The modern world has a multitude of issues it is facing, most of which filter through and incorporate agriculture in some fashion. Whether speaking of climate change, overpopulation, deforestation, or any number of other topics, the food supply and how we produce it is involved.

For a significant chunk of the countries in the world, access to more productive crops and protection from pests, stresses, and weather conditions is paramount to feeding not just the farmers themselves, but also their communities. The good thing is that these problems are being tackled and it is expected over the next two decades that world hunger will be practically eradicated.

As a part of these efforts, methods to control fungi, both positively and negatively, are of high importance. Fungi as a whole play an essential role in the web of life in basically every ecosystem in the world, where they act mutualistically or parasitically with neighboring plants. Nutrients are just as important for fungi as with all other life.

Some have evolved to work with, as one example, networks of trees where they trade soil minerals to them in order to receive necessary sugars from the photosynthetic capabilities of the trees. Others instead just infest or tap into plants in order to steal the nutrients they need.

We will be talking about both in this article, with a focus on how they interact with plants biologically and what biotech measures have recently been taken in regards to them. Before that though, let’s talk some history.

The History of Fungi Biotechnology

The Origins of the Origins

The use of fungi for human purposes is hardly a new phenomenon. In reality, its historical usage stretches back thousands of years. Food applications for fungi have existed, for example, with the cultivation of wine and the necessity of nurturing the fungi population in the soil in order to create the particular essence that high quality vineyards are uniquely famous for. Bread-making, as well, wouldn’t exist without the wide family of fungi known as yeasts.

Fungi have been used in fermentation processes around the world and as medicine, staking a claim in the cultural practices of many a society. In fact, there is an entire field of study dedicated to investigating the cultural history of fungi within folklore and elsewhere. This field is known as ethnomycology.

Indeed, even the origins of microscopy with the infamous Anton van Leeuwenhoek involved him using his crude device to observe yeast cells. Though he, at the time, thought that they were a part of a plant, assuming they came from the cereal grains used to make the beer he was observing.

Creating Pure Samples And Expanding

The other necessity beyond just being able to see fungal cells directly was being able to grow them in a lab. This entailed not just a growth mechanism, but the ability to make pure samples containing only the fungal cells of interest. Older methods of mushroom cultivation started hundreds of years ago and was considered a method of fine art in much of Europe at the time.

It wasn’t until 1890 that the work of Constantin and Matruchot were able to develop pure culture offspring of a fungus, with the scientist Duggar continuing the work in 1905 of succeeding at creating new growth from a tissue sample of an adult mushroom. This opened the doorway, admittedly at a much later date, to true fungal biotechnology. Without access to pure samples, the field would have never been possible.

Much of the later expansion of fungi farming coincided with scientific expansions in the ability to cultivate bacteria. This included the need to make antibiotics to help deal with dangerous infectious bacteria. That was where the Penicillin group of antibiotics come into play, derived from the fungal genus Penicillium.

Harvesting of Fungal Enzymes

Another important discovery happened in that fast-paced decade of the 1890’s. A German chemist named Eduard Buchner found in his experiments on yeast that it was possible to induce fermentation without the yeast having to be present at all, just their extracts.

In short, what he had discovered was fungal enzymes, the actual working proteins that conduct the chemical reactions for fermentation. He even won the Nobel Prize for this discovery, as monumental as it was.

Now, all those uses for fungi that were mentioned before could be done without necessarily needing any live fungus at all. This allowed for industrial up-sizing in the processes of making bread, cheese, sugar, starches, and so much more.

Though it would still take most of a century before another scientist in the 1960’s would discover how to immobilize enzymes to make them easier to collect for them to last in a more stable condition. This resulted in lowering production costs tremendously.

The Introduction of Biotech Techniques

All of this newly uncovered science would lead directly to biotech methods of improving fungi usage. One of the especially needed things in using fungi was creating better strains with more desired traits, much like in agriculture and growing crops.

Basic methods like artificial selection were also used in the growing of fungi, but the ability to induce development of these traits with chemical mutagenesis was quickly adopted in the field. The very different reproductive life cycle of fungi actually made them more open to modification and improvement at different stages of life.

No direct sexual reproduction also meant that there was less genetic variability to interfere with breeding techniques. Strains of Penicillium chrysogenum that originally produced only 100 units per milliliter of penicillin were morphed to being able to make 50,000 units in the same amount.

Protoplast fusion as a type of genetic modification also came into use. This involved taking a cell from each of the different species (fungi species in this case) and using enzymes, chemicals, and electricity to cause them to fuse together into a single cell, creating what is called a somatic hybrid.

These are then grown into a mass of cells called a callus and then into the final organism. This is usually done with plants, resulting in a young plantlet, but fungi can instead just grow into their usual masses of cells, hardly different from calluses in the end.

Molecular Biology and Genetic Transformations

The last step in the rise of fungal biotechnology has been the revolutions within molecular biology itself. The discovery of plasmids in the middle of the 20th century blew open the capabilities of yeast in particular. Being able to transform its genome with such a remote method enabled it to be used to grow essentially any molecule desired.

It was sometimes referred to as the “E. coli of the eukaryotes” afterwards due to its similar versatility. Methods to transform all the other fungal species in use had to be made as well over time. Some of the highlights from this include finding a number of new types of antibiotics, improving yields for fungal production of specific substances, and of course using yeast as a surrogate for the production of things much more complicated creatures originally had to be used for making.

And that is the history of fungal biotechnology in a nutshell. A bit broad in its description of events, but we have much more to cover and specific advancements in the past decade to discuss.

Beneficial Fungi And Their Hosts

Due to how fungi can be found around the world in essentially every type of soil out there, it is no surprise that there are very different kinds of fungi that one can find. Many simply act as decomposers within the food web, breaking down dead organic matter into constituent parts to be used as food for themselves and as fertilizer for all other plants.

Other fungi, however, are not content with this basic lifestyle. Some live their lives connected to other organisms, either in a mutualistic relationship that benefits both or in a parasitic one that leaches away the nutrients and sometimes life force of their host. For now, we’ll be focusing on the former.

Root Relationships

Though even this grouping isn’t as simple to pin down, since different parts of their life cycle are spent doing different things. The fungi that do spend some part of this cycle connected to plants for sustenance are called mycorrhizal fungi, after the fungal root connection they form. These types of fungi, even while being a smaller subset of fungi overall, are still numerous enough that they form interactions with over 90% of plant species in the world.

They are commonly involved in processes such as nutrient cycling between the soil and the plant roots, the collection of minerals for use by the plant, and assistance in the uptake of water. These fungal species even help protect their mutualistic host plants by helping drive off pathogenic bacteria and other disease causing microorganisms.

Due to all of this, if they were suddenly removed from the global equation, most of the plant life worldwide would likely die in a matter of days or weeks. The so-called “wood-wide web” is a requirement for plant life and connects even different species of plants together through sharing a fungal hyphae network within the soil.

Connecting With Cells

These mutualistic fungi are then further broken down into two groups called ectomycorrhizal and endomycorrhizal fungi. The former are fungi that build themselves around the tips of roots and within the intercellular spaces surrounding the plant cells. The latter actually enter the plant cells themselves and grow within them, allowing a more direct link for nutritional and liquid transfer in and out of the plant roots.

These two groups then break down into a wide number of other more specifically termed organizations related to exactly how they form their hyphae filament networks and the structures therein, but we won’t be covering that in this article. The basics are enough. There are pages and pages more that could be said on how these relationships form and function, but we shall now move onto specific biotech research done recently with beneficial fungi.

Recent Biotechnology Efforts For Beneficial Fungi Manipulation

The Field of Biocontrol

The simplest place to start is to mention the field of biocontrol. Written fully as biological pest control, it covers all the methods used to try and combat the pests that destroy crops and the biodiversity of ecosystems in general. With as broad of a definition as that, you can expect that it covers quite a few things and likely extends back hundreds of years. And you’d be right, even if scholarly reviews of it have only come about in recent memory.

For the topic of fungi, one of the earliest discovered fungal agents used in biocontrol was the genus of Trichoderma. This group of fungi were first scientifically described in 1794, but it wasn’t until the 1930’s that their abilities as a biocontrol agent came to the forefront. The interesting part of a fair number of fungal biocontrol agents is that they do not directly attack or harm their opposing fungus. Instead, they seek to compete for the same space and resources as them, while also not harming the host plant at the same time.

In short, they trade a parasitic relationship for a mutualistic one, forcing out the harmful fungal species. The Trichoderma genus is made up of species that span the various possibilities of biocontrol. Some do the space stealing method just described, others do that and also release enzymes that actively harm the other fungus, and others still actively parasitize the other fungus. The latter is referred to as mycoparasitism.

With the recent development of genetic mapping technologies, Trichoderma has become an even more important part of the biocontrol field, as scientists have broken down the genes and compounds that make up its activities. Gene editing options have allowed these researchers to amplify desired activities of the fungi in order to be more effective against whatever parasitic fungus it is being pitted against.

And a final way to sidestep even the need for Trichoderma at all has been the transgenic addition of these fungal genes into plant species, allowing them to actively produce agents that protect them from the parasitic fungi. In many cases, this ability may be preferable, though there is the possibility of it increasing stress on the plant due to the extra production it requires. These benefits and negatives have to be weighed when making any such agricultural biocontrol decision.

The Virulence of Mycoinsecticides

Beyond controlling for dangerous fungi themselves, beneficial fungi have also been used to target insects that prey upon crops. They act as natural mycoinsecticides, as the phrase has been termed, that do not harm or affect the plants at all. Instead, acting as dangerous ingested toxins for the specific species of insects they target.

A primary problem in the past for using these mycoinsecticides has been that they have a low virulence, only really killing the insects long after they have decimated a farmer’s crops. Improving their lethality toward insects has been a main focus of research. Transgenic crossing of genes between different fungi types has seen remarkable success in accomplishing this, creating a recombinatory genome that evolution likely would have eventually gotten to if there had been any selective pressure to protect agricultural crops. But, lacking that, we can do the work for nature ourselves.

Another tactic has been to add genes from the target insects into the fungi. This results in a sort of targeting system, when properly set up, that has the fungus attacking a specific regulatory system in the insects, whether their ability to digest food, their ability to maintain cellular osmotic pressure, or to just go directly after their neural system. Fungi in the wild on their own have gotten quite creative and we’ve picked up on some of their tricks.

A final application has been to use genes from organisms that are predators to the insects in question. This often means arthropods like spiders and scorpions. Moving venom producing genes into the fungi results in a tremendous boost to their virulence. Since, even though the amount of venom produced by the fungi is so small that anything larger than even a rather large insect would be unaffected, the very small size of most insects leaves them susceptible.

All of these showcase how beneficial fungi can be used against multiple kinds of pests, from other fungi to insects to even weeds if it came to it. Since many of these species of fungi naturally prey on such pests, they are already well suited to helping farmers protect their livelihoods. We just need to use a little science sometimes to enhance this effect to make them more effective at defeating the pests before they’ve harmed any crops.

Unfortunately, the parasitic fungi in particular often find ways to evade even these attempts to stop them. Agriculture is a continuing and ongoing war against pests. Below, we’ll discuss the fungal parasitism process and how scientists are working on other methods beyond beneficial fungi in order to fight back.

What Is Fungal Parasitism?

Much of what there is to say on the topic of parasitism works closely with the mutualistic relationships already described. But rather than a beneficial relationship that has the fungus working together with its host plant for nutrients, parasitic fungi invade and take the nutrients they desire. The first step to this is to directly invade the plant root cells and set up shop.

A Fungi Invasion

Fungi have several means to do so. The simplest is to just physically invade the cell walls by adhering to the outside of the roots and using a specialized cell called an appressorium. This is designed to apply more and more pressure against its outer cell wall in a pointed direction, causing it to puncture through the plant cell wall in turn. This pressure is so strong, it has been found in some fungal species to even be capable of puncturing strengthened mylar sheets.

Another way is to use previously mentioned chemical tactics, such as enzymes that break down opposing cell walls. This is an easy way for the fungi to just remove any strengthened root wall blocking its passage. Since the polymer cutin is often used to reinforce the outer cuticle of plants, especially on leaves and other such structures, some fungi have been found to secrete cutinases that break down this polymer and give access to the invader. The same can be done by applying cellulases to dissolve any cellulose within plant cells themselves, rapidly enhancing the degradation of their host plant, as is sometimes the goal of a fungal parasite.

Lastly, toxic compounds can be applied to infect and weaken a plant overall, letting the fungus just wait until the cells are shriveled and weakened enough to enter and consume the remaining nutrients. Fungal toxins are often host-specific, allowing a high pathogenicity, meaning the capability to cause disease, for that specific target species.

Other fungi use more broad toxins that don’t target a specific host. These are sometimes the most dangerous, because in order to be effective, the fungi must often make these general toxins extremely harmful for all plant organisms. This sometimes even means extreme harm for other organisms, such as humans, that accidentally ingest them.

Inside The Plants

Once inside and having overcome the plant’s immune system, a whole other topic that we won’t be covering here, the fungi are free to consume sugars and other nutrient products of the plant. Some do this slowly in order to keep their host alive and allow them to parasitize their host for years to come.

Others actively seek to take nutrients as quickly as possible and kill their host in the process. Fungi that prey on plant species that grow in large amounts in close proximity to each other often take this path, as there are plenty of other hosts to target afterwards.

All of this might sound quite scary and for farmers and those that work in agriculture, it is. A widespread outbreak of some plant pathogenic fungi can result in mass famines and the starvation of thousands. So ways to control for these fungi attacks is important, as has been already emphasized many times.

So, you’ve seen discussed the methods of fighting these fungi with other fungi. Now, let’s look at other ways to use modern biotechnology and genomics to learn about fungi and give plants the ability to protect themselves.

Biotech Techniques For Fighting Fungal Infections

The first step in many things in molecular biology, and biotechnology is no exception, is to run a genetic sequencing. In order to find ways to fight against an organism, it must first be understood genetically and have its composite genes deciphered. This allows for opposing forces to be identified.

But, to run such a sequence, a pure sample must first be obtained. This process was explained back in the history section, but it also allows for a special trick. When growing samples of a pathogenic fungus on a leaf disc culture, this can also be used to find other species of fungi, bacteria, or other microorganisms, that might prey upon the parasitic fungus.

Cultures and Transfers

Last year, researchers decided to try this approach with coffee rust (Hemileia vastatrix), a fungus that routinely destroys more than half of the coffee market worldwide. Or, at least it did in the past. What the scientists found in their cultures was a rather diverse community. For fungi alone, there were more than 60 different species growing in the same region.

By comparing the species growing on the control culture and the rust infected culture, they were able to find 15 mycoparasitic fungi that prey upon coffee rust. Such a simple method as comparing cultured leaf discs can be very powerful in finding tools to fight against parasitic fungi.

These tools are necessary since, much like bacteria, fungi are rampant in combining genes from other fungal species and beyond. Horizontal gene transfer between fungi and other organisms is fairly common, with any average genome likely having hundreds of such obtained genes. This variety gives the fungi versatility in their actions and makes them harder to combat, at least from the agricultural side of things.

Though this abundance of obtained genes also gives scientists an opening to attack. Finding out which genes are transgenically taken from others means that biotech has the capability to target those genes in particular. Since a number of them apply to fungal abilities to invade plants, animals, and others, they are a prime genome location to try and disrupt with any available technology.

On that note, let’s turn to our final topic, giving plants the ability to resist and fight back against fungi.

The Evolution of Non-Host Resistance

A remarkable innovation over the past several decades has been the advancement of knowledge on the topic of non-host resistance (NHR). Namely, the ability of plants to resist pathogens that are specifically evolved to target a particular species of plant that is not that plant in question. It is not the natural host for the pathogen and thus is resistant against it, hence the name.

NHR genes can take many forms. This include pre-invasion genes that stop the parasitic fungi from getting into the plant cells in the first place and post-invasion genes that have the plant immune system target and eradicate the invading fungus. The model organism Arabidopsis has many of these genes and continues to serve as a model for this as well in scientific testing.

All of this raises one direct question. Can these NHR genes be transgenically transferred to the actual host plant and give it the same NHR resistance? As you might guess, the answer is an emphatic yes. For the horrors of Asian soybean rust, transferring select Arabidopsis NHR genes into soybeans was found to give significant resistance to these transgenic lines.

Activating Gene Silencing

The other stratagem used by scientists is the process of gene silencing either through shutting down of a gene itself or through RNA interference to stop the end product protein from being made.

Funny enough, plants already use this process themselves. In a study in 2016, scientists were able to view how cotton plants reacted to infection by the fungus Verticillium dahliae. What they found is that the plants, as one ploy, start production of a particular set of microRNA and export them into the fungal hyphae invading the plant.

Once inside of the fungus, these microRNA began targeting specific gene sequences in order to silence it. The two types of microRNA created focused on two genes related to fungal virulence and, upon shutting them down, gave the cotton plants the upper hand in their fight against the fungus. This essentially increased their overall disease resistance and they did it all on their own.

As you can see, there is still much we can learn from plants themselves.

This wily action on the part of plants can also be exploited and improved by researchers. In a test involving the disease brown patch caused by the fungus Rhizoctonia solani, scientists took four essential genes from the fungus and inserted them into a test plant of tall fescue. When the fungus tried to attack the fescue, the plant used the genes as pre-written targets for it to attack and silence, improving its resistance chances considerably.

This allows steps to be taken against even the most intractable fungi that science has not been able to find counters for on its own. Instead, we can just give some assistance to the plants themselves and let them do the rest.

New Technologies And More Potential

With more recent improved technologies allowing for greater than ever precision and targeting in genomes, we will be sure to see even better mechanisms unfolding within agricultural biotechnology in the coming years. Though this may include giving us the ability to learn far more about the depths of what plants can do on their own with just a little nudge from us.

Since that’s how science and agriculture have to work, in tandem, in order to protect the plants against pathogens like parasitic fungi and help them save themselves.

References

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Photo CCs: Mycena inclinata, Clustered Bonnet, UK from Wikimedia Commons

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