WO2012006519A2

WO2012006519A2 - Dinoflagellate-specific algicidal compound and uses thereof

Info

Publication number: WO2012006519A2
Application number: PCT/US2011/043350
Authority: WO
Inventors: Kathryn J. Coyne; Mark E. Warner
Original assignee: University Of Delaware
Priority date: 2010-07-09
Filing date: 2011-07-08
Publication date: 2012-01-12
Also published as: WO2012006519A3

Abstract

The present invention relates to a dinoflagellate-specific algicidal compound secreted by Shewanella strain IRI-160, and uses thereof for controlling the growth of a dinoflagellate.

Description

DINOFLAGELLATE-SPECIFIC ALGICIDAL COMPOUND AND USES THEREOF

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application No.

61/362,881, filed July 9, 2010, the content of which is incorporated herein by reference in its entirety for all purposes.

REFERENCE TO U.S. GOVERNMENT SUPPORT

This work is supported by a grant from the U.S. Environmental Protection Agency (Grant Nos. EPA STAR ECOHAB R83-3221 AND R83-1041) and a grant from the National Oceanic and Atmospheric Administration (NOAA) (Grant No.

NA10NOS4780136). The United States has certain rights in the invention.

FIELD OF THE INVENTION

The invention relates generally to the use of a natural bacterial product for preventing or mitigating dinoflagellate blooms.

BACKGROUND OF THE INVENTION

Blooms of harmful dinoflagellate species can cause significant and permanent damage to the environment and pose a serious threat to human health and marine life. Although deaths are rare, the costs associated with monitoring, loss of revenue for local fisheries, and other human health-related impacts during dinoflagellate bloom events can reach into the tens of millions of dollars for a single event. An historic bloom of Alexandrium fundyense in 2005, for example, extended from Maine to Massachusetts, resulting in the closure of shellfish beds with economic losses in shellfish sales for Massachusetts alone costing an estimated $18 million. Other examples include the Pfiesteria outbreak of 1997 in mid-Atlantic estuaries resulting in an estimated $37-$72 million in lost revenue to the seafood and recreational fishing industries, and annual blooms of Karenia brevis, with an estimated cost approaching $4 million each year just in emergency room visits in Sarasota County, Florida. These numbers represent only a fraction of the costs associated with these blooms, and the full impact of dinoflagellate blooms in terms of lost revenue, monitoring efforts, human illness and long term decline in ecosystem health over the past few decades has been conservatively estimated at greater than $ 1 billion.

Hare et al . (Harmful Algae 4 : 221-234) describe the algicidal effect of bacterium Shewanella IRI-160 against three dinoflagellate species, Pfiesteria piscicida,

Prorocentrum minimum and Gyrodinium uncatenum. In the experiments conducted by Hare et al ., the bacterial cells were added directly to the algal cu ltures. Over a period of time, the growth rates of the dinoflagellates decreased compared to control cultures. It is inconvenient and cumbersome to use the bacterial cells directly to reduce the growth of dinoflagellate species .

There remains a need for a natural and convenient product suitable for use to prevent or mitigate dinoflagellate blooms.

SUMMARY OF THE INVENTION

The present invention relates to a dinoflagellate-specific algicidal compound secreted by Shewanella strain IRI-160.

A composition comprising a compound secreted by Shewanella strain IRI-160 is provided . The compound is purified from a culture of the Shewanella strain IRI- 160, and exhibits an algicidal activity towards a dinoflagellate.

The dinoflagellate may be selected from the group consisting of genus

Karlodinium, Gyrodiunium, Pfiesteria, Alexandrium, Cochlodinium, Dinophysis, Karenia and Prorocentrum. The dinoflagellate may also be selected from the group consisting of species Pfiesteria piscicida, Karlodinium veneficum, Alexandrium fundyense,

Cochlodinium polykrikoides, Prorocentrum minimum, Gyrodinium instriatum, and Gyrodinium uncatenum.

The culture of the Shewanella strain IRI- 160 may be at a mid-log growth stage.

The compound may be purified from a filtrate of the culture of the Shewanella strain IRI-160. The compound may also be purified from a hydrophilic fraction of the filtrate. The compound may be hydrophilic.

A method for controlling the growth of a dinoflagellate in an environment, particularly an aqueous environment such as a body of water, is also provided. The method comprises applying an effective amount of a composition comprising a compound to the environment. The compound is secreted by Shewanella strain IRI- 160, purified from a culture of the Shewanella strain IRI-160, and exhibits an algicidal activity towards the dinoflagellate. The growth rate of the dinoflagellate may be maintained at no more than 0% per day.

Another method for controlling the growth of a dinoflagellate in an environment is provided. The method comprises applying an effective amount of a composition comprising a filtrate to the environment. The filtrate is obtained from a culture of the Shewanella strain IRI-160, and exhibits an algicidal activity towards the dinoflagellate. The growth rate of the dinoflagellate may be maintained at no more than 0% per day.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the relative fluorescence of K. veneficum cultures after addition of Shewanella IRI-160 (squares) or cell-free filtrate (open triangles). Error bars represent standard deviation of triplicate cultures.

Figure 2 shows the relative in vivo fluorescence of Karlodinium veneficum culture after addition of IRI-160 filtrate that had been passed through a C18 column. Fluorescence is expressed as a ratio of the fluorescence of the treatment culture to the fluorescence of the control cultures. "Pass through" is treatment with the aqueous phase that passed through the column. The 20%, 40%, 60%, 80% and 100% treatments refer to the concentration of methanol in the methanol gradient used to elute the column after the aqueous phase passed through.

Figure 3 shows the in vivo fluorescence (Y-axis) of cultures of dinoflagellates (A)

Rhodomonas sp., (B) Prorocentrum minimum, (C) Karlodinium veneficum, and (D) Gyrodinium instriatum) after addition of IRI-160 filtrate compared to control cultures. Fluorescence was measured every day for 3 days (TO - T72) . Error bars represent standard deviation of triplicate cultures.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the discovery of a compound (IRI- 106AA) secreted by Shewanella strain IRI-160 and capable of inducing cell death in

dinoflagellates by, for example, inhibiting cell cycle progression and/or by inducing programmed cell death.

According to one aspect of the invention, a composition comprising a compound (IRI- 160AA) secreted by Shewanella strain IRI- 160 is provided. Compound IRI- 160AA is purified from a culture of the Shewanella strain IRI- 160 (or Shewanella strain IRI- 160 culture), and exhibits an algicidal activity towards a dinoflagellate.

The dinoflagellate may be any dinoflagel late species, for example, a harmful dinoflagellate species. A harmful dinoflagellate species can cause significant and permanent damage to an environment, and pose a serious threat to human health and marine life. The dinoflagellate may be selected from genus Karlodinium, Gyrodiunium, Pfiesteria, Alexandrium, Cochlodinium, Dinophysis, Karenia and Prorocentrum. As an example, the dinoflagellate species may be Pfiesteria piscicida, Karlodinium veneficum, Alexandrium fundyense, Cochlodinium polykrikoides, Prorocentrum minimum,

Gyrodinium instriatum, and Gyrodinium uncatenum.

The Shewanella strain IRI-160 is a bacterial isolate from the Delaware Inland

Bays. The preparation and maintenance of the Shewanella strain IRI- 160 have been described previously by Hare et al . in Harmful Algae 4 : 221-34 (2005), the content of which is incorporated herein by reference in its entirety. A Shewanella strain IRI- 160 culture may be obtained using conventional techniq ues. For example, a colony of the Shewanella strain IRI- 160 may be transferred to any suitable medium (e.g . , LM mediuma) and incubated under suitable conditions (e.g ., at 20-24 °C) until, for example, a mid to late exponential phase. The culture may be centrifuged, and the supernatant may be passed through a filter (e.g ., Mixed Cellu lose Easters (MCE) membrane filter) having a size of no more than 0.2 μηη. As a result, a filtrate of the Shewanella strain IRI-160 culture is obtained. In one embodiment, the filtrate is aqueous (i.e., the compound is present in an aqueous medium). The filtrate may be essentially free of bacterial cells.

For example, Shewanella sp. IRI-160 was grown in LM medium to mid-log growth stage. The mixture was centrifuged to remove the medium. The cell pellet was washed twice with seawater medium (amended with nutrients), then resuspended in seawater medium and incubated at 30°C for 1 week. The cell culture was then filtered through a 0.2 pm membrane filter to remove the cells.

The filtrate containing IRI-160AA may be stored at -80°C until use. The filtrate containing IRI-160AA may be separated into hydrophobic and hydrophilic fractions. IRI-160AA remains in the hydrophilic fraction. IRI-160AA may be further purified or isolated from the filtrate, and used to control or suppress the growth of a dinoflagellate.

In a composition in accordance with the present invention, compound IRI-160AA may be purified from a Shewanella strain IRI-160 culture at a mid-log growth stage. Compound IRI-160AA may also be purified from a filtrate of the Shewanella strain IRI- 160 culture, or a hydrophilic fraction of the filtrate. Compound IRI-160AA is preferably hydrophilic.

The algicidal compound, IRI-160AA, effectively kills dinoflagellates (e.g .,

Karlodinium, Gyrodinium and Prorocentrum) in a manner consistent with programmed cell death, while having no significant effect on other algal species tested (e.g.,

Rhodomonas). IRI-160AA may be used as a dinoflagellate-specific algicide, for example, by distributing it directly into areas (e.g., bodies of water) where

dinoflagellate blooms, or by incorporating into a device that releases the algicide into areas (e.g., bodies of water) at risk of dinoflagellate blooms to prevent them from occurring. Application of the filtrate comprising compound IRI-160AA may be carried out by any suitable method such as spraying, injection or the like.

The composition of the present invention may also be used as an anti-cancer drug by inhibiting the growth of cancer cells.

According to another aspect of the invention, a method for controlling the growth of a dinoflagellate in an environment is provided. The method comprises applying an effective amount of a composition comprising a compound to the

environment. The compound is secreted by Shewanella strain IRI-160, purified from a culture of the Shewanella strain IRI-160, and exhibits an algicidal activity towards the dinoflagellate.

The term "controlling the growth of a dinoflagellate in an environment" as used herein means controlling the population or growth rate of the dinoflagellate in the environment. The environment is an aquatic system, for example, a body of fresh water or a marine environment. The population of the dinoflagellate may be

maintained at no more than about 10¹⁰ cells per ml (e.g., about 10¹⁰, 10⁹, 10⁸, 10⁷, 10⁶, 10⁵, or 10⁴ cells per ml). The growth rate of the dinoflagellate may be maintained at no more than about 0%, 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30% 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, or 500% per day.

In a method in accordance with the present invention, the dinoflagellate may be any dinoflagellate species, for example, a harmful dinoflagellate species. Examples of dinoflagellates may include those in genus Karlodinium, Gyrodiunium, Pfiesteria, Alexandrium, Cochlodinium, Dinophysis, Karenia and Prorocentrum. Other examples include the dinoflagellate species Pfiesteria piscicida, Karlodinium veneficum,

Alexandrium fundyense, Cochlodinium polykrikoides, Prorocentrum minimum,

Gyrodinium instriatum, and Gyrodinium uncatenum.

In a method in accordance with the present invention, compound IRI-160AA is secreted by the Shewanella strain IRI-160, and purified from a Shewanella strain IRI- 160 culture. Compound IRI-160AA is preferably purified from Shewanella strain IRI- 160 culture at a mid-log growth stage. Compound IRI-160AA may also be purified from a filtrate of the Shewanella strain IRI-160 culture, or a hydrophilic fraction of the filtrate. Compound IRI-160AA is preferably hydrophilic. According to yet another aspect of the invention, a method for controlling the growth of a dinoflagellate in an environment is provided. The method comprises applying an effective amount of a composition comprising a filtrate to the environment. The filtrate is obtained from a culture of the Shewanella strain IRI-160, and exhibits an algicidal activity towards the dinoflagellate. The population of the dinoflagellate may be maintained at no more than about 10¹⁰ cells per ml (e.g ., about 10¹⁰, 10⁹, 10⁸, 10⁷, 10⁶, 10⁵, or 10⁴ cells per ml). The growth rate of the dinoflagellate may be maintained at no more than about 0%, 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30% 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, or 500% per day.

In a method in accordance with the present invention, the filtrate may be obtained from a Shewanella strain IRI-160 culture at a mid-log growth stage. The filtrate may be the supernatant of a Shewanella strain IRI-160 culture. The filtrate may also be a hydrophilic fraction of the supernatant.

The initially obtained filtrate may be concentrated, if desired, by removing at least a portion of the water or other volatile substances present so as to increase the concentration of compound IRI-160AA. The filtrate and compound IRI-160AA may be dried, and used for a method in accordance with the present invention. Drying may be carried out by any suitable method such as, for example, spray drying, freeze drying or the like. The dried filtrate or compound may be in the form of, for example, a dust, a powder, or granules, and introduced into an environment (e.g ., a body of water) to control the growth of dinoflagellate in the environment. Such introduction may be accomplished by any suitable method such as spraying, broadcast spreading, or the like. For ease of handling, the dried filtrate or compound may be coated onto or admixed with a carrier.

The term "about" as used herein when referring to a measurable value such as an amount, a percentage, and the like, is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1% from the specified value, as such variations are appropriate. Example 1. Algicidal activity of a Shewanella strain IRI-160 filtrate (IRI-160AA) on Karlodinium veneficum cultures

The impact of compound IRI-160AA purified from a Shewanella strain IRI-160 culture on Karlodinium veneficum cultures was studied. The methodologies for preparing the Karlodinium veneficum cultures and the Shewanella strain IRI-160 cells, as well as for the test are described in details by Hare et al. in Harmful Algae 4: 221-34 (2005), which is incorporated herein by reference.

Shewanella sp. IRI-160 was grown in LM medium to mid-log growth stage. The mixture was centrifuged to remove cells. The cell culture was then filtered through a 0.2 urn filter to further remove the cells. The filtrate containing IRI-160AA (or IRI-160 filtrate) was stored at -80°C until use.

The IRI-160AA filtrate and Shewanella IRI-160 cells were added at 10% of the total volume of the Karlodinium veneficum cultures and at 10⁹ cells per ml_,

respectively, and incubated at 25 °C. A control with no addition was included. In vivo fluorescence was measured on the Karlodinium veneficum cultures on days 0, 1, 2, 3 and 4. Like Shewanella IRI- 160 cells, IRI-160AA was found effective in killing

Karlodinium veneficum on days 1, 2, 3 and 4 (Fig. 1).

Example 2. Algicidal activity of a hydrophilic fraction of a Shewanella strain IRI-160 filtrate (IRI-160AA) on Karlodinium veneficum cultures

A study was carried out to characterize compound IRI-160AA in the Shewanella strain IRI-160 filtrate. Filtrates were obtained from Shewanella strain IRI-160 cultures and were passed through a C18 column, and eluted with methanol/water mixtures. A hydrophilic fraction (Pass through) of the Shewanella strain IRI-160 filtrates showed an algicidal activity on days 1 and 2 after addition to K. veneficum cultures while factions collected by elution with 20%, 40%, 60%, 80% and 100% methanol/water mixtures did not (Fig. 2).

Example 3. Dinoflagellate-specific algicidal activity of a Shewanella strain IRI-160 filtrate (IRI-160AA) A study was carried out to determine whether the algicidal activity of the

Shewanelia strain IRI-160 filtrate (IRI-160AA) was dinoflagellate specific. The

Shewanelia strain IRI-160 filtrate (IRI-160AA) was prepared and added to Kariodinium veneficum, Gyrodinium instriatum, Rhodomonas sp., and Prorocentrum minimum cultures as described in Example 1. Viable cells in the cultures measured by in vivo fluorescence on days 0 (TO), 1 (T24), 2 (T48) and 3 (T72). Dinoflagellate-specific algicidal activity was observed in Kariodinium veneficum, Gyrodinium uncatenum, and Prorocentrum minimum cultures, but not the non-dinoflagellate species Rhodomonas sp. (Fig. 3).

All documents, books, manuals, papers, patents, published patent applications, guides, abstracts, and/or other references cited herein are incorporated by reference in their entirety. Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with the true scope and spirit of the invention being indicated by the following claims.

Claims

What is Claimed:

1. A composition comprising a compound secreted by Shewanella strain IRI- 160, wherein the compound is purified from a culture of the Shewanella strain IRI-160, and exhibits an algicidal activity towards a dinoflagellate.

2. The composition of claim 1, wherein the dinoflagellate is selected from the group consisting of genus Karlodinium, Gyrodiunium, Pfieste a, Alexandrium, Cochlodinium, Dinophysis, Karenia and Prorocentrum.

3. The composition of claim 1, wherein the dinoflagellate is selected from the group consisting of species Pfiesteha piscicida, Karlodinium veneficum, Alexandrium fundyense, Cochlodinium polykrikoides, Prorocentrum minimum, Gyrodinium instriatum and Gyrodinium uncatenum.

4. The composition of claim 1, wherein the culture of the Shewanella strain IRI-160 is at a mid-log growth stage.

5. The composition of claim 1, wherein the compound is purified from a filtrate of the culture.

6. The composition of claim 5, wherein the compound is purified from a hydrophilic fraction of the filtrate.

7. The composition of claim 1, wherein the compound is hydrophilic.

8. A method for controlling the growth of a dinoflagellate in an environment, comprising applying an effective amount of a composition comprising a compound to the environment, wherein the compound is secreted by Shewanella strain IRI-160, purified from a culture of the Shewanella strain IRI-160, and exhibits an algicidal activity towards the dinoflagellate.

9. The method of claim 8, wherein the dinoflagellate is selected from the group consisting of genus Karlodinium, Gyrodiunium, Pfiesteha, Alexandrium,

Cochlodinium, Dinophysis, Karenia and Prorocentrum.

10. The method of claim 8, wherein the dinoflagellate is selected from the group consisting of species Pfiesteha piscicida, Karlodinium veneficum, Alexandrium fundyense, Cochlodinium polykrikoides, Prorocentrum minimum, Gyrodinium instriatum and Gyrodinium uncatenum.

11. The method of claim 8, wherein the culture of the Shewanella strain IRI- 160 is at the mid-log growth stage.

12. The method of claim 8, wherein the compound is purified from a filtrate of the culture.

13. The method of claim 12, wherein the compound is purified from a hydrophilic fraction of the filtrate.

14. The method of claim 8, wherein the growth rate of the dinoflagellate is maintained at no more than 0% per day.

15. A method for controlling the growth of a dinoflagellate in an environment, comprising applying an effective amount of a composition comprising a filtrate to the environment, wherein the filtrate is obtained from a culture of the Shewanella strain IRI- 160, and exhibits an algicidal activity towards the dinoflagellate.

16. The method of claim 15, wherein the dinoflagellate is selected from the group consisting of genus Karlodinium, Gyrodiunium, Pfiesteria, Alexandrium,

Cochlodinium, Dinophysis, Karenia and Prorocentrum.

17. The method of claim 15, wherein the dinoflagellate is selected from the group consisting of species Pfiesteria piscicida, Karlodinium veneficum, Alexandrium fundyense, Cochlodinium polykrikoides, Prorocentrum minimum, Gyrodinium instriatum and Gyrodinium uncatenum.

18. The method of claim 15, wherein the growth rate of the dinoflagellate is maintained at no more than 0% per day.

19. The method of claim 15, wherein the culture of the Shewanella strain IRI-160 is at the mid-log growth stage.

20. The method of claim 15, wherein the filtrate is a supernatant of the culture.