Biocontrol agent Fusarium oxysporum f.sp. strigae has no adverse effect on indigenous total fungal communities and specific AMF taxa in contrasting maize rhizospheres

We studied the effects of Fusarium oxysporum f.sp. strigae (Fos), a soil-borne biocontrol agent (BCA) against Striga hermonthica, on total fungal and arbuscular mycorrhizal fungal (AMF) taxa in rhizospheres of maize in both clayey and sandy soil. Effects of Fos-BCA ‘Foxy-2’ were evaluated against (1) S. hermonthica presence, and (2) organic fertilization with Tithonia diversifolia residues at 14, 28 and 42 d after ‘Foxy-2’ inoculation, via DNA-based quantitative PCR and TRFLP fingerprinting. In both soils, ‘Foxy-2’ occasionally promoted total fungal abundance, while the community composition was mainly altered by T. diversifolia and S. hermonthica. Notably, ‘Foxy-2’ stimulated AMF Gigaspora margarita abundance, while G. margarita was suppressed by S. hermonthica. Total fungal and AMF abundance were promoted by T. diversifolia residues. In conclusion, ‘Foxy-2’ resulted in no adverse effects on indigenous rhizosphere fungal communities substantiating its environmental safety as BCA against S. hermonthica.     

Tissue specific reactions of sorghum roots to the mycoherbicide Fusarium oxysporum f. sp. strigae versus the pathogenic F. proliferatum

Fusarium oxysporum f. sp. strigae (Foxy 2) is a mycoherbicide against Striga hermonthica. To ensure the safe use of this biocontrol agent, and as part of the risk assessment, this study was aimed at providing cytological evidence that Foxy 2 does not possess pathogenic behavior towards the non-target host sorghum. Therefore, we compared the infection processes and sorghum root tissue reactions towards the pathogenic F. proliferatum to that of Foxy 2 using light- and transmission electron microscopy. Given that during the growth process, hyphae could get into the central cylinder, tissue specific reactions of sorghum to Foxy 2 were also investigated by wounding the roots (exposing the vascular system), and testing for proliferation of hyphae within the vessels. Results showed that 2 weeks after sowing, F. proliferatum had invaded and destroyed all cell types including the central cylinder while Foxy 2 hyphae were located around the outer endodermal layer and were not able to penetrate the latter. There was an increase in blue autoflourescence in the central cylinder and especially the endodermis, probably due to increased phenolics in Foxy 2 infected roots which was not the case for F. proliferatum. This might contribute to the inability of Foxy 2 to penetrate the endodermis. Transmission electron microscopy showed extensive degradation of endodermis and vessel walls into thin translucent layers by F. proliferatum but not by Foxy 2. In the mechanically wounded and infected roots, Foxy 2 could invade the central cylinder close to the wound but was not identified a few millimeters away from the wound. This implies that it was not able to grow within or destroy the central cylinder even when already present within it; probably due to the observed increased phenolics. Thus, exposure of the vascular system did not serve as a route for the invasion of Foxy 2 which therefore could not further cause tracheomycosis. Therefore, Foxy 2 could be seen as a fungus well suited for biocontrol.     

Promoting effect of Fusarium oxysporum [f.sp. strigae] on abundance of nitrifying prokaryotes in a maize rhizosphere across soil types

The integrated application of resistant crop varieties with biological control agents (BCAs) such as the Fusarium oxysporum [f.sp. strigae] strain “Foxy-2” has shown to be effective in fighting off the weed Striga hermonthica which is parasitic to several cereals cultivated in Sub-Saharan Africa (Schaub et al., 2006; Venne et al., 2009). “Foxy-2” proliferates in the rhizosphere and has been mainly studied for its virulence and mode of action. Contrary, no understanding is available regarding its interactions with key rhizosphere microorganisms steering relevant nutrient cycles in soils including nitrogen (N). In this study, we tested the hypothesis that “Foxy-2” displaces indigenous prokaryotic, N cycling communities in the maize rhizosphere due to competition for organic resources. Consequently, we evaluated if the application of an N-rich organic residue (i.e., Tithonia diversifolia with C/N ratio = 13, lignin content = 8.9%, polyphenol content = 1.7%) compensates these presumed competition effects. In a rhizobox experiment, quantitative polymerase chain reaction was used to follow the response of rhizosphere ammonia-oxidizing archaea (AOA) and bacteria (AOB) as well as total bacteria and archaea following “Foxy-2” inoculation in two physico-chemically contrasting soils (sandy Ferric Alisol versus clayey Humic Nitisol). Soils were treated with or without “Foxy-2”, S. hermonthica seeds, and T. diversifolia residues. Contrary to our expectations, we observed a distinct soil texture dependent, promoting effect of “Foxy-2” on rhizosphere prokaryotes. Abundance of AOA and total prokaryotic communities increased in response to “Foxy-2” in the sandy soil, while AOB remained unaffected. This effect on AOA was accelerated when T. diversifolia residues were incorporated. Further, in the clayey soil, AOA abundance was promoted when exposed to S. hermonthica infestation of maize. This suggested their capability to adapt to this biotic stress situation. It was concluded that “Foxy-2” did not pose a negative effect on targeted indigenous microorganisms, but the underlying mechanisms for the observed promoting effect of AOA abundance by “Foxy-2” inoculation are yet to be understood.     

Development and assessment of fusarium oxysporum-based mycoherbicide for control of Striga Hermonthica in sorghum

Abstract

Experiments were carried out from 2002 to 2003 to determine the most suitable form of fungal delivery for possible use by farmers in biological control of Striga hermonthica. Six mycoherbicides were developed, based on Fusarium oxysporum isolated from wilted S. hermonthica. In mycoherbicide formulation, rock phosphate powder, sorghum bran and gum arabic powder were used as carriers. Besides its role as a carrier, gum arabic powder was used as a sticker. There were three carriers with two formulations each, making six treatments altogether. Living propagule studies were based on colony, mycelium and conidium number of F. oxysporum. In greenhouse evaluation of mycoherbicides, each kg sorghum seed was coated with 10 g mycoherbicide before sowing. Carrier rock phosphate powder with gum arabic powder as a sticking agent was the most suitable form of its delivery for use by peasant farmers.     

Presence of Fusarium oxysporum f. sp. melonis Race 1 in Soils Cultivated with Melon in the State of Colima (Mexico)

During years 2001, 2002 and 2003 the gravity of the Fusarium wilt in 1000 hectares of melon culture was evaluated in Colima (Mexico). In spite of the soil disinfections with methyl bromide, the losses could reach 25% of the final production. The analysis of 4 soil samples from the fields with ill plants, in a selective medium for Fusarium, allowed to detect the presence of F. oxysporum. By means of the presented technique “soil phytopathometry”, 31 isolates of F. oxysporum f. sp. melonis were obtained from the soil samples. The isolates were inoculated on melon plants to evaluate their pathogenicity. The 31 isolates inoculated, produced the symptoms of chlorosis and wilting, in melon cultivars that allowed us to affirm that all isolates were race 1 of F. oxysporum f. sp. melonis. Being this the first news of the presence of F. oxysporum f. sp. melonis in the state of Colima (Mexico).     

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.     

Biological control ofFusarium oxysporum f.sp.cubense in banana by inoculation withPseudomonas fluorescens

Native strains ofPseudomonas fluorescens exhibitedin vitro antibiosis towards isolates of races 1 and 4 ofFusarium oxysporum f.sp.cubense, the Panama wilt pathogen of banana. The seedlings ofMusa balbisiana seedlings treated withP. fluorescens showed less severe wilting and internal discolouration due toF. oxysporum f.sp.cubense infection in greenhouse experiments. In addition to suppressing Panama wilt, bacterized seedlings ofM. balbisiana also showed better root growth and enhanced plant height.     

Incidence of Fusarium spp. and Levels of Fumonisin B1 in Maize in Western Kenya

Maize kernel samples were collected in 1996 from smallholder farm storages in the districts of Bomet, Bungoma, Kakamega, Kericho, Kisii, Nandi, Siaya, Trans Nzoia, and Vihiga in the tropical highlands of western Kenya. Two-thirds of the samples were good-quality maize, and one-third were poor-quality maize with a high incidence of visibly diseased kernels. One hundred fifty-three maize samples were assessed for Fusarium infection by culturing kernels on a selective medium. The isolates obtained were identified to the species level based on morphology and on formation of the sexual stage in Gibberella fujikuroi mating population tests. Fusarium moniliforme (G. fujikuroi mating population A) was isolated most frequently, but F. subglutinans (G. fujikuroi mating population E), F. graminearum, F. oxysporum, F. solani, and other Fusarium species were also isolated. The high incidence of kernel infection with the fumonisin-producing species F. moniliforme indicated a potential for fumonisin contamination of Kenyan maize. However, analysis of 197 maize kernel samples by high-performance liquid chromatography found little fumonisin B₁ in most of the samples. Forty-seven percent of the samples contained fumonisin B₁ at levels above the detection limit (100 ng/g), but only 5% were above 1,000 ng/g, a proposed level of concern for human consumption. The four most-contaminated samples, with fumonisin B₁ levels ranging from 3,600 to 11,600 ng/g, were from poor-quality maize collected in the Kisii district. Many samples with a high incidence of visibly diseased kernels contained little or no fumonisin B₁, despite the presence of F. moniliforme. This result may be attributable to the inability of F. moniliforme isolates present in Kenyan maize to produce fumonisins, to the presence of other ear rot fungi, and/or to environmental conditions unfavorable for fumonisin production.     

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.     

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.     

Races and virulence of Fusarium oxysporum f.sp. cubense on local banana cultivars in Kenya

Virulence of 31 Kenyan isolates of Fusarium oxysporum obtained from bananas showing symptoms of Panama disease was tested against the differential banana cvs Bluggoe, Gros Michel, Dwarf Cavendish, and two other local cvs Muraru and Wang'ae. Seventeen isolates were assigned to either race 1 or race 2 of F. oxysporum f.sp. cubense (FOC). Race 4 was not apparent in this sample of 31 isolates from Kenya as none were pathogenic to cv. Cavendish, and no wilted Cavendish have been observed in field surveys in Kenya. Races could not be assigned to 12 isolates as they were virulent on more than one differential cultivar, and two were apparently not pathogenic. All isolates assigned to races 1 and 2 belonged to the VCG bridging complex 0124/5/8/20, but some other isolates belonging to this VCG complex could not be assigned to race. All five isolates assigned to VCG 01212 could not be assigned to known races. Considerable variability thus exists within FOC isolates within this region. Local cultivars of banana showed differential resistance to the pathogen. The interaction of cultivars and isolates on the level of disease was significant. Overall, cv. Wang'ae was the most susceptible to most of the isolates tested, regardless of their race, and could therefore be used as a reference cultivar in pathogenicity tests of isolates of FOC in the East African region. Of the cultivars tested that are widely grown on smallholder farms in Kenya, Muraru was the least susceptible.     

Winter wheat roots grow twice as deep as spring wheat roots, is this important for N uptake and N leaching losses?

Nitrogen (N)-deficiency and lack of phosphorus (P) availability are major constraints to maize yields in Western Kenya. In a two-season field study in the lake Victoria basin, we tested the capacity of white lupin (Lupinus albus (L.), cv. Ultra), as a nitrogen-fixing crop with a highly efficient P-acquisition capacity, to increase maize yields when used as a companion or cover crop, or as a source of organic matter. Each experiment was performed on three different fields (Vertisols) differing in N/P availability, previous cropping history and in levels of infestation by the parasitic weed Striga hermonthica (Del.) Benth. Our results show that white lupin led to significantly higher yields of maize when used as a cover crop. When lupin was grown as a companion crop, it also slightly enhanced the yield of the co-cultivated maize. When lupin shoots were incorporated to the soil, the positive effect of lupin on maize growth was field-dependent and only occurred in the field most heavily infested with S. hermonthica. Despite the beneficial impact on maize yield, no clear effect of lupin on soil N and P availability or on maize N/P uptake were observed. In contrast, lupin significantly inhibited infestation of maize by S. hermonthica: when lupin was grown together with maize in pots inoculated with S. hermonthica, the emergence of the weed was strongly reduced compared to the pots with maize only. This work opens a new range of questions for further research on white lupin and its potential beneficial impact as a S. hermonthica-inhibiting crop.     

Comparative root colonisation of strawberry cultivars Camarosa and Festival by Fusarium oxysporum f. sp. fragariae

Background and aims

Strawberry (Fragaria x ananassa) is a high-value crop worldwide. Fusarium oxysporum f. sp. fragariae causes rapid wilting and death of strawberry plants and severe economic losses worldwide. To date, no studies have been conducted to determine colonisation of either susceptible or resistant strawberry plants by F. oxysporum f. sp. fragariae, or whether plant colonisation by F. oxysporum f. sp. fragariae differs between susceptible and resistant cultivars.

Methods

Colonisation of strawberry plants by a pathogenic isolate of F. oxysporum f. sp. fragariae was examined both on the root surface and within root tissue of one resistant cv. Festival and one susceptible cv. Camarosa using light and scanning electron microscopy from 4?h to 7?d post inoculation (pi).

Results

Resistant cv. Festival significantly impeded the spore germination and penetration from 4 to 12 hpi and subsequent growth and colonisation by this pathogen until 7 dpi compared with susceptible cv. Camarosa. At 7 dpi, fungal colonisation in resistant cv. Festival remained mainly confined to the epidermal layer of the root, while in susceptible cv. Camarosa, hyphae not only had heavily colonised the cortical tissue throughout but had also colonised vascular tissues.

Conclusions

This study demonstrates for the first time that resistance of a strawberry cultivar to F. oxysporum f. sp. fragariae is a result of impedance of pathogen growth and colonisation both on the plant surface and within host tissues. Resistance mechanisms identified in this study will be of high value for breeding programmes in developing new disease-resistant cultivars to manage this serious strawberry disorder.     

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).     

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.     

Pathological Relationship of Ditylenchus dipsaci and Fusarium oxysporum f. sp. medicaginis on alfalfa

Ditylenchus dipsaci and Fusarium oxysporum f. sp. medicaginis synergistically affected the mortality and plant growth of Ranger alfalfa, a cultivar susceptible to stem nematode and Fusarium wilt. The nematode-fungus relationship had an additive effect on mortality and plant growth of Lahontan (nematode resistant and Fusarium wilt susceptible) and of Moapa 69 (nematode susceptible and Fusarium wilt resistant). Mortality rates were 13, 16, 46, and 49% for Ranger; 4, 18, 26, and 28% for Lahontan; and 19, 10, 32, and 30% for Moapa 69 inoculated with D. dipsaci, F. oxysporum f. sp. medicaginis, and simultaneously and sequentially with D. dipsaci and F. oxysporum f. sp. medicaginis, respectively. Shoot weights as a percentage of uninoculated controls for the same treatments were 52, 84, 26, and 28%, for Ranger; 74, 86, 64, and 64% for Lahontan; and 50, 95, 44, and 39% for Moapa 69. Plant growth suppression was related to vascular bundle infection and discoloration of alfalfa root tissue. Disease severity and plant growth of alfalfa did not differ with simultaneous or sequential inoculations of the two pathogens. Fusarium oxysporum f. sp. medicaginis affected alfalfa growth but not nematode reproduction.     

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.     

Molecular markers reflect differentiation of Fusarium oxysporum forma speciales on tomato and forma on eggplant

Selective pressure induces pathogens to change their method of infection and, sometimes, causes species to become infectious. Pathogenic fungi must differentiate different morphological and physiological properties during the process of host specialization in their life cycle. In the present study, we conducted a genetic investigation and compared similarities within a generation of Fusarium oxysporum forma speciales (f. sp.) infecting tomato and forma (f.) infecting eggplants using selected ISSR and RAPD markers, two horticultural commodities belonging to the same taxon of the Solanaceae. Interestingly, genetic data showed that fungi belonging to F. oxysporum f. sp. infecting tomato have a close genetic relationship with the fungi f. infecting eggplant. Furthermore, F. oxysporum f. sp. infecting tomato showed less genetic variation than F. oxysporum f. melongenae, suggesting that it could be developed more recently during host adaptation. On the other hand, the gene sequence of inter-simple sequence repeat (ISSR) markers resulting in high polymorphism showed matches with gene sequences encoding specific proteins related to pathogenicity of F. oxysporum species. These findings support the notion that selected ISSR markers can be used to follow host-associated divergence of F. oxysporum species infecting tomato and eggplant and that differentiation of their specific genes can also be related to pathogenicity and development as predictive studies before initiating detailed sequencing analysis.     

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.