Histology and biochemistry of the interaction between resistant and susceptible citrus rootstocks to Phytophthora nicotianae

No Brasil Phytophthora nicotianae é um dos principais agentes causadores da podridão do pé e de raízes e radicelas em citros. As doenças provocadas pelo gênero ocasionam danos elevados à produção agrícola e o uso de porta-enxertos resistentes é medida importante para controle. Nesse trabalho objetivou-se estudar aspectos relacionados à histologia e bioquímica da interação entre P. nicotianae e porta-enxerto de tangerineira Sunki (suscetível) e citrumeleiro Swingle (resistente). Para tal, raízes de plântulas desses genótipos com dois, três ou seis meses foram inoculadas com suspensão de 105 zoósporos/mL de P. nicotianae e mantidas a 25°C. As análises foram realizadas em microscópio de luz (ML) e confocal (MC), em cromatografia líquida de alta eficiência (CLAE), em cromatógrafo a gás (CG) e em microscópio eletrônico de varredura (MEV). Em ML, as secções de raízes coradas com azul de toluidina um, dois, quatro e seis dias após a inoculação (dai) indicaram diferenças entre os porta-enxertos no modo e na velocidade de colonização do patógeno. O hospedeiro resistente apresentou menor número de hifas nos tecidos e essas se localizaram principalmente nos espaços intercelulares. Em MC, analisou-se a distribuição da elicitina do patógeno em secções de raízes um, dois e quatro dai. A elicitina foi detectada em menor quantidade e mostrou concentração constante em raízes de citrumeleiro Swingle e em quantidade maior e em gradual aumento em raízes tangerineira Sunki. Análises da superfície das raízes em MEV, 1, 2 e 4 horas após a inoculação, revelaram menor número de cistos do patógeno sobre o hospedeiro resistente no tempo de 2 horas. Testes histoquímicos com cloreto de zinco iodado e cloreto férrico para a detecção, respectivamente, de lignina e compostos fenólicos, em secções a fresco de raízes dos porta-enxertos um, três e seis dai foram visualizadas em ML e evidenciaram ausência de alteração nos níveis desses compostos entre raízes inoculadas e não inoculadas. A análise em CLAE, três dai, demonstrou que ambos os porta-enxertos, possuem compostos fenólicos em suas raízes. Entretanto, não houve diferença qualitativa e quantitativa destes compostos em plantas de um mesmo genótipo não inoculado e inoculado com P. nicotianae. Porém, diferenças quantitativas foram observadas entre ambos os genótipos. Em citrumeleiro Swingle encontrou-se menor quantidade de equivalentes em ácido clorogênico e apigenina e maior quantidade de equivalentes em rutina, quando comparado à tangerineira Sunki. A produção de etileno dos genótipos, analisada em CG, foi avaliada durante dez dai. O hospedeiro suscetível inoculado apresentou maior produção do gás comparado ao controle, do primeiro ao sexto dia. O hospedeiro resistente inoculado e não inoculado não apresentaram diferenças na produção do gás durante o ensaio. Esses resultados indicam diferenças na interação entre P. nicotianae e plântulas de citrumeleiro Swingle e tangerineira Sunki. Todavia, não esclarecem os mecanismos pelos quais essas diferenças ocorrem. Tais resultados fornecem subsídios para estudos sobre os mecanismos envolvidos na resistência de genótipos de citros à P. nicotianae.In Brazil Phytophthora nicotianae is one of the main causal agents of foot and root rot in citrus. Diseases caused by this genus are responsible for significant losses in agricultural production and the use of resistant rootstocks is an important control procedure. This work aimed to study aspects related to histology and biochemistry of the interaction between P. nicotianae and Swingle citrumelo (resistant) and Sunki tangerine (susceptible) rootstocks. For this purpose, roots of two, three or six months old seedlings of both genotypes were inoculated with a suspension of 105 zoospores/mL of P. nicotianae and kept at 25°C. Analyses were performed with light (LM) and confocal (CM) microscope, with high performance liquid chromatograph (HPLC), with gas chromatograph (GC) and, with scanning electron microscope (SEM). In LM, root sections stained with toluidine blue one, two, four and six days after inoculation (dai) indicated differences in the mode and speed of colonization of the pathogen between the rootstocks. The resistant host showed a lower number of hyphae inside its tissue, mainly in the intercellular spaces. In CM, the pathogen elicitin distribution was analyzed in root sections one, two and four dai. The elicitin amount was lower and apparently stable in Swingle citrumelo root and it was higher and increasing gradually in Sunki tangerine roots. Roots surface analysis by SEM, 1, 2 and 4 hours after inoculation, indicated fewer pathogen cysts on resistant host at 2 hours. Histochemical tests in fresh root sections with iodized zinc chloride and ferric chloride for detection, respectively, of lignin and phenolic compounds were seen one, three and six dai in LM. The results showed no change in levels of these compounds in roots of inoculated and uninoculated rootstocks. HPLC root analysis, three dai, revealed that both rootstocks, inoculated and uninoculated, had phenolic compounds. However, there was no qualitative and quantitative difference in phenolic compounds between inoculated and uninoculated plants of the same genotype. Quantitative differences were observed between both hosts. There was lower concentration of apigenin and chlorogenic acid equivalents and higher concentration of rutin equivalents in Swingle citrumelo as compared to Sunki tangerine. Production of ethylene by the genotypes was analyzed in GC during 10 dai. The susceptible host, when inoculated, showed higher ethylene production compared to control from the first to the sixth day. The resistant host, inoculated or not, showed no difference in ethylene production during the test. These results indicate differences in the interaction between P. nicotianae and seedlings of Swingle citrumelo and Sunki tangerine. Nevertheless, they do not clarify the mechanisms through which these differences occur. These results indicate some points where further studies should concentrate on the resistance mechanisms of citrus genotypes against P. nicotianae

Pre-penetration of Guignardia psidii in guava: effect of temperature, wetness duration, fruit age and concentrations of ethylene and carbon dioxide

A pinta preta provocada pelo fungo Guignardia psidii Ullasa e Rawal (Phyllosticta psidiicola (Petrak) van der Aa) vem gerando perdas pós-colheita na produção de goiaba para consumo in natura. Este trabalho buscou maiores informações sobre esta doença por meio do estudo do efeito da temperatura, da duração do período de molhamento, do efeito dos gases dióxido de carbono e etileno, e da idade dos frutos de goiabeira na pré-penetração de conídios de G. psidii. Os estudos in vitro foram avaliados por meio de microscópio de luz e os in vivo por meio de microscópio eletrônico de varredura, registrando-se a porcentagem de conídios germinados, apressórios formados e apressórios melanizados (in vitro). O efeito da temperatura (10 a 40ºC) e da duração do período de molhamento (6 a 48 in vitro e 6 a 24 horas in vivo) na germinação, formação de apressórios e apressórios melanizados (in vitro) foram avaliados em superfície de poliestireno e de frutos de goiabeira. Em ambas as superfícies, à medida que a temperatura aumentou, observou-se o aumento gradual da germinação, formação de apressório e apressório melanizado atingindo o máximo a 25ºC e duração de período de molhamento de 24 horas in vivo e 48 horas in vitro. Nessas condições, os valores obtidos foram 38% de conídios germinados, 37,7% de apressórios formados e 32,7% de apressórios melanizados in vitro e 45,6% de conídios germinados e 11,2% de apressórios formados in vivo. As taxas de germinação e formação de apressório foram maiores no experimento in vivo. A germinação e formação de apressório de conídios de G. psidii foram avaliadas em cinco diferentes idades de frutos: 10 dias, 35 dias, 60 dias, 85 dias e 110 dias. Observou-se o aumento gradual das variáveis avaliadas com o aumento da idade do fruto, apresentando, em frutos com 10 dias, 8,4% de conídios germinados e 3,2% de apressórios formados, enquanto os frutos com 110 dias apresentaram 43,2% de conídios germinados e 26,4% de apressórios formados. O efeito das concentrações 0, 3, 6 e 12% de dióxido de carbono e 0, 1, 3 e 6 ppm de etileno na pré-penetração de conídios de G. psidii foram avaliadas in vitro e in vivo. No experimento in vitro, o aumento das concentrações de dióxido de carbono provocou queda na germinação dos conídios de G. psidii quando comparado ao controle. No entanto, entre as concentrações deste gás a taxa de germinação manteve-se estável. Portanto, no controle esta variável apresentou valor médio de 45,1% enquanto que nas concentrações 3, 6 e 12% do gás passou para, respectivamente, 24,6%, 22,6% e 25,8%. Entretanto, o aumento das concentrações deste gás provocou decréscimo progressivo da taxa de formação de apressórios e apressórios melanizados. Na concentração de 3% de dióxido de carbono houve 10,3% de apressórios formados e 5,4% de apressórios melanizados e na concentração de 12%, esses valores foram reduzidos para 4,1% e 0,5%, respectivamente. No entanto, no experimento in vivo, as taxas de germinação e formação de apressórios apresentaram redução gradativa entre as concentrações 3 e 6% de dióxido de carbono, elevando-se novamente a 12%, passando de 28,2% de conídios germinados e 20,5% de apressórios formados na concentração de 6% de dióxido de carbono para, respectivamente, 49,3% e 38,2% a 12% de concentração desse gás. Esse comportamento pode ser explicado devido à remoção dos conídios não germinados durante o preparo das amostras para visualização em microscópio eletrônico. O etileno, tanto nos experimento in vitro quanto in vivo não apresentou efeito sobre a germinação e formação de apressórios de G. psidii. Apresentando no controle in vitro 34,6% de conídios 9 germinados, 34,2% de apressórios formados e 29,9% de apressórios melanizados e na maior concentração de etileno estudada (6 ppm) 26,7% de conídios germinados, 26,3% de apressórios formados e 20,2% de apressórios melanizados. Nos ensaios in vivo, o controle apresentou 40% de conídios germinados e 32,7% de apressórios formados e na maior concentração de etileno estudada 49,3% de conídios germinados e 38,2% de apressórios formados.The black spot caused by the fungus Guignardia psidii Ullasa and Rawal (Phyllosticta psidiicola (Petrak) van der Aa) has been causing post-harvest losses of guava fruit for consumption in natura. This work was designed to investigate the effects of temperature, wetness duration, carbon dioxide and ethylene concentration, and guava fruit age on the pre-penetration of G. psidii. The percentages of germinated conidia, formed appressoria and melanized appressoria were evaluated in both in vitro and in vivo experiments, using a light microscope in the former and a scanning electron microscope in the later. The effects of temperature (10 to 40ºC) and wetness duration (6 to 48 hours in vitro and 6 to 24 hours in vivo) on the germination, appressoria formation and melanized appressoria (in vitro) were evaluated on a polystyrene surface and on the fruit\'s surface. In both surfaces, as the temperature increased a gradual increase in conidia germination, appressoria formation and melanized appressoria was observed, reaching maximum at 25ºC with wetness duration being 24 hours in vivo and 48 hours in vitro. In these conditions, the values obtained were 38.0% germinated conidia, 37.7% formed appressoria and 32.7% melanized appressoria in vitro and 45.6% germinated conidia and 11.2% formed appressoria in vivo. Conidia germination and appressoria formation rates were higher in the in vivo experiment. G. psidii conidia germination and appressoria formation were evaluated at five different fruit ages: 10 days, 35 days, 60 days, 85 days and 110 days. A gradual increase of the evaluated variables was observed as the fruit\'s age increased, presenting in fruits with 10 days, 8.4% germinated conidia and 3.2% formed appressoria, while the fruits with 110 days presented 43.2% germinated conidia and 26.4% formed appressoria. The effects of carbon dioxide concentration at 0, 3, 6 and 12% and ethylene at 0, 1, 3 and 6 ppm on the pre-penetration of G. psidii were evaluated in vitro and in vivo. In the in vitro experiment an increase in the carbon dioxide concentration provoked a decrease in G. psidii conidia germination when compared to the control. However, between the gas concentrations tested, the germination rate was stable. Therefore, in the control this variable presented a mean value of 45.1% while in the gas concentration of 3, 6 and 12%, the mean values were, respectively, 24.6%, 22.6% and 25.8%. However, increasing carbon dioxide concentration caused a progressive decrease in appressoria formation and melanized appressoria rates. At a concentration of 3% carbon dioxide, there was 10.3% formed appressoria and 5.4% melanized appressoria and at 12% concentration these values were reduced to 4.1% and 0.5%, respectively. Nonetheless, in the in vivo experiment, the rates of germination and appressorium formation presented a gradual reduction between the 3 and 6% dioxide carbon concentrations, increasing again at 12%, increasing from 28.2% germinated conidia and 20.5% formed appressoria at 6% carbon dioxide concentration to, respectively, 49.3% and 38.2% at 12% concentration of this gas. This behavior could be due to the removal of the conidia that did not germinate, during the preparation of the sample for electron microscope observation. Ethylene, in both the in vitro and in the in vivo experiments, showed no effect on G. psidii germination and appressoria formation, presenting in the in vitro control, 34.6% germinated conidia, 34.2% formed appressoria and 29.9% melanized appressoria and in the highest ethylene concentration studied (6 ppm) 26.7% germinated conidia, 26.3% formed appressoria and 20.2% melanized appressoria. In the in vivo assays, the control presented 40.0% 11 germinated conidia and 32.7% formed appressoria and in the highest ethylene concentration studied 49.3% germinated conidia and 38.2% formed appressoria

Important Insect and Disease Threats to United States Tree Species and Geographic Patterns of Their Potential Impacts

Diseases and insects, particularly those that are non-native and invasive, arguably pose the most destructive threat to North American forests. Currently, both exotic and native insects and diseases are producing extensive ecological damage and economic impacts. As part of an effort to identify United States tree species and forests most vulnerable to these epidemics, we compiled a list of the most serious insect and disease threats for 419 native tree species and assigned a severity rating for each of the 1378 combinations between mature tree hosts and 339 distinct insect and disease agents. We then joined this list with data from a spatially unbiased and nationally consistent forest inventory to assess the potential ecological impacts of insect and disease infestations. Specifically, potential host species mortality for each host/agent combination was used to weight species importance values on approximately 132,000 Forest Inventory and Analysis (FIA) plots across the conterminous 48 United States. When summed on each plot, these weighted importance values represent an estimate of the proportion of the plot’s existing importance value at risk of being lost. These plot estimates were then used to identify statistically significant geographic hotspots and coldspots and of potential forest impacts associated with insects and diseases in total, and for different agent types. In general, the potential impacts of insects and diseases were greater in the West, where there are both fewer agents and less diverse forests. The impact of non-native invasive agents, however, was potentially greater in the East. Indeed, the impacts of current exotic pests could be greatly magnified across much of the Eastern United States if these agents are able to reach the entirety of their hosts’ ranges. Both the list of agent/host severities and the spatially explicit results can inform species-level vulnerability assessments and broad-scale forest sustainability reporting efforts, and should provide valuable information for decision-makers who need to determine which tree species and locations to target for monitoring efforts and pro-active management activities

Viability and dissemination of Pantoea ananatis, etiological agent of Maize White Spot disease.

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Viability and dissemination of Pantoea ananatis, etiological agent of Maize White Spot disease.

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Prepenetration Stages of Guignardia psidii in Guava: Effects of Temperature, Wetness Duration and Fruit Age

This study examined the effects of temperature and wetness duration in vitro and in vivo as well as the effects of fruit age on germination and appressoria formation by conidia of Guignardia psidii, the causal agent of black spot disease in guava fruit. The temperatures tested for in vitro and in vivo experiments were 10, 15, 20, 25, 30, 35 and 40 degrees C. The wetness periods studied were 6, 12, 24, 36 and 48 h in vitro and 6, 12 and 24 h in vivo. Fruit 10, 35, 60, 85 and 110-days old were inoculated and maintained at 25 degrees C, with a wetness period of 24 h. Temperature and wetness duration affected the variables evaluated in vitro and in vivo. All variables reached their maximum values at between 25 and 30 degrees C with a wetness duration of 24 h in vivo and 48 h in vitro. These conditions resulted in 31.3% conidia germination, 33.6% appressoria formation and 32.5% appressoria melanization in vitro, and 50.4% conidia germination and 9.5% appressoria formation in vivo. Fruit age also influenced these factors. As fruit age increased, conidia germination and appressoria formation gradually increased. Conidia germination and appressoria formation were 10.8% and 2.3%, respectively, in 10-day-old fruits. In 110-day-old fruits, conidia germination and appressoria formation were 42.5% and 23.2% respectively