Assessment of the knowledge, attitudes and practices toward respiratory tract infections among the Trinidadian population

Respiratory Tract Infections (RTIs) are currently at the forefront of discussions as the world battles the COVID-19 pandemic. It is important that more awareness is raised on RTIs, their causes, the different types, how they are contracted and spread and complications of and risk factors for RTIs. Prevention measures towards RTIs should also be assessed and encouraged, such as proper hand washing, mask wearing, sneezing and coughing etiquette as well as vaccination. Therefore, this investigation was undertaken to assess the knowledge, attitudes, and practices towards Respiratory Tract Infections (RTIs) among Trinidadian population.

A rare report on 18-month survival of a dog born with multiple anomalies including atresia ani.

Long term survival of animals with major congenital anomalies is very rare. This report documents the 18-month survival of a dog with multiple anomalies including atresia ani. An 18-month-old black Cocker Spaniel bitch was presented for evaluation of prolapsed glands of the third eyelid involving both the eyes. Clinical examination revealed a single perineal opening, fecal matter in the vestibule, distended abdomen, hypoplastic vulva, and the absence of a tail without any neurological deficits. Abdominal contrast radiography revealed a distended colon with fecal stasis, rectovestibular fistula, termination of the rectum as a blind pouch, lumbar scoliosis due to block vertebrae, and the presence of only two hypoplastic coccygeal vertebrae. The case was diagnosed as atresia ani type II with rectovestibular fistula, hypoplastic vulva, lumbar scoliosis, and anury, in the global context of a caudal regression syndrome. The wide aperture fistula, connected to the vestibule, undamaged spinal cord and sacrum without any neurological deficits were the favorable prognostic factors that maintained continence and allowed the dog to survive to adult life with these anomalies. Thus, an appropriate bowel management program and specialty care can improve the quality of life and longevity of this animal.

Predictors of mortality in the elderly patient with a fracture of the odontoid process.

AIMS: Fracture of the odontoid process (OP) in the elderly is associated with mortality rates similar to those of hip fracture. The aim of this study was to identify variables that predict mortality in patients with a fracture of the OP, and to assess whether established hip fracture scoring systems such as the Nottingham Hip Fracture Score (NHFS) or Sernbo Score might also be used as predictors of mortality in these patients. PATIENTS AND METHODS: We conducted a retrospective review of patients aged 65 and over with an acute fracture of the OP from two hospitals. Data collected included demographics, medical history, residence, mobility status, admission blood tests, abbreviated mental test score, presence of other injuries, and head injury. All patients were treated in a semi-rigid cervical orthosis. Univariate and multivariate analysis were undertaken to identify predictors of mortality at 30 days and one year. A total of 82 patients were identified. There were 32 men and 50 women with a mean age of 83.7 years (67 to 100). RESULTS: Overall mortality was 14.6% at 30 days and 34.1% at one year. Univariate analysis revealed head injury and the NHFS to be significant predictors of mortality at 30 days and one year. Multivariate analysis showed that head injury is an independent predictor of mortality at 30 days and at one year. The NHFS was an independent predictor of mortality at one year. The presence of other spinal injuries was an independent predictor at 30 days. Following survival analysis, an NHFS score greater than 5 stratified patients into a significantly higher risk group at both 30 days and one year. CONCLUSION: The NHFS may be used to identify high-risk patients with a fracture of the OP. Head injury increases the risk of mortality in patients with a fracture of the OP. This may help to guide multidisciplinary management and to inform patients. This paper provides evidence to suggest that frailty rather than age alone may be important as a predictor of mortality in elderly patients with a fracture of the odontoid process. Cite this article: Bone Joint J 2019;101-B:253-259.

Synthetic and antimicrobial studies on natural cyclopolypeptide from Caribbean marine-sponge and its analogs

Objective: A natural cyclic peptide, rolloamide, previously isolated from marine-sponge, was synthesized by coupling of tri and tetrapeptide units Boc­Phe-Pro- Val­OMe and Boc­Pro-Leu-Pro-Ile­OMe after proper deprotection at carboxyl and amino terminals followed by cyclization of linear heptapeptide segment. Design and Methodology: Solution phase technique was adopted for the synthesis of cycloheptapeptide. Required tri and tetrapeptide units were prepared by coupling of Boc-protected dipeptides viz. Boc­Phe-Pro­OH and Boc­Pro-Leu­OH with respective amino acid methyl ester hydrochloride Val-OMe.HCl and dipeptide methyl ester Pro-Ile-OMe. Cyclization of linear heptapeptide unit was done by p-nitrophenyl ester method. Similarly, two analogs of rolloamide were prepared by modification of tripeptide unit. The structures of synthesized cyclopeptide and its analogs were elucidated by spectral and elemental analysis. The newly synthesized peptide was subjected to antimicrobial screening and compared with biopotential of analogs. Results: Synthesis of cyclopeptide was accomplished with >84% yield utilizing diisopropylcarbodiimide (DIPC) as coupling agent. Newly synthesized peptide possessed promising activity against C. albicans and P. aeruginosa, K. pneumonia as compared to standard drugs, in addition to moderate activity against dermatophytes. Synthesized peptide analogs showed better antimicrobial potential against C. albicans and dermatophytes. Conclusions: Solution phase technique employing N,Ndiisopropylcarbodiimide (DIPC) and triethylamine (TEA) proved to be effective for the synthesis of natural cycloheptapeptide. N-methyl morpholine (NMM) was found to be a better base for cyclization of linear heptapeptide unit in comparison to TEA and pyridine. Promising antimicrobial potential was seen for newly synthesized cyclic peptide and its analogs.

Correlates and prevalence of hypogonadism in patients with early- and late-onset type 2 diabetes.

This study aims to compare the prevalence of hypogonadism between male patients with early-onset type 2 diabetes mellitus (T2DM) and late-onset type 2 diabetes. A total of 122 male patients with early-onset T2DM (diagnosis age ≤40 years) and 100 male patients with late-onset T2DM (diagnosis age >40 years) were recruited from our in-patient department between 1 January 2013 and 28 December 2015. Serum FSH, LH, testosterone, lipid profile, uric acid, HbA1c, and beta-cell function were determined in blood samples. The diagnosis of hypogonadism was based on the levels of LH, FSH, and total testosterone. The mean onset age was 29.86 ± 6.31 and 54.47 ± 9.97 years old in the early-onset group and late-onset group, respectively. Compared with late-onset T2DM, those with early-onset T2DM had a higher proportion of new-onset diabetes, were more likely to be obese, and had worse glycemic control, lipid control, and lower sex hormone-binding globulin (SHBG). The prevalence of hypogonadism was much higher in the early-onset group than in the late-onset group (48.0% vs. 26.7%, p < 0.05). The rate of secondary hypogonadism in the early-onset group and late-onset group were 44.3% and 25.0%, respectively (p < 0.05). Obesity, waist circumference, and SHBG were significantly associated with serum total testosterone level in all, early-onset, and late-onset T2DM. Both all and early-onset T2DM groups had positive correlations between total testosterone and fasting C-peptide, total cholesterol, triglycerides, and uric acid. Our results indicate that in a population of admission to a large urban hospital in China, the prevalence of hypogonadism was higher in the patients with early-onset T2DM than that of late-onset T2DM. This prevalence might be attributable to greater obesity, worse lipid control, and lower SHBG levels in those patients.

Phylogeny and Haplotype Analysis of Fungi Within the Fusarium incarnatum-equiseti Species Complex.

Fusarium spp. are ranked among the top 10 most economically and scientifically important plant-pathogenic fungi in the world and are associated with plant diseases that include fruit decay of a number of crops. Fusarium isolates infecting bell pepper in Trinidad were identified based on sequence comparisons of the translation elongation factor gene (EF-1a) with sequences of Fusarium incarnatum-equiseti species complex (FIESC) verified in the FUSARIUM-ID database. Eighty-two isolates were identified as belonging to one of four phylogenetic species within the subclades FIESC-1, FIESC-15, FIESC-16, and FIESC-26, with the majority of isolates belonging to FIESC-15. A comparison of the level of DNA polymorphism and phylogenetic inference for sequences of the internal transcribed spacer region (ITS1-5.8S-ITS2) and EF-1a sequences for Trinidad and FUSARIUM-ID type species was carried out. The ITS sequences were less informative, had lower haplotype diversity and restricted haplotype distribution, and resulted in poor resolution and taxa placement in the consensus maximum-likelihood tree. EF-1a sequences enabled strongly supported phylogenetic inference with highly resolved branching patterns of the 30 phylogenetic species within the FIESC and placement of representative Trinidad isolates. Therefore, global phylogeny was inferred from EF-1a sequences representing 11 countries, and separation into distinct Incarnatum and Equiseti clades was again evident. In total, 42 haplotypes were identified: 12 were shared and the remaining were unique haplotypes. The most diverse haplotype was represented by sequences from China, Indonesia, Malaysia, and Trinidad and consisted exclusively of F. incarnatum isolates. Spain had the highest haplotype diversity, perhaps because both F. equiseti and F. incarnatum sequences were represented; followed by the United States, which contributed both F. equiseti and F. incarnatum sequences to the data set; then by countries representing Southeast Asia (China, Indonesia, Malaysia, Thailand, and Philippines) and Trinidad; both of these regions were represented by only F. incarnatum sequences. Trinidad shared two haplotypes with China and one haplotype with the United States for only F. incarnatum isolates. The findings of this study are important for devising disease management strategies and for understanding the phylogenetic relationships among members of the FIESC.

Metabolic Characteristics in Obese Patients Complicated by Mild Thyroid Hormone Deficiency.

The prevalence of subclinical hypothyroidism (SH) is increasing, especially in obese people. The purpose of this study was to evaluate the difference in metabolic profiles between obese patients with mild increased thyrotropin (TSH) or with normal TSH. A total of 219 obese patients were recruited in this cross-sectional study. They were divided into 2 groups: obese patients with normal TSH (0.35-2.5 mU/l) and age-, and body mass index (BMI)-matched obese patients with higher-normal TSH (2.5-5.5 mU/l). We have named it compensatory hypothyroidism or mild thyroid hormone deficiency. Anthropometric data, glucose-lipid metabolism, markers of inflammation, body composition, and thyroid function parameters were measured. Results showed that: 1) The levels of fasting plasma glucose (FPG), high density lipoprotein cholesterol (HDL-C), and 25-hydroxyvitamin D levels were significantly lower in obese patients complicated by mild increased TSH than in obese patients with normal TSH (p<0.05). The fasting insulin (FINS) and C reactive protein (CRP) levels were significantly higher in obese patients complicated by mild increased TSH when compared to the obese patients with normal TSH (p<0.01). Jostel's TSH index (TSHI) and standard TSH index (sTSHI) were significantly higher in obesity with mild increased TSH when compared to obesity with normal TSH (both p<0.001). Thyroid's secretory capacity (GT) was significantly lower in obesity with mild increased TSH when compared to obesity with normal TSH (p<0.001). 2) In the obese patients complicated by mild increased TSH group, serum TSH was significantly positively correlated with ALT, AST and CP (p<0.05). In the obese patients with normal TSH group, serum TSH was significantly positively correlated with UA (p<0.05). In all subjects, serum TSH was significantly positively correlated with ALT, AST, FINS, CP, CRP, and UA (p<0.05), but negatively with DBP, FPG, and HDL-C (p<0.05). 3) There was significant difference of the prevalence of hyperinsulinemia and impaired fasting glucose (IFG) between the 2 groups (p<0.05). In Conclusion, obesity complicated by mild increased TSH manifested higher fasting insulin levels, more severe chronic low-grade inflammation, and lower HDL-C levels than obesity with normal TSH.

Detection and Molecular Characterization of Benzimidazole Resistance Among Colletotrichum truncatum Isolates Infecting Bell Pepper in Trinidad.

Anthracnose is an economically important disease that affects pepper (Capsicum spp.) production worldwide. Eighty-seven Colletotrichum truncatum isolates infecting bell pepper in Trinidad were isolated and screened for resistance to benomyl. All isolates were found to be highly resistant at the discriminatory dose of 10.0 µg/ml. The effective concentration required to achieve 50% colony growth inhibition values were found to be significantly higher (P ≤ 0.05) for isolates collected in South Trinidad compared with those collected in North Trinidad. Isolates with the resistant phenotype had a single amino acid substitution from glutamic acid to alanine at position 198 (E198A) within the ß-tubulin 2 gene. Single-nucleotide polymorphisms that result in amino acid substitutions in the ß-tubulin 2 protein are associated with high resistance to benzimidazole chemistries. There were also two other deduced amino acid changes at nucleotide positions 359 to 361 (ATA/TTG [F270Y]) and at 362 to 364 (CGC/GCC [A271S]). Genetic analysis revealed that benomyl-sensitive isolates clustered separately from the resistant isolates regardless of species, with strong bootstrap support (85%). Within the resistance cluster, there was an apparent differentiation among those isolates with the F200Y, E198A, and E198K genotypes, with moderate support (>60%) for clustering of the F200Y and E198K genotypes. C. truncatum clustered separately (97%) from the other resistant genotypes due to the additional amino acid substitutions detected. The findings also indicated that consistent benzimidazole fungicide use may explain the predominance of the C. truncatum pathogen populations in bell pepper fields in Trinidad because sensitive C. gloeosporioides sensu lato isolates were selectively minimized. This underlines the importance of accurate identification of Colletotrichum spp. associated with anthracnose disease and routine monitoring for development of fungicide resistance.

Investigation of tDCS volume conduction effects in a highly realistic head model.

OBJECTIVE: We investigate volume conduction effects in transcranial direct current stimulation (tDCS) and present a guideline for efficient and yet accurate volume conductor modeling in tDCS using our newly-developed finite element (FE) approach. APPROACH: We developed a new, accurate and fast isoparametric FE approach for high-resolution geometry-adapted hexahedral meshes and tissue anisotropy. To attain a deeper insight into tDCS, we performed computer simulations, starting with a homogenized three-compartment head model and extending this step by step to a six-compartment anisotropic model. MAIN RESULTS: We are able to demonstrate important tDCS effects. First, we find channeling effects of the skin, the skull spongiosa and the cerebrospinal fluid compartments. Second, current vectors tend to be oriented towards the closest higher conducting region. Third, anisotropic WM conductivity causes current flow in directions more parallel to the WM fiber tracts. Fourth, the highest cortical current magnitudes are not only found close to the stimulation sites. Fifth, the median brain current density decreases with increasing distance from the electrodes. SIGNIFICANCE: Our results allow us to formulate a guideline for volume conductor modeling in tDCS. We recommend to accurately model the major tissues between the stimulating electrodes and the target areas, while for efficient yet accurate modeling, an exact representation of other tissues is less important. Because for the low-frequency regime in electrophysiology the quasi-static approach is justified, our results should also be valid for at least low-frequency (e.g., below 100 Hz) transcranial alternating current stimulation.

The influence of sulcus width on simulated electric fields induced by transcranial magnetic stimulation.

Volume conduction models can help in acquiring knowledge about the distribution of the electric field induced by transcranial magnetic stimulation. One aspect of a detailed model is an accurate description of the cortical surface geometry. Since its estimation is difficult, it is important to know how accurate the geometry has to be represented. Previous studies only looked at the differences caused by neglecting the complete boundary between cerebrospinal fluid (CSF) and grey matter (Thielscher et al 2011 NeuroImage 54 234-43, Bijsterbosch et al 2012 Med. Biol. Eng. Comput. 50 671-81), or by resizing the whole brain (Wagner et al 2008 Exp. Brain Res. 186 539-50). However, due to the high conductive properties of the CSF, it can be expected that alterations in sulcus width can already have a significant effect on the distribution of the electric field. To answer this question, the sulcus width of a highly realistic head model, based on T1-, T2- and diffusion-weighted magnetic resonance images, was altered systematically. This study shows that alterations in the sulcus width do not cause large differences in the majority of the electric field values. However, considerable overestimation of sulcus width produces an overestimation of the calculated field strength, also at locations distant from the target location.

First Report of Fusarium proliferatum Infecting Pimento Chili Peppers in Trinidad.

In Trinidad, pimento chili peppers (Capsicum annuum L.) are grown for large domestic and regional export markets. Production is intensive during the rainy season (June to December). In August 2010, pimento fruits with symptoms of fruit rot were collected from fields located in Tableland, Valencia, Aranguez-North and -South, and Macoya. Symptoms began as a discoloration and soft rot of the peduncle and calyx (green to brown then black); a tan, watery lesion (with irregular margins) developed and expanded rapidly from the calyx down the sides of the fruit with internal rot of the placenta. Excessive fruit drop was also common. Estimated yield loss was ~20 to 60% for each field. Symptoms were observed on green and red fruits. Fruits were surface disinfected (2 min in 70% ethanol, 2 min in 0.5% NaOCl, followed by three rinses with sterile distilled water) and then a 4-mm3 block of tissue was taken from the lesion edge and placed on water agar. After 7 days at 25 ± 1°C, a 4-mm3 block of agar that contained the advancing hyphal edge of each colony was transferred to selective fusarium agar (3) and incubated as previously described. Colonies were fast growing with white, fluffy, aerial mycelia; hyphae densely branched; polyphialides abundant; microconidia abundant, thin walled, hyaline, ovoid, aseptate or 1-celled, and 5.5 to 12.2 × 2.0 to 3.2 µm. Macroconidia were moderately curved to straight, hyaline, 3- to 4-celled, thick walled, and 20.5 to 35.0 × 3.5 to 5.0 µm. Molecular characterization was based on a two-loci approach. PCR amplification was carried out with universal primers (ITS4/5) and translation elongation factor primers (EF1/2) (2). Sequences of the ITS1-5.8S-ITS2 region of rDNA (GenBank Accession No. HQ333547) and partial EF-1α gene (GenBank Accession No. HQ333548) were compared to cognate sequences available in GenBank and the FUSARIUM-ID databases (2). Comparisons revealed 100% similarity to Fusarium proliferatum (Matsush.) Nirenberg ex Gerlach & Nirenberg 1982. F. proliferatum (synonym Gibberella intermedia) is the anamorphic form of the G. fujikuroi complex that belongs to the Nectriaceae family (4). Pathogenicity tests were conducted by dispensing 10 µl of a prepared spore suspension (106 spores/ml) onto nonwounded and wounded sites of pimento fruits (landrace 'Trinidad seasoning', 10 fruits per isolate, 8 isolates). Negative controls were fruits inoculated with sterile distilled water. Inoculated fruits were kept at 25 ± 1°C in partially sealed plastic containers and monitored for the onset of symptoms for 7 days. The test was conducted twice. Lesions, similar to those recorded on field infected fruit, developed on inoculated fruits that were wounded and nonwounded, but not on water controls. The pathogen was reisolated from infected tissues, thereby fulfilling Koch's postulates. F. proliferatum is associated with disease of a number of economically important crops and ornamental plants worldwide (1). Fusarium fruit rot of pepper has been shown to significantly reduce marketable yield and shelf life of infected fruits. To our knowledge, this is the first report of Fusarium fruit rot of pimento chili peppers caused by F. proliferatum in Trinidad. References: (1) J. Armengol et al. Eur. J. Plant Pathol. 112:123, 2005. (2) D. M. Geiser et al. Eur. J. Plant Pathol. 110:473, 2004. (3) J. Leslie and B. Summerell. Page 1 in: The Fusarium Laboratory Manual. Blackwell Publishing, Oxford, UK, 2006. (4) H. Nirenberg and K. O'Donnell. Mycologia 90:434, 1998.

First Report of Anthracnose Caused by Colletotrichum gloeosporioides in Pumpkin in Trinidad.

In Trinidad, pumpkin (Cucurbita pepo L. and C. moschata L.) is extensively grown for local and international export markets. In November 2008, symptoms of foliar chlorosis and necrosis were observed in 15 commercial pumpkin fields located in the main production areas of St. George East, Caroni, Victoria, and St. Patrick counties. Severely infected plants were unable to support fruit maturation, which resulted in yield loss. The pathogen was isolated from surface-sterilized tissues of symptomatic plants. Colonies on potato dextrose agar (PDA) were white to cream with gray spore masses in the center. Conidia were hyaline, cylindrical with rounded ends, aseptate, and measured 12.5 to 16.5 µm × 3.5 to 5.0 µm. PCR amplification was carried out with ITS4/5 universal primers (4) and species-specific primers, CgInt/ITS4 (1), using a positive control of Colletotrichum gloeosporioides (courtesy of D. Perez-Brito). Species-specific primers generated a single amplicon, ~450 bp long, which corresponded with the positive control. The ITS1 region (1) of pumpkin isolates (GenBank No. GU320190) was 100% identical to cognate sequences of C. gloeosporioides isolates (GenBank Nos. AY841136 and FJ624257). Phylogenetic analyses (MEGA 4 - Molecular Evolutionary Genetic Analysis Software version 4 for Windows) using the neighbor-joining (NJ) algorithm placed the pumpkin isolates in a well-supported cluster (>90% bootstrap value based on 1,000 replicates) with other C. gloeosporioides isolates. The tree was rooted with C. crassipes (GenBank No. AJ536230). The pathogen was similar to C. gloeosporioides (Penz.) Penz. & Sacc. (3). In pathogenicity tests, six plants (cv. Jamaican squash) for each of five isolates were spray inoculated to runoff with a conidial suspension (1.0 × 106 conidia/ml). Negative controls were sprayed with sterile distilled water. In repeated tests, plants were symptomatic of infection 7 days postinoculation. There were no symptoms on control plants. Koch's postulates were fulfilled with the reisolation of the pathogen from symptomatic leaf tissues. Anthracnose is a serious threat to cucurbit production; however, infection is not common in pumpkin and squash (2). To my knowledge, this is the first report of C. gloeosporioides causing widespread anthracnose infection in pumpkin in Trinidad. References: (1) A. E. Brown et al. Phytopathology 86:523, 1996. (2) G. Kelly. Acta Hortic. (ISHS) 731:479, 2007. (3) B. C. Sutton. Page 1 in: Colletotrichum: Biology, Pathology and Control. CAB International. Wallingford, UK, 1992. (4) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, 1990.

First Report of Fusarium solani Causing Fruit Rot of Sweet Pepper in Trinidad.

In Trinidad, sweet pepper (Capsicum annuum L.) is an important crop that is produced for local markets and regional export. From February to April 2010, severe fruit rot was observed in 9 of 11 commercial fields located in North Trinidad in the major production areas of North and South Aranguez. All fields were in the late harvesting stage and the most commonly grown cultivars were Aristotle and Canape. Disease incidence for each field was estimated to be 80% with a yield loss of 40 to 60%. Symptoms appeared on mature red fruits but growers reported that disease can also occur on green fruit. Symptoms began as soft lesions that turned dark brown to black. Lesions usually originated at the calyx end of the fruit and extended down the sides. Fruits were surface sterilized by rinsing with 70% ethanol for 2 min, followed by three rinses with sterile distilled water. Two 4-mm3 blocks of tissue from the opposite sides of fruit lesions were transferred to water agar and incubated for 5 to 7 days at 25 ± 1°C. A 4-mm3 agar block consisting of the leading mycelial edge was then transferred to potato dextrose agar (PDA) and incubated under the same conditions. Colonies on PDA were fast growing with white, fluffy, aerial mycelia; hyphae were septate and hyaline; conidiophores were unbranched; microconidia were abundant, thin walled, hyaline, ovoid, one to two celled, and measured 6 to 10 × 2 to 4 µm. Macroconidia were hyaline, three to four celled, curved, thick walled, and measured 20 to 30 × 4 to 6 µm. PCR amplification was carried out utilizing universal primers ITS4/5 and translation elongation factor primers EF1/2 (2). Sequence comparisons of the internal transcribed spacer (ITS) region (HM157262) and EF-1α gene (HQ014854) with cognate sequences available in GenBank and the FUSARIUM-ID databases revealed 100 and 99.6% sequence identity, respectively, to Fusarium solani (Mart.) Sacc. Pathogenicity tests were conducted by drop inoculating 10-µl of spore suspension (106 spores/ml) of each of four isolates on wounded and unwounded sites of mature sweet pepper fruits (five per isolate of cvs. Aristotle, Canape, Century, Destra, and Paladin). Control fruits were inoculated with sterile distilled water. Inoculated fruits were kept at 25 ± 1°C in loosely sealed plastic containers and monitored for the onset of symptoms for 6 days. The experiment was conducted twice. Lesions (8.0 to 15.2 mm in diameter) developed on wounded fruit of Aristotle, Canape, and Century. No symptoms were seen on Destra, Paladin, or the water controls. No symptoms developed on nonwounded fruits. Koch's postulates were fulfilled by reisolating the pathogen from infected tissues. Fruit rot caused by F. solani has been reported to be a serious constraint to sweet pepper production in Canada (4), the United Kingdom (1), and New Zealand (3). To our knowledge, this is the first report of Fusarium fruit rot of sweet pepper in Trinidad. References: (1) J. T. Fletcher. Plant Pathol. 43:225, 1994. (2) D. M. Geiser et al. Eur. J. Plant Pathol. 110:473, 2004. (3) J. L. Tyson. Aust. Plant Pathol. 30:375, 2001. (4) R. Utkhede and S. Mathur. Plant Dis. 87:100, 2003.

First Report of Fusarium solani Fruit Rot of Pumpkin (Cucurbita pepo) in Trinidad.

Trinidad is a major exporter of pumpkins (Cucurbita pepo L.) to other Caribbean countries, Canada, and the United States. Producers and exporters have reported 50 to 80% yield losses because of soft rot and overnight collapse of fruit at the pre- and postharvest stages. Severe fruit rot occurred in fields in Victoria County in South Trinidad between April and May 2006 (mid-to-late dry season) with an increase in the severity and number of affected fruit in the rainy season (July to December). Symptoms began as water-soaked lesions on the fruit of any age at the point of contact with the soil. The disease progressed to a soft rot with leakage and whole fruit collapse. A dark brown, soft decay also developed at the base of the main vines. Fusarium solani was isolated on selective fusarium agar and potato dextrose agar (PDA) (1) after 7 to 10 days of incubation at 25°C. The pathogen was identified by morphological characteristics and pathogenicity tests. Colonies were fast growing with white aerial mycelia and a cream color on the reverse side; hyphae were septate and hyaline, conidiophores were unbranched, and microconidia were abundant, thin walled, hyaline, fusiform to ovoid, generally one to two celled, and 8 to 10 × 2 to 4 µm. Macroconidia were hyaline, two to three celled, moderately curved, thick walled, and 25 to 30 × 4 to 6 µm. Pathogenicity tests for 10 isolates were conducted on 2-week-old pumpkin seedlings (cv. Jamaican squash; seven plants per isolate) and mature pumpkin fruit (2). Briefly, seedlings were inoculated by dipping their roots in a spore suspension (1 × 104 spores per ml) for 20 min. The plants were repotted in sterile potting soil. For negative controls, plant roots were dipped in sterile water. After the rind of fruit was swabbed with 70% ethanol followed by three rinses with sterile distilled water, 0.4-cm-diameter agar plugs of the isolates were inserted into wounds made with a sterile 1-cm-diameter borer. Sterile PDA plugs served as negative controls. Fruit were placed in sealed, clear, plastic bags. Inoculated plants and fruit were placed on greenhouse benches (30 to 32°C day and 25 to 27°C night temperatures) and monitored over a 30-day period. Tests were repeated once. Inoculated fruit developed a brown, spongy lesion that expanded from the initial wound site over a period of approximately 17 days after inoculation. White mycelia grew diffusely over the lesion. Inoculated plants developed yellow and finally necrotic leaves and lesions developed on stems at the soil line approximately 21 days after inoculation. No symptoms developed on the control plants. The fungus was reisolated from symptomatic tissue, fulfilling Koch's postulates. To my knowledge, this is the first report of Fusarium fruit rot of pumpkin in Trinidad. References: (1) J. Leslie and B. Summerell. Page 1 in: The Fusarium Laboratory Manual. Blackwell Publishing, Oxford, 2006. (2) W. H. Elmer. Plant Dis. 80:131, 1996.

First Report of Verticillium dahliae Causing Wilt in Pumpkin in Trinidad.

Pumpkin (Cucurbita pepo L.) is an economically important crop in Trinidad. Production supplies local and export markets in Barbados, Antigua and Barbuda, Canada, the United States, and the United Kingdom. However, there has been a marked decline in pumpkin export over the last 3 years. Field infections and development of disease in transit have been identified as major factors in decreased export. Between November 2006 and December 2007 (day/night temperatures of 32 to 34°C and 25 to 27°C, respectively), symptoms of vascular wilt were observed in at least 17 pumpkin fields. Symptoms included discoloration (yellowing with subsequent browning) of the older leaves and stems followed by wilting, girdling, and dieback of vines prior to fruit maturity. Necrotic leaves remained attached to the stems. A light brown vascular discoloration in the absence of a sticky exudate was observed in the stems. Onset of symptoms was most apparent at the late flowering to early fruit development stage. All C. pepo lines appeared equally affected by the disease. Yield losses (reduction of fresh weight and proportion of immature fruits at harvest time) as a result of this disease were estimated at 30 to 80%. Accurate diagnosis and pathogen identification were based on symptomology, colony morphology on potato dextrose agar (PDA) and semiselective media (including presence of microsclerotia), pathogenicity tests, and molecular characterization of the rDNA region. Pathogenicity tests with five isolates (randomly selected but geographically distinct) were conducted on healthy pumpkin plants at the two-true-leaf stage. A spore suspension of 2.1 × 106 conidia/ml was used to inoculate eight seedlings grown in sterile potting mix (1). Plants inoculated with sterile distilled water served as negative controls. Plants inoculated with the spore suspension developed a vascular wilt and general decline 35 days postinoculation. The pathogen was reisolated from all inoculated pumpkin seedlings. Pathogenicity tests were repeated once. Colony morphology consistently conformed to established criteria for Verticillium dahliae (Kleb.) (2). PCR amplification with universal primers ITS4 and ITS5 (3) was carried out on fungal DNA extracted from pure cultures. Sequence comparison of amplicons of approximately 600 bp long (GenBank Accession No. EF377335) was carried out using MEGABLAST search ( www.ncbi.nlm.nih.gov/blast/ ) for highly similar sequences and Lasergene v7.2 (DNASTAR Inc. Madison, WI) software. Alignment data revealed the highest and most significant homology to Verticillium dahliae (GenBank Accession No. DQ282123) at 98.2%. V. dahliae has a wide host range and causes vascular wilt in a large number of economically important crops (2). Control strategies are complicated by the ability of the microsclerotia produced by V. dahliae to survive for more than a decade in soil despite the absence of a host (4). To my knowledge, this is the first report of Verticillium wilt affecting pumpkin in Trinidad. References: (1) S. T. Koike et al. Plant Dis. 78:1116, 1994. (2) G. F. Pegg. Phytopathol. Mediterr. 23:176, 1984. (3) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, 1990. (4) S. Wilhelm. Phytopathology. 45:180, 1955.

Detection of begomoviruses in clarified plant extracts: a comparison of standard, direct-binding, and immunocapture polymerase chain reaction techniques.

ABSTRACT Three polymerase chain reaction (PCR) techniques-standard PCR (Std-PCR), direct-binding PCR (DB-PCR), and immunocapture PCR (IC-PCR)-using degenerate primers were optimized and evaluated for the detection of begomoviruses. Tomato leaf samples were ground in three different extraction buffers and subjected to Std-PCR. The effect of the buffers on the detection limits of amplification of the virus (detection of the initial and end points of dilution) was determined. With the optimal extraction buffer determined by the first experiment, the antibody concentration and incubation conditions for IC-PCR were evaluated to determine the requirements for maximum capture of antigens during the capture phase of the technique. The incubation conditions of DB-PCR were also investigated to determine the most favorable conditions for adsorption of the viral template. The reproducibility of all assays was evaluated. With the results of the optimization experiments, the applicability of the three techniques to different plant species was assessed. Extracts of plant species belonging to three families were subjected to the optimized Std-, DB-, and IC-PCR protocols. Std- and IC-PCR both achieved reproducible detection of begomoviruses, but the detection limits and amplified band intensity for all plant species tested were superior for the latter. DB-PCR was an unreliable method of detection, because of poor reproducibility and low intensity of amplified bands. These results indicate that the optimized IC-PCR detection system using degenerate primers is the most effective for the detection of begomoviruses in clarified plant extracts.

Identification of Resistance to Potato yellow mosaic virus-Trinidad Isolate (PYMV-TT) Among Lycopersicon Species.

Three studies were carried out with the objective of identifying resistance to Potato yellow mosaic virus-Trinidad isolate (PYMV-TT) among Lycopersicon species through field screening and using field-inoculated infector rows. The presence of PYMV-TT was confirmed using dot blot hybridization assays. In the first study, eight commercial cultivars of tomato were tested for resistance. In a subsequent study, 11 breeding lines showing high levels of resistance to Tomato mottle virus (ToMoV-Florida) and six lines resistant to Tomato yellow leaf curl virus (TYLCV-Old World Begomovirus) were screened for resistance to PYMV-TT. All breeding lines and the commercial cultivars tested were susceptible to PYMV-TT infection. The third study involved screening 92 accessions from a representative core collection of wild Lycopersicon germ plasm. PYMV-TT was not detected in individuals of 22 accessions.

Detection of Two Bipartite Geminiviruses Infecting Dicotyledonous Weeds in Trinidad.

Severe symptoms of suspected geminivirus etiology were manifested as intense yellow or golden mottling or mosaic of the lamina accompanied by mild leaf margin distortion on dicotyledonous weed species, Sida rhombifolia (L.) and Rhynchosia minima (L.), collected from 1999 to 2002 from the northeastern and central regions of Trinidad. S. rhombifolia is a common roadside weed while R. minima may have been introduced through restricted cultivation as a forage legume for livestock. Potato yellow mosaic virus-Trinidad isolate (PYMV-TT) has been implicated as the primary causal agent of begomoviral disease in large-scale tomato cultivation in Trinidad (2). It has been suggested that these weeds may be alternative hosts to PYMV-TT. However, all samples tested negative for PYMV-TT in dot blot hybridization assays using a PYMV-TT-specific DNA-A probe under high stringency. These results excluded the presence of PYMV-TT in these weeds. Polymerase chain reaction (PCR) amplification using clarified leaf extracts with degenerate primers for DNA-A (MP16 and MP82, PAL1v1978 and PAR1c715, and prV324 and prC889) and for DNA-B (PBL1v2040 and PCRc1) was performed on the weed samples (S. N. Rampersad and P. Umaharan, unpublished). Degenerate primers MP16 and MP82 target the 5' terminal region of the coat protein (cp) (2); PAL1v1978 and PAR1c715 direct amplification of the replication-associated protein gene (rep) and part of the cp gene (1); prV324 and prC889 amplify the core cp sequence (3). Primers PBL1v2040 and PCRc1 target the intergenic region and the 5' terminal of the BL1 ORF (1). PCR fragments obtained through amplification using this primer pair confirmed the presence of a DNA-B component for the unknown viruses. PCR fragments were sequenced and alignments were performed using DNASTAR (DNASTAR Inc., Madison, WI) and BLASTN ( www.ncbi.nlm.nih.gov/blast/ ) programs. None of the partial nucleotide sequences obtained for the viruses produced significant alignments with each other (5' terminal cp: 74% identity; core cp sequence: 78% identity), suggesting the detection of two distinct viruses. In addition, the partial sequences obtained were aligned to sequences of homologous regions of 11 New World begomoviruses (from the major representative clusters). The nearest match for R. minima, using alignments with 5' terminal cp (GenBank Accession No. AY221124), core cp (GenBank Accession No. AY217344), and 5' terminal BL1 region (GenBank Accession No. AY220490) was obtained for Rhynchosia golden mosaic virus (RhGMV, GenBank Accession Nos. AF408199 and AF442117) with 84 and 88% identity. There were no significant similarities found for sequence comparisons of the BL1 ORF. For S. rhombifolia, the highest homology using the 5' terminal cp (GenBank Accession No. AY220489), core cp (GenBank Accession No. AY217345), rep/cp region (GenBank Accession No. AY220488), and the 5' terminal BL1 region (GenBank Accession No. AY221125) was obtained for Sida golden mosaic virus (SiGMV, GenBank Accession Nos. AF049336, AF070923, and Y11100), with 82, 89, 84, and 87% identity. To our knowledge, this is the first report of geminivirus infection in these weed species in Trinidad. This may have substantial implications to future geminivirus disease outbreaks especially if there is expansion of the host range of these viruses to include economically important crops. References: (1) M. R. Rojas et al. Plant Dis. 77:340, 1993. (2) P. Umaharan et al. Phytopathology 88:1262, 1998. (3) S. D. Wyatt and J. K. Brown. Phytopathology 86:1288, 1996.

Genomic organization, sequence and transcriptional regulation of the human CXCL 11(1) gene.

CXCL 11, encoded by the cDNA sequences designated beta-R1, H-174, or I-TAC, is a CXC chemokine ligand for CXCR3 and assumed to be involved in inflammatory diseases characterized by the presence of activated T-cells. We here describe the genomic organization (four exons interrupted by three introns of 585, 98 and 230 bp) and sequence including 960 bp from the immediate 5'-upstream region of the human CXCL 11 gene. Within the promoter region, consensus sequences for regulatory elements (ISRE, GAS, NF-kappaB) important for cytokine-induced gene transcription were identified. The effect of (pro)inflammatory cytokines on CXCL 11 mRNA expression in monocytic cell lines (THP-1, U937) and primary cultures of dermal fibroblasts and endothelial cells were examined using Northern blot analysis. For these cell types, IFN-gamma was a potent inducer of CXCL 11 transcription, which was synergistically enhanced by TNF-alpha.