Saprophytic Species Research Articles

Development of a multiplex PCR diagnostic assay for the detection of Stagonosporopsis species associated with ray blight of Asteraceae

Ray blight, a destructive disease of Asteraceae worldwide, is caused by three morphologically similar but phylogenetically distinct species; Stagonosporopsis chrysanthemi, S. inoxydabilis and S. tanaceti. Stagonosporopsis chrysanthemi has been reported as a specific pathogen of chrysanthemum while S. inoxydabilis has been found associated with various Asteraceae. Stagonosporopsis tanaceti has only been reported in Australia, causing substantial crop loss on pyrethrum. All three species were shown to infect and cause disease on in vitro grown pyrethrum plants, hence, S. chrysanthemi and S. inoxydabilis may pose a significant biosecurity threat to the Australian pyrethrum industry. All these Stagonosporopsis species are also Level 2 quarantine pathogens in Europe. Rapid and accurate detection and differentiation of these species is a priority for ray blight management in Australia and in Europe. Accordingly, three species-specific PCR-based assays, targeted to the intergenic spacer of the nuclear ribosomal DNA, were developed. The specificity of each assay was confirmed against 21 Stagonosporopsis spp. as well as 14 pathogenic and saprophytic fungal species commonly found in association with pyrethrum in Australia. The primers were highly sensitive and specific to the target species, detecting down to 4 fg of genomic DNA. These primers were further used in a multiplex PCR to differentiate the presence of the three Stagonosporopsis spp. based on variable sized amplicons in a single reaction.

Acquisition of negative complement regulators by the saprophyte Leptospira biflexa expressing LigA or LigB confers enhanced survival in human serum

Leptospiral immunoglobulin-like (Lig) proteins are surface exposed molecules present in pathogenic but not in saprophytic Leptospira species. We have previously shown that Lig proteins interact with the soluble complement regulators Factor H (FH), FH like-1 (FHL-1), FH related-1 (FHR-1) and C4b Binding Protein (C4BP). In this study, we used the saprophyte L. biflexa serovar Patoc as a surrogate host to address the specific role of LigA and LigB proteins in leptospiral complement evasion. L. biflexa expressing LigA or LigB was able to acquire FH and C4BP. Bound complement regulators retained their cofactor activities of FI in the proteolytic cleavage of C3b and C4b. Moreover, heterologous expression of ligA and ligB genes in the saprophyte L. biflexa enhanced bacterial survival in human serum. Complement deposition on lig-transformed L. biflexa was assessed by flow cytometry analysis. With regard to MAC deposition, L. biflexa expressing LigA or LigB presented an intermediate profile: MAC deposition levels were greater than those found in the pathogenic L. interrogans, but lower than those observed for L. biflexa wildtype. In conclusion, Lig proteins contribute to in vitro control of complement activation on the leptospiral surface, promoting an increased bacterial survival in human serum.

Destruction of Opportunistic Pathogens via Polymer Nanoparticle-Mediated Release of Plant-Based Antimicrobial Payloads.

The synthesis of antimicrobial thymol/carvacrol-loaded polythioether nanoparticles (NPs) via a one-pot, solvent-free miniemulsion thiol-ene photopolymerization process is reported. The active antimicrobial agents, thymol and carvacrol, are employed as "solvents" for the thiol-ene monomer phase in the miniemulsion to enable facile high capacity loading (≈50% w/w), excellent encapsulation efficiencies (>95%), and elimination of all postpolymerization purification processes. The NPs serve as high capacity reservoirs for slow-release and delivery of thymol/carvacrol-combination payloads that exhibit inhibitory and bactericidal activity (>99.9% kill efficiency at 24 h) against gram-positive and gram-negative bacteria, including both saprophytic (Bacillus subtilis ATCC 6633 and Escherichia coli ATCC 25922) and pathogenic species (E. coli ATCC 43895, Staphylococcus aureus RN6390, and Burkholderia cenocepacia K56-2). This report is among the first to demonstrate antimicrobial efficacy of essential oil-loaded nanoparticles against B. cenocepacia - an innately resistant opportunistic pathogen commonly associated with debilitating respiratory infections in cystic fibrosis. Although a model platform, these results point to promising pathways to particle-based delivery of plant-derived extracts for a range of antimicrobial applications, including active packaging materials, topical antiseptics, and innovative therapeutics.

Ancestral state reconstruction infers phytopathogenic origins of sooty blotch and flyspeck fungi on apple

Members of the sooty blotch and flyspeck (SBFS) complex are epiphytic fungi in the Ascomycota that cause economically damaging blemishes of apples worldwide. SBFS fungi are polyphyletic, but approx. 96% of SBFS species are in the Capnodiales. Evolutionary origins of SBFS fungi remain unclear, so we attempted to infer their origins by means of ancestral state reconstruction on a phylogenetic tree built utilizing genes for the nuc 28S rDNA (approx. 830 bp from near the 59 end) and the second largest subunit of RNA polymerase II (RPB2). The analyzed taxa included the well-known genera of SBFS as well as non-SBFS fungi from seven families within the Capnodiales. The non-SBFS taxa were selected based on their distinct ecological niches, including plant-parasitic and saprophytic species. The phylogenetic analyses revealed that most SBFS species in the Capnodiales are closely related to plant-parasitic fungi. Ancestral state reconstruction provided strong evidence that plant-parasitic fungi were the ancestors of the major SBFS lineages. Knowledge gained from this study may help to better understand the ecology and evolution of epiphytic fungi.

Track analysis of agaricoid fungi of the Patagonian forests

Agaricoid fungi from Patagonia have been vastly studied taxonomically since 1887, and more recently ecologically. We found five generalised tracks and three nodes for a selection of nine ectomycorrhizal and nine saprophytic species. Two areas are supernodes, complex areas supported by many nodes. One of these supernodes could be a result of a lack of sampling in the Strait of Magellan area. The other could imply a biotic radiation and a differential tolerance to more arid climate conditions in the Andes mountain chain around 44.3°S, 71.5°W. Two important areas to focus future sampling of agaricoid fungi are suggested. Generalised tracks obtained match those found for weevils (Coleoptera: Curculionidae) and oribatid mites (Acari: Oribatida) distributed along the Magellanic Forest and Magellanic Moorland provinces of the Andean region. Overlap of generalised tracks among unrelated taxa supports the idea that common processes might have caused the observed patterns. The most significant and undeniable fact is that fungal species present ecological traits that can be vital for studying geological events that have marked the biotic development.

Diversidad de orquídeas en la isla de Hainan en China: distribución y conservación

Las Orchidaceae se distribuyen ampliamente en muchos ecosistemas terrestres con excepción de las zonas polares y desérticas, y constituyen una suerte de «buque insignia» de la conservación biológica. Siendo como es la mayor isla tropical de China, Hainan tiene cinco formaciones vegetales de bosques tropicales, a saber, bosque monzónico caducifolio, selva tropical de tierras bajas, bosque pluvial montano, bosque siempreverde montano y bosque mesófilo de montaña. Hay 317 especies de orquídeas en la isla, incluyendo 33 endémicas, 158 epífitas, 148 terrestres y 11 especies saprófitas. La mayoría de las orquídeas, que se localizan principalmente en el centro y el sur de la isla, se distribuyen típicamente en condiciones húmedas de los bosques tropicales en las montañas a una altitud de 500 a 1500 m. El máximo nivel de endemismo también se centra en estas áreas. Las orquídeas están especialmente amenazadas por la fragmentación del hábitat debido a que crecen en pequeñas poblaciones, y la fragmentación puede bloquear el flujo de genes, lo que resulta en una menor diversidad genética. Además, debido a su valor ornamental y medicinal, muchas orquídeas son recolectadas de forma excesiva. Por lo tanto, la conservación de las orquídeas en la isla de Hainan es muy urgente. Nuestro objetivo en este artículo es determinar el patrón de distribución de las orquídeas y exponer el estado de investigación y conservación en la isla de Hainan y, además, proponer futuras estrategias de conservación.

Isolation of a Seawater Tolerant Leptospira spp. from a Southern Right Whale (Eubalaena australis).

Leptospirosis is the most widespread zoonotic disease in the world. It is caused by pathogenic spirochetes of the genus Leptospira spp. and is maintained in nature through chronic renal infection of carrier animals. Rodents and other small mammals are the main reservoirs. Information on leptospirosis in marine mammals is scarce; however, cases of leptospirosis have been documented in pinniped populations from the Pacific coast of North America from southern California to British Columbia. We report the isolation of a Leptospira spp. strain, here named Manara, from a kidney sample obtained from a Southern Right Whale (Eubalaena australis) calf, which stranded dead in Playa Manara, Península Valdés, Argentina. This strain showed motility and morphology typical of the genus Leptospira spp. under dark-field microscopy; and grew in Ellinghausen-McCullough-Johnson-Harris (EMJH) medium and Fletcher medium after 90 days of incubation at 28°C. Considering the source of this bacterium, we tested its ability to grow in Fletcher medium diluted with seawater at different percentages (1%, 3%, 5%, 7% and 10% v/v). Bacterial growth was detected 48 h after inoculation of Fletcher medium supplemented with 5% sea water, demonstrating the halophilic nature of the strain Manara. Phylogenetic analysis of 16S rRNA gene sequences placed this novel strain within the radiation of the pathogenic species of the genus Leptospira spp., with sequence similarities within the range 97–100%, and closely related to L. interrogans. Two different PCR protocols targeting genus-specific pathogenic genes (G1-G2, B64I-B64II and LigB) gave positive results, which indicates that the strain Manara is likely pathogenic. Further studies are needed to confirm this possibility as well as determine its serogroup. These results could modify our understanding of the epidemiology of this zoonosis. Until now, the resistance and ability to grow in seawater for long periods of time had been proven for the strain Muggia of L. biflexa, a saprophytic species. To the best of our knowledge, this is the first isolation of a Leptospira sp. from cetaceans. Our phenotypic data indicate that strain Manara represents a novel species of the genus Leptospira, for which the name Leptospira brihuegai sp. nov. is proposed.

Detection of Airborne Inoculum of Hymenoscyphus fraxineus and H. albidus during Seasonal Fluctuations Associated with Absence of Apothecia

Hymenoscyphus fraxineus is an invasive fungal species causing the most serious disease of ashes (Fraxinus spp.) in Europe—ash dieback. The biology of this fungus is not totally elucidated, neither its relation to the saprophytic species Hymenoscyphus albidus, native in Europe. Our study is focused on the description of seasonal spore dispersal of both fungi and its relation to meteorological conditions, which is needed for more precise and effective control of the disease. For this experiment one long time infected mixed forest in the SE Czech Republic was chosen. A seven-day automatic volumetric spore trap and a weather station were installed to continuously sample the aerospora from April to October 2014. In seven periods a rotating arm spore trap was also used to obtain 48-h air samples to compare the efficiency of these two types of air samplers. Air samples were evaluated solely by qPCR with a very low detection limit. Results show co-occurrence of inoculum of both fungi throughout the entire sampling period with peak levels in August. The origin of the inoculum sampled in the periods without apothecia is discussed. Air-inoculum occurrence of both fungi is significantly correlated with each other, suggesting their coexistence in this forest.

Distribution of medically important saprophytic Zygomycetes species in Bushenyi District, Uganda

Distribution of medically important saprophytic Zygomycetes species in Bushenyi District, Uganda

Saprophytic and pathogenic fungi in the Ceratocystidaceae differ in their ability to metabolize plant-derived sucrose.

BackgroundProteins in the Glycoside Hydrolase family 32 (GH32) are carbohydrate-active enzymes known as invertases that hydrolyse the glycosidic bonds of complex saccharides. Fungi rely on these enzymes to gain access to and utilize plant-derived sucrose. In fungi, GH32 invertase genes are found in higher copy numbers in the genomes of pathogens when compared to closely related saprophytes, suggesting an association between invertases and ecological strategy. The aim of this study was to investigate the distribution and evolution of GH32 invertases in the Ceratocystidaceae using a comparative genomics approach. This fungal family provides an interesting model to study the evolution of these genes, because it includes economically important pathogenic species such as Ceratocystis fimbriata, C. manginecans and C. albifundus, as well as saprophytic species such as Huntiella moniliformis, H. omanensis and H. savannae.ResultsThe publicly available Ceratocystidaceae genome sequences, as well as the H. savannae genome sequenced here, allowed for the identification of novel GH32-like sequences. The de novo assembly of the H. savannae draft genome consisted of 28.54 megabases that coded for 7 687 putative genes of which one represented a GH32 family member. The number of GH32 gene family members appeared to be related to the ecological adaptations of these fungi. The pathogenic Ceratocystis species all contained two GH32 family genes (a putative cell wall and a putative vacuolar invertase), while the saprophytic Huntiella species had only one of these genes (a putative cell wall invertase). Further analysis showed that the evolution of the GH32 gene family in the Ceratocystidaceae involved transposable element-based retro-transposition and translocation. As an example, the activity of a Fot5-like element likely facilitated the assembly of the genomic regions harbouring the GH32 family genes in Ceratocystis.ConclusionsThis study provides insight into the evolutionary history of the GH32 gene family in Ceratocystidaceae. Our findings suggest that transposable elements shaped the evolution of the GH32 gene family, which in turn determines the sucrolytic activities and related ecological strategies of the Ceratocystidaceae species that harbour them. The study also provides insights into the role of carbohydrate-active enzymes in plant-fungal interactions and adds to our understanding of the evolution of these enzymes and their role in the life style of these fungi.Electronic supplementary materialThe online version of this article (doi:10.1186/s12862-015-0550-7) contains supplementary material, which is available to authorized users.

ORCHID RE-INVENTORY IN PEGUNUNGAN MENOREH, PREFECTURE PROVINCE OF KULONPROGO, D.I. YOGYAKARTA

<p>Pegunungan Menoreh is one area with high orchid diversity. In this area, have a suitable climate for growing orchids. The last research was conducted by Nugroho et al. (2010) in Pegunungan Menoreh that found 11 species orchid. However, it was still potentially to found more orchid. This research has been carried out on May 2011- November 2012 after last inventory on 2010. The aim of this research is to complete the basic data of orchid diversity in Pegunungan Menoreh, Kulonprogo, D.I. Yogyakarta. This research was done by explorative method in Baladewa, Bukit Krengseng, Ngasinan and Watublencong. The results showed 19 orchid species from 17 genera consists of 6 terrestrial species, 2 saprophyte species, 10 epiphytic species and 1 amoebophyte species. The terrestrial species were Arundina graminifolia (D. Don) Hochr., Dienia ophrydis(J. König) Seidenf., Malaxis sp., Nervilia aragoana Commons ex Gaudich., Spathoglottis plicata Blume and Vanilla planifolia Jacks. Ex Andrews. The saprophyte species were Didymoplexis pallens Griff. and Epipogium roseum(D. Don) Lindl. The epiphyte species were Acriopsis lilifolia (Koenig) Ormerod, Appendicula ramosa Blume.,Cymbidium sp., Dendrobium mutabile Blume (Lindl.), D. crumenatum Swartz, Eria retusa (Blume) Rchb.f., E.Javanica (Sw.) Blume, Flickingeria fimbriata(Blume) A.D. Hawkes, Liparis condylobulbon Rchb.f. and Oberonia sp. The amoebophytes was Zeuxine gracillis. </p><p><strong>Keywords</strong> : orchid diversity, Pegunungan Menoreh, re-inventory</p>

English

Chemical treatment is known to effectively eradicate or at least reduce the presence of fungi in seeds. However, this treatment should not impair seed quality. The purpose of the present study was to evaluate the physiological and sanitary quality of Cozumel hybrid melon seeds treated with the fungicide Thiabendazole. The experiments were conducted under a completely randomized design, consisting of 15 treatments in a 3×5 factorial system (three seed lots × five Thiabendazole doses: 0, 0.12, 0.2, 0.3 and 0.4% a.i.). The commercial product used was Tecto® SC (485 g L-1 of Thiabendazole). Four replications of 50 seeds were used for germination and vigor tests (evaluated by germination test first count: GFC). Eight replications of 50 seeds were performed for seed pathology analysis, amounting to 400 seeds per treatment. The average results were compared by Tukey test at 5% probability. The results showed differences in germination between lots, with lower total germination in lot 84538 (90.8%). There was no difference in Thiabendazole dosage regarding both total germination (94.8% on average) and GFC (93.2% on average), showing that the fungicide did not affect the physiological quality of seeds. Pathogenic species were not detected in the sanitary analysis, only saprophytic fungal species (Alternaria sp., Aspergillus spp., Curvularia spp and Penicillium spp.) and a general reduction in fungi incidence was observed with the increase of Thiabendazole doses. Key words: Cucumis melo, seed treatment, fungicide.

Alternaria section Alternaria: Species, formae speciales or pathotypes?

The cosmopolitan fungal genus Alternaria consists of multiple saprophytic and pathogenic species. Based on phylogenetic and morphological studies, the genus is currently divided into 26 sections. Alternaria sect. Alternaria contains most of the small-spored Alternaria species with concatenated conidia, including important plant, human and postharvest pathogens. Species within sect. Alternaria have been mostly described based on morphology and / or host-specificity, yet molecular variation between them is minimal. To investigate whether the described morphospecies within sect. Alternaria are supported by molecular data, whole-genome sequencing of nine Alternaria morphospecies supplemented with transcriptome sequencing of 12 Alternaria morphospecies as well as multi-gene sequencing of 168 Alternaria isolates was performed. The assembled genomes ranged in size from 33.3–35.2 Mb within sect. Alternaria and from 32.0–39.1 Mb for all Alternaria genomes. The number of repetitive sequences differed significantly between the different Alternaria genomes; ranging from 1.4–16.5 %. The repeat content within sect. Alternaria was relatively low with only 1.4–2.7 % of repeats. Whole-genome alignments revealed 96.7–98.2 % genome identity between sect. Alternaria isolates, compared to 85.1–89.3 % genome identity for isolates from other sections to the A. alternata reference genome. Similarly, 1.4–2.8 % and 0.8–1.8 % single nucleotide polymorphisms (SNPs) were observed in genomic and transcriptomic sequences, respectively, between isolates from sect. Alternaria, while the percentage of SNPs found in isolates from different sections compared to the A. alternata reference genome was considerably higher; 8.0–10.3 % and 6.1–8.5 %. The topology of a phylogenetic tree based on the whole-genome and transcriptome reads was congruent with multi-gene phylogenies based on commonly used gene regions. Based on the genome and transcriptome data, a set of core proteins was extracted, and primers were designed on two gene regions with a relatively low degree of conservation within sect. Alternaria (96.8 and 97.3 % conservation). Their potential discriminatory power within sect. Alternaria was tested next to nine commonly used gene regions in sect. Alternaria, namely the SSU, LSU, ITS, gapdh, rpb2, tef1, Alt a 1, endoPG and OPA10-2 gene regions. The phylogenies from the two gene regions with a relatively low conservation, KOG1058 and KOG1077, could not distinguish the described morphospecies within sect. Alternaria more effectively than the phylogenies based on the commonly used gene regions for Alternaria. Based on genome and transcriptome comparisons and molecular phylogenies, Alternaria sect. Alternaria consists of only 11 phylogenetic species and one species complex. Thirty-five morphospecies, which cannot be distinguished based on the multi-gene phylogeny, are synonymised under A. alternata. By providing guidelines for the naming and identification of phylogenetic species in Alternaria sect. Alternaria, this manuscript provides a clear and stable species classification in this section.

Sporothrix chilensis sp. nov. (Ascomycota: Ophiostomatales), a soil-borne agent of human sporotrichosis with mild-pathogenic potential to mammals

A combination of phylogeny, evolution, morphologies and ecologies has enabled major advances in understanding the taxonomy of Sporothrix species, including members exhibiting distinct lifestyles such as saprobes, human/animal pathogens, and insect symbionts. Phylogenetic analyses of ITS1/2 + 5.8s sequences split Sporothrix genus in two well-defined groups with dissimilar ecologies. Species embedded in the Sporothrix schenckii complex are frequently agents of human and animal sporotrichosis, and some of these are responsible for large sapronoses and zoonoses around the warmer temperate regions of the world. At the other extreme, basal saprophytic species evolved in association with decaying wood and soil, and are rarely found to cause human disease. We propose to create a new taxa, Sporothrix chilensis sp. nov., to accommodate strains collected from a clinical case of onychomycosis as well as from environmental origins in Chile. Multigene analyses based on ITS1/2 + 5.8s region, beta-tubulin, calmodulin and translation elongation factor 1α revealed that S. chilensis is a member of the Sporothrix pallida complex, and the nearest taxon is Sporothrix mexicana, a rare soil-borne species, non-pathogenic to humans. The ITS region serves as a primary barcode marker, while each one of the protein-coding loci easily recognized species boundaries providing sufficient information for species identification. A disseminated model of murine sporotrichosis revealed a mild-pathogenic potential, with lung invasion. Although S. chilensis is not a primary pathogen, accidental infection may have an impact in the immunosuppressed population. With the introduction of distinct species with similar routes of transmission but different virulence, identification of Sporothrix agents at the species level is mandatory.

Analysis of the metabolome of Anopheles gambiae mosquito after exposure to Mycobacterium ulcerans.

Infection with Mycobacterium ulcerans causes Buruli Ulcer, a neglected tropical disease. Mosquito vectors are suspected to participate in the transmission and environmental maintenance of the bacterium. However, mechanisms and consequences of mosquito contamination by M. ulcerans are not well understood. We evaluated the metabolome of the Anopheles gambiae mosquito to profile the metabolic changes associated with bacterial colonization. Contamination of mosquitoes with live M. ulcerans bacilli results in disruptions to lipid metabolic pathways of the mosquito, specifically the utilization of glycerolipid molecules, an affect that was not observed in mosquitoes exposed to dead M. ulcerans. These results are consistent with aberrations of lipid metabolism described in other mycobacterial infections, implying global host-pathogen interactions shared across diverse saprophytic and pathogenic mycobacterial species. This study implicates features of the bacterium, such as the putative M. ulcerans encoded phospholipase enzyme, which promote virulence, survival, and active adaptation in concert with mosquito development, and provides significant groundwork for enhanced studies of the vector-pathogen interactions using metabolomics profiling. Lastly, metabolic and survival data suggest an interaction which is unlikely to contribute to transmission of M. ulcerans by A. gambiae and more likely to contribute to persistence of M. ulcerans in waters cohabitated by both organisms.

Identification of three extra-chromosomal replicons in Leptospira pathogenic strain and development of new shuttle vectors.

BackgroundThe genome of pathogenic Leptospira interrogans contains two chromosomes. Plasmids and prophages are known to play specific roles in gene transfer in bacteria and can potentially serve as efficient genetic tools in these organisms. Although plasmids and prophage remnants have recently been reported in Leptospira species, their characteristics and potential applications in leptospiral genetic transformation systems have not been fully evaluated.ResultsThree extrachromosomal replicons designated lcp1 (65,732 bp), lcp2 (56,757 bp), and lcp3 (54,986 bp) in the L. interrogans serovar Linhai strain 56609 were identified through whole genome sequencing. All three replicons were stable outside of the bacterial chromosomes. Phage particles were observed in the culture supernatant of 56609 after mitomycin C induction, and lcp3, which contained phage-related genes, was considered to be an inducible prophage. L. interrogans–Escherichia coli shuttle vectors, constructed with the predicted replication elements of single rep or rep combined with parAB loci from the three plasmids were shown to successfully transform into both saprophytic and pathogenic Leptospira species, suggesting an essential function for rep genes in supporting auto-replication of the plasmids. Additionally, a wide distribution of homologs of the three rep genes was identified in L. interrogans isolates, and correlation tests showed that the transformability of the shuttle vectors in L. interrogans isolates depended, to certain extent, on genetic compatibility between the rep sequences of both plasmid and host.ConclusionsThree extrachromosomal replicons co-exist in L. interrogans, one of which we consider to be an inducible prophage. The vectors constructed with the rep genes of the three replicons successfully transformed into saprophytic and pathogenic Leptospira species alike, but this was partly dependent on genetic compatibility between the rep sequences of both plasmid and host.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1321-y) contains supplementary material, which is available to authorized users.

Armillaria novae‐zelandiae and other basidiomycete wood decay fungi in New Zealand Pinus radiata thinning stumps

SummaryTwo studies were undertaken in a young New Zealand Pinus radiata stand to find a biological control agent for armillaria root disease caused by Armillaria novae‐zelandiae. Fresh thinning stumps were inoculated with saprophytic basidiomycete species as wooden dowel cultures or as homogenized aqueous mycelial suspensions, while all stumps were also inoculated with dowel cultures of A. novae‐zelandiae. After a period of between one and two years, no differences were found among test fungi in the percentage of Armillaria species isolated from stumps. However, inoculation of Stereum sanguinolentum as homogenate, and of Phlebiopsis gigantea, Sistotrema brinkmannii, Hypholoma acutum and Rigidoporus concrescens as dowel cultures, significantly increased their isolation incidence in comparison with stumps not treated by these species. Phlebiopsis gigantea, Resinicium bicolor and R. concrescens were also isolated with greater frequency nearer to their dowel inoculation points. Results suggest that P. gigantea and S. sanguinolentum should be included in further testing. However, a future trial is likely to be more effective if evaluation is directed towards the portion of the stump below the soil surface, particularly the root system.

An improved high throughput sequencing method for studying oomycete communities

Culture-independent studies using next generation sequencing have revolutionized microbial ecology, however, oomycete ecology in soils is severely lagging behind. The aim of this study was to improve and validate standard techniques for using high throughput sequencing as a tool for studying oomycete communities. The well-known primer sets ITS4, ITS6 and ITS7 were used in the study in a semi-nested PCR approach to target the internal transcribed spacer (ITS) 1 of ribosomal DNA in a next generation sequencing protocol. These primers have been used in similar studies before, but with limited success. We were able to increase the proportion of retrieved oomycete sequences dramatically mainly by increasing the annealing temperature during PCR. The optimized protocol was validated using three mock communities and the method was further evaluated using total DNA from 26 soil samples collected from different agricultural fields in Denmark, and 11 samples from carrot tissue with symptoms of Pythium infection. Sequence data from the Pythium and Phytophthora mock communities showed that our strategy successfully detected all included species. Taxonomic assignments of OTUs from 26 soil sample showed that 95% of the sequences could be assigned to oomycetes including Pythium, Aphanomyces, Peronospora, Saprolegnia and Phytophthora. A high proportion of oomycete reads was consistently present in all 26 soil samples showing the versatility of the strategy. A large diversity of Pythium species including pathogenic and saprophytic species were dominating in cultivated soil. Finally, we analyzed amplicons from carrots with symptoms of cavity spot. This resulted in 94% of the reads belonging to oomycetes with a dominance of species of Pythium that are known to be involved in causing cavity spot, thus demonstrating the usefulness of the method not only in soil DNA but also in a plant DNA background. In conclusion, we demonstrate a successful approach for pyrosequencing of oomycete communities using ITS1 as the barcode sequence with well-known primers for oomycete DNA amplification.

Chameleon of head and neck diseases: Intraoral actinomycosis

Actinomycosis is a chronic infectious granulomatous disease caused by saprophytic Actinomyces species. The case discussed in this report is of an adult female patient with a large actinomycotic lesion in the region of the mandibular left second premolar, first and second molars. Provisional diagnosis was made as extragingival pyogenic granuloma. Definitive diagnosis was based on histopathological examination. Since actinomycotic infection has opportunistic characteristics, early diagnosis and proper management of the disease is mandatory to prevent further complications.

English

The purpose of treating seeds chemically is to eradicate their pathogens and/or protect them against soil pathogens, mainly by germination time. However, there is little research on vegetables investigating the effect of this treatment on seed quality. Therefore, this study evaluates the effects of Carboxin + Thiram doses on germination and vigor of three lots of broccoli seeds, as well as on the incidence of fungi in treated seed. The 15 treatments were evaluated in a factorial system (3x5), with the first factor consisting of three lots of 'Avenger' broccoli seeds (lots 82744, 82745 and 82749), and the second factor consisting of five doses (0, 0.04, 0.06, 0.10 and 0.12% of a.i.) of Carboxin + Thiram fungicide (commercial name Vitavax-Thiran). The germination and seed vigor were evaluated, in addition to the presence of pathogens in seeds after treatment (blotter test). All lots showed high levels of germination and vigor. The lot 82749, however, showed higher value in plug test in substrate emergence (99%) than lot 82745 (95%). Regarding the treatment with Carboxin + Thiram, no changes in germination average (98%) and vigor were noticed (average for the first germination count, length, and dry weight of seedling, plug test at 10 days after sowing of 97%, 4.9 cm, 4.0 mg and 96%, respectively), showing that this fungicide, in the evaluated doses, does not affect the quality of broccoli seeds. As to seeds health, the pathogens Alternaria spp. and Fusarium spp. were detected, in addition to saprophytic species such as Penicillium, Aspergillus, Trichoderma, and Rhizopus. The higher incidence of Fusarium spp. was noticed in lot 82744, and the lowest in lot 82749. As to Penicillium spp., lot 82479 was the most contaminated. Regarding other fungi, the general incidence was very low and there was no difference between lots and doses used.   Key words: Brassica oleracea, seed treatment, fungicides.