Pod Rot Of Cacao Research Articles

First report of Diaporthe tulliensis causing leaf spot on Millettia speciosa in China.

Millettia speciosa Champ, renowned for its diverse applications in traditional medicine, is extensively cultivated in the Guangxi region of China, spanning roughly 5,973 hectares. In July 2021, a plantation in Yulin, Guangxi, China (22°64'N; 110°29'E), exhibited severe leaf spot disease on M. speciosa. Notably, a 46,690 square meters area had over 40% leaf spot incidence. Initially, symptoms appeared as small, circular, pale-yellow lesions on the leaves, then turned into irregular, dark brown spots with yellow halos, leading to the wilt and defoliation of leaves. To identify the responsible pathogen, a total of five symptomatic leaves were collected and sterilized systematically. Small tissue segments (5×5 mm) from lesion peripheries were aseptically excised, then surface sterilized with 75% ethanol for 10 s, and 1% sodium hypochlorite (NaClO) for 3 min. Following this, the sterilized tissues were triple-rinsed with sterile water and cultured on potato dextrose agar (PDA) at 28 °C in the dark for 7 d. A total of seven isolates were obtained through single-spore isolation, and one representative isolate, N2-3, was selected for further analysis. After 7 d of incubation, colonies displayed flat, white, and extensively branched aerial hyphae. Over time, the reverse side of the colony changed from white to yellowish-white. The pycnidia were black with conidial droplets ranging from cream to pale yellow exuding from their ostioles. The α-conidia were one-celled, hyaline, ovoid to cylindrical, typically with one or two droplets, 2.6 to 5.9 ×1.4 to 3.9 μm (n=50). These morphological traits align with those of the genus Diaporthe, as reported by Li et al. (2022) and Crous et al. (2015). To identify the species, isolate N2-3 underwent sequencing of the internal transcribed spacer (ITS), β-tubulin (BT), and translation elongation factor 1 alpha (EF1-α) sections (Huang et al. 2021). Obtained sequences of ITS, BT and EF1-α (Genebank accessions nos. OR600532, OR662169 and OR662168) displayed a 99% similarity to Diaporthe tulliensis (Genebank accessions nos. OP219651, ON932382, OL412437, respectively). Based on the concatenated ITS, BT and EF1-α, a neighbor-joining phylogenetic analyses using MEGA7.0 clustered with D. tulliensis. Therefore, the fungus was identified as D. tulliensis (teleomorph name) based on morphological and molecular features. A pathogenicity test was conducted on 1-year-old M. speciosa seedlings by gently abrading healthy leaves with sterilized toothpicks to create superficial wounds. Wounded leaves were then inoculated with 5 mm diameter mycelial plugs, while control seedlings received PDA plugs. Three leaves per plant and five plants per treatment were selected for assessment. All seedlings were kept in a controlled greenhouse (12/12h light/dark, 25 ± 2 °C, 90% humidity). After 7 d, the inoculated leaves showed symptoms like those in the field, while control plants remained healthy. The fungus was consistently reisolated from the infected leaves, satisfying Koch's postulates. Notably, D. tulliensis has caused Boston ivy leaf spot, bodhi tree leaf spot, cacao pod rot, and jasmine stem canker (Huang et al. 2021; Li et al. 2022; Serrato-Diaz et al. 2022; Hsu et al. 2023). This discovery is significant as it marks the first report of Diaporthe tulliensis causing leaf spot on Millettia speciossa in China, which has direct implications for the development of diagnostic tools and research into potential disease management strategies.

Description and Pathogenicity of Colletotrichum kapreanum sp. nov, a Cherelle Wilt Pathogen Belonging to the Gigasporum Species Complex.

Similar to cacao pod rot, cherelle wilt decreases production from cacao fields. Among all known fungal pathogens of the cacao, Colletotrichum spp. are common infectious agents that affect the cherelles and pods of cacao; thus, cacao diseases are often classified by stage. Therefore, knowing whether these pathogens are common in both fruit stages is necessary for implementing disease control measures. Symptoms of cherelle wilt were found in cacao plants in Pangasinan, Philippines, in 2022. The fungal strain obtained from the lesion was found to be pathogenic towards cherelles, but not towards pods. The strain was classified as an unknown species belonging to the gigasporum species complex, based on the morphological and molecular phylogenetic analyses of ITS, GAPDH, CHS1, ACT, and TUB2. We propose Colletotrichum kapreanum sp. nov. as a causal agent of cacao cherelle wilt, but not pod rot.

DNA Barcoding of Cacao Pod Rot Fungi in Papua Province

Cocoa (Theobroma cacao) pod rot fungi have various types. In general, cause cocoa pod rot are fungi of the species Fusarium sp, Rhizoctonia sp, Phytium sp, Colletotricum sp, Amerosporium sp, Phomopsis sp and Phytopthora sp. Various species of fungi that can rot cocoa pods have high similarity in morphology characters even though the treatment for each species of fungi is different. This problem occurs in the people's nucleus cocoa plantations in the Yapsi district, Jayapura, Papua province. Samples of cacao pods infected with the fungus were taken and the isolates obtained were reinoculated into uninfected cacao pods. Isolates showing dominant infectivity were taken and identified molecularly. Identification was carried out by creating DNA barcoding by sequencing the Internal Transcribed Spacer (ITS) region of the 18SrDNA marker gene from the fungal isolates SP4DYAPSI-1, SP4 DYAPSI-2, SP3DYAPSI-1 and SP6DAUN3. Sequencing data in the form of isolate fungi DNA barcodes were analyzed with the ClustalX program analyses. The resulting phylogenetic tree construction shows that the four isolates are related to the fungus species Fusarium solani with a nucleotide similarity index above 95%. This shows that the cause of damage to cocoa pods in the Yapsi area, Jayapura, Papua, Indonesia is the fungus Fusarium solani.

First Report of Diaporthe tulliensis Causing Necrotic Spots and Leaf Blight on Rambutan in Puerto Rico.

Rambutan (Nepehelium lappaceum) is a tropical exotic fruit belonging to the Sapindaceae family. Several pathogens have been identified in rambutan causing different diseases on fruits, inflorescences, and branches (Serrato-Diaz et al., 2015, 2017, 2020) but few on leaves. From 2015 to 2021, a disease survey was conducted in one greenhouse in Mayaguez, Puerto Rico and experimental rambutan field orchards of the USDA-ARS Tropical Agriculture Research Station located at Isabela, Corozal, Santa Isabel, and Adjuntas, Puerto Rico (Latitude: 18°12'28"N, 18°34'10'' N, 18°00'47''N, 18°16'35''N and Longitude: 67°08'17"W, 66°31'74'' W, 66°38'98''W, 66°72'32''W, respectively). Varieties Benjai, Gula Batu, Jitlee, R-134, R-156Y, R-162, R-167 and Rongren were sampled. Necrotic spots and leaf blight were commonly observed with a disease incidence of 80%. Diseased leaves showed necrosis starting from the apex and spreading through the lamina. Ten diseased leaves were collected from each location and sections of symptomatic tissue (5mm2) were disinfected and plated on potato dextrose agar (PDA) and oatmeal agar (OA). Two representative isolates of Diaporthe tulliensis, A3 and A4, were obtained, purified, and identified morphologically and by PCR amplifications of three nuclear genes of the Internal Transcribed Spacer ITS1-5.8S-ITS2 region of the ribosomal DNA primers ITS5/ITS4, portions of the β tubulin (BT) primers Bt2a/Bt2b and the translation elongation factor 1-α (TEF1-α) primers EF1728F/EF1986R. On PDA and OA colonies of isolates A3 and A4 were initially white and flat with sparse mycelia that turned yellowish-white to grey with age. Pycnidia were black with cream to pale yellow conidial droplets that exuded from ostioles. Hyaline, unicellular alpha conidia were oval to cylindrical, rounded at apex and obconically truncate at base. Alpha conidia (n = 50) for isolates of D. tulliensis were 4.9 to 5.9 μm long by 2.2 - 2.5 μm wide. DNA sequences of the ITS region and partial sequences of TEF1-α and BT genes were compared by BLASTN with Diaporthe sequences deposited in GenBank. ITS, BT and EF1-α sequences of isolates A3 and A4 (OP219651 and OP161553 for ITS region; OP222137 and OP168832 for TEF1-α; OP222136 and OP168831 for BT, respectively) were grouped to the holotype BRIP 62248a (Bootstrap BS=100) of Diaporthe tulliensis R.G. Shivas, Vawdrey & Y.P. Tan. Pathogenicity tests were conducted on six of six-months-old rambutan seedlings of R-167 variety. Three unwounded healthy non-detached leaves were inoculated per isolate with one 5mm mycelial disk from pure cultures grown on PDA. Rambutan seedlings were kept in a humid chamber using plastic bags for 8 days under greenhouse conditions. Two of six seedlings were used as controls and inoculated with PDA disks only. Eight and 14 days after inoculation (DAI), D. tulliensis isolates caused necrotic spots and leaf blight, on leaves. Diseased leaves turned from light to dark brown starting from the apex and spreading through the lamina with necrotic lesions ranging in size from 5 - 10 mm. Untreated controls showed no symptoms, and no fungi were re-isolated from tissue. D. tulliensis was re-isolated from diseased leaves, fulfilling Koch's postulates. D. tulliensis has been reported in Taiwan causing Diaporthe leaf spot in Boston Ivy (Huang, C. et al., 2021) and Bodhi trees (Li, K.Y. et al., 2022), as well as Jasmin stem canker (Ching Hsu, C. et al., 2022). It has been reported as causing leaf blight of coffee (Gong, J. L., et al., 2019), kiwifruit stem canker in China (Bai et al., 2017), and most recently causing cacao pod rot in Puerto Rico (Serrato-Diaz, L.M. et al., 2022). To our knowledge, this is the first report of Diaporthe tulliensis causing necrotic spots and leaf light on rambutan in Puerto Rico and often associated with a potassium deficiency in many parts of the world. It will be important to establish an adequate and effective control management of this disease in rambutan producing countries worldwide. References and doi hyperlinks: 1. Huang, C. et al. Plant Dis. 105:2718, 2021 https://doi.org/10.1094/PDIS-12-20-2652-PDN 2. Li, K.Y. et al. Plant Dis. 0:ja, 2022 https://doi.org/10.1094/PDIS-01-22-0211-PDN 3. Ching Hsu, C. et al. Plant Dis. 0:ja, 2022 https://doi.org/10.1094/PDIS-09-21-1908-PDN 4. Gong, J. L., et al. Plant Dis. 104:570, 2019 https://doi.org/10.1094/PDIS-09-19-1833-PDN 5. Bai et al. Plant Dis. 101:508, 2017 https://doi.org/10.1094/PDIS-10-16-1445-PDN 6. Serrato-Diaz L.M., et al. 2015. Plant Dis. 99: 1187. https://doi.org/10.1094/PDIS-09-14-0923-PDN 7. Serrato-Diaz L.M. et al. 2017. Plant Dis. 101: 1043. https://doi.org/10.1094/PDIS-11-16-1557-PDN 8. Serrato-Diaz, L.M., et al. 2020. Plant Dis. 104: 105-115. https://doi.org/10.1094/PDIS-02-19-0295-RE 9. Serrato-Diaz, L.M. et al. 2022 Plant Dis. 106: 2530. https://doi.org/10.1094/PDIS-12-21-2634-PDN.

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.

First report of Diaporthe tulliensis causing leaf spot of betel vine

The betel vine (Piper betle) is grown in protected areas under the humid and shaded conditions required for the plant's growth. This damp, shady environment favours the spread of numerous diseases, particularly leaf spot disease (Chattopadhayay & Maity, 1990). During 2019–2022, diseased betel leaves were collected from the East Khasi Hills and Ri Bhoi districts of Meghalaya, India. The leaves had spots that were irregular in shape and size, light to dark brown and surrounded by a chlorotic yellow halo, which gradually spread to the leaf centre followed by yellowing, drying and drooping of the whole leaf. An area of approximately of 8 m2 was affected and 5–20% of the plants had symptoms, depending on the year. The number of leaf spots increased with temperature and humidity. To identify the causal agent, lesions (5-10 mm in diameter) were cut from ten infected leaves (Figure 1), surface disinfected with 70% ethanol, rinsed three times in sterile distilled water and then plated on potato dextrose agar (PDA) amended with 30mg/ml of streptomycin sulphate. The same colony type was obtained from all leaves which was white producing yellowish-green pigments and the reverse olive green to brown after seven days (Figure 2A-B) and after 30 days of incubation at 30°C, produced semi-immersed black pycnidial conidiomata which exuded pale yellow to black conidial droplets (Figure 2C). The α-conidia were one to two-celled, hyaline, oval to fusiform, guttulate and 5.3 × 2.5 μm (Figure 2D), conidiophores hyaline but no β- and γ-conidia were observed (Li et al., 2022), and conidiogenous cells were hyaline and septate (Figure 2E-F). The identification of the fungus was confirmed by sequence analysis using ITS1 and ITS4 primers (White et al., 1990) (GenBank Accession No. OQ402579). BLAST analysis showed 100% identity with sequences of Diaporthe tulliensis (e.g. MN911384). In phylogenetic analysis, the isolate in the current study clustered with D. tulliensis using the neighbour-joining method, showing 100% identity (Figure 3). To confirm pathogenicity, the isolate was grown in potato dextrose broth at 28°C for seven days, then inoculated onto five healthy betel leaves using pin-prick inoculation with 10 μl spore suspension (1×105 spores/ml). The untreated control did not show any symptoms, however, the inoculated plants had brown necrotic spots followed by yellowing after four days (Figure 4). On PDA, cultures isolated from infected plant leaf lesions exhibited the same morphology as D. tulliensis and similar conidia were discovered during microscopic analysis subsequently satisfying Koch's postulates. To the best of our knowledge, this is the first report of D. tulliensis as a fungal pathogen of betel leaves. Diaporthe tulliensis has been reported to cause leaf spots on coffee in China (Gorg et al., 2020) and on Parthenocissus tricuspidata in Taiwan (Huang et al., 2021), cacao pod rot in Puerto Rico (Serrato-Diaz et al., 2022), and stem canker of kiwifruit in China (Bai et al., 2017) and of jasmine in Taiwan (Hsu et al., 2023). The authors thank the Head of the Department of Botany, North-Eastern Hill University for providing laboratory facilities. The first author is also grateful to the Non-Net Fellowship, New Delhi, for financial support in the form of a research fellowship.

First Report of Diaporthe tulliensis and D. pseudomangiferae causing cacao pod rot in Puerto Rico.

Worldwide cacao pod rot is a devastating disease of Theobroma cacao, infected cacao pods turn necrotic reducing yield up to 30%. From July 2020 to August 2021, a survey was conducted at the USDA-ARS cacao germplasm collection located at Mayaguez, Puerto Rico. Incidence of cacao pod rot was 73.9%, observed in 142 of the 196 accessions sampled. The disease was observed at different stages of pod development (small, green, mature pods, and dry mummified large pods). Diseased tissue from three cacao pods (1 mm2) per each cacao accession was surface disinfested by immersion in 70% ethanol for one minute, rinsed with sterile-distilled-water and plated on potato dextrose agar (PDA) amended with 250 mg/L ampicillin and 60 mg/L streptomycin. After 30 days of incubation at 25°C, seven isolates developing white fast-growing colonies with black-globose pycnidia were observed. All isolates produced hyaline, one-celled, biguttulate, and cylindrical and rounded at the apex α conidia of 5.1 to 7.3 µm × 2.5 to 3.0 µm in size and were identified as Diaporthe spp. (Gomes et al. 2013; Crous et al. 2015). To determine the species identity, seven isolates were sequenced of the internal transcribed spacer (ITS), sections of β-tubulin (BT) and translation elongation factor 1 alpha (EF1-α) and compared using the BLASTn with Diaporthe spp. type specimens deposited in NCBI GenBank. ITS, BT and EF1-α sequences of Phomocac16, Phomcac17, Phomcac18 and Phomcac21 isolates (GenBank accession nos. OL353698 to OL353701, OL412430 to OL412433, and OL412437 to OL412440 for ITS, BT and EF1-α, respectively) were grouped to the holotype BRIP 62248a (Bootstrap BS=100) of Diaporthe tulliensis R.G. Shivas, Vawdrey & Y.P. Tan. The other three isolates (Phomcac8P1, Phomcac8P3 and Phomcac8P4) were grouped to the ex-type (CBS 101339) of Diaporthe pseudomangiferae R.R. Gomes, Glienke & Crous, ITS, BT and EF1-α sequences of (GenBank accessions nos. OL353702 to OL353704, OL412434 to OL412436, and OL412441 to OL412443, for ITS, BT and EF1-α, respectively). Pathogenicity tests were conducted using isolates Phomocac16, Phomcac17, Phomcac18 and Phomcac21 of D. tulliensis and isolates Phomcac8P1, Phomcac 8P3 and Phomcac8P4 of D. pseudomangiferae on five healthy detached green, yellow and red pods of the following cacao varieties: TARS27, ICS16, ICS1, GS29, UF601, SIAL56, Amelonado, SIAL98, EET94, ICS129 and GNV58. Cacao pods were wounded and inoculated with 5-mm mycelial disks from 8-day-old pure cultures grown on PDA of each isolate and wrapped with parafilm. Untreated controls were inoculated with PDA disks only. Fruits were kept in a humid chamber for 8 days at 25°C. Tests were repeated twice. Eight days after inoculation with D. tulliensis and D. pseudomangiferae, all cacao pods turned dark brown, untreated controls showed no symptoms of pod rot, and no fungi were isolated from tissue. Both species, D. tulliensis and D. pseudomangiferae were reisolated from their respective diseased tissues fulfilling Koch's postulates. Diaporthe tulliensis has been reported from rotted stem ends of cacao pods in Australia (Crous et al. 2015), and D. pseudomangiferae was reported in a shipment of cacao seed pods in California; however, pathogenicity tests were not conducted at either location. In California D. pseudomangiferae is considered a quarantine pathogen with a temporary Q rating (Chitambar 2017). To our knowledge, this is the first report of D. tulliensis and D. pseudomangiferae causing cacao pod rot in Puerto Rico. Knowing the identity and incidence of these new cacao pathogens is the first step for developing specific control measures and potential sources for resistance to cacao pod rot caused by Diaporthe spp.

Phylogenetic analyses and cross‐infection studies of Phytophthora species infecting cacao and durian in South‐Central Mindanao, Philippines

AbstractPhytophthora pathogens have contributed largely to the production losses of plantation crops such as cacao and durian. Correct identification and knowledge of the diversity of the pathogen is a requisite in the formulation of effective management strategies. Fifty‐eight putative Phytophthora isolates were collected from cacao and durian orchards in South‐Central Mindanao, Philippines, with 43 from cacao and 15 from durian. Molecular identification of isolates based on DNA sequences of the internal transcribed spacer (ITS), partial β‐tubulin (β‐tub) and mitochondrial gene (cox2+spacer) regions combined with morphocultural characterization identified 57 Phytophthora palmivora isolates from cacao and durian typically showing variations in colony morphology and sporangial shape. However, these variations were not phylogenetically informative. One Phytophthora meadii (CDDS‐P3) causing pod rot of cacao was phylogenetically isolated from P. palmivora. Phylogenetic analysis of isolates using combined ITS, β‐tubulin (β‐tub) and mitochondrial (cox2+spacer) DNA regions showed an intraspecific variation within P. palmivora. Representative isolates of P. palmivora used for cross‐inoculation showed more aggressiveness on its original host with higher lesion diameter and less aggressiveness on its potential hosts with smaller lesion diameter on artificially inoculated detached plant part. All Phytophthora isolates were tested pathogenic to cacao, durian and its potential hosts (rubber and coconut); therefore, cross‐infection in the field is possible in an intercrop system. Implementation of combined control strategies should be done against highly diverse pathogens such as P. palmivora and the potentially destructive pathogen P. meadii, which was tested pathogenic and virulent to its original (cacao) and potential hosts (coconut, durian and rubber).

Morphological, molecular and pathogenic characterization of Phytophthora palmivora isolates causing black pod rot of cacao in Colombia

Aim of study: To characterize isolates of Phytophthora sp. causing black pod rot (BPR) of cacao (Theobroma cacao L.).Area of study: Eight cocoa-growing regions in Colombia.Material and methods: Sixty isolates of Phytophthora sp. were obtained from tissues of cacao pods showing symptoms of BPR. Isolates were characterized using the morphology of sporangia and chlamydospores, molecular sequencing of regions of nuclear DNA (rDNA-ITS) and mitochondrial (COX) and virulence in different genotypes of cocoa pods.Main results: A high phenotypic variability between the isolates was determined, being the pedicel length and the length/width ratio (L/W) the most stable characters for species identification. Short pedicels with an average of 3.13 μm ± 0.28 and a length/width ratio of sporangia (L/W) with an average of 1.55 μm ± 0.11 were established as the most consistent morphological characteristics within palmivora species.Research highlights: Phytophthora pamivora was the only species associated to BPR, identified using morphology together with sequence analyses.

PENGARUH PENYARUNGAN DENGAN BERBAGAI KETEBALAN PLASTIK DAN DIAMETER BUAH TERHADAP SERANGAN PENGGEREK BUAH KAKAO Conopomorha cramerella (Snellen) (LEPIDOPTERA : GRACILLARIDAE)

This research is aimed to find out the preference of cocoa pod borer to diameter, biophysical, and biochemistry cocoa pod; the percentage of infested pods, percentage of seed damaged, and percentage of yield lost. In addition, this research is also aimed to assess the effect of plastic thickness on cacao pod rot disease, the effect of sheathing on the black ant and mealybug populations on pods. This research was conducted at Selemadeg District, Tabanan Regency from October 2009 until January 2010. The field trial was arranged into randomized complete block design (RCBD) by 15 treatments were sheathing with various thickness of plastics (S0-S4) and cocoa pod diameters (B1-B3). The results indicated that, based on the percentage of the infested pod, cocoa pod borer has preferred the fruit with diameter > 4.5-7.5 cm. This condition may be contrasted, the smallest pod size (3,5-4,5 cm) was the healthiest pod, even though it contains more nutritionally compound. The smaller pod rot disease in red and green types. Sheathing by plastic of > 0.02 mm thick in medium size of pods was the most effective to prevent cocoa pod borer infestation. Sheathing with plastic was not affecting the population of both black ants and mealy bugs. Based on these results, it can be suggested that sheathing with plastic of > 0.02 mm thick on medium size of the pod is promising control measure to prevent yield lost caused by cocoa pod borer and cocoa pot rot disease.

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.

Field Application of Trichoderma Suspension to Control Cacao Pod Rot (Phytophthora palmivora)

Cacao pod rot caused by Phytophthora palmivora , is an important disease and contributes significant disease losses to global cocoa production. This research objective was to determine the effect of Trichoderma harzianum and T. virens suspensions to cacao pod rot disease on the field. This research was carried out in Pulo Hagu village, Pidie Regency, Aceh, Indonesia from March to July 2017. The single pattern randomized block design was adopted to evaluate three treatments, i.e. without suspension (control), suspensions of T. Harzianum , and T. virens for eight replications. Each replication consisted of three of experimental units. The result showed that both of Trichoderma species contained only Alkaloid metabolite based on Phytochemical test. On the field, the application of T. harzianum suspension reduced the percentage of fruit infection and disease intensity for 48.57 %, 46.04 % at 12 weeks after application (WAA) respectively. Based on the percentage reduction in the area of the spot between the metabolites T. harzianum suspension and control and T. virens and control are 47.24 % and 27.46 % at 87 WAA respectively. In addition, T. virens suppressed the percentage of infected fruit and the intensity of infected fruit for 40.61 % and 38.02 % at 12 WAA.

The impact of soil treatments on black pod rot (caused by Phytophthora megakarya) of cacao in Cameroon

In Cameroon, Phytophthora megakarya is the causal agent of black pod rot of cacao (Theobroma cacao L). Primary inoculum of P. megakarya is located in the soil. With the onset of the rainy season, this primary inoculum is activated and through rain splash cacao pods become infected. From there repeated cycles of pod infection, sporulation, spore dispersal and pod infection, result in losses that can reach up to 70–80% when no control measures are in place. Thus, if this soil-borne inoculum could be eliminated or prevented from reaching cacao pods, losses from black pod could possibly be reduced considerably.Biological control of cacao black pod disease, using Trichoderma asperellum PR11, has proven successful in Cameroon. Moreover, T. asperellum PR11 has also been proven to be useful against soilborne inoculum of Pythium myriotylum and Phytophthora ramorum. The objective of this study therefore, was to use soil applications of T. asperellum PR11 to reduce black pod disease due to P. megakarya.This study was undertaken under farmers’ field condition in the Centre region of Cameroon. Trichoderma asperellum PRR11, together with a water and fungicide (Ridomil Gold 66 WP) control, were applied on a three weeks basis over two consecutive production seasons. Weekly data collection consisted of counting healthy and diseased pods. For each treatment six replicate plots, each containing 16 trees, were used. Soil samples were taken at the beginning and end of the second season to determine the impact of the treatments on P. megakarya primary inoculum levels as well as the presence of fungal antagonists of P. megakarya.Although soil applications did not significantly reduce the total number of diseased pods, nor overall pod rot rate, soil applications did change disease dynamics by reducing the speed in which the disease progressed. Moreover, soil applications did impact the presence of fungal antagonists and most importantly significantly reduced the amount of P. megakarya primary inoculum present in the soil. Given the effectiveness of T. asperellum in controlling black pod rot, combined aerial and soil applications should have additive effects when combined simultaneously for the control of black pod disease in cacao.

The differential influence of temperature on Phytophthora megakarya and Phytophthora palmivora pod lesion expansion, mycelia growth, gene expression, and metabolite profiles

Phytophthora megakarya and Phytophthora palmivora cause black pod rot of cacao. P. megakarya occurs in Africa while P. palmivora is distributed world-wide. P. palmivora has a higher temperature maximum (34 °C) than P. megakarya (30 °C). Factors contributing to temperature maxima in Phytophthora species have not been studied in detail. In culture, P. megakarya lost viability after 3 days at 32 °C while P. palmivora viability began decreasing after 3 days at 36 °C. When exposed to heat, genes encoding heat shock proteins and chaperones were most commonly induced in both species. Heat responsive genes in P. palmivora also tended to be induced at low temperatures (9 °C and 11 °C). In general, P. palmivora maintained higher metabolite pools than P. megakarya, although some temperature induced differences were recorded. When recovering from heat stress, P. megakarya produced a coralloid form of mycelia not previously observed in any Phytophthora species. P. megakarya's temperature range mimics the cacao production conditions in Africa and elevated seasonal field temperatures there are unlikely to persist long enough to eliminate P. megakarya although Phytophthora induced disease epidemiology may be influenced.

The cacao pathogen Moniliophthora roreri (Marasmiaceae) produces rhexolytic thallic conidia and their size is influenced by nuclear condition

Moniliophthora roreri, the causal agent of frosty pod rot of cacao, is a member of the mushroom-forming family Marasmiaceae (Marasmiineae, Agaricales, Basidiomycota). Yet, M. roreri has never been observed to produce a mushroom fruiting body, but rather produces billions of spores on the surface of infected pods. The question of whether these spores are produced via meiosis or mitosis has been the subject of some speculation. However, numerous molecular-based studies have been unable to support a hypothesis of sexual recombination for this fungus. We re-examined sporogenesis and the nuclear condition of hyphae and spores in M. roreri via nuclear staining and spore germination studies. Conidia are produced asexually in a thallic and rhexolytic manner as is true for other Marasmiineae species such as M. perniciosa, Flammulina velutipes and Marasmius puerariae. We also found that hyphal cells as well as spores harbor one or two nuclei, rarely three, that conidium size is influenced by number of nuclei within, and that individual isolates produced consistently and significantly different proportions of binucleate and mononucleate spores regardless of varietal group.

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.

Trichoderma martiale sp. nov., a new endophyte from sapwood of Theobroma cacao with a potential for biological control

The new species Trichoderma martiale was isolated as an endophyte from sapwood in trunks of Theobroma cacao (cacao, Malvaceae) in Brazil. Based on sequences of translation-elongation factor 1-alpha ( tef1) and RNA polymerase II subunit ( rpb2) T. martiale is a close relative of, and morphologically similar to, T. viride, but differs in the production of discrete pustules on corn meal–dextrose agar (CMD) and SNA, in having a faster rate of growth, and in being a tropical endophyte. This new species was shown, in small-scale, in situ field assays, to limit black pod rot of cacao caused by Phytophthora palmivora, the cause of black pod disease.

Bacterial endophytes: Bacillus spp. from annual crops as potential biological control agents of black pod rot of cacao

Diseases are the most important factors limiting production of Theobroma cacao in South America. Because of high disease pressure and environmental concerns, biological control is a pertinent area of research for cacao disease management. In this work, we evaluated the ability of four Bacillus spp. isolated from vegetable crops, for their ability to colonize T. cacao seedlings and reduce the severity of black pod rot ( Phytophthora capsici). Of the Bacillus spp. tested, application of B. cereus isolates BT8 (from tomato) or BP24 (from potato) together with the polysilicon surfactant Silwet L-77 (0.24% vol/vol) resulted in long-term (>68 days) stable colonization of cacao leaves. Further investigation revealed that foliar colonization by BT8 and BP24 was primarily epiphytic, with endophytic populations typically representing 5–15% of total foliar bacteria. Significant reductions of disease severity ( P ⩽ 0.05) on cacao leaf disks challenged with P. capsici were recorded from after day 26, and through 68 days following colonization with BT8. No bacterial colonists were observed in or on leaves that developed after bacteria application, suggesting that the bacteria were not capable of systemic movement through vascular tissues. These newly developed, non-colonized leaves from colonized plants exhibited disease suppression, which supports a probable disease suppression mechanism of induced systemic resistance for the BT8 isolate.

Expected selection efficiency for resistance to cacao pod rot (Phytophthora palmivora) comparing leaf disc inoculations with field observations

A genetic analysis of resistance of cacao to Phytophthora palmivora was carried out in a 5 × 5 diallel and in a 4 × 2 NC II factorial design, involving cross-progenies and parental clones planted in the nursery and field, respectively. Resistance was scored in the laboratory by inoculation of leaf discs with P. palmivora spores with four replicates and, for the factorial design, results were compared with the percentage of rotten pods in the field assessed over a seven-year period. Significant differences between genotypes were observed for both laboratory and field evaluation methods. For the laboratory test, no reciprocal effect was observed and plant effects within seedling progenies were more significant than plant effects within clones. General combining ability was the main source of variation but specific combining ability was also significant for the leaf disc test applied to the diallel. Heritabilities increased with the number of replicates, reaching values of 0.34 and 0.67 for narrow sense heritability, and 0.60 and 0.67 for broad sense heritability, for the diallel and the factorial design, respectively. These values were higher than observed for the percentage of rotten pods in the field in the factorial design (0.42 and 0.47, respectively). For the leaf test, the expected genetic gains were around 30% with a selection intensity of 5%. The existence of significant genetic (0.71) and phenotypic (0.39) coefficients of correlation between resistance on leaves and percentage of rotten pods in the field in the factorial design confirms the feasibility of using the leaf disc test for early selection of resistance to Phytophthora pod rot of cacao.

The causal agents of witches' broom and frosty pod rot of cacao (chocolate, Theobroma cacao) form a new lineage of Marasmiaceae

The two most devastating diseases of cacao (Theobroma cacao)--the source of chocolate--in tropical America are caused by the fungi Crinipellis perniciosa (witches' broom disease) and Moniliophthora roreri (frosty pod rot or moniliasis disease). Despite the agricultural, socio-economic and environmental impact of these fungi, most aspects of their life cycles are unknown, and the phylogenetic relationships of M. roreri have yet to be conclusively established. In this paper, extensive phylogenetic analyses of five nuclear gene regions (28S rDNA, 18S rDNA, ITS, RPB1, and EF1-alpha) confirm that C. perniciosa and M. roreri are sister taxa that belong in the Marasmiaceae (euagarics). Furthermore, these taxa form part of a separate and distinct lineage within the family. This lineage includes the biotrophic fungi Moniliophthora perniciosa comb. nov. and M. roreri, as well as one undescribed endophytic species. The sister genera to Moniliophthora are Marasmius, Crinipellis and Chaetocalathus, which consist mainly of saprotrophic litter fungi.