Rust (Puccinia psidii) recorded in Indonesia poses a threat to forests and forestry in South-East Asia

Abstract

Over the past decade, Puccinia psidii, which causes rust on species of Myrtaceae, has spread rapidly to new areas and is now widespread. Quarantine has done little to prevent its movement through the Americas, the Pacific, Africa, and in this report, South-East Asia. Puccinia psidii is reported for the first time from Indonesia on two genera of Myrtaceae, namely Eucalyptus and Melaleuca. Its identity was confirmed using morphology, a molecular barcode comparison to an epitype specimen, and with a molecular phylogenetic approach. Comparison of seven microsatellite markers indicates that the rust genotype in Indonesia and Australia is identical. The potential impacts of P. psidii in South-East Asia to the natural environment and plantation forestry are discussed in light of this first report from the region.

Full text

Rust (Puccinia psidii) recorded in Indonesia poses a threat to forests
and forestry in southeast Asia
Alistair R. McTaggart
1
, Jolanda Roux
2
, Abdul Gafur
3
, Marthin Tarrigan
3,
Santhakumar
3
, Michael J. Wingfield
1
1
Department of Microbiology and Plant Pathology, Tree Protection Co-
operative Programme (TPCP), Forestry and Agricultural Biotechnology
Institute (FABI), Private Bag X20, University of Pretoria, Pretoria, 0028, South
Africa
2
Department of Plant Sciences, Tree Protection Co-operative Programme
(TPCP), Forestry and Agricultural Biotechnology Institute (FABI), Private Bag
X20, University of Pretoria, Pretoria, 0028, South Africa
3
Riau Andalan Paper and Pulp ****
corresponding author e-mail: Alistair.McTaggart@fabi.up.ac.za
Abstract
Over the past decade, Puccinia psidii, which causes rust on species of
Myrtaceae, has spread rapidly to new areas and is now widespread.
Quarantine has done little to prevent its movement through America, the
Pacific, Africa, and in this report, South-East Asia. Puccinia psidii is reported
for the first time from Indonesia on two genera of Myrtaceae, namely
Eucalyptus and Melaleuca. The identity was confirmed by morphology, a
molecular barcode comparison to an epitype specimen, and with a molecular
phylogenetic approach. The potential impacts of P. psidii in South-East Asia
to the natural environment and plantation forestry are discussed in light of this
first report from the region.
Keywords: disease report, Melaleuca, Myrtaceae, plant disease, plant
pathogen, Pucciniales, quarantine, Sphaerophragmiaceae, Uredinales
INTRODUCTION
Puccinia psidii (Pucciniales, Pucciniomycotina) causes rust on plants in the
Myrtaceae and is a serious threat to Eucalyptus plantations and native
ecosystems with myrtaceous species (Coutinho et al. 1998, Glen et al. 2007).
The pathogen has a wide host range and is reported from approximately 244
species in 56 genera (Machado et al. 2015). In this regard, it is unusual in
being one of only a few rust fungi that occur on multiple host genera. It was
first described on Psidium guajava from Brazil, and was believed to have
undergone a host shift (see Slippers et al. 2005) to species of Eucalyptus and
Syzygium jambos (Castro et al. 1983). However, the populations of P. psidii
on Eucalyptus and Psidium diverged more than 1000 years ago, and a host
shift most likely did not occur after the introduction of Eucalyptus to Brazil
(Graça et al. 2013). The genotype of P. psidii on Eucalyptus and S. jambos
has an unexplained origin (Graça et al. 2013).

Puccinia psidii is known from two lifecycle stages, uredinia and telia. The
mitotic, uredinial stage produces masses of yellow urediniospores that
defoliate juvenile trees, cause severe stem and foliage blight, and can affect
developing fruits and inflorescences (Pegg et al. 2014). The symptoms
produced by the uredinia of P. psidii are characteristic and useful to
distinguish it from other rusts on Myrtaceae, such as Phakopsora
myrtacearum (Maier et al. 2015). A study on the lifecycle of P. psidii could not
conclusively determine the function of the meiotic, telial stage, and indicated
that populations of P. psidii are clonal and spread by uredinia (Morin et al.
2014).
Unmitigated spread of P. psidii occurred through South America (Telechea et
al. 2003; Rodas et al., 2015), North America (Marlatt & Kimbrough 1979),
Hawaii (Uchida et al. 2006), Japan (Kawanishi et al. 2009), Australia (as
Uredo rangelii) (Carnegie et al. 2010), China (Zhuang & Wei 2011), South
Africa (Roux et al. 2013) and New Caledonia (Giblin 2013). Wingfield et al.
(2001) observed a trend in the increased movement of plant material with new
reports of plant disease, despite the efforts of quarantine; this trend is
highlighted by the global spread of P. psidii.
In July 2015, symptoms of rust similar to P. psidii were observed on
Eucalyptus pellita and Melaleuca leucadendra in north and south Sumatra,
Indonesia. Morphology, and a molecular barcoding and phylogenetic
approach with markers from ribosomal DNA (rDNA) were used to identify the
cause of rust on these myrtaceous hosts in Indonesia. The potential impact of
P. psidii in South-East Asia is discussed.
MATERIALS AND METHODS
DNA was extracted from pressed and dried rust specimens. Uredinia were
selectively removed from plant material and DNA was extracted with the
UltraClean Microbial DNA Isolation Kit (MoBio Laboratories Inc., Solana
Beach, CA, USA).
The internal transcribed spacer region (ITS) of ribosomal DNA (rDNA) was
amplified with primers ITS1F (Gardes & Bruns 1993)/ITS4rust (Beenken et al.
2012). The ITS2-Large Subunit (LSU) region of rDNA was amplified with
Rust2inv (Aime 2006)/LR6 (Vilgalys & Hester 1990). PCRs were performed
with FastStart Taq (Roche Diagnostics Corporation, Indianapolis, USA)
according to the manufacturer’s instructions. The PCRs were performed with
the following annealing temperatures: ITS at 55°C and LSU at 62°C. PCR
products were cleaned by an ethanol precipitation and sequenced in both
directions using an ABI PRISM Dye-Terminator Cycle Sequencing Kit
(Applied Biosystems) on an automated ABI 3130xl sequencer at the DNA
Sequencing Facility of the Faculty of Natural and Agricultural Sciences,
University of Pretoria. Sequences were assembled using the CLC Main
Workbench (Qiagen).
A phylogenetic species concept was tested for the rust collected in Indonesia.
Puccinia psidii, rusts on Myrtaceae, and members of the

Sphaerophragmiaceae that have an affinity with P. psidii (Beenken & Wood
2015, Maier et al. 2015) were included in the analyses (Table 1). The LSU
region of all taxa was aligned with the MAFFT algorithm (Katoh et al. 2009) in
SATe (Liu et al. 2012), and run under two phylogenetic criteria, Bayesian
inference and maximum likelihood. GTRGAMMA with an estimate of
invariable sites was used as the model of evolution in both criteria. MrBayes
was used to conduct a Markov Chain Monte Carlo (MCMC) search with
Bayesian inference (Ronquist & Huelsenbeck 2003). Four runs, each
consisting of four chains, were implemented for 10 million generations. The
cold chain was heated at a temperature of 0.25. Substitution model
parameters were sampled every 1000 generations and trees were saved
every 1000 generations. Convergence of the Bayesian analysis was
confirmed using the cumulative and compare functions in AWTY (Nylander et
al. 2008) (available at: ceb.csit.fsu.edu/awty/) and 30,004 trees were
summarized. Maximum likelihood (ML) was implemented as a search criterion
in RAxML (Stamatakis 2014). The RAxML analyses were run with a rapid
Bootstrap analysis (command -f a) using a random starting tree and 1000
maximum likelihood bootstrap replicates.
RESULTS
The urediniospores and teliospores matched the morphology of P. psidii
reported in Australia (available at: http://collections.daff.qld.gov.au/web/key/
rustfungi/Media/Html/pucciniapsidii.html; Shivas et al. 2014). Uredinia
occurred on leaves and petioles, single or gregarious, amphigenous on young
shoots and leaves, erumpent, round, up to 0.5 mm, bright yellow to yellowish
brown (Fig. 1A). Urediniospores were globose to ovoid, pyriform, yellowish
brown, 13‒20 × 12‒ 16 µm; wall 1.5‒2.0 µm thick, finely echinulate, germ pore
absent or inconspicuous (Fig. 1B, C). Telia occurred on leaves, up to 0.5 mm
diam., abaxial, erumpent, pulvinate, yellowish brown to brown. Teliospores
were cylindrical or ellipsoidal, apex rounded, pale yellowish brown, 22‒38 ×
14‒18 µm; wall 1‒2 µm thick, smooth, 2–3 celled, short remnant of pedicel
attached up to 15 µm long, phragmobasidium up to 60 µm long (Fig. 1D, E).
Specimens examined: INDONESIA, North Sumatra, Porsea, on Eucalyptus
grandis x E. pellita clone collected by M.J. Wingfield, 10 July 2015, PREM
****** (GenBank ITS: ********, LSU: ********); on Melaleuca leucadendra,
collected by M.J. Wingfield, 01 July 2015, PREM ****** (GenBank ITS:
********, LSU: ********); Sumatra, Pelalawan, on M. leucadendra, collected by
M.J. Wingfield, 10 July 2015, PREM ****** (GenBank ITS: ********, LSU:
********).
The ITS regions of the specimens from Indonesia on Eucalyptus (GB******)
and Melaleuca (GB****** and GB******) had 563/567 identities to the epitype
of P. psidii (KM282154) (Machado et al. 2015) in a BLASTn search on
GenBank. Three of the mismatched bases were degenerate bases caused by
intra-individual single nucleotide polymorphisms, and one was caused by a
nucleotide mismatch. The LSU region had high identity to P. psidii (KM282159
1098/1098 identities and KF318436 1013/1013 identities) in a BLASTn
search.

All isolates of P. psidii, which included the specimens from Indonesia, were
recovered in a monophyletic group under both phylogenetic criteria (Fig. 2).
Puccinia psidii was recovered as sister to species of Dasyspora in the
Sphaerophragmiaceae.
DISCUSSION
The present study is the first report of P. psidii in Indonesia and the first report
of this pathogen from South-East Asia. Indonesia, Malaysia, Thailand and
Vietnam have Eucalyptus plantations on a combined area of approximately
1,020,000 ha (Harwood & Nambiar 2014). The discovery of P. psidii in
Indonesia and its importance as a Eucalyptus pathogen is likely to have
implications for forest industries in the region. The pathogen also poses a
potential threat to native species of Myrtaceae, which are represented by
approximately 30 genera in Indonesia (Craven et al. 2003).
Puccinia psidii has spread globally during the past decade. The pathogen
reached northern Asia (Kawanishi et al. 2009) and Australia (Carnegie et al.
2010) relatively recently. Countries in South-East Asia have native and
introduced species of Myrtaceae suitable for infection by P. psidii, and its
discovery in Indonesia is perhaps not surprising. It is unknown how long P.
psidii has been present in Sumatra but it was undetected in active surveys in
the region during the past 20 years (Wingfield, unpublished). This suggests
that its establishment in Sumatra is relatively recent. Thus far, P. psidii has
been found only on Eucalyptus and a native species of Myrtaceae, M.
leucadendra. It is likely to infect many native and non-native Myrtaceae in
Indonesia in the future.
The present study recovered P. psidii within the Sphaerophragmiaceae sister
to the Pucciniaceae, a relationship that was previously shown by Maier et al.
(2015) and Beenken & Wood (2015). Puccinioid spores are a homoplasious
character within the Pucciniales (Beenken & Wood 2015). The generic
placement of P. psidii will require the resolution of other puccinioid genera
within the Sphaerophragmiaceae and Uropyxidaceae sensu Cummins &
Hiratsuka (2003).
The genotype of P. psidii on Eucalyptus has an unknown origin (Graça et al.
2013). New disease reports from countries with susceptible hosts eliminate
these origins as potential sources, as the rust would be expected to occur
there already. Previous studies have indicated the origin of P. psidii may be
from a location with shared plants in the Annonaceae and Myrtaceae (Maier
et al. 2015). Future studies will determine the genotype of P. psidii in
Indonesia and its likely pathway of entry.
Tropical Asia is one of the centers of diversity for species of Myrtaceae with
over 800 described species of Syzygium (Govaerts et al. 2008). One of these
species, S. jambos, is among the most susceptible plants to infection by P.
psidii. While commercial interests such as those relating to Eucalyptus
propagation are of concern, the greatest threat of P. psidii will likely reside in
the damage caused to the native environment. This is consistent with

observations in Australia where the rust has brought 12 species of native
Myrtaceae to the brink of extinction (Pegg et al. 2014).
Pathogens of native Myrtaceae in Asia are already recognized as threats to
Eucalyptus propagation in the region. For example, various species of the
Chryphonectriaceae found on native Syzygium spp. in China were shown to
be potential, important pathogens of Eucalyptus spp. grown in plantations
(Chen et al. 2010). The extensive and growing Eucalyptus plantation industry
in Indonesia and South-East Asia is likely to be affected by P. psidii in the
future. However, there are many opportunities to manage the negative
impacts of this and other pathogens in plantations. In this regard, breeding,
selection and the propagation of resistant genotypes will be amongst the most
effective strategies (Wingfield et al. 2008, Wingfield et al. 2013; Wingfield et
al. 2015).
The rapid, global spread of P. psidii is of concern and illustrates the ease with
which tree pests and pathogens move Wingfield et al., 2015). Pathways of
spread differ depending on the ecology of the organisms involved. In the case
of P. psidii, local spread is likely to occur rapidly via wind-borne spores. Long-
distance spread has most likely occurred via transport of living plant tissue.
Species of Myrtaceae susceptible to P. psidii produce edible fruits, which
could be infected and moved to new areas. However, horticultural trade in
living plant material including eucalypts and other Myrtaceae, represents one
of the most important potential pathways of introduction into new areas. Great
care should thus be taken to discourage exchange of potentially hazardous
plant material between countries and continents.
ACKNOWLEDGEMENTS
We thank the members of the Tree Protection Co-operative Programme
(TPCP), the THRIP initiative of the Department of Trade and Industry, and the
Department of Science and Technology (DST) / National Research
Foundation (NRF) Centre of Excellence in Tree Health Biotechnology (CTHB)
for financial assistance that made this study possible.
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psidii%2$52=%3/:-(%]=56/3%G"-3-68%EEMD%)PKF)+K(%
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Table 1. Taxon names and LSU GenBank numbers for isolates used in the
phylogenetic analyses.
Taxon
GenBank number
Reference
Allodus podophylli
JQ423285
Minnis et al. (2012)
Caeoma torreyae
AF522183
Szaro and Bruns unpublished
Coleosporium plumeriae
KM249866
McTaggart et al. (2014b)
Cronartium ribicola
DQ354560
Aime (2006)
Dasyspora echinata
JF263462
Beenken et al. (2012)
Dasyspora gregaria
JF263474
Beenken et al. (2012)
Dasyspora guianensis
JF263479
Beenken et al. (2012)
Dasyspora winteri
JF263492
Beenken et al. (2012)
Endoraecium phyllodiorum
KJ862324
McTaggart et al. (2015)
Endoraecium tierneyi
KJ862335
McTaggart et al. (2015)
Hamaspora acutissima
KT199398
McTaggart et al. unpublished
Hemileia vastatrix
DQ354566
Aime (2006)
Kernkampella breyniae
KJ862346
McTaggart et al. (2015)
Maravalia cryptostegiae
KT199401
McTaggart et al. unpublished
Masseeëlla capparis
JX136798
Liberato et al. (2014)
Phakopsora annonae-sylvaticae
KF528998
Beenken (2014)
Phakopsora cherimoliae
KF528012
Beenken (2014)
Phakopsora myrtacearum
KP729473
Maier et al. (2015)
Phakopsora pistila
KF528028
Beenken (2014)
Phakopsora rolliniae
KF528036
Beenken (2014)
Phragmidium mexicanum
DQ354553
Aime (2006)
Prospodium lippiae
DQ354555
Aime (2006)
Prospodium tuberculatum
KJ396195
Pegg et al. (2014)
Puccinia graminis
KM249852
McTaggart et al. (2014b)
Puccinia lagenophorae
KF690700
McTaggart et al. (2014a)
Puccinia psidii
Puccinia psidii
Puccinia psidii
Puccinia psidii
KF318447
Pegg et al. (2014)
Puccinia stylidii
KJ622215
McTaggart et al. (2014a)
Puccinia ursiniae
KF690705
McTaggart et al. (2014a)

Ravenelia neocaldoniensis
KJ862348
McTaggart et al. (2015)
Sphaerophragmium sp.
KJ862350
McTaggart et al. (2015)
Sphenorchidium polyalthiae
JF263493
Beenken et al. (2012)
Thekopsora minima
KC763340
McTaggart et al. (2013)
Uredinopsis pteridis
KM249869
McTaggart et al. (2014b)
Uromycladium acaciae
KR612235
McTaggart et al. unpublished
Uromycladium simplex
KJ632990
Doungsa-ard et al. (2015)
Fig. 1. Phylogram obtained from a maximum likelihood search in RAxML with
a dataset of the large subunit region of ribosomal DNA. Bootstrap values
(≥70%) from 1000 replicates above nodes. Posterior probabilities (≥0.95)
summarized from 30,004 trees in a Bayesian search in MrBayes below nodes.
GTRGAMMA with an estimate of invariable sites was the model of evolution
0. 05
Caeoma torreyae
Maravalia cryptostegiae
Hemileia vastatrix
Allodus podophylli
Thekopsora minima
Uredinopsis pteridis
Cronartium ribicola
Coleosporium plumeriae
Hamaspora acutissima
Phragmidium potentillae
Phragmidium mexicanum
Endoraecium tierneyi
Endoraecium phyllodiorum
Ravenelia neocaledoniensis
Kernkampella breyniae
Phakopsora pachyrhizi
Masseeëlla capparis
Phakopsora myrtacearum
Phakopsora crucis-filii
Phakopsora annonae-sylvaticae
Prospodium lippiae
Prospodium tuberculatum
Uromycladium simplex
Uromycladium acaciae
Puccinia graminis
Puccinia lagenophorae
Puccinia ursiniae
Sphaerophragmium sp.
Sphenorchidium polyalthiae
Dasyspora gregaria
Dasyspora echinata
Dasyspora winteri
Dasyspora guianensis
Puccinia psidii BRIP 58517
Puccinia psidii BRIP 58164
Puccinia psidii
Puccinia psidii
Puccinia psidii
100
100
100
100
96
99
91
100
100
99
76
100
100
96
72
94
77
87
94
Mikronegeriaceae
Incertae sedis
Coleosporiaceae sensu Aime (2006)
Phragmidiaceae
Raveneliaceae
Phakopsoraceae
Uropyxidaceae
Incertae sedis
Pucciniaceae
Sphaerophrag-
miaceae

for both phylogenetic criteria. Taxon name and GenBank numbers listed in
Table 1.
Fig. 2. Puccinia psidii on Melaleuca leucadendron (PREM ******). a. host
symptoms b. equatorial plane of urediniospores. c. surface of urediniospores.
d-e. teliopspores. Scale bars = 10 µm.