Cacao pod transcriptome profiling of seven genotypes identifies features associated with post-penetration resistance to Phytophthora palmivora.

The oomycete Phytophthora palmivora infects the fruit of cacao trees (Theobroma cacao) causing black pod rot and reducing yields. Cacao genotypes vary in their resistance levels to P. palmivora, yet our understanding of how cacao fruit respond to the pathogen at the molecular level during disease establishment is limited. To address this issue, disease development and RNA-Seq studies were conducted on pods of seven cacao genotypes (ICS1, WFT, Gu133, Spa9, CCN51, Sca6 and Pound7) to better understand their reactions to the post-penetration stage of P. palmivora infection. The pod tissue-P. palmivora pathogen assay resulted in the genotypes being classified as susceptible (ICS1, WFT, Gu133 and Spa9) or resistant (CCN51, Sca6 and Pound7). The number of differentially expressed genes (DEGs) ranged from 1625 to 6957 depending on genotype. A custom gene correlation approach identified 34 correlation groups. De novo motif analysis was conducted on upstream promoter sequences of differentially expressed genes, identifying 76 novel motifs, 31 of which were over-represented in the upstream sequences of correlation groups and associated with gene ontology terms related to oxidative stress response, defense against fungal pathogens, general metabolism and cell function. Genes in one correlation group (Group 6) were strongly induced in all genotypes and enriched in genes annotated with defense-responsive terms. Expression pattern profiling revealed that genes in Group 6 were induced to higher levels in the resistant genotypes. An additional analysis allowed the identification of 17 candidate cis-regulatory modules likely to be involved in cacao defense against P. palmivora. This study is a comprehensive exploration of the cacao pod transcriptional response to P. palmivora spread after infection. We identified cacao genes, promoter motifs, and promoter motif combinations associated with post-penetration resistance to P. palmivora in cacao pods and provide this information as a resource to support future and ongoing efforts to breed P. palmivora-resistant cacao.

Clonal reproduction of Moniliophthora roreri and the emergence of unique lineages with distinct genomes during range expansion.

The basidiomycete Moniliophthora roreri causes frosty pod rot of cacao (Theobroma cacao) in the western hemisphere. Moniliophthora roreri is considered asexual and haploid throughout its hemibiotrophic life cycle. To understand the processes driving genome modification, using long-read sequencing technology, we sequenced and assembled 5 high-quality M. roreri genomes out of a collection of 99 isolates collected throughout the pathogen's range. We obtained chromosome-scale assemblies composed of 11 scaffolds. We used short-read technology to sequence the genomes of 22 similarly chosen isolates. Alignments among the 5 reference assemblies revealed inversions, translocations, and duplications between and within scaffolds. Isolates at the front of the pathogens' expanding range tend to share lineage-specific structural variants, as confirmed by short-read sequencing. We identified, for the first time, 3 new mating type A locus alleles (5 in total) and 1 new potential mating type B locus allele (3 in total). Currently, only 2 mating type combinations, A1B1 and A2B2, are known to exist outside of Colombia. A systematic survey of the M. roreri transcriptome across 2 isolates identified an expanded candidate effector pool and provided evidence that effector candidate genes unique to the Moniliophthoras are preferentially expressed during the biotrophic phase of disease. Notably, M. roreri isolates in Costa Rica carry a chromosome segment duplication that has doubled the associated gene complement and includes secreted proteins and candidate effectors. Clonal reproduction of the haploid M. roreri genome has allowed lineages with unique genome structures and compositions to dominate as it expands its range, displaying a significant founder effect.

Comparative Genomic Analysis of 31 Phytophthora Genomes Reveals Genome Plasticity and Horizontal Gene Transfer.

Phytophthora species are oomycete plant pathogens that cause great economic and ecological impacts. The Phytophthora genus includes over 180 known species, infecting a wide range of plant hosts, including crops, trees, and ornamentals. We sequenced the genomes of 31 individual Phytophthora species and 24 individual transcriptomes to study genetic relationships across the genus. De novo genome assemblies revealed variation in genome sizes, numbers of predicted genes, and in repetitive element content across the Phytophthora genus. A genus-wide comparison evaluated orthologous groups of genes. Predicted effector gene counts varied across Phytophthora species by effector family, genome size, and plant host range. Predicted numbers of apoplastic effectors increased as the host range of Phytophthora species increased. Predicted numbers of cytoplasmic effectors also increased with host range but leveled off or decreased in Phytophthora species that have enormous host ranges. With extensive sequencing across the Phytophthora genus, we now have the genomic resources to evaluate horizontal gene transfer events across the oomycetes. Using a machine-learning approach to identify horizontally transferred genes with bacterial or fungal origin, we identified 44 candidates over 36 Phytophthora species genomes. Phylogenetic reconstruction indicates that the transfers of most of these 44 candidates happened in parallel to major advances in the evolution of the oomycetes and Phytophthora spp. We conclude that the 31 genomes presented here are essential for investigating genus-wide genomic associations in genus Phytophthora. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Mitochondrial Genomics of Six Cacao Pathogens From the Basidiomycete Family Marasmiaceae.

Thread blight disease has recently been described as an emerging disease on cacao (Theobroma cacao) in Ghana. In Ghana, thread blight disease is caused by multiple species of the Marasmiaceae family: Marasmius tenuissimus, M. crinis-equi, M. palmivorus, and Marasmiellus scandens. Interestingly, two additional members of the Marasmiaceae; Moniliophthora roreri (frosty pod rot) and Moniliophthora perniciosa (witches' broom disease), are major pathogens of cacao in the Western hemisphere. It is important to accurately characterize the genetic relationships among these economically important species in support of their disease management. We used data from Illumina NGS-based genome sequencing efforts to study the mitochondrial genomes (mitogenomes) of the four cacao thread blight associated pathogens from Ghana and compared them with published mitogenomes of Mon. roreri and Mon. perniciosa. There is a remarkable interspecies variation in mitogenome size within the six cacao-associated Marasmiaceae species, ranging from 43,121 to 109,103 bp. The differences in genome lengths are primarily due to the number and lengths of introns, differences in intergenic space, and differences in the size and numbers of unidentified ORFs (uORF). Among seven M. tenuissimus mitogenomes sequenced, there is variation in size and sequence pointing to divergent evolution patterns within the species. The intronic regions show a high degree of sequence variation compared to the conserved sequences of the 14 core genes. The intronic ORFs identified, regardless of species, encode GIY-YIG or LAGLIDADG domain-containing homing endonuclease genes. Phylogenetic relationships using the 14 core proteins largely mimic the phylogenetic relationships observed in gene order patterns, grouping M. tenuissimus with M. crinis-equi, and M. palmivorus with Mon. roreri and Mon. perniciosa, leaving Mar. scandens as an outlier. The results from this study provide evidence of independent expansion/contraction events and sequence diversification in each species and establish a foundation for further exploration of the evolutionary trajectory of the fungi in Marasmiaceae family.

Changes in Gene Expression in Leaves of Cacao Genotypes Resistant and Susceptible to Phytophthora palmivora Infection.

Black pod rot, caused by Phytophthora palmivora, is a devastating disease of Theobroma cacao L. (cacao) leading to huge losses for farmers and limiting chocolate industry supplies. To understand resistance responses of cacao leaves to P. palmivora, Stage 2 leaves of genotypes Imperial College Selection 1 (ICS1), Colección Castro Naranjal 51 (CCN51), and Pound7 were inoculated with zoospores and monitored for symptoms up to 48 h. Pound7 consistently showed less necrosis than ICS1 and CCN51 48 h after inoculation. RNA-Seq was carried out on samples 24 h post inoculation. A total of 24,672 expressed cacao genes were identified, and 2,521 transcripts showed induction in at least one P. palmivora-treated genotype compared to controls. There were 115 genes induced in the P. palmivora-treated samples in all three genotypes. Many of the differentially expressed genes were components of KEGG pathways important in plant defense signal perception (the plant MAPK signaling pathway, plant hormone signal transduction, and plant pathogen interactions), and plant defense metabolite biosynthesis (phenylpropanoid biosynthesis, α-linolenic acid metabolism, ethylene biosynthesis, and terpenoid backbone biosynthesis). A search of putative cacao resistance genes within the cacao transcriptome identified 89 genes with prominent leucine-rich repeat (LRR) domains, 170 protein kinases encoding genes, 210 genes with prominent NB-ARC domains, 305 lectin-related genes, and 97 cysteine-rich RK genes. We further analyzed the cacao leaf transcriptome in detail focusing on gene families-encoding proteins important in signal transduction (MAP kinases and transcription factors) and direct plant defense (Germin-like, ubiquitin-associated, lectin-related, pathogenesis-related, glutathione-S-transferases, and proteases). There was a massive reprogramming of defense gene processes in susceptible cacao leaf tissue after infection, which was restricted in the resistant genotype Pound7. Most genes induced in Pound7 were induced in ICS1/CCN51. The level of induction was not always proportional to the infection level, raising the possibility that genes are responding to infection more strongly in Pound7. There were also defense-associated genes constitutively differentially expressed at higher levels in specific genotypes, possibly providing a prepositioned defense. Many of the defense genes occur in blocks where members are constitutively expressed at different levels, and some members are induced by Ppal infection. With further study, the identified candidate genes and gene blocks may be useful as markers for breeding disease-resistant cacao genotypes against P. palmivora.

Identification and Characterization of Fungi Causing Thread Blight Diseases on Cacao in Ghana.

Theobroma cacao (chocolate tree) is currently under serious threat from thread blight disease (TBD), which has been attributed to the causal agent Marasmiellus scandens in other regions of the world. TBD in Ghana has similar symptomology but variable signs. This study sought to determine whether TBD in Ghana was caused by a single agent and whether Marasmiellus scandens was a significant agent of TBD. Forty-eight isolates were collected from eight geographical locations in Ghana for morphological and molecular characterization. Disease signs occurred as vegetative rhizomorphs or hyphal aggregates, which were classified into five morphotypes: A, abundant thin, black, "horse hair"-type rhizomorphs; B, scattered brown rhizomorphs; C, whitish to brownish-white; D, faint cream or dull white; and E, aggregates of shiny or silky white hyphae. Sequencing and analyses of three loci-the internal transcribed spacer region of the nuclear ribosomal repeat, nuclear large subunit, and mitochondrial small subunit-detected four species, all members of the Marasmiaceae, causing TBD-like disease. These were identified as Marasmius crinis-equi (morphotype A), Marasmius tenuissimus (morphotypes B and C), Marasmiellus palmivorus (morphotype E), and Marasmiellus scandens (morphotype D). Marasmius tenuissimus, the most frequently isolated TBD fungus in this study, is primarily an Asian fungus and not previously associated with diseases of cacao. Marasmiellus palmivorus, the second most frequently isolated fungus, is a pan-tropical pathogen with a broad host range; this is the first report of the fungus causing TBD on cacao. Marasmius crinis-equi also has a broad pan-tropical distribution and host range and causes thread blight on several tropical tree crops. Surprisingly, Marasmiellus scandens, the most frequently cited agent of TBD in cacao, made up only 8% of the isolates.

Independent Whole-Genome Duplications Define the Architecture of the Genomes of the Devastating West African Cacao Black Pod Pathogen Phytophthora megakarya and Its Close Relative Phytophthora palmivora.

Phytophthora megakarya and P. palmivora are oomycete pathogens that cause black pod rot of cacao (Theobroma cacao), the most economically important disease on cacao globally. While P. palmivora is a cosmopolitan pathogen, P. megakarya, which is more aggressive on cacao than P. palmivora, has been reported only in West and Central Africa where it has been spreading and devastating cacao farms since the 1950s. In this study, we reconstructed the complete diploid genomes of multiple isolates of both species using single-molecule real-time sequencing. Thirty-one additional genotypes were sequenced to analyze inter- and intra-species genomic diversity. The P. megakarya genome is exceptionally large (222 Mbp) and nearly twice the size of P. palmivora (135 Mbp) and most known Phytophthora species (∼100 Mbp on average). Previous reports pointed toward a whole-genome duplication (WGD) in P. palmivora In this study, we demonstrate that both species underwent independent and relatively recent WGD events. In P. megakarya we identified a unique combination of WGD and large-scale transposable element driven genome expansion, which places this genome in the upper range of Phytophthora genome sizes, as well as effector pools with 1,382 predicted RxLR effectors. Finally, this study provides evidence of adaptive evolution of effectors like RxLRs and Crinklers, and discusses the implications of effector expansion and diversification.

Genome and transcriptome analysis of the latent pathogen Lasiodiplodia theobromae, an emerging threat to the cacao industry.

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.

Draft genome sequence of fastidious pathogen Ceratobasidium theobromae, which causes vascular-streak dieback in Theobroma cacao.

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.

Chocolate Under Threat from Old and New Cacao Diseases.

Theobroma cacao, the source of chocolate, is affected by destructive diseases wherever it is grown. Some diseases are endemic; however, as cacao was disseminated from the Amazon rain forest to new cultivation sites it encountered new pathogens. Two well-established diseases cause the greatest losses: black pod rot, caused by several species of Phytophthora, and witches' broom of cacao, caused by Moniliophthora perniciosa. Phytophthora megakarya causes the severest damage in the main cacao producing countries in West Africa, while P. palmivora causes significant losses globally. M. perniciosa is related to a sister basidiomycete species, M. roreri which causes frosty pod rot. These Moniliophthora species only occur in South and Central America, where they have significantly limited production since the beginnings of cacao cultivation. The basidiomycete Ceratobasidium theobromae causing vascular-streak dieback occurs only in South-East Asia and remains poorly understood. Cacao swollen shoot disease caused by Cacao swollen shoot virus is rapidly spreading in West Africa. This review presents contemporary research on the biology, taxonomy and genomics of what are often new-encounter pathogens, as well as the management of the diseases they cause.

Morphological variants of Moniliophthora roreri on artificial media and the biotroph/necrotroph shift.

Moniliophthora roreri (Mr) causes frosty pod rot of Theobroma cacao in a hemibiotrophic association. The Mr biotroph-like phase has not been studied in culture. Mr spores (isolates Co12, Co52, and B3) were germinated on high (V8) and low (BPMM) nutrients with different media hardness (0.5% to 3% agarose). Germination was high on V8 media. Hardness affected germination on BPMM. Most colonies on V8 were slow-growing, failing to sporulate. Colony morphology depended on the isolate. On BPMM, exaggerated mycelia formed of limited length with enlarged cells. On agarose, rapidly expanding sporulating necrotrophic colonies formed rarely. Co12 and B3 spores were germinated on V8 and BPMM with low melting point (LMP) agarose. Slow-growing colonies of B3 on BPMM were unstable on LMP agarose, often forming slow-growing/rapidly expanding hybrids. Slow-growing colonies are hypothesized to represent the biotrophic phase. One nucleus was common in Mr cells, other than spores. Binucleate cells were occasionally observed in aged cells of slow-growing mycelia. Co52 cells often had more than two nuclei per cell after germination. Mr mycelia cells typically carry a single nucleus, being considered haploid. Biotroph- and necrotroph-like mycelia displayed differential gene expression but results were inconsistent with published in vivo results and require further study.

Moniliophthora roreri, causal agent of cacao frosty pod rot.

Taxonomy: Moniliophthora roreri (Cif.) H.C. Evans et al. ; Phylum Basidiomycota; Class Agaricomycetes; Order Agaricales; Family Marasmiaceae; Genus Moniliophthora. Biology: Moniliophthora roreri attacks Theobroma and Herrania species causing frosty pod rot. Theobroma cacao (cacao) is the host of major economic concern. Moniliophthora roreri is a hemibiotroph with a long biotrophic phase (45-90 days). Spore masses, of apparent asexual origin, are produced on the pod surface after initiation of the necrotrophic phase. Spores are spread by wind, rain and human activity. Symptoms of the biotrophic phase can include necrotic flecks and, in some cases, pod malformation, but pods otherwise remain asymptomatic. Relationship to Moniliophthora perniciosa: Moniliophthora roreri and Moniliophthora perniciosa, causal agent of witches' broom disease of cacao, are closely related. Their genomes are similar, including many of the genes they carry which are considered to be important in the disease process. Moniliophthora perniciosa, also a hemibiotroph, has a typical basidiomycete lifestyle and morphology, forming clamp connections and producing mushrooms. Basidiospores infect meristematic tissues including flower cushions, stem tips and pods. Moniliophthora roreri does not form clamp connections or mushrooms and infects pods only. Both pathogens are limited to the Western Hemisphere and are a threat to cacao production around the world. Agronomic importance: Disease losses caused by frosty pod rot can reach 90% and result in field abandonment. Moniliophthora roreri remains in the invasive phase in the Western Hemisphere, not having reached Brazil, some islands within the Caribbean and a few specific regions within otherwise invaded countries. DISEASE MANAGEMENT: The disease can be managed by a combination of cultural (for example, maintenance of tree height and removal of infected pods) and chemical methods. These methods benefit from regional application, but can be cost prohibitive. Breeding for disease resistance offers the greatest potential for frosty pod rot management and new tolerant materials are becoming available.

Phytophthora megakarya and P. palmivora, Causal Agents of Black Pod Rot, Induce Similar Plant Defense Responses Late during Infection of Susceptible Cacao Pods.

Phytophthora megakarya (Pmeg) and Phytophthora palmivora (Ppal) cause black pod rot of Theobroma cacao L. (cacao). Of these two clade 4 species, Pmeg is more virulent and is displacing Ppal in many cacao production areas in Africa. Symptoms and species specific sporangia production were compared when the two species were co-inoculated onto pod pieces in staggered 24 h time intervals. Pmeg sporangia were predominantly recovered from pod pieces with unwounded surfaces even when inoculated 24 h after Ppal. On wounded surfaces, sporangia of Ppal were predominantly recovered if the two species were simultaneously applied or Ppal was applied first but not if Pmeg was applied first. Pmeg demonstrated an advantage over Ppal when infecting un-wounded surfaces while Ppal had the advantage when infecting wounded surfaces. RNA-Seq was carried out on RNA isolated from control and Pmeg and Ppal infected pod pieces 3 days post inoculation to assess their abilities to alter/suppress cacao defense. Expression of 4,482 and 5,264 cacao genes was altered after Pmeg and Ppal infection, respectively, with most genes responding to both species. Neural network self-organizing map analyses separated the cacao RNA-Seq gene expression profiles into 24 classes, 6 of which were largely induced in response to infection. Using KEGG analysis, subsets of genes composing interrelated pathways leading to phenylpropanoid biosynthesis, ethylene and jasmonic acid biosynthesis and action, plant defense signal transduction, and endocytosis showed induction in response to infection. A large subset of genes encoding putative Pr-proteins also showed differential expression in response to infection. A subset of 36 cacao genes was used to validate the RNA-Seq expression data and compare infection induced gene expression patterns in leaves and wounded and unwounded pod husks. Expression patterns between RNA-Seq and RT-qPCR were generally reproducible. The level and timing of altered gene expression was influenced by the tissues studied and by wounding. Although, in these susceptible interactions gene expression patterns were similar, some genes did show differential expression in a Phytophthora species dependent manner. The biggest difference was the more intense changes in expression in Ppal inoculated wounded pod pieces further demonstrating its rapid progression when penetrating through wounds.

Combination of RNAseq and SNP nanofluidic array reveals the center of genetic diversity of cacao pathogen Moniliophthora roreri in the upper Magdalena Valley of Colombia and its clonality.

Moniliophthora roreri is the fungal pathogen that causes frosty pod rot (FPR) disease of Theobroma cacao L., the source of chocolate. FPR occurs in most of the cacao producing countries in the Western Hemisphere, causing yield losses up to 80%. Genetic diversity within the FPR pathogen population may allow the population to adapt to changing environmental conditions and adapt to enhanced resistance in the host plant. The present study developed single nucleotide polymorphism (SNP) markers from RNASeq results for 13 M. roreri isolates and validated the markers for their ability to reveal genetic diversity in an international M. roreri collection. The SNP resources reported herein represent the first study of RNA sequencing (RNASeq)-derived SNP validation in M. roreri and demonstrates the utility of RNASeq as an approach for de novo SNP identification in M. roreri. A total of 88 polymorphic SNPs were used to evaluate the genetic diversity of 172 M. roreri cacao isolates resulting in 37 distinct genotypes (including 14 synonymous groups). Absence of heterozygosity for the 88 SNP markers indicates reproduction in M. roreri is clonal and likely due to a homothallic life style. The upper Magdalena Valley of Colombia showed the highest levels of genetic diversity with 20 distinct genotypes of which 13 were limited to this region, and indicates this region as the possible center of origin for M. roreri.

Differential gene expression by Moniliophthora roreri while overcoming cacao tolerance in the field.

Frosty pod rot (FPR) of Theobroma cacao (cacao) is caused by the hemibiotrophic fungus Moniliophthora roreri. Cacao clones tolerant to FPR are being planted throughout Central America. To determine whether M. roreri shows a differential molecular response during successful infections of tolerant clones, we collected field-infected pods at all stages of symptomatology for two highly susceptible clones (Pound-7 and CATIE-1000) and three tolerant clones (UF-273, CATIE-R7 and CATIE-R4). Metabolite analysis was carried out on clones Pound-7, CATIE-1000, CATIE-R7 and CATIE-R4. As FPR progressed, the concentrations of sugars in pods dropped, whereas the levels of trehalose and mannitol increased. Associations between symptoms and fungal loads and some organic and amino acid concentrations varied depending on the clone. RNA-Seq analysis identified 873 M. roreri genes that were differentially expressed between clones, with the primary difference being whether the clone was susceptible or tolerant. Genes encoding transcription factors, heat shock proteins, transporters, enzymes modifying membranes or cell walls and metabolic enzymes, such as malate synthase and alternative oxidase, were differentially expressed. The differential expression between clones of 43 M. roreri genes was validated by real-time quantitative reverse transcription polymerase chain reaction. The expression profiles of some genes were similar in susceptible and tolerant clones (other than CATIE-R4) and varied with the biotrophic/necrotropic shift. Moniliophthora roreri genes associated with stress metabolism and responses to heat shock and anoxia were induced early in tolerant clones, their expression profiles resembling that of the necrotrophic phase. Moniliophthora roreri stress response genes, induced during the infection of tolerant clones, may benefit the fungus in overcoming cacao defense mechanisms.

Successful pod infections by Moniliophthora roreri result in differential Theobroma cacao gene expression depending on the clone's level of tolerance.

An understanding of the tolerance mechanisms of Theobroma cacao used against Moniliophthora roreri, the causal agent of frosty pod rot, is important for the generation of stable disease-tolerant clones. A comparative view was obtained of transcript populations of infected pods from two susceptible and two tolerant clones using RNA sequence (RNA-Seq) analysis. A total of 3009 transcripts showed differential expression among clones. KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis of differentially expressed genes indicated shifts in 152 different metabolic pathways between the tolerant and susceptible clones. Real-time quantitative reverse transcription polymerase chain reaction (real-time qRT-PCR) analyses of 36 genes verified the differential expression. Regression analysis validated a uniform progression in gene expression in association with infection levels and fungal loads in the susceptible clones. Expression patterns observed in the susceptible clones diverged in tolerant clones, with many genes showing higher expression at a low level of infection and fungal load. Principal coordinate analyses of real-time qRT-PCR data separated the gene expression patterns between susceptible and tolerant clones for pods showing malformation. Although some genes were constitutively differentially expressed between clones, most results suggested that defence responses were induced at low fungal load in the tolerant clones. Several elicitor-responsive genes were highly expressed in tolerant clones, suggesting rapid recognition of the pathogen and induction of defence genes. Expression patterns suggested that the jasmonic acid-ethylene- and/or salicylic acid-mediated defence pathways were activated in the tolerant clones, being enhanced by reduced brassinosteroid (BR) biosynthesis and catabolic inactivation of both BR and abscisic acids. Finally, several genes associated with hypersensitive response-like cell death were also induced in tolerant clones.

Genome and secretome analysis of the hemibiotrophic fungal pathogen, Moniliophthora roreri, which causes frosty pod rot disease of cacao: mechanisms of the biotrophic and necrotrophic phases.

BACKGROUND: The basidiomycete Moniliophthora roreri is the causal agent of Frosty pod rot (FPR) disease of cacao (Theobroma cacao), the source of chocolate, and FPR is one of the most destructive diseases of this important perennial crop in the Americas. This hemibiotroph infects only cacao pods and has an extended biotrophic phase lasting up to sixty days, culminating in plant necrosis and sporulation of the fungus without the formation of a basidiocarp. RESULTS: We sequenced and assembled 52.3 Mb into 3,298 contigs that represent the M. roreri genome. Of the 17,920 predicted open reading frames (OFRs), 13,760 were validated by RNA-Seq. Using read count data from RNA sequencing of cacao pods at 30 and 60 days post infection, differential gene expression was estimated for the biotrophic and necrotrophic phases of this plant-pathogen interaction. The sequencing data were used to develop a genome based secretome for the infected pods. Of the 1,535 genes encoding putative secreted proteins, 1,355 were expressed in the biotrophic and necrotrophic phases. Analysis of the data revealed secretome gene expression that correlated with infection and intercellular growth in the biotrophic phase and invasive growth and plant cellular death in the necrotrophic phase. CONCLUSIONS: Genome sequencing and RNA-Seq was used to determine and validate the Moniliophthora roreri genome and secretome. High sequence identity between Moniliophthora roreri genes and Moniliophthora perniciosa genes supports the taxonomic relationship with Moniliophthora perniciosa and the relatedness of this fungus to other basidiomycetes. Analysis of RNA-Seq data from infected plant tissues revealed differentially expressed genes in the biotrophic and necrotrophic phases. The secreted protein genes that were upregulated in the biotrophic phase are primarily associated with breakdown of the intercellular matrix and modification of the fungal mycelia, possibly to mask the fungus from plant defenses. Based on the transcriptome data, the upregulated secreted proteins in the necrotrophic phase are hypothesized to be actively attacking the plant cell walls and plant cellular components resulting in necrosis. These genes are being used to develop a new understanding of how this disease interaction progresses and to identify potential targets to reduce the impact of this devastating disease.

The mitochondrial genome of Moniliophthora roreri, the frosty pod rot pathogen of cacao.

In this study, we report the sequence of the mitochondrial (mt) genome of the Basidiomycete fungus Moniliophthora roreri, which is the etiologic agent of frosty pod rot of cacao (Theobroma cacao L.). We also compare it to the mtDNA from the closely-related species Moniliophthora perniciosa, which causes witches' broom disease of cacao. The 94 Kb mtDNA genome of M. roreri has a circular topology and codes for the typical 14 mt genes involved in oxidative phosphorylation. It also codes for both rRNA genes, a ribosomal protein subunit, 13 intronic open reading frames (ORFs), and a full complement of 27 tRNA genes. The conserved genes of M. roreri mtDNA are completely syntenic with homologous genes of the 109 Kb mtDNA of M. perniciosa. As in M. perniciosa, M. roreri mtDNA contains a high number of hypothetical ORFs (28), a remarkable feature that make Moniliophthoras the largest reservoir of hypothetical ORFs among sequenced fungal mtDNA. Additionally, the mt genome of M. roreri has three free invertron-like linear mt plasmids, one of which is very similar to that previously described as integrated into the main M. perniciosa mtDNA molecule. Moniliophthora roreri mtDNA also has a region of suspected plasmid origin containing 15 hypothetical ORFs distributed in both strands. One of these ORFs is similar to an ORF in the mtDNA gene encoding DNA polymerase in Pleurotus ostreatus. The comparison to M. perniciosa showed that the 15 Kb difference in mtDNA sizes is mainly attributed to a lower abundance of repetitive regions in M. roreri (5.8 Kb vs 20.7 Kb). The most notable differences between M. roreri and M. perniciosa mtDNA are attributed to repeats and regions of plasmid origin. These elements might have contributed to the rapid evolution of mtDNA. Since M. roreri is the second species of the genus Moniliophthora whose mtDNA genome has been sequenced, the data presented here contribute valuable information for understanding the evolution of fungal mt genomes among closely-related species.

Endophytic Trichoderma isolates from tropical environments delay disease onset and induce resistance against Phytophthora capsici in hot pepper using multiple mechanisms.

Endophytic Trichoderma isolates collected in tropical environments were evaluated for biocontrol activity against Phytophthora capsici in hot pepper (Capsicum annuum). Six isolates were tested for parasitic and antimicrobial activity against P. capsici and for endophytic and induced resistance capabilities in pepper. Isolates DIS 70a, DIS 219b, and DIS 376f were P. capsici parasites, while DIS 70a, DIS 259j, DIS 320c, and DIS 376f metabolites inhibited P. capsici. All six isolates colonized roots but were inefficient stem colonizers. DIS 259j, DIS 320c, and DIS 376f induced defense-related expressed sequence tags (EST) in 32-day-old peppers. DIS 70a, DIS 259j, and DIS 376f delayed disease development. Initial colonization of roots by DIS 259j or DIS 376f induced EST with potential to impact Trichoderma endophytic colonization and disease development, including multiple lipid transferase protein (LTP)-like family members. The timing and intensity of induction varied between isolates. Expression of CaLTP-N, encoding a LTP-like protein in pepper, in N. benthamiana leaves reduced disease development in response to P. nicotianae inoculation, suggesting LTP are functional components of resistance induced by Trichoderma species. Trichoderma isolates were endophytic on pepper roots in which, depending on the isolate, they delayed disease development by P. capsici and induced strong and divergent defense reactions.