First Report of Anthracnose Caused by Colletotrichum nymphaeae on Strawberry Fruits in Central Argentina.

Strawberry (Fragaria x ananassa) production in Argentina extends to around 1700 hectares. Coronda city, located in Santa Fe province, is an important strawberry producer due to ideal agroecological conditions for culture and a high specialization for production. In November 2021, anthracnose symptoms were observed on strawberries cvs. 'San Andreas' and 'Splendor' in Coronda (31°58'S, 60°55'W), central Argentina. During these years, the incidence of the disease reached 40% of the production. Symptoms included 2-3 mm circular to irregular dark brown spots which enlarged rapidly and became sunken. Under high humidity conditions, concentric rings of pinhead-size salmon-colored acervuli developed on the lesions. The causal agent was isolated by touching acervuli with a sterile needle and monosporic cultures were obtained on PDA after 10 days at 25°C, with a 12-h light period. Colonies were white to gray on the top and orange on the underside, where concentric rings of salmon acervuli were clearly distinguished. The width and length of one hundred conidia were examined in three isolates (CF1, CF2, and CF3), ranging from 3.27 to 5.53 µm (avg.= 4.3 µm), and from 10.37 to 19.52 µm (avg.= 14.27 µm), respectively. The conidia were hyaline, smooth-walled, aseptate, and cylindric-clavate with one end round and one end acute. These morphological characteristics correspond to species belonging to the C. acutatum complex (Damm et al. 2012; Liu et al. 2022). To accurately identify the species, DNA was extracted from isolates, and ß-tubulin (TUB2), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and histone (HIS3) genes were partially amplified and sequenced (Vieira et al. 2020). TUB2, GAPDH, and HIS3 sequences presented a 100% of identity with species of Colletotrichum nymphaeae. The nucleotide sequences were deposited in GenBank (OR271556-OR271558, TUB2; OR271559-OR271561, GAPDH; and OR271562-OR271564, HIS3). Multilocus phylogenetic analyses performed with reference sequences (Damm et al. 2012) showed that the three isolates clustered with C. nymphaeae, in accordance with BLAST results. To confirm pathogenicity, each isolate was inoculated in eight detached fruits of the cultivar from which it was originally obtained. Two drops of 10 µl of conidial suspension (1x105 conidia per ml) were deposited in non-wounded areas on fruits previously disinfested with 1% sodium hypochlorite solution for 1 min and rinsed twice with sterile distilled water. Drops of sterile water were deposited in eight fruits as control. Pathogenicity tests were repeated twice. Fruits were kept in moist chamber (80+5% relative humidity) at 25°C for ten days. First symptoms appeared 4 days after inoculation. After that, all of the isolates produced symptoms identical to those previously described, whereas the controls remain symptomless. The pathogen was re-isolated from lesions, and identified as C. nymphaeae by morphological characteristics and based on the TUB2 sequences, as previously described. Strawberry anthracnose in Argentina was previously associated with Colletotrichum acutatum, C. gloeosporioides and C. fragariae species based on morphological characteristics (Ramallo et al. 2000; Monaco et al. 2000) but molecular identification was not performed until today. To our knowledge, this is the first report of C. nymphaeae causing anthracnose on strawberry in Argentina. This accurate identification will help to develop more efficient management strategies.

Genomic and Experimental Analysis of the Biostimulant and Antagonistic Properties of Phytopathogens of Bacillus safensis and Bacillus siamensis.

The B. safensis RGM 2450 and B. siamensis RGM 2529 strains were isolated from the rhizosphere of plants presenting resilience to abiotic and biotic stress conditions. To understand the implications of bacteria in resilience, a genomic and experimental analysis was carried out on their biostimulant and phytopathogenic antagonist properties. Genome analyses of both strains indicated that they have the potential to synthesize bioactive compounds such as the battery of non-ribosomal peptides, polyketides, extracellular enzymes and phytohormones. These results were consistent with the antagonistic activities of both strains against the phytopathogens Botrytis cinerea, Colletotrichum acutatum, Fusarium oxysporum and Phytophtora cinnamomi. They also showed the capacity to solubilize phosphorus, fix nitrogen and produce indole acetic acid. This was observed in tomato seedlings grown from seeds inoculated with the mixture of strains which presented significantly greater length as well as wet and dry weight in comparison with the treatments individually inoculated with each strain and the control. Accordingly, the combination of B. safensis RGM 2450 and B. siamensis RGM 2529 showed synergistic biostimulant activity. These findings contribute new knowledge of the genomic and metabolomic properties taking part in the symbiotic interactions between these strains and the plants and uphold the combined use of both strains as a biostimulant.

CgNPG1 as a Novel Pathogenic Gene of Colletotrichum gloeosporioides From Hevea brasiliensis in Mycelial Growth, Conidiation, and the Invasive Structures Development.

The rubber tree (Hevea brasiliensis) is a tropical perennial crop for the primary source of natural rubber. Colletotrichum gloeosporioides from Hevea brasiliensis (C. gloeosporioides Hb) and Colletotrichum acutatum from Hevea brasiliensis (C. acutatum Hb) are the causal agents of rubber tree anthracnose and lead to serious loss of natural rubber production. Inoculation tests showed that C. gloeosporioides Hb possessed higher pathogenicity than C. acutatum Hb to the rubber tree. Genomic analysis revealed that an unknown gene, named CgNPG1 (a Novel Pathogenic Gene 1), was presented in the genome of C. gloeosporioides Hb but not identified in C. acutatum Hb. CgNPG1 was predicted to encode a small secretory protein without any conserved domain. To investigate the functions of CgNPG1 in C. gloeosporioides Hb and in C. acutatum Hb, the gene deletion and overexpression mutants were generated. The phenotype analysis showed that deletion of CgNPG1 led to changed conidia morphology, decreased mycelial growth, conidiation, conidia germination rate, appressorium formation rate, and pathogenicity of C. gloeosporioides Hb to the rubber tree. Meanwhile, heterogeneous expression of CgNPG1 in C. acutatum Hb significantly changed the conidia morphology and improved the mycelial growth rate, conidiation, conidia germination rate, appressorium formation rate, and the pathogenicity of C. acutatum Hb to the rubber tree. Consistently, CgNPG1 increased the expression level of CaCRZ1 and CaCMK1 in C. acutatum Hb. These data suggested that CgNPG1 contributed to mycelial growth, conidiation, the development of invasive structures, and the pathogenicity of Colletotrichum to the rubber tree, which might be related to the modulation of CaCRZ1 and mitogen-activated protein kinase CMK1. Our results provided new insight into CgNPG1 in regulating growth and pathogenicity of the Colletotrichum spp.

In vitro antifungal activity of Myrcia ovata essential oils and their major compounds against pathogens of citrus, sweet potato, and coconut / Atividade antifungica in vitro de óleos essenciais de myrcia ovata e seus compostos majoritários sobre patógenos de citros, batata-doce e coqueiro

Myrcia ovata, an endemic species to the Brazilian Atlantic Forest, presents antifungal properties. The phytopathogens Colletotrichum acutatum, Plenodomus destruens,and Thielaviopsis paradoxa are responsible for the diseases citrus postbloom fruit drop, sweet potato foot rot, and coconut stem bleeding, respectively. The antifungal activity of the essential oils of five M. ovata chemotypes (MYRO-159, nerolic acid chemotype; MYRO-180, nerolic acid + linalool chemotype; MYRO-388, geraniol chemotype; MYRO-157, citral + (E)-nerolidol chemotype; and MYRO-174, isopulegol + linalool chemotype), four major compounds (nerolic acid, nerolic acid + linalool, geraniol, and citral + (E)-nerolidol), and threepure compounds (citral, (E)-nerolidol, and linalool) against the fungi C. acutatum, P. destruens,and T. paradoxawere evaluated. For this, in vitro tests were conducted in a completely randomized design with three replications, testing concentrations (v/v) ranging from 0.01 to 1.0 µL.mL-1. All treatments presented toxicity at different levels to the three fungi. For C. acutatum,the essential oil from the individual MYRO-180 (nerolic acid + linalool chemotype) and its major compound showed the lowest Minimal Inhibitory Concentration (MIC) and Minimum Fungicidal Concentration (MFC) of 0.03 and 0.1 µL.mL-1, respectively. For P. destruens, the essential oil from the individual MYRO-159 (nerolic acid chemotype) presented the lowest MIC of 0.05 µL.mL-1. The nerolic acid + linalool chemotype and its major compound presented an MFC of 0.07 µL.mL-1. For T. paradoxa, the major compound citral + (E)-nerolidol stood out with the lowest MIC and MFC of 0.03 and 0.2 µL.mL-1, respectively. Linalool presented the lowest toxicity to the three tested fungi.

Myrcia ovata, uma espécie nativa de fitofisionomia de Restinga, possui atividade antifúngica. Os fitopatógenos Colletotrichum acutatum, Plenodomus destruens e Thielaviopsis paradoxa são responsáveis pelas doenças podridão floral de citros, mal-do-pé da batata doce e resinose do coqueiro, respectivamente. A atividade antifúngica de cinco quimiotipos de M. ovata (MYRO-159, quimiotipo ácido nerólico; MYRO-180, ácido nerólico + linalol; MYRO-388, quimiotipo geraniol; MYRO-157, quimiotipo citral + (E)-nerolidol; e, MYRO-174, quimiotipo isopulegol + linalol), quatro compostos majoritários (ácido nerólico, ácido nerólico + linalol, geraniol e citral + (E)-nerolidol) e três compostos isolados (citral, (E)-nerolidol e linalol) foram avaliados sobre os fungos C. acutatum, P. destruens e T. paradoxa. Testes in vitro foram conduzidos em delineamento inteiramente casualizado com três repetições e concentrações (v/v), que variaram de 0,01 a 1,0 µL.mL-1. Todos os tratamentos testados apresentaram atividade antifúngica. Para o fungo C. acutatum, o óleo essencial do indivíduo MYRO-180, de quimiotipo ácido nerólico + linalol, e seu composto majoritário apresentaram menores Concentração Mínima Inibitória (CMI) e Concentração Mínima Fungicida (CMF) de 0,03 e 0,1 µL.mL-1, respectivamente. Para o fungo P. destruens, o óleo essencial do indivíduo MYRO-159, de quimiotipo ácido nerólico, apresentou menor CMI de 0,05 µL.mL-1, e o quimiotipo ácido nerólico + linalol e seu composto majoritário apresentaram a menor CMF de 0,07 µL.mL-1. Para o fungo T. paradoxa,a combinação de citral + (E)-nerolidol destacou-se com CMI e CMF de 0,03 e 0,2 µL.mL-1, respectivamente. Linalol foi o menos tóxico sobre os três fungos testados.

Alternative control of grape rots by essential oils of two Eucalyptus species.

BACKGROUND: Essential oils (EOs) are volatile natural compounds produced by plant secondary metabolism, and some of them exhibit antimicrobial activity. The objective of the present study was to determine the chemical composition the EOs of Eucalyptus staigeriana and Eucalyptus globulus, and their effect in vitro and in vivo against Botrytis cinerea and Colletotrichum acutatum, the most important fungal rot diseases of grapes. Moreover, grapes collected from field experiments were used to evaluate the impact of the alternative control on the alcoholic fermentation and wine composition. RESULTS: The major compound of E. staigeriana EO were citral 30.91% (19.74% geranial, 11.17% neral), 1.8-cineole (24.59%) and limonene (19.47%), while 1.8-cineole represented 68.26% of E. globulus EO. The two EOs showed in vitro antifungal activity against both pathogens. Eucalyptus staigeriana EO exhibited the highest activity inhibiting mycelial growth (MG) and conidial germination at 0.5 µL mL-1 . Moreover, this EO was able to reduce the incidence and severity of grey rot caused by B. cinerea and the severity of ripe rot caused by C. acutatum The alternative control did not significantly influence alcoholic fermentation, the physicochemical characteristics, and the volatile composition of wines. CONCLUSION: These results are promising and indicate that E. staigeriana EO might be further investigated as a natural alternative for the control of fungal rots on wine grapes. © 2019 Society of Chemical Industry.

A diagramatic scale to quantify severity of antracnose in Rubus glaucus Benth / Uma escala diagramática para quantificar a severidade da antracnose em Rubus glaucus Benth

Rubus glaucus Benth, commonly referred to as mora de Castilla, is affected by Colletotrichum acutatum, as it induces anthracnose in many of the plant organs. Generally, it affects the fruits during the post-harvest phase and damages them, causing economic losses due to the poor crop quality. At present, no standardized methods are available for this pathosystem that can be used to characterize quantitatively the epidemic and to permit the prediction and comparison of the disease management techniques. In this research, we proposed a logarithmic diagrammatic scale of the severity of anthracnose induced by C. acutatum in the fruits of the thornless variety of R. glaucus Benth. This scale is constructed on the adjustment of the Weber-Fechner law and includes six classes, viz., 0%, 1-6%, 7-25%, 26-50%, 51-75%, 76-100%. The scale was validated using 14 evaluators, which entailed measuring the affected fruits with and without utilizing the scale; this improved the precision, accuracy and reproducibility of the calculations whenever the scale was used. We concluded that the scale proposed can be used to assess the severity of anthracnose induced by the fungus in the R. glaucus Benth fruits.(AU)

Mora de Castilla (Rubus glaucus Benth) é afetado por Colletotrichum acutatum, causando antracnose em órgãos diferentes da planta. Nos frutos geralmente é em pós-colheita, causando danos que geram perdas econômicas relacionadas com a qualidade das culturas. Ainda não existem para esse patossistema métodos padronizados para caracterizar quantitativamente a epidemia e que pode prever e comparar os métodos de manejo da doença. Por esta razão, neste trabalho foi criada uma escala logarítmica diagramática da severidade da antracnose causada pelo fungo C. acutatum em frutos de R. glaucus Benth com base na lei de Weber-Fechnere composto por 6 niveis: 0%, 1-6%, 7-25%, 26-50%, 51-75% e 76-100%. A escala foi validada por 14 evaluadores, fazendo medições de frutos afetados com e sem o uso da escala, que mostrou melhor precisão, exatitude e reproducibilidade nas avaliações em que o uso da escala foi feito. Isto permite concluir que a escala proposta pode ser usada na estimação da severidade da antracnose causada em frutos de R. glaucus Benth.(AU)

A diagramatic scale to quantify severity of antracnose in Rubus glaucus Benth / Uma escala diagramática para quantificar a severidade da antracnose em Rubus glaucus Benth

ABSTRACT: Rubus glaucus Benth, commonly referred to as mora de Castilla, is affected by Colletotrichum acutatum, as it induces anthracnose in many of the plant organs. Generally, it affects the fruits during the post-harvest phase and damages them, causing economic losses due to the poor crop quality. At present, no standardized methods are available for this pathosystem that can be used to characterize quantitatively the epidemic and to permit the prediction and comparison of the disease management techniques. In this research, we proposed a logarithmic diagrammatic scale of the severity of anthracnose induced by C. acutatum in the fruits of the thornless variety of R. glaucus Benth. This scale is constructed on the adjustment of the Weber-Fechner law and includes six classes, viz., 0%, 1-6%, 7-25%, 26-50%, 51-75%, 76-100%. The scale was validated using 14 evaluators, which entailed measuring the affected fruits with and without utilizing the scale; this improved the precision, accuracy and reproducibility of the calculations whenever the scale was used. We concluded that the scale proposed can be used to assess the severity of anthracnose induced by the fungus in the R. glaucus Benth fruits.

RESUMO: Mora de Castilla (Rubus glaucus Benth) é afetado por Colletotrichum acutatum, causando antracnose em órgãos diferentes da planta. Nos frutos geralmente é em pós-colheita, causando danos que geram perdas econômicas relacionadas com a qualidade das culturas. Ainda não existem para esse patossistema métodos padronizados para caracterizar quantitativamente a epidemia e que pode prever e comparar os métodos de manejo da doença. Por esta razão, neste trabalho foi criada uma escala logarítmica diagramática da severidade da antracnose causada pelo fungo C. acutatum em frutos de R. glaucus Benth com base na lei de Weber-Fechnere composto por 6 niveis: 0%, 1-6%, 7-25%, 26-50%, 51-75% e 76-100%. A escala foi validada por 14 evaluadores, fazendo medições de frutos afetados com e sem o uso da escala, que mostrou melhor precisão, exatitude e reproducibilidade nas avaliações em que o uso da escala foi feito. Isto permite concluir que a escala proposta pode ser usada na estimação da severidade da antracnose causada em frutos de R. glaucus Benth.

Monoterpene biotransformation by Colletotrichum species.

OBJECTIVE: To investigate the biocatalytic potential of Colletotrichum acutatum and Colletotrichum nymphaeae for monoterpene biotransformation. RESULTS: C. acutatum and C. nymphaeae used limonene, α-pinene, ß-pinene, farnesene, citronellol, linalool, geraniol, perillyl alcohol, and carveol as sole carbon and energy sources. Both species biotransformed limonene and linalool, accumulating limonene-1,2-diol and linalool oxides, respectively. α-Pinene was only biotransformed by C. nymphaeae producing campholenic aldehyde, pinanone and verbenone. The biotransformation of limonene by C. nymphaeae yielded 3.34-4.01 g limonene-1,2-diol l-1, depending on the substrate (R-(+)-limonene, S-(-)-limonene or citrus terpene (an agro-industrial by-product). This is among the highest concentrations already reported for this product. CONCLUSIONS: This is the first report on the biotransformation of these terpenes by Colletotrichum spp. and the biotransformation of limonene to limonene-1,2-diol possibly involves enzymes similar to those found in Grosmannia clavigera.

Bacillus subtilis based-formulation for the control of postbloom fruit drop of citrus.

Postbloom fruit drop (PFD) caused by Colletotrichum acutatum affects flowers and causes early fruit drop in all commercial varieties of citrus. Biological control with the isolate ACB-69 of Bacillus subtilis has been considered as a potential method for controlling this disease. This study aimed to develop and optimize a B. subtilis based-formulation with a potential for large-scale applications and evaluate its effect on C. acutatum in vitro and in vivo. Bacillus subtilis based-formulations were developed using different carrier materials, and their ability to control PFD was evaluated. The results of the assays led to the selection of the B. subtilis based-formulation with talc + urea (0.02 %) and talc + ammonium molybdate (1 mM), which inhibited mycelial growth and germination of C. acutatum. Studies with detached citrus flowers showed that the formulations were effective in controlling the pathogen. In field conditions, talc + urea (0.02 %) provided 73 % asymptomatic citrus flowers and 56 % of the average number of effective fruit (ANEF), equating with fungicide treatment. On the contrary, non-treated trees had 8.8 % of asymptomatic citrus flowers and 0.83 % ANEF. The results suggest that B. subtilis based-formulations with talc as the carrier supplemented with a nitrogen source had a high potential for PFD control.

Inactivation of plant-pathogenic fungus Colletotrichum acutatum with natural plant-produced photosensitizers under solar radiation.

The increasing tolerance to currently used fungicides and the need for environmentally friendly antimicrobial approaches have stimulated the development of novel strategies to control plant-pathogenic fungi such as antimicrobial phototreatment (APT). We investigated the in vitro APT of the plant-pathogenic fungus Colletotrichum acutatum with furocoumarins and coumarins and solar radiation. The compounds used were: furocoumarins 8-methoxypsoralen (8-MOP) and 5,8-dimethoxypsoralen (isopimpinellin), coumarins 2H-chromen-2-one (coumarin), 7-hydroxycoumarin, 5,7-dimethoxycoumarin (citropten) and a mixture (3:1) of 7-methoxycoumarin and 5,7-dimethoxycoumarin. APT of conidia with crude extracts from 'Tahiti' acid lime, red and white grapefruit were also performed. Pure compounds were tested at 50µM concentration and mixtures and extracts at 12.5mgL(-1). The C. acutatum conidia suspension with or without the compounds was exposed to solar radiation for 1h. In addition, the effects of APT on the leaves of the plant host Citrus sinensis were determined. APT with 8-MOP was the most effective treatment, killing 100% of the conidia followed by the mixture of two coumarins and isopimpinellin that killed 99% and 64% of the conidia, respectively. APT with the extracts killed from 20% to 70% of the conidia, and the extract from 'Tahiti' lime was the most effective. No damage to sweet orange leaves was observed after APT with any of the compounds or extracts.

Metabolism and antifungal activity of safrole, dillapiole, and derivatives against botryodiplodia theobromae and colletotrichum acutatum / Metabolismo y actividad antifúngica de safrol, dilapiol, y derivados contra botryodiplodia theobromae y colletotrichum acutatum

The direct in vitro fungitoxicity and metabolism of safrole and dillapiole (isolated from Piper auritum and Piper holtonii, respectively) by Botryodiplodia theobromae and Colletotrichum acutatum were investigated. Higher values of mycelial growth inhibition for both fungi were obtained for dillapiole, as compared with safrole. B. theobromae was able to metabolize both compounds to their respective vicinal diols, reaching 65 percent relative abundance during the biotransformation of dillapiole; while C. acutatum only transformed safrole to various metabolites with relative abundances under 5 percent. According to the low antifungal activity of the major metabolic products (< 5 percent for vicinal diols), a detoxification process was implied. Studies on the influence of some substituents in the aromatic ring of safrole and dillapiole on the antifungal activity against B. theobromae were also carried out. As result, the safrole nitrated derivative, 6-nitrosafrole, showed a fungitoxicity level similar to that displayed by the commercial fungicide Carbendazim® under the conditions used. In light of this, safrole and dillapiole could be suggested as feasible structural templates for developing new antifungal agents.

Se investigó la fungitoxicidad directa in vitro y el metabolismo de safrol y dilapiol (obtenidos desde Piper auritum and Piper holtonii, respectivamente) por Botryodiplodia theobromae y Colletotrichum acutatum. Los valores mayores de inhibición del crecimiento micelial de ambos hongos se obtuvieron para dilapiol, en comparación con safrol. B. theobromae metabolizó ambos compuestos a sus respectivos dioles vecinales, alcanzando abundancias relativas del 65 por ciento durante la biotransformación del dilapiol; mientras que C. acutatum solo transformó safrol en varios metabolitos con abundancias relativas menores al 5 por ciento. De acuerdo con la baja actividad antifúngica de los productos metabólicos mayoritarios (< 5 por ciento para los dioles vecinales), se sugiere un proceso de desintoxicación. Adicionalmente, se evaluó la influencia de algunos sustituyentes en el anillo aromático de safrol y dilapiol sobre la actividad antifúngica contra B. theobromae. Como resultado, el derivado nitrado del safrol, el 6–nitro safrol, presentó un nivel de fungitoxicidad similar al exhibido por el fungicida comercial Carbendazim® bajo las condiciones usadas. A la luz de lo anterior, safrol y dilapiol podrían ser sugeridos como plantillas estructurales adecuadas para el desarrollo de nuevos agentes antifúngicos.

Structural and biochemical characteristics of citrus flowers associated with defence against a fungal pathogen.

The constitutive characters of plants can be structural or biochemical and play an important role in their defence against pathogens. Citrus postbloom fruit drop (PFD) caused by Colletotrichum spp. is one of the most important fungal diseases of citrus. The pathogen infects the flowers, leading to premature fruit drop and reducing citrus production. However, flower buds smaller than 8 mm long are usually not infected by Colletotrichum spp. Thus, this study investigated whether there are constitutive mechanisms in flower buds related to Colletotrichum spp. infection. We studied flower buds that were 2, 3, 4, 8, 12 and 15 mm long and petals, after anthesis, of sweet orange 'Valência' using light and scanning electron microscopy and histochemistry. We evaluated the effect of volatile organic compounds (VOCs) in flowers (R-limonene and linalool) on the in vitro growth of Colletotrichum acutatum. We found that the arrangement of the epidermal papillae in the petal primordia, the occurrence of prismatic crystals and the distribution of oil glands are the main differences between buds smaller than 8 mm and buds 8-15 mm long. Osmophores at the tips of petals produced and accumulated phenols, terpenes and lipophilic compounds. Flower buds smaller than 8 mm long have constitutive structural and biochemical barriers to Colletotrichum spp. infection. In addition, this is the first time that osmophores have been reported in citrus. Our study shows that natural terpenes of Citrus flowers inhibit the fungal growth in vitro, highlighting the potential use of terpenes for the chemical control of PFD in citrus.

Furocoumarins and coumarins photoinactivate Colletotrichum acutatum and Aspergillus nidulans fungi under solar radiation.

The increasing tolerance to currently-used fungicides is a major problem both in clinical and agricultural areas leading to an urgent need for the development of novel antifungal strategies. This study investigated the in vitro antimicrobial photo treatment (APT) of conidia of the plant-pathogenic fungus Colletotrichum acutatum and the ascomycete Aspergillus nidulans with the furocoumarins 8-methoxypsoralen (8-MOP) and isopimpinellin, and a mixture of two coumarins (7-methoxy coumarin and citropten). Subcellular localization of the photosensitizer 8-MOP was also determined in C. acutatum conidia. Additionally, the effects of APT on the leaves of the plant host Citrus sinensis were determined. APT with 8-MOP (50µM) led to a reduction of approximately 4 logs in the survival of the conidia of both species, and the mixture of the two coumarins (12.5mgL(-1)) resulted in a reduction of approximately 4 logs for A. nidulans and 3 logs for C. acutatum. Isopimpinellin (50µM) displayed a reduction of 4 logs for A. nidulans but less than 2 logs for C. acutatum. Washing the conidia to remove unbound photosensitizers before light exposure reduced the photodynamic inactivation of C. acutatum both with 8-MOP and the mixture of the two coumarins. The reduction was smaller for A. nidulans. 8-MOP spread throughout the cytoplasm and accumulated in structures such as lipid bodies of C. acutatum conidia. No damage to orange tree leaves was observed after APT with any of the photosensitizers.

INDUCCIÓN DE ACTIVIDAD PEROXIDASA Y DE FENOLES TOTALES COMO RESPUESTA DEL FRUTO DE LULO (Solatium quitoense L.) AL PATÓGENO CAUSAL DE LA ANTRACNOSIS / INDUCTION OF PEROXIDASE AND TOTAL PHENOLS AS A RESPONSE OF LULO (Solanum quitoense L.) FRUIT TO PATHOGEN CAUSING ANTHRACNOSE / INDUÇÃO DA ACTMDADE PEROXIDASA E FENÓIS TOTAIS COMO RESPOSTA DO FRUTO DE LULO (Solanum quitoense L.) AO PATÓGENO QUE CAUSA ANTRACNOSE

Se evaluó la inducción de peroxidasa en frutos de lulo con el fin de determinar su participación en las respuestas bioquímicas hacia el patógeno Colletotrichum acutatum, causante de la antracnosis. Se establecieron como mejores condiciones para su extracción y para determinación de la actividad: buffer fosfatos 100 mM pH 7, 1% SDS y 1 % PVPP; sustrato guayacol 15 mM, peróxido de hidrógeno 10 mM, pH 6,5, 55 °C y 30 µL de extracto. Se realizó un ensayo in vivo usando frutos verdes, pintones y maduros, inoculados con el hongo o con agua estéril. Se determinó la actividad peroxidasa a diferentes horas a partir de la inoculación, encontrándose una respuesta diferencial con el tiempo por efecto de la presencia del patógeno, y según el estado de madurez de los frutos. En lulos verdes inoculados con el hongo se observó aumento en la actividad al cabo de 6 y 144 horas. En lulos pintones no se observó efecto notable, mientras que en maduros el aumento en actividad fue prácticamente a todos los tiempos. Los resultados del contenido de fenoles totales mostraron que hubo acumulación a 96 y 144 horas por efecto del patógeno, para lulos en estado verde y maduro, mientras que para pintones, en los que se presentaron más rápido y con mayor severidad los síntomas de la antracnosis, no se observó aumento a ninguno de los tiempos. En los frutos más enfermos, el cambio en actividad peroxidasa y en contenido total de fenoles fue menos evidente, por lo que se sugiere una relación inversa de estos con el desarrollo de la antracnosis.

The induction of peroxidase in lulo fruits was evaluated in order to determine its participation in the biochemical responses towards the pathogen Colletotrichum acutatum, which causes the antrachnosis disease. For extraction and activity determination, these conditions were established as the best: buffer phosphates 100 mM pH 7, 1% SDS y 1 % PVPP; substrate guaiacol 15 mM, hydrogen peroxide 10 mM, pH 6,5, 55 °C and 30 µL of the extract. An in vivo test was developed using unripe, semi-ripe and ripe fruits, inoculated with the fungus or sterile water. The peroxidase activity was measured at different hours starting from the inoculation, finding a time differential response caused by the pathogen presence and according to the maturity state of fruits; in unripe lulo fruits inoculated with the fungus, an increase of the activity was observed after 6 and 144 hours. In semi-ripe fruits no considerable effect was seen, while in ripe fruits the increase of the activity was found practically at all times. The results of the measured total phenol content, showed accumulation at 96 and 144 hours as a result of the pathogen presence in unripe and ripe fruits, while for semi-ripe fruits, in which the antrachnosis symptoms were noticed faster and more severely, no phenol increase was found at any time. The less evident changes seen in peroxidase and phenol content, using severely affected fruits by the disease, suggest an inverse relationship between these parameters and the development of the antrachnosis.

A indução da peroxidasa foi avaliada na casca de frutos de lulo com a finalidade de determinar a sua possível participação em reacções de defesa contra o patógeno Colletotrichum acutatum, causador da doença antracnose. Foram estabelecidas as melhores condições para a sua extracção e para medir a actividade da enzima extraída, encontrando-se que com tampão fosfato 100 mM pH 7, 1% SDS y 1% PVPP, substrato guaicol 15 mM, peróxido de hidrogeno 10 mM, pH 6,5, 55 °C y 30 µL de extracto enzimático se conseguiram as actividades enzimáticas mais elevadas. Foi realizado um ensaio in vivo usando frutos verdes, em processo de maduração e maduros, inoculados com o fungo ou com água estéril. A actividade peroxidasa foi determinada a diferentes horas a partir da inoculação encontrando-se uma resposta diferencial com o tempo pelo efeito da presença ao patógeno e de acordo ao estado de madurez dos frutos. Em lulos verdes observou-se um aumento da actividade nos frutos inoculados com o fungo ao fim de 6 e 144 horas. Em lulos em processo de maduração, o patógeno não teve maior efeito, enquanto que em lulos maduros o aumento na actividade da enzima, como resposta à presença do patógeno, foi praticamente a todos os tempos avaliados. Os resultados do conteúdo de fenóis totais mostraram que ouve uma acumulação significativa a 96 e 144 horas por efeito da inoculação com o patógeno para lulos em estado verde e maduro, enquanto que para os lulos em processo de maduração, nos quais se apresentaram mais rápido e com maior severidade os sintomas de antracnose, não se observou aumento em nenhum dos tempo avaliados. Nos frutos mais afectados pelos sintomas, a mudança na actividade POD e no conteúdo total de fenóis foi menos evidente, motivo pelo qual se sugere uma relação inversa de estes com o desenvolvimento de antracnose.

INDUCCIÓN DE POLIFENOLOXIDASA EN FRUTOS DE LULO (Solanum quitoense) COMO RESPUESTA A LA INFECCIÓN CON Colletotrichum acutatum / Polyphenol Oxidase Induction in Lulo Fruits (Solanum quitoense) Infected by Colletotrichum acutatum

Se evaluó la actividad polifenoloxidasa (PFO) en corteza de frutos de lulo con el fin de determinar su participación en respuestas hacia el patógeno Colletotrichum acutatum, causante de la antracnosis. Se estudiaron condiciones para la adecuada extracción de esta enzima, encontrándose que con buffer fosfatos 100 mM pH 7, 1% SDS y 1% PVPP se logran las mayores actividades. Se determinaron como mejores parámetros para medir la actividad de la enzima extraída, sustrato catecol 40 mM, pH 7,0, 23 °C y 30 µL de extracto. Para determinar su posible inducción en la interacción con el patógeno, se realizó un ensayo in vivo usando frutos verdes, pintones y maduros, inoculados con el hongo o con agua estéril. A nueve tiempos postinoculación se determinó la actividad PFO encontrándose que hay una respuesta diferencial con el tiempo y la madurez de los frutos y por efecto del patógeno. Se obtuvo aumento de actividad en lulos verdes a 48, 96 y 144 horas postinoculación (hpi) y en maduros a la mayoría de los tiempos evaluados, siendo éste estado en el que se presentó la respuesta más notable de inducción. En pintones aumentó solo a 72 y 144 hpi. Los mayores valores se registraron en general para frutos en estado verde. Los frutos respondieron al estrés ocasionado por la herida activando también esta enzima. La inducción de actividad se presentó a tiempos más rápidos en los frutos menos afectados por la enfermedad (verdes y maduros), por lo que se puede postular una relación positiva entre inducción de PFO y respuesta de tolerancia a la antracnosis.

Polyphenol oxidase (PFO) activity induction was evaluated in lulo fruits to determine the role of this enzyme in resistance responses towards the pathogen Colletotrichum acutatum which causes anthracnose disease. We studied the experimental conditions to obtain the enzyme, using lulo peel, and found that extraction with phosphates buffer 100 mM pH 7, 1% SDS y 1% PVPP showed higher activities. The adequate parameters for activity measurement was also evaluated and established as cathecol 40 mM, pH 7,0, 23 °C and 30 µL of extract. Then, we performed an in vivo assay using lulo fruits in three maturity stages, green, semimature and mature, which were inoculated with the fungus or with sterile water. Enzymatic induction of this protein was studied at nine postinoculation times, and it was found that the induction was differential according to the time, the maturity stage, and as consequence of pathogen presence. The PFO activity increased in green fruits at 48, 96 y 144 (hours postinoculation (hpi), and in mature lulos for the majority of times studied, with the most significant induction response at this stage. In semimature lulo, the induction was observed only at 72 and 144 hpi. The highest nominal value of activity was found in green fruits. Fruits responded to incision with enzyme activation. The increase in the activity of the enzyme was faster in the fruits with the minor anthracnose symptoms than the ones that were more affected. Therefore, it is possible to postulate a positive relation between PFO induction and tolerance to anthracnose symptoms.

Contribuição ao estudo da atividade antifúngica de Maytenus ilicifolia Mart. ex Reiss., Celastraceae / Study of antifungal activity of Maytenus ilicifolia Mart. ex Reiss., Celastraceae

Maytenus ilicifolia Mart. ex Reiss, Celastraceae, espinheira-santa, é nativa em muitas partes da América do Sul, sendo encontrada no sul do Brasil. É utilizada no tratamento de gastrite, úlceras e outras desordens do estômago, na forma de decocção das folhas ou extrato liofilizado em cápsulas. Neste trabalho, estudou-se a atividade antifúngica do seu extrato etanólico sobre o crescimento micelial dos fungos fitopatogênicos Colletotrichum acutatum, Fusarium oxysporum e Cylindrocladium spathulatum. Na comparação do crescimento micelial de isolados desses fungos em BDA (Batata Dextrose Agar), adicionados do extrato etanólico da espinheira-santa, foi observado que o mesmo inibiu em mais de 10 por cento o crescimento micelial de Fusarium oxysporum, nas três concentrações avaliadas (0,2; 0,4 e 0,6mg/mL), estimulou o crescimento micelial de Colletotrichum acutatum em mais de 30 por cento na concentração de 0,2mg/mL, e algumas de suas frações inibiram o desenvolvimento de Cylindrocladium spathulatum.

Study of antifungal activity of Maytenus ilicifolia Mart. ex Reiss., Celastraceae. Maytenus ilicifolia Mart. ex Reiss., Celastraceae, known as espinheira-santa, is native to South America, and in southern Brazil its leaves decoction is traditionally used for the treatment of gastritis, ulcers and other gastrointestinal disorders. The objective of this work was to investigate the effect of the crude ethanolic extract of espinheira-santa on the mycelial growth of three phytopathogenical fungi: Colletotrichum acutatum, Fusarium oxysporum and Cylindrocladium spathulatum. It was observed that this extract inhibited in more than 10 percent the mycelial growth of Fusarium oxysporum at all used concentrations (0.2; 0.4 and 0.6 mg/mL), stimulated the Colletotrichum acutatum mycelial growth in more than 30 percent at 0.2 mg/mL and some of its fractions inhibited the Cylindrocladium spathulatum development.