Plant Pathology (2010) 59, 703–711 Doi: 10.1111/j.1365-3059.2010.02299.

PCR assays for the sugarcane rust pathogens Puccinia

kuehnii and P. melanocephala and detection of a SNP
associated with geographical distribution in P. kuehnii

N. C. Glynna*, L. J. Dixonb, L. A. Castleburyb, L. J. Szaboc and J. C. Comstocka

a
USDA-ARS Sugarcane Field Station, 12990 US Hwy 441N, Canal Point, FL 33438; bUSDA-ARS Systematic Mycology and
Microbiology Laboratory, 10300 Baltimore Ave., Beltsville, MD 20705; and cUSDA-ARS Cereal Disease Laboratory, 1551 Lindig
Street, St. Paul, MN 55108, USA

Puccinia kuehnii and P. melanocephala cause orange and brown rust of sugarcane, respectively. Puccinia kuehnii has been
confirmed in Asia, Australia and recently, the Caribbean basin, whereas P. melanocephala is distributed among the majority
of sugarcane growing regions. Differentiating these two economically significant pathogens visually is problematic and lim-
ited to material exhibiting mature disease symptoms or spores. Partial ITS1, ITS2 and complete 5Æ8S sequences were gener-
ated from P. kuehnii and P. melanocephala isolates from around the world. PCR primers and dual labelled hydrolysis
probes were designed for each pathogen for use in real-time PCR and optimized using locked nucleic acids (LNA). The prim-
ers amplified DNA from their target pathogens and not from other species of Puccinia or fungal species isolated from sugar-
cane leaves. Optimized real-time PCR conditions allowed the detection of 0Æ19 pg of P. kuehnii or P. melanocephala
genomic DNA and differentiated the pathogens on sugarcane leaves prior to observing typical symptoms in the field. Pri-
mer-introduced restriction analysis-PCR (PIRA-PCR) was used to detect a single nucleotide polymorphism (Pk ITS1
183A>G) in ITS1 of P. kuehnii. Allele 183A was observed in all samples, whereas 183G was detected in 52% of samples
from Asia and Australia yet absent from all Caribbean basin samples. Long distance spore dispersal, dispersal through an
intermediate location or improper movement of contaminated material could explain the introduction of P. kuehnii to the
Western hemisphere. However, the current proliferation of the pathogen in the Americas is limited to isolates which contain
only the 183A allele.

Keywords: brown rust of sugarcane, locked nucleic acids, orange rust of sugarcane, PIRA-PCR, real-time PCR,
Saccharum spp.

(Comstock & Shine, 1992). The first occurrence of P.

Introduction
melanocephala in the Western hemisphere was in the
Two species of Puccinia can cause rust diseases of sug- Dominican Republic in 1978 (Presley et al., 1978). Fol-
arcane (a complex hybrid of Saccharum spp.). Puccinia lowing this introduction, it spread rapidly through the
kuehnii is the causal agent of orange rust of sugarcane sugarcane growing regions of North, Central and South
whereas P. melanocephala causes brown rust of sugar- America as well as the Caribbean and within a few
cane. Brown rust occurs in the majority of sugarcane years was present in almost all sugarcane-growing areas
growing regions: Africa, North and South America, in the Americas (Purdy et al., 1985). Orange rust is less
South-East Asia, Southern Asia and Australasia (Ryan widespread than brown rust and was considered by
& Egan, 1989). Yield losses of 10–40% due to brown Ryan & Egan (1989) as only occurring in the sugarcane
rust have been reported on susceptible varieties industries in Asia and Australia. The recent report of
orange rust in Florida was the first confirmed occur-
*E-mail: [email protected] rence of the pathogen in the Western hemisphere (Com-
Product names and trademarks are mentioned to report stock et al., 2008). The disease has since been
factually on available data; however, the USDA-ARS neither confirmed in Guatemala (Ovalle et al., 2008), Nicara-
guarantees nor warrants the standard of the product, and the gua, Costa Rica (Chavarria et al., 2009), El Salvador,
use of the name by USDA-ARS does not imply approval of the Panama and Mexico (Flores et al., 2009) and recently
product to the exclusion of others that may also be suitable. Brazil (Barbasso et al., 2010).
The experiments reported comply with current US laws.
Economic losses caused by orange rust were estimated
at AUST $150–200 M in an epidemic in Australia in
Published online 9 May 2010

No claim to original US government works

Journal compilation ª 2010 BSPP 703
 13653059, 2010, 4, Downloaded from https://bsppjournals.onlinelibrary.wiley.com/doi/10.1111/j.1365-3059.2010.02299.x by Nat Prov Indonesia, Wiley Online Library on [25/08/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
704 N. C. Glynn et al.

2001 (Magarey et al., 2001). The spread of orange rust

Materials and methods
has so far been less rapid than that of brown rust. Cur-
rently with the exception of Brazil, South America and
Puccinia kuehnii and P. melanocephala rDNA
the sugarcane growing regions of the USA other than
sequences
Florida remain free from orange rust. Additionally, there
are no confirmed occurrences of orange rust in Africa. Sources of P. kuehnii and P. melanocephala were chosen
These areas are under considerable threat from this eco- to represent a geographically diverse population by incor-
nomically important pathogen. This makes the develop- porating most of the world sugarcane growing regions
ment of rapid and sensitive methods for detecting where these pathogens have been reported (Table 1). Sug-
P. kuehnii critical in order to allow the rapid implementa- arcane leaves exhibiting symptoms typical of orange rust
tion of strategies that will limit the spread of the disease. caused by P. kuehnii or brown rust caused by P. melano-
Since both P. kuehnii and P. melanocephala can occur cephala were removed from the plants, cut into sections
together on sugarcane leaves and in the sugarcane grow- approximately 15 cm in length, pressed flat between
ing environment, it is important to be able to accurately absorbent paper and placed in paper envelopes. After dry-
and reliably distinguish between the two pathogens. ing at room temperature for 1–2 days, samples were
Differences in urediniospore coloration on uredinial placed in paper envelopes and sent to the USDA, ARS,
lesions (pustules) are the most distinguishing morpho- Systematic Mycology and Microbiology Laboratory,
logical features between the two pathogens, with pustules Beltsville, MD, USA.
of P. kuehnii typically light orange and those of DNA was isolated from 100 mg of dried leaf material
P. melanocephala usually dark brown. Additionally, pus- containing uredinial lesions. Leaf material was chopped
tules caused by P. kuehnii are typically shorter and initially using a scalpel into pieces about 2 · 4 mm or
appear oval in shape whilst those caused by P. melano- smaller, placed in a tube containing lysing matrix ‘C’ (MP
cephala are usually longer and narrower in appearance. Biomedicals) and disrupted in a FastPrep (MP Biomedi-
These general differences in both urediniospore colour cals) using a speed setting of 4Æ5 for 30 s. Disruption was
and shape of uredinia are often subtle and can vary repeated up to four times until the leaf material took on
depending upon age of infection, location and host the appearance of a fine powder. The remainder of the
cultivar. Microscopic examination of spore morphology extraction procedure was performed using OmniPrep
can be used to identify urediniospores of P. kuehnii and (G Biosciences) kits according to the manufacturer’s
differentiate them from those of P. melanocephala in instructions. The resulting DNA pellets were re-sus-
well-preserved infected material that exhibit mature pended in the supplied TE buffer (G Biosciences). DNA
disease symptoms. However, these methods require a concentration was determined by spectrophotometry
high power microscope and are less discriminatory when and diluted to a final concentration of 40 ng lL)1.
samples are preserved under sub-optimal conditions or Primers PkPm-F and PkPm-R, located in ITS1 and
when samples are taken during the initial stages of infec- ITS2, respectively, (Table 2) were designed from align-
tion. Furthermore, these methods are difficult to apply to ments of sequences accessioned in the National Center
environmental samples, such as rain or soil, where the for Biotechnology Information (NCBI) GenBank data-
number of spores may be extremely low. Accurate identi- base and are conserved between the two pathogens.
fication of infection by sugarcane rust pathogens in the These primers were used to amplify fragments of 606 bp
field is only possible when spores are produced from (P. kuehnii) or 585 bp (P. melanocephala) from each
uredinial lesions. DNA extract. PCR reactions were performed in volumes
Accurate identification can be achieved using molecu- of 50 lL and contained 2Æ5 mM MgCl2, 0Æ25 mM dNTPs,
lar methods involving sequencing regions of rDNA. 0Æ5 lM of each primer and 2Æ5 units Taq (Promega) and
However, this can be time consuming and expensive to respective buffer. Thermocycling consisted of initial
apply on a large scale. The development of quick, less denaturation at 94C for 5 min, followed by 35 cycles of
expensive diagnostic methods are needed to allow for the 94C for 30 s, 56C for 30 s and 72C for 30 s and a final
accurate and sensitive discrimination between P. kuehnii extension of 72C for 7 min.
and P. melanocephala. Regions of rDNA sequence have Amplified fragments were excised from agarose gels
been widely exploited for the development of diagnostic following electrophoresis, purified, and cloned. At least
methods for plant pathogens including several species of 10 positive clones were purified and sequenced for each
rust pathogens (Fraaije et al., 2001; Barnes & Szabo, sample. Sequences from each pathogen ⁄ location combi-
2007; Barnes et al., 2009). The advantage of using this nation were edited and aligned using SEQUENCHER version
region is that, due to the high number of tandem repeats 4.7 (Genecodes).
of rDNA, detection methods targeting this region are
highly sensitive.
Species-specific primers
The aims of this study were (i) to develop both conven-
tional and real-time PCR assays for P. kuehnii and P. mel- Two sets of species-specific primers were designed. The
anocephala, and (ii) to develop an assay that allows the first set was developed for use with standard thermocy-
detection of a single nucleotide polymorphism (SNP) cling equipment and fragment visualization using agarose
identified in ITS1 of P. kuehnii. gel electrophoresis. The primers Pk1-F and Pk1-R

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PCR assays for sugarcane rusts 705

Table 1 Sources and number of Puccinia kuehnii and P. melanocephala-infected sugarcane leaf samples used in this study and GenBank accession
numbers for resulting rDNA sequences

No.
Country Pathogen samples GenBank Acc. No. Yeara Cultivara Locationa

Australia P. kuehnii 2 GU564415, GU564419 2001 Q124 Mackay, QLD

China P. kuehnii 2 GU564408, GU564417 2008 RB72–454 Guangxi Province
Costa Rica P. kuehnii 8 GU564409 2008 CP 72–2086 Curts Canas, Guanacaste
Guatemala P. kuehnii 2 GU564410 2007 BT 65–152 Santa Lucia, Cotzumalguapa
Jamaica P. kuehnii 1 GU564411 2008 BT 9186 St. Catherine Parish, Middlesex County
Japan P. kuehnii 8 GU564416, GU564420 2007 NCo310 Makabe, Okinawa
Mexico P. kuehnii 3 GU564414 2008 NCo 301 Chiapas
Nicaragua P. kuehnii 3 GU564412 2007 ISA 00–1000 Ingenio San Antonio
Philippines P. kuehnii 14 GU564413, GU564418 2007 56–226 Silay City, Negros Occidental
USA P. kuehnii 6 GU564421 2009 CP 72–2086 Palm Beach County, Florida
Australia P. melanocephala 2 GU564422 2007 Q117 Woodford, QLD
Brazil P. melanocephala 6 GU564423 2007 RB 92579 Pedro Alfonso, Tocantins
Colombia P. melanocephala 13 GU564424 2008 MZC74–275 San Antonio de Los Caballeros,
Cauca Valley
D.R. Congo P. melanocephala 3 GU564434 2008 SP70–1284 200 km S. Kinshasa
Dominican Republic P. melanocephala 4 GU564425 2008 CR9303 Higueral
Guadeloupe P. melanocephala 4 GU564435 2008 B86517 Petit-bourg
Guatemala P. melanocephala 3 GU564426 2007 CG97–97 Santa Lucia, Cotzumalguapa
Jamaica P. melanocephala 1 GU564431 2008 BT 9087 Scarlette Piece
Mauritius P. melanocephala 5 GU564430 2008 M 1202 ⁄ 01 Le Re’duit
Nicaragua P. melanocephala 1 GU564427 2008 CP 72–2086 El Viejo, Puerto Morazán
Pakistan P. melanocephala 4 GU564428 2007 SPF-234 Lalazar, Punjab
Panama P. melanocephala 1 GU564432 2008 BT-34152 Natá, Coclé Province
Reunion P. melanocephala 9 GU564433 2008 R 92 ⁄ 6261 St. Pierre
South Africa P. melanocephala 6 GU564429 2007 N29 Mount Edgecombe, KwaZulu-Natal
USA P. melanocephala 11 GU564436 2007 CP 05–1501 Palm Beach County, Florida

a
Year, cultivar and location within country given for samples with GenBank accession numbers.

Table 2 Targets for the primers and probes used in this study of Puccinia kuehnii and P. melanocephala and sizes of amplified products

Name and sequencea

Assay Target Forward Reverse Probeb Product size

PkPm P. kuehnii or PkPmF-aagagtgcacttaattgtggctc PkPmR-tcccacctgatttgaggtct N⁄A 606 or 585

P. melanocephala
Pk1 P. kuehnii Pk1F-aagagtgcacttaattgtggctc Pk1R-caggtaacaccttccttgatgtg N⁄A 527
Pk2 P. kuehnii Pk2F-gGgaaAcctcatTattaac Pk2R-gcctagagatcCattgtta Pk2P-tataa 142
CATTatCCCC
Pk-PIRA P. kuehnii Pk-PIRAF-ggaaacctcattattaacaagt Pk-PIRAR-atctatataacttttaacaatggatct N⁄A 136
Pm1 P. melanocephala Pm1F-aattgtggctcgaaccatcttc Pm1R-ttgctactttccttgatgctc N⁄A 480
Pm2 P. melanocephala Pm2F-gtaatCaggtaTaagtggc Pm2R-gCctagagaTccattgtta Pm2P-cCCC 130
AttTTAaaCA

a
Bases consisting of 2¢-O, 4¢-C methylene bridge (LNA bases) indicated in capitals, mismatch base for PIRA-PCR assay underlined.
b
Probes were dual labelled with 6FAM (5¢) and BHQ1 (3¢).

(Table 2) generated a 527 bp product from P. kuehnii The primers Pk1, Pk2, Pm1 and Pm2 were tested
and Pm1-F and Pm1-R generated a 480 bp product from against DNA isolated from additional P. kuehnii and
P. melanocephala DNA. Two additional forward primers P. melanocephala infected leaves (Table 1) and DNA
were designed for use in real-time PCR assays, one spe- isolated from the rust causing pathogens P. coronata,
cific to P. kuehnii (Pk2-F) and one to P. melanocephala P. graminis, P. recondita and Phakopsora pachyrhizi.
(Pm2-F). These were used with a reverse primer con- Since secondary colonizing fungi are found on sugarcane
served between the two pathogens (Pk2-R and Pm2-R). leaves and among uredinia, the primers were also tested
The primers Pk2-F and Pk2-R (Table 2) generated a against fungi isolated from sugarcane leaves and from
142 bp product from P. kuehnii DNA and primers Pm2-F collections of urediniospores using the following proce-
and Pm2-R (Table 2) generated a 130 bp product from dure. Leaf pieces containing uredinial lesions were
P. melanocephala DNA. excised from P. kuehnii and P. melanocephala-infected

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706 N. C. Glynn et al.

sugarcane leaves and plated onto potato dextrose agar tion of DNA concentration and change in cycle threshold
(PDA). In addition, sugarcane leaves with orange or (Ct) value calculated for each reaction (Gallup & Acker-
brown rust pustules were vacuumed to remove uredini- mann, 2006), efficiency slopes closest to )3Æ3 were
ospores together with any contaminating organisms pres- selected as the optimum. Optimized concentrations of
ent on the leaf surface or associated with the spores. The reaction ingredients were 0Æ5 lM (primers) and 0Æ1 and
vacuumed material was retained in an Eppendorf tube, 0Æ2 lM, respectively, for the P. kuehnii and P. melanocep-
diluted in sterile distilled water (SDW) and plated onto hala probes. The sensitivity and detection range of the
PDA. Plates were incubated at room temperature for assays was then determined using P. kuehnii and P. mela-
7 days and the resulting colonies were plated onto fresh nocephala genomic DNA extracted from approximately
PDA plates, incubated for a further 7 days at room tem- 30 mg of urediniospores of each pathogen according to
perature and identified to genus level based on colony the procedures described for the leaf samples above.
characteristics and morphology of fungal structures. DNA was diluted to an initial concentration of 40 ng
DNA was extracted from the mycelium using the Omni- lL)1 and then along a 10-fold serial dilution series.
Prep (G Biosciences) kits described above and DNA
was diluted to 20 ng lL)1. The P. kuehnii and P. melano-
Application of real-time PCR assays
cephala primers were tested against DNA from these col-
onies using the above reaction conditions. DNA from Field inspections of sugarcane cultivars in the Canal Point
each sample was tested using primers ITS4 and ITS5 breeding programme in South Florida were performed in
(White et al., 1990) to confirm the quality of the extracted June 2007 (when P. kuehnii was first observed in the
DNA. region) and again in April 2009. The plots of several dif-
ferent cultivars showed a range of symptoms consistent
with infection by sugarcane rust pathogens. Symptoms
Real-time PCR assays
ranged from hypersensitive flecking to uredinial lesions
Dual-labelled hydrolysis probes specific for each patho- on the underside of leaves. Where possible, the putative
gen were designed for use with primers Pk2 and Pm2 for causal agent of the observed symptoms was recorded.
use in real-time assays. Probes consisted of the dye Leaf parts from up to three separate leaves exhibiting
quencher BHQ1 attached at the 3¢-end and the fluores- these symptoms were excised from the leaves, dried and
cent label FAM at the 5¢-end (Table 2). Primers Pk2 and the DNA extracted from a 50 mg sample as described
Pm2 and the respective probes were designed manually above. Further surveys for sugarcane rusts were per-
from conserved positions within the sequence alignment formed in the same field plots several weeks following the
of each pathogen and differing between P. kuehnii and P. taking of leaf samples. The susceptibility status of each
melanocephala. Nucleotides with a 2¢-O, 4¢-C methylene cultivar towards both orange and brown rust was deter-
bridge, known as locked nucleic acids (LNA) were used in mined according to a rating scale originally described for
the primers and probes. The number and positions of the brown rust by Tai et al. (1981) and recently adapted for
LNA bases was optimized using EXIQON software avail- both brown and orange rust (Comstock et al., 2010). A
able at http://lnatools.com/, with positions chosen to 0–4 rating scale was used where no pustules visible = 0
minimize self-hybridization and the generation of sec- (highly resistant, HR), few poorly developed pustules = 1
ondary structures. The primer and probe sequences (Resistant, R), more abundant pustules, larger in size = 2
including the positions of LNA bases are given in Table 2. (moderately resistant, MR), numerous large pustules
Real-time PCR reactions were performed using a spread over entire plant = 3 (moderately susceptible,
Chromo4 real-time detection system (Bio-Rad) in 20 lL MS), and numerous large-coalescing pustules, dead
volumes containing 1· Premix Ex Taq (Perfect Real leaves = 4 (very susceptible, VS). DNA extracted from
Time) (TaKaRa Bio. Inc.). Reaction conditions were opti- infected leaves was used in real-time PCR assays to detect
mized by performing gradients of annealing temperature P. kuehnii and P. melanocephala. Real-time PCR reac-
(54–64C), primer concentration (0Æ05–0Æ5 lM), and tions were performed in triplicate using 100 ng of tem-
probe concentration (0Æ1–0Æ2 lM). The rDNA fragment plate genomic DNA and conditions as described above.
amplified with primers PkPm-F ⁄ R and containing the Ct values for each reaction were recorded.
species specific primer sites nested internally was excised
from the gel following electrophoresis using Wizard SV
PIRA-PCR assay
gel Clean-Up System (Promega). This was used as tem-
plate DNA to optimize the real-time reaction conditions. A SNP in which nucleotide A is substituted for G was
The concentration of purified DNA was determined by identified in ITS1 of P. kuehnii at position 183 of the
spectrophotometry, diluted to an initial concentration of 606 bp fragment generated using primers PkPm-F ⁄ R and
approximately 20 ng lL)1 then along a serial dilution of is referred to as PkITS1 )183A>G. No restriction enzyme
12 10-fold dilutions. Thermocycling conditions consisted was available that recognizes this polymorphism, there-
of 98C for 5 min followed by 45 cycles of 95C for 30 s fore a primer-introduced restriction analysis-PCR (PIRA-
and the optimized annealing temperature for both prim- PCR) assay was developed. The primers Pk-PIRAF and
ers of 60C for 30 s followed by 72C for 30 s. Reaction Pk-PIRAR (Table 2) were designed using software
efficiencies for each condition were determined as a func- described by Ke et al. (2001) and available at http://

Plant Pathology (2010) 59, 703–711

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PCR assays for sugarcane rusts 707

cedar.genetics.soton.ac.uk/public_html/primer2.html. cane leaves, vacuumed spore collections, or with host

The PIRA-PCR assay is a nested design involving an DNA isolated from sugarcane stalks (Table 3).
initial amplification reaction using the primers PkPm and Reaction efficiencies for both assays and the dynamic
reaction conditions described above. This product was range of amplification were similar using the purified
diluted 50-fold and 1 lL used for re-amplification using rDNA or genomic DNA from P. kuehnii or P. melanocep-
the primers Pk-PIRAF and Pk-PIRAR. Reaction volumes hala as a template (Fig. 1). Optimized reaction conditions
of 20 lL were used and reagent concentrations and for the P. kuehnii and P. melanocephala assays allowed
thermocycling conditions were as described for the amplification along a standard curve from 0Æ002 fg to
PkPm primers except that an annealing temperature of 2000 fg using rDNA as template and 0Æ00019 ng to
52C was used. The resulting 136 bp product was 190 ng using genomic DNA as a template. The standard
digested at position 109 bp by the restriction enzyme curves for both assays together with reaction efficiencies
BglII (A.GATCT) only if PkITS1 )183G was present and slopes are given in Fig. 1.
and resulted in products of 109 and 27 bp. Digestions
were performed in 20 lL volumes containing 8 lL of
Application of real-time PCR assays
re-amplified product and 12 lL of a master mix contain-
ing 20 units of BglII (New England Biolabs), appropriate The sugarcane leaf samples that exhibited disease symp-
buffer and molecular biology grade water. Digestion toms identified as orange rust in 2007 yielded Ct values
incubation was performed according to the manufac- ranging from 17 to 21 using the P. kuehnii real-time assay
turer’s recommendations of 37C for 1 h. The resulting (Table 4). Assays were negative using the P. melanocep-
products were visualized following electrophoresis on a hala specific primer and probe, confirming P. kuehnii as
3% agarose gel. the casual agent of disease. Orange rust pustules were
subsequently observed on all cultivars sampled which
were classified as resistant or moderately resistant to
Results
orange rust. Although no brown rust symptoms were
Primer specificity and real-time assays
Fragments of rDNA were successfully isolated from all (a) 40
the P. kuehnii and P. melanocephala-infected samples
35
tested using primers PkPm-F and -R. The putative
y = –3·70 + 36·59x
P. kuehnii and P. melanocephala specific primers 30
Cycle threshold (Ct)

Efficiency= 90%
designed from the alignments of these sequences only 25
r2= 1·00
amplified DNA from each respective pathogen. Neither
20
primer set cross-reacted with several other rust fungi
tested or with fungal species isolated from infected sugar- 15

10

5
Table 3 Fungal species or genera used to test the Puccinia kuehnii and
P. melanocephala specific primers developed in this study. The primers 0
ITS4 and ITS5 (White et al., 1990) were used to confirm that the extracted 0·1 1 10 100 1000 10000 100000 1000000
DNA was amplifiable Picograms of template DNA (Log scale)

Assay tested ⁄ result (b) 40

Species or genus P. kuehnii P. melanocephala ITS 4 ⁄ 5 35
y = –3·35 + 35·01x
Alternaria sp. ) ) + 30
Cycle threshold (Ct)

Efficiency= 107%
Botrytis sp. ) ) + r2= 1·00
25
Cercospora sp. ) ) +
Cochliobolus sativus ) ) + 20
Darluca sp. ) ) +
15
Epicoccum sp. ) ) +
Fusarium sp. ) ) + 10
Penicillium sp. ) ) +
5
Phakopsora pachyrhizia ) ) +
Puccinia coronataa ) ) + 0
Puccinia graminisa ) ) + 0·1 1 10 100 1000 10000 100000 1000000
Puccinia striiformisa ) ) + Picograms of template DNA (Log scale)
Pyricularia sp. ) ) +
Rhizoctonia sp. ) ) + Figure 1 Standard curves for (a) Puccinia kuehnii and
Zygomycota sp. ) ) + (b) P. melanocephala real-time PCR assays using primers Pk2 and
Pm2. Data points are DNA amount plotted against the mean cycle
a
DNA provided by the USDA-ARS Cereal Disease Laboratory. threshold (Ct).

Plant Pathology (2010) 59, 703–711

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708 N. C. Glynn et al.

Table 4 Real-time detection of Puccinia kuehnii and P. melanocephala using primers Pk2 and Pm2, respectively, in sugarcane leaves sampled in South
Florida showing a range of symptoms

Real-time PCR result (Ct) Subsequent resistance rating

Year Cultivar Visual observations P. kuehnii P. melanocephala P. kuehnii P. melanocephala

a b
2007 CPCL 02–2632 Orange rust 19Æ0 ± 0Æ32 – R (R1) R (R1)
2007 CPCL 02–0320 Orange rust 17Æ4 ± 0Æ29 – R (R1) HR (R0)
2007 CPCL 99–4295 Orange rust 18Æ0 ± 0Æ10 – R (R1) R (R1)
2007 CP 03–1491 Orange rust 21Æ3 ± 0Æ40 – MR (R2) MR (R2)
2007 CP 72–2086 Orange rust 21Æ4 ± 0Æ11 – MR (R2) MR (R2)
2007 CP 89–2143 Orange rust 18Æ7 ± 0Æ41 – MR (R2) R (R1)
2009 CP 88–1762 Orange rust 17Æ7 ± 0Æ 27 – MS (R3) HR (R0)
2009 SP 89–1115 Orange rust 20Æ4 ± 0Æ06 – MS (R3) MR (R2)
2009 N 29 Brown rust – 25Æ7 ± 0Æ22 HR (R0) MS (R3)
2009 CP 05–1658 Unidentified pustules 19Æ4 ± 0Æ38 – MS (R3) HR (R0)
2009 CP 05–1463 Flecking 21Æ8 ± 0Æ08 – MS (R3) MR (R2)
2009 CPCL 05–6415 Unidentified pustules 25Æ3 ± 0Æ18 – R (R1) R (R1)
2009 CP 04–1367 Unidentified pustules – 20Æ7 ± 0Æ14 HR (R0) R (R1)
2009 CP 07–2393 Flecking 33Æ3 ± 0Æ74 – HR (R0) HR (R0)
2009 CP 03–1491 Flecking 36Æ0 ± 0Æ43 – MR (R2) HR (R0)
2009 CPCL 02–0908 Flecking 35Æ2 ± 0Æ38 – MR (R2) HR (R0)
2009 CPCL 06–3316 Flecking 34Æ7 ± 0Æ12 – MR (R2) HR (R0)
2009 CP 06–2664 Flecking 35Æ9 ± 0Æ60 – HR (R0) HR (R0)

a
– = no Ct value observed.
b
HS, highly susceptible; S, susceptible; MS, moderately susceptible; MR, moderately resistant; R, resistant; HR, highly resistant.
Number in brackets refers to 0–4 susceptibility rating scale.

observed on and no P. melanocephala DNA was detected cloning and sequencing the respective fragments (Fig. 2).
in the leaves tested, brown rust pustules were observed on The frequency of these two alleles in the sequences gener-
some of the cultivars later in the season and were classi- ated in this study together with results from the
fied as resistant or moderately resistant to brown rust PIRA-PCR assay, and the frequency in P. kuehnii
(Table 4). sequences accessioned in the NCBI database, are given in
Puccinia kuehnii was detected in leaf samples from all Table 5. Allele )183A was observed in all 53 samples
six cultivars that exhibited flecking symptoms sampled in examined using the PIRA-PCR assay whereas )183G
April 2009. Mean Ct values for these samples ranged was observed only in 15 (52%) of the 29 samples from
from 22 to 36 (Table 4). Cultivars CP 07–2393 and CP Australia, Japan, Philippines and China and absent from
06–2664 were subsequently classified as highly resistant, all samples obtained from the Western hemisphere. Simi-
CP 03–1491, CPCL 02–0908 and CPCL 06–3316 moder- larly, )183G was absent from all sequences generated in
ately resistant and CP 05–1463 moderately susceptible to this study and in the NCBI database from the Western
orange rust based on visual symptoms. Puccinia kuehnii hemisphere (Table 2). The detection frequencies of
was also detected in two samples on which pustules were 183G:183A among samples from the Eastern hemisphere
observed but could not be speciated visually, giving mean were 89% in the sequences generated in this study, 52%
Ct values of 19 and 25 (Table 4). Puccinia melanocephala using the PIRA-PCR assay and 29% among sequences in
DNA was detected in two samples in 2009. Symptoms on the NCBI database.
cv. N 29 were identified as brown rust however, pustules
on CP 04–1367 could not be identified, these cultivars
Discussion
were subsequently classified as moderately susceptible
and resistant respectively to brown rust. No orange rust The first objective of this study was to develop conven-
symptoms were observed on either of these cultivars. tional and real-time PCR assays for each of the two spe-
Puccinia melanocephala was not detected on any of the cies of Puccinia which cause rust diseases of sugarcane.
other cultivars which were subsequently classified as Sequencing isolates from almost all sugarcane growing
highly resistant (seven cultivars), resistant (two cultivars) regions combined with a strategy of sequencing multiple
or moderately resistant (two cultivars) based on visual colonies from the cloning reactions increased the likeli-
symptoms (Table 4). hood that the assays developed detect all isolates of the
two pathogens. Testing the assays on DNA from fungal
pathogens and saprophytes isolated from sugarcane
SNP detection using PIRA-PCR
leaves and spore collections also reduced the potential
The PIRA-PCR assay allowed the discrimination of the for cross reactions with other organisms associated with
alleles of SNP PkITS1 )183A>G without the need for sugarcane.

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PCR assays for sugarcane rusts 709

Table 5 Frequency of the two alleles of a single nucleotide polymorphism

1 2 3 4
identified in ITS1 of Puccinia kuehnii. Data is separated by hemisphere of
sample origin and method of detection: number of colonies sequenced,
analysis of herbaria using PIRA-PCR assay and sequences accessioned in
the NCBI database

Allele ⁄ number of
observations

Region Sample detection )183A )183G

c
Western Sequences 73 0
hemispherea Dried samples (PIRA-PCR) 24 0
NCBI 11 0
Eastern Sequencesc 45 40
hemisphereb Dried samples (PIRA-PCR) 29 15
NCBI 24 7

a
Includes data for samples from USA, Guatemala, Nicaragua, Costa
Rica, Mexico, Jamaica, Panama and El Salvador.
b
Includes data for samples from China, Philippines, Australia,
Japan, Indonesia and Papua New Guinea.
c
From sequencing positive colonies from cloning reactions.

monitoring the spread of the pathogen in these regions.

Quantitative real-time PCR has been used to monitor the
spread of the soybean rust pathogen Phakopsora
pachyrhizi (Barnes et al., 2009) and several cereal rust
pathogens (Barnes & Szabo, 2007) in rain collected in
remote samplers. A similar strategy could be imple-
mented for P. kuehnii in the sugarcane growing regions of
the Americas.
Puccinia kuehnii and P. melanocephala were detected
Figure 2 Example of results from PIRA-PCR assay of Puccinia
in this study prior to uredinia development. Several previ-
kuehnii DNA. Lane 1: 20 bp ladder molecular marker; Lane 2:
ous studies have examined relationships between early
a 606 bp product amplified using PkPm primers; Lane 3:
re-amplification of the PkPm product with primers PIRA-Pk and
detection of foliar pathogens and visual symptoms
digested with the restriction enzyme BglII (two bands are evident (Fraaije et al., 2001; Guo et al., 2006; Jackson et al.,
corresponding to the 136 bp undigested product (A allele) and the 2006; Lihua et al., 2008). This feature could prove useful
109 bp digested product (G allele); Lane 4: the re-amplified, for disease control strategies as it would allow control
digested product for a sample which only contains the A allele. measures such as protective fungicides to be deployed,
thereby limiting the spread of inoculum.
The PIRA-PCR assay developed in this study proved to
Locked nucleic acid (LNA) nucleosides were incor- be an effective method for detecting the SNP PkITS1
porated into the primers and probes used in the real-time )183A>G in P. kuehnii. This was not possible using
assays. LNAs provide equivalent sensitivity and specific- hydrolysis probes (data not shown) possibly due to the A-
ity as minor groove binding modifications (Letertre et al., T rich nature of the surrounding bases, and no restriction
2003), by increasing the duplex melting temperatures. enzyme recognizing either of the SNP alleles could be
LNAs allow probes to be shorter (15–25 nucleotides) identified.
than unmodified probes which are typically 25–35 nucle- One of the two SNP alleles was detected only in sam-
otides. The probes developed in this study were up to half ples from Asia and Australia and was absent from all sam-
the length of fluorescent probes reported by Barnes & ples from the Caribbean basin tested in this study. This
Szabo (2007) for four species of cereal rust pathogens. was further confirmed by P. kuehnii sequences in the
This feature is advantageous for detection methods based NCBI database in which the minor allele was only present
on variable sequences such as the ITS regions of rDNA in samples from Asia and Australia. The frequency of
since the portions of conserved sequence may be limited. )183A and )183G varied with method of detection
Since the first confirmed occurrence of P. kuehnii in the (sequencing, PIRA-PCR and NCBI accessioned
Western hemisphere, the pathogen has been detected sequences). This may reflect differences between the
throughout Central America and the Caribbean basin. populations of P. kuehnii isolates represented in each
The sugarcane industries of Louisiana, Texas and South source. Alternatively this may be due to additional varia-
America, which so far remain free from infection, are tions existing close to the 183A>G loci resulting in the
under considerable threat from this pathogen. The non-detection of the two alleles by the PIRA-PCR assay.
P. kuehnii assay developed in this study could be used for A possible explanation for this absence among Caribbean

Plant Pathology (2010) 59, 703–711

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710 N. C. Glynn et al.

samples is that the allele was present at an extremely low Barnes CW, Szabo LJ, 2007. Detection and identification of
frequency. However, this is unlikely since over 70 four common rust pathogens of cereals and grasses using
colonies were sequenced from samples collected in the real-time polymerase chain reaction. Phytopathology 97,
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Two forms of natural plant pathogen dispersal occur: quantifying Phakopsora pachyrhizi spores in rain.
single step invasions, where pathogen introduction Phytopathology 99, 328–38.
Brown JKM, Hovmøller MS, 2002. Aerial dispersal of pathogens
occurs over a large area in a single event, and range
on the global and continental scales and its impact on plant
expansions, where spread occurs gradually through
disease. Science 297, 537–41.
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2002). Puccinia melanocephala was found initially in the
rust of sugarcane caused by Puccinia kuehnii in Costa Rica and
Dominican Republic following a single step invasion by Nicaragua. Plant Disease 93, 425.
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Several possible mechanisms could explain the recent agent of orange rust of sugarcane, in the United States and
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sources of P. kuehnii before 2007, it is possible that the orange rust, an emerging disease in North and Central America:
pathogen was initially aerial dispersed from this region to its impact and comparison to sugarcane brown rust. The
an intermediate location(s), remaining undetected or Americas Journal of Plant Science and Biotechnology.
undiscovered due to confusion with the widely distrib- In press.
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ducted movement of vegetative planting material which Salvador and Panama. Plant Disease 93, 1347.
also contained P. kuehnii-infected leaves could have Fraaije BA, Lovell DJ, Coelho JM, Baldwin S, Hollomon DW,
resulted in an initial spread of the pathogen from Asia or 2001. PCR-based assays to assess wheat varietal resistance to
blotch (Septoria tritici and Stagonospora nodorum) and rust
Australia followed by rapid and unnoticed spread.
(Puccinia striiformis and Puccinia recondita) diseases. European
Recently, the rapid, global spread of the wheat yellow
Journal of Plant Pathology 107, 905–17.
rust fungus P. striiformis f. sp. tritici has been reported
Gallup JM, Ackermann MR, 2006. Addressing fluorogenic real-
(Hovmøller et al., 2008). The same strains of the patho-
time qPCR inhibition using the novel custom Excel file system
gen that first appeared in North America in 2000 were ‘FocusField2-6GallupqPCRSet-upTool-001’ to attain consistently
detected in Western Australia just 2 years later. The high fidelity qPCR reactions. Biological Procedures Online 8,
spread of P. kuehnii to the Western hemisphere could 87–153.
therefore have taken place in a similar, rapid and long dis- Guo JR, Schnieder F, Verreet JA, 2006. Presymptomatic and
tance dispersal event. Whichever mechanism led to the quantitative detection of Mycosphaerella graminicola
spread of P. kuehnii, only )183A type isolates predomi- development in wheat using a real-time PCR assay. FEMS
nate in the Western hemisphere. This has important Microbiology Letters 262, 223–9.
implications for the development of sugarcane cultivars Hovmøller MS, Yahyaoui AH, Milus EA, Justesen AF, 2008.
with durable resistance to P. kuehnii. Sugarcane breeding Rapid global spread of two aggressive strains of a wheat rust
for orange rust resistance must attempt to incorporate fungus. Molecular Ecology 17, 3818–26.
germplasm screening against isolates harbouring both Jackson E, Avant J, Overturf K, Bonman J, 2006. A quantitative
)183A and )183G to combat any differences in virulence assay of Puccinia coronata f. sp. avenae DNA in Avena sativa.
that may be associated with the two isolate types identi- Plant Disease 90, 629–36.
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Acknowledgements Letertre C, Perelle S, Dilasser F, Arar K, Fach P, 2003. Evaluation
of the performance of LNA and MGB probes in 5¢-nuclease PCR
The authors thank Kay McCorkle for technical assis-
assays. Molecular and Cellular Probes 17, 307–11.
tance in the laboratory and Charles Barnes and Jerry
Lihua C, Shichang X, Ruiming L, Taiguo L, Wanquan C, 2008.
Johnson at the Cereal Disease Laboratory for technical
Early molecular diagnosis and detection of Puccinia striiformis
support and for providing Puccinia spp. DNA.
f. sp. tritici in China. Letters in Applied Microbiology 46,
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