1. Field of the Invention
Head scab, also known as Fusarium head blight (FHB), is a devastating disease of wheat and barley that is primarily caused by the fungus Gibberella zeae (anamorph=Fusarium graminearum). This disease can reach epidemic levels and causes extensive damage to wheat and barley in humid and semi-humid wheat growing areas of the world. In recent growing seasons, the disease has caused large scale devastation in the United States, Canada and China. FHB was responsible for almost 500 million bushels of wheat lost in the United States from 1991 until present. Economic loss has been estimated at between 1.3 to 2.6 billion during this time period. In an epidemic in Indiana in 1986, grain samples from 43 of 44 counties had scab [Tuite et al., (1990) Plant Dis. 74:959-962]. Other countries of the world that produce large amounts of wheat in humid and semi-humid regions and would be susceptible to major outbreaks of FHB include India, Russia, France, Germany and the United Kingdom.
The infection of seed by G. zeae reduces seed germination, seedling vigor and plant emergence [Bechtel et al., (1985) Cereal Chem. 62:191-197]. Infection of wheat kernels by G. zeae reduces grain yield and affects grain quality [Clear et al., (1990) Can. J. Plant Sci. 70:1057-1069]. Reductions in grain yield are at least partially attributable to the pathogen producing the vomitoxin deoxynivalenol (DON) [Snijders, (1990) Neth J. Plant Pathol. 96:187-198; Proctor et al., (1995) MPMI 8:593-601] which can inhibit amino acid incorporation and protein production in plant tissues [Casale et al., (1988) Phytopathology 78:1673-1677]. This toxin is also implicated in adversely affecting the growth of mammalian cells [Knasmxc3xcller et al., (1997) Mutation Research 391:39-48]. DON is retained in semolina at approximately 50% and F. graminearum has a strong adverse effect on pasta color when Fusarium damaged kernels make up as little as 2% of a lot [Dexter et al., (1997) Cereal Chem. 74:519-525]. Additionally, G. zeae infected kernels can contain the estrogenic toxin zearalenone. Grain contaminated with either of these mycotoxins often is downgraded or can not be sold [Tuite et al., (1990)]. Contaminated grain is frequently unsuitable for human consumption and may be refused as feed [Vesonder et al., (1980) Process Biochem. 16:12-15]. The importance of FHB was recognized by the 105th U.S. Congress when it adopted the xe2x80x9cWheat and Barley Protection Actxe2x80x9d that authorized expenditure of 26 million dollars for the study of FHB.
This invention relates to five microbial antagonists that reduce FHB.
2. Description of the Prior Art
Though some success in controlling FHB can be expected by plowing fields to bury crop residues infested with F. graminearum after harvest [Bai et al., (1994) Plant Dis. 78:760-766], minimal tillage practices render this alternative unacceptable. Some progress has been made in finding and analyzing scab resistance in wheat, though all cultivars in current production are susceptible [Bai et al., (1994)]. Foliar fungicides applied at anthesis can be useful in reducing scab [McMullen, (1998) Fungicide technology network of the National FHB initiativexe2x80x941998 Report. Proceedings of the 1998 Head Scab Forum, Michigan State University, October 26-27, pp.47-49], but few fungicides are registered for use on wheat this late in the growing season [Shaner et al., (1992) Fungic. Nematicide Tests. 47:206-207]. Additionally, costs and concerns in the public and private sectors over pesticide residues in the environment and in food products render this disease control alternative less attractive.
Biological control, though currently not available, would be an environmentally acceptable method for substantially decreasing the level of disease incited by G. zeae. Though biological control agents (BCA""s) have become a more acceptable control alternative for plant pathogens and BCA products are being marketed to a greater extent than ever before [Fravel et al., (1996) Biological and Cultural Tests 11:1-7] to date there have been few attempts to develop strategies and microorganisms for biologically controlling FHB [Stockwell et al., (1997) Phytopathology 87(6):S94; Perondi et al, (1996) Fitopatologia Brasiliera 21:243-249]. The life cycle of G. zeae suggests that the pathogen is especially susceptible to control using applied microorganisms at anthesis through the soft dough stage of kernel development, when the majority of wheat head infection by G. zeae is generally considered to occur [Andersen, (1948) Phytopathology 38:595-611; Arthur, (1981) Indiana Agric. Exp. Stn. Bull 36:129-138]; Fernando et al., (1997) Phytopathology 87(6):S30 (Abstr.)].
Luz et al. [5th International Congress of Plant Pathology, Abstracts of Papers, p. 348 (1988)] reports in vitro screening in excess of 300 bacteria and yeasts isolated from wheat against F. graminearum. Likewise, Perondi et al. [Anais do 2xc2x0 Simposio de Controle Biolxc3x3gico, Brasilia, DF, p. 128 (Abstr., 1990); Fitopatologia Brasiliera 21:243-249 (1996)] reported testing microbial strains as possible antagonists against F. graminearum. Promising strains selected by the funnel method and tested in greenhouse studies were shown by Luz et al. [Fitopatologia Brasiliera 15(3)246-247 (1990)] to diminish the severity of wheat scab between 7 and 31% when compared to the control.
We have now discovered 4 yeasts and 1 bacterium as being superior antagonists of F. graminearum. These antagonists suppress FHB in cereals, particularly in wheat and barley. The antagonists were selected from a pool of more than 700 microbial strains obtained from anthers of wheat. Initial selection of specific anther colonists for further study was based on random selection or the ability of a colonist to utilize a compound of potential use in formulating the colonist. Selected microbes were then bioassayed on seed heads of a cereal plant, inoculated with F. graminearum, for the ability of the strain to reduce the severity of FHB. The five antagonists selected in this manner were superior in reducing FHB severity in greenhouse and in field trials.
In accordance with this discovery, it is an object of this invention to provide novel microbial strains that suppress the profusion of F. graminearum in seed heads of wheat and barley.
This and other objects of the invention will become readily apparent from the ensuing description.
Purified cultures of four yeasts and one bacteria identified as being effective antagonists of F. graminearum have been deposited on Sep. 7, 1999, in the U.S. Department of Agriculture, Agricultural Research Service Culture Collection in Peoria, Ill., under the terms of the Budapest Treaty. Accession Numbers for these deposits are as follows:
For purposes of this invention it is understood that the use of term xe2x80x9cFusariumxe2x80x9d is intended to include both the sexual (teleomorphic) stage of this organism and also the asexual (anamorphic) stage, also referred to as the perfect and imperfect fungal stages, respectively. For example, the anamorphic stage of Gibbereblla zeae is known as Fusarium graminearum, the causative agent of FHB. This disease results when the flower or seed head becomes inoculated with conidia produced by the imperfect form OR ascospores produced by the perfect form of this fungus.
The expression xe2x80x9csuperior antagonistxe2x80x9d used herein in reference to a microorganism is intended to mean that the subject strain exhibits a degree of inhibition of Fusarium-induced head blight exceeding, at a statistically significant level, that of an untreated control.
The term xe2x80x9ccerealxe2x80x9d as used herein is intended to refer to any cereal species that is normally susceptible to FHB. Cereals reported to be susceptible include wheat, barley, oats, and triticale, though wheat and barley are the two crops in which this disease presents a significant economic problem. Tests in the Examples, below, with one variety of hard red spring wheat, two varieties of soft red winter wheat and one variety of durum wheat demonstrate that antagonist strains of this invention are efficacious in reducing FHB on all these types and varieties of wheat. Any of these cereals may be target species for FHB control.
F. graminearum primarily infects the heads (flower heads, seed heads, or seed spikes) of cereal plants from the time of flowering through the soft dough stage of head development. Germinated conidia or ascospores of F. graminearum penetrate through anthers and associated tissues to initiate infection of the host and the development of symptoms of FHB.
This invention emanated from the postulation that some of the microorganisms present on (and opportunistically colonizing) cereal anthers may be effective in biologically controlling FHB. Though it is likely that some or all of these same organisms would be present in the head after dehiscence of the anthers, it is considered preferred to collect samples from anthers prior to dehiscence in order to maximize the possibility that a given colonist in the sample is instrumental in the biocontrol of F. graminearum. Samples may be subjected to immediate isolation, or alternatively may be frozen in 10% glycerol or the like until use.
Strains may be obtained from the collected anther samples by conventional methods as known in the art. Aqueous or glycerol suspensions of the samples are preferably mixed under conditions of shear to liberate the microorganisms from anther surfaces. Suspensions containing the microorganisms are then serially diluted onto suitable media. Malt extract agar and Tryptic soy broth are exemplary media for use in preferentially isolating yeasts and bacteria, respectively. Corn steep liquor (CSL) medium represents a general purpose isolation medium composed of inexpensive nutrient sources. Microorganisms isolated from CSL would, by the fact that they grew on this medium, be preselected as likely to be amenable to production on a medium that is economically feasible for commercial producers of a prospective biological control product.
Candidate organisms are passed through a plant bioassay in which cells of the microbial strain are introduced to a cereal plant seed head inoculated with conidia of F. graminearum. Typically, the F. graminearum will be produced on a solidified growth medium and the level of harvested inoculum should be on the order of about 104-106 conidia/ml, preferably about 105 conidia/ml of aqueous suspension. The cells of candidate antagonist in medium or a suitable buffer are introduced at a level of approximately 107-108 cfu/ml. In one embodiment of the invention, the conidia of F. graminearum and cells of the test strain are combined in a weak phosphate buffer and approximately 10 xcexcL of the suspension are used to inoculate the plant seed head. The plants are then cultivated under conditions of near 100% relative humidity conducive to infection by the fungus for a period of about 3 days. After a period of time sufficient for noticeable development of the disease (usually at least about 2 weeks post inoculation), microbes used to treat seed heads that do not develop visible symptoms of FHB are selected for subsequent evaluation.
Organisms selected in the plant bioassay described above are optionally subjected to a second, more highly replicated plant seed head bioassay similar to the first. The organisms are again grown on a suitable medium until sufficiently expanded for use in the bioassay. However, in this second plant bioassay, it is preferable to grow the strains in liquid culture since this practice is widely used in industry and, antagonists must show bioefficacy when grown under liquid culture conditions. Colonized broth containing cells of individual strains and a conidial suspension of F. graminearum are used to inoculate seed heads as previously described. The cells and conidial suspension may be precombined prior to inoculation. As in the first plant bioassay, microbes used to treat seed heads that do not develop visible symptoms of FHB are selected as candidate antagonists.
Confirmation of antagonist efficacy in controlling F. graminearum can be made in scaled-up greenhouse studies or in field studies in which flowering plants are treated with cells of the test strains, before, during, or after inoculation with conidia of F. graminearum. The plant treatment can be conducted in the same manner as a bona fide field application as described in more detail in Examples 7-9, below.
The aformentioned method was used to isolate and identify five strains of FHB antagonist: OH 71.4 (NRRL Y-30213), Torula aurea; OH 72.4 (NRRL Y-30214), an unidentified yeast; OH 131.1 (NRRL B-30212), Bacillus sp.; OH 181.1 (NRRL Y-30215), Torula sp.; and OH 182.9 (NRRL Y-30216), Cryptococcus nodaensis. OH 181.1 is considered to be a new species of Torula.
Optimal conditions for the cultivation of antagonists of the invention will, of course, depend upon the particular strain. However, by virtue of the conditions applied in the selection process and general requirements of most microorganisms, a person of ordinary skill in the art would be able to determine essential nutrients and conditions.
The antagonists would typically be grown in aerobic liquid cultures on media which contain sources of carbon, nitrogen, and inorganic salts assimilable by the microorganism and supportive of efficient cell growth. Preferred carbon sources are hexoses such as glucose, but other assimilable sources such as amino acids, may be substituted. Many inorganic and proteinaceous materials may be used as nitrogen sources in the growth process. Preferred nitrogen sources are amino acids and urea but others include gaseous ammonia, inorganic salts of nitrate and ammonium, vitamins, purines, pyrimidines, yeast extract, beef extract, proteose peptone, soybean meal, hydrolysates of casein, distiller""s solubles, and the like. Among the inorganic minerals that can be incorporated into the nutrient medium are the customary salts capable of yielding calcium, zinc, iron, manganese, magnesium, copper, cobalt, potassium, sodium, molybdate, phosphate, sulfate, chloride, borate, and like ions.
For the organisms of the invention, cell growth can be achieved at temperatures between 1 and 36xc2x0 C., with the preferred temperature being in the range of 15-30xc2x0 C. The pH of the nutrient medium can vary between 4 and 9, but the preferred operating range is pH 6-8. ordinarily, maximal cell yield is obtained in 20-72 hours after inoculation.
The antagonists of the invention can be applied by any conventional method to the surfaces of cereal heads. For example, they can be applied as an aqueous spray or dip, as a wettable powder, or as a dust. Formulations designed for these modes of application will usually include a suitable liquid or solid carrier together with other adjuvants, such as wetting agents, sticking agents and the like. Starch, polysaccharides, sodium alginate, cellulose, etc. are often used in such formulations as carriers and sticking agents.
The expressions xe2x80x9can effective amountxe2x80x9d and xe2x80x9ca suppressive amountxe2x80x9d are used herein in reference to that quantity of antagonist treatment which is necessary to obtain a reduction in the level of disease relative to that occurring in an untreated control under suitable conditions of treatment as described herein. The actual rate of application of a liquid formulation will usually vary from a minimum of about 1xc3x97103 to about 1xc3x971010 viable cells/ml and preferably from about 1xc3x97106 to about 5xc3x97109 viable cells/ml. Under most conditions, the strains of the invention described in the examples, below, would be optimally effective at application rates in the range of about 1xc3x97106 to 1xc3x97109 viable cells/ml, assuming a mode of application which would achieve substantially uniform contact of at least about 50% of the wheat head. If the antagonists are applied as a solid formulation, the rate of application should be controlled to result in a comparable number of viable cells per unit area of cereal head surface as obtained by the aforementioned rates of liquid treatment.
It is envisioned that the temperatures at which the antagonists are effective would range from about 5xc2x0 C. to about 35xc2x0 C. The preferred temperature range is 15-30xc2x0 C., and the optimal range is considered to be 18-28xc2x0 C.
The antagonists can theoretically be applied to the seed head at any time after the boot stage and before the hard dough stage of cereal development. The cereal head is only susceptible to infection by F. graminearum from the onset of flowering (anthesis) through the soft dough stage of kernel development. Thus, the best time to apply the biological control agents would be from the time immediately preceding flowering until as late as the soft dough stage of kernel development. Application of antagonists to heads before flowering would allow antagonists to have colonized wheat head parts prior to the wheat head becoming susceptible to infection. Additionally, antagonists would be well positioned to colonize and protect anthers as they emerge from florets. However, it is expected that the antagonists would still be effective if applied after flowering has begun, but before the hard dough stage of development. Though Example 5, below, demonstrates that delays of 4 h between pathogen and antagonist inoculation did not significantly affect antagonist performance, it is anticipated that longer delays may decrease the effectiveness of the microbial treatment depending on methods of cell formulation and application.
The following examples are intended only to further illustrate the invention and are not intended to limit the scope of the invention which is defined by the claims.