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.     

Pathogenicity of Fusarium oxysporum Isolates from Malaysia and F. oxysporuin f. sp. elaeidis from Africa to Seedlings of Oil Palms (Elaeis guineensis)

Pathogenicity tests with Fusarium oxysporum isolated form Malaysian oil palm were made with oil palms seedlings raised form Malaysian seed as well s with wilt-susceptible seedlings gown from African seed. Oil palm seedlings grown form Malaysian seed were also inoculated with African isolates of F. oxysporum f. sp. elaeidis and F. oxysporum var. redolens. The experiments were made under normal soil moisture conditions and under water stress. F. oxysporum f. sp. elaeidis isolates form Africa were pathogenic to oil palm seedlings from Malaysian seeds but the Malaysian F oxysporum isolates were non-pathogenic to plams grown from Malaysian seed or the wilt-susceptible palms from African seed. Seedlings from Malaysian seed proved to be highly susceptible to the vascular wilt disease caused by F. oxysporum f. sp. elaeidis as 75–90% of the palms were infected. The susceptibility of the palms from Malaysian seed varied with different African isolates tested. The Yaligimba isolate from Zaire which was found to be F. oxysporum var. redolens was the most virulent. Disease was more severe when oil palm seedlings were subjected to a period of water stress. The incidence of death in the seedlings under stress conditions was 45% as compared with only 15% for palms grown under normal conditions.     

Optimization of RAPD-PCR Fingerprinting to Analyse Genetic Variation Within Populations of Fusarium oxysporum f.sp. cubense

Genetic variation among 11 isolates of Fusarium oxysporum f.sp. cubense (FOC) was analysed by random amplification of polymorphic DNA using the polymerase chain reaction (RAPD-PCR). The isolates represented three of the four FOC races and the seven vegetative compatibility groups (VCGs) known to occur in Australia. Isolates of F. oxysporum f.sp. cubense were also compared to isolates of F. oxysporum f.sp. gladioli, F. oxysporum f.sp. zingiberi, F. oxysporum f.sp. lycopersici, F. moniliforme, Aspergillus niger and Colletotrichum gloeosporioides. DNA was extracted from fungal mycelium and amplified by RAPD-PCR using one of two single random 10-mer primers; the primer sequences were chosen arbitrarily. The RAPD-PCR products were separated by polyacrylamide gel electrophoresis producing a characteristic banding pattern for each isolate. The genetic relatedness of the F. oxysporum f.sp. cubense isolates was determined by comparing the banding patterns generated by RAPD-PCR. This RAPD-PCR analysis revealed variation at all five levels of possible genetic relatedness examined. F. oxysporum f.sp. cubense could very easily be distinguished from the other fungi, and the three races and five VCGs of F. oxysporum f.sp. cubense could also be differentiated. Within F. oxysporum f.sp. cubense, each isolate was scored for the presence or absence of each band (50 different bands were produced for primer SS01 and 59 different bands for primer RC09) and these data were clustered using the UPGMA method (unweighted pair-group method, arithmetic average). UPGMA cluster analysis of the data generated by primer SS01 revealed two distinct clusters. One cluster contained race 4 isolates (VCGs 0120, 0129 and 01211) and the other cluster contained both race 1 (VCGs 0124, 0124/5 and 0125) and race 2 isolates (VCG 0128). Similar results were obtained with primer RC09. The banding patterns for each isolate were reproducible between experiments. These results indicated that RAPD-PCR was a useful method for analysing genetic variation within F. oxysporum f.sp. cubense. Some of the advantages of this technique were that it was rapid, no sequence data were required to design the primers and no radioisotopes were required.     

Effet de L'acide Caféoylshikimique des Racines du Palmier Dattier sur L'activité et la Production des Enzymes Hydrolytiques de Fusarium oxysporum f. sp. albedinis

Effect of caffeoylshikimic acid of date palm roots on activity and production of Fusarium oxysporum f. sp. albedinis cell wall‐degrading enzymes The caffeoylshikimic acid (CSA), a major phenolic compound of date palm roots, represents one of the resistance factors of the host to Fusarium oxysporum f. sp. albedinis. The CSA was tested at various concentrations (0,25 to 3 µ mol/ml) on the activity and the production of F. oxysporum f. sp. albediniscell wall‐degrading enzymes (CWDE): proteases, cellulases, pectinemethyl‐esterases (PME), polygalacturonases (PG) and polygalacturonate trans‐eliminases (PGTE). The results obtained show that CSA had very little effect on the activity of the various enzymes although it greatly reduced their production. The mycelial growth was also affected by CSA, but this does not explain why only the production of CWDE was noticeably reduced. In order to explain this differential effect of CSA on the activity and production of CWDE, in one group of experiments the effects of the products of hydrolysis of CSA (caffeic acid and shikimic acid) was tested and in another, the effect of the products of CSA (quinones) obtained by tyrosinase oxidation was investigated. The results obtained show that the shikimic acid did not have a significant effect on the activity of the CWDE but presented a weak inhibition of their production.The caffeic acid showed a larger inhibition of the activity of the various CWDE that was more than that of CSA and its inhibiting effect appeared to be more important during their production. The oxidation of CSA by tyrosinase was accompanied by a greater inhibition of the activity of the various CWDE. This inhibition was appreciable in comparison with that observed due to the effect of non‐oxidized CSA on CWDE production. In the same way, oxidation of caffeic acid provoked a greater inhibiting effect on the activity of CWDE than unoxidized caffeic acid. These results suggests that CSA generates products of hydrolysis (in particular caffeic acid) and products of oxidation (quinones) which inhibit the activity of the proteolytic, cellulolytic and pectinolytic enzymes produced by F. oxysporum f. sp. albedinis in the culture medium.     

Protein and Esterase Patterns of Pathogenic, Fusarium oxysporum f. sp. elaeidis and F. oxysporum var. redolens from Africa, and Non-Pathogenic K oxysporum from Malaysia

Protein and esterase patterns of eleven isolates of F. oxysporum f. sp. elaeidis, one isolate of F. oxysporum var. redolens pathogenic to oil palm from Africa and six non-pathogenic isolates of F. oxysporum from oil palm soils in Malaysia were studied by vertical disc-electrophoresis and isoelectric focusing, to determine whether the pathogenic and saprophytic forms of F. oxysporum could be distinguished using these two methods. The protein patterns of all the isolates studied by the two methods were almost identical qualitatively and it was impossible to distinguish between the pathogenic isolates of F. oxysporum f. sp. elaeidis and F. oxysporum var. redolens from Africa and saprophytic isolates of F. oxysporum from Malaysia. Esterase zymograms of the isolates produced by the two methods were different. Esterase zymograms produced by vertical disc-electrophoresis showed great variations between and within the African and Malaysian isolates, but the esterase patterns produced by isoelectric focusing were almost identical qualitatively.     

Acremonium as an endophytic bioagent against date palm Fusarium wilt

A total of 250 endophytic fungal isolates, representing 30 morphotaxa, were isolated and characterised, they were collected from the different living symptomless parts of date palm trees of orchards of six Egyptian governorates. Colonisation was greater in samples from the midrib than in those from laminar tissue and slightly greater at the tip of the lamina compared with the base of the leaf. Acremonium spp. were frequently isolated as date palm root endophytes. Acremonium isolates were screened in Petri dishes to select the highest antagonistic one against an Algerian isolate of Fusarium oxysporum f.sp. albedinis. Two-week-old axenically reared date palm seedlings grown in Petri dishes were directly injected with spore suspension (1.5?×?10⁷ spores/ml) of a pure culture of the virulent antagonistic isolate of Acremonium sp. One week after endophytic colonisation, date palm seedlings were then challenged with the pathogen, Fusarium albedinis. The challenged seedlings exhibited a significant reduction in wilt symptom percentage (by 87.0%), while the seedlings exposed to Fusarial toxin without pathogen exhibited the wilt disease symptoms. This indicates that the endophyte ably depresses any toxic action of F. albedinis. The endophytic fungus was recovered from sites distant from the point of inoculation after six?months from the application, indicating that the Acremonium sp. has the potential to move throughout the tissue plant, even the end time of trial. The Acremonium mode of action, as a biocontrol agent, was discussed.     

Host-specific Fusarium oxysporum Causes Wilt of Jojoba

Jojoba [Simmondsia chinensis (Link) Schneider] plantations in Israel originated from vegetative propagation, planted during 1991–92, have shown symptoms of wilting and subsequent death. Verticillium dahliae was only rarely isolated from these plants and artificial inoculation showed only mild disease symptoms. Fusarium oxysporum caused severe chlorosis, desiccation, defoliation and wilt in leaves of jojoba plants, resulting in plant death. Recovery of the fungus from artificially inoculated stem cuttings and seedlings showed for the first time that F. oxysporum was the primary pathogen. Inoculated cuttings exhibited wilt within 3 weeks, while in seedlings wilt occurred 10–24 weeks after inoculation. Seedlings and cuttings of jojoba which were inoculated with other Fusarium isolates originating from different crops (F. oxysporum f. sp. vasinfectum from cotton, F. oxysporum f. sp. dianthi from carnation, F. oxysporum f. sp. lycopersici from tomato and F. oxysporum f. sp. basilicum from basil) did not develop symptoms. Moreover, cotton, tomato, melon and cucumber seedlings inoculated with several virulent F. oxysporum isolates from jojoba did not show any symptoms of wilt or defoliation. These results indicate a high degree of specificity of the Fusarium isolates from jojoba; therefore, it is suggested that this isolate be defined as F. oxysporum f. sp. simmondsia.     

Molecular detection and genotyping of Fusarium oxysporum f. sp. psidii isolates from different agro-ecological regions of India

Twenty one isolates of Fusarium oxysporum f. sp. psidii (Fop), causing a vascular wilt in guava (Psidium guajava L.), were collected from different agro-ecological regions of India. The pathogenicity test was performed in guava seedlings, where the Fop isolates were found to be highly pathogenic. All 21 isolates were confirmed as F. oxysporum f. sp. psidii by a newly developed, species-specific primer against the conserved regions of 28S rDNA and the intergenic spacer region. RAPD and PCR-RFLP were used for genotyping the isolates to determine their genetic relationships. Fifteen RAPD primers were tested, of which five primers produced prominent, polymorphic, and reproducible bands. RAPD yielded an average of 6.5 polymorphic bands per primer, with the amplified DNA fragments ranging from 200–2,000 bp in size. A dendrogram constructed from these data indicated a 22–74% level of homology. In RFLP analysis, two major bands (350 and 220 bp) were commonly present in all isolates of F. oxysporum. These findings provide new insight for rapid, specific, and sensitive disease diagnosis. However, genotyping could be useful in strain-level discrimination of isolates from different agro-ecological regions of India.     

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.     

Discriminant analysis of volatile organic compounds of Fusarium oxysporum f. sp. cepae and Fusarium proliferatum isolates from onions as indicators of fungal growth

Basal rot is a common onion disease and is mainly caused by Fusarium oxysporum f. sp. cepae and Fusarium proliferatum. To study the possibility of using volatile organic compounds (VOCs) as biomarkers for these fungi, pathogenic isolates of F. oxysporum and F. proliferatum from onions were cultivated in onion medium and VOCs were measured by solid phase microextraction (SPME). Forty-two compounds were detected, and thirty of these compounds were highly related to fungal metabolic activity. Allyl mercaptan was specific to F. oxysporum isolate Fox006. Analysis of the VOCs showed significant differences between the two species and among different isolates within the same species. Sixteen of the VOCs showed were highly positively correlated with the fungal biomass estimated by real-time polymerase chain reaction (PCR). Ethanol, ethyl formate, ethyl acetate, 2-methyl-1-propanol, methyl thioacetate, n-propyl acetate and 3-methyl-1-butanol are volatile metabolites that were potential indicators of Fusarium growth on onions.     

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.     

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.     

Biological Control Activity of Three Trichoderma Isolates Against Fusarium Wilts of Cotton and Muskmelon and Lack of Correlation with their Lytic Enzymes

The enzymatic activity and the biocontrol ability of two new isolates of Trichoderma spp. (T-68 and Gh-2) were compared in laboratory and glasshouse experiments with a previously studied T. harzianum strain (T-35). In dual culture tests with Fusarium oxysporum f. sp. melonis and F. oxysporum f. sp. vasinfectum, isolates T-68 and Gh-2 overgrew the colonies of Fusarium, whereas T-35 failed to parasitize both wilt pathogens. Under glasshouse conditions, the three isolates of Trichoderma were effective in controlling Fusarium wilt of cotton but only T-35 was effective against F. oxysporum f. sp. melonis on muskmelon. When the three Trichoderma isolates were grown on liquid media containing laminarin, colloidal chitin or F. oxysporum f. sp. melonis cell walls as sole carbon sources, maximum β-1,3-glucanase and chitinase specific activity in the culture filtrates of all fungi was reached after 72h of incubation. When culture filtrates of the three Trichoderma isolates were incubated with freeze-dried mycelium of F. oxysporum f. sp. melonis or F. oxysporum f. sp. vasinfectum, different concentrations of glucose and N-acetyl-D-glucosamine were released. Overall no correlation was found between enzymatic activity and the biocontrol capability against Fusarium wilt on muskmelon and cotton.     

Management Fusarium wilt on melon and watermelon by Penicillium oxalicum

The potential of the biological control fungus Penicillium oxalicum to suppress wilt caused by Fusarium oxysporum f. sp. melonis and F. oxysporum f. sp. niveum on melon and watermelon, respectively, was tested under different growth conditions. The area under disease progress curve of F. oxysporum f. sp. melonis infected melon plants was significantly reduced in growth chamber and field experiments. In glasshouse experiments, it was necessary to apply P. oxalicum and dazomet in order to reduce Fusarium wilt severity in melons caused by F. oxysporum f. sp. melonis. For watermelons, we found that P. oxalicum alone reduced the area under the disease progress curve by 58% in the growth chamber experiments and 54% in the glasshouse experiments. From these results, we suggested that P. oxalicum may be effective for the management of Fusarium wilt in melon and watermelon plants.     

Endopolygalacturonase PG1 in Different Formae Speciales of Fusarium oxysporum

PG1, the major endopolygalacturonase of the vascular wilt pathogen Fusarium oxysporum, was secreted during growth on pectin by 10 of 12 isolates belonging to seven formae speciales, as determined with isoelectric focusing zymograms and sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels. A Southern analysis of genomic DNA and PCR performed with gene-specific primers revealed that the pg1 locus was highly conserved structurally in most isolates. Two PG1-deficient isolates were identified; one lacked the encoding gene, and the other carried a pg1 allele disrupted by a 3.2-kb insertion with sequence homology to hAT transposases. The virulence for muskmelon of different F. oxysporum f. sp. melonis isolates was not correlated with PG1 production in vitro. We concluded that PG1 is widely distributed in F. oxysporum and that it is not essential for pathogenicity.     

Evolutionary Relationships among the Fusarium oxysporum f. sp. cubense Vegetative Compatibility Groups

Fusarium oxysporum f. sp. cubense, the causal agent of fusarium wilt of banana (Musa spp.), is one of the most destructive strains of the vascular wilt fungus F. oxysporum. Genetic relatedness among and within vegetative compatibility groups (VCGs) of F. oxysporum f. sp. cubense was studied by sequencing two nuclear and two mitochondrial DNA regions in a collection of 70 F. oxysporum isolates that include representatives of 20 VCGs of F. oxysporum f. sp. cubense, other formae speciales, and nonpathogens. To determine the ability of F. oxysporum f. sp. cubense to sexually recombine, crosses were made between isolates of opposite mating types. Phylogenetic analysis separated the F. oxysporum isolates into two clades and eight lineages. Phylogenetic relationships between F. oxysporum f. sp. cubense and other formae speciales of F. oxysporum and the relationships among VCGs and races of F. oxysporum f. sp. cubense clearly showed that F. oxysporum f. sp. cubense''s ability to cause disease on banana has emerged multiple times, independently, and that the ability to cause disease to a specific banana cultivar is also a polyphyletic trait. These analyses further suggest that both coevolution with the host and horizontal gene transfer may have played important roles in the evolutionary history of the pathogen. All examined isolates harbored one of the two mating-type idiomorphs, but never both, which suggests a heterothallic mating system should sexual reproduction occur. Although, no sexual structures were observed, some lineages of F. oxysporum f. sp. cubense harbored MAT-1 and MAT-2 isolates, suggesting a potential that these lineages have a sexual origin that might be more recent than initially anticipated.Fusarium oxysporum Schlechtendahl emend. Snyder and Hansen is a cosmopolitan species (9) comprised of both pathogenic and nonpathogenic isolates (20). The pathogenic isolates of F. oxysporum cause fusarium wilt of several agricultural crops, and are accordingly subdivided into formae speciales (3, 26, 55). One of the economically more important and destructive formae speciales is the causal agent of fusarium wilt (Panama disease) of banana (Musa spp.), F. oxysporum f. sp. cubense (E. F. Smith) Snyder et Hansen. This disease has been reported in all banana production regions of the world, except those bordering the Mediterranean, Melanesia, Somalia, and some islands in the South Pacific (66, 77).A range of approaches are typically employed for the characterization of F. oxysporum f. sp. cubense isolates. Based on virulence to specific banana cultivars (66, 67), the pathogen may be classified into one of three races (i.e., races 1, 2, and 4), although this designation may be contingent on environmental conditions. For instance, genetically identical isolates of F. oxysporum f. sp. cubense are classified as race 4 isolates in the subtropics and as race 1 isolates in the tropics because they cause disease to Cavendish bananas under subtropical conditions only (67, 86). Based on vegetative compatibility, F. oxysporum f. sp. cubense isolates have been separated into 24 so-called vegetative compatibility groups (VCGs) (5, 29, 47, 68). Finally, various DNA-based tools have been used to separate F. oxysporum f. sp. cubense into a number of clonal lineages that more or less correspond to their grouping based on VCGs (6, 22, 38, 59).The evolutionary history of F. oxysporum f. sp. cubense is complex. Based on the results of phylogenetic studies (4-7, 22, 38, 57, 59). F. oxysporum f. sp. cubense represent multiple unrelated lineages, some of which are more closely related to other formae speciales of F. oxysporum than to other F. oxysporum f. sp. cubense lineages (3, 57, 59). This has lead to speculations that new pathogenic forms of F. oxysporum may be derived from other pathogenic and nonpathogenic members of this species (21). Factors such as coevolution with the plant host and the spread of virulence determinants via processes such as parasexuality, heterokaryosis, and sexual recombination also have been implicated in the evolution of this pathogen (11, 36, 37, 39, 64, 65, 69). Although parasexuality and heterokaryosis are known to occur in F. oxysporum (11, 39), sexual fruiting structures have never been observed in the species and only indirect evidence for sexual recombination has been detected (82). Indeed, the organization of the F. oxysporum f. sp. cubense mating type locus (MAT) is similar to those found in the closely related Gibberella fujikuroi (Sawada) Ito in Ito et K. Kimura complex and other heterothallic ascomycetes (2, 90).Development of appropriate disease management strategies and the selection of F. oxysporum f. sp. cubense-resistant banana cultivars may benefit from a better understanding of the diversity and evolutionary history of the pathogen. Although most previous DNA-based studies provided knowledge regarding the diversity of F. oxysporum f. sp. cubense, the genetic relatedness among the lineages identified in these studies remains uncertain (22). It is also not clear how the different races and VCGs of F. oxysporum f. sp. cubense are related to one another and to other isolates of F. oxysporum. Therefore, the main objective of this study was to resolve the relationships among the F. oxysporum f. sp. cubense VCGs and determine their relationships with other formae speciales and nonpathogenic members of F. oxysporum by using a multigene phylogenetic approach (8, 32, 52, 53, 62, 75, 91). To facilitate the rapid differentiation of the various F. oxysporum f. sp. cubense lineages, we also aimed to develop a diagnostic PCR-restriction fragment length polymorphism (RFLP) procedure. To evaluate the potential of F. oxysporum f. sp. cubense to reproduce sexually, sexual crosses among isolates of opposite mating types were attempted after PCR-based detection of the MAT-1 and MAT-2 idiomorphs (34).     

Race Differentiation in Fusarium oxysporum f.sp. chrysanthemi

The pathogenicity of different isolates of Fusarium oxysporum obtained from plants of Gerbera (Gerbera jamesonii), Chrysanthemum (Chrysanthemum morifolium), Paris daisy (Argyranthemum frutescens) and African daisy (Osteospermum sp.), all in the family Asteraceae, was tested on different cultivars of these hosts, to assess their pathogenicity. The reactions were compared with those of isolates of F. oxysporum f. sp. chrysanthemi and of f.sp. tracheiphilum obtained from the American Type Culture Collection. We found that isolates of F. oxysporum f. sp. chrysanthemi can be distinguished as three physiological races on the basis of their pathogenicity to the panel of differential cultivars. Sequencing of the intergenic spacer (IGS) region of ribosomal DNA (rDNA) and phylogenetic analysis showed that the Fusarium races fell into three phylogenetic groups, which coincided with those observed in pathogenicity tests. Analysis of the IGS sequences revealed a high degree of similarity among strains from Italy and Spain from different host species, suggesting that recent outbreaks in these ornamentals were probably caused by introduction of infected nursery material from a common origin.     

Specific PCR-based marker for detection of pathogenic groups of Fusarium oxysporum f. sp. cucumerinum in India

For the detection of Fusarium oxysporum f. sp. cucumerinum pathogenic groups, a specific PCR-based marker was developed. Specific random amplified polymorphic DNA (RAPD) markers which identified in four pathogenic groups I, II, III, and IV were cloned into P^Gem-Teasy vector. Cloned fragments were sequenced, and used for developing sequence characterized amplified regions (SCAR) primers for detection of pathogenic groups. F. oxysporum f. sp. cucumerinum isolates belonging to four pathogenic groups in India, cucumber nonpathogenic F. oxysporum, F. oxysporum f. sp. moniliforme and melonis, Fusarium udum, and isolate of Alternaria sp. were tested using developed specific primers. A single 1.320 kb, 770 bp, 1.119 kb, and 771 bp fragment were amplified from pathogenic group I, II, III, and IV isolates, respectively. Results showed the PCR based marker, which used in this research work, could detect up to 1 ng of fungal genomic DNA. The specific SCAR primers and PCR technique developed in this research easily detect and differentiate isolates of each F. oxysporum f. sp. cucumerinum pathogenic groups.     

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.     

Biological control of bayoud disease in date palm: Selection of microorganisms inhibiting the causal agent and inducing defense reactions

Twenty-one isolates of microorganisms, including Bacillus spp., Rhizobium spp., Ulocladium atrum, Candida guilliermondii, Pseudomonas sp., Rahnella aquatilis and other bacteria not yet identified, were tested to determine their effects on the mycelial growth and the sporulation of Fusarium oxysporum f.sp. albedinis (Foa), the causal agent of bayoud on date palm. The potential of these antagonists in the induction of defense reactions in date palm seedlings was also studied. Four bacteria, B. pumilus W1, R. aquatilis W2, B. cereus X16 and n.d. S1, have exhibited a high inhibition toward mycelial growth of Foa (70–77%), and its sporulation (80–95% of the control). Moreover, cytological alterations have been detected in the Foa mycelium grown in the inhibition zone. Application of these antagonists into date palm seedlings has led to trigger defense reactions with an accumulation of non-constitutive hydroxycinnamic acid derivatives, such as the sinapic derivative I2, known to play a crucial role in resistance of date palm to Foa. This reaction was more pronounced in resistant cultivar (BSTN) than in susceptible (JHL). The combined effects of direct and indirect actions of Foa antagonists are discussed in the hope of providing a biocontrol strategy against bayoud.