P. Panda1, M.A.W.J. Fiers2, K. Armstrong1 and A.R. Pitman1,2*
1 Bio-Protection Research Centre, Lincoln University, PO Box 84, Canterbury, New Zealand
2 New Zealand Institute for Plant & Food Research Ltd, Private Bag 4704, Christchurch, New Zealand
Received: 14 Jun 2012; Published: 19 Sep 2012
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.
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©2012 The Authors