Volatile Substances Produced by Fusarium oxysporum from Coffee Rhizosphere and Other Microbes affect Meloidogyne incognita and Arthrobotrys conoides

Microorganisms produce volatile organic compounds (VOCs) which mediate interactions with other organisms and may be the basis for the development of new methods to control plant-parasitic nematodes that damage coffee plants. In the present work, 35 fungal isolates were isolated from coffee plant rhizosphere, Meloidogyne exigua eggs and egg masses. Most of the fungal isolates belonged to the genus Fusarium and presented in vitro antagonism classified as mutual exclusion and parasitism against the nematode-predator fungus Arthrobotrys conoides (isolated from coffee roots). These results and the stronger activity of VOCs against this fungus by 12 endophytic bacteria may account for the failure of A. conoides to reduce plant-parasitic nematodes in coffee fields. VOCs from 13 fungal isolates caused more than 40% immobility to Meloidogyne incognita second stage juveniles (J₂), and those of three isolates (two Fusarium oxysporum isolates and an F. solani isolate) also led to 88-96% J₂ mortality. M. incognita J₂ infectivity decreased as a function of increased exposure time to F. oxysporum isolate 21 VOCs. Gas chromatography-mass spectrometry (GC-MS) analysis lead to the detection of 38 VOCs produced by F. oxysporum is. 21 culture. Only five were present in amounts above 1% of the total: dioctyl disulfide (it may also be 2-propyldecan-1-ol or 1-(2-hydroxyethoxy) tridecane); caryophyllene; 4-methyl-2,6-di-tert-butylphenol; and acoradiene. One of them was not identified. Volatiles toxic to nematodes make a difference among interacting microorganisms in coffee rhizosphere defining an additional attribute of a biocontrol agent against plant-parasitic nematodes.     

The role of onion-associated fungi in bulb mite infestation and damage to onion seedlings

In Israel Rhizoglyphus robini is considered to be a pest in its own right, even though the mite is usually found in association with fungal pathogens. Plant protection recommendations are therefore to treat germinating onions seedlings, clearly a crucial phase in crop production, when mites are discovered. The aim of this study was to determine the role of fungi in bulb mite infestation and damage to germinating onion seedlings. Accordingly we (1) evaluated the effect of the mite on onion seedling germination and survival without fungi, (2) compared the attraction of the mite to species and isolates of various fungi, (3) assessed the effect of a relatively non-pathogenic isolate of Fusarium oxysporum on mite fecundity, and (4) determined the effects of the mite and of F. oxysporum separately and together, on onion seedling germination and sprout development. A significant reduction of seedling survival was recorded only in the 1,000 mites/pot treatment, after 4 weeks. Mites were attracted to 6 out of 7 collected fungi isolates. Mite fecundity on onion sprouts infested with F. oxysporum was higher than on non-infested sprouts. Survival of seedlings was affected by mites, fungi, and their combination. Sprouts on Petri dishes after 5 days were significantly longer in the control and mite treatments than both fungi treatments. During the 5-day experiment more mites were always found on the fungi-infected sprouts than on the non-infected sprouts. Future research using suppressive soils to suppress soil pathogens and subsequent mite damage is proposed.     

Production of volatile organic compounds by an antagonistic strain Paenibacillus polymyxa WR-2 in the presence of root exudates and organic fertilizer and their antifungal activity against Fusarium oxysporum f. sp. niveum

The volatile organic compounds (VOCs) produced by antagonistic microbes have great antifungal potential against soil-borne fungal pathogens. The VOCs produced by Paenibacillus polymyxa strain WR-2 in the presence of root exudates and organic fertilizer were identified and their effects on the growth and spore germination of Fusarium oxysporum f. sp. niveum were evaluated. The VOCs produced by WR-2 inhibited the growth of F. oxysporum by 38%, 36% and 40% in agar medium, sterilized soil and natural soil, respectively. This inhibitory effect was increased to 60%, 58% and 64% with the addition of organic fertilizer in agar medium, sterilized soil and natural soil, respectively. The addition of root exudates did not affect the production of antifungal VOCs by WR-2. The VOCs produced by WR-2 completely inhibited the germination of F. oxysporum spores. Out of 42 identified VOCs, seven VOCs; benzothiazole, benzaldehyde, undecanal, dodecanal, hexadecanal, 2-tridecanone and phenol were found to inhibit the growth of F. oxysporum. The results of these experiments suggest another significance of using organic fertilizer as a carrier material with the biocontrol agents to control soil-borne fungal pathogens.     

Naphthoquinones produced by Fusarium oxysporum isolated from citrus

Six naphthoquinone pigments are described which were produced by Fusarium oxysporum isolates obtained from roots of diseased citrus trees. These were 8-O-methylbostrycoidin, 9-O-methylfusarubin, 9-O-methylanhydro-fusarubin, 5-O-methyljavanicin, 5-O-methylsolaniol and 1,4-naphthalenedione-3,8-dihydroxy-5,7-dimethoxy-2-(2-oxopropyl). These naphthoquinones had not been previously identified from F. oxysporum isolates.     

Induction of defence-related enzymes in onion by combined application of fungal and bacterial biocontrol agents against Fusarium oxysporum f. sp. cepae

Basal rot disease of onion is a major problem in different onion growing regions of Tamil Nadu, India. Fungal and bacterial cultures were isolated and tested their efficiency against Fusarium oxysporum f. sp. cepae under in vitro conditions. Effective bacterial and fungal antagonists were tested alone and in combinations for the control of F. oxysporum f. sp. cepae in glasshouse experiments. Defence-related enzymes such as peroxidase, polyphenol oxidase and phenylalanine ammonia-lyase were induced and accumulated in onion treated with fungal and bacterial antagonists. Defence-related enzymes were significantly higher in onion pretreated with consortial formulation of Pf12 + Pf27 + TH3 at 5 days after the challenge inoculation with F. oxysporum f. sp. cepae and gave resistance to onion against basal rot disease.     

A multiplex real-time PCR method using hybridization probes for the detection and the quantification of Fusarium proliferatum, F. subglutinans, F. temperatum, and F. verticillioides

Maize contamination with Fusarium species is one of the major sources of mycotoxins in food and feed derivates. In the present study, a LightCycler^® real-time PCR method using hybridization probes was developed for the specific identification, detection, and quantification of Fusarium proliferatum, Fusarium subglutinans, Fusarium temperatum, and Fusarium verticillioides, four mycotoxin-producing pathogens of maize. Primers and hybridization probes were designed to target the translation elongation factor 1α (EF-1α) gene of F. subglutinans and F. temperatum or the calmodulin (Cal) gene of F. proliferatum and F. verticillioides. The specificity of the real-time PCR assays was confirmed for the four Fusarium species, giving no amplification with DNA from other fungal species commonly recovered from maize. The assays were found to be sensitive, detecting down to 5 pg and 50 pg of Fusarium DNA in simplex and multiplex conditions respectively, and were able to quantify pg-amounts of Fusarium DNA in artificially Fusarium-contaminated maize samples. The real-time PCR method developed provides a useful tool for routine identification, detection, and quantification of toxigenic Fusarium species in maize.     

Glucosinolate profiling of Brassica rapa cultivars after infection by Leptosphaeria maculans and Fusarium oxysporum

The glucosinolate contents of two different cultivars of Brassica rapa (Herfstraap and Oleifera) infected with Leptosphaeria maculans and Fusarium oxysporum were determined. Infection triggered the accumulation of aliphatic glucosinolates (gluconapin, progoitrin, glucobrassicanapin and gluconapoleiferin) and indole glucosinolate (4-hydroxy-glucobrassicin) in Herfstraap and of two indole glucosinolates (glucobrassicin and 4-hydroxy-glucobrassicin) in Oleifera. While total and aliphatic glucosinolates decreased significantly in Oleifera, a large increase was observed in Herfstraap after fungal infection. The indole glucosinolate glucobrassicin accumulated in Oleifera at a higher rate than Herfstraap especially after infection with F. oxysporum. Apparently the interaction between fungus and B. rapa is cultivar and fungal species specific.     

The ITS region as a taxonomic discriminator between Fusarium verticillioides and Fusarium proliferatum

The maize pathogens Fusarium verticillioides (Fv) and Fusarium proliferatum (Fp) are morphologically very similar to one another, so Fp isolates have been often mistaken as Fusarium moniliforme (the former name of Fv). The only presently accepted morphological discriminator between these species is the presence/absence of polyphialides. Here, a collection of 100 Fusarium strains, isolated from infected maize kernels on plants grown in north-western Italy, were assigned as Fv or Fp on the basis of the presence/absence of polyphialides. This classification was tested on a subset of isolates by sexual crosses, ITS and calmodulin sequencing and AFLP profiling. An ITS-RFLP assay was extended to the full collection and to a number of Fv and Fp isolates of different geographical origin and hosts. The ITS region is proposed as taxonomically informative for distinguishing between Fp and Fv.     

Effect of a novel antifungal peptide P852 on cell morphology and membrane permeability of Fusarium oxysporum

P852, a novel cyclic peptide isolated from Bacillus amyloliquefaciens L-H15, showed potent antifungal activity against several major plant fungal pathogens including Fusarium oxysporum. To elucidate the antifungal mechanism, the impact of P852 on the cell morphology and membrane permeabilization of F. oxysporum was studied. By applying electron microscopy and fluorescent techniques, we showed that P852 treatment caused the morphological change of F. oxysporum cells and disrupted its cell structure, including formation of blebs, broken hyphae, deformation of membrane, intracellular organization disruption, pore formation, and cell lysis. Our findings provide insights into the mode of action of P852, which laying a foundation to develop P852 as a novel antifungal agent to control plant fungal pathogens.     

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.     

Bulb and Root Rot in Lily (Lilium longiflorum) and Onion (Allium cepa) in Israel

In the past 10 years, there has been a substantial increase in reports, from growers and extension personnel, on bulb and root rots in lily (Lilium longiflorum) in Israel. Rot in these plants, when grown as cut flowers, caused serious economic damage expressed in reduction in yield and quality. In lily, the fungal pathogens involved in the rot were characterized as binucleate Rhizoctonia AG‐A, Rhizoctonia solani, Pythium oligandrum, Fusarium proliferatum (white and purple isolates) and F. oxysporum, using morphological and molecular criteria. These fungi were the prevalent pathogens in diseased plants collected from commercial greenhouses. Pathogenicity trials were conducted on lily bulbs and onion seedlings under controlled conditions in a greenhouse to complete Koch's postulates. Disease symptoms on lily were most severe in treatments inoculated with binucleate Rhizoctonia AG‐A, P. oligandrum and F. proliferatum. Plant height was lower in the above treatments compared with the control plants. The least aggressive fungus was R. solani. In artificial inoculations of onion, seedling survival was significantly affected by all fungi. The most pathogenic fungus was F. proliferatum w and the least were isolates of F. oxysporum (II and III). All fungi were successfully re‐isolated from the inoculated plants.     

Identification of Fusarium species causing basal rot of onion in East Azarbaijan province,Iran and evaluation of their virulence on onion bulbs and seedlings

One of the economically important diseases of onion is the basal rot caused by various Fusarium species. Identification of the pathogenic species prevalent in a region is indispensable for designing management strategies, especially to develop resistant cultivars. Eighty Fusarium isolates are obtained from red onion bulbs on infected fields of East Azarbaijan province. Inoculating the onion bulbs with 38 selective isolates indicated that 17 isolates were pathogenic on onion. According to the morphological and molecular characteristics, these isolates were identified as F. oxysporum, F. solani, F. proliferatum and F. redolens. This is the first report of F. redolens on onion in Iran. On the other hand, the virulence of each pathogenic isolate was evaluated on onion bulbs and seedlings. F. oxysporum which causes severe rot and damping-off was considered as a highly virulent species in both conditions. While, F. proliferatum was considered as the most destructive on onion bulbs. Rot ability of F. solani was not considerable, and only the 4S isolate caused pre- and post-emergence damping-off more than 50%. Finally, F. redolens with less pathogenicity on onion bulbs was identified as the most virulent isolate on onion seedlings, which was explanatory of its importance on farm.     

There it is! Fusarium pseudograminearum did not lose the fusaristatin gene cluster after all

Fusarium pseudograminearum is a significant pathogen of cereals in arid regions worldwide and has the ability to produce numerous bioactive secondary metabolites. The genome sequences of seven F. pseudograminearum strains have been published and in one of these strains, C5834, we identified an intact gene cluster responsible for biosynthesis of the cyclic lipopeptide fusaristatin A. The high level of sequence identity of the fusaristatin cluster remnant in strains that do not produce fusaristatin suggests that the absence of the cluster evolved once, and subsequently the resulting locus with the cluster fragments became widely dispersed among strains of F. pseudograminearum in Australia. We examined a selection of 99 Australian F. pseudograminearum isolates to determine how widespread the ability to produce fusaristatin A is in F. pseudograminearum. We identified 15 fusaristatin producing strains, all originating from Western Australia. Phylogenetic analyses could not support a division of F. pseudograminearum into fusaristatin producing and nonproducing populations, which could indicate the loss has occurred relatively recent.     

Genealogical concordance phylogenetic species recognition in the Fusarium oxysporum species complex

Fusarium oxysporum is an important plant and human pathogenic ascomycetous group, with near ubiquity in agricultural and non-cultivated ecosystems. Phylogenetic evidence suggests that F. oxysporum is a complex of multiple morphologically cryptic species. Species boundaries and limits of genetic exchange within this complex are poorly defined, largely due to the absence of a sexual state and the paucity of morphological characters. This study determined species boundaries within the F. oxysporum species complex using Genealogical Concordance Phylogenetic Species Recognition (GCPSR) with eight protein coding loci. GCPSR criteria were used firstly to identify independent evolutionary lineages (IEL), which were subsequently collapsed into phylogenetic species. Seventeen IELs were initially identified resulting in the recognition of two phylogenetic species. Further evidence supporting this delineation is discussed.     

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.     

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.     

Antagonistic activity and hyphal interactions of Trichoderma spp. against Fusarium proliferatum and F. oxysporum in vitro

Trichoderma is a well-known antagonist against soilborne plant pathogens. However, the species and even various isolates have different biocontrol potential. To evaluate the antagonistic activities of Trichoderma harzianum, T. harzianum strain T100 (T100), T. viride and T. haematum against Fusarium oxysporum and F. proliferatum, we used dual culture and productions of volatile and non-volatile metabolites in three different phases in vitro. An analysis of the data in dual culture tests represented T. viride, T. haematum and T100 as effective antagonists of Fusarium while T100 was the only fungus being able to lyse the confronting mycelia. Similar results were obtained in the volatile metabolites tests also. In contrast with the two previous tests, the non-volatile metabolites produced by T. harzianum inhibited Fusarium mycelial growth the most, and T100 acted moderately. It was also clearly showed that the antagonistic effect of Trichoderma spp. was more on F. proliferatum than on F. oxysporum. Finally, because Trichoderma spp. was most effective in the second phase, we recommend to use T100 against F. proliferatum at the initial stages of infection as its mycoparasitism on F. oxysporum was observed microscopically through forming apressoria structures without any coiling around the pathogen.     

Genetic and virulence variation in Fusarium oxysporum f. sp. cepae causing root and basal rot of common onion in Iran

Root and basal rot of common onion (Allium cepae L.) caused by Fusarium oxysporum f. sp. cepae is one of the most important diseases causing tremendous losses in onion‐growing areas worldwide. In this study, random amplified polymorphic DNA (RAPD), intersimple sequence repeats (ISSR) and virulence studies were conducted to analyse 26 F. oxysporum f. sp. cepae isolates obtained from the main onion‐growing regions of Iran, including Fars, Azerbaijan and Isfahan states. Cluster analysis using UPGMA method for both RAPD and ISSR markers revealed no clear grouping of the isolates obtained from different geographical regions, and the isolates were observed to derive probably from the same clonal lineage. Pathogenicity test indicated that all F. oxysporum f. sp. cepae isolates were pathogenic on onion; however, virulence variability was observed among the isolates. The grouping based on virulence variability was not correlated with the results of RAPD and ISSR analyses.     

Quick and accurate detection of <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f. sp. <Emphasis Type="Italic">carthami</Emphasis> in host tissue and soil using conventional and real-time PCR assay

Safflower wilt, caused by Fusarium oxysporum f. sp. carthami (Foc) is a major limiting factor for safflower (Carthamus tinctorius) production worldwide. In India alone, about 40–80% disease incidence has been reported. A rapid, efficient, specific, and sensitive diagnostic technique for Foc is therefore crucial to manage Fusarium wilt of safflower. Twenty-five isolates of F. oxysporum formae speciales infecting other crops, 17 isolates of Fusarium spp. and seven isolates of other fungal pathogens of safflower along with 75 Foc isolates were used for identification of band specific to Foc using inter-simple sequence repeat (ISSR) analysis. Out of 70 ISSR primers, the one that specifically amplified a 490 bp fragment from all the Foc isolates was selected. Sequence of the amplified fragment was utilized to design sequence characterized amplified region (SCAR) primers (FocScF/FocScR). The primer pair unambiguously and exclusively amplified a DNA fragment of approximately 213 bp in all the 75 Foc isolates. The primer set was able to detect as low as 10 pg of Foc genomic DNA using conventional PCR, while the SCAR primers when coupled with real-time qPCR demonstrated detection limits of 1 pg for Foc genomic DNA and 1000 conidia/g for soil. The assay enabled reliable diagnosis of Foc DNA in contaminated safflower fields and expedited Foc detection at 72 h post inoculation in asymptomatic seedlings. This method facilitates quick and precise detection of Foc in plant and soil samples and can be exploited for timely surveillance and sustainable management of the disease.