Transcriptome Analysis of Eucalyptus grandis Implicates Brassinosteroid Signaling in Defense Against Myrtle Rust (Austropuccinia psidii)

Eucalyptus grandis, in its native Australian range, varies in resistance to Austropuccinia psidii (syn. Puccinia psidii). The biotrophic rust fungus, A. psidii is the causal agent of myrtle rust and poses a serious threat to Australian biodiversity. The pathogen produces yellow pustules of urediniospores on young leaves and shoots, resulting in shoot tip dieback, stunted growth, and death. Dissecting the underlying mechanisms of resistance against this pathogen will contribute to improved breeding and control strategies to mitigate its devastating effects. The aim of this study was to determine the molecular dialogue between E. grandis and A. psidii, using an RNA-sequencing approach. Resistant and susceptible E. grandis seedlings grown from seed collected across its natural range were inoculated with the pandemic biotype of A. psidii. The leaf tissue was harvested at 12-h post inoculation (hpi), 1-day post inoculation (dpi), 2-dpi and 5-dpi and subjected to RNA-sequencing using Illumina 50 bp PE reads to a depth of 40 million reads per sample. Differential gene expression and gene ontology enrichment indicated that the resistant seedlings showed controlled, coordinated responses with a hypersensitive response, while the susceptible seedlings showed no systemic response against myrtle rust. Brassinosteroid signaling was apparent as an enriched term in the resistant interaction at 2-dpi, suggesting an important role of this phytohormone in defense against the pathogen. Brassinosteroid mediated signaling genes were also among the candidate genes within two major disease resistance loci (Puccinia psidii resistance), Ppr3 and Ppr5. While brassinosteroids have been tagged as positive regulators in other plant disease resistance interactions, this is the first report in the Eucalyptus – Austropuccinia psidii interaction. Furthermore, several putative resistance genes, underlying known resistance loci and implicated in the interaction have been identified and highlighted for future functional studies. This study provided further insights into the molecular interactions between E. grandis and A. psidii, contributing to our understanding of this pathosystem.

The presence of Austropuccinia psidii and the threat to Myrtaceae plantations in Indonesia

Austropuccinia psidii is an invasive pathogenic rust that infects the Myrtaceae family. This rust is a threat to Myrtaceae plantations around the world due to its widespread distribution. In this study, we observed the presence of A. psidii in three species of Myrtaceae, i.e. Melaleuca cajuputi, Syzygium myrtifolium, and Syzygium polyanthum planted in Yogyakarta and Sukabumi. The symptoms of infection were yellow-reddish spot in young leaves, presence of urediniospores in infected spot, foliage, and branch dieback. To confirm the presence of A. psidii on those trees, a molecular detection was performed using specific primer for A. psidii (Ppsi1/Ppsi6) on DNA samples extracted from diseased leaves. The presence of A. psidii was proved by the presence of DNA amplicon sized around 500bp in all samples collected from three different hosts. In this study, S. myrtifolium was firstly reported to be infected by this rust in Indonesia. Further study about the presence and the economic impact of this pathogen in Indonesia should be conducted. Indonesia has many species numbers of Myrtaceae and some species are important for medicines, herbs, foods, and as industrial plants. A strategy to control this pathogen should be established to avoid large economic losses in Myrtaceae plantations in Indonesia.

Evaluating the efficacy of potential fungicide-adjuvant combinations for control of myrtle rust in New Zealand

Myrtle rust is a serious fungal disease caused by Austropuccinia psidii affecting a number of Myrtaceae species in New Zealand and elsewhere. Control with fungicides or biologicals provides a mechanism to reduce the build-up of inoculum in the short-term while other strategies are being developed or deployed for long-term management. This study evaluated the efficacy of fungicides for control of myrtle rust under controlled conditions and identified adjuvants that would promote spreading of fungicidal active ingredients across the leaf surface. The spread of fungicide on detached M. excelsa leaves was assessed by applying three different adjuvants in combination with seven fungicides. Subsequently, M. excelsa plants were treated with three fungicides/mixes, (azoxystrobin + epoxiconazole, triademinol or a natural tea-extract) at a single rate followed by inoculation with A. psidii urediniospores on day 0, 7, 14 or 21 days after spraying. The response to infection in M. excelsa plants based on different inoculation timings at days 0, 7 and 21 significantly differed among fungicide treatments. The fungicide azoxystrobin + epoxiconazole was the most effective with infections significantly lower on the adaxial leaf surface than abaxial, despite good surface coverage of fungicide being achieved on both leaf surfaces. There were significant differences among fungicides based on the proportion of infected leaves on M. excelsa plants. Day 21 post-spray inoculation indicated a significant interaction between inoculation time and fungicide on leaf disease ratings. However, this was not the case at either 28 or 35 days post-inoculation. This research contributes to fungicide options for myrtle rust control in New Zealand.

Leptospermum repo (Myrtaceae), a new species from northern Aotearoa / New Zealand peat bog habitats, segregated from Leptospermum scoparium s. l.

Leptospermum repo de Lange & L.M.H.Schmid sp. nov. (Myrtaceae) is segregated from L. scoparium J.R.Forst. & G.Forst. (sensu lato). The new species is endemic to the peat bogs of the Waikato, Bay of Plenty, and adjacent eastern ranges of the northern portion of Te Ika a Maui / North Island, Aotearoa / New Zealand. The new species belongs to the northern Te Ika a Maui / North Island clade of L. scoparium s. l., from whose other members it is morphologically distinguished by its gracile, spindly, open-branched growth habit; widely divergent, longer, linear, linear-lanceolate (rarely filiform), shortly cuspidate leaves; flowers with white petals, stamens mostly with white filaments (very rarely tinged pink near base), and by the style and stigma which are usually green (very rarely pink). Leptospermum repo now occupies ca. 10% of its former peat bog habitat, where aside from a few protected peat bogs the species is still in decline through loss of its habitat. A more recent potential threat to L. repo is myrtle rust disease, caused by Austropuccinia psidii, an exotic rust first detected in Aotearoa / New Zealand in May 2017. This rust poses a serious threat to the Myrtaceae of Aotearoa / New Zealand, for which there is at present no known effective treatment to prevent the death of infected hosts. Therefore, due to the historic loss of habitat, a threat which is still ongoing, causing the decline of many L. repo populations; and the potential threat of Austropuccinia psidii to this species, the listing proposed for the species (as Leptospermum aff. scoparium (c) (AK191319; "Waikato peat bog") by the New Zealand Indigenous Vascular Plant Threat Listing Panel of "Threatened / Nationally Critical DP, De" is here upheld.

Austropuccinia psidii (myrtle rust).

Austropuccinia psidii is a rust fungus with a wide and expanding host range within the Myrtaceae, with over 440 host species currently known (Carnegie and Lidbetter, 2012; Morin et al., 2012; Pegg et al., 2014). Like many rusts, urediniospores of A. psidii can be wind-dispersed over long distances. Viable spores have been detected on clothing and personal effects following visits to rust-affected plantations (Langrell et al., 2003), and this is a viable pathway for dispersal. Furthermore, there are several instances of (accidental) long-distance movement of A. psidii on diseased plants, both within and between continents (Loope et al., 2007; Kawanishi et al., 2009; Carnegie and Cooper, 2011; Zambino and Nolan, 2012). Under sub-optimal conditions, the rust can remain un-symptomatic within plants for more than a month (Carnegie and Lidbetter, 2012). This combination of wide host range and ease of long-distance dispersal make A. psidii a successful invasive pathogen. It has spread quickly once established in new countries, including Jamaica (MacLachlan, 1938), Hawaii (Uchida and Loope, 2009), Australia (Carnegie and Cooper, 2011; Pegg et al., 2014) and New Caledonia (DAVAR Nouvelle-Calédonie, 2014). Severe impact on a range of Myrtaceae has been recorded in amenity plantings, commercial plantations and the native environment. A. psidii was first identified as an invasive pathogen in the 1930s when it caused extensive damage to allspice (Pimenta dioica) plantations in Jamaica (Smith, 1935; MacLachlan, 1938). A. psidii has been identified as a quarantine risk for some time in many countries including Australia (Australian Quarantine Service, 1985; Grgurinovic et al., 2006), South Africa (Coutinho et al., 1998) and New Zealand (Kriticos and Leriche, 2008).

Revealing the high variability on nonconserved core and mobile elements of Austropuccinia psidii and other rust mitochondrial genomes

Mitochondrial genomes are highly conserved in many fungal groups, and they can help characterize the phylogenetic relationships and evolutionary biology of plant pathogenic fungi. Rust fungi are among the most devastating diseases for economically important crops around the world. Here, we report the complete sequence and annotation of the mitochondrial genome of Austropuccinia psidii (syn. Puccinia psidii), the causal agent of myrtle rust. We performed a phylogenomic analysis including the complete mitochondrial sequences from other rust fungi. The genome composed of 93.299 bp has 73 predicted genes, 33 of which encoded nonconserved proteins (ncORFs), representing almost 45% of all predicted genes. A. psidii mtDNA is one of the largest rust mtDNA sequenced to date, most likely due to the abundance of ncORFs. Among them, 33% were within intronic regions of diverse intron groups. Mobile genetic elements invading intron sequences may have played significant roles in size but not shaping of the rust mitochondrial genome structure. The mtDNAs from rust fungi are highly syntenic. Phylogenetic inferences with 14 concatenated mitochondrial proteins encoded by the core genes placed A. psidii according to phylogenetic analysis based on 18S rDNA. Interestingly, cox1, the gene with the greatest number of introns, provided phylogenies not congruent with the core set. For the first time, we identified the proteins encoded by three A. psidii ncORFs using proteomics analyses. Also, the orf208 encoded a transmembrane protein repressed during in vitro morphogenesis. To the best of our knowledge, we presented the first report of a complete mtDNA sequence of a member of the family Sphaerophragmiacea.