First report of Phomopsis longicolla causing soybean stem blight in China

In late August 2006, severe stem blight of soybean was observed in several fields in Heilongjiang Province. The characteristic symptoms were dry rot with discoloration on the lower stem at the junction of a branch or petiole, which caused plant wilt and death. Fungi were isolated from diseased stem tissue in order to identify the causal agent.The isolates were cultured on acidified potato dextrose agar (PDA, pH 4.5) at 25^°C with a 12/12 h light/dark regime. Colonies were floccose, dense, and white with occasional green-yellow areas. The undersides of the cultures were colourless. Stromata were large, black, and widespread. In order to induce fruiting bodies, the isolates were grown on autoclaved soybean stems on water agar at 25^°C in the dark. Pycnidia with long beaks were observed on the stem and produced both alpha-conidia and beta-conidia. Alpha-conidia were hyaline, ellipsoidal to fusiform, and guttulate, 4.05-7.57x 1.48-3.25 μm. Beta-conidia were rare, hyaline, filiform, and hamate. The cultural and morphological characteristics of all the tested isolates fit the description of Phomopsis longicolla (Hobbs et al., 1985). Pathogenicity was confirmed by inoculation of two-week-old seedlings (six to eight replicates) of cv. Hefeng 25 using mycelia plugs placed into cuts in the stems. All inoculated plants showed dry rot lesions with discoloration. Black pycnidia could be observed on the lesions about two weeks later. For each isolate tested, P. longicolla was reisolated from inoculated plants.

To confirm the morphological identification, DNA of 79 isolates was extracted from seven day-old cultures grown in potato dextrose broth. The region of rDNA-ITS was amplified with universal primers ITS4 and ITS5, and digested with AluI, RsaI and HhaI restriction enzymes (Zhang et al., 1998). All isolates produced the characteristic banding pattern of P. longicolla (Zhang et al., 1998). The ITS sequences of five isolates were obtained (one typical sequence being GenBank Accession No. EU650789), and comparisons with GenBank showed 100% similarity with nine isolates of P. longicolla (EF026104, AF000206, AF000207, AF000208, AF000209, AF000210, AF000211, U97658, and AY857868).

P. longicolla is an important soybean pathogen causing phomopsis seed decay. This fungus has a wide distribution across the world (CAB International, 2005), and can cause severe yield losses and poor seed quality in soybean. Besides infecting soybean, P. longicolla has been reported to infect some weed species in the following genera: Abutilon, Ambrosia, Cannabis, Convolvulus, Digitaria, Euphorbia, Ipomoea, Rumex, Sesbania, Xanthium (Mengistu et al., 2007). However, to our knowledge, P. longicolla has not been reported in soybean nor any weed hosts in China.