Clematis terniflora var. mandshurica (Rupr.) Ohwi, a traditional Chinese medicinal plant, is widely distributed in northeast China, such as Heilongjiang, Jilin and Liaoning provinces. In August 2019, C. terniflora var. mandshurica (Rupr.) Ohwi leaf blight was found in Harbin (45.72°N, 126.68°E), Heilongjiang, China. The incidence was up to 90% on the 0.4 ha plantation, and almost every plant leaf was withered. Initial symptoms were chlorosis at the leaf apex or margin, and the leaf apex gradually formed brown lesions. As the infection progressed, the lesions expanded and leaves withered. Ten symptomatic leaves were randomly collected from ten plants in Clematis plantation. The leaves (5mm×5mm) between symptomatic and healthy tissue were cut and disinfected with 75% ethanol for 1 min and 1% NaClO for 2 min, rinsed three times in distilled water, plated on potato dextrose agar (PDA: the diameter of Petri dish is 90 mm) and incubated for 7 days at 25℃, further purified by single-spore isolation method. The morphology of all the ten isolates was similar. The colonies were round or sub round, 60–68 mm diameter in size, velvety, brownish green with distinct concentric rings. Conidiophores were solitary or clustered, suberect, brown, 30.3–82.4×2.5–5 μm (n=150) in size, and with septum. Conidia were solitary or in chains, smooth surface, obclavate, ellipsoid, brown, 12.5–46.25×10–16.25 μm (n=150) in size, with 2–7 transverse septa, 0–3 longitudinal septa. Conidia were produced continuously and extended in a synaxial manner. Based on the morphological characteristics, the isolates were identified as Alternaria alternata The morphological characters matched those of Alternaria alternata (Simmons 2007). Two representative strains TXL0816 and TXL0916 were used for molecular identification. The internal transcribed spacer (ITS) region, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), translation elongation factor 1-alpha (tef1), RNA polymerase second largest subunit (RPB2), and Alternaria major allergen (Alt a 1) were amplified with the primers ITS1/ITS4 (Iturrieta-González et al. 2020), RPB2-5F2/RPB2-7CR (Khodaei and Arzanlou 2013), Alt-for/Alt-rev (Hong et al. 2005), gpd1/gpd2 and EF1-728F/EF1-986R (Nishikawa and Nakashima 2020). The obtained sequences were deposited in the GenBank (ITS: MW403865 and MT508666, GAPDH: MZ298638 and MZ298639, tef1: MZ298640 and MZ298641, RPB2: MW432545 and MT510746, Alt a 1: MW432544 and MT501719). The phylogenetic tree of combined sequences showed that isolates TXL0816, TXL0916 and A. alternata CBS916.96 were clustered together with 100% bootstrap values. clustered into one branch supported with 100% bootstrap values. To verify pathogenicity, twenty leaves of eight plants were sprayed with spore suspensions (1×106 spores /mL) of the 7 days-old isolates TXL0816 and TXL0916 (four plants were used as replicates for each isolate)each isolate infected four plants). As a control, four plants were sprayed with sterile distilled water. The plants were incubated at 25℃ in a greenhouse. Twenty days after inoculation, brown lesions appeared on the leaf apex. The pathogenicity test was repeated three times. The same fungi were re-isolated from the inoculated leaves, The fungi were re-isolated from inoculated leaves with the same morphological and molecular traits as A. alternata, fulfilling Koch's postulates. No fungi were isolated from the control group. To our knowledge, this is the first report of A. alternata causing leaf blight on C. terniflora var. mandshurica (Rupr.) Ohwi. This study provides a basis for the pathogenesis of the leaf blight on C. terniflora var. mandshurica (Rupr.) Ohwi, and helps to formulate control measures to reduce future economic losses.
Menispermum dauricum DC. is an ornamental plant used in traditional Chinese medicine. (Tang et al. 1992). In September 2019, a leaf spot on M. dauricum DC. was first found in a medicinal plant plantation in Harbin city (45.80°N, 126.53°E), Heilongjiang Province, China. The incidence was 76-90% on the 0.02 ha plantation. The initial symptoms were irregular black and brown spots on the leaves. The lesions expanded and coalesced, eventually leading to blight. Fresh leaf samples from ten M. dauricum plants with typical symptoms were collected. The areas of leaf between symptomatic and healthy tissue (5㎜×5㎜) were cut and surface disinfeated in 75% ethanol for 2 min, and with 1% HgCl2 for 1 min, and then rinsed three times with sterile water. Small lesion pieces were incubated on potato dextrose agar (PDA) for 7 days at 25℃, in the dark. Ten fungal isolates were obtained and transferred onto new PDA and potato carrot agar (PCA) plates to establish pure cultures. After 8 days, the colonies on PDA were 75-86㎜ in diameter, circular, with distinct concentric rings and a whitish aerial-mycelium margin, cottony, light gray to dark bluish brown. The colonies on the PCA were olive-green and bordered by white aerial hyphae. A total of 150 conidia were single or in short chains, obclavate, oval or inverted pear, light brown to brown, smooth or slightly spiny, with 1 to 6 transverse septa, 0 to 4 longitudinal or oblique septa, not narrow or slightly narrowed at the separation, 22.5-42.5×7.5-15.5㎛, and rostrate. Conidiophores were simple, erect, or ascending, dark brown, geniculate, septate, and with one or several conidial scars, 32.5-77.5×3.0-5.0㎛. Beaks were columnar or conical, 7.5-22.5×2.5-3.5㎛. Morphologically, all ten isolates were most similar to Alternaria alternata (Simmons 2007). For further identification of the fungus at the molecular level, internal transcribed spacer rDNA regions (ITS), RNA polymerase second largest subunit gene (RPB2) and Alternaria major allergen (Alt a 1) were amplified and sequenced using the primers ITS1 and ITS4, RPB2-5F2 and RPB2-7CR, Alt-for and Alt-rev (Woudenberg et al. 2015). The resulting sequences were deposited in GenBank (ITS: MT995193, MZ150794, RPB2: MT999483, MZ170963, Alt a 1: MT802122, MZ170962). BLAST search of these sequences showed 99%-100% homology with the ITS (FJ196306), RPB2 (KC584375) and Alt a 1 (KT315515) of the type strain CBS 916.96 of A. alternata, respectively. Thus, the fungus was identified as A. alternata based on the morphology and molecular analysis. For the pathogenicity test, spore suspensions (1×106 spores/mL) of the representative isolates BFG001 and BFG002 were sprayed onto the leaves of six healthy plants, separately. As a control, six plants were treated with sterile distilled water. The plants used in the experiment were covered with plastic bags and incubated at 25℃ for 10 days. Eight days after inoculation, irregular, slightly sunken black leaf spots appeared at the leaf margin. The experiment was repeated three times with the same method. The same fungus was successfully re-isolated from the leaves of the inoculated plants, fulfilling Koch’s postulates. No symptoms were observed on control plants. To our knowledge, this is the first report of leaf spot disease on M. dauricum DC. caused by A. alternata in the world. The appearance of leaf spot disease reduces the yield and quality of Chinese medicinal materials. This report has laid the foundation for the further research and control of leaf spot disease.
Phedimus aizoon is native to east Asian countries that including China, Siberia, Korea, Mongolia, and Japan. In China, the plant is highly valued for use in folk medicine, for detoxification and analgesia, blood pressure, hemostasis, and used as an ornamental. In August 2021, a leaf spot and blight disease were observed on P. aizoon in a 120-ha field in Pizhou, Jiangsu Province, China where disease incidence reached 90%, and almost every leaf was withered. Early symptoms appeared as dark brown lesions on leaf margins that enlarged and coalesced to form large necrotic areas. In efforts to determine the cause of the disease, ten symptomatic leaves were randomly collected from ten different plants at the site. Diseased leaf pieces that measured 5 mm2 were disinfected in 75% ethyl alcohol for 30 s and 7% NaOCl for 60 s, rinsed three times in sterile distilled water, and placed on potato dextrose agar (PDA). Ten fungal isolates obtained by single-spore isolations were selected for further study. These isolates produced colonies that measured 70 to 82 mm in diameter after 7 days growth on PDA. Colonies were black to brown in color with gray-white aerial hyphae on their surfaces. The isolates produced conidia that were ovate to pear-shaped, brown to black in color, with 1 to 4 transverse septa and 0 to 1 oblique septa, smooth surfaced, parietal cells extending into the beak, and measured 10 to 35.5 × 5.0 to 12.5 μm. Conidiophores were brown, erect or curved, branched, with pronounced spore marks, and measured 7.5 to 37.5 × 2.5 to 5.0 μm. All ten fungal isolates were morphologically similar to Alternaria alternata (Simmons 2007). Two representative isolates FC01 and FC02 were used for molecular identification. The internal transcribed spacer (ITS) region, RNA polymerase second largest subunit (RPB2), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), translation elongation factor 1-alpha (TEF1), and Alternaria major allergen (Alt a 1) were amplified with the primers ITS4/ITS5, RPB2-5F2/RPB2-7CR (Khodaei and Arzanlou 2013), gpd1/gpd2, EF1-728F/EF1-986R (Nishikawa and Nakashima 2020) and Alt-for/Alt-rev (Woudenberg et al. 2015). The resulting sequences were deposited in GenBank (ITS, ON584560, ON564492; RPB2, ON729984, ON703241; GAPDH, ON652866, ON652867; TEF1, ON652868, ON652869; Alta1, ON652870, ON652871). Phylogenetic analyses showed 100% identity between FC01 and FC02 and the type strain CBS 916.96. Thus, the fungus was identified as A. alternata based on morphology and molecular analysis. Pathogenicity tests were done by spraying conidial suspensions containing 106 conidia per ml of A. alternata isolates FC01 and FC02 on leaves of five healthy P. aizoon plants, separately. Five control plants were sprayed with distilled water and both sets of plants covered with plastic bags and placed in a greenhouse maintained at 25⁰ C. Plastic bags were removed from plants after 48 h. Dark brown lesions developed on inoculated plants after 16 days and control plants remained symptomless. The pathogenicity tests were conducted three times. A. alternata was reisolated and identified based on morphological and molecular traits, thus fulfilling Koch’s postulates. To our knowledge, this is the first report of A. alternata causing leaf blight on P. aizoon in China and worldwide. Based on the plant's medicinal value, further studies should be directed toward control of this disease.
Fraxinus rhynchophylla Hance, is a deciduous trees cultivated on a commercial scale focused on medicinal and wood production. In September 2021, leaf spot was observed on F. rhynchophylla in Heilongjiang Province (127.34°E, 45.19°N), China. These symptoms were observed on 100% F. rhynchophylla plants and the incidence of diseased leaves per plant reached 70% in fields measuring 90 ha. Disease symptoms were small yellow flecks initially, and then turned to gray necrotic spot. Ten diseased leaves were collected randomly from 5 plants and surface disinfested. Tissue samples (2 × 2 mm) were cut at the disease-health junction of the leaves, surface sterilized in 75% ethanol for 30 s, submerged in a 7% NaOCl solution for 3 mins, and rinsed three times with sterile water. Leaf segments were placed onto potato dextrose agar (PDA) and incubated at 26℃ for 5 days. After isolation and purification of monospore, the colonies of the all isolates were inky black, with aerial fluffy mycelium, and concentric whorls on PDA. The conidiophore is septate, single-branched, brown, smooth and 35 - 313 × 2 - 5 μm in size (n = 50), while the conidia are brown, bow-shaped, mostly four cells, with three septa and 12 - 385 × 5 - 20 μm in size (n = 150). The morphological characters matched those of Curvularia muehlenbeckiae (Madrid et al. 2014). DNA was extracted from isolates HQLa and HQLb and used for PCR amplification of RNA polymerase II gene (RPB2) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene sequences using the primer fRPB2-SF/fRPB2-7Cr (Schoch et al. 2009), gpd1/gpd2 (Berbee et al. 1999), respectively. The RPB2 (OM984674, OM984675) and GAPDH (OM984672, OM984673) were deposited in GenBank. The phylogenetic tree was constructed by combining other published sequences of RPB2 and GAPDH genes using the maximum likelihood method, and the results showed that the obtained isolates clustered into the same clear branch as C. muehlenbeckiae CBS 144.63 (HG779180, HG779108), with 100% bootstrap support. Combining morphological characteristics and phylogenetic analysis of the fungus, the obtained isolates were identified as C. Muehlenbeckiae. To fulfill the Koch’s postulates, pathogenicity tests were carried out on newly grown leaves of F. rhynchophylla. Conidia of the selected isolates grown on PDA plates were flooded with sterile distilled water. Spore suspension was adjusted to 105 spores/mL with the hemocytometer. Three leaves of each plant were disinfected with 1% NaOCl for 2 min, washed with sterilized distilled water three times, and dried with sterile paper towels. Three plants were randomly selected for inoculation under field conditions and each leaf was sprayed with 2 mL of the spore suspension for a total of nine leaves, then the plants were bagged and moistened for 48 h. However, control leaves were sprayed with distilled water. Symptoms were observed nine days after inoculation. No symptoms were observed on control leaves. The same fungus was successfully re-isolated from the lesions. The experiment was replicated three times with the same results and C. muehlenbeckiae identification was confirmed by morphological observations and RPB2 and GAPDH sequencing, indicating that the fungus is the causal pathogen of leaf spot disease on F. rhynchophylla. This is the first report of C. muehlenbeckiae determined as fungal pathogens on F. rhynchophylla plant in China. The results of the study laid the foundation for the future occurrence and epidemiological pattern of the disease and scientific control.
Clematis brevicaudata DC. is distributed in China, Korea, Mongolia, Russia and Japan. This plant is both ornamental and medical, used in the treatment of nervous disease, dyskinesia and other diseases. In September, 2019, a leaf spot on C. brevicaudata was first found in a 5 ha C. brevicaudata plantation in Harbin, Heilongjiang Province, China. The incidence was about 80%. The symptoms were elliptical, circular, or irregular brown to black necrotic lesions in leaf apex and leaf margin. Ten fresh sample leaves with typical symptoms were collected from ten C. brevicaudata plants. The tissues (5mm×5mm) between symptomatic and healthy junction were cut and surface disinfected in 75% ethanol, and with 7% NaClO for 1 min, then rinsed three times with sterilized water, 30s each time. The sterilized tissues were inoculated on potato dextrose agar (PDA) plates for 7 days at 25℃. The colonies were obtained and transferred onto new PDA and potato carrot agar (PCA) plates by single spore method to further purify. After 7 days, the colonies on PDA were 50 to 63 mm in diameter, circular, grayish brown, with white aerial hyphae. A total of 150 conidia on PCA were single or in chains, ovoid, inverted pear, 2 to 7 transverse septa, 0 to 3 longitudinal or oblique septa, 17.5 to 57.5 × 7.5 to 17.5 μm. Beaks and supposititious beaks were mostly columnar, rarely conical, 2.5 to 6.0 × 2.0 to 3.0 μm. Conidiophores were solitary or clustered, pale brown, erect or bent, branched or unbranched, separated, 112.0 to 151.0 × 5.1 to 14.7 μm. Ten isolates purified on PDA were obtained. Morphological identification showed the ten isolates were similar and appeared to be Alternaria alternata (Simmons, 2007). Two strains from ten isolates were selected for molecular identification. Genomic DNA was extracted from mycelia of two isolates (LD2020520 and LD2020521) on PDA using a modified CTAB method. Internal transcribed spacer rDNA regions (ITS), RNA polymerase II second largest subunit gene (RPB2), Alternaria major allergen (Alt a 1), endopolygalacturonase (endoPG) and glyceraldehyde 3-phosphate dehydrogenase (gpd) were amplified and sequenced using two directional sequencing with the primers ITS1/ITS4, RPB2-F/RPB2-R, Alt-F/Alt-R, end-F/end-R and gpd-F/gpd-R (Woudenberg et al. 2015). The sequences obtained were deposited in GenBank (ITS: MT501762, OK571395; RPB2: MT506027, OK631891; Alt a 1: MT506026, OK631890; endoPG: ON054189, ON054188; gpd: ON054191, ON054190). The phylogenetic analysis of maximum-likelihood tree by MEGA 7 software showed that the two isolates had 99% identity with the A. alternata CBS 916.96. For pathogenicity testing, eighteen leaves of six 5-week-old plants were sprayed with spore suspensions (1×106 spores /mL) of the 7 days-old isolates LD2020521 and LD2020520 (Each isolate infected three plants and each infected three leaves). Three plants were sprayed with sterile distilled water as a control group. The plants were incubated at 25℃. After 15 days, taupe irregular spots appeared on the leaves. The pathogenicity test was repeated three times. The same fungi were re-isolated from the inoculated leaves and with the same morphological and molecular characteristics as LD2020520 and LD 2020521, fulfilling Koch's postulates. No fungi were isolated from the control group. This is the first report of leaf spot on C. brevicaudata caused by A. alternata. Leaf spot can reduce the yields of C. brevicaudata. This study provides a reference for the prevention and treatment to the leaf spot of C. brevicaudata.
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