First report of Chrysanthemum morifolium leaf spot caused by Alternaria species in Mexico.

Chrysanthemum foliar spots were first observed in Mexico in 2010, reaching up to 30 per cent incidence in some greenhouses. The symptoms were spots on the edges of the leaves: 1 to 2 × 1 cm, rounded to oval, brown to blackish, some zonated and/or with halos, and others with entire yellowish leaves. The aims of this study were to identify the Alternaria species isolated, to test their pathogenicity, and to carry out their molecular characterization. During June 2011, 180 leaves with spots were collected from 12 greenhouses located in the State of Mexico. For colony morphology and sporulation branching pattern (Simmons 2007), the isolated monosporic colonies were divided in 2 groups and 2 isolates were selected per group. The identification was done according to the Simmons (Simmons 2007)protocols. The isolates were grown on potato carrot agar (PCA)for 7 days and used to inoculate leaves of 5 varieties (Eleonora, Godorniz, Holandesa, Moreliana, and Polar)by spraying with a 2.5 × 104 conidia/ml suspension. The inoculated leaves were incubated in a moist chamber at 23.5 to 25°C and 90 to 100 per cent RH. Characterization of the 4 selected isolates was done by sequencing 5 regions: ITS; OPA (OPA1-3 and OPA2-1); and ?-Tubulin (TUB1 and TUB2). Twenty sequences (5 per isolate and 1 per region)were deposited at GenBank. Two isolates were identified as A. alternata (Fr.)Keissl., and the other two as A. tenuissima (Nees)Wiltshire. Both species were previously reported for the same host in other countries with A. alternata in the United States (Farr et al. 1989)and A. tenuissima in China (Xu et al. 2009). Five-day colonies of A. alternata (HA041 and HA042)on PCA were dark green to blackish and zonated, and reverse slightly blackish. They produced abundant simple conidiophores with apical clusters of branched chains of 10 to 37 conidia and intercalary secondary conidiophores. Conidia were light brown with a light yellowish tint, tuberculate to verrucose, and dictyospore of three forms: elliptic (at the chain base), ellipsoid and ovoid, 15 to 35 × 7.5 to 12.5 µm. As for A. tenuissima (HA043 and HA044), on V8-juice agar at 5 days old, colonies were dark green to blackish and zonated, with reverse blackish coloration. They produced abundant simple conidiophores with mainly a single apical chain of 5 to 12 conidia and intercalary secondary conidiophores. Conidia were light golden to brown, tuberculate and of 4 shapes. These were obclavate and ovoid dictyospore (few conidia with the middle septum constricted)and fragmospores with or without long apical cells, and 30 to 67.5 (rare 75)× 7.5 to 12.5 µm. After 4 days of incubation period, the 4 isolates were pathogenic in the 5 chrysanthemum varieties. Both species of Alternaria were reisolated from the spots induced. There were no symptom differences between the 2 inoculated Alternaria species nor in diseased plants in greenhouses. The alignment in GenBank by ITS (KF728748 to KF728751); OPA1-3 (KF728756 to KF728758, KP064315); OPA2-1 (KF728759 to KF728761, KP064316); TUB1 (KF728752 to KF728755); and TUB2 (KF728762 to KF728765)[mainly 100 Query cover or less, until 90 (query bp length was more than the subject); 99 to 100 Identity, and 0.0 E-value; except for TUB2 with 2e-169 to 5e-171 E-value]was with each species of this study (Andrew et al. 2009). This disease is a direct threat to production of chrysanthemum in Mexico.