First report of Gymnosporangium clavipes Cooke & Peck affecting Crataegus mexicana var. Chapeado and C. gracilior in Mexico

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Abstract

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Species of the genus, Crataegus, are commonly called ‘’tejocote’’ in Mexico. This country is the largest producer of tejocote with 3,675 t in 2014 (SIAP 2015). Gymnosporangium species are heteroecious, producing the aecial phase on leaves and fruit of pome hosts and the telial phase on leaves, stems and branches of cypress (Cupressus spp.) and juniper (Juniperus spp.) (Sinclair and Lyon, 2005). In Mexico, there have been several species of Gymnosporangium reported, including G. clavipes in Crataegus mexicana and Crataegus sp. (León-Gallegos and Cummins, 1981). During May to September in 2012 and 2013, aecia fruits (Fig. 1A) were collected from Crataegus mexicana Moc. & Sessé var. Chapeado (SNICS TEJ-002-160709) and C. gracilior Phipps plantations in the Trans-Mexican Neovolcanic Belt in the State of Puebla. These samples were deposited as ENCB 121364 Crataegus mexicana var. Chapeado and C. gracilior as ENCB 121365 at the fungus collection of Escuela Nacional de Ciencias Biológicas at the Instituto Politécnico Nacional. Peridium and aeciospores measurements were taken using the software Images Plus 2.0 (Motic, Hong Kong, CH). The width and length of the aecia were measured using the Image Tool v 3.0 software (UTHSCSA, USA). Key (Kern, 1973) and the diagnostic protocol (EPPO, 2006) were used. These specimens were observed using a scanning electron microscope (SEM) XL 30 ESEM (Philips, A’dam, NED) with a range of 2.5-20 Kv. The ornamentation of the outer, side and inner walls of the peridial cells of aecia and the ornamentation of the aeciospores were observed based on the classification (Lee and Kakishima, 1999ab). Aecia were roestelioid and white in colour, measuring 7.59-15.25 × 0.54-0.79 μm, with little dehiscent peridium (Fig. 1B). On the peridial cell walls of aecia, outer (Fig. 1C) and side walls were smooth (Fig. 1D). However, the inner walls were tuberculate (Fig. 1D). Aeciospores were ellipsoid and orange in colour, measuring 18.8-37.6 × 24.0-57.2 μm (Fig. 1E). The ornamentation of aeciospores presented small annulations (Fig. 1F). Based on morphological characteristics, the fungus was identified as Gymnosporangium clavipes (EPPO, 2006; Kern, 1973). For confirmation of morpho)logy-based identification, the specimens (ENCB 121364 and 121365) were subjected to molecular analysis. Aecia samples amplified with the primers D1/D2 and Rust18S-R/NS1, resulted in the expected band of 700 bp and 1750 bp, respectively. The PCR products were purified and sequenced. The resulting sequence generating primer pair, D1/D2, was deposited to the GeneBank database as tejocote fruit aecia ENCB 121364 (GenBank accession No. KM382067) and analysed for similarity to known sequences in the database. The sequence showed high similarity to G. clavipes up to 99% (GenBank accession Nos. DQ354545 and HQ317528). In addition, the resulting sequence generating primer pair, Rust18S-R/NS1, the resulting sequence was deposited to the GeneBank database as tejocote fruit aecia ENCB 121365 (GenBank accession No. KM382070). The sequence also showed high similarity to G. clavipes up to 99% (GenBank accession No. DQ354546). In most reports of the species discussing about Gymnosporangium rust, the pathogenicity test was not carried out (Dervis et al., 2010; Lee and Kakishima, 1999a, b; Yun et al., 2008; Yun et al., 2009). Although G. clavipes had been identified previously (León-Gallegos and Cummins, 1981) in C. mexicana and Crataegus sp., in this study, we have additionally employed SEM to identify infection on the specific tejocote species and varieties and molecular analysis. To our knowledge, this is the first confirmed report of Gymnosporangium clavipes affecting Crataegus mexicana var. Chapeado and C. gracilior in Puebla, Mexico.

Fig. 1

Aecial phase of Gymnosporangium clavipes. (A) Aecia fruits. (B) Apex dehiscent aecia (Scale bar=1 mm). (C) The outer walls (OW) of peridial cells are smooth (Scale bar=10 μm). (D) The side walls (SW) of peridial cells are smooth and the inner walls (IW) of peridial cells are tuberculate (Scale bar=10 μm). (E) Orange aeciospores have thick walls (Scale bar=20 μm). (F) Ornamentation of aeciospores has small annulations (Scale bar= 10 μm).

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