Isolation of Diaporthe phaseolorum var. caulivora from Abutilon theophrasti in Croatia

During 2000-2003, a weed survey was carried out in fields of soybean, sunflower and sugar beet in Croatia. Abutilon theophrasti (velvetleaf), found in soybean fields, showed symptoms of black lesions, several centimetres in length on the stems and side-branches (Fig. 1).

A fungus was isolated from stem lesions on potato dextrose agar (PDA) and incubated at 25°C, with a 12-hour-dark/light regime. On PDA, the fungus quickly formed a dense white mycelium, which later became fluffy. The mycelium was yellow-white at first; turning yellow-brown later. After 10 days black stromata were produced. These were irregularly shaped and measured 2-5 mm in diameter. Numerous black perithecia were formed within the stromata (Fig. 2). Ten perithecia, fifty asci and one hundred ascospores were measured. The sizes of perithecia were 200-360 x 295-450 µm and these had a protruding neck which was highly variable in length (300-1200 µm). The sizes of the asci were 25.1-42.0 x 4.2-8.3 µm. The ascospores were two-celled, hyaline, elongate to ellipsoidal, bi-guttulate, constricted at the septum, and measured 8.0-13.1 x 2.5-4.4 µm. Based on the morphological characteristics, the pathogen was identified as Diaporthe phaseolorum var. caulivora.

A. theophrasti plants were infected in the field, by applying a mycelium plug to the plant stem. The inoculation point was covered with a piece of wet cotton wool and aluminium foil, in order to retain moisture. Lesions of 5 to 19 mm long were observed, 10 days post infection. Diaporthe phaseolorum var. caulivora was reisolated out of the lesions of inoculated plants, fulfilling Koch's postulates. A pathogenicity test was also done on soybean seedlings. Mycelium plugs were put on the hypocotyl, which was previously wounded with a sterile scalpel. After 10 days, the pathogen was reisolated from the stem lesion of inoculated plants. Uninfected control plants did not exhibit any symptoms.

To confirm the pathogen identification, molecular analysis was performed. DNA was extracted from a monoconidial culture and amplified with ITS4 and ITS5 universal primers (White et al., 1990). The ITS was sequenced and submitted to GenBank (accession number AY857867). Comparison of the ITS sequences available on the database revealed that it was almost identical, with only two deletions in positions 24 and 542, to D. phaseolorum var. caulivora (AJ312360).

It is known that weeds can act as a reservoir for infection by D. phaseolorum (Black et al., 1996). A. theophrasti has been recorded before as a host for D. phaseolorum var. sojae (Hepperly et al., 1980) and Phomopsis longicolla (Li et al., 2001). Each year in Croatia, the presence of fungi in the Diaporthe/Phomopsis complex can be observed on soybean, but these fungi do not appear to cause great economic damage under local conditions. This is however the first report of A. theophrasti as a host for Diaporthe phaseolorum var. caulivora in Croatia.