First report of Clonostachys rosea causing root rot of soybean in the United States

Multiple fungal species have been associated with root rot of soybean (Glycine max) in the United States, but root rot in Minnesota (MN) also occurs in plants not known to be infected with previously reported pathogens (1). Soybean plants that lacked foliar symptoms, but exhibited taproot and lateral root necrosis were observed in 15 fields from nine counties in MN during 2007 and 2008. Plants were arbitrarily dug up at the R3 growth stage in July as part of a root rot study. Roots were washed, surface disinfested in 0.5% NaOCl for 3 min, rinsed in deionized water, dried, and embedded in potato dextrose agar (PDA). Thirty isolates with morphological characteristics consistent with those of Clonostachys rosea were recovered in total from necrotic lesions on different plants from all fields (3). For further morphological characterization, cultures were grown on PDA for 1 week at 24°C in the dark. Colonies were 39 to 46 mm in diameter, yellowish-white, and the surface was felty to tomentose with thick aerial hyphae. Primary conidiophores were Verticillium-like with two to three levels. The stipe length measured 65 to 105 μm and the base width was 5 μm. Primary conidia were smooth, hyaline, slightly curved, with an average length and width of 7 to 9 × 2.6 to 3 μm. Secondary conidiophores were penicillate with two or three whorls of phialides. The stipe length measured 50 to 75 μm, base width was 5 μm, and penicillus height was 25 to 35 μm. Secondary conidia were 5 to 6 × 2.5 μm. Perithecia were not produced. The identity of isolates was confirmed by sequencing the internal transcribed spacer (ITS) locus using the primers ITS1F/ITS4. BLAST analysis of the sequences in the NCBI database resulted in a 99.8 to 100% match for both C. rosea and its teleomorph Bionectria ochroleuca (e.g., HM751081, GU256766). Each isolate was tested for pathogenicity on soybean by initially growing it on sterile sorghum grain for 2 weeks at 23°C. Sterile sorghum was used for control plants. Seeds of soybean 'AG2107' were planted in 11.4-cm square pots containing pasteurized potting mix and a 25-cm3 layer of infested or sterile sorghum placed ~1 cm below the seeds. Two replicate pots containing four plants each were used per treatment and the experiment was repeated once. Root rot was assessed 28 days after planting in a greenhouse at 23°C day and 18°C night with a 14-h photoperiod. Twenty-eight of 30 C. rosea isolates caused taproot necrosis on inoculated plants in both experiments, whereas control roots did not exhibit necrosis. Approximately 6% of inoculated plants also developed interveinal chlorosis and marginal necrosis on trifoliates. Isolations were attempted from roots of all plants, and the isolates recovered from inoculated plants were identified as C. rosea based on morphology and ITS sequences. This fungus was not isolated from control plants. C. rosea was also isolated from petioles of symptomatic trifoliates, indicating systemic colonization of the plants. To our knowledge, this is the first report of C. rosea causing root rot of soybean and systemically colonizing soybean. This fungus may have been previously isolated from asymptomatic soybean plants and identified as Gliocladium roseum (2). The impact of this fungus on soybean production is unknown.