KR100463773B1 - Lycopersicon pimpinellifolium as a source of resistance to the plant pathogen phytophthora infestans - Google Patents

Abstract

The present invention includes a tomato plant (lycopericone esculentum) that is resistant to the tomato bacterium pi infestans 0 and 1. Lycopperic cone esculentum plants, which are resistant to the tomato bacterium P. infestans 0 and 1, are used to produce the tomato plants of the present invention. Preferred lycophericone esculentum for use in the present invention is designated LA2533.

Description

LYCOPERSICON PIMPINELLIFOLIUM AS A SOURCE OF RESISTANCE TO THE PLANT PATHOGEN PHYTOPHTHORA INFESTANS} As a Source of Resistance to the Plant Pathogen Phytophthora Infestans

Since the 1st century, late blight in tomato and potato plants has been of interest to plant breeders. Potato plague is common on cold, wet climates and can devastate lands during wet and wet seasons. Potato plague is a global problem, but is particularly prevalent in the eastern United States, Mexico and the Far East.

Potato plague is caused by a fungus called Phytophthora infestans (" P. infestans "). Phytophthora infestans can be found in plant tissues, potato tubers and solanaceous weeds. Spores of the fungus typically spread widely during windy rainy seasons, but the fungus can grow widely on infected plant tissues and fruits. If the spores settle on the leaves, stems and fruits of the tomato plant, the spores can quickly invade the plant tissues, disrupt the tissues and eventually kill the plants.

At present, there are two physiological varieties described in blood infestans in tomatoes. The first is blood infestans 0, which is pathogenic for commercial strains that do not have a resistance gene. The second is P. infestans 1, which is pathogenic for commercial strains with Ph ₁ resistance genes.

In 1952, resistance to blood infestans was reported in Lycopersicon Pimpinellifolium , a type of wild tomato. This resistance was found to be conferred by one dominant gene (Ph ₁ , formerly known as TR ₁ ) that provided resistance to the tomato bacterium P. infestans 0. See Gallegly, ME, "Resistance to the Late Blight Fungus in Tomato", (1960) Campbell Plant Science Seminar, p. 116. In addition, multigene resistance was found in tomato varieties West Virginia 700 (P1204996). This resistance is not complete and confers only resistance to blood infestans 0 and 1. If environmental conditions are favorable for the development of P. infestans 1, these resistant plants tolerated severe disease.

The cultivated tomato Lycopersicon esculentum is one of the most important plant crops in the United States and in the world, and millions of tonnes are produced annually in the United States alone. Due to the commercial importance of these crops, constant efforts have been required to improve the cultivars. Thus, there is a need for cultivated tomato varieties that exhibit disease resistance to blood infestans 1.

It is an object of the present invention to disclose a novel lycopperic cone pimpinifolium cultiva that can be used as a source of resistance to the tomato bacterium P. infestans 1. It is another object of the present invention to provide a lycopperic cone esculentum plant that is resistant to tomato strains P. infestans 0 and 1 and maintains resistance to P. infestans 1 even when the disease is high. To provide.

Summary of the Invention

The present invention includes a method for producing a tomato plant (lycopericone esculentum) resistant to the tomato bacterium pi infestans 0 and 1. This plant is produced by crossing lycoppericum pimpinifolium plants and lycoppericcone esculentum which have been found to contain novel allele (s) that confer resistance to blood infestans 0 and 1. After mating, the seeds are collected and regenerated into plants. The obtained plants were evaluated for resistance to tomato bacteria pi infestans 0 and 1. Identify and select resistant plants. Selected resistant plants are backcrossed with other lycophericone esculentum varieties that exhibit the desired phenotype to obtain commercially acceptable strains resistant to the tomato strains P. infestans 0 and 1. This method can also be used to produce seeds that occur with tomato plants (lycopericone esculentum) that are resistant to the tomato bacteria P. infestans 0 and 1.

It was found to have new resistance to P. infestans 0 and 1, and the selected lycopperic pimfinellifolium used for cross-breeding with L. esculentum was named LA 2533. This is also called the Hope 84 by the inventors.

Plants of the invention can also be produced by protoplast fusion. In order to produce the plant by protoplast fusion, protoplasts from lycoppericcon pimfinellifoliium plants resistant to the tomato strains P. infestans 0 and 1 are obtained along with protoplasts from lycoppericcone esculentum. The protoplasts are then fused using standard protoplast fusion procedures well known in the art. As a result, allogeneic cells are obtained, regenerated into plants, and the plants are evaluated for resistance to the tomato bacteria P. infestans 0 and 1. Identify and select resistant plants.

Lycopericon esculentum plants produced according to the method of the present invention are resistant to the tomato bacteria P. infestans 0 and 1, and tolerate P. infestans 1 even in the field with high onset of disease.

The present invention also relates to tomato plants containing allele (s) conferring resistance to the tomato bacterium pi infestans 0 and 1, and seeds produced by the tomato plants.

The present invention relates to the production of tomato plants (lycopericone esculentum) resistant to the tomato bacterium P. infestans. When a plant is exposed to the tomato bacterium P. infestans, the plant is considered to be disease resistant if it does not show disease symptoms or is significantly reduced compared to plants that are less resistant. The plant of the present invention is new and novel because it is resistant to the tomato bacterium pi infestans 0 and 1.

The inventors of the present invention have found a lycoppericcon pimpinellifoliium plant containing allele (s) conferring resistance to the tomato bacterium pi infestans 0 and 1. This lycoppericum pimfinellifolium plant can be used to produce tomato plants (lycopericone esculentum) that are resistant to the tomato bacterium P. infestans 0 and 1. For example, to produce the plant of the present invention, a lycoppericum pimpinifolium plant designated LA 2533 is used. The inventors also called the lycoppericicon pimfinellifolium plant, designated LA 2533, as hop 84. Seeds from LA 2533 (Hoff 84) were deposited with the American Type Culture Collection (ATCC), 12301 Parkron Drive 12301, Maryland, in accordance with the provisions of the Budapest Treaty. The seed was deposited on December 14, 1995 and was assigned ATCC Accession No. 97382. In addition, LA 2533 (Hope 84) is available upon request from the Rick Center of the University of California-Davis, Davis, CA.

We found that the LA 2533 plant was resistant to the tomato bacterium P. infestans 0 and 1. Prior to this discovery, resistance to blood infestans 0 and 1 in plants named LA 2533 was unknown. Lycopericone pimfinellifoliium plants, designated LA 2533, contain allele (s) conferring resistance to the tomato bacterium P. infestans 0 and 1. However, one of ordinary skill in the art will appreciate that lycopericone pimpinifolium, which contains the allele (s) and is resistant to the tomato bacterium P. infestans 0 and 1, may be used in the present invention.

The plants of the present invention can be produced by traditional breeding techniques. For example, one may use seeds of the lycophericone pimpinifolium plant designated LA 2533. Seeds of LA2533 are planted in greenhouses or outdoors. The obtained plant is exposed to the tomato bacteria pi infestans 0 and 1. After being left in a highly diseased environment, the plants with the highest resistance are selected. Resistance to P. infestans 0 and 1 can be measured in two ways. A first way of measuring resistance to blood infestans involves using the so-called "greenhouse screen" method. In the greenhouse screening method, tomato seedlings are grown until five sesame leaves begin to sprout. At this time, the plants are sprayed with a suspension of blood infestans 1 spore sac (concentration is 10,000 spore sacs per ml of solution). After inoculation, the plants are placed in a chamber at 100% humidity for 24 hours. The plant is then removed from the humidity chamber and transferred to a greenhouse bench for disease to occur. At about 7 days resistant plants are identified and selected. The suspension of blood infestans 0 can then be secondary sprayed to the plant in the same manner. However, plants do not need to be tested for resistance to blood infestans 0 several times, which is generally known to those skilled in the art that tomato plants that are resistant to blood infestans 1 are necessarily resistant to blood infestans 0. Because.

A second way of measuring resistance to 0 and 1 to P. infestans is the field screen method. Field screening involves transplanting seedlings into field strips where climatic conditions are beneficial for natural infections and disease outbreaks. After the disease has occurred, when the plant begins to bear fruit, the resistant plant is identified and selected.

The inventors of the present invention devised a disease assessment grade to evaluate whether the plants produced by the method of the present invention are resistant to or less resistant to blood infestans 0 and 1. Disease ratings are based on the numbers 1 to 5, more specifically:

1 corresponds to no or small lesion on the leaf;

2 corresponds to a state in which there are no or small lesions on the leaves, the leaf lesions are enlarged, and the petioles have small lesions (diameter 2 mm);

3 corresponds to a state in which there are no or small lesions on the leaves, the leaf lesions are enlarged, the leaf lesions have small lesions (diameter 2 mm), and the leaf diseases are enlarged;

4 corresponds to a state in which there are no or small lesions on the leaves, the leaf lesions are enlarged, the leaf lesions have small lesions (diameter 2 mm), the leaf diseases are enlarged, and the stems have small lesions (diameter 2 mm);

5 has no or small lesions on the leaves, enlarged leaf lesions, small lesions on the leaf disease (diameter 2 mm), enlarged lesions on the leaf disease, small lesions on the stem (diameter 2 mm), enlarged lesions on the stem, Or dead plants.

Plants rated at disease ratings less than 3 are considered resistant, and plants rated at disease rating 3 or higher are considered to be less resistant.

Traditionally, most plant breeders do not evaluate, identify, and screen tomatoes that are resistant to blood infestans. Most breeders do not study blood infestans because of the fear that the fungus will kill the entire crop. Instead, most breeders control blood infestans by spraying fungicides to prevent the fungus from invading crops.

Next, the most resistant plant crosses the commercial tomato plant (El Esculentum) by placing its pollen on the pistil of another plant. After the tomato fruit has developed, the seeds are collected. Seeds are grown into plants and assessed for disease resistance. Resistant plants are screened and backcrossed with commercial tomato plants with the desired expressive traits, such as plant type (determinate or indeterminate), fruit size, shape, aroma, color, and long shelf life. It may be. Backcrossing may be continued until a strain with the desired expressing trait and resistant to the tomato strains P. infestans 0 and 1 is obtained. The plant obtained is stable against P. infestans 1 even in high open areas.

It is interesting that lycophericone esculentum is cleistogamous. Therefore, there is a high possibility that cross-pollination between lycoppericcon pimfinellifoliium and lycoppericcone esculentum occurs in the wild without human intervention. In addition, there is no possibility that identification, selection and backcrossing of allele (s) conferring resistance to blood infestans 0 and 1 will occur in nature.

Protoplast fusion can be used to produce the plants of the invention. For example, a first protoplast is obtained from lycophericone pimpinifolium, designated LA 2533, and a second protoplast is obtained from lycophericone esculentum. The protoplasts are then fused using traditional protoplast fusion procedures well known in the art.

For example, protoplast fusion can be achieved using polyethylene glycol (PEG), which causes aggregation in the presence of fusion buffers (ie, high pH solutions that allow cell membranes to fuse). Such somatic hybridization can also be performed under conditions for producing interspecies hybrids or variants thereof, as disclosed in Sundberg et al., Plant Science , 43, (1986) 155, which is incorporated herein by reference. However, those skilled in the art will appreciate that protoplast fusion can be performed in a manner other than the use of polyethylene glycol (PEG). For example, protoplasts are described in Koop et al., “Electric Field-Induced Fusion and Cell Reconstruction with Preselected Single Protoplasts and Subprotoplasts of Higher Plants,” Electroporation and Electrofusion in Cell Biology , Numan et al. Fusing using the field inductive fusion technique described in Edit, pp. 355-365. Protoplast fusion is also described in Hauptmann et al., "Carrot x Tobacco Somatic Cell Hybrids Selected by Amino Acid Analog Resistance Complementation", 6th International Protoplast Symposium, Basel, Aug. 12, 1983. 1-16 days], using dextran and polyvinyl alcohol.

In the following, examples of the present invention are provided, which are for illustrative purposes and do not limit the present invention.

Example 1

Several different plants were evaluated for their resistance to possible blood infestans. The following seeds were obtained: El Pimfinellifolium, LA 373, El Esculentum Bar, LA 1338. L. esculentum var.cerasiforme , El Pimfinellipolium , LA 1375, L. hirsutum , LA 2650, and El Pimfinellipoli , LA 2533 (Hope 84). cellar. All of these seeds were obtained from Rick Center, University of California-David, Davis, CA. Hope 33 is a tomato variety that is resistant to pi infestans 0 and 1. Celebrity and Fandango are commercial varieties that show no resistance to blood infestans.

Seeds were sown in a greenhouse. Tomato seeds were sown in soil (50% moss: 50% sand), placed on a bench in a greenhouse (daytime 75 ° F. and night temperature 70 ° F.) and watered as needed. When five seedlings of tomato seedlings were inoculated, they were inoculated with blood infestans 1. Seedlings were sprayed at a concentration of 10,000 spore sacs per ml of solution until all of the suspension of blood infestans spore sacs was depleted. After inoculation, the plants were placed in a chamber at 100% humidity for 24 hours. Next, the plants were exposed to cause disease. Approximately 7 days after inoculation, resistant plants were identified and selected using the following disease grades. These same varieties described above were evaluated in the same way using P. infestans O.

Disease grade

1: no lesion on the leaves or small

2: no or small lesion on the leaf, enlarged leaf lesion, and small lesion (diameter 2mm) on the leaf disease

3: no leaf on the leaf or small, the leaf lesion is enlarged, the leaf disease has a small lesion (diameter 2mm) and the leaf disease is enlarged

4: no or small lesions on the leaves, enlarged leaf lesions, small lesions on the leaf disease (diameter 2 mm), enlarged lesions on the leaf disease, and small lesions (diameter 2 mm) on the stem

5: no or small lesions on the leaves, enlarged leaf lesions, small lesions on the leaf disease (diameter 2 mm), enlarged lesions on the leaf disease, small lesions (diameter 2 mm) on the stem, enlarged lesions on the stem, Or dead plants.

Disease grading results are shown in Table 1 below:

The table above shows that the El Pimfinellifoliium plant, designated LA 2533, has a high level of resistance to P. infestans 0 and 1, while the El Pimfinellifoliium strain, designated LA 373 and 1375, has a high level of resistance. It does not show.

Example 2

A. El Pimfinellifolium plants, named LA 2533 and also called Hope 84, were crossed with tomato (L. Esculentum) plants, named Fla 7065. The plant Fla 7065 is a finite inflorescence, large, fruity, and homogeneous green breeding strain that is bred by Dr. Jay Scott of the University of Florida, Florida, USA. It was obtained by. Seeds obtained from this cross (F1) were sown in soil as described in Example 1, grown in greenhouses and inoculated with blood infestans 1. When using the same disease grade as described in Example 1, all seeds obtained above had a grade of 1 or 2 indicating that they were resistant to blood infestans 0 and 1. This suggests that the gene (s) involved in resistance are dominantly inherited.

Example 3

The F1 plants described in Example 2 were self-pollinated. Seeds from this F2 were sown in soil and grown in greenhouses. After several weeks, the seeds were transferred outdoors and water was supplied by dropping irrigation and grown to adulthood. Before flowering, it was cut, transferred to soil and grown in a greenhouse. Two weeks after cleavage, the spores of blood infestans 1 (concentration: 10,000 spores per ml) were inoculated and placed in a chamber at 100% humidity for 24 hours. After 24 hours, the disease was advanced by exposing the plants. After 14 days the cuts were graded 1-5. The disease grade used here is as follows:

1: no lesion on the leaves or small

2: no or small lesion on the leaf, enlarged leaf lesion, and small lesion (diameter 2mm) on the leaf disease

3: no leaf on the leaf or small, the leaf lesion is enlarged, the leaf disease has a small lesion (diameter 2mm) and the leaf disease is enlarged

4: no or small lesions on the leaves, enlarged leaf lesions, small lesions on the leaf disease (diameter 2 mm), enlarged lesions on the leaf disease, and small lesions (diameter 2 mm) on the stem

5: no or small lesions on the leaves, enlarged leaf lesions, small lesions on the leaf disease (diameter 2 mm), enlarged lesions on the leaf disease, small lesions (diameter 2 mm) on the stem, enlarged lesions on the stem, Or dead plants.

The grades for the F2 plants are shown in Tables 2a to 2d below.

Breeds with a disease grade of 2.9 or lower were considered resistant, while those with a disease grade of 3.0 or higher were considered poor (71 varieties were resistant and 24 varieties were weak). The data in Table 2 shows that the ratio of resistant varieties: less resistant varieties is 3: 1, which is expected when the resistant locus is dominant. However, many varieties have disease grades 2.0 to 2.8. The inventors believe that these varieties may be heterozygous. If this is true, this suggests that resistance is partially dominant inheritance when homozygous plants have a higher level of resistance than heterozygous plants.

Example 4

The F2 plants described in Example 3 were self-pollinated. Seeds from this F3 were sown in soil and grown in greenhouses. A few weeks later, the seeds were transferred outdoors with climatic conditions favorable for natural infection and disease progression of P. infestans 1. Serious disease outbreaks became more apparent as dying weak control plants (celebrities) died. The disease was graded from 1 to 5 when the plants began to bear fruit. The disease grade used here is as follows:

1: no lesion on the leaves or small

2: no or small lesion on the leaf, enlarged leaf lesion, and small lesion (diameter 2mm) on the leaf disease

3: no leaf on the leaf or small, the leaf lesion is enlarged, the leaf disease has a small lesion (diameter 2mm) and the leaf disease is enlarged

4: no or small lesions on the leaves, enlarged leaf lesions, small lesions on the leaf disease (diameter 2 mm), enlarged lesions on the leaf disease, and small lesions (diameter 2 mm) on the stem

5: no or small lesions on the leaves, enlarged leaf lesions, small lesions on the leaf disease (diameter 2 mm), enlarged lesions on the leaf disease, small lesions (diameter 2 mm) on the stem, enlarged lesions on the stem, Or dead plants.

Disease graded results are shown in Tables 3A and 3B below.

The above results confirm the results from the F2 plants described in Example 3. Plants with homozygous resistance identified in F2 plants (94FH98, 94FH99, 94FH114, 94FH120, 94FH121, 94FH148, 94FH162, 94FH170) and less resistant plants (94FH95, 94FH110, 94FH116, 94FH128, 94FH129, 94FH155, 94F188) Showed the same disease class in F3 plants. If resistance is conferred from numerous genes (multigenic complex), it will not be isolated in a 3: 1 ratio and homozygous resistant plants will not be identified as easily and quickly as demonstrated.

The invention described and claimed herein is not limited to the specific embodiments disclosed herein, because these embodiments illustrate several aspects of the invention. Any equivalent embodiment would fall within the scope of the present invention. Indeed, it will be apparent to those skilled in the art that various modifications may be made to the present invention from the foregoing description, in addition to those described herein. Such modifications are also considered to be within the scope of the appended claims.

Claims (7)

a. Providing a Lycopersicon pimpinellifolium plant, LA 2533 having ATCC Accession No. 97382; b. Mating the lycoppericcon pimfinellifolium plant provided in step a with a lycopersicon esculentum plant; c. Collecting the seeds resulting from the crossing in step b; d. Regenerating the seed of step c into a plant; e. Inoculating the plant of step d with the tomato bacteria Phytophthora infestans races 0 and 1; f. Evaluating the plant of step e for resistance to phytophthora infestans 0 and 1; g. Identifying plants resistant to phytophthora infestans 0 and 1; And h. Screening plants resistant to phytophthora infestans 0 and 1 A method of producing a tomato plant that is resistant to the tomato bacterium Phytophthora infestans 0 and 1. The method of claim 1, The plants selected in step h are lyco having the desired expression trait over generations sufficient to obtain lycopperic cone esculentum plants resistant to phytophthora infestans 0 and 1 and having the desired expression trait. Further backcrossing with the pericone esculentum plant. The method of claim 1, Methods for evaluating plants using the following disease grade criteria 1-5: 1: no lesion on the leaves or small 2: no or small lesion on leaf, enlarged leaf lesion, petiole with small lesion (diameter 2mm) 3: no leaf on the leaf or small, the leaf lesion is enlarged, the leaf disease has a small lesion (diameter 2mm) and the leaf disease is enlarged 4: no or small lesions on the leaves, enlarged leaf lesions, small lesions on the leaf disease (diameter 2 mm), enlarged lesions on the leaf disease, and small lesions (diameter 2 mm) on the stem 5: no or small lesions on the leaves, enlarged leaf lesions, small lesions on the leaf disease (diameter 2 mm), enlarged lesions on the leaf disease, small lesions (diameter 2 mm) on the stem, enlarged lesions on the stem, Or dead plants. a. Providing a lycopericone pimpinifolium plant of LA 2533 having ATCC accession number 97382; b. Mating the lycoppericcon pimpinifolium plant provided in step a with the lycoppericcone esculentum plant; And c. Harvesting seeds produced from the cross in step b, which occurs in tomato plants resistant to the tomato bacterium Phytoptotora infestans 0 and 1 at sowing A method of producing a seed that occurs with a tomato plant resistant to the tomato bacteria phytoptotora infestans 0 and 1 comprising. a. Providing a lycopericone pimpinifolium plant of LA 2533 having ATCC accession number 97382; b. Mating the lycoppericcon pimpinifolium plant provided in step a with the lycoppericcone esculentum plant; c. Collecting the seeds resulting from the crossing in step b; d. Regenerating the seed of step c into a plant; e. Inoculating 0 and 1 phytophthora infestans which are tomato bacteria to the plant of step d; f. Evaluating the plant of step e for resistance to phytophthora infestans 0 and 1; g. Identifying plants resistant to phytophthora infestans 0 and 1; h. Selecting plants resistant to phytophthora infestans 0 and 1; i. The plants selected in step h have the desired expression traits over generations sufficient to obtain lycopperic cone esculentum plants that are resistant to phytophthora infestans 0 and 1 and have the desired expression traits. Backcrossing with the lycophericone esculentum plant; And j. Harvesting seeds generated from the cross in step i, which occurs in tomato plants resistant to the tomato bacterium phytoptotora infestans 0 and 1 at sowing A method of producing a seed that occurs with a tomato plant resistant to the tomato bacteria phytoptotora infestans 0 and 1 comprising. The method of claim 1, self-crossing the plant selected in step h over generations sufficient to obtain a plant that is immobilized to an allele that confers resistance to the tomato strain Phytophthora infestans 0 and 1; And The plants are cross-crossed with lycophericone esculentum plants having the desired expression traits to obtain lycophericone esculentum plants resistant to phytophthora infestans 0 and 1 and having the desired expression traits. Steps to How to further include. a. Providing a lycopericone pimpinifolium plant of LA 2533 having ATCC accession number 97382; b. Mating the lycoppericcon pimpinifolium plant provided in step a with the lycoppericcone esculentum plant; c. Collecting the seeds resulting from the crossing in step b; d. Regenerating the seed of step c into a plant; e. Inoculating 0 and 1 phytophthora infestans which are tomato bacteria to the plant of step d; f. Evaluating the plant of step e for resistance to phytophthora infestans 0 and 1; g. Selecting plants resistant to phytophthora infestans 0 and 1; h. Self-crossing the plant selected in step g over generations sufficient to obtain a plant that is immobilized to an allele that confers resistance to the tomato strain Phytophthora infestans 0 and 1; And i. The plant produced in step h is cross-crossed with the lycoppericcon esculentum plant having the desired expression trait, thereby making it resistant to phytophthora infestans 0 and 1 and having the desired expression trait. Obtaining Coolentum Plants A method of producing a seed that occurs with a tomato plant resistant to the tomato bacteria phytoptotora infestans 0 and 1 comprising.