JOBNAME: horts 43#5 2008 PAGE: 1 OUTPUT: June 19 20:24:03 2008 tsp/horts/167620/02855 MISCELLANEOUS HORTSCIENCE 43(5):1471–1478. 2008. pathogen from the stigma to the hypanthium where infection occurs through nectarthodes (Thomson, 1986). Erwinia amylovora ap- Characterization of Stigma Exudates pears to be well adapted epiphytically to the stigmatic surfaces of even nondisease– in Aqueous Extracts from Apple host rosaceous species (Johnson et al., 2006). As an alternative to the use of antibiotics for and Pear Flowers suppression of E. amylovora on apple and pear blossoms, researchers have evaluated P. Lawrence Pusey1,3, David R. Rudell2, Eric A. Curry2, and other naturally occurring microbial residents James P. Mattheis2 on flowers as potential biological control U.S. Department of Agriculture, Agricultural Research Service, Tree Fruit agents for fire blight (Johnson and Stockwell, 2000). Beneficial strains exhibiting antago- Research Laboratory, 1104 North Western Avenue, Wenatchee, WA 98801- nism toward E. amylovora suppress the 1230 pathogen by competing for space and nutri- Additional index words. Pyrus communis, Malus pumila, Malus mandshurica, Erwinia ents and producing antibiotic compounds. In 1996, Pseudomonas fluorescens (Trevison) amylovora, Rosaceae Migula strain A506 was the first microbial Abstract. The stigmatic secretions of pomaceous flowers serve as a natural medium not agent to become available commercially only for pollen, but also for the pathogen Erwinia amylovora (Burr.) Winslow et al. and for fire blight management (Johnson and other microorganisms. To understand the microecology on the stigma, exudates from Stockwell, 2000). Others have since been cultivars of pear (Pyrus communis L.), apple (Malus pumila P. Mill.), and crab apple developed, and in at least one case, farm [Malus mandshurica (Maxim.) Kom.] were analyzed for free sugars and free amino acids managers are given the option of delivering as available carbon and nitrogen sources. Extracts were obtained at different stages of the product to flowers using honeybees anthesis by submerging and sonicating stigmas in water. Certain free sugars (glucose and (Vanneste, 1996; Vanneste et al., 2002). fructose) and free amino acids (proline, asparagine, glutamic acid, and glutamine) were Knowledge of the biochemistry of consistently predominant and increased during anthesis. Apple stigma extracts were also stigma exudates is not only a prerequisite analyzed for polysaccharides and proteins. Of major components identified for apple, to understanding pollen–stigma interactions free sugars made up 4.5% by mass; polysaccharides (composed of arabinose and (Kandasamy and Vivekanandan, 1986; galactose), 49.6%; and proteins, 45.9%. The two largest components are likely present Knox, 1984), but it is necessary for under- as glycoproteins. This may be the first report on characteristics of rosaceous stigma standing microbial interactions that may be exudates that includes the identity of specific free sugars, free amino acids, and poly- advantageous for preventing fire blight of saccharide subcomponents. Discussion includes the comparison of pomaceous stigma apple and pear. Chemical analyses of the exudates to those of other plants and the microecological implications. pomaceous exudates provide basic informa- tion related to reproduction in an important plant group not yet given such attention and Given the importance of the stigma to Knox, 1984). Exudates produced by flower may reveal major nutrient sources supporting fruit development in agriculturally prominent stigmas of pome fruit trees are also an both disease-causing and beneficial micro- species in Rosaceae, there is surprisingly excellent medium for many microorganisms organisms on stigmas. The latter objective little information about the composition of (Pusey, 1997, 1999b; Stockwell et al., 1999). was the primary motivation for the current stigma exudates of plants in this group. Pomaceous stigmas and their exudates not study. Stigma exudates from apple and pear Although such data are extensive for a only sustain a wide diversity of bacteria, at different stages of anthesis were first number of other flowering plants (Knox, yeasts, and other fungi, but they also support analyzed for free sugars and free amino 1984), studies involving rosaceous species microbial population densities far exceeding acids that may serve as available carbon are limited to histological investigations in- those of most other aerial plant surfaces. In a and nitrogen sources for microbial activity. dicating the general presence of intercellu- review by Johnson and Stockwell (1998), this Further analyses were performed with apple lar carbohydrates, proteins, and lipids in point was illustrated by comparing maximum to identify other major components of the stigmatic tissue (Cresti et al., 1980; Stant, bacterial populations on bean leaves to those exudates. 1981; Uwate and Lin, 1981) and to the recent on pear stigmas. Pseudomonas syringae pv. focus on S-RNases (Certal et al., 1999; syringae is reported to attain populations Materials and Methods Ishimizu et al., 1996; McClure and Franklin- of 106 colony-forming units (cfu) per gram Tong, 2006; Sonneveld et al., 2005). The fresh weight of bean leaves (Beattie and Extraction procedure. Stigma exudates deficiency of information for tree fruit species Lindow, 1994), but the carrying capacity were collected from flowers of pear (Pyrus may be partly the result of the relatively small of bacteria on pear stigmas is estimated at communis L.) and apple (Malus pumila P. size of stigmas and brevity of the bloom 109 to 1010 cfu/g. Another unique aspect of Mill.) in 2003 and 2004. Tree cultivars were period. the stigma as a microbial habitat is its fre- ‘Anjou’ and ‘Bartlett’ pear and ‘Fuji,’ ‘Gala’, The stigma exudates of flowering plants quent contact with pollinating insects that and ‘Golden Delicious’ apple, all located in serve as a natural medium for pollen germi- carry microorganisms from flower to flower research orchards near Wenatchee, WA. nation and other functions in the pollen– (Johnson et al., 1993; Thomson et al., 1992; Blossoms were not protected from indige- stigma interaction (Heslop-Harrison, 2000; Vanneste, 1996). nous microorganisms or insect visitors dur- Microbial colonization on stigmas has ing the sampling period. Crab apple [Malus been given considerable attention in connec- mandshurica (Maxim.) Kom.], maintained tion with fire blight, a serious disease of in a greenhouse and used routinely as a Received for publication 25 Feb. 2008. Accepted apple, pear, and other rosaceous plants. The source of blossoms for laboratory assays for publication 3 Apr. 2008. stigma is the primary site supporting estab- (Pusey, 1997), was evaluated along with We thank Janet Duffy, Brenda Steady, and Dave lishment of the causal bacterium, Erwinia apple and pear cultivars in 2004. Flowers Buchanan for technical assistance and Van Well Nursery for providing trees. amylovora (Burr.) Winslow et al. (Johnson were sampled at three stages based on anther 1Research Plant Pathologist. and Stockwell, 1998). Typically, epiphytic dehiscence: nondehisced, 40% to 60% 2Research Plant Physiologist. populations increase on stigmas during warm dehisced, and 100% dehisced. For each 3To whom reprint requests should be addressed; periods but usually do not cause disease until cultivar and stage per year, a minimum of e-mail [email protected] rain or dew facilitates movement of the two groups of 50 flowers was sampled. Each HORTSCIENCE VOL. 43(5) AUGUST 2008 1471 JOBNAME: horts 43#5 2008 PAGE: 2 OUTPUT: June 19 20:24:04 2008 tsp/horts/167620/02855 Speed Vac Concentrator; Savant Instruments Inc., Holbrook, NY). Afterward, 5 mL2- dimethylaminoethanol and 125 mL methoxy- amine hydrochloride in pyridine (20 mgÁmL–1) were added to the dry extract, and the solution was heated at 85 °C for 1 h. After cooling to room temperature, 125 mL hexamethyldisilazane and 5 mL trifluoroace- tic acid were added. The sample solution was centrifuged for 5 min at 5900 gn, then the clear supernatant was withdrawn and trans- ferred to a GC vial. The analyses were performed with an Agilent Model 6890N GC (Agilent Technol- ogies, Palo Alto, CA) equipped with a flame ionization detector. One microliter of each sample was injected on a 2.5% phenyl methyl siloxane capillary column (25 m · 0.2 mm i.d., 0.33-mm film thickness; Agilent 19091B-102E). Hydrogen was used as the carrier gas at a flow rate of 0.5 mLÁmin–1. The injector temperature was held at 250 °Cin the split mode (split ratio 50:1). The column oven temperature started at 180 °C for 2 min, increased to 240 °Cat10°CÁmin–1, held at 240 °C for 3.5 min, increased to 270 °Cat 30 °CÁmin–1, and held at 270 °C for 12 min. Identification and quantification of sugars were based on retention times and peak areas, respectively, of reference sugars (Sigma- Aldrich Co., St. Louis, MO). Amino acid analysis. Amino acids were converted to derivatives with o-phthaldialde- hyde (OPA) and 9-fluorenylmethoxycar- bonyl chloride (FMOC) using the method of Herbert et al. (2000), with several minor modifications, before analysis by high-per- formance liquid chromatography (HPLC). The HPLC system consisted of model HP1090L (Hewlett Packard Co., Rancho Fig. 1. Free sugars detected in stigma exudates from ‘Anjou’ and ‘Bartlett’ pear (Pyrus communis); ‘Fuji’, Bernardo, CA) fitted with a HypersilAA ‘Gala’, and ‘Golden’ apple (Malus pumila) and ‘Manchurian’ crab apple (Malus mandshurica)in(A) column (Agilent 79916AA) and a tunable 2003 and (B) 2004. Each bar represents mean (±SE) of at least six samples, each derived from fluorescence detector (HP1046A; Hewlett 50 flowers. Packard Co.). For derivatization of primary amino acids, 5 mL of 0.4 N borate buffer (pH 10.2) 1 mL OPA reagent (Agilent p/n 5061– excised flower was temporarily maintained To substantiate how the free sugar com- 3337), and 1 mL of the extract were intro- with cut pedicel supported in a 2-mL vial position of stigma exudates compares with duced into the sample loop and allowed to filled with water. The flowers were trans- nectar sugar composition, which has already mix.