Stepan Company: Rhamnolipids from Fermentation — An Emerging Biofungicide for Zoosporic Diseases

As the agricultural industry continues to look for less hazardous, bio-derived and biological solutions for crop protection, the search to identify new and effective options continues to expand. The search for effective fungicides has persisted throughout history, with the practice of using sulfur to treat pests documented as far back as the ancient Greeks and Romans. The first organic fungicide was patented in the 1930s and the improved analytical and manufacturing techniques of the 20th century ushered in a new era of organic fungicides.¹  

Rhamnolipids from Fermentation

Rhamnolipids are a class of glycolipid bio-surfactant composed of a mono- or di- rhamnose carbohydrate attached to mono- or di-fatty acid chains. Rhamnolipids were first identified in 1946 and the focus on their utility as a biosurfactant continued for some time. ² For fermentation products, the final composition can be tailored through the selection of the microorganism and organic feedstock used in production. 

Rhamnolipid Fungicide Mode of Action

There are more than 100 different species of zoosporic pathogens, which can make it quite daunting when searching for a means to control the spread of disease on crops. The commonality these pathogens share is their namesake zoospore stage, identified as the part of the life cycle when the pathogen can spread and infect a plant host most easily. Fortunately, the zoospore stage has a weakness in the form of a pesticide-susceptible plasma membrane, as opposed to a more robust cell wall it develops in later stages of its life cycle.³  Target pathogens in this family include downy mildews, Botrytis spp., Pythium spp., Phytophthora spp., and many more. By the 1990s, it was discovered that rhamnolipids exhibited activity against zoosporic plant pathogens.

Additionally, rhamnolipids have the unique feature of exhibiting surfactant properties, due to the hydrophilic nature of the rhamnose moiety and the hydrophobic nonpolar fatty acid tails. These amphiphilic features allow rhamnolipids to reduce surface and interfacial tension, thereby increasing the permeability of the zoospores. This behavior has been suggested as the mechanism for activity for rhamnolipids as a biofungicide by allowing them to penetrate and disrupt the zoospore cell membrane. ⁴

Rhamnolipid Biofungicide Safety and Efficacy

Rhamnolipids have been shown to be readily biodegradable and environmentally friendly. The safety of these materials has been recognized by the United States Environmental Protection Agency, which has listed the material as exempt from the requirement of a tolerance when used in accordance with good agricultural practices. ⁵

Several studies have been published reporting the efficacy of rhamnolipids as a biofungicide against a variety of common pathogens of horticultural and agricultural crops. This article provides examples of the work that has been published demonstrating the utility of rhamnolipids for biological control of these damaging plant pathogens.

Case Study #1: Rhamnolipid Treatment Against Tomato Late Blight ⁶

This trial by Johnson, Jordan and Gevens tested the efficacy of rhamnolipids, along with other biofungicides, before and after plants were inoculated with Phytophthora infestans. Plants were compared to untreated areas and use rates were determined by label recommendations. Three different P. infestans isolates were studied in this program: US-22, US-23 and US-24. The leaves of each plant were evaluated seven days following inoculation and a disease rating assigned, from which a percent severity calculation was made.

In the preventative study (treatment applied one hour prior to inoculation), the rhamnolipid biofungicide treatment resulted in much lower disease severity compared to the untreated plants (Figure 1). 

Figure 1. Efficacy of Rhamnolipid Against Tomato Late Blight (Preventative)

Alternatively, when the rhamnolipid biofungicide was applied after inoculation (two hours prior), it did not demonstrate an effective level of control. This study suggests that rhamnolipids can be an effective preventative treatment for late blight, but further optimization and possible combination with additional control methods post-inoculation may be necessary. 

Interesting to note, this work also included a microscopic study of the impact of rhamnolipid on zoospores. When a drop of rhamnolipid was added to a suspension of zoospores, it was observed that all swimming zoospores were eliminated, supporting the mode of action suggested above.

Case Study #2: Rhamnolipid Treatment Against Downy Mildew of Cucumbers ⁷

In this study by Gugino and Grove, six treatments of various fungicides and fungicide combinations were applied and compared to an untreated plot. The rhamnolipid alone was found to have a significant impact on the calculated area under the disease pressure curve (AUDPC) compared to the untreated plot. The rhamnolipid showed even more improvement when the six treatments alternated between the rhamnolipid and a copper hydroxide fungicide (Figure 2).

Figure 2. Rhamnolipid in Combination with Copper Hydroxide

Stepan Innovation Center and Greenhouse 

Ongoing research at Stepan Company’s new facility, located in Winder, Georgia, US, has shown positive crop tolerance to rhamnolipid treatments. A variety of crops have been treated with rhamnolipid with no phytotoxicity observed (Figure 3).

Figure 3. (l to r) Basil, Pepper & Carrot Plants Treated with Rhamnolipid 

Stepan has a long history in agricultural formulation development and crop protection. In addition to phytotoxicity, the Agricultural Innovation Center supports research into efficacy, sprayability, in-vitro studies, stability, tank mix compatibility and more. Stepan scientists are able to carry out rapid prototyping and development of rhamnolipid biofungicides within our unique formulation and greenhouse facilities.

Summary

Trials with rhamnolipid as a biofungicide against zoospore pathogens show the product is effective in controlling disease and can also be used in combination with other conventional fungicides and biofungicides. Stepan Agricultural Solutions’ research in this area includes optimizing use rates, application methods and product formulations to provide effective control of zoospore diseases.

The demand for more sustainable products and the rise in organic farming have only further accelerated the use of biofungicides. Rhamnolipids have been studied against a wide range of pathogens for horticultural and agricultural crops with promising results. Fermentation as a commercial technology platform offers a wide variety of new and exciting solutions for farmers and consumers worldwide.

Stepan Agricultural Solutions believes in growing through science and has invested in fermentation technology and capabilities to develop novel solutions for the agriculture market. 

Reference:

1. McCallan, S. E. A. (1967). “History of Fungicides.” Fungicides: An Advanced Treatise (Volume 1), edited by D Torgeson, Academic Press Inc, pp 1-37.

2. Kumar R., Das A.J. (2018). “Rhamnolipid Biosurfactants and Their Properties”. Rhamnolipid Biosurfactant, Springer, pp 1-14. 

3. Stanghellini ME, Miller RM. (1997). “Biosurfactants: Their identity and potential efficacy in the biological control of zoosporic plant pathogen.” Plant Disease, 81(1), 4–12.

4. Sotirova, A., et al. (2009). “Effects of rhamnolipid-biosurfactant on cell surface of Pseudomonas aeruginosa.” Microbiological Research, 164(3),  297-303.

5. United States Environmental Protection Agency. (2004, March 31) Washington, D.C. 40 CRF, Part 180, Vol 69, No. 62.

6. Johnson, S, A, C, Jordan, S and Gevens, A. (2015). Efficacy of Organic and Conventional Fungicides and Impact of Application Timing on Control of Tomato Late Blight Caused by US-22, US-23 and US-24 Isolates of Phytophthora infestans. Plant Disease , 99(5), 641-647.  https://doi.org/10.1094/PDIS-04-14-0427-RE

7. Gugino B.K. and Grove, T.L. (2015). Evaluation of select biofungicides and fungicide programs for the management of downy mildew of cucumber, 2014. Plant Disease Management Reports, 9:V014. https://doi.org/10.1094/PDMR09