Irregular Brown Spots Research Articles

A new species of Pachyrhabda Meyrick (Lepidoptera: Stathmopodidae) from the Korean Peninsula

The Stathmopodidae, belonging to the Gelechioidea, was first reported by Edward Meyrick in 1913. Since then, species with various morphological characters and various ecological adaptations such as feeding on crops, mosses, and aphids have been reported. In this study, we report the genus Pachyrhabda Meyrick, 1897, for the first time in the Korean Peninsula. This genus of moths is known for consuming spores of ferns. In the present article, we report the discovery of a new species, Pachyrhabda benearena sp. nov. It is diagnosed from the congeneric species by irregular brown spots on the forewings. The new species' description and geographic distribution information are provided. Additionally, a world checklist of the genus is provided.

First Report of Colletotrichum gloeosporioides Causing Leaf Blight on Cercis chinensis Bunge in China.

Cercis chinensis Bunge, commonly used as an ornamental plant, is native to southeastern China and extensively cultivated in gardens across major cities in the country. In August 2023, a new high-incidence disease was discovered at Huangshan University in Huangshan, Anhui Province, China. The symptoms initially began as small brown spots, which gradually expanded into large irregular brown spots with black-brown edges. The disease was investigated at both Jilingshan Park and Huangshan University, where C. chinensis Bunge was planted, revealing an average incidence rate of was 85 % at these sites. Seventy two leaf tissue samples (3 to 4 mm²) were collected from the margins of the lesion and subjected to surface sterilization with 75% ethanol for 30 seconds followed by 1% sodium hypochlorite for 90 seconds. Subsequently, the tissues were rinsed with sterile H2O, placed on potato dextrose agar (PDA) medium, and incubated at 25℃ for 5 days. The same fungus was isolated from 90% of the tissues, and pure cultures were obtained by monosporic isolation. Representative isolates ZJ 2-1, ZJ 2-2 and ZJ 2-3 were selected for morphological and molecular characterization. The colonies displayed a color range from white to gray, with white margins and aerial hyphae, while the reverse side of the colonies appeared gray to brown. Conidia were cylindrical, aseptate, with obtuse to slightly rounded ends, measuring 15.8±1.8×4.7±0.56 μm (n = 50). The morphological characteristics were generally consistent with those of Colletotrichum gloeosporioides species complex (Weir et al. 2012). Five conserved regions of isolates (ZJ 2-1, ZJ 2-2 and ZJ 2-3), including the internal transcribed spacer (ITS), glutamine synthase (GS), calmodulin (CAL), actin (ACT), and chitin synthase 1(CHS1) gene regions, were amplified using specific primers ITS1/ITS4 (Gardes et al. 1993), GSR1/GSF1 (Guerber et al. 2003), CL1C/CL2C (Li et al. 2018), ACT-512F/ACT-783R, and CHS-79F/CHS-345R (Zhu et al. 2019), respectively. Using the BLAST, ITS, GS, CAL, ACT and CHS1 gene sequences (GenBank accession nos. PP514751, PP448025, PP448026, PP448027 and PP448028, respectively) were 100% (594 out of 594 bp), 100% (864 out of 864 bp), 100% (299 out of 299 bp), 100% (732 out of 732 bp) and 100% (282 out of 282 bp) identical to C. gloeosporioides (GenBank accession nos. JX010152, JX010085, JX009818, JX009731 and JX009531, respectively). A Maximum Likelihood phylogenetic tree, constructed by combining all sequenced loci in MEGA7, showed that the isolates ZJ 2-1, ZJ 2-2 and ZJ 2-3 clustered within the C. gloeosporioides clade with 99% bootstrap support (Fig. S1). To fulfill Koch's postulates, five C. chinensis Bunge plants were tested for pathogenicity in the field with isolates ZJ 2-1, ZJ 2-2 and ZJ 2-3 at Huangshan University. Twelve leaves from each tree were wounded and inoculated with mycelial plugs (approximately 4 mm in diameter) and 10 μl of a spore suspension (1.0 × 106 conidia/ml) of C. gloeosporioides. Inoculation with sterile PDA plugs and pure water on leaves of each tree served as negative controls. Plastic bags were used to wrap the leaves, and sterile H2O was sprayed into the bags to maintain moisture conditions (Zhang et al.2020). The experiment was repeated two times, and within 5 days, all inoculated points displayed lesions similar to those observed in the field, whereas controls remained asymptomatic (Fig. S2). The same fungus was reisolated from these lesions with a frequency of 100%. Consequently, the pathogen responsible the disease in C. chinensis Bunge was identified as C. gloeosporioides. To the best of our knowledge, this is the first report of C. gloeosporioides causing leaf blight on C. chinensis Bunge in China. This study provides valuable insights for implementing targeted measures to control leaf blight on C. chinensis Bunge and lays a foundation for the prevention and treatment of the disease.

First Report of Leaf Blight of Osmanthus fragrans Caused by Diaporthe eres in China.

Osmanthus fragrans is an evergreen garden tree species, with high ecological, social, and economic benefits (Lan et al. 2023), which is widely planted in Guizhou Province. From late April to June 2023, a leaf blight disease was observed on O. fragrans in a bauxite mining area in Qingzhen City, with an incidence of ~50%. Symptoms first appeared at the leaf tip or margin, as irregular brown spots, which gradually coalesced into dark brown patches until the leaves withered and fell off. Symptomatic leaves were collected and surface disinfected with 2% NaClO for 30 s, 75% ethanol for 30 s, rinsed 3 times in sterile ddH2O, air-dried and placed on potato dextrose agar (PDA) medium and incubated at 25°C for 7 d. Fungal colonies on PDA of 9 similar obtained isolates were white, with at least one concentric ring. The reverse was light yellow and gradually turned brown. At 12 d, the pycnidia on PDA was gray to black, spherical or conical, with a diameter of 305.15 μm (n=20). The conidial horns oozed out from pycnidia after 25 d of incubation on Pinus massoniana needles. The alpha conidia were unicellular, fusiform, hyaline, had a guttule at each end, and measured 6.24 ± 0.10 μm × 2.48 ± 0.04 μm (n=50). No beta or gamma conidia were observed. The morphological characteristics were likely to Diaporthe spp. (Gomes et al. 2013). DNA of isolates GH02, GH06 and GH08 was extracted. The internal transcribed spacer region (ITS) and partial sequences of translation elongation factor 1-alpha (TEF1-α), calmodulin (CAL), beta-tubulin (TUB2), and histone H3 (HIS) genes were amplified with primers ITS1/ITS4 (White et al. 1990), EF1-728F/EF1-986R, CAL228F/CAL737R (Carbone and Kohn, 1999), βt2a/βt2b and CYLH3F/H3-1b (Crous et al. 2004; Glass and Donaldson, 1995), respectively. The sequences of ITS, TEF-1α, TUB2, CAL and HIS were deposited in GenBank (GH02: PP813499, PP813844, PP813846, PP813848 and PP813850; GH06: PP813500, PP813845, PP813847, PP813849 and PP813851; GH08: PP507168 and PP529956 to PP529959). BLAST results showed the sequences of GH08 were highly identical to sequences of Phomopsis mahothocarpi (NR147522 [ITS], 527/530), P. mahothocarpi (MW700277 [TEF-1α], 367/372), D. eres (OR885862 [TUB], 513/513), D. celeris (ON221721 [CAL], 484/486), and D. eres (OP968956 [HIS], 477/477). A phylogenetic tree constructed with MEGA X using Neighbor-Joining algorithm (Felsenstein, 1985) indicated the isolate GH02, GH06 and GH08 separated from D. eres CBS 297.77 previously reported from O. aquifolium in Netherlands, as well as D. osmanthi and D. fusicola from O. fragrans in China (Gomes et al. 2013; Long et al. 2019; Si et al. 2021). Based on these results, the three isolates were identified as D. eres (Chaisiri et al., 2021). The isolate GH08 was deposited in the Forest Protection Laboratory, Guizhou University. To confirm pathogenicity, spore suspensions (1×105 spores/mL) of GH08 were sprayed on healthy detached leaves (n=10) and leaves of 3-year-old potted O. fragrans seedlings (n=8). An equal volume of sterile water was sprayed for the control. Then they were placed at 20°C and 70-80% RH. Similar leaf blight symptoms appeared after 5 and 15d on the inoculated leaves and seedlings, respectively. The re-isolated fungus, was identical to D. eres based on morphological and molecular analysis, thus fulfilling Koch's postulates. To our knowledge, this is the first report of D. eres causing leaf blight of O. fragrans in China, supporting a basis for developing effective methods to manage this disease.

First report of Alternaria alternata causing leaf spot on Polygonatum kingianum in China.

Polygonatum kingianum is a Chinese herbal medicine that belongs to the genus Polygonatum of the family Liliaceae. In June 2023, Polygonatum kingianum Coll. et Hemsl. in nurseries in Qujing, Yunnan Province, China, showed irregular brown spots on the leaves, whole leaf necrosis, and plant death in serious cases, with an incidence of 10-20% (Fig. S1). To identify the pathogens of P. kingianum, six diseased samples were collected from nurseries with 0.6 acre. These diseased sample leaves were soaked in 0.1% HgCl2 for 1 min and 75% ethanol for 2 min and then rinsed thrice with sterile water. Treated leaves were cut into small pieces (5×5 mm) and cultured on potato dextrose agar (PDA) for five days at 28°C. Total thirteen fungal strains were isolated from PDA medium. The nuclear ribosomal internal transcribed spacer of ribosomal DNA (ITS rDNA) region of these 13 strains was amplified by polymerase chain reaction (PCR) using universal primers ITSI/ITS4 (White et al. 1990). Sequencing and BLAST of the ITS region on NCBI showed that 11 out of 13 fungal strains belonged to the genus Alternaria, with an identity ≥99%. We selected one of the Alternaria strains, HJ-A1, for further study. The HJ-A1 colony appeared grayish brown white-to-gray with a flocculent texture on the front side and a dark gray underside on the PDA medium (Fig. S1). The conidiophores appeared brown, either single or branched, and produced numerous short conidial chains. The conidia were obclavate to obpyriform or ellipsoid in shape and contained 1-4 transverse septa and 0-2 oblique septa. The conidial diameter was 27.30µm in length and 12.27µm in width. (Fig. S1). To further determine the species of HJA1, the genomic DNA of HJ-A1 was extracted using the Lysis Buffer for PCR (AG, Hunan, China). Four Alternaria genomic DNA regions including the ITS, translation elongation factor 1-α gene (TEF1-α), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and Alternaria major allergen gene (Alt a1) were amplified by PCR using the primers as previously reported (Woudenberg et al. 2013, Hong et al. 2005). Sequence analysis revealed that the ITS (484bp) of HJ-A1 (NCBI No. PP082633), TEF1-α (267bp) of HJ-A1 (NCBI No. PP419893), GAPDH (582bp) of HJ-A1 (NCBI No. PP419892), and Alt a1 (522bp) of HJ-A1 (NCBI No. PP228046) shared the highest identity with A. alternata respectively (99≥%). A maximum likelihood phylogenetic tree was constructed with the combined sequence data sets of ITS, GAPDH, TEF, and Alt a1 using MEGA 7. The results showed that HJ-A1 strain clustered with A. alternate (Fig. S2). The pathogenicity of HJ-A1 was tested according to Koch's postulates by inoculating HJ-A1 conidia suspension (2×105 conidia/mL) into leaves of 1-year-old P. kingianum, with sterile water as a control. Each treatment group included 3 plants with 3 replicates. The tested plants were planted in a phytotron at 28℃ and 90% humidity. Three days after inoculation, symptoms similar to those under natural conditions were observed in the HJ-A1-inoculated plants, whereas no symptoms were observed in the control plants (Fig. S1). The same fungal strains were re-isolated from inoculated leaves and identified by morphologically and sequence of ITS. Previous studies showed that Alternaria alternata funji cause many plant diseases, such as fig fruit rot (Latinović N et al. 2014)，daylily leaf spot (Huang D et al. 2022), fruit blight on sesame (Cheng H et al. 2021)，leaf spot of Cynanchum atratum Bunge (Sun H et al. 2021) and so on. To our knowledge, this is the first report of A. alternata causing P. kingianum leaf spot in China. The discovery of this pathogen will help to guide the protection and control of P. kingianum disease.

First Report of Leaf Rot Caused by Apiospora paraphaeosperma on Lily in Wuhan, China.

Lily (Lilium spp.) is a valuable ornamental bulb flower plant in Liliaceae, and its bulbs have high edible and medicinal value. Compared with bulb propagation of other lilies, seed propagation and short growth period are the most significant characteristics of Lilium×formolongi. In 2023, leaf rot disease (LRD) was observed on approximately 70% of the Lilium×formolongi seedlings sown in an experimental greenhouse in Wuhan, Hubei province, China. Irregular brown water-soaked spots were discovered in the early stages of infected seedlings. Then, spots spread throughout the leaves and caused the leaves to brown, soften, and wilted. A pathogen associated with symptoms was isolated by incubating sterilized leaves on potato dextrose agar plates at 25 ℃ for 2-3 days. Then, a pure single colony was isolated through a single hyphal tip isolation method. The fungal colony was white with abundant aerial mycelium and produced a yellow pigment diffusible into the agar. Microscopically, isolated mycelia were reticulate and pale yellow, while conidia were dark brown, smooth, and spherical, 7.31 to 6.98 × 4.03 to 3.87μm (average 5.44×5.41μm; n=30); oval in lateral view, and had a light stripe in the middle. To identify the species of the fungus at the molecular level, ITS and EF-1α genes were amplified and sequenced using primers ITS1/ITS4 (M Gardes et al. 1993) and 758F/986R (Carbone and Kohn 1999). The BLAST results in GenBank showed that the ITS(OR523578) and EF-1α(PP066842) sequences of LRD shared 99.82% and 99.24% identity with the distinct Apiospora paraphaeosperma strains (GenBank accession MT040110, ON806628.1, respectively). Combined with the morphology of the colony and conidium, the fungus was identified as Ap. paraphaeosperma. In the pathogenicity test, six healthy leaves were inoculated with mycelium disc and then kept in an incubator (22 ℃, 90% humidity, 16h light /8h darkness). The inoculated leaves showed necrosis and wilt symptoms similar to those observed in the greenhouse, while the control leaves were asymptomatic. A re-isolation, morphology identification and DNA sequencing of the fungus confirmed its infection with Ap. paraphaeosperma in Lilium spp. At present, rot caused by Ap. paraphaeosperma has only been reported in Thailand and South Korea, both of which are found on bamboo stems (Hyde et al. 2016; Sun Lul Kwon et al. 2022). As far as we know, this is the first report of leaf rot of lily caused by Ap. paraphaeosperma in China. This report can help identify this disease and further develop effective control measures.

First report of Fusarium commune Causing Bulb Rot on Lily (Lilium brownii var. Viridulum Baker) in Hunan Province of China.

Lily (Lilium brownii var. Viridulum Baker) is a well-known edible plant with large, white and sweet bulb scales that has important medicinal value (Zhou et al. 2021) and is grown mainly in the Hebei, Shanxi and Henan provinces of China. In May 2021, a case of bulb rot was discovered in a 3.33 hm2 plantation in Huaihua, Hunan Province, affecting 20% of the area (27°59'30″N, 110°32'20″E). The disease is most severe during the rainy season in May and June. In the early stage, irregular brown spots appeared on the lily scales, the necrosis was depressed and gradually enlarges, and in the later stage, the scales were scattered from the base of the disc and slough off. Ten samples were taken randomly from different plants in the plantation area to isolate the pathogens. After washing with sterile water, they were cut into small pieces and sterilised with 3% hydrogen peroxide for 30 s, 75% ethanol for 90 s, rinsed three times with sterile water and dried on sterile filter paper, then placed on a water agar plate and incubated in the dark in a constant temperature incubator at 28℃ for 3 to 5 days. After 2 days, the mycelium at the edge of the colony was transferred to a PDA plate and incubated for 3-5 days at 28°C in the dark to obtain pure fungal isolates. Eighteen purified fungal isolates were obtained, of which sixteen looked like Fusarium (88.9% isolation rate) and three representative isolates (BHBR2, BHBR3 and BHBR5) were selected for further study. The surface of this fungus was white with dense aerial mycelium. Some had an orange centre in the medium. Microconidia were oval in shape and appeared either straight or slightly curved. These microconidia were colourless, had 0-1 septa and measured 3.334 to 14.724 × 2.216 to 5.385 μm (n=100). Macroconidia were predominantly three-septate, crescent-shaped structures that were thin-walled and slightly curved. Cells at the apex and base were similarly curved. Macroconidia measured 17.956 to 32.150 × 2.788 to 4.492 μm (n=100). The mitochondrial small subunit (mtSSU) and translation elongation factor 1-α (TEF1) genes were amplified and sequenced using the NMS1/NMS2 and TEF-R/TEF-F primers to verify the identity of the pathogens (Stewart et al. 2006). The sequences were submitted to GenBank (BHBR2: mtSSU, PP273435; TEF, OR900976; BHBR3: mtSSU, PP277729; TEF, OR900977; BHBR5: mtSSU, PP277728; TEF, OR900978). A concatenated phylogenetic tree of the two genes was constructed and analysis showed that BHBR2, BHBR3 and BHBR5 were significantly clustered with Fusarium commune. Based on the results of morphological identification and phylogenetic tree analysis, the three isolates were identified as Fusarium commune. We carried out pathogenicity tests using two methods, one in which 6 × 6 mm fungal blocks were inoculated on lily (L. brownie var. viridulum Baker) scales and controls inoculated with sterile blocks, and the other in which strain BHBR2 was selected to carry out pathogenicity tests on bulbs of live plants soaked with 50 ml of a 1 × 106 conidial suspension and bulbs of control plants soaked with sterile water, all in three replicates. They were placed in a growth chamber at 28°C and 80% relative humidity, and the scales were moistened with moistened sterile filter paper. After 3 days of rearing treated scales, lesions appeared on lily scales inoculated with mycelial blocks and expanded with time, whereas no lesions appeared on lily scales inoculated with sterile blocks. One month later, whole plants soaked in the spore suspension wilted, while the control plants grew well. The pathogens re-isolated from the diseased tissues had the same morphological characteristics as representative isolates. This confirms Koch's hypothesis. Fusarium commune has been shown to be the most important pathogenic fungus causing root rot in Alfalfa (Medicago sativa) (Yang et al. 2022) and blueberry (Vaccinium uliginosum L.) (Li et al. 2023) in China. To our knowledge, this is the first report of Fusarium commune causing lily bulb rot in the world, which will lay the foundation for future control of lily bulb rot.

First Report of Leaf Spot on Taraxacum mongolicum Caused by Alternaria solani in China.

Taraxacum mongolicum is a perennial herbaceous plant in the family Asteraceae, with a high edible and medicinal value and is widely planted in China. In August 2022, leaf spots were found on T. mongolicum in Tianjiazhai Town, Xining City, Qinghai Province, China (36°27'17.65″N, 101°47'19.65E, elevation: 2,408 m). The plants exhibited round or irregular brown spots, and the centers of some of the spots were gray (Fig. S1A). An investigation was performed over a hectare area, and the incidence of leaf spot reached 15%-30%, seriously affecting the quality and yield of T. mongolicum. Eleven T. mongolicum leaf spot samples were collected. To isolate the pathogenic fungus, approximately 0.5 cm×0.5 cm pieces of tissues were obtained using sterile scissors from the junction of infected and healthy tissues. The symptomatic leaves were surface-disinfected with 3% NaClO for 1.5 min and washed three times with sterile water. The disinfected pieces were dried and placed on water agar plates in an incubator for 2 days at 25°C. Subsequently, the leaf surface exhibited conidiophores and conidia. Eleven isolates were obtained by single spore isolation. The sparse aerial mycelia were dark grey to black brown in color on potato dextrose agar (PDA) (Fig. S2A), and produced dark, multi-septate conidia with 7-11 transverse septa and 1-2 longitudinal septa (Fig. S2C). Conidia with one or two beaks were long-ovoid, with an average length and width of 103.4 × 21.2 μm, and 80.7 × 3.9 μm of the beaks. One hundred and ten conidia were measured. The identification of 11 isolates was confirmed by multilocus sequence analyses of the internal transcribed spacer of ribosomal DNA (rDNA ITS) (White et al. 1990), and the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Xu et al. 2022), actin (ACT) (Yang et al. 2020), histone 3 (HIS3) (Zheng et al. 2015), translation elongation factor 1-α (TEF1-α) (Carbone. 1999), and the second largest subunit of RNA polymerase II (RPB2) (Liu et al. 1999) genes. The sequences of all the isolates were deposited in Genbank (NCBI Accession Nos. ITS: OR105029-OR105039, ACT: OR135220-OR135230, GAPDH: OR135231-OR135241, HIS3: OR122992-OR123002, TEF1-α: PP055972-PP055982, and RPB2: PP055983-PP055993), and the sequence similarity of ITS, ACT, GAPDH, HIS3，TEF1-α and RPB2 were 100%, 98%, 100%, 99%, 100%, and 99% to the sequences of Alternaria solani, respectively. Combined sequences of ITS, GAPDH, TEF1-α, and RPB2 genes were concatenated and a maximum parsimony tree was constructed with PAUP* v. 4.0 alpha. The results indicated that 11 isolates were clustered together with A. solani (Fig. S2D). Therefore, 11 isolates were identified as A. solani based on their morphological and molecular characteristics. Eleven isolates were inoculated on their host to perform Koch's postulates. The isolates were grown on PDA for six days. Healthy one month old T. mongolicum seedlings were planted in 10 cm flowerpots (Fig. S1B) or the seedlings were moved to Petri dish (Fig. S1C), and their leaves were inoculated with 5 mL of hyphae suspension by smearing method. In addition, seedlings of the same age were treated with sterile water to serve as the control. The inoculated seedlings were moved into an artificial climatic box at 25℃, relative humidity was 70%, with 12 h light/12 h dark condition. Totally 80 seedlings were inoculated with isolates and 15 were used as the control. After 7 days, similar symptoms were observed on the plants inoculated with isolates, while control plants did not produce symptoms. The assays were conducted three times. Furthermore, isolates were re-isolated from the symptomatic leaves, and the colonial morphology was the same as the original isolates (Fig S2 A and B). The recovered isolates were identified as A. solani by amplifying and sequencing a portion of the HIS3 gene. Alternaria solani has been previously reported to cause early blight of potato and other Solanum crops (van der Waals et al. 2004; Zheng et al. 2015). To our knowledge, this is the first report of A. solani causing leaf spot of T. mongolicum in China. This disease must be considered in management practices, and our finding provided a basis for disease prevention and management.

First Report of Colletotrichum siamense Causing Anthracnose on Syzygium grijsii in China.

Syzygium grijsii is an evergreen shrub belonging to the family Myrtaceae, and widely cultivated in southern China as an ornamental medicinal plant. In May 2022, anthracnose symptoms were observed on leaves of S. grijsii planted in a nursery (N22°55'46″, E108°22'11″) in Nanning, Guangxi Province, China. More than 30% of leaves were infected. Initially, irregular brown spots (1 to 2 mm in diameter) formed on the leaves, with a slight depression in the center, then expanded into large, dark-brown lesions. In severe infections, lesions coalesced and covered the entire leaf, causing wilt and fall off the plant. To identify the pathogen, 30 diseased leaves were collected from five plants. Leaf tissues (5 × 5 mm) were cut from the infected margins, surface sterilized (75% ethanol 10 s, 2% NaClO 5 min, rinsed three times with sterile water), then placed on potato dextrose agar (PDA), and incubated at 28℃ in darkness. After 5 days, 16 fungal isolates with similar morphology were obtained from 30 plated tissues. Colonies on PDA were abundant with grayish-white fluffy mycelia, and yellowish-white on the back. Conidia were one-celled, hyaline, smooth-walled, cylindrical with narrowing at the center, blunt at the ends, and ranged from 11.35 to 22.14 × 4.88 to 7.67 μm (n=100). Morphological characteristics of the isolates were similar to the descriptions of Colletotrichum sp. (Prihastuti et al. 2009). Five representative isolates (Cs34, Cs31, Cs32, Cs33 and Cs35), which were preserved in the Guangxi Key Laboratory of Biology for Crop Diseases and Insect Pests, were selected for molecular identification. The ITS (Nos. OQ618199, OR539576 to OR539579), TUB2 (Nos. OQ630972, OR545076 to OR545079), ACT (Nos. OQ685919, OR545060 to OR545063), CHS-1 (Nos. OQ685917, OR545068 to OR545071), GAPDH (Nos. OQ685916, OR545072 to OR545075), and CAL (Nos. OQ685918, OR545064 to OR545067) sequences showed >99% identity to those of Colletotrichum siamense ex-type culture ICPM 18578 (Nos. JX010171, JX009924, JX009714 and JX009518) and strain C1315.2 (Nos. JX009865 and JX010404) in GenBank. Multigene phylogenetic analyses (ITS, TUB, ACT, CHS-1, GAPDH, and CAL) using the Maximum likelihood method indicated that the 5 isolates were clustered with C. siamense. To perform pathogenicity tests, three one-year-old healthy S. grijsii plants were inoculated with conidial suspension (1 × 106 conidia/ml) of isolate Cs34 by brushing gently with a soft paintbrush, each plant was inoculated with 3 leaves. The same number of plants were inoculated with sterile water as control, and pathogenicity tests were performed three times. All plants were kept in an artificial climatic box at 28℃, with a 90% humidity and a 12 h light/dark cycle. Similar symptoms to those of the field were observed on all inoculated leaves after 5 days, whereas controls remained symptomless. Reisolated fungi from the diseased leaves were confirmed to be C. siamense by morphology and molecular characterization, confirming Koch's postulates. C. siamense has been reported causing anthracnose on Crinum asiaticum (Khoo et al. 2022) in Malaysia, and Erythrina crista-galli in China (Li et al. 2021). To our knowledge, this is the first report of C. siamense causing anthracnose on S. grijsii in China. The results of pathogen identification provide crucial information for control strategies of the disease.

Integrated Management of Alternaria Leaf Spot of Cauliflower under Pot Conditions

Cauliflower is widely grown throughout the world. Alternaria leaf spot of Cauliflower caused by Alternaria brassicae is the most destructive disease at the curd stage and seed setting stage, attributed to causing 30-50% losses and in severe cases reaching up to 98% in seed crop. In the present investigation, the pathogen Alternaria brassicae was found to produce typical symptoms of the disease as small, irregular brown spots with concentric rings, also surrounded by a yellow chlorotic halo. Later on, lesions were found to coalesce forming irregular, brown to dark brown patches with concentric rings. The disease is difficult to manage alone with fungicides or bio agents or botanicals. Hence the present study was undertaken to manage the disease effectively by application of fungicides, bio agents, botanicals and their combinations. During the investigation on integrated management under sick pot conditions, the combination of T. viride + Neem + Azoxystrobin (10.81% Percent Disease Index) was found most effective with inhibition of disease by 79.02%. This was followed by T. viride + Azoxystrobin (11.63% Percent Disease Index) with inhibition of the disease by 77.40% compared to the control 51.52% Percent Disease Index.

First Report of Epicoccum nigrum Causing Brown Leaf Spot of Sweet Cherry (Prunus avium) in China

Sweet cherry (Prunus avium L.) has become an important economic fruit in China, mainly produced in Shandong Province. In recent years, the planting area of Aba Prefecture in Sichuan Province has increased. In June 2022, sweet cherry brown leaf spot was found in a cherry plantation (100ha) in Wenchuan County (30°54′50.21″N, 103°24′49.10″E), with an incidence of 50 - 70%. The symptoms appeared as brown circular spots on the leaf, gradually expanding until multiple lesions coalesced to form large irregular brown spots; eventually entire leaves were killed. To isolate the causal pathogens, 10 diseased trees were randomly selected from an orchard, one diseased leaf was taken from each tree, and samples (4×4 mm2) were cut from the border between diseased and healthy tissues of 10 diseased leaves, surface sterilized with 75% ethanol for 30 sec, washed three times with sterilized water, dried on sterilized filter paper and placed on potato dextrose agar (PDA). After 5d at 25℃, five morphologically similar colonies were obtained, colony appears yellow fluffy and released a large amount of red-orangepigment. Microscopy revealed circular to ovoid, verrucose, and multicellular conidia measuring 20×25 μm diameter (n = 30) were produced on the mycelia. The morphological characteristics were consistent with the description of Epicoccum nigrum (Lima et al 2011). To further identify the strains, the internal transcribed spacer (ITS), β-tubulin, and RNA polymerase second largest subunit (RPB2) gene regions were amplified with ITS1/ITS4 , Bt2a/Bt2b, and 5f2/7cr (White et al. 1990; Glass and Donaldson 1995; Sung et al. 2007), respectively. BLAST analysis revealed that the ITS, β-tubulin, and RPB2 sequences were 99.2%, 100% and 99.6% homologous, with those of E. nigrum (KU204750.1, OL782123.1, and MW602294.1), respectively. The sequences of the five isolates were identical; and those of representative strain TY3 were deposited in GenBank (ITS, OP410968; β-tubulin, OR502448; RPB2, OP484927). Maximum likelihood phylogenetic analyses were performed for the combined data set with ITS , β-tubulin and RPB2 using MEGA6 under the Tamura–Nei model (Tamura et al. 1993). Isolate TY3 clustered with E. nigrum type strain CBS 505.85. The pathogenicity of TY3 was tested on 10 sweet cherry trees aged 3 years (there were about 50 leaves per plant). Five plants were sprayed with 50 mL of spore suspension (1×105 spores/mL), while the controls (Five plants) were sprayed with 50 mL of sterile water. All plants were in closed plastic bags to maintain high humidity, placed in a greenhouse, and incubated at 25℃with a 12-h photoperiod. Twelve days after inoculation, 35% of the inoculated leaves showed lesions; that were consistent with those observed in the field, and the control group was asymptomatic. To confirm Koch´s postulates, two isolates were taken from the margins of leaf lesions and both were confirmed to be E. nigrum based on morphological observations and molecular identification using ITS β-tubulin, and RPB2 sequences. This is the first report of brown leaf spot caused by E. nigrum on P. avium in China. This discovery needs to be considered in developing and implementing disease management programs in sweet cherry production.

First Report of Colletotrichum aenigma Causing Anthracnose on Bromus japonicus in Tianjin, China.

Japanese brome (Bromus japonicus Thunb.) is a weed commonly found in roadsides, floodplain wetlands, and farmlands. During September 2020 and 2021, a leaf spot disease was observed on B. japonicus in greenhouses of Baodi district, Tianjin, China (117°15'E, 39°47'N). More than 10% of the weeds were infected. Initial irregular brown spots on leaf apices continued to expand until adjacent spots coalesced. Eventually, severely infected leaves became yellow, thinner, drier and withered. Small patches (3×3 mm) were cut from symptomatic leaves, sterilized with 75% ethanol for 30s, rinsed three times with sterile water and incubated on Petri dishes with potato dextrose agar at 25°C in darkness for 7 days. Three isolates, with uniform morphology were selected for further analysis. Colonies were cottony with entire edges and aerial white mycelia; and average growth rate of 4.5 mm/day. The upper side was pale white, and the reverse side was grayish-green. Conidia were aseptate, hyaline, subcylindrical with rounded ends, 8.6 to 18.7×4.4 to 8.3 μm (n = 50). Appressoria were dark brown, oval or irregular shaped with a few lobes, 5.7 to 9.4×4.5 to 7.8 μm (n = 50). Total genomic DNA of isolates was extracted with Fungal DNA Kit (GBCBIO, Guangzhou, China). Primers for sequences of internal transcribed spacer (ITS) regions, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), β-tubulin (TUB2), and calmodulin (CAL) genes were amplified and sequenced (Weir et al., 2012). After aligned and trimmed, the sequences of TJBDA1, TJBDA2, and TJBDA3 were identical. TJBDA1 representative isolate sequences were deposited in GenBank ITS OP247554 with 99.83% (576/577) similarity to MT476809, GAPDH OP414834 with 99.59% (244/245) similarity to MT501009, TUB2 OP414836 with 100% (703/703) to MT501053, and CAL OP414835 with 100% (601/601) to MT500921. Maximum likelihood trees based on concatenated sequences of the four genes were constructed using MEGA7.0. The results showed that the strains isolated from B. japonicus were closely related to C. aenigma with 99% bootstrap support. Pathogenicity tests were conducted on 3-leaf stage B. japonicus seedlings. Conidial suspension of TJBDA1 (1×106 conidia/ml) brushed from a 7-day-old culture of the fungus were sprayed on 9 B. japonicus seedlings. Control plants were sprayed with sterile water. All treatments were replicated four times. The treatment plants were placed in an incubator (25°C, relative humidity > 80%, 12-h photoperiod). Typical leaf spot symptoms resembling ones in the fields were observed on inoculated leaves after 7 days, but control leaves remained symptomless. The fungi reisolated from diseased leaves were morphologically and molecularly identical to the inoculated isolatescompleting Koch's postulates. According to morphological, pathological characteristics and multilocus phylogenetic analysis, the isolated strains from B. japonicus were identified as C. aenigma. To our knowledge, this is a new host record for C. aenigma causing anthracnose on B. japonicus in China. Currently, B. japonicus has evolved a high level of resistance to herbicides in some regions of China (Li et al, 2022) and C. aenigma caused serious disease to B. japonicus. We hope to discover a biocontrol method against weed on non-host cultivated plants through the production of secondary metabolites by C. aenigma.

First Report of Leaf Spot Caused by Alternaria alternata on Cyclocodon lancifolius in China.

Cyclocodon lancifolius Bunge in the family Campanulaceae, and commonly known as Hong Guo Ginseng, is found in the Indonesia, Philippines, Vietnam, Japan, and China. The leaves and roots of C. lancifolius are widely used as tonics by ethnic minorities in Guizhou and Hunan Provinces in China. In addition, the fruit is edible, and it is a new resource for both medicine and food. In June 2022, symptoms of leaf spot (Fig 1 A and B.) were observed on C. lancifolius plants in the medicinal plant greenhouse of Guizhou University of Traditional Chinese Medicine (106°61'E, 26°39'N), Guizhou Province. The incidence of leaf spot on C. lancifolius was approximately 40 to 70% of all leaves in canopy. Early symptoms on leaves were small circular or irregular brown spots. As the disease progressed the lesions gradually expanded, and multiple lesions coalesced to form large irregular brown spots. Eventually the seedlings died and leaves of mature plants wilted. In order to isolate the pathogen, ten leaf pieces (5×5 mm) were cut from the junction of the diseased and the healthy tissues, surface sterilized with 75% ethanol for 30 s, 0.1% mercuric chloride (HgCl2) solution for 60 s, rinsed in sterile water three times, finally dried and placed on potato dextrose agar (PDA) and cultured in the dark at 27°C for 4 days. Five purified fungal isolates were obtained by single spore isolation. The colonies were olivaceous to dark olive with white margins and abundant aerial mycelia. On potato carrot agar (PCA) medium, these fungi produced septate conidiophores. Conidia were obclavate or ellipsoid, brown, with one to four transverse septa and one to two longitudinal septa. Spores measured 7.64 to 14.20 × 3.32 to 6.38 µm (n=50). These morphological characteristics are consistent with Alternaria alternata (S. P. Wiltshire. 1933). To further confirm the identification, four genomic DNA regions including the ribosomal DNA internal transcribed spacer (ITS), translation elongation factor 1-a gene (TEF), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), RNA polymerase II second largest subunit (RPB2), and Alternaria major allergen gene (Alt a1) were amplified and sequenced with primers ITS1/ITS4 (White et al. 1990), TEF1-728F/TEF1-986R (Carbone and Kohn 1999), gpd1/gpd2 (Berbee et al. 1999), RPB2-5F/RPB2-7cR (Liu et al. 1999), and Alt-for/Alt-rev (Hong et al. 2005), respectively. Sequences were deposited in GenBank with accession Nos. ITS: OQ128111, OQ690707, and OQ690708; TEF: OQ200380, OQ700996, and OQ700998; GAPDH: OQ200378, OQ700993, and OQ700995; RPB2:OQ200379, OQ701002, and OQ701004; Alt: OQ675614, OQ700999, and OQ701001. In a BLAST search, the sequences were 99-100% identical with corresponding sequences of A. alternata. A maximum likelihood phylogenetic tree was constructed with the combined sequence data sets of ITS, TEF, GAPDH, RPB2, and Alt a1 using MEGA 11. The isolate DHY0, DHY1, and DHY3 clustered with A. alternata (J. H. C. Woudenberg et al. 2015) (Fig. 2). To fulfill Koch's postulates, leaves on three healthy 3-month-old potted C. lancifolius seedlings were wounded with sterile needles and inoculated with 5 mm diameter mycelium, which was covered moist by sterile cotton for 24 h. Sterile water was used as the control. After inoculation, the plants were incubated at 27°C, 85% relative humidity, and a 12 h photoperiod. The experiment was repeated three times. Fifteen days after inoculation, all the leaves showed leaf spot symptoms that were similar to those observed in the greenhouse, while control leaves were asymptomatic (Fig. 1). A. alternata was successfully re-isolated from the symptomatic leaves and identified by morphology and the molecular methods described above. This pathogen has been reported to cause a leaf disease in a wide range of vegetables (Zhang et al. 2021), flowers (Zhang et al. 2022), and medicinal plants (Xing et al. 2020). To the best of our knowledge, this is the first report of A. alternata causing leaf spot on C. lancifolius in China. The accurate identification of this pathogen will provide a basis for the prevention and control of C. lancifolius leaf spot disease in the future.

First Report of Leaf Spot Caused by Clonostachys rosea on Tea (Camellia sinensis) in China.

Tea (Camellia sinensis), which originated in southwest of China 60 - 70 million years ago, is widely consumed as a beverage for its potential enhancing effect on human health with rich polyphenol content (Pan et al. 2022).From October to December in 2021, a disease with symptoms similar to leaf spot affected the quality and yield of teaPuer (102°73 'E, 25°07' N), Yunnan province, China. Based on the survey, leaf spot symptoms were observed on approximately 60% of tea plants in a 5,700 m2 field. The symptoms initially appeared as shrinking, yellowing, and later became circular or irregular brown spots. To isolate the pathogen, 10 symptomatic leaves were collected from 10 trees, and portions of the diseased tissue (0.5×0.5 cm) were cut at the junction of infected and healthy tissues. After surface sterilization (0.5 min with 75% ethanol and 2 min with 3% NaOCl, washed three times with sterilized distilled water), the disinfected pieces were dried and plated onto potato dextrose agar (PDA) and incubated at 25°C in the dark for 5 days. Four single-spore isolates, FH-1, FH-5, FH-6 and FH-7, were obtained, these isolates were identical in morphology and in the sequences of internal transcribed spacer region [ITS] and translation elongation factor 1-alpha [TEF] genes. Therefore, the representative isolate FH-5 was used for further study. Fungal colonies were white or light yellow on PDA after 7 days incubated at 28ºC. Conidia were hyaline, round or oval, aseptate, occur singly or in clusters on hyphae or conidia stalks, and measured as 2.94 ± 1.79 × 1.82 ± 0.2 μm (n = 50). Primary conidiophores is Verticillium-like (Fig1.K,L), which generally formed first, 1-3-level verticillate, mostly with divergent branches and phialides, and measured as 16.67 ± 4.39 µm (n = 50). Secondary conidiophores is penicillate (Fig1.I,J), which generally appearing after one week, sometimes even more often branched, and with a length of 16.02 ± 3.83 µm (n = 50). The morphological features were consistent with the descriptions of Clonostachys rosea Schroers H.J. (Schroers et al. 1999). The pathogen was confirmed to be C. rosea by amplification and sequencing of the internal transcribed spacer region (ITS) and translation elongation factor 1-alpha (TEF) genes using primers ITS1/ITS4 and EF1-728F/EF1-986R, respectively (Fu Rongtao 2019). The sequences of PCR products were deposited in GenBank with accession numbers ON332533 (ITS) and OP080234 (TEF). BLAST searches of the obtained sequences revealed 99.22% (510/514 nucleotides) and 98.37% (241/245 nucleotides) homology with those of C. rosea HQ-9-1 form GenBank (MZ433177 and MZ451399, respectively). Phylogenetic analysis (MEGA 7.0) using the maximum likelihood method placed the isolate FH-5 in a well-supported cluster with C. rosea. The pathogenicity of FH-5 was tested through a pot assay. Ten healthy tea plants were scratched with a sterilized needle on the leaves. Plants were inoculated by spraying a spore suspension (105 spores·mL-1) of FH-5 onto leaves until runoff, and the control leaves sprayed with sterile water. Inoculated plants were put in an artificial climate box at 25℃, 70% relative humidity. The pathogenicity test was replicated three times. Symptoms developed on all inoculated leaves but not on the control leaves. Lesions around the wound edge became pale yellow, and brown spots were first observed at 72 h after inoculation, and typical lesions similar to those observed on field plants appeared after two weeks. The same fungus was reisolated and identifiedbased on themorphological characterization and molecular analyses (ITSand TEF)from the infected leaves but not from the noninoculated leaves. In addition, C. rosea has also been reported to cause diseases to broad bean (N. Afshari et al. 2017 ), garlic (Diaz et al. 2022), beet (Haque M.E et al. 2020) and other plants. To our knowledge, this is the first report of leaf spot on tea caused by C. rosea in China. This study provides valuable information for the identification and control of the leaf spot on tea.

First Report of Botrytis deweyae Causing Gray mold on Polygonatum cyrtonema in China.

Polygonatum cyrtonema Hua., is one of the cultivated varieties of Polygonatum sibiricum Redouté., which also an important cash crop in China (Chen, J., et al. 2021). From 2021 to 2022, symptoms resembling gray mold were observed on P. cyrtonema leaves with 30 to 45% disease incidence in Wanzhou District (30°38'1″N, 108°42'27″E) of Chongqing. The symptoms started to occur from April to June and more than 39% of leaves were infected from July to September. Symptoms started as irregular brown spots and progressed to the leaf edges or tips and stems. In dry conditions, the infected tissue appeared dry and thin, light brown in color, and became dry and cracked in the later stages of disease development. When the relative humidity was high, infected leaves developed water-soaked decay with a brown stripe around the lesion, and a gray mold layer appeared. To identify the causal agent, 8 typical diseased leaves were collected, leaf tissues were chopped into small pieces (3×5 mm), surface sterilized for 1 min in 70% ethanol and 5 minutes in 3% sodium hypochlorite, rinsed three times using sterile water, placed onto potato dextrose agar (PDA) amended with streptomycin sulfate (50 μg/ml) and incubated at 25°C for 3 days in dark conditions. Then 6 colonies (3.5 to 4 cm diameter) with similar morphology were transferred onto new plates. In the initial stage of growth of isolates, all hyphal colonies were white, dense, and clustered, and dispersed in all directions. After 21 days, brown to black-colored sclerotia (2.3 to 5.8 mm diameter) were observed embedded on the bottom of the medium. The six colonies were confirmed to be Botrytis sp. based on the morphological characteristics. The conidia were attached in branches on the conidiophores in grape-like clusters. Conidiophores were straight and 150 to 500 μm in length, and the conidia were single-celled, long ellipsoidal, or oval-like, with no septa and 7.5 to 20 × 3.5 to 14 μm (n=50). For molecular identification, DNA was extracted from representative strains 4-2 and 1-5. The internal transcribed spacer (ITS) region and sequences from the RNA polymerase II second largest subunit (RPB2), and the heat-shock protein 60 (HSP60) genes were amplified using primers ITS1/ITS4, RPB2for/RPB2rev, and HSP60for/HSP60rev, respectively (White T.J., et al.1990; Staats, M., et al. 2005). The sequences were deposited in GenBank: 4-2 [ITS; OM655229: RPB2; OM960678: HSP60; OM960679] and 1-5 [ITS; OQ160236: RPB2; OQ164790: HSP60; OQ164791]. These sequences from isolates 4-2 and 1-5 had 100% similarity to the B. deweyae CBS 134649/ MK-2013 [ITS; HG799538.1: RPB2; HG799518.1: HSP60; HG799519.1] ex-type sequences, and phylogenetic analyses based on multi-locus alignment demonstrated strains 4-2 and 1-5 as B. deweyae. Isolate 4-2 was used to verify whether B. deweyae can cause gray mold on P. cyrtonema, by conducting Koch's postulates experiments (Gradmann, C., 2014). The leaves of P. cyrtonema planted in pots were washed with sterile water, and brushed with 10 mL of hyphal tissue in 55% glycerin. Leaves of another plant were brushed with 10 mL 55% glycerin as control, and Kochs' postulates experiments were conducted three times. Inoculated plants were kept in a chamber with 80% relative humidity at 20 ± 1°C. Seven days after inoculation, disease symptoms similar to those in the field were observed on leaves, whereas control plants remained asymptomatic. The fungus was reisolated from inoculated plants and identified as B. deweyae based on multi-locus phylogenetic analysis. To our knowledge, B. deweyae is mostly found on Hemerocallis, is likely to be an important contributor to the development of 'spring sickness' symptoms (Grant-Downton, R.T., et al. 2014.), and this is the first report of B. deweyae causing gray mold on P. cyrtonema in China. Although B. deweyae has a limited host range, it might also become a potential threat to P. cyrtonema. This work will provide a basis for the prevention and treatment of the disease in the future.

A new species of green tree frog of the genus Gracixalus (Anura: Rhacophoridae) from the evergreen forest of Northeast India

Abstract We describe a new species of rhacophorid frog of the genus Gracixalus from northeast India based on molecular, morphological and acoustic evidence. The new species, formally described herein as Gracixalus patkaiensissp. nov., is morphologically distinct from other congeners by a suite of morphological characters such as snout-vent length 23.6–26.5 mm in adult males; green dorsum with irregular brown spots; dorsal skin shagreened with numerous spinules; snout shape nearly acuminate in dorsal and ventral view; a prominent dark streak along the cranial margins; white reticulations along lateral side and ventrum distinct in life. Genetically, the new taxon is found to differ from all the recognized Gracixalus species by 4–14.8% divergence in the 16S mitochondrial gene. The discovery confirms the presence of genus Gracixalus from the Indian state of Arunachal Pradesh underlines the importance of biological exploration even in well-known protected areas of India.

First report of wildfire disease caused by Pseudomonas amygdali pv. tabaci on perilla in Korea

Perilla (Perilla frutescens) is an important crop which produces edible leaves and oil from its seeds which is used as a seasoning in Korean food (Kim et al., 2009). Perilla is cultivated throughout the year in greenhouses in southern Korea. In November 2020, irregular brown spots with halos that led to necrosis on most upper leaves were observed in Miryang (35°32´04.6˝N; 128°46´01.9˝E) (Figure 1-3). The symptoms were very similar to wildfire disease in soybeans. The disease spread to up to 20–30% of the perilla plants cultivated in two greenhouses. Five diseased plants were collected and the leaves of each plant were removed. To isolate the causal pathogen, supernatants of surface-sterilised and homogenised leaves were plated on King's B medium (Sigma-Aldrich, USA) and incubated at 28°C for two days. Fluorescent colonies occupied approximately 60% of the area covered by bacterial colonies, indicating that a fluorescent bacterium was the predominant isolate from the diseased perilla. One isolate (designated strain MY1) was collected for analyses of 16S rRNA gene sequences and multilocus sequence analysis (MLSA) using sequences of the gltA, gyrB, and rpoD genes (Hwang et al., 2005). A BLAST search showed that the 16S rRNA gene sequence of strain MY1 (GenBank Accession No. ON810536) corresponded to Pseudomonas amygdali strain DEB with 99% identity. Moreover, strain MY1 was closest to P. amygdali pv. tabaci strain ATCC 11528 in a phylogenetic tree constructed using MLSA (Yun & Kim, 2021) (Figure 4). The physiological and biochemical characteristics of strain MY1 were determined based on levan production, oxidase activity, potato soft rot, arginine dihydrolase activity and tobacco hypersensitive response (LOPAT) tests. In the tests, strain MY1 was positive for levan production and tobacco hypersensitivity, but negative for oxidase activity, potato soft rot and arginine dihydrolase activity. Based on the LOPAT and MLSA analyses, strain MY1 belongs to P. amygdali pv. tabaci 1a (Sun et al., 2021). To substantiate the pathogenicity of strain MY1, a bacterial suspension of the strain (adjusted to 108 CFU/ml) was inoculated onto ten four-week-old perilla and incubated at 28°C. After seven days, the inoculated perilla leaves showed similar symptoms to those observed originally in the greenhouse. Completing Koch's postulates, bacteria with fluorescent colonies were re-isolated from symptomatic tissue and the 16S rRNA sequence of the isolates was identical to P. amygdali. To the best of our knowledge, this is the first report of wildfire disease caused by P. amygdali pv. tabaci on perilla in Korea and globally. Perilla wildfire disease reduces the quality of leaves, so early detection and prevention are essential to reduce the spread of disease. S.Y. Choi and J.I. Kim contributed equally to this work. This work was supported by “Cooperative Research Program for Agriculture Science & Technology Development project (PJ01425901)” Rural Development Administration, Korea.

First Report of Leaf Spot Caused by Alternaria tenuissima on Panicle Hydrangea (Hydrangea paniculata) in China.

Panicle hydrangea (Hydrangea paniculata), belonging to the Saxifragaceae family (Wu et al. 2001), is an ornamental flowering plant which is native to China and Japan. In July 2021, brown leaf spots (diameter ranged from 3 to 5 mm) were observed on panicle hydrangea plants in a 0.1 ha field of Northeast Agriculture University (126.72°E, 45.74°N), Heilongjiang Province. The incidence was approximately 30%. Small circular or irregular brown spots initially appeared on the older leaves, and these lesions gradually expanded with time. In some serious cases, lesions joint together and caused leaf wilting. To identify the causal agent, ten symptomatic leaves from different panicle hydrangea plants were collected, and surface sterilized with 70% ethanol for 30 s, followed by 0.5% NaClO treatment for 4 min, and then rinsed with sterile water three times. Tissues between healthy and necrotic area were cut into 5×5 mm pieces after air drying on sterile filter paper. The pieces were placed on potato dextrose agar (PDA) amended with streptomycin sulfate (50 mg/liter) and incubated at 25°C for 5 days. Fifteen pure isolates were obtained using the hyphal tip technique and cultured on PDA for 7 days at 25°C for morphological and molecular identification. Colonies of all isolates were dark olivaceous with white margin. Conidiophores were septate, singly arising, and light brown, with a size range of 10.2-60.1 × 1.4-5.6 µm (n=50). Conidia were obclavate to obpyriform, brown to dark brown, and in a size range of 15.0-25.2 × 5.1-15.3 µm (n=50). To further identify these isolates, four different genomic DNA regions including ribosomal DNA internal transcribed spacer (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), RNA polymerase II second largest subunit (rpb2), and histone 3 (HIS3) were amplified and sequenced with primers ITS1/ITS4 (White et al. 1990), gpd1/gpd2 (Berbee et al. 1999), RPB2-5F2/RPB2-7R (Sung et al. 2007), and H3-1a/H3-1b (Xu et al. 2022) for three representative isolates (DXQ2-2, DXQ2-3, and DXQ2-4), respectively. The sequences of ITS, GADPH, rpb2, and HIS3 for these three isolates were identical and only sequences of DXQ2-2 were deposited in GenBank (GenBank accession nos. OL305828, OL333601, OL333602, and OL436242). These sequences showed 100%, 100%, 100%, and 99.61% identity with A. tenuissima BJ-CX-1 (GenBank accession nos. MK683974, MK683784, MK684069, and MK683879), respectively. Based on morphological features and DNA sequences analyses, these isolates were identified as Alternaria tenuissima (Simmons 2007). To fulfill Koch's postulates, ten healthy and surface disinfected leaves of one panicle hydrangea plant grown in pot were sprayed with a conidial suspension (1×106 conidia/mL) of isolate DXQ2-2. Meanwhile, ten surface disinfected leaves of another panicle hydrangea plant grown in pot sprayed with sterilize water served as the control. All plants were maintained in a greenhouse at 25℃ and 70% relative humidity. Five days after inoculation, leaves inoculated with conidial suspension showed leaf spot symptoms that were similar to those observed in the field, whereas no symptom was observed on the control leaves. The experiment was conducted twice. The Alternaria tenuissima isolate was successfully re-isolated from the symptomatic leaves and confirmed based on above morphological and molecular methods. To our knowledge, this is the first report of leaf spot on panicle hydrangea caused by A. tenuissima. Leaf spot has a negative effect on the aesthetic value of panicle hydrangea, so further investigation and management is needed to control this disease.

First Report of Alternaria tenuissima Causing Leaf spot on Luffa cylindrica in China.

In August 2020-2021, symptoms of leaf spot were observed in luffa (Luffa cylindrical) fields in Qingdao city, Shandong Province. In all the 10 fields investigated, leaf spot occurred. The incidence (% luffa plants with symptoms from a defined number of plants assessed) was 35 to 60%. Early symptoms of infected leaves were small and irregular chlorotic lesions which later became irregular brown spots. As the disease progressed, the lesions gradually spread from the edge to the center of leaves to the middle, and became dark brown. The enlarged spots coalesced and eventually led to the withering and death of the leaves. In order to isolate the pathogen, 30 symptomatic leaves were collected from different planting fields. Small pieces of leaf tissues (5×5 mm) were cut from the junction of healthy and diseased tissues, sanitized with 2% NaClO for 1 min, rinsed three times with sterile distilled water. The tissue samples were then placed on potato dextrose agar (PDA) amended with 50 mg/L streptomycin sulfate, and incubated at 28℃ for 5 days in the dark. Ten purified fungal isolates were obtained by single spore isolation method. Colonies of these fungal isolates on the PDA medium were initially grayish-white, and then turned olive green with abundant cotton-like aerial hyphae. On potato carrot agar (PCA) medium, these fungi produced light brown and solitary conidiophore with septum. Conidia were obclavate or ellipsoid, brown, with 1-5 transverse septa and 0-3 longitudinal septa, and measured 13.2 to 49.5 × 9.5 to 21.6 µm (n=50). The morphological characteristics of these isolates were consistent with that of Alternaria spp. (Simmons 2007). The representative isolate NEAU-SG-1 was selected for molecular identification. The internal transcribed spacer (ITS) region of ribosomal DNA, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), translation elongation factor 1-α gene (TEF), histone 3 (HIS3), and RNA polymerase II second largest subunit (RPB2) were amplified using primer pairs ITS1/ITS4 (White et al. 1990), gpd1/gpd2 (Berbee et al. 1999), EF1-728F/EF1-986R (Carbone and Kohn 1999), H3-1a/H3-1b (Glass and Donaldson 1995), and RPB2-5F2/fRPB2-7cR (Sung et al. 2007), respectively. Sequences of these genes of isolate NEAU-SG-1 were deposited into GenBank database with the accession numbers of OL307719, OL415166, OL415169, OL415167, and OL415168. BLAST analysis of these sequences showed 99-100% homology with sequence homology with Alternaria tenuissima strains (ITS, MH824269; GAPDH, MK683783; TEF, MN056178; HIS3, MH824371; RPB2, LC621694). To fulfill Koch's postulates, ten surface disinfected 30-day-old luffa seedlings were inoculated by spraying conidia suspension (106 conidia/ml) of isolate NEAU-SG-1. The other ten surface disinfected seedlings inoculated with sterile distilled water served as the control group. After inoculation, each plant was covered with plastic bags for three days and cultured in greenhouse at 25℃. One week later, leaves inoculated with conidia suspension were observed with the same symptoms as described above, while the leaves of the control group were asymptomatic. Pathogenicity test was repeated twice. The Alternaria isolates were successfully re-isolated from those infected leaves and identified using the morphological and molecular methods described above. A. tenuissima has a wide host range in the world, and is the pathogen of leaf spot of many crops (Ma et al. 2021). To our knowledge, this is the first report of A. tenuissima causing leaf spot on luffa in China. This report will provide basic information for the diagnosis and prevention and control strategies of luffa leaf spot.

First report of Diaporthe eres causing leaf spot on Platanus acerifolia in China.

Platanus acerifolia Willd. is one of the world famous urban greening trees, known as "the king of street trees" (Loretta et al. 2020). In August 2021, severe leaf spot disease was observed on P. acerifolia with 15% incidence on a street of Haidian district (116°29'E, 39°95'N), Beijing municipality, China. Typical symptoms were small and irregular brown spots with or without yellow haloes, which gradually expanded, coalesced and became necrotic lesions. For pathogen isolation, the leaf lesions were cut into small tissue pieces, disinfected by 0.3% sodium hypochlorite for 2 min and 70% ethanol for 40 s, rinsed in sterile distilled water, and then incubated on potato dextrose agar (PDA) plates. After incubation at 28°C for 4 days, three fungal isolates (FTDX2, FTDX3, and FTDX6) with similar colony characteristics were obtained after single spore isolation. Colonies were white with fluffy aerial mycelia, abundant pycnidia with black stomata appeared and cream-white liquid oozed after 20 days. Alpha conidia were 7.9 ± 0.6 × 2.5 ± 0.3 μm (n= 30), aseptate, hyaline, fusiform to ellipsoidal, and often biguttulate, while beta conidia were 22.7 ± 1.3 × 1.1 ± 0.1 μm (n= 30), aseptate, hyaline, linear, curved or hamate. The morphological characteristics were consistent with those of Diaporthe sp. (Udayanga et al. 2014). For further identification, total DNA was extracted from the three isolates. The internal transcribed spacer (ITS) region, translation elongation factor 1-α (EF1-α), beta-tubulin (TUB), calmodulin (CAL) and histone (HIS) genes were amplified and sequenced with primers ITS1/ITS4 (White et al. 1990), EF1-728F/EF1-986R (Carbone and Kohn 1999), BT2a/BT2b (Glass and Donaldson 1995), CL1/CL2A (O'Donnell et al. 2000) and CYLH3F/H3-1b (Crous et al. 2014), respectively. The sequences were all deposited in GenBank (accession nos. OL870615 - OL870617 for ITS, OL870618 - OL870620 for EF1-α, OL870621 - OL870623 for TUB, OL870624 - OL870626 for CAL, and OL870627 - OL870629 for HIS) and aligned using BLASTn, obtaining 99-100% homology with the corresponding sequences of known Diaportheeresstrains in NCBI. Phylogenetic analysis of the combined sequences attributed the three isolates to the Diaportheeres clade. Pathogenicity tests were performed on three healthy one-year-old P. acerifolia plants using the randomly selected isolate FTDX2. The leaves were inoculated with 20 µl of spore suspension (106conidia/ml), with or without wound pretreatment, sterilized water inoculation under the same condition was used as control. All the treated plants were incubated in the greenhouse at 25°Cand 90% RH with a 12-h photoperiod. After 8 days,the inoculated plants showed spot symptoms on leaves similar to those previously observed, whereas the control leaves remained symptomless. Lesions on the wounded leaves were much larger in size compared with those unwounded. The same pathogen was re-isolated and identified based on morphological characteristics and gene sequencing data, fulfilling Koch's postulates. Diaporthe ereshas been reported to cause leaf spot on many horticultural plants, such as Photinia fraseri (Song et al. 2019) andPodocarpus macrophyllus (Zheng et al. 2020). To our knowledge, this is the first report ofD. erescausing leaf spot onPlatanus acerifoliain China. This finding is a valuable contribution to the knowledge on leaf spot disease development in horticultural plants.

First report of Macrophomina phaseolina causing leaf blight on Pometia pinnata in Malaysia.

Pometia pinnata (family Sapindaceae), locally known as 'Kasai', is a tropical hardwood and fruit tree species grown in Malaysia. The decoction of the bark is used for the treatment of fever, sores and colds, while the fruits are edible (Adema et al. 1996). In May 2021, irregular brown spots and necrotic lesions were observed on 'Kasai' with an incidence and severity of approximately 60% and 10% on 10 plants in a nursery (5°55'30.7"N 116°04'36.2"E) in Penampang, Sabah province. When the disease progressed, the spots coalesced into extended patches, blightening the leaves and, gradually, the entire foliage. Small pieces (5 x 5 mm) of infected leaves were excised from the infected margin, and then surface sterilized according to Khoo et al. (2022b), and plated on potato dextrose agar (PDA), and cultured at 25 °C. for 6 days. Colonies were dark brown in color initially whitish on the PDA. The color of fungal colony was dark as the culture aged. Semi-appressed mycelia were observed on the plates with abundant microsclerotia engrossed in the agar. Aggregation of hyphae formed black and round to oblong or irregular shaped microsclerotia. Thirty sclerotia from a representative isolate measured average 63-171 μM length x 57-128 μM wide. The morphological features matched the description of Macrophomina phaseolina (Abd Rahim et al. 2019). The fungal genomic DNA was extracted based on Khoo et al. (2022a and 2022b). PCR was performed using primer sets ITS1/ITS4 (White et al. 1990), EF1-728/EF2 (O'Donnell et al. 1998; Carbone and Kohn, 1999) and T1/T22 (O'Donnell and Cigelnik 1997) to amplify the internal transcribed spacer (ITS) region of rDNA, translation elongation factor 1-α (TEF-1α) region and partial β-tubulin (TUB) gene. PCR products with positive amplicons were sent to Apical Scientific Sdn. Bhd. in Malaysia for sequencing. According to results (GenBank Accession No. OK465197, OM677767, ON237461), they were 100% identical with the reference sequence of Macrophomina phaseolina containing approximately 537 bp, 438 bp and 659 bp of the presented ITS, TEF-1α and TUB region (GenBank Accession No. MN629245, MN136199 and KF952208, respectively). The pathogen was identified as M. phaseolina based on its morphological and molecular data (Abd Rahim et al. 2019). To confirm the pathogenicity test, three non-wounded and healthy leaves of one-month-old 'Kasai' seedlings were inoculated with mycelium plug (1 x 1 cm) of M. phaseolina. Additional three 'Kasai' seedlings were inoculated with sterile PDA agar plug (1 x 1 cm) to serve as controls. The seedlings were monitored and incubated in a greenhouse at ambient temperature based on Iftikhar et al. (2022). After 6 days of inoculation, all infected leaves exhibited the symptoms as observed in the nursery, while the controls remained asymptomatic. The experiment was repeated twice. Re-isolation was performed from the symptomatic leaves and controls. The reisolated fungal isolates were identical to M. phaseolina morphologically and molecularly. No pathogens were isolated from the mock controls. M. phaseolina has been reported to cause leaf blight on Jasminium multiflorum in India (Mahadevakumar and Janardhana, 2016), and Crinum asiaticum and Hymenocallis littoralis in Malaysia (Abd Rahim et al. 2019). To our knowledge, this is the first report of M. phaseolina causing leaf blight on 'Kasai' in Malaysia and worldwide. Our findings serve as a warning for the authorities and farmers that the disease threat has appeared for 'Kasai' in Malaysia.