Development of PCR-RFLP Technique for Identify Several Members of Fusarium incarnatum-equiseti Species Complex and Fusarium fujikuroi Species Complex

Fusarium incarnatum-equiseti species complex (FIESC) contain over 40 members. The primer pair Smibo1FM/Semi1RM on the RPB2 partial gene has been reported to be able to identify Fusarium semitectum. The F. fujikuroi species complex (FFSC) contains more than 50 members. The F. verticillioides as a member of this complex can be identified by using VER1/VER2 primer pair on the CaM partial gene. In this research, the Smibo1FM/Semi1RM can amplify F. sulawesiense, F. hainanense, F. bubalinum, and F. tanahbumbuense, members of FIESC at 424 bp. The VER1/VER2 can amplify F. verticillioides, F. andiyazi, and F. pseudocircinatum, members of FFSC at 578 bp. Polymerase chain reaction-restriction fragment length polymorphism by using the combination of three restriction enzymes EcoRV, MspI, and HpyAV can differentiate each species of FIESC used. The two restriction enzymes HpaII and NspI can distinguish each species of FFSC used. The proper identification process is required for pathogen control in the field in order to reduce crop yield loss.     

Molecular Identification,Mycotoxin Production and Comparative Pathogenicity of Fusarium temperatum Isolated from Maize in China

A recently isolated Fusarium population from maize in Belgium was identified as a new species, Fusarium temperatum. From a survey of Fusarium species associated with maize ear rot in nineteen provinces in 2009 in China, ten strains isolated from Guizhou and Hubei provinces were identified as F. temperatum. Morphological and molecular phylogenetic analyses based on the DNA sequences of individual translation elongation factor 1‐alpha and β‐tubulin genes revealed that the recovered isolates produced macroconidia typical of four‐septate with a foot‐shaped basal cell and belonged to F. temperatum that is distinctly different from its most closely related species F. subglutinans and others within Gibberella fujikuroi complex species from maize. All the strains from this newly isolated species were able to infect maize and wheat in field, with higher pathogenicity on maize. Mycotoxin determination of maize grains infected by the strains under natural field condition by ultra‐high‐performance liquid chromatography–tandem mass spectrometry and gas chromatography–mass spectrometry analyses showed that among fifteen mycotoxins assayed, two mycotoxins fumonisin B₁ and B₂ ranging from 9.26 to 166.89 μg/g were detected, with massively more FB₂ mycotoxin (2.8‐ to 108.8‐fold) than FB₁. This mycotoxin production profile is different from that of the Belgian population in which only fumonisin B₁ was barely detected in one of eleven strains assayed. Comparative analyses of the F. temperatum and F. subglutinans strains showed that the highest fumonisin producers were present among the F. temperatum population, which were also the most pathogenic to maize. These results suggested a need for proper monitoring and controlling this species in the relevant maize‐growing regions.     

Morphological and Molecular Characterization of Fusarium Isolated From Maize in Syria

Maize is the third most important cereal after wheat and barley in Syria. Maize plants are attacked by several Fusarium species causing mainly stalk and ear rot of maize which poses a major impact worldwide. Identification of Fusarium species is important for disease control and for assessment of exposure risk to mycotoxines. To identify Fusarium species attacking maize in Syria, a total of 32 Fusarium isolates were recovered from maize ears collected from four different geographical regions, mainly from Ghouta surrounding Damascus. Fusarium isolates were identified based on morphology and on partial DNA sequencing of the TEF1‐α and rDNA/ITS genes. The majority (26 of 32) of these isolates was identified as F. verticillioides (subdivided into four groups), whereas three isolates turned out to be F. thapsinum, F. equiseti and F. andiyazi. The remaining three isolates were close to F. andiyazi, although further investigation is needed to confirm whether they represent a yet undescribed species. Furthermore, our results showed that sequencing the TEF1‐α gene is much more informative than sequencing of the rDNA/ITS region for Fusarium identification at the species level. PCR analysis showed that only F. verticillioides isolates were potentially fumonisin producers and that only the F. equiseti isolate was potentially trichotecene producer. This is the first report on Fusarium thapsinum, F. equiseti and F. andiyazi attacking maize in Syria.     

Fusarium Species Associated with Mango Malformation in Peninsular Malaysia

Mango malformation has become the most important global disease on mango. Fusarium species previously associated with this disease include F. mangiferae, F. mexicanum, F. sterilihyphosum, F. proliferatum, F. subglutinans and F. tupiense. A few strains of F. proliferatum have been reported from Malaysia, but in this study, we report the results of more extensive sampling. The recovered strains were evaluated with morphology, mating tester strain cross‐fertility, amplified fragment length polymorphisms (AFLPs), and partial DNA sequences of the genes encoding translation elongation factor 1‐α (tef‐1α) and β‐tubulin (tub‐2). Amongst the 43 strains evaluated, three species were identified – F. proliferatum, F. mangiferae and F. subglutinans – with F. proliferatum being the most frequent (69%). None of the Fusarium species that appear to originate in the Americas were recovered in Malaysia, which suggests special measures may be warranted to keep these species from entering the country.     

Reactive Oxygen Species Activity and Antioxidant Properties of Fusarium Infected Bananas

Fusarium infection of bananas is a global problem that threatens the production of bananas. This study looks at the effects of the infection upon the reactive oxygen species (ROS) system, as well as the induced antioxidant properties in the roots, stems, leaves and fruits. Results show that there is a greater amount of damage in infected tissue samples as opposed to non‐infected. The damage was observed to be higher in the root samples. ROS assays were divided into two classes: ROS assays and ROS‐scavenging assays. Of the ROS assays, lipoxygenase was observed to be higher in the infected samples, while peroxidase (POD) and polyphenol oxidase (PPO) were significantly higher in infected stem, leaf and fruit samples. Among root samples, there was no significant difference in POD activity and PPO was lower in infected samples. Induction of ROS is important for the hypersensitive response (HR) to function properly. The ROS‐scavenging enzymes, namely ascorbate peroxidase, guaiacol peroxidase and superoxide dismutase, exhibited higher levels in the infected tissue. This is most likely to counter the build‐up of the ROS enzymes and to prevent further cell death. The increase in ROS‐scavenging assays also correlates with higher antioxidant properties as antioxidants play a critical role in regulating the HR free radicals.     

Fusarium Species From Plant Debris Associated With Soils From Maize Production Areas in the Transkei Region of South Africa

Fusarium species were isolated from plant debris in soil samples collected from cultivated maize fields and from undisturbed grasslands in two areas of the Transkei region. A total of 1205 Fusarium isolates were recovered from 27 soil samples. Fifteen Fusarium species were recovered from plant debris from Bizana soils and 13 Fusarium species from plant debris from Centane soils. The two dominant Fusarium species in both areas were F. oxysporum and F. equiseti. Very few isolates of F. moniliforme and F. subglutinans were recovered, but both of these species had significantly higher relative densities in cultivated soils than in undisturbed soils. This revised version was published online in June 2006 with corrections to the Cover Date.     

Ergosterol and Fusarium mycotoxins content in two maize cultivars under different forms of nitrogen fertilizers

Mycotoxins are secondary metabolites produced by many filamentous fungi, such as Fusarium, and are widespread in nature and can adversely affect almost all organisms. Mycotoxins can be formed in very different conditions such as when plants are growing or in crops that are stored in improper conditions. One of the methods to prevent the formation of mycotoxins in powdered feed is using varieties of appropriate quality and proper mineral fertilization, primarily nitrogen. In this study, the concentration of ergosterol and mycotoxins in the kernels of two maize cultivars from different maturity groups in Poland during the harvest of 2011 and 2012 was investigated using HPLC methods. We aimed to analyse the concentration of ergosterol as a fungal biomass indicator and mycotoxins from two maize cultivars under six different forms of nitrogen fertilizers. The “stay‐green” hybrid ES Paroli was characterized by a significantly lower ergosterol and the ability to accumulate the mycotoxins, compared to the classic cultivar ES Palazzo. The varieties of maize that remain green for a longer time period, the so‐called stay‐green type, are characterized by a different coefficient of nitrogen remobilization compared to the classic hybrids. Their dynamics of growth and accumulation of nutrients indicates a nitrogen fertilization system, indicating that slow‐release fertilizers are potentially more adapted to the cycles of maize vegetation. Hence, compared to the classic variant, the “stay‐green” variety uses nitrogen more effectively from mineral fertilizers. Such hybrids are healthier and remain in good condition for a longer time; therefore, they are characterized by a lower pathogen pressure. Thus, the cultivation of such varieties can be considered as one of the elements of integrated maize production. We observed identical reactions of the studied experimental factors for both vegetation seasons, which indicates the lack of environmental impact on the functioning and interaction of experimental factors.     

我国东北地区玉米穗腐镰孢菌的种类及其分离频率

从辽宁、吉林、黑龙江三省采集的43份玉米病穗上共分离获得327株镰孢菌,根据形态特征鉴定属于7个种,其分离频率分别为半裸镰孢菌(Fusarium semitecturrt)37.77%,尖孢镰孢菌(F.oxysporum)3.51%,拟轮枝镰孢菌(F.verticillioides)3.57%,克鲁克威尔镰孢菌(F.c...     

Rapid diagnosis of rice bakanae caused by Fusarium fujikuroi and F. proliferatum using loop‐mediated isothermal amplification assays

Rice bakanae is an important disease that causes serious rice production loss worldwide. We describe a new method for rapid diagnosis of rice bakanae caused by Fusarium fujikuroi and F. proliferatum, based on loop‐mediated isothermal amplification (LAMP) assays. After screening, primers were selected to target FusariumDNA sequences, that is, the intergenic spacer (IGS) region of the nuclear ribosomal operon and reductase‐coding region (RED1) in F. fujikuroi and F. proliferatum, respectively. Both LAMP assays efficiently amplified target genes in 70 min at 62°C. A colour change from purple to sky blue (visible to the unaided eye) was observed in the presence of the DNA of the targeted pathogens only, by adding hydroxynaphthol blue to the reaction system prior to amplification. The minimum of genomic DNA needed in the assays was 67 and 346 pg/μl for F. fujikuroi and F. proliferatum, respectively. Using the two assays described here, we successfully and rapidly diagnosed suspected diseased rice plant and seed samples collected from Jiangsu Province.     

Quantification of Alternaria,Cladosporium, Fusarium and Penicillium verrucosum in Conventional and Organic Grains by qPCR

We analysed the levels of Alternaria, Cladosporium, Fusarium and Penicillium verrucosum in grain samples harvested in 2011 and 2012 from conventional and organic farms using qPCR. In general, both Alternaria and Cladosporium occurred in all cereal grains in the highest quantities, followed by P. verrucosum and Fusarium. Alternaria, Cladosporium and P. verrucosum had the highest levels in crop mixtures, barley and rye and lower levels in wheat, while Fusarium levels were the highest in crop mixtures and wheat. The levels of Alternaria and P. verrucosum were higher in organic rye and wheat than conventional grains. Although the level of Fusarium was higher in conventional than organic rye, opposite results were obtained for crop mixtures. A positive correlation was found between Alternaria, Cladosporium and P. verrucosum, indicating that similar factors might affect the distribution of these fungi in grains.     

Genetic Diversity and Pathogenicity of Fusarium oxysporum f.sp. melonis Races from Different Areas of Italy

Fusarium wilt caused by Fusarium oxysporum f.sp. melonis (FOM) is a devastating disease of melon worldwide. Pathogenicity tests performed with F. oxysporum isolates obtained from Italian melon‐growing areas allowed to identify thirty‐four FOM isolates and the presence of all four races. The aims of this work were to examine genetic relatedness among FOM isolates by race determination and to perform phylogenetic analyses of identified FOM races including also other formae speciales of F. oxysporum of cucurbits. Results showed that FOM race 1,2 was the most numerous with a total of eighteen isolates, while six and nine isolates were identified as race 0 and 1, respectively, and just one isolate was assigned to race 2. Phylogenetic analysis was performed by random amplified polymorphic DNA (RAPD) profiling and by translation elongation factor‐1α (TEF‐1α) sequencing. The analysis of RAPD profiles separated FOM races into two distinct clades. Clade 1, which included races 0, 1 and 1,2, was further divided into ‘subclade a’ which grouped almost all race 1,2 isolates, and into ‘subclade b’ which included race 0 and 1 isolates. Clade 2 comprised only race 2 isolates. The phylogenetic analysis based on TEF‐1α separated FOM from the other formae speciales of F. oxysporum. Also with TEF‐1α analysis, FOM races 0, 1 and 1,2 isolates grouped in one single clade clearly separated from FOM race 2 isolates which grouped closer to F. oxysporum f.sp. cucumerinum. RAPD technique was more effective than TEF‐1α in differentiating FOM race 1,2 isolates from those belonging to the closely related races 0 and 1. Both phylogenetic analyses supported the close relationship between the three different FOM races which might imply the derivation from one another and the different origin of FOM race 2.     

Identification and genetic diversity of Fusarium sacchari associated with pokkah boeng disease of sugarcane in Northeast Brazil

Isolates from the Fusarium fujikuroi species complex, mainly F. sacchari, have been reported to be the causal agents of pokkah boeng in sugarcane in Brazil. However, inadequate information was available on the occurrence and genetic diversity of F. sacchari in Northeast Brazil, which is a limiting factor on management. Thus, isolates of F. subglutinans sensu lato from sugarcane plants with symptoms of pokkah boeng were evaluated using the sexual cross-fertility to determine species. All the isolates produced black perithecia when they were crossed with the test isolates of F. sacchari. Three weeks after the crossing, the formation of fertile ascospores cirri was observed. Thirty-four isolates were self-sterile hermaphrodites, while 21 were fertile only as males. Five isolates were homothallic. The effective size [N_e(f)] of the population as a function of the frequency of hermaphrodites and female sterile strains was 95.5%. The F. sacchari isolates were separated into four genetic groups independent of geographic location. The mean of polymorphism among all populations was 79%, and the average unbiased genetic diversity (uh) was considered moderate (0.31). This study in addition to confirming that F. sacchari as the main species associated with pokkah boeng in sugarcane in Northeast Brazil, reveals the relationship of mating type and genetic diversity of F. sacchari. The unrestricted gene flow between regions is probably the best explanation for the low geographic correlation. This knowledge will help in the adoption of management measures with fungicides or resistant cultivars.     

A multigene phylogeny of theGibberella fujikuroi species complex: Detection of additional phylogenetically distinct species

Phylogenetic relationships within theGibberella fujikuroi species complex were extended to newly discovered strains using nucleotide characters obtained by sequencing polymerase chain reaction (PCR) amplified DNA from 4 loci used in a previous study [nuclear large subunit 28S rDNA, nuclear ribosomal internal transcribed spacer (ITS) region, mitochondriaal small subunit (mtSSU) ribosomal DNA, and β-tubulin] together with two newly sampled protein-encoding nuclear genes, translation elongation factor EF-1α and calmodulin. Sequences from the ribosomal ITS region were analyzed separately and found to contain of two highly divergent, nonorthologous ITS2 types. Phylogenetic analysis of the individual and combined datasets identified 10 new phylogenetically distinct species distributed among the following three areas: 2 within Asia and 4 within both Africa and South America. Hypotheses of the monophyly ofFusarium subglutinans and its two formae speciales, f. sp.pini and f. sp.ananas, were strongly rejected by a likelihood analysis. Maximum parsimony results further indicate that the protein-encoding nuclear genes provide considerably more phylogenetic signal that the ribosomal genes sequenced. Relative apparent synapomorphy analysis was used to detect long-branch attraction taxa and to obtain a statistical measure of phylogenetic signal in the individual and combined datasets.     

Identification and Genetic Division of Fusarium graminearum and Fusarium asiaticum by Species‐Specific SCAR Markers

Fusarium head blight (FHB), also called scab, is a devastating and insidious disease of cereals including wheat (Triticum spp.) and barley (Hordeum vulgare L.) worldwide. Apart from direct yield losses, the most serious concern about FHB is the contamination of the crop with mycotoxins, which pose a health risk to human and livestock. Recent research reported that phylogenetic species F. asiaticum (Fa) and F. graminearum (Fg) were the major causal agents of FHB from infected wheat heads in China. To investigate the population structure of Fusarium species in China by species‐specific as well as the chemotype‐specific markers, sequence‐related amplified polymorphism (SRAP) markers were screened on representative isolates of F. asiaticum‐NIV, F. asiaticum‐ 3ADON and F. graminearum‐15ADON to find amplification products characteristic of either species or chemotypes. Selected amplified fragments were cloned and sequenced so that sequence‐characterized amplified region (SCAR) primer pairs could be developed which permit specific detection of Fusarium species using conventional PCR. Primer pairs SCAR‐Fa1 and SCAR‐Fg1 were confirmed to be able to amplify specific products only in F. asiaticum and F. graminearum isolates, respectively. These species‐specific primers were applied to determine genetic division of F. asiaticum and F. graminearum isolates collected in Yangtze–Huaihe valley. The results indicated that F. asiaticum was the predominant species causing FHB in this wheat production area. It is the first report that SRAP markers were adapted for species characterization in Fusarium isolates.     

中国的膨孢组镰孢菌(英文)

膨孢组镰孢菌Fusarium在自然界中广泛分布。该组包括4个种:木贼镰孢菌F.equiseti,藨草镰孢菌F.scirpi,长脚镰孢菌F.longipes和紧致镰孢菌F.compactum。这4个种均产生腹背不平行弯曲的大孢子。分离获得并描述了其中的3个种。木贼镰孢菌F.equiseti是镰孢菌中最常见的种之一,它产生典型的腹背不平行弯曲的大孢子,大孢子的顶细胞和足跟状基细胞明显伸长,菌落因缺乏红色素而呈黄褐色。藨草镰孢菌F.scirpi是比较少见的种类,它的典型特征是在典型的十字形产孢细胞上产生大量的小型分生孢子。由于它在PDA培养基上容易发生小孢子缺乏型的变异,因此,常常被错误地鉴定为木贼镰孢菌F.equiseti。长脚镰孢菌F.longipes的大孢子最容易与其他种的大孢子区分,它的顶细胞和基细胞均极度延长。当长脚镰孢菌F.longipes菌落因为变异而失去产生红色素的能力时,也容易与木贼镰孢菌F.equiseti混淆。基于该组大孢子的典型特征,作者将锐顶镰孢菌F.acuminatum排除在该组之外。     

Survival of Phytophthora cinnamomi and Fusarium verticillioides in commercial potting substrates for ornamental plants

Live plants, particularly when accompanied by soil or potting substrates, are considered the main pathway for international spread of plant pathogens. Modern, rapid shipping technologies for international plant trade increase the probability of plant pathogen survival during transport and the subsequent chances of disease outbreaks in new locations. The survival of two model pathogens, an Oomycete, Phytophthora cinnamomi, and a filamentous fungus, Fusarium verticillioides, was studied in two different commercial potting substrates (peat and peat‐free) under glasshouse conditions in the absence of a plant host. Survival rates were analysed at 2, 7, 12 and 17 months after substrate inoculation. Fusarium verticillioides had the longest survival rate, and was still present at 17 months. In contrast, P. cinnamomi survived up to 7 months but was not recovered after 12 or 17 months. There was no significant difference in the number of colony‐forming units (CFUs) of either pathogen in the two substrates, except at 2 months, when higher numbers were recovered from peat substrates.     

Identification and antibacterial characteristics of an endophytic fungus Fusarium oxysporum from Lilium lancifolium

Aims: The aim of this study is to isolate and identify an endophytic fungus with antibacterial activity from the Asian medicinal and culinary plant Lilium lancifolium and to study the characteristics of its major antibacterial fractions. Methods and Results: After strict sample sterilization, an endophytic fungus BH‐3 with great antibacterial activity against Leuconostoc mesenteroides was isolated from the bulbs of L. lancifolium and was identified as Fusarium oxysporum on the basis of internal transcribed spacer (ITS) rDNA sequence and morphological traits. After partial purification including superfiltration and gel filtration, the major antibacterial fractions were found to be the substances with the molecular mass ranging from 35 to 60 kDa, mainly 55 kDa. The partially purified antibacterial fractions were stable at thermal processes, with more than 80% of activity left at 60°C for 1 h, and even 70·75% left at 121°C for 15 min. 90·33–98·97% of activity was observed in the pH range of 4·0–7·0. But the fractions were sensitive to different proteases. Conclusions: Endophytic strain F. oxysporum BH‐3 isolated from the bulbs of L. lancifolium produced protein‐like antibacterial metabolites. The antibacterial assay against Leuc. mesenteroides indicated that the fractions were stable at thermal processes and wide pH conditions, but sensitive to proteolyses. Significance and Impact of the Study: This study provides an increasing understanding of endophytic F. oxysporum in L. lancifolium and its metabolites, which have a great potential in food industry as antibacterial agents.     

Fusarium spp. are Responsible for Shoot Canker of Kumquat in China

Shoot and branch canker and tree decline of kumquat (Fortunella margarita cv. Guban) were recorded in Yangshuo County, Guilin City, in the Guangxi Zhuang Autonomous Region of China during 2008–2011. Fusarium oxysporum and a new Fusarium species within the Gibberella fujikuroi complex (Fusarium sp. GLB1) were isolated repeatedly from the infected shoots and branches. Species identifications were verified by their high translation elongation factor 1‐alpha (TEF1) sequence similarity with those of the species epitypes. Koch's postulates were fulfilled on kumquat (cv. Guban) and mandarin establishing pathogenicity. To our knowledge, this is the first report of Fusarium shoot canker disease caused by F. oxysporum and Fusarium sp. on kumquat.