Diplodia pinea (Desm.) Kickx y Fusarium circinatum Niremberg & O'Donell, principales hongos de chancro de las masas forestales de Pinus radiata D. Don del País Vasco

228 p.

Susceptibilidad a Fusarium verticillioides, Gibberella zeae y Diplodia maydis en líneas e híbridos pre-comerciales de maíces templados y tropicales

Las podredumbres de espiga (causada por Fusarium verticillioides y Gibberella zeae) y tallo (causada por F.verticillioides) provocan importantes pérdidas de rendimiento y calidad del grano en maíz (Zea mays L.), por lo que es importante mantener actualizado el conocimiento sobre la variabilidad genotípica de la especie para la tolerancia a las mismas así como la posible relación entre la presencia de una y otra podredumbre. Con estos objetivos se evaluaron (i) 438 líneas y 84 híbridos precomerciales de origen templado para resistencia a ambas podredumbres, y (ii) 84 líneas de origen templado y tropical para resistencia a podredumbre de la espiga por Diplodia maydis. Ambos ensayos fueron conducidos en un DBCA en Fontezuela (33.9 ºS, 60.47º O), Argentina, durante 2009-2010. Los tratamientos empleados fueron inoculación artificial e infección natural. Se encontraron diferencias significativas entre ambas para incidencia (I: porcentaje de plantas afectadas en el surco), Severidad (S: porcentaje promedio de podredumbre de las espigas sobre el total de espigas afectadas) y S×I, tanto para híbridos como para líneas. Existió una correlación positiva entre el tratamiento control sin inocular y el inoculado para todas las enfermedades. Se detectó una pequeña influencia de los caracteres morfológicos de la espiga y el grano sobre la resistencia a podredumbre de espiga por F.verticillioides y D. maydis, respectivamente. No se encontró G. zeae en el control, por lo que no pudieron realizarse los análisis correspondientes para esta enfermedad. La asociación entre el tratamiento inoculado con fusariosis de espiga y el control para fusariosis de tallo, y viceversa, nunca fue consistente, por lo tanto puede emplearse un tratamiento como control del otro. Los materiales de origen tropical fueron más resistentes a D. maydis que los templados, aunque las diferencias entre grupos heteróticos no resultaron altamente significativas. Se determinó que la inoculación artificial empleando infección natural como control es una práctica recomendable en el mejoramiento de maíz por resistencia a enfermedades. La inoculación artificial puede generar mayor variación en las respuestas, permitiendo observar y comparar el comportamiento de distintos materiales frente a la infección de patógenos.

Infection mechanism of Diplodia corticola

Diplodia corticola is regarded as the most virulent fungus involved in cork oak decline, being able to infect not only Quercus species (mainly Q. suber and Q. ilex), but also grapevines (Vitis vinifera) and eucalypts (Eucalyptus sp.). This endophytic fungus is also a pathogen whose virulence usually manifests with the onset of plant stress. Considering that the infection normally culminates in host death, there is a growing ecologic and socio-economic concern about D. corticola propagation. The molecular mechanisms of infection are hitherto largely unknown. Accordingly, the aim of this study was to unveil potential virulence effectors implicated in D. corticola infection. This knowledge is fundamental to outline the molecular framework that permits the fungal invasion and proliferation in plant hosts, causing disease. Since the effectors deployed are mostly proteins, we adopted a proteomic approach. We performed in planta pathogenicity tests to select two D. corticola strains with distinct virulence degrees for our studies. Like other filamentous fungi D. corticola secretes protein at low concentrations in vitro in the presence of high levels of polysaccharides, two characteristics that hamper the fungal secretome analysis. Therefore, we first compared several methods of extracellular protein extraction to assess their performance and compatibility with 1D and 2D electrophoretic separation. TCA-Acetone and TCA-phenol protein precipitation were the most efficient methods and the former was adopted for further studies. The proteins were extracted and separated by 2D-PAGE, proteins were digested with trypsin and the resulting peptides were further analysed by MS/MS. Their identification was performed by de novo sequencing and/or MASCOT search. We were able to identify 80 extracellular and 162 intracellular proteins, a milestone for the Botryosphaeriaceae family that contains only one member with the proteome characterized. We also performed an extensive comparative 2D gel analysis to highlight the differentially expressed proteins during the host mimicry. Moreover, we compared the protein profiles of the two strains with different degrees of virulence. In short, we characterized for the first time the secretome and proteome of D. corticola. The obtained results contribute to the elucidation of some aspects of the biology of the fungus. The avirulent strain contains an assortment of proteins that facilitate the adaptation to diverse substrates and the identified proteins suggest that the fungus degrades the host tissues through Fenton reactions. On the other hand, the virulent strain seems to have adapted its secretome to the host characteristics. Furthermore, the results indicate that this strain metabolizes aminobutyric acid, a molecule that might be the triggering factor of the transition from a latent to a pathogenic state. Lastly, the secretome includes potential pathogenicity effectors, such as deuterolysin (peptidase M35) and cerato-platanin, proteins that might play an active role in the phytopathogenic lifestyle of the fungus. Overall, our results suggest that D. corticola has a hemibiotrophic lifestyle, switching from a biotrophic to a necrotrophic interaction after plant physiologic disturbances.This understanding is essential for further development of effective plant protection measures.

Effective Use in Livestock Feeds of Mouldy and Weather-damaged Grain Containing Mycotoxins-Case Histories and Economic Assessments Pertaining to Pig and Poultry Industries of Queensland

Mould growth in field crops or stored grain reduces starch and lipid content, with consequent increases in fibre, and an overall reduction in digestible energy; palatability is often adversely affected. If these factors are allowed for, and mycotoxin concentrations are low, there are sound economic reasons for using this cheaper grain. Mycotoxins are common in stock feed but their effects on animal productivity are usually slight because either the concentration is too low or the animal is tolerant to the toxin. In Australia, aflatoxins occur in peanut by-products and in maize and sorghum if the grain is moist when stored. Zearalenone is found in maize and in sorghum and wheat in wetter regions. Nivalenol and deoxynivalenol are found in maize and wheat but at concentrations that rarely affect pigs, with chickens and cattle being even more tolerant. Other mycotoxins including cyclopiazonic acid, T-2 toxin, cytochalasins and tenuazonic acid are produced by Australian fungi in culture but are not found to be significant grain contaminants. Extremely mouldy sorghum containing Alternaria and Fusarium mycotoxins decreased feed conversion in pigs and chickens by up to 14%. However, E moniliforme- and Diplodia maydis-infected maize produced only slight reductions in feed intake by pigs and Ustilago- infected barley produced no ill effects. Use of these grains would substantially increase profits if the grain can be purchased cheaply.

Three new Lasiodiplodia spp. from the tropics, recognized based on DNA sequence comparisons and morphology

Botryosphaeria rhodina (anamorph Lasiodiplodia theobromae) is a common endophyte and opportunistic pathogen on more than 500 tree species in the tropics and subtropics. During routine disease surveys of plantations in Australia and Venezuela several isolates differing from L. theobromae were identified and subsequently characterized based upon morphology and ITS and EF1-a nucleotide sequences. These isolates grouped into three strongly supported clades related to but different from the known taxa, B. rhodina and L. gonubiensis, These have been described here as three new species L. venezuelensis sp. nov., L. crassispora sp. nov. and L. rubropurpurea sp. nov. The three could be distinguished easily from each other and the two described species of Lasiodiplodia, thus confirming phylogenetic separations. Furthermore all five Lasiodiplodia spp. now recognized separated from Diplodia spp. and Dothiorella spp. with 100% bootstrap support.

引起橡胶和马尾松木材蓝变的真菌—可可球二孢

<正> 可可球二泡(Botryodiplodia theobromae Pat.)早在1879年就被Cooke发现,目前,至少拥有48个异名,常见的异名是Diplodia theobromae(Pat.)Nowell,Diplodia natalensis PoleEvans,Lasiodiplodia theobromae(Pat.)Griff.＆ Maubl。Punithalingam和Sutton分别对该菌作过详细的评述。它广泛分布于热带、亚热带和温带。其寄主植物已知约500种,它不但引起植物病害,

湿地松色二孢菌和可可球二孢菌根腐病研究

松树根腐病在我国首次发现于雷州半岛10年生左右的湿地松林分。病原菌被鉴定为松色二孢菌Diplodia pinea，另外还发现可可球二孢菌Botryodiplodia theobromae不侵染马尾松，但对湿地松、火炬松和加勒比松也是一种病原菌。 松色二孢菌在林间土病根内以菌丝体方式可存活150天以上，在室内5～50%含水量自然土中，菌丝体和分生孢子可存活120天以上。 松色二孢菌在含水量为40%的湿地松离体根内扩展较快.在含水量为50～70%的离体根内扩展较慢。不论接种前或接种后调节土壤的含水量，一年生松苗的死亡率在含水量为50%土壤中较低，在含水量为5～25%土壤中较高。苗木要经过约20天的干旱影响后，才显示出较易受病菌的侵染。

Phenotypical characterization in "Plasmopara halstedii" (sunflower downy mildew) isolates of several races

Phenotypic variation (morphological and pathogenic characters), and genetic variability were studied in 50 isolates of seven Plasmopara halstedii (sunflower downy mildew) races 100, 300, 304, 314, 710, 704 and 714. There were significant morphological, aggressiveness, and genetic differences for pathogen isolates. However, there was no relationship between morphology of zoosporangia and sporangiophores and pathogenic and genetic characteristics for the races used in our study. Also, our results provided evidence that no relation between pathogenic traits and multilocus haplotypes may be established in P. halstedii. The hypothesis explaining the absence of relationships among phenotypic and genetic characteristics is discussed.

Investigation of regio- and sterochemistries of microbial biotransformation /

Epoxides can be hydrolyzed by fungi to produce chiral diols. The first part of this thesis presents an investigation of the microbial hydrolysis of aziridines comparable in structure to epoxide biotransformation substrates. Biotransformation of the aziridines 1 -methyl-2-phenyl aziridine, 2- phenylaziridine and N-methyl-7-aza bicyclo[4.1.0] heptane was studied using Beauveria sulfurescens, Aspergillus niger and Diplodia gossypina but no evidence for enzymic hydrolysis was obtained. The hydroxylation reaction performed by the fungus Beauveria sulfurescens ATCC 7159 has been studied for many years and several models describing the hydroxylating pattern exhibited by this fungus have been proposed. The second part of this thesis presents a test of the proposed models. The ability of Beauveria sulfurescens to hydroxylate thirty potential substrates was examined, and the data suggest that none of the earlier proposed models accounts for all of the bioconversion results. A possible explanation is proposed, suggesting that there is more than one enzyme responsible for the hydroxylation reactions performed by Beauveria sulfurescens.

Desempenho de fungicidas no controle da mancha-de-macrospora na cultura do milho

Fungicide application is one of the control strategies of fungal diseases in corn leaves. In Brazil, there are no fungicides recorded for the control of corn macrospora leaf spot (MLS). The aim of this study was to assess the efficacy of 16 fungicides on MLS control in a protective, curative and eradicant form. Fungicides of the chemical groups of benzimidazoles, strobilurins and triazoles were used alone or in mixture, in completely randomized block design, with six replicates of five plants, totaling 30 plants per treatment. The experiments were carried out in a greenhouse with the single-cross hybrid AS 1565 in phenological stage of two to six expanded leaves, using an isolate of S. macrospsora from the same hybrid. The inoculum was deposited into the cartridge of plants at 48 hours after, 48 hours before and 10 days before fungicide applications for preventive, curative and eradicant action, respectively. The experiment was repeated twice. The data underwent analysis of variance (p<0.05), and the means of treatments were compared by using the Scott-Knott test (p<0.05). Severity was estimated at 21 days after inoculations. All fungicides significantly differed from the control treatment in the preventive, curative and eradicant action. For the preventive action, mean disease control was 85%. The mixture of triazoles plus strobilurins controlled, on average, 75% of the disease severity, while the isolated products such as strobilurins reduced it by 62%, benzimidazoles by 55% and triazoles by 38% for the curative action. The lowest control was obtained for the eradicant action, with mean reduction of 40.1% in the disease severity and no significant difference among fungicides.

Sensibilidade de stenocarpella macrospora a fungicidas

The application of fungicides in the aerial organs is control strategy to macrospora spot caused by fungus Stenocarpella macrospora. The objective of this study was to determine the sensitivity of S. macrospora to fungicides by inhibition of mycelial growth (MG) and conidial germination (CG). It was eval uated 12 fungicides belonging to the chemical groups of the benzimidazoles, triazoles and strobilurins, six concentrations and two isolates of the fungus (SC and MT). The fungicides were diluted in sterile distilled water and added to the culture medium of potato dextrose agar (mycelium) and water-agar (spore) after sterilization. The percentage of inhibition of MC and CG was calculed in comparison with control, estimating of 50% inhibitory concentration (IC50). The fungicides tested were effective in inhibiting the MC. The IC50 was less than 1 ppm for all fungicides. There was no difference between isolates. The inhibition of CG had higher fungitoxicity strobilurins, and the IC50 was between 0.0035 and 0.03 ppm, and the isolated SC showed the higher sensitivity to the fungicides. The IC50 values obtained for fungicides and specific S. macrospora will be useful in monitoring the sensitivity of the fungus, especially in regions with intense demand for fungicides in corn.

Reação de híbridos de milho à mancha-de-macrospora

Macrospora leaf spot, caused by the fungus Stenocarpella macrospora, has shown to be frequent and important among corn fields in Brazil. Genetic resistance is one of the main strategies to control corn leaf diseases. In Brazil, there is scarce information on the resistance of hybrids to Stenocarpella macrospora. The aim of this study was to evaluate the reaction of 25 corn hybrids to macrospora leaf spot. The experiment was conducted in 2011, in a greenhouse under controlled temperature and relative humidity conditions. Experimental design was completely randomized, with five replicates, each experimental unit consisting of a pot with five plants. Inoculation was done in the V2 growth stage (two fully expanded leaves), and the whorl of each plant received 2.0 mL suspension of 1.8 x 10(4) conidia mL-1 pathogen. The four used fungal isolates were obtained from infected crop residues at the municipalities Lages and Quilombo, Santa Catarina State, and Campinas do Sul and Vacaria, Rio Grande do Sul State. Disease severity was assessed at 21 days after inoculation in the V4 stage (four fully expanded leaves). No tested hybrid was totally resistant to the fungus S. macrospora. There was a significant difference in the disease severity between hybrids and fungal isolates. Hybrids inoculated with Quilombo isolate showed four reaction groups, while the isolates Vacaria, Lages and Campinas do Sul showed two groups. Some hybrids had varied behaviors against the isolates, suggesting different aggressiveness levels. There were hybrids that showed similar reaction to the isolates, suggesting greater stability for macrospora leaf spot.

Levantamento de doenças foliares em cultivares e híbridos de milho no sistema plantio direto no estado do Pará.

Objetivou-se avaliar a ocorrência e identificar doenças foliares de milho (Zea mays) em 13 cultivares e híbridos plantados em Sistema de Plantio Direto, em área de Pesquisa da Embrapa Amazônia Oriental, no município de Paragominas, PA. O levantamento das principais doenças foi realizado no início do florescimento, estádio reprodutivo do milho. Amostras de tecido foliar com sintomas de doença foram analisadas no Laboratório de Fitopatologia da Embrapa Amazônia Oriental e observou-se que na maioria dos cultivares e híbridos houve a ocorrência de doenças causadas por fungos dos gêneros Colletotrichum, Bipolaris e Diplodia , agentes etiológicos de doenças que podem causar redução na produtividade da cultura e são amplamente distribuídos nas regiões produtoras de milho do Brasil. O aumento da dispersão do milho pode ser um fator contribuinte para o aumento das doenças, principalmente em Sistema de Plantio Direto. Concluiu-se que houve a incidência de doenças foliares de importância econômica na maioria dos cultivares de milho no Sistema de Plantio Direto em estudo, as quais podem atuar potencialmente para a diminuição da produtividade da cultura nesse Sistema.