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The mystery of the sexual jungle fungus.

The Mystery of the Sexual Jungle Fungus

He trudges through the tropics of South America and brings back fungi. Lots of fungi. More than a thousand specimens from a month spent in French Guiana. And then there was that other trip - yes, the one where he brought back the probable solution to the Trichoderma fungus mystery.

It's been a good trip if Gary J. Samuels returns from South America with a suitcase full of fungi that are new to science or that have potential uses in biological control. These beneficial fungi may naturally kill those fungi that cause plant diseases.

"Besides being biological controls, fungi have a very important place in the ecosystem," he says. "They degrade organic matter, serving as nature's recyclers to provide nutrients from dead organisms for use by living plants. Without them we'd be waist deep in dead wood, manure, and other detritus."

Samuels, a mycologist (fungus scientist) with the Agricultural Research Service, brings back both living and dried specimens of fungi to the Systematic Botany and Mycology Laboratory in Beltsville, Maryland. There he catalogs them.

To be able to catalog the specimens, he has to identify them by species. And in order to properly identify fungi, he has to determine how alike or different they are from one another.

And herein lies the mystery of the Trichoderma fungus.

Most fungi reproduce asexually, meaning the spores they release from the fruiting body (the part we see) are all alike. Since each generation is like its parent, taxonomists - scientists who specialize in identifying organisms - have used their morphology (what they look like) to tell these fruiting bodies apart.

But in addition to the asexual bodies, Trichoderma also have a form that produces microscopic sexual spores with genes that have recombined. So different are the two forms that 100 years ago, when the fungus was first found, the two forms of spores - sexual and asexual - were classified as belonging to different species of fungi.

Fungi such as Trichoderma are dear to agricultural scientists because they can help control diseases that are caused by other fungi. For example, when painted onto the pruned areas of fruit trees, one species can control the development of the fungus that causes silver leaf disease of stone fruit.

Samuels identifies Trichoderma by its morphology, providing other scientists with needed information to study its values as a biological control. These researchers in turn will study the chemicals these fungi produce such as enzymes, toxins, or antibiotics. They may even distribute the fungal spores in soil, in very much the same way fungicide might be distributed, and see how well they control pathogenic fungi.

In the soil, beneficial fungi work in one of three ways: They are parasites and kill the pathogenic fungi outright, they produce chemicals that are toxic to the pathogens and so protect plant seeds, or their physical presence prevents the development of harmful fungi by simply using up all of the nutrients.

From three trips to South America, Samuels brought back the sexual stage of a species of tropical Trichoderma. But when he grew out the spores in the laboratory, they produced the asexual stage of the fungus. Since these cultures have recombined genes, they should have had different characteristics.

But could they be completely different species?

Collections made throughout the world show there is only one species of Trichoderma - when it is defined by the morphology of the sexual stage. But the asexual stage that came from the laboratory-grown sexual stage shows great morphological variation.

What kind of differences does it to make up a separate species, anyway? The answer is not as cut and dried as one might expect.

"The questions are: Which morphological characteristics are reliable? Which should we base our species on, especially if we only have one stage, usually only the asexual Trichoderma?" asks Samuels.

Samuels and colleagues are taking a unique approach - using biotechnology to look inside these fungi - to sexual Trichoderma really should be depicted as the same species.

Robert J. Meyer, a research associate also with the laboratory, is checking the fungus' DNA to identify patterns that could serve as a "fingerprint" for each species of Trichoderma.

He has been examining a part of the total DNA, called ribosomal DNA (rDNA), that carries instructions for part of the machinery that makes the fungus' proteins. Meyer treats the rDNA with enzymes and then examines it on agarose gels.

The rDNA produces characteristic patterns on the gels that vary according to the strain of fungus the DNA came from. Using these patterns, a computer program determines the similarity of the strains and diagrams them in a "tree" by how closely they are related.

"The rDNA separates strains that are different morphologically," says Meyer. "But these rDNA characteristics are more sensitive indicators than the morphological ones because the rDNA patterns also group the strains at the sub-species level.

"The DNA studies have done what could not be done by other methods: Link strains for which the sexual stage was unknown to strains that were derived from the sexual stage," he adds. "This is an important step in clarifying the taxonomy of Trichoderma. Ultimately, the DNA patterns could be used to identify a strain regardless of whether it came from the sexual or asexual stage."

A visiting entomologist from France to Beltsville's Beneficial Insects Laboratory has also been drawn into the problem. Silvie Manguin is checking out the enzymes the fungus produces; she's found that some of these enzymes can inhibit the development of other fungi - possibly making Trichoderma useful as a biocontrol against harmful fungi.

Using starch gel electrophoresis, she is also determining the genetic variation among the Trichoderma species. These variations are expressed by differences in the amino acids that make up each of the 24 enzymes she tested.

"Related species of fungi can have slight differences in the sequences of their amino acids," says Manguin. "From these differences, I am creating a cladistic tree - a schematic that tells how closely related they are.

"My results, so far, have been similar to Meyer's," she adds. "Samuels found that morphologically these were closely related, and the DNA and enzyme studies are helping us clarify how close."

"The more characteristics we have to identify the Trichoderma fungi, the easier it is," says Samuels. "Because we have three detectives on the trail, we've got a whole other suite of characteristics that we can use for classification."

"This particular Trichoderma is known species," says Samuels. "Next we will look at a soil-dwelling species where we don't yet know about its sexual stage.

"Our goal is to see if DNA and enzyme studies are also good techniques to identify fungi," adds Samuels. "For example, is spore color really an important characteristic to identify a fungus? It may turn out that spores of each color produce the same enzymes, and therefore the color isn't a good way to identify fungi."

Isolates of the Trichoderma are kept at the laboratory in Beltsville for further studies. The rest are sent to the American Type Culture Collection in Rockville, Maryland. About 100,000 species of fungi have been described by taxonomists; an estimated 200,000 exist worldwide. - By Dvora Aksler Konstant, ARS.

Gary J. Samuels is at the USDA-ARS Systematic Botany and Mycology Laboratory, Bldg. 011A, Beltsville Agricultural Research Center, Beltsville, MD 20705 (301) 344-2279.

PHOTO : Following electrophoresis and staining, the number and locations of red-stained bands give an indication of the number of species of Trichoderma. (K-3811-2)

PHOTO : Entomologist Sylvie Manguin prepares enzyme extracts from Trichoderma in preparation for electrophoresis. (K-3812-6)

PHOTO : In the Herbarium of the National Fungus Collection, Gary Samuels studies specimens of fungi growing on sticks. (K-3810-8)
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Title Annotation:Trichoderma fungus reproduction research
Author:Konstant, Dvora Aksler
Publication:Agricultural Research
Date:Feb 1, 1991
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