Parvodontia relampaga sp. nov.: A Cystostereaceae fungal pathogen that is the causal agent of relampago blight of woody plants in Florida, USA.

Starting in the fall of 2019, mortality, blight symptoms, and signs of white fungal mycelia were observed on external host tissues of non-native landscape trees as well as numerous native trees, understory shrubs, and vines throughout northern and central Florida, USA. We determined that the fungus is an undescribed species of Basidiomycota based on morphological characteristics and DNA sequence analysis. Phylogenetic analyses of the internal transcribed spacer (ITS), large subunit (LSU), and translation elongation factor 1-alpha (tef1) regions revealed that this novel plant pathogen is an undescribed taxon of the genus Parvodontia (Cystostereaceae, Agaricales). We propose the name Parvodontia relampaga sp. nov. which describes its unique morphological features and phylogenetic placement. We confirmed the pathogenicity of P. relampaga in greenhouse inoculations on host plants from which strains of this novel pathogen were isolated, including the non-native gymnosperm Afrocarpus falcatus, the non-native and commercially important Ligustrum japonicum, and the native tree Quercus hemisphaerica. P. relampaga was also detected on a total of 27 different species of woody host plants, including such economically and ecologically important hosts as Fraxinus, Ilex, Magnolia, Persea, Prunus, Salix, Vitis, and Vaccinium. For this new plant disease, we propose the name "relampago blight," which refers to the lightning-like rhizomorph growth (relámpago means 'lightning' in Spanish). This study presents a newly discovered fungal taxon with a wide host range on both angiosperms and gymnosperms that may be an emerging pathogen of concern in Florida and the Gulf Coast region.

Southern blight of water lily: The first host record of Agroathelia rolfsii on Nelumbo nucifera discovered in Florida, USA.

Nelumbo nucifera Gaertn. (Nelumbonaceae, Eudicots), also known as water lily or sacred lotus, is a nonnative and invasive plant commonly found in artificial ponds and natural lakes throughout Florida (UF-IFAS 2023; Wunderlin et al. 2023). In August 2020, a single sample of water lily plants showing large leaf spots were collected at a residence in Dunnellon, Marion County, Florida (80% disease prevalence with 40% leaf coverage). Symptoms and signs of the disease were necrotized adaxial leaf spots only, bordered by whitish mycelia and hyphae with clamp connections, and whitish to light brown sclerotia formed in the center (<0.7 mm diameter). Symptomatic tissue was plated on acid potato dextrose agar (APDA) amended with chloramphenicol (100 mg/L) and ampicillin (30mg/L), and incubated at 20 °C for one week. Data supporting the molecular identification of this putative pathogen were gathered by PCR amplification and Sanger sequencing of the complete internal transcribed spacer (ITS) and a fragment of the large subunit (LSU) of the rRNA gene (~1.5 kb) using primers ITS1F and LR5 (FDACS-DPI PPST 2020-105211, GenBank OR492009) (White et al. 1990). The identification of the host was confirmed by Sanger sequencing of three plant barcode fragments: ITS2 (ITS2-S2F/ITS4, OR492008), ribulose 1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL) (rbcLa-F/rbcLa-R, GenBank OR502388), and Maturase K (matK) (matK-KIM1R/matK-KIM3F, GenBank OR502389) (Fazekas et al. 2012). MegaBLAST queries of the ITS/LSU sequence obtained here recovered a 99.61% match to the fungal pathogen Agroathelia (=Athelia) rolfsii (Sacc.) Redhead & Mullineux. (Redhead and Mullineux 2023) (Amylocorticiaceae, Agaricomycotina) strain GP3 (GenBank JABRWF010000005) (Yan et al. 2021). MegaBLAST queries of three host plant DNA barcodes recovered matches of greater than 99.62% similarity to N. nucifera sequences. After diagnosis, symptomatic dried leaf samples were deposited at Plant Industry Herbarium Gainesville (PIHG 17807) and an axenic culture was deposited at the Agricultural Research Services Culture Collection (NRRL 66964). Koch's postulates were fulfilled by the inoculation of sclerotia (as in Terrones-Salgado et al. 2022) on adaxial leaf surface of four-week- old water lily transplants obtained from an artificial pond on campus (two plants with five leaves each). One additional transplant was not inoculated and served as a control; this plant remained asymptomatic during the experimentation period. Each transplant was kept in a 27-gallon plastic container (21W × 30L × 14H in) filled with tap water containing one tablespoon of 20-20-20 all-purpose-water-soluble plant fertilizer (VPG, TX, USA) in a plant biosafety level 2 greenhouse (23 °C, >50% relative humidity, and a 12-h/12-h photoperiod). All inoculated leaves showed necrotized areas after one week and new sclerotia were observed floating on the water surface after three weeks. Fungal pathogen was reisolated and reidentified subsequently. Agroathelia rolfsii is the causal agent of southern blight, also known as grey rot, and is reported from at least in 260 plant genera, including specialty crops such as citrus, cucumber, pepper, peanuts, pumpkin, and strawberry (Farr and Rossman 2018). Agroathelia rolfsii usually causes lower stem, crown, and root rots; consequently, leaf spots are a noteworthy presentation of symptoms for this fungus.

Taxonomy and phylogeny of the genus Ganoderma (Polyporales, Basidiomycota) in Costa Rica.

Ganoderma species are well recognised by their significant role in the recycling of nutrients in ecosystems and by their production of secondary metabolites of medical and biotechnological importance. Ganoderma spp. are characterised by laccate and non-laccate, woody basidiocarps, polypore hymenophores and double-walled basidiospores generally with truncate apex. Despite the importance of this genus, its taxonomy is unclear and it includes several species' complexes with few circumscribed species and incorrect geographic distributions. The aim of this work was to provide detailed morphological descriptions together with phylogenetic analyses using ITS sequences to confirm the presence of seven species of Ganoderma in Costa Rica: G.amazonense, G.applanatum s.l., G.australe, G.curtisii, G.ecuadorense, G.oerstedii and G.parvulum. This is the first study that integrates morphological and phylogenetic data of Ganoderma from Central America and a key of the neotropical species. Besides, the distribution range of G.curtisii, previously reported from North America and G.ecuadorense from South America, is expanded to Central America.

Susceptibility of centipede tongavine (Epipremnum pinnatum) commercially grown in nurseries in Florida to aroid leaf rust (Pseudocerradoa paullula).

Epipremnum pinnatum (L.) Engl., (Araceae, Monocots) known as dragon-tail plant or centipede tongavine, is the most cultivated aroid species worldwide (Boyce 2004). In 2022, symptomatic dragon-tail plants, collected from plant nurseries in south Florida (e-Xtra Fig.1). Symptoms included round leaf spots often with a yellow halo and erupting pustules mainly distributed in the underside of the leaves. Visits to the nurseries revealed a 60% incidence of approximability 50 mature plants, with some leaves showing up to 30% of tissue damage. The putative pathogen was identified morphologically as Pseudocerradoa paullula (Syd. & P. Syd.) M. Ebinghaus & Dianese (Pucciniaceae, Basidiomycota) (Ebinghaus et al. 2022), characterized by the production of pseudosuprastomatal uredinia with globose to subglobose urediniospores, light-brown, echinulate (1 µm height), 24-31 µm diam with thick walls, 1.5-2.5 µm in height (n=30). Identical morphological features reported by Urbina et al. (2023) (e-Xtra Fig. 1). PCR amplification followed by Sanger sequencing of the internal transcribed spacer (ITS) and large subunit (LSU) of the ribosomal RNA genes (Aime 2006) together with LSU internal species specific primer (Urbina et al. 2023) were used to confirm the identification of the pathogen (GenBank ON887194-ON887196). MegaBlast (Chen et al. 2015) searches resulted in a >99% sequence similarity to a P. paullula specimen collected in Florida (2019-101665, GenBank ON887197). Host identification was made by using the Ribulose-1,5-bisphosphate carboxylase-oxygenase (rbcL. GenBank ON887186, ON887187) and Maturase K (matK) loci (GenBank ON887190, ON887191) (Fazekas et al. 2012). Both barcodes resulted in >99.13% sequence similarity to voucher J.R. Abbott 24912 FLAS (GenBank GU135198 and GU135036, respectively). Symptomatic dried specimens were deposited in the Plant Industry Herbarium (PIHG 16229 - 16232). Koch's postulates were fulfilled using urediniospores collected from an infected E. pinnatum sample that was kept in darkness at 4°C for seven days until inoculation. Eight potted dragon-tail plants were inoculated by hand rubbing urediniospores against upper and lower leaf surfaces and three plants were used as controls. All plants were misted with sterile water and covered with plastic bags (23 °C, >90% RH, 12/12 h daylight). Bags were removed 48 h after inoculation, plants were set in a climate-controlled greenhouse (~30 °C, ~65% RH, 12/12 h light cycle) and monitored daily for symptoms. Chlorotic spots appeared after 10 days, and pustules after 25 days while the non-inoculated controls remained symptomless. Aroid leaf rust is known to infect several aroid species, including dragon-tail (Shaw 1995), which some varieties capable to outdoors in USDA 9a hardiness zones (Wunderlin et al. 2023), but the rust fungus has not been observed on any species of Epipremnum in the landscape yet, suggesting that its susceptibility could be driven by plant growth conditions that favor pathogen infection (e.g., excess of humidity and nutrients, dense planting, overhead irrigation, etc.). Here we encourage dragon-tail plant growers to be aware of its susceptibility to P. paullula and to stay vigilant of the culture conditions to avoid plants from getting infected with this airborne pathogen.

The first USA continental record of coffee leaf rust (Hemileia vastatrix) on coffee (Coffea arabica) in southwest Florida, USA.

Coffee leaf rust (CLR), caused by Hemileia vastatrix Berk. & Broome (Zaghouaniaceae) is considered the most significant fungal disease of Coffea arabica L. (Rubiaceae), from which berries are harvested and processed to obtain coffee beverage (Talhinhas et al. 2017). In Florida, coffee plants are mainly used as ornamentals due to their fragrant flowers; however, there are ongoing field trials evaluating the adaptability of plants for coffee production to climate conditions in the state (Crane et al. 2005). In November 2021, young seedlings of C. arabica var. caturra from a residence in Naples (Collier County) in southwest Florida were discovered with signs of rust fungus. Minute, yellow, suprastomatal sori 53-81 µm in diam were formed on the abaxial leaf surface, forming blotches. Light-yellow urediniospores measured 29-31 × 20-29 µm, with a reniform or "hunchbacked" shape, had thick walls measuring 1.5-2.5 µm in height, and were dorsally echinulate, the individual spikes measuring 2.5-3.3 µm in height. Spikes were scattered over most of the dorsal surface and form a dense ridge separating the dorsal from the smooth ventral side. (e-Xtra Fig. 1). Symptoms and signs are consistent with published descriptions of CLR produced by H. vastatrix (Ritschel 2005). To confirm the identification, DNA sequencing of the large subunit (LSU) of the ribosomal repeat was done following the protocols of Aime (2006) (GenBank accession number OR296753-OR296754). The Florida specimen shares 100% sequence identity (887/728 bp) with other accessions of H. vastatrix in congruence with maximum likelihood phylogenetic analysis performed in RAxMLv8.0.0 (Stamatakis 2014) (e-Xtra Fig. 2). In addition to CLR, Hemileia coffeicola Maubl. & Roger, causal agent of powdery rust of coffee, produces similar leaf spots on coffee but has a restricted geographical distribution. This agent is found only above 500 m a.s.l. in central Africa (Silva et al. 2006) and produces larger urediniospores (34-40 × 20-28 µm) (Maublanc & Roger 1934) in sori are scattered in abaxial leaf surface giving a powdery appearance. Hemileia vastatrix has been reported from almost every major coffee growing country of the world as well as Hawaii and Puerto Rico (Keith et al. 2022, Ramirez-Camejo et al., 2022). This is the first report of CLR in the continental USA, however, CLR poses a limited threat to the USA agriculture in view of the fact coffee is not commercially grown within the continental USA. A voucher was made of dried symptomatic leaves and deposited at Plant Industry Gainesville Herbarium (PIHG 15712, 16332) and the Arthur Fungarium at Purdue University (PUR N23473). The remaining infested coffee seedlings were destroyed after phytopathological diagnosis, and the pathogen has been absent from all additional screenings since November 2021.

First Report of Leaf Blight Caused by Curvularia aeria and C. senegalensis on Tomato (Solanum lycopersicum L.) in Florida, USA.

Tomato (Solanum lycopersicum L.) is an important economic crop in Florida and worldwide. In November 2021, a leaf blight was reported on tomato plants (hybrid cherry and artisan tomatoes) from a small farm in Miami-Dade County, Florida. About 100 plants showed symptoms with disease severity of 15% and disease incidence of 80%. Symptoms on the leaves started as small dark spots and coalesced to form larger necrotic lesions over time. Symptomatic leaf tissues were cut into 5-mm pieces, surface disinfected with 70% ethanol for 30 s and 1% NaClO for 5 min, then cultured on PDA for 3 to 5 days at 25°C. Isolations were conducted in three rounds, with 15 samples in each round. Except for the saprophytes, fungal isolates of Curvularia were consistently recovered from tissues in each round. Single spore isolates grouped in two morphotypes (CT1 and CT3, CT2 and CT4) were examined for morphological and molecular identification. Colonies on PDA were dark yellow-green, with a fluffy surface, then both morphotypes turned black, although CT2 and CT4 were light yellow at the edges. CT1 and CT3 produced light-brown, straight to curved conidia with smooth walls and 1 to 3 septa, 18 to 28 ✕ 9 to 12 µm (n=60), and dark-brown stromatic synnemata (> 200 µm in length) in the center of the colony after ~30 days of incubation in PDA. CT2 and CT4 produced brown, mostly curved conidia, 14 to 23 ✕ 8 to 9 µm (n=60), with slightly rugose walls and 3-4 septa, without synnemata. A dehydrated culture of each isolate was deposited in the Plant Industry Gainesville Herbarium [(PIGH, accession numbers 17443 (CT1), 17444 (CT2), 17445 (CT3), 17446 (CT4)]. Total DNA was extracted using DNeasy Plant Pro Kit (Qiagen, Germantown, MD) followed by PCR amplification and Sanger sequencing of the internal transcribed spacers (ITS) and the Large Subunit (LSU) of the rRNA gene, together with the protein coding gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH), elongation factor 1- (TEF1) and -tubulin (TUB) (Marin-Felix et al. 2020; Himashi et al. 2021; Manamgoda et al. 2012; Myllys et al. 2002) (GenBank accession numbers ITS-LSU: OQ657944-OQ657947, GAPDH: OQ689438 to OQ689441, TEF1: OQ689442 to OQ689445, TUB: OQ689446 to OQ689449). Curvularia clavata as the molecular marker (96% identity) was used for identification. Sequence similarity of 100% in GAPDH, ITS and LSU was obtained in megaBLAST searches for both groups of morphotypes, CT1 and CT3 to Curvularia aeria (Bat., J.A. Lima & C.T. Vasconc.) Tsuda type culture CBS 294.61, and CT2 and CT4 to Curvularia senegalensis (Speg.) Subram. culture CBS 149.71. Pathogenicity tests were conducted with each isolate on six tomato plants that were 6- weeks-old. The seeds used in the tests were provided by the farm, and the variety 'Red Bounty' was also used. Inoculation was accomplished by spraying a spore suspension (1 x 106 spores/ml) of each of the four isolates (CT1 to CT4) and by placing 6-mm PDA plugs of the isolates on the leaves. Six tomato plants were used as the control. All plants were covered by plastic bags and placed in a greenhouse at 23-27°C. The inoculated plants developed small dark spots on leaves 2 weeks after inoculation, and the leaves inoculated by plugs of the fungal isolates had large necrotic lesions, which were similar to those observed on tomato plants from the field. The pathogenicity tests were repeated three times, Curvularia was consistently isolated from inoculated leaves after the symptoms developed, and they were confirmed morphologically in each test. No symptoms were observed from the control plants. Curvularia aeria and C. senegalensis are known foliar pathogens on several important crops, but not tomatoes. To our knowledge, this is the first report of C. aeria and C. senegalensis causing leaf blight in tomatoes worldwide. This finding is important because it will extend the host range of C. aeria and C. senegalensis to tomato, it also implied the essentiality of crop rotation in disease management.

Detection of aroid leaf rust Pseudocerradoa paullula on Swiss cheese plant Monstera deliciosa in the continental USA.

Monstera deliciosa Liebm. (Araceae, Monocots), sometimes referred to as Swiss cheese plant, is one of the most common aroids used as an indoor and landscape ornamental plant (Cedeño et al. 2020). Production of M. deliciosa and other closely related Araceae species represents an important sector of the ornamental nursery business worldwide. Swiss cheese plant is believed to have originated in the tropical forests of southern Mexico, where its fruit is considered a delicacy due to its sweet, exotic flavor (Cedeño et al. 2020). Since 2019, symptomatic Monstera plants from two plant nurseries and residential properties in South Florida were submitted for disease diagnosis to the Florida Department of Agriculture and Consumer Services, Division of Plant Industry (FDACS-DPI) in Gainesville, Florida, and to the University of Florida, Tropical Research and Education Center Plant Clinic in Homestead, Florida. Symptoms included small chlorotic spots on the leaf surface, which expanded and became brown to reddish-brown often with a yellow halo and produced uredinia with abundant urediniospores. The pathogen was identified morphologically as the rust fungus Pseudocerradoa (=Puccinia) paullula (Syd. & P. Syd.) M. Ebinghaus & Dianese (Pucciniaceae, Basidiomycota) (Ebinghaus et al. 2022), characterized by the production of pseudosuprastomatal uredinia. Uredinospores light-brown and globose, echinulate (1 µm height), reddish to light brown, 24 - 31 µm diameter, with thick walls, 1.5 - 2.5 µm height (n=15). Teliospores 2-celled, light-yellow and ellipsoidal, 23 - 28 × 19 - 24 µm (n =15) were observed in sori appearing as dark-brown leaf spots on the adaxial side of the leaves (e-Xtra Fig. 1). Molecular characterization of the fungal pathogen was based on the small subunit (SSU), internal transcribed spacer (ITS), and large subunit (LSU) of the ribosomal RNA genes (Aime 2006) with the addition of a LSU internal primer specific for the rust species Ppaullula_int-forward 5'ATAGTTATTGGCTTTGATTTACA-3' designed in this study to increase the quality and the sequence read length due to a 3'- ~21-Ts-homopolymer (e-Xtra Fig. 2) (GenBank accession number ON887196, ON887197, OQ275200, OQ275201). In addition to morphological identification, the host plant was identified using the Ribulose-1,5-bisphosphate carboxylase-oxygenase (rbcL) and Maturase K (matK) genes (Fazekas et al. 2012) (GenBank accession numbers ON887189, ON887193, respectively). MegaBlast searches confirmed the morphological identification with 100% identity to M. deliciosa vouchers GQ436772 and MK206496, respectively (Chen et al. 2015). Dried specimens were deposited in the Plant Industry Herbarium Gainesville (PIHG 16226, 16227, 17154, 17155). Molecular identification of the rust pathogen P. paullula was carried out through megaBlast (Chen et al. 2015) searches together with a phylogenetic analysis performed in RAxML v8 (Stamatakis 2014) (e-Xtra Fig. 3). Koch's postulates were performed by using urediniospores, collected from an infected sample and were kept for 7 days at 4 C, as an inoculum source. Healthy rooted M. deliciosa plants were inoculated by rubbing the inoculum on both leaf surfaces at >90% RH, room temperature, 12/12 light cycle. After the incubation period (48 h), plants were placed in a climate-controlled greenhouse and watered twice a week, ~30 C, ~65 RH, 12/12 light cycle. After three weeks, all inoculated plants developed symptoms resembling those observed on the samples submitted for disease diagnosis. Controls did not show symptoms. Spores from the pustules of inoculated plants were identified as P. paullula by both morphology and molecular means. The genus Pseudocerradoa comprises P. paullula and its sister species P. rhaphidophorae (Syd.) M. Ebinghaus & Dianese. Both species can be distinguished by size and coloration of urediniospores and their host range within the Araceae. Pseudocerradoa rhaphidophorae produces smaller urediniospores and only occurs on Rhaphidophora species (Shaw 1995). Pseudocerradoa paullula is not considered fully established in Florida, since the host distribution is mainly restricted to indoors and M. deliciosa is rarely used as an outdoor ornamental (Wunderlin et al. 2023). Here we name the disease caused by P. paullula as "aroid leaf rust", due to its ability to infect several species in this plant family. Other closely related hosts reported as susceptible to this pathogen are Monstera standleyana G.S.Bunting (as M.s. cv. variegata), Monstera adansonii var. laniata (Schott) Mayo & I.M. Andrade, Monstera subpinnata (Schott) Engl., Typhonodorum lindleyanum Schott, and Stenospermation sp. (Shaw 1991, 1992, 1995). To date, the aroid leaf rust was only known from Australia, China, Japan, Malaysia, and Philippines (Lee et al. 2012; Shaw 1991). Based on our review, P. paullulla was intercepted once from Malaysia in 2014 at the port of Los Angeles, USA (BPI voucher 893085). This present study reports the establishment of P. paullula in Florida, USA infecting M. deliciosa.

Provenance of rhizobial symbionts is similar for invasive and noninvasive acacias introduced to California.

Plant-soil interactions can be important drivers of biological invasions. In particular, the symbiotic relationship between legumes and nitrogen-fixing soil bacteria (i.e. rhizobia) may be influential in invasion success. Legumes, including Australian acacias, have been introduced into novel ranges around the world. Our goal was to examine the acacia-rhizobia symbiosis to determine whether cointroduction of non-native mutualists plays a role in invasiveness of introduced legumes. To determine whether acacias were introduced abroad concurrently with native symbionts, we selected four species introduced to California (two invasive and two noninvasive in the region) and identified rhizobial strains associating with each species in their native and novel ranges. We amplified three genes to examine phylogenetic placement (16S rRNA) and provenance (nifD and nodC) of rhizobia associating with acacias in California and Australia. We found that all Acacia species, regardless of invasive status, are associating with rhizobia of Australian origin in their introduced ranges, indicating that concurrent acacia-rhizobia introductions have occurred for all species tested. Our results suggest that cointroduction of rhizobial symbionts may be involved in the establishment of non-native acacias in their introduced ranges, but do not contribute to the differential invasiveness of Acacia species introduced abroad.

Lasiodiplodia iraniensis, a New Causal Agent of Tuber Rot on Yam (Dioscorea Species) Imported into the United States and Implications for Quarantine Decisions.

One negative consequence of international trade of agricultural commodities is the inadvertent global spread of crop diseases. Yam (Dioscorea spp.) is a staple food crop in many countries and is traded globally. Most of the commercially traded yams in the United States are imported. In late 2020, samples of yam tubers from a commercial facility were submitted to the plant diagnostic clinic at the UF/IFAS Tropical Research and Education Center in Homestead, Florida. Samples showed rotten symptoms and were drawn from lots that were marked to be destroyed because the source of the rotting symptoms was unknown. Preliminary isolation showed that a fungus was consistently associated with the symptoms and was confirmed in the subsequent pathogenicity test as the causal agent. The fungus grew profusely on potato dextrose agar (PDA) with highly melanized hyphae. Matured conidia showed longitudinal striations. Based on its growth pattern and morphology, it was suspected that this fungus may be in the genus Lasiodiplodia. DNA-based identification using partial sequences of the internal transcribed spacer (ITS), ß-tubulin (TUB2), 28S rDNA (LSU), and elongation factor alpha (EF1-α) genes confirmed the identity of the isolates as Lasiodiplodia iraniensis Abdollahz., Zare & A.J.L. Phillips (synonym: L. iranensis). This is the first report of L. iraniensis affecting yam and has implications for international trade. This finding will provide an important foundation for making quarantine decisions to prevent spread of this disease.

First report of cedar-quince rust Gymnosporangium clavipes on fruit of dwarf hawthorn Crataegus uniflora in Florida, USA.

The dwarf hawthorn Crataegus uniflora Münchh. (Rosaceae, Maloideae) is a small deciduous tree species native to the central and eastern US and south into northern Mexico. Dwarf hawthorn is drought tolerant and commonly found in disturbed areas (e.g., hedges and roadsides). In May 2021, we observed several individuals of dwarf hawthorn growing on the border of an empty field in the Natural Area Teaching Laboratory at The University of Florida main campus in Gainesville, Alachua County, Florida, USA (N29.633382, W82.368350) that were severely infected by fruit galls with visible, whitish aecia (e-Xtra Fig.1). The affected fruit were collected and transported to the Florida Department of Agriculture and Consumer Services - Division of Plant Industry headquarters in Gainesville for identification (FDACS-DPI, 2021-107788). The conspicuous rust fungus, occurring on the fruit (fructicolous), consisted of tubular aecia (roestelioid), 4 - 5 mm in length × 0.5 mm in diameter, with whitish peridia containing bright orange spores in masses. Aeciospores were semigloboid to globoid, some with an angular side, with bright orange contents, 26 - 31 µm in diameter (n= 20). The wall was densely verrucose, hyaline, 3 - 4 µm wide. Side and wall ornamentation are considered diagnostic features (EPPO Bulletin, 2006). Peridial cells of the aecia were hyaline, angular (pentagonal to hexagonal) to irregular, with a thin, convoluted wall, 41- 57 × 30 - 35 µm (n =10). Aeciospores were detected on blackish, mummified berries five months after the initial collection and aecium disappearance. This persistence demonstrates one of the adaptations allowing the pathogen to remain in a given location (e-Xtra Fig.1). The morphological characteristics are consistent with those of Gymnosporangium clavipes Cooke & Peck (Gymnosporangiaceace, Pucciniaceae, Pucciniomycotina) described by Kern (1973). A voucher was deposited in the DPI Herbarium (PIHG, specimen number 15618). The morphological identification was confirmed by molecular identification: following DNA extraction (DNeasy Plant Pro extraction kit, Qiagen Corporation, Hilden, Germany), we amplified a fragment of the internal transcribed spacer (ITS) and the large subunit (LSU) via PCR using the primer pairs Rust2inv/LR6 and Rust28S/LR5 (OK337508) (Aime, 2006); amplicons were then Sanger sequenced. NCBI megaBlast searches (Chen et al. 2015) of the resulting fungal sequences revealed high identity (ITS and LSU) to two G. clavipes vouchers: NYBG461394 (99.86%, Genbank accession no. MN605691) sequenced in the latest publication addressing species delimitation in Gymnosporangium (Zhao et al., 2020), and PPST 2020-104160 (99.72%, GenBank MW148514): the first report of this rust occurring on Crataegus marshallii Eggleston (McVay et al., 2021), also recently found in Gainesville, Florida. Phylogenetic analyses were carried out in the phylogenetic package RAxMLv8.0.0 (Stamatakis, 2014) (e-Xtra Fig. 2) further supports placement of 2021-107788 within G. clavipes. The heteroecious nature of this rust fungus precludes Koch's postulates. Based on exhaustive reviews of collection indices and literature, a specimen of G. clavipes on C. uniflora exists at the U.S. National Fungus collections (BPI 117783A) collected in Newfield, New Jersey in 1888 (Farr & Rossman, 2022); this rust fungus has a host range of at least 18 other species of Crataegus (Farr and Rossman, 2022; McVay et al., 2021; Zhao et al., 2020). This report represents the first published record of G. clavipes on dwarf hawthorn, and the first report in Florida.

Sporobolomyces lactucae sp. nov. (Pucciniomycotina, Microbotryomycetes, Sporidiobolales): An Abundant Component of Romaine Lettuce Phylloplanes.

Shifts in food microbiomes may impact the establishment of human pathogens, such as virulent lineages of Escherichia coli, and thus are important to investigate. Foods that are often consumed raw, such as lettuce, are particularly susceptible to such outbreaks. We have previously found that an undescribed Sporobolomyces yeast is an abundant component of the mycobiome of commercial romaine lettuce (Lactuca sativa). Here, we formally describe this species as Sporobolomyces lactucae sp. nov. (Pucciniomycotina, Microbotryomycetes, and Sporidiobolales). We isolated multiple strains of this yeast from commercial romaine lettuce purchased from supermarkets in Illinois and Indiana; additional isolates were obtained from various plant phylloplanes in California. S. lactucae is a red-pigmented species that is similar in appearance to other members of the genus Sporobolomyces. However, it can be differentiated by its ability to assimilate glucuronate and D-glucosamine. Gene genealogical concordance supports S. lactucae as a new species. The phylogenetic reconstruction of a four-locus dataset, comprising the internal transcribed spacer and large ribosomal subunit D1/D2 domain of the ribosomal RNA gene, translation elongation factor 1-α, and cytochrome B, places S. lactucae as a sister to the S. roseus clade. Sporobolomyces lactucae is one of the most common fungi in the lettuce microbiome.

Isolation and Molecular Characterization of the Romaine Lettuce Phylloplane Mycobiome.

Romaine lettuce (Lactuca sativa) is an important staple of American agriculture. Unlike many vegetables, romaine lettuce is typically consumed raw. Phylloplane microbes occur naturally on plant leaves; consumption of uncooked leaves includes consumption of phylloplane microbes. Despite this fact, the microbes that naturally occur on produce such as romaine lettuce are for the most part uncharacterized. In this study, we conducted culture-based studies of the fungal romaine lettuce phylloplane community from organic and conventionally grown samples. In addition to an enumeration of all such microbes, we define and provide a discussion of the genera that form the "core" romaine lettuce mycobiome, which represent 85.5% of all obtained isolates: Alternaria, Aureobasidium, Cladosporium, Filobasidium, Naganishia, Papiliotrema, Rhodotorula, Sampaiozyma, Sporobolomyces, Symmetrospora and Vishniacozyma. We highlight the need for additional mycological expertise in that 23% of species in these core genera appear to be new to science and resolve some taxonomic issues we encountered during our work with new combinations for Aureobasidiumbupleuri and Curvibasidium nothofagi. Finally, our work lays the ground for future studies that seek to understand the effect these communities may have on preventing or facilitating establishment of exogenous microbes, such as food spoilage microbes and plant or human pathogens.

Naming the untouchable - environmental sequences and niche partitioning as taxonomical evidence in fungi.

Due to their submerged and cryptic lifestyle, the vast majority of fungal species are difficult to observe and describe morphologically, and many remain known to science only from sequences detected in environmental samples. The lack of practices to delimit and name most fungal species is a staggering limitation to communication and interpretation of ecology and evolution in kingdom Fungi. Here, we use environmental sequence data as taxonomical evidence and combine phylogenetic and ecological data to generate and test species hypotheses in the class Archaeorhizomycetes (Taphrinomycotina, Ascomycota). Based on environmental amplicon sequencing from a well-studied Swedish pine forest podzol soil, we generate 68 distinct species hypotheses of Archaeorhizomycetes, of which two correspond to the only described species in the class. Nine of the species hypotheses represent 78% of the sequenced Archaeorhizomycetes community, and are supported by long read data that form the backbone for delimiting species hypothesis based on phylogenetic branch lengths.Soil fungal communities are shaped by environmental filtering and competitive exclusion so that closely related species are less likely to co-occur in a niche if adaptive traits are evolutionarily conserved. In soil profiles, distinct vertical horizons represent a testable niche dimension, and we found significantly differential distribution across samples for a well-supported pair of sister species hypotheses. Based on the combination of phylogenetic and ecological evidence, we identify two novel species for which we provide molecular diagnostics and propose names. While environmental sequences cannot be automatically translated to species, they can be used to generate phylogenetically distinct species hypotheses that can be further tested using sequences as ecological evidence. We conclude that in the case of abundantly and frequently observed species, environmental sequences can support species recognition in the absences of physical specimens, while rare taxa remain uncaptured at our sampling and sequencing intensity.

The Suhomyces clade: from single isolate to multiple species to disintegrating sex loci.

Candida tanzawaensis clade members are now placed in Suhomyces. The group was virtually unknown until the early 2000s. Here, we review progress made on Suhomyces over the last two decades and provide data from reports of new members of the group from distant localities worldwide, their habitats and a new study of mating loci that helps explain earlier failed compatibility tests. Phylogenetic studies indicate early diverging members are mostly associated with plants, but later diverging species are usually fungus-feeding insect associates. The genome of S. tanzawaensis was known to have a heterothallic mating allele arrangement with a single MAT α idiomorph. For this review, we generate sequence data and compare the MAT gene arrangement of 30 strains from nine Suhomyces species. These varied from MAT α loci containing mating genes α1 and α2, hypothetical MAT a loci without detectable mating genes a1 and a2 to truncated, possibly completely dissociated MAT loci with intraspecific variation. The absence of a second MAT in a genome locus precludes the possibility of mating type switching. Sympatric speciation likely occurred after MAT locus deterioration began in isolated habitats. Although asexual reproduction may be an effective short-term strategy, theory predicts it will not endure over the extreme long term.

Two new endophytic Atractiellomycetes, Atractidochium hillariae and Proceropycnis hameedii.

Sterile fungal isolates are often recovered in leaf and root endophytic studies, although these seldom play a significant role in downstream analyses. The authors sought to identify and characterize two such endophytes-one representing the most commonly recovered fungal isolate in recent studies of needle endophytes of Pinus taeda and the other representing a rarely isolated root endophyte of Populus trichocarpa. Both are shown by DNA sequencing to be undescribed species of Atractiellomycetes (Pucciniomycotina, Basidiomycota), a poorly characterized class of mostly plant-associated and presumably saprobic microfungi. The authors describe the new genus and species Atractidochium hillariae (Phleogenaceae) and the new species Proceropycnis hameedii (Hoehnelomycetaceae), both in the Atractiellales, to accommodate these unusual isolates. Following incubations of 1-2 mo, A. hillariae produces minute white sporodochia, similar to those produced by several other members of Atractiellales, whereas Pr. hameedii forms conidia singly or in chains in a manner similar to its sister species Pr. pinicola. Additionally, we provide a taxonomic revision of Atractiellomycetes based on multilocus analyses and propose the new genera Neogloea (Helicogloeaceae) and Bourdotigloea (Phleogenaceae) to accommodate ex-Helicogloea species that are not congeneric with the type H. lagerheimii. Atractiellomycetes consists of a single order, Atractiellales, and three families, Hoehnelomycetaceae, Phleogenaceae, and Helicogloeaceae. Accumulated evidence suggests that Atractiellomycetes species are common but infrequently isolated members of plant foliar and root endobiomes.

A closer look at Sporidiobolales: Ubiquitous microbial community members of plant and food biospheres.

Carotenoid-containing yeasts in Sporidiobolales (Microbotryomycetes, Pucciniomycotina, Basidiomycota) have been reported from contrasting ecosystems, including marine, soil, phylloplane, polar ice, and many others. Here, we present several analyses drawing on 583 new isolates collected from various substrates around the globe and publicly available sequences from numerous published environmental studies. We provide a multilocus phylogenetic reconstruction of the order, estimates for total species richness, a snapshot of global distribution patterns, and analysis of niche preferences in Sporidiobolales, emphasizing their occurrence in commercial crops and food products. We evaluated loci commonly used in fungal phylogenetics, finding that RNA polymerase II subunits 1 and 2 (RPB1, RPB2) are of little utility in this group. We have reconfirmed the monophyly of Sporidiobolales with three well-supported genera, which are, in descending order of number of species, Rhodotorula, Sporobolomyces, and Rhodosporidiobolus. From our data, we estimate ca. 260 species in Sporidiobolales, of which 42 are described, and ca. 52,000 species in Pucciniomycotina. The majority of data regarding Sporidiobolales are from North America and Europe, highlighting severe knowledge gaps for most of South and Central America and Africa.

Xylaria karyophthora: a new seed-inhabiting fungus of Greenheart from Guyana.

In 2010-2011, a Xylaria sp. was documented growing from seeds of both Chlorocardium rodiei and Chlorocardium venenosum, a commercially valuable timber in Guyana. We conducted extensive surveys in 2015-2016, where this Xylaria sp. was observed fruiting from ca. 80% of dispersed seeds in both natural and logged forests in the Upper Demerara-Berbice, Potaro-Siparuni, and the Cuyuni-Mazaruni districts of central Guyana. Species of Xylaria are ascomycetous fungi generally characterized by black, carbonaceous, multiperitheciate ascoma commonly found growing on dead wood. Combined teleomorphic and molecular data indicate that the fungus represents a novel species, described here as Xylaria karyophthora.

Specificity in Arabidopsis thaliana recruitment of root fungal communities from soil and rhizosphere.

Biotic and abiotic conditions in soil pose major constraints on growth and reproductive success of plants. Fungi are important agents in plant soil interactions but the belowground mycobiota associated with plants remains poorly understood. We grew one genotype each from Sweden and Italy of the widely-studied plant model Arabidopsis thaliana. Plants were grown under controlled conditions in organic topsoil local to the Swedish genotype, and harvested after ten weeks. Total DNA was extracted from three belowground compartments: endosphere (sonicated roots), rhizosphere and bulk soil, and fungal communities were characterized from each by amplification and sequencing of the fungal barcode region ITS2. Fungal species diversity was found to decrease from bulk soil to rhizosphere to endosphere. A significant effect of plant genotype on fungal community composition was detected only in the endosphere compartment. Despite A. thaliana being a non-mycorrhizal plant, it hosts a number of known mycorrhiza fungi in its endosphere compartment, which is also colonized by endophytic, pathogenic and saprotrophic fungi. Species in the Archaeorhizomycetes were most abundant in rhizosphere samples suggesting an adaptation to environments with high nutrient turnover for some of these species. We conclude that A. thaliana endosphere fungal communities represent a selected subset of fungi recruited from soil and that plant genotype has small but significant quantitative and qualitative effects on these communities.

Phosphorus cycling in deciduous forest soil differs between stands dominated by ecto- and arbuscular mycorrhizal trees.

Although much is known about how trees and their associated microbes influence nitrogen cycling in temperate forest soils, less is known about biotic controls over phosphorus (P) cycling. Given that mycorrhizal fungi are instrumental for P acquisition and that the two dominant associations - arbuscular mycorrhizal (AM) and ectomycorrhizal (ECM) fungi - possess different strategies for acquiring P, we hypothesized that P cycling would differ in stands dominated by trees associated with AM vs ECM fungi. We quantified soil solution P, microbial biomass P, and sequentially extracted inorganic and organic P pools from May to November in plots dominated by trees forming either AM or ECM associations in south-central Indiana, USA. Overall, fungal communities in AM and ECM plots were functionally different and soils exhibited fundamental differences in P cycling. Organic forms of P were more available in ECM plots than in AM plots. Yet inorganic P decreased and organic P accumulated over the growing season in both ECM and AM plots, resulting in increasingly P-limited microbial biomass. Collectively, our results suggest that P cycling in hardwood forests is strongly influenced by biotic processes in soil and that these are driven by plant-associated fungal communities.

Archaeorhizomyces borealis sp. nov. and a sequence-based classification of related soil fungal species.

The class Archaeorhizomycetes (Taphrinomycotina, Ascomycota) was introduced to accommodate an ancient lineage of soil-inhabiting fungi found in association with plant roots. Based on environmental sequencing data Archaeorhizomycetes may comprise a significant proportion of the total fungal community in soils. Yet the only species described and cultivated in this class is Archaeorhizomyces finlayi. In this paper, we describe a second species from a pure culture, Archaeorhizomyces borealis NS99-600(T) (=CBS138755(ExT)) based on morphological, physiological, and multi-locus molecular characterization. Archaeorhizomyces borealis was isolated from a root tip of a Pinus sylvestris seedling grown in a forest nursery in Lithuania. Analysis of Archaeorhizomycete species from environmental samples shows that it has a Eurasian distribution and is the most commonly observed species. Archaeorhizomyces borealis shows slow growth in culture and forms yellowish creamy colonies, characteristics that distinguish A. borealis from its closest relative A. finlayi. Here we also propose a sequence-based taxonomic classification of Archaeorhizomycetes and predict that approximately 500 species in this class remain to be isolated and described.