Cladistic and phenetic analyses of relationships among Fusarium spp. in Dongtan wetland by morphology and isozymes

Variations of 12 morphological characters and 78 isozymic bands among 78 isolates of five Fusarium spp. from Dongtan wetland were described and analysed with cladistic parsimony and phenetic UPGMA methods. Hierarchical cluster analysis of 12 morphological characters grouped 78 strains into five defined species with a high overlap between isolates. Hierarchical cluster analysis of isozyme patterns showed a higher degree of relationship among five Fusarium spp., in which Fusarium nivale, Fusarium semitectum and Fusarium oxysporum clustered as one group, and F. semitectum was closer to F. nivale than to F. oxysporum; Fusarium graminearum and Fusarium moniliforme formed one group and showed clearly distinct from the first group. Groups of individual isolates indicated by a plot of principal component analysis were consistent with these findings. The comparison of two different data sets revealed that isozyme patterns showed higher variations between species and among individual isolates than morphological characters. Parsimony analysis of morphological characters yielded unresolved cladograms. Parsimony analysis of isozymes as presence/absence characters revealed the same five species in general as the results indicated by phenetic analysis, differing in the relative position of species in subclusters.     

Frequency and diversity of Fusarium spp. colonizing roots of Egyptian cottons

Abstract

Fusarium species are known to play a role in several diseases of cotton including the seedling disease complex, wilt, and boll rot. Therefore, a mycoflora study was conducted in 1998 in order to identify Fusarium species found in association with cotton roots. A total of 109 samples of cotton seedlings infected with post-emergence damping-off or rotted roots of adult plants were obtained from different cotton-growing areas in Egypt. Forty-six isolates were recovered and were identified as follows: F. oxysporum (28 isolates), F. moniliforme (9), F. solani (6), F. avenaceum (2), F. chlamydosporum (1). F. oxysporum, F. moniliforme and F. solani, the dominant species, accounted for 60.9%, 19.6% and 13% of the total isolates, respectively in 1998. F. oxysporum showed the highest isolation frequency in Beharia and Minufiya while F. moniliforme showed the most isolation frequency in Minufiya and Gharbiya. F. oxysporum was one of the major taxa of the Fusarium assemblage from Giza 70. F. oxysporum showed the most frequently isolated fungus in May while F. moniliforme and F. solani were the most frequently isolated fungi in August. Isolation frequency of Fusarium spp. during July and August was significantly greater than that of April or June. This implies that cotton roots are subjected more to colonization by Fusarium spp. as plants mature. Regarding pathogenicity, of the 46 isolates of Fusarium spp. tested under greenhouse conditions, 38 isolates (82.4%) were pathogenic to seedlings of Giza 89. This study indicates that F. oxysporum and F. moniliforme are important pathogens in the etiology of cotton damping-off in Egypt.     

Identification, pathogenicity and comparative virulence of Fusarium spp. associated with insect-damaged, diseased Centaurea spp. in Europe

Fusarium spp. isolated from insect-infested, diseased Centaurea diffusa and Centaurea maculosa in Europe were assessed for pathogenicity to North American plants of their respective original hosts: either C. diffusa or C. maculosa. Of the ten isolates of Fusarium spp. isolated from diseased Centaurea spp. in the Caucasus region of Russia and eastern Europe, all caused one or more disease symptoms or reductions in fresh weight of North American accessions of their original host species. In three instances, these reductions were statistically significant (p = 0.05). Symptoms included overall stunting, root lesions, and crown rot. Reductions in fresh weight of C. diffusa ranged from 17–78%, and C. maculosa exhibited reductions of 18–82%. The pathogenic cultures were identified as F. solani, F. tricinctum and F. oxysporum. Six of seven other cultures were identified as F. oxysporum, and one as F. tricinctum. It was concluded that further screening of a larger set of isolates of foreign Fusarium spp. under quarantine conditions stateside or in limited USDA-ARS overseas facilities is justified and promising.     

Assessment of Parasitic Activity of Fusarium Strains Obtained from a Heterodera schachtii-Suppressive Soil

This study assessed the potential impact of various Fusarium strains on the population development of sugarbeet cyst nematodes. Fungi were isolated from cysts or eggs of Heterodera schachtii Schmidt that were obtained from a field suppressive to that nematode. Twenty-six strains of Fusarium spp. were subjected to a phylogenic analysis of their rRNA-ITS nucleotide sequences. Seven genetically distinct Fusarium strains were evaluated for their ability to influence population development of H. schachtii and crop performance in greenhouse trials. Swiss chard (Beta vulgaris) seedlings were transplanted into fumigated field soil amended with a single fungal strain at 1,000 propagules/g soil. One week later, the soil was infested with 250 H. schachtii J2/100 cm³ soil. Parasitized eggs were present in all seven Fusarium treatments at 1,180 degree-days after fungal infestation. The percentage of parasitism ranged from 17 to 34%. Although the most efficacious F. oxysporum strain 471 produced as many parasitized eggs as occurred in the original suppressive soil, none of the Fusarium strains reduced the population density of H. schachtii compared to the conducive check. This supports prior results that Fusarium spp. were not the primary cause of the population suppression of sugarbeet cyst nematodes at this location.     

Silver nanoparticles synthesized by Nigrospora oryzae showed antifungal activity

In this investigation, an alternate green-route based on myco-synthesised silver nanoparticles (Ag NPs) was evaluated to control plant disease to reduce the usage of synthetic chemicals. Here, we described biologically synthesised Ag NPs using the corn grain contaminant, Nigrospora oryzae, and were well-characterised by UV–visible spectrophotometer, X-ray powder diffraction (XRD), transmission electron microscopy (TEM), Energy Dispersive Spectroscopy (EDS) and particle size analyzer. The pathogenic behaviour of the Fusarium spp. were checked on Giza 86 and Giza 90 cultivars under greenhouse conditions. F. moniliforme and F. oxysporum exhibited high pathogenecity against Giza 90 and Giza 86 cultivars respectively. The antifungal activity of biosynthesised Ag NPs was evaluated against eight species of Fusaria causing damping-off of cotton seedlings. In vitro treatments with different concentrations of Ag NPs were achieved on Czapek Dox agar and Potato dextrose agar plates. Fungal growth was drastically retarded from 25 to 200 ppm of Ag NPs interaction. The antifungal activity of Ag NPs against the Fusarium spp. was clearly proven.     

Identification of pathogenicity‐related genes in Fusarium oxysporum f. sp. cepae

Pathogenic isolates of Fusarium oxysporum, distinguished as formae speciales (f. spp.) on the basis of their host specificity, cause crown rots, root rots and vascular wilts on many important crops worldwide. Fusarium oxysporum f. sp. cepae (FOC) is particularly problematic to onion growers worldwide and is increasing in prevalence in the UK. We characterized 31 F. oxysporum isolates collected from UK onions using pathogenicity tests, sequencing of housekeeping genes and identification of effectors. In onion seedling and bulb tests, 21 isolates were pathogenic and 10 were non‐pathogenic. The molecular characterization of these isolates, and 21 additional isolates comprising other f. spp. and different Fusarium species, was carried out by sequencing three housekeeping genes. A concatenated tree separated the F. oxysporum isolates into six clades, but did not distinguish between pathogenic and non‐pathogenic isolates. Ten putative effectors were identified within FOC, including seven Secreted In Xylem (SIX) genes first reported in F. oxysporum f. sp. lycopersici. Two highly homologous proteins with signal peptides and RxLR motifs (CRX1/CRX2) and a gene with no previously characterized domains (C5) were also identified. The presence/absence of nine of these genes was strongly related to pathogenicity against onion and all were shown to be expressed in planta. Different SIX gene complements were identified in other f. spp., but none were identified in three other Fusarium species from onion. Although the FOC SIX genes had a high level of homology with other f. spp., there were clear differences in sequences which were unique to FOC, whereas CRX1 and C5 genes appear to be largely FOC specific.     

Production of Intraspecific Hybrids of Fusarium oxysporum f. sp. radicis-lycoperisici and Fusarium oxysporum f. sp. lycopersici by Protoplast Fusions

The fusion of protoplasts from the cycloheximide-resistant mutant FOL(C) of Fusarium oxysporum f. sp. lycopersici (FOL) and the mycostatin-resistant mutant FORL(M) of F. oxysporum f. sp. radicis-lycopersici (FORL), produced hybrids which expressed significant differences from the parents in their pathogenicity and growth and in the electrophoretic separation patterns of their proteins, enzymes and isoenzymes. The results suggest a transformed genetic basis for these altered expressions and the feasibility of using protoplast fusion technology for examining the biology of pathogenicity genes and for elucidating the disease and virulence potential for new races from within hybridisable taxa of Fusarium spp. Such information would be useful for the design and development of long-term control systems for Fusarium diseases, particularly in breeding programs for disease resistance in crops.     

Immunoelectrophorecal differences between phytopathogenic Fusarium species

Immunoelectrograms of certain tested Fusarium spp revealed that each has a characteristic pattern and showed the relatedness between F. oxysporum on the one hand and F. culmorum and F. semitectum on the other. The B₄ protein fraction is common to the former pair, while α_1a protein fraction links the second.     

Fot 1 Insertions in the Fusarium oxysporum f. sp. albedinis Genome Provide Diagnostic PCR Targets for Detection of the Date Palm Pathogen

Populations of Fusarium oxysporum f. sp. albedinis, the causal agent of Bayoud disease of date palm, are derivatives of a single clonal lineage and exhibit very similar Fot 1 hybridization patterns. In order to develop a sensitive diagnostic tool for F. oxysporum f. sp. albedinis detection, we isolated several DNA clones containing a copy of the transposable element Fot 1 from a genomic library of the date palm pathogen. Regions flanking the insertion sites were sequenced, and these sequences were used to design PCR primers that amplify the DNA regions at several Fot 1 insertion sites. When tested on a large sample of Fusarium isolates, including 286 F. oxysporum f. sp. albedinis isolates, 17 other special forms, nonpathogenic F. oxysporum isolates from palm grove soils, and 8 other Fusarium species, the primer pair TL3-FOA28 allowed amplification of a 400-bp fragment found only in F. oxysporum f. sp. albedinis. Sequence analysis showed that one of the Fot 1 copies was truncated, lacking 182 bp at its 3′ terminus. The primer pair BI03-FOA1 amplified a 204-bp fragment which overlapped the Fot 1 truncated copy and its 3′ site of insertion in the F. oxysporum f. sp. albedinis genome and identified 95% of the isolates. The primer pairs BIO3-FOA1 and TL3-FOA28 used in PCR assays thus provide a useful diagnostic tool for F. oxysporum f. sp. albedinis isolates.     

Dispersal of <Emphasis Type="Italic">Fusarium</Emphasis> spp. by rainwater and pathogenicity on four plant species

Research focused on the occurrence of Fusarium spp. in atmospheric dust or rainwater is not common. Preliminary studies with four sampling dates in 2007 revealed that several species of Fusarium may also be conveyed by rainwater. In order to determine the regular presence of Fusarium spp. in rainfall water, samples were systematically collected for a year (from October 2009 to October 2010) in three points on the Mediterranean coast of the province of Granada (Spain) 10-km distance between them. Throughout the year of sampling, a total of 179 rainwater samples were collected during every significant rainfall event. Eight different Fusarium species were isolated from the rainwater samples: F. oxysporum (32 %), F. proliferatum (26 %) and F. equiseti (20 %) coincide with previous studies, while F. dimerum (3 %), F. semitectum (4.7 %), F. solani (8 %), F. avenaceum (0.5 %) and F. chlamydosporum (3.7 %) were isolated for the first time from rainwater. Results were consistent with previous surveys conducted 100 km away from the sampling sites. Inoculation of 39 different isolates from five different Fusarium species showed pathogenicity on plants. Disease severity differed depending on the inoculated plant species, which means that rain water can be an effective vector to transport new pathogens into new cultivated areas. This work reveals some epidemiological aspects of Fusarium genus in natural environments. Some of the isolated Fusarium spp. are potential mycotoxin producers, such as zearalenone, fumonisin, moniliformin or nivalenol.     

Effects of Management Practices on Nematode and Fungi Populations and Okra Yield

Okra was grown in field plots of Tifton loamy sand naturally infested with the nematodes Meloidogyne incognita and Criconemoides ornalus and the pathogenic fungi Fusarium oxysporum, F. solani, F. roseum, and Pythium spp. Plots were treated with various soil pesticides and left exposed or covered with biodegradable paper film mulch under trickle irrigation. Soil was assayed for nematodes and fungi, and plant roots were examined for root-rot and insect damage. Fewer nematodes and fungi generally were recovered from soil treated with DD-MENCS (with and without film mulch) or methyl bromide-chloropicrin (2:1) (MBC) and film mulch than from nontreated soil. Funfigation with DD-MENCS or MBC suppressed populations of M. incognita, C. ornatus, F. oxysporum, F. solani, F. roseum, and Pythium spp. Ethoprop (alone or combined with other pesticides), sodium azide, and chloroneb were less effective than DD-MENCS and MBC. Plant growth anti yield were greatest when nematodes and pathogenic fungi were controlled. Yield was increased 3-fold by DD-MENCS + film mulch or MBC + film mulch in comparison with the average yield of okra produced in Georgia. The root-knot nematode-Fusarium wilt complex was most severe in nonfuntigated soil.     

Fungal microbiota from rain water and pathogenicity of <Emphasis Type="Italic">Fusarium</Emphasis> species isolated from atmospheric dust and rainfall dust

In order to determine the presence of Fusarium spp. in atmospheric dust and rainfall dust, samples were collected during September 2007, and July, August, and October 2008. The results reveal the prevalence of airborne Fusarium species coming from the atmosphere of the South East coast of Spain. Five different Fusarium species were isolated from the settling dust: Fusarium oxysporum, F. solani, F. equiseti, F. dimerum, and F. proliferatum. Moreover, rainwater samples were obtained during significant rainfall events in January and February 2009. Using the dilution-plate method, 12 fungal genera were identified from these rainwater samples. Specific analyses of the rainwater revealed the presence of three species of Fusarium: F. oxysporum, F. proliferatum and F. equiseti. A total of 57 isolates of Fusarium spp. obtained from both rainwater and atmospheric rainfall dust sampling were inoculated onto melon (Cucumis melo L.) cv. Piñonet and tomato (Lycopersicon esculentum Mill.) cv. San Pedro. These species were chosen because they are the main herbaceous crops in Almeria province. The results presented in this work indicate strongly that spores or propagules of Fusarium are able to cross the continental barrier carried by winds from the Sahara (Africa) to crop or coastal lands in Europe. Results show differences in the pathogenicity of the isolates tested. Both hosts showed root rot when inoculated with different species of Fusarium, although fresh weight measurements did not bring any information about the pathogenicity. The findings presented above are strong indications that long-distance transmission of Fusarium propagules may occur. Diseases caused by species of Fusarium are common in these areas. They were in the past, and are still today, a problem for greenhouses crops in Almería, and many species have been listed as pathogens on agricultural crops in this region. Saharan air masses dominate the Mediterranean regions. The evidence of long distance dispersal of Fusarium spp. by atmospheric dust and rainwater together with their proved pathogenicity must be taken into account in epidemiological studies.     

Occurrence and distribution of Fusarium species in maize fields in New Zealand

Fusarium populations were investigated in maize grains and their husks about six weeks before harvest in three maize fields in the Manawatu region of New Zealand. The role of litter and soil as reservoirs for these fungi was also examined. Two techniques were used to examine populations, dilution plating and direct plating. Using the dilution plating technique the highest overall populations were found in husks (mean 2.2 × 10⁵/g) and litter (mean 1.4 × 10⁵/g), while similar lower numbers of viable propagules were obtained from grain (mean 2.1 × 10³/g) and soil (2.8 × 10³/g). With this technique five Fusarium spp. were commonly isolated; F. graminearum (Gibberella zeae), F. culmorum, F. subglutinans, F. oxysporum and F. acuminatum, of which F. graminearum was the most abundant. With the direct plating technique 87% of grains were infected with Fusarium spp., with some grains being infected with more than one species. Segments from husks and litter, 70% and 43% respectively, were colonised by Fusarium spp. F. graminearum was the most frequent species isolated from maize grain and husk segments(48.3 and 37.7% colonisation respectively). Other species, particularly F. culmorum and F. acuminatum, were also found to be common contaminants. A total of 15 Fusarium spp. was recovered from all material examined by both techniques. Cultures with characteristics resembling those of F. moniliforme were rarely observed.This revised version was published online in October 2005 with corrections to the Cover Date.     

Hyphal Growth of Phagocytosed Fusarium oxysporum Causes Cell Lysis and Death of Murine Macrophages

Fusarium oxysporum is an important plant pathogen and an opportunistic pathogen of humans. Here we investigated phagocytosis of F. oxysporum by J774.1 murine cell line macrophages using live cell video microscopy. Macrophages avidly migrated towards F. oxysporum germlings and were rapidly engulfed after cell-cell contact was established. F. oxysporum germlings continued hyphal growth after engulfment by macrophages, leading to associated macrophage lysis and escape. Macrophage killing depended on the multiplicity of infection. After engulfment, F. oxysporum inhibited macrophages from completing mitosis, resulting in large daughter cells fused together by means of a F. oxysporum hypha. These results shed new light on the initial stages of Fusarium infection and the innate immune response of the mammalian host.     

Association of soybean root mycoflora with root modulation, root necrosis and plant fresh weight

Fungi were isolated from soybean plants in ten fields in south-west France. Correspondence analysis (COA) was used to find associations among taxa (species, genera, groups) and sampling units (i.e. one field at one sampling date). The first axis of the COA demonstrated a dominant sampling date effect; the second indicated an association among certain sampling units and specific taxa. Regression analysis of COA coordinate values indicated that fields with high relative isolation frequencies of Fusarium spp., F. oxysporum and ‘other species’ (mostly Chaetomium spp. and sterile fungi) were characterised by greater plant fresh weight and nodule number and lower ratings for root necrosis. Cylindrocarpon spp., Cephalosporium spp. and F. solanihad high relative isolation frequencies in fields with lower plant weight, lower nodule number and higher root necrosis ratings. The relative importance of fungal taxa was also related to soil type.     

The role of root-knot nematode on the wilt disease of Telfairiaoccidentalis in northern Nigeria

Cultivation of Telfairia occidentalis, an important vegetable grown in central and southern Nigeria, has gradually diffused to northern Nigeria where it has gained an economic importance. Its cultivation is, however, threatened by wilt disease occurring in farmers’ fields. A preliminary survey of farmers’ fields indicated that the disease was severe in fields with high root-knot population compared to the fields with less root-knot. Using Koch's postulate, the wilt causative organism was identified as Fusarium spp. The objective of this study was to determine therole of Meloidogyne incognita and its interaction with Fusarium oxysporum onwilt development in T. occidentalis. Four-week-old seedlings, raised in heat sterilised soil, were inoculated with Fusarium spp. and M. incognita under screenhouse condition. Seedlings were either inoculated with M. incognita and/or F.oxysporum as sole infection and as combined infection in a complex. Combined infection with both pathogens produced wilt symptom on the plant and gave significantly lower vegetative yield (p = 0.05) than sole inoculation with either M.incognita or F. oxysporum, except where the seedlings were mechanically inoculated with F. oxysporum. Results from the screenhouse studies were consistent with the field observations.     

Development of a Loop‐Mediated Isothermal Amplification Assay to Detect Fusarium oxysporum

We report the development of a loop‐mediated isothermal amplification (LAMP) assay targeting the CYP51C element for visual detection of F. oxysporum which caused Fusarium wilt in soybean. The CYP51C‐LAMP assay efficiently amplified the target gene in 60 min at 62°C. And specificity was evaluated against F. oxysporum, Fusarium spp. and other fungal species. The detection limit of the CYP51C‐specific LAMP assay for F. oxysporum was four conidia per gram soil. The assay also detected F. oxysporum from inoculated soybean tissues and residues. These results suggest that this CYP51C‐LAMP assay can be used to detect residues on plants in the field.     

Molecular characterization of pathogenic Fusarium species in cucurbit plants from Kermanshah province, Iran

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.     

Identification and evaluation of a potential biocontrol agent,Bacillus subtilis,against Fusarium sp. in apple seedlings

To find a potential biocontrol agent against Fusarium sp. in apple seedlings, an endophytic bacterium strain was isolated from apple tree tissues. The inhibitive efficiency of the isolated strain against the hyphal growth of Fusarium sp. and Rhizoctonia solani was tested. Strain Y-1 showed significant inhibitory effects against Fusarium oxysporum, F. moniliforme, F. proliferatum, F. solani and R. solani. Its antifungal activity against F. oxysporum was the highest, reaching up to 64.90 %. In vivo tests indicated that strain Y-1 effectively protects apple from F. oxysporum infections. The control effect reached 92.26 % when bacterial inoculation was performed 3 days prior to pathogen inoculation. Strain Y-1 could colonize the rhizosphere and tissues within 30 days. It was also able to induce systemic resistance in apple seedlings as shown by the activities of SOD and POD. Strain Y-1 significantly increased the root length, root wet and dry weights, and plant height of the apple seedlings compared with the control group. The homology analysis of the 16S rRNA sequence, together with morphological, physical, and biochemical analyses, revealed that strain Y-1 is Bacillus subtilis.