Allozyme Differentiation of Intersterility Groups of Heterobasidion annosum Isolated from Conifers in the Western United States

Otrosina, W. J., Chase. T. E.. and Cobb, F. W., Jr. 1992. Allozyme differentiation of intersterility groups of Hererobasidion isolated from conifers in the western United States. Phytopathology 82:540-545. Allozyme analysis was conducted on 64 isolates from basidiocarps of Heterobusidion unnosum. The isolates belonged lo the “S” and “P” intersterility groups and were collected from live conifer species found in the western United States. Ten allozyme loci distributed among eight enzyme systems were examined. Intersterility groups differed at nine loci. Few alleles were common lo both intersterility groups, and only one locus, MDH-I, was monomorphic. Several loci, including MDH-2, GDH, and PGI, were diagnostic for the S or P intersterility groups, suggesting fixation for alternative or null alleles. Distribution of alleles between Addiricmd keywords: Basidiomycetes, biological species, isozymes. intersterility groups indicates an extremely high degree of genetic divergence; Nei’s genetic distance was equal to 0.926. Isolates from diseased pine trees had only alleles consistent with those of P group, and isolates from diseased true fir had S group alleles, indicating a complete association among intersterility groups, allozyme profiles, and host species. These data support a hypothesis of genetic isolation between intersterility groups in nature. Allozyme genotypes provide a rapid test for identifying intersterility groups of the fungus in North America. Heterobasidion anbosum (Fr.:Fr.) Bref. (=Fomes annosus (Fr.:Fr.) Cooke) is a destructive pathogen that causes root and butt rot of coniferous tree species in temperate zone forests throughout the world. In California alone, about 3.3 million hectares of commercial forest lands is estimated to be affected by this pathogen (32). In pine species, the fungus colonizes recently cut s tumps via airborne deposit ion of basidiospores. The fungus then colonizes adjacent healthy trees through root contacts and &tfts. In addi t ion to stump infect ions, basal wounds and other infect?on courts may account for infection of Abies spp. and possibly other nonpine species. Once present in a stand, the fungus can survive for decades in resinous woody t issues and can cause continued mortal i ty via spread through root contacts to adjacent trees and in new seedling reproduction (21). Until recently, H. annosum was regarded by forest pathologists as a s ingle taxonomic unit . Korhonen (17), however, demonstrated two intersterility groups (ISGs) or biological species of the fungus. He designated these “S” and “P”. The S group was isolated T h i s a r t i c l e i s i n t h e p u b l i c d o m a i n a n d n o t c o p y r i g h t a b l e . I t m a y b e f r e e l y reprinted with customary crediting of the source. The American Phytopathological Society, 1992. 5 4 0 P H Y T O P A T H O L O G Y primarily from butt-rotted Picea abies (L.) H. Karst. and Pinus sylvestris L. saplings. The P group was isolated from P. sylvesrris and a variety of other conifers and hardwood species. Later, Chase (6) and Chase and Ullrich (9) showed ihat both the S and P ISGs are present in the western United States. Similar results were obtained by Harrington et al (15). Numerous field obs&vations and some experimental inoculation studies with seedlings of different host species (11,35) indicate considerable differentiat ion in the fungus relat ive to host range or host special izat ion. Allotyme analyses have been used by many researchers to determine interand intraspecific variation in various fungal species (e.g., l&19,37). However, very few studies have dealt with interrelationships between intersterility barriers and allozyme differentiation. In the genus Pleurotus, intersterility studies have demonstrated complete reproductive isolat ion between morphological ly indist inguishable or barely dis t inguishable species (3) . Relationships between intersterility barriers and isozymes have been studied in the genus Armillaria, a taxon containing important forest tree root pathogens that have undergone comparatively little morphological differentiation (22). Similarly, the S and P ISGs of H. annosum in the United States are not easily distinguishable on the basis of macroscopic cri teria, and further comparat ive morphological s tudies remain to be done. May and Royse (I 8) employed allozyme analysis to separate isolates of Pfeurotus species into specific classes, and they also have provided evidence of species misidentif icat ion among certain groups of this fungus. This study examines the relationships between ISGs of H. annosum and allozyme structure of populations of this fungus in California and Oregon, where there are numerous conifer hosts of this pathogen. Preliminary results of this work have been publ ished (10,27). MATERIALS AND METHODS Fungus isolates and hosts. Collections were made from disease centers in both pine and true fir stands and in other host tree species in the area ranging from the Fremont National Forest in southern Oregon to the San Bernardino National Forest in southern California. A small number of isolates was obtained from sites in Montana and Arizona. Host tree species included Jeffrey pine (Pinus jeffreyi E. Murray), ponderosa pine (Pinus ponderosa Douglas ex P. Laws. & C. Laws), white fir (Abies concolor (Gordon & Glend.) Lindl ex Hildebr.), red fir (Abies magntjica Andr. Murray), Coulter pine (Pinus coulteri D. Don), single-leaf pinyon (Pinus monophylla Torr. & F&m.), giant sequoia (Sequoiadendron giganreum (Lindl.) Buchholz), western juniper (Juniperus occidentalis Hook.), incense cedar (Libocedrus decurrens Torr.), and manzanita (Arcrosraphylos uva-ursi (L.) Spreng.). Isolates of H. annosum were obtained from the context t issue of basidiocarps or from infected root t issue of symptomatic seedlings, larger trees, or stumps. In a few instances, polybasidiospore isolates were obtained from spore castings of individual basidiocarps. Geographic origins, host substrate, and number of isolates obtained for both allozyme analysis and intersterility test ing are given in Table 1. TABLE 1. Conifer hosts, sample origins, numbers of isolates identified by intersterility groups used in isozyme study, and MDH-2 diagnostic alleles used in the study of Hererobusidion annosum’ Host substrate Sample oriainb Number of isolates MDH-2 allele S group isolates White fir stumps