Fungal Diversity (2013) 61:181–193
DOI 10.1007/s13225-013-0231-z
Species of Lasiodiplodia associated with mango in Brazil
Marília W. Marques & Nelson B. Lima & Marcos Antônio de Morais Jr &
Maria Angélica G. Barbosa & Breno O. Souza & Sami J. Michereff &
Alan J. L. Phillips & Marcos P. S. Câmara
Received: 23 January 2013 / Accepted: 27 March 2013 / Published online: 7 April 2013
# Mushroom Research Foundation 2013
Abstract Mango (Mangifera indica) is a major tropical
fruit species cultivated in Brazil. The objective of this study
was to identify species of Lasiodiplodia associated with
dieback and stem-end rot of mango in the semi-arid region
of Northeastern Brazil, and compare the species in relation to
mycelial growth, pathogenicity and virulence. A total of 120
isolates of Lasiodiplodia were used and identifications were
made using a combination of morphology and phylogenetic
analysis based on partial translation elongation factor 1-α
sequence (EF1-α) and internal transcribed spacers (ITS).
The following species were identified: Lasiodiplodia
crassispora, L. egyptiacae, L. hormozganensis, L. iraniensis,
L. pseudotheobromae, L. theobromae and Lasiodiplodia sp..
Lasiodiplodia theobromae was the most frequently isolated
species, which represented 41 % of all the isolates. Only this
species had been previously reported on mango in Brazil,
while the other species represent the first report associated
with mango tree diseases in this country. Lasiodiplodia
crassispora is reported for the first time associated with man-
go diseases worldwide. There were significant differences in
mycelial growth rates among the Lasiodiplodia species and
M. W. Marques : N. B. Lima : B. O. Souza : S. J. Michereff :
M. P. S. Câmara (*)
Departamento de Agronomia, Universidade Federal Rural de
Pernambuco, 52171-900 Recife, Brazil
e-mail: mcamara@depa.ufrpe.br
M. A. de Morais Jr
Departamento de Genética, Universidade Federal de Pernambuco,
50732-970 Recife, Brazil
M. A. G. Barbosa
Embrapa Semi-Árido, 56302-970 Petrolina, Brazil
A. J. L. Phillips
Centro de Recursos Microbiológicos, Departamento de Ciências
da Vida, Faculdade de Ciências e Tecnologia, Universidade Nova
de Lisboa, 2829-516 Caparica, Portugal
also in the optimum temperature for growth. All species of
Lasiodiplodia were pathogenic on mango fruit. There were
significant differences in virulence among the species, where-
in L. hormozganensis and Lasiodiplodia sp.were the most
virulent, while the least virulent were L. iraniensis, L.
pseudotheobromae, L. crassispora and L. egyptiacae.
Keywords Botryosphaeriaceae . Mangifera indica . ITS .
EF1-α . Phylogeny . Virulence
Introduction
Mango tree (Mangifera indica L.) is a major tropical fruit
species cultivated in Brazil. In 2010, Brazilian production
reached 1,197,694 t of fruit in an area of approximately
75,416 ha, generating about US$ 334 million. Considering
both income and exported volume, mango occupied the
third place in the segment of fresh fruit, with revenues of
US$ 119,929 million and 124,694 t of fruit, respectively
(Agrianual 2012). All mango exported is produced in the
semiarid area of the Northeast region, mainly in the São
Francisco Valley and Assú Valley (Costa et al. 2010). This
crop also plays an important social role, generating over
25,000 direct and 75,000 indirect jobs in the São Franscico
Valley alone (Souza et al. 2002). Among the wide range of
diseases that impact on mango production in Brazil, dieback
and stem-end rot have become increasingly important
(Costa et al. 2010). The first report of mango dieback in
Brazil was in 1947 (Batista 1947) and since then the inten-
sity of the disease has increased leading, in some cases, to
the complete loss of production and elimination of entire
orchards (Tavares et al. 1991; Tavares 2002).
Mango dieback and stem-end rot are caused by a com-
plex of fungi, but members of the Botryosphaeriaceae are
considered to be the most important (Johnson 1992; Al
182
Adawi et al. 2003; Slippers et al. 2005; Javier-Alva et al. 2009;
Costa et al. 2010; Ismail et al. 2012). The Botryosphaeriaceae
is a genus-rich family in the Dothideomycetes, comprising
numerous species with a cosmopolitan distribution (Crous et
al. 2006; Phillips et al. 2008; Liu et al. 2012). Species of the
Botryosphaeriaceae are associated with several host plants,
and can act as primary or secondary pathogens, or as endo-
phytes that under stress conditions of the host plant can
become pathogenic (Denman et al. 2000; Crous et al. 2006).
Lasiodiplodia theobromae (Pat.) Griff. & Maubl, a member of
the Botryosphaeriaceae, occurs mainly in tropical and sub-
tropical regions, causing severe damage in almost 500 host
plants (Punithalingam 1980; Burgess et al. 2006). The fungus
has been reported as a mango pathogen worldwide associated
with several disease symptoms including dieback, stem-end
rot, decline, gummosis and canker (Ploetz et al. 1996; Jacobs
2002; Khanzada et al. 2004; Abdollahzadeh et al. 2010; Costa
et al. 2010; Sakalidis et al. 2011a; Ismail et al. 2012). Besides
mango, several other crops of economic importance are af-
fected by L. theobromae in Brazil, especially avocado (Persea
americana Mill.), banana (Musa spp.), barbados cherry
(Malpighia glabra L.), cashew (Anacardium occidentale L.),
citrus (Citrus spp.), coconut palm (Cocos nucifera L.), custard
apple (Annona squamosa L.), grapevine (Vitis sp.), guava
(Psidium guajava L.), muskmelon (Cucumis melo L.), papaya
(Carica papaya L.), passion fruit (Passiflora edulis Sims),
soursop (Annona muricata L.) and watermelon (Citrullus
lanatus (Thunb.) Matsum. & Nakai) (Tavares 2002; Freire et
al. 2003).
According to Sutton (1980) and Phillips et al. (2008) the
main features that distinguish Lasiodiplodia from other
closely related genera are the presence of pycnidial paraph-
yses and longitudinal striations on mature conidia (Sutton
1980). Recent studies based on ITS and EF-1α sequence
data have led to the identification of cryptic species within
the L. theobromae species complex (Pavlic et al. 2004;
Burgess et al. 2006; Damm et al. 2007; Alves et al. 2008;
Pavlic et al. 2008; Abdollahzadeh et al. 2010; Begoude et al.
2010; Úrbez-Torres et al. 2012; Ismail et al. 2012). Presently,
17 species are recognized in Lasiodiplodia.
Several species of Lasiodiplodia have been associated
with mango diseases worldwide. Lasiodiplodia theobromae
was recorded in Australia (Slippers et al. 2005), Brazil
(Costa et al. 2010), Egypt (Ismail et al. 2012), Iran
(Abdollahzadeh et al. 2010), South Africa (Jacobs 2002)
and USA (Ploetz et al. 1996). Recently, four new species
of Lasiodiplodia (L. citricola Abdollahzadeh, Zare & A.J.L.
Phillips, L. gilanensis Abdollahzadeh, Javadi & A.J.L.
Phillips, L. hormozganensis Abdollahzadeh, Zare & A.J.L.
Phillips and L. iraniensis Abdollahzadeh, Zare & A.J.L.
Phillips) and L. pseudotheobromae A.J.L. Phillips, A.
Alves & Crous were associated with this host in Iran
(Abdollahzadeh et al. 2010). In Australia, L. iraniensis
Fungal Diversity (2013) 61:181–193
and L. pseudotheobromae were isolated from cankers and
dieback of mango (Sakalidis et al. 2011a). In 2012, L.
pseudotheobromae and a new species (L. egyptiacae A.M.
Ismail, L. Lombard & Crous) were recorded in Egypt caus-
ing dieback of mango (Ismail et al. 2012).
In Brazil, for a long time, dieback and stem-end rot of
mango were attributed exclusively to L. theobromae, but
recent studies based on molecular methods revealed the pres-
ence of other Botryosphaeriaceae species Neofusicoccum
parvum (Pennycook & Samuels) Crous, Slippers & A.J.L.
Phillips, Fusicoccum aesculi (Corda) Crous, Slippers &
A.J.L. Phillips and Pseudofusicoccum stromaticum (Mohali,
Slippers & M.J. Wingf.) Mohali, Slippers & M.J. Wingf.
associated with these diseases (Costa et al. 2010; Marques et
al. 2012).
The increasing economic importance of plant diseases
caused by Lasiodiplodia and the recent discovery of several
new species of fungus associated with tropical plants led us to
speculate that more than one species of Lasiodiplodia may be
associated with mango diseases in Northeastern Brazil. The
disease etiology is crucial for epidemiological studies and for
a better understanding of the distribution and importance of
individual species, as well as finding effective management
strategies to each pathogen. Therefore, the objective of this
study was to identify species of Lasiodiplodia from a large
number of isolates associated with dieback and stem-end rot
of mango in the semi-arid region of Northeastern Brazil.
Materials and methods
Sampling and fungal isolation
From January to February 2010, stems and fruits showing
dieback and stem-end rot symptoms were collected from 14
mango orchards (25 samples per orchard) located in São
Francisco Valley and Assú Valley, Northeastern Brazil.
Samples were recovered from the cultivars Tommy Atkins,
Keitt, Haden and Palmer. Plant tissues were surface
disinfested in 70 % ethanol for 30 s and 1 % NaOCl for
1 min. Samples were then rinsed in sterile distilled water for
30 s and dried before small pieces (4–5 mm) of tissue were
taken from the margin between necrotic and apparently
healthy tissue and plated onto potato dextrose agar (PDA)
(Acumedia, Lansing, USA) amended with 0.5 g l−1 strepto-
mycin sulfate (PDAS). Plates were incubated at 25 °C in the
dark for 3 to 4 days. Fungal colonies emerging from plant
tissue pieces that were morphologically similar to species of
Botryosphaeriaceae (Sutton 1980; Phillips 2006) were trans-
ferred to PDA plates and incubated at 25 °C in the dark, with
observation after 3, 5 and 15 day. To obtain single-spore
isolates, pycnidia were obtained on 2 % water agar (WA)
with autoclaved pine needles as a substrate after 3-week
Fungal Diversity (2013) 61:181–193
incubation at 25 °C under a 12 h daily photoperiod with
near-ultraviolet light (Slippers et al. 2004). A single pycnid-
ium was cut from each isolate under a stereo microscope
(Zeiss Stemi DV4; Carl Zeiss, Berlin, Germany) and placed
in 250 μl of sterile water to produce a conidial suspension.
A 20 μl aliquot was spread on PDAS and incubated at 28 °C
in the dark for 24 h. A single-pycnidiospore isolate was
recovered for an individual sample and transferred to a fresh
PDA plate. All isolates were morphologically identified as
Lasiodiplodia when characteristics typical of genus were
present, namely conidiomatal paraphyses, conidia that were
initially hyaline and aseptate, but in time developed a single
median septum, the wall became dark brown and melanin
granules deposited longitudinally on the inner surface of the
wall gave the conidia a striate appearance (Sutton 1980;
Alves et al. 2008). Stock cultures were stored in PDA slants
at 5 °C in the dark.
DNA isolation, PCR amplification and sequencing
A portion of the translation elongation factor 1α (EF1-α)
gene was sequenced for all the Lasiodiplodia isolates col-
lected from mango orchards. The internal transcribed spacer
(ITS) region of rDNA was sequenced to confirm the identity
of representative isolates within each EF1-α identified spe-
cies. Using a sterile 10 μl pipette tip, a small amount of
aerial mycelium was scraped from the surface of a 5 day old
culture on PDA at 25 °C and genomic DNA was extracted
using the AxyPrep™ Multisource Genomic DNA Miniprep
Kit (Axygen Scientific Inc., Union City, USA) following the
manufacturer’s instructions. The ITS region was amplified
using the primers ITS1 and ITS4 (White et al. 1990) as
described by Slippers et al (2004) and EF1-α gene was
amplified using the primers EF1-688F and EF1-1251R
(Alves et al. 2008) as described by Phillips et al. (2005).
Each 50 μl polymerase chain reaction (PCR) mixture in-
cluded 21 μl of PCR-grade water, 1 μl of DNA template,
1.5 μM of each primer, and 1 μl of PCR Master Mix (2X)
(0.05 u μl−1 de Taq DNA polimerase, reaction buffer, 4 mM
MgCl2, 0.4 mM of each dNTP; Thermo Scientific, Waltham,
USA). PCR reactions were carried out in a thermal cycler
(Biocycler MJ 96; Applied Biosystems, Foster City, USA).
The PCR amplification products were separated by electro-
phoresis in 1.5 % agarose gels in 1.0× Tris-acetate acid
EDTA (TAE) buffer and were photographed under UV light
after staining with with ethidium bromide (0.5 μg ml−1) for
1 min. PCR products were purified using the AxyPrep™
PCR Cleanup Kit (Axygen) following the manufacturer’s
instructions. ITS and EF1-α regions were sequenced in both
directions using a ABI PRISM® 3100-Avant Genetic
Analyzer (Applied Biosystems) at the Sequencing Platform
LABCEN/CCB in the Universidade Federal de Pernambuco
(Recife, Brazil).
183
Phylogenetic analyses
Sequences were edited with Chromas v. 2.32 (Technelysium
Pty Lda, Brisbane, Australia). Sequences of both DNA regions
of additional isolates were retrieved from GenBank. Sequences
were aligned with ClustalX v. 1.83 (Thompson et al. 1997) and
manually adjusted when necessary. Phylogenetic information
contained in indels (gaps) was incorporated into the phyloge-
netic analyses using simple indel coding as implemented by
GapCoder (Young and Healey 2003). A partition homogeneity
test determined the possibility of combining the ITS and EF1-α
datasets (Farris et al. 1995; Huelsenbeck et al. 1996).
Sequences of Lasiodiplodia species obtained from GenBank
were included in the analyses (Table 1). Diplodia seriata De
Not. (CBS 112555) and D. mutila Fr. (CBS CBS 112553) were
used as outgroup.
Phylogenetic analyses were performed using PAUP v.
4.0b10 (Swofford 2003) for maximum-parsimony and
MrBayes v. 3.0b4 (Ronquist and Huelsenbeck 2003) for
Bayesian analyses. Maximum-parsimony analyses were
performed using the heuristic search option with 1,000
random taxa addition and tree bisection and reconnection
(TBR) as the branch-swapping algorithm. All characters
were unordered and of equal weight and gaps were treated
as missing data. Branches of zero length were collapsed and
all multiple, equally parsimonious trees were saved. The
robustness of the most parsimonious trees was evaluated
from 1,000 bootstrap replications (Hillis and Bull 1993).
Other measures used were consistency index (CI), retention
index (RI) and homoplasy index (HI).
Bayesian analyses employing a Markov Chain Monte
Carlo method were performed. The general time-reversible
model of evolution (Rodriguez et al. 1990), including esti-
mation of invariable sites and assuming a discrete gamma
distribution with six rate categories (GTR+Γ+G) was used.
Four MCMC chains were run simultaneously, starting from
random trees for 1,000,000 generations. Trees were sampled
every 100th generation for a total of 10,000 trees. The first
1,000 trees were discarded as the burn-in phase of each
analysis. Posterior probabilities (Rannala and Yang 1996)
were determined from a majority-rule consensus tree gener-
ated with the remaining 9,000 trees. This analysis was
repeated three times starting from different random trees to
ensure trees from the same tree space were sampled during
each analysis.
Phylogenetic trees were viewed with Treeview (Page
1996). Sequences generated in this study were deposited in
GenBank and the alignment in TreeBASE (S13281).
Representative isolates of different Lasiodiplodia species
obtained in this study were deposited in the Culture
Collection of Phytopathogenic Fungi “Prof. Maria Menezes”
(CMM) at the Universidade Federal Rural de Pernambuco
(Recife, Brazil).
184 Fungal Diversity (2013) 61:181–193

Table 1 Isolates of Lasiodiplodia species used in this study

Taxon Culture Host Location Collector GenBank Accession No.b

Accession No. a
ITS EF-1α

Diplodia mutila CBS 112553 Vitis vinifera Portugal A.J.L. Phillips AY259093 AY573219
D. seriata CBS 12555 Vitis vinifera Portugal A.J.L. Phillips AY259094 AY573220
Lasiodiplodia citricola CBS 124707 Citrus sp. Iran J. Abdollahzadeh GU945354 GU945340
& A. Javadi
L. citricola IRAN 1521C Citrus sp. Iran A. Shekari GU945353 GU945339
L. crassispora CMW 13488 Eucalyptus urophylla Venezuela S. Mohali DQ103552 DQ103559
L. crassispora CMW 14691 Santalum album Australia T.I. Burgess & G. Pegg DQ103550 DQ103557
L. crassispora CMM 3982 Mangifera indica Brazil M.W. Marques JX464058 JX464015
L. egyptiacae CBS 130992 M. indica Egypt A.M. Ismail JN814397 JN814424
L. egyptiacae BOT 29 M. indica Egypt A.M. Ismail JN814401 JN814428
L. egyptiacae CMM 3981 M. indica Brazil M.W. Marques JX464072 JX464035
L. egyptiacae CMM 1485 M. indica Brazil V.S.O. Costa JX464076 JX464044
L. gilaniensis IRAN 1501C Unknown Iran J. Abdollahzadeh GU945352 GU945341
& A. Javadi
L. gilaniensis CBS 124704 Unknown Iran J. Abdollahzadeh GU945351 GU945342
& A. Javadi
L. gonubiensis CMW 14078 Syzigium cordatum South Africa D. Pavlic AY639594 DQ103567
L. gonubiensis CBS 115812 S. cordatum South Africa D. Pavlic DQ458892 DQ458860
L. hormozganensis CBS 124709 Olea sp. Iran J. Abdollahzadeh GU945355 GU945340
& A. Javadi
L. hormozganensis IRAN 1498C M. indica Iran J. Abdollahzadeh GU945356 GU945344
& A. Javadi
L. hormozganensis CMM 1546 M. indica Brazil V.S.O. Costa JX464077 JX464053
L. hormozganensis CMM 3983 M. indica Brazil M.W. Marques JX464069 JX464033
L. hormozganensis CMM 3984 M. indica Brazil M.W. Marques JX464080 JX464052
L. hormozganensis CMM 3985 M. indica Brazil M.W. Marques JX464084 JX464101
L. hormozganensis CMM 3986 M. indica Brazil M.W. Marques JX464085 JX464022
L. hormozganensis CMM 3987 M. indica Brazil M.W. Marques JX464094 JX464034
L. iraniensis CBS 124710 Salvadora persica Iran J. Abdollahzadeh GU945348 GU945336
& A. Javadi
L. iraniensis IRAN 1502C Juglans sp. Iran A. Javadi GU945347 GU945335
L. iraniensis CMM 3990 M. indica Brazil M.W. Marques JX464067 JX464028
L. iraniensis CMM 1483 M. indica Brazil V.S.O. Costa JX464073 JX464023
L. iraniensis CMM 3993 M. indica Brazil M.W. Marques JX464068 JX464029
L. iraniensis CMM 3995 M. indica Brazil M.W. Marques JX464097 JX464046
L. iraniensis CMM 3996 M. indica Brazil M.W. Marques JX464100 JX464019
L. iraniensis CMM 3998 M. indica Brazil M.W. Marques JX464059 JX464045
L. iraniensis CMM 4051 M. indica Brazil M.W. Marques JX464071 JX464030
L. mahajangana CBS 124927 Terminalia catappa Madagascar J. Roux FJ900597 FJ900643
L. mahajangana CMW 27801 T. catappa Madagascar J. Roux FJ900595 FJ900641
L. margaritaceae CBS 122065 Adansonia gibbosa Western Australia T.I. Burgess EU144051 EU144066
L. margaritaceae CBS 122519 A. gibbosa Western Australia T.I. Burgess EU144050 EU144065
L. missouriana UCD 2199MO Vitis vinifera Missouri, USA K. Striegler HQ288226 HQ288268
& G.M. Leavitt
L. missouriana CBS 128311 V. vinifera Missouri, USA K. Striegler HQ288225 HQ288267
& G.M. Leavitt
L. parva CBS 494.78 Cassava-field soil Colombia O. Rangel EF622084 EF622064
L. parva CBS 456.78 Cassava-field soil Colombia O. Rangel EF622083 EF622063
L. plurivora CBS 120832 Prunus salicina South Africa U. Damm EF445362 EF445395
L. plurivora STE-U4583 V. vinifera South Africa F. Halleen AY343482 EF445396
L. pseudotheobromae CBS 116459 Gmelina arborea Costa Rica J. Carranza-Velásquez EF622077 EF622057
L. pseudotheobromae IRAN 1518C Citrus sp. Iran J. Abdollahzadeh/ GU973874 GU973866
A. Javadi
Fungal Diversity (2013) 61:181–193 185

Table 1 (continued)

Taxon Culture Host Location Collector GenBank Accession No.b

Accession No. a
ITS EF-1α

L. pseudotheobromae CMM 3999 M. indica Brazil M.W. Marques JX464075 JX464018

L. pseudotheobromae CBS 116459 G. arborea Costa Rica J. Carranza-Velásquez EF622077 EF622057
L. pseudotheobromae CMM 4001 M. indica Brazil M.W. Marques JX464092 JX464039
L. pseudotheobromae CMM 4002 M. indica Brazil M.W. Marques JX464086 JX464020
L. pseudotheobromae CMM 4008 M. indica Brazil M.W. Marques JX464090 JX464016
L. gonubiensis CMW 14078 S. cordatum South Africa D. Pavlic AY639594 DQ103567
L. gonubiensis CBS 115812 S. cordatum South Africa D. Pavlic DQ458892 DQ458877
L. rubropurpurea WAC 12535 E. grandis Queensland T.I. Burgess & G. Pegg DQ103553 DQ103571
L. rubropurpurea WAC 12536 E. grandis Queensland T.I. Burgess & G. Pegg DQ103554 DQ103572
L. theobromae CBS 164.96 Fruit on coral New Guinea A. Aptroot AY640255 AY640258
reef coast
L. theobromae CBS 111530 Unknown Unknown Unknown AY622074 AY622054
L. theobromae CMM 1476 M. indica Brazil M.W. Marques JX464083 JX464057
L. theobromae CMM 1481 M. indica Brazil M.W. Marques JX464095 JX464021
L. theobromae CMM 1517 M. indica Brazil M.W. Marques JX464060 JX464054
L. theobromae CMM 4019 M. indica Brazil M.W. Marques JX464096 JX464026
L. theobromae CMM 4033 M. indica Brazil M.W. Marques JX464081 JX464032
L. theobromae CMM 4039 M. indica Brazil M.W. Marques JX464065 JX464041
L. theobromae CMM 4041 M. indica Brazil M.W. Marques KC184891 JX464042
L. theobromae CMM 4042 M. indica Brazil M.W. Marques JX464070 JX464017
L. theobromae CMM 4043 M. indica Brazil M.W. Marques JX464087 JX464056
L. theobromae CMM 4046 M. indica Brazil M.W. Marques JX464091 JX464027
L. theobromae CMM 4047 M. indica Brazil M.W. Marques JX464082 JX464025
L. theobromae CMM 4048 M. indica Brazil M.W. Marques JX464093 JX464048
L. theobromae CMM 4050 M. indica Brazil M.W. Marques JX464062 JX464024
L. theobromae CMM 4052 M. indica Brazil M.W. Marques JX464088 JX464050
L. theobromae CMM 4053 M. indica Brazil M.W. Marques JX464089 JX464051
L. theobromae CMM 4054 M. indica Brazil M.W. Marques JX464099 JX464038
L. theobromae CMM 4021 M. indica Brazil M.W. Marques JX464064 JX464047
L. venezuelensis WAC 12539 Acacia mangium Venezuela S. Mohali DQ103547 DQ103568
L. venezuelensis WAC 12540 A. mangium Venezuela S. Mohali DQ103548 DQ103569
L. viticola UCD 2604MO V. vinifera USA K. Striegler HQ288228 HQ288270
& G.M. Leavitt
L. viticola CBS 128313 V. vinifera USA R.D. Cartwright HQ288227 HQ288269
& W.D. Gubler
Lasiodiplodia sp. CMM 1472 M. indica Brazil V.S.O. Costa JX464061 JX464040
Lasiodiplodia sp. CMM 1491 M. indica Brazil V.S.O. Costa JX464079 JX464036
Lasiodiplodia sp. CMM 4010 M. indica Brazil M.W. Marques JX464078 JX464043
Lasiodiplodia sp. CMM 4011 M. indica Brazil M.W. Marques JX464074 JX464037
Lasiodiplodia sp. CMM 4013 M. indica Brazil M.W. Marques JX464066 JX464055
Lasiodiplodia sp. CMM 4014 M. indica Brazil M.W. Marques JX464098 JX464031
Lasiodiplodia sp. CMM 4015 M. indica Brazil M.W. Marques JX464063 JX464049
a
CBS Centraalbureau voor Schimmelcultures, Utrecht, Netherlands; CMW Forestry and Agricultural Biotechnology Institute, University of
Pretoria, South Africa; WAC Department of Agriculture Western Australia Plant Pathogen Collection, University of Western Australia, Perth,
Australia; CMM Culture Collection of Phytopathogenic Fungi “Prof. Maria Menezes”, Universidade Federal Rural de Pernambuco, Recife, Brazil;
STE-U Culture Collection of the Department of Plant Pathology, University of Stellenbosch, Stellenbosch, South Africa; UCD Phaff Yeast Culture
Collection, Department of Food Science and Technology, University of California, Davis, USA; BOT A. M. Ismail, Plant Pathology Research
Institute, Giza, Egypt; IRAN Culture Collection of the Iranian Research Institute of Plant Protection, Tehran, Iran. Isolate numbers in bold represents
ex-type specimens
b
Sequence numbers in bold were obtained in the present study
186 Fungal Diversity (2013) 61:181–193

CMM 4039
61/-- CMM 4047
CMM 4042
CMM 4050
CMM 4043
CMM 4033
59/0.55 CMM 1481
CMM1476
L. theobromae CBS 164.96
58/0.93 CMM 4019
CMM 4021
CMM 1517
67/-- CMM 4046
CMM 4053
CMM 4054
CMM 4052
CMM 4048
L. theobromae CBS 111530
CMM 4041
CMM 4011
CMM 1472
CMM 4013
CMM 1491
CMM 4010
CMM 4014
CMM 4015
64/1.00 L. viticola CBS 128313
99/1.00 L. viticola UCD 2604MO
L. iraniensis CBS 124710
CMM 3993
CMM 3996
CMM 3990
CMM 3998
99/1.00 CMM 1483
CMM 3995
97/0.65 CMM 4051
68/0.90 L. iraniensis IRAN1502C
L. gilanensis CBS 124704
L. gilanensis IRAN 1501C
L. missouriana UCD 2199MO
L. missouriana CBS 128311
L. plurivora STE-U 4583
99/0.99 L. plurivora CBS 120832
97/0.99 L. mahajangana CBS 124927
100/1.00 L. mahajangana CMW 27801
CMM 3986
94/1.00 L. hormozganensis IRAN 1498C
80/0.72 CMM 3987
CMM 3983
CMM 3985
CMM 1546
L. hormozganensis CBS 124709
CMM 3984
99/1.00 CMM 4008
L. pseudotheobromae CBS 116459
L. pseudotheobromae IRAN 1518C
CMM 3999
CMM 4002
74/0.90 CMM 4001
L. egyptiacae BOT-29
CMM 3981
L. egyptiacae CBS130992
CMM1485
L. citricola IRAN 1521C
L. citricola CBS 124707
97/1.00 L. parva CBS 456.78
L. parva CBS 494.78
65/0.95 L. rubropurpurea WAC 12535
100/1.00 L. rubropurpurea WAC 12536
L. venezuelensis WAC 12539
99/1.00 L. venezuelensis WAC 12540
100/1.00 L. crassispora CMW 13488
CMM 3982
100/1.00 L. crassispora CMW 14691
L. margaritacea CBS 122519
99/1.00 L. margaritacea CBS 122065
100/1.00 L. gonubiensis CBS 115812
L. gonubiensis CMW 14078
D. seriata CBS 112555
D. mutila CBS 112553
10
 Fungal Diversity (2013) 61:181–193
ƒFig. 1 One of 4 most parsimonious trees (length = 327; CI = 0.758; RI =
0.918; HI = 0.242) obtained from combined ITS and EF1-α sequence
data. Maximum parsimony bootstrap support values from 1,000 replica-
tions and Bayesian posterior probability scores are shown at the nodes.
Ex-type isolates are in bold
Morphological characterization
The isolates that were identified in the phylogenetic analysis
were used to study colony morphology and conidial char-
acteristics. The color and aerial hyphal growth from isolates
were recorded during 15 days of growth on PDA 2 % at
25 °C in the dark. Conidial characteristics were observed
after placing cultures on 2 % WA containing autoclaved pine
needles and incubation under near-ultraviolet light, as previ-
ously described. Conidia and other structures were mounted in
100 % lactic acid and digital images recorded with a Leica
DFC320 camera on a Leica DMR HC microscope fitted with
Nomarski differential interference contrast optics (Leica
Microsystems Imaging Solutions Ltd., Cambridge, UK). The
length and width of 50 conidia per isolate were measured with
the Leica IM500 measurement module. Mean and standard
errors of the conidial measurements, including mean
length to width ratio (L/W) of the conidial measurements were
calculated.
Isolates were also used to determine the effect of tempera-
ture on colony growth of different species. A 3-mm-diameter
mycelial plug from the growing margin of a 3-day-old colony
was placed in the center of a 90-mm-diameter 2 % PDA plate,
and four replicates of each isolate were incubated at temper-
atures ranging from 5 °C to 35 °C in 5 °C intervals in the dark.
After a 2-days incubation period, the colony diameter (mm)
was measured in two perpendicular directions. The experi-
ment was done twice. Colony diameters were plotted against
temperature and a curve was fitted by a cubic polynomial
regression (y=a+bx+cx2 +dx3). Optimal temperature was es-
timated from the regression equation and numeric summary
with TableCurve™ 2D v. 5.01 (SYSTAT Software Inc.,
Chicago, USA). Optimum temperature was defined as the
temperature that produced the maximum mycelial growth rate.
The colony diameter data at 30 °C were used to calculate the
mycelial growth rate (mm/day). One-way analyses of variance
(ANOVA) were conducted with data obtained from optimum
temperature and mycelial growth rate experiments, and means
were compared by Fisher’s least significant difference (LSD)
test at the 5 % significance level using STATISTIX v. 9.0
(Analytical Software, Tallahassee, USA).
Pathogenicity and virulence in fruits
The isolates used in the morphological characterization were
selected for this test. Mango fruits (cv. Tommy Atkins) at
stage three of maturation (Assis 2004), which were not
treated with fungicides, were washed in running water,
187
surface disinfested in 70 % ethanol for 1 min and 1 %
NaOCl for 5 min, then rinsed in sterile distilled water.
After drying, the fruits were placed on plastic trays, on the
base of each were four layers of paper towels wetted with
distilled water to increase humidity. Each fruit was put on a
sterilized Petri plate to avoid direct contact with water. Since
non-wounded treatment caused no lesions of Lasiodiplodia
(non published data), each fruit was wounded at the medium
region by pushing the tip of four sterile pins through the
surface of the skin to a depth of 3 mm. A mycelial plug
(5 mm in diameter) removed from the margin of a 5-day-old
PDA culture grown at 28 °C in the dark of each isolate was
immediately placed on the wound. A non-colonized agar
plug was used for the control. The trays were enclosed in
plastic bags and incubated at 25 °C in the dark. The plastic
bags and paper towels were removed after 48 h and the fruits
were kept at the same temperature. Isolates were considered
pathogenic when the lesioned area advanced beyond the 5-mm
diameter initial injury. The virulence of the isolates was evalu-
ated by measurement of the lesion length at 72 h after inocu-
lation in two perpendicular directions on each fruit. The
experiment was arranged in a completely randomized design
with six replicates per treatment (isolate) and one fruit per
replicate. The experiment was conducted twice. Differences
in virulence caused by Lasiodiplodia species were determined
by one-way ANOVA and means were compared by LSD test at
the 5 % significance level using STATISTIX.
Results
DNA sequencing and phylogenetic analyses
A total of 120 isolates of Lasiodiplodia spp. were obtained
from mango stems and fruits. From this total, seven species
of Lasiodiplodia were identified based on phylogenetic
analysis of the partial translation elongation factor 1α
(EF1-α) gene: L. crassispora Burgess & Barber, L. egyptiacae,
L. hormozganensis, L. iraniensis, L. pseudotheobromae, L.
theobromae and Lasiodiplodia sp.. To confirm the identity of
the isolates, the internal transcribed spacer (ITS) sequence was
obtained for 44 isolates representing each putative species.
PCR fragments for the ITS were approximately 580 bp in size,
while those for EF1-α was 450 bp. A partition homogeneity
test showed no significant difference between the data from the
different gene regions, indicating that they could be combined
in a single dataset (P=0.08). The combined data set consisted
of 78 taxa and 2 outgroup species. The alignment contained
738 characters including coded alignment gaps. Of these char-
acters 548 were constant, 43 were variable and parsimony
uninformative and 147 were parsimony-informative. Heuristic
search generated four equally parsimonious trees with (TL =
327; CI = 0.758; RI = 0.918; HI = 0.242). The phylogenetic
188
analyses of Maximum-parsimony and Bayesian methods pro-
duced nearly identical topologies (Bayesian tree not shown).
Sequences of ex-type isolates of various Lasiodiplodia species
from GenBank were included in the analysis together with
isolates obtained in this study (Table 1). The combined dataset
resulted in 17 well supported clades. Each clade corresponds
to previously described species. The isolates from this study
grouped in seven clades. The majority of isolates (17 isolates)
clustered together in a large clade containing the species L.
theobromae (CBS 164.96; CBS111530). These isolates
subdivided into five sub-clades, with low support. The second
group with seven isolates formed a sub-clade that clustered
together with L. viticola, with low bootstrap support (64 %)
but high Bayesian support (100 %). Seven isolates that clus-
tered with L. iraniensis presented a well-supported clade (MP /
MB: 99/1.00), subdivided into two other sub-clades. Six iso-
lates clustered together with L. hormozganensis (MP/MB:
80/0.72). Four isolates showed sequences nearly identical to
the ex-type isolate of L. pseudotheobromae strongly supported
by bootstrap values (MP/MB: 99/1.00). Two Isolates grouped
with a species recently described from mango, L. egyptiacae
(MP/MB: 74/0.90) and one isolate resided in a clade with the
ex-type isolate of L. crassipora strongly supported by a boot-
strap value of 100 % (Fig. 1).
Lasiodiplodia theobromae was the predominant species
isolated (44 %) followed by Lasiodiplodia sp. (18 %), L.
iraniensis (17 %), L. pseudotheobromae (13 %), L.
hormozganensis (5 %), L. egyptiacae (2 %) and L. crassispora
(1 %). The distribution of Lasiodiplodia species differed be-
tween Assú Valley and São FranciscoValley. Lasiodiplodia
theobromae was the predominant species in the two regions.
L. pseudotheobromae, L. iraniensis, L. hormozganensis and
Lasiodiplodia sp. were also found in both regions, while L.
crassipora and L. egyptiacae were found only in São
Francisco Valley (Fig. 2).
Morphology and cultural characteristics
The isolates that were identified in the phylogenetic analysis
using the combined data set were used to study colony
morphology and conidial characteristics. Anamorphic struc-
tures formed on the pine needles on WA within 2–4 week.
No sexual (teleomorph) structures were observed during this
study. All species showed morphological features typical of
the genus, namely slowly maturing conidia with thick walls
and longitudinal striations resulting from melanin deposi-
tion on the inner surface of the wall (Punithalingam 1976,
1980). All isolates on PDA grew rapidly, covering the entire
surface of the Petri dishes within 4 days. The aerial myce-
lium was initially white, turning dark greenish-grey or
greyish after 4–5 days at 25 °C in the dark. There were
differences in conidial dimensions between the
Lasiodiplodia spp. (Table 2). The conidia produced by
Fungal Diversity (2013) 61:181–193
species obtained in this study were similar to conidial di-
mensions of Lasiodiplodia species previously described in
the literature, although in all cases they were slightly larger
(Table 2).
There were significant differences (P≤0.05) in growth
rate among the Lasiodiplodia species and differences in the
optimum temperature for mycelial growth. The optimum
temperature for growth of L. iraniensis (28.3 °C) was sig-
nificantly lower than that of L. hormozganensis (31.1 °C),
Lasiodiplodia sp. (29.9 °C) and L. theobromae (29.9 °C).
The other species (L. crassispora, L. egyptiacae and L.
pseudotheobromae) presented intermediate values of opti-
mum temperature for growth, without differing of observed
extremes. The mycelial growth rates of L. hormozganensis
and Lasiodiplodia sp. (49.1 mm/day) were significantly
higher than L. crassispora, L. iraniensis, L.
pseudotheobromae and L. theobromae, which varied from
35.9 to 41.4 mm/day. Only L. egyptiacae (41.9 mm/day) did
not differ significantly from these extremes (Table 3).
Pathogenicity and virulence in fruits
All isolates of Lasiodiplodia were pathogenic to mango
fruits. Observed symptoms on the fruit surface were dark
brown necrotic lesions with roughly circular shape around
the inoculation sites. There were significant (P≤0.05) differ-
ences in virulence among the species, wherein Lasiodiplodia
sp. and L. hormozganensis were most virulent, causing the
largest lesions (33.8 mm and 33.6 mm, respectively). The less
virulent species were L. iraniensis, L. pseudotheobromae, L.
crassispora and L. egyptiacae, with lesions varying from 22.5
to 17.2 mm. The lesion length induced by L. theobromae
differs from these extremes, and represented intermediate
virulence (Fig. 3).
Discussion
Seven species of Lasiodiplodia associated with dieback and
stem-end rot of mango in Brazilian Northeast were identi-
fied in the present study: L. crassispora, L. egyptiacae, L.
hormozganensis, L. iraniensis, L. pseudotheobromae,
Lasiodiplodia sp. and L. theobromae. Currently, several
Lasiodiplodia species have been reported in mango world-
wide (Jacobs 2002; Slippers et al. 2005; Abdollahzadeh et
al. 2010; Costa et al. 2010; Sakalidis et al. 2011a; Ismail et
al. 2012).
In this study, L. theobromae was the most frequently
isolated species with 48 % and 42 % of all the isolates in
the São Francisco Valley and Assú Valley, respectively,
indicating it is the most widespread Lasiodiplodia species
in Northeastern of the Brazil. This species is known for its
cosmopolitan distribution and its wide range of hosts
Fungal Diversity (2013) 61:181–193 189

Fig. 2 Frequency (%) of Assú Valley (n = 72 )

Lasiodiplodia species
associated with dieback and L. hormozganensis
stem-end rot of mango in Assú 3%
Valley and São Francisco
L. iraniensis
Valley, Northeastern Brazil
22%

L. theobromae
42%

L. pseudotheobromae
14%

Lasiodiplodia sp.
19%

São Francisco Valley (n = 48)

L. crassispora L. egyptiacae
2% 4%
L. hormozganensis
8%

L. iraniensis
8%
L. theobromae
48%

L. pseudotheobromae
13%

Lasiodiplodia sp.
17%

Table 2 Comparison of conidial

dimensions of Lasiodiplodia Species Conidial size (μm) L/W ratio References
species examined in this study
and previous studies Lasiodiplodia crassispora 26.61–28.61×16.34–18.84 1.6 Present study
27–30×14–17 1.8 Burgess et al. 2006
L. egyptiacae 20.69–22.96×10.86–13.14 1.8 Present study
20–24×11–13 1.8 Ismail et al. 2012
L. hormozganensis 20.62–22.89×11.8–13.09 1.8 Present study
19.6–23.4×11.7–13.3 1.7 Abdollahzadeh et al. 2010
L. iraniensis 22.51–26.09×12.75–14.97 1.7 Present study
18.7–23.0×12.1–13.9 1.6 Abdollahzadeh et al. 2010
L. pseudotheobromae 25.07–28.23×13.4–15.6 1.8 Present study
25.5–30.5×14.8–17.2 1.7 Alves et al. 2008
L. theobromae 24.49–27.49×13.3–14.79 1.8 Present study
23.6–28.8×13–15.4 1.9 Alves et al. 2008
Lasiodiplodia sp. 25.1–27.3×14.1–15.0 1.8 Present study
190 Fungal Diversity (2013) 61:181–193

Table 3 Optimum temperature

for mycelial growth and myceli- Species n Optimum temperature (°C) ± SE Mycelial growth rate (mm/day) ± SE
al growth rate at 30 °C of
Lasiodiplodia species associated Lasiodiplodia 1 29.0±1.27 ab 35.9±4.20 b
with dieback and stem-end rot of crassispora
mango in Northeastern Brazil L. egyptiacae 1 29.3±1.27 ab 41.9±4.20 ab
L. hormozganensis 5 31.1±0.57 a 49.1±1.88 a
L. iraniensis 7 28.2±0.48 b 41.4±1.67 b
Mean ± standard error. Values
within columns followed by the L. pseudotheobromae 4 29.8±0.64 ab 36.4±2.30 b
same letter do not differ signifi- L. theobromae 20 29.9±0.28 a 41.3±0.96 b
cantly according to Fisher’s LSD Lasiodiplodia sp. 6 29.9±0.52 a 49.0±1.71 a
test (P≤0.05)

(Punithalingam 1980; Burgess et al. 2006), and is reported
commonly as an important pathogen associated with disease
of mango trees in tropical and subtropical regions (Ploetz et
al. 1996; Jacobs 2002; Khanzada et al. 2004; Abdollahzadeh
et al. 2010; Costa et al. 2010; Sakalidis et al. 2011a; Ismail
et al. 2012). The phylogenetic analysis of the L. theobromae
clade reveals low support in the analyses of Maximum-
parsimony and Bayesian method, indicating a great intra-
specific diversity. This intraspecific variation was observed
also in other studies with this species (Pavlic et al. 2004;
Begoude et al. 2010; Ismail et al. 2012). During the past
150 years this fungus has had many names and has been
treated as many different species. This trend ended with the
monograph of Punithalingam (1976) which reduced most
species to synonymy with L. theobromae. However, lately
several species have been described in the L. theobromae
complex, mostly because of the increase in the application of
DNA sequence data, but also because of the increased sam-
pling of relatively unexplored areas, including Venezuela
(Burgess et al. 2006), Australia (Pavlic et al. 2008), Iran
(Abdollahzadeh et al. 2010), Egypt (Ismail et al. 2012) and
Brazil (this study).
Lasiodiplodia sp. was the second most prevalent species
in the São Francisco Valley with 17 % and the third in the
Assú Valley with 19 % of all isolate. Considering the phy-
logenetic data, the mango isolates that grouped together
with L. viticola formed a subgroup, strongly supported in
the Bayesian analysis (1.00) but with only moderate support
in MP (64 %). Since these isolates have sequences in the
EF-1α that were identical to L. viticola additional analysis is
needed to clarify this subgroup. Such a study is currently in
progress and will be addressed in a future paper.
The species L. hormozganensis and L. iraniensis were
recently described in Iran associated with several hosts,
including mango (Abdollahzadeh et al. 2010). In this work,
L. iraniensis was the third most prevalent species, and L.
hormozganensis together with Lasiodiplodia sp. were the
most virulent species in mango fruit. A similar result was
found by Sakalidis et al. (2011a), where L. hormozganensis
isolates produced the largest lesions in mango branches.
In our study L. crassispora was the least abundant spe-
cies with only one isolate. This species has notably thicker
cell walls in the immature spores and the striations appear to
be wider and the cytoplasm wart-like in appearance, which

Fig. 3 Mean lesion lengths 40

(mm) caused by Lasiodiplodia
species associated with dieback a a
and stem-end rot of mango in
30 b
Northeastern Brazil, 72 h after
Lesion length (mm)

inoculation with mycelium

colonized agar plugs onto c
c
wounded fruits of Tommy 20 c c
Atkins cultivar. Bars above
columns are the standard error
of the mean. Columns with
same letter do not differ 10
significantly according to
Fisher’s LSD test (P≤0.05)
0
ora cae sis nsi
s ae ae sp.
ss isp p tia anen nie ob rom obrom lodia
cra egy ozg ai
ra
the the dip
ia dia ho
rm odi do ia sio
ip lod di plo ia ipl seu lod La
iod sio lod od p ip
s La dip asi dia s iod
La sio
L plo La
La si odi
La

Species
Fungal Diversity (2013) 61:181–193
is different from all other species (Burgess et al. 2006). At
present the host range is considered to be limited and this
species has been reported only on Santalum album L.,
Eucalyptus urophylla S. T. Blake., V. vinifera, Corymbia
sp. Hook., and Syzygium spp. Gaertn. (Sakalidis et al.
2011b; Perez et al. 2010; Burgess et al. 2006; Úrbez-
Torres et al. 2010). Information about pathogenicity of this
species is scarce. Lasiodiplodia crassispora was isolated as
a stem endophyte from Adansonia gregorii F. Muell. and the
pathogenicity trials demonstrated that it is pathogenic
(Sakalidis et al. 2011b). It is likely that this fungus also
occurs as an endophyte on mango but with the ability to
cause disease. This study represents the first report of this
species associated with mango in the world, with proven
pathogenicity but low virulence compared to other species
found in Northeastern Brazil.
Lasiodplodia pseudotheobromae differs from L.
theobromae in its bigger conidia that are more ellipsoid and
do not taper as strongly towards the base. This species was
described from Acacia, Citrus, Coffea, Gmelina and Rosa
species (Alves et al. 2008). Worldwide, L. pseudotheobromae
has been reported on numerous hosts, but in Brazil it has been
reported only on Vitis spp. (Correia et al. 2012). The present
work represents the first report on mango in Brazil. Regarding
the pathogenicity, Sakalidis et al. (2011a) reports L.
pseudotheobromae as the most virulent species in mango
fruits in Australia. In another work, a pathogenicity test on
mango tree saplings revealed that some isolates of L.
pseudotheobromae were more virulent than L. theobromae
(Ismail et al. 2012). In Terminalia catappa L., this species
was also considered to be the most aggressive (Begoude et al.
2010).
However, in the present work, compared with other spe-
cies, L. pseudotheobromae did not show much virulence in
mango fruit. Pathogenicity data in many crops is still scarce,
as for a long time this species was identified as L. theobromae.
However, recent works suggest that, like L. theobromae, L.
pseudotheobromae also has worldwide distribution and a
wide range of hosts (Abdollahzadeh et al. 2010; Begoude et
al. 2010; Sakalidis et al. 2011a; Ismail et al 2012).
Another species found in this study was L. egyptiacae,
with only two isolates. This species was recently described
on mango in Egypt. This is the first report of L. egyptiacae
outside Egypt. Pathogenicity tests done by Ismail et al.
(2012) showed that this species is less virulent on mango
fruit than other Lasiodiplodia species. In this study, L.
egyptiacae showed low levels of virulence on mango fruit.
More sampling is necessary to understand the host range,
distribution and variability of this species.
Regarding cultural characteristics, the optimum tempera-
ture for mycelial growth for the Lasiodiplodia species varied
between 28 and 31 °C. In addition, all the species in this
study grew at a temperature of 10 °C. This growth at low
191
temperature corroborates the work of Abdollahzadeh et al.
(2010) and is in contrast to other studies that show only L.
pseudotheobromae as capable of growing at this tempera-
ture (Alves et al. 2008; Ismail et al. 2012). As can be
observed, cultural characteristics vary greatly amongst iso-
lates of the same species and are therefore, of limited value
in the determination of species.
All the species found in Northeastern Brazil have potential
to cause disease to mango, but L. hormozganensis and
Lasiodiplodia sp. were the most virulent species. Information
about these species is scarce because of its recent descriptions.
Studies are needed on the epidemiology and impact on mango
production together with information referring to ecology, dis-
tribution and host range of all species of Lasiodiplodia found in
this study.
The correct identification of plant pathogenic fungi is of
utmost importance for quarantine and control measures, once
the control measures are specific for each pathogen. This
study found seven species of Lasiodiplodia associated with
dieback and stem end rot in mango tree. Future research on the
distribution and epidemiology are important and could aid in
finding effective management strategies of these pathogens
that represent a threat to the mango crop in Brazil.
Acknowledgments We are grateful to Sequencing Platform
LABCEN/CCB in the Universidade Federal de Pernambuco for use
of its facilities. This work was financed by Conselho Nacional de
Desenvolvimento Científico e Tecnológico (CNPq 141275/2009-0)
and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
(CAPES/BEX 0245/12-7). M. P. S. Câmara, Marcos A. Morais Junior
and S. J. Michereff also acknowledge the CNPq research fellowship. A.
J.L. Phillips thanks Fundação para a Ciência e a Tecnologia (Portugal) for
financial support through grant PEst-OE/BIA/UI0457/2011
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