Isolates Of Fusarium Solani Research Articles

Phytotoxicity of Culture Filtrate fromFusarium solani,the Causal Agent of Sudden Death Syndrome of Soybean

An isolate of Fusarium solani that caused sudden death syndrome (SDS) of soybean was grown in a semidefined liquid medium. The phytotoxicity of cell-free fungal culture filtrates was determined on soybean calli grown on tissue culture medium amended with fungal culture filtrate. The mean calli brown rating increased as culture filtrate increased in the medium. There were significant (P < 0.05) positive correlations between SDS severity from field microplot data and calli browning due to culture filtrates. Seedlings of five soybean cultivars inoculated with the fungus under greenhouse conditions had significantly (P < 0.05) different foliar severity ratings and relative plant fresh weights. Sensitivity of calli of the same five cultivars to culture filtrate also had significantly (P < 0.05) different brown ratings and relative fresh weights. There were significant (P < 0.05) correlations between variables measured on inoculated soybean seedlings and calli sensitivity. Pathogenicity and phytotoxicity of F. solani isolates from different hosts were tested on soybean plants and calli, respectively. Isolates from hosts other than soybean did not cause SDS symptoms, and their culture filtrates had significantly lower toxicity to soybean calli than did that of soybean SDS-causing isolates.

Severity of Foliar Symptoms and Root and Crown Rot of Soybean Inoculated with Various Isolates and Inoculum Rates ofFusarium solani

Soybean plants (cvs. Spencer and Ripley) were grown in Cone-Tainers infested with different rates of sudden death syndrome (SDS) isolates of Fusarium solani. Soil inoculum rate significantly affected severity of root rot and percent leaflets of inoculated plants with SDS symptoms with isolate Mont-1. Leaf symptoms of SDS on Ripley were significantly less than on Spencer. When SDS isolate Cora-7 was used, only the soil inoculum rate was significant for percent leaflets with SDS symptoms and root rot severity. Nine SDS fungal isolates differed in the amount of root rot and severity of leaf symptoms that they produced on inoculated Spencer and Ripley plants.

Effect of increasing inoculum sizes of pathogenic filamentous fungi on MICs of antifungal agents by broth microdilution method

Inoculum size is a critical variable in development of methods for antifungal susceptibility testing for filamentous fungi. In order to investigate the influence of different inoculum sizes on MICs of amphotericin B, 5-fluorocytosine, itraconazole, and miconazole, 32 clinical isolates (8 Aspergillus fumigatus, 8 Aspergillus flavus, 5 Rhizopus arrhizus, 8 Pseudallescheria boydii, and 3 Fusarium solani isolates) were studied by the broth microdilution method. Four inoculum sizes were studied: 1 x 10(2) to 5 x 10(2), 1 x 10(3) to 5 x 10(3), 1 x 10(4) to 5 x 10(4), and 1 x 10(5) to 5 x 10(5) CFU/ml. The National Committee for Clinical Laboratory Standards reference method for antifungal susceptibility testing in yeasts was modified and applied to filamentous fungi. The inoculum was spectrophotometrically adjusted, and all tests were performed in buffered medium (RPMI 1640) at pH 7.0 with incubation at 35 degrees C for 72 h. MICs were read at 24, 48, and 72 h. Amphotericin B showed a minimum effect of inoculum size on MICs for all species with the exception of P. boydii (P < 0.05). A significant effect of inoculum size on MICs was observed with 5-fluorocytosine, for which there was an increase of more than 10-fold in MICs against all Aspergillus spp. between inoculum concentrations of 10(2) and 10(4) CFU/ml (P < 0.001). For itraconazole, the results showed a more species-dependent increase of MICs, most strikingly for R. arrhizus and P. boydii. Miconazole, which was tested only with P. boydii, did not demonstrate a significant effect of inoculum size on MICs. In summary, the effect of inoculum size on MICs for filamentous fungi was dependent upon the organism and antifungal compound tested. Thus, among antifungal compounds, itraconazole and 5-fluorocytosine demonstrated significant inoculum effects, while amphotericin B and miconazole showed comparatively minimum inoculum effects against pathogenic filamentous fungi. Moreover, among filamentous fungi, P. boydii and R. arrhizus exhibited the greatest inoculum effect.

Genetic analysis of variation of pathogenicity in Nectria haematococca ( Fusarium solani) on Cucurbita sp.

Pathogenicity phenotypes for over 30 isolates of Fusarium solani, from Cucurbita sp., were measured as lesion sizes on both unwounded and wounded seedling hypocotyls and on fruit mesocarp tissue. These tests detected different capacities in both the pre-and post-penetration phases of infection. The isolates were grouped, based on their mating behaviour, as non-mating, or members of either mating population I (MPI) or MPV of Nectria haematococca. The MPI isolates readily infected unwounded hypocotyls whereas MPV and non-mating isolates were either incapable of infecting or were very weakly pathogenic on this issue. In contrast all isolates were capable of colonizing both wounded hypocotyls and fruit mesocarp tissue, although there was a wide range in the size of lesions. Segregation for pathogenicity on unwounded hypocotyls was studied in the progeny from seven crosses between MPI isolates of differing pathogenicity. Pathogenicity was a polygenically controlled character with estimates of the number of effective factors, k, involved ranging from 3 to 15 for the seven crosses. In three of the crosses there was non-additive genetic interaction, and in the other four crosses the genetic variation was predominantly additive. There was evidence for a weak association between female sex and pathogenicity in several crosses.

Comparison of Fusarium solani and F. oxysporum as Causal Agents of Fruit Rot and Root Rot of Muskmelon

Rotting muskmelon fruits commonly are associated with commercial fields that are affected by the root rot/vine decline disease syndrome found in southern Texas. Four isolates of Fusarium solani previously shown to be either weakly pathogenic or nonpathogenic to muskmelon seedlings caused extensive rot on mechanically wounded muskmelon fruits. Two of these isolates caused more extensive fruit rot than either F. solani (Mart.) Sacc. f. sp. cucurbitae W.C. Snyder &amp; H.N. Hans. or F. oxysporum Schlechtend.:Fr. melonis (Leach &amp; Currence) W.C. Snyder &amp; H.N. Hans., causal agents of fusarium crown and foot rot of cucurbits and fusarium wilt of muskmelon, respectively. In other tests, root-dip inoculation of seedlings showed that all muskmelon cultigens included in this study and the breeding line MR-1 were susceptible to a California and an Arkansas strain of F. s. f. sp. cucurbitae race 1.

Fusarium solani from Ascospores of Nectria haematococca Causes Sudden Death Syndrome of Soybean

This is the first report of a teleomorph connection for isolates of Fusarium solani that infect soybean. Nectria haematococca isolated from roots of soybean plants with sudden death syndrome proved to be the ascoma stage of F. solani. A single ascospore culture IN-2X-11A from IN-2X-11 produced sporodochia with masses of blue macroconidia and essentially no microconidia. Another single asco? spore culture IN-2X-11B from IN-2X-11 did not produce macroconidia, but it did produce the perfect state and microconidia. Hypocotyl inoculation of soybeans with macroconidia plus mycelia of IN-2X11A caused severe foliar symptoms typical of sudden death syndrome. In contrast, no foliar symptoms of sudden death syndrome developed in plants inoculated with microconidia plus mycelia. Pathogenicity ofthe N. haematococca isolate producing abundant F. solani macroconidia has been stable in inoculation tests since 1989, and it is more virulent than several other Indiana isolates of F. solani from soybean plants with severe sudden death syndrome.

Pseudomonas cepacia and Pseudomonas putida as winter wheat inoculants for biocontrol of Rhizoctonia solani

Pseudomonas cepacia R55 and R85 and Pseudomonas putida R104, antagonistic towards plant pathogenic fungi in vitro, were assessed as seed inoculants for winter wheat (cv. Norstar) grown in a growth chamber in soil infested with Fusarium solani or Rhizoctonia solani isolate AG-1, AG 2-1, or AG-3. Infestation of soil with R. solani AG-1 or AG 2-1 reduced root dry weight of uninoculated plants by 62 and 78%, respectively, whereas R. solani AG-3 or F. solani had no effect on plant biomass. Pseudomonad inoculants increased (relative to plants subjected to disease) the winter wheat root dry weight by 92–128% and shoot dry weight by 28–48% in the soil infested with R. solani AG-1. The shoot material of all plants inoculated with pseudomonads also had significantly (P &lt; 0.05) higher total Fe contents than the uninoculated treatment in the R. solani AG-1 infested soil. Pseudomonas cepacia R55 produced the highest (P &lt; 0.01) total Fe contents in the shoots, but it had no effect on N and P content. Pseudomonas cepacia R85 significantly increased total N (P &lt; 0.05) and total P (P &lt; 0.01) of wheat shoots, and P. putida R104 increased the percentage (P &lt; 0.05) and (or) total P content (P &lt; 0.01) in the soil infested with R. solani AG-1. Pseudomonas cepacia R85 also significantly (P &lt; 0.05) increased wheat shoot biomass in R. solani AG-3 infested soil. All three pseudomonads produced fluorescent siderophores when cultured in a low-iron medium. These results suggest an in situ antibiosis activity of three fluorescent pseudomonad strains towards phytopathogenic fungi and suggest that the plant growth response was probably due to protection against damage caused by R. solani. Key words: biocontrol, pseudomonads, Rhizoctonia solani, winter wheat, siderophores.

Antifungal susceptibility of 44 clinical isolates of Fusarium species determined by using a broth microdilution method.

The MICs and minimum fungicidal concentrations of amphotericin B, natamycin, miconazole, itraconazole, and flucytosine against 17 isolates of Fusarium solani, 14 isolates of Fusarium moniliforme, 10 isolates of Fusarium oxysporum, and 3 isolates of Fusarium semitectum were determined by a broth microdilution method. Amphotericin B and natamycin were the most active agents tested and failed to show any inoculum size effect. In contrast, miconazole and itraconazole showed poor inhibitory and fungicidal activities, and the inoculum size had a major effect on the results. Flucytosine had no activity against any of the isolates tested.

Two linear plasmids in mitochondria of Fusarium solani f. sp. cucurbitae

Two linear plasmid-like DNAs designated pFSC1 (9.2 kbp) and pFSC2 (8.3 kbp) were found in an isolate of the plant pathogenic fungus Fusarium solani f. sp. cucurbitae race 1. The plasmids were maternally inherited and copurified with mitochondrial DNA obtained from a mitochondria-enriched cell fraction suggesting that they are located in mitochondria. The plasmids did not share extensive sequence similarity. No homology was detected between either plasmid and the nuclear or mitochondrial genome when cloned plasmids were used as probes in Southern hybridization analyses. The fungus was cured of plasmids by ethidium bromide treatment. Compared to the plasmid-containing isolate, plasmid-cured derivatives had reduced pathogenicity on a susceptible plant host, Cucurbita maxima “Pink Banana.”

Comparative characteristics of a kievitone hydratase-producing isolate of Fusarium solani f. sp. phaseoli and of two strains deficient in this enzyme

Fusarium solani f. sp. phaseoli, isolate FB1-S, is a wild-type virulent pathogen of Phaseolus vulgaris and a good source of kievitone hydratase (KHase), which catalyses detoxification of the isoflavonoid phytoalexin, kievitone. Two strains derived from the wild-type, one an induced mutant (FB1-S4) and the other a colony variant (FB1-SX), produced little or no detectable KHase and were more sensitive to kievitone than the original isolate. The strains were also more vulnerable to phaseollin and phaseollinisoflavan treatments than FB1-S. In addition, FB1-S4 and FB1-SX were also deficient in their abilities to convert these two isoflavonoid phytoalexins to the known products, la-hydroxyphaseollone and metabolite-1. Several differences were apparent in the extracellular protein-banding patterns, revealed by polyacrylamide gel electrophoresis, of acetone precipitates from the three fungi. Although FB1-S4 and FB1-SX were more sensitive to kievitone, phaseollin and phaseollinisoflavan than the wild-type, they were less susceptible to osmotic stress. The several differences between FB1-S, FB1-S4 and FB1-SX, in addition to KHase activity, rendered the association between KHase and virulence in F. solani f. sp. phaseoli less certain.

Isolation of a Fusarium solani mutant reduced in cutinase activity and virulence.

Fusarium solani isolate T-8 produces an extracellular enzyme, cutinase, which catalyzes the degradation of cutin in the plant cuticle. Cutinase activity can be measured by the hydrolysis of either the artifical substrate, p-nitrophenylbutyrate (PNB), or radioactive cutin containing [14C]palmitic acid. In the present study, the culture filtrate contained basal levels of cutinase when T-8 was grown on acetate as a sole source of carbon. After mutagenesis, a cutinase-defective mutant (PNB-1) was identified by screening acetate-grown colonies for a loss of PNBase activity. The mutant possessed an 80 to 90% reduction in cutinase activity when grown for 3 to 5 days on acetate- or cutin-containing medium. Induction of cutinase by cutin or hydrolyzed cutin after growth on glucose medium was similarly reduced. Kinetic analysis indicated that cutinase from the mutant possessed a near normal Km for PNB and a 92% reduction in Vmax. Fluorography and Western blotting of 15% sodium dodecyl sulfate-polyacrylamide gels of separated 35S-labeled proteins from cutin induction medium revealed that in the mutant the 22,000-molecular-weight band corresponding to cutinase was reduced approximately 85%. The virulence of the mutant in a pea stem bioassay was decreased by 55% and was restored to nearly the parental level by the addition of purified cutinase. The data suggest that the mutant synthesizes reduced quantities of a functional and immunoreactive cutinase enzyme and that cutinase plays a critical role in infection. The PNB1 mutation may be within a regulatory gene or a promoter for cutinase.

PHYTOTOXIC EFFECTS OF CELL-FREE CULTURAL FILTRATES OF Fusarium solani ISOLATES ON VIRULENCE, HOST SPECIFICITY AND RESISTANCE

No relationship was found between degrees of virulence and the phytotoxic properties of cell-free cultural filtrates of 21 isolates of Fusarium solani f. sp. pisi. Two media (Czapek-Dox, Kern’s) were compared for production of phytotoxins and the specific naphthazarin compounds fusarubin, isomarticin and javanicin. Incubation of cultures in Czapek-Dox resulted in more phytotoxic activity than incubation in Kern’s medium. In addition, isomarticin (previously reported to be more phytotoxic than fusarubin or javanicin) was produced in larger amounts in Czapek-Dox than Kern’s medium. Virulence of F. solani f. sp. pisi was not correlated with production of isomarticin. However, the two isolates of F. solani, not pathogenic to peas (S-1, S-2), did not produce any detectable isomarticin in either medium. Plant introduction accessions with resistance to F. solani f. sp. pisi were as severely affected by toxin activity as the susceptible cultivar Dark Skin Perfection. Phytotoxic activity was not confined to peas because toxin injection into seedling roots of several other leguminous and nonleguminous hosts resulted in lesion formation.

Role of cutinase and cell wall degrading enzymes in infection of Pisum sativum by Fusarium solani f. sp. pisi

Virulence of several isolates of Fusarium solani f. sp. pisi (Nectria haematococca) was measured by bioassays using pea stems with or without intact cuticular and cell wall barriers. Isolate T-8 was highly virulent in both assays whereas isolate T-30, which was almost avirulent on the intact surface, was highly virulent only on wounded stems. With a highly sensitive radioassay for cutinase the germinating spores of T-8 were shown to release this enzyme which was similar, if not identical to, that produced during saprophytic growth on cutin. The amount of cutinase released by germinating spores of Fusarium isolates correlated with the degree of virulence expressed on intact surfaces. The low virulence of T-30 shown on stems possessing intact cuticles and cell walls was increased to the level expressed on wounded stems by addition of cutinase, pectinesterase, pectinase and cellulase. These results strongly suggest that the level of cutinase and cell wall degrading enzymes produced by germinating spores can determine the efficiency of infection.

Effect of soil temperature on root rot of rough lemon caused by Fusarium solani

Rough lemon seedlings were root dip-inoculated in Fries liquid cultures of two Fusarium solani isolates, one from California and one from Florida. Plants were potted and placed in soil temperature tanks at 15, 21, 27, and 33 C. Plant height, trunk diameter, and fresh weight of inoculated plants significantly differed from the control at 15 and 21 C. Plant height was the only growth parameter significantly affected by inoculation at 27 and 33 C. Height of plants inoculated with the California isolate was significantly less than the control at 27 but not at 33 C. Height of plants inoculated with the Florida isolate did not differ from the controls at 27 and 33 C. Significantly more root rot occurred on inoculated plants at 15 and 21 C, but not at 27 and 33 C.

Effect of Fusarium isolates and their filtrates on respiratory rate and chemical analysis of squash plants

The highly pathogenic isolate stimulated the emergence of the squash seedlings first, caused, however, the highest death rate of the seedlings finally. Fusarium isolates and their culture filtrates inhibited the respiratory rate of squash plants significantly. However, F. oxysporum isolates inhibited respiration more than F. solani isolates. Seasonal changes of respiration decline show that the respiratory rate decreased with plant growth in the case of infested soil and of plants injected with culture filtrates. However, spraying Fusarium culture filtrates on the foliage gave opposite results when the plants grew older. Fusarium solani isolates decreased nitrogen content of squash stems and leaves, while F. oxysporum isolates gave reverse results. Injecting Fusarium culture filtrate into the plant decreased nitrogen content of both stems and leaves, while spraying the foliage with the filtrates increased nitrogen content more than that of the control. Phosphorus content of the stems of squash plants, sown in infested soil, was less than in the control when the plants were treated with F. solani and higher when they were treated with F. oxysporum isolates. On the other hand, the phosphorus content of squash leaves was higher than in the control. In the case of injected plants, however, the phosphorus content in stems and leaves was equal to that of the control or less, and with sprayed plants it was higher than in the control. Infesting the soil with Fusarium isolates and spraying the foliage with their culture filtrates increased potassium content of squash stems and leaves, while injecting the filtrates into the plants decreased potassium content of both stems and leaves.

Photoinduced perithecial formation by Nectria haematococca on media containing either L-tyrosine; L-phenylalanine; or D-glucose+NaNO3 as sole carbon and nitrogen sources.

A homothallic isolate of Fusarium solani produced red perithecia when illuminated and grown on a simple basal medium containing L-tyrosine; L-phenylalanine; or D-glucose + NaNO3 as a sole carbon and nitrogen source. Few or no perithecia formed in darkness on these media. Quantitative comparisons of perithecial formation in illuminated treatments indicated that L-tyrosine was a significantly better sole source of carbon and nitrogen than either L-phenylalanine or D-glucose + NaNO3. There was no significant difference between the responses on media containing L-phenylalanine and D-glucose + NaNO3. If dark-grown treatments were subsequently illuminated, perithecia formed on media containing L-tyrosine and D-glucose + NaNO3 but not on L-phenylalanine. The initial pH of the medium containing L-tyrosine did not seem to affect the fruiting response in illuminated cultures. A reduction in the amount of L-tyrosine in the basal medium resulted in a corresponding decrease in perithecial formation.The results are discussed in connection with a possible relationship of sexual reproduction, the light requirement, and tyrosine metabolism.

Utilization of carbon compounds byFusarium Solani F.Aurantifoliae Bhat. and Prasad

The two isolates ofFusarium solani f.aurantifoliae responsible for lime twig disease were found to utilise carbon from a wide variety of compounds. In general, monosaccharides proved to be better than other sources tested for growth and sporulation though maltose exhibited best growth.