Molecular Characterization of Fusarium oxysporum f. melongenae by ISSR and RAPD Markers on Eggplant

Fusarium oxysporum f. melongenae is a major soil-borne pathogen of eggplant (Solanum melongena). ISSR and RAPD markers were used to characterize Fusarium oxysporum f. melongenae isolates collected from eggplant fields in southern Turkey. Those isolates were not pathogenic to tomato. Pathogens were identified by their morphology, and their identity was confirmed by PCR amplification using the specific primer PF02-3. The isolates were classified into groups on the basis of ISSR and RAPD fingerprints, which showed a level of genetic specificity and diversity not previously identified in Fusarium oxysporum f. melongenae, suggesting that genetic differences are related to the pathogen in the Mediterranean region. The primers selected to characterize Fusarium oxysporum f. melongenae may be used to determine genetic differences and pathogen virulence. This study is the first to characterize eggplant F. oxysporum species using ISSR and RAPD.     

Use of RAPD and ISSR Markers in Detection of Genetic Variation and Population Structure among Fusarium oxysporum f. sp. ciceris Isolates on Chickpea in Turkey

Genetic variation among the isolates of Fusarium oxysporum f. sp. ciceris, the causal agent of chickpea wilt worldwide, was analysed using pathogenicity tests and molecular markers – random amplified polymorphic DNA (RAPD) and inter‐simple sequence repeat (ISSR) polymorphism. Hundred and eight isolates were obtained from diseased chickpea plants in 13 different provinces of Turkey, out of which 74 isolates were assessed using 30 arbitrary decamer primers and 20 ISSR primers. Unweighted pair‐grouped method by arithmetic average cluster analysis of RAPD, ISSR and RAPD + ISSR datasets provided a substantially similar discrimination among Turkish isolates and divided into three major groups. Group 1, 2 and 3 consisted of 41, 18 and 15 isolates, respectively. These methods revealed a considerable genetic variation among Turkish isolates, but no correlation with regard to the clustering of isolates from different geographic regions. Analysis of molecular variance confirmed that most genetic variability resulted from the differences among isolates within regions. Our results also indicated that the low‐genetic differentiation (F_ST) and high gene flow (Nm) among populations had a significant effect on the emergence and evolutionary development of F. oxysporum f. sp. ciceris. This is the first report on genetic diversity and population structure of F. oxysporum isolates on chickpea in Turkey.     

Kinetic of the production of polygalacturonase and pectin lyase by two closely relatedFormae speciales ofFusarium oxysporum

The kinetic of thein vitro production of polygalacturonase and pectin lyase of two closely related fungi,Fusarium oxysporum f.sp.lycopersici andF. oxysporum f.sp.radicis-lycopersici, was examined under various culture conditions such as the source of carbon, the pH, and the age of cultures. Over a 5-day period, the production of these enzymes by various isolates of the sameforma specialis (f. sp.) ofF. oxysporum was not significantly different (P ≥ 0.05). However, the amount of the enzymes produced differed markedly between both f. sp. The different carbon sources added to the culture media, such as citrus pectin, apple pectin, tomato cell wall fragments, andd-galacturonic acid, proved to be higher pectinase inducible substrates than sucrose and glucose. For both fungi, polygalacturonase and pectin lyase activities were optimal at pH 5.0 and 8.0, respectively. Furthermore, pectin lyase production had a partial Ca²⁺ requirement in contrast to polygalacturonase production which was limited by Ca²⁺. In most experiments performed, the production of polygalacturonase appeared superior withF. oxysporum f.sp.radicislycopersici than withF. oxysporum f.sp.lycopersici. On the other hand, pectin lyase production ofF. oxysporum f.sp.lycopersici was approximately 10-fold greater than that byF. oxysporum f.sp.radicis-lycopersici in media supplemented withd-galacturonic acid.     

The intercropping partner affects arbuscular mycorrhizal fungi and Fusarium oxysporum f. sp. lycopersici interactions in tomato

Arbuscular mycorrhizal fungi (AMF) and their bioprotective aspects are of great interest in the context of sustainable agriculture. Combining the benefits of AMF with the utilisation of plant species diversity shows great promise for the management of plant diseases in environmentally compatible agriculture. In the present study, AMF were tested against Fusarium oxysporum f. sp. lycopersici with tomato intercropped with either leek, cucumber, basil, fennel or tomato itself. Arbuscular mycorrhizal (AM) root colonisation of tomato was clearly affected by its intercropping partners. Tomato intercropped with leek showed even a 20 % higher AM colonisation rate than tomato intercropped with tomato. Positive effects of AMF expressed as an increase of tomato biomass compared to the untreated control treatment could be observed in root as well as in shoot weights. A compensation of negative effects of F. oxysporum f. sp. lycopersici on tomato biomass by AMF was observed in the tomato/leek combination. The intercropping partners leek, cucumber, basil and tomato had no effect on F. oxysporum f. sp. lycopersici disease incidence or disease severity indicating no allelopathic suppression; however, tomato co-cultivated with tomato clearly showed a negative effect on one plant/pot with regard to biomass and disease severity of F. oxysporum f. sp. lycopersici. Nonetheless, bioprotective effects of AMF resulting in the decrease of F. oxysporum f. sp. lycopersici disease severity were evident in treatments with AMF and F. oxysporum f. sp. lycopersici co-inoculation. However, these bioprotective effects depended on the intercropping partner since these effects were only observed in the tomato/leek and tomato/basil combination and for the better developed plant of tomato/tomato. In conclusion, the effects of the intercropping partner on AMF colonisation of tomato are of great interest for crop plant communities and for the influences on each other. The outcome of the bioprotective effects of AMF resulting in the decrease on F. oxysporum f. sp. lycopersici disease severity and/or compensation of plant biomass does not depend on the degree of AM colonisation but more on the intercropping partner.     

Genetic Diversity of Fusarium oxysporum Populations Isolated from Tomato Plants in Tunisia

Fusarium crown and root rot of tomato (Lycopersicon esculentum) caused by Fusarium oxysporum f. sp. radicis‐lycopersici is a new devastative disease of tomato greenhouse crops in Tunisia. Nothing is known neither about the population of this pathogen in this region, nor about the population of F. oxysporum f. sp. lycopersici the causal agent of Fusarium wilt of tomato. In order to examine the genetic relatedness among the F. oxysporum isolates by intergenic spacer restriction fragment length polymorphism (IGS‐RFLP) analysis and to elucidate the origin of the formae specialesradicis‐lycopersici in Tunisia by looking for genetic similarity of Tunisians isolates with isolates from a foreign source, the genetic diversity among F. oxysporum f. sp. radicis‐lycopersici and F. oxysporum f. sp. lycopersici populations was investigated. A total of 62 isolates of F. oxysporum, obtained from symptomless tomato plants, were characterized using IGS typing and pathogenicity tests on tomato plants. All Fusarium isolates were highly pathogenic on tomato. Fusarium oxysporum f. sp. radicis‐lycopersici isolates were separated into five IGS types. From the 53 F. oxysporum f. sp. radicis‐lycopersici isolates, 34 isolates have the same IGS types (IGS type 25), and the remaining 19 isolates were distributed into four IGS types. However, the only nine isolates of F. oxysporum f. sp. lycopersici have six different IGS types. This difference of diversity between the two formae speciales suggests that F. oxysporum f. sp. radicis‐lycopersici isolates have a foreign origin and may have been accidentally introduced into Tunisia.     

Genetic Diversity of Fusarium oxysporum Strains from Common Bean Fields in Spain

Fusarium wilt is an endemic disease in El Barco de Avila (Castilla y León, west-central Spain), where high-quality common bean cultivars have been cultured for the last century. We used intergenic spacer (IGS) region polymorphism of ribosomal DNA, electrophoretic karyotype patterns, and vegetative compatibility and pathogenicity analyses to assess the genetic diversity within Fusarium oxysporum isolates recovered from common bean plants growing in fields around El Barco de Avila. Ninety-six vegetative compatibility groups (VCGs) were found among 128 isolates analyzed; most of these VCGs contained only a single isolate. The strains belonging to pathogenic VCGs and the most abundant nonpathogenic VCGs were further examined for polymorphisms in the IGS region and electrophoretic karyotype patterns. Isolates belonging to the same VCG exhibited the same IGS haplotype and very similar electrophoretic karyotype patterns. These findings are consistent with the hypothesis that VCGs represent clonal lineages that rarely, if ever, reproduce sexually. The F. oxysporum f. sp. phaseoli strains recovered had the same IGS haplotype and similar electrophoretic karyotype patterns, different from those found for F. oxysporum f. sp. phaseoli from the Americas, and were assigned to three new VCGs (VCGs 0166, 0167, and 0168). Based on our results, we do not consider the strains belonging to F. oxysporum f. sp. phaseoli to be a monophyletic group within F. oxysporum, as there is no correlation between pathogenicity and VCG, IGS restriction fragment length polymorphism, or electrophoretic karyotype.     

Comparison of two fatty acid analysis protocols to characterize and differentiate Fusarium oxysporum f. sp. lycopersici and F. oxysporum f. sp. radicis-lycopersici

The utility of fatty acid methyl ester (FAME) profiles for characterization and differentiation of isolates of Fusarium oxysporum f. sp. lycopersici and F. oxysporum f. sp. radicis-lycopersici was investigated. Two fatty acid analysis protocols of the normal (MIDI) and a modified MIDI method were used for their utility. Only the modified MIDI method allowed a clear differentiation between F. oxysporum f. sp. lycopersici and F. oxysporum f. sp. radicislycopersici. FAME profiles using the modified MIDI method gave the most consistent and reproducible analyzed fatty acid data. Evaluation of the FAME profiles based on cluster analysis and principal-component analysis revealed that FAME profiles from tested isolates were correlated with the same vegetative compatibility groups (VCGs) compared to the same races in F. oxysporum f. sp. lycopersici. Results indicated that FAME profiles could be an additional tool useful for characterizing isolates and forma species of F. oxysporum obtained from tomato.     

Host perception of jasmonates promotes infection by Fusarium oxysporum formae speciales that produce isoleucine‐ and leucine‐conjugated jasmonates

Three pathogenic forms, or formae speciales (f. spp.), of Fusarium oxysporum infect the roots of Arabidopsis thaliana below ground, instigating symptoms of wilt disease in leaves above ground. In previous reports, Arabidopsis mutants that are deficient in the biosynthesis of abscisic acid or salicylic acid or insensitive to ethylene or jasmonates exhibited either more or less wilt disease, than the wild‐type, implicating the involvement of hormones in the normal host response to F. oxysporum. Our analysis of hormone‐related mutants finds no evidence that endogenous hormones contribute to infection in roots. Mutants that are deficient in abscisic acid and insensitive to ethylene show no less infection than the wild‐type, although they exhibit less disease. Whether a mutant that is insensitive to jasmonates affects infection depends on which forma specialis (f. sp.) is infecting the roots. Insensitivity to jasmonates suppresses infection by F. oxysporum f. sp. conglutinans and F. oxysporum f. sp. matthioli, which produce isoleucine‐ and leucine‐conjugated jasmonate (JA‐Ile/Leu), respectively, in culture filtrates, whereas insensitivity to jasmonates has no effect on infection by F. oxysporum f. sp. raphani, which produces no detectable JA‐Ile/Leu. Furthermore, insensitivity to jasmonates has no effect on wilt disease of tomato, and the tomato pathogen F. oxysporum f. sp. lycopersici produces no detectable jasmonates. Thus, some, but not all, F. oxysporum pathogens appear to utilize jasmonates as effectors, promoting infection in roots and/or the development of symptoms in shoots. Only when the infection of roots is promoted by jasmonates is wilt disease enhanced in a mutant deficient in salicylic acid biosynthesis.     

Fusarium oxysporum f. sp. fatshederae, a new forma specialis causing wilt of x Fatskedera lizei

A Fusarium oxysporum sensu Snyder & Hansen was pathogenic in the vascular tissue and caused a severe wilt and dieback of ×Fatshedera lizei, Fatsia japonica and Hedera helix. The fungus appeared nonpathogenic on plants of 29 species in numerous genera. Benomyl soil drenches were effective in preventing infection. A new forma specialis, Fusarium oxysporum f. sp. fatshederae, f. sp. nov. is proposed for this pathogen.     

The Nuclear Protein Sge1 of Fusarium oxysporum Is Required for Parasitic Growth

Dimorphism or morphogenic conversion is exploited by several pathogenic fungi and is required for tissue invasion and/or survival in the host. We have identified a homolog of a master regulator of this morphological switch in the plant pathogenic fungus Fusarium oxysporum f. sp. lycopersici. This non-dimorphic fungus causes vascular wilt disease in tomato by penetrating the plant roots and colonizing the vascular tissue. Gene knock-out and complementation studies established that the gene for this putative regulator, SGE1 (SIX Gene Expression 1), is essential for pathogenicity. In addition, microscopic analysis using fluorescent proteins revealed that Sge1 is localized in the nucleus, is not required for root colonization and penetration, but is required for parasitic growth. Furthermore, Sge1 is required for expression of genes encoding effectors that are secreted during infection. We propose that Sge1 is required in F. oxysporum and other non-dimorphic (plant) pathogenic fungi for parasitic growth.     

Population structure and linkage disequilibrium in a large collection of Fusarium oxysporum strains analysed through iPBS markers

In the current study, 160 pathogenic strains of Fusarium oxysporum collected from tomato, eggplant and pepper were studied. Eighteen inter‐primer binding site (iPBS)‐retrotransposon primers were used, and these primers generated 205 scorable polymorphic bands. The number of polymorphic bands per primer varied between 9 and 19, with a mean of 11 bands per primer. The highest polymorphism information content (PIC) value was determined as 0.27, and the lowest was 0.05. The unweighted pair‐group method with arithmetic averages (UPGMA) dendrogram including a heat map revealed that the 160 pathogenic strains of F. oxysporum were divided into two main clusters. The first cluster mainly included F. oxysporum f. sp. capsici (FOC) and F. oxysporum f. sp. melongenae (FOMG) isolates. The second cluster mainly comprised F. oxysporum f. sp. lycopersici (FOL) and F. oxysporum f. sp. radicis lycopersici (FORL) isolates. The highest percentage of loci in significant linkage disequilibrium (LD) was detected for FOL, whereas the lowest level of LD was found for FOC, and 95.2%, 99.4%, 99.1% and 97.4% of the relative kinship estimates were less than 0.4 for FOL, FOMG, FORL and FOC, respectively. LD differences were detected among formae speciales, and LD was higher in FOL as compare to FOC species. The findings of this study confirm that iPBS‐retrotransposon markers are highly polymorphic at the intraspecific level in Fusarium spp.     

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.     

Characterization of Fusarium oxysporum Isolates from Common Bean and Sugar Beet Using Pathogenicity Assays and Random-amplified Polymorphic DNA Markers

Fusarium wilt is an economically important fungal disease of common bean and sugar beet in the Central High Plains (CHP) region of the USA, with yield losses approaching 30% under appropriate environmental conditions. The objective of this study was to characterize genetic diversity and pathogenicity of isolates of Fusarium oxysporum obtained from common bean and sugar beet plants in the CHP that exhibited Fusarium wilt symptoms. A total of 166 isolates of F. oxysporum isolated from diseased common bean plants were screened for pathogenicity on the universal susceptible common bean cultivar ‘UI 114’. Only four of 166 isolates were pathogenic and were designated F. oxysporum f.sp. phaseoli (Fop). A set of 34 isolates, including pathogenic Fop, F. oxysporum f.sp. betae (Fob) isolates pathogenic on sugar beet, and non‐pathogenic (Fo) isolates, were selected for random‐amplified polymorphic DNA (RAPD) analysis. A total of 12 RAPD primers, which generated 105 polymorphic bands, were used to construct an unweighted paired group method with arithmetic averages dendrogram based on Jaccard's coefficient of similarity. All CHP Fop isolates had identical RAPD banding patterns, suggesting low genetic diversity for Fop in this region. CHP Fob isolates showed a greater degree of diversity, but in general clustered together in a grouping distinct from Fop isolates. As RAPD markers revealed such a high level of genetic diversity across all isolates examined, we conclude that RAPD markers had only limited usefulness in correlating pathogenicity among the isolates and races in this study.     

Chromatographic Characterization and Phytotoxic Activity of Fusarium oxysporum f. sp. albedinis and Saprophytic Strain Toxins

The bayoud disease, vascular fusariosis of date palm tree (Phoenix dactylifera L.), is caused by the pathogenic fungus Fusarium oxysporum f. sp. albedinis. The characteristic symptoms of the bayoud disease were elicited on detached leaves of F. oxysporum f. sp. albedinis‐susceptible cultivars of date palm trees, which were treated either with the F_II (F. oxysporum f. sp. albedinis) fraction purified from the organic extracts of a F. oxysporum f. sp. albedinis liquid culture, or with a solution of fusaric acid. Enniatins, which are secreted by several Fusarium species, were tested at different concentrations and were not capable of inducing symptoms on such detached leaves. The F_II (F. oxysporum f. sp. albedinis) fraction was unable to induce necrosis of potato slices, which indicates that it does not contain significant amounts of enniatins. The high‐performance liquid chromatography (HPLC) profiles of the F_II (F. oxysporum f. sp. albedinis) fraction showed toxic peaks different from fusaric acid. A fraction, named F_II (AZ₄), was obtained from culture filtrates of a saprophytic Fusarium strain maintained in the same cultural conditions as for the F. oxysporum f. sp. albedinis. The HPLC profile of the F_II (AZ₄) fraction did not show the characteristic phytotoxic peaks present in the F_II (F. oxysporum f. sp. albedinis) fraction. This finding well agrees with the fact that the F_II (AZ₄) fraction is not toxic to detached date palm leaves. Moreover, the HPLC profiles of F_II fractions obtained from other special forms of F. oxysporum are different the F_II (F. oxysporum f. sp. albedinis) profile. The phytotoxic compounds purified from the F_II (F. oxysporum f. sp. albedinis) fraction are probably new molecules that may help in understanding the pathogenesis of bayoud disease.     

Fusarium Species Isolated from Common Weeds in Eggplant Fields and Symptomless Hosts of Fusarium oxysporum f. sp. melongenae in Turkey

Thirteen species of weed plants were collected between May and September in 2010 and 2011 from eggplant fields representing 11 distinct locations covering a wide geographical area of Turkey. Weeds are potential hosts of many plant pathogens and may not exhibit disease symptoms when colonized. Fusarium spp. were isolated from five monocotyledonous species and eight dicotyledonous species. A total of 212 isolates recovered from weeds were assigned to eight Fusarium species on the basis of morphological characteristics. F. oxysporum was the most frequently isolated species (29.7%), followed by F. solani (19.8%), F. graminearum (13.7%), F. verticillioides (12.7%), F.equiseti (9.9%), F. avenacearum (8.0%), F. proliferatum (3.8%) and F. subglutinans (2.4%). The F. oxysporum isolates from different weed hosts were characterized by means of pathogenicity and vegetative compatibility grouping (VCG) tests. Among these, 29 isolates were found to be pathogenic to eggplant cv. Kemer and re‐isolated as Fusarium oxysporum Schlecht. f. sp. melongenae (Fomg) as evidenced. These isolates from weed hosts were assigned to VCG 0320. This study is the first report of Fomg isolated from weeds in eggplant fields in Turkey. None of the weed species tested showed symptoms of wilting in pot experiments, and F. oxysporum was isolated with greater frequency from all inoculated weeds. The results of this study indicate that several weed plants may serve as alternative sources of inoculum for Fomg, during the growing season.     

Fungal phytopathogens encode functional homologues of plant rapid alkalinization factor (RALF) peptides

In this article, we describe the presence of genes encoding close homologues of an endogenous plant peptide, rapid alkalinization factor (RALF), within the genomes of 26 species of phytopathogenic fungi. Members of the RALF family are key growth factors in plants, and the sequence of the RALF active region is well conserved between plant and fungal proteins. RALF1‐like sequences were observed in most cases; however, RALF27‐like sequences were present in the Sphaerulina musiva and Septoria populicola genomes. These two species are pathogens of poplar and, interestingly, the closest relative to their respective RALF genes is a poplar RALF27‐like sequence. RALF peptides control cellular expansion during plant development, but were originally defined on the basis of their ability to induce rapid alkalinization in tobacco cell cultures. To test whether the fungal RALF peptides were biologically active in plants, we synthesized RALF peptides corresponding to those encoded by two sequenced genomes of the tomato pathogen Fusarium oxysporum f. sp. lycopersici. One of these peptides inhibited the growth of tomato seedlings and elicited responses in tomato and Nicotiana benthamiana typical of endogenous plant RALF peptides (reactive oxygen species burst, induced alkalinization and mitogen‐activated protein kinase activation). Gene expression analysis confirmed that a RALF‐encoding gene in F. oxysporum f. sp. lycopersici was expressed during infection on tomato. However, a subsequent reverse genetics approach revealed that the RALF peptide was not required by F. oxysporum f. sp. lycopersici for infection on tomato roots. This study has demonstrated the presence of functionally active RALF peptides encoded within phytopathogens that harbour an as yet undetermined role in plant–pathogen interactions.     

Microsatellite marker-based detection of Fusarium wilt pathogens of Psidium guajava L.

Wilt of Psidium guajava L., incited by Fusarium oxysporum f. sp. psidii and Fusarium solani is a serious soil-borne disease of guava in India. Forty-two isolates each of F. oxysporum f. sp. psidii (Fop) and F. solani (Fs) collected from different agro climatic zones of India showing pathogenicity were subjected to estimate the genetic and molecular characterisation in terms of analysis of microsatellite marker studies. Out of eight microsatellite markers, only four microsatellite markers, viz. MB 13, MB 17, RE 102 and AY212027 were amplified with single band pattern showing the character of identical marker for molecular characterisation and genetic identification. Microsatellite marker MB 13 was amplified in F. oxysporum f. sp. psidii and F. solani isolates. Product size of 296 bps and 1018 bps were exactly amplified with a single banding pattern in all the isolates of F. oxysporum f. sp. psidii and F. solani, respectively. Microsatellite markers, viz. MB 17, RE 102 and AY212027 were also exactly amplified with a single banding pattern. MB 17 was amplified in F. oxysporum f. sp. psidii isolates with a product size of 300 bp. RE 102 and AY212027 were amplified in F. solani isolates with the product size of 153 bp and 300 bp, respectively. Therefore, amplified microsatellite marker may be used as identifying DNA marker.     

Two xylanase genes of the vascular wilt pathogen Fusarium oxysporum are differentially expressed during infection of tomato plants

Two genes encoding putative family F xylanases from the tomato vascular wilt pathogen Fusarium oxysporum f.sp. lycopersici have been cloned and sequenced. The two genes, designated xyl2 and xyl3, encode proteins with calculated molecular masses of 33 and 39.3?kDa and isoelectric points of 8.9 and 6.7, respectively. The predicted amino acid sequences show significant homology to other family F xylanases. XYL3 contains a cellulose-binding domain in its N-terminal region. Southern analysis suggested that xyl2 and xyl3 homologs are also present in other formae speciales of F. oxysporum. Both genes were expressed during growth on oat spelt xylan and tomato vascular tissue in vitro. RT-PCR revealed that xyl3 is expressed in roots and in the lower stems of tomato plants infected by F. oxysporum f.sp. lycopersici throughout the whole disease cycle, whereas xyl2 is only expressed during the final stages of disease.