Drug – bio-affecting and body treating compositions – Whole live micro-organism – cell – or virus containing – Fungus
Reexamination Certificate
2000-03-02
2003-04-08
Naff, David M. (Department: 1651)
Drug, bio-affecting and body treating compositions
Whole live micro-organism, cell, or virus containing
Fungus
C424S900000, C424S405000, C435S261000, C435S254100, C435S267000, C435S911000
Reexamination Certificate
active
06544512
ABSTRACT:
BACKGROUND OF THE INVENTION
Currently a single strain of fungus, identified as
Ampelomyces quisqualis
, AQ10 Biofungicide, is in commercial use for biocontrol of powdery mildew on grapes and other crops (Hofstein, R. and B. Fridlender, 1994, Development of production, formulation and delivery systems, p. 1273-1280. In Brighton Crop Protection Conference-Pests and Diseases, vol. 3. British Crop Protection Council, Farnham, U.K.). The market for this biofungicide was over $1 million in 1998 and is expected to grow. Phylogenetically this strain is grouped within the divergent
Ampelomyces lade.
A few reports have also identified Phoma species that are antagonistic to fungal plant pathogens. A Phoma sp (P66A) significantly reduced conidial germination of apple scab (
Venturia inequalis
) (Ouimet et al., 1997
, Can. J. Bot.
75: 632-639) and
Phoma etheridgei
produced antifungal compounds inhibitory to the tree pathogen
Phellinus tremulae
(Hutchinson et al., 1994
, Can. J. Bot
., 72: 1424-1431).
Several isolates of fungi, previously attributed to
genus Ampelomyces
, have now been shown to belong to genus Phoma through study of morphology and rDNA ITS1 sequence analysis. The species identified as
Phoma glomerata
has now been demonstrated to effectively colonize and suppress development of powdery mildew.
SUMMARY OF THE INVENTION
An object of the present invention is to provide a biocontrol agent which comprises
Phoma glomerata
for suppression of the development of fungal diseases such as powdery mildew on plants.
Another object of the present invention is to provide a method of suppressing development of fungal diseases such as powdery mildew on plants which comprises applying
Phoma glomerata
to a plant.
DETAILED DESCRIPTION OF THE INVENTION
Cultures isolated from several different genera of powdery mildews in both Europe and the United States have now been identified as
P. glomerata.
These fungi are demonstrated herein to have a number of characteristics which render them readily distinguishable from Ampelomyces.
For example, stipitate pycnidia that develop into slow-growing colonies characterize
A. quisqualis
(Galper, S., A. Sztejnberg, and N. Lisker, 1985
, Can. J. Microbiol
., 31: 961-964; and Kiss, L. and Nakasone,: K. K., 1998
, Current Genetics
33: 362-367). Sessile pycnidia that develop into rapidly-growing colonies characterize
Phoma glomerata
(Boerema et al., 1965
, Persoonia
, 4: 47-68.; and White, J. F., Jr. and Morgan-Jones, G., 1987
, Mycotaxon
, 28: 437-445). For cultural growth rates, an average of 8±1 mm/day for the
P. glomerata
isolates was measured while an average of 0.8±0.1 mm/day for the Ampelomyces isolates were measured.
The process of pycnidia formation in association with the powdery mildew is also different between the two genera. Ampelomyces infects conidiogenous cells of the powdery mildew, internally colonizing them and forming pycnidia within the conidiophore so that the pycnidia appear stipitate. Phoma does not appear to internally-infect conidiogenous cells and pycnidia are formed directly on the leaf surface so that they are sessile. While many species of Phoma are known to be plant pathogens (Sutton, B. C., :1980, The Coelomycetes, Commonwealth Mycological Institute, Kew, U. K.),
P. glomerata
is not known to be a pathogen of plants. However, it is adept at saprophytic growth in tissues of plants and is known to be a secondary invader of diseased tissues, perhaps feeding on fungal saprophytes or pathogens of diseased tissues (Morgan-Jones, G., 1967
, Phoma glomerata, CMI Desc. Pathol. Fungi Bact
., No. 134; Commonwealth Mycological Institute; Sutton, B. C., 1980, The Coelomycetes, Commonwealth Mycological Institute, Kew, U.K.; and White, J. F., Jr. and Morgan-Jones, G., 1987, Studies in the genus Phoma. VII. Concerning
Phoma glomerata
, Mycotaxon, 28: 437-445).
Dictyochlmydospores was observed in only
A. humuli
and the South River
P. glomerata
. However, the similarity of colonies and pycnidia of fungi identified as
A. heraclei, A. humuli
and
A. quercinus
, and the very, close sequence homology among these isolates suggests that all are isolates of
P. glomerata
that have been misidentified as species of Ampelomyces.
Further, phylogenetic analysis revealed considerable divergence in the sequences included in the Ampelomyces lade as compared to those of the
Didymella/Phoma
clade. Internal transcribed spacer region 1, also referred to herein as ITS1 sequences, of the South River
P. glomerata
(GenBank AF126816; SEQ ID NO:1) isolate and
A. heraclei
(ATCC 36804) were identical, while
P. glomerata
differed from
A. humuli
(GenBank AF035779; SEQ ID NO:5) in a single indel change. Because of their near identity with
P. glomerata
, both
A. heraclei
and
A. humuli
were excluded from the maximum likelihood analysis.
A. quercinus
differed in three changes: an indel and two nucleotide substitutions. Maximum likelihood analysis produced two nearly identical trees (tree length=204, consistency index=0.784, rescaled consistency index=0.608, homoplasy index=0.216, retention index=0.776). Thus, South River
P. glomerata
and
A. quercinus
are grouped together in the
Didymella/Phoma
lade, while the ITS1 sequence of putative South River
Ampelomyces quisqualis
isolate grouped in the Ampelomyces lade demonstrated close similarity to
Ampelomyces quisqualis
(ATCC 200245) isolated from grapes.
P. glomerata
was demonstrated to be an effective biocontrol agent for suppressing development of fungal diseases such as powdery mildew on plants. In inoculation experiments, examination of inoculation sites after 30 days revealed that control leaves showed development of the powdery mildew cleistothecia while all leaves treated with
P. glomerata
conidia showed development of abundant pycnidia and suppression of powdery mildew cleistothecia in the white mycelium in and around the inoculation sites. Pycnidia were removed from leaves using fine needles and plated onto PDA+3. Colonies that developed were confirmed by observation of dictyochlamydospores, pycnidia and by subsequent sequence analysis to be
P. glomerata.
The very slow growth in culture media, internal colonization of powdery mildews, and. apparent limitation to powdery mildew as a substrate, are all indicative that Ampelomyces are obligate parasites of powdery mildews. In contrast, the rapid growth of
P. glomerata
in culture and its known occurrence on many types of substrates are indicative that it is a facultative parasite of powdery mildew, infecting powdery mildew when it is available. Thus, from the widespread occurrence of this hyperparasite, it is believed that
P. glomerata
has untapped potential as a biocontrol agent. Further, with its rapid development in culture and its broader potential to grow on various substrates, it is believed that of
P. glomerata
is even more useful than species of Ampelomyces as biocontrol agents of fungal diseases, and in particular powdery mildew, of plants.
Accordingly,
P. glomerata
can be incorporated into a biocontrol agent for suppression of fungal diseases on plants. In a preferred embodiment, this biocontrol agent is used to suppress the development of powdery mildew plants. This biocontrol agent can be applied to plants, preferably on the leaves of plants, to suppress the development of fungal disease such as powdery mildew on the plant.
The following nonlimiting examples are provided to further illustrate the present invention.
REFERENCES:
patent: 5288488 (1994-02-01), Backman et al.
patent: 5432147 (1995-07-01), Winston et al.
patent: 5856002 (1999-01-01), Mori
patent: 5985795 (1999-11-01), Suganuma
Arie et al., Control of soilborne clubroot disease of cruciferous plants by epoxydon fromPhoma glomerata1998, Plant Pathol. 47, No. 6, pp. 743-748 Abstract only.*
Brighton Crop Protection Conference—Pests and Diseases, vol. 3. British Crop Protection Council, Franham, U.K.
Desai, U. J. and P. K. Pfaffle, “Single-Step Purification of a Thermostable DNA Polymerase E
Licata & Tyrrell P.C.
Naff David M.
Rutgers, the State University of New Jersey
Ware Deborah K.
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