Buban t cost864 manuscript

Efficacy of Pantoea agglomerans strain HIP32 against Erwinia amylovora
Tamás Bubán1*, Tamás Lakatos1, Tímea Tóth1, László Dorgai 2, Ildikó Hudák3, Mária Hevesi4
1Research and Extension Centre for Fruit Growing, 4244 Újfehértó P.O.Box 38., Hungary 3Biotechn. Laboratory, Res. Centre of the Debrecen University, 4400 Nyíregyháza, Hungary 4Corvinus University, Faculty of Horticult. Sci., Dept. of Pomology, 1118 Budapest, Hungary
*corresponding author: bubant@ujfehertokutato.hu
Abstract. When a high level of P. agglomerans HIP32 population is present in the blossoms
- steadily established after the first-, or rebuilt by the second treatment - a sizable native
Erwinia amylovora population is measurable only within a few hours after the treatment. The
consequent decrease in the antagonist population results in the continuous presence of the pathogen at relatively high level. Results of inoculation experiments under control ed conditions prove that P. agglomerans HIP32 treatment results in a significant reduction in the
E. amylovora population size, associated with convincing decrease in the index of infection.

Biological control of fire blight, the bacterial disease caused by E. amylovora
can be achieved by applying bacterial antagonists onto the flowers before a sizable epiphytic population of the pathogen is established [1], [2]. The Pantoea agglomerans strain HIP32 (HIP32) isolated from apple cv. Starking Delicious leaves in Hungary [4] is a potential
Material and Methods. Idared (in 2005) as wel as Jonica, Pinova, Idared and Royal Gala
(in 2006) apple trees on M.9 rootstock were sprayed with HIP32 twice during bloom time in the years of 2005 and 2006 (109 and 108 cfu/ml in 10 mM PBS, respectively). Check trees were sprayed with PBS. Estimation of the bacterial population size in the flowers was done by dilution plating on King B medium (HIP32) and Mil er-Schrott medium (native E. amylovora) in 2005, but on Luria-Bertani medium (HIP32) and Chromocult coliform agar (E. amylovora) in 2006. Incidence of blossom blight and shoot blight in the field was monitored in Inoculation experiments were carried out under control ed conditions. Flowers from trees treated with HIP32 in the field (2005), or treated with HIP32 ChlR in the laboratory (108 cfu/ml, 2006) were inoculated by E. amylovora strain Ea1 KanR, (106 cfu/ml in 2005 and 105 cfu/ml in 2006). Bacterial population sizes were measured by dilution plating on mediums amended with chloramphenicol and kanamycin, respectively. The index of infection was calculated by an equation [3] considering both the occurrence and severity of blossom blight scored by the – partly modified – rating scale of Pusey [5]. Analysis of variance was done with SPSS program package, mean separation by Tukey’s. While comparing population sizes, the ln transformed data were used.
Results and Discussion. A rainfal of 7.4 mm a day after the first spraying in 2005 caused a
decrease in the ratio of flowers with sizable antagonist population merely to 20%. The 2nd
and 4th day fol owing the second spraying, however, the population size of HIP32 remained at a steady level of about 106 cfu/flower. Population size of E. amylovora on the 4th day after the second treatment was 3-4x103 cfu/flower in two of the 15 samples from the check trees and was not detectable in flowers of treated ones. Probably the pathogen could not be establish due to the cold weather. The pathogen population in the flowers of trees treated in the field and inoculated in the laboratory with E. amylovora Ea1 KanR was significantly (p<0.01) less than in the check flowers (3.75x104 vs. 1.00x106 cfu/flower). The results of field trials in 2006 (Tab. 1) indicate that a stably established, high HIP32 population (see cv. Royal Gala), or a population rebuilt by the second treatment (Jonica) coincides with a native population of E. amylovora measurable at the beginning of the experiment only (Jonica), after that it is reduced to, or stays at a very low, though detectable level (Royal Gala, Jonica). A steadily decreasing antagonist population is paral eled with the continuous presence of the pathogen at a measurable level (Pinova). As a function of HIP32 ChlR treatment, the significant reduction in the E. amylovora population size is associated with a convincing decrease in the index of infection (Tab. 2). Due to the low temperature in both years, the incidence of blossom blight in the field was not high enough to estimate the Population sizes of Pantoea agglomerans HIP32 and native Erwinia amylovora in blossoms 2 hours 2nd day/A1 2nd day/B2 2 hours 2nd day/A1 2nd day/B2
after treatments________________________ Jonica
P.a.HIP32 1.0x107a 3,2x104b 3.7x106a 1.3x103 3 6/6
36/6 36/6
P.a.HIP32 6.0x106a 2.7x105b 8.9x104c 1.3x103 1.3x102 2.0x103
– 5.8x102 35/6
P.a.HIP32 6.8x105a 8.5x105a 3.7x104b 2.0x101 1.4x103 36/6
Royal Gala
P.a.HIP32 2.09x106a 1.07x106b 1.35x106ab 33/6 36/6
31/6 31/6
12nd day after the 1st spraying, just before the 2nd treatment
22nd day after the 2nd spraying
3number of replications from 6 ones with detectable level (<1x101 cfu/blossom) of pathogen
Data with various letters differ significantly at p<0.05 with the cv. Royal Gala and at p<0.001 Bacterial populations and disease rating in artificially infected blossoms of apple cultivars Population sizes (cfu/blossom)1 of Index
amylovora infection2
Jonica P. agglomerans strain HIP32 ChlR 8.45x105 5.5x105a 0.21a
1.04x108b 2.17b
Pinova P. agglomerans strain HIP32 ChlR 2.9 x106 1.5x105a 0.00a
3.74x107b 2.17b
1the 4th day after inoculation 2the 5th day after inoculation
Data with various letters within a cultivar differ significantly at p<0.05


[1] Johnson K.B. et al. (1993) Phytopathology 83: 995-1002.
[2] Stockwel W.O. (2002) In Lindow S.E. et al. (Eds) Phyl osphere Microbiology, APS Press, [3] Bertrand P.F., Gottwald T.R. (1978) In Zehr E.I. (Ed.) Methods for evaluating plant fungi- cides, nematicides and bactericides, APS Press, St. Paul, pp. 179-181.
[4] Hevesi M., El-Arabi K.F. (1999) Acta Horticulturae 489: 619-622. [5] Pusey P.L. (1999) Acta Horticulturae 489: 521-524. This project of BAROSS-2-2005-0013 is supported by the National Office for Research and Technology;

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