Report Of Leaf Spot Research Articles

First report of leaf spot caused by Phoma matteuciicola on Commelina communis in Liaoning, China

First report of leaf spot caused by Phoma matteuciicola on Commelina communis in Liaoning, China

First report of leaf spot and stem canker on Malania oleifera caused by Neofusicoccum parvum in China

First report of leaf spot and stem canker on Malania oleifera caused by Neofusicoccum parvum in China

First Report of Leaf Spot Caused by Alternaria brassicae on Avena sativa in China.

Oat (Avena sativa) is an annual gramineous crop, which contains a source of soluble dietary fiber, β-glucan, unsaturated fatty acids, vitamins, minerals, phenolic acids and avenanthramides. It widely cultivated in cool and semi-arid areas in northern China (Li et al, 2017). In July 2018, a severe leaf spot infection was observed in the Forage Germplasm Nursery (31°17'22″N, 103°40'15″E, 2885 m elevation) in Tianzhu County, Wuwei City of Gansu Province in China. Disease incidence (total number of diseased leaves / total number of surveyed leaves X 100%) was 93% over 300 m2 planting area. Symptoms initially appeared as small circular to irregular, gray-green, water-soaked spots on the leaves in the middle or along the margin of leaves, that enlarged and coalesced. The center of the leaf spots turned brown to reddish-brown. Infected tissues from symptomatic leaves were cut into small pieces (5×5 mm), surface sterilized with 70% ethanol for 60 s, soaked in 5% commercial bleach (~0.275% NaClO) for 5 min (Xue et al, 2018), rinsed five times with distilled water, plated on potato dextrose agar (PDA) medium, and incubated for 3 days in the dark at 25°C (Zhang, 2003; Blagojević et al, 2020; Humpherson et al, 1989). Hyphae emerging from the tissue were subcultured on fresh PDA medium for purification. All colonies were light brown with intensive sporulation in rings that was grayish white, and later became grayish brown. The back of the colony was dark brown. Conidiophores were light brown, unbranched, grew vertically on hyphae, and each conidiophore produced 3 to 7 conidia (mostly 6). Conidia were light brown, septate, straight to slightly curved, single or in chains, oval or obclavate, measured 17 to 32 µm wide and 63 to 106 µm long with the conical beak cell, 7 to 12 transverse septa, 0 to 5 longitudinal septa. These morphological characteristics were similar to the descriptions of Alternaria spp. (Simmons, 2008). A single isolate, YMZZ1, was selected for molecular identification. The ITS region of rDNA, partial GAPDH and Tef1-α gene sequences were amplified by PCR with the primer pairs of ITS1/ITS4, gpd1/gpd2 and EF1-728F/EF1-986R, respectively (Woudenberg et al, 2013). Sequences were deposited in GeneBank under accessions MN446739 (ITS), MN481462 (GAPDH), and MN464104 (Tef1-α). A nucleotide BLAST search revealed ITS, GAPDH and Tef1-α sequences to be 99% similar to accessions numbers MN856410 (565/573 bp), MK026431 (575/575 bp), and MH754531 (211/211 bp), respectively) of A. brassicae. Neighbor-joining (NJ) and Maximum Likelihood (ML) phylogenetic analysis were conducted based on ITS, GAPDH and TEF-1α sequences using MEGA7.0 under Kimura 2-parameter model. The isolate YMZZ1 clustered with a representative strain A. brassicae LGBA22 with 100% bootstrap support. To test its pathogenicity, six healthy 3 week old plants were spray-inoculated with a suspension of 3×105 conidia/mL of YMZZ1. The same number of plants were sprayed with sterilized water as control. All plants were covered with transparent plastic bags for 48 h to maintain high relative humidity and incubated in a 25°C growth chamber (16/8 h light/dark) for observation. Ten days after inoculation, leaf spot symptoms were observed on leaves similar to those previously observed in nursery; no symptoms were observed on the control. The pathogenicity test was repeated twice under the same conditions and A. brassicae was re-isolated from inoculated plants each time fulfilling Koch's postulates. A. brassicae has not been previously reported as a pathogen of A. sativa in the world, but has been mentioned as a pathogen of horse radish (Armoracia rusticana) in Serbia (Blagojevic et al, 2015). To our knowledge, this is the first report of leaf spot caused by A. brassicae on A. sativa in China. This study stresses an urgent need to identify appropriate management strategies of A. brassicae that help in preventing losses in quality and yield of oats in northern China.

First report of leaf spot disease caused by Corynespora cassiicola on American sweetgum (Liquidambar styraciflua L.) in China.

American sweetgum (Liquidambar styraciflua L.) is a forest plant native to North America, which has been introduced into other countries due to its ornamental and medicinal values. In June 2019, symptoms of leaf spots on sweetgum were observed in a field (5 ha) located in Xuzhou, Jiangsu Province, China. On this field, approximately 45% of 1,000 trees showed the same symptoms. Symptoms were observed showing irregular or circular dark brown necrotic lesions approximately 5 to 15 mm in diameter with a yellowish margin on the leaves. To isolate the pathogen, diseased leaf sections (4×4mm) were excised from the margin of the lesion, surface-sterilized with 0.1% NaOCl for 90 s, rinsed 4 times in sterile distilled water, air dried and then transferred on potato dextrose agar (PDA) medium at 25°C in the dark. Pure cultures were obtained by monospore isolation after subculture. Ten purified isolates, named FXI to FXR, were transferred to fresh PDA and incubated as above to allow for morphological and molecular identification. After 7 days, the aerial mycelium was abundant, fluffy and exhibited white to greyish-green coloration. The conidia were dark brown or olive, solitary or produced in chains, obclavate, with 1 to 15 pseudosepta, and measured 45 to 200µm  10 to 18µm. Based on morphological features, these 10 isolates were identified as Corynespora cassiicola (Ellis et al. 1971). Genomic DNA of each isolate was extracted from mycelia using the cetyltrimethylammonium bromide (CTAB) method. The EF-1α gene and ITS region were amplified and sequenced with the primer pairs rDNA ITS primers (ITS4/ITS5) (White et al. 1990) and EF1-728F/EF-986R (Carbone et al.1999) respectively. The sequences were deposited in GenBank. BLAST analysis revealed that the ITS sequence had 99.66% similarity to C. cassiicola MH255527 and that the EF-1α sequence had 100% similarity to C. cassiicola KX429668A. maximum likelihood phylogenetic analysis based on EF-1α and ITS sequences using MEGA 7 revealed that ten isolates were placed in the same clade as C. cassiicola (Isolate: XQ3-1; accession numbers: MH572687 and MH569606, respectively) at 98% bootstrap support. Based on the morphological characteristics and phylogenetic analyses, all isolates were identified as C. cassiicola. For the pathogenicity test, a 10 µl conidial suspension (1×105 spores/ml) of each isolate was dripped onto healthy leaves of 2-year-old sweetgum potted seedlings respectively. Leaves inoculated with sterile water served as controls. Three plants (3 leaves per plant) were conducted for each treatment. The experiment was repeat twice. All seedlings were enclosed in plastic transparent incubators to maintain high relative humidity (90% to 100%) and incubated in a greenhouse at 25°C with a 12-h photoperiod. After 10 days, leaves inoculated with conidial suspension of each isolate showed symptoms of leaf spots, similar to those observed in the field. Control plants were remained healthy. In order to reisolate the pathogen, surface-sterilized and monosporic isolation was conducted as described above. The same fungus was reisolated from the lesions of symptomatic leaves, and its identity was confirmed by molecular and morphological approaches, thus fulfilling Koch's postulates. Chlorothalonil and Boscalid can be used to effectively control Corynespora leaf spot (Chairin T et al.2017). To our knowledge, this is the first report of leaf spot caused by C. cassiicola on L. styraciflua in China.

First Report of Diaporthe longicolla Causing Leaf Spot on Kalanchoe pinnata in China.

Kalanchoe pinnata (Lam.) Pers. [syn.: Bryophyllum pinnatum (Lam.) Oken] is an important medicinal agent in southern China. The succulent leaves of this plant are used in the treatment of cholera, bruises, uri-nary diseases and whitlow. In Oct. 2019, leaf spots were detected on K. pinnata plants in Chengmai County, Hainan Province, China. Lesions with brown to black margins were irregularly shaped and associated with leaf margins. Spots coalesced to form larger lesions (Fig. S1-A), with black pycnidia present in more mature lesions. Symptomatic K. pinnata were found with 10-20% incidence during the humid winters of Hainan Province. Leaf tissues of 10 symptomatic plants were collected and surface sterilized in 70% ETOH for 30s, 0.1% HgCl2 for 30 s, rinsed 3x with sterile distilled water for 30s, placed on potato dextrose agar (PDA) amended with 30mg/L of kanamycin sulfate, and incubated at 25°C in the dark for 3-5 days. Four fungal isolates were obtained using a single-spore isolation method. The colonies were floccose, dense, and white with forming on older colonies grown on PDA (Fig. S1-B-1&2). Alpha conidia exuded from ostiole, rostrate, long-beaked pycnidia in creamy-to-yellowish drops. Alpha conidia were hyaline, ellipsoidal, separated and averaged 6.3μm (SD ± 1.13) long × 1.9μm (SD ± 0.33) wide (n=50). Beta conidia were not seen. The morphological characteristics matched the previous description of Diaporthe longicolla (syn. Phomopsis longicolla) (Hobbs et al. 1985). Mycelial genomic DNA of the representative isolate LDSG3-2 was extracted as template. The internal transcribed spacer (ITS) , translation elongation factor 1α gene (TEF) and β-tubulin (TUB2) regions were amplified. These loci were amplified using primer pairs ITS4/ITS5 (White, et al. 1990), EF1-728F/EF1-986R (Carbone and Kohn 1999) and Bt2a/Bt2b (Glass and Donaldson 1995), respectively. A BLAST search of GenBank showed ITS (MN960195), TEF (MN974483) and TUB2 (MN974482) sequences of the isolate were 99%, 100%, and 99% homologous with D. longicolla strains DL11 (MF125048, 557/563 bp), D55 (MN584792, 347/347 bp) and DPC-HOH-32 (MK161506, 502/504 bp). Maximum likelihood trees based on concatenated nucleotide sequences of the three genes were constructed using MEGA 7.0, and bootstrap values indicated the isolate was D. longicolla (Fig. S1-D). Pathogenicity testing was performed using isolate LDSG3-2 by depositing 5µl droplets of a conidial suspension (1 × 106 ml-1) into 5 artificially wounded leaves (using a sterile needle) of 10 healthy 3-month-old K. pinnata plants. An equal number of artificially wounded control leaves were inoculated with sterile water to serve as a negative control. The test was conducted three times. Plants were kept at 25°C in 80% relative humidity and observed for symptoms. Two weeks after inoculation, no symptoms were observed on control plants (Fig. S1-C-1) and all inoculated plants showed symptoms (Fig. S1-C-2) similar to those observed in the field. The fungus was re-isolated from the infected tissues and showed the same cultural and morphological characteristics of the strain inoculated and could not be isolated from the controls fulfilling Koch's postulates. To our knowledge, this is the first report of leaf spot on K. pinnata caused by D. longicolla in China. This disease is of concern since Phomopsis diseases are common in K. pinnata fields and can cause significant reduction in yield.

First Report of Leaf Spot caused by Curvularia lunata on Wild Rice in China.

Wild rice (Oryza rufipogon), a species only recently cultivated in China, is an invaluable resource for rice breeding and basic research. In June 2019, a leaf spot disease on wild rice (O. rufipogon cv. 'Haihong-12') was observed in a 3.3 ha field in Zhanjiang (20.93 N, 109.79 E), China. The early symptoms were the presence of small, brown, and circular to oval spots that eventually turned reddish brown. The size of the spots varied from 1.0-5.0 mm × 1.0-3.0 mm. Disease incidence was higher than 20%. High temperature and high humidity climate were favorable for the disease occurrence. Twenty diseased leaves were collected from the field. The margin of the diseased tissues was cut into 2 mm × 2 mm pieces, surface-disinfected with 75% ethanol for 30 s and 2% sodium hypochlorite for 60 s, then rinsed three times with sterile water before isolation. The tissues were plated onto potato dextrose agar (PDA) medium and incubated at 28 °C in the dark for 4 days. Pure cultures were produced by transferring hyphal tips to new PDA plates. Three isolates, namely, Cls-1, Cls-2, and Cls-3, were subjected to further morphological and molecular studies. The colonies of the three isolates on PDA were initially light gray later becoming dark green. Conidiophores were erect, dark brown, geniculate, and unbranched. Conidia were fusiform, geniculate or hook-shaped, smooth-walled, dark-brown, 3-septate, with the second curved cell about 13.4-18.2 μm × 6.5-8.6 μm in size (n = 30). These morphological features agreed with previous descriptions of Curvularia lunata (Wakker) Boed (Macri and Lenna 1974). The ITS region, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and translation elongation factor (EF-1α) were amplified using primers ITS1/ITS4, gpp1/gdp2 (Berbee et al. 1999), and EF-1/EF-2 (O'Donnell 1997), respectively. Amplicons of the three isolates were sequenced and submitted to GenBank (accession nos. MW042182, MW042183, and MW042184; MW091453, MW091454, and MW091455; MW090049, MW090050, and MW090051). The sequences of the two isolates were 100% identical to those of C. lunata (accession nos. MG971304, MG979801, MG979800) according to the results of BLAST analysis. A phylogenetic tree was built on the basis of concatenated data from the sequences of ITS, GAPDH, and EF-1α via the maximum likelihood method. The tree clustered Cls-1, Cls-2, and Cls-3 with C. lunata. The three isolates were determined as C. lunata by combining morphological and molecular characteristics. Pathogenicity tests were performed on Cls-1 in a greenhouse at 24 °C-30 °C with 80% relative humidity. Individual rice plants (cv. 'Haihong-12') with three leaves were grown in 10 pots, with approximately 50 plants per pot. Five pots were inoculated by spraying a spore suspension (105 spores/mL) onto leaves until runoff occurred, and another five pots were sprayed with sterile water and used as controls. The test was conducted three times. Disease symptoms were observed on the leaves after 10 days, but the controls remained healthy. C. lunata occurs on O. sativa (rice) (Liu et al. 2014; Majeed et al. 2016), but it has not been reported on O. rufipogon until now. To the best of our knowledge, this study is the first to report that C. lunata causes leaf spots on O. rufipogon in China. Thus, vigilance is required for breeding O. rufipogon.

First Report of Leaf Spots caused by Nigrospora oryzae on Wild Rice in China.

In recent years, wild rice (Oryza rufipogon Griff) has been widely cultivated because of its health-promoting effects. In May 2019, leaf spot lesions on cv. Haihong-12 were observed in Zhanjiang (20.93N, 109.79E), China. Leaf symptoms were yellow-to-brown, oval or circular with a verydistinctive, large yellow halo. Black spores appeared on the leaves with advanced symptoms. The lesions coalesced, causing the entire leaf to become blighted and die. Disease incidence reached approximately 10% in the fields (8 ha) surveyed. Twenty leaves with symptoms were collected and cut into pieces of 2 ×2 cm in size. They were surface-disinfected with 75% ethanol for 30 s and 2% sodium hypochlorite (NaOCl) for 60 s, rinsed three times with sterile water, blotted dry on sterile paper, plated on potato dextrose agar (PDA) medium, and incubated at 28°C in the dark for 4 days. Ten pure cultures were obtained by transferring hyphal tips to new PDA plates, and monosporic cultures were obtained from three isolates (Nos-1, Nos-2, and Nos-3). Those isolates exhibited very similar morphological characteristics on PDA. Colony of isolate Nos-1 was white at the early stage and became dark gray after 7 days. Conidia were produced from clusters of conidiophores, single celled, black, smooth, spherical, and 9.5 to 14.2 µm (average 10.6 µm ± 0.42) in diameter. Morphological characteristics of the isolates matched the description of Nigrospora oryzae Petch (Wang et al. 2017). The ITS region was amplified using primers ITS1 and ITS4 (White et al. 1990). Nucleotide sequences of isolates Nos-1, Nos-2, and Nos-3 deposited in GenBank under acc. nos. MW042173, MW042174, and MW042175, respectively, were 100% identical to N. oryzae (acc. nos. KX985944, KX985962; and KX986007). A phylogenetic tree generated based on the ITS sequences and using a Maximum Likelihood method with 1,000 bootstraps showed that these three isolates from wild rice were grouped with other N. oryzae isolates downloaded from GenBank (bootstrap = 100%) but away from other Nigrospora spp. Pathogenicity test was performed with these three isolates in a greenhouse at 24 to 30°C. Approximately 50 seedling of wild rice cv. Haihong-12 were grown in each pot. At the 3-leaf stage, plants in three pots were inoculated with each isolate by spraying a spore suspension (105 spores/ml) until runoff. Three pots sprayed with sterile water served as the controls. Each 3-pot treatment was separately covered with a plastic bag. The test was conducted three times. Diseased symptoms were observed on the inoculated leaves after 10 days while no disease was observed in the control plants. Morphological characteristics and the ITS sequences of fungal isolates re-isolated from the diseased leaves were identical to those of N. oryzae. N. oryzae has been reported to cause leaf spot on O. sativa (Wang et al. 2017), but not on O. rufipogon. Thus, this is the first report of N. oryzae causing leaf spot of O. rufipogon in China. The finding provides the information important for further studies to develop management strategies for control of this disease.

First report of leaf spot on Elaeagnus pungens caused by Epicoccum latusicollum in China

AbstractElaeagnus pungens is an ornamental plant used in landscapes and is native to China and Japan. In Nov. 2015, a leaf spot was found on El. pungens in the Sheshan National Forest Park located at Shanghai. A fungus was isolated from the margins of the lesions and one isolate, EP8, selected for identification and pathogenicity tests. Morphological characteristics of the fungus were similar to Epicoccum latusicollum. Phylogenetic analysis using concatenated sequences of ITS, LSU, TUB, and RPB2 also showed that isolate EP8 clustered with strains of E. latusicollum, a relationship supported with a high bootstrap value. This report is the first of E. latusicollum causing leaf spot on El. pungens.

First Report of Leaf Spot and Stem Canker on Watermelon Caused by Paramyrothecium foliicola in Mexico.

Watermelon is an important vegetable crop in Mexico and produced on 358,105 ha, with nearly 1.5 x 106 tons. In September 2019, brown, irregular shape to round lesions with concentric rings were observed on the leaves and stems of watermelon plants in Sonora State. The surface of the lesions contained abundant cup-shaped sporodochia covered by masses of olive-green to black conidia. Edge sections of symptomatic tissues were cut from the leaves, disinfected in 70% ethanol for 1 min and subsequently washed twice with distilled water. Disinfected tissue samples were transferred to PDA medium and incubated at 27°C for 15 days. White colonies were observed with spordochia arranged in concentric rings with characteristic of greenish-black masses of conidia. Spore masses stained with lactophenol blue were examined microscopically. Conidia were nonseptate and rod-shaped with rounded ends that measured 6.65 ± 0.54 x 1.56 ± 0.25 μm (n = 100). The characteristics of the fungus were similar to those reported for Paramyrothecium foliicola (Rennberger and Keinath, 2020). Molecular identification was performed on a representative isolate. RNA polymerase II second largest subunit (RPB2), calmodulin (CmdA) and the β-tubulin (B-tub) genes were amplified and sequenced with the primer sets RPB2-5F2-RPB2-7cR, CAL228F-CAL737R and Bt2a-Bt2b, respectively. These sequences were submitted to GenBank with the acc. nos. MW116070 for RPB2, MW116071 for CmdA and MW116072 for B-tub. BLASTn analysis of the sequences demonstrated 99.34 to 100% identity with Paramyrothecium foliicola (acc. nos. MN398043, MN593713 and MN398138). Koch's postulates were verified on 15-day-old watermelon seedlings and mature fruit. One point of each of ten watermelon seedlings and six points of each of five fruit were marked for inoculation. A plug of mycelium obtained from a monosporic pure culture (grown for 15 days in PDA) was applied to each point without wounds. PDA only medium was included as the controls. The pathogenicity tests were repeated twice. Treated seedlings and fruit were kept in plastic bags at 27°C for 15 days. The first symptoms appeared 4 days after inoculation on the seedlings and 3 days after inoculation on the fruit. At the end of the test, the symptoms were similar to those observed initially in the field. The pathogen was re-isolated from lesion edges, and the morphological characteristics of the pathogen were determined to correspond with those of the inoculated fungus. Control seedlings and fruits remained healthy. P. foliicola has been reported to cause leaf spot disease on wild rocket and basil (Matić et al., 2019) and, recently, on watermelon in South Carolina (Rennberger and Keinath, 2020). To the best of our knowledge, this report is the first to describe P. foliicola causing leaf spot and stem canker on watermelon in Mexico.

First Report of Leaf Spot Caused by Colletotrichum fructicola and C. siamense on Zizyphus mauritiana in Guangxi, China.

Zizyphus mauritiana Lam. is an important tropical fruit tree and has significant economic value. It is widely planted in Hainan, Guangdong, Guangxi and Fujian provinces in China (Yang et al. 2017). In March 2019, leaf spot was observed on leaves of Z. mauritiana at Bagui fields in Nanning, Guangxi, China, with incidence exceeding 50%. Symptomatic leaves developed a yellow to tan-brown sunken lesion and finally abscised. To isolate the pathogen causing the symptoms, small pieces (5 × 5 mm) of infected leaves were surface sterilized by exposure to 75% ethanol for 10 sec, 1% sodium hypochlorite for 1 min and rinsed three times in sterile water. Fifty pieces were isolated, surface sterilized, and pieces were plated onto potato dextrose agar (PDA) and grown at 28°C for 7 days. The isolation rate of Colletotrichum species was 100%. Three representative isolates (DQZ3-1, DQZ3-2 and DQZ3-3) were selected for further study. Mycelia were greyish-white for all three isolates, with isolate DQZ3-1 also appearing dark green in the center of the colony. Conidia were elliptical, aseptate and hyaline, with sizes of 13.4 ± 0.12 µm × 5.7 ± 0.1 µm, 14.8 ± 0.1 µm × 5.8 ± 0.1 µm and 15.1 ± 0.1 µm × 5.5 ± 0.1 µm for DQZ3-1, DQZ3-2 and DQZ3-3, respectively. Genomic DNA was extracted using the DNAsecure Plant Kit [Tiangen Biotech (Beijing) Co., Ltd] and the internal transcribed spacer (ITS), partial actin (ACT), calmodulin (CAL), chitin synthase (CHS-1), beta-tubulin (TUB2), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes were sequenced (Weir et al. 2012). Phylogenetic analysis of the three isolates was performed with MEGA-X (Version 10.0) based on sequences of multiple loci (ITS, ACT, CAL, CHS-1, TUB2 and GAPDH) using Maximum Likelihood analysis. Isolate DQZ3-1 was identified as C. fructicola, and the other two isolates, DQZ3-2 and DQZ3-3, were identified as C. siamense (accessions MT039396 to MT039410, for ACT, CAL, CHS-1, GAPDH and TUB2 of DQZ3-1, DQZ3-2 and DQZ3-3; MT041651 to MT041653 for ITS of DQZ3-1, DQZ3-2 and DQZ3-3). Pathogenicity tests were conducted on 1-year-old plants. Young, healthy leaves were artificially wounded by gently scratching with a sterile needle and 10 µl droplets of conidial suspension (106 spores/ml) applied per wound site for each isolate. Some wounded leaves were inoculated with 10 µl droplets of water as controls. Each isolate was inoculated onto three plants, with 15 leaves at least for each plant, same as controls. All inoculated plants were sprayed with water and covered with plastic bags to maintain high humidity. Symptomatic lesions were observed on the inoculated leaves after 7 days at 28°C, whereas no symptoms were observed on the control leaves. To fulfill Koch's postulates, fungi were re-isolated from 50 symptomatic leaf pieces and fungi re-isolated from each leaf piece were morphologically identical to the inoculated isolates, for a 100% isolation frequency. To our knowledge, this is the first report of leaf spot caused by C. fructicola and C. siamense on Z. mauritiana worldwide. This research may accelerate the development of future epidemiological studies and management strategies for anthracnose caused by C. fructicola and C. siamense on Z. mauritiana.

First report of leaf spots on Gentiana rhodantha caused by Pestalotiopsis trachicarpicola

First report of leaf spots on Gentiana rhodantha caused by Pestalotiopsis trachicarpicola

First report of leaf spot on sorghum caused by Pestalotiopsis trachycarpicola in China

First report of leaf spot on sorghum caused by Pestalotiopsis trachycarpicola in China

First report of leaf spot on Prunus salicina caused by Alternaria alternata in China

First report of leaf spot on Prunus salicina caused by Alternaria alternata in China

First report of leaf spot caused by Colletotrichum siamense on Sophora tonkinensis

A leaf spot, found on Sophora tonkinensis in Hechi city, Guangxi province, China was identified as Colletotrichum siamense based on morphological and molecular phylogenetic analysis of the internal transcribed spacer (ITS) of ribosomal DNA, β-tublin (TUB2), the translation elongation factor 1-alpha (TEF1-α), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin (ACT), chitin synthase 1 (CHS-1), calmodulin (CAL) and glutamine synthetase (GS) genes. Koch’s postulates were satisfied by successful reisolation of C. siamense only from plants inoculated with the pathogen. This is the first report of leaf spot caused by C. siamense on Sophora tonkinensis.

Correction to: First report of leaf spot caused by Albifimbria verrucaria on spinach in Pakistan

Correction to: First report of leaf spot caused by Albifimbria verrucaria on spinach in Pakistan

First Report of Leaf Spot Caused by Nigrospora aurantiaca in Tobacco in China.

Tobacco (Nicotiana tabacum L.) is one of the most important cash crops in China. In June 2019, tobacco (cv. Yunyan 87) samples with gray spots surrounded by yellowish ring were collected in Zhengan (107.43° N, 28.55° E), Guizhou province, China. Pieces of leaf tissue (3 mm × 3 mm) that were cut at the junction of diseased and healthy portion were surface sterilized and plated on potato dextrose agar (PDA). After incubation at 25°C in the dark for 7 days, an isolate (T22) was chosen and used for pathogen identification. The colonies had aerial hyphae, initially white and then turned grey, and produced a soluble red pigmen on PDA. The colonies were floccose aerial mycelia, dark grey, with pale brown hyphae, and produced conidia on oatmeal agar. Conidia were ovoid or ampulliform, black, smooth. Based on morphological characteristics, isolate T22 was identified as Nigrospora aurantiaca (Wang et al. 2017). For molecular identification, the large subunit (LSU) and internal transcribed spacer (ITS) of ribosomal RNA, β-tubulin (TUB) and translation elongation factor 1-alpha (TEF1) genes of T22 were amplified by PCR with the primer sets LROR/LR5, ITS1/ITS4, Bt2a/Bt2b and EF1-728F/EF2 (Suwannarach et al.2019), then PCR products were sequenced. Their GenBank accession numbers were MT341787, MT328649, MT348395 and MT348394, respectively. Phylogenetic tree of combined LSU, ITS, TUB, and TEF sequences showed that isolate T22 was assigned to N. aurantiaca strain (CGMCC 3.18130 and LC 7034) with 100% bootstrap support. Based on morphological characteristics and multi-gene molecular analysis, isolate T22 was identified as N. aurantiaca. To fulfill Koch's postulates, PDA plugs grown with N. aurantiaca were placed on the leaves of four tobacco plants (cv. Yunyan 87) at the 10-leaf stage. Leaves inoculated with PDA only plugs served as the controls. Treated plants were maintained in a greenhouse with temperatures ranging from 18 to 28 °C. Five days after inoculation, typical symptoms were observed on inoculated leaves but not on the controls. N. aurantiaca was re-isolated from the diseased leaves but not from the controls. To our best of knowledge, this is the first report of N. aurantiaca causing leaf spot on tobacco in China. N. aurantiaca has been reported to cause leaf spot on Castanea mollissima in China (Luo et al. 2020). Due to potential serious damage caused by the disease in this region, proper disease management practices should be developed and implemented.

First report of leaf spot caused by Albifimbria verrucaria on spinach in Pakistan

First report of leaf spot caused by Albifimbria verrucaria on spinach in Pakistan

The First Report of Leaf Spot Caused by Alternaria alternata on Italian Ryegrass (Lolium multiflorum) in China.

Italian ryegrass (Lolium multiflorum Lam.) is one of the most important forage crops in southwestern China. In 2018, a leaf spot was observed in a field of Italian ryegrass in Mengyang, Sichuan province, China (30.96925°N, 104.10223°E). From January to early March, this leaf spot developed sporadically and appeared as brown to dark brown lesions. In late May, this disease reached a peak with incidence up to 80% and appeared as reddish-brown necrotic spots with a grayish white to brown center. To isolate the pathogen, sections (0.5 × 1 cm2) of 30 diseased leaves collected from 10 plants were surface-disinfested in 70% ethanol solution for 30 s, 5% NaOCl solution for 5 min, rinsed thrice in sterilized distilled water, air dried, plated on potato dextrose agar (PDA), and incubated at 25°C in the dark for 4 days. To obtain pure isolates, the single-spore isolation technique (Cai et al. 2009) was used. The conidial suspensions were diluted to a reasonable concentration, spread onto PDA, and incubated at 25°C in dark for 24 to 48 h, and then single germinated conidia were transferred onto new PDA plates (Cai et al. 2009). Nine pure isolates showing similar morphology were obtained for further study. Colonies on PDA were dark gray in the center surrounded by white to gray, with gossypine mycelia on the upper side, and red to dark red on the reverse side. Conidia were obclavate or pyriform, olivaceous to dark brown, with 0 to 6 transverse septa and 0 to 4 longitudinal septa, 13.2 to 55.0 (27.9) × 6.3 to 12.5 (9.8) µm. Conidiophores were septate, hyaline to olivaceous brown, either branched or unbranched, geniculate at the tip, 2.5 to 5.9 μm wide and up to 70 μm long. These morphological and cultural characteristics were consistent with the descriptions of Alternaria alternata (Fr.) Keissl. isolated from Apple (Elfar et al. 2018). To confirm the pathogenicity on Italian ryegrass, healthy plants (8-week-old) of cultivar Splendor grown in five pots filled with potting soil were spray-inoculated with conidial suspension (1 × 106 conidia/ml). Plants in another, five pots were sprayed with sterilized distilled water as controls. All pots were individually covered with transparent polyethylene bags for 5 days to maintain high relative humidity and placed in a greenhouse at 18 to 25°C. At 14 days post inoculation, symptoms typical of brown to dark brown leaf spots developed on the plants inoculated with conidial suspension, whereas no symptoms on the control plants. The pathogenicity tests were carried out three times. The same pathogen was consistently re-isolated from inoculated leaves and confirmed by morphological characterization as described above. To further identify this pathogen, isolate HMCH-9 (=CGMCC 3.19924) was selected as a representative for molecular characterization. Following Woudenberg et al. (2015), the internal transcribed spacer regions 1 and 2 and intervening 5.8S rDNA (ITS), glyceraldehyde-3-phosphate dehydrogenase (GPD), translation elongation factor 1-alpha (TEF), RNA polymerase second largest subunit (RPB2), and Alternaria major allergen (Alt) genes were partially amplified and sequenced. Sequences were deposited in GenBank (accession nos. MH567106 for ITS, MH567107 for GPD, MH567109 for TEF, MH567110 for RPB2, and MH567108 for Alt). BLAST analysis of all these five segments showed >99.8% identity with those sequences of ex-type isolate CBS 916.96 of A. alternata (Woudenberg et al. 2015). To our knowledge, this is the first report of A. alternata causing leaf spot on Italian ryegrass in China. The accurate identification of this pathogen would be useful for the prevention and control of leaf spot on Italian ryegrass in the future.

First Report of Leaf Spot on White Chrysanthemum (Chrysanthemum morifolium) Caused by Epicoccum sorghinum in Hubei province, China.

White Chrysanthemum (Chrysanthemum morifolium), a perennial herb of the Compositae family, is used for traditional medicine. The planting area of white chrysanthemum in Macheng city, Hubei Province is about 3333 ha and the annual output can reach more than 5000 tons. In 2019, leaf spot disease appeared on almost all middle and lower leaves of white chrysanthemum in most fields of Fengshumiao county, Macheng city (N31°29'57″, E115°05'49″). This county has 33 acres white chrysanthemum planting area, and most of the plants in the county were infected with the leaf spot disease. The average incidence of leaf spot disease was 65%, and incidence in some areas was 100%. In our observations, leaf spot disease can occur throughout the whole growth period of white chrysanthemum, and it will become more serious under the high temperature and humidity condition. Usually, the diseased leaves account for 30 to 80% of the total leaves on the plant. Leaf spot initially manifests as necrotic lesions on the edge and tip of the leaf, and then the lesions coalesce and gradually expand to form irregular light-brown to brown-black spots, eventually leading to necrosis and curling of the entire leaf. This disease seriously affects the growth and development of plants, resulting in the decline of yield and quality of white chrysanthemum. Ten symptomatic leaf samples were collected, the surfaces were disinfected with 0.1% mercuric chloride (HgCl2) for 3 min, and washed with sterile distilled water three times. Ten tissue samples at the junction of diseased and healthy areas (0.5 × 0.5 cm2) were cut and placed on potato dextrose agar (PDA) medium containing 100 µg/ml cefotaxime sodium and incubated in a dark chamber at 28°C. After 2 days, the hyphal tips from the edges of growing colonies were transferred to fresh PDA plates for further purification. Finally, eight isolates were obtained and these isolates were similar in morphology. The color of purified isolates was initially white to pale yellow. After six days of incubation, colonies had a diameter of 8 cm and the cultures were pale gray and starting to secrete scarlet pigment. After 15 days incubation, the colonies were grayish brown, while the backside was reddish-brown. Gray to tan chlamydospores were observed, nearly spherical, with a wart-like surface. Unicellular chlamydospores were 7.91 to 32.23 × 12.03 to 38.42 µm (n=30) and multicellular chlamydospores were 6.32 to 25.10 × 21.75 to 100.05 µm (n=30). The morphological characteristics were similar to Epicoccum sorghinum (Kang et al. 2019). The isolate FDY-5 was chosen for molecular identification. The sequence of rDNA-ITS, TUB, and LSU of the FDY-5 were amplified (GenBank MT800929, MT799852, and MT800935, respectively) (White et al. 1990; Carbone and Kohn 1999; Lumbsch et al. 2000). BLAST results showed that the rDNA-ITS sequences, the TUB gene sequences, and LSU gene sequences of strain FDY-5 shared 99% identity with the sequences of E. sorghinum (syn. Phoma sorghina) in GenBank (MN555348.1, MF987525.1, MK516207.1, respectively). Moreover, a phylogenetic tree of the LSU gene sequence of FDY-5 was constructed based on the Neighbor-Joining (NJ) method in MEGA6 software (Tamura et al. 2013) and revealed that strain FDY-5 was closest to E. sorghinum. Based on morphological and molecular characteristics, the fungus was identified as E. sorghinum. Pathogenicity tests were conducted on two-month-old white chrysanthemum plants. The upper three leaves of three plants were randomly selected for stab treatment and were inoculated with 5 × 5 mm mycelial discs produced from a fifteen-day-old colony on PDA. The inoculated and control (treated with sterile PDA disks) plants were incubated in a moist chamber (25 ± 2 °C, RH 85%). The first lesions appeared 1 day after inoculation on leaves, and the necrotic lesion area expanded outward and showed typical symptoms 3 days later. To fulfill Koch's postulates, the pathogen was reisolated from nine inoculated leaves by repeating the above isolating operation, and confirmed as E. sorghinum by morphology. To the best of our knowledge, this is the first report of E. sorghinum causing leaf spot on white chrysanthemum in China. E. sorghinum has a wide host range worldwide and often causes crop yield reduction. This report will facilitate the diagnosis of white chrysanthemum leaf spot of white chrysanthemum allowing control measures to be adopted to manage this disease in a timely manner. References Carbone, I., and Kohn, L. M. 1999. Mycologia 91:553. Kang, Y., et al. 2019. Plant Dis. 103 (7):1787. Lumbsch, H., et al. 2000. Plant Biol. 2:525. Tamura, K., et al. 2013. Mol. Biol. Evol. 30:2725-2729. White, T. J., et al. 1990. Page 315 in:PCR protocols:a guide to methods and applications. Academic Press, San Diego, CA. Funding Funding was supported by Major Increase and Decrease Projects at the Central Level of China (2060302) and the National Key Research and Development Program (2017FYC1700704).

First report of Bipolaris yamadae leaf spot disease on Guinea grass (Panicum maximum) in Florida.

Guinea grass is an invasive perennial C4 grass and is a common weed around agricultural crops in Louisiana, Texas, and Hawaii, USA (Overholt and Franck 2019). In November 2018, leaf spots were observed on Guinea grass occurring in an organic garden located in Gainesville, Florida, USA. Lesions were oblong to irregular, dark grey to brownish center with pale-yellow to brownish black margin. Lesions had coalesced, forming necrotic margins that spread from the leaf tip, resulting in leaf blight and collapse of the canopy. Pieces of symptomatic leaf blades (5 sq cm) were surface sterilized (1 min), washed with sterile distilled water and plated onto water agar media plates. Plates were incubated at 27°C under 12-h light/dark for 3 to 5 days. Grey to black cottony mycelium was consistent on all plates and produced conidia characteristic of Bipolaris spp. Conidia were transferred to potato dextrose agar (PDA) plates with a 0.5 mm diameter sterile needle. Three isolates GG1, GG2 and GG3 were successfully grown on PDA. Conidia were black to brown colored, distoseptate with 3 to 8 septa and measured from (60.6- )70-105(-139.8) × (16.0-)17-23(-25.9) μm (avg: 93.3 μm, n=35, SD = 20.6; avg = 21.3 μm, n = 35, SD = 2.89). Conidiophores were in groups or single, brown, smooth and straight, septate and swollen at upper tip. Sigma Extract-N-Amp was used for genomic DNA extraction. Primers ITS1/ITS4 and GPD1/GPD2 (Berbee et al. 1999) were used to amplify and sequence the internal transcribed spacer region (ITS) and partial glyceraldehyde-3-phosphate dehydrogenase (GPDH) gene, respectively. Sequences were aligned using MUSCLE and alignment was trimmed for length. Maximum likelihood phylogenetic trees were constructed with 1,000 bootstrap samples based on the K2+G substitution model, selected by BIC for these two loci using Mega X (Kumar et al. 2018). The ITS and GPDH sequences of GG1, GG2 and GG3 (Genbank accessions MT514518-20, MT576654-56), grouped with B. yamadae isolates CPC_28807 and CBS_202.29 in phylogenetic trees (Marin-Felix et al. 2017). All three isolates from Guinea grass were inoculated on Sach's agar (Luttrell 1958) at 27°C under 12-h light/dark for a week, but no sexual morph was observed, and consistent for two repeated inoculations. To fulfill Koch's postulates, one isolate, GG1, was used. Conidia were harvested from a one-week-old colony grown on PDA incubated at 27°C and 12-h light/dark cycle. The concentration of the conidial suspension was adjusted to 105 conidia/ml using a hemocytometer. Using a Passche H-202S airbrush sprayer, five-week-old seedlings of Guinea grass were sprayed until runoff with the conidia suspension or 0.5% tween water only. Each treatment included four replicates and the experiment was repeated. Leaf spot symptoms were observed on the seedlings inoculated with conidia, whereas seedlings sprayed with water were asymptomatic. Cultures with the expected morphology were isolated from symptomatic leaf blades and absent from control plants. To our knowledge, this is the first report of leaf spot on Guinea grass caused by B. yamadae in Florida, USA. B. yamadae was previously reported from Guinea grass in India, and from other Panicum species in the northern USA (Farr and Rossman 2019). B. yamadae was also isolated from sugarcane in Cuba and China, and corn in Japan (Manamgoda et al. 2014, Raza et al. 2019), which suggests that it has the potential to impact agronomic crops in Florida, such as sugarcane and corn.