Interrelationship of Temperature, Flower Development and Biological Control of Fire Blight

BlightBan (a.i., Pseudomonas fluorescens strain A506) has been available in recent years and other biological agents (e.g., Pantoea agglomerans strains C9-1 and E325) are being developed for fire blight control. Advances will partly depend on an understanding of interrelationships involving environment, flower development and senescence, and microorganisms. Laboratory experiments were performed with detached blossoms of crab apple (Malus sp. ‘Manchurian’). When inoculated stigmas were held at various temperatures, strain A506 failed to grow at the upper temperature range of Erwinia amylovora, but strains C9-1 and E325 had ranges extending beyond that of the pathogen. When flowers were subject to various temperatures and incubation periods before inoculation, the duration of stigma conduciveness to bacterial multiplication decreased as temperature increased, and it was shorter when flowers were pollinated versus non-pollinated. These interactions were confirmed with mature apple trees (Malus x domestica Borkh.‘Gala’) surrounded by plastic enclosures with heating and cooling to maintain different temperature ranges. In trials in 2000 and 2001, temperatures during bloom averaged 13.4 and 14.1 C at the low range and 21.5 and 21.9C at the high range, respectively. The longest period stigmas retained the capacity to support bacterial growth when inoculated was about 14 days, occurring with non-pollinated flowers at low temperatures. The shortest period was 3 or 4 days, occurring with pollinated flowers at high temperatures. Results were similar with different bacteria. However, strain A506 failed to multiply at late stages of stigma senescence, which did allow growth of the pathogen and strain E325. On crab apple flowers, antagonist strains differed in their capacity to reduce pathogen populations and these differences were consistent during the period when stigmas were conducive to pathogen growth. INTRODUCTION Biological control is still a relatively new tool for managing fire blight of apple and pear caused by Erwinia amylovora. The product BlightBan (a.i., Pseudomonas fluorescens strain A506) has been available in recent years and other biological agents (e.g., Pantoea agglomerans strains C9-1 and E325) are currently being developed commercially for this disease. Experience with these beneficial bacteria indicates an inconsistency possibly related to environment and the plant host. Improvements in efficacy will partly depend on an understanding of these factors. The flower part of greatest interest is the stigma, where E. amylovora can become established even under dry western conditions. It is known that temperature affects the duration that flower stigmas are receptive to pollen and that stigma senescence is accelerated after pollination (Soltesz et al., 1996). Also, a relationship between stigma age and colonization by E. amylovora has been reported (Gouk and Thomson, 1999). Information is lacking, however, regarding the relationship of these factors to biological control. This study examines the interrelationship of temperature, pollination, stigma age, and microbial colonization and interactions. Proc. 9th Int. Workshop on Fire Blight Eds. C. Hale and R. Mitchell Acta Hort. 590, ISHS 2002 248 MATERIALS AND METHODS Plant Material Laboratory experiments were performed with detached blossoms of ‘Manchurian’ crab apple trees (1.6 cm minimum stem diameter) received from a local nursery and induced to bloom in a greenhouse as described previously (Pusey, 1997). Newly opened flowers with non-dehisced anthers were collected and maintained by submerging the cut end of the pedicle in 10% sucrose contained in a 2-ml vial. Vials with flowers were supported in 4-liter chambers and, unless otherwise indicated, relative humidity (RH) was established at 90% by flooding the bottom of the containers with 1 L of a glycerol solution (Johnson, 1940). Field experiments were performed in 2000 and 2001 with ‘Gala’ apple trees in an experimental block at Columbia View near Wenatchee, Washington. Trees were 6-years old in the first year. Bacterial Strains and Inoculation Bacterial strains used were E. amylovora strain Ea153 (Johnson et al., 1993) and antagonists P. fluorescens strain A506 (Lindow et al., 1996) and P. agglomerans strains E325 (Pusey, 1997 and 1999) and C9-1 (Ishimaru, 1984; Johnson et al., 1993). Strain Ea153 was previously marked with nalidixic acid resistance and antagonist strains were marked with rifampicin resistance. Bacteria were cultured on nutrient yeast dextrose agar (NYDA; nutrient broth, 8 g; yeast, 5 g; dextrose, 5 g; agar, 15 g; and deionized water, 1 liter) for 24 h at 24° C , and inoculum suspensions were prepared in 10 mM potassium phosphate buffer (pH 7.0) and 0.03% Tween-20. Flowers were inoculated with micropipette by applying about 0.1 ul of the suspension (10, 10 or 10 cfu/ml) to the stigmatic surfaces of each flower. Population size of bacterial strains on flower stigmas was determined by placing the stigmas, along with portions of the supporting styles, in sterile microcentrifuge tubes containing 1 ml of sterile buffer (10 mM potassium phosphate, pH 7.0). Tubes were vortexed briefly and placed in a sonication bath for 60 s. Samples were again vortexed, and serial dilutions were spread on CCT medium (Ishimaru and Klos, 1984) amended with nalidixic acid (100 μg/ml) for detection of strain Ea153 or on NYDA amended with 25 ppm rifampicin and 50 ppm cycloheximide for detection of strains E325, C9-1 and A506. Plates were incubated at 24C. Fluctuating versus Constant Diurnal Temperatures To study the effects of fluctuating versus constant diurnal temperatures on microbial growth on the stigma, detached crab apple flowers were inoculated with Ea153 by applying a suspension of 10 cfu/ml to the stigmatic surfaces of each flower. Flowers were supported in open containers placed in four different controlled environment chambers, each programmed for a different diurnal temperature range (14° C constant, 10 to 18° C, 7 to 21° C or 4 to 24° C). In each chamber, the daily average temperature was 14° C. All chambers were set at a constant of 90% relative humidity. Ten flowers were sampled after 0, 24, 48, and 72 h to determine bacterial population size. Data from two trials were pooled. Temperature Range and Optimum for Microbial Growth To determine the temperature range and optimum for growth of bacterial strains on flower stigmas, stigmas of detached crab apple flowers were inoculated with strain Ea153, E325, C9-1 or A506 using a suspension of 10 cfu/ml. The estimated starting population size on each flower was about 10 cfu. Flowers were incubated for 24 h at temperatures ranging from 4 to 40° C and at increments of 4° C before determining bacterial population size. Five flowers were used per strain per temperature. Data from three trials were pooled.