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Monday, April 16, 2012

Black mark for cave biocides: Identifying cave fungi with denaturing gradient gel electrophoresis

Painting of an auroch (ancestor of
domestic cattle) in Lascaux cave.
Black stains
The use of biocides to control a fungal outbreak in the Lascaux cave in France, home to some of the best known Paleolithic cave art, probably just made the problem worse, conclude European scientists. Rather than controlling the outbreak, the biocide treatment merely encouraged the growth of fungi that are now covering the cave in black stains, they say.

Discovered by a group of teenagers in 1940, the Lascaux caves contain hundreds of paintings, comprising a mix of abstract symbols, human figures and animals such as horses, cattle, birds and felines, estimated to be over 17,000 years old. The cave soon became a popular tourist attraction, but this changed the microclimate in the cave and caused green algae to start growing on the walls. As a consequence, the cave was closed to the public in 1963.

Nevertheless, the cave was still open to scientific visitors and in 2001 it experienced an outbreak of the fungi Fusarium solani, which was controlled with a benzalkonium chloride (BC) biocide. Soon afterwards, black stains started to appear in the caves. These stains have since spread over many of the ceilings and walls, and now present the main threat to the cave's paintings, as biocides have failed to control them.

Finding the culprits
So scientists are now hard at work trying to determine exactly what fungal species are responsible for the black stains. A recent study identified two new species of the fungal genus Ochroconis in the stains, but now a team of scientists led by Cesareo Saiz-Jimenez at the Spanish Institute for Natural Resources and Agrobiology in Seville show that this is probably just the tip of the iceberg.


Saiz-Jimenez and his team used a combination of culturing and denaturing gradient gel electrophoresis (DGGE) to analyse the black stains and to try to identify the component fungal species from their DNA and RNA. In DGGE, strands of DNA or RNA travel through a column filled with a gel containing a steadily increasing concentration of some denaturing agent (such as formamide and urea), which causes the DNA or RNA strands to break down. The rate at which a DNA or RNA strand breaks down depends on its precise base sequence, while its speed through the column depends on how extensively it is broken down. This means that the length of time a DNA or RNA strand travels through the column provides information about its sequence, which can in turn be used to identify the fungal species it comes from.

Encouraging diversity
Saiz-Jimenez and his team began their study in 2008 by analyzing black stains both before and after they were treated with BC. This revealed that while the two Ochroconis species were indeed the main fungi in the black stains before treatment, after treatment the black stains contained a much wider range of fungal species. It appears that the BC treatment was reducing the Ochroconispopulations, although by no means completely eradicating them, but this simply caused them to be replaced by fungal species from genera such as Aspergillus and Trichoderma.

What is more, the scientists also found that the fungal species making up the black stains are changing over time. In 2008, the two Ochroconis species were the dominant fungi in the stains. By 2010, several species belonging to the Herpotrichiellacaeae family, commonly known as black yeast, had become dominant. Not only are these species resistant to the biocide, but they actually appear to be feeding on it, using the biocide as a source of carbon and nitrogen.

'What my study demonstrated was the uselessness of BC applications and how this treatment increased the fungal diversity in the treated stains, showing clear adverse effects,' Saiz-Jimenez toldseparationsNOW. This is an important finding, as fungal outbreaks are actually quite common in show caves and need to be controlled, but just not with biocides.

'In other caves we have proposed preventive conservation by controlling the microclimate and other environmental parameters and by modifying the cave management, because the use of biocides is not appropriate in a subterranean environment,' says Saiz-Jimenez.