First Report of Phyllosticta capitalensis Causing Leaf spot in Hymenocallis littoralis in China.

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 of Phyllosticta capitalensis (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 for ITS, OM831376 - OM831378 for tef1-α, OM831346 - OM831348 for ACT, 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 be P. 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. capitalensis was successfully re-isolated from the inoculated leaves. Previously, P. capitalensis has 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. capitalensis on H. littoralis in China. This study provides an important reference for the control of the disease.

First Report of Pseudocercospora oenotherae Causing Leaf spot in Rhinacanthus nasutus in China.

Rhinacanthus nasutus (L.) Kurz is a traditional Chinese medicine in China. In August 2022, leaf spots were observed on R. nasutus in a Chinese herbal garden in Zhanjiang, Guangdong Province, China (21°17'30″N, 110°18'25″E). Disease incidence was 90% (n = 100 investigated plants from about 800 plants). The yellow spots were round, gray in the center and scattered on the leaves. 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 (RNPO-1, RNPO-2, and RNPO-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, geniculate-sinuous, tapered toward the apex, and 10 to 20 × 3 µm (n = 20). Conidia were solitary, smooth, straight or curved, pale brown, 3 to 8-septate, apex acute, base truncate, and 50.5 to 88.5 × 2.0 to 3.5 µm (n = 50). 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), actin (ACT) , and RNA polymerase II second largest subunit (RPB2) loci of the isolates using primer pairs ITS1/ITS4, EF1/EF2, ACT-512F/ACT-783R, and RPB2-7CF/fRPB2-11aR, respectively (O'Donnell et al. 1998; O'Donnell et al. 2010). Their sequences were deposited in GenBank under nos. OP963568 to OP963570 (ITS), OP998376 to OP998378 (TEF1), OP998373 to OP998375 (ACT), and OP998379 to OP998381 (RPB2). By using the Maximum Likelihood method, a phylogenetic tree was generated on the basis of the concatenated data from the sequences of ITS, TEF1, ACT, and RPB2 that clustered the isolates with P. oenotherae (the type strain CBS 131920). The fungus was thus identified as P. oenotherae basing on these morphological and molecular characteristics (Guo and Liu. 1992; Kirschner. 2015). Pathogenicity testing was performed in a greenhouse with 80% relative humidity at 28 °C to 30 °C. Healthy plants of R. nasutus were grown in pots, with one plant in each pot. Sterile cotton balls were immersed in the spore suspension (1 × 105 per mL) of the isolates for about 15 s before they were adhered to the leaves for 3 days. Each isolate was inoculated with three plants (2 month old), and each plant was inoculated with five leaves. Sterile distilled water was as the control and same treatment. The test was performed three times. Symptom were found on the inoculated plants after 2 weeks with the disease incidence 100%, 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 pathogen was isolated from the control plants. P. oenotherae caused leaf spot on Oenothera biennis L. (Guo and Liu. 1992). To the best of our knowledge, this is the first report that R. nasutus is the new host of P. oenotherae (Crous et al. 2013). Thus, this work provides an important reference for the control of this disease in the future.

First Report of Colletotrichum truncatum Causing leaf spot on Clausena lansium in China.

Wampee (Clausena lansium [Lour.] Skeels) is a tropical fruit. In July 2022, leaf spot symptom was observed in wampee (cv. JIXIN) in a field ((21°25'N, 110°10'E, about 100 ha ), Guangdong Province, China. Disease incidence was around 70% (n = 100 investigated plants from about 2 ha). Leaf spots were round or irregular with a clear yellow halo around a brown, necrotic lesion. 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 potato dextrose agar (PDA), and incubated at 28 °C in the dark for 3 days. Pure cultures were obtained by transferring hyphal tips to new PDA plates. Twenty isolates were obtained. Three representative single-spore isolates (CLCT-1, CLCT-2, and CLCT-3) from the twenty isolates were confirmed to be identical based on morphological characteristics and ITS analysis and used for further study. The colonies on PDA were gray white at first, subsequently turning grayish to dark gray, with numerous black microsclerotia and setae. Conidia were hyaline, aseptate, falcate with pointed ends, and 16.5 to 22.3 × 2.5 to 3.2 µm (n = 30). Morphological characteristics of the isolates were consistent with the description of Colletotrichum truncatum (Schwein.) Andrus & W. D. Moore (Sawant et al. 2012). For molecular identification, the colony PCR method (Lu et al., 2012) was used to amplify the internal transcribed spacer (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and actin (ACT) loci of the isolates using primer pairs ITS1/ITS4, GDF1/GDR1, and ACT-512F/ACT-783R, respectively (Weir et al. 2012). The sequences were submitted to GenBank under accession numbers OP740964 to OP740966 (ITS), OP800837 to OP800839 (GAPDH), and OP800843 to OP800845 (ACT). The sequences of the three isolates were 100% identical (ITS, 547/547 bp; GAPDH, 290/290 bp; and ACT, 266/266 bp) with those of C. truncatum (accession nos. GU227869, GU228261, and GU227967) through BLAST analysis.. In addition, a phylogenetic tree was generated on the basis of the concatenated data from sequences of ITS, GAPDH, and ACT that nested within the clade containing C. truncatum (the type strain CBS 112998) by the maximum likelihood method. From the combination of the morphological and molecular characteristics, the isolates were determined to be C. truncatum. A pathogenicity test was performed in a greenhouse at 24 to 30°C with 80% relative humidity. Wampee plants (cv. JIXIN, n =5, 1-month-old) were inoculated with a spore solution (1 × 105 per mL) until it run-off. Whereas control plants were sprayed with sterile distilled water. Leaf spots were observed on the inoculated plants after 10 days while the control ones remained healthy. The pathogen re-isolated from all the symptomatic leaves was identical to the inoculation isolates in terms of morphology and just ITS analysis, but unsuccessful from the control plants. C. truncatum has also been reported to be the causal agent of anthracnose disease in multiple crops (Diao et al. 2014; Villafana et al. 2018; Stella de et al. 2021), thus, this is the first to report C. truncatum causing leaf spot on C. lansium in China. This study provides an important reference for the control of the disease due to the high host range of C. truncatum.

First report of Rhizoctonia solani AG-1 IA causing rice sheath blight of Oryza rufipogon in China.

Red rice (Oryza rufipogon Griff.) is a valuable source of important agronomic traits as well as genes for biotic and abiotic stress tolerance. In June 2020, rice sheath blight on O. rufipogon cv. Bin09 was observed in Zhanjiang (20.93N, 109.79E), China. Initial symptoms on sheaths were water-soaked and light green lesions. Then, the lesions gradually expanded into oval or cloud shaped lesions with a gray white center. The lesions coalesced, causing the entire sheath to become blighted. Disease incidence reached approximately 30% in the fields (10 ha) surveyed. Twenty sheaths 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), and incubated at 28°C in the dark for 4 days. Thirty-six pure cultures were obtained by transferring hyphal tips to new PDA plates, and three isolates (ORRS-1, ORRS-2, and ORRS-3) with similar morphological characteristics on PDA were selected as the representative isolates for study. Colony of isolate ORRS-1 was white initially, then turned brown with brown sclerotia. Septate hyphae were hyaline, smooth, and branched at right angles with a septum near the point of branching. Based on these morphological characteristics, the fungus was identified as Rhizoctonia solani Kuhn (Sneh et al. 1991). The isolates were deposited in the fungus collection of the Aquatic Organisms Museum of Guangdong Ocean University. For molecular identification, genomic DNA from each of the three isolates was extracted, and the internal transcribed spacer (ITS) region was amplified, and sequenced with the primer pair ITS5/ITS4 (White et al. 1990). The sequences were deposited in GenBank (accession nos. OP497977 to OP497979). The three isolates were 100% identical (716/716 bp; 716/716 bp; and 716/716 bp) with those of R. solani AG-1 IA (accession nos. KX674518, MK481078, and MK480532) through BLAST analysis. The phylogenetic tree grouped the three isolates within the R. solani AG-1 IA clade with high bootstrap support (99%) by the maximum likelihood method. A pathogenicity test was performed with these three isolates in a greenhouse at 24 to 30°C. Approximately 50 seedling of red rice cv. Bin09 were grown in each cup ( 250 ml in size with sterile soil 50 cm3). At the 3-leaf stage, plants in five cups were inoculated with each isolate by spraying a mycelial suspension (106 mycelial fragments/ml) until runoff. The mycelial suspension was prepared by adding sterile distilled water to the cultures and gently scraping the surface with a sterilized scalpel blade. Five plants sprayed with sterile water served as the controls. The test was conducted three times. Sheath blight was observed on the inoculated leaves after 15 days while no disease was observed in the control plants. Morphological characteristics and the ITS sequences of fungal isolates re-isolated from the diseased sheaths were identical to those of R. solani AG-1 IA. R. solani AG-1 IA is one of the most important plant pathogens worldwide, causing foliar diseases on maize, rice (O. sativa L.), and soybean (Joana et al. 2009). To our knowledge, this is the first report of R. solani AG-1 IA causing rice sheath blight of O. rufipogon in China (Farr and Rossman, 2022). With the spread of the pathogen on weedy populations of red rice, resistant races or pathotypes may evolve that could spread to cultivated rice.

First Report of Shoot Blight on Clausena lansium Caused By Fusarium proliferatum in China.

Wampee (Clausena lansium [Lour.] Skeels) is a tropical fruit. In July 2022, shoot rot symptom was observed in wampee (cv. JIXIN) in a field ((21°25'N, 110°10'E, about 100 ha ), Guangdong Province, China. The most obvious symptom of the disease was the rotting and withering of the tops. Disease incidence was approximately 90% (n = 500). Twenty diseased samples were randomly collected from the field and cut into 2 mm × 2 mm pieces next to the margins of diseased tissues. These pieces were then sterilized with 75% alcohol for 30 s and 2% sodium hypochlorite for 3 min and subsequently washed with sterile water three times. Tissue pieces were placed onto potato dextrose agar (PDA) and incubated at 25℃ for 3 days. Pure cultures were obtained by transferring hyphal tips to new PDA plates. Sixty isolates of Fusarium ssp. (60/80 = 75%) were obtained. Three representative single-spore isolates (CLFP-1, CLFP-2, and CLFP-3) were used for further study. Colonies were white to pink on PDA. Conidiogenous cells were monophialidic or polyphialidic. Macroconidia were slightly curved, tapering apically with 3 to 5 septa, and measured from 31.7 to 55.5 µm × 2.5 to 5.0 µm in size (n=50). The morphological features of these fungi were analogous to F. proliferatum (Leslie and Summerell 2006). For molecular identification, a colony PCR method (Lu et al. 2012) was used to amplify the internal transcribed spacer (ITS) and portions of elongation factor 1-α (EF1-α), RNA polymerase II largest subunit (RPB1), and RNA polymerase II second largest subunit (RPB2) genes using primers ITS1/ITS4, EF1-728F/EF1-986R, RPB1-R8/RPB1-F5, and RPB2-7CF/fRPB2-11aR, respectively (O'Donnell et al. 1998; 2010). The sequences were submitted to GenBank under accession numbers OP740961 to OP740963 (ITS), and OP800846 to OP800854 (RPB1, RPB2, EF1-α). The BLAST comparison of the sequences showed the three isolates were 100% similar to F. proliferatum (ITS: MT378328; TEF1: MH582344; RPB1: MN193921; RPB2: MN892349). The sequences of the three isolates were 100% identical (ITS, 537/537 bp; RPB1, 1606/1606 bp; RPB2, 770/770 bp and EF1-α, 683/683 bp) with those of F. proliferatum (accession nos. MT378328, MN193921, MH582196, and MH582344) through BLAST analysis. Analysis of the concatenated sequences revealed a 99.87 to 100% identity with the isolates of the F. proliferatum (F. fujikuroi species complex, Asian clade) by polyphasic identification using the FUSARIUM-ID database (Yilmaz et al. 2021). The sequences were also concatenated for phylogenetic analysis by the maximum likelihood method. The isolates clustered with F. proliferatum. Pathogenicity was tested through in vivo experiments. The inoculated and control plants (n = 5, 3 months old, cv. JIXIN) were sprayed with a spore suspension (1 × 105 per mL) of the three isolates and sterile distilled water, respectively, until run-off (Feng and Li. 2019). The test was performed three times. The plants were grown in pots in a greenhouse at 25℃ to 28℃, with relative humidity of approximately 80%. Symptoms were observed on the inoculated plants with disease incidence 100% after 2 weeks, while the control plants remained healthy. The pathogen re-isolated from all the inoculated plants was identical to the inoculated isolates in morphology and ITS sequences. No pathogen were isolated from the control plants. To the best of our knowledge, this study is the first to report F. proliferatum causing shoot blight symptom in wampee (cv. JIXIN). This disease has caused severe losses and will provide the foundation for management strategies.

First Report of Pseudocercospora paraguayensis Causing Leaf Spots on Bixa orellana from Chinese Mainland.

Bixa orellana L. is a traditional Chinese medicine. In December 2019, a leaf spot disease was observed on B. orellana from a field in Zhanjiang (21°18'12''N, 110°17'22''E), China. Disease incidence was around 85% (n = 100 investigated plants from about 30 hectares). Initial leaf spots were circular, and the center of the lesions was grayish-white with a purple black border. The coalescence of individual spots 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, and the surfaces were disinfected with 75% ethanol for 30 sec, and 2% sodium hypochlorite for 60 sec. The the samples were then rinsed three times in sterile water, plated on potato dextrose agar (PDA), and incubated at 28 °C. Pure cultures were obtained by transferring the hyphal tips to new PDA plates. Three representative isolates (BOPP-1, BOPP-2, and BOPP-3) were used for further study. The colonies of isolates on PDA were dark olive green with off-white aerial mycelia after 7 days at 28 °C. Conidia were solitary, smooth to verrucous, olive to light brown, slightly curved, narrowly obclavate, apex obtuse, base obconic-truncate, had 2-4 septa, and 30.4-55.5×2.0-3.5 µm in size.. These morphological characteristics showed did not differ from the description of Pseudocercospora paraguayensis (Crous et al. 1997). For molecular identification, the internal transcribed spacer (ITS) region, the translation elongation factor 1-α (TEF1) gene, and actin (ACT) gene were amplified using primer pairs ITS1/ITS4 (White et al., 1990), EF1/EF2 ( O'Donnell et al. 1998), and ACT-512F/ACT-783R (Carbone and Kohn, 1999) and sequenced from DNA extracted from the three isolates, respectively. Sequences were deposited in GenBank under accession no. MZ363823-MZ363825 (ITS), MZ614954-MZ614956 (TEF1), and MZ614951-MZ614953 (ACT). A phylogenetic tree was generated on the basis of the concatenated data from the sequences of ITS, TEF1, and ACT that the three isolates were nested within the clade containing the type specimen of P. paraguayensis (CBS 111286) but not within P. bixae (the type specimen CPC 25244). Pathogenicity was tested through in vivo experiments. Inoculation and control seedlings (n = 5, 1-month-old) were sprayed with a spore suspension (1 × 105 per ml) of P. paraguayensis and sterile distilled water (control), respectively, until run-off (Fang. 1998). The plants were grown in pots in a greenhouse at 28°C, with at approximately 80% RH. The test was performed three times. Symptoms similar to those in the field were observed on the inoculated plants after two weeks. The control plants remained healthy. The fungus was re-isolated from the infected leaves and confirmed as the same isolates by morphological and comparison of ITS sequences with 100% identical to those of isolates. No original fungi were isolated from the control plants. A previous study reported that P. paraguayensis caused leaf spots on pistachio and eucalypts, and the fungus causing the leaf spots of B. orellana was redescribed as P. bixae (Crous et al. 2019). However, multilocus phylogenetic analyses differentiated P. paraguayensis from P. bixae. In the present study, P. paraguayensis was distinguished from P. bixae due to the absence of catenulate conidia and the presence of finely verruculose conidia (Crous et al. 2013). P. eucalypti as a synonyms was reported in Taiwan (www.MycoBank.org). The current study is the first to report P. paraguayensis causing leaf spots on B. orellana from Chinese Mainland. This finding will help to provide a scientific basis for the disease detection.

First Report of Pseudocercospora oenotherae Causing Leaf spot in Hymenocallis littoralis in China.

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 Oenothera biennis L. (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.

First Report of Mucor irregularis Causing Flower Rot on Selenicereus undatus in China.

Dragon fruit (Selenicereus undatus (Haw.) D.R.Hunt is a famous tropical fruit (Korotkova et al. 2017). In May 2021, a flower rot disease was found on Dragon fruit in a field (21˚19'42''N, 110˚28'32''E), Zhanjiang, Guangdong Province, China. The incidence rate was approximately 30% (n=500 investigated plants from about 30 hectares). Flower rot was evident, and was light brown, watery, soft, and covered with white mycelia. The pathogen could continue to infect the fruit during the fruit ripening stage with about 20% rot rate. Ten samples of symptomatic flowers were collected in the field. Margins of the diseased tissue 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. Pure cultures were obtained by transferring hyphal tips to new PDA plates. Three representative isolates (HUM-1,HUM-2, and HUM-3) by single-spore isolation were randomly selected for further study. Colonies on PDA were circular with massive aerial hyphae, white to ochraceous in color. Nonseptate hyphae were hyaline. Sporangiophores arose from hyphae. Sporangiospores were hyaline, smooth-walled, mostly subspherical to ellipsoidal, and measured 3.15 to 6.55 µm × 1.35 to 2.85 µm (n =50). Morphological characteristics of isolates were consistent with the description of Mucor irregularis (Lima et al. 2018). Molecular identification was done using the colony PCR method with MightyAmp DNA Polymerase (Takara-Bio, Dalian, China) (Lu et al. 2012) used to amplify the internal transcribed spacer (ITS) region and large subunit (LSU) with ITS1/ITS4 and LR0R1/LR5 (Vilgalys et al. 1990). The amplicons were sequenced and the sequences were deposited in GenBank with accession numbers ITS, OL376751-OL376753, and LSU, OM672239-OM672241. BLAST analysis of these sequences revealed a 100% identity with M. irregularis in GenBank. The sequences were also concatenated for phylogenetic analysis by the maximum likelihood method. The isolates clustered with M. irregularis (the type strain CBS 103.93).The pathogenicity was tested through in vivo experiments. Nine healthy flowers of Dragon fruit were inoculated with 3-day-old mycelial plugs (5 × 5 mm) of isolates, while another five healthy flowers were treated with PDA plugs (controls). Those plugs were embedded inside the calyxes, and each flower was inoculated with one plug in one calyx. Besides, the inoculated and control flowers (n = 5) were sprayed with a spore suspension (1 × 105 per mL) of the three isolates individually and sterile distilled water, respectively, until run-off (Feng and Li. 2019). The plants were grown in pots in a greenhouse at 28°C, with relative humility approximately 80%. The test was repeated three times. After 3 days of incubation, rot symptoms developed on the inoculated flowers, which were similar to those observed on the naturally samples in the field. The control flowers remained healthy. The fungus was reisolated from the inoculated flowers and confirmed as M. irregularis by morphology and ITS analysis. M. irregularis was reported as a pathogen causing human skin diseases and post-harvest diseases of crop (Álvarez et al. 2011; Lima et al. 2018; Wang et al. 2022). This is the first report of M. irregularis causing flower rot of Dragon fruit and reduce yield in China. This research can provide a theoretical basis for the fruit industry to maintain yield.

First report of Alternaria alternata causing leaf yellow spot on Heteropanax fragrans in China.

Heteropanax fragrans (Roxb.) Seem is a common garden landscape tree in China. In December 2020, a leaf disease on H. fragrans was observed in a 2 ha field in Zhanjiang (20.85° N, 109.28° E), Guangdong province, China. Early symptoms were small yellow spots on leaves. Later, the spots gradually expanded and turned into necrotic tissues with a clear yellow halo and a white center. The disease incidence on plants was 100%. 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 and 60 s, respectively, and rinsed thrice with sterile water before isolation. The tissues were plated onto potato dextrose agar (PDA) medium and incubated at 28 ℃. After 2-day incubation, grayish fungal colonies appeared on the PDA, then pure cultures were produced by transferring hyphal tips to new PDA plates. Single-spore isolation method was used to recover pure cultures for three isolates (HFA-1, HFA-2, and HFA-3). The colonies first produced a light-grayish aerial mycelia, which turned dark grayish upon maturity. Conidiophores were branched. Conidia numbered from two to four in chains, were dark brown, ovoid, or ellipsoid and mostly beakless; had 1-4 transverse and 0-3 longitudinal septa; measured within 7.2-17.8 (average = 10.2) × 2.5-7.5 (average = 4.3) µm (n = 30). Molecular identification was performed using the colony polymerase chain reaction method with MightyAmp DNA Polymerase (Takara-Bio, Dalian, China) (Lu et al. 2012) to amplify the large subunit (LSU), internal transcribed spacer (ITS) region, translation elongation factor (TEF) , and Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) with NL1/LR3, ITS1/ITS4, EF-1α-F/EF-1α-R, and GDF1/GDR1 (Walther et al. 2013;Woudenberg et al. 2015; Nishikawa and Nakashima. 2020). Amplicons of the isolates were sequenced and submitted to GenBank (LSU, ON088978-ON088980; ITS, MW629797, ON417005 and ON417006; TEF, MW654167, ON497264,and ON497265;GAPDH, MW654166, ON497262,and ON497263). The obtained sequences were 100% identical with those of Alternaria alternata strain CBS 102600 upon BLAST analysis . The sequences were also concatenated for phylogenetic analysis by maximum likelihood. The isolates clustered with A. alternata (CBS 102600, CBS 102598, CBS 118814, CBS 918.96,CBS 106.24, CBS 119543, CBS 916.96). The fungus associated with leaf yellow spot on H. fragrans was thus identified as A. alternata. Pathogenicity tests were conducted in a greenhouse at 24 â„ƒ-30 â„ƒ with 80% relative humidity. Individual plants were grown in pots (n = 5, 1 month old). The unwounded leaflets were inoculated with 5 mm-diameter mycelial plugs of the isolates or agar plugs (as control). The test was performed thrice. Disease symptoms were found on the leaves after 7 days, whereas the controls remained healthy. The pathogen was re-isolated from infected leaves and phenotypically identical to the original isolates to fulfill Koch's postulates. To our knowledge, this report is the first one on A. alternata causing leaf yellow spot on H. fragrans. Thus, this work provides an important reference for the control of this disease in the future.

First report of Neopestalotiopsis clavispora Causing Leaf Spots on Garcinia mangostana in China.

Garcinia mangostana L. is a famous tropical fruit in Asia. In April 2021, a leaf disease on G. mangostana cv. Huazhu was observed in Zhanjiang (21.17° N, 110.18° E), Guangdong province, China. Symptoms was on new leaves of 2 year old plants. The spots were circular to irregular, gray in the center, and brown on the lesion margin. The disease incidence was estimated 25% (n = 500 investigated plants from about 50-ha). Twenty diseased leaves were collected from the orchard. 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 and 60 s, respectively; and rinsed thrice with sterile water. The tissues were plated onto potato dextrose agar (PDA) medium and incubated at 28 ℃. Twenty-eight isolates were obtained (isolation frequency = 28/4×20 = 35%). Single-spore isolation method was used to recover pure cultures for three isolates (GMN-1, GMN-2, and GMN-3) (Liu et al. 2021). The colonies were initially white with cottony aerial mycelium at 7 days on PDA. Then, they developed black acervular conidiomata at 10 days. Conidia were clavate to fusiform, four-septate, straight or slightly curved, and measured 16.5 to 21.4 µm long (average 19.5 µm; n = 40) × 4.5 to 6.5 µm wide (average 5.2 µm; n = 40). The three median cells were versicolored, whereas the basal and apical cells were hyaline. Conidia had a single basal appendage (4.5 to 5.5 µm long; n = 40) and three apical appendages (19.2 to 24.5 µm long; n = 40). The morphological characteristics of the isolates are comparable with those of the genus Neopestalotiopsis (Sajeewa et al. 2012). Molecular identification was performed using the colony polymerase chain reaction method with MightyAmp DNA Polymerase (Takara-Bio, Dalian, China) (Lu et al. 2012). Sequences were generated from the isolates using primers for the rDNA ITS (ITS1/ITS4), TEF1-α (EF1-728F/EF1-986R), and ß-tubulin (T1/ßt2b) loci (Sajeewa et al. 2012). The sequences of the isolates were submitted to GenBank (ITS, MZ026535-MZ026537; TEF, MZ032203-MZ032205; ß-tubulin, MZ032206-MZ032208). The sequences of the isolates were 100% identical to the type strain MFLUCC12-0281 (accession nos. JX398979, JX399014, and JX399045) through BLAST analysis. The isolates clustered with N. clavispora (MFLUCC12-0280 and MFLUCC12-0281). The pathogenicity was tested in vivo. Individual plants (cv. Huazhu) were grown (n = 2, 1-1.5 year old) in a greenhouse at 24 â„ƒ-30 â„ƒ with 80% relative humidity. Wounded leaflets were inoculated with 5-mm-diameter mycelial plugs or agar plugs (as control). Besides, sterile cotton balls were immersed in the spore suspension (1 × 105 per mL) and sterile distilled water (control) for about 15 s before they were fixed on the leaves for 3 days. One plant employed for each isolate with nine leaves. The test was performed thrice. Disease symptoms were found on the leaflets after 10 days, whereas the controls remained healthy. The pathogen was re-isolated from infected leaves and phenotypically identical to the original isolates to fulfill Koch's postulates. Neopestalotiopsis clavispora and Pestalotiopsis clavispora are synonyms. The fungus appeared to have a wide host range and distribution including in Thailand, Malaysia, North Queensland, and Australia (Sajeewa et al. 2012;Shahriar et al. 2022). Thus, this is the first report of N. clavispora causing leaf spot on G. mangostana in China. This finding will help improve management strategies against the leaf spots on G. mangostana in China.

Pseudocercospora rhododendricola Causing Leaf dark Spot on Rhododendron pulchrum by the first phylogenetic analyses.

Rhododendron pulchrum Sweet is a famous ornamental flower in China. In December 2020, a leaf spot disease was observed on cv. Maojuan in Zhanjiang (21.17 N, 110.18 E), Guangdong, China. The spots were irregular and distributed on both sides of the main vein. They were dark to black, and their borders were obvious. The coalescence of the spots eventually led to leaf wilt. The disease incidence was 100% (n = 100, about 50 ha ). Thirty infected leaves were collected from the field, and the margin of the diseased tissues was cut into 2 mm × 2 mm pieces. Samples were surface disinfected with 75% ethanol and 2% sodium hypochlorite for 30 and 60 s, respectively. They were rinsed thrice with sterile water before isolation. The tissues were plated on potato dextrose agar (PDA) medium and incubated at 28 ℃. After 5 days, fungal colonies appeared on the PDA. Pure cultures were produced by transferring hyphal tips to new PDA plates. Three isolates (RSP-1, RSP-2, and RSP-3) were obtained and the colonies of isolates were preserved in glycerol (15%) at -80 °C deposited at the Museum of Guangdong Ocean University. The morphology of these three isolates was consistent, and their sequences showed 100% homology according to ITS, TEF1, and ACT analysis results. The colonies grew to approximately 5 cm in diameter after 10 days. They showed olive green with off-white aerial mycelia. Stromata and conidia were observed on leaf lesions. Stromata were olivaceous brown. Conidia were solitary, cylindrical to narrowly obclavate, mildly curved, obtuse to rounded at the apex, and 1- to 3-septate; they had dimensions of 20 to 60 × 2.0 to 3.0 µm (n = 30). These morphological characteristics were not different from the description of Pseudocercospora rhododendricola (J.M. Yen) Deighton (Liu et al. 1998). For molecular identification, the colony PCR method with MightyAmp DNA Polymerase (Takara-Bio, Dalian, China) (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 ITS4/ITS5, EF1/EF2, and ACT-512F/ACT-783R, respectively (White et al., 1990; O'Donnell et al. 1997). The sequences of the isolate RSP-1 were deposited in the GenBank (ITS, MW629798; TEF1, MW654168; and ACT, MW654170). BLAST analysis showed that the sequences of P. rhododendricola were submitted to GenBank for the first time by the author of this paper. A phylogenetic tree was generated based on the concatenated data of ITS, TEF1, and ACT sequences from GenBank by the Maximum Likelihood method. The isolates were closest to Pseudocercospora sp. CPC 14711 (Crous et al., 2013). Phylogenetic and morphological analyses identified the isolates as P. rhododendricola. Pathogenicity tests were conducted in a greenhouse at 24 °C-30 â„ƒ with 80% relative humidity. Healthy cv. Maojuan were grown in pots. Unwounded leaflets were inoculated with 5 mm-diameter mycelial plugs of the isolates or agar plugs (as control) (5 leaflets per plant, 3 plants, 2-month-old plants). The test was performed thrice. Disease symptoms were found on the leaves after 2 weeks, 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. P. rhododendricola was the cause of leaf spot of Rhododendron sp. from Singapore (Liu et al., 1998). For the first time, this pathogen was identified by combining phylogenetic and morphological analyses. The sequences in this study would be used as the reference sequences for further studies.

First Report of Fusarium proliferatum Associated with Leaf Yellow on Alternanthera philoxeroides in China.

Alternanthera philoxeroides (Mart.) Griseb is a highly invasive weed commonly found in rice fields in China. In May 2021, leaf yellowing was observed on this weed (about 10 ha) in Zhanjiang (21°19'N, 110°20'E), Guangdong Province, China. Disease incidence was approximately 20% (n = 100 investigated plants). Ten yellow leaves from 10 plants were sampled, surface-sterilized with 75% ethanol for 30 s, followed by 2% NaClO for 5 min. The leaves were rinsed three times in sterile distilled water and four sections of each leaf were placed onto potato dextrose agar (PDA). Pure cultures were obtained by transferring hyphal tips to new PDA plates. Twenty-two isolates of Fusarium ssp. (69% of the isolates) were obtained from 55% of the leaf samples. Three representative single-spore isolates (APF-1, APF-2, and APF-3) were used for further study. Colonies were white to pink on PDA. Conidiogenous cells were monophialidic or polyphialidic. Macroconidia were slightly curved, tapering apically with three to five septa, and measured from 32.5-55.8 µm × 2.5-5.1 µm in size (n=50). The morphological features of these fungi were noted to be in line with those of Fusarium proliferatum (Leslie and Summerell, 2006). For molecular identification, a colony PCR method (Lu et al. 2012) was used to amplify the internal transcribed spacer (ITS) and portions of elongation factor 1-α (EF1-α), RNA polymerase II largest subunit (RPB1), and RNA polymerase II second largest subunit (RPB2) genes using primers ITS1/ITS4, EF1-728F/EF1-986R, RPB1-R8/RPB1-F5, and RPB2-7CF/fRPB2-11aR, respectively (O'Donnell et al. 1998; O'Donnell et al. 2010). The sequences were submitted to GenBank under accession numbers MZ026797-MZ026799 (ITS) and MZ032209-MZ032217 (RPB1, RPB2, EF1-α). The sequences of the three isolates were 100% identical (ITS, 537/537 bp; RPB1, 1606/1606 bp; RPB2, 770/770 bp and EF1-α, 683/683 bp) with those of F. proliferatum (accession nos. MT378328, MN193921, MH582196, and MH582344) through BLAST analysis. Analysis of the sequences revealed a 99.87 - 100% identity with the isolates of the F. proliferatum (F. fujikuroi species complex, Asian clade) by polyphasic identification using the FUSARIUM-ID database (Yilmaz et al. 2021). The sequences were also concatenated for phylogenetic analysis by the maximum likelihood method. The isolates clustered with F. proliferatum. Pathogenicity was tested through in vivo experiments. The inoculated and control plants (n = 5, 30 days old) were sprayed with a spore suspension (1 × 105 per mL) of the three isolates individually and sterile distilled water, respectively, until run-off (Feng and Li. 2019). The test was performed three times. The plants were grown in pots in a greenhouse at 25 °C to 28 °C, with relative humidity of approximately 80%. Yellowing was observed on the inoculated plants after 7 days, while the control plants remained healthy. The pathogen re-isolated from all the inoculated plants was identical to the inoculated isolates in terms of morphology and ITS sequences. No fungi were isolated from the control plants. To the best of our knowledge, this study is the first to report F. proliferatum causing yellow symptoms on A. philoxeroides. The fungus has some potential biological control properties, but its host range needs to be further determined.

First Report of Seedling Rot of Castor (Ricinus communis) caused by Choanephora cucurbitarum in China.

Castor (Ricinus communis L.) oil is used in the manufacture of cosmetics, lubricants, plastics, pharmaceuticals, and soaps and is grown in more than 40 countries with India and China leading in oil production(Tunaru et al. 2012). In June 2021, a seedling rot disease was observed on castor cv. Zibi-5 in a plant nursery in Zhanjiang (21°17' N, 110°18' E), China. Initial symptoms on leaves and stems were water-soaked and dark green lesions that resulted in rapid rotting. Disease incidence was 25% and resulted in seedling death. White fungal mycelia developed on the rotting plant tissues. Leaves and stems were collected from 10 diseased plants, surface disinfected in 0.5% sodium hypochlorite and 75% ethyl alcohol, and tissue pieces placed in plates of potato dextrose agar (PDA) which were maintained at 28℃. Hyphal tips from fungal mycelia that developed in the PDA plates were selected to establish pure cultures and three representative fungal isolates, designated RCC-1, RCC-2, and RCC-3, were selected for further study. The fungal isolates produced sporangiophores that were smooth, hyaline, aseptate, and apically swollen. Sporangiophores bore monosporous sporangiola that were broadly ellipsoidal, longitudinally coarsely striate, brown to dark brown, and measured 6.2 to 14.8 x 10.5 to 26.5 um (n=30). Sporangia contained few to many spores that were spherical, brown, and measured 59 to 150 um in diameter (n=20). Sporangiospores were ellipsoid, striate, and brown with multiple hyaline polar appendages and measured 6.6 to 12.3 x 10.6 to 25.5 um (n=30) in size. Based on these morphological characteristics, the fungus was identified as Choanephora cucurbitarum (Berk. & Ravenel) Thaxt. (Kirk, 1984). Molecular identification was done using the colony PCR method with MightyAmp DNA Polymerase (Takara-Bio, Dalian, China) (Lu et al. 2012) used to amplify the internal transcribed spacer (ITS) region and large subunit (LSU) with ITS1/ITS4 and NL1/LR3 (Walther et al. 2013). The amplicons were sequenced and the sequences were deposited in GenBank with accession numbers ITS, OL376748-OL376750, and LSU, OL763430-OL763432. BLAST analysis of these sequences revealed a 100% to 99% identity with the sequences (ITS, MG650194; 573/573, 573/573, and 573/573; LSU, AF157181; 673/676, 673/676, and 673/676) of C. cucurbitarum in GenBank. Pathogenicity tests, to fulfill Koch's postulates, were performed in a greenhouse with a temperature range of 24℃ to 30℃ and 80% relative humidity. Thirty-day-old cv. Zibi-5 castor plants were grown in pots and used for inoculation tests. Ten plants were inoculated by placing agar plugs with mycelia of fungal isolate RCC-1 on leaves or stems. Ten control plants were inoculated with agar plugs only and the test was repeated three times in total. Five days after inoculation, all plants, with either leaf or stem inoculations, became infected and began rotting. Symptom progression was consistent with that observed in the nursery and all control plants remained healthy. C. cucurbitarum was successfully reisolated from all inoculated plants and identified by morphological characteristics and by sequence analysis. This fungus is known to cause serious damage on a wide range of hosts (Liu et al. 2019) and previously was reported on castor in India (Shaw 1984) and Papua New Guinea (Peregrin and Ahmad 1982). We observed that the pathogen grows very rapidly and causes serious damage to castor seedlings, warranting further investigation on the epidemiology and control of this disease.

First Report of Pyricularia oryzae Causing Blast on Wild Rice (Oryza rufipogon) in China.

Wild rice (Oryza rufipogon) is an excellent genetic resource for rice breeding programs. In June 2019, typical symptoms of blast on the leaves of wild rice cv. 'Haihong-12' were observed in a 3.3-ha field in Zhanjiang (20.93° N, 109.79° E), China. The symptoms included fusiform lesions with yellowish halo at the age of lesion, grayish-white color at the center, brown and elongated central veins at both ends of lesion, and grayish-white mold layer formed on the back of lesion under humid weather conditions. Disease incidence was more than 10%. Thirty diseased leaves were collected, and infected tissues were cut into 2 × 2 mm pieces, surface disinfected with 75% ethanol for 30 s and 2% sodium hypochlorite for 60 s and rinsed three times with sterile water. The tissues were plated onto potato dextrose agar (PDA) medium and incubated at 28 °C in darkness for 3 days. Three single-spore isolates (Pos-1, Pos-2, and Pos-3) were obtained using the method described by Jia (Jia 2009) and were subjected to further morphological and molecular identification. Colonies on PDA were light grey, with cottony mycelium. Conidiophores were solitary, erect, straight or curved, septate, and pale brown and measured 68 to 125 × 3 to 4 µm. Conidiogenous cells were sympodial and denticulate. Conidia were pale brown, pyriform, and 18.2 to 42.4 × 5.1 to 8.5 µm (n = 30) in size, with two septa. Appressoria were spherical and had the size ranging 4.3 to 6.5 × 4.7 to 6.5 µm (n = 20). These morphological features agreed with the previous description of Pyricularia oryzae Cavara (Klaubauf et al. 2014). For molecular identification, the colony PCR method with MightyAmp DNA Polymerase (Lu et al. 2012) was used to amplify the internal transcribed spacer (ITS), calmodulin (CAL), actin (ACT), -tubulin (TUB) loci of the isolates using primer pairs ITS1/ITS4, CL1C/CL2C, ACT-512F/ACT-783R, and T1/ßt2b, respectively (O'Donnell et al. 1997; Weir et al. 2012; White et al. 1990). Analysis of ITS (acc. nos. MW042176 to MW042178), ACT (MW091444 to MW091446), CAL (MW091447 to MW091449), and TUB (MW091441 to MW091443) sequences revealed 100% identity with the corresponding ITS (MH859782), ACT (MH589787), CAL (MH589663), and TUB (MH589547) sequences of P. oryzae in GenBank. A phylogenetic tree was generated based on the ITS sequences using maximum likelihood method that clustered Isolates Pos-1, Pos-2, and Pos-3 with known P. oryzae. Thus, the isolates were identified as P. oryzae. Pathogenicity tests were performed using Isolate Pos-1 in a greenhouse at 24 to 30 °C with 80% relative humidity. Individual rice plants (cv. 'Haihong-12') with three leaves were grown in 10 pots, with 50 plants per pot (40 × 60 cm). Five pots were spray inoculated with a spore suspension (105 spores/ml) until runoff from leaves, and the remaining five pots were sprayed with sterile water to serve as the controls. The test was conducted three times. Disease symptoms were observed on 10% of leaves at 10 days after inoculation, but the control plants remained healthy. The fungus was re-isolated from the diseased plants and morphologically identified as P. oryzae. Thus, this is the first report of P. oryzae causing blast on O. rufipogon in China. The results provide the information that can be used by rice breeders and fungal geneticists for further studies.

First Report of Colletotrichum siamense Causing Anthracnose in Osmanthus fragrans in China.

Osmanthus fragrans Lin. is widely cultivated in China. Its flower is precious spices. It is also a garden ornamental plant. In March 2021, anthracnose-type lesions were observed on the leaves of O. fragrans in a public garden in Zhanjiang, Guangdong Province, China (21˚17'47''N, 110˚18'58''E). Disease incidence was around 50% (n = 100 investigated plants from about 30 hectares). The early symptoms were yellow spots on the edge or tip of the leaves. The spots gradually expanded and became dark brown, eventually coalescing into large irregular or circular lesions. 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 sec and 2% sodium hypochlorite for 60 sec . Thereafter, the samples were rinsed thrice in sterile water, placed on PDA, and incubated at 28 ℃. Pure cultures were obtained by transferring hyphal tips to new PDA plates. Thirty-two isolates of Colletotrichum ssp. were obtained (isolation frequency = 32/4×10 = 80%). Three representative single-spore isolates (OFC-1, OFC-2, and OFC-3) were used for further study. Colonies on PDA were white to gray with cottony mycelia in 6 days at 28 ℃. Conidia were one-celled, hyaline, cylindrical, clavate, and obtuse at both ends; they measured 10.5 to 17.5 µm × 3.5 to 5.0 µm (n = 50). Appressoria were oval to irregular in shape and dark brown in color, and they measured 6.5 to 8.5 µm × 4.5 to 7.5 µm (n = 20). Morphological characteristics matched the description of Colletotrichum siamense (Prihastuti et al. 2009; Sharma et al. 2013). For molecular identification, the colony PCR method (Lu et al., 2012) was used to amplify the internal transcribed spacer (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), chitin synthase (CHS-1), and actin (ACT) loci of the isolates using primer pairs ITS1/ITS4, GDF1/GDR1, CHS-79F/CHS-354R, and ACT-512F/ACT-783R, respectively (Weir et al. 2012). Sequences of them deposited in GenBank under nos. MZ047368-MZ047370 (ITS), MZ126925-MZ126927 (GAPDH), MZ126895-MZ126897 (CHS-1), and MZ126835-MZ126837 (ACT). A phylogenetic tree was generated on the basis of the concatenated data from sequences of ITS, GAPDH, CHS-1, and ACT that clustered the isolates with C. siamense (the type strain MFLU 090230), while distanced the isolates with C. gloeosporioides (the type strain CBS 112999). The pathogenicity was tested through in vivo experiments. In group 1, the inoculation and control plants (n = 5, 3-month-old) were sprayed with a spore suspension (1 × 105 per mL) of the isolates and sterile distilled water, respectively, until run-off. In group 2, the unwounded leaflets were inoculated with mycelial plugs of the isolates or agar plugs (as control). Three plugs were for each leaflet ( n = 5). The plants were grown in pots in a greenhouse at 25°C to 28°C, with relative humidities approximately 80%. Anthracnose lesions were observed on the inoculated leaves after 10 days while the control plants remained healthy. The pathogen re-isolated from all the inoculated leaves was identical to the inoculation isolates in terms of morphology and just ITS analysis, but unsuccessful from the control plants. C. gloeosporioides has been reported to cause leaf spot on O. fragrans in Jiangxi Province of China (Tanget al., 2018), but not by C. siamense. To the best of our knowledge, this study is the first to report C. siamense causing anthracnose on O. fragrans. Thus, this work provides a foundation for controlling anthracnose in O. fragrans in the future.

First Report of Phoma herbarum Causing Leaf Spot on Rhapis humilis in China.

Rhapis humilis Blume is an ornamental plant for landscaping that is widely distributed in China. In February 2020, a leaf spot disease was observed on R. humilis in a nursery shed in Zhanjiang (21.17 N, 110.18 E), Guangdong, China. The disease incidence was more than 90%. The early symptom was small water-soaked lesions, which then turned into black necrotic spots. Eventually, the individual lesions coalesced into larger ones, leading to the death of diseased leaves. Ten diseased leaves were collected from the nursery. The diseased tissues were cut into 2 × 2 mm 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 pathogen 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. Three isolates (RHPH-1, RHPH-2, and RHPH-3) were obtained. The colonies of the isolates were approximately 5 cm in diameter after 7 days. They were initially whitish and later became grayish white. The NaOH testing on MEA cultures was negative. No sporulation was detected after 30 days. The fertile structures of the specimens collected in the nursery were examined. Pycnidia were globose, measured 68 to 265 × 72 to 360 µm (n = 20), and mostly embedded. Conidia were aseptate, hyaline, and ellipsoid, measuring 3.6 to 6.5 × 2.2 to 2.7 µm (n = 30). Based on the morphological characteristics, the fungus was identified as in genus Phoma (Boerema et al. 2004). For molecular identification, the colony PCR method with MightyAmp DNA Polymerase (Takara-Bio, Dalian, China) (Lu et al. 2012) was used to amplify the internal transcribed spacer (ITS), partial RNA polymerase II largest subunit (RPB2), and beta-tubulin (ß-tub) loci of three isolates using primer pairs ITS4/ITS5, RPB2-6F/RPB2-7R, and BT2a/BT2b, respectively (Chen et al, 2015; White et al, 1990). The sequences were deposited in GenBank (ITS, MZ419364-MZ419366; RPB2, MZ562293-MZ562295; and ß-tub, MZ562296-MZ562298). Based on BLAST analysis, the sequences of the ITS, RPB2, and ß-tub all showed 100% similarity to Phoma herbarum Westend. (CBS 377.92, accession nos. KT389536 for ITS; KT389663 for RPB2; and KT389837 for ß-tub). Pathogenicity testing was performed in a greenhouse with 80% relative humidity at 25 to 30°C. Ten healthy plants of R. humilis were grown in pots, with one plant in each pot. The leaves were pinpricked with sterile needles before inoculation. They were inoculated with mycelial plugs of the isolates or sterile agar plugs (as control), with four plugs for each leaf. Five plants were used in each treatment. Disease symptoms similar to those in the nursery were observed on the inoculated plants 2 weeks after inoculation, whereas the control plants remained healthy. The fungus was reisolated from the symptomatic leaves and confirmed as P. herbarum by morphology and ITS analysis. P. herbarum was reported to cause leaf spot on Atractylodes lancea, Camellia sinensis, Elaeis guineensis, Lilium brownii, and Vetiveria zizanioides in China; Bituminaria bituminosa, Glycine max, Medicago sativa, and Pisum sativum in Australia; and Salvia nemorosa in Italy (Li et al. 2011; Li et al. 2012; Thangaraj et al. 2018). To our knowledge, the present study was the first to report P. herbarum causing leaf spot on R. humilis in China. P. herbarum seriously affects the supply of seedlings in R. humilis, and its epidemiology on R. humilis should be further studied.

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

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.

First Report of Podosphaera xanthii Causing Powdery Mildew on Melothria indica in China.

Melothria indica Lour. is a wild ornamental plant widely distributed in South China. In November 2020, powdery mildew symptoms with 100% (60 plants) incidence were observed on M. indica climbing on a fence in Zhanjiang (21.17N,110.18E), Guangdong, China. The symptoms were typical for powdery mildew with white colonies on leaf surfaces and stems. Conidiophores appeared in all symptomatic tissues. Chasmothecia were observed only during the late stage of disease. Hyphae were hyaline, branched, and septate. Conidiophores were erect, hyaline, smooth, and had a dimension of 61.5 to 185.6 µm × 8.5 to 14.5 µm (n=20) and a cylindrical, flexuous foot cell, followed by 1 to 5 (-6) shorter cells. Conidia were ellipsoid to ovoid and had a dimension of 24.5 to 38.5 µm×15.5 to 21.8 µm (n=50) with well-developed fibrosin bodies. Germ tubes were in the lateral position. Chasmothecia were gregarious or scattered, subglobose, (64.8-) 65.5 µm to 115.5 (-120.5) µm (n=20) in diameter. The appendages were few, and hyphoid. Ascus one per ascomas, clavate, or subglobose, 56.5 to 78.3 (-90) µm×52.5 to 60.5 (-72) (n=20) µm. Each ascus had eight ascospores that were broadly ellipsoid and sized 13.8 to 18.6 µm×12.5 to 16.5 µm (n=30). The morphological characteristics were consistent with the previous description of Podosphaera xanthii (Castagne) U. Braun & Shishkoff (Braun and Cook 2012). Three voucher specimens, Ms-1, Ms-2, and Ms-3, were deposited in the fungus collection at Aquatic Organisms Museum of Guangdong Ocean University, and were used for molecular analysis. Their internal transcribed spacer (ITS) regions were amplified using primers ITS1/ITS4. Amplicons were sequenced and submitted to GenBank (accession no. MW512919, MW512920, and MW512921). The sequences were identical to each other and 100% similar to two of P. xanthii (Accession No. MT472035 and MN818563). On the basis of the morphological and molecular characteristics, the fungus was identified as P. xanthii. Pathogenicity was examined through inoculation by gently pressing the naturally infected leaves onto healthy ones of three potted M. indica plants with three leaves. Healthy leaves were leaves of three further plants which served as the control. White powdery mildew colonies with an incidence of 100% were similarly observed after 7 days at 28 °C and 80% relative humidity in a greenhouse. The fungal colonies on diseased leaves were morphologically identical to the specimen, and the control plants developed no symptoms. The Koch's postulates have completed. Golovinomyces cichoracearum is known to cause powdery mildew on M. indica in China (Liu et al. 2015). P. xanthii (synonym:P. fusca p.p.) is the cause of powdery mildew on cucurbits worldwide (Braun and Cook 2000), including M. indica (synonym:M. japonica) in Korea (Kwon et al. 2015) and Japan (Takamatsu et al. 2005), but hitherto not for China. While, the teleomorph of the fungus on cucurbits is seldom found worldwide and in China only in the north (Liu et al. 2011), chasmothecia are recorded for here southern China (21.17N,110.18E).

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 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.

First Report on Colletotrichum siamense Causing Anthracnose of Castor Bean in Zhanjiang, China.

Castor bean (Ricinus communis L.) is an important oil crop. Anthracnose lesions were observed on leaves of castor bean at the stage of budding and fruiting in field (21˚17'51''N, 110˚18'16''E), Zhanjiang, Guangdong Province, China in August 2019. The incidence rate was approximately 40% (n=600 investigated plants). Early symptoms were yellow spots appearing from the edge or the tip of the leaves. Later, the spots gradually expanded and became dark brown, which coalesced into larger irregular or circular lesions (Supplementary Figure 1). Seven diseased leaves were collected from seven plants. Margins of the diseased tissue were cut into 2 mm × 2 mm pieces. The surfaces were disinfested 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 PDA, and incubated at 28 °C. Pure cultures were obtained by transferring hyphal tips to new PDA plates. A single-spore isolate (RLC-1) was used for further study. The colony of isolate RLC-1 on PDA was white to gray in color with cottony mycelia in 6 days at 28 °C. Conidia were one-celled, hyaline, cylindrical, clavate, obtuse at both ends and measured 14.2 to 18.5 µm × 3.8 to 5.5 µm (n =50). Appressoria were oval to irregular in shape, dark brown, and ranged from 7.3 to 10.5 µm × 5.7 to 6.5 µm (n = 20). Morphological characteristics of isolate RLC-1 were consistent with the description of Colletotrichum siamense (Prihastuti et al. 2009; Sharma et al. 2013). DNA of the isolate RLC-1 was extracted for PCR sequencing using primers for the rDNA ITS (ITS1/ITS4), GAPDH (GDF1/GDR1), and ACT (ACT-512F/ACT-783R) (Weir et al. 2012). Analysis of the ITS (accession no. MN880199), GAPDH (MN884048), and ACT (MN891766) sequences revealed a 99%-100% identity with the corresponding ITS (JX010250), GAPDH (KX578786), ACT (JX009541) sequences of C. siamense in GenBank. A phylogenetic tree was generated on the basis of the concatenated data from sequences of ITS, GAPDH, and ACT that clustered the isolate RLC-1 with C. siamense with the type strain ICMP 19118 (Supplementary Figure 2). Morphological characteristic and phylogenetic analysis identified the isolate RLC-1 associated with anthracnose of castor bean as C. siamense. Pathogenicity test was performed in a greenhouse at 24 °C to 30 °C with 80% relative humidity. Twenty healthy plants of Zi Bi No. 5 castor bean (2 month old) were grown in pots with one plant in each pot. Inoculation was conducted on leaves with mycelial plugs of RLC-1 or agar plugs (as control). Three plugs were considered for each leaflet. Ten plants were used in each treatment (five for wounded inoculation and five for unwound inoculation). Anthracnose lesions as earlier were observed on the leaves after 2 weeks, while the control plants remained healthy. The pathogen re-isolated from all inoculated leaves was identical to the isolate RLC-1 by morphology and ITS analysis but not from control plants. C. siamense causes anthracnose on various plant hosts, including mango in Colombia (Pardo-De la Hoz et al. 2016) and Rosa chinensis in China (Feng et al. 2019) but not including castor bean. To the best of our knowledge, this study is the first to report C. siamense causing anthracnose on castor bean. Thus, this work provides a basis for focusing on the management of the disease in future.