Sari R. Mohali, Centro de Estudios Forestales y ...

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INTRODUCTION. Cacao trees were present in Venezuela long before the Spaniard people arrived. Much before their arrival, diseases on this crop had been.
Microfungi associated to diseases on Theobroma cacao in Merida state-Venezuela Sari R. Mohali, Centro de Estudios Forestales y Ambientales de Postgrado (CEFAP), Laboratorio de Fitopatología, Facultad de Ciencias Forestales y Ambientales, Universidad de Los Andes, Mérida 5101-A, Venezuela Jane E. Stewart, Department of Bioagricultural Sciences and Pest Management, Colorado State University, 307 University Avenue, Ft. Collins, Colorado, 80523, USA .

Sari Mohali

INTRODUCTION

Cacao trees were present in Venezuela long before the Spaniard people arrived. Much before their arrival, diseases on this crop had been reported during the mid-1630s, such as the blight "alhorra". However, very little is known about fungi that cause disease on cacao in Venezuela, and especially in the small cacao plantations distributed in Merida state. The principal goal of this work was to identify fungi associated with die-back and sudden death of cacao and anthracnose on fruits in cacao trees plantations in 5 different locations in Merida State, Venezuela (Figs. 1 and 2).

RESULTS

Jane Stewart

Three species within the Botryosphaeriaceae family were identified, including Lasiodiplodia theobromae, L. brasiliense and Cophinforma atrovirens. Five new reports of fungal pathogens on cacao in Venezuela have been identified including L. brasiliense, C. atrovirens, Fusarium oxysporum, F. solani and Hypoxylon investiens, as well as, two new species have been identified. Colletotrichum sp. nov, was found associated to anthracnose on Cacao fruits and Cylindrocladium sp. nov. was found associated with cankers. • Cophinforma atrovirens, Fusarium solani and F. oxysporum were found associated to fruit anthracnose. • Lasiodiplodia theobromae, L. brasiliensis, Hypoxylon investiens and Cylindrocladium sp. nov. were isolated from stems and branches of cacao trees in association with dieback or sudden death symptoms.

FUNGI

ITS

SSU

LSU

TEF1

BT

cmdA

RPB2

Colletotrichum

F: AMF1 R: AMR1

Cylindrocladium Lasiodiplodia Cophinforma

GS F: GSLF2 R: GSLR1

F: EF1-728F F: T1 F: CAL-228F R: EF-2 R: CYLTUB1R R: CAL-737R F:ITS1 R:ITS4 F:ITS1 R:ITS4

F: NS1 R: NS4

F: ITS1 R: NL4

Fusarium Hypoxylon

ApMat

F: EF1-688 R: EF1-1251 F: EF1-688 R: EF1-1251 F: EF1 R: EF2

F:ITS1 R:ITS4

F: BT2a R: BT2b F: BT2a R: BT2b F: fRPB2-5f R:fRPB2-7cr F: T1 R: T22

Table 1. Regions sequenced for each fungal genera, including ITS: internal transcribed spacer region and flanking regions, SSU: subunit of rDNA, LSU: units D1/D2 of the rDNA large subunit, TEF1: translation elongation factor 1-α, BT: Beta-tubulin, CmdA: Calmodulin, RPB2: RNA polymerase II second largest subunit, ApMat: intergenic spacer and partial mating type gene, GS: Glutamine synthetase.

Fungi associated with cankers Phylogenetic trees for all species. Bootstrap values and posterior probabilities (MP/BI) are shown above branches, with bootstrap values ≤ 50 % or absent values are marked with -. All species identified from Cacao are highlighted in the phylogenetic trees.

Fig. 1. Geographic location of Merida State, Venezuela. The red stars highlighted the sites of Cacao sampling in the five municipalities in Merida state Venezuela.

Fig. 2. A. Sudden death or Die-back of Cacao tree. B. Stained stem of the dead tree. C. Anthracnose on the fruits.

Combined EF & RPB2

Lasiodiplodia spp.

Hypoxylon investiens

Combined ITS & BT

Combined ITS, TEF1, BT

Fungi associated with fruit anthracnose

MATERIALS AND METHODS

Cophinforma atrovirens

Isolates Samples were collected and transported to the phytopathology laboratory of the School of Forestry Engineering, University of Los Andes (ULA) Venezuela. Symptomatic tissues were cut and surface disinfected with 10% sodium hypochlorite, washed three times with sterilized water, plated onto 2% malt extract agar (MEA) and incubated at 25°C for 7 days or until mycelia were observed on the samples. Fruits with anthracnose were initially checked for the presence of fruiting bodies (ascomata and/or conidiomata) and conidia. Fungal mycelium was sub-cultured onto MEA. Single spore isolations were made and hyphal tipping methods were performed obtain pure culture.

Fusarium spp.

ITS

EF1

Cylindrocladium sp. nov

BT

BT

cmdA

TEF1

DNA extraction, PCR amplification and Sequencing Total DNA were extracted from axenic 7 day old cultures on PDA (Difco Laboratories, Detroit, MI) using ZR Fungal/Bacterial DNA Kits (Zymo Research) following manufacturer’s instructions. A total of nine loci were used to identify species and were amplified and sequenced (Table 1). Based on previous studies, these regions provided the best phylogenetic signal at species level for the genera, including: • internal transcribed spacer region and flanking regions (5.8S + ITS + 18S) • Large subunit - LSU (D1/D2 domains of the 28S rRNA gene), • Translation elongation factor 1-α (TEF 1-alpha), • Glutamine synthetase (GS), • Intergenic spacer and partial mating type gene (ApMAT: Apn2-Mat12), • Calmodulin (CmdA), • Beta-tubulin (TUB2), and • RNA polymerase II second largest subunit (RPB2) Models of evolution for each locus were determined using DT-ModSel. Phylogenies for fungal groups were produced using maximumparsimony (MP) and Bayesian Inference (BI) analyses using PAUP and MrBayes. If congruent topologies were observed among loci, these were combined for phylogenetic analyses.

Colletotrichum sp. nov

ApMAT

LSU

GS

SSU

CONCLUSIONS This survey has shown that there are a large diversity of fungi, specially ascomycetes, in cacao plantations associated different diseases in Merida state. This research will result in several new reports and species descriptions novel to Venezuela. Based on the diversity we observed, future collections are warranted.