RESUMO
Cylindrocladium buxicola (syn. C. pseudonaviculatum; teleomorph Calonectria pseudonaviculata) is an important fungal pathogen of Buxus spp. Although widespread in Western Europe, this pathogen has only recently been introduced into North America, where it represents a significant threat to the U.S. and Canadian boxwood industries. Trade of latently infected nursery stock is an important mode of long-distance dissemination and introduction of this pathogen but no methods for detection of latently infected material are available. Also, the pathways for short-distance dispersal of C. buxicola have not been adequately studied. Improved detection methods of this pathogen in air and water samples would benefit future research in this area. We have developed real-time polymerase chain reaction assays for the detection of C. buxicola based on the ribosomal DNA internal transcribed spacer 1 (ITS) and the ß-tubulin 2 gene (TUB). Using a TaqMan probe conjugated with a 3' minor groove binding group (TaqMan MGB probe), the ITS-based assay could reliably detect as little as 10 fg of genomic DNA or 20 copies of cloned target DNA and was approximately 70 times more sensitive than the SYBR Green TUB-based assay. The ITS-based assay provided good but not complete specificity, and is well suited for epidemiological studies. The TUB-based assay, however, proved to be fully specific and can be used for diagnostics. We developed and optimized sample processing and DNA extraction methods for detection of latently present C. buxicola in boxwood plants and quantification of conidia in water and air samples. C. buxicola could be detected in 20 g of plant material, of which only 1 ppm of the tissue was infected, in 10-ml water samples containing as low as 1 conidium/ml, and on Melinex tape pieces representing 12 h of air sampling containing 10 or more conidia. The applicability of the techniques to plant, water, and air samples of practical size was demonstrated.
RESUMO
Belgium is one of the most important Rhododendron-producing areas in Europe, with an annual sale of approximately 1.6 million plants. In June 2010, an outbreak of leaf spots on several thousands of Rhododendron cv. Marcel Menard plants took place at a nursery near Gent. Diseased plants showed dark brown leaf spots that enlarged and finally resulted in leaf drop. Symptoms developed most explicitly on this cultivar, especially after standard repotting during May or June and when repotting was followed by a few days of unusually warm temperatures (30 to 35°C). The leading edge of diseased leaf tissue was excised, surface disinfected with 1% NaOCl for 60 s, and rinsed twice with sterile distilled water before being plated onto potato dextrose agar (PDA). After 5 days of incubation at 21°C in the dark, Cylindrocladium-like fungal colonies with white aerial mycelium and amber-brown growth within the agar consistently developed. Mycelium was transferred aseptically to fresh plates of PDA and incubated for 10 to 14 days at 17°C under a 12-h fluorescent light regimen to study the morphological characteristics. Conidiophores showed a penicillate arrangement of fertile branches, producing two to six phialides. They arose from a stipe and terminated in a clavate vesicle (3 to 5 µm). Conidia were straight, cylindrical, rounded at both ends, three septate, and measured 60 to 70 × 4 to 6 µm. Yellow subglobose to oval perithecia were abundantly produced. Asci were clavate, four spored, and measured 100 to 150 × 15 to 30 µm. Ascospores were hyaline, three septate, and measured 50 to 65 × 5 to 6 µm. These characteristics are consistent with those of Calonectria colhounii Peerally (anamorph Cylindrocladium colhounii) (1). The ß-tubulin gene was PCR-amplified with DNA extracted from the mycelium and the T1 and T2 primers (3), sequenced directly with a BigDye Terminator Cycle Sequencing Kit (Applied Biosystems, Carlsbad, CA), and the DNA sequence was deposited (GenBank Accession No. JF802784). BLASTn alignment showed 99% identity (525 of 526 nucleotides) with the ß-tubulin DNA sequence derived from Calonectria colhounii CBS 293.79 (GenBank Accession No. DQ190564). A spore suspension (105 conidia per ml) was prepared from a 1-week-old culture, and 50-µl drops were used to inoculate the abaxial side of 10 detached 1-year-old leaves from Rhododendron cv. Cunningham's White. Ten control leaves were inoculated with water. The leaves were placed in a moist chamber and incubated at 21°C in the dark. After 5 to 6 days, all spore-inoculated leaves showed lesions identical to those on the naturally infected leaves, while the water-inoculated leaves remained symptom free. Following the original procedure, the fungus was reisolated from the diseased leaves and the morphological characteristics of the resulting culture were the same as those of the inoculated isolate, completing Koch's postulates. This fungus has been described on Rhododendron in the United States (2), but to our knowledge, this is the first record of Calonectria colhounii on Rhododendron in Belgium. References: (1) P. W. Crous. Taxonomy and Pathology of Cylindrocladium (Calonectria) and Allied Genera. The American Phytopathological Society, St. Paul, MN, 2002. (2) P. W. Crous et al. Stud. Mycol. 55:213, 2006. (3) K. O'Donnell and E. Cigelnik. Mol. Phylogenet. Evol. 7:103, 1997.
RESUMO
Macrolophus pygmaeus is a heteropteran predator that is widely used in European glasshouses for the biological control of whiteflies, aphids, thrips and spider mites. We have demonstrated that the insect is infected with the endosymbiotic bacterium Wolbachia pipientis. Several gene fragments of the endosymbiont were sequenced and subsequently used for phylogenetic analysis, revealing that it belongs to the Wolbachia supergroup B. The endosymbiont was visualized within the ovarioles using immunolocalization. Tetracycline treatments were used to cure M. pygmaeus from its infection. Although a completely cured line could not be obtained by this approach, the application of a constant antibiotic pressure over 13 generations resulted in a line with a significantly reduced Wolbachia concentration. Crosses performed with this tetracycline-treated line revealed that the endosymbiont causes severe cytoplasmic incompatibility. This is the first report of a reproductive effect induced by Wolbachia in an economically important heteropteran predator that may have vital implications for its commercial production and use in biological control.
