L. N. Yang

Genetic diversity of Microdochium nivale isolates from turfgrass

Conserved primers were used in a polymerase chain reaction to amplify the ITS region of the rDNA of 100 Microdochium nivale isolates collected from different turfgrasses in southern Ontario. The profile of the restriction digestion of the amplified ITS region revealed that all the M. nivale isolates analysed belonged to var. nivale. RAPD profiling and RFLP analyses of the IGS regions of rDNA revealed extensive genetic diversity within var. nivale. With RAPD markers, the average similarity coefficient was 66% and the estimate of genotypic diversity was 0.179. Population subdivision analysis showed that 92.2% of the total genetic diversity was found among individuals within populations compared to 7.8% among populations. In dendrograms derived from genetic distances using RAPD and IGS-RFLP markers, there was some evidence for host specialization. Most RAPD markers were shared by individuals from different turfgrasses, and the populations were not highly differentiated. The high level of genotypic diversity detected within populations and the low level of genetic differentiation among populations show that recombination and migration are likely playing important roles in the population biology of M. nivale var. nivale.

First Report of Botrytis cinerea Causing Stem Blight of Scutellaria baicalensis in Jilin Province, China

Chinese skullcap (Scutellaria baicalensis) is a perennial herb in the Lamiaceae, and is grown in Jilin and 10 other provinces in China. Skullcap root extracts are used for treating allergies and respiratory conditions (5), and have inhibitory effects on some plant pathogens (2). In 2012, stem blight on 3-year-old Chinese skullcap plants were observed from mid-June to late-September in a herb garden at Jilin Agricultural University, Jilin Province, China. More than 90% of the 15 ha of skullcap plants were infected, and almost 60% of the infected plants died. A similar situation occurred in other regions of Jilin in 2012 and 2013. Oblong to irregular stem lesions, each 3 to 5 cm long, appeared on the basal stem. Lesions were initially light brown, and turned dark brown because of velvety, thick, gray fungal sporulation that formed over the lesions. The lesions eventually encircled the stems, preventing water and nutrient uptake. The infected plants often turned chlorotic and wilted. Symptomatic stem tissues were collected from three gardens in Jilin Province. Small pieces of diseased stems were surface-disinfested in 3% NaOCl for 2 min, rinsed twice in sterilized distilled water, plated on potato dextrose agar (PDA) medium, and incubated at 22°C for 5 days. Ten isolates were recovered, and all produced gray hyphae and dark sclerotia on PDA. The conidia formed on tree-like, branched conidiophores and were colorless, single, lemon-shaped, smooth-walled, and 9.0 to 16.9 × 5.7 to 9.7 μm. Sclerotia formed when cultures on PDA plates were incubated for 20 days at 22°C. The sclerotia were dark, irregular, and 2.2 to 3.8 × 1.1 to 2.6 mm. To confirm pathogenicity of each of the 10 isolates, five 3-year-old Chinese skullcap plants were each inoculated on the basal stem with an 8-mm-diameter colonized PDA plug of the appropriate isolate. Five plants were inoculated similarly with non-colonized PDA plugs as a control treatment. All plants were maintained at 22°C in a greenhouse. The first lesions appeared on stems 5 days after inoculation, whereas control plants remained healthy. The same fungus was consistently recovered from inoculated lesions as the original isolates, and no fungus was re-isolated from control plants. DNA was extracted from isolate RSL-1 and amplified using the ribosomal DNA (rDNA) internal transcribed spacer (ITS) region primers ITS5/ITS4 (1), glyceraldehyde-3-phosphate dehydrogenase gene (G3PDH) primers G-F/G-R, heat-shock protein 60 gene (HSP 60) primers H-F/H-R, and DNA-dependent RNA polymerase subunit II gene (RPB2) primers R-F/R-R (3). The ITS, G3PDH, HSP 60, and RPB2 sequences (GenBank Accession Nos. JX840480, KJ018760, KJ018758, and KJ018756, respectively) of isolate RSL-1 showed 100% similarity to the ITS sequence of strain WM6 of Botryotinia fuckeliana (anamorph Botrytis cinerea) (JN164269) (1), 100% identity to the G3PDH sequence of isolate Ice-2 of B. fuckeliana (AB546620) (3), 100% identity to the HSP60 sequence of isolate MUCL1152 of B. fuckeliana (AJ716090) (4), and 99.8% identity to the RPB2 sequence of isolate WM6 of B. fuckeliana (JN164272) (1). Based on these characteristics, the fungus was identified as B. cinerea. To the best of our knowledge, this is the first report of B. cinerea causing stem blight on S. baicalensis in Jilin Province, China. This disease may potentially cause great losses under favorable conditions. References: (1) X. Li. Plant Dis. 95:1592, 2011. (2) J. Y. Liu et al. Hubei Agric. Sci. 50:1809, 2011. (3) K. Maeda et al. J. Gen. Plant Pathol. 76:303, 2010. (4) M. Staats et al. Mol. Biol. Evol. 22:333, 2005. (5) K. Zandi et al. BMC Compl. Alternat. Medicine 13:1472, 2013.

First report of ginseng (Panax ginseng) as a natural host of dodder (Cuscuta japonica) in China.

Ginseng (Panax ginseng C.A. Meyer) belongs to the Araliaceae family and is a high-value medicinal herb grown mainly in Jilin Province, China. China is the worlds oldest and largest producer of ginseng. Annual yield and exports account for more than 78 and 60% for the world, respectively. In July 2009 and August 2010, dodder (Cuscuta sp.), a parasitic seed plant, was observed on 4- and 5-year-old plants that were being cultivated on raised beds under artificial shading in three separate locations approximately 200 kilometers apart in Jilin Province. The infested area was approximately one-third of the field (300 m2 of 10,000 m2), which was previously planted with pine trees and herbaceous plants. Initially, there were no obvious symptoms on ginseng plants, but later, symptoms consisted of poor growth, chlorosis, and wilting, eventually followed by death in large areas throughout the field. One typical representative of 10 samples collected was identified as Cuscuta japonica based on several morphological characteristics, including yellow stems with purplish spots with branches 1.2 to 2.5 mm in diameter. Inflorescences were spicate and measured 3 cm with broadly ovate, scale-like bracts. Flowers were sessile with deeply divided calyxes. The parasite had purplish, ovate sepals with pink or greenish white corollas. The plant had five stamens with yellow, ovate-circular anther. Ovaries were globose and smooth. Capsules contained three to four pale yellow or brown ovate seeds measuring 1.9 to 2.7 mm (3,4). The internal transcribed spacer (ITS) region of rDNA of the representative samples was amplified using primers ITS4/ITS5 (2) and sequenced. BLASTn analysis of the 650-bp amplicon (GenBank Accession No. JF431541) showed 99% sequence identity with C. japonica (Accession Nos. DQ924571 and EU330320). Phylogenetic trees constructed by utilizing the neighbor-joining method with software MEGA 4.0 (1) placed the dodder specimens and C. japonica (DQ924571 and EU330320) into one group. The reported hosts of C. japonica include plants belonging to the Leguminosae, Salicaceae, Polygonaceae, and Compositae. Therefore, to our knowledge, this is the first report of C. japonica naturally infecting P. ginseng in the world. Reference: (1) K. Tamura et al. Mol. Biol. Evol. 24:1596, 2007. (2) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, 1990. (3) Z. Y. Wu. Flora Reipublicae Popularis Sinicae 64:147, 1979. (4) L. P. Yin. Plant Quarantine (in Chinese) 9:292, 1995.

First Report of Botrytis Leaf Blight on Eleutherococcus senticosus Caused by Botrytis cinerea in China

Eleutherococcus senticosus (Acanthopanax senticosus, manyprickle acathopanax) is a perennial herb belonging to the family Araliaceae and is mainly distributed in northeastern China, Siberia, Korea, and Japan. It is used for the treatment of rheumatism and neurasthenia. With the development of its cultivation, many diseases began to occur (2) and a previously unknown leaf blight on manyprickle was first observed in July of 2010 in Linjiang City, Jilin Province. The same symptoms were detected in other areas of Jilin Province, such as Baishan and Hunchun cities. The disease has resulted in serious loss of production of manyprickle acanthopanax, with 5 to 10% of leaves infected. The infection initially manifested as irregular lesions on the tips or margins of the leaves, which gradually developed into a V-shaped blight with concentric rings that was grayish brown in the center and dark brown at the margins. The blight eventually spread to cover one third of the entire leaf. Severely infected leaves were rolled or distorted, eventually desiccated and became brittle. Under continuously humid conditions, scattered gray mycelium and conidia appeared on the surface of affected leaf tissue. To isolate the causal agent, tissues were excised from diseased leaves, immersed in 0.1% mercuric chloride, suspended in sterile water, and plated on potato dextrose agar (PDA). Conidiophores arose singly or in groups, straight or flexuous, septate, with an inflated basal cell and dendriform near the apex, brown to light brown, and measured 5.0 to 10.0 × 100.0 to 150.0 μm (n = 50). Conidia were single-celled, globoid or oval-shaped, colorless, measuring 6.0 to 10.0 × 7.0 to 13.0 μm (n = 50). In culture, dark, irregular sclerotia were produced. The morphological descriptions and measurements of the fungi were similar to Botrytis cinerea (4). The ITS region of rDNA was amplified and sequenced. BLAST analysis of the 567-bp segment (JX840481) showed 100% identity with the sequence of Botryotinia fuckeliana (perfect stage of B. cinerea). To further identify the species of B. cinerea, three nuclear protein-coding genes (G3PDH, HSP60, and RPB2) (3) were sequenced and the sequences (KJ018759, KJ018757, and KJ018755) all showed 100% identity with those of B. fuckeliana. Pathogenicity tests were carried out on potted, healthy, 1-year-old plants (n = 10). A conidial suspension of 105 conidia/ml was sprayed with each strain (five strains total) on five leaves still on plants, and five plants were sprayed with water as controls. Plants were covered with polyethylene bags and incubated for 3 days at 25°C in a greenhouse. Symptoms appeared 7 days after inoculation, and were similar to those originally observed on plants under natural conditions, whereas control plants remained healthy. The pathogen was successfully re-isolated from inoculated leaves and was identified as B. cinerea on the basis of its morphological characteristics and related gene sequences. B. cinerea has been previously reported on E. senticosus in Korea (1). However, to our knowledge, this is the first report of Botrytis leaf blight of E. senticosus caused by B. cinerea in China. These results lay the foundation for the disease control. References: (1) K. J. Choi et al. Korean J. Med. Crop Sci. 15:199, 2007. (2) J. Gao et al. Plant Dis. 95:493, 2011. (3) M. Staats et al. Mol. Biol. Evol. 22:333, 2005. (4) Z. Y. Zhang. Flora Fungorum Sinicorum. 26. Botrytis, Ramularia. Science Press, Beijing, 2006.

First Report of Botrytis pelargonii Causing Postharvest Gray Mold on Fresh Ginseng Roots in China

Chinese ginseng (Panax ginseng) is a perennial Chinese herb of the Araliaceae family. It is found in the three northeast provinces of China. As a valuable restorative tonic, it is widely used in traditional Chinese medicine (Radad et al. 2006). In November 2015 and 2016, gray mold was observed on 10 to 35% of fresh ginseng roots in two storage facilities under complete darkness at 4°C in Ji An and Fu Song City, Jilin Province. Initial symptoms included light brown, water-soaked lesions on ginseng roots. Within 30 days, the diseased areas enlarged, with dense, abundant gray mycelia forming on lesions. To isolate the pathogen, small pieces of diseased roots were surface disinfested in 3% NaClO for 2 min, rinsed twice in distilled water, plated on potato dextrose agar (PDA), and incubated at 22°C for 5 days. Sixteen isolates were recovered by single-spore isolation. Colony morphology was almost identical among all isolates. Representative isolate PG-S015 was grown on PDA under constant fluorescent light at 22°C. After 5 days, abundant mycelia with branched conidiophores were produced. Conidia were single, aseptate, pale brown, and globose to subglobose. Conidial size ranged from 8.2 to 17.3 μm in length and from 5.8 to 12.2 μm in width, averaging 13.6 ± 0.6 × 8.7 ± 0.3 μm (n = 50). The conidia were slightly longer than B. cinerea (Zhang et al. 2006). After 9 days, black, scattered, irregular sclerotia were produced. Sclerotial size measured 1.0 to 3.0 × 1.3 to 6.0 mm with the average of 2.1 × 3.8 mm (n = 50). DNA was extracted from all isolates. All 16 isolates were sequenced for the rDNA internal transcribed spacer (ITS), glyceraldehyde-3-phosphate dehydrogenase (G3PDH), DNA-dependent RNA polymerase subunit II (RPB2), and heat-shock protein 60 (HSP60) genetic markers. Their ITS sequences (KY810515 to KY810530) were identical to those of B. pelargonii (AJ716290) and B. cinerea (KY319171). The G3PDH (KY827182 to KY827197) and HSP60 (KY827198 to KY827213) sequences were 137 and 42 bp shorter than those of B. cinerea (GenBank KY275256 and KX229753) and identical to those of B. pelargonii (AJ704990 and AJ716046). The RPB2 sequences (KY827214 to KY827229) were also 99% identical to that of B. pelargonii (AJ745662). The fungus was identified as B. pelargonii based on its morphological and molecular characteristics (Staats et al. 2005; Zhang et al. 2006). Pathogenicity tests were performed by pipetting a 100 μl of conidial suspension (1 × 10⁵ conidia/ml) of each selected isolate PG-S015 and PG-S-FS-017 onto a 5-year-old fresh ginseng root, three replicates per isolate. Sterilized water was used as the noninoculated control treatment. The experiment was carried out three times. All inoculated roots were maintained at 4°C and 90% relative humidity, similar to the environmental conditions in which the disease was observed. Within 30 days, all inoculated ginseng roots developed similar symptoms to those of the naturally occurring disease described above, whereas all noninoculated roots remained healthy. Isolates with identical morphological and molecular characteristics were recovered from the diseased roots. Prior to this report, B. pelargonii was exclusively recovered from Pelargonium spp. (Strider 1985). This is the first report of B. pelargonii causing postharvest gray mold on stored fresh ginseng roots. Proper management should be taken to reduce the losses from this disease to the quality and marketability of ginseng as a highly valuable traditional Chinese medicine.

Fusarium solani, a New Pathogen Causing Postharvest Fruit Rot on Kiwifruit (Actinidia deliciosa) in China

China is the top kiwifruit producing country worldwide, approximately 10,000 ha of commercial production (Li et al. 2016; Wang et al. 2017). About 10% of the kiwifruits harvested from several Chinese cultivars including Jinyan, Hongyang, and Qinmei in several fruit markets from Shaanxi (Xi’an city) and Sichuan (Dujiangyan city) provinces of China had postharvest rot in 2015 and 2016. All fruits were collected from the fruit markets and the symptoms were observed. Symptoms appeared as soft, light to dark brown, sunken lesions which gradually became round or oval. The lesions often started from the pericarp and then extended to the whole fruit. To isolate the pathogen, small pieces (5 mm length) of the lesion margin collected from ten diseased fruits were surface-sterilized in 0.1% HgCl2 for 1 min, 75% ethanol for 30 s, rinsed twice with sterile distilled water, placed on potato dextrose agar (PDA), and incubated for 4 days at 25°C. Then, eight pure cultures were obtained by single-spore culturing (Fs-c-1 t...