B. Henricot¹ and P. Beales²

¹Department of Plant Pathology, The Royal Horticultural Society, Wisley, Woking, Surrey GU23 6QB
²Plant Disease Diagnosis, Central Science Laboratory, Sand Hutton, York, YO41 1LZ

*beatriceh@rhs.org.uk

Accepted for publication 3/12/02

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 myrtles. 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 hour 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 of 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 Cylindrocladium 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 of 42-60 x 3-4 µm. The morphological characteristics fitted the description of C. pauciramosum.

Cylindrocladium pauciramosum isolated from myrtle
showing the conidiophores with stipe extensions terminating
in obpyriform to broadly ellipsoidal vesicles

To confirm the identity of the fungus, the 5’end of the ß-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 AY162320). Comparison of the sequence of the ß-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 ß-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 x10⁵ spores per ml). 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 hours which were 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 re-isolated from the leading edge of necrosis of the infected crown fulfilling Koch’s postulates. Control plants did not develop any symptoms

Infection assays on 1-year old myrtle plants. Symptoms 14 days
post infection on 1-year old myrtle plants (right plant), control (left plant)

This root and crown rot disease was first identified on Myrtus 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 of Italy had been reported from Europe. This is therefore the first record of Cylindrocladium pauciramosum in Portugal.

References

Crous PW, Wingfield MJ, 1994. A monograph of Cylindrocladium, including anamorphs of Calonectria. Mycotaxon 51, 341-45.

Henricot B, Culham A, 2002. Cylindrocladium buxicola, a new species affecting Buxus spp., and its phylogenetic status. Mycologia 94, 980-997.

Koike ST, Crous PW, 2001 First report of a root and crown rot disease of myrtle in California caused by Cylindrocladium pauciramosum. Plant Disease 85, 448.

Polizzi G, Crous PW, 1999. Root and collar rot of milkwort caused by Cylindrocladium pauciramosum, a new record for Europe. European Journal of Plant Pathology 105, 407-411.

Schoch CL, Crous PW, Polizzi G, Koike S, 2001. Female fertility and single nucleotide polymorphism comparisons in Cylindrocladium pauciramosum. Plant Disease 85, 941-946.

The British Society for Plant Pathology