Gene Cloning and Tissue Expression Analysis of a PR-5 Thaumatin-Like Protein in Phellinus weirii-Infected Douglas-Fir

In western North America, Douglas-fir (Pseudotsuga menziesii) is the most economically important conifer species susceptible to laminated root rot caused by Phellinus weirii. While attempting to internally sequence an endochitinase found to be up-regulated in P. weirii-infected Douglas-fir roots, we obtained overlapping peptide fragments showing 28% similarity with a PR-5 thaumatin-like protein (TLP) designated PmTLP (Pm for Pseudotsuga menziesi). A rabbit polyclonal antibody was reared against a synthetic peptide composed of a 29-amino-acid-long, conserved, internal sequence of PmTLP and purified by immunoaffinity. Western immunoblot analysis of infected roots of 24-year-old coastalfir showed significantly higher amounts of PmTLP (P < 0.01) closest to the point of P. weirii inoculation and infection than in uninfected regions of the same root. The antibody was also used to screen for PmTLP in roots of 25-year-old interior Douglas-firs naturally infected with a related pathogen, Armillaria ostoyae, and results showed significantly higher levels of PmTLP in bark tissues adjacent to infection (P < 0.05) than in uninfected tissue. Using polymerase chain reaction (PCR)-based cloning, the cDNA of PmTLP was shown to have a 702-bp open reading frame with a signal peptide cleavage site at 155 bp corresponding to a 29-amino-acid-long residue prior to the start of the N-terminal. Based on the deduced amino acid sequence, the molecular mass of the putative PmTLP was calculated to be 21.0 kDa with an isoelectric point of 3.71. Alignment analysis of PmTLP cDNA with a representative genomic DNA PCR sequence showed presence of one intron of variable size, within the coding region. The induction of PmTLP at the site of root infection and its presence in needle tissue suggests a general role for this protein in adaptation to stress and may be part of an integrated defense response initiated by the host to impede further pathogen spread.

First Report of Laminated Root Rot on Sabina przewalskii Caused by Phellinus weirii sensu stricto in China

Members of the Phellinus weirii complex cause laminated root rot of living conifers. The cedar type (P. weirii (Murrill) Gilb. sensu stricto) of the complex is usually found on species of the Cupressaceae family, especially Thuja plicata in western North America, and the Douglas-fir type (P. sulphurascens Pilát) is found on species of the Pinaceae family (1,2,3). Outside North America, P. weirii occurs on species of Juniperus in the Ural Mountains, and P. sulphurascens occurs on other conifers in eastern Asia, including China (1). During a field inventory of wood-decay fungi in western China in 2003, laminated root rot of Sabina przewalskii (synonym Juniperus przewalskii) was found in natural forests of the Qilian Mountains in Qinghai Province (37°36′N and 102°15′E). Trees were approximately 80 to 150 years old and occurred in pure stands. Distinct disease patches that were as much as one hectare consisted of dead-standing and symptomatic trees, suggesting that the fungus spread by root contact. Symptomatic trees showed slow growth, thin crowns, and chlorotic foliage. After cutting several of the symptomatic trees, cambial necrosis and wood decay were found, and the trees apparently died when the cambial necrosis girdled the base of the trees. The wood of infected trees was reddish brown at the early stages of decay and later had numerous small cavities and separated into sheets at the junction of annual rings. Perennial, poroid, resupinate, dark brown basidiocarps formed on the root surface of dead trees. The basidiocarps had a monomitic hyphal system, hyphae without clamp connections, trama tissue with hyphoid setae, and thin-walled, hyaline, smooth, ellipsoid basidiospores. The fungus was identified as P. weirii and distinguished from P. sulphurascens by its perennial basidiocarps, smaller pores (5 to 8 versus 4 to 5 per mm), and narrower hyphoid setae (4 to 6 versus 5 to 10 μm in diameter). P. weirii also resembles P. ferrugineofuscus (P. Karst.) Bourdot in macro-morphology, but the latter species is a saprophyte and has allantoid to almost lunate basidiospores. The studied specimens of P. weirii and P. sulphurascens are preserved at the herbaria of the Botanical Museum of the University of Helsinki, the Institute of Applied Ecology, and the Chinese Academy of Sciences as DAOM 8734, Lowe 6960, TAA 52744, 55644, and 103812, and Dai 988, 2053, 2061, 2527, and 5067. To my knowledge, this is the first report of P. weirii sensu stricto from China and the first report of laminated root rot on S. przewalskii. References: (1) Y. C. Dai and G. F. Qin. Fungal Sci. 13:101, 1998. (2) E. M. Hansen et al. Species limits for Phellinus weirii. Pages 119–127 in: Root and butt rots of forest tress. Int. Conf. Root and Butt Rots, INRA, France, 1998. (3) M. J. Larsen et al. Mycologia 86:121, 1994.

Laminated Root Rot: New Considerations For Surveys

In 1981, Douglas-fir (Pseudotsuga menziesii) trees were placed into 3 disease classes of 45 trees each based on signs and symptoms of Phellinus weirii infection: infected, probably infected, and probably not infected. Trees that died during the course of the study were felled and their stumps and roots removed from the soil, cleaned carefully, dissected, and examined. In August 1991, the remaining study trees were similarly treated. Eight trees recorded as infected in 1981, based on the presence of ectotrophic mycelium, were found to be not infected in 1991. Only one-third of the trees near inoculum sources, and thus expected to be infected, were infected. Of the trees thought to be not infected, one-third were infected. Based on limited observations, laminated root rot appears to be distributed both as "pockets" (aggregated) that appear as openings early in stand development and in a more "diffuse" manner that may lead to openings late in stand development. These findings have implications for disease survey and disease management. West. J. Appl. For. 12(2):49-51.