Carnation Leaf Agar Medium Research Articles

First Report of Strawberry Root Rot Caused by Fusarium falciforme in China.

Root rot caused by Fusarium spp. is a destructive disease affecting agricultural regions worldwide. Strawberries (Fragaria × ananassa Duch.) are an economically important crop in China. In March 2023, root rot was observed in strawberries grown in Jinan, Shandong Province, China. Symptoms included leaf wilt, necrotic roots, and plant death (Figure 1). Four strawberry samples (two symptomatic and two asymptomatic) were collected from ~2-acre fields where the disease incidence rate ranged from 2 to 3%. Tissue pieces (5 mm × 5 mm × 5 mm) from two healthy and two diseased strawberry root tissues were surface-disinfected with 75% ethanol for 3 min, treated with 10% sodium hypochlorite for 5 min, and washed three times with sterile water. These pieces were cultured for 5 days at 28°C on potato dextrose agar (PDA) containing 200 mg/L timentin. Typical Fusarium spp. like growth was observed on plates with the two symptomatic samples. Two representative fungal isolates (CM1 and CM2) with similar morphological characteristics were purified using the single-spore method (Figure 1). CM1 showed an average growth rate of 5 mm/d in PDA and comprised of several white-to-cream aerial mycelia after 5 d. After cultivation in carnation leaf agar medium for 7 d, falciform macroconidia, with blunt apical cells and slightly hooked basal cells comprising 3 to 4 septa of varying sizes (20 to 39)×(3.6 to 6.7 μm) were observed (n=50) (Figure 1). The chlamydospores were spherical, terminal or intercalary, solitary or chain-forming, and 3.1-10.5 μm in diameter (Figure 1). The microconidia on PDA were (5.8 to 13.6)× (2.5 to 3.3) μm in size (n=50). These morphological characteristics are consistent with previous descriptions of the Fusarium solani species complex (FSSC). DNA was extracted using the CTAB method (Stenglein and Balatti 2006). The internal transcribed spacer (ITS), translation elongation factor 1-α gene (tef1), RNA polymerase II largest subunit (rpb1), and RNA polymerase II second largest subunit (rpb2) were amplified and sequenced using specific primers (O'Donnell et al. 2010). The ITS (OR526528, OR526529), tef1 (OR536947, OR536948), rpb1 (OR536949, OR536950), and rpb2 (OR536951, OR536952) sequences of the CM1 and CM2 isolates were uploaded to the NCBI database. BLASTn analysis revealed that the ITS, tef1, rpb1, and rpb2 sequences were 99.1-100% identical to those of the Fusarium falciforme reference strains NRRL 54989 and NRRL 54978. A phylogenetic tree based on the ITS, tef1, rpb1, and rpb2 sequences was generated using MEGA v.11 via the maximum-likelihood method (Tamura et al. 2021). CM1 clustered with the Fusarium falciforme reference strains NRRL 54989 and NRRL 54978 and belonged to the FSSC based on its morphological and molecular characteristics (Figure 2). To test for pathogenicity, the roots of nine 3-month-old healthy strawberry (cv. Akihime) plants were exposed to conidial suspensions (1×108 spores/mL) of the CM1 isolate. Another nine root samples were treated with sterile water and used as controls. All strawberry plants were maintained in a growth chamber under a 12/12 h light/dark cycle at 28°C and 90% relative humidity and the experiment was repeated three times. After one month, the inoculated plants had withered and died, and the pith became dark red (similar to field plants) (Figure 1). The fungi isolated from the experimental plants were confirmed as F. falciforme using morphological and sequence analyses. F. falciforme causes root rot in several species including Nicotiana tabacum (Qiu et al. 2023) and Weigela florida (Shen et al. 2020); however, this study is the first to report root rot caused by F. falciforme in strawberries in China. Overall, F. falciforme infection poses a threat to strawberry production and breeding.

First report of Fusarium cugenangense causing root rot of tea plants (Camellia sinensis) in China.

Root rot is an important disease of tea plants owing to its unobvious early symptoms and permanent damage (Huu et al. 2016). In 2019, 5% of tea plants displayed symptoms consistent with root rot in a tea plantation (28°09'N, 113°13'E) located in Changsha city, Hunan province of China. The symptoms of the diseased tea plants ranged from wilting leaves to entirely dead. The roots had black lesions and rot typical of this disease. Symptomatic roots were collected, washed with water and disinfected with 75% ethanol, then cut into pieces and sterilized with 0.1% mercuric chloride for 30 s, 75% ethanol for 1 min, and rinsed with sterile water five times. After drying on sterilized filter paper, root tissues were cultured on potato dextrose agar (PDA) medium at 25 oC for 7 days in the dark. Four isolates, CAGF1, CAGF2, CAGF3, and CAGF4 were purified by selecting single spores. All isolates were subjected to a pathogenicity test. A conidial suspension of each strain was collected at a concentration of 2×106 conidia/mL. For the pathogenicity test, two-year-old field grown tea plants were transplanted in plastic pots containing 240 g of the rice grain-bran mixture (inoculated with 4 mL of conidial suspension and cultured for 14 days) and 960 g of sterilized soil (Huu et al. 2016). The pots without inoculated mixture served as control group. All the pots were kept in illumination incubators at 25 oC and a 12L:12D photoperiod. The pathogenicity test for each strain was repeated three times with three repetitions. Only strain CAGF1 exhibited pathogenicity to tea plants. Symptoms appeared on the third day post inoculation (dpi) and gradually worsened by the 7 dpi. On the 14 dpi, most leaves had died and the roots were black and partially rotten, similar to field symptoms. The reisolated fungus from potted roots was identified as CAGF1 based on ITS region and colony morphology, while isolation was attempted, CAGF1 was not isolated from the control plants, which fulfilled Koch's postulates. On PDA, the colony center of CAGF1 was purple with white margin, while on carnation leaf agar (CLA) medium was white. On CLA medium, macroconidia have 0 to 3 septa, measured 19.1 μm to 41.2 μm × 4.2 μm to 5.4 μm (mean= 31.2 μm × 4.8 μm, n=30). The microconidia were measured as 6.7 μm to 12.8 μm × 2.4 μm to 4.9 μm (mean= 10.1 μm × 3.3 μm, n=30), with 0 to 1 septa. And the chlamydospores were measured as 6.0 to 9.7μm (mean= 7.7μm, n=30). Morphologically, strain CAGF1 was identified as Fusarium oxysporum (Leslie and Summerell 2006). Additionally, the genomic DNA of strain CAGF1 was extracted by cetyltrimethylammonium bromide (CTAB) method, the internal transcribed spacer (ITS), elongation factor 1 alpha (EF-1α) and second largest subunit of RNA polymerase II (RPB2) were amplified using the primers ITS1/ITS4 (White et al. 1990), EF-1/EF-2 (Geiser et al. 2004) and fRPB2-5F/fRPB2-7cR (Liu et al. 1999), respectively. Sequences were deposited in GenBank (ITS, OK178562.1; EF-1α, OK598121.1; RPB2, OP381476.1). BLASTn searches revealed that strain CAGF1 was 100% (ON075522.1 for ITS and JX885464.1 for RPB2) and 99.6% (JQ965440.1 for EF-1α) identical to Fusarium oxysporum species complex (FOSC). Based on phylogenetic analysis, the strain CAGF1 was identified as Fusarium cugenangense, belonging to FOSC. To our knowledge, this is the first report of F. cugenangense causing root rot of tea plants in China. The findings are important for the management of this root rot and the improvement of economic benefits of tea cultivation.

First report of Calonectria pseudopteridis causing brown spot on Eriocaulon buergerianum in Zhejiang province, China.

Eriocaulon buergerianum is a traditional Chinese herb, used to treat eye diseases. In July 2020, a severe brown spot disease occurred on E. buergerianuim in Yongjia county (120°19'E, 27°58'N), Zhejiang province, China. Seventy-three plants from a survey of about 150 plants showed brown leaf spots. The spots were yellowish-brown to brown, and primarily affected leaves. As the disease progressed, the spots expanded, and fused. Sixty of the 150 plants wilted (Fig. 1 A-D). Diseased tissues were surface-sterilized in 75% ethanol (30 s), rinsed three times with sterile distilled water, air-dried (5 min), placed on potato dextrose agar (PDA) at 26°C (12-h light/dark cycle), and cultured for 4 days. Hyphal tip technique was used to obtain five isolates, which were transferred to malt extract agar (MEA), oatmeal agar (OA), and PDA. After seven days of growth at 26°C, the colonies had light, yellowish-brown centers with gray-white edges; the reverse sides had reddish- to yellowish-brown centers. Seven-day-old colonies grown on oatmeal agar (OA) produced a sparse aerial mycelium with yellowish-brown centers, and light yellowish-brown centers on the reverse side. Seven-day-old colonies grown on PDA exhibited yellowish-brown, floccose, aerial mycelia with reddish-brown to yellowish-brown pigment on the back (Fig. 1 E-G). The colonies also produced microsclerotia on OA and PDA media. On carnation leaf agar (CLA) and water agar (WA), only macroconidia were produced. Macroconidiophores comprised a stipe bearing penicillate, fertile branches, and a clavate vesicle. Macroconidia were cylindrical, rounded at both ends, colorless, hyaline, 1- to 2-septate, but mainly one, and 71.86 to 96.11 µm (mean = 82.27 µm, n = 50) × 4.13 to 5.21 µm (mean = 4.63 µm, n = 50) (Fig. 1 H-K). Morphological characteristics of the isolate on CLA or WA medium were similar to the Calonectria petridis species complex (Alfenas et al. 2015; Liu et al. 2020). The actin (Act), calmodulin (Cal), internal transcribed spacer (ITS), Histone3 (His3), a fragment of the large subunit nuclear ribosomal DNA (LSU), RNA polymerase II subunit 2 (Rpb2), translation elongation factor 1α (Tef1), and β-tubulin 2 (Tub2) genes were sequenced. The sequences were deposited in GenBank as: His3: Not detected; Act, OM933603, MZ770831, OM933604-OM933606; Cal, OM933607, MZ770832, OM933608-OM933610; ITS, OM955624, MZ720794, OM955625-OM955627; LSU, OM955640, MZ720792, OM955641-OM955643; Rpb2, OM933615, MZ770834, OM933616-OM933618; Tef1, OM933611, MZ770833, OM933612-OM933614; and Tub2, OM933619, MZ770835, OM933620-OM933622. Sequences of the Act, Cal, ITS, LSU, Rpb2, and Tef1 genes of these strains show a 99% match to the ex-type strain Ca. pteridis CBS111793 and Ca. pseudopteridis CBS163.28 (Act, GQ280494, MT335112; Cal, GQ267413, MT335347; ITS, GQ280616, MT359808; LSU, GQ280738, MT359568; Rpb2, KY653438, MT412640; Tef1, FJ918563, MT412878). The Tub2 sequences showed 99% identity with sequences of the ex-holotype Ca. pteridis CBS111793 (Tub2, DQ190578) (Alfenas et al. 2015; Liu et al. 2020; Lombard et al. 2016). Phylogenetic analysis showed that these strains and Ca. pseudopteridis CBS163.28 were clustered in a high-support bootstrap value clade (bootstrap = 85) (Fig. 2). In August 2020, healthy E. buergerianum plants in the blooming-fruiting stage were used to test the pathogenicity of the isolates. The mycelium of isolate GJC3 was cultured on CLA medium at 26°C (12-h light/dark cycle) for ten days. Eight healthy plants were inoculated by spraying with a conidial suspension (10 mL of 1 × 105 conidia/mL). Another eight plants were sprayed with sterilized water 10 mL of sterilized water as controls. The experiment was repeated three times. Pathogenicity tests were performed in the greenhouse (60% relative humidity, 28 to 20°C day/night, temperature range, and natural sunlight). After 15 days, inoculated plants showed yellowish-brown to brown spots and withering of the leaves, whereas the control plants remained healthy (Fig. 1 L-N). Ca. pseudopteridis was reisolated from the inoculated plants, and its morphology and gene sequences were similar to the original isolate GJC3. Ca. pseudopteridis was not isolated from the control plants. Morphological characteristics, molecular data and pathogenicity test identified these organisms as Ca. pseudopteridis. This report provides a basis for further research on biology and management of this disease.

First report of Fusarium keratoplasticum causing strawberry root rot in Sinaloa, Mexico.

Strawberry (Fragaria × ananassa) is a fruit of economic importance for Mexico, occupying the third place in world production, with an approximate production of 861, 337 t (SIAP, 2021). In January 2021, in Culiacan, Sinaloa, Mexico (24°46'46″N; 107°27'04″ W), wilting symptoms (stunted growth, leaf yellowing and wilting, necrosis in vascular bundles, root rot and wilting) were observed on commercial strawberry crops, with an incidence of 5 to 10 %. Tissue samples from symptomatic roots were cut and disinfected with alcohol, sodium hypochlorite and sterile water, to later be plated on potato dextrose agar (PDA). Fifteen monosporic isolates were obtained by single-spore culturing (Hansen and Smith, 1932). Typical Fusarium spp. colonies were obtained from all root samples. On PDA the colonies were abundant with white aerial mycelium, hyphae were branched and septate, and light-yellow pigmentation was observed in the center of old cultures (Leslie and Summerell 2006). From 10-day-old cultures grown on carnation leaf agar medium, macroconidia were slightly curved, showing three marked septa, broad central cells, slightly tapered apices, foot-shaped basal cells and measured 59.6 - 73.4 (x̄ = 68.7) x 10.4 - 14.9 μm (x̄ = 13.6) (n = 40). The microconidia (n = 40) were thin-walled, hyaline, ovoid unicellular that measured 19.7 - 32.2 (x̄ = 26.6) x 8.8 - 11.8 μm (x̄ = 10.2). The translation elongation factor 1 alpha (EF1-α) gene (O'Donnell et al. 1998) was amplified by polymerase chain reaction and sequenced. Maximum likelihood analysis was carried out using the EF1-α sequence from the isolate FKTFRESCULSIN (GenBank accession no. OK491929) and other Neocosmospora and Fusarium species. Phylogenetic analysis revealed the isolate was Fusarium keratoplasticum (currently named Neocosmospora keratoplastica) belonging to the Fusarium solani species complex (FSSC). Pathogenicity tests were performed on strawberry plants (cultivar Camarosa) grown on autoclaved sandy loam soil mix. Twenty plants were inoculated by drenching with 20 ml of a conidial suspension (1 × 105 CFU/ml) in an isotonic saline solution of FKTFRESCULSIN grown on PDA. Ten uninoculated plants served as controls. Plants were maintained for 60 days under greenhouse conditions (25 to 30°C). The assay was conducted twice. Root and stem rot similar to that observed on the infected plants in the field was observed. No symptoms were observed on uninoculated control plants after 60 days. The pathogen was reisolated from necrotic tissue from all inoculated plants and identified as F. keratoplasticum by sequencing the partial EF1-α gene and based on its morphological characteristics, thus fulfilling Koch's postulates. To our knowledge, this is the first report of root rot and wilt of strawberry caused by F. keratoplasticum in Mexico; it also contributes knowledge to the scientific community, since there is little information about this pathogen causing damage to plants in the world. Strawberry is an important crop in Mexico, and the occurrence of this disease could threaten strawberry production.

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.

Fusarium verticillioides Causing Root and Stem Rot in Papaya (Carica papaya) in Mexico.

Mexico is the fifth largest producer of papaya in the world with an estimated production of 1, 134, 753 metric tons per year (FAOSTAT 2022). In February 2022, in the center zone of Sinaloa State (Mexico), in a seedling-producing greenhouse, papaya seedlings were observed with an incidence (20%) of root and stem rot and necrotic tissue. Symptomatic tissues were collected from 10 papaya plants, which were cut into small pieces and surface sterilized sequentially with 70% alcohol for 20 s and 1% sodium hypochlorite for 2 min, dried, placed on potato dextrose agar (PDA), and incubated at 26°C in darkness for 5 days. Typical Fusarium spp. colonies were obtained from all root samples. Ten pure cultures were obtained by single-spore culturing and morphologically characterized on PDA and carnation leaf agar (CLA) media. On PDA, the colonies produced abundant white aerial mycelium, and the center of old cultures was yellow pigmentation (Leslie and Summerell 2006). From 10-day-old cultures grown on CLA medium, macroconidia were slightly curved, which showed zero to three septa, with some slightly sharp apices, and basal cells with notches, the measurements were from 22.53 to 48.94 x 6.9 to 13.73 µm (n= 50). The microconidia were presented in abundant chains of microconidia. The microconidia presented thin walls, oval and hyaline in shape, forming long chains, measuring 10.4 to 14.25 x 2.4 to 6.8 µm (n= 50). Chlamydospores were not observed. The translation elongation factor 1 alpha (EF1-α) gene (O'Donnell et al. 1998) was amplified by polymerase chain reaction and sequenced from isolate FVTPPYCULSIN (GenBank accession no. OM966892). Maximum likelihood analysis was carried out using the EF1-α sequence (OM966892) and other species from the genus Fusarium. Phylogenetic analysis revealed that the isolate was Fusarium verticillioides (100% bootstrap). Furthermore, the isolate FVTPPYCULSIN was 100 % similar with other reported Fusarium verticillioides sequence (GenBank accession nos. MN657268) (Dharanendra et al. 2019). Pathogenicity tests were performed on 60-day-old papaya plants (cultivar Maradol) grown on autoclaved sandy loam soil mix. Ten plants per isolate (n = 10) were inoculated by drenching with 20 ml of a conidial suspension (1 × 105 CFU/ml) of each isolate per plant. The suspension was obtained by collecting the spores of each isolate grown on PDA with 10 ml of an isotonic saline solution. Ten noninoculated plants served as controls. Plants were maintained for 60 days under greenhouse conditions (25 to 30°C). The assay was conducted twice. Root and stem rot similar to that observed on the infected plants in the greenhouse was observed on the papaya plants. No symptoms were observed on noninoculated control plants after 60 days. The pathogen was reisolated from the necrotic tissue from all inoculated plants and was identified again as Fusarium verticillioides by sequencing the partial EF1-α gene again and based on its morphological characteristics, genetic analysis, and pathogenicity test, fulfilling Koch's postulates. The molecular identification was confirmed via BLAST on the Fusarium ID and Fusarium MLST databases. The isolate FVTPPYCULSIN was deposited in the fungal collection of the Faculty of Agronomy of the Autonomous University of Sinaloa. To our knowledge, this is the first report of root and stem rot of papaya caused by F. verticillioides. Papaya is an important fruit crop in Mexico, and the occurrence of this disease needs to be taken into account in papaya production.

Leaf spot on Alocasia macrorrhizos caused by Fusarium asiaticum in Sichuan, China.

Alocasia macrorrhizos (Giant elephant's ear), a perennial herb in the Araceae family, is native to South Asia and the Asia-Pacific (Takano, et al. 2012). It is cultivated as a medicinal and ornamental plant, and has a considerable economic importance in China. In September 2020, a severe infection of unknown leaf spot disease was observed on these plants at the Sichuan Agricultural University, Sichuan, China. The leaf spots first appeared as yellow dots. As these lesions expanded, they became circular to oval and light brown with darker brown edges. Around the lesions, the leaf tissue was chlorotic, thereby creating a yellow halo. When the infection became severe, spots merged into larger irregular lesions. Eventually, the diseased leaves senesced and dried. To identify the pathogen, five leaf samples of diseased plants were collected, and symptomatic tissues were surface-disinfected with 75% ethanol for 30 s followed by 3% NaCl solution for 30 s. Samples were rinsed three times in sterilized water, placed on potato dextrose agar (PDA), and incubated at 25°C ± 1°C in the dark. The colony grown on PDA was white (3 days), the center was brown (5 days), turned pink to dark red (8 days) with fluffy aerial mycelium and pigmentation with age. Ten pure cultures were inoculated into carnation leaf agar (CLA) medium and incubated at 25°C in an incubator (12 h for one light-dark cycle). In CLA medium, pathogen produced hyaline, sickle-shaped, macroconidia with 3 to 5 septa, and an average size of 30 to 50 × 4 to 5 µm (n = 30) macroconidia but no microconidia in 10 days. Chlamydospores were spherical to subspherical (5.4 to 13.8 µm). Morphological characteristics of the all isolates were consistent with the description of the Fusarium asiaticum (Leslie and Summerell 2006). To validate this identification, RNA polymerase II (RPB2) (Liu et al. 1999), translation elongation factor (EF-1) (Geiser et al. 2004), and β-tubulin (TUB2) gene region of five isolates were amplified and sequenced (O' Donnell et al. 2015; White et al. 1990). The sequence of one representative isolate (ZL10) sequence was submitted to GenBank (ON215729, ON215730, and ON215731). The NCBI BLAST identified the top hits, 100%, 100%, and 99.87% for RPB2, EF, and TUB gene sequences, respectively, all indicating to Fusarium asiaticum. Pairwise matched of RPB2 and EF genes by MycoBank Fusarium MSIL showed the top hit rate of 100% for F. asiaticum (MH582120 and MH582249). For Koch's postulate and pathogenicity test, spore suspensions (1 × 10^7 conidia/ml) collected from PDA and CLA cultures with 0.05% Tween 80 buffer were used to inoculate with a spray bottle on leaves of a one year old A. macrorrhizos plants. Two leaves of each plant (20 pots in total) were inoculated with the spore suspension (approximately 2000 µl per leaf). An equal number of control leaves were applied with water and 0.05% Tween 80 buffer. Twenty days later, the inoculated plants showed similar symptoms to those of the original diseased plants while the controls remained asymptomatic. Fusarium asiaticum was reisolated from the infected leaves and confirmed using morphological characteristics and DNA sequence analysis. The pathogenicity test was repeated three times with similar results. This first report raises awareness of a new leaf spot disease infecting a commercial A. macrorrhizos in China. It provides an insight for a need of systematic survey identifying current spread, disease origin, and ultimately developing disease management strategies. Funding: Funding was provided by Sichuan Agricultural University Subject Dual Support Program (Grant No. 2121993055). Funding was provided by Deyang Science and Technology Bureau (Sichuan Province) for key R&D projects in agriculture and rural areas (Grant No. 2021NZ048). Funding was provided by the Sichuan Provincial Department of science and technology for the Sichuan Provincial Science and technology project for connecting and Promoting Rural Revitalization (Grant No, 2022ZHXC0007) References： Geiser, D. M., et al. 2004. Eur. J. Plant Pathol. 110:473. https://doi.org/10.1023/B:EJPP.0000032386.75915.a0 Crossref, ISI, Google Scholar Leslie, J. F., and Summerall, B. A., eds. 2006. Page 176 in The Fusarium Laboratory Manual. Blackwell Publishing, Ames, IA. https://doi.org/10.1002/9780470278376 Liu, Y. J., et al. 1999. Mol. Biol. Evol. 16:1799. https://doi.org/10.1093/oxfordjournals.molbev.a026092 O'Donnell, K., and Cigelnik, E. 1997. Mol. Phylogenet. Evol. 7:103. https://doi.org/10.1006/mpev.1996.0376 Takano K T, et al. 2012, Plant Bio., 14(4). https://doi.org/10.1111/j.1438-8677.2011.00541.x.

First Report of Mango malformation disease caused by Fusarium proliferatum in Mexico.

Mango (Mangifera indica L.) is the most economically important fruit in the tropical and subtropical regions of the world. Mexico is ranked the fourth largest mango producer worldwide with an approximate production of 2 396 675 t in 2019 (FAO 2020). Sinaloa is the principal mango production state in Mexico with 410,147 t in 2020 (SIAP 2021). Mango malformation disease (MMD) is one of the main limitations in the production of this crop worldwide, causing serious losses in yield. During December 2017 to April 2018, symptoms of MMD were observed in commercial mango in the municipality of El Rosario (Sinaloa, Mexico). These symptoms included malformed and compacted inflorescences, abnormal development of vegetative shoots with shortened internodes at an incidence of 25 %. Tissue from 15 symptomatic trees were superficially disinfested with 2% sodium hypochlorite and transferred to potato dextrose agar (PDA). Typical Fusarium spp. colonies were obtained from all samples. Fifteen pure cultures were obtained by single spore culturing. White to cream-colored aerial mycelia of typical Fusarium colonies were observed from all samples on PDA (Leslie and Summerell 2006). From 10-day-old cultures grown on carnation leaf agar medium, macroconidia (n = 50) were hyaline, relatively slender with a curve, 4 to 5 septate, and measured 39.5 to 76.8 x 5.7 to 9.5 μm. The microconidia (n = 50) were hyaline and pyriform, without septa, and measured 8.1 to 10.6 x 5.1 to 6.9 μm. Chlamydospores were observed. The EF1-α gene (O'Donnell et al. 1998) was amplified by PCR and sequenced from the isolates. The EF1-α sequence from one representative isolate (128FRSIN) was deposited in GenBank with the accession number MK932806. Maximum likelihood analysis was carried out using the representative EF1-α sequence for F. proliferatum (MK932806) and other Fusarium species. Phylogenetic analysis revealed the isolate most closely related was F. proliferatum (100% bootstrap). The molecular identification was also confirmed via BLAST on the Fusarium ID and Fusarium MLST databases. The pathogenicity tests were carried out on healthy three-month-old mango plants. Twenty plants and five shoots per plant were inoculated with 20 µl of the conidial suspension (1 x 106 conidia/ml) (Freeman et al. 1999). Twenty plants served as noninoculated controls. Plants were maintained for 365 days under greenhouse conditions (25 to 30°C). The assay was conducted twice. Symptoms of multiple vegetative shoots and shortened internodes were observed four months after inoculation on the infected plants with an average disease of 4.5 in the first trial and 4.4 in the second assay according to the disease severity scale outlined by Iqbal et al., (2006). No symptoms were observed on non inoculated control plants after 365 days. One isolate per plant was isolated again from the plants with malformation symptoms (n=20), and identified again as F. proliferatum, by morphological and molecular characteristics. F. proliferatum was identified as the causal agent of MMD in China by Zhan et al. (2010). To our knowledge, this is the first report of F. proliferatum causing MMD in Mexico. The development of management strategies to prevent crop loss is required in this important mango production area.

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

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

Caracterización de especies de Fusarium que afectan al cultivo de espárrago en Veracruz, México

Asparagus cultivation is highly profitable and is appreciated in international gourmet cuisine. In Mexico, the production of this vegetable is mainly for export, in the state of Veracruz it has been recently introduced. The objective of the research was to characterize by morphology, molecular techniques and pathogenicity test the Fusarium species isolated from symptomatic tissues of asparagus shoot. A directed sampling was carried out on plants that showed rot, wilting and dark brown spots. Fungal isolates were obtained from diseased shoots, which were disinfected and seeded on potato dextrose agar. Isolates were purified in monosporic cultures by hyphal tip. Four species of Fusarium were identified by taxonomic morphology, in a selective carnation leaf agar medium, then pathogenicity tests were carried out, complying with Koch's postulates. Next, the strains that were pathogenic were characterized by molecular techniques, and their identity as Fusarium oxysporum and Fusarium proliferatum was confirmed. The information obtained allows the implementation of appropriate management strategies for pathogenic species and thereby avoids economic losses in the short, medium, and long term in the asparagus crop.

Fusarium proliferatum Associated with Basal Rot Disease of Bambusa pervariabilis × Dendrocalamopsis grandis in China.

Fusarium proliferatum Associated with Basal Rot Disease of Bambusa pervariabilis × Dendrocalamopsis grandis in China.

Fusarium incarnatum-equiseti Species Complex Causing Root Rot Disease on Leymus chinensis in China.

Leymus chinensis (Trin.) Tzvel. is a rhizomatous grass widely grown in the grasslands of Eurasia. With strong fertility and stress resistance, L. chinensis makes an excellent pasture and mowing grass, contributing to animal husbandry and thus playing an important role in the local economy of the northern grassland area in China (Baoyin et al. 2014). During August to September 2019, diseased roots of L. chinensis were collected from an artificially planted grassland (40°47'44" N, 111°43'58″ E, alt. 1049 m) in Shaerqin County, Hohhot, China. Infected plants were scattered across the field with disease incidence up to 2%. Symptoms observed were wilted plants and rotten roots. In order to identify the causal pathogen of root rot on L. chinensis, symptomatic pieces (5 × 5 mm) of grass roots were excised and surface sterilized with 75% ethanol for 3-5 s followed by 1% NaClO for 2-3 min, rinsed three times with sterile distilled water, and placed on water agar and incubated at 25°C for 3 days. The mycelia were cut and transferred onto potato dextrose agar (PDA) for subculture. A fungus was consistently isolated, and a strain, named LCH054, was obtained by hyphal tip culture. Culture developed as white and fluffy aerial mycelia, with diffused pink pigment on the reverse side of PDA after culturing at 25℃ for 7 days. A culture of LCH054 was transferred to carnation leaf agar (CLA) (Li et al. 2014) and incubated at 25°C for 10 days. Microconidia were absent but macroconidia were produced. Macroconidia were hyaline, sickle-shaped, and had 4 to 7 septa, 19.8 to 63.6 (mean 43.8) × 1.8 to 5.7 (mean 3.2) μm (n = 100). Chlamydospores were ellipsoidal or subglobose, with thick walls in clumps or chains. All morphological characteristics of LCH054 resembled Fusarium equiseti (Leslie and Summerell 2006). The primers of the internal transcribed spacer (ITS) region (White et al. 1990) and translation elongation factor 1α gene (TEF-1α) (O'Donnell et al. 1998) were used to amplify the isolate, and the fragments were sequenced. BLASTn search in the NCBI database using the ITS and TEF-1α sequences revealed 99 to 100% similarities with F. equiseti. BLAST analysis of the ITS and TEF-1α sequencies in the FUSARIUM-ID database showed them to have 99.21% (500 bp out of 504 bp) and 99.52% (622 bp out of 625 bp) similarities with the Fusarium incarnatum-equiseti species complex (FIESC) (strain NRRL 45997) (O'Donnell et al. 2009), respectively. The ITS and TEF1-α sequences were deposited in GenBank as accession numbers MT937067 and MT947530, respectively. The strain LCH054 was identified as a member of the FIESC based on morphological and molecular characteristics. For the pathogenicity test, one hundred of L. chinensis seeds were planted into five pots (12 cm [diameter]) × 15 cm [high]) and kept in a greenhouse under a 16-h photoperiod with temperatures of 20-25°C and 40% relative humidity. The conidial suspension of LCH054 was prepared by washing 7-day old fungal culture grown on CLA medium using sterile deionized water. Conidia were filtered through three layers of sterile cheese cloth, counted, and adjusted to 1 × 105 conidia/ml with a hemocytometer. Forty 1-month-old healthy plants (four pots) were inoculated with 400 ml of conidia suspension using the root drenching method, whereas the inoculum was replaced with 100 ml sterile water on control plants (one pot). Fourteen days after inoculation, all inoculated plants showed the typical symptoms of root rot identical to those observed in the field, whereas the control plants remained healthy. LCH054 was re-isolated from the inoculated plants and identified by the morphological and molecular approaches as described above. To the best of our knowledge, this is the first report of root rot caused by F. incarnatum-equiseti on L. chinensis in China as well as worldwide. The presence of the pathogen could cause significant economic losses in L. chinensis production. For this reason, strategies for the management and control of this disease should be developed and implemented.

First report of Fusarium falciforme (FSSC 3+4) causing root rot on chickpea in Mexico.

Chickpea (Cicer aretinium L.) is a legume crop of great importance worldwide. In January 2019, wilting symptoms on chickpea (stunted grow, withered leaves, root rot and wilted plants) were observed in three fields of Culiacan Sinaloa Mexico, with an incidence of 3 to 5%. To identify the cause, eighty symptomatic chickpea plants were sampled. Tissue from roots was plated on potato dextrose agar (PDA) medium. Typical Fusarium spp. colonies were obtained from all root samples. Ten pure cultures were obtained by single-spore culturing (Ff01 to Ff10). On PDA the colonies were abundant with white aerial mycelium, hyphae were branched and septae and light purple pigmentation was observed in the center of old cultures (Leslie and Summerell 2006). From 10-day-old cultures grown on carnation leaf agar medium, macroconidias were falciform, hyaline, with slightly curved apexes, three to five septate, with well-developed foot cells and blunt apical cells, and measured 26.6 to 45.8 × 2.2 to 7.0 μm (n = 40). The microconidia (n = 40) were hyaline, one to two celled, produced in false heads that measured 7.4 to 20.1 (average 13.7) μm × 2.4 to 8.9 (average 5.3) μm (n = 40) at the tips of long monophialides, and were oval or reniform, with apexes rounded, 8.3 to 12.1 × 1.6 to 4.7 μm; chlamydospores were not evident. These characteristics fit those of the Fusarium solani (Mart.) Sacc. species complex, FSSC (Summerell et al. 2003). The internal transcribed spacer and the translation elongation factor 1 alpha (EF1-α) genes (O'Donnell et al. 1998) were amplified by polymerase chain reaction and sequenced from the isolate Ff02 and Ff08 (GenBank accession nos. KJ501093 and MN082369). Maximum likelihood analysis was carried out using the EF1-α sequences (KJ501093 and MN082369) from the Ff02 and Ff08 isolates and other species from the Fusarium solani species complex (FSSC). Phylogenetic analysis revealed the isolate most closely related with F. falciforme (100% bootstrap). For pathogenicity testing, a conidial suspension (1x106 conidia/ml) was prepared by harvesting spores from 10-days-old cultures on PDA. Twenty 2-week-old chickpea seedlings from two cultivars (P-2245 and WR-315) were inoculated by dipping roots into the conidial suspension for 20 min. The inoculated plants were transplanted into a 50-hole plastic tray containing sterilized soil and maintained in a growth chamber at 25°C, with a relative humidity of >80% and a 12-h/12-h light/dark cycle. After 8 days, the first root rot symptoms were observed on inoculating seedlings and the infected plants eventually died within 3 to 4 weeks after inoculation. No symptoms were observed plants inoculated with sterilized distilled water. The fungus was reisolated from symptomatic tissues of inoculated plants and was identified by sequencing the partial EF1-α gene again and was identified as F. falciforme (FSSC 3 + 4) (O'Donnell et al. 2008) based on its morphological characteristics, genetic analysis, and pathogenicity test, fulfilling Koch's postulates. The molecular identification was confirmed via BLAST on the FusariumID and Fusarium MLST databases. Although FSSC has been previously reported causing root rot in chickpea in USA, Chile, Spain, Cuba, Iran, Poland, Israel, Pakistan and Brazil, to our knowledge this is the first report of root rot in chickpea caused by F. falciforme in Mexico. This is important for chickpea producers and chickpea breeding programs.

First Report of Fusarium oxysporum Causing Root Rot in Ophiopogon bodinieri in China

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

First Report of Leaf Spot of Chenopodium album Caused by Fusarium equiseti in China

Chenopodium album L. (commonly known as bathu) is one of the most troublesome weeds associated with crops in the world. In June 2018, leaf spot on C. album was observed in soybean and corn fields in Yanshou County (45°26′N, 128°18′E), Heilongjiang Province, China, with 20 to 40% incidence. The disease was not observed on soybean or maize. Initial symptoms were brown necrotic spots with a white center. Lesions rapidly expanded (1 to 10 mm diameter), contained concentric rings, and became necrotic. Ten C. album leaves were collected for fungal isolations. Diseased tissues were surface sterilized with 0.1% HgCl₂ for 1 min, rinsed in sterile distilled water, and cultured on potato dextrose agar at 26°C. After 5 days, six fungal isolates were obtained and subcultured by transferring hyphal tips. The colony had abundant white aerial mycelia and became peach-orange in color with age on potato dextrose agar. Macroconidia were mostly five- to seven-septate, slightly curved with a tapered and elongated apical cell and prominent foot-shaped basal cell, 23.2 to 31.8 μm in length, and 3.1 to 4.92 μm in width on carnation leaf agar medium. No microconidia were observed. Chlamydospores were abundant in clumps or chains, ellipsoidal or subglobose. Morphological characteristics of the isolates were consistent with those of Fusarium equiseti (Jacobs et al. 2018). Genomic DNA was extracted from single conidial cultures of the representative isolate H5, and the internal transcribed spacer regions (ITS) and translation elongation factor 1-α gene (TEF1) were amplified with primers ITS1/ITS4 (White et al. 1990) and EF1-728F/EF1-986R (Akbar et al. 2018), respectively, and sequenced. The DNA sequence of the isolate was identical, and the representative isolate H5 was deposited in GenBank (accession nos. MN241064 and MK990601). BLAST analysis showed that the obtained sequence for the ITS amplicon shared 99.6% similarity with F. equiseti isolate KA (accession no. JQ690085.1), whereas the sequence of the TEF1 amplicon shared 99% similarity with F. equiseti strain G17WX1-5-2 (accession no. MG670196.1). To determine pathogenicity, C. album plants were grown in 15-cm pots containing a commercial potting mix (one plant/pot). After the eight-leaf stage, three plants (two leaves/plant) were inoculated with 50 μl of a conidial suspension (4 × 10⁶ spores/ml). Three plants treated with sterile distilled water served as a control. All plants were placed in a humidity chamber (>95% relative humidity) at 26°C for 48 h after inoculation and then kept in a greenhouse at 22/28°C (night/day). After 10 days, all inoculated leaves showed symptoms similar to those observed in the field. No disease occurred on control plants. Five same fungal isolates were reisolated from five diseased leaves and confirmed to be F. equiseti. To our knowledge, this is the first report of leaf spot on C. album caused by F. equiseti in China.

First Report of Onion Basal Rot Caused by Fusarium falciforme in Mexico

Basal rot is one of the most destructive diseases in onion (Allium cepa) growing regions worldwide. This disease can reduce crop yields by interfering with the absorption and translocation of water and nutrients, causing premature plant death (Delgado-Ortiz et al. 2016). In August 2016, unusual disease symptoms were observed in commercial onion crops (cv. Carta Blanca) within municipality of Angostura (Sinaloa, Mexico). These included yellowish and chlorotic leaves from the tip, progressively developing downward, dwarfing in the plants, soft consistency in bulbs, necrosis in the basal part of the bulb, and decrease in roots. Forty symptomatic onion plants were sampled. Tissue from roots and bulbs was plated on potato dextrose agar (PDA) medium. Typical Fusarium spp. colonies were obtained from all root and bulb samples. Three pure cultures were obtained by single-spore culturing (Fs05 to Fs07). On PDA the colonies were abundant white aerial mycelium, and light purple pigmentation was observed in the center of old cultures (Leslie and Summerell 2006). From 10-day-old cultures grown on carnation leaf agar medium, macroconidia were falciform, hyaline, three to five septate, with well-developed foot cells and blunt apical cells, and measured 27.3 to 46.3 × 2.1 to 7.1 μm (n = 40). The microconidia (n = 40) were hyaline, one to two celled, oval or reniform, 8.2 to 12.3 × 1.6 to 4.8 μm, and borne in false heads that measured 7.5 to 20 (average 13.75) μm × 2.3 to 9.0 (average 5.3) μm (n = 40); chlamydospores were not evident. The internal transcribed spacer and the translation elongation factor 1 alpha (EF1-α) genes (O’Donnell et al. 1998) were amplified by polymerase chain reaction and sequenced from the isolate Fs05 (GenBank accession nos. MH038177 and MH041264). Maximum likelihood analysis was carried out using the EF1-α sequence (MH041264) from the Fs05 isolate and other species from the Fusarium solani species complex (FSSC). Phylogenetic analysis revealed the isolate most closely related with F. falciforme (100% bootstrap). Pathogenicity tests were performed on onion plants (cv. Carta Blanca) grown on autoclaved vermiculite. Ten plants per isolate (n = 3) were inoculated by drenching with 20 ml of a conidial suspension (1 × 10⁵ CFU/ml) of each isolate per plant. The suspension was obtained by collecting the spores of each isolate grown on PDA, with 10 ml of an isotonic saline solution. Ten noninoculated plants served as controls. Plants were maintained for 60 days under greenhouse conditions. The assay was conducted twice. Bulb rot and necrosis similar to those observed on the infected plants in the field were observed on the bulbs. The pathogen was reisolated from the necrotic tissue from all inoculated plants and was identified by sequencing the partial EF1-α gene again and was identified as F. falciforme (FSSC 3 + 4) (O’Donnell et al. 2008) based on its morphological characteristics, genetic analysis, and pathogenicity test, fulfilling Koch’s postulates. The molecular identification was confirmed via BLAST on the FusariumID and Fusarium MLST databases. No symptoms were observed on noninoculated controls after 60 days. Although FSSC has been previously reported causing bulb rot in onion in Tukey, Japan, and Iran, to our knowledge this is the first report of basal rot in onion caused by F. falciforme in Mexico. This is important for onion producers and the Fusarium worldwide community. Also, Fusarium mycotoxin contamination of onion bulb could pose a public health risk.

First Report of Fusarium brasiliense Causing Root Rot of Dry Bean in the United States

Michigan is the second largest dry bean (Phaseolus vulgaris L.) producer in the United States and is ranked first in the production of black beans. Stand establishment and root rots are constant constraints in dry bean cultivation owing to susceptibility of germplasm. To determine which pathogens are responsible, surveys were conducted on Michigan State University research farms and in commercial dry bean fields in Michigan during 2014 to 2017. Fungi were isolated from symptomatic dry bean roots on water agar amended with metalaxyl (15 µg/ml) and streptomycin (300 µg/ml) at room temperature (22°C) under laboratory lighting conditions for 7 to 10 days. Single spore isolates were obtained from sporodochia, which developed on cultured root tissue. Fusarium isolates F-14-42, F-15-46, and F-15-174 were collected from Montcalm, Huron, and Gratiot Counties in Michigan, respectively. Colony morphology and color of isolates grown on full-strength potato dextrose agar at 25°C under dark conditions for 10 days displayed undulated margins, with variations in color of white, with tints of yellow, pink, or greyish-blue. On spezieller nahrstoffarmer agar or carnation leaf agar medium at 25°C under dark conditions for 10 days, sporodochia were a yellowish-white to pale yellow, and macroconidia were falcate, mostly four-septate, apex rounded or hooked ventrally, and base with a distinct foot cell (45.8 to 52.0 × 5.0 to 6.4 μm on average). DNA of each isolate was extracted using a cetyltrimethylammonium bromide method (Cubero et al. 1999), and the ribosomal intergenic spacer (IGS) region and partial translation elongation factor (EF-1α) were amplified by polymerase chain reaction with primer pairs NL11/CNS1 (Aoki et al. 2003) and EF1/EF2 (O’Donnell et al. 1998), respectively. A BLAST search of IGS and EF-1α sequences from the three isolates showed they were 100 and 99% identical with Fusarium brasiliense NRRL 22678 (GenBank accession nos. FJ919501 and AY320158). Phylogenetic analysis of IGS sequences of Fusarium solani species complex clade 2 revealed that the sequences of three isolates and two strains of F. brasiliense (NRRL 22678 and NRRL 31757) were clustered together in a group, which was supported by a bootstrap value of 97%. Based on morphological and sequence analysis, the isolates were identified as F. brasiliense (Aoki et al. 2005). All sequences obtained in this study were deposited into GenBank (accession nos. MG967556 to MG967561). To determine pathogenicity of isolates to dry bean, a seedling pathogenicity screening was performed on dark red kidney (cv. Red Hawk) and black (cv. Zorro) seed types. Each isolate was grown on Nash–Snyder medium at room temperature under laboratory lighting conditions for 21 days and inoculated to sterilized sorghum grain and incubated for 4 weeks to generate inoculum. Twelve grams of infested inoculum mixed with 250 ml of vermiculite was poured into a pot, with an additional 70 ml of vermiculite placed over the inoculum mixture. Six bean seeds were sown in each pot and covered with 70 ml of vermiculite. After 14 days, roots were harvested and washed for disease severity rating and measurement of dried root mass. Roots infected by F. brasiliense exhibited a large lesion of rot on the tap root, 20 to 50% reduction of root mass, and a disease severity rating of 4 on a 1 to 7 scale. The experiment was conducted twice with five replicate pots (six seeds in each pot) per experiment. The fungus was reisolated from the diseased roots and confirmed to be F. brasiliense. Soybean sudden death syndrome caused by F. brasiliense has been reported in Brazil and Argentina (Aoki et al. 2005; O’Donnell et al. 2010) and South Africa (Tewoldemedhin et al. 2017). Significantly, this is the first record of F. brasiliense causing root rot of dry bean in the United States.

FIRST REPORT OF FUSARIUM WILT OF LICORICE BY FUSARIUM OXYSPORUM IN IRAN

In September 2011, licorice (Glycyrrhiza glabra) plants were observed in a field in the Iranian province of Yazd that showed symptoms consisting of wilting, leaf necrosis, vascular discoloration, and death of the plant. Fragments of vascular tissues plated on potato dextrose agar amended with 0.5 g/l streptomycin sulfate yielded white to pale-violet fungal colonies that produced macro- and microconidia. In carnation leaf agar medium, the macroconidia were three-septate and sickle-shaped whereas microconidia were elliptical, and formed abundantly in false heads. The 5.8S subunit and flanking internal transcribed spacer of the rDNA gene were amplified using the ITS1/ITS4 primers (White et al., 1990) and sequenced. The ITS sequence was 99% similar to that of F. oxysporum (GenBank accession No. JQ045558.1), hence confirming the observed morphological traits typical of this species (Nelson et al., 1983). Wounded roots of 4-week- old plants of G. glabra were dipped for 10 min in a conidial suspension (1x105 spores/ml), while control plants were dipped in sterile tap water. Seedlings were transplanted into pots (13x15 cm) and maintained in a growth chamber at 25°C. Typical symptoms on the leaves developed after five weeks and F. oxysporum was successfully re-isolated from artificially infected plants. Control plants remained symptomless. Previously, F. oxysporum had been reported as a root rot agent of Russian licorice (Glycyrrhiza uralensis) in China (Cao et al., 2013). To our knowledge, this is the first report of F. oxysporum causing wilt of G. glabra in Iran.

First Report of Fusarium succisae Causing Flower Rot on Thread-leaf Coreopsis.

In July 2010, flower rot of thread-leaf coreopsis (Coreopsis verticillata) was found in a garden in the Icheon City, Korea. The disease affected about 20 to 50% of a 100 m2 area. The disease was characterized by the appearance of pinkish mycelia on the stigmata and inflorescences of flowers. In some cases, flowers failed to bloom or turned brown before opening fully. Fragments (each 5 × 5 mm) of the symptomatic tissue were surface-sterilized with 1% NaOCl for 1 min, and then rinsed twice in sterilized distilled water. The tissue pieces were placed on water agar (WA) and incubated at 25°C for 4 to 6 days. Twenty-two isolates of Fusarium species were obtained from the diseased flowers. All isolates were identified as Fusarium succisae based on their morphological characteristics on carnation leaf agar (CLA) medium and DNA sequences of the translation elongation factor 1-alpha gene (1). Macroconidia and sporodochia were sparsely produced on CLA medium. Microconidia were abundant, borne in false heads, oval or allantoid and sometimes pyriform, and measured 4.2 to 13 × 2.2 to 5.4 μm. Chlamydospores were absent. The EF-1α gene was amplified from three isolates by PCR assay and the amplification products were sequenced (2). The nucleotide sequences obtained were deposited in GenBank with accession numbers KF514658, KF514659, and KF514660. BLASTn analysis showed 99% homology with the EF-1α sequence of F. succisae NRRL13613 (GenBank Accession No. AF160291). Pathogenicity tests were conducted with inoculation of flowers on Coreopsis verticillata. Spore suspension was prepared by flooding 7-day-old cultures on potato dextrose agar with sterilized 2% (w/v) sugar solution. When the plants started to have buds, the isolates were inoculated by placing one drop (20 μl) of spore suspension (1 × 106 spores ml-1) into the buds. Fifteen buds of the plants were arranged into three replications. The control was treated with sterilized 2% sugar solution. Inoculated plants were kept in a greenhouse at 25/20°C (12 h/12 h). Three weeks after inoculation, the symptoms were observed on buds with mycelial production. Control plants had no mycelia on buds. F. succisae was re-isolated from the inoculated flowers. To our knowledge, this is the first report of flower rot of thread-leaf coreopsis caused by F. succisae.

First Report of Fusarium oxysporum f. sp. lycopersici Race 3 Causing Fusarium Wilt on Tomato in Korea.

In July 2010, fusarium wilt symptoms of tomato (Lycopersicon esculentum Mill.) plants were found in two commercial greenhouses in the Damyang area of Korea. Approximately 1% of 7,000 to 8,000 tomato plants were wilted and chlorotic in each greenhouse. The vascular tissue was usually dark brown and the discoloration extended to the apex. Fragments (each 5 × 5 mm) of the symptomatic tissue were surface-sterilized with 1% NaOCl for 1 min, then rinsed twice in sterilized distilled water (SDW). The tissue pieces were placed on water agar and incubated at 25°C for 4 to 6 days. Nine Fusarium isolates were obtained from four diseased plants, of which three isolates were identified as F. oxysporum based on morphological characteristics on carnation leaf agar medium and DNA sequences of the translation elongation factor 1-alpha (EF-1α) gene (2). Macroconidia were mostly 3- to 5-septate, slightly curved, and 28 to 53 × 2.8 to 5.2 μm. Microconidia were abundant, borne in false heads or short monophialides, generally single-celled, oval to kidney shaped, and 5 to 23 × 3 to 5 μm. Chlamydospores were single or in short chains. The EF-1α gene was amplified from three isolates by PCR assay using ef1 and ef2 primers (3), and the amplification products were sequenced. The nucleotide sequences obtained were deposited in GenBank (Accession Nos. KC491844, KC491845, and KC491846). BLASTn analysis showed 99% homology with the EF-1α sequence of F. oxysporum f. sp. lycopersici MN-24 (HM057331). Pathogenicity tests and race determination were conducted using root-dip inoculation (4) on seedlings of tomato differential cultivars: Ponderosa (susceptible to all races), Momotaro (resistant to race 1), Walter (resistant to races 1 and 2), and I3R-1 (resistant to all races). A spore suspension was prepared by flooding 5-day-old cultures on potato dextrose agar with SDW. Plants at the first true-leaf stage were inoculated by dipping the roots in the spore suspension (1 × 106 conidia/ml) for 10 min. Inoculated plants were transplanted into pots containing sterilized soil, and maintained in the greenhouse at 25/20°C (12/12 h). Twenty-four seedlings of each cultivar were arranged into three replications. An equal number of plants of each cultivar dipped in water were used as control treatments. Disease reaction was evaluated 3 weeks after inoculation, using a disease index on a scale of 0 to 4 (0 = no symptoms, 1 = slightly swollen and/or bent hypocotyl, 2 = one or two brown vascular bundles in the hypocotyl, 3 = at least two brown vascular bundles and growth distortion, 4 = all vascular bundles brown and the plant either dead or very small and wilted). All isolates caused symptoms of fusarium wilt on all cultivars except I3R-1, indicating that the isolates were race 3. The pathogen was reisolated from the discolored vascular tissue of symptomatic plants. Control plants remained asymptomatic, and the pathogen was not reisolated from the vascular tissue. Fusarium wilt of tomato caused by isolates of F. oxysporum f. sp. lycopersici races 1 and 2 has been reported previously; however, race 3 has not been reported in Korea (1). To our knowledge, this is the first report of isolates of F. oxysporum f. sp. lycopersici race 3 on tomato in Korea.