SUMMARY OF PHD THESES UNIVERSITY OF VESZPRÉM GEORGIKON FACULTY OF AGRICULTURE Institute of Plant Protection Head of PhD Program: Supervisor: DR. GYULA SÁRINGER MHA DR. habil. GÉZA FISCHL CSc EFFECTS OF ENVIRONMENTAL FACTORS ON FRUIT ROT DISEASE OF TOMATO SHAPED PEPPER By: DR. JÁNOS KOVÁCS KESZTHELY 2001.
2 Introduction and aims of research The tomato shaped type of sweet pepper is one of the favorite products for the fresh market in Hungary. This type of pepper was an important Hungarian export product in the 1970s. Since that time, however, exports have decreased because of Alternaria fruit rot of pepper, which is the most serious disease of tomato-shaped sweet peppers. The causal organism of this disease is the fungus Alternaria alternata FR. KEISSLER (Syn.: Alternaria tenuis C. G. NEES). This fungus produces several toxins, of which tentoxin is the best known in Hungarian and foreign literature. Infection of the pepper fruits is only detectable when they are cut open. The symptom is a large grayishbrown velvety mold inside the ripe fruit. Using of fungicides does not give enough protection, this is the reason why to breed resistant cultivars. The aims of this study were: Comparing Alternaria alternata isolates by morphology and habit of colonies. Determining the effects of ph, quality and quantity of sugar and nitrogen in media, visible light and UV radiation on growth of colonies of Alternaria alternata. Examining sensibility of pepper seedlings to tentoxin. Screening susceptibility of pepper genotypes and work out a new method of testing the resistance on cotyledon leaves.
3 Proving the connection between rate of infected fruits and quantity of dry material and sugar, and ph value in fruit juice. Determining the connection between fruit length and rate of infected fruits. Breeding Alternaria alternata resistant cultivars with high yield productivity.
4 Materials and methods Trials were made at Pannon University of Agricultural Sciences (now University of Veszprém) Georgikon Faculty of Agriculture, Department of Phytopathology and Plant Virology, and Department of Horticulture. Alternaria alternata isolates were isolated from infected ripe pepper fruits and sown on potato dextrose agar (PDA) for isolation by the Institute for Plant Protection. The isolates were incubated for four days at 24 C and the most typical isolates were used for inoculation. During autecological experiments we examined the effect of ph value of potato dextrose agar (PDA) media on Alternaria alternata. The ph values were between 4-10. After inoculation the colonies were incubated at 24 C. Evaluation was made on 3 d, 6 th and 9 th days after inoculation. We determined the diameters of colonies, sporulation and reproductive marks. In vitro trials were made to check the effects of sugar forms (fructose, glucose and saccharose /sucrose/) and sugar concentration (10-50 g/l) of Czapek media on Alternaria alternata. The effect of nitrogen concentration of media was determined on Czapek media, in which the normal nitrogen dose is 3 g/l NaNO 3. Trials were 0, 0.5, 1, 1,5 and 2 times the normal dose. We measured the diameters of colonies on the 5 th and 8 th days, and made visual and microscopic observation. Different nitrogen sources also were used in equal concentration as the normal dose. N-sources were: sodium-nitrate /NaNO 3 /, potassium-
5 nitrate /KNO 3 /, ammonium-nitrate /NH 4 NO 3 /, carbamide CO(NH 2 ) 2, and Calcium-nitrate /Ca(NO 3 )2*4H 2 O/. Using a typical Alternaria alternata isolate, we examined the effects of visible light and UV radiation. The fungus was grown on PDA media. After the 3-4 days growing in darkness the colonies were illuminated with light from different wavelength region (white, red, yellow, green, blue, near UV and UV contains the 254 nm Hg line), and with different exposition time (1h, 2h s, 4h s ). The control colonies were grown in darkness, in thermostate. We measured the diameter of colonies, examined the intensity of sporulation, and recorded the habit of colonies. At the Department of Horticulture, breeding of tomato shaped pepper lines resistant to fruit rot started in 1985. The pepper lines and cultivars were collected in Hungary and abroad during 1985-87. After making groups, we crossed the lines to get new hybrids. For screening the Alternaria fruit rot resistance of pepper, we made artificial inoculation of flowers and small fruit at the first part of the examination (1985-88). The pepper seedlings were grown in a heated plastic covered greenhouse for six weeks and then were transplanted into unheated ones. When the plants reached flowering stage they were inoculated by spraying with an aqueous suspension of mycelia and conidia of the pathogen. Alternaria alternata was isolated from ripe peppers and streaked on potato dextrose agar (PDA) for isolation. Colonies were incubated for four days at 24 C and the most typical isolates were used for the experiments. In the second part of the examination (from 1989.), the artificial inoculation with Alternaria
6 alternata was performed twice: seeds and flowers. 50 seeds of each genotype were incubated at 22 C and germinated on plates containing the Alternaria alternata mycelia. Forty of the germinated seeds per genotype were planted into plastic pots using four replications with ten seeds each. The other conditions were the same as in the 1 st part of trials. Alternaria alternata produces several phytotoxins, of which tentoxin has an effect on mitochondria and affects the metabolism of CO 2 and photosynthesis. As a result, the chlorophyll production of the germinating plant can already be inhibited, showing the typical symptoms of chlorophyll deficiency. The green, i. e. resistant plants can be easily selected. Measuring of chlorophyll content of leaves and comparing with control, the toxin sensitivity can be indexed. Some of the ripe, red fruits contained fungal mycelia. The susceptibility to capsule mold was evaluated as the percent of infected fruits. We made fruit juice from ripe red fruits, and measured the volume of dry material by Abbé refractometer, the sugar content by Bertrand method and the ph value by electrical ph meter.
7 Results Characterization of Alternaria alternata isolates We made 285 isolations from infected pepper fruits during 1992-94., from them 92,6% of isolates were Alternaria alternata. Colonies were screened at 10 th day after inoculation. The colonies were weak in growth (diameter under 4 cm), medium (4-7 cm) and vigorous (over 7 cm). Colony colors were light, medium or dark gray. Most of the colonies had velvety mycelia sometimes we found thick mycelia. Type and intensity of sporulation was different at colonies. In 1993, we found 17,2% tentoxin producer colonies. Autecological examination of Alternaria alternata There was a very little difference at ph 6-9, but we found significant difference at ph 5 and 10 at growth of colonies. The ph below 5 prevents growing mycelia. The fungus is able to utilize all the tree forms of sugar, lower sugar concentration modifies the appearance, color, morphology of colony and intensity of sporulation. The quantity of nitrogen in media and nitrogen forms had influenced growth of mycelia. Higher concentration increased, lower decreased the diameters of colonies. Ammonium-nitrate and carbamide prevented growth of colonies.
8 Light and UV radiation had influence on fungus. Irradiation with UV caused the greatest changing at colonies, it decreased speed of growing. Pepper lines and cultivars After treating of seeds with tentoxin or tentoxin producer Alternaria alternata isolates, the cotyledon leaves and ordinary leaves of seedling showed chlorotic and necrotic spots. Sometimes cotyledon leaves were albinistic and these plants did not contain chlorophyll at all. We found that 20 µg/ml clean tentoxin caused very strong symptoms. Those plants, which were not so sensible, survived the treatment, and grew up in greenhouse. Summarize our results we determined which cultivars are susceptible or resistant to Alternaria fruit rot of pepper. The susceptibility to capsule mould was based on the percentage of infected fruits. The least susceptible cultivars proved to be the following: Rotund zelena, PAZ Pallagi and Piknik. The Pimiento tropical proved to be the most susceptible to the disease, but could tolerate the treatment with tentoxin. Lower volume of dry material and sugar in fruit juice may reduce the number of infected fruits. The ph value below 5 also can reduce the infection of fruits.
9 New scientific results 1. We worked out a new method of resistance breeding, based on an artificial inoculation and selection system. 2. Non host specific tentoxin can be used in selection of pepper seedlings. 3. This new technique is applicable in whole year in laboratories, and lots of young plats can be screened. 4. Screening the Alternaria alternata isolates in 1993, we found 17,2% tentoxin producer isolates from them. Trials with tentoxin and artificial inoculation with tentoxin producer Alternaria alternata isolates give same symptoms on pepper seedlings. 5. We verified, that the components and the ph of media have an effect on growth and sporulation of fungus. Lower sugar concentration modifies the sporulation becomes more intensive. NH 4 NO 3 as a nitrogen source has an impending effect on growth of Alternaria alternata. UV radiation increased the sporulation but reduced growth of mycelia. 6. Lower sugar content in pepper fruit juice effects better resistance against the pathogen. 7. Cultivar Piknik using in hybrid combinations gives a better resistance for progenies. 8. Breeding of Turbán new variety candidate.
10 Suggestions Department of Plant Pathology and Plant Virology, and the Department of Horticulture at University of Veszprém, Georgikon Faculty of Agriculture made first time the selection of resistant cultivars with seed inoculation method in Hungary. Our trials demonstrated, that non host specific tentoxin can be used in selection of pepper seedlings. This kind of the screening can be made on large population in labs in the whole year in a short time. Lower sugar content in pepper fruit juice effects better resistance against the pathogen. We suggest using these types of cultivars in production. Cultivar Piknik, using it in hybrid combinations, gives a better resistance for progeny. So we can suggest using of Piknik in different crosses for another breeders. From screened pepper genotypes the least susceptible cultivars proved to be Piknik, where the number of infected fruits was lower, than 10% in average of years. It gives a good alternative in using cultivars in horticultural production.
11 Publications Main publications: GÁBORJÁNYI, R., HORVÁTH, J., KOVÁCS, J. and KAZINCZI, G.: 1998. Role of virus- and phytoplasma infections in pepper decline in Hungary: An owerview. Acta Phytopathol. Entomol. Hung. 33: 229-236. KOVÁCS, J. and FISCHL, G.: 1996. Resistant cultivars to Alternaria fruit rot of pepper (Alternaria alternata KEISSLER). 1 st Egyptian- Hungarian Horticultural Conference, Kafr El-Sheikh, Egypt. 15-17. September 1996. Proc. Vol II. pp. 206-212. KOVÁCS, J., FISCHL, G. and KOVÁCS, J.: 1995. Breeding pepper cultivars for resistance to Alternaria fruit rot of pepper (Alternaria alternata KEISSLER). IX th Meeting on Genetics and Breeding on Capsicum and Eggplant. 21-25 August, 1995. Budapest. Proc. pp 225-228. FISCHL, G. és KOVÁCS, J.: 1990. Paprika genotípusok és Alternaria alternata izolátumok kölcsönhatása. Növényvédelem. 26. 9. 391-396. KOVÁCS J., FISCHL G. és NAGY P.: 1999. A cukorformák és a cukorkoncentráció hatása az Alternaria alternata (FR.) Keissler gombafajra in vitro körülmények között. Növényvédelem 35. (2): 53-56.
12 Other publications: FISCHL, G. és KOVÁCS, J.: 1989. Paprika genotípusok reakciója Alternaria fertőzésre csíranövény korban. Vetőmag. 1989. Október 10-13. KOVÁCS, J.: 1993. Paradicsomalakú paprikafajták magház penészesedésének okai és a megelőzés lehetősége. Kertészeti és Élelmiszeripari Egyetem. Szakdolgozat, Budapest. KOVÁCS, J.: 1994. Paradicsomalakú paprikák nemesítése magház penészesedés megszűntetése érdekében. Debreceni Agrártudományi Egyetem. Egyetemi doktori értekezés, Debrecen. KOVÁCS, J. és FISCHL, G.: 1988. A paprika magházpenész betegsége. Vetőmag. 1988. Január. 4-7. KOVÁCS, J., FISCHL, G. és KOVÁCS, J.: 1990. A csíranövény korban létrejött Alternaria fertőzés átvészelésének esélye pritamin típusú paprika vonalaknál, Vetőmag. 1990. Március. 4-7. KOVÁCS, J., FISCHL, G. és KOVÁCS, J.: 1994. Magházpenész betegség paradicsomalakú paprikán. Hajtatás, korai termesztés. 25 (4.): 9-10.
13 Oral presentations and posters: FISCHL, G., KOVÁCS, J. és KOVÁCS, J.: 1990. Módszertani lehetőségek a paradicsomalakú paprika magházpenésze elleni rezisztenciára nemesítésben. Lippay J. Tud. Ü. Kertészeti és Élelmiszeripari Egyetem Kiadványai, Budapest. 185-186. FISCHL G., KOVÁCS J., NAGY P. és SURMANN Á.: 2000. In-vitro vizsgálatok a paprika magházpenészét okozó Alternaria alternata gombafajjal. Lippay J. - Vas K. Tud. Ü. Budapest. Abs. 378-379. FISCHL G., SZUNICS L., KOVÁCS J. és NAGY T.: 1995. Néhány növényfaj Alternaria fajok okozta betegségéről. V. Keszthelyi Növényvédelmi Fórum előadásai. 1995. I. 25-27. Keszthely. Abs. 9. GÁBORJÁNYI, R., HORVÁTH, J., KOVÁCS, J. AND KAZINCZI, G.: 1998. Role of viruses in pepper decline in Hungary. X th Eucarpia Meeting on Genetics and Breeding of Capsicum and Eggplant. 1998. Avignon, France. Abs. 129-132. KOVÁCS J.: 1999. Paprika fajták rezisztencia nemesítése. Előadás. PATE Kert. Tansz. Szak. Nap., Keszthely, 1999. márc. 11. KOVÁCS, J. and FISCHL, G.:1996. Alternaria fruit rot tolerant pepper cultivars. National Pepper Conference, Naples, Florida, USA. 8-11. December 1996.
14 KOVÁCS, J. és FISCHL, G.: 1997. Abiotikus és biotikus tényezők hatása a paprika alternáriás termésrothadására. Növ. Véd. Tud. Napok, Budapest, 1997. KOVÁCS, J. and FISCHL, G.:1997. Data for fruit rot disease of tomatoshape pepper. 1 st International Plant Protection Symposium at Debrecen AU. 18-22. August 1997.Debrecen, Hungary. Abs. 19. KOVÁCS J., és FISCHL G. (1998): Újabb adatok a pritamin típusú paprika magházpenészét és rothadását okozó Alternaria alternata gombafaj ökológiai jellemzéséhez. Növényvédelmi Tudományos Napok. Budapest, 1998. feb. 24-25. Abs. 111. KOVÁCS, J., és FISCHL, G.: 1998. A paradicsom alakú paprika magházpenészedés elleni rezisztencia nemesítése. Biológiai Alapok a Növénytermesztésben, OMMI, Budapest. KOVÁCS, J. és PFEIFFER, ZS.: 1992. Termesztett paradicsomalakú paprikafajták magházpenész érzékenysége. A "Lippay J." Tud. Ü. Kertészeti és Élelmiszerip. Egy. Kiadványai, Budapest. 924-929. NAGY P., FISCHL G. and KOVÁCS J.: 2000. The effect of light illumination on growth and sporulation of some microscopic fungi. ESNA. XXX. Annual Meeting, Keszthely, Hungary. Abs. 117.