Mycobiota associated with anthracnose and dieback symptoms on Theobroma cacao L. in Mérida State, Venezuela

Micobiota associada a antracnose e sintomas de morte regressiva em Theobroma cacao L. no estado de Mérida, Venezuela

Micobiota asociada con antracnosis y síntomas de muerte regresiva en Theobroma cacao L. en el estado Mérida, Venezuela

ABSTRACT

Diverse fungi collected from symptomatic fruit, stem and branch tissues of Theobroma cacao in five T. cacao-producing localities or municipalities of Mérida State, Venezuela, were identified using both morphological methods and sequencing of multiple loci (ITS, LSU, SSU, TEF1, BTUB, RPB2). Cophinforma atrovirens, Lasiodiplodia brasiliensis and Hypoxylon investiens are reported for the first time on T. cacao in Venezuela. Fungi found in association with fruit anthracnose included Cophinforma atrovirens, Fusarium solani and F. oxysporum, whereas species associated with dieback or sudden death symptoms include Cophinforma atrovirens, L. theobromae, L. brasiliensis and H. investiens. All of the aforementioned fungi are considered putative pathogens of T. cacao, which warrant further pathogenicity tests.

Keywords
Botryosphaeria ; Cophinforma ; Fusarium ; Hypoxylon ; Lasiodiplodia ; fungal genera

RESUMEN

Diversos fungos coletados de tecidos sintomáticos de frutos, caules e ramos de Theobroma cacao em cinco localidades ou municípios produtores de T. cacao no estado de Mérida, Venezuela, foram identificados usando métodos morfológicos e sequenciamento de múltiplos loci (ITS, LSU, SSU, TEF1, BTUB, RPB2). Cophinforma atrovirens, Lasiodiplodia brasiliensis e Hypoxylon investiens foram relatados pela primeira vez em T. cacao na Venezuela. Dentre os fungos encontrados em associação com antracnose nos frutos estavam Cophinforma atrovirens, Fusarium solani e F. oxysporum, enquanto que espécies associadas a sintomas de morte regressiva ou morte súbita incluem Cophinforma atrovirens, L. theobromae, L. brasiliensis e H. investiens. Todos os fungos mencionados acima são considerados patógenos putativos de T. cacao, o que justifica testes de patogenicidade adicionais.

Palabras clave
Botryosphaeria ; Cophinforma ; Fusarium ; Hypoxylon ; Lasiodiplodia ; gêneros fúngicos

RESUMO

Se identificaron diversos hongos recolectados desde tejidos sintomáticos de frutos, tallos y ramas de Theobroma cacao en cinco localidades o municipios productores de T.cacao en el estado Mérida, Venezuela, utilizando tanto métodos morfológicos como secuenciación de múltiples loci (ITS, LSU, SSU, TEF1, BTUB, RPB2). Cophinforma atrovirens, Lasiodiplodia brasiliensis e Hypoxylon investiens son reportados por primera vez en T. cacao en Venezuela. Los hongos encontrados en asociación con la antracnosis del fruto incluyeron a Cophinforma atrovirens, Fusarium solani y F. oxysporum, mientras que las especies asociadas con síntomas de muerte súbita o muerte regresiva incluyeron a Cophinforma atrovirens, Lasiodiplodia theobromae, L. brasiliensis, Hypoxylon investiens. Todos los hongos antes mencionados se consideran patógenos putativos de T. cacao, que justifican más pruebas de patogenicidad.

Palavras-chave
Botryosphaeria ; Cophinforma ; Fusarium ; Hypoxylon ; Lasiodiplodia ; Géneros de hongos

The name given by Linnaeus to the genus of Theobroma cacao L. [theos (God) + broma (beverage) = beverage of the Gods] recognized the Mayan belief that the plant had divine origins (⁵). Cheesman (⁴) suggested that T. cacao has been cultivated in Mexico and Central America for over 2,000 years and that no truly wild populations were present in this region, indicating that T. cacao was introduced into Central America and Mexico. It has been hypothesized that T. cacao was naturally present throughout the Amazon Valley and may have subsequently been dispersed along two routes: one leading north and the other one heading west (⁴²). Domestication of T. cacao began during this dispersal process in South America, before migrating indigenous human populations spread the seed to Central America and southern Mexico (⁴²). Molecular analyses, combined with the hypotheses of Cheesman (⁴) and Schultes (⁴²), uphold the theories that T. cacao originated in South America and was later introduced to Central America by human activities (²⁶).

Historical records on the origin of T. cacao in Venezuela indicate wild T. cacao trees were first recorded in Aug 5, 1602, and highlight the discovery of 100,000 T. cacao trees close to Maracaibo Lake in western Venezuela (³⁹). Subsequently, propagation of T. cacao occurred throughout Venezuela between 1600 and 1700, spreading the cultivar ‘sweet cacao’ or ‘Criollo’ (= ‘Creole’) (³⁹).

The oldest recorded disease affecting T. cacao worldwide was documented in Venezuela in the mid-1630s on the “haciendas” upwind of La Guaira, where ‘blight’ (alhorra) had destroyed over half of all T. cacao on the coast within a decade (⁷, ⁴⁹). In 1824, the high-yielding T. cacao cultivar ‘Forastero Trinitario’ was introduced to Venezuela. The longer fermentation period required for beans of ‘Forastero Trinitario’ negatively influenced flavor, resulting in cacao of a lower quality compared to the ‘Criollo’ cultivar (³⁹, ⁴⁹). The ‘Forastero Trinitario’ cultivar was rapidly distributed westward from the Paria peninsula to Barlovento and Tuy, where T. cacao ‘Criollo’ had practically disappeared because of disease pressures. Later, ‘Forastero Trinitario’ was transported into central Venezuela, but not to the western states of the country (³⁴).

Information on diseases affecting T. cacao fruits and trees in Venezuela is typically sparse and dispersed in technical reports or meeting proceedings, and the fungi are mostly described using only morphology. Among the economically most important pathogens on T. cacao are Lasiodiplodia sp., causing pod rot and dieback in Ghana, Cameroon, Cuba, Ecuador and Venezuela (¹⁴, ²², ³⁹, ⁴⁷), and Fusarium sp., as pathogens, endophytes and/or antagonists against other fungal pathogens of plants (¹², ²², ³⁹).

Though several fungal diseases have been reported attacking T. cacao in Venezuela, little research has been conducted to detect and identify the causal agents since the advent of molecular technologies. For closely related fungi, DNA-based analyses coupled with phylogenetic methods can be used to separate cryptic species. The aim of the present study is to use a combination of morphological and molecular techniques to determine the diversity of fungi associated with anthracnose and dieback of T. cacao in five important production localities of Mérida State, Venezuela.

Materials and Methods

Isolation of putative fungal pathogens

Stems, branches and fruits with symptoms of diseases, including dieback, sudden death and anthracnose, were collected from T. cacao from five municipalities in Mérida State (Table 1, Figure 1). Samples were transported to the laboratory and initially checked for the presence of fungal fruiting bodies; conidia and single spore isolations were performed as previously described (³⁷). For additional fungal isolations, small pieces of stem, branch or fruit tissues (5 mm²) were excised from the margins of necrotic lesions. Tissue pieces were surface disinfested for 30 s in 70% ethanol and 60 s in 0.5% sodium hypochlorite (NaOCl-bleach); then, they were washed in three changes of sterile water for 60 s each and dried on sterile filter paper. Surface-disinfested samples were plated onto 2% malt extract agar (MEA; Difco Laboratories, Detroit, MI, USA) and incubated at 25 °C for 7 days or until mycelia were observed growing from the plant tissues. Emerging fungal mycelium was sub-cultured to fresh MEA and hyphal tip methods were used to obtain pure cultures.

Figure 1
(A) Sites of sampling (red stars) for Theobroma cacao in the five municipalities in Mérida State, Venezuela; map in grey scale shows additional T. cacao-producing states in Venezuela (55); (B, C) Dieback or sudden death symptoms; (D) Anthracnose on T. cacao fruits.

Table 1
Municipalities and localities within Mérida State, Venezuela, where fungal samples were collected from Theobroma cacao showing different disease symptoms.

Municipality	Capital	Locality	Symptom
Alberto Adriani	El Vigía	Kilometro 15	Dieback or sudden death; canker in the tree Anthracnose on fruits
Andrés Bello	La Azulita	La Azulita La Pueblita	Dieback or Sudden death
Antonio Pinto Salinas	Santa Cruz de Mora	San Rafael Los Bocadillos San Miguel Mesa de las Palmas Mesa Bolivar	Dieback or sudden death
Obispo Ramos de Lora	Santa Elena de Arenales	Eloy Paredes-Las Honduras Sector	Dieback or sudden death
Sucre	Lagunillas	Estanques-Santo Domingo Sector San juan de Lagunillas-Instituto Nacional de Investigaciones Agrícolas (INIA) Sector	Anthracnose on fruits Dieback or sudden death

Mycelial plugs (5-mm) were taken from the periphery of actively growing cultures and transferred to 9-cm Petri dishes containing potato dextrose agar (PDA; Difco Laboratories, Detroit, MI, USA), MEA or synthetic nutrient-poor agar medium (SNA, ²⁸). Following incubation at room temperature (25 °C) for 7 days, colony characteristics and pigment production were noted; colony diameters were measured after 7- to 10-day growth. Conidia produced on PDA and MEA were mounted in lacto-phenol for measurement of dimensions and morphological analyses.

DNA extraction and sequencing

Total DNA was extracted from 7-day-old cultures, grown on half-strength PDA amended with 100 mg/L streptomycin sulphate (Sigma-Aldrich, USA). Isolates were sub-cultured to sterilized MF-47-mm Millipore membrane filters (Millipore Sigma, Burlington, MA, USA) overlaid on the surface of the half-strength PDA and incubated at 25 ˚C. DNA was extracted from cultured mycelia using the ZR Fungal/Bacterial DNA MiniPrep (Zymo Research, Irvine, CA, USA), following the manufacturer’s protocol. Polymerase chain reactions (PCR) were performed in a reaction mixture comprising 30 ng template genomic DNA, 2.5 μL10 standard Taq reaction buffer (New England BioLabs; NEB, Ipswich, MA, USA), 0.5 μL10 mM dNTP (Roche Applied Science, Penzberg, Germany), 1 μL of each 10 μM primer, 0.125 μL (0.6 units) Taq DNA polymerase (NEB), and sterile deionized water to a total volume of 25 μL. PCR products were cleaned with ExoSAP-IT^® PCR Product Cleanup (Thermo Fisher Scientific, Grand Island, NY, USA) and sequenced at Eurofins Scientific (www.eurofinsus.com).

Six loci were used for DNA-sequence analyses of each isolate: 5.8S rDNA with the two flanking internal transcribed spacers (ITS), a portion of the nuclear ribosomal 18S rRNA gene (small subunit; SSU), a portion of the nuclear rRNA cluster comprising the ITS region plus the D1/D2 variable domains of the ribosomal 28S rRNA gene (large subunit; LSU), and partial sequences of translation elongation factor 1-α (TEF1), β-tubulin (BTUB) and RNA polymerase II second largest subunit (RPB2) genes. Primer sets for amplification included 1) ITS-1F (⁸) and ITS4 (⁴⁸) for ITS (Cophinforma, Hypoxylon and Lasiodiplodia); 2) NS1 and NS4 (⁴⁸) for SSU (Cophinforma); 3) ITS1 (⁴⁸) and NL4 (²⁹) for LSU (Cophinforma); 4) EF1-688 and EF1-1252 (¹) for TEF1 (Cophinforma and Lasiodiplodia), EF-1 and EF-2 (³¹) for TEF1 (Fusarium); 5) Bt2a and Bt2b (¹⁰) for BTUB (Cophinforma and Lasiodiplodia); T1 and T22 (³⁰) for BTUB (Hypoxylon), and 6) 5F2 and 7eR (²¹) for RPB2 (Fusarium). PCR thermal cycle programs were performed as previously described (⁸, ²¹, ²⁹, ³⁰, ³¹, ⁴⁸).

Phylogenetic analyses

Sequence data from closely related species for phylogenetic analyses were obtained from GenBank (https://blast.ncbi.nlm.nih.gov/Blast.cgi). Sequence alignments, including manual adjustments and concatenation of loci, were conducted using MAFFT (¹⁷) within Geneious Pro v. 9.0.5.

Phylogenetic analyses compared DNA sequences of isolates from the current study with sequences from closely related taxa. Phylogenies were estimated for each locus separately and, if necessary, multiple loci were assessed together for each genus. Maximum likelihood (ML) and Bayesian inference (BI) algorithms were used to reconstruct phylogenies for alignments using PhyML (¹¹) and MrBayes v. 3.2.1 (⁴¹) in Geneious v6.1.3 (Biomatters Inc.). A best-fit substitution model for each dataset was selected using the Bayesian Information Criterion (BIC) implemented in DT ModSel (²⁴): 1) Cophinforma - K80+G for ITS, TIM+G for SSU, K80 for LSU, HKY+I for TEF1, TVMef+I+G for BTUB; 2) Fusarium - GTR+I+G for TEF1, TrNef+I+G for RPB2; 3) Hypoxylon - TVM+I+G for ITS+BTUB, and 4) Lasiodiplodia - TrN+I+G for ITS+BTUB+TEF1.

For ML analyses within PhyML, phylogenies were run with 1,000 bootstrap pseudo-replicates. Bayesian Markov Chain Monte Carlo analyses were carried out using MrBayes (¹⁵). Six chains were run for 2,000,000 generations and trees sampled every 100 generations. The first 25,000 trees were discarded as burn-in and the remaining trees used for estimating posterior probabilities (PP) for the majority rule consensus tree.

Phylogenies for multiple loci datasets were estimated using Bayesian analyses implemented in Bayesian Evolutionary Analysis Sampling Trees (BEAST) (⁶). BEAST does not use concatenation but rather co-estimates the individual gene trees embedded inside the summary species tree. Bayesian Evolutionary Analysis Utility (BEAUti), version 1.7.5, was used to create XML-formatted input files for BEAST v1.7.5. In BEAST, a Markov Chain Monte Carlo algorithm was used to sample the posterior distribution of trees by conducting five independent runs of 100 million generations, each using a constant size tree prior, strict molecular clock, and uniform priors. Trees were sampled every 1,000 generations and the first 20% discarded as burn-in. Post burn-in trees were combined with the program LogCombiner (BEAST v1.7.5); chains were assumed to converge when the average standard deviation of split frequencies was < 0.01. The maximum clade credibility tree with PP of each node was computed with Tree Annotator (BEAST v 1.7.5). Log files and tree files were visualized in Tracer v1.5 (http://tree.bio.ed.ac.uk/software/tracer/) and FigTree v1.3.1 (http://tree.bio.ed.ac.uk/software/figtree/), respectively.

Results AND Discussion

Morphological characterization

Morphological characteristics were evaluated to separate and group the fungi at the genus level. Three genera were separated and identified on the basis of morphology: 1) Cophinforma sp., conidiophores and paraphyses absent; conidia hyaline, unicellular, rarely becoming septate, mostly fusoid to ellipsoidal, most conidia longer than 30 μm; 2) Lasiodiplodia sp., conidia hyaline when young, later becoming 1-septate, dark brown with longitudinal striations, thick-walled, oblong to ellipsoid, straight, broadly rounded at the apex, base truncate; and 3) Fusarium sp., chlamydospores abundant, macroconidia septate, apical cell curved and tapering to a point, basal cell foot-shaped. Hypoxylon sp. could not be identified by morphology and was identified with DNA sequence-based analyses (Figure 2).

Figure 2
(A) Cophinforma atrovirens conidia; (B) Fusarium oxysporum microconidia (false heads) on short monophialides; Lasiodiplodia sp. mature (C) and inmature (D) conidia. A, C, D, scale bar = 5 μm, and B, scale bar = 10 μm.

Phylogenetic analyses

ML and BI analyses of all examined loci produced similar results. Representative sequences for fungal isolates from Venezuela are highlighted in bold type in Figures 3-6. GenBank numbers are listed next to each sequence for reference. Posterior probability support for each clade ranged from 1 to 0.80. For internal nodes, however, PP was often lower. Bootstrap (BS) values below 50% are marked with a hyphen (-) on the phylogenetic trees. Fungal genera are presented based on their potential pathogenic importance as species newly reported on T. cacao, based on field observations.

Fungal species associated with diseased T. cacao

Lasiodiplodia brasiliensis M.S.B. Netto, M.W. Marques & A.J.L. Phillips and L. theobromae (Pat.) Griffon & Maubl.

A total of 28 taxa and 56 isolates were included in the phylogenetic analyses; Macrophomina phaseolina was included as the outgroup (Table 2). A concatenation of three gene regions (ITS+TEF1+ BTUB) was used to separate Lasiodiplodia isolates from T. cacao from other species of Lasiodiplodia. Approximately 570 base pairs were sequenced at the ITS, 540 bp at the TEF1, and 430 bp at the BTUB, for a total of 1,540 bp (Figure 3). The concatenated phylogeny highlighted the separation of L. brasiliensis and L. theobromae (BS/PP = 86%/1.00 and 65%/0.82).

Table 2
GenBank and culture collection accession numbers for Lasiodiplodia spp. isolates included in this study. Sequences that represented the isolates from Venezuela in this study are highlighted in bold.

Species	Accession numbera	Host	Locality	Collector	GenBank accession numberb
					ITS	TFE1	BTUB
Lasiodiplodia americana	CERC1960	Pistachia vera	USA	T. J. Michailides	KP217058	KP217066	KP217074
L. americana	CERC1961	Pistachia vera	USA	T. J. Michailides	KP217059	KP217067	KP217075
L. brasiliensis	CMW 35884	Adansonia madagascariensis	Madagascar	M. J. Wingfield	KU887094	KU886972	KU887466
L. brasiliensis	IBL415	Manilkara zapota	Brazil	I. B. L. Coutinho	KT151806	KT151800	KT151803
L. brasiliensis	CSM 11	Theobroma cacao	Venezuela	S. R. Mohali	MF436018	MF436006	MF435998
L. brasiliensis	CSM 15	Theobroma cacao	Venezuela	S. R. Mohali	MF436019	MF436007	MF435997
L. brasiliensis	CSM 16	Theobroma cacao	Venezuela	S. R. Mohali	MF436020	MF436008	MF435996
L. brasiliensis	CSM 232	Theobroma cacao	Venezuela	S. R. Mohali	MF436021	MF436009	MF435995
L brasiliensis	CSM 235	Theobroma cacao	Venezuela	S. R. Mohali	MF436022	MF436010	MF435994
L. caatinguensis	IBL366	Spondias purpurea	Brazil	I. B. L. Coutinho/J. S. Lima	KT154760	KT008006	KT154767
L. caatinguensis	IBL381	Spondias purpurea	Brazil	I. B. L. Coutinho/J. S. Lima	KT154757	KT154751	KT154764
L. citricola	IRAN1521C	Citrus sp.	Iran	A. Shekari	GU945353	GU945339	KU887504
L. citricola	IRAN1522C	Citrus sp.	Iran	J. Abdollahzadeh/A. Javadi	GU945354	GU945340	KU887505
L. crassispora	WAC 12533	Santalum album	Australia	T.I. Burgess	DQ103550	DQ103557	KU887506
L. crassispora	CMW 13488	Eucalyptus urophylla	Venezuela	S.R. Mohali	DQ103552	DQ103559	KU887507
L. egyptiacae	CMM3611	Jatropha curcas	Brazil	A. R. Machado	KF234545	KF226691	KF254928
L. egyptiacae	CMM3648	Jatropha curcas	Brazil	A. R. Machado	KF234549	KF226705	KF254933
L. euphorbicola	CMM3609	Jatropha curcas	Brazil	A.R. Machado/O.L. Pereira	KF234543	KF226689	KF254926
L. euphorbicola	CMM3651	Jatropha curcas	Brazil	A.R. Machado/O.L. Pereira	KF234553	KF226711	KF254937
L. exigua	CBS 137785=BL104	Retama raetam	Tunisia	B.T. Linaldeddu	KJ638317	KJ638336	KU887509
L. gilanensis	IRAN1501C	Unknown	Iran	J. Abdollahzadeh/A. Javadi	GU945352	GU945341	KU887510
L. gilanensis	IRAN1523C	Unknown	Iran	J. Abdollahzadeh/A. Javadi	GU945351	GU945342	KU887511
L. gonubiensis	CBS 115812	Syzygium cordatum	South Africa	D. Pavlic	KF766191	DQ458877	KU887512
L. hormozganensis	IRAN1498C	Mangifera indica	Iran	J. Abdollahzadeh/A. Javadi	GU945356	GU945344	KU887514
L. hormozganensis	IRAN1500C	Olea sp.	Iran	J. Abdollahzadeh/A. Javadi	GU945355	GU945343	KU887515
Lasiodiplodia iraniensis	IRAN1520C	Salvadora persica	Iran	J. Abdollahzadeh/A. Javadi	GU945348	GU945336	KU887516
L. iraniensis	IRAN1502C	Juglans sp.	Iran	J. Abdollahzadeh/A. Javadi	GU945347	GU945335	KU887517
L. jatrophicola	CMM3610	Jatropha curcas	Brazil	A. R. Machado	KF234544	KF226690	KF254927
L. macrospora	CMM3833	Jatropha curcas	Brazil	A.R. Machado/O.L. Pereira	KF234557	KF226718	KF254941
L. mahajangana	CMW27801	Terminalia catappa	Madagascar	J. Roux	FJ900595	FJ900641	FJ900630
L. mahajangana	CMW27820	Terminalia catappa	Madagascar	J. Roux	FJ900597	FJ900643	FJ900632
L. margaritacea	CBS122519	Adansonia gibbosa	Australia	T.I. Burgess	EU144050	EU144065	KU887520
L. mediterranea	CBS 137783	Quercus ilex	Italy	B. T. Linaldeddu	KJ638312	KJ638331	KU887521
L. missouriana	UCD2193MO	Vitis vinifera	USA	K. Striegler/G. M. Leavitt	HQ288225	HQ288267	HQ288304
Lasiodiplodia missouriana	UCD2199MO	Vitis vinifera	USA	K. Striegler/G. M. Leavitt	HQ288226	HQ288268	HQ288305
L. parva	CBS456.78	Cassava-field soil	Colombia	O. Rangel	EF622083	EF622063	KU887523
L. plurivora	STE-U 5803	Vitis vinifera	Africa	F. Halleen	EF445362	EF445395	KU887524
L. pontae	CMM 1277	Spondias purpurea	Brazil	J. S. Lima/F. C. O. Freire	KT151794	KT151791	KT151797
L. pseudotheobromae	IBL134	Annona x atemoya	Brazil	I. B. L. Coutinho/J. S. Lima	KT247481	KT247485	KT247487
L. pseudotheobromae	IBL266	Anacardium occidentale	Brazil	I. B. L. Coutinho/J. S. Lima	KT247480	KT247484	KT247486
L. rubropurpurea	WAC12536	Eucalyptus grandis	Australia	T.I. Burgess/G. Pegg	DQ103554	DQ103572	KU887530
L. subglobosa	CMM3872	Jatropha curcas	Brazil	A.R. Machado/O.L. Pereira	KF234558	KF226721	KF254942
L. subglobosa	CMM4046	Jatropha curcas	Brazil	A.R. Machado/O.L. Pereira	KF234560	KF226723	KF254944
L. theobromae	IBL340	Spondias purpurea	Brazil	I. B. L. Coutinho/J. S. Lima	KT247466	KT247472	KT247475
L. theobromae	IBL375	Talisia esculenta	Brazil	I. B. L. Coutinho/J. S. Lima	KT247467	KT247473	KT247474
L. theobromae	CSM 33	Theobroma cacao	Venezuela	S. R. Mohali	MF436024	MF436012	MF436004
L. theobromae	CSM 34	Theobroma cacao	Venezuela	S. R. Mohali	MF436025	MF436013	MF436003
L. theobromae	CSM 36	Theobroma cacao	Venezuela	S. R. Mohali	MF436026	MF436014	MF436002
L. theobromae	CSM 53	Theobroma cacao	Venezuela	S. R. Mohali	MF436027	MF436015	MF436001
L. theobromae	CSM 54	Theobroma cacao	Venezuela	S. R. Mohali	MF436028	MF436016	MF436000
Lasiodiplodia theobromae	CSM 57	Theobroma cacao	Venezuela	S. R. Mohali	MF436029	MF436017	MF435999
L. venezuelensis	WAC12539	Acacia mangium	Venezuela	S.R. Mohali	DQ103547	DQ103568	KU887533
L. viticola	UCD2553AR	Vitis vinífera	USA	K. Striegler/G. M. Leavitt	HQ288227	HQ288269	HQ288306
L. viticola	UCD2604MO	Vitis vinifera	USA	K. Striegler/G. M. Leavitt	HQ288228	HQ288270	HQ288307
Macrophomina phaseolina	CMM3615	Jatropha curcas	Brazil	A. R. Machado	KF234547	KF226693	KF254930
M. phaseolina	PD112	Prunus dulcis	USA	TJM, HR, PI	GU251105	GU251237	GU251765

• ^a
  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; IRAN: Iranian Fungal Culture Collection, Iranian Research Institute of Plant Protection, Iran; WAC: Department of Agriculture, Western Australia Plant Pathogen Collection, South Perth, Western Australia; STE-U: Culture Collection of the Department of Plant Pathology, University of Stellenbosch, South Africa; CMM: Bradford Art Galleries and Museums U.K. England. BRADFORD; PD: Culture Collection, University of California, Davis, USA; CERC, Culture Collection of China Eucalypt Research Centre, Chinese Academy of Forestry, ZhanJiang, GuangDong, China; CSM: Personal culture collection deposited in the Department of Bioagricultural Sciences & Pest Management, Colorado State University, USA.
• ^b
  See Table 2 for full locus name.

Figure 3
Phylogeny of Lasiodiplodia species generated from Bayesian analysis based on the concatenated ITS, BTUB, and TEF1 sequences. The phylogeny was rooted with Macrophomina phaseolina (CMM 3615 and PD 112). Node support (bootstrap ≥ 60%:1,000 replicates and Bayesian posterior probabilities ≥ 0.80, BS/PP) are highlighted by arrows. The new isolates from this study are in bold italics.

Species of Lasiodiplodia are known to cause diseases on T. cacao in plantations across diverse global regions. Based on morphological descriptions, L. theobromae and L. citricola were associated with branch and trunk cankers on T. cacao in Nigeria (¹⁶), while L. theobromae was isolated from rotting T. cacao fruits in Ghana and Cuba (²², ⁴⁶) and from branches and twigs of healthy T. cacao in Brazil (¹²). In addition, microsatellite markers were used to identify L. theobromae and L. pseudotheobromae associated with dieback symptoms on T. cacao in Cameroon (²).

In Venezuela, L. theobromae was identified morphologically in association with T. cacao showing various symptoms, including fruit rot, dieback and cankers on branches, twigs and roots (³⁵, ³⁹). Lasiodiplodia theobromae, identified with ITS sequencing, was also reported as an isolate from T. cacao with cushion galls (⁴⁵); however, ITS sequences appear unreliable as a sole method for identifying currently recognized species of Lasiodiplodia. Several well-established cryptic species are recognized in the Lasiodiplodia complex, all with nearly identical ITS sequences and/or similar morphology (⁴⁴). For this reason, multiple loci, including ITS, TEF1, BTUB and RPB2, are necessary for Lasiodiplodia species identification, and sequences of these loci are well-represented in databases. Importantly, these loci provide sufficiently stringent phylogenetic signals to distinguish most known cryptic species within Lasiodiplodia (⁴³). Lasiodiplodia brasiliensis, first described in Brazil as causing stem-end rot of papaya (²⁷), was also reported causing dieback of mango in Peru (⁴⁰) and postharvest fruit rot of custard apple (Annona squamosa) in Brazil (³). Similar methodology was used to isolate Lasiodiplodia brasiliensis from branches and twigs of T. cacao with dieback or sudden death symptoms in Mérida State. This paper is the first to report L. brasiliensis potentially associated with dieback or sudden death on T. cacao in Venezuela; however, further studies are needed to confirm pathogenicity of L. brasiliensis on T. cacao.

Cophinforma atrovirens (Mehl & Slippers) A. Alves & A.J.L. Phillips.

Three or four taxa and 18 isolates (except 20 isolates for ITS sequencing) were included in the phylogenetic analyses using five gene regions, including ITS: 550 bp; TEF1: 550 bp; SSU: 1020-1025 bp; LSU: 1090 bp, and BTUB: 440 bp. The outgroup taxon for all loci was Macrophomina phaseolina (CBS 162.25). Cophinforma mamane (= Botryosphaeria mamane isolates 97-58 and 97-59) was included in the ITS dataset but not for subsequent phylogenic analyses because sequence data for other loci is lacking for C. mamane. The remaining loci (TEF1, SSU, LSU and BTUB) included 18 isolates comprised within three taxa: C. atrovirens, Botryosphaeria dothidea and M. phaseolina (Table 3). Phylogenies of the ITS and TEF1 showed that isolates of Cophinforma spp. from T. cacao fruits and stem grouped with known isolates of C. atrovirens in well-supported clades (BS/PP = -/0.98 and 100%/1.00); phylogenies of the LSU and BTUB loci had moderate levels of support (BS/PP = 82%/0.65 and 80%/0.57); however, no support was identified at the SSU locus (BS/PP = -/-) (Figure 4).

Table 3
GenBank and culture collection accession numbers of Cophinforma spp. isolates included in this study. Sequences that represented the isolates from Venezuela in this study are highlighted in bold.

Species	Accession numbera	Host	Locality	Collector	GenBank accession numberb
					ITS	TEF1	BTUB	SSU	LSU
Cophinforma atrovirens	MFLUCC 11-0425	Eucalyptus sp.	Thailand	M. Doilom	JX646800	JX646865	JX646848	JX646833	JX646817
C. atrovirens	MFLUCC 11-0655	Eucalyptus sp.	Thailand	M. Doilom	JX646801	JX646866	JX646849	JX646834	JX646818
C. atrovirens	CBS 117444	Eucalyptus urophylla	Venezuela	S. R. Mohali	KF531822	KF531801	KF531802	KF531821	DQ377855
C. atrovirens	CSM 72	Theobroma cacao	Venezuela	S.R Mohali	MF436087	MF436099	MF436111	MF436134	MF436146
C. atrovirens	CSM 74	Theobroma cacao	Venezuela	S.R Mohali	MF436088	MF436100	MF436112	MF436133	MF436145
C. atrovirens	CSM 80	Theobroma cacao	Venezuela	S.R Mohali	MF436089	MF436101	MF436113	MF436132	MF436144
C. atrovirens	CSM 81	Theobroma cacao	Venezuela	S.R Mohali	MF436090	MF436102	MF436114	MF436131	MF436143
C. atrovirens	CSM 82	Theobroma cacao	Venezuela	S.R Mohali	MF436091	MF436103	MF436115	MF436130	MF436142
C. atrovirens	CSM 185	Theobroma cacao	Venezuela	S.R Mohali	MF436092	MF436104	MF436116	MF436129	MF436141
C. atrovirens	CSM 191	Theobroma cacao	Venezuela	S.R Mohali	MF436093	MF436105	MF436117	MF436128	MF436140
C. atrovirens	CSM 192	Theobroma cacao	Venezuela	S.R Mohali	MF436094	MF436106	MF436118	MF436127	MF436139
C. atrovirens	CSM 256	Theobroma cacao	Venezuela	S.R Mohali	MF436095	MF436107	MF436119	MF436126	MF436138
C. atrovirens	CSM 259	Theobroma cacao	Venezuela	S.R Mohali	MF436096	MF436108	MF436120	MF436125	MF436137
C. atrovirens	CSM 262	Theobroma cacao	Venezuela	S.R Mohali	MF436097	MF436109	MF436121	MF436124	MF436136
C. atrovirens	CSM 263	Theobroma cacao	Venezuela	S.R Mohali	MF436098	MF436110	MF436122	MF436123	MF436135
C. mamane	97-58	Sophora chrysophylla	Hawaii	D.E. Gardner	AF246929	-	-	-	-
C. mamane	97-59	S. chrysophylla	Hawaii	D.E. Gardner	AF246930	-	-	-	-
Botryosphaeria dothidea	CBS 110302	Vitis vinifera	Portugal	A.J.L. Phillips	AY259092	AY573218	EU673106	EU673174	EU673243
B. dothidea	CBS 115476	Prunus sp.	Switzerland	B. Slippers	AY236949	AY236898	AY236927	EU673173	AY928047
Macrophom. phaseolina	CBS 162.25	Eucalyptus sp.	Uganda	Goig T	KF531826	KF531803	KF531805	KF531824	DQ377905

• ^a
  CBS: Centraalbureau voor Schimmelcultures, Fungal Biodiversity Centre, Utrecht, The Netherlands; MFLUCC: Mae Fah Luang University Culture Collection, Chiang Rai, Thailand; CSM: Personal culture collection deposited in the Department of Bioagricultural Sciences & Pest Management, Colorado State University, USA.
• ^b
  See Table 2 for full locus name.
• - : Data not found.

Figure 4
Phylogenies of Cophinforma species generated from Bayesian analyses using A: ITS, B: TEF1, C: BTUB, D: LSU, and E: SSU loci. Phylogenies were rooted with Macrophomina phaseolina (CBS 162.25). Node support (bootstrap ≥ 50%:1,000 replicates and Bayesian posterior probabilities ≥ 0.50, BS/PP) are highlighted by arrows. The new isolates from this study are in bold italics.

The genus Cophinforma was introduced within Botryosphaeriaceae by Liu et al. (²⁰) as a monotypic genus that comprised C. eucalypti. Phillips et al. (³⁷), however, showed that two species previously included in Botryosphaeria were better accommodated in Cophinforma: Botryosphaeria mamane as Cophinforma mamane, and Cophinforma eucalypti renamed as Cophinforma atrovirens. The conidia produced by Cophinforma are longer than those of any known species of Botryosphaeria. Although Botryosphaeria and Cophinforma share other morphological similarities, these genera are phylogenetically distinct. Based on phylogenetic relationships, C. mamane and C. atrovirens are currently recognized within Cophinforma (³⁷).

Little published information is available for Cophinforma spp. Cophinforma atrovirens (= Cophinforma eucalypti = Fusicoccum atrovirens) was reported as a saprophyte on a dead branch of Eucalyptus sp. in Thailand (²⁰), whereas C. atrovirens (as F. atrovirens) was isolated from asymptomatic branches and twigs of Pterocarpus angolensis in South Africa (²³). In Venezuela, C. mamane (as B. mamane) was isolated from stems and branches of Acacia mangium and Eucalyptus hybrids (²⁵), and it was also isolated from Sophora chrysophylla in Hawaii (⁹). In the present study, C. atrovirens was isolated from fruit anthracnose- and stem/branch dieback-associated tissues of T. cacao, and it was accompanied by L. theobromae and L. brasiliensis in Mérida State. Despite these findings, the capacity of C. atrovirens to cause disease is not verified. This report is the first showing that C. atrovirens is associated with fruit anthracnose, dieback or sudden death on T. cacao in Venezuela; however, inoculation work is required to conclusively determine if C. atrovirens is a pathogen of T. cacao.

Fusarium oxysporum Schlechtendahl emend. Snyder & Hansen. and Fusarium solani (Martius) Appel & Wollenweber emend. Snyder & Hansen/sexual morph Neocosmospora solani (Mart.) L. Lombard & Crous.

A total of 41 isolates comprised within 26 taxa of Fusarium spp. were used in the phylogenetic analyses with F. lyarnte RBG 5331 as the outgroup (Table 4). A total of 1,680 bp were sequenced at the TEF1 (710 bp) and RPB2 (970 bp) loci to determine species identity of 13 isolates of Fusarium from T. cacao. Phylogenetic analyses of the TEF1 and RPB2 showed that 11 isolates belonged to Neocosmospora solani, while two isolates belonged to Fusarium oxysporum; both species clades possessed well-supported nodes (BS/PP = 100%/1.00; Figure 5).

Table 4
GenBank and culture collection accession numbers of Fusarium spp. included in this study. Sequences that represented the isolates from Venezuela in this study are highlighted in bold.

Species	Accession numbera	Host	Locality	Collector	GenBank accession numberb
					TEF1	RPB2
Fusarium avenaceum	FRC R-09495	-	-	-	GQ915502	GQ915486
F. aywerte	RBG5743	-	Australia	RBG	KP083250	KP083278
F. beomiforme	RBG4549	-	Australia	RBG	HQ667157	HQ646396
F. burgessii	RBG5319	Soil	Australia	B.Wang/A. Fraser	HQ667149	HQ646392
F. coicis	RBG5368	Coix gasteenii	Australia	RBG	KP083251	KP083274
F. commune	NRRL28387	Dianthus caryophyllus	The Netherlands	NRRL	AF246832	JX171638
F. culmorum	RBG3558	-	Australia	RBG	HQ667167	HQ646401
F. fujikuroi	NRRL13566	Oryza sativa	Taiwan	NRRL	AF160279	EF470116
F. gaditjirri	F15048	Heteropogon triticeus	Australia	L.W. Burgess	AY639634	HQ662690
F. goolgardi	RBG5411	Xanthorrhoea glauca	Australia	D.M Robinson/M.H. Laurence	KP101123	KP083280
F. hostae	RBG3994	-	Australia	RBG	HQ667160	HQ646390
F. lyarnte	RBG5331	-	Australia	RBG	EF107118	HQ662691
F. miscanthi	RBG4177	-	Australia	RBG	HQ667166	HQ662693
F. moniliforme	NRRL43656	Contact lens	USA	NRRL	EF452987	EF470026
F. mundagurra	RBG5717	From soil Carnarvon Gorge National Park	Australia	S.M. Dunstan/M.H. Laurence	KP083256	KP083276
F. napiforme	NRRL13604	Pennisetum typhoides	Namibia	A.Lübben	AF160266	EF470117
F. nelsonii	NRRL13338	Soil	Australia	NRRL	GQ505402	GQ505466
F. newnesense	RBG5443	-	Australia	RBG	KJ397074	KP083277
F. nisikadoi	RBG4043	-	Australia	RBG	HQ667165	HQ662692
F. oxysporum	RBG5413	-	Australia	RBG	HQ667161	HQ646385
F. oxysporum	RBG5414	-	Australia	RBG	HQ667163	HQ646388
F. oxysporum	CSM 105	Theobroma cacao	Venezuela	S. R. Mohali	MF436042	MF447156
F. oxysporum	CSM 109	Theobroma cacao	Venezuela	S. R. Mohali	MF436041	MF447155
F. proliferatum	NRRL43665	Contact lens	USA	NRRL	EF452996	EF470035
F. pseudograminearum	RBG3580	-	Australia	RBG	HQ667168	HQ646400
Fusarium redolens	RBG4173	-	Australia	RBG	HQ667159	HQ646391
F. sibiricum	NRRL53429	Avena sativa	Russia	L. S. Malinovskaya/E.A.Pirjazeva	HM744683	HQ154471
F. solani	CBS 138564	Homo sapiens	Turkey	-	KT272100	KT272102
F. solani	CBS 131775	wheat	Iran	M. Davari	JX118990	JX237778
F. solani	CSM 103	Theobroma cacao	Venezuela	S. R. Mohali	MF436040	MF436050
F. solani	CSM 104	Theobroma cacao	Venezuela	S. R. Mohali	MF436039	MF436052
F. solani	CSM 108	Theobroma cacao	Venezuela	S. R. Mohali	MF436038	MF436051
F. solani	CSM 111	Theobroma cacao	Venezuela	S. R. Mohali	MF436037	MF436053
F. solani	CSM 208	Theobroma cacao	Venezuela	S. R. Mohali	MF436036	MF436049
F. solani	CSM 246	Theobroma cacao	Venezuela	S. R. Mohali	MF436035	MF436048
F. solani	CSM 247	Theobroma cacao	Venezuela	S. R. Mohali	MF436034	MF436047
F. solani	CSM 250	Theobroma cacao	Venezuela	S. R. Mohali	MF436033	MF436046
F. solani	CSM 254	Theobroma cacao	Venezuela	S. R. Mohali	MF436032	MF436045
F. solani	CSM 255	Theobroma cacao	Venezuela	S. R. Mohali	MF436031	MF436044
F. solani	CSM 261	Theobroma cacao	Venezuela	S. R. Mohali	MF436030	MF436043
F. tjaetaba	RBG5361	Sorghum interjectum	Australia	J.L. Walsh.	KP083263	KP083275

• ^a
  RBG: Royal Botanic Gardens Trust, Sydney, New South Wales, Australia; NRRL: Agricultural Research Service Culture Collection, Peoria, Illinois, USA; F: University of Sydney, Sydney, New South Wales, Australia; CBS: Centraalbureau voor Schimmelcultures, Fungal Biodiversity Centre, Utrecht, The Netherlands; CSM: Personal culture collection deposited in the Department of Bioagricultural Sciences & Pest Management, Colorado State University, USA.
• ^b
  See Table 2 for full locus name.
• - : Data not found.

Figure 5
Phylogenies of Fusarium species generated from Bayesian analysis based on A: TEF1 and B: RPB2 sequences. Phylogenies were rooted with Fusarium lyarnte (RBG 5331). Node support (bootstrap ≥ 90%:1,000 replicates and Bayesian posterior probabilities ≥ 0.90, BS/PP) are highlighted by arrows. The new isolates from this study are in bold italics.

Taxa within the genus Fusarium are known as pathogens, endophytes and/or antagonists against other fungal pathogens of plants (¹²). Fusarium decemcellulare, the cause of cushion disease, is a weak pathogen that requires injuries to infect T. cacao (¹⁴). Cushion disease occurs in Sri Lanka, West Africa and the Western Hemisphere, and it causes economically important damage in Colombia, Costa Rica and Nicaragua (¹³). Fusarium decemcellulare has also been reported in Cuba and Venezuela, where it occurs with other diseases and pathogens of T. cacao, including fruit rots and stem/branch/root cankers associated with Phytophthora spp., Lasiodiplodia spp. and M. perniciosa (²², ³⁶, ³⁸, ³⁹).

In the present study, isolates of F. oxysporum and F. solani were obtained from T. cacao fruits with anthracnose symptoms. In Mérida State, these same Fusarium species were also isolated from T. cacao tissues with dieback or sudden death symptoms in association with L. brasiliensis and L. theobromae.

Fusarium oxysporum, F. solani and other Fusarium species, such as F. incarnatum, F. equiseti, F. camptoceras, F. crokwellense, F. moniliforme, F. proliferatum and F. decemcellulare, were previously identified on T. cacao in Venezuela, based on morphological and ITS sequence data (³⁹, ⁴⁵, ⁴⁷). The use of the ITS region alone, however, does not provide sufficient resolution to distinguish some species of Fusarium that diverged relatively recently from common ancestors (³²), and ITS is less informative than TEF1, RPB2 and BTUB (³³) for phylogenetic analyses. The current paper confirmed the presence of F. oxysporum and F. solani on T. cacao in Venezuela based on multilocus sequencing.

Hypoxylon investiens (Schwein.) M. A. Curtis.

Two gene regions, ITS and BTUB, were used to identify the two isolates of Hypoxylon collected from T. cacao. A total of 44 isolates representing 41 Hypoxylon species were used in phylogenetic analyses, and Nemania serpens voucher 235 was used as the outgroup (Table 5). A total of 2,108 bp were sequenced: 1,448 bp for BTUB and 660 pb for ITS. Using a concatenated dataset of the BTUB and the ITS regions, a well-supported node grouped the two isolates collected from T. cacao into the Hypoxylon investiens clade (BS/PP = 100%/1), strongly supporting this identity for these isolates (Figure 6).

Table 5
GenBank and culture collection accession numbers of Hypoxylon spp. included in this study. Sequences that represented the isolates from Venezuela in this study are highlighted in bold.

Species	Accession numbera	Host	Locality	Collector	GenBank accession numberb
					ITS	BTUB
Hypoxylon addis	MUCL 52797	-	Ethiopia	Holotype	KC968931	KC977287
H. brevisporum	voucher 36 (JDR)	on fallen tree	Puerto Rico	D. J. Lodge	JN660821	AY951705
H. carneum	voucher YMJ 30	Malus sp.	France	J. Fournier	JN660822	AY951706
H. cercidicola	CBS 119009	Fraxinus excelsior	France	J. Fournier	KC968908	KC977263
H. cinnabarinum	voucher YMJ 8	Branch	Mexico	F. San Martin	JN979409	AY951709
H. crocopeplum	voucher YMJ 11	Decorticated rotten wood	USA	J. D. Rogers	JN979410	AY951710
H. dieckmannii	voucher YMJ 45	Wood	Mexico	F. San Martin	JN979412	AY951712
H. erythrostroma	MUCL 53759	-	Martinique	-	KC968910	KC977296
H. fendleri	YMJ 92092016	Wood	Taiwan	Y. M. Ju	JN979418	AY951718
H. fraxinophilum	MUCL 54176	Fraxinus excelsior	France	J. Fournier	KC968938	KC977301
H. fuscopurpureum	voucher YMJ 67	Quercus sp.	USA	J. D. Rogers	JN979421	AY951721
H. fuscum	voucher YMJ 91010205	Alnus formosana	Taiwan	Y. M. Ju	JN979423	AY951723
H. griseobrunneum	MUCL 53754	On wood and bark	Martinique	J. Fournier	KC968909	KC977289
H. haematostroma	MUCL 53301	Dicot bark	Martinique	J. Fournier	KC968911	KC977291
H. hypomiltum	CBS 129036	corticated branch	Guadeloupe	J. Fournier	KC968914	KC977298
H. investiens	CBS 118183	-	Malaysia	T. Læssoe	KC968925	KC977270
H. investiens	CSM 173	Theobroma cacao	Venezuela	S. R. Mohali	MF435993	MF435991
H. investiens	CSM 174	Theobroma cacao	Venezuela	S. R. Mohali	MF435992	MF435990
H. isabellinum	CBS 129035	Swietenia macrophylla	Martinique	J. Fournier	KC968935	KC977295
H. jecorinum	voucher YMJ 39	Diospyros texana Scheele	Mexico	F. San Martin	JN979429	AY951731
H. laminosum	CBS 129032	Bamboo	Martinique	J. Fournier	KC968934	KC977292
H. lividicolor	voucher YMJ 70	On decorticated wood	Taiwan	Holotype	JN979432	AY951734
H. macrosporum	voucher YMJ 47	Populus sp.	Canada	B. E. Callan	JN979434	AY951736
H. monticulosum	MUCL54626	-	Martinique	MUCL	KC968939	KC977302
H. nicaraguense	CBS 117739	-	Burkina Faso	J. H. Petersen	AM749922	KC977272
H. ochraceum	MUCL 54625	bark (coastal xerophilic forest)	Martinique	MUCL	KC968937	KC977300
H. papillatum	ATCC 58729	-	USA	Holotype	KC968919	KC977258
Hypoxylon perforatum	MUCL 54174	-	Japan	MUCL	KC968936	KC977299
H. polyporus	MUCL 49209	-	Ivory Coast	MUCL	AM749941	KC977283
H. porphyreum	CBS 119022	Quercus petraea	France	J. Fournier	KC968921	KC977264
H. pulicicidum	MUCL53764	on rotten wood	Martinique	MUCL	JX183077	JX183073
H. pulicicidum	CBS 122622	on rotten wood (coastal mesophilic forest)	Martinique	J. Fournier	JX183075	JX183072
H. rickii	YMJ 25 (JDR)	Wood	Mexico	F. San Martin	JQ009313	AY951750
H. samuelsii	MUCL 51843	on dicot. wood and bark	Guadeloupe	C. Lechat	KC968916	KC977286
Hypoxylon shearii var. minor	voucher YMJ 29	on branch of Quercus	Mexico	Isotype	EF026142	AY951753
H. subgilvum	voucher YMJ 88113007	-	Taiwan	Y. M. Ju	JQ009315	AY951755
H. sublenormandii	CBS 138917	-	Sri Lanka	-	KM610291	KM610303
H. submonticulosum	CBS 115280	-	France	-	KC968923	KC977267
H. cf. subticinense	MUCL 53752	-	French Guiana	MUCL	KC968913	KC977297
H. tortisporum	STMA06079	-	Thailand	not yet deposited	KF234420	KF300546
H. trugodes	voucher YMJ 57 (JDR)	Wood	Taiwan	Y. M. Ju	JQ009319	AY951759
H. ulmophilum	voucher YMJ 350 (JDR)	Ulmus japonica	Russia	L. Vasilyeva	JQ009320	AY951760
Nemania serpens	voucher 235 (HAST)	Soil	Canada	G. Barron	GU292820	GQ470223

• ^a
  CBS: Centraalbureau voor Schimmelcultures, Fungal Biodiversity Centre, Utrecht, The Netherlands; ATCC: American Type Culture Collection, Virginia, USA; MUCL: Université Catholique de Louvain Belgium. LOUVAIN-LA-NEUVE; JF: Jonkershoek Forestry Research Centre South Africa. Western Cape Province. STELLENBOSCH; YMJ and J.R: Ju and Rogers cultures accumulated from previous studies; CSM: Personal culture collection deposited in the Department of Bioagricultural Sciences & Pest Management, Colorado State University, USA.
• ^b
  See Table 2 for full locus name.
• - : Data not found

Figure 6
Phylogeny of Hypoxylon species generated from Bayesian analysis based on combined ITS and BTUB sequences. The phylogeny was rooted with Nemania serpens (235). Node support (bootstrap ≥ 90%:1,000 replicates and Bayesian posterior probabilities ≥ 0.90, BS/PP) are highlighted by arrows. The new isolates from this study are in bold italics.

Hypoxylon is one of the largest genera in Xylariaceae, including 159 currently accepted taxa with a global distribution, the highest diversity of which occurs in the tropics (¹⁹). The sexual morphs are usually associated with dead hardwood species, often occurring along with their respective asexual morphs (¹⁹). Hypoxylon is most diverse in warmer climates, and Hypoxylon spp. can live as saprophytes or as facultative parasites on stressed or diseased hosts (¹⁸). In this study, H. investiens was isolated from stems and branches of T. cacao in Mérida State, where it appeared to co-exist with other fungi as a potential endophyte; however, more studies are needed to confirm the ecological role of H. investiens in association with T. cacao. Hypoxylon investiens is reported for the first time on T. cacao in Venezuela, and identification of this fungus increases our knowledge of the mycoflora on T. cacao.

Historically, fungi reported from T. cacao plantations in Venezuela were identified using morphological characteristics or, more recently, through ITS sequence data. This paper presents the first detailed study examining putative disease-causing fungi on T. cacao from Venezuela based on morphology and DNA sequence-based analyses of multiple loci. DNA sequencing enabled the identification of several fungi that are reported for the first time in Venezuela.

The current study demonstrates a large diversity of fungi, especially ascomycetes, associated with diverse disease symptoms on T. cacao in plantations of Mérida State, Venezuela. Since samples were collected only in western Venezuela, it is likely that collections from other T. cacao-producing areas in eastern Venezuela could further increase the known diversity of fungi associated with T. cacao.

Declaration of competing interest

The authors declare no potential conflict of interest for this study, and this study has not been published previously.

ACKNOWLEDGEMENTS

We are grateful to the U. S. Department of State Fulbright Scholar Program for supporting a 1-year exchange visit and providing financial support to Prof. Sari R. Mohali Castillo, ID assigned: PS00236102, as a researcher in the Department Agricultural Biology, Colorado State University. We also thank Kristen Otto for excellent laboratory assistance. This study was partially supported by the employees of the USDA Forest Service, and the findings and conclusions in this publication are those of the authors and should not be construed to represent any official USDA determination or policy.

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Edited by

Editor associado para este artigo: Wagner Bettiol

Publication Dates

• Publication in this collection
  28 Aug 2023
• Date of issue
  2023

History

• Received
  24 Nov 2020
• Accepted
  28 Sept 2022