In silico prediction of the enzymes involved in the degradation of the herbicide molinate by Gulosibacter molinativorax ON4T.

Gulosibacter molinativorax ON4T is the only known organism to produce molinate hydrolase (MolA), which catalyses the breakdown of the thiocarbamate herbicide into azepane-1-carboxylic acid (ACA) and ethanethiol. A combined genomic and transcriptomic strategy was used to fully characterize the strain ON4T genome, particularly the molA genetic environment, to identify the potential genes encoding ACA degradation enzymes. Genomic data revealed that molA is the only catabolic gene of a novel composite transposon (Tn6311), located in a novel low copy number plasmid (pARLON1) harbouring a putative T4SS of the class FATA. pARLON1 had an ANI value of 88.2% with contig 18 from Agrococcus casei LMG 22410T draft genome. Such results suggest that pARLON1 is related to genomic elements of other Actinobacteria, although Tn6311 was observed only in strain ON4T. Furthermore, genomic and transcriptomic data demonstrated that the genes involved in ACA degradation are chromosomal. Based on their overexpression when growing in the presence of molinate, the enzymes potentially involved in the heterocyclic ring breakdown were predicted. Among these, the activity of a protein related to caprolactone hydrolase was demonstrated using heterologous expression. However, further studies are needed to confirm the role of the other putative enzymes.

Phylogeography and population structure analysis reveals diversity by mutations in Lasiodiplodia theobromae with distinct sources of selection.

Lasiodiplodia theobromae is a plant pathogen with a worldwide distribution, with low host specificity, causing stem cankers, dieback diseases, and fruit rot in several species of plants. In coconut, this pathogen is reported as the etiological agent of "coconut leaf blight" (CLB) disease, causing several losses in fruit production. The CLB is an important disease for this crop in Brazil. In our study, we used a phylogeographic approach through the molecular characterization of the translation elongation factor 1-α (TEF1-α) to elucidate the pathogen distribution in Brazil and other countries, besides, search information about diversity sources of this pathogen in coconut palm tree at Brazilian northern, northeast, and southeast. We found that L. theobromae diversity is within populations (locations), and populations that are located closest to the center of the tropical zone have more variability as Central Africa, Brazilian Southeast, and Northeast. The widespread distribution could be in part related with long-distance dispersal via global trade of plants and plant products. The entrance route of L. theobromae in Brazil probably occurred from Africa route and not occurred once. In Brazil, the diversity of this pathogen in coconut tree could be linked to two agents of selection: high host diversity (in Northeast) and distinct management measures adopted in Southeast. These different sources of selection, mainly the mutations, could be one of the reasons that we found distinct reactions to "coconut leaf blight" chemical control in these regions.

Generation of micro-sized PDMS particles by a flow focusing technique for biomicrofluidics applications.

Polydimethylsiloxane (PDMS), due to its remarkable properties, is one of the most widely used polymers in many industrial and medical applications. In this work, a technique based on a flow focusing technique is used to produce PDMS spherical particles with sizes of a few microns. PDMS precursor is injected through a hypodermic needle to form a film/reservoir over the needle's outer surface. This film flows towards the needle tip until a liquid ligament is steadily ejected thanks to the action of a coflowing viscous liquid stream. The outcome is a capillary jet which breaks up into PDMS precursor droplets due to the growth of capillary waves producing a micrometer emulsion. The PDMS liquid droplets in the solution are thermally cured into solid microparticles. The size distribution of the particles is analyzed before and after curing, showing an acceptable degree of monodispersity. The PDMS liquid droplets suffer shrinkage while curing. These microparticles can be used in very varied technological fields, such as biomedicine, biotechnology, pharmacy, and industrial engineering.

Magnetic resonance imaging for planning intracavitary brachytherapy for the treatment of locally advanced cervical cancer.

Cervical cancer is the third most common gynecological cancer. Its treatment depends on tumor staging at the time of diagnosis, and a combination of chemotherapy and radiotherapy is the treatment of choice in locally advanced cervical cancers. The combined use of external beam radiotherapy and brachytherapy increases survival in these patients. Brachytherapy enables a larger dose of radiation to be delivered to the tumor with less toxicity for neighboring tissues with less toxicity for neighboring tissues compared to the use of external beam radiotherapy alone. For years, brachytherapy was planned exclusively using computed tomography (CT). The recent incorporation of magnetic resonance imaging (MRI) provides essential information about the tumor and neighboring structures making possible to better define the target volumes. Nevertheless, MRI has limitations, some of which can be compensated for by fusing CT and MRI. Fusing the images from the two techniques ensures optimal planning by combining the advantages of each technique.

Re-orientation of human resources for health: a great challenge for the Brazilian National Health System.

OBJECTIVES: To present the data available and discuss the progress, current advances and challenges of the initiatives, current policies and guidance implemented by the Health and Education Ministries in Brazil to target transformation of health teaching in order to improve the health care offered by the Brazilian National Health System. STUDY DESIGN: Literature review. METHODS: Documentary analysis and review of articles identified in a search of electronic databases, along with reports and documents acquired from the Health and Education Ministries between 1988 and 2013. RESULTS: This study identified some important initiatives, including the Programme for the Encouragement of Curricular Changes in Medical Courses (PROMED), implemented in 2002 for medical courses alone. Inspired by PROMED and covering a wider range of graduate courses, the National Programme for Re-orientation of Health Professionals was implemented in 2005. This initiative launched its third edition in 2012, covering 14 health professional areas. Another relevant innovation was the National Policy of Permanent Health Education, implemented in 2007, with the goal of transforming public health services into a locus of teaching-learning through working. The Unified Health System Open University was also implemented. CONCLUSIONS: There is general concern and ongoing actions involving different sectors in Brazil in an attempt to improve the health of the Brazilian population in the future. However, the changes pursued involve deep transformations and may take considerable time.

First Report of Colletotrichum karstii Causing Anthracnose on Blueberry Leaves in Brazil.

Anthracnose or ripe rot of blueberry (Vaccinium spp.) is caused predominantly by Colletotrichum fioriniae, which belongs to the C. acutatum complex since it has cylindrical conidia with both ends acute (2). In May 2013, an isolate typical of the C. boninense complex (cylindrical conidia with both ends rounded) (1) was obtained from leaves of southern highbush blueberry seedlings in a nursery located in the municipality of Pelotas, Rio Grande do Sul, Brazil. The symptoms initially appear as circular, necrotic lesions 10 to 30 mm in diameter. At high humidity, lesions expand rapidly to cover the entire foliar surface, leading to severe defoliation of the seedlings. This disease occurred in 100% of the seedlings, causing serious losses in the nursery. A single-conidium culture was obtained on potato-dextrose-agar (PDA) medium followed by morphological and molecular characterization. This culture was deposited at the culture collection of the Universidade Federal de Viçosa, Brazil (Accession No. COAD 1741). Conidia were cylindrical, aseptate, hyaline, rounded at both ends, and 11 to 16 µm (µ = 13) long and 5 to 6 µm (µ = 5.5) wide (n = 100). For the molecular characterization, sequences of the internal transcribed spacer (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and ß-tubulin 2 (ßt) regions were obtained and deposited in GenBank (KM055653 to KM055655). A search of the Q-bank Fungi database using the ITS, ßt, and GAPDH sequences retrieved C. karstii with 100, 99, and 100% identities, respectively. Based on morphological and molecular data, the fungus was identified as C. karstii. To verify pathogenicity, 20 leaves from the upper branches of 1-m tall blueberry seedlings were inoculated with 6-mm-diameter plugs from a 7-day-old culture. PDA plugs were placed on the leaves of seedlings to serve as the control. Initially, seedlings were maintained at 25 ± 2°C in the dark. Thereafter, seedlings were covered with plastic bags and transferred to a greenhouse. Anthracnose symptoms on the leaves were observed at 5 days after inoculation. Seedlings from the control treatment remained symptomless. The fungus was re-isolated from the necrotic lesions, confirming Koch's postulates. C. karstii has a wide host range and in Brazil has been previously reported on Bombax aquaticum, Carica papaya, Eugenia uniflora, Malus domestica, and Mangifera indica (1,3,4). To the best of our knowledge, this is the first report of C. karstii causing anthracnose on the leaves of blueberry seedlings in Brazil or worldwide. Due to the high disease severity and intense defoliation of blueberry seedlings, this pathogen represents a new threat for nurseries. Therefore, control strategies should be investigated for this disease. References: (1) U. Damm et al. Stud. Mycol. 73:1, 2012. (2) U. Damm et al. Stud. Mycol. 73:37, 2012. (3) Lima et al. Plant Dis. 97:1248, 2014. (4) Velho et al. Plant Dis. 98:157, 2014.

First Report of Curvularia eragrostidis Causing Postharvest Rot on Pineapple in Brazil.

Pineapple (Ananas comosus L. Merril.) is the main plant of the Bromeliaceae, cultivated economically for the fruits' appealing flavor and a refreshing sugar-acid balance. In 2013, fruits with no initially visible symptoms began to show a postharvest rot after 3 days in a market in the municipality of Viçosa, Minas Gerais, Brazil. The rot can rarely be detected from the outside of the fruit, but a longitudinal section allows observation of extension of the affected area toward the center of the fruit. The symptoms initially appear as a dark brown to black rot on surface of the fruits, which gradually enlarges in size, leading to increased rot and disposal of infected fruits. Until now, this disease occurred sporadically and caused small losses. A fungus was isolated from rot observed in fruits from cultivar Pérola and a single-spore culture was deposited in the culture collection of the Universidade Federal de Viçosa (Accession No. COAD 1588). After 7 days of incubation at 25°C, the strain displayed radial growth and gray-white to black colonies. Microscopic observations revealed brown to light brown conidiophores present singly or in groups. The septate, simple or rarely branched conidiophores are straight or curved, up to 245 µm long and 5 µm wide, and some have a geniculate growth pattern near the apex. The conidia are ellipsoidal or barrel-shaped and 22 to 25 µm long and 10 to 12.5 µm wide. The median septum appears as a black band and the cells at each end of the conidia are pale, whereas the intermediate cells are brown or dark brown. Based on morphological characteristics, the fungus was identified as Curvularia eragrostidis (4). To confirm this identification, DNA was extracted and sequences of the internal transcribed spacer (ITS), 28S and 18S rDNA regions were obtained and deposited in GenBank (Accession Nos. KJ541818 to KJ541820). The sequence of the ITS region exhibited 99% identity over 530 bp with other C. eragrostidis sequence in GenBank (JN943449) and Bayesian inference analysis placed our isolate in the same clade with others C. eragrostidis (study S15670 deposited in TreeBASE). Koch's postulates were conducted by inoculating six fruits of pineapple previously disinfected with 2% sodium hypochlorite and washed in sterile distilled water. For inoculation, the isolate was grown in potato dextrose agar (PDA) for 15 days at 25°C. Six millimeter diameter disks were removed from the surface of fruits with a sterile cork borer and replaced with PDA disks containing mycelia from the margins of the culture. An agar plug was deposited in three control fruits and all fruits were maintained at 25°C in plastic trays. Inoculated fruits showed symptoms 7 days after inoculation that were similar to those initially observed in the infected fruits, while control fruits showed no symptoms. C. eragrostidis is a cosmopolitan pathogen that infects hosts from several botanical families (2,4). In Brazil, this fungus causes leaf spot on A. comosus (3) and also infects Allium sativum, Dioscorea alata, D. cayenensis, Oryza sativa, Sorghum bicolor, Vigna unguiculata, and Zea mays (1). To our knowledge, this is the first report of C. eragrostidis causing postharvest rot disease in pineapple in Brazil. Because invasion of the fungus can occur through minute fractures, fruits should be carefully handled to avoid mechanical damage. References: (1) D. F. Farr and A. Y. Rossman. Fungal Databases. Systematic Mycology and Microbiology Laboratory, ARS, USDA. Retrieved from http://nt.ars-grin.gov/fungaldatabases , 18 February 2014. (2) D. S. Manamgoda et al. Fungal Divers. 51:3, 2011. (3) J. J. Ponte et al. Fitopatologia 10:21, 1975. (4) A. Sivanesan. Mycological Papers 158:113, 1987.

First Report of Leaf Spot Caused by Myrothecium roridum on Coffea canephora in Brazil.

Coffea canephora (conilon coffee) represents approximately 30% of the coffee marketed worldwide. The state of Espírito Santo is the largest conilon coffee-producing state in Brazil. In 2013 and 2014, leaves with a leaf spot were observed on most of the conilon coffee seedlings in a commercial nursery in Laranja da Terra, Espírito Santo, Brazil. The infected leaves were deposited in the VIC Herbarium (VIC 42482) and a pure single-spore culture of the pathogen was deposited in the culture collection of the Universidade Federal de Viçosa (Accession No. COAD 1729). The initial symptoms were circular, brown to dark brown lesions with yellow margins occurring on both leaf surfaces. In high humidity, concentric rings formed and the lesions expanded rapidly to reach up to 30 mm in diameter, and later became dark brown with a grayish center. Black sporodochia with white, and marginal mycelial tuffs bearing black spore masses were observed in the older lesions. These symptoms were consistent with those of Myrothecium leaf spot reported on Coffea spp. (3). Microscopic observation revealed aseptate, hyaline, and cylindrical conidia, rounded at both ends, greenish to black in mass, and 5 to 6 µm long and 1 to 2 µm wide. The symptoms and morphological characteristics described above matched the description of Myrothecium roridum Tode (4). To confirm this identification, DNA was extracted using a Wizard Genomic DNA Purification Kit and the sequence of an internal transcribed spacer (ITS) region was obtained and deposited in GenBank (Accession No. KJ815095). The sequence of the ITS region exhibited 100% identity over 561 bp with another M. roridum sequence in GenBank (JF343832). To verify the pathogenicity of the fungus, healthy leaves of the C. canephora clones 12v and 14 (four seedlings each) were wounded superficially with a sterilized needle and inoculated by spraying them with a suspension of M. roridum conidia (106 conidia ml-1). The seedlings were covered with plastic bags and incubated in a growth chamber at 25°C under a photoperiod of 12 h light/12 h dark for 5 days. The control seedlings were sprayed with distilled water and incubated similarly. Fifteen days after inoculation, symptoms in all inoculated seedlings were consistent with those initially observed on the naturally infected seedlings, whereas the controls remained healthy. Re-isolation and identification confirmed Koch's postulates. M. roridum has a wide host range, and symptoms were similar to those reported in other hosts of the pathogen in Brazil (2,3). There is only one report of M. roridum on C. canephora in Colombia (1); however, this pathogen was previously reported on C. arabica in Brazil, Colombia, Costa Rica, Guatemala, India, Indonesia, Puerto Rico, and the Virgin Islands (1,3). To our knowledge, this is the first report of a leaf spot caused by M. roridum on conilon coffee in Brazil. The cultivation of conilon coffee is increasing and the reported leaf spot disease affects the quality of the seedlings in nurseries. It is therefore important to conduct a thorough study of management strategies for this disease. References: (1) D. F. Farr and A. Y. Rossman. Fungal Databases. Syst. Mycol. Microbiol. Lab. ARS, USDA. Retrieved from http://nt.ars-grin.gov/fungaldatabases , 27 May 2014. (2) A. M. Quezado Duval et al. Braz. J. Microbiol. 41:246, 2010. (3) S. F. Silveira et al. Fitopatol. Bras. 32:440, 2007. (4) M. Tulloch. Mycol. Pap. No. 130. CMI, Wallingford, UK, 1972.

First Report of Anthracnose on Pepper Fruit Caused by Colletotrichum scovillei in Brazil.

Anthracnose is major disease of pepper (Capsicum annum) in the tropics and causes severe damage both in the field and postharvest. In Brazil, this disease is caused by Colletotrichum acutatum, C. boninense, C. capsici, C. coccodes, and C. gloeosporioides, where the first species is responsible for 70% of all occurrences (3). Recently, C. acutatum has been considered a species complex (1); thus, the aim of this study was to verify the etiology of anthracnose on peppers using a morphological and molecular approaches. In 2011, pepper fruits with typical symptoms of anthracnose (dark, sunken spots with concentric rings of orange conidial masses) were collected in Viçosa, Minas Gerais, Brazil. A single spore isolate was obtained on potato dextrose agar (PDA), and the derived culture was deposited in the Coleção de Culturas de Fungos Fitopatogênicos "Prof. Maria Menezes" (code CMM-4200). The upper side colonies on PDA were gray, cotton-like, and pale gray to pale orange. Conidia were hyaline, aseptate, smooth, straight, cylindrical with round ends or occasionally with end ± acute, 12.5 to 17 µm long and 3.5 to 4 µm wide on synthetic nutrient deficient agar. The isolate was morphologically typical of species belonging to the C. acutatum complex. Molecular identification of the pathogen was carried out and sequences of the regions internal transcribed spacer (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and ß-tubulin (ßt) were obtained and deposited in GenBank (Accession Nos. KJ541821 to KJ541823). A search in the Q-bank fungi database using the ITS, ßt, and GAPDH sequences retrieved C. scovillei with 100% identity for all three genes. This pathogen was previously reported in Capsicum spp. only in Thailand, Indonesia, and Japan (1,2). To confirm pathogenicity, drops with 105 spores/ml were deposited in 10 artificially wounded fruits (cv. Itapuã 501 and Melina). In control fruits, drops of sterilized water were deposited onto wounds. The fruits were covered for one day with a transparent plastic bag with moisture supplied by a wet filter paper. The fruits were detached and mature. The bags were removed, and the fruits were incubated for 10 days in a growth chamber at 25°C with a photoperiod of 12 h. After 4 days, gray-brown to black sunken spots with concentric rings were observed on 100% of the wounded fruits that had been inoculated. No disease was observed on the control fruits. The fungus C. scovillei was successfully re-isolated from symptomatic fruits to fulfill Koch's postulates. To our knowledge, this is the first report of anthracnose on pepper fruit caused by C. scovillei in Brazil. Due to the diversity of species that cause anthracnose in Capsicum, future studies using morphological and molecular tools are essential for the correct identification of Colletotrichum spp. on pepper in Brazil. References: (1) U. Damm et al. Stud. Mycol. 73:37, 2012. (2) T. Kanto et al. J. Gen. Plant. Pathol. 80:73, 2014. (3) M. J. Z. Pereira et al. Hortic. Bras. 29:569, 2011.

First Report of Leaf Spot Caused by Phyllosticta yuccae on Yucca filamentosa in Brazil.

Yucca filamentosa L. (Agavaceae), commonly known as Adam's needle, is known in Brazil as "agulha-de-adão." It is an ornamental garden plant with medicinal properties (4). In 2010, 100% of Y. filamentosa seedlings and plants were observed with a severe leaf spot disease in two ornamental nurseries located in the municipality of Viçosa, Minas Gerais, Brazil. Initially, lesions were dark brown, elliptical, and scattered, and later became grayish at the center with a reddish brown margin, irregular and coalescent. Infected leaf samples were deposited in the herbarium at the Universidade Federal de Viçosa (Accession Nos. VIC32054 and VIC32055). A fungus was isolated from the leaf spots and single-spore pure cultures were obtained on potato dextrose agar (PDA). The sporulating single-spore cultures were deposited at the Coleção de Culturas de Fungos Fitopatogênicos "Prof. Maria Menezes" (CMM 1843 and CMM 1844). On the leaf, the fungus produced pycnidial conidiomata that were scattered or gregarious, usually epiphyllous, immersed, dark brown, unilocular, subglobose, and 95 to 158 × 108 to 175 µm, with a minute, subcircular ostiole. Conidiogenous cells were blastic, hyaline, conoidal, or short cylindrical. Conidia were aseptate, hyaline, smooth walled, coarsely granular, broadly ellipsoidal to subglobose or obovate, usually broadly rounded at both ends, occasionally truncate at the base or indented slightly at the apex, and 7.5 to 13.5 × 6 to 10 µm. Conidia were also surrounded by a slime layer, usually with a hyaline, flexuous, narrowly conoidal or cylindrical, mucilaginous apical appendage that was 10 to 16 µm long. Spermatia were hyaline, dumbbell shaped to cylindrical, both ends bluntly rounded, and 3 to 5 × 1 to 1.5 µm. These characteristics matched well with the description of Phyllosticta yuccae Bissett (1). To confirm this identification, DNA was extracted using a Wizard Genomic DNA Purification Kit and amplified using primers ITS1 and ITS4 (2) for the ITS region (GenBank Accession Nos. JX227945 and JX227946) and EF1-F and EF2-R (3) for the TEF-1α (JX227947 and JX227948). The sequencing was performed by Macrogen, South Korea. The ITS sequence matched sequence No. JN692541, P. yuccae, with 100% identity. To confirm Koch's postulates, four leaves of Y. filamentosa (five plants) were inoculated with 6-mm-diameter plugs from a 7-day-old culture growing on PDA. The leaves were covered with plastic sack and plants were maintained at 25°C. In a similar manner, fungus-free PDA plugs were placed on five control plants. Symptoms were consistently similar to those initially observed in the nurseries and all plants developed leaf spots by 15 days after inoculation. P. yuccae was successfully reisolated from the symptomatic tissue and control plants remained symptomless. P. yuccae has been previously reported in Canada, the Dominican Republic, Guatemala, Iran, and the United States of America. To our knowledge, this is the first report of P. yuccae causing disease in Y. filamentosa in Brazil and it may become a serious problem for the nurseries, due to the severity of the disease and the lack of chemical products to control this pathogen. References: (1) J. Bissett. Can. J. Bot. 64:1720, 1986. (2) M. A. Innis et al. PCR Protocols: A guide to methods and applications. Academic Press, 1990. (3) Jacobs et al. Mycol. Res. 108:411, 2004. (4) H. Lorenzi and H. M. Souza. Plantas Ornamentais no Brasil. Instituto Plantarum, 2001.

First Report of Curvularia gladioli Causing a Leaf Spot on Gladiolus grandiflorus in Brazil.

Gladiolus (Iridaceae) is a popular bulbous plant grown worldwide as an ornamental garden plant or cut flower due to its attractive color, size, and flower shape. In April 2012, leaf spots were observed on plants of Gladiolus grandiflorus varieties T-704 and Amsterdam growing in a production area of cut flowers located in the city of Viçosa, Minas Gerais. The oval to round leaf spots were brown with a dark border surrounded by a halo of yellow tissue. Infected leaf samples were deposited in the herbarium at the Universidade Federal de Viçosa (VIC31897). A fungus was isolated from the leaf spots and a single-spore pure culture was initiated and grown on corn meal carrot agar (CCA) medium in petri dishes incubated at 25°C under a 12-h photoperiod for 4 weeks. A sporulating single-spore culture was deposited at the Coleção de Culturas de fungos fitopatogênicos "Prof. Maria Menezes" (UFRPE, Brazil) code CMM 4055. On CCA medium, the fungal isolate initially appeared white, becoming dark after 14 days. Thirty conidia and conidiophores were measured for identification to species. The septate, smooth to pale brown conidiophores were present singly or in groups. The simple, straight or flexuous conidiophores were 42.5 to 82.5 × 3.5 to 7.5 µm and some had a geniculate growth pattern. The majority of conidia were curved at the third (central) cell from the base, which was usually enlarged compared to the end cells. The cells at each end of the 3-distoseptate conidia were pale brown, the intermediate cell brown or dark brown, and the third (central) cell was often the darkest. The basal cell had a protuberant hilum. Conidia were smooth and 20.0 to 33.5 × 10 to 17.5 µm. These characteristics matched well with the description of Curvularia gladioli (1). To confirm this identification, DNA was extracted using a Wizard Genomic DNA Purification Kit and the internal transcribed spacer region (ITS) of rDNA was amplified using ITS1 and ITS4 primers and the partial 28S rDNA region using primers LR0R and LR5. The sequences were deposited in GenBank as accession nos. JX995106 and JX995107, respectively. The ITS sequence matched sequence AF071337, C. gladioli, with 100% identity. This pathogen was first identified as C. lunata, but based on the characteristic of the hilum, spore size, and pathogenicity testing, the fungus was renamed C. trifolii f. sp. gladioli (3). Due to the explicit curvature of the conidia at the third cell and molecular data, the fungus was reclassified as C. gladioli (1,2). To confirm Koch's postulates, 1-month-old healthy plants of G. grandiflorus var. T-704 and Amsterdam (five plants each) were inoculated with a conidial suspension (2 × 104 conidia mL-1) by spraying the foliage and then placed on a growth chamber at 25°C. The control plants were sprayed with distilled water. Symptoms were consistent with those initially observed and all plants developed leaf spots by 4 days post-inoculation. C. gladioli was consistently recovered from the symptomatic tissue and control plants remained symptomless. To our knowledge, this is the first report of C. gladioli causing leaf spot on G. grandiflorus in Brazil. Due to a lack of chemical fungicides for management of this pathogen, further studies to evaluate the susceptibility of the main varieties of gladiolus grown in Brazil to C. gladioli may be necessary. References: (1) G. H. Boerema and M. E. C. Hamers. Neth. J. Plant Pathol. 95:1, 1989. (2) D. S. Manamgoda et al. Fungal Divers. 56:131, 2012. (3) J. A. Parmelee. Mycologia 48:558, 1956.

Decrease in the expression of N-methyl-D-aspartate receptors in the nucleus tractus solitarii induces antinociception and increases blood pressure.

N-methyl-D-aspartate receptors (NMDAR) have a role in cardiovascular control at the nucleus tractus solitarii (NTS), eliciting increases or decreases in blood pressure (BP), depending on the area injected with the agonists. In spite of the association between cardiovascular control and pain modulation, the effects of manipulating NMDAR in pain responses have never been evaluated. In this study, we decreased the expression of NMDAR in the NTS using gene transfer to target receptor subunits and evaluate long-term effects. Seven days after the injection of lentiviral vectors containing the NR1a subunit cDNA of NMDAR, in antisense orientation, into the intermediate NTS of Wistar rats, BP was measured, and the formalin test of nociception was performed. The antisense vector induced a decrease of NR1 expression in the NTS and elicited BP rises and hypoalgesia. Antisense vectors inhibited formalin-evoked c-Fos expression in the spinal cord, indicating decreased nociceptive activity of spinal neurons. Using a time-course approach, we verified that the onset of both the increases in BP and the hypoalgesia was at 4 days after vector injection into the NTS. The injection of NMDA into the NTS reversed the effects of antisense vectors in pain behavioral responses and spinal neuronal activation and decreased BP and heart rate. The present study shows that the NR1 subunit of the NMDAR at the NTS is critical in the regulation of tonic cardiovascular and nociceptive control and shows an involvement of the nucleus in the modulation of sustained pain.

First Report of Collar and Root Rot of Physic Nut (Jatropha curcas) Caused by Neoscytalidium dimidiatum in Brazil.

Physic nut (Jatropha curcas L.; Euphorbiaeae) has become important in Brazil due to its potential as a feedstock for biodiesel production. In October 2010, during routine monitoring of fields in the state of Piauí, several plants were observed with symptoms of collar and root rot. Initially, plants appeared withered and chlorotic, and later became defoliated. Roots and collars of affected plants also appeared rotten with black fungal structures directly observed. Symptomatic tissue fragments of 5 mm diameter were washed with 70% ethanol, 1% sodium hypochlorite, and sterilized water, before being placed in petri dishes containing potato dextrose agar (PDA) and incubated at 25°C. Pure cultures were then obtained by single-spore isolation. The fungus isolated was grown on plates containing 2% water agar overlaid with sterilized corn straw or pine twigs and incubated at 25°C under a photoperiod of 12 h for 4 weeks to induce the formation of fruiting bodies. Thirty measurements of all of the relevant morphological characters were made using a light microscope for the identification of the species. On PDA, isolates initially appeared white and became dark after 7 days. The aerial mycelia formed chains of zero- to one-septate arthroconidia, oblong to globose, initially hyaline that became brown and with a thick wall with age. The dimensions of arthroconidia were 4 to 12 × 2.5 to 8 µm. The formation of pycnidia was observed on the plates with corn straw and pines twigs. These were dark, with a globose base up to 250 µm and a neck up to 810 µm. Conidiogenous cells were holoblastic, lageniform to ampulliform, hyaline, and 6 to 10 × 1.5 to 2.5 µm. Conidia were hyaline, ellipsoid to nearly fusiform, and 8 to 12 × 4 to 5 µm. Septate and dark conidia were not observed. DNA was extracted from one isolate following Wizard Genomic DNA Purification Kit procedures and amplified using primers ITS1 and ITS4. Products were directly sequencing by Macrogen, Korea. The 856-bp sequence obtained was deposited in GenBank (Accession No. JQ927342). The sequence was 99% similar to Neoscytalidium dimidiatum (Penz.) Crous & Slippers, further supporting the identification by morphology. Pathogenicity tests were conducted by using 6 mm disks removed from the outer bark of the collar region of healthy plants using a sterile cork borer, and 6-mm diameter plugs were placed in each wound. Five plants were inoculated with the isolate and five plants inoculated with an isolate-free agar plug. Below these, pieces of moistened cotton were placed and covered with Parafilm. After 60 days, all inoculated plants reproduced the symptoms observed in the field, and the pathogen was successfully reisolated. All non-inoculated plants remained healthy. The genus Neoscytalidium includes species having Scytalidium-like synanamorphs in the aerial mycelia and Fusicoccum-like conidia in the pycnidia (1). Currently, this species is reported to cause diseases in fig, mango, and orange (2,3). To our knowledge, this is the first report of collar and rot root caused by N. dimidiatum in J. curcas and of this fungus in Brazil. It seems likely the disease exists in areas beyond Piauí and could cause important losses for biodiesel production. References: (1) P. W. Crous et al. Stud. Mycol. 55:235, 2006. (2) G. Polizzi et al. Plant Dis. 93:1215, 2009. (3) J. D. Ray et al. Austral. Plant Dis. Notes 5:48, 2010.

First Report of Leaf Spot Disease Caused by Cercosporella pfaffiae on Brazilian Ginseng (Pfaffia glomerata) in Brazil.

Pfaffia glomerata (Spreng) Pedersen (Amaranthaceae) and other species in this genus, popularly known as "Brazilian ginseng," have been marketed and used for many years in folk medicine for the treatment of various diseases (1). In January 2012, samples of P. glomerata with leaf spots were collected in the city of Viçosa, state of Minas Gerais, Brazil. Two samples were deposited in the herbarium at the Universidade Federal de Viçosa (VIC31849 and VIC31851). The diseased leaves were examined using a stereomicroscope (75×). The fungal structures were scraped with a scalpel from the plant surface and mounted in lactophenol. Thirty measurements of all of the relevant morphological characters were obtained using light microscopy for the identification of the species. To confirm the identification, fungal DNA from single-spore pure culture was isolated from the diseased leaves on PDA, and the DNA was amplified using primers ITS1 and ITS4 for the ITS region (GenBank Accession No. JQ990331) and LR0R and LR5 for partial 28S rDNA (Accession No. JQ990330). Sequencing was performed by Macrogen, Korea. The symptoms observed were leaf spots, subcircular, usually up to 6 mm diameter, initially yellowish becoming brown to reddish, margin indefinite, with the formation of fungal structures, hypophyllous, white, scattered, or grouped. Conidiophores were very numerous in dense subsynnematal fascicles, moderately brown at the base but for most of the length subhyaline, 42.5 to 350 × 2.5 to 3.5 µm, showing conidial scars. Conidia formed singly, 22.5 to 77.5 × 5 to 6 µm, hyaline, hilum slightly thickened, and refractive. These characteristics show that the fungus found on P. glomerata matched well with the description of Cercosporella pfaffiae (2). Koch's postulates were fulfilled by inoculation of 6-mm-diameter PDA plugs with the isolate mycelia on leaves of P. glomerata. Six plants were inoculated with the isolate and six plants were inoculated with an isolate-free agar plug. Inoculated plants were maintained in a moist chamber for 24 hours and subsequently in a greenhouse at 26°C. Leaf spot was observed in inoculated plants 15 days after inoculation, and symptoms were similar to those in the field. All non-inoculated plants remained healthy. A Megablast search of the NCBI GenBank nucleotide sequence database using the ITS sequence retrieved C. virgaureae as the closest match [GenBank GU214658; Identity = 458/476 (96%), Gaps = 2/476 (0%)]. To confirm the identification, Bayesian inference analyses were employed, and the tree was deposited in TreeBASE (Study S12680). The analysis placed our isolate in the same clade with the type species of Cercosporella. Molecular studies and morphological characteristics confirm our identification. C. pfaffiae has been previously reported in P. iresinoides (H.B.K.) Spreng. in Trinidad and Gomphrena glomerata L. in Argentina (2). To our knowledge, this is the first report of C. pfaffiae causing disease in P. glomerata in Brazil and it may become a serious problem for some medicinal plant growers, due to the severity of the disease and the lack of chemical products for this pathogen. References: (1) Neto et al. J. Ethnopharmacol. 96:87, 2005. (2) U. Braun. A Monograph of Cercosporella, Ramularia and Allied Genera (Phytopathogenic Hyphomycetes). Eching bei Müchen, IHW-Verlage. Vol. 1, p. 68, 1995.

[Mushroom poisoning in Portugal]. / Intoxicação por cogumelos em Portugal.

The renewed interest in mycology has been reflected in growing use of wild mushrooms in culinary, driven by its nutritional, organoleptic and commercial value. However, the international scientific literature describes several syndromes of poisoning by mushrooms. We live, therefore, a paradigm conducive to an increase of mycetism, whose diagnosis requires a high level of suspicion and knowledge of clinical profiles. In Portugal, the real dimension of this problem is unknown. Although some mycetisms, such as the hepatotoxic syndrome, have high morbidity and mortality, their relative incidences are unknown. Add up to the shortage of international scientific literature, often outdated and inappropriate to clinical practice. In this context, this article provides an updated epidemiological and clinical perspective emphasizing a narrative and descriptive information on the forms of presentation, differential diagnosis and therapeutic approach, with the ultimate goal of the elaboration of a national diagram-oriented approach to decision-making diagnosis. We analyzed all the clinical records of patients admitted into ten hospitals between 1990 and 2008, notified with the code 988.1 of GDH (acute poisoning by mushrooms). There were registered demographic data, way of presentation, time between ingestion and onset of symptoms, the annual distribution, clinical profile, clinical and analytical treatment performed and complications. We identified 93 cases of acute poisoning by mushrooms, with equal gender distribution and inclusion of individuals of all age groups (from 1 to 85 years), but with greater representation from 21 to 50 years. There was a bimodal seasonal pattern, with a higher peak between September and December and a second in the spring. The hepatotoxic profile presentation corresponded to 63.4% and 31.7% of the cases to gastroenteritis syndrome. The mortality in cases of hepatotoxicity was 11.8%. The developmental profile of the rate of prothrombin time (PT), aspartate aminotransferase (AST) and bilirubin, was an important setting for diagnosis and prognosis. TGO increases early, always within 48 hours, having an essential role in the diagnosis of hepatotoxicity. Despite the late elevation of bilirubin, the cases of death revealed that there was an earlier increase, reaching higher values, which seems to have a prognostic value, to be evaluated with further studies. Finally, we propose a diagram of diagnostic performance, considerating the generalized lack of mycological diagnosis in Portugal, which emphasizes the need for a careful history, focused on quantifying the latency period.

First Report of Cercospora apii Leaf Spot on Capsicum chinense in Brazil.

In Brazil, Capsicum chinense Jacq. is the predominant species of commercial hot peppers because of its popular citrus-like aroma and adaptability to different soils and climates (4). In June 2010, 30 samples of C. chinense with severe leaf spot were collected from a field in the city of Viçosa, state of Minas Gerais, Brazil. Symptoms were observed on leaves, calyxes, fruits, and stems on most of the plants found in the area. On leaves, symptoms included amphigenous lesions that were initially circular to ellipsoid, 1 to 5 mm in diameter, whitish to tan in the center, and surrounded by a dark brown or reddish purple border. Lesions coalesce and turned necrotic with age. A fungus isolated from the lesions matched well with the description of Cercospora apii Fresen. It formed erumpent stromata that were dark brown and spherical to irregular; fascicule conidiophores were clear brown or pale, straight or curved, unbranched, geniculate, 22.5 to 80 × 5 to 7.5 µm, 0 to 3 septate, subtruncate apex; and conidia were solitary, hyaline to subhyaline, filiform, base truncate, tip acute, straight to curved, 12.5 to 140 × 3.5 to 5 µm, and 0 to 11 septate (1,2). A sample was deposited in the herbarium of the Universidade Federal de Viçosa, Minas Gerais, Brazil (VIC 31415). Identity was confirmed by amplifying part of the calmodulin gene with species-specific primers CercoCal-apii and CercoCal-R (3) of fungal DNA from a single-spore culture. In amplification reaction, initial denaturation step was done at 94°C for 5 min, followed by 40 cycles of denaturation at 94°C (30 s), annealing at 56°C (30 s), and elongation at 72°C (30 s). Primers CercoCal-apii and CercoCal-R amplified a single DNA product of 176 bp, and coupled with the morphological characteristics, confirmed the identity of the fungus as Cercospora apii. To check pathogenicity, a 6-mm-diameter plug of the isolate was removed from the expanding edge of a 21-day-old culture grown on potato dextrose agar (PDA) and placed in contact with the adaxial face of the leaves of 8-week-old C. chinense grown in 2-liter plastic pots with soil substrate. Six plants, one per pot, were inoculated with the isolate and six plants were inoculated with the fungus-free PDA plug. Inoculated plants were maintained in a moist chamber for 24 h and then subsequently kept in a greenhouse at 26°C. Leaf spot was observed in all inoculated plants 15 days after inoculation and symptoms were similar to those expressed in the field. The fungus was reisolated from the inoculated plants and matched well with the description of Cercospora apii. All fungus-free PDA inoculated plants remained healthy. Cercospora apii comprises a complex of 281 morphologically indistinguishable species that can infect an extremely wide host range (2). To our knowledge, this pathogen has the potential to cause significant damage to the hot pepper industry of Brazil. References: (1) C. Chupp. A Monograph of the Fungus Cercospora. Cornell University Press, Ithaca, NY, 1954. (2) P. W. Crous and U. Braun. CBS Biodivers. Ser. 1:1, 2003. (3) M. Groenewald et al. Phytopathology 95:951, 2005. (4) S. D. Lannes et al. Sci. Hortic. 112:266, 2007.

Microinjection of angiotensin II in the caudal ventrolateral medulla induces hyperalgesia.

Nociceptive transmission from the spinal cord is controlled by supraspinal pain modulating systems that include the caudal ventrolateral medulla (CVLM). The neuropeptide angiotensin II (Ang II) has multiple effects in the CNS and at the medulla oblongata. Here we evaluated the expression of angiotensin type 1 (AT(1)) receptors in spinally-projecting CVLM neurons, and tested the effect of direct application of exogenous Ang II in the CVLM on nociceptive behaviors. Although AT(1)-immunoreactive neurons occurred in the CVLM, only 3% of AT(1)-positive neurons were found to project to the dorsal horn, using double-immunodetection of the retrograde tracer cholera toxin subunit B. In behavioral studies, administration of Ang II (100 pmol) in the CVLM gave rise to hyperalgesia in both the tail-flick and formalin tests. This hyperalgesia was significantly attenuated by local administration of the AT(1) antagonist losartan. The present study demonstrates that Ang II can act on AT(1) receptors in the CVLM to modulate nociception. The effect on spinal nociceptive processing is likely indirect, since few AT(1)-expressing CVLM neurons were found to project to the spinal cord. The renin-angiotensin system may also play a role in other supraspinal areas implicated in pain modulation.