Fusarium Fujikuroi Species Complex Research Articles

First report of Fusarium verticillioides causing blight on Ammopiptanthus mongolicus in China.

Ammopiptanthus mongolicus is the only evergreen broadleaf shrub and constructive species in the northwest desert of China (Hu et al. 2021). It also is listed as one of the national second-class endangered plants. Ammopiptanthus mongolicus has a good effect of water conservation, windbreak and sand fixation because its deep root system (Zhou et al. 2012; Dong et al. 2023). A large number of dead plants of Ammopiptanthus mongolicus were found in Etuoke county, Inner Mongolia Autonomous Region, China (40°4'28″-40°4'34″ N, 106°53'5″-106°53'31″ E). In September 2023, the investigation and research in the region found that the incidence of diseased plants in this field was about 30%, and for individual plant, the incidence of diseased branches was about 60%. The leaves of diseased branches initially became from green to yellow and then wilt and fall. Eventually the plant dies. (Figure 1). The miter cut of the root showed that the root steles of diseased plants had obvious black and brown color (Figure 2). For isolation, the 30 tissue blocks (10×10 mm) of from 10 symptomatic roots diseased were surface sterilized with 70% ethanol for 3 minutes and sodium hypochlorite (2.5% available chlorine) for 5 minutes, and rinsed three times with sterilized distilled water. Then, these tissue blocks were placed on potato dextrose agar (PDA) medium, and incubated from 3 to 5 days at 25°C. After 3 days on PDA, the surface of the colony was rough, the color were white-pink at the beginning, and deep purple pigment were produced in the later stage, making the colony bluish-purple to gray-purple, their undersides were bluish-purple. Mycelia were white. After 7 days on SNA, Microconidia were typical of the clavicular type, 8.5 ± 2.5 μm × 2.3 ± 0.2 μm(×400). Microconidia were usually very long conidial chains, sometimes the spore chain collapses and the conidia clump together to form an approximate pseudocephaly. The macroconidia were slender and long, slightly falcate or straight, 42.8 ± 3.4 μm × 3.8 ± 0.7 μm(×400) (Figure 4). Species identity was confirmed by sequencing the EF1-α gene (EF1 and EF2 primers)(O'Donnell et al. 1998), RPB1 (F5 and G2R primers)(O'Donnell et al. 2022) and RPB2 (5F2 and 11AR)(O'Donnell et al. 2022). The amplified sequences of a representative isolate (AmP10) have deposited in GenBank with accession number OR594338 (EF1-α), OR841329 (RPB1) and OR841331 (RPB2). Thee results of pairwise alignment in Fusarioid-ID datebase(Crous et al. 2021) showed that EF1-α sequence was 99.54% similarity and 89.96% overlap to the corresponding sequence KF499582 of ex-epitype CBS 218.76 of Fusarium verticillioides, Fusarium fujikuroi species complex (FFSC, previously GFSC) (Lecellier et al. 2014), RPB1 sequence was 100% similarity and 100% overlap to the corresponding sequence MW402638 of ex-epitype CBS 218.76 of Fusarium verticillioides, Fusarium fujikuroi species complex (FFSC, previously GFSC) (Yilmaz et al. 2021), RPB2 sequence was 99.94% similarity and 87.83% overlap to the corresponding sequence MW928835 of ex-epitype CBS 218.76 of Fusarium verticillioides, Fusarium fujikuroi species complex (FFSC, previously GFSC) (Crous et al. 2021). Moreover, the result of polyphasic identification in Fusarioid-ID datebase also showed EF1-α, RPB1 and RPB2 sequences were 100% similarity to the corresponding sequences of ex-epitype CBS 218.76 of Fusarium verticillioides, Fusarium fujikuroi species complex (FFSC, previously GFSC). To test the pathogenicity, the healthy green seedlings (64 days old) were planted into plastic pots containing sterilized soil in the greenhouse after the seeds of Ammopiptanthus mongolicus were surface sterilized with 70% ethanol for 3 minutes and 2.5% sodium hypochlorite for 3 minutes. The roots of 3 seedlings were inoculated with 1×106 /ml of the conidial suspension, and another 3 used as controls with inoculated sterile water. Then, all pots were placed in a greenhouse maintained at 18°C to 25°C. After incubation for 3-5 days, the typical symptoms similar to the symptoms in the field (Figure 5), brown root steles (Figure 6), developed on the plants inoculated with conidial suspension, whereas no symptoms were observed on the control plants. The same pathogen was consistently reisolated from the inoculated roots and confirmed as Fusarium verticillioides based on morphological and molecular analyses. To our knowledge, this is the first report of Fusarium verticillioides on Ammopiptanthus mongolicus in China. This study provides a basis for identifying pathogens causing blight on Ammopiptanthus mongolicus and managing the disease.

Fusarium amaranthuense sp. nov. from amaranth is an emergent species closely related to Fusarium circinatum.

Amaranth (Amaranthus spp. L) is not native to South Korea but cultivated in small scales for ornamental purposes as well as leafy vegetables and pseudo cereals. In this study, a new species within the genus Fusarium was isolated from amaranth, showing stem rot symptoms from a farmer field in Hwaseong, South Korea. The disease is characterized by dark-brown spots with black borders, leading to withering. Phylogenetic analysis based on concatenated sequences of translation elongation factor 1-alpha (TEF1), beta-tubulin (tub2), calmodulin (cmdA), RNA polymerase largest subunit (RPB1) and RNA polymerase II second largest subunit (RPB2) genes, revealed that the obtained isolates were formed a distinct clade within the Fusarium fujikuroi species complex and closely related to F. circinatum. Cultural and morphological characteristics and pathogenicity on healthy amaranth plants (stem and leaves) were examined. The isolates readily differentiated from F. circinatum based on 1-5 septate macroconidia and the absence of sterile hyphae. Based on molecular and morphological characteristics, this fungus is demonstrated to be a new species and is described here as Fusarium amaranthuense, the causal agent of stem rot of amaranth in South Korea.

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.

Maize–Fusarium associations and their mycotoxins: Insights from South Africa

For maize, a staple food in South Africa, there is a lack of comprehensive knowledge on the mycotoxin-producing fungal diversity. In this study, a fungal community profile was established using culture-dependent methods for 56 maize seed samples that were also analysed for 13 mycotoxins. The fungal isolates were identified by morphology and DNA sequencing. A total of 723 fungal isolates from 21 genera and 99 species were obtained and characterised. Fusarium was the most common genus (isolated from 52 samples), followed by Cladosporium (n = 45), Aspergillus (n = 41), Talaromyces (n = 40), and Penicillium (n = 38). Fusarium communities were dominated by the Fusarium fujikuroi species complex, which includes species such as Fusarium verticillioides and Fusarium temperatum, while Fusarium awaxy and Fusarium mirum are reported here for the first time from South Africa. As for the deoxynivalenol (DON) producing species, only Fusarium boothii and Fusarium graminearum were isolated to a lesser extent. DON (n = 37), fumonisins (FUM) (n = 32), and zearalenone (ZEA) (n = 6) were detected. The presence of a particular species did not guarantee the presence of the corresponding mycotoxins, while the inverse was also true. The occurrence of DON and/or FUM in South African maize remains a health concern, so continuous monitoring of both fungal species and their mycotoxins is important.

Species diversity of fusarioid genera associated with sweet potato in Brazil, including the description of a new species

AbstractThe sweet potato is of great socioeconomic importance in Brazil and throughout the world. Among the fungal pathogens associated with root diseases of this crop, Fusarium is of particular importance due to the diversity of species, the pathogenic potential in their various hosts and their ability to survive inside plants as endophytes without apparently causing damage. The objective of this work was to identify fusarioid genera from stems and roots of symptomatic and asymptomatic plants. The identification of fungal species was based on multilocus phylogenetic analysis and morphology. A total of 51 isolates, distributed in the Fusarium incarnatum‐equiseti species complex (FIESC), Fusarium fujikuroi species complex (FFSC), Fusarium oxysporum species complex (FOSC)—with a taxonomic novelty—and Neocosmospora were identified. The pathogenicity test on roots showed that 13 of the 14 tested isolates obtained from symptomatic roots and stems and from asymptomatic stems were capable of reproducing the rot symptoms observed in the field. The confirmation of the pathogenicity of isolates from asymptomatic stems corroborates the hypothesis that these fungi can live endophytically in asymptomatic tissue and are capable of causing damage to their hosts in stress situations. This is the first report of Fusarium agrestense, Fusarium annulatum, Fusarium caatingaense, Fusarium elaeidis, Fusarium pernambucanum, Fusarium pseudocircinatum, Neocosmospora falciformis and Neocosmospora suttoniana associated with root and stem rot of sweet potato.

H3K27me3 is vital for fungal development and secondary metabolite gene silencing, and substitutes for the loss of H3K9me3 in the plant pathogen Fusarium proliferatum.

Facultative heterochromatin marked by histone H3 lysine 27 trimethylation (H3K27me3) is an important regulatory layer involved in secondary metabolite (SM) gene silencing and crucial for fungal development in the genus Fusarium. While this histone mark is essential in some (e.g., the rice pathogen Fusarium fujikuroi), it appears dispensable in other fusaria. Here, we show that deletion of FpKMT6 is detrimental but not lethal in the plant pathogen Fusarium proliferatum, a member of the Fusarium fujikuroi species complex (FFSC). Loss of FpKmt6 results in aberrant growth, and expression of a large set of previously H3K27me3-silenced genes is accompanied by increased H3K27 acetylation (H3K27ac) and an altered H3K36me3 pattern. Next, H3K9me3 patterns are affected in Δfpkmt6, indicating crosstalk between both heterochromatic marks that became even more obvious in a strain deleted for FpKMT1 encoding the H3K9-specific histone methyltransferase. In Δfpkmt1, all H3K9me3 marks present in the wild-type strain are replaced by H3K27me3, a finding that may explain the subtle phenotype of the Δfpkmt1 strain which stands in marked contrast to other filamentous fungi. A large proportion of SM-encoding genes is allocated with H3K27me3 in the wild-type strain and loss of H3K27me3 results in elevated expression of 49% of them. Interestingly, genes involved in the biosynthesis of the phytohormones gibberellins (GA) are among the most upregulated genes in Δfpkmt6. Although several FFSC members harbor GA biosynthetic genes, its production is largely restricted to F. fujikuroi, possibly outlining the distinct lifestyles of these notorious plant pathogens. We show that H3K27me3 is involved in GA gene silencing in F. proliferatum and at least one additional FFSC member, and thus, may serve as a regulatory layer for gene silencing under non-favoring conditions.

Rapid detection of Fusarium fujikuroi in rice seeds and soaking water samples based on recombinase polymerase amplification‐lateral flow dipstick

AbstractBakanae disease is a rice seedborne disease caused by the Fusarium (Gibberella) fujikuroi species complex (FFSC), among which F. fujikuroi is the dominant pathogen. Pathogens usually hide inside or on the surface of seeds, and infection occurs mainly at the germination stage. In this study, a method for the detection of F. fujikuroi in rice seeds and seed soaking water samples was established using recombinase polymerase amplification (RPA) technology with lateral flow device (LFD) chromatography test strips. A pair of specific primers and probes based on the cyp51c gene were screened. RPA‐LFD was used to detect 10 F. fujikuroi strains, and the results showed that all of them tested positive and there was no cross‐reaction with other Fusarium or non‐Fusarium species. The target production of the RPA‐LFD assay was obtained at 35–45°C for 8–14 min, and optimal reaction conditions of amplification at 39°C for 8 min is recommended. The sensitivity test showed that the detection limit of the RPA‐LFD test for F. fujikuroi genomic DNA in rice seeds was 100 fg/μL, and the detection limit for F. fujikuroi spores in submerged water samples was 100 spores/mL. In the assay for field samples, it successfully detected F. fujikuroi carried in the seeds of three out of five rice varieties. In addition, the whole RPA‐LFD assay can specifically detect F. fujikuroi within 30 min. This method is expected to become an early field monitoring tool for rice bakanae disease.

Fusarium and Sarocladium Species Associated with Rice Sheath Rot Disease in Sub-Saharan Africa

Sarocladium and Fusarium species are commonly identified as causal agents of rice sheath rot disease worldwide. However, limited knowledge exists about their genetic, pathogenic, and toxigenic diversity in sub-Saharan African (SSA) countries, where an increasing incidence of this disease has been observed. In this study, seventy fungal isolates were obtained from rice plants displaying disease symptoms in rice research programs and farmer fields in Mali, Nigeria, and Rwanda. Thus, an extensive comparative analysis was conducted to assess their genetic, pathogenic, and toxigenic diversity. The Fusarium spp. were characterized using the translation elongation factor (EF-1α) region, while a concatenation of Internal Transcribed Spacer (ITS) and Actin-encoding regions were used to resolve Sarocladium species. Phylogenetic analysis revealed four Fusarium species complexes. The dominant complex in Nigeria was the Fusarium incarnatum-equiseti species complex (FIESC), comprising F. hainanense, F. sulawesiense, F. pernambucatum, and F. tanahbumbuense, while F. incarnatum was found in Rwanda. The Fusarium fujikuroi species complex (FFSC) was predominant in Rwanda and Mali, with species such as F. andiyazi, F. madaense, and F. casha in Rwanda and F. annulatum and F. nygamai in Mali. F. marum was found in Nigeria. Furthermore, Fusarium oxysporum species complex (FOSC) members, F. callistephi and F. triseptatum, were found in Rwanda and Mali, respectively. Two isolates of F. acasiae-mearnsii, belonging to the Fusarium sambucinum species complex (FSAMSC), were obtained in Rwanda. Isolates of Sarocladium, which were previously classified into three phylogenetic groups, were resolved into three species, which are attenuatum, oryzae, and sparsum. S. attenuatum was dominant in Rwanda, while S. oryzae and S. sparsum were found in Nigeria. Also, the susceptibility of FARO44, a rice cultivar released by Africa Rice Centre (AfricaRice), was tested against isolates from the four Fusarium species complexes and the three Sarocladium species. All isolates evaluated could induce typical sheath rot symptoms, albeit with varying disease development levels. In addition, liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to determine variation in the in vitro mycotoxins of the Fusarium species. Regional differences were observed in the in vitro mycotoxins profiling. Out of the forty-six isolates tested, nineteen were able to produce one to four mycotoxins. Notably, very high zearalenone (ZEN) production was specific to the two F. hainanense isolates from Ibadan, Nigeria, while Fusarium nygamai isolates from Mali produced high amounts of fumonisins. To the best of our knowledge, it seems that this study is the first to elucidate the genetic, pathogenic, and toxigenic diversity of Fusarium species associated with the rice sheath rot disease complex in selected countries in SSA.

First report of Rice panicle blight caused by Fusarium sporotrichioides in China.

Rice (Oryza sativa L.) panicle blight, caused by various fungal and bacterial pathogens, is an emerging threat to rice production, due to the impact on rice yield and quality. In the autumn of 2020, a survey was conducted to understand the etiology of the disease in Liaoning province, an important rice growing area in northeastern China. Rice seeds with typical reddish or brown spots on the glumes were collected from various rice fields. Symptomatic seeds were sterilized with 5% sodium hypochlorite and 75% ethanol, rinsed in sterile distilled water, and placed on potato dextrose agar (PDA) plates. After 3 to 5 days of incubation at 25°C, suspected Fusarium strains showing cream to salmon colonies on PDA were purified by the single-spore isolation method. The identification of these strains were performed based on morphological and molecular characteristics. Fusarium incarnatum-equiseti species complex was the most frequently, followed by the members of Fusarium graminearum species complex and Fusarium fujikuroi species complex. However, one strain was identified as F. sporotrichioides Sherb. based on the following results: (I) Colonies on PDA produced dense mycelia and typical red pigment on the undersurface. Macroconidia were usually 3- to 5-septate, moderately curved to straight, and 27.46 ± 7.16 × 3.78 ± 0.8 μm (n = 50). Microconidia were ellipsoid to fusoid and 9.77 ± 2.29 × 2.99 ± 0.65 μm (n = 50). (II) Genomic DNA was extracted by AxyPrep Multisource Genomic DNA Miniprep Kit according to the manufacturer's instructions; the sequence analysis of partial translation elongation factor-1α (TEF-1α) and RNA polymerase II second largest subunit (RPB2) were accomplished with the primers EF1/2 and RPB5F/7CR, respectively. BLAST searches of the obtained sequences had 99-100% homology with several F. sporotrichioides strains from GenBank. DNA sequences of partial TEF-1α and RPB2 were deposited in GenBank as OQ068267 and OQ068269, respectively. (III) This strain can synthesis T-2, HT-2, diacetoxyscirpenol (DAS), and Neosolaniol (NEO) toxin at the concentration of about 5000, 600, 700 and 8000 μg/kg in rice culture, respectively, according to the previous culture and analysis methods (1,2). (IV) Pathogenicity tests were conducted with the rice variety Nanjing 9108 by spraying spore suspension (106 conidia/ml) on rice spikes (5 mL per spike) at the flowering stage. Control spikes were inoculated with sterile distilled water. Three weeks after inoculation, the inoculated rice glumes showed similar symptoms with the original samples in the field. No symptoms were observed on controls. Besides, F. sporotrichioides was successfully recovered from the inoculated rice spikes but not from controls. To our knowledge, this is the first report of F. sporotrichioides causing rice panicle blight in China and this disease appears to be a serious risk to food safety and human health. Funding: This work was supported by Jiangsu Agriculture Science and Technology (CX(21)1005).

Fusarium Species and Mycotoxins Associated with Sorghum Grains in Uruguay.

Grain mold and stalk rot are among the fungal diseases that cause significant losses in sorghum worldwide and are caused by different Fusarium spp. The presence of Fusarium species in sorghum grains causes yield losses and mycotoxin contamination, which represents a risk to consumers. In this study, Fusarium graminearum species complex (FGSC) had a high incidence, followed by Fusarium fujikuroi species complex (FFSC) and F. incarnatum-equiseti species complex. Within FFSC, F. proliferatum, F. andiyazi, F. fujikuroi, F. thapsinum, F. verticillioides and F. subglutinans were identified, and this was the first report of F. fujikuroi in sorghum. The most frequent toxins found in sorghum samples were deoxynivalenol (DON) and zearalenone (ZEN). The presence of fumonisins and nivalenol (NIV) was detected at low levels. This study adds new knowledge about the occurrence of Fusarium species and mycotoxins in sorghum grains. Furthermore, this is the first report in Uruguay on fungicide sensitivity for Fusarium isolates from sorghum, which constitutes an important starting point for defining management practices to minimize fungal infection and mycotoxin contamination.

Fusarium Keratitis From a Comprehensive Eye Health Care Facility in South India: Molecular Characterization by MALDI-TOF Versus Polymerase Chain Reaction Sequencing, Species Complex Distribution, and Clinical Correlation.

Fusarium keratitis possesses significant diagnostic and therapeutic challenges. Medically relevant Fusaria belong to various species complexes and show prominent differences in their antifungal susceptibility profile which may influence the clinical outcome. Rapid diagnostic methods are warranted for precise identification of species complexes for prompt initiation of correct antifungals. The aim of the study was to compare between matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) and polymerase chain reaction sequencing for correct species-level identification and to analyze the clinical outcome among different Fusarium species complexes. Twenty-nine culture-proven Fusarium keratitis cases were included in this study. A phylogenetic tree was constructed after TEF1α gene sequencing and isolates were subjected to MALDI-TOF MS, followed by database expansion and identification. Clinical outcome and risk association among species complexes were analyzed retrospectively. Maximum likelihood phylogeny categorized 68.9% isolates as Fusarium solani species complex (FSSC), 17.2% as Fusarium dimerum species complex (FDSC), followed by 13.7% as Fusarium fujikuroi species complex (FFSC). With extended database, MALDI-TOF MS could correctly speciate 96.5% (28/29) isolates. Previous antibiotic usage ( P = 0.034) and preoperative antifungal treatment with natamycin, voriconazole, or ketoconazole ( P = 0.025) were significantly higher in the FSSC group. The patients in the FFSC group had a significantly longer duration of symptoms at the time of clinical presentation to the clinic (15 days vs. 5 days, P = 0.030). Among 11 patients with a clinically poor outcome, 9 (31%) had FSSC infection. Patients infected with the FSSC had more aggressive infection with poor prognosis. MALDI-TOF MS can serve as the best alternative method to conventional molecular identification with reduced turnaround time, which may help the ophthalmologists to consider the appropriate antifungals or early surgical intervention for improved outcome.

Genetic Diversity and Pathogenicity of Fusarium fujikuroi Species Complex (FFSC) Causing Sugarcane Pokkah Boeng Disease (PBD) in China.

Pokkah boeng disease (PBD), a sugarcane foliar disease, is caused by various Fusarium spp. within the Fusarium fujikuroi species complex (FFSC). In the current study, we investigated the diversity of Fusarium spp. associated with PBD in China. In total, 320 leaf samples displaying PBD symptoms were collected over 10 consecutive years (2012 to 2021), during winter and summer, from six various sugarcane-growing regions (Guangxi, Yunnan, Guangdong, Zhejiang, Hainan, and Fujian) in China. Phylogenetic analysis of Fusarium spp. was reconstructed using translation elongation factor 1-α, and DNA-directed RNA polymerase II largest subunit and second-largest subunit multigene sequences. Evolutionary studies of these regions categorized the isolates into four FFSC species (F. sacchari, F. proliferatum, F. verticillioides, and F. andiyazi). The identified isolates, which developed irregular necrotic patches and rotting symptoms on the sugarcane plant after approximately 30 days were tested for their pathogenicity. Symptoms that appeared during pathogenicity testing were consistent with those observed under field conditions. Each strain of the pathogenic Fusarium spp. belonged to different vegetative compatibility groups (VCGs), and there was no affinity between VCGs. Our results contribute to understanding FFSC and accurately identifying Fusarium spp. associated with the sugarcane crop.

Effect of Abiotic Conditions on Growth, Mycotoxin Production, and Gene Expression by Fusarium fujikuroi Species Complex Strains from Maize.

Fusarium fujikuroi species complex (FFSC) strains are a major concern for food quantity and quality due to their strong ability to synthesize mycotoxins. The effects of interacting conditions of water activity, temperature, and incubation time on the growth rate, toxin production, and expression level of biosynthetic genes were examined. High temperature and water availability increased fungal growth. Higher water activity was in favor of toxin accumulation. The maximum amounts of fusaric acid (FA) and fumonisin B1 (FB1) were usually observed at 20-25 °C. F. andiyazi could produce a higher content of moniliformin (MON) in the cool environment than F. fujikuroi. The expression profile of biosynthetic genes under environmental conditions varied wildly; it was suggested that these genes might be expressed in a strain-dependent manner. FB1 concentration was positively related to the expression of FUM1, while a similar correlation of FUB8 and FUB12 with FA production could be observed in F. andiyazi, F. fujikuroi, and F. subglutinans. This study provides useful information in the monitoring and prevention of such toxins entering the maize production chain.

Morphological characterization, pathogenicity screening, and molecular identification of Fusarium spp. isolates causing post-flowering stalk rot in maize.

Post flowering stalk rot (PFSR) of maize caused by the Fusarium species complex is a serious threat to maize production worldwide. The identification of Fusarium species causing PFSR based on morphology traditionally relies on a small set of phenomic characteristics with only minor morphological variations among distinct Fusarium species. Seventy-one isolates were collected from 40 sites in five agro-climatic zones of India to assess the diversity of Fusarium spp. associated with maize crops showing symptoms of PFSR in the field. To investigate the pathogenicity of Fusarium spp. causing PFSR sixty isolates were toothpick inoculated between the first and second node at 55 days after sowing during the tassel formation stage of the crop in Kharif (Rainy season), and Rabi (Winter season) season field trials. Ten most virulent Fusarium isolates, based on the highest observed disease index, were identified by homology and phylogenetic analyses of partial sequences of the translation elongation factor 1 α (Tef-1α). Based on morphological traits such as mycelial growth patterns and mycelial pigmentation, Fusarium isolates were divided into nine clusters. The isolates were judged to be virulent based on their ability to decrease seedling vigour in in-vivo situations and high disease severity in field experiments. Pathogenicity test during the Kharif season showed 12 isolates with virulent disease symptoms with a mean severity ranging between 50 to 67 percent disease index (PDI) whereas in Rabi season, only five isolates were considered virulent, and the mean severity ranged between 52 to 67 PDI. Based on pathological characterization and molecular identification, 10 strains of Fusarium species namely, Fusarium acutatum (2/10), Fusarium verticillioides (Syn. Gibberella fujikuroi var. moniliformis) (7/10), Fusarium andiyazi (2/10) recorded the highest diseases index. All these species are part of the Fusarium fujikuroi species complex (FFSC). The distribution of virulent isolates is specific to a geographical location with a hot humid climate. Increased knowledge regarding the variability of Fusarium spp. responsible for PFSR of maize occurring across wide geographical locations of India will enable more informed decisions to be made to support the management of the disease, including screening for resistance in maize-inbred lines.

Relationship between Dubas Bug (Ommatissus lybicus) Infestation and the Development of Fungal-Induced Leaf Spots in Date Palms (Phoenix dactylifera)

The dubas bug (Ommatissus lybicus) (Hemiptera: Tropiduchidae) is a serious pest in date palms in several date-producing countries, including Oman. Infestation results in a severe reduction in yield and a weakening of date palm growth. In addition, egg laying, which causes injuries to date palm leaves, results in the development of necrotic lesions on the leaves. This study aimed at investigating the role of fungi in the development of necrotic leaf spots following dubas bug infestation. Leaf samples developing leaf spot symptoms were collected from dubas-bug-infested leaves, as the leaf spot symptoms were not observed on the non-infested leaves. Isolation from date palm leaves collected from 52 different farms yielded 74 fungal isolates. Molecular identification of the isolates revealed that they belonged to 31 fungal species, 16 genera, and 10 families. Among the isolated fungi, there were five Alternaria species, four species each of Penicillium and Fusarium, three species each of Cladosporium and Phaeoacremonium, and two species each of Quambalaria and Trichoderma. Out of the thirty-one fungal species, nine were pathogenic on date palm leaves and induced varying levels of leaf spot symptoms. The pathogenic species were Alternaria destruens, Fusarium fujikuroi species complex, F. humuli, F. microconidium, Cladosporium pseudochalastosporoides, C. endophyticum, Quambalaria cyanescens, Phaeoacremonium krajdenii, and P. venezuelense, which were reported for the first time as leaf spot causal agents in date palms. The study provided novel information on the effect of dubas bug infestation in date palms on the development of fungal infection and associated leaf spot symptoms.

Application of direct PCR for phylogenetic analysis of Fusarium fujikuroi species complex isolated from rice seeds.

Plant pathogenic fungi cause severe yield losses and mycotoxin contamination in crops. The precise and rapid detection of fungal pathogens is essential for effective disease management. Sequencing universal DNA barcodes has become the standard method for the diagnosis of fungal diseases, as well as for identification and phylogenetic analysis. A major bottleneck in obtaining DNA sequence data from many samples was the laborious and time-consuming process of sample preparation for genomic DNA. Here, we describe a direct PCR approach that bypasses the DNA extraction steps to streamline the molecular identification of fungal species. Using a direct PCR approach, we successfully sequenced the nuclear ribosomal internal transcribed spacer (ITS) region for the representatives of major fungal lineages. To demonstrate the usefulness of this approach, we performed a phylogenetic analysis of the Fusarium fujikuroi species complex, which causes bakanae ("foolish seedling") disease of rice and mycotoxin contamination. A total of 28 candidate strains were isolated from rice seeds in the Republic of Korea, and the identity of the isolates was determined using the DNA sequence of both ITS and translation elongation factor 1-α regions. In addition, 17F. fujikuroi isolates were examined for fumonisin (FB) production in rice medium using an enzyme-linked immunosorbent assay. Phylogenetic and toxigenic analyses showed that the F. fujikuroi strains could be distinguished into two groups: FB producers (B14-type) and non-producers (B20-type). These results will accelerate the molecular identification of fungal pathogens and facilitate the effective management of fungal diseases.

First Report of Strawberry Leaf Blight Caused by Fusarium Fujikuroi Species Complex in Bangladesh

Strawberry leaf blight is one of the devastating fungal diseases, responsible for reducing the quality of strawberry production worldwide. The present experiment was conducted to determine the pathogenic fungal association with strawberry leaves obtained from the experimental site of Botanical Garden, Jahangirnagar University, Bangladesh. A fungal pathogen causing leaf blight disease of the strawberry plant was detected as Fusarium fujikuroi species complex through classical and molecular approaches. Molecular characterization showed that nucleotide sequences of the internal transcribed region of the studied fungus (MH368119.1) were genetically identical with Fusarium fujikuroi species complex. Different culture media, pH, light regimes, temperatures were assayed to know the growth pattern of the identified pathogenic fungus. The optimum vegetative growth of Fusarium fujikuroi species complex was recorded on Potato Dextrose Agar, Potato Sucrose Agar, and Honey Peptone Agar media, at 25°C temperature, pH seven and under complete dark conditions. Among the three antagonistic fungi for the assessment as a biocontrol agent, Trichoderma asperellum was comparative effective against Fusarium fujikuroi species complex under in vitro conditions. Among systemic chemical fungicides, the highest mycelial inhibition (89.42%) of the isolated fungus was found by Tilt 250 EC (500 ppm). The leaf blight of strawberry caused by Fusarium fujikuroi species complex is the new record in Bangladesh. J Bio-Sci. 30(1): 11-20, 2022 (June)

Fusarium pseudocircinatum causing stunting and malformation of sunflower plants in Brazil.

Sunflower (Helianthus annuus L.) is among the main oleaginous crops used in Brazil. During January, 2017, at CCA/UFPB laboratory and greenhouses (Areia/Brazil, 6°58'12″ S; 35°42'15″ W), we observed various sunflower seeds (cultivar Olisun 3, 2017-2018 crop) highly infested with Fusarium. Those seeds were from crops in the municipality of Alagoinha -PB/Brazil (06º57'00'' S; 35º32'42'' W), supplied by Empresa Brasileira de Pesquisa Agropecuária/EMBRAPA. The emerged seedlings from these seeds were also contaminated, with 5% to 26% of them exhibiting stunting and malformation. Fusarium strains were isolated from symptomatic plants, and a single spore was used to grow pure colonies on potato-dextrose-agar (PDA) and synthetic-nutrient-poor-agar (SNA) media. Mycelia of PDA colonies were floccous and dense varying from yellow to orange. Fungal colonies developed aerial mycelium, producing orange pigments. On SNA, hyaline macroconidia, measuring 2.9-4.1 x 32.4-65.0 μm, slightly falcate with three to six septa. Oval microconidia, measuring 2.4-3.6 x 5.1-9.0 μm, were abundant in false heads forming on monophyalides. Chlamydospores were absent. Sterile hyphae were rarely formed. Colectively, the morphological features corresponded to species that belong to the Fusarium fujikuroi species complex (Leslie & Summerell, 2006). To assure the species identity, we sequenced the elongation factor 1α region of two representative isolates (i.e., F2 and F3, GenBank access numbers: MZ666934 and MZ666935, respectively) and compared them to the other Fusarium species found at Fusarium-ID and GenBank databases. Subsequently, we performed a maximum likelihood phylogenetic analysis including previously published sequences (Nicolli et al., 2020). Both isolates exhibited 100% similarity with Fusarium pseudocircinatum (MN386745), and clustered with its ex-type at 100% bootstrap values. The isolates were then grown on PDA amended with manitol to adjust the osmotic pressure to -1.0 Mpa, at 25 ± 2 ° C, for seven days (Sousa et al., 2008). A total of 100 disinfested sunflower seeds (cultivar Olisun 3, 2018-2019 crop) were distributed over the colonies and 48h later they were sown on sterile substrate maintained inside a greenhouse. About 30 days after inoculation, the emerged plants exhibited symptoms of stunting and malformation (60%) compared to controls, which were healthy. F. pseudocircinatum was reisolated from the symptomatic plants, completing Koch's postulates and identified based on above morphological and molecular biological methods. This test was performed twice. Fusarium pseudocircinatum is a broadly distributed and ecologicaly diverse species that infects several wild and cultivated plants. For instance, it was reported on seeds of the wild 'Peroba Rosa' (Aspidosperma polyneuron Muell. Arg.) in Brazil (Mazarotto et al. 2020). Infection of sunflowers may cause plant stand failures, thus resulting in yield and economic losses for Brazilian growers. The correct identification of any pathogen, especialy a generalist one such as F. pseudocircinatum, is crucial to develop eficient management strategies. To our best knowledge, this is the first report of F. pseudocircinatum causing stunting and malformation of sunflower plants in Brazil.

Identification and Characterization of a QTL for Growth of Fusarium circinatum on Pine-Based Medium.

Fusarium circinatum is an economically important pathogen of pine and resides in the Fusarium fujikuroi species complex. Here we investigated the molecular processes underlying growth in F. circinatum by exploring the association between growth and the nutritional environment provided by the pine host. For this purpose, we subjected a mapping population consisting of F. circinatum X F. temperatum hybrid progeny to an analysis of growth rate on a pine-tissue derived medium. These data, together with the available genetic linkage map for F. circinatum, were then used to identify Quantitative Trait Loci (QTLs) associated with growth. The single significant QTL identified was then characterized using the available genome sequences for the hybrid progeny's parental isolates. This revealed that the QTL localized to two non-homologous regions in the F. circinatum and F. temperatum genomes. For one of these, the F. circinatum parent contained a two-gene deletion relative to the F. temperatum parent. For the other region, the two parental isolates encoded different protein products. Analysis of repeats, G+C content, and repeat-induced point (RIP) mutations further suggested a retrotransposon origin for the two-gene deletion in F. circinatum. Nevertheless, subsequent genome and PCR-based analyses showed that both regions were similarly polymorphic within a collection of diverse F. circinatum. However, we observed no clear correlation between the respective polymorphism patterns and growth rate in culture. These findings support the notion that growth is a complex multilocus trait and raise the possibility that the identified QTL contains multiple small-effect QTLs, of which some might be dependent on the genetic backgrounds. This study improved our current knowledge of the genetic determinants of vegetative growth in F. circinatum and provided an important foundation for determining the genes and processes underpinning its ability to colonize its host environment.

Evaluation of Pathogenic Fusarium spp. Associated with Soybean Seed (Glycine max) in Hubei Province, China.

Soybean (Glycine max L.) seeds showing serious symptoms from rotted pods were collected from fields during the harvesting period (July to August 2020) in Taihu Farm, Jingzhou City, Hubei Province, China. Fusarium strains were frequently encountered during fungal isolation. According to the morphology and prepathogenicity tests, six strains showing variable effects on the seeds were selected for identification based on morphology and multilocus phylogenetic analysis of the internal transcribed spacer (ITS) region of the ribosomal DNA, translation elongation factor (EF-1α), calmodulin (CAM), β-tubulin (TUB), and partial RNA polymerase second largest subunit (RPB2), and to evaluate the pathogenic abilities on seed, root, and pod. The results indicated that the strains contained two species (Fusarium fujikuroi and F. proliferatum) in the Fusarium fujikuroi species complex (FFSC) and two species (F. luffae and F. sulawense) from the Fusarium incarnatum-equiseti species complex (FIESC). The two species of FFSC were more aggressive than those of FIESC on soybean seed, root, and pod. Among the strains, F. proliferatum YZU 201408 exhibited the most pathogenicity on all tests, with 72.2 to 90% disease severity.