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.

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.

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.

Passive marijuana usage and water intoxication.

This article reports a case of a 3-month-old male presenting with hyponatremic seizure and hypothermia. The presence of marijuana in the infant's urine was remarkable. An etiologic role for the drug is questioned. A review of the literature is provided and the hypothesis of child neglect in the genesis of water intoxication is explored.