Potato Dextrose Agar Cultures Research Articles

First Report of Diaporthe tectonendophytica Causing Leaf Spot on Dalbergia odorifera in Guangxi, China.

Dalbergia odorifera T. Chen (Family: Fabaceae) is a national level II protected plant in China, with extremely high economic value and medical properties (Zhao et al. 2020). In June 2023, an unknown leaf spot was found in a garden land of Pingxiang city, Guangxi, China, and approximately 80% of the plants covered an area of 500 m2 displayed similar symptoms. The spots were grey to white, 4~6 mm in diameter (n=30) with black pycnida on the spots surface (Fig S1, A-D). Multiple disease spots were observed on a single leaf. The pycnida on the lesion were picked and mashed, to make a conidia suspension using sterile water. The conidial solution was then spread onto a potato dextrose agar (PDA) plate containing streptomycin, with 10 mg of streptomycin per 100 mL, and incubated for 3 days at 28°C with a 12 hour photoperiod. Three isolates (GXPX01, GXPX02 and GXPX03) were obtained by re-culturing the colonies on fresh PDA plates. The colony on PDA were white with aerial mycelia (Fig S1, E-F). Black conidiomata developed at 28°C with a 12 hour photoperiod in 20 days (Fig S1, G-H). Alpha conidia were 4.2~6.4 μm × 1.8~2.6 μm (average =5.1 × 2.3 μm, n = 30), mostly bi-guttulate, hyaline, ellipsoid, apex bluntly rounded, base obtuse to subtruncate, smooth (Fig S1, I). Beta conidia were 15.1~33.5 μm × 1~1.8 μm (average = 24.5 × 1.5 μm, n = 30), filiform, hyaline, curved or hamate, aseptate, base subtruncate (Fig S1, J). Morphological characteristics of the three isolates matched those of Diaporthe spp.(Gomes et al. 2013). The rDNA internal transcribed spacer (ITS) region, the translation elongation factor 1-α (TEF1), the calmodulin (CAL), the histone H3 (HIS) and the β-tubulin (TUB2) genes of the three isolates were amplified using the primer pairs ITS4/ITS5, EF1-728F/EF1-986R, CAL-228F/CAL2Rd, CYLH3F/H3-1B, and T1 /CYLTUB1R, respectively (Crous et al. 2004, Sun et al. 2021). The sequences were all deposited in GenBank (accession numbers OR437511 to OR437513 for ITS, OR454965 to OR454967 for TEF1, OR454968 to OR454970 for CAL, OR454971 to OR454973 for TUB2, OR454974 to OR454976 for H3). Sequences had 98.36% to 100% homology with the corresponding sequences of known Diaporthe tectonendophytica strains MFLUCC 13-0471 in the NCBI database. Phylogenetic analysis was based on combined ITS, TEF1, TUB2 and CAL sequences data using MEGA 11 software to construct phylogenetic tree with Maximum Likelihood (Doilom et al. 2017). In the phylogenetic tree, the combined sequences attributed the three isolates to the D. tectonendophytica (Fig S2). The pathogenicity was tested on leaves of 1.5-year-old D. odorifera seedlings. Three leaves were wounded with a sterile needle and individually inoculated with a 5 mm mycelial disk of PDA culture from each isolate. Sterile PDA disks inoculated leaves as a control. The test was repeated three times. The inoculated plants were placed in a greenhouse at 25℃ and 90% humidity, with a photoperiod of 12 hours. Five days after inoculation, necrotic lesions appeared on inoculated leaves and symptoms from all three isolates were the same as those form natural infections ( Fig S1, K-N), whereas all the control remained symptomless (Fig S1, P). The pathogen was reisolated from the inoculated leaves and again identified as D. tectonendophytica, with the same methodology used for the initial identification. D. tectonendophytica was reported to cause plant diseases, such as stem gray blight of red-fleshed dragon fruit (Hylocereus polyrhizus) (Rahim et al. 2021), leaf spots disease on Elaeagnus conferta and Pometia pinnata (Sun et al. 2021). To our knowledge, this is the first report of D. ctonendophytica causing leaf spot disease on D. odorifera.

Zinc Oxide:Gold Nanoparticles (ZnO:Au NPs) Exhibited Antifungal Efficacy Against Aspergillus niger and Aspergillus candidus

Fungal pathogens are a major health issue that threatens the era of antifungal drugs commonly used in the treatment of infections. An effective approach of biosynthetic nanoparticles can be used as antifungal agents owing to their intrinsic features such as their simplicity, non-toxic, and physicochemical properties. Therefore, this study was aimed to molecularly ascertain Aspergillus species known to cause aspergillosis and investigate the potency of zinc oxide:gold nanoparticles (ZnO:Au NPs) against the fungal pathogens. Two Aspergillus strains retrieved with potato dextrose agar (PDA) culture media from commercial food products in South Africa were molecularly identified using calmodulin (CaM) gene region. DNA sequence phylogeny of the gene showed that the strains were A. niger and A. candidus. ZnO:Au (1%) NPs were synthesised and characterised using X-ray diffraction (XRD), scanning electron microscopy (SEM), and X-ray photoelectron spectroscopy (XPS). Two distinct peak plasmon bands for ZnO and ZnO:Au NPs were 390 nm and 565 nm, respectively. FE-SEM images demonstrated the presence of Au on the surface of ZnO nanoparticles in the ZnO:Au nanocomposites. The ZnO:Au NPs antifungal activity of 10 µg/mL and 50 µg/mL concentrations were evaluated against the two Aspergillus spp. ZnO:Au NPs at 50 µg/mL exhibited a maximum antifungal activity against A. candidus and A. niger, with zones of inhibition (ZoI) of 31.2 ± 0.15 mm and 25.0 ± 0.06, respectively. When the ZoI was observed by SEM, major morphological damages on the conidia were observed for both strains, indicating that the antifungal activity may have been enhanced by the ZnO:Au NPs. Therefore, due to these outstanding properties, ZnO:Au NPs can be utilised as potential antifungal agents to inhibit the proliferation of fungal pathogens.

First Report of Fusarium proliferatum Causing Basal Rot and Dry Rot on Onion Bulbs (Allium cepa) in Spain.

Spain is the second largest onion-producing country in Europe with 1,465,430 tons and an export value of 584 million € in 2021 (MAPA 2022). In summer 2022 rot bulb symptoms were observed in five commercial fields and during the storage of cultivars 'Orlenda', 'Veleta', 'Mallory', 'Citation' and 'Pantano' from La Roda in Albacete (Castilla-La Mancha, Spain). Approximately 20% of sampled bulbs (113 bulbs analyzed) were affected with dry scales showing brown to dark brown rot on the top and basal plate of the onion bulbs. Occasionally, white to light pink fungal mycelium was observed between rotten scales and the plate basal. Sections of dry scales (5-10 mm) of the apical and basal plate were cut and placed on potato dextrose agar (PDA) and Komada medium (Komada 1975). From 5-day-old cultures typical white to light pink mycelium with microconidia in chains formed on polyphialides and macroconidia resembling Fusarium proliferatum (Nelson et al. 1983). To confirm the pathogen identity, partial translation elongation factor 1-alpha (TEF1) and RNA polymerase II subunit 2 (RPB2) genes were amplified and sequenced using primers reported in O´Donnell et al. (1998) and Samuels et al. (2002) for TEF1 and Liu et al. (1999) for RPB2. In BLAST analyses, the sequences showed 100% identity to the corresponding region of F. proliferatum (KP964908 and JF740801). Sequences were submitted to GenBank, and registered accession numbers are OR061014-16 for TEF1 and OR061017-19 for RPB2. Pathogenicity tests were conducted by inoculating healthy onion bulbs (five replicates per treatment) on the apical and basal plate by placing a 7-day old mycelial plug (10 mm diameter) from PDA cultures. Two onion cultivars ('Pandero' by Nunhems USA and 'Mallory' by Bejo The Netherlands) were inoculated separately with three isolates (PRO1, PRO9, PRO12). Control bulbs were inoculated with sterile PDA. The experiment was carried out twice. All bulbs were placed in a moist chamber and incubated at 25°C in the dark. After 15 days, bulbs inoculated with mycelial plugs showed similar symptoms to those of the original diseased bulbs. Browning dry rot was observed on the apical and basal plate of bulbs. When bulbs were cut longitudinally inner progressing rot was observed. Control bulbs remained symptomless. In both experiments, F. proliferatum was successfully re-isolated and morphologically confirmed from symptomatic bulbs to fulfill Koch's postulates. These results confirmed that isolates PRO1, PRO9 and PRO12 were the pathogen causing basal and dry rot on onion bulbs. This pathogen has recently been identified in China on Allium cepa L. var. agrogatum (Liu et al. 2022) and Idaho on onion (Beck et al. 2020) and could become a serious threat to onion production in Spain, reducing the quality and yield of onion.

First description of adenosine production by Gnomoniopsis smithogilvyi, causal agent of chestnut brown rot

Gnomoniopsis smithogilvyi (Gnomoniaceae, Diaporthales) is the main causal agent of chestnut brown rot on sweet chestnut worldwide. The rotting of nuts leads to alterations in the organoleptic qualities and decreased fruit production, resulting in significant economic losses. In 2021, there was an important outbreak of chestnut rot in southern Galicia (Spanish northwest). The profile of secondary metabolites from G. smithogilvyi was studied, especially to determine its capability for producing mycotoxins, as happens with other rotting fungi, due to the possible consequences on the safety of chestnut consumption. Secondary metabolites produced by isolates of G. smithogilvyi growing in potato dextrose agar (PDA) medium were identified using liquid chromatography coupled with high-resolution mass spectrometry. Three metabolites with interesting pharmacological and phyto-toxicological properties were identified based on their exact mass and fragmentation patterns, namely adenosine, oxasetin, and phytosphingosine. The capacity of G. smithogilvyi to produce adenosine in PDA cultures was assessed, finding concentrations ranging from 176 to 834 µg/kg. Similarly, the production of mycotoxins was ruled out, indicating that the consumption of chestnuts with necrotic lesions does not pose a health risk to the consumer in terms of mycotoxins.

First report of Phaeosphaeria caricicola causing leaf spot on Belamcanda chinensis in China.

Belamcanda chinensis (L.) Redouté, a member of the Iridaceae family, is globally well-known for its medicinal value as clearing away heat, detoxifying, detumescence and pain (Qin 2000). In 2021, spots were observed on 40% B. chinensis leaves and about 28 disease index in Wanzhou District (30°32'N; 108°22'E) of Chongqing. Initial symptoms appeared as circular yellow white, sunken spots lesions, and then expanded into irregular lesions, the center of the spots was beige, external layer was light brown and surrounded by yellow halo. Symptomatic leaf tissues (5 × 5 mm) were cut from the infected margin, surface sterilized with 75% ethanol for 1 min, washed with 3% sodium hypochlorite for 3 min, rinsed three times with sterile water, placed on potato dextrose agar (PDA) medium incubated at 25°C for 7 days in the dark, forty isolates with similar morphology were obtained. Three isolates (SG9、SG20 and SG33) was selected for subsequent research. Colonies color changed from beige to light brown color after 14 days on PDA medium. Fungal colonies transformed from beige to brown at the edges after 28 days and light brown on top. Ascomata dark brown, ellipsoidal to globose 116.6 to 253.3 × 89.6 to 172.6 μm in diamensions. Asci stipitate, cylindrical with obtuse ends, and 69.1 to 114.7 × 10.2 to 24.1 μm (n = 30) in size, with eight overlapping linearly biseriate ascospores. Ascospores brown, narrowly fusiform, straight or slightly curved with three transversely septate, slightly constricted at septa, and 9.7 to 12.6 × 27.6 to 32.6 μm (n = 30). These characteristics are consistent with Phaeosphaeria sp. reported by Quaedvlieg et al in 2013. DNA was extracted from representative isolates. The internal transcribed spacer (ITS) region, the large subunit rDNA (LSU), the small subunit rDNA (SSU) and the RNA polymerase II second largest subunit (RPB2) genes were amplified for Polymerase chain reaction (PCR) by used ITS1/ITS4, LR5/LROR, NS1/NS4, and RPB2-5f2/RPB2-7cr primers (White et al. 1990; Vilgalys et al. 1990; Qi M W. et al. 2008; De G. J. et al. 1992). The sequences were submitted to NCBI GenBank: SG-G9 (ITS, OR701701; LSU, OR701699; SSU, OR701700; RPB2, OR738464); SG-G20 (ITS, OQ748032; LSU, OQ780728; SSU, OQ780723; RPB2, OQ779979); SG-G33 (ITS, OQ748033; LSU, OQ780729; SSU, OQ780722; RPB2, OQ779980). A phylogenetic analysis revealed a 99% similarity to the Phaeosphaeria caricicola CBS 603.86 (ITS, KF251182; LSU, GQ387590; SSU, GQ387529; RPB2, KF252189) sequences. Mycelial agar plugs (5-mm diameter) from a 7-day-old PDA culture of a fungal isolate were placed onto pinpricked leaves of three two-year-old B. chinensis plants. While the sterile PDA plugs inoculated in pinpricked leaves of B. chinensis as controls. Inoculated plants were placed in a greenhouse at 25°C and remained 95±1% relative humidity. The inoculated leaves of treatment developed symptoms after 20 days, whereas no symptoms occurred on controls, fulfilling Koch's postulates. The experiments were repeated three times. The fungus was re-isolated and was identical to original isolate by morphologically and molecularly. As far as we know, P. caricicola can cause diseases on carex plants and has been found in Switzerland. This is the first report of P. caricicola causing leaf spot on B. chinensis in China. Along with recording the occurrence of this disease, plant disease management strategies need to be established to reduce losses.

Essential oils from Mediterranean aromatic plants Artemisia herba-alba, Artemisia absinthium and Mentha longifolia to improve blueberry shelf life

The Mediterranean Basin is particularly rich in aromatic plants with essential oils whose composition and properties are still to be investigated. The aims of this work were to: investigate the chemical composition of essential oils from three typical Spanish aromatic species: Artemisia herba-alba, Artemisia absinthium and Mentha longifolia; evaluate the antifungal potential of essential oils against seven plant pathogenic and postharvest fungi; analyse the effectiveness of the Mentha longifolia essential oil in extending the blueberry fruit shelf life. The main compounds for the Artemisia herba-alba essential oil were camphor (29.8%), trans-thujone (22%), 1,8-cineole (14.5%) and cis-thujone (10.8%). In the A. absithium essential oil, they were cis-cryshanthemyl acetate (35.4%), cis-β-epoxiocimene (36%) and trans-β-epoxiocimene (10.4%). α-Terpineol acetate (32.6%), pulegone (14.2%) and 8-acetoxy carvotanacetone (10.3%) were the most important molecules in the obtained composition of the Mentha longifolia essential oil. Different bioassays were performed at the 300 μg/mL and 600 μg/mL doses using the distinct essential oils in potato dextrose agar culture medium. The Artemisia herba-alba and Artemisia absinthium essential oils at 300 μg/mL were not very effective. The Mentha longifolia essential oil strongly inhibited mycelial growth at the two tested doses, but it totally inhibited the growth of Curvularia hawaiiensis, Fusarium nygamai and Verticilliun dahliae at 600 μg/mL. The blueberries coated with the Mentha longifolia essential oil solution obtained a statistically higher percentage of healthy fruit than the control. They could be considered an ecofriendly alternative for postharvest treatment, and for maintaining the quality and turgidity of these berries during storage.

First Report of Colletotrichum aenigma causing Anthracnose on Pecan in China.

Pecan (Carya illinoinensis) is an important economic forest crops widely cultivated in China. From June to September in both 2021 and 2022, severe leaf disease resembling anthracnose was observed in 6.6-ha pecan orchard in Jintan (31°42'23.84″ N, 119°21'22.90″ E), Jiangsu Province. The disease severity was about 15 to 25% with 5 to 12% incidence on 100 surveyed trees of the orchard in 2022. Symptoms initially appeared as small gray-bark sunken lesions, which gradually developed to big sunken lesions with brown edges and irregular-shaped. Small fragments (4 × 4 mm) from the necrotic borders of infected leaves were surfaced sterilized, plated on potato dextrose agar (PDA) and then incubated in darkness at 25°C for 3 days. Pure cultures were obtained by monosporic isolation. Twenty-one isolates with similar characteristics were obtained from the infected leaves (isolation frequency about 90%). The upper side of colonies on the PDA plates was milky, and the reverse side was pale yellow at the center and pale white at the margin. After 10 days of growth on the PDA medium, these isolates produced spores separately. . Through electron microscopic observation, conidia were smooth walled, hyaline, aseptate, guttulate, cylindrical with rounded ends with 15 to 20.5 × 5.3 to 6.7 μm (mean 18.5 × 5.8 μm, n = 50) in size. These morphological characteristics were similar to those of the species of Colletotrichumspp (Weir et al. 2012, Fu et al. 2019). To further identify the isolates, the regions of internal transcribed spacer (ITS), actin (ACT), calmodulin (CAL), chitin synthase (CHSI), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and beta-tubulin 2 (TUB2) loci of the three representative isolates (JSJT-1, JSJT-2, and JSJT-3) were amplified and sequenced with the primer pairs ITS-1F/ITS-4, ACT-512F/ACT-783R, CL1/CL2A, CHS-79F/CHS-345R, GDF/GDR and T1/T2 primers, respectively (Weir et al. 2012). Sequences of them were deposited in GenBank under nos. OR214960 to OR214962 (ITS), OR228543 to OR228545 (ACT)，OR228546 to OR228548 (CAL), OR228549 to OR228551 (CHSI), OR228552 to OR228554 (GAPDH), and OR228555 to OR228557 (TUB2). Multilocus phylogenetic analysis revealed that the three isolates and C. aenigma were clustered in the same clade. Based on the results of morphological and molecular analysis, these isolates were identified as C. aenigma. The pathogenicity of three isolates was tested on leaves of pecan seedlings. Suspensions of conidia were obtained by scraping the surface of a 10-day-old sporulated petri dish PDA cultures into sterile water. Suspensions were adjusted to a density of 2 × 106 conidia/ml with a hemocytometer.The conidial suspension of each isolate was sprayed evenly on the surface of leaves from three healthy pecan seedlings. Sterilized distilled water was used for negative controls. The pathogenicity experiment was repeated three times. Finally, all inoculated plants were kept in a light-incubator at 28°C under 100% relative humidity and 12 h photoperiod. Two weeks after inoculation, the inoculated plants developed symptoms similar to those of the original diseased plants, while controls remained asymptomatic. C. aenigma were re-isolated from from inoculated leaves. C. aenigma has been reported as the causal agent of anthracnose on several economically important plants, such as grape ( Kim et al. 2021), tree peonies (Wang et al.2023), chili (Diao et al. 2017), and pear (Fu et al. 2019), but this is the first report of C. aenigma causing anthracnose on pecan in China. Identification of C. aenigma as a pathogen of pecan is important for implementing control management strategies for pecan disease.

First Report of Panicle Wilting on Rice (Oryza sativa L.) Caused by Fusarium kyushuense in Heilongjiang Province, China.

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.

Evaluation of Genetic Diversity and Agronomic Traits of Germplasm Resources of Stropharia rugosoannulata

China possesses abundant wild germplasm resources and a wide range of cultivated strains of Stropharia rugosoannulata. However, issues such as genetic diversity and unclear genetic relationships have had an impact on the classification and preservation of germplasm resources, the breeding of new varieties, and the promotion of superior strains. There is an urgent need for genetic diversity analysis and assessment of germplasm resources. In this study, we conducted whole-genome resequencing of 50 cultivated and wild strains collected from various regions across the country. After applying a series of filtering parameters, we obtained 888,536 high-quality Single Nucleotide Polymorphism (SNP) markers. Using these SNP markers, we performed principal component analysis, population structure analysis, and phylogenetic clusters analysis on the 50 strains. Most cultivated strains exhibited high genetic similarity, while significant genetic diversity was observed among wild strains. Based on factors such as marker distribution throughout the entire genome and marker quality, we selected 358 core SNP markers to construct SNP fingerprints. Two-dimensional barcodes were generated for each strain to enable specific identification. Additionally, the mycelial growth rate of strains was evaluated on Potato Dextrose Agar (PDA) and substrate culture media. We also assessed their lignin degradation capability using guaiacol agar plates assay. It was observed that the mycelial growth rate on PDA and substrate culture medium exhibited a significant correlation with the diameter of the mycelial colony on guaiacol agar medium. Additionally, the correlation between the mycelial growth rate on the substrate medium and that on the guaiacol agar plate was stronger than that on PDA medium. This study provided molecular-level identification and assessment of germplasm resources. It clarified the genetic relationships among strains and the characteristics of mycelium growth-related agronomic traits of each strain. This research contributed to the enrichment and development of utilizable germplasm resources and breeding materials for S. rugosoannulata, offering a scientific basis for further research.

First Report of Leaf Spot Caused by Arthrinium arundinis on Amaranthus hybridus in Korea.

Amaranthus hybridus (=A. patulus), often called green amaranth, is an annual herbaceous plant of the Amaranthaceae. This plant is considered a harmful weed in the agricultural context of North America and has expanded its distribution to Asia and Europe. In Korea, it has become a problematic invasive issue, leading to economic losses due to reduced crop yields and rising weed management costs (Park et al., 2014), although its seeds and young leaves are edible and frequently consumed. In October 2020, we observed leaf spot symptoms on A. hybridus plants that were growing within perilla farms (Perilla frutescens var. japonica) located in Damyang (35°14'07"N, 126°59'40"E), Korea, with a disease incidence of 20 to 30% of the inspected plants. The initial signs appeared as grey to brown dots on the leaves, which gradually expanded into irregular, brown patches with a diameter of 2-3 cm. A single spore was isolated from the diseased leaf under a dissecting microscope, placed onto a 2% water agar plate, and incubated in darkness at 25°C for three days. Pure cultures were obtained by transferring single hyphal tips onto potato dextrose agar (PDA) plates. Five single-spore isolates were the same in the cultural and morphological examination, and a representative isolate (P309) was preserved at the Korean Agricultural Culture Collection (KACC49813), Korea. Colonies appeared light gray to white with regular margins and reached 4 to 5 cm in diameter after a week. After two weeks, black patches of spores were often visible in the aerial mycelia. Conidiophores were brown to pale brown, often branched, thick-walled, and measured 6.8 × 2.7 µm (n = 30). Conidia were single-celled, dark brown, globose to ellipsoid, and measured 6.8 × 5.0 µm (n = 50), with a ratio of length/width of 1.1 to 1.6 (n = 50). These morphological characteristics matched those of Arthrinium arundinis (Crous et al., 2013). For molecular identification, genomic DNA was extracted from conidia and mycelia on two-week-old PDA culture of the KACC49813. PCR was performed for the internal transcribed spacer (ITS) (primers ITS1/ITS4, White et al. 1990), the large subunit (LSU) rDNA (primers LROR/LR5, Vilgalys et al. 1990), the beta-tubulin gene (TUB) (primers T1/Bt-2b, O'Donnell and Cigelnik 1997), and the translation elongation factor 1-alpha (TEF) (primers EF1-728F/EF-2, Crous et al. 2013). A BLASTn search of the resulting sequences of ITS (560 bp; OL744431), LSU (881 bp; OL744432), TUB (790 bp; PP084934), and TEF (445 bp; PP084935) revealed 100 % similarity (e-value=0.0, coverage=100%) to previously reported sequences of Arthrinium arundinis (e.g. MF627422 for ITS, KF144930 for LSU, KF144973 for TUB, and KY705146 for TEF), confirming the identity of the Korean isolate. Pathogenicity assays were performed twice by spraying 1 ml of a conidial suspension (1.1 × 104 conidia per mL) onto the leaf surface of sixteen healthy A. hybridus plants. Sixteen control plants were sprayed with sterile water. All plants were kept in a growth chamber at 80% relative humidity and 23 °C with a 12-h light/dark cycle. Three weeks after the inoculation, initial symptoms mirroring the aforementioned ones appeared, while the control plants remained symptomless. Fungal isolates were successfully re-isolated from the inoculated leaves, and their identity as A. arundinis was confirmed by DNA sequencing, thus fulfilling Koch's postulates. To our knowledge, this is the first report of leaf spot caused by A. arundinis on Amaranthus hybridus, not only in Korea but globally. Arthrinium arundinis has also been reported as a plant pathogen on some agricultural crops (Ji et al. 2020; Liao et al. 2022; Farr and Rossman 2023), suggesting its polyphagous behavior. Then, this pathogen could represent a potential risk to the cultivation of edible amaranth in Korea and other crops where Amaranthus species are spread as weeds. For this reason, continuous monitoring is necessary to assess the impact of this fungus on Amaranthus and other crops.

First Report of Botrytis cinerea Causing Gray Mold on Tea (Camellia sinensis) in China.

Tea (Camellia sinensis (L.) O. Kuntze) is an important economic crop cultivated in China. In March 2019, leaf blight symptoms were observed on tea trees (C. sinensis cv. Fuding-dabaicha) in Enshi City (30º02'66" S, 109º01'56" W), Hubei Province (Fig. 1A). The disease occurred on the senescent flowers and mature leaves. The average disease incidence was approximately 10% of plants in the surveyed 30 ha tea garden. Initial symptoms consisted of brown lesions, which expanded and became covered with gray mold, followed by abscission. To identify the pathogen, 20 symptomatic leaves were collected and 0.5 cm2 diseased leaf pieces were excised and surface sterilized by immersion in 1.5% sodium hypochlorite (NaOCl) for 2 min, and rinsed three times in sterilized distilled water. The leaf pieces were allowed to dry, placed on potato dextrose agar (PDA) plates, and incubated at 20°C under 12h fluorescent light. The fungus formed gray to grayish brown colonies (Fig. 1B) and produced conidia after 1 week. The isolate was purified by single spore isolation. The conidia were one-celled, ellipsoid or ovoid, almost colorless, with a size range of 5.3 to 10.24 × 5.2 to 8.1 μm (n = 50) (Fig. 1C). The sclerotia produced in culture were black, round or irregular in shape and 1.3 to 2.2 × 2.0 to 3.3 mm (average 1.8 × 2.3 mm) in size. Morphological characteristics of these isolates matched the description of Botrytis spp. (Hong et al. 2001). Representative isolate LCHM was selected for molecular identification based on DNA sequencing of the ITS region of rDNA and three nuclear protein-coding genes (G3PDH, HSP60 and RPB2) (Staats et al. 2005). BLAST analysis showed that isolate LCHM (GenBank Acc. Nos. MN448502, MN448500, MN433708 and MN448501 for ITS, G3PDH, HSP60 and RPB2, respectively) shared 99 to 100% identity with B. cinerea (GenBank Acc. Nos. MH316147.1, MG846500.1, MG846504.1 and MG846510.1, respectively), which suggested that isolate LCHM belongs to B. cinerea. This identification was further confirmed by phylogenetic analysis based on combined DNA sequence data of G3PDH, HSP60 and RPB2 (Fig. 2). For the pathogenicity test, needle-wounded, attached leaves on 1-year-old C. sinensis cv. Fuding-dabaicha plants were inoculated with mycelial plugs (6 mm diameter from 2-day-old PDA cultures) or a conidial suspension (20 μL, 1×106 conidia/ml in half strength potato dextrose broth) of isolate LCHM on three plants per treatment (six leaves). The control treatments were also wounded, but only treated with agar plugs or half strength PDB. All inoculated plants and controls were incubated in a growth chamber (20°C, 90 ± 10% RH). Leaves inoculated with mycelial plugs, whether wounded or not, showed brown necrotic lesions around the agar plugs after 2 days (Fig. 1D, E), whereas conidial inoculations of wound sites showed necrotic lesions after 6 days (Fig. 1 F, G). Non-wounded leaves inoculated with conidia and all the control treatments remained symptomless. B. cinerea was reisolated from the inoculated leaves and isolates were morphologically similar to the original cultures. Gray mold of C. sinensis caused by B. cinerea has been recorded in Japan, Turkey, and Brazil (Hamaya 1981, Aziz and Harun 2010, Pereira and Mio, 2020). To our knowledge, this is the first report of gray mold caused by B. cinerea on C. sinensis in China. B. cinerea may cause economic losses of tea, therefore, more surveys in other tea-growing regions should be done to specifically search for this disease.

Antifungal Activity of Some Inorganic Salts on the Development of Fusarium spp., Causal Agents of Potato Tuber Dry Rot

Potato dry rot caused by Fusarium spp. is one of the main fungal diseases hampering the production and marketing of potato tubers in Cameroon. This study was carried out in order to improve the health status of potato tubers and their preservation. To achieve this objective, Fusarium spp. were isolated on the Potato Dextrose Agar (PDA) culture medium and identified on the basis of morphological and microscopic characteristics using reference documents. Antifungal activity of six inorganic salts was evaluated in vitro on the development of Fusarium spp. and on potato tubers artificially infected with these fungal species. The results showed that all the inorganic salts had a depressant effect on the development of Fusarium spp. Copper sulfate inhibited at 100%, the radial growth and sporulation of Fusarium spp. at all concentrations. Sodium metabisulfite and sodium carbonate inhibited at 100%, the growth and sporulation of all fungal species from a concentration of 6.4 mg/ml. No lesions were observed on inoculated potato tubers when they were treated with copper sulfate. These results suggest the implementation of a strategy to control dry rot of potato tubers through the use of inorganic salts, in order to reduce the losses caused by Fusarium spp.

First report of Leptosphaeria biglobosa 'brassicae' causing leaf spots in Eutrema japonicum in Yunnan, China.

Wasabi (Eutrema japonicum) is a root vegetable that is cultivated at large scales in southwestern China. In November 2021, approximately 40% of plants in a forested plantation in Dadishui, Yunnan Province, China (25.47°N, 103.22°E), showed leaf spot symptoms. The early symptoms were small black spots that gradually expanded into irregular brown to black lesions (0.5-1.5 cm), which were restricted by leaf veins. Yellow halos were observed at the outer edges of necrotic lesions. To identify the causal agent, we collected 20 diseased leaves and obtained fungal isolates from symptomatic leaf tissues. Following surface sterilization with 75% ethanol for 30 s, the tissues were cultured on potato dextrose agar (PDA) plates and incubated at 25°C under a 12 h light/12 h dark light cycle. After 7 days of incubation, a total of 12 isolates were obtained through single-spore culture. All isolates had similar colony morphology, and produced fluffy white mycelia and yellow pigment after 1 week of PDA culture at 25°C, and blackish- brown mycelium, tan pigment, and conidia after 2 weeks. The conidia were hyaline and cylindrical, with an average size of 4.6 μm × 2.2 μm. These morphological characteristics similar to the description of Leptosphaeria biglobosa (Shoemaker et. al, 2001) and Leptosphaeria maculans (Vincenot et al. 2008). Genomic DNA was extracted from mycelium of isolate SK-1, which was harvested from 10-day-old PDA culture using a FAST plant genomic DNA Extraction Kit (Biomed, China), following the manufacturer's instructions. The species-specific primers LbigF, LmacF, and LmacR (Liu et al. 2006) were used for identification via polymerase chain reaction (PCR). A 444-bp fragment characteristic of L. biglobosa 'brassicae' (Lbb), and a 330-bp of L. maculans 'brassicae' (Lmb) were amplified, respectively. Internal transcribed spacer (ITS) sequences (592 bp), part of the 5' end of beta-tubulin (968 bp), and actin (899 bp) were also amplified using the primers ITS1/ITS4, BT1/BT2, and ACTF/ACTR (Vincenot et al. 2008), respectively. PCR was performed in a volume of 25 μL containing 12.5 μL 2 × T5 Super PCR Mix (Tsingke Biotech, Beijing, China), 1 μL 10 μM primer (Tsingke Biotech), 1 μL DNA template, and an aliquot of sterile water to attain the total volume. The thermal cycler settings were 5 min at 98°C; 35 cycles of 10 s at 98°C, 10 s at 58°C, and 30 s at 72°C; and extension for 2 min at 72°C. The ITS sequence of isolate SK-1 (GenBank accession no. OQ216838), the partial β-tubulin gene sequence (OQ241183), and the actin gene sequence (OQ241184) indicated 100% query cover and 100% identity with L. biglobosa (DQ458906), Lbb strain B3.6 (AY748995), and Lbb strain 2379-4 (AY748949), respectively. Phylogenetic analysis (King et al. 2022) also identified of isolate SK-1 as Lbb. To determinate its pathogenicity, isolate SK-1 was grown on PDA incubated at 28°C for 2 weeks, and conidial suspensions were prepared at a concentration of 106 conidia/mL. Then, 15 leaves of 4-month-old E. japonicum seedlings were needle-wounded on the front and inoculated by syringe injection of 10 μL of the appropriate conidial suspension. We used 10 μL of the sterilized distilled water as the control under forest growth conditions. All inoculation sites were covered with cotton strips and moistened with 1.0 mL sterile water to maintain humidity. After 12 days of incubation, the leaves developed symptoms similar to those observed in the field, and the fungus was reisolated from diseased leaves, whereas the controls remained healthy. Based on these results, we identified L. biglobosa 'brassicae' as the causal agent of leaf spot on E. japonicum in China. This fungus has been reported to cause blackleg in many Brassica crops in China such as Brassica napus (Fitt et al. 2006), Brassica oleracea (Zhou et al. 2019), B. juncea var. tumida (Deng et al. 2020), Brassica rapa subsp. pekinensis (Yu et al. 2021). To the best of our knowledge, this is the first report of L. biglobosa causing leaf spots in E. japonicum in China. Our data provide a basis for disease management in E. japonicum production in China.

First Report of Cladosporium ramotenellum Causing Sooty Spot on Postharvest Clementines in the United States.

In 2022, post-harvest symptoms of black spots were observed at an incidence of 2-5% on easy peeling clementines (citrus × clementina) in a fresh fruit market in Phoenix metropolitan area, Arizona. Black lesions on the fruit rind were superficial, circular, dry, and firm with gray sporulation. Gray or black aerial mycelium was also noticed on top of the black spots. Black spots were distributed over the entire fruit surface without any regular patterns. Maceration of rind and flesh was also noticed under refrigerated storage conditions. Two isolates were obtained (S13 and S14) and cultured on potato dextrose agar (PDA) at 25oC for 14 days. The colony color and texture of both isolates were identical on PDA: olivaceous black with abundant sporulation, margin entire edge to slightly undulate. Conidia were 0-1 septate and globose or ellipsoid with sizes ranging from 4-10 × 2-4 μm (n = 15). Ramoconida were also 0-1 septate and ellipsoid to cylindrical with sizes ranging from 15-30 × 2-5 μm (n = 15). Conidiophore had a width of 3-6 μm (n = 15). Isolates were identified as Cladosporium ramotenellum based on these morphological features (Bensch et al. 2012). The IDs of these two isolates were further confirmed by genomic DNA isolation, PCR amplification and sequencing of the ITS1-5S-ITS2-28S region of rDNA (V9G/LR5 de Hoog and Gerrits van den 1998, primers ITS4/ITS5 White et al.), actin gene (ACT-512F/ACT-783R, Carbone and Kohn 1999), and elongation factor 1α gene (EF1-1018F/EF1-1620R, Stielow 2015). Sequences of the two isolates were identical and thus only one sequence of each gene was deposited in the GenBank. A BLASTn search of actin sequence (233-bp, OQ185511) revealed 99.1% match with ex-type sequence EF 679538 (strain CBS:121628) of C. ramotenellum holotype (query coverage: 97%). BLASTn analysis of a portion of EF-1α gene (579-bp, OQ185512) revealed more than 99.7% similarity with sequences KU933429 (ATCC strain 16022) and MT881827 (strain 18EPLE003) of C. ramotenellum (query coverage: 100%). The ITS sequence (1519-bp, OQ236707) was identical to the ITS sequences of C. ramotenellum strains in easy peeler mandarins from Peru (Murciano et al. 2021). Pathogenicity tests were carried out twice on fresh easy-peeling clementine fruit. The inocula (1 x 105 spores/ml) were prepared in sterile distilled water containing 0.1% Tween 20 (TW) by mixing the conidial suspensions of two isolates from 7-day-old PDA cultures. Ten fruit were washed, surface sanitized with 70% ethanol, and wound-inoculated by immersing five fruit in spore suspension for 1 min. Five control fruit were wound-inoculated with TW. Inoculated and control fruit were stored in separate zip-lock bags for one week. Black spots resembling those observed on naturally infected fruit were present on inoculated fruit, while control fruit remained symptomless. C. ramotenellum was reisolated and was morphologically identical to the original isolates, thus completing Koch's postulates. C. ramotenellum has been reported as a fungal pathogen causing sooty spots on easy peeler mandarins from Peru (Murciano et al. 2021). To our knowledge, this is the first report of C. ramotenellum causing postharvest sooty spot and decay on clementines in the United States. Results show that infected fruit is potentially a pathogen source for long-distance dispersal. This occurrence was communicated to the state regulatory agencies for regulatory actions on imports of citrus fruit from countries with the occurrence of C. ramotenellum.

First report of Nalanthamala vermoesenii causing pink rot of Phoenix canariensis in Mexico.

In Mexico City, the Canary Island date palm (Phoenix canariensis Chabaud) is an important plant forming part of its landscape identity. In February 2022, pink rot disease symptoms were observed on 16 P. canariensis plants in Mexico City (19°25'43.98"N, 99° 9'49.41"W). The incidence was 27%, while the severity 12%. External symptoms included necrotic lesions that advanced from the petiole towards the rachis. Internal symptoms were rotted, dark brown discoloration in bud, petiole, and rachis. Abundant conidial masses were developed on the infected tissues. Pieces of diseased tissues (5-mm cubes) were surface sterilized for 2 min in 3% sodium hypochlorite, rinsed with sterilized distilled water, plated onto potato dextrose agar (PDA), and incubated at 24°C and 12-h photoperiod, 20 pink fungal colonies were developed with sparse aerial mycelia on PDA. Conidiophores were hyaline, dimorphic, penicillate, and Acremonium-like. Conidia were dimorphic, typically with somewhat truncated ends, 4.5 to 5.7 × 1.9 to 2.3 μm (mean 4.99 × 2.15, n = 100), borne in long chains on penicillate conidiophores; on Acremonium-like conidiophores conidia were cylindrical, straight, and slightly curved, 4.55 to 10.1 × 1.2 to 2.35 μm (mean 8.2 × 1.7, n = 100). These morphological characteristics resembled those of Nalanthamala vermoesenii (Biourge) Schroers (Schroers et al. 2005). Genomic DNA was extracted from the mycelia of a representative isolate CP-SP53. The internal transcribed spacer (ITS) region and the large subunit of ribosomal ribonucleic acid (LSU) were amplified and sequenced. The sequences were deposited in GenBank with accession numbers OQ581472 (ITS) and OQ581465 (LSU). Phylogenetic trees based on ITS and LSU sequences of Nalanthamala species were reconstructed using maximum likelihood and Bayesian inference methods. Isolate CP-SP53 was placed in the clade of Nalanthamala vermoesenii. The pathogenicity test was carried out twice with isolate CP-SP53 on five 3-year-old P. canariensis plants. Four petioles per plant were surface disinfected with 75% ethanol, and wounded with a sterilized scalpel (shallow cuts 0.5 cm wide). A mycelial plug (5 mm in diam.) of a 1-week-old PDA culture was placed on each wounded site. Sterile PDA plugs were used for five non-inoculated control plants. All plants were maintained at 22 ± 2°C and a 12-h photoperiod. Twenty-five days after inoculation (dai), wounded petioles showed the same symptoms observed in the field, whereas control plants remained healthy. Forty-five dai, all inoculated plants died. Pink conidial masses developed on symptomatic tissues. To fulfill Koch's postulates, the pathogen was reisolated by placing the pink conidial masses onto PDA. The colony characteristics and morphometric measurements were identical to those of isolate CP-SP53. Nalanthamala vermoesenii has been reported on P. canariensis in Greece and United States (Feather et al. 1979; Ligoxigakis et al. 2013) and Syagrus romanzoffiana in Egypt (Mohamed et al. 2016). To our knowledge, this is the first report of Nalanthamala vermoesenii as the causal agent of pink rot on P. canariensis in Mexico. This plant is the most commonly planted ornamental palm in Mexico City. The spread of N. vermoesenii could be a threat for the estimated 15 thousand palms, therefore dramatically change the urban landscape.

First Report of Colletotrichum gloeosporioides and Colletotrichum siamense, Causing Anthracnose Disease on coffee trees, in Saudi Arabia.

Coffee (Coffea arabica L.) is a promising agricultural commodity in many countries including Saudi Arabia, but crop production is often constrained by diseases. In December 2021, coffee trees had symptoms of anthracnose disease (CAD) were observed in Jazan Province, Saudi Arabia (17°19'00.8"N 43°11'26.8"E), and the incidence was 55%. Affected trees showed dieback and leaves necrosis. On green and ripening berries, slightly sunken and dark brown lesions were occurred; the berries finally become mummified (Fig. S1). For pathogen isolation, symptomatic tissues (4×4mm) of 30 diseased branches and berries samples were surface-sterilized in 1% sodium hypochlorite for 2 min, followed by 70% ethanol for 20 s, rinsed in sterile distilled water and placed on potato dextrose agar (PDA). Cultures were incubated at 26℃ for 8 days in the dark. Eighteen isolates were recovered, and 2 representative single spore isolates (KSU-CgM17, KSU-CsM42) were used for further study. PDA culture of KSU-CgM17 had aerial white mycelium at first and later became gray to grayish black; light salmon to orange conidial masses were observed on the mycelium plate surface as the cultures aged (Fig. S2). Colony produced by KSU-CsM42 was off-white to gray with cottony mycelia and grayish-white on the undersides of the culture after 10 days at 28° (Fig. S2). Conidial shape of these two isolates were both aseptate, cylindrical to nearly straight, hyaline, rounded at both ends. Conidia (n = 50) measurements were 16 to 18.0 µm long × 4.8 to 6.4 µm wide for KSU-CgM17 and 12.6 to 17.5 µm long × 3.2 to 4.5 μm wide for KSU-CsM42. The microscopic and culture features fitted those for Colletotrichum gloeosporioides species complex (Weir et al. 2012). To further identify these isolates, four genomic DNA loci including the partial ITS rDNA region, and CAL, TUB2, and GAPDH genes were amplified and sequenced (Hu et al., 2015). All sequences were deposited into GenBank under accession numbers: OQ791412 & OQ791413 (ITS), OQ786847 & OQ786851 (CAL), OQ786849 & OQ786850 (TUB2), and OQ786848 & OQ786852 (GAPDH) for KSU-CgM17and KSU-CsM42, respectively (Tables S1& S2). A BLAST search of GenBank showed that these pathogens were identified as C. gloeosporioides (KSU-CgM17) and C. siamense (KSU-CsM42). The pathogenicity was tested on detached coffee leaves or green and red berries (Coa et al., 2019). For inoculation, healthy leaves and berries were wounded with a sterilized needle, placed inside petri dishes containing moist filter paper, and then inoculated with a 10-µl droplet of conidial suspension (106 spores/ ml). Sterile distilled water was used as a negative control. Six replicates were tested per isolate and the experiment was repeated once. The inoculated materials were incubated at 25°C and 100% relative humidity for 8 days. Necrotic lesions developed on 100% of the inoculated coffee materials 6 days later, whereas the negative controls were asymptomatic (Fig. S2). Koch's postulates were fulfilled when typical colonies of these species were successfully re-isolated from the from symptomatic tissues. These pathogens were reported previously to affect coffee in Vietnam (Nguyen et al., 2010), China (Cao et al., 2019), and Puerto Rico (Serrato-Diaz et al., 2020). To our knowledge, this is the first record of C. gloeosporioides and C. siamense causing CAD in Saudi Arabia. Further studies on the epidemiology of CAD on arabica coffee plantations as well as effective strategies for managing this disease are needed.

First Report of Cladosporium cladosporioides Causing Leaf Spot of Aralia cordata var. continentalis in Korea.

Aralia cordata var. continentalis (Kitag), commonly known as Japanese spikenard, is an upright herbaceous perennial medicinal plant effective in relieving pain. It is also consumed as a leafy vegetable. Leaf spots and blight symptoms on A. cordata resulting in defoliation were observed in July 2021 from a research field with a disease incidence of nearly 40-50% from 80 plants in Yeongju, Korea. Brown spots with chlorotic halos first appear on the upper leaf surface (Fig. 1A). In the later stage, spots enlarge and coalesce; resulting in the leaves to dry-off (Fig. 1B). To isolate the causal agent, small pieces of diseased leaves displaying the lesion were surface-sterilized by 70% ethanol for 30 s and rinsed twice with sterile distilled water (SDW). Later, the tissues were crushed in a sterile 2.0-ml Eppendorf tube with a rubber pestle in SDW. The suspension was serially diluted and spread on potato dextrose agar (PDA) medium, incubated at 25°C for 3 days. A total of 3 isolates were obtained from the infected leaves. Pure cultures were obtained by the monosporic culture technique (Choi et al. 1999). After 2 to 3 days of incubation with a 12-h photoperiod, the fungus initially produced gray mold colonies in olive color, and the edges of the mold appeared white with a velvety texture after 20 days (Fig. 1C). Microscopic observations revealed small, single-celled, rounded, and pointed conidia that measured 6.67 ± 0.23 µm × 4.18 ± 0.12 µm (length × width) (n=40 spores) (Fig. 1D). On the basis of its morphology, the causal organism was identified as Cladosporium cladosporioides (Torres et al. 2017). For molecular identification, pure colonies of three single-spore isolates were used for DNA extraction. A fragment of the ITS, ACT, and TEF1-α were amplified using the primers ITS1/ITS4 (Zarrin et al. 2016), ACT-512F/ACT-783R, and EF1-728F/EF1-986R, respectively, by PCR (Carbone et al. 1999). The DNA sequences from all three isolates (GYUN-10727, GYUN-10776, and GYUN-10777) were identical. The resulting ITS (ON005144), ACT (ON014518), and TEF1-α (OQ286396) sequences from the representative isolate GYUN-10727 were 99 to 100% identical to the C. cladosporioides (ITS: KX664404, MF077224; ACT: HM148509; TEF1-α: HM148268, HM148266). The phylogenetic dendrogram was constructed from the comparative analysis of ITS, ACT, and TEF1-α gene sequences, showing the relationship between Cladosporium cladosporioides and related Cladosporium species (Fig. 2). The isolate GYUN-10727 has been deposited in Korean Agricultural Culture Collection (KACC 410009), and used as a representative strain in this study. For the pathogenicity test, healthy fresh leaves (3 leaves per plant) of 3-months-old A. cordata plants in pots were spray inoculated with conidial suspensions (1 × 10⁴ conidia/mL) of GYUN-10727, which was obtained from a 7-day-old PDA culture. Leaves sprayed with SDW were considered as control. After 15 days of incubation at 25°C ± 5°C under greenhouse conditions, necrotic lesions were observed on the inoculated A. cordata leaves, while control leaves did not develop any disease symptoms. The experiment was performed twice with three replicates (pots) per treatment. The pathogen was re-isolated from the symptomatic A. cordata leaves, but not from control plants, to fulfill Koch's postulates. The re-isolated pathogen was identified by PCR. Cladosporium cladosporioides has been reported to cause diseases in sweet pepper (Krasnow et al. 2022) and garden peas (Gubler et al. 1999). To our knowledge, this is the first report of C. cladosporioides causing leaf spots of A. cordata in Korea. The identification of this pathogen will help develop strategies to efficiently control the disease in A. cordata.

First report of Diaporthe sojae causing stem canker on blueberry in China.

Blueberry (Vaccinium corymbosum) plants are popular all over the world due to their high nutritional value and health benefits. In October 2020, blueberry stems (cv. O'Neal) displaying reddish brown necrotic lesions were observed from a blueberry field in Anqing (Anhui, China), with the incidence of approximately 90%. The affected plants were somewhat stunted that had smaller fruit, and in severe cases, partial or whole plant died. We randomly selected three sampling sites to collect stems with the symptoms. Samples at the margin between diseased and healthy tissues were taken out, cut into 5 mm pieces in length，and then mixed them together. Twenty small samples were surface-sterilized, and plated onto potato dextrose agar (PDA). The plates were incubated at 25°C in the dark until fungal colonies were observed. After subculturing single hyphal tips, 9 out of 12 fungal isolates with similar morphologies were obtained. The representative isolate, LMKY12 was selected for further identification. The colonies on PDA showed white, fluffy aerial mycelia with 7.9  0.2 mm (n=5) diameter after inoculation in darkness at 25°C for one week. The colony darkens in color with age, yellowish pigmentation in reverse were observed. After 15 days of incubation, dark brown, irregular hard particles (fruiting bodies in sexual stage) accumulated on the surface of the colonies. Asci were 8-spored, sessile, club-like, hyaline, and 35-46 x 6-9 μm (n=30) in size. The ascospores were oval or spindle shaped, two-celled, constricted at division, and containing four guttulates with larger guttules at centre and smaller one at ends, measured 9-11 x 2-4 um (n=50). No sporulation observed on blueberry stems after inoculated 30 days. In order to induce the production of conidiophores, mycelial plugs were placed on blueberry leaves and cultured in darkness at 25°C. There are two types of conidia observed after 20 days of inoculation. Alpha conidia were aseptate, hyaline, smooth, ovate to ellipsoidal, often biguttulate, measured 5.33-7.26 x 1.65-2.53 μm (n=50). Beta conidia were hyaline, linear, measured 12.60-17.91 x 0.81-1.38 μm (n=30). The morphological characteristics matched the previous description of D. sojae (Udayanga et al. 2015; Guo et al. 2020). To confirm the identification, the mycelial genomic DNA of LMKY12 was extracted as a template. The rDNA internal transcribed spacer (ITS), translation elongation factor 1-α gene (TEF1-α), and calmodulin (CAL) were amplified and sequenced using primers ITS1/ITS4 (White et al. 1990), EF1-728F/EF1-986R, and CAL-228F/CAL-737R (Carbone and Kohn 1999), respectively. BLAST analysis revealed that the ITS (ON545758), CAL (OP886852), and TEF1-α (OP886853) sequences were 100% (527/527 base pairs), 99.21% (504/508 base pairs), and 99.41% (336/338 base pairs) similar to the strain FAU636 of D. sojae (KJ590718, KJ612115, KJ590761), respectively. Phylogenetic analysis based on concatenated sequences of ITS, TEF1-α, and CAL using MEGA 7.0 by maximum likelihood attributed the isolate LMKY12 to the D. sojae clade. Pathogenicity tests were performed on blueberry cv. O'Neal using detached stems (n=8) in laboratory, one-year-old potted plants (n=4) in greenhouse. Inoculations were done by placing mycelial plugs (7 mm in diameter) taken from a 7-day-old PDA culture on wounded stems. Inoculations with uncolonized agar plugs served as negative controls. Reddish dark brown lesions similar to the symptoms were observed on all inoculated stems 7 days after inoculation. No symptoms developed on control stems. Reisolations were successfully made from all the inoculated stems, and the pathogen was confirmed by the presence of pycnidia, alpha conidia and beta conidia. To our knowledge, this is the first report of D. sojae causing blueberry stem canker in China.

First Report of Cucurbita Blossom Blight and Fruit Rot Caused by Choanephora cucurbitarum in Mexico.

In December 2022, blossom blight, abortion, and soft rot of fruits were observed on Cucurbita pepo L. var. Zucchini in Mexico under greenhouse conditions (temperatures of 10 to 32°C and relative humidity up to 90%). The disease incidence in about 50 plants analyzed was around 70% with a severity of nearly 90%. Mycelial growth on flower petals and fruit rot with brown sporangiophores was observed. Ten disinfested fruit tissues in 1% NaClO for 5 min and then rinsed twice in distilled water from the lesion edges were placed on potato dextrose agar culture medium (PDA) supplemented with acid lactic and then, the morphological characterization was carried out in V8 agar medium. After 48 h of growth at 27°C, the colonies were pale yellow with diffuse cottony mycelia that were non-septate and hyaline and produced both sporangiophores bearing sporangiola and sporangia. The sporangiola were brown, ranged from ellipsoid to ovoid, and had longitudinal striations that measured 22.7 to 40.5 (29.8) μm x 16.08 to 21.9 (14.5) μm long and wide, respectively (n=100). The sporangia were subglobose, had a diameter of 127.2 to 281.09 (201.7) μm (n=50), and contained ovoid sporangiospores that measured 26.5 to 63.1 (46.7) μm x 20.07 to 34.7 (26.3) μm long and wide, respectively (n=100) which had hyaline appendages at the ends. Based on these characteristics, the fungus was identified as Choanephora cucurbitarum (Ji-Hyun et al. 2016). For molecular identification, DNA fragments for the internal transcribed spacer (ITS) and the large subunit rRNA 28S (LSU) regions for two representative strains (CCCFMx01 and CCCFMx02) were amplified and sequenced with the primer pairs ITS1-ITS4 and NL1-LR3 (White et al. 1990; Vilgalys and Hester 1990). The ITS and LSU sequences were deposited in GenBank database (Accession numbers OQ269823-24 and OQ269827-28, respectively) for both strains. The Blast alignment showed from 99.84 to 100% identity with Choanephora cucurbitarum strains JPC1 (MH041502, MH041504), CCUB1293 (MN897836), PLR2 (OL790293), and CBS 178.76 (JN206235, MT523842). To confirm the specie identification, the evolutionary analyses were conducted from the concatenated sequences of the ITS and LSU of C. cucurbitarum and other mucoralean species with the Maximum Likelihood method and Tamura-Nei model included in the software MEGA11. The pathogenicity test was demonstrated using five surface-sterilized zucchini fruits inoculated with a sporangiospores suspension containing a concentration of 1 x 105 esp/mL on two sites per fruit (20 µL each) that previously were wounded with a sterile needle. For fruit control, 20 µL of sterile water was used. Three days after inoculation under humidity conditions at 27°C, white mycelia and sporangiola growth with a soaked lesion were observed. That fruit damage was not observed on the control fruits. C.cucurbitarum was reisolated from lesions on PDA and V8 medium which was confirmed by morphological characterization fulfilling Koch's postulates. Blossom blight, abortion, and soft rot of fruits caused by C. cucurbitarum were observed on Cucurbita pepo and C. moschata in Slovenia and Sri Lanka (Žerjav and Schroers 2019; Emmanuel et al. 2021). This pathogen has the capability to infect a wide variety of plants worldwide (Kumar et al. 2022; Ryu et al. 2022). There are no reports of C. cucurbitarum causing agricultural losses in Mexico, and this is the first report causing the disease symptoms in Cucurbita pepo in this country; however, this fungus was found in the soil of papaya-producing areas and it is considered an important plant pathogenic fungus. Therefore, strategies for their control are highly recommended to avoid spreading the disease (Cruz-Lachica et al. 2018).

First report of Fusarium verticillioides causing safflower root rot in Sinaloa, Mexico.

Safflower cultivation is of great socioeconomic importance worldwide. Production is intended for the extraction of oil from the seeds. In 2021 Mexico ranked fifth in world production with approximately 52,553.28 tons (SIAP, 2021). In April 2022, in the north-central zone of Sinaloa, Mexico, diseased plants were reported in fields planted with safflower. Symptoms included chlorotic plants, necrosis and rot in vascular bundles, dwarfed plants and reflexed plants bent towards the ground. The disease caused estimated losses of 15% of seed production, with respect to the production obtained from the previous year in the safflower fields surveyed. Twenty-five plants with symptoms were sampled to isolate the pathogen. Plants were cut at the base of the stem near the roots and roots cut into 5 mm2 pieces. Tissue samples were superficially disinfected by immersing in 70% alcohol for 10 sec, 2% sodium hypochlorite for 1 min, washed in sterile water, and placed on potato dextrose agar (PDA) at 28 ºC for 7 days in the dark. Twelve monosporic isolates derived from the PDA culture were morphologically characterized. Abundant white aerial mycelium and small pink to dark violet pigments in the center of the culture were observed. From 10-day-old cultures grown on carnation leaf agar medium microconidia and macroconidia were produced. Microconidia were hyaline, had zero to two septa, and were oval or ellipsoidal, 4.6 to 14 x 1.8 to 4.2 μm (n = 40). The macroconidia were hyaline, were slightly curved with three to five septa, and measured from 26 to 69 x 3 to 6.1 μm (n = 40). No chlamydospores were observed. According to the morphological characteristics, the isolates were identified as Fusarium verticillioides (Leslie and Summerell, 2006). DNA was extracted from one isolate and the Translation Elongation Factor 1-α (EF1) gene was amplified and sequenced (O'Donnell et al. 2010). The sequence obtained from isolate FV3CARCULSIN with 645 base pairs was submitted to NCBI GenBank with accession number OQ262963. The BLAST search revealed 100% similarity with F. verticillioides isolate 13 (KM598773) (Lizárraga et al. 2015). Identification in FUSARIUM ID resulted in a 99.85% similarity with isolate F. verticillioides CBS 131389 (MN534047) (Yilmaz et al. 2021). A phylogenetic tree, made with sequences of the EF1 gene, revealed that FV3CARCULSIN was most closely related to F. verticillioides (100% bootstrap). Pathogenicity tests were carried out on safflower plants (cv. Oleico) grown in sterile vermiculite. Plants were inoculated with a conidial suspension (1 × 105 conidia/ml) obtained from FV3CARCULSIN grown on PDA for 7 days. A total of 45 plants were inoculated by drenching the roots with 20 ml of inoculum when the plants were 20 days old. Fifteen plants served as negative controls without inoculation. Plants were kept for 60 days in greenhouse conditions; however, after 45 days the plants began to die. The assay was conducted twice. Rotting and necrosis was observed in the roots of the plants. The pathogen was reisolated from the tissue of all the plants with symptoms and identified as F. verticillioides using morphological characteristics and EF1 sequences, completing Koch's postulates. No symptoms were observed in control plants after 60 days. This is the first report of root rot in safflower caused by F. verticillioides in Mexico. The fungus has been reported in maize (Figueroa et al. 2010), but it is unknown if it could be the same pathogen of safflower. Identification of the pathogen is important for implementing management methods to reduce yield losses and for additional studies on the impact of the disease on oil quality extracted from safflower seeds.