Potential of osmoadaptation for improving Pantoea agglomerans E325 as biocontrol agent for fire blight of apple and pear

Biological Control 62 (2012) 29–37

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Potential of osmoadaptation for improving Pantoea agglomerans E325 as biocontrol agent for fire blight of apple and pear P.L. Pusey a,⇑, C. Wend b a b

US Department of Agriculture, ARS, Tree Fruit Research Laboratory, Wenatchee, WA 98801, USA Northwest Agricultural Products, 821 S. Chestnut, Pasco, WA, USA

g r a p h i c a l a b s t r a c t

h i g h l i g h t s " Growth/survival on flowers not increased under dry field conditions (<50% avg RH). " Detached flower tests indicated advantage in hypanthium at critical RH (70%). " Increased stability of freeze dried E325 during storage prior to application. " Complemented effects of cryoprotectants added before freeze drying.

a r t i c l e

i n f o

Article history: Received 30 September 2011 Accepted 2 March 2012 Available online 12 March 2012 Keywords: Erwinia amylovora Malus domestica Trehalose Xanthan gum

a b s t r a c t Pantoea agglomerans biocontrol strain E325 is the active ingredient in a commercial product for fire blight, a destructive disease of apple and pear initiated by Erwinia amylovora in flowers. Osmoadaptation, involving the combination of saline osmotic stress and osmolyte amendment to growth media, was investigated to improve the epiphytic colonization by E325 on apple flowers, particularly in dry climates. E325 was osmoadapted in nutrient yeast dextrose broth and in the commercial fermentation medium, both amended with NaCl and glycine betaine according to previous research. The bacterium was cultured and freeze dried with cryoprotectants at an ARS-USDA laboratory (Wenatchee, WA) and a commercial facility (Pasco, WA) prior to treating apple flowers in an orchard where relative humidity (RH) averaged <50%. On orchard flowers and on detached crab apple flowers, osmoadaptation generally did not affect colonization of E325 on flower stigmas or nectar-rich hypanthia. The exception was the significant advantage of osmoadapted E325 on hypanthia of detached flowers at 70% RH, resulting in osmotic conditions marginally conducive for bacteria. Osmoadaptation proved most beneficial for increasing E325 survival during freeze drying and storage prior to orchard application. It also complemented cryoprotection, improving overall stability of freeze dried preparations of this biocontrol agent. Published by Elsevier Inc.

1. Introduction Fire blight, a destructive disease of apple and pear trees, is generally initiated by epiphytic populations of Erwinia amylovora that ⇑ Corresponding author. Fax: +1 509 664 2287. E-mail (P.L. Pusey).

addresses:

[email protected],

1049-9644/$ - see front matter Published by Elsevier Inc. http://dx.doi.org/10.1016/j.biocontrol.2012.03.002

[email protected]

become established on flower stigmas during warm weather (Vanneste, 2000). Rain or heavy dew facilitates the movement of bacterial cells from the stigma to the cup-shaped hypanthium, where infection occurs through natural openings from which nectar is secreted (Thomson, 1986). The disease has been managed in recent decades through risk assessment (Billing, 2000) and the application of antibiotics to suppress E. amylovora on floral parts; however, these agents are being phased out because of resistance

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in the pathogen and concerns related to human health (Psallidas and Tsiantos, 2000). Biological control with microbial antagonists is an alternative approach to suppressing E. amylovora on flowers (Johnson and Stockwell, 2000). In 1996 Pseudomonas fluorescens strain A506, initially selected for its pre-emptive exclusion of an ice-nucleating strain of Pseudomonas syringae on corn leaves (Lindow, 1985), was the first commercialized antagonist for fire blight management (BlightBan A506; Nufarm Americas Inc., Burr Ridge, IL). Other microorganisms have since been commercially developed for fire blight management, including the following Pantoea strains: Pantoea agglomerans P10c marketed as BlossomBless in New Zealand (Vanneste et al., 2002); Pantoea vagans C9-1 (syn. P. agglomerans C9-1) (Ishimaru et al., 1988; Smits et al., 2010) registered as BlightBan C9-1 in the USA and Canada (Johnson and Stockwell, 2000; Pusey et al., 2008b); and P. agglomerans E325 marketed as Bloomtime FD in the USA and Canada (Pusey et al., 2008b). P. agglomerans strain E325 was selected from more than 1000 isolates of bacteria and yeasts evaluated for their capacity to suppress E. amylovora on the stigmatic surfaces of crab apple flowers (Pusey, 1997; Pusey et al., 2009). Like other microbial strains studied (Johnson and Stockwell, 2000), E325 colonizes stigmas (Pusey, 2002) and likely competes with the pathogen for space and nutrients. Antibiosis, as another mechanism was not detected consistently in vitro until assays were done with partial stigma-based medium (PSBM) low in phosphate and consisting of nutrients identified in stigma exudates from apple and pear flowers (Pusey et al., 2008a,b). The E325 antibiotic exhibited characteristics unique from other Pantoea antibiotics (Pusey et al., 2008b) and was shown to have a role in biological control on apple flower stigmas (Pusey et al., 2011). Plant surfaces exposed to rapidly fluctuating temperature and relative humidity conditions are generally considered as hostile environments for bacterial colonists (Lindow and Brandl, 2003). The flower stigma, however, is unusual as a habitat because aqueous secretions support microbial colonization. It is unknown whether conditions in arid fruit-growing regions lead to stigma solute concentrations high enough to cause osmotic stress in bacteria. Present knowledge of water relations in the flower hypanthium as a microbial habitat exceeds what we know about stigmas, probably because nectar quantities in the hypanthium are more visible and measurable. Under low relative humidity, sugar levels in apple nectar commonly range from 35% to 55% (Campbell et al., 1990; Ivanoff and Keitt, 1941). In this high-sugar environment, E. amylovora grows minimally or declines over time (Hildebrand and Phillips, 1936; Thomas and Ark, 1934); as nectar is diluted by water, the pathogen multiplies and the likelihood of infection increases dramatically (Ivanoff and Keitt, 1941). On detached flowers held under controlled environment conditions (Pusey, 2000), population size of E. amylovora was closely related to relative humidity and water potential of the nectar. Experiments with synthetic nectar (Pusey, 1999) revealed that strains of E. amylovora were more tolerant of osmotic conditions in the concentrated sugar solutions than strains identified as species of Pantoea, Bacillus, and Pseudomonas. To improve the survival of P. agglomerans E325 on floral surfaces under typical dry orchard conditions in the western USA, we investigated the process known as osmoadaptation (Galinski, 1995; Sleator and Hill, 2001) involving the combination of saline osmotic stress and osmolyte amendment to growth media. This approach increased the tolerance of P. agglomerans EPS125 to desiccation on apple fruit surfaces and enhanced its biocontrol efficacy against postharvest fungal decay (Bonaterra et al., 2005). In a subsequent study (Bonaterra et al., 2007), osmoadapted cells of P. fluorescens EPS62e, an antagonist selected for fire blight control,

survived better on apple leaves than non-osmoadapted cells. However, such differences were not demonstrated with detached flowers held at 50% relative humidity (RH) in the laboratory or with flowers in the orchard where average RH was between 70% and 80%. In the study with P. fluorescens (Bonaterra et al., 2007), whole flowers were treated and sampled, and so bacterial population sizes on specific floral parts were not examined. In the present study, osmoadaptation was induced in P. agglomerans E325 using the methods of Bonaterra et al. (2007) and evaluated for its effect on the population size of E325 on flower stigmas and hypanthia. This was done first in an apple orchard and later in the laboratory with detached crab apple flowers held under controlled RH conditions. Early results with flowers showing differences in population size immediately after treatment led us to also examine the effects of osmoadaptation on E325 survival during freeze drying and storage prior to the orchard applications.

2. Materials and methods 2.1. Culture and preparation of bacterial cells P. agglomerans strain E325 used in this study is a spontaneous rifampicin- and streptomycin-resistant mutant of the wild-type strain isolated from ‘Gala’ apple (Pusey, 1997) and the active ingredient in Bloomtime FD, a product of Northwest Agricultural Products (NAP) in Pasco, WA. E325 was cultured on nutrient yeast dextrose agar (NYDA; 8 g nutrient broth, 5 g yeast extract, 5 g dextrose, and 15 g agar in 1 l of deionized water) at 24 °C for 24 h prior to starting cultures in broth media amended or not amended (control) with 0.5 M NaCl and 0.1 mM glycine betaine for osmoadaptation according to Bonaterra et al. (2005, 2007). Instead of using a defined medium as described by previous researchers, E325 was cultured in complex media to simulate commercial production. Nutrient yeast dextrose broth (NYDB; same as NYDA but without agar) was used in all field and laboratory experiments. In addition to NYDB, field trials involved the commercial production medium (CPM) of NAP (ingredients not disclosed). At the Wenatchee USDA-ARS laboratory, E325 was grown in NYDB or CPM, with and without amendments, in 4 ml of broth per 40-ml tube, and incubated on a rotary shaker at 125 rpm and 24 or 26 °C for 24 h. At NAP, E325 was grown in CPM, with and without amendments, in a commercial fermenter at 30 °C for about 15 h. Pre-cultures were prepared at both facilities with the same broth medium used in the experiment. At the USDA laboratory, the pre-culture was started as follows. A suspension of E325 cells from a 24-h NYDA culture was suspended in 0.1 M phosphate buffer (pH 7) and adjusted to 0.1 optical density at 600 nm with a spectrophotometer (Spectronic 20; Milton Roy Co., Rochester, NY) giving a cell concentration of approximately 108 CFU/ml. The suspension was diluted to 4  107 CFU/ml, and 10 ll was transferred to 4 ml of liquid medium to obtain a starting level of 105 CFU/ml in each pre-culture tube. After 24-h incubation, concentration of cells from the preculture was adjusted in buffer as before to obtain a starting level of 105 CFU/ml in each new culture tube. After another 24 h, cultures in separate tubes were combined and centrifuged at 6000 rpm for 10 min. Pelleted cells were combined with cryoprotectants xanthan gum and milk (Stockwell et al., 1998) or trehalose (Samuel et al., 1995). To every 1 mg of wet pelleted cells, approximately 3 ll of 0.7% xanthan gum and 12.7% powdered milk, or 3 ll of 5% trehalose, was added. The mixtures were vortexed, held at 80 °C for 1 h, and then lyophilized in a Virtis Unitrap II freeze dryer (Gardiner, NY) for a minimum of 4 h. Preparations were generally stored at 20 °C prior to experiments.

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2.2. Orchard experiments Field trials were conducted with ‘Gala’ apple trees in research orchards shared by Washington State University (WSU) and US Department of Agriculture at Columbia View (CV) north of Wenatchee, WA and at the WSU, Tree Fruit Research and Extension Center (TFREC) located in Wenatchee 17 miles from CV. Prior to treatment branches were tagged for use, and older flowers (>50% of anthers dehisced) and all unopened flowers or buds were removed to ensure that only newly-opened flowers were treated in each experiment. Treatment preparations of P. agglomerans E325 were from cultures grown in NYDB or CM, and amended or not amended with NaCl and betaine glycine, as already described. They were also derived from cultures grown in CM at the commercial NAP facility, as well as the USDA laboratory. All bacterial treatments involved cells that were freeze-dried with cryoprotectants (xanthan gum and milk were used at Wenatchee) and stored at 20 °C, except for one treatment with cells from fresh cultures grown in nonamended NYDB. Suspensions of E325 were made to 107 CFU/ml in 10 mM phosphate buffer (pH 7) and 0.3% Tween-20. The concentration of fresh-cultured E325 cells was adjusted using a spectrophotometer as already described, and the concentration of E325 cells from freeze-dried preparations was based on dilution plating of samples 1–4 days prior to field application. In the years 2009 and 2010, treatments were applied specifically to the stigmas of apple flowers using an artist brush (2009) or sterile swab (2010). Flowers were sampled after 0, 1, 2, 4, 6, and 8 days. The ‘‘0-day’’ sample was collected at between 2 and 4 h after treatment. To estimate population size, stigmas and styles of individual flowers were placed in sterile microcentrifuge tubes containing 1 ml of sterile buffer (10 mM potassium phosphate, pH 7). Tubes were vortexed briefly, placed in sonication bath for 60 s, again vortexed, and serial dilutions spread on trypticase soy agar (TSA; 15 g of tryptone, 5 g of soytone (Difco Laboratories, Detroit), 5 g of NaCl, and 15 g agar in 1 l of deionized water) amended with rifampicin (25 lg ml/ml) and cycloheximide (50 lg/ml). Six single-tree replicates were used per treatment in a completely randomized block, and five flowers were collected per tree on each sampling date. The experiment was performed from 28 April to 6 May in 2009 and from 19 April to 27 April in 2010. In 2011, the above treatments were targeted to the flower hypanthia rather than stigmas (with the exclusion of those involving preparations generated at NAP) by pipetting 10 ll of the E325 suspension (107 CFU/ml) into each flower hypanthium. The experiment was performed twice in 2011, i.e., from 2 May to 10 May at CV and from 7 May to 15 May at TFREC. To estimate population size, the hypanthium was isolated by removing other flower parts prior to the buffer wash and dilution plating. Experimental design and other procedures matched those described for the 2009 and 2010 trials. 2.3. Detached flower experiments To better interpret field results, experiments were performed under controlled conditions with detached flowers of Manchurian crab apple (Malus mandshurica (Maxim.) Kom.) as previously described (Pusey, 1997). The cut end of the flower pedicle was submerged in 10% sucrose contained in a 2-ml vial, and vials were supported in tube racks enclosed in a 4-l container. RH was maintained at 50%, 70%, or 90% by flooding the bottom of the containers with 1 l of a glycerol solution (Johnson, 1940) and temperature was held at 18 °C. The newly-opened crab apple flowers were treated with preparations of E325 cells osmoadapted or non-osmoadapted in NYDB. Freeze-dried cells were suspended in phosphate buffer at

107 CFU/ml as already described and applied with a pipet, 0.1– 0.2 ll per flower on stigmas or 1 ll per flower in the hypanthium. On each sampling date (0, 1, 2, 3, 4, and 6 days), five flowers were collected per treatment and flower part, and population size of E325 estimated as described for field experiments. The experiment was performed twice at each RH. 2.4. Stability of biocontrol preparations Field results in 2009 prompted investigation of the effect of osmoadaptation on E325 during and after freeze drying. Viable levels of E325 were estimated in cultures before freeze drying and periodically afterwards in samples held at 22 or 20 °C. In the stability test at 22 °C, cultures of E325 grown in NYDB were combined and redistributed into microfuge tubes, 0.6 ml into each 1.7-ml tube. E325 cells were maintained in the same microfuge tube for centrifugation, addition of cryoprotectant, freeze drying, and storage in a vacuum chamber at 22 °C. Sample tubes were prepared in four ways prior to freeze drying: (1) non-osmoadapted without cryoprotectant, (2) osmoadapted with cryoprotectant, (3) osmoadapted with xanthan gum and milk, and (4) osmoadapted with trehalose. On each sampling date (0, 1, 3, 7, 14, 28, 56, and 112 days after freeze drying), E325 cells in each of three tubes were resuspended with 0.6 ml of 10 mM potassium phosphate buffer (pH 7) and viable levels estimated by dilution plating on TSA. The 0-day samples were usually plated within one hour after freeze drying. The test was performed three times. Our experiment at 20 °C was set up like the previous test. In addition, non-osmoadapted cultures were freeze dried with trehalose, or with xanthan gum and milk. The test was performed three times, sampling at 0, 1, 2, 3, 5, 7, 10, and 14 days. 2.5. Data analysis Population data was transformed to log10 prior to analysis. For orchard tests, population sizes on flowers of each tree were averaged before calculating the mean and standard error for replicate trees. For laboratory experiments, data from separate trials were pooled and mean values and standard errors were calculated by averaging values for single flowers sampled on one date. The relative area under the population curve (RAUPC) of bacteria was calculated for each replicate tree or flower set, using the following formula (Shaner and Finney, 1977; Stockwell et al., 2002): n   P yi þyi1

RAUPC ¼ i¼1

2

 ðt i  t i1 Þ

tn  ti

where y is the mean population size of a bacterial strain on the ith sample date and t is the corresponding sample time. Data were subjected to analysis of variance using SAS version 9.2 (SAS Institute, Cary, NC) and mean values were separated according to least significant difference test (P = 0.05). In some experiments the size of starting populations of E325 on flowers varied significantly, possibly due to different viable levels in preparations prior to treatment. To compare changes in population sizes of osmoadapted and non-osmoadapted E325 cells on floral surfaces after the day of treatment, values for mean population size were adjusted to offset the initial variability. Thus, population size immediately after treatment (0 days) was set at zero. With y as mean population size, all values used in place of mean population size on later sampling days were equal to yi  y0. The adjusted RAUPC (A-RAUPC) was calculated by replacing yi and yi1 in the above equation with (yi  y0) and (yi1  y0), respectively. Population values used in calculating A-RAUPC were plotted only for the first field experiment (Fig. 2) to illustrate how A-RAUPC

P.L. Pusey, C. Wend / Biological Control 62 (2012) 29–37

Rain (mm) RH (%)

30 20

A

B

2009

2010

6

10 100

A

4

4

C

D

Fresh NYDB NYDB+S

50 0 1 0 30 20

F

E 0 1

H

2 3

4 5 CV

6

7 8 0 1

2 3

I

4 5 6 TFREC

7 8

10 100

J

K

0 6

C

D

2

2

6

0 4

4

0

B

2

2

Log CFU per flower

Temp. (oC)

Rain (mm) RH (%)

Temp. (oC)

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CPM-lab CPM-lab+S

E

0 4

F

50 0 10 0

4

L 0 1

M

T 2 3 4

5

6 7

8 0 1 Days

2

T 2 3

4 5

2

6

CPM-cf CPM-cf+S

7 8

Fig. 1. Environmental conditions in apple orchards in Central Washington State used for comparing osmoadapted and non-osmoadapted preparations of Pantoea agglomerans E325. Data are for Columbia View (CV) in 2009 (A, C, and E), 2010 (B, D, and F), and 2011 (H, J, and L), and for Tree Fruit Research and Extension Center (TFREC) in 2011 (I, K, and M). Temperature (A, B, H, and I) and relative humidity (C, D, J, and K) data are presented at hourly intervals (thin line) and as daily averages (bold). Rainfall (E, F, L, and M) is presented as daily totals; T = trace precipitation.

alters the treatment curves. For later experiments, only the mean values of A-RAUPC and statistical data are presented.

3. Results

0

0

2

4 Days

6

0

8

0

2

4 Days

6

8

Fig. 2. Growth of osmoadapted and non-osmoadapted P. agglomerans E325 on stigmas of apple flowers in orchard in 2009. E325 was cultured in nutrient yeast dextrose broth (NYDB) or commercial production medium (CPM) at USDA-ARS laboratory (lab), or in CPM in commercial fermentor (cf). E325 was osmoadapted by amending the media with NaCl salt (+S). One treatment involved non-osmoadapted cells from fresh NYDB cultures (fresh); all others involved freeze dried preparations. Data are presented as mean population size for preparations from cultures in NYDB (A) or CPM (C and E); adjusted population values in corresponding panels at right (B, D, and F), with populations set at zero on 0 day, are used to offset variation at time of treatment, allowing comparisons of subsequent bacterial fitness. (Adjusted data presented here for illustration were used to calculate A-RAUPC in other flower experiments.) Each point is the mean of six single-tree replicates, using the average of five flowers per tree, and vertical lines represent standard error.

3.1. Orchard experiments Field tests performed during apple bloom in years from 2009 to 2011 met dry conditions typical for the area (Fig. 1). Overall average temperature at the CV location in 2009, 2010, and 2011 was 10.8, 13.6, and 12.6 °C , respectively; average RH was 54%, 42%, and 45%, respectively (daily RH minimum averaged 25%, 23%, and 25%, respectively); and total rainfall amounted to only 1.5, 3.0, and 0.3 mm, respectively. Average temperature and RH at TFREC in 2011 was 13.4 °C and 58%, respectively (daily RH minimum averaged 38%). During the first 7 days of the experiment at TFREC, RH averaged 50% and rainfall totaled 2.8 mm; however, during the last two days, RH averaged 86% and total rainfall totaled 28.7 mm. In 2009, treatment of apple flower stigmas with osmoadapted E325 resulted in larger (P = 0.05) population sizes of the antagonist than treatment with non-osmoadapted E325 (Fig. 2A, C, and E) based on a comparison of RAUPC mean values (Table 1). However, when values for population size and RAUPC were adjusted (A-RAUPC) to offset the variation soon after treatment, no differences in treatments were indicated (Fig. 2B, D, and F; Table 1). Also, results with non-osmoadapted fresh-cultured cells and osmoadapted freeze-dried cells, both produced in NYDB, were not different (Fig. 2A, Table 1). In 2010, concentrations of viable E325 in freeze-dried preparations were established by dilution plating closer to the day of application (1 day versus 4 days in 2009). Consequently, the starting population levels on flowers were similar (Fig. 3), and no differences were indicated by RAUPC or A-RAUPC comparisons (Table 1).

Table 1 Relative area under population curve (RAUPC) or adjusted RAUPC (A-RAUPC) of P. agglomerans E325 on stigmas of ‘Gala’ apple flowers in orchard treated with cells osmoadapted in high-saline medium.x Facilityy

Mediumy

USDA

NYDB (Fresh) NYDB NYDB + S CPM CPM + S CPM CPM + S

USDA NAP

2009z

2010z

RAUPC

A-RAUPC

RAUPC

A-RAUPC

4.9a 2.9b 4.8a 3.0b 4.5a 3.1b 4.8a

2.0a 2.0a 2.3a 2.3a 2.5a 2.5a 1.8a

6.0a 5.5a 5.5a 5.5a 5.2a 5.7a 5.6a

3.6a 3.4a 3.5a 3.9a 4.3a 3.6a 3.4a

x Trials were performed in orchard with six single-tree replications. New flowers were treated on stigmas with E325 at 107 CFU/ml using brush or swab, and sampled periodically to estimate population size. y Liquid complex media used were nutrient yeast dextrose broth (NYDB) and commercial production medium (CPM) of Northwest Agricultural Products (NAP). One treatment suspension was prepared from fresh cultures; all others were from freeze-dried preparations. E325 was cultured in CPM at USDA-ARS laboratory, Wenatchee, WA, or in CPM at NAP, Pasco, WA. Media were amended with NaCl salt (+S) to induce osmoadaptation or not amended. z RAUPC and A-RAUPC were calculated for all sample dates as described in Section 2. The latter equation was used to offset variation at time of treatment due to differences in preparation stability, allowing comparative evaluation of bacterial fitness after the day of treatment. Means within column followed by the same letter are not different according to least significant difference test (P = 0.05).

In 2011, E325 population sizes on the hypanthia of flowers were shown to decrease soon after treatment and then later increase,

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6

A

6

4

B

4 CPM-lab CPM-lab+S

2 0 6

C

Log CFU per flower

Log CFU per flower

6

B

A

2 0

Fresh NYDB NYDB+S

C

D

4 2 CPM CPM+S

0 0

2

4

6

8

0

2

4

6

8

Days

4 CPM-cf CPM-cf+S

2 0

TFREC

4 Fresh NYDB NYDB+S

2 0

CV

0

2

4

6

8

Days Fig. 3. Growth of osmoadapted and non-osmoadapted P. agglomerans E325 on stigmas of apple flowers in orchard in 2010. E325 was cultured in nutrient yeast dextrose broth (NYDB) or commercial production medium (CPM) at USDA-ARS laboratory (lab), or in CPM in commercial fermentor (cf). E325 was osmoadapted by amending the media with NaCl salt (+S). One treatment involved non-osmoadapted cells from fresh NYDB cultures (fresh); all others involved freeze dried preparations. Data are presented as mean population size for preparations from cultures in NYDB (A) or CPM (B and C). Each point is the mean of six single-tree replicates, using the average of five flowers per tree, and vertical lines represent standard error.

but usually stay below initial levels (Fig. 4) as reflected by negative mean values for A-RAUPC (Table 2). Osmoadapted and non-osmoadapted preparations differed (P = 0.05) in cases involving NYDB at CV and CPM at TFREC (Fig. 4A and D) based on a comparison of RAUPC mean values (Table 2), though differences were not indicated by A-RAUPC comparisons. Cells osmoadapted in NYDB and freeze-dried had slightly higher (P = 0.05) A-RAUPC mean values than non-osmoadapted cells from fresh NYDB cultures (Table 2). Population increases in the hypanthium on the last sampling day at TFREC (Fig. 4B and D) corresponded to the occurrence of rain and elevated RH (Fig. 1K and M); adjusted mean population size on this date was higher (P = 0.05) for osmoadapted than for nonosmoadapted cells from CPM (Fig. 4D), but not for cells from NYDB (Fig. 4B). 3.2. Detached flower experiments When E325 preparations freeze dried with xanthan gum and milk were applied to detached flowers held under different RH conditions, osmoadapted and non-osmoadapted cells grew at similar rates on stigmas regardless of RH (Fig. 5A, C, and E). RAUPC mean values were not different at 50 or 90% RH, but were shown to be slightly different (P = 0.05) at 70% RH (Table 3); however, no differences were indicated according to A-RAUPC comparisons. On the hypanthia of flowers held at 50% RH, E325 population size generally declined over the 6-day sampling period (Fig. 5F); in contrast, at 90% RH (Fig. 5D), bacterial populations on hypanthia increased by 2 log units over 3 days to a stationary phase. Under both the low and high RH conditions, osmoadaptation showed slight or no effect on hypanthial populations (Table 3). At the

Fig. 4. Comparison of osmoadapted and non-osmoadapted population levels of P. agglomerans E325 on hypanthia of apple flowers in orchard in 2011. E325 was osmoadapted in nutrient yeast dextrose broth (NYDB) or commercial production medium (CPM) at USDA-ARS laboratory by amending the media with NaCl salt (+S). One treatment involved non-osmoadapted cells from fresh NYDB cultures (fresh); all others involved freeze dried preparations. Data are presented as mean population size for treatment preparations from NYDB (A and B) and CPM (C and D) applied at CV (A and C) and TFREC (B and D) orchards. Each point is the mean of six single-tree replicates, using the average of five flowers per tree, and vertical lines represent standard error.

Table 2 Relative area under population curve (RAUPC) and adjusted RAUPC (A-RAUPC) of Pantoea agglomerans E325 on hypanthia of ‘Gala’ apple flowers at two orchard locations treated with cells osmoadapted in high-saline medium in 2011.x Mediumy

NYDB (fresh) NYDB NYDB + S CPM CPM + S

CVz

TFRECz

RAUPC

A-RAUPC

RAUPC

A-RAUPC

3.7a 2.3b 3.9a 3.3a 3.8a

0.7a 0.7a 0.9a 0.8a 0.5a

3.3a 3.2a 3.6a 1.4b 2.9a

1.3b 1.1ab 0.5a 1.4a 0.9a

x Trials were performed at USDA-ARS, Columbia View (CV) experimental orchards and Washington State University, Tree Fruit Research and Extension Center (TFREC) with six single-tree replications. Flowers were each treated on hypanthium with 10 ll of E325 suspension at 107 CFU/ml using pipet, and sampled periodically to estimate population size. y Liquid complex media used were nutrient yeast dextrose broth (NYDB) and commercial production medium (CPM) of Northwest Agricultural Products. One treatment suspension was from fresh cultures; all others were from freeze-dried preparations. E325 was cultured and freeze dried at USDA-ARS laboratory. Media were amended with NaCl (+S) to induce osmoadaptation or not amended. z RAUPC and A-RAUPC were calculated for all sample dates as described in Materials and Methods. The latter equation was used to offset variation at time of treatment due to differences in preparation stability, allowing comparative evaluation of bacterial fitness after the day of treatment. Means within column followed by the same letter are not different according to least significant difference test (P = 0.05).

intermediate RH of 70%, osmoadapted and non-osmoadapted E325 populations decreased by about 2 log units during the first 24 h (Fig. 5D), then increased after 3 days. At 6 days, the osmoadapted population size had increased by more than one log unit beyond the starting population; whereas, the non-osmoadapted population size had decreased by about 1.7 log units compared to the starting population. Two trials at 70% RH yielded similar results, and pooled data indicated differences (P = 0.05) based on both RAUPC and A-RAUPC comparisons (Table 3).

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Stigma 6

A

B 90% RH

4

Log CFU per flower

2 0 6

Hypanthium

90% RH

NYDB NYDB+S

C

D

4

70% RH

70% RH

2 0 6

E

F 50% RH

4 50% RH 2 0 0

1

2

3

4

5

6 0 Days

1

2

3

4

5

6

Fig. 5. Comparison of osmoadapted and non-osmoadapted population levels of P. agglomerans E325 on stigmas (left panels) and hypanthia (right panels) of detached crab apple flowers held at 18 °C and relative humidity of 90% (A and B), 70% (C and D), and 50% (E and F). E325 was osmoadapted in nutrient yeast dextrose broth (NYDB) by amending the medium with NaCl salt (+S). Each point is the mean of 10 flowers and vertical lines represent standard error.

Table 3 Relative area under population curve (RAUPC) and adjusted RAUPC (A-RAUPC) of Pantoea agglomerans E325 on crab apple flowers treated with cells osmoadapted in high-saline medium and incubated under different relative humidity conditions.y Tissue

NaCl

Relative humidityz 50%

Stigma Hypanthium

 +  +

70%

90%

RAUPC

ARAUPC

RAUPC

ARAUPC

RAUPC

ARAUPC

4.60a 4.71a 2.45a 2.80a

2.43a 2.20a 0.74a 0.54a

4.71b 5.01a 1.53b 2.84a

2.84a 2.79a 1.83b 0.36a

5.20a 5.42a 4.87a 4.86a

3.94a 3.53a 1.63a 1.19b

y E325 was cultured in nutrient yeast dextrose broth with or without additional NaCl to induce osmoadaptation, then freeze dried. About 0.1–0.2 ll of E325 suspension (107 CFU/ml) was applied per flower to stigmas, and 1 ll of the suspension was applied to the hypanthium. Flowers were incubated at 18 °C and sampled periodically to estimate population size. z RAUPC and A-RAUPC were calculated as described in Section 2. The latter equation was used to offset variation at time of treatment due to differences in preparation stability, allowing comparative evaluation of bacterial fitness after the day of treatment. Means within column for each tissue type followed by the same letter are not different according to least significant difference test (P = 0.05).

3.3. Stability of biocontrol preparations The population sizes of osmoadapted and non-osmoadapted E325 in broth cultures prior to freeze drying did not differ (P = 0.05), with mean log values of 10.43 ± 0.1 and 10.38 ± 0.1 CFU/ml, respectively, for cultures used in stability tests at 22 °C; values were 10.3 ± 0.1 and 10.5 ± 0.1 CFU/ml, respectively, for cultures used in tests at 20 °C. Immediately after freeze drying, re-suspension in the original aqueous volume always resulted in

lower (P = 0.05) viable levels, with the exception of the non-osmoadapted preparation freeze dried with trehalose and held at 20 °C (Table 4). Preparations that were neither osmoadapted or cryoprotected in the first stability test (22 °C) were most dramatically affected, with the mean viable level 2.4 log units lower than in the culture and1.6 log units lower (P = 0.05) compared to the osmoadapted non-cryoprotected preparation (Table 4). Not surprisingly, the second stability test showed that bacteria were least affected by freeze drying when cryoprotectants were present. At 22 °C, levels of viable non-osmoadapted and non-cryoprotected E325 continued a rapid decline during the first 2 or 3 weeks after freeze drying, stabilizing by 8 weeks at about 103 CFU/ml of original culture medium (Fig. 6). Osmoadapted population levels declined more gradually to 105 CFU/ml by 8 weeks, 104 CFU/ml by 16 weeks. Osmoadaption combined with cryoprotection resulted in greater survival than osmoadaptation alone (Fig. 6). After sixteen weeks at 22 °C, levels of osmoadapted E325 prepared with xanthan gum and milk were at about 107 CFU/ml, and those prepared with trehalose were at 109 CFU/ml. RAUPC mean values differed (P = 0.05) among all E325 preparations (Table 4). At 20 °C, non-osmoadapted and non-cryoprotected E325 levels declined significantly during the first sampling week (Fig. 7A) and appeared to stabilize after 10 days at about 108 CFU/ml. In contrast, osmoadapted levels declined similarly but stabilized near 109 CFU/ml. In the presence of xanthan gum and milk, non-osmoadapted E325 stabilized after 1 week at about log 9.0 CFU/ml (Fig. 7B), and osmoadaped E325 stabilized after the same period at around log 9.6 CFU/ml. For preparations without cryoprotectants, or that include the cryoprotectants xanthan gum and milk, osmoadapted cells survived better (P = 0.05) than non-osmoadapted cells based on RAUPC comparisons (Table 4). In preparations with trehalose as a cryoprotectant, osmoadapted and non-osmoadapted E325 stabilized at around log 9.8 and log 9.6 CFU/ml, respectively (Fig. 7C), and RAUPC mean values were not different (Table 4). 4. Discussion Osmoadaptation was induced in the biocontrol strain P. agglomerans E325 to improve colonization on apple floral surfaces under dry weather conditions. Orchard experiments yielded disappointing results, leading to confirmatory tests with detached flowers held under controlled environment conditions and to evaluations of E325 survival in treatment preparations. The detached-flower experiments revealed that osmoadapted cells of E325 may colonize the hypanthium better than non-osmoadapted cells under specific RH conditions. Also, osmoadaption was shown to boost E325 survival during freeze drying and storage prior to its use on flowers. In research by Bonaterra et al. (2005, 2007), 0.5 M NaCl and 0.1 mM glycine betaine proved optimal in glucose minimal medium for osmoadaptation. Consistent with these previous studies, preliminary tests with strain E325 (unpublished data) indicated that 0.5 M is near the maximum NaCl level that does not impede growth in liquid media; thus, this concentration was adopted for the present study. The addition of glycine betaine, along with NaCl, to complex media NYDB and CPM did not alter E325 growth, possibly because glycine betaine is already present in yeast or beef extract components in these media (Robert et al., 2000). Nevertheless, since 0.1 mM glycine betaine did not show a negative effect on E325, it was added to the media. In preparation for the 2009 orchard trial, E325 was cultured and freeze dried with xanthan gum and milk, previously effective as cryoprotectants of the antagonists P. fluorescens A506 and P. agglomerans C9-1 (Johnson et al., 1993; Stockwell et al., 1998). Results indicated a wide separation between population sizes of osmoadapted and non-osmoadapted E325 on flower stigmas

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P.L. Pusey, C. Wend / Biological Control 62 (2012) 29–37

Table 4 Effect of combined osmoadaptation and cryoprotection on the survival of Pantoea agglomerans E325 based on bacterial levels before and after freeze drying and on relative area under population curve (RAUPC). Treatmentx

NaCl

Control S XM S + XM T S+T

Cryoprotectant

 +  +  +

  + + + +

22 °C

20 °C

Post-FD (CFU/ml)y

RAUPCz

Post-FD (CFU/ml)y

RAUPCz

8.0c 9.6b ND 9.9ab ND 10.1a

3.8d 5.2c ND 7.9b ND 9.2a

9.3c 9.2c 10.0b 10.1b 10.2ab 10.0b

8.4c 9.0b 9.1b 9.8a 9.8a 9.8a

x E325 was cultured in nutrient yeast dextrose broth (NYDB) with or without additional NaCl salt (S) to induce osmoadaption prior to freeze drying with or without cryoprotectants xanthan gum and milk (XM) or trehalose (T) and stored in vacuum at 22 °C for 112 days or 20 °C in sealed tube for 14 days. Samples were collected periodically to estimate viable E325 levels. y Dry preparations were plated immediately after freeze drying (FD) to determine CFU/ml in same aqueous volume as pre-FD culture, which grew to approximately log 10.4 CFU/ml. Population levels in all post-FD preparations were lower (P = 0.05) than those in pre-FD culture, except non-osmoadapted trehalose-cryoprotected preparation in the 20 °C stability test. z RAUPC was calculated as described in Section 2. ND = no data. Means within column are not different according to least significant difference test (P = 0.05).

A

Log CFU/ ml

10 8

NS S

10

6 4

9

NS S S+t S+xm

2 0 0

8

20 40 60 80 100

B

Fig. 6. Stability of osmoadapted P. agglomerans E325 freeze dried with or without cryoprotectants and held in vacuum chamber at 22 °C. E325 was cultured in nutrient yeast dextrose broth amended with NaCl salt (S) to induce osmoadaptation or not amended (NS). Two osmoadapted preparations were cryoprotected by the addition of trehalose (+t) or xanthan gum and milk (+xm) prior to freeze drying. Each point is the mean of nine preparation samples, each stored in separate microfuge tube, and vertical lines represent standard error.

2–4 h after treatment (Fig. 2). Treatment suspensions were plated at the time of application to confirm concentrations of approximately 107 CFU/ml, based on viable levels in freeze dried preparations 4 days earlier. Treatment concentrations ranged from log 6.7 to log 7.6 CFU/ml, with the lowest levels indicated for the two non-osmoadapted E325 suspensions from CPM cultures (log 6.7 and 6.8 CFU/ml). This explained, at least in part, the variation in population size of E325 soon after treatment, and deflected attention to the effects of osmoadaptation during freeze drying and storage, which will be discussed later. After the initial sampling day in the 2009 field experiment, populations of osmoadapted and non-osmoadapted E325 appeared to increase in size at similar rates. For this reason the A-RAUPC equation was devised to offset the variation in starting population size, allowing for comparative analysis after the day of treatment. Comparison of A-RAUPC mean values indicated no differences between the growth of osmoadapted and non-osmoadapted E325 on flower stigmas, as later confirmed in 2010. Although variation in the starting population size was reduced by estimating CFU per gram in preparations closer to the time of treatment, the A-RAUPC equation remained useful for circumventing the dissimilar stability among E325 preparations. Bonaterra et al. (2005, 2007) found that osmoadapted bacterial cells survived better than non-osmoadapted cells on fruit and leaf surfaces in controlled environments at low RH (45–50%) but not high RH (90%). Under field conditions (Bonaterra et al., 2007), population levels of osmoadapted and non-osmoadapted P. fluorescens EPS62e on apple and pear flowers were not different, presumably due to favorable RH averaging between 70% and 80%. In the current

Log CFU/ml

Days 10 9 8

C 10 9 8 0

2

4

6 8 10 12 14 Days

Fig. 7. Effect of osmoadaptation and cryoprotection, combined and separate, on the stability of P. agglomerans E325 preparations freeze dried and held at 21 °C. E325 was cultured in nutrient yeast dextrose broth amended with NaCl salt (S) to induce osmoadaptation or not amended (NS). The osmoadapted and non-osmoadapted preparations were freeze dried with no cryoprotectant (A) or cryoprotected with xanthan gum and milk (B) or trehalose (C). Each point is the mean of nine preparation samples, each stored in separate microfuge tube, and vertical lines represent standard error.

study with P. agglomerans E325 conducted in an arid fruit production region, RH during bloom experiments in three different years averaged <50%, with the daily average minimum at <30%. Even under these relatively low RH levels in the orchard, and constant 50% RH in the laboratory, population size of osmoadapted and nonosmoadapted E325 increased similarly on flower stigmas. The field population data in relation to RH must be interpreted with caution because wide diurnal fluctuations in RH corresponded to variations in temperature, which also affect bacterial cell multiplication and longevity. RH sometimes exceeded 90%, a level highly

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P.L. Pusey, C. Wend / Biological Control 62 (2012) 29–37

conducive to bacteria, but this usually occurred at night when temperatures as low as <5 °C limited bacterial growth. There is also uncertainty about what RH value is more important, the average or the minimum; thus, both are presented here. During the period from 0 to 4 days in 2010, when RH averaged 39% (daily average minimum was 22%), both osmoadapted and non-osmoadapted E325 populations increased in size by 4 log units and exceeded 106 CFU per flower, which is maximal for bacterial epiphytes on apple or pear stigmas (Johnson and Stockwell, 2000). This is consistent with recent studies (Pusey et al., 2011) showing that a hypermucoid mutant of E325 had no growth advantage on flower stigmas under dry conditions, as might be expected (Danhorn and Fuqua, 2007). Such results may be instructive regarding the nature of the stigma microenvironment. Although the volume of stigma secretions fluctuates as dependent on temperature and RH (Soltesz et al., 1996), possibly the stigma maintains osmotic conditions favorable for pollen germination and bacterial growth. In contrast to the high multiplication rate of E325 on stigmas under low average RH (650%), E325 populations in the hypanthium often showed a net size reduction under these conditions in the field and laboratory. The hypanthium as a microhabitat is dramatically affected by changes in RH and free moisture. This was recognized early by fire blight researchers (Hildebrand and Phillips, 1936; Thomas and Ark, 1934) who suggested that water availability affects sugar concentrations in nectar secretions, which in turn affects growth and survival of the pathogen. More recent research with flowers (Pusey, 2000) demonstrated the close interrelationships of RH, nectar volume, nectar water potential, sugar concentration, and bacterial growth in the hypanthium. Laboratory experiments demonstrated the conduciveness of stigma and hypanthial surfaces to osmoadapted and non-osmoadapted E325 under a wide RH range. On stigmas of detached flowers, E325 increased to high levels, regardless of RH, and no differences were indicated between treatment preparations. Results with hypanthia were more complicated. At the low RH of 50%, E325 never exceeded the starting population size; whereas at the high RH of 90%, E325 reached a maximum of 2 log units above the starting level. At these two extremes, little or no difference was indicated between osmoadapted and non-osmoadapted preparations. Results at the intermediate RH of 70% are of greater interest. In repeated trials, bacterial populations from the treatment preparations similarly decreased in size during the first 24 h, but subsequently exhibited differing capacities to recover; osmoadapted E325 increased and eventually surpassed the starting population size by nearly 1 log unit, whereas non-osmoadapted E325 showed a net decrease of about 1.5 log units at the end of the sampling period. Thus, it appears that the osmoadapted E325, pre-conditioned in a high-saline medium, had an advantage over non-osmoadapted E325 in the hypanthium when the RH level produced osmotic conditions marginally conducive for bacterial growth. In a previous study with detached flowers (Pusey, 2000) maintained as in the present, but incubated for only 24 h at 24 °C, even the pathogen (E. amylovora) did not grow in the hypanthium at 685% RH; yet, raising RH to 90% or 95% decreased nectar osmolality in the hypanthium resulting in bacterial population increases of 100- or 1000-fold, respectively. Thus, the RH range for minimal or optimal growth of bacteria in the hypanthium is relatively narrow and shifts as dependent on other factors such as temperature and water potential in the floral tissues (Pusey, 2000). Within a critical RH range, osmoadapted E325 will likely be more effective against E. amylovora than non-osmoadapted E325. However, this does not mean disease control will be measurably improved in arid regions, where certain RH levels may occur only briefly during daily extreme fluctuations. Wetting events preceding fire blight

development are often also brief, allowing bacteria to migrate from stigmas to hypanthium, bypassing further multiplication and directly invading flowers (Billing, 2000; Thomson, 1986); in this scenario, suppression on the stigma is critical and an increased antagonist adaptability to the hypanthium may not matter. It is possible that osmoadaptation may provide a greater advantage to bacterial antagonists on the hypanthium in more humid fruit production regions (e.g., the eastern USA) and contribute to disease control. The benefits of osmoadaptation subsequent to field application of E325, or other biocontrol agents for fire blight, may be minimal. A possible advantage of this approach yet to be investigated, however, is the prospect of increasing bacterial survival on dry nonsecretory flower parts that come in contact with pollinating insects. Conceivably, this could improve bacterial spread to flowers emerging after or between spray treatments. Further insight into the limits of osmoadaptation for enhancing biological control of fire blight might be gained through the use of GFP-based biosensors to visualize water-stressed bacterial cells (Axtell and Beattie, 2002) on floral surfaces. Although osmoadaptation was not shown to be advantageous in the orchard subsequent to E325 applications, there are no apparent drawbacks to this approach. The inclusion of NaCl in commercial production media is a relatively cheap modification, and growth of E325 in amended and non-amended media was comparable. Also, the high-saline medium did not diminish the capacity of E325 to suppress E. amylovora on flower stigmas (unpublished data). The greatest benefit of osmoadapting E325 in a high-saline medium appears to be the increase in cell survival during freeze drying and storage prior to orchard applications. Stability experiments simulated storage of E325 in a vacuum pack at room temperature (22 °C) or sealed container at 20 °C. The greater instability of non-osmoadapted compared to osmoadapted E325 preparations was most evident immediately or within a few days after freeze drying, with viable concentrations differing by margins between 0.5 and 1.6 log units. The addition of cryoprotectants to osmoadapted E325 cells prior to freeze drying resulted in even higher survival rates than what was obtained through osmoadaptation alone. Hence, osmoadaptation could complement cryoprotection to increase overall stability of freeze dried preparations of E325 or other biocontrol agents. Acknowledgments The authors thank Brenda Steady, Janet Duffy, Tara Culbertson, Lora Dunlap, Cindy Kahn, and Nancy Buchanan for technical assistance; Van Well Nursery for trees; and USDA-CSREES-SCRI for grant funds. References Axtell, C.A., Beattie, G.A., 2002. Construction and characterization of a proU-gfp transcriptional fusion that measures water availability in a microbial habitat. Applied and Environment Microbiology 68, 4604–4612. Billing, E., 2000. Fire blight risk assessment systems and models. In: Vanneste, J.L. (Ed.), Fire Blight: The Disease and Its Causative Agent, Erwinia amylovora. CAB International, Wallingford, UK, pp. 235–252. Bonaterra, A., Camps, J., Montesinos, E., 2005. Osmotically induced trehalose and glycine betaine accumulation improves tolerance to desiccation, survival and efficacy of the postharvest biocontrol agent Pantoea agglomerans EPS125. FEMS Microbiology Letters 250, 1–8. Bonaterra, A., Cabrefiga, J., Camps, J., Montesinos, E., 2007. Increasing survival and efficacy of a bacterial biocontrol agent of fire blight of rosaceous plants by means of osmoadaptation. FEMS Microbiology Ecology 61, 185–195. Campbell, R.J., Fell, R.D., Marini, R.P., 1990. Characterization of apple nectar sugars in selected commercial and crab apple cultivars. Fruit Varieties Journal 44, 136– 141. Danhorn, T., Fuqua, C., 2007. Biofilm formation by plant-associated bacteria. Annual Review of Microbiology 61, 401–422.

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