Synthesis and Fungicide Activity on Asperisporium caricae of Glycerol Derivatives Bearing 1,2,3-Triazole Fragments.

In agriculture, the control of fungal infections is essential to improve crop quality and productivity. This study describes the preparation and fungicidal activity evaluation of 12 glycerol derivatives bearing 1,2,3-triazole fragments. The derivatives were prepared from glycerol in four steps. The key step corresponded to the Cu(I)-catalyzed alkyne-azide cycloaddition (CuAAC) click reaction between the azide 4-(azidomethyl)-2,2-dimethyl-1,3-dioxolane (3) and different terminal alkynes (57-91% yield). The compounds were characterized by infrared spectroscopy, nuclear magnetic resonance (1H and 13C), and high-resolution mass spectrometry. The in vitro assessment of the compounds on Asperisporium caricae, that is, the etiological agent of papaya black spot, at 750 mg L-1 showed that the glycerol derivatives significantly inhibited conidial germination with different degrees of efficacy. The most active compound 4-(3-chlorophenyl)-1-((2,2-dimethyl-1,3-dioxolan-4-yl) methyl)-1H-1,2,3-triazole (4c) presented a 91.92% inhibition. In vivo assays revealed that 4c reduced the final severity (70.7%) and area under the disease severity progress curve of black spots on papaya fruits 10 days after inoculation. The glycerol-bearing 1,2,3-triazole derivatives also present agrochemical-likeness properties. Our in silico study using molecular docking calculations show that all triazole derivatives bind favorably to the sterol 14α-demethylase (CYP51) active site at the same region of the substrate lanosterol (LAN) and fungicide propiconazole (PRO). Thus, the mechanism of action of the compounds 4a-4l may be the same as the fungicide PRO, blocking the entrance/approximation of the LAN into the CYP51 active site by steric effects. The reported results point to the fact that the glycerol derivatives may represent a scaffold to be explored for the development of new chemical agents to control papaya black spot.

First report of Crassiphiala sp. (Trematoda: Diplostomidae) as an etiological agent of black spot disease in commercial ornamental fish from Brazil.

Ornamental fish are becoming increasingly popular, but the lack of knowledge regarding their various diseases is a major challenge. Skin diseases commonly found in freshwater fish include black spot disease (BSD), which is characterized by melanin deposits around the metacercariae of some trematode species. Since BSD remains poorly understood, this study describes an outbreak of BSD in Etroplus maculatus raised in outdoor ponds at a Brazilian fish farm. Metacercariae samples were collected, examined, and subjected to molecular phylogenetic analysis. The parasites were conspecific to an unnamed species, Crassiphiala lineage 5, recently found in Brazilian birds (Megaceryle torquata). Sequences obtained for longifurcate cercariae of the planorbid snail Biomphalaria straminea from the same region were identical to our metacercariae of Crassiphiala sp. These results suggest that Biompahalaria snails are likely an intermediate host of this parasite on farms where E. maculatus was found to be infected. We provide the first molecular evidence that Crassiphiala are the causative agents of BSD in fish from Brazil. Combatting snails and preventing access of fish-eating birds to outdoor ponds are strategies to control this disease in ornamental fish farms.

Occurrence of citrus brown and black spot diseases and their control using pre-harvest approaches

Mild brown and black spot diseases symptoms were detected on citrus varieties, Valencia and Navel fruits during season 2018 in some citrus orchards at North Egypt. Collected diseased fruit samples revealed isolation of Alternaria alternata and Phyllosticta citricarpa (McAlpine) van der Aa. Some organic acids, salts and Saccharomyces cerevisiae were evaluated against the diseases incidents in vitro and in vivo. Complete growth inhibition was recorded for fungi tested at 2g/L of salicylic acid, Potassium dihydrogen phosphate, Tri-Sodium polyphosphate and 2.5 g/L of S. cerevisiae. All pre-harvest treated Valencia trees, inoculated fruits revealed no diseases symptoms up to 10 days of storage period. Meanwhile, S. cerevisiae and salicylic acid treatments had extended protective effect up to 20 days. Control strategy through spraying Valencia orange trees with yeast or some organic acids and salts as pre-harvest approaches should be taken in consideration especially these diseases began to occur in North region of Egypt.

Morphological characterization and molecular identification of Colletotrichum species associated to sweet persimmon anthracnose in Southern Brazil / Caracterização morfológica e identificação molecular de espécies de Colletotrichum associadas à antracnose de caqui doce no Sul do Brasil

The highlands of Southern Brazil contribute with 40% of Brazilian persimmon production. Although expanding, persimmon production faces major problems caused by anthracnose disease (black spot), including fruit rot and necrosis of leaves. Several Colletotrichum species (C. horii, C. gloeosporioides, among others) are implicated in persimmon anthracnose around the world. To identify Colletotrichum species associated with persimmon anthracnose in the highlands of Southern Brazil, 34 isolates were analyzed by ITS-rDNA partial region, GAPDH, and TUB2 partial gene sequences, morphological characteristics, and virulence on persimmon fruits and leaves. Data showed a high prevalence of C. horii (85.3%), that associated with its high virulence on fruits and leaves, confirm a considerable degree of host preference. Moreover, other species C. aenigma, C. asianum, C. fructicola, and C. nymphaeae, were identified, but the last three ones exhibited low virulence on fruits and were not able to produce symptoms on leaves. As far as we know this is the first reference on C. asianum in persimmon. The present data may contribute to a better understanding of the etiology of anthracnose in sweet persimmon in Southern Brazil, and it will be useful for epidemiological studies and the development of disease management measures.(AU)

As terras altas do sul do Brasil contribuem com 40% da produção brasileira de caqui. Embora em expansão, a produção de caqui enfrenta grandes problemas causados ​​pela antracnose (mancha preta), incluindo o apodrecimento dos frutos e necrose das folhas. Várias espécies de Colletotrichum (C. horii, C. gloeosporioides, entre outras) estão envolvidas com a antracnose do caqui em todo mundo. Para identificar espécies de Colletotrichum associadas à antracnose de caqui nas terras altas do sul do Brasil, 34 isolados foram analisados ​​através da região parcial de ITS-rDNA e sequências parciais dos genes GAPDH e TUB2, características morfológicas e virulência em frutos e folhas de caqui. Os dados mostraram uma alta prevalência de C. horii (85,3%), que associada à sua alta virulência em frutos e folhas, confirma um grau considerável de preferência pelo hospedeiro. Além disso, foram identificadas outras espécies C. aenigma, C. asianum, C. fructicola, e C. nymphaeae, mas as três últimas exibiram baixa virulência nos frutos e não foram capazes de produzir sintomas nas folhas. Até onde sabemos, esta é a primeira referência sobre C. asianum em caqui. Os presentes dados podem contribuir para uma melhor compreensão da etiologia da antracnose em caqui doce no sul do Brasil e isso pode ser útil para estudos epidemiológicos e para o desenvolvimento de medidas de controle da doença.(AU)

Morphological characterization and molecular identification of Colletotrichum species associated to sweet persimmon anthracnose in Southern Brazil / Caracterização morfológica e identificação molecular de espécies de Colletotrichum associadas à antracnose de caqui doce no Sul do Brasil

ABSTRACT: The highlands of Southern Brazil contribute with 40% of Brazilian persimmon production. Although expanding, persimmon production faces major problems caused by anthracnose disease (black spot), including fruit rot and necrosis of leaves. Several Colletotrichum species (C. horii, C. gloeosporioides, among others) are implicated in persimmon anthracnose around the world. To identify Colletotrichum species associated with persimmon anthracnose in the highlands of Southern Brazil, 34 isolates were analyzed by ITS-rDNA partial region, GAPDH, and TUB2 partial gene sequences, morphological characteristics, and virulence on persimmon fruits and leaves. Data showed a high prevalence of C. horii (85.3%), that associated with its high virulence on fruits and leaves, confirm a considerable degree of host preference. Moreover, other species C. aenigma, C. asianum, C. fructicola, and C. nymphaeae, were identified, but the last three ones exhibited low virulence on fruits and were not able to produce symptoms on leaves. As far as we know this is the first reference on C. asianum in persimmon. The present data may contribute to a better understanding of the etiology of anthracnose in sweet persimmon in Southern Brazil, and it will be useful for epidemiological studies and the development of disease management measures.

RESUMO: As terras altas do sul do Brasil contribuem com 40% da produção brasileira de caqui. Embora em expansão, a produção de caqui enfrenta grandes problemas causados ​​pela antracnose (mancha preta), incluindo o apodrecimento dos frutos e necrose das folhas. Várias espécies de Colletotrichum (C. horii, C. gloeosporioides, entre outras) estão envolvidas com a antracnose do caqui em todo mundo. Para identificar espécies de Colletotrichum associadas à antracnose de caqui nas terras altas do sul do Brasil, 34 isolados foram analisados ​​através da região parcial de ITS-rDNA e sequências parciais dos genes GAPDH e TUB2, características morfológicas e virulência em frutos e folhas de caqui. Os dados mostraram uma alta prevalência de C. horii (85,3%), que associada à sua alta virulência em frutos e folhas, confirma um grau considerável de preferência pelo hospedeiro. Além disso, foram identificadas outras espécies C. aenigma, C. asianum, C. fructicola, e C. nymphaeae, mas as três últimas exibiram baixa virulência nos frutos e não foram capazes de produzir sintomas nas folhas. Até onde sabemos, esta é a primeira referência sobre C. asianum em caqui. Os presentes dados podem contribuir para uma melhor compreensão da etiologia da antracnose em caqui doce no sul do Brasil e isso pode ser útil para estudos epidemiológicos e para o desenvolvimento de medidas de controle da doença.

Morphological characterization and molecular identification of Colletotrichum species associated to sweet persimmon anthracnose in Southern Brazil / Caracterização morfológica e identificação molecular de espécies de Colletotrichum associadas à antracnose de caqui doce no Sul do Brasil

The highlands of Southern Brazil contribute with 40% of Brazilian persimmon production. Although expanding, persimmon production faces major problems caused by anthracnose disease (black spot), including fruit rot and necrosis of leaves. Several Colletotrichum species (C. horii, C. gloeosporioides, among others) are implicated in persimmon anthracnose around the world. To identify Colletotrichum species associated with persimmon anthracnose in the highlands of Southern Brazil, 34 isolates were analyzed by ITS-rDNA partial region, GAPDH, and TUB2 partial gene sequences, morphological characteristics, and virulence on persimmon fruits and leaves. Data showed a high prevalence of C. horii (85.3%), that associated with its high virulence on fruits and leaves, confirm a considerable degree of host preference. Moreover, other species C. aenigma, C. asianum, C. fructicola, and C. nymphaeae, were identified, but the last three ones exhibited low virulence on fruits and were not able to produce symptoms on leaves. As far as we know this is the first reference on C. asianum in persimmon. The present data may contribute to a better understanding of the etiology of anthracnose in sweet persimmon in Southern Brazil, and it will be useful for epidemiological studies and the development of disease management measures.

As terras altas do sul do Brasil contribuem com 40% da produção brasileira de caqui. Embora em expansão, a produção de caqui enfrenta grandes problemas causados ​​pela antracnose (mancha preta), incluindo o apodrecimento dos frutos e necrose das folhas. Várias espécies de Colletotrichum (C. horii, C. gloeosporioides, entre outras) estão envolvidas com a antracnose do caqui em todo mundo. Para identificar espécies de Colletotrichum associadas à antracnose de caqui nas terras altas do sul do Brasil, 34 isolados foram analisados ​​através da região parcial de ITS-rDNA e sequências parciais dos genes GAPDH e TUB2, características morfológicas e virulência em frutos e folhas de caqui. Os dados mostraram uma alta prevalência de C. horii (85,3%), que associada à sua alta virulência em frutos e folhas, confirma um grau considerável de preferência pelo hospedeiro. Além disso, foram identificadas outras espécies C. aenigma, C. asianum, C. fructicola, e C. nymphaeae, mas as três últimas exibiram baixa virulência nos frutos e não foram capazes de produzir sintomas nas folhas. Até onde sabemos, esta é a primeira referência sobre C. asianum em caqui. Os presentes dados podem contribuir para uma melhor compreensão da etiologia da antracnose em caqui doce no sul do Brasil e isso pode ser útil para estudos epidemiológicos e para o desenvolvimento de medidas de controle da doença.

Scaphanocephalus-associated dermatitis as the basis for black spot disease in Acanthuridae of St. Kitts, West Indies.

Acanthurus spp. of St. Kitts and other Caribbean islands, including ocean surgeonfish A. bahianus, doctorfish A. chirurgus, and blue tang A. coeruleus, frequently show multifocal cutaneous pigmentation. Initial reports from the Leeward Antilles raised suspicion of a parasitic etiology. The aim of this study was to quantify the prevalence of the disease in St. Kitts' Acanthuridae and describe its pathology and etiology. Visual surveys demonstrated consistently high adjusted mean prevalence at 3 shallow reefs in St. Kitts in 2017 (38.9%, 95% CI: 33.8-43.9) and 2018 (51.5%; 95% CI: 46.2-56.9). There were no differences in prevalence across species or reefs, but juvenile fish were less commonly affected than adults. A total of 29 dermatopathy-affected acanthurids were sampled by spearfishing for comprehensive postmortem examination. Digenean metacercariae were dissected from <1 mm cysts within pigmented lesions. Using partial 28S rDNA sequence data they were classified as Family Heterophyidae, members of which are commonly implicated in black spot disease of other fishes. Morphological features of the parasite were most typical of Scaphanocephalus spp. (Creplin, 1842), and 2 genetic profiles were obtained suggesting more than 1 digenean species. Histologically, pigmented lesions had mild chronic perivascular dermatitis and increased melanophores and melanin density, often centered on encysted digenean metacercariae. In 1 affected A. chirurgus, similar metacercariae were histologically identified in skeletal and cardiac muscle. Further research is needed to clarify impact on host fitness, establish the number of heterophyid digenean species that cause black spots on Caribbean fishes and to determine the intermediate and definitive host species.

Comparative genome analysis of Phyllosticta citricarpa and Phyllosticta capitalensis, two fungi species that share the same host.

BACKGROUND: Citrus are among the most important crops in the world. However, there are many diseases that affect Citrus caused by different pathogens. Citrus also hosts many symbiotic microorganisms in a relationship that may be advantageous for both organisms. The fungi Phyllosticta citricarpa, responsible for citrus black spot, and Phyllosticta capitalensis, an endophytic species, are examples of closely related species with different behavior in citrus. Both species are always biologically associated and are morphologically very similar, and comparing their genomes could help understanding the different lifestyles. In this study, a comparison was carried to identify genetic differences that could help us to understand the biology of P. citricarpa and P. capitalensis. RESULTS: Drafts genomes were assembled with sizes close to 33 Mb for both fungi, carrying 15,206 and 14,797 coding sequences for P. citricarpa and P. capitalensis, respectively. Even though the functional categories of these coding sequences is similar, enrichment analysis showed that the pathogenic species presents growth and development genes that may be necessary for the pathogenicity of P. citricarpa. On the other hand, family expansion analyses showed the plasticity of the genome of these species. Particular families are expanded in the genome of an ancestor of P. capitalensis and a recent expansion can also be detected among this species. Additionally, evolution could be driven by environmental cues in P. citricarpa. CONCLUSIONS: This work demonstrated genomic differences between P. citricarpa and P. capitalensis. Although the idea that these differences could explain the different lifestyles of these fungi, we were not able to confirm this hypothesis. Genome evolution seems to be of real importance among the Phyllosticta isolates and it is leading to different biological characteristics of these species.

Phylogeographic distribution of asperisporium caricae isolates in brazilian southeastern and northeastern coastal regions / Distribuição filogeográfica de isolados de Asperisporium caricae no litoral do sudeste e nordeste brasileiro

Papaya (Carica papaya L.) is one of the main tropical fruits consumed in Brazil. The country is also one of the main papaya exporters, but one of the factors hindering its production lies on foliar diseases such as papaya black spot, which is caused by fungus Asperisporium caricae. This pathogen is widely distributed in the main producing regions of the Brazilian coastal area. Phylogeographic studies contribute to the knowledge about the genetic diversity and geographical distribution of genealogical lineages (haplotypes) and help better understanding the reproductive and evolutionary processes of closely related species or of a single species. Such information is useful in studies about phytopathogenic fungi because it enables identifying the most prevalent genealogical lineages in a given location, as well as inferring dispersal routes and providing information on the origin and frequency of exotic material introduction events. Results in this type of study can significantly help developing new disease control strategies. Literature still lacks studies on the Papaya x A. caricae pathosystem. Based on the phylogenetic and phylogeographic analysis applied to nucleotide sequences of the Internal transcribed spacer (ITS) gene, we herein address the genealogical and dispersal events recorded for this pathogen in order to better understand its evolution in, and adaptation to, Brazilian orchards. Three haplotypes were identified among the A. caricae isolates; their distribution was mostly related to the geographic distance between sample collection regions rather than to any reproductive or evolutionary processes presented by the species. The low variability among the herein studied isolates may result from the physiological specialization (survival exclusively associated with the host plant) and from the regional transport of contaminated fruits (with lesions and spores), besides the low contribution of reproductive events, which corroborate the lack of knowledge about the sexual stages of A. caricae.

O mamoeiro (Carica papaya L.), é uma das principais frutas tropicais consumidas no Brasil. O país é um dos principais exportadores de mamão e um dos gargalos da produção mundial está diretamente ligado à ocorrência de doenças foliares, podendo-se destacar a pinta-preta do mamoeiro, causada pelo fungo Asperisporium caricae. O patógeno está amplamente distribuído nas principais regiões produtoras, as quais englobam grande parte do litoral Brasileiro. Estudos filogeográficos contribuem não só para oconhecimento da diversidade genética e da distribuição geográfica das linhagens genealógicas (haplótipos), como também contribuem para o conhecimento dos processos reprodutivos e evolutivos de uma espécie ou de espécies estreitamente relacionadas. No estudo de fungos fitopatogênicos, tais informações são úteis para identificar linhagens genealógicas mais prevalentes em um determinado local, inferir as rotas de dispersão e fornecer informações sobre a origem e frequência dos eventos de introdução de material exótico. Este tipo de estudo produz resultados que podem colaborar significativamente na elaboração de novas estratégias de controle da doença. Não existe, até o presente momento, este tipo de estudo para o patossistema Mamoeiro x A. caricae. Neste trabalho, por meio da análise filogenética e filogeográfica, a partir de sequências nucleotídicas do gene Internal transcribed spacer (ITS), discutimos os eventos genealógicos e de dispersão deste patógeno com o intuito de compreender melhor sua evolução e adaptação nos pomares brasileiros. Dentre os isolados de A. caricae foram identificados 3 haplótipos, sendo sua distribuição relacionada mais ao distanciamento geográfico das regiões das coletas das amostras do que a eventuais processos reprodutivos ou evolutivos da espécie. Acredita-se que a baixa variabilidade dentre os isolados estudados seja explicada pela especialização fisiológica (sobrevivência exclusivamente associada à planta hospedeira) e ao transporte regional de frutos contaminados (com lesões e esporos), sendo baixa a contribuição de eventos reprodutivos, o que corrobora o desconhecimento de fase sexual de A. caricae.

Biological activity of Diaporthe terebinthifolii extracts against Phyllosticta citricarpa.

Citrus black spot disease, caused by the phytopathogen Phyllosticta citricarpa, depreciates the market value of citrus fruits and prevents their exportation to disease-free regions. It may also reduce the productivity of citrus fruit orchards. To identify an alternative to conventional disease control measures, isolates of Diaporthe terebinthifolii, active against P. citricarpa, were selected from an endophytic fungal population of Schinus terebinthifolia leaves. Different culture media were screened to identify the culture medium that afforded the most efficient production of biologically active extracts. A particular fraction (fraction VI) of the extract completely protected orange leaves by inhibiting the germination of P. citricarpa conidia with a minimum inhibitory concentration of 0.003 µg.mL-1. The active constituents in D. terebinthifolii extract fractions were identified by gas chromatography coupled to mass spectrometry as verbanol, phenylethyl alcohol, verbenyl acetate and methyl hexadecanoate. The results obtained strongly suggest the existence of a synergistic effect among the metabolites produced. Thus, these fungal metabolites could be used to control the CBS disease. As the asexual spores of P. citricarpa play an important role in fruit lesion development and disease dispersion, fungal extracts that inhibit the spore germination can be used as an effective alternative for directional disease control.

Desenvolvimento de Alternaria solani em diferentes meios de cultura, fotoperíodo e temperatura / Alternaria solani development in diferent culture media, photoperiod and temperature

O tomateiro está entre as hortaliças mais cultivadas, porém é altamente susceptível ao ataque de patógenos. A pinta-preta é uma das doenças mais frequentes sendo constatada em condições de alta umidade e temperatura, ou seja, ideais ao desenvolvimento do patógeno. O objetivo do presente trabalho foi avaliar o efeito dos meios de cultura, temperatura e fotoperíodo no crescimento micelial e esporulação de Alternaria solani. Foram realizados diferentes ensaios. O primeiro, de diferentes meios de cultura com os tratamentos: 1) BDA; 2) BDA+Polpa de Tomate; 3) BDA+Folha de Tomate; 4) BDA+Cenoura e 5) Aveia+Ágar, com 4 repetições. O segundo de luminosidade: 1) escuro, 2) fotoperíodo alternado e 3) claro contínuo, com 7 repetições. E, por fim, o de temperatura, constituído por: 1) 25 °C; 2) temperatura ambiente e 3) 30 °C, com 7 repetições. Foram realizadas avaliações diárias de crescimento micelial e esporulação do patógeno ao final de cada ensaio. Dados foram submetidos à análise pelo teste de Tukey a 5%. O meio de cultura contendo BDA+polpa de tomate apres entou o melhor crescimento micelial e esporulação da Alternaria solani, apresentando-se como mais adequado para seu isolamento. A luminosidade interferiu significativamente na esporulação do fungo, sendo o fotoperíodo escuro o que apresentou a maior esporulação, não havendo diferença para crescimento micelial. A temperatura de 30 °C proporcionou o melhor tratamento para isolamento do patógeno, tanto em crescimento micelial, quanto em esporulação.(AU)

Tomato is among the most cultivated vegetables, but is highly susceptible to attack of pathogens. The blight is one of the most frequent diseases and is observed in high humidity and temperature, ideal environmental conditions for the development of the pathogen. The objective of this study was to evaluate the effect of culture media, temperature and photoperiod on Alternaria solani mycelia growth and sporulation. Different assays were performed. The first, of different culture media with the treatments: 1) PDA; 2) PDA+Tomato pulp; 3) PDA+Tomato Leaf; 4) PDA+Carrot and 5) Oatmeal+Agar with 4 replications. The second of light influence: 1) dark, 2) alternating photoperiod and 3) continuous light, with 7 repetitions. And, temperature, constituted by: 1) 25 °C; 2) 30 °C and 3) ambient temperature with 7 replicates. Daily reviews of mycelia growth and sporulation of the pathogen were performed. Data were analyzed by Tukey test at 5%. All variables showed interference on growth and sporulation of Alternaria solani, highlighting the PDA culture medium + Tomato Pulp, dark photoperiod and 30 °C. It can be concluded that in studies of the of pathogen etiology, climatic and environmental factors may influence their growth and reproduction.(AU)

Desenvolvimento de Alternaria solani em diferentes meios de cultura, fotoperíodo e temperatura / Alternaria solani development in diferent culture media, photoperiod and temperature

O tomateiro está entre as hortaliças mais cultivadas, porém é altamente susceptível ao ataque de patógenos. A pinta-preta é uma das doenças mais frequentes sendo constatada em condições de alta umidade e temperatura, ou seja, ideais ao desenvolvimento do patógeno. O objetivo do presente trabalho foi avaliar o efeito dos meios de cultura, temperatura e fotoperíodo no crescimento micelial e esporulação de Alternaria solani. Foram realizados diferentes ensaios. O primeiro, de diferentes meios de cultura com os tratamentos: 1) BDA; 2) BDA+Polpa de Tomate; 3) BDA+Folha de Tomate; 4) BDA+Cenoura e 5) Aveia+Ágar, com 4 repetições. O segundo de luminosidade: 1) escuro, 2) fotoperíodo alternado e 3) claro contínuo, com 7 repetições. E, por fim, o de temperatura, constituído por: 1) 25 °C; 2) temperatura ambiente e 3) 30 °C, com 7 repetições. Foram realizadas avaliações diárias de crescimento micelial e esporulação do patógeno ao final de cada ensaio. Dados foram submetidos à análise pelo teste de Tukey a 5%. O meio de cultura contendo BDA+polpa de tomate apres entou o melhor crescimento micelial e esporulação da Alternaria solani, apresentando-se como mais adequado para seu isolamento. A luminosidade interferiu significativamente na esporulação do fungo, sendo o fotoperíodo escuro o que apresentou a maior esporulação, não havendo diferença para crescimento micelial. A temperatura de 30 °C proporcionou o melhor tratamento para isolamento do patógeno, tanto em crescimento micelial, quanto em esporulação.

Tomato is among the most cultivated vegetables, but is highly susceptible to attack of pathogens. The blight is one of the most frequent diseases and is observed in high humidity and temperature, ideal environmental conditions for the development of the pathogen. The objective of this study was to evaluate the effect of culture media, temperature and photoperiod on Alternaria solani mycelia growth and sporulation. Different assays were performed. The first, of different culture media with the treatments: 1) PDA; 2) PDA+Tomato pulp; 3) PDA+Tomato Leaf; 4) PDA+Carrot and 5) Oatmeal+Agar with 4 replications. The second of light influence: 1) dark, 2) alternating photoperiod and 3) continuous light, with 7 repetitions. And, temperature, constituted by: 1) 25 °C; 2) 30 °C and 3) ambient temperature with 7 replicates. Daily reviews of mycelia growth and sporulation of the pathogen were performed. Data were analyzed by Tukey test at 5%. All variables showed interference on growth and sporulation of Alternaria solani, highlighting the PDA culture medium + Tomato Pulp, dark photoperiod and 30 °C. It can be concluded that in studies of the of pathogen etiology, climatic and environmental factors may influence their growth and reproduction.

Diaporthe endophytica and D. terebinthifolii from medicinal plants for biological control of Phyllosticta citricarpa.

The citrus industry is severely affected by citrus black spot (CBS), a disease caused by the pathogen Phyllosticta citricarpa. This disease causes loss of production, decrease in the market price of the fruit, and reduction in its export to the European Union. Currently, CBS disease is being treated in orchards with various pesticides and fungicides every year. One alternative to CBS disease control without harming the environment is the use of microorganisms for biological control. Diaporthe endophytica and D. terebinthifolii, isolated from the medicinal plants Maytenus ilicifolia and Schinus terebinthifolius have an inhibitory effect against P. citricarpa in vitro and in detached fruits. Moreover, D. endophytica and D. terebinthifolii were transformed by Agrobacterium tumefaciens for in vivo studies. The transformants retained the ability to control of phytopathogenic fungus P. citricarpa after transformation process. Furthermore, D. endophytica and D. terebinthifolii were able to infect and colonize citrus plants, which is confirmed by reisolation of transformants from inoculated and uninoculated leaves. Light microscopic analysis showed fungus mycelium colonizing intercellular region and oil glands of citrus, suggesting that these two new species are capable of colonizing citrus plants, in addition to controlling the pathogen P. citricarpa.

Agrobacterium-mediated transformation of Guignardia citricarpa: an efficient tool to gene transfer and random mutagenesis.

Guignardia citricarpa is the causal agent of Citrus Black Spot (CBS), an important disease in Citriculture. Due to the expressive value of this activity worldwide, especially in Brazil, understanding more about the functioning of this fungus is of utmost relevance, making possible the elucidation of its infection mechanisms, and providing tools to control CBS. This work describes for the first time an efficient and successful methodology for genetic transformation of G. citricarpa mycelia, which generated transformants expressing the gene encoding for the gfp (green fluorescent protein) and also their interaction with citrus plant. Mycelia of G. citricarpa were transformed via Agrobacterium tumefaciens, which carried the plasmid pFAT-gfp, contains the genes for hygromycin resistance (hph) as well as gfp. The optimization of the agrotransformation protocol was performed testing different conditions (type of membrane; inductor agent concentration [acetosyringone - AS] and cocultivation time). Results demonstrated that the best condition occurred with the utilization of cellulose's ester membrane; 200 µM of AS and 96 h as cocultivation time. High mitotic stability (82 %) was displayed by transformants using Polymerase Chain Reaction (PCR) technique to confirm the hph gene insertion. In addition, the presence of gfp was observed inside mycelia by epifluorescence optical microscopy. This technique easy visualization of the behaviour of the pathogen interacting with the plant for the first time, allowing future studies on the pathogenesis of this fungus. The establishment of a transformation method for G. citricarpa opens a range of possibilities and facilitates the study of insertional mutagenesis and genetic knockouts, in order to identify the most important genes involved in the pathogenesis mechanisms and plant-pathogen interaction.

Injuries in Valência oranges cultivated in organic and conventional systems / Injúrias em laranja Valência produzida em sistemas orgânico e convencional

The organic plantation of citrus comes increasing annually, searching to take care of the increasing demand for healthful foods and sustainably produced. This study aimed to characterize the injuries of biotic (diseases and pests) and abiotic origin (mechanical damage and physiological disorders) in fruits of Valencia orange collected in orchards of organic and conventional crops in two locations (Borborema and Itápolis, State of Sao Paulo), as well as in organic Valencia orange commercialized in the Companhia de Entrepostos e Armazéns Gerais de São Paulo (Ceagesp). Fruits were evaluated visually for the incidence of injuries and stored during 14 days at 25 ºC and 85% of UR, when were evaluated the increase in the incidence of black spot citrus (BSC) and of dehydrated fruits. The MPC was the main disease affecting more than 50% in the sampled fruits in the orchards and with an average increase of 17% after storage. The incidence of melanose was higher in organic fruits, however, for the other diseases the results varied in relation to the cropping system in the localities. The incidence of symptoms of pesticide phytotoxicity was higher in fruits from the conventional system. Five species of scale insects were observed, especially black parlatore, with incidences ranging depending on the farming system in the localities. Major damage by hopes were observed in organic fruits,

O plantio de citros sob sistema orgânico vem aumentando anualmente, buscando atender a demanda crescente por alimentos saudáveis e produzidos de forma sustentável. Este trabalho objetivou caracterizar as injúrias de origem biótica (doenças e pragas) e abiótica (distúrbios fisiológicos e danos mecânicos) em frutos de laranja Valência coletados em pomares cultivados orgânica e convencionalmente em duas localidades (Borborema e Itápolis, Estado de São Paulo), assim como em frutos de laranja Valência orgânicos, comercializados na Companhia de Entrepostos e Armazéns Gerais de São Paulo (Ceagesp). Os frutos foram avaliados visualmente quanto à incidência de injúrias e submetidos a 25ºC e 85% de umidade relativa, por 14 dias, quando avaliou-se o aumento na incidência da mancha preta dos citros (MPC) e a incidência de frutos desidratados. A MPC foi a principal doença com incidência superior a 50% nos frutos amostrados nos pomares e com um aumento médio de 17% após o armazenamento. Maior incidência de melanose foi observada nos frutos orgânicos, entretanto, para as demais doenças os resultados foram variáveis em relação ao sistema de cultivo nas localidades. Maior incidência de sintomas de fitotoxidez por defensivos foi observada nos frutos do sistema convencional. Foram observadas cinco espécies de cochonilhas, com destaque para parlatória-preta, apresentando incidências variáveis

Injuries in Valência oranges cultivated in organic and conventional systems / Injúrias em laranja Valência produzida em sistemas orgânico e convencional

The organic plantation of citrus comes increasing annually, searching to take care of the increasing demand for healthful foods and sustainably produced. This study aimed to characterize the injuries of biotic (diseases and pests) and abiotic origin (mechanical damage and physiological disorders) in fruits of Valencia orange collected in orchards of organic and conventional crops in two locations (Borborema and Itápolis, State of Sao Paulo), as well as in organic Valencia orange commercialized in the Companhia de Entrepostos e Armazéns Gerais de São Paulo (Ceagesp). Fruits were evaluated visually for the incidence of injuries and stored during 14 days at 25 ºC and 85% of UR, when were evaluated the increase in the incidence of black spot citrus (BSC) and of dehydrated fruits. The MPC was the main disease affecting more than 50% in the sampled fruits in the orchards and with an average increase of 17% after storage. The incidence of melanose was higher in organic fruits, however, for the other diseases the results varied in relation to the cropping system in the localities. The incidence of symptoms of pesticide phytotoxicity was higher in fruits from the conventional system. Five species of scale insects were observed, especially black parlatore, with incidences ranging depending on the farming system in the localities. Major damage by hopes were observed in organic fruits,

O plantio de citros sob sistema orgânico vem aumentando anualmente, buscando atender a demanda crescente por alimentos saudáveis e produzidos de forma sustentável. Este trabalho objetivou caracterizar as injúrias de origem biótica (doenças e pragas) e abiótica (distúrbios fisiológicos e danos mecânicos) em frutos de laranja Valência coletados em pomares cultivados orgânica e convencionalmente em duas localidades (Borborema e Itápolis, Estado de São Paulo), assim como em frutos de laranja Valência orgânicos, comercializados na Companhia de Entrepostos e Armazéns Gerais de São Paulo (Ceagesp). Os frutos foram avaliados visualmente quanto à incidência de injúrias e submetidos a 25ºC e 85% de umidade relativa, por 14 dias, quando avaliou-se o aumento na incidência da mancha preta dos citros (MPC) e a incidência de frutos desidratados. A MPC foi a principal doença com incidência superior a 50% nos frutos amostrados nos pomares e com um aumento médio de 17% após o armazenamento. Maior incidência de melanose foi observada nos frutos orgânicos, entretanto, para as demais doenças os resultados foram variáveis em relação ao sistema de cultivo nas localidades. Maior incidência de sintomas de fitotoxidez por defensivos foi observada nos frutos do sistema convencional. Foram observadas cinco espécies de cochonilhas, com destaque para parlatória-preta, apresentando incidências variáveis

Assessment of environmental quality through analysis of frequency of the black spot disease in an assemblage of fish, Guaíba lake, RS, Brazil / Avaliação da qualidade ambiental através da análise da frequência da doença dos pontos pretos em assembléias de peixes, Lago Guaíba, RS, Brasil

The frequency of the black spot disease caused by digenetic trematodes in fish was tested as an indicator of the quality of water in the watershed of Lake Guaíba. Samples were standardised and quarterly made using a seine net at eleven sites in the basin. A total of 53,408 individuals of 66 specimens pertaining to 22 families and 8 orders were collected and analysed. The highest frequency of the disease was found in Astyanax fasciatus. The simple Chi-Squared test applied to the species showed statistically significant frequency of occurrence for three sampled sites. Two of these sites showed the highest frequencies for two consecutive sampling periods of one year and the best levels of water quality. Results of this study suggest that the association between high frequency of infestation by a parasite that causes the black spot disease in fish, and environments with less degradation in water quality, recorded from physical, chemicals and microbiological variables, could be used as an indicator of water quality in these environments.

A frequência da doença dos pontos pretos, provocada por trematódeos digenéticos em peixes, foi testada como indicador da qualidade da água na bacia hidrográfica do lago Guaíba (RS). As amostragens foram padronizadas e realizadas trimestralmente com redes do tipo picaré em onze pontos desta bacia. Foram coletados e analisados 53.408 exemplares, totalizando 66 espécies pertencentes a 22 famílias e oito ordens. A maior frequência de ocorrência da doença foi verificada em Astyanax fasciatus. Um Qui-Quadrado simples aplicado para essa espécie indicou frequência estatisticamente significativa de ocorrência da doença para três locais amostrados. Dois destes locais apresentaram as maiores frequências durante dois períodos consecutivos de um ano de amostragem e os melhores níveis de qualidade de água. Os resultados deste trabalho sugerem que a associação entre a elevada frequência de infestação pelo parasito que causa a doença dos pontos pretos e os ecossistemas com menor degradação na qualidade da água, com registros de variáveis físicas, químicas e microbiológicas, pode ser utilizada como indicador de melhor qualidade ambiental.

Assessment of environmental quality through analysis of frequency of the black spot disease in an assemblage of fish, Guaíba lake, RS, Brazil

The frequency of the black spot disease caused by digenetic trematodes in fish was tested as an indicator of the quality of water in the watershed of Lake Guaíba. Samples were standardised and quarterly made using a seine net at eleven sites in the basin. A total of 53,408 individuals of 66 specimens pertaining to 22 families and 8 orders were collected and analysed. The highest frequency of the disease was found in Astyanax fasciatus. The simple Chi-Squared test applied to the species showed statistically significant frequency of occurrence for three sampled sites. Two of these sites showed the highest frequencies for two consecutive sampling periods of one year and the best levels of water quality. Results of this study suggest that the association between high frequency of infestation by a parasite that causes the black spot disease in fish, and environments with less degradation in water quality, recorded from physical, chemicals and microbiological variables, could be used as an indicator of water quality in these environments.

A frequência da doença dos pontos pretos, provocada por trematódeos digenéticos em peixes, foi testada como indicador da qualidade da água na bacia hidrográfica do lago Guaíba (RS). As amostragens foram padronizadas e realizadas trimestralmente com redes do tipo picaré em onze pontos desta bacia. Foram coletados e analisados 53.408 exemplares, totalizando 66 espécies pertencentes a 22 famílias e oito ordens. A maior frequência de ocorrência da doença foi verificada em Astyanax fasciatus. Um Qui-Quadrado simples aplicado para essa espécie indicou frequência estatisticamente significativa de ocorrência da doença para três locais amostrados. Dois destes locais apresentaram as maiores frequências durante dois períodos consecutivos de um ano de amostragem e os melhores níveis de qualidade de água. Os resultados deste trabalho sugerem que a associação entre a elevada frequência de infestação pelo parasito que causa a doença dos pontos pretos e os ecossistemas com menor degradação na qualidade da água, com registros de variáveis físicas, químicas e microbiológicas, pode ser utilizada como indicador de melhor qualidade ambiental.

High molecular diversity of the fungus Guignardia citricarpa and Guignardia mangiferae and new primers for the diagnosis of the citrus black spot

RAPD markers were used to investigate the distribution of genetic variability among a group of Guignardia citricarpa, G. mangiferae, and Phyllosticta spinarum isolates obtained from several hosts in Brazil, Argentina, Mexico, Costa Rica, Thailand, Japan, United States and South Africa. Pathogenic isolates G. citricarpa Kiely (anamorph form P. citricarpa McAlp Van Der Aa) are the etiological agent of the Citrus Black Spot (CBS), a disease that affects several citric plants and causes substantial injuries to the appearance of their fruits, thus preventing their export. Several previous studies have demonstrated the existence of an endophytic species with high morphological similarity to the causal agent of CBS that could remain latent in the same hosts. Consequently, the identification of the plants and fruits free from the causal agent of the disease is severely hampered. The RAPD analysis showed a clear discrimination among the pathogenic isolates of G. citricarpa and endophytic isolates (G. mangiferae and P. spinarum). In addition, a Principal Coordinate Analysis (PCO) based on a matrix of genetic similarity estimated by the RAPD markers showed four clusters, irrespective of their host or geographical origin. An Analysis of Molecular Variance (AMOVA) indicated that 62.8 percent of the genetic variation was found between the populations (G. citricarpa, G. mangiferae, P. spinarum and Phyllosticta sp.). Substantial variation was found in the populations (37.2 percent). Exclusive RAPD markers of isolates of G. citricarpa were cloned, sequenced and used to obtain SCARS (Sequence Characterized Amplified Regions), which allowed the development of new specific primers for the identification of G. citricarpa PCR (Polymerase Chain Reaction) analysis using a pair of primers specific to pathogenic isolates corroborating the groupings obtained by the RAPD markers, underscoring its efficiency in the identification of the causal agent of CBS.

Marcadores de RAPD foram utilizados para investigar a distribuição da variabilidade genética de linhagens de Guignardia citricarpa, G. mangiferae, e Phyllosticta spinarum isolados em diversos hospedeiros no Brasil, Argentina, México, Costa Rica, Tailândia, Japão, EUA e África do Sul. O fungo Guignardia citricarpa Kiely (Phyllosticta citricarpa McAlp Van Der Aa) é o agente causal da Mancha Preta dos Citros (CBS), uma doença que afeta diversas plantas cítricas, causando dano a aparência dos frutos, prejudicando a exportação. Diversos estudos têm demonstrado a existência de uma espécie endofítica muito semelhante morfologicamente a G. citricarpa, e que permanece de forma endofítica no mesmo hospedeiro. Dificultando assim, a identificação de plantas e frutos livres do agente causa da CBS. A análise do perfil de RAPD revelou uma clara discriminação entre isolados patogênicos de G. citricarpa e isolados endofíticos (G. mangiferae e P. spinarum). A Análise de Coordenadas Principais (PCO) baseada na matriz de similaridade genética dos marcadores RAPD, demonstrou a formação de quatro grupos, sem relação com origem geográfica ou com hospedeiros utilizados. A análise de Variância de Marcadores Moleculares (AMOVA) indicou que 62,8 por cento da variação genética é encontrada entre as populações (G. citricarpa, G. mangiferae, P. spinarum and Phyllosticta sp.). Entretanto, variação substancial foi encontrada dentro destas populações (37,2 por cento). Bandas de RAPD exclusivas de isolados de G. citricarpa foram clonadas, sequenciadas e utilizadas na obtenção de SCARS (Sequence Characterized Amplified Regions), que permitiram o desenvolvimento de novos primers específicos para a identificação de G. citricarpa. Reações de PCR (Polymerase Chain Reaction) utilizando este par de primers corroboraram os agrupamentos obtidos pelos marcadores de RAPD, revelando sua eficiência na identificação do agente causal da CBS.

Fungicide resistance and genetic variability in plant pathogenic strains of Guignardia citricarpa / Resistência a fungicidas e variabilidade genética em linhagens patogênicas de plantas Guignardiacitricarpa

Citrus black spot (CBS) is a plant disease of worldwide occurrence, affecting crops in Africa, Oceania, and South America. In Brazil, climate provides favorable conditions and CBS has spread to the Southeast and South regions. CBS is caused by the fungus Guignardia citricarpa (anamorph: Phyllosticta citricarpa) and its control is based on the use of fungicides, such as benzimidazoles. In South Africa, the disease was kept under control for 10 years with benomyl, until cases of resistance to high concentrations of this fungicide were reported from all citrus-producing areas. Azoxystrobin (a strobilurin) has been found effective in controlling phytopathogens, including CBS, in a wide range of economically important crops. The present study investigated in vitro the effects of the fungicides benomyl and azoxystrobin on 10 strains of G. citricarpa isolated from lesions in citrus plants from Brazil and South Africa. Benomyl at 0.5 µg/mL inhibited mycelial growth in all strains except PC3C, of African origin, which exhibited resistance to concentrations of up to 100.0 µg/mL. The spontaneous mutation frequency for resistance to benomyl was 1.25 ï 10-7. Azoxystrobin, even at high concentrations, did not inhibit mycelial growth in any of the strains, but significantly reduced sporulation rates, by as much as 100%, at a concentration of 5.0 µg/mL. Variations in sensitivity across strains, particularly to the strobilurin azoxystrobin, are possibly related to genetic variability in G. citricarpa isolates.

A Mancha Preta dos Citros (MPC) tem ocorrência mundial afetando a produção de citros na África, Oceania e América do Sul. No Brasil, onde o clima é favorável ao seu desenvolvimento, a doença está espalhada nas regiões Sul e Sudeste. O controle da MPC, causada pelo fungo Guignardia citricarpa (anamorfo: Phyllosticta citricarpa) é baseado na aplicação de fungicidas, como os benzimidazóis. Na África do Sul, após 10 anos de controle da doença com o fungicida benomil, os casos de resistência a altas concentrações deste fungicida atingiram todas as áreas produtoras. O fungicida estrolilurina chamado azoxistrobina tem se mostrado eficiente no controle dos fitopatógenos de uma grande variedade de culturas economicamente importantes, incluindo a MPC. Neste trabalho foram investigados os efeitos in vitro dos fungicidas benomil e azoxistrobina em 10 linhagens de G. citricarpa isoladas de lesões em plantas cítricas no Brasil e na África do Sul. Houve inibição do crescimento micelial a 0,5 µg/mL do fungicida benomil entre as linhagens testadas, com exceção de PC3C de origem sul-africana, que apresentou resistência até a concentração de 100,0 µg/mL de benomil. A freqüência de mutação espontânea para resistência ao benomil foi de 1,25 ï 10-7. A estrobilurina azoxistrobina, mesmo em altas concentrações, não inibiu o crescimento micelial dos isolados, entretanto reduziu significativamente a produção de esporos, chegando a 100% de inibição em concentrações de 5,0 µg/mL de azoxistrobina. A variação na sensibilidade das linhagens, principalmente com a estrobilurina azoxistrobina, possivelmente está relacionada com a variabilidade genética dos isolados de G. citricarpa.