Single-spore Isolates Research Articles

First Report of Fusarium incarnatum-equiseti Species Complex Causing Fruit Rot on Muskmelon in Taiwan.

Muskmelon (Cucumis melo L.) is an economically important fruit crop in Taiwan. In March 2020, the symptoms of fruit rot were observed in approximately 10% of mature muskmelon fruits in a field located in Wuri (24.043585, 120.657588), Taichung City, Taiwan. Symptoms including water-soaked lesions were initially observed on the lwer side of fruit, extending with time to cover most of the fruit area, and internal dissolution with white to brown mycelia on the surface was also observed. Ten rotted fruits were disinfested with 70% ethanol for 1 min followed by 1% NaOCl for 5 min, then rinsed three times with sterile distilled water (SDW). Fifteen sterilized symptomatic fruit fragments were cut into 1-cm3 pieces, placed on potato dextrose agar (PDA) amended with 35 mg/liter of streptomycin sulfate and incubated at 28°C in the dark for 1 week. Ten isolates with similar morphology were obtained and the representative isolate FOS-1 was characterized further. Single-spore isolates were used for morphological and molecular analyses. Isolates grown on PDA had dense, cottony white aerial mycelium, changed to light brown, and with the time yellowish-brown pigmentation appeared. Microconidia were ovoid, fusiform, or slightly curved, 0 to1 septate, and ranged between 7.9 to 16.5 × 2.8 to 3.5 μm. Macroconidia were 3 to 5 septate, with a slightly curved and tapering apical cell, and ranged between 18.7 to 35.1 × 3.3 to 4.1 μm. Spherical chlamydospores with thick walls were abundant and single, being produced in terminal or intercalary position. Based on morphological characteristics, the fungus was identified as Fusarium sp. (Leslie and Summerell 2006). PCR amplification and DNA sequencing were performed using primers ITS1/ITS4 (White et al. 1990) and EF1-728F/EF1-986R (Carbone and Kohn 1999) to amplify the complete internal transcribed spacer (ITS) region and the partial translation elongation factor 1-alpha (TEF1-α) gene, respectively. The ITS and TEF1-α gene sequences of Isolate FOS-1 were deposited in GenBank database with acc. nos. MZ749694.1 and MZ782277.1, respectively. BLAST analysis showed 99.64% and 100% sequence identity with F. incanatum-equiseti species complex (FIESC) with MT563419.1 for ITS and MW034437.1 for EF-1α, respectively. BLAST analysis of TEF1-α gene sequence in FUSARIUM-ID database (Geiser et al. 2004), showed 99.31% sequence identity with FIESC (NRRL34070). Pathogenicity was confirmed by fulfilling Koch's postulates. Three healthy muskmelon fruit were disinfested using 70% ethanol for 30 s and 1% NaOCl for 5 min, and followed by three rinses with SDW. Then, the fruit were wounded using a sterile needle and inoculated with an 8 mm-mycelium agar plug. Three sites per fruit were inoculated, and three other fruits treated with mycelium-free PDA plugs served as the controls. The inoculated and control fruit were placed in a plastic box and incubated at 25°C under a 12 h photoperiod for 1 week. All inoculated fruit showed symptoms similar to those observed in the field, whereas no symptoms occurred on the controls. The fungus was re-isolated from the infected fruit, and identified as FIESC by the morphological and molecular methods described above. This pathogen could cause great losses in muskmelon. Members of the FIESC have been reported to cause leaf spot and fruit rot in muskmelon (Cao et al. 2019; Ismail et al. 2021). To our knowledge, this is the first report of the FIESC causing fruit rot of muskmelon in Taiwan.

First Report of Corynespora cassiicola Causing Leaf spot of Strobilanthes cusia in China.

Strobilanthes cusia (Nees) Kuntze is a vital medicinal and industrial herb, planted extensively in southern China (Hu, et al. 2011.). In July and August of 2021, leaf spot incidence on >60% plants and reduced yields >20% for fresh leaves were observed in S. cusia cultivar 'Malan No.1' across the Shufeng whole Township, Xianyou County, Fujian province. Initial symptoms on leaves were observed as small, dark-brown, spots surrounded by a yellow halo, expanding irregularly or into semicircular spots. As symptoms developed, the spots became dark brown, thin and fragile, forming small holes. In severe cases plants were defoliated. The pathogen was isolated from the margin of 60 symptomatic leaf lesions， surfacesterilized with 75% ethanol for 45 s, rinsed three times with sterile water, air dried, and cultured on PDA at 25°C in the dark. Pure cultures were obtained by single-spore isolation after subculture. Ten representative single-spore isolates (MY-1 to MY-10) from 154 pathogens in 10 sampling points were selected for morphological characterization and identification. After 7 days, mycelial colonies were gray to dark gray with few aerial hyphae. Conidia (32.3 to 132.8 × 5.8 to 8.4 μm, average 81.4 × 6.3 μm, n=50) were pale to brown, erect or curved, solitary or in chains, with 0 to 15 pseudosepta. Based on morphological characteristics, the isolates were preliminarily identified as Corynespora cassiicola. Genomic DNA of isolate MY-2 (randomly selected from 10 isolates as representative) was extracted from mycelia using the Ezup DNA extraction kit (Sangon Biotech Co., Ltd. Shanghai, China). The ITS (internal transcribed spacer) region of rDNA, TEF1-α (translation elongation factor 1 alpha) and TUB2 (beta-tubulin) genes were amplified and sequenced with primers ITS4/ITS5, EF1-728F/EF-986R (Wang et al. 2021) and Bt2a/Bt2b (Glass et al. 1995), respectively. BLASTN sequence analyses of ITS (538 bp), TEF1-α (302 bp) and TUB2 (436 bp) of isolate MY-2 (GenBank accessions OK355515, OM339443, OM339442) showed 100%, 97.6%, 100% identity with C. cassiicola in GenBank (Accession numbers JX908713, MW961421, AB539228). A neighbor-joining phylogenetic analysis based on ITS and TEF1-α sequences using MEGA7 showed that MY-2 clustered in the same clade with C. cassiicola. For pathogenicity tests, five S. cusia plants were inoculated onto the adaxial surface of leaves with mycelial plugs from ten isolates of 8-day-oldcultures on PDA. Five leaves per plant were inoculated, covered with wet cotton, and kept in a controlled greenhouse (26~33 °C, RH 80% ~ 90%). Leaves inoculated with sterile PDA plugs served as a negative control. At 3-5 days post inoculation, all 25 inoculated leaves of each isolate showed leaf spot lesions similar to those observed in the field, and control leaves were symptomless. C. cassiicola was successfully reisolated from the diseased leaves. The pathogenicity tests were repeated three times under the same conditions and similar results were observed. In view of morphology, pathogenicity and sequence results, the isolates were identified as C. cassiicola, a pathogen reported from many important crops (Lu et al. 2021). This is the first report of C. cassiicola as a pathogen in China which poses a potential threat to leaf production and S. cusia processing.

First Report of Diaporthe phoenicicola causing leaf spot on blueberry (Vaccinium virgatum) in China.

Blueberry (Vaccinium virgatum), a member of the Ericaceae family, is an increasingly important crop in China because of its abundant nutritional benefits and economic value (Kuzmanović et al. 2019). In October 2021, leaf spots were detected on 'Rabbiteye' blueberry at the Agricultural Science and Technology Park of Jiangxi Agricultural University in Nanchang, China (28°45'51"N, 115°50'52"E), which caused severe defoliation of the crop and fruit yield losses of 25% (Figure 1A). Disease surveys were conducted at that time; the results showed that disease incidence was 75.5%, observed in 151 of the 200 accessions sampled, and this disease had not been found at other cultivation fields in Nanchang. Lesions with taupe to dark brown margins were irregularly shaped and associated with leaf margins. Spots coalesced to form larger lesions, with black pycnidia present in more mature lesions. To identify the causal agent, 10 small pieces (5 mm2) of leaf tissue excised from the lesion margins were surface sterilized in 75% ethanol solution for 30 s and 0.1% mercuric chloride solution for 2 min, rinsed three times with sterile distilled water, then placed on potato dextrose agar (PDA) at 25°C for 5 to 7 days in darkness. Five fungal isolates showing similar morphological characteristics were obtained as pure cultures by single-spore isolation. All fungal colonies on PDA were floccose, dense, and white (Figure 1B.C). Black pycnidia developed on PDA at 25°C under a 12/12 h light/dark cycle for 30 days. Alpha conidia were 6.17 to 8.53 × 1.64 to 3.20 µm (average 6.94 × 2.52 µm, n = 100), aseptate, hyaline, fusiform to ellipsoidal, often biguttulate. Beta conidia were 15.26 to 25.41 × 0.92 to 1.40 µm (average 20.14 × 1.27 µm, n = 30), aseptate, hyaline, linear to hamate (Figure 1D). Based on morphological characteristics, the fungal isolates were suspected to be Diaporthe spp. (Gomes et al. 2013). To further confirm the identity of this putative pathogen, two representative isolates (LGM1 and LGM2) were selected for molecular identification. The internal transcribed spacer region (ITS), translation elongation factor 1α (EF1-α), histone H3 (HIS), calmodulin (CAL), and β-tubulin (TUB2) genes were amplified from gDNA and sequenced using primers ITS1/ITS4 (Peever et al. 2004), EF1-728F/EF1-986R and CAL228F/CAL737R (Carbone et al. 1999), CYLH3F/H3-1b (Crous et al. 2004), Bt2a/Bt2b (Glass and Donaldson 1995), respectively. GenBank accession numbers of isolate LGM1 and LGM2 were OM778771 to 72 for the ITS region, OM868228 to 29 for EF1-α, OM837771 to 72 for TUB2, ON206971 to 72 for CAL, ON206973 to 74 for HIS. BLAST results showed that the ITS, EF1-α, TUB2, HIS, and CAL sequences showed 99% (538/545 bp), 100% (322/322 bp), 99% (480/484 bp), 99% (459/460 bp), 99% (430/433 bp) identity, respectively, with those of Diaporthe phoenicicola (GenBank accession no. MW504735, MW514099, MW514142, MW514067, MT409304). Two maximum likelihood phylogenetic trees were built based on the sequences of ITS, EF1-α, HIS, CAL, and TUB2 by using MEGA 5. The two isolates LGM1 and LGM2 clustered with D. phoenicicola (Figure 2 and 3). The fungus was identified as D. phoenicicola by combining morphological and molecular characteristics. To evaluate the pathogenicity, three healthy young potted V. virgatum plants were spray inoculated with a conidial suspension of 106 conidia/ml. Another set of three plants that were sprayed with sterilized distilled water served as the controls. The experiment was repeated three times, and all plants were maintained in a climate box (12 h light/dark) at 25°C with 80% relative humidity. Five days after inoculation, no symptoms were observed on control plants (Figure 1F), and all inoculated plants showed symptoms (brown flecks) similar to those observed in the field (Figure 1E). The fungus was reisolated from the infected tissues and confirmed as D. phoenicicola by morphological and molecular identification, and could not be isolated from the controls, fulfilling Koch's postulates. To our knowledge, this is the first report of D. phoenicicola causing leaf spot on blueberry in China. The discovery of this new disease and the identification of the pathogen will provide useful information for developing specific control measures and potential sources for resistance to this disease caused by D. phoenicicola.

First Report of Brown Spot of Dekopon Fruit Caused by Cladosporium tenuissimum in China.

Dekopon citrus (Citrus reticulata Shiranui) is a three-way hybrid (Citrus unshiu Marcov. × C. sinensis Osbeck × C. reticulata Blanco) developed in Japan in 1972. This citrus is popular in China due to its sweet and tender taste (Lim 2012). In November of 2021, a brown spot disease on Dekopon fruits with about 20% disease incidence was observed in an orchard of the Institute of Citrus Research in Ganzhou, Jiangxi Province, China. Initially, the symptoms appeared as slightly sunken deep red to purple spots on the fruit surface, with the disease progression, lesions became brown to brown-black large necrotic regions covered with a fluffy layer of gray spores. Infected fruits were surface sterilized with 70% ethanol for 30 sec and rinsed three times with sterile distilled water. Diseased tissues from the edge of lesions were cut into small segments, placed onto potato dextrose agar and incubated at 25℃ for 7 days. Ten single-spore isolates were obtained in total. Fungal colonies were olive green to dark green, velvet-like in texture and sporulated abundantly, surrounded by grayish-white hyphae. Conidiophores were subcylindrical, straight, septate, solitary or in clusters of two to three, and ranged in size from 65 to 550 × 3.8 to 6.3 µm (x ̅= 261.7 ± 60.5 × 5.2 ± 0.4 µm, n=50). Ramoconidia were cylindrical，aseptate, and 10 to 22 × 2.8 to 4.5 µm (x ̅= 15.5 ± 1.4 × 4.0 ± 0.9 µm, n=50). Conidia were lemon-shaped to oval-shaped, smooth-walled, and 1.8 to 5.0 × 1.4 to 2.5 µm (x ̅= 3.9 ± 0.4 × 2.2 ± 0.2 µm, n=50). The morphological characteristics of the pathogen were consistent with those of Cladosporium tenuissimum Cooke (Li et al. 2021). For further identification, DNA was extracted from two representative isolates. The internal transcribed spacer (ITS) region, translation elongation factor (EF1-α), and actin (ACT) were amplified by using primers ITS1/ITS4, EF1-728F/EF1-986R, and ACT-512F/ACT-783R (Bensch et al. 2012), respectively. ITS (OM232067, OM232068), EF1-α (OM256525, OM256526) and ACT (OM256529, OM256530) sequences were deposited in GenBank. Multi-gene (combined data set of ITS, EF1-α and ACT) phylogenetic analysis was conducted using the Maximum Likelihood method (Nguyen et al. 2015). Based on the morphological characteristics and the molecular data, two fungal isolates were identified as C. tenuissimum. To evaluate pathogenicity, fifteen fruits were surface sterilized with 1% NaClO solution for 30 sec, rinsed twice with sterile distilled water and dried. Dekopon fruits (n=10) were wounded with a sterile needle and inoculated with a 10 µL drop of conidial suspension (1 × 106 conidia/mL) of isolate GZCJ-1, followed by incubation at 25℃ and 80% relative humidity. The controls (n=5) were treated with sterile water and maintained under the same conditions. Five days after inoculation, small brown sunken spots were observed on the wounded and inoculated fruits. After 7 days, lesions were coated by a layer of brown conidia that were similar to those described above, whereas control remained symptomless. Pathogenicity test was repeated twice. Cladosporium tenuissimum was consistently re-isolated from inoculated fruits and confirmed by morphological and molecular data, fulfilling the Koch's postulates. To our knowledge, this is the first report of C. tenuissimum causing the brown spot of dekopon fruit in China and perhaps the world. The disease may become the potential risk for fruit production, making fruits unfit for marketing purposes, and the appropriate management actions will be necessary.

Alternaria alternata Causing Brown Spot Disease on Walnut in Xinjiang, China.

Walnut is one of the Xinjiang's characteristic dried fruits and the main source of income for farmers in walnut growing areas. In September 2019, Juglans regia leaves with brown spots were observed in a 10 hm2 orchard in Hotan area, the diseased leaf rate reached more than 25%. The leaf lesions were suborbicular to irregular, black-brown, 3 to 8 mm in diameter, with distinct dark borders. Colonies were isolated from 10 diseased leaves collected from two trees in the orchard. Leaf sections (4 × 4 mm) from diseased leaves were surface disinfested with 75% ethyl alcohol for 30 s and 2% NaClO for 3 min, washed with sterile water three times and then plated on potato dextrose agar (PDA) and incubated at 27℃ with a 12h/12h light/dark photoperiod for 4 days. A total of 7 fungal isolates were obtained by single-spore isolation. All the colonies were dark olivaceous on the PDA plates, with loose, cottony mycelium. On potato carrot agar (PCA), all fungal isolates produced conidial chains with numerous secondary chains. The conidia were ellipsoid or obpyriform with 0-3 longitudinal septa and 2-4 transverse septa, measuring 20.6 to 35.8 × 6.8 to 11.2 μm (25.5 ± 0.4 × 8.7 ± 0.2 μm, n=50). The morphological characteristics of the seven fungal isolates were consistent with the A. alternata descriptions of Simmons (2007). DNA was extracted from 50 mg of mycelia for the representative isolate HLP17-7. The internal transcribed spacer (ITS) region was PCR amplified using the universal primers ITS1 / ITS4 (White et al.1990), the partial coding sequence of endopolygalacturonase (endoPG) and the partial region of the histone 3 (H3) were amplified using primers PG2b / PG3 (Andrew et al. 2009) and H3-1a / H3-1b (Glass and Donaldson 1995) respectively. The products were sequenced and deposited in GenBank database under the accession numbers MW514319 [ITS], ON806938 endoPG, MW489301 [H3]. ITS, endoPG and H3 sequences had 99.81% (1/535 nt difference), 99.78% (1/448 nt difference) and 100% (0/417 nt difference) homology with homologous sequences of A. alternata strains (KP124306 [ITS], KP124006 [endoPG], MK085979 [H3]), respectively. During the early autumn, pathogenicity tests were carried out on the healthy mature leaves of seven-year-old Juglans regia plants in the field. Thirty leaves (five leaves per plant) were wounded with a sterile needle and then sprayed with a spore suspension prepared from 10-day-old PDA culture. Five wounded leaves per plant were sprayed with sterile water as control. All the treated leaves were covered with clear plastic bags for 3 days, and the experiment was replicated three times. On the 8th day after inoculation, brown spots appeared on the inoculated leaves, but no spots were observed in the control. Morphological observation and gene sequencing confirmed that the original fungal pathogen was re-isolated from the inoculated leaves. No colony was isolated from the control leaves. The pathogen causing the brown spot was identified as A. alternata based on morphological features and sequence analysis. A. alternata has been reported previously in Sichuan (Yang et al., 2017) causing brown spot in walnut. Xinjiang is dry with little rain and abundant sunshine, so there are few diseases on walnuts. However, the occurrence of brown spot disease has alarmed fruit farmers, walnuts are still at the risk of A. alternata infections even in dry environment with little rain. To our knowledge, this is the first report of A. alternata causing brown spot in walnut in Xinjiang, China.

First report of anthracnose on sugar beet (Beta vulgaris) caused by Colletotrichum incanum in Michigan, USA

In 2016, sugar beet leaf samples were received at the Michigan State University plant diagnostic laboratory with distinctive oblong lesions on the petiole (Fig. 1). The lesions contained acervuli with dark setae (Fig. 2). Lesions on the blade had light and dark rings that showed similar acervuli in a 90% humidity chamber. Pure cultures from single spore isolation were grown on malt extract agar (MEA). The resulting cultures produced acervuli-like structures with dark setae distributed throughout the conidiomata. Spores were curved and had a distinct gutule (Fig. 3). Based on these characteristics, isolates were identified as a Colletotrichum sp. A representative isolate, Co16-4, was grown in liquid V8 medium with shaking for seven days. Fungal tissue was lyophilised and DNA extracted using a DNeasy Plant Mini Kit (Qiagen, Germany). Partial sequences of the internal transcribed spacer (ITS), actin (ACT), and β-tubulin (TUB2) genes were amplified according to Damm et al. (2009) and submitted to the Research Technology Support Facility (East Lansing, USA) for Sanger sequencing. Sequences were compared to data in GenBank where ITS gave the closest match to C. spaethianum (>98%), while ACT and TUB2 gave the closest match to C. incanum (99 and 96%, respectively). Sequences were submitted to GenBank as Accession Nos. OP628186 (ITS), OP627081 (ACT) and OP627082 (TUB2). Due to the BLAST search ambiguity, sequences of 19 Colletotrichum species were retrieved from GenBank as identified in Yang et al. (2014). Sequences were concatenated and manually adjusted using Geneious Prime 2021.1.1 (Biomatters Ltd., Auckland, New Zealand). Alignment and a maximum-likelihood tree were generated in Mega version 11.0.13 (Tamura et al., 2021) using the Tamura-Nei substitution model with 1000 bootstrap replicates (Fig. 4). Based on this data, isolate Co16-4 was identified as Colletotrichum incanum. Koch's postulates were fulfilled by spraying sugar beet foliage (C869) at the 10- to 12-leaf growth stage with 10ml of a spore suspension (1×104/ml with 0.1% Tween 20). After seven days, an oblong lesion was observed on the petiole and irregular lesions developed on inoculated leaves. No lesions were observed on control plants. A morphologically identical Colletotrichum sp. was isolated from lesions. Colletotrichum incanum has been reported on soybean (Yang et al., 2014), but to our knowledge, no Colletotrichum spp. have been reported causing anthracnose on beet in the United States. The symptoms and morphology of the lesions on sugar beets are consistent with beet anthracnose reported in Japan and Canada which was attributed to C. dematium (Chiko et al., 1984; Gourley, 1966). In Japan, a radish isolate, originally identified as C. dematium, was recently identified as C. incanum (Gan et al., 2016). Leaves with similar lesions were found in commercial sugarbeet fields in 2021 and 2022, indicative of continuing disease presence, but yield effects are unknown.

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

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

First report of Rhizopus oryzae causing rhizome rot on ginger in China.

Ginger (Zingiber officinale Rosc.) is a herbal plant, widely grown in China for its medicinal and culinary purposes. In July 2020, a new rhizome rot disease was observed on ginger in Laiwu, Shandong Province, China. The disease symptoms were observed on both above-ground and underground plant parts. The above ground stems and leaves becoming withered and yellow, and water-soaked symptoms were observed on the collar region. The diseased rhizomes were poorly developed with brown lesion and eventually they would rot, without offensive odors. Disease incidence was estimated at approximately 5% across the survey area. To isolate the pathogen, tissues from 30 rhizomes were cut from the border between diseased and healthy tissue, surface sterilized in 75% alcohol for 15 s, soaked in 0.1% mercuric chloride for 1 min, washed with sterile distilled water three times, and plated on potato dextrose agar (PDA) at 25°C for 2-3 days. Twenty nine fungal isolates with similar morphological characteristics were obtained and pure cultures were obtained using single spore isolation. The colony of AQJ-1, a representative isolate, on PDA was cottony, fluffy, white, and beige coloration on the reverse side at first, and subsequently many black sporangia were produced. The sporangia were black, sub-globose, and 45.2-181.7 μm (n = 50) in diameter. The sporangiospores were unequal, globose or sub-globose, about 3.2-8.7 × 4.6-12.3μm (n = 50) in diameter. For the molecular characterization, genomic DNA was extracted by modified CTAB method (Niu et al., 2008). Internal transcribed spacer (ITS) region and translation elongation factor 1-alpha (EF-1α) gene were amplified using the primer pairs ITS1/ITS4 (White et al., 1990) and MEF10/MEF4 (Abe et al., 2007), respectively. The ITS and EF-1α sequences of isolate AQJ-1 were submitted to GenBank (MN606288 and MN735220, respectively). The BLASTn analysis of the sequences showed 99%-100% similarity to the sequences of R. oryzae strain CBS 120.12 (MH854609, AB281529, respectively). Therefore, based on morphological and molecular characteristics, isolate AQJ-1 was identified as R. oryzae. For pathogenicity tests, thirty ginger seedlings (Laiwu Big Ginger) were grown for 30 days in plastic pots and removed from the pots and the rhizomes washed in running tap water. The rhizomes of fifteen ginger seedlings were attached to a 7 mm agar disk from a plate containing 2-day-old mycelium, and the other fifteen seedlings were attached to agar disk without mycelium as control. Then the inoculated and control seedlings were planted in pots and were kept in separate chambers in a greenhouse at 25±2 °C. After 14 days, the same symptoms of rhizome rot were observed in all inoculated plants as previously described, and no symptoms were observed on the control plants. The pathogen was re-isolated from symptomatic tissues, and was identified as R. oryzae, which full-filled the Koch's postulates. To our knowledge, this is the first report of R. oryzae causing rhizome rot on ginger in China. This disease may pose a potential threat to ginger production in China.

First Report of Epicoccum sorghinum Causing Leaf Spot on Paeonia suffruticosa in China.

Tree peony (Paeonia suffruticosa Andr.; Paeoniaceae) is highly valued in Chinese culture for their ornamental and medicinal benefits in anti-inflammation, anti-arrhythmia, activating the immune system and protecting the cardiovascular system (Zhai et al. 2020). In May from 2019 to 2021, leaf spot symptoms were observed on tree peony in an area of 0.8 hm2 in Bozhou, Anhui Province, China; with a disease incidence of 35%. Initially, black-brown spots (0.5-2.4 mm) appeared on the leaves, as the disease developed, the spots enlarged to form round, black lesions. Small pieces of tissue taken from the margins of lesions were surface-disinfected by 75% ethanol for 20 s followed by 0.1% mercuric chloride for 3 min, rinsed with distilled sterile water for three times, placed on potato dextrose agar (PDA) at 25 ºC for 7 days in the dark.Fifteen single spore isolates were obtained as described (Dong 2009). When cultured on PDA for 10 days, colonies were white, villose, then became gray-brown, with the reverse side becoming reddish-brown; Chlamydospores globose or irregular shaped, dark brown, unicellular or multicellular (4.3 to 12.2 × 13.1 to 33.7 µm) (n = 200). Pycnidia were black-brown, mostly spheroid, and 86 to 128 × 110 to 197 µm (n = 50); Conidia were hyaline, ellipsoidal, unicellular, aseptate, and 4.1 to 5.2 × 1.8 to 2.4 µm (n = 200) . The DNA of a representative isolate AF21-1-2 was extracted using the CTAB protocol (Doyle and Doyle 1987). The rDNA internal transcribed spacer (ITS), β-tubulin (TUB) and 28s nrDNA (LSU) genes were amplified and sequenced using the primers ITS1/ITS4 (White et al. 1990), Bt2a/Bt2b (Glass et al. 1995), and LR5/LR0R (Vilgalys and Hester, 1990), respectively (OK485136, OK631973, and ON358331). BLASTn analysis showed that the ITS, TUB, and LSU sequences had 99% (497/501 bp), 98% (530/539 bp), and 99% (878/882 bp) identity with the Epicoccum sorghinum isolates ESCZO20 (MN944541), CZ01 (MT254851) and Dl16-338 (LN907481), respectively. Maximum likelihood phylogenetic tree revealed that AF21-1-2 clustered with E. sorghinum isolates. For pathogenicity test, leaves of 3-year-old healthy tree peony plant in the field were inoculated with 105 conidia/ml spore suspension (prepared with 30-day-old cultures on PDA), added with 0.1% (v/v) Tween-80. Ten surface-sterilized leaves per plant were sprayed with 30 ml suspension until runoff, a total of 3 plants were inoculated. Leaves sprayed with sterilized water serve as a negative control. Each plant was covered with a plastic bag to maintain high relative humidity at 15 to 23°C and was monitored daily for symptom development. Typical symptoms appeared on more than 80% of the leaves after 7 days of inoculation, while control leaves remained symptomless. Cultures isolated from inoculated leaves had the same morphological and molecular traits as those that were previously used to test Koch's postulates. Based on the morphological and molecular characteristics, the causal agent on tree peony was identified as E. sorghinum (Aveskamp et al. 2010). To our knowledge, this is the first report of E. sorghinum causing leaf spot on tree peony in China. The disease will cause serious economic loss for tree peony if it is not managed properly. This report may provide the basis for diagnosis and control of the disease.

First report of Fusarium falciforme causing basal rot of onion (Allium cepa) in India.

Onion (Allium cepa, L) is a very important vegetable crop in India. India is the second largest producer of onion in the world and the crop is grown on more than 1.22 million hectares. Fusarium Basal Rot (FBR) is an economically important diseasef onion that causes considerable losses in onion production up to 50% in field and 30-40% during post-harvest storage of bulbs (Gupta and Gupta 2013; Rajamohan et al. 2019). Onion plants showing chlorosis, twisting, wilting, necrosis, bulb discoloration, rot in the basal parts of bulb and roots typical to FBR were observed, in a field trial of 36 onion cultivars during October 2020 in Bangalore, Karnataka. FBR incidence varied from 30-100% in this field (Fig 1 a-d). Symptomatic bulbs were washed with water, basal plate and fleshy leaves cut into 0.5 to 1 cm-size, surface disinfected with 1.5% sodium hypochlorite (NaOCl) for 3 min, and rinsed with sterile distilled water. Twenty pieces were placed on potato dextrose agar (PDA) in Petri plates and incubated at 25°C for 7 days. Colonies from single-spore isolates on PDA showed abundant white aerial mycelium. Colonies showed light pink or purple coloration on the reverse side of the culture plate with brown center (Fig 1e-f). Macroconidia were 19.13 to 28.35 (mean= 24.2) × 4.29 to 6.06 (mean= 5.05) µm, hyaline, falcate, with slightly curved apexes, and three to five septa. Microconidia were cylindrical to ellipsoid, aseptate, hyaline 8.20 to 12 (mean=10.0) × 3.55 to 4.79 (mean= 4.29) µm (Fig 1g). Chlamydospores were round, intercalary, hyaline, single or in chains (Fig 1h). Two isolates (IBFF-09 & IBFF-10) were analyzed for internal transcribed spacer-ITS (White et al. 1990) and translation elongation factor 1-α (tef1) gene (O'Donnell et al. 1998) by polymerase chain reaction (PCR) and sequenced. ITS and partial tef1 gene sequences of isolates IBFF-09 and IBFF-10 were submitted to the NCBI database (GenBank accession # ON394614&ON026859; # ON409480, ON093166 respectively). Phylogenetic analysis of tef1 gene placed the isolates with F. falciforme (Fig 1i). A pathogenicity test was performed by dipping roots of 28 days old healthy onion seedlings of a susceptible genotype 16/7Y GR3 into a conidial suspension (1 × 104 conidia/ml) of isolate IBFF-10 for 15 min and then transplanting the plants into pots containing sterilized potting mix. Inoculated plants developed typical symptoms of FBR and were all dead by 20 days post inoculation (Fig 1j) while the non-inoculated controls remained healthy. Pathogen was re-isolated from infected plants and showed the same morphology, ITS and tef1 sequence similarity as the original isolate, thus fulfilling Koch's postulates. Basal rot of onion by F. falciforme is reported from Mexico (Tirado-Ramírez et al. 2021). Till date, only F .oxysporum, F. proliferatum and F. solani have been implicated in onion FBR in India (Lee et al. 2021; Rathore and Patil, 2019). F. falciforme however, i prevalent in India and is reported to infect other crops (Gangaraj et al. 2022; Gupta et al. 2019; Homa et al. 2018). There is a high probability that this pathogen is contributing significantly to basal rot disease but it has not been reported yet. To our knowledge, this is the first report of F. falciforme infecting onions in India. In order to develop FBR resistant onion cultivars it is critical to identify and study the response of onion genotypes to different Fusarium spp causing the disease.

Leaf spot on Photinia × fraseri caused by Neopestalotiopsis asiatica in China.

Photinia × fraseri is a well-known evergreeen ornamental tree. Owing to its flower-like red leaves and its ability to tolerate stressful environments, P. fraseri is widely cultured as a fast-growing hedge in southern China. From JulytoSeptemberin2021, a disease with symptoms similar to leaf spot was extensively observed on P. fraseri in Daozhen county (28° 51 'N, 107° 57 'E), Zunyi, Guizhou province, China. About 500 plants were surveyed and the incidence of leaf spot on P. fraseri leaves was 35% to 70%, significantly reducing the ornamentalandeconomicvalue. The symptomatic leaves displayed irregular, watery dark brown lesions with black conidiomata in gray centers, and 10 symptomatic leaves were collected from 10 trees.After surface sterilization (0.5 min in 75% ethanol and 2 min in 3% NaOCl, washed three times with sterilized distilled water) (Fang 2007), small pieces of symptomatic leaf tissue (0.2 × 0.2 cm) were plated on potato dextrose agar (PDA) and incubated at 25°C for about 7 days. Three single-spore isolates, GZAAS21-0327, GZAAS21-0328 and GZAAS21-0329, were obtained, which were identical in morphology and molecular analysis. Therefore, the representative isolate GZAAS21-0328 was used for further study. The pathogenicity of GZAAS21-0328 was tested through a pot assay. Ten healthy plants were scratched with a sterilized needle on the leaves. Plants were inoculated by spraying a spore suspension (106spores mL-1) of GZAAS21-0328 onto leaves until runoff, and the control leaves sprayed with sterile water. The plants were maintained at 28°C with high relative humidity (95%) in a growth chamber. The pathogenicity test was carried out three times (Fang 2007). The symptoms developed on all inoculated leaves but not on the control leaves. The lesions were first visible 72 h after inoculation, and typical lesions similar to those observed on field plants appeared after 15 days. The same fungus was reisolated and identified based on the morphological characterization and molecular analyses (ITS, TUB and TEF) from the infected leaves but not from the noninoculated leaves. Results of pathogenicity experiments of isolated fungi fulfilled Koch's postulates. Fungal colonies on PDA were villiform, creamy-white and sparse aerial mycelium on the surface with black, gregarious conidiomata. The conidia were fusoid, ellipsoid, straight to slightly curved, 4-septate, septa darker than the rest of the cell, and 23.0 (21.0 to 27.0) × 6.0 (5.0 to 7.0) µm (n=50). The morphological features were consistent with the descriptions ofNeopestalotiopsis asiatica Maharachch. & K.D. Hyde (Maharachchikumbura et al. 2012; Maharachchikumbura et al. 2014; Farr et al. 2022). The pathogen was confirmed to beN. asiaticaby amplification and sequencing of the internal transcribed spacer region (ITS), the partial β-tubulin (TUB) and partial translation elongation factor 1-alpha (TEF) genes using primers ITS4/ITS5, T1/Bt-2b and EF1-728F/EF-2, respectively (Maharachchikumbura et al. 2014). The sequences of PCR products were deposited in GenBank with accession numbers OK563071 (ITS), OK584020 (TUB) and OK663023 (TEF). BLAST searches of the obtained sequences revealed 100% (482/482 nucleotides), 99.05% (419/421 nucleotides), and 99.33% (891/897 nucleotides) homology with those ofN. asiaticain GenBank (JX398983, JX399018 and JX399049, respectively). Phylogenetic analysis (MEGA 6.0) using the maximum likelihood method placed the isolate GZAAS21-0328 in a well-supported cluster withN. asiatica. The pathogen was thus identified asN. asiaticabased on the morphological characterization and molecular analyses. To our knowledge, this is the first report of leaf spot on P. fraseri caused by N. asiatica in China. This study provides valuable information for the identification and control of the leaf spot on Photinia × fraseri.

Occurrence of Brown Rot of American Plum Caused by Monilinia fructicola in Missouri

Prunus americana (wild plum) is commonly found in Missouri and many other U.S. states from Texas to Massachusetts. In May 2022, P. americana fruits exhibiting brown rot symptoms were observed in Cole County, Missouri. The symptoms include brown rot and mummified fruits. Advanced symptoms include fruits covered with fungal growth. A single-spore isolate (PAM-1) was recovered from fruits showing brow rot symptoms. The PAM-1 isolate cultured on potato dextrose agar (PDA) grew a 100 mm diameter Petri dish in seven days. The colony was off-white to light brown in color, circular, and showed concentric rings. The colony had entire margins and produced abundant conidia on PDA. The conidia ( n = 50) were 12.07 µm long and 7.64 µm wide. The conidia are oblong to lemon-shaped and hyaline. The polymerase chain reaction with ITS1 and ITS4 primers generated 508 bp DNA sequence which showed 100% identity to Monilinia fructicola (GenBank accession nos. FJ515894.1 and EF207419.1). Pathogenicity tests on both wounded and unwounded fruits exhibited similar symptoms. Control fruits did not show any symptoms. Based on the disease symptoms, pathogen morphology, molecular characterization, and pathogenicity test the pathogen was identified as M. fructicola. To our knowledge, this is the first scientific report showing M. fructicola as a causal organism of brown rot of wild plum in Missouri.

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

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

First Report of Yeast-Spot Disease of Soybean Seeds Caused by Eremothecium coryli in Serbia.

Over the last 15 years, the area planted with soybeans (Glycine max) in Serbia has increased drastically, from 131,000 hectares in 2005 to 230,000 in 2019, and the average yield reached 3.2 t/ha in 2020. The Province of Vojvodina is the most important soybean production region with 95% of the total soybean area in Serbia (www.stat.gov.rs). During the 2021 growing season, soybean seeds with various kinds of symptoms including colour changes, light and dark brown spots, blotching, necrosis, and shriveling were collected from soybean field before harvest of soybean cv. Dukat in the Tamiš locality (South Banat District, Vojvodina Province: GPS: 44°56'12.936"N 20°43'24.216"E) in Serbia. The incidence of symptomatic seeds was estimated at 6.4%. Symptomatic soybean seeds were surface disinfected with 2% NaOCl for 2 min, rinsed in sterile water, dried on sterile filter paper, placed on potato dextrose agar (PDA) and were incubated at 25°C in the dark for 10 to 14 days. The identification of fungi at the genus level based on morphological characteristics revealed the presence of species of Macrophomina, Botrytis, Cercospora and Alternaria, which were previously reported as pathogens of soybean seed in Serbia (Krsmanović et al. 2020). Also, seven white to slightly creamy colonies with yeast-like morphology were observed around seeds expressing discoloration and necrotic and sunken spots. Ten days later, microscopic observations of yeast-like colonies revealed the presence of globose budding cells (diameter of 20 to 28 μm) mostly single or rarely in short chains. Also, two to eight needle-shaped ascospores (52 to 80 μm in length) were arranged lengthwise in many cylindrical to naviculate asci (60 to 96 x 8 to 12, avg. 72.4 x 9.2 µm). Ascospores were with a unilateral, slender, flexuous, whip-like appendage. The morphology of the different fungal structures indicated that the pathogen was Eremothecium coryli (Pelgion) Kurtzman and it was further supported by molecular identification. Total DNA was extracted directly from fungal mycelium with a DNeasy Plant Mini Kit (Qiagen, Hilden, Germany) and PCR amplification performed with primers ITS1F (Gardes and Bruns 1993) and ITS4 (White et al. 1990). Sequence analysis of ITS region revealed that the Serbian isolate ND2/21 (GenBank Accession No. OL958602) shared the highest nucleotide identity of 100% with E. coryli isolate (Accession No. KY103387). For pathogenicity test, fresh soybean seeds (cv. Sava) were surface-disinfected with 2% NaOCl and rinsed in sterile water before inoculation. The seeds were pierced 3-4 times with a sterile insect pin through a drop of yeast suspension (concentration 106 ascospores/ml) of one selected single-spore isolate (ND2-21). Similarly, control seeds were pierced with sterile insect pins through a drop of sterile distilled water. Five inoculated seeds and control (five replicates per treatment) were arranged uniformly in a Petri dish (9 cm diameter) and incubated at 22 to 25°C in the dark and kept under >95% relative humidity during the first 48 h. Twenty days after inoculation, small brown necrotic lesions were visible on the soybean seeds. Re-isolation from symptomatic seeds on PDA dishes yielded yeast-like colonies with the same morphological characteristics as those used for inoculation, thus confirming Koch's postulates. The control seeds had no symptoms. This fungus is widely known as a pathogen of yeast spot disease on soybean seeds (Heinrichs et al. 1976; Kimura et al. 2008), but to our knowledge, it has never been reported in Serbia. Considering that invasive species Nezara viridula L. and Halyomorpha halys (STÅL, 1855), the vectors of this fungus, were reported in our country (Kereši et al. 2012; Šeat 2015) and that their mass appearance has been documented in recent years (Konjević et al. 2020), the presence of this pathogen has the potential to cause considerable damage and severe yield losses, resulting in significant economic impact on soybean production in Serbia.

Cultural and morphological characteristics of Colletotrichum sublineolum isolates infecting sorghum in eastern Ethiopia

Colletotrichum sublineolum is the most destructive pathogen causing sorghum anthracnose worldwide. The fungus is known to have highly variable pathotypes. A characteristic study of pathogen is important to document the change occurring in population as variability in morphology indicates the existence of different pathotypes. Controlled condition experiment was conducted to examine cultural and morphological characteristics of C. sublineolum isolates infecting sorghum in eastern Ethiopia. Sorghum leaves showing symptoms of anthracnose were collected from five districts through survey. To study the characteristics of C. sublineolum, single-spore isolates representing isolate collection districts were selected from the stock cultures and cultivated on potato dextrose agar. Culture growth, colony color, elevations, texture and margin, conidial diameter and shape were used to characterize isolates. The isolates were varied significantly in many aspects. Colony colors were differed from light-gray to gray, purple-gray to cottony-gray, white to salmon-whit, plum-pink to beige and rosy brown on upper side of the petri dishes. The mean culture growth of C. sublineolum isolates showed highly significant (P < 0.01) variations among each other and ranged from 15 to 44 mm eight-days after incubation. Most of the isolates were produced hyaline, smooth walled, falcate conidia but without septa. Conidial diameter of C. sublineolum isolates showed variations with width and length ranged from 2.97 to 6.01 μm and 10.01–27.75 μm, respectively. Most isolates had smooth colony margin and few had undulated margin. This finding revealed that substantial variations were observed among C. sublineolum isolates and the existence of variable characteristic showed the presence of several sub-species of the pathogen infecting sorghum in different agro-ecologies of eastern Ethiopia.

A simplified spore-drop technique for rapid isolation of rice blast pathogen Magnaporthe oryzae from the infected rice leaf

Single spore isolation from a diseased sample is an essential step in obtaining a pure culture of a fungal pathogen. Rice blast disease caused by Magnaporthe oryzae is an inferior saprophytic competitor, and therefore, many fast-growing fungal or bacterial contaminants are predominant during its isolation. For isolation of M. oryzae, several methods are being followed; however, they are complex and often lead to contamination. In the present study, we have standardized an efficient method for rapid isolation of M. oryzae from the blast disease infected rice-leaf using single spore isolation by spore-drop technique. Following the spore-drop technique, pure culture for an isolate of M. oryzae was obtained quickly with the least contamination (4%), whereas the conventional spore-dilution and leaf-press method recorded 26.12 and 45.50% contamination, respectively. The spore-drop approach has yielded the single spore isolates in the shortest time (10 days) and can be used for regular rice blast pathogen isolation. This method can also be used for other sporulating pathogens successfully.

First report of Fusarium equiseti causing leaf spots of Bitter gourd (Momordica charantia) in Pakistan.

Bitter gourd (Momordica charantia L.) is an important vegetable crop of the Cucurbitaceae family widely cultivated in Pakistan and around the world. In October 2020, a nutrition management trial of Bitter gourd cv. Seminis-200) was conducted on an area of 10,860 sq. ft. (99×110 feet) at the Agricultural Research farm of Bahauddin Zakariya University, Multan (30.2601° N, 71.5158° E), Pakistan. Symptoms of large, brown necrotic leaf spots were observed on the leaves of bitter gourd vines. The disease started from the yellowing of leaves within the reticulate venation and turned brown. Irregular brown leaf spots coalesced to form large necrotic areas followed by foliar chlorosis then wilting that occurred very late. There were no crown rot symptoms although there was slight discoloration of roots and when cut longitudinally, browning of tissues was observed. The disease was assessed visually with 37% incidence which resulted in poor quality and yield in terms of reduced size and yellowing of fruit. Infected vines along with the roots were collected for the isolation of pathogen. A total of 34 leaves and 22 root samples were collected from the field for isolation. The leaf, collar and root portions were cut into 0.5 to 1 cm in length and surface disinfected with 1% sodium hypochlorite (NaOCl) for 2-3 minutes followed by washing twice with autoclaved distilled water and after drying, placed on potato dextrose agar (PDA) medium, and incubated at 25±2 °C for one week. The fungal colonies of fluffy white growth with light orange pigment were isolated. For morphological characterization, a total of 4 pure cultures were isolated from leaves, collar region and root by single spore technique on carnation leaf agar (CLA) medium after 15 days of incubation at 25±2℃. Curved and thick-walled macroconidia with elongated or pointed apical characteristic foot-shaped basal cells were produced in sporodochia. Macroconidia with 5-7 septa measured 22.50-41.80 μm × 2.90-4.20 μm (n = 60). Thick, brown with roughened walls and subglobose ellipsoidal chlamydospores were observed in clumps or chains with the dimension of 5.8 to 10.8 μm (n = 20). On morphological characteristics, the fungus was identified as Fusarium equiseti (Corda) Sacc. according to Leslie and Summerell (2006). Two single spore isolates were used for molecular identification by amplifying ribosomal DNA of the internal transcribed spacer (ITS) region with ITS1/ITS4 primers (White et al. 1990) and for β-tubulin gene region, primers T1/Bt-2b (O'Donnell and Cigelnik, 1997) were used. The obtained sequences were deposited in GenBank with accession numbers MW880179 and MW880198 from the ITS region and BLAST search in GenBank showed 100 and 98.11% alignment with previously published sequences of F. equiseti with accessions OM992323.1and MT558569.1 respectively. Accession number OM867571from the β-tubulin region showed 100% sequence similarity with F. equiseti with accession MN653163.1. For pathogenicity, macroconidia from 2-week-old cultures on CLA medium were harvested to prepare spore suspension (1 × 106 conidia/ml). Koch's postulates were confirmed on nine bitter gourd plants (cv. Seminis-200) by applying spore suspension of fungal inoculum at 3-4 leaf stage separately on leaves by automizer, on collar region after making incision spore suspension was applied and in the root zone, 20ml spore suspension was added whereas distilled water was used as a control with three replications. Plants were kept under controlled conditions in the greenhouse with 65% to 75% humidity and the temperature was maintained at 32±2 °C for one week. After 7-8 days, inoculated plants began to exhibit symptoms of brown, necrotic leaf spots on the leaves of bitter gourd vines followed by yellowing of leaves that eventually turned brown. Roots showed slight discoloration and browning of vascular bundles and finally, the plants wilted after four weeks. while control plants remained symptomless. The symptoms resembled those noticed in the field. The fungus was re-isolated from leaves, collar region and roots, followed by morphological identification, and finally confirmed as F. equiseti. To the best of our knowledge, this is the first report of a leaf spot caused by F. equiseti in a bitter gourd from Pakistan. If the disease is not managed properly, it may cause a drastic effect on yield under favorable environmental conditions. The pathogen may also damage other cucurbitaceous crops cultivated in the area.

First report of Alfalfa root rot caused by Fusarium commune in China.

Alfalfa (Medicago sativa) is the most important perennial forage legume worldwide, with high yields and nutrient quality. Diseases are significant threats to alfalfa and cause substantial yield losses (Fang et al. 2021). In March 2022, a disease with symptoms similar to fusarium wilt was observed in an alfalfa (cultivar Aurora) field in Baima teaching and research base (119°18'07″E, 31°61'47″N) in Nanjing, China. Reddish-brown discoloration of the roots, stele, basal stems and the withering symptoms on the aerial portions are the specific symptoms of fusarium wilt. The disease incidence varied from 3 to 6% in around 0.3 hectares of alfalfa fields surveyed. Fresh tissues from symptomatic alfalfa were cut into pieces (3-5 mm) and surface sterilized with 75% ethanol for 30 seconds, followed by 1% sodium hypochlorite (NaClO) for 120 seconds, and three rinses with sterile distilled water. Tissue pieces were placed on selective potato dextrose agar (PDA) containing 50 mg/L rifampicin and ampicillin. Plates were sealed and incubated at 25°C for 48 to 72 hours. Over 70 tissue fragments plated, 20 isolates from different fragments showed similar colonies. These isolates were purified by single spore isolation and grown on PDA and mung bean medium for morphology identification, molecular identification and pathogenicity test. The colony showed white to pink, abundant, densely aerial mycelium, while its backside was light violet. Macroconidia were hyaline, falcate with septa, and ranged in size from 25.5 to 61.5 × 3.8 to 6.2 μm (n=50). Microconidia were non-septate, hyaline, oval, straight to slightly curved, and were 5.6 to 10.7 × 2.4 to 3.2 μm (n=50). Spherical chlamydospores were 10.6 ± 1.3 μm in size. The rDNA internal transcribed spacers (ITS), RNA polymerase II subunit 2 (RPB2) and translation elongation factor 1-α (TEF) genes were amplified and sequenced (White et al. 1990; Carbone et al. 1999; Wang et al. 2019). Blast analysis showed 99.41% to 100% identity to F. commune sequences in the GenBank database (KU341324.1, MN892350.1, MH341219.1). The pathogenicity test was conducted using the dipping method in roots. Fresh F. commune hyphae were cut into 3 x 3mm agar plugs from a 7 cm PDA plate and inoculated in 200 mL mung bean medium on a shaker at 160 rpm, 25°C for 5 days. Spores were filtered through a cheese cloth, adjusted to 1 × 106 spores/mL with distilled water and used immediately. Roots in two-week-old alfalfa seedlings were soaked in spore suspension for 30 mins before being transplanted to sterile vermiculate with four repeats, while those in sterile water were used as non-inoculated control. The plants were placed in the green house at 25°C with 16 h of light and 8 h of darkness. The symptoms were observed two weeks following the inoculation. 30% of the seedlings withered, and reddish-brown discoloration symptoms were visible on the roots and stem of the unwithered plant. F. commune was isolated from these lesions again, and no disease symptoms were observed in the control plant. F. commune has been reported to cause root and stem rot in many plants worldwide, including tobacco (Zhong et al. 2021), maize (Mezzalama et al. 2021; Xi et al. 2019), soybean (Detranaltes et al. 2022) and sugarcane (Wang et al. 2018). Our results show that F. commune was the causative agent of alfalfa root rot. To our knowledge, this is the first report of F. commune causing root rot in alfalfa. The finding provides insights for disease diagnosis of alfalfa root rot disease and management in the region.

A Novel Gammapartitivirus That Causes Changes in Fungal Development and Multi-Stress Tolerance to Important Medicinal Fungus Cordyceps chanhua.

Cicada flower, scientifically named Cordyceps chanhua, is an important and well-known Chinese cordycipitoid medicinal mushroom. Although most mycoviruses seem to induce latent infections, some mycoviruses cause host effects. However, the effects of mycovirus on the fungal development and stress tolerance of C. chanhua remain unknown. In this study, we report a novel mycovirus designated Cordyceps chanhua partitivirus 1 (CchPV1) from C. chanhua isolate RCEF5997. The CchPV1 genome comprises dsRNA 1 and dsRNA 2, 1784 and 1563 bp in length, respectively. Phylogenetic analysis using the aa sequences of RdRp revealed that CchPV1 grouped with members of the genus Gammapartitivirus in the family Partitiviridae. We further co-cultivated on PDA donor strain RCEF5997 and recipient C. chanhua strain RCEF5833 (Vf) for 7 days, and we successfully obtained an isogenic line of strain RCEF5833 with CchPV1 (Vi) through single-spore isolation, along with ISSR marker and dsRNA extraction. The biological comparison revealed that CchPV1 infection slows the growth rate of the host, but increases the conidiation and formation of fruiting bodies of the host. Furthermore, the assessment of fungal tolerance demonstrated that CchPV1 weakens the multi-stress tolerance of the host. Thus, CchPV1 infection cause changes in fungal development and multi-stress tolerance of the host C. chanhua. The findings of this study elucidate the effects of gammapartitivirus on host entomogenous fungi and provide a novel strategy for producing high-quality fruiting bodies of C. chanhua.

Genetic diversity of Venturia carpophila populations from different hosts and geographic regions in China.

Venturia carpophila, the causal agent of scab disease of peach, mume, and apricot, is widely distributed around the world. Scab of stone fruits is an important disease in China. However, little is known about the population biology and genetic diversity of the V. carpophila. To better understand the genetic diversity and population structure of V. carpophila, 186 single-spore isolates from different hosts and geographic regions were obtained and analyzed by using 31 simple sequence repeat (SSR) markers. This included 156 isolates from peach spanning 14 provinces, 15 isolates from mume and 15 isolates from apricot in Huazhong Agricultural University (HZAU). Diversity analysis with SSR markers showed a low incidence of polymorphisms within mume isolates (32.59% of markers), but a higher incidence of polymorphisms within peach isolates (42.96%) and apricot isolates (57.04%). Within peach isolates, Nei's average gene diversity ranged from 0.07 for Hebei population to 0.18 for Hubei population. AMOVA analysis revealed that 13% of the observed genetic diversity was partitioned among the geographic populations, while 40% of the observed genetic diversity was partitioned among the host populations. Other analyses (PCoA, STRUCTURE, DAPC, MSN, and UPGMA) indicated that the Chinese V. carpophila populations could be clustered into three distinct genetic groups, which correspond to the host boundaries of peach, mume and apricot. The genetic identity of V. carpophila isolates throughout the range is dependent on hosts, but not geographic regions.