The'root and stem rot'is a new disease of cucumber (Cucumis sativus L.), which appeared for the first time worldwide in Crete, Greece during the growing period 1989-90 in greenhouse crops in the lerapetra and Sitia areas, Lasithi (Vakalounakis & Fragkiadakis 1999a, b). In the next few years the disease spread to all the main cucumber production areas of Crete, as well as in the Trifilia and Olympia areas Peloponnese, where nowadays it causes severe losses in the yield. In addition, the disease appeared in Canada (Fraser Valley of British Colombia) in 1994 and in Northeastern France in 1999, where it causes significant losses in greenhouse cucumber crops (Punja et al. 1998, Claude Alabouvette, personal communication).

The'root and stem rot'disease of cucumber (C. sativus) is caused by Fusarium oxysporum Schlechtend.: Fr. f. sp. radicis-cucumerinum D. J. Vakalounakis

(Vakalounakis & Fragkiadakis 1999a, b). At 23°C the colonies of the fungus on APDA are white with prolific aerial mycelium and form numerous green-blue sclerotia and large numbers of macroconidia on sporodochia in a small area around the plug. At 27 to 33°C, the colonies are white with no sclerotia, but with progressively increasing areas of slimy sporodochia with numerous macroconidia around the plug as the temperature increases. At 35°C, slimy sporodochia occupy the whole dish. At 20°C, the lower surface of the colony is orange-yellow, with a small violet area around the plug. At 20°C the growth of the colonies on APDA is rapid (0.8 cm/day) (Vakalounakis & Fragkiadakis 1999a). Microconidia and macroconidia are formed abundantly on lesions of the hypocotyl. Microconidia are hyaline with the following shapes: oval (7%) 6,31,45 (4,8-9,7) X 3,0+0,48 (2,4-3,6) nm, slightly curved (28%) 9,42,18 (6,1-15,7) X 2,90,48 (2,4-4,4) Fm, cylindrical (65%) 8,22,18 (4,3-12,1) X 2,70,48 (1,9-3,6) llm (average of 325 conidia). Macroconidia are hyaline, fusiform, slightly to strongly curved, foot cells generally inconspicuous, and under microscopic examinations they ranged from 1 to 4 septate in the following frequency: 1 septate (12%) 24,43,15 (24,4-31,5) X 3,40,73 (2,4-4,8) urn, 2 septate (3%) 28,66,53 (16,9-36,3) X 4,4+1,2 (2,4-5,3) film, 3 septate (84%) 33,23,15 (21,8- 41,1) X 4,60,48 (3,6-6.1) Rm, 4 septate (1%) 38,22,66 (36,3-39,9) X 5,40,97 (4,8- 6,1) urn (average of 200 conidia) (Vakalounakis & Fragkiadakis 1999a).

Of 18 cultivated species from the botanical families Apiaceae, Brassicaceae, Chenopodiaceae, Cucurbitaceae, Fabaceae and Solanaceae, that were artificially inoculated in greenhouse experiments, two species of Cucurbitaceae, melon (C. melo L.) and sponge gourd (Luffa aegyptiaca Mill.) showed root and stem rot symptoms similar to those caused by isolates of F. oxysporum that attack cucumber (Vakalounakis & Fragkiadakis 1999a).

In greenhouse crops symptoms first appear on plants about one month old. Collar rot and later hypocotyl rot develop, usually on one side of the stem, which ranges in color from very light green to amber and brown. Progressively, the hypocotyl rot becomes more severe and a white-orangish fungal growth accompanied by fructifications may appear on the affected tissue under high humidity conditions. In an advanced disease stage the crown and the roots of the plants usually appear a

complete rot. Plants with these symptoms have a stunted growth, wilt and die.

Although young plants can be killed, usually plants reach cropping size and symptoms do not appear until first-fruiting stage. Adult plants then undergo slow wilting with a progressive yellowing. These symptoms usually follow a unilateral cortical rot with a longitudinal canker at the hypocotyl and a white-orangish fungal growth accompanied by fructifications of the pathogen, that may extend upward for 20 to 40 cm, and downward to the root system. On the stem a vascular brown discoloration may appear (Figure 4), extending for 40 to 200 cm above the soil line. Primary, secondary, and tertiary roots have brown lesions, many confluent with hypocotyl lesions. Isolated unilateral cracks with rots varying in length from 5 to 15 cm or more, usually with white growth of the pathogen, might also appear on the upper portion of the stem.

During the winter, when plant vigor within greenhouses is reduced due to unfavorable microclimatic conditions (reduced illumination and average air temperature lower than 15°C), the disease progresses fast, causing severe damage (Vakalounakis & Fragkiadakis 1999a).

The cultivated cucumber (C. sativus), is one of the most important vegetable crops in Greece and many other countries. The commercial importance of the crop has necessitated a constant effort to develop cultivars with excellent agronomic characteristics and resistance to the most serious parasites. To date, genetic resistances to many plant pathogens have been incorporated into commercial varieties and hybrids, like: Cladosporium cucumerinum, Fusarium oxysporum f. sp. cucumerinum, Corynespora cassiicola, Sphaerotheca fusca, Erysiphe, orontii, E. cucurbitacearum, Pseudoperonospora cubensis, Pseudomonas syringae pv. lachrymans, CMV, ZYMV, PRSV etc. is a very destructive disease of greenhouse cucumber in Greece and other countries. However, none commercial cultivar has been developed up to now with resistance to the'root and stem rot'disease of cucumber caused by F. oxysporum f. sp. radicis-cucumerinum, despite the disease severity in Greece and other countries. The use of resistant cucumber cultivars to F. oxysporum f. sp. radicis-cucumerinum would contribute to the control of'root and stem rot', reduce the use of fungicides and increase quantity and quality of the fruits as well as would contribute to the better protection of the environment.

The grafting of commercial cucumber onto resistant rootstocks is at present the most effective control method against'root and stem rot'disease of cucumber caused by F. oxysporum f. sp. radicis-cucumerinum. The resistant rootstocks, which have been used successfully in practice, are Cucurbita ficifolia, C. moschata and C. maxima x C. moschata from which the last one has given the highest yield (Pavlou & Vakalounakis 1998). However, the use of a resistant C. sativus rootstock to graft commercial cucumber varieties and hybrids would offer an excellent compatibility between the rootstock and the scion possibly resulting in increased quantity and improved quality of fruits.

It is an object of the present invention to: (a) disclose a novel cucumber (C. sativus) cultivar that can be used as a source of resistance to'root and stem rot' disease caused F. oxysporum f. sp. radicis-cucumerinum, (b) create commercial cucumber (C. sativus) plants that are resistant to'root and stem rot'disease caused F. oxysporum f. sp. radicis-cucumerinum and remain resistant to this pathogen in the greenhouse or the open field crops when the disease pressure is high and (c) provide grafted commercial cucumber (C. sativus) plants onto resistant cucumber (C. sativus) rootstocks against F. oxysporum f. sp. radicis-cucumerinum for greenhouse and open field cucumber crops.

> Summary of the invention The present invention involves a method for producing cucumber plants (C. sativus) which are resistant to the'root and stem rot'disease caused by F. oxysporum f. sp. radicis-cucumerinum. These plants are produced by crossing a cucumber (C. sativus) plant, which was discovered to contain genes, which confer resistance to F. oxysporum f. sp. radicis-cucumerinum with a Cucumis sativus plant which displays desirable phenotypic characteristics. After the cross is made, the seed is collected and regenerated into plants. The resulting plants are backcrossed with plants that display desirable phenotypic characteristics. After the backcross is made, the seed is collected and regenerated into plants. The resulting plants are self-fertilized and the seed is collected and regenerated into plants, which are evaluated for resistance to the'root and stem rot'disease caused by F. oxysporum f. sp. radicis-cucumerinum. Plants that demonstrate resistance are identified and selected. These selected resistant plants are backcrossed with other cucumber (C. sativus) plants that display desirable phenotypic

characteristics. After the backcross is made, the seed is collected and regenerated into plants. The resulting plants are self-fertilized and the seed is collected and regenerated into plants, which are evaluated for resistance to the'root and stem rot'disease caused by F. oxysporum f. sp. radicis-cucumerinum. This procedure is followed for several generations to obtain commercially acceptable varieties, which are resistant to the 'root and stem rot'disease caused by F. oxysporum f. sp. radicis-cueumerinum. This method can also be used to produce seeds that result in cucumber (C. sativus) plants that are resistant to the'root and stem rot'disease caused by F. oxysporum f. sp. radicis-cucumerinum. In the above-described methods the plants with desirable phenotypic characteristics are used as female parents during the crosses. In addition, the evaluation of the resistant genotypes against the'root and stem rot'disease caused by F. oxysporum f. sp. radicis-cucumerinum is carried out either in greenhouses or in growth chambers with young plants which are artificially inoculated with the pathogen, or in open fields artificially or naturally infested with the pathogen.

The present invention also involves a method for producing cucumber (C. sativus) plants, which are resistant to the'root and stem rot'disease caused by F. oxysporum f. sp. radicis-cucumerinum with simultaneous self-fertilizations and backcrosses with the purpose to shorten the time of the breeding procedure for producing cucumber (C. sativus) plants, which are resistant to the'root and stem rot'disease caused by F. oxysporum f. sp. radicis-cucumerinum. The self-fertilizations are made to ascertain whether the resistant genes to F. oxysporum f. sp. radicis-cucumerinum have been transferred to the backcross populations. The evaluations of the self-fertilization progenies are made on young plants after an artificial inoculation. To be exact, a cucumber (C. sativus) plant which was discovered to contain genes, which confer resistance to the'root and stem rot'disease caused by F. oxysporum f. sp. radicis- cucumerinum is crossed with a cucumber (C. sativus) plant which display desirable phenotypic characteristics. After the cross is made, the seed is collected and regenerated into plants. The resulting plants are backcrossed with plants that display desirable phenotypic characteristics. After the backcross is made, the seed is collected and regenerated into plants. The resulting plants are self-fertilized and backcrossed simultaneously with plants which display desirable phenotypic characteristics. The self-fertilization progenies are evaluated for possible resistance against F. oxysporum f. sp. radicis-cucumerinum after an artificial inoculation in the young stage with the

purpose to ascertain whether the resistant genes against the pathogen are transferred to the backcross progenies. In a positive case a number of resistant self-fertilization progenies should be found during this evaluation. In that case the seed of the backcross which comes from the same plant which was self-fertilized is collected, while in a negative case is discarded. This procedure is followed for several generations to obtain commercially acceptable varieties, which are resistant to the 'root and stem rot'disease caused by F. oxysporum f. sp. radicis-cucumerinum. This method can also be used to produce seeds that result in cucumber (C. sativus) plants that are resistant to the'root and stem rot'disease caused by F. oxysporum f. sp. radicis-cucumerinum.

The cucumber (C. sativus) selection that was discovered to contain novel resistance genes against the'root and stem rot'disease caused by F. oxysporum f. sp. radicis- cucumerinum and subsequently used in crosses with cucumber (Cucumis sativus) cultivars displaying desirable characteristics is designated as C566.

The resistant cucumber (C. sativus) plants against the'root and stem rot'disease caused by F. oxysporum f. sp. radicis-cucumerinum of the present invention can also be produced by protoplast fusion, despite the disadvantage of this method to change the cytoplasm and miss characteristics of the recurrent parent. To produce plants by protoplast fusion, a protoplast from a cucumber (C. sativus) plant that is resistant to the'root and stem rot'disease caused by F. oxysporum f. sp. radicis-cucumerinum is obtained along with a protoplast from cucumber (C. sativus) plant which displays desirable phenotypic characteristics (recurrent parent). The protoplasts are then fused using standard protoplast fusion procedures, which are well known in the art. The resulting allogenic cells are obtained and regenerated into plants. From these plants protoplasts are obtained which are fused with protoplasts from plants of the recurrent parent. The resulting cells are obtained and regenerated into plants. From these plants protoplasts are obtained which are fused with protoplasts from the same plant. The resulting cells are obtained and regenerated into plants, which are evaluated for resistance to the'root and stem rot'disease caused by F. oxysporum f. sp. radicis- cucumerinum. Resistant plants are identified and selected. This procedure is followed to obtain commercially acceptable varieties, which are resistant to the'root and stem

rot'disease caused by F. oxysporum f. sp. radicis-cucumerinum and display the desirable phenotypic characteristics of the recurrent parent.

The cucumber (C. sativus) plants produced according to the method of this invention are resistant to the'root and stem rot'disease caused by F. oxysporum f. sp. radicis-cucumerinum in the young plant stage in growth chambers as well as in greenhouse crops under natural infection conditions when the disease pressure is high.

The present invention also involves cucumber (C. sativus) plants that contain genes, which confer resistance to the'root and stem rot'disease caused by F. oxysporum fsp. radicis-cucumerinum and seed produced by said cucumber (C. sativus) plants.

In addition, the present invention involves techniques of grafting cucumber (C. sativus) plants from any commercial cultivar onto the selection C566 or any other cucumber (C. sativus) genetic material with resistance against the'root and stem rot' disease caused by F. oxysporum f. sp. radicis-cucumerinum.

> Advantages of the invention To date, genetic resistances to many plant pathogens have been incorporated into various commercial cucumber varieties and hybrids. However, none commercial cultivar has been developed with resistance to the'root and stem rot'disease of cucumber caused by F. oxysporum f. sp. radicis-cucumerinum, despite the high disease severity in Greece and other countries. The development of resistant cucumber cultivars to F. oxysporum f. sp. radicis-cucumerinum would contribute to the control of'root and stem rot', reduce the use of fungicides and increase quantity and quality of the fruits as well as would contribute to the better protection of the environment.

The grafting of cucumber onto resistant rootstocks is the most effective control way against F. oxysporum Esp. radicis-cucumerinum. The rootstocks used in practice are cucurbits belonging to the genus Cucurbita. However, the use of rootstocks belonging to Cucumis sativus will offer better compatibility with the grafted cucumber resulting in increased fruit quantity and quality.

> Detailed description of the invention The present invention involves the creation of cucumber (C. sativus) plants that are resistant to the'root and stem rot'disease caused by F. oxysporum f. sp. radicis- cucumerinum. Plants are said to be resistant if, when exposed to environmental conditions that favor their infection by F. oxysporum f. sp. radicis-cucumerinum, the plants either fail to exhibit disease symptoms or exhibit substantially reduced symptoms compared to susceptible plants. The plants of the present invention are new and novel because they are resistant to the'root and stem rot'disease caused by F. oxysporum f. sp. radicis-cucumerinum.

The inventor of the present invention has discovered cucumber (C. sativus) plants, which contain resistance genes against the'root and stem rot'disease caused by F. oxysporum f. sp. radicis-cucumerinum. These cucumber (C. sativus) plants can be used to create cucumber (C. sativus) plants that are resistant to the'root and stem rot' disease caused by F. oxysporum f. sp. radicis-cucumerinum and display desirable phenotypic characteristics. For example, cucumber (C. sativus) plants designated as C566 were used to create the plants of the present invention. Seeds of C566 have been deposited with the National Agricultural Research Foundation (N. AG. RE. F.), Plant Protection Institute, Kastorias Street, Heraklio, Crete, Greece.

The inventor of the present invention has discovered that plants of C566 exhibit resistance to the'root and stem rot'disease caused by F. oxysporum f. sp. radicis- cucumerinum. Prior to this discovery, resistance to the'root and stem rot'disease caused by F. oxysporum f. sp. radicis-cucumerinum in plants designated as C566 as well as in any other genetic material of cucumber (C. sativus) had been unknown.

Cucumber (C. sativus) plants of C566 contain resistance genes against the'root and stem rot'disease caused by F. oxysporum f. sp. radicis-cucumerinum. However, one skilled in the art would recognize that any cucumber (C. sativus) plant which contain the resistance genes of C566 against the'root and stem rot'disease caused by F. oxysporum f. sp. radicis-cucumerinum can be used in this invention.

The selection of plants with resistance to the'root and stem rot'disease caused by F. oxysporum f. sp. radicis-cucumerinum of this invention is carried out in growth chambers or in soiless cultures (preferably in greenhouses) infested with the pathogen.

After sufficient disease pressure, those plants exhibiting the best resistance are selected. In the first case (growth chambers): young cucumber plants in the 1-3 true leaf stage are inoculated artificially with the pathogen by the root dipping technique.

The roots are dipped in a spore suspension (approximately 106 spores/ml) of the fungus for about 30 min and then seedlings are transplanted in sterile organic substrate and kept in growth chambers for 20-30 days at 17-22°C with a 12-h photoperiod. In the second case (preferably in greenhouse): cucumber plants are transplanted in the soil or in soiless culture, which previously have been artificially infested with the pathogen. The plants are grown until about fruit harvest.

The inventor of the present invention devised a disease rating scale for evaluating whether the plants produced by the method of this invention are resistant or susceptible to F. oxysporum f. sp. radicis-cucumerinum. The disease rating scale is based on a numerical designation from 0 to 3, which more specifically described as follows: 0=no disease symptoms (healthy plants); 1=light yellowing of cotyledons, light-medium rot in the tap and secondary roots, crown rot; 2=medium-heavy yellowing of cotyledons with or without wilt, stunting, heavy rot in the tap and secondary roots, crown rot with or without hypocotyl rot, vascular discoloration of the stem and 3=dead or almost dead plants. Plants with a disease rating of 0 are considered resistant, while plants with a disease rating of 1-3 are considered susceptible.

The transfer of resistance against the'root and stem rot'disease caused by F. oxysporum f. sp. radicis-cucumerinum from the resistant to the susceptible plant, which displays desirable phenotypic characteristics is achieved by placing the pollen from the one plant on the stigma to the other plant according to the procedure, which is well known to all skilled in the art. After the cucumber fruit develops, seed is collected. The seed is grown into plants, which are backcrossed with plants displaying desirable phenotypic characteristics. During backcrosses the plants displaying desirable phenotypic characteristics are always used as female parents. After the backcross, the seed is collected and regenerated into plants, which are self-fertilized.

Then, the seed is collected and regenerated into plants, which are evaluated for resistance to the'root and stem rot'disease caused by F. oxysporum f. sp. radicis- cucumerinum, as it appears in Figure 1. Resistance to the'root and stem rot'disease No segregation for Segregation for b is No segregation for Segregation for b is b is observed observed b is observed observed It is discarded During backcross It is discarded During backcross it is used as a it is used as a parent parent

caused by F. oxysporum f. sp. radicis-cucumerinum is oligogenic and for simplification is presented in the Figure 1 by the gene b.

Figure 1 Parent possesses desirable phenotvpic traits (recurrent parent) : AABB Resistant selection (donor parent)): aabb AABB X aabb F : AaBb Backcross: AABB x AaBb Bl AABB AABb AaBB AaBb Self-fertilization

Resistant plants are backcrossed with plants, which possess desirable phenotypic traits and then the resulting plants are self-fertilized. The self-fertilization progenies are evaluated for the selection of the resistant genotypes. The backcrossing and self- fertilizing may be continued until a variety is obtained that possesses desirable phenotypic traits and is resistant to the'root and stem rot'disease caused by F. oxysporum f. sp. radicis-cucumerinum. The resulting plants are resistant to F.

oxysporum f. sp. radicis-cucumerinum in the field (greenhouse conditions) under high disease pressure.

An alternative method which could be used to shorten the time of the breeding procedure is the simultaneous self-fertilization and backcross, as it appears in the following Figure 2. After the cross between the resistant plant against the'root and stem rot'disease caused by F. oxysporum f. sp. radicis-cucumerinum and the plant displaying desirable phenotypic traits, the seed collection and the first backcross of the resulting hybrid to the plant displaying desirable phenotypic traits, simultaneous self-fertilization and backcross is carried out. The self-fertilization is made to ascertain whether the resistant genes to F. oxysporum f. sp. radicis-cucumerinum have been transferred to the backcross populations. If during evaluation the self- fertilization progenies are susceptible then the backcross progenies of the respective backcross are discarded. In the case that the self-fertilization progenies are susceptible and resistant simultaneously then the progenies of the respective backcross are backcrossed again with the plants possessing desirable phenotypic traits. These backcrosses and self-fertilizations are continued until a variety is obtained that possesses desirable phenotypic traits and is resistant to the'root and stem rot'disease caused by F. oxysporum f. sp. radicis-cucumerinum. The resulting plants are resistant to F. oxysporum f. sp. radicis-cucumerinum in the field (greenhouse conditions) under high disease pressure. During backcrosses the plants displaying desirable phenotypic characteristics are used as female parents: The resistant plants against the'root and stem rot'disease caused by F. oxysporum f. sp. radicis-cucumerinum of the present invention can also be produced by protoplast fusion, despite the disadvantage of this method to change the cytoplasm and miss characteristics of the recurrent parent. For example, a protoplast from a plant of the selection C566 is obtained along with a protoplast from a cucumber (C. sativus) plant that displays desirable phenotypic characteristics (recurrent parent). The protoplasts are then fused using standard protoplast fusion procedures, which are well known in the art. The resulting allogenic cells are obtained and regenerated into plants. From these plants protoplasts are obtained which are fused with protoplasts from plants of the recurrent parent. The resulting cells are obtained and regenerated into plants. From these plants protoplasts are obtained which are fused with protoplasts from the same plant. The resulting cells are obtained and regenerated into plants, which are evaluated

Figure 2 Parent possesses desirable phenotvpic traits (recurrent parent): AABB Resistant selection (donor parent)): aabb AABB x aabb Ft : AaBb Backcross: AABB xAaBb (X) BC2 (X) BC2 (X) BC2 (X) BC2 In self-fertilization pro-n self-fertilization pro-n self-fertilization n self-fertilization pro- genies no segregation genies segregation for b progenies no segrega-genies segregation for b for b is observed is observed tion for b is observed is observed The backcross The back-cross The backcross The back-cross progenies are progenies are pre-progenies are progenies are pre- discarded served discarded served for resistance to the'root and stem rot'disease caused by F. oxysporum f. sp. radicis- cucumerinum. Resistant plants are identified and selected. This procedure is followed to obtain commercially acceptable varieties, which are resistant to the'root and stem rot'disease caused by F. oxysporum f. sp. radicis-cucumerinum and display the desirable phenotypic characteristics of the recurrent parent. The protoplast fusion is made with various ways, like the use of electric-field induced fusion techniques as described by Koop et al. (1989)"Electric Field-Induced Fusion and Cell Reconstruction-with Preselected Single Protoplasts and Subprotoplasts of Higher Plants"in Electroporation and Electrofusion in Cell Biology, Neuman et al. editors, pgs. 355-365, herewith incorporated by reference. Additionally, protoplast fusion can be accomplished by employing polyethylene glycol (PEG) causing agglutination, in

the presence of a fusion buffer, i. e., a high pH solution to let the membranes fuse.

Such somatic hybridization may be effected under the conditions disclosed by Sundberg et al. (Plant Science, 43, (1986) 155), hereby incorporated by reference, for the production of interspecific hybrids or modifications thereof. However, one skilled in the art would recognize that the protoplast fusion can be accomplished in other ways. For example, the protoplasts can be fused by using dextran and polyvinyl alcohol as described by Hauptmann et al.,"Carrot x Tobacco Somatic Cell Hybrids Selected by Amino Acid Analog Resistance Complementation", 6th International Protoplast Symposium, Basel, Aug. 12-16,1983, herewith incorporated by reference The present invention also involves methods of grafting cucumber (C. sativus) plants from any commercial genetic material onto C566 used as a rootstock or any other genetic material of cucumber (C. sativus), which possesses resistance as C566 against the'root and stem rot'disease of cucumber caused by F. oxysporum f. sp. radicis-cucumerinum.

According to the first method: (a) The growing tip of a young (beginning of the first-true leaf stage) plant (rootstock) from the selection C566 or any other cucumber (C. sativus) genetic material with resistance to the'root and stem rot'disease caused by F. oxysporum f. sp. radicis-cucumerinum is removed. (b) In the rootstock a toothpick is introduced. (c) By using a razor two transverse sections are made on the scion, which converge symmetrically. (d) The scion is placed inside the section of the rootstock and in a depth about 0,5-1 cm, so that the cotyledons of the scion to be vertical to the cotyledons of the rootstock. The grafted plants are kept in a shady place (no direct view to the sunlight) with a high relative humidity (about 80-90%) and temperature not more than 30°C. The grafted plants are transplanted in the greenhouse when they are in the 3-4 true-leaf stage.

According to the second method: (a) On the hypocotyl of a young cucumber plant of the selection C566 or any other cucumber (C. sativus) genetic material with resistance to the'root and stem rot'disease caused by F. oxysporum f. sp. radicis- cucumerinum, which is at the fully developed first-true leaf stage and has a hypocotyl of at least 10 cm in diameter, a transverse section is made by a razor in about a 20° angle. This section is made on the hypocotyl in a distance of about 1 cm from the

cotyledons and in the opposite site of that of the first leaf. The section reaches the 1/2 to 2/3 of the hypocotyl axis, with a downward direction. (b) In the scion and in the side of the first true leaf a transverse section is made by a razor in about a 20° angle and in a distance of about 2-3 cm from the cotyledons, which usually reaches the middle of the hypocotyl axis and it has a downward direction. (c) The two plants (scion and rootstock) are placed each close to each other so that the two sectioned surfaces to be in touch (Figure 7) and fastened using either a plastic pin or a piece of aluminum foil or plastic tape and transplanted in a pot with garden soil or organic substrate. The further procedure concerning the cultural practice is similar to that of the first technique. Eight to ten days after the grafting and in the case that it has been succeeded the upper and the lower parts of the rootstock and the scion are removed, respectively and in a distance about 2 cm from the grafting point.

Examples By way of example and not limitation, examples of the present invention will now be given.

Example 1 Thirty young cucumber plants from each of the cultivars Ashley, Straight-8, Kvcosós and C566 were inoculated artificially at the first true leaf stage by the root dipping technique in a spore suspension of F. oxysporum f. sp. radicis-cucumerinum adjusted to 5 X 106 spores/ml for 30 min. After inoculation plants were transplanted to pots 8.5 in diameter with organic substrate Belplanto (Klasmann-Deilmann GmbH, Geeste, Germany) and kept in a growth chamber at 19°C with a 12-h photoperiod and light intensity at plant level approximately (pMioq 150 umol. s'. m'. Plants of the same cultivars, which were not inoculated artificially but dipped in sterile water served as controls. Symptom readings were taken daily. Twenty days after inoculation all the plants of the cultivars Ashley, Straight-8 and Knosos which had been inoculated artificially with F. oxysporum f. sp. radicis-cucumerinum were dead or almost dead, while the plants of the selection C566 as well the plants of the cultivars Ashley, Straight-8 and Knosos, which had been dipped in sterile water were absolutely healthy.

Example 2 Young cucumber plants of the cultivar Knosos (P1) and the inbred line C566 as well as of the crosses Knosos X C566 (F1), (C566 X Knosos) X Knosos (BCP), (C566 X Knosos) X C566 (BCP2) and (C566 X Knosos) (X) (F2) were inoculated artificially at the two true-leaf stage by the root dipping technique in a spore suspension of F. oxysporum f. sp. radicis-cucumerinum adjusted to 107 spores/ml for 30 min. After inoculation plants were transplanted to pots 8.5 in diameter with organic substrate Belplanto (Klasmann-Deilmann GmbH, Geeste, Germany) and kept in a growth chamber at 19°C with a 12-h photoperiod and light intensity at plant level approximately ##### 150 µmol. s-1.m-2. Plants of the cultivar Knosos and C566, which were not inoculated artificially but dipped in sterile water served as controls.

Symptom readings were taken daily. Twenty five days after inoculation all the plants which had been dipped in sterile water were absolutely healthy, while those which had been inoculated artificially by the fungus during evaluation gave disease reactions appeared in Table 1.

Table 1. Resistance to Fusarium oxysporum f. sp. radicis-cucumerinum in progenies of crosses of a resistant selection C566 and a susceptible cultivar Knosos Pedigree Gene-Disease index Disease reaction ration 0 1 2 3 Resistant Susceptible Knosos Pl 0* 1 I 14 0 16 C566 P2 8 0 0 0 8 0 Knosos X C566 Fl 4 7 2 22 4 31 (C566 X Knosos) X BCP1 0 2 6 19 0 27 K##### (C566 X Knosos) X C566 BCP2 11 4 3 34 11 41 (Knosos X C566) (X) F2 0 0 1 22 0 23 * Number of plants Example 3 Young cucumber plants of the cultivar C461 (P,) and the inbred line C566 (P2) as well as of the crosses C461 X C566 (Fi), C461 X (C461 X C566) (BCP) and (C461

X C566) (X) (F2) were inoculated artificially at the two true-leaf stage by the root dipping technique in a spore suspension of F. oxysporum f. sp. radicis-cucumerinum adjusted to 5 X 106 spores/ml for 30 min. After inoculation plants were transplanted to pots 8.5 in diameter with organic substrate Belplanto (Klasmann-Deilmann GmbH, Geeste, Germany) and kept in a growth chamber at 19°C with a 12-h photoperiod and light intensity at plant level approximately ##### 150 umol. s~l. m~2. Plants which were not inoculated artificially but dipped in sterile water served as controls. Symptom readings were taken daily. Thirty three days after inoculation all the plants which had been dipped in sterile water were absolutely healthy, while those which had been inoculated artificially by the fungus during evaluation gave disease reactions appeared in Table 2.

Table 2. Resistance to Fusarium oxysporum f. sp. radicis-cucumerinum in progenies of crosses of a resistant selection C566 and a susceptible inbred line C461 Pedigree Gene-Disease index Disease reaction ration 0 1 2 3 resistant Susceptible C461 Pi 0* 0 0 12 0 12 C566 P2 8 0 0 0 8 0 C461 X C566 Fx 1 1 3 22 1 26 C461 X (C461 X C566) BCPI 0 0 4 41 0 45 (C461 X C566) (X) F2 4 5 5 21 4 31 * Number of plants Example4 Thirty cucumber plants of the hybrid Brunex F, and the selection C566 were transplanted on 28 September 1995 in a glasshouse in the Heraklio area, Crete. Before planting the glasshouse soil had been disinfected with Methyl Bromide and then the soil surface was sprayed with a spore suspension of F. oxysporum f. sp. radicis- cucumerinum adjusted to 10'° spores/ml. During the cultivation, the plants were grown according to the local adopted system. Symptom readings were taken every two days. First disease symptoms appeared at the beginning of November 1995 and consisted of yellowing of the leaves at the basal portion of the stem. Later these

symptoms were accompanied by wilt and rot consisted of an orange mass of conidia on infected tissue. Infected plants were progressively dead. The first dead plants of the hybrid Brunex appeared on 30 November 1995 and the last ones on 18 January 1966.

Some plants of the selection C566 appeared a yellowing of the leaves on 11 January 1996, while the first six dead plants of C566 appeared on 6 February 1996, but without the characteristic appearance of the symptoms (crown and stem rot, vascular brown discoloration of the stem). On 12 March 1996 (end of the experiment), 14 C566 dead plants were counted, but without appearing any characteristic disease symptom and without the pathogen to be isolated from the vascular system of the stem.

Example5 Sixteen cucumber plants from each of the hybrids Brunex F, and Rayo Fi and the selection C566 were transplanted on 8 September 1999 in a glasshouse in the Heraklio area, Crete. Before planting the glasshouse soil had been disinfected with Methyl Bromide and then the soil surface was sprayed with a spore suspension of F. oxysporum f. sp. radicis-cucumerinum adjusted to 600 X 107 spores/plot. During the cultivation, the plants were grown according to the local adopted system. Symptom readings were taken every two days. First disease symptoms appeared on 6 October 1999 on plants of the hybrids Brunex and Rayo. Later these symptoms were accompanied by wilt and rot with an orange mass of conidia on infected tissue.

Infected plants were progressively dead. The first dead plants of the hybrid Brunex and Rayo appeared on 30 October 1999 and the last ones on 3 December 1999. In contrast, the C566 plants were all healthy and no symptoms appeared until the middle of February 2000. At the end of March (end of the experiment) the most C566 plants were dead, but without appearing the characteristic disease symptoms and without the pathogen to be isolated from the vascular system of the stem. However, two plants appeared the characteristic rot of the disease accompanied by an orange mass of conidia of Fusarium.

> Deposit information Cucumber seeds designated C566 have been placed on deposit with the National Agricultural Research Foundation (N. AG. RE. F.), Plant Protection Institute of Heraklio, Kastorias Street, Mesa Katsampas, Heraklio, Crete, Greece.

> Literature Hauptmann et al. (1983)."Carrot X Tobacco Somatic Cell Hybrids Selected by Amino Acid Analog Resistance Complementation", 6 International Protoplast Symposium, Basel Aug. 12-16,1983.

Koop, H-U. et al. (1989)."Electric Field-Induced Fusion and Cell Reconstruction- with Preselected Single Protoplasts and Subprotoplasts of Higher Plants"in Electroporation and Electrofusion in Cell Biology, Neuman et al. Editors, pgs.

355-365,1989.

Pavlou, G. C. & D. J. Vakalounakis (1998). Control of the'root and stem rot'of greenhouse cucumber caused by Fusarium oxysporum f. sp. radicis-cucumerinum with grafting onto resistant rootstocks. Agriculture Crop & Animal Husbandry 6 : 19-26.

Punja, Z. K., M. Parker, S. Rose & D. Louie (1998). Occurrence of Fusarium crown and root rot, a new disease on greenhouse cucumbers in British Columbia, and methods for disease control. Cucurbitaceae 98 : 174-185.

Vakalounakis, D. J. & G. A. Fragkiadakis (1999a).'Root and stem rot'of greenhouse cucumber. A new worldwide disease, which threatens seriously the crops in Greece. Control methods. Agriculture Crop & Animal Husbandry 7: 36-48.

Vakalounakis, D. J. & G. A. Fragkiadakis (1999b). Genetic diversity of Fusarium oxysporum isolates from cucumber: Differentiation by pathogenicity, vegetative compatibility and RAPD fingerprinting. Phytopathology 89: 161-168.