DESC:FIELD OF THE INVENTION
[0001] The present invention relates to a method of isolation of microbes. More specifically, the present invention relates to a method of isolation of Phytophthora infestans. Particularly, the present invention discloses a method of selective isolation, maintenance, and sporangial formation of pathogen Phytophthora infestans, causing late blight of potato and tomato.

BACKGROUND OF THE INVENTION
[0002] Late blight is caused by oomycetes fungal pathogen Phytophthora infestans (P. infestans). This devastating pathogen can infect potato foliage and tubers at any stage of crop growth and development as shown in Fig.1. The primary host is potato, but P. infestans can also, infect other solanaceous plants, including tomato, petunia and hairy nightshade, that can act as secondary source of inoculum for causing late blight in potato. Late blight of potato was responsible for the Irish potato famine in the mid-nineteenth century. P. infestans can infect and destroy the leaves, stems, fruits, and tubers of potato and tomato plants. Besides pathogen also survive in soil and can cause infection to subsequent crops. Before the disease appeared in Ireland it caused a devastating epidemic in the early 1840s in the north-eastern United States.
[0003] The pathogen P. infestans is favored by moisture, high relative humidity, low temperature ranging from 15°C to 24°C. Sporangial formation is optimal at 12°C–18°C (54°F –64°F) in water-saturated or nearly saturated environments, and zoospore production is favored at temperatures below 15°C (59°F). Lesions and mycelial growth are typically optimal at a slightly warmer temperature ranging from 20°C to 24°C (68°F to 75°F).
[0004] The knowledge on the local population structure, migrations and evolution of P. infestans is essential for the understanding of late blight epidemiology to take appropriate chemical or biological control and resistance breeding.
[0005] Basic literature search provides various media used for the isolation and sub-culturing of Phytophthora infestans. Media such as Rye media, V8 juice Media, Water agar media, Cornmeal agar, P10VP Medium (Tsao and Ocana 1969), 3-P Medium (Eckert and Tsao 1960, 1962), P10ARP Medium (Kannwischer and Mitchell 1978), P5ARP Medium (Jeffers and Martin 1986; Papavizas et al. 1981), Hymexazol-amended Medium (Masago et al. 1977) are used for the regular isolation and sub-culturing of Phytophthora infestans.
[0006] The culture dish contains nutrients within the media that cells need to thrive and prosper. Apart from the regular constituents in the media antibiotics and fungicides played a crucial role in the selective isolation of Phytophthora infestans. Antibiotics and fungicides such as Pimaricin, Penicillin, Polymixin B, Vancomycin, Pentachloronitrobenzene, Hymexazol, Benomyl, Nystatin, Rifampicin were also used for the selective isolation in various combinations and concentrations in the above-mentioned media. Antibiotics are used because of their active role in the fight against bacterial contamination and selective suppression of non-oomycetous pathogens in the culture dish. However, antibiotics affect cell biochemistry and may modify the differentiation potential of cultured cells.
[0007] Further, the culture media frequently used in state of art does not support both mycelium growth and sporulation of Phytophthora infestans. When Phytophthora infestans is grown in any of the above-stated medium, the pathogen has to be infected into any of the host plants such as potato/tomato/apple and re-isolated from plants to obtain a virulent strain. Thus, the stated culture media does not support the direct use of the pathogen from an in-vitro set up to a field infection study.
[0008] Hence, there is a need in the art to develop a method for isolation of Phytophthora infestans that does not require antibiotics and provides suitable conditions for both mycelium growth and sporangial formation of Phytophthora infestans.

OBJECT OF THE INVENTION
[0009] A primary object of the present invention is to provide a method for culture and isolation of Phytophthora infestans that does not require antibiotics.
[0010] In another object of the present invention is to provide a culture medium that supports both mycelium growth and sporulation of Phytophthora infestans.
[0011] In yet another object of the present invention is to provide a culture medium through which virulence of Phytophthora infestans is maintained for extended period after isolation from host.

SUMMARY OF THE INVENTION
[0012] The present invention provides a method for isolation of Phytophthora infestans comprising the steps of: preparation of culture medium; aseptically transferring of hyphal tips of the fungus on to the culture medium in a petri dish; and incubating petri dish in a shaker cum incubator; wherein the culture medium comprises of potato, sugar, agar and water. The culture medium of the present invention does not contain antibiotics. The sugar suitable for use in present invention is sucrose.
[0013] The potato, sucrose and agar are present in ratio of 25:2:2 in the culture medium and potato is used in form of potato infusion. The potato infusion is prepared by boiling unpeeled potatoes in water for 30 minutes and filtering it with cheesecloth. Sucrose and agar is added to potato infusion followed by addition of water and boiling to dissolve all contents completely. The solution is then autoclaved at 121ºC for 15 minutes.
[0014] The incubator suitable for the purpose of the present invention is BOD incubator (Bio-Oxygen Demand) for multiplication of P. infestans and incubation takes place at temperature 19°C+2°C, relative humidity 95-100% and under dark conditions.
[0015] The method of isolation and culture of the present invention helps in maintaining virulence of P. infestans in the culture medium by sub-culturing regularly at an interval of 30 days into potato sucrose agar at temperature 19°C±2?, Relative humidity (RH) at 95-100%.

BRIEF DESCRIPTION OF FIGURES ON THE INVENTION
Fig. 1 shows late blight infected potato leaf.
Fig. 2 shows surface sterilization of half infected and half healthy leaf tissues with 70% ethanol.
Fig. 3 shows inoculation of infected leaf tissues into potato slices and incubation at dark conditions.
Fig. 4 shows white mycelium with sporangia production of P. infestans on potato slices.
Fig. 5 shows pure culture of P. infestans on culture medium
Fig. 6 shows sporangia and aseptate hypha of P. infestans.
Fig. 7 (a) shows 0th day of incubation and (b) shows 14th day of incubation.
Fig. 8 shows P. infestans growth– incubation from 0th day to 14th day.
Fig. 9 (a) Medium-1: P. infestans growth 2 cm; (b) Medium-2: P. infestans growth 0.5 cm; (c) Medium-3: P. infestans growth 9 cm.
Fig. 10 shows P. infestans growth on different culture medium (14 days incubation).
Fig. 11 shows successful symptom expression on potato leaf by P. infestans originally isolated from host during 2019.
Fig. 12 shows P. infestans culture-successful recovery from 3 months old plate.
DESCRIPTION OF THE INVENTION
[0016] The present invention relates to a method of isolation of Phytophthora infestans on a culture media without the use of antibiotics. The present invention uses a culture media of potato, sucrose, agar and distilled water.
[0017] It is to be noted, as used in the specification and claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. It should also be noted that the term “or” is generally employed in its sense including “and/or” unless the content clearly dictates otherwise. The expression of various quantities in the terms of “% w/w” or “%” means the percentage by weight, relative to the weight of the total composition unless otherwise specified.
[0018] Similarly, the words "comprise," "comprises," and "comprising" are to be interpreted inclusively rather than exclusively. Likewise, the terms "include," "including" and "or" should all be construed to be inclusive, unless such a construction is clearly prohibited from the context. However, the embodiments provided by the present disclosure may lack any element that is not specifically disclosed herein. Thus, a disclosure of an embodiment defined using the term "comprising" is also a disclosure of embodiments "consisting essentially of’ and "consisting of’ the disclosed components. Where used herein, the term "example," particularly when followed by a listing of terms, is merely exemplary and illustrative, and should not be deemed to be exclusive or comprehensive. Any embodiment disclosed herein can be combined with any other embodiment disclosed herein unless explicitly indicated otherwise.
[0019] As used in the present invention “culture media”, also known as growth media, are specific mixtures of nutrients and other substances that support the growth of microorganisms such as bacteria and fungi (yeasts and molds). Most commonly, culture media are based on a nutrient broth (liquid), often mixed with agar and prepared in petri dishes (they may be semi-solid or solid). For enriched or selective media, further specific components are added. Culture media preparation must be carried out accurately to ensure the growth of desired fungal pathogen of interest. The individual ingredients of the culture media (powders, gels and liquids) must be carefully weighed out according to the culture media formulation recipe.
[0020] Mineral salts and carbohydrates are used as major carbon sources in in vitro culture medium. Carbohydrate is an important component for plant growth and development in vitro where the conditions are not suitable for photosynthesis; thus, carbohydrate is supplemented as a carbon source to maintain carbon supply as well as to maintain osmotic potential of cells. Types of carbohydrate commonly used are sucrose, fructose, and glucose. For the purpose of the present invention sucrose is preferred due to its most favorable effect on growth of Phytophthora infestans and relatively low cost.
[0021] Agar is the most commonly used growth medium for microorganisms. Agar or agar-agar, is a jelly-like substance, obtained from red algae. Agar is a mixture of two components: the linear polysaccharide agarose, and a heterogeneous mixture of smaller molecules called agaropectin. Due to the ease with which agar can be transported (dry, dissolved, and gelled), it is ubiquitous in the modern-day laboratory. Solid agar plates can support microbial growth when supplemented with appropriate nutrients or when used for antibiotic selection. Agar media is essential for the study of microorganisms and molecular biology and is widely used in the culture and detection of pathogens from contaminated food and water.
[0022] In an embodiment, the culture media comprises of potato extract, sucrose, agar and water. Potato sucrose agar media has not been utilized for the growth and maintenance of Phytophthora infestans. The culture medium of potato, sucrose, agar and water can be also used to grow Phytophthora infestans under proper condition.
[0023] In another embodiment, the culture media is prepared by preparing a potato infusion (extract). A potato infusion is prepared by boiling sliced unpeeled potatoes with water for 30 minutes and then filtering it with cheese cloth. To this potato infusion sucrose and agar is added and the final volume of the extract is made up to 1 Liter and boiled to dissolve completely all sucrose and agar. The solution is then autoclaved for a definite time and at definite temperature.
[0024] Potato sucrose medium is more suitable for Phytophthora infestans growth. The speed of growth of Phytophthora infestans is fast, and growth of mycelium is good as shown in Fig. 4 and Fig. 6. Also potatoes and sucrose are readily available and are of low cost.
[0025] In yet another embodiment, the present invention also describes a method for growing Phytophthora infestans on the culture medium under incubation in dark at definite temperature and relative humidity.
[0026] In yet another embodiment, the present invention describes optimum conditions for maintaining the virulence of Phytophthora infestans.
[0027] The following examples illustrate, but in no way are intended to limit the present invention.

EXAMPLE 1: Method for preparing culture medium
[0028] The components of culture media Potato, Sucrose, Agar are taken in respective amount in the ratio of 25:2:2 and is diluted with distilled water to make the final volume of 1 Liter. A person skilled in art may vary the amount of potato, sucrose and agar according to his need for culturing Phytophthora infestans. The first step is preparation of potato infusion. Potatoes are taken washed with water and were sliced. 200 g of sliced unpeeled potatoes is boiled in 1 liter of water for 30 minutes. The boiled water is then filtered through cheesecloth, saving effluent, which is potato infusion. Next step is addition of sucrose 20 gm (2%), agar 20 gm (2%) to potato infusion of 250gm (25%), and adding distilled water to make the volume up to 1000 ml. The obtained mixture is then boiled to dissolve the sucrose and agar completely. The obtained media is then sterilized by autoclaving at 121ºC for 15 minutes. The amount of potato, sucrose and agar may vary depending upon the amount of culture media required by a person skilled in art and autoclaving conditions may also vary accordingly.

Example 2: Culturing of isolates
[0029] Petri dish (90 mm diameter) is taken and sterilized. The culture media from Example 1 is transferred to petri dish. A late blight infected leaf is taken and surface sterilization of half infected and half healthy leaf tissue with 70% ethanol is done as shown in Fig. 2. The infected leaf tissue is inoculated into potato slices and incubated under dark as shown in Fig.3. The hyphal tips of the fungus from above said potato slices are aseptically transferred onto the petri dish using sterile needle made of nichrome wire under laminar air flow chamber (sterile environment).
[0030] Culturing of P. infestans: From 14 days old pure culture of P. infestans as shown in Fig. 5, 5 mm disc will be excised using sterile cork borer, and it will be transferred aseptically using sterile needle into fresh petri plate containing culture medium. The petri plate is kept in a shaker cum BOD incubator (Bio-Oxygen Demand) for multiplication of P. infestans. The conditions during incubation are maintained at temperature 19°C+2°C, Relative humidity 95-100%, and incubation is done under dark. It is observed that it took 14 days for the P. infestans to cover full petri plate (90 mm diameter) as shown in Fig. 7 and Fig. 8. The virulence of P. infestans in the culture medium may be maintained by sub-culturing regularly at an interval of 30 days into potato sucrose agar at temperature of 19°C±2?, Relative humidity of 95-100%.
[0031] Confirmation of P. infestans through molecular characterization:
Genomic DNA has been extracted from P. infestans cultured in Example 2 and used for PCR amplification. Details of primers used and GenBankR accession numbers are mentioned in Table 1. GenBankR is a comprehensive public database of nucleotide sequences and supporting bibliographic and biological annotation. GenBankR is built and distributed by the National Center for Biotechnology Information (NCBI), a division of the National Library of Medicine, located on the campus of the U.S. National Institutes of Health (NIH) in Bethesda, MD, USA. NCBI builds GenBank primarily from the submission of sequence data from authors and from the bulk submission of expressed sequence tag (EST), genome survey sequence (GSS), whole-genome shotgun (WGS) and other high throughput data from sequencing centers. The U.S. Patent and Trademark Office also contributes sequences from issued patents. GenBank participates with the EMBL European Nucleotide Archive (ENA), and the DNA Data Bank of Japan (DDBJ) as a partner in the International Nucleotide Sequence Database Collaboration (INSDC). The INSDC partners exchange data daily to ensure that a uniform and comprehensive collection of sequence information is available worldwide.

Table 1: Molecular characterization of P. infestans
S. No. Gene details Primers used GenBankR accession number
1 ITS (Universal primers) ITS1 & ITS4 ON087277
2 ITS (Universal primers) ITS3 & ITS4 ON087543
3 ITS (Universal primers) ITS4 & ITS6 MW285753
4 Avr 3A (Species specific) Avr3AF & Avr3AR ON093168
5 Beta tubulin (Species specific) BtubF & BtubR ON093169
6 RXLR (Species specific) RXLRF & RXLRR ON093170
7 Glucan 1,3 beta glucosidase [GBG] (Species specific) GBGF & GBGR ON093171
8 G- protein beta subunit-1 [GPB] (Species specific) GPBF & GPBR ON093172

[0032] The pathogen is confirmed as P. infestans by universal primers (ITS) and species-specific primers (avr3A, beta tubulin, RXLR, GBG and GPB).

Example 3: Efficacy of ratio of potato extract: sucrose: agar and water on culture of P. infestans
[0033] To study the effect of ratio of potato extract: sucrose: agar on culture of P. infestans three culture media are studied having different ratio of potato extract(infusion): sucrose: agar. As illustrated in Table 2, culture media 1 has potato extract: sucrose: agar in ratio of 10:10:2; culture media 2 has potato extract: sucrose: agar in ratio of 5:5:2; culture media 3 has potato extract: sucrose: agar in ratio of 25:2:2. As is illustrated in Table 2, potato extract (infusion): sucrose: agar in ratio of 25:2:2 covers a diameter of 9 cm of petri plate, which is comparatively very high when compared to 2 cm diameter in potato extract: sucrose: agar in ratio of 10:10:2 and 0.5 cm diameter growth in potato extract: sucrose: agar in ratio of 5:5:2. Diameter of petri plate is 9cm, so if the growth covers whole plate, it indicates P. infestans can cover whole plate within 14 days after incubation as shown in Fig.9.

Table 2: Effect of ratio of potato extract: sucrose: agar on fungal mycelial growth
Medium Media constituents Ingredient (%) Fungal mycelial growth in diameter (cm) after 14 days of incubation at 19?±2?, RH - 95-100%
1 Potato infusion 10 2.0
Sucrose 10
Agar 2
Water q.s.to 100
2 Potato infusion 5 0.5
Sucrose 5
Agar 2
Water q.s.to 100
3 Potato infusion 25 9.0 (complete petri plate)
Sucrose 2
Agar 2
Water q.s. to 100

Example 4: Effect of different culture media on mycelium growth and sporulation of P. infestans
[0034] A comparative study is done to study the effect of different growth medium on mycelium growth and sporulation of P. infestans. Table 3 shows that among the nine culture media studied it is only potato, sucrose and agar medium of the present invention which supports both mycelium growth and sporulation of P. infestans as shown in Fig. 10 after 14 days of incubation. Table 4 provides composition of different culture media as shown in Table 3.

Table 3: Effect of different culture media on mycelium growth and sporulation of P. infestans
S. No. Medium Mycelium production Sporangia production
1 Potato Dextrose Agar Yes No
2 Corn Meal Agar Yes No
3 Rye agar A Yes No
4 Wheat agar Yes No
5 Oat meal agar Yes No
6 Pea sucrose agar Yes No
7 Pea agar Yes No
8 V8 agar Yes No
9 Potato Sucrose Agar Yes Yes

Table 4: Composition of different culture media of Table 3
S. No. Medium Composition
1 Potato Dextrose Agar 200g of potato infusion+20g of dextrose+ 20g of agar in 1L distilled water
2 Corn Meal Agar 50g of Corn meal infusion+ 20g of agar in 1L distilled water
3 Rye agar A 60g of Rye, 20g of sucrose, 20g of agar in 1L of distilled water
4 Wheat agar 20g of wheat infusion, 20g of agar in 1L of distilled water
5 Oat meal agar 60g of Oat meal, 15g of Agar in 1L of distilled water
6 Pea sucrose agar 160g peas (infusion form), 5g sucrose, 20g agar into 1L distilled water
7 Pea agar 160g peas (infusion form), 20g agar into 1L distilled water
8 V8 agar 8.3g of V8 juice,10g of L-Asparagine, 2g of yeast extract, 2g of calcium carbonate, 2g of glucose, 20g of agar in 1L distilled water
9 Potato Sucrose Agar 250g of potato infusion, 20g of sucrose, 20g of agar in 1L distilled water

[0035] The present invention isolation method provides following advantages:
[0036] Firstly, in comparison to regular medium which is used for culturing fugal microorganisms P. infestans produced both mycelium and sporangia (both asexual propagules) in potato sucrose agar medium as shown in Fig.4. Secondly, virulence of pathogen is maintained even after 2 years of isolation from host as shown in Fig. 11 of the specification. Thirdly, recovery of pathogen is successful even after 3 - 4 months from single plate as shown in Fig. 12 of the specification. The culture media of the present invention does not require the usage of any antibiotic.
[0037] The method disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the method of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the method and in the steps or in the sequence of steps of the method described herein without departing from the concept and scope of the invention. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the scope and concept of the invention as defined by the appended claims.
,CLAIMS:1. A method for isolation of Phytophthora infestans comprising the steps of:
- preparation of culture medium;
- aseptically transferring of hyphal tips of the fungus on to the culture medium in a petri dish; and
- incubating petri dish in a shaker cum incubator, wherein the culture medium comprises of potato, sugar, agar and water,
wherein the culture medium is free of antibiotics.

2. The method as claimed in claim 1, wherein the sugar is sucrose.

3. The method as claimed in claim 1, wherein potato, sucrose and agar are present in ratio of 25:2:2.

4. The method as claimed in claim 1, wherein the potato is in form of potato infusion.

5. The method as claimed in claim 4, wherein the potato infusion is prepared by boiling sliced unpeeled potatoes in water for 30 minutes and filtering.

6. The method as claimed in claim 1, wherein culture medium is prepared by adding sucrose, agar and water to potato infusion and boiling to dissolve sucrose and agar.