FUSARIUM-ID Database Research Articles

First Report of Fusarium oxysporum Causing Root Rot on Salvia yunnanensis in China.

The roots of Salvia yunnanensis, an herbaceous perennial widely distributed in Southwest China, is often used as a substitute for S. miltiorrhiza, a highly valued plant in traditional Chinese medicine (Wu et al. 2014). In June 2023, wilted plants of S. yunnanensis were observed in Wenshan City (23.20°N, 104.01°E), China. The average disease incidence was 40% to 65% and the total area affected by the disease was approximately 50 ha. The infected plants displayed wilted leaves, black necrotic lesions on roots, and eventually plant death. Fungal colonies with similar morphology were consistently isolated from the symptomatic roots. Eighteen monosporic isolates were individually cultured on potato dextrose agar (PDA) in separate petri dishes at 25 ± 1°C in darkness. After 7 days, the mycelia within the colonies exhibited a cottony texture and the colors ranged from white to pink or purple, while their reverse sides were white to purple. After 20 days incubation on carnation leaf agar (CLA) medium, spore characteristics of the isolates were evaluated (Zheng et al. 2024). On CLA medium, macroconidia had 2 to 5 septa, usually 3 septa, measuring 21.7 to 39.8 × 4.0 to 6.5 μm (n = 100). Microconidia were falciform, slightly curved or straight, measuring 6.8 to 15.4 × 2.5 to 5.4 μm (n = 100), with 0 to 1 septa. Chlamydospores were globose to subglobose, intercalary or terminal, with an average diameter of 8.9 μm (n = 100). Morphologically, the isolates were identified as Fusarium oxysporum (Lombard et al. 2019; Zheng et al. 2024). To confirm the identification, the translation elongation factor 1-α (EF1α) region was amplified with the primers EF-1/EF-2 (O'Donnell et al. 1998) and the RNA polymerase second largest subunit region (RPB2) was amplified with the primers fRPB2-6f/fRPB2-7cr (Eddouzi et al. 2013). The EF1α (GenBank accession no. PP805676) and RPB2 (PQ383276) sequences of isolate DS10-1 were compared with all sequences in the FUSARIUM ID database (O'Donnell et al. 2022) using polyphasic identification. The highest similarity (100%) was with F. oxysporum isolates, including the ex-epitype of Fusarium cugennagense isolate InaCC F984 (100% similarity). To further assess the phylogenetic relationships, a phylogenetic tree was constructed based on the Neighbor-Joining method in MEGA-X (Kumar et al. 2018). The tree confirmed that the isolate DS10-1 was closely related to F. oxysporum. Pathogenicity tests of the 18 isolates were conducted on five healthy one-year-old S. yunnanensis plants per isolate. Inoculum (1 ml of 106 conidia/ml) of each strain was brushed onto the roots of individual plants with a paintbrush. As controls, five plants were inoculated with sterile water. All plants were potted in plastic containers (diameter = 25 cm, five plants per pot) filled with a sterilized substrate mixture of sand and vermiculite (1:1, v/v), and maintained in the greenhouse at 20 to 26°C with 80% relative humidity. After 45 days, symptoms similar to those observed in the field were present on the roots of all plants inoculated with the 18 isolates, whereas the controls remained symptomless. The experiment was repeated two times with similar results. The same pathogens were consistently reisolated from inoculated roots and confirmed as F. oxysporum based on morphological and molecular analyses. F. oxysporum has been previously reported to cause root disease in various hosts, and to our knowledge, this is the first report of F. oxysporum causing root rot on S. yunnanensis in China. Therefore, subsequent crops should be grown to circumvent this disease.

Fusarium spp. causing invasive disease in humans: A case series from north India.

Owing to their inherent resistance to different classes of antifungals, early identification of Fusarium spp. is crucial. In this study, 10 clinical isolates were included from patients with invasive fusariosis involving lungs, sinuses, or both. Clinico-radiological data were collected. Samples were processed by standard laboratory procedures. Three gene regions (ITS, TEF1, and RPB2) were amplified by PCR for multilocus sequencing. Fusarium MLST, FUSARIUM-ID, and FUSARIOID-ID databases were used for final identification. Antifungal susceptibility testing was performed by broth microdilution following CLSI M38-A3 and Sensititre™ YeastOne™ YO9 plate. Pulmonary involvement was seen in all patients, and sino-nasal involvement was present in six. Radiologically, consolidations and cavitations were present in eight and six cases, respectively. Halo sign was present in six; reverse halo sign was also found in three of them. Direct microscopy showed septate hyphae that were morphologically different from those found in aspergillosis. Results of the molecular identification were as follows: two Fusarium irregulare, one Fusarium pernambucanum, one Fusarium incarnatum, one Fusarium sp. FIESC 30, two Fusarium keratoplasticum, one Fusarium falciforme, one Fusarium pseudonygamai, and one Fusarium delphinoides. For both Fusarium solani (FSSC) and Fusarium incarnatum-equiseti (FIESC) species complexes, amphotericin B had the lowest minimum inhibitory concentrations (MICs). Importantly, for terbinafine, all FIESC isolates had low MICs, while FSSC isolates had high MICs. In some cases, early identification of Fusarium spp. is possible by means of morphology of hyphae on direct microscopy and findings on radiology. Molecular identification, at least to the species complex level, is crucial for the choice of antifungals.

Characterization of Fusarium solani Associated with Tobacco (Nicotiana tabacum) Root Rot in Henan, China.

The aim of this study was to characterize the Fusarium solani species complex (FSSC) population obtained from tobacco roots with root rot symptoms by morphological characteristics, molecular tests, and assessment of pathogenicity. Cultures isolated from roots were white to cream with sparse mycelium on potato dextrose agar, with colony growth of 21.5 ± 0.5 to 29.5 ± 0.5 mm after 3 days. Sporodochia were cream on carnation leaf agar (CLA) and Spezieller Nährstoffarmer agar (SNA), and macroconidia formed in sporodochia were 3 to 6 septate and straight to slightly curved, with wide central cells, a slightly short blunt apical cell, and a straight to almost cylindrical basal cell with a distinct foot shape, ranging in size from 20.92 to 64.37 × 3.91 to 6.57 μm. Microconidia formed on CLA were reniform and fusiform, with 0 or 1 to occasionally 2 septa, that formed on long monophialidic conidiogenous cells, with a size range of 5.99 to 32.32 × 1.76 to 5.84 μm. Globose to oval chlamydospores were smooth- to rough-walled, 6.5 to 13.3 ± 0.37 μm in diameter, and terminal or intercalary and occurred singly, in pairs, or occasionally in short chains on SNA. Molecular tests consisted of sequencing and phylogenetic analysis of the translation elongation factor-1 alpha (EF-1α), RNA polymerase II largest subunit, and second largest subunit regions. All the obtained sequences revealed 98.14 to 100% identity to F. solani in both Fusarium ID and Fusarium MLST databases. Phylogenetic trees of the EF-1α gene and concatenated three-locus data showed that isolates from tobacco in Henan grouped in the proposed group 5, which is nested within FSSC clade 3 (FSSC 5). Twenty-seven of the 28 isolates caused root rot in artificially inoculated tobacco seedlings, with a disease severity index ranging from 15.00 ± 1.67 to 91.11 ± 2.22. Cross-pathogenicity tests showed that three representative isolates were virulent to six species of Solanaceae and two species of Poaceae, with disease severity indexes ranging from 6.12 ± 0.56 to 84.44 ± 0.00, indicating that these isolates have a wide host range. The results may inform the control of tobacco root rot through improved crop rotations.

Identification of Fusarium spp. Associated with Chickpea Root Rot in Montana

Root rot caused by Fusarium spp. is a significant issue in the chickpea-growing regions of Montana. The specific Fusarium species responsible for the disease and their prevalence remain uncertain. A survey was conducted in 2020 and 2021 to identify Montana’s Fusarium species associated with chickpea. Four hundred and twenty-six Fusarium isolates were recovered from symptomatic chickpea roots across ten counties in the state. Isolates were identified by comparing translation elongation factor 1-α (TEF1-α) sequences in the FUSARIUM-ID database. Among the recovered isolates, Fusarium oxysporum was the most prevalent species (33%), followed by F. acuminatum (21%), F. avenaceum (15%), F. redolens (14%), F. culmorum (6%), F. sporotrichioides (6%), Neocosmospora solani (6%), F. equiseti (2%), F. torulosum (0.9%), F. gamsii (0.8%), F. proliferatum (0.2%), F. pseudograminearum (0.2%), and F. brachygibbosum (0.1%). The aggressiveness of a subset of 51 isolates representing various Fusarium spp. was tested on chickpea cv. ‘CDC Frontier’. A non-parametric variance analysis conducted on disease severity ranks indicated that F. avenaceum isolates were highly aggressive. This study reports for the first time that F. gamsii, F. proliferatum and F. brachygibbosum are causal agents of root rot in chickpea in the United States. This knowledge is invaluable for making informed decisions regarding crop rotation, disease management, and developing resistant chickpea varieties against economically significant Fusarium pathogens.

First report of Fusarium falciforme causing root rot and wilt on strawberry in Sinaloa, Mexico.

In Mexico strawberry production has great economic importance for the local and export markets as the country is the main strawberry supplier to the United States (SIAP, 2020). In 2022, strawberry plants with yellowing and wilting leaves, root rot and wilting, necrosis of vascular bundles and small fruits symptoms were observed in different commercial fields in the north-central Mexican state of Sinaloa, causing yield losses of about 10%. Typical Fusarium spp. colonies were recovered from all samples. They produced abundant white aerial mycelium with cream to orange pigment and branched septate hyphae (Fig. 1) (Leslie and Summerell, 2006). A total of 18 monosporic isolates were obtained by serial dilutions. The 18 isolates grown for 10 days on carnation leaf agar (CLA) produced hyaline microconidia with 0-2 septa, measuring 9.2 - 15.4 by 4.5 - 6.5 μm (n = 40) and hyaline macroconidia with three septa that measured 28.4 - 53.5 by 4.5 - 9 μm (n = 40). Chlamydospores were not observed. A fragment of the translation elongation factor 1-alpha (EF1-alpha) gene was amplified by polymerase chain reaction (PCR) using the primer pair EF-1/EF-2 (O'Donnell et al. 1998) from two monosporic isolates. The sequences were registered in the NCBI GenBank under accession numbers OR878541 and OR878543 (FRESIN178 and FRESIN194). BLASTn queries of NCBI GenBank identified the sequences as F. falciforme with 98% and 100% similarity to accession numbers OQ262968 and DQ246941 respectively. Fusarium ID database also identified the sequences as F. falciforme, is a member of the F. solani species complex (FSSC). Phylogenetic analysis revealed the partial EF1 sequences grouped with F. falciforme (Fig. 2). A pathogenicity test was performed on thirty strawberry plants (cv. Cabrillo) grown in sterile vermiculite. The plants were inoculated by immersing roots in 20 mL of a conidial suspension (1 × 105 conidia/mL) of isolate FRESIN194. Twelve uninoculated plants served as the control. All plants were grown for 60 days under greenhouse conditions (28 to 35°C). The assay was repeated twice. After 50 days, symptoms of root rot and wilting leaves like those observed in the field were observed. Uninoculated control plants did not develop symptoms. The fungus was reisolated from necrotic tissues of the inoculated plants and identified as F. falciforme by sequencing the EF1-alpha gene and morphological characteristics, completing Koch's postulates. Fusarium falciforme has been reported as the causal agent of root rot, stem rot, and wilt of tomato, papaya, chickpea, onion, common bean, and maize in Mexico (Díaz-Najera et al. 2021, Douriet-Angulo et al. 2021, Felix et al 2022, Tirado-Ramírez et al 2018, Vega-Gutiérrez et al. 2019a, Vega-Gutiérrez et al. 2019b). To our knowledge this is the first report of F. falciforme causing root rot and wilt on strawberry in Sinaloa, Mexico. This result provides useful information for the development and implementation of disease control strategies to mitigate damage caused by F. falciforme.

First Report of Fusarium pernambucanum Causing Fruit Rot of Melon in China.

In May of 2020, November of 2021 and May of 2022, a preharvest fruit rot with white mycelia was observed inside and outside of the fruits of thick skin muskmelon (Cucumis melo L.) growing in about ten greenhouses (each greenhouse had about 320 muskmelons) with disease incidence of 70% in Ningbo, Zhejiang Province of China. In order to identify the causal agent, plant tissues from the margin of the symptomatic tissue were sterilized for 1 min with 1% sodium hypochlorite (NaClO), 2 min with 75% ethyl alcohol, rinsed in sterile distilled water three times (Zhou et al 2019), and then placed on potato dextrose agar (PDA) plates containing streptomycin sulfate (100 μg/mL) at 25℃ for 4 days. Only Fusarium colonies were isolated from all the plant tissues. The growing hyphae were transferred to new PDA plates using the hyphal tip method, putative Fusarium colonies were purified by single-sporing. Six fungal isolates (Fi-1~6) were obtained. The average radial mycelial growth rate of Fusarium isolate Fi-3 was 4.6 mm/day at 25℃ in the dark on PDA, and like other five isolates. The colonies are abnormal, producing lots of aerial hyphae, each isolate was white to light orange. Isolate Fi-3 produced macroconidia with 4 to 6 septa, tapered with pronounced dorsiventral curvature and measured 21 to 30 μm long 4 to 5 μm wide on Spezieller Nährstoffarmer Agar (SNA) medium at 25℃ for 10 days (Leslie and Summerell 2006), but polyphialides and chlamydospores were still not available for 30 days. The pathogen species was further identified by translation elongation factor-1 alpha (EF-1α) sequencing. The EF-1α of six isolates were sequenced, and their EF-1α sequences were 100% identical to each other, and the sequence of strain Fi-3 was deposited in GenBank with accession no. OL782040 and was also compared with sequences in the FUSARIUM-ID database (Geiser et al. 2004), which indicated that it was 100% identical to those of F. pernambucanum strain NRRL 32864 (GenBank accession GQ505613), F. pernambucanum strain LC7040 (GenBank accession MK289626), and F. pernambucanum strain LC12149 (GenBank accession MK289588) within the Fusarium incarnatum - F. equiseti species complex 17 (FIESC17). Two phylogenetic trees were established based on the TEF1-α sequences of Fi-1~6 and other Fusarium spp., Fi-1~6 was clustered with the sequences of F. pernambucanum within the FIESC17. Thus, both morphological and molecular criteria supported identification of the strain as F. pernambucanum. A pathogenicity test was conducted to verify Koch's postulates, mycelium agar plugs (6 mm in diameter) were removed from the colony margin of a 3-day-old culture of strain Fi-3, healthy melon fruits were surface-sterilized with 70% ethanol and rinsed twice with sterile-distilled water. Then, the melons were wounded using a sterile inoculating needle to stab and inoculated by a mycelium agar plug of strain Fi-3 on the wound sites. 5 fruits were inoculated in each treatment, and a mycelium-free PDA plug was used as a negative control, repeated 3 times, at 25℃ with high relative humidity for 10 days. The results show disease symptoms similar to those naturally infected fruits on all inoculated melon fruits. The fungus re-isolated from the diseased fruits, showed the same colony morphology as the original isolate. Koch's postulates were repeated three times with the same results. Strain Fi-3 inoculated fruits without wounding remained healthy. To our knowledge, this is the first report of fruit rot of melon caused by F. pernambucanum in China.

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

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

First Report of Fusarium proliferatum Causing Bulb Rot on Lilium davidii var. willmottiae in China.

Lilium davidii var. willmottiae, known as Lanzhou lily, is widely cultivated in China for its edible bulbs. In July 2023, symptoms of bulb rot were observed on Lanzhou lilies harvested from Lanzhou, Gansu Province, during storage at the Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences (Beijing, China), at an incidence of nearly 70%. The surface of the lily scales had dark water-stained spots, after the development of which the color gradually darkened, the bulbs became soft, accompanied by a pungent smell. Finally, the whole bulb became ruined and rotten, and there were thick mycelium layers on the bulbs. The infected bulbs were washed with clean water, sterilized with 75% ethanol for 30 s and 2% sodium hypochlorite for 5 min, and then rinsed three times with sterile distilled water. The 5 mm×5 mm tissue pieces from the junction of the diseased part and the healthy part were clipped, placed on potato dextrose agar (PDA) medium and subsequently incubated at 25 °C. Pure cultures were obtained by transferring hyphal tips to new PDA plates. A total of 10 fungal isolates were obtained, all of which exhibited typical Fusarium characteristics. The colonies were white to pink with white to cream-colored aerial mycelia. After 10 to 15 days of incubation, the macroconidia (n = 50) were hyaline, relatively slender with a curve, three to five septate, and 8.73 to 33.24 × 2.16 to 4.19 μm in length. The microconidia (n = 50) were hyaline and pyriform, without septa, and measured 4.04 to 8.48 × 1.24 to 2.65 μm. These morphological characteristics were similar to those described for Fusarium proliferatum (Leslie and Summerell 2006). For molecular identification, a cetyltrimethylammonium bromide (CTAB) protocol was used to extract total genomic DNA (O'Donnell et al., 1998), after which the internal transcribed spacer (ITS), translation elongation factor subunit 1-alpha (TEF1-α) and RNA polymerase Ⅱ subunit 2 (RPB2) genes were amplified using the universal primers ITS1/ITS4, EF1/EF2 and RPB2-5f2/fRPB2-7cr, respectively, and subsequently sequenced (White et al., 1990; O'Donnell et al., 1998; Liu et al., 1999; Reeb et al., 2004; O'Donnell et al., 2007; Jiang et al., 2018). The sequences of a representative isolate (CAAS01) were analyzed and submitted to GenBank under accession numbers OR554007 (ITS), OR594233 (TEF1-α) and OR603932 (RPB2). A BLAST analysis revealed that the sequences of the ITS, TEF1-α, and RPB2 genes shared 100%, 100%, and 100% identity, respectively, with those of Fusarium proliferatum (MT466521.1, MK952792.1, and LT841266.1) in GenBank. In addition, the ITS, TEF1-α and RPB2 sequences shared 100%, 100%, and 100% identity with those of Fusarium annulatum (LC13675, the Fusarium fujikuroi species complex; previously known as the Gibberella fujikuroi species complex) in the Fusarium-ID database. Fusarium proliferatum, whose common synonyms are Gibberella fujikuroi mating population D and Gibberella fujikuroi var. intermedia, is the anamorphic form of the Gibberella fujikuroi complex that belongs to the Nectriaceae family. A phylogenetic tree was constructed based on the combined TEF1-α and RPB2 sequences of CAAS01 and other Fusarium isolates, revealing that CAAS01 was grouped with Fusarium proliferatum. Based on sequence alignment and phylogenetic analysis, the isolate was identified as Fusarium proliferatum. To determine the pathogenicity of the isolated fungi, healthy bulbs were punctured with disposable sterilized needles and soaked in equal amounts of sterile water and conidial suspension (1×107 conidia/mL) for 30 min respectively. The pathogenicity experiment was repeated three times. After 7 days of inoculation at 25 °C and 80% relative humidity, the surface of the inoculated bulbs produced water-stained spots and mycelium layers consistent with the symptoms exhibited by Lilium davidii var. willmottiae bulbs during storage, while the uninoculated lily bulbs remained symptomless. Fusarium proliferatum was reisolated from the infected bulbs and identified based on morphological and molecular characteristics, fulfilling Koch's postulates. To our knowledge, this is the first report of bulb rot on Lilium davidii var. willmottiae caused by Fusarium proliferatum in China. This study will contribute to the development of management strategies for this postharvest disease in Lilium davidii var. willmottiae.

First report of Fusarium equiseti causing bulb rot on lily (Lilium 'White planet' ) in China.

Lily (Lilium spp.) is one of the main ornamental plants grown in the world. In addition, bulbs of lily have been extensively used as edible and medicinal herbs in northern and eastern Asia, especially in China (Yu et al. 2015; China Pharmacopoeia Committee 2020; Tang et al. 2021). In August of 2021, a disease of stem and leaf rot was observed on lily cultivar 'White planet' with approximately 25% disease incidence in the greenhouse and fields at the Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences (Beijing, China). The bulbs of symptomatic plants were brown and rotten, with sunken lesions. Symptomatic plants showed short, discolored leaves, and eventually lead to stem wilt and death of the whole plants. Infected bulbs were surface sterilized in 75% ethanol for 30 s, then in 2% sodium hypochlorite for 5 min, and rinsed three times with sterile distilled water. A 0.5×0.5 cm2 tissue piece was then placed on potato dextrose agar (PDA) medium and incubated at 25±1℃. After 5 days, the isolate was purified by single spore isolation technique. The singled-spored fungal colony was characterized by fluffy white aerial mycelia, and produced orange pigments with age. After seven days on Spezieller Nahrstoffarmer agar (SNA), conidia produced from simple lateral phialides. Macroconidia have pronounced dorsiventral curvature typical, significantly enlarged in the middle, a tapered whip-liked pointed apical cell and characteristic foot-shaped basal cell, 3 to 6 septate, measuring 18.71 to 43.01×2.89 to 5.56 μm with an average size of 26.98×3.90 μm (n=30). Microconidia were not observed. Typical verrucose thick chlamydospore with rough walls were profuse in chains or clumps, ellipsoidal to subglobose. These morphological characteristics were consistent with Fusarium spp. (Leslie et al. 2006). For molecular identification, the internal transcribed spacer (ITS), translation elongation factor subunit 1-alpha (TEF1-α) and RNA polymeraseⅡsubunit 2 (RPB2) genes were amplified using primers ITS1/ITS4, EF1/EF2 and 5F2/7cR respectively and sequenced (White et al. 1990; Jiang et al. 2018; O'Donnell et al. 2007). Sequences were submitted to GenBank under accession numbers OM078499 (ITS), Accession OM638086 (TEF1-α) and OM638085 (RPB2). BLAST analysis showed that ITS, TEF1-α and RPB2 sequences shared 100%, 99.8%, 99.2% identity to F. equiseti (OM956073, KY081599, MW364892) in GenBank, respectively. In addition, ITS, TEF1-α and RPB2 sequences shared 100%, 99.53%, 100% identity with Fusarium lacertarum (LC7927, Fusarium incarnatum-equiseti species complex) in the Fusarium-ID database. Based on the morphological characteristics and molecular sequences, the isolates were identified as Fusarium equiseti. A pathogenicity test was performed on potted lily ('White planet') under greenhouse conditions (25±1℃ with a 16 h light and 8 h dark cycle). Three healthy lily bulbs were selected and one bulb was planted in each pot filled with sterilized soil. Each pot was inoculated with 5 mL of conidia suspension (1×107 conidia/mL) in te soil around bulbs with a stem length of 3 cm, with an equal amount of sterilized water as a control. This test had three replicates. After 15 days of inoculation, typical symptoms of bulb rotten, like those observed in the greenhouse and fields, developed on the inoculated plants but not on the controls. The same fungus was consistently reisolated from the diseased plants. To our knowledge, this is the first report that F. equiseti caused bulb rot on Lilium in China. Our result should help with future monitoring and control of lily wilt disease.

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.

First report of Fusarium oxysporum causing stem spots on Polygonatum odoratum in China.

First report of Fusarium oxysporum causing stem spots on Polygonatum odoratum in China.

Fusarium diversity from the Golden Gate Highlands National Park.

Members from the genus Fusarium can infect a broad range of plants and threaten agricultural and horticultural production. Studies on the diversity of Fusarium occurring in natural ecosystems have received less attention than the better known phytopathogenic members of the genus. This study identified Fusarium species from soils with low anthropogenic disturbance found in the Golden Gate Highlands National Park (GGHNP), a part of the Drakensberg system in South Africa. Selective techniques were implemented to obtain 257 individual isolates from the selected soil samples for which the translation elongation factor 1α (tef-1α) gene region was sequenced and compared against the Fusarium MLST and FUSARIUM-ID databases. Phylogenetic analyses, based on maximum likelihood and Bayesian inference, were used to determine species diversity in relation to reference isolates. Species level identifications were made within three of the seven species complexes and identified F. brachygibbosum, F. sporotrichioides, F. andiyazi, and F. gaditjirri based on the FUSARIUM-ID database, with F. transvaalense and F. lyarnte identified against the Fusarium MLST database. This indicated highly diverse populations of Fusarium from soils with low anthropogenic disturbance from the Afromontane grassland region found in mountain ranges.

First Report of Fusarium falciforme Causing Leaf Blight in Acacia mangium in China.

Acacia mangium Willd. is an important economic tree species with diverse uses and high ecological value, used in agriculture, restoration of degraded lands, for ornamental purposes, and commercial forestry (Leroy et al. 2009). This tree is widely cultivated in tropical and subtropical regions of China, such as Hainan, Guangdong, and Guangxi Provinces. In July 2021, a leaf blight of A. mangium saplings was observed with widespread distribution in the cultivation base of Hainan University (20° 3' 33″ N, 110°18' 56″ E) in Hainan Province, China. The initial symptoms were brown-to-black, irregular-shaped lesions on the leaf margin or tip. The entire leaf wilted and turned brown, leading to death and defoliation in the later periods of the disease (Fig. 1A-C). Of the 50 trees surveyed, the incidence rate was 68%. Small pieces of diseased tissue were collected from six different infected trees. The tissue surface was disinfested with 75% ethanol for 30 s, and 1% mercury chloride for 1 min, then rinsed three times with sterile distilled water. Infected tissues were placed onto potato dextrose agar (PDA) containing 0.01% streptomycin and incubated at 25°C for 7 days. Seventeen isolates were obtained by single-spore isolation on fresh PDA plates, and 15 isolates were identified morphologically as Fusarium spp. (Leslie et al. 2006). The colony surface was white, and the reverse side yellowish (Fig. 1D-E). Microconidia were oval or kidney-shaped, 6.2 to 9.6 × 2.5 to 6.3 μm (n=60). Macroconidia were sickle-shaped, with three to five septa, 19.5 to 41.2 × 3.7 to 6.8 μm (n=60) (Fig. 1F). For molecular identification, genomic DNA of three isolates (HNKFS01, HNKFS02, and HNKFS03) was extracted using E.Z.N.A.® HP Plant DNA kit (Omega Bio-Tek) and the internal transcribed spacer of rDNA (ITS), translation elongation factor 1-α (EF1-α), RNA polymerase II beta subunit (RPB2), and β-tubulin (TUB) regions were amplified and sequenced using primers ITS1F/ITS4, EF-1/EF-2, RPB2-5F2/11aR, and T1/T2, respectively (O'Donnell et al. 2010; O'Donnell and Cigelnik 1997; White et al. 1990). Sequences were deposited in GenBank for ITS (OM289152 to OM289154), EF1-α (OM289155 to OM289157), RPB2 (ON193365 to ON193367), and TUB (ON193314 to ON193316). Using BLASTn, ITS sequences matched 98.88% (532/538 bp) to F. falciforme (MT114705), EF1-α sequences matched 99.58% (703/706 bp; MK752502), RPB2 sequences matched 98.31% (988/1005 bp; MW691193), and TUB gene sequences matched 99.10% (332/335 bp; OK087483). Polyphasic identification with ITS, EF1-α, RPB2, and TUB sequences revealed a 98.51% match with F. falciforme (NRRL 32729) sequences in Fusarium-ID databases. Pathogenicity of 15 isolates was determined by using healthy one-year-old A. mangium seedlings. Three leaves from each seedling were selected to test. Mycelial plugs of each isolate were inoculated on one leaf (three plugs per leaf). An agar plug without the fungus was placed on a leaf as a control. The pathogenicity tests were repeated twice. After 7 days of incubation in a greenhouse, all leaves inoculated with the pathogen showed black lesions with white flocculent hyphae (Fig. 1H), while control leaves were asymptomatic (Fig. 1G). Typical colonies of F. falciforme were isolated from all inoculated leaves, and identified by morphology and ITS, EF1-α, RPB2, and TUB sequence analysis, fulfilling Koch´s postulates. To our knowledge, this is the first report of F. falciforme causing leaf blight in A. mangium in China and the world. This disease can lead to death of A. mangium, which seriously restricts commercialization, requiring that management strategies be adopted.

First Report of Fusarium proliferatum Associated with Leaf Yellow on Alternanthera philoxeroides in China.

Alternanthera philoxeroides (Mart.) Griseb is a highly invasive weed commonly found in rice fields in China. In May 2021, leafyellowing was observed on this weed (about 10 ha) in Zhanjiang (21°19'N, 110°20'E), Guangdong Province, China. Disease incidence was approximately 20% (n = 100 investigated plants). Ten yellow leaves from 10 plants were sampled, surface-sterilized with 75% ethanol for 30 s, followed by 2% NaClO for 5 min. The leaves were rinsed three times in sterile distilled water and four sections of each leaf were placed onto potato dextrose agar (PDA). Pure cultures were obtained by transferring hyphal tips to new PDA plates. Twenty-two isolates of Fusarium ssp. (69% of the isolates) were obtained from 55% of the leaf samples. Three representative single-spore isolates (APF-1, APF-2, and APF-3) were used for further study. Colonies were white to pink on PDA. Conidiogenous cells were monophialidic or polyphialidic. Macroconidia were slightly curved, tapering apically with three to five septa, and measured from 32.5-55.8 μm × 2.5-5.1 μm in size (n=50). The morphological features of these fungi were noted to be in line with those ofFusariumproliferatum(Leslie and Summerell, 2006). For molecular identification, a colony PCR method (Lu et al. 2012) was used to amplify the internal transcribed spacer (ITS) and portions of elongation factor 1-α (EF1-α), RNA polymerase II largest subunit (RPB1), and RNA polymerase II second largest subunit (RPB2) genes using primers ITS1/ITS4, EF1-728F/EF1-986R,RPB1-R8/RPB1-F5, and RPB2-7CF/fRPB2-11aR, respectively (O'Donnell et al. 1998; O'Donnell et al. 2010). The sequences were submitted to GenBank under accession numbers MZ026797-MZ026799 (ITS) and MZ032209-MZ032217 (RPB1, RPB2, EF1-α). The sequences of the three isolates were 100% identical (ITS, 537/537 bp; RPB1, 1606/1606 bp; RPB2, 770/770 bp and EF1-α, 683/683 bp) with those of F. proliferatum (accession nos. MT378328, MN193921, MH582196, and MH582344) through BLAST analysis. Analysis of the sequences revealed a 99.87 - 100% identity with the isolates of theF. proliferatum (F. fujikuroi species complex, Asian clade) by polyphasic identification using theFUSARIUM-ID database (Yilmaz et al. 2021). The sequences were also concatenated for phylogenetic analysis by the maximum likelihood method. The isolates clustered withF. proliferatum. Pathogenicity was tested through in vivo experiments. The inoculated and control plants (n = 5, 30 days old) were sprayed with a spore suspension (1 × 105 per mL) of the three isolates individually and sterile distilled water, respectively, until run-off (Feng and Li. 2019). The test was performed three times. The plants were grown in pots in a greenhouse at 25 °C to 28 °C, with relativehumidity of approximately80%. Yellowing was observed on the inoculated plants after 7 days, while the control plants remained healthy. The pathogen re-isolated from all the inoculated plants was identical to the inoculated isolates in terms of morphology and ITS sequences. No fungi were isolated from the control plants. To the best of our knowledge, this study is the first to report F. proliferatum causing yellow symptoms on A. philoxeroides. The fungus has some potential biological control properties, but its host range needs to be further determined.

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

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

First Report of stem rot in Cymbidium sinense Caused by Fusarium oxysporum (FOSC) in China.

Cymbidium sinense (Jackson ex Andr.) Willd is a perennial terrestrial plant in the orchid family mainly distributed in China, Japan, India and Southeast Asia that occupies a strong position in the flower market due to its bright green leaves and fragrant flowers (Zhang et al. 2013). Cymbidium sinense is not only valued by people for its ornamental and economic value, but its roots have antiasthmatic medicinal properties (Ke et al. 2004). In August 2020, about 15% stem rot on two-year old C. sinense with varying severity was observed in five nursery gardens located in Enshi city (N 30° 16', E 109° 29'), Hubei province, China. Typical symptoms of C. sinense included roots and inner part of the pseudobulbs changing from white to brown and rotting. Leaves became brown and withered from bottom to top, and there was an obvious blight yellow halo at the junction of diseased and healthy tissue, which eventually caused the whole plant to wilt and die (Fig. 1d). To isolate the pathogen, a total of 15 leaf tissues from the disease-health junction (3 × 3 mm) from 5 individual plants (3 leaves/plant) with symptoms were surface sterilized with 75% ethanol for 30 s and 2% sodium hypochlorite (NaOCl) for 3 min. The sterilized tissue was rinsed three times with sterilized water, and then placed on potato dextrose agar (PDA) for incubation at 28°C in the dark for 5 days. Isolated colonies were subcultured by a hyphal tip protocol. Thirteen fungal isolates were obtained. Through preliminary pathogenicity tests, we found that ten isolates induced leaf blight. These ten isolates with pathogenicity showed similar morphological characteristics, with initial white-flocculent aerial mycelium that secreted a lavender pigment and produced colonies with an irregular edge after 3 days on PDA. The ten strains were cultured on PDA plates at 28℃ for 5 and 15 days to observe colony and conidial characteristics. The ten strains were identified as Fusarium based on morphological characteristics (Leslie and Summerell 2006). Strain ML0303 was selected for further identification. Macroconidia were falciform, hyaline, slightly pointed at both ends with two to four septa, 24.0 ± 5.6 µm × 4.7 ± 0.8 µm (n = 50). Microconidia were hyaline, oval, globose, with zero to one septum, 5.5 ± 1.3 µm × 2.2 ± 0.5 µm (n = 50) (Fig. 1c). Total genomic DNA of strain ML0303 was extracted with a CTAB protocol (Stenglein and Balatti 2006). The translation elongation factor (EF-1α), RNA polymerase II second largest subunit (RPB2) and β-tubulin (Tub2) genes were amplified respectively using primer pairs EF1/EF2, RPB2-5F2/RPB2-7cR and T1/T22 respectively (O'Donnell. et al. 2010, O'Donnell. et al. 1997). The EF-1α, RPB2 and Tub2 (accession numbers-MW719874, OL614838, OL689398, respectively) gene sequences were submitted to GenBank. EF-1α, RPB2 and Tub2 sequences of ML0303 showed 99.5% - 100% identity respectively with Fusarium oxysporum in the Genbank and FUSARIUM-ID databases. The multilocus sequence data was used to infer a phylogenetic tree via a Neighbor-joining (NJ), Maximum-likelihood (ML) and Maximum-Parsimony(MP) together with reference sequences from GenBank. The topology of the three trees was similar; only the NJ tree is presented here. Strain ML0303 and F. oxysporum formed a clade supported with high values (NJ/ML/MP: 96,95,97). The results indicated that the fungus was F. oxysporum based on the phylogenetic analysis and BLASTn queries. For pathogenicity tests, conidia of strain ML0303 were collected by rinsing PDA plates. Two-year-old C. sinense grown in plastic pots filled with sterilized autoclaved sandy loam soil were used for the tests. Three pots (two plants/pot) were included in each treatment. Spore suspensions (106spores/ml) of strain ML0303 were used to irrigate the stem-zone of the plants, and sterile water was used as control. The two treatments were placed in a greenhouse and incubated at 28±2℃ with a 14-hour light/10-hour dark cycle. The experiment was repeated twice. After three weeks, stem rot symptoms were observed on C. sinense inoculated with ML0303, that were the as same as observed in the nursery (Fig. 1e-h). No symptoms were observed on the negative control. Fusarium oxysporum was re-isolated from the infected plants to fulfill Koch's postulates. Partial EF-1α and RPB2 gene sequences were used for molecular identification. Members of the FOSC are notorious for causing many diseases, which includes stem rot of Sulcorebutia heliosa and root rot of Torreya grandis (Garibaldi et al. 2020; Zhang et al. 2016). To our knowledge, this is the first report of stem rot by F. oxysporum on C. sinense in China. The finding of this pathogen provides a clear target for stem rot control.

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 Damping off and Seedling Rot of Hemp (Cannabis sativa L.) caused by Fusarium solani (Mart.) Sacc. in North Dakota, USA.

Hemp (Cannabis sativa L.) is grown for medicinal and industrial uses. Symptomatic hemp (Mountain Mango) seedlings were received from a grower's greenhouse in Towner County (48.7486° N, 99.2761° W), North Dakota (ND), USA in July 2020. Seedlings had brown to blackish root tips, thread-like hypocotyl rot and seedling collapse, with about 8 to 10% disease incidence. Roots were surface disinfested in 1% sodium hypochlorite for 1 min, rinsed thrice with sterile distilled water, and blotted dry. About 1-cm sectioned root tips were plated on water agar (WA) and acidified potato dextrose agar (PDA, pH: 4.8) media and incubated under fluorescent light with a 12-h photoperiod at 25° C. After 7 days, single spores were isolated and sub-cultured on PDA and carnation leaf agar for morphological observations (Dhingra and Sinclair, 1995). Colonies had uniform appearances and produced white, thick and floccose mycelium. Conidiophores produced from lateral hyphae were simple to branched. Phialides were slender, smooth, hyaline and septate. Macro-conidia were 12.5 to 30.2 x 2.2 to 3.6 µm, septate (3-5), thick walled, hyaline and moderately curved shaped. Micro-conidia were oval to ellipsoid, smooth walled, no septa and measured 3.4 to 8.8 μm and 1.3 to 4.3 μm. Chlamydospores were round shaped, thick-walled, and produced singly or in pairs. Based on morphological characteristics, isolates were identified as Fusarium solani (Mart.) (Carbone and Kohn 1999; Leslie and Summerell 2006). For molecular identification, genomic DNA of three representative fungal isolates were extracted using DNeasy Plant Mini Kit (Qiagen, Hilden, Germany). PCR was done using both the primers (EF1/EF2) of the translation elongation factor (TEF-1α) and primers 5F2/7cR and 7cF/11aR of the RNA polymerase II (RPB 2) for Fusarium species (O'Donnell et al. 2009 and 2010). All isolates had identical PCR product sequences for the respective primer sets. The DNA sequences were deposited to NCBI GenBank with accession No. OK880264 (TEF-1α), OK880266 (RBP 5F2/7cR), and OK880265 (RBP 7cF/11aR). The NCBI Megablast search of the OK880264, OK880266, and OK880265 showed 100% similarity with respective homologue sequences from F. solani species complex (GU170628, KC808344, and EU329608). Similar results were obtained by BLASTN search in the FUSARIUM ID database (Geiser et al. 2004). For pathogenicity testing, 200 µl conidial suspension (1 x 106 conidia/ml) was pipetted, without wounding roots, onto the soil around the base of four plants individually potted in peat mix (Sunshine mix 1, Sun Gro Horticulture Ltd.; Alberta, Canada) and maintained in the greenhouse with 12 h photoperiod and temperature of 23 ± 2°C (Argus Control Systems Ltd.; British Columbia, Canada). Four plants inoculated with distilled water served as control. The test was conducted twice. At 10 days post inoculation (dpi), yellowing of leaves and damping off were observed in all inoculated plants. Re-isolated fungi from infected plant samples were morphologically identical to the isolate used for root inoculation. F. solani has been reported to cause damping off and root rot in several states in the U.S., Canada and Italy (Gauthier et al. 2019; Iriarte et al. 2021; Sorrentino et al. 2019). This is the first report of F. solani causing seedling damping off and root rot on hemp in ND. Hemp acreage has decreased in ND because of diseases (Buetow et al. 2020). Information on identification and management of diseases affecting hemp will be useful to producers.

First Report of Fusarium Sheath Rot of Rice Caused by Fusarium incarnatum-equiseti Species Complex in the United States.

Fungal diseases, including sheath rot (Sarocladium oryzae), cause significant losses of yield and milling quality of rice (Oryza sativa). In August 2021, symptoms like sheath rot were observed on 20% of rice plants (cv. Presidio) in 1-hectare field in Eagle Lake, Texas. Initial lesions occurred on the upper flag leaf sheaths and were oblong or irregular oval, with gray to light brown centers, and a dark reddish-brown diffuse margin. Lesions enlarged, coalesced, and covered a large area of the sheath. Infection led to panicle rot with kernels turning dark brown. Unlike sheath rot, sheath infection also led to inside culm infection with irregular dark brown lesions. Infected tissue pieces were sterilized with 1% NaOCl for 2 min, followed by 75% ethanol for 30 s, washed in sterile H2O three times, air dried and incubated on PDA at 27℃. Fungal isolates were obtained from 15 diseased plant samples and their singled-spored fungal colonies were whitish, loosely floccose and produced light yellow pigmentation. On carnation leaf agar, macroconidia were slightly curved and tapered at the ends, with 3 to 5 septa, and measured 17.5 to 34.3 × 3.1 to 5.0 µm. Microconidia were ovoid, usually with 0 to 1 septum and were 4.0 to 15.5 × 2.5 to 4.5 µm. Spherical shaped chlamydospores were produced in chain. These morphological characteristics were consistent to those described for Fusarium incarnatum-equiseti species complex (O'Donnell et al. 2009), including F. incarnatum (Wang et al. 2021) and F. equiseti (Avila et al. 2019). For molecular identification, DNA of a representative isolate was extracted and ITS, LSU, and EF1 of the fungus were amplified using the primers of ITS1/ITS4 (Wang et al. 2014), D1/D2 domain region of LSU (Fell et al. 2000), and EF1 (Wang et al. 2014), respectively, and sequenced. The ITS sequence (OL344049) was 99.61% identical to F. incarnatum-equiseti species complex (FD_01692) in Fusarium-ID database and 99.61% identical to F. equiseti (LC514690, KY523100, MW016539) and F. incarnatum (MH979697) in NCBI database. The LSU sequence (OK559512) was 98.77% similar to F. equiseti (MN877913, MN368509) and F. incarnatum (MH877332, MH877326); the EF1 sequence (OK570044) was 99.27% similar to F. equiseti (MK278902) in NCBI database. A phylogenetic analysis based on the concatenated nucleotide sequences grouped this isolate in the F. incarnatum-equiseti species complex clade at 100% bootstrap support. To evaluate pathogenicity, a conidial suspension of 1 x 106 conidia/ml or sterilized water (the controls) was injected into the sheaths and young panicles of three rice plants (cv. Presidio) at boot. Treated plants were maintained in a greenhouse at 25 to 30℃. After 3 weeks, typical symptoms, like those observed in the field, developed on the inoculated plants but not on the controls. The same fungus was consistently re-isolated from the diseased plants. To our knowledge, this is the first report of Fusarium sheath rot caused by F. incarnatum-equiseti species complex in rice in the U. S. F. incarnatum-equiseti species complex has been reported to be associated with panicle infection in wild rice (O. latifolia) in Brazil (Tralamazza et al. 2021). F. incarnatum has also been reported to cause panicle rot in China (Wang et al. 2021). F. proliferatum has been reported to cause Fusarium sheath rot in India (Prabhukarthikeyan et al. 2021) and the U. S. (Cartwright et al. 1995). This research demonstrates the potential of different pathogens being involved in causing sheath rot of rice.

First report of Fusarium solani (FSSC 6) associated with the root rot of Magnolia denudata in China.

Magnolia denudata (Lilytree or Yulan magnolia) is an important ornamental species of the genus Magnolia. It has considerable economical value because of its beautiful fragrant flowers and excellent tree structure (Wang et al. 2010). In Beijing, nurseries cultivate M. denudata as an ornamental plant and traditional medicine. In May 2020, patches of root rotted plants were observed in a field in Beijing, China, with an estimated incidence of approximately 31%. Early symptoms comprised leaves melanocratic shrunken, and the vascular tissue of roots turned brown. Progressively, the roots rotted and the whole plant died (Fig. 1 a-d). Infected roots tissue was surface disinfested and plated on potato dextrose agar (PDA) medium at 25±2 °C and incubated in the dark for 7 days. Pure cultures were obtained by hyphal tip excision (strain MFR1215.4). Fungal colonies were entire margins, and the aerial mycelium was copious, early white, and gradually developed into cream white. Colonies developed to 45.1 mm in 4 days at 25±2 °C on PDA media. On Spezieller Nährstoffarmer Agar (SNA) medium at 25±2 °C for 10 days. The morphological characteristics including macroconidia, microconidia, and chlamydospores were shown in Fig.1 (i-p). These morphological characteristics of the isolate corresponded to the description given for Fusarium solani sensu lato (Nelson et al. 1983, Summerell, 2003). Molecular identification was confirmed via amplification of translation elongation factor 1α (EF-1α), RNA polymerase I beta subunit gene (RPB1), and RNA polymerase II beta subunit gene (RPB2) regions using EF1/EF2, RPB1-Fa/G2R, RPB2-5f2/7cR, and RPB2-7cF/11aR primers (O'Donnell, 2010). Sequences were registered in GenBank. In the Fusarium-ID database, the EF-1α, RPB1, and RPB2 sequences showed 100% (677/677 bp), 99.8% (1568/1571 bp), and 100% (1457/1457 bp) identity with the F. solani species complex (FSSC). The same species-level identification was also found using Fusarium MLST. A best maximum likelihood tree was constructed using PhyloSuite v1.2.2 (Zhang et al. 2020), and the sequences of the MFR1215.4 isolation showed the same homology with FSSC 6. Pathogenicity tests were conducted on healthy one-year-old M. denudata potted seedlings. 200 ml spore suspension (1×106 spores/ml) was poured over the roots of twenty seedlings, and sterile distilled water was irrigated into twenty seedlings as controls in a greenhouse with 25/15°C day/night temperature and 80% relative humidity. The experiment was repeated three times. All inoculated seedlings showed similar symptoms to those in the field after 65 days, whereas the controls remained symptomless. The reisolating pathogens from symptomatic tissues were identical to the original isolates by morphology and EF-1α sequence identification. Based on morphological, molecular, and pathogenic characterization, the isolated pathogen was identified as FSSC 6. Fusarium species have been recorded in various places of the world and are known to be harmful to numerous plants (Trabelsi et al. 2017). It has been reported that FSSC has infected soybeans (Coleman, 2016, Nelson et al. 1989), oil palm (Hafizi et al. 2013), tobacco (Yang et al. 2020), resulting in sudden death syndrome, crown disease, and root rot. To our knowledge, this is the first report of FSSC-induced root rot in M. denudata in China. This research may contribute to the development of epidemiology and management strategies for root rot caused by FSSC on M. denudata.