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Microbes in a soil sample

This article originally appeared in the July 2017 edition of Grapegrower & Winemaker.

 

Science has always gone hand-in-hand with nature when it comes to making wine, and Australian wine has long been at the forefront of developments in wine science. In this article, prepared for Wine Australia, Dr Jamie Goode – British wine writer and plant biology PhD, looks at whether microflora can help shape a vineyard’s terroir.


There’s an unseen world all around us. The world of microbes. Bacteria and fungi are everywhere: even inside us and on our skin. In our guts alone it’s estimated that we harbour around 10 to the power of 14 bacteria (as a guide, a million is 106, and a billion is 109, so this is a lot), which means we have about 10 times as many bacterial cells in our bodies as we do of our own. Altogether, there are around 1,100 species of bacteria living in us and they play a vital role, for which we reward them by giving them somewhere to grow. Many of these are termed ‘commensal’ (they just live alongside us without harming or helping), but some are actually mutualistic, doing things to help us.

Now, with the emergence of next generation DNA sequencers, this unseen world is becoming a little clearer. And, with regard to wine, the picture that is developing is that the microbes present in the vineyard, both in the soil and on the aerial parts of the grapevine, are playing a much more important part in wine character and quality than previously thought. The Australian Wine Research Institute (AWRI) is in the middle of a four-year project funded by Wine Australia examining the population of yeasts in wine and how it might be at least in part responsible for regional differences in wines.

No microbes, no wine

But first, some background… Wine is a microbial product, and since the middle of the 19th century we’ve known that it’s yeasts that are responsible for the conversion of sugar to alcohol which is the fundamental act in the creation of wine. And the role of bacteria, both as spoilage organisms and also in carrying out malolactic fermentation, has been appreciated since the turn of the 20th century. But it’s only with modern sequencing technology that the picture has become clearer.

“It’s something we’ve not been able to get a handle on until fairly recently,” said Dr Paul Chambers, AWRI research manager, who is an expert on yeasts and bacteria.

“Traditional methods of sampling microbial environments typically require taking a sample of the environment of interest.

Now there is a new approach that tells us what is actually present. It is called metagenomics, and it involves going into mixed communities of microbes and sampling them to see what is there.

 

“You filter out all the lumpy bits leaving the microbial life forms behind, and then you culture.”

This involves a process called ‘plating’ onto selected culture media, and then seeing what grows. The single colonies that result are all the same species, and you can then identify them using microscopy, biochemical tests, or more recently DNA technology.

“But the culturing step where we plate things out is incredibly limiting,” Chambers said. “Most microorganisms will not grow on the media that we have in the laboratory. So we miss out by a factor of 10, or 100 or even 1000 on what is there.”

Now there is a new approach that tells us what is actually present. It is called metagenomics, and it involves going into mixed communities of microbes and sampling them to see what is there. The first step is the same as before: sampling the environment of interest and getting rid of the lumpy bits. But then, instead of culturing – the bottle neck step – you just extract DNA from the mixed community.

“This DNA is prepared for sequencing and put through a sequencing machine,” Chambers said. “These are phenomenal these days.”

DNA Sequencing: the next generation

Indeed, one of the things that makes these new studies possible is the fact that the cost of DNA sequencing has fallen dramatically with what is called ‘next generation’ DNA sequencing. When I visited the AWRI recently, one of Chambers’ colleagues, Dr Anthony Borneman, showed me a DNA sequencer called MinIon, which is made by a UK-based company called Oxford Nanopore. It’s the size of bar of chocolate, can fit in your pocket, and plugs directly into the USB port of a computer. With this technology, you could sequence in real-time around a winery, for example.

DNA Sequencing tools assist with microflora research

“It is then left to computing,” Chambers said. “Bioinformatics is used to interpret the massive data sets from these experiments, and tell us what strains and species of bacteria and fungi are present. The important thing is that there’s no requirement for plating or culturing so we don’t have to know how to grow these things: we isolate them from their environment and sequence them.”

Now, several laboratories around the world are using metagenomic approaches to understand the microbial diversity of vineyards and wineries. Dr Borneman and his colleagues at the AWRI recently began looking at whether there are regional microbes in Australian vineyards that might be capable of producing regionally distinct wine styles with funding from Wine Australia, and in the 2016 vintage they looked at 100 ferments from 30 wineries across the country.

The main finding was that there was there was a defined biogeography of these different musts

 

Nick Bokulich, a PhD student in the lab of David Mills at the University of California, Davis, has some interesting results. He looked at eight vineyards across four regions in California, studying 273 separate musts, with Chardonnay, Zinfandel and Cabernet Sauvignon the varieties involved. The main finding was that there was there was a defined biogeography of these different musts: the microbes present in the fermentation and also the metabolites produced singled out specific regions, and even single vineyards. There were also different microbes associated with the three different varieties. Although there were some differences with vintages, there was a core fingerprint each time.

Similar studies have been carried out elsewhere. Cátia Pinto, Diogo Pinho and colleagues from Portugal have looked at fermentation microbiome from six Portuguese regions and have also demonstrated that the initial musts show biogeographic differences from one region to another. In New Zealand, Matthew Goddard has been studying the yeasts responsible for un-inoculated fermentations across several New Zealand wine regions, and has shown evidence suggesting that each vineyard has its native populations that are then responsible for carrying out the fermentation.

Microbes: there’s a world underground

But it’s not just about fermentations. The microbes present in the soil and on the vines have an important effect on the way that the vines grow, and this is something that is only just becoming apparent. They transform the availability of organic matter and nutrients, this shaping the soil quality. They can help mitigate the effect of environmental stresses, and can compete with pathogens for nutrient and space. And plants and microbes ‘talk’ to each other through chemical signals, for example in inducing plant defence.

A recent study using the ‘model’ plant Arabidopsis thaliana looked at the effects of different experimental microbiomes, and found that they had significant effects on plant growth and the chemicals produced on the leaves. They also found out that the microbiomes altered the feeding behaviour of a species of larva.

“If regionality of microbiota is confirmed – if we do find that there is a genuine biogeography with the microflora – then we have to ask whether it is a cause or effect.

 

“Does microbial diversity contribute to terroir? We don’t know yet, but the data we have are really tantalising,” Chambers said. “It looks like there is biogeography of microbial composition of vineyards, and that this is carrying over into spontaneous fermentations, and is shaping these fermentations.

“If regionality of microbiota is confirmed – if we do find that there is a genuine biogeography with the microflora – then we have to ask whether it is a cause or effect. Is the microflora bringing in aspects of the terroir, or is the terroir shaping the microflora? More likely is that they’ll influence each other.”

With all the studies underway around the wine world, it’s likely we’ll have answers to these questions sooner rather than later. And we may well have to alter our understanding of the mechanisms underlying terroir.

Disclaimer

This information is presented in good faith and on the basis that Wine Australia, nor their agents or employees, are liable (whether by reason of error, omission, negligence, lack of care or otherwise) to any person for any damage or loss whatsoever which has occurred or may occur in relation to that person taking or not taking (as the case may be) action in respect of any statement, information or advice given.

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