'Bacterial canker of cherry - methods of susceptibility testing'

Monika Kałużna, Piotr Sobiczewski Research Institute of Horticulture Skierniewice, Poland

The disease

It occurs in all regions of cherry cultivation in the world causing the greatest damages in young orchards and nurseries

Cankers developing on branches and trunk sometimes lead to death of the trees

Disease development

The source of pathogen: primary and secondary infections Two phases in disease cycle: summer and winter

Pathogen survives also as epiphyte

Cycle disease of bacterial canker

Agrios, 2005

Pathogenicity and virulence factors

The hrp (hypersensitive response and pathogenicity) cluster genes encoding the components of a so-called type III secretion system(TTSS)

Phytotoxins: coronatine and syringomycine

Siderophores that can play a role in virulence

Synergism of bacteria with frost

Symptoms

Symptoms

Causal agents

Pseudomonas syringae pv. syringae Pseudomonas syringae pv. morsprunorum race 1 and 2

Pseudomonas syringae pv. avii

Pseudomonas syringae pv. persicae

Testing of sweet cherry terminal shoots susceptibility to bacterial canker after exposure to frost - Sobiczewski and Jones, 1992:

Bacteria: 108 cfu/ml in 0.01M Potassium Phosphate Buffer (7.2)-Pss and Psm

Cut shoots in January terminal and lateral shoots 25-30 cm long of 12-yr-old sweet cherry cv. Napoleon, Emperor Francis, Gold, Nelson, Ulster, Sam, Vega, Windsor,

Schmidt, Hedelfingen, Valera, Vic, Viva Disinfection - 0.02% sodium hypochloride 5 sec.; sterile distilled water; 96%

ethanol 5 sec. and water again basal end dipped about 2 cm in melted paraffin

Inoculation: 20µl of bacterial suspension (drop contained1.5x106 cfu/ml) was placed

on cut end of the tip below apical bud after absorption of inoculum the shoot tip was wrapped with parafilm

Experiment conditions

shoots were placed in jars with water and cotton at the bottom and covered with plastic to maintain moist atmosphere

15ºC, 7 days; -10ºC, 1.5 days; 15ºC, 10 days or in 15ºC

throughout the experiment

measurements of necrosis in the bark (phloem) and shoots (cambium or xylem)

comparison on two cv. 10ºC, 10days, half of inoculated and

noninoculated were placed in -5ºC, 3days and then all 10ºC 12 days(this temp increase development of bacterial canker on apricot in Hungary and peach shoots in France)

Results

exposure of inoculated shoots of sweet cherry to -5ºC did not increase the development of bacterial canker, however selection of assay degrees should be connected with natural condition in winter

greatest necrosis after inoculations with Pss: Cv. Napoleon, Emperor, Francis, Gold, Nelson and Ulster (in decreasing older)

greatest necrosis after inoculations with Psm: Ulster, Vega And Napoleon

extend of necrosis with Psm was less than with Pss susceptibility can be connected with degree of dormancy (higher during full dormancy than in other time (but this is connected with cv)

selection of cv. resistant to both pv. is difficult some cv. give moderate to both pv but some more susceptible to Pss than Psm

Results

Pss nonfrozen

Psm and Pss – Effect of exposure to freezing temperature

The size of necrosis caused by bacteria

Days after inoculation

Frozen at -5°C

Inoculation with 1.5x106 bacteria

7 18.5 18.5

No No Yes

Pss 1590* 16900 19400

1 cm

Psm 169 12100 26600

7 18.5 18.5

No No Yes

8 44 1000

2 cm

0.4 250 2900

7 18.5 18.5

No No Yes

10 2 80

3 cm

0.1 100 280

7 18.5 18.5

No No Yes

2 0.1 30

4 cm

0 70 80

7 18.5 18.5

No No Yes

0.1 0.0 1

5cm

0 5 30

*numer of bacteria (x102)/1 cm of shoot

Multiplication and distribution of bacteria

Resistance testing of sour cherry clones – Santi et al., 2004

Bacteria: 108 -109 cfu/ml in STW; pvs.: avii, persicae, syringae and P. fluorescens

Shoots cut in February, December, January Disinfection (0.5% sodium hypochloride 5 min.); rinsed under running

water, then soaked 3 times 5 min in clean running water

25-100µl of bacterial suspension (depend of size of shoot) were placed on freshly cut shoot tip-20-60 shoots per isolate

Parafilm placed over the tip

Experimental conditions - laboratory test

shoots were placed in bucked with water (2cm) and placed in room without controlling humidity

Incubation 15ºC, 7 days (16h of light); -5ºC, 7 days (in plastic bag in the dark); 15ºC, 4 weeks (16h of light) in closed transparent boxes suspended above 5cm of water

Experiment conditions-field test

cambial inoculations of 25µl of bacterial suspension into holes 1-2mm in deph 7-10 mm in width by removing the cortex and the phloem by cutting twice obliquely-on the stem (3-year old) and 5 shoots (2-year old)- in November-16 trees per clone

The inoculated holes not covered

After 6 weeks the cortex and partly the floem were peeled from infected tip of each shoots for necrosis lesions observation

0-no symptom, 1-less than1/4 affected, 2-1/4 to1/2 affected …5-total girdling

Results-laboratory and field test order and larger shoots were more susceptible, 2 years old shoots

was better for clonal discrimination

in the field test size and age of the shoots were not correlated to girdling

good agreement was found for the best clones in the labolatory and in fiels test however but worst clones in field test was the best in lab in some years of lab analysis (only 10 the same clones in field was analysed comparing to 29 in lab)

at least 2 independent lab tests are neccesary to evaluate resistance

lab test is good for positive selection but for final selection also field test because of more natural conditions shoud be included

Results-laboratory and field test

Good agreement was found for the best clones in the laboratory and in field test however but worst clones in field test was the best in lab in some years of lab analysis (only 10 the same clones in field was analyzed comparing to 29 in lab)

Detached leaf bioassay for sweet cherry - Bedford et al., 2003

Bacteria Pss and saprophytic P.fluorescens 108 cfu/ml in SDW

Leaves surface sterilized: running tap water 70% etanol, 30 sec. Twice in SDW 1 min and placed on petri dishes containing 0.5% water agar, incubation 2 weeks 25°C (16h photoperiod)

Leaf infiltration or midrib leaf infection

cv. Merpet and Merchant(resistant) Royal Ann, Sweetheart, Lapins and Bing

Results

young leaf proved HR to leaf washing procedure so fully mature leaves were chosen

rating provided after 14 days provided the best results (after 7 days no differences between susceptible and resistant cv)

infiltration successful for confirming the pathogenicity but not allowed for discrimination of resistance of cv tested-statistical analysis did not showed any differences e.g

the midrib wound infection was better-susceptible cv. Sweetheart was significantly more susceptible that resistant Merchant and Merpet

significant difference in reaction to virulent Pss in comparison to P.fluorescens and water

108 cfu/ml necessary for good symptoms development

Wild cherry micropropagated plantlets - Vicente and Roberts, 2003

Bacteria: Pss, Psm races 1 and 2; 108 -109 cfu/ml water suspension

Plantlets: 2-3 weeks old cultures of wild cherry clones: FD1-57-4/17, 1900, 1912, F12/1, Charger

Methods: dipping the plantlets in in bacterial suspensions without wounding;

Conditions: Freezing at -5°C, 5 min or no freezing;

Scale: 0-no symptoms; 1-small chlorotic or necrotic area in 1-2 leaves;3- less

than half of the plantlet with chlorotic or necrotic lesions; 5-half of plantlet…; 7 more than half…; 9death plantlet

0-1 resistant; 3-5 intermadiate, 7-9 susceptible

Results

Symptoms after 4 weeks

Cherry clones showed partial level of resistance to Psm and were more susceptible to race 1 than to race 2

Wild cherry clones FD1-57-4/166 and Cobtree highest level to races 1 and 2 respectively

No clear differences between reactions of sweet cherry cvs. Napoleon and Roundel when inoculated with both races of Psm (Roundel was more resistant to race1 in studies of Freigoun and Crosse 1975)

Leaf spot observed only after inoculation with Psm (more with race1)

Author conclusion

Plantlets are good for resistance testing-better than field or twig

Clearly differentiated Psm and Pss

Preliminary results of rooted plants showed that they was more susceptible to Pss than to Psm

Screening should always include both pathovars

Our conclusions

Screening of cultivars or clones for resistance should always include freezing - the temperature should be also adjusted

Both pathovars (Pss, Psm) should be included or at least highly virulent Pss strain INA+

Dormancy period is important (depending of cv.)

Susceptibility of cultivars Sweet cherry

susceptible: ‘Hedelfińska’, ‘Van’, ‘Czarna Późna’, ‘Napoleon’ Less susceptible/resistant: ‘Rivan’, ‘Riversa Wczesna’,

‘Buttnera Czerwona’, ‘Burlat’, ‘Merton Premier’, ‘Kunzego’, ‘Vega’, ‘Sam’, ‘Karesova’, ‘Schneidera Późna’, ‘Ulster’

Sour cherry susceptible: ‘Nefris’, ‘Wanda’ Less susceptible/resistant : ‘North Star’, ‘Łutówka’,

‘Groniasta z Ujfeherto’, ‘Pandy 103’, ‘Lucyna’, ‘Sabina’

Thank you very much for your attention