Friday, December 9, 2011

Stopping the Insatiable Sclerotinia sclerotiorum

Sclerotinia sclerotiorum is a necrotrophic (requires dead host tissue to survive) fungal plant-pathogen that causes significant crop losses throughout the world [1]. The cost in the United States of crop loss due to S. sclerotiorum exceeds $200 million annually whereas yield losses in China of the oilseed rape crop can approach a staggering 80% [1,2]. The host range of this fungus includes over 400 species, most of which include agronomic crops and horticulture plants [3]. Finding a realistic solution to this fungal infection is currently of great importance, especially when considering the growing food shortages around the globe.
The defining feature of this fungus is the development of sclerotia (Figure 1). Sclerotia are long-term survival and dissemination structures (functionally similar to that of the common endospore of Bacillus species) that can withstand adverse environmental conditions such as extreme temperature, UV light, desiccation, or antagonistic organisms [3]. The survival and eventual germination of sclerotia depends on various environmental factors such as aeration and moisture [3]. The structure of sclerotia—which consist of carbohydrates, enzymes, free amino acids, and fatty acids—includes a pigmented rind, a thin-walled cortex, and a large central medulla [6]. There are two general types of germination in this fungal pathogen. Myceliogenic germination results in the production of hyphae (branching filamentous structures) that directly infect plant tissues whereas carpogenic germination results in the release of ascospores [3]. Regardless of the type, both germination methods results in initiation of disease.
Figure 1. Three distinct stages in the formation of sclerotia of Sclerotinia sclerotiorum(A)
initiation (4 d of growth); (B) development (5 d of growth); and (C) maturation (8 d of growth).
The enlarged panels on the right corners show enlarged view of a typical sclerotium (arrow). [4]
Activity of fungi is greatly affected by environmental factors such as pH, temperature, water availability (perhaps most important), and soil water potential [4]. Total soil water potential, or how much energy is required to extract water from a substrate, is a sum of matric, osmotic, pressure, and gravitational potentials [4]. Osmotic potential is due to solutes (i.e. dissolved particles) in soil water and figures prominently in growth within tissues [4]. Matric potential includes both adsorption and capillary effects and is relevant to growth in soil or on root surfaces [4]. Understanding how environmental factors effect fungal growth is important for potentially producing a local soil environment not conducive to S. sclerotiorum.
Although fungicide resistance in S. sclerotiorum has drastically increased over the past decade, there are still several fungicides available [2]. However, these inhibitors are expensive, largely ineffective, and can be extremely hazardous to humans and the environment [2]. Crop rotation, which has been effective in fighting other pathogenic fungi, does not work in stopping S. sclerotiorum due to its wide host range of pathogenicity [2]. In addition, this fungus is capable of persisting in soil for many years due to these sclerotia structures [3].
Factors limiting commercial microbial biological control products include the stability of antimicrobials, spectrum of activity, consistency, and efficacy [4]. A simple standard for analyzing a potential antimicrobial product is measuring how it stacks up with chemical pesticide alternatives, specifically on cost and effectiveness. Temperature, soil moisture, soil type, host cultivar, and other factors have been shown to impact biological control [4].
Recent research by Hu et. al describes a new biological control technique using the bacterium Bacillus subtilis to suppress Sclerotinia sclerotiorum on oilseed rape. Isolates of B. subtilis are commercially attractive because they produce endospores that aid in the overall stability of their biomass. In addition, B. subtilis produce a number of broad-spectrum antibiotics which makes them that much more desirable [2]. Hu et. al. found that B. subtilis produced the lipopeptide antibiotic iturin and contained the genes necessary for the biosynthetic pathway of the antibiotic bacilysin. Iturin exhibits broad spectrum inhibitory activity, which includes fungi that produce sclerotia.
The research by Hu et. al. was broken into two trials. In both trials, oilseed treatments were done by two distinct formulations. In the first trial, the researchers found that formulations of Tu-100 (the Bacillus subtilis bacterium used in the experiment) resulted in greater plant dry mass and seed yield while reducing disease relative to their controls. In the second trial, one distinct formulation of Tu-100 performed significantly better than the other in reducing the incidence of disease, but both performed similarly in plant dry mass and seed yield.
Both formulations were found to have good seed germination (>85%) and stable Tu-100 biomass over a 6 month storage period at room temperature. These results were attained with small-scale field trials at two locations. It has yet to be shown if these formulations could be effectively integrated in large-scale, real-world conditions. A major issue with the viability of this technique to use Bacillus isolates to suppress fungal infections lies in how the antibiotic interacts with its target. Tu-100 must colonize the oilseed rape shoot so that the produced antibiotics are in close proximity to the invading pathogen. There are also unresolved problems with how the plant protects itself against Tu-100, thereby nullifying this potential biological control method.
Past research has found that the protein saccharopine dehydrogenase is vital in early sclerotial development. This protein catalyzes the biosynthesis of lysine, an important amino acid in the synthesis of the fungal cell wall. Previous studies have found that deleting the lysine biosynthesis gene severely reduces virulence of the fungus Aspergillus fumigatus [3]. However, more research must be conducted to tell whether this is a viable target to inhibit S. sclerotiorum pathogenesis.
Current research by Liang et. al. investigated these proteins involved in sclerotial development [3]. Liang et. al. found that proteins involved in energy metabolism decreased between the initiation of sclerotium formation and subsequent development stages. Some key proteins looked at were isocitrate dehydrogenase, fumarate hydratase, and aconitate hydratase (all three of which are important enzymes in the citric acid cycle). In addition, the researchers found GAPD drastically decreased during sclerotial development. This suggests that GAPD would be a critical protein during sclerotial initiation. This research on key proteins in sclerotial formation is important for finding viable targets for inhibitors as well as understanding overall fungal pathogenic function.
Although there is no clearly viable solution to the costly fungus Sclerotinia sclerotiorum, recent research is somewhat promising. Liang et. al. provided a plethora of data into key proteins in the sclerotial development process. Hopefully, at least one of these proteins will be a realistic target for inhibitory drugs. In addition, Hu et. al. illustrated the promise of utilizing Bacillus isolates to possibly suppress Sclerotinia outbreaks and slow its spread.

This blog post was contributed by J. M. Luby.

[1]   Williams, B., M. Kabbage, H. Kim, R. Britt, M. Dickman. (2011). Tipping the balance: Sclerotinia sclerotiorum secreted oxalic acid suppresses host defenses by manipulating the host redox environment. PLoS Pathog. 7(6):e1002107.
[2]   Hu, X., D. Roberts, J. Maul, S. Emche, X. Liao, X. Guo, Y. Liu, L. McKenna, J. Buyer, S. Liu. (2011). Formulations of the endophytic bacterium Bacillus subtilis Tu-100 suppress Sclerotinia sclerotiorum on oilseed rape and improve plant vigor in field trails conducted at separate locations. Can. J. Microbiol. 57:539-546.
[3]   Liang, Y., M. Rahman, S. Strelkov, N. Nat, V. Kav. (2010). Developmentally induced changes in the sclerotial proteome of Sclerotinia sclerotiorum. Fungal Biology. 114:619-627.
[4]   Jones, E., A. Stewart, J. Whipps. (2011). Water potential affects Coniothyrium minitans growth, germination and parasitism of Sclerotinia sclerotiorum sclerotia.

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