Humidity

Example 12

Plant transformation—The Arabidopsis thaliana var Columbia (To plants) were transformed according to the Floral Dip procedure [Clough S J, Bent A F. (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 16(6): 735-43; and Desfeux C, Clough S J, Bent A F. (2000) Female reproductive tissues were the primary targets of Agrobacterium-mediated transformation by the Arabidopsis floral-dip method. Plant Physiol. 123(3): 895-904] with minor modifications. Briefly, Arabidopsis thaliana Columbia (C010) T₀ plants were sown in 250 ml pots filled with wet peat-based growth mix. The pots were covered with aluminum foil and a plastic dome, kept at 4° C. for 3-4 days, then uncovered and incubated in a growth chamber at 18-24° C. under 16/8 hours light/dark cycles. The T₀ plants were ready for transformation six days before anthesis.

Single colonies of Agrobacterium carrying the binary vectors harboring the genes of some embodiments of the invention were cultured in YEBS medium (Yeast extract 1 gr/L, Beef extract 5 gr/L, MgSO₄*7H₂O, Bacto peptone 5 gr/L) supplemented with kanamycin (50 mg/L) and gentamycin (50 mg/L). The cultures were incubated at 28° C. for 48 hours under vigorous shaking to desired optical density at 600 nm of 0.85 to 1.1. Before transformation into plants, 60 μl of Silwet L-77 was added into 300 ml of the Agrobacterium suspension.

Transformation of T₀ plants was performed by inverting each plant into an Agrobacterium suspension such that the above ground plant tissue was submerged for 1 minute. Each inoculated T₀ plant was immediately placed in a plastic tray, then covered with clear plastic dome to maintain humidity and was kept in the dark at room temperature for 18 hours to facilitate infection and transformation. Transformed (transgenic) plants were then uncovered and transferred to a greenhouse for recovery and maturation. The transgenic T₀ plants were grown in the greenhouse for 3-5 weeks until siliques were brown and dry, then seeds were harvested from plants and kept at room temperature until sowing.

For generating T₁ and T₂ transgenic plants harboring the genes of some embodiments of the invention, seeds collected from transgenic T₀ plants were surface-sterilized by exposing to chlorine fumes (6% sodium hypochlorite with 1.3% HCl) for 100 minutes. The surface-sterilized seeds were sown on culture plates containing half-strength Murashig-Skoog (Duchefa); 2% sucrose; 0.5% plant agar; 50 mg/L kanamycin; and 200 mg/L carbenicylin (Duchefa). The culture plates were incubated at 4° C. for 48 hours and then were transferred to a growth room at 25° C. for three weeks. Following incubation, the T₁ plants were removed from culture plates and planted in growth mix contained in 250 ml pots. The transgenic plants were allowed to grow in a greenhouse to maturity. Seeds harvested from T₁ plants were cultured and grown to maturity as T2 plants under the same conditions as used for culturing and growing the T₁ plants.

Example 1

119 Dicty strains were screened for their ability to feed on Dickeya (Dd) or Pectobacterium (Pcc) at 10° C. This assay was performed by inoculating Dd or Pcc on a low nutrient medium (SM2 agar) that supports both bacterial and Dicty growth. Dicty spores from individual strains were then inoculated on top of the bacterial growth and incubated at 10° C. to mimic potato storage temperatures. Dicty strains that successfully fed on Dd or Pcc created visible clearings in the lawn of bacterial growth and ultimately produced sporangia (fruiting bodies) that rose from the agar surface. An example of the phenotype that was considered successful clearing of bacteria is shown in FIG. 3A. From this initial screen, 36 Dicty strains that were capable of feeding on both Dd and Pcc at 10° C. were identified (FIG. 1B).

Of the 36 strains capable of feeding on both Dd and Pcc, 34 came from the Group 4 Dictyostelids (FIG. 1). This group includes D. discoideum, D. giganteum, D. minutum, D. mucoroides, D. purpureum, and D. sphaerocephalum (72). The results indicate that this group is particularly enriched in Dd and Pcc-feeding strains.

A further experiment was performed to identify Dicty species capable of feeding on biofilms of Dd and Pcc. Microporous polycarbonate membranes (MPMs) are widely reported to support biofilm formation of numerous Enterobacteriaceae species (2, 63, 70, 71). It was determined if Dd and Pcc formed biofilms on MPMs and determined if Dicty strains were capable of feeding on these biofilms. Membranes were placed on top of SM2 agar to provide Dd and Pcc with nutrients for growth. Bacteria were then inoculated on the surface of the MPMs and growth was monitored over the course of 1 week by washing bacteria off the membranes and performing dilution plating for colony counting. Growth of both bacterial strains plateaued around 4 dpi (FIG. 2).

From these results, it was determined that the best time to collect inoculated MPMs for biofilm analysis was at 2 dpi. Scanning electron microscopy (SEM) is commonly used to confirm biofilm formation by detecting extracellular polymeric substance (EPS) that forms the biofilm matrix (2). Samples of Dd and Pcc after 2 days of growth on MPMs in the presence and absence of Dicty are analyzed using SEM.

19 Dicty strains identified as active were tested for their ability to feed on Dd and Pcc growing on MPMs. These experiments were performed by establishing Dd and Pcc growth on MPMs overlaid on SM2 agar at 37° C. for 24 hr. Dicty spores were then applied to the center of bacterial growth in a 5 uL drop containing 1000 spores. Bacteria and Dicty were incubated at 10° C. for 2 weeks before remaining bacteria were washed off and colonies were counted. Representative images of Dicty growing on Dd and Pcc on MPMs are shown in FIG. 3A.

No Dicty strains produced a statistically significant reduction in Dd viability compared to the non-treated control. However, treating Dd lawns with Cohen 36, Cohen 9, WS-15, WS-20, and WS-69 consistently reduced the number of viable bacteria by approximately 100,000-fold compared to the non-treated control (FIG. 3B). Cohen 9 was the only Dicty strain that produced a statistically significant reduction in viability of Pcc compared to the non-treated control (FIG. 3C). Other Dicty strains capable of reducing the number of viable Pcc by at least 100,000-fold were Cohen 35, Cohen 36, WS-647, and WS-69 (FIG. 3C).

It was observed that Dicty strains Cohen 9, Cohen 36, and WS-69 were capable of feeding on both Dd and Pcc when these bacteria were cultured on SM2 agar and MPMs (FIGS. 1 and 3). These strains were also particularly effective feeders as all three reduced the number of viable Dd and Pcc on MPMs at 10° C. by 100,000-fold compared to the non-treated control (FIGS. 3B and 3C).

To determine if these strains could suppress soft rot development on seed potato tubers, tubers were tab-inoculated with Dd or Pcc and treated with spores from each Dicty strain. Seed potatoes were surface-sterilized and punctured using a sterile screw to a depth of 1.5 mm. Overnight cultures of Dd and Pcc were suspended in 10 mM potassium phosphate buffer, diluted to an OD600 of approximately 0.003, and administered as a 5 μL drop into the wound. Next, 5 of a Dicty spore suspension (100,000 spores) was added to the wound. Inoculated seed potatoes were placed in a plastic container with moist paper towels and were misted with water twice a day to maintain a high humidity. After 3 days at room temperature, seed potatoes were sliced in half and the area of macerated tissue was quantified using ImageJ.

All three strains reduced the severity of soft rot caused by Dd and Pcc (FIG. 4). Cohen 36 was the most effective strain on both Dd and Pcc: reducing the area of tissue maceration by 60% and 35%, respectively (FIG. 4B). Treating seed potatoes with WS-69 reduced the area of tissue maceration by 50% and 30% for Dd and Pcc, respectively (FIG. 4B). Finally, Cohen 9 was the least effective, but still able to reduce tissue maceration caused by Dd and Pcc by 25% and 20%, respectively (FIG. 4B).

FIG. 7 shows that three Dicty isolates control Dd and Pcc in seed tubers (at 25° C.). Two sets of data from different weeks were normalized to the Dickeya or Pectobacterium only bacterial control. The average area of macerated potato tissue measured in mm² was set as “1” or “100%”. The average of all the other treatments including Dicty were divided by bacteria only control and multiplied by 100 to obtain a percentage. Each set contained 5 tubers per treatment.

Dicty should be capable of sporulating at temperatures as cold as 10° C. on a potato surface if they are applied as a one-time pre-planting or post-harvest treatment. Sporulation was assessed by inoculating small potato discs (5×6 mm) with 10 μL of Dd or Pcc suspensions at an OD600 of 3×10⁻⁵ and Dicty spores at a concentration of 1×10⁷ spores/mL. Potato discs were kept in a covered 96-well plate for two weeks at 10° C. followed by visual inspection for son using a dissecting microscope. Representative images of a strain producing many sori (WS-517) and a strain producing few sori (WS-69) are shown in FIG. 5. Of the 11 strains evaluated, only Cohen 9 and WS-20 were unable to sporulate in the presence of both pathogens (Table 1).

Example 2

This example describes the use of a high throughput screening assay to identify Dicty strains from Alaska (e.g., BAC10A, BAF6A, BAC3A, NW2, KB4A (ATCC® MYA-4262™) SO8B, SO3A, BAF9B, IC2A (ATCC® MYA-4259™), AK1A1 (ATCC® MYA-4272™) PBF4B (ATCC® MYA-4263), PBF8B, BSB1A, SO5B (ATCC® MYA-4249), PBF3C, PBF6B, NW2B, NW10B (ATCC® MYA-4271™), PBF9A, IC5A (ATCC® MYA-4256TH), ABC8A (ATCC® MYA-4260), NW16B, ABC10B, ABB6B (ATCC® MYA-4261), BA4A (ATCC® MYA-4252), AKK5A, AKK52C, HP4 (ATCC® MYA-4286), HP8 (ATCC® MYA-4284), or NW9A) that feed on Dd and Pcc at 10° C. on potatoes.

Results from 11 Dicty strains screened against Dd at 10° C. are presented in FIG. 6. Data was analyzed for significance using a one-way analysis of variance (ANOVA; alpha =0.05) with Tukey's honest significant difference (HSD) test to compare means between the treatments and the No Dicty control. A reduction in Dd proliferation when potato discs were treated with Dicty strains Cohen 9, Cohen 36, WS-15, Maryland 18a, BAF6A, NW2, and SO3A.

The Alaskan Dicty strains, and those identified in Example 1, are further tested against coinfections of Dd and Pcc. It is useful to identify Dicty strains that can suppress Dd and Pcc coinfections as these two pathogens have been isolated together from diseased potatoes (15). The ability of Dicty strains with different feeding preferences (Dd vs. Pcc) to complement each other when administered as a cotreatment is assayed.

Example 13

Batch analytical data for Formula 21 was determined and recorded in Table 15. Results were recorded at time, T=0 and again at time, T=1 month at a temperature of 40° C. and 75% relative humidity (RH).