Molecular Phylogeny and Morphology of Mycosphaerella nawae, the Causal Agent of Circular Leaf Spot on Persimmon.
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Abstract
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Molecular Phylogeny and Morphology of Mycosphaerella nawae, the Causal Agent of Circular Leaf Spot on Persimmon
1,3Abstract
In this study, the phylogeny and morphology of Mycosphaerella nawae (Dothideomycetes, Ascomycota) were examined using Korean and Japanese isolates, to establish the phylogenetic relationship between M. nawae and its allied species. Korean and Japanese isolates of M. nawae were collected from circular leaf spot-diseased leaves and were confirmed based on internal transcribed spacer (ITS) sequence data. Phylogenetic analysis was conducted using multiple genes, including the ITS region, 28S rDNA, β-tubulin, translation elongation factor-1α, and actin genes. Our results revealed that M. nawae is closely related to members of the genus Phaeophleospora but are distant from the Ramularia spp. In addition, microscopic analysis revealed pseudothecia on the adaxial and abaxial surface of overwintered diseased leaves (ODL) and only on the abaxial surface of diseased leaves. Ascospores are oval to fusiform, one-septate, tapered at both ends, 1.7~3.1 × 8.1~14.1 µm, and were observed in ODL. Conidia are oval, guttulate, one-septate, 3.5~4.9 × 12.8~19.8 µm, and barely discernable on 30-day cultures. To our knowledge, this is the first report on the phylogeny of M. nawae, which is closely related to the genus Phaeophleospora, especially P. scytalidii.
Circular leaf spot (CLS) that is caused by Mycosphaerella nawae Hiura & Ikata, occurs only on persimmons (Diospyros kaki Thunb.) and has been reported in Japan, Korea, and Spain [1,2,3]. The typical symptoms of CLS include necrotic spots on leaves, chlorosis, red discoloration, and early defoliation [4]. This disease consequently leads to premature fruit maturation and abscission, ultimately resulting in economic losses [3,5]. Previous studies have shown that M. nawae has a long latent period and that typical symptoms on leaves appear approximately 4 mon after infection [6,7]. Similarly, it grows very slowly on cultured media [5]. Therefore, isolation of M. nawae from diseased leaves (DL) has proven difficult [4]. According to Kwon et al. [5,6,7], the anamorph of M. nawae is of Ramularia-type and can be observed in a 90-day-old growth on potato dextrose agar (PDA) media as well as in circular leaf spots. The authors identified the anamorphic type of M. nawae as Ramularia sp. based on their morphological characteristics; however, its classification was not supported by their phylogenetic analysis based on molecular marker genes [7].
The genus Mycosphaerella includes numerous fungal pathogens mainly associated with foliar diseases of various host plants [8,9]. Classification of the genus Mycosphaerella has relied on host plant symptoms, morphological and cultural characteristics [10,11,12,13,14], and phylogenetic analyses using molecular markers [12,13,14], or molecular markers along with morphological characteristics [15]. Reassessment of taxonomic status has been performed for many fungal species in the genus Mycosphaerella, and most of these studies have used morphological characteristics and molecular methods [16,17,18]. Recently, new genera and combinations have been reported in Mycosphaerellaceae and Teratosphaeriaceae based on molecular marker genes and morphological characteristics, whereas several combinations only occurred based on phylogenetic analysis [19,20].
This study aimed to examine the morphological characteristics and compare the phylogenetic position of Korean and Japanese M. nawae isolates, based on the internal transcribed spacer (ITS) region, 28S rDNA, β-tubulin, and actin genes, in relation to fourteen allied species and Ramularia spp. which was reported as an anamorph of M. nawae. These comprehensive experiments were conducted to enhance our understanding of the phylogenetic position of M. nawae.
MATERIALS AND METHODS
Isolation of Mycosphaerella nawae from DL and their microscopic observation.
CLS-diseased persimmon leaves were collected from seven different regions, including Sangju-si, Gumi-si, Gimhae-si, Miryang-si, and Changwonsi in Korea, and the Wakayama prefecture in Japan, from August to October 2014. To isolate M. nawae from the DL, dark green necrotic spots were sterilized in 70% ethanol for 30 sec and 1% sodium hypochlorite for 60 sec. The samples were then washed thrice in double distilled water (DDW). The sterilized samples were dried on filter paper at room temperature for 30 min, and DDW (50 µL) was then added on the back of the symptomatic spots, which were then spread on a PDA plate and then incubated at 25![[degree celsius]](/corehtml/pmc/pmcents/x2103.gif)
until colonies appeared. After 2~3 days, small black colonies were transferred onto a new PDA plate.
Genomic DNA preparation and PCR amplification of molecular markers.
Total genomic DNA was extracted from the isolated M. nawae according to the cetyltrimethylammonium bromide method [21]. Using the genomic DNA of M. nawae isolates and their allied species, the ITS region, the partial region of 28S rDNA, Tub, and Act were amplified using the corresponding primer pairs [22,23,24,25]. A total reaction volume of 20 µL contained 1 µL of genomic DNA, 2 µL of 10× Taq buffer, 0.4 µL of 10 mM dNTP, 0.5 µL each of 10 pM forward and reverse primer, and 0.2 µL of Taq DNA polymerase (Solgent Co., Daejeon, Korea). PCR was performed in a Veriti 96-well Thermal Cycler (Applied Biosystems, Carlsbad, CA, USA). The obtained PCR products were electrophoresed on 1% agarose gel, stained with ethidium bromide, and observed under a UV illuminator. All the amplified PCR products were purified using ExoSAP-IT (USB Co., Cleveland, OH, USA) and were directly sequenced (Solgent Co.).
Nucleotide sequences and phylogenetic analyses.
All the obtained sequences of ITS, partial 28S rDNA, Tub, and Act were compared with the available sequence data, using BLAST search against the NCBI GenBank database to identify the sequences, and multiple sequence alignments were performed using CLUSTAL W [26]. Phylogenetic trees were constructed according to the maximum likelihood method with 1,000 bootstrap replications, using the MEGA 7 software ver. 7.0.14. Moreover, each of the homosynonyms and heterosynonyms of the allied species of M. nawae were surveyed through the MycoBank Database (http://www.mycobank.org).
Microscopic observation.
Isolated colonies were observed under a light microscope (BX-50; Olympus, Tokyo, Japan) after 30 days of cultivation. To observe the conidia, aerial mycelia were collected from 30-day-old colonies, using DDW, and the suspension was spread onto a PDA plate. The PDA was observed under a light microscope to determine conidia before germination.
Observation of pseudothecia on diseased and overwintered DL
To observe the pseudothecia on DL, DL and overwintered diseased leave (ODL) were collected from diseased trees and the leaf litter around the diseased trees in Sangju-si. The adaxial and abaxial sides of the DL and ODL were observed under a stereoscopic microscope (DIMIS-M; Siwon Optical Technology, Co., Ltd., Anyang, Korea) and a light microscope (BX-50; Olympus) after staining with 1% methylene blue. To prepare semi-thin sections, the diseased part was excised using a sterilized surgical blade. Samples were then treated with Karnovsky's fixative (2% paraformaldehyde and 2.5% glutaraldehyde in 0.05 M cacodylate buffer, pH 7.2) for 24 hr. The fixed samples were dehydrated in a graded ethanol series of 30%, 50%, 70%, 80%, 90%, and absolute ethanol for 20 min at each concentration and were then infiltrated with propylene oxide. Finally, the samples were embedded in Spurr's resin and polymerized at 70 for 10 hr. The embedded samples were cut using an ultra-microtome (MT-7000; RMC Boeckeler, Tuscon, AZ, USA) and each section was observed using a light microscope after staining with 1% methylene blue.
RESULTS
Isolation of Mycosphaerella nawae from CLS-DL.
Twenty isolates of M. nawae were obtained from collected leaves with CLS-disease from each region of collection. At first, the colonies appeared white, dense, and round, and grew slowly on the PDA plates compared to other fungi. After 5 to 7 days, the colonies turned dark green toward the middle. After 4 wk, they transformed into grayish brown or dark brown colonies that were raised in the center, had a wave pattern with a wrinkled surface, and ranged from 19 to 21mm in diameter at 25 (Fig. 1A and 1B).
Molecular identification based on ITS sequences.
The obtained ITS region sequences from 20 Korean and Japanese M. nawae isolates were searched in the NCBI database, using the BLAST search. All the obtained sequences from the Korean and Japanese isolates were 665 bp long and were identical (data not shown). We observed that all the isolate sequences were identical to those of the Spanish M. nawae isolates (GQ465767 and GQ465768). The phylogenetic analysis showed that they were indistinguishable from the Spanish M. nawae isolates (GQ465767 and GQ465768) but were distinct from those of Ramularia spp. whereas the sequences of Phaeophleospora gregaria were not distinguished from M. nawae (Fig. 2).
Phylogenetic analysis based on molecular markers.
To examine the phylogenetic relationship of M. nawae with its allied species, a maximum likelihood tree was constructed based on the combined dataset composed of concatenated sequences of ITS, 28S rDNA, Tub, and Act. The obtained sequences of all molecular markers were deposited in the NCBI database (LC121109~LC121232). The combined dataset was approximately 2,450 bp and included sequences from 20 M. nawae isolates and the derived allied species from the NCBI (Table 1). In the resulting tree topology, the Korean and Japanese M. nawae isolates were clustered together forming a single sister clade to the clade containing the genus Phaeophleospora (Fig. 3). In addition, we tested the phylogenetic relationship between the newly introduced species in the Mycosphaerellaceae and the Korean and Japanese isolates of M. nawae based on ITS, partial of 28S rDNA and translation elongation factor-1α (TEF-1α) genes (Table 1). The combined dataset was approximately 1,200 bp and a phylogenetic tree was constructed using the maximum likelihood method with 1,000 replicates. The results showed that the Korean and Japanese M. nawae isolates were closest to the genus Phaeophleospora spp. especially P. scytalidii (Fig. 4).
Table 1
aThe synonym was searched in Mycobank.
bIt indicates the reference that newly reclassified the species belonging to the genus Mycosphaerella.
Observation of pseudothecia on DL.
The upper and lower surfaces of CLS-DL were observed using a stereoscopic microscope. Pseudothecia were observed on both surfaces side of DL (Fig. 5). The structures were observed on the cross sections of the leaves. The pseudothecia where located between the epidermal cells and the palisade parenchyma of the ODL. They were mostly flask- and pear-shaped structures, 55.1~62.2 µm wide (average 58.3 µm), and 70.8~80.3 µm high (average 76.0 µm) (Fig. 6). The pseudothecia on the DL were located between the palisade and spongy parenchyma, and were mostly ovoid and flask-shaped, 55.6~69.2 µm wide (average 60.7 µm), and 55.6~69.9 µm high (average 64.8 µm) (Fig. 6). The morphology of the asci and ascospores observed on the structures in the ODL confirmed that these structures represented the pseudothecia of M. nawae.
Observation of ascospores and conidia.
Mature asci were observed in ODL collected from leaf litter between May and July 2015 (Fig. 7). These were cylindrical to clavate and banana-shaped structures, 8-spored, straight or curved, 4.6~6.8 µm wide (average 5.7 µm), and 25.9~34.1 µm high (average 31.1 µm) (Fig. 7A and 7B). The ascospores were oval to fusiform, hyaline, one-septate or aseptate, mostly tapering at both ends, 1.7~3.1 µm wide (average 2.5 µm), and 8.1~14.1 µm high (average 10.3 µm) (Fig. 7C). Furthermore, because conidia were rarely observed in M. nawae cultured on PDA, a suspension of the aerial mycelia was spread on the PDA. Thereafter, very few conidia including germinated conidia and hyphae were observed on the PDA. The conidia were oval, guttulate, hyaline, one-septate, 12.8~19.8 µm high (average 17.1 µm), and 3.5~4.9 µm wide (average 4.3 µm) (Fig. 7F).
DISCUSSION
A previous study reported that the M. nawae anamorph was similar to that of Ramularia spp. in its morphological characteristics [7], whereas the M. nawae isolates in the present study were distinct from those of the Ramularia spp., as indicated in our phylogenetic analysis of the ITS region (Fig. 2). The phylogenetic placement of M. nawae using the combined dataset revealed that the M. nawae group was closest to the genus Phaeophleospora, especially P. scytalidii (Figs. 3 and and4).4). This suggests that M. nawae had a high degree of similarity with the genus Phaeophleospora.
Recently, new combinations and genera were introduced in Mycosphaerellaceae, based on phylogenetic analysis, such as genus Amycosphaerella, Xenomycosphaerella, and Phaeophleospora [19,20]. Among these, several species belonged to the genus Phaeophleospora, Xenomycosphaerella, and Paramycosphaerella; they combined the species or changed the genus name based on only phylogenetic analysis results, without morphological comparison [19]. In this study, we constructed a phylogenetic tree comparing the allied species of M. nawae and the Korean and Japanese M. nawae isolates, based on combined ITS region, 28S rDNA, Tub, and Act sequences (approximately 2,450 bp) and the combined ITS region, 28S rDNA and TEF-1α gene sequences (approximately 1,200 bp). The results showed that the Korean and Japanese M. nawae isolates were closest to Phaeophleospora spp., and that P. scytalidii was the closest species of the genus Phaeophloeospora (Figs. 3 and and4).4). According to Videira et al., M. nawae has a Ramularia-like anamorph and is close to the genus Phaeophleospora, based on the ITS region [27]. Our results confirmed that i) M. nawae could be differentiated from Ramularia spp. by its morphological characteristics, ii) although it was close to the genus Phaeophleospora, it was closest to P. scytalidii.
The conidia of M. nawae were previously observed only in 1929 [1], and later, Kwon et al. [7] reported the anamorph stage of M. nawae as that of Ramularia spp. because of their similar morphological characteristics. One of the major characteristics of Ramularia spp. is the presence of scar structures on conidia [27]. In this study, the structures were not observed during microscopic observation (Fig. 7D~7F). Furthermore, phylogenetic analyses based on the ITS sequence data revealed that the Ramularia spp. were not closely related to the M. nawae isolates. These results indicate that M. nawae is distinct from Ramularia spp. Phylogenetic analysis showed that the genus Phaeophleospora was closely related to M. nawae (Fig. 3 and and4).4). The morphology of M. scytalidii (= P. scytalidii) has many similarities with M. nawae, such as pseudothecium production, similar size of ascospores, and conidia tapering at both ends, guttulate, and septate, among others [17]. Interestingly, mycelial structures of P. scytalidii and M. nawae share similarities (Fig. 7D and 7E), including being solitary or branched, septate, and peanut- or bottle gourd-shaped [7,17].
Many recent studies have been conducted on the genus Mycosphaerella and its anamorph [19,20,28,29,30]. Many species belonging to the genus Mycosphaerella have been segregated into other groups based on the morphology of their particular anamorph [31,32], as well as based on teleomorph features such as asci and ascospores [12]. However, these classifications have not always been correlated with phylogenetic analysis [15,32]. Recent studies on the genus Phaeophleospora indicated that species that were newly transferred into the genus based on phylogenetic inference, including P. gregaria, P. scytalidii, and P. stramenti, reproduce sexually and lack the asexual state [19]. In addition, phylogenetic analysis based on the multi-locus result revealed that most of the heterosynonym or homosynonym species are included in the genus Mycosphaerella (Table 1, Fig. 3). Furthermore, these current species names were not reflected in the anamorph stage, except for M. graminicola (anamorph: Zymoseptoria tritici). Since phylogenetic data revealed that the M. nawae cluster was closely related to Phaeophleospora spp. and especially to P. scytalidii, there is a possibility that M. nawae could be accommodated in the genus Phaeophleospora according to previous reports [19,20]. Nonetheless, the common morphological features, cultural characteristics, and classification of the Phaeophleospora spp. and M. nawae need to be re-evaluated.
ACKNOWLEDGEMENTS
This work was supported by the Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries (IPET) through the Advanced Production Technology Development Program, funded by the Ministry of Agriculture, Food and Rural Affairs (MAFRA) (314031-3).
References
Articles from Mycobiology are provided here courtesy of Korean Society of Mycology
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BioStudies: supplemental material and supporting data
Nucleotide Sequences (4)
- (1 citation) ENA - GQ465767
- (1 citation) ENA - LC121232
- (1 citation) ENA - GQ465768
- (1 citation) ENA - LC121109
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