Production of Pectic Enzymes by Fusarium oxysporum f. sp. cepae and its Involvement in Onion Bulb Rot

Fusarium oxysporum f. sp. cepae produced an exo-polygalacturonase (exo-PG) and endopectin-trans-eliminase (endo-PTE) in a mineral medium supplemented with a restricted supply of either D-galacturonic acid or onion cell walls. These enzymes were also extracted from infected onion tissue, but only endo-PTE caused tissue maceration and cell death. The patterns of host tissue colonization and pectic enzyme production were followed during bulb rot development. Stem plates were invaded within two weeks of inoculation. The pathogen then remained confined to the stem plates for several weeks or months, before spreading to the outer fleshy scales to initiate a basal rot. In most cases the inner leaf sheaths containing the lateral bud remained healthy. Exo-PG activity m stem plate tissue was greatest at two weeks after inoculation, then it declined. Endo-PTE was not detected in newly invaded stem plate tissue, but was recovered from infected stem plates before decay and from the bases of bulb scales and leaf sheaths at the onset of bulb rot. There was no pectic enzyme activity in uninvaded onion tissue. Spread of the fungus and pectic enzyme production in two Caledon Globe genotypes susceptible or tolerant to F. oxysporum f. sp. cepae were similar, but the onset of bulb rot in tolerant genotypes was considerably delayed.     

Fungi associated with storage rots of cocoyams (Colocasia spp.) in Nsukka,Nigeria

Cocoyam (Colocasia spp.) corms and cormels showing spoilage symptoms were collected from many stores in Nsukka locality and examined for rot and associated fungal pathogens. Aspergillus niger, Botryodiplodia theobromae, Corticium rolfsii, Geotrichum candidum, Fusarium oxysporum, and F. solani were recovered from rotten cocoyams. The representative isolates of these species caused cocoyam rot in pathogenicity tests. The rot due to A. niger, B. theobromae and C. rolfsii was extensive resulting in complete maceration of cocoyam tissue. Potassium sorbate (0.1 mg/ml) protected cocoyams from fungal rot with the exception of C. rolfsii.     

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.     

THE ENZYMATIC MACERATION OF PLANT TISSUES: OBSERVATIONS USING A NEW METHOD OF MEASUREMENT

Mc Clendon , J. H., and G. F. Somers . (U. Delaware, Newark.) The enzymatic maceration of plant tissues: observations using a new method of measurement. Amer. Jour. Bot. 47(1) :1-7. Illus. 1960.—An apparatus is described for measuring the breaking strength of tissue slices. The apparatus was used in the measurement of maceration of potato tuber slices by fungal and tomato enzymes. During the enzymatic maceration of the slices, the strength fell in an approximately logarithmic manner to a stable value less than 5% of the initial strength. Calcium ion did not prevent enzymatic maceration, although it increased the strength of the tissue. Chelating agents used alone did not macerate but facilitated the enzymatic maceration. There was a pH optimum at 3.0—3.5 with a commercial “Pectinase” and enzymes from Botryosphaeria ribis but near 4.7 for a preparation from tomato fruit. The reciprocal of the time for a set strength reduction was proportional to the square root of the enzyme concentration. The relative strength remaining [(initial strength/final strength)–1] after an arbitrary reaction time was proportional to the enzyme concentration raised to the 0.8 power. The temperature coefficient was about 2.5, but other evidence indicated some limitation by diffusion. Non-enzymatic maceration increased rapidly below pH 3 and was especially prominent after subsequent neutralization.     

On Growth-inhibition against Aspergillus oryzae No 40 by the Compound III-3 in Rice Grain

Partially purified lipoxygenase (LOX) extracts were obtained from Fusarium proliferatum, Fusarium oxysporum, Chlorella pyrenoidosa, and Saccharomyces cerevisiae; the enzymatic extract of F. proliferatum showed the highest LOX activity while those of F. oxysporum and S. cerevisiae demonstrated only 27.8 and 16.5% of the activity at pH 8.0, and 61.2 and 9.7% of the enzyme activity at pH 10.0, respectively. The lowest LOX activity was that in the C. pyrenoidosa extract. The microbial enzymatic preparations were assayed with linoleic acid as substrate, which was bioconverted into 9- and 13-hydroperoxides (HPODEs) by all four extracts; in additon, the LOX activity in the F. oxysporum extract produced the 10- and 12-HPODEs from linoleic acid while that of the C. pyrenoidosa extract produced only the 10- HPODE. When assayed with the 9- and 13-HPODEs as substrates, the selected microbial extracts had secondary enzyme activities, one of which produced hexanal. The highest hexanal-producing activity was 1.51 and 1.39 nmol hexanol/mg protein/min in the F. proliferatum and C. pyrenoidosa extracts, respectively, while those of F. oxysporum and S. cerevisiae had approximately 15% of the HPODE-cleaving enzyme activity. The C. pyrenoidosa extract had the highest proportion of pentanone, which was produced at only one-fourth the concentration by the HPODE-cleaving enzyme activity in the three other microbial enzymatic extracts.     

Production of ethanol from sugars in wood hydrolysate byFusarium oxysporum

Wood hydrolysate used for ethanol production by two strains ofFusarium oxysporum contained 2.3% (w/v) reducing sugars (xylose and glucose). Ethanol production at the optimum reducing sugar concentration of 54.8 g/l medium, at pH 5.5, and 30°C was 12.3 g/l and 11.7 g/l byF. oxysporum D-140 and NCIM-1072, respectively in shake flasks during 96 h fermentation. The maximum production of ethanol under optimum cultural conditions, and in the presence of yeast extract plus minerals, was 13.2 g/l medium byF. oxysporum D-140 over 108 h fermentation.

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Résumé L'hydrolysat de bois utilisé pour la production d'éthanol par deux souches deFusarium oxysporum contenait 2.3% (poids/vol.) de sucres réducteurs (xylose et glucose). La production d'éthanol, à la concentration optimum en sucres réducleurs de 54.8 g par litre de milieu à pH 5.5 et à 30°C était de 12.3 g/l et 11.7 g/l respectivement chezF. oxysporum D-140 et NCIM-1072, en flacons agités pendant 96 h de fermentation. La production maximum d'éthanol, dans les conditions optimum de culture, et en prosence d'extrait de levure et de minéraux a mit de 13.2 g par litre de milieu chezF. oxysporum D-140 en 108 h de lermentation.

     

Effects of treatment with phenylthiosemicarbazide and 4'-chlorophenylthiosemicarbazide on Fusarium wilt of pea and tomato plants

Effects of treatment with phenylthiosemicarbazide (PTS) and its 4′-chloro-derivative (4′-chloro-PTS) on Fusarium wilt of pea and tomato plants were investigated. Depending on pH and availability of oxygen, PTS and 4′-chloro-PTS are converted to their corresponding phenylazothioformamides and phenylazothioformamide-S-oxides, which are the actual fungitoxic compounds. PTS and 4′-chloro-PTS were shown to inhibit growth of Fusarium oxysporum f. pisi and F. oxysporum f. lycopersici in liquid media as well as on agar plates at concentrations of 50–100 mg/1. Inhibition was greater at pH 7 than at pH 5. When administered to pea and tomato plants, both compounds caused severe phytotoxic effects, especially at temperatures favouring Fusarium wilt, thus almost entirely obscuring any protective activity against the diseases. All compounds were strongly adsorbed to loam, but readily released from sand. Neither in pea nor in tomato plants were PTS and 4′-chloro-PTS converted to any fungitoxic substance, not already present in the aqueous solutions administered.     

Comparative studies on the effect of vitamins on sporulation in Fusarium oxysporum schlecht. ex. Fr. and Fusarium moniliforme V. subglutinans Wr. & Rg.

On the basis of sporulation (total output of all the three spore forms taken together) and fungal mat production bothF. oxysporum Schlecht ex.Fr. andF. moniliforme v.subglutinans Wr. &Rg. are auxoheterotrophic for thiamine, biotin, inositol, riboflavin and pyridoxine. The first three vitamins are selective in accelerating macro-conidial production also inF. moniliforme, which otherwise shows decrease with advance in days of incubation.F. moniliforme is an auxo-autotroph for nicotinic acid, Ca-pantothenate and folic acid and auxoheterotroph for ascorbic acid. Auxoautotrophy for Ca-pantothenate, folic acid, l-ascorbic acid and p-aminobenzoic acid cannot be suggested forF. oxysporum. Whereas nicotinic acid is a depressent of sporulation inF. oxysporum, inF. moniliforme another vitamin p-aminobenzoic acid depresses sporulation. As the two species ofFusarium show differences in preference as well as inhibition to at least five of the vitamins studied and also varied trends of pH changes exists there is full justification for their separate taxonomic placements.     

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.     

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.     

Screenhouse and Field Persistence of Nonpathogenic Endophytic Fusarium oxysporum in Musa Tissue Culture Plants

Two major biotic constraints to highland cooking banana (Musa spp., genome group AAA-EA) production in Uganda are the banana weevil Cosmopolites sordidus and the burrowing nematode Radopholus similis. Endophytic Fusarium oxysporum strains inoculated into tissue culture banana plantlets have shown control of the banana weevil and the nematode. We conducted screenhouse and field experiments to investigate persistence in the roots and rhizome of two endophytic Fusarium oxysporum strains, V2w2 and III4w1, inoculated into tissue-culture banana plantlets of highland cooking banana cultivars Kibuzi and Nabusa. Re-isolation of F. oxysporum showed that endophyte colonization decreased faster from the rhizomes than from the roots of inoculated plants, both in the screenhouse and in the field. Whereas rhizome colonization by F. oxysporum decreased in the screenhouse (4–16 weeks after inoculation), root colonization did not. However, in the field (17–33 weeks after inoculation), a decrease was observed in both rhizome and root colonization. The results show a better persistence in the roots than rhizomes of endophytic F. oxysporum strains V2w2 and III4w1.     

Fusarium oxysporum and post-emergence herbicide for the control of the parasitic plant Striga hermonthica in maize

Two trials were conducted to evaluate the efficacy of a granular mycoherbicide formulation based on Fusarium oxysporum and post-emergence herbicide for the control of the parasitic plant, Striga hermonthica in the Nigerian Savanna. Four fungal treatments were used: F. oxysporum followed by 2,4-D, F. oxysporum followed by supplementary hoe weeding, F. oxysporum followed by Triclopyr and a control (No F. oxysporum but hoe-weeded). The experiments were laid out in a split plot design with three replications in the two locations. The two varieties (Across 97 TZL and farmer's local variety) formed the main plot treatments, while the Striga fungal treatments formed the sub-plot treatments. At the Lafia location, the emergence of Striga was delayed by 7 days (46 days) as compared to the Makurdi location, which germinated earlier 39 days after sowing. Maize variety Across 97 TZL similarly delayed the time to Striga emergence when compared to the farmer's local variety. However, the different Striga control methods did not have any significant effect on the time of Striga emergence. Generally, number of maize plants infected with Striga was highest with the farmer's local variety throughout the period of observation, while in the Striga control treatments, hoe-weeded check recorded the highest; the minimum was obtained with plots treated with F. oxysporum followed by postemergence application of Triclopyr at the rate of 0.36 kg a.i./ha at 6 weeks after sowing. Highest maize grain yields were obtained at Lafia with Across 97 TZL and plots treated with F. oxysporum followed by either post-emergence 2,4-D or Triclopyr at 0.36 kg a.i./ha each. The results demonstrate the high potentiality of using F. oxysporum as a spot application at planting followed by post-emergence herbicide (2,4-D or Triclopyr) application at 6 weeks after sowing for the control of the parasitic plant S. hermonthica in the Nigerian Savanna.     

Effect of Placement and Sequence of Inoculation with Fusarium oxysporum on its Interaction with Meloidogyne arenaria on Subterranean Clover

The effect of the placement of inoculum of Fusarium oxysporum at two soil depths, and the sequences of inoculations with Meloidogyne arenaria and Fusarium oxysporum on root growth and development of root disease in Trifolium subterraneum L. (subterranean clover) were investigated. The timing of infection and the proximity of root tips of the host root system to infection by M. arenaria and F. oxysporum appeared to be the major determining factors of root growth and of disease development in plants exposed to the pathogens. Immediate contact of roots with F. oxysporum (where the fungus was placed at seed level of 10 mm depth) appeared to result in more severe effects on roots in the presence of the nematode than later infection by the fungus placed at 30 mm depth. The production of galls by the nematode and early infection by F. oxysporum at 10 mm depth resulted in a severe inhibition of root growth, particularly of the lateral roots. But no such growth inhibition was evident when F. oxysporum and M. arenaria were introduced together at the lower depth of 30 mm. The lowest density of M. arenaria inoculum was sufficient to cause severe root rot if F. oxysporum was present at the host seed level. With the fungus at 30 mm depth, however, the expression of root rot appeared to be influenced by the inoculum level of the nematode. In sequential inoculation with F. oxysporum or M. arenaria, the organism added 2 weeks later had little or no effect on root development. The first organism (M. arenaria or F. oxysporum) to infect the germinated seedlings was the main cause of root growth inhibition. The organism that came into contact with the roots 2 weeks later had little or no effect on the roots. Concurrent infection by F. oxysporum and M. arenaria resulted in less M. arenaria gall production on the tap root system than those added with the nematode alone or in advance of the fungus.     

Production of pectolytic and cellulolytic enzymes byfusarium oxysporum andF. moniliforme under different cultivation conditions

Six-day incubation was most suitable for production of pectolytic and cellulolytic enzymes byFusarium on different culture media. Czapek’s medium favoured maximum production of polygalacturonase (PG) and cellulase (C_x), peptone dextrose gave highest yields of pectin methyl galacturonase (PMG) withF. oxysporum. Cole’s medium was found to be poor for the enzyme production by both organisms. A positive correlation was observed between the growth rate of the pathogenic forms and their enzyme production. InF. oxysporum the PG secretion was maximum at pH 4.5 and inF. moniliforme at pH 5.0. PMG production optimum was at pH 5.5. No PG and PMG were produced above pH 7. InF. oxysporum the C_x activity was highest at pH 5.5 and inF. moniliforme at pH 4.5. Maximum PG and PMG activities were recorded at 35 °C in both pathogens. The C_x activity of both organisms was maximum at 45 °C but some carboxymethyl cellulose hydrolysis was found even at 60 °C.     

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.     

Influence of the ectomycorrhizal fungus <Emphasis Type="Italic">Laccaria laccata</Emphasis> on pre-emergence,post-emergence and late damping-off by <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> and <Emphasis Type="Italic">F. verticillioides</Emphasis> on Stone pine seedlings

In greenhouse experiments, the ectomycorrhizal fungus Laccaria laccata was evaluated for biological control of preemergence, post-emergence and late damping-off of Pinus pinea caused by Fusarium verticillioides and F. oxysporum. In pre-emergence damping-off assays, preinoculation with Laccaria laccata did not significantly improve germination of seeds and no statistical significant differences were found in Fusarium treatments when compared with controls. At 18 weeks after sowing, inoculation with L. laccata reduced the incidence of post-emergence damping-off but differences were significant only in F. oxysporum treatments. Pinus pinea transplanted plants were used in late damping off assays, and only F. oxysporum produced significant damage. Inoculation with L. laccata did not attenuate significantly the virulence of F. oxysporum. However, the percentage of mycorrhization did not reached significant level, so the amount of mycorrhizal fungus was insufficient for effective protection. Although very low percentages of mycorrhization were recorded in all mycorrhized treatments, and Fusarium occurrence significantly reduced mycorrhization, those levels have been efficient to reduce damage in F. oxysporum post-emergence damping-off assays. In short, pre-emergence damping-off was not found; only F. oxysporum produced significant damage on P. pinea seedlings and L. laccata reduced damage when the percentage of mycorrhization reached a significant level. These results have been compared with previous work on P. sylvestris inoculated with the same mycorrhizae isolate and Fusarium pathogens.     

Fusarium oxysporum Forms Heterokaryons with Fusarium redolens

Vegetative compatibility among three isolates of Fusarium oxysporum f. sp. lupini and two isolates of F. oxysporum var. redolens from diseased lupins was investigated. Pairings between five mutants originated from each isolate revealed two compatibility groups. The first VCG comprised race 1 of F. oxysporum f. sp. lupini and one isolate of F. oxysporum var. redolens; the second VCG comprised race 2 of F. oxysporum f. sp. lupini and two isolates of F. oxysporum var. redolens. Heterokaryon formation was observed in many pairings involving mutants of both taxa. These findings provide evidence of the conspecificity of these two taxa and they support Gordon 's classification (1952) according to which F. redolens is actually F. oxysporum.     

Screening of zearalenone-producing Fusarium species in Egypt and chemically defined medium for production of the toxin

296 isolates of Fusarium spp. from 100 samples of cereale grains were examined for their ability to produce zearalenone on liquid culture medium. Thin layer Chromatographic analysis revealed that the mycotoxin was detected from 45 isolates, (F. oxysporum, 36; F. moniliforme, 8; and F. equiseti, one isolate). A suitable liquid medium and some optimal conditions for the biosynthesis of zearalenone were reported. Glutamine and riboflavin stimulated the production of the toxin. The maximum amount of zearalenone appeared at pH 7, after 12 days of incubation at 20 °C.