Project Description

Background

Walking into any supermarket in the world, one is sure to find bunches of ripened gold laying on fruit racks, unabashedly declaring its presence. Even as it loses its brilliant yellow, the brown blotches signal its excitement to participate in pastry. Each level of ripeness has its own faction of advocates with praise that would turn anything else blushing red. This fruit, of course, is the banana.

Bananas, beyond being simply a popular food, hold a far more important place in the modern world than one may expect. With around 20-25 billion dollars in market value, bananas are crucial to the economy of nations such as Ecuador, Colombia, and Costa Rica; they are the top suppliers of bananas to the European Union (Eurostat 2022). As one of the most popular fruits in the western world, bananas are predominantly imported from Latin American nations into the EU and the United States. The banana market benefits all levels of the supply chain – the low cost, convenient consumption, and high nutrition of the banana give consumers enjoyment; the large sums of revenue generated from bananas offer job opportunities for millions due to high market demands (FAO of UN 2022). Bananas have held this important place even prior to the 1950s when it was called the “poor man’s fruit” because of their affordability and high starch and sugar content.

Furthermore, about 400 million people depend on bananas for food security, and in some rural areas, a quarter of calorie intake every day comes from bananas. The banana industry also supports over two million jobs in Ecuador, where bananas account for 10% of Ecuador’s exports in terms of value - about 2.8 billion USD in 2018 (Reinventing Banana Production in Ecuador, 2019).

The place of the banana thus makes the crisis that threatens its existence all the more serious. In truth, the existence of the banana has always been one of struggle against numerous pathogens - this vulnerability stems from the very genetic characteristics that give the banana its sweet, seedless flesh.

Banana Cultivation

As with most vegetables and fruits that the modern world is accustomed to, the modern banana is the result of extensive artificial selection. Specifically, during the process of selecting for favorable traits, a nondisjunction event occurred and made subsequent bananas triploid due to the acquisition of an additional chromosome set. This drastic alteration resulted in seedlessness and sterility. To overcome the inability of banana plants to naturally reproduce, farmers utilized asexual propagation (Burr and Burr, 2000). This practice led to the emergence of cultivars: despite all plantation banana trees being genetic clones, they nevertheless originate from different parent trees; small genetic differences in the parent trees manifest as different phenotypes. The name given to the cultivar identifies the parent tree of any given banana tree and its characteristics. Modern plantations often plant singular cultivars en masse; while this ensures the consistency of the fruit, it leaves banana trees extremely vulnerable to disease due to low genetic diversity and proximity to each other. This threat lacks no precedence - in fact, the banana nearly met its end in the 1950s.

The cultivar that met its end in the 1950s was the Gros Michel cultivar of the Musa acuminata AAA group. Its murderer is the infamous Panama Disease, henceforth referred to as Fusarium wilt; the pathogen responsible was the fungus Fusarium oxysporum f. sp. cubense Race 1(foc R1). Due to Gros Michel’s utter lack of resistance to this pathogen as well as the lack of measures taken to mitigate its outbreak, the cultivar was rapidly destroyed. It had previously been the major export cultivar of Central America, but due to the devastation caused by Fusarium wilt, the world was forced to switch to an alternative. Here, the modern banana enters the story - the Cavendish cultivar was the essential antidote to the banana crisis. It possessed resistance to foc R1 and was thus immune to the ravaging of Fusarium wilt. This provided much-needed stability to the turmoil, and the world soon began a slow and costly switch to Cavendish bananas.

TR4 & the Approaching Extinction of Cavendish bananas

Yet history repeats itself, and the stability that Cavendish bananas brought about is drawing to a close as the threat of Fusarium wilt rises again - a new variant of the pathogen called foc Tropical Race 4(foc TR4) or Fusarium odoratissimum looms over banana plantations worldwide. The outbreak of this new wave of Fusarium wilt was first reported in Taiwan around the 1970s (Maymon et al., 2020). Foc TR4 possesses far greater virulence than R1, giving it the ability to devastate even the Cavendish cultivar. The fungus spread globally through the informal exchange of planting materials, the movement of spore-bearing soil, and the export of monocrop bananas in the 1900s (Dita et al., 2018).

Foc is a vascular wilt fungus that spreads through the contaminated soil and penetrates the roots of the banana plant to cause infection. Before Foc recognizes the host in the surrounding area, it remains dormant as chlamydospores (Kurtsmen et al., 2011). Once Foc is stimulated to germinate by susceptible root tissues or root exudates, it activates signaling pathways that increase virulence and colonization of the root surface (Husaini et al., 2018). Then, Foc penetrates the root epidermis with infective hyphae and progresses further into the tissue of the corm root. Once root penetration is achieved, Foc is able to colonize the xylem vessel through the production of macroconidia and microconidia, causing congestion and blockage of water flow and resulting in Fusarium Wilt’s namesake wilting (Dita et al., 2018).

The prognosis of the disease, in most cases, is plant death (Husaini et al., 2018). Foc TR4 poses a major crisis as 80% of banana and plantain production is based on Foc TR4-susceptible cultivars. Furthermore, 99% of exported bananas are of the Cavendish cultivar; it is thus evident that an overwhelming portion of bananas and plantains are vulnerable to the devastation of Foc TR4 (Pérez-Vicente et al., 2014). Due to the world’s economic and nutritional dependence on bananas, the aftermath of an uncontrollable foc TR4 pandemic would be too much for the world to bear.

Current solutions & limitations

Made vigilant by the destruction of Gros Michel, the world has taken numerous measures to quarantine and control the spread and impact of TR4 Fusarium wilt so that the catastrophe doesn’t repeat with the Cavendish. Although numerous publications indicate that different methods can be used to manage Fusarium Wilt, very few are effective in field application (Thangavelu and Mustaff, 2010). Much of the literature on this topic reports results from short-term in-vitro or greenhouse studies with no indications that the result would be useful in field application. For example, although rice hull burning (heat sterilization of the soil) has been recommended in southern Mindanao in the Philippines and Indonesia, there is a lack of data on its use (Ploetz, 2015). Among the many current solutions attempting to curb the impacts of Fusarium wilt, the general consensus favors chemical treatment and the development of resistant cultivars. Both solutions have been proven to be impactful to some degree, but are ultimately flawed.

Chemical Treatments

Chemicals are commonly used to prevent and inhibit Fusarium Wilt. A wide range of chemicals was historically used to control the disease with relative success. These chemicals can be classified into four categories: fungicides, surface sterilants, fumigants, and activators. These chemical control methods serve to inhibit virulence, induce resistance, improve soil quality, and activate plants’ self-defense mechanisms, known as systemic acquired resistance (SAR). The downsides of chemical treatments are obvious - consequences such as chemical accumulation in fruit and environmental pollution have long been in the public’s attention. Many chemicals once used for soil treatment have proven to be harmful to humans and to the environment.

Resistant cultivars

We analyze the current solutions to have primarily focused on prevention and quarantine - to stave off or destroy the fungus before it can reach plantation soil. This leaves a glaring gap in the scenario where foc TR4 inevitably reaches plantation soil but has not yet taken hold. Without a response to this stage of Fusarium wilt spread, the last line of defense against this disease would be quarantine, past which foc TR4 is free to ravage plantations. With how easy it is for the pathogen to accidentally spread, we assess the current repertoire of responses to be insufficient; we thus sought to engineer another line of defense in the inevitable case that quarantine fails.

Our Theory

The vulnerability of Cavendish bananas to foc TR4 is fundamental to its genetic composition - it is an impossibility to cure the Cavendish banana of this deathly disease. Our solution aims not to achieve this absurd task, but rather to buy time for the ongoing search for a genetically distinct, TR4-resistant banana cultivar. Thus, our task is to minimize the devastation caused by Fusarium Wilt to the banana industry. To tackle the persistent threat of Fusarium Wilt, we chose to engineer an equally persistent method of protection in the form of a soil probiotic. Gut probiotics are known to prevent opportunistic infections through various methods such as niche competition; taking inspiration from this, our soil probiotic will take a more aggressive approach to prevent disease by attacking foc itself. B. subtilis was chosen as our chassis since it is an established Plant-Growth Promoting Rhizobacteria(PGPR), meaning it possesses the capacity to adapt well to soil conditions and the ability to aid the banana plant in growth and defense against pathogens.

SEC secretion of Chitinase

With a chassis in mind, we needed a weapon against foc. Foc TR4, being a fungus, possesses a chitin cell wall and is thus vulnerable to chitinase, an enzyme specialized in the hydrolysis of chitin. The antifungal properties of chitinase are well known and characterized. We will couple this with the SEC secretion in order to allow extracellular secretion of chitinase, thus allowing our bacteria to attack the foc’s vulnerable hyphae.

We’d be able to engineer bacteria to secrete chitinase into the environment through fusion with the YebF secretion tag. We can achieve improved antifungal protein production through overexpression with a strong T7 promoter.

The construct illustrated above would allow us to deliver a conventional antifungal effector in a conventional manner to combat foc. Despite these advantages stemming from simplicity, chitinase fails a major criterion for application in biocontrol - specificity. Thus, in addition to a system to attack foc, we also needed to engineer a biocontainment system to properly control our bacteria.

Root-localization Kill Switch System

By limiting the bacteria’s presence to being exclusively around the root, we would be able to control the effects of our engineered bacteria spatially. This constraint on bacterial growth would allow us to contain the bacteria to its intended use case - to combat foc TR4. Though chitinase may not be able to distinguish mycorrhizal friend from fusarium foe, by preventing our bacteria from proliferating in all soil we can limit harm to harmless fungi. To do all this, we plan to modify the kill switch system from 2018 Team Pasteur Paris. To adapt their system to our purposes, we will change the controlling factor from temperature to malate concentration. The purpose of making our bacteria dependent on malate is based on the findings of Yuan et al., who found that organic acids such as malic acid were present in banana root exudate and induced chemical responses in bacteria. By comparing the extent of chemotaxis induced by the banana root exudate and different concentrations of malic acid, we were able to approximate the concentration of malic acid in banana root exudate at . In essence, the dependence of our bacteria on malate will cause it to be dependent on the root exudate of banana trees, and thus limited to its immediate surroundings; this achieves our goal of using spacial specificity to control our biofungicide bacteria.

References

Burr, B., Burr, F. (2000). How do seedless fruits arise and how are they propagated? Scientific American. Link to Source

D’Hont, A. (2012). Domestication of the banana. Musarama. CIRAD field collection, Guadeloupe, France. Link to Source

Dita, M., Barquero, M., Heck, D., Mizubuti, E. S. G., & Staver, C. P. (1AD, January 1). Fusarium wilt of banana: Current knowledge on epidemiology and research needs toward sustainable disease management. Frontiers. Retrieved July 19, 2022 Link to Source

Food and Agriculture Organization of the United Nations. (2022). Banana facts and figures. United Nations. Link to Source

Gros Michel Banana. Miami Fruit. Link to Source

Hashem, A., Tabassum, B., & Fathi Abd_Allah, E. (2019). Bacillus subtilis: A plant-growth promoting rhizobacterium that also impacts biotic stress. Saudi Journal of Biological Sciences, 26(6), 1291–1297. Link to Source

Husaini, A. M., Sakina, A., & Cambay, S. R. (2018). Host–pathogen interaction in fusarium oxysporum infections: Where do we stand? Molecular Plant-Microbe Interactions®, 31(9), 889–898. Link to Source

Kema, G., et al. (2021). Editorial: Fusarium Wilt of Banana, a Recurring Threat to Global Banana Production. Front. Plant Sci, 11, Link to Source

Kurtzman, C. P., Fell, J. W., Boekhout, T., & Robert, V. (2011). Methods for isolation, phenotypic characterization and maintenance of yeasts. The Yeasts, 87–110. Link to Source

Mach, A., & Reed, C. (2016, January 24). 8 things you didn't know about bananas. PBS. Retrieved July 21, 2022 Link to Source

Maryani, N., Lombard, L., Poerba, Y. S., Subandiyah, S., Crous, P. W., & Kema, G. H. J. (2019). Phylogeny and genetic diversity of the banana fusarium wilt pathogen fusarium oxysporum f. sp. cubense in the Indonesian centre of origin. Studies in Mycology, 92, 155–194. Link to Source

Pérez-Vicente, L., Dita, M. A., & Parte, E. M. de la. (2014, May). Technical manual prevention and diagnostic of fusarium wilt (Panama disease) of banana caused by Fusarium oxysporum f. sp. cubense Tropical Race 4 (TR4). Technical Manual. Retrieved July 19, 2022, from Link to Source

Yuan, J., Zhang, N., Huang, Q., Raza, W., Li, R., Vivanco, J. M., & Shen, Q. (2015). Organic acids from root exudates of banana help root colonization of PGPR strain Bacillus amyloliquefaciens NJN-6. Scientific reports, 5, 13438. Link to Source