Drug – bio-affecting and body treating compositions – Whole live micro-organism – cell – or virus containing – Fungus
Reexamination Certificate
2001-04-03
2004-10-19
Marx, Irene (Department: 1651)
Drug, bio-affecting and body treating compositions
Whole live micro-organism, cell, or virus containing
Fungus
C435S254100, C504S117000
Reexamination Certificate
active
06805859
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to symbiotic fungi, plants into which a symbiotic fungus is artificially introduced, and a method of introducing a symbiotic fungus into plants. More particularly, this invention relates to a fungus, i.e., an endophyte, whose final metabolic product is chanoclavine, to a plant which can be used for pasture which such an endophyte is caused to infect, and to a method of infecting the plant.
2. Description of the Related Art
Japanese Patent Opening Heisei 5-317092 (Gazette), for example, discloses a method wherein a test is performed to discover whether or not an endophyte is living in plant tissue, e.g., callus derived from perennial rye grass, and after introducing an endophyte into callus which is confirmed not to contain endophyte, the endophyte is introduced to perennial rye grass wherein the plant tissue is reproduced.
A symbiotic fungus, i.e. an endophyte, is a filamentous fungus which lives in plant tissue, and it is know that the plants infected by such filamentous fungus have improved resistance to pests, resistance to disease, better growth rate, and resistance to environmental stresses such as heat and dryness, as compared with individuals which are not so infected. Thus, artificial infection with an endophyte leads to an improvement of plant characteristics.
Symbiotic fungi which can infect and live in plants successively biosynthesize the metabolic products shown in FIG.
1
. The end product of this biosynthesis is an ergopeptine such as ergovaline or ergotamine.
It is known that grasses which are symbiotically infected by such endophytes show toxicity due to the presence of ergoto alkaloids. This toxicity is considered to be a problem in husbandry as it affects livestock. In the case of pasture, for example, grasses infected with endophytes such as perennial rye grass and tall fescue are very commonly distributed, and cases of poisoning have been reported due to ergoto alkaloids.
Rye grass slugger was first reported by New Zealand in 1906, and occurs mainly in two countries in Oceania, i.e., New Zealand and Australia. Rye grass slugger occurs when endophytes infect perennial rye grass which has withered in the severity of the dry conditions from summer to autumn, and livestock such as sheep eat the grass.
In rye grass slugger, livestock which ate grass show various symptoms. i.e, their limbs stiffen, and the neck, shoulder and flank shake. In other words, a kind of spasm occurs, and if this state continues, the animals become weaker. In the worst case, they are no longer able to move and may starve to death. It has now become clear that the substance responsible for this is Lolitrum B, which is a Lolitrum alkaloid.
Fescue toxicosis mainly occurred in the American southeast. Cows which consumed tall fescue suffered serious weight loss, and did not eat sufficient fodder. Their milk production declined, and their birth rate also fell. Endophytes were found to have infected the grass which the cows had eaten, and ergovaline which is an ergoto alkaloid was specifically detected from endophyte-infected plant individuals. It thus became clear that ergovaline causes fescue toxicosis.
Although resistance to pests, resistance to disease, growth rate and resistance to environmental factors such as heat and dryness are improved by infecting plants, especially pastures, with an endophyte, the endophytes biosynthesized substances which are poisonous to livestock, and gave rise to the problem of rye grass slugger, fescue toxicosis, etc.
OBJECTS AND SUMMARY OF THE INVENTION
It is therefore an object of this invention to solve the above problem.
It is an another object of this invention to provide a symbiotic fungus which improves plant characteristics without synthesizing poisonous substances when applied to grass.
A further object of this invention is to provide plants into which said symbiotic fungus is artificially introduced, and to provide a method of artificially introducing the symbiotic fungus.
A still further object of this invention is to provide a plant not showing livestock toxicity and having strong resistance to pest.
According to one aspect of this invention, there is provided a symbiotic fungus comprising a filamentous fungus whose final metabolic product is chanoclavine. The symbiotic fungus meant here may belong to the genus Neotyphodium. The symbiotic fungus may also be one, two or more types of fungi deposited at the Japanese National Institute of Bioscience and Human Technology under the numbers FERM P-17672 (BP-08480), FERM P-17673 (BP-08481) and FERM P-17674 (BP-08482).
The invention relating to a symbiotic fungus whose final metabolic product is chanoclavine, is selected by screening for using chanoclavine as a marker. Here, the screening may be performed using thin layer chromatography or liquid chromatography using chanoclavine as a marker.
The invention relating to a plant, concerns a plant into which the symbiotic fungus whose final metabolic product is chanoclavine, is introduced. Here, the symbiotic fungus may be a filamentous fungus belonging to the genus Neotyphodium. The plant into which the symbiotic fungus is artificially introduced may be a grass which is any of Agrostis, Festuca, Poa and Lolium. Later generations of seeds taken from these plants, plants grown from later generations of seeds, or hybrid plants having these plants and seeds as parents, are also within the scope of this invention.
One invention relating to a method of introducing a symbiotic fungus, comprises a step for isolating symbiotic fungi from plants which exist naturally, a step for artificially cultivating the isolated symbiotic fungi, a step for introducing the cultivated symbiotic fungi into target plants, a step for infecting the plants with the introduced symbiotic fungi, and a step for selecting a plant infected with a symbiotic fungus whose final metabolic product is chanoclavine.
Another invention relating to a method of introducing a symbiotic fungus, comprises a step for isolating symbiotic fungi from plants which exist naturally, a step for artificially cultivating the isolated symbiotic fungi, a step for selecting a symbiotic fungus whose final metabolic product is chanoclavine from the cultivated symbiotic fungi, a step for introducing the selected symbiotic fungus into a target plant, and a step for infecting the plant with the introduced symbiotic fungus.
The alkaloid metabolism of the symbiotic fungus is shown in FIG.
1
. Chanoclavine is an intermediate product of such metabolism. Chanoclavine is a clavine alkaloid having the chemical structure shown in
FIG. 2
, and it has various pharmacological effects. Ergotoalkaloids such as ergovaline are produced from chanoclavine. However, in the symbiotic fungi searched by the Inventor, the ergotoalkaloids were not biosynthesized and chanoclavine was produced as the final metabolic product. The symbiotic fungus whose final metabolic product is chanoclavine does not show ergovaline toxicity. Therefore, plants which do not show ergovaline toxicity can now be provided by making such a symbiotic fungus infect the plant.
The present Application, which specifically concerns a symbiotic fungus which biosynthesizes chanoclavine as its final metabolic product, involves screening before and after introduction of the fungus into the plant, followed by infection of the plant with the fungus. Filamentous fungi belonging to the genus Neotyphodium are examples of symbiotic fungi which infect grasses.
Using this type of filamentous fungi, filamentous fungi which biosynthesize chanoclavine as their final metabolic product infect the plants. Hence plants can be produced which, although they do not show ergovaline toxicity, still have improved characteristics. Also, as plants infected with filamentous fungi which biosynthesize chanoclavine as a final metabolic product, accumulate large amounts of chanoclavine in their tissues, it is possible to provide a fungus which does not biosynthesize ergovaline or Lolitrum B by screening using chanoclavine as a marke
Hiruma Naoya
Imada Takahiro
Kurihara Yousuke
Mizutani Junya
Shimaike Miho
Kanesaka & Takeuchi
Marx Irene
Mayekawa Manufacturing Co., Ltd.
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