Molecular Identification of Fungi Associated with Vascular Discoloration of Soybean in the North Central United States

Brown stem rot (BSR) is one of the most important diseases of soybean (Glycine max (L.) Merr.) in the north-central soybean-production region (10), yet the causal agent of the disease is poorly known. The disease is characterized by a browning of the vascular system and pith, and characteristic chlorosis and necrosis of the foliage develops late in the season, usually in August (2). However, similar vascular discoloration is found late in the season with other diseases of soybean. Some BSR-resistant germ plasm is available (3,24), mostly identified by the extent of vascular browning, but development of resistant cultivars and deployment of resistance have been hampered by imprecise resistance rating, a poor understanding of the causal agent of the disease, and uncertainty about the variation in aggressiveness in the pathogen (16,24,29,33). The pathogen was described as Cephalosporium gregatum in 1948 by Allington and Chamberlain (2). Unfortunately, the fungus forms rather nondescript conidiophores, phialides, and conidia, and many unrelated species are morphologically similar. Many Cephalosporium spp. are now placed in the heterogeneous genus Acremonium, but the proper generic placement of C. gregatum is still in question. Gams (14) transferred the species to the genus Phialophora, though the fungus generally produces non-pigmented conidiophores. Sequences of ribosomal DNA suggest that Phialophora gregata is an anamorph of a discomycete (T. C. Harrington, unpublished). Two or more Acremonium-type fungi that are similar to P. gregata have been associated with brown stem rot in soybean and adzuki bean (5,18,23), and at least one of these Acremonium spp. causes vascular discoloration in soybean (23). Gray (16) reported that the disease in soybean is caused by either of two forms of P. gregata, a defoliating form (type I) and a nondefoliating form (type II), with the latter causing vascular discoloration but no foliar symptoms. Gray and Hepburn (18) did not distinguish type I and type II isolates of P. gregata. Phillips (29) found at least three virulence genotypes of P. gregata based on vascular discoloration, and Willmot et al. (33) distinguished type I and type II isolates based on foliar symptoms. Mengistu and Grau (23) also recognized type II isolates of P. gregata and isolates of an unidentified Acremonium sp. that caused vascular discoloration but limited foliar symptoms. Later, Mengistu et al. (25) characterized type II isolates as growing faster than type I isolates, while the opposite was reported by Gray (16), and their (25) illustration of type II colonies show them to be atypical of P. gregata. Although it is not clear if type II isolates (sensu Gray, 16; Willmot et al., 33; and Mengistu et al., 25) are P. gregata or another Acremonium-type sp., we examined isolates of the Acremonium spp. of Mengistu and Grau (23) and found them to be Plectosphaerella cucumerina (Lindf.) Kleb. (anamorph = Plectosporium tabacinum (van Beyma) M. E. Palm), a common pyrenomycete in soybean and other plants that may be pathogenic to some hosts (6,27,28). Because its anamorph is similar in morphology to Phialophora gregata, it is possible that Plectosphaerella cucumerina was misidentified as the so-called “non-defoliating” form of Phialophora gregata in earlier studies (16,25). Gray and Hepburn (18) also found that some adzuki bean isolates identified as P. gregata were likely an unrelated Acremonium sp., and conidia and conidiophores of their fungus look similar to the anamorph of Plectosphaerella cucumerina. The brown stem rot pathogen in adzuki has recently been designated as a distinct specialized form, Phialophora gregata f. sp. adzukicola (21). With the advent of polymerase chain reaction (PCR), inexpensive DNA sequencing, and a relatively large databank of ribosomal DNA sequences, it is now possible to more objectively characterize and identify fungal species. We applied these techniques to fungi associated with brown stem rot of soybeans. Our objectives were to clarify the etiology of this disease, more clearly characterize the causal agent, and delineate the pathogen from similar fungi isolated from vascular discoloration in soybean, using a larger number of isolates and sampling a wider geographic area than had been utilized in earlier studies. ABSTRACT Harrington, T. C., Steimel, J., Workneh, F., and Yang, X. B. 2000. Molecular identification of fungi associated with vascular discoloration of soybean in the north central United States. Plant Dis. 84:83-89.