Adhesion of Fusarium oxysporum conidia to tomato roots

The occurence of a binding process between Fusarium oxysporum conidia and the surface of tomato roots was demonstrated in vitro by using a quantitative assay and a serial washing procedure. The number of conidia bound per root unit increased with increasing the concentration of the spores in solution until binding reached saturation. The attachment could be described accurately in terms of the Langmuir adsorption isotherm, indicating the existence of a single class of specific, high-affinity adherence sites on the root surface. No differences were detected in the extent of binding of several strains of F. oxysporum differing either in pathogenicity or in host range. Site-specific binding of F. oxysporum conidia may be important in securing the fungal spores at the root surface, after which germination and other processes required for colonization can proceed.     

In vitro selection for Fusarium wilt resistance in Gladiolus

Cormels pieces of four Fusarium susceptible Gladiolus cultivara (Friendship, Peter Pears, Victor Borge and Novalux) formed friable calli when cultured in vitro on Murashige and Skoog basal medium containing various concentrations of auxin and cytokinin. The friable calli established cell suspensions. Plantlet regeneration was obtained from the control callus, control cell suspension derived callus and in vitro selected Fusarium oxysporum Schlecht. resistant cell-lines of Friendship. The in vitro cormlets showed 85-95% germination after breaking dormancy of 8weeks at 4℃. Cell suspensions of all four Gladiolus cultivara were found to be highly sensitive to fusaric acid. Gradual Increase in fusaric acid concentrations to the cell-suspension cultures decreased cell growth considerably. One albino plant was found from the second generation of the In vitro selected cell line of Friendship. The albino plant was found to be highly susceptible to F. oxysporum. The cormlets of all in vitro selected call lines of Friendship were inoculated with a conidial suspension of the F. oxysporum before planting and were also sprayed with the same spore suspension for further characterization when the height of plants was about 6cm. The four selected cell lines showed the same response whether or not they were Inoculated with conidia of the F. oxysporum. Plantlets of all of the selected call lines exhibited significant growth as compared with the control after application of conidia of the F.oxysporum.     

Regularities in the Germination of Conidia of Phytopathogenic Fungi

The autoregulation of conidium germination in phytopathogenic micromycetes of the genera Fusarium, Botrytis, and Bipolaris was studied. It was shown that Trichoderma longibrachiatum was less competitive than Fusarium oxysporum after their simultaneous inoculation but inhibited the phytopathogen growth in the case of earlier introduction. In the latter case, no autoinhibition of the germination of F. oxysporum conidia occurred; moreover, a cooperative effect was observed, i.e., the number of germinated F. oxysporum conidia increased with an increase in their density.     

Suppression of Fusarium oxysporum and induced resistance of plants involved in the biocontrol of Cucumber Fusarium Wilt by Streptomyces bikiniensis HD-087

Cucumber Fusarium Wilt, caused by Fusarium oxysporum f. sp. cucumerinum, which usually leads to severe economic damage, is a common destructive disease worldwide. To date, no effective method has yet been found to counteract this disease. A fungal isolate, designated HD-087, which was identified as Streptomyces bikiniensis using physiological-biochemical identification and 16S rRNA sequence analysis, is shown to possess distinctive inhibitory activity against F. oxysporum. The fermentation broth of HD-087 leads to certain abnormalities in pathogen hyphae. It peroxidizes cell membrane lipids, which leads to membrane destruction along with cytoplasm leakage. This broth also restrains germination of the conidia. The activities of the enzymes peroxidase, phenylalanine ammonia-lyase, and β-1,3-glucanase in cucumber leaves were dramatically increased after treated with fermentation broth of HD-087. The levels of chlorophyll and soluble sugars were also found to be increased, with the relative conductivity of leaves being reduced. In short, the metabolites of strain HD-087 can effectively suppress F. oxysporum and trigger induced resistance in cucumber.     

A novel enzyme immunoassay for conidia of Fusarium oxysporum f.sp. cucumerinum F504

This study was undertaken to develop a new tool to study fusarial diseases of plants. Micro- and macro-conidia of a strain (F504) of Fusarium oxysporum were isolated and antiserum against the conidia was elicited in rabbits. A highly specific and sensitive competitive-type enzyme-linked immunosorbent assay (ELISA) for conidia of the strain was developed using the antiserum with beta-D-galactosidase-labeled anti-rabbit IgG as the secondary antibody and conidia fragments of the strain as antigen attached to Amino-Dylark solid-phase balls. The assay was highly specific to conidia of the strain F504, while conidia-free hypha of the strain F504 as well as all other microorganisms tested including nine other strains of Fusarium species showed little cross-reactivity. Application of the ELISA to following the growth rates of conidia in hyphae of the strain F504 under several conditions are also reported.     

Effect of dsRNA-containing and dsRNA-free hypovirulent isolates of Fusarium oxysporum on severity of Fusarium seedling disease of soybean in naturally infested soil

In the sandy soils of eastern Virginia, soybean seedlings are colonized by hypovirulent and virulent isolates of Fusarium oxysporum and F. solani. Our objectives were to determine if prior inoculation of soybean seeds with hypovirulent F. oxysporum isolates reduced severity of seedling disease in naturally infested soil, and to determine if there was an association between the presence of dsRNA mycovirus and hypovirulence in isolates of F. oxysporum and F. solani from soybean plants. The presence of dsRNA was not associated with hypovirulence in F. oxysporum since some hypovirulent isolates contained dsRNA while other hypovirulent isolates did not. Furthermore, of six dsRNA-containing F. oxysporum isolates, three were hypovirulent and three were virulent. Four segments of dsRNA, with sizes of 4.0, 3.1, 2.7 and 2.2 kb were detected in extracts of all six F. oxysporum isolates. No hypovirulent or dsRNA-containing of F. solani isolates were found. Prior inoculation of cv. Essex soybean seeds with conidia of dsRNA-free hypovirulent F. oxysporum isolates significantly (P < 0.05) reduced disease severity on cotyledons and hypocotyls, and increased the rate of seedling emergence in field soil, compared to control plants. No significant (P > 0.05) differences were found between dsRNA-containing and dsRNA-free hypovirulent F. oxysporum isolates in their effects on reducing disease severity. Hypovirulent isolates that colonize soybean tissues may play a role in reducing Fusarium seedling disease of soybean in natural soils.     

Effect of volatile and gaseous metabolites of germinating pea seeds on micromycetes

Differences in the effect of volatile and gaseous metabolites of germinating pea seeds on the germination of spores of Mucor racemosus and macroconidia of Fusarium oxysporum are described. Germination of spores of M. racemosus was inhibited by seed metabolites whereas germination of macroconidia of F. oxysporum was stimulated during the first two days and inhibition occurred only after further two days of germination of the seeds. A pronounced inhibition of germination of spores of both micromycetes took place due to absorption of CO2 from volatile and gaseous metabolites. Absorption of some components of seed metabolites in a KMnO4 solution led to a decrease of the inhibitory effect on germination of spores of M. racemosus and stimulatory effect on germination of macroconidia of F. oxysporum.     

尖孢镰刀菌四个专化型细胞核的初步研究

本文对尖孢镰刀菌(Fusarium oxysporum)的4个专化型12株菌的孢子萌发、核分裂时间及核DNA含量进行了比较,结果表明:不同专化型菌株孢子萌发速度基本一致,而萌发过程中发生第一次核分裂的时间不同。黄瓜、西瓜、荸荠、大豆各专化型第一次核分裂时间分别为:5.67hr、5.45hr、7.35hr、7.82hr,其核DNA含量分别为:0.321pg、o.306pg、0.177pg 0.174pg。测定结果显示出不同专化型菌株的孢子核DNA含量可能存在倍数关系,并推测F.oxysporum的黄瓜专化型与西瓜专化型为同一类型,而荸荠专化型与大豆专化型为同一类型。     

Differentiation of allergenic fungal spores by image analysis, with application to aerobiological counts

The ability of an image analysis routine to differentiate between spores of eleven allergenic fungal genera was tested using analysis based on seven basic and up to 17 more complex features, extracted from digitised images. Fungal spores of Alternaria, Cladosporium, Fusarium, Aspergillus, Penicillium, Botrytis, Epicoccum, Exserohilum, Ustilago, Coprinus and Psilocybe were examined in a series of experiments designed to differentiate between spores at the genus and species level. Linear and Quadratic Discriminant Analysis of feature measurements, recorded for 100 to 1600 spores per taxon, differentiated between genera and species with a high level of accuracy. Genus comparisons using only seven basic features resulted in 98% accuracy for the recognition of conidia belonging to Cladosporium, Fusarium and Epicoccum. Differentiation between conidia of Aspergillus and Penicillium was the least reliable, with 56% of Aspergillus conidia correctly identified and 41% misidentified as Penicillium. At the species level, conidia of Cladosporium macrocarpum, Fusarium moniliforme (microconidia), F. oxysporum (microconidia), F. solani (macroconidia), Alternaria helianthi and A. brassicae were consistently identified with 86--100% accuracy. Reduced levels of accuracy in the identification of spores by image analysis reflected similarities between species in their spore morphology. The application of image analysis to aerobiological counting methods is discussed in relation to the results obtained.     

Bacterial ectosymbionts and virulence silencing in a Fusarium oxysporum strain

In the present article we have ascertained the presence of a consortium of ectosymbiotic bacteria belonging to Serratia, Achromobacter, Bacillus and Stenotrophomonas genera associated to the mycelium of the antagonistic Fusarium oxysporum MSA 35 [wild-type (WT) strain]. Morphological characterization carried out on the WT strain, on the F. oxysporum MSA 35 without ectosymbionts [cured (CU) strain] and on the pathogenic F. oxysporum f.sp. lactucae (Fuslat 10) showed that the ectosymbionts, present only in the WT strain, caused a depleted production of micro conidia and aerial hyphae, and a change in shape and dimension of the latter. Virulence tests showed that the cured Fusarium was a pathogenic strain and, as shown by polymerase chain reaction and microscope analysis, pathogenicity was correlated with the capability of the cured hyphae of penetrating lettuce roots. Accordingly, the hyphae of the WT strain were impaired in entering the plant roots. Typing experiments provided evidence that both CU and WT strains belong to F. oxysporum f.sp. lactucae. This implies that the antagonistic effect of WT Fusarium is not a fungal trait, but it is due to the interaction with the ectosymbiotic bacteria. Expression analysis showed that fmk1, chsV and pl1 genes involved in F. oxysporum pathogenicity are not expressed in the WT strain whereas they are expressed in the cured fungus. These results, together with the hyphal characteristics, suggest that the inability of WT strain to penetrate the plant roots could be due to alterations in the expression profile of cell wall-degrading enzymes. In conclusion, we demonstrated a modulation of F. oxysporum gene expression in response to the interaction with the ectosymbiotic bacteria. Preliminary researches indicated that the presence of bacteria attached to the hyphae of antagonistic F. oxysporum is not an isolated phenomenon. Further investigations are necessary to better understand the rule and the diffusion of ectosymbiotic bacteria among antagonistic Fusarium.     

Xylem Fluids of White Lupin (Lupinus albus) Cultivars Susceptible and Resistant to Fusarium oxysporum f. sp. Lupini Race 3

Xylem fluids from white lupin cv. Neuland, susceptible to wilt causes by Fusarium oxysporum f. sp. lupini , promoted germination of conidia and chlamydospores of he pathogen, and the production of micro-conidia, to a greater extent than did xylem fluids from the resistant cv. Primorskij No sugars, organic acids or phenolic compounds were detected in the xylem fluids, but a number of amino acids were found, and the concentration of some differed in xylem fluids from the two cultivars; on the whole, concentrations were greater in the susceptible cv. Neuland. Synthetic amino acids were supplied to the fungus in vitro and both stimulatory and inhibitory effects (according to the compound and its concentration) were noted upon the germination of conidia and chlamydospores and on mycelial growth and the production of conidia. The role of amino acids and other compounds in the susceptibility of white lupin to wilt is discussed.     

Hybridization and breeding of the benomyl resistant mutant, Trichoderma harziantum antagonized to phytopathogenic fungi by protoplast fusion

A diploid strain obtained from heterokaryons of Trichoderma harzianum by protoplast fusion grew on minimal medium containing 100ppm benomyl. This strain inhibited the growth of the phytopathogenic fungus Fusarium oxysporum f. sp. raphani on paired cultures and also protected against radish yellows and a drop in germination induced by F. oxysporum f. sp. raphani.     

大蒜根系分泌物的化感作用

以苍山白蒜和蔡家坡紫蒜为材料,采用水培方法收集根系分泌物,研究了2个大蒜品种的根系分泌物对莴苣种子发芽和幼苗生长及对黄瓜枯萎病菌、西瓜枯萎病菌的化感效应.结果表明:2个大蒜品种的根系分泌物对莴苣种子发芽和幼苗生长均表现为低浓度(0.1、0.2 g·mL-1)促进、高浓度(0.4、0.6 g·mL-1)抑制,高浓度时蔡家坡紫蒜的抑制作用大于苍山白蒜;对黄瓜枯萎病菌和西瓜枯萎病菌的菌丝生长及孢子萌发均表现为抑制作用,随着根系分泌物浓度的提高,抑制作用增强,其中黄瓜枯萎病菌较敏感,且蔡家坡紫蒜的抑制作用大于苍山白蒜.     

唾液乳杆菌抑制镰孢霉的研究

目的 研究唾液乳杆菌抑制产毒镰孢霉的生物学性能,初步探索抑菌机制.方法 以禾谷镰孢霉和尖孢镰孢霉2种典型霉菌为指示菌,唾液乳杆菌为测试对象,对霉菌孢子萌芽、孢子生长和菌丝体生长3个生理阶段进行抑制效应观察.结果 10%的唾液乳杆菌耗尽上清就能抑制83%的禾谷镰孢霉孢子和50%尖孢镰孢霉孢子萌芽;耗尽上清24 h内能显著抑制镰孢霉孢子的生长;96 h内孢霉菌丝体的生长.结论 唾液乳杆菌产生的有机酸对禾谷镰孢霉和尖孢镰孢霉生长起主要抑制作用.     

Vibrational, 1H-NMR spectroscopic,and thermal characterization of gladiolus root exudates in relation to Fusarium oxysporum f. sp. gladioli resistance

Fourier transform Raman (FT Raman) and IR (FTIR) and (1)H-NMR spectroscopies coupled with differential scanning calorimetry (DSC) were applied to the characterization of root exudates from two cultivars of gladiolus (Spic Span and White Prosperity) with different degrees of resistance and susceptibility to Fusarium oxysporum gladioli, the main pathogen of gladiolus. This work was aimed at correlating the composition of root exudates with the varietal resistance to the pathogen. Spectroscopic analysis showed that White Prosperity root exudate differs from Spic Span root exudate by a higher relative amount of the aromatic-phenolic and sugarlike components and a lower relative amount of carbonylic and aliphatic compounds. DSC analysis confirmed the spectroscopic results and showed that White Prosperity root exudate is characterized by an aromatic component that is present in a higher amount than in the Spic Span root exudate. The results are discussed in relation to the spore germination tests showing that White Prosperity, which is characterized by a remarkable resistance toward F. oxysporum gladioli, exudes substances having a negative influence on microconidial germination of the pathogen; root exudates from Spic Span, one of the most susceptible cultivars to F. oxysporum gladioli, proved to have no effect. White Prosperity's ability to inhibit conidial germination of F. oxysporum gladioli can be mainly related to the presence of a higher relative amount of aromatic-phenolic compounds.     

Antifungal activity and enhancement of plant growth by Bacillus cereus grown on shellfish chitin wastes

Bacillus cereus QQ308 produced antifungal hydrolytic enzymes, comprising chitinase, chitosanase and protease, when grown in a medium containing shrimp and crab shell powder (SCSP) produced from marine waste. The growth of the plant-pathogenic fungi Fusarium oxysporum, Fusarium solani, and Pythium ultimum were considerably affected by the presence of the QQ308 culture supernatant. The supernatant inhibited spore germination and germ tube elongation of F. oxysporum, F. solani, and P. ultimum. The increase in the growth time of the fungal culture was associated with a gradual decrease in inhibition. Besides antifungal activity, QQ308 enhanced growth of Chinese cabbage. These characteristics were unique among known strains of B. cereus. To our knowledge, this is the first report on the antifungal and Chinese cabbage growth enhancing compounds produced by B. cereus.     

Interactions of Fusarium species during prepenetration development

Interspecies interactions between Fusarium avenaceum, Fusarium culmorum, Fusarium graminearum, Fusarium poae, and Fusarium tricinctum were studied during early growth stages of isolates on model surfaces. Additionally, germination and germ tube growth of the pathogens were studied on attached and detached wheat leaves at 10 °C and 22 °C. Two-species interactions between Fusarium isolates during germination and germ tube growth were assessed after 8 hours of incubation. All species except F. tricinctum germinated and grew faster at higher than lower temperature. All species were able to germinate with more than one germ tube per conidium cell; and germination and germ tube growth were faster on leaves than on glass surface. Interactions among Fusarium species during germination and germ tube growth were predominantly competitive with macroconidia-producing species being more competitive. It is concluded that the type of conidia as well as environmental factors influence the competitiveness of Fusarium species during early stages of growth.     

Photodamage to spores of Fusarium fungi, sensitized by protoporphyrin IX

The influence of photodynamic action with protoporphyrin IX as a sensitizer on the state of the components of hydrated spores of Fusarium fungi and germination of conidia in growth medium was investigated. It was shown, that protoporphyrin IX in micromole concentrations sensitizes the photooxidation of proteins and lipids from hydrated spores of Fusarium poae and Fusarium culmorum under illumination of their suspensions in doses of 50 - 200 kJ/m2. It was found that the photosensitized oxidation of cellular components leads to the disturbance of conidium membrane permeability and inhibition of spore germination during their subsequent cultivation in growth medium.     

Species-specific primers for Fusarium redolens and a PCR-RFLP technique to distinguish among three clades of Fusarium oxysporum

The currently available morphological and molecular diagnostic techniques for Fusarium redolens and the three phylogenetic clades of Fusarium oxysporum are problematic. Aligned translation elongation factor 1 alpha (TEF-1 alpha) gene sequences from these species and their close relatives were used to design F. redolens-specific primers, and to identify restriction sites that discriminate among the three clades of F. oxysporum. The F. redolens-specific primers distinguished this species from all others included in the study. There were three TEF-1 alpha-RFLP patterns among formae speciales of F. oxysporum. These PCR-RFLP patterns corresponded with the three clades. These techniques provide simple and inexpensive diagnostic methods for the identification of F. redolens and members of the three clades of F. oxysporum.     

Visualization of interactions between a pathogenic and a beneficial Fusarium strain during biocontrol of tomato foot and root rot

The soilborne fungus Fusarium oxysporum f. sp. radicis-lycopersici causes tomato foot and root rot (TFRR), which can be controlled by the addition of the nonpathogenic fungus F. oxysporum Fo47 to the soil. To improve our understanding of the interactions between the two Fusarium strains on tomato roots during biocontrol, the fungi were labeled using different autofluorescent proteins as markers and subsequently visualized using confocal laser scanning microscopy. The results were as follows. i) An at least 50-fold excess of Fo47over F. oxysporum f. sp. radicis-lycopersici was required to obtain control of TFRR. ii) When seedlings were planted in sand infested with spores of a single fungus, Fo47 hyphae attached to the root earlier than those of F. oxysporum f. sp. radicis-lycopersici. iii) Subsequent root colonization by F. oxysporum f. sp. radicis-lycopersici was faster and to a larger extent than that by Fo47. iv) Under disease-controlling conditions, colonization of tomato roots by the pathogenic fungus was significantly reduced. v) When the inoculum concentration of Fo47 was increased, root colonization by the pathogen was arrested at the stage of initial attachment to the root. vi) The percentage of spores of Fo47 that germinates in tomato root exudate in vitro is higher than that of the pathogen F. oxysporum f. sp. radicis-lycopersici. Based on these results, the mechanisms by which Fo47 controls TFRR are discussed in terms of i) rate of spore germination and competition for nutrients before the two fungi reach the rhizoplane; ii) competition for initial sites of attachment, intercellular junctions, and nutrients on the tomato root surface; and iii) inducing systemic resistance.