The Ontario Apple Growers in partnership with the Ontario Tender Fruit Growers completed a joint apple and pear fire blight research project with grant funding through Growing Forward 2. The goal of the project was to provide growers with tools to respond quickly and effectively to fire blight outbreaks, including the following objectives:

  • Gain information on the prevalence and distribution of fire blight resistance to streptomycin in pome fruit orchards across Ontario.
  • Develop strategies for integrating biologicals, antibiotics and copper for management of fire blight.

Objective 1: Determine the prevalence and distribution of resistance to streptomycin in the fire blight pathogen, Erwinia amylovora, in commercial apple and pear orchards across Ontario.

The bacterial pathogen, Erwinia amylovora, is commonly controlled by the antibiotic streptomycin which is applied during bloom. Streptomycin resistance has been detected in E. amylovora populations in California (1971), Oregon/Washington (1972), Michigan (1991) and New York (1972) (1). The most recent Canadian survey was conducted in British Columbia where the results revealed the presence of streptomycin resistance in the pathogen population (2).

In 2016, 48 apple and 18 pear orchards were surveyed from all 5 apple growing districts across Ontario. At each orchard, 25 fire blight infected shoots were collected from throughout the block. Variety and age of planting were recorded.

The following steps summarize the screening protocol used:

  • Step 1 - Five infected shoots per orchard were selected, plated onto semi-selective medium and, if possible, approximately 250 bacterial colonies or 50 colonies/shoot were processed.
  • Step 2 - The artificial media on which the pathogen was isolated is semi selective; therefore each colony had to be identified as E. amylovora by molecular technologies.
  • Step 3 - The positively identified colonies were grown on 0, 100 and 1000 ppm streptomycin amended growth media.
  • Step 4 - Bacterial colonies that survived on 100 and/or 1000 ppm were further tested by molecular protocols to identify and characterise the nature of the resistance. Moderate streptomycin resistance (100 ppm) is characterised by the presence of StrAB genes while high streptomycin resistance (1000 ppm) results from a single point mutation, or rspL (3).

To complete the screening of all the farms in 2016, the decision was made to screen for the antibiotic resistance using the same protocol; however the identification of E. amylovora by molecular technologies was omitted from the process due to time and material limitations. Sites that had growth on streptomycin amended media were screened by PCR to confirm that they were actually E. amylovora. Any positive E. amylovora were tested for the presence of resistance genes.

In the 32 farms where real time PCR was used to identify the fire blight pathogen (Table 1), the results found:

  • 5,707 bacterial colonies of E. amylovora were streptomycin sensitive.
  • There were no colonies with medium resistance and 4 colonies (3 orchards total) with high resistance.
  • The rspL mutation was confirmed through both droplet digital PCR and sequencing.

Isolates from the remaining 41 farms were screened for streptomycin resistance (Table 2); however the bacterial colonies should be considered as tentative E. amylovora. For these results:

  • 7,753 bacterial colonies of E. amylovora were streptomycin sensitive.
  • There were 42 colonies (20 orchards total) with medium resistance and 17 colonies (10 orchards total) with high resistance, albeit at very low levels.
  • Frozen samples from farm 136 and 137 (1 high and 8 moderate resistant isolates, respectively) were reprocessed and no streptomycin resistant colonies were obtained.

The problem with not initially identifying E. amylovora using PCR was that the common orchard epiphyte Pantoea agglomerans can have the same appearance on the semi-selective artificial medium. P. agglomerans is common in the orchard as an epiphyte. Interestingly, in a recent study, Tancos and Cox (2017) showed that there was a significant increase in streptomycin resistance in the P. agglomerans epiphytic populations in orchards where streptomycin is applied on a continual basis (4).

Samples from 7 farms were not processed due to poor condition and/or inability to isolate the pathogen on the artificial medium.

Table 1: Growth of Erwinia amylovora colonies (identified by real-time PCR) from Ontario apple and pear farms on streptomycin-amended media
Orchard Variety Age No. of colonies
testeda		 Streptomycin concentrationb (ppm)
0		 Streptomycin concentrationb (ppm)
100		 Streptomycin concentrationb (ppm)
1000
District 1 (Southwest)
1
 IdaRed
5-10
193
193
0
0
2
 IdaRed, Crispin
5-10
154
154
0
0
3
 IdaRed, Fuji
4
137
137
0
0
4
 GingerGold
3
178
178
0
0
District 2 (Central)
1
 Ida Red
25
150
150
0
0
2
 Ambrosia
n/a
197
197
0
0
3
 PaulaRed
15
123
123
0
0
District 3 (Georgian Bay)
1
 Early Golden
10
243
243
0
0
2
 Spy, Russet, IdaRed
25+
191
191
0
0
3
 Mutsu, Gala
10+
176
176
0
0
4
 Bartlett
10
221
221
0
0
District 4 (Niagara)
1
 Courtland
3
150
150
0
0
2
 Gala
1
167
166
0
1c
3
 Mixed Apple
42
223
223
0
0
4
 Gala, Mutsu
13
209
209
0
0
5
 Gala
3
214
214
0
0
6
 Mixed pear
15
199
199
0
0
7
 Mixed pear
n/a
249
249
0
0
8
 Mixed pear
20
250
250
0
0
9
 Bosc, Bartlett
20
250
250
0
0
10
 Mixed pear
10
249
249
0
0
11
 Clapp
40
250
248
0
2c
12
 Mixed pear
6
150
150
0
0
13
 Michellin, Courtland
n/a
12
12
0
0
14
 Gala
7
133
133
0
0
District 5 (East)
1
 Paula Red
26
247
246
0
1c
2
 Gala
9
100
100
0
0
3
 Gala
3
201
201
0
0
4
 Mixed apple
n/a
249
249
0
0
5
 IdaRed
4
50
50
0
0
6
 Gala
n/a
150
150
0
0
7
 Honeycrisp
20
50
50
0
0
Total
5,711
5,707
0
4

a Erwinia amylovora colonies confirmed by real-time PCR
b Streptomycin amended medium; 0 indicates no addition of streptomycin
c High resistance, presence of rspL mutation, was confirmed by sequencing and digital drop PCR
Italicized text - colonies were obtained from Table 2 and retested from frozen samples

Table 2: Growth of tentative Erwinia amylovora colonies (not identified by real-time PCR) from
Ontario apple and pear farms on Streptomycin-amended media
Orchard Variety Age No. of colonies
testeda		 Streptomycin concentrationb (ppm)
0		 Streptomycin concentrationb (ppm)
100		 Streptomycin concentrationb (ppm)
1000
District 1 (Southwest)
1
 Silken
3
50
50
0
0
2
 Empire, Fuji
20
250
250
0
0
3
 IdaRed
5-10
252
251
0
1
4
 IdaRed, Crispin
5-10
259
251
8
0
5
 IdaRed, Fuji
5-10
257
252
5
0
6
 Golden Delicious, Gala
4
250
250
0
0
7
 Mixed apple
30-60
252
250
1
1
8
 GingerGold
3
250
250
0
0
District 2 (Central)
1
 Ambrosia
1
251
250
1
0
2
 IdaRed
24
150
150
0
0
3
 Mixed pear
30
253
250
1
2
4
 Mixed pear
13
250
250
0
0
5
 Cortland
10-13
253
250
3
0
6
 PaulaRed
15
202
200
2
0
District 3 (Georgian Bay)
1
 Mixed apple
25
255
252
3
0
2
 Mixed apples
3-25
250
250
0
0
3
 Gala, Ambrosia
5
250
250
0
0
4
Spy
10+
100
100
0
0
5
Mutsu, Gala
10+
250
250
0
0
6
Mixed
10
50
50
0
0
7
 Gala
4
200
200
0
0
District 4 (Niagara)
1
 Harrow Crisp, Cold Snap
6
101
100
1
0
2
 Bosc
30
200
200
0
0
3
 Coldsnap
3
201
200
1
0
4
 Mixed pear
6
201
200
1
0
5
 Bosc, French Bartlett
15-20
255
250
3
2
6
 Coldsnap, Harrow Crisp, Bounty
6
154
150
1
3
7
 Coldsnap, Harrow Crisp, Bossc
6
51
50
1
0
8
 Michelin, Cortland
10
253
250
1
2
9
 Gala
7-8
252
250
2
0
10
 Gala, Paulared
4/45
200
200
0
0
District 5 (East)
1
 Mixed apple
10
252
250
1
1
2
 Gala
28
251
250
0
1
3
 IdaRed
4
200
200
0
0
4
 Gala
n/a
250
250
0
0
5
 Gala
n/a
150
150
0
0
6
 Mixed apple
4-6
257
250
4
3
7
 Honeycrisp
20
201
200
1
0
8
 McIntosh
30
251
250
1
0
9
 Mutsu
20
151
150
0
1
Total
7,812
7,753
42
17

a Tentative E. amylovora colonies
b Medium and high streptomycin resistance were not confirmed by molecular technologies.
Italicized text - colonies were retested by identification by real time PCR and screening on amended media

Objective 2: Develop a strategy for integrating biologicals, antibiotics and copper for management of fire blight in apple and pear.

Pear, c.v. Bosc on Swiss Bartlett rootstock, and apple, c.v. Gala on M9 rootstock, were planted in early-mid May, 2016 in an experimental orchard at Agriculture & Agri-Food Canada (AAFC) Jordan Research Station. Tree spacing was 1.2 m and row spacing was 2.45 m. Each plot consisted of 5 trees. The experimental design was a randomized complete block with 4 replicates. The pears did not have a sufficient consistent bloom to allow for the trial so only apples were included in the treatments and inoculation. The number of blossoms on each tree was recorded before treatments were applied.

Year 1 - 2016

The targeted bloom stages for spray applications were trace bloom, 25-50% bloom and 50-80% bloom. However, bloom on the young trees was very inconsistent and high temperatures pushed bloom rapidly so treatments were applied with a calibrated CO2 backpack sprayer to first drip the mornings of May 30, 31 and June 1. All plots were inoculated with a mixture of cultures of E. amylovora at 1 x 106 cfu/L the evening of June 1. Trees were monitored daily after inoculation and the % trees infected was determined. As infected blossoms were identified and counted, they were removed in order to preserve the trees for the following year's experiments.

Weather conditions were not conducive to the development of blossom blight symptoms and many of the blossoms were at petal fall stage by June 1 when inoculation took place; however, shoot blight symptoms continued to develop through the end of June. As shoot blight symptoms were observed, affected parts of the trees were noted, removed and destroyed. If entire trees were infected, they were removed and replaced in November 2016.

Since bloom was very inconsistent with many trees not having bloom in the first year after planting and variable numbers of trees per individual plot with bloom, statistical analysis was not done.

Year 2 - 2017

Treatments were applied at 5-10% bloom (May 12), 25-50% bloom (May 15) and 50-80% bloom (May 19). The second and third applications were made at 09:00 and 07:30, respectively. Inoculum of E. amylovora was applied at 106 cfu/L following the second and third treatment applications.

Trees were observed daily for symptoms of blossom blight or shoot blight. Infected blossoms were removed upon detection and shoots pruned back to prevent spread of the infection into the tree.

Conditions were very favourable for blossom blight as well as shoot blight in 2017. None of the treatments had statistically significant lower fire blight than the untreated control (Figure 1). However, there were interesting trends:

  • Control at the 50-80% stage appears most important.
  • The lowest incidence of fire blight was with Streptomycin applied at 50-80% bloom regardless of what was applied at 5-10% or 25-50% bloom.
  • Full season Kasumin was comparable to full season Streptomycin.
  • Full season treatments of the biologicals alone did not provide protection comparable to Streptomycin.
  • Blossom Protect was the most effective biological used in rotation with Streptomycin.
  • Double Nickel full season had significantly higher fire blight incidence than the untreated control.
  • The addition of Cueva to Double Nickel appeared to improve control; however, the difference was not statistically significant.

Figure 1: Incidence of fire blight (% blossoms infected/tree) with different treatments on Gala, 2017
*Treatments included under the same colour bar to the right are not significantly different.

What does this mean for Ontario growers?

Based on the 2016 survey results, streptomycin resistance was not present at significant levels in any orchard tested. Of the 32 farms that were fully processed using molecular technologies for pathogen identification, only 4 colonies from 3 orchards in the province were classified as high resistance. As well, the resistance in these 4 colonies was determined to be from a mutation rather than actually reflecting the presence of a population within an orchard. In addition, very low levels of resistance were detected in the 41 orchards that were screened for streptomycin resistance on amended media only. Therefore, streptomycin appears to remain a valuable tool in Ontario fire blight management.

Under optimum conditions for fire blight development, the biologicals Blossom Protect and Double Nickel alone or in combination with Cueva can be effective rotational partners with Streptomycin when positioned earlier in the infection period (before 50-80% bloom). Use of these alternatives to Streptomycin earlier in bloom would provide effective resistance management by reducing Streptomycin use to critical infection times. However, multiple years of study are necessary in order to accurately assess the effectiveness of biologicals under varied environmental conditions.

Since Kasumin provides control of fire blight comparable to Streptomycin, rotating between these products during the high infection periods later in bloom would also be an effective resistance management strategy and help extend the use of Streptomycin within a season.

Fire blight management should:

  • Consist of a season-long program, including pruning out infected material as soon as possible (see Summer management of fire blight) and maintaining good insect control to prevent spread of bacteria.
  • Include a dormant copper spray at silver tip to ¼ inch green.
  • Delaying copper after ½ inch green can result in phytotoxicity and severe fruit russetting, depending on the copper product used, especially on lighter skinned apples and some pear varieties such as Anjou.
  • Do not apply under slow drying conditions or just prior to a predicted frost to also help minimize the risk of phytotoxicity.
  • Use a sound rotational program of registered products during bloom, including antibiotics (Streptomycin, Kasumin), copper (Cueva) and/or biologicals (Blossom Protect, Double Nickel, Serenade OPTI).
  • Ensure adequate water volumes are used and that sprays are deposited into blossoms. Do not use excessive air speed on sprayers as this will propel sprays past the flower targets. For more information on calibrating an airblast sprayer or troubleshooting coverage, refer to Airblast 101 Handbook.
  • Products are most effective when applied just prior to an infection period. Since biologicals have a preventative action, these products should be applied when a forecasting model (see below) says risk is coming in the next 3-4 days. Antibiotics will provide activity 24 hours before and after a wetting event.
  • Products should be re-applied every 2-3 days during times of infection risk.
  • Copper and some fungicides are not compatible with biologicals. Check the label.
  • Apply control products when infection conditions are predicted using a forecasting model such as:
  • Consider managing growth of succulent shoots by limiting excess nitrogen and incorporating Apogee beginning at king bloom petal fall. Trials in Ontario are on-going to determine the impact of cultural practices on the development of shoot blight in young trees. Stay tuned for more information.

We would like to sincerely thank all of the growers who participated in providing samples for testing and who offered their knowledge and assistance in establishing the experimental orchards. As well, our sincere gratitude to Antonet Svircev, Darlene Nesbitt and Kinga Bodnar at Agriculture & Agri-Food Canada for laboratory testing; Leslie Huffman, Lindsay Pink, Kathy Hoshkiw, Margaret Appleby and Kevin Schooley who conducted the sampling, as well as OAG and OMAFRA summer students for their assistance in carrying out the project.

Generous support was provided by NuFarm Agriculture Inc., Bayer CropScience, Bio-Ferm GmbH, Certis USA, Engage Agro Corp., Arysta LifeScience, Niagara Peninsula Fruit & Vegetable Growers Association, Ontario Orchard Supply, and Vanden Bussche Irrigation. This project is funded in part by Growing Forward 2 (GF2), a federal-provincial-territorial initiative. The Agricultural Adaptation Council assists in the delivery of GF2 in Ontario.

References

  • Tancos, KA, Villani, S, Keuhne S, Borejsza-Wysocka E, Breth D, Carol, J, Aldwinckle HS, Cox KD. 2016. Prevalence of streptomycin-resistant Erwining amylovora in New York apple orchards. Plant Dis. 100:802-809.
  • Sholberg PL, Bedford KE, Haag P, Randall P. 2001. Survey of Erwinia amylovora isolates from British Columbia for resistance to bactericides and virulence on apple. Can. J. Plant Pathol. 23:60-67.
  • McGhee, GC, Bellomo L, Blumer SE, Sundin GW. 2008. Emergence and progression of streptomycin resistance in Erwinia amylovora in Michigan, p. 371-374, International Workshop on Fire Blight, vol. 793, Portland OR.
  • Tancos, KA, Cox, KD. 2017. The effects of consecutive streptomycin and kasugamycin applications on bacteria in apple in the phyllosphere. Plant Dis. 101:158-164.