Chlamydospores Of Fusarium Oxysporum Research Articles

First Report of Fusarium oxysporum f. sp. lactucae Race 4 Causing Lettuce Wilt in Spain.

Spain is the fourth largest lettuce-producing country in the world and the leading European producer. Much of the production, mainly grow in open field, is dedicated to export with a value of 887 million U.S. dollars per year. In summer 2021 wilting symptoms were observed in a commercial field crop on butterhead lettuce 'Amible' in Albacete (Castilla-La Mancha, Spain). Approximately 15% of plants were affected, but losses were even more severe on subsequent crops. Vascular tissue of affected plants showed a brown to red discoloration. Sections of infected vascular tissue (3 to 5 mm long) were surface sterilized in 70% ethanol for 30 s, washed three times with sterile water, and plated on potato dextrose agar (PDA) amended with streptomycin sulfate (100 mg/liter). From 5-day-old cultures typical pale cream to purplish mycelia with microconidia, macroconidia, and chlamydospores of Fusarium oxysporum were observed. Microconidia were abundant on carnation leaf agar and measured 6.1 to 9.2 μm (mean 7.1 ± 0.7 μm; n=50)). Macroconidia were sparse, three-septate, straight to slightly curved, 23.3 to 34.8 × 4 to 5.2 µm (mean 31.5 ± 2.8 × 4.2 ± 0.3 µm; n=50). Chlamydospores were terminal and intercalary, rough walled, and measured 7.2 to 10.1 μm (mean 9.5 ± 0.6 μm; n=50) µm. DNA was extracted from three single-spore isolates using the protocol of Querol et al. (1992) and the translation elongation factor 1-α gene (TEF) was sequenced with exTEF-F/FUexTEF-R primers as described by Taylor et al. (2016). All TEF sequences (GenBank accession no. OP903519) were identical. In BLAST analyses, the isolates showed 100% identity to the corresponding region of Fusarium oxysporum f. sp. lactucae (FOL) race 4 (MK059958). All Spanish isolates were identified as FOL race 4 using a race-specific polymerase chain reaction (PCR) with the primers FPUF/FPUR (Gilardi et al. 2017), and a previously identified FOL race 4 isolate Fus 1.01 as a positive control. Pathogenicity tests were conducted to confirm the positive result of the race 4-specific PCR and to complete Koch's postulates. Three differential lettuce cultivars ('Costa Rica No. 4', 'Banchu Red Fire', and 'Romana Romabella 30 CN') provided by Rijk Zwaan (The Netherlands) were inoculated with three Spanish isolates (Al1A1, Al1D, Al2B) and the Fus 1.01 isolate used as FOL race 4 positive control (Claerbout et al., 2018). Roots of 3-week-old plants (five replicates per treatment) were dipped in a spore suspension (1 × 106 conidia/ml) for 10 min before transplanting into 250-ml pots with sterile substrate. Non-inoculated control plants were dipped in sterile water for 10 min. The experiment was carried out twice. Inoculated lettuce seedlings were planted and maintained in a growth chamber (25°C day, 18°C night). Plants were slightly watered every other day. After 21 days, wilting was observed in the cultivars 'Costa Rica No. 4' and 'Romana Romabella 30 CN'. Moreover, taproots were cut longitudinally, and vascular browning was observed in the taproot. No discoloration could be observed in the taproot of 'Banchu Red Fire' plants, coinciding with the result of isolate Fus 1.01 used as FOL race 4 positive control. Non-inoculated control plants remained healthy and vascular browning was not observed. In both experiments, F. oxysporum f.sp. lactucae was consistently reisolated using PDA medium. These results confirmed that the isolates Al1A1, Al1D and Al2B were FOL race 4. This race has recently been identified in The Netherlands (Gilardi et al. 2017), Belgium (Claerbout et al. 2018), United Kingdom, Ireland (Taylor et al. 2019) and Italy (Gilardi et al. 2019) and could become a serious threat to Spain lettuce production.

Study of the Differentiation of Fusarium oxysporum f.sp. albedinis Chlamydospores on Different Culture Media

Chlamydospores of Fusarium oxysporum f.sp. albedinis are well known as fungus survival spores in soil, also act as a causal agent of Bayoud disease of the date palm. Till now, there are a little study in the literature about the favorable media for the formation of chlamydospores and the stages of chlamydogenesis. This study shows that rice flour based medium (45 chlamydospores per mm 2), oatmeal (31 chlamydospores per mm 2) and bean flour (27 chlamydospores per mm 2) tested for the first time, are among the media inducing the formation of chlamydospores in large numbers. The optimum formation of chlamydospores is observed on rice flour-based medium after 7 days of Original Research Article Smail et al.; ARRB, 18(4): 1-9, 2017; Article no.ARRB.35653 2 incubation at 18°C and pH 7. The effect of light does not seem very important on chlamydogenesis. However, relative humidity has a significant effect on the growth and chlamydogenesis of F. oxysporum f.sp. albedinis. The tested G 1 isolate grows and forms chlamydospores at relative humidities of 70 and 100%. The number of chlamydospores varies between 40.01 and 44.05 chlamydospores/mm 2. To form chlamydospores, cultures of F. oxysporum f.sp. albedinis need to be in contact with the favorable culture medium. Thus, the cultures develop and form more chlamydospores on a single layer of cellophane deposited on a culture medium, but show slower growth and do not form chlamydospores on two layers of cellophane. Chlamydospores of Fusarium oxysporum f.sp. albedinis are always at the extremity of the mycelial filaments. They differ in one to four cells in superimposed position. A terminal hyphal bulge leads to the formation of the terminal cell. The second cell is formed at the base of the first, and the process continues so that one can sometimes observed chlamydospores in bead-like chains.

Effects of heat stress on loss of C, germination and pathogenicity from chlamydospores of Fusarium oxysporum f. sp. ciceri

Chlamydospores of Fusarium oxysporum f. sp. ciceri lost different amounts of organic C when exposed to continuous lethal heating (45 or 50°C for 6–72 h), short-duration sublethal heating (40 or 45°C for 30 min) and a daily intermittent heating cycle (2 h at 40°C for 1–15 d). Loss of 14C was detected as 14CO 2 evolved from the chlamydospores and by 14C exudation in soil. Chlamydospores showed highest C losses (32.5%) under intermittent heating, followed by the sublethal (15.2%) and continuous lethal (11.9%) heat treatments. Most of the 14CO 2 evolution, and 14C exudation occurred, during the first 24 h of exposure to soil after treatment. Loss of ability to germinate was observed for all heat-treated chlamydospores. Pathogenic aggressiveness of the chlamydospores on chickpeas exposed to various heat-stress conditions was less than that of unheated chlamydospores. Chlamydospores which were subjected to various heat-stress conditions and then colonized by bacteria, fungi and actinomycetes in soil, sustained a further loss of viability and pathogenic aggressiveness. The latter results demonstrate an important mechanism involved in biological control of this pathogen.

Biochemical studies on the cell wall degradation of Fusarium oxysporum f.sp. lycopersici race 2 by lytic enzymes from Mucorales for its biocontrol

The lysis of cell wall, cell wall fractions (F1S—alkali and water soluble, F1I—alkali soluble and water insoluble, and F4—β-chitin-glucan) and chlamydospores of Fusarium oxysporum f.sp. lycopersici race 2 (Fol 2) with crude enzyme preparations from Mucorales (Absidia californica, Mortierella vinacea, Mucor hiemalis, Phycomyces blakesleeanus, Rhizopus stolonifer and Syncephalastrum racemosum) has been studied during an incubation period of 72 h. The lysis of the F1S fraction and chlamydospores was followed by the release of reducing substances. The lysis of the other fractions and cell walls was followed by percentage of decrease in optical density (lysis degree) and release of reducing substances. A correlation between the degradation of cell walls and cell wall fractions was observed. The soluble fraction was the most resistant to lysis by these enzymes, with the highest release of reducing substances at 4% of the material as glucose equivalent using the crude enzyme preparation from Mucor hiemalis. Crude enzyme preparations from Rhizopus stolonifer produced the highest lysis (33.4%) and release of reducing substances (22.1%) from the Fol2 cell wall. The enzymes from M. hiemalis produced the highest degradation of chlamydospores. A possible biological control of Fol 2 with lytic enzymes from R. stolonifer or M. hiemalis is indicated. We wish to thank Dr J. Tello for the Fol 2 strain, Dr P. Melgarejo for her valuable discussion and Mrs Carmelina Rodriguez for her technical assistance. This work was supported by the Spanish programmes ‘Plan Nacional de Investigacion Cientifica y Desarrollo Tecnologico’ (AGR91-0014-C02-02) and ‘Plan Nacional de Investigacion de la Comunidad de Madrid’ (C052/91) and DGICYT PB92–0001. We also wish to thank Norel S.A. for their contract to one of us (O.M. Nuero).

Effect of different environmental conditions on bacterial chemotaxis toward fungal spores

The chemotactic response of four common soil bacteria, Agrobacterium radiobacter, Bacillus subtilis, Pseudomonas fluorescens, and Xanthomonas malvacearum, was observed in natural soil to conidia of Cochliobolus sativus, chlamydospores of Fusarium oxysporum f.sp. ciceri, sclerotia of Macrophomina phaseolina, and oospores of Phytophthora drechsleri f.sp. cajani. All bacteria migrated through soil toward fungal spores. Chemical analysis of fungal exudates demonstrated the presence of various amino acids and sugars that served as chemoattractants. The effect of temperature, pH, soil water matric potential, and soil texture on bacterial chemotaxis toward fungal spores was also investigated. In general, the response of bacteria to different types of fungal spores was significantly greater (P = 0.05) at higher water matric potential (0 and −5 kPa) than at lower soil water potential (−10 and −20 kPa). The chemotactic response was greatest in sandy soil, followed by sandy loam and clay loam soil. High temperature (40 °C) was not favourable for bacterial chemotaxis. The chemotactic response decreased by 4 to 28 times when soil pH decreased from 7 to 5 or increased from 7 to 9. The relative concentration response of the exudate of fungal spores and the motility or chemotaxis of bacteria were assessed. A significant positive correlation (P = 0.05; r = 0.89–0.92) was recorded between motility and relative concentrations of exudate. The swimming speed and tumbling frequency of Pseudomonas fluorescens cells was observed at different pH values and temperatures. The swimming speed of bacteria increased with increasing temperature, but decreased with an increase in pH from 5 to 7. The tumbling frequency had a sharp peak at 30 °C and pH 7. Bacteria were able to stimulate the release of exudate from the fungal spores. The amount of exudation was also affected by temperature, pH, and soil texture. A positive significant correlation (P = 0.05; r = 0.79–0.85) was recorded between exudation and a progressive increase in temperature. The amount of exudation also increased with an increase in soil pH from 5 to 8, but further increases in pH decreased the rate of exudation. In general, fungal spores exuded the greatest amounts of carbon compounds in sandy soil, followed by sandy loam and clay loam soil. Exudation in sandy soil was often greatest in the presence of A. radiobacter cells, while the greatest exudation in sandy loam and clay loam soils was induced by B. subtilis cells.Key words: chemotaxis, chemoattractants, fungal spores, motility.

Influence of host and non-host rhizospheres and organic amendments on survival of Fusarium oxysporum f. sp. elaeidis

Chlamydospores of Fusarium oxysporum f. sp. elaeidis germinated and produced mycelium in the rhizospheres of susceptible and tolerant cultivars of the host ( Elaeis guineensis ) and four non-host plants. Higher chlamydospore germination was recorded in the non-host rhizospheres than in host rhizospheres and no significant differences were recorded in germination response to susceptible and tolerant host rhizospheres. Increase in the soil population of F. oxysporum f. sp. elaeidis was stimulated by all test plants. Of the five plants tested, Zea mays was the most active in the stimulation of fungal growth and survival. All organic amendments tested except farmyard manure, applied at 1 and 5 % (w/w) to infested soils, stimulated chlamydospore germination and initial increases in the population of the pathogen. Germination was, however, soon followed by lysis of germlings and subsequent reduction in the population of the fungus. All amendments except Manihot esculenta leaves caused significant reduction in pathogen populations of amended soils below those in non-amended soils, 4 wk after application. Farmyard manure was the most effective in reducing the population of F. oxysporum f. sp. elaeidis .

The Possible Role of Competition betweenTrichoderma harzianumandFusarium oxysporumon Rhizosphere Colonization

Soil was enriched with chlamydospores of Fusarium oxysporum f.sp. asinfectum and F. oxysporum f. sp. melonis and amended with increasing concentrations of glucose and asparagine. Maximal germination of chlamydospores was obtained in soil amended with 0.4 mg of glucose and 0.8 mg of asparagine per gram of soil. Addition of conidia of the biocontrol agent Trichoderma harzianum (T-35) significantly (P=0.05) reduced the chlamydospore germination rate of both Fusaria. (...)

Weakening Effect on Propagules ofFusariumby Sublethal Heating

Sublethal heating of conidia and chlamydospores of Fusarium oxysporum f.sp. niveum at 38-42 C caused 0-33% reduction in propagule viability and resulted in a weakening effect in the surviving propagules. This weakening effect was expressed as a delay in germination, in reduction in growth of conidial and chlamydospore germ tubes, and in enhanced decline of the population density of viable conidia in soil. Viability of conidia that were heat-treated or exposed to solarized soil declined faster than unheated conidia in a soil suspension culture

Reduced chlamydospore formation and enhanced lysis of chlamydospores ofFusarium oxysporum in soil with added urea or ammonium chloride

Addition of 0.1% NH4Cl or 0.1% urea to one of two soils tested had little or no effect in one soil, but resulted in fewer chlamydospores ofFusarium oxysporum present after 6 weeks in the other soil. This was due to reduced formation of chlamydospores and, particularly, to enhanced lysis of chlamydospores. The hypothesis that ammonia was the causal factor was tested. Ammonia evolved from NH4Cl and urea in both soils to the same amount. Since the effects of NH4Cl and urea were different in the two soils, it is concluded that ammonia was not responsible for the reduced number of chlamydospores. Possibly nitrite is involved.

Abiotic factors and chlamydospore formation in Fusarium oxysporum f.sp. apii

Production of chlamydospores of Fusarium oxysporum f.sp. apii , race 2, was optimum at 24–27°C, pH 7.1, a matric potential of −25 centibars and an osmotic potential of zero bars.

The fine structure of mature and germinating chlamydospores of Fusarium oxysporum.

A number of features not described previously has been revealed in electron-microscope studies of mature chlamydospores of Fusarium oxysporum. On the maturation of one isolate, many spores formed a thick matrix-like layer containing electron-dense particles on the exterior surface of the spore wall. In thin sections of mature chlamydospores of the same isolate, cisternae of endoplasmic reticulum surrounding, and in close apposition to, the limiting boundary of the lipid bodies were revealed. The germination of chlamydospores was accompanied by (a) the rapid appearance of polysaccharide deposits and changes in the configuration of some subcellular organelles, (b) the formation of a new wall layer between the plasma membrane and the innermost layer of the spore wall, (c) the rupture of the outermost coats of the spore wall, and (d) the emergence of the germ tube as an extension of the new wall layer.

Fine structure of Chlamydospore germination in Fusarium oxysporum

Twelve-month-old chlamydospores of Fusarium oxysporum germinated readily in distilled water, but newly developed chlamydospores required a carbohydrate source before germination occurred. In the presence of growing tomato roots, germinating chlamydospores developed germ-tubes that were often wider than the chlamydospore. Germ-tubes arose as an extension of a tertiary wall incorporated within the chlamydospore prior to germination.

Germination of chlamydospores of Fusarium oxysporum f. sp. pisi race 1 in the rhizosphere, and penetration of the pathogen into roots of a susceptible and a resistant pea cultivar

Germination of chlamydospores ofFusarium oxysporum f. sp.pisi race 1 in the rhizosphere of pea seedlings and red clover seedlings grown in natural soil heavily infested with the pathogen, was highest in percentage along the actively growing parts of the roots. At these sites, exudation of ninhydrinpositive substances and reducing sugars was most intense with seedlings grown in vitro. No significant difference in the percentage of germinating chlamydospores ofFusarium oxysporum f. sp.pisi race 1 were observed in the rhizosphere soil and on the root surface of homologous parts of roots of seedlings and mature plants of a susceptible ‘Rondo’ and a resistant ‘Rovar’ pea cultivar grown in natural soil heavily infested with the pathogen. Differences in the growth of mycelium of the pathogen on the root surface, or in the attachment of the mycelium to the root surface of both cultivars were not observed. Epidermis and cortex cells of roots of both cultivars reacted on penetration by the pathogen by producing a cellulose thickening of the cell wall, which later became infiltrated with a ligning-like material. A selective effect on the activities of the pathogen in the rhizosphere, on the root surface and in the epidermis and cortex in relation to resistance thus could not be demonstrated. Formation of new chlamydospores from germ tubes of germinating chlamydospores was frequently observed in the rhizosphere of the susceptible and resistant pea cultivar and in the rhizosphere of red clover seedlings.