Rapid screening of commercial forestry species to Uredo rangelii

SUSTAINABILITY & RESOURCES
PROJECT NUMBER: PRC179-0910
AUGUST 2010
Rapid screening of commercial
forestry species to Uredo rangelii
(myrtle rust) and distinguishing U.
rangelii from Puccinia psidii
(guava rust)
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Rapid screening of commercial forestry species to
Uredo rangelii (myrtle rust) and distinguishing U.
rangelii from Puccinia psidii (guava rust)
Prepared for
Forest & Wood Products Australia
by
Angus J. Carnegie, Morag Glen & Caroline Mohammed
Publication: Rapid screening of commercial forestry species to
Uredo rangelii (myrtle rust) and distinguishing U.rangelii from
Puccinia psidii (guava rust)
Project No: PRC179-0910
This work is supported by funding provided to FWPA by the Australian Government Department
of Agriculture, Fisheries and Forestry (DAFF).
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ISBN: 978-1-921763-10-6
Principal Researcher:
A. J. Carnegie & J. R. Lidbetter
Forest Science Centre, Industry & Investment NSW
PO Box 100, Beecroft, NSW, 2119
M. Glen & C. Mohammed
Tasmanian Institute of Agricultural research
University of Tasmania, Hobart, Tasmania
Final report received by FWPA in August, 2010
Forest & Wood Products Australia Limited
Level 4, 10-16 Queen St, Melbourne, Victoria, 3000
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W www.fwpa.com.au
Executive Summary
A fungal disease was detected on 23 April 2010 on Agonis flexuosa (willow myrtle)
grown for cut flowers at a property on the central coast of NSW. The pathogen causing
the disease was identified by its spore morphology as myrtle rust (Uredo rangelii), a
fungal species first described in 2006 as belonging to a complex of fungal species similar
to guava rust (Puccinia psidii).
Guava or eucalypt rust caused by Puccinia psidii is one of the most serious disease threats
to Australian native flora. Of most significance to commercial forestry, guava rust is a
serious disease in species of Eucalyptus, Corymbia and Melaleuca.
Myrtle rust is described from a very small number of specimens with a limited host range
and does not have any associated biological information. Guava rust has received
significant attention overseas and has a reputation as a damaging pathogen.
The identification of the incursion pathogen as an unknown quantity - myrtle rust - was
one factor that contributed towards a confused and slow response to the pathogen. The
pathogen should have been immediately eradicated.
This project therefore was aimed at bringing clarity to the situation, to provide
information which would highlight the need for speedy eradication measures. The
objectives were;
•
To see if molecular analyses could discriminate between myrtle rust and guava
rust
•
To test the susceptibility of key commercial eucalypt species to myrtle rust
The outcomes of the project are clear and indisputable.
•
None of the gene regions sequenced support the distinction of Uredo rangelii
from Puccinia psidii, or provide any evidence that a “species complex” exists.
•
Key commercial forestry species are susceptible to “myrtle” rust, including those
used in plantations and native forestry in NSW and Queensland.
These knowledge outcomes benefit the industry by providing the correct information
about the pathogen detected in NSW, its biology and the level of biosecurity threat posed
i to commercial forestry. Without this knowledge adequate pressure may not be exerted to
eradicate/contain the pathogen. Forestry can consider the full spectrum of possible
outcomes of this incursion including trade implications, germplasm movement embargos
and the cost of management strategies if the pathogen becomes widespread.
To avoid confusion myrtle rust should now be referred to as guava rust (Puccinia psdii)
although the guava rust in NSW may represent a variety of guava rust. It is evident that
the rust in NSW does not have a restricted host range (as was originally claimed for
myrtle rust) and at least 15 species in 11 genera are susceptible to this rust that has
entered NSW. In the fight against any pathogen of damaging importance “Know your
enemy” is a concept of paramount importance.
Given the high biosecurity threat posed by guava rust to the industry, it should be vigilant
about the progress of the eradication campaign in NSW and the decisions that may drive
the stand down of this campaign.
The industry should be proactive in planning for the worst case scenario that this
pathogen is not eradicated or contained around the initial infection sites in NSW.
ii Table of Contents
Executive Summary .................................................................................................................... i
Introduction ................................................................................................................................ 1
Methodology .............................................................................................................................. 3
Rapid screening of commercial forestry species to U. rangelii ............................................. 3
Test plants and inoculation ................................................................................................. 3
Experiment I ....................................................................................................................... 3
Experiment II...................................................................................................................... 3
Experiment III .................................................................................................................... 4
Experiment IV .................................................................................................................... 4
Morphological confirmation of infection by U. rangelii ................................................... 5
Distinguishing U. rangelii from P. psidii ............................................................................... 5
DNA amplification and phylogenetic analysis................................................................... 5
Results ........................................................................................................................................ 6
Host Testing ........................................................................................................................... 6
Experiment I & Experiment II ........................................................................................... 6
Experiment III .................................................................................................................... 6
Experiment IV .................................................................................................................... 6
Distinguishing U. rangelii from P. psidii ............................................................................... 7
Discussion ................................................................................................................................ 16
Conclusions .............................................................................................................................. 18
Recommendations .................................................................................................................... 19
References ................................................................................................................................ 20
Acknowledgements .................................................................................................................. 22
Introduction
Native to South and Central America, Puccinia psidii infects a broad range of hosts in the
Myrtaceae, including introduced species such as Syzygium jambos and Eucalyptus spp. It is
recognised as a threat to Australia’s environment and economy if it were to become
established in Australia (Glen et al. 2007a). Simpson et al (2006) described a new species,
Uredo rangelii, distinguished from Uredo psidii, the anamorph of P. psidii, based on the
presence of a tonsure on the urediniospore, though this character is considered to be a variable
characteristic of P. psidii by Brazilian plant pathologists. The description was based on two
old herbarium specimens and no new collections were included. No DNA sequences were
provided to support the species separation.
Uredo rangelii (myrtle rust) was detected in Australia in April 2010 on the Central
Coast of NSW (Carnegie et al. 2010). U. rangelii is a taxon within the Puccinia psidii s.l.
(guava rust) complex; it is morphologically distinct from P. psidii s.s. (Simpson et al. 2006),
though the ribosomal DNA (rDNA) internal transcribed spacer (ITS) sequence was
indistinguishable from that of P. psidii (Carnegie et al. 2010). This gene region is known to
discriminate species of Puccinia, including in species complexes (Szabo 2006).
Known hosts of U. rangelii in Australia are Agonis flexuosa, Callistemon viminalis
and Syncarpia glomulifera (Carnegie et al. 2010), while overseas U. rangelii is known from
Myrtus communis and Syzygium jambos (Simpson et al. 2006). The five known hosts of U.
rangelii are from five separate “tribes” within Myrtaceae (Wilson et al. 2005), and as such the
host range is expected to be larger (Carnegie et al. 2010). P. psidii s.l. has a large host range,
being reported on over 75 species from 16 genera within Myrtaceae (Coutinho et al. 1998;
Simpson et al. 2006; Office of the Chief Plant Protection Officer 2007; Zauza et al. 2010).
As U. rangelii has only recently been separated from P. psidii s.l. (Simpson et al. 2006), and
is not considered a valid species by Brazilian pathologists (Alfena, pers. comm..), it is likely
that literature pertaining to P. psidii s.l., such as host range and impact, includes reference to
disease caused by U. rangelii (Carnegie et al. 2010).
Numerous authors have conducted host susceptibility tests for P. psidii s.l. with a
range of myrtaceous species, including Dianese et al. (1984, 1986), Rayachhetry et al. (2001),
Alfenas et al. (2003), Tommerup et al. (2003) and Zauza et al. (2010). Several studies have
determined conditions for spore germination and infection (e.g., Ruiz et al. 1989; Piza and
Ribeiro 1988), and although there is some variation amongst studies, it is generally agreed
that optimum conditions are 15–25° C, high humidity or leaf wetness for up to 8 h, low light
or darkness, with infection only occurring on immature foliage. Continued ambient
temperatures and high humidity are then generally required, with uredinial pustules and
sporulation observed within 10–12 days (Marlatt & Kimbrough 1979; Rayachhetry et al.
1997; Alfenas et al. 2003).
The current work has two aims. Due in part to the ambiguity between the historical
host range of U. rangelii and P. psidii s.l., the first aim of this study was to test a range of
myrtaceous species for susceptibility to U. rangelii to ascertain whether myrtle rust could be a
threat to forestry in Australia. The second aim was to determine whether other gene regions
support the distinction of U. rangelii from P. psidii. Van der Merwe et al. (2007)
reconstructed a phylogeny for species of Puccinia and Uromyces based on sequences from
transcription elongation factor 1 (tef-1) and β-tubulin genes. Both of these gene regions were
able to discriminate closely related species, and indicated the possible presence of cryptic
species in some lineages. Several DNA samples of P. psidii were available in Australia from
previous studies (Langrell et al. 2008) and also from Hawaii. The only DNA sample linked to
a herbarium specimen is the Hawaiian sample, and its urediniospores lack the tonsure that has
1
been used to separate U. rangelii. The Brazilian samples came from a range of different hosts
and geographic locations.
2
Methodology
Rapid screening of commercial forestry species to U. rangelii
Test plants and inoculation
The species we selected were key commercial eucalypt species in Australia: Eucalyptus
agglomerata, E. cloeziana, E. globulus, E. grandis, E. pilularis and Corymbia maculata. We
were, however, restricted by availability, so not all key species were tested (e.g., E. dunnii, E.
nitens, E. regnans, C. variegata). Melaleuca quinquenervia was also tested, as it is a known
highly susceptible host of P. psidii s.l. (Rayachhetry et al. 2001). Agonis flexuosa cv.
‘Afterdark’ was used as a control, as it is known to be highly susceptible to U. rangelii in
Australia (Carnegie et al. 2010). Eucalypt seedlings were potted into 150 mm pots using
regular native potting mix, and kept in a glass house until used, with watering twice a day. A.
flexuosa cv. ‘Afterdark’ plants were sourced as either 150 mm pots or 200 mm pots. Two 200
mm pots of Melaleuca quinquenervia were also used. Plants to be tested were selected to
ensure they had fresh new growth (immature leaves).
The eight species were tested in four experiments. Due to biosecurity concerns, three
experiments were conducted in situ at IP 1 [Infected Premise 1 – the first property where
myrtle rust was detected in Australia, on the Central Coast of NSW] and the fourth in a
Biological Safety Cabinet (Class II) at the Industry & Investment NSW (forestry) laboratories
at West Pennant Hills. Diseased A. flexuosa cv. ‘Afterdark’ and/or Syncarpia glomulifera
leaves and shoots, with fresh uredinial pustules containing urediniospores, were collected
from IP 1 for inoculations. In most cases, material was either collected the same day as
inoculations or a day or two previously and kept in a fridge prior to inoculations.
Urediniospores were scraped from uredinial pustules using a clean scalpel such that they
“dusted” onto immature leaves; in some cases scraped urediniospores were gently wiped onto
immature leaves with the scalpel. In most cases, yellow urediniospores could be seen on
inoculated leaves either with the naked eye or a 10× hand lens.
Experiment I
This experiment relied on natural infection from diseased trees at IP 1, and began in May
2010. Nine potted plants each of E. agglomerata, E. cloeziana, E. grandis, E. pilularis, C.
maculata and A. flexuosa cv. ‘Afterdark’ were placed under infected S. glomulifera and
misted with water a single time (Fig. 1). Although fungicide spraying had occurred at IP 1
prior to this experiment, the western side of the S. glomulifera windbreak had not been
sprayed, and so plants were located under diseased branches – with freshly sporulating
uredinial pustules present on leaves – on the western side of trees. Test plants were moved
after 48 h into a shade house (due to an impending fungicide spray operation), where they
were misted twice-daily for 30 mins in the morning and early evening. Plants were inspected
weekly for evidence of myrtle rust infection.
Experiment II
In this experiment we attempted to increase the chance of infection by artificially inoculating
plants with urediniospores freshly collected from diseased S. glomulifera and by maintaining
leaf wetness for up to 48h. It was again conducted at IP 1, beginning in May 2010. Three
potted plants each of E. agglomerata, E. cloeziana, E. grandis, E. pilularis, C. maculata and
A. flexuosa cv. ‘Afterdark’ were misted with water to ensure leaf wetness on immature leaves
and inoculated as described above. Inoculated plants were then covered with clear plastic
3
bags, which were misted with water inside to increase humidity, and left outside adjacent A.
flexuosa cv. ‘Afterdark’ plants (Fig. 2). After 48h, most plants (and plastic bags) were still
moist, and plants were moved into a shade house, where they were misted twice-daily for 30
mins in the morning and early evening. Plants were inspected weekly for evidence of myrtle
rust infection.
Experiment III
This was again performed at IP 1, beginning in late May 2010. A temporary inoculation
chamber was constructed out of black plastic and a wooden frame (Fig. 3) and housed in a
greenhouse (Fig. 4) located at IP 1. Three plants each of E. agglomerata, E. cloeziana, E.
globulus, E. grandis, E. pilularis, C. maculata, A. flexuosa cv. ‘Afterdark’ and a single plant
of Melaleuca quinquenervia were misted with water to ensure leaf wetness on immature
leaves and inoculated as above. Plants were then placed inside the temporary inoculation
chamber, the walls of the chamber misted with water to increase humidity, and the door
sealed. Temperature within the temporary inoculation chamber during this time ranged from
7–25° C. This method allowed optimum temperatures for infection (which only occur during
the daytime in May) to be matched with darkness and leaf wetness. After 36 h, most plants
still had moist leaves and were moved from the temporary inoculation chamber to a bench
within the greenhouse and misted for 5 seconds every hour. Plants were inspected weekly for
evidence of myrtle rust infection. Temperatures within the greenhouse during early June
ranged from 5–25° C and in late June from 0–29° C.
Experiment IV
In this experiment, conducted in June 2010, we tried what we believe is a new method: using
cuttings in tissue culture jars. Young shoots from actively growing seedlings were cut and
immediately inserted into 1.5% or 2% water agar in 500 ml polycarbonate jars (tissue culture
jars) (Fig. 5). Six species were used for this experiment: E. globulus, E. cloeziana, E.
grandis, C. maculata, M. quinquenervia and A. flexuosa cv. ‘Afterdark’. Initially we used
“old” inoculum of U. rangelii from both A. flexuosa cv. ‘Afterdark’ and S. glomulifera leaves
collected a month or so previously from IP 1 and air dried and stored in herbarium envelopes.
We then repeated the experiment using “fresh” inoculum (actively sporulating pustules)
collected from potted plants of A. flexuosa cv. ‘Afterdark’ at IP 1 and kept in a paper bag in
the fridge overnight. Viability of inoculum was tested by brushing urediniospores onto 2%
water agar and observing germination at ~25° C after 24 h.
Cuttings were first misted with sterilised water (with Tween 20 added) to moisten
actively growing leaves and inoculated as above. Jars were sealed with Parafilm®, and placed
into plastic zip-lock bags which were misted with water then sealed (Fig. 5–6). Due to
limited inoculum, only two cuttings of each species were inoculated, with three cuttings of the
A. flexuosa cv. ‘Afterdark’ inoculated. To enhance biosecurity, inoculations were conducted
in a Biological Safety Cabinet Class II (Fig. 6) at the Forest Science Centre, Industry &
Investment NSW, West Pennant Hills. Inoculated cuttings were then left in the Biological
Safety Cabinet, with the glass covered to ensure darkness, for 24 h. The temperature within
the Biological Safety Cabinet over this period ranged from 17–26° C, with the first 8 hrs
being 21–26° C. After 24 h, leaves on most cuttings were still moist. The sealed jars (still
within zip-lock bags) were then placed on a laboratory bench in a secure room (i.e. restricted
access) with natural light and a temperature range of 14–26° C. Cuttings were inspected
regularly for presence of rust pustules for two weeks.
4
Morphological confirmation of infection by U. rangelii
Suspect positive samples were initially inspected under a binocular microscope, and then sent
to Dr Michael Priest at the NSW Plant Pathology Herbarium (DAR) in Orange, NSW for
confirmation. Positive samples are lodged at DAR.
Distinguishing U. rangelii from P. psidii
DNA amplification and phylogenetic analysis
DNA samples, with plant hosts and geographic origin, are provided (Table 1). DNA was
amplified by PCR targeting genes for transcription elongation factor 1, beta-tubulin and intergenic spacers. Additional sequences from GenBank were included in phylogenetic analyses.
Sequences were aligned with ClustalX (Thompson et al. 1997) and maximum likelihood
phylogenetic analysis was carried out using DNAML (Felsenstein 1989).
Table 1. DNA samples used in the phylogenetic study.
Sample
DAR80678
UFV1
UFV3
UFV8
UFV9
UFV26
SZ1
SZ2
SZ3
Host
Agonis flexuosa cv. Afterdark
Biotype 00*
Biotype 02*
Psidium guajava (guava)
Syzygium jambos
Psidium guajava
Syzygium jambos
Melaleuca quinquenervia
Metrosideros polymorpha
Geographic Location
NSW (IP1)
Brazil
Brazil
Viçosa, Brazil
Viçosa, Brazil
Campos, Brazil
Hawai’i
Hawai’i
Hawai’i
*Indicates single urediniospore isolates from the collection of Prof. Acelino Alfenas, Universidade Federale
Viçosa (Langrell et al. 2008).
5
Results
Host Testing
Experiment I & Experiment II
No evidence of infection (purpling or uredinial pustules) was observed on any plant, including
the A. flexuosa cv. ‘Afterdark’, at one, two, three or four weeks, or at eight weeks after
inoculation. The fact that the A. flexuosa cv. ‘Afterdark’ was not infected, and that there was
evidence of infection on planted A. flexuosa cv. ‘Afterdark’ at IP 1 during this time, indicates
that neither of these techniques worked, suggesting the optimum conditions for infection were
not obtained. All plant material used in these two experiments was sprayed with the fungicide
Bayfidan® after 8 weeks and then destroyed, following biosecurity procedures.
Experiment III
After two weeks there was no evidence of infection on the A. flexuosa cv. ‘Afterdark’, but
chlorotic to purple spots and blotches were observed on leaves of one plant each of E.
pilularis and E. cloeziana. No rust pustules were associated with this discolouration though at
this time. No change had occurred after three weeks; but at four weeks, uredinial pustules
were observed on the three inoculated A. flexuosa cv. ‘Afterdark’. There was still no
evidence (using a 10× hand lens) of uredinial pustules on the now purple spots and blotches
on the E. pilularis and E. cloeziana at four weeks. Plants were not inspected in week five; but
after six weeks uredinial pustules were observed on the purple spots and blotches of the E.
pilularis (Figs 7–8) and E. cloeziana (Figs 9–10). On both hosts sporulation was more
prevalent on the lower (abaxial) leaf surface. Also at six weeks, purple spots were now
observed on two plants of E. agglomerata (Figs 11–12) and a single plant of E. grandis, but
there was no evidence (using a 10× hand lens) of uredinial pustules on these plants. Later
examination under a dissecting then compound microscope identified these spots on E.
agglomerata and E. grandis as being associated with U. rangelii urediniospores, and rust on
the A. flexuosa cv. ‘Afterdark’, E. cloeziana and E. pilularis was confirmed as U. rangelii.
No other species inoculated showed any evidence of infection (Table 2). Samples were
collected from E. pilularis, E. cloeziana, E. agglomerata, E. grandis and A. flexuosa cv.
‘Afterdark’ for confirmation, then all plants were sprayed with Bayfidan® and then destroyed,
following biosecurity procedures.
Experiment IV
In most instances the cuttings remained “healthy” and “alive” for the duration of the
experiment. However, some plants lost a few leaves prematurely, and some plants became
infected with fungi that were likely inhabiting plants prior to cutting and placing in the tissue
culture jars. After 24 h on water agar, ~5% of urediniospores from the “old”, air-dried
inoculum had germinated, while ~25% had germinated from the “fresh” inoculum. No
evidence of infection (purpling or uredinial pustules) was observed after two weeks using the
“old” inoculum. However, using the “fresh” inoculum, uredinial pustules were observed after
two weeks on two of the three cuttings of A. flexuosa cv. ‘Afterdark’ (Figs 13–14), one
cutting of E. cloeziana (Figs 15–16) and one of the M. quinquenervia cuttings (Figs 17–18).
No evidence of infection (purpling or uredinial pustules) was observed on any other host
tested (Table 2). Rust pustules on A. flexuosa cv. ‘Afterdark’, E. cloeziana and M.
quinquenervia were confirmed as U. rangelii.
6
Distinguishing U. rangelii from P. psidii
Sequences from the β-tubulin and tef-1 genes were successfully obtained for nine samples.
Phylogenetic analyses were conducted separately as the sequences from each gene region
came from different collections and the other rust species (out-groups) clustered differently
for each gene region (Figs 19 and 20). ITS phylogenies are also included from previous work
(Figs 21 and 22, Glen unpublished). Given the lack of sequence variation in these three gene
regions, sequencing of the rDNA LSU was not pursued as this region is more conserved
(Maier et al. 2003) and so is highly unlikely to provide any additional information.
7
Table 2. Results of inoculation experiments (Experiment III & IV) with Uredo rangelii on a range of hosts
Species
Provenance/Cultivar
Agonis flexuosa
Eucalyptus agglomerata
E. cloeziana
E. globulus
E. grandis
E. pilularis
Corymbia maculata
Melaleuca quinquenervia
‘Afterdark’
Olney State Forest, NSW
Gympie, Qld
unknown
Buladelah, NSW
Newry State Forest, NSW
unkown
unkonw
1
2
Inoculation
Chamber at IP 1
(Experiment III)
3/31
2/3
1/3
0/3
1/3
1/3
0/3
0/1
Number of plants infected per plants inoculated
Using “fresh” inoculum
8
Cuttings in tissue
culture jars2
(Experiment IV)
2/3
1/2
0/2
0/2
0/2
1/2
Susceptible
+
+
+
+
+
+
1
2
4
3
5
6
Figures 1–6. Methodology for host testing experiments. 1. Potted plants placed under infected trees at
IP 1 (Experiment I). 2. Pots in plastic bags inoculated with myrtle rust (Experiment II). 3–4. Artificial
inoculation chamber within greenhouse at IP 1 (Experiment III). 5–6. Tissue culture jars in Biological
Safety Cabinet (Experiment IV).
9
7
8
10
9
11
12
Figures 7–12. Infection on various hosts following artificial inoculation with Uredo rangelii during
Experiment III. 7–8. Purple spots and blotches and uredinial pustules on Eucalyptus pilularis
(adaxial and abaxial leaf surface, respectively). 9–10. Purple spots and blotches and uredinial
pustules on E. cloeziana (adaxial and abaxial leaf surface, respectively). 11–12. Purple spots on E.
agglomerata (two separate plants).
10
13
14
15
16
17
18
Figures 13–18. Infection on various hosts following artificial inoculation with Uredo rangelii during
Experiment IV. 13–14. Agonis flexuosa cv. ‘Afterdark’ (uredinial pustules arrowed). 15–16. Eucalyptus
cloeziana (uredinial pustules arrowed). 17–18. Melaleuca quinquenervia (uredinial pustules arrowed).
11
DQ925240 Cumminsiella mirabilissima
EU487237 Melampsora reticulata
EU487218 Pucciniastrum epilobii
EF560586 Phakopsora pachyrhizi
P. psidii Brazil UFV-1
P. psidii Brazil UFV-8 Psidium guajava
P. psidii Brazil UFV-26 Psidium guajava
P. psidii Brazil UFV-3
P. psidii Viçosa Brazil UFV-9 Syzygium jambos
EU812463 P. psidii Colombia Syzygium jambos
DAR80678 Uredo rangelii NSW Agonis flexuosa
P. psidii Hawaii Syzygium jambos
EU812464 P. psidii Uruguay Eucalyptus grandis
P. psidii Hawaii Melaleuca quinquenervia
P. psidii Hawaii Metrosideros polymorpha
DQ925255 Puccinia alpina
DQ925285 Puccinia saccardoi
DQ925271 Puccinia lagenophorae
DQ925249 Puccinia perplexans
DQ925248 Puccinia agropyrina
DQ925246 Puccinia actaeae-agropyri
DQ925265 Puccinia graminis
DQ925275 Puccinia malvacearum
0.1
Figure 19. Maximum Likelihood tree based on partial translation elongation factor 1
sequences, bar represents 10% expected nucleotide variation.
12
AC188862 Phakopsora pachyrhizi
DQ983208 Puccinia lagenophorae
EF570835 Puccinia saccardoi
EF570844 Puccinia stylidii
AF317682 Melampsora lini
DQ983197 Phakopsora apoda
EU487246 P. psidii Colombia Syzygium jambos
P. psidii Brazil UFV26 Psidium guajava
P. psidii Hawaii Metrosideros polymorpha
P. psidii Hawaii Melaleuca quinquenervia
DAR80678 Uredo rangelii NSW Agonis flexuosa
P. psidii Hawaii Syzygium jambos
P. psidii Brazil UFV8 Psidium guajava
P. psidii Brazil UFV3
P. psidii Brazil UFV1
P. psidii Brazil UFV9 Syzygium jambos
EU487247 P. psidii Uruguay Eucalyptus grandis
EU487248 P. psidii Uruguay Eucalyptus grandis
DQ983198 Puccinia alpina
DQ983199 Puccinia malvacearum
DQ983214 Cumminsiella mirabilissima
EF570827 Puccinia poae-nemoralis
EF570810 Puccinia graminis
DQ983223 Puccinia perplexans
DQ983222 Puccinia actaeae-agropyri
EF570812 Puccinia triticina
0.1
EF570831 Puccinia agropyrina
Figure 20. Maximum Likelihood tree based on partial beta tubulin 1 sequences, bar
represents 10% expected nucleotide variation.
13
AF087710 Melampsora occidentalis
UCPRGBAA Pucciniastrum goeppertianum
PBU75987 Peridermium bethelii
L76491 Cronartium quercuum
AJ406048 P. kuehnii
AJ406052 P. sp.
0.1
AF333489 Phakopsora pachyrhiza
DAR80678 Uredo rangelii
EF210140 P. psidii
AF180201 U. striolatus
PCU88234 P. caricina
PCU57351 P. carduorum
AF468042 P. obscura
AF468040 P. distincta
AF468041 P. lagenophorae
AF511083 P. triticina
AF511082 P. recondita
AF511086 P. hordei
AF511079 P. allii
AF511076 P. allii
AF511084 U. reichertii
AF511085 U. scillarum
AF468043 P. poarum
AY114289 P. graminis
AY114292 P. striiformis
AY114290 P. coronata f. sp.
avenae
PDU88228 P. drabae
PDU88230 P. drabae
EF490601 P. cygnorum
AY348707 P. boroniae
AY114291 P. sorghi
PCU88216 P. consimilis
PCU88233 P. codyi
PAU88232 P. aberrans
PTU88220 P. thlaspeos
AF182965 P. thlaspeos
AF182998 P. monoica
PSP406055 P. sp.
PRU406071 P. rufipes
PME406064 P. melanocephala
PMI406072 P. miscanthi
Figure 21. Maximum Likelihood tree based on rDNA internal transcribed spacer sequences,
bar represents 10% expected nucleotide variation. Abbreviations: P = Puccinia, U =
Uromyces.
14
DQ354543 P. podophyllii
DAR80678 Uredo rangelii
EU348742 HNR
EU711422
EF210143 Eugenia uniflora, Br.
EF210144 Eugenia uniflora, Br.
AJ421808 S. jambos, Viçosa, Br.
AJ421803 E. grandis, Viçosa, Br.
AJ535658 M. quinquenervia, Fl.
AJ421805 Myrciaria cauliflora, Viçosa,
AJ536601 Psidium guajava Santa Catarina,
EU348743 HNR Uruguay
EF210142 S. jambos, Viçosa, Br.
AJ535661 Psidium guajava, South Bahia, Br.
EU071048 HNR biotype 02
EU711421 Metrosideros polymorpha Hi.
EU071046 M. quinquenervia Hi.
EF210141 Psidium guajava, Viçosa, Br.
EU439921 HNR Uruguay
EU439920 HNR Uruguay
EU711419 M. quinquenervia Fl.
EF599767 HNR Fl.
EU071045 M. quinquenervia Hi.
EU071047 HNR biotype 00
AJ421806 S. jambos Viçosa, Br.
AJ535659 Pimenta dioica Fl.
EU348744 HNR Uruguay
AJ421801 Eugenia uniflora Viçosa, Br.
AJ536603 E. grandis Bahia, Br.
EU711423 S. jambos Colombia
AJ535657 S. jambos Fl.
AJ421802 M. quinquenervia Viçosa, Br
Puccinia psidii from
various hosts and
geographic locations
.
AJ421807 S. jambos, Viçosa, Br.
AJ535660 E. sp. Br.
AJ536602 E. grandis Bahia Br.
AJ421800 S. jambos, Viçosa, Br.
AJ421804 E. grandis, Viçosa, Br.
EF599768 HNR Hi.
EU711420 S. jambos Hi.
DQ021883 Uredo baruensis
0.1
Figure 22: Maximum Likelihood tree based on rDNA internal transcribed spacer sequences,
including all P. psidii sequences from GenBank. Bar represents 10% expected nucleotide
variation. Abbreviations: Br. = Brazil, E. = Eucalyptus, Hi. = Hawaii, HNR = Host not
recorded (in GenBank), M. = Melaleuca, P. = Puccinia, S. = Syzygium.
15
Discussion
This work expands the host range of U. rangelii to include seven genera: Myrtus, Syzigium,
Agonis, Callistemon, Syncarpia and now Eucalyptus and Melaleuca. Another five hosts have
recently been identified during surveys for myrtle rust on the Central Coast of NSW
(Carnegie, Lidbetter & Priest, unpublished), with species in Leptospermum, Tristania,
Syzygium, Metrosideros and Gossia (=Austromyrtus). Discussions to date related to the
current incursion of U. rangelii in Australia have emphasised the “restricted” host range
(Office of the Chief Plant Protection Officer 2010). The host range now includes 15 species
in eight of the 17 “tribes” within Myrtaceae (Wilson et al. 2005): Eucalypteae,
Leptospermeae, Melaleuceae, Metrosiderae, Myrteae, Syncarpieae, Syzigieae and Tristaniae
(Table 3). This rapidly expanding host range is likely to increase, with further host testing
underway at CSIRO in Canberra (L. Morin, unpublished). As such, the host range for U.
rangelii (myrtle rust) is likely to more closely resemble that for P. psidii (guava rust).
Experiments in situ at IP 1 revealed that during the colder winter months the infection
period increases significantly. Under optimum conditions for guava rust (15–25°C, Ruiz et
al. 1989; Piza and Ribeiro 1988) infection occurs within two weeks (Marlatt & Kimbrough
1979; Rayachhetry et al. 1997; Alfenas et al. 2003). Likewise, we observed infection within
two weeks in Experiment IV in the laboratory, where temperatures were within the optimum
range. However, in Experiment III (in situ at IP 1), where temperatures were much colder,
the infection period was four weeks on A. flexuosa and five weeks on the Eucalyptus spp.
This has implications for myrtle rust surveillance and domestic quarantine, as the time from
infection to observation of symptoms is significantly greater during cooler temperatures.
Although there is no doubt that the species identified here are susceptible to U.
rangelii, results need to be used with caution. The conditions under which infection occurred
in these experiments are less likely to occur on a regular basis in the native environment. We
have not observed myrtle rust on species of Eucalyptus or M. quinquenervia in the field as
yet. However, artificial inoculation studies are commonly used to identify the risk of
introduction of exotic pests into countries (e.g. Office of the Chief Plant Protection Officer
2007; Matsuki et al. 2001).
None of the three gene regions sequenced support the distinction of U. rangelii from
P. psidii, or provide any evidence that a “species complex” exists. As these regions have all
been shown to be discriminatory at species level for rusts, including the genus Puccinia, we
conclude that the morphological distinction of U. rangelii does not represent a genetically
distinct species. Discussion of these results with rust taxonomists indicate that U. rangelii
may be better placed as a variety of P. psidii (Puccinia psidii var. rangelii).
16
Table 3. Current known hosts of Uredo rangelii based on literature, surveys and host testing
Tribes
EUCALYPTEAE
″
″
″
LEPTOSPERMEAE
″
″
″
MELALEUCEAE
″
″
MESTROSIDERAE
MYRTEAE
″
SYNCARPIEAE
SYZYGIEAE
″
TRISTANIAE
Host of Uredo rangelii
Eucalyptus agglomerata
E. cloeziana
E. grandis
E. pilularis
Agonis flexuosa
A. flexuosa
A. flexuosa
Leptospermum rotundifolium
Callistemon viminalis
Callistemon sp.
Melaleuca quinquenervia
Metrosideros collina
Gossia (=Austromyrtus) inophloia
G. inophloia
Myrtus communis
Syncarpia glomulifera
Syzygium jambos
S. wilsonii subsp. wilsonii ×
S. leuhmanii
Tristania neriifolia
Cultivars/Varieties
‘Afterdark’
‘Burgundy’
‘Jedda’s Dream’
‘Hannah Ray’
‘Kings Park Special’
‘Dwarf’
‘Aurora’
‘Blushing Beauty’
‘Cascade’
Reference
Present Study
Present Study
Present Study
Present Study
Carnegie et al. (2010)
Carnegie et al. (2010)
Carnegie et al. unpublished
Carnegie et al. unpublished
Carnegie et al. (2010)
Carnegie et al. unpublished
Present Study
Carnegie et al. unpublished
Carnegie et al. unpublished
Carnegie et al. unpublished
Simpson et al. (2006)
Carnegie et al. (2010)
Simpson et al. (2006)
Carnegie et al. unpublished
Carnegie et al. unpublished
17
Conclusions
Uredo rangelii does not have a restricted host range. The present study, and observations
from recent surveys, reveals at least 15 species in 11 genera are susceptible to U. rangelii.
This supports the observation that U. rangelii is part of the guava rust complex, and likely to
have a similar host range.
Key commercial forestry species are susceptible to U. rangelii, including those used in
plantations and native forestry in NSW and Queensland.
None of the three gene regions sequenced support the distinction of Uredo rangelii from
Puccinia psidii, or provide any evidence that a “species complex” exists. As these regions
have all been shown to be discriminatory at species level for rusts, including the genus
Puccinia, we conclude that the morphological distinction of U. rangelii does not represent a
genetically distinct species.
18
Recommendations
1. Forestry organisations (both commercial and environmental) to provide a prioritised
list of host species for ongoing host testing. Initially this will be at a species level, but
in the future would include provenances, families etc.
2. Forest production nurseries to be made aware of the implications of myrtle rust
affecting the production of seedlings of susceptible species, and potential management
options if/when myrtle rust expands its geographic range in Australia.
3. Awareness campaign of forest industries regarding the change in status of myrtle rust
from a disease with a restricted host range to a disease with an expanding host range.
19
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21
Acknowledgements
This work was funded by Forest and Wood Products Australia and Forests NSW. Seedlings
were supplied by Forests NSW. We thank the owners of IP 1 for the use of their greenhouse
and cooperation during the in situ experiments and Kerrie Simms for assistance with
microscope photography.
22