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Effect of harpin ea to fruit production and control of phytophthora infestans in greenhouse tomato

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V.A. Bourbos and E.A. Barbopoulou NAGREF, Institute of Olive Tree and Subtropical Plants of Chania, Lab. of Plant Pathology and Ecotoxicology of Plant Protection Products, Agrokipio, 73100 Chania, Crete, Greece

Protein harpin Ea considered today as an effective stimulator of self-defense system as well as of other physiological mechanisms in many plant species. This work studies the effect of harpin Ea in fruit production as well as in the possibility to control Phytophthora infestans, a serious pathogen for open-field and greenhouse tomato cultivation. Harpin Ea was applied at a dose of 50,6 g/hl of the commercial product Messenger®. In order to study the possibility to control tomato late blight with harpin Ea, the fungicide fosetyl-Al was chosen as reference product at a dose of 200 g/hl of the commercial product Aliette 80 WP. Daily growth, number of flowers and fruits per cluster, fruit set percentage and total yield per plant were measured in order to estimate the effect on production. Estimation of the effectiveness to control the pathogen was based on the measurement of leaf spots and stem lesions. In the conditions of the experiment harpin Ea increased fruit set percentage at 6,92-7,73% and yield at 47,83-50,33% as well as it restricted pathogen with an effectiveness that ranged 98,83-100% in respect to the control.

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Effect of harpin ea to fruit production and control of phytophthora infestans in greenhouse tomato

  1. 1. Effect of Harpin Ea to Fruit Production and Control of Phytophthora infestans in Greenhouse Tomato V.A. Bourbos and E.A. Barbopoulou NAGREF, Institute of Olive Tree and Subtropical Plants of Chania, Lab. of Plant Pathology and Ecotoxicology of Plant Protection Products, Agrokipio, 73100 Chania, Crete, Greece Keywords: stimulator, self-defense system, late blight, growth, flowers, fruit set, yield, leaf spots, stem lesions Abstract Protein harpin Ea considered today as an effective stimulator of self-defense system as well as of other physiological mechanisms in many plant species. This work studies the effect of harpin Ea in fruit production as well as in the possibility to control Phytophthora infestans, a serious pathogen for open-field and greenhouse tomato cultivation. Harpin Ea was applied at a dose of 50,6 g/hl of the commercial product Messenger® . In order to study the possibility to control tomato late blight with harpin Ea, the fungicide fosetyl-Al was chosen as reference product at a dose of 200 g/hl of the commercial product Aliette 80 WP. Daily growth, number of flowers and fruits per cluster, fruit set percentage and total yield per plant were measured in order to estimate the effect on production. Estimation of the effectiveness to control the pathogen was based on the measurement of leaf spots and stem lesions. In the conditions of the experiment harpin Ea increased fruit set percentage at 6,92-7,73% and yield at 47,83-50,33% as well as it restricted pathogen with an effectiveness that ranged 98,83-100% in respect to the control. INTRODUCTION Research has been made during the last years on harpins produced by various plant or non-plant pathogenic bacteria, which are characterized for their self-stimulating properties. Harpins that have been produced include harpin Ea (Wei and Parker, 2001) by Erwinia amylovora, harpin Pss by Pseudomonas syringae pv. syringae (Hoyos et al., 1996), harpin Ecc by Erwinia carotovora subsp. carotovora (strain Ecc71) (Mukherjee et al., 1997), harpin Pst by Pseudononas syringae pv. tomato, harpin Psph by Ps. syringae pv. phaseolicola, harpin Rs by Ralstonia (Pseudomonas) salanacearum, harpin Pa by Pseudomonas avellanae (Loreti et al., 2001), harpin Pnss by Pantoea stewartii subsp. stewartii (Ahmad et al., 2001), harpin Pst by Pseudomonas syringae pv. tabaci (Huang et al., 2004), harpin Ech by Erwinia chrysanthemi and others. Harpin Ea originated by Erwinia amylovora is already produced on a commercial scale from a weakened strain of Eschericia coli. Harpin not only stimulates plant defense system against various pathogens but at the same time increases photosynthesis (13-15%) and other plant responses providing improved germination, enhanced plant growth of foliar and underground parts, accelerated flowering, increased yield (up to 22,4% in tomato) and fruit quality, advanced maturity and recovery after stress. It is a low cost product applied through foliar spraying or irrigation. Application is repeated at 14-day intervals. Once a plant is treated, activation is generally initiated within 5 to 10 minutes and full response generally occurs
  2. 2. within 3 to 5 days, while the effects may continue for several weeks (Wei and Parker, 2001). After the application, harpin Ea is not detected in plants (even if they are infected by Erwinia amylovora) (Perino et al., 1999), soil and water. It is non-toxic for human and workers may enter the field 4 hours after application. It is also non-toxic to beneficial fauna, bees, fishes, aquatic plants, birds, plants, and algae. Mode of action does not allow any possibility of plant pathogen resistant stains or races to develop. It has been used in Integrated Pest Management programs decreasing usage of conventional pesticides up to 70 (tomato) or 75% (strawberry). Successful results have been obtained by using harpin Ea in cucumber, other cucurbits, pepper, aubergine, potato, okras, strawberry, broccoli, cauliflower, chinese cabbage, radish, carrot, lettuce, celery, spinach, onion, garlic, leek, beetroot, asparagus, artichoke, maize, citrus, olive, avocado, apple, pear, peach, kiwi, almond, walnut, macadamia, pecan, peanut, cotton, sugar beet, sugar cane, maize, tobacco, rice, wheat, barley, groundnut, legumes, grape, raspberry, blackberry, gooseberry, conifer seedlings, ornamentals (Wei and Parker, 2001; EPA, 2002a; EPA, 2002b) and post harvest treatments (Capdeville et al., 2002). This trial studies the effect of harpin Ea on daily growth, number of flowers and fruits per cluster, fruit set percentage and total yield as well as the possibility to control late blight [Phytophthora infestans (Mont.) de Bary] in greenhouse tomato. MATERIALS AND METHODS The experiment was conducted in an unheated plastic greenhouse with tomato cultivation of ‘Bella Dona’. Experimental design was based on randomized blocks with 10 replications. Each experimental plot included 10 plants. The fungicide fosetyl-Al was used as reference product at a dose of 200 g/hl of the commercial product Aliette 80 WP. The specific biostimulator harpin Ea was applied at a dose of 50,6 g/hl of the commercial product Messenger® . Products were applied 3 times in 12-day interval when the plants were fully-developed with the use of a low-pressure hand operated sprayer. Daily growth, number of flowers and fruits per cluster, fruit set percentage and total yield per plant were measured in order to estimate the effect on fruit production. Estimation of the effectiveness to control the pathogen was based on the measurement of leaf spots at 10 leaves per plant and stem lesions, before the first and one day after each spraying. Effectiveness was estimated with the help of Hederson-Tilton equation regarding leaf spots and Abbott equation regarding stem lesions (Puntener, 1981). Statistical analysis of the experimental results was based on Duncan test (p=0,05). RESULTS Daily growth was significantly higher (3.01 - 3.05 cm) in the experimental plots where harpin Ea was used compared to control. At the same plots, it was observed statistically significant difference regarding the number of flowers and fruits per cluster as well as the fruit set percentage (Table 1). Total yield was notably increased from 10.120 to 10.178 Kg per plant in the plots where the tested product was used. The results showed that regarding the number of spots per leaf, harpin Ea considerably restricted late blight with an effectiveness ranging from 98.83% at the first to 100% at the second cultivation period that was significantly higher from that of the reference product (97.36 and 72.61% respectively) (Table 2).
  3. 3. Harpin Ea controlled also efficiently stem lesions with 100% effectiveness during both cultivation periods while for the conventional fungicide ranged 99.36-99.53%. DISCUSSION The protein harpin Ea is produced by Erwinia amylovora, while the non- pathogenic to human bacterium Escherichia coli has been modified to produce harpin on a commercial scale. E. coli cells are killed, lysed and filtered at the end of the fermentation process. Harpin Ea opens up important prospects in fruit quality improvement and fruit yield increase as well as in conventional pesticide reduction. After first contact of harpin Ea with plant cells and binding on cellular wall receptors, plants identify it as pathogen attack. Several biochemical responses follow, resulting in some species like Nicotiana tabacum and N. sylvestris (Garmiera et al., 2001) and Arabinopsis thaliana (Wei et al, 1992; El-Maaroufa, 2001; Peng et al., 2003; Huang et al., 2004) in the development of the phenomenon of hypersensitivity cell death (HCD) and mainly the phenomenon of systemic acquired resistance (SAR). In the last case, the enzymes for the production of protein kinase group (kinase 4 and 6) are activated, particularly myelin, affecting the endogenous stimulators of plant defense system salicylic acid, jasmonic acid and ethylene. The common presence of these stimulators provides plant resistance to fungi, bacteria, viruses, insects, mites and nematodes (Desikan et al., 2001; Wei and Parker, 2001). In the conditions of the experiment, harpin Ea in the form of the commercial product Messenger® significantly increased daily growth as well as flower and fruit number, fruit set and total yield. These results should be attributed to the positive effects that harpin Ea induces in growth of foliar and underground parts of the plant, photosynthesis, recovery after stress and flowering acceleration. According to the obtained results, tomato late blight [Phytophthora infestans (Mont.) de Bary] was effectively controlled in the treatments where harpin was used. Remarkable is the observation that plants treated with harpin Ea were not infected by other pathogens and had increased vigor during the whole cultivation period. Same results have been noted in cucumber powdery mildew (Bourbos and Barbopoulou, 2004) Literature Cited Ahmad, M. Majerczak, D.R. Pike, S. Hoyos, M.E. Novacky, A and Coplin, D.L. 2001. Biological activity of harpin produced by Pantoea stewartii subsp stewartii. Molecular Plant-Microbe Interactions, 14 (10):1223-1234. Bourbos, V.A. and Barbopoulou, E.A. 2004. Possibility to control Sphaerotheca fuliginea in cucumber greenhouse cultivation with the use of a specific biostimulator. 12th Hellenic Phytopathological Symposium, 12-15 Oct., Kastoria, Greece. Capdeville (de), G. Wilson, C.L. Beer, S.V. and Aist, J.R. 2002. Alternative disease control agents induce resistance to blue mold in harvested 'Red Delicious' apple fruit. Phytopathology, 92 (8): 900-908. Desikan, R. Hancock, J.T. Ichimura, K. Shinozaki, K. and Neill S.J., 2001. Harpin induces activation of the Arabidopsis MAP kinases AtMPK4 and AtMPK6. Plant Physiology 126, 1579–1587. El-Maaroufa, H. Barnya, M.A. Ronab, J.P. and Bouteaub F., 2001. Harpin, a hypersensitive response elicitor from Erwinia amylovora, regulates ion channel activities in Arabidopsis thaliana suspension cells. FEBS Letters, 497 (2-3), 82-84.
  4. 4. Environmental Protection Agency (EPA). 2002a. Biopesticide regulatory action document: Harpin protein (PC Code 006477). 32pp. http://www.epa.gov/oppbppd1/ biopesticides/ingredients/tech_docs/ brad_006477.pdf Environmental Protection Agency (EPA). 2002b. Harpin protein (006477) Fact sheet. http://www.epa.gov/oppbppd1/biopesticides/ingredients/factsheets/factsheet_006477. htm Garmiera, M. Dutilleula, C. Mathieua, C. Chétrita, P. Boccarab, M. and De Paepe, R. 2001. Changes in antioxidant expression and harpin -induced hypersensitive response in a Nicotiana sylvestris mitochondrial mutant. Plant Physiology and Biochemistry, 40 (6-8), 561-566. Hoyos, M.E. Stanley, C.W. He, S.Y. Pike, S. Pu, X.A. and Novacky, A. 1996. The interaction of harpin (Pss), with plant cell walls. Molecular Plant-Microbe Interactions, 9 (7): 608-616. Huang, H.E. Ger, M.J. Yip, M.K. Chen, C.Y. Pandey A.K. and Feng, T.Y. 2004. A hypersensitive response was induced by virulent bacteria in transgenic tobacco plants overexpressing a plant ferredoxin-like protein (PFLP). Physiological and Molecular Plant Pathology (in Press). Loreti, S. Sarrocco, S. and Gallelli, A. 2001. Identification of hrp genes, encoding harpin protein, in Pseudomonas avellanae (Psallidas) Janse et al. Journal of Phytopathology- Phytopathologische Zeitschrift, 149 (3-4): 219-226. Mukherjee, A. Cui, Y.Y. Liu, Y. and Chatterjee, A.K. 1997. Molecular characterization and expression of the Erwinia carotovora hrpN (Ecc) gene, which encodes an elicitor of the hypersensitive reaction. Molecular Plant-Microbe Interactions, 10 (4): 462-471. Peng, J.L. Dong, H.S. Dong, H.P. Delaney, T.P. Bonasera, J.M. and Beer, S.V. 2003. Harpin-elicited hypersensitive cell death and pathogen resistance require the NDR1 and EDS1 genes. Physiological and Molecular Plant Pathology, 62 (6): 317-326. Perino, C. Gaudriault, S. Vian, B. and Barny, M.A. 1999. Visualization of harpin secretion in planta during infection of apple seedlings by Erwinia amylovora. Cellular Microbiology, 1 (2): 131-141. Puntener, W. 1981. Manual pour essays de plein champ. Protection des vegetaux. CIBA- GEIGY Documenta. 205 pp. Wei, Z. and Parker, S.P. 2001. Messenger a new tool for IPM. Proceedings from the 75th Annual Western Orchard Pest and Disease Management Conference, January 10- 12, Imperial Hotel, Portland, Oregon, USA. Wei, Z.M. Laby, R.J. Zumoff, C.H. Bauer, D.W. He S.Y. Collmer, A. Beer, S.V. 1992. Harpin, elicitor of the hypersensitive response produced by the plant pathogen Erwinia amylovora. Science, 257: 85-88.
  5. 5. Tables Table 1. Effect of protein harpin Ea in daily growth (cm), number of flowers and fruits per cluster, fruit set percentage (%) and total yield per plant (Kg) in greenhouse tomato. Daily growth Flowers per cluster Fruits per cluster Fruit set Yield per plant 1st period 2nd period 1st period 2nd period 1st period 2nd period 1st period 2nd period 1st period 2nd period Control 2.21 c 2.18 c 6.00 c 5.89 c 5.03 c 5.00 c 83.83 c 83.10 c 6.885 c 6.732 c Fosetyl-Al 2.29 b 2.27 b 6.23 b 6.22 b 5.30 b 5.28 b 84.42 b 84.30 b 7.920 b 7.840 b Harpin Ea 3.05 a 3.01 a 6.60 a 6.52 a 5.95 a 5.91 a 89.63 a 89.52 a 10.178 a 10.120 a Table 2. Effectiveness of protein harpin Ea to control late blight in greenhouse tomato. Number of leaf spots/plant Number of stem lesions/plant 1st period……. 2nd period…….. 1st period… 2nd period… before after % effective- ness before after % effective- ness after % effective- ness after % effective- ness Control 0.052 2.216 0.00 c 0.052 1.553 0.00 c 2.576 0.00 c 1.554 0.00 c Fosetyl-Al 0.063 0.134 97.36 b 0.022 0.202 72.61 b 0.012 99.53 b 0.010 99.36 b Harpin Ea 0.024 0.012 98.83 a 0.010 0.000 100.00 a 0.000 100.00 a 0.000 100.00 a

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