First Report of Root Rot Caused by Plectosphaerella cucumerina on Indigofera pseudotinctoria in China

Alfalfa (Medicago sativa L.) is an important forage crop with high nutrition for animal feed. In May 2016, a disease showing brown root rot was observed on alfalfa collected from several farms in Tongliao City (44°17' N; 121°29' E), Inner Mongolia Autonomous Region of China. The incidence of brown root rot was approximately 50 to 70% in the 2-year-old alfalfa field. Infected alfalfa exhibited varying degrees of decay in the tap root. Symptomatic roots were cut into 0.5-cm pieces, surface disinfected with 70% ethanol for 5 s and 0.1% HgCl2 for 35 s, then rinsed with sterilize distilled water three times, and placed onto potato dextrose agar (PDA) at 26°C in the dark. After 5 days, hyphal tips of the growing colonies were transferred onto PDA plates for purification. Forty-four isolates belonging to five fungal species were obtained from 20 diseased root samples. Six of the isolates resembled the genus Plectosphaerella. Colonies of these isolates were white to cream in color with sparse aerial mycelium, and then gradually became salmon pink with slimy or moist mycelium. The hyphae were transparent and branched. Colonies produced numerous hyphal coils with conidiophores. Conidiogenous cells and conidia were both hyaline, solitary, and smooth. Conidia were 4 to 8.5 ×1.2 to 4.8 µm (n= 100), 0 to 1 septum, elliptical and ovoid, and aggregating to form a head (Palm et al. 1995). According to these morphological characteristics, the fungus was identified as P. cucumerina (Lindf.) (Carlucci et al. 2012). To confirm the identification, the genomic DNA of two representative isolates was extracted and their internal transcribed spacer (ITS) region was amplified and sequenced with the primer pair ITS1/ITS4 (White et al. 1990). The ITS sequences of the two isolates were deposited in GenBank (acc. nos. MN915126 and MN915127). The two ITS sequences showed 99 to 100% identical to known P. cucumerina strains CBS 131739 (acc. no. KY662258.1) (Su et al. 2017) and MP313 (acc. no. KC756835.1) from alfalfa in China (Wen et al. 2015). To test for pathogenicity, a set of 15 alfalfa seedlings (cv. Aohan) were root-dipped in the conidial suspension of one of the isolates (1×105 conidia /ml) prepared from 7-day-old cultures on PDA. Inoculated seedlings were transplanted in three pots (10×15 cm) with sterilized nursery soil. Another set of five alfalfa seedlings inoculated with sterile water only served as the controls. Treated alfalfa seedlings were maintained in a greenhouse at 25°C to 28°C under a 12-h photoperiod. After 25 days, the roots of all inoculated plants showed brown lesions. P. cucumerina was reisolated from symptomatic tissue. No symptoms were observed on the control plants. P. cucumerina was previously reported on alfalfa in the fields of Huanxi Country (36°20' N; 107°21'), Gansu Province, China (Wen et al. 2015). To our knowledge, this is the first report of P. cucumerina causing root rot of alfalfa in Inner Mongolia Autonomous Region, China. This disease may cause serious economic losses in the region. It is needed to develop effective management strategies for control of this disease.

HomePlant DiseaseVol. 102, No. 9First Report of Common Bean (Phaseolus vulgaris) Root Rot Caused by Plectosphaerella cucumerina in China PreviousNext DISEASE NOTES OPENOpen Access licenseFirst Report of Common Bean (Phaseolus vulgaris) Root Rot Caused by Plectosphaerella cucumerina in ChinaL. Yang, X. H. Lu, S. D. Li, and B. M. WuL. Yanghttp://orcid.org/0000-0002-7673-679XSearch for more papers by this author, X. H. LuSearch for more papers by this author, S. D. Li†Corresponding authors: S. D. Li, E-mail: E-mail Address: [email protected]; and B. M. Wu, E-mail: E-mail Address: [email protected]Search for more papers by this author, and B. M. Wu†Corresponding authors: S. D. Li, E-mail: E-mail Address: [email protected]; and B. M. Wu, E-mail: E-mail Address: [email protected]Search for more papers by this authorAffiliationsAuthors and Affiliations L. Yang , Department of Plant Pathology, China Agricultural University, Beijing 100193, China X. H. Lu S. D. Li † , Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193 B. M. Wu † , Department of Plant Pathology, China Agricultural University, Beijing 100193. Published Online:21 Jun 2018https://doi.org/10.1094/PDIS-10-17-1659-PDNAboutSectionsSupplemental ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat In March 2016, root rot was observed on common bean (Phaseolus vulgaris L.) samples (cultivar Shuangqing 38) collected from a commercial farm in Sanya City, Hainan Province, China. The incidence of this disease in the field was about 30%. The symptoms of infected plants included dwarf and yellowing aboveground; irregular, scaled, black-brown lesions on roots; and lack of lateral roots. Symptomatic root tissues were excised from four plants, cut into 1-cm pieces, surface sterilized with 1% NaClO for 2 min, rinsed in sterilized distilled water three times, and then placed on potato dextrose agar (PDA) plates. After incubation at 25°C for 7 days, hyphal tips from the colony edge were transferred to a fresh PDA plate, and a total of 13 isolates were obtained. Morphological and molecular identification indicated that 11 of these isolates from four plants were fungal species previously reported on common bean, and the other two from two individual plants were Plectosphaerella cucumerina. The colonies of these two isolates were pink and moist with appressed and slimy mycelium. Hyphae were transparent and branched. Numerous hyphae anastomosed frequently and formed hyphal coils. Conidiogenous cells were monophialides. Conidia were hyaline, measured 4.0 to 9.8 × 2.0 to 4.3 μm, 0 to 1 septate, ellipsoid or long ellipsoid dwindling gradually from the middle toward the apex, gathering together to form a capitulum (Palm et al. 1995). The internal transcribed spacer (ITS) regions of ribosomal DNA were amplified from the two isolates with the primer pair ITS1/ITS4 (White et al. 1990) and then sequenced. The sequences of the two isolates were identical (accession no. MF669723) and showed 99% identity to sequence KP407871 of P. cucumerina (strain KX37). To test the pathogenicity of these isolates, seeds of common bean (cultivar Baifeng) were sown in 4 × 3-cm plastic pots with sterilized nursery soil and were inoculated with a wheat kernel colonized with the fungi placed adjacent to each seed. Seeds sown with sterilized, noncolonized wheat kernels were used as controls. Three replicates with a total of 15 plants for each isolate were included. The trays were then maintained in a greenhouse with a 12-h photoperiod at 25°C. No symptoms developed on the aboveground parts in 10 days, but the roots of all plants inoculated showed black lesions similar to those originally observed in the field. Using the same methods described above, P. cucumerina was reisolated from these lesions. Control plants remained healthy, and no P. cucumerina was reisolated. In 2017, P. cucumerina was also isolated from common bean roots (cultivar English Red) with similar symptoms in two fields in Kelan County, Shanxi Province, China. To our knowledge, this is the first report of P. cucumerina causing root rot of common bean in China, although it has been reported on other crops (Gao et al. 2016; Li et al. 2017; Yan et al. 2016). It is important to study the impacts of this new disease on common bean production in China for the potential infestation of the pathogen.

Aloe genus plants are perennial evergreen herb belonging to Liliaceae family which is widely used in food, medicine, beauty, and health care (Kumar et al. 2019). In August 2021, symptoms of root and stem rot was observed in approximately 20% of Aloe vera plantings in Yuanjiang County, Yunnan Province, China (23° 64' 53" N, 101° 99' 84" E). The most typical symptoms were stem and root rot, browning and necrosis of vascular tissues, gradual greening, and reddish-browning of leaves from bottom to top, abscission, and eventual plant death (Fig. S1). Therefore, to isolate and identify the pathogen, the plants showing the above symptoms were collected. The plant tissues were cut from the edges of root and stem lesions, followed by disinfection with 75% ethanol for 1 min, rinsed three times with sterilized distilled water, and cut into 3 × 3 mm small squares after excision of marginal tissues. The tissues were transferred to the oomycetes selective medium (Liu et al. 2022) and incubated at 28 °C in the dark for 3~5 days, and suspected colonies were purified. The colonies were then inoculated onto potato dextrose agar (PDA), V8-juice agar (V8), and oatmeal agar (OA) medium plates for morphological characteristics. Finally, 18 isolates with the same colonial and morphological characteristics were obtained from 30 lesioned tissue and one of them was named as ARP1. On PDA, V8 and OA medium plates, the ARP1 colonies were white. On PDA plate, the mycelia were dense and the colonies were petal-like; on V8 plate, the mycelia were cashmere and the colonies were radial or star-like. Whereas, on OA plate, the mycelia were cotton-like and the colonies were fluffy and radial (Fig. S2 A~C). Mycelium did not have septum with high branching and swelling. Sporangia were abundant, semi-papillate, varying in shape from ovoid-ellipsoid to long-ellipsoid, 18-26 × 45-63 μm (average: 22 × 54 μm, n = 30), sporangia released numerous zoospores from the papillate after maturation. The chlamydospores were spherical, 20-35 μm in diameter (average: 27.5 μm, n = 30) (Fig. S2 D~F). These morphological features were like those of the pathogenic species of the oomycetes (Chen et al. 2022). For the molecular characterization, the genomic DNA of the isolate was extracted using the cetyl trimethyl ammonium bromide method, and the translation elongation factor 1α (tef-1α) (Stielow et al. 2015), β-tubulin (β-tub) (Kroon et al. 2004) and internal transcribed spacer (ITS) (White et al. 1990) of isolated strain ARP1 were amplified using primer pairs EF1-1018F/EF1-1620R, TUBUF2/TUBUR1 and ITS1/ITS4, respectively. The tef-1α, β-tub genes and ITS region of ARP1 were directly sequenced and their sequence information was deposited in GenBank under accession numbers OQ506129, OQ506127 and OQ449628. ARP1 was clustered on the same evolutionary branch with Phytophthora palmivora (Fig. S3). To confirm the pathogenicity of ARP1, the main root of A. vera was wounded to 1 cm long and 2 mm deep with a scalpel blade followed by inoculation with 50 ml suspension of ARP1 zoospores at a concentration of 1 × 106 spores / ml per potted plant, and an equal volume of water as control. All inoculated plants were placed in the greenhouse at 28°C, 12 h / 12 h light / dark. After 15 dpi, the inoculated plants showed typical symptoms of wilted and drooping leaves and stem and root rot, same as observed in the field condition (Fig. S4). After inoculation with ARP1, a strain with the same morphological and molecular characteristics as the original isolate was re-isolated, confirming Koch's postulates. To our knowledge, this is the first report of P. palmivora causing root and stem rot of A. vera in the study region. This disease could be a potential risk for aloe production and therefore appropriate management measures should be taken.

Fusarium species are associated with stalk rot disease of maize, which can result in significant yield losses. The typical symptom of the disease is rotting of the plant stem at lower internodes. The color of the plant changes to grayish-green, which later turns tan, and the plant wilts. When the stem is split, the inside of the stalk shows a light pink to tan discoloration and the pith is disintegrated, but the vascular bundles remain intact. During the 2016 crop season, symptomatic maize tissues were collected from four different locations in Dandong city, Liaoning province, China. The disease was found to occur on approximately 5% of the surveyed area. Plant tissues exhibiting typical stalk rot symptoms were cut into small pieces (approximately 5 mm²), placed onto potato dextrose agar (PDA) amended with streptomycin sulfate (150 μg/ml) and kanamycin (150 μg/ml), and incubated at 25°C in darkness for 4 days, as described by Shan et al. (2017). Fungal colonies showing morphological characteristics of Fusarium spp. were subcultured from single conidia onto PDA and carnation leaf agar (CLA) plates for identification and DNA sequence analyses. Microscopic and DNA analyses confirmed the presence of many Fusarium species known to cause ear and stalk rot diseases in maize, such as F. verticillioides, F. graminearum, and F. proliferatum. However, F. commune, a relatively new species described by Skovgaard et al. (2003), was also identified in 12 diseased samples collected from various locations in Dandong city. On PDA plates, colonies of F. commune were white to orange-white in color with densely flocculate to fluffy aerial mycelium and dark violet pigmentation. On CLA plates, macroconidia were slightly curved, hyaline, usually contained three or five septa, with an obvious foot-shaped basal cell and tapering gradually to a point at the tip. Three-septate macroconidia were 39.4 ± 6.2 × 3.7 ± 0.6 μm, whereas five-septate macroconidia were 48.6 ± 4.5 × 4.3 ± 0.3 μm. Microconidia were generally cylindrical, straight to slightly curved, aseptate, and measuring 11.5 ± 3.1 × 3.1 ± 0.7 μm in size. Spherical chlamydospores were 8.6 ± 1.4 × 8.4 ± 1.4 µm in size and were produced singly or in pairs from mycelium. Species identification was also confirmed by partial sequences of the translation elongation factor gene (TEF1-α, EF1 and EF2 primers), the mitochondrial small subunit gene (mtSSU, NMS1 and NMS2 primers), and the second largest subunit of the RNA polymerase gene (RPB2, 5f2 and 7cr primers) (O’Donnell et al. 2010). Partial sequences of the TEF1-α, mtSSU, and RBP2 showed 100, 100, and 99% sequence identity to those of F. commune (GenBank accession nos. KX500397, KY439901, and KU171700, respectively). DNA sequences of partial TEF1-α, mtSSU, and RBP2 from two different isolates were deposited in GenBank (accession nos. MH716808 to MH716813). Pathogenicity tests of two representative isolates were carried out by individually inoculating five maize plants at the five-leaf stage. Maize stalks, at second or third internode, were injected with 20 μl of spore suspension having concentration of 1 × 10⁶ spores/ml as described by Zhang et al. (2016). Five plants inoculated with sterilized water were used as a control. The inoculated plants were maintained at 25 ± 0.5°C in a greenhouse with a 12-h photoperiod. After 10 days, all inoculated plants developed internal dark brown necrotic regions around the inoculated site. The control plants were symptomless. F. commune was reisolated from the symptomatic plants but not from the control. In China, F. commune was also reported to cause root rot diseases in different plant species, for example, sugarcane (Wang et al. 2018) and Gentiana scabra (Guan et al. 2016). To our knowledge, this is the first report of F. commune as a pathogen on maize in China.

Triticale (×Triticosecale Wittmack) is obtained from wheat × rye crossing. It is positioned between wheat and rye in terms of resistance to soilborne pathogens including Gaeumannomyces graminis var. tritici, Fusarium culmorum, F. avenaceum, and Bipolaris sorokiniana (Arseniuk and Góral 2015). In 2019, seven triticale fields were surveyed in Almaty Province, Kazakhstan to examine soil-borne fungal pathogens. A total of 28 symptomatic plants with stunting, rot or discolored root were collected to identify causal agents. The overall disease incidence was approximately 8 to 10% in the fields. Fungi were isolated from 3-5 mm pieces excised from symptomatic tissues. The pieces were exposed to surface disinfection in 1% sodium hypochlorite solution for 2 min, rinsed three times with sterile distilled water, blotted dry, and plated on 1/5 strength potato dextrose agar (PDA) amended with 0.01% streptomycin. Plates were left in the dark at 23°C for 7 days. A total of 34 fungal colonies were isolated of which nineteen isolates, originally from six fields showed the cultural characteristics of B. sorokiniana. This species was previously reported to cause common root rot on triticale in Kazakhstan (Özer et al. 2020). Ten isolates from four fields produced pale orange and cottony mycelium with red pigmentation on the agar, which is typical of Fusarium-like growth. The remaining isolates (n=5) from two fields produced salmon-colored and scarce aerial mycelium with no soluble pigmentation, similar to Microdochium spp. Fusarium isolates produced thick-walled and curved macroconidia with 3-4 septa (n=50, 25.7 to 37.6 × 4.1 to 7.3 μm in size) and notched basal cell on PDA, but microconidia were absent, which matches the description of F. culmorum (Wm.G. Sm.) Sacc. (Leslie and Summerell 2006). Microdochium isolates produced swollen, brown, and thick-walled chlamydospores and hyaline, one-celled, and thin-walled conidia (n=50, 5.4 to 9.3 × 1.5 to 3.0 μm in size) formed on ampullate and cylindrical conidiogenous cells on oatmeal agar (OA). These morphological features are consistent with previous observations for Microdochium bolleyi (R. Sprague) de Hoog & Herm.-Nijh. (Hong et al. 2008). To confirm morphological preliminary identifications, the portion of the translation elongation factor 1-alpha (EF1-α) gene was amplified with EF1/EF2 primers (O'Donnell et al. 1998) for representative Fusarium isolates (n=4) for each field. Additionally, the internal transcribed spacer (ITS) of ribosomal DNA was amplified with ITS1/ITS4 primers (White et al. 1990) for representative Microdochium isolates (n=2) for each field. BLASTn queries against NCBI GenBank revealed that the EF1-α sequences of Fusarium isolates (MW311081-MW311084) shared 100% identity with F. culmorum strain CBS 110262 (KT008433). The ITS sequences of M. bolleyi isolates (MW301448-MW301449) matched that of M. bolleyi strain CBS 137.64 (AM502264) with 100% sequence similarity. Pathogenicity test was conducted on pregerminated seeds of triticale cv. Balausa. A plastic pot (17 cm height, 9 cm in diam) was filled with a sterile mixture of vermiculite, peat, and soil (1:1:1, v/v/v). Mycelial plugs (1 cm in diam) were cut from the margin of a growing culture of representative isolates (Kaz_Fus123 and Kaz_Mb01) and placed onto the mixture in the pot. A sterile agar plug was employed as a control treatment. One pregerminated seed was put on the plug and covered with the mixture. The pots were transferred to a growth chamber set at 23 ± 2°C and a photoperiod of 14 hours. The experiment was performed twice using 5 replication pots per isolate. Four weeks after inoculation, discoloration of the crown was observed on all the inoculated roots, whereas no symptoms were observed on the control plants. Koch's postulates were fulfilled by reisolating and identifying the pathogen based on the morphology described above. This is the first report of M. bolleyi and F. culmorum causing root rot on triticale in Kazakhstan. Although B. sorokiniana is the most primary pathogen that may limit yield in the production of triticale in Kazakhstan, F. culmorum and M. bolleyi have been found to be less frequent and less aggressive pathogens, respectively. Further studies are needed to better understand the potential distribution and impact of these pathogens on triticale.

Mustard (Brassica juncea) plants grown on the premises of the Indian Agricultural Research Institute, New Delhi, showing wilting of leaves and twigs and necrosis of stem, crown and roots were observed in four consecutive years (2013 to 2016), with an incidence of up to 20%. To determine the causal agent of the disease, small pieces of stems and roots were surface-sterilized with 3% sodium hypochlorite washed in sterile distilled water, placed on potato dextrose agar (PDA) plates and incubated at 25°C for seven days. A dense white mycelium developed, that turned first beige, finally buff brown. Single-spore cultures produced falcate macroconidia with a pedicillate foot cell, and 4-7 distinct septa 21-58 x 3-6 µm in size, developing from lateral phialides. Chlamydospores were intercalary, in chains or solitary, brown-pigmented, 6-9 µm in diameter. The fungus was morphologically identified as Fusarium equiseti (Corda) Sacc. (Booth, 1977; Leslie and Summerell, 2004). To confirm the identity at the molecular level, the internal transcribed spacer (ITS) region and the translation elongation factor (tef-1) were amplified using the respective primers (White et al., 1990; Leslie and Summerell, 2004). The sequences (GenBank accession Nos. KT380024 for ITS and KY271154 for tef-1) matched those of F. equiseti in BLAST analysis. For pathogenicity tests mustard seedlings pot-grown in a glasshouse at 23±2°C with 75% relative humidity and 16 h light cycle/day were injected with 5 ml of a conidial suspension (104 conidia ml-1 ) at the root and collar region. Seedlings treated with sterile water served as control. The same symptoms seen in the field were shown within 30 days by inoculated plants only, from which F. equiseti was re-isolated. To the best of our knowledge, this is the first report of F. equiseti causing stem and root rot disease on mustard.

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

Polygonatum cyrtonema Hua is a traditional Chinese medicine, which has anti-oxidant, anti-inflammatory, immunomodulatory, and other pharmacological effects (Lu et al. 2023). In June 2022, A disease of root rot was observed on P. cyrtonema plants in Tonggu County (28°63'89″N, 114°48'07″E), Jiangxi Province, China. The disease incidence was approximately 30% in a surveyed area of 3 hectares, which contained approximately 20,000 plants. Initially, the above-ground parts of the plants did not show any obvious symptoms. However, the underground roots exhibited red-brown spots that gradually expanded and sunken areas appeared, and the diseased plants presented leaf chlorosis and red-brown discoloration of the tubers, eventually leading to plant death. To identify the pathogen, symptomatic root tissues (0.5×0.5×0.5cm) from the lesion margin surface were sterilized with 75% ethanol for 30s, 3% NaClO for 3 min followed by rinsing three times with sterile water. The sterile root pieces were placed on potato dextrose agar (PDA) and incubated at 25℃. Thirteen pure fungal isolates with the same morphological characteristics were obtained by monosporic isolation from 20 pieces of roots, and the representative isolates, HJGF1-1, HJGF1-2 were used for morphological studies and phylogenetic analyses. Initially, the two colonies on PDA appeared white with cotton-shaped aerial hyphae, which later turned light green to green and formed concentric rings. At the end of the conidiophores, 3 to 6 pear-shaped branches are irregularly gathered, and the angles between the branches are acute. The conidia were mostly solitary ellipsoid or obovate with the size of (3.7-5.9) × (3.6-4.5) μm (n=100). These morphological characteristics are consistent with the description of a Trichoderma spp. (Kamala et al. 2015). For molecular identification, the internal transcribed spacer (ITS), translation elongation factor 1-alpha (TEF-1α) and RNA polymerase II second largest subunit (RPB2) sequences were PCR amplified using primer pairs of ITS1/ITS4 (White et al. 1990), EF1-728F/EF1-986R (Carbone et al. 1999), and RPB2-5F2/RPB2-7cR (O'Donnell et al. 2022), respectively. BLAST analysis showed that the ITS, TEF-1α and RPB2 sequences of isolates HJGF1-1 (GenBank accession nos. OR229621, OR290791, OR334600) and HJGF1-2 (GenBank accession nos. OR229622, OR290792, OR334600) showed 99%-100% identity with multiple GenBank sequences of Trichoderma virens. A phylogenetic tree based on concatenated sequences of ITS, TEF-1α and RPB2 using maximum-likelihood analyses revealed that the two isolates HJGF1-1 and HJGF1-2 were in the same clade with T. virens strains. The two isolates were identified as T. virens based on the morphological characteristics and molecular phylogeny. To test pathogenicity, ten healthy P. cyrtonema plants (one tuber each, 5 tubers per isolate, n=10) in the field were pin-pricked with a sterile needle and pour-inoculated with 5 mL spore suspension per tuber (1× 107 conidia/ mL) with a temperature of about 28℃. Another five tubers were were pin-pricked with a sterile needle, inoculated with sterile water and served as controls. The resulting symptoms were similar to those on the original infected plants in the field, and control tubers remained symptomless fourteen days after inoculation. T. virens was reisolated from the diseased tubers, nevertheless no pathogenic fungus was isolated from the control tubers. T. virens has been reported causing disease on tulip bulb (Lou et al. 2003) but has not previously been reported causing disease on P. cyrtonema. Although several species of Trichoderma are known to be beneficial microorganisms, the beneficial fungus may have had an evolutionary period of interaction with plants in which it behaved as a plant pathogen (Poveda et al. 2022). To our knowledge, this is the first report of T. virens infecting P. cyrtonema in China. This result may expanded the etiological study of T. virens and the control strategy of P. cyrtonema root rot.

As an important landscaping plant, Syringa oblata is widely planted in northern China with high ornamental, medicinal, and edible value (Men et al. 2023). In September 2023, a new leaf blight disease on S. oblata was observed in Tianjin (39.0916°N, 117.1019°E), China. The onset of foliar symptoms was marked by the appearance of yellow-brown spots that originated from the tip and margin, subsequently evolving into irregularly shaped brown lesions. Finally, the lesions are distributed throughout the leaf surface, causing the leaf to wilt and seriously affecting photosynthesis. To identify the pathogen responsible for leaf blight of S. oblata, symptomatic leaves were collected and cut into square leaf blocks with a size of 0.3 cm², which were sterilized by immersion in 75% ethanol for 60 s and 5% NaClO for 30 s, and rinsed three times with sterile distilled water. The sterilized leaf pieces were then placed on potato dextrose agar (PDA) and incubated at 25 °C for 3-5 days. The peripheral hyphae of the fungal colony which developed from the infected tissues were isolated onto PDA plates. The fungal cultures on PDA plates were used for morphological observation and identification of the fungus. Colonies of B. dothidea on PDA medium were initially off-white, batting-shaped and gradually grayish-black. Aerial hyphae were well developed and could reach the tip of the petri dish. To induce sporulation, the hyphae were picked into medium containing sterilized pine needles. Conidia were found on pine needles after 30 d of incubation at 25°C. Conidia were hyaline, unicellular, oblong to spindle-shaped, and 17.5-23.0 µm in size × 6.1-8.5 µm(n=50). Based on these characteristics, the isolates were preliminarily identified as B. dothidea (Phillips et al. 2005). To provide additional evidence for the classification of the isolate, genomic DNA was extracted from the isolates of B. dothidea and used for Polymerase Chain Reaction (PCR). The internal transcribed spacer (ITS), glyceraldehyde-3-phosphate dehydrogenase (GPDH), and actin (ACT) were amplified with the primer pairs ITS1/ITS4, GDP1/GDP2, and ACT-512F/ACT-783R, respectively (Jiang et al. 2022). A BLAST search of sequences showed the ITS, GPDH, and ACT sequences had >99% identity with homologous sequences from B. dothidea isolates Bb158-4(HQ392696.1), PPO-46523(MG761771.1), and CMW7779 (AY972117.1), respectively. Phylogenetic analysis determined that the isolate was in the same clade position as B. dothidea, which confirmed the above morphological identification. To assess pathogenicity, the fungal cakes (6 mm diameter) were obtained from the edge of a fresh colony (cultured on a PDA plate for 7 days) using a sterile perforator. Ten surface-sterilized leaves of healthy S. oblata with uniform growth condition were collected and inoculated with fungal cakes after wounding. Ten leaves were inoculated with sterile PDA medium blocks as control. The test was repeated three times. All leaves were kept at 25°C and sterile H2O was sprayed daily to keep leaves surface moist. After five days, all vaccination sites showed lesions similar to those of the S. oblata diseased leaves in the field, while the controls were asymptomatic. B. dothidea was reisolated from symptomatic tissues, thus fulfilling Koch's postulates. B. dothidea is a member of Botryosphaeriaceae. Currently, B. dothidea infection of Syringa spp. plants has not been reported. However, in China, it has been reported to cause stem rot on Forsythia suspense (He et al. 2022) and leaf dieback on sweet osmanthus (Ling et al. 2010), demonstrating that B. dothidea can infect Oleaceae species. This study found that S. oblata could be infected by B. dothidea. To the best of our knowledge, this is the first report of B. dothidea causing leaf blight on S. oblata in China. Identifying the pathogen of S. oblata leaf blight is essential for the prevention and management of disease associated with S. oblata.

Heilongjiang is the largest rice-producing province in China, with annual yield of 28.9 million tons cultivated on 3.8 million hectares (Liu et al. 2021). During field surveys from July to August (2021-2022), symptoms of wilting were observed on rice panicles across Baoqing county (46.32°N, 132.20°E), Shuangyashan city, Heilongjiang province, China. Disease incidence ranged from 10 to 35%, and yield losses were estimated to be 5 to 20% over 7 surveyed fields of 18.5 ha in total. Initially, infected panicles exhibited carmine to brownish spots at the flowering and early grain-filling stages, which gradually merged into large and irregular lesions and spread to the entire panicle surface. Eventually, panicles became wilting and decayed at the ripening stage. To identify the etiological agent, thirty-five symptomatic panicles were collected randomly from 35 plants at different positions in 7 fields. The fragments (approximately 3 mm2) were dissected from margins of individual lesions, surface-disinfested with 70% ethanol for 30 s followed by 2% sodium hypochlorite for 2 min, and rinsed three times in sterilized water. The pieces were then dried and placed onto half-strength potato dextrose agar (PDA) supplemented with 50 μg/mL of streptomycin sulfate. After incubation at 28°C for 4 days, nineteen cultures were obtained and purified using the single-spore isolation method. On PDA plates, the colonies produced fluffy and cottony aerial mycelia and were white to yellowish with deep-yellow to red-brown pigments. The microconidia were hyaline, elliptical or clavate, zero to one septum, measuring 6.3 to 19.2 × 2.6 to 5.1 μm in size (n = 50). On carnation leaf agar (CLA), the macroconidia were thick-walled, falcate to almost straight, three to five septa, apical cell hooked to tapering, basal cell foot-shaped, measuring 27.4 to 47.8 × 3.6 to 5.4 μm in size (n = 50). No chlamydospore was observed. The internal transcribed spacer (ITS) region of ribosomal RNA, translation elongation factor (TEF-1α) gene, and β-tubulin (β-TUB) gene were amplified and sequenced using primers ITS1/ITS4 (White et al. 1990), EF1/EF2 (O'Donnell 2000), and T1/T22 (O'Donnell and Cigelnik 1997) from three representative isolates (PJ58, PJ69 and PJ83), respectively. The obtained sequences were deposited in GenBank (accession nos. ON527509, OQ772202 and OQ777725 for ITS; ON573222, OQ784926 and OQ784927 for TEF-1α; ON573223, OQ784928 and OQ784929 for β-TUB, respectively). BLASTn analysis revealed 99.8 to 100% homology with the corresponding sequences of Fusarium kyushuense (MH892849 for ITS, AB674297 for TEF-1α, and GQ915442 for β-TUB, respectively) in GenBank. Maximum likelihood phylogeny based on the concatenated sequences of ITS, TEF-1α and β-TUB grouped three representative isolates in the F. kyushuense clade. Combined with the morphological and molecular characteristics, the fungus was identified to be F. kyushuense. Pathogenicity of the three isolates of F. kyushuense was evaluated on a susceptible rice cultivar Nanjing 46 at the booting stage. The upper part of a healthy panicle was inoculated by injecting 2 ml of a conidial suspension (1 × 106 spore/ml) obtained from a 7-day-old PDA culture of each isolate. The negative control was treated with sterile distilled water. The experiment was performed thrice with ten replicated plants for each treatment. All plants were placed in a humid chamber at 25°C with a 12-h photoperiod and 80% relative humidity. Twenty days after inoculation, it was found that the inoculated panicles showed typical reddish to brownish lesions, whereas control plants remained symptomless. Pathogens were reisolated from the artificially inoculated panicles and confirmed by morphological and molecular tests, fulfilling Koch's postulates. In recent years, this species has been associated with stalk rot and ear rot of maize (Cao et al. 2021; Wang et al. 2014) and wilt of tobacco (Wang et al. 2013). Also, it was mentioned as a producer of mycotoxins, especially trichothecenes and HT-2 toxin (Varga et al. 2016). To our knowledge, this is the first report of F. kyushuense causing panicle wilting on rice in China. The appropriate control strategies should be made to reduce the risk of disease due to food security concerns and potential threats to rice production.

In March 2021, unusual plant stuning, collar, and wet root rot of lettuce (Lactuca sativa L.) during the rosette stage was observed in two commercial fields in Serbia (44°58'N, 20°32'E; 44°45'N, 20°43'E). Disease incidence in the fields (≈ 0.9 ha each) was approximately 15 and 20%, respectively. Initial above-ground symptoms were yellowing and wilting of leaves, while below-ground symptoms were collar, wet root rot, and lesions becoming necrotic. Eventually, whole plants wilted, collapsed, and died. A total of 35 symptomatic plants were collected from the fields, and diseased tissues were cut into small pieces, surface sterilized, and plated on potato dextrose agar (PDA). Isolation resulted in 20 morphologically uniform monoconidial isolates. The isolates formed white to creamy colonies, gradually becoming salmon pink, slimy, or moist in appearance, with sparse aerial mycelia. Numerous hyphal coils with conidiophores and hyaline, smooth-surfaced, ellipsoid to ovoid, septate or aseptate conidia were formed (4.5 to 10.1×1.2 to 3.7 μm (n = 100)). To confirm the species identity, the internal transcribed spacer (ITS) region and the D1/D2 region of a selected representative isolate 13-3-c were amplified and sequenced by using primer pairs ITS1/ITS4 (White et al. 1990) and N1/N2 (O'Donnell and Gray 1995), respectively. The sequences were deposited in GenBank (ITS: OR880564 and D1/D2: OR880567). Sequence analysis revealed 100% nucleotide identity with P. cucumerina isolates from different countries deposited in the NCBI GenBank, including isolate MH860704 (Vu et al. 2019) (ITS region) and isolate KY662256 (Su et al. 2017) (D1/D2 region). Neighbor-joining analysis was conducted based on the combined ITS and D1/D2 regions, and the tree was constructed with the substitution models (1,000 bootstrap). The combined phylogeny confirmed that the sequences shared a common clade with P. cucumerina. Hence, morphological, microscopic, and molecular characterization confirmed the pathogen as P. cucumerina (Palm et al., 1995; Carlucci et al., 2012). In a pathogenicity assay, 10 isolates were tested. Five 30-day-old lettuce plants (cv. Majska Kraljica) per isolate were root-dipped in the conidial suspensions (1×105 conidia/ml). The 10 inoculated plants were transplanted into 1 L pots containing sterile substrate (Floragard, Germany). Plants treated with sterile distilled water were used as controls. Plants were maintained in a greenhouse at 25 to 28°C under a 12-hour photoperiod (Cai et al., 2021). Four weeks after inoculation, stunting, chlorosis, and wilting of plants were observed, while collars and roots exhibited typical decaying symptoms. No symptoms were observed on the control plants. The pathogen was reisolated from symptomatic tissue as previously described. Koch's postulates were completed by confirming the identity of reisolates based on morphological features. To our knowledge, this is the first report of P. cucumerina on lettuce or any other crop in Serbia. P. cucumerina is already known as a pathogen of lettuce and other hosts grown in many countries worldwide, as well as in some European countries (Belgium, England, Italy, the Netherlands, and Switzerland) (Zhang et al. 2019). This emerging pathogen may cause significant economic losses in lettuce production in Serbia and in the entire Balkan region. Our results may help to develop effective management strategies based on accurate and timely identification and regular pathogen monitoring.

Pear (Pyrus communis) is an important fruit crop in the Netherlands, with a total production of 400,000 tons in 2020, and 'Conference' is the main pear cultivar that comprises 80% of total pear production area. In the Netherlands, pears are kept in controlled atmosphere cold storage (-0.5°C) up to 11 months after harvest. Calyx-end rot incidences of 1% to 5% were observed on 'Conference' pears from different orchards in surveys from 2019-2021 in packing houses in the Netherlands. Infections showed 1 to 3 cm brown necrosis. Lesions were round, slightly sunken and next to or including part of the calyx. To isolate the causal agent, fruit were rinsed with sterile water, lesions were sprayed with 70% ethanol until droplet runoff, the skin was removed aseptically with a scalpel, and tissue under the lesion was isolated and placed onto Potato Dextrose Agar (PDA) (Oxoid, UK). The PDA plates were incubated at 20°C in the dark, and hyphal tip isolates were transferred to fresh PDA plates. Colonies on PDA were rosy-whitish to peach-colored. Colonies grown on oat meal agar (OA) under UV light were peach to red color, aerial mycelium sparse, and produced a pink to salmon colored conidial matrix. Conidia were irregular-ellipsoidal to allantoid, smooth, hyaline and usually with one or several gutulles. Conidia were sometimes one septate and measured 15.2±2.8 x 4.0±0.7 μm (n =14), but mostly aseptate and measured 7.9±1.7 x 3.2±0.6 μm (n =100). The fungus was morphologically identical to Didymella macrostoma (syn. Phoma macrostoma) (Boerema et al. 2004; Hou et al. 2020). The identity of four representative isolates, WURR-206, WURR-223, WURR-227 and WURR-308, from affected pears from four orchards in the Netherlands, was determined by multilocus gene sequencing. To this end, genomic DNA was extracted using the LGC Mag Plant Kit (Berlin, Germany) in combination with the Kingfisher method (Waltham, MA). Sequences of the internal transcribed spacer (ITS) region of ribosomal DNA, the large-subunit rRNA (LSU) region, partial sequences of beta-tubulin (TUB) and the translation elongation factor 1-alpha (TEF1) gene region were amplified with primers ITS1/ITS4 (White et al. 1990), LROR/LR5 (Vilgalys and Hester 1990), Btub2Fd/Btub4Rd (Woudenberg et al. 2009) and EF1-983F/EF1-1567R (Rehner and Buckley 2005), respectively. Sequences were deposited under GenBank accession numbers ON077588-ON077591 (ITS), ON113487-ON113490 (LSU), ON098515-ON098518 (TUB) and ON098519-ON098522 (TEF1). MegaBLAST analysis revealed that the ITS, LSU, TUB sequences matched with 100% identity to culture collection sequences of Didymella macrostoma in GenBank MH854841 (ITS), MH866341 (LSU), MN983895 (TUB). The TEF1 sequences matched with 99.7% identity to TEF sequence of Didymella macrostoma MT454020. Subsequently, Koch's postulates were performed on 10 'Conference' pears per isolate (WURR-206, WURR-223, WURR-227 and WURR-308). Fruits wiped with 70% ethanol were inoculated in pathogenicity tests with an agar disk (5 mm diameter) of D. macrostoma prepared from the actively growing edge of 14-day-old cultures grown on PDA. Inoculated fruits were sealed in plastic bags and were incubated in darkness at 20°C. Typical symptoms appeared 7-10 days after inoculation on all pears. PDA-only controls remained symptomless. Fungal colonies isolated from the lesions and cultured on PDA morphologically resembled the original isolate from the infected pears. The identity of the re-isolations was confirmed as D. macrostoma by sequencing, thus completing Koch's postulates. To the best of our knowledge, this is the first report of D. macrostoma causing calyx-end rot of pears. The identification of this causal agent is important knowledge necessary for developing control measures for postharvest diseases of pear.

First Report of Root Rot Caused by Fusarium incarnatum on Mongolian Snake gourd in China.

In July 2022, stem lesions, approximately 4 to 5 cm in length as well as leaf wilt and dark brown necrosis on stems and roots were observed in two fields in Southwest Idaho on 20 to 30% of watermelons (Citrullus lanatus). To determine the causal agent, isolations were attempted from symptomatic tissue. The surface of the affected material was disinfected with 0.6% sodium hypochlorite for 1 min and rinsed three times with sterile water. Approximately 2 mm3 sections of tissue were plated on water agar amended with 0.02% penicillin and 0.08% streptomycin and incubated at room temperature for 7 days. Fungal colonies were tentatively identified as Rhizoctonia from right-angle branching and septate hyphal structures, slight constriction and septum near the branch base, and the production of 1 to 2 mm white to light brown irregularly shaped sclerotia. Single hyphal tips were transferred to potato dextrose agar (PDA) and grown at room temperature. Approximately ten isolates from each field with a consistent macromorphology were observed. These isolates had light brown mycelia, produced sclerotia at ambient temperature with no exposure to continuous light, and a representative isolate, designated D22-110 was selected for sequencing and pathogenicity testing. For isolate D22-110, mycelia were removed with a scalpel after 7 days of growth, for DNA extraction and sequencing of the rDNA internal transcribed spacer (ITS) region as previously described (White et al., 1990). A 726 bp product was generated and the sequence was submitted to GenBank (Accession No. OQ794049). NCBI-BLAST indicated this sequence was 99% identical (631 of 634 bp and 632 of 634 bp identical) with known reference isolates previously identified as R. solani AG 4 HG-III (Accession No. AF354075 and AF354076, respectively) from a phylogenetic study (Gonzalez et al., 2001). Pathogenicity testing was performed twice on two-week-old seedlings of watermelon cultivars Endless Summer and Wingman in greenhouse conditions (29oC, 12 h daylight). Two disks (3 mm diam) from 7-day-old plates of PDA were placed around each seedling at the root and stem convergence point. Ten seedlings were mock-inoculated with sterile PDA plugs as a control. Approximately 35% damping-off incidence was observed on inoculated seedlings six days post-inoculation, while control seedlings remained healthy. At 20 days post-inoculation, 20 (first trial) and 34 seedlings (second trial) were assessed for visible stem and root lesions. Incidence of stem lesions occurred on 90% of seedlings, with 80% of seedlings possessing lesions greater than 10 mm in diameter. Seedlings without R. solani inoculation were free of stem and root lesions. R. solani was re-isolated from symptomatic tissue, with 40% frequency of isolation, identified by right-angle branching of the hyphae thus confirming Koch's postulates. R. solani AG 4 has been reported in watermelon in the US since 1994 (Hall and Summer, 1994) but the AG 4 subgroup was not reported. AG 4 HG-III was reported in melon seedlings causing damping-off in Kyrgyzstan (Erper et al. 2016). In other hosts, AG 4 HG-III was found in potatoes in South Africa (Muzhinji et al., 2014), buckwheat and foxtail millet in China (Zhou et al., 2015; Hao et al., 2023), broccoli and spinach (Kuramae et al., 2003) and turnip green (Sekiguchi et al., 2015). To the best of our knowledge, this is the first report of R. solani AG 4 HG-III causing disease in watermelon in Idaho. Given the the rate of disease incidence observed in the field, growers should consider avoiding planting alternative host crops to minimize inoculum buildup.

Pepino (Solanum muricatum L.) is a popular solanaceous crop that is native to South America and is commercially grown in many countries including China for its attractive, sweet and flavorful fruits. In September 2023, a postharvest fruit rot was observed at an incidence of 7% to 10% on pepino at supermarket in Nanchang, Jiangxi, China (28.69°N, 115.81°E). Symptoms on fruits initially appeared as small black spots that later enlarged and became necrotic. To isolate the pathogen, symptomatic tissues were surface-sterilized using 75% ethanol for 15 s, then 1% sodium hypochlorite for 30 s, rinsed three times in sterile water, air dried, finally placed on potato dextrose agar (PDA) plates and incubated at 25℃ for 4 days. Ten strains (about 83% isolationfrequencyfromsymptomaticpepinofruits) with similar morphological characteristics were isolated. The colonies on PDA were initially white, gradually turning gray and eventually becoming black, and had abundant aerial mycelia. Conidia were fusiform to linetype, dark brown, measuring 50 to 100 × 10 to 28 μm (n = 30) with 5 to 10 transverse septa and 0 to 3 longitudinal septa. Based on the morphological characteristics, the pathogen was identified as Alternaria sp. (Ma et al. 2021). To further confirm species, two representative isolates (JXAL-1 and JXAL-2) were selected for molecular identification. The internal transcribed spacer (ITS) region, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), elongation factor 1-alpha (TEF1), Alternaria major allergen gene (Alt a 1) and DNA-directed RNA polymerase II core subunit (RPB2) were amplified and sequenced by using primers ITS5/ITS4, gpd1/gpd2, Alt-for/Alt-rev, EF1-728F/EF1-986R and fRPB2-5F2/fRPB2-7cR (Woudenberg et al. 2013; Woudenberg et al. 2014), respectively. These sequences were deposited into GenBank with accession number PP231808-PP231809 (ITS), PP238480-PP238481 (GAPDH), PP238482-PP238483 (Alt a 1), PP238484-PP238485 (TEF1) and PP238486-PP238487 (RPB2). A BLASTn homology search for these nucleotides showed 100% identity to ITS (KJ718182, 525 nt/525 nt), GAPDH (KJ718026, 579 nt/579 nt), Alt a 1 (KJ718694, 472 nt/472 nt), TEF1 (KJ718530, 334 nt/334 nt) and RPB2 (KJ718355, 772 nt/772 nt) sequences of Alternaria linariae CBS 107.61. The maximum likelihood analyses were performed for the combined ITS, GAPDH, Alt a 1, TEF1 and RPB2 using the IQtree web server (Trifinopoulos et al. 2016). In the phylogenetic tree, the isolates and isolates of A. linariae clustered together with 100% bootstrap support. Therefore, the fungus was identified as A. linariae. To evaluate pathogenicity, five healthy fruits of pepino were surface-sterilized with 75% ethanol, then wounded and a 5 mm diameter agar with isolate JXAL-1 was put on the wound. Another five fruits was inoculated with sterile agar plugs as control. All treated fruits were incubated at 25 ℃ with 80% humidity , and repeated twice. Five days later, all the wounded fruits inoculated with A. linariae showed similar symptoms and A. linariae was reisolated, while the control fruits remained healthy and no pathogen was isolated, fulfilling Koch's postulates. A. linariae is known as an important pathogen causing early blight of tomato and potato(Adhikari et al. 2020). To our knowledge, this is the first report of A. linariae causing postharvest fruit rot on S. muricatum in China, which expands the natural host range of A. linariae and will be helpful to develop efficient management strategies on pepino.