Target Spot on Menispermum dauricum Caused by Streptobotrys (≡Streptotinia) caulophylli, a New Disease in China.

Menispermum dauricum (moonseed) (family Menispermaceae), a perennial twining vine, is an ornamental plant traditionally used in Chinese medicine. M. dauricum is distributed mostly in northeastern, northern, and eastern China, Japan, Korea, and southern Siberia (4). Rhizoma menispermi is the dry root of M. dauricum, which has detoxifying and dehumidifying effects, and is mainly used for the treatment of sore throat, enteritis, diarrhea, and rheumatism. From June to September in 2012 and 2013, target spots were observed on moonseed plants, with an incidence above 30% in the medicinal herb garden of the Institute of Special Animal and Plant Sciences at the Chinese Academy of Agricultural Science (44°02' N, 126°05' E) in Jilin Province. Lesions on the leaves were roughly circular, forming concentric rings of alternating light and dark brown bands with yellow halos at the margins, and up to 50 mm in diameter. Lesions coalesced to a large area capable of destroying the leaf. Under humid conditions, the lesions enlarged rapidly. Occasionally, grayish-white mycelia appeared on the lesions. Subsequently, grayish-brown conidiophores arose, singly or in dense groups, up to 700 µm high, with large loose conidial heads. Side branches and branchlets were tightly twisted, brown and 7 to 11 µm in width. Conidiogenous cells were inflated at the apex of the branches and delimited by a septum. Conidia were globose to subglobose, pale brown, unicellular, minutely echinulate, and rounding to 7.8 to 16.9 µm in diameter. Four isolates were obtained from necrotic tissue from leaf spots and cultured on potato dextrose agar (PDA) at 25°C. Mycelia grown on PDA were sparse, whitish-gray, and produced small black sclerotia within 3 to 5 days. Sclerotia were round or oval to oblong and 0.3 to 1.7 × 0.8 to 1.7 mm. No conidiophores or conidia were produced on PDA. The similar species Streptobotrys streptothrix had smooth conidia and small sclerotia. So, all isolates were identified as S. caulophylli based on their morphological and cultural characteristics (2,3). The internal transcribed spacer (ITS) region was amplified by using the primers ITS4 and ITS5 (1). The ITS sequences (529 bp) were identical in these four isolates (Accession No. HG918042). Pathogenicity tests were performed on healthy 2-year-old moonseed plants. Ten leaves were inoculated with a 0.6-cm diameter mycelial plug from 3-day-old PDA cultures for each isolate, and the inoculation sites covered with moistened sterile absorbent cotton. Another 10 leaves were inoculated with sterile PDA plugs as controls. All plants in the experiments were covered with plastic bags and maintained in a greenhouse at 20 to 25°C for 24 h. After 3 days, dark brown spots were observed on all leaves inoculated with these isolates. After 7 days, the classical symptoms were evident, while control plants remained healthy. The re-isolated pathogen was identified as S. caulophylli based on morphological analysis. The pathogenicity test was repeated with similar results. Currently, the economic importance of this disease is limited, but it may become a more significant problem with the cultivation area of M. dauricum increasing. To our knowledge, this is the first report of S. caulophylli causing target spot on M. dauricum in China. References: (1) D. E. L. Cooke et al. Mycol. Res. 101:667, 1997. (2) M. E. Elliott, Can. J. Bot. 40:1197, 1962. (3) S. K. Hong et al. Plant Pathol. J. 20:192, 2004. (4) Y. H. Liu. Page 39 in: Flora Republicae Popularis Sinicae, vol. 30. Sciences Press, Beijing, 1996.

Summer Blight of White Clover (Trifolium repens) Caused by Rhizoctonia solani AG-1-IB in China.

White clover (Trifolium repens), also known as Dutch clover (family Fabaceae), is an herbaceous, perennial plant, widely planted as pasture crop and occasionally used as lawn plant. From June to September in 2012 and 2013, approximately 5 to 8% of the plants as garden lawn were infected in the areas surveyed in Tonghua County, Jinlin Province. Ash green and water-soaked lesions appeared initially on the petiole and leaves. Subsequently, petioles collapsed with soft watery rot followed by collapse of leaves and eventually the entire plant. Aerial hyphae appeared on all the infected parts, followed by production of light brown to brown sclerotia. Seven isolates with the morphological characteristics of Rhizoctonia solani Kühn were isolated from symptomatic petioles and leaves which were surface disinfested in 70% alcohol for 30 s and 0.5% sodium hypochlorite for 1 min and plated on potato dextrose agar (PDA). Hyphal tips were transferred to a fresh plate of PDA and the cultures were examined for morphological characters microscopically. Mycelia of all isolates were branched at right angles with a septum near the branch and a slight constriction at the branch base. Hyphal cells were determined to be multinucleate when stained with 1% safranin O and 3% KOH solution (1) and examined at 400× magnification with a microscope. The internal transcribed spacer (ITS) region of the nuclear rDNA was amplified by using the primers ITS4 and ITS5 (2). The ITS sequences of isolates BSYJ14 (GenBank Accession No. HF678123), BSYJ31 (HF571130), BSYY21 (HF678126), and BSY22 (KC572140) exhibited 100% identity with that of R. solani AG 1-1B (AB122138 and HQ185364). ITS sequences of another three isolates, BSYJ11 (HF678122), BSYJ32 (HF678125), and BSYJ12 (HF678121) exhibited 99% identity with the ITS sequence of R. solani AG 1-1B. Pathogenicity tests were performed on healthy, potted T. repens. Five potted plants were inoculated at the base of the petiole with a 0.6-cm diameter mycelial plug from 3-day-old PDA cultures for each isolate, and the inoculation sites were covered with moistened sterile absorbent cotton. Another five potted plants were inoculated with sterile PDA plugs as controls. All plants in the experiments were covered with plastic bags and kept in a greenhouse at 20 to 25°C for 72 h, then the plastic bags were removed. After 5 to 7 days, the symptoms of watery rot were observed on petioles and leaves of all plants inoculated with these isolates, while control plants remained healthy. R. solani AG 1-IB was re-isolated from all plants inoculated with the isolates. The isolates were confirmed by morphological characteristics of the hyphae and hyphal fusions with the original isolates. The pathogenicity test was carried out twice with similar results. R. solani has been reported to cause root rot on T. pratense in northwestern China (4) and summer blight on T. pratense in Japan (3). To our knowledge, this is the first report of R. solani AG 1-IB causing summer blight on T. repens in China. References: (1) R. J. Bandoni. Mycologia 71:873, 1979. (2) D. E. L. Cooke et al. Mycol. Res. 101:667, 1997. (3) T. Tsukiboshi et al. Bull. Natl. Grassl. Res. Inst. 39:50, 1988. (4) W.-Y. Zhuang (ed.). Fungi of Northwestern China. Mycotaxon Ltd. Ithaca, NY, 2005.

First Report of Phoma rhei as a Pathogen of Rheum rhabarbarum in China.

Rheum rhabarbarum L., rhubarb, is a perennial herb planted mainly in Hebei, Hubei, Shanxi, Heilongjiang, and Jilin provinces as well as Inner Mongolia, China. The plant grows about 1,000 meters above sea level (4), and is used widely in China to treat constipation and gout. From June to September 2012, a leaf spot was observed on R. rhabarbarum in the medicinal garden of Jilin Agricultural University, Changchun, Jilin Province, causing significant effects on the leaves of all infected plants. In the early stage of disease development, small red lesions were visible on infected leaves, which subsequently developed into irregularly shaped or circular necrotic spots, each with a light colored center, pink-red alternating concentric rings, and surrounded by a chlorotic halo. Some lesions became perforated in the center. Lesions ranged from 1 to 15 mm in diameter. Extensive spotting resulted in general browning and yellowing of entire leaves. As lesions enlarged and coalesced, some leaves died from the margin inwards. Lesions on the stem were fusiform and sunken. Small pieces of diseased leaves and stems were surface-disinfested in 75% ethanol for 60 s, rinsed twice in distilled water, dried, and plated on potato dextrose agar (PDA). A Phoma species was isolated that produced a gray or dark gray colony after 5 to 7 days. The isolate was transferred to oatmeal agar (OA) (3). Pycnidia were dark brown to black, globose to subglobose, and 121 to 354 × 100 to 262 µm. Conidia were ellipsoidal or reniform, colorless, unicellular, and 3.8 to 6.5 × 1.7 to 4.1 µm. On the basis of these characteristics, the fungus was identified as Phoma rhei (1). A PCR assay with the ITS4 and ITS5 primers was used to amplify the internal transcribed spacer (ITS) region of ribosomal DNA (rDNA) (2). The amplified product (567 bp) was sequenced and the sequence submitted to GenBank (Accession No. KF531831). The ITS sequence exhibited 99% identity to that of a P. rhei isolate in GenBank (GU237743.1), confirming the morphological identification. Pathogenicity of eight isolates on rhubarb was confirmed by spraying a spore suspension (1 × 106 spores/ml) produced on PDA on the leaves of each 6-year-old R. rhabarbarum (cv. Boyedahuang) plant. Each isolate was inoculated onto five plants, and five plants were sprayed similarly with sterilized water as a control treatment. The plants were then covered with plastic bags for 48 h, and kept in a greenhouse (20 to 30°C with a 12-h photoperiod/day). Initial symptoms on inoculated leaves were observed 3 to 4 days after inoculation, while the control plants remained healthy. Re-isolations from lesions on the inoculated leaves, using the same protocol as the original isolations, produced fungal colonies with the same morphological characteristics as the original isolates of P. rhei, but no fungi were re-isolated from the control plants. This fungus has been found on R. rhaponticum in New Zealand (1), but to our knowledge this is the first report of P. rhei on R. rhabarbarum in China. References: (1) G. H. Boerema et al. Phoma Identification Manual. Diffferentiation of Specific and Infra-Specific Taxa in Culture. CABI Publishing. Wallingford, UK, 2004. (2) D. E. L. Cooke et al. Mycol. Res. 101:667, 1997. (3) Z. D. Fang. Research Method of Phytopathology. China Agricultural Press (In Chinese), 1998. (4) A. J. Li et al. Flora Reipublicae Popularis Sinicae. Tomus 25:171, 1998.

Veronica sibirica Leaf Spots Caused by Phacellium veronicae, a New Disease in China.

Veronica sibirica (Veronicastrum sibiricum) is an erect perennial herb, an ornamental, and a traditional Chinese medicine plant distributed mostly in northeastern, northern, and northwestern China. It has dehumidifying and detoxifying properties, and is mainly used for the treatment of cold, sore throat, mumps, rheumatism, and insect bites (4). In June 2008 through 2012, leaf spots of V. sibirica were observed in the Medicinal Herb Garden of Jilin Agricultural University (43°48'N, 125°23'E) and the medicinal plantations of Antu County (43°6'N, 128°53'E), Jilin Province. Leaf spots were amphigenous, subcircular, angular-irregular, brown, and 1 to 10 mm in diameter; they occasionally merged into a larger spot with an indefinite margin or with a pale center and dark border. Pale conidiomata were hypophyllous and scattered on the spots. The conidiophores were 100 to 400 µm high and clustered together to form synnemata 20 to 50 µm in diameter, which splayed out apically and formed loose to dense capitula. Conidiophores occasionally emerged through the stomata individually and produced conidia on the surface of the infected leaves. The conidiogenous cell terminal was geniculate-sinuous with somewhat thickened and darkened conidial scars. Conidia were solitary or catenulate, ellipsoid-ovoid or subcylindric-fusiform, hyaline and spinulose, 4.01 to 7.18 × 11.16 to 20.62 µm with obtuse to somewhat attenuated ends, and slightly thickened, darkened hila. Six isolates were obtained from necrotic tissue of leaf spots and cultured on potato dextrose agar at 25°C. After incubation for 14 days, colony surfaces were white to pinkish. The colony diameter increased by 12 mm after 21 days' incubation. Hyphae were hyaline, septate, and branched. Conidiophores grew individually or fascicularly. The symptoms and morphological characteristics were consistent with previous descriptions (1,2), and the fungus was identified as Phacellium veronicae (Pass.) (U. Braun 1990). The internal transcribed spacer (ITS) region of the nuclear rDNA was amplified using primers ITS4/ITS5 (3). The ITS was identical among all six isolates (HE995799) and 98% identical to that of P. veronicae (JQ920427, HQ690097). Pathogenicity was confirmed by spraying five 1-year-old V. sibirica seedlings with a conidial suspension (106 conidia/ml) of each isolate and five seedlings with sterile water as a control treatment. Plants were grown in the greenhouse at 20 to 25°C and were covered with plastic bags to maintain humidity on the foliage for 72 h. After 15 days, the same symptoms appeared on the leaves as described earlier for the field-grown plants; the control plants remained healthy. The same fungus was reisolated from the leaf spots of inoculated plants. Currently, the economic importance of this disease is limited, but it may become a more significant problem, as the cultivated area of V. sibirica is increasing. To our knowledge, although P. veronicae was recorded on the other species of Veronica (V. austriaca, V. chamaedrys, V. grandis, V. longifolia, V. paniculata, and V. spicata ssp. incana) in Europe (Germany, Denmark, Ireland, Romania) and V. wormskjoldii in North America (Canada) (1), this is the first report of V. sibirica leaf spots caused by P. veronicae in the world, and it is a new disease in China. References: (1) U. Braun. A monograph of Cercosporella, Ramularia and allied genera (phytopathogenic Hyphomycetes) 2, IHW-Verlag, Germany, 1998. (2) U. Braun. Nova Hedwigia 50:499, 1990. (3) D. E. L. Cooke et al. Mycol. Res. 101:667, 1997. (4) Jiangsu New Medical College. Dictionary of Chinese Materia Medica. Shanghai: Shanghai Scientific and Technical Publishers, China, 1977.

First Report of Daylily Leaf Streak Caused by Kabatiella microsticta in China.

Daylily (Hemerocallis spp.) is an herbaceous, perennial plant, cultivated for its flowers. Daylily is sold in Asian markets as fresh or dried flowers (the flowers of some species, e.g., Hemerocallis citrina, are edible) or as the corm, which is used for medicinal purposes. In June 2011, daylily leaf streak was found in a nursery of Jilin Agricultural University, Jilin Province, China. Symptoms included water-soaked, irregular spots along the leaf midvein that turned orange to reddish brown and eventually enlarged to coalesce into extensive, necrotic streaks along the length of the leaf, as previously reported (2). Heavily infected leaves often withered and died. Four isolates were recovered from necrotic tissue of leaf spots and cultured on potato dextrose agar (PDA) at 25°C. All colonies were initially cream to peach colored and appeared slimy. With the maturation of the culture, the colonies became dark brown to black with sparse aerial hyphae. Blastic conidia formed simultaneously on intercalary or terminal, undifferentiated conidiogenous cells, and were scattered in dense sections on culture surface. When the conidia dropped from conidiogenous cell, an indistinct scar or a denticle remained. Conidia were hyaline, one-celled, smooth, ellipsoidal, and variable in size (2.73 to 6.01 × 8.45 to 19.36 µm), and all morphological characteristics were consistent with Kabatiella microsticta Bubak (syn. Aureobasidium microstictum; 2,4). The internal transcribed spacer (ITS) region of the nuclear rDNA was amplified using primers ITS4/ITS5 (1). ITS (534 bp) was identical among all four isolates (GenBank Accession No. HE798117) and 100% identical to that of K. microsticta CBS 114.64 (FJ150873). Pathogenicity was confirmed by spraying 20 seedlings of daylily, propagated in tissue-culture medium, with a conidial suspension (106 conidia/ml) of each isolate. A second set of 20 seedlings was sprayed with the same volume of sterile water as the noninoculated control treatment. Plants were grown in the greenhouse at 20 to 25°C and were covered with plastic bags to maintain humidity on the foliage for 72 h. After 5 days, the foliar symptoms described earlier for the field plants appeared on the leaves, whereas the control plants remained healthy. K. microsticta was reisolated from the leaf spots of all 20 inoculated plants. Leaf streak is the most destructive disease of daylily, and was previously reported in Japan and the United States (Illinois, Kentucky, Mississippi, Louisiana, Pennsylvania, Maryland, Virginia, Florida, North Carolina, and Georgia) (3). To our knowledge, this is the first report of the disease caused by K. microsticta in China. References: (1) D. E. L. Cooke et al. Mycol. Res. 101:667, 1997. (2) E. J. Hermanides-Nijhof. Stud. Mycol. 15:153, 1977. (3) R. M. Leahy et al. Plant Pathology Circular No. 376, 1996. (4) P. Zalar et al. Stud. Mycol. 61:21, 2008.

First Report of Alternaria Leaf Spot on Eleutherococcus senticosus Caused by Alternaria tenuissima in China.

Eleutherococcus senticosus (Ruper et Maxim) Maxim, a very important potential medicinal plant used for the treatments of neurasthenia, anti-aging, and kidney deficiency, is a perennial herb belonging to Araliaceae and mainly distributed in northeast China. With the development of its cultivation, many diseases start to occur and a previously unknown leaf spot was observed on this plant in July 2007 in Linjiang City, Jilin Province, China. This disease incidence reached 100% in some planting grounds and it has resulted in serious loss of acanthopanax production. This disease generally happens during July and August in Jilin Province, China. At the initial stage of the infection, some small, light brown spots appeared on the leaves that gradually become round or irregular, dark brown, concentrically zonate with a dark brown margin, frequently surrounded by light yellow haloes and conspicuous black brown concentric rings in the advanced stage of the infection. The necrotic areas often coalesce and result in the appearance of larger spots with a diameter of 13.0 to 15.0 mm. Severely affected plants were defoliated. On leaf spots, conidia, generally in short chains, were straight, multicellular, obclavate or obpyriform, olivaceous brown or dark brown, with three to eight transverse and rarely zero to four longitudinal or zero to three oblique septa, and measured 8.3 to 27.5 × 17.3 to 55.0 µm. Conidiophores arose singly or in groups, straight or flexuous, cylindrical, expand in base cell, branch occasionally, septate, pale to olivaceous brown, 25.0 to 75.0 µm long, 3.0 to 6.0 µm wide; beak or false beak cylindrical, septate, colorless or light brown, and measured 2.0 to 5.1 × 10.4 to 47.7 µm. The morphological descriptions and measurements of the fungi are similar to Alternaria tenuissima (2). Six single cultures from the infected leaves were isolated on potato dextrose agar. Pathogenicity tests were carried out on the potted, healthy, 1-year-old plants (n = 10). These plants were divided into two groups, one group was sprayed with a conidial suspension of 105 conidia per ml and the other was sprayed with sterilized water as control plants. All plants were covered with polyethylene bags for 3 days. Symptoms of the disease appeared 5 days after inoculation. Symptoms on the inoculated leaves were similar to those that naturally occurred on the plants. The fungal pathogen was consistently reisolated from the inoculated plants but not from the control plants. The internal transcribed spacer (ITS) region of rDNA was amplified from DNA extracted from single-spore isolate cwz-2 of the pathogen using the ITS1/ITS4 and sequenced (GenBank Accession No. HQ402558). The ITS sequence had 99% identity with that of A. tenuissima strain XSD-83 (GenBank Accession No. EU326185). Therefore, the pathogen was identified as A. tenuissima on the basis of its morphological characteristics and ITS sequence. A. tenuissima was reported to occur on many plants such as blueberry in China (1). However, to our knowledge, this is the first report of A. tenuissima occurring on E. senticosus in China. References: (1) Y. S. Luan et al. Plant Dis. 91:464, 2007. (2) T. Y. Zhang et al. Fungi Notes-Genera Alternaria in China, 16:19, 38, 2003.