Scientific communication is facilitated by a data-driven, scientifically sound taxonomy that considers the end-user's needs and established successful practice. In 2013, the Fusarium community voiced near unanimous support for a concept of Fusarium that represented a clade comprising all agriculturally and clinically important Fusarium species, including the F. solani species complex (FSSC). Subsequently, this concept was challenged in 2015 by one research group who proposed dividing the genus Fusarium into seven genera, including the FSSC described as members of the genus Neocosmospora, with subsequent justification in 2018 based on claims that the 2013 concept of Fusarium is polyphyletic. Here, we test this claim and provide a phylogeny based on exonic nucleotide sequences of 19 orthologous protein-coding genes that strongly support the monophyly of Fusarium including the FSSC. We reassert the practical and scientific argument in support of a genus Fusarium that includes the FSSC and several other basal lineages, consistent with the longstanding use of this name among plant pathologists, medical mycologists, quarantine officials, regulatory agencies, students, and researchers with a stake in its taxonomy. In recognition of this monophyly, 40 species described as genus Neocosmospora were recombined in genus Fusarium, and nine others were renamed Fusarium. Here the global Fusarium community voices strong support for the inclusion of the FSSC in Fusarium, as it remains the best scientific, nomenclatural, and practical taxonomic option available.
A single step extraction-cleanup procedure using porous membrane-protected micro-solid phase extraction (μ-SPE) in conjunction with liquid chromatography-tandem mass spectrometry for the extraction and determination of aflatoxins (AFs) B1, B2, G1 and G2 from food was successfully developed. After the extraction, AFs were desorbed from the μ-SPE device by ultrasonication using acetonitrile. The optimum extraction conditions were: sorbent material, C8; sorbent mass, 20mg; extraction time, 90 min; stirring speed, 1,000 rpm; sample volume, 10 mL; desorption solvent, acetonitrile; solvent volume, 350 μL and ultrasonication period, 25 min without salt addition. Under the optimum conditions, enrichment factor of 11, 9, 9 and 10 for AFG2, AFG1, AFB2 and AFB1, respectively were achieved. Good linearity and correlation coefficient was obtained over the concentration range of 0.4-50 ng g(-1) (r(2) 0.9988-0.9999). Good recoveries for AFs ranging from 86.0-109% were obtained. The method was applied to 40 samples involving malt beverage (19) and canned coffee (21). No AFs were detected in the selected samples.
The development of a reversed phase high performance liquid chromatography fluorescence method for the determination of the mycotoxins fumonisin B(1) and fumonisin B(2) by using silica-based monolithic column is described. The samples were first extracted using acetonitrile:water (50:50, v/v) and purified by using a C(18) solid phase extraction-based clean-up column. Then, pre-column derivatization for the analyte using ortho-phthaldialdehyde in the presence of 2-mercaptoethanol was carried out. The developed method involved optimization of mobile phase composition using methanol and phosphate buffer, injection volume, temperature and flow rate. The liquid chromatographic separation was performed using a reversed phase Chromolith(®) RP-18e column (100 mm × 4.6 mm) at 30 °C and eluted with a mobile phase of a mixture of methanol and phosphate buffer pH 3.35 (78:22, v/v) at a flow rate of 1.0 mL min(-1). The fumonisins separation was achieved in about 4 min, compared to approximately 20 min by using a C(18) particle-packed column. The fluorescence excitation and emission were at 335 nm and 440 nm, respectively. The limits of detections were 0.01-0.04 μg g(-1) fumonisin B(1) and fumonisin B(2), respectively. Good recoveries were found for spiked samples (0.1, 0.5, 1.5 μg g(-1) fumonisins B(1) and B(2)), ranging from 84.0 to 106.0% for fumonisin B(1) and from 81.0 to 103.0% for fumonisin B(2). Fifty-three samples were analyzed including 39 food and feeds and 14 inoculated corn and rice. Results show that 12.8% of the food and feed samples were contaminated with fumonisin B(1) (range, 0.01-0.51 μg g(-1)) and fumonisin B(2) (0.05 μg g(-1)). The total fumonisins in these samples however, do not exceed the legal limits established by the European Union of 0.8 μg g(-1). Of the 14 inoculated samples, 57.1% contained fumonisin B(1) (0.16-41.0 μg g(-1)) and fumonisin B(2) (range, 0.22-50.0 μg g(-1)). Positive confirmation of selected samples was carried out using liquid chromatography-tandem mass spectrometry, using triple quadrupole analyzer and operated in the multiple reaction monitoring mode.
A reversed-phase high-performance liquid chromatographic method with capacitively coupled contactless conductivity detector (C(4)D) has been developed for the separation and the simultaneous determination of five underivatized long chain fatty acids (FAs), namely myristic, palmitic, stearic, oleic, and linoleic acids. An isocratic elution mode using methanol/1mM sodium acetate (78:22, v/v) as mobile phase with a flow rate of 0.6 mL min(-1) was used. The separation was effected by using a Hypersil ODS C(18) analytical column (250 mm x 4.6 mm x 5 microm) and was operated at 45 degrees C. Calibration curves of the five FAs were well correlated (r(2)>0.999) within the range of 5- 200 microg mL(-1) for stearic acid, and 2-200 microg mL(-1) for the other FAs. The proposed method was tested on four vegetable oils, i.e., pumpkin, soybean, rice bran and palm olein oils; good agreement was found with the standard gas chromatographic (GC) method. The proposed method offers distinct advantages over the official GC method, especially in terms of simplicity, faster separation times and sensitivity.
Zeolite Linde Type L (LTL) crystals with different length, diameter and particle size (nanosized LTL, rod LTL, cylinder LTL and needle LTL) were synthesized, characterized and were used as sorbent in the micro-solid phase extraction of ochratoxin A (OTA) before the high performance liquid chromatography detection. Under the optimized conditions, the detection limits of OTA for coffee and cereal were 0.09 ng g(-1) and 0.03 ng g(-1), respectively, while the quantification limits were 0.28 ng g(-1) and 0.08 ng g(-1), respectively. The recoveries of OTA of coffee and cereal spiked at 0.5, 10 and 25 ng g(-1) ranged from 91.7 to 101.0%. The proposed method was applied to forty-five samples of coffee and cereal. The presence of OTA was found in twenty-five samples, ranging from 0.28 to 9.33 ng g(-1).
A simple and rapid high performance liquid chromatographic with fluorescence detection method for the determination of the aflatoxin B1, B2, G1 and G2 in peanuts, rice and chilli was developed. The sample was extracted using acetonitrile:water (90:10, v/v%) and then purified by using ISOLUTE® multimode solid phase extraction. After the pre-column derivatisation, the analytes were separated within 3.7 min using Chromolith® performance RP-18e (100-4.6mm) monolithic column. To assess the possible effects of endogenous components in the food items, matrix-matched calibration was used for the quantification and validation. The recoveries of aflatoxins that were spiked into food samples were 86.38-104.5% and RSDs were <4.4%. The method was applied to the determination of aflatoxins in peanut (9), rice (5) and chilli (10) samples. Liquid chromatography-tandem mass spectrometry analysis using triple quadruple analyser and operated in the multiple reaction monitoring modes on the contaminated samples was performed for confirmation.
A simple, environmental friendly and selective sample preparation technique employing porous membrane protected micro-solid phase extraction (μ-SPE) loaded with molecularly imprinted polymer (MIP) for the determination of ochratoxin A (OTA) is described. After the extraction, the analyte was desorbed using ultrasonication and was analyzed using high performance liquid chromatography. Under the optimized conditions, the detection limits of OTA for coffee, grape juice and urine were 0.06 ng g(-1), 0.02 and 0.02 ng mL(-1), respectively while the quantification limits were 0.19 ng g(-1), 0.06 and 0.08 ng mL(-1), respectively. The recoveries of OTA from coffee spiked at 1, 25 and 50 ng g(-1), grape juice and urine samples at 1, 25 and 50 ng mL(-1) ranged from 90.6 to 101.5%. The proposed method was applied to thirty-eight samples of coffee, grape juice and urine and the presence of OTA was found in eighteen samples. The levels found, however, were all below the legal limits.
A total of 82 isolates of microfungi were isolated from 6 sandy soil samples collected from Teluk Aling beach, Pulau Pinang. The soil microfungi were isolated by using direct isolation, debris isolation and soil dilution techniques. Based on morphological characteristics, seven genera of microfungi were identified namely, Fusarium (42%), Aspergillus (24%), Trichoderma (13%), Curvularia (9%), Colletotrichum (6%), Helminthosporium (4%) and Penicillium (2%). The most common species isolated was Fusarium solani followed by Fusarium semitecum, Aspergillus niger, Trichoderma viride, Curvularia clavata, Curvularia lunata, Helminthosporium velutinum, Colletotrichum sp. and Penicillium chrysogenum. From the present study, it appears that the sandy beach contains a microfungi reservoir comprising of a variety of genera which contributes significantly to the ecological functioning of a marine ecosystem.
Sorghum is the fifth most important cereal worldwide, spreading from Africa throughout the world. It is particularly important in the semi-arid tropics due to its drought tolerance, and when cultivated in Southeast Asia commonly occurs as a second crop during the dry season. We recovered Fusarium from sorghum in Thailand and found F. proliferatum, F. thapsinum and F. verticillioides most frequently, and intermittent isolates of F. sacchari and F. beomiforme. The relatively high frequencies of F. proliferatum and F. verticillioides, suggest mycotoxin contamination, particularly fumonisins and moniliformin, should be evaluated. Genetic variation within the three commonly recovered species was characterized with vegetative compatibility, mating type, Amplified Fragment Length Polymorphisms (AFLPs), and female fertility. Effective population number (N e ) was highest for F. verticillioides and lowest for F. thapsinum with values based on mating type allele frequencies higher than those based on female fertility. Based on AFLP genetic variation, the F. thapsinum populations were the most closely related, the F. verticillioides populations were the most distantly related, and the F. proliferatum populations were in an intermediate position. The genetic variation observed could result if F. thapsinum is introduced primarily with seed, while F. proliferatum and F. verticillioides could arrive with seed or be carried over from previous crops, e.g., rice or maize, which sorghum is following. Confirmation of species transmission patterns is needed to understand the agricultural systems in which sorghum is grown in Southeast Asia, which are quite different from the systems found in Africa, Australia, India and the Americas.
The aim of this study was to identify the foodborne pathogens mainly, Aspergillus sp. colonizing rice grains using cultural and microscopic methods. Four differential media (Czapek Dox Agar (CZA), Czapek Yeast Agar (CYA), Malt Extract Agar (MEA) and Czapek yeast 20% sucrose agar (CYA20S)) were used for differentiation of five Aspergillus sp., colonizing rice grains comparing with standard cultures. We studied macroscopic (colony color and diameter, conidia color, exudates, sclerotia and colony texture) and microscopic (conidiophore color, length and breadth, conidia size, shape and surface texture, vesicle diameter and phialides length and breadth) characteristics for identification of 110 isolates of Aspergillus sp. isolated from 65 rice grain samples collected from various countries in South Asia (Cambodia, India, Indonesia, Malaysia and Thailand). According to morphological characters, all these isolates were belonging to Aspergillus flavus (45), A. fumigatus (8), A. ochraceus (7), A. niger (42) and A. tamarii (8). This is the first report on identification of large number of Aspergillus strains isolated from rice grains in South Asia.
Fusarium is one of the most important pathogenic and toxigenic fungi widely distributed all over the world, including Iran. Fusarium species are found frequently in stored agriculture products especially wheat. The objective of this study was to identify Fusarium species associated with stored wheat seeds and their pathogenicity on root and head of wheat in Kermanshah, the leading province in wheat production in Iran. In this survey 75 seed samples of stored wheat were collected from 10 different regions during 2006-2008 and tested for the presence of Fusarium. Fusarium spp. were found in 51 (68%) of 75 samples. A total of 580 Fusarium strains were isolated, identified and preserved. All these strains belong to 20 Fusarium spp. according to morphological characters. Each conidial suspension of selected strains representing all species was evaluated for their pathogenicity on roots and spikes of healthy wheat var. Fallat in the greenhouse. F. graminearum, F. crookwellense, F. trichothecioides, F. culmorum and F. verticillioides were the most pathogenic to wheat's head. Foot rot assessment revealed that F. pseudograminearum and F. culmorum were the most damaging species. Of the Fusarium isolates, F. graminearum was the most prevalent followed by F. verticillioides and F. proliferatum. This is the first comprehensive report on identity and distribution of Fusarium spp. from stored wheat seeds in Iran while F. nelsonii was reported for the first time from wheat seeds in Iran.
Fusarium is a cosmopolitan and highly diversified genus of saprophytic, phytopathogenic and toxigenic fungi. However, the existence and diversity of a few species of Fusarium are restricted to a certain area or climatic condition. The present study was conducted to determine the occurrence and diversity of Fusarium species in tropical highland areas in Malaysia and to compare with those in temperate and subtropical regions. A series of sampling was carried out in 2005 to 2009 at several tropical highland areas in Malaysia that is: Cameron Highlands, Fraser Hills and Genting Highlands in Pahang; Penang Hill in Penang; Gunung Jerai in Kedah; Kundasang and Kinabalu Park in Sabah; Kubah National Park and Begunan Hill in Sarawak. Sampling was done randomly from various hosts and substrates. Isolation of Fusarium isolates was done by using pentachloronitrobenzene (PCNB) agar and 1449 isolates of Fusarium were successfully recovered. Based on morphological characteristics, 20 species of Fusarium were identified. The most prevalent species occurring on the highlands areas was F. solani (66.1%) followed by F. graminearum (8.5%), F. oxysporum (7.8%), F. semitectum (5.7%), F. subglutinans (3.5%) and F. proliferatum (3.4%). Other Fusarium species, namely F. avenaceum, F. camptoceras, F. chlamydosporum, F. compactum, F. crookwellense, F. culmorum, F. decemcellulare, F. equiseti, F. nygamai, F. poae, F. proliferatum, F. sacchari, F. sporotrichioides, F. sterilihyphosum and F. verticillioides accounted for 1% recoveries. The present study was the first report on the occurrences of Fusarium species on highland areas in Malaysia.
Fusarium is one of the important phytopathogenic genera of microfungi causing serious losses on cucurbit plants in Kermanshah province, the largest area of cucurbits plantation in Iran. Therefore, the objectives in this study were to isolate and identify disease-causing Fusarium spp. from infected cucurbit plants, to ascertain their pathogenicity, and to determine their phylogenetic relationships. A total of 100 Fusarium isolates were obtained from diseased cucurbit plants collected from fields in different geographic regions in Kermanshah province, Iran. According to morphological characters, all isolates were identified as Fusarium oxysporum, Fusarium proliferatum, Fusarium equiseti, Fusarium semitectum and Fusarium solani. All isolates of the five Fusarium spp. were evaluated for their pathogenicity on healthy cucumber (Cucumis sativus) and honeydew melon (Cucumis melo) seedlings in the glasshouse. F. oxysporum caused damping-off in 20-35 days on both cucurbit seedlings tested. Typical stem rot symptoms were observed within 15 days after inoculation with F. solani on both seedlings. Based on the internal transcribed spacer (ITS) regions of ribosomal DNA (rDNA) restriction fragment length polymorphism (RFLP) analysis, the five Fusarium species were divided into two major groups. In particular, isolates belonging to the F. solani species complex (FSSC) were separated into two RFLP types. Grouping among Fusarium strains derived from restriction analysis was in agreement with criteria used in morphological classification. Therefore, the PCR-ITS-RFLP method provides a simple and rapid procedure for the differentiation of Fusarium strains at species level. This is the first report on identification and pathogenicity of major plant pathogenic Fusarium spp. causing root and stem rot on cucurbits in Iran.
Endophytic fungi were isolated from different parts of healthy paddy plants (Oryza sativa). The most common endophytic fungal genus recovered was Fusarium, followed by Aspergillus, Curvularia, Penicillium, Gilmaniella and Arthrobotrys foliicola. Fusarium and Curvularia had higher occurrences in the seeds compared with the other fungi. Aspergillus was recovered mostly from leaf blades and Penicillium from the leaf sheath. Gilmaniella and A. foliicola were isolated only from the roots and leaf blade, respectively. The assemblage of endophytic fungi in healthy tissues of paddy plants may indicate that some of the fungi are possible latent pathogens and some may become saprophytic.
Malaysian population widely consumes the cereal-based foods, oilseeds, nuts, and spices in their daily diet. Mycotoxigenic fungi are well known to invade food products under storage conditions and produce mycotoxins that have threat to human and animal health. Therefore, determining toxigenic fungi and aflatoxin B(1) (AFB1) in foods used for human consumption is of prime importance to develop suitable management strategies and to minimize risk. Ninety-five food products marketed in Penang, Malaysia were randomly collected from different supermarkets and were analyzed for presence of Aspergillus spp. by agar plate assay and AFB1 by enzyme-linked immunosorbent assay (ELISA). A. flavus was the dominant fungi in all foods followed by A. niger. Fifty-five A. flavus strains were tested for their ability to produce aflatoxins on rice grain substrate. Thirty-six (65.4%) strains out of 55 produced AFB1 ranging from 1700 to 4400 μg/kg and 17 strains (31%) produced AFB2 ranging from 620 to 1670 μg/kg. Natural occurrence of AFB1 could be detected in 72.6% food products ranging from 0.54 to 15.33 μg/kg with a mean of 1.95 μg/kg. Maximum AFB1 levels were detected in peanut products ranging from 1.47 to 15.33 μg/kg. AFB1 levels detected in all food products were below the Malaysian permissible limits (<35 μg/kg). Aspergillus spp. and AFB1 was not detected in any cookies tested. Although this survey was not comprehensive, it provides valuable information on aflatoxin levels in foods marketed in Malaysia.
Members of Fusarium solani species complex (FSSC) have been known as plant, animal, and human pathogens. Nevertheless, the taxonomic status of such an important group of fungi is still very confusing and many new species as well as lineages have been elucidated recently. Unfortunately, most of the new taxa came from temperate and subtropical regions. Therefore, the objectives of the present study were to identify strains of FSSC recovered from different sources in Malaysia. In the present study, 55 strains belonging to the FSSC were examined and phylogenetically analyzed on the basis of internal transcribed spacer (ITS) regions and partial translation elongation factor-1 (TEF-1α) sequences. Based on morphological features, a total of 55 strains were selected for molecular studies. Based on morphological features, the strains were classified into four described Fusarium species, namely Fusarium keratoplasticum, Fusarium falciforme, FSSC 5, and Fusarium cf. ensiforme, and one unknown phylogenetic species was introduced. Although the data obtained from morphological and molecular studies sufficiently supported each other, the phylogenetic trees based on ITS and TEF-1α dataset clearly distinguished closely related species and distinctly separated all morphological taxa. All members of FSSC in this research were reported for the first time for Malaysian mycoflora.
Mating studies were conducted on 78 isolates of Fusarium species section Liseola from rice, sugarcane and maize. From the crosses with tester strains of Gibberella fujikuroi species complex, 64.1% (50 out of 78 isolates) were cross-fertile with tester strains of mating populations A to E. The results of the mating studies showed that of the 50 isolates, 19 belonged to mating population A (Gibberella moniliformis), 18 to mating population B (Gibberella sacchari), 4 to mating population E (Gibberella subglutinans), 6 to mating population D (Gibberella intermedia) and 3 to mating population C (G. fujikuroi). Identification of several mating populations from rice, sugarcane and maize could be important biological entities under field conditions.
A total of 13 Colletotrichum isolates were obtained from different banana cultivars (Musa spp.) with symptoms of anthracnose. Colletotrichum isolates from anthracnose of guava (Psidium guajava) and water apple (Syzygium aqueum) were also included in this study. Based on cultural and morphological characteristics, isolates from banana and guava were identified as Colletotrichum musae and from water apple as Colletotrichum gloeosporiodes. Isolates of C. musae from banana and guava had similar banding patterns in a randomly amplified polymorphic DNA (RAPD) analysis with four random primers, and they clustered together in a UPGMA analysis. C. gloeosporiodes from water apple was clustered in a separate cluster. Based on the present study, C. musae was frequently isolated from anthracnose of different banana cultivars and the RAPD banding patterns of C. musae isolates were highly similar but showed intraspecific variations.
The objective of this study was to identify Fusarium species in the Gibberella fujikuroi species complex from rice, sugarcane and maize as most of the Fusarium species in the species complex are found on the three crops. Isolates used were collected from the field and obtained from culture collection. The Fusarium isolates were initially sorted based on morphology and identifications confirmed based on the DNA sequence of the translation elongation factor 1-α (TEF-1α) gene. Based on the closest match of BLAST analysis, five species were recovered, namely, F. sacchari, F. fujikuroi, F. proliferatum, F. andiyazi and F. verticillioides. This is the first report regarding F. andiyazi from rice in Malaysia and Southeast Asia. The phylogenetic tree generated by using the neighbor joining method showed that isolates from the same species were grouped in the same clade. The present study indicated that Fusarium species in the G. fujikuroi species complex are widespread in rice, sugarcane and maize in Peninsular Malaysia. The findings also suggest that the use of morphological characters for identification of Fusarium species in the G. fujikuroi species complex from the three crops will lead to incorrect species designation.