First report of Phytophthora hedraiandra on Viburnum tinus in Spain

E. Moralejo¹*, L. Belbahri², G. Calmin², F. Lefort², J. A. García¹ and E. Descals¹

¹ Instituto Mediterráneo de Estudios Avanzados, IMEDEA (CSIC-UIB), Miquel Marquès 21, 07190, Esporles, Balearic Islands, Spain
² Laboratory of Applied Genetics, School of Engineering of Lullier, University of Applied Sciences of Western Switzerland, 150 Route de Presinge, Jussy 1254, Switzerland

Accepted for publication 21/10/05

During Phytophthora ramorum surveys carried out at garden centres in Majorca, Spain in 2002, several potted plants of Viburnum tinus showing branch die-back were inspected. The origin of the lesions was traced to basal stem cankers ca. 10 cm above ground. The outer bark was aseptically removed and small pieces of tissue from the lesion front were plated onto a P_5ARP selective medium (Erwin & Ribeiro, 1997). Two isolates, P3842 (= CBS 117808) and P3942 (= CBS 117809), were obtained and putatively identified from morphological characters as Phytophthora cactorum. However, when sequenced in 2005, the ITS region of the rDNA of P3842 (GenBank Acc. No. AY881005) was identical, whilst P3942 (AY961608) had a 1 bp substitution to that of Phytophthora hedraiandra (AY707987) (de Cock & Man in’t Veld, 2004), a recently described species recovered from V. tinus leaves in The Netherlands. The isolates P3842 and P3942 had 3 and 4 bp substitutions respectively in the ITS compared to that of P. cactorum (AY943299). Furthermore, the partial sequence of the Cox1 mitochondrial gene of isolate P3842 (DQ220015) and P3942 (DQ220016) showed a 1 bp substitution to that of P. hedraiandra culture ex-type CBS 111725 (AY769115). The morphology of both isolates was therefore re-examined and compared with P. cactorum isolate P1611 (= CBS 117810) (AY943299).

Figure 1: Colony pattern of P. hedraiandra (P3842) on CA (A), CMA (B), MEA (C) and PDA (D).	Figure 2: Morphology of P. hedraiandra (P3842) and P. cactorum (P1611). A) characteristic subglobose, papillate sporangia of P. hedraiandra; B) P. cactorum sporangial shapes; C) sessile, paragynous antheridium of P. hedraiandra; and D) amphigynous antheridium of P. hedraiandra.

Colonies on carrot agar (CA; Brasier, 1967) were slightly stellate with appressed to low tufted aerial mycelium (Fig. 1); on corn meal agar (CMA) radiate and submerged; on malt extract agar (MEA) uniform or faintly stolonate and velvet; and on potato dextrose agar (PDA) uniform to slightly petaloid with felted to appressed mycelium. Radial growth rates on CA and CMA at 20ºC were 8.5 and 5.5 mm per day respectively. No chlamydospores or sporangia were formed on any media, but sporangia did appear when mycelial plugs on CA were submerged in soil extract (Moralejo et al., 2004) for 3 days at 20ºC. Gametangia formed profusely within 4 days on CA even on the aerial mycelium. The morphology of sporangia and gametangia of both isolates (Fig. 2) agreed with the species description of de Cock & Lévesque (2004). The isolates differed from P. cactorum mainly in their slightly higher colony growth rates at 20ºC, formation of aerial tufts on CA, predominance of subglobose sporangia, abundance of sessile antheridia with near absence of tangled hyphae below, and larger oogonia (mean diameter 31.2 μm vs. 28.1 μm for P.cactorum) and oospores (mean diam. 28.4 μm vs. 24.7 μm for P. cactorum).

Figure 3: (Left) Symptoms on V. tinus leaves seven days after wound-inoculation with P. hedraiandra (P3842); and (right) symptom on a twig of V. tinus 10 days after wound-inoculation with P. hedraiandra (P3942)

Pathogenicity was determined by wound-inoculating the underside of detached leaves and twigs of V. tinus (wound made ca. 10 cm below the apex) with mycelial plugs. Controls were wound-inoculated with sterile CA plugs. Both inoculated plant material and controls were incubated under continuous fluorescent white light at 20ºC. Excluding the controls, all leaves and twigs (Fig. 3) developed extensive necrotic lesions seven and 10 days after inoculating respectively, from which the fungus was re-isolated. This is the first finding of P. hedraiandra in Spain.

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Erwin DC, Ribeiro OK, 1996. Phytophthora diseases worldwide. St. Paul, MN, USA: APS Press.

Moralejo E, Puig M, Man in’t Veld WA, 2005. First report of Phytophthora tentaculata on Verbena spp. in Spain. Plant Pathology 53, 806.

The British Society for Plant Pathology