First report of Puccinia xanthii causing rust disease on Xanthium orientale in Korea.

Puccinia xanthii Schw. is a microcyclic rust fungus, first found on Xanthium strumarium Lour in North Carolina, the United States. This rust fungus is native to the continental United States, Hawaii, Mexico, and the West Indies (Arthur 1934). It has become notoriously invasive and is now distributed in the Europe (Bulgaria, France, Hungary, Italy, Romania, Spain, and the former Yugoslavia), India, Indonesia, Australia, and South Africa (Parmelee 1969; Alcorn 1976; Wahyuno 2012). In East Asia, the fungus has been reported in Japan (Hiratsuka et al. 1992) and China (Zhao et al. 2014) but not in Korea. It has been reported mainly on the invasive weeds Xanthium and Ambrosia species. In addition, it rarely occurs on sunflowers (Helianthus spp.) in Australia (Alcorn 1976), South Africa (Pretorius et al. 2000), and North America (Gulya and Charlet 2002). In Korea, rust disease symptoms caused by a Puccinia fungus were first found on X. orientale L. at the roadside of Okcheon-gun, Chungcheongbuk-do (36 27'95.428"N 127 66'26.378"E) in October 2021 and were repeatedly observed in the same site in 2022. The similar symptom was additionally found on X. orientale in Yesan-gun, Oct. 2022. The symptoms were brown spots on round chlorotic haloes on the adaxial leaf surface and dark brown pustules on the abaxial leaf surface. Telia were brown to dark brown, round, mostly grouped, 0.28-0.61 mm in diameter, and mainly formed on the abaxial leaf surface but sometimes on the adaxial leaf surface. Teliospores were two-celled, pedicellate, and measured 37.6-110 × 12.4-21.5 µm in size; the wall was yellowish or almost colorless, smooth, 1.2-2.6 µm thick at the sides, and up to 7.4 µm thick at the apex. The morphological characteristics of the teliospores were identical to those of P. xanthii described by Arthur (1934) and Parmelee (1969). Based on phylogenetic analyses (e-Xtra 2) of the internal transcribed spacer (ITS) and partial large subunit (LSU) rDNA extracted from the teliospores, they were identified as P. xanthii. BLAST analysis showed that the sequences had high homologies (over 99.82%) with the reference strains of P. xanthii (EF635903 and KX999896). The representative specimens were preserved at the Animal and Plant Quarantine Agency Herbarium (PQK211005 for Okcheon-gun isolate and PQK220913 for Yesan-gun) and the sequences were deposited in GenBank (OR958716 and OR958692). A pathogenicity test was performed by dropping a suspension of germinating teliospores and basidiospores onto the adaxial leaf surfaces of apparently healthy X. orientale plants in Oct. 2022, using the isolate PQK220913 (OR958692). The three inoculated plants were placed together with three controls treated with only distilled water, in the dark at saturated humidity for 24 hours in an isolated greenhouse. After two weeks, typical rust symptoms were observed in the three infected plants, whereas no symptoms appeared in the control plants (e-Xtra 1). The causal fungus was identified as P. xanthii based on host relationships, successful experimental inoculation, morphological characteristics, and sequence similarity of partial DNA fragments. To our knowledge, this is the first report of P. xanthii on X. orientale in Korea. P. xanthii was additionally confirmed on X. orientale in Gumi-si, Boeun-gun, Seongju-gun, Naju-si, and Gunsan-si in 2023, indicating its wide distribution in Korea. It is expected that P. xanthii could be a candidate as a biological agent for controlling the invasive weed, X. orientale.

First report of a rust disease of Lycium chinense caused by Puccinia tumidipes in Korea.

Lycium chinense Mill is a deciduous broad-leaved shrub belonging to the Solanaceae family and, is widely distributed throughout Korea. This plant is native to, or cultivated for various oriental medicinal purposes in, multiple regions of Asia, including Korea, China, and Japan (Lee 1982; Kim et al. 1994). Eleven Puccinia species have been reported to infect Lycium species (Otálora et al. 2018). In May and October 2022, symptoms of rust disease caused by Puccinia sp. were observed on almost all the leaves of about 60 sprawling stems of L. chinense plants on the seashore of Jeju island, Korea (33°14'15.0835″N, 126°30'53.40E). Brownish red (uredinium) or blackish brown (telium) pustules were observed on upper and lower surfaces of infected leaves. These symptoms were observed on about 40 L. chinense plants, barely growing between rocks on the seashore of Ulsan Metropolitan City, and on the about 20 L. chinense plants on a small home garden of Jindo-gun, Korea, in June and October 2023, respectively. Uredinia were amphigenous, individually scattered, but sometimes formed groups of two or three on leaves and sepals, ferruginous, pulverulent, and surrounded by a ruptured epidermis, often developing into blackish telia. Urediniospores were either ellipsoid or ovoid, approximately 29.3-34.9 × 17-24.3 µm, with yellowish walls, 1-2 µm thick. The germ pores were bizonate, and each band contained four pores covered by low papillae. Blackish-brown telia were observed on both leaf surfaces. Teliospores were broadly ellipsoidal, and rounded at the apex and towards the base. They were measured approximately 37.1-53.4 × 25-34.5 µm. The walls were light chestnut-brown and 2-3.7 µm thick, apically up to 5-9 µm thick. The swollen pedicel was persistent, basal, hyaline, smooth, and similar in length to the spores (Fig. 1). These morphological characteristics were similar to those of P. tumidipes, as described by Otálora et al. (2018). The representative specimens were preserved at the Animal and Plant Quarantine Agency Herbarium (PQK220531, -230605, and -231026). The fungal internal transcribed spacer (ITS2) and cytochrome oxidase subunit 3(CO3) regions were amplified from the total DNA of the isolates, using the primer pairs ITS5, ITS4, CO3F1, and CO3R1 for phylogenetic analysis (White et al. 1990; Vialle et al. 2009). PCR products were sequenced (Celemics, Seoul, Korea), and deposited in GenBank (Accession numbers are shown in Fig. 2.). The combined ITS2 and CO3 sequences were grouped with those of other isolates of P. tumidipes in the phylogenetic tree (Fig. 2). In November 2022, three pathogenicity tests were conducted using a urediniospore suspension made with the PQK220531 isolate in sterile distilled water. The suspension was smeared onto the upper surface of healthy L. chinense leaves. The three inoculated plants were kept in the dark at saturated moisture levels for 24 hours and placed in an isolated glasshouse together with the three control plants. After two weeks, uredinia of P. tumidipes were observed on the leaves of the inoculated plants, but not on the control plants. Although no spermogonial or aecial stage has been observed in Korea, our study has proven that P. tumidipes is the causal fungus of rust disease in L. chinense. This result is also the first discovery of the New-World P. tumidipes in Asia, showing this fungus is not limitedly distributed in America and suggesting further surveys be done on its exact geographical distribution.

Malva sylvestris, a new host of Puccinia modiolae in Korea.

Malva sylvestris (Malvaceae), known as common mallow, is native to Europe, western Asia, and northern Africa. It was intentionally introduced to Korea as an ornamental plant in the early 20th century, and has become partly naturalized in several areas including the woods (Jung et al. 2017). Among nine microcylic Puccinia species attacking the Malvaceae plants, three species of P. heterospora, P. malvacearum, and P. modiolae have been reported on M. sylvestris (Classen et al. 2000, Colenso 1885, McKenzie 1998 and Melo et al. 2012). Only P. modiolae has been found on Alcea rosea and M. verticillata, and not M. sylvestris in Korea (Lee et al. 2022; Ryu et al. 2022). In August 2022, rust disease symptoms of a Puccinia fungus were observed on some overgrown seedlings of M. sylvestris, which were neglected in containers after sales at a wholesale nursery (36°50'19.8″N, 128°55'28.7″E) in Bonghwa, Korea. Typical rust spots were observed around 60% (on 111 seedlings of the 186 seedlings of M. sylvestris). The brown spots were produced on round chlorotic haloes on the adaxial leaf surface, and brown to dark brown pustules on the abaxial. Subepidermal spermogonia on the adaxial, were obovoid, and 112.1-160.0 × 88.7-149.3 µm in size. Telia were golden-brown to dark brown, round, mostly grouped, and 0.30-0.72 mm in diameter, and mainly hypophyllus. Fusoid teliospores were two-celled, rarely one- or three-celled, 36.2-92.3 × 10.6-19.3 µm in size, with many anomalies appearing notched at apex; wall was yellowish or almost colorless, smooth, 1.0-2.6 µm thick at the sides, and up to 6.8 µm thick at the apex; pedicel was hyaline, thick wall, persistent, and (39.3-)60.4-154.6(-189.9) µm long. Based on these morphological features together with the results of the phylogenetic analyses (e-Xtra 2) using internal transcribed spacer (ITS) and partial large subunit (LSU) sequences according to the method described by Ryu et al. (2022), the fungus was identified as an autoecious P. modiolae, recently reported on M. verticillate and A. rosea in Korea (Lee et al. 2022; Ryu et al. 2022). A representative sample was deposited in the Animal and Plant Quarantine Agency Herbarium (PQK220818). Pathogenicity tests were done using three host plants: M. sylvestris, M. verticillate and A. rosea. Three to four leaf discs with basidiospore-bearing telia were placed on the upper surfaces of healthy young leaves of the seedlings. Three replicates of each host plant set including an untreated control were tested. The plants were kept in an isolated glass house. At ten to twelve days after inoculation, typical telial spots of P. modiolae were recovered, but not in the control plants, showing all three tested species were highly susceptible (e-Xtra 1). The ITS and LSU sequences obtained from the genomic DNAs of each newly recovered rust spot were consistent with that of the inoculum (accession no. OQ542745). The previous A. rosea isolate (OP369290 by Ryu et al. 2022) also showed the pathogenesis on M. sylvestris and M. verticillata by the same tests, mentioned above (e-Xtra 1). To date, only one collection of P. modiolae on M. sylvestris has been reported in Louisiana, the United States (Aime and Abbasi 2018). The results of this study show that P. modiolae is firstly confirmed as the causal rust fungus of M. sylvestris and the same causal agent of M. verticillate and A. rosea rust disease, recently reported in Korea.

First Report of Rust Disease on Alcea rosea Caused by Puccinia modiolae in Korea.

Alcea rosea, in the family Malvaceae, is a biennial plant native to China and is grown typically for gardening in Korea (Lee 2003). Seven microcyclic Puccinia species have been reported on A. rosea: P. heterogenea, P. heterospora, P. lobata, P. malvacearum, P. platyspora, P. sherardiana, and P. modiolae (Demers et al. 2015; Aime and Abbasi 2018). In early May 2022, characteristic symptoms of rust were observed on four of ten seedlings of A. rosea purchased at a wholesale nursery (36°50'19.8″N, 128°55'28.7″E) in Bonghwa, Korea. Rust spots were present on almost 90% of the 1,000 seedlings of A. rosea in that nursery during our survey in late May. Through a distribution survey from June to July 2022, similar symptomatic leaves were additionally collected from the leaves of A. rosea grown in gardens at five sites in Gimcheon (two sites), Gumi (one), Seongju (one), and Busan (one). Spots were yellow-orange the center surrounded by chlorotic haloes on the adaxial leaf surface, and reddish-brown or dark brown pustules on the abaxial leaf surface. Over time, the spots enlarged and coalesced, causing the decay of large sections of the leaves, and heavily infected leaves fell early. Spermogonia, produced at the center of the chlorotic spot on the adaxial leaf surface, were subepidermal, obovoid, and 113.2-164.5 × 97.6-153.3 µm in size. Telia were reddish-brown to dark brown, round, mostly grouped, 0.28-0.61 mm in diameter, and mainly formed on the abaxial leaf surface but sometimes on the adaxial leaf surface also. Teliospores were two-celled, but rarely one- or three-celled, and were fusoid and 37. 6-110 × 12.4-21.5 µm in size; the wall was yellowish or almost colorless, smooth, 1.2-2.6 µm thick at the sides, and up to 7.4 µm thick at the apex. The morphological characteristics were similar to those of P. modiolae, although the teliospores in our study were longer than those observed by Aime and Abbasi (2018). For phylogenetic analysis, genomic DNA was extracted from the teliospores of each regional specimen. Partial 18S, internal transcribed spacer (ITS), and partial 28S sequences were amplified using primers NS1, ITS4, ITS5, and LR11. The PCR products were sequenced (Celemics, Seoul, Korea) and deposited in GenBank. The ITS-partial large subunit (LSU) sequence and 28S sequences had 100% homology with other P. modiolae sequences deposited in GenBank (accession numbers are shown in Fig. 2). In the phylogenetic trees of the ITS and LSU sequences, the isolates collected in this study were grouped with the reference sequences of P. modiolae, including the Korean isolate (ON631218) recently reported on Malva verticillata by Lee et al. (2022). For the pathogenicity test, the teliospores with germinating basidiospores were suspended in sterile distilled water and smeared on the upper surface of asymptomatic A. rosea leaves in August. The inoculated plants were sprayed with distilled water and kept in the dark with saturated moisture for 24 h in an isolated glass house of the Animal & Plant Quarantine Agent. After 2 weeks, typical rust spots and telia of P. modiolae were observed on the leaves of the inoculated plants, but not in the control plants, which were only sprayed with distilled, sterilized water and otherwise treated similarly to the inoculated plants. The results of this study show that the casual fungus is P. modiolae, which has been commonly found in A. rosea in Korea. To the best of our knowledge, this is the first report of P. modiolae in A. rosea in Korea.

First report of mume virus A infecting Prunus salicina worldwide and Prunus mume in Korea.

Mume virus A (MuVA) of the genus Capillovirus in the family Betaflexiviridae was first isolated from a Japanese apricot tree (Prunus mume) exhibiting symptoms of diffuse chlorotic spots (Marais et al. 2018). MuVA infection has been reported in Japanese apricot trees in Japan as well as in peach (P. persica) and Japanese apricot trees in China (Marais et al. 2018; Zhang et al. 2021; Zheng et al. 2020). In the present study, the diversity of viruses and viroids infecting Chinese plum trees (P. salicina) was investigated using high-throughput sequencing (HTS). Ten flowers each from 50 trees without obvious symptoms related to virus and/or viroid infection were randomly collected from five orchards in Gimcheon, Korea, in April 2020. The samples from each Chinese plum tree were pooled, and the same amounts of 50 individual samples prepared in advance were pooled for the extraction of total RNA using the RNeasy Plant Mini Kit (QIAGEN, Hilden, Germany). Removal of ribosomal RNA and construction of cDNA library from the extracted total RNA were conducted using the TruSeq Stranded Total RNA with Ribo-Zero Plant kit (Illumina, San Diego, CA, USA). Paired-end RNA sequencing using Illumina NovaSeq 6000 System (paired-end reads of 101 bp and a total of 162,845,322 reads) and data analysis were performed at Macrogen (Daejeon, Korea). Adaptor and low-quality sequences of reads were removed using Trimmomatic program. Trimmed reads were assembled into contigs using Trinity program, and several databases including NCBI Nucleotide and Kyoto Encyclopedia of Genes and Genomes were used for functional annotation. HTS identified plum bark necrosis stem pitting-associated virus (PBNSPaV; four contigs ranging from 2081 to 3202 nucleotides) and hop stunt viroid (HSVd; one contig of 618 nucleotides). PBNSPaV and HSVd were also detected by RT-PCR (PBNSPaV det-F and PBNSPaV det-R for PBNSPaV [Al Rwahnih et al. 2007]; VP-19 and VP-20 for HSVd [Astruc et al. 1996]) and confirmed by Sanger sequencing of the amplified products. Interestingly, one contig derived from MuVA, which was not previously reported in Korea, was also detected. The contig was 7,618-nucleotide long (15,205 reads), and NCBI BLASTN search revealed 98.74% homology (100% query coverage) with the MuVA isolate pm14 (GenBank accession number MG783575). To design diagnostic primers for reverse transcription (RT)-polymerase chain reaction (PCR), the contig sequence and MuVA sequences available in NCBI GenBank (GenBank accession numbers MG783575 and MN412555) were aligned using CLC Main Workbench 6.9.1 (QIAGEN, Redwood, CA, USA). The following primer set (expected size of 1,143 bp) was prepared: MuVA-2F (5'-CAGCTTTGTGACTCYAACCC-3') and MuVA-2R (5'-AATGGCTTGAGGRCCTGCAG-3'). The primers target a partial region (nt position 1185 to 2327 on the basis of the reference genome sequence of MuVA, GenBank accession no. NC_040568) of the polyprotein gene (ORF1). Each of the 50 samples was tested for the presence of MuVA using the above-mentioned RT-PCR primers with SuPrimeScript RT-PCR Premix (GeNet Bio, Daejeon, Korea). MuVA was detected in three samples collected from the same orchard. The three amplicons were inserted into a T&A cloning vector (RBC Bioscience, Taipei, Taiwan) and sequenced at Macrogen. Three consensus sequences obtained by Sanger sequencing were registered in NCBI GenBank under accession numbers MW589492, MW589493, and MW589494. NCBI BLASTN search revealed that the Korean isolates of MuVA shared high homology with isolate pm14 [98.16%, 98.08%, and 98.16% (100% query coverage), respectively]. To confirm additional MuVA infections, leaf samples of Chinese plum trees were collected from orchards in Uiseong (70 trees) and Seongju (50 trees) as well as a Japanese apricot tree in Chuncheon, from April to July 2021. RT-PCR confirmed additional MuVA infections from Uiseong (one tree) and Seongju (one tree) as well as from the Japanese apricot tree in Chuncheon. NCBI BLASTN search of the three additional amplicons (GenBank accession numbers OM210030, OM210031, and OM210032) revealed high homology with isolate pm14 [98.25%, 98.08%, and 97.90% (100% query coverage)]. To the best of our knowledge, this is the first report of MuVA infecting P. mume in Korea and P. salicina worldwide. Further research is needed to investigate MuVA infections on various Prunus spp. including P. persica in Korea.