The genus Neopestalotiopsis Maharachch was recently segregated from Pestalotiopsis Steyaert, which was separated into three genera, Neopestalotiopsis, Pestalotiopsis, and Pseudopestalotiopsis, based on morphological data and phylogenetic analysis of internal transcribed spacer (ITS), 28S nrRNA (LSU), β-tubulin (BT), and translation elongation factor 1-alpha (TEF) gene sequences [1]. The genus Pestalotiopsis was a heterogeneous group of coelomycetous fungi differentiated primarily based on conidial characteristics such as size, septation, presence or absence of appendages, and color of the median cells [2, 3]. Neopestalotiopsis species can be easily distinguished from the other two genera by its versicolorous median cells and indistinct conidiophores, which are often reduced to conidiogenous cells [1]. Numerous studies have shown that the relationship between plants and Pestalotiopsis-like fungi can be parasitic, symbiotic, or saprophytic [3, 4].
Pathogenic Pestalotiopsis species cause a variety of plant diseases worldwide, including fruit rot disease on grape, gray blight of tea plant, and canker on the medicinal plant Acanthopanax divaricatus in Korea [4-6]. Neopestalotiopsis species also exhibit pathogenicity and cause economic loss to various crops such as palm, coconut, and mango [1]. The type species of the Neopestalotiopsis genus, N. protearum, was isolated from leaf spot on Leucospermum cuneiforme and N. vitis was recently reported as the causal agent of grape leaf spot in China [7, 8].
During our studies of microbial communities in the field soil of Wanju, Jeollabuk-do, the Korea fungal strain KNU16-005 was isolated. Based on its morphological characteristics and phylogenetic analysis, this isolate was identified as Neopestalotiopsis australis Maharachch and designated as KNU16-005. This fungus has not been previously reported in Korea.
Collected soil samples (1 g) were suspended in 10 mL of sterile distilled water and the prepared suspension was vortexed, serially diluted, and spread onto potato dextrose agar (PDA; Difco, Detroit, MI, USA) plates. The plates were incubated at 25°C for 3 days. Single colonies on these plates were purified by transferring colonies onto new plates followed by incubation on PDA at 25°C. One isolate, KNU16-005, was selected for further morphological and molecular phylogenetic analyses.
The isolate KNU16-005 was cultured at 25°C and colony characteristics such as color, shape, and size were recorded. After 12 days of incubation on PDA agar, the colony was 6.5~6.8 cm in diameter, whitish to yellowishwhite in color and had irregular edges, dense aerial mycelium on the surface, and black conidiomata (Fig. 1A,1B, 1E). The morphology of the isolate was examined under an Olympus CX31 light microscope (Tokyo, Japan). Conidia were 21~25 × 6~8 μm, 4-septate, and straight to slightly curved (Fig. 1F). The basal cell was conical in shape with an obtuse end, hyaline, and thin-walled. Three median cells were versicolorus. The second cell was brownish, the third cell was dark brown, and the fourth cell was brown. Three apical appendages arising from the apex of the apical cell were 20~30 μm in length, while the basal appendage was small hyaline pedicel and 3~6 μm in length. As shown in Table 1, these morphological characteristics of isolate KNU16-005 agreed well with those for representatives of the genus Neopestalotiopsis and were most closely matched to the characteristics of N. australis [1].
Table 1. Morphological characteristics of Neopestalotiopsis australis isolated in this study
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| PDA, potato dextrose agar. aSource of description [1]. |
To confirm the reliability of morphological identification, the isolate KNU16-005 was subjected to molecular identification based on amplification of three DNA regions that are widely used for phylogenetic analysis. For this purpose, genomic DNA was extracted from the mycelia using the HiGene Genomic DNA prep kit (BIOFACT, Daejeon, Korea). The ITS regions, including 5.8S, were amplified with the primers ITS1F and ITS4 [9]. To amplify the partial BT and TEF genes, the T1/Bt2b and EF-728F/EF2 primer pairs were used [10-13]. The amplified PCR products were sequenced using an ABI 3730XL DNA analyzer (Applied Biosystems, Foster City, CA, USA). ITS, BT, and TEF gene sequences of related Neopestalotiopsis and Pestalotiopsis strains were obtained from GenBank and used for phylogenetic analysis (Table 2). Evolutionary distance matrices for the neighbor-joining algorithm were calculated using Kimura’s two-parameter model [14]. Tree topology was inferred by the neighbor-joining method in the program MEGA7 [15], with bootstrap values based on 1,000 replications. GenBank BLAST searching revealed that the ITS regions and BT and TEF gene sequences of KNU16-005 matched those of N. australis CBS 114159 (KM199348, KM199432, and KM199537) with 99.63%, 99.04%, and 99.59% similarities, respectively, clearly indicating that both strains belong to same Neopestalotiopsis species. The isolate KNU16-005 clustered together with N. australis CBS 114159 in the phylogenetic tree constructed based on the combined ITS, BT, and TEF sequences, confirming their close relationship at the species level (Fig. 2).
Fig. 2
Neighbor-joining phylogenetic tree based on the combined internal transcribed spacer (ITS), β-tubulin (BT), and translation elongation factor 1-alpha (TEF) sequences, showing the relationship between Neopestalotiopsis australis KNU16-005 and closest Neopestalotiopsis and Pestalotiopsis taxa. Bootstrap values (based on 1,000 replications) greater than 70% are shown at the branch points. Bar, 0.02 substitutions per nucleotide position.
The results of morphological and phylogenetic characterizations clearly indicate that isolate KNU16-005 is N. australis. This fungal species has not been previously detected in Korea. Previous studies showed that Pestalotiopsislike fungi produce many important bioactive secondary metabolites such as the anti-cancer drug taxol or antimycotic pestacin [3, 16]. Thus, further investigations of N. australis KNU16-005 as a producer of bioactive compounds should be conducted.
