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A molecular diagnostic for tropical race 4 of the banana
fusarium wilt pathogen
M. A. Dita
a,b
, C. Waalwijk
b
, I. W. Buddenhagen
c
, M. T. Souza Jr
b,d
and G. H. J. Kema
b
*
a
Embrapa Cassava & Tropical Fruits, Cruz das Almas, 44380-000, Bahia, Brazil;
b
Plant Research International B.V., PO Box 16, 6700
AA Wageningen, the Netherlands;
c
1012 Plum Lane, Davis, California, USA; and
d
Embrapa LABEX Europe, PO Box 16, 6700 AA
Wageningen, the Netherlands
This study analysed genomic variation of the tran slation elongation factor 1a (TEF-1a) and the intergenic spacer region
(IGS) of the nuclear ribosomal operon of Fusarium oxysporum f. sp. cubense (Foc) isolates, from different banana produc-
tion areas, representing strains within the known races, comprising 20 vegetative compatibility groups (VCG). Based on two
single nucleotide polymorphisms present in the IGS region, a PCR-based diagnostic tool was developed to specifically detect
isolates from VCG 01213, also called tropical race 4 (TR4), which is currently a major concern in global banana production.
Validation involved TR4 isolates, as well as Foc isolates from 19 other VCGs, other fungal plant pathogens and DNA sam-
ples from infected tissues of the Cavendish banana cultivar Grand Naine (AAA). Subsequently, a multiplex PCR was devel-
oped for fungal or plant samp les that also discriminated Mu sa acuminata and M. balbisiana genotypes. It was concluded
that this diagnostic procedure is currently the best option for the rapid and reliable detection and monitoring of TR4 to sup-
port eradication and quarantine strategies.
Keywords: Fusarium oxysporum f. sp. cubense in planta detection, Musa spp., Panama disease, PCR-based diag-
nostic, vegetative compatibility groups
Introduction
Banana and plantain (Musa spp.) are among the most
important crops in the world, serving as a staple food and
source of income in many developing countries. Banana
is also the world’s leading fruit crop and consequently an
important export commodity for several agricultural-
based economies in Latin America, Africa and Asia, and
represents the fifth most important agricultural crop in
world trade (Aurore et al., 2009). Among the major glo-
bal constraints on production are several diseases such
as black Sigatoka or black leaf streak disease caused by
Mycosphaerella fijiensis and Panama disease or fusarium
wilt caused by Fusarium oxysporum f. sp. cubense (Foc)
(Stover, 1962; Ploetz, 2006). Symptoms of fusarium wilt
start with yellowing and wilting of the older leaves, which
progresses to the younger leaves until the death of the
entire plant. Internally, plants with advanced infection
show discolora tion of the rhizome and necrosis of xylem
vessels in the pseudostem. Foc is a soilborne pathogen
that produces chlamydospores, enabling the fungus to
persist in soil in the absence of the host. Hence, once soil
is infested with Foc, susceptible varieties cannot be suc-
cessfully replanted for up to 30 years (Stover, 1962,
1990). As a result, fusarium wilt wiped out the banana
industry based on cv. Gros Michel in Central America in
the middle of the last cen tury. This forced the trade to
shift to resistant cultivars of the Cavendish subgroup
(AAA) (Stover, 1962, 1990; Ploetz, 2006). Cavendish
cultivars solved the problems for the banana export trade
from Latin America, where tropical race 4 (TR4, see
below) is absent, but not in Asian countries, where TR4 is
present. Hence, fusarium wilt continues to be a constraint
to susceptible varieties and is still considered a major
threat to banana production because, unlike black leaf
streak disease, it cannot be controlled with fungicides.
Early attempts to rationalize pathogen diversity
resulted in the design ation of race 1 and race 2, differ-
entially pathogenic on cvs Gros Michel (AAA) and
Bluggoe (ABB) from observations in Honduras (Waite
& Stover, 1960). Later, in Taiwan, Cavendish bananas
were affected and a race 4 was designated. However,
this pathotype could also cause disease in banana culti-
vars susceptible to races 1 and 2 (Su et al., 1986).
Before 1990, isolates that were classified as race 4 only
caused serious losses in Cavendish genotypes in sub-
tropical regions of Australia, the Canary Islands and
Taiwan (Su et al., 1986; Pegg et al., 1996). Since then,
*E-mail: gert.kema@wur.nl
ª 2010 Plant Research International
Journal compilation ª 2010 BSPP
1
Plant Pathology (2010) Doi: 10.1111/j.1365-3059.2009.02221.x
a new variant that severely affects Cavendish cultivars
in the tropics was identified. Thus, two types of Foc
race 4, viz. subtropical race 4 (ST4) and tro pical race 4
(TR4) were designated. However, while ST4 isolates
cause disease in Cavendish in the subtropics, mainly
when plants are exposed to abiotic stress, TR4 isolates
are pathogenic under both tropical and subtropical
conditions (Buddenhagen, 2009).
Since its appearance, TR4 has caused severe damage to
Cavendish cultivars in Malaysia, Indonesia, South China,
the Philippines and the Northern Territory of Australia
(Ploetz, 2006; Molina et al., 2008; Buddenhagen, 2009).
Control strategies of TR4 are based on visual monitoring
for early symptom appearance, eradication of infected
plants and isolation of infested areas to reduce pathogen
dissemination. However, these strategies are often
impractical and therefore not carried out. Additionally,
identification is further complicated by the above
mentioned race concept, which does not adequately
capture genetic variation. Therefore, alternative charac-
terization strategies have been implemented. Vegetative
compatibility group (VCG) analyses (Co rrell et al., 198 7;
Ploetz & Correll, 1988; Moore et al., 1993) and phylo-
genetic studies based on molecular data (Koenig et al.,
1997; Bentley et al., 1998; O’Donnell et al., 1998;
Groenewald et al., 2006; Fourie et al., 2009) revealed
more genetic variation in Foc. At least 21 different VCGs
of Foc have been characterized, with the majority of
groups present in Asia, where the pathogen is thought to
have evolved (Ploetz & Pegg, 1997; Fourie et al., 2009).
While TR4 isolates are designated as VCG 01213 (or
VCG 01216, which is a different designation for the same
VCG) isolates classified as ST4 belong to VCGs 0120,
0121, 0122, 0129 and 01211 (Buddenhagen, 2009).
Therefore, VCG tests are useful for TR4 diagnosis, but
require time-consuming generation and characterization
of nit mutants and the availability of testers.
This paper describes the development of a rapid and
reliable PCR diagnostic for Foc TR4 ⁄ VCG 01213 that
can also be used for in planta detection. It is anticipated
that it will be used to support national and international
quarantine measures in order to avoid further dissemina-
tion of TR4.
Materials and methods
Fusarium oxysporum isolates and cultural conditions
In total, 82 Foc isolates originating from different banana
production areas and comprising 20 VCGs were analysed
(Table 1). Samples from geographic regions known to be
infested by TR4 were received as dry pseudostem strands
and were sectioned into pieces (2 cm long and 0Æ5cm
wide), transferred to Komada’s medium (Komada, 1975)
and incubated at 25C. After 3–5 days, when fungal
growth appeared as white and pink aerial mycelia, iso-
lated colonies were examined by light microscopy for the
presence of macroconidia and micro conidia diagnostic of
F. oxysporum. Positive samples were transferred to plates
with potato-dextrose agar (PDA) and stored for further
analyses.
Vegetative compatibility group analyses
Nitrate-nonutilizing (nit) mutants of the wild-type Foc
strains were generated in minimal medium (MM)
(Puhalla, 1985) amended with 1Æ5–4Æ 5% KCl0
3
and
incubating for 7–14 days at 25C. Spontaneous
KClO
3
-resistant sectors were transferred to MM.
Those that grew as thin colonies with no aerial myce-
lium were classified as nit mutants and were further
characterized on media containing one of four different
sources of nitrogen (Correll et al., 1987). Finally,
VCGs of all mutants were determined by pairing on
MM with tester nit mutants from strains with known
VCGs (Correll et al., 1987). Complementation between
different nit mut ants resulted in dense aerial growth at
the contact zone between the two colonies. None of
the isolates tested was self-incompatible.
DNA isolation, PCR amplification and sequencing
For DNA isolation, a single-spore culture of each isolate
(Table 1) was grown in Petri plates (6 cm diameter)
containing PDA and incubated at 25C for 5 days. To
facilitate the harvest of mycelia, a cellophane disc (5Æ5cm
diameter) was placed on the medium surface prior to
inoculation. Mycelium was harvested by scraping the
cellophane disc and was subsequently stored in 2-mL
tubes at )80C. After addition of a tungsten bead, the
mycelium was lyophilized and ground by vigorous shak-
ing of the tubes in a MM300 mixer mill (Retch). Total
genomic DNA was extracted using the Wizard Magnetic
DNA Purification System for Food kit (Promega) accord-
ing to the manufacturer’s instructions. DNA samples
were diluted to 10 ng lL
)1
and stored at )20C until use.
DNA samples from isolates of Fusarium oxysporum f. sp.
passiflorae, F. guttiforme, F. graminearum and F. verticil-
lioides (Table 1) were used for specificity tests. The trans-
lation elongation facto r 1a gene, TEF-1 a, was amplified
with primers EF-1 and EF-2 (O’Donnell et al., 1998)
using the following programme: 95C for 2 min and 35
cycles of 95C for 30 s, 57C for 30 s and 72C for 1 min,
followed by an additional extension time for 10 min at
72C. The intergenic spacer (IGS) region of the nuclear
ribosomal operon was amplified using primers iNL11
(5¢-AGGCTTCGGCTTAGCGTCTTAG-3¢) and iCNS1
(5¢-TTTCGCAGTGAGGTCGGCAG-3¢) and the fol-
lowing programme: 95C for 5 min and 30 cycles of 95C
for 1 min, 62C for 1 min and 72C for 3 min, followed
by an additional extension time for 10 min at 72C. PCR
products were directly sequenced using Big Dye Termina-
tor (v3.1; Applied Bio systems). The TEF-1a gene was
sequenced using the aforementioned primers. The IGS
regions of the nuclear ribosomal operons were sequenced
with primers iNL11, iCNS1, NLa (5¢-TCTA
GGGTAGGCKRGTTTGTC-3¢ ) and CNSa (5¢-TCTCA
TRTACCCTCCGAGACC-3¢).
2 M. A. Dita et al.
Plant Pathology (2010)
Table 1 Origin of isolates of Fusarium oxysporum f.sp. cubense and other species, their known or determined vegetative compatibility groups (VCG), race
classification and r esponse to known and newly develope d PCR diagnostics
Code Received as VCG
b
Race
c
Host
d
Location Source
e
PCR diagnostic
Foc-1 ⁄
Foc-2
FocTR4-F ⁄
FocTR4-R
Focu1 Foc 0120 Mons Mari Queensland Australia, 2 + )
Focu2 Foc 0121 Gros Michel Costa Rica, 2 + )
NRRL36102 Foc 0121 Cavendish Taiwan, 3 + )
NRRL25603 Foc 0122 Cavendish Australia, 3 + )
NRRL36103 Foc 0122 Cavendish Philippines, 3 + )
NRRL26022 Foc 0123 Pisang Awak Thailand, 3 ))
NRRL36101 Foc 0123 R1 Mons Mari Australia, 3 + )
NRRL36104 Foc 0123 Kluai Namwa Sai Deng Thailand, 3 ))
Focu3 Foc 0124 Bluggoe Honduras, 2 ))
Focu4 Foc 0124 Bluggoe Jamaica, 2 ))
NRRL25607 Foc 0124 R2 Bluggoe USA, 3 ))
NRRL36105 Foc 0124 Bluggoe Honduras, 3 ))
Focu5 Foc 0125 Lady Finger Currumbin,
Queensland
Australia, 2 ))
NRRL36106 Foc 0125 Pome Australia, 3 ))
Focu6 Foc 0126 Maquen
˜
o Honduras, 2 + )
NRRL36107 Foc 0126 Maquen
˜
o Honduras, 3 + )
NRRL36111 Foc 0128 Bluggoe Australia, 3 ))
NRRL36110 Foc 0129 Cavendish Australia, 3 + )
Focu7 Foc 01210 Apple Florida USA, 2 + )
NRRL26029 Foc 01210 R1 Silk Florida USA, 3 + )
NRRL36109 Foc 01211 SH3142 Australia, 3 + )
NRRL36108 Foc 01212 Ney Poovan Tanzania, 3 ))
NRRL36114
a
Foc 01213 TR4 Pisang Manurung Indonesia, 5 + +
Focu8 Foc 01214 Harare Misuki Hills, Karonga, Malawi, 2 ))
NRRL25609 Foc 01214 Harare Malawi, 3 ))
NRRL36113 Foc 01214 Bluggoe Malawi, 3 ))
NRRL36112 Foc 01215 Cavendish South Africa, 3 + )
NRRL36120 Foc 01218 Pisang Awak Thailand, 3 ))
NRRL36118 Foc 01221 Pisang Awak Thailand, 3 ))
NRRL36117 Foc 01222 Pisang Awak Legor Malaysia, 3 ))
NRRL36116 Foc 01223 Pisang Keling Malaysia, 3 ))
NRRL36115 Foc 01224 Pisang Ambon Malaysia, 3 ))
BPI-0901 Field samples
(petiole)
0120* Cavendish Java Indonesia, 6 + )
Foc19508 Foc 0120* R1 Gros Michel Guapiles Costa Rica, 4 + )
FocST498 Foc 0120* ST4 Dwarf Cavendish Canary Islands Spain, 1 + )
BPS1.1 Field samples
(pseudostem)
01213* Cavendish Kuta-village Bali Indonesia, 6 + +
BPS3.1
a
Field samples
(pseudostem)
01213* Cavendish Darwin Australia, 6 + +
BPS3.2
a
Field samples
(pseudostem)
01213* Cavendish Darwin Australia, 6 + +
BPS3.3
a
Field samples
(pseudostem)
01213* Cavendish Darwin Australia, 6 + +
BPS3.4
a
Field samples
(pseudostem)
01213* Cavendish Darwin Australia, 6 + +
Foc-T105 Foc 01213* R4 Cavendish Nantow Taiwan, 7 + +
Foc-T14 Foc 01213* R4 Cavendish Taitung Taiwan, 7 + +
Foc-T202 Foc 01213* R4 Cavendish Nantow Taiwan, 7 + +
II-5
a
Foc 01213* TR4 Pisang Manurung Indonesia, 5 + +
BPI-0902 Field samples
(pseudostem)
† Silk Mariana Islands (Saipan),
Farm: Lucy Norita
Indonesia, 6 ))
BPI-0903 Field samples
(pseudostem)
† Silk Mariana Islands (Rota CNMI)
Farm: Frank Calvo
Indonesia, 6 ))
BPI-0904 Field samples
(pseudostem)
† Silk Mariana Islands (Rota CNMI) Indonesia, 6 ))
Diagnostic test for F. oxysporum f. sp. cubense TR4 3
Plant Pathology (2010)
Table 1 Continued
Code Received as VCG
b
Race
c
Host
d
Location Source
e
PCR diagnostic
Foc-1 ⁄
Foc-2
FocTR4-F ⁄
FocTR4-R
BPI-0905 Field samples (pseudostem) † Silk Mariana Islands
(Tinian Island),
Indonesia, 6 ))
Foc_R1 Foc † R1 Silk Cruz das Almas,
Bahia
Brazil, 9 ))
Foc_R2 Foc † R2 Monthan Cruz das Almas,
Bahia
Brazil, 9 ))
BPS4.1 Field samples (pseudostem) Awak Namulon Uganda, 4 ))
BPS5.1 Field samples (pseudostem) Sukara NE Kampala Uganda, 6 ))
BPS5.2 Field samples (pseudostem) Sukara NE Kampala Uganda, 6 ))
BPS5.3 Field samples (pseudostem) Sukara NE Kampala Uganda, 6 ))
BPS5.4 Field samples (pseudostem) Sukara NE Kampala Uganda, 6 ))
BPS5.5 Field samples (pseudostem) Sukara NE Kampala Uganda, 6 ))
Foc05 Foc R1 Prata Janau
´
ba Minas
Gerais
Brazil, 8 ))
Foc49 Foc R1 Prata Ana
˜
Cruz das Almas,
Bahia
Brazil, 9 ))
Foc97 Foc R1 Silk Botucatu, SP Brazil, 9 ))
FocYB Foc R1 Yamgambi Botucatu, SP Brazil, 9 + )
FT1 Foc Pisang Awak Uganda, 8 ))
FT12 Foc Pelipita Uganda, 8 ))
FT13 Foc Pelipita Uganda, 8 ))
FT14 Foc Gros Michel Uganda, 8 ))
FT23 Foc Pisang Ceylan Uganda, 8 ))
FT24 Foc Pisang Ceylan Uganda, 8 ))
FT3 Foc Pisang Awak Uganda, 8 ))
IMI 141103 Foc R2 10 ))
IMI 141109 Foc R1 10 ))
T91-1A Foc Taiwan, 2 + )
T91-1B Foc Taiwan, 2 + )
T91-1C Foc Taiwan, 2 + )
T91-2 Foc Taiwan, 2 + )
T91-4A Foc Taiwan, 2 + )
T91-4B Foc Taiwan, 2 + )
T91-4C Foc Taiwan, 2 + )
T91-5A Foc Taiwan, 2
+ )
T91-5C Foc Taiwan, 2 + )
T91-6A Foc Taiwan, 2 + )
T91-6B Foc Taiwan, 2 + )
T91-6C Foc Taiwan, 2 + )
T91-7 Foc Taiwan, 2 + )
Fop-08-1 F. o. f. sp. passiflorae Passion fruit Brazil, 9 ))
Fgt-08-1 F. guttiforme Pineapple Brazil, 9 ))
Fg820 F. graminearum Wheat Netherlands, 11 ))
M2 F. verticillioides Maize Netherlands, 11 ))
a
Isolates BPS3.1, BPS3.2, BPS3.3, BPS3.4 came from different pseudos tem strands of the same plant; isolates II-5 and NRRl36114
were obtained from different sources, but were thought to be clones.
b
Vegetative compatibility groups (VCGs) were assigned using nit mutants according to Correll et al. (1987). *Isolates with VCG
determined in this study; †Isolates not complemented with VCG 01213 testers.
c
Race designation as provided by supplier. R1, race 1; R2, race 2; ST4, subtropical race 4; TR4, tropical race 4.
d
Banana cultivars are inter- and intraspecific diploid or triploid hybrids of M. acuminata (AA) and M. balbisiana (BB). Ploidy levels and
constitutions of cultivars as follows: AA, SH3132; AAA, Cavendish, Dwarf Cavendish, Gros Michel, Lady Finger, Mons Mari, Pisang
Ambon, Yamgambi; AAB, Apple, Maquen
˜
o, Pisang Ceylan, Pisang Keling, Pisang Manurung, Pome, Prata, Prata Ana
˜
, Silk, Sukara; AB,
Ney Poovan; ABB, Awak, Bluggoe, Harare, Kluai Namwa Sai Deng, Monthan, Pelipita, Pisang Awak, Pisang Awak Legor.
e
Source: 1, Julio Hernandez, Instituto de Investigaciones Canarias, Spain; 2, Marie-Jo-Daboussi, Universite
´
Paris Sud, Paris, France; 3,
Kerry O’Donnell, National Center for Agricultural Utilization Research, USDA, Peoria, IL, USA; 4, Mauricio Guzma
´
n, Corbana, Guapiles,
Costa Rica; 5, Corby Kistler, ARS-USDA, Cereal Disease Laborat ory, St Paul, MN, USA; 6, Ivan Buddenhagen; 7, Pi-Fang Linda Chang,
Department of Plant Pathology, National Chung Hsing University, Taiwan; 8, Jim Lorenzen, International Institute of Tropical Agriculture,
Uganda; 9, Embrapa Cassava & Tropical Fruits, Brazil; 10, Myc otheque de l’Universite Catholique de Louvain, Belgium; 11, Plant
Research International, Wageningen University, the Netherlands.
4 M. A. Dita et al.
Plant Pathology (2010)
Sequence analyses and TR4 primer design
Sequences were manually edited using the SEQMAN mod-
ule of
DNASTAR 6.0 to generate a consensus sequence. Align-
ment was performed using the
CLUSTALW tool in the
MEGALIGN module of DNASTAR 6.0. DNA sequences of the
IGS region and the TEF-1a gene were used, both as indi-
viduals and as a combined dataset for the 82 Foc isolates.
In addition, a dataset containing TEF-1a and IGS
sequences from 848 F. oxysporum isolates (O’Donnell
et al., 2009) was used for comparative anal yses. Single
nucleotide polymorphisms (SNPs) were identified and
used for primer design . The pri mer set FocTR4-F ⁄ Fo-
cTR4-R for specific detection of TR4 (VCG 01213) was
designed to generate a unique amplicon of 463 base pairs
(bp). Amplification conditions were as described above
for IGS amplification, except the annealing temperature,
which was fixed to 60C. In addition, the Foc-1 ⁄ Foc-2
primer set (5¢-CAGGGGATGTATGAGGAGGCT-3¢⁄
5¢-GTGACAGCGTCGTCTAGTTCC-3¢) reported for
specific detection of Foc race 4 was tested (Lin et al.,
2008).
Plant inoculation and in planta detection
Hardened 3-month-old tissue-cultured banana plants of
cv. Grand Naine were inoculated with three TR4 isolates
(NRRL36114, BPS3.4 and II-5) and with one race-1
isolate (Foc_R1) that is pathogenic on cv. Silk (AAB)
(Table 1). Plants were inoculated by root dipping
(30 min, 10
6
conidia per mL) and then transferred to pots
(8 L) partially filled with sand supplemented with
20 maize kernels colonized (after sterilization) with each
isolate for 10 days. During acclimatization and after inoc-
ulation plants were maintained in a greenhouse at 28C,
80% relativity humidity and 16 h light. Rhizome and
pseudostem samples collected 40 days after inoculation
(d.a.i.), were cut in half, with one half pla ted on Komad-
a’s medium for selective isolation of Foc and the other
half used for DNA extraction. Total genomic DNA from
plant tissues was extracted using the aforementioned kit.
In planta detection for TR4 was performed using the
FocTR4-F ⁄ FocTR4-R primer set as described above for
fungal DNA on cv. Grand Naine and additionally on six
AA diploid, five BB diploid and two AAB triploid banana
genotypes (Table 2).
Multiplex PCRs
Using the amplification conditions fixed for the
FocTR4-F ⁄ FocTR4-R primers, multiplex PCRs were
developed to detect in one single reaction false negatives
in either fungal or plant samples. For fungal DNA, the
multiplex PCR incorporated the TEF-1a primer set
(EF-1 and EF-2) as internal positive control. For plant
samples, the banana actin gene AF285176.1 (http://
www.ncbi.nlm.nih.gov) was used to design the Ban-
Actin2-F (5¢-ACAGTGTCTGGATTGGAGGC-3¢) and
BanActin2-R (5¢-GCACTTCATGTGGACAATGG-3¢)
primers that amplified a 217-bp product as internal
positive control .
Results
Genetic diversity of Fusarium oxysporum f. sp.
cubense
Foc was not recovered from some samples received as dry
pseudostem from the field, but most samples produced
typical Fusarium colonies on Komada’s medium. This
resulted in 16 field isolates being selected for further anal-
yses in this study (Table 1). VCG tests were performed for
most of the isolates from areas where TR4 is reported,
which were suspected to belong to VCG 01213 (Table 1).
Several isolates (NRRL36110, NRRL36111, NRRL
36112, Foc_R1 and Foc_R2) clearly showed increased
levels of resistance to KCLO
3
even at 4Æ5%, but eventu-
ally nit mutant s could be generated. Foc isolates Foc_R2
and Foc_R1 did not complement any VCG tester of the
collection.
High-quality genomic DNA was obtained for all iso-
lates and the primers and amplification conditions
resulted in high-quality DNA sequences of the TEF-1 a
gene and IGS region. Phylogenetic analyses of IGS and
TEF-1a revealed polymorphisms between the Foc iso-
lates, but for the TEF-1a gene these were insufficient to
allow a reliable discrimination of VCG 01213 from other
VCGs. For instance, isolates of VCGs 0120, 0121, 0129
and 01211 showed 100% similarity with VCG 01213
isolates representative of TR4 (data not shown). Com-
parative analysis of the IGS region showed a higher SNP
frequency (Fig. 1) and was therefore, along with data
from 848 isolates of Fusarium spp. (O’Donnell et al.,
2009), used for primer desig n. These analyses revealed
that VCG 01213 iso lates are closely related to VCG 1210
(NRRL26029), VCG 0129 (NRRL36110), VCG 012 0
(NRRL25603) and VCG0126 (NRRL36107) isolates,
but differences were sufficient for specific primer design
(Fig. 1).
Table 2 Banana genotypes used for PCR amplifications
Cultivar
Genome
composition Species
Borneo AA Musa acuminata
Mandang AA Musa acuminata
Born Pisan Mas AA Musa acuminata
Calcutta 4 AA Musa acuminata
Selangor AA Musa acuminata
Z6Fb AA Musa acuminata
Etikehel BB Musa balbisiana
Singapuri BB Musa balbisiana
Tani BB Musa balbisiana
Buthonan BB Musa balbisiana
MPL BB Musa balbisiana
Grand Naine AAA Musa acuminata
Silk AAB Musa spp.
Prata Ana
˜
AAB Musa spp.
Diagnostic test for F. oxysporum f. sp. cubense TR4 5
Plant Pathology (2010)
Specificity of the FocTR4-F ⁄ FocTR4-R primer set
The designed primer set for TR4, FocTR4-F (5¢-CAC
GTTTAAGGTGCCATGAGAG-3¢) and FocTR4-R (5¢-
CGCACGCCAGGACTGCCTCGTGA-3¢), produced
the predicted 463-bp amplicon that was confirmed by gel
electrophoresis (Fig. 2). PCR amplification only gener-
ated this diagnostic 463-bp amplic on in VCG 01213 iso-
lates (Table 1, Fig. 2). The Foc-1 ⁄ Foc-2 primer set of Lin
et al. (2008) amplified bands in Foc isolates belonging to
at least nine VCGs. These comprised VCG 01213, as well
as VCGs 0120, 0121, 0122, 0126, 0129, 01210, 01211
and 01215, plus isolates of unknown VCGs from Brazil
(FocYB) and Taiwan (Table 1).
Disease development and in planta detection
In TR4-inoculat ed plants, typical external yellowing
appeared 7 d.a.i. and internal rhizome discoloration
occurred 14 d.a.i. At 40 d.a.i. TR4-inoculated plants
showed severe wilting and internal necrosis, even in the
pseudostem (Fig. 3). No symptoms were observed in
plants inoculated with Foc_R1 or in those used as non-
inoculated controls. The three TR4 isolates caused simi-
lar symptoms, with no differences regarding incubation
period or severity. All three TR4 isolates were success-
fully recovered from rhizomes with symptoms on Ko-
mada’s medium. DNA (20 ng) from infected plants was
successfully used for PCR amplification of the diagnostic
Figure 2 Amplification of PCR products of 20 representative vegetative compatibility groups (VCGs) of Fusarium oxysporum f. sp. cubense
(Foc) using primer set Foc-1 ⁄ Foc-2 (upper panel), FocTR4-F ⁄ FocTR4-R (middle panel) and EF-1 ⁄ EF-2 (lower panel). Lane 1, NRRL3610 1
(0120); 2, NRRL36102 (0121); 3, NRRL36103 (0122); 4, NRRL36104 (0123); 5, NRRL36105 (0124); 6, NRRL36106 (0125); 7, NRRL36107
(0126); 8, NRRL36111(0128); 9, NRRL36110 (0129); 10, NRRL26029 (01210); 11, NRRL36109 (01211); 12, NRRL36108 (01212); 13,
NRRL36114 (01213); 14, NRRL36113(01214); 15, NRRL36112 (01215); 16, NRRL36120 (01218); 17, NRRL36118 (01221); 18, NRRL36117
(01222); 19, NRRL36116 (01223); 20, NRRL36115 (01224). Numbers in parentheses are VCGs. Specific DNA bands for Foc race 4 (242 bp),
Foc TR4 (463 bp) and elongation factor 1a (648 bp) are indicated on the left. M, molecular marker 1-kb DNA ladder plus.
Figure 1 Genetic relationship of representative isolates of Fusarium oxysporum f. sp. cubense (Foc) related to NRRL36114 (VCG 01213; TR4)
based on DNA sequences of the intergenic spacer region of the ribosomal operon (upper panel). Isolates positive for the Foc-1 ⁄ Foc-2 primer
set (Lin et al., 2008) are indicated by asterisks. The alignment of the representative IGS sequences of Foc isolates related to NRRL36114
shows the two single nucleotide polymorphisms that were used for primer design (lower panel).
6 M. A. Dita et al.
Plant Pathology (2010)
463-bp amplicon using the FocTR4-F ⁄ FocTR4-R primer
set. No amplicons were observed from samples of non-
inoculated cv. Grand Naine plants and the 13 additional
banana genotypes that were tested (data not shown).
Duplex PCR using fungal DNA generated two frag-
ments in TR4 isolates, one belonging to the TEF-1a gene
(648 bp) and the VCG 01213 diagnostic 463-bp ampli-
con. Samples from isolates of other VCGs only generated
the TEF-1a amplicon (Fig. 2). For in planta detection, the
duplex PCR generated the VCG 01213 diagnostic 463-bp
amplicon only in TR4-infected samples (Fig. 4). The
amplicon derived from the banana actin gene was suc-
cessfully amplified in all the banana DNA samples. Inter-
estingly, the banana actin amplicons were specific for
either Musa acuminata (A genome, 217 bp) or M. balbisi-
ana (B genome, 280 bp) banana genotypes, whereas
AAB triploids showed both fragments (Fig. 4).
Discussion
Considering the history of Panama disease (Stover,
1962, 1990; Ploetz, 1994) and the Cavendish-depen-
dence of export trades, TR4 is currently a major threat
to the global banana industry. If TR4 enters the major
banana plantations in Latin America, the Caribbean
and West Africa, a multibillion dollar production and
export industry will be facing devastation. Moreover,
the food security of millions of people depending on
smallholder production will be in danger. In the
absence of resistant cultivars, delimiting the dissemina-
tion of the disease is a top priority that relies on accu-
rate diagnosis. Fusarium oxysporum comprises
morphologically indistinguishable pathogenic as well
as non-pathogenic strains. Therefore, identification to
the species, forma specialis and strain levels is highly
desired (Lievens et al., 2008), particularly for quaran-
tine pathogens that are of high economic importance,
such as Foc TR4. This study reports a new PCR diag-
nostic that uniquely amplifies a 463-bp amplicon in
Figure 4 Amplification products of duplex PCRs using DNA from
pure-culture Fusarium oxysporum f. sp. cubense (Foc; upper panel)
or banana plants (lower panel) as templates. Duplex PCRs for Foc
cultures were performed using the elongation factor-1a (EF-1 ⁄ EF-2)
primer set as internal control in combination with the TR4-specific
primers FocTR4-F ⁄ FocTR4-R (upper panel). Duplex PCRs of
banana samples used the banana actin (BanAct2-F ⁄ BanAct2-r) and
TR4-specific (FocTR4-F ⁄ FocTR4-R) primer sets (lower panel). Lane 1,
Musa balbisiana cv. Buthohan (BB); 2, Musa acuminata cv. Pisang
Mas (AA); 3, Grand Naine (from leaf of tissue-cultured plants); 4, Silk
(AAB); 5, Prata Ana
˜
(AAB); 6, rhizomes from non-inoculated Grand
Naine plants; 7–9, infected rhizomes from Grand Naine plants
inoculated with Foc TR4 isolates NRRL36114, BPS3.4 and II-5; 10-11,
Infected pseudostems from cv. Grand Naine plants inoculated with
Foc TR4 isolates NRRL36114 and BPS3.4; 12, positive control using
DNA from a pure culture of isolate NRRL36114. Specific DNA bands
for Foc TR4 (463 bp), elongation factor 1a (648 bp) and the banana
actin gene (217 bp) are indicated on the left. M, molecular marker
1-kb DNA ladder plus.
(a)
(b) (c)
(e)
(d)
Figure 3 Banana cv. Grand Naine 40 days after inoculation with TR4 isolate NRRL36114 of Fusarium oxysporum f. sp. cubense (Foc). (a)
Plant showing fusarium wilt symptoms; bar = 10 cm. (b–d) Cross sections of pseudostem (b, c) and rhizome (d, e) of inoculated (b, d) and
non-inoculated (c, e) plants; arrows show necrosis caused by Foc TR4; bar = 1 cm.
Diagnostic test for F. oxysporum f. sp. cubense TR4 7
Plant Pathology (2010)
isolates belonging to Foc VCG 01213, which encom-
passes TR4 (Ploetz, 2006; Buddenhagen, 2009).
Until now, Foc race diagnosis relied exclusively on
pathogenicity trials and VCG testing. It has been repeat-
edly stated that the lack of a universally acceptable green-
house inoculation technique is an important bottleneck
for the characterization of Foc isolates (Bentley et al.,
1998; Groenewald et al., 2006; Smith et al., 2008). The
inoculation procedure used in this study was efficient and
reliable, not only with TR4 isolates on cv. Grand Naine,
but also for race 1. When more differentials become avail-
able, the Foc complex of banana might be better resolved
by testing a range of Foc isolates from different VCGs on
a panel of diverse banana genotypes with different ploidy
levels, as was shown for F. oxysporum f. sp. dianthi in
carnation (Aloi & Baayen, 1993).
Field data for TR4 or ST4 occurrence should be inter-
preted with caution. TR4 is more aggressive than ST4
(Ploetz, 2006; Buddenhagen, 2009), but the latter can
also cause severe damage in Cavendish cultivars, particu-
larly under abiotic stress, such as low temperatures and
waterlogging (Su et al., 1986; Pegg et al., 1996; Budden-
hagen, 2009). This is not always known by growers and
extension officers, who may consider such infections as
TR4, resulti ng in false alarms and needless eradication
measures. An example is isolate BPI-0901, a suspect TR4
isolate obtained from Indonesian Cavendish samples. It
was negative in the present PCR-based diagnosis and only
after time-consumin g (6 months) successive attempts
were nit mutants obtained. Subsequent VCG character-
ization resulted in VCG 0120, further validating the
molecular TR4 diagnostic.
VCG analyses have contrib uted to an improved under-
standing of genetic variation in Foc, but the lack of an
accessible international VCG tester collection compli-
cates its use for diagnosis. Molecular studies have shown
the existence of different genotypic groups and clonal lin-
eages of Foc that were largely VCG-specific, but no corre-
lation between these data and race designations was
observed (Koenig et al., 1997; Bentley et al., 1998;
O’Donnell et al., 1998; Gerlach et al., 2000; Groenewald
et al., 2006; Fourie et al., 2009). Mutations in the vic
locus, could, however, render isolates within the same
VCG inco mpatible (Bentley et al., 1995, 1998). In addi-
tion, some VCGs of Foc can produce heterokaryons
between separate groups, such as VCGs 0120 and 01215
(Bentley et al., 1998; Gerlach et al., 2000; Groenewald et
al., 2006). Therefore, TR4 diagnostics should focus on
genetic specificity based on molecular data.
Sequences of IGS and TEF-1a were used to study
genetic diversity of Foc, with the aim of identifying SNPs
for specific primer design. The TEF-1a gene has been
widely used in Fusarium spp. for both phy logenic
(O’Donnell et al., 1998; Fourie et al., 2009) and identifi-
cation purposes (Bogale et al., 2007; Mehl & Epstein,
2007). In the present study, however, TEF-1a revealed
insufficient polymorph isms for reliable dis crimination of
VCG 01213 from other VCGs. Instead, the results
showed that the higher SNP frequency of the IGS region
provides a rich source of genetic diversity in this
forma
specialis, which was successfully exploited to develop a
Foc TR4 diagnostic PCR. Moreover, it can also be used to
further eluci date phylogenetic relationships among Foc
populations. This confirms the results of Fourie et al.
(2009), who also showed that restriction fragment length
polymorphisms of the IGS region (IGS-RFLP) were more
discriminative than three other genome regions, includ-
ing TEF-1a, for Foc lineages. As the higher copy number
of IGS increases the sensitivity of PCR-based diagnostics,
this region also has been used to develop diagnostics for
other plant pathogenic Fus arium spp., such as F. circina-
tum (Schweigkofler et al., 2004) and F. oxysporum f. sp.
vasinfectum (Zambounis et al., 2007).
Specificity of diagnostics is required for the unequivo-
cal detection of quarantine organisms. The diagnostic
developed here was specific for TR4 on pure-culture
DNAs of VCG 01213 isolates that were either character-
ized prior to or after the PCR test, the latter including iso-
lates from infected banana tissues (BPS1.1, BPS3.1) from
Indonesia and pure cultures from Taiwan (T-14, T105
and T-202). The Foc-1 ⁄ Foc-2 primer set recently pub-
lished by Lin et al. (2008), considered to be specific for
Foc race 4 (both ST4 and TR4), was also tested. However,
this primer set reacted with isolates of 10 different VCGs,
including those belonging to the 01213 group (TR4).
These results are in agreement with those of Buddenha-
gen (2009), who reported that ST4 isolates belong to
VCGs 0120, 0121, 0122, 0129 and 01211. In addition,
the present results suggest that isolates of VCG 01215
also affect Cavendish in subtropical areas. Interestingly,
isolates from Brazil, Costa Rica, Honduras and the USA
were also positive with the Foc-1 ⁄ Foc-2 primer set.
Whilst positive results for isolate s from Taiwan or other
countries where ST4 is present (Su et al., 1986; Ploetz,
2006; Lin et al., 2008) were expected, it was intriguing to
also find positives in areas where ST4 is not officially
reported. This suggests that ST4 is present in Central and
Latin America, as well as the USA. This ambiguity illus-
trates the drawback of the current race designatio n sys-
tem for Foc in banana. An isolate may be classified as ST4
in subtropical areas (where it affects Cavendish), but as
race 1 in tropical areas, such as Brazil, Costa Rica and
Honduras (where it is unable to affect Cavendish).
Although this system initially helped to discriminate
Foc populations, it is currently outdated and leads to
erroneous conclusions, hampering decision making.
From a diagnostic and regulatory perspective, methods
that are repeatable, highly specific, sensitive for the
target pathogen and also can be used on infected plant
tissue, without the need for pathogen isolation and
culture, would be strongly preferred (Martin et al.,
2009). The TR4 diagnostic developed here unambigu-
ously detected TR4 in infected tissues of banana cv.
Grand Naine. In comparison with traditional agar plat-
ing and pathogen purification from infected samples,
VCG analysis and pathogenicity tests, which may take
weeks or months, the in planta detection metho d
described here provides a receipt-to-result efficiency of
8 M. A. Dita et al.
Plant Pathology (2010)
about 6 h. This is comparable to in planta detection
methods previously reported for other plant pathogenic
fungi and oomycetes (Alves-Santos et al., 2002; Wang
et al., 2007; Vincelli & Ti sserat, 2008). It is concluded
that this PCR diagnostic is currently the only option for
rapid, reliable and specific detection of TR4. Applica-
tion enables the monitoring of the disease and supports
management and eradication strategies.
Acknowledgements
We thank Drs Jim Lorenzen (IITA, Uganda), Julio Her-
na
´
ndez (Instituto de Investigaciones Cana
´
rias, Spain),
Kerry O’Donnell (National Center for Agricultural Utili-
zation Research, USA), Marie-Jo Daboussi (Universite
´
Paris Sud, France), Mauricio Guzma
´
n (Corbana, Costa
Rica), Pi-Fang Linda Chang (National Chung Hsing Uni-
versity, Taiwan) and Corby Kistler (ARS-USDA, USA)
for providing isolates or samples used in this study. We
also thank Dr Irie Vroh (IITA, Nigeria) for supplying part
of the banana DNA used for PCRs, Caucasella Diaz
(Plant Research International, the Netherlands) for
tissue-culture acclimatized cv. Grand Naine plants and
Ineke de Vries for technical support. MAD is grateful to
CAPES (Coordenac¸a
˜
o de Aperfeic¸oamento de Pessoal de
Nı
´vel
Superior) for the post-doctoral fellowsh ip. This
research was partially funded by the Dutch Dioraphte
Foundation and the EU Endure prog ramme.
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