Effect of azole fungicide mixtures, alternations and dose on azole sensitivity in the wheat pathogen Zymoseptoria tritici

The evolution of fungicide resistance in the cereal pathogen Zymoseptoria tritici, is a serious threat to the sustainability and profitability of wheat production in Europe. Application of azole fungicides has been shown to affect fitness of Z. tritici variants differentially, so it has been hypothesised that combinations of azoles could slow the evolution of resistance. This work was initiated to assess the effects of dose, mixtures and alternations of two azoles on selection for isolates with reduced sensitivity and on disease control. Naturally infected field trials were carried out at six sites across Ireland and the sensitivity of Z. tritici isolates monitored pre- and post-treatment. The azoles epoxiconazole and metconazole were applied as solo products, in alternation with each other and as a pre-formulated mixture. Full and half label doses were tested. The two azoles were partially cross-resistant, with a common azole resistance principal component accounting for 75% of the variation between isolates. Selection for isolates with reduced azole sensitivity was correlated with disease control. Decreased doses were related to decreases in sensitivity but the effect was barely significant (P = 0.1) and control was reduced. Single applications of an active ingredient (a.i.) caused smaller decreases in sensitivity than double applications. Shifts in sensitivity to the a.i. applied to a plot were greater than to the a.i. not applied, and the decrease in sensitivity was greater to the a.i. applied at the second timing. These results confirm the need to mix a.i.s with different modes of action.

The GATA-type transcriptional activator Gat1 regulates nitrogen uptake and metabolism in the human pathogen Cryptococcus neoformans

Nitrogen uptake and metabolism are essential to microbial growth. Gat1 belongs to a conserved family of zinc finger containing transcriptional regulators known as GATA-factors. These factors activate the transcription of Nitrogen Catabolite Repression (NCR) sensitive genes when preferred nitrogen sources are absent or limiting. Cryptococcus neoformans GAT1 is an ortholog to the Aspergillus nidulans AreA and Candida albicans GAD genes. In an attempt to define the function of this transcriptional regulator in C. neoformans, we generated null mutants (gat1 Delta) of this gene. The gat 1 mutant exhibited impaired growth on all amino acids tested as sole nitrogen sources, with the exception of arginine and proline. Furthermore, the gat1 mutant did not display resistance to rapamycin, an immunosuppressant drug that transiently mimics a low-quality nitrogen source. Gal is not required for C. neoformans survival during macrophage infection or for virulence in a mouse model of cryptococcosis. Microarray analysis allowed the identification of target genes that are regulated by Gat1 in the presence of proline, a poor and non-repressing nitrogen source. Genes involved in ergosterol biosynthesis, iron uptake, cell wall organization and capsule biosynthesis, in addition to NCR-sensitive genes, are Gat1-regulated in C. neoformans. (C) 2010 Elsevier Inc. All rights reserved.

A Broad-spectrum mer Operon in a Multi-drug Resistant Strain of the Fish Pathogen, Aeromonas salmonicida.

Aeromonas salmonicida AS03, a potential fish pathogen, was isolated from Atlantic salmon, Salmo salar, in 2003. This strain was found to be resistant to ≥1000 mM HgCl2 and ≥32 mM phenylmercuric acetate as well as multiple antimicrobials. Mercury (Hg) and antibiotic resistance genes are often located on the same mobile genetic elements, so the genetic determinants of both resistances and the possibility of horizontal gene transfer were examined. Specific PCR primers were used to amplify and sequence distinctive regions of the mer operon. A. salmonicida AS03 was found to have a pDU1358-like broad-spectrum mer operon, containing merB as well as merA, merD, merP, merR and merT, most similar to Klebsiella pneumonaie plasmid pRMH760. To our knowledge, the mer operon has never before been documented in Aeromonas spp. PCR and gene sequencing were used to identify class 1 integron associated antibiotic resistance determinants and the Tet A tetracycline resistance gene. The transposase and resolvase genes of Tn1696 were identified through PCR and sequencing with Tn21 specific PCR primers. We provide phenotypic and genotypic evidence that the mer operon, the aforementioned antibiotic resistances, and the Tn1696 transposition module are located on a single plasmid or conjugative transposon that can be transferred to E. coli DH5α through conjugation in the presence of low level Hg and absence of any antibiotic selective pressure. Additionally, the presence of low-level Hg or chloramphenicol in the mating media was found to stimulate conjugation, significantly increasing the transfer frequency of conjugation above the transfer frequency measured with mating media lacking both antibiotics and Hg. This research demonstrates that mercury indirectly selects for the dissemination of the antibiotic resistance genes of A. salmonicida AS03.

A broad-spectrum mer operon in a multi-drug resistant strain of the fish pathogen, Aeromonas salmonicida

Aeromonas salmonicida AS03, a potential fish pathogen, was isolated from Atlantic salmon, Salmo salar, in 2003. This strain was found to be resistant to ≥1000 mM HgCl2 and ≥32 mM phenylmercuric acetate as well as multiple antimicrobials. Mercury (Hg) and antibiotic resistance genes are often located on the same mobile genetic elements, so the genetic determinants of both resistances and the possibility of horizontal gene transfer were examined. Specific PCR primers were used to amplify and sequence distinctive regions of the mer operon. A. salmonicida AS03 was found to have a pDU1358-like broad-spectrum mer operon, containing merB as well as merA, merD, merP, merR and merT, most similar to Klebsiella pneumonaie plasmid pRMH760. To our knowledge, the mer operon has never before been documented in Aeromonas spp. PCR and gene sequencing were used to identify class 1 integron associated antibiotic resistance determinants and the Tet A tetracycline resistance gene. The transposase and resolvase genes of Tn1696 were identified through PCR and sequencing with Tn21 specific PCR primers. We provide phenotypic and genotypic evidence that the mer operon, the aforementioned antibiotic resistances, and the Tn1696 transposition module are located on a single plasmid or conjugative transposon that can be transferred to E. coli DH5α through conjugation in the presence of low level Hg and absence of any antibiotic selective pressure. Additionally, the presence of low-level Hg or chloramphenicol in the mating media was found to stimulate conjugation, significantly increasing the transfer frequency of conjugation above the transfer frequency measured with mating media lacking both antibiotics and Hg. This research demonstrates that mercury indirectly selects for the dissemination of the antibiotic resistance genes of A. salmonicida AS03.

Defining plant resistance against Phytophthora Cinnamomi and application of resistance to revegetation

Phytophthora cinnamomi is a soil borne plant pathogen that causes devastating disease in many Australian ecosystems and threatens the survival of native flora. Compared with the number of plant species that are susceptible to P. cinnamomi, only a few species are known to be resistant and control of this pathogen by chemicals is difficult and undesirable in natural systems. The major aim of our research is therefore to characterise natural resistance and determine which signalling pathways and defence responses are involved. Our examination of resistance is being approached at several levels, one of which is through the use of the model plant, Arabidopsis. Previously, Arabidopsis had been shown to display ecotypic variation in responses to P. cinnamomi and we are exploring this further in conjunction with the analysis of a bank of Arabidopsis defence pathway mutants for their responses to the pathogen. These experiments will provide a fundamental basis for further analysis of the defence responses of native plants. Native species (susceptible and resistant) are being assessed for their responses to P. cinnamomi at morphological, biochemical and molecular levels. This research also involves field-based studies of plants under challenge at various sites throughout Victoria, Australia. The focus of this field-based research is to assess the responses of individual species to P. cinnamomi in the natural environment with the goal of identifying individuals within susceptible species that display 'resistance'. Understanding how plants are able to resist this pathogen will enable strategies to be developed to enhance species survival and to restore structure and biodiversity to the ecosystems under threat.

Abscisic acid regulation of plant defence responses during pathogen attack

The plant hormone, abscisic acid (ABA), has previously been shown to have an impact on the resistance or susceptibility of plants to pathogens. In this thesis, it was shown that ABA had a regulatory effect on an extensive array of plant defence responses in three different plant and pathogen interaction combinations as well as following the application of an abiotic elicitor. In unique studies using ABA deficient mutants of Arabidopsis, exogenous ABA addition or ABA biosynthesis inhibitor application and simulated drought stress, ABA was shown to have a profound effect on the outcome of interactions between plants and pathogens of differing lifestyles and from different kingdoms. The systems used included a model plant and an important agricultural species: Arabidopsis thaliana (Arabidopsis) and Peronospora parasitica (a biotrophic Oomycete pathogen), Arabidopsis and Pseudomonas syringae pathovar tomato (a biotrophic bacterial pathogen) and an unrelated plant species, soybean (Glycine max) and Phytophthora sojae (a hemibiotrophic Oomycete pathogen), Generally, a higher than basal endogenous ABA concentration within plant tissues at the time of avirulent pathogen inoculation, caused an interaction shift towards what phenotypically resembled susceptibility. Conversely, a lower than basal endogenous ABA concentration in plants inoculated with a virulent pathogen caused a shift towards resistance. An extensive suppressive effect of ABA on defence responses was revealed by a range of techniques that included histochemical, biochemical and molecular approaches. A universal effect of ABA on suppression or induction of the phenylpropanoid pathway via regulation of the key entry point gene, phenylalanine ammonia-lyase (PAL), when stimulated by biotic or abiotic elicitors was shown. ABA also influenced a wide variety of other defence-related components such as: the development of a hypersensitive response (HR), the accumulation of the reactive oxyden species, hydrogen peroxide and the cell wall strengthening compounds lignin and callose, accumulation of SA and the phytoalexin, glyceollin and the transcription of the SA-dependent pathogenesis- related gene (PR-1). The near genome-wide microarray gene expression analysis of an ABA induced susceptible interaction also revealed an yet unprecedented insight into the great diversity of defence responses that were influenced by ABA that included: disease resistance like proteins, antimicrobial proteins as well as phenylpropanoid and tryptophan pathway enzymes. Subtle differences were found in the number and type of defence responses that were regulated by ABA in each type of plant and pathogen interaction that was studied. This thesis has clearly identified in plant/pathogen interactions previously unknown and important roles for ABA in the regulation of many defence responses.

Defining plant resistance to Phytophthora cinnamomi : a standardized approach to assessment

Phytophthora cinnamomi is a soil-borne plant pathogen that causes devastating disease in agricultural and natural systems worldwide. While a small number of species survive infection by the pathogen without producing disease symptoms, the nature of resistance, especially under controlled conditions, remains poorly understood. At present, there are no standardized criteria by which resistance or susceptibility to P. cinnamomi can be assessed, and we have used five parameters consisting of plant fresh weight, root growth, lesion length, relative chlorophyll content of leaves and pathogen colonization of roots to analyse responses to the pathogen. The parameters were tested using two plant species, Zea mays and Lupinus angustifolius, through a time course study of the interactions and resistance and susceptibility defined 7days after inoculation. A scoring system was devised to enable differentiation of these responses. In the resistant interaction with Z. mays, there was no significant difference in fresh weight, root length and relative chlorophyll content in inoculated compared with control plants. Both lesion size and pathogen colonization of root tissues were limited to the site of inoculation. Following inoculation L. angustifolius showed a significant reduction in plant fresh weight and relative leaf chlorophyll content, cessation of root growth and increased lesion lengths and pathogen colonization. We propose that this technique provides a standardized method for plant-P. cinnamomi interactions that could be widely used to differentiate resistant from susceptible species.

Insulin sensitivity not modulated 24 to 78 h after acute resistance exercise in type 2 diabetes patients

Background
The aim of this study was to describe the clinical characteristics, causative pathogens, clinical management and outcomes of patients presenting to a tertiary adult Australian intensive care unit (ICU) with a diagnosis of necrotizing fasciitis (NF).
Methods
This retrospective observational study was conducted in a 19-bed, level III, adult ICU in a 450-bed tertiary, regional hospital. Clinical databases were accessed for patients diagnosed with NF and admitted to The Geelong Hospital ICU between 1 February 2000 and 1 June 2011. Information on severity of sepsis, surgical procedures and microbiological results were collected.
Results
Twenty patients with NF were identified. The median age was 52.5 years and 38% were female. The overall mortality rate was 8.3%. Common co-morbidities were diabetes (21%) and heart failure (17%), although 50% of patients had no co-morbidities. Group A Streptococcus was the identified pathogen in 11 (46%) patients, and Streptococcus milleri group in 5 (21%) patients. Hyperbaric oxygen therapy was not used in the majority of patients. The initial antibiotics administered were active against subsequently cultured bacteria in 83% of patients. Median time to surgical debridement was 20 h. Diagnosis and management was delayed in the nosocomial group.
Conclusions
This study reports physiological data, aetiology and therapeutic interventions in NF for an adult tertiary hospital. We demonstrate one of the lowest reported mortality rates, with early surgical debridement being achieved in the majority of patients. The main delay was found to be in the diagnosis of NF.

The field resistance of potato cultivars to foliar late blight and its relationship with foliage maturity type and skin type in Brazil

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Resistance to experimental autoimmune encephalomyelitis development in Lewis rats from a conventional animal facility

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory disease of the brain and spinal cord that is mediated by CD4+ T lymphocytes specific to myelin components. In this study we compared development of EAE in Lewis rats from two colonies, one kept in pathogen-free conditions (CEMIB colony) and the other (Botucatu colony) kept in a conventional animal facility. Female Lewis rats were immunized with 100 µl of an emulsion containing 50 µg of myelin, associated with incomplete Freund's adjuvant plus Mycobacterium butyricum. Animals were daily evaluated for clinical score and weight. CEMIB colony presented high EAE incidence with clinical scores that varied from three to four along with significant weight losses. A variable disease incidence was observed in the Botucatu colony with clinical scores not higher than one and no weight loss. Immunological and histopathological characteristics were also compared after 20 days of immunization. Significant amounts of IFN-gamma, TNF-alpha and IL-10 were induced by myelin in cultures from CEMIB animals but not from the Botucatu colony. Significantly higher levels of anti-myelin IgG1 were detected in the CEMIB colony. Clear histopathological differences were also found. Cervical spinal cord sections from CEMIB animals showed typical perivascular inflammatory foci whereas samples from the Botucatu colony showed a scanty inflammatory infiltration. Helminths were found in animals from Botucatu colony but not, as expected, in the CEMIB pathogen-free animals. As the animals maintained in a conventional animal facility developed a very discrete clinical, and histopathological EAE in comparison to the rats kept in pathogen-free conditions, we believe that environmental factors such as intestinal parasites could underlie this resistance to EAE development, supporting the applicability of the hygiene hypothesis to EAE.

Inheritance of resistance to powdery mildew in pea and pathogenesis-related aspects

A herança da resistência ao oídio na cultivar de ervilha MK-10 e alguns aspectos histológicos da infecção foram estudados. Para o estudo da herança, as gerações F1, F2, retrocuzamentos e geração F3 de MK-10 com duas populações suscetíveis foram avaliadas. Nas avaliações histológicas observou-se a porcentagem de conídios germinados, porcentagem de conídios que formaram apressório, porcentagem de conídios que estabeleceram colônia e número de haustórios por colônia. Para comparar as razões de segregação obtidas no estudo da herança da resistência, adotou-se o teste do Qui-quadrado (X²) e para os dados das análises histológicas, utilizou-se o teste Tukey a 5% de probabilidade. Concluiu-se que a resistência de MK-10 ao oídio é devida a um par de alelos recessivos e que a resistência é expressa na fase de pré-penetração, completada por uma morte celular localizada pós-penetração, característica da presença do par de alelos recessivos er1er1.

Diallel analysis of maize resistance to Phaeosphaeria maydis

Em programas de melhoramento visando resistência genética a doenças, a estimativa de parâmetros genéticos que governam a resistência permite direcionar a introdução de resistência em germoplasmas. O objetivo deste trabalho foi estimar os efeitos heteróticos, a capacidade geral (CGC) e específica (CEC) de combinação, utilizando-se de dois métodos de avaliação da resistência, à Phaeosphaeria maydis através da análise dialélica de 36 híbridos F1 e de suas nove linhagens genitoras, em experimentos conduzidos em três ambientes. Foi utilizado um delineamento experimental em blocos casualizados com três repetições e a parcela experimental foi representada por uma fileira de 5 m. As diferenças entre as estimativas da capacidade de combinação, em diferentes ambientes e para os dois métodos de avaliação, apresentaram efeitos significativos (P < 0.01) para ambientes (E), CGC e CGC x E. O efeito de CEC e a interação CEC x E não foi significativa para os dois métodos de avaliação. Os efeitos de CGC foram mais importantes que CEC nesse conjunto de linhagens, sugerindo que efeitos genéticos aditivos são mais importantes como fonte de variação para resistência a esta doença. Efeitos heteróticos para resistência foram estimados, sendo possível identificar combinações híbridas específicas entre linhagens com alto potencial para o controle genético deste patógeno. Resultados para os dois métodos de avaliação foram praticamente idênticos, embora o método PI seja de maior praticidade de uso.

Molecular mapping of two loci that confer resistance to Asian rust in soybean

Asian soybean rust (ASR) is caused by the fungal pathogen Phakopsora pachyrhizi Sydow & Sydow. It was first identified in Brazil in 2001 and quickly infected soybean areas in several countries in South America. Primary efforts to combat this disease must involve the development of resistant cultivars. Four distinct genes that confer resistance against ASR have been reported: Rpp1, Rpp2, Rpp3, and Rpp4. However, no cultivar carrying any of those resistance loci has been released. The main objective of this study was to genetically map Rpp2 and Rpp4 resistance genes. Two F(2:3) populations, derived from the crosses between the resistant lines PI 230970 (Rpp2), PI 459025 (Rpp4) and the susceptible cultivar BRS 184, were used in this study. The mapping populations and parental lines were inoculated with a field isolate of P. pachyrhizi and evaluated for lesion type as resistant (RB lesions) or susceptible (TAN lesions). The mapping populations were screened with SSR markers, using the bulk segregant analysis (BSA) to expedite the identification of linked markers. Both resistance genes showed an expected segregation ratio for a dominant trait. This study allowed mapping Rpp2 and Rpp4 loci on the linkage groups J and G, respectively. The associated markers will be of great value on marker assisted selection for this trait.

An alternative field method for screening soybean genotypes for resistance to Heterodera glycines

The soybean cyst nematode (Heterodera glycines) has become an increasingly severe problem in soybean production areas in Brazil. The development and use of resistant cultivars is the most efficient method of minimizing losses due to this pathogen. Our objective was to test the efficiency of an alternative method for screening soybean genotypes for resistance to H. glycines in field plots. The alternative method was compared to the standard method of sowing the test genotypes in fields found to be infested during the previous crop season. In the alternative method, the test genotypes are sown in the furrow following the uprooting of 45-day-old infected plants. The alternative method resulted in twice the cyst population and fewer escapes, and more consistent results than the standard method. The major advantage of the alternative method is that it permits screening in a more homogeneous distribution of H. glycines in the soil.

Cryptococcosis: epidemiology, fungal resistance, and new alternatives for treatment

Cryptococcosis is an important systemic mycosis and the third most prevalent disease in human immunodeficiency virus (HIV)-positive individuals. The incidence of cryptococcosis is high among the 25 million people with HIV/acquired immunodeficiency syndrome (AIDS), with recent estimates indicating that there are one million cases of cryptococcal meningitis globally per year in AIDS patients. In Cryptococcus neoformans, resistance to azoles may be associated with alterations in the target enzyme encoded by the gene ERG11, lanosterol 14α-demethylase. These alterations are obtained through mutations, or by overexpressing the gene encoding. In addition, C. gattii and C. neoformans present a heteroresistance phenotype, which may be related to increased virulence. Other species beyond C. neoformans and C. gattii, such as C. laurentii, have been diagnosed mainly in patients with immunosuppression. Infections of C. albidus have been isolated in cats and marine mammals. Recent evidence suggests that the majority of infections produced by this pathogen are associated with biofilm growth, which is also related with increased resistance to antifungal agents. Therefore, there is a great need to search for alternative antifungal agents for these fungi. The search for new molecules is currently occurring from nanoparticle drugs of plant peptide origin. This article presents a brief review of the literature regarding the epidemiology of cryptococcosis, as well as fungal resistance and new alternatives for treatment. © 2013 Springer-Verlag Berlin Heidelberg.