Lasiodiplodia theobromae (Pat.) Griffon & Maubl., a member of the family Botryosphaeriaceae, is becoming a significant threat to crops and woody plants in many parts of the world, including the major cacao growing areas. While attempting to isolate Ceratobasidium theobromae, a causal agent of vascular streak dieback (VSD), from symptomatic cacao stems, 74% of isolated fungi were Lasiodiplodia spp. Sequence-based identification of 52 putative isolates of L. theobromae indicated that diverse species of Lasiodiplodia were associated with cacao in the studied areas, and the isolates showed variation in aggressiveness when assayed using cacao leaf discs. The present study reports a 43.75 Mb de novo assembled genome of an isolate of L. theobromae from cacao. Ab initio gene prediction generated 13 061 protein-coding genes, of which 2862 are unique to L. theobromae, when compared with other closely related Botryosphaeriaceae. Transcriptome analysis revealed that 11 860 predicted genes were transcriptionally active and 1255 were more highly expressed in planta compared with cultured mycelia. The predicted genes differentially expressed during infection were mainly those involved in carbohydrate, pectin, and lignin catabolism, cytochrome P450, necrosis-inducing proteins, and putative effectors. These findings significantly expand our knowledge of the genome of L. theobromae and the genes involved in virulence and pathogenicity.
Background
Ceratobasidium theobromae, a member of the Ceratobasidiaceae family, is the causal agent of vascular-streak dieback (VSD) of cacao, a major threat to the chocolate industry in the South-East Asia. The fastidious pathogen is very hard to isolate and maintain in pure culture, which is a major bottleneck in the study of its genetic diversity and genome.
Result
This study describes for the first time, a 33.90 Mbp de novo assembled genome of a putative C. theobromae isolate from cacao. Ab initio gene prediction identified 9264 protein-coding genes, of which 800 are unique to C. theobromae when compared to Rhizoctonia spp., a closely related group. Transcriptome analysis using RNA isolated from 4 independent VSD symptomatic cacao stems identified 3550 transcriptionally active genes when compared to the assembled C. theobromae genome while transcripts for only 4 C. theobromae genes were detected in 2 asymptomatic stems. De novo assembly of the non-cacao associated reads from the VSD symptomatic stems uniformly produced genes with high identity to predicted genes in the C. theobromae genome as compared to Rhizoctonia spp. or genes found in Genbank. Further analysis of the predicted C. theobromae transcriptome was carried out identifying CAZy gene classes, KEGG-pathway associated genes, and 138 putative effector proteins.
Conclusion
These findings put forth, for the first time, a predicted genome for the fastidious basidiomycete C. theobromae causing VSD on cacao providing a model for testing and comparison in the future. The C. theobromae genome predicts a pathogenesis model involving secreted effector proteins to suppress plant defense mechanisms and plant cell wall degrading enzymes.
Trichoderma asperellum, composted plant residues, and its combination can control vascular-streak dieback (VSD) disease caused by fungus Ceratobasidium theobromae in laboratory conditions. In this trial, we evaluated these treatments in two years through the application of foliar spraying and stem infusion for T. asperellum alone, through soil amendment for compost alone, and T. asperellum plus this organic fertilizer in the field on susceptible cacao clone. The disease is characterized by full-leaf chlorosis and necrosis that can develop rapidly to the entire branch, with around 70% incidence in seven months, and we detected the pathogen in branches showing light symptoms. All treatments except for T. asperellum plus composted plant residues three and seven months post application did not have any impact on the reduction of VSD incidence in the first year. In the second year, we observed a significant reduction of the disease by both T. asperellum in combination with compost and compost alone in a time span of three to seven months, and with T. asperellum spraying and infusion in a time span of five to seven months. By comparing to the control, the efficacy of these treatments in controlling the VSD disease seven months post-first application in the last year was 44.4%, 23.5%, 23.1%, and 15.1%, respectively. Detection of trees inoculated with T. asperellum indicated that this fungus was present in root and branch tissues except for treatment through infusions, while in the uninoculated trees, Trichoderma was not present or was present at a very low level. These data showed that combination of T. asperellum and composted plant residues applied through soil amendment was able to control VSD disease effectively and could potentially be used at large scale to control this disease and other diseases infesting aerial parts of cacao, and to improve soil fertility.
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