New Disease Reports
New Disease Reports (2012) 26, 15. [http://dx.doi.org/10.5197/j.2044-0588.2012.026.015]
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First report of blackleg and soft rot of potato caused by Pectobacterium carotovorum subsp. brasiliensis in New Zealand

P. Panda 1, M.A.W.J. Fiers 2, K. Armstrong 1 and A.R. Pitman 1,2*

*Andrew.Pitman@lincoln.ac.nz

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Received: 14 Jun 2012; Published: 19 Sep 2012

Keywords: Erwinia, enterobacteria, multi-locus sequence analysis, 16S rRNA gene

Blackleg and stem rot of potato occur sporadically in New Zealand, causing economic damage under optimal temperature and humidity conditions for disease development. Both Pectobacterium atrosepticum (Pba) and P. carotovorum subsp. carotovorum (Pcc) have previously been isolated from potato tubers with soft rot symptoms in New Zealand (Crowhurst & Wright, 1998) whereas only Pba has been shown to cause blackleg. A collection of 89 enterobacteria were recently isolated from potato tubers from commercial crops in the Auckland, Waikato, Manawatu-Wanganui and Canterbury regions of New Zealand. The majority were initially assigned as Pcc by their growth at 37°C, carbon utilisation profiles and restriction fragment length polymorphisms (Pitman et al., 2008). These isolates were mostly unable to cause blackleg symptoms, although several were shown to be highly aggressive upon stem infection. Further characterisation of these aggressive isolates using PCR assays to distinguish Pcc from P.  carotovorum subsp. brasiliensis (Pbr) was carried out using primers EXPCCF and EXPCCR (Kang et al., 2003) and Br1f and L1r (Duarte et al., 2004), respectively. From 18 isolates, occurring throughout the four major growing localities (Fig. 1), a 322-bp fragment specific to Pbr was amplified using Br1f and L1r (Fig. 2), whereas no PCR product (550-bp) was obtained typical of Pcc. The 16S rRNA gene was amplified (Wang & Wang, 1996) from one of the putative Pbr isolates, NZEC1 (deposited in the International Collection of Microbes from Plants as ICMP 19477). BLAST analysis of the 16S rRNA DNA sequence (GenBank Accession No. JQ771053) showed 100% identity to the 16S rRNA of Pbr.

Multi-locus sequence analysis was performed using concatenated DNA sequences of acnA, gapA, icdA, mdh, mtlD, pgi and proA from NZEC1 (JQ820114-JQ820120) and 46 related taxa (Ma et al., 2007). The resulting majority rule consensus tree constructed using MrBayes v. 3.0b4 (Fig. 3) clustered NZEC1 with Pbr, readily distinguishing it from the closely related subspecies Pcc. Pathogenicity assays were conducted using potato plants (cv. ‘Ilam Hardy’) by injecting 10 μl of a bacterial suspension (10 cfu/ml) into either stems or tubers. Each time, severe blackleg (Fig. 4) or tuber soft rot (Fig. 5) were observed on plants inoculated with Pbr whereas Pcc strains were unable to cause blackleg. The results were consistent with observations throughout the world that suggest Pcc is largely unable to cause this stem disease.

To our knowledge, this is the first report of Pbr infecting potato in New Zealand.The prevalence of Pbr isolates in Canterbury is of particular note due to the high proportion of seed tuber production that takes place in this region. As Pcc is usually unable to cause blackleg and Pba is only rarely isolated from tubers in New Zealand (Pitman et al., 2008), Pbr is likely to be an important component of the blackleg syndrome in New Zealand. Consequently, this pathogen is expected to contribute to economic losses resulting from both blackleg and tuber soft rot. Pbr also causes severe economic impacts both in Brazil and South Africa.

 

 

Figure1+
Figure 1: Locations in New Zealand (indicated by pie charts) from which potato tubers infected with Pectobacterium carotovorum subsp. brasiliensis (Pbr) were collected. Shading distinguishes major geographical localities where potato is cultivated. Total area (ha) of commercial crops in each region is indicated. Each pie chart defines the proportion of enterobacterial isolates found to be Pbr (black) or another Pectobacterium species (white).
Figure 1: Locations in New Zealand (indicated by pie charts) from which potato tubers infected with Pectobacterium carotovorum subsp. brasiliensis (Pbr) were collected. Shading distinguishes major geographical localities where potato is cultivated. Total area (ha) of commercial crops in each region is indicated. Each pie chart defines the proportion of enterobacterial isolates found to be Pbr (black) or another Pectobacterium species (white).
Figure2+
Figure 2: PCR detection of Pbr using the Br1f and Lr1 primers. Lanes: 1, Pbr CFBP 6619; 2, Pbr CFBP 6617; 3, Pbr NZEC1; 4, Pbr NZEC150; 5, Pcc ICMP5702T; 6, Pba ICMP1526T; 7, P. betavasculorum ICMP4226T; 8, P. wasabiae ICMP9121T; 9, P. carotovorum subsp. odoriferum ICMP11533T; 10, No template control (NTC); 11, Marker (1000-bp ladder), (T, type strain). See text for abbreviations of bacterial species names.
Figure 2: PCR detection of Pbr using the Br1f and Lr1 primers. Lanes: 1, Pbr CFBP 6619; 2, Pbr CFBP 6617; 3, Pbr NZEC1; 4, Pbr NZEC150; 5, Pcc ICMP5702T; 6, Pba ICMP1526T; 7, P. betavasculorum ICMP4226T; 8, P. wasabiae ICMP9121T; 9, P. carotovorum subsp. odoriferum ICMP11533T; 10, No template control (NTC); 11, Marker (1000-bp ladder), (T, type strain). See text for abbreviations of bacterial species names.
Figure3+
Figure 3: Bayesian phylogenetic tree using concatenated partial gene sequences of acnA, gapA, icdA, mdh, mtlD, pgi and proA for 46 Enterobacteriaceae taxa.
Figure 3: Bayesian phylogenetic tree using concatenated partial gene sequences of acnA, gapA, icdA, mdh, mtlD, pgi and proA for 46 Enterobacteriaceae taxa.
Figure4+
Figure 4: Blackleg symptoms three days post-inoculation of a potato stem (‘Ilam Hardy’) with Pectobacterium carotovorum subsp. brasiliensis NZEC1.
Figure 4: Blackleg symptoms three days post-inoculation of a potato stem (‘Ilam Hardy’) with Pectobacterium carotovorum subsp. brasiliensis NZEC1.
Figure5+
Figure 5: Soft rotting of a potato tuber (‘Ilam Hardy’) five days post-inoculation with Pectobacterium carotovorum subsp. brasiliensis NZEC1.
Figure 5: Soft rotting of a potato tuber (‘Ilam Hardy’) five days post-inoculation with Pectobacterium carotovorum subsp. brasiliensis NZEC1.

References

  1. Crowhurst RN, Wright PJ, 1998. Subspecies of Erwinia carotovora causing blackleg of potato in Pukekohe and Pukekawa and their survival in soils.  New Zealand Potato Bulletin 109, 20-22.
  2. Duarte V, De Boer SH, Ward LJ, de Oliveira AMR, 2004. Characterization of atypical Erwinia carotovora strains causing blackleg of potato in Brazil.  Journal of Applied Microbiology 96, 96:535. [http://dx.doi.org/10.1111/j.1365-2672.2004.02173.x]
  3. Kang HW, Kwon SW, Go SJ, 2003. PCR-based specific and sensitive detection of Pectobacterium carotovorum ssp. carotovorum by primers generated from a URP-PCR fingerprinting-derived polymorphic band. Plant Pathology 52, 127. [http://dx.doi.org/10.1046/j.1365-3059.2003.00822.x]
  4. Ma B, Hibbing ME, Kim HS, Reedy RM, Yedidia I, Breuer Jane, Breuer Jeffrey, Glasner JD, Perna NT, Kelman A, Charkowski AO, 2007. Host range and molecular phylogenies of the soft rot Enterobacterial genera Pectobacterium and Dickeya. Phytopathology 97, 1150-1163. [http://dx.doi.org/10.1094/PHYTO-97-9-1150 ]
  5. Pitman AR, Wright PJ, Galbraith MD, Harrow SA, 2008. Biochemical and genetic diversity of pectolytic enterobacteria causing soft rot disease of potatoes in New Zealand. Australasian Plant Pathology 37, 559-568. [http://dx.doi.org/10.1071/AP08056]
  6. Wang GCY, Wang Y, 1996.  The frequency of chimeric molecules as a consequence of PCR co-amplification of 16S rRNA genes from different bacterial species. Microbiology 142, 1107-1114. [http://dx.doi.org/10.1099/13500872-142-5-1107]
To cite this report: Panda P, Fiers MAWJ, Armstrong K, Pitman AR, 2012. First report of blackleg and soft rot of potato caused by Pectobacterium carotovorum subsp. brasiliensis in New Zealand. New Disease Reports 26, 15. [http://dx.doi.org/10.5197/j.2044-0588.2012.026.015]

©2012 The Authors