Differentiation of Fusarium verticillioides from Banana Fruits by IGS and EF-1α Sequence Analyses

Fusarium verticillioides(Gibberella moniliformis, G. fujikuroi mating population A) is an important pathogen of maize and produces several mycotoxins, including fumonisins, which cause diseases in humans and animals. The partial sequences of the IGS region (Intergenic Spacer of rDNA units) and the translation elongation factor EF-1α gene of a representative sample (48 strains) of F. verticillioides isolated from diverse hosts, geographical origins and with different levels of fumonisin production were analyzed. A phylogenetic approach by PAUP was used to evaluate the genetic variability in this species and to detect the occurrence of lineages which could be associated with different hosts or produced different toxin profiles within this species. Genetic variability detected by both sequences was high, especially with the IGS sequence which showed a high number of parsimony-informative sites and nucleotide diversity. The results of the phylogenetic analysis indicated that F. verticillioides occurs as (i) a major fumonisin-producing population with a wide geographical distribution, wide host preferences (cereals), showing variability and considerable incidence of sexual reproduction and (ii) a minor fumonisin non-producing population, with restricted host preference (banana), low variability and clonal reproductive strategy.     

Toxin Profile, Fertility and AFLP Analysis of Fusarium verticillioides from Banana Fruits

Gibberella fujikuroi is composed of at least nine mating populations (MPs), corresponding to biological species and assigned letters (from A to I). Each MP possesses a specific toxicological profile and a preferential host. Members of Fusarium verticillioides and F. thapsinum, anamorphs respectively of MPs A (G. moniliformis) and F (G. thapsina), share identical morphological traits, but they have a different preferential hosts (maize and sorghum, respectively) and toxin profiles, beingable the only member of MP A to produce fumonisins and the only member of MP F to produce moniliformin. Isolates from banana fruits were identified morphologically as F. verticillioides. The isolates were analyzed for fumonisin and moniliformin production. While none of the isolates produced fumonisin, all the isolates produced moniliformin. The isolates were crossed with tester strains of MPs A and F, showing ability to produce fertile perithecia only when crossed by MP A tester strains isolated from maize. However, the time required for the formation of fertile perithecia and their size differed significantly from the usual fertile crosses of strains belonging to MP A. Pathogenicity tests using such isolates of F. verticillioides isolated from banana and a set of F. verticillioides isolates isolated from maize were also performed on banana fruits. The data showed that the isolates from banana were more pathogenic. Finally, isolates from banana and maize were compared using AFLP. The results revealed that isolates from banana and maize produced two distinctly different clusters. In conclusion, isolates of F. verticillioides from banana showed specific traits (toxin production, in vitro fertility, pathogenicity and molecular profiles), that were different to those of the same species from maize. This could reflect important differences in the ecology and natural history of the population from banana and should encourage further investigations into the mechanisms of toxin production and pathogenicity within the same MP.     

Synteny in Toxigenic Fusarium Species: The Fumonisin Gene Cluster and the Mating Type Region as Examples

A comparative genomic approach was used to study the mating type locus and the gene cluster involved in toxin production (fumonisin) in Fusarium proliferatum, a pathogen with a wide host range and a complex toxin profile. A BAC library, generated from F. proliferatum isolate ITEM 2287, was used to identify chromosomal regions flanking the mating type locus and the gene cluster involved in the biosynthesis of fumonisin. These regions were sequenced and compared with corresponding sequences in other ascomycetes. The results demonstrated that the level of synteny between ascomycetes can vary greatly for different genomic regions and that the level of similarity of genes within a region can also fluctuate strongly. Synteny was found in the regions flanking the mating type idiomorph among ascomycetes that supposedly diverged 280 million years ago. The fumonisin gene clusters of F. proliferatum and F. verticillioides were completely syntenic but absent in F. graminearum. The regions flanking the fumonisin gene clusters were highly dissimilar between F. proliferatum and F. verticillioides, whereas they formed a continuous region in F. graminearum. This indicates that the fumonisin gene cluster has been inserted at different genome locations in both species. Surprisingly low similarity was found between the corresponding genes within the fumonisin cluster of F. proliferatum and F. verticillioides, compared to other genomic sequences indicative for two independent acquisition events from distinct genetic sources. The results demonstrate the power of comparative genomics for gene annotation and for studies on the evolution of genes, gene-clusters and species.     

Genetic structure of <Emphasis Type="Italic">Fusarium verticillioides</Emphasis> populations isolated from maize in Argentina

Fusarium verticillioides (sexual stage Gibberella moniliformis) is a common fungal pathogen of maize worldwide that also produces fumonisin mycotoxins. Populations of this fungus can be diverse with respect to neutral and selectable genetic markers. We used vegetative compatibility groups (VCGs) and amplified fragment length polymorphisms (AFLPs) to evaluate the genetic structure of three F. verticillioides populations from commercial maize fields in Argentina. Based on work with similar populations from outside South America, we expected individuals within the populations to be genetically diverse, that genotypic variation would be distributed in a manner consistent with random mating, and that populations from different locations would be genetically indistinguishable from one another. We analysed 62 AFLP loci for 133 fungal isolates. All three populations were genotypically diverse but genetically similar and potentially part of a larger, randomly mating population, with significant genetic exchange occurring between the three subpopulations. There was no evidence for linkage disequilibrium at P = 0.05. The low values of G _ST , the lack of frequent private alleles, and the lack of a systemic pattern of linkage disequilibrium all suggest that sexual reproduction is sufficiently common in F. verticillioides and that the dispersal of strains is sufficiently efficient for the population of F. verticillioides in the main maize growing region to be a single randomly mating population with no detectable genetic subdivision. Thus differences in disease and/or toxin production observed in this region are best attributed to differences other than the genetic composition of the population.     

Epidemiology of Fusarium Diseases and their Mycotoxins in Maize Ears

Fusarium species cause two distinct diseases on ears of maize, Fusarium ear rot (or pink ear rot) and Gibberella ear rot (or red ear rot), both of which can result in mycotoxin contamination of maize grain. The primary causal agent for Fusarium ear rot is Fusarium verticillioides, but F. subglutinans and F. proliferatum are also important. Gibberella ear rot is caused primarily by F. graminearum, but F. culmorum can also be important, especially in Europe. Aspects of the epidemiology of both diseases have been studied for decades, but only recently have efforts been made to synthesize this information into comprehensive models of disease development. Much of the work on F. graminearum has focused on Fusarium head blight of small-grain crops, but some of the results obtained are also relevant to maize. The primary mycotoxins produced by these fungi, fumonisins and deoxynivalenol, have differing roles in the disease-cycle, and these roles are not completely understood, especially in the case of fumonisins. Progress is being made toward accurate models for risk assessment of both diseases, but key challenges remain in terms of integrating models of pre- and post-infection events, quantifying the roles of insects in these diseases, and characterizing interactions among competing fungi and the environment.     

Genetic characteristics of <Emphasis Type="Italic">Fusarium verticillioides</Emphasis> from corn in the Philippines

Fusarium verticillioides (teleomorph: Gibberella moniliformis = G. fujikuroi mating population A) is one of the most important fungal pathogens of corn worldwide. The pathogen produces fumonisins, mycotoxins that are potentially harmful to humans and animals. Thirty-five Fusarium isolates from Laguna and Isabela, Philippines were identified morphologically and molecularly as F. verticillioides and characterized by PCR for mating type (MAT). Twenty-six isolates were MAT1-2, while nine isolates were MAT1-1. The isolates from Isabela were tested for aggressiveness, rated according to a disease index (%) on ‘Super Sweet’ corn IPB variety 1 under field conditions across two trials using the toothpick inoculation method. Other aggressiveness traits such as inhibition of seedling emergence, decrease of seedling height, fresh and dry mass were also determined in two greenhouse trials. All isolates were pathogenic to corn seedlings and mature plants compared to the noninoculated control. Significant genotypic variation was observed (P = 0.01) in trial, isolate, and isolate × trial interaction for all traits across two greenhouse trials and that aggressiveness was highly influenced by the trial conditions. Similarly, significant genotypic variation was observed in trial, replication within trial and isolate × trial interaction. Heritability was high for the five traits in the greenhouse (h ² = 0.80–0.90) but moderate for disease index in the field (h ² = 0.49). In an analysis of fumonisin production in corn culture by high performance liquid chromatography, 30 of 35 strains produced a detectable level of fumonisins, varying from 0.44 to 742 μg FB₁/g corn, 0.51–222 μg FB₂/g and 0.12–37 μg FB₃/g. Isabela isolates produced more fumonisins than the Laguna isolates did. In vitro fumonisin production had little correlation with the field disease index (r = 0.32) or with greenhouse seedling germination (0.25).     

Ear Rot Susceptibility and Mycotoxin Contamination of Maize Hybrids Inoculated with Fusarium Species Under Field Conditions

The development of new maize hybrids with resistance to Fusarium infection is an effective means of minimizing the risk of mycotoxin contamination. Several maize hybrids have been investigated for Fusarium ear rot and accumulation of fumonisin B₁ (FB₁), fumonisin B₂ (FB₂), beauvericin (BEA) and fusaproliferin (FP) after artificial inoculation in the field with toxigenic strains of Fusarium verticillioides and Fusarium proliferatum. The year of inoculation had a significant influence on the disease severity and mycotoxin accumulation in maize kernels. Of all the hybrids tested, only Mona exhibited resistance to ear rot caused by F. verticillioides and produced low levels of fumonisins during three years of experiments. In Fusarium-damaged kernels (FDK), fumonisin B₁, fumonisin B₂, beauvericin and fusaproliferin were detected at concentrations much higher (up to 10–20 times) than in healthy-looking kernels (HLK). Animal and human exposure to these mycotoxins can be drastically reduced by removing mouldy and visibly damaged kernels from the commodity.     

Control of Fusarium verticillioides,cause of ear rot of maize,by Pseudomonas fluorescens

BACKGROUND: Maize is one of the staple food crops grown in India. Fusarium verticillioides (Sacc.) Nirenberg is the most important fungal pathogen of maize, associated with diseases such as ear rot and kernel rot. Apart from the disease, it is capable of producing fumonisins, which have elicited considerable attention over the past decade owing to their association with animal disease syndromes. Hence, the present study was conducted to evaluate ecofriendly approaches by using a maize rhizosphere isolate of Pseudomonas fluorescens (Trev.) Mig. and its formulation to control ear rot disease and fumonisin accumulation, and also to study the capacity to promote growth and yield of maize. In vitro assays were conducted to test the efficacy of P. fluorescens as a seed treatment on seed germination, seedling vigour and also the incidence of F. verticillioides in different maize cultivars. The field trials included both seed treatment and foliar spray. For all the experiments, P. fluorescens was formulated using corn starch, wheat bran and talc powder. In each case there were three different treatments of P. fluorescens, a non‐treated control and chemical control. RESULTS: Pure culture and the formulations, in comparison with the control, increased plant growth and vigour as measured by seed germination, seedling vigour, plant height, 1000 seed weight and yield. P. fluorescens pure culture used as seed treatment and as spray treatment enhanced the growth parameters and reduced the incidence of F. verticillioides and the level of fumonisins to a maximum extent compared with the other treatments. CONCLUSION: The study demonstrates the potential role of P. fluorescens and its formulations in ear rot disease management. The biocontrol potential of this isolate is more suited for fumonisin reduction in maize kernels intended for human and animal feed. Copyright © 2009 Society of Chemical Industry     

Toxigenic Fusarium Species and Mycotoxins Associated with Maize Ear Rot in Europe

Several Fusarium species occurring worldwide on maize as causal agents of ear rot, are capable of producing mycotoxins in infected kernels, some of which have a notable impact on human and animal health. The main groups of Fusarium toxins commonly found are: trichothecenes, zearalenones, fumonisins, and moniliformin. In addition, beauvericin and fusaproliferin have been found in Fusarium-infected maize ears. Zearalenone and deoxynivalenol are commonly found in maize red ear rot, which is essentially caused by species of the Discolour section, particularly F. graminearum. Moreover, nivalenol and fusarenone-X were often found associated with the occasional occurrence of F. cerealis, and diacetoxyscirpenol and T-2 toxin with the occurrence of F. poae and F. sporotrichioides, respectively. In addition, the occurrence of F. avenaceum and F. subglutinans usually led to the accumulation of moniliformin. In maize pink ear rot, which is mainly caused by F. verticillioides, there is increasing evidence of the wide occurrence of fumonisin B₁. This carcinogenic toxin is usually found in association with moniliformin, beauvericin, and fusaproliferin, both in central Europe due to the co-occurrence of F. subglutinans, and in southern Europe where the spread of F. verticillioides is reinforced by the widespread presence of F. proliferatum capable of producing fumonisin B₁, moniliformin, beauvericin, and fusaproliferin.     

Complementary host–pathogen genetic analyses of the role of fumonisins in the Zea mays–Gibberella moniliformis interaction

Zea mays often is colonized with the fungus Gibberella moniliformis, which produces fumonisin toxins. The role of fumonisins in seedling colonization and blight was studied using complementary genetic analyses of host and pathogen. Only one of two fumonisin B1 (FB1)-insensitive maize backcross lines was more resistant than the FB1-sensitive parent to seedling blight, indicating that the increase in FB1-insensitivity was not associated with an increase in resistance. FB1-producing and nonproducing isogenic fungal strains did not differ in ability to cause seedling blight, but the FB1-producing strain was more effective in systemic colonization of seedlings in reciprocal strain challenge tests. Together, these and previous results indicate that the role of fumonisins depends on complex environmental and genetic contexts in this host–pathogen interaction.     

拟轮枝镰孢菌EST-SSR信息分析与标记开发

为开发可用于拟轮枝镰孢菌Fusarium verticillioides及其近缘种遗传多样性分析的SSR引物,利用生物信息学方法和PCR技术,通过对从NCBI下载的87 086条拟轮枝镰孢菌的EST序列信息进行分析,设计EST-SSR引物,检测其在拟轮枝镰孢菌及其近缘种中的扩增情况,并用筛选出的多态性引物对15株拟轮枝镰孢菌进行SSR遗传多样性分析。结果表明,在EST序列中,共查找到11 952个SSR位点,592种重复基元,SSR出现频率为1.09%,重复基元出现数量最多的为三核苷酸(54.00%),其中(CAA/TTG)n基元出现频率最高。设计的25对EST-SSR引物在拟轮枝镰孢菌种内的有效扩增率和多态率分别为80.00%与32.00%,对5种近缘镰孢菌种的通用率和多态率分别为40.00%和8.00%。遗传多样性分析结果表明,在相似系数为0.664水平下,供试菌株可划分为4个SSR类群,但类群的划分与菌株的地理来源无关;不同菌株间存在明显的遗传分化。表明基于拟轮枝镰孢菌EST序列开发的SSR引物可用于拟轮枝镰孢菌及其近缘种的遗传多样性分析。     

Morphological and molecular identification and PCR amplification to determine the toxigenic potential of Fusarium spp. isolated from maize ears in southern Poland

The average amount of precipitation in spring and summer 2010 and 2011 coupled with relatively high temperatures caused massive Fusarium spp. infection of maize and yield losses in southern Poland. In order to examine the cause of this disease outbreak, Fusarium spp. were isolated and fungal strains were identified based on morphological characters and species-specific PCR assays. A total of 200 maize samples were processed, resulting in the obtention of 71 strains, which belonged to five Fusarium species, F. poae being the predominant one (74.56%). Other isolates were identified as F. graminearum, F. oxysporum, F. verticillioides and F. proliferatum. PCR-based detection of mycotoxin-synthesis-pathway genes was also used to determine the potential of the analyzed strains to produce trichothecenes (DON and NIV) and fumonisins (FUM). Only 14 isolates revealed the potential to produce DON (11 strains) and FUM (3 strains). HPLC analyses of grain samples revealed the presence of DON only – other mycotoxins were not detected. Moreover, 57.1% of potentially mycotoxin-producing isolates indicated the toxicity in a biological test.     

Fusarium temperatum, a mycotoxin-producing pathogen of maize

In a recent study, a population of Fusarium strains isolated from maize in Belgium was described as a new species, F. temperatum, that is morphologically similar and phylogenetically closely related to F. subglutinans, a species in the American clade of the Gibberella fujikuroi species complex. In fields, the F. temperatum:F. subglutinans ratio was very high, suggesting that F. temperatum outcompetes its sister species F. subglutinans. This raised the question whether this novel species contributes to the final rot symptoms observed on maize plants at harvest, as well as to the potential mycotoxin contamination. Results of the pathogenicity tests by soil and toothpick inoculation demonstrate the ability of F. temperatum to cause seedling malformation and stalk rot under greenhouse conditions. Screening of 15 Fusarium mycotoxins showed the ability of F. temperatum to produce moniliformin, beauvericin, enniatins and fumonisin B₁. The results indicate that F. temperatum can produce mycotoxins and cause maize diseases and, therefore, poses a potential risk to maize production and to the safety of human food and animal feed.     

Interactions between Fusarium verticillioides and Fusarium graminearum in maize ears and consequences for fungal development and mycotoxin accumulation

Fungal interactions of Fusarium verticillioides and F. graminearum in maize ears and the impact on fungal development and toxin accumulation were investigated in a 2‐year field study at two locations in France. Maize ears were inoculated either with a spore mixture of F. graminearum and F. verticillioides or using a sequential inoculation procedure consisting of a first inoculation with F. graminearum followed by a second with F. verticillioides 1 week later. Toxin and fungal biomass were assessed on mature kernels, using HPLC and quantitative PCR. Correlation between the levels of DNA and toxin was high concerning F. graminearum DNA and deoxynivalenol (R² = 0·73) and moderate for F. verticillioides DNA and fumonisin (R² = 0·44). Fusarium graminearum DNA either decreased in mixed inoculations or was not influenced by subsequent inoculations with F. verticillioides, compared to single inoculations. In contrast, F. verticillioides DNA either significantly increased or was not affected in mixed and sequential inoculations. In two of the replicates, it can be assumed that natural contamination by F. verticillioides was favoured by previous contamination with F. graminearum. Overall, the results suggest that F. verticillioides has competitive advantages over the F. graminearum strains. Additionally, the data provide, for the first time, key evidence that previous contamination by F. graminearum in maize ears can facilitate subsequent infections by F. verticillioides.     

Natural occurrence of <Emphasis Type="Italic">Fusarium</Emphasis> species and fumonisin on maize grains in Ethiopia

Fusarium species causing maize kernel rot are major threats to maize production, due to reduction in yield as well as contamination of kernels by mycotoxins that poses a health risk to humans and animals. Two-hundred maize kernel samples, collected from 20 major maize growing areas in Ethiopia were analyzed for the identity, species composition and prevalence of Fusarium species and fumonisin contamination. On average, 38 % (range: 16 to 68 %) of maize kernels were found to be contaminated by different fungal species. Total of eleven Fusarium spp. were identified based on morphological characteristics and by sequencing the partial region of translation elongation factor 1-alpha (EF-1α) gene. Fusarium verticillioides was the dominant species associated with maize kernels (42 %), followed by F. graminearum species complex (22.5 %) and F. pseudoanthophilium (13.4 %). The species composition and prevalence of Fusarium species differed among the areas investigated. Fusarium species composition was as many as eight and as few as four in some growing area. The majority of the maize samples (77 %) were found positive for fumonisin, with concentrations ranging from 25 μg kg^?1 to 4500 μg kg^?1 (mean: 348 μg kg^?1 and median: 258 μg kg^?1). Slight variation in fumonisin concentration was also observed among areas. Overall results indicate widespread occurrence of several Fusarium species and contamination by fumonisin mycotoxins. These findings are useful for intervention measures to reduce the impact of the main fungal species and their associated mycotoxins, by creating awareness and implementation of good agricultural practices.     

Toxigenic Fusarium Species of Liseola Section in Pre-harvest Maize Ear Rot, and Associated Mycotoxins in Slovakia

The occurrence of Fusarium species of Liseola section and related toxins was investigated for two years (1996 and 1998) on maize ear rot samples collected in the most important areas for maize growing in Slovakia. The species most frequently isolated was F. verticillioides, followed by F. proliferatum in 1996 and F. subglutinans in 1998. Most of the strains belonged to mating populations A, D, and E of the teleomorph Gibberella fujikuroi. Fusarium graminearum was also frequently recovered in both the years of investigations. Toxin analysis of maize ears showed that most of the samples (21 out of 22) were contaminated with at least one toxin. In particular, the concentration of fumonisin B₁, and fumonisin ₂ was up to 26.9 and 5.1gg^-1, respectively in 1996, and up to 12.1 and 6.3gg^-1, respectively in 1998. Beauvericin was detected only in one sample in 1996. Seven samples in 1996 were contaminated by fusaproliferin up to 8.2gg^-1, but just traces of the toxin were found in one sample in 1998. All 29 strains of F. verticillioides, two of three strains of F. proliferatum and none of eight F. subglutinans strains isolated from samples produced fumonisin B₁ in culture on whole maize kernels (0.1–5646 and 940–1200ugg^-1, respectively). Two strains of F. subglutinans and two of F. proliferatum produced beauvericin (up to 65 and 70gg^-1, respectively). Ten strains of F. verticillioides produced beauvericin: 9 strains produced a low amount (up to 3gg^-1), while only one of them produced a high level of toxin (375gg^-1). Fusaproliferin was produced by two F. proliferatum strains (220 and 370gg^-1), by seven F. subglutinans (20–1335gg^-1) and by three F. verticillioides (10–35gg^-1). This is the first report on fusaproliferin production by F. verticillioides, although at low level.     

A Species-Specific PCR Assay Based on the Calmodulin Partial Gene for Identification of Fusarium Verticillioides, F. Proliferatum and F. Subglutinans

Fusarium proliferatum, F. subglutinans and F. verticillioides are the most important Fusarium species occurring on maize world-wide, capable of producing a wide range of mycotoxins which are a potential health hazard for animals and humans. The ribosomal internal transcribed spacer and a portion of the calmodulin gene were sequenced and analysed in order to design species-specific primers useful for diagnosis. The primer pairs were based on a partial calmodulin gene sequence. Three pairs of primers (PRO1/2, SUB1/2 and VER 1/2) produced PCR products of 585, 631 and 578bp for F. proliferatum, F. subglutinans and F. verticillioides, respectively. Primer specificity was confirmed by analyzing DNA of 150 strains of these species, mostly isolated from maize in Europe and USA. The sensitivity of primers was 12.5 pg when the pure total genomic DNA of each species was analyzed. The developed PCR assay should provide a powerful tool for the detection of toxigenic fungi in maize kernels.     

Pathological evaluation of host-specific AAL-toxins and fumonisin mycotoxins produced by Alternaria and Fusarium species

Host-specific AAL-toxins and mycotoxin fumonisins are structurally related and were originally isolated from the tomato pathotype of Alternaria alternata and from Fusarium verticillioides, respectively. Previous reports on the production of fumonisin derivatives by the tomato pathotype suggested a possible involvement in the pathogenicity of the pathogen. Here, we have evaluated the role of fumonisin in A. alternata–tomato interactions. The results indicate that highly pathogenic isolates of A. alternata tomato pathotype produce AAL-toxin as the sole toxin, strongly implicating it as a pathogenicity factor. The related compound, fumonisin, is also toxigenic and has infection-inducing activity on susceptible tomato plants.