ArticlePDF Available

Abstract and Figures

This study analysed genomic variation of the translation elongation factor 1 (TEF-1) and the intergenic spacer region (IGS) of the nuclear ribosomal operon of Fusarium oxysporum f. sp. cubense (Foc) isolates, from different banana production areas, representing strains within the known races, comprising 20 vegetative compatibility groups.
Content may be subject to copyright.
A molecular diagnostic for tropical race 4 of the banana
fusarium wilt pathogen
M. A. Dita
, C. Waalwijk
, I. W. Buddenhagen
, M. T. Souza Jr
and G. H. J. Kema
Embrapa Cassava & Tropical Fruits, Cruz das Almas, 44380-000, Bahia, Brazil;
Plant Research International B.V., PO Box 16, 6700
AA Wageningen, the Netherlands;
1012 Plum Lane, Davis, California, USA; and
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
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,
ª 2010 Plant Research International
Journal compilation ª 2010 BSPP
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
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
incubating for 7–14 days at 25C. Spontaneous
-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
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¢-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
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
Location Source
PCR diagnostic
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,
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 ))
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
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
01213* Cavendish Kuta-village Bali Indonesia, 6 + +
Field samples
01213* Cavendish Darwin Australia, 6 + +
Field samples
01213* Cavendish Darwin Australia, 6 + +
Field samples
01213* Cavendish Darwin Australia, 6 + +
Field samples
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 + +
Foc 01213* TR4 Pisang Manurung Indonesia, 5 + +
BPI-0902 Field samples
Silk Mariana Islands (Saipan),
Farm: Lucy Norita
Indonesia, 6 ))
BPI-0903 Field samples
Silk Mariana Islands (Rota CNMI)
Farm: Frank Calvo
Indonesia, 6 ))
BPI-0904 Field samples
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
Location Source
PCR diagnostic
BPI-0905 Field samples (pseudostem) Silk Mariana Islands
(Tinian Island),
Indonesia, 6 ))
Foc_R1 Foc R1 Silk Cruz das Almas,
Brazil, 9 ))
Foc_R2 Foc R2 Monthan Cruz das Almas,
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
Brazil, 8 ))
Foc49 Foc R1 Prata Ana
Cruz das Almas,
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 ))
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.
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.
Race designation as provided by supplier. R1, race 1; R2, race 2; ST4, subtropical race 4; TR4, tropical race 4.
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.
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
specific detection of Foc race 4 was tested (Lin et al.,
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
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:// was used to design the Ban-
primers that amplified a 217-bp product as internal
positive control .
Genetic diversity of Fusarium oxysporum f. sp.
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
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
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
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
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).
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.
(b) (c)
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
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.
We thank Drs Jim Lorenzen (IITA, Uganda), Julio Her-
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
Superior) for the post-doctoral fellowsh ip. This
research was partially funded by the Dutch Dioraphte
Foundation and the EU Endure prog ramme.
Aloi C, Baayen RP, 1993. Examination of the relationships
between vegetative compatibility groups and races in Fu sarium
oxysporum f. sp. dian thi. Plant Pathology 42, 839–50.
Alves-Santos FM, Ramos B, Garcia-S anchez MA, Eslava AP, Diaz-
Minguez JM, 2002. A DNA-based procedure for in planta
detection of Fusarium oxysporum f. sp phaseoli.
Phytopathology 92, 237–44.
Aurore G, Parfait B, Fahrasman e L, 2009. Bananas, raw materials
for making processed food products. Trends in Food Science
& Technology 20, 78–91.
Bentley S, Pegg KG, Dale JL, 1995. Genetic-variation among a
world-wide collection of isolates of Fusarium oxysporum f. sp.
cubense analyzed by RAPD-PCR fingerprinting. Mycological
Research 99, 1378–84.
Bentley S, Pegg KG, Moore NY, Davis RD, Buddenhagen IW,
1998. Genetic variation among vegetative compatibility groups
of Fusarium oxysporum f. sp. cubense analyzed by DNA
fingerprinting. Phytopathology 88, 1283–93.
Bogale M, Wingfield BD, Wingfield MJ, Steenkamp ET, 2007.
Species-specific primers for Fusarium redolens and a PCR-RFL P
technique to distinguish among three clades of Fusarium
oxysporum. FEMS Microbiology Letters 271, 27–32.
Buddenhagen IW, 2009. Understanding stra in diversity in Fusarium
oxysporum f. sp. cubense and history of introduction of ‘tropical
race 4’ to better manage banana production. In: Jones D, Van
Den Bergh I, eds. Proceedings of the International Symposium
on Recent Advances in Banana Crop Protection for Sustainable
Production and Improved Livelihoods, White River, South
Africa. ISHS Acta Horticulturae 828, 193–204.
Correll JC, Klittich CJR, Leslie JF, 1987. Nitrate non-utilizing
mutants of Fusarium oxysporum and their use in vegetative
compatibility tests. Phytopathology 77, 1640–6.
Fourie G, Steenkamp ET, Gordon TR, Viljoen A, 2009.
Evolutionary relationships among the vegetative compatibility
groups of Fusarium oxysporum f. sp. cubense. Applied and
Environmental Microbiology 75, 4770–81.
Gerlach KS, Bentley S, Moore NY, Pegg KG, Aitken EAB, 2000.
Characterisati on of Australian isolates of Fusarium oxysporum f.
sp. cubense by DNA fingerprinting analysis.
Australian Journal
of Agricultural Research 51, 945–53.
Groenewald S, Van den Berg N, Marasas WFO, Viljoen A, 2006.
The application of high-throughput AFLP’s in assessing genetic
diversity in Fusarium oxysporum f. sp . cubense. Mycological
Research 110, 297–305.
Koenig RL, Ploetz RC, Kistler HC, 1997. Fusarium oxysporum f.
sp. cubense consists of a small number of divergent and globally
distributed clonal linea ges. Phytopathology 87, 915–23.
Komada H, 1975. Development of a selective medium for
quantitative isolation of Fusarium oxysporum from natural soil.
Plant Protection Research 8, 114–25.
Lievens B, Rep M, Thomma B, 2008. Recent developments in the
molecular discrimination of formae speciales of Fusa rium
oxysporum. Pest Management Science 64, 781–8.
Lin YH, Chang JY, Liu ET, Chao CP, Huang JW, Chang PFL,
2008. Development of a molecular marker for specific detection
of Fusarium oxysporum f. sp. cubense race 4. European Journal
of Plant Pathology 123, 353–65.
Martin FN, Coffey MD, Zeller K et al., 2009. Evaluatio n of
molecular markers for Phytophthora ramorum detection and
identification: testing for specificity using a standardized library
of isolates. Phytopathology 99, 390–403.
Mehl HL, Epstein L, 2007. Identification of Fusarium solani f. sp
cucurbitae race 1 and race 2 with PCR and production of
disease-free pumpkin seeds. Plant Disease 91, 1288–92.
Molina AB, Fabregar EG, Sinohin V, Fourie G, Viljoen A, 2008.
Tropical race 4 of Fusarium oxysporum f. sp. cubense caus ing
new Panama wilt epidemics in Cavend ish varieties in the
Philippines. Phytopathology 98(Suppl.), S108.
Moore NY, Pegg KG, Allen RN, Irwin JAG, 1993. Vegetative
compatibility and distribution of Fusarium oxysporum f. sp.
cubense in Australia. Australian Journal of Experimental
Agriculture 33, 797–802.
O’Donnell K, Kistler HC, Cigelnik E, Ploetz RC, 1998. Multiple
evolutionary origins of the fungus causing Panama disease of
banana: concordant evidence from nuclear and mitochondrial
gene genealogies. Proceedings of the National Academy of
Sciences, USA 95
, 2044–9.
O’Donnell K, Gueidan C, Sink S et al., 2009. A two-locus DNA
sequence database for typing plant and human pathogens within
the Fusarium oxysporum species complex. Fungal Genetics and
Biology 46, 936–48.
Pegg KG, Moore NY, Bentley S, 1996. Fusarium wilt of banana in
Australia: a review. Australian Journal of Agricultural
Research 47, 637–50.
Ploetz RC, 1994. Panama-disease return of the first banana
menace. International Journal of Pest Management 40,
Ploetz RC, 2006. Fusarium wilt of banana is caused by several
pathogens referred to as Fusarium oxysporum f. sp. cubense.
Phytopathology 96, 653–6.
Diagnostic test for F. oxysporum f. sp. cubense TR4 9
Plant Pathology (2010)
Ploetz RC, Correll J C, 1988. Vegetative compatibility among races
of Fusarium oxysporum f. sp. cubense. Plant Disease 72, 325–8.
Ploetz R, Pegg K, 1997. Fusarium wilt of banana and W allace’s
line: was the disease originally restricted to his Ind o-Malayan
region? Australasian Plant Pathology 26, 239–49.
Puhalla JE, 1985. Classification of strains of Fusarium oxysporum
on the basis of vegetative compatibility. Canadian Journal of
Botany 63, 179–83.
Schweigkofler W, O’Donnell K, Garbelotto M, 2004. Detection
and quantification of airborne conidia of Fusarium circinatum,
the causal agent of pine pitch canker, from two California sites
by using a real-time PCR approach combined with a simple
spore trapping method. Applied and Environmental
Microbiology 70, 3512–20.
Smith LJ, Smith MK, Tree D, O’Keefe D, Galea VJ, 2008.
Development of a small-plant bioassay to assess banana grown
from tissue culture for consistent infection by Fusarium
oxysporum f. sp. cubense. Australasian Plant Pathology 37,
Stover RH, 1962. Fusarial Wi lt (Panama Disease) of B ananas and
Other Musa Species. Kew, UK: Commonwealth Mycological
Stover RH, 1990. Fusarium wilt of banana: some history
and current status of the disease. In: Ploetz RC, ed.
Fusarium Wilt of Banana.StPaul,MN,USA:APSPress,
Su HJ, Hwang SC, Ko WH, 1986. Fusarial wilt of Cavendish
bananas in Taiwan. Plant Disease 70, 814–8.
Vincelli P, Tisserat N, 2008. Nucleic acid-based pathogen
detection in applied plant pathology. Plant Disease 92,
Waite BH, Stover RH, 1960. Studies on Fusarium wilt of
bananas, VI. Variability and cultivar concept in Fusarium
oxysporum f. sp. cubense. Canadian Journal of Botany 38,
Wang Y, Ren Z, Zheng XB, Wang YC, 2007. Detection of
Phytophthora melonis in samples of soil, water, and plant tissue
with polymerase chain reaction. Canadian Journal of Plant
Pathology 29, 172–81.
Zambounis AG, Paplomatas E, Tsaftaris AS, 2007. Intergenic
spacer-RFLP analysis and direct quantification of
Australian Fusarium oxysporum f. sp vasinfectum isolates
from soil and infected cotton tissues. Plant Disease 91,
10 M. A. Dita et al.
Plant Pathology (2010)
... starts with infection of the root system and subsequent colonization of the vascular tissue, leading to water stress, severe chlorosis and wilting (Beckman, 1987;Ploetz, 2015). Infected plants frequently die before they produce bunches, hence Fusarium wilt significantly reduces yields in infested fields (Stover and Ploetz, 1990;Dita et al., 2010). Race 1 strains caused one of the worst botanical epidemics in history and decimated the commercial Gros Michel banana based industry in Central America in the 1950s (Ploetz, 2005a). ...
... For Fusarium spp. several protocols were developed (Sun and Su, 1984;Adesemoye and Adedire, 2005;Amorim et al., 2009;Dita et al., 2011;García- Bastidas et al., 2014;Li et al., 2014;Ordonez et al., 2015a), of which many are based on the use of commercial growth media but also natural sources such as beans (Vigna radiata L.) ( Bai and Shaner, 1996;Li et al., 2001;Yuan and Zhou, 2005;Mudge et al., 2006;Amorim et al., 2009;Dita et al., 2010;Dita et al., 2011;García-Bastidas et al., 2014;Li et al., 2014;Ordonez et al., 2015a). However, these methods cannot be up scaled to the large volumes of inoculum required for extensive phenotyping experiments ( Burgess et al., 1991;Leslie et al., 2006), due to either large quantities of expensive culture medium or costly infrastructure. ...
... Hitherto protocols facilitated the mere screening of approximately 15 plants hour −1 person −1 ( Dita et al., 2010;Dita et al., 2011;Ordonez et al., 2015a). Clearly, this hampers throughput and potential automation during phenotyping mutant panels or segregating populations that usually comprise hundreds or even thousands of plants. ...
Full-text available
Fusarium oxysporum (Fo) belongs to a group of soil-borne hyphomycetes that are taxonomically collated in the Fusarium oxysporum Species Complex (FOSC). Hitherto, those infecting bananas were placed in the forma specialis cubense (Foc). Recently, however, these genetically different Foc lineages were recognized as new Fusarium spp. placed in the Fusarium of Banana Complex (FOBC). A member of this complex F. odoratissimum II-5 that uniquely comprises the so-called Tropical Race 4 (TR4), is a major problem sweeping through production zones of Cavendish banana in several regions of the world. Because of this, there is an urgent need for a phenotyping method that allows the screening for resistance to TR4 of large numbers of banana genotypes. Most Fusarium species produce three types of spores: macroconidia, microconidia and the persistent chlamydospores that can contaminate soils for many years. Inoculum production has been an important bottleneck for efficient phenotyping due to the low or variable number of conidia and the elaborate laboratory procedures requiring specific infrastructure. Here, we report a rapid, simple and high-yielding spore production method for nine F. oxysporum formae speciales as well as the biocontrol species Fo47 and Fo618-12. For Fusarium spp. causing Fusarium wilt or Panama disease of banana, we used the protocol for four species comprising the recognized physiological races, including Tropical Race 4 (TR4). We subsequently tested the produced inoculum in comparative inoculation trials on banana plants to evaluate their efficiency. All assays resulted in typical symptoms within 10 weeks; significant differences in final disease ratings were observed, depending on inoculum concentration. Pouring inoculum directly onto banana plants showed the most consistent and reproducible results, as expressed in external wilting, internal discoloration and determined by real-time PCR assays on entire rhizomes. Moreover, this method allows the inoculation of 250 plants per hour by one individual thereby facilitating the phenotyping of large mutant and breeding populations.
... cubense. Most of them have focused on the rapid identification of race 4 including VCGs associated to both the tropical as well as subtropical races (Dita et al., 2010;Li et al., 2013;Lin et al., 2013). ...
... , the primers reported byDita et al. (2010) known as Foc R4T F and R4T R, which target the Intergenic Spacer (IGS) region and Internal Transcribed Spacer (ITS) of the ribosomal operon.Aguayo et al. (2017) reported the development of a real-time PCR test to detect VCGs 01213/16 and 0121, based on a putative virulence gene previously described byLin et al. (2013). The methodologies mentioned above have been included as an initial part of a pre-diagnosis in pre-symptomatic and symptomatic plants and have been considered as part of the diagnosis in a large number of reports and scientific articles, including the most recent incursions of Fusarium tropical race 4(García-Bastidas et al., 2014;Ordóñez et al., 2016;Chittarath et al., 2018;Hung et al., 2018;Zheng et al., 2018). ...
This datasheet on Fusarium oxysporum f.sp. cubense tropical race 4 covers Identity, Overview, Distribution, Dispersal, Hosts/Species Affected, Diagnosis, Biology & Ecology, Impacts, Prevention/Control, Further Information.
... The antagonistic activity of the 96 isolates was evaluated against Foc TR4 strain NRRL36114 (= CBS 102025) (Dita et al., 2010), which was obtained from the Westerdijk Fungal Biodiversity Institute (Utrecht, Netherlands). The dual culture method was used to evaluate the effect of diffusible metabolites and volatile compounds of Pseudomonas spp., Bacillus spp., and Streptomyces spp. ...
... The cellophane agar method and the overlapping plate assay described above were also used to evaluate the effect of metabolites produced by Foc against the seven isolates constituting SynCom 1.1. Besides Foc TR4, Foc R1 (NRRL36110 = CBS102021; obtained from the Westerdijk Fungal Biodiversity Institute) (Dita et al., 2010) was also included in these experiments. ...
Full-text available
Fusarium oxysporum f. sp. cubense ( Foc ) tropical race 4 (TR4) is threatening banana production because of its increasing spread. Biological control approaches have been widely studied and constitute interesting complementary measures to integrated disease management strategies. They have been based mainly on the use of single biological control agents (BCAs). In this study, we moved a step forward by designing a synthetic microbial community (SynCom) for the control of Fusarium wilt of banana (FWB). Ninety-six isolates of Pseudomonas spp., Bacillus spp., Streptomyces spp., and Trichoderma spp. were obtained from the banana rhizosphere and selected in vitro for the antagonism against Foc TR4. In pot experiments, a large community such as SynCom 1.0 (44 isolates with moderate to high antagonistic activity) or a small one such as SynCom 1.1 (seven highly effective isolates) provided similar disease control (35% symptom severity reduction). An in vitro study of the interactions among SynCom 1.1 isolates and between them and Foc revealed that beneficial microorganisms not only antagonized the pathogen but also some of the SynCom constituents. Furthermore, Foc defended itself by antagonizing the beneficial microbes. We also demonstrated that fusaric acid, known as one of the secondary metabolites of Fusarium species, might be involved in such an interaction. With this knowledge, SynCom 1.2 was then designed with three isolates: Pseudomonas chlororaphis subsp. piscium PS5, Bacillus velezensis BN8.2, and Trichoderma virens T2C1.4. A non-simultaneous soil application of these isolates (to diminish cross-inhibition) delayed FWB progress over time, with significant reductions in incidence and severity. SynCom 1.2 also performed better than two commercial BCAs, BioPak ® and T-Gro. Eventually, SynCom 1.2 isolates were characterized for several biocontrol traits and their genome was sequenced. Our data showed that assembling a SynCom for biocontrol is not an easy task. The mere mixtures of antagonists (e.g., SynCom 1.0 and 1.1) might provide effective biocontrol, but an accurate investigation of the interactions among beneficial microorganisms is needed to improve the results (e.g., SynCom 1.2). SynCom 1.2 is a valuable tool to be further developed for the biological control of FWB.
... Fusarium wilt of cowpea is a global problem and F.oxysporum is also problematic in the production of tomato and some other vegetable crops in India (Ghag 2019 andNirmaladevi et al.,2016). Molecular diagnostic methods has now been used advantageously in solving the complex situation of host pathogen relationship involving F. oxysporium and crops such as banana in tropical agro ecosystem (Dita et al., 2010). Since research focus is diverted towards lasting solution in no distance time, this present study is expected to make its own contribution in solving the Fusarium wilt problem. ...
... To date, TR4 belongs to a single group of vegetative compatibility (VCG 01213) while 9 vegetative compatibility groups associated with SR4. TR4 with VCG 1216 or 1213/16 complex has also been reported but current evidence indicates that it is the same group as 1213 (Dita et al., 2010;Ploetz and Viljoen personal communication, 2009). Foc isolates are divided into different lineages (at least 8), with closely-related compatible vegetative groups (VCGs), even when are distributed over a wide geographic area. ...
Full-text available
Meenakshi Mukhopadhyay*1, Surmilita Biswas2, Mongistha Ghoshal3, Sayani Basu4, Supriya Kumar Ghosh5, Sudeshna Shyam Chowdhury6 and Arup Kumar Mitra7 1Department of Botany, Vivekananda College, 269 Diamond Harbour Road, Kolkata-700063 E-mail: 2-7 Department of Microbiology, St. Xavier’s College (Autonomous) Kolkata,30, Mother Teresa Sarani (Park Street) Kolkata-700016 ABSTRACT Soybean, Glycine max(L.) Meril. is a miracle crop containing over 40% proteins and 20% oil contributing about 25% of total global edible oil and two thirds of the protein concentrate for livestock feeding. This legume meets its own Nitrogen requirement through biological N2- fixation andalso leaves a considerable amount in soil for successive crops.Soil degradation and persistent decline in fertility is a major global issue in the 20th century for its serious impact on world food and environmental security. To ensure sustained productivity the natural ways of feeding the soil with microbial inoculants are now practiced. Accordingly the investigation was undertaken to upgrade a low productive soil by the use of resident microorganisms. The soil sample was collected from a fallow land of Bahadurpur village, Diamond Harbour, South 24 Parganas District, West Bengal. It had poor nutrient status with 48.7 mg/kg of Nitrogen, 80.1 mg/kg of Phosphate, 134.6 mg/kg of Potassium and 0.73% organic carbon content. Microbes were isolated from it; six bacterial isolates were detected to be Nitrogen fixer as well as Phosphate solubilizer and one of them can mobilize potassium. Based on their nutrient harnessing ability and interaction study, three microbial consortia were formulated for application to the soil; their efficacies were evaluated growing soybean as a test crop in pots under natural condition. Among them one combination of consortium was observed to be most promising in improving productivity of the potted soybean plants.
... cubense tropical race 4 is still not well understood [ 47 , 10 ]. Besides, a Fusarium wilt-like disorder once called ' Matooke wilt' has existed in East African Highland banana cultivars in Uganda [ 31,32 ] Unfortunately, there has been insufficient information on pathogenicity tests on F. oxysporum V5w2 [ 38,56 ]), especially those that utilize differential host cultivars [ 14,21,106 ]. ...
Full-text available
Fusarium oxysporum V5w2 is a fungal agent, which was originally intended for biological control of the root pathogenic nematode Radopholus similis among other banana pests. Information on some of its actual effects on banana plants and pests has been scarce. A study was conducted to assess the effects of F. oxysporum V5w2 and mulch on R. similis and banana plants in the field. The experiment was a 3-factor (2 × 2 × 2) complete randomized block design with or without F. oxysporum V5w2, R. similis or mulch. Root damage was higher when plants were inoculated with R. similis compared to non-inoculated plants, while plant height, leaf size, bunch size and banana yields were lower in R. similis-inoculated plants. Plant growth and bunch size were greater in mulched than in non-mulched plots, with banana yields on mulched plots increasing by 131 % to reach 37 tonnes per hectare. F. oxysporum V5w2-inoculated plants were shorter, with fewer leaves that were smaller, and had shorter duration to harvest than non-inoculated ones. When R. similis-inoculated mulched plants received F. oxysporum V5w2 inoculum, plant toppling-over occurred less frequently compared to plants that were not inoculated with the fungus, probably because the plants were smaller in size during growth. The benefits of mulching in banana production were evident, while the claimed enhancing effect of inoculating tissue culture plants with F. oxysporum V5w2 as a biological control agent could not be verified. I conclude by suggesting that, the use of F. oxysporum V5w2 as a biological control agent of R. similis and other pests should be reconsidered, because this fungal strain has the potential of invading xylem vessels and becoming pathogenic to banana plants.
... Foln were tested in the field, and the disease of the plants was observed after maturity, the infected samples were selected for fungal isolation [18]. In order to confirm that symptoms were a result of the fungal infection, the inoculated plants were sampled for fungal isolation from the flax roots. ...
A plant’s early response to pathogen stress is a vital indicator of its disease resistance. In order to study the response mechanism of resistant and susceptible flax cultivars to Fusarium oxysporum f. sp. lini (Foln), we applied RNA-sequencing to analyze transcriptomes of flax with Foln 0.5, 2 and 8 hours post inoculation (hpi). We found a significant difference in the number of differential expression genes (DEGs) between resistant and susceptible flax clutivars. The number of DEGs in the Fusarium-resistant cultivar increased dramatically at 2 hpi, and a large number of DEGs participated in the Fusarium-susceptible cultivar response to Foln infection 0.5 hpi. GO enrichment analysis determined that the up-regulated DEGs of both flax cultivars were enriched such as oxidoreductase activity and oxidation-reduction process. At the same time, the genes involved in diterpenoid synthesis were up-regulated in resistant cultivar, while those involved in extracellular region, cell wall and organophosphate ester transport were down-regulated in susceptible cultivar. KEGG enrichment analysis showed the genes encoded WRKY 22 and WRKY33 which involved in MAPK signaling pathway were up-regulated expressed in S-29 and down-regulated expressed in R-7, negatively regulated the disease resistance of flax; The genes encoded Hsp 90 family which in involved in plant pathogen interaction pathway were up-regulated in R-7 and down-regulated in S-29, which positively regulated the disease resistance of flax; The genes encoded MYC2 transcription factor and TIFY proteins which involved in plant hormone signaling pathway were up-regulated in R-7, and regulated the jasmonic acid metabolism of flax and the signal transduction of plant hormones. Meanwhile seven regulatory genes with the most correlation were screened out, Among Lus10025000.g and Lus10026447.g regulated other genes expressed both in plant hormone signal transduction pathway and MAPK signal pathway. In conclusion, these findings will facilitate further studies on the function of these candidate genes in flax of response to Fusarium stress, and the breeding of disease-resistant flax cultivar.
... The pathogen has spread in different banana production systems in Asia, Africa, and the Middle East (Ploetz, 1994;Jeger et al., 1996;Magnaye, 2001;Singh, 2008;Buddenhagen, 2009;García-Bastidas et al., 2013;Ordoñez et al., 2015;Ploetz, 2015). The TR4 is a soil-borne pathogen, microscopic, lacks visible symptoms on suckers and fruits (Brunschot, 2006), displays observable symptoms in the plant during its advanced stage (Ploetz, 1994;Buddenhagen, 2009), and cannot be controlled by a fungicide ( Dita et al., 2010). Methods of contamination are not exhaustive but some observations indicated that it may be transferred through infected suckers, soil, surface water, and farm implements (Ploetz, 2015). ...
Full-text available
An integrative management approach to the spread and emergence of global plant diseases, such as the soil-borne fungus Fusarium oxysporum f. sp. cubense tropical race 4 (Foc TR4), entails a combination of technical measures and the responsiveness and awareness of area-specific constellations supporting conditions conducive to interactions and coordination among organizations and actors with different resources and diverse interests. Responses to banana diseases are mostly studied through technical and epidemiological lenses and reflect a bias to the export industry. Some authors, however, indicate that cross-sector collaboration is crucial in responding to a disease outbreak. Earlier studies on the outbreak of diseases and natural disasters suggest that shared cognition and effective partnerships increased the success rate of response. Hence, it is important not to focus exclusively on the impacts of a pathogen at farm or field level and to shift attention to how tasks and knowledge are coordinated and shared. This paper aims to detect whether and how the emergence of Foc TR4 is a driver of coordination. The case study focuses on the interactions between a variety of banana producers and among a range of public and private actors in southern Philippines. The analysis identifies distinct forms of coordination emerging in the context of three organizational fields responding to Foc TR4, which underlie shared capacity to handle and understand the spread of a global plant disease. The research is based on qualitative key informant interviews and document analysis and on observations of instructive events in 2014–2017. Analysis of the composition and actions developed in three organizational fields leads to distinguishing three theory-driven forms of coordination: rule-based, cognition-based, and skill-based. The combination of these three forms constitutes the possibility of a collaborative community, which conditions the implementation of an integrative management approach to mitigate Foc TR4.
Full-text available
Fusarium wilt caused by Fusarium oxysporum f. sp. cubense (Foc), is the most lethal soil-borne fungal pathogen infecting bananas. Foc race 1 (R1) and 4 (R4) are the two most predominant races affecting the economically important Cavendish groups of bananas in India. A total of seven vegetative compatibility groups (VCGs) from three pathogenic races has been isolated during our field survey and were found to be highly virulent towards cv. Grande Naine. According to comparative genome analyses, these Indian Foc VCGs were diverse in genomic organization and effector gene profiles. As a result, false-positive results were obtained with currently available molecular markers. In this context, the study has been initiated to develop PCR-based molecular markers for the unambiguous identification of Indian Foc R1 and R4 isolates. Whole-genomic sequences of Foc R1 (GCA_011316005.3), Foc TR4 (GCA_014282265.3), and Foc STR4 (GCA_016802205.1), as well as the reference genomes of Foc (ASM799451v1) and F. oxysporum f. sp. lycopersici (Fol; ASM14995v2), were aligned to identify unique variable regions among the Foc races. Using putative chromosome and predicted gene comparison, race-specific unique Foc virulence genes were identified. The pu-tative lineage-specific DNA identified genes encoding products secreted in xylem (SIX) that may be necessary for disease development in the banana. An in silico analysis was performed and primers were designed from a region where sequences were dissimilar with other races to develop a specific marker for Foc R1, R4, TR4, and STR4. These race-specific markers allowed target amplification in the characterized highly virulent Foc isolates, and did not show any cross-amplification to any other Foc races, VCGs or banana pathogens, Fusarium species, and nonpathogenic Fusarium oxysporum isolates. The study demonstrated that the molecular markers developed for all the three Foc races of India could detect the pathogen in planta and up to 0.025 pg µL-1 DNA levels. Thus, the markers developed in this study are novel and could potentially be useful for the accurate diagnosis and detection of the Indian Foc races which is important for the effective management of the disease.
Full-text available
Fusarium wilt is caused by the fungus Fusarium oxysporum f. sp. cubense (Foc), the most serious disease affecting banana (Musa spp.) classified into Foc race 1 (R1), Foc race 2, and Foc race 4 based on the host specificity. As the rate of spread and the ranges of the devastation of the Foc races are higher than the center of the banana’s origin, even in non-targeted cultivars, there could be a chance of variation in virulence-associated genes. Therefore, the present study investigates the genome assembly of Indian Foc races belonging to the vegetative compatibility group (VCG) 0120 (sub-tropical race 4), 0124 (race 1), and 01213/16 (tropical race 4) infecting the Cavendish (AAA) banana group in India. In addition to the GO, SNPs, and InDels, the study looked at virulence-associated genes, specifically effector genes, and sought insights into race-specific molecular mechanisms of infection based on the presence of unique genes. The result of the analyses revealed that there is a variation in the organisation of genome assembly and virulence-associated genes, specifically secreted in xylem (SIX) genes when compared to the reference genome. The findings help to un-derstand and will help to design effective Foc management practices for different Foc races in India and beyond.
Plant domestication is the process of adapting plants to human use by selecting specific traits. The selection process often involves the modification of some components of the plant reproductive mechanisms. Allelic variants of genes associated with flowering time, vernalization, and the circadian clock are responsible for the adaptation of crops, such as rice, maize, barley, wheat, and tomato, to non-native latitudes. Modifications in the plant architecture and branching have been selected for higher yields and easier harvests. These phenotypes are often produced by alterations in the regulation of the transition of shoot apical meristems to inflorescences, and then to floral meri-stems. Floral homeotic mutants are responsible for popular double-flower phenotypes in Japanese cherries, roses, camellias, and lilies. The rise of peloric flowers in ornamentals such as snapdragon and florists' gloxinia is associated with non-functional alleles that control the relative expansion of lateral and ventral petals. Mechanisms to force outcrossing such as self-incompatibility have been removed in some tree crops cultivars such as almonds and peaches. In this review, we revisit some of these important concepts from the plant domestication perspective, fo-cusing on four topics related to the pre-fertilization mechanisms: flowering time, inflorescence architecture, flower development, and pre-fertilization self-incompatibility mechanisms.
Full-text available
Tropical Race 4 (TR4), the virulent strain of Fusarium oxysporum f. sp. cubense, is a serious threat to the export Cavendish-based banana industry. TR4 has not been reported in the Philippines until recently when Panama wilt infections were observed in several commercial Cavendish farms. To confirm the identity of the new epidemics, a survey was conducted from September to December 2005. Infected plants showing the typical Panama wilt symptoms were collected from several commercial farms. Samples were sent to the Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, South Africa for Vegetative Compatibility Group (VCG) analyses. Results confirmed that the pathogens isolated from infected samples were identified to belong to VCG 1213/16 complex, the VCG known to be associated with TR4. To quantify the severity of epidemics, the incidence of Panama wilt cases were monitored in two farms. The incidence of Panama wilt increased from 700 cases in 2005 to 15,000 cases in 2007. The new TR4 epidemic threatens the long term sustainability of the Philippine banana industry and its dominance in the banana exports in Asia. The spread of TR4 is also a threat as a transboundary pathogen to other Cavendish producing countries and continents.
Fusarium wilt of banana is caused by 35 different strains or genotypes of Fusarium oxysporum f. sp. cubense. VCG 01213, so-called 'tropical race 4', is just one of six distinct strains that can attack Cavendish, but it is much more aggressive on Cavendish than strains known earlier in Australia and South Africa. New plantations established in the 1990s in peninsula Malaysia and Sumatra soon succumbed. VCG 01213 was later found to be common in village banana plants in those areas. It is only one of eight strains present in the villages. It is not a new mutant strain but is only newly recognised as unique. The combination of many strains and many different cultivars in mixed plantings allows sufficient banana production for home use. Yet attempts to grow Cavendish in plantation fail within a few years of establishment. Commercial Cavendish production in Taiwan, where strain VCG 01213 is present, is possible only by planting partially resistant Cavendish mutants in rotation with paddy rice and ratooning only twice. Even so, losses continue and costs are high. Permanent commercial plantations are no longer economic in areas where VCG 01213 is found. However, surveys indicate this pathogen is not ubiquitous. VCG 01213 was recently introduced into the Northern Territory of Australia, where eradication was attempted. This involved investigation of source, elimination of entire banana fields, isolation of sites, no replanting and tight quarantine. The situation here is not confounded by 'village banana plants' and is simplified by isolation and distance from the country's main banana-growing areas. In the Philippines and China, the disease now occurs in plantations situated within large areas of commercial banana production. The threat was at first not taken seriously and strain identification was delayed. Spread has occurred very rapidly in South China and less so in the Philippines. The VCG 01213 pathosystem is virtually unstudied, and its biology and epidemiology unknown. The results of experimental studies in Sumatra on incidence, treatment with endophytes and antagonists, cassava rotation, timing of infection, root invasion and breeding for resistance are reported. Rotation with cassava appears promising.
Fusarium wilt of cotton, caused by Fusarium oxysporum f. sp. vasinfectum, can have devastating effects on the vascular system of cotton plants and is a major threat to cotton production throughout the world. Accurate characterization and improved detection of these pathogenic isolates is needed for the implementation of a disease prevention program and the development of disease management strategies. Polymerase chain reaction (PCR) amplification of the ribosomal intergenic spacer (IGS) regions combined with digestion with three restriction enzymes (AluI. HaeIII, RsaI) resulted in three unique restriction profiles (IGS-restriction fragment length polymorphism [RFLP] haplotypes) for Australian F. oxysporum f. sp. vasinfectum isolates, which were capable of distinguishing them from other formae speciales of F. oxysporum. Furthermore, a portion of the IGS region flanking the 5' end was sequenced and single nucleotide polymorphisms (SNPs) were revealed. Using these sequence data. two specific real-time PCR-based assays were developed for the absolute quantification of genomic DNA from isolates obtained from soil substrates and infected cotton tissues. Standard curves of real-time PCR-based assays showed a linear relation (R-2 = 0.993 to 0.994) between log values of fungal genomic DNA and real-time PCR cycle thresholds. Using these assays, it was possible to detect fungal DNA as low as 5 pg/mu l. The detection sensitivity for inoculum added to sterile soils was lower than 10(4) conidia/g soil. In plant samples, the quantified fungal DNA varied from 30 pg to 1 ng/100 ng of total plant genomic DNA.