In organic apple-growing control agents are necessary to prevent the blossoms being infected by

the fire blight pathogen Erwinia amylovora. Detached apple blossoms were used as an experimental

model to develop preparations based on yeast isolates for use in the control of fire blight. Several

yeast isolates reduced disease incidence in apple blossoms. The efficiency of yeast isolates

was increased by developing a suitable formulation. This yeast preparation exhibited high efficiency

in the control of fire blight in field-trials and will be marketed under the tradename "Blossom-

Protect"

Kunz, Stefan

English

Organic Eprints

Bio-Protect GmbH, Lohnerhofstraße 7, 78467 Konstanz, s.kunz@bio-protect.de  Development of "Blossom-Protect" - a yeast preparation for the reduction of blossom infections by fire blight  Entwicklung von "Blossom-Protect"- ein Hefepräparat zur Reduktion von Blüteninfek-tionen durch Feuerbrand Stefan Kunz  Abstract In organic apple-growing control agents are necessary to prevent the blossoms being infected by the fire blight pathogen Erwinia amylovora. Detached apple blossoms were used as an experimen-tal model to develop preparations based on yeast isolates for use in the control of fire blight.  Sev-eral yeast isolates reduced disease incidence in apple blossoms. The efficiency of yeast isolates was increased by developing a suitable formulation. This yeast preparation exhibited high efficiency in  the  control  of  fire  blight  in  field-trials  and  will  be  marketed  under  the  tradename  "Blossom-Protect".  Keywords:  Fire blight, Blossom-Protect, Biopro Erwinia amylovora, Aureobasidium pullulans,  Introduction: Fire blight caused by Erwinia amylovora is the most serious bacterial disease in apples. During the last three decades it has spread through nearly all European countries. Since pruning of diseased material and other sanitation methods are not sufficient to stop the spread of the disease, efficient control  agents  are  needed.  Primary infections occur in the blossom where the pathogen enters through natural openings after multiplying on the stigmas. To prevent the epiphytic multiplication by the pathogen several micro organisms were examined for their ability to colonise apple blossoms (3, 4, 6)..  Yeast isolates showed a promising ability to colonise apple stigmas and nectar (7)..  In this study yeast strains, isolated from apple fruits, were tested for their ability to suppress symp-tom  development  on  detached  apple  blossoms  after  inoculation  with  E.  amylovora..  For  these strains, production procedures were developed, providing storable powder formulations with high numbers of viable blastospores. Additives were found, which increased the efficiency of the yeast isolates on detached blossoms and in field trials.   Material and Methods: Micro organisms used and composition of test preparations Names and origin of the isolates used are given in table 2. From these isolates three test prepara-tions were produced (tab. 1).   Archived at http://orgprints.org/14453/      Table 1: Composition of Test- preparations  Name  Microbial strains (spores/g)  Formulation  Conc. for appli-cation (%) BIOPRO®  BsBD170 (2x1010)  WP  0,1 BPAxxx  ApCF10 (1x1010) + ApCF40 (1x1010)  WP  0,2 BPASxxx  ApCF10 (1,5x109) + ApCF40 (1,5x109)  WP+buffer  1,3   Reduction of fire blight symptoms on detached blossoms An in vivo test-system with detached apple blossoms was established according to Pusey (6, 7). From January to August apple trees (`Gala`) were stored at 2° C in the dark. Weekly a group of trees was transferred to the greenhouse to force them to bloom.  The blossoms were cut and maintained with cut peduncle submerged in 10 % sucrose solution in plastic racks (23° C, 100% rH). Blossoms were spraye d with a suspension of  E. amylovora (106 cfu/ml) in water until runoff. Dependant on the experiment, treatments were sprayed 24h before, 1h after or 22h after inoculation. The number of blossoms with bacterial ooze at the peduncle was counted 6 days after the inoculation.   Reduction of fire blight symptoms on blossoms taken from a field trial The field trial was performed on the variety `Jonagold` and was arranged in a completely random-ised block design with 4 replicates per treatment. Applications were timed according to the devel-opment of the blossoms and the infection risk predicted with the Maryblyt model. An infection risk day was predicted for the 29th April. Therefore, the first application was made on  28th April when approx. 50% of the blossoms were open. From the 4th May onwards the temperature necessary for infection was reached. On the 6th May the second application was made , when approx. 95% of the blossoms were open, as rain was predicted for the following days. During blossoming (26.04.03-08.05.03) no other pesticides were applied in the test orchard. The orchard was controlled for fire blight symptoms regularly from the end of July.  To see, if the blossoms were protected by treatment at a certain time, 4x24 blossoms per treatment were cut at the 28th, 29th , 30th of April and the 5th and 6th of May and inoculated with E. amylovora in the laboratory as described above.    Results: Reduction of fire blight symptoms on detached blossoms The ability of bacterial or yeast isolates to reduce fire blight symptoms was determined on detached apple blossoms. The microbial strains were cultivated in shake cultures and applied to the blossoms 24h before inoculation. The yeast isolates were more effective in reducing fire blight symptoms than the bacterial isolates (tab. 3). It was possible to develop production procedures for  ApCF10 and ApCF40 resulting in storable powders with a high number of viable blastospores. SrCF35 was not appropriate for mass production. Therefore ApCF10 and APCF40 were used for further testing and to mix test preparations.  Archived at http://orgprints.org/14453/   Table 2: Reduction of fire blight symptoms on detached apple blossoms by different microbial strains or streptomycin. Bacteria (108 cfu/ml) or yeast (107 cfu/ml) was suspended in water and sprayed on the blossoms 24h before inoculation with E. amylovora (106 cfu/ml).    Name  Species  Origin  No. *  Reduction of symptoms (%) Bs BD170  Bacillus subtilis  BBA, Darmstadt  8  60 Bs Hg77  Bacillus amyloliquefa-ciens University of Kon-stanz 2  67 Bacteria Ra 39  Rahnella aquatilis  BBA, Darmstadt  2  64 Ap CF10  Aureobasidium pullu-lans University of Kon-stanz 2  88 Ap CF40  Aureobasidium pullu-lans University of Kon-stanz 6  93 Yeast Sr CF35  Sporobolomyces roseus University of Kon-stanz 3  79 Antibiotic    Streptomycin-sulfat  (0,02%)   6  89 * No. of experiments      025507510024 -1 -22Time between treatment and inoculation (h)Symptom reuction (%)0,5% BIOPRO 0,2% BPAxxx1.3% BPASxxx 0,025% Streptomycinsulfatn. e.. Figure  1: Reduction of fire blight symptoms by different test preparations on de-tached apple blossoms. The blossoms were treated 24h before, 1h after or 22h after the inoculation with E. amylovora (1x 106 Zellen/ml). BPASxxx was not examined at 24h before inoculation. Biopro reduced fire blight symptoms on detached apple blossoms by 60%, when applied 24h prior to inocula-tion with E. amylovora whereas BBAxxx reduced it by 92% and streptomycinsulfat by 86% when applied at Archived at http://orgprints.org/14453/  the same time (fig.1). The treatment with BPAxxx 1h after the inoculation led to a symptom reduction of 53%. Additives in BPASxxx, which made the application suspension acidic, increased the symptom reduction to 66%.  Following the Maryblight model E. amylovora needs a growth phase on the stigma characterised by a sum of degree hours (> 18,3° C) of 110.  The incubation of  the blossoms at 23° C for 22h leads to a sum of degr ee hours of 103. Applications at this time with BPAxxx or BPASxxx reduced disease symptoms by approx. 30% whereas a treatment with streptomycin was more efficient (fig. 1). This shows, that the yeast preparations were able to prevent the growth of E. amylovora on the blossom surface, but were not able to prevent infec-tions after the pathogen was able to multiply on the stigmas.  Reduction of fire blight symptoms on blossoms taken from a field trial   02040608010024.425.426.427.428.429.430.41.52.53.54.55.56.57.58.59.510.511.5Symptom reduction (%); Open Blossoms (%)0510152025Mean Temp. (°C); Rain (mm)BPAxxx BPASxxx application dateopen blossoms rain mean temp.I I I I Figure 2: Reduction of fire blight symptoms on blossoms taken from the field trial by 0,2% BPAxxx or 1,3% BPASxxx. The Application was done at the marked dates (X). Per sampling date and treatment 4x24 blossoms were cut, put in 10% sucrose solution and inoculated with E. amylovora in the laboratory. After 6d incubation (23° C ; 100%  rH) blossoms with bacterial ooze at the peduncle were counted. The columns indicate the symptom reduction in com-parison to the untreated control. Infection risk days (Maryblight) are marked with I.    In the test orchard no fire-blight symptoms were found in 2003.  One reason for this, is that on 6th May, 100% of the blossoms were already open and infection risk days did not occurr  until 8th May (fig. 2).  The artificial inoculation of the detached blossoms taken from the field trial shows, that the yeast preparations BPAxxx und BPASxxx reduced the fire blight symptoms noticeably  (fig.. 2). After the first application the symptom reduction reached 60% over a period of three days. The 4th sampling date was 7 days after application. In the meantime, the treated blossoms faded and new blossoms had opened - the symptom reduction was correspondingly low. In the samples from the day of the second  application  and  on  the  following  day  a  symptom  reduction  of  70%  was  reached  with BPASxxx (fig. 2). Discussion: Archived at http://orgprints.org/14453/  The experiments on detached apple blossoms showed, that spray treatments with yeast isolates reduced fire blight symptoms significantly. The yeast isolates needed less time to build up a de-fence barrier on the blossoms than the tested bacteria and the efficiency of the yeast isolates was not dependent on the incubation temperature. The use of a suitable formulation increased the effi-ciency of the yeast strains.  The symptom reduction shown here on detached blossoms was confirmed by field trials, in which some trees were inoculated with E. amylovora. In 2002 a preparation corresponding to BPAxxx achieved a symptom reduction of 37% averaged over 4 trials (1, 2, 5).. In 2003 the use of the for-mulated preparation BPASxxx resulted in a symptom reduction of 74% (8). As a result of the dem-onstrated effectiveness of  this yeast preparation as a biological control agent against fire blight, it will be marketed under the trade name "Blossom-Protect". Further investigations to integrate "Blos-som-Protect" into spray schedules of organic - and integrated apple production will be carried out.  Acknowledgements: I thank K. Mendgen, University of Konstanz, for providing the yeast strains and the greenhouse and G. Mögel and M. Schwarz for their valuable technical assistance. This work was partly supported by the Ministry of Nutrition and Agriculture of Baden-Württemberg, Germany.   Literature Cited 1.  Fried A. 2002. Feuerbrandbekämpfungsversuch 2002. Obstbau 27: 551-5 2.  Harzer U. 2002. Feuerbrand-Bekämpfungsversuch 2002. Obstbau 27: 555-7 3.  Johnson KB, Stockwell VO. 1998. Management of fire blight: a case study in microbial ecol-ogy. Annual Review of Phytopathology 36: 227-48 4.  Kunz S, Laux P. 2001. Entwicklung eines Verfahrens zum biologischen Pflanzenschutz durch den Einsatz bakterieller Antagonisten gegen Bakteriosen im Pflanzenbau. http://edok01.tib.uni-hannover.de/edoks/e01fb02/357261569l.pdf: Bio-System GmbH, Kon-stanz. 48 pp. 5.  Moltmann E, Lange E, Trautmann M. 2002. Eine neue Methode zur Durchführung von Feu-erbrandversuchen. Obstbau 27: 557-60 6.  Pusey PL. 1997. Crab apple blossoms as a model for research on biological control of fire blight. Phytopathology 87: 1096-102 7.  Pusey PL. 1999. Effect of nectar on microbial antagonists evaluated for use in control of fire blight of pome fruits. Phytopathology 89: 39-46 8.  Seibold A, Fried A, Kunz S, Moltmann E, Lange E, Jelkmann W. 2004. Yeasts as Antago-nists against Fire Blight. EPPO Bulletin 34: in press Archived at http://orgprints.org/14453/

Development of "Blossom-Protect" - a yeast preparation for the reduction of blossom infections by fire blight

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