Strains of streptomyces and relevant uses thereof

Details Strains of streptomyces and relevant uses thereof Strains of streptomyces and relevant uses thereof

2001-04-25

2001-09-11

Tate, Christopher R. (Department: 1651)

Chemistry: molecular biology and microbiology

Micro-organism, per se ; compositions thereof; proces of...

Bacteria or actinomycetales; media therefor

C435S041000

Reexamination Certificate

active

06287845

ABSTRACT:

FIELD OF THE INVENTION
The invention relates to novel strains of
Streptomyces orientalis
and
Streptomyces melanogenes;
and the use of the strains.
BACKGROUND OF THE INVENTION
There are a variety of insects that cause major economic losses in agriculture and spread diseases among plants. Whitefly is one of the most notorious agricultural pests in the world. For example, in 1981, the sweet potato whitefly,
Bemisia tabaci
(Gennadius), caused 100 million US dollar damage in cotton, cucurbits, and lettuce in the United States. In 1986, whitefly became a problem in Florida where
B. tabaci
caused approximately US$2 million of damage to Florida's US$8 to 10 million poinsettia crop.
Whitefly is now known to feed on more than 500 different plants. For example, sweet potato, tomato, beans, cotton, carrot, cassava, squash, lettuce, pepper, egg plant, watermelon, and cucumber are all known hosts to the pest. It is also known that sweet potato whitefly may transmit more than 70 diseases caused by virus and microorganisms.
Silverleaf whitefly (
B. argentifolii
Bellows and Perring) was first found in Taiwan in 1991. In 1995, silverleaf whitefly was responsible for 1000 hectares of agriculture losses in Taiwan.
Whitefly is very difficult to control with conventional pesticide application. Many factors contribute to the lack of control obtained with pesticides. Only few commercially available pesticides are effective against whiteflies. However, these pesticides are only effective if care is taken in a very thorough application several times a week. In addition, whiteflies spend most of their life on the undersides of leaves; therefore, growers must adjust their management practices to permit increased pesticide coverage there. The spacing of the plants must be such that the chemical spray can penetrate the canopy and reach all surfaces of the plants.
Further, the ineffective use of the chemical pesticides may be costly, and has other significant drawbacks, such as the pollution to the environment, and the potential health hazards to agricultural workers and to consumers.
Therefore, safer and more effective methods for controlling whiteflies are needed. Although biological control agents have been studied for years, to date no biological control agent has been commercially successful for the control of whiteflies.
U.S. Pat. Nos. 4,942,030 and 5,413,784 disclosed the utilization of fungi
Paecilomyces fumosoroseus
and
Beauveria bassiana
in controlling sweet potato whitefly.
Streptomyces sps. have been widely used in the production of antibiotic materials. However, thus far, no Streptomyces sp. had been identified as having biopesticidal activity against whiteflies.
SUMMARY OF THE INVENTION
New strains in the Streptomyces genus, designated
Streptomyces orientalis
Y31014 and
Streptomyces melanogenes
Y31042-1, surprisingly have been found. The strains are found to be active against whiteflies.
Therefore, in the first aspect of the invention, the novel strains
Streptomyces orientalis
Y31014 and
Streptomyces melanogenes
Y31042-1 are provided.
In the second aspect of the invention, a biopesticide composition comprising the novel strains is provided.
In a further aspect of the invention, the method for controlling pests by contacting pests with the novel strains is provided.
IDENTIFICATION AND CHARACTERIZATION OF THE MICROORGANISMS
The novel Y31014 and Y31042-1 strains were isolated from a soil sample taken form Miaoli, Taiwan, ROC. These microorganisms have been identified by the Food Industry Research and Development Institute, Shin-Chu, Taiwan, ROC as strains of
Streptomyses orientalis
and
Streptomyces melanogenes,
respectively. The methods and results are as follows:
Taxonomic and morphologic characterization was made using the methods recommended by the International Streptomyces Project (ISP) for characterizing Streptomyces species.
1. Cell Wall Analysis
Dried cells (10 mg) were put into a test tube containing 1 ml of 6N HCl, and were hydrolyzed in 100° C. for 18 hours. Hydrolysate was filtered and dried. The dry powder was dissolved in 0.4 ml of distilled water, and poured on a PTLC plate. Methanol-H
2
O—6N HCl-pyridine was used as the developing solution. Ninhydrin was then applied after an air-drying process. Diaminopimelic acid (DAP) produces a yellowish green color, while other amino acids produce a purple red color. See Komagata, et al.,“Lipid and Cell-Wall Analysis in Bacterial Systematics,” Meth. Microbiol. 19:161-207 (1987).
2. Whole Cell Sugar Analysis
Dried cells (50 mg) were put into a test tube containing 1 ml of 1N H
2
SO
4
, and were hydrolyzed in 100° C. for 2 hours. The pH of the hydrolysate was adjusted to the range of 5.0-5.5 by saturated Ba(OH)
2
. The hydrolysate was then centrifuged. The upper suspension was collected and concentrated. The concentrate was dissolved in 0.4 ml of distilled water and then poured on a filter. N-butanol-H
2
O-pyridine-toluene was uses as developing solution. Aniline phthalate was applied to develop the color of the sugars. Six-carbon sugars will result in a brown color, and five-carbon sugars will result in pink color after an air-drying process. See Komagata, et al.,“Lipid and Cell-Wall Analysis in Bacterial Systematics,” Meth. Microbiol. 19:161-207 (1987).
3. Cultural Characteristics Analysis
Cells were cultured on yeast extract-malt extract agar (ISP# 2 medium), oatmeal agar (ISP# 3 medium), inorganic salts starch agar (ISP# 4 medium), and glycerol-asparagine agar (ISP# 5 medium), respectively for 14 days to observe the vegetative mass, aerial mass, spore production and pigment production. See Shiring et al., “Methods for Characterization of Streptomyce species,” Int. J. Syst. Bacteriol., 16:313-340 (1966).
4. Melanoid Pigment Production Analysis
Cells were cultured on tryptone-yeast extract broth (ISP# 1 medium), peptone-yeast extract iron agar (ISP# 6 medium) and tyrosine agar (ISP# 7 medium) for 7 and 14 days respectively, to observe the melanoid pigment production. See Shiring et al., “Methods for Characterization of Streptomyce species,” Int. J. Syst. Bacteriol., 16:313-340 (1966).
5. Morphological Characteristics
Cells together with agar were cut from ISP# 2, 3, 4, and 5 media, dehydrated in oven, and coated with gold in an ion-coater. The morphology was studied using a scanning electron microscope (SEM).
6. Sugar Utilization and Physical Characteristics Analysis
Cells were cultured on ISP# 9 medium containing 1% sugar (D-glucose, L-arabinose, D-xylose, sucrose, D-fructose, raffinose, rhamnose, I-inositol, D-mannitol, cellulose, salicin) for 7 and 14 days to observe cell growth. See Shiring et al., “Methods for Characterization of Streptomyce species,” Int. J. Syst. Bacteriol., 16:313-340 (1966).
The results are shown in Tables 1 to 4.
TABLE 1
Cultural characteristics of Y31014
Characteristic
Medium
Growth
Substrate mycelium
Aerial mycelium
Sporulation
Soluble pigment
Yeast extract-malt
Well
Deep orange yellow
Yellowish white
Good
None
extract agar (ISP #2
medium)
Oat meal agar (ISP #
Well
Brilliant yellow
Yellowish white
Moderate
None
3 medium)
Inorganic salt agar
Well
Strong yellow
Yellowish white
Good
None
(ISP #4 medium)
Glycerol asparagine
Moder-
Brilliant yellow
Pale yellow
Poor
None
agar (ISP #5
ate
medium)
TABLE 2
Physiological characteristics of Y31014
Cabon source
Y31014
D-glucose
+*
D-xylose
+
D-fructose
+
Sucrose
−
L-arabinose
+
Rhamnose
−
Raffinose
+
D-mannitol
+
I-inositol
+
Cellulose
−
Salicin
+
*+: positive reaction, −: negative reaction
TABLE 3
Cultural characteristics of Y31042-1
Characteristic
Medium
Growth
Substrate mycelium
Aerial mycelium
Sporulation
Soluble pigment
Yeast extract-malt
Moder-
Strong brown
Light gray
Moderate
None
extract agar (ISP #2
ate
medium)
Oat meal agar (ISP #
Moder-
Strong yellowish
Grayish yellowish
Poor
None
3 medium)
ate
pink
pink
Inorganic salts starch
Moder-
Deep orange
Light gray
Poor
None
agar (ISP #4
ate
medium)
Glycerol asp

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