Water Agar Research Articles

First Report of Leaf Spot Disease on Avocado Caused by Bipolaris setariae in China.

Avocado (Persea americana), which is native to Latin America, is mostly planted in southwest China. In November 2021, leaf spot symptoms were observed in a nursery in Chongzuo (22.2019°N, 106.4723°E), Guangxi, China. Approximately 90% of avocado seedlings in the nursery were affected. Symptomatic plant fully expanded leaves showed small brown spots that ranged from 1 to 3 mm, with a yellow halo around (Fig.1). Lesions gradually expanded and became nearly round and dark brown. Finally, leaves withered or curled. For pathogen isolation, 15 symptomatic leaves were randomly sampled from different plants of the nursery, five leaves were selected and four samples size 4×4mm were taken from each leaf and were plated on potato glucose agar. Identical fungus colonies were observed in 80% of the samples, and no bacteria were isolated. Single conidial isolation was performed. After 4 days, the colony diameter reached 74.6 mm, colonies appeared gray, and developed aerial hyphae. Conidiophores were mostly solitary with a few clustered erect or slightly curved, knee shaped, and 3.89 to 5.24 µm wide. Conidia were 39.33 -96.88 × 9.96 - 15.59 µm, slightly curved, rarely straight, light brown to yellowish brown, fusoid or navicular, and truncated at the base with 4 to 10 septa. Based on morphological and cultural characteristics, the fungus was identified as Bipolaris sp. (Manamgoda et al. 2014). An isolate named MP211122 was grown on Sachs' ager at 27℃ under 12-h light/dark for 1 week and consistently with Adhikari et al. (2021) no sexual from was observed. To confirm the tentative identification, genomic DNA was extracted, ITS and GAPDH gene were amplified and sequenced using primers ITS1/ITS4 and GPD/GPD2, respectively (Tan et al. 2022). The ITS sequence (GenBank ON248469) shared 100% identity with B. setariae (MN215632.1), and the GAPDH sequence (ON642344) shared 99.82% identity with B. setariae (MF490833.1, MK144540.1) and B. yamadae (MK026428.1). A maximum likelihood phylogenetic analysis based on GAPDH and ITS sequences using MEGA 7.0 revealed that the isolate clustered with B. setariae with 100% bootstrap support(Fig. 2). Healthy 11-month old potted avocado seedlings from disease-free nursery were selected , the conidial suspension (1 × 105 conidia/mL) of MP211122 isolate was prepared by harvesting conidia from a 10-day-old culture on water agar. Conidia were sprayed onto young leaves of six potted plants. Three additional seedlings sprayed with sterile distilled water served as controls. All plants were covered with plastic bags for 3 days to maintain high humidity and then maintained in a greenhouse at 30℃ with a 12-h/12-h light/dark cycle. After 5 days, typical symptoms of small brown spots were observed on all inoculated leaves (Fig.3). All leaves on control plants remained asymptomatic. The reisolated fungus was morphologically identical to the original isolate used for inoculation, fulfilling Koch's postulates. This is the first report of B. setariae as a pathogen causing leaf spot on avocado in China. This information will facilitate further studies, monitoring and control of the disease as accurate identification of the causal agent is a primary requisite for designing management strategies.

First Report of Anthracnose on Banana Peppers Caused By Colletotrichum scovillei in New York.

In early August 2023, a disease outbreak on hot banana peppers (Capsicum annuum cv. Golden Dagger) was reported in Cattaraugus County, New York (NY). Disease incidence was at least 60%. Affected developing and mature fruit had at least one tan, soft, sunken lesion with salmon-colored spore masses surrounded by brown, necrotic margins. Microscopic observation of the lesions identified acervuli and setae typical of Colletotrichum spp. Isolations were made from these lesions by spreading conidia from the acervuli on 2% water agar (WA) + 0.02% (w/v) ampicillin. Colonies were hyphal tipped and transferred onto clarified V8 juice agar (CV8) and incubated at 20°C. The isolation frequency was 100% and a total of six isolates were obtained: Coll23Pep001, Coll23Pep003, Coll23Pep005, Coll23Pep007, Coll23Pep008, and Coll23Pep010. After 10 days, colonies were subcultured to potato dextrose agar (PDA) and CV8. On PDA, colonies appeared off-white to dark gray with sparse aerial mycelia. On CV8, the colony was pale gray with acervuli and orange-colored spore masses in the center. Conidia were hyaline, smooth and fusiform to round, and tapered at both ends. Mean conidial dimensions (n = 20) were 20.2 (13.75 to 25) µm long × 4.7 (3 to 6.25) µm wide. To confirm the identity of the isolates, DNA was extracted, and PCR performed to amplify the internal transcriber spacer (ITS) region (primers ITS1/ITS4; White et al. 1990), and actin (ACT) (primers ACT-512F/ACT-783R; Carbone and Kohn 1999) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes (primers GDF1/GDR1; Guerber et al. 2003). Pairwise alignment of the sequences showed all isolates had 100% similarity to C. scovillei ex. holotype CBS 126529 (Damm et al. 2012). Sequences from all isolates were deposited in GenBank with accessions PP556967 to PP556972 (ITS), PP565766 to PP565771 (ACT), and PP565772 to PP565777 (GAPDH). For pathogenicity testing, all isolates were grown on CV8 at 20°C in the dark for 10 days. Conidia were harvested by flooding the plates of each isolate with sterile distilled water and filtering the suspension through a double layer of cheesecloth. The concentration of the conidial suspension was adjusted to 5 × 105 spores per ml. Pathogenicity of the six isolates was tested on banana pepper fruit by using a sterile toothpick to pierce the skin at the two opposite ends. A droplet (10 µl) of the conidial suspension was placed on each wound. The same number of fruit were inoculated without wounding, and non-inoculated control fruit received a droplet of sterile distilled water (either wounded or unwounded). The experiment was repeated twice. All fruit were placed in a humid box at room temperature for 7 days. All wounded and inoculated fruit developed sunken lesions filled with salmon-colored conidial masses. Disease did not occur on the unwounded, inoculated fruit nor the non-inoculated controls. C. scovillei was recovered from all inoculated fruit by reisolating onto CV8 media and isolates had similar morphology and conidial dimensions to the original isolates. To the best of our knowledge, this is the first report of C. scovillei causing anthracnose on pepper in NY. C. scovillei has been reported in South Carolina (Toporek and Keinath 2021), Brazil (Caires et al. 2014), eastern Asia (de Silva et al. 2019), and Kosovo (Xhemali et al. 2023). The pathogen is particularly aggressive on pepper and poses substantial threats to pepper production around the world.

First Report of Neofusicoccum parvum causing leaf spot on Camellia japonica in China.

Camellia japonica is an important garden landscape plant in southern China. In April 2022, leaf spot symptoms were observed at the camellia garden of Jiaying University (24°32'83″N, 17 116°12'31″E) in Meizhou city, Guangdong Province, China. The initial symptoms were grayish brown spots on the leaves, as the disease progressed, the lesions were enlarged and affected the whole leaf and eventually led to the loss of its ornamental value. The disease incidence was above 15%. Leaf pieces (5 × 5 mm) from 3 diseased Camellia leaves were sterilized in 75% ethanol for 1 min, then in 1% NaOCl for 1 min; and rinsed three times with sterile water. Leaf pieces were inoculated on potato dextrose agar (PDA) medium and incubated at 25 °C. Three days later, fungal colonies initially showed a white aerial mycelium, turning gray after 5 days, and dark gray after 7 days of incubation. Conidia were single-celled, hyaline, ellipsoidal and without septa. Dimensions of conidia (n≥50) were 14.27 to 20.65 × 4.28 to 6.56 μm. The morphological characteristics matched the genus Neofusicoccum (Pavlic et al. 2009). For molecular identification, the rDNA internal transcribed spacer (ITS1, 5.8S and ITS2) region, translation elongation factor 1-alpha (tef1-α), and beta-tubulin (tub2) of a representative isolate SC6-2 were amplified using the primer pairs ITS1/ITS4, EF1/EF2 and BT2a/-BT2b, respectively (Golzar and Burgess，2011). The sequences obtained were deposited in GenBank (accession nos. PP064173, PP479650 and PP082457 for ITS, tef1-α and tub2, respectively). Nucleotide BLAST analysis showed a 99.81% homology with N. parvum (519/520 bp, OQ509869; 519/520 bp, KF294003; 518/519 bp KF293989) for ITS, 100% homology with N. parvum (398/398 bp, MN318108; 398/398 bp, MK294085; 398/398 bp, MH936021) for tub2, and >99% homology with N. parvum (259/259 bp, 100%, MW390561; 263/265 bp, 99.25%，MN175952; 263/265 bp, 99.25%, MK781982) for tef1-α. The combined phylogenetic analyses (ITS, tef1-α, and tub2) showed that the sequence of the tested isolate and the corresponding sequence of N. parvum (CMW9081, SHSJ1-2) in GenBank grouped in the same branch of the phylogenetic tree. Based on morphological characters, DNA sequencing, and the phylogenetic tree, it can be determined that the pathogen was Neofusicoccum parvum. Inoculation on Camellia leaves was performed to confirm pathogenicity. Nine healthy camellia leaves were pin-pricked with a sterile needle and inoculated with mycelial plugs of isolate SC6-2. Nine other healthy leaves were pin-pricked and inoculated with noncolonized PDA plugs as control leaves. The inoculated leaves were maintained on water agar solid medium at 25°C. To keep a high-humidity environment the inoculation sites were covered by moistened cotton for 2 days. The experiment was repeated three times. Five days after inoculation, all the inoculated leaves showed similar symptoms to those observed in the field, whereas control leaves were asymptomatic for 6 days. The fungal isolates recovered from inoculated leaves were morphologically identical to the N. parvum isolates originally recovered from symptomatic leaves collected in the field, fulfilling Koch's postulates. Neofusicoccum parvum is an aggressive pathogen that causes severe disease on important tree and woody species (Liddle et al. 2019). It has been reported that N. parvum can infect the leaves and branches of grapes (Otoya-Martinez et al. 2023), dieback on Camellia japonica (Pintos, et al. 2012), Brazilian pepperwood (Bertetti et al. 2022), mango (Giancarlo et al. 2023) and other plants. To our knowledge, this is the first report of N. parvum causing leaf spot on Camellia japonica in China.

Root rot of spinach caused by Pythium myriotylum and P. aphanidermatum in Taiwan.

Spinach (Spinacia oleracea) is a commonly used green vegetable. During September and October in both 2022 and 2023, a vegetable nursery company located among paddy rice fields in Taichung City, Taiwan, reported significant failures in spinach seedling production in net-houses with mean outdoor temperatures of 28.7℃. Abnormal growth was observed in approximately 30% of the spinach seedlings in each batch (n = 2,000 to 3,000), with aboveground tissues showing stunting, yellowing, and wilt, and underground tissues displaying root rot. The symptoms resembled the spinach damping-off documented in Taiwan in extension articles but which lacked complete pathogen identification. A total of 110 plants from two batches were used for pathogen isolation by placing roots on water agar incubated at 25℃ or were examined for the presence of oospores in diseased roots. Eighty-one percent of these plants were associated with Pythium. Nine Pythium isolates were used in subsequent analyses. Genomic DNA from these isolates was subjected to amplification of ITS, β-tubulin gene (TUB2), and cytochrome C oxidase subunit Ⅱ (COXII) gene with primer pairs ITS1 / ITS4, BT5 / BT6, and FM58 / FM66 (Villa et al. 2006). Sequences of ITS (PP209187-PP209195), TUB2 (PP212864-PP212872), and COXII (PP212855-PP212863) were deposited in GenBank. Four isolates (sp01, sp02, sp03, and sp04) were 100% identical to the neotype strain (CBS 118.80) of Pythium aphanidermatum (Edson) Fitzp. for the ITS (761 bp), TUB2 (583 bp), and COXII (547 bp). Five isolates (2sp, 3sp, ND2-4sp, D3-4sp, and ND3-3sp) were 99.87%, 100%, and 99% identical to the reference strain (CBS 254.70) of Pythium myriotylum Drechsler for the ITS (762 bp), TUB2 (602 bp), and COXII (556 bp), respectively. Phylogenetic analysis of Pythium isolates inferred from concatenated sequences of the three genes (LéVesque and De Cock 2004; Villa et al. 2006) revealed that the same four isolates grouped with the neotype strain of P. aphanidermatum, and the five isolates clustered with the reference strain of P. myriotylum, each with a 100% bootstrap support. Morphological features of isolates ND3-3sp and sp01 were used for identification. Isolate ND3-3sp produced inflated lobulate sporangia and aplerotic and smooth oospores (16.3 to 25.1 um; n = 30) attached with three to five antheridia, consistent with identification as P. myriotylum. Isolate sp01 produced inflated lobulate sporangia and aplerotic and smooth oospores (17.0 to 24.0 um; n= 30) attached with a single intercalary antheridium, agreeing with the morphology of P. aphanidermatum (Van der Plaats-Niterink 1981). To investigate the pathogenicity of the nine Pythium isolates on spinach, 20 mycelial agar discs (4 mm in diameter) from a 2-day-old V8 culture of each isolate were used to induce sporangia and zoospores in 20 ml sterilized water at 25℃ with a 12 h light / dark regime. A 1.5 ml zoospore suspension (6 × 103 zoospores / ml) was dropped into BVB growth substrate of two spinach seedlings in 2-week-old at 25℃ with 12 h light / dark regime, resulting in symptoms resembling those observed in commercial nurseries at 7 days post-inoculation (dpi). Each Pythium isolate inoculated 20 seedlings in 10 cells of a planting tray. At 14 dpi, disease incidences were 95 to 100% for P. myriotylum isolates and 60 to 85% for P. aphanidermatum isolates, while control plants treated with water showed no symptoms. Re-isolated pathogens from the inoculated plants were morphologically identical to the inoculated isolates, completing Koch's postulates. Results of the pathogenicity assay, along with molecular and morphological identification, conclude that the root rot of spinach was caused by P. myriotylum and P. aphanidermatum. The two oomycetes were not formally documented to cause spinach diseases in Taiwan. Although P. myriotylum has been isolated from spinach (Wang et al. 2003), its pathogenicity to spinach was not documented worldwide. Root rot of spinach caused by P. aphanidermatum has been reported in the United States (Bates and Stanghellini 1984), Korea (Cho and Shin 2004), and Italy (Garibaldi et al. 2015). These pathogens thrive in humid and hot weather (Littrell and McCarter, 1970). Producing spinach in cooler weather or in a temperature-controlled environment may help prevent severe occurrence of the disease.

First Report of Neofusicoccum parvum Causing Fruit Rot on Psidium guajava in China.

Guava (Psidium guajava L.), a nutrient-rich and economically significant fruit, is extensively cultivated in southern China. In six continuous years (from 2018 to 2023), dark-purple rotted guava with sunken lesion were observed on guava trees (variety 'Zhenzhu', aged over 5 years) in Dongguan and Panyu districts, Guangdong Province. Annually, the incidence of fruit rot disease in the affected fields reached 30% to 50% and significantly reduced the yield and quality of harvest guava. The initial symptoms on the epicarp of the fruits were black, needle-like dots that rapidly spread, causing partial or complete fruit rot within two to three weeks. To identify the causative agent, six symptomatic fruits were collected from two different orchards. Samples of 0.5 cm³ were excised from the lesion margins of each fruit. These samples were surface-sterilized with 70% ethanol for 30 seconds, followed by 0.2% NaClO for 2-3 minutes, and rinsed in sterile water three times. The samples were then cultured on potato dextrose agar (PDA) at 25°C for five days. This process yielded eight fungal isolates with similar morphological. Initially, the colonies were white with dense aerial mycelium becoming dark gray after 4 to 5 days. The mycelia were septate and branched. No spores were observed on PDA. To induce spore formation, the isolates were cultured on water agar for 20 days. This process led to the production of hyaline, aseptate, ellipsoid conidia, which were thin-walled, smooth-surfaced, and measured 3.7-5.1 × 1.6-2.2 μm (n = 100). Three isolates, including at least one from each orchard (Np1, Np2, Np3), were selected for further analysis. Genomic DNA was extracted using Axygen MAG-FRAG-I-50 (Axygen Bio-Tek). The internal transcribed spacer of rDNAs (ITS), beta-tubulin (tub2), the nuclear ribosomal large subunit (LSU), and translation elongation factor 1-α (tef1-α) gene regions were amplified using the primers ITS1/4, Bt2a/Bt2b, LR5/LR0R, and EF1-728F/EF1-986R (Golzar and Burgess 2011) and sequenced. Sequence analysis using MEGA 7.0 (Kumar et al., 2018) revealed 100% similarity among the isolates. BLAST searches of the ITS, tub2, LSU, and tef1-α sequences (accession nos. MN907637, MT537938, MT528156, MT537939) showed the highest nucleotide similarities (99.24 to 100%) to Neofusicoccum parvum strains (Crous et al. 2006). A phylogenetic tree was constructed with MEGA 7.0 based on the nucleotide sequence tub2 using the maximum likelihood method. Pathogenicity tests on 10 healthy guava fruits using mycelium-inoculated and control fruits confirmed the causative agent. The inoculated fruits, maintained at 25°C under a 12-h light/dark cycle, exhibited symptoms identical to the field infections within four to seven days, while control fruits remained symptomless. The fungus, reisolated from the inoculated fruits, was morphologically identical to the original isolates, fulfilling Koch's postulates. In conclusion, based on molecular, morphological, and pathogenic analysis, N. parvum as the causal agent of the fruit rot disease on guava. Previously, N. parvum has been reported in association with fruit rot on Eriobotrya japonica and Juglans regia (Zhai et al. 2019; Chen et al. 2019). To our knowledge, this is the first report of N. parvum affecting guava in China.

First Report of Leaf Spot on Eriobotrya japonica Caused by Alternaria alternata in Southwest Korea.

Loquat (Eriobotrya japonica) is a crop cultivated in Southwest Korea, covering an area of 101 ha and yielding 120 tons at harvest (KASS, 2024). Due to its high-income potential, the cultivation area is gradually expanding. In May 2023, 30% of leaf brown spots were observed on all three trees in the Suncheonman National Garden, Suncheon (3488'57.97" N, 12750'92.83" E). As the disease progressed, the brown spot gradually enlarged, turning greyish-ivory inside and forming concentric circles. Three leaf lesions from each tree were cut into 5 x 5 mm pieces, surface-sterilized with 70% ethanol for 1 min, and washed in sterile water three times to isolate the pathogen potentially responsible for these symptoms. The samples obtained were subsequently cultured on 1.5% water agar and then incubated in the dark at 25℃. A total of nine isolates were obtained, with three isolates from each of the three trees through single-spore isolation, namely SYP-1202-1 to 3, SYP-1202-4 to 6, and SYP-1202-7 to 9. The colonies reached 90 mm in diameter after 10 days on potato dextrose agar (PDA), initially dark green, and turned sooty gray after 2 weeks. The hyphae grown on a 0.6% KCl medium for 3 days produced long chains containing three to twelve conidia. The conidia were ellipsoidal or obpyriform in shape and light brown. The conidiophores were straight or curved, measuring 12.1-75.3 x 1.6-4.8 μm (n = 100). The primary and secondary conidia measured length × width of 19.1-60.6 × 6.1-14.4 μm and 8.4-27.8 × 3.5-9.5 μm (n = 100), respectively. The conidia had 1 to 7 transverse and 0 to 3 vertical septa. The morphology of the nine isolates was identical and consistent with Alternaria species (van der Waals et al., 2011; Woudenberg et al., 2015). For molecular identification, ITS (OR844500 to OR844508), GAPDH (OR866383 to OR866391), TEF1 (OR866392 to OR866400), RPB2 (OR866401 to OR866409), Alt a1 (OR866410 to OR866418), endoPG (OR866419 to OR866427), and OPA10-2 (OR866428 to OR866436) sequences from SYP-1202-1 to 9 showed a 100% (515 bp/515 bp), 100% (579/579), 100% (240/240), 100% (753/753), 95.1% (449/472), 100% (448/448), and 100% (634/634) identity with that of type strain A. alternata CBS 115152 (KP124348, KP124202, KP125124, KP124816, KP123896, KP124049, and KP124658, respectively). A pathogenicity test was conducted on three 5-year-old E. japonica cultivar Daebang trees in pots. The surface of the five leaves per tree was sterilized with 70% ethanol for 1 min. Before inoculation, the leaves were wounded with sterile needles and sprayed with the conidial suspension (1×106 conidia/ml) produced from a 1-week-old culture grown on PDA. In contrast, control leaves were sprayed with sterile distilled water. The inoculated leaves were wrapped with black plastic bags and kept at 100% relative humidity for two days. At seven days post-inoculation, symptoms were observed on the wounded leaves, whereas the nonwounded and control leaves did not exhibit any symptoms. The experiment was performed three times in the greenhouse. For each experiment, pathogens were reisolated from the two symptomatic leaves per plant. The identity of the reisolated pathogens was then confirmed via analysis of ITS and RPB2 genes, thereby confirming adherence to Koch's postulates. To the best of our knowledge, this is the first report of E. japonica being infected by A. alternata in Korea. This report provides important information to support effective disease control strategies for E. japonica in orchards in southern Korea.

Direct Isolation Method for Tomato-Infecting Phytophthora infestans

Infection of Phytophthora infestans at high altitudes in tropical regions causes symptoms of tomato late blight throughout the year. Even though it is very easy to find in the field, P. infestans is often very difficult to isolate aseptically in the laboratory. This study aims to evaluate direct isolation techniques that can increase the success of isolating P. infestans. Isolation was carried out on a non-specific medium, consisting of potato dextrose agar (PDA), corn meal agar (CMA), oatmeal agar (OMA), and water agar (WA) with three alternative types of antibiotics, i.e. chloramphenicol, amoxicillin, and rifampicin. Observations were carried out to determine the effect of the medium on sporangia induction and the age of the original colony’s growth in CMA and OMA media. The results showed that the isolation of P. infestans using the direct method was successfully carried out on non-specific PDA, CMA, OMA and WA medium. The highest isolation success rate was obtained on CMA medium with the addition of 50 mg L-1 rifampicin. The fastest sporangia induction (8 days) was shown by colonies grown on OMA medium with the addition of 50 mg L-1 rifampicin based on the category of abundant sporangia after 20 days of incubation. Rejuvenation of P. infestans colonies for research purposes in the laboratory is recommended to be carried out routinely twice a month. This research provides practical guidance for understanding the bioecology of P. infestans infecting tomato plants, especially for further study on oomycetes fungi.

Potentialities of Technosol-isolated PGPB consortium in promoting plant growth in lettuce seedlings

Abstract Background and aims Reducing land degradation and safeguarding agricultural productions ensures the provision of ecosystem services and economic welfare, as highlighted by the 2030 Sustainable Development Goals. Among the promising solutions to tackle these issues, the study investigates the use of Technosol-isolated PGPB, as a novel approach for enhancing plant growth and the capability to cope with soil salinization. Methods Several bacteria have been isolated from a Technosol in Naples (Italy), selected to produce a consortium, based on their PGP features and tested on lettuce. The promotion of lettuce growth was evaluated both in soil (sterilized and not-sterilized Technosols) and in water agar media differing in NaCl concentrations, focusing on total polyphenols, 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity, soluble proteins, and sugars, which were evaluated on seedlings. Results Germination kinetics differed among treatments in both soil and artificial media setups. In the Technosol trial, the PGPB treated seeds were the quickest to emerge, showing a higher radical scavenging activity and lower soluble carbohydrate content. In the salinity trial, total soluble proteins were significantly higher in primed seeds at null saline cocentration. Conclusion Our results indicate that the PGPB application has a beneficial outcome on the Technosol but under saline stress produces an inhibitory or a costly effect on the biochemistry and germination kinetics. However, Technosol PGPBs employement may open new research scenarios on their potential application considering Nature-Based Solutions and/or plant growth in degraded environments.

First report of soybean root rot caused by Fusarium proliferatum in the Republic of Korea.

Fusarium species are widespread soilborne pathogens that can cause damping-off, root rot, and wilting in soybean [Glycine max (L.) Merrill], subsequently leading to significant yield suppression. Several Fusarium spp. have already been documented for their pathogenicity on soybean plants in the Republic of Korea. The nationwide monitoring of soybean diseases continues to identify new pathogenic Fusarium spp. In 2016, five plant samples at R3-R4 growth stages, showing symptoms of wilting in the upper parts and root rot, were collected in Suwon, Gyeonggi, Republic of Korea. Fungal colonies were obtained from the diseased root samples, with the surface sterilized in 1% sodium hypochlorite for 2 min, rinsed thrice with sterile distilled water, and placed on water agar at 25°C. Five isolates were collected and purified by single-spore isolation. The fungal mycelium was subsequently cultivated on potato dextrose agar for ten days. The isolates produced abundant, aerial, and white mycelium and became purple in old cultures. Macroconidia were slender, falcate to almost straight, usually 3 to 5 septated, and thin-walled. Microconidia were formed in chains from polyphalides, clavate or oval, usually single-celled with a flattened base. These characteristics of isolates were consistent with the description of F. proliferatum (Leslie and Summerrell 2006), and the representative isolate 16-19 was selected for molecular identification to confirm its identity as F. proliferatum. Two evolutionarily conserved genes, the translation elongation factor 1-alpha (EF-1α) and the second-largest subunit of RNA polymerase II (RPB2) genes, were partially amplified using the primers described by O'Donnell et al. (2008), resulting in nucleotide sequences of 680 and 382 base pairs, respectively. These two sequences (GenBank accession numbers: OQ992720 and OR060666) showed 100 and 99.5% identity to the EF-1α and RPB2 of F. proliferatum A40 (GenBank accession numbers: KP964907 and KP964842). For the Petri-dish pathogenicity assay (Broders et al. 2007), five surface-sterilized seeds were placed on water agar media with either sterile water or actively growing '16-19' culture. After 7 days of incubation in a growth chamber (25°C; 12-hour photoperiod), brown lesions were observed on the roots of the inoculated plants, while no symptoms were observed in the sterile water-treated controls. The experiment was conducted three times. For root-cut pathogenicity assay, conidial suspension (1×106 conidia/ml) of the isolate '16-19' was prepared with harvested mycelia cultured on PDA for 10 days with sterile water. The roots of 10-day-old soybean seedlings were partially cut and soaked in either the suspension or sterile water for 2 hours. The seedlings were transplanted into 12 cm plastic pots (11 cm in height) and grew in a greenhouse (26 ± 3°C, 13-h photoperiod). The experiment followed a completely randomized design with three replicates (i.e. three plants in a pot), and it was repeated twice. The inoculated plants began to wilt 7 days after inoculation, while the sterile water-treated controls remained healthy. Ten days after inoculation, all plants were collected, washed under running tap water, and evaluated for the presence and severity of root rot using a 0-4 scale (Chang et al. 2015). The inoculated plants exhibited reduced vigor and developed dark brown lesions on their roots. F. proliferatum was reisolated from symptomatic root tissues of the infected plants, while not from those of the controls. Its colony and spores were morphologically identical to those of the original isolate. F. proliferatum was previously reported as a causative agent of soybean root rot in the United States (Díaz Arias et al. 2011) and Canada (Chang et al. 2015). This is the first report of soybean root rot caused by F. proliferatum in the Republic of Korea. This finding implies that F. proliferatum may potentially threaten soybean production in the Republic of Korea and suggests that effective disease management strategies should be established for soybean protection against the disease, along with continuous surveillance.

Disease prevalence, incidence, morphological and molecular characterisation of Lasiodiplodia pseudotheobromae causing collar rot disease on peanut plants in Turkey

AbstractPeanut (Arachis hypogaea L.) holds significant commercial and dietary importance as a major source of edible oil and protein in Turkey. Stem, collar or root rot, caused by several fungal disease agent, are serious soil-borne diseases of peanut. Accurate and precise identification of the disease agent provides fundamental and precise information for integrated plant management. During the period from June to September 2021, symptoms consistent with collar rot disease, including dark-brown stem rot, chlorotic leaves, wilting, and eventual whole plant death, were observed on peanut plants cultivated in the different districts of Osmaniye Province of Turkey. The disease incidence ranged from 8.0 to 45.0% in the inspected fields with an average of 3.4% overall. Twenty-four single-spore representative isolates were obtained from surface-disinfected symptomatic tissues. Morphological characteristics of fungal mycelium, conidial and pycnidial structures on potato sucrose agar (PSA) and water agar (WA) closely resembled those described for Lasiodiplodia spp. All isolates caused typical collar rot symptoms upon artificial inoculation of peanut seedlings. Morphological identification of Lasiodiplodia spp. isolates was corroborated by MALDI-TOF and molecular analyses utilizing sequences from the internal transcribed spacer (ITS), β-tubulin 2 (tub2) and translation elongation factor-1 alpha (TEF1-α) loci. Phylogenetic analysis confirmed that the representative fungal isolates (MKUBK-B1 and MKUBK-K22) belong to Lasiodiplodia pseudotheobromae. To the best of our knowledge, this is the first report of L. pseudotheobromae infecting peanut plants in Turkey. This work is expected to contribute to previously limited knowledge regarding the host range, incidence and prevalence of L. pseudotheobromae as a soilborne pathogen of peanuts. Due to the potential destructiveness and broad host range of this pathogen, it is essential to develop new strategies to establish more reliable, environmentally sustainable, and cost-effective management approaches for this disease.

First Report of Alternaria alternata Causing Leaf Spot on Smooth Bromegrass (Bromus inermis Leyss.) in China.

Smooth bromegrass (Bromus inermis Leyss.) is an important forage crop in northern China. In July 2021, leaf spot symptoms were observed on smooth bromegrass in Ewenki Banner, Hulunbuir, Inner Mongolia. In an area of approximately 0.12 hectares, 95% disease incidence was observed. Ten diseased plants were collected for pathogen isolation. Leaf tissues near the lesions were cut into 5 × 5 mm pieces, surface-disinfested in 75% ethanol for 3 min, and rinsed with sterile distilled water. The pieces were placed on water agar in petri plates and incubated at 25℃ for three days. The resulting colonies were flushed with sterile water and a spore suspension was serially diluted and plated on potato dextrose agar (PDA). A single-spore colony was obtained. Ten isolates were obtained and designated HE1 to HE10. The colony morphology was identical for all isolates, grayish white in color on the upper surface and light black on the underside. The mycelia were light gray and velvety. Conidia were light brown to brown in color and oblate, oblong or oval. The conidial dimensions were typically between 15 to 43 μm by 8 to 9 μm in size. The conidia possessed one to six transverse septa, with slight to distinct constrictions at each division, and zero to two longitudinal septa. These morphological characteristics resembled Alternaria alternata (Fr.) Keissl.. DNA was extracted from three isolates, HE3, HE4 and HE5, using the CTAB method. Polymerase chain reaction (PCR) was performed on the extracted DNA with a set of primers ITS1/ITS4, H31a/H31b, gpd1/gpd2, TEF1-728F/TEF1-986R, and RPB2-5F2/fRPB2-7cR. The amplicon sequences from the three isolates were analyzed using the BLAST in GenBank (https://www.ncbi.nlm.nih.gov/). The results showed a high sequence identity, ranging from 99 to 100%, with the A. alternata strain YTMZ-20-2 across all the genetic markers tested. The strong match reinforced the identification of the strains as A. alternata. The sequences were deposited in GenBank (Table S1). The three fungal isolates were identified as A. alternata based on their morphological and genetic data. To conduct Koch's postulates, the representative isolate HE4 was used. Smooth bromegrass seed was soaked in water for four days and sown in potting soil contained in plastic pots (10 cm diameter × 15 cm height, five seeds/pot) in a greenhouse under a 16-h photoperiod at temperatures between 20 to 25°C and 60% relative humidity. When the plants reached a height of approximately 20 cm, the plants in three pots (replicates) were sprayed with a spore suspension (106 conidial/ml) at 10 ml/pot, and three pots were sprayed with sterile water for control. Five days after inoculation, the plants exhibited leaf spot symptoms similar to those previously described, while the control plants remained unaffected. The causative fungus was successfully re-isolated from the diseased plants and confirmed morphologically and molecularly on its identity as described above. This experiment was independently conducted three times. This is the first report of A. alternata causing leaf spot on smooth bromegrass in China. Since there is risk that the disease could seriously reduce the yield of the forage crop smooth bromegrass, further research is needed.

First Report of Root and Collar Rot caused by Phytophthora nicotianae on ⨯Graptoveria 'Silver Star' in Korea.

⨯Graptoveria 'Silver Star' (a cross between Graptopetalum filiferum and Echeveria agavoides) from the Crassulaceae family, are an evergreen succulent with lotus constellation-shaped flowers, making it consumer favorite ornamental plant in Korea. In 2019, Korea's ornamental production was estimated at KRW 517.4 billion (EUR 382 million), from 4,244 ha of farming area according to the Ministry of Agriculture, Food and Rural Affairs of Korea. In July 2023, ⨯Graptoveria 'Silver Star' plants with chlorotic leaves, root and collar rot were observed in a greenhouse in Yongin (37°14'27.9"N, 127°10'39.19"E), Korea. To isolate the causal agent, small pieces (1 mm2) of symptomatic tissues were surface-sterilized using 1% NaOCl for 1 min, then put onto a water agar (WA) plate and incubated in the dark at 25℃ for five days. Two isolates (FD00202, FD00203) were obtained from diseased leaves, stem and roots by isolating single sporangium. To investigate the morphological characteristics of the isolates, the mycelium from potato dextrose agar (PDA) were transferred to V8 agar (V8A) followed by incubation at 25°C in the dark for 7 days. The isolates produced dense cottony mycelium, with slightly petaloid and light rossette pattern, with coralloid edges measuring 70 to 83 mm diameter. Sporangium were spheroid (30.0-48.0 µm long, 25.0-35.0 µm wide) with globose chlamydospores (17.0-50.0 µm long, 18.0-38.0 µm wide). Oogonia were not observed. Morphological and cultural characteristics of these isolates were phenotypically similar to that of Phytophthora nicotianae (Faedda et al. 2013; Abad et al. 2023). For molecular identification, genomic DNA was extracted from 5 days old cultures using the Maxwell® RSC PureFood GMO and Authentication Kit (Promega). Two gene regions, the rDNA-ITS, COX I were amplified and sequenced using primers ITS1/ITS4 and FM83/FM84, respectively (White et al. 1990; Martin and Tooley 2003). The resulting sequences were deposited in GenBank with accession no. LC783858 to LC783861. A BLASTn search of the DNA sequences from ITS, COX I showed 99.81 and 98.94% identity to P. nicotianae isolate IMI 398853, respectively. Maximum likelihood phylogenetic analyses were performed for the combined data set with ITS, COX I using MEGA7 under the Tamura-Nei model (Kumar et al. 2016). The isolates formed a monophyletic group with P. nicotianae isolate IMI 398853, CPHST BL162, and CPHST BL 44. Based on morphological characteristics and molecular analysis, the isolates were identified as P. nicotianae. T confirm their pathogenicity, inoculum was prepared in accordance with Ann (2000). Artificially wounded healthy plant roots were dipped in zoospore suspension (3.0 × 106 zoospore/ml) for 24 hours, with mock-treated plants (control) dipped in sterile distilled water (Ann. 2000). Thereafter, the plants were transplanted into new medium and kept under high humidity. Symptoms were observed after 10 days of incubation. The plants inoculated with P. nicotianae showed similar symptoms of chlorotic leaves with root and collar rot, while control remained symptomless. The pathogen was re-isolated from all inoculated plants and confirmed as P. nicotianae by morphological and molecular analysis. but not from controls, fulfilling Koch's postulates. Phytophthora nicotianae was previously report on Echeveria derenbergii and Kalanchoe blossfeldiana causing brown spot on stems and roots in California and Korea, respectively (French 1989; Oh and Son 2008). To best of our knowledge, this is the first report of P. nicotianae causing root and collar rot on ⨯Graptoveria 'Silver Star' plants in the Korea.

Occurrence of Powdery Mildew Caused by Erysiphe corylacearum on Hazelnuts in Israel

Hazelnut (Corylus avellana L) is an emerging crop in Israel, primarily cultivated as a host plant to establish truffle plantations through symbiosis with ectomycorrhizal fungi. A significant damage and yield reduction is caused by the prevalent occurrence of powdery mildew in hazelnut trees (Sezer et al., 2017). Until recently, Phyllactinia guttata was considered the primary pathogen in Western Asia, the Caucasus region, and Europe (Abasova et al. 2018; Arzanlou et al. 2018; Mezzalama et al. 2021). However, in the last years, a new destructive species Erysiphe corylacearum has been identified as the pathogen of powdery mildew on hazelnuts in these regions (Meparishvili et al. 2019; Mezzalama et al. 2021; Kalmár et al. 2022; Zajc et al. 2023). In May 2022, powdery mildew symptoms were observed on hazelnut plants in the Ein-Zivan truffle plantation, gardens of Merom-Golan, and the adjacent garden of the packing house Pri-Beresheet in the northern Golan region of Israel. Symptoms were observed on the abaxial and adaxial leaf surfaces, fruits, and husks. Disease incidence and severity ranged between 30-70% and 5-90%, respectively. Disease severity was significantly greater on the leaves of the offshoots compared to those on the tree canopy. Morphological characterization of leaf samples from ten different trees showed the following characteristics: hyphal appressoria were lobed, solitary, 1–4 μm in diameter; mycelium was amphigenous, hyaline, and septate; conidiophores vertically elevated from the mycelium 50– 80 μm long. Conidia (n= 30) on conidiophores were hyaline, ellipsoid to ovoid, 24 – 34 μm long and 15.5 – 23 μm wide. Chasmothecia in several maturation degrees appeared in October on both sides of the leaves. They were spherical (n= 30) 86 – 125 μm in diameter, with 7 - 14 aseptate straight appendages, 67 - 96 μm long, 4.9 - 7.1 μm wide, dichotomous branched at the end 42 - 56 μm wide. In each chasmothecia, there were 3-5 asci (n=30) with a width of 38 – 43 μm and a length of 48–64 μm of oval-ellipsoid shape. Asci contained 4-8 ascospores (n=30), 18 - 26 μm long and 11 - 15.5 μm wide. A pathogenicity test was conducted to fulfill Koсh’s postulates. Both detached leaves and plants of C. avellana were artificially infected by brushing conidia from infected leaves. Inoculated leaves in Petri dishes on 2% water agar (n= 5), and plants (n= 5) were incubated under 25°C and 12-h photoperiod/day. Untreated leaves and plants served as control. Typical symptoms appeared on the upper surface of the leaves within 7-10 days after inoculation. No symptoms were found on untreated control plants or detached leaves. The fungus isolated from the inoculated leaves was morphologically identical to the original isolates from natural diseased plants. DNA was extracted and the rDNA internal transcribed spacer region of five isolates originated from leaves of the tree canopy and offshoots was amplified using primers ITS1 and ITS4, and sequenced. BLAST analysis of 595 bp fragments (all identical and represented by isolate Cora1, GenBank Accession No. OR752437) showed 99% identity to ITS rDNA sequences of E. corylacearum from Georgia (MK157199) and 100% identity to isolates from Azerbaijan, Turkey and Italy (LC270863, KY082910 and MW045425, respectively) and only 83% similarity to P. guttata (accession number AB080558). To the best of my knowledge this is the first report on E. corylacearum causing powdery mildew in Israel. Future control measures to manage the disease on hazelnuts in truffle plantations in Israel should be considered.

Abundance and diversity of fungal endophytes isolated from monk fruit (Siraitia grosvenorii) grown in a Canadian research greenhouse.

Monk fruit (Siraitia grosvenorii) is an herbaceous perennial vine of the Cucurbitaceae family cultivated commercially mainly in southern China. There is very little information available about the fungal endophytes in monk fruit. In this study, monk fruit plants were grown from seeds in a research greenhouse at Kwantlen Polytechnic University in British Columbia, Canada to explore the abundance and diversity of their fungal endophytes. Fungal endophytes were isolated from seeds, seedlings, mature monk fruit plants, and fruits, and cultured on potato dextrose agar and water agar media. Isolates were identified by microscopic examination and BLAST comparison of ITS sequences to published sequences in GenBank. At least 150 species of fungal endophytes representing 60 genera and 20 orders were recovered from monk fruit tissues. Non-metric multidimensional scaling (NMDS) was carried out to explore the similarity of fungal communities among roots, stems, leaves, flowers, fruits, and seeds based on fungal orders. Our study showed that monk fruit plants are a rich source of fungal endophytes with the greatest abundance and diversity in leaves. This work has deepened our understanding of the intricate interactions between plants and fungi that sustain ecosystems and underpin plant health and resilience.

Identification of Diagnosis Fungi that Cause Potato Root Rot

Results of collecting samples from different regions of Anbar Governorate (Al-Amiriyah, Al-Khalidya, Fallujah, Heet and Ramadi) showed that potato root rot disease is widespread in all regions collected. The results of isolation and phenotypic and molecular diagnosis using the polymerase chain reaction (PCR) technique indicated showed the presence of fungus Rhizoctonia spp., and Fungus Fusarium spp. Accompanying potato root rot disease and the pathogenicity test using radish seeds on water Agar (W.R.) culture media, all tested isolates achieved a significant reduction in radish seed plants compared with control treatment uncontaminated by any of the isolates of fungi, which recorded infection rate 0%. Keywords: Potato Root Rot, Diagnosis, Fungi, Rhizoctonia solani , Fusarium solani.

61 Determining in vivo and in vitro predatory activity of Duddingtonia flagrans and interaction with dietary coccidiostat

Abstract A nematode trapping fungus, Duddingtonia flagrans (Df), fed to sheep is useful for biocontrol of gastrointestinal nematodes on pasture. The objectives were: 1) validate methods to determine viability of a commercial source of Df before beginning research studies, and 2) examine the interaction between Df and dietary coccidiostat (as decoquinate). Methods: Study 1: In Jan 2023, weaned Katahdin lambs (97.3 ± 1.2 d of age; 19.5 ± 1.3 kg BW; packed cell volume of 26.5 ± 1.4 %), naturally infected with predominantly Haemonchus contortus (89%) and mean fecal egg count (FEC) of 6,860 ± 1,535 eggs/g, were randomly assigned to: 1) no Df (-g)/(FEC-g)×100]. Study 2: In Aug 2023, 7 mo old anthelmintic-treated ram lambs (FEC < 50 egg/s; 51.6 ± 3.9 kg in BW) were fed grain supplement daily with decoquinate (Deccox; +C) or without (-C; n = 3/diet) on d -7 to 1, and, within diet, administered 3 g BioWorma/d in 6 gel capsules (+DF; n = 2/diet) or not (-DF; n = 1) on d -1 and -2. Fecal samples were collected on d 0 (2 g/plate + 300 L3 of H. contortus) and d 1 (6 g/plate + 1000 L3) and placed on water agar plates (n = 3/lamb) at 25°C. Trapping structures were visually assessed after 24, 48, and 144 h (6 d), and cultures Baermannized on d 7 for L3 recovery. Relative reduction was (x̅-DF-C-x̅+DF+C/ x̅-DF-C)×100%. Data for variables collected one time were analyzed by Proc GLM (SAS), and for those collected serially, a Proc Mixed with repeated measures. A 2×2 factorial was used in Study 2 and included treatments, day and interaction in model. Results: Study 1: As expected, L3 recovery was reduced (P = 0.005) in DF lambs by 67-78% relative to control. Study 2: DF formed loops and traps around L3 by d 6 and reduced L3 recovery on both days (P < 0.001) without an interaction by decoquinate (P = 0.69). The L3 reduction relative to -DF-C was between 60.2-73.5%. The coccidiostat, decoquinate, can be added to the diet in the presence of BioWorma without antagonism. In conclusion, the in vitro studies allow detection of adverse issues with viability of Df and can reduce the number of animals needed for research studies.

Occurence of leaf spot on Narrow-head ragwort (Ligularia stenocephala) caused by Alternaria cinerariae in Korea.

Narrow-head ragwort (Ligularia stenocephala (Maxim.) Matsum. & Koidz.) has been used for medicinal purposes and a leafy vegetable in South Korea (Choi et al., 2007; Debnath et al., 2017). In May 2022, brown spots and blight were observed on the leaves in a farmer's field in Namwon (35°26'58.9"N, 127°28'55.9"E), Korea. About 80% of the plants in a 3000 m2 cultivation area were infected. To isolate the causal agent, small pieces (1 mm2) surface-sterilized (1% NaOCl for 1 min) symptomatic leaf tissues were put onto a water agar (WA) plate and incubated in the dark at 25℃. After five days of incubation, two isolates (FD00021, FD00022) were obtained from diseased leaves using a single spore isolation technique. Morphological characteristics were examined after seven days of incubation at 25℃ in the dark. Colonies were 51.6 to 65.3 mm in diameter, gray-green in the center, and ivory at the edge. Conidiophores were straight or curved and 10 - 34 × 4 - 5 μm. Conidia were solitary or two to four in a chain, long ellipsoid to obclavate, one to thirteen transverse septa, 52 - 169 ×14 - 34 μm, blunt-tapered beak variable in size 4 - 56 × 3 - 10 μm (n=75). The morphological and cultural characteristics of the isolates were consistent with that of Alternaria cinerariae (Nishikawa and Nakashima, 2015; Simmons, 2007). For molecular identification, genomic DNA was extracted from 5-day-old cultures using the Maxwell® RSC PureFood GMO and Authentication Kit (Promega). Five gene regions, including rDNA ITS, GPD, Alt a, RPB2, and EF1-α were amplified and sequenced using ITS1/ITS4, gpd1/gpd2, Alt-a1-for/ Alt-a1-rev, RPB2-5F2/RPB2-7cR, and EF1-728F/EF1-986R primer sets respectively (Wang et al., 2022; Garibaldi et al., 2022). The resulting sequences were deposited in GenBank with accession no. OP785152, OP785153 and OP832000 to OP832007). The concatenated genes (rDNA ITS, GPD, Alt a, RPB2, and EF1-α) sequence identity of the FD00021 and FD00022 against the reference strain A. cinerariae CBS 116495 is 99.92% (2572/2574) and 99.84% (2570/2574), respectively. Maximum Likelihood tree was inferred based on the concatenated sequences of the five gene regions using the Kimura 2-parameter model with 1,000 bootstrap replications. The phylogenetic tree showed that the present strains and A. cinerariae CBS 116495 fell into the same clade with high bootstrap support (100%). Based on morphological characteristics and molecular analysis, the isolates were identified as A. cinerariae. To confirm their pathogenicity, drops (70 μl) of conidial suspension (1×104 spores/ml) were applied on intact healthy leaves (3 leaves/plant) of plants (3 plants/isolate) that had been cultivated for one month after transplantation as seedlings. Controls were treated with sterile distilled water. The treated plants were covered with plastic boxes to maintain humidity around 90% and were maintained in an incubator at 25℃ in a 12-hour light-dark cycle. Symptoms appeared only on inoculated leaves after four days of inoculation, while controls remained asymptomatic. The A. cinerariae isolates were re-isolated from infected tissue of the inoculated leaves, thus fulfilling Koch's postulates. Alternaria cinerariae is an important plant pathogen that can cause leaf spot and blight on a variety of host plants including Cineraria spp. and Tussilago farfara (He et al. 2020). This is the first report on leaf spot on Narrow-head ragwort caused by A. cinerariae in the world. Leaf spot disease caused by Alternaria cinerariae is a significant threat to narrow-head ragwort agriculture in South Korea. Therefore, its control strategies are necessary for increasing productivity.

Factors affecting the germination of teliospores of Sporisorium scitamineum and the development of sugarcane smut in the field

AbstractTemperature and water potential are critical factors influencing the germination of Sporisorium scitamineum teliospores and subsequent crop infection. This paper reports on the effect of temperature, water potential and incubation time on S. scitamineum teliospore germination. A field trial was conducted to determine the influence of temperature and length of exposure to teliospores on smut incidence. A 200 μL aliquot of teliospore (1.5 × 106 teliospores/mL) was cultured on water agar (WA) amended by potassium chloride (KCl) or sucrose. The plates were incubated across a range of temperatures and incubation times. For the field trial, sugarcane setts were inoculated and incubated at different temperatures and for varying incubation periods. The results demonstrated that germination decreased with declining water potential with either KCl or sucrose. The greatest germination (95.8%) occurred on unamended WA incubated at 30°C for 6 h. Even at the lowest water potential of −45 bars and 30°C, 50% teliospores were still able to germinate. These results indicate that 30°C is the optimum temperature for teliospore germination and that S. scitamineum may still germinate in fairly dry conditions. However, high temperatures (40°C), greatly reduce S. scitamineum germination. These results are consistent with field trial outcomes, which showed that the highest disease incidence (40.4%) occurred when temperature of inoculation was maintained at 30°C. On the other hand, the incidence was significantly reduced to 5.7% at 35°C. The results suggested consistent conditions for teliospore germination and sugarcane bud infection, with a temperature of 30°C being optimal for both.

Impact of Foliar Fungicide Application on the Culturable Fungal Endophyte Community of Soybean Seed in the Midwest United States.

The purpose of our study was to determine whether the application of quinone outside inhibitor (QoI) and pyrazole-carboxamide fungicides as a tank mix would impact the endophyte community of soybean seed. Field trials during 2018 in Iowa, South Dakota, and Wisconsin, U.S.A., investigated the impact of a single combination fungicide spray at early pod set in soybeans. The composition of culturable endophytic fungi in mature soybean seed was assessed on three cultivars per state, with maturity groups (MGs) ranging from 1.1 to 4.7. An unusually wet 2018 season delayed harvest, which led to a high level of fungal growth in grain. The survey included 1,080 asymptomatic seeds that were disinfested and individually placed on 5-cm-diameter Petri plates of acidified water agar. The survey yielded 721 fungal isolates belonging to 24 putative species in seven genera; taxa were grouped into genera based on a combination of morphological and molecular evidence. The dominant genera encountered in the survey were Alternaria, Diaporthe, and Fusarium. The study showed that the fungicide treatment reduced the incidence of Fusarium in Wisconsin seed, increased the incidence of Diaporthe in seed from all states, and had no impact on the incidence of Alternaria. This is one of the first attempts to characterize the diversity of seed endophytes in soybean and the first to characterize the impacts of fungicide spraying on these endophyte communities across three states. Our study provides evidence that the impact of a fungicide spray on soybean seed endophyte communities may be influenced by site, weather, and cultivar maturity group.

First Report of Leaf Spot on Taraxacum mongolicum Caused by Alternaria solani in China.

Taraxacum mongolicum is a perennial herbaceous plant in the family Asteraceae, with a high edible and medicinal value and is widely planted in China. In August 2022, leaf spots were found on T. mongolicum in Tianjiazhai Town, Xining City, Qinghai Province, China (36°27'17.65″N, 101°47'19.65E, elevation: 2,408 m). The plants exhibited round or irregular brown spots, and the centers of some of the spots were gray (Fig. S1A). An investigation was performed over a hectare area, and the incidence of leaf spot reached 15%-30%, seriously affecting the quality and yield of T. mongolicum. Eleven T. mongolicum leaf spot samples were collected. To isolate the pathogenic fungus, approximately 0.5 cm×0.5 cm pieces of tissues were obtained using sterile scissors from the junction of infected and healthy tissues. The symptomatic leaves were surface-disinfected with 3% NaClO for 1.5 min and washed three times with sterile water. The disinfected pieces were dried and placed on water agar plates in an incubator for 2 days at 25°C. Subsequently, the leaf surface exhibited conidiophores and conidia. Eleven isolates were obtained by single spore isolation. The sparse aerial mycelia were dark grey to black brown in color on potato dextrose agar (PDA) (Fig. S2A), and produced dark, multi-septate conidia with 7-11 transverse septa and 1-2 longitudinal septa (Fig. S2C). Conidia with one or two beaks were long-ovoid, with an average length and width of 103.4 × 21.2 μm, and 80.7 × 3.9 μm of the beaks. One hundred and ten conidia were measured. The identification of 11 isolates was confirmed by multilocus sequence analyses of the internal transcribed spacer of ribosomal DNA (rDNA ITS) (White et al. 1990), and the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Xu et al. 2022), actin (ACT) (Yang et al. 2020), histone 3 (HIS3) (Zheng et al. 2015), translation elongation factor 1-α (TEF1-α) (Carbone. 1999), and the second largest subunit of RNA polymerase II (RPB2) (Liu et al. 1999) genes. The sequences of all the isolates were deposited in Genbank (NCBI Accession Nos. ITS: OR105029-OR105039, ACT: OR135220-OR135230, GAPDH: OR135231-OR135241, HIS3: OR122992-OR123002, TEF1-α: PP055972-PP055982, and RPB2: PP055983-PP055993), and the sequence similarity of ITS, ACT, GAPDH, HIS3，TEF1-α and RPB2 were 100%, 98%, 100%, 99%, 100%, and 99% to the sequences of Alternaria solani, respectively. Combined sequences of ITS, GAPDH, TEF1-α, and RPB2 genes were concatenated and a maximum parsimony tree was constructed with PAUP* v. 4.0 alpha. The results indicated that 11 isolates were clustered together with A. solani (Fig. S2D). Therefore, 11 isolates were identified as A. solani based on their morphological and molecular characteristics. Eleven isolates were inoculated on their host to perform Koch's postulates. The isolates were grown on PDA for six days. Healthy one month old T. mongolicum seedlings were planted in 10 cm flowerpots (Fig. S1B) or the seedlings were moved to Petri dish (Fig. S1C), and their leaves were inoculated with 5 mL of hyphae suspension by smearing method. In addition, seedlings of the same age were treated with sterile water to serve as the control. The inoculated seedlings were moved into an artificial climatic box at 25℃, relative humidity was 70%, with 12 h light/12 h dark condition. Totally 80 seedlings were inoculated with isolates and 15 were used as the control. After 7 days, similar symptoms were observed on the plants inoculated with isolates, while control plants did not produce symptoms. The assays were conducted three times. Furthermore, isolates were re-isolated from the symptomatic leaves, and the colonial morphology was the same as the original isolates (Fig S2 A and B). The recovered isolates were identified as A. solani by amplifying and sequencing a portion of the HIS3 gene. Alternaria solani has been previously reported to cause early blight of potato and other Solanum crops (van der Waals et al. 2004; Zheng et al. 2015). To our knowledge, this is the first report of A. solani causing leaf spot of T. mongolicum in China. This disease must be considered in management practices, and our finding provided a basis for disease prevention and management.