Morphology, Phylogeny and Pathogenicity of Colletotrichum menglaense sp. nov., Isolated from Air in China

A new species, Colletotrichum menglaense, isolated from air in Mengla, Xishuangbanna, Yunnan Province, China, was characterized and described combining morphological characteristics and multigene phylogenetic analysis. Morphologically, it is characterized by oblong, sometimes slightly constricted, micro-guttulate conidia and simple obovoid to ellipsoidal appressoria. Phylogenetic analysis of the ITS, ACT, CHS, and GAPDH sequences showed that C. menglaense belongs to the C. gloeosporioides complex. The pathogenicity of C. menglaense on fruits of several crop plants, including strawberry, orange, grape, tomato, and blueberry, was tested and confirmed by the re-isolation of C. menglaense.


Introduction
There are numerous bacteria and fungi in the near-surface atmosphere and upper troposphere, with millions of cells per cubic metre of air [1]. More than 40,000 species of fungi live in the air, mainly from soil, animals and plants, human activities, and excreta [2]. Fungi eject spores into the atmosphere by water jets or droplets, and become an important part of aerosols [3,4]. Hence, airborne fungi are closely related to air pollution, environmental quality, and human health.
Earlier investigations of microorganisms in air were connected with dust [5,6] or aerosol particles [7][8][9][10][11] to detect the fungal distribution or characterisation. Other studies suggested that a considerable proportion of indoor airborne fungi were derived from outdoor fungi [12,13], and fungi had a great impact on human health [14][15][16][17][18]. With the advance of molecular approaches and the awareness of the importance of mycobiota diversity research, the number of studies about outdoor mycobiota is increasing. Many investigations recovered fungi from various places: the atmosphere in the urban environment of the USA [19], a rural area in India [20], at an altitude of 20,000 m in Earth's atmosphere [5], in southeastern Austria [16], and from the Himalayan region [21]. Most of these studies monitored remote, extreme, sparsely populated sites to learn the diversity, distribution, and seasonal variations of fungal species [22]. Several genera were often detected in air samples, Curviclavula G. Delgado et al. [23], Aspergillus P. Micheli, Penicillium Link, and Talaromyces C.R. Benj [13], and occasionally also species of Colletotrichum Corda [24,25].
Colletotrichum (Sexual morph: Glomerella) is a genus of the family Glomerellaceae, Glomerellales, Sordariomycetes, which was established with C. lineola Corda as the type species. Anthracnose, caused by the Colletotrichum species, is a major disease of plants, mainly fruit, worldwide. It causes significant yield losses and reduces the marketability of the fruit [26][27][28][29]. There is growing evidence showing that Colletotrichum spp. is ubiquitous and widespread. As one of the top ten plant pathogenic fungi in the world [30], many Colletotrichum species were isolated from diseased plants, e.g., C. miaoliense P.C. Chung & H.Y. Wu and C. australianum W. Wang et al. were isolated from anthracnose symptoms on strawberry and citrus fruits [31,32]. In addition, Colletotrichum spp. were often reported as endophytes, including from healthy leaves of Bletilla ochracea [33]. Species of Colletotrichum are occasionally found as saprobes [34,35]. Some species were also isolated simultaneously as an endophyte, pathogen, and saprobe [36,37]. As for Colletotrichum spp. from air, an unidentified and a known species were reported. Colletotrichum sp. was isolated from the air of Dhaka, Bangladesh by Sultana [24]. Lal also trapped propagules of C. falcatum in air and confirmed that this species infected healthy plants [25].
When we investigated the fungi diversity of air samples in the town of Mengla, Xishuangbanna, a new species of Colletotrichum was identified based on morphological characteristics and DNA sequence data from four loci, and we named it C. menglaense. Its pathogenicity to several fruits was tested and confirmed by re-isolating the fungus.

Phylogenetic Analysis
In the phylogenetic tree inferred from ITS, the strain is well clustered in the Colletotrichum gloeosporioides complex (not shown here). Therefore, we downloaded ITS, ACT, CHS, and GAPDH sequences in the C. gloeosporioides complex species. The dataset comprised 47 species, 67 isolates, and 1 outgroup taxa Monilochaetes infuscans (Table 1). A total of 1534 characters (ACT: 305, CHS: 300, GAPDH: 306, ITS: 623) were analysed by using Bayesian. The topologies of the tree were shown with the Bayesian posterior probability values for the analysed clades ( Figure 1). In this tree, C. menglense is a sister clade to C. aeschynomenes B.S. Weir & P.R. Johns and C. dianesei N.B. Lima, M.P.S. Câmara & Michereff, and formed a single clade with high Bayesian inference posterior probability values ( Figure 1). Therefore, we determined that our strain belonged to a novel species of Colletotrichum.

Pathogenicity Test
After 7 days, five kinds of fruit inoculated with conidia suspension developed pale white hyphae around the wounds, and typical dark brown anthracnose lesions appeared on the strawberries, but no symptoms developed on the controls. Strawberry and tomato were the most susceptible, with disease scores from 7 to 9 (Table 2). Then, after 14 days, there were obvious anthracnose lesions around the wounds of the strawberry, orange, and tomato fruits. In fact, all of the fruits were susceptible to YMF 1.04960. The results showed that C. menglaense YMF 1.04960 is not host-specific ( Figure 2).
The conidia isolated from the infected fruits are the same as those of YMF 1.04960 ( Figure 3F), and the ITS sequence is also the same as YMF 1.04960. So, the pathogenicity was confirmed. Table 2. Disease Score (DS) on a 0-9 scale of different fruits for C. menglaense inoculated by wounding or non-wounding methods.

Pathogenicity Test
After 7 days, five kinds of fruit inoculated with conidia suspension developed pale white hyphae around the wounds, and typical dark brown anthracnose lesions appeared on the strawberries, but no symptoms developed on the controls. Strawberry and tomato were the most susceptible, with disease scores from 7 to 9 (Table 2). Then, after 14 days, there were obvious anthracnose lesions around the wounds of the strawberry, orange, and tomato fruits. In fact, all of the fruits were susceptible to YMF 1.04960. The results showed that C. menglaense YMF 1.04960 is not host-specific ( Figure 2).   The conidia isolated from the infected fruits are the same as those of YMF 1.04960 ( Figure 3F), and the ITS sequence is also the same as YMF 1.04960. So, the pathogenicity was confirmed.  Description: Colonies growing on CMA with entire margin, 28-32 mm diameter d at 28 °C; aerial mycelia medium grey to pale buff in centre, light grey to greyish w  Description: Colonies growing on CMA with entire margin, 28-32 mm diameter in 4 d at 28 • C; aerial mycelia medium grey to pale buff in centre, light grey to greyish white in the margin, entirely covered with floccose to dense. Reverse dark white to grey with white margin. Conidiomata acervular, with orange conidial masses. No setae observed. Conidiophores cylindrical, unbranched or branched, straight or flexuous, 0-1-septate, hyaline, branched, 14.9-59.7 µm × 1.4-3.3 µm. Conidiogenous cells monophialidic, subulate, integrated, determinate, terminal, hyaline. Conidia acrogenous, oblong, sometimes slightly constricted at the middle, micro-guttulate, hyaline, unicellular, smooth-walled, 12.2-17.1 µm × 4.2-6.4 µm (av. = 14.4 µm × 5.1µm, n = 30). Appressoria simple, brown to dark brown, aseptate, mostly ellipsoidal to broadly obovoid, entire or irregular, somewhat crenate to lobed at margin, 6.7-20.0 µm × 4.8-11.0 µm, L/W ratio = 2.7.
Notes: C. menglaense can be distinguished from phylogenetically closely related C. aeschynomenes  For each sample, we used a surface air system (SAS) Super ISO 180 (VWR European Cat.No.710-0870, San Giusto, Italy) that takes five minutes to capture 1,000 L of air. A 90 mm Petri dish containing RBA (5 g peptone, 10 g dextrose, 1 g potassium dihydrogen phosphate, 0.5 g magnesium sulfate, 15 g agar, 0.033 g rose bengal, 0.1 g chloramphenicol, 1000 mL distilled water) was put on the sampler for a few seconds to collect air. The Petri dishes were immediately sealed after air collection and brought back to the laboratory. Petri dishes were incubated in the continuous light at outdoor ambient temperature (mean 25 • C) and examined periodically. When a few mycelium appeared, it was picked up and transferred to PDA (200 g potato, 20 g glucose, 18 g agar, 40 mg streptomycin, 30 mg ampicillin, 1000 mL distilled water) dishes for incubation at 25 • C. The pure cultures were further incubated on CMA (20 g cornmeal, 18 g agar, 40 mg streptomycin, 30 mg ampicillin, 1000 mL distilled water) dishes to induce sporulation. Colony morphology and microscopic characteristics were examined, measured, and photographed after incubation for 7 days by using the aid of a BX51 microscope.

Sample Collection and Morphological Characterisation
The pure culture was deposited in the Herbarium of the Laboratory for Conservation and Utilization of Bio-resources, Yunnan University, Kunming, Yunnan, China (YMF, formerly Key Laboratory of Industrial Microbiology) and the China General Microbiological Culture Collection Centre (CGMCC).

DNA Extraction, PCR Amplification, and Sequencing
Detailed protocols for Genomic DNA extraction are described previously [40,41]. The relative quantity of total genomic DNA was observed on a 1% TAE agarose gel stained with ethidium bromide. The following loci were amplified with the indicated primers: the internal transcribed spacer (ITS) region and actin gene (ACT) with primers ITS4/ITS5 [42,43]; ACT512F/ACT783R [40], respectively. The thermo-cycling parameters were used: initial denaturation at 95 • C for 3 min, followed by 34 cycles of 95 • C for 1 min, 52 • C for 30 s, 72 • C for 1 min, with a final extension step of 72 • C for 10 min. Chitin synthase 1 (CHS-1) were amplified with CHS-79F/CHS-354R [44]. The cycling parameters consisted of 94 • C for 5 min, followed by 35 cycles at 94 • C for 30 s, 56 • C for 30 s, 72 • C for 90 s, and a final extension step of 72 • C for 7 min. Partial sequences of the glyceraldehyde -3-phosphate dehydrogenase (GAPDH) were amplified with primers GDF1/GDR1 [45]. The cycling parameters consisted of a denaturation step at 94 • C for 4 min, followed by 34 cycles at 94 • C for 45 s, 60 • C for 45 s, 72 • C for 1 min, and a final cycle at 72 • C for 10 min. Amplification was performed in a total of 25 µL reaction volume, which contained 1.0 µL DNA template, 1.0 µL of each forward and reverses primer, 12.5 µL 2 × Master Mix (Tiangen Biotech, Beijing, China), and 9.5 µL dd H 2 O. The sequencing reactions were carried out by TsingKe Biological Technology, Kunming, China using the same primers as for amplification. The new sequences were submitted to the GenBank database at the National Center for Biotechnology Information (NCBI), and the accession numbers are listed in Table 1.

Phylogenetic Analysis
The obtained ITS sequences were compared with those in GenBank using BLAST searches to determine the primary phylogenetic placement of the fungus. The results indicated that our strain belongs to Colletotrichum. Neighbour-joining analysis of ITS sequence was used to determine further phylogenetic placement. Then, we retrieved ITS, ACT, GAPDH, and CHS sequences of representative species and additional species belonging to this complex species. All sequences used in this study are listed in Table 1 [46]. The resulting alignments were subsequently manually adjusted and linked by BioEdit version v. 7.0(Borland, Austin, TX, United States) [47]. To ensure that all sequences are of the same length, the missing base was replaced with "?". Then, the combined alignment was converted to a NEXUS file using the program mega7(Mega Limited, Auckland, New Zealand) [48]. Phylogenetic analyses were performed for Bayesian inference (BI) analysis using MrBayes v.3.2.2 (Department of Biodiversity Informatics, Swedish Museum of Natural History, Stockholm, Sweden) [49].
For BI analysis, the best nucleotide substitution model for each locus was determined using Mrmodeltest v. 2.3 (Department of Systematic Zoology, Evolutionary Biology Centre, Uppsala University, Uppsala, Sweden) [50]. The analyses of four MCMC chains were run from random trees for 1,000,000 generations, and trees were sampled every 100 generations, resulting in 20,000 total trees. The first 25 % of the trees were discarded as the burn-in phase of each analysis, and the remaining trees were used to calculate posterior probabilities. Sequences derived in this study were deposited in GenBank (Table 1), and the concatenated alignments were deposited in TreeBASE with http://purl.org/phylo/treebase/phylows/ study/TB2:S27941, and the descriptions and nomenclature in MycoBank (www.mycobank.org).

Pathogenicity Assay and Confirmation
In order to test the pathogenicity of the new species, detached fruits inoculations were conducted. Healthy Fragaria ananassa Duch. (strawberry), Citrus tachibana (Makino) Tanaka (orange), Vitis vinifera L. (grape), Lycopersicon esculentum Mill. (tomato), and Vaccinium uliginosum Linn. (blueberry) were used for the pathogenicity test. All fruits were immersed in 70% ethanol for 3 min and 1% sodium hypochlorite for 3 min, then rinsed three times in sterile distilled water and air dried in the laminar flow cabinet.
Prior to the inoculation, holotype strain YMF 1.04960 of new species was cultivated on CMA for 7 days at 28 • C, adding 0.4 g yeast extract per 100 mL to induce sporulation. After incubation, conidia were harvested by adding 10 mL sterile water to each culture followed by scraping the surface with a sterile brush. The resulting conidia suspensions were filtered through sterile six layers of filter paper. Then, conidia were diluted to 10 6 /mL using sterile water (concentration was adjusted by using a haemocytometer). Fruit were wounded with a sterilised insect needle and inoculated with 10 µL conidium suspension. Control fruits were inoculated with sterilised water. Five replications were set. The inoculated fruits with the controls were put into plastic containers, covered with plastic wrap to maintain humidity, sealed and stored in a constant temperature incubator, and examined periodically.
Seven days and 14 days after inoculation, the virulence was evaluated as described by Montri et al. [51]. In particular, 0 (highly resistant), no infection; 1 (resistant), 1-2% of the fruit with a necrotic lesion or a larger water soaked lesion surrounding the infection site; 3 (moderately resistant), >2 to 5% of the fruit with a necrotic lesion, possibly acervuli, may be present, or a watery lesion covering up to 5% of the fruit surface; 5 (susceptible), >5 to 10% of the fruit showing a necrotic lesion, possibly acervuli, or a water-soaked lesion covering up to 25% of the fruit surface; 7 (very susceptible), >10 to 25% of the fruit covered with a necrotic lesion with acervuli; and 9 (highly susceptible), >25% of the fruit showing necrosis, lesion often encircling the fruit, abundant acervuli. Symptomatic fruits were surface-sterilised as described above. The symptomatic tissue segments were cut with a sterilised scalpel about 5 mm × 5 mm × 5 mm and then placed on the PDA to re-isolate the fungus. The identity of obtained isolates was confirmed on the basis of morphological characteristics and ITS sequence.

Discussion
ITS has been proposed as the official fungal barcoding marker [52], but phylogenetic analysis using only ITS sequences could not confidently resolve the phylogenetic placement of some species within Colletotrichum. In this study, phylogenetic analysis based on ITS showed that C. menglaense could not be distinguished from C. queenslandicum, C. salasolae, and C. siamense, but the results showed that C. menglaense is well clustered in the C. gloeosporioides species complex, so we further used multi-locus phylogeny to distinguish closely related species. Combining sequences of ITS, ACT, CHS, and GAPDH, the phylogenetic position of C. menglaense was determined. In the phylogenetic tree, the species relationships were well defined, with all of the major clades supported by high Bayesian inference posterior probabilities (Figure 1). C. menglaense grouped together with C. aeschynomenes and C. dianesei, and the ITS similarity between C. menglaense and C. aeschynomenes (KU239115) is 99.08%, while, between C. menglaense and C. dianesei (KC329775), it is 99.47%. Morphologically, C. menglaense obviously differs from C. aeschynomenes and C. dianesei in the shape and size of the conidia.
The pathogenicity test showed that C. menglaense may be a potential pathogen to fruit. Among all of the test fruits, C. menglaense was very aggressive on strawberries, while it was less aggressive on blueberries and grapes. This is in agreement with Xavier et al., who reported that the C. gloeosporioides species complex was more aggressive to strawberry than other Colletotrichum species complex organisms [53]. The degree of fruit infection may be related to fruit condition, humidity, temperature, inoculum concentration, and inoculation method [54], so, among fruits tested here, strawberry with softer tissue showed the highest disease scores of 5 to 9. Several fruits inoculated with C. menglaense presented different degrees of anthracnose, indicating that C. menglaense was non-host specific. In fact, many Colletotrichum species present on a wide range of host plants [27,55]. For example, C. karstiiwas was reported from diseased black plum (Diospyros australis), strawberry (Fragaria xananassa), and banana (Musa nana Lour). C. gloeosporioides species complex organisms were also frequently isolated from a variety of hosts, including kumquat, finger lime, grapefruit, lemon, lime, mandarin, orange, and Persian lime [37,56].
Previously, it was reported that Cladosporium was the most frequent fungus in the air; the next were Fusarium, Alternaria, and Epicoccum [57]. Some leaf surface fungi are major contributors to air spores through the action of wind or rain spatter, and the canopy is closer to the leaves of the plant, so there are more fungal spores in the air below the canopy [58]. Similar to previous reports, the air samples that we obtained were collected in the lower part of the canopy. Besides, some airborne spores have been reported to be pathogenic fungi. Alternaria alternata airborne spores might be sufficient to cause human spore-related asthma symptoms to people even with only a limited concentration [59]. Here, C. menglaense is an airborne fungus that has certain pathogenicity to plants. Previous studies also showed that Colletotrichum spp. from air were pathogenic fungi to plants [25]. Due to limited study, we do not know how many pathogenic fungi are present in the air and how they contribute to the spread of plant diseases. In this respect, the present article provides new information.