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(11) Patent Number: KE 103
(45) Date of grant: 14/05/2005
(12) PATENT
 
(51) Int.C1.6: A 01G 7/00
(21) Application Number: 1998/ 000231
(22) Filing Date: 20/01/1998
(30) Priority data:4958/86 01/12/1986 IN
 
(73) Owner:COUNCIL OF SCIENTIFIC & INDUSTRIAL RESEARCH of, Rafi Marg, New Delhi 110 001, India
(72) Inventor:OM PARKASH; ANIL SOOD; MADHU SHARMA and PARAMVIR SINGH AHUJA
(74) Agent/address for correspondence: W.R.McA SPENCE, P.o.Box 43984 Nairobi
 
(54) Title: AN IMPROVED METHOD OF TEA PROPAGATION.
(57) Abstract:
The invention relates to a method of tea propagation, which comprises (a) raising in vivo juvenile stock plants, (b) in vitro raising of micro-shoots of tea as scions, (c) decapitation of stock plantlets, (d) treatment of cut ends of stock and scions with plant growth regulators, (e) grafting of in vitro raised micro-shoots on to in vivo raised juvenile stoic plants, (1) holding of the said graft union tightly using any biocompatible material, and (g) hardening of the said micro-grafted plants under controlled conditions which include a light source providing a minimum of 1000 luc intensity, a minimum carbon dioxide concentration of 400 ppm and relative humidity in the range of 50-95 percent to obtain field transferable plants.
 
FIELD OF THE INVENTION
The present invention relates to an improved method of tea propagation. Particularly, this invention relates to a method for the efficient transfer of micro-propagated tea shoots to the fields. More particularly, the method provides simultaneous grafting and hardening of micro-shoots on root stocks.
BACKGROUND OF THE INVENTION
Tea is one of the most important commercial crops. In order to increase productivity per unit area, it is essential to either put more area under tea cultivation or to replace the existing old tea bushes" with young plants of high potential cultivars. This requires a regular bulk supply of high quality plants of elite cultivars.
Tea plants can be propagated sexually or asexually. Traditionally, tea is propagated by single node cuttings or through seeds. Tea being an outbreeding species, the seed progenies are heterogeneous and are not replicas of the parent plant. Propagation through single node cuttings is quite a common practice with nurserymen. However, the major factor in the asexual reproduction through single node cuttings is the non-availability of cuttings for large scale propagation which requires a lot of time and space.
PRIOR ART REFERENCES
In tissue culture techniques, very small number of nodal cuttings are required for initiating aseptic cultures. Micro-propagation is one of the most suitable methods for rapid propagation because of its advantages like mass multiplication, production of disease free clones, product uniformity, and economy of space. However, these methods suffer from the recalcitrant nature of woody plants, including tea, which are generally poor rooters. Tea micro-propagation involves the production of multiple micro-shoots by using single node cuttings as explants (Arulpragasam, P.V. 1990. Proc. Intl. Conf. on Tea Research:Global Perspective, pp. 1-5, Tea Res. Association, Calcutta; Agarwal, B., Sing, U. and Banerjee, M., 1992; Plant Cell, Tissue and Organ Culture 30: 1-5; Nakamura, Y. 1989, Bull. Shizuoka Tea Exptl. Station 14: 1-9) followed by rooting. Root induction is an essential step. Because of the difficulties in root induction and hardening thereof, the success rate of micro-propagation in woody species including tea is limited (Paranjothy, K., Saxena, S., Banerjee, M., Jaiswal, V.S. and Bhojwani, S.S. 1990. In: Plant Tissue Culture: Application and Limitations, S.S. Bhojwani (ed.), Elsevier, pp. 190-219). Moreover, the rooting is always adventitious which perform poorly during hardening and establishment in the field (Jain, S.M., Das, S.C. and Barman, T.S. 1991. Acta Hortic. 289: 339-340; ha, T. and Sen, S.K. 1992. Plant Cell Reports 11: 101-104).

Grafting of nodal cuttings on seed raised plants has been used to provide a tap root advantage to the asexually propagated plants through single node cuttings, but this method also has the bottleneck of the number of nodal cuttings available at any given time.
An improvement over this method is the in vitro micro-grafting (Jonard, R. 1986. In: Biotechnology in Agriculture and Forestry, Vol. I Trees I, Y.P.S. Bajaj (ed), Springer-Verlag, N.Y. pp. 31-48; Ewald, D. and Kretzschmar, U. 1996, Plant Cell Tissue and Organ Culture, 44: 249-252; Ozzambak, E. and Schmidt, H. 1991. Gartenbauwissenschaft 56: 221-223; Starrantino, A. 1992. Petria (2) Supplemento: 27-35) that involves grafting of tissue culture raised micro-shoots or active meristem on to a tissue culture raised root stocks. However, the method involves dextrous handling and maintenance of aseptic conditions throughout. Hardening, an essential step in micro-propagation, of these micro-grafted plants has to be done essentially after ensuring the successful union of scion and stock, thus delaying their transfer to soil.
Thus, none of these methods can achieve rapid clonal propagation cum establishment of plants with a tap root system. The present invention overcomes the aforesaid limitation of the rate of clonal propagation and achievable success in the field.
OBJECTS OF THE INVENTION
The main object of the present invention is to provide method of tea propagation.
Another object of the present invention is to provide a quicker method of assessing stock scion compatibility.

Still another object of the present invention is the reduction in time for field transfer of micro-propagated tea shoots.
Yet another object of the present invention is to provide a method for simultaneous grafting and hardening of tea micro shoots on root stocks.
During the course of present investigation, the applicants have observed that the time required for getting field transferable micro-propagated plants having tap root system can be reduced from 18-24 months to 10 months. The present method involves grafting of in vitro raised tea micro-shoots on in vitro raised juvenile stock plants after plant growth regulators treatment tothe cut ends of both scion and stock followed by simultaneous hardening of tissue culture raised micro-shoots and establishment of the resulting plants in soil.
DETAILED DESCRIPTION OF THE INVENTION
Accordingly, the present investigation provides an improved method of tea propagation, which comprises (a) raising in vivo juvenile stock plants, (b) in vitro raising of micro-shoots of tea as scions, (c) decapitation of stock plantlets, (d) treatment of cut ends of stocks and scions with plant growth regulators, (e) grafting of in vitro raised micro-shoots on to in vivo raised juvenile stock plants, (f) holding of the said graft union tightly using any bio-compatible material (g) hardening of the said micro-grafted plants under controlled conditions which include a light source providing a minimum of 1000 lux intensity, a minimum carbon dioxide concentration of 400 ppm and relative humidity in the range of 50-95 percent, to obtain field transferable plants.
In an embodiment of the present invention, tea micro-shoots of a china hybrid were used.

 In another embodiment of, the present invention, tea micro-shoots of an assamica hybrid were used.

In yet another embodiment of the present invention, tea micro-shoots of a combod hybrid were used.
The juvenile stock plantlets may be produced by direct sowing of seeds in soil or in containers.
The tissue culture protocols for raising micro-shoots as scions may be selected out of preexisting meristems like apices, axillary buds, root tips or by inducing meristematic activity in cells of different explants like cotyledon, leaf, stem or root segments.
The plant growth regulator treatment given to cut ends of stock and scion may comprise auxin(s) or a combination of the cytokinin(s) and auxin(s) at the concentration of 0.01-10.0 ppm.
The auxins used as plant growth regulators for treatment to cut ends of stock and scions may be selected out of Indole-3-acetic acid, Indole-3-butyric acid, a-Naphthalene acetic acid, 2,4Dichlorophenoxy acetic acid.

The cytokinins used for treatment to cut ends of stock and scion may be selected out of 6-Benzyl amino purine, Kinetin, 2-Isopentenyl adenide or Zeatin.
 
The treatment to the cut ends of the stock and scion may last for at least one second.
The grafting of the micro-shoots as scions on decapitated juvenile root stocks may be done by any conventional method like cleft grafting, splice grafting, wedge grafting, veneer grafting or whip grafting.
The material for holding graft union point may be selected out of polythene, moist cotton, moss fastened with a string, parafilm, wax or clips, plastic films or pastes.
The light source may be selected out of incandescent, fluorescent or optic fiber illuminator or under direct/diffused sunlight.
The humidity may be maintained using any one or more of the water tanks, humidifiers, foggers, misters, with and without timers.
The carbon dioxide concentration may be maintained by supplying additional carbon dioxide from external sources.
Tea micro-propagation has been widely attempted and there are sporadic reports of successful plantlet development (Kato, M. 1985. Jpn. J. Breed. 35: 317-322; Kato, M. 1989. In: YPS Bajaj (ed) Biotechnology in Agriculture and Forestry, Vol 7. Springer-Verlag pp. 82-98; Arulpragasm, P.V., Latiff, R., Seneviratne, P. 1988. Sri Lankan J. Tea Sci., 57: 20-23; Palni, L.M.S; Sood, A., Chand, G., Sharma, M., Rao, D.V. and Jain, N.K. 1992. Tissue culture of tea: Possibilities and limitations. In: Tea Culture, Processing and Marketing, M.J. Mulky and V.S. Sharma (Eds), Oxford & IBH Publishing Co. Pvt. Ltd., New Delhi, pp 21-31). However, the developments thus far limit commercial application due to problems at the root induction phase and a long gestation period between hardening and field transfer. Moreover, only adventitious roots which are fragile and generally formed in vitro preceded by a small callus formation at the base of tea micro-shoots and such shoots show poor survival in soil. (Seneviratne, P., Latiff R. and Arulpragasm, P.V. 1988. Sri Lankan J. Tea Sci. 57: 16-19; Nakamura, Y. 1988. Tea Res. J. 68: 1-7). Lateron, cut ends of micro-shoots were treated with varying concentrations of IBA; low (2.0 mg/I, Seneviratne et al., 1988; Nakamura Y. 1991. JARQ, 25: 185-194) or very high (50-500 mg/1); followed by culturing in hormone free media or by direct planting in peat/soil mixture (Jain, S.M., Das, S.C. and Barman, T.S. 1991. Acta Hortic. 289: 339-340; Jha, T. and Sen, S.K. 1992. Plant Cell Reports, 11: 101-104). It, therefore, becomes quite obvious that exploitation of tea micro-propagation for commercial purposes has been delimited by lack of a systematic approach for rooting and establishment in the soil. The plantlets thus produced take longer i.e. 18-24 months before these can actually become ready for transfer to soil and initially show a slow growth.
On the other hand, the invention explained herein involves grafting of in vitro raised juvenile tea micro-shoots as scions on to soil raised juvenile seedlings after PGR treatment offer a system whereby, tap root advantage of the root stock plants can be fully exploited for early field establishment and it takes only 8-10 months in this case. There is no published report on the use of grafting of micro-shoots of tea on in vivo raised rooters which are already being grown in the soil. Although micro-grafting has been used successfully for many important fruit trees in the past such as oranges, cashew, grapes, apple, pecannuts, etc. (Burger, D.W. 1984. Proceedings Intl. Plant Propagators Soc. 34: 244-248) but nobody has used a combination of micro-shoots as scions, juvenile seedlings growing in vivo as stocks, treatment of cut ends with PGRs, hardening of grafted plants under controlled conditions of high CO, concentration, high light intensity and increased relative humidity in hardening chambers leading to around 90 percent survival. The grafted plants showed increased vigor as compared to micro-shoots rooted in vitro or even by direct planting in a soil mixture. This method can also determine the stock scion compatibility in a comparatively shorter time to try new combinations with more compatible tea clones. Finally, by this method, hardening of tea micro-shoots and establishment in soil is done as a single step procedure with tap root advantage of the juvenile seedlings.
 
BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS
Fig.1 shows the manner the seedlings are prepared and used as root stocks.
Fig.2 shows how a matching slanting cut can be given at the lower end of the tea micro-shoot which is used as a scion.
Fig.3 shows how the root stock and scion are placed together at the point of matching cuts.
Fig.4 shows the manner of securing of the root stock and scion together along with moist cotton or moss and thereafter, rapping them tightly from outside with parafilm.
Fig.5 shows the healthier and larger leaves of the grafted plants hardened under high Co2 concentration, high humidity and light.
Fig.6 shows the rate of growth of grafted tea on juvenile shoots and then the plant raised through direct rooting of micro-shoots. The picture clearly shows that the health of one year old tea graft was better than the micro-shoots which were directly rooted in soil.
Fig.7 shows the grafted plants in polysleeves kept on perforated support in a closed hardening chambers.
Fig.8 shows grafted plants shifted to polytunnel after 60 days in hardening chamber.
Fig.9 shows a field comprising grafted plants which were transplanted in said field after 8 months.
The following examples are given by way of illustration of the present invention and should not be construed to limit the scope of the present invention.

Example 1:
Tea seeds of china hybrids were collected from experimental farm and placed in moist sand medium (river bed sand garden soil: farm yard manure (FYM): 9:1:1) in pits covered over by polythene sheets for stratification for 3 weeks. The semi sprouted seeds were transferred into polysleeves containing a mixture of tea garden soil and sand in 9:1 ratio with soil pH at 5.4 and kept under a polytunnel. After 8 weeks in polysleeves, seedlings with 3, 4 and 5 leaves were used (Fig.1) as root stocks. Four months and one year old seedlings were also used as root stock in order to compare graft compatibility at different stages of development.
Multiple shoot cultures were established using nodal segments each containing an axillary bud of a china hybrid (BS-96). Nodal segments of 2-3 cm length were excised from the tea bush and washed thoroughly with distilled water and a laboratory detergent, teepol. Subsequently, these were surface disinfected with 2.0% (w/v) calcium hypochlorite for 10 minutes and washed with water again. Finally, these were treated with 0.04% (w/v) mercuric chloride solution for 6 minutes and repeatedly washed with sterilized distilled water so as to remove the traces of sterilizing agent. The cut ends of explains were cut with a sharp surgical blade and inoculated aseptically in a laminar flow cabinet on half strength Murashige and Skoog (MS, 1962) medium containing 3% (w/v) sucrose and 0.8% (w/v) agar powder for initial screening for any fungal or bacterial contamination. The cultures were incubated at 25+2°C under 12 hour light (3,500 lux) and equal dark cycles. Aseptic cultures after one week were transferred to MS medium supplemented with 6-Benzyl amino purine (BAP, 2.0 mg/1), indole-3-acetic acid (IAA, 0.2 mg/1), sucrose (2.0 % w/v) and agar powder (0.8%, w/v). Shoot proliferation took place in this medium and young micro-shoots for grafting were formed in 5-6 weeks.
After decapitating the hypocotyl region of the 8 weeks, 4 months and 1 year old seedlings to be used as root stocks at 1.0, 1.5 and 2.0 cm respectively above the soil surface with a sharp surgical blade, another slanting cut was given so as to expose the cambium. A matching slanting cut was also given at the lower end of the tea micro-shoot (Fig.2) which was to be used as a scion. The cut ends of both stock and scion were treated with BAP (5.0 mg/I) and NAA (5.0 mg/1) for 10 minutes and untreated ones served as control. The root stock and the scion were held together at the point of matching cuts with moist cotton (Fig.3) or moss and then wrapped tightly from outside with parafilm (Fig.4).
The grafted plants in polysleeves were kept in closed hardening chambers. These chambers were made of concrete provided with glass fitted lids joined on hinges. The bottom of the chamber was filled with water to increase humidity (90%) and grafted plants were kept on the perforated steel platform (Fig.7). Carbon dioxide was supplied through a side port with the help of a rubber tube and its level was maintained at 800 ppm. After putting the plants in, the lids were closed and light was provided by the overhung electric bulbs (1600 lux). The lights were kept on for 9 hours.
The graft union between the compatible clones took 40days. After 60 days in hardening chambers (Fig.7), the plants were shifted to polytunnel (Fig.8) and after 8 months, the grafted plants were translated in the fields (Fig.9). Up to 92 percent survival in the field has been achieved.
On comparing the rate of growth of grafted tea on juvenile shoots and tea raised through direct rooting of micro-shoots (Fig.6), it was found that the health of one year old tea graft was better than the micro-shoots which were directly rooted in soil.
The above example shows the usefulness of this method for auto-grafting that means both scions and stocks belonging to the same clone.
In these experiments, beneficial effects of PGR treatments to both scion and stock for early new leaf development, compatibility studies on different clones and suitability of young seedlings as root stocks has been established. Besides, the time of grafting during the year had a profound effect on the overall results as the grafts made during spring (2nd week, of February to 1st week of April) established relatively faster and with higher percentage of success (92.00%).
Hardening of the grafted plants under high CO, concentration, high humidity and light proved beneficial for preparing the plants for field transfer. The leaves of the grafted plants (Fig.5) were healthier and larger in size and were transferred to fields within 10 months.
Example 2:
Juvenile tea seedlings of china hybrids were prepared as explained in Example 1 with all the parameters and conditions remaining exactly the same.
However, multiple shoot cultures were established from another high yielding tea clone of commercial importance, TV-18 a combod hybrid using the same protocols as for china hybrid the details of which were given under Example 1.
The grafting was then done using the micro-shoots of TV-18 as scions and decapitated juvenile seedlings of china hybrid. The graft union took place in 40 days’ time and after 60 days in hardening chambers, the grafted tea plants were shifted first to the polytunnels and then to the field. The is an example of successful heterograft union where scion belonged to combod hybrid and stock was that of china hybrid.
Example 3:
Juvenile tea seedlings of china hybrids were prepared as explained in Examples 1 and 2 with all the parameters and other conditions remaining exactly the same.

Multiple shoot cultures were established from yet another high yielding and commercially important clone from South India, UPASI-9, an assamica hybrid using the same protocols as for china hybrid and TV-18 in the examples 1 and 2.
The grafting was then done using the micro-shoots of UPASI-9 as scions and decapitated juvenile seedlings of china hybrid.
In this case also graft union took place in 40 days and after 60 days in polytunnels, the grafted plants were transferred to the fields in 10 months’ time. This again is a case of successful heterograft. However, the percent success was up to 80 percent depending upon the age of the root stock (In this case, 4 months old seedlings were used).
Example 4:
Juvenile tea seedlings of china hybrids were prepared as explained in Examples 1, 2 and 3 with all the parameters and other conditions remaining exactly the same.
In this case, multiple shoot cultures were established from a bi-clonal tea hybrid using two well-known commercial clones Phoobseering-312 (0) X TV-1 (0) by the embryo rescue technique. Crosses of the above mentioned two tea clones were made by sprinkling the pollen of Phoobseering-312 (0) on the stigmas of V-1 (0) and covering with butter paper bags. The young seedlings showed a tendency towards embryo abortion if retained on the bushes. Therefore, the seedlings with immature embryos were harvested after 350 days. The outer seed coats were removed from the seeds and surface sterilized with calcium hypochlorite (2% w/v) solution for 6 minutes. The seeds were again surface sterilized with mercuric chloride solution (0.04% w/v) for 5 minutes and washed repeatedly with sterilized distilled water in a laminar flow cabinet. The hard testa was then removed carefully with sharp stainless steel scalpel and fine needles and immature embryos were taken out from inbetween the cotyledons and placed on a half strength Murashige and Skoog (1962) medium supplemented with 3% (w/v) sucrose and 0.8% (w/v) agar. After initial screening on this medium, the embryos were transferred to the basal MS medium with 2% (w/v) sucrose and activated charcoal (0.2%, w/v). These showed germination in 3 weeks and young seedlings when grew to about 1-1.5 cm were transferred to MS medium supplemented with BAP (2.0 mg/3), IAA (0.2 mg/1), sucrose (2% w/v) and agar powder (0.8% w/v). Multiple shoot formation took place in 5 weeks and young micro-shoots could be used for grafting. The grafting was done using the micro-shoots as scions of the biclonal tea hybrids as produced above and decapitated juvenile seedlings of china hybrid. The graft union took place in 40¬45 days and after 60 days in hardening chambers, as explained in examples 1, 2 and 3, the grafted plants were shifted first to the polytunnels and then to the fields. This again is an example of the usefulness of the invention in bringing about successful transfer of biclonal hybrids as novelty to the field conditions in a comparatively shorter time of 10 months.

 Therefore, it is amply clear from the above examples that the present method has proved to be successful not only for auto-grafts but for heterograft’s as well involving scions from other well-known high yielding clones and even new bicional hybrids where embryo abortion would otherwise take place if seeds are left intact on the tea bushes.
In these examples, beneficial effect of PGR treatment to both scion and stock for early new leaf development, compatibility studies on different clones and suitability of young seedlings as root stocks has been established. Besides, the time of grafting during the year had a profound effect on the overall results as the grafts made during spring (2nd week of February to 1st week of April) established relatively faster and with higher percentage of success (92%).
Hardening of the grafted plants under high CO2 concentration, high humidity and light proved beneficial for preparing the plants for field transfer. The leaves of the grafted plants were healthier and larger in size and the plants thus produced could be transferred to fields within 10 months.
ADVANTAGES OF THE INVENTION
The main advantages of the present invention are:
 
1. The micro-grafting helps in the early detection of compatibility factors between any two tea clones/hybrids for carrying out studies on varietal improvements;
2.The methods can also be employed for disease and drought resistance studies by choosing stocks from drought resistant clones and scions from the disease resistant varieties.
3.The micro-grafting method ensures hardening and establishment of rooted plants as a one-step procedure thereby, saving time.
4. Establishment in the fields gets easier with well hardened plants having a tap root system.
5. Micro-grafting also helps in the commercial scale production of superior planting materials for an early release.
6. The method can be applied to all the tea clones and a number of new compatible grafts with increased vigor and adaptability can be made.
7. A nearly non-stop production schedule can be maintained throughout the year.
8. The grafted plants can be tested in the field with a reasonable amount of assurance of their success using any one or more of the water tanks, humidifiers, foggers, misters with and without timers.

9.    A method as claimed in claims 1 to 11, wherein the carbon dioxide concentration is maintained by supplying additional carbon dioxide from external sources.
10. An improved method of tea propagation substantially as herein described with reference to the examples and photographs accompanying this specification,
 
Claims:
1. An improved method of tea propagation, which comprises (a) raising in vivo juvenile stock plants, (b) in vitro raising of micro-shoots of tea as scions, (c) decapitation of stock plantlets, (d) treatment of cut ends of stock and scions with plant growth regulators, (e) grafting of in vitro raised micro-shoots on to in vivo raised juvenile stock plants, (f) holding of the said graft union tightly using any biocompatible material, (g) hardening of the said micro-grafted plants under controlled conditions, which include a light source providing a minimum of 1000 lux intensity, a minimum carbon dioxide concentration of 400 ppm and relative humidity in the range of 50-95 percent to obtain field transferable plants.
2. A method as claimed in claim 1, wherein the juvenile stock plantlets are produced by directly sowing of tea seeds in soil or in containers.
3. A method as claimed in claim 1 and 2, wherein the explants for raising micro-shoots as scions using pre-existing meristems like apices, axillary buds, root tips or by inducing meristematic activity in cells of different explants like cotyledon, leaf, stem or root segments.
4. A method as claimed in claims 1 to 3, wherein the plant growth regulator treatment given to cut ends of stock and scion comprises auxin(s) or a combination of the cytokinin(s) and auxin(s) at the concentration of 0.01-10.0 ppm.
5. A method as claimed in claims 1 to 4, wherein auxins used as plant growth regulators for treatment to cut ends of stock and scions is selected out of Indole-3-acetic acid, Indole-3-butyric acid, a-Naphthalene acetic acid, 2,4- Dichlorophenoxy acetic acid.
6. A method as claimed in claims 1 to 5, wherein the cytokinins used for treatment to cut ends of stock and scion is selected out of 6-Benzyl amino purine, Kinetin, 2-Isopentenyl adenine or Zeatin.
7. A method as claimed in claims 1 to 6, wherein the treatment to the cut ends of the stock and scion lasts for at least one second.
8. A method as claimed in claims 1 to 7, wherein the grafting of the micro-shoots as scions on decapitated juvenile root stocks is done by any conventional method like cleft grafting, splice grafting, wedge grafting, veneer grafting or whip grafting.
9. A method as claimed in claims 1 to 8, wherein the material for holding graft union point is selected out of polythene, moist cotton, moss fastened with a string, parafilm, wax or clips, plastic films or pastes.
10. A method as claimed in claims 1 to 9, wherein the light source is selected out of incandescent, fluorescent or optic fiber illuminators or under direct/diffused sunlight.
11. A method as claimed in claims 1 to 10, wherein the humidity is maintained using any one or more of the water tanks, humidifiers, foggers, misters with and without timers.
12. A method as claimed in claims 1 to 11, wherein the carbon dioxide concentration is maintained by supplying additional carbon dioxide from external sources.
13. An improved method of tea propagation substantially as herein described with reference to the examples and photographs accompanying this specification.

 
III. an aqueous dispersion consisting of 20 parts by wt. of compound 9, 40 parts by wt. cyclohexanone, 30 parts by wt. isobutanol, 20 parts by wt. of the addition product of 40 mol ethylene oxide in 1 mol castor oil;
IV. an aqueous dispersion consisting of 20 parts by wt. of compound 24, 25 parts by wt. cyclohexene, 65 parts by wt. of a mineral-oil fraction of boiling point 210 to 280°C and 10 parts by wt. of the addition product of 40 mol ethylene oxide in 1 mol castor oil;
V. a mixture ground in a hammer mill of 80 parts by wt. of compound 35, 3 parts by wt. of the sodium salt of diisobutylnaphthalene-a-sulphonic acid, 10 parts by wt. of the sodium salt of a lignosulphonic acid from a sulphite lye and 7 parts by wt, of powdered silica gel; by fine distribution of the mixture in water a spraying-mixture is obtained;
VI. an intimate blend of 3 parts by wt. of compound 56 and 97 parts by wt. of finely-particulate kaolin; this dust contains 3% by wt. of active substance;
VII. an intimate blend of 30 parts by wt. of compound 108, 92[*2] parts by wt.
powder-form silica gel and 8 parts by wt. paraffin oil which has been sprayed on to the surface of this silica gel; this process gives the product good retention;
VIII. a stable aqueous dispersion of 40 parts by wt, of compound 166, 10 parts by wt. of the sodium salt of a phenosulphonic urea-formaldehyde condensation product, 2 parts by wt. silica gel and 48 parts by wt. water, which may be further diluted;
IX. a stable oily dispersion consisting of 20 parts by wt. of compound 214, 2 parts by wt. of the calcium salt of dodecyl benzene sulphonic acid, 8 parts by wt. fatty-alcohol polyglycol ether, 20 parts by wt, of the sodium salt of a phenolsulphonic urea-formaldehyde condensation product and 68 parts by wt. of a paraffin mineral oil.
In these preparations the active substances according to the invention may also be combined with other active substances, e.g. herbicides, insecticides, growth regulators, fungicides or fertilizers.
 
In many cases mixing with fungicides results in a broadening of the spectrum of fungicidal activity.
The following list of fungicides with which the invention compounds may be jointly applied is intended to indicate the possible combinations, but is not exhaustive:
sulphur,
dithiocarbamates and derivatives of them such as ferridimethyldithiocarbamate, dimethyl zinc
dithiocarbamate,

ethylene zinc bis-dithiocarbamate,
ethylene manganese bis-dithiocarbamate,
manganese-zinc-ethylenediamine bis-dithiocarbamate,
tetramethyl tiara disulphide,
ammonia complex of zinc-(N,N-ethylene-bis-dithiocarbamate),
ammonia complex of zinc-(N,N'-propylene-bis dithiocarbamate),
Zinc-(N, N.-propylene-bis-(thiocarbamoyi)-disulphide;
Nitroderivatives such as
dinitro-(1-methylhepty1)-phenylcrotonate,
2-sec-butyl-4, 6-dinitrophenyl-3, 3-dimethyl acrylate,
2-sec-butyl-4, 6-dinitrophenyl-isopropyl carbonate;
5-nitro-isophthalic acid di-isopropyl ester heterocyclic substances such as2-heptadecy1-2-imidazoline-acetate,
 2, 4-dichloro-6-(o-chloroanilino)-s-thiazine, o, o-diethyl-phthalimido-phosphonothioate,
2-arnino-1-[bis-(dimethylamino)-phosphinyl]-3-pheny1-1, 2, 4-triazole, 2,3-dicyano-1,4-dithioanthroquinone,
 2-thio-1, 3-dithiolo[4, 5-b]-quinoxaline, 1-(butylcarbamoy1)-2-benzimidazole-methyl carbamate,
2-methoxycarbonylamino-benzimidazole,
2-(fury1-(2))-benzimidazole,
2-(thiozoly1-(4))-benzimidazole,
N-(1, 1, 2, 2-tetrachloroethylthio)-tetrahydrophthalimide,
N-trichloromethylthio tetrahydrophthalimide, N-trichloromethylthio phthalimide,
 
N-dichlorofluoromethylthio-N, N-dimethyl-N-phenyl sulphuric diamide,
 5-ethoxy-3-trichloromethy1-1, 2, 3-thiadiazole,
2-rhodan methylthiobenzthiazole,
1, 4-dichloro-2, 5-dimethox0enzene,
4-(2-chlorophenylhydroazano)-3-methy1-5-isoxazalone,
Pyridin-2-thio-1-oxide,
8-hydroxyquinoline or its copper salt,
2, 3-dihydro-5-carboxanilido-6-methy1-1, 4-oxathiine,
 2, 3-dihydro-5-carboxanilido-6-methyl-1, 4-oxathiine-4,4-dioxide,
 2-methyl-5, 6-dihydro-4H-pyran-3-carbonic anilide,
2-methyl-furan-3-carbonic anilide,
2, 5-dimethyl-furan-3-carbonic anilide,
2, 4, 5-trimethylfuran-3-carbonic anilide
2, 5-dimethyl-furan-3-carbonic cyclohexylamide
N-cyclohexyl-N-methoxy-2, 5-dimethyl-furan-3-carbonamide,
2-methyl-benzoic anilide,
2-iodine-benzoic anilide,
N-formyl-N-morpholine-2, 2, 2-trichloroethyl acetal,
Piperazine-1, 4-diyl bis-(1, (Z2, 2-trichloroethyl)-formamide,
1-(3, 4-dichloroanito)-1-formylamino-2, 2, 2-trichloroethane,
2, 6-dimethyl-N-tridecyl morpholine or its salts,

2, 6-dimethyl-N-cyclodedecyl morpholine or its salts,
N-[3-(p-tert.-butylpheny1)-2-methylpropyl]-cis-2, 6-dimethyl morpholine,
 N-[3-(p-tart: butylpheny1)-2-methylpropyl]-piperdine,
142-(2, 4-dichloropheny1-4-ethy1-1, 3-d foxolan-2-yl-ethyl]-1 H-1, 2, 4-triazole,
142-(2, 4-clichloropheny1)-4n-propy1-1, 3-d ioxolan-2-yl-ethyll-1 H-1, 2, 4-triazole,
N-(n-propy1)-N-(2, 4, 6-trichlorophenoxyethyl)-N'-imidazole-yl-urea
1 - (4-chlorophenoxy)-3, 3-d methyl-1 - (1 H-1, 2, 4-triazol-1-y1)-2-butanone,
1-(4-chlorophenoxy)-3, 3-d I methyl-1 - (1 H-1, 2, 4-triazol-1 -y1)-2-butanol,
a-(2-chlorophenyl)-a-(4-chlorophenyl)-5-pyrimidine methanol,
5-butyl-2-dimethylamino-4-hydroxy-6-methyl pyrimidine,
 bis-(p-chloropheny1)-3-pyridine methanol,
 1, 2-bis-(3-ethoxycarbony1-2-thioureido)-benzene,
 1, 2-bis-(3-methoxycarbony1-2-thioureido-benzene,
And various fungicides such as dodecyl guanidine acetate,
3-(3-(3, 5-dimethy1-2-oxycyclohexyl)-2-hydroxyethylFglutarimide
Hexachlorobenzene,

 DL-methyl-N-(2, 6-dimethylphenyl) N-furoyl (2) alanine,
DL-N-(2, 6-dimethylphenyl)-N-(2-methoxyacetyl)-alanine methyl ester,
 N-(2, 6-dimethyl phenyl)-N-chloroacetyl-D, L-2-aminobutyrolactone,
 DL-N-(2, 6-dimethylphenyi)-N-phenylacetyl) alanine methyl ester,
 5-methy1-5-viny1-3-(3, 5-dichlorophenyI)-2, 4-dioxo-1, 3-oxazolidine,
343, 5-d ichlorophenyl (-5-methy1-5-methoxmethyl]-1, 3-oxazolidine-2, 4-dione,
 3-(3, 5-dichlorophenyI)-1-isopropylcarbamoyl hydration, N-(3, 5-dichlorophenyI)-1,2-dimethylcyclopropane-1,2-dicarboxylic imide,
2-cyano [N-(ethylaminocarbony1)-2-methoximinol acetamide,
 42-(2, 4-dichloropheny1)-penty11-1H-1, 2, 4-triazole,
2, 4-difluoro-a-(1H-1, 2, 4-triazoly1-1-methyl)-benzhydryl alcohol,
 N-(3-chloro-2, 6-dinitro-4-trifluoromethyl-pheny1)-5-trifluoromethy1-3-chloro-2- aminopyridine,

1-((bis-(4-fluoropheny1)-methylsily1)-methyl)-1H-1, 2, 4-triazole
Effectiveness against Pvrenophora teres
At the two-leaf stage barley seedlings of the "Igri" variety were sprayed until dripping wet with 0.05% aqueous suspensions containing 80% active substance from those listed in tabulated examples 1, 56 and 166 and 20% emulsifier in dry-solid form. After 24 hours the plants were infected were a suspension of the fungus Pyrenophora teres and placed for 48 hours in a climatic chamber with high humidity at 18°C.
The plants were then cultivated for a further 5 days in a greenhouse at 20 to 22°C and 70% relative humidity. The extent of fungal infection was then assessed.
The treated plants were found to have fungal infection of only 5-15% in contrast to a control test (no treatment, 80% fungal infection).

 Example 2
Effectiveness against vine peronospora Leaves of "Muller-Thurgau" tub-vines were sprayed with 0.025% aqueous suspensions containing 80% active substance and 20% dry-solid emulsifier. To assess the duration of the 1, 2-naphthoquinones' action the plants were placed in a greenhouse for 8 days after the sprayed-on coating had dried. Only then were the leaves infected with a zoospore suspension of Plasmopara viticota (vine peronospora) and the plants placed for 48 hours in a steam-saturated greenhouse at 24°C. The vines were then cultivated for 5 days in a greenhouse at temperatures between 20 and 30°C and in a moist chamber for another 16 hours to accelerate sporangia outburst. The extent of fungal infection on the underside of the leaves was then assessed.
In contrast to a control test (no treatment, 80% fungal infection) the result showed active substances 2, 56, 166 and 214 to have only 5-15% fungal infection when applied as a 0.025% solution.
Claims
1. Fungicide containing carrier substances and a 1,2-naphthoquinone of the general formula I
[Graphic: please see source text] in which the substituents R1 to IV have the following meanings:
(a) hydrogen or halogen;
(b)  up to 3 of the substituents R1 to RO denote the following groups: cyano, nitro, partially or completely halogenated C1-C10-alkyl, C2-C10-alkenyl with up to 2 halogen substituents, C2-C10alkinyl, C3-C6-cycloalkyl, C2-C6alkoxy-alkoxycarbonyl, C1-C10-alkyl or C2-C10alkenyl, the last two groups possibly carrying up to 2 of the following substituents: hydroxy, cyano, C1-C4-alkylthio or C1-C4alkoxycarbonyl;
(c) up to 3 of the substituents R1-R6 denote a group of the general formula: MR7, where X denotes oxygen, sulphur, a -CO-, -C0-0-, -0-C0-, -NR8-,-CO-NR5- or C1-C4-CO-NR5 group and R7 and R8 denote hydrogen, C1-C6-alkyl which may be partially or wholly halogenated, C3-C6-alkenyl or C3-C6-alkinyl;
(d) up to 2 of the substituents R1-R6 which should not be vicinal, denote the following groups: aziridinyl, pyrrolidino, piperidino or morpholino, where the last two groups may carry up to 2 of the following substituents: C1-C10-alkyl, C2 C10 alkoxyalkyl, C2-C10-alkylthioalkyl, phenyl or benzyl;
(e) up to 2 of the substituents R1-R5, which should not be vicinal, denote the following groups:
phenyl, benzyl or C5-C6cheteroaryl, to which a benzene ring may be annellated, the aryl groups carrying up to 3 of the following groups: halogen, nitro, cyano, trifluoromethyl, C1-C4alkylamino alkylamino, C1-C4alkoxy, C1-C4-haloalkoxy, C2-C4-alkoxyalkyl, C1-C4-alkylthio or C1-C4-haloalkylthio;
(f) R2, R° denote a group of the general formula: YR9, where Y denotes oxygen, sulphur, a -0-CO- or -NR5- group and R9 denotes phenyl or benzyl, which in the phenyl residue may carry up to 3 of the following substituents: halogen, cyano, nitro or trifluoromethyl;
(g) R2, R6:
groups of the general formulas IIa or IIb
[Graphic: please see source text] IIa    IIb
 
where R10 denotes hydrogen, Cl-C10-alkyl, C1-C10-haloalkyl, C3-C10-ralkenyl, C3-C10 alkinyl, C2-C14-alkoxyalkyl, C2-C14-alkyl thioalkyl or C3-C7-cycloalkyl and R11 denotes Cl-C10-alkyl, C2-C8-alkenyl, trifluoromethyl, cyclopropyl or a phenyl group, which in turn may carry up to three of the following substituents: halogen, cyano, nitro, C1-C8; alkylor trifluoromethyl;
(h) two vicinal substituents of the substituents R1 to R5 denote together an annellated 5- or 6-membered unsaturated ring to whose C-atoms a further benzene ring may be annellated and which may contain sulphur or up to 2 non-vicinal oxygen- or up to 3 nitrogen-atoms as heteroatoms: halogen, hydroxyl, nitro, cyano, amino, Cl-C4alkylamino, C1-C8-dialkylamino, trifluoromethyl, C1-C5-alkyl, C1-C5 alkoxy, C1-C5haloalkoxy, C1-C5-alkylthio, Cl -C5,haloalkyithio or phenyl, except for 3,8-climethy1-5-isopropyl-1,2- naphthoquinone.
2. Fungicides containing inert carrier substances and a 1, 2-naphthoquinone I as per Claim 1, where R° denotes a hydroxyl group.
3. Fungicides containing inert carrier substances and a 1, 2-naphthoquinone I as per Claim 2, where R2 denotes fluorine, chlorine, bromine, a trifluoromethyl group or a C1-C4alkoxy group.
4. Fungicides containing inert carrier substances and a 1,2-naphthoquinone 1 as per Claim 1, where R2 stands for a methoxy group, a methylthio group, a CH(CN)- COOMe group or a 4-(4-tert.-butylphenyl)-piperidin-1-yl group and the remaining residues denote hydrogen.
5. Method of controlling fungi characterized in that a fungicidally effective quantity of an active substance containing a 1,2-naphthoquinone of the formula I as per Claim 1 is caused to act on fungi or materials, areas, plants or seedcorn at risk of fungal infection.
 
EP 0 413 224 Al
CATEGORY OF NAMED DOCUMENTS
X: of particular importance considered alone
Y: of particular importance in conjunction with another publication of the same category
A: technological background
0: unwritten disclosure
P: recent publication
T: theories or principles underlying the invention
E: older patent document, but published only on or after the date of filing
D: document cited in the Application
L: document cited for other reasons &member of the same patent family, congruent document

 
Translators Notes:
[*1] These notes have been added owing to shortage of space and uncertainty of interpretation.
[*2] Sic: possibly a misprint in the original for "62".

 

 

 

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