CZ2007119A3 - Method of making Crataegus sp. clones with proved biological properties and resistance behavior to Erwinia amylovora - Google Patents

Abstract

The essence of the solution is an original method of using methodical procedures, which allows the creation of clone Crataegus sp.s verified biological properties and resistant behavior to Erwinia amylovora (EA). The method includes procedures for selection of donor plants, derivation of primary culture in vitro, micropropagation in vitro, conversion of regenerant to ex vitro, growing of young plants in vivo, laboratory tests of resistance to EA, planting of young clone plants in situ, verification of biological properties and evaluation of field resistance, preserving maternal clones under in vitro conditions.

Description

A method for producing Crataegus sp. with proven biological properties and resistant behavior to Envinia amylovora.

Technical field:

The invention relates to a unique system of protection of plants of the Rosacea family against infection by the quarantine bacterium Envinia amylovora (Burrill) as the causative agent of the "pink scorch" disease.

BACKGROUND OF THE INVENTION:

In 1986 (Kítdela, 1988) the first occurrence of a significant disease in the Czech Republic, named after manifestations and hosts of “pink scarlet plant - the causative agent of Erwinia amylovora (Burrill) - (EA), was recorded in the Czech Republic. To date, this dangerous quarantine infection has spread to most of the Czech Republic. Methods of spreading EA bacterial cells, so-called propagules (slime form, air ropes) can be very diverse - wind, rain, insects, birds, transport corridors and the like. (Kiideia, 1990). The most important way to passively spread EA is by moving infected plants or parts thereof in vegetative reproduction and trade in plant material.

The occurrence of EA has been proven around 200 species of the Rosacea family in the world (Kůdela, 1990), in the Czech Republic the vast majority of occurrences are related to the genus Crataegus.

Hawthorn (Crataegus sp.) Is an ecologically important woody species that helps to create a suitable environment for many herbaceous plant species, serves as a shelter and food for small animals, is a beekeeping and medicinal plant. It is one of the most widespread tree species in the flora of the Czech Republic. It is important especially in drying habitats and it occurs very often even in the landscape significantly modified by man - ie in forest forests, on boundaries, weights and ruderal areas.

In the Czech Republic, the genus Crataegus is the most important in terms of long-term persistence of EA and therefore a source of infection for other hosts of the rose family, especially for fruit production trees. For these reasons, within the framework of eradication measures applied by the State Phytosanitary Administration, up to 1997, more than 25 ths. hawthorn (Korba et al., 1998), these measures have had a negative impact on the biodiversity of hawthorn communities. Under the new law, since 2004 the eradication of infected plants in natural communities has been abandoned, with the exception of the surroundings of the production areas of host fruit trees. By reducing eradication, direct devastation of hawthorn habitats has been stopped, but on the other hand, there is an increase in the pathogen (EA) infection pressure and thus an increase in the risk of local spread of infection and the intensity of damage to these stands. New plantings of hawthorn susceptible to EA may also have a negative effect on the development of this situation.

L) Crataegus sp. the disease manifests in rapid burn of shoots and brachyblasts, sometimes even whole branches, and the intensity of damage may vary from year to year. Initial direct harmful effects on hawthorn vital plants were not great, and the importance of EA binding to this taxon was largely based on the formation of reservoirs of infection in the landscape. Reversal of the patosystem occurred in 2004, when it was first recorded a complex damage to hawthorn of irreversible character and therefore the actual threat to the state of the taxon. This development was due to the interaction of a strong manifestation of EA infection with other bionoses and abionoses, with the development of weather patterns making a very predisposition. Currently, the damage in some shrubs, especially on drying-out of typical hawthorn habitats, is quite pronounced in the form of burn symptoms of EA infections, drying of the crown from its periphery, high fructification, minimizing the formation of annual shoots. This cycle of damage is repeated and in a number of investigated cases it has reached the complete death of the shrub. Continuation

- Even these developments may lead to a decrease in the biodiversity of plant communities in ecosystems where hawthorn plays an irreplaceable role.

Direct effective ecologically favorable protection of host plants against EA is not yet known. A very effective option is to use sources of natural resistance to EA {Patáková, Korba. 2000 /, which was already in the Czech Republic at the hawthorn Crutaegus sp. proved.

Systematic search for sources of EA resistance in natural communities, using in vitro multiplication methods to create clonal offspring, and laboratory tests to verify the resistance of EA clones obtained in hawthorn have not been performed worldwide. Repatriation and reintroduction of EA-resistant hawthorn clone plants in laboratory tests into natural hawthorn communities to verify EA field resistance and assessment of its biological properties worldwide has also not been performed.

SUMMARY OF THE INVENTION:

SUMMARY OF THE INVENTION The present invention is based on an original method of using methodologies which allow the production of Crataegus sp. with proven biological properties and resistant behavior to Erwinia amylovora (EA).

The method includes procedures for selecting donor plants, deriving a primoculture in vitro. in vitro micropropagation, exogenous regeneration, in vivo cultivation of young plants, EA resistance laboratory tests, in vitro sowing of young clone plants, verification of biological properties and evaluation of field resistance, in vitro preservation of parent clones in the VUKOZ genobank, v.v.i.

Examples:

In a locality with natural hawthorn communities (Crutaegus spj by], following the development of Erwinia amylovora (EA) infection in 1994-2006, a hawthorn shrub was selected as a donor plant to establish a primary in vitro culture. The occurrence of EA infection in the monitored locality was proved in the individual years in the infected bushes in the laboratory.

Annual shoots were taken from the donor shrub and their apical and axillary buds were used to establish the primary culture in vitro. The primary in vitro culture was based on 7.5 g.l-1 agar-strengthened WPM medium supplemented with 0.5 mg.lI benzyladenine, 20 g.l-1 sucrose and vitamins. Cultivation was carried out in a 16-hour photoperiod at 22 ° C. The derived culture was gradually multiplied in vitro. the top cuttings were taken and rooted in the steamed propagating substrate under in vivo conditions in a culture box. After reaching a size of about 1 (J: m), the young clone plants were placed in a cubicle greenhouse, gradually plated into a peat substrate of type A and grown in hotbeds up to a size of about 20 - 40 cm. 20 pieces were transferred to a specialized bacteriological laboratory for EA resistance testing Laboratory tests were performed by inoculating decapitated shoots with a drop of a mixture of EA strains, the resistance was determined on a USD A scale (Korba et al. 1998; Mohan et al .. 2002) .

Young clone plants with resistance demonstrated in laboratory tests were repatriated to their locus of origin in the vicinity of their donor plant. At the same time, they were reintroduced to four other localities in different areas of the Czech Republic with natural hawthorn communities (Crataegus sp.y long-term infested EA). Hawthorn seedlings of the size corresponding to clone plants were also planted at all five sites. The occurrence of EA symptoms was observed in young clone plants, control seedlings and naturally growing shrubs, samples with suspected symptoms were always analyzed by laboratory methods using standard methods for EA presence.

T

The prevalence of EA infection in naturally growing hawthorn bushes with symptoms of scarlet fever at five sites was always demonstrated by laboratory tests in each year. The spread of EA to new plantations at the site was demonstrated by detection of EA in some seedlings of control seedlings. At the same time, the overall growth and development of clone plants was evaluated and compared with the growth and development of control seedlings, donor plants and other naturally growing hawthorn bushes in the locality.

In five growing seasons, clone plants were not found to be infected with EA in the natural conditions of EA infection pressure at the site, and their good growth and development was also verified. Field cleavage to EA was also demonstrated in clonal plants, while maintaining biological properties.

The clone showing the stability of the desired traits becomes the parent clone and is maintained under in vitro conditions in the VÚKOZ genobank, v.v.i. Průhonice.

Industrial Applicability;

The use of hawthorn (Crataegus sp.) Clone plants with proven biological properties and resistant behavior to Erwinia amylovora (EA) in new plantations is the most effective way of preventive protection against this quarantine bacterial infection.

From the point of view of the genus Crataegus, these clone plants will allow new plantations to be realized, minimizing the risk of their devastation, loss of biological functionality and utility value. From an epidemiological point of view, these plantings will make it possible to reduce the reservoirs of EA infection in the landscape and reduce the resources of the EA inoculum for other host plants of the Rosacea family at the site.

Consequently, the clone plants formed in this way solve the problem of planting hawthorns, particularly in the protection zones of the Pyrus and Malus production units, in urban greenery and in the cladding of fast-growing tree plantations.

List of references cited.

KŮDĚLA V. (1988): Envinia amylovora, the causal agent of the scarlet rose in Czechoslovakia. Choir, ÚVTIZ - Ochr. Plant., 24: 173-182.

KUDELA V. (1990): The scent of pink plants. Ministry of Agriculture and Food of the Czechoslovak Republic. Agrodat, ISBN 80-7084-018-8, 163 pp.

KORBA J., PATÁKOVÁ S., KÚDELA V. (1998): Resistance of hawthorns to rosacea. Plant Protection Science, 34: 53-58.

MOHAN, S.K., FALLAHI, E. AND BIJMAN, V.P. (2002): Evaluation of Apple Varieties for Susceptibility to Erwinia amylovora by Artificial Inoculation under Eield Conditions. Acta Hort. (ISHS) 590: 373-375

PATÁKOVÁ S., KORBA J. (2000): Determination of the degree of resistance of apple and pear varieties to rosacea. Proceedings of papers from XV. Czech and Slovak conferences on plant protection. Brno, 2000, pp. 36 37

Claims (1)

Claims: A method for producing Crataegus sp. with proven biological properties and resistant behavior to Envinia amylovora (Burrill) (EA), characterized in that: (a) in a locality with natural hawthorn communities (Crataegus spj long-term infested EA), a hawthorn bush not infected with EA is selected and with good growth and development as a donor plant for the establishment of a primary in vitro culture; (b) a primary in vitro culture is established from both the apical and axillary buds of annual shoots, (c) the derived culture is progressively multiplied in vitro, d) harvested top cuttings are rooted in in vivo conditions in a culture box, e) rooted clonal plants are grown to a size of approx. 20 - 40 cm, (f) in the case of young clone plants, the degree of resistance shall be determined in laboratory tests; (g) clone plants with resistance demonstrated in laboratory tests are replanted at the original site (repatriation) and at other EA infested sites (reintroduction), (h) hawthorn seedlings (Crataegus sp.) f (i) at each planting site in each vegetation, naturally growing hawthorn shrubs, donor plants, planted hawthorn seedlings and clone plants shall be assessed for the presence of scarlet fever, laboratory evidence of the presence of EA, monitoring for all growth and development abnormalities and determining their cause; j) for clone plants, their growth and development at individual planting sites are compared and clones that show stability of the desired traits are identified, k) selected parent clones are maintained under in v / 7ro, y genubance conditions. VÚKOZ, v.v.i. Průhonice ^