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The Pennsylvania State University The Graduate School Department of Plant Pathology WIDESPREAD OCCURRENCE AND EVOLUTION OF HUMAN PATHOGENIC FUSARIUM A Dissertation in Plant Pathology by Dylan Short Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy December 2011 The dissertation of Dylan Short was reviewed and approved* by the following: David M Geiser Professor of Plant Pathology Dissertation Advisor Chair of Committee Maria del Mar Jiminez Gasco Professor of Plant Pathology Eddie C Holmes Professor of Biology Seogchang Kang Professor of Plant Pathology Gary W Moorman Professor of Plant Pathology Frederick E Gildow Jr. Professor of Plant Pathology Head of the Department of Plant Pathology *Signatures are on file in the Graduate School iii ABSTRACT We tested the hypothesis that plumbing systems might serve as a significant environmental reservoir of human pathogenic isolates of Fusarium performing the first extensive multilocus sequence typing (MLST) survey of plumbing drain-associated Fusarium isolates, and comparing the diversity observed to the known diversity of clinical Fusarium isolates. We found that 66% of 471 sinks and 80% of 131 buildings surveyed yielded at least one Fusarium culture. The 297 isolates of Fusarium collected were subjected to MLST to identify the phylogenetic species and sequence types (STs) of these isolates. The six most common STs in sinks were identical to those most frequently associated with human infections. We speculate that the most prevalent STs, by virtue of their ability to form and grow in biofilms, are well adapted to plumbing systems. The most common group observed, the Fusarium solani species complex, (FSSC) is the most common group of fusaria associated with life- threatening opportunistic human infections as well as infections of the cornea. Here we present the description and taxonomy of Fusarium fistularum sp. nov., the single most common human pathogen in the genus Fusarium, that was previously known as FSSC Group 2. F. fistularum is genetically diverse, cosmopolitan and associated with biofilms on plumbing surfaces in the environment. Morphologically, F. fistularum isolates show high levels of variation in a range of characteristics that are typical for most concepts of ‘F. solani,’ with many isolates failing to produce sporodochia in culture and possessing aberrant iv morphological characters. Similar ranges of morphology were observed in three other commonly encountered phylogenetic species in FSSC. Secondary metabolites produced by F. fistularum include anhydrofusarubin, fusarubin, solaniol, and javanicin. A haematonectria-like heterothallic sexual stage was produced for F. fistularum; an epitype was provided for the sexual stage, and described as an addendum to the description of F. fistularum. Most pairings of isolates indicated possible high levels of infertility. In addition, based on a DNA sequence connection with an ex-Type culture, we apply the name F. petroliphilum, elevated to species status from F. solani var. petroliphilum, to another common Fusarium species associated with human infections and biofilms, FSSC 1. To better understand the population dynamics of this F. fistularum for purposes of epidemiology and control, we expanded an existing 3- locus MLST system by adding six novel sequence-based markers developed based on the complete genome sequence of Nectria haematococca Mating Population VI (FSSC 11). 9-locus MLSTs were generated for 231 isolates from six continents and from a variety of sources, including plumbing and from human and animal infections. High levels of genetic diversity and evidence for both recombination and clonality were detected among 111 unique 9-locus STs. Inclusion of the mating type as a tenth marker revealed ten additional STs, indicating that true clones are not well resolved even with ten loci. The most common ST (2-d2), with 49 members, was found in plumbing, contact lenses and lens cases, as well as fusarial keratitis. No evidence for population differentiation v between clinical isolates and isolates from environmental sources was found. Cryptic speciation with F. fistularum suggested in the previous three-locus MLST system was not supported with the addition of new loci, but evidence of introgression of ribosomal RNA genes from another strongly supported phylogenetic species also known from plumbing and human infections (FSSC 9), was detected in two isolates of F. fistularum. vi TABLE OF CONTENTS LIST OF FIGURES ......................................................................................... vii LIST OF TABLES ........................................................................................... x ACKNOWLEDGEMENTS ............................................................................... xi Chapter 1 Literature Review and Summary of Findings ................................. 1 Chapter 2 Widespread occurrence of pathogenic types of the fungus Fuasrium in bathroom sinks .................................................................... 28 Chapter 3 Fusarium fistularum sp. nov., a common human pathogen and inhabitant of plumbing-associated biofilms ............................................... 93 Chapter 4 Recombination, clonality, and hybridization in the polyextremophilic human pathogen Fusarium fistularum ......................... 136 Chapter 5 Future Directions........................................................................... 216 vii LIST OF FIGURES Figure 2-1: Frequencies of Fusarium species complexes characterized by MLST from A. clinical sources based on previous studies and B. drains based on the present study .......................................................... 34 Figure 2-2: Frequencies of 59 MLSTs represented by 297 fusaria isolated from drains in the present study .............................................................. 36 Figure 2-3: Frequencies of FOSC STs isolated from A. clinical sources from previous studies and B. from drains in this study. ............................ 37 Figure 2-4: Cladogram of the FSSC based on TEF, rDNA and RPB2 highlighting the spectrum of FSSC diversity found in drains ................... 38 Figure 2-5: Phylogram of the FDSC based on TEF, rDNA, and TUB highlighting the spectrum of FDSC diversity found in drains ................... 40 Figure 2-6: Phylogram of the FIESC based on TEF, rDNA, RPB2 and CAM highlighting the spectrum of diversity forund in drains ..................... 42 Figure 2-7: Generalized schematic map showing 57 collection areas (black dots) containing buildings yielding Fusarium ................................ 44 Figure 3-1: Phylogram of the FSSC based on TEF, rDNA, and RPB2 ......... 107 Figure 3-2: Sporodochial conidia (macroconidia) of five isolates of F. fistularum ................................................................................................. 112 Figure 3-3: Aerial conidia, monophialides, and chalmydospore of five isolates of F. fistularum ............................................................................ 113 Figure 3-4: Sporodochial conidia of three isolates of F. petroliphilum ........... 113 Figure 3-5: Aerial conidia of two isolates of F. petroliphilum .......................... 114 Figure 3-6: Colony pigmentation of eight isolates of F. fistularum grown on PDA. .................................................................................................... 114 Figure 3-7: Colony pigmentation of eight isolates of F. petroliphilum grown on PDA. .................................................................................................... 115 Figure 3-8: Red to scarlet perithecia from the cross of FRC S-2477 and FRC S-2391 ............................................................................................. 116 viii Figure 3-9: Asci and ascospores of the cross of FRC S-2477 and FRC S- 2391 ......................................................................................................... 117 Figure 4-1: Plot of mean number of genotypes vs. number of loci sampled created using Multilocus 1.2b. .................................................................. 166 Figure 4-2: Nine individual locus unrooted maximum parsimony trees created using the parsimony ratchet ........................................................ 170 Figure 4-3: Neighbor net of 111 unique 9-locus haplotypes created using SplitsTree4. .............................................................................................. 172 Figure 4-4: Population snapshots of F.fistularum created using Phyloviz Beta. ......................................................................................................... 175 Figure 4-S1a: First half of a maximum parsimony bootstrap tree for locus TEF. ......................................................................................................... 186 Figure 4-S1b: Second half of a maximum parsimony bootstrap tree for locus TEF. ................................................................................................ 187 Figure 4-S2a: First half of a maximum parsimony bootstrap tree for locus rDNA. ....................................................................................................... 188 Figure 4-S2b: Second half of a maximum parsimony bootstrap tree for locus rDNA ............................................................................................... 189 Figure 4-S3a: First half of a maximum parsimony bootstrap tree for locus RPB2. ......................................................................................................