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Nonionic Surfactants and Biosurfactants as Root Disease Preventatives

 

Surfactants (surface active agents) are compounds that you come across in your daily life such as foaming agents, emulsifiers, detergents, dispersants and wetting agents. They act to lower the surface tension of the liquid it is dissolved in or the interfacial tension between two liquids or a liquid and a solid.

 

Surfactants were initially used in agriculture to enhance the penetration and effectiveness of foliar-applied herbicides, defoliants and insecticides by decreasing surface tension of aqueous systems. Now surfactants have broader and more intensive use in immunoassays, biosynthesis of nucleic acids, floral induction, soil wetting, fruit thinning, hormone interactions, and photoperiodicity.

 

Surfactants have been shown to exert growth benefits in soilless/hydroponic systems. For example, Tween 20 (a nonionic surfactant) stimulated growth of solution-cultured tobacco[1] and promoted hypocotyl growth of pea by 90% hydroponically.[2] Méndez et al. (1967) demonstrated that 0.01% Tween 20 and IAA increased coleoptile elongation more than IAA alone. [3] Low concentrations of Tween 20 have been shown to accentuate the activity of growth hormones.[4], [5], [6], [7] The most likely mode of action of surfactants exerting growth benefits is that they make root membranes more permeable thereby allowing more nutrients (or other compounds) to be absorbed by the roots. However, the plant growth promoting effects of nonionic surfactants probably go even further than this and, as the research suggests, these compounds can also act as synergists with plant hormones such as IAA (and other auxins often used on cuttings for root formation) giving further growth stimulating effects.

 

In more recent times surfactants have also been shown to be effective for controlling/eliminating zoosporic plant pathogens.

Surfactants and Zoosporic Plant Pathogens


Zoosporic plant pathogens are associated with destructive root and foliar diseases of numerous crops. The latter include certain downy mildews and numerous species in the genera Pythium and Phytophthora. There are approximately 143 species of zoosporic plant pathogens. What makes these particularly damaging in hydroponics is that diseases producing zoospores release these into the nutrient solution or irrigation water. Zoospores survive easily in water and are able to swim, locate and infest new root systems. Therefore, zoospores in hydroponic systems can spread an isolated disease outbreak fairly rapidly through this highly efficient system of zoospore infection. However, zoospores are the weak link in the life cycle of these pathogens because they have no cell wall. The absence of a cell wall imparts a high degree of vulnerability to this spore stage and surfactants are demonstrated to rupture the plasma membrane which encases the zoospore, resulting in the rapid death of the zoospore. For example, one study showed that the motile zoospores of P. capsici were lysed within one minute of contact with a nonionic surfactant while the surfactant had little or no effect on other life stages of the pathogen.[8]

 

Biosurfactants

 

Natural surfactants (‘biosurfactants’) are produced by plants, bacteria and fungi and can be extracted for use as substitutes for synthetic surfactants. Several plant-based natural surfactants, for example saponins, lecithins and soy proteins have excellent emulsification properties but are expensive to produce and have other debatable issues such as solubility and hydrophobicity.[9] Microbial compounds that exhibit pronounced surface and emulsifying activities are classified as biosurfactants.

The discovery of biosurfactants as zoospore pathogen control agents began as many scientific breakthroughs do… it was an accident. In 1995, Michael Stanghellini, a plant pathologist, was looking for ways to control fungal diseases in plants. In a greenhouse experiment, he injected hydroponic plant specimens with different pathogens and then added fungicides. During a routine check he noticed that in one unit inoculated with the pathogen, not a single plant had died. He also noticed the nutrient solution was foaming extensively.

After considering and dismissing several causes, he contacted Raina Miller, a microbiologist. They isolated a bacterium from the recirculating nutrient solution of the hydroponic unit, and identified it as Pseudomonas aeruginosa. They found that the bacterium was synthesizing a biosurfactant that was responsible for the foaming in the nutrient solution.

Miller and Stanghellini wanted to know just how the protective bacteria were killing the pathogens so they isolated the active material and tested it on a series of different zoosporic plant pathogens. They found that the biosurfactant produced by the bacteria was able to lyse, or destroy cell membranes with dramatic results.

Rhamnolipid (RL) Biosurfactants


From an agricultural perspective, among the various categories of biosurfactants the glycolipid biosurfactants “rhamnolipids” (RL) stand apart. Rhamnolipid, primarily a crystalline acid, is composed of β-hydroxy fatty acid connected by the carboxyl end to a rhamnose sugar molecule. Rhamnolipids are predominantly produced by Pseudomonas aeruginosa (bacteria) and classified as mono and di-rhamnolipids. Other Pseudomonas species that have been reported to produce rhamnolipids are P. chlororaphis, P. plantarii, P. putida and P. fluorescens.

RL Biosurfactants present as a reasonably good option for controlling root zone pathogens in hydroponic systems. They have been demonstrated to be effective in controlling Rhizoctonia sp., Pythium sp., Phytophtora sp., and Fusarium sp. Other than RL, some biosurfactants such as lipodepsipeptides (LDPs), produced by Pseudomonas syringae, have been shown to act synergistically with ‘cell wall degrading enzymes’ (CWDEs) to increase pathogen control activity. These findings clearly indicate that the synergistic interaction LDPs—CWDEs is involved in the antagonistic mechanism of Pseudomonas syringae and they support the concept that a more effective disease control is given by the combined action of the two agents.[10] These findings also suggest that RL and CWDEs may also work synergistically to better control plant pathogens.

RL are shown to have biocidal activity towards pathogenic microorganisms while at the same time promoting beneficial bacteria numbers. Additionally, Vatsa et al. (2010) state that RL not only work as biocides but are also capable of stimulating the innate immunity in plant cells.[11] This immune response has been shown to participate to resistance against the hemibiotrophic bacterium Pseudomonas syringae pv, the biotrophic oomycete Hyaloperonospora arabidopsidis and the necrotrophic fungus Botrytis cinerea.[12]

 

Therefore RL act on three levels to protect plants from disease; firstly, they kill/antagonize plant pathogens through rupturing (lysing) their cell membranes; secondly, they promote beneficial bacteria numbers – these beneficial bacteria then act to further suppress disease; thirdly, they stimulate the immune system which makes plants less susceptible to disease.

Several studies have described antifungal activity of RL mainly against phytopathogens including Botrytis sp., Rhizoctonia sp., Pythium sp., Phytophtora sp. and Plasmopara sp.

Yoo et al. (2005)[13] investigated rhamnolipid biosurfactants as alternative antifungal agents against typical plant pathogenic oomycetes, including Phytophthora sp. and Pythium sp. They showed that RL significantly decrease the incidence of water-borne damping-off disease. Sharma et al. (2007)[14] obtained similar results in field trials on chili pepper and tomato. Using bacterial mutants, Perneel et al. (2008) [15]clearly showed that phenazine and RL interact in the biological control of soil-borne diseases caused by Pythium spp. Recent studies also demonstrated that a combination mixture of SRE (Syringomycin E, a class of lipodepsinonapeptide molecule) and RL is efficient against pathogenic and opportunistic fungi recovered from diseased grape.[16]

 

Biosurfactant producing rhizospheric isolates of Pseudomonas and Bacillus have exhibited biocontrol of soft rot causing Pectobacterium and Dickeya spp.[17] RL have demonstrated inhibition of zoospore forming plant pathogens that have acquired resistance to commercial chemical pesticides.[18]

 

One study demonstrated the efficacy of RL and saponin biosurfactants in the control of root rot in trials with hydroponically grown pepper. Amending the recirculating nutrient solution with either a rhamnolipid or a saponin biosurfactant selectively killed zoospores, resulting in 100% control of the spread of the pathogen. Disease control was achieved in both ebb and flow and top-irrigated growing systems with either an organic potting mix or rockwool as the planting medium. In the absence of either biosurfactant all plants within the growing system died within 6–7 weeks following pathogen inoculation of a single plant in the system which served as the source of infection. Injecting the RL biosurfactant into the irrigation line during every irrigation resulted in 100% control of the disease.[19]

 

Another study showed that continuous application of 150 mg·L-1 of RL controlled 100% of disease caused by Phytophthora capsici in pepper plants (Capsicum annuum). Nutrient solutions of pepper plants treated with RL and nitrapyrin biosurfactant resulted in two orders of magnitude higher concentration of aerobic bacteria[20] and Pseudomonas putida than the untreated solution.[21]

 

A 2014 study showed that the treatment of pea seeds and seedlings under natural conditions of light, temperature and humidity with the RL at a concentration of 25 µg ml−1 prior to sowing or planting in pathogen laden soil resulted in complete suppression of characteristic wilt symptoms. The results demonstrated the possibility to develop a sustainable and eco-friendly control measure against F. oxysporum.[22]

 

Surfactant issues/Considerations

 

Different surfactant products require different dosage rates depending on the concentration of active ingredients. Many biosurfactant products designed for hydroponic use give dose rates on the product label and these should be carefully followed. As with most products and compounds used in hydroponic solutions excessive use/application does not give better results and in many cases may prove phytotoxic to plants. For example, reduction in plant biomass of pepper plants was observed when 150 mg·L-1 rhamnolipid biosurfactant was applied in overhead and ebb and flow irrigation in organic or rockwool media.[23] For this reason when working with surfactants follow manufacturer recommendations (usage rates) carefully.  Another issue to consider with dosage rates is that surfactants are gradually broken down by microbial action over time so determining how often to re-dose may be difficult and require some trial and error.

 

Foaming in the nutrient reservoir is common where using surfactants is concerned. Foaming is to be expected when what is essentially a strong detergent is added to moving water; however, the degree of bubble formation differs between surfactant products.

 

Author’s Note: To access more information on surfactants and biosurfactants as water treatment technologies there is a searchable database that outlines various disinfection water treatment (pathogen eradication) technologies (ppm required, phytotoxicity thresholds etc) at watereducationalliance.org under the “grower tools” section. This tool summarizes published research that tests control of plant pathogens and algae using water treatment technologies. Among others, surfactant and biosurfactant efficiency data can be searched.

 

References

 

[1] MacDowall, F.D.H. 1963. Effects of nonionic surfactants on tobacco roots. Can. J.Bot. 41:1281–1287.

[2] Stowe, B.B. 1958. Growth promotion in pea epicotyl sections by fatty acid esters.Sci. 128:421–423.

[3] Méndez, J., A. Vieitez, C. Mato, and A. Vázquez. 1967. Direct and synergistic influence of tweens on Avena coleoptile section elongation. Physiol. Plant.20:437–441.

[4] Stowe, B.B. 1959. Similar activating effects of lipids on cytochromes and on plant hormones. Biochem. Biophyl. Res. Comm. 1:86–90.

[5] Stowe, B.B. 1960. Growth promotion in pea stem sections. I. Stimulation of auxin and gibberellin action by alkyl lipids. Plant Physiol. 35:262.

[6] Stowe, B.B. and J.B. Obreiter. 1962. Growth promotion in pea-stem sections. II. By neutral oils and isoprenoid vitamins. Plant Physiol. 37:158–164.

[7] Stowe, B.B. and M.A. Dotts. 1971. Probing a membrane matrix regulating hormone action. I. The molecular length of effective lipids. Plant Physiol. 48:559–565.

[8] Stanghellini, M.E., Kim, D. H., Russmussen, S.L., and Rorabaugh, P. A. (1996) Control of Root Rot of Pepper Caused by Phytophthora capsici with a nonionic surfactant

[9] Xu Q., Nakajima M., Kiu Z., Shiina T. (2011). Biosurfactants for microbubble preparation and application. Int. J. Mol. Sci. 12, 462–475 10.3390/ijms12010462

[10] Fogliano, V. Ballio, A. Gallo, M. Woo, S. Scala, F. and Lorito, M (2002) Pseudomonas Lipodepsipeptides and Fungal Cell Wall-Degrading Enzymes Act Synergistically in Biological Control. Molecular Plant-Microbe Interactions. April 2002, Volume 15, Number 4, Pages 323-333

[11] Vatsa P, Sanchez L, Clement C, Baillieul F, Dorey S. Rhamnolipid biosurfactants as new players in animal and plant defense against microbes. Int J Mol Sci. 2010;11:5095–5108

[12] Sanchez, L. Courteaux, B.  Hubert, J. Kauffmann, S. Renault, JH. Clément, C. Baillieul, F and Dorey, S. (2012) Rhamnolipids Elicit Defense Responses and Induce Disease Resistance against Biotrophic, Hemibiotrophic, and Necrotrophic Pathogens That Require Different Signaling Pathways in Arabidopsis and Highlight a Central Role for Salicylic Acid

[13] Yoo, D.S.; Lee, B.S.; Kim, E.K. Characteristics of microbial biosurfactant as an antifungal agent against plant pathogenic fungus. J. Microbiol. Biotechnol. 2005, 15, 1164–1169.

[14] Sharma, A.; Jansen, R.; Nimtz, M.; Johri, B.N.; Wray, V. Rhamnolipids from the rhizosphere bacterium Pseudomonas sp. GRP(3) that reduces damping-off disease in Chilli and tomato nurseries. J. Nat. Prod. 2007, 70, 941–947.

[15] Perneel, M.; D’Hondt, L.; De Maeyer, K.; Adiobo, A.; Rabaey, K.; Hofte, M. Phenazines and biosurfactants interact in the biological control of soil-borne diseases caused by Pythium spp. Environ. Microbiol. 2008, 10, 778–788.

[16] Takemoto, J.Y.; Bensaci, M.; De Lucca, A.J.; Cleveland, T.E.; Gandhi, N.R.; Skebba, V.P. Inhibition of fungi from diseased grapeby syringomycin E-rhamnolipid mixture. Am. J. Enol. Vitic. 2010, 61, 120–124.

[17] Krzyzanowska DM, Potrykus M, Golanowska M, Polonis K, GwizdekWisniewska A, Lojkowska E, Jafra S (2012) Rhizosphere bacteria as potential biocontrol agents against soft rot caused by various Pectobacterium and Dickeya spp. strains. J plant pathol 94

[18] Sha R, Jiang L, Meng Q, Zhang G, Song Z. Producing cell-free culture broth of rhamnolipids as a cost-effective fungicide against plant pathogens. J Basic Microbiol. 2011;52:458–466.

[19] Nielsen,C.J. Ferrin D.M.  & Stanghellini, M.E. (2006) Efficacy of biosurfactants in the management of Phytophthora capsici on pepper in recirculating hydroponic systems. Canadian Journal of Plant Pathology Volume 28, Issue 3 2006

[20] Nielsen, C.J., Ferrin, D.M., Stanghellini, M.E., 2006. Efficacy of biosurfactants in the management of Phytophthora capsici on pepper in recirculating hydroponic systems. Canadian Journal of Plant Pathology 28, 450-460.

[21] Paggliaccia, D., Ferrin, D., Stanghellini, M.E., 2007. Chemo-biological suppression of root infecting zoosporic pathogens in recirculating hydroponic systems. Plant and Soil 299, 163- 179.

[22] Borah, S. N. Goswami, D. Lahkar, J. Sarma, H. K. Khan, M. R. and Deka, S (2014) Rhamnolipid produced by Pseudomonas aeruginosa SS14 causes complete suppression of wilt by Fusarium oxysporum f. sp. pisi in Pisum sativum. BioControlJune 2015, Volume 60, Issue 3, pp 375-385

[23] Nielsen, C.J., Ferrin, D.M., Stanghellini, M.E., 2006. Efficacy of biosurfactants in the management of Phytophthora capsici on pepper in recirculating hydroponic systems. Canadian Journal of Plant Pathology 28, 450-460.