Cladosporium Species Associated with Fruit Trees in Guizhou Province, China

During an investigation of fungal diversity on fruit trees in Guizhou Province, 23 Cladosporium strains were isolated from various locations in Guizhou Province. Culture characteristics, morphology and molecular phylogenetic analysis of three genetic markers, namely, the internal transcribed spacer regions (ITS) of the rDNA, partial fragments of actin (act), and the translation elongation factor 1-α (tef1-ɑ) loci were used to characterize these isolates. Seven new Cladosporium species and new host records for five other species were introduced, with detailed descriptions and illustrations. This study showed that there is a rich diversity of Cladosporium spp. in fruit trees in Guizhou Province.


Introduction
Cladosporium is accommodated in a large family, Cladosporiaceae, with its asexual morphs being dematiaceous hyphomycetes [1]. The conidia of Cladosporium usually form in branched chains and are so small that they can spread easily as one of the most common air-borne microorganisms [2][3][4]. This genus includes more than 878 epithets in the Index Fungorum database (https://www.indexfungorum.org/; accessed on 4 December 2022). The species are commonly discovered as endophytes, plant pathogens, human pathogens, and hyperparasites of other fungi [5][6][7][8][9]. A strain of C. cladosporioides may have potential as a biocontrol agent for reducing apple scab caused by Venturia inaequalis in leaves and fruit [10]. Cladosporium spp. can also produce compounds of medical interest or as potential biocontrol agents for other plant diseases [11,12].
The present study uses three gene loci to establish a phylogenetic tree for our new strains and provides a morphological comparison and colony characteristics for 23 Cladosporium isolates obtained from fruit trees in Guizhou Province, China.
In the phylogenetic tree, all 96 known Cladosporium taxa and our 23 strains formed a strong clade (100% ML/100% MP/1 PP). Our fungal strains were scattered in six phylogenetic Clades ( Figure 1). Over half of our strains (12)  . Only one of our strains (GUCC 21262.1) in clade 3 had no phylogenetic distance from the ex-type strain of C. eucommiae. Clade 4 included two taxa (C. cucumerinum and C. subuliformae) and three of our strains (GUCC 21212.1, GUCC 21208.1 and GUCC 21208.2), which formed a branch (99% ML/98% MP/1 PP) with an ex-type culture of C. subuliformae. For clade 5, strain GUCC 21260.3 had a close relationship to C. xylophilum (CBS 125997T and CBS 113749) (99% ML/99% MP/1 PP), but with some phylogenetic distance. The two remaining strains (GUCC 21267.1 and GUCC 21271.5) were accommodated in clade 6, with GUCC 21267.1 being C. xanthochromaticum and GUCC 21271.5 forming a branch with an ex-type strain of C. perangustum (CBS 125996) (94% ML/100% MP/1 PP). The DNA base differences on different gene loci between our Cladosporium strains and their relatives are summarized in Table 3.
Culture characteristics: Colonies on PDA reaching 48-62 mm diam. after 2 weeks at 25 • C, olivaceous brown or pale olivaceous brown, dull-yellow toward the margins, reverse deep black-yellow, dull yellow toward the margins, fluffy-felty, margin broad, feathery, somewhat undulate, aerial mycelium abundant, loose to dense, low to high, sporulating. Colonies on MEA reaching 47-55 mm diam. after 2 weeks at 25 • C, olivaceous brown at margins where sporulation is profuse, reverse dark brown, fluffy-felt, margin pale yellow, feathery, aerial mycelium abundant, loose to high, colony center folded and wrinkled, radially furrowed, without prominent exudates. Colonies on SNA reaching 48-61 mm diam. after 2 weeks at 25 • C, light brown to dark brown, flat, velvety, sparse, margin regular, aerial mycelium loose diffuse, pale yellow and uniform and regular color. Without prominent exudates, sporulation profuse on all media.
Notes: Phylogenetically, Cladosporium kaiyangensis was placed in the root of clade 1 but only with the BP value (0.96) (Figure 1). Cladosporium kaiyangensis is morphologically comparable with C. colocasiae, but it has generally shorter conidiophores (23.5-123.0 × 3.0-7.0 vs. 110-180 × 4-6 µm), slightly shorter and narrower secondary ramoconidia and conidia, and somewhat wider conidiogenous loci and hila. The conidia of C. kaiyangensis are narrower than those of C. tenuissimum, C. colocasiae, C. oxysporum, C. prunisalicina and C. congjiangedsis, which are all located close to each other in the phylogenetic tree. Cladosporium colocasiae, C. tenuissimum and C. oxysporum have gray-olivaceous to olivaceous or dull green colonies on PDA [15], while those of C. kaiyangensis are olivaceous brown or pale olivaceous brown with dull-yellow toward the margins. Additionally, the margin of C. kaiyangensis's colony did not have a light white ring or dark brown ring similar to C. pruni-salicina and C. congjiangedsis, nor was there an obvious radial furrow. Notes: Phylogenetically, Cladosporium kaiyangensis was placed in the root of clade 1 but only with the BP value (0.96) (Figure 1). Cladosporium kaiyangensis is morphologically comparable with C. colocasiae, but it has generally shorter conidiophores (23.5-123.0 × 3.0-7.0 vs 110-180 × 4-6 µm), slightly shorter and narrower secondary ramoconidia and conidia, and somewhat wider conidiogenous loci and hila. The conidia of C. kaiyangensis are narrower than those of C. tenuissimum, C. colocasiae, C. oxysporum, C. pruni-salicina and C. congjiangedsis, which are all located close to each other in the phylogenetic tree. Cladosporium colocasiae, C. tenuissimum and C. oxysporum have gray-olivaceous to olivaceous or dull green colonies on PDA [15], while those of C. kaiyangensis are olivaceous brown or pale olivaceous brown with dull-yellow toward the margins. Additionally, the margin of C. kaiyangensis's colony did not have a light white ring or dark brown ring similar to C. pruni-salicina and C. congjiangedsis, nor was there an obvious radial furrow.   Index Fungorum number: IF900110 Etymology: ribus, in reference to the plant genus (Ribes burejense), from which the fungus was isolated.
Culture characteristics: Colonies on PDA reaching 50-65 mm diam. after 2 weeks at 25 °C, smoke-gray and olivaceous due to abundant and dense aerial mycelium, olivaceous gray toward the margins, reverse dull olive-brown, with a whitish narrow final edge, fluffy, margins narrow, white, somewhat feathery, regular or slightly undulate, growth flat, sporulation loose. Colonies on MEA reaching 40-52 mm diam. after 2 weeks at 25 °C, smoke-gray to light olive-gray due to abundant aerial mycelium, reverse dull olivebrown, velvety or fluffy, with a dark olivaceous brown narrow final edge. Colonies on Culture characteristics: Colonies on PDA reaching 50-65 mm diam. after 2 weeks at 25 • C, smoke-gray and olivaceous due to abundant and dense aerial mycelium, olivaceous gray toward the margins, reverse dull olive-brown, with a whitish narrow final edge, fluffy, margins narrow, white, somewhat feathery, regular or slightly undulate, growth flat, sporulation loose. Colonies on MEA reaching 40-52 mm diam. after 2 weeks at 25 • C, smoke-gray to light olive-gray due to abundant aerial mycelium, reverse dull olive-brown, velvety or fluffy, with a dark olivaceous brown narrow final edge. Colonies on SNA reaching 45-55 mm diam. after 2 weeks at 25 • C, gray-olivaceous to olivaceous, olivaceousgray reverse, flat, velvety, sparse mycelium, margin regularly. Without prominent exudates, sporulation profuse on all media.
Culture characteristics: Colonies on PDA reaching 55-68 mm diam. after 2 weeks at 25 • C, dull yellow brown due to abundant and dense aerial mycelium, dull brown to pale yellow brown, with white margins, reverse dull olive-brown, with a pale yellow narrow final edge, fluffy, margins narrow, somewhat feathery, regular or slightly undulate, growth flat, somewhat radially furrowed, sporulation loose. Colonies on MEA reaching 52-65 mm diam. after 2 weeks at 25 • C, pale yellow brown due to abundant aerial mycelium, reverse dull olive-brown, velvety or fluffy, with a pale white-yellow narrow final edge, glabrous to somewhat feathery, aerial mycelium brown, floccose, abundant, dense, somewhat radially furrowed. Colonies on SNA reaching 65-78 mm diam. after 2 weeks at 25 • C, pale yellow, flat, velvety, margin regular, growth effuse to low convex, reverse light yellow. Without prominent exudates, sporulation profuse on all media.
Culture characteristics: Colonies on PDA reaching 58-70 mm diam. after 2 weeks at 25 • C, pale yellow brown due to abundant and dense aerial mycelium, reverse dull olive-brown, with a pale yellow narrow final edge, fluffy, margins narrow, feathery, aerial mycelium loose, diffuse, growth flat, radially furrowed. Colonies on MEA reaching 60-75 mm diam. after 2 weeks at 25 • C, pale yellow brown due to abundant aerial mycelium, reverse dull olive-brown, sometimes yellowish white at margins, margins narrow, glabrous or feathery, radially furrowed, folded and wrinkled in colony center, aerial mycelium sparse, diffuse. Colonies on SNA reaching 46-58 mm diam. after 2 weeks at 25 • C, pale yellow-brown, flat, powdery to felty-floccose, velvety, margin regularly, growth effuse to low convex, reverse light yellow. Without prominent exudates, sporulation profuse on all media.
Notes: The phylogenetic position of Cladosporium punicae (GUCC 21271.5) was sister to the ex-type culture of C. perangustum (CBS 125996) with high statistical support (ML/MP/BI = 94/100/1) (Figure 1). The comparison of DNA base composition indicated that between GUCC 21271.5 and CBS 125996, there were only 2 base differences in the ITS region, but 31 base differences in the tef 1-A region and 7 base differences in the act region ( Table 3). The colony of C. punicae is pale yellow-brown on PDA and dull olive-brown on MEA, but C. perangustum is olivaceous-gray or iron-gray on PDA and pale olivaceous-gray to glaucous-gray or dull gray-olivaceous on MEA. Additionally, C. punicae produces slightly wider conidia and secondary conidia than C. perangustum [15]. Thus, Cladosporium punicae was proposed as a new species. that between GUCC 21271.5 and CBS 125996, there were only 2 base differences in the ITS region, but 31 base differences in the tef1-ɑ region and 7 base differences in the act region ( Table 3). The colony of C. punicae is pale yellow-brown on PDA and dull olive-brown on MEA, but C. perangustum is olivaceous-gray or iron-gray on PDA and pale olivaceousgray to glaucous-gray or dull gray-olivaceous on MEA. Additionally, C. punicae produces slightly wider conidia and secondary conidia than C. perangustum [15]. Thus, Cladosporium punicae was proposed as a new species.
Culture characteristics: Colonies on PDA reaching 58−70 mm diam. after 2 weeks at 25 • C, gray olivaceous or olivaceous, with yellowish-white margins, margin narrowed, reverse dull olive-brown, with yellow margins, margin broad, regular, glabrous to feathery, fluffy, aerial mycelium loose, diffuse, growth flat. Colonies on MEA reaching 40−55 mm diam. after 2 weeks at 25 • C, olivaceous, brown olivaceous and pale whitish-yellow toward the margins, reverse dull olive-brown, margin broad, radially furrowed, with raised, cratershaped colony center, with white, undulate, submerged margin. Colonies on SNA reaching 45−62 mm diam. after 2 weeks at 25 • C, dull olive-brown, flat, powdery to felty-floccose, velvety, margin regular, growth effuse to low convex, reverse light yellowish. Without prominent exudates, sporulation profuse on all media. Notes: Our strain (GUCC 21267.1) formed a branch of the ex-type strain of C. xanthochromaticum (CBS 140691) with high statistical support (ML/MP/BI = 100/100/1) in Figure 1. The comparison of DNA base composition (Table 3) indicated that GUCC 21267.1 and CBS 140691 had identical sequences in the act region and only three base differences in the tef 1-A region and three in the ITS region. In morphology, GUCC 21267.1 shares almost the same colony morphology, apical conidia and secondary conidia as the description of C. xanthochromaticum [17]. Thus, GUCC 21267.1 is a new Chinese record of Hylocereus undatus 'Foo-Lon'.

Discussion
Twenty-three Cladosporium isolates were obtained from ten plant hosts in six counties in Guizhou Province. Phylogenetic analyses indicated that they belonged to six clades (Clade 1 to Clade 6), which did not suggest powerful host speciation. The most prominent morphological feature typical of Cladosporium spp. is a thick refractive to darkened cladosporioid or coronate scar, defined as a raised periclinal rim with a central convex dome. The conidia are frequently 2-3-septate, regularly verrucose, short conidial chains and pronounced prolongations of the conidiophores [6,7,[14][15][16]. However, in the present study, the morphological features of Cladosporium spp. were insufficient to support taxonomic conclusions. Thus, the taxonomy of this group needs phylogenetic analyses.
Seven novel species were introduced along with five other taxa that were newly recorded on various hosts, mainly based on phylogenetic analyses of three gene loci. Interestingly, all of our taxa belonged to the C. cladosporioides species complex [15]. This fungal group was also reported as fruit rot pathogens of red raspberries in the mid-Atlantic and co-occurrence with Drosophila suzukii [52], which provided a clue to clarify the association between our strains and diseased plant samples.
Cladosporium spp. are distributed widely as saprobic or endophytic fungi, which are often isolated from air, soil, textiles or many other substrates. Occasionally, they occur as opportunistic pathogens invading the dead or rotten issues of many plants [53]. For example, C. sphaerospermum has been reported to cause diseases of Aloe vera in India, and one Cladosporium sp. can cause strawberry rot in Brazil [54,55]. Bautista et al. identified C. cladosporioides as a microorganism associated with the anthracnose of Musa paradisiaca in the Philippines [56]. All our strains were isolated from diseased samples of ten plant hosts, and four taxa (C. congjiangedsis, C. ribus, C. subuliforme and C. tenuissimum) were found on two plant hosts in the meantime. At the same time, different Cladosporium taxa can also be discovered on one plant sample, similar to the previous study on Eucommia ulmoides [20]. We believed Cladosporium spp. on Passiflora edulis were able to cause one leaf blight symptom but belonged to an opportunistic pathogen because it was only found in greenhouse environments with a high temperature and humidity. Because of abundant plant diversity in Guizhou Province, after comprehensive investigation, there will be an overwhelming number and diversity of Cladosporium spp. and other fungi.