A Unique Genotype of the Rust Pathogen, Puccinia Psidii, on Myrtaceae in South Africa

Australasian Plant Pathol. DOI 10.1007/s13313-016-0447-y ORIGINAL PAPER A unique genotype of the rust pathogen, Puccinia psidii, on Myrtaceae in South Africa J. Roux1 & G. M. Granados1 & L. Shuey 2 & I. Barnes2 & M. J. Wingfield1 & A. R. McTaggart1 Received: 11 July 2016 /Accepted: 23 September 2016 # Australasian Plant Pathology Society Inc. 2016 Abstract The rust pathogen, Puccinia psidii, was first detect- Keywords Eugenia natalitia . Guava rust . Myrtle rust . ed in South Africa in 2013 on a single non-native ornamental Myrtus communis Myrtus communis tree. This prompted surveys of the country to determine its geographic distribution and host range. Previously developed microsatellite markers where used to Introduction characterize the genetic diversity of P. psidii isolates collected from these surveys. In addition, artificial inoculation studies The rust pathogen, Puccinia psidii Winter (Urediniales, and field observations were used to evaluate the susceptibility Sphaerophragmiaceae), has been described as Bthe of native Myrtaceae to infection by P. psidii. The pathogen greatest threat to the ecosystem^ by Australian scientists was found on native Myrtaceae in isolated natural situations and newspapers (Dayton and Higgins 2011;Glenetal. and it was also common on exotic Myrtaceae in nurseries and 2007;Peggetal.2014b). Subsequent to its first detection gardens. Marker analysis showed that a single genotype of in Australia (Carnegie et al. 2010), P. psidii has led to the the rust is present in South Africa and that this is different near extinction of 12 species of native Australian to the so-called Bpandemic^ strain recorded in countries Myrtaceae (Carnegie et al. 2016;Peggetal.2014b). outside Brazil. It was found to have a broad distribution in The pathogen has also had a significant impact on agri- South Africa with collections as far as 1500 km apart. The culture and commercial plantationforestryinotherre- data provide firm evidence for a single introduction of the gions where it occurs, devastating the allspice (Pimenta pathogen from an as yet unkown source. Its wide distri- dioica) industry in Jamaica (MacLachlan 1938)and bution, particularly in relatively isolated natural areas, resulting in considerable management costs to the euca- suggests that P. psidii has been present in South Africa lypt industry in Brazil (Alfenas et al. 2004;Ferreira1983; for much longer than implied by its first detection in the Graça et al. 2011). country. Puccinia psidii was first reported in 1884 from southern Brazil, infecting Psidium guajava (Winter 1884). The fungus has subsequently been reported from North America (Marlatt and Kimbrough 1979) and Hawaii (Uchida et al. 2006), China (Zhuang and Wei 2011), Japan (Kawanishi et al. 2009), * J. Roux Australia (Carnegie et al. 2010), Africa (Roux et al. 2013), [email protected] New Caledonia (Giblin 2013; Machado et al. 2015)and Indonesia (McTaggart et al. 2016). All of these reports, out- 1 side Brazil, were attributed to a single genoptype of Department of Plant and Soil Sciences, Forestry and Agricultural ‘ Biotechnology Institute (FABI), University of Pretoria, Private Bag P. psidii (Machado et al. 2015), referred to as the pan- X20, Pretoria 0028, South Africa demic’ genotype (Ross-Davis et al. 2013). It is widely 2 Department of Genetics, Forestry and Agricultural Biotechnology believed that P. psidii is native to Latin America due to Institute (FABI), University of Pretoria, Private Bag X20, its long history in that region and the fact that native Pretoria 0028, South Africa Myrtaceae in the region are generally tolerant to infection, J. Roux et al. as opposed to the susceptible introduced species of Myrtaceae to P. psidii using inoculation studies under Myrtaceae (Coutinho et al. 1998;Glenetal.2007). greenhouse conditions. Recent population genetic studies on collections of P. psidii from the Americas also suggest that host specific genotypes are native to Central and South America (Graça Materials and methods et al. 2013; Zhong et al. 2008, 2011). For many years it was believed that P. guajava (guava) Disease surveys and sample collections was the host of origin of P. psidii and that it had under- gone a host jump to non-native Eucalyptus and other Surveys of Eucalyptus plantations, nurseries and native stands Myrtaceae (Alfenas et al. 2005; Castro de et al. 1983; of Myrtaceae were conducted in 2013, 2014 and 2015 in order Tommerup et al. 2003). However, a study by Graça to assess the current distribution of P. psidii in South Africa. et al. (2013) showed that isolates from guava and euca- Sites were selected based on occurrence maps of native lypts in Brazil represented distinct genotypes/biotypes of Myrtaceae (www.sanbi.org) published by the South African the pathogen. The source population of the rust that oc- National Biodiversity Institute (SANBI) and as identified by curs on eucalypts remains unknown although it seems Roux et al. (2015) as high risk areas for the occurrence of P. likely that this would be in South or Central America. psidii in the country. These included areas along the coast of the Puccinia psidii hasbeenreportedfrommorethan73gen- KwaZulu-Natal Province, the Mbombela region in the era and 450 species of plants in the Myrtaceae (Pegg et al. Mpumalanga Province and the Tzaneen and Soutpansberg re- 2014b; Giblin and Carnegie 2014; Zauza et al. 2015). This gions in the Limpopo Province. Reports of possible infected plant family includes approximately 140 genera accommodat- plants in private gardens, made by the general public, were ing about 5500 species and it occurs worldwide (Biffin et al. investigated after awareness campaigns in the media and garden 2010). Three genera of the Myrtaceae occur in South forums. Eucalypt nurseries and plantations in high risk areas Africa, namely Eugenia, Metrosideros and Syzygium werevisitedonanadhocbasis,andmonitoredbyforestrystaff. (Coates Palgrave 2002). A closely related genus, Heteropyxis, is endemic to southern Africa and has been Fragment analyses and genotyping classified either in its own family, the Heteropyxidaceae, or a sub-family within the Myrtaceae (Stevens 2012; Seven P. psidii samples collected from countrywide surveys, Wilson et al. 2005). Of the South African species of as well as four isolates from Australia, were used in the genetic Myrtaceae, only H. natalensis has been tested for its sus- analyses (Table 1). Urediniospores were harvested from in- ceptibility to P. psidii. Artificial inoculation trials in Brazil fected leaf samples using a vacuum pump and stored in showed that H. natalensis was highly susceptible to the 2 mL screw- cap vials at −80 °C prior to DNA extraction. genotype of rust fungus used in that study (Alfenas et al. The genotypes of samples were determined using seven mi- 2005). Despite the relatively low number of native crosatellite markers (PpSS012, PpSS014, PpSS018, Myrtaceae in South Africa and other African countries, PpSS022, PpSS102, PpSS161 and PpSS195) developed by compared to Australia and South America, these plants are important components of the natural ecosystem, pro- Table 1 Puccinia psidii samples used for microsatellite analyses viding fruits for humans and animals, timber and medic- Location inal compounds (Coates Palgrave 2002). Puccinia psidii was first detected in South Africa in Host – South Africa May 2013, on a single Myrtus communis tree, in a private Backhousia citriodora Private Garden, Irene, Gauteng garden on the south coast of KwaZulu-Natal (Roux et al. Eugenia erythrophylla Nursery, Port Edward, KwaZulu-Natal 2013). Subsequent to its detection, many articles have been Eugenia natalitia Natural forest, Grootbos, Modjadjiskloof, published in garden and botanical magazines as well as in the Limpopo media to raise awareness about the disease in South Africa E. natalitia Natural forest, Wolkberg, Limpopo (Roux 2015;RouxandPegg2014). Disease alerts and regular Heteropyxis natalensis Nursery, Port Edward, KwaZulu-Natal updates of Myrtle rust have also been provided to the South Myrtus communis Private Garden, Pennington, KwaZulu-Natal African forestry industry through newsletters and the web M. communis Nursery, Tshwane, Gauteng (http://fabinet.up.ac.za/archive). The aims of this study were Host – Australia to determine the distribution of P. psidii subsequent to its first Backhousia citriodora The Channon, New South Wales report in South Africa; determine whether the pandemic Gossia inophloia Salisbury, Queensland genotype of P. psidii is present in South Africa by comparison Melaleuca viminalis Chapel Hill, Queensland with specimens from Australia using microsatellite markers, and Rhodamnia rubescens Chapel Hill, Queensland evaluate the relative susceptibility of native South African A unique genotype of the rust pathogen, Puccinia psidii Zhong et al. (2008) and modified by Graça et al. (2013). mixed to ensure an even distribution of the inoculant. Spore Genomic DNAwas extracted from a single uredinium per host counts were made using a haemocytometer and the suspen- using the Ultraclean® Microbial DNA Isolation Kit (MoBio sion adjusted to a concentration of 1 × 105 spores/ml. Native Laboratories, Carlsbad, California, USA). PCR mixtures in- plants were inoculated with a fine mist of the spore suspension cluded 1× PCR FastStart Taq Buffer with MgCl2 (Sigma- using an artist airbrush (Iwata LPH-80) powered by a 1/6 HP Aldrich, St. Louis, Missouri, USA), 200 μMdNTPs, tubular compressor (Iwata IS 875HT Smart Jet Plus). The 0.1 μM forward (labeled with

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