First Report of Globisporangium ultimum Causing Pythium Damping-Off on Aleppo Pine in Algeria, Africa, and the Mediterranean Region.

Abstract

Globisporangium ultimum (Trow) Uzuhashi, Tojo & Kakish. (syn. Pythium ultimum Trow, syn. P. ultimum Trow var. ultimum) is a known oomycetal species from Pythium s.l. causing damping-off and/or root rot on a great variety of plants throughout the world, including some pine species (Pinus L.) and conifers (2,3). Aleppo pine (Pinus halepensis Mill.) is a common native forest tree in the Mediterranean region. Pre- and post-emergence damping-off disease symptoms were observed during 2008 and 2009 in four forest nurseries from northwestern Algeria (Relizane, Sidi Belabes, and Tlemcen departments). This disease occurred under cool conditions, and Aleppo pines were significantly affected, reducing seedling emergence. Disinfected segments, about 5 mm in length, from decayed root and collar, were cultured on CMA at 25°C. This oomycetal species was identified based on the species description in Pythium keys (3,4). For the molecular identification, PCR was used to amplify the ITS region of Pythium isolates. It was amplified with the flanking primers ITS1 and ITS4, and these products were directly sequenced. Sequence data were compared to known sequences deposited in the NCBI non redundant database to confirm morphological identification. A BLAST search identified U3CR, U7CR, U1RT, U2CR, U4CR, U14CR, U7RT, and U17RT isolates (GenBank Accession Nos. JX191921, 22, 27, 29, 31, and 33 to 35, respectively) as G. ultimum based on 100% similarity with corresponding sequence of the reference isolate no. UZ056 MAFF240024 (AB468781) (3). Phytopathogenicity testing was conducted in a petri dish and pot experiment. In the petri dish experiment, a 3 mm diameter plug was transferred from a 7-day-old CMA colony to the center of a CMA petri dish, with three replicates per isolate, and three control plates were inoculated with sterile agar plugs. After 72 h, 10 Aleppo pine seeds were placed equally spaced to 1 cm from the edge of each plug. After 7 days at 22°C in the dark, germination inhibition (46.1 to 87.6%) and root growth inhibition (62.3 to 92.2%) were calculated. In the control plates, germination failure (13.4%) and root length (27.7 cm) were observed. For the pot experiment, inocula were produced by adding a 5 mm diameter plug from a 7-day-old CMA culture to a previously sterilized 500 ml flask containing 237.5 g sand, 12.5 g cornmeal, and 80 ml SDW. Nine-day-old inoculum was mixed with sterile soil at a rate of 1:3 (v:v). Inoculum was transferred to 500 ml pot, and 10 Aleppo pine seeds were planted, with three replicates per isolate, and three control pots were used. After 2 weeks, all of the isolates tested caused typical symptoms of Aleppo pine Pythium damping-off, the percentage of inoculated plants that became infected was 36.6 to 83.3%. In the control pots, no infected plants were observed. To our knowledge, this is the first report of G. ultimum causing damping-off on Aleppo pine in Algeria, Africa, and the Mediterranean Region. Before, Aleppo pine damping-off caused by G. ultimum was reported in Australia (1).