First report of Macrophomina tecta causing charcoal rot in Sesamum indicum L. in India

Stevia (Stevia rebaudiana Bertoni) is an important medicinal crop grown worldwide. Leaves of stevia contain a non-caloric sweetener, stevioside, which is used as a substitute to artificial sweeteners. In August 2022, symptoms of chlorosis, wilting, and root rot were observed in about 30 % of stevia plants growing at the Agricultural Station at Yuma Agricultural Center, Yuma, AZ, USA (32.7125° N, 114.7067° W). Infected plants initially showed chlorosis and wilting, and the plants eventually died with foliage remaining intact to the plant. Cross sections of the crown tissue of affected stevia plants showed necrotic tissue and a dark brown discoloration in areas of the vascular and cortical tissues. Dark brown microsclerotia were observed on stem bases and necrotic roots of the infected plants. Five symptomatic plants were sampled to isolate the pathogen. Root and crown tissues (0.5 to 1 cm) were surface disinfested with 1% sodium hypochlorite for 2 min, rinsed three times with sterile water, and plated onto potato dextrose agar (PDA). All the five isolates displayed rapid mycelial growth on PDA at 28°C with a 12-h photoperiod. The mycelia were initially hyaline and turned from gray to black after 7 days. Masses of dark spherical to oblong microsclerotia were observed after 3 days on PDA, measuring an average of 75 µm width × 114 µm length (n=30). For molecular identification, genomic DNA was extracted from mycelia and microsclerotia of a representative isolate (Yuma) using the DNeasy Plant Pro kit (Qiagen, Hilden, Germany). The internal transcribed spacer (ITS), translation elongation factor-1α (TEF-1α), calmodulin (CAL), and β-tubulin (β-TUB) regions were amplified using the primer sets, ITS1/ITS4 (White et al. 1990), EF1-728F/EF1-986R (Carbone and Kohn 1999), MpCalF/MpCalR (Santos et al. 2020), and T1/T22 (O'Donnell and Cigelink 1997), respectively. A BLAST search of sequences revealed 98.7 to 100% identity to Macrophomina phaseolina sequences (MK757624, KT261797, MK447823, MK447918). Both morphological and molecular characteristics confirmed the fungus as M. phaseolina (Holliday and Punithaligam 1970). Sequences were submitted in the GenBank under accession numbers OP599770 (ITS), OP690156 (TEF-1α), OP612814 (CAL), and OP690157 (β-TUB). Pathogenicity assay was performed on 9-week-old stevia plants (var. SW2267), grown in 4-inch planters in the greenhouse. The inoculum was made from a 14-day-old culture of M. phaseolina grown in conical flasks (250 ml) in potato dextrose broth at 28°C. Mycelial mats of the fungus were blended in 250 ml of sterile distilled water, filtered through four layers of cheesecloth, and then calibrated to 105 microsclerotia/ml using a hemocytometer. Twenty healthy plants were inoculated by soil drenching 50 ml of the inoculum per pot. Soil drenching using sterile distilled water was done on 5 non-inoculated control plants. Plants were maintained in the greenhouse at 28 ± 3°C with 12 h photoperiod. After 6 weeks, necrosis at the base of petioles and chlorosis of the leaves, followed by wilting were noticed on all 20 inoculated plants, whereas all the 5 control plants remained healthy. The fungus was reisolated and identified as M. phaseolina based on the morphology and sequences of ITS, TEF-1α, CAL and β-TUB regions. Although M. phaseolina has been reported earlier on stevia in NC, USA (Koehler and Shew 2018), this is a first report from AZ, USA. M. phaseolina is known to be favored by high soil temperatures (Zveibil et al. 2011), thus represents a potential threat to stevia production in AZ, USA in coming years.

Cowpea (Vigna unguiculata (L.) Walp) is an important legume crop cultivated in arid and semi-arid regions in underdeveloped and developing countries. India is a leading cowpea producer. In addition to India, Nigeria and Niger are the predominant producers of cowpea in the world. Brazil, Haiti, Myanmar, Sri Lanka, and the United States are also significant producers of cowpea. This is a drought-tolerant annual crop that thrives in warm weather (3), and is more well-adapted to the drier regions of the tropics than any other legume. Cowpea fields (190 ha) surveyed in Mysore district (Karnataka State) from 2010 to 2012 were found affected by a new leaf spot disease. Over 60% of surveyed fields were affected by this disease, with individual fields ranging from 30 to 75% disease incidence. Individual fields experienced an estimated 10 to 15% yield loss. Initially, leaf spot symptoms appeared as small, dark, necrotic lesions that increased to a diameter of 0.5 to 1.0 cm. These spots later enlarged to form brown, circular, elliptical, and irregular spots with halo margins. Symptoms persisted throughout the cropping season. Under severe infection, defoliation occurred. Black, sessile, discoid conidiomata were observed in lesions and exuded a pink spore mass that later turned brown. The fungus was isolated from affected leaf tissues that were surface sterilized with 2% NaOCl2 solution, washed thrice with sterile water, blotter dried, and inoculated onto potato dextrose agar (PDA). White mycelia produced black globular acervuli with conidia on PDA after 7 days of incubation at 28 ± 2°C with a 12-h alternate light and dark period. Conidia had 5-celled (21.37 to 24.89 × 6.3 to 6.9 μm) segmentation with darker median cells and hyaline end cells. The apical cell typically had three appendages (sometimes 2 to 4) measuring 22.0 to 27.3 μm long and the basal appendage was 3.47 to 6.2 μm long. Based on these morphological features, the fungal pathogen was identified as Pestalotiopsis species. The isolated fungus was tested for pathogenecity on 30-day-old healthy cowpea plants grown under greenhouse conditions. A conidial suspension was prepared from 7-day-old culture by flooding with 2 to 4 ml of sterile distilled water. Spores were collected with a sterile micropipette and spore concentration was adjusted to 3 × 106 conidia/ml and applied as foliar spray onto 15 plants each in three replicates. Non-inoculated control plants were sprayed with sterile water. The plants were kept under high humidity (80%) for 5 days and at ambient temperature (28 ± 2°C). After 10 to 12 days post-inoculation, leaf spot symptoms appeared on inoculated plants, and the fungal pathogen was re-isolated and no such symptoms were found on control plants. The pathogen was confirmed by micro-morphological features. The ITS region of the ribosomal RNA gene was amplified using primers ITS1 and ITS4 (2). The amplified PCR product was purified and sequenced. nBLAST search comparison of sequences revealed 99% homology to Pestalotiopsis photiniae (AY682946.1). A representative sequence was deposited in GenBank (KC568288.1). Pestalotiopsis is an important pathogen on many crop plants and has been recorded on a wide variety of hosts, primarily on leaves, fruits, and in the rhizosphere. In recent times, cowpea is susceptible to a wide range of fungal pathogens causing severe yield loss at all stages of growth and development (1). Leaf spot caused by Pestalotiopsis species are becoming a major constraint for cowpea production in India. No previous reports are available on Pestalotiopsis species causing leaf spot of cowpea in India.

Plumeria alba L. is a flowering plant in the family Apocynaceae and widely cultivated in Malaysia as a cosmopolitan ornamental plant. In January 2020, anthracnose lesions were observed on leaves of Plumeria alba planted in Agricultural Farm, Universiti Putra Malaysia, in Selangor state, Malaysia. The disease mainly affected the leaves with symptoms occurring with approximately a 60% disease incidence. Ten symptomatic leaves were sampled from 3 different trees in the farm. Symptoms initiated as small circular necrotic spots that rapidly enlarged into black lesions with pale brown borders. Diseased tissues (5×5 mm) were surface-sterilized with 70% ethanol for 1 min, rinsed three times with sterile distilled water, dried on sterile filter papers, plated on PDA and, incubated at 25 °C with a 12-h photoperiod. A total of seven single-spore isolates with similar colony morphologies were obtained from tissue samples. After 7 days, the colonies raised the entire margin and showed white-to-gray aerial mycelium, orange conidial masses in the center and appeared dark brown at the center of the reverse view. The conidia were 1-celled, hyaline, smooth-walled, cylindrical with narrowing at the center, averaged (13-15 μm × 3 - 4 μm) (n=40) in size. Morphological characteristics of the isolates were similar to those detailed in taxonomic description of Colletotrichum sp. (Prihastuti et al. 2009). For molecular identification, genomic DNA of two representative isolates, PL3 and PL4 was extracted from fresh mycelium using DNeasy Plant Mini Kit (Qiagen, USA). The internal transcribed spacer (ITS) region, actin (ACT) and calmodulin (CAL) genes were amplified using ITS5/ITS4 (White et al. 1990), ACT-512F/783R (Carbone and Kohn 1999) and CL1C/CL2C primer sets (Weir et al. 2012). A BLAST nucleotide search of GenBank using ITS sequences showed 100% identity to Colletotrichum siamense ex-type culture ICMP 18578 (GenBank accession no. JX010171). ACT and CAL sequences showed 100% identity with C. siamense ex-type isolate BPD-I2 (GenBank accession no. FJ907423 and FJ917505). The sequences were deposited in GenBank (ITS: accession nos. MW335128, MT912574), ACT: accession nos. MW341257, MW341256, CAL: accession nos. MW341255 and MT919260). Based on these morphological and molecular characteristics, the fungus was identified as C. siamense. Pathogenicity of PL3 and PL4 isolates was verified using four healthy detached leaves of Plumeria alba. The leaves were surface-sterilized using 70% ethanol and rinsed twice with sterile water before inoculation. The leaves (three inoculation sites/leaf) were wounded by puncturing with a sterile needle through the leaf cuticle and inoculated in the wound site with 10-μl of conidial suspension (1×106 conidia/ml) from 7-days-old culture on PDA. Four leaves were used as a control and were inoculated only with 10-μl of sterile distilled water. Inoculated leaves were kept in humid chambers for 2 weeks at 25 °C with 98% relative humidity on a 12-h fluorescent light/dark period. The experiment was repeated three times. Anthracnose symptoms were observed on all inoculated leaves after 3 days, whereas controls showed no symptoms. Fungal isolates from the diseased leaves showed the same morphological characteristics as isolates PL3 and PL4, confirming Koch's postulates. C. siamense has been reported causing anthracnose on rose (Rosa chinensis) in China (Feng et al. 2019), Coffea arabica in Thailand (Prihastuti et al. 2009) and mango leaf anthracnose in Vietnam (Li et al. 2020). To our knowledge, this is the first report of Colletrotrichum siamense causing leaf anthracnose on Plumeria alba in Malaysia. Accurate identification of this pathogen provides a foundation in controlling anthracnose disease on Plumeria alba.

A countrywide survey of fungal diseases of barley (Hordeum vulgare L.) was conducted from 2005 to 2009. Unusual leaf necrosis varying in shape from 1 × 2 mm necrotic flecks to 15 × 20 mm ovoid spots was found. Sometimes a chlorotic halo surrounding the dead area was observed. Lesions appeared on various cultivars in many commercial fields and experimental plots at a number of sampling sites. Symptomatic leaves were taken to the laboratory and incubated in a moist chamber at room temperature on the bench to induce sporulation of the pathogen. Conidiophores on the diseased tissues were single or in small groups, dark brown, and bore several hyaline-to-olive brown, almost cylindrical conidia with three to seven pseudosepta. Dimensions of conidia were 75.2 to 100.9 × 16.5 to 18.8 μm. Under a stereo microscope, single conidia were transferred aseptically from the leaves onto potato dextrose agar (PDA) with a sterile needle. Plates were kept in the dark at 20°C for 2 weeks. Cultures were gray to olive green, cottony, and did not form conidia and sexual structures. These characteristics indicated that the pathogens belonged to the genus Pyrenophora. Species identity was confirmed by PCR assays with specific primers developed for the barley pathogenic Pyrenophora spp. (3,4). Of 169 isolates, 41 were identified as P. teres Drechs. f. maculata Smed.-Pet., the spot form of net blotch pathogen (2), and two of them have been deposited at an international culture collection under accession nos. CBS 123929 and CBS 123930. The remaining isolates were either P. graminea or P. teres f. teres, the leaf stripe and net form of net blotch pathogens of barley, respectively. Pathogenicity of four P. teres f. maculata and two P. teres f. teres isolates from different regions was confirmed by Koch's postulates. Each isolate was grown on two 9-cm PDA plates at 22°C in darkness. After 10 days, aerial mycelia were scraped off, blended in 100 ml of sterile distilled water, and filtered through two layers of cheesecloth. Ten seedlings of cv. Botond were sprayed at the two-leaf stage with the mycelium suspension of each isolate and a water control until runoff. Seedlings were kept in a growth chamber at 100% relative humidity and 20°C in the dark for 24 h, then at 70% relative humidity and 24/20°C (day/night) with a 12-h photoperiod. Within 3 weeks, one to four brownish ovoid spots, typical of the spot form of net blotch symptoms, developed on the leaves inoculated with P. teres f. maculata. In contrast, the seedlings inoculated with P. teres f. teres exhibited characteristic net-like lesions, whereas the control plants sprayed with sterile water remained healthy. All strains were reisolated and identified by specific PCRs as described above. To our knowledge, this is the first report of the occurrence of P. teres f. maculata in Hungary. Resistance of barley against P. teres f. maculata and P. teres f. teres is inherited independently (1). Therefore, knowledge regarding the frequency and distribution of these pathogens is important for disease management and resistance breeding.

Rhizoma paridis is a perennial, traditional Chinese medicinal herb. In May 2013, a disease was observed in an approximately 10 ha cultivated field in Enshi, Hubei Province, China. Approximately 80% of plants in the field were affected. Symptoms were visible on the basal leaves of affected plants. Chlorosis followed by necrosis started at the leaf tips and margins and gradually spread inward until the entire leaf was necrotic. Thick, gray mycelium and conidia were visible on both sides surface of leaves under wet, humid conditions. The leading edge of the chlorotic leaves was excised from 20 plant samples surface disinfested with 1% NaOCl solution for 1 min, rinsed in sterile water, air dried, and placed on potato dextrose agar (PDA). Plates were incubated at 22°C in the dark. Mycelia were initially hyaline and white, and became dark gray after 72 h. Mycelia were septate with dark branched conidiophores. Conidia were smooth, hyaline, ovoid, aseptate, and ranged from 8 to 14.5 × 7 to 8.5 μm. Numerous hard, small, irregular, and black sclerotia that were 1 to 3 × 2 to 5 mm were visible on PDA plates after 12 days. The fungus was identified as Botrytis cinerea on the basis of these characters (1). The internal transcribed spacer (ITS) region of rDNA was amplified using the ITS1 and ITS4 primer and sequenced (GenBank Accession No. KF265499). BLAST analysis of the PCR product showed 99% identity to Botryotinia fuckeliana (perfect stage of B. cinerea) (EF207415.1, EF207414.1). The pathogen was further identified to the species level as B. cinerea using gene sequences from glyceraldehyde-3-phosphate dehydrogenase (G3PDH), heat-shock protein 60 (HSP60), and DNA-dependent RNA polymerase subunit II (RPB2) (2) (KJ638600, KJ638602, and KJ638601). Pathogenicity was tested by spraying the foliage of 40 two-year-old plants with a suspension of 106 conidia per ml of sterile distilled water. Each plant received 30 ml of the inoculum. Ten healthy potted plants were inoculated with sterilized water as control. All plants were covered with plastic bags for 5 days after inoculation to maintain high relative humidity and were placed in a growth chamber at 22°C. The first foliar lesions developed on leaves 7 days after inoculation and were similar to those observed in the field. No symptoms developed on the control plants. B. cinerea was consistently re-isolated from all artificially inoculated plants. The pathogenicity test was completed twice. To our knowledge, this is the first report of gray mold of R. paridis caused by B. cinerea in China. The root of R. paridis is the most commonly used Chinese herbal medicine to treat viper bites. In recent years, cultivation of this herb has increased in China because of its high value. Consequently, the economic importance of this disease is likely to increase with the greater prevalence of this host species.

EUCAST Definitive Document EDef 7.1: method for the determination of broth dilution MICs of antifungal agents for fermentative yeasts: Subcommittee on Antifungal Susceptibility Testing (AFST) of the ESCMID European Committee for Antimicrobial Susceptibility Testing (EUCAST)∗

Muskmelon(Cucumis meloL.) is a widely cultivated and economically important fruit crop worldwide. In June 2022, fruitrot symptoms were observed on ripening muskmelons (cv. Boyang) in Shouguang City (36.81°N 118.90°E) of China. To determine the causal agent, we surveyed 200 muskmelon plants in about 1000 m2 of planting area and collected diseased muskmelons. Approximately 20% of muskmelon fruits had symptoms, and yield loss averaged 20%. Water-soaked lesions were observed on the surface and the fruit rotted from inside. Lesions were covered with white mycelium. Rotted fruit were surface-disinfested with 1% NaOCl for 1 min, 75% ethanol for 30 s, and washed three times with sterile water. Pieces (1 cm3) were cut from the disinfested fruit, placed on potato dextrose agar (PDA), and incubated at 25°C for 1 week. Ten isolates with similar morphology were obtained and isolates SG66 and SG68 were selected for further characterization. Colonies maintained on PDA in the dark had an average radial growth rate of 10-12 mm/d at 25°C. Surface was white, velvety to felty mycelium. Reverse was white to pale wheat. Diffusible pigments were absent. On carnation leaf agar, sporodochia appeared as slimy dots, macroconidia were 3- to 5-septate, 20-35 × 3-5 μm, falcate, with a pronounced dorsiventral curvature, with blunt to papillate apical cell, and barely to distinctly notched basal cell. Microconidia and chlamydospores were not observed. These morphological characteristics were consistent with descriptions of Fusarium sp. DNA was extracted from isolates SG66 and SG68 using a CTAB method. Nucleotide sequences of the internal transcribed spacers (ITS) (White et al. 1990), calmodulin (CAM), RNA polymerase II second largest subunit (RPB2), and translation elongation factor 1-α gene (TEF1) (Xia et al. 2019) were amplified using generic primers, the products sequenced, and sequences deposited in GenBank (ITS: OP251362, OP251363; CAM: OP266024, OP266025; RPB2: OP266028, OP266029; TEF1: OP266026, OP266027). Isolates SG66 and SG68 clustered with Fusarium sulawesiense (85% bootstrap) (Maryani et al. 2019). The Fusarioid-ID database pairwise alignment of ITS (526 bp), CAM (534 bp), RPB2 (861 bp), and TEF1 (636 bp) sequences from isolate SG66 showed 99.6% (98.9% coverage), 100% (100% coverage), 100% (100% coverage) and 100% (98.4% coverage) similarity with the corresponding sequences (GQ505730, LS479422, LS479855 and GQ505641), respectively, of the reference strains of F. sulawesiense (InaCC F940 and NRRL 34059). To perform a pathogenicity test, 10 μl of conidial suspensions (1 × 106 conidia/ml) were injected into ten muskmelon fruit using a syringe, and ten control fruit were inoculated with 10 μl of sterile distilled water. The test was repeated three times. After 7 days at 25°C, the pulp of all inoculated muskmelons began to rot, and the lesion expanded from the inside to the fruit surface at the injection site and became covered with white mycelia. No symptoms developed on the control fruit. The fungus was successfully re-isolated from infected tissues and confirmed as F. sulawesiense by morphological and phylogenetic analyses. F. sulawesiense has previously been reported onyellow melon (Canary) in Brazil (Lima et al. 2021) and on a range of hosts, including Luffa aegyptiaca, in China (Wang et al. 2019). To our knowledge, this is the first report of muskmelon fruit rot caused by F. sulawesiense in China.

Fig (Ficus carica L.) holds economic significance in Atushi, Xinjiang, but as fig cultivation expands, disease prevalence has risen. In July 2024, approximately 22% of harvested fig (cv. Xinjiang Zaohuang) from 20 commercial orchards (covering 40 hectares) in Atushi (39°39'37.65" N, 76°14'3.62" E) showed varying degrees of fruit rot symptoms. The initial symptoms were characterized by the appearance of small, brown lesions on the fruit surface. These lesions rapidly progressed into water-soaked spots, which expanded quickly. As the disease advanced, the affected areas became covered with dense, white, fluffy mycelia, accompanied by prominent black sporulation. In later stages, the infected tissues softened further, ultimately resulting in the complete decay of the fruit. Twenty diseased fig were collected from the sampling site. Tissue samples (5×5×5 mm) were cut at the diseased-healthy junction, surface-sterilized in 0.5% NaClO for 1 minute, rinsed twice in sterile distilled water, air-dried, and transferred aseptically onto potato dextrose agar (PDA), and incubated at 25°C for 5 days with a 12-hour photoperiod. Fifteen isolates were obtained from the infected tissues, with two representative isolates (WH 12 and WH 23) selected for further study due to morphological similarity. The fungal colonies initially appeared as white mycelium, later turning olive green to grayish-black. Colony growth was rapid (32 mm/day). Arthrospores were colorless to light brown, short columnar, aseptate, with a truncated base, 0 to 1 septate, averaging 11.9±2.3×3.6±0.8 μm (n = 50), and sometimes formed arthric chains. Chlamydospores were dark brown, round or oval, 0 to 1 septate, averaging 7.26±1.7×5.05±1.0 μm (n = 50). Genomic DNA was extracted from the two isolates. The internal transcribed spacer (ITS), translation elongation factor 1-alpha (TEF1-α), and beta-tubulin (TUB2) genes were amplified using primers ITS1/ITS4 (White et al. 1990), EF1-728F/EF1-986R (Carbone & Kohn. 1999), and BT2a/BT2b (Glass & Donaldson. 1995), respectively, and sequences were deposited in GenBank (ITS: PQ555020, PQ555021; TUB2: PQ557519, PQ557521; TEF1-α: PQ557520, PQ557522). BLAST analysis revealed 99-100% similarity to Neoscytalidium dimidiatum Arp2-D (ITS: MK813852; TUB2: MK816354; TEF1-α: MK816355). Phylogenetic analysis using IQ-Tree and MrBayes3.2.7 based on concatenated ITS-TEF1-TUB sequences showed WH 12 and WH 23 clustering with N. dimidiatum Arp2-D (99% bootstrap). Morphological and molecular data identified the isolates as N. dimidiatum (Penz.) Crous & Slippers (Crous et al. 2006). Pathogenicity tests were conducted on 20 healthy fig (cv. Xinjiang Zaohuang) by inoculating each fruit with 10 µl of a WH 12 conidial suspension (1 × 10⁶ conidia/ml) using sterile needles. The Control were treated with 10 µl of sterile distilled water. All fruits were placed in sterile plastic containers and incubated at 25 ± 1°C, 90% relative humidity, and a 12-hour light cycle. This experiment was performed twice. On the 1st day post-inoculation, brown lesions began to develop on the fruit. By the 4th day post-inoculation, the entire fruit was completely decayed and covered with white mycelia and black spores, while the control fruit showed no symptoms. The fungus was successfully reisolated from the inoculated fruits and identified as N. dimidiatum following the methods described above, fulfilling Koch's postulates. N. dimidiatum has been reported to have a wide range of hosts in China, such as Jacaranda mimosifolia, Hylocereus megalanthus, Hylocereus undatus, and Styphnolobium japonicum (Li et al. 2024; Zeng et al. 2024; Lan et al. 2022; Luo et al. 2024). To our knowledge, this study is the first report of N. dimidiatum as the causal agent of fruit rot in fig in China. Our findings have expanded the host range of N. dimidiatum in China and provide a theoretical basis for the diagnosis and treatment of the disease.

In southern Xinjiang, walnut (Juglans regia L.) is the primary source of income for farmers and a key crop in the region's specialty fruit and forestry industries. In September 2024, leaf spotting was observed in several walnut orchards in Aral, Xinjiang. In the early stages of the disease, small brown spots appeared on the leaf tips and margins, which later expanded irregularly across the entire leaf, eventually causing the leaves to wither and necrotize completely. Diseased leaves were collected from four walnut (Xinwen185) orchards in Aral (81.2997°E, 40.5583°N) for pathogen isolation. Leaf sections (4×4 mm) were excised from lesion margins and subjected to surface sterilization through sequential treatment with 75% ethanol for 30 s and 1% sodium hypochlorite for 3 min. They were then rinsed eight times by immersion in 50 mL of sterile distilled water with gentle agitation for 30 s each time. After drying with sterile filter paper, the segments were transferred onto potato dextrose agar (PDA) plates and incubated at 28 ℃ for 5-7 days. Twelve single-spore strains were obtained through single-spore isolation technique. After 6 days of incubation, the isolates produced abundant aerial white mycelia and acquired a purple pigmentation. The hyphae were hyaline with septation. The isolates produced numerous oval unicellular microconidia without septa, 4.8 to 18.6 × 1.5 to 4.2 µm (n = 50) and very few macroconidia with three to four septa (21.6 to 46 × 3.1 to 4.3 µm [n = 30]), narrowed at both ends (Fig.S1). These morphological characteristics are consistent with those described for F. proliferatum (Leslie and Summerell, 2006). Genomic DNA was extracted from a representative isolate KY7 using the cetyltrimethylammonium bromide (CTAB) protocol. Four genetic loci were amplified and sequenced: the elongation factor 1-α (TEF1) gene, internal transcribed spacer (ITS) region, 28S large ribosomal subunit (LSU) rRNA gene, and calmodulin (CAL) gene, along with the second largest subunit of RNA polymerase II (RPB2) gene. Amplification was performed using the following primer pairs: TEF1: EF1-728F/EF1-986R, ITS: ITS1/ITS4, LSU: LR0R/LR5, CAL: CL1/CL2A, RPB2: fRPB2-5F/fRPB2-7Cr. The sequences of the isolate KY7, were deposited in GenBank under the following accession numbers: ITS (PV441515), TEF1 (PV524931), LSU (PV441477), CAL (PV524935), RPB2(PV524933). BLASTn analysis revealed that isolate KY7 showed 99.62% (ITS: EF453150.1), 98.84% (TEF1:KX656210.1), 99.89% (LSU: PP336543.1),99.73% (CAL: AF291057.1) and 99.66% (RPB2: MN193893.1) with F. proliferatum. A maximum likelihood phylogenetic tree was constructed based on concatenated sequences of EF-1α, CAL, and RPB2 using Fast tree 2.2 software. The isolate KY7 clustered within the same evolutionary clade as F. proliferatum. Based on morphological and molecular analyses, isolate KY7 was identified as F. proliferatum (Fig.S2) (Leslie and Summerell, 2006). Three-month-old walnut seedlings were selected for pathogenicity testing of isolate KY7. The leaves were surface-sterilized with 75% ethanol, rinsed three times with sterile water, and then wounded with a sterile needle. For the inoculation group, each plant was sprayed with 5 mL of KY7 spore suspension (1×106 spores/mL), while the control group was sprayed with an equal volume of sterile water. All plants were maintained at 28℃ with 80% relative humidity under a 12 h photoperiod. The experiment consisted of two independent trials, with each trial including five inoculated seedlings and five control seedlings (resulting in a total of ten inoculated and ten control plants). Ten days after inoculation, irregular brown spots consistent with field observations appeared on the inoculated leaves, whereas the control plants remained asymptomatic (Fig.S1). The same pathogen was re-isolated from the diseased leaves, fulfilling Koch's postulates. F. proliferatum has been reported to cause leaf blight in Phoenix dactylifera in Tunisia (Namsi et al., 2021) and leaf spot in Juglans regia in Hebei Province, China (Wang et al., 2022). In Xinjiang, characterized by arid conditions, low rainfall, and abundant sunlight (Chang et al., 2022), the pathogen's ability to infect walnuts has raised concerns among local farmers. In contrast, disease management practices in Hebei Province are likely more targeted. The identification of F. proliferatum in this study enables the development of tailored management strategies, providing a critical foundation for precise disease control. This will assist growers in selecting effective fungicides and formulating optimized application protocols. To our knowledge, this is the first report of F. proliferatum causing walnut leaf spot in Xinjiang, China.

Atractylodes lancea Thunb. DC (cangzhu) is a traditional Chinese medicinal plant (Cai et al., 2020). In June 2020, leaf spots were observed in A. lancea plants at the Chongqing Institute of Medicinal Plant Cultivation located in Nanchuan District, Chongqing, China (29°8'26.46″ N, 107°13'23'21″ E). Approximately 75% of the plants displayed leaf spot, partial leaf wilting, and stunted growth, and some plants died. To determine the cause of this disease, five typical leaf spots were cut into small pieces. The pieces were successively surface-disinfected with 0.5% NaClO for 1 min and 75% ethanol for 30 s, washed thrice with sterile water, and placed on potato dextrose agar (PDA) to incubate at 25 ℃. These isolates initially formed abundant white aerial mycelium, then gradually developed a rose pigmentation with a brownish color in the center and grayish rose at the periphery of the colony (Li etal. 2019).Mycelial tipswere picked and placed on carnation leaf agar (CLA) and inoculated for 7 days. The macroconidia of the isolates were slender, distinctively curved in the bottom half of the apical cell, and sickle-shaped, with 3-4 septa. They ranged in size from 16.68-26.49 × 1.48-2.34 μm (n=50). The microconidia were fusiform with or without one septum. Their size ranged from 6.19-11.02 × 1.25-1.43 μm (n=50) (Li etal. 2019). The morphological characteristics of the isolates were consistent with those of Fusarium spp. PCR amplification and DNA sequencing of the internal transcribed spacer (ITS) region and β-tubulin (TUB2) gene were performed using the primers ITS1/ITS4 (White etal. 1990) and Bt-2a/Bt-2b (Robideau etal. 2011), respectively. BLASTn analysis revealed that the ITS sequences of the isolates were 100% identical to those of the F. acuminatum isolates from the Fusarium MLST database (http://isolate.fusariumdb.org/guide.php). Furtheranalysisrevealed that the TUB2 sequences were 99.14% identical to those of the F. acuminatum strain S16 isolates (MF662644) from the GeneBank database of the NCBI server. Based on the morphology and sequence analyses, the isolates were identified as F. acuminatum. Pathogenicity tests were conducted on 1.5-year-oldA.lanceaplants by inoculating spore suspensionsunder greenhouse conditions (25°C). For this, wound were made on leaves by piercing with sterilized toothpicks. 30 μl of spore suspension containing 2 × 106 conidia/ml was placed on each wound. Wounds on the leaves of control plants were inoculated with 10 μl of sterile distilled water. There were three plants for each treatment. After incubation at 25 °C for 5 days in a greenhouse, the leaves of the treated plants all showed partial wilting, consistent with the field observations. No symptoms were observed in controlled plants. The fungi were again isolated from the symptomatic tissues and were identical to the original isolate. The experiment was repeated twice with similar results. Pathogenicity symptoms were similar to what was first observed in the field and the isolated fungi were verified based on morphological characteristics, thus fulfillingKoch'spostulate. To the best of our knowledge, this is the first time that A. lancea leaf spot caused by F. acuminatum has been discovered in China. The leaf spot caused by F. acuminatum on A. lancea has serious yield loss, and proper control measures should be applied.

Wurfbainia villosa var. villosa is a traditional Chinese herbal medicine under the family Zingiberaceae, and its ripe fruits (called Fructus Amomi) are widely used clinically for the treatment of gastrointestinal disorders (Yang et al. 2023; Chen et al. 2023). In September 2023, plants of W. villosa var. villosa exhibited anthracnose-like symptoms on leaf with a disease incidence of 35% (n = 100 investigated plants) in an approximately 90 m2 field in Guangning, China (N23°42'51.70″, E112°26'35.75″). Light yellowish-green spots (~2 mm diameter) initially appeared on the infected leaves, gradually formed sub-circular or irregular spots, then fused and expanded, resulting in wilting of the leaves. To identify the causal agent, 10 symptomatic leaves were collected and transferred to the laboratory. The symptomatic leaf samples were surface sterilized in 0.5% NaClO for 2 min, and in 70% ethanol for 30 s, then washed three times with sterile water and air-dried on sterile filter paper. The leaf tissues were placed on potato dextrose agar (PDA) medium containing 100 μg mL-1 of ampicillin (Sigma-Aldrich, St. Louis, MO) and incubated for 7 days at 28°C in darkness. Nine isolates with similar colony morphology were isolated from the 10 plated leaves. Three representative isolates (GNAF03, GNAF06, GNAF09 with approximately 3.5 cm in diameter after 3 days of incubation) appeared gray to dark brown with dense aerial hyphae at the front and gray to black colonies on the reverse of the plates. Conidia were cylindrical and measured 21.2 to 29.3 μm long × 7.1 to 9.6 μm wide (n = 50). Appressoria were formed by the tips of germ tubes or hyphae and were brown, ellipsoid, thick-walled, and smooth-margined, measuring 10.2 to 12.3 μm long × 6.4 to 8.2 μm wide (n = 50). Morphologically, the fungal isolates resembled Colletotrichum sp. (Weir et al. 2012). For molecular analysis, genomic DNA was extracted from fresh mycelia of the three isolates, and the primers ACT-512F/ACT-783R, CL1/CL2A, GDF/GDR, and ITS1/ITS4 were used to amplify partial regions of rDNA-ITS, actin (ACT), calmodulin (CAL), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) regions, respectively (Weir et al. 2012). The resulting sequences with more than 99% nucleotide identity to C. gloeosporioides were submitted to GenBank (accession numbers PP552725, PP552726, and OR827444 for ACT; PP552727, PP552728, and OR827443 for CAL; PP552729, PP552730, and OR827445 for GAPDH; PP549996, PP549999, and OR841394 for ITS). A phylogenetic tree was generated by the maximum likelihood method using the concatenated sequences of ACT, CAL, GADPH, and ITS by Polysuite software (Damm et al. 2020). Based on morphological and molecular analysis, the three isolates were characterized as C. gloeosporioides. The pathogenicity of the GNAF09 isolate was assessed on W. villosa var. villosa seedling leaves inoculated by spraying with 40 μL of conidial suspension at 106 conidia mL-1 or wounded with a sterile toothpick then inoculated with mycelial agar plugs (5 mm diameter). Control leaves were inoculated with 40 μL of sterile distilled water or agar plugs without mycelia. The inoculated plants were placed in a humid chamber at 28°C with 80% humidity and a 12 h light-dark photoperiod. Symptoms similar to those seen on naturally infected leaves were observed on all inoculated leaves after 7 days inoculation. Re-isolation was performed from 80% of the inoculated leaves and isolates were confirmed as C. gloeosporioides morphologically, confirming Koch's postulates, and by sequencing the ACT, CAL, GADPH, and ITS regions. The control groups remained asymptomatic. In previous studies, C. gloeosporioides has also caused anthracnose on Chinese medicinal plants, including Baishao (Radix paeoniae alba) (Zhang et al. 2017) and Rubia cordifolia L. (Tang et al. 2020). To our knowledge, this is the first report of C. gloeosporioides causing anthracnose on W. villosa var. villosa in China. The results of our report serve as valuable references for further research on this disease.

In August 2006, leaf spots were observed on half-high blueberry (Vaccinium corymbosum) in a plant nursery in Dalian, China. The symptomatic potted 1-year-old blueberry plants were located in parts of a plant nursery with poor ventilation. The primary symptom was a leaf spot, 0.4 to 0.8 cm in diameter, with brown margins that enlarged and coalesced. Mycelium grew from symptomatic and green leaf tissue removed from the margin of a necrotic leaf spot. Plant tissues were surface disinfested with 0.1% mercuric chloride for 3 min and 70% ethyl alcohol for 30 s before plating onto potato dextrose agar. The resulting colonies were white with a regular margin and a rough surface. The cultures were covered with black and globular acervuli with a diameter of 100 to 200 μm. The base of each conidiophore was swollen and globose with phialides growing from the apical end. Mature conidia were straight to fusiform, measuring 19.0 to 27.5 × 6.3 to 9.2 μm, and five-celled with the three middle cells brown and darker than the end cells. The apical cell was triangular and hyaline with three simple setulae that were 17.2 to 29.7 μm long. The base cell terminated in a point 4.0 to 8.6 μm long. Koch's postulates were fulfilled for the fungus by spray inoculating two healthy young plants with 2 × 105 conidia per ml of sterile distilled water. As a control, two similar plants were sprayed with sterile water. Plants were placed inside plastic bags to maintain humidity and incubated in a growth chamber at 26°C under fluorescent light for 14 h and at 20°C in darkness for 10 h. After 3 days, the plastic bags were removed and plants were maintained under the same conditions. More than 20 days after inoculation, symptoms on inoculated plants were similar to those previously described in the nursery. Control plants did not show any symptoms. Cultures isolated from the lesions were similar to those isolated previously from plants in the nursery. The morphological descriptions and measurements were similar to Pestalotiopsis clavispora (1). The 5.8S subunit and flanking internal transcribed spacers (ITS1 and ITS2) of rDNA and partial β-tubulin gene were amplified from DNA extracted from single-spore cultures using the ITS1/ITS4 and T1/Bt2b primers (2) respectively, and sequenced (GenBank Accession Nos. EF119336 and EF152585). The ITS sequences were most similar to the ITS regions of P. clavispora TA-8 (98%; GenBank Accession No. AY924264), P. clavispora TA-6 (98%; GenBank Accession No. AY924263), and P. clavispora PSHI 2002 Endo 389 (96%; GenBank Accession No. AY682929). The partial β-tubulin gene sequence was identical to Pestalotiopsis sp. isolate PSHI 2004 Endo 86 (100%; GenBank Accession No. DQ657901). The morphology and sequence data support the identity of the causal fungus as P. clavispora. To our knowledge, this is the first report on the presence of a Pestalotiopsis sp. causing a disease of blueberry in China.

Lily is an economically important ornamental crop in Korea. In August 2008, severe leaf spot symptoms were observed on an oriental Lily 'Action' in a plant nursery in Daegu, Korea. Disease incidence was 20 to 30%. Initial symptoms were olive green-to-brown lesions on the leaf that developed into tan, elliptical, necrotic lesions. On severely infected leaves, lesions coalesced and killed the entire leaf blade. Infected leaves were surface disinfested with 70% ethanol for 30 s and 2% chlorox for 15 min before plating 1 cm2 sections onto potato dextrose agar. Hyphae appeared 5 days after inoculation and pure culture. Conidia were hyaline, transversely septate with one to three septa; most had two. Conidia were obpyriform and measured 29 to 46 μm long and 7 to 17 μm wide. Mycelia morphology and conidia production were consistent with that described previously for Pyricularia grisea (1). Koch's postulates were fulfilled by spraying five, healthy, vegetative-stage plants with 2 × 105 conidia per ml of sterile distilled water plus 0.05% Tween 20. As a control, five similar plants were sprayed with sterile water plus 0.05% Tween 20 only. Plants were placed inside plastic bags to maintain high relative humidity and incubated in a growth chamber at 25°C under fluorescent light for 14 h and at 20°C in darkness for 10 h. After 3 days, the plastic bags were removed and plants were maintained under the same conditions. Initial symptoms were observed 7 days after inoculation. Ten days after inoculation, disease symptoms on inoculated plants were similar to those previously described in the nursery. Control plants did not show any symptoms. Fungi isolated from these lesions had the same morphological characteristics as the ones isolated previously from plants in the nursery. To our knowledge, this is the first report of gray leaf spot on lily caused by P. grisea in Korea.

Mexico is the largest avocado (Persea americana) producer and exporter in the world. In January of 2019, typical symptoms of fruit anthracnose were observed on approximately 90% of avocado trees in backyards localized in Leonardo Bravo municipality in Guerrero, Mexico. Lesions on avocado fruits were circular, necrotic, and sunken, whereas the mesocarp showed a soft rot with dark brown discoloration. To perform fungal isolation, small pieces from adjacent tissue to lesions of five symptomatic fruits were surface disinfested by immersion in a 1% sodium hypochlorite solution for 2 min, rinsed in sterile distilled water, and placed in Petri dish containing potato dextrose agar (PDA). Plates were incubated at 25 ºC for 5 days in darkness. Colletotrichum-like colonies were consistently isolated and seven monoconidial isolates were obtained. An isolate was selected as a representative for morphological characterization, molecular analysis, and pathogenicity tests. The isolate was deposited in the Culture Collection of Phytopathogenic Fungi at the Colegio Superior Agropecuario del Estado de Guerrero (Accession No. CSAEG-CJ19). After 8 days on PDA, the colonies were gray on the upper surface, and with orange conidial masses. Conidia (n= 100) were cylindrical, hyaline, aseptate, with rounded ends, 14.4 to 18.5 × 4.5 to 6.2 μm. Based on morphological features, the isolate was tentatively identified in the C. gloeosporioides species complex (Weir et al. 2012). For molecular identification, genomic DNA was extracted and the internal transcribed spacer (ITS) region of rDNA, and partial sequences of actin (ACT), β-tubulin (TUB2), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes were amplified by PCR, and sequenced with primers ITS5/ITS4 (White et al. 1990), ACT-512F/ACT-783R (Carbone and Kohn 1999), Bt2A/Bt2B (Glass and Donaldson 1995), and GDF/GDR (Templeton et al. 1992), respectively. BLAST analysis of the obtained sequences of the ITS, ACT, TUB2, and GAPDH genes revealed 100%, 99.63%, 99.77% and 100% identity with those of isolate LF687 of C. jiangxiense in GenBank (Accession numbers KJ955201, KJ954471, KJ955348, and KJ954902). A phylogenetic tree based on Bayesian inference and including published ITS, ACT, TUB2, and GAPDH data for Colletotrichum species was constructed. The multilocus phylogenetic analysis clearly distinguished the isolate CSAEG-CJ19 as C. jiangxiense separating it from all other species within the C. gloeosporioides species complex. The sequences were deposited in GenBank (accession numbers ITS:MT011397; ACT:MN968784, TUB2:MN968786, and GAPDH:MN968785). To conduct Koch's postulates, 20 healthy avocado fruits (cv. Hass) were wounded with a sterile toothpick (2 mm in depth) and a drop of 15 µl of conidial suspension (1 × 105 spores/mL) was placed on each wound. Ten control fruit were wounded and treated with sterilized water. All the fruits were kept in a moist plastic chamber at 25°C for 8 days. All inoculated fruits developed circular and necrotic lesions (12 to 18 mm in diameter), 5 days after inoculation, whereas control fruits remained healthy. The fungus was consistently re-isolated from the inoculated fruits. Previously, C. jiangxiense has been reported as a pathogen on Camellia sinensis and Citrus sinensis in China (Farr and Rossman 2020). To our knowledge, this is the first report of C. jiangxiense causing anthracnose on avocado worldwide. This study shown another species in the C. gloeosporioides complex associated with avocado diseases in Mexico. Therefore, it is necessary to explore the diversity of Colletotrichum species in detail through subsequent phylogenetic studies as well as to monitor the distribution of this pathogen into other Mexican regions.

During the spring of 2003, flower spots were observed on French hydrangea (Hydrangea macrophylla (Thunb.) DC) in CETEFFHO-INTA-JICA experimental greenhouses in Castelar, Argentina. Brown, irregular spots randomly distributed on petals were detected on an old, whiteflowering variety of unknown origin, cultivated by growers. Small pieces of diseased tissue were surface disinfested with 2% NaOCl, plated on 2% potato dextrose agar (PDA) with pH 7, and incubated at 22 to 24°C. Dense, whitish mycelium developed within 48 h and turned gray after 72 h. Conidia were ellipsoid, hyaline, nonseptate, and formed in botryose heads. Spores from 10-day-old colonies that were developed on PDA in test tubes were removed with 4 ml of sterile water per tube. Prior to inoculation, inflorescences were detached and placed in water-filled glass vases. To test pathogenicity, eight healthy inflorescences were sprayed with a 5-ml suspension (2 × 104 conidia per ml of sterile distilled water). Another eight healthy inflorescences were sprayed with sterile distilled water. The inflorescences were maintained at 21°C and covered with polyethylene bags that were removed after 3 days. Brown, circular-to-irregular spots appeared on petals 5 days after inoculation, became coalescent, and covered 50 to 60% of each inflorescence in 8 days. Gray mold consisting of black conidiophores and gray-in-mass conidia was observed 3 days after the development of the symptoms. Controls remained symptomless. The same pathogen was recovered from inoculated flowers and was identified as Botrytis cinerea Pers.:Fr. (1). To our knowledge, this is the first report of this fungus on Hydrangea macrophylla in Argentina. Reference: (1) M. V. Ellis and J. M. Waller. Sclerotinia fuckeliana (condial state: Botrytis cinerea).No. 431 in: Descriptions of Pathogenic Fungi and Bacteria, CMI, Kew, Surrey, UK, 1974.