Fusarium udum parasitic onAspergillus luchuensis andSyncephalastrum racemosum

Summary Mycoparasitic behaviour ofF. udum on two soil-inhabiting microfungi,Aspergillus luchuensis andSyncephalastrum racemosum, was studied in dual cultures.Fusarium udum coiled and penetrated the host fungi and formed chlamydospores inside their hyphae and reproductive structures. The vegetative hyphae ofA. luchuensis showed swellings due to diffusible toxic substances ofF. udum in the medium and formed vesicle-like structures. This is the first record of these fungi being hosts of the mycoparasiteF. udum.     

The infection process ofFusarium oxysporum in cotton root tips

Summary Conidia ofFusarium oxysporum f. sp.vasinfectum started to germinate on the roots of cotton (Gossypium barbadense L.) 6 h after inoculation and formed a compact mycelium covering the root surface. 18 h later, penetration hyphae branched off and infected the root. The number of penetration hyphae increased with the number of conidia used for inoculation. The optimal temperature for penetration was between 28 and 30 °C. The highest numbers of penetration hyphae were found in the meristematic zone, 40 percent less in the elongation and root hair zones, and none in the lateral root zone. The fine structure of the infection process was studied in protodermal cells of the meristematic zone and in rhizodermal cells of the elongation zone. The penetration hyphae were well preserved after freeze substitution and showed a Golgi equivalent consisting of three populations of smooth cisternae. Plant reactions were found already during fungal growth on the root surface. In the meristematic zone, a thickening of the plant cell wall due to an apposition of dark and lightly staining material below the hyphae occurred. This wall apposition increased in size around the hypha invading the plant cell and led to the formation of a prominent wall apposition with finger-like projections into the host cytoplasm. In the elongation zone, the deposits around the penetration hypha appeared less thick and the dark inclusions were less pronounced. High pressure freezing of infected cells revealed, thatF. oxysporum penetrates and grows within the host cells without inducing damages such as plasmolysis, cell degeneration or even host necrosis. We suggest thatF. oxysporum has an endophytic or biotrophic phase during colonization of the root tips.Abbreviation Ph penetration hyphae     

Formation of resting body byCephalosporium roseo-griseum Saksena inside the conidiophore ofAspergillus flavus Link. Ex Fries during mycoparasitism

Summary Mycoparasitic behaviour ofCephalosporium roseo-griseum Saksena withAspergillus flavus Link. ex Fries was studiedin vitro. Frequent coiling of hyphae ofC. roseo-griseum around the conidiophores ofA. flavus, penetration of hyphae ofC. roseo-griseum inside those ofA. flavus, and formation of resting bodies byC. roseo-griseum in the conidiophores ofA. flavus were observed.     

Purification and characterisation of NAD+-dependent xylitol dehydrogenase from Fusarium oxysporum

An NAD⁺-dependent xylitol dehydrogenase (XDH) from Fusarium oxysporum, a key enzyme in the conversion of xylose to ethanol, was purified to homogeneity and characterised. It was homodimeric with a subunit of M _r 48 000, and pI 3.6. It was optimally active at 45 °C and pH 9–10. It was fully stable at pH 6–7 for 24 h and 30 °C. K _m values for d-xylitol and NAD⁺ were 94 mM and 0.14 mM, respectively. Mn²⁺ at 10 mM increased XDH activity 2-fold and Cu²⁺ at 10 mM inhibited activity completely.     

Lysis of mycelium ofFusarium oxysporum f. sp.udum in soil amended with organic matters

Summary Autoclaved or natural field soil amended with 0.1 to 5.0 per cent (W/W) of margosa cake, rice husk and sawdust with or without supplemental nitrogen were tested for lytic activity and bacterial numbers. Generally, non-amended autoclaved soil caused little or no lysis of mycelium ofF. oxysporum f. sp.udum; non-amended natural soil caused more lysis. Amendment of soil with margosa cake, rice husk or saw-dust with or without supplemental nitrogen greatly enhanced its lytic effect on the fungus. The degree of lysis depended on the dosage of amendment used and the stage of its decomposition in the soil. The extent of lysis increased as the bacterial population increased. Amongst bacteria,Bacillus subtilis was very common in most lytic zones.     

Production and partial characterization of alkaline feruloyl esterases by Fusarium oxysporum during submerged batch cultivation

Production of feruloyl esterases (FAEs) by Fusarium oxysporum was enhanced by optimization of initial pH of the culture medium, the type and concentration of nitrogen and carbon source. Submerged batch cultivation in a laboratory bioreactor (17 l) produced activity at 82 nkat g⁻¹ dry substrate (corn cobs) which compared favorably to those reported for the other microorganisms. Use of de-esterified corn cobs as carbon source decreased FAE production by 5.5-fold compared to untreated corn cobs even though ferulic acid (FA) was added to the concentration found in alkali-extracts of corn cobs. Production of FAE does not therefore, require FA, however, production is diminished by the removal of esterified FA from the growth substrate. Optimal FAE activity was observed at pH 7 and 50 °C with 68 and 55% activity at pH 8 and pH 9, respectively. The esterase was fully stable at pH 5–8 and up to 40 °C and retained 72 and 40% of its activity after 6 h at pH 9 and pH 10, respectively. After separation by isoelectric focusing electrophoresis, a zymogram indicated one major FAE activity exhibiting pI value of 10.5. This revised version was published online in August 2006 with corrections to the Cover Date.     

Occurrence and toxicity ofFusarium subglutinans from Peruvian maize

Twenty-five samples of maize kernels collected at harvest time from geographically different corn fields in Peru, were examined for the occurrence of toxigenicFusarium species. The most frequently recovered species wereF. subglutinans (48%),F. moniliforme (46%), andF. equiseti (5%). OtherFusarium species isolated (up to 1%) includedF. graminearum, F. acuminatum, F. solani, F. oxysporum, andF. culmorum. Assays ofFusarium culture extracts usingArtemia salina larvae, showedF. subglutinans as one of the most toxigenic species, and its toxicity was mostly correlated to the capability to produce beauvericin (BEA). All eight tested isolates ofF. subglutinans grown on autoclaved corn kernels produced BEA (from 50 to 250 mg/Kg) as well as moniliformin (M) (from 70 to 270 mg/Kg). This is the first report on BEA and M production by maize isolates ofF. subglutinans from South America.     

Uptake of protoplasts from the filamentous fungiAspergillus nidulans andFusarium oxysporum into protoplasts from celery (Apium graveolens L.)

Summary Protoplasts isolated from celery cell suspension cultures, were mixed with fungal protoplasts, from either the saprophytic speciesAspergillus nidulans or the pathogenic speciesFusarium oxysporum. The incubation of protoplast mixtures with PEG caused close adhesion between plant and fungal protoplasts. Subsequent dilution of PEG resulted in the uptake of protoplasts from either fungal species into the plant protoplast cytoplasm. A range of PEG concentrations, incubation times and dilution rates were tested to maximise adhesion and uptake frequencies. Identification of uptake was achieved either by fluorescent staining of nuclei or by electron-microscopy. A maximum of 10% celery protoplasts had taken upA. nidulans protoplasts after PEG treatment. Fungal protoplasts were taken up into celery protoplast cytoplasm by endocytosis, and were maintained within vesicles; two bounding membranes were observed by electron microscopy. Plant protoplast viability was determined during prolonged incubation following fungal protoplast uptake. The presence ofA. nidulans protoplasts tended to maintain celery protoplast viability and although some morphological disintegration occurred intact celery protoplasts remained for at least 92 h after uptake. The uptake ofF. oxysporum protoplasts markedly depressed celery protoplast viability after 24 h incubation and greater celery protoplast disintegration occurred.Abbreviations PEG Polyethylene glycol - DAPI 4,6-diaminido-2-phenylindole - 2,4-D 2,4-dichlorophenoxyacetic acid     

The possible involvement of peroxidase in defense of yellow lupine embryo axes against Fusarium oxysporum

Peroxidase activity (EC 1.11.1.7) towards phenolic substrates, i.e. pyrogallol, syringaldazine and guaiacol, and ascorbate peroxidase activity (EC 1.11.1.11) were analyzed in embryo axes of Lupinus luteus L. cv. Polo cultured on Heller medium for 96h after inoculation with the necrotrophic fungus Fusarium oxysporum f.sp. Schlecht lupini. Four variants were compared: inoculated embryo axes cultured with 60mM sucrose (+Si) or without it (-Si), and non-inoculated embryo axes cultured with 60mM sucrose (+Sn) or without it (-Sn). Between 0 and 96h of culture, peroxidase activity towards the phenolic substrates increased in all variants except -Si, where a decrease was noted in peroxidase activity towards syringaldazine and guaiacol, but not towards pyrogallol. In +Si tissues, a considerable increase in enzyme activity towards these substrates was recorded starting from 72h of culture. Lignin content of +Si tissues increased already at the first stage of infection, i.e. 24h after inoculation. Additionally, in +Sn tissues, high ascorbate peroxidase activity was observed during the culture. Its activity increased in +Si tissues, beginning at 72h after inoculation. However, this was lower than in +Sn tissues. At 72h after inoculation, a considerably stronger development of the infection was observed in -Si than in +Si tissues during our earlier research [Morkunas, I. et al., 2005. Sucrose-stimulated accumulation of isoflavonoids as a defense response of lupine to Fusarium oxysporum. Plant Physiol Biochem 2005; 43: 363-73]. Both peroxidases assayed towards phenolic substrates and ascorbate peroxidase was less active in -Si tissues than in -Sn tissues. Hydrogen peroxide concentration was much higher in -Si than in +Si tissues. These results indicate that peroxidases may be some of the elements of the defense system that are stimulated by sucrose in yellow lupine embryo axes in response to infection caused by F. oxysporum.     

In vitro selection of alfalfa plants resistant to Fusarium oxysporum f. sp. medicaginis

Summary From two lines of Medicago sativa characterized by a high regeneration capability, calli resistant to culture filtrate of Fusarium oxysporum f. sp. medicaginis have been selected. In these calli regeneration capability was greatly reduced and only one plant per callus was recovered. Regenerated plants have been evaluated for resistance to culture filtrate and for in vivo resistance to the pathogen. Three plants out of eight were resistant to the fungus and a high correlation between resistance to culture filtrate and in vivo resistance was observed.Research work supported by C.N.R., Italy. Special grant I.P.R.A. Subproject 1, paper no. 1468     

Isolation and identification of an endophytic strain of Fusarium oxysporum producing podophyllotoxin from Juniperus recurva

Podophyllotoxin, a well-known naturally occurring aryltetralin lignan occurs in few plant species that is used as a precursor for the chemical synthesis of the anticancer drugs like etoposide, teniposide and etopophose phosphate. The availiability of this lignan is becoming increasingly limited because of the scarce occurance of its natural sources and also because synthetic approaches for its production are still commercially unacceptable. This paper reports first time the production of podophyllotoxin by an endophytic fungus Fusarium oxysporum isolated from the medicinal plant Juniperus recurva. Further confirmation and quantification of podophyllotoxin was performed by HPLC, LC-MS, and LC-MS/MS.     

Influence of Plant Root Exudates, Germ Tube Orientation and Passive Conidia Transport on Biological Control of Fusarium Wilt by Strains of Nonpathogenic Fusarium oxysporum

In earlier studies, biological control of Fusarium wilt of cucumber induced by Fusarium oxysporum f. sp. cucumerinum was demonstrated using nonpathogenic strains C5 and C14 of Fusarium oxysporum. Strain C14 induced resistance and competed for infection sites whether roots were wounded or intact, whereas strain C5 required wounds to achieve biocontrol. In the current work, additional attributes involved in enhanced resistance by nonpathogenic biocontrol agents strains to Fusarium wilt of cucumber and pea were further investigated. In pre-penetration assays, pathogenic formae specials exhibited a significantly higher percentage of spore germination in 4-day-old root exudates of cucumber and pea than nonpathogens. Also, strain C5 exhibited the lowest significant reduction in spore germination in contrast to strain C14 or control. One-day-old cucumber roots injected with strain C14 resulted in significant reduction in germ tube orientation towards the root surface, 48–96 h after inoculation with F. o. cucumerinum spores, whereas strain C5 induced significantly lower spore orientation of the pathogen and only at 72 and 96 h after inoculation. In post-penetration tests, passive transport of microconidia of pathogenic and nonpathogens in stems from base to apex were examined when severed plant roots were immersed in spore suspension. In repeated trials, strain C5, F. o. cucumerinum and F. o. pisi were consistently isolated from stem tissues of both cucumber and pea at increasing heights over a 17 days incubation period. Strain C14 however, was recovered at a maximum translocation distance of 4.6 cm at day 6 and later height of isolation significantly declined thereafter to 1.2 cm at day 17. In pea stem, the decline was even less. Significant induction of resistance to challenge inoculation by the pathogen in cucumber occurred 72 and 96 h after pre-inoculation with biocontrol agents. Nonetheless, strain C14 induced protection as early as 48 h and the maximum resistance was reached at 96 h. The presented data confirm the previous findings that attributes important for nonpathogenic fusaria to induce resistant are: rapid spore germination and orientation in response to root exudate; active root penetration and passive conidia transport in stem to initiate defence reaction without pathogenicity and enough lag period between induction and challenge inoculation. Strain C14 possesses all these qualifications and hence its ability to enhance host resistance is superior than strain C5.     

Differentiation ofFusarium sambucinum Fuckel sensu lato and related species by RAPD PCR

DNA polymorphisms generated by the random amplified polymorphic DNA polymerase chain reaction (RAPD PCR) were used to analyse 41 isolates investigated in the EuropeanFusarium sambucinum Project (EFSP). Employing ten arbitrary (10-mer) oligonucleotides and simple repeat sequences (M13, (GACA)₄) as single primers, informative banding patterns typical for identifying European populations ofFusarium sambucinum Fuckel s. str.,F. torulosum (Berk. & Curt.) Nirenberg andF. venenatum Nirenberg were obtained.     

The effects of Penicillium digitatum and Fusarium oxysporum rots on nutritional content of pawpaw (Carica papaya L.)

Investigations were carried out on the effects of Penicillium digitatum and Fusarium oxysporum on the nutritional value of pawpaw (Carica papaya). Decreases were observed in ash content, phosphorus, sodium, reducing sugars and ascorbic acid levels of fruits infected with P. digitatum, but increases in calcium and potassium content. In fruits infected by F. oxysporium, there were decreases in phosphorus, calcium, sodium ascorbic acid and reducing sugar levels; but the levels of ash content increased. The total protein level increased in the fruits infected with both fungi. These results revealed a reduction in fruit quality.     

An RFLP marker in tomato linked to the Fusarium oxysporum resistance gene I2

Summary The locus, I2, which in tomato confers resistance against Fusarium oxysporum f. sp. lycopersici race 2, was introgressed into Lycopersicon esculentum from the wild species L. pimpinellifolium (P.I. 126915). We searched for restriction fragment length polymorphisms (RFLPs) between nearly isogenic lines (NILs) in clones that map to the region introgressed from the wild species. Since I2 maps to chromosome 11, we used DNA clones from this chromosome as hybridization probes to Southern blots containing bound DNA of the NILs digested with 23 restriction enzymes. Of the 14 chromosome 11 clones, 9 exhibited polymorphism. These clones were further hybridized to verification filters that contained DNA from resistant and susceptible L. esculentum varieties digested with the enzymes that gave the polymorphism. One clone, TG105, was found to be associated with I2; 19 susceptible lines showed a different RFLP with this probe than 16 resistant lines, including the original L. pimpinellifolium accession used as a source for the resistance gene. These results together with our mapping analysis indicate that TG105 is closely linked to the resistance gene.     

Effect of Fusarium oxysporum f. sp. glycines and Sclerotium rolfsii on the pathogenicity of Meloidogyne incognita race 2 to soybean

Screenhouse studies were conducted to investigate the effects of Fusarium oxysporum f. sp. glycines and Sclerotium rolfsii on the pathogenicity of Meloidogyne incognita race 2 on soybean and the influence of the nematode on wilt incidence and growth of soybean. The interaction of each fungus with the nematode resulted in reduced shoot and root growth. Final nematode population was also reduced with concomitant inoculation of nematode and fungus or inoculation of fungus before nematode. While M. incognita suppressed wilt incidence in two nematode-susceptible cultivars of soybean (TGX 1485-2D and TGX 1440-IE), it had limited effect on wilt incidence in the nematode resistant cultivar of soybean (TGX 1448-2E). When F. oxysporumwas inoculated with the nematode, the mean number of nematodes that penetrated soybean roots decreased by 75% in TGX 1448-2E, 68% in TGX 1485-1D and 65% in TGX 1440-1E. Similarly when the soil was treated with S. rolfsii, the number decreased by 78% in TGX 1448-2E, 77% in TGX 1485-1D and 68% in TGX 1440-1E. The nematode did not develop beyond second-stage juvenile in TGX-1448-2E.     

Two granular formulations of Fusarium oxysporum f.sp. orthoceras to mitigate sunflower broomrape Orobanche cumana

Fusarium oxysporum Schlecht. f.sp. orthoceras (Appel & Wollenw.) Bilai, a potential biocontrol agent against Orobanche cumana Wallr.,was formulated into two granular forms, wheatflour kaolin (`Pesta') granules and sodium alginatepellets. The formulations were compared in terms ofeffectiveness for mitigating O. cumanaparasitism in sunflower and shelf-life forstorage. `Pesta' granules reduced the emergence of O. cumana shoots by 64% while sodium alginatepellets did not reduce the emergence rate but increased thepercentage of diseased O. cumana plants.Calculated efficacy of the application was better for`Pesta' granules. Viability of the formulatedmaterial tested in the laboratory was higher in sodium alginatepellets than in the `Pesta' formulation.However, a loss of virulence after six months of storage wasalso observed in sodium alginate pellets in agreenhouse experiment.     

Effects of supplementary UV-B radiation on development of damping-off in spinach caused by the soil-borne fungusFusarium oxysporum

The effects of UV-B radiation (290–320 nm) on development of damping-off of spinach (Spinacia oleracea) caused by the fungusFusarium oxysporum were examined in a growth cabinet. The incidence of disease greatly increased when experimental plants were grown in visible radiation with supplementary UV-B radiation. This increase was suppressed by increasing the irradiation of visible radiation.Fusarium oxysporum was isolated from the roots of all damping-off plants and the roots of some unwilted plants, indicating that spinach infected with the pathogen did not necessarily suffer from damping-off in 15d. Supplementary UV-B radiation suppressed the increase in growth components such as the number of leaves, the plant height and the fresh weight of aboveground plant parts, but did not affect the fresh weight of roots. The ratio of the number of plants infected with pathogen to the total number of plants was over 80% irrespective of light conditions. It was suggested that the defense response of spinach to this pathogen was greatly influenced by the physiological state of aboveground plant parts resulting from supplementary UV-B radiation.