A Second Spore Stage Confirmed for Apioplagiostoma populi, the Causal Agent of Bronze Leaf Disease of Populus in Minnesota

Abstract

Bronze leaf disease (BLD) affects several Populus species in North America but is particularly damaging to hybrids in section Populus (4). BLD, caused by the fungus Apioplagiostoma populi (syn. Plagiostoma populi) described by Cash and Waterman (1), takes its name from the characteristic dark purple to brown pigmentation of infected leaves. A. populi has not been cultured on artificial media either from diseased tissues or cast ascospores. An anamorph has not been conclusively identified but spores from blister-like acervuli on symptomatic leaves have been suggested to function as spermatia (3). In an attempt to describe the imperfect stage of A. populi, collections of diseased leaves of P. alba × sieboldii and P. alba × grandidentata‘Crandon’ growing in a plantation near Rosemount, MN, were made on September 14, 2011 (leaves attached to shoots, rolled inward), October 12, 2011 (leaves attached or on the ground, tightly rolled inward), and November 1, 2011 (most leaves on the ground), and examined in the laboratory for fungal development. Leaf laminae from the September 14 collection were uniformly covered with erumpent, subcuticular blister-like acervuli on the adaxial surface only, containing unicellular, colorless spores ranging in size from 2.2 to 10.0 × 2.0 to 5.0 μm (mean 3.6 × 6.2 μm) (n = 100). Attempts to germinate and obtain cultures from these spores on common artificial media were unsuccessful. On leaves collected October 12, the blister-like acervuli were predominantly empty or releasing spores and immature perithecia of A. populi were present. Leaves collected on November 1 contained immature perithecia but were heavily colonized by surface saprophytic fungi and no intact blister-like acervuli were present. DNA was extracted from bulked samples of spores removed from the blister-like acervuli in leaves from multiple trees collected on September 14 using the DNeasy Plant Mini Kit (Qiagen Sciences, Germantown, MD). The internal transcribed spacer (ITS2) region of rDNA was PCR amplified and sequenced with primers ITS3 and ITS4. BLASTn searches revealed that sequences from three independent bulked samples were homologous (99% identity, 353 of 355 nucleotides) to A. populi from isolated perithecia (GenBank Accession No. GU205341) (2). The inability of the spores to germinate, the timing of their development and release, and the tight, inward roll of infected leaves facilitating their spread across the upper leaf surface suggest that these spores function as spermatia in the life cycle of A. populi and the blister-like acervuli in which they develop are spermogonia. References: (1) E. K. Cash and A. M. Waterman. Mycologia 49:756, 1957. (2) L. M. Kawchuk et al. Plant Disease 94:377, 2010. (3) W. A. Sinclair and H. H. Lyon, Comstock Publishing Associates, Cornell University Press, Ithaca, NY, 2005. (4) J. A. Smith et al. Plant Disease 86:462, 2002.