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.

It is now known that yeasts previously placed in form genus Sporobolomyces can be found across the three subphyla of Basidiomycota [6,7] and even in Ascomycota [8]. Sporobolomyces salmonicolor, however, belongs to Sporidiobolales (Pucciniomycotina, Microbotryomycetes), and Sporidiobolus is now considered a synonym of Sporobolomyces [9]. Currently, 22 species of Sporobolomyces sensu stricto (s.s.) are accepted [3,10,11], although estimates are that the genus contains upwards of 60 species [12]. All known species in the genus produce an asexual morph and reproduce by ballistoconidia. Some species produce a sexual morph and pseudohyphae in addition to yeast cells [13].
Yeasts in the genus Sporobolomyces are known for their bright red, orange, or pink appearance in culture [2] and have been studied for a variety of applications. The red pigmentation of the yeasts is due to the production of carotenoids such as beta-carotene [14,15]. The carotenoid-producing capability of Sporobolomyces yeasts has been of interest to the field of biotechnology to develop commodities such as pigments [14][15][16]. Sporobolomyces roseus exhibits antimicrobial activity inhibiting the growth of Pseudomonas fluorescens and Staphylococcus aureus, two bacteria that are both known to infect humans as opportunistic pathogens [17]. As a biological control agent, S. roseus has been effective against Cochliobolus sativus (common root rot) [18].
Sporobolomyces yeasts grow in various habitats, such as aquatic systems, soil, and plant phylloplanes [2,7,10,12]. They are best known for their association with plant leaves, with many organisms from the genus first isolated from the phylloplane [12,19]. Sporobolomyces yeasts are cosmopolitan in this regard and are capable of growing on a wide variety of plants, including agricultural crops [12,[20][21][22][23][24]. The yeasts of Sporidiobolales are known to inhabit vegetable surfaces with little, if any, association with food spoilage [25,26].
We have previously found that the yeasts of Sporidiobolales are represented in the phylloplane of commercially grown romaine lettuce [12], which is consistent with a previous study on the lettuce microbiome [23]. We then characterized the mycobiome (fungi of the microbiome) of romaine lettuce obtained in Urbina and Aime [12]. By conducting this characterization, we found that over 25% of the mycobiome is represented by a single Sporobolomyces species, which is previously undescribed [24]. Although basidiomycetous yeasts comprise a small fraction of the romaine lettuce phylloplane microbiome, Sporidiobolales yeasts are the most common fungi present [12] and are represented overwhelmingly by Sporobolomyces spp. [24]. Here, we describe the most abundant of these red yeasts, Sporobolomyces lactucae sp. nov., and discuss its ecology and natural range.

Lettuce Leaf Preparation and Culturing of Fungal Isolates
Lettuce leaf homogenization and culture plating were completed as described in previous work [12,24]. In brief, commercial lettuce was purchased from grocery stores in Illinois and Indiana. Lettuce leaves were homogenized in 225 mL of 100 µM phosphate buffer (5.4 g monosodium phosphate L −1 and 8.7 g disodium phosphate L −1 ). Aliquots were plated on yeast extract-peptone-glucose agar with 25 µg chloramphenicol mL −1 and 50 µg ampicillin mL −1 to inhibit bacterial growth (BD, Franklin Lakes, NJ, USA; Thermo Fisher, Waltham, MA, USA). Isolates collected in California were obtained by using the ballistospore drop method [27]. Sections from leaves collected in the field were secured with petroleum jelly to the inside of a Petri plate lid. Ballistospores that dropped from the leaf-inhabiting yeasts grew on either potato dextrose agar (PDA) or yeast malt agar (YMA) (BD; Thermo Fisher). Subculturing was repeated until axenic cultures were obtained and maintained on PDA. Back-up cultures for long-term preservation were prepared in 40% (v/v) glycerol for −80 • C storage and on PDA slants for 4 • C storage. Working cultures were incubated on PDA at room temperature (25 • C). Live cultures were deposited in the Agricultural Research Service Culture Collection (NRRL) and the Westerdijk Fungal Biodiversity Institute (CBS). All isolates obtained for this study and their origins are presented in Supplementary Table S1.

Morphological and Physiological Characterization
The morphological and physiological description was performed according to Suh et al. [28]; their protocols conform to the standard outlined in The Yeasts [29]. Culture morphology was observed on YMA, corn meal agar (CMA), and YM broth (BD; Thermo Fisher). After seven-day incubation, colonies were described in terms of elevation, margin, color (oac; [30]), form, and surface texture. Individual cells were examined under a compound microscope (Olympus BH-2; Tokyo, Japan). Micrographs were captured using an Olympus SC30 camera. A total of 30-180 cells were measured per isolate per treatment using Piximètre v5.10 (http://www.piximetre.fr/, accessed on 4 May 2021). Carbon and nitrogen assimilations were performed according to Suh et al. [28]. Positive assimilations marked with "+++" or "++" were reassigned as "+" while negative assimilations were assigned as "−"; weak growth was denoted as "(w)", and delayed growth was denoted as "(d)". This modified scale was used to normalize our data and allow for comparison across previously published data with varying scales.
PCR protocols followed Toome et al. [37] for SSU, ITS, and LSU. For tef1, we used a touchdown PCR protocol as in Wang et al. [38]. For cytb, we followed Wang and Bai [39]. Purification and Sanger sequencing with amplification primers were outsourced to Genewiz, Inc. (South Plainfield, NJ, USA). Raw sequence reads were assembled and edited in Sequencher v. 5.2.3 (Gene Codes, Ann Arbor, MI, USA). Newly generated sequences were submitted to NCBI GenBank (accession numbers in Supplemental Table S1).

Phylogenetic Inferences and Species Concepts
Generated sequences of each locus were blasted against the NCBI GenBank standard nr/nt nucleotide database (http://ncbi.nlm.nih.gov/blast/Blast.cgi, accessed on 27 January 2022) to confirm identity [40]. For phylogenetic placement of our isolates, we downloaded ITS, LSU, tef1, and cytb ex-type sequences of Sporobolomyces from GenBank, following Urbina and Aime [12], Li et al. [11], and Tan et al. [41] as a guide for taxon selection to represent the 22 currently accepted species (Table 1). Rhodosporidiobolus microsporus and Rhodotorula babjevae were selected as outgroup taxa [12]. Sequences were aligned using MUSCLE v.3.8.1551 via the CIPRES Science Gateway [42,43]. The newly created multiple sequence alignment for each dataset was trimmed using TrimAI v.1.2.59 via CIPRES [43,44]. After trimming, single-locus trees were constructed using the Random Axelerated Maximum Likelihood (RAxML) v.8 program [45] available through CIPRES [43]. We applied a genealogical gene concordance hypothesis for species delimitation [54] by using information from multiple loci to establish overlapping phylogenies that are in consensus. Single-locus trees were constructed as above. Concordance was determined by hand. The SSU region was not informative and, thus, was not used in the multi-locus phylogenetic reconstruction.

Environmental Determination of S. lactucae
To infer the broader distribution and habitats of S. lactucae, a second dataset was constructed of ITS sequences. The holotype strain of S. lactucae sp. nov. was pairwise aligned with environmental sequences deposited in GenBank that shared ≥98.0% identity. Environmental sequences were edited, aligned, and trimmed as above. We reconstructed an ITS-based phylogeny of the environmental sequences via IQ-TREE and inferred matching S. lactucae isolates based on this phylogeny. The final environmental dataset included 118 ITS sequences, 19 of which are Sporobolomyces s.s. type species, and was evaluated by using the K3Pu + F + G4 model (−lnL = 2533.283).

Phylogenetic Analyses
By performing gene genealogical concordance tests, each single-locus phylogenetic tree supported the delineation of S. lactucae as a new species. A congruence test of the four loci (ITS, LSU, tef1, and cytb) showed similar results with respect to our isolates. These single-locus phylogenies can be found in Supplemental Figures S1-S4. After determining concordance, a multi-locus phylogeny was constructed.
The final four-locus dataset (Figure 1)   In total, we recovered 66 S. lactucae isolates from commercial romaine lettuce. Additionally, we recovered 27 S. lactucae isolates from the phylloplanes of Rhamnaceae (Ceanothus arboreus), Plantaginaceae (Antirrhinum majus), and Lilaceae in two different years, al within the San Francisco Bay Area region of California (Figure 2; Supplemental Table S1) To better infer true S. lactucae environmental sequences, we reconstructed an ITS-based phylogeny. For our environmental dataset, we found 99 Sporobolomyces ITS sequences from ten studies or surveys (including the present study) that shared at least 98% sequence identity with S. lactucae. In total, we recovered 66 S. lactucae isolates from commercial romaine lettuce. Additionally, we recovered 27 S. lactucae isolates from the phylloplanes of Rhamnaceae (Ceanothus arboreus), Plantaginaceae (Antirrhinum majus), and Lilaceae in two different years, all within the San Francisco Bay Area region of California (Figure 2; Supplemental Table S1). To better infer true S. lactucae environmental sequences, we reconstructed an ITS-based phylogeny. For our environmental dataset, we found 99 Sporobolomyces ITS sequences from ten studies or surveys (including the present study) that shared at least 98% sequence identity with S. lactucae.
Etymology: lactucae (Latin), referring to the genus of the lettuce plant from which the holotype isolate was sourced.
Diagnosis: Similar to S. jilinensis and S. roseus but differing in the ability to assimilate glucoronate and D-glucosamine but not lactate or citrate.
Habitat and distribution: on leaf surfaces, particularly those of agricultural products, in mild or Mediterranean climates.
Description: In the asexual state, colonies are orange-pink in color (oac616) after 7 d incubation at 25 • C on PDA and YMA. Colonies are smooth and glistening, varying between circular and irregular with the entire margin. Colonies are raised in elevation. After 7 d incubation in YM broth, single cells appeared ellipsoidal, 5-11 µm × 3-5 µm, and uninucleate. On CMA, cells measured 5-10 µm × 3-6 µm. A single large vacuole forms in the cells. The formation of ballistoconidia was observed on CMA; new cells arise from sterigmata on mother cells. Pseudohyphae were not observed, but small chains of cells (usually about three cells) were rarely observed. No sexual stage was observed.

Discussion
Sporobolomyces lactucae was the most frequently isolated yeast from phylloplanes of commercial romaine lettuce purchased from grocery stores in Illinois and Indiana, USA [24]. Of the thousands of phylloplane isolates of Sporidiobolales we have made throughout the world ( [12]; M.C. Aime, unpubl.), we have recovered S. lactucae only from plant samples in the San Francisco and Monterrey Bay areas of California (Supplemental Table S2); identical sequences have been detected by others in areas across the world that have similar climates ( Table 3). The majority of lettuce produced in the USA is grown in California [60], and we were able to trace the romaine lettuce origins for most of our samples to the Salinas Valley in California (Supplemental Table S3). It is well-documented that pathogenic microorganisms can spread through the production, distribution, and preparation of food, with greater risks of foodborne illnesses for foods consumed raw such as lettuce [61]. It stands to reason that commensal organisms are moved through our food distribution systems as well. In this case, S. lactucae isolated from lettuce purchased in the Midwest may very well have originated in California. The ability to trace commensal microbial organisms through food distribution may improve our ability to trace pathogenic outbreaks. Much like other members of Sporidiobolales, S. lactucae appears to be widespread, occupying various habitats [12]. We found sequences consistent with S. lactucae in public databases from regions that experience a Mediterranean climate ( Table 3). Half of the environmental samples were isolated in Egypt. The other half are distributed across Réunion Island (Indian Ocean), South Africa, Greece, Portugal, Granada, and Pullman (Washington, USA). Additionally, our environmental analysis indicates the potential presence of S. lactucae in Antarctica as well as Yunnan Province, China. Although the substrate from which environmental sequences were obtained vary, most of these originated from agricultural samples. Substrates were either agricultural crops, such as grape berries; in food products such as strawberry or guava juice; or flowers of agricultural or horticultural plants.
Supplementary Materials: The following supporting information can be downloaded at the following website: https://www.mdpi.com/article/10.3390/jof8030302/s1, Figure S1: Maximum likelihood tree of Sporobolomyces s.s. species using the cyt-b locus, Figure S2: Maximum likelihood tree of Sporobolomyces s.s. species using the ITS locus, Figure S3: Maximum likelihood tree of Sporobolomyces s.s. species using the LSU D1/D2 locus, Figure S4: Maximum likelihood tree of Sporobolomyces s.s. species using the tef-1 locus, Table S1: Isolates that were examined in the description of the new species Sporobolomyces lactucae. All HU strains were isolated via lettuce leaf homogenization and plated. All MCA strains were isolated via spore drop method, noted as SDC (spore drop culture) in the table below [27]. Holotype is denoted in bold, Table S2: Carbon and nitrogen assimilations and high osmotic pressure tests for all Sporobolomyces s.s. species included in phylogenetic analysis in this manuscript. Weak = w; delayed = d; variable = v, and Table S3: Lettuce origins of samples from which S. lactucae was isolated based on publicly available data. Lettuce samples were cross-referenced with brand and retailer. Suppliers of identified brands and/or retailers were determined via a public search engine.