flowers were sampled per tree to procure sufficient nectar for the analyses.

After collection, the analysis was broken down into two components: microbial analysis and ethanol analysis, which were performed for each nectar sample. This allowed direct comparison of the relationship between presence of yeasts and ethanol.

Microbial Analysis

For each nectar sample (thus each plant), a loop was inoculated with nectar using appropriate sterile technique and streaked onto an agar plate. The medium on the plate was Sabouraud's Dextrose Agar (Oxoid CM41) as this medium is designed for culturing Ascomycota (Sac fungi) and Basidiomycota (Club fungi), thus ideal for culturing yeasts.

Plates were sealed with Parafilm and incubated for 14 days at 18[degrees]C inside a culture cabinet. Colonies were then sub-cultured, purified and sent to the Mycology Laboratory, Pathology North, Royal Northshore Hospital (PaLMS), NSW, for identification, where possible (only one per cent of yeast taxa has been identified [Barnett et al. 2000]). The remaining nectar was then used for ethanol analysis.

Ethanol Analysis

The presence of ethanol was examined using gas chromatographic mass spectroscopy. Due to the small amount of material available for analysis, injection by auto loader was not feasible, thus samples were manually injected (0.5 [??]L) into an Agilent Gas Chromatograph (Agilent Technologies 6890N Network GC system; column; Agilent 19091J-413 HP-5 5% phenyl methyl siloxane capillary, 30 [micro]m x 320 [micro]m x 0.25 [micro]m).

A calibration curve was produced using solutions of ethanol of known concentrations. The chromatograph gives retention times (the time which the vaporised solution takes to move through the column when pushed by energy generated from an internal oven) of volatile substances in the solution. Having the retention times of the known solutions gives a baseline for comparison with unknown solutions of nectar and thus the ethanol concentrations (%v/v) of the nectar samples can be determined.

Results

Microbial Analysis

Yeasts occurred in G. 'Robyn Gordon' at all sites examined, although not all shrubs within a site had yeasts present in the nectar. No yeasts grew in cultures of E. sideroxylon nectar. Only three identifiable yeasts were found for G. 'Robyn Gordon' (Table 1), two were Cryptococcus species and one was a Candida (Table 1). Unidentified yeasts were most frequent and generally are referred to as 'environmental' yeasts, probably because most taxonomically known yeasts are linked to human conditions.

Ethanol Analysis

Ethanol was found in the nectar produced by both G. 'Robyn Gordon' and E. sideroxylon but the number of trees for which this occurred varied from site to site (Figs. 1 and 2). High variability of ethanol concentration occurred between sites (Figs. 3 and 4) although this was not statistically significant (G. 'Robyn Gordon': F = 1.03; df = 9; p = 0.44. E. sideroxylon: t = -1.4; p = 0.09).

Discussion

Only one of the three yeasts identified in this study, Candida globosa, is known to ferment sugars found in nectar. Indeed, it can ferment all nectar sugars with the exception of xylose (Barnett et al. 2000). This yeast occurred in a nectar sample containing alcohol, thus could have been responsible for fermentation of the nectar. Candida globosa also has been found in fermenting sugar cane in Spain and in fermenting fruiting bodies in the UK. This study has presented the first record of its presence in nectar of Australian flora.

Cryptococcus laurentii is a non-fermenting yeast and has been found on species of the Proteaceae and Myrtaceae in Australia, suggesting a common affinity with taxa of the same lineage (Barnett et al. 2000). It also is known to have a pathogenic relationship with humans, colonising the bronchi of immune suppressed patients (Barnett et al. 2000). Cryptococcus albidus was another non-fermenting yeast. Most yeasts found in this study were not able to be identified and were broadly categorised as environmental yeasts. This came as no surprise as less than 1% of the yeast taxa have been classified (Mrak and Praff 1948; Barnett et al. 2000). Therefore, it is hard to ascertain the role of environmental yeasts in nectar fermentation, although several of these were found in the presence of alcoholic nectar. Further microbial investigation would have allowed more conclusive results but this study was of an opportunistic nature and funding was very limited.

Flowers of everal trees contained ethanol but no yeasts were isolated. Yeasts are only one of various types of micororganisms that could be responsible for fermentation of nectar. Several anaerobic bacteria and lignocellulosic fungi also have the potential to ferment sugars occurring in nectar. Certain Eschericha and Salmonella species can ferment sugars and are widespread in the environment. They also have been found to occur in nectar and honey (Snowdon and Cliver 1995). A number of trees examined contained bacterial and fungal growths in the presence of alcoholic nectar but, as this study examined only yeasts, these growths were not identified.

Nectar composition differs from species to species and even within a species. This can be due to a number of factors including temperature, nutrient availability, water availability and target pollinator species (Perret et al. 2000; Baker and Baker 1983). It is hypothesised that the sugar composition of nectar may influence the yeast or other microorganism distribution, thus affecting fermentation potential (Nicolson and Van Wyk 1998; Freeman and Wilkin 1987). Future studies could examine sugar composition, identify other microorganisms present in nectar and, possibly, fermentation experiments under laboratory conditions to determine whether specific microorganisms could ferment sugar under different humidities and temperatures--the two environmental variables considered to trigger fermentation in floral cups (Birtchnell and Gibson 2008).

Acknowledgements

We would like to thank: Linda Moon, Heidi Rees, Maria Amodio and Timothy Sanders, all from Deakin University, for technical help; Peter Vlahandreas of the Eastern Golf Club and grounds staff at the Montmorency Football Club for allowing collection of nectar from plants on these premises. Melanie Birtchnell also is thanked for discussions of her work and for comments on the manuscript, as is Christine Tyshing.

References

Baker HG and Baker I (1983) Floral nectar sugar constituents in relation to pollinator type. In Handbook of Experimental Pollination Biology, pp. 117-141. Eds CE Jones and RJ Little. (Van Nostrand-Reinhold: New York)

Barnett JA, Payne RW and Yarrow D (2000) Yeasts: Characteristics and identification. 3rd edn. (Cambridge University Press: Cambridge)

Birtchnell M, Tyshing C and Gibson M (2005) Drunken Honey Bees. The Victorian Naturalist 122, 120.

Birtchnell M and Gibson M (2008) Flowering ecology of south-east Australian melliferous flora. Rural Industries Research and Development Corporation Report, RIRDC Project No. UD-3A, Canberra.

Boutroux L (1884) Sur la conservation des ferments alcooliques dans la nature. Annales des Sciences Naturelles Botanique 17, 145-209.

Chalcoff VR, Aizen MA and Galetto L (2006) Nectar concentration and composition of 26 species from the Temperate Forest of South America. Annals of Botany 97, 413-421.

Ecroyd CE, Franich RA, Kroese HW and Steward D (1995) Volatile constituents of Dactylanthus taylorii nectar in relation to flower pollination and browsing by animals. Phytochemistry 40, 1387-1389.

Ernst WHO and Bast-Cammer WB (1980) The effect of lead contamination of soils and air on its accumulation in pollen. Plant and Soil 57 (2-3), 491-496.

Fleet G (2001) Yeast interactions and wine flavour. International Journal of Food Microbiology 86, 11-22.

Freeman E and Wilken DH (1987) Variation in nectar sugar composition at the intraplant level in Ipomopsis longiflora (Polemoniaceae). American Journal of Botany 74, 1681-1689.

Hassan E (1992) Driven to drink: a sorry tale of bees' boozy life. New Scientist. 1992, 14.

Herzberg M (2004) Ecology of yeasts in plant-bumblebee mutualism in Central Europe. Microbiology Ecology 50, 87-100.

Jackson S and Nicolson SW (2001) Xylose as a nectar sugar: from biochemistry to ecology. Comparative Biochemistry and Physiology 131, 613-620.

Lachance MA, Bowles JM, Starmer WT and Barker JSF (1999) Kodamaea kakaduensis and Candida tolerans, two new ascomycetous yeast species from Australian Hibiscus flowers. Canadian Journal of Microbiology 45, 172-177.

Lawton RO, Alexander L, Setzer WN and Byler KG (1993) Floral essential oil of Guettarda poasana inhibits yeast growth. The Association for Tropical Biology and Conservation 25, 483-486.

Mittlebach M, Yurko AM, Nocentini D, Nepi M, Weigend M and Begerow D (2015) Nectar sugars and bird visitation define a floral niche for basidiomycetous yeast on the Canary Islands. BMC Ecology 15, 2. doi:10.1186/s12898015-0036-x.

Mrak EM and Phaff HJ (1948) Yeasts. Annual Review of Microbiology 2, 1-46.

Nicolson SW and Van Wyk B-E (1998) Nectar sugars in Proteaceae: patterns and processes. Australian Journal of Botany 46, 489-504.

Perret M, Chautems A, Spichiger R, Piexoto M and Savolainen V (2001) Nectar sugar composition in relation to pollination syndromes in Sinningieae (Gesneriaceae). Annals of Botany 87, 267-273.

Snowdon JA and Cliver DO (1995) Microorganisms in honey. International Journal of Food Microbiology 31, 1-26.

Received 6 August 2015; accepted 24 September 2015

Nicholas Evans (1) and Maria Gibson (1,2)

(1) School of Life and Environmental Sciences, Deakin University, 221 Burwood Highway, Burwood, Victoria 3125

(2) Contact author

Table 1. Yeasts of Grevillea 'Robyn Gordon'.

Yeast species                               Number of
                                      plants in which
                                      yeasts occurred
                                               (n=30)

Unidentified (environmental) yeasts                17
Cryptococcus laurentii                              4
Cryptococcus albidus                                3
Candida globosa                                     1
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Title Annotation:Research Reports
Author:Evans, Nicholas; Gibson, Maria
Publication:The Victorian Naturalist
Article Type:Report
Date:Oct 1, 2015
Words:2581
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