Yarrowia lipolytica increased the activities of disease defense related enzymes and anti-fungal compounds in asparagus (Asparagus officinalis).

Fungal diseases pose significant threats to the production of asparagus, resulting in economic losses and decreased crop quality. The potential of the yeast Yarrowia lipolytica as a biocontrol agent against Fusarium proliferatum, a common pathogen of asparagus, was investigated in this study. The effects of Y. lipolytica treatment on decay incidence, disease index, and activities of major disease defense-related enzymes were investigated. In addition, we examined the levels of antifungal compounds such as total phenols, flavonoids, and lignin in asparagus plants exposed to Y. lipolytica. The results showed that Y. lipolytica treatment significantly reduced decay incidence and disease index caused by F. proliferatum when compared to the control group. Furthermore, Y. lipolytica-treated plants showed increased activity of disease defense-related enzymes, indicating that defense responses were activated. The activities of all evaluated enzymes were significantly higher in Y. lipolytica-treated asparagus, indicating an improved ability to combat fungal pathogens. Furthermore, Y. lipolytica treatment increased the content of antifungal compounds such as total phenols, flavonoids, and lignin, which are known to possess antimicrobial properties. These findings highlight the potential of Y. lipolytica as a biocontrol agent for fungal diseases in asparagus crops. The ability of Y. lipolytica to reduce disease incidence, boost disease defense-related enzymes, and increase antifungal compound content provides valuable insights into its efficacy as a natural and sustainable approach to disease management. However, further investigations are needed to optimize application methods and determine its efficacy under field conditions.

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

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

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.

Rapid and Sensitive On-site Nucleic Acid Detection of Three Main Fusarium Pathogens of Maize Stalk Rot Based on RPA-CRISPR/Cas12a.

Maize stalk rot is a soil-borne disease that poses a serious threat to maize production worldwide, with the most significant cause being fungal stalk rot. The development of a visual and rapid detection method for the maize stalk rot pathogen is significant for its prompt and accurate identification, enhancing agricultural production efficiency, and implementing timely preventive measures. These measures will help safeguard the maize yield and quality, ultimately reducing agricultural losses. In this study, we aimed to develop an efficient method to detect maize stalk rot pathogens. We focused on three pathogenic fungi commonly found in maize-producing regions worldwide: Fusarium verticillioides, Fusarium proliferatum, and Fusarium graminearum. Based on TEF-1α, we developed a rapid detection technique using RPA-CRISPR/Cas12a, combined with test strips to develop an on-site rapid visual detection test for these pathogens. The method showed detection sensitivity for F. verticillioides, F. proliferatum, and F. graminearum within 20 min at concentrations of 7.8 pg/µL, 0.11 ng/µL, and 0.13 ng/µL, respectively. The sensitivity increased with increasing reaction time. Testing of field disease samples indicated that the method is effective in detecting nucleic acids obtained through crude extraction methods. In conclusion, we developed a visually rapid detection technology that does not rely on complex instruments and equipment for the on-site early detection of F. verticillioides, F. proliferatum, and F. graminearum in the field to implement effective control measures, ensuring stable and high maize yields.

Transcriptomic and Metabolomic Analyses Reveal the Role of Phenylalanine Metabolism in the Maize Response to Stalk Rot Caused by Fusarium proliferatum.

Stalk rot is a prevalent disease of maize (Zea mays L.) that severely affects maize yield and quality worldwide. The ascomycete fungus Fusarium spp. is the most common pathogen of maize stalk rot. At present, the molecular mechanism of Fusarium proliferation during the maize stalk infection that causes maize stalk rot has rarely been reported. In this study, we investigated the response of maize to F. proliferatum infestation by analyzing the phenotypic, transcriptomic, and metabolomic data of inbred lines ZC17 (resistant) and CH72 (susceptible) with different levels of resistance to stalk rot. Physiological and phenotypic results showed that the infection CH72 was significantly more severe than ZC17 after inoculation. Transcriptome analysis showed that after inoculation, the number of differentially expressed genes (DEGs) was higher in CH72 than in ZC17. Nearly half of these DEGs showed the same expression trend in the two inbred lines. Functional annotation and enrichment analyses indicated that the major pathways enriched for DEGs and DEMs included the biosynthesis of plant secondary metabolites, phenylalanine metabolism, biosynthesis of plant hormones, and plant-pathogen interactions. The comprehensive analysis of transcriptome and metabolome data indicated that phenylalanine metabolism and the phenylalanine, tyrosine, and tryptophan biosynthesis pathways played a crucial role in maize resistance to F. proliferatum infection. In addition, a transcription factor (TF) analysis of the DEGs showed that several TF families, including MYB, bHLH, NAC, and WRKY, were significantly activated after inoculation, suggesting that these TFs play important roles in the molecular regulatory network of maize disease resistance. The findings of this study provide valuable insights into the molecular basis of the response of maize to Fusarium proliferatum infection and highlight the importance of combining multiple approaches, such as phenotyping, transcriptomics, and metabolomics, to gain a comprehensive understanding of plant-pathogen interactions.

First Report of Shoot Blight on Clausena lansium Caused By Fusarium proliferatum in China.

Wampee (Clausena lansium [Lour.] Skeels) is a tropical fruit. In July 2022, shoot rot symptom was observed in wampee (cv. JIXIN) in a field ((21°25'N, 110°10'E, about 100 ha ), Guangdong Province, China. The most obvious symptom of the disease was the rotting and withering of the tops. Disease incidence was approximately 90% (n = 500). Twenty diseased samples were randomly collected from the field and cut into 2 mm × 2 mm pieces next to the margins of diseased tissues. These pieces were then sterilized with 75% alcohol for 30 s and 2% sodium hypochlorite for 3 min and subsequently washed with sterile water three times. Tissue pieces were placed onto potato dextrose agar (PDA) and incubated at 25℃ for 3 days. Pure cultures were obtained by transferring hyphal tips to new PDA plates. Sixty isolates of Fusarium ssp. (60/80 = 75%) were obtained. Three representative single-spore isolates (CLFP-1, CLFP-2, and CLFP-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 3 to 5 septa, and measured from 31.7 to 55.5 µm × 2.5 to 5.0 µm in size (n=50). The morphological features of these fungi were analogous to F. proliferatum (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; 2010). The sequences were submitted to GenBank under accession numbers OP740961 to OP740963 (ITS), and OP800846 to OP800854 (RPB1, RPB2, EF1-α). The BLAST comparison of the sequences showed the three isolates were 100% similar to F. proliferatum (ITS: MT378328; TEF1: MH582344; RPB1: MN193921; RPB2: MN892349). 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 concatenated sequences revealed a 99.87 to 100% identity with the isolates of the F. proliferatum (F. fujikuroi species complex, Asian clade) by polyphasic identification using the FUSARIUM-ID database (Yilmaz et al. 2021). The sequences were also concatenated for phylogenetic analysis by the maximum likelihood method. The isolates clustered with F. proliferatum. Pathogenicity was tested through in vivo experiments. The inoculated and control plants (n = 5, 3 months old, cv. JIXIN) were sprayed with a spore suspension (1 × 105 per mL) of the three isolates 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℃ to 28℃, with relative humidity of approximately 80%. Symptoms were observed on the inoculated plants with disease incidence 100% after 2 weeks, while the control plants remained healthy. The pathogen re-isolated from all the inoculated plants was identical to the inoculated isolates in morphology and ITS sequences. No pathogen were isolated from the control plants. To the best of our knowledge, this study is the first to report F. proliferatum causing shoot blight symptom in wampee (cv. JIXIN). This disease has caused severe losses and will provide the foundation for management strategies.

Genome Sequence Resource of Fusarium proliferatum f. sp. malus domestica MR5, the Causative Agent of Apple Replant Disease.

Apple replant disease (ARD) caused by the fungal pathogen Fusarium proliferatum f. sp. malus domestica (Fpmd) MR5 brings annual losses to apple production within China. However, the genomic information of the pathogen is not yet available. Here, we obtained the whole-genome sequence of the highly virulent Fpmd MR5 using the Illumina PE150 platform. The genome size was 42.76 Mb and consisted of 9,047 genes. The GC content was 48.80%, and several genes potentially associated with pathogenicity were identified, such as carbohydrate-active enzymes, secreted proteins, and secondary metabolite gene clusters. There were 260 specific virulence factor genes, mainly related to fungal vegetative growth and the production of cell wall-degrading enzymes. These data will aid future studies investigating host-pathogen interactions and help us develop suitable disease management strategies.

Transcriptome changes associated with apple (Malus domestica) root defense response after Fusarium proliferatum f. sp. malus domestica infection.

BACKGROUND: Apple replant disease is a soilborne disease caused by Fusarium proliferatum f. sp. malus domestica strain MR5 (abbreviated hereafter as Fpmd MR5) in China. This pathogen causes root tissue rot and wilting leaves in apple seedlings, leading to plant death. A comparative transcriptome analysis was conducted using the Illumina Novaseq platform to identify the molecular defense mechanisms of the susceptible M.26 and the resistant M9T337 apple rootstocks to Fpmd MR5 infection. RESULTS: Approximately 518.1 million high-quality reads were generated using RNA sequencing (RNA-seq). Comparative analysis between the mock-inoculated and Fpmd MR5 infected apple rootstocks revealed 28,196 significantly differentially expressed genes (DEGs), including 14,572 up-regulated and 13,624 down-regulated genes. Among them, the transcriptomes in the roots of the susceptible genotype M.26 were reflected by overrepresented DEGs. MapMan analysis indicated that a large number of DEGs were involved in the response of apple plants to Fpmd MR5 stress. The important functional groups identified via gene ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment were responsible for fundamental biological regulation, secondary metabolism, plant-pathogen recognition, and plant hormone signal transduction (ethylene and jasmonate). Furthermore, the expression of 33 up-regulated candidate genes (12 related to WRKY DNA-binding proteins, one encoding endochitinase, two encoding beta-glucosidases, ten related to pathogenesis-related proteins, and eight encoding ethylene-responsive transcription factors) were validated by quantitative real-time PCR. CONCLUSION: RNA-seq profiling was performed for the first time to analyze response of apple root to Fpmd MR5 infection. We found that the production of antimicrobial compounds and antioxidants enhanced plant resistance to pathogens, and pathogenesis-related protein (PR10 homologs, chitinase, and beta-glucosidase) may play unique roles in the defense response. These results provide new insights into the mechanisms of the apple root response to Fpmd MR5 infection.

Preinoculation with Endophytic fungus Phomopsis liquidambaris reduced rice bakanae disease caused by Fusarium proliferatum via enhanced plant resistance.

AIMS: This study evaluated the control effect of the endophytic fungus Phomopsis liquidambaris B3 against rice bakanae disease (RBD) caused by Fusarium proliferatum and the disease control result of different inoculation times of beneficial micro-organisms. METHODS AND RESULTS: Rice seedlings preinoculated, coinoculated and noninoculated with B3 were exposed to F. proliferatum stress and grown under controlled conditions. Greenhouse experimental results showed that rice preinoculation with B3 significantly reduced rice bakanae disease by 21.45%, inhibited the colonization of F. proliferatum, increased defence-related enzyme activities, upregulated the expression of defence genes and promoted plant photosynthesis. However, bakanae disease in rice coinoculation with B3 increased by 11.45%, resulted in excessive reactive oxygen species (ROS) bursts and plant cell death. CONCLUSIONS: Preinoculation with the endophytic fungus P. liquidambaris B3 significantly reduced rice bakanae disease by triggering the SA-dependent defence pathways of plants, and promoted plant growth. However, coinoculatiton with P. liquidambaris B3 activated excessive defence responses, resulting in plants cell death and aggravation of bakanae disease. SIGNIFICANCE AND IMPACT OF THE STUDY: This study indicated that P. liquidambaris B3 was an effective method for agricultural control against rice bakanae disease caused by F. proliferatum, and provides an experimental basis for the development of sustainable endophytic fungal resources to effectively control plant diseases caused by pathogenic fungi, and suggests that precise application of beneficial micro-organisms may be become a key factor in farmland crop disease management.

Proliferatins suppress lipopolysaccharide-induced inflammation via inhibition of the NF-κB and MAPK signaling pathways.

Three previously undescribed polyketides [proliferatin A-C (1-3)] with anti-inflammatory activity were isolated from Fusarium proliferatum. 1-3 attenuated the production of inflammatory signal messengers including nitric oxide (NO), reactive oxygen species, proinflammatory cytokines interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and interleukin-1ß (IL-1ß), as well as the related proteins nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in lipopolysaccharide (LPS)-induced RAW264.7 macrophages. Transcriptome analyses based on RNA-seq indicated the potential anti-inflammatory mechanism of 1-3 involved in the nuclear factor kappa-B (NF-κB) and mitogen activated protein kinases (MAPKs) signaling pathways. Experimental evaluation of the protein levels revealed that 1-3 can inhibit the phosphorylation of IκB kinase (IKK), the degradation of NF-κB Inhibitor-α (IκBα), the phosphorylation of nuclear factor-κB (NF-κB) and can reduce NF-κB transportation to the nucleus. Interestingly, 1-3 decreased the phosphorylation of MAPKs including p-p38, p-ERK, and p-JNK. Molecular docking models suggest that binding of 1-3 to TLR4-MD-2 complex may lead to inhibition of NF-κB and MAPK signaling pathways, which was confirmed in vitro by surface plasmon resonance (SPR) assays. 1-3 can thus constitute potential therapeutic candidates for the treatment of inflammation-associated diseases.

Prevalence, genetic diversity and antifungal susceptibility profiles of F. fujikuroi, F. solani and Fusarium incarnatum-equiseti species complexes from onychomycosis in north of Iran.

Onychomycosis, a nail fungal infection, is normally caused by dermatophytes. However, yeasts and non-dermatophyte moulds (NDM) are among pathogens that cause nail disease. Regarding, this study aimed to describe the molecular epidemiology of Fusarium onychomycosis in the North of Iran. Two hundred and fifty seven nail samples collected from the patients clinically suspected of onychomycosis were subjected to direct microscopy, calcofluor white staining and culture. Fusarium isolates were identified at a species level through determination of multi-locus sequences for internal transcribed spacer and translation elongation factor 1 alpha. Based on the findings, Fusarium species were isolated from onychomycosis patients (n = 27). According to a previous partial genes analysis, the species in the recent study belonged to the members of F. fujikuroi species complex (n = 14), Fusarium incarnatum-equiseti species complex (n = 1) and F. solani species complex (n = 12). In this study, F. proliferatum was the dominant Fusarium species collected from the samples. The correct identification of Fusarium species is essential regarding the increased prevalence of Fusarium onychomycosis and the inherent resistance of these agents to a wide spectrum of antifungals. The obtained results indicated variation in the epidemiology of Fusarium species isolated from onychomycosis. Moreover, the minimum inhibitory concentration (MIC) of luliconazole and lanoconazole was in the range of 0.001-1 µg/ml, with the geometric mean of MICs obtained at 0.0103 and 0.0343 µg/ml against Fusarium species, respectively. These findings can increase researchers' knowledge regarding diversity of species, distribution of onychomycosis and the choice of a proper treatment.

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 proliferatum causing leaf spot on tea plants in Jiangxi Province, China.

Tea (Camellia sinensis (L.) O. Kuntze), a perennial evergreen shrub, is one of the most important cash crops in China. In September 2021, leaf spot symptoms were observed on approximately 30% of tea plants in a 2 ha commercial field of Lushan (29°33'0" N, 115°58'48" E), Jiangxi Province, China. The symptoms initially appeared as small, gray lesions, and later became larger (10-15 mm in diameter) circular to irregular spots with light brown centers and gray borders. To isolate the pathogen, small pieces (3×3 mm) cut from the margins of lesions were sterilized with 75% ethanol for 10 s, 0.1% HgCl2 for 20 s, and then rinsed three times with sterile water. The pieces were placed onto acidified potato dextrose agar (APDA) plates, and incubated in darkness at 28℃. Pure cultures were prepared by subculturing hyphal tips. A total of 16 fungal isolates were obtained, and the colonies of 15 isolates (isolation rate 93.8%) looked identical, resembling those of the genus Fusarium. The colonies were white to pink with purple woolly mycelium. After 10 to 15 days incubation, slightly curved macroconidia with three to four septa measuring 14.0 to 34.5 × 2.0 to 3.5 µm (n = 50), and oval, unicellar microconidia measuring 4.0 to 9.0 × 1.5 to 3.5 µm (n = 50) were observed. These morphological characteristics were similar to that described for Fusarium proliferatum (Leslie and Summerell 2006). Genomic DNA of representative isolates (LSZWY, LSZWY2, LSZWY3) was extracted with the Ezup Column Fungi Genomic DNA Purification Kit (Sangon Biotech Co., Ltd, Shanghai). The translation elongation factor 1 alpha gene (EF-1ɑ) was amplified using primers EF-1H / EF-2T (O'Donnell, et al. 2015). PCR product was sequenced and the sequence was 709 bp (Accession No. OL614004, ON357634, ON595710). BLAST search results showed that it had 99.9% identity with the EF-1ɑ gene sequence of F. proliferatum (MH341215, MT371378). To test pathogenicity, nine leaves from 5-year-old healthy tea plants (Ca. Luyun 3) were wounded using a sterilized needle and inoculated with a 20µl conidial suspension (2 × 107 conidia·mL-1) on one side of the plants and the other side with sterilized distilled water as a control. All leaves were incubated in a growth chamber at 28℃ and 80% relative humidity with a 12 h light/dark photoperiod. Seven days later, all inoculated treatments showed symptoms identical to those observed in the field, while the control remained asymptomatic. The experiment was repeated three times with similar results. Koch's postulates were fulfilled by successful re-isolation and morphological and molecular identification of F. proliferatum from the inoculated leaves. This pathogen can cause diseases of many crops, e.g. tobacco, Polygonatum cyrtonema and others (Li, et al 2017; Zhou, et al. 2021). However, this is the first report of F. proliferatum causing leaf spot on tea plants in China. This new disease poses a threat to the yield and quality of tea and methods need to be developed for its control and to prevent further spread.

First report of spotting disease caused by Fusarium proliferatum infection of Alisma orientale in China.

Alisma orientale (Alismatidae) is highly valued for both its pharmaceutical and nutritional properties. The tubers are in Chinese herbal medicine and the leaves and stems for several Chinese delicacies. Intercropping A. orientale and Nelumbo nucifera may increase quality, yield, and other economic benefits. In July 2021, a novel spotting disease was observed in these plants in the White Lotus Science and Technology Expo Park in Guangchang County, Fuzhou City, Jiangxi Province (26.79°N, 116.31°E). The symptom was round to regular black spots on the stems during the early stage of infection. Over time, the larger spots merged, resulting in stem breakage and eventually death. A. orientale spot disease arose in July of 2021, causing approximately 50% of leaves to die, and leading to 10 to 25% yield loss. To identify the pathogenic organism, 5×5 mm samples were taken from affected tissue adjoining healthy tissue, sterilized in 75% ethanol for 30 s, and immersed in 0.1% mercury chloride for a further 30 s, before washing in sterile water and transfer to potato glucose agar (PDA) plates. After culturing at 28℃±1℃ for seven days, aerial mycelia were identified. At the start of culture, the mycelia were white but later turned purple-red. Three to five straight or partially bent septa were visible on the macroconidia, which were 28.8(19.1~38.6) ×2.9(1.9~4.0) µm in size (n=50). In contrast, the microconidia appeared glassy and elliptical, with sizes of 9.8(4.9~14.8) ×2.7(1.2~4.1) µm (n=50). These features suggested F. proliferatum (Zhao et al., 2019). To verify this, various primers, including universal ITS1/ITS4, Fusarium-specific EF1T/EF2T, PRO1/PRO2 (Mulè et al., 2004), and Bt2a/Bt2b (Glass and Donaldson, 1995; O'Donnell and Cigelnik, 1998) primers were used for amplification of the 5.8S rRNA/ITS, α-elongation factor, calmodulin, and ß-tubulin genes. The resulting sequences were between 99% and 100% identical to those of F. proliferatum in GenBank (accession numbers MW721116.1, KR071735.1, KU604008.1, and MH398186.1, respectively). The present sequences were uploaded with accession numbers of OK047496, OL448294, OL448295, and OM280358, with sequence lengths of 549 bp, 725 bp, 594 bp, and 325bp, respectively. A maximum likelihood-phylogenetic tree was created in MEGA5 based on ITS+TEF+PRO sequences. Pathogenicity was tested by hyphal inoculation. Needles, cotton, and water were sterilized under high temperature and pressure. Five-millimeter punches were taken from infected and uninfected PDA plates and three uninfected stems of A. orientale were inoculated with the pathogen with a fourth used as the control. Plants were maintained in experimental field of the Bailian Science and Technology Expo Park. Infected wounds were gently wetted with sterile water and sealed with sellotape. After 10 days, the infected stems displayed symptoms while the controls did not. The same pathogen was recovered from the infected stems, fulfilling Koch's requirements. This appears to be the only report describing F. proliferatum infection of A. orientale stems. These results are useful for the recognition and avoidance of F. proliferatum infections in A. orientale and other plants.

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, leaf yellowing 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 of Fusarium proliferatum (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 the F. proliferatum (F. fujikuroi species complex, Asian clade) by polyphasic identification using the FUSARIUM-ID database (Yilmaz et al. 2021). The sequences were also concatenated for phylogenetic analysis by the maximum likelihood method. The isolates clustered with F. 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 relative humidity of approximately 80%. 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.

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

Bambusa pervariabilis × Dendrocalamopsis grandis is the main cultivated bamboo species used for ecological construction in the Yangtze River basin. This species has the advantages of easy reproduction, wide adaptability and strong resistance and has high economic, ecological and social benefits (Peng et al. 2020). One area of B. pervariabilis × D. grandis with basal rot disease was discovered in Renshou County, Sichuan Province, China (29°41'N, 104°11'E) in June 2020. The disease occurrence area was 68 hm2 in Renshou County, with an incidence rate of 34.8%, and 5% of the B. pervariabilis × D. grandis with basal rot disease died. The pathogen initially invaded from the first section of the base of the bamboo stalk, appearing as black to yellowish brown strips or lumps of disease spots, and rapidly developed horizontally and vertically, which caused the whole plant to wither in severe cases. Diseased tissues were collected from the base of a 4-year-old bamboo stalk with a sterile blade. 100 pieces (5 × 5 × 2 mm) of diseased tissues were sterilized with 3% NaClO for 30 s and in 75% ethanol for 90 s, rinsed three times with sterile distilled water, dried with sterile surface water on sterile filter paper, plated onto potato dextrose agar amended with streptomycin sulfate (Solarbio, 50 µg/ml), and incubated at 25 °C for 7 days with light. A total of five isolates were obtained, of which four isolates were similar in morphology. Using the method of monospore isolation (Leslie and Summerell 2006) and culturing it on PDA, the fungus produced round colonies with a diameter of approximately 8.4 mm and a surface color ranging from white to purple within 7 days at 25 °C. For identification by typical spores, the fungus was cultured on carnation leaf agar (CLA) medium at 25 °C for 7 days. The microconidia by the isolates BD2002, BD2004, BD2008 and BD2010 cultured on CLA medium were elliptical, ovoid, without septum, and measured 4.56 to 15.53 µm long × 1.36 to 6.98 µm wide (n=100). The macroconidia were rod-shaped or slightly curved, tapering apically with three to five septa, and measured 18.86 to 52.99 × 1.56 to 6.42 µm in size (n=100). According to the morphological characteristics of macroconidia and microconidia, the isolates were identified as Fusarium sp. (Leslie and Summerell 2006). For molecular identification, fungal DNA of isolates BD2002, BD2004, BD2008 and BD2010 was extracted by a fungal genomic DNA extraction kit. Polymerase chain reactions (PCRs) were performed with primers ITS1/ITS4 for the internal transcribed spacer (ITS) rDNA region (White et al. 1990), primers Bt2a/Bt2b for the ß-tubulin (TUB) region (Glass and Donaldson 1995), primers EF1F/EF2R for the translation elongation factor 1α (TEF) region (Carbone et al. 1999), primers 5f2/7cr for the RNA polymerase II genes (RPB2) region (O'Donnell et al. 2010), primers H3-1a/H3-1b for the histone H3 (HIS) region (Jacobs et al. 2010), and primers NMS1/NMS2 for the mitochondrial small subunit (mtSSU) rDNA region (Stenglein et al. 2010). Using BLASTn to search GenBank for ITS, TUB, TEF, RPB2, HIS and mtSSU sequences, all isolates showed the highest similarity with Fusarium proliferatum (Matsushima) Nirenberg. The representative isolate BD2010 showed that ITS had 99.61% similarity to F. proliferatum Z23-28 (FJ648201.1); HIS had 99.57% similarity to F. proliferatum M06A_4G_4 (KX681532.1); and the TUB, TEF, RPB2, and mtSSU sequences showed 99.67%, 99.10%, 99.06%, and 99.57% similarity, respectively, to F. proliferatum ITEM2287 (accession numbers LT841243.1, LT841245.1, LT841252.1, and LT841247.1 in GenBank). The GenBank numbers of the representative isolate BD2010 were ITS, OK325614; TUB, OK377026; TEF, OK377027; RPB2, OK377028; HIS, OK377029; and mtSSU, OK338638. To confirm the pathogenicity, thirty 4-year-old healthy bamboo plants were grown in 30 pots. Each five plants were inoculated with one isolate, and a total of twenty-five plants were inoculated with five isolates. A conidia suspension (1 × 106 conidia/ml) of the fungus was inoculated (100 µl each) into plants that had been acupunctured at the base by a sterile syringe. Five control plants were inoculated only with the same amount of sterile distilled water. The inoculation site was wrapped with wet gauze to maintain moisture. All bamboo plants were watered every seven days. The illumination conditions were 12 h light and 12 h dark. All plants were cultured in a greenhouse at 25-28 °C and 70-80% relative humidity. One month later, twenty plants inoculated with conidial suspensions of BD2002, BD2004, BD2008 and BD2010 showed the same symptoms as those observed in the field, whereas plants inoculated with the other fungus and the control treatment remained asymptomatic. The pathogenicity test was conducted three times, and the experimental results were consistent. Furthermore, the fungi were reisolated from the diseased part and were identified as F. proliferatum by morphological and molecular comparison. To our knowledge, this is the first report of basal rot disease caused by F. proliferatum on B. pervariabilis × D. grandis in China. This research is conducive to laying the foundation for the development of effective control strategies for basal rot disease in this species.

First Report of Leaf Spot on Basella rubra Caused by Fusarium proliferatum in Malaysia.

Basella rubra (family Basellaceae), locally known as 'Remayong Merah', is the edible perennial vine served as leafy vegetable in Malaysia. In May 2021, B. rubra's leaves with circular to subcircular purple spots (ranging from 1-10 mm wide) were collected in Lido (5°56'44.6"N 116°04'46.5"E), Sabah province. The disease severity was about 60% with 20% disease incidence on fifty plants. As disease developed, the spots grew larger and necrosis were formed within the purple spots. Small pieces (5 x 5 mm) of five diseased spots were excised, and then surface sterilized based on Khoo et al. (2022b) before plating on water agar at 25°C. Once obtained the pure cultures from all diseased spots, they were incubated on potato dextrose agar at 25°C. After 7 days, white aerial mycelium with light violet pigmentation on lower side were observed on PDA. Then, the fungi were cultured on Carnation leaf agar (CLA) at 25°C and 12-h light/dark photoperiod for 10 days. Thin-walled slender and slightly curved macroconidia (n= 20) with 3 to 5 septa were ranged from 2.3 to 2.6 µm wide by 26.8 to 44.5 µm long in size. Oval microconidia (n= 20) with no septa were 2 to 2.2 µm wide by 9.5 to 15 µm long in size. Chlamydospores were absent. Monophialids with false head were observed. Isolate Lido and Lido02 were kept in the Laboratory of Genetics, Faculty of Science and Natural Resources, Universiti Malaysia Sabah for public request. Their genomic DNA were extracted from fresh mycelia of isolates based on Khoo et al. (2022a). EF1/EF2, RPB1-Fa/RPB1-G2R and RPB2-5f2/RPB2-7cr (Jiang et al. 2021) were used to amplify the translation elongation factor 1-α (TEF1) region, RNA polymerase largest subunit gene (RPB1) and RNA polymerase second largest subunit gene (RPB2) based on PCR condition in Khoo et al. (2022b). The isolate's sequences were deposited in GenBank as OM048109, OM634654 (TEF1), OM634655, OM634657 (RPB1) and OM634656, OM634658 (RPB2). They were 99 to 100% homology to TEF1 of isolate DPCT0102-2 (LC581453) (657/657 bp), RPB1 of strain ZJ05 (MT560605) (1558/1558 bp) and RPB2 of isolate GR_FP248 (MT305154) (1867/1869 bp) sequences. These sequences were polyphasic identified at the Fusarium MLST (https://fusarium.mycobank.org/), and were more than 99% similarity to Gibberella fujikuroi species complex (NRRL 25200). Gibberella fujikuroi and Fusarium fujikuroi are synonymous with Fusarium proliferatum (Leslie and Summerell 2006). The pathogen was identified as F. proliferatum based on morphological characterization, molecular data and phylogenetic analysis. Two non-wounded leaves of three one-month-old B. rubra seedlings were inoculated with mycelium plug (10 x 10 mm). Additional three B. rubra seedlings received sterile PDA agar plug (10 x 10 mm) to serve as controls. They were incubated in a glasshouse at room temperature 25°C with a relative humidity of 80 to 90%. After 8 days of inoculation, all inoculated leaves exhibited the symptoms as observed in the field, while the controls showed no symptoms, thus confirming the Koch's postulates. The experiment was repeated two more times. The reisolated pathogens were identified as F. proliferatum via PDA macroscopically, CLA microscopically and PCR amplification. F. proliferatum was reported previously causing leaf spot disease on Cymbidium orchids (Wang et al. 2018), tobacco (Li et al. 2017) and tomato (Gao et al. 2017). To our knowledge, this is the first report of F. proliferatum causing leaf spot on B. rubra in Malaysia. Infections of leaves reduce plant vigor and marketability. The identification of leaf spot caused by F. proliferatun will enable plant health authorities and farmers to identify practices to minimize disease on this important crop.

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 Lotus Seedpod Withering Caused by Fusarium proliferatum on Nelumbo nucifera Plants in China.

Nelumbo nucifera (Nymphaeaceae family) is a well-known plant in China and with the increasing value of this crop, the planting area of lotus is expanding. In May 2019, an unknown withering lotus seedpod was obtained in Guangchang County of Jiangxi Province (26.79°N, 116.31°E). The disease arose between May and July of each year, resulted in the withering and consequent death of ~10% of lotus seedpods, with the disease being most serious during the rainy season. The initial symptoms of this disease include the shrinking of young lotus seedpods with concomitant yellowing of the epidermal tissue layer. These pods failed to grow normally and could to wither and die within one week, with the withering symptoms gradually spreading to associated stem tissues. To characterize the pathogens responsible for this disease, ten diseases seedpods were collected and cut into pieces of ~5×5 mm, then sterilized with 75% ethanol for 30 s, and treated with 0.1% mercuric chloride for 5 min. After being washed four times under sterilized water, samples were then transferred onto potato dextrose agar (PDA) and incubated for 7 d at 28℃ in the dark. Eight purified isolates yielded large numbers of aerial mycelium that were initially white in color, but then changed to a purple-red color over the course of this incubation period. The average mycelial growth rate was 6.3 mm per day (n=5). On PDA, macroconidia exhibited 3-5 septa and were straight or slightly curved, with a size of 21.6-47.4×2.5-4.6 µm (average: 31.9×3.5 µm, n=50). The microconidia were hyaline, ovoid or ellipse and 4.6-13.5×2.2-4.3 µm in size (average: 8.7×3.1 µm, n=50). The morphological features of these fungi were noted to be in line with those of Fusarium proliferatum (Leslie and Summerell, 2006; Zhao et al., 2019). To confirm the identity of this putative pathogen at the molecular level, the universal ITS4/ITS5 primers (White et al., 1990), the Fusarium specific pair PRO1/PRO2 (Mulè et al., 2004), EF1T/EF2T (O'Donnell et a., 1998) and RPB2F/R (O'Donnell et al., 2010) primers were utilized to amplify the internal transcribed spacer 1 (ITS1)-5.8S rRNA gene-internal transcribed spacer 2 (ITS2), calmodulin, alpha elongation factor genes, and RNA-dependent DNA polymerase II subunit from these isolates. Following alignment of the resultant sequences with GenBank via a BLAST analysis, the sequences (GenBank accession numbers: MW862499, MW762531, MW767988, MW831311, respectively.) showed 100% identities to the corresponding DNA sequences in F. proliferatum (GenBank accession numbers: MW817705, LS423443, MH153750, and MW091308, respectively.). Based upon these morphological and molecular findings, this pathogen was identified as F. proliferatum. Pathogenicity testing was then performed using five plump healthy lotus seedpods. Sterile needles were used to generate wounds (2 mm deep, 1 mm in diameter) a 10 µL suspension of prepared spores (1.0×106 spores/mL) derived from a 7-day-old culture grown on PDA was injected into the wound sites of the lotus seedpod. As a control, give seedpods were additionally wounded and injected as the same as treated with 10 µL of sterile water. The experiments were repeated three times with five biological replicates. All seedpods were then incubated at 28℃ in a growth chamber (12 h light/dark) with 80% relative humidity. After a 3-day incubation period, wounded sites injected with spore suspensions exhibited browning. Following a 5-day incubation period, a mean lesion diameter of 9.8 mm was observed, with white mycelia growing on the wound surface and with evident withering of the internal and external tissues near the wounded site. In contrast, blank control wound sites remained healthy. We were again able to isolate F. proliferatum from the infected lotus seedpods. Finally, eight isolates were obtained were identified as the pathogen based on these morphological and molecular analyses, thus fulfilling Koch's postulates. This is the first report to our knowledge to have described a case of F. proliferatum causing lotus seedpod withering in China, providing a foundation for future research efforts aimed at presenting diseases caused by this pathogen.

First report of Fusarium proliferatum on date palm (Phoenix dactylifera L.) in Jordan.

Date palm (Phoenix dactylifera L.) is one of the world's oldest cultivated fruit crops. In Jordan, date palm farming started in the 1990s. The major date palm planting areas are Jordan valley, Aqaba, and Azraq (Al Antary et al., 2015). 'Medjool' and 'Barhi' are the two major cultivars in Jordan. In early 2018, some 18- to 24- month old date palm trees (cv 'Medjool') showed light brownish discoloration and dryness symptoms on the leaves and branches of infected date palm trees at the Jordan University Agricultural Research Station (JUARS) at the Jordan Valley (GPS coordinates 32.086871, 35.597219) (Figure 1). All the leaf parts including leaf base, spines, and leaflets were wrinkled and malformed. The infection led to a loss of 1-2% out of 1100 total Medjool trees at the station. Similar symptoms were observed in many date palm farms in the Jordan Valley. Diseased samples from rachis tissue from the JUARS were collected, surface sterilized with 5% sodium hypochlorite for five minutes, rinsed with distilled water for three times, dried, and plated on potato dextrose agar (PDA) medium (HIMEDIA). The plates were incubated at 25°C for seven days. After that, different fungal colonies were purified using the hyphal tip method. Mycelium of a representative isolate (FpDP2018JO-01) was harvested, DNA extracted using the CTAB protocol (Doyle and Doyle, 1990), amplified with three primers: ITS1/4 (White et al., 1990), ß-tubulin and the elongation factor 1-alpha (EF1) gene regions. Amplicons were sequenced at Macrogen Inc, South Korea. Sequences were edited via MEGA 7 software (Kumar et al., 2016) and Blastn at the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov) which was used to search for similar accessions. The sequences were submitted to the GenBank and accession numbers were received for ITS1/2 (MK522076), ß-tubulin (MK720958) and elongation factor 1 alpha (MW533146). The sequences were further used at the Fusarium MLST (https://fusarium.mycobank.org/) for identity confirmation. ITS1/4 and ß-tubulin could not discriminate the species Fusarium proliferatum but EF1 - alpha could (Figure 2a-c; Supplement 1). For morphological identification, four representative F. proliferatum isolates (FpDP2018JO-01- FpDP2018JO-04) were used. Mycelium were white to dark purple in color, macroconidia (20.5 - 44.5 × 3.3 - 7.5 µm) were thin, slender, with 3-5 septa, and microconidia (4.3 - 12.1 × 2.5 - 4.3 µm) were thin and aseptate (Figure 3). Koch's postulate was performed on one-year-old seedlings according to Abdalla et al., 2000 method using the same sequenced isolate (FpDP2018JO-01). Five plants were inoculated by injecting 2 mlof inoculum into the crown area using a hypodermic needle and syringe. The inoculum was prepared according to Abdalla et al. (2000). The control set of seedlings (n=5) were injected with sterile distilled water. The experiment was arranged in a CRD design. Symptoms were evaluated three months after inoculation. On seedlings, yellowing of leaflets, discoloration of spines and rachis, and dryness of leaves were observed. Control seedlings showed no symptoms. Re-isolation form the detached leaves and infected seedlings was conducted to satisfy Koch's postulates. Fusarium sp. was confirmed to be F. proliferatum based on their microscopic characteristics. To our knowledge, this is the first record of F. proliferatum on date palm in Jordan. Date palm in Jordan especially 'Medjool' is an important cash crop. Fusarium spp. is an important pathogen that could cause huge losses on date palm and other crops. In Jordan, the pathogen has been isolated from samples from six farms so far, but detailed studies have not been conducted. It would be of importance to survey date palm farms for fungal diseases, test their pathogenicity using several isolates, and characterize them for proper management strategies. F. proliferatum was isolated from roots and leaves of declining date palm trees from many regions of Saudi Arabia and caused symptoms similar to those of F. oxysporum f. sp. albedinis, the causal agent of Bayoud (Abdalla et al. 2000; Saleh et al. 2016). Notonly that, but F. proliferatum was found to have the highest colonization abilities on date palm leaflets and is becoming serious pathogen on date palm (Saleh et al. 2016.