12.16: THIS IS HOW WE DO IT: Can beneficial fungi save our chocolate?

People love chocolate. It’s a plant product at the heart of a huge economic industry. More than 3.5 million tons of cocoa beans are produced each year, and the global chocolate market is valued at close to $100 billion per year.

Chocolate is made from the seeds of the cacao tree (Theobroma cacao), a plant that is native to Central and South America. Twenty to 60 seeds are harvested from the large fruits, called cacao pods. The seeds are fermented, dried, roasted, cracked, and crushed, after which they may be combined with other ingredients, depending on the product.

Worldwide production of chocolate, unfortunately, is significantly reduced each year by fungal diseases that affect cacao plants. The fungi reduce the ability of trees to grow and to produce pods, as well as rotting the interior of the seed pods. In Peru, for example, the “frosty pod rot” fungus alone has reduced cacao production by one-third.

Because farmers would like alternatives to the high use of pesticides—which are expensive and not very effective—researchers are looking for other ways to reduce the impact of these fungal pathogens.

Fungi often live within plants. In fact every single plant species examined has been found to harbor fungi inside its tissue. These fungi are extremely diverse and, in many cases, do not seem to harm their plant host. For this reason, researchers have taken the approach of searching for species of fungi that might be able to outcompete or even harm the pathogenic fungi responsible for three of the most common fungal diseases.

To get started, the cacao researchers identified three distinct issues or questions requiring investigation.

1. Are there fungal species with anti-pathogen activity?

2. Can those species be introduced into cacao plants (without harming the plants)?

3. Within cacao plants, do these fungi actually limit damage by the pathogenic fungi and improve the plant’s productivity?

To investigate the first question, researchers isolated 110 different types (called morphospecies) of fungi that can live in cacao trees and seem not to harm them. They then cultivated these fungi in Petri dishes, along with the pathogenic fungi. In each case, they evaluated which fungi “won” the interaction—which of two interacting species survived in the Petri dish.

This approach made it possible to efficiently identify fungi with anti-pathogen activity to serve as “biocontrol agents.” In fact, the researchers discovered 53 different fungi that showed antagonism toward the major pathogenic fungi. Of these, they identified three candidates that had both anti-pathogen activity and good colonizing ability.

The next step, to investigate the second question, was to attempt to deliver the helpful fungi into cacao seedlings. The researchers first germinated cacao seeds in sterile soil. They then grew the seedlings in plastic shade houses that prevented the leaves from being exposed to any spores of airborne pathogenic fungi, and sprayed the plants with a suspension containing spores of a potentially beneficial fungus (while other plants were not sprayed, serving as controls).

Evaluating leaf tissue after 10, 25, and 33 days, the researchers identified fungal growth and estimated the percentage of fungal colonization. After 33 days, they found that colonization had occurred in 39%, 62%, and 92% of the cacao plants, depending on which of the three potentially beneficial fungi they had used for inoculation.

The third, and possibly most important, issue examined was whether the beneficial species would reduce the impact of the pest species on cacao trees’ productivity. Here, the results were a bit mixed.

In a greenhouse study, the researchers found 25% leaf mortality among trees infected with one of the pathogens. Among trees infected with both the pathogen and the potentially beneficial fungus, leaf mortality was just under 10%, a statistically significant reduction.

In the field, however, the results were less dramatic. Across four farms, researchers observed 320 experimental trees (inoculated with the potentially beneficial fungus) and 320 control trees (not inoculated with the potentially beneficial fungus). They found no reduction in the incidence of pod loss due to the pathogenic fungi. This may have been a consequence of an unusually low rate—less than 20%—of infection by the pathogenic fungi on these farms. The researchers did note, however, that in plants treated with the potentially beneficial fungus there was no reduction in plant growth and there was a 10% reduction in lesions caused by the pathogenic fungus.

It’s clear that—at least using current methods—biocontrol isn’t a magic bullet that wipes out the major fungal diseases of cacao trees. But researchers are optimistic.

The researchers’ optimism stems from several of their results. (1) They were able to identify potentially “good” fungi quickly in lab experiments. (2) They were able to inoculate cacao trees with “good” fungi. And (3) there seems to be at least some inhibition of pathogen activity in trees inoculated with “good” fungi.

The researchers are continuing their work, evaluating the impact of other factors on the anti-pathogen impact of the “good” fungi. These factors include (1) the timing of the spraying of seedlings to inoculate them, (2) the impact of using multiple potentially “good” fungal morphospecies simultaneously, (3) the amount of shade versus sun the trees are exposed to, and (4) the extent of pathogen infection.