Microsatellite analysis indicates that Puccinia psidii in Australia is mutating but not recombining

Abstract

Puccinia psidii is considered a biosecurity threat in Australia because of its broad host range that includes many species of Myrtaceae which dominate Australian ecosystems. Since it was first reported in this country, in April 2010, there has been little information about the population structure of the pathogen. In this study, six microsatellite loci were analysed to determine the genetic relationship among rust specimens from different hosts and locations in Australia, New Caledonia, Hawaii and China. The Chinese and New Caledonian specimens share a multi-locus genotype with the majority of the Australian specimens. The results also indicated a close relationship between Australian and Hawaiian samples. At present, the P. psidii population in Australia is genetically uniform with no evidence of sexual recombination. Five of the 104 collections varied by one allele at single loci, indicating that mutations are common but persistence of the mutants in the population may be less common.

Full text

ORIGINAL PAPER
Microsatellite analysis indicates that Puccinia psidii in Australia
is mutating but not recombining
Patrícia da S. Machado
1
& Acelino C. Alfenas
1
& Rafael F. Alfenas
1
&
Caroline L. Mohammed
2
& Morag Glen
2
Received: 26 June 2015 /Accepted: 3 July 2015 /Published online: 30 July 2015
#
Australasian Plant Pathology Society Inc. 2015
Abstract Puccinia psidii is considered a biosecurity threat in
Australia because of its broad host range that includes many
species of Myrtaceae which dominate Australian ecosystems.
Since it was first reported in this country, in April 2010, there
has been little information about the population structure of
the pathogen. In this study, six microsatellite loci were
analysed to determine the genetic relationship among rust
specimen s from different hosts and locations in Australia,
New Caledonia, Hawaii and China. The Chinese and New
Caledonian specimens share a multi-locus genotype with the
majority of the Australian specimens. The results also indicat-
ed a close relationship between Australian and Hawaiian sam-
ples. At present, the P. psidii population in Australia is genet-
ically uniform with no evidence of sexual recombination. Five
of the 104 collections varied by one allele at single loci, indi-
cating that mutations are common but persistence of the mu-
tants in the population may be less common.
Keywords Myrtaceae
.
Myrtle rust
.
Plant disease
.
Genotyping
Introduction
Australia is the centre of origin of eucalypt and melaleuca
biodiversity (Ladiges et al. 2003; Crisp and Cook 2013)and
has over 1,646 species of Myrtaceae (Australian National
Botanic Gardens and Centre for Australian National
Biodiversity Research 2012) Australia n Flora Statistic,
https://www.anbg.gov.au/aust-veg/australian-flora-statistics.
html). Eucalyptus, one of the largest genera within Myrtaceae
and including several industrially important species, is
susceptible to two types of rust fungi, Phakopsora
myrtacearum and Puccina psidii Win t er. Phakopsora
myrtacearum was recently reported infecting Eucalyptus
species in three countries from Africa (Maier et al. 2015). P.
psidii, known colloquially as guava rust, eucalyptus rust or
myrtle rust, is considered a biosecurity threat to many
Myrtaceae species worldwide, especially Australia. Plants
belonging to the Myrtaceae are dominant in this country in
ecosystems ranging from tall forests to swamps and wetlands
(Australian Government Department of Agriculture 2015).
Puccinia psidii was first reported in Australia in April 2010,
on Agonis flexuosa on the Central Coast of New South Wales
(Carnegie et al. 2010) and spread rapidly along the east coast
where it was detected in Queensland in December 2010 and
1 year later in Victoria (Pegg et al. 2013). It was not detected in
Tasmania until February 2015 and in the Northern Territory
(Tiwi Islands) until May 2015, and is still (May 2015) not
recorded from South Australia or Western Australia.
Up to 2015, the pathogen has been reported on about 56
genera and 244 species of host in the Myrtaceae distributed in
different continents: from South and North America (Carnegie
and Lidbetter 2012), Asia (Kawanishi et al. 2009; Zhuang and
Wei 2011), Oceania (Carnegie et al. 2010;Giblin2013)and
Africa (Roux et al. 2013). In Brazil, the pathogen is consid-
ered endemic (Tommerup et al. 2003) and is not usually severe
on native hosts with the exception of occasional epidemics in
guava orchards (de Goes et al. 2004; Ribeiro and Pommer
2004), but it can be a problem in eucalypt plantations, an
important industrial crop for the country (Alfe nas et al.
* Morag Glen
Morag.Glen@utas.edu.au
1
Department of Plant Pathology, BIOAGRO, Federal University
of Viçosa, Viçosa, MG 36570-900, Brazil
2
Tasmanian Institute of Agriculture, University of Tasmania,
Hobart, TAS 7001, Australia
Australasian Plant Pathol. (2015) 44:455–462
DOI 10.1007/s13313-015-0364-5

2009). After introduction into new areas with Myrtaceae spe-
cies that have not previously been exposed to this pathogen,
P. psidii can rapidly expand its host range. This has bee n
observed in Jamaica, Florida, Hawaii and Australia
(MacLachlan 1938; Rayachhetry et al. 2001; Loope 2010;
Pegg et al. 2013).
The ultimate impact of the pathogen on Australian biodi-
versity is yet to be determined, but considering its rapid dis-
semination, wide host range and the severe damage reported
to some species such as Rhodamnia rubescens (Benth.) Miq.,
Rhodomyrtus psidioides (G.Don) Benth., Syzygium anisatum
(Vicke ry) Crav en & B iffen and Melaleuca quinquenervia
(Cav.) S.T.Blake (Carnegie and Cooper 2011), P. psidii is a
threat not only for the vegetation but also animal species
which may depend on native plant species (Tommerup et al.
2003;Glenetal.2007). Besides biodiversity, the pathogen can
have a commercial impact on the forest and timber industry,
lemon myrtle plantations (Plant Health Australia 2007)and
commercial nurseries (Plant Health Australia 2010).
Since the pathogen was reported in 2010, studies have been
conducted to identify vulnerable areas (Booth and Jovanovic
2012; Elith et al. 2013) and the host range of susceptible and
resistant plant species (Carnegie and Lidbetter 2012). Howev-
er, there is no information about the genetic variation of the
pathogen population, how the rust was introduced into the
country and how it spreads. Recent studies using microsatel-
lite markers have determined the population structure and the
host specificity of P. psidii in different areas such as Hawaii
and Brazil (Zhong et al. 2011; Graça et al. 2013). In Hawaii,
P. psidii collections are genetically uniform, indicating that the
population consists of a single clonal lineage originating from
one introduction (Zhong et al. 2011). In Brazil, host species
provide strong selection pressure on P. psidii populations, re-
gardless of geographic location (Graça et al. 2013). Principal
coordinate analysis also indicated a high degree of genetic
differentiation among collections from nine Brazilian states
on different host species, revealing five major groups, the first
formed by specimens from Eucalyptus spp. and Syzygium
jambos, the second included collections from Psidium
guajava and Psidium guineense, and three weakly separated
groups formed by specimens collected from Syzygium cumini,
Myrciaria cauliflora and Eugenia uniflora (Graça et al. 2013).
The existence of host-specific genotypes may indicate the
occurrence of cryptic species within the P. psidii complex or
potential evolution to the level of
Bfo
rmae speciales^. Genetic
analysis of pathogen populations is required to understand the
mechanisms generating genetic variation, host-pathogen co-
evolution, and in the management of resistance (Keiper et al.
2003). An initial study based on a small number of collections
of P. psidii soon after its introduction demonstrated the pres-
ence of a single multi-locus genotype in Australia (Glen and
Mohammed 2011), consistent with the Hawaiian population,
but did not include specimens from other countries where the
pathogen was also recently introduced, such as China and
New Caledonia. In this study microsatellite loci were analysed
to determine the genetic relationship among rust specimens
from different hosts and locations from Australia and recent
incursions in other countries.
Material and methods
Sampling
A total of 104 single uredinial pustules of P. psidii were col-
lected on 55 Myrtaceae species in Australia, New Caledonia
(Fig. 1) China and Hawaii (Table 1). The samples were col-
lected in mainland Australia in 2010 and 2013 and from
Tasmania in 2015. The survey points were geo-referenced
and most collections were deposited in the Queensland plant
pathology herbarium (BRIP). A portion of each specimen was
preserved in ethanol and retained for DNA extraction. Single
pustules w ere excised and placed separately into 1.5-mL
microcentrifuge tubes and stored at −80 °C prior to DNA
extraction. Samples from New Caledonia, consisting of
urediniospores collected from multiple Syzygium jamb os
plants in New Caledonia, were preserved in 70 % ethanol
and imported into Australian under import permit
IP13103123. DNA from the Chinese and Hawaiian collec-
tions was extracted and imported under IP13007011 and
IP07020087.
DNA extraction
Genomic DNA was extracted directly from a single P. psidii
pustule (fungus+host tissue) using one metal bead placed in a
1.5-ml microcentrifuge tube followed by two rounds of mac-
eration using a TissueLyser II (Qiagen) for 2 min at the fre-
quency 30 Hz. A total of 250 μl extraction buffer (Raeder and
Broda 1985) was added and the tubes incubated at 65 °C for
1 h. Tubes were centrifuged at 14,000 rpm for 15 min and the
supernatant removed. DNA was purified by binding to silica
(Boyle and Lew 1995). Briefly, 600 μlof100%NaIand10μl
silica were added to 200 μl of the supernatant and vortexed
briefly. The mixture was incubated on ice for 15 min with
occasional shaking. Tubes were centrifuged for 10 s at 14,
000 rpm, the supernatant removed, and the pellet resuspended
in 600 μl of wash buffer (100 mM NaCl, 10 mM Tris HCl
pH 7.5, 1 mM EDTA in 50 % ethanol). Following centrifuga-
tion for 10 s at 14,000 rpm, the supernatant was removed, the
pellet suspended in 600 μl 100 % ethanol and centrifuged as
before. Finally, the supernatant was removed and the pellet
dried for 20 min. DNA was eluted by adding 20 μlofTE
buffer, vortexing briefly and incubating at 45 °C for 10 min.
Supernatant containing DNA was removed following centri-
fugation for 2 min at 14,000 rpm and stored at –20 °C.
456 P. da S. Machado et al.

Microsatellite genotyping
The samples were genotyped at 6 microsatellite loci
(EF523503, EF523504, EF523507, EF523508, EF523510,
EF523513) (Zhong et al. 2008; Graça et al. 2013). For each
10 μL PCR reaction we used 5 μL of 2× Master Mix (Type-It
Microsatellite PCR kit, Qiagen), 0.1 μL(20μM) of forward
(labelled with either D2, D3 or D4 Well-RED fluorescent dye,
Sigma-Aldrich) and reverse primers, 0.2 mg/mL of Bovine
Serum Alb umin (BSA, Fisher BioReagents™), 3.6 μLof
nuclease-free water and 1 μL genomic DNA. PCR amplifica-
tions were performed using a thermal cycler (model 2720,
Applied Biosystems) and the following program: 95 °C for
3 min, then 34 cycles of 94 °C for 15 s, 45 to 50 °C (depending
on the locus) for 15 s, 72 °C for 45 s, ending with a hold at
60 °C for 30 min, then 14 °C. Fragment analysis was conduct-
ed on a CEQ™ 8000 Genetic Analysis System (Beckman
Coulter), using 1 μL of PCR product mixed with 38.5 μL
Sample Loading Solution (Beckman Coulter) and 0.5 μLsize
marker (DNA Size Standard Kit – 400, Beckman Coulter).
Results
Very little genetic variability was found among the 104 spec-
imens of P. psidii; a single multilocus genotype was observed
Fig. 1 Sampling locations of
Puccinia psidii in Australia (a)
and in New Caledonia (b)
Puccinia psidii in Australia is mutating but not recombining 457

Tabl e 1 Host and geographic origin of Puccinia psidii collections
Herbarium or specimen code
a,b
Host Location
c
Latitude Longitude
MR1 Syzygium jambos NSW, Australia −33,868 151,207
MR2 Syzygium jambos Qld, Australia −27,919 153,051
MR3 Syzygium australe Qld, Australia −27,475 152,973
MR4 Rhodomyrtus canescens Qld, Australia −27,475 152,973
MR5 Rhodomyrtus pervagata Qld, Australia −27,475 152,973
MR6 Syzygium nervosum Qld, Australia −27,475 152,973
MR7 Syzygium macilwraithianum Qld, Australia −27,475 152,973
BRIP59494a Rhodamnia blairiana Qld, Australia −27,475 152,973
BRIP59495a Chamelaucium uncinatum Qld, Australia −27,475 152,973
BRIP59496a Backhousia oligantha Qld, Australia −27,475 152,973
BRIP59497a Austromyrtus dulcis NSW, Australia −28,605 153,572
BRIP59499a Melaleuca quinquenervia NSW, Australia −28,605 153,572
BRIP59500a Rhodomyrtus psidioides NSW, Australia −28,605 153,572
BRIP59502a Agonis flexuosa NSW, Australia −28,672 153,279
BRIP59503a Syzygium jambos Qld, Australia −27,485 152,992
BRIP59504a Melaleuca fluviatilis Qld, Australia −27,404 153,073
BRIP59505a Melaleuca quinquenervia Qld, Australia −26,333 152,82
BRIP59506a Rhodamnia rubescens Qld, Australia −26,333 152,82
BRIP59507a Melaleuca quinquenervia Qld, Australia −26,941 152,974
BRIP59508a Leptospermum trinervium Qld, Australia −26,941 152,974
BRIP59509a Me
laleuca quinquenervia Qld, Australia −26,941 152,974
BRIP59510a Melaleuca quinquenervia Qld, Australia −26,941 152,974
BRIP59511a Rhodamnia sessiliflora Qld, Australia −27,494 152,944
BRIP59512a Eugenia reinwardtiana Qld, Australia −16,912 145,767
BRIP59513a Melaleuca quinquenervia Qld, Australia −17,222 145, 664
BRIP59514a Eugenia reinwardtiana Qld, Australia −17,222 145, 664
BRIP59515a Syzygium sp. Qld, Australia −17,222 145, 664
BRIP59516a Melaleuca sp. Qld, Australia −17,222 145, 664
BRIP59517a Gossia sp. Qld, Australia −16,816 145,686
BRIP59518a Eugenia reinwardtiana Qld, Australia −16,816 145,686
BRIP59519a Melaleuca leucadendron Qld, Australia −16,816 145,686
BRIP59520a Austromyrtus dulcis Qld, Australia −16,816 145,686
BRIP59521a Gossia inophloia Qld, Australia −16,816 145,686
BRIP59522a Hybrid Syzygium leuhmannii x S. wilsonii Qld, Australia −16,816 145,686
BRIP59523a Leptospermum sp. Qld, Australia −16,816 145,686
BRIP59524a Xanthostemon sp. Qld, Australia −16,816 145,686
BRIP59525a Gossia myrsinocarpa Qld, Australia −16,812 145,685
BRIP59526a Syzygium cormiflorum Qld, Australia −16,812 145,685
BRIP59527a Rhodamnia sessiliflora Qld, Australia −16,812 145,685
BRIP59528a Gossia sp. Qld, Australia −16,812 145,685
BRIP59529a
Rhodamnia spongiosa Ql
d, Australia −16,812 145,685
BRIP59530a Leptospermum madidum subsp. sativum Qld, Australia −16,82 145,642
BRIP59531a Tristaniopsis exiliflora Qld, Australia −16,82 145,642
BRIP59532a Leptospermum petersonii Qld, Australia −16,825 145,624
BRIP59533a Syzygium wilsonii subsp. wilsonii Qld, Australia −16,825 145,624
BRIP59534a Melaleuca viminalis Qld, Australia −16,825 145,624
BRIP59535a Gossia sp. Qld, Australia −16,825 145,624
BRIP59536a Eugenia reinwardtiana Qld, Australia −16,825 145,624
BRIP59537a Rhodamnia sessiliflora Qld, Australia −17,037 145,613
458 P. da S. Machado et al.

Tabl e 1 (continued)
Herbarium or specimen code
a,b
Host Location
c
Latitude Longitude
BRIP59538a Rhodamnia sp. Qld, Australia −17,037 145,613
BRIP59539a Gossia myrsinocarpa Qld, Australia −17,037 145,613
BRIP59540a Syzygium sayeri Qld, Australia −17,037 145,613
BRIP59541a Melaleuca viridiflora Qld, Australia −16,98 145,552
BRIP59542a Melaleuca sp. Qld, Australia −16,98 145,552
BRIP59543a Syzygium jambos Qld, Australia −16,876 145,757
BRIP59544a Syzygium kuranda Qld, Australia −16,831 145,667
BRIP59545a Gossia myrsinocarpa Qld, Australia −16,804 145,636
BRIP59546a Tristaniopsis exiliflora Qld, Australia −16,804 145,636
BRIP59547a Rhodamnia spongiosa Qld, Australia −16,804 145,636
BRIP59548a Decaspermum humile Qld, Australia −16,804 145,636
BRIP59549a Rhodomyrtus pervagata Qld, Australia −16,796 145,622
BRIP59550a Eucalyptus tereticornis Qld, Australia −16,699 145,529
BRIP59551a Melaleuca nervosa Qld, Australia −16,699 145,529
BRIP59552a Backhousia hughesii Qld, Australia −16,681 145,519
BRIP59553a Melaleuca nervosa Qld, Australia −16,658 145,477
BRIP59554a Rhodomyrtus effusa Qld, Australia −16,582 145,321
BRIP59555a Rhodomyrtus pervagata Qld, Australia −16,582 145,321
BRIP59556a Gossia lewisensis Qld, Australia −16,594 145,284
BRIP59557a Rhodomyrtus canescens Qld, Australia −16,588 145,275
DAR80674 Ag
onis flexuosa NSW, Australia −33,224 151,219
DAR80675 Syncarpia glomuifera NSW, Australia −33,224 151,219
DAR80678 Agonis flexuosa NSW, Australia −33,224 151,219
MR73 Syzygium apodophyllum Qld, Australia −16,898 145,747
MR74 Myrtus communis Vic, Australia −37,807 144,953
MR75 Melaleuca quinquenervia Qld, Australia −25,191 153,151
MR77 Rhodamnia maideniana Qld, Australia −28,209 153,270
MR78 Syzygium jambos Qld, Australia −28,209 153,270
M10-13851 Melaleuca quinquinervia NSW, Australia −33,389 151,469
M10-14107 Melaleuca quinquinervia NSW, Australia −33,271 151,422
M10-15047 Rhodamnia rubescens NSW, Australia −33,173 151,261
ON10/0304 Tristania neriifolia NSW, Australia np
d
np
ON10/0307 Tristania neriifolia NSW, Australia np np
O10-00033 Metrosideros collina NSW, Australia −33,320 151,179
O10-00113 Tristania neriifolia NSW, Australia −33,683 151,227
PPS001 Austromyrtus inophloia Qld, Australia −27,345 153,010
PPS002 Austromyrtus inophloia Qld, Australia −27,364 153,016
PPS003 Austromyrtus inophloia Qld, Australia −27,080 152,933
PPS004 Syzygium australe ‘Golden Hedge’ Qld, Australia −27,080 152,933
PPS009 Austromyrtus inophloia Qld, Australia −27,108 152,947
PPS010 Austromyrtus inophloia Qld, Australia −27,584 153,302
PPS013 Austromyrtus inophloia Qld, Australia −27,471 153,095
PPS021 Austromyrtus inophloia Qld, Australia −26,654 153,081
PPS022 Austromyrtus inophloia Qld, Australia −26,804 153,124
PPS023 Austromyrtus inophloia Qld, Australia −27,207 153,052
35-15 Lophomyrtus sp. Tas, Australia np np
42-15 Lophomyrtus sp. Tas, Australia np np
65-15 Ugni molinae Tas, Australia np np
87-15 Lophomyrtus sp. Tas, Australia np np
Puccinia psidii in Australia is mutating but not recombining 459

in the majority of the Australian collections and samples from
New Caledonia, Hawaii, and China (Table 2). However, in
five collections from Australia (BRIP59525a, BRIP59529a,
BRIP59543a, BRIP59545a and 65-15) an unusual allele was
detected at four loci (Table 2). The first four of these collec-
tions were from Cairns and surrounds on three different hosts
and the fifth from y et another host species in Tasmania
(Table 2). Genotyping was repeated 2–3 times for these col-
lections from the original DNA, providing the same result
each time. Further DNA was extracted from additional pus-
tules from the same collections, with variable genotyping re-
sults (Table 2). In three instances, further variation at the
variable locus was detected; in the other two, the common
MLG was detected.
Discussion
Low genetic variation was demonstrated in P. psidii collec-
tions from Australia, New Caledonia and China using six
microsatellite loci previously shown to be polymorphic
among different P. p s id i i populations (Zhong et al. 2008,
2011; Graça et al. 2013). The same heterozygous genotype
found among the majority of collections in Australia indicates
Tabl e 1 (continued)
Herbarium or specimen code
a,b
Host Location
c
Latitude Longitude
NC3 Syzygium jambos Farino, NC np np
NC4 Syzygium jambos Farino, NC np np
NCC Syzygium jambos Farino, NC np np
NCE Eugenia gacognei Mare, NC np np
HMAS242567 Syzygium jambos Hainan, China np np
HAW45011 Syzygium jambos Oahu, USA np np
a
BRIP=Queensland Plant Pathology Herbarium, DAR=Orange Agricultural Institute, HMAS=Herbarium Mycologicum Academiae Sinicae, HAW=
Joseph F. Rock Herbarium
b
Herbarium specimens with a code not beginning with BRP, DAR, HMAS or HAW were not retained
c
Qld Queensland, NC New Caledonia, NSW New South Wales, Tas Tasmania, USA United States of America
d
np not provided
Tabl e 2 Allele sizes for microsatellite loci of collections from Australia, New Caledonia (NC) and China. Variant allele sizes are in bold text
Collection Locus
503 504 507 508 510 513
MR1
a
217, 219 155, 157 162, 171 173, 179 68, 78 215, 227
NC3
b
217, 219 155, 157 162, 171 173, 179 68, 78 215, 227
HMAS242567 217, 219 155, 157 162, 171 173, 179 68, 78 215, 227
HAW45011 217,219 155, 157 162,171 173,179 68,78 215, 227
BRIP59525a (pustule 1) 215, 217 155, 157 162, 171 173, 179 68, 78 215, 227
BRIP59525a (pustule 2) 213, 215 155, 157 162, 171 173, 179 68, 78 215, 227
BRIP59525a (pustule 3) 217, 219 155, 157 162, 171 173, 179 68, 78 215, 227
BRIP59529a (pustule 1) 217, 219 155, 157 162, 171 173, 179 68, 78 215, 221
BRIP59529a (pustule 2) 217, 219 155, 157 162, 171 173, 179 68, 78 215, 223
BRIP59543a (pustule1) 217, 219 155, 157 162, 171 175, 177 68,78 215,227
BRIP59543a (pustule2) 217, 219 155, 157 162, 171 177, 179 68,78 215,227
BRIP59545a (pustule 1) 217, 219 155, 157 162, 171 173, 179 68, 78 215, 215
BRIP59545a (pustule 2) 217, 219 155, 157 162, 171 173, 179 68, 78 215, 227
65-15 (pustule 1) 217, 219 155, 157 162, 171 173, 179 68, 78 215, 227
65-15 (pustule 2) 217, 219 155, 157 162, 173 173, 179 68, 78 215, 227
Four collections from the Tiwi Islands, Northern Territory, had multilocus genotypes consistent with MR1 and the majority of collections from mainland
Australia
a
MR1 and the majority of collections (=99) from Australia have this same genotype
b
All collections from New Caledonia have the same genotype
460 P. da S. Machado et al.

a la ck of genetic recombination and no selection by host,
consistent with a recent introduction of a single, clonally-
reproducing rust genotype in Australia. Although teliospores
were identified in 20 % of the samples in a survey in Queens-
land (Pegg et al. 2013), the lack of recombination and struc-
ture of Australian collections is consistent with the lack of
recombination in the Hawaiian rust population (Zhong et al.
2011; Graça 2011), where the pathogen was reported 9 years
ago (Uchida et al. 2006). The low variability in the Australian
population is consistent with clonal reproduction, precluding
analysis with GenAlex 6.4 (Peakall and Smouse 2006). The
collections that showed an unusual allele size were from
Cairns and surrounds from three different hosts as well as
from a fourth host species in Tasmania and no correlation
was found among host, allele size or loci, indicating that these
mutations are random occurrences. Similar levels of mutation
have been observed in clonal populations of Puccinia triticina
in wheat cultivars, where a strong correlation between geno-
type and pathotype has been demonstrated (Goyeau et al.
2007).
Microsatellite markers have been used to infer the origin of
the P. psidii incursion in Hawaii. A unique genotype found in
four Hawaiian Islands (Maui, Oahu, Kauai, and Big Island)
was also found in two collections from different hosts in Cal-
ifornia, indicating that California may have been the source of
the P. psidii introduction into Hawaii, probably by the trade of
Myrtaceae plant between both states (Graça 2011). The origin
of the genotype in California is unknown.
The collections from New Caledonia and China and the
majority of Australian collections have the same genotype as
that present in Hawaii. Although P. psidii was reported first in
Hawaii followed by China, Australia and most recently, New
Caledonia, it is not possible to confirm the origin of the incur-
sion in these countries, unlike in Japan where the rust was
detect ed on Metrosideros plants imported from Hawaii
(Kawanishi et al. 2009). The rust may have been distributed
from California to all of the other countries or may have trav-
elled from e.g., California to Hawaii, from Hawaii to China,
from China to Australia, and finally from Australia to New
Caledonia. The multilocus genotypes of the P. psidii popula-
tion in South Africa is unknown.
In contrast with the rust populations in Australia and Ha-
waii, the genetic variability of P. psidii collections in Brazil is
high. In a recent study based on analysis of 10 microsatellite
loci in 148 P. psidii collections from seven host species (Graça
et al. 2013), all loci were polymorphic and strong selection by
host species regardless of geographic location was demon-
strated. As no evidence of recent sexual recombination among
the host populations on guava and eucalypts, it is likely that
they have become differentiated by a series of mutations sim-
ilar to those observed in the Australian population. As the
mutations accumulate, the mutants that are better adapted to
a particular host species would have a better chance of
survival and eventually a strain that has a MLG quite different
to the original would evolve. Despite the high genetic vari-
ability and broad distribution of this pathogen in Brazil, the
genotype present in Australia, Hawaii and California has not
been detected in Brazil (Graça 2011). It may be present at low
levels in native vegetation in Brazil or may have arisen outside
of Brazil.
The source and pathway of the incursion in Australia is
unknown. Although the country has a continental size, wind
combined with susceptible host and suitable climatic condi-
tions provided a near-continuous corridor where the spores of
thepathogenwerespreadalongtheeastcoast,alsoassistedby
human movement of host plants (Carnegie and Lidbetter
2012). There is also evidence of aerial dispersal of two other
rust sp ecies, Melampsora larici-populina Klebah and
Melampsora medusa Thümenth, from the east coast of Aus-
tralia to New Zealand across the Tasman Sea (Close et al.
1978). Whether this also occurred with P. psidii moving be-
tween Australia and New Caledonia is unknown. Besides air-
flows, the commercial trade of plants and movements of peo-
ple and commodities are likely to be the other long-distance
di
spersal pathways for pathogen spores (Sheridan 1989; Wil-
liams et al. 2000).
In Brazil the populations of P. psidii collected on different
host species are genetically distinct (Graça et al. 2013). This
contra st with th e population of the pathogen in Australia,
where 5 years after the first report of the pathogen in this
country a few mutants of the dominant genotype were ob-
served. Artificial inoculations showed that at least 107 native
host species in 30 genera are susceptible to this predominant
genotype (Carnegie and Lidbetter 2012). While mutations in
microsatellite loci are unlikely to affect host range, and the
persistence of mutant genotypes in the population has not
yet been demonstrated, this indicates the potential for genetic
changes in genomic regions that may affect host adaptation
and the possible emergence of new pathotypes. This has been
demonstrated in Brazil where the genotype that is widespread
on eucalyptus has mutated to create a new race that has over-
come rust resistance (Graça et al. 2011). Thus, avoiding the
introduction of new P. psidii genotypes into, and dispersal
around the country in areas of high Myrtaceae biodiversity
which have not previously been exposed to this rust, is highly
desirable.
Acknowledgments The project was supported by Conselho Nacional
de Desenvolvimento Científico e Tecnológico, Brasil (CNPq), Fundação
de Amparo a Pesquisa do Estado de Minas Gerais (FAPEMIG) and the
Australian Department of Agriculture. We thank Adam Smolenski for
assistance with genotyping and Geoff Pegg, Angus Carnegie, Richard
Davis, Stephen McKenna, Elodie Nakamura and Yuan Ziqing for assis-
tance with specimen collection and host species identification.
Puccinia psidii in Australia is mutating but not recombining 461

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