A Novel Yeast Genus and Two Novel Species Isolated from Pineapple Leaves in Thailand: Savitreella phatthalungensis gen. nov., sp. nov. and Goffeauzyma siamensis sp. nov.

Four yeast strains, representing one genus and two novel anamorphic yeast species, were isolated from pineapple leaves collected in Thailand. Analysis of the sequences of the D1/D2 domains of the large subunit (LSU) rRNA gene and the internal transcribed spacer (ITS) regions showed the two strains (DMKU-PAL186 and DMKU-PAL178) were closely related to the type strains of the Protomyces and Taphrina species, but with high nucleotide divergence. Two strains (DMKU-PAL39 and DMKU-PAL18) were found to be closely related to the type strains of Goffeauzyma iberica, but with eight nucleotide substitutions in the D1/D2 domains and 26 nucleotide substitutions in the ITS regions. In phylogenetic analyses, the strains DMKU-PAL186 and DMKU-PAL178 formed a well-separated lineage from Protomyces and Taphrina genera, confirming that they represented a distinct genus, while the strains DMKU-PAL39 and DMKU-PAL18 represented a species in the genus Goffeauzyma, which was phylogenetically distinct from other recognized species of the genus. Based on molecular analyses and phenotypic characteristics, the names Savitreella gen. nov. (Taphrinomycetes, Ascomycota) and Savitreella phatthalungensis sp. nov. are proposed to accommodate the strains DMKU-PAL186 and DMKU-PAL178, and the name Goffeauzyma siamensis sp. nov. (Tremellomycetes, Basidiomycota) is proposed to accommodate the strains DMKU-PAL39 and DMKU-PAL18.


Introduction
Pineapple (Ananas comosus), belonging to the family Bromeliaceae, is a well-known and readily available tropical fruit. The global production of pineapple is projected to grow at 1.9 percent annually, to reach 31 million tonnes in 2028 [1]. In Thailand, the pineapple is one of the most important plants to be grown commercially. Pineapple is a source of functional ingredients (fiber) [2], anti-inflammatory agents (bromelain) [3], antioxidants (flavonoids and phenolic acids), and vitamins (vitamin C) [4]. It has a high sugar content (sucrose, glucose, and fructose) [5] and is consequently the habitat of several microorganisms, including lactic acid bacteria (i.e., Lactobacillus plantarum and L. rossiae) and yeasts (i.e., Meyerozyma caribbica and Pichia guilliermondii) [6,7]. However, there are only a few reports of yeast being associated with the pineapple phyllosphere. Pineapple leaves exhibit an extreme environment involving such factors as high temperature, variable relative humidity, radiation, and acidic conditions [8]. Therefore, microorganisms that can grow on pineapple leaves need to be adapted to survive under variable and severe conditions. gen., sp. nov. (DMKU-PAL186 and DMKU-PAL178) and Goffeauzyma siamensis sp. nov. (DMKU-PAL39 and DMKU-PAL18) were proposed based on a polyphasic taxonomy.

Sample Collection and Yeast Isolation
Green and healthy leaves of pineapple (Ananas comosus) were randomly collected from cultivated fields in Chon Buri province (21 samples), eastern region of Thailand, on December 26, 2020, and Phatthalung province (10 samples) southern region of Thailand, on January 13, 2021. Leaf samples (five leaves per sample) were put in plastic bags, sealed, and kept in icebox for 6-12 h during transfer to the laboratory. The samples were stored at 8 • C until subjected to yeast isolation, which was no longer than 48 h. The yeasts were isolated by a dilution plate technique from the healthy pineapple leaves. The leaves (5 g) were suspended in 50 mL of 0.85% saline solution in a 250 mL Erlenmeyer flask and shaken on a rotary shaker at 150 rpm, 25 • C for 60 min to detach yeast cells from the surface. The suspension was serially diluted tenfold (1:10 to 1:10 3 ) by sterile 0.85% (w/v) saline solution; then, 100 µL of each dilution was spread onto yeast extract-malt extract (YM) agar (0.3% (w/v) yeast extract, 0.3% (w/v) malt extract, 0.5% (w/v) peptone, 1% (w/v) glucose, and 2% (w/v) agar) supplemented with 0.02% (w/v) chloramphenicol and incubated at 25 • C for seven days. The different yeast morphotypes were picked and purified by streaking on YM agar. All purified yeast strains were suspended in YM broth supplemented with 10% (v/v) glycerol and stored at −80 • C.

Biochemical and Physiological Studies
Four strains (DMKU-PAL186, DMKU-PAL178, DMKU-PAL39, and DMKU-PAL18) were characterized biochemically and physiologically, according to the standard methods described by Kurtzman et al. [24]. Fermentation of carbohydrates was carried out in a liquid medium using Durham fermentation tubes. Carbon source and nitrogen source assimilation tests were conducted in liquid medium and starved inocula were used in nitrogen assimilation tests [24]. Cycloheximide resistance was also performed in a liquid medium, while urea hydrolysis was conducted on agar slants. Acid production and the diazonium blue B (DBB) reaction were investigated on a solid medium in Petri dishes. Growth at various temperatures (15,30,35,37, and 40 • C) was determined by cultivation on YM agar.

Molecular Study
Each yeast strain was grown on YM agar for two days. The target gene sequences were determined from polymerase chain reaction (PCR) products amplified from genomic DNA extracted from yeast cells. The methods used for DNA extraction and amplification were as described by Limtong et al. [25]. The D1/D2 domains of the LSU rRNA gene, ITS regions, SSU rRNA gene, TEF1, and RPB2 genes were amplified with the primer pairs NL1 and NL4 (for D1/D2 domains) [26], ITS1 and ITS4 (for ITS regions) [27], SSU1f and SSU4r, SSU3f and SSU2r (for SSU) [28], EF1-983f and EF1-2218r (for TEF1) [29], and RBP2-5f and RPB2-7r (for RPB2) [30]. The PCR products were checked by agarose gel electrophoresis and purified using the TIANgel Midi Purification kit (TIANGEN Biotech), according to the manufacturer's protocol. The purified PCR product was sequenced with the same primers used in PCR amplification at the First BASE Laboratories Sdn Bhd, Malaysia. The newly generated sequences were submitted to GenBank (http://www.ncbi.nlm.nih.gov/genbank, accessed on 30 November 2021) and the GenBank accession number of the sequences used in this study are shown in Tables 1 and 2.
* Gene sequences: SSU, nuclear small subunit rRNA gene; ITS, internal transcribed spacer regions; LSU, D1/D2 domains of the nuclear large subunit rRNA gene; RPB2, RNA polymerase II; TEF1, translation elongation factor 1-alpha. The newly generated sequences are in boldface. Type strains indicated by "T".
Sequences generated from the forward and reverse primers were aligned and assembled with MEGA software, version 11 (MEGA11) [31], to get full length of the gene sequence. Then, the assembled sequences were compared with the GenBank database using the BLASTn search tool [32]. Multiple sequence alignment was performed using the MUSCLE algorithm. Phylogenetic analyses, based on the concatenated sequences of the ITS regions and the D1/D2 domains of the LSU rRNA gene and the multilocus datasets, using the neighbor-joining (NJ) and the maximum likelihood (ML) methods were performed with MEGA11 software. The Kimura-2 parameter distance correction and the general time reversible (GTR) models were respectively used for the NJ and ML analyses. Confidence levels of the clades were estimated from bootstrap analysis (1000 replicates) [33].
During the investigation of yeasts associated with pineapple leaves collected from cultivated fields in Thailand, two yeast strains (DMKU-PAL186 and DMKU-PAL178) representing a novel yeast taxon of the Taphrinomycetes, Ascomycota were discovered. These two strains were found to be closely related to the type strains of the Protomyces and Taphrina species. In addition, another two strains (DMKU-PAL39 and DMKU-PAL18) representing a novel species of the genus Goffeauzyma (Tremellomycetes, Basidiomycota) were also obtained.

Strain DMKU-PAL186 and DMKU-PAL178
Analysis of the sequences of the D1/D2 domains of the LSU rRNA gene and ITS regions demonstrated that the strains DMKU-PAL186 and DMKU-PAL178 were identical. BLASTn searches of the GenBank database revealed that the D1/D2 sequence of the two strains DMKU-PAL186 T and DMKU-PAL178 are closely related to the type strains of Protomyces, whereas the ITS sequences of these two strains are closely related to the type strains of the Taphrina species, followed by the Protomyces species. Consequently, all Protomyces species (P. inouyei, P. pachydermus, P. lactucaedebilis, P. macrosporus, P. gravidus, P. inundatus, and P. arabidopsidicola) and six of the Taphrina species (T. virginica, T. wiesneri, T. letifera, T. communis, T. deformans, and T. carnea) showing a high degree of similarity in the BLAST searches were selected to compare genetic divergences. In terms of pairwise sequence similarities, the two strains (DMKU-PAL186 and DMKU-PAL178) differed from the type strains of the Protomyces species by 52-66 nucleotide (nt) substitutions in the D1/D2 domains and differed from a group of the related Taphrina species, by 63-65 nt substitutions in the D1/D2 domains (Table 3). In addition, the ITS sequences of the two strains differed from the Protomyces species and the Taphrina species by 57-106 and 89-114 nt substitutions, respectively (Table 3). In phylogenetic analysis based on the concatenated sequences of the ITS and the D1/D2 domains of the LSU rRNA gene, the trees derived from neighbor-joining (NJ) and maximum likelihood (ML) analysis were found to be similar and showed that the two strains formed a monophyletic clade closely related to Protomyces and Taphrina species, with strong statistical support (Figure 1). To clarify the placement of the two strains, the phylogenetic analysis of a multilocus dataset (SSU, ITS, LSU, TEF1, ACT1, and RPB2) needs to be conducted. However, the sequence data of the other marker genes (e.g., RPB2, TEF1, and ACT1) of several Protomyces and Taphrina species are not available in the public online databases. Consequently, the phylogenetic analysis based on the combined sequences of the SSU, ITS, and the D1/D2 domains of the two strains, and the type strains of representative species in the Taphrinomycotina with available SSU rRNA data, were performed in this study. From these analyses, the NJ and ML trees made it clear that the two strains formed a well-separated lineage from the neighboring genera, Protomyces and Taphrina (Figure 2), and confirmed the presence of the monophyletic clade that was phylogenetically distinct from any recognized families with yeast state in the Taphrinomycotina (Protomycetaceae, Schizosaccharomycetaceae, and Taphrinaceae) as well as two recognized genera in the Taphrinomycotina incertae sedis (Saitoella and Novakomyces), providing deeper level support (100% bootstrap support) for considering them as a distinct species and genus (Figures 1  and 2). Since the family assignment of the Taphrinomycotina is still unclear, we chose not to assign the family of the new genus and have left open the family assignment until the existing family structure within the Taphrinomycotina is better clarified, based on eventual phylogenomic data.  In the comparison between morphological and phenotypic characteristics of the proposed novel species and type strains of the related species in genera Protomyces and Taphrina, we found the colonies of the strain DMKU-PAL186 T on YM agar are cream to light pinkishred, convex and with entire margins ( Figure 3B), which is consistent with the colony color of Protomyces species reported by Kurtzman [9] and Wang et al. [12]. However, these two strains can be distinguished from their phylogenetically closest recognized neighbors by some phenotypic characteristics, as shown in Table 4. The proposed novel species assimilates D-gluconate, while the related Protomyces and Taphrina species do not. Several Protomyces and Taphrina species assimilate D-arabinose, L-arabinose, and potassium nitrate, but the proposed novel species does not. Starch formation is negative for the proposed novel species but is positive for the Protomyces (weak positive) and Taphrina species. Growth at 30 • C is present for the proposed novel species but is absent for the related Protomyces and Taphrina species. Based on polyphasic analyses (genetic divergent, phylogenetic, and phenotypic characteristics), we concluded that these two strains, DMKU-PAL186 and DMKU-PAL178, represent a novel genus and species of the subphylum Taphrinomycotina. The name Savitreella phatthalungensis gen. nov., sp. nov. is proposed to accommodate these yeast strains.  Assimilation of carbon compound D-Galactose - Data for species 1 is from the present study, for species 2-7 and 9-15 from Kurtzman [9] and for species 8 from Wang et al. [12].

DMKU-PAL39 and DMKU-PAL18
The sequence analyses of the D1/D2 domains of the LSU rRNA gene and ITS regions revealed that the two strains (DMKU-PAL39 and DMKU-PAL18) had identical sequences, indicating that the two strains represented a single species. Pairwise sequence comparison of the D1/D2 domains and ITS regions of strain DMKU-PAL39 with respect to its related species showed that these strains were distinct from their closest species, Goffeauzyma iberica CBS 10871 T , which differed by 8 nt substitutions (1.4%) of 570 nt in the D1/D2 domains and 26 nt substitutions of 454 nt (6.2%) in the ITS regions.
A phylogenetic analysis based on the combined sequences of the ITS regions and the D1/D2 domains of the LSU rRNA gene demonstrated that the strains DMKU-PAL39 and DMKU-PAL18 placed in the Goffeauzyma species, but in a distinct position from the other Goffeauzyma species in the clade, with relatively high bootstrap support (Figure 4). To confirm the placement of these two strains, phylogenetic analyses of the combined sequences of the SSU rRNA, ITS regions, D1/D2 domains of the LSU rRNA, RPB2 and TEF genes of these two strains and the type strains of the Goffeauzyma species were performed by using the NJ and ML methods. From these analyses, the NJ and ML trees were found to be similar, making it clear that the two strains were placed in the Goffeauzyma clade and formed a well-separated lineage closely related to Goffeauzyma iberica ( Figure 4). In practice, Goffeauzyma siamensis nov. is not only distinguishable on the basis of molecular analyses but also by some phenotypic characteristics and growth abilities (Table 5). G. siamensis sp. nov. assimilates glycerol, ribitol, D-gluconate and DL-lactate, whereas G. iberica does not. Growth on medium containing 0.01% cycloheximide is absent for G. siamensis but is present for G. iberica. Based on this evidence, we therefore concluded that the strains DMKU-PAL39 and DMKU-PAL18 represent a single novel species of the genus Goffeauzyma. The name Goffeauzyma siamensis sp. nov. is proposed to accommodate these yeast strains. Data for species G. siamensis from this study and for G. iberica is from Gadanho and Sampaio [20]. Description: The genus can be separated from the neighboring genera, Protomyces and Taphrina, by phylogenetic analyses (Figures 1 and 2) and phenotypic characteristics (Table 4). Yeast cells divide by multilateral budding. Pseudohyphae are not produced. Ascospores have not been observed in individual or in mixed cultures. Glucose is not fermented. Diazonium blue B reaction, urease activity and acid production are negative. The genus is phylogenetically related to Protomyces and Taphrina.

Taxonomic Description of Genus and New Species
Classification: Taphrinales, Taphrinomycetes, Taphrinomycotina, Ascomycota. Holotype: DMKU-PAL186 T is the holotype of Savitreella phatthalungensis. It was isolated from a pineapple leaf collected from pineapple cultivated field, Phatthalung province, Thailand. It has been preserved as a metabolically inactive state in the culture collection of the Department of Microbiology, Faculty of Science, Kasetsart University, Bangkok, Thailand (DMKU). The culture ex-type has been deposited in a metabolically inactivate state in the Thailand Bioresource Research Center (TBRC), National Center for Genetic Engineering and Biotechnology, Thailand as TBRC 15109 and the Portuguese Yeast Culture Collection (PYCC), Portugal as PYCC 9005.

Discussion
In accordance with the guidelines for ascomycetous yeast identification based on nucleotide sequence divergences, the two compared strains with 0-3 nucleotide differences in the D1/D2 domains are designated to be the same species, whereas the strains that differed by greater than 6 nucleotides (1%) are recognized as different species [26]. For basidiomycetous yeasts, the strains that differed by two or more nucleotides in the D1/D2 regions represent different taxa [34]. Later, Vu et al. [35] proposed the threshold to consider a strain to belong to a different species from its close relatives is of less than 98.31% similarity (Ascomycota) or 98.61% similarity (Basidiomycota) in terms of ITS region and less than 99.41% similarity (Ascomycota) or 99.51% similarity (Basidiomycota) when considering the D1/D2 domains. In the present study, the sequences of the D1/D2 of the LSU rRNA gene and ITS regions of the four strains were compared with their closely related species. Two strains (DMKU-PAL186 and DMKU-PAL178) were identical in the D1/D2 and ITS regions but differed from the type strains of the closely related species by 52-66 nucleotide substitutions in the D1/D2, and by 57-114 nucleotide substitutions in the ITS regions, while two strains (DMKU-PAL39 and DMKU-PAL18) were identical in both rRNA regions but differed from the type strains of the closely related species by eight nucleotide substitutions in the D1/D2, and by 10 nucleotide substitutions in the ITS regions. According to the criteria mentioned above, these strains are sufficiently separated from known species. Consequently, we were justified in assigning the two strains (DMKU-PAL186 and DMKU-PAL178) as a novel genus and species (namely, Savitreella phatthalungensis gen., sp. nov.) of the Taphrinomycetes (phylum Ascomycota), and two strains (DMKU-PAL39 and DMKU-PAL18) as a novel species (namely, Goffeauzyma siamensis sp. nov.) of the Tremellomycetes (phylum Basidiomycota). In practice, the two novel species can be distinguished from their closest related species not only by the analysis of nucleotide sequence divergence but also by phylogenetic analyses and phenotypic characteristics.
The members of Protomyces and Taphrina have been isolated from various plants, such as giant ragweed (Ambrosia trifida), hawk's-beard (Crepis japonica), wild celery (Apium nodiflorum), the leaf gall of hedge parsley (Torilis japonica), Arabidopsis (Arabidopsis thaliana) [9,11], grey alder (Alnus incana), pear (Pyrus communis), damson plums (Prunus insititia), and white or silver-leaved poplars (Populus alba) [13]. Based on the evidence provided above, it has been suggested that the Protomyces and Taphrina species are plant-associated genera. However, there is a member of the Protomyces species (P. inouyei) that has not only been obtained from a plant but also isolated from ice in a glacier cave [36]. In addition, the members of both Protomyces and Taphrina are generally recognized as phytopathogens. Most Protomyces species are plant parasites causing gall symptoms in the flowers, stems, leaves, and fruits of Compositiae and Umbelliferae [9,11,37]. A large number of Taphrina species cause symptoms similar to those seen as a result of Protomyces infections, such as galls on stems, leaf curls, and witches' brooms of various plants, especially economically important fruit trees, viz., peach, plum and cherry [17]. In the present study, the two strains (DMKU-PAL186 T and DMKU-PAL178) were isolated from healthy cultivated pineapple leaves. The pathogenicity of the strains DMKU-PAL186 T and DMKU-PAL178 were evaluated on pineapple leaves in a greenhouse for three weeks and the results revealed no damage or disease symptoms on the tested leaves. Thus, it could be hypothesized that these two strains might be a saprobic stage, which allows them to survive outside the host plant, or that the pineapples are non-host plants for this species. The distribution of this species may be due to human or insect transmission. In this respect, these strains (DMKU-PAL186 T and DMKU-PAL178) differ from the genus Protomyces and Taphrina.
The members of genus Goffeauzyma have been isolated from a wide range of habitats associated with acidic conditions, such as the acidic water of a volcanic environment in Argentina (G. agrionensis) [21], acid rock drainage in Iberian pyrite belt ecosystems in Portugal (G. aciditolerans, G. iberica and G. metallitolerans) [20], stomach lavage of a tuberculosis patient in Norway (G. gastrica) [22], soil and litter of an acidophilous beech forest in Austria (G. gastrica), and in soil from a tundra wetland at Cape Barrow in Alaska (G. gilvescens) [22]. In this study, the strains DMKU-PAL39 and DMKU-PAL18 were isolated from pineapple leaves, which were considered to be an acidic environment (pH 3-4), in Thailand. Thus, the species in genus Goffeauzyma appeared to occur commonly in diverse acidic environments and different geographical locations. In the samples from which the strains of the novel species were obtained, other yeast species were also isolated, namely, Hannaella pagnoccae, Papiliotrema sp., Rhodotorula toruloides, Rhodosporidiobolus ruineniae, Symmetrospora suhii, Saitozyma sp., and Tremella sp. This likely means that these yeast species are adapted to survive under acidic conditions. In order to evaluate differences in their genome that justify their adaptation to these environments, the genome of these new isolates should be sequenced.

Conclusions
A novel yeast genus and two novel species were described and illustrated. Based on the molecular analyses and phenotypic characteristics, the name Savitreella phatthalungensis gen. nov., sp. nov. is proposed in the phylum Ascomycota, and the name Goffeauzyma siamensis sp. nov. is proposed in the phylum Basidiomycota. In the case of Savitreella phatthalungensis, the pathogenicity of this species on pineapple leaves was examined, and no damage or disease symptoms were observed on the tested leaves.