First report of leaf spot caused by Curvularia asiatica on water spinach ( Ipomoea aquatica ) in the United States

Water convolvulus (Ipomoea aquatica Forsk.), a member of the Convolvulaceae family, is an important tropical vegetable cultivated in China (Liu et al. 2017). From 2016 to 2020, dark-brown leaf spots were observed in major water convolvulus (cv. Large leaf) growing areas (2 ha) in Honghe City (24°12' N, 103°6' E), Yunnan Province, China. Field investigations showed that a leaf spot disease occurred on water convolvulus in four fields with 15% incidence (50 plants in each field were investigated) and resulted in up to a 10% decrease in its total production. Symptoms on water convolvulus plants appeared as small lesions, yellowish-green and circular on the leaves. Ten plants were selected randomly from the growing area, with three diseased leaves collected from each plant. Symptomatic tissues were excised, surface sterilized with 75% ethanol for 30 s, washed in sterile-distilled water three times, and placed on the Potato Dextrose Agar (PDA) followed by incubation at 25°C in the dark for 7 days. Colonies on PDA were gray to green in color and fuzzy in the middle, with irregular borders. Conidiophore morphology showed single, yellowish-brown or brown structures with 1~6 septa, and long 22~125 µm, wide 3.5~5.5 µm. Conidia were elliptical, black-brow, solitary, with a smooth surface, 1~6 longitudinal septa and 1~3 transverse septa, 20~30 µm in length, and 15~22 µm in width. The morphological characteristics of the fungus were consistent with the description of Stemphylium solani (Chai et al. 2014; Weber, 1930). To further confirm the identity of the 30 isolates, the partial gapdh (glyceraldehyde-3-phosphate dehydrogenase), tef1 (translation elongation factor 1-alpha), cmdA (Calmodulin) and ITS (intemal transcribed spacers) sequences were amplified by PCR with the primer pairs of gpd1/gpd2, EF1-728F/EF1-986R, CALDF1/CALDR2 and ITS1/ITS4, respectively (Berbee et al. 1999; Carbone & Kohn. 1999; Lawrence et al. 2013; White et al. 1990). Multiple sequence alignments were generated using MEGA7, and phylogenetic analysis was conducted with the neighbor-joining (NJ) method (Tamura et al. 2007), the results indicated that all sequences from the 30 isolates were identical. Thus, one representative isolate, KXC11033003 was chosen for further analysis. The ITS, gapdh, cmdA and tef1 sequences of this isolate were submitted to the NCBI GenBank database (accession nos. OL444947~OL444950). The strain KXC11033003 and S. solani (CBS-408.54) formed a clade with 82% bootstrap value (Figure S2). To fulfill Koch's postulates, 30 plants were inoculated for each of the thirty isolates. Conidia were sprayed on leaves of water convolvulus (8-true-leaf stage) in a suspension of 107 conidia/mL or water as a healthy control in a greenhouse at 15~18℃ (night) / 25~28℃ (day) with 95% humidity. Symptoms of dark brown spots appeared on the leaves after 7 days, whereas controls remained healthy The pathogens were reisolated from the lesions and confirmed identical to the original isolate by gene sequences. No pathogens were isolated from the control plants. To our knowledge, this is the first report of leaf spot caused by S. solani on water convolvulus in Yunnan Province, China. Further, Stemphylium leaf spot caused by S. solani has been reported previously on tomato, garlic, pepper (Zheng et al.2008; Nasehi et al.2018). This study stresses the need to identify appropriate management strategies for S. solani that help prevent quality and yield losses in water convolvulus in China.

Water spinach (Ipomoea aquatica Forsk.) is important for its nutritional, economic, and environmental benefits. However, pathogen infections can impact its production. This study aimed to identify the fungal pathogen responsible for leaf spots on lowland water spinach. Infected leaves showed brown necrotic spots surrounded by yellow halos. Pathogenicity assays confirmed the virulence of the fungal pathogen WS1 in both lowland and upland water spinach. Through morphological, cultural, and molecular characterization, the pathogen was identified as Diaporthe batatas Harter & E.C. Field. Key characteristics of the fungal isolate included hyaline, septated, and branched hyphae, with conidia measuring 4.1 to 8.3 µm in length and 1.8 to 3.2 µm in width. The isolate displayed distinct growth patterns on various artificial media with transparent, flat, rhizoid mycelium on water agar; white, velvety mycelium with black pycnidia on malt extract agar and potato dextrose agar; and dark brown to black filamentous colonies on Sabouraud dextrose agar. Sequencing of the internal transcribed spacer (ITS) and β-tubulin genes matched reference sequences of D. batatas. The study identifies D. batatas as a fungal pathogen of water spinach.

Pogostemon cablin (Blanco) Benth. is a vital source of patchouli oil, utilized in traditional Chinese medicine, Ayurveda, cosmetics, and hygiene products(Swamy et al. 2016). In 2022, a leaf spot disease outbreak on patchouli plants occurred nearly 10 acres in Yunfu, Guangdong (21°2' N; 110°3' E), with an average incidence rate of 50%. Infected leaves initially showed circular spots with tan centers and yellow halos. Within five to ten days, these spots expanded, crossed leaf veins, and became polygonal with rough surfaces. As the disease progressed, the spots merged, darkening the veins and eventually causing leaf loss. Five symptomatic leaves were soaked in 75% ethanol for 10 s, and 2.5% sodium hypochlorite for 30 s, followed by a final rinse in sterile water. These samples were then placed on potato dextrose agar (PDA) plates. After 3-5 days of incubation at 28 °C, the mycelial growth was transferred to fresh PDA plates and purified by isolating the hyphal tip three times. Four of the five isolates had similar morphology and caused leaf spot symptoms upon inoculation. During culture on PDA, these isolates formed colonies with downy, gray-brown mycelium and darker centers. Microscopic examination revealed branched, septate mycelium; solitary, erect, unbranched grayish-brown conidiophores; and club-shaped conidia. Conidia were faint brown, segmented, contained 2 to 15 septa(predominant number being 3 to 4), ranged from 32 to 220 μm in length and 8.4 to 22.4 μm in width (n = 30). The morphology of these isolates was identical to that of Corynespora cassiicola (Ellis 1971). Pathogenicity tests were conducted on 3-month-old seedlings of P. cablin using the conidium infection method. A suspension of conidia (1 × 106 conidia/ml) was prepared from cultures induced to sporulate by 90-minute near-UV exposure followed by 2-day dark incubation at 28 °C, and 30 ml of this suspension was sprayed onto leaves of each seedling. Inoculated plants were incubated at 28 °C in an incubator. Potted plants treated only with sterile water were used as controls. Each treatment was inoculated into five potted plants. After seven days, all the inoculated leaves displayed symptoms similar to those observed in the fields, whereas the control leaves did not exhibit these symptoms. The pathogenicity test was repeated three times. Following Koch's postulates, the pathogen was re-isolated and identified as C. cassiicola through morphological and ITS sequence each time. To further identify, we selected a representative isolate, LD-TJ, for multi-locus sequence analysis of its ITS, LSU, and TEF1-α genes (GenBank Accession Nos. PQ042036, PQ035023, PQ060235)(Voglmayr et al. 2017). BLASTN analysis of the sequences obtained showed a high similarity of 99 to 100% with the ITS (JAEMHE010000031.1:51214-51777, 481/481 nucleotides), LSU (JAEMHE010000018.1:728464-729373, 910/910 nucleotides), and TEF1-α (JAEMHE010000005.1:656736-657712, 969/970 nucleotides) sequences of C. cassiicola CC01. The phylogenetic tree showed that the LD-TJ strain, C. cassiicola CC01(isolated from rubber trees), and C. cassiicola CC_29(isolated from soybean leaves) clustered into a clade with a 99% bootstrap value. C. cassiicola was identified as the cause of patchouli leaf spot in Hainan(Chen et al. 2010), but it has not yet been reported in Guangdong. Identifying P. cablin leaf spot disease is crucial in Guangdong Province because it is the main growing area for P. cablin in China (Yan et al. 2021).

Crop productivity around the world is being seriously affected by adverse environmental conditions. High temperature (HT) stress has severely hampered plant growth, yield, and quality. Water spinach is a significant heat-resilient green leafy vegetable that can mitigate prolonged HT stress. However, the morphological, physiological, and biochemical alterations that occur in its response to heat stress remain unknown. In this study, the physiological response to HT stress in water spinach plants with different temperature (25-control, 30, 35, 40, 45 °C) tolerances was investigated. When plants were subjected to HT over a long period of time, their growth was stunted. The results showed that no significant difference was seen between the control (25 °C) and 30 °C for some traits (root shoot fresh weight, root morphological traits, and leaf gas exchanges parameters). Further, HT (35, 40, and 45 °C) stress significantly reduced the growth status, the gas exchange parameters, the pigment content, the photosystem function, and the root architecture system of water spinach. Conversely, HT stress considerably enhanced secondary metabolites in terms of total phenolics, flavonoids, soluble sugars, and anthocyanin content. Furthermore, heat stress remarkably increased the accumulation of reactive oxygen species (ROS) and caused cellular membrane damage. HT stress effectively altered the antioxidant defense system and caused oxidative damage. Generally, HT has an adverse effect on the enzyme activity of water spinach, leading to cell death. However, the current study found that temperatures ≥35 °C had an adverse effect on the growth of water spinach. Further research will be needed to examine the mechanism and the gene expression involved in the cell death that is caused by temperature stress in water spinach plants.

Hymenocallis littoralis (Jacq.) Salisb. is a common ornamental plant in China. In November 2021, leaf spots were observed on H. littoralis in a public garden in Zhanjiang, Guangdong Province, China (21°17'25″N, 110°18'12″E). Disease incidence was around 60% (n = 100 investigated plants from about 1 ha). Leaf symptoms were round spots with collapse centers, surrounded by yellow halos. Ten symptomatic leaves from 10 plants were sampled. The margins of the samples were cut into 2 mm × 2 mm pieces. The surfaces were disinfected with 75% ethanol for 30 s and 2% sodium hypochlorite for 60 s. Thereafter, the samples were rinsed thrice in sterile water, placed on PDA, and incubated at 28 °C in dark. Pure cultures were obtained by transferring hyphal tips to new PDA plates. Twenty pure cultures were obtained. Single-spore isolation method (Liu et al. 2021) was used to recover the cultures of three isolates (HPC-1, HPC-2, and HPC-3). Colonies of the isolates were dark green with a granular surface, and irregular white (later turning black) edge. Pycnidia were black, globose and 96 -140 μm in diameter. Conidia were single-celled, oval, 7.5 to 13.5 × 4.0 to 7.5 µm (n= 40), with a single apical appendage. Morphological characteristics of the isolates were consistent with the description ofPhyllostictacapitalensis(Wikee et al. 2013). Molecular identification was performed using PCR method with MightyAmp DNA Polymerase (Takara-Bio, Dalian, China) (Lu et al. 2012). The internal transcribed spacer (ITS) region, translation elongation factor (TEF1), actin (ACT), and glyceradehyde-3-phosphate dehydrogenase (GAPDH) were amplified using primers ITS1/ITS4, EF1-728F/EF1-986R (Druzhinina et al. 2005), ACT-512F/ACT-783R, and Gpd1-LM/Gpd2-LM (Wikee et al. 2013), respectively. Sequences were deposited in GenBank with accession numbers OM654570 - OM654572 forITS, OM831376 - OM831378 fortef1-α, OM831346 - OM831348 forACT, and OM831364 - OM831366 for GAPDH. BLASTn analysis showed that these sequences were 99 to 100% similarity with those of P.capitalensis(ITS, FJ538339; TEF1, FJ538397; ACT, FJ538455; and GAPDH, JF343723). Besides, a phylogenetic tree was generated on the basis of the concatenated data from the sequences of ITS, TEF1, ACT, and GAPDH that nested within the clade containing P.capitalensis (CBS 117118, CPC20510,CPC20267, and CPC18848). From the combination of the morphological and molecular characteristics, the isolates were determined to beP.capitalensis. Pathogenicity testing was performed in a greenhouse with 80% relative humidity at 25 to 30°C. Ten healthy plants of H. littoralis (2 month old with 4 leaves) were grown in pots with one plant in each pot. Three leaves on one plant per isolate were inoculated with three mycelial plugs obtained from 7-day cultures, totaling five plants. Five plants treated with PDA plugs served as the controls. Wet cotton balls were fixed on the leaves with transparent tape for five days to keep it from drying out. The test was conducted three times. After 15 days, similar symptoms were observed in the inoculated leaves as in the garden, whereas control leaves remained asymptomatic and P.capitalensiswas successfully re-isolated from the inoculated leaves. Previously, P.capitalensishas been reported to cause leaf spot disease of various host plants around the world (Wikee et al. 2013). However, to our knowledge, this is the first report of leaf spot caused by P.capitalensison H. littoralis in China. This study provides an important reference for the control of the disease.

Cucumber (Cucumis sativus L.) is one of the most important vegetables cultivated in the world. It is widely cultivated and mostly grown under greenhouse conditions (Sallam et al. 2021). Cucumber has a long growth cycle and is particularly susceptible to bacterial diseases. In May 2021, bacterial leaf spot was found on cucumbers of the variety Lyuyou NO.3 in Hainan Province, China. In the early stage of the disease, the leaves showed small yellow-brown spots in the shape of water stains. When exposed to light, a yellow halo around the disease spots could be seen. In later stages, the lesions gradually become larger and more yellow. The leaf veins around the disease site also gradually turned yellow (Figure 2a). In serious cases, the whole leaf turned yellow, resulting in leaf death. We collected plants with the same symptoms from 25 different farms in Hainan Province. Five plants were selected from each farm by the classic five-point sampling method and three leaves were selected from each plant, for a total of 15 leaves collected from each farm. Then three leaves were randomly selected from the 15 leaves on each farm for isolation of the pathogen, and a total of 75 leaves were isolated. We found that the incidence of the disease was from 20% to 30% based on a diagnostic test, which conducted on 75 cucumber leaves samples suspected of same symptom of cucumber, collected from Hainan Province. Using microscopy, bacterial streaming was observed to tentatively identify the causal agent as a bacteria. Tissue isolation was used to isolate the responsible pathogens. A 5 mm × 5 mm sample of tissue at the junction of diseased and healthy sections was collected. First, the surface of the tissue was disinfected in a 75% ethanol solution for 30 sec; then it was soaked in 2% NaOCl for 5-7 min, and finally, it was washed thrice in sterile distilled water. The tissues were inoculated onto lysogen broth culture media (LB) and cultured in a 28℃ incubator for 2 days. Bacterial colonies that emerged from the tissues were cultured in LB. Four isolated colonies were selected for verification. The colonies of isolated from the diseased leaves of cucumber are round, egg yellow and slightly sticky (Figure 2c). The isolate named PA-1 was identified by PCR amplification and sequencing of the partial 16S rRNA gene with the primer 27F/1492R (Lane 1991) and gyrB gene (Li et al. 2019). Sequences were stored in GenBank with the accession numbers OK576932.1 (16S rRNA, PA-1) and OL978577 (gyrB); BLASTn was used to compare these with other GenBank sequences. Sequencing of the 16S rRNA gene showed that PA-1 had a sequence length of 1403bp, with 99.78% genetic similarity to Pantoea ananatis strain MZ007857.1. Sequencing of the gyrB gene showed that the sequence length of PA-1 was 1136bp, with 99.29% genetic similarity to P. ananatis strain MW981331.1. Then, a pathogenicity text was conducted to verify Koch's postulates, which was done by first inoculating P. ananatis into LB liquid medium (shake culture at 28°C, 180 r/min). The log phase cell was collected by centrifugation (5,000 r/min for 2 min at 4°C), and inoculated strains were resuspended in sterile water at OD600 = 0.5. The bacterial suspension was inoculated on healthy cucumber leaves with a syringe. The control was sterile water, which was injected onto healthy cucumber leaves using the same methodology. The plants were placed in a greenhouse with a diurnal temperature difference of 21- 27°C and were observed daily. After two weeks, all bacterial inoculated plants developed symptoms of shriveling and necrosis (Figure 2b), while the control group showed no symptoms. From the symptomatic plants, the pathogen was isolated again and identified by morphological and molecular characterization. The sequences of the isolates recovered from the inoculated experiment matched 100% the sequences of the isolate PA-1. Koch's postulates were completed by following the previously described method. To our knowledge, this is the first report of P. ananatis causing leaf spot of cucumber.

ABSTRACT. Phytoremediation with mono and polyculture systems of giant salvinia (Salvinia molesta) and water spinach (Ipomoea aquatica) plants for the treatment of batik wastewater has been carried out. This research aims to study the effect of giant salvinia and water spinach plants in reducing pollutant levels in batik wastewater, determine the order kinetics of Cu, TDS, and BOD reduction in monoculture and polyculture systems, and determine the effectiveness of Cu, TDS, and BOD reduction in monoculture against polyculture system. The research methodology included two main treatments, namely phytoremediation and data analysis. Phytoremediation was carried out by varying the combination of water spinach and giant salvinia plants with ratio of 0:100, 50:50, and 100:0 with a total plant mass of 100 gr. Analysis was conducted based on spectrophotometric and gravimetric principles. The results of the analysis were tested for significance by ANOVA test. Research data showed that polyculture of giant salvinia (Salvinia molesta) and water spinach (Ipomoea aquatica) could increase the effectiveness of reducing Cu metal by 91%, dye by 90%, and TDS by 36%. While polyculture system has better effectiveness in reducing Cu metal, TDS and dyes concentration than monoculture system, but the difference in effectiveness is not statistically significant. Keywords: Batik wastewater, Ipomoea aquatica, mono and polyculture, phytoremediation, Salvinia molesta.

Bletilla striata (named "Bai Ji" in Chinese) is a plant from the Orchidaceae family that has been employed in traditional Chinese medicine for thousands of years in China. Polysaccharides extracted from B. striata have been shown to have an effect on Alzheimer's disease (Lin et al. 2021). Since 2021, leaf spots have been observed in the B. striata plantation in Chongqing, China. Out of 200 plants, the disease incidence was estimated at 56%, and the disease index was estimated at 32%. The symptoms were necrotic lesions with brown edges and yellow halos; severe infection caused the infected leaves to become blighted, dry and fall off. To identify the causal agent, eighteen leaves with typical symptoms were collected from the B. striata plantation (30.60°N, 108.64°E). The margins of infected tissue areas were cut into small pieces (5×5 mm), surface sterilized with 70% ethanol for 1 min, and rinsed twice with sterile distilled water. The tissue was then surface sterilized in 3% sodium hypochlorite for 2 min, followed by three rinses with sterile water. The tissue was then placed onto potato dextrose agar (PDA) plates and incubated at 25°C for 3 days, pure cultures of fungal isolates were obtained by single-spore isolation, stored on PDA slants and maintained at 4°C. Colonies of the fungal isolates showed three color types, ranging from grayish white to green above with olive green on the reverse, but conidial characteristics were more similar and indicated this was a single fungus. Conidiophores were single, lateral from hyphae or terminal; straight or curved; smooth-walled with 1 to 8 septa; pale brown; usually with only one pigmented terminal conidiogenous site, sometimes with one additional lateral conidiogenous locus; sometimes slightly swollen at the apex; and 15 to 170 μm long, 2.5 to 4.5 μm wide. Conidia were in short or moderately long chains of 2-8 conidia normally, sometimes with more; rarely branched; normally 14.07 to 50 × 5.24 to 10 μm in size; ellipsoid, fusiform, long ellipsoid, obclavate or ovoid with 1 to 11 transverse septa and 2 to 4 longitudinal septa; beakless or with subcylindric or cylindric secondary conidiophores, analogous to the beak 4.25 to 58.6 μm long, 3.2 to 4.8 μm wide. The fungal isolates were tentatively identified as Alternaria sp. The representative isolate BJ8 was selected for the pathogenicity test. The leaves of six healthy plants of B. striata (two years old) grown in pots were washed with sterile water. Ten mL of conidial suspension (1×106 conidia mL-1) contained in 0.05% Tween 80 buffer was brushed onto upper and lower surfaces of all the leaves on three plants, while other plants were brushed with 10 mL 0.05% Tween 80 buffer to serve as controls. Plants were placed in a greenhouse at 25°C and 95±1% relative humidity after inoculation and observed for symptoms. The symptoms initially developed as irregular brown necrotic lesions on the inoculated leaves after 7 days, with a yellow halo around the lesions, consistent with the symptoms in the field. Leaves on the control plants did not produce any symptoms. For molecular identification, the genomic DNAs of representative isolates BJ5, BJ6, and BJ8 were extracted. The internal transcribed spacer (ITS) region and RNA polymerase II second largest subunit (RPB2), translation elongation factor 1-alpha (TEF1), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes were used for polymerase chain reaction (PCR), using primers ITS5/ITS4, GPD1/GPD2, EF-1F/EF-1B and RPB27cR/RPB25F2, respectively (White et al. 1990; Berbee and Pirseyedi et al. 1999; Carbone and Kohn 1999; Liu et al. 1999). The neighbor-joining tree revealed that these isolates are clustered together with the reference strain of A. burnsii. The sequences were deposited in NCBI GenBank BJ5 [ITS: OP897263; GAPDH: OQ544937; TEF1: OQ544941; RPB2: OQ544939], BJ6 [ITS: OP897262; GAPDH: OQ544938; TEF1: OQ544942; RPB2: OQ544940], and BJ8 [ITS: OK285209; GAPDH: OK340046; TEF1: OK340047; RPB2: OQ544936]. All three isolates showed 100% similarity with A. burnsii CBS 107.38 [ITS: KP124420; GAPDH: JQ646305; TEF1: KP125198; RPB2: JQ646457] ex-type sequence, thus the pathogen causing the leaf spot on B. striata was identified as A. burnsii. A. burnsii is an important pathogenic fungus causing blight of cumin (Shekhawat et al. 2013). Furthermore, Al-Nadabi et al. (2018) found that A. burnsii can cause leaf spots on wheat and date palms, and Sunapao et al. (2022) reported that A. burnsii can infect coconuts (Cocos nucifera), causing dirty panicle disease. This is the first report of A. burnsii causing leaf spot on B. striata in China. The new discovery shows that since A. burnsii can readily adapt to a variety of climatic conditions, controlling the fungus is crucial for the healthy growth of B. striata in the future. This study will provide a basis for further elucidating the pathogenic mechanism and development of effective control measures for this disease.

Alpinia oxyphylla Miq. is mainly distributed in Hainan, Guangdong and Guangxi provinces of China. Between July and August 2021, a leaf spot disease was observed in Ledong, Hainan Province, China (18°70'20.50″ N, 109°25'25.47″E) on A.oxyphylla. The incidence of infected leaves ranged from 8% to 10%, and the incidence rate of infected plants was about 50%. Symptoms appeared as primary yellow-brown withered spots on the diseased leaves, which further developed into irregular red-brown spots. The center of the lesions was gray-black, and the tissue was irregularly necrotic, ruptured or perforated, and there were yellow chlorotic halos around the edges of the lesions (Figure 1A). Tissues 5 mm in diameter were taken from the junction of diseased and healthy tissue for pathogen isolation, Successively, a total of 8 isolates were obtained from the affected leaves. Three single spore isolates (YZ-HN-001, YZ-HN-043 and YZ-HN-051) were obtained and confirmed to be identical based on morphological characteristics. Therefore, the representative isolate YZ-HN-001 was selected for morphological and molecular identification. On Potato Dextrose Agar(PDA), the colony was gray-white at first and gradually turned dark green to dark brown with lead gray on the back, growth was slow, and mycelium was short and dense (Figure 1B and Figure 1C). Pycnidia were epiphyllous, globose, brown (about 120-140 µm in diameter), and conidia were elliptical, colorless, single celled and smooth (8-12×4-7 µm) (Figure 1D). Molecular identification was performed by partially sequencing the internal transcribed spacer gene (ITS), 18S rRNA gene and the actin gene (ACT) by using the primers ITS1/ITS4 (White et al. 1990), EF4/Fungi5 (Khodaparase et al. 2005) and ACT-512F/ACT-783R (Carbone and Kohn. 1999). The sequences of the amplified fragments were deposited in GenBank, the ITS sequence (ON005130, 616 bp) showed 100% identity with Phyllosticta capitalensis strain CGMCC3.14345 (JN791605.1), the 18S rRNA sequence (ON005129, 541 bp) showed 99% identity with P. capitalensis isolate MUCC0029 (AB454185.1) and the ACT sequence (ON049348, 251 bp) showed 100% identity with P. capitalensis strain DZSN202005-2 (MW533248.1). A phylogenetic analysis was conducted in MEGA X using the neighbor-joining method and showed that isolate YZ-HN-001 clustered together with P. capitalensis (Figure 2). Based on the above morphological and molecular characteristics, the isolate was determined to be P. capitalensis. Pathogenicity tests were conducted in three replicates by inoculating surface-sterilized leaves of A. oxyphylla. The leaves were wounded and inoculated with colonized PDA plugs (5×5 mm) from 15-day-old cultures. Control leaves wounded in the same way and were inoculated with sterile PDA plugs (5×5 mm). Leaves were moisturized by spraying with sterile water every three days. After 20 days at room temperature (23 to 28℃), similar symptoms were observed in the inoculated leaves as in the field (Figure 1E), but no symptoms were observed on the control leaves (Figure 1F). The same P. capitalensis was reisolated in the inoculated leaves, confirming Koch's postulates. Phyllosticta capitalensis has been reported to cause leaf spots or black spots on various host plants around the world (Wikee et al. 2013), including on oil palm (Nasehi et al. 2020), tea plant (Cheng et al. 2019 ), and castor (Tang et al. 2020). Nevertheless, to our knowledge, this is the first report of leaf spot caused by P. capitalensis on A. oxyphylla worldwide.

Tomato (Solanum lycopersicum) is a staple vegetable across the world. In October 2019, leaf spots were observed on tomato (cv. Tianmi) in a greenhouse in JiZhou District Tianjin, China(117°10 'E; 39°55 'N). Symptoms initially appeared as small brown spots, which gradually expanded and turned into circular, oval or irregular spots (some spots with distinct concentric zones). In severe cases, some spots coalesced and eventually covered the whole leaf. Disease incidence ranged between 12 and 18%. Twenty symptomatic leaves from five plants were collected and cut into small pieces, surface disinfested in 2% NaClO for 60 s, rinsed three times in sterile water, and subsequently plated on potato dextrose agar (PDA). Plates were incubated at 25°C in the dark for 7 days. A total of 102 isolates were obtained and 92 isolates had the same morphology. Colonies were initially white with abundant aerial mycelia and formed sporodochia with conidial masses in olivaceous green concentric rings. All isolates formed single-celled, hyaline, and rod-shaped conidia were 4.91 to 7.43 (avg. 6.53±0.72) × 1.41 to 2.45 （avg. 2.11±0.30）μm with rounded ends (n=50). Conidiophores were highly branched. These characteristics resembled a Paramyrothecium-like fungus (Lombard et al. 2016). The genomic DNA of three representative single-spored isolates TJJXPF1-3 were extracted and the internal transcribed spacer (ITS) region, β-tubulin (tub2), large subunit ribosomal RNA (LSU), calmodulin (cmdA) and translation elongation factor 1-alpha (tef1) genes were amplified and sequenced using the primer pairs ITS4/ITS5 (White et al. 1990), Bt2a/Bt2b (Glass and Donaldson 1995), LR0R/LR5 (Rehner and Samuels 1995; Vilgalys and Hester 1990), CAL-228F/CAL2Rd (Carbone and Kohn 1999; Groenewald et al. 2013) and EF1-728F/EF2 (O'Donnell et al. 1998), respectively. All sequences were deposited in GenBank (ITS: MW463444, OM368178, OM368179; tub2: MW269542，OM714930，OM714931; LSU: OM349050, OM397398, OM390582; cmdA: MW280443, OM350474, OM350476; tef1: MW560083, OM350475, OM350477). BLASTN analysis showed 99.3-100% similarity with reference isolate QB1 of P. foliicola (MK335967, MT415353, MT415362, MT415356 and MT415359). Multilocus phylogenetic analysis showed that TJJXPF1-3 best grouped with the P. foliicola clade, which was identified by morphological characteristics and phylogenetic analysis. To fulfill Koch's postulates, pathogenicity tests were conducted by spray-inoculation with a conidial suspension of isolate TJJXPF1 prepared with distilled water (1×105 conidia/mL) on five 45-day old tomato plants. Three healthy plants were sprayed with sterile water as control. All treatments were incubated in an artificial climate chamber (25°C, 80% RH, 12h light/12h dark ). After two weeks, leaf spots were observed on all inoculated plants, which were similar to those in the greenhouse of JiZhou District, while control plants remained asymptomatic. Additionally, the pathogens were reisolated from symptomatic leaves and three representative isolates TJJXPF4-6 were identified as P. foliicola. The pathogenicity tests were repeated thrice. To our knowledge, this is the first report of leaf spot caused by P. foliicola on tomato in China. This disease could be a serious threat to tomato production in the future. Our findings will help to differentiate this disease from other leaf spot-like diseases and develop disease control strategies.

White clover (Trifolium repens L.) is a perennial legume widely cultivated as forage, lawn grass, a green manure crop, and soil conservation plant worldwide (Zhang et al. 2016). In March 2018, necrotic leaf lesions were observed in several white clover fields in Chongqing, southwest China. The disease incidence varied from 10 to 65% between fields. Infected leaves had small, brown, circular spots that became elliptic to irregular. The spots were 1 to 15 mm in diameter with dark brown margins, yellow halos, and sometimes concentric rings. As the disease progressed, lesions gradually coalesced, forming large necrotic patches. In advanced infections, severe disease led to defoliation. Symptomatic leaves were collected from eight different fields in the Rongchang and Qianjiang districts of Chongqing, surface sterilized with 70% ethanol for 30 s followed by 0.1% HgCl₂ treatment for 3 min, and rinsed in sterile water three times. Thereafter, tissue samples (2 × 2 mm) from margins of individual lesions were placed on potato dextrose agar (PDA) amended with 50 mg/liter of chloramphenicol and incubated at 25°C in the dark. Fungal colonies consistently isolated on PDA were initially white and then turned grayish black (93.75% isolation frequency). Conidia were light to dark brown, ovoid to long ellipsoid, with one to four transverse and zero to two longitudinal septa, slightly constricted near some septa, and approximately 12 to 29 × 6 to 17 µm in size. Conidiophores were straight or flexuous, brown, and 11 to 60 × 3 to 8 µm. Morphological characteristics matched the descriptions of Alternaria alternata (Woudenberg et al. 2013). To confirm the identity, the internal transcribed spacer region (ITS), partial sequencing of 28S large subunit ribosomal RNA (LSU), major allergen Alt a 1 (Alt), and glyceraldehyde-3-phosphate dehydrogenase (GPD) genes of eight representative isolates were amplified with primers ITS1/4, LSU1Fd/LR5, Alt-for/rev, and gpd1/2, respectively, and were sequenced (Woudenberg et al. 2015). BLAST results showed 100% identity of the ITS, LSU, and Alt sequences and 99.83% identity of the GPD sequences with those of A. alternata strain CBS 104.26 (KP124299, KP124450, KP123848, and KP124156). Based on morphology and DNA sequence analysis, the associated fungus was identified as A. alternata. Representative sequences of one isolate (ATH1-2) were deposited in GenBank (MK894882, MK903026, MK903027, and MK903028). Pathogenicity tests were carried out on potted white clover, cultivar Gede. Ten healthy plants were sprayed with a conidial suspension (approximately 10⁵ conidia/ml), and the control plants were inoculated with sterile distilled water. Plants were incubated in a greenhouse at 20 to 24°C under natural light and enclosed in plastic bags for the first 2 days to maintain high humidity. After 7 days, characteristic dark brown spots were observed on the inoculated leaves and not on the control plants. A. alternata was reisolated from inoculated symptomatic leaves, thus completing Koch’s postulates. To our knowledge, this is the first report of leaf spot on white clover caused by A. alternata in China. This disease not only seriously affects lawn appearance and quality but also greatly reduces forage quality and yield, resulting in economic losses to animal husbandry.

Sweet cherry, Prunus avium (L.) L., is not much cultivated in Korea, with only 150 ha planted for domestic consumption. In September 2012, a previously unknown leaf spot was observed with nearly 100% incidence on trees (cv. Seneca) planted in a plastic greenhouse in Iksan City of Korea. Interestingly, the same cultivar as well as other cultivars planted outdoors did not show these symptoms. Leaf spots were irregular to subcircular, dark brown with or without a yellow halo, and becoming coalesced to cause leaf blight and premature defoliation. A cercosporoid fungus was consistently associated with disease symptoms. Fungal structures within the lesion developed on both leaf sides but mostly on the upper side. Stromata were well-developed, globular, dark brown, composed of textura angularis-globosa, and 30 to 80 μm in diameter. Conidiophores were densely fasciculate, pale olivaceous to pale brown, subcylindrical, geniculate-sinuous, 8 to 24 × 3 to 4 μm, and aseptate to 2-septate. Conidiogenous loci were inconspicuous, neither thickened nor darkened. Conidia were olivaceous, generally darker than conidiophores, cylindrical to obclavate, almost straight to mildly curved, short obconically truncate at the base, obtuse at the apex, 1- to 10-septate, constricted at the septa, 12 to 86 × 3.5 to 5 μm, guttulate, and had unthickened, not darkened hila. Morphological characteristics of the fungus were consistent with previous descriptions of Pseudocercospora pruni-persicicola (J.M. Yen) J.M. Yen (1,3). A voucher specimen was deposited in the Korea University herbarium (Accession No. KUS-F27264) and a monoconidial isolate was deposited in the Korean Agricultural Culture Collection (Accession No. KACC47019). The complete internal transcribed spacer (ITS) region of rDNA was amplified with the primers ITS1/ITS4 (4) and sequenced. The resulting 505-bp sequence was deposited in GenBank (Accession No. KF670713). A BLAST search in GenBank revealed that the sequence showed >99% similarity with sequences of many Pseudocercospora species, indicating the close phylogenetic relationship of species in this genus. To conduct a pathogenicity test, a conidial suspension (~1 × 104 conidia/ml) was prepared in sterile water by harvesting conidia from 2-week-old cultures on V8 juice agar, and the suspension was sprayed until runoff onto the leaves of five healthy seedlings. Control plants were sprayed with sterile water. The plants were covered with plastic bags to maintain a relative humidity of 100% for 48 h and then transferred to a greenhouse. Necrotic spots appeared on the inoculated leaves 20 days after inoculation, and were identical to the ones observed in the field. P. pruni-persicicola was re-isolated from symptomatic leaf tissues, fulfilling Koch's postulates. Control plants remained symptomless. The fungus has previously been recorded on Prunus persica (L.) Stokes in Taiwan (2,3). To our knowledge, this is the first report of this fungus on P. avium globally as well as in Korea. The disease poses a new threat to the sweet cherry industry in Korea.

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.

Illicium difengpi B. N. Changet al., a shrub with aromatic odor in the Illicium genus, is extensively used as a medicinal plant in China. In June of 2020, a leaf spot on I. difengpi with incidence of about sixty percent was observed in a field located in Guilin (25°4'40"N; 110°18'21"E), Guangxi Province, China. Initialleafsymptoms were roundspotswith gray centers, surrounded by yellow halos. The spots gradually spread and merged. Six samples of symptomaticleaveswere collected from six diseased plants, and they were surface disinfested before isolation. Potato dextrose agar (PDA) was used to culture pathogens. Successively, pure cultures were obtained by transferring hyphal tips to new PDA plates. A total of 10 isolates were obtained from the affectedleaves.Two single-spore isolates (GX-1 and GX-2) were obtained and confirmed to be identical based on morphological characteristics. The representative isolate GX-2 was selected for further study on morphological and molecular characteristics. The colony of isolate GX-2 was about 4 cm in diameter on a PDA plate in 5 days, dark green with a granular surface, and irregular white edge. Conidia were hyaline, unicellular, oval, narrow at the end with a single apical appendage, and 8.2 to 13.8 × 3.7 to 7.2 µm (n= 50). Spermatia were hyaline, bacilliform with swollen ends, 3.8 to 8.9 × 1.3 to 1.9 µm (n= 50). Morphological characteristics of isolate GX-2 were consistent with the description of Phyllostictacapitalensis (Wikee et al. 2013). The internal transcribed spacer (ITS) region, translation elongation factor 1-α (tef1-α), glyceraldehyde-3-phosphate dehydrogenase (GPDH) and actin (ACT) were amplified using primers ITS1/ITS4, EF-728F/EF-986R, Gpd1-LM/Gpd2-LM and ACT-512F/ACT-783R, respectively (Wikee et al. 2013). Sequences were deposited in GenBank with accession numbers OL505439 forITS, OL539429 forACT, OL539430 fortef1-α and OL539431 for GPDH. BLAST analysis in GenBank showed that these sequences were 99 to 100% similar to the correspondingITS(MT649668),ACT(MN958710), tef1-α(MN958711) andGPDH (KU716077) sequences ofP. capitalensis. Also, the phylogenetic tree based on genes of ITS, tef1-α, GPDH and ACTby the maximum likelihood method showed that isolate GX-2 clustered together withP. capitalensis.The pathogenicity tests were carried out on a healthy 3 year-old plant in the greenhouse with 80% relative humidity at 25 °C. Four sterilized leaves were wounded with a needle and inoculated with 20 μL spore suspension (1 × 106spores/ml). Another four sterilized leaves were inoculated with 20 μL sterile water as a control. All plants were incubated in a chamber with 98% relative humidity at 25 ± 1°C. After 12 days, disease symptoms similar to the field were observed on leaves, whereas control plants remained healthy.P. capitalensiswas successfully reisolated only from the inoculated leaves and identified based on morphological characters. P. capitalensiscausedleafspotson various host plants around the world (Wikee et al. 2013), including on tea plants in China (Cheng et al. 2019) and oil palm in Malaysia (Nasehi et al. 2020), but it has not been reported on I. difengpi. Thus, this is the first report ofP. capitalensiscausingleafspoton I. difengpi. This study will provide an important reference for the control of the disease. The epidemiology ofthis disease should be investigated in further research.

Hymenocallis littoralis (Jacq.) Salisb. is a common ornamental plant in China. In November 2021, leaf spots were observed on H. littoralis in a public garden in Zhanjiang, Guangdong Province, China (21°17'25″N, 110°18'12″E). Disease incidence was 82% (n = 100 investigated plants from about 10 ha). Initial small white dots densely distributed on the leaves and gradually expanded into round lesions with purple centers typically surrounded by yellow halos. The coalescence of the individual spot eventually led to leaf wilt. Ten symptomatic leaves from 10 plants were sampled. The margins of the samples were cut into 2 mm × 2 mm pieces. The tissue surface was disinfected with 75% ethanol for 30 s and 2% sodium hypochlorite for 60 s. Thereafter, the samples were rinsed three times in sterile water, placed on potato dextrose agar (PDA), and incubated at 28 °C. Pure cultures were obtained by transferring hyphal tips to new PDA plates. Twenty-eight isolates were obtained (isolation frequency = 28/4 × 10 = 70%). Three representative single-spore isolates (HPO-1, HPO-2, and HPO-3) by a single-spore isolation method (Fang. 1998) were used for further study. The colonies of isolates on PDA were olive green in 7 days at 28 °C. Conidiogenous cells were unbranched, straight to geniculate-sinuous, tapered toward the apex, and 12-19 × 3 μm (n = 20). Conidia were solitary, smooth, straight or curved, pale brown, 3-8-septate, apex acute, base truncate, and 55.3-86.5 × 2.0-3.5 μm (n = 50). The morphological characteristics were consistent with the description of Pseudocercospora oenotherae (Guo and Liu. 1992; Kirschner. 2015). For molecular identification, the colony PCR method with Taq DNA polymerase and MightyAmp DNA Polymerase (Lu et al. 2012) was used to amplify the internal transcribed spacer (ITS), translation elongation factor 1-α gene (TEF1), and actin (ACT) loci of the isolates using primer pairs ITS1/ITS4, EF1/EF2, and ACT-512F/ACT-783R, respectively (O'Donnell et al. 1998). Their sequences were deposited in GenBank under nos. OM654573-OM654575 (ITS), OM831379-OM831381 (TEF1), and OM831349-OM831351 (ACT). A phylogenetic tree was generated on the basis of the concatenated data from the sequences of ITS, TEF1, and ACT that clustered the isolates with P. oenotherae (the type strain CBS 131920). Pathogenicity testing was performed in a greenhouse with 80% relative humidity at 28 °C to 30 °C. Healthy plants of H. littoralis were grown in pots, with one plant in each pot. They were inoculated with a spore suspension (1 × 105 per mL) of the isolates and sterile distilled water (control). Sterile cotton balls were immersed in the spore suspension and sterile distilled water for about 15 s before they were adhered to the leaves for 3 days. Each isolate was inoculated with three plants (1 month old), and each plant was inoculated with two leaves. The test was performed three times. Symptom were found on the inoculated plants after 2 weeks with the disease incidence 88.89%, whereas the control plants remained healthy. The fungus was re-isolated from the infected leaves and confirmed as the same isolates by morphological and ITS analyses. No fungus was isolated from the control plants. P. oenotherae caused leaf spot on OenotherabiennisL. (Guo and Liu. 1992). H. littoralis is the second host of the fungus investigated in this study firstly (Crous, et al. 2013). Thus, this work provides an important reference for the control of this disease in the future.