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

K. Vrandecic¹*, J. Cosic¹, L. Riccioni², T. Duvnjak³ and D. Jurkovic¹

¹ Faculty of Agriculture in Osijek, PO Box 719, 31000 Osijek, Croatia
² Istituto Sperimentale per la Patologia Vegetale, Via C.G. Bertero 22, 00156 Rome, Italy
³ Agricultural Institute of Osijek, PO Box 149, 31000 Osijek, Croatia

*kvrandecic@pfos.hr

Accepted for publication 04/01/05

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).

Figure 1: Symptoms on the stem of Abutilon theophrasti

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.

Figure 2: Perithecia on Abutilon theophrasti

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.

References

Black BD, Padgett GB, Russin JS, Griffin JL, Berggren GT, 1996. Potential weed hosts for Diaporthe phaseolorum var. caulivora, casual agent for soybean stem cancer. Plant Disease 80, 763-765.

Hepperly PR, Kirkpatrick BL, Sinclair JB, 1980. Abutilon theophrasti: wild host for three fungal parasites of soybean. Phytopathology 70, 307-310.

Li S, Bradley CA, Hartman GL, Pedersen WL, 2001. First report of Phomopsis longicolla from velvetleaf - causing stem lesions on inoculated soybean and velvetleaf plants. Plant Disease 85, 1031.

White TJ, Bruns T, Lee S, Taylor JW, 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ, eds. PCR Protocols: A Guide to Methods and Applications. New York, USA: Academic Press Inc., 315-322. 

The British Society for Plant Pathology