Yellow Halo Research Articles

First Report of Tobacco Leaf Tip Blight Caused by Alternaria gossypina in China.

Tobacco (Nicotiana tabacum L.) belongs to the family Solanaceae, an economically significant crop (Zhou et al. 2023). Twelve samples with leaf spots were collected in Keti Village, Changshun County, Zunyi City, Guizhou province, China in 2022. Twenty-five percent of the samples had dry lesions near the leaf tip which resulted leaf tip blight after development. Fungi were isolated by a previous method (Wei et al. 2022). Six Alternaria strains were obtained and preserved in the Fungal Herbarium of Yangtze University (YZU), Jingzhou, Hubei, China. Among them, one strain YZU 221477 showed distinct cultural characteristics out of five A. alternata strains, which was again determined by growing on potato dextrose agar (PDA) at 25°C for 7 days in dark to evaluate. The colonies (60 mm in diameter) were white cottony in the center surrounded by vinaceous purple. To examine the morphology, mycelia were inoculated onto potato carrot agar (PCA) at 22°C, following an 8 h light/16 h dark photoperiod (Simmons 2007). Conidia were obclavate or ovoid, normally 3-5 conidial units per chain, 20-38 × 10-16.5 μm, 3 to 5 transverse septa, beakless or a short beak (4-30 μm). The observation results were consistent with those of A. gossypina (Zhang 2003). Total genomic DNA was extracted using the CTAB method and seven gene regions including internal transcribed spacer of rDNA (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), translation elongation factor 1 alpha (TEF1), RNA polymerase second largest subunit (RPB2), Alternaria major allergen gene (Alt a 1), endopolygalacturonase (EndoPG) and an anonymous gene region (OPA10-2) were amplified with ITS5/ITS4, gpd1/gpd2, EF1-728F/EF1-986R, RPB2-5F/RPB2-7cR, Alt-for/Alt-rev, PG3/PG2b and OPA10-2L/OPA10-2R primers, respectively. All sequences were deposited in GenBank (ITS: OR710806; GAPDH: PP057862; TEF1: PP158601; RPB2: PP057863; Alt a 1: PP057865; EndoPG: PP057861; OPA10-2: PP057864). Combining with relevant sequences retrieved from the NCBI database were used for the phylogenetic analysis. Maximum Likelihood (ML) tree was constructed with RAxML v.7.2.8 employing GTRCAT model using 1000 bootstrap (BS) replicates to assess statistical support. The results indicated that the present strain grouped with A. gossypina (type strain of CBS 104.32) supported with 73% bootstrap values, also having a support of 0.83 Bayesian posterior probabilities values. Based on morphology and molecular evidence, the strain YZU 221477 is identified as Alternaria gossypina. Pathogenicity was examined to fulfill Koch's postulates. Mycelial plugs (6 mm diameter) of the present strain and A. alternata cultivated on PDA were taken from the margin and inoculated onto viable tobacco leaves (Cultivar: Yunyan 87, n=3) growing forty days, while controls were inoculated with sterile PDA. The assay was conducted three times. The plants were maintained at 25°C with humidity levels over 85% in a greenhouse. Leaves were evaluated after 7 days, necrotic spots encircled by yellow halos were on both inoculums, except controls. Pathogen re-isolation confirmed that it was the same as inoculated fungus based on morphology. A. gossypina was firstly found on cotton (Hopkins 1931), late reported to induce disease on Minneola, Nopalea, Hibiscus, Citrus, Solanum and Ageratina. To our knowledge, this is the first report of A. gossypina causing tobacco leaf tip blight in China, and it also provides a basis for controlling of tobacco leaf tip blight.

Leaf Spot Disease of Michelia alba caused by Alternaria alternata in China.

The perennial deciduous tree Michelia alba is a widely cultivated street plant in China. In June 2021 and March 2022, M. alba trees with leaf spots were found in the green belt of the approximately 200,000 m2 community (32.62°N, 116.98°E) of Tianjia'an District, Huainan, Anhui, China, where approximately half of the M. alba trees had brown leaf spots surrounded with irregular yellow halos ranging from 2 to 6 mm in diameter (Fig S1A). The leaves of M. alba trees with multiple lesions became blighted. To isolate the potential pathogens causing leaf spot symptoms in M. alba trees, twenty fragments (2 cm2) were excised from the margin of the necrosis on symptomatic leaves, immersed in 1% sodium hypochlorite for 45 s, and then washed three times in sterile water. The fragments were plated and incubated on potato dextrose agar (PDA) at 25 °C and 15 dark green fungal colonies were obtained 5 days later. Single-spore isolates of the fungal colonies plated on potato carrot agar (PCA, Simmons 2007) produced gray, floccose colonies, which reached 71 mm after 7 days at 25 °C were obtained 5 days later (Fig S1C). Optical microscopy analysis showed that single-spore isolates formed sparsely branched chains with pale brown conidiophores on PCA after incubation at 25 °C in darkness for 7 days. The conidia were ellipsoidal, inverted rod, or ovoid, light brown, and 10.0 to 52.5 × 4.5 to 22.7 μm, with zero to four longitudinal or oblique and zero to eight transverse septa (n = 50). Partial conidia are 2.5 to 27.5 × 0.6 to 3.7 μm with cylindrical light brown beaks (n = 50) (Fig S1D, E). The cultural and morphological characteristics of the isolated fungi were consistent with the description of Alternaria alternata (Woudenberg et al. 2015). To further characterize the isolated fungi, the genomic DNA of three representative strains (BYL-1, BYL-2 and BYL-3) were extracted from their mycelia, respectively. ITS region and housekeeping genes GPD, and TEF, were amplified and sequenced using ITS4/ITS5 (White et al. 1990), Gpd1/Gpd2 (Berbee et al. 1999), and EF1-728F/EF1-986R (Carbone and Kohn 1999), primer pairs, respectively. BLAST analysis showed that the isolates BYL-1 (GenBank accession nos. OP325693, OP405008, and OP405009), BYL-2 (GenBank accession nos. PP057859, PP138442, and PP138444), and BYL-3 (GenBank accession nos. PP057860, PP138443, and PP138445) shared 99 to 100% identity with Alternaria alternata (GenBank accession nos. AF347032.1, AY278809.1, KC584693.1), which suggested that all the three isolates belong to A. alternata. The identifications were further confirmed by phylogenetic analysis based on combined DNA sequences data of ITS, GPD, and TEF. As showed in Fig S2, the strains of BYL-1 , BYL-2 and BYL-3 formed a robust clade with A. alternata CBS918.96. Taken together, the morphology and molecular assays suggest that strain BYL-1 is A. alternata. To test pathogenicity, the isolate BYL-1 was cultured on PCA for 7 days to prepare conidial suspensions, and the spore concentration was adjust to a final concentration of 105 spores/ml. The leaves of 3-5-leaf stage of six 5-years-old natural planting M. alba plants were sprayed with conidial suspensions and sterile distilled water, respectively. The petiole of each inoculated leaves of M. alba were secured with sterile wet cotton, and covered with plastic bags to prevent moisture evaporation after incubation. After a 3- to 5- day of inoculation, necrotic lesions appeared on the leaves inoculated with conidial suspensions, whereas no necrotic lesion was observed in the control leaves inoculated with sterile distilled water (Fig S1B). To fulfill the Koch,s postulates, fungi were re-isolated from the margin of necrotic lesions and identified as A. alternata by DNA sequencing the ITS gene. To our knowledge, this is the first report of A. alternata causing leaf spot on M. alba. Because the disease could cause damage to the foliage influencing the greening and ornamental effects of these trees, control measures may need to be implemented during daily management.

First Report of Fusarium falciforme Causing Root and Rhizome Rot of Epimedium sagittatum in China.

Epimedium sagittatum (Sieb.et Zucc.) Maxim., a perennial herb, is an important medicinal plant, rich in flavonoids, and widely used in the treatment of sexual dysfunction, rheumatic disease, and cancers (Tan et al. 2016). In July 2022, a disease of root and rhizome was found on E. sagittatum aged 1-8 years in a planting area (266 ha) of Zhumadian City (32°58'12" N, 114°37'48" E), Henan Province, China. The disease incidence per field (660 m2) was around 10-15% in six randomly surveyed fields planted with about 10,000 E. sagittatum plants each. Symptoms included leaf yellowing, root and rhizome browning, rotting and necrosis, and eventually the whole plant wilted and died. Fifteen plants with symptoms were sampled to isolate the pathogen. Symptomatic tissues were cut into small pieces of 5×5 mm, surface sterilized with 75% ethanol for 30 s, followed by three rinses with sterile double-distilled water (ddH2O). The pieces were then surface disinfected with 0.1% HgCl2 for 30 s, rinsed three times with sterile ddH2O, placed onto potato dextrose agar (PDA) plates and incubated at 28°C in the dark for 5 days. Twelve deferent Fusarium spp. colonies were purified by excising hyphal tip onto PDA for cultivation. Pathogenicity test of all strains was performed. Only isolate GY2 could result in root and rhizome rot of host plant. Colonies of GY2 on PDA had abundant white aerial mycelia with yellow halo. Macroconidia were hyaline, falciform, with a slightly curved apical cell and blunt basal cell, 29.7~45.0 (average 38.3) × 4.5~6.6 (average 5.3) µm (n =50), with 2-3 septa. Microconidia were oval, or reniform, hyaline, 8.4~26.5 (average 16.5) × 2.7~6.0 (average 4.5) µm (n =50), with 0-2 septa. Morphological characteristics of isolate GY2 were consistent with those of the Fusarium solani species complex (FSSC) (Chehri et al. 2015). For molecular identification, a region of the translation elongation factor 1-α (TEF) and RNA polymerase second largest subunit (RPB2) of GY2 were PCR-amplified and sequenced using the primers EF1-728F/986R (Carbone et al. 1999) and RPB2-5f2/7cr (O'Donnell et al. 2010), respectively. The TEF and RPB2 sequences (GenBank accession nos. OR978135.1 and OR978136.1) of GY2 were concatenated for a phylogenetic analysis using the Bayesian method (Zhang et al. 2020). The phylogenetic tree revealed that isolate GY2 clustered with F. falciforme with a credibility value of 99%. Morphological and molecular results support identification of isolate GY2 as F. falciforme. A pathogenicity test was performed on 4-year-old healthy plants grown in pots. Twenty healthy plants were inoculated by pouring a 200 mL conidial suspension (1×106 conidia/mL) around the rhizome. Control plants received 200 mL of sterile ddH2O. All treatments were maintained in a greenhouse at 25±1°C and 80% relative humidity. The assay was conducted three times. After 20 days, similar symptoms as those in the field were observed on the inoculated plants, whereas controls remained asymptomatic. Fusarium falciforme was reisolated from the symptomatic plants and showed the same morphological and molecular characteristics as isolate GY2, fulfilling Koch's postulates. Fusarium falciforme was reported to cause root rot of tobacco (Qiu et al. 2023) and industrial hemp (Paugh et al. 2022). However, this is the first report of F. falciforme causing root and rhizome rot of E. sagittatum. Our study will contribute to the development of strategies for the effective management of this disease on E. sagittatum.

First Report of Paramyrothecium breviseta Causing Target Spot on Amorphophallus muelleri in Yunnan, China.

Amorphophallus muelleri is an Araceae plant with perennial tuber, widely used in food, pharmaceutical and chemical industry due to its richness in glucomannan. In April 2022, an outbreak of a target spot on A. muelleri plantlets was observed in a nursery in Ruili, Yunnan, China. The leafstalks of the diseased plantlets in the nursery turned brown and decayed (Fig.1 A-B), then gradually some water-soaked spots on the true leaves developed along the veins (Fig.1 A). Subquencely, the spots on the true leaves turned dark green to white-grayish in the center, which formed light to dark brown concentric rings with a target-like appearance surrounded by a yellow halo (Fig.1 C). When the temperature was 20-34℃ and the relatively humidity was 25-80%, dark-green to black sporodochia with white hypha appeared on the lower and upper leaf surfaces. Finally, 5-8% of the plants surveyed on 800 m2 of one-year-old plantlets in the nursery showed the symptoms and some plants with infected leafstalks would be death. Similar symptoms were also observed on about 10% of the transplanted plants surveyed on 12000 m2 (1.2 ha) of two-year-old plantlets in the field. Five diseased leaves from five distinct plantlets in the nursery were collected for pathogen isolation. Leaf pieces(5 x 5 mm) were cut from the edge of necrotic lesions, and surface-sterilized with 2.5% sodium hypochlorite for 1 min, 75% ethanol for 30 s, then rinsed 5 times by sterilized distilled water, finally put the leaf pieces on sterilized filter paper for 3-5 minutes to dry them and transferred onto potato dextrose agar (PDA) in petri dishes at 25℃ for three days. Five pure cultures identical to colony and conidial characteristics were isolated from five individual plants. The representative pure culture (M1) was grayish-white and circular colonies were 7.50 cm in diamter after 15 days at 25℃, with dark green concentric rings of sporodochia, the dorsal view of the colonies were yellowish. Conidia were aseptate, smooth, cylindrical, 5.00-6.25 (5.71) x 1.25-1.67 (1.63) µm (n = 20) rounded at both ends. A spore suspension (1 x 106 spores/ml) was prepared by harvesting spores from 15-day-old cultures grown in the dark at 25℃, then a thirty-ml of spore suspension was sprayed on the healthy leaves of 10 two-year-old plantlets. Thirty-ml of sterile water was sprayed on the healthy leaves of another 10 seedlings and used as the control. All seedlings were placed in a nursery at 20 to 34℃ and a relative humidity of 25 to 80%. Similar symptoms (Fig.1 D-F) to those observed in the nursery and field developed on all the 10 seedlings inoculated with M1 after two days, but not on the control leaves. The pathogenicity tests were repeated for three times. Fungal cultures reisolated from the infected leaves were identical to the original colonies and conidia, completing Koch's postulates. The internal transcribed spacer (ITS, primers ITS1 and ITS4) region of ribosomal DNA (OQ553785), calmodulin (cmdA, primers CAL-228F and CAL2Rd)(OQ559103), RNA polymerase II second largest subunit (rpb2, primers RPB2-5F2 and RPB2-7cR) (OQ559104) and β-tubulin (tub2, primers Bt2a and Bt2b) (OQ559105) of M1 had 100%, 98.52%, 98.98% and 98.98% identity with the sequences of Paramyrothecium breviseta CBS544.75 (KU846289 for ITS, KU846262 for cmdA, KU846351 for rpb2, and KU846406 for tub2), respectively. In the phylogenic tree based on ITS, cmdA, rpb2 and tub2 gene sequences, the pure culture M1 clustered with P. breviseta CBS544.75, SDBR-CMU387, DRL4 and DRL3, which has been reported as the pathogen of leaf spot of Coffea arabica in China, C. canephora in China and Thailand (Wu et al. 2021; Withee et al. 2022). Molecular and morphological observations showed the pure culture M1 were P. breviseta (Withee et al. 2022), in addition the disease was named as target spot dueing to the typical target symptom on the leaves. To our knowledge, this is the first report of P. breviseta on A. muelleri from Yunnan, China, as well as worldwide. This disease can caused serious economic losses of A. muelleri dueing to that it can result 5-8% death of the plants in the nursery.

First repot of Chaetomium globosum causing leaf spot on Averrhoa carambola in India.

The Star fruit (Averrhoa carambola L.) was globally distributed, particularly in countries like China, India, Indonesia and was renowned for its abundant vitamin, mineral and antioxidant content (Reddy et al., 2023). In early February 2023, leaf spot symptoms were observed on A. carambola at 2 hectare model orchard, College farm, Agricultural College, Aswaraopet (17.252038 latitude, 81.109574 longitude) and Horticulture nurseries of Aswaraopet, Bhadradri Kothagudem (Dist), Telangana, India. In the surveyed fields (February-2023 to February 2024), the disease was prevalent year round, with varying incidence i.e., July to February (35% to 40% with a yield loss of 35%) and from March to June (20% to 30% with a yield loss of 20%). The disease was initiated as small reddish spots, which grew over 8-10 days to 1-5 mm spots with a necrotic center, dark reddish brown margin and a prominent yellow halo around them, within 17 to 20 days, all spots coalesced, resulting in leaf yellowing and defoliation (SF 1). To isolate pathogen, diseased leaf tissues (n=20) (5 × 5 mm) were surface sterilized (70% alcohol (30 s), 1% sodium hypochlorite (30 s) and sterile distilled water (3 × 60 s), inoculated to PDA media and incubated at 26 ± 2°C with 12 hours photoperiod for 72 hours (Chi et al. 2022). The emerging hyphae from the diseased tissues were sub cultured and incubated on PDA at 26 ± 2 °C. Initially, the fungal colonies appeared white, later transitioning to light brown and finally developed into olivaceous grey colour (SF 2A). The ascospores (n=20) were lemon shaped, pointed at both ends, with a length of 10.3 μm (9.1 to 12.1 μm) and width of 8.6 μm (7.2 to 10.2 μm) (SF 2B and 2C). For further identification of the pathogen, four fungal isolates were cultured in potato dextrose broth and genomic DNA was extracted using the CTAB method. Identification of the pathogen was confirmed through amplification and sequencing the Internal Transcribed Spacer region (ITS), Translation Elongation Factor 1-α (TEF1) and RNA polymerase subunit (RPB1) genes. The resulting sequences were deposited in Gen Bank with accession numbers (OR337915, OR337916, OR337893 and OR337892 for ITS, OR669280, OR669281, OR669282, and OR669283 for TEF, and PP092153, PP092154, PP092155 and PP092156 for RPB1). To study pathogenicity of fungus, five isolates of C. globosum were isolated from five A. carambola plants, grown in potato dextrose broth. Spore suspension of 1x106 spores/mL were prepared by adjusting with hemacytometer and were sprayed onto the leaves of healthy, surface sterilized (50% ethanol), 3 months old A. carambola plants and incubated in greenhouse (T: 26°C; RH: 80%). For each of the five isolates, the spore suspension from each individual isolate was inoculated into three plants and three control plants were maintained for each isolate. The experiment was replicated thrice. After a period of 10 to 12 days, symptoms appeared on the inoculated leaves in the form of reddish spots, similar to original symptoms (Alam et al. 2021) (SF 1H). The fungus isolated from the inoculated leaves was morphologically similarities to C. globosum. Notably, C. globosum, a widespread leaf spot pathogen in crops like A. hypogaea, C. sativa and Pomegranate (Chaffin et al. 2020). To our knowledge, this is the first report of A. carambola leaf spot caused by C. globosum in India and worldwide. The result will be helpful for providing a basis for further research on the control of the disease.

Leaf Spot caused by Alternaria tenuissima on Rhamnella franguloides in China.

The small tree species Rhamnella franguloides, belonging to the genus Rhamnella in the tribe Rhamneae Hook. f. of Rhamnaceae (Hauenschild et al. 2016), is an important medicinal plant commonly used for making tea in China. In August 2023, leaf spot symptoms were observed on R. franguloides in Shangyao county, Yantai, Shandong, China, with a disease incidence of 45-65%. Initially appearing as small dark brown spots on the tip lesions, they later expanded and merged into irregular-shaped brown necrotic lesions with yellowish halos. To isolate pathogen, 20 symptomatic tissue fragments (5 × 5 mm) from ten sampling randomly plants were surface sterilized, placed on potato dextrose agar (PDA) plates, and incubated at 25°C in darkness for 3 days to obtain colonies.10 purified isolates with similar morphological characteristics were obtained by single-spore isolation from the colonies. The representative isolate MR13 was chosen for morphological and molecular analysis. The colonies On PDA medium initially appear as a circular yellow-brown ring with white round margins, ultimately turning into olive green with fluffy aerial hyphae. The conidiophores displayed brown pigmentation, solitary or branched, producing abundant short chains of conidia. The conidia were typically obclavate to obpyriform or ellipsoid in shape, 22.5-64.5 × 12.5-23.6μm in size, with a short conical beak at the apex, zero to three longitudinal septa and one to five transverse septa. Based on cultural and morphological characteristics, the fungus was identified as Alternaria spp (Simmons 2007). Due to morphological traits, five genes (the internal transcribed spacer [ITS], actin [ACT], plasma membrane ATPase [ATP], Alternaria major allergen [Alt a1] and histone 3 [H3]) form MR13 were amplified using primer pairs ITSI / ITS4, ACTDF1/R1, ATPDF1 / ATPDRI, Alt-for / Alt-rev, and H3-1a/1b, respectively (Hong et al. 2005; Lawrence et al. 2013; Lousie and Donaldson 1995). BLASTn analysis failed to confirm the identification of MR13 species based on ITS, ACT, ATP and Alt a1(ITS, OR668512; ACT, OR676918; ATP, OR676917; Alt a1, OR676919). The phylogenetic tree showed that it was closely related to Alternaria alternate, A. tenuissima, and A. destruens. The H3 sequence (OR676920) exhibited 100% similarity to A. tenuissima (OR485421). The phylogenetic tree constructed solely with H3 further confirmed MR13 as A. tenuissima. Pathogenicity tests were conducted by introducing the fungus onto healthy R. franguloides leaves in the field. Fifty leaves (five per plant) were treated with a 20ml suspension containing around 1x10^4 spores/ml, while an equal number of control samples were sprayed with distilled water. Transparent plastic bags were used to cover the treated leaves for 48 hours and maintain humidity. After fourteen days of inoculation, consistent leaf spotting symptoms were observed. In contrast, the control leaves showed no sign of infection. The fungal pathogen was successfully reisolated and identified as A. tenuissima through morphological and sequence analysis, fulfilling Koch's postulates. To our knowledge, this is the first report of A. tenuissima causing leaf spot disease on R. franguloides in China. Identifying the disease's causal agent is crucial for developing effective management strategies.

First Report of Alternaria alternata Causing Leaf Spot on Coffea arabica in China.

Yunnan Province is the major region for coffee (Coffea arabica) cultivation in China, contributing to over 98% of the national yield and total production value (Ma et al. 2022). In May 2023, brown spot symptoms were observed only on the leaves of coffee plants in a field located in Baoshan City (98°52'37.988400"E, 24°58'17.673600"N), Yunnan Province. Notably, brown and irregularly shaped spots initially started on the leaf bases. The spots enlarged and developed concentric rings with dark brown margins, which are often surrounded by yellow halos. Finally, the necrotic spots spread across the entire leaf and caused the leaf to curl and fall off. The incidence of the disease was approximately 3% of the coffee plants (n = 600). The symptomatic leaves collected from 10 plants were sectioned (5 × 5 mm), subjected to surface sterilization with 70% ethanol for 40 s, rinsed with sterile distilled water, air-dried, and transferred to potato dextrose agar (PDA). Fungi with grayish-white, cotton-like aerial mycelia grew after 7 days at 28°C. The older mycelia of these isolates displayed dark gray pigmentation. Single conidia were cultivated on PDA, and 15 morphologically similar monosporic isolates were ultimately obtained. Microscopic observation revealed that these isolates produced branched, septate, transparent and amber mycelium. Brown, elliptical or pear-shaped conidia with 2 to 4 transversal septa and 0 to 3 longitudinal septa, measuring 9.6 to 33.3 long × 6.0 to 15.0 μm wide (n = 30), were observed on potato carrot agar (PCA). Molecular identification of multiple genes, such as ITS (Schoch et al. 2012), RPB2 (O'Donnell et al. 2010) and GAPDH (Berbee et al. 1999), indicated consistent 100% identity among these isolates. Sequences of the representative isolates CFSY1-CFSY5 were deposited in GenBank (acc. nos.: OR351112, PP188577, PP188578, PP294863, PP294864, OR509742, PP215341-PP215344, OR509740 and PP239378-PP239381), revealing 98.35% - 100% homology with distinct Alternaria alternata strains previously deposited in GenBank (acc. nos.: PP110780, MN649031 and OR485338). The multigene phylogenetic analysis positioned isolates CFSY1-CFSY5 within a distinct cluster, alongside diverse A. alternata isolates. Based on morphological and molecular characterizations, the pathogen was identified as A. alternata. To verify its pathogenicity, a conidial suspension (1×106 conidia/mL) of isolate CFSY1 was sprayed on six leaves of three healthy one-year-old C. arabica seedlings. Subsequently, the inoculated seedlings were covered with plastic bags and placed in a growth chamber under controlled conditions (a 14 h daylight period and a 10 h dark period at 28°C). The experiment was repeated three times. After 20 days, typical brown spot symptoms analogous to those originally observed in the field appeared on the leaves in all inoculated plants. Reisolation, morphology identification and DNA sequencing substantiated Koch's postulates. In contrast, control plants treated with sterilized water remained asymptomatic, and no pathogen was reisolated from them. Significantly, A. alternata has been previously reported as the causal agent for leaf spot disease in a diverse variety of woody plant species in China, including Prunus avium (Ahmad et al. 2020), Magnolia grandiflora (Liu et al. 2019) and citrus (Wang et al. 2010). This study represents the first report of brown leaf spot caused by A. alternata specifically on C. arabica in China, enriching the contents of fungal pathogens under Chinese coffee cultivation conditions.

First Report of Black Leaf Spot Caused by Epicoccum latusicollum on Chinese Yam in China.

Chinese yam (Dioscorea polystachya Turczaninow cv. Tiegun), which belongs to the family Dioscoreaceae, is widely cultivated throughout China due to its high economic and medicinal value. In June 2023, black leaf spots on Chinese yam (cv. Purple 1) were observed in Nanchang city (28.45° N, 115.49° E) of Jiangxi province, southeastern China. The incidence of the disease ranged between 70 and 85% of plants, and up to 30% of the leaves per plant were affected in the field over a 2-week period of study. Infected foliage displayed brown necrotic lesions, elliptical or irregular, with yellow halo at the edge of the lesion (0.5 to 3 cm diam.). To identify the causal agent, 32 symptomatic leaves of eight symptomatic plants were collected. Small pieces from the margin of necrotic leaf tissue (about 3 x 3 mm) were surface sterilized in 75% ethanol for 30 s followed in 0.1% HgCl2 for 1 min, and washed three times with ddH2O. Then, the pieces were transferred onto potato dextrose agar (PDA) plates and incubated at 26°C for 3 days with a 12-h light-dark cycle. From the 32 isolates, 21 exhibited similar morphology after hyphal tipping resulting in an isolation frequency of 65.6%. Colonies on PDA were initially white aerial hyphae but became grayish with age, and a reddish orange pigment on the underside. After 16 days of incubation, pycnidia were observed, which were dark, spherical or flat spherical, and 64.1 to 172.5 µm (n = 25) in diameter. Conidia were ellipsoidal, aseptate, hyaline, and 4.1 to 5.6 × 1.8 to 2.7 µm (n = 80). In addition, a blackish green discoloration was produced on malt extract agar (MEA) using the NaOH spot test. The isolates were tentatively identified as Epicoccum spp. based on morphological characteristics (Chen et al. 2017). Isolate AYZ-1 was randomly selected for identification and pathogenicity testing. Genomic DNA of the isolate (AYZ-1) was extracted and amplified by polymerase chain reaction (PCR) using ITS1/ITS4 for the internal transcribed spacer (ITS) region (White et al. 1990), Btub2Fd/Btub4Rd for the β-tubulin (TUB) region (Woudenberg et al. 2009), LROR/LR7 for the large ribosomal RNA gene (LSU) region (Rehner and Samuels 1994), and RPB2-5F2/fRPB2-7cR for RNA polymerase II second largest subunit (RPB2) region(Liu et al. 1999), respectively. The concatenated sequences (GenBank Accession No. OR574165, OR567827, OR574166, OR567828, respectively) shared 99.8 to 100% identity withEpicoccum latusicollum(OP788080, MN329871, OR428532, and OL422485, respectively). A neighbor-joining phylogenetic tree was generated based on the concatenated sequences in MEGA7, placed isolate (AYZ-1) within E. latusicollum. To fulfill Koch's postulates, healthy leaflets from three one-year-old Chinese yam (cv. Purple 1) were used as inoculation materials, using isolate AYZ-1. Two sites of each leaf were wounded with a sterile needle and covered with a piece of cotton drenched with 200 µL spore suspension (106 conidia/mL) on the left sides, while sterilized water served as the control on the right sides of leaves. All inoculated leaves were covered with clear polyethylene bags for 24 h. Plants were grown outdoors at a daily average temperature of 26°C with relative humidity over 45%. After 7 days of incubation, the leaves showed the same symptoms as the original diseased leaves. The E.latusicollumisolate was re-isolated from diseased leaves and confirmed by morphology and sequencing analysis, fulfilling Koch's postulates. E.latusicollumhas beenpreviously reportedtocauseblack root on yam inChina'ssouth-westernprovince ofSichuan(Han et al. 2019). Meanwhile,leafspothave been reportedon many plants by this genus, such astobacco(Guo et al. 2020) andbanana(Liu et al. 2023). According to our knowledge, this is thefirstreportofE.latusicollumcausingblack leafspoton Chinese yam inChina. This finding will provide an important reference for understanding the biology of E. latusicollum and the distribution of the disease, but more research is needed to determine if management is warranted.

First Report of Colletotrichum cordylinicola Causing Anthracnose on Cordyline fruticosa in China.

Cordyline fruticosa is a shrub plant, commonly used in landscape, and distributed in the tropical regions of southern China. In September 2022, anthracnose symptoms were found on this species in Nanning, Guangxi, China. The disease incidence was between 30% to 80% and disease severity was 10% to 30% in five surveyed planting areas. The symptoms initially appeared as small, round, brown spots on leaves. As the disease developed, the lesions turned gray-white with brown borders and yellow halos. Some spots coalesced into larger irregular shapes and even leading to leaf blight. Small segments of the diseased tissues (3×3 mm) were cut from the leaves, surface-sterilized by dipping in a 1% sodium hypochlorite solution for 1 min, rinsed three times with sterile distilled water, and plated on potato dextrose agar (PDA). These plates were incubated at 28°C in the dark for 5 days. Ten fungal isolates with similar morphology were consistently isolated from these diseased tissues. The colonies on PDA were initially white with sparse aerial mycelia and turned pale orange with abundant orange conidial masses on the center after 8 days of culture. The reverse color was pale orange. No sclerotia or setae were found in culture. Conidia were single-celled, hyaline, straight, cylindrical with round ends, and 12.2 to 17.8 µm long (mean 14.9 µm) and 3.9 to 7.3 µm wide (mean 4.8 µm, n=50). The morphological characteristics of these isolates were similar to the Colletotrichum cordylinicola (Sharma et al., 2014). Genomic DNA of two isolates Z3 and Z4 generated from monospore culture was extracted using a fungal DNA extraction kit (Solarbio, Beijing, China). Partial sequences of internal transcribed spacer (ITS), partial actin (ACT), chitin synthase (CHS-1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and beta-tubulin (TUB2) were amplified using the primer pairs ITS1/ITS4, ACT-512F/ACT-783R, CHS-79F/CHS-345R, GDF1/GDR1, and BT2A/BT2B (Lin et al., 2022), respectively. All the sequences (GenBank accession nos. OQ509909, OQ509910, OQ658690, OQ658691, and OK649310 to OK649314) showed 99% to 100% identity with those of C. cordylinicola in GenBank database. A phylogenetic tree based on concatenated sequences of ITS, ACT, CHS-1, TUB, and GAPDH using maximum likelihood analysis by MEGA X software revealed that Z3 and Z4 clade with reference strains of C. cordylinicola (OJX010226 and MK935473). Based on morphological observation and multi-gene sequence analysis, the isolates were identified as C. cordylinicola (Phoulivong et al., 2010). To assess their pathogenicity, conidial suspensions (106 conidia/ml) of C. cordylinicola were inoculated onto 10 healthy living leaves wounded by slight puncturing (10 μl/wounded spot). Control leaves were treated with sterile water. All inoculated and control plants were maintained under high relative humidity (~90%) and 28℃ in a climate chamber. After 8 days, all the inoculated leaves showed brown lesions resembling natural symptoms, whereas the control group remained symptom-free. The same fungus was re-isolated from the symptomatic leaves, thus completing Koch's postulates. C. cordylinicola is a species of the C. gloeosporioides complex (Weir et al., 2012). It has been reported to cause anthracnose on C. fruticosa in USA and Thailand (Phoulivong et al., 2010; Sharma et al., 2014). To our knowledge, this is the first report of C. cordylinicola causing anthracnose on C. fruticosa in China. Knowing the causal agent is essential to control the serious disease effectively.

First Report ofLasiodiplodia hormozganensisCausing Leaf Spot Disease onAreca catechuin China.

The areca palm (Areca catechu L.) is one of the significant cash crops in Hainan Province (China), and a valuable tropical medicinal plant (Cao et al. 2020). In September 2020, spots were found on about 80% of the area of the leaves in a 1,000-acre plantation of areca palms in Haikou City, Hainan Province, and the average incidence was 25%. Initially, Elliptical or irregular dark brown spots appeared on the leaves, with an average size of about 1.5 cm2. With the further expansion of the disease, the spot turned light brown in the center with dark brown edges and a prominent yellow halo. Later stage of the disease, the spots became grayish-white in the center, with obvious whorls, on which many small black spots (pycnidia) were scattered. Eventually the leaves dried out. Ten leaves with typical symptoms were collected from the field. Lesion marginal tissues (5×5 mm2) were surfaced sterilized in 75% ethanol for 20 s, followed by 4 min in 1% NaClO, rinsed 3 times with sterile water, plated on PDA and incubated at 28 ℃. A fungus was isolated with a 98% isolation rate. This strain was named HNAC-5. Subcultures were 80 mm in diameter, white, villous, and neatly edged, after two days of incubation at 28 ℃ in dark. Pycnidia were solitary or clustered in stromata, with orifices that oozed black liquid. Conidiogenous cells were colorless and short cylindrical. Conidia unicellular, initially hyaline, aseptate, ellipsoid to ovoid with granular content, becoming pigmented, 1-septate with longitudinal striations, and measuring 20-31×10-13 μm (n=100). These morphological characteristics were similar to Lasiodiplodia spp. (Abdollahzadeh et al. 2010). The internal transcribed spacer region of rDNA, β-tubulin gene, and translation elongation gene were amplified using ITS1/ITS4, Bt2a/Bt2b, and EF1-728F/EF1-986R primers, respectively (Alves et al. 2008; Glass and Donaldson 1995; White et al. 1990). The resulting sequences were deposited in GenBank under accession numbers OR272043, OR282568, and OR282567. BLAST analysis showed that the three sequences of HNAC-5 were more than 99% similar to strain CBS 124709 of L. hormozganensis. Phylogenetic analysis was performed using the maximum likelihood method based on the three-gene combined dataset with MEGA 7.0 software. The results indicated that HNAC-5 was grouped in the same clade as otherL. hormozganensis Abdollahzadeh, Zare & A.J.L. Phillips. Pathogenicity test was carried out on 15 healthy leaves by in vivo inoculation. Ten leaves were pricked with a sterile needle and divided into group 1 and 2. The remaining five uninjured leaves were group 3. Group 1 and 3 were inoculated with 5-mm-diameter mycelial plugs obtained from 3-day cultures, and group 2 treated with PDA plugs served as controls. Fifteen leaves were cultured at 28°C and 100% relative humidity. After 5 days, leaves of group 1 showed symptoms of the disease and on the tenth day showed the same symptoms as the initial onset of the disease in the field, while leaves of Group 2 and 3 showed no symptoms. Pathogenicity tests were conducted three times with the same results. L. hormozganensiswas re-isolated from the inoculated symptomatic leaves, thus, Koch's postulates were confirmed. In China, L. hormozganensis has been reported to cause Bougainvillea spectabilis Willd. branch blight disease (Li et al. 2015), and Scaevola taccada (Gaertn.) Roxb. leaf spot disease (Zhang et al. 2020). To our knowledge, this is the first report of L. hormozganensiscausing leaf spot disease onA. catechuin China.

First report of Phaeosphaeria caricicola causing leaf spot on Belamcanda chinensis in China.

Belamcanda chinensis (L.) Redouté, a member of the Iridaceae family, is globally well-known for its medicinal value as clearing away heat, detoxifying, detumescence and pain (Qin 2000). In 2021, spots were observed on 40% B. chinensis leaves and about 28 disease index in Wanzhou District (30°32'N; 108°22'E) of Chongqing. Initial symptoms appeared as circular yellow white, sunken spots lesions, and then expanded into irregular lesions, the center of the spots was beige, external layer was light brown and surrounded by yellow halo. Symptomatic leaf tissues (5 × 5 mm) were cut from the infected margin, surface sterilized with 75% ethanol for 1 min, washed with 3% sodium hypochlorite for 3 min, rinsed three times with sterile water, placed on potato dextrose agar (PDA) medium incubated at 25°C for 7 days in the dark, forty isolates with similar morphology were obtained. Three isolates (SG9、SG20 and SG33) was selected for subsequent research. Colonies color changed from beige to light brown color after 14 days on PDA medium. Fungal colonies transformed from beige to brown at the edges after 28 days and light brown on top. Ascomata dark brown, ellipsoidal to globose 116.6 to 253.3 × 89.6 to 172.6 μm in diamensions. Asci stipitate, cylindrical with obtuse ends, and 69.1 to 114.7 × 10.2 to 24.1 μm (n = 30) in size, with eight overlapping linearly biseriate ascospores. Ascospores brown, narrowly fusiform, straight or slightly curved with three transversely septate, slightly constricted at septa, and 9.7 to 12.6 × 27.6 to 32.6 μm (n = 30). These characteristics are consistent with Phaeosphaeria sp. reported by Quaedvlieg et al in 2013. DNA was extracted from representative isolates. The internal transcribed spacer (ITS) region, the large subunit rDNA (LSU), the small subunit rDNA (SSU) and the RNA polymerase II second largest subunit (RPB2) genes were amplified for Polymerase chain reaction (PCR) by used ITS1/ITS4, LR5/LROR, NS1/NS4, and RPB2-5f2/RPB2-7cr primers (White et al. 1990; Vilgalys et al. 1990; Qi M W. et al. 2008; De G. J. et al. 1992). The sequences were submitted to NCBI GenBank: SG-G9 (ITS, OR701701; LSU, OR701699; SSU, OR701700; RPB2, OR738464); SG-G20 (ITS, OQ748032; LSU, OQ780728; SSU, OQ780723; RPB2, OQ779979); SG-G33 (ITS, OQ748033; LSU, OQ780729; SSU, OQ780722; RPB2, OQ779980). A phylogenetic analysis revealed a 99% similarity to the Phaeosphaeria caricicola CBS 603.86 (ITS, KF251182; LSU, GQ387590; SSU, GQ387529; RPB2, KF252189) sequences. Mycelial agar plugs (5-mm diameter) from a 7-day-old PDA culture of a fungal isolate were placed onto pinpricked leaves of three two-year-old B. chinensis plants. While the sterile PDA plugs inoculated in pinpricked leaves of B. chinensis as controls. Inoculated plants were placed in a greenhouse at 25°C and remained 95±1% relative humidity. The inoculated leaves of treatment developed symptoms after 20 days, whereas no symptoms occurred on controls, fulfilling Koch's postulates. The experiments were repeated three times. The fungus was re-isolated and was identical to original isolate by morphologically and molecularly. As far as we know, P. caricicola can cause diseases on carex plants and has been found in Switzerland. This is the first report of P. caricicola causing leaf spot on B. chinensis in China. Along with recording the occurrence of this disease, plant disease management strategies need to be established to reduce losses.

First report of Colletotrichum siamense causing leaf spot on silver date palm in China

Silver date palm (Phoenix sylvestris Roxb.) is an important ornamental plant in southern China. In October 2021, a serious anthracnose-like leaf spot of silver date palm was observed at a park in Haikou City, China. Some brown spots with yellow halos around the lesions appeared on the middle and lower leaves of the tree crown. The disease incidence reached 100% of trees and 32.8% of leaves. The pathogen was identified as Colletotrichum siamense by morphological characters, pathogenicity tests and multi-locus phylogenetic analyses combined with ITS, gapdh, tub2, cal and ApMat sequences. This is the first report of C. siamense causing the leaf spot of silver date palm in China.

First Report of Colletotrichum fructicola Causing Anthracnose on Epimedium sagittatum (Sieb. et Zucc.) Maxim. in China.

Epimedium sagittatum (Sieb.et Zucc.) Maxim., belonging to the family Berberidaceae and genus Epimedium, is a perennial herb widely studied for its anti-osteoporosis, anti-cancer, and anti-sexual-dysfunction effects in Asian countries (Tan et al. 2016; Zhang et al. 2016). High levels of bioactive chemicals in Epimedium spp. has endowed it with important clinical and commercial values (Liu et al. 2013). In September 2021, a leaf disease was found in Zhumadian City, China (32°58'12" N, 114°37'48" E). Survey statistics indicated that disease prevalence in a 266-ha planting area was approximately 29.6%. The lesions appeared at the leaf tips, gradually enlarged, and were brown with a yellow halo. Further, the lesions were dry with distributed black spots. Thirty infected leaves collected from five sites within the planting base . The collected leaves were cut into 5×5 mm pieces , surface-sterilized in 75% alcohol for 15 s, triple washed with sterile ddH2O, disinfested with 0.1% HgCl2 solution for 30 s (Liu et al. 2021), triple washed again with sterilized ddH2O, and then placed onto PDA and incubated in the dark for 3 d at 28°C. Subsequently, five fungal strains were purified; among them, only the isolate HY3-2 infected the host plant and was selected for further morphological characterization. The colonies of HY3-2 initially appeared white, their mycelia became gray at the center after 4 d, and orange-red conidial clumps appeared in them after 7 d. Conidia (10.0-19.5 μm × 4.5-5.6 μm, n=50) were single celled, nearly spherical or stick-shaped and colorless. Morphological characteristics of the isolate were consistent with those of Colletotrichum species. Additionally, glycerol-3-phosphate dehydrogenase (gapdh), actin (act), calmodulin (cal), β-tubulin 2 (tub2), and chitin synthase-1 (chs-1), (Weir et al. 2012) were amplified and sequenced using the primers GDF/GDR, ACT-512F/783R, CL1C/CL2C, T1/Bt2b, and CHS-79F/354R, respectively for molecular identification. The resulting sequences were deposited in GenBank: gapdh (ON351609), act (ON351608), tub2 (ON351610), chs-1 (ON532788), and cal (ON532787). Phylogenetic analyses were performed by concatenating all the sequenced loci using the Bayesian method (Zhang et al. 2020). The phylogenetic tree showed that the isolate belongs to C. fructicola clade with a credibility value of 85%.To satisfy Koch's postulates, a conidial suspension (106 conidia/mL) of the isolate HY3-2 were prepared with sterile ddH2O to infect the leaves. Ninety healthy leaves from 30 plants in pots were punctured using a sterilized needle (Huang et al. 2022), and inoculated by spraying the conidial suspension on the leaves in a greenhouse at 25°C and 80% relative humidity. In the control plants, the suspension was replaced with water. After 7 d, the inoculated plants showed symptoms similar to those of the original infected plant, whereas the control showed no symptoms. C. fructicola was isolated and identified again as previously described. A pathogenicity test was also conducted in the field using the same method as that used in the greenhouse in July 2022, the results of which were consistent with those of the greenhouse. In China, C. fructicola has been reported on Walnut (Wang et al. 2022), Punica granatum (Hu et al. 2023) and others. To our knowledge, this is the first report of C. fructicola causing anthracnose in E. sagittatum in China. This report provides an important basis for further disease control research.

First Report of Cercospora Leaf Spot Caused by Cercospora cf. flagellaris on Lonicera japonica Thunb. in Henan, China.

Honeysuckle flower (Lonicera japonica Thunb.) is a traditional Chinese medicinal plant. It is perennial and widely cultivated in China, Japan and Korea. From late August to October in 2021 and 2022, leaf spots symptoms were observed on L. japonica in different planting fields in Yuzhou, Yuanyang and Fenqiu districts, Henan province, China. The disease incidence was above 85% which reduce photosynthesis. Early disease symptoms appeared as small, circular to elliptical, brown spots on the leaves and later the lesions (1 to 5 mm × 1 to 4 mm) slowly developed yellow haloes. The different brown lesions seldom merge and form larger irregular lesions. Small fragments (3 to 5 mm) of leave tissue were excised from the lesion margins and surface-sterilized in 3% NaClO for 3 min, followed by three washes with sterile distilled water, and then placed on potato dextrose agar (PDA) and incubated at 25°C in the dark for 5 days. A total number of 8 cultures were obtained and purified by single-spore subcultures on PDA for morphological identification. The colonies on PDA were whitish to gray, with cottony aerial mycelium. Conidiophores were fasciculate, olivaceous brown, straight or geniculate, uniform in width, multiseptate, and ranged from 290 to 700 μm (560 μm on average, n = 20). Conidia were hyaline, slightly curved or straight, needle shaped, truncate at the base, and terminal at the tip, 3 to 17-septate, and measuring 150 to 240 μm (180 μm on average, n = 20). The morphological features were consistent with Cercospora cf. flagellaris Ellis & G. Martin (Groenewald et al. 2013). The genomic DNA was extracted using CTAB method. The nuclear ribosomal internal transcribed spacer region (ITS), portions of the actin (ACT), histone H3 (HIS3), and translation elongation factor 1-α (TEF1) genes were amplified using primers ITS1/ITS4 (Groenewald et al. 2013), ACT-512F/ACT-783R (Carbone and Kohn 1999), CYLH3F/CYLH3R (Crous et al. 2006), and EF1-728F/EF1-986R (Carbone and Kohn 1999). The resulting 537-bp ITS, 226-bp ACT, 410-bp HIS3, and 306-bp TEF1 sequences of isolate JDJ002 were deposited in GenBank (accession nos. OR492367, OR548247, OR548248 and OR548248, respectively). Sequence analysis revealed that ITS, ACT, HIS3 and TEF1α sequences exhibited ≥99% of identity with the ITS (KP896013), ACT(KP895965), HIS3(MK991295) and TEF1 (MN180408) sequences of C. cf. flagellaris, respectively. A pathogenicity test was conducted on healthy of L. japonica leaves. The healthy leaves pricked from L. japonica plants, rinsed in autoclaved distilled water three times and dried with distilled filter paper. Then twelve healthy leave were inoculated with a mycelial plug (0.4 cm diameter) harvested from the periphery of two week-old colony. As negative control, leaves inoculated with PDA medium plugs. Inoculated leaves were covered with plastic bags to maintain high relative humidity and incubated at 25°C in growth chamber. After 7 days, the inoculated leaves showed symptoms identical to those observed in the field under natural conditions, whereas negative control remained symptom-free. Re-isolation of the fungus from lesions on inoculated leaves confirmed that the causal agent was C. cf. flagellaris. Pathogenicity tests were repeated three times by the same methods with the same results. To our knowledge, this is the first report of C. cf. flagellaris except Cercospora rhamni Fack., Alternaria alternata, Corynespora cassiicola or Phomopsis sp. causing leave spots on L. japonica in China.

Métodos extractivos y aislamiento de ácido aristolóquico a partir de matrices naturales.

Introduction: Medicinal plants from the Aristolochia genus have traditionally been employed to treat various pathologies. However, these plants are known to contain substantial quantities of aristolochic acids, both type I and II, which have been linked to the development of nephropathies and high-risk cancers due to their inherent toxicity. Thus, the utilization of these plants is considered a serious health risk for individuals. Methods: This study aimed to identify effective extraction and isolation methods for aristolochic acid from natural matrices to obtain a standardized extract of the Aristolochia plant. The Zaragoza liana (Aristolochia) from Puyo and Macas- Ecuador, was selected for analysis. The extraction methods employed included supercritical fluids and ultrasound-assisted extraction, with methanol as the solvent. Subsequently, metabolites obtained from these processes were identified using thin-layer chromatography (TLC) to confirm the presence of aristolochic acid. Results: The outcomes revealed that aristolochic acid exhibited a retention factor within Rf 0.61-0.77. These results were observable through yellow-colored bands under white light, appearing dark or gray under UV light of 254 nm and displaying black coloration with a yellow halo under UV light of 366 nm. These distinctive characteristics provided conclusive evidence of the presence of aristolochic acid in the analyzed samples. Conclusion: This study has established effective methods for extracting and isolating aristolochic acid from Aristolochia plants. The results obtained through thin-layer chromatography confirm the presence of this compound in the analyzed samples, underscoring the concern regarding the toxicity associated with these medicinal plants. These findings emphasize the importance of standardizing Aristolochia extracts and highlight the potential risks linked to their consumption in human health.

First Report of Apiospora arundinis Causing Leaf Spot on Polygonatum cyrtonema Hua in China.

Polygonatum cyrtonema Hua, which is a perennial herb of Liliaceae, can be used as clinical Chinese medicine for treating diabetes and asthma. It is widely cultivated in China, with 700 ha planted in Tonggu County, Jiangxi province (Chen et al. 2022). In June 2022, leaf spot symptoms were observed on P. cyrtonema in Tonggu county (28°71'42″N, 114°56'19″E), and the disease incidence was estimated to be above 35%. In the early stages of infection, small brown spots appear on the edge or tip of the leaves. As the lesion matures, the spots gradually expand to form wedge-shaped or elliptic to irregular lesions with brown edges and yellow halos. To identify the pathogen species, leaf pieces (5 × 5 mm) from the lesion borders were surface sterilized in 75% ethanol for 30 s, followed by 2% NaClO for 2 min, then rinsed with sterile distilled water for 3 times and dried with sterile filter paper. The tissues were placed on potato dextrose agar (PDA) and incubated at 25°C. Pure cultures were obtained by monosporic isolation, and the representative isolates, HJYB2-1, HJYB2-2 were used for morphological studies and phylogenetic analyses. Colonies on PDA of the two isolates were white with fluffy aerial mycelia. The hyphae were smooth, hyaline, and branched. The conidiophores were hyaline or pale brown and produced conidiogenous cells. The conidiogenous cells were pale brown, smooth, and ampulliform, 5.8-11.7 × 3.0-4.9 μm (n=50). The conidia were brown, smooth, and ellipsoidal to spherical, 4.7-7.3 × 2.3-4.5 μm (n=50). Morphological features were similar to Apiospora arundinis species complex (Crous et al. 2013, Pintos et al. 2021). For molecular identification, the ITS, TUB2 and TEF 1-α genes were amplified with the primer pairs ITS1/ITS4, T1/Bt-2b and EF1-728F/EF-2 (White et al. 1990), respectively. The generated sequences were deposited in GenBank with accession numbers OR229623, OR229624 (ITS), OR260104, OR260105 (TUB2), and OR290789, OR290790 (TEF1-α). Maximum likelihood and Bayesian posterior probability analyses using IQtree v. 1.6.8 and Mr. Bayes v. 3.2.6 with the concatenated sequences placed HJYB2-1 and HJYB2-2 in the clade of A. arundinis. The two isolates were identified as A. arundinis based on morphological and multilocus phylogenetic analyses. For pathogenicity testing, healthy leaves of six P. cyrtonema plants (five leaves each, n=30) in the field were pin-pricked with a sterile needle and inoculated with 100 µL spore suspension on the wound (1×106 conidia per mL). Healthy leaves of another three P. cyrtonema plants (five leaves each, n=15) in the field were pin-pricked with a sterile needle, inoculated with sterile distilled water and served as the control. All the inoculated leaves were covered with plastic bags to keep a high-humidity environment with a temperature of about 28℃. The test was repeated three times. More than 90% of inoculated leaves showed similar symptoms to those observed in the field, whereas control leaves were asymptomatic for 7 days. Apiospora arundinis was reisolated from the leaf lesions on the inoculated plants. No pathogenic fungus was isolated from the control leaves. A. arundinis has been reported causing disease on Camellia sinensis (Thangaraj et al. 2019), Prunus persica (Ji et al. 2020), Saccharum officinarum (Liao et al. 2022) but has not previously been reported causing disease on P. cyrtonema. To our knowledge, this is the first report that A. arundinis can cause leaf spot on P. cyrtonema in China. Our result should help with future monitoring and control of this 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.

Colletotrichum kahawae subsp. cigarro causing leaf spot disease on arecanut, Areca catechu L. in India: A first report

Arecanut is a prominent cash crop in India, known for its potential to significantly boost per capita income. The presence of fungal diseases poses a significant constraint to arecanut cultivation. In October 2019, a severe outbreak of leaf spot disease with 90°% disease incidence was recorded in the farmer's arecanut gardens from Kalasa hobli, Chikkamangaluru, Karnataka, India. Leaf spot symptoms started as small, irregular, light-dark brown spots (3–10 mm in diameter) with a yellow halo on the leaf lamina, which later coalesced to form larger necrotic lesions and a blighted appearance. The symptoms were also observed on leaf sheaths and nuts. In the present study, we identified and characterized the pathogen causing leaf spot disease using cultural, morphological, molecular characterization using multi-gene phylogeny, and pathogenicity assays. Microscopic, and molecular identification of CPCRICkc7G isolate retrieved from the leaf spot infected sample established the association of Colletotrichum kahawae subsp. cigarro as the causal agent. Pathogenicity assays confirmed the Koch's postulate by reproducing the field observed symptoms. To best of our knowledge, this confirms the first report of C. kahawae subsp. cigarro causing leaf spot disease on arecanut in India. The findings from this current study play a pivotal role in enhancing the knowledge of arecanut diseases in India, and lay an essential foundation for devising effective disease management approaches to curtail the disease spread.

Identifying the fungal diseases of African Yam Bean (Sphenostylis stenocarpa) and their incidence in south-west Nigeria

The incidence of fungi infecting Sphenostylis stenocarpa ([Hochst ex. A. Rich.] Harms) commonly known as African yam bean (AYB) were surveyed in Oyo, Ondo, Ekiti, Osun, and Ogun states within SW Nigeria during the 2018 planting season. The common field symptoms across all sites were tiny spot, brown spot, leaf blight, brown spot with yellow halo, necrotic lesion on foliage, and brown spot on the pods. Of these 1005 isolations, 14 fungal species were commonly identified after their purification: Aspergillus sp, Botrytis sp, Colletotrichum gloeosporioides, Curvularia lunata, Trichoderma harzianum, Macrophomina phaseolina, Pestalotia sp, Phoma sp, Fusarium verticillioides, F. oxysporum, F. solani, Botryodiplodia theobromae, Choanephora curcubitarium and Nigrospora spp. based on morphological characteristics. Phoma sp and C. gleosporoides had highest frequency of occurrence 69.9% and 51.9% at early and mature stages, respectively. To conform to Koch's postulates, the pathogenicity of 15 exemplar strains of the most abundant fungal species were confirmed under greenhouse conditions. The identities of Colletotrichum sp., Aspergillus sp., Curvularia aeria, Macrophomina phaseolina, Didymella sp., Pestalopsis sp, Lasiodiplodia theobromae, F. solani and F. oxysporum were confirmed based on internal transcribed spacer (ITS) sequencing and comparisons with the Genbank database. AYB germplasm from curated seed banks and farmer donated landraces were grown at the same site in 2020 and identical fungi were isolated. Further, genotypes with reduced disease incidence were identified. This first study to reveal the diversity of fungi associated with AYB in SW Nigeria that could inform disease management practices.

First report of Epicoccum nigrum causing leaf spots on Campomanesia guazumifolia (Camb.) Berg in Brazil.

Campomanesia guazumifolia is a native tree that produces fruit that can be consumed fresh or used by industry (Donadio et al., 2002). In February 2022, in the experimental area of the Universidade Tecnológica Federal do Paraná - Brazil, disease was observed in 22 trees, with 50% to 80% severity in crown leaves. Symptoms were small, irregular, or circular-shaped, dark-brown lesions with yellow halos (Figure S1). As the disease progressed, the lesions increased in size, without distinction between mature and young tissues, causing complete leaf wilting. Twenty symptomatic leaves from 11 trees grown in the same orchard line were collected. For fungal isolation, the leaf surfaces were disinfected with 0.5% NaOCl solution for 1 min, rinsed in sterile distilled water, and dried on sterile filter paper. Five fragments of diseased leaf tissue were placed on a potato dextrose agar medium. The morphological characteristics of the colony, such as filamentous mycelium and golden yellow on the upper part, with the presence of circular to ovoid and multicellular conidia (mean 21.00 µm x 24.45 µm, n = 30) of the nine isolates, coincided with the description of the fungus of the genus Epicoccum (Valenzuela-Lopez et al., 2018). Further identification of one of these nine isolates was confirmed by amplifying and sequencing three loci (ITS, β-tubulin, and RPB2) using the ITS1/ITS4, Bt2a/Bt2b, and 5F2/7cR primer pairs, respectively (White et al., 1990, Glass and Donaldson, 1995, O'Donnell et al., 2007). A single representative isolate (Cgen01) was analyzed and submitted to GenBank (OR020968, OR079879, and OR079878). The Bayesian Inference was used to reconstruct the phylogenetic trees (Figure S2), starting from random trees for 5,000,000 generations, using MrBayes v. 3.2.1 (Ronquist et al., 2012). The isolate clustered together with the isolate of Epicoccum nigrum (Chen et al., 2017) with a high posterior probability (0.98). For the pathogenicity tests, four young, healthy branches containing 20 leaves were spray-inoculated with 1.5 mL of conidia suspension of Cgen01 (106 conidia mL-1), covered with perforated transparent plastic bags, and moistened with distilled water in the orchard. The air temperature ranged from 14ºC to 25ºC. Sterile distilled water was used as a control. Three replicates (pathogen and control) on different trees were evaluated. After five days, the fungus was re-isolated from the symptomatic lesion, showing morphological characteristics similar to those of Cgen01. Control branches did not show fungal growth. The inoculation test was conducted twice and similar symptoms were observed. This is the first report of leaf spots caused by E. nigrum on C. guazumifolia in Brazil. E. nigrum, an endophytic fungus described as a mycoparasite, showed phytopathogenic behavior in this study, causing spots and loss of leaves in C. guazumifolia, drastically reducing the production of photoassimilates and affecting the quality of the fruits.