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.

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. 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. 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. 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. 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