First Report of Leaf Spot in Farfugium japonicum Caused by Alternaria alternata in China

Farfugium japonicum (L.) Kitam (with the common name leopard plant) is known as a garden and medical herb, and belongs to the family Asteraceae. In May 2019, a leaf spot disease was observed on the upper leaf surface of F. japonicum in Changsha city, Hunan province, China. More than 98% of the F. japonicum plants were infected in a garden of Donghu district (28°13′ N; 112°56′ E). Leaf symptoms included small (1 to 10 mm in diameter), brown spots that were circular, tan to gray in the center and distinct brownish-yellow margins. Severely affected leaves were blighted and plants were dying. For isolation, symptomatic leaf tissue was surface sterilized, rinsed in sterile distilled water, and plated on potato dextrose agar (PDA) amended with a 50 μg/ml streptomycin sulfate followed by incubation at 25°C in darkness. By a single-spore isolation technique, pure fungal cultures were obtained and displayed gray-brown and gray-white aerial mycelia after five days of incubation. One representative isolate (HnAa-1) was selected for further studies. Conidia of HnAa-1 were olive brown, obpyriform, either branched or unbranched with a short beak, 1 to 5 transverse septa, and 0 to 3 longitudinal or oblique septa. The conidia were 10 to 35 μm long and 2 to 12 μm wide. HnAa-1 was identified as an Alternaria sp. on the basis on morphological characterization by Simmons (1). Further identification to species level was made by molecular analyses. DNA of HnAa-1 was extracted from the regions internal transcribed spacer (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and partial Alt a 1 major allergen (ALT) gene. Amplification and sequencing was carried out with the method described by Woudenberg et al.(2) . BLASTn searches showed that the ITS, GAPDH and ALT sequences had the highest similarity with A. alternata strains, with 100% (548/548) identities for ITS (GQ169728), 100% (567/567) identities for GAPDH (MK903028) and 99.36% (466/469) identities for ALT (MN184998). Moreover, the ITS, GAPDH and ALT sequences had more than 99% identities with the epitype CBS 916.96 of A. alternata (ITS: AF347031; GAPDH: AY278808; ALT: AY563301). The ITS, GAPDH and ALT sequences of HnAa-1 were submitted to GenBank (Accession No. MT767170, No. MW115639 and No. MW316727). Pathogenicity tests were conducted by spraying a 10 ml conidial suspension (1.0 ×105 conidia /mL) on surfaces of leaves of three healthy plants (8-week-old). Leaves of three healthy plants were sprayed with sterile distilled water as a control treatment. All inoculated plants were maintained in growth chamber at 25°C with a 12-h photoperiod. The pathogenicity test was repeated twice. After five days inoculation, typical brown spots and necrotic lesions similar to those observed in the field, had developed on all inoculated plants but not on water-treated control plants. Alternaria alternata was re-isolated from the symptomatic tissue of inoculated plants but not from the control plants, and re-identified with morphological and molecular methods, which fulfilled Koch's postulates. This host-pathogen association has been reported in Korea (3), but it is the first report of A. alternata causing leaf spots on F. japonicum in China. Since A. alternata is a ubiquitous and very important plant pathogen causing leaf spot diseases in over 100 species plant, the occurrence of this disease is a serious threat to F.japonicum and might lead to economic losses. Therefore, appropriate prevention strategies to F.japonicum should be adopted.

The taxonomic status of Aphelenchoides besseyi Christie, 1942 (Nematoda: Aphelenchoididae) populations from the southeastern USA, and description of Aphelenchoides pseudobesseyi sp. n.

Summary Populations reviously identified as Aphelenchoides besseyi were studied. Using an integrated approach, the A. besseyi species complex contains several cryptic species: A. besseyi sensu stricto, A. oryzae, A. pseudobesseyi sp. n. and other putative undescribed species. A population from Florida strawberry morphologically fits the A. besseyi of both Christie and Allen and is considered the only representative of this species. A Louisiana rice population fitted the descriptions of A. oryzae of both Yokoo and Fortuner; PUS length was consistently less than one-third of VA. Aphelenchoides oryzae, parasitising rice and other monocots, was re-established based on morphological and molecular datasets. Three populations from Florida ornamental plants (Dryopteris erythrosora, Echinacea sp. and Farfugium japonicum) differed from those of the two above-mentioned species and are described as A. pseudobesseyi sp. n. Populations previously identified as ‘A. besseyi’ from several countries were considered representatives of this new species, which usually had a large and conspicuous PUS, 8-14 μm wide and with a length greater than one-third of VA in 40-70% of studied specimens. Morphological variability made separation of A. pseudobesseyi sp. n. from A. oryzae and A. besseyi unreliable without the examination of numerous specimens and molecular analysis.

First Report of Leaf Spot in Farfugium japonicum Caused by Alternaria tenuissima in Korea

Farfugium japonicum (L.) Kitam (common name: Leopard plant) is known as a medical herb and belongs to family Asteraceae (1). In June 2012, a leaf spot disease was observed on the leaf surface of F. japonicum at a forest research plot Jinju, Gyeongnam province, Korea. More than 95% of F. japonicum plants were infected and leaf spot symptom appeared in the regions under our investigation. Light brownish symptoms initially developed and the spot size gradually increased and turned dark brown with an irregular shape as the disease progressed (spot size 1 to 10 mm in diameter). At the late stage of disease, spots became hollow and completely dehydrated. The infected leaves were easily crumbled, possibly due to dryness. To isolate the causal agent, the infected leaves were surface disinfected and pieces of leaves were placed on water agar (WA). Nine isolates were isolated from 10 pieces of the infected leaves. Fungi mycelia from the WA were transferred on potato dextrose agar (PDA) and incubated at 28°C for 7 days. The colonies were purple navy to black and conidia spores developed on the media. The morphological characteristics of spores were multi-septate, dark brown, pyriform, and 6.7 to 12.8 × 22.2 to 38.4 μm. The spores had 1 to 4 transverse and 0 to 3 longitudinal septa. The morphological characteristics of the isolates showed considerably similar to well-known Alternaria tenuissima (2). The leaf spot disease caused by A. cinerariae of F. japonicum was reported from Japan (3). Spores of A. cinerariae are golden brown to brown with 3 to 9 transverse and 0 to 6 longitudinal septa and are 87.5 × 28.7 μm (avg.) (3). To verify pathogenicity of the isolate, the pure cultured fungi on the PDA medium was taken (4 mm in diameter) and placed on healthy leaves of Leopard plant. The artificially inoculated leaves were placed on wet filter paper in Petri dishes and incubated at 25°C and 80% humidity. At 7 days after inoculation, similar disease symptoms developed on 8 out of 10 infected Leopard plant leaves. The pathogen was reisolated from artificially infected leaves. To identify in molecular biology level, genomic DNA was extracted and the ITS-rDNA region was amplified using universal primers ITS1 and ITS4. The amplified PCR product was purified and sequenced (528 bp) with ITS1 and ITS4 primers for both directions and then deposited in GenBank (Accession No. KC415611.1). The BLAST search showed that it matched previously reported A. tenuissima with 100% identity. To the best of our knowledge, this is the first report of Leopard plant leaf spot disease in Korea. References: (1) E. Y. Kim et al. J. Ethnopharmacol. 146:40, 2013. (2) E. G. Simmons. Page 1 in: Alternaria Biology, Plant Diseases and Metabolites. J. Chelchowski and A. Visconti, eds. Elsevier, Amsterdam, 1992. (3) T. Sakoda et al. Res. Bull. Pl. Prot. Japan 46:73, 2010.

First Confirmed Report of Powdery Mildew Caused by Podosphaera xanthii on Farfugium japonicum in Korea

Farfugium japonicum (L.) Kitam., known as Japanese silver leaf, is native to Japan, Korea, and Taiwan. It is grown as an ornamental plant for garden plantings and containers not only in East Asia but more recently also in Europe and North America. Since 2003, powdery mildew infections of F. japonicum ‘Gigantea’ have been consistently found in the southern part of Korea, including the districts of Jeju, Seogwipo, Busan, Wando, and Ulleungdo. Specimens have been deposited in the Korea University Herbarium (KUS). Signs of powdery mildew first appeared as circular to irregular white patches on both sides of the leaves. The infections were usually severe on young leaves and caused malformation and browning. Appressoria on the mycelium were nipple-shaped or nearly absent. Conidiophores, measuring 160 to 280 × 10 to 12.5 μm, were simple and produced 2 to 12 immature conidia in chains, followed by 2 to 3 cells. Foot-cells in conidiophores were relatively short, 50 to 95 μm long, and constricted at the base. Conidia were hyaline, ellipsoid to ovate, 32 to 48 × 17.5 to 25 μm (length/width ratio = 1.4 to 2.3), had distinct fibrosin bodies, and produced germ tubes on the lateral position. No chasmothecia were observed. The morphology and dimentions of reproductive structures were compatible with those of Podosphaera xanthii (Castagne) U. Braun & Shishkoff (1). To confirm the identity of the causal fungus, the complete ITS region of rDNA from isolate KUS-F26469 was amplified with primers ITS5 and P3 (4) and directly sequenced. The resulting sequence of 475 bp was deposited in GenBank (Accession No. KC155426). A GenBank BLAST search of this sequence revealed 100% identity (475/475 bp) with those of many P. fusca isolates on plants in the Aster family plants including Calendula officinalis, Euryops pectinatus, Syneilesis palmata, and F. japonicum from Japan (e.g., AB040346). The P. fusca isolates listed above are now placed in P. xanthii (1). Pathogenicity was confirmed through inoculation by gently pressing diseased leaves onto leaves of three healthy potted plants of the same cultivar. Three non-inoculated plants served as controls. Plants were maintained in a greenhouse at 25 ± 2°C. Inoculated plants developed typical signs and symptoms of powdery mildew after 8 days, whereas the control plants remained symptomless. The fungus present on the inoculated leaves was morphologically identical to that originally observed on diseased plants. Powdery mildew infections of F. japonicum caused by P. fusca (syn. P. fuliginea) have been reported previously in both Japan and Korea (2). In Korea, it was listed simply as a host fungus of Ampelomyces quisqualis, which is hyperparasitic to powdery midlews, without any data on its identity (3). To our knowledge, this is the first confirmed report of powdery mildew caused by P. xanthii on F. japonicum in Korea. References: (1) U. Braun and R. T. A. Cook. Taxonomic Manual of the Erysiphales (Powdery Mildews), CBS Biodiversity Series No.11. CBS, Utrecht, 2012. (2) D. F. Farr and A. Y. Rossman. Fungal Databases. Syst. Mycol. Microbiol. Lab., Online publication, ARS, USDA, Retrieved November 14, 2012. (3) M. J. Park et al. Fungal Biol. 114:235, 2010. (4) S. Takamatsu et al. Mycol. Res. 113:117, 2009.