Australasian Biotechnology (backfiles) AusBiotech ISSN: 1036-7128 Vol. 8, Num. 6, 1998

Australasian Biotechnology, Vol.8 No.6, December 1998

Identification and Characterisation of Isolates of Erwinia amylovora from
 Cotoneaster in Australia





R.K.Taylor and C.N.Hale, Horticulture and Food research
 Institute of New Zealand Ltd, Mt Albert Research centre, Private Bag 92169, Auckland, New
 Zealand


Code Number:AU98042


The results of tests conducted on cotoneaster samples showing typical fire blight


symptoms in the Royal Botanic Gardens, Melbourne, suggest that the causal organism is Erwinia


amylovora. Isolates from affected plant tissue were indistinguishable from authentic


cultures of Erwinia amylovora in their morphology, in PCR, in DNA hybridisation


tests using an Erwinia amylovora specific DNA probe, in tobacco hypersensitivity


tests, and in pathogenicity tests on immature pear fruit and apple seedlings. 





Introduction 





In April 1997, samples of cotoneaster (Cotoneaster sp.) exhibiting typical fire


blight symptoms were collected and tested for the presence of 
Erwinia amylovora


from the Royal Botanic Gardens, Melbourne, Australia. The most obvious symptoms typical of


E. amylovora infections were scorched leaves on affected branches and, in some


cases, the typical shepherd's crook on shoots. The symptoms were widespread on cotoneaster


bushes throughout the gardens. Specific polymerase chain reaction (PCR) analytical and DNA


hybridisation methods were recently developed to detect E. amylovora at very low


levels on the surface or in the calyxes of apples (Hale and Clark 1990, Guilford et al.


1996). These techniques, as well as DNA fingerprinting and standard bacteriological tests


were used, to characterise the isolates from cotoneaster. 





Materials and Methods 





Bacterial isolation 





Internal stem tissue samples were taken at the junction where dark brown stained tissue


adjoined apparently healthy cotoneaster tissue. Samples were macerated in 500ml of


extraction buffer (4g NaCl, 0.2g KCl, 0.25ml Tween 20, 10g polyvinyl pyrrolidone and 2g


bovine serum albumin per 500ml sterile distilled water (dH2O)). Five ml samples


of the resultant suspensions were mixed in a 0.2ml PCR tube with 15ml of GeneReleaserTM


to bind inhibitors of the PCR, vortexed for 10-20 seconds, heated in a microwave


oven at full power for five minutes and incubated at 80oC for five minutes


prior to PCR analysis. Routine isolations of bacteria from cotoneaster internal stem


tissue were also made on CCT medium (Ishimaru and Klos, 1984) and incubated at 27oC


for 24 hours. 





PCR analysis 





The PCR analyses of plant tissue and of bacterial colonies were performed using the


methods of Guilford et al. (1996) and Bereswill et al. (1995). Positive


controls using authentic cultures of e. amylovora (Table 1) and negative controls


containing dH2O, extraction buffer, bacteriological saline (0.85% w/v


NaCl), and GeneReleaserTM were included in each set of reactions. PCR analyses


were performed on bacterial colonies, grown on CCT plates which exhibited pitting when


viewed microscopically (X32). 





DNA Hybridisation 





Colonies from pure cultures on CCT were tested using a specific and sensitive DNA


hybridisation method (Hale and Clark, 1990) using a radioactive labeled DNA probe prepared


from E. amylovora ICMP 1501. Controls consisted of colonies of E. amylovora


ICMP* 1501, 1532, and 8865, Pseudomonas viridiflava ICMP 8952 (from kiwifruit) and


isolates of saprophytic bacteria from apple and kiwifruit. 





* International Collection of Microorganisms from Plants, Landcare/Manaaki Whenua


research, Auckland, New Zealand. 





DNA fingerprinting 





Bacterial colonies from pure cultures on CCT medium were cultured in nutrient broth and


incubated at 27oC for 16 hours. Three ml samples of bacterial culture were


centrifuged at 13,000 rpm for five minutes, the supernatant discarded and the pellet


washed twice in 1M NaCl. DNA was isolated using the method of Rudner et al. (1994)


and quantified spectrophotometrically. Positive controls using authentic cultures of E.


amylovora ICMP 1501, 1532 and 8865, and a negative control of dH2O were


included in each set of reactions. Random amplified polymorphic DNA (RAPD) analyses of the


bacterial cultures were performed using the method of Momol et al. (1997). For


arbitrarily primed-PCR (AP-PCR) analysis the method of bereswill et al. (1995) was


used. 











Table 1. Erwinia amylovora strains used in this study











  



    
Species Name


    
Strain Number ICMP


    
Host


    
Location


  


  



    
Erwinia amylovora


    
1505


    
Crataegus monogyna


    
New Zealand 


  


  



    



    
1532


    
Cotoneaster melanocarpus


    
United Kingdom


  


  



    



    
1493


    
Pyrus communis


    
New Zealand


  


  



    



    
1540


    
Pyrus communis


    
United Kingdom


  


  



    



    
1501


    
Malus


    
New Zealand 


  


  



    



    
1392


    
Malus


    
United States


  


  



    
Test Isolates


    
13293


    
Cotoneaster sp.


    
Australia


  


  



    



    
13294


    
Cotoneaster sp.


    
Australia


  


  



    



    
13295


    
Cotoneaster sp.


    
Australia


  


  



    



    
13296


    
Cotoneaster sp.


    
Australia


  


  



    



    
13297


    
Cotoneaster sp.


    
Australia


  


  



    



    
13298


    
Cotoneaster sp.


    
Australia


  


  



    



    
13299


    
Cotoneaster sp.


    
Australia


  


  



    



    
13300


    
Cotoneaster sp.


    
Australia


  


  



    
All strains listed are deposited in the international


    Collection of Micro-organisms from plants (icmp), Landcare / manaaki Whenua Research,


    Auckland, New Zealand.


  








Pathogenicity 





Immature pear fruit were surface-sterilised with a 10% v/v sodium


hypochlorite solution and stab-inoculated with saline suspensions of pure cultures from


cotoneaster (ICMP 13293, 13294). The inoculated pears were enclosed in humid chambers (RH


> 95%) and incubated for 72 hours at c. 20oC. Controls included immature


pear fruit inoculated with either E. amylovora ICMP 1501 or bacteriological saline.








Apple seedlings were inoculated, by pin-prick into stems, with colonies from pure


cultures of ICMP 13293, 13294, 13295 and 13296. The inoculated seedlings were enclosed in


polyethylene bags and held at 22oC in humidity chambers (RH c.100%) for 48


hours and then grown in a glasshouse at c. 20oC. Controls included apple


seedlings inoculated with cultures of either E. amylovora, bacteriological saline,


or dH2O. 





tobacco leaves (cv. White Burley) were infiltrated using the method of Klement (1963),


with suspensions of pure cultures of ICMP 13293, 13294, 13295 



and 13296 in bacteriological saline containing c. 108 colony forming units/ml


and held in a glasshouse at c. 20oC. Controls included tobacco leaves


infiltrated with either E. amylovora ICMP 1501 or bacteriological saline. 





isolations were made on CCT medium from ooze which appeared on inoculated immature pear


fruit after incubation for 72 hours at c. 200C and from apple seedlings after


five days. Colonies were checked for pitting and analysed by PCR as described previously. 





Results 





PCR analysis 





The PCR products from the plant tissue samples using Ea71 primers (Guilford et al.


1996), contained a discrete 187bp DNA band which was identical to the specific band


produced by reactions containing authentic cultures of E. amylovora. No DNA bands


were produced in PCRs containing extraction buffer, bacteriological saline, and


GeneReleaserTM. 





After 24 hours incubation on CCT numerous white colonies with pitted surfaces were


visible on plates from cotoneaster tissue samples. These colonies were morphologically


similar to colonies of authentic cultures of E. amylovora. 





The PCR products from the test isolates using Ea71 primers (Guilford et al.


1996), contained a 187bp DNA band and using AMSb primers (Bereswill et al. 1995)


contained a discrete 1.6 kb DNA band (Figure 1). These were identical to the specific


bands produced by reactions containing authentic cultures of E. amylovora. No


specific bands were produced in control PCR reactions containing dH2O. 

 Figure 1. PCR detection using Ea71 and AMSb primers



DNA hybridisation





The radioactively-labeled DNA probe, specific for E. amylovora , produced a


strong hybridisation signal with the isolates from cotoneaster (ICMP 13293, 13294, 13295


and 13296) and the known cultures of E. amylovora. However, no signal was produced


with either saprophytic bacteria from apple and kiwifruit, or with P. viridiflava


ICMP 8952. 





DNA fingerprinting 





RAPD analysis of the test isolates from cotoneaster produced fingerprints that were


identical to those produced by the authentic cultures of E. amylovora (Figure 2).


No nucleic acid bands were produced in PCRs containing dH2O. 

 Figure 2. RAPD fingerprints of genomic DNA from Erwinia amylovora strains



AP-PCR analysis of the test isolates from cotoneaster produced fingerprints which are


typical for E. amylovora (Bereswill et al. 1995). Identical fingerprints


were also produced in analyses of the authentic cultures of E. amylovora. No


nucleic acid bands were produced in PCRs containing dH2O. 





Pathogenicity 





After 72 hours incubation of immature pears in humid chambers bacterial ooze was seen


on fruit inoculated with the test isolates from cotoneaster (ICMP 13293, 13294). ooze was


also seen on immature pear fruit inoculated with an authentic culture of E. amylovora ICMP


1501, but not on those inoculated with bacteriological saline (Figure 3). 





After five days blackening of the petioles and some leaf necrosis was observed on apple


seedlings inoculated with the cultures from cotoneaster (ICMP 13293, 13294, 13295 and


13296) and the authentic cultures of E. amylovora ICMP 1501. No symptoms were seen


on the seedlings inoculated with either bacteriological saline or dH2O. 





A hypersensitive reaction was produced in the infiltrated tobacco leaves by the test


isolates (ICMP 13293, 13294, 13295 and 13296) and the authentic cultures of E.


amylovora ICMP 1501, 8865 and 1532. No hypersensitive reaction was produced in leaves


infiltrated with bacteriological saline. 





Numerous white colonies with pitted surfaces were produced on CCT from isolations from


ooze and lesions which appeared on inoculated immature pear fruit and apple seedlings. The


PCR products from cultures produced from the ooze and lesions contained a discrete 187bp


DNA band using Ea 71 primers and a 1.6kb DNA band using AMSb primers identical to those


produced by the test isolates and the authentic cultures of E. amylovora ICMP 1392,


1492, 1501, 1505, 1532, and 1540. 

 Figure 3 Immature pears inoculated with test isolates



Discussion 





PCR was used to test for the presence of E. amylovora in cotoneaster material,


from the Royal Botanic Gardens in Melbourne. The cotoneaster isolates were compared to


authentic cultures of e. amylovora using a number of criteria: by morphology on a


selective medium, DNA hybridisation, DNA fingerprinting, specific primers for PCR


amplification, tobacco plant hypersensitivity and pathogenicity tests on apple and pear.


Results showed that the bacteria isolated from cotoneaster were identical to authentic


cultures of E. amylovora. 





DNA fingerprinting of the cotoneaster isolates using RAPDs and AP-PCR indicated a high


degree of similarity with authentic cultures of E. amylovora from New Zealand,


United States and the United Kingdom. This type of similarity between isolates from


different geographical regions is characteristic of E. amylovora. This is


consistent with earlier reports on the genetic diversity of E. amylovora which


indicated that the species is very homogeneous(Momol et al. 1997, McManus and Jones


1995). 





The conclusion that E. amylovora is the cause of fire blight symptoms in


cotoneaster in the Royal Botanic Gardens, Melbourne has been further verified by Dr Klaus


Geider, Max Planck Horticultural Research institute, Heidleburg, Germany (pers. comm.).


The disease may have been established in the Royal Botanic Gardens, Melbourne for some


time, as the symptoms were seen on mature plants. The fact that E. amylovora has


only been found so far in the Royal Botanic Gardens, Melbourne does not imply that this is


an isolated incident. Although it is possible that E. amylovora is present in other


parts of Australia, symptoms of the disease have not been found in a recent survey of


pipfruit production areas. 





Acknowledgements 





Aspects of this article have been reproduced from an article originally published in


the New Zealand Biotechnology Association newsletter No. 35. 





References 





Bereswill, S., Bugert, P., Bruchmuller, I., & Geider, K. (1995) Identification of


the fire blight pathogen, Erwinia amylovora , by PCR assays with chromosomal DNA. Applied


and Environmental Microbiology., 61, 2636-2642. 





Guilford, P.J., Taylor, R.K., Clark, R.G., Hale, C.N., & Forster, R.L.S. (1996)


PCR-based techniques for the detection of Erwinia amylovora. Acta


Horticulturae., 411, 53-56. 





Hale, C.N., & Clark, R.G. (1990) Detection of Erwinia amylovora from apple


tissue by DNA hybridisation. Acta Horticulturae., 273, 51-55. 





Ishimaru, C., & Klos, E.J. (1984) New medium for detecting Erwinia amylovora


and its use in epidemiological studies. Phytopathology., 74, 1342-1345. 





Klement, Z. (1963) Rapid detection of the pathogenicity of phytopathogenic pseudomonads.


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McManus, P.S., & Jones, A.L. (1995) Genetic fingerprinting of Erwinia amylovora


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1547-1553. 





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Copyright 1998 Australian Biotechnology Association Ltd.

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