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.

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First Report of Bipolaris oryzae Causing Leaf Spot on Cultivated Wild Rice (Oryza rufipogon) in China

Plant Disease ◽

10.1094/pdis-11-20-2529-pdn ◽

2021 ◽

Author(s):

Yue Lian Liu ◽

Jian Rong Tang ◽

Ya Li ◽

Hong Kai Zhou

Keyword(s):

Leaf Spot ◽

Wild Rice ◽

Morphological Characteristics ◽

Oryza Rufipogon ◽

Likelihood Method ◽

Leaf Spot Disease ◽

Pathogenicity Test ◽

Sterile Water ◽

Bipolaris Oryzae ◽

First Report

Wild rice (Oryza rufipogon) has been widely studied and cultivated in China in recent years due to its antioxidant activities and health-promoting effects. In December 2018, leaf spot disease on wild rice (O. rufipogon cv. Haihong-12) was observed in Zhanjiang (20.93 N, 109.79 E), China. The early symptom was small purple-brown lesions on the leaves. Then, the once-localized lesions coalesced into a larger lesion with a tan to brown necrotic center surrounded by a chlorotic halo. The diseased leaves eventually died. Disease incidence was higher than 30%. Twenty diseased leaves were collected from the fields. The margin of diseased tissues was cut into 2 × 2 mm2 pieces, surface-disinfected with 75% ethanol for 30 s and 2% sodium hypochlorite for 60 s, and then rinsed three times with sterile water before isolation. The tissues were plated on 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. Fifteen isolates were obtained. Two isolates (OrL-1 and OrL-2) were subjected to further morphological and molecular studies. The colonies of OrL-1 and OrL-1 on PDA were initially light gray, but it became dark gray with age. Conidiophores were single, straight to flexuous, multiseptate, and brown. Conidia were oblong, slightly curved, and light brown with four to nine septa, and measured 35.2–120.3 µm × 10.3–22.5 µm (n = 30). The morphological characteristics of OrL-1 and OrL-2 were consistent with the description on Bipolaris oryzae (Breda de Haan) Shoemaker (Manamgoda et al. 2014). The ITS region, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and translation elongation factor (EF-1α) were amplified using primers ITS1/ITS4, GDF1gpp1/GDR1 gdp2 (Berbee et al. 1999), and EF-1α-F/EF-1α-R EF-1/EF-2 (O’Donnell 2000), respectively. Amplicons of OrL-1 and OrL-2 were sequenced and submitted to GenBank (accession nos. MN880261 and MN880262, MT027091 and MT027092, and MT027093 and MT027094). The sequences of the two isolates were 99.83%–100% identical to that of B. oryzae (accession nos. MF490854，MF490831，MF490810) in accordance with BLAST analysis. A phylogenetic tree was generated on the basis of concatenated data from the sequences of ITS, GAPDH, and EF-1α via Maximum Likelihood method, which clustered OrL-1 and OrL-2 with B. oryzae. The two isolates were determined as B. oryzae by combining morphological and molecular characteristics. Pathogenicity test was performed on OrL-1 in a greenhouse at 24 °C to 30 °C with 80% relative humidity. Rice (cv. Haihong-12) with 3 leaves was 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 five pots were sprayed with sterile water and used as controls. The test was conducted three times. Disease symptoms were observed on leaves after 10 days, but the controls remained healthy. The morphological characteristics and ITS sequences of the fungal isolates re-isolated from the diseased leaves were identical to those of B. oryzae. B. oryzae has been confirmed to cause leaf spot on Oryza sativa (Barnwal et al. 2013), but as an endophyte has been reported in O. rufipogon (Wang et al. 2015).. Thus, this study is the first report of B. oryzae causing leaf spot in O. rufipogon in China. This disease has become a risk for cultivated wild rice with the expansion of cultivation areas. Thus, vigilance is required.

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

Plant Disease ◽

10.1094/pdis-11-20-2522-pdn ◽

2021 ◽

Author(s):

Yue Lian Liu ◽

Jian Rong Tang ◽

Ya Li ◽

Hong Kai Zhou

Keyword(s):

Leaf Spot ◽

Wild Rice ◽

Morphological Characteristics ◽

Oryza Rufipogon ◽

Its Sequences ◽

Likelihood Method ◽

Pathogenicity Test ◽

Sterile Water ◽

First Report ◽

Nigrospora Oryzae

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 very distinctive, 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.

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First report of Alternaria alternata causing brown leaf spot on wild rice (Oryza rufipogon) in China

Plant Disease ◽

10.1094/pdis-05-21-1024-pdn ◽

2021 ◽

Author(s):

Fei Teng Zhong ◽

Yue Lian Liu ◽

Dianfeng Zheng ◽

Shili Lu

Keyword(s):

Rna Polymerase Ii ◽

Alternaria Alternata ◽

Leaf Spot ◽

Wild Rice ◽

Disease Incidence ◽

Morphological Characteristics ◽

Oryza Rufipogon ◽

Sterile Water ◽

Brown Leaf Spot ◽

Brown Leaf

Oryza rufipogon Griff is a wild rice germplasm that might contain genes valuable for rice breeding. In May to June 2019, a leaf 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), Guangdong, China. Early symptoms were yellow spots from the tip of leaves. Later, the spots gradually expanded downward the entire leaf to turn brown in turn. Symptoms were found in the tillering to the grain-filling stages (Supplementary Figure 1). The disease incidence on plants was between 10% and 40%. 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 and 2% sodium hypochlorite for 30 s and 60 s, respectively, and rinsed three times with sterile water before isolation. The tissues were plated onto potato dextrose agar (PDA) medium and incubated at 28 °C. After 5-day incubation, grayish fungal colonies appeared on PDA. Single-spore isolation method was used to recover pure cultures for three isolates (Aas-1, Aas-2, and Aas-3). The colonies first produced light-grayish aerial mycelia, which turned dark grayish upon maturity. Conidiophores were branched. Conidia were two to four in chains, dark brown, ovoid or ellipsoid, and mostly beakless; had one to four transverse and zero to three longitudinal septa; and measured within 7.0–18.5 (average = 12.5) × 3.0–8.8 (average = 4.5) μm (n = 30). Morphological characteristics of the isolates were consistent with the description of Alternaria alternata (Fr.) Keissler (Simmons 2007). The internal transcribed spacer (ITS) region, partial RNA polymerase II largest subunit (RPB2) gene, translation elongation factor, and glyceraldehyde-3-phosphate dehydrogenase were amplified with primers ITS1/ITS4, RPB2-6F/RPB2-7R, EF-1α-F/EF-1α-R, and GDF1/GDR1, respectively (Woudenberg et al. 2015). Amplicons were sequenced and submitted to GenBank (accession nos. MW042179 to MW042181, MW090034 to MW090036, MW090046 to MW090048, and MW091450 to MW091452, respectively). The sequences of the three isolates were 100% identical (ITS, 570/570 bp; RPB2, 1006/1006 bp; TEF, 254/254 bp and GADPH, 587/587 bp) with those of CBS 479.90 (accession nos. KP124319, KP124787, KP125095, and KP124174) through BLAST analysis. The sequences were also concatenated for phylogenetic analysis by maximum likelihood. The isolates clustered with A. alternata CBS 479.90 (Supplementary Figure 2). The fungus associated with brown leaf spot on wild rice was thus identified as A. alternata. Pathogenicity tests were done 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 around 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 to serve as controls. The test was done three times. Disease symptoms were found on the leaves after 7 days. The tips of the leaves turned yellow and spread downward. Then, the whole leaf turned brown and dried out, but the controls stayed healthy. The pathogen was re-isolated from infected leaves and phenotypically identical to the original isolate Aas-1 to fulfill Koch’s postulates. To our knowledge, this report is the first one on A. alternata causing brown leaf spot on wild rice (O. rufipogon). The pathogen has the potential to reduce wild rice yields and future breeding should consider resistance to this pathogen.

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First Report of Epicoccum layuense Causing Leaf Brown Spot on Camellia oleifera in Hefei, China

Plant Disease ◽

10.1094/pdis-08-21-1726-pdn ◽

2022 ◽

Author(s):

Xiang Xie ◽

Shiqiang Zhang ◽

Qingjie Yu ◽

Xinye Li ◽

Yongsheng Liu ◽

...

Keyword(s):

Leaf Spot ◽

Brown Spot ◽

Likelihood Method ◽

Leaf Spot Disease ◽

Original Strain ◽

Conidial Suspension ◽

Camellia Oleifera ◽

Sterile Water ◽

Beta Tubulin ◽

First Report

Camellia oleifera, a major tree species for producing edible oil, is originated in China. Its oil is also called ‘‘eastern olive oil’’ with high economic value due to richness in a variety of healthy fatty acids (Lin et al. 218). However, leaves are susceptible to leaf spot disease (Zhu et al. 2014). In May 2021, we found circular to irregular reddish-brown lesions, 4-11 mm in diameter, near the leaf veins or leaf edges on 30%-50% leaves of 1/3 oil tea trees in a garden of Hefei City, Anhui Province, China (East longitude 117.27, North latitude 31.86) (Figure S1 A). To isolate the causal agents, symptomatic leaves were cut from the junction of diseased and healthy tissues (5X5 mm) and treated with 70 % alcohol for 30 secs and 1 % NaClO for 5 min, and subsequently inoculated onto PDA medium for culture. After 3 days, hyphal tips were transferred to PDA. Eventually, five isolates were obtained. Then the isolates were cultured on PDA at 25°C for 7 days and the mycelia appeared yellow with a white edge and secreted a large amount of orange-red material to the PDA (Figure S1 B and C). Twenty days later, the mycelium appeared reddish-brown, and sub-circular (3-10 mm) raised white or yellow mycelium was commonly seen on the Petri dish, and black particles were occasionally seen. Meanwhile, the colonies on the PDA produced abundant conidia. Microscopy revealed that conidia were globular to pyriform, dark, verrucose, and multicellular with 14.2 to 25.3 μm (=19.34 μm, n = 30) diameter (Figure S1 D). The morphological characteristics of mycelial and conidia from these isolates are similar to that of Epicoccum layuense (Chen et al.2020). To further determine the species classification of the isolates, DNA was extracted from 7-day-old mycelia cultures and the PCR-amplified fragments were sequenced for internal transcribed spacer (ITS), beta-tubulin and 28S large subunit ribosomal RNA (LSU) gene regions ITS1/ITS4, Bt2a/Bt2b and LR0R/LR5, followed by sequencing and molecular phylogenetic analysis of the sequences analysis (White et al. 1990; Glass and Donaldson 1995; Vilgalys and Hester 1990). Sequence analysis revealed that ITS, beta-tubulin, and LSU divided these isolates into two groups. The isolates AAU-NCY1 and AAU-NCY2, representing the first group (AAU-NCY1 and AAU-NCY5) and the second group (AAU-NCY2, AAU-NCY3 and AAU-NCY4), respectively, were used for further studies. Based on BLASTn analysis, the ITS sequences of AAU-NCY1 (MZ477250) and AAU-NCY2 (MZ477251) showed 100 and 99.6% identity with E. layuense accessions MN396393 and KY742108, respectively. And, the beta-tubulin sequences (MZ552310; MZ552311) showed 99.03 and 99.35% identity with E. layuense accessions MN397247 and MN397248, respectively. Consistently, their LSU (MZ477254; MZ477255) showed 99.88 and 99.77% identity with E. layuense accessions MN328724 and MN396395, respectively. Phylogenetic trees were built by maximum likelihood method (1,000 replicates) using MEGA v.6.0 based on the concatenated sequences of ITS, beta-tubulin and LSU (Figure S2). Phylogenetic tree analysis confirmed that AAU-NCY1 and AAU-NCY2 are closely clustered with E. layuense stains (Figure S2). To test the pathogenicity, conidial suspension of AAU-NCY2 (106 spores/mL) was prepared and sterile water was used as the control. Twelve healthy leaves (six for each treatment) on C. oleifera tree were punched with sterile needle (0.8-1mm), the sterile water or spore suspension was added dropwise at the pinhole respectively (Figure S1 E and F). The experiment was repeated three times. By ten-day post inoculation, the leaves infected by the conidia gradually developed reddish-brown necrotic spots that were similar to those observed in the garden, while the control leaves remained asymptomatic (Figure S1 G and H). DNA sequences derived from the strain re-isolated from the infected leaves was identical to that of the original strain. E. layuense has been reported to cause leaf spot on C. sinensis (Chen et al. 2020), and similar pathogenic phenotypes were reported on Weigela florida (Tian et al. 2021) and Prunus x yedoensis Matsumura in Korea ( Han et al. 2021). To our knowledge, this is the first report of E. layuense causing leaf spot on C. oleifera in Hefei, China.

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First Report of Leaf Spot on Industrial Hemp (Cannabis sativa L.) Caused by Alternaria alternata in China

Plant Disease ◽

10.1094/pdis-01-21-0130-pdn ◽

2021 ◽

Author(s):

Lili Tang ◽

Xixia Song ◽

Liguo Zhang ◽

Jing Wang ◽

Shuquan Zhang

Keyword(s):

Leaf Spot ◽

Cannabis Sativa ◽

Leaf Spot Disease ◽

Molecular Characteristics ◽

Pathogenicity Test ◽

Conidial Suspension ◽

Sterile Water ◽

First Report ◽

Leaf Spots ◽

Industrial Hemp

Industrial hemp is an economically important plant with traditional uses for textiles, paper, building materials, food and medicine (Li 1974; Russo et al. 2008; Zlas et al. 1993). In August 2020, an estimated 80% of the industrial hemp plants with leaf spots were observed in greenhouse in Minzhu town, Harbin City, Heilongjiang Province, China (45.8554°N, 126.8167°E), resulting in yield losses of 20%. Leaf symptoms began as small spots on the upper surface of leaves and gradually developed into brown spots with light yellow halos. These irregular spots expanded gradually and eventually covered the entire leaf; the center of the spots was easily perforated. To identify the pathogen, 20 diseased leaves were collected, and small sections of (3 to 5 mm) were taken from the margins of lesions of infected leaves. The pieces were sterilized with 75% alcohol for 30 s, a 0.1% mercuric chloride solution for 1 min, and then rinsed three times with sterile water. Samples were then cultured on potato dextrose agar at 28℃ in darkness for 4 days. A single-spore culture was obtained by monosporic isolation. Conidiophores were simple or branched, straight or flexuous, brown, and measured 22 to 61 μm long × 4 to 5 μm wide (n = 50). Conidia were solitary or in chains, brown or dark brown, obclavate, obpyriform or ellipsoid. Conidia ranged from 23 to 55 μm long × 10 to 15 μm wide (n = 50) with one to eight transverse and several longitudinal septa. For molecular identification (Jayawardena et al. 2019), genomic DNA of pathogenic isolate (MZ1287) was extracted by a cetyltrimethylammonium bromide protocol. Four gene regions including the rDNA internal transcribed spacer (ITS), glyceraldehyde-3-phosplate dehydrogenase (GAPDH), translation elongation factor 1-alpha (TEF1) and RNA polymerase II beta subunit (RPB2) were amplified with primers ITS1/ITS4, GDF1/GDR1, EF1-728F/EF1-986R and RPB2-5F/RPB2-7cR, respectively (White et al. 1990). Resulting sequences were deposited in GenBank with accession numbers of MW272539.1, MW303956.1, MW415414.1 and MW415413.1, respectively. A BLASTn analysis showed 100% homology with A. alternata (GenBank accession nos. MN615420.1, MH926018.1, MN615423.1 and KP124770.1), respectively. A neighbor-joining phylogenetic tree was constructed by combining all sequenced loci in MEGA7. The isolate MZ1287 clustered in the A. alternata clade with 100% bootstrap support. Thus, based on morphological (Simmons 2007) and molecular characteristics, the pathogen was identified as A. alternata. To test pathogenicity, leaves of ten healthy, 2-month-old potted industrial hemp plants were sprayed using a conidial suspension (1×106 spores/ml). Control plants were sprayed with sterile water. All plants were incubated in a greenhouse at 25℃ for a 16 h light and 8 h dark period at 90% relative humidity. The experiment was repeated three times. After two weeks, leaf spots of industrial hemp developed on the inoculated leaves while the control plants remained asymptomatic. The A. alternata pathogen was re-isolated from the diseased leaves on inoculated plants, fulfilling Koch's postulates. Based on morphology, sequencing, and pathogenicity test, the pathogen was identified as A. alternata. To our knowledge, this is the first report of A. alternata causing leaf spot disease of industrial hemp (Cannabis sativa L.) in China and is worthy of our attention for the harm it may cause to industrial hemp production.

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First report of leaf spot caused by Colletotrichum fructicola on Dalbergia hupeana in China

Plant Disease ◽

10.1094/pdis-08-21-1842-pdn ◽

2021 ◽

Author(s):

Yang Zhou ◽

Rou Ye ◽

Qin Ying ◽

Yang Zhang ◽

Linping Zhang

Keyword(s):

Phylogenetic Tree ◽

Leaf Spot ◽

Disease Incidence ◽

Morphological Characteristics ◽

Jiangxi Province ◽

Leaf Spot Disease ◽

Sterile Water ◽

First Report ◽

Spot Disease ◽

Colletotrichum Fructicola

Dalbergia hupeana is a kind of wood and medicinal tree widely distributed in southern China. Since 2019, a leaf spot disease was observed on the leaves of D. hupeana in Gangxia village, Luoting town in Jiangxi Province, China (28°52′53″N, 115°44′58″E). The disease incidence was estimated to be above 50%. The symptoms began as small spots that gradually expanded, developing a brown central and dark brown to black margin. The spots ranged from 4 to 6 mm in diameter. Leaf pieces (5 × 5 mm) from lesion margins were surface sterilized in 70% ethanol for 30 s followed by 2% NaOCl for 1 min and then rinsed three times with sterile water. Tissues were placed on potato dextrose agar (PDA) and incubated at 25°C. Pure cultures were obtained by monosporic isolation. Fifteen strains with similar morphological characterizations were isolated, and three representative isolates (JHT-1, JHT-2, and JHT-3) were chosen and used for further study. Colonies on PDA of three isolates were grayish-green with white edges and dark green on the reverse side. Conidia were transparent, cylindrical with rounded ends, and measured 3.6-5.3 µm × 9.5-15.2 µm (3.7 ± 0.2 × 13.6 ± 1.1 µm, n = 100). Appressoria were dark brown, globose or subcylindrical, and ranged from 6.2-9.2 µm× 5.1-6.8 µm (7.9 ± 0.4 × 5.9 ± 0.3 µm, n=100). The morphological characteristics of the three strains were consistent with the description of species in the Colletotrichum gloeosporioides complex (Weir et al. 2012). The internal transcribed spacer (ITS) regions, actin (ACT), calmodulin (CAL), chitin synthase (CHS-1) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and beta-tubulin 2 (TUB2) were amplified from genomic DNA for the three isolates using primers ITS1/ITS4, ACT-512F/ACT-783R, CL1/CL2, CHS-79F/CHS-345R, GDF/GDR and T1/Bt2b (Weir et al. 2012), respectively. The sequences were deposited in GenBank (Accession Nos. MZ482016 - MZ482018 for ITS; MZ463636 - MZ463638 for ACT; MZ463648- MZ463650 for CAL; MZ463639 - MZ463641 for CHS-1; MZ463642 - MZ463644 for GAPDH; MZ463645 - MZ463647 for TUB2). A neighbor-joining phylogenetic tree was constructed with MEGA 7.0 using the concatenation of multiple sequences (ITS, ACT, GAPDH, TUB2, CHS-1, CAL) (Kumar et al. 2016). According to the phylogenetic tree, three isolates fall within the Colletotrichum fructicola clade (boot support 99%). Based on morphological characteristics and phylogenetic analysis, three isolates were identified as C. fructicola. The pathogenicity of three isolates was conducted on two-yr-old seedlings (30 cm tall) of D. hupeana. Healthy leaves were wounded with a sterile needle and then inoculated with 10 μL spore suspension (106 conidia per mL). Controls were treated with sterile water. All plants were covered with transparent plastic bags and incubated in a greenhouse at 28°C with a 12 h photoperiod (relative humidity > 80%). Within five days, the inoculated leaves developed lesions similar to those observed in the field, whereas controls were asymptomatic. The experiments repeated three times showed similar results. The infection rate was 100%. C. fructicola was re-isolated from the lesions, whereas no fungus was isolated from control leaves. C. fructicola can cause leaf diseases in a variety of hosts, including Aesculus chinensis (Sun et al. 2020), Peucedanum praeruptorum (Ma et al. 2020), and Mandevilla × amabilis (Sun et al. 2020). C. brevisporum and C. gigasporum were also reported to infect Dalbergia odorifera (Chen et al. 2021; Wan et al. 2018). However, This is the first report of C. fructicola associated with leaf spot disease on D. hupeana in China. These results will help to develop effective strategies for appropriately managing this newly emerging disease.

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First Report of Gray Leaf Spot of Maize Caused by Cercospora zeina in China

Plant Disease ◽

10.1094/pdis-03-13-0280-pdn ◽

2013 ◽

Vol 97 (12) ◽

pp. 1656-1656 ◽

Cited By ~ 9

Author(s):

K.-J. Liu ◽

X.-D. Xu

Keyword(s):

Leaf Spot ◽

Its Region ◽

Gray Leaf Spot ◽

Molecular Characteristics ◽

Maturity Stage ◽

Sterile Water ◽

Causal Organism ◽

First Report ◽

Single Well ◽

Species Specific

Gray leaf spot of maize (Zea mays L.) is an important foliar disease in many parts of China. The causal organism of gray leaf spot in China is generally regarded as Cercospora zeae-maydis (3). In October 2011, symptoms similar to gray leaf spot were observed on 77% of maize plants in 25 locations (about 3,000 ha.) of Yunnan Province, China, and the disease could cause yield losses of 35 to 50%. The symptoms of leaf spot were different from those caused by C. zeae-maydis. The lesions on leaves were oblong, pale gray to pale brown, 2 to 3 × 5 to 40 mm, and confined by leaf veins that eventually coalesced. To identify the pathogen, 75 leaf samples were collected from 25 fields (three leaf samples for each field) at the kernel maturity stage. Single, well-separated lesions were excised and surface-sterilized by placing them in 75% ethanol for 5 s, then disinfested in 2% sodium hypochlorite for 5 min and rinsed with sterilized water. The lesions were incubated on water agar (WA) at 24°C for 48 to 72 h to allow sporulation. Seventy-five single-conidial isolates were obtained and cultured as described in Crous (1). Morphology of the isolates was determined on plates containing maize leaf powder agar (MLPA). After 5 days, isolates produced pale brown mycelia that consisted of 3- to 4-μm-wide, septate, branched hyphae. Conidiophores were 5 to 7 × 55 to 100 μm, straight to slightly flexuous, and usually 1- to 5-septate. Conidia were average 7.5 × 68 μm, fusiform, apex subobtuse, base subtruncate, and 3- to 6-septate. These characteristics are similar to C. zeina (2). The internal transcribed spacer (ITS) region of rDNA was amplified from each of the 75 isolates using primers ITS1/ITS4 and sequenced. The same sequences were obtained and the sequence of isolate YNGLS was submitted to GenBank (Accession No. KC878692). BLAST analysis of the sequence showed 100% confirmation to C. zeina (DQ185081). Additionally, a PCR-based diagnostic test using species-specific primers (2) confirmed the identification of the 75 isolates as C. zeina. The pathogenicity of the isolates was tested on greenhouse grown maize variety Huidan 4. The test was performed on 40 plants and replicated three times. The plants were inoculated at the 10 leaf stage by injecting 2 ml of conidial suspensions (2,500 conidia ml–1) into leaf whorl using a hypodermic syringe, and control plants were injected with sterile water. Conidia were collected from 5-day-old cultures grown on MLPA and suspended in sterile water. Forty days after inoculation, all inoculated plants showed characteristic lesions on leaves, but control plants remained asymptomatic. C. zeina was reisolated from the lesions, and the identity of the reisolates was confirmed by the morphological and molecular characteristics as stated above. C. zeina was previously reported as the causal agent of maize gray leaf spot (2). To our knowledge, this is the first report of C. zeina causing gray leaf spot of maize in China. References: (1) P. W. Crous. Mycologia Memoir. 21:1, 1998. (2) P. W. Crous et al. Stud. Mycol. 55:189, 2006. (3) C. H. Lu et al. J. Southwest China Normal Univ. 37:51, 2012.

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First Report of Corynespora Leaf Spot Caused by Corynespora cassiicola on Gynura in China

Plant Disease ◽

10.1094/pdis-12-13-1284-pdn ◽

2014 ◽

Vol 98 (7) ◽

pp. 1007-1007 ◽

Cited By ~ 1

Author(s):

B. J. Li ◽

J. X. Chuan ◽

M. Yang ◽

G. F. Du

Keyword(s):

Leaf Spot ◽

Disease Incidence ◽

Morphological Characteristics ◽

Its Region ◽

Leaf Spot Disease ◽

Herbaceous Plant ◽

Distilled Water ◽

Corynespora Cassiicola ◽

Single Spore ◽

First Report

Gynura (Gynura bicolor DC.) is a perennial herbaceous plant in the family Compositae. It is an important Chinese vegetable, and is commonly used as a Chinese herbal medicine. In 2010, a severe leaf spot disease was observed on gynura grown in the main production areas in Tong Nan County, Chongqing City, China. Some farms experienced 60% disease incidence. Symptoms usually began on the lower leaves, as circular to elliptical or irregular spots with concentric rings. Individual spots were dark brown with grayish centers, sometimes coalescing and leading to extensive necrosis. The fungus associated with lesions was characterized as follows: Conidiophores were single or in clusters, straight or flexuous, unbranched, percurrent, cylindrical, pale to dark brown, 87.5 to 375.0 μm long and 5.0 to 10.5 μm wide. Conidia were solitary or catenate, straight to slightly curved, obclavate to cylindrical, 3 to 14 pseudoseptate, 82.8 to 237.5 μm long and 7.0 to 7.8 μm wide, and pale brown. The morphological characteristics of the conidia and conidiophores agreed with the descriptions for Corynespora cassiicola (1). To isolate the causal pathogen, surface-sterilized tissue at the margin of lesions was immersed in 75% ethanol for 30 s, rinsed in sterile water, dried in a laminar flow bench, transferred to PDA, and incubated at 28°C. Four single-spore cultures of the isolates were obtained and named from ZBTK10110637 to ZBTK10110640. All strains were identified as C. cassiicola. The isolate ZBTK10110637 was selected as representative for molecular identification. Genomic DNA was extracted by CTAB (2). The internal transcribed spacer (ITS) region of the rDNA was amplified using primers with ITS1 (5′-TCCGATGGTGAACCTGCGG-3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′). Amplicons were 433 bp (GenBank Accession No. JX867272) and shared 100% similarity with that of C. cassiicola (NRC2-1 No. AB539285.1). To confirm pathogenicity, four isolates were used to inoculate 12 gynura plants (6 weeks old) by mist spray-inoculation with 108 spores/ml suspension in sterile distilled water on the leaves. Control plants were misted with sterile distilled water. After inoculation, all plants were incubated in a greenhouse maintained at 20 to 28°C with relative humidity of 80 to 85%. Five days after inoculation, dark brown spots with a grayish center typical of field symptoms were observed on all inoculated plants. No symptoms were seen on water-treated control plants. The fungus was re-isolated from inoculated plants. The morphological characteristics of isolates were identical with the pathogen recovered originally. This is the first report of C. cassiicola on gynura. References: (1) M. B. Ellis. CMI Mycological Papers 65(9):1-15, 1957. (2) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA, 1990.

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Screening of Cowpea (Vigna unguiculata ( L.)Walp) Varieties for Resistance to Leaf Spot in Southern Guinea Savannah Agro- Ecology of Nigeria

Nigerian Journal of Biotechnology ◽

10.4314/njb.v36i2.2 ◽

2020 ◽

Vol 36 (2) ◽

pp. 9-20

Author(s):

C. Ekhuemelo ◽

H.U. Igbor ◽

S.J. Ocheje

Keyword(s):

Vigna Unguiculata ◽

Leaf Spot ◽

Disease Incidence ◽

Natural Infection ◽

Growth And Yield ◽

Leaf Spot Disease ◽

Curvularia Lunata ◽

Aspergillus Tamarii ◽

Spot Disease ◽

Cowpea Varieties

Five cowpea varieties (Vigna unguiculata (L.) Walp) namely UAM 09 1055-6, UAM 09 1051-1, IT 99k-573-1-1, IT 90k-277-2 and IT 99k-573-2-1 were investigated for fungi associated with the seed, leaf spot disease incidence and severity in Makurdi (07o 45’- 7o 50’N and 08o 45’ - 08o 50’E ; 98 m)and Otobi (7°07’ - 7°11'N and 8° 05- 8°10'E) in Benue State, Nigeria under natural infection. The effect ofleaf spot incidence and severity on the growth and yield of cowpea varieties were also evaluated. Fungi associated with the seeds and leaf spot lesions were isolated and identified. Diseased leaf samples from the study locations were found to be infected with Fusarium verticillioides, Curvularia lunata, Aspergillus tamarii Kite, Lasiodiplodia theobromae, Aspergillus flavus Link and Aspergillus niger van Tiegh, Pythium spp, Fusarium solani, Macrophomina phaseolina and Phoma sp. Otobi field had significantly higher incidence and severity of leaf spot disease than the Makurdi field. Cowpea variety IT 99k-573-1-1, IT 99k-573-2-1 and IT 90k-277-2 were classified as moderately resistant in Makurdi with a mean incidence of 22.23%, 16.97% and 16.67% respectively while varieties UAM 09 1051-1 and UAM 09 1055-6 were classified as Moderately susceptible and Highly susceptible with mean leaf spot incidence of 41.67% and 99.17% respectively. In Otobi, all the cowpea varieties screened were classified as Susceptible to leaf spot incidence with the exception of variety IT 90k-277-2 which was classified as moderately susceptible to leaf spot disease. Key words: Cowpea, leaf spot, screening, resistant, susceptible.

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First Report of Nigrospora oryzae Causing Leaf Spot on Ginger in China

Plant Disease ◽

10.1094/pdis-02-21-0260-pdn ◽

2021 ◽

Author(s):

Zeng-Liang LIU ◽

Shuangyun Zhou ◽

Liangliang Qi ◽

Xiaoguo Wang ◽

Juan Song ◽

...

Keyword(s):

Leaf Spot ◽

Disease Incidence ◽

Morphological Characteristics ◽

Control Measures ◽

Likelihood Method ◽

Effective Control ◽

Economic Losses ◽

Leaf Spot Disease ◽

First Report ◽

Nigrospora Oryzae

Ginger (Zingiber officinale Rosc.) is an herbal crop widely grown in China for its medicinal and savory qualities of rhizomes. In August 2018, leaf spot symptoms were observed on ginger plants grown in a field in Nanning, Guangxi Province (E108°3'54", N23°14'48"). Disease incidence was above 50%, and in a Nanning field, rhizome yield loss was almost 30%. Early symptoms appeared as circular, necrotic areas that later developed into circular or irregular spots. The centers of the lesions were white and often surrounded by chlorotic halos (Figure S1A). In severe infections, the spots frequently coalesced, causing the entire leaf to become withered and curved. Small pieces (3 to 4 mm2) from the margin of infected lesions were surface sterilized in 75% ethanol for 40 s followed by 1% NaOCl for 90 s, placed on potato dextrose agar (PDA) and incubated at 28°C in the dark for 4 days. Hyphal tips from the leading edge of colonies were transferred to fresh PDA plates to obtain pure cultures. Fungal colonies were initially white, then turned black/grayish brown when maintained in the dark at 28°C after 5 days (Figure S1B). Conidia were single-celled, brown, or black, smooth, spherical, or subspherical with diameters varying from 9.5 to 15 μm (mean = 13.5 ± 0.72 µm, n = 50) (Figure S1C). Based on these morphological characteristics, the isolates were provisionally identified as Nigrospora oryzae (Ellis 1971; Hudson 1963). Genomic DNA was extracted from a representative isolate Sjb-2. The internal transcribed spacer (ITS) region, beta-tubulin (TUB2), and the translation elongation factor 1-alpha (TEF1-α) were amplified using primer pairs including ITS1/ITS4 (White et al. 1990), Bt-2a/Bt-2b (Glass and Donaldson 1995), and EF1-728F/EF1-986R (Carbone et al. 1999), respectively. The obtained ITS sequence (GenBank accession no. MW555242), TUB2 sequence (MZ048644), and TEF1-α sequence (MZ048645) showed >99% similarity with several GenBank sequences of N. oryzae (KF516962 for ITS; MK550707 for TUB2; and KY019425 for TEF1-α, respectively). Based on the combined sequences of ITS, TUB2 and TEF1-α sequences, a phylogenetic tree was constructed using the maximum likelihood method and confirmed that the isolates were N. oryzae (Figure S2). Pathogenicity of the isolate was confirmed by fulfilling Koch’s postulates. Agar blocks (3 mm diameter) containing a fungal mycelium were placed on detached healthy leaves of ginger. The leaves were then wrapped with sterile polyethylene and incubated in a greenhouse at 25°C with 60% RH. Within 7 days, symptoms appeared on inoculated leaves similar to spots observed in the field, whereas controls remained symptomless. The same pathogen was reisolated from the spots. Pathogenicity tests were performed twice with three replications, indicating that N. oryzae is responsible for leaf spot disease on ginger. The disease in ginger caused by N. oryzae had been reported in Southern Africa (Grech et al. 1989). To our knowledge, this is the first report of N. oryzae causing leaf spot of ginger in China. In the field, this pathogen can substantially affect ginger's health and rhizome yield if no effective control measures are implemented. Therefore, management of the disease should be further investigated to avoid major economic losses.

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