First record of Cylindrocladium pauciramosum on myrtle (Myrtus communis) in Portugal

Abstract

During March 2002, young myrtle plants growing in Portugal were received at the Central Science Laboratory. These plants were showing a range of disease symptoms, from mild wilting and chlorosis of growing tips through to severe crown and root rot leading to eventual plant death. A Cylindrocladium sp. was consistently isolated from the crown of diseased plants. To determine the species, a hyphal tip culture of the fungus was grown on carnation leaf agar for 7 days at 25°C under near-ultraviolet light (12 h cycle). The identification of the fungus was achieved using the keys of Crous & Wingfield (1994), complemented by the publication of Polizzi & Crous (1999). Only the material growing on the carnation leaves was examined. Thirty structures were measured. The width of the vesicles was in the range 4–8 µm (mean 5·7 µm). The widest part of the vesicles was always observed below the middle, a characteristic feature that separates Cylindrocladium pauciramosum from the morphologically similar species C. scoparium (Polizzi & Crous, 1999). The conidiophores had stipe extensions terminating in obpyriform to broadly ellipsoidal vesicles. The conidia were all one-septate and their measurements were in the range 42–60 × 3–4 µm. The morphological characteristics fitted the description of C. pauciramosum. To confirm the identity of the fungus, the 5′ end of the β-tubulin gene was amplified using the primers T1 and Bt2b as described by Henricot & Culham (2002). The sequence of the amplified product was deposited in GenBank (accession number AY162230). Comparison of the sequence of the β-tubulin region with other sequences available in the GenBank database revealed that it was identical to the C. pauciramosum DISTEF-G 60 isolate from Myrtus communis in Italy. This isolate forms a monophyletic group with other C. pauciramosum isolates which all have identical β-tubulin sequences (Schoch et al., 2001). These isolates originate from California, South Africa, Italy and Australia (Schoch et al., 2001). Koch's postulates were fulfilled by inoculating 1-year-old plants grown from cuttings with a spore suspension of the fungus (2 × 105 spores mL−1). The spore suspension was added to a wound made at the base of the stem with a scalpel. Sterile distilled water was used as a control. The wound was sealed with Parafilm and a polythene bag (sprayed on the inside with water to maintain high humidity) was placed over the plants for 48 h, which were then held at room temperature. Symptoms appeared 4 days post-infection as tip browning of the new leaves and wilting of the young shoots. Cylindrocladium pauciramosum was successfully reisolated from the leading edge of necrosis of the infected crown, fulfilling Koch's postulates. Control plants did not develop any symptoms. This root and crown rot disease was first identified on M. communis and other hosts in Italy in 1993 (Polizzi & Crous, 1999) and also in the USA in 2000 (Koike & Crous, 2001). To date no other cases on myrtle outside Italy have been reported from Europe. This is therefore the first record of Cylindrocladium pauciramosum in Portugal.