Herbicide comprising phytotoxins of Lasiodiplodia theobromae...

Plant protecting and regulating compositions – Plant growth regulating compositions – Micro-organisms or from micro-organisms

Reexamination Certificate

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Reexamination Certificate

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06277786

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to the biological control of unwanted herbs by the use of a pathogenic toxin from
Lasiodiplodia theobromae
(LT). The invention also provides a novel herbicidal composition, a process for the preparation of the composition and its use in controlling weeds.
BACKGROUND AND DESCRIPTION OF THE PRIOR ART
The Phytotoxins are host-specific because they are toxic only to the host that is, susceptible to the pathogen (which produces the toxin), and if they induce nearly all symptoms of the disease then they are considered to be definitive chemical probes in a study of disease susceptibility and physiological stress at the molecular level (Bottinii A. T., and Gilchrist, D. G. 1981. Tetrahedron lett. 22:2719-2722). Physiological, biochemical, genetic and histological data all confirm that these toxic compounds produced by the fungi are the key determinants of disease and host selection (Scheffer, R. P., Kohmoto, K. and Durbin, R. D., 1989. ed. Host-Specific Toxins, pp.1-17).
The tolerance and sensitivity to a toxin is controlled by the same genes in the same way as they control resistance and susceptibility to the fungus (Scheffer, Robert P. and Livingston, Robert S.,1984. Science, 223:17-21). If a fungus does not grow on a plant, the phytotoxin produced by the fungus will not affect that plant, depending on whether or not it has dominant or recessive alleles (Grogan, R. G., Kimble, K. A., and Misaghi, I., 1975. Phytopathology, 65:880-886).
Members of the genus Lasiodiplodia are known to produce a wide range of phytotoxic compounds which affect a large number of plants on which the fungus is found (Domsch, K. H., and Ganms, W., 1980. Compendium of soil fungi, vol.1, Academic press, pp:143-145 ).
Lasiodiplodia theobromae
is a widespread soil-borne saprophyte or wound parasite in the tropics of all continents but has not been reported from the temperate zones of Europe and the U.S.A. It is common on fruits, stems and roots of numerous tropical and subtropical plants, particularly Theobromae, Castilla, Hevea, Citrus, Mangifera, Ficus and Musa (Goss, R. D., Cox, E. A. and Strotzky, 1961. Mycologia, 53:262-277; Laskin, A. I. and Lechevalier, H. A. (EDS) 1973, Handbook of Microbiology, Vol. 3, Microbial Products, CRC Press, Cleveland)
There are many fungi which are pathogenic to weeds because they produce phytotoxins and, therefore, could be used as herbicides (Abbas, H. K., Boyette, C. D., Hoagland, R. E., and Vesonder, R. F., 1991. Weed Sci. 39:673-677. The genus Aternaria is also known to produce a wide range of phytotoxins (Bruce, V. R., Stack, M. E., and Mislivec, P. B., 1984. J. Food Sci. 49:1626-1627. An isolate of
Fusarium moniliforme
, obtained from infected jimsonweed, was found to produce fumonisin phytotoxin.
The applicants in the course of their studies on
Coleus forskohlii
Briq. (Labiate) found that this plant is susceptible to attack by micro-organisms and most of the pathogens attacking the plant have not been identified. The applicants have also found that a fungus
Lasiodiplodia theobromae
which is responsible for the rot disease in coleus plants, releases certain toxins which exhibit phytotoxicity against weeds like Parthenium.
OBJECTS
The main object of this invention is to provide a novel herbicide effective against a wide range of herbs including weeds.
Another object of the invention is to provide a herbicide comprising a phytotoxically effective amount of the toxin produced by
Lasiodiplodia theobromae
together with an additive or a carrier compatible with the said toxin and soil environment.
A further object of the invention is to provide a method for the preparation of the herbicide.
Yet another object is to provide a method for controlling weeds using the toxins produced by
Lasiodiplodia theobromae.
SUMMARY
In accordance with the foregoing objects, the invention provides a herbicide comprising phytotoxins produced by
Lasiodiplodia theobromae
, optionally, with appropriate carriers and additives. The invention also provides methods for the preparation of the herbicidal composition and methods for control of herbs including weeds in the field.
DETAILED DESCRIPTION OF THE INVENTION
In the present invention, the applicants describe a method for controlling weeds such as
Parthenium hysterophorus
, duckweeds, jimsonweed, black nightshade,
Euphorbia hirta
, prickly sida etc. using a phytotoxic amount of LT-toxin produced by the fungus
Lasiodiplodia theobromae
. The LT-toxin can be applied to the weeds in any suitable form including cultures of
L. theobromae
fungus in any suitable solvent such as water, and culture filtrate. The LT-toxin can be applied as a post-emergent or pre-emergent herbicide.
Coleus forskohlii
Briq. (Labiatae) is an useful perennial herb growing in many parts of the world including India and in subtropical Himalayan regions. This herb has been used since ancient times for medical treatment in Hindu and Ayurvedic traditional medicine. Screening of extracts of roots of this plant led to the isolation of a group of diterpenoids possessing the basic skeleton of 11-oxo-manoyl oxide (Tandon et al, 1977. Indian J. Chem. 15B, 880), the main constituent of which being forskolin. Forskolin possesses positive inotropic, antihypertensive and adenylate cyclase stimulating activities. Clinical applications of this diterpenoid as a hypertensive, spasmolytic, lipolytic or antithrombotic agent and for the treatment of glaucoma and cardiac insufficiency are studied. (DeSouza et al, 1983. Mol. Res. Reviews, 201). Forskolin is an approved drug for cancer treatment. Reports are available on the isolation of forskolin from the root of
C. forskohlii
and on the in vitro culture of the same (Yanagihara et al, 1998. Plant Med. 54, 200-204). Because of its pharmacological importance (Mersinger et al, 1995, Plant Med. 62, 169-172) farmers have been encouraged to cultivate
C. forskohlii
in large areas. However, this plant is susceptible to attacks by microorganisms causing severe loss to the farmers, and most of the pathogens have not been identified until the date of this patent application is made.
To isolate the phytotoxin, the applicants cultured the fungus
Lasiodiplodia theobromae
in a defined medium for 1 to 7 days and the cell free filtrate was analyzed for phytotoxic activity using leaf disks of Coleus and Cucumber seeds as pre and post-emergents. The cell free filtrate exhibited phytotoxicity. However, the applicants during the course of their research, found that the phytotoxin did not show growth inhibition in
E.coli
and
Saccharomyces cerevisiae
cultures.
The pathogen was isolated and identified. Isolation of the causative organism was done by taking the infected tissues and culturing them on a suitable medium. Among the few isolates, only one was observed to cause severe damage when introduced into healthy Coleus plant. The isolated organism was identified as a fungus,
Lasiodiplodia theobromae
. This pathogen acts by releasing extracellular toxins, which are, hereafter, referred as LT-toxins.
Studies were carried out with the filtrate (free of organisms and spores) which showed inhibitory effect on the root elongation in cucumber seedlings; necrotic effect on
C. forskohlii
, ground nut and Parthenium in leaf disk and leaf puncture assays; necrosis was also seen in Coleus leaf callus.
This fungal extracellular toxin exhibits phytotoxicity, so field trials were carried out on Parthenium, which showed promising results. These studies suggested that the fungal filtrate (free of live organisms or spores) can be used as a herbicide to eradicate weeds like Parthenium.
L. theobromae
can be easily isolated from susceptible Coleus plants exhibiting symptoms of root rot disease by known procedures. Isolates can be grown on potato-dextrose agar and identified based on conidial morphology (Von Arx, J. A., 1981. The genera of fungi sporulating in pure culture, J. Cramer (EDS)., Germany, pp: 206-207). The fungus is characterized with dark brown hyphae, thick walled fruiting bodies, striate and slowly m

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