Rapid Molecular Identification of Scolytinae (Coleoptera: Curculionidae).

Routine identification of bark and ambrosia beetles is done using morphology. For people lacking the necessary taxonomic knowledge, proper identification of a novel specimen can be challenging and time consuming. This study compares the usefulness of four genetic markers (28S, EF-1a, ITS2, and COI) and five primer pairs (D2F1/D3R2, eflafor1/eflarev1, ets149/efa754, ITS2F/ITS2R, and LCO1490/HCO2198) to identify Scolytinae beetles, and outlines a molecular identification strategy, with results possible in two days. Markers COI and EF-1a were selected based on the ability of the respective primers to amplify DNA from multiple genera (Coptoborus, Xyleborus, Hypothenemus, Theoborus, and Araptus) and the ability of the resulting sequences to provide accurate and unambiguous matches in GenBank. BLASTn analysis of EF-1a sequences (both primer pairs) correctly identified four out of the five genera and COI sequences identified at least one sample of every genus tested and was the only primer pair to correctly identify Araptus specimens. Further, 28S sequences successfully identified Coptoborus, Xyleborus, and Theoborus but not Hypothenemus or Araptus. The low number of EF-1a (1), 28S (7), and ITS2 (0) sequences from Araptus individuals present in GenBank compared with COI (137) is likely the reason that only the latter marker was capable of identifying members of this genus. ITS2 sequences were insufficient to identify any of the samples tested. This study also determined the minimum quantity of DNA that could be used for molecular identification. Primers D2F1 and D3R2, which had the highest rate of amplification in all genera tested, could yield an informative sequence with as little as 0.00048 ng of DNA, however, at least 0.0024 ng was needed for reliable amplification.

Fungal Pathogens of Cacao in Puerto Rico.

Cacao production is a rapidly expanding industry in Puerto Rico, with new farmers planting ~20,000 trees in the past few years. To determine the etiology and extent of diseases affecting cacao in Puerto Rico, a survey was performed at eight sites around the island. Pod rot and/or branch dieback were observed at all sites. Most organisms isolated from symptomatic pod and stem samples were identified as Diaporthe spp. (48%) and Lasiodiplodia spp. (25%) based on sequences of the internal transcribed spacer and large subunit regions. Within these genera, Diaporthe tulliensis and Lasiodiplodia theobromae were the most prevalent species and were used in inoculation studies to determine their relative virulence on pods and stems. Phytophthora palmivora served as a positive control due to its well-established pathogenicity in all tissues. On pods, L. theobromae and P. palmivora caused significantly larger lesions (6.1 and 5.9 cm, respectively) than D. tulliensis (2.7 cm) four days post-inoculation. All three species caused disease on stems, with no differences found among species. Although P. palmivora was thought to be the primary pathogen affecting cacao in Puerto Rico, this study identifies L. theobromae and D. tulliensis as the common pathogens on the island. This improved understanding will help scientists and farmers control disease by selecting fungicides effective against both oomycetes and fungi.

Phytophthora theobromicola sp. nov.: A New Species Causing Black Pod Disease on Cacao in Brazil.

Black pod disease, caused by Phytophthora species, is among the main limiting factors of cacao (Theobroma cacao L.) production. High incidence levels of black pod disease have been reported in Brazil, being induced by Phytophthora capsici, Phytophthora citrophthora, Phytophthora heveae, and Phytophthora palmivora. To assess the diversity of Phytophthora species affecting cacao in Brazil, 40 new isolates were obtained from cacao pods exhibiting symptoms of black pod disease collected in different smallholder farms in 2017. Further, ten cacao-infecting isolates morphologically identified as P. citrophthora and P. palmivora were molecularly characterized. The genomic regions beta-tubulin, elongation factor 1 alpha, heat shock protein 90, and internal transcribed spacer, and the mitochondrially encoded cytochrome c oxidase I and II genes were PCR-amplified and Sanger-sequenced from the cacao-infecting Phytophthora isolates. The morphological characterization and evaluation of the mycelial growth rates for the Phytophthora isolates were performed in vitro. Based on the molecular analysis and morphological comparisons, 19 isolates were identified as P. palmivora (clade 4). Interestingly, 31 isolates grouped together in the phylogenetic tree and were placed apart from previously known species in Phytophthora clade 2. Therefore, these isolates are considered as a new species herein referred to as Phytophthora theobromicola sp. nov., which produced papillate, semipapillate, and persistent sporangia on simple sporangiophores. The P. palmivora isolates were identified as A1 mating type by pairing each isolate with known A1 and A2 tester strains of P. capsici, but no oogonia/antheridia were observed when P. theobromicola was paired with the different tester strains. The P. theobromicola and P. citrophthora isolates showed higher mycelial growth rates, when compared to P. palmivora, on different media at 10, 15, and 20°C, but similar values were observed when grown on clarified CA media at 25 and 30°C. The pathogenicity tests carried out on pods of four cacao clones (CCN51, PS1319, Cepec2004, and CP49) showed significant variability among the isolates of both Phytophthora species, with P. theobromicola inducing higher rates of necrotic lesion expansion, when compared to P. palmivora. Here, two Phytophthora species were found associated with black pod disease in the state of Bahia, Brazil, and the previously undescribed P. theobromicola seems to be prevalent in field conditions. This is the first report of P. theobromicola on T. cacao. Also, these findings are crucial to improve the disease control strategies, and for the development of cacao materials genetically resistant to Phytophthora.