A Review of Botryosphaeriales in Venezuela with Special Reference to Woody Plants

Abstract

The Botryosphaeriales order are best known for the diseases they cause in woody plants, as primary pathogens or latent pathogens residing in the woody tissue of asymptomatic hosts. In the first instance, Botryosphaeriales species have been identified in Venezuela using morphological de-scriptions in the 80's and 90's, and later, the mid-2000s using molecular techniques. The mor-phological descriptions of the asexual morphs were initially used for the identification of Botry-osphaeriales genera and species. Lasiodiplodia spp., (as L. theobromae) was the most isolated fungus in Venezuela within the Botryosphaeriales and has been found in more than 50% of the hosts in native and non-native plants, followed by Diplodia, Dothiorella, Fusicoccum, Lasiodiplodia, Micro-diplodia, Macrophomina, Neofusicoccum, Sphaeropsis, and Botryosphaeria, considered all of them cosmopolitan group. With molecular studies, that included DNA sequence data from multiple genes, such as the internal transcribed spacer of rDNA (ITS), translation elongation factor-1α (tef1), and β-tubulin (btub) used on the fungi isolated from woody plants, mainly trees or forest species, resulted in the presence of two families within the Botryosphaeriales order for Venezuela. Botryosphaeriaceae family with the genera: Botryosphaeria, Cophinforma, Diplodia, Lasiodiplodia and Neofusicoccum, and the Pseudofusicoccumaceae family that includes the genus Pseudofusicoccum. In Botryosphaeriaceae family was again the Lasiodiplodia genus the most predominant in most hosts, and the specie L. theobromae the most isolated in native and non-native plants; Botryosphaeria dothidea, Cophinforma atrovirens, Diplodia scrobiculata (syn. Diplodia guayanensis), Lasiodiplodia brasi-liensis, L. crassispora, L. pseudotheobromae, Neofusicoccum arbuti (syn. N. andinum), N. parvum, and N. ribis are cosmopolitan species, and they were isolated from native and non-native plants; while Pseudofusicoccum stromaticum was found in plantations non-native of Acacia mangium, E. urophylla x E. grandis, Eucalyptus urophylla, and reported exclusively in South America; Lasiodiplodia venezue-lensis has only been reported in Venezuela, from native and non-native plants. The presence, distribution, diversity, and symptoms of these fungi, mainly of the new genus, new species, and reports found in Venezuela and other parts of the world, were also reviewed.

Full text

Review Not peer-reviewed version
A Review of
Botryosphaeriales
in
Venezuela with Special Reference
to Woody Plants
Sari Ramon Mohali-Castillo *
Posted Date: 26 June 2023
doi: 10.20944/preprints202306.1753.v1
Keywords: Botryosphaeriaceae; DNA sequence; Forest; Pseudofusicoccumaceae; Fungal Taxonomy
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Review
A Review of Botryosphaeriales in Venezuela with
Special Reference to Woody Plants
Sari Ramon Mohali-Castillo
Centro de Estudios Forestales y Ambientales de Postgrado (CEFAP), Laboratorio de Fitopatología,
Facultad de Ciencias Forestales y Ambientales, Universidad de Los Andes, Mérida 5101, Venezuela;
sarirmohali@gmail.com
Abstract: The Botryosphaeriales order are best known for the diseases they cause in woody plants, as
primary pathogens or latent pathogens residing in the woody tissue of asymptomatic hosts. In the
first instance, Botryosphaeriales species have been identified in Venezuela using morphological
descriptions in the 80's and 90's, and later, the mid-2000s using molecular techniques. The
morphological descriptions of the asexual morphs were initially used for the identification of
Botryosphaeriales genera and species. Lasiodiplodia spp., (as L. theobromae) was the most isolated
fungus in Venezuela within the Botryosphaeriales and has been found in more than 50% of the hosts
in native and non-native plants, followed by Diplodia, Dothiorella, Fusicoccum, Lasiodiplodia,
Microdiplodia, Macrophomina, Neofusicoccum, Sphaeropsis, and Botryosphaeria, considered all of them
cosmopolitan group. With molecular studies, that included DNA sequence data from multiple
genes, such as the internal transcribed spacer of rDNA (ITS), translation elongation factor-1α (tef1),
and β-tubulin (btub) used on the fungi isolated from woody plants, mainly trees or forest species,
resulted in the presence of two families within the Botryosphaeriales order for Venezuela.
Botryosphaeriaceae family with the genera: Botryosphaeria, Cophinforma, Diplodia, Lasiodiplodia and
Neofusicoccum, and the Pseudofusicoccumaceae family that includes the genus Pseudofusicoccum. In
Botryosphaeriaceae family was again the Lasiodiplodia genus the most predominant in most hosts, and
the specie L. theobromae the most isolated in native and non-native plants; Botryosphaeria dothidea,
Cophinforma atrovirens, Diplodia scrobiculata (syn. Diplodia guayanensis), Lasiodiplodia brasiliensis, L.
crassispora, L. pseudotheobromae, Neofusicoccum arbuti (syn. N. andinum), N. parvum, and N. ribis are
cosmopolitan species, and they were isolated from native and non-native plants; while
Pseudofusicoccum stromaticum was found in plantations non-native of Acacia mangium, E. urophylla x
E. grandis, Eucalyptus urophylla, and reported exclusively in South America; Lasiodiplodia
venezuelensis has only been reported in Venezuela, from native and non-native plants. The presence,
distribution, diversity, and symptoms of these fungi, mainly of the new genus, new species, and
reports found in Venezuela and other parts of the world, were also reviewed.
Keywords: Botryosphaeriaceae; DNA sequence; Forest; Pseudofusicoccumaceae; Fungal Taxonomy
1. Introduction
Forest ecosystems are a natural resource of great importance to humanity, since many people
depend on them for their survival, in addition to other benefits such as human and environmental
health, carbon sequestration, and genetic resources that underpin important wood and wood
products-based industries [1]. At present, the health of forests, both natural and managed, is more
heavily threatened, and these threats arise from direct and indirect anthropogenic influences on
fungal pathogens, and insect pests [1,2]. Plantations in the tropics (planted forests of a single species)
are usually of non-native species, such as the genera of Pinus, Eucalyptus, and Acacia, the main forest
species planted in Venezuela.
Non-native trees in plantations are in part successful because they have been separated from
their natural enemies, but when plantation trees are reunited with their coevolved pests, which may
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2
be introduced accidentally, or when they encounter novel pests to which they have no resistance,
substantial damage or loss can ensue [3]. The longer non-native trees are planted in an area, the more
threatened they become by native pests. Where the trees are of native species, they can be vulnerable
to introduced pests. But the relative species uniformity of monoculture stands in intensively managed
native plantation forests can make them especially susceptible to the many native pests occurring in
the surrounding natural forests [4,5].
An example of an epidemic of native pathogens moving onto an exotic species is provided by
the shoot pathogen Gremmeniella abietina (Lagerberg) Morelet, endemic and not particularly
damaging on Scots pine in Sweden but causing widespread destruction of Swedish plantations of the
extensively planted exotic lodgepole pine [6].
The Botryosphaeriales contains numerous fungal species that occur as saprophytes, parasites, or
endophytes on a diverse range of plant hosts [7,8], as well as opportunistic pathogens of woody
plants, especially when host plants are stressed [9]. Different species within of order Botryosphaeriales
are well known pathogens on forest trees and other woody plants associated with branch and trunk
cankers, dieback, decline and mortality, and represent a growing threat to forest ecosystems
worldwide [7,8]. An ecological and biological characteristic of the species in the Botryosphaeriales
order is the lack of host specificity able to colonize and cause disease in diverse native and introduced
plant hosts [7,10].
Different Botryosphaeriales genera can infect multiple hosts, increases the threat that they pose as
potential economic and ecological important pathogens of native and cultivated trees around the
world. Examples of inter-host exchanges of the Botryosphaeriales, and that include those amongst and
between native and non-native trees, we have Botryosphaeriales species have moved between trees in
native stands of Eucalyptus (Myrtaceae) and adjacent plantations of these trees [11], between native
waterberry trees (Syzygium cordatum; Myrtaceae) and related eucalypt plantations (Myrtaceae) [12],
from Pinus resinosa windbreaks to pine nurseries [13], among various tree hosts in the Casuarinaceae,
Cupressaceae, Fabaceae, Myrtaceae, Proteaceae, Santalaceae [14], and among native Terminalia spp.
(Combretaceae) and between these trees and Theobroma cacao (Malvaceae) [15]. The epidemiology of
Botryosphaeriales species is complex. These fungi can be monocyclic or oligocyclic pathogens that
cause polyetic epidemics. As monocyclic pathogens, they complete one disease cycle, or even part of
one, in one season. Depending on the weather conditions, these species can be oligocyclic pathogens,
i.e., polycyclic pathogens with a few (two or three) disease cycles per season [16]. In Venezuela, the
Botryosphaeriales species are polycyclic since there are no marked seasons in the tropics as in the
temperate regions. In the tropic, high temperatures and humidity are present almost all year round,
therefore, these fungi will present several disease cycles and produce constant inoculum or spores
throughout the year.
Species identification in Botryosphaeriales has been largely based on the asexual morphs due to
the lack of diversity among sexual morph features within this order and the difficulty of finding the
sexual morphs in nature or obtaining them under laboratory conditions [17,18]. Different species
within the same genera of Botryosphaeriales frequently possess overlapping morphological features
[19] that can cause confusion in their accurate identification. In recent decades, several researchers
began using identification techniques based on DNA sequencing and phylogenetic analyses to
resolve the taxonomic problems associated with overlapping morphological characteristics among
the species asexual morphs within Botryosphaeriales genera [17,18,20–22]. The phylogenetic analyses
of DNA sequence data have significantly impacted all aspects of the systematics and taxonomy of the
Botryosphaeriales, including a redefinition of families and genera, identification of new species, cryptic
species, and more recently hybrids [23]. Crous et al. [24] defined all genera in the Botryosphaeriales
based predominantly on phylogenetic inference and characteristics of their asexual morphs, and
without morphological evidence of a sexual morph. In various cases, genera were thus established in
the family based on asexual names.
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3
The Botryosphaeriales order has undergone changes in its systematics, mainly at the family level.
A total of nine families have been included in the last 5 years within the Botryosphaeriales order, based
on phylogenetic, morphological, and ecological differences [23]. These families are: Aplosporellaceae
Slippers et al. 2013 [25], Botryosphaeriaceae Theiss. & Syd., 1918 [24], Endomelanopsisaceae TaoYang &
Crous, 2016 [26], Melanopsaceae Phillips et al. 2013 [25], Phyllostictaceae Fr., 1849 [27], Planistromellaceae
M.E. Barr, 1996 [28], Pseudofusicoccumaceae Tao Yang & Crous, 2016 [26], Saccharataceae Slippers et al.
2013 [25] and Septorioideaceae Wyka & Broders, 2016 [29], being the Botryosphaeriaceae family with the
largest number of genera within it.
The purpose of this review is to update all relevant information on morphological descriptions
and DNA sequencing data on the Botryoshaeriales fungi that produce different diseases on cultivated
and wild plants, as well as their distribution and diversity on woody plants in Venezuela.
2. Genera and species of Botryosphaeriales identified with morphological descriptions in
Venezuela
Few general morphological features of within Botryosphaeriales species have been reported in
agricultural crops, forest plantations, and natural forests in Venezuela, where the taxonomic
identification and associated reports have been initially based on morphological descriptions of the
asexual morph. Such morphological descriptions are frequently based on 1) conidial features, such
as septation, presence/absence of pigmentation, and wall thickness, and 2) presence/absence of
conidiophores, conidiogenous cells, and paraphyses in the conidiomata [30–37].
Lasiodiplodia Ellis & Everh., species are well-known and widespread plant pathogens, occurring
mostly in tropical and subtropical regions [38]. Lasiodiplodia theobromae (Pat.) Griffon & Maubl., has
been widely reported and commonly occurs on different crops in Venezuela [39]. In a published list
of plant diseases in Venezuela [39], L. theobromae was the common fungal pathogen. This list from
Urtiaga [39] was updated using website records of fungi from 1998-2001 with specimens from the
fungal collection of the Simon Bolivar University, Caracas-Venezuela [40], together with reports from
Mohali and other authors during the 1990s through the mid-2000s (Table 1). In addition to
Lasiodiplodia, other reported genera include Diplodia Fr., Dothiorella Sacc., Botryosphaeria Ces. & De
Not., Microdiplodia Allesch., and Macrophomina Petr. [39,40]. In addition to two Neofusicoccum Crous,
Slippers & A.J.L. Phillips species isolated from M. indica in 2012 and 2016, where identified through
their morphological descriptions [41,42] (Table 1).
In Venezuela, at least eight genera of the Botryosphaeriales order within two families can be
differentiated through the asexual morph. Seven genera belong to Botryosphaeriaceae family, five with
dark-conidia when mature age: Diplodia Fr., Dothiorella Sacc., Lasiodiplodia Ellis & Everh.,
Macrophomina Petr., Sphaeropsis Sacc., and two genera with hyaline conidia: Cophinforma Doilom, J.K.
Liu & K.D. Hyde and Neofusicoccum Crous, Slippers & A.J.L. Phillips; and one genus in the
Pseudofusicoccumaceae family, Pseudofusicoccum Mohali, Slippers & M.J. Wingf., with hyaline-conidia
surrounded by a persistent mucous sheath (Table 2).
Table 1. Different genera and species within Botryosphaeriales identified by their asexual morph in
Venezuela.
Fungi Host Place Reference
Diplodia Fr. Ceiba pentandra (L.) Gaertn-old leaves Buena vista, Lara state [39]
Diplodia sp. Cassia L.- root - [40]
Diplodia ochromae Pat. Ochroma lagopus Sw. -trunk - [40]
Diplodia mutila Fr. Apud
Mont.
Pinus caribaea morelet var. hondurensis (Barr.
and Golf.)-
blue stain on wood
Chaguaramas,
Anzoátegui state
[36]
Dothiorella Sacc. Delonix regia (Bojer ex Hook) Raf. -branches El Tocuyo, Lara state [39]
Dothiorella sp. Psidium guajava L.-fruit rot Merida and Zulia states [33]
Dothiorella dothidea (=
Botryosphaeria dothidea)
Prunus pérsica (L.) Batsch- brown rot of fruits El Arenal, Merida state [31]
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F
usicoccum Corda Echinodorus berteroi (Spreng) Fassett-leaves Guanare, Portuguesa
state
[39]
Lasiodiplodia
theobromae (Pat.) Griffon
& Maubl.
Pachystachys lutea Nees-branches Barquisimeto, Lara state [39]
L. theobromae
A
nacardium occidentale L- terminal branch
death
Barquisimeto, Lara state [39]
L. theobromae
M
angifera indica L-branches and seeds La Calzada de Páez,
Barinas state
[39]
L. theobromae
A
nnona reticulata L.-old leaves Barquisimeto, Lara state [39]
L. theobromae Catharanthus roseus (L.) G. Don-Leaves Barquisimeto, Lara state [39]
L. theobromae Crescentia cujete L.- branches and leaves Wide distribution in
Venezuela
[39]
L. theobromae Citrullus lanatus (Thunb.) Matsum. & Nakai-
fruits rot and branches
La Miel, Lara state [39]
L. theobromae
J
uniperus lucayana Britton- twigs Barquisimeto, Lara state [39]
L. theobromae Curatella Americana L.-old leaves La Calzada de Páez,
Barinas state
[39]
L. theobromae Codiaeum variegatum (L.) Blume var. pictum
(Lodd.) Muell
- [39]
L. theobromae Hura crepitans L.-old leaves La Calzada de Páez,
Barinas state
[39]
L. theobromae
M
anihot esculenta Crantz-branches Urachiche, Yaracuy state [39]
Lasiodiplodia
theobromae
Duranta repens L.-branches Ureña, Táchira [39]
L. theobromae
A
rachis hypogaea L.-root Buría Londres, Lara
state
[39]
L. theobromae Phaseolus lunatus L.-branches Sabana de Parra,
Yaracuy state
[39]
L. theobromae Sansevieria trifasciata Prain.-old leaves Barquisimeto, Lara state [39]
L. theobromae Cedrela odorata L.-branches Chivacoa, Yaracuy state [39]
L. theobromae Cecropia peltata L.-branches Chivacoa, Yaracuy [39]
L. theobromae Ficus pumila L.-old leaves and galls on the
leaves
Barquisimeto, Lara state [39]
L. theobromae
M
axillaria Ruiz & Pavon-old leaves Duaca, Lara [39]
L. theobromae Passiflora edulis Sims. form flavicarpa Degener El Eneal, Lara [39]
L. theobromae Salix babylonica L.- black root rot Barquisimeto, Lara state [39]
L. theobromae Pachystachys lutea Nees-branches Barquisimeto, Lara state [39]
L. theobromae Cajanus indicus Spreng.-branches Lara state [39]
L. theobromae Duranta repens L.-branches Tachira state [39]
L. theobromae Theobroma cacao L. - [40]
L. theobromae Vinca rosea L.-leaf and branch Lara state [39]
L. theobromae Persea Americana Mill.-fruits Yaracuy state [41]
L. theobromae Citrus latifolia Tanaka-fruits Yaracuy state [41]
L. theobromae Citrus sinensis (L.) Osbeck-fruits Yaracuy state [41]
Lasiodiplodia
theobromae
C. sinensis-Lesion and Gummosis on the
b
ranches
Caño Amarillo, Tachira
state
[30]
L. theobromae Citrus aurantiifolia-Lesion and Gummosis
on the branches
Caño Amarillo, Tachira
state
[30]
L. theobromae Passiflora edulis Sims f. flavicarpa-Dieback
on the branches
South of Maracaibo
Lake,
Zulia and Merida states
[32]
L. theobromae Pinus caribaea var. hondurensis-blue stain on
wood
Uverito plantation and
Uverito sawmill,
Monagas state
[123]
L. theobromae
A
zadirachta indica
A. Juss-blue stain on wood
Cojedes state [37]
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L. theobromae Pinus oocarpa Schiede ex Schltdl Merida state [37]
L. theobromae
M
angifera indica-branches dieback Maracay (INIA-
CENIAP), Aragua state
[42]
M
icrodiplodia buddleiae
Gucevicz
Opuntia caracasana Salm.-spot leaves Humocaro Bajo, Lara
state
[39]
M
acrophomina
p
haseolina (Tassi)
Goidanich
Begonia sp.- spot on the leaf Barquisimeto, Lara state [39]
M
acrophomina
p
haseolina
Calendula officinalis L.-stem and inflorescence Barinas state [39]
M
. phaseolina Ipomoea batata (L.) Lam.-stolons at the roots Siquisique, Lara state [39]
M
. phaseolina Phaseolus vulgaris L.-stem and basal rot Moroturo, Lara state [39]
M
. phaseolina Glycine Willd. - [40]
M
. phaseolina Gossypium L. - [40]
M
. phaseolina Ipomoea L. - [40]
M
. phaseolina Nicotiana L. - [40]
M
. phaseolina Phaseolus L. - [40]
M
. phaseolina Psidium guajava L.-Fruits - [40]
M
. phaseolina Solanum melongena L. - [40]
M
. phaseolina Vigna Savi - [40]
Neofusicoccum
mangiferae (Syd. & P.
Syd.) Crous, Slippers &
A.J.L. Phillips
M
angifera indica- death of branches Maracay (INIA-
CENIAP), Aragua state
[42]
Neofusicoccum parvum
(Pennycook & Samuels)
Crous, Slippers & A.J.L.
Phillips
M
angifera indica- death of branches Maracay (INIA-
CENIAP), Aragua state
[42]
Sphaeropsis Sacc. Cecropia peltata L.-branch and trunk knots Reserva Forestal de
Ticoporo, Mirí, Barinas
state
[39]
Sphaeropsis sp. Phthirusa paniculata (Kunth) J.F.Macbr.-lea
f
Lara state [39]
Sphaeropsis palmarum
Cooke
Cocos nucifera L.-old leaves Cumanacoa, Sucre state [39]
Sphaeropsis sapinea (Fr.)
Dyko & B. Sutton
Pinus caribaea Morelet- Chlorosis in the
needles and discoloration lesions on the stem
Nirgua, Yaracuy state [35]
S. sapinea Pinus oocarpa Schiede-blue stain on wood Andes region (1600
meters
above sea level), Merida
state
[37]
Sphaeropsis sapinea Pinus caribaea var. hondurensis-Shoot blight,
dieback and canker on trunks, branches, and
roots (Plantations), and death at the tips of the
needles (seedlings in nurseries)
Uverito (Monagas state),
and Coloradito y Los
Hachos (Anzoátegui
state)
[34]
Sphaeropsis tumefaciens
Hedges
Citrus L.-gall - [40]
Botryosphaeria festucae
(Lib.) Arx & E. Müll
Zea mays L.-bract, leaf, and seed - [40]
Botryosphaeria dothidea
(Moug. ex Fr.) Ces. & De
Not
Compositae-stem Aragua state [40]
Botryosphaeria ribis
Grossenb. & Duggar
Rosa canina L.-branch Lara state [39]
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Table 2. Morphological differentiation between the Botryosphaeriaceae genera and a genus in
Pseudofusicoccumaceae both belonging to the order Botryosphaeriales found in Venezuela.
Genera Conidia Conidiomata Conidiophores Conidiogenesis
cells
Paraphyses
Cophinforma
Doilom, J.K. Liu &
K.D. Hyde
Hyaline, thin walled,
unicellular, aseptate, rarely
b
ecoming septate, mostly fusoid
to ellipsoidal. Most conidia
longer than 30 μm
Material pycnidial,
superficial,
multilocular, dark
b
rown to black,
eustromatic
Absent Enteroblastic,
hyaline, cylindrical
Absent
Diplodia Fr. Initially hyaline, aseptate, thick-
walled, becoming 1–septate only
rarely becoming 2-septate, pale
transluscent brown after
discharge from the pycnidia.
Some species the conidia
b
ecome pigmented while still
enclosed in the conidioma and
these species the conidia rarely
b
ecome septate.
Pycnidial, ostiolate,
formed in uni- or
multiloculate
stromata
When present:
hyaline,simple,
occasionally
septate, rarely
b
ranched,
cylindrical,
Holoblastic,
hyaline, cylindrical
Absent
Dothiorella Sacc. Initially hyaline, becoming dark
b
rown and one-euseptate within
the pycnidial cavity, ellipsoid to
ovoid, thick-walled, externally
smooth, or striate, internally
verruculose
Stromatic, ostiolate,
individual or in
loose clusters of up
to 10 conidiomata,
immersed, breaking
through the bark
when mature.
Absent Holoblastic,
hyaline, smooth-
walled, cylindrical
Absent
Lasiodiplodia Ellis
& Everh.
Hyaline when young, later
b
ecoming medianly 1-euseptate,
dark brown with longitudinal
striations, thick-walled, oblong
to ellipsoid, straight, broadly
rounded at the apex, base
truncate
Stromatic, immersed
or superficial,
separate, or
aggregated and
confluent, globose,
dark brown, uni- or
multilocular
Often reduced to
conidiogenous
cells, if present
hyaline, simple,
sometimes
septate, rarely
b
ranched
Holoblastic,
hyaline, smooth,
cylindrical to
subobpyriform,
discrete,
determinate, or
indeterminate
Present
M
acrophomina
Petr.
Aseptate, obtuse at each end,
straight, cylindrical to fusiform,
thin-walled, smooth, guttulate,
enclosed in mucoid sheath.
Immature conidia hyaline,
mature conidia becoming
medium to dark brown.
Pycnidial, stromatic,
dark brown to black,
solitary, or
gregarious
Reduced to
conidiogenous
cells
Enteroblastic,
phialidic,
determinate,
discrete,
lageniform to
doliiform, hyaline,
smooth, with wide
aperture and
minute collarette,
formed from the
inner cells of the
pycnidial wall,
enclosed in mucoid
sheath
Absent
N
eofusicoccum
Crous, Slippers &
A.J.L. Phillips
Mostly fusoid to ellipsoidal,
hyaline.
Stromatic, pycnidial,
solitary or
aggregated, often
occurring within the
same stroma as the
ascomata, walls
composed of dark
b
rown
When present
hyaline,
cylindrical,
b
ranched at the
b
ase, smooth, 0–1
septate
Enteroblastic,
integrated, hyaline,
smooth, cylindrical
Absent
Pseudofusicoccuma
ceae Tao Yang &
Crous,
Pseudofusicoccum
Mohali, Slippers &
M.J. Wingf.
Conidia are more cylindrical
than in Noefusicoccum species
and surrounded by a persistent
mucous sheath, hyaline.
Large, superficial,
unilocular or
multilocular locule
Reduced to
conidiogenous
cells
Holoblastic,
smooth,
cylindrical to
subcylindrical,
hyaline
Present or
absent
Notes. Macrophomina has sclerotia black, smooth, hard, formed of dark brown, thick-walled cells [8].
Neofusicoccum was introduced by Crous et al. [24] for species that are morphologically similar to Fusicoccum,
but phylogenetically distinct from them, and thus could no longer be accommodated in that genus. The presence
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of paraphyses in Sphaeropsis differentiates this genus from Diplodia, which does not have pycnidial paraphyses
and striate conidia of Lasiodiplodia differentiate it from Sphaeropsis, which has smooth-walled conidia [8]. Also,
the absence of septa (aseptate) in mature conidia of Sphaeropsis separates it from the genus Diplodia which is
characterized by conidia septate [8].
3. DNA Sequence-based identification of Botryosphaeriales in Venezuela
In the early 2000s, publications began appearing for identifying species within the
Botryosphaeriales using DNA sequence data. DNA-based approaches helped to solve the problem of
identifying species with overlapping morphology, and the combination of morphological
characteristics and DNA sequence data became a powerful tool to separate and identify new genera
and species [18,21]. However, single-gene genealogies were not always useful for resolving closely
related or cryptic species of the Botryosphaeriales; moreover, comparisons of DNA sequence data from
multiple genes or different gene regions were exceptionally useful for discriminating among several
closely related species [19,43,44].
From mid-2000s through 2022, different species and genera within the Botryosphaeriales in
Venezuela were isolated. Analysis of the morphological characteristics and DNA sequences were
used for identifying a new genus and four new species. Multiple DNA loci were used to identify
these Botryosphaeriales isolates from Venezuela including, the internal transcribed spacer of rDNA
(ITS), translation elongation factor-1α (tef1), and β-tubulin (btub) (Table 3).
Table 3. Genera and species within Botryosphaeriales order identified with DNA sequence data in
Venezuela.
Species Accession
number
Host Locality GenBank accession numbe
r
References
ITS ITS BTUB
Botryosphaeria
dothidea
CMW8000
Ex-type
Prunus sp. Switzerland AY236949 AY236898 AY236927 [43]
B. dothidea CMW13390=
CBS117919
Eucalyptus
urophylla
x E.grandis
CR and WCR EF118044 - - [53]
Cophinforma
atrovirens
CMW13416=
CBS117444
E.urophylla
x E.grandis
CR and WCR EF118050 GU134938 - [53]
C. atrovirens CMW13425=
CBS117445
A
cacia
mangium
CR and WCR EF118046 GU134939 - [53]
C. atrovirens CSM 72 Theobroma
cacao
AR MF436087 MF436099 MF436111 [54]
C. atrovirens MFLUCC
11-0425
Ex-type
Eucalyptus sp Thailand
J
X646800
J
X646865
J
X646848 [56]
Diplodia
scrobiculata (syn.
D. guayanensis)
CBS129749
A
cacia
mangium
NER
J
X545106
J
X545126
J
X545146 [59]
D. scrobiculata
(syn. D.
g
uayanensis)
CBS129750
A
cacia
mangium
NER
J
X545108
J
X545128
J
X545148 [59]
D. scrobiculata CMW189 =
CBS 118110
Ex-type
Pinus
banksiana
United States KF766160 KF766399 AY624258 [60,127]
Lasiodiplodia
brasiliense
CMM4015
Ex-type
M
angifera
indica
Brazil
J
X464063
J
X464049 - [69]
L. brasiliensis CSM 11 Theobroma
cacao
AR MF436018 MF436006 MF435998 [54]
L. brasiliensis CSM 15 Theobroma
cacao
AR MF436019 MF436007 MF435997 [54]
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L. crassispora WAC 12533=
CBS118741
Ex-type
Santalum
album
Australia DQ103550 EU673303 KU887506 [72,80]
L. crassispora CMW 13488 Eucalyptus
urophylla
CR and WCR DQ103552 DQ103559 KU887507 [72,80]
L.
p
seudotheobromae
CBS 129752
A
cacia
mangium
NER
J
X545091
J
X545111
J
X545131 [90]
L.
p
seudotheobromae
CBS116459
Ex-type
Gmelina
arborea
Costa Rica KF766193 EF622057 EU673111 [25,128]
Lasiodiplodia
theobromae
CBS 164.96
Ex-neotype
From
unidentified
fruit along
coral reef
coast
Papua New
Guinea,
Madang
AY640255 AY640258 KU887532 [72,129]
L. theobromae CSM 22 Theobroma
cacao
AR MF436023 MF436011 MF436005 [54]
L. theobromae CBS129751
A
cacia
mangium
NER
J
X545096
J
X545116
J
X545136 [59]
L. theobromae CMW13487 Europhylla
urophylla
x E. grandis
CR and WCR EF118053 - - [53]
L. theobromae CBS129754 Pinus caribaea
var.
hondurensis
NER
J
X545099
J
X545119
J
X545139 [59]
L. theobromae CMW13489=
CBS117922
Eucalyptus
urophylla x E.
g
randis
CR and WCR DQ103525 - - [53]
L. theobromae CMW13510
A
cacia
mangium
CR and WCR DQ103526 - - [43]
L. theobromae CMW13520 Pinus caribaea CR and WCR DQ103527 - - [43]
L. theobromae CAA006 Ficus insipida GR DQ458891 DQ458876 DQ458859 [106]
L. venezuelensis CBS129755 Pinus caribaea
var.
hondurensis
NER
J
X545104
J
X545124
J
X545144 [59]
L. venezuelensis CBS129757 Ficus insípida GR
J
X545102
J
X545122 - [106]
L. venezuelensis WAC12539=
CBS118739
Ex-type
A
cacia
mangium
CR and WCR DQ103547 DQ103568 KU887533 [72,80]
L. venezuelensis CBS 129759
J
acaranda
copaia
GR
J
X545101
J
X545121
J
X545141 F. Castro-
Medina/
S.R. Mohali-
unpublished
Neofusicoccum
arbuti
CBS
116131=AR
4014 Ex-type
A
rbutus
menziesii
USA AY819720 KF531792 KF531793 [8,108]
Neofusicoccum
arbuti (syn. N.
andinum)
CMW13455=
CBS117453
Eucalyptus sp.AR AY693976 AY693977 KX464923 [26,105]
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Neofusicoccum
arbuti (syn. N.
andinum)
CMW13446=
CBS117452
Eucalyptus sp.AR DQ306263 DQ306264 KX464922 [26,105]
N. parvum CMW9081
Ex-type
Eucalyptus
g
randis
South Africa AY236943 AY236888 [43]
N. parvum CMW13350=
CBS117923
Psidium
g
uajava
ZR EF118036 - - [53]
N. parvum CMW13355=
CBS117915
Eucalyptus
urophylla
CR and WCR EF118035 - - [53]
N. ribis CMW7772
Ex-type
Ribes sp. New York,
United States
AY236935 AY236877 - [43]
N. ribis CMW13360=
CBS117916
Eucalyptus
urophylla
CR and WCR EF118037 - - [53]
N. ribis CMW13410=
CBS117443
Eucalyptus
urophylla
CR and WCR EF118038 - - [53]
Pseudofusicoccum
stromaticum
CMW13434=
CBS117448
Ex-type
Eucalyptus
urophylla
x E. grandis
CR and WCR AY693974 AY693975 EU673094 [105,128]
P. stromaticum CMW13426=
PREM
58513
A
cacia
mangium
CR and WCR EF118041 - - [53]
Acronyms of culture collections: CBS: Centraalbureau voor Schimmelcultures, Fungal Biodiversity Centre,
Utrecht, The Netherlands; IBL: Independent Biological Laboratories Israel. KEFAR MALAL; CMW: Tree
Patholgy Co-operative Program, Forestry and Agricultural Biotechnology Institute, University of Pretoria, South
Africa; WAC: Department of Agriculture, Western Australia Plant Pathogen Collection, South Perth, Western
Australia; CSM: Personal culture collection deposited in the Department of Bioagricultural Sciences & Pest
Management, Colorado State University, USA. MFLUCC: Mae Fah Luang University Culture Collection,
ChiangRai, Thailand. CAA: A. Alves, Universidade de Aveiro, Portugal. Locality in Venezuela (see map-Figure
3): Central Region=CR (Cojedes state) and Western Central Region=CR and WCR (Falcon, Lara, and Portuguesa
states). Los Andes Region (Mérida state) = AR. Northeastern Region (Anzoátegui and Monagas states) = NER.
Guayana Region (Bolívar and Delta Amacuro states) = GR. Zulia Region (Zulia state) = ZR. (-) = No sequences.
3.1. Phylogenetic analysis
For this review, a phylogenetic analysis was carried out for those genera and species of
Botryosphaeriales in Venezuela that were identified by partial gene sequences available in NCBI
GenBank Database (http://www.ncbi.nlm.nih.gov). For this analysis, Lasiodiplodia genus was
analyzed separately from the remaining of the genera and species of Botryosphaeriales because it has
the largest number of species reported for different hosts in Venezuela.
For the phylogenetic study were internal transcribed spacers 1 and 2 including the intervening
5.8S nrDNA gene (ITS) [45], the translation elongation factor 1-alpha gene (tef1) [46] and the beta-
tubulin gene (tub2) [47]. The 3-loci concatenated alignment contained 1232 characters including gaps
for Lasiodiplodia group (526 from ITS, 328 from tef1 and 378 from tub2) and 1295 characters including
gaps for Botryosphaeriales remaining (532 from ITS, 333 from tef1 and 430 from tub2) (Table 3).
Phylogenetic analyses were performed for the combined datasets using two different methods:
Maximum Likelihood (ML) and Bayesian Inference (BI). A partition homogeneity test (PHT) [48,49]
was conducted to determine whether the datasets for the three gene regions could be combined. The
PHT performed on the concatenated dataset of three gene regions yielded a P-value = 0.01. The value
P-value was significant, datasets for multiple gene regions were combined for phylogenetic analysis.
The ML phylogenies were evaluated with a bootstrapping (BS) method. ML phylogenies were
performed with MEGA-X [50], and BI phylogenies were performed with MRBAYES v3.2.1 [51]. All
sequences from representative isolates were aligned using MUSCLE that along with BI phylogenies
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were used in association with the Geneious Prime software version 2020.1.2. The best-fit nucleotide
substitution models for the combined datasets (ITS, tef1, and btub) were identified separately for ML
and BI. For BI analyses, the best-fit nucleotide substitution models were determined with jModeltest
2.1.10 [52] using the Akaike Information Criterion (AIC) and for ML were determined with MEGA-
X [50], with HKY+G substitution model used as the best model for both. Phylogenetic species were
determined with ML ≥50%, and BI ≥0.6 for Lasiodiplodia group (Figure 1), and ML ≥ 90% and BI ≥ 0.90
for the remaining of Botryosphaeriales (Figure 2).
Figure 1. Phylogenetic tree of Lasiodiplodia genus in Venezuela results from Bayesian analysis (BI) of
the combined ITS, tef1, and tub2 sequence alignment. Maximum likelihood (ML) bootstrap support
values (ML ≥50%) and Bayesian posterior probabilities (BI ≥0.6) are shown at the nodes (ML/BI). Ex-
type strains are indicated and all hosts named in the tree belong to Venezuela. The tree was rooted to
Botryosphaeria dothidea CMW8000 Ex-type.
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Figure 2. Phylogenetic tree of Botryosphaeriales remaining group in Venezuela results from Bayesian
analysis (BI) of the combined ITS, tef1, and tub2 sequence alignment. Maximum likelihood (ML)
bootstrap support values (ML ≥ 90%) and Bayesian posterior probabilities (BI ≥ 0.90) are shown at the
nodes (ML/BI). Ex-type strains are indicated and all hosts named in the tree belong to Venezuela. The
tree was rooted to Lasiodiplodia brasiliense CMM4015 Ex-type.
4. Taxonomy, diversity and distribution of a new genus, new species and reports found in
Venezuela and other regions of the world
The taxonomy of a new genus, new species, and reports of Botryosphaeriales identified by DNA
sequences and their hosts in Venezuela are discussed below (Table 3, Figures 1–3).
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Figure 3. Localities or states in Venezuela where Botryosphaeriales has been reported using sequences
data (bold) and morphological methods (dark red).
Cophinforma atrovirens (Mehl & Slippers) A. Alves & A.J.L. Phillips (Basionym: Fusicoccum
atrovirens Mehl & Slippers) was isolated from stems and branches of A. mangium, Eucalyptus urophylla-
hybrids, E. urophylla x E. grandis and reported for the first time in Cojedes (CR) and Portuguesa (WCR)
states, [53], and from fruits and trees of Theobroma cacao L., in Merida state (AR) [54], Venezuela.
Initially, Mohali et al. [53] reported this fungus as Botryosphaeria mamane D.E. Gardner (asexual morph
Cophinforma mamane (D.E. Gardner) A.J.L. Phillips & A. Alves), but Phillips et al. [8] found that ITS
sequences of the Venezuelan isolates of C. mamane are the same as the ITS sequence of C. atrovirens,
therefore they consider the Venezuelan isolates to represent C. atrovirens.
In other regions of the world C. atrovirens was isolated from asymptomatic branches and twigs
of Pterocarpus angolensis, in South Africa [55]; dead branch of Eucalyptus sp., in Thailand [56] as
Cophinforma eucalypti Doilom, J.K. Liu & K.D. Hyde; it was also isolated from Dimocarpus longan Lour.,
but producing lesions on inoculated seedlings of Eucalyptus sp., in China [57]; and stem rot and
dieback on Cashew tree (Anacardium occidentale) in Brazil [58].
Diplodia scrobiculata J. de Wet, Slippers & M.J. Wingf., (syn. Diplodia guayanensis F. Castro-
Medina, J.R. Úrbez-Torres, S.R. Mohali & W.D. Gubler sp. nov., MycoBank 812480) was isolated from
the trunk of A. mangium in plantations of Monagas state, North Eastern Region (NER), Venezuela
[59]. Diplodia guayanensis was distinguished from D. scrobiculata by its larger conidia [59]. Later,
sequence alignment ITS, tef1 and btub of D. scrobiculata and combining two (ITS and tef1) [60] and
three loci (ITS, tef1 and btub) [61] for phylogenetic analysis both concluded that D. guayanensis is
indistinguishable from D. scrobiculata based on phylogenetic analyses, and considered it to be a
synonym for D. scrobiculata, and this was further supported on the basis that Úrbez-Torres et al. [59]
used older sequences for D. scrobiculata in their phylogenetic analyses [60], although Zhang et al. [61]
used the old sequences and obtained the same results as Linaldeddu et al. [60]. Furthermore,
morphological variability is common in these fungi [8,60]; however, distinctive RFLP patterns were
obtained for D. guayanensis compared against their closely related species D. scrobiculata and D.
sapinea (Fr.) Fuckel (A and B) using Cfol restriction fragments in tef1 PCR products [59]. The PCR-
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RFLP fingerprinting profiles have been useful in this study to distinguish Botryosphaeriales, although
overlapping RFLP patterns may be observed between some species using one, two, or more RE [62].
Diplodia scrobiculata was isolated and identified for the first time from needles of Pinus banksiana
Lamb., P. resinosa Aiton, P. greggii Engelm. ex Parl., in USA (Wisconsin, Minnesota, California),
Mexico, and Europe (France, Italy) [63]; wilted twigs, branch dieback, necrosis and stem cankers on
Pinus halepensis Mill., trees, in Tunisia [64]; symptomless and die-back on Pinus patula Schiede ex
Schltdl. & Cham., in South Africa [65]; asymptomatic trees in Pinus radiata D. Don plantations but
producing lesions on inoculated P. radiata seedlings in Spain [66]; Pinus sp., in Canada [67]; and
dieback on Coast redwood (Sequoia sempervirens (Lamb. ex. D. Don) Endl.) in California, USA [68].
Lasiodiplodia brasiliensis M.S.B. Netto, M.W. Marques & A.J.L. Phillips was isolated for the
first time in Venezuela from T. cacao plantations in the state of Merida (AR), Venezuela [54], although
Zhang et al. [61], reported to L. brasiliensis on P. caribaea var. hondurensis, F. insipida and J. copaia wood
in Venezuela, these authors taken by mistake these sequences from GenBank that belong to L.
theobromae from Venezuela (see Table S1 of these authors).
Lasiodiplodia brasiliensis was identified and reported for the first time in Brazil on stems of Mango
(Mangifera indica L.) and fruits of Carica papaya L. [69] and other hosts in Brazil; saprobic on dead
branch of teak (Tectona grandis L.f.), in Thailand [70]; Mango dieback, in Peru [71]; Adansonia
madagascariensis Baill., in Madagascar [72]; Eucalyptus sp., in China [56]; as endophytic fungus isolated
from healthy, brown, and ligaloes tissue of evergreen trees (Aquilaria crassna Pierre ex Lecomte), in
Laos [73]; symptoms of gummosis, stem cankers, and dieback on Persian lime (Citrus latifolia Tan.),
in Mexico [74]; Gossypium hirsutum L., in Australia [75]; leaf blight of Sansevieria trifasciata Prain
(mother-in-law’s tongue or snake plant), ornamental plant, in Malaysia [76]; dieback and corky bark
on longan trees (Dimocarpus longan L.), in Puerto Rico [77]; branch dieback, T. cacao, Cameroon and
Psychotria tutcheri Dunn fruits, in China [61].
Cruywagen et al. [72] and Farr & Rossman [78] mistakenly cited to L. brasiliensis as the causing
of dieback in strawberries (Fragaria x ananassa Duchesne), in Turkey, but the pathogen reported was
L. theobromae [79].
Lasiodiplodia crassispora Burgess, Barber sp. nov., was isolated for the first time from wood of
living E. urophylla in Acarigua, Portuguesa State (WCR), Venezuela and canker of Santalum album L.,
(sandalwood) in Western Australia, Australia [80]. The sandalwood is native to southern India,
eastern Indonesia, and northern Australia (https://en.wikipedia.org/wiki/Santalum_album),
therefore, L. crassispora found in central-western Venezuela (WCR) could have been introduced
through imported eucalyptus seeds used for the plantations in Venezuela.
Lasiodiplodia crassispora was associated the internal wood decay symptoms observed in the
cordon samples on grapevine (Vitis vinifera L.), in South Africa [81]; E. urophylla, in Uruguay [82];
perennial cankers in the vascular tissue of grapevines, in California, USA [83]; endophytic in Corymbia
sp. Hook, and minor lesions in inoculations on 4-month-old baobab seedlings (Adansonia gregorii
F.Muell.), in Australia [84]; dieback and stem-end rot of mango, fresh fruit of table grape (Vitis spp.),
and causing dieback on Annonaceae in Brazil [85–87]; dieback symptoms from trunks and branches
on grapevines in Sonora and Baja California, Mexico [88]. Lasiodiplodia crassispora (syn. Lasiodiplodia
pyriformis F.J.J. van der Walt, Slippers & G.J.Marais) isolated from the leading edges of lesions on
branches of Acacia mellifera (M.Vahl) Benth., in Namibia [61,89].
Lasiodiplodia pseudotheobromae A.J.L. Phillips, A. Alves & Crous was reported for the first
time in Uverito plantations, Monagas State (NER), Venezuela in A. mangium [90].
Lasiodiplodia pseudotheobromae was identified for the first time from Gmelina arborea Roxb.,
(Melina) and A. mangium in Costa Rica, Rosa sp., in Netherlands, Coffea sp., in Zaire and Citrus
aurantium L., Suriname [91]; isolated from trees apparently healthy or showing canker and dieback
symptoms of Acacia confuse Merr., Albizia falcataria (L.) Fosberg, Eucalyptus sp., Mangifera sylvatica
Roxb., and Paulownia fortunei (Seem.) Hemsl., in China [92]; dieback on blackthorn (Acacia mellifera
(M.Vahl) Benth.), in Namibia [89]; Adansonia digitata L., in Mozambique and South Africa [71];
Cashew gummosis (Anacardium humile A.St.-Hil.), in Brazil [93]; Annona muricata L., in Australia [75];
Bouea burmanica Griff., Hevea brasiliensis (Willd. ex A.Juss.) Müll.Arg., Persea americana Mill., Coffea
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arabica L., Mangifera indica, Ficus racemosa L., Syzygium samarangense (Blume) Merr. & L.M.Perry,
Dimocarpus longan Lour., in Thailand [94]; stem cankers, gummosis, and branches dieback Citrus
latifolia Tan., in Mexico [74]; trunk cankers, Citrus reticulata Blanco, in Pakistan [95]; symptoms of
branch dieback, cankers and fruit rot in Citrus sp., in Iran [96]; dieback, Mango, in Egypt, Peru and
South Korea [71,97,98]; stem canker on the native Uruguayan tree, Myrcianthes pungens (O.Berg) D.
Legrand and pathogenic in inoculated 4 month-old Eucalyptus grandis seedlings, in Uruguay [82];
dieback and fruit rot on Rambutan trees (Nephelium lappaceum L.), in Puerto Rico [77]; symptoms of
branch dieback and cankers, and shoot and panicle blight in pistachio (Pistacia sp. and Pistacia vera
L.), in Spain, [99]; shoot-dieback, gummosis, and sunken necrotic bark lesions in young nectarine
(Prunus persica) trees, in Turkey [100]; Rosa sp., in Netherlands [91]; leaf blight of Sansevieria trifasciata,
in Malaysia [76]; die-back disease on Schizolobium parahyba (Vell.) S. F. Blake var. amazonicum (Ducke)
Barneby trees, in Ecuador [101]; trunk Diseases in Vitis vinifera, in Tunisia [102]; and post flowering
stalk rot of maize (Zea mays L.), in India [103].
Lasiodiplodia theobromae (Pat.) Griffon & Maubl., is a cosmopolitan fungus occurring
predominantly throughout tropical and subtropical regions [38,80]. It has also been known as a
human pathogen causing keratomycosis and phaeohyphomycosis [103], and as a plant pathogen
associated with about 500 plant hosts causing numerous diseases, including dieback, root rot, fruit
rots, leaf spot and cankers of many others [38], and it also occurs as an endophyte [104].
Lasiodiplodia theobromae has been reported in Venezuela on A. mangium, and E. urophylla, in
Portuguesa State (WCR) [105]; P. caribaea var. hondurensis, E. urophylla x E. grandis, and A. mangium,
in Cojedes (CR), Falcon and Portuguesa States (WCR) [53]; Pinus caribaea and A. mangium, in Monagas
State (NER) [59]; Ficus insipida, logs yard located within the natural forest of the Imataca Forest
Reserve, between the Bolivar and Delta Amacuro States (GR) ([106]; Theobroma cacao, in Merida State
(AR) [54].
In Venezuela, regarding the population structure of L. theobromae isolated from forest tree
plantations was of a high gene flow between populations and a lack of population differentiation
from the three host types considered, A. mangium and Eucalyptus urophylla, in Cojedes and Portuguesa
State, and P. caribaea var. hondurensis in Falcon State, therefore the reproduction was predominantly
clonal, and all three Venezuelan populations were pooled [104].
Lasiodiplodia venezuelensis Burgess, Barber, Mohali, sp. nov., MB500237 was isolated and
described for the first time from wood of living Acacia mangium Willd., in Acarigua, Portuguesa State
(WCR), Venezuela. Later, was found causing blue stain on Pinus caribaea Morelet var. hondurensis
(Sénécl.) W.H.Barrett & Golfari wood and light-brown cankers with a black exudate on A. mangium
in Monagas State (NER), and blue stain on Ficus insipida Willd., wood, Imataca Forest Reserve (natural
forests), between the Bolivar and Delta Amacuro States (GR) [59,80,106]. To date, L. venezuelensis has
only been reported in Venezuela, and found in the natural forest causing blue stain wood of F.
insipida, and as a pathogen in A. mangium plantations. L. venezuelensis could be an endemic native
fungus causing blue stain in light wood species native for Venezuela as is the case of F. insipida and
moving onto an exotic species as a pathogen in A. mangium plantations [59].
Neofusicoccum arbuti (D.F. Farr & M. Elliott) Crous, Slippers & A.J.L. Phillips (syn.
Neofusicoccum andinum (Mohali, Slippers & M.J. Wingf.) Mohali, Slippers & M.J. Wingf. comb. nov.
MycoBank MB500871. Basionym: Fusicoccum andinum Mohali, Slippers & M.J. Wingf.) [8,24,105], was
isolated from asymptomatic branches of mature Eucalyptus sp., trees in Mucuchies (3140 m),
Cordillera Los Andes mountains (AR), Venezuela [105].
Li et al. [57,107] using combination of ITS, tef1, tub2, and rpb2 regions, with maximum parsimony
(MP)/maximum likelihood (ML) tests analyses, they could separate cryptic species between N.
andinum and N. arbuti. Later, Zhang et al. [61], evaluated the species in Botryosphaeriales, and
performed Bayesian analysis of the combined ITS, tef1, tub2 and rpb2 sequence alignment to obtain a
new phylogenetic tree of Neofusicoccum spp. They found that the ex-type culture of N. arbuti had
nucleotide similarities with the sequences of the ex-type of N. andinum [(ITS: 466/471 (98.94 %), rpb2:
536/537 (99.81 %), tef1: 240/241 (99.59 %) and tub2: 376/376 (100 %), respectively], therefore N.
andinum was reduced to synonymy with N. arbuti. Mohali et al. [105] did not include the N. arbuti
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sequences in the phylogenetic tree because these were not available at that time [61]. Neofusicoccum
arbuti was isolated from cankers of Arbutus menziesii Pursh (Pacific madrone), in Washington and
California, USA, and Canada [108], and stem canker and dieback of Vaccinium spp. (Blueberry), in
Chile [109].
Neofusicoccum parvum (Pennycook & Samuels) Crous, Slippers & A.J.L. Phillips and
Neofusicoccum ribis (Slippers, Crous & M.J. Wingf.) Crous, Slippers & A.J.L. Phillips both were
isolated on E. urophylla S.T. Blake, and Botryosphaeria dothidea (Moug. ex Fr.) Ces. & De Not.,
isolated on E. urophylla x E. grandis W. Hill ex Maiden hybrids all from asymptomatic plant tissue, as
well as trees exhibiting blue stain and die-back and from entirely dead trees in Portuguesa State, and
N. parvum on Psidium guajava L., in Zulia State (ZR) [53].
Inoculation trial was conducted on E. urophylla x E. grandis hybrid stems in Portuguesa State
with the fungi B. dothidea, N. parvum and N. ribis, and after 7 weeks lesions development was
recorded. Botryosphaeria dothidea produced very small lesions in comparison to N. ribis and N. parvum
which produced significantly larger lesions, bark swelling around the inoculation points and in some
cases the bark was cracked producing black kino exudation when the outer bark was removed from
the points of inoculation [110].
Information on the wide geographic distribution and host range of L. theobromae, N. parvum, N.
ribis and B. dothidea can be found in Fungal Database (https://nt.ars-grin.gov/fungaldatabases/) and
Mycobank Database (https://www.mycobank.org/).
Pseudofusicoccum Mohali, Slippers & M.J. Wingf. gen. nov. MycoBank MB500884;
Pseudofusicoccum stromaticum (Mohali, Slippers & M.J. Wingf.) Mohali, Slippers & M.J. Wingf.,
comb. nov. MycoBank MB500885, Basionym: Fusicoccum stromaticum Mohali, Slippers & M.J. Wingf.,
[8,24,105], was isolated from branches of Eucalyptus urophylla S.T.Blake and E. urophylla x E. grandis
W. Mill ex Maiden-hybrids, and from branches and stems of Acacia mangium Willd., in Western
Central Region (WCR) of Venezuela [105].
Crous et al. [24] introduced to Pseudofusicoccum genus for species that are morphologically
similar to Fusicoccum and Neofusicoccum but phylogenetically distinct from both of these genera.
Pseudofusicoccum genus resembles species of Fusicoccum but distinct in having conidia encased in a
persistent mucous sheath, and conidia are also more cylindrical than in Fusicoccum species [24]. Yang
et al. [26] using robust backbone phylogeny for Botryosphaeriales (LSU and rpb2 genes) described and
raised this genus as a new family, Pseudofusicoccumaceae Tao Yang & Crous where morphologically
the family, is typified by Pseudofusicoccum.
In Venezuela, inoculations with P. stromaticum were made on 2-year-old trees in plantations of
E. urophylla x E. grandis hybrid clones. Seven weeks after inoculation produced small lesions on the
stems, but at the same time it was observed that the inoculation points had started to heal and
produce callus by the end of the trial [110].
Pseudofusicoccum stromaticum has been widely reported in Brazil causing diseases in different
hosts such as: dieback on mango (Mangifera indica L.) stems, pathogenic on 5-month-old mango
seedlings, and producing the small lesions on inoculated mango fruits [111,112]; dieback, wilting of
branches, discoloration of the vascular system, decline and subsequent death of Malay apple
(Syzygium malaccense L.) trees [113]; associated with gummosis on native cashew (Anacardium
othonianum Rizzinin) [93]; dieback and stem and branch cankers on cashew (Anacardium occidentale
L.), guava (Psidium guajava L.) and caja-umbu (Spondias mombin L. x S. tuberosa Arruda) trees [114]; as
endophyte in Myracrodruon urundeuva Fr. All. (Anacardiaceae) [115], and dieback of the Annonaceae
[87]. In Uruguay, P. stromaticum was associated with cankers showing gummosis in peach shoots and
showed moderate virulence on both inoculated apple and peach shoots [116].
In addition to P. stromaticum, eight species have subsequently been added to the genus, such as
Pseudofusicoccum adansoniae Pavlic, T.I. Burgess, M.J. Wingf., on Adansonia gibbosa (A.Cunn.) Guymer
ex D.A.Baum, Acacia synchronicia Maslin, Eucalyptus L'Hér., and Ficus opposite Miq., in Australia and,
Ficus krishnae L. and Jatropha podagrica Hook, in India [117,118]; P. africanum Marinc., Jami & M.J.
Wingf., on twigs of Mimusops caffra E.Mey. ex A.DC. (coastal red milkwood), in Eastern Cape
Province, Haga Haga, South Africa [119]; P. ardesiacum Pavlic, T.I. Burgess, M.J. Wingf., on A. gibbosa
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and Eucalyptus sp., in Australia; P. artocarpi T. Trakunyingcharoen, L. Lombard & Crous, on twigs of
Artocarpus heterophyllus Lam., in Chiang Mai Province, Thailand [94]; P. calophylli Jayasiri, E.B.G.
Jones & K.D. Hyde on decaying fruit pericarp of Calophyllum inophyllum L., in Krabi Province,
Mueang Krabi District, Thailand [120]; P. kimberleyense Pavlic, T.I. Burgess, M.J. Wingf., on Acacia
synchronicia Maslin, Adansonia gibbosa, Eucalyptus sp., and Ficus opposite Miq. in Australia [121] and
Persea americana Mill., USA [61]; P. olivaceum Mehl & Slippers on asymptomatic branches and twigs
of Pterocarpus angolensis (Kiaat), in Mpumalanga Province, Kruger National Park, Pretoriuskop,
Terminalia sericea Burch. ex DC., and Terminalia prunioides M.A.Lawson, in South Africa [55,61]; P.
violaceum Mehl & Slippers on asymptomatic branch of P. angolensis Mpumalanga Province, Mawewe
Nature Reserve, in South Africa [55], and Microcos paniculatus, in Hong Kong, China [61].
This genus is known only as the asexual morph and thus far nine species have been reported
[61]. To date, P. stromaticum has been reported exclusively from South America while the remaining
of the Pseudofusicoccum species have been reported from other regions, such as South Africa,
Australia, Thailand, China, USA, and India [61,117,118].
5. Symptoms associated with species from Botryosphaeriales in Venezuela
Botryosphaeriales species infect plants via wounds or through natural plant openings, such as
buds, lenticels, and stomata, resulting in diverse symptoms, such as twig, branch, and main stem
cankers; die-back of leaders, shoots, or whole branches; seed capsule abortion; collar rot; damping off
or blight of seedlings; root cankers; blue-stain; decline; and death of whole trees in severe cases [7].
The Table 1, different genera within the Botryosphaeriales were found and isolated from different hosts
and locations in Venezuela, associated with diverse symptoms, and identified through its asexual
morph, and other were identified using DNA sequence data (Table 3, Figures 1 and 2).
Diplodia spp. and Lasiodiplodia spp., have been reported to cause different symptoms, such as
blue stain (synonymous sap stain), which is a result of melanin, a pigment produced by the fungal
pathogen [122]. The blue color of the wood develops as an optical effect due to refraction of light
[123], such as observed in the following examples: Lasiodiplodia theobromae, L. venezuelensis and
Diplodia mutila (Fr.) Mont., on Pinus caribaea var. hondurensis (Figure 4a–i); L. theobromae and L.
venezuelensis on Ficus insípida (Figure 4j,k). The discolorations in the wood of living trees/woody
plants or dead logs are the result of diverse biotic and abiotic causes [124,125]. Wood discoloration
and decay are often the result from wounding, such as those caused by animal chewing, branch
breaking, pruning, mechanized wood harvest, construction injury, motor traffic, etc. [126], and
insects. Further discolorations can result from tree-produced substances, such as deposition of
heartwood substances developed by living tree cells, later microbial stains, and finally colored
derivatives of wood decay processes [124], examples of tree/wood discolorations include the
following: sudden death or die-back in E. urophylla and Eucalyptus hybrid of Portuguesa State caused
by Lasiodiplodia crassispora, L. theobromae, Neofusicoccum parvum, N. ribis, Botryosphaeria dothidea,
Pseudofusicoccum stromaticum, and Cophinforma atrovirens (Figure 4l,m); discolorations on Acacia
mangium in Cojedes and Portuguesa States caused by Lasiodiplodia theobromae, L. venezuelensis,
Cophinforma atrovirens, and Pseudofusicoccum stromaticum (Figure 4n,o).
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Figure 4. (a) Pinus caribaea var. hondurensis plantations (Maderas del Orinoco C.A) located in Uverito,
Monagas state, Venezuela. (b) Blue stain of the wood, observed in fallen trees inside the plantations.
(c) Log yard to be processed at the sawmill in Maderas del Orinoco company. (d) Blue stain in the
logs at the sawmill. (e) Pycnidium. (f) Conidia of Lasiodiplodia spp. (g, h) Hyphae of Lasiodiplodia spp.
invading the medullary rays of Pinus caribaea var. hondurensis (red arrows), g=80X and h=Scanning
Electron Microscopy. (i) Hypha found between tracheid or intercellular space of the wood of
Caribbean pine (red arrow), Transmission Electron Microscope (19000X). (j) Blue stain in lumber of
Ficus insipida; (k) Cross section Ficus insipida lumber with blue stain; (l) Eucalyptus urophylla trees
exhibiting dieback or entirely dead trees (sudden death) in Portuguesa state. (m) Eucalyptus urophylla
tree dead with blue stain. (n) Acacia mangium plantations in Portuguesa State. (o) Discoloration (red
arrow) on Acacia mangium trees in Portuguesa State. Pictures e, f [32]; g [123]; h, i [130]; j, k [106].
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Discolorations and canker in the stem of A. mangium caused by Lasiodiplodia pseudotheobromae, L.
theobromae, L. venezuelensis, and Diplodia scrobiculata (syn. D. guayanensis) in plantations of Maderas
del Orinoco Company (Figure 5a–d), as well decline symptoms observed in A. mangium, and P.
caribaea var. hondurensis. Pathogenicity tests were carried out in commercial plantations at the
company Maderas del Orinoco to investigate the status of Botryosphaeriales associated with decline
symptoms observed in A. mangium and P. caribaea var. hondurensis. Three Lasiodiplodia spp. and one
Diplodia sp., were inoculated in A. mangium, and two Lasiodiplodia spp., on P. caribaea var. hondurensis.
A. mangium showed bark swelling, vascular discoloration, necrosis, and cankering around the
inoculation points (Figure 5e–g), while in P. caribaea var. hondurensis did not cause any lesions [59,90].
This study showed that Lasiodiplodia spp., and Diplodia sp., are highly virulent to A. mangium,
showing. Other pathogenicity tests were carried out in the field, which gave us information about the
susceptibility or tolerance to diseases, such as is the case of Eucalyptus spp., a forest species introduced
in Venezuela to obtain fibers for cardboard production. These assessments of inoculations were made
with different genera and species of Botryosphaeriales in commercial plantations of Eucalyptus at the
company Smurfit Kappa Reforestadora Dos, Portuguesa State on different commercial clones of
Eucalyptus-hybrids (E. urophylla x E. grandis), and where these clones were shown to be tolerant of
Botryosphaeriaceae were observed [110]. Cophinforma atrovirens was isolated from T. cacao fruits with
anthracnose and together with Lasiodiplodia theobromae, and L. brasiliensis were found in association
with dieback or sudden death symptoms on T. cacao trees [54] in Merida State (Figure 5h), producing
discolorations in branches (Figure 5i) and stems (Figure 5j,k). These discolorations were mainly
associated with wounds caused by bark beetles- Scolytinae (Figure 5l). Stems, branches, and roots
with cankers and dieback on P. caribaea var. hondurensis trees in plantations from 4 to15 years old and
in nurseries on 8-month-old seedlings in displaying completely browned needles (Figure 5m,n) were
observed at the Maderas del Orinoco Company, and the main fungal pathogen reported as causing
these diseases was Sphaeropsis sapinea (Fr.) Dyko & B. Sutton [34]. Cedeño et al. [34] based their
identification on the asexual morph, conidia 39,8 (37-45) x 12,7 (11-16) μm, one septum and rarely
two or three septa (Figure 5o,p); measurements close to Diplodia sapinea (25.5-) 30.5-52.5 (-54) x (10-)
12.5-20 (-21) μm, D. scrobiculata (37.5-) 39.5 (-41.5) x (13-) 14 (-15.5) μm, and D. scrobiculata (syn. D.
guayanensis) (33.5-) 40.6-42.4 (56) x (12-) 15.8-16.7 (-18.5) μm [59]. The absence of septa (aseptate) in
mature conidia of Sphaeropsis separates it from the Diplodia genus, which is characterized by septate
conidia [8] (Table 2), therefore, the diseases observed by Cedeño et al. [34] in the nurseries and
plantations of P. caribaea var. hondurensis could have been caused by a fungal species in the Diplodia
genus.
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Figure 5. (a) Acacia mangium tree with canker in the stem (red arrow) in Maderas del Orinoco
plantations. (b, c) Discoloration in the A. mangium stem. (d) Discoloration from A. mangium tree base.
(e) Pathogenicity test with different Botryosphaeria species using standing trees of A. mangium in a
commercial plantation, and where A. mangium tree showed bark swelling around the inoculation
points and necrosis of the vascular system below the bark (canker) 12 weeks after inoculation. (f, g)
Black exudation was observed when the outer bark was removed from inoculation points (red
arrows). (h) Dieback or sudden death symptoms in Theobroma cacao tree. (i) Discoloration in branch;
(j, k) Discolorations in stems of T. cacao with dieback or sudden death symptoms in Merida State. (l)
Bark beetle (Scolytinae) collected from cacao tree stem with discoloration. (m) Control seedling (left)
and, tip death symptom in a seedling inoculated with Sphaeropsis sapinea (right) in Caribbean pine
three weeks after inoculation. (n) Seedling with longitudinal stem section inoculated with S. sapinea
where the discoloration within the stem can be observed. (o) Pycnidium and, (p) S. sapinea Conidia
(possible Diplodia sp.). Pictures e-g [59]; m-p [34].
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Neofusicoccum arbuti (syn. N. andinum) was collected from asymptomatic branches of mature
Eucalyptus sp. trees growing in the Cordillera Los Andes Mountains of Venezuela at an altitude of ca.
3000 m (Figure 6a–d). Photographs of other Botryosphaeriales genera; Diplodia scrobiculata (syn.
Diplodia guayanensis) (Figure 6e,f), Pseudofusicoccum stromaticum (Figure 6g–i); Neofusicoccum
ribis/Neofusicoccum parvum (Figure 6j,k), Cophinforma atrovirens (Figure 6l, m), and B. dothidea (Figures
6n-p).
Figure 6. (a, b) Eucalyptus sp., at the Cordillera Los Andes Mountains, Merida state, Venezuela at an
altitude of approx. 3140 meters above sea level. The black arrows show old Eucalyptus trees without
apparent damage. (c) Conidiogenous cells and (d) Conidia of Neofusicoccum arbuti (syn. Neofusicoccum
andinum) isolated from Eucalyptus sp., asymptomatic branches at the Cordillera Los Andes
Mountains. (e) Conidia immature and mature of Diplodia scrobiculata (syn. Diplodia guayanensis). (f)
Mature conidia of D. guayanensis with two and three septa. (g) Pseudofusicoccum stromaticum
producing big conidioma on 2 % Malt Extract Agar. (h) Multilocular conidiomata of P. stromaticum
without ostioles and embedded locule. (i) Pseudofusicoccum stromaticum conidia encased in a persistent
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mucous sheath (red arrow). (j) Neofusicoccum ribis/parvum complex conidiogenous cells. (k)
Neofusicoccum ribis/parvum complex conidia. (l) Cophinforma atrovirens, asci bitunicate with ascospores
aseptate, hyaline, with granular textured contents. (m) Cophinforma atrovirens conidia with one and
two septa. (n-p) Botryosphaeria dothidea conidia with 0-2 septa. Pictures e, f [59]; g-I [105]; j-p [53].
6. Conclusions
This is a review and update of information that represents almost 40 years of research work with
species pertaining to the order Botryosphaeriales that cause diseases, with special reference to woody
plants. The nomenclature of the different species and genera found within the Botryosphaeriales order
have been updated, including the identification of the news species of Lasiodiplodia, a new genus and
specie of Pseudofusiccocum, and new reports for Venezuela using molecular tools.
At the morphological level, nine genera were isolated and identified within Botryosphaeriales
order, where Lasiodiplodia spp is the most abundant of all genera. This was isolated from fruit
plantations such as citrus, mango, cacao, avocado, and forest tree plantations of exotic species such
as pine, and from native forest species.
With molecular tools, it was possible to define exactly the name of the species that produce or
are associated with forest diseases, especially in forest plantations of exotic species. Lasiodiplodia
theobromae and L. venezuelensis both didn't cause lesions when they were inoculated in Pinus caribaea
var. hondurensis trees, but they were routinely reisolated from asymptomatic wood which indicates
the latent pathogen status of these species in this host, as well causing of blue stain on pine wood
observed in fallen trees and in log yards at sawmills.
Lasiodiplodia pseudotheobromae, L. theobromae, L. venezuelensis, and Diplodia scrobiculata (=D.
guayanensis) were isolated from trunks with symptoms light-brown cankers with a black exudate in
Acacia mangium plantations. Inoculation tests carried out on this host showed bark swelling around
the inoculation points and necrosis of the vascular system below the bark and black exudation,
showing these four species their high virulence on A. mangium.
The fungi B. dothidea, C. atrovirens, L. theobromae, N. arbuti (=N. andinum), N. parvum, N. ribis and
P. stromaticum isolated from Eucalyptus spp., plantations, were inoculated on hybrid Eucalyptus trees,
where N. ribis and N. parvum produced significantly large lesions (canker) on the trunk, therefore
these pathogens can be considered as new emerging diseases on these forest species introduced in
the country; B. dothidea produced very small lesions, and the remainder of the fungi did not.
Regarding natural tropical forests in Venezuela, the blue stain of the wood on Ficus insipida in
lumber yards was caused by L. theobromae and L. venezuelensis.
Plantations of the non-native forest species, Pinus caribaea var. hondurensis, in the East of
Venezuela (between the States of Anzoátegui and Monagas), began in 1961. This plantation had a
planted area of approximately 600,000 ha, but currently there are 112,000 ha. Later, and on a smaller
scale, non-native species, Acacia mangium, was planted. These forest plantations border one of the
largest natural forest reserves in South America, The Imataca Forest Reserve occupying
approximately 3.7 million ha, and located between the Bolivar and Delta Amacuro States in
Venezuela [106]. The proximity between non-native and native species has allowed native pathogens,
such as Lasiodiplodia venezuelensis, found so far only in Venezuela, and together with L. theobromae, L.
pseudotheobromae, D. arbuti (=D. guayanensis) to be transferred to these specie exotics causing blue stain
on pine wood, and canker in A. mangium plantations.
Batista et al. [2] have assumed that human movement and trade are the main routes of dispersal
for all species within the order Botryosphaeriales with worldwide distribution across all continents,
with the exception of Antarctica, with climatic variability being the main limitations for the
appearance of new stable populations and additionally, they also highlight that the disease
expression is mainly due to occasional climatic events that can affect the susceptibility of the host.
Botryosphaeriales are reported as saprophytic, parasites, endophytic, and opportunistic
pathogens in different crops, natural forests, and plantations, causing significant losses to the
Venezuelan economy, but these losses are not quantified. Information on diseases caused by fungi of
the Botryosphaeriales order and their description at the morphological level in Venezuela is very
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scarce, scattered, and with little information, in addition to future research in plant pathology,
phylogenetic studies and fungal taxonomy, and the rest of the other areas of science that are
developed in Venezuela is in great uncertainty due to current economic and political problems.
Author Contributions: Not applicable
Funding: Not applicable
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Acknowledgments: I gratefully acknowledge Dr. Ned B. Klopfenstein of Forest Service, Rocky Mountain
Research Station (USDA) and Prof. Leslie Holland Department of Plant Pathology, University of Wisconsin-
Madison, USA for their cooperation and comments for the improvement of this paper.
Conflicts of Interest: The author declares no conflict of interest.
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