APPLIED ANDENVIRONMENTALMICROBIOLOGY, 0099-2240/00/$04.0010Oct. 2000, p. 43564360Vol. 66, No. 10Copyright 2000, American Society for Microbiology. All Rights Reserved.PCR Primers That Amplify Fungal rRNA Genes from Environmental SamplesJAMES BORNEMAN*ANDR. JACK HARTINDepartment of Plant Pathology, University of California, Riverside, California 92521Received 28 March 2000/Accepted 9 July 2000Two PCR primer pairs were designed to amplify rRNA genes (rDNA) from all four major phyla of fungi: Ascomycota, Basidiomycota, Chytridomycota, and Zygomycota. PCRs performed with these primers showed that both pairs amplify DNA from organisms representing the major taxonomic groups of fungi but not from nonfungal sources. To test the ability of the primers to amplify fungal rDNA from environment samples, clone libraries from two avocado grove soils were constructed and analyzed. These soils possess different abilities to inhibit avocado root rot caused by Phythophthora cinnamomi. Analysis of the two rDNA clone libraries revealed differences in the two fungal communities. It also revealed a markedly different depiction of the soil fungalcommunity than that generated by a culture-based analysis, confirming the value of rDNA-based approaches for identifying organisms that may not readily grow on agar media. Additional evidence of the usefulness of the primers was obtained by identifying fungi associated with avocado leaves. In both the soil and leaf analyses,no nonfungal rDNA sequences were identified, illustrating the selectivity of these PCR primers. This work demonstrates the ability of two newly developed PCR primer sets to amplify fungal rDNA from soil and plant tissue, thereby providing unique tools to examine this vast and mostly undescribed community of organisms.Fungi play complex and diverse roles in ecosystems and human society. They comprise the majority of the biomass in soil, decompose organic material, provide nutrients to plants, and act as indicators of ecosystem health (2, 7). In agriculture, fungi can both devastate crop yields and provide a means to control plant pests, including other fungi (29). In humans, fungi can cause a range of conditions from psoriasis to men- ingitis. They have also proven to be effective curative agents; for example, Saccharomyces boulardii can prevent Clostridiumdifficile toxicity and other intestinal disturbances caused by antibiotic usage (6). In the biotechnology arena, fungi produce numerous secondary metabolites that have valuable pharma- ceutical properties (24). Their importance to this industry and other bioprospecting endeavors is enhanced by the vast diver- sity of extant fungi (10). Although considerable knowledge about fungi has been amassed, most of these organisms remain uncharacterized. Es- timates suggest that there are 1.5 million species of fungi on Earth; however, only approximately 70,000 species have been described, leaving 95% of the species undescribed (16). Thereasons for this deficiency include habitats that have not beenwell investigated, organisms that are difficult to culture axeni- cally, such as obligately associated fungi, and inaccurate iden-tification of catalogued samples (16). Strategies used to rectifythis deficit should include the use of comparative sequence analysis of rRNA and rRNA genes (rDNA), which has led to the discovery of many new bacterial and archaeal phylotypes in environments such as Yellowstone hot springs, soil, and rock (3, 22). Several PCR primers that amplify fungal rDNA from a wide range of taxonomic groups have been described (31), but few of these were designed for use with environmental samples.Such a tool must have high specificity, as fungal DNA may be rare compared to DNA from other sources, such as bacteria,plants, or other eukaryotes (14). The ITS1-F and ITS4-B prim- ers have been used to amplify basidiomycete ITS1, ITS2, and 5.8S rDNA sequences from plant tissues containing fungi (12). Similarly, the VANS1 primer has been used in combination with other primers to amplify rDNA from vesicular-arbuscular endomycorrhizal fungi (27). To identify disease-causing fungi,PCR primers have been designed to specifically amplify both human (4, 18, 21) and plant (17) pathogens. In addition, three PCR primer pairs described by Smit et al. were recently used to amplify fungal rDNA from wheat rhizosphere samples (28). In this report, we describe two new PCR primer pairs designed to amplify rDNA from all major taxonomic groups of fungi, and in this study we demonstrated the use of these primer pairs by examining the fungal communities of two avocado grove soils.MATERIALS AND METHODSPrimer design. A total of 213 fungal small-subunit rDNA sequences of rep- resentatives of all major phylogenetic groups were obtained from GenBank (National Center for Biotechnology Information NCBI) and were aligned with PILEUP (Genetics Computer Group, Madison, Wis.). Conserved sequenceswithin this group were identified with PRETTY (Genetics Co