Further Investigation of the Specificity of Interactions Between Wild Lactuca spp. and Bremia lactucae Isolates from Lactuca serriola

Callus cultures derived from isogenic lines of the tomato cultivars Moneymaker and Craigella, resistant or susceptible to F. oxysporum f. sp. lycopersici, were inoculated with Fusarium oxysporum f. sp. lycopersici race 1. Fungal growth was restricted on callus derived from resistant plants, after inoculation with a conidial suspension, whereas callus derived from susceptible plants was totally overgrown by the fungus within 7 days. The concentration of the phytoalexin rishitin was significantly higher in the callus culture derived from a resistant tomato line compared with the callus culture from a susceptible line, 2 and 3 days after inoculation with mycelium. The results of the experiments were compared with experiments with whole plants. Rishitin production as well as growth of the fungus was comparable with responses in plant-fungus interaction. Therefore callus culture may be useful in studying the interaction between tomato plants and race 1 of F. oxysporum f. sp. lycopersici.     

Invasion of a Habitat-Forming Seaweed: Effects on Associated Biota

Fucus evanescens is a brown alga of arctic origin that has invaded European coasts. The epiphytic community of F. evanescens in southern Sweden was compared with that of the native Fucus vesiculosus, to examine to what extent an invading seaweed can modify local biodiversity. F. evanescens was much less fouled than F. vesiculosus, supporting both less biomass and fewer species of epiphytes. Multivariate analysis of the most common epiphyte taxa showed that the epiphytic community composition of F. evanescens was not entirely separated from that of F. vesiculosus, but host species contributed significantly to explain the variation in community composition. The biomass of free-living invertebrates was also lower on F. evanescens, although the pattern differed between taxonomic groups. While the biomass of amphipods was lower on F. evanescens, there was no significant difference in biomass of isopods or gastropods between the Fucus species. The good correlation between biomass of epiphytes and free-living animals suggests that the epiphytes play an important role in providing a suitable habitat for many species of free-living epifauna. The study shows that the invasion of F. evanescens affects the environmental conditions for many species associated with the Fucus community but that the direct effect on biodiversity is probably low.     

The secreted ribonuclease T2 protein FoRnt2 contributes to Fusarium oxysporum virulence

Secreted RNase proteins have been reported from only a few pathogens, and relatively little is known about their biological functions. Fusarium oxysporum is a soilborne fungal pathogen that causes Fusarium wilt, one of the most important diseases on tomato. During the infection of F. oxysporum, some proteins are secreted that modulate host plant immunity and promote pathogen invasion. In this study, we identify an RNase, FoRnt2, from the F. oxysporum secretome that belongs to the ribonuclease T2 family. FoRnt2 possesses an N-terminal signal peptide and can be secreted from F. oxysporum. FoRnt2 exhibited ribonuclease activity and was able to degrade the host plant total RNA in vitro dependent on the active site residues H80 and H142. Deletion of the FoRnt2 gene reduced fungal virulence but had no obvious effect on mycelial growth and conidial production. The expression of FoRnt2 in tomato significantly enhanced plant susceptibility to pathogens. These data indicate that FoRnt2 is an important contributor to the virulence of F. oxysporum, possibly through the degradation of plant RNA.     

In vitro selection of yellow passion fruit genotypes for resistance to <Emphasis Type="Italic">Fusarium</Emphasis> vascular wilt

Fusarium vascular wilt (caused by Fusarium oxysporum f. sp. passiflorae) is a limiting factor in the cultivation of yellow passion fruit (Passiflora edulis). Since there is no effective and economically viable control available, development of resistant or at least tolerant cultivars are in demand. A number of procedures have been used for the initial selection of plant genotypes resistant to various fungal pathogens by means of a fungal culture filtrate or purified toxin. In this study, seeds and in vitro-grown plantlets of passion fruit were screened with different concentrations of either Fusarium oxysporum f. sp. passiflorae (FOP) culture filtrate (0, 20, 30, 40 or 50%, v/v) or fusaric acid (0.10, 0.20, 0.30 or 0.40 mM) supplemented in Murashige and Skoog (MS) basal media. Subsequently, selected plants were inoculated with a conidial suspension of FOP to assess correlation between in vivo and in vitro responses. In vitro sensitivity to the selective agents and the resistance response to the pathogen were also compared. Root growth was markedly influenced by FA, culture filtrate, and conidial suspension culture treatments. Observations indicated that roots were primary targets for attack by F. oxysporum. Successful in vitro selection of resistant genotypes by both FA and culture filtrate treatments suggested that this strategy was viable for accelerating breeding of passion fruit for resistance to the Fusarium vascular wilt.     

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.     

Fusarium spp. are Responsible for Shoot Canker of Kumquat in China

Shoot and branch canker and tree decline of kumquat (Fortunella margarita cv. Guban) were recorded in Yangshuo County, Guilin City, in the Guangxi Zhuang Autonomous Region of China during 2008–2011. Fusarium oxysporum and a new Fusarium species within the Gibberella fujikuroi complex (Fusarium sp. GLB1) were isolated repeatedly from the infected shoots and branches. Species identifications were verified by their high translation elongation factor 1‐alpha (TEF1) sequence similarity with those of the species epitypes. Koch's postulates were fulfilled on kumquat (cv. Guban) and mandarin establishing pathogenicity. To our knowledge, this is the first report of Fusarium shoot canker disease caused by F. oxysporum and Fusarium sp. on kumquat.     

Variability and characterization of mycotoxin-producing Fusarium spp isolates by PCR-RFLP analysis of the IGS-rDNA region

In the present report, a total of 75 Fusarium spp isolates (35 of the Gibberella fujikuroi species complex, 26 of F. oxysporum, 7 of F. graminearum, 5 of F. culmorum, 1 of F. cerealis, and 1 of F. poae) from different hosts were characterized morphologically, physiologically and genetically. Morphological characterization was performed according to macroscopic and microscopic aspects. Physiological characterization was based on their ability to produce fumonisin B₁ (FB₁), fumonisin B₂ (FB₂), zearalenone (ZEA) and type B trichothecenes (deoxynivalenol, nivalenol and 3-acetyldeoxynivalenol). FB₁, FB₂, and ZEA were determined by liquid chromatography and trichothecenes by gas chromatography. Molecular characterization of isolates was carried out using an optimized and simple method for isolation of DNA from filamentous fungi and polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLP) of the intergenic spacer region (IGS) of the rDNA. The results indicated that G. fujikuroi complex isolates can be␣divided into low and high fumonisin producers. The haplotypes obtained with HhaI, EcoRI, AluI, PstI and XhoI enzymes provided very characteristic groupings of G. fujikuroi isolates as a function of host type and fumonisin producing capacity. F. graminearum, F. culmorum and F. cerealis isolates were high ZEA␣and type B trichothecene producers, while F. oxysporum and the G. fujikuroi complex isolates did not show this ability. The haplotypes obtained with CfoI, AluI, HapII, XhoI, EcoRI and PstI enzymes permitted to discern these five Fusarium species and G. fujikuroi complex isolates but the restriction patterns of the IGS region did not show any relationship with the geographic origin of isolates.     

The biosynthesis of gibberellic acids by the transformants of orchid-associated <Emphasis Type="Italic">Fusarium oxysporum</Emphasis>

The genus Fusarium, including multiple strains in the Gibberella fujikuroi species complex (GFC), is well known for its production of diverse secondary metabolites. F. fujikuroi, associated with the “bakanae” disease of rice, is an active producer of gibberellins (GAs), a wide class of plant hormones. In addition to some members of the GFC, the GA biosynthetic gene cluster, or parts of it, occurs also in some isolates of the closely related species of F. oxysporum, which does not belong to the GFC. However, production of GAs has never been observed in any F. oxysporum strain. In this study, we report on the GA biosynthetic activity in an orchid-associated F. oxysporum strain by transforming a cosmid with the entire F. fujikuroi GA gene cluster. Southern and Northern blot analyses confirmed not only the integration of the entire gene cluster into the genome but also the active expression of the seven GA biosynthetic genes under nitrogen-limiting conditions. The transformants produced GAs at levels similar to those of F. fujikuroi. These data show that the regulatory network for expression of GA genes is fully active in the F. oxysporum background.     

Gibberellin biosynthesis and gibberellin oxidase activities in Fusarium sacchari,Fusarium konzum and Fusarium subglutinans strains

Several isolates of three Fusarium species associated with the Gibberella fujikuroi species complex were characterized for their ability to synthesize gibberellins (GAs): Fusarium sacchari (mating population B), Fusarium konzum (mating population I) and Fusarium subglutinans (mating population E). Of these, F. sacchari is phylogenetically related to Fusarium fujikuroi and is grouped in the Asian clade of the complex, while F. konzum and F. subglutinans are only distantly related to Fusarium fujikuroi and belong to the American clade. Variability was found between the different F. sacchari strains tested. Five isolates (B-12756; B-1732, B-7610, B-1721 and B-1797) were active in GA biosynthesis and accumulated GA₃ in the culture fluid (2.76–28.4 μg/mL), while two others (B-3828 and B-1725) were inactive. GA₃ levels in strain B-12756 increased by 2.9 times upon complementation with ggs2 and cps-ks genes from F. fujikuroi. Of six F. konzum isolates tested, three (I-10653; I-11616; I-11893) synthesized GAs, mainly GA₁, at a low level (less than 0.1 μg/mL). Non-producing F. konzum strains contained no GA oxidase activities as found for the two F. subglutinans strains tested. These results indicate that the ability to produce GAs is present in other species of the G. fujikuroi complex beside F. fujikuroi, but might differ significantly in different isolates of the same species.     

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.     

Regeneration of plants from Ipomoea cairica L. protoplasts and production of somatic hybrids between I. cairica L. and sweetpotato, I. batatas (L.) Lam.

Plants were regenerated from mesophyll protoplasts of Ipomoea cairica L., a wild relative of sweetpotato (Ipomoea batatas (L.) Lam.), and somatic hybrids between I. cairica L. and sweetpotato cv. Xushu 18 were obtained by PEG-mediated method. I. cairica L. protoplasts were isolated from the leaves of in vitro grown plants and cultured in a modified MS medium containing 0.05 mg l⁻¹ 2,4-D and 0.5 mg l⁻¹ kinetin. Nine weeks after plating, the obtained small calluses up to about 2 mm in diameter were transferred to solid MS medium supplemented with 0.05 mg l⁻¹ 2,4-D and 0.5 mg l⁻¹ kinetin for callus proliferation. Three weeks after transfer, the calluses were transferred to MS medium supplemented with 0–1.0 mg l⁻¹ IAA and 1.0–3.0 mg l⁻¹ BAP and further to hormone-free MS medium for plant regeneration. The frequencies of calluses forming plants ranged from 6.0% to 41.3% based on the different concentrations of IAA and BAP, and 2.0 mg l⁻¹ BAP gave the highest regeneration frequency of protoplast-derived calluses in I. cairica L.. The regenerated plants, when transferred to soil, showed 100% survival. No morphological variations were observed. Mesophyll protoplasts of I. cairica L. were fused with protoplasts isolated from embryogenic suspension cultures of Xushu 18 by PEG-mediated method. The fused products were cultured with the best protoplast culture system of I. cairica L.. Finally, 114 plants were produced from 63 of the 182 calluses derived from the fused protoplasts, and 46 plants of them were confirmed to be somatic hybrids through peroxidase isozyme, RAPD, morphological and cytological analyses.     

Medium pH in submerged cultivation modulates differences in the intracellular protein profile of Fusarium oxysporum

Fusarium oxysporum is a filamentous fungus that damages a wide range of plants and thus causes severe crop losses. In fungal pathogens, the genes and proteins involved in virulence are known to be controlled by environmental pH. Here, we report the influence of culture-medium pH (5, 6, 7, and 8) on the production of degradative enzymes involved in the pathogenesis of F. oxysporum URM 7401 and on the 2D-electrophoresis profile of intracellular proteins in this fungus. F. oxysporum URM 7401 was grown in acidic, neutral, and alkaline culture media in a submerged bioprocess. After 96?hr, the crude extract was processed to enzyme activity assays, while the intracellular proteins were obtained from mycelium and analyzed using 2D electrophoresis and mass spectrometry. We note that the diversity of secreted enzymes was changed quantitatively in different culture-medium pH. Also, the highest accumulated biomass and the intracellular protein profile of F. oxysporum URM 7401 indicate an increase in metabolism in neutral–alkaline conditions. The differential profiles of secreted enzymes and intracellular proteins under the evaluated conditions indicate that the global protein content in F. oxysporum URM 7401 is modulated by extracellular pH.     

Biomass accumulation and shading effects of epiphytes on leaves of the seagrass, Heterozostera tasmanica, in Victoria,Australia

A method is described for estimating the rate of accumulation of epiphyte biomass on leaves of the seagrass, Heterozostera tasmanica (Martens ex Aschers.) den Hartog and for estimating the effect of epiphyte biomass on photosynthesis of the seagrass. Epiphyte biomass was determined by comparison of the weight per unit area of epiphyte-covered and epiphyte-free leaf blades. Epiphyte weight increased as age of the seagrass leaves increased. Linear regression on epiphyte biomass vs. leaf age estimated the rate of biomass accumulation. Rates varied from 5.7 to 104 μg epiphyte dry weight per cm² of leaf surface per day at three sites in Western Port and Port Phillip Bay, Victoria. Rates of accumulation of epiphyte biomass were generally higher during December through March (summer) than in May (autumn), August (winter) or October (Spring). Light attenuation by epiphytes increase linearly with biomass. The rate of biomass accumulation of epiphytes was compared with leaf growth rate, ambient photon flux density in H. tasmanica beds and the photosynthesis—photon flux density curve of H. tasmanica. This comparison demonstrated that epiphyte biomass can accumulate fast enough to shade H. tasmanica leaves and significantly reduce the time (to less than one half of the leaf life span) in which positive net photosynthesis of the leaf blade is possible.     

Fusaric acid contributes to virulence of Fusarium oxysporum on plant and mammalian hosts

Fusaric acid (FA) is amongst the oldest identified secondary metabolites produced by Fusarium species, known for a long time to display strong phytotoxicity and moderate toxicity to animal cells; however, the cellular targets of FA and its function in fungal pathogenicity remain unknown. Here, we investigated the role of FA in Fusarium oxysporum, a soil‐borne cross‐kingdom pathogen that causes vascular wilt on more than 100 plant species and opportunistic infections in humans. Targeted deletion of fub1, encoding a predicted orthologue of the polyketide synthase involved in FA biosynthesis in F. verticillioides and F. fujikuroi, abolished the production of FA and its derivatives in F. oxysporum. We further showed that the expression of fub1 was positively controlled by the master regulator of secondary metabolism LaeA and the alkaline pH regulator PacC through the modulation of chromatin accessibility at the fub1 locus. FA exhibited strong phytotoxicity on tomato plants, which was rescued by the exogenous supply of copper, iron or zinc, suggesting a possible function of FA as a chelating agent of these metal ions. Importantly, the severity of vascular wilt symptoms on tomato plants and the mortality of immunosuppressed mice were significantly reduced in fub1Δ mutants and fully restored in the complemented strains. Collectively, these results provide new insights into the regulation and mode of action of FA, as well as on the function of this phytotoxin during the infection process of F. oxysporum.     

Antagonistic effects of soil bacteria onFusarium oxysporum Schlecht f.sp.dianthii (Prill and Del.) Snyd. and Hans.

Summary Thirty two bacteria antagonistic to a number of phytopathogenic fungi were isolated from soil samples. One bacterial strain, designated as M 51, appeared to be particularly active towardsF. oxysporum f. sp.dianthii, in vitro andin vivo and it was inhibitoryin vitro to three otherFusarium spp. used. Tests to find if there was protection against fusarium wilt were carried out by three different methods of inoculation of the cuttings: a) dipping of cuttings for ten minutes in bacterial suspension; b) spraying of suspension on perlite where the rooted cuttings were planted; c) spraying the greenhouse bench rooting boxes, where the non-rooted cuttings were planted, with bacterial suspension. Following this all the cuttings were transplanted into soil naturally highly infested withFusarium oxysporum f. sp.dianthii (3000 units/g). Good protection against fusarium wilt was obtained for cuttings inoculated by method (b). However protection decreased gradually about 60 days after they were transplanted; both control and inoculated cuttings showed a comparable mortality rate. Method of inoculation and the development of the protective effect are discussed.     

Effect of nutrient availability, grazer assemblage and seagrass source population on the interaction between Thalassia testudinum (turtle grass) and its algal epiphytes

Seagrass leaves are often densely covered by epiphytic algae which can suppress seagrass productivity and has been implicated in declines of seagrass meadows worldwide. The net effect of epiphytes on seagrass growth and morphology depends on the independent and interactive effects of a variety of factors, including nutrient availability and the intensity of grazing on epiphytes. Here I report the results of a mesocosm experiment designed to test the effects of nutrient addition and within-functional group variation (grazer species composition and the source population of seagrass) on the strength of the interactions among grazers, epiphytes, and turtle grass (Thalassia testudinum). Turtle grass ramets from two sites in the northern Gulf of Mexico were cleared of epiphytes and transplanted into common-garden mesocosms. Replicate ramets were grown in a split-split plot design with two levels of dissolved nutrients and four different grazer species combinations (Tozeuma carolinense alone, Pagurus maclaughlinae alone, both species together, and no grazers present). As expected, grazers had a significant negative effect on epiphyte biomass/leaf area and a significant positive effect on turtle grass growth in the mesocosms. The two species were more similar in their direct effects on epiphyte biomass than in their indirect effects on turtle grass growth; this may reflect differences in epiphyte community composition under different grazer treatments. The effect of nutrient addition on turtle grass growth depended critically on the intensity of grazing: in the presence of grazers, turtle grass tended to produce a greater biomass of new leaf tissue in the tanks with nutrients added than in the control tanks. However, when grazers were absent, the direction of the effect was reversed, and plants with nutrients added grew less than the control plants. The two source populations of turtle grass differed significantly in epiphyte biomass/leaf area accrued in the mesocosms as well as in the strength of the effect of grazers on turtle grass growth. This suggests that population differentiation in seagrass interactions with epiphytes, as well as spatial and temporal variation in resources and grazer community composition, can greatly effect the role of epiphytes in limiting seagrass productivity.     

Effects of Auxin and Gibberellin on Conidial Germination and Elongation of Young Hyphae in Gibberella fujikuroi and Penicillium notatum

In Gibberella fujikuroi and Penicillium notatum, IAA, 2,4-Dand GA₃ promoted conidial germination and the elongation ofyoung hyphae. The promotive effects of IAA and GA₃ were additive.In both fungi, the concentrations of endogenous auxin and gibberellinin the culture media were 10^–10 to 610^–12M. (Received April 27, 1985; Accepted August 12, 1985)     

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).     

Influence of Endotrophic Mycorrhizae on the Fusarial Wilt of Cassia tora L.

The wilting of Cassia tora caused by Fusarium oxysporum was reduced significantly when inoculated with VAM. Mycorrhizal fungi highly influenced growth stimulation of the seedlings. The population of F. oxysporum was reduced in the presence of VAM.