Metabolite mass spectrometry profiling of cacao genotypes reveals contrasting resistances to Ceratocystis cacaofunesta phytopathogen.

Ceratocystis wilt is a lethal disease of cacao, and the search for resistant genotypes may provide the best way to deal with the disease. Resistance or susceptibility behavior of some cacao genotypes when infected by Ceratocystis cacaofunesta is not yet understood. Herein, we report an LC-MS metabolomic screening analysis based on high-resolution MS to obtain comprehensive metabolic profile associated with multivariate data analysis of PLS-DA, which was effective to classify CCN-51 and TSH-1188 as resistant genotypes to C. cacaofunesta fungus, while CEPEC2002 was classified as a susceptible one. Using reversed-phase LC method, electrospray interface, and high-resolution tandem MS by the quadrupole-TOF analyzer, the typical profiles of metabolites, such as phenylpropanoids, flavonoids, lipids, alkaloids, and amino acids, were obtained. Untargeted metabolite profiles were used to construct discriminant analysis by partial least squares (PLS-DA)-derived loading plots, which placed the cacao genotypes into two major clusters related to susceptible or resistant groups. Linolenic, linoleic, oleic, stearic, arachidonic, and asiatic acids were annotated metabolites of infected, susceptible, and resistant genotypes, while methyl jasmonate, jasmonic acid, hydroxylated jasmonic acid, caffeine, and theobromine were annotated as constituents of the resistant genotypes. Trends of these typical metabolites levels revealed that CCN51 is susceptible, CEPEC2002 is moderately susceptible, and TSH1188 is resistant to C. cacaofunesta. Therefore, profiles of major metabolites as screened by LC-MS offer an efficient tool to reveal the level of resistance of cacao genotypes to C. cacaofunesta present in any farm around the world.

Structural and Functional Genomics of the Resistance of Cacao to Phytophthora palmivora.

Black pod disease, caused by Phytophthora spp., is one of the main diseases that attack cocoa plantations. This study validated, by association mapping, 29 SSR molecular markers flanking to QTL (Quantitative Trait Loci) associated with Phytophthora palmivora Butler (Butler) (PP) resistance, in three local ancient varieties of the Bahia (Comum, Pará, and Maranhão), varieties that have a high potential in the production of gourmet chocolate. Four SSR loci associated with resistance to PP were detected, two on chromosome 8, explaining 7.43% and 3.72% of the Phenotypic Variation (%PV), one on chromosome 2 explaining 2.71%PV and one on chromosome 3 explaining 1.93%PV. A functional domains-based annotation was carried out, in two Theobroma cacao (CRIOLLO and MATINA) reference genomes, of 20 QTL regions associated with cocoa resistance to the pathogen. It was identified 164 (genome CRIOLLO) and 160 (genome MATINA) candidate genes, hypothetically involved in the recognition and activation of responses in the interaction with the pathogen. Genomic regions rich in genes with Coiled-coils (CC), nucleotide binding sites (NBS) and Leucine-rich repeat (LRR) domains were identified on chromosomes 1, 3, 6, 8, and 10, likewise, regions rich in Receptor-like Kinase domain (RLK) and Ginkbilobin2 (GNK2) domains were identified in chromosomes 4 and 6.

Phytophthora theobromicola sp. nov.: A New Species Causing Black Pod Disease on Cacao in Brazil.

Black pod disease, caused by Phytophthora species, is among the main limiting factors of cacao (Theobroma cacao L.) production. High incidence levels of black pod disease have been reported in Brazil, being induced by Phytophthora capsici, Phytophthora citrophthora, Phytophthora heveae, and Phytophthora palmivora. To assess the diversity of Phytophthora species affecting cacao in Brazil, 40 new isolates were obtained from cacao pods exhibiting symptoms of black pod disease collected in different smallholder farms in 2017. Further, ten cacao-infecting isolates morphologically identified as P. citrophthora and P. palmivora were molecularly characterized. The genomic regions beta-tubulin, elongation factor 1 alpha, heat shock protein 90, and internal transcribed spacer, and the mitochondrially encoded cytochrome c oxidase I and II genes were PCR-amplified and Sanger-sequenced from the cacao-infecting Phytophthora isolates. The morphological characterization and evaluation of the mycelial growth rates for the Phytophthora isolates were performed in vitro. Based on the molecular analysis and morphological comparisons, 19 isolates were identified as P. palmivora (clade 4). Interestingly, 31 isolates grouped together in the phylogenetic tree and were placed apart from previously known species in Phytophthora clade 2. Therefore, these isolates are considered as a new species herein referred to as Phytophthora theobromicola sp. nov., which produced papillate, semipapillate, and persistent sporangia on simple sporangiophores. The P. palmivora isolates were identified as A1 mating type by pairing each isolate with known A1 and A2 tester strains of P. capsici, but no oogonia/antheridia were observed when P. theobromicola was paired with the different tester strains. The P. theobromicola and P. citrophthora isolates showed higher mycelial growth rates, when compared to P. palmivora, on different media at 10, 15, and 20°C, but similar values were observed when grown on clarified CA media at 25 and 30°C. The pathogenicity tests carried out on pods of four cacao clones (CCN51, PS1319, Cepec2004, and CP49) showed significant variability among the isolates of both Phytophthora species, with P. theobromicola inducing higher rates of necrotic lesion expansion, when compared to P. palmivora. Here, two Phytophthora species were found associated with black pod disease in the state of Bahia, Brazil, and the previously undescribed P. theobromicola seems to be prevalent in field conditions. This is the first report of P. theobromicola on T. cacao. Also, these findings are crucial to improve the disease control strategies, and for the development of cacao materials genetically resistant to Phytophthora.

Morphologic, molecular, and pathogenic characterization of Phytophthora palmivora isolates causing flower rot on azalea.

The objective of this work was to characterize two Phytophthora palmivora isolates causing floral blight and rot in azalea plants and to evaluate the pathogenicity of this oomycete pathogen on several plant species. Azalea plants with symptoms of flower blight and rot were obtained in the municipality of Holambra-SP. After an attempt of isolation, colonies with Phytophthora characteristics grown only on selective V8 medium. Molecular identification of the isolates was done by amplification and sequencing of ITS and COX2 regions. In the phylogenetic analysis, the azalea isolates clustered with reference isolates of P. palmivora. Morphological characteristics were similar to those described for P. palmivora. Isolates were inoculated in healthy azalea plants and caused leaf blight and floral rot. The pathogen was re-isolated from symptomatic plants completing Koch's postulates. In a host range test, the azalea isolates were able to cause lesions on leaves of vinca, snapdragon, basil, and tomato, and affected both leaves and flowers of geranium. Fruit rot was observed on tomato, potato, sweet pepper, scarlet eggplant, zucchini, cucumber, maroon cucumber, onion, apple, papaya, guava, and carrot. This is the first report of the species P. palmivora causing flower blight and rot in azalea plants in Brazil and probably in the world.

Aflatoxins and ochratoxin A: occurrence and contamination levels in cocoa beans from Brazil.

In the present study, the occurrence of aflatoxins (AFs) and ochratoxin A (OTA) was evaluated in 123 samples of cocoa beans produced in five Brazilian states. The presence of these mycotoxins was determined by high-performance liquid chromatography with fluorescence detection (HPLC-FLD) after immunoaffinity column clean-up. The mean level of total AFs in cocoa beans samples was 5.7 µg.kg-1. Four (3.3%) samples exceeded the maximum limit of 10 µg.kg-1 established by the Brazilian legislation for total AFs. The mean level of OTA contamination was 1.2 µg.kg-1, and none of the samples exceeded the maximum limit established by the Brazilian legislation. The co-occurrence of AFs and OTA was observed in 4.9% of the samples. The results of the present study demonstrated that, in relation to the levels of AFs and OTA established by the Brazilian legislation, most samples of cocoa beans analyzed are safe for consumption. This is the first report on the occurrence and levels of AFs and OTA in cocoa beans from the five main Brazilian states producing cocoa. The data in this study provide important information for farmers, traders, industry, consumers and law enforcement agencies.

Control of Phytophthora palmivora on postharvest papaya with Trichoderma asperellum, T. virens, T. harzianum and T. longibrachiatum / Controle de Phytophthora palmivora em mamão na pós-colheita por Trichoderma asperellum, T. virens, T. harzianum e T. longibrachiatum

Papaya (Carica papaya) is one of the most cultivated and consumed tropical fruit worldwide. Its production might be limited by preharvest and postharvest diseases. The fruit rot caused by Phytophthora palmivora is one of the most important postharvest diseases of papaya in Brazil. The control of these diseases is usually made with fungicide applications. Therefore, studies concerning biocontrol of postharvest diseases might generate data that may reduce the environmental impacts caused by pesticides. Thus, the biological control by Trichoderma in postharvest diseases is an alternative to the use of fungicides for the postharvest control of P. palmivora in the papaya fruit. Four antagonists [T. asperellum (SF04), T. virens (255C1), T. harzianum (THP) and T. longibrachiatum (4088)] were tested, as follow: 1) Trichoderma spp. applied 1 hour after inoculation of P. palmivora and; 2) Trichoderma spp. applied 24 hours after inoculation of P. palmivora; 3) Trichoderma spp. applied 1 hour before inoculation of P. palmivora, and 4) Trichoderma spp. applied 24 hours before inoculation of P. palmivora. All Trichoderma significantly (P£0,05) reduced the incidence and severity of disease. The 4088 (T. longibrachiatum) isolate was the best controller agent of P. palmivora in postharvest.

O mamão é uma fruta muito cultivada e consumida nas regiões tropicais e subtropicais do mundo e apresenta diversos problemas fitossanitários. Sendo assim, estudos de doenças pós-colheita com biocontroladores viabilizam a diminuição de impactos causados pelo uso de fungicidas. A podridão-dos-frutos (Phytophthora palmivora) é uma importante doença pós-colheita em mamão no Brasil. Neste contexto, o controle biológico desta doença na póscolheita com Trichoderma é uma alternativa viável ao uso de fungicidas e foi aplicado neste estudo para avaliar a eficácia de Trichoderma spp. para o biocontrole de P. palmivora em mamão na pós-colheita. Foram utilizados quatro potenciais antagonistas: T. asperellum (SF04), T. virens (255C1), T. harzianum (THP) e T. longibrachiatum (4088). E as frutas foram submetidas aos seguintes tratamentos: Inoculação de P. palmivora e 1 hora depois inoculação do Trichoderma spp.; Inoculação de P. palmivora e 24 horas depois inoculação do Trichoderma spp.; Inoculação de Trichoderma spp. e 1 hora depois inoculação do P. palmivora e; Inoculação de Trichoderma spp. e 24 horas depois inoculação do P. palmivora. Todos os isolados de Trichoderma reduziram significativamente tanto na incidência como na severidade da doença. O isolado 4088 (T. longibrachiatum) foi o melhor no controle da podridão.

Reduction of papaya rot (Phytophthora palmivora) with phosphite and Acibenzolar-S-Methyl in preharvest and postharvest / Redução da podridão do mamão (Phytophthora palmivora) com fosfitos e Acibenzolar-S-Metil em pré e pós-colheita

The papaya fruit rot (Phytophthora palmivora) is responsible for significant losses. To reduce diseases, especially in areas with climate and humidity favorable to pathogens, are adopted chemical methods, which sometimes increase the cost of production and cause severe environmental impacts. Alternatively, there are products, such as, phosphites of potassium and acibenzolar-S-methyl (ASM) that might be efficient on disease control and less aggressive to environment. Phosphites of K and ASM were evaluated in this study on the control effectiveness of papaya fruit rot at different dosages in preharvest and postharvest. The severity and percentage of disease control were evaluated for each treatment. For the pre-harvest treatments (applied six days before harvest), the phosphite of K [240 g L-1 K2O, 340 g L-1 P2O5 and 50 g L-1 (Reforce® + Salicylic Acid)] at 3 or 6 mL L-1significantly reduced disease severity, and, reduced fruit ripening. On postharvest application, ASM reduced disease severity.

A podridão dos frutos do mamoeiro (Phytophthora palmivora) é responsável por perdas significativas e para minimizar a doença, principalmente em locais com clima favorável ao patógeno, medidas de controle químico são adotadas. Porém, estas medidas podem elevar o custo da produção e causar severos impactos ambientais. Alternativamente, existem produtos como os fosfitos de potássio (K) e acibenzolar-S-metil (ASM) que podem ser eficientes no controle de doenças e menos agressivos ao ambiente. Fosfitos de K e ASM foram avaliados neste trabalho quanto à eficácia do controle da podridão de frutos de mamão 'Sunrise Solo' tipo exportação, cultivados no Sul da Bahia, em diferentes doses e formulações comerciais na pré e pós-colheita. Para cada tratamento foi avaliada a severidade da doença e o percentual de controle da doença, sendo também verificada a influência nas características fisioquímicas na fruta. Para os tratamentos pré-colheita, aplicados aos seis dias antes da colheita, o fosfito de K [240 g L-1 K2O, 340 g L-1 P2O5 e 50 g L-1 C7H6O3 (Reforce® + Ácido Salicílico)] a 3 ou 6 mL L-1 reduziu significativamente a doença, e influenciou diretamente no atraso da maturação da fruta. Na pós-colheita, ASM incitou redução na severidade da doença. Todos os tratamentos não alterarão as características fisioquímicas da fruta.

Major phytopathogens and strains from cocoa (Theobroma cacao L.) are differentiated by MALDI-MS lipid and/or peptide/protein profiles.

Phytopathogens are the main disease agents that promote attack of cocoa plantations in all tropical countries. The similarity of the symptoms caused by different phytopathogens makes the reliable identification of the diverse species a challenge. Correct identification is important in the monitoring and management of these pests. Here we show that matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) in combination with multivariate data analysis is able to rapidly and reliably differentiate cocoa phytopathogens, namely Moniliophthora perniciosa, Phytophthora palmivora, P. capsici, P. citrophthora, P. heveae, Ceratocystis cacaofunesta, C. paradoxa, and C. fimbriata. MALDI-MS reveals unique peptide/protein and lipid profiles which differentiate these phytopathogens at the level of genus, species, and single strain coming from different hosts or cocoa tissues collected in several plantations/places. This fast methodology based on molecular biomarkers is also shown to be sufficiently reproducible and selective and therefore seems to offer a suitable tool to guide the correct application of sanitary defense approaches for infected cocoa plantations. International trading of cocoa plants and products could also be efficiently monitored by MALDI-MS. It could, for instance, prevent the entry of new phytopathogens into a country, e.g., as in the case of Moniliophthora roreri fungus that is present in all cocoa plantations of countries bordering Brazil, but that has not yet attacked Brazilian plantations. Graphical Abstract Secure identification of phytopathogens attacking cocoa plantations has been demonstrated via typical chemical profiles provided by mass spectrometric screening.