Commercial plantations of teak (Tectona grandis L.f.) are affected by many economically important fungal diseases under Brazilian conditions. Teak plants exhibiting distinctive vascular wilt symptoms were observed in Mirassol do Oeste (MT), Brazil. Trunk samples of the affected trees were collected, disinfected, and plated onto potato dextrose agar. Fungal cultures obtained displayed morphological characteristics typical of the Fusarium oxysporum species complex. A representative F. oxysporum isolate was used in pathogenicity assays. Teak plants displayed symptoms similar to those observed under field conditions approx. 60 d after root-dipping inoculation. Amplicons corresponding to segments of the translation elongation factor 1-α (TEF-1α) and RNA polymerase II second largest subunit (RPB2) genes were obtained using as template the genomic DNA extracted from two Fusarium isolates obtained from teak. Phylogenetic analyses of the amplicon sequences placed the isolates into the same cluster of isolates belonging to the F. oxysporum species complex. To our knowledge, this is the first report of vascular wilt of teak caused by F. oxysporum in the Neotropical region

BOITEUX, Leonardo S.

BORGES, Rafaela C.F.

CABRAL, Cléia S.

FERREIRA, Maria A.

FONSECA, Maria E.N.

FONTES, Maria G.

MACEDO, Mônica A.

REIS, Ailton

ROSSATO, Maurício

SANTOS, Maria D.M.

Firenze University Press: E-Journals

Phytopathologia Mediterranea (2018), 57, 1, 115−121
www.fupress.com/pm ISSN (print): 0031-9465
Firenze University Press ISSN (online): 1593-2095
DOI: 10.14601/Phytopathol_Mediterr-20896
Corresponding author: R.C.F. Borges 
E-mail: rafaelafal@hotmail.com
© 2018 Author(s). This is an open access article distributed under the terms of the Creative Commons Attribution License (CC-BY-4.0), 
which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
NEW OR UNUSUAL DISEASE REPORTS
Vascular wilt of teak (Tectona grandis) caused by Fusarium oxysporum 
in Brazil
Rafaela C. f. BORGeS1, MôniCa a. MaCeDO1, Cléia S. CABRAL1, MauRíCiO ROSSATO1, MaRia G. FONTES1, MaRia D. 
M. SanTOS1, MaRia a. feRReiRa2, MaRia e. n. fOnSeCa3, ailTOn ReiS3 and leOnaRDO S. BOiTeuX1,3
1 Plant Pathology Department, University of Brasília, Brasília–DF, Brazil
2 Plant Pathology Department, University of Lavras–MG, Brazil
3 National Center for Vegetable Crops Research (CNPH), Embrapa Hortaliças, Brasília–DF, Brazil
Summary. Commercial plantations of teak (Tectona grandis L.f.) are affected by many economically important fun-
gal diseases under Brazilian conditions. Teak plants exhibiting distinctive vascular wilt symptoms were observed 
in Mirassol do Oeste (MT), Brazil. Trunk samples of the affected trees were collected, disinfected, and plated onto 
potato dextrose agar. Fungal cultures obtained displayed morphological characteristics typical of the Fusarium 
oxysporum species complex. A representative F. oxysporum isolate was used in pathogenicity assays. Teak plants 
displayed symptoms similar to those observed under field conditions approx. 60 d after root-dipping inoculation. 
Amplicons corresponding to segments of the translation elongation factor 1-α (TEF-1α) and RNA polymerase 
II second largest subunit (RPB2) genes were obtained using as template the genomic DNA extracted from two 
Fusarium isolates obtained from teak. Phylogenetic analyses of the amplicon sequences placed the isolates into the 
same cluster of isolates belonging to the F. oxysporum species complex. To our knowledge, this is the first report of 
vascular wilt of teak caused by F. oxysporum in the Neotropical region.
Key words: molecular diagnosis vascular disease, t.
Introduction
Teak (Tectona grandis Lf; Lamiaceae family) is a 
tropical forest species, which is native from the mon-
soon areas of India, Myanmar (Burma), Laos, and 
Thailand (Narayanan et al., 2007). Teak wood has high 
commercial value due to its aesthetic qualities, dura-
bility, and resistance to environmental factors (Mes-
quita et al., 2017).
Teak introduction and commercial cultivation in 
Brazil was initiated in the early 1970s, in the bounda-
ries as well as within the Legal Amazon region. Since 
then, several economically important diseases of fun-
gal etiology, associated with extensive yield and qual-
ity losses, have been reported in commercial planta-
tions in this warm tropical region. These include rust 
caused by Olivea tectonae (Cabral et al., 2010); vascular 
wilt caused by Ceratocystis fimbriata (Firmino et al., 
2012) and, more recently, trunk canker disease caused 
by Lasiodiplodia theobromae isolates (Borges et al., 2015). 
In field surveys carried out in 2014, vascular dis-
ease of teak, with a peculiar set of symptoms, was 
found in commercial forests in Mirassol do Oeste, 
Mato Grosso State-MT, Brazil. Fusarium oxysporum 
isolates were consistently isolated from these affected 
trees. In the present study, we carried out morpho-
metrical analyses and pathogenicity assays of these 
isolates, aiming to characterize the causal agents as-
sociated with this vascular disease, which was previ-
ously unreported in Brazil. To confirm the identity of 
isolated fungi at the species level, molecular analy-
ses were carried out using genomic information from 
the partial translation elongation factor 1-α (TEF1-α) 
and RNA polymerase second largest subunit (RPB2) 
Phytopathologia Mediterranea116
R.C.F. Borges et al.
genes. These are the most phylogenetically informa-
tive genomic regions and the best represented se-
quence databases of Fusarium (O’Donnel et al., 2010). 
Materials and methods
In 2014, teak trees with typical vascular wilt symp-
toms were observed in field inspections in Mirassol 
do Oeste (MT), Brazil. Trunks disks with dark heart-
wood and sapwood dark lesions (Figure 1) were col-
lected and subjected to a series of disinfection steps 
with 70% alcohol for 1 min, followed by submersion 
of the plant tissues in 2% of sodium hypochlorite for 
1 min and a final washing step in double distilled wa-
ter for 1 min. The disinfected trunk fragments were 
then transferred to Petri plates containing 2% water 
agar, and maintained at 25 ± 1°C with a photoperiod 
12 h during 3 d. Agar plugs (0.5 mm diam.) contain-
ing fungal structures were then transferred to Petri 
plates containing Saltwater Nutrient Agar (SNA), and 
these plates were then incubated under the same con-
ditions. Pure fungal cultures were examined under a 
light microscope (Leica DM 2500) and photographed 
using a Leica DFC 490 camera. Morphometrical anal-
yses of the fungi were performed using the Leica 
QWin-Plus program. The type of phialides as well 
as the lengths and widths of macroconidia, microco-
nidia, and chlamydospores (n = 30) were annotated 
using fungal samples from 25 d-old cultures.
To confirm the identity of the vascular wilt causal 
agent at the species level, a set of analyses were carried 
out for two representative fungal isolates (‘FusTeca2’ 
and ‘FusTeca3’), using the genomic information from 
(TEF1-α) and (RPB2) genes (O’Donnel et al., 2010). 
Genomic DNA from fungal samples was extracted 
using the 2× CTAB buffer and organic solvents with 
minor modifications (Boiteux et al., 1999). PCR assays 
were carried out using total genomic DNA with the 
primers EF1 and EF2 targeting the TEF-1α region, 
and fRPB2-5f and fRPB2 targeting the RPB2 region 
(O’Donnel et al., 2010).
The TEF-1α and RPB2 amplicons were gel-purified 
and directly sequenced in both directions. The identi-
ties of the sequences were confirmed to correspond 
to the TEF-1α and RPB2 regions via BLAST, and their 
alignments were conducted using MAFFT software 
(Katoh et al., 2013) as a plugin of Geneious R8 (Kearse 
et al., 2012). A phylogenetic tree was constructed 
using the Bayesian inference method (two million 
chains; 25% burn-in), using concatenated sequences 
Figure 1. Cross sections of teak (Tectona grandis) tree trunks displaying heartwood and sapwood (xylem) darkening as well 
as black vascular areas (a and b).
117Vol. 57, No. 1, April, 2018
Fusarium oxysporum on teak trees
of partial TEF-1α (420 bp alignments) and RPB2 (298 
bp alignments) of two Fusarium isolates from teak 
(‘FusTeca2’ and ‘FusTeca3’) as well as 20 reference se-
quences of Fusarium isolates available at the GenBank 
(Table 1). Fusarium solani was employed as outgroup 
in the analyses. The analyses employed GTR model 
(Tavaré, 1986) with gamma-distributed variation rate 
across sites chosen by Mega 7.0 (Kumar et al., 2016). 
For pathogenicity assays, three elite teak clones 
(named clones A, B, and C) were inoculated with one 
representative fungal isolate (‘FusTeca2’). Inoculation 
of teak plants (80 d after transplanting) was carried 
out via root-dipping, using a suspension of F. oxyspo-
rum microconidia (adjusted to 106 mL-1). Sterile water 
suspension was used in the inoculation of the control 
plants. The inoculated plants were maintained under 
greenhouse conditions and visually evaluated for dis-
ease up to 90 d after inoculation.
Results
The symptoms of previously unreported disease 
were initially found in tree trunks, which displayed 
black darkening of heartwood and sapwood vascular 
areas (Figure 1). External symptoms of leaf yellowing, 
leaf abscission and wilting were also observed in the 
analyzed trees.
On culture medium, the microconidia of the fun-
gal isolates had lengths of 5–10 μm and widths of 
2–4 μm (Figure 2a, b, c, d and h). The microconidia 
Table 1.
Species Strain RPB2 TEF
Fusarium anthophilum NRRL 25214 KU171696 KU171716
Fusarium babinda NRRL 25539 KU171698 KU171718
Fusarium bulbicola NRRL 22947 KU171699 KU171719
Fusarium dlaminii NRRL 13164 KU171701 KU171721
Fusarium euwallaceae NRRL 62626 KU171702 KU171722
Fusarium foetens NRRL 38302 KU171703 KU171723
Fusarium gaditjirri NRRL 45417 KU171704 KU171724
Fusarium miscanthi NRRL 26231 KU171705 KU171725
Fusarium oxysporum FusTeca2 MF170550 MF170551
Fusarium oxysporum FusTeca3 MF170552 MF170553
Fusarium oxysporum CIB_11 LN828100 LN828039
Fusarium oxysporum CIB_13 LN828102 LN828041
Fusarium oxysporum CIB_23 LN828106 LN828045
Fusarium oxysporum LEMM_110248 LN828073 LN828011
Fusarium oxysporum LEMM_110414 LN828079 LN828017
Fusarium oxysporum LEMM_122066 LN828096 LN828034
Fusarium proliferatum NRRL 62905 KU171707 KU171727
Fusarium redolens NRRL 22901 KU171708 KU171728
Fusarium solani LEMM_110029 LN828048 LN827959
Fusarium solani LEMM_111347 LN828058 LN827970
Fusarium succisae NRRL 13298 KU171712 KU171732
Fusarium thapsinum NRRL 22049 KU171713 KU171733
Phytopathologia Mediterranea118
R.C.F. Borges et al.
were predominantly oval to ellipsoid, slightly curved 
(without septae) (Figure 2a, b, c and d), and organ-
ized in false heads produced in short monophialides 
(Figure 2a and b). Macroconidia (lengths 19–45 μm 
and widths 3–6 μm) were falcate, moderately curved, 
and each with three to five septae (Figure 2c, d and 
e). Chlamydospores were globular and formed at the 
ends or interspersed within the hyphae occurring sin-
gly, in pairs, or in chains (Figure 2f and g).
The three teak clones used in the pathogenicity as-
says exhibited wilting, and associated symptoms of 
premature leaf dropping and vascular browning, fol-
lowed by plant collapse and death about 60 d after 
inoculation (Figure 3). Fungi isolated from affected 
plants onto PDA displayed the same morphological 
and molecular characteristics of the inoculated isolate 
(see above).
The TEF-1α sequences obtained from the Fusarium 
isolates ‘FusTeca2’ and ‘FusTeca3’ were deposited in 
the GenBank database as ‘FusTeca2’ (MF170551) and 
‘FusTeca3’ (MF170553). RPB2 sequences of these two 
isolates were also deposited in the GenBank database 
as ‘FusTeca2’ (MF170550) and ‘FusTeca3’ (MF170552). 
Both of these Fusarium isolates clustered with 
other F. oxysporum isolates, with maximum values of 
posterior probability (Figure 4). These phylogenetic 
analyses confirmed that the causal agent of this vas-
cular wilt of teak belonged to the F. oxysporum species 
complex.
Discussion
The Fusarium oxysporum species complex (FOSC) 
is a cosmopolitan group of soil-borne pathogens that 
has been reported in association with vascular wilt 
diseases in a wide range of plant species (O’Donnell 
et al., 2010). Approximately 80 host-specific formae 
speciales are described within the FOSC. The three 
teak clones used in the pathogenicity assays exhib-
ited wilting symptoms after inoculation with one 
isolate morphologically and molecularly identical to 
the Fusarium isolates re-isolated on PDA, thus fulfill-
ing the Koch’s postulates for this pathogen. Molecu-
lar markers have been developed to identify various 
formae speciales (Lievens et al., 2008). Given the high 
level of phylogenetic diversity and large number of 
Figure 2. Morphological characteristics of a teak-infecting Fusarium oxysporum isolate. Conidiophores and phialides (a and 
b). Microconidia (oval to ellipsoid and slightly curved) with no septae (a, b, c, d, and h). Falcate and slightly curved macro-
conidia with three to five septae (c, d, and e). Globose to oval chlamydospores formed at the ends of hyphae or interspersed 
in the mycelium, which were found single, in pairs, or in chains (f and g).
119Vol. 57, No. 1, April, 2018
Fusarium oxysporum on teak trees
formae speciales, multilocus DNA sequence typing cur-
rently represents the most robust approach for char-
acterizing these fungal variants (Lievens et al., 2008; 
O’Donnell et al., 2009; 2010). However, pathogenicity 
assays are still the standard technique for identifying 
host-specific pathogens within members of the FOSC. 
In the present study, the infectivity of this F. oxyspo-
rum isolate was only confirmed in teak. A host range 
assessment needs to be performed to confirm if this iso-
late represents a new forma specialis of F. oxysporum. 
The TEF-1α and RPB2 genomic regions are usu-
ally employed in formae speciales discrimination as-
says, mainly because of their adequate levels of 
phylogenetic signal (O’Donnell et al., 2010). Due to 
their importance and utility, these regions were used 
in the present study in concatenated analyses (with 
Figure 3. Pathogenicity assay of one Fusarium oxysporum isolate (‘FusTeca2’) from teak (Tectona grandis): seedlings of teak 
(80 d after transplanting) were inoculated via root-dipping using microconidium suspension (a). Typical damage induced 
by the F. oxysporum isolate in teak seedlings (b). Overview of teak seedlings in the pathogenicity assay with the F. oxysporum 
isolate (c). Distinct levels of vascular browning symptoms after inoculation of teak seedlings with the F. oxysporum isolate 
(c, d, e, and f).
Phytopathologia Mediterranea120
R.C.F. Borges et al.
three phylogenetic methods). The results strongly 
indicated a clustering of Fusarium isolates from teak 
with a set of reference F. oxysporum isolates. No base 
differences were found in alignments of the isolates 
‘FusTeca2’ and ‘FusTeca3’ with other reference F. ox-
ysporum isolates, corroborating these high levels of 
genetic similarity.
There are no formal reports of FOSC members in-
fecting teak in other continents, including Asia. To our 
knowledge, this is the first report of F. oxysporum in 
teak in the Neotropics, and probably the first world-
wide report of vascular wilt caused by F. oxysporum 
in this host species. The geographic origins of the F. 
oxysporum isolates for which we found the greatest 
sequence similarities from the BLASTn search were 
mainly from countries in Asia (data not presented). 
This suggests that F. oxysporum from teak in Brazil is 
more likely to be exotic than endemic. Teak is a tropi-
cal forest species found dispersed across India, My-
anmar, Laos, and Thailand, and several members of 
FOSC are well-known to be transmitted via contami-
nated seeds and/or seedlings (Reis et al., 2008; Cabral 
et al., 2014) or as endophytes. Therefore, this poten-
tially new forma specialis of F. oxysporum from teak was 
likely to be introduced into Brazil via contaminated 
plant material.
The commercial teak production areas in Brazil are 
expanding, but some soil-borne diseases have already 
caused serious yield and quality losses. Description 
of this new vascular disease caused by F. oxysporum 
provides important new knowledge to stimulate the 
teak production industry and researchers to search 
for development of effective vascular wilt manage-
ment strategies.
Literature cited
Boiteux L.S., M.E.N. Fonseca, and P.W. Simon, 1999. Effects of 
plant tissue and DNA purification method on randomly 
amplified polymorphic DNA-based genetic fingerprinting 
analysis in carrot. Journal of the American Society for Horticul-
tural Science 124, 32–36. 
Borges R.C.F., M. D. M. Santos, M.A. Macedo, I. Martins, A.G. 
Nascimento, A.C. Cafe-Filho, L.S. Boiteux, M.E.N. Fonseca, 
C.A. Inácio and S.C. M. Mello, 2015. A trunk canker disease 
of Tectona grandis induced by Lasiodiplodia theobromae in Bra-
zil. New Disease Report 31, 26. 
Cabral P. G. C., A. S. Capucho, O.L. Pereira, E. Maciel-Zam-
bolim, R.L. Freitas and L. Zambolim, 2010. First report of 
teak leaf rust disease caused by Olivea tectonae in Brazil. 
Australasian Plant Disease Notes 5, 113–114. 
Cabral C.S., K.R. Brunelli, H. Costa, M.E.N. Fonseca, L.S. Boi-
teux, and A. Reis, 2014. Identification of Fusarium oxyspo-
rum f. sp. lactucae race 1 as the causal agent of lettuce wilt in 
Brazil. Tropical Plant Pathology 39, 197–202.
Firmino A.C., H.J. Tozze Jr, and E.L. Furtado, 2012. First report 
of Ceratocystis fimbriata causing wilt in Tectona grandis in 
Brazil. New Disease Reports 25, 24.
Katoh K and D.M. Standley, 2013. MAFFT multiple sequence 
alignment software version 7: improvements in perfor-
mance and usability. Molecular Biology and Evolution 30, 
772–780.
Kearse M., R. Moir, A. Wilson, S. Stones-Havas, M. Cheung, S. 
Sturrock, S. Buxton, A. Cooper, S. Markowitz, C. Duran, T. 
Thierer, B. Ashton, P. Meintjes and A. Drummond, 2012. 
Geneious Basic: an integrated and extendable desktop 
software platform for the organization and analysis of se-
quence data. Bioinformatics 28, 1647–1649.
Kumar S., G. Stecher and K. Tamura, (2016). MEGA7: molec-
ular evolutionary genetics analysis version 7.0 for bigger 
datasets. Molecular Biology and Evolution 33, 1870–1874.
Lievens B., M. Rep and B.P.H.J Thomma, 2008. Recent develop-
ments in the molecular discrimination of formae speciales 
of Fusarium oxysporum. Pest Management Science 64, 781–788.
Figure 4. Phylogenetic tree obtained by Bayesian inference 
(GTR+G model) of the concatenated partial translation 
elongation factor1-α (TEF1-α) and RNA polymerase second 
largest subunit (RPB2) genes sequences along with other 20 
reference Fusarium isolates available at the GenBank.
121Vol. 57, No. 1, April, 2018
Fusarium oxysporum on teak trees
Mesquita R.R.S., J.C. Gonçalez and M.H. de Paula, 2017. Com-
portamento da madeira de Tectona grandis frente ao intem-
perismo. Revista Floresta 47, 29–36.
Narayanan C., S.A. Wali, N. Shukla, R. Kumar, A.K. Mandal 
and S.A. Ansari, 2007. RAPD and ISSR markers for molecu-
lar characterization of teak (Tectona grandis) plus trees. Jour-
nal of Tropical Forest Science 19, 218–225.
O’Donnell K., C. Gueidan, S. Sink, P.R. Johnston, P.W. Crous, A. 
Glenn, R. Riley, N.C. Zitomer, P. Colyer, C. Waalwijk, T. Lee, 
A. Moretti, S. Kang, H. S. Kim, D. M. Geiser, J. H. Juba, R. P. 
Baayen, M. G. Cromey, S. Bithell, D. A. Sutton, K. Skovgaard, 
R. Ploetz, H. Corby Kistler, M. Elliott, M. Davis and B.A.J. 
Sarver, 2009. A two-locus DNA sequence database for typing 
plant and human pathogens within the Fusarium oxysporum 
species complex. Fungal Genetics and Biology 46, 936–948.
O’Donnell K., D.A. Sutton, M.G. Rinaldi, B.A. Sarver, S.A. 
Balajee, H.J. Schroers, R.C. Summerbel, V.A.R.G. Robert, P. 
W. Crous, N. Zhang, T. Aoki, K. Jung, J. Park, Y. H. Lee, S. 
Kang, B. Park and D. M. Geiser, 2010. Internet-accessible 
DNA sequence database for identifying fusaria from hu-
man and animal infections. Journal of Clinical Microbiology 
48, 3708–3718.
Reis A., L.S. Boiteux and R.F. Vieira, 2008. Search for sources 
of wide-spectrum resistance to Fusarium oxysporum f. sp. 
basilici isolates in accessions of Ocimum species. Journal of 
General Plant Pathology 74, 375–381.
Tavaré S. 1986. Some probabilistic and statistical problems in 
the analysis of DNA sequences. Lectures on Mathematics in 
the Life Sciences 17, 57–86.
Accepted for publication: March 15, 2018
Published online: May 15, 2018


Vascular wilt of teak (Tectona grandis) caused by Fusarium oxysporum in Brazil

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Vascular wilt of teak (Tectona grandis) caused by Fusarium oxysporum in Brazil

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