First Report of Brown Rot Caused by Monilinia fructicola in Peach Orchards in Ebro Valley, Spain.

Monilinia fructicola causes brown rot of stone fruit in India, Japan, the Republic of Korea, Oceania, and North and South America and is in the A2 list of quarantine organisms for Europe. M. fructicola was found in peach orchards for the first time in Europe in 2001 in France (4) and later in the Czech Republic (2). M. fructicola was not detected among 428 isolates of Monilinia spp. collected from Spanish peach orchards from 1998 to 2005. In March of 2006, M. fructicola was detected to be overwintering in three mummified peach fruit (cv. Autumn Free) trees in an orchard located in Sudanell (Lleida, Spain). Morphological and molecular identification of isolates were performed according to protocols previously described (1,3). The characteristics of these isolates were: i) colonies were entire and showing concentric rings of spores when grown on potato dextrose agar (PDA); ii) sporogenous tissues were gray to buff; iii) single and nearly straight germ tubes were at least 220 µm long before branching; and iv) growth rates on PDA under long-wave UV/darkness were as much as 20 × 10 mm2. Isolates were further identified by a PCR test using primers developed with sequence-characterized amplification region markers obtained by random amplified polymorphic DNA for M. fructicola: IColaS (GAGACGCACACAGAGTCAG) and IColaAS (GAGACGCACATAGCATTGG) (3). The expected PCR product of 386 bp was produced only in M. fructicola isolates. Koch's postulates were fulfilled with the three isolates by inoculating five healthy fruit with a conidial suspension of each isolate (104 conidia ml-1). Symptoms similar to those observed in the field were small brown spots, which rapidly showed brown rot. Noninoculated control fruit did not show symptoms. The fungus was reisolated on PDA from inoculated fruit after 4 days of incubation at 22°C, 80 to 100% relative humidity, and 16 h under fluorescent lighting, 100 µE·m-2·s-1. To our knowledge, this is the first report of M. fructicola in peach orchards in Spain. References: (1) A. De Cal and P. Melgarejo. Plant Dis. 83:62, 1999. (2) J. Duchoslavová et al. Plant Dis. 91:907, 2007. (3) I. Gell et al. J. Appl. Microbiol. 103:2629, 2007. (4) J. Lichou et al. Phytoma 547:22, 2002.

Two different PCR approaches for universal diagnosis of brown rot and identification of Monilinia spp. in stone fruit trees.

AIMS: To design a protocol for the universal diagnosis of brown rot by polymerase chain reaction (PCR) in plant material and subsequently Monilinia spp. identification. METHODS AND RESULTS: Primers for discrimination of Monilinia spp. from other fungal genera by PCR were designed following a ribosomal DNA analysis. Discrimination among species of Monilinia was subsequently achieved by developing primers using SCAR (Sequence Characterised Amplified Region) markers obtained after a random amplified polymorphic DNA study. In addition, an internal control (IC) based on the utilization of a mimic plasmid was designed to be used in the diagnostic protocol of brown rot to recognize false negatives due to the inhibition of PCR. CONCLUSIONS: The four sets of primers designed allowed detection and discrimination of all Monilinia spp. causing brown rot in fruit trees. Addition of an IC in each PCR reaction performed increased the reliability of the diagnostic protocol. SIGNIFICANCE AND IMPACT OF THE STUDY: The detection protocol presented here, that combined a set of universal primers and the inclusion of the plasmid pGMON as an IC for diagnosis of all Monilinia spp., and three sets of primers to discriminate the most important species of Monilinia, could be an useful and valuable tool for epidemiological studies. The method developed could be used in programmes to avoid the spread and introduction of this serious disease in new areas.

Awake fibreoptic intubation in the semi-prone position following facial trauma.

A fit 27-year-old man presented with severe facial trauma following an industrial accident. Initial assessment showed severe swelling around the lower jaw and haemorrhage from the mouth, nose, scalp and left ear. The patient was conscious with a Glasgow Coma Score of 13 but in respiratory distress. Following adoption of the prone position his airway improved. Relief of the patient's airway obstruction was a priority and the patient underwent awake fibreoptic intubation in the prone position prior to induction of anaesthesia. Computed tomography scans of his head and neck were unremarkable and after fixation of a bilateral mandibular fracture he made an uneventful recovery. Intubation in the semi-prone position may be a useful technique in injuries of this type.