Molecular detection of Phytophthora pluvialis, the causal agent of red needle cast in Pinus radiata.

BACKGROUND: Phytophthora pluvialis was first described in 2013 and is the causal agent of red needle cast (RNC) in Pinus radiata as well as infection in Douglas fir (Pseudotsuga menziesii). A species-specific PCR is necessary for detection of this pathogen and diagnosis of RNC. OBJECTIVE: To design and validate a species-specific molecular assay for P. pluvialis using isolates from infected pine needles. METHODS: Species-specific PCR primers were generated from the ras-related GTP-binding protein 1 gene (ypt1) gene sequence, concentrating on DNA regions unique to P. pluvialis, and real-time and quantitative polymerase chain reaction (qPCR) were used to detect P. pluvialis from both artificially inoculated and naturally infected samples. RESULTS: The species-specific PCR assay was generated following P. pluvialis DNA sequence analysis. In vitro tests of the specificity of the probe-based, quantitative, polymerase chain reaction (qPCR) assay showed that no amplification was observed with other Phytophthora species including other closely-related clade 3 species, or with fungal species associated with pine or with pine DNA. The limit of detection of the qPCR assay was 2 pg/µl. When the qPCR assay was used to detect P. pluvialis in artificially-inoculated and naturally infected P. radiata needles, a PCR product was detected in all inoculated samples; the mean concentration ranges of P. pluvialis DNA in the inoculated and naturally infected samples tested were 5.9-124.5 pg/µl and 8.1-340.2 pg/µl, respectively. The assays described herein were used with serological diagnostic strips, providing the ability to identify to species level. CONCLUSIONS: The assay described herein detects P. pluvialis with high specificity and sensitivity from a range of DNA samples, including those extracted from infected plant material and serological diagnostic strips. The ability to detect and identify P. pluvialis, from infected tissues directly, provides value and practicality to diagnostics, biosecurity and research.

Genome sequences of six Phytophthora species associated with forests in New Zealand.

In New Zealand there has been a long association of Phytophthora diseases in forests, nurseries, remnant plantings and horticultural crops. However, new Phytophthora diseases of trees have recently emerged. Genome sequencing has been performed for 12 Phytophthora isolates, from six species: Phytophthora pluvialis, Phytophthora kernoviae, Phytophthora cinnamomi, Phytophthora agathidicida, Phytophthora multivora and Phytophthora taxon Totara. These sequences will enable comparative analyses to identify potential virulence strategies and ultimately facilitate better control strategies. This Whole Genome Shotgun data have been deposited in DDBJ/ENA/GenBank under the accession numbers LGTT00000000, LGTU00000000, JPWV00000000, JPWU00000000, LGSK00000000, LGSJ00000000, LGTR00000000, LGTS00000000, LGSM00000000, LGSL00000000, LGSO00000000, and LGSN00000000.

Real-Time PCR Assays for the Detection of Puccinia psidii.

Puccinia psidii (Myrtle rust) is an emerging pathogen that has a wide host range in the Myrtaceae family; it continues to show an increase in geographic range and is considered to be a significant threat to Myrtaceae plants worldwide. In this study, we describe the development and validation of three novel real-time polymerase reaction (qPCR) assays using ribosomal DNA and ß-tubulin gene sequences to detect P. psidii. All qPCR assays were able to detect P. psidii DNA extracted from urediniospores and from infected plants, including asymptomatic leaf tissues. Depending on the gene target, qPCR was able to detect down to 0.011 pg of P. psidii DNA. The most optimum qPCR assay was shown to be highly specific, repeatable, and reproducible following testing using different qPCR reagents and real-time PCR platforms in different laboratories. In addition, a duplex qPCR assay was developed to allow coamplification of the cytochrome oxidase gene from host plants for use as an internal PCR control. The most optimum qPCR assay proved to be faster and more sensitive than the previously published nested PCR assay and will be particularly useful for high-throughput testing and to detect P. psidii at the early stages of infection, before the development of sporulating rust pustules.

North American prairie wetlands are important non-forested land-based carbon storage sites.

We evaluated the potential of prairie wetlands in North America as carbon sinks. Agricultural conversion has resulted in the average loss of 10.1 Mg ha(-1) of soil organic carbon on over 16 million ha of wetlands in this region. Wetland restoration has potential to sequester 378 Tg of organic carbon over a 10-year period. Wetlands can sequester over twice the organic carbon as no-till cropland on only about 17% of the total land area in the region. We estimate that wetland restoration has potential to offset 2.4% of the annual fossil CO(2) emission reported for North America in 1990.

Ocular penetration of amikacin following intramuscular injection.

Amikacin sulfate was given intramuscularly (IM) (7.5 mg/kg) to study its ocular penetration in man. Seventy-three patients with cataracts received a single dose and 35 received two doses given 5 1/2 to 12 hours apart. After a single dose the aqueous humor levels of the antibiotic between two and ten hours ranged from 0.15 to 3.10 mg/L (average and median, 1.0 mg/L). Two doses given 5 1/2 to eight hours apart produced an average level of 3.5 mg/L (range, 0.91 to 8.31 mg/L). When the second dose was given nine to 12 hours after the first, the aqueous humor levels were similar to those found for a single dose. Aqueous humor concentrations of 1.0 mg/L of amikacin would be expected to be bactericidal for most gram-negative bacterial pathogens, whereas levels of 3.5 mg/L would inhibit most strains of Staphylococcus aureus and many strains of Pseudomonas aeruginosa.