Fungal trunk pathogens associated with wood decay of almond trees on Mallorca (Spain).

Severe decline of almond trees has recently been observed in several orchards on the island of Mallorca (Balearic Islands, western Mediterranean Sea). However, the identity of the causal agents has not yet been investigated. Between August 2008 and June 2010, wood samples from branches of almond trees showing internal necroses and brown to black vascular streaking were collected in the Llevant region on the island of Mallorca. Several fungal species were subsequently isolated from the margin between healthy and symptomatic tissue. Five species of Botryosphaeriaceae (namely Botryosphaeria dothidea, Diplodia olivarum, D. seriata, Neofusicoccum australe and N. parvum), Eutypa lata, Phaeoacremonium iranianum and Phomopsis amygdali were identified based on morphology, culture characteristics and DNA sequence comparisons. Neofusicoccum parvum was the dominant species, followed by E. lata, D. olivarum and N. australe. First reports from almond include D. olivarum and Pm. iranianum. Two species are newly described, namely Collophora hispanica sp. nov. and Phaeoacremonium amygdalinum sp. nov.

First Report of Phaeoacremonium inflatipes, P. iranianum, and P. sicilianum Causing Petri Disease of Grapevine in Spain.

In 2008, four isolates of Phaeoacremonium, morphologically and genetically different from known Phaeoacremonium spp. in Spain, were isolated from rootstocks of young grapevine (Vitis vinifera) plants showing Petri disease symptoms including low vigor, reduced foliage, and dark streaking of the xylem in Badajoz Province (western Spain; cv. Syrah on SO4 rootstock), Tarragona Province (eastern Spain; cv. Garnacha on 161 49 C rootstock), and Balearic Islands (eastern Spain; cv. Tempranillo on Rupestris de Lot rootstock). Single-conidial isolates were obtained and grown on potato dextrose agar (PDA) and malt extract agar (MEA) at 25°C for 2 to 3 weeks in the dark until colonies sporulated (3). Identification was based on morphological characteristics (1-3). Phaeoacremonium inflatipes W. Gams, Crous & M. J. Wingf. and P. iranianum L. Mostert, Gräf., W. Gams & Crous were detected in Badajoz Province and P. sicilianum Essakhi, Mugnai, Surico & Crous in Tarragona Province and Balearic Islands. Colonies of P. inflatipes were gray on PDA and gray-brown on MEA. Conidiophores were branched, 15 to 37 (mean 25) µm long. Conidia were hyaline, oblong-ellipsoidal or obovoid, 3 to 5.5 (mean 4) µm long, and 1.2 to 1.9 (mean 1.6) µm wide. Colonies of P. iranianum were brownish gray on PDA and pale brown on MEA. Conidiophores were unbranched and 18 to 47.5 (mean 29) µm long. Conidia were hyaline, oblong-ellipsoidal, 3 to 5 (mean 4) µm long, and 1 to 1.8 (mean 1.5) µm wide. Colonies of P. sicilianum were pale brown on PDA and brown to pale orange on MEA. Conidiophores were branched and 13 to 55 (mean 32.5) µm long. Conidia were hyaline, allantoid, 3 to 8.5 (mean 6) µm long, and 1.5 to 2 (mean 1.8) µm wide. Identity of isolates Pin-2, Pir-4, Psi-1, and Psi-2 was confirmed by sequencing a fragment of the beta-tubulin gene with primers T1 and Bt2b (P. inflatipes, isolate Pin-2: GenBank Accession No. FJ872407, 100% similarity to Accession No. AY579323; P. iranianum, isolate Pir-4: GenBank Accession No. FJ872406, 99% similarity to Accession No. EU128077; P. sicilianum isolates Psi-1 and Psi-2: GenBank Accession Nos. FJ872408 and No. FJ872409, 100% similarity to Accession No. EU863489). Pathogenicity tests were conducted using Pin-2, Pir-4, and Psi-1 isolates. One-year-old callused and rooted cuttings of 110 R rootstock cultivated in sterile peat were wounded at the uppermost internode with an 8-mm cork borer. An 8-mm mycelium plug from a 2-week-old culture was placed into the wound. Wounds were wrapped with Parafilm. Ten cuttings per fungal isolate were used. Ten control plants were inoculated with 8-mm noncolonized PDA plugs. Plants were maintained in a greenhouse at 25°C. Within 2 months, all Phaeoacremonium-inoculated cuttings exhibited shoots with poor growth, small leaves, short internodes, and black streaks in the xylem. The mean shoot weight per plant was 1.8 g in P. inflatipes-inoculated plants, 1.9 g in P. iranianum-inoculated plants, and 1.6 g in P. sicilianum-inoculated plants, all lower than the control treatment (6.8 g). Control plants did not show any symptoms. All fungal species were reisolated from wood of all inoculated cuttings, completing Koch's postulates. Their identity was confirmed with the methods described above. To our knowledge, this is the first report of P. inflatipes, P. iranianum, and P. sicilianum causing Petri disease in Spain. References: (1) P. W. Crous et al. Mycologia 88:786, 1996. (2) S. Essakhi et al. Persoonia 21:119, 2008. (3) L. Mostert et al. Stud. Mycol. 54:1, 2006.

First Report of Phytophthora ramorum on Rhododendron sp. in Spain.

Phytophthora ramorum has been recently described (4) from isolates collected since approximately 1993, from Rhododendron and Viburnum in the Netherlands and Germany. Since 2001 and 2002, respectively, there have been unconfirmed reports from Poland and Great Britain (1). There are growing concerns about this oomycete since it has been recognized as the causal agent of sudden oak death in California, where thousands of oaks and tanoaks (Quercus agrifolia, Q. kelloggii, Q. parvula var. shrevei, and Lithocarpus densiflorus) have died. Since the first outbreak in Marin County, California in 1995, (3) the epidemic has remained concentrated mainly along the Pacific Coast near San Francisco but has been reported in Oregon (2). There have been no reports of oaks or other trees affected in Europe. During February 2002, E. Moralejo surveyed several nurseries in Mallorca, (Balearic Islands, Spain) to verify the presence of P. ramorum on rhododendron. Two shipments of rhododendrons, from the towns of Palma and Santa Maria del Camí, showed twig blight and necrotic spots at leaf tips and margins. Less than 0.5% of the aerial parts of the potted plants of both sets were affected. Isolations from symptomatic tissue consistently yielded cultures of Phytophthora using selective media (corn meal agar supplemented with nystatin, rifampicin, and ampicillin). Pure cultures were obtained from three isolates by hyphal-tip transfers on carrot piece agar (CPA) and identified as P. ramorum on the basis of morphological and cultural characters (4). Colonies grown on CPA at 17°C were appressed with sparse aerial mycelium and formed typical concentric growth rings under 16 h fluorescent light. Cardinal temperatures for vegetative growth were 2°C (minimum), 20°C (optimum), and 28°C (maximum). Daily radial growth was 3 to 3.2 mm at 20°C. Sporangia formed singly or in clusters on aerial sporangiophores in the absence of free water, and were mainly semipapillate, caducous, with a very short or no pedicel, ellipsoidal to elongated-ovoid, with a length/breath ratio of 1.9 to 2.1. Large spherical, terminal, and intercalary chlamydospores, 32 to 72 µm in diameter, were readily formed in agar. The mating type of these isolates from Mallorca was A1 based on dual cultures on CPA with four different heterothallic Phytophthora species. Crossing was successful with P. cryptogea BBA 63651 mating type A2. Oogonia were subglobose and measured 24 to 32 µm in diameter. Oospores were plerotic. Antheridia were always amphigynous, and mainly barrel-shaped to spherical, 14 to 22 µm in diameter One of the three isolates also produced a very small number of gametangia with the A2 tester strain P. cinnamomi BBA 62660, but the oospores appeared degenerate. Only A1 mating type strains of P. ramorum have been found in Europe. The Mallorca isolates have been deposited in the culture collection of the Federal Biological Research Centre for Agriculture and Forestry (BBA), Germany. To our knowledge, this is the first report of P. ramorum in Spain. Further research is urgently needed to assess whether the Mediterranean evergreen oak ecosystem is in danger. References: (1) EPPO Reporting Service. Published on-line, http://www.eppo.org/PUBLICATIONS/EPPO_RS/reporting_service.html Publication no. 2002/040 and 2002/077. (2) E. M. Goheen et al. Plant Dis. 86:441, 2002. (3) D. M. Rizzo et al. Plant Dis. 86:205, 2002. (4) S. Werres et al. Mycol. Res. 105:1155, 2001.