First report of Colletotrichum fioriniae causing Leaf Anthracnose on Spiraea japonica in China

Aucuba japonica is widely planted in China for landscape use, particularly in gardens and parks. In September–October 2024, leaf blight symptoms were observed on A. japonica in Meicheng Park, Nanyang City, Henan Province (32°59′21″ N, 112°32′54″ E). A subsequent survey across different sections of the park recorded a disease incidence of 39% (n = 100 plants). Initial symptoms consisted of small dark-brown spots on leaves that enlarged into irregular leaf-blight lesions; in severe cases, lesions coalesced and caused defoliation and noticeable aesthetic decline. Diseased leaves (n = 20) were collected and surface sterilized. From each leaf, two lesion margins (3–3 mm²) at the interface of healthy and diseased tissue were excised and placed on potato dextrose agar (PDA). In total, 40 fungal isolates were obtained from symptomatic tissues. Thirty-six isolates showed typical Colletotrichum morphology. Isolates shared similar morphology, and three strains (SJSH09, SJSH12, SJSH23) obtained from symptomatic plants located in different sectors of Meicheng Park were selected for detailed analyses. Colonies on PDA were white to pale gray with a cottony texture, typically exhibiting subtle concentric zoning on the colony surface. Conidia were hyaline, aseptate, smooth-walled, and cylindrical to fusiform, measuring 7.3–22.8 × 2.6–5.3 μm (n = 100), consistent with Colletotrichum morphology (Talhinhas et al. 2023). For molecular identification, five loci—the rDNA internal transcribed spacer (ITS), glyceraldehyde-3-phosphate dehydrogenase ( gapdh ), chitin synthase (chs-1), actin (act), and β-tubulin (tub2)—were amplified from the three strains (Zhang et al. 2023). Sequences have been deposited in GenBank under accession numbers PV636156–PV636158 (ITS), PV641785–PV641787 (gapdh), PV641782–PV641784 (chs-1), PV641788–PV641790 (act), and PV641794–PV641796 (tub2). BLASTn analyses showed that all loci had 99–100% identity to Colletotrichum aenigma reference strains, with no conflicting top hits to other species. A maximum-likelihood phylogeny based on the concatenated dataset (ITS, gapdh, chs-1, act, tub2) and ex-type reference sequences was inferred in MEGA. The three strains formed a clade with the C. aenigma ex-type ICMP 18608, TJ325, and XZC02, clearly distinguishing them from other Colletotrichum species. Morphological and molecular evidence together support identification of all three strains as C. aenigma. Pathogenicity was assessed by spraying conidial suspensions (10⁶ conidia mL⁻¹) onto unwounded leaves of five A. japonica plants; five additional plants were sprayed with sterile water as mock-inoculated controls. Plants were incubated at 28 °C and ~90% relative humidity. 14 days post-inoculation, lesions resembling those in the field developed on all inoculated plants, whereas no symptoms appeared on controls. The pathogen was re-isolated. ITS sequences of these re-isolated were 100% identical to those of the inoculated strains, confirming that the same pathogen were recovered and thereby fulfilling Koch’s postulates. Anthracnose of A. japonica caused by Colletotrichum boninense has been reported previously from Guizhou Province, China (Liu et al. 2022). This is the first report of C. aenigma causing leaf anthracnose on A. japonica. Given the widespread use of A. japonica as an ornamental shrub in urban landscapes, this newly recognized anthracnose disease poses a potential threat to plant health and aesthetic value. Accurate identification provides a basis for reliable diagnosis and for the development of targeted fungicide applications and other management strategies to protect ornamental plantings.

Buckwheat (Fagopyrum esculentum), belonging to the Polygonaceae family, is one of the most important "functional food" crops in China. In fall 2020, buckwheat plants grown in field exhibiting stem canker symptoms were found in Tongxin county, Ningxia province, China. Symptoms included stem canker, dieback and extensive vascular discoloration. Cankers were bleached, silvery-white to dark gray, slightly sunken, oval to linear with slightly tapered tips, pycnidia formation was also observed within the cankers. Disease incidence was approximately 30% and moderate to high severity across the field. Symptomatic tissues were cut into 1-2 cm pieces, surface sterilized (75% ethanol for 30 s and 0.1% NaClO for 2 min) and washed four times with sterile distilled water, dried in sterile filter paper for 3 times, and placed on potato dextrose agar (PDA) at 25 ℃. Fluffy mycelium was visible for all isolates after 48 h of incubation. Twenty-five single isolates were hyphal-tip purified on PDA. Six representative isolates were used for further study. The fungal colonies on PDA were flat with an entire margin, gray aerial hyphae, light brown pigmentation, appressed slimy mycelium within which numerous brown-black perithecia formed. Colonies on oatmeal agar (OA) were flat, with flocculent mycelium, conidiomata and conidia and the reverse side was black to smoke-grey. Sparse brown-black perithecia were observed within the mycelium. Conidia were hyaline, one-celled, smooth-walled, rarely finely verruculose, aseptate, slightly curved, both sides gradually tapering towards the round to slightly acute apex and truncate base, measured (15.7-23.7) µm (length) × (2.8-5.7) µm (width), (avg. 20.2 µm×4.2 µm, n=100). Genomic DNA was extracted from the same six isolates, the internal transcribed spacer (ITS) region and the genes encoding beta-tubulin (TUB), chitin synthase (CHS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and histone H3 (HIS3) were independently amplified with primers V9G/ITS4, T1/Bt-2b, CHS-354R/CHS-79F, GDF1/GDR1 and CYLH3F/CYLH3R, respectively (Damm et al., 2019). Sequences for all six isolates were identical. The sequences of the representative isolate 9J1 were deposited in GenBank (accession nos. MW819604, MW836580, MW836577, MW836578 and MW836579). The results of BLASTn showed that the ITS, TUB, CHS, GAPDH and HIS3 sequences of 9J1 were greater than 98% (555/557bp, 477/486bp, 258/259bp, 242/248bp and 339/345bp, respectively) identical to those of Colletotrichum liriopes (GenBank: MT645674 (ITS), GU228098 (TUB), MT663546 (CHS), MH291255 (GAPDH) and MH292811 (HIS3)). MrBayes phylogenetic analysis using concatenated sequences of ITS, TUB, CHS, GAPDH and HIS3 showed that the isolate clustered monophyletically with strains of C. liriopes. Based on morphological and molecular characteristics, the isolate was identified as C. liriopes. To fulfill Koch's postulates, spores of the isolate 9J1 grown on OA for 14 days were harvested in 0.01% Tween 20, and the suspension were adjusted to 104 spores/ml. Six one-month-old potted plants of buckwheat were inoculated by spraying the spore suspension until run-off. Plants were kept inside a plastic bag for 3 d to maintain high relative humidity and maintained in the greenhouse. Six control plants were sprayed with sterile deionized water and kept under the same conditions. Two weeks after inoculation, all inoculated plants showed stem canker symptoms as described above, whereas control plants remained healthy. The pathogen was successfully reisolated from leaf and stem symptomatic tissue, and identified as C. liriopes based on morphological features and DNA sequence analysis, thereby fulfilling Koch's postulates. C. liriopes has been reported causing anthracnose on Eria coronaria (Yang et al., 2011), Liriope spicata (Chen et al., 2019) in China, Liriope muscari in Mexico (Damm et al., 2009), Rohdea japonica in Korea (Kwon and Kim, 2013) and in the United States (Trigiano et al., 2018). To our knowledge, this is the first report of C. liriopes causing anthracnose on buckwheat worldwide. Occurrence of this disease may represent a significant impact for buckwheat production because this crop is the major agricultural commodity in some parts of China. More studies are needed to understand the epidemiology of this disease and foster disease management programs in China.

Aucuba japonica var. variegata Dombrain is a common evergreen cultivated ornamental in China (Li et al. 2016). In December 2022, severe leaf blight on A. japonica was observed next to the Meishiyuan of Zhejiang Normal University (29°8'4″N, 119°37'54″E) in Jinhua City, Zhejiang Province, China. There were seven plants in the surveyed area, and over 50% of leaves were affected. The early symptoms were small gray spot parts with brown borders on the tip of the leaves. Then the grey parts gradually expanded and became brownish black. In severe cases, the whole leaves became black and blighted. To identify the pathogen, 5 symptomatic leaves were randomly collected from 5 plants and cut into small pieces (5 mm × 5 mm), surface disinfected in 1% sodium hypochlorite solution for 3 min, followed by 75% alcohol for 30 s, then rinsed in sterile distilled water thrice. Tissues were cultured on potato dextrose agar (PDA) and incubated at 28°C for 7 days. Pure cultures were obtained by the single-spore method. Thirteen strains were isolates from the tissues, and nine of them showed similar morphological characteristics. Colonies were white initially, then became gray. The undersides of the colonies became black gradually. Hyaline, fusiform conidia (n = 30) were 17.1 to 24.76 µm (average 20.39 ± 1.906 µm) in length and 5.4 to 6.61 µm (average 6.19 ± 0.434 µm) in width. The DNA of nine isolates were extracted by Ezup Column Bacteria Genomic DNA Purification Kit, and their sequences were identical, so they were named QM1. The internal transcribed spacer (ITS) region, translation elongation factor 1-α (TEF1), and β-tubulin (TUB2) genes were amplified with primer pairs ITS1/ITS4, TEF1-728F/TEF1-986R and βt2a/βt2b (Slippers et al. 2004), respectively. The BLAST analysis indicated that ITS (OR215464), TEF1 (OR243689), and TUB2 (OR243688) of the isolate QM1 were 99 to 100% identical to those of Botryosphaeria dothidea (GenBank accession nos. MH329646 for ITS sequences; OL891702 for TEF1 sequences; MK511445 for TUB2 sequences). In addition, the phylogenetic tree based on sequences from ITS, TEF1 and TUB2 was constructed with MEGA 11 by use of the maximum likelihood method with 1,000 bootstrapping iterations. Based on the multi-locus phylogeny and morphological features, the isolate QM1 was identified as B. dothidea. To test the Koch's postulates, ten leaves from three healthy two- to three-year-old A. japonica plants were surface disinfested with 75% ethanol for 30 s, rinsed with ddH2O three times. The leaves were wounded with a sterile needle and inoculated with 2ml drop of the isolate QM1 conidial suspension (106 spores/mL), with sterile distilled water as a control. All plants were placed in a greenhouse at 28°C, >70% relative humidity and 12 h light/day. The experiment was repeated three times. After 7 days, leaves of the inoculated group showed symptoms similar to those observed on the naturally infected leaves, while leaves of the control group remained asymptomatic. The pathogen was reisolated from inoculated leaves and was confirmed as B. dothidea based on morphological and molecular analyses. It has been reported B. dothidea cause leaf disease in a wide range of hosts in China, such as Camellia oleifera (Hao et al. 2023), Kadsura coccinea (Su et al. 2021). To our knowledge, this is the first report of Botryosphaeria dothidea causing leaf blight on Aucuba japonica in Zhejiang Province of China. B. dothidea are usually secondary invaders and are known to cause diseases in stressed plants. The results further expand the host-range of B. dothidea, and would help to establish control strategy against the disease.

Sugarcane (Saccharum officinarum L.) is one of the most important crops in the world. However, several factors can interfere with production, including the occurrence of diseases. In February 2024, symptoms of leaf blight were observed in the CTC4 and RB92579 cultivars in sugarcane fields located in the city of Carpina, in the state of Pernambuco, Brazil (7° 3’0.776” S / 35°14’16.555” W). The symptoms initially observed included leaf blight and slight leaf rolling. The burn displayed a straw-colored appearance with reddish borders and progressed in a v-shape, producing visible fungal fruiting bodies on the lesions. The incidence of symptoms was estimated to range between 70 and 80%. Symptomatic leaves, of ten plants were collected, in an area of ​​five hectares, but a similar symptom occurs in a large area of ​​sugarcane cultivation on the coastal strip of Brazilian northeastern. The possible causal agent was isolated using an indirect method on potato dextrose agar (PDA) culture medium. Plates were incubated at 28°C, and after seven days of cultivation, the colonies on PDA displayed a reddish-brown color, with dense growth of compact aerial mycelia, a felty appearance with white to pink coloration, and forming several mycelial pellets with the same coloration, visible on the underside. The chlamydospores were brown, uni- or multicellular, measuring 12.5 to 25.8 x 9.8 to 21.5 µm (n = 60). The pycnidia were dark brown to brown, predominantly semispherical, measuring 96 to 118.39 x 64 to 111 µm (n = 10). The conidia were ellipsoidal, hyaline, and aseptate, measuring 4.56 to 6.11 x 1.56 to 2.57 µm (n = 50). The morphological characteristics were similar to those of Epicoccum latusicollum (Chen et al. 2017). From the isolation, ten strains were obtained and deposited in the microorganism culture collection at the Plant Pathology Laboratory of the Sugarcane Experimental Station in Carpina–CMLABFITO, from all plants collected with symptoms, strains were obtained, however, two strains were randomly selected used for characterization in this work. To verify the pathogenicity of the (CMLABFITO 2.17 and CMLABFITO 2.18), 60-day-old plants of the CTC4 variety were inoculated with 10 mL of the conidial suspension (106 conidia/ mL) per plant using a manual sprayer until runoff. Four replicates per isolate were used, each containing two plants. Sterilized distilled water was used as a negative control. The plants were maintained in a humid chamber for 48 h at 28°C. The experiment was repeated twice. Eight days after inoculation, the plants exhibited subtle tip burn on the leaves, and the symptoms progressed over time. No symptoms were observed in the control plants. Koch's postulates were fulfilled through the reisolation of the isolates from the lesions. The reisolated fungus corresponded morphologically to the inoculated pathogen. For molecular identification of the pathogen, internal transcribed spacer (ITS) of region of ribosomal RNA (rDNA), as well as the β-tubulin (TUB2) and Actin (ACT) genes were amplified by PCR and sequenced using the Sanger method (Sanger et al. 1977). The sequences were deposited in GenBank database, ITS sequences (GenBank accession PQ412528 and PQ412529), TUB2 sequences (GenBank accession PQ416614 and PQ416615) and Actin gene sequences (GenBank accession PV178719 and PV178720) for CMLABFITO 2.17 and CMLABFITO 2.18, respectively. For sequences obtained from strain CMLABFITO 2.17, the ITS sequence showed identity 99.05% (438/441), TUB2 showed identity 99.32% (415/419) with E. latusicollum and ACT showed identity 100% to E. sorghinum. For sequences obtained from strain CMLABFITO 2.18 the ITS sequence showed identity 99.31% (432/435), TUB2 showed identity 100% (458/458) to E. latusicollum and ACT showed identity 100% to E. sorghinum. For actin the only closest sequeces available on NCBI belong to the E. sorghinum, because do not exist actin sequences from E. latusicollum on database. The phylogenetic analysis showed a very defined clade comprising CMLABFITO 2.17, CMLABFITO 2.18 and worldwide E. latusicollum strains. E. latusicollum has been reported causing disease in other plants, such as banana, Curcuma kwangsiensis and Hemerocallis citrina (Li et al. 2023; Liu et al. 2023; Wang et al. 2023). This is the first report of E. latusicollum causing leaf blight in sugarcane in Brazil.

Grape (Vitis spp.) is one of the most profitable fruit crops in Taiwan because of its delicacy and high nutritious value. Fruits of grape are harvested two times a year (summer and winter). In July 2015, a ripe rot disease was observed on grape berries (cv. Black queen) planted in a vineyard in Erlin Township of Changhua County (23°53'19" N, 120°24'40" E). The problem caused great concerns to the vine farmers because of its wide distribution and serious damage on berries, especially in rainy weather. Symptoms observed on ripe and nearly ripe berries showed reddish brown, irregular lesions covered with salmon-colored spore masses. Four fungal isolates were single spore isolated from four diseased berries by a hand-made glass needle. Fungal isolates were grown on potato dextrose agar (PDA) at 24 to 28°C with diffused light. All four strains produced salmon-colored conidial masses with few whitish mycelia around the colony on PDA. The conidia were hyaline, single-celled, round cylindrical on both ends, thin-walled and the contents guttulate. The sizes of conidia were 13.0±0.2 (11.0 to 15.0) ×4.5±0.1 (3.0 to 5.0) μm (L/W ratio=3.0±0.1, n=40). DNA was isolated from GC9 and used for amplification of partial sequences of the internal transcribed spacer (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin (ACT), β-tubulin (TUB2), chitin synthase 1 (CHS-1) and apn2/MAT1-2-1 (ApMAT) genes (Silva et al. 2012; Weir et al. 2012). A BLAST search against the NCBI database revealed that GC9 gene sequences (GenBank accession nos. MT613359 [ITS], MT648518 [GAPDH], MT815915 [ACT], MT648525 [TUB2], MW684718 [CHS-1], MT648530 [ApMAT]) displayed 99.6%, 100.0%, 99.5%, 99.5%, 99.2% and 100.0% nucleotide identity to the respective gene sequences of Colletotrichum viniferum GZAAS5.08601 (JN412804, JN412798, JN412795, JN412813, JX009413) and GZAAS5.08608 (KJ623242). Bayesian inference analysis (Noireung et al. 2012) of the concatenated sequences of ITS, GAPDH, ACT, CHS-1 and TUB2 revealed that isolate GC9 and C. viniferum GZAAS5.08601 were grouped in the same clade, which was clearly separated from the other five closely related species of Colletotrichum. Conidial suspensions (1 ×106 conidia/mL) were prepared from a mixture of the four isolates of C. viniferum and inoculated by spraying onto detached, ripe, healthy, nonwounded and surface-disinfected grape berries (cv. Kyoho, n=4). Four bunches of berries were sprayed with sterile water as control. Berries were kept in a moist chamber (>90% relative humidity, 24 to 28°C) for 24 h and maintained in the lab for additional 5 days. The inoculated fruit showed small light brown-colored spots, which eventually developed into brown, water-soaked lesions, similar to the symptoms in the vineyard. No symptom was observed on berries treated with water. C. viniferum was reisolated from symptomatic fruit, showing similar morphological characteristics to those collected from the field, thus fulfilling Koch's postulates. The experiment was repeated once showing similar results. The GC9 isolate of C. viniferum with the identification number BCRC FU31518 has been deposited at Taiwan Bioresource Collection and Research Center. C. viniferum has been reported to infect grape in China, Korea, Brazil and Japan (Farr and Rossman 2021). To our knowledge, this is the first report of C. viniferum causing grape ripe rot in Taiwan.

Clausena lansium (Lour.) Skeels is a tropical and subtropical plant species, it is not only a fruit but also has medicinal value. Its cultivated areas are mainly distributed in Guangdong, Guangxi, Fujian, and Hainan provinces in China. A severe anthracnose disease was observed during surveys of Clausena lansium disease in Yanfeng town of Hainan province in June 2024. The acreage of plantation was 2 acres. The percentage of symptomatic plants was about 70%. Symptoms appeared both on leaves and young shoots. Lesions appeared as brown point-sized at first on the leaf, and then enlarged to dark brown irregular lesions lined by a yellow halo. Diseased leaves became distorted and wrinkled at last. On young shoots, lesions appeared as water-soaked at first, and then enlarged to dark brown and sunken, ultimately leading to dieback. Five symptomatic leaves were sampled from different trees for pathogen isolation. 15 small pieces (5 mm) of necrotic tissue were removed from the border between symptomatic and healthy tissue, surface sterilized for 1 min in 1.5% NaOCl, washed three times with sterile distilled water, and plated onto potato dextrose agar (PDA). The plates were incubated at 28°C for five days. We obtained 10 Colletotrichum isolates with similar colony morphology. For a pure culture of the isolate, spore masses were picked off with a sterilized wire loop and streaked on the surface of water agar. After incubation overnight at 25 °C, single germinated spores were picked up with a sterile needle and transferred to PDA. Identification of the isolate was based on morphological as well as molecular characterisation. Colony characters and microscopic morphology characteristics of isolates were observed after growth on PDA at 28°C under dark for 7 days. The colonies on PDA were white at first, and then turned dark gray, cottony, with orange conidiomata, reverse dark gray to black at the center. Conidiophores formed on a cushion of medium brown roundish cells. Conidiophores were hyaline, Conidiogenous cells were cylindrical to ampulliform, straight to flexuous, 33-51 μm. Conidia were hyaline, aseptate, cylindrical with round ends, smooth-walled, guttulate, 9.9-19.9× 4.7-6.0 μm (mean ± SD = 12.8 ± 2.8 × 5.4 ± 0.4 μm, n=50). Genomic DNA of the 10 isolates was extracted from the culture, and DNA sequencing of an intron of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH), beta-tubulin (TUB2), actin (ACT), chitin synthase 1 (CHS-1), and the internal transcribed spacer (ITS) was conducted to accurately identify the species. ITS, TUB2 and ACT sequences from the ten isolates shared 100% identity, GAPDH and CHS-1 shared 99.6% identity. Sequences of the isolate named HPCS1 were deposited in GenBank (ITS Accession Nos. PQ113778; TUB2 Accession Nos: PQ133411; GAPDH Accession Nos: PQ137484; ACT Accession Nos: PQ782238, CHS-1 Accession Nos: PQ782239, respectively). ITS (513bp) of the Clausena lansium isolate HPCS1 were 100%, and GAPDH (232bp/234bp), ACT (256bp/257bp), CHS-1 (215bp/216bp) and TUB2 (419bp/420bp) were more than 99% similar with the Colletotrichum siamense type strain CBS130417 (Prihastuti et al. 2009) (GenBank Accession Nos. JX010171, JX009924, and JX010404) (Weir et al. 2012). In the phylogenetic analysis, the 10 isolates formed a monophyletic group and clustered within a clade with the ex-type isolate of Colletotrichum siamense (Sharma et al. 2015; Liu et al. 2016). Based on morphological and multilocus phylogeny, the Clausena lansium isolates were identified as C. siamense. To confirm pathogenicity, a conidial suspension (1 × 106 conidia/ml) of HPCS1 isolate was prepared by harvesting conidia from 10-day-old cultures growing on PDA and 5 mL was sprayed onto the healthy young shoots with tender leaves without wounding. All 9 young shoots inoculated were covered with plastic bags for 48 h to maintain high humidity and incubated at 28°C (Lin et al. 2020). After 5 days, symptoms were observed on the C. lansium leaves and were similar to those observed on the naturally infected plants. No symptoms were observed on the control leaves (inoculated with sterilized water in the same way). The pathogen was re-isolated from symptomatic tissues, but not from controls. These re-isolates matched the morphological and molecular characteristics of the original isolates, fulfilling Koch's postulates. C. siamense was described from Coffea arabica in Thailand (Prihastuti et al. 2009). In China, Colletotrichum scovillei was reported as a pathogen causing anthracnose on C. lansium (Lin et al. 2020). To our knowledge, this is the first report of C. lansium anthracnose causing by C. siamense.

First Report of Species of <i>Colletotrichum</i> Causing Leaf Spot of <i>Liriodendron chinense</i> × <i>tulipifera</i> in China

Japanese camellia (Camellia japonica), is an important ornamental species that has an increasing economic value in China, Japan, Australia and the USA (Vela et al. 2013). Leaf blight symptoms were observed on 20-year-old C. japonica 'April Tryst' leaves collected from a research plot in McMinnville, TN in March 2022. Leaf blight first appeared in the leaf tips and was irregular in shape (2 to 3 cm in diameter). Affected areas displayed gray color discoloration with a deep black margin and gradually expanded in size along the leaf margin, eventually causing leaf death and defoliation. Dark brown globose to subglobose conidiomata (pycnidia) were observed abundantly on the infected leaves (Fig. 1a). Disease severity was 25 to 50% of leaf area and incidence was 10% out of 60 plants. Three leaves were collected from each symptomatic plant and the surface disinfected with 10% NaOCl for 60 s, washed thrice with distilled water, and plated on potato dextrose agar (PDA). Colony growth of the isolates FBG4744 and FBG6184 on PDA, 15 days after incubation at 25°C (light/dark: 12/12h) were white to pale grey with dense and felted mycelium with concentric zonation. Spherical black pycnidia were observed on the concentric rings 2-3 weeks after incubation. Alpha conidia were on average 7.15 × 4.82 µm (4.89 to 9.37 µm × 2.91 to 6.74 µm) in size and were aseptate, hyaline, smooth, and ellipsoidal (n=50). Beta conidia were not observed. Pathogen identity was confirmed by extracting total DNA using the DNeasy PowerLyzer Microbial Kit from 7-day-old cultures. Primer pairs ITS1/ITS4 (White et al. 1990), T1/T222 and EF1/EF2 (Stefańczyk et al. 2016) were used to amplify and sequence the ribosomal internal transcribed spacer (ITS), beta-tubulin (BT), and translation elongation factors 1-α (EF1-α) genetic markers, respectively. The sequences (GenBank accession nos. OR607729, ITS; OR608485, BT; OR608487, EF1-α) were 100% similar to Diaporthe fukushii (=Phomopsis fukushii) in the NCBI nr/nt database (JQ807450: ITS; MG812590: BT, and MG281573: EF1-α). A phylogenetic analysis was performed using concatenated sequences of ITS, BT, and EF1-α of D. fukushii and other closely related taxa retrieved from GenBank (Fig. 2). Pathogenicity tests were performed on 1-year-old 10 healthy potted plants of C. japonica 'April Tryst' per isolate (Mathew et al. 2015; Yang et al. 2019). One leaf per plant was wounded with a sterilized 0.2-mm needle. PDA plugs (5 mm) taken from 7-day old cultures of FBG4744 and FBG6184 isolates were deposited on the wounded leaves and covered with moist cotton (Yang et al. 2019; Zhao et al. 2020). Ten additional plants were used as control and sterile PDA plugs were placed on the wounded leaves. Plants were covered with clear plastic bags and kept inside a greenhouse at 21 to 23°C, 70% RH, 16 h photoperiod. All inoculated leaves exhibited blight symptoms 14 days after inoculation (Fig. 1b) while control plants remained asymptomatic (Fig. 1c). The pathogen was reisolated from all the inoculated leaves and was confirmed as D. fukushii using morphological and molecular tools. Diaporthe species (D. tulliensis, D. passiflorae and D. perseae) have been previously reported to cause leaf spot on Camellia sinensis in Taiwan (Ariyawansa et al. 2021), but to our knowledge, this is the first report of leaf blight of C. japonica caused by Diaporthe fukushii in Tennessee and the United States. Identification of this novel disease is important in developing necessary management approaches.

In recent years, postharvest rot diseases of kiwifruit (Actinidia sp.) have caused severe damage in China (Li et al. 2017). Infected fruit were obtained from a commercial farm (30°98′N) after 4 months cold storage. Nineteen fruits (21.1%) out of one package showed rot symptoms with dark-brown, sour-smelling lesions. The lesions began as small pale yellow or light brown spots and then enlarged rapidly and formed dark or dark brown lesions. The margins between symptomatic and healthy tissues (4 × 4 mm) were cut from five rotted fruits, surface disinfested in 1% NaClO and 70% ethanol solution, washed, dried, plated on potato dextrose agar (PDA) containing 50 mg/liter of streptomycin sulfate, and incubated at 25°C for 3 days. Hyphal tips were transferred to PDA to obtain pure cultures. After 7 days, a total of 20 fungal isolates were obtained, including 16 previously reported Botrytis spp. (Xue et al. 2017), Penicillium spp. (Prodromou et al. 2018), and Alternaria spp. (Li et al. 2017), and four unknown isolates (DJY16A1-4, DJY16A1-5, DJY16A5-1, and DJY16A5-2). Strains DJY16A1-4 and DJY16A1-5 were incubated at 25°C for 7 days on PDA, wherein abundant white, fluffy aerial mycelium were grown on the dish. After 25 days, colonies produced black, spherical, or bluntly conical pycnidia. The conidia were hyaline, unicellular, ellipsoidal or fusiform in shape, and 6.52 × 3.43 µm in size. Similarly, incubated DJY16A5-1 and DJY16A5-2 formed white to light brown aerial mycelial mats with gray concentric rings. Pycnidia formed after 26 days, and α-conidia of the isolates were similar to those of the DJY16A1-4 and DJY16A1-5. β-Conidia were filiform or hamate, 23.9 × 1.2 µm in size. To identify these four isolates to species, the internal transcribed spacer (ITS), β-tubulin (BT), and translation elongation factor-1 alpha regions (TEF-1α) were amplified using specific primer pairs (You et al. 2015) and sequenced. By BLASTn analysis, DJY16A1-4 and DJY16A1-5 were 98% homologous to Diaporthe passiflorae CBS 132527 (NR_120155.1) based on ITS sequences (MH595928 or MH595929), 99 or 98% to D. passiflorae CBS 132527 (KY435674.1) based on BT sequences (MH621348 or MH621349), and 91% to D. passiflorae CBS 132527 (KY435633.1) based on the TEF-1α sequences (MH621352 or MH621353). DJY16A5-1 and DJY16A5-2 were 99% homologous to D. nobilis strain JL1 (KT163359.1) based on ITS sequences (MH595930 or MH595931), 97% to D. nobilis strain JL1(KX016113.1) based on BT sequences (MH621350 or MH621351), and 99% to D. nobilis strain 3JW-01S3 (KJ623308.1) based on TEF-1α sequences (MH621354 and MH621355). A phylogenetic tree was constructed using the method of maximum parsimony (MEGA7) with a combined dataset of ITS, BT, and TEF-1α sequences. Molecular phylogenetic analysis confirmed that DJY16A1-4 and DJY16A1-5 were D. passiflorae, and DJY16A5-1 and DJY16A5-2 were D. nobilis. The pathogenicity of the isolates was tested on the fruits of cultivars Xuxiang (A. deliciosa) and Zespri SunGold kiwifruit (A. chinensis). Ripe, healthy fruit were surface disinfected with 1% NaClO solution, rinsed in sterile distilled water, and dried. The middle of the fruit was stabbed with a sterile needle. Five wounded or unwounded fruits were inoculated with a 5-mm-diameter PDA plug with actively growing mycelium for individual isolates. Five wounded and five unwounded fruits were treated with sterile PDA plugs to serve as controls. Inoculated fruit were kept in sterilized transparent plastic boxes for 7 days at 25°C with 12-h light and 12-h dark. The wound-inoculated fruits produced the same symptoms as the rotted fruits collected initially from cold storage. Fruit rot was not observed on control fruit. The test was performed twice, and the fungi were reisolated and identified as either D. passiflorae or D. nobilis morphologically. D. lithocarpus was recently reported to cause kiwifruit rot (Li et al. 2016). But, to our knowledge, this is the first report of D. passiflorae and D. nobilis causing the postharvest rot in China.

In October 2022, typical symptoms of anthracnose were observed on apple (Malus ⅹ domestica cv. Fuji) fruits collected from Pocheon in Gyeonggi province, South Korea (N37.98074°, E127.33995°). In the surveyed orchard, the incidence rate of apple anthracnose was less than 1%. The initial symptoms were brown-to-dark brown lesions, and with disease progression, they enlarged and the pulp became soft, forming a brown band. In total 29 apple fruits were collected, and the causal agent was isolated by removing the peel, and the diseased tissues were directly transferred onto potato dextrose agar (PDA), followed by incubation for 7 days at 25°C. As the results, two isolates (GgPc22-1-11 and GgPc22-1-13) were obtained. For describing morphological and cultural characteristics, isolate GgPc22-1-11 was cultured on PDA and synthetic nutrient-poor agar (SNA) at 25°C under near-UV light with a 12-h photoperiod for 10 days. The colonies of GgPc22-1-11 on PDA were initially white and subsequently appeared light gray to olivaceous with white margins. The reverse side of the plates were dark brown and slate blue (Supplementary Fig. S1). Colonies on SNA were flat with an entire margin and short sparse white aerial mycelium. No setae were observed. Conidia on PDA were hyaline, straight, aseptate with a rounded apex, clavate to cylindrical, and measured 16.4 ± 2.4 (10.8-23.8) × 5.5 ± 0.7 (3.6-7.7) μm (n = 200). Appressoria were medium-to-dark brown, aseptate, solitary or in groups with irregular outlines, and lobate or having undulate margins (Supplementary Fig. S1). These morphological and cultural characteristics of GgPc22-1-11 were consistent with those of Colletotrichum grevilleae F. Liu, Damm, L. Cai & Crous, pathogens of Proteaceae and Punica granatum (Liu et al. 2013; Huang et al. 2023). DNA was extracted from GgPc22-1-11, PCR was performed and Phylogenetic analysis of concatenated partial sequences of the internal transcribed spacer (ITS) of rDNA, β-tubulin (TUB2), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), chitin synthase 1 (CHS-1), and actin (ACT) genes was conducted (Weir et al. 2012). The resulting sequences were deposited in GenBank under the accession numbers LC773710-LC773714. A nucleotide BLAST search revealed that the ITS sequences of the isolates were 98.95% identical to those of C. grossum CAUG7 (KP890165.1). The TUB2, GAPDH, CHS-1, and ACT sequences of the isolates were 99.79%, 99.24%, 100%, and 100%, respectively, identical to those of C. grevilleae WP4. GgPc22-1-11 was clustered with C. grevilleae WP4 using neighbor joining analysis conducted with MEGA X software (Kumar et al. 2018) (Supplementary Fig. S2). Pathogenicity tests were conducted using GgPc22-1-11 and repeated three times. A total of 12 symptomless apples of each variety were selected, including Fuji, Hongro, Tsugaru, and RubyS. The apples were surface-sterilized with 70% ethanol and wounded using a sterile needle. Both wounded and unwounded apples were inoculated with mycelium plugs and paper disks containing a conidial suspension (1 × 106 conidia/ml) and placed in a plastic box with moist paper towels (>90% relative humidity) at 25°C in dark. At 5 days after inoculation, all artificially wounded fruits exhibited symptoms and 30% (4 out of 12) of unwounded inoculated fruits showed symptoms in each apple variety while control fruits were asymptomatic both the unwounded and wounded inoculations (Supplementary Fig. S1). To fulfill Koch's postulates, the fungi were reisolated from symptomatic tissues and were identical to GgPc22-1-11 confirmed by morphological and molecular analysis. To the best of our knowledge, C. grevilleae has been reported in Protea sp. and pomegranate (Liu et al. 2013; Huang et al. 2023) but not in apples to date, and this is the first report of C. grevilleae causing anthracnose in apple fruits. This research of the newly emerged unreported Colletotrichum species can offer valuable information for development of an effective fungicide spray program to control apple anthracnose.

Ophiopogon bodinieri H. Lev. is an important ornamental groundcover widely used in urban gardens in southern China (Liu et al. 2011). In September 2017, a disease occurred on approximately 20% of O. bodinieri in 9 ha in Guangzhou, Guangdong province in China. Symptoms included etiolation in the leaves, wilt, root rot, and necrotic vascular systems. Three diseased plants were sampled for pathogen isolation. Portions (about 5 mm²) of symptomatic root tissues were dissected and surface disinfected (3% NaClO for 10 s and 70% ethanol for 30 s). Tissues were rinsed three times using sterile distilled water, dried on sterile filter paper, and transferred to Petri plates with potato dextrose agar (PDA) supplemented with streptomycin sulfate (150 µg/ml). Petri plates were incubated at 28°C for 5 days (Dita et al. 2010). Only one isolate was obtained from all the plates and was subcultured to new PDA plates. A single-spore isolate was obtained from a hyphal tip, and the culture characteristics and conidial morphology were studied on PDA and carnation leaf agar (CLA) (Neish 1983). The isolate grown on PDA formed abundant white-colored fungal colonies with radial mycelium in 5 days at 28°C. Microscopic observations from CLA medium revealed the curved macroconidia were usually three- to five-septate, with the size of 2.2 to 4.0 × 18.3 to 42.4 μm. Microconidia were kidney shaped with the size of 4.7 to 6.8 × 7.3 to 12.1 μm. Chlamydospores were single or in clusters, with the size of 9.1 to 11.0 μm in diameter. The elongation factor 1-alpha (EF1α) gene (accession no. MN026924, 686 bp), amplified and sequenced using primer pair EF-1/EF-2 (O’Donnell et al. 1998), showed 100% identification to a Fusarium oxysporum strain (accession no. KY508353.1) (Geiser et al. 2004). The molecular identification was confirmed via BLAST on the Fusarium ID and Fusarium MLST databases. Ten-week-old plants were used for pathogenicity tests. First, the plants were wounded by cutting off 1 cm of the roots. Then, 10 plants were inoculated by root dipping (30 min, 10⁴ spores/ml), and another 10 plants were treated with sterile water as a control. The plants were then repotted in potting mix and incubated at 28°C. The assay was conducted three times. After 15 days, the plants showed symptoms of leaf wilting, root rot, and necrosis in vascular tissues. After 40 days, all the inoculated plants were dead, whereas no symptoms were observed in the controls. Subsequently, the F. oxysporum isolate was successfully reisolated from the inoculated plants and was identified again by sequencing the EF1α. The pathogen was further identified by PCR amplification and sequencing the internal transcribed spacer (ITS) gene region using the primers ITS5/ITS4 and the 18s nuclear ribosomal small subunit (SSU) using the primers NS1/NS4 (Schoch et al. 2012). The isolate showed 100% and 99% identity to those of F. oxysporum (accession KF498869.1 for ITS and KU512835.1 for SSU). The sequences of ITS (accession no. MH752745.1), SSU (accession no. MH752591.1), and EF1α (accession no. MN026924) were deposited in GenBank. The first F. oxysporum causing disease on another Ophiopogon species (O. japonicus) was discovered in Florida in 1991 (Farr and Rossman 2019). To our knowledge, this is the first report of root rot disease of O. bodinieri caused by F. oxysporum in the world. This disease may pose a risk for urban landscapes in China.

Data processing can mask biology: towards better reporting of fungal barcoding data?

Thai basil (Ocimum basilicum L.) is widely cultivated in Malaysia and commonly used for culinary purposes. In March 2019, necrotic lesions were observed on the inflorescences of Thai basil plants with a disease incidence of 60% in Organic Edible Garden Unit, Faculty of Agriculture in the Serdang district (2°59'05.5"N 101°43'59.5"E) of Selangor province, Malaysia. Symptoms appeared as sudden, extensive brown spotting on the inflorescences of Thai basil that coalesced and rapidly expanded to cover the entire inflorescences. Diseased tissues (4×4 mm) were cut from the infected lesions, surface disinfected with 0.5% NaOCl for 1 min, rinsed three times with sterile distilled water, placed onto potato dextrose agar (PDA) plates and incubated at 25°C under 12-h photoperiod for 5 days. A total of 8 single-spore isolates were obtained from all sampled inflorescence tissues. The fungal colonies appeared white, turned grayish black with age and pale yellow on the reverse side. Conidia were one-celled, hyaline, subcylindrical with rounded end and 3 to 4 μm (width) and 13 to 15 μm (length) in size. For fungal identification to species level, genomic DNA of representative isolate (isolate C) was extracted using DNeasy Plant Mini Kit (Qiagen, USA). Internal transcribed spacer (ITS) region, calmodulin (CAL), actin (ACT), and chitin synthase-1 (CHS-1) were amplified using ITS5/ITS4 (White et al. 1990), CL1C/CL2C (Weir et al. 2012), ACT-512F/783R, and CHS-79F/CHS-345R primer sets (Carbone and Kohn 1999), respectively. A BLAST nucleotide search of ITS, CHS-1, CAL and ACT sequences showed 100% similarity to Colletotrichum siamense ex-type cultures strain C1315.2 (GenBank accession nos. ITS: JX010171 and CHS-1: JX009865) and isolate BPDI2 (CAL: FJ917505, ACT: FJ907423). The ITS, CHS-1, CAL and ACT sequences were deposited in GenBank as accession numbers MT571330, MW192791, MW192792 and MW140016. Pathogenicity was confirmed by spraying a spore suspension (1×106 spores/ml) of 7-day-old culture of isolate C onto 10 healthy inflorescences on five healthy Thai basil plants. Ten infloresences from an additional five control plants were only sprayed with sterile distilled water and the inoculated plants were covered with plastic bags for 2 days and maintained in a greenhouse at 28 ± 1°C, 98% relative humidity with a photoperiod of 12-h. Blossom blight symptoms resembling those observed in the field developed after 7 days on all inoculated inflorescences, while inflorescences on control plants remained asymptomatic. The experiment was repeated twice. C. siamense was successfully re-isolated from the infected inflorescences fulfilling Koch's postulates. C. siamense has been reported causing blossom blight of Uraria in India (Srivastava et al. 2017), anthracnose on dragon fruit in India and fruits of Acca sellowiana in Brazil (Abirami et al. 2019; Fantinel et al. 2017). This pathogen can cause a serious threat to cultivation of Thai basil and there is currently no effective disease management strategy to control this disease. To our knowledge, this is the first report of blossom blight caused by C. siamense on Thai basil in Malaysia.

Zephyranthes candida, an bulbous perennial plant, are planted in almost every park. In October 2023, anthracnose symptoms were observed on Z. candida leaves in Jiangxi Agricultural University (28.75° N, 115.83°E), Nanchang, Jiangxi Province, China, and the incidence of disease were up to 35% (140 of 400 plants). The lesions extended fromtheleafapextothebase, appearing as a dark brown color, and later changed to yellow and became dry. To isolate the pathogen, 20 symptomatic leaves were collected and cut into small pieces (4×4 mm, one pieces per leave), surface-sterilized with 70% ethanol for 10 s and 1% NaClO for 30 s, rinsed thrice with sterile water, placed onto potato dextrose agar (PDA) plates and incubated at 25℃ for 5 days. Fifteen isolates (15 out of 20) with similar morphological characteristics were obtained. The colonies on PDA presented effuse mycelium, initially white and later pale gray. Conidia were hyaline, curved or slightly curved, aseptate, with a truncate base and acute apex, measuring 17 to 23 × 3 to 6 μm (n = 50), and were matched to Colletotrichum species (Damm et al. 2009). To further confirm species, two representative isolates (JFRL 03-2873 and JFRL 03-2874) were selected for molecular identification. The internal transcribed spacer (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), chitin synthase (CHS), histone 3 (HIS3), actin (ACT) and β-tubulin 2 (TUB2) regions were amplified and sequenced by using primers sets ITS5/ITS4, Gpd1/Gpd2, CHS-79F/CHS354R, CYLH3F/CYLH3R, ACT-512F/ACT-783R and T1/Bt2b (Tan et al. 2022), respectively. These sequences were deposited into GenBank with accession number PP425890-PP425891 (ITS), PP437551-PP437552 (GAPDH), PP437549-PP437550 (CHS), PP480643-PP480644 (HIS3), PP437547-PP437548 (ACT) and PP437553-PP437554 (TUB2). A BLASTN search revealed high similarity of 99%-100% to ITS (GU227807, 518 nt/519 nt), GAPDH (GU228199, 525 nt/526 nt), CHS (GU228297, 251 nt/251 nt), HIS3 (GU228003, 372 nt/373 nt), ACT (GU227905, 236 nt/237 nt) and TUB2 (GU228101, 490 nt/490 nt) sequences of Colletotrichum spaethianum CBS 167.49. A maximum likelihood phylogenetic tree was constructed by combining ITS, GAPDH, CHS , HIS3, ACT and TUB2 sequences in IQtree web server (Ngugen et al. 2015). The result indicated that the two representative isolates were clustered together with Colletotrichum spaethianum in a clade with 100% bootstrap support. Based on morphologicalobservationandsequenceanalysis, the isolates were identified as C. spaethianum. To confirm pathogenicity, six surface-sterilized leaves of Z. candida were wounded and inoculated with 1 × 106 conidia/ml conidial suspension of JFRL 03-2873, and control leaves were inoculated with sterile water. They were incubated at 25 ℃ with 12 h photoperiod and 80% humidity, theexperimentwasrepeatedtwice. After five days, all leaves inoculated with JFRL 03-2873 displayed anthracnose symptom, whereas the control leaves remained unaffected. We re-isolated C. spaethianum from the symptomatic leaves and identified it based on morphological and molecular characteristics. Previous studies reported that C. spaethianum caused anthracnose on various common herbaceous plants in China (Vieira et al. 2014, Guo et al. 2013), but to our knowledge, this is the first report of C. spaethianum causing anthracnose on Z. candida in China. Anthracnose disease caused great economic loss to the cultivation of landscape plant Z. candida. Therefore, it is necessary to pay more attention to the anthracnose disease of herbaceous plants caused by C. spaethianum and develop appropriate control strategies.

In August 2022, two-month-old maize plants (Zea mays cv. 'Zihei'; "Chinese purple corn") exhibited irregular lesions on leaves and leaf blight symptoms (Figure 1). Although the lesions were yellow at the early infection stages, they turned brown during the pathogen advancement and culminated in leaf blight. Nearly 60% of plants from a non-commercial maize field (0.2 ha) in south-eastern Jiangsu (Nantong municipality, China; 120.54º E, 31.58º N) exhibited brown lesions, and about 4% of the diseased plants showed advanced leaf blight symptoms. The disease resulted in approximately a 9% yield loss compared to previous years when no disease symptoms were observed. Thirty small leaf pieces, approximately 0.3 cm2 in size and showing disease symptoms, were surface sterilized in 1.5% NaOCl for 1 min and washed twice with sterile ddH2O. The pathogen was cultured on PDA medium in the dark at 25 ºC, with grayish colonies observed after 5 days. Morphological analysis showed the presence of round/oval conidia (8.81 ± 0.50 μm diameter; n = 86) and branched conidiophores, which was consistent with the morphology of Penicillium spp. (Visagie et al. 2014). Nine representative isolates were obtained from different leaf pieces via single spore isolation, and the internal transcribed spacer (ITS), β-tubulin (TUB2) and calmodulin (CMD) genes were amplified using ITS1/ITS4, BT2a/BT2b and CMD5/CMD6 primers, respectively. The obtained ITS (OP954496-OP954497 and OP942428-OP942434), TUB2 (OP966781-OP966784 and OQ025045-OQ025049) and CMD (OQ078664-OQ078672) sequences were submitted in GenBank. Two isolates belonged to the P. citrinum species, while seven of the isolates belonged to the P. oxalicum species. A blast search revealed that the obtained P. citrinum ITS and CMD sequences had 99.39% and 100% homology to the ex-type strain P. citrinum NRRL 1841; GenBank numbers: AF033422 and GU944638 (Peterson & Horn 2009). Additionally, the obtained P. oxalicum ITS and CMD sequences had 99.82-100% and 94.64-95.49% homology to the ex-type strain P. oxalicum NRRL 787; GenBank numbers: AF033438 and KF296367 (Visagie et al. 2015). A molecular phylogenetic tree was constructed using MEGA7 to confirm the identity of the pathogen (Figure 2). To confirm pathogenicity, 3-week-old healthy 'Zihei' plants were used. The leaves were sprayed with aqueous solutions (sterilized ddH2O) that contained 1 × 106 spores/mL of each isolate. For the control experiment, sterilized ddH2O was used. After 5 days in a growth chamber at 25 ºC and 70% relative humidity, yellow lesions were observed. The number of lesions was higher when inoculating with P. oxalicum than when inoculating with P. citrinum. This result, together with the higher occurrence of P. oxalicum isolates, suggests that P. oxalicum is the main species causing the disease symptoms. The pathogen was recovered from the infected plants, and its identity was confirmed by ITS sequencing and morphological analysis. As far as we know, this is the first report of P. citrinum and P. oxalicum causing maize leaf blight worldwide. These species have previously been associated with maize kernels, as a source of mycotoxins posing relevant hazards to human health (Keller et al. 2013; Yang et al. 2020). P. citrinum was recently identified as the causal agent of green mold on Dictyophora rubrovalvata in China (Qin et al. 2022), while P. oxalicum was reported to cause citrus rot, pineapple leaf spot, and blue mold on Gastrodia elata, Astralagus membranaceus and muskmelon (Tang et al. 2020; Wu et al. 2022; Zheng et al. 2022). China is one of the world's largest producers of maize, harvesting more than 171 million tons in 2021. This report will help to better understand the pathogens that affect China's maize production.