Fusarium oxysporum (Schlecht.) Snyd. et Hans. — A dominant fungus species on the root-surface of woody plant seedlings

Summary and conclusions The imperfect fungusFusarium oxysporum dominated the root surface of first-year seedlings of woody plants grown in the forest nursery in eany near Pardubice, Czechoslovakia. The second most frequent species wasCylindrocarpon radicicola whose relative frequency increased towards the end of the growing season. The morphological variability of isolated strains ofFusarium oxysporum was studied; notwithstanding their great variability they all fall within the range of this complex species. The ecological conditions for the occurrence ofFusarium oxysporum on the root surface and the saprophytic or parasitic relations between this fungus and the higher plants are discussed on the basis of personal observations and reports in the literature.     

Barriers to the radial diffusion of ions in maize roots

Summary The water-extractable and ion-exchangeable fractions of the free space of maize roots for sodium ions has been determined. The free space of whole roots, excised roots and isolated stelar and cortical tissues, has been compared and the results examined for any evidence of a barrier between the cortex and the stele. Similarly the free space of whole roots and excised roots, from which the epidermal and outer cortical cells have been removed by shaving, has been compared and the results examined for any evidence of an epidermal barrier.Whole roots gave a free space value some 20% lower than excised roots. It was calculated that this difference could be accounted for if the cortical tissues only were considered in estimating the whole root value, that is if the stele was considered as participating in the excised root but not in the whole root. Samples in which isolated cortical and stelar material were measured together, or separately and the value calculated, gave similar values to those obtained for excised roots. These results are interpreted as evidence that a barrier to free diffusion exists between the cortical and the stelar tissues at or near the endodermis. Shaving both whole and excised roots increased the free space by about 35%. However, as this value was similar for both, it was concluded that the increase was due to the contribution of damaged cortical cells and does not indicate that the epidermis is an effective barrier to the diffusive entry of sodium ions into the root.     

Membrane ATPase Activity of Barley Roots and Potassium Uptake by Isolated Stele and Cortex

Uptake of potassium ions by isolated stelar tissues of barley from 0.5 and 10 mM K⁺ was respectively 13 and 3.6% of that of the cortical tissues. 0.1 mM H₂PO₄, LO mM ATP and 10 mM Ca(NO₃)₂ did not increase the potassium uptake of either stele or cortex during 5 h of uptake period. A time-course incubation for histological demonstration of the ATPase activity of the plasmalemma and tonoplast of the matured sections of the roots demonstrated a greater activity for the cortical than the stelar tissue. In the stelar parenchyma cells, the plasma lemma showed a higher activity than the tonoplast. These results, which support the “leakiness hypothesis” of the stele, are discussed in relation to the proposed mechanisms of radial ion transport in roots.     

The effect of CCCP on ion fluxes in the stele and cortex of maize roots

Summary The accumulation of ⁸⁶Rb labelled potassium by isolated stelar and cortical tissues from 7-day-old roots of Zea mays has been compared with the levels accumulated by these tissues in the intact root. Cortical tissues have similar uptake eapacities in these two conditions whereas stelar tissues only exhibit an uptake capacity in the intact root system. The uncoupler carbonylcyanide m-chlorophenylhydrazone caused a considerable decrease in the uptake of potassium by these tissues. In the intact root system it prevented ions from the bathing medium reaching the stelar tissues. The efflux pattern from preloaded isolated stelar and cortical tissues was considerably altered by the inhibitor, a promotion of the efflux occurring in both of these tissues.It is concluded that stelar tissues only accumulated ions when these are supplied through the root symplasm and that the stelar plasmalemma has only a limited uptake capacity per se. Stelar uptake is thus a reflection of vacuolar accumulation across the tonoplast. There is no evidence in the present study of a carrier-mediated active secretion of ions across the stelar plasmalemma. The fact that the efflux was promoted rather than depressed by the uncoupler supports the postulate that a passive leakage is the final stage in the transport of ions across the plant root.     

Localization and identification of auxin in roots of Zea mays

Summary Roots of 3.5-day-old seedlings of Zea mays cv. Giant White Horsetooth contain an extractable auxin which has chromatographic properties and reactions to chromogenic sprays identical with those of indole-3-acetic acid (IAA). By separating stele from cortex (and root tips) before extraction it was shown that the auxin is localized predominantly in the stele, with little being found in the cortex. Whole roots, isolated cortices and isolated steles accumulate and metabolize exogenously applied IAA-1-¹⁴C. The stelar tissue is distinguished from whole roots and cortical tissue in having a different pattern of IAA metabolism.     

Phenology and fungus-nematode relations of corky root disease of Douglas-fir

Symptoms of corky root disease of Douglas-fir seedlings include: (I) swollen tap- and lateral roots; (2) paucity of lateral roots; and (3) stunted shoot growth. In diseased roots, the primary cortex persisted and enlarged, but secondary vascular tissues, periderm, pericycle and endodermis were reduced. Cylindrocarpon destructans was the fungus most frequently isolated from cortical cells and symptoms were induced by massive inoculum of some isolates but not by dispersed inoculum nor by extracts from cultures. Whereas symptoms first appeared 2 months after seeds sown in May germinated, C. destructans was rarely isolated until 4 months after germination. Relatively more colonies of C. destructans were isolated from corky roots in the winter than in the summer, when Fusarium oxysporum or Mycelium radicis atrovirens were more abundant. Populations of the plant-parasitic nematode Xiphinema bakeri on roots and in adhering soil were highest 2 months after germination and during the summer, correlating positively with the per cent of tap-root affected and negatively with numbers of lateral roots.     

Evidence for proteins specific for vascular elements in intact and cultured tissues and cells of maize

A group of antigenically distinct proteins characteristic for the tissue complex of the vascular cylinders was found in maize (Zea mays L.) seedlings using an immunofiltration technique. Specific stelar antigens present in the fully developed stele (vascular cylinder) of the primary root were also found in steles extracted from adventitious roots and from the mesocotyl but were absent, within the limits of sensitivity of the immunodiffusion tests employed, in root cortex and epidermis. Some of the stelar antigens were also evident in the meristem of the primary root and were present in traces in the scutellum, the mesocotyl node, and the primary leaves plus coleoptile. The specific stelar antigens could be traced in 13- and 15-day-old developing embryos and were definitely expressed by the 21 st day after pollination. Several stelar-specific antigens were found in embryo-derived callus tissues and in stem-derived cells maintained in serial suspension culture. Higher resolution of the stelar antigens by a modified technique of crossed immunoelectrophoresis was used to demonstrate several minor stelar antigens that were presumably characteristic exclusively of the completely differentiated stele. This technique along with sequential immunoprecipitation of labelled proteins provided a semiquantitative estimate of the specific stelar antigens in the meristem and the stele of the primary root, and in suspension-cultured cells which were devoid of noticeable signs of vascular differentiation.     

Chemical control of root disease of Douglas–fir seedlings in relation to fungus and nematode populations

Seed treatment with thiram reduced post–emergence damping–off, while fumigating forest nursery soils with methyl bromide or DD improved Douglas-fir seedling emergence, shoot and root growth, and decreased the incidence of root disease. At an old site, where corky root develops, the benefits from these fumigants were associated with fewer (a) Xiphinema bakeri and (b) isolates of Cylindrocarpon radicicola. At a new site, decrease in Fusarium root rot and increase in shoot growth were related to reduction of populations of Paratylenchus and Pratylenchus, respectively. In unfumigated soils, Fusarium oxysporum was isolated from diseased and healthy roots.     

Cell marking in Arabidopsis thaliana and its application to patch–clamp studies

Ion transport processes at the plasma membrane of plant cells are frequently studied by applying membrane-patch voltage-clamp (patch–clamp) electrophysiological techniques to isolated protoplasts. As plants are composed of many tissues and cell types, and each tissue and cell type may be specialized to a particular function and possess a unique complement of transport proteins, it is important to certify the anatomical origin of the protoplasts used for patch–clamp studies. This paper describes a general molecular genetic approach to marking specific cell types for subsequent patch–clamp studies and presents a specific example: a comparison of the K⁺ currents in protoplasts from cortical and stelar cells of Arabidopsis roots. Transgenic Arabidopsis were generated in which the expression of green fluorescent protein (GFP) from Aequoria victoria was driven by the CaMV 35S promoter (line mGFP3). In roots of the transgenic mGFP3 line, visible fluorescence was restricted to the stele. Protoplasts were generated from roots of the mGFP3 line and K⁺ currents in non-fluorescent (cortical/epidermal) and fluorescent (stelar) protoplasts were assayed using patch–clamp techniques. It was found that both the frequency of observing inward rectifying K⁺ channel (IRC) activity and the relative occurrence of IRC compared to outward rectifying K⁺ channels were significantly lower in protoplasts from cortical/epidermal cells compared to cells of the stele. The presence of GFP did not affect the occurrence or biophysical properties of K⁺ channels. It is concluded that the generation of transgenic Arabidopsis expressing GFP in a cell-specific fashion is a convenient and reliable way to mark protoplasts derived from contrasting cell types for subsequent patch–clamp studies.     

The production of a phytotoxin,nectrolide, by some root-surface isolates ofCylindrocarpon radicicola,Wr.

Summary A phytotoxic compound, for which the name nectrolide has been proposed, was isolated fromCylindrocarpon radicicola. Analysis of nectrolide and its physical and chemical properties show that the compound is identical with brefeldin A, an antibiotic produced byPenicillium brefeldianum.Twenty different isolates ofC. radicicola from plant roots were examined for their ability to produce nectrolide in liquid culture. Twelve of these yielded nectrolide at concentrations varying from 31 to 82 µg per ml when grown for 14 days on Czapek-Dox liquid medium. Eight isolates did not yield the metabolite in culture.At a concentration of 6 µg per ml nectrolide arrested the growth of germinated blackbutt seedlings, whilst at lower concentrations (2–4 µg per ml) the compound caused severe stunting and blackening of the roots of seedling plants. Nectrolide watered onto blackbutt seedlings growing in forest soils can be detected in the roots after 24 hrs. The compound has been crystallised from ether extracts of tomato roots supplied with pure nectrolide and can be detected in stem tissues within 24 hours of watering it onto the roots.As a result of the widespread association ofC. radicicola with unthrifty plants and the phytotoxic nature of nectrolide, it is suggested that this compound may function in a number of plant diseases, the causes of which have not been elucidated.     

Interaction of Fusarium Oxysporum f.sp. Cubense with Pseudomonas Fluorescens Precolonized to Banana Roots

Effect of precolonization of banana cv Neeypovan roots with Pseudomonas fluorescens on infection with Fusarium oxysporum f.sp. cubense was studied. Under in vitro conditions Pseudomonas fluorescens clearly inhibited Fusarium oxysporum f.sp. cubense. Fluorescein isothiocyanate-tagged antibodies raised in a rabbit system for Pseudomonas fluorescens and Fusarium oxysporum f.sp. cubense separately were used to study the spread of both organisms in banana root. It was observed that precolonization with Pseudomonas fluorescens could reduce Fusarium oxysporum f.sp. cubense colonization by 72%, and also correlated with a number of structural changes in the cortical cells, mainly with densely stained amorphous material and polymorphic wall thickenings as revealed by light and electron microscopic studies. Massive depositions of unusual structures at sites of fungal entry was also noticed, which clearly indicated that bacterized root cells were signalled to mobilize a number of defence structures for preventing the spread of pathogen in the tissue. This revised version was published online in July 2006 with corrections to the Cover Date.     

A morphological study of the mycorrhiza ofEntoloma clypeatum f.hybridum onRosa multiflora

Mycorrhizas ofEntoloma clypeatum f.hybridum onRosa multiflora in the field in Japan were studied by stereo, light and electron microscopy. In most mycorrhizas, the root cap, meristem, and apical region of the cortex disappeared, but in a few mycorrhizas, these tissues remained. Fungal hyphae of the mycorrhizas invaded root tissues and branched palmately. Hyphae in contact with cortical cells were larger than those far from the root cells and contained many mitochondria, cisternae of endoplasmic reticulum and transitional vesicles. Invading hyphae were undulate in the apical part of the mycorrhiza, and some of them lacked distinct organelles. Electron-dense granules accumulated in the root cells adjacent to the fungal hyphae. Both the remnants of the plant cells and the fungal hyphae were included in the amorphous materials on the tip of the stele. These observations suggest the destructive infection by fungal hyphae of the root cells and their collapse near the tip of the stele.     

Water use and phosphorus and potassium status of wheat seedlings colonized byGaeumannomyces graminis orPhialophora radicicola

Summary The water consumption and levels of phosphorus, potassium, and total minerals were measured for wheat seedlings colonized byGaeumannomyces graminis var.tritici, Phialophora radicicola var.radicicola, orPhialophora radicicola var.graminicola. Infection byG. graminis resulted in a considerable reduction in water consumption, and reduced level of phosphorus when the supply of phosphorus to the seedlings was plentiful. Colonization byP. radicicola var.radicicola increased levels of phosphorus and potassium, but these increases varied according to the isolate of the fungus and the supply of phosphorus and potassium available to the seedlings. Colonization byP. radicicola var.graminicola resulted in reduced water consumption by the seedlings.The results are discussed in relation to stelar cell wall thickening in wheat roots colonized byP. radicicola, and the effects on nutrient uptake of mycorrhizal root systems.     

Loss of pectin is an early event during infection of cocoyam roots by Pythium myriotylum

Cocoyam (Xanthosoma sagittifolium) is an important tuber crop in most tropical zones of Africa and America. In Cameroon, its cultivation is hampered by a soil-borne fungus Pythium myriotylum which is responsible for root rot disease. The mechanism of root colonisation by the fungus has yet to be elucidated. In this study, using microscopical and immunocytochemical methods, we provide a new evidence regarding the mode of action of the fungus and we describe the reaction of the plant to the early stages of fungal invasion. We show that the fungal attack begins with the colonisation of the peripheral and epidermal cells of the root apex. These cells are rapidly lost upon infection, while cortical and stele cells are not. Labelling with the cationic gold, which binds to negatively charged wall polymers such as pectins, is absent in cortical cells and in the interfacial zone of the infected roots while it is abundant in the cell walls of stele cells. A similar pattern of labelling is also found when using the anti-pectin monoclonal antibody JIM5, but not with anti-xyloglucan antibodies. This suggests that early during infection, the fungus causes a significant loss of pectin probably via degradation by hydrolytic enzymes that diffuse and act away from the site of attack. Additional support for pectin loss is the demonstration, via sugar analysis, that a significant decrease in galacturonic acid content occurred in infected root cell walls. In addition, we demonstrate that one of the early reactions of X. sagittifolium to the fungal invasion is the formation of wall appositions that are rich in callose and cellulose.     

Sequential depolarization of root cortical and stelar cells induced by an acute salt shock - implications for Na(+) and K(+) transport into xylem vessels

Early events in NaCl-induced root ion and water transport were investigated in maize (Zea mays L) roots using a range of microelectrode and imaging techniques. Addition of 100 mm NaCl to the bath resulted in an exponential drop in root xylem pressure, rapid depolarization of trans-root potential and a transient drop in xylem K(+) activity (A(K+) ) within ～1 min after stress onset. At this time, no detectable amounts of Na(+) were released into the xylem vessels. The observed drop in A(K+) was unexpected, given the fact that application of the physiologically relevant concentrations of Na(+) to isolated stele has caused rapid plasma membrane depolarization and a subsequent K(+) efflux from the stelar tissues. This controversy was explained by the difference in kinetics of NaCl-induced depolarization between cortical and stelar cells. As root cortical cells are first to be depolarized and lose K(+) to the environment, this is associated with some K(+) shift from the stelar symplast to the cortex, resulting in K(+) being transiently removed from the xylem. Once Na(+) is loaded into the xylem (between 1 and 5 min of root exposure to NaCl), stelar cells become more depolarized, and a gradual recovery in A(K+) occurs.     

Studies on microbial antagonism in the establishment of clover pasture

Summary Fungi inhabiting soil recently cleared of the native vegetation have been isolated and compared with those isolated from under established clover pastures, Three methods of isolation of the fungi were used in each situation, and profoundly influenced the population obtained.In newly cleared soils the composite fungal population pattern deduced all three isolation methods comprised species of Mortierella, Phoma, Trichoderma, Penicillium, Fusarium, Sclerotium and non-sporing mycelia. In soil supporting established clover pastures the main rhizosphere fungi were species of Fusarium, Aspergillus, Phoma, Curvularia, Macrophomina, Sclerotium and one Ascomycete.Fusarium oxysporum was the most frequently isolated fungus.Conventional plating techniques suggested that the dominant fungi in the rhizosphere of clover wereFusarium oxysporum andAspergillus versicolor, whereas isolation of fungi from hyphae adhering to the clover roots indicated that species of Macrophomina, Curvularia and an Ascomycete were the most abundantly occurring organisms. In the case of the root residues of the native vegetation in newly cleared soils plating techniques resulted in species of the freely sporing Mortierella, Trichoderma, Aspergillus, Fusarium and Penicillium occurring most frequently, whereas the isolation of fungi from hyphal strands on the residues gave a population pattern dominated by species of Mortierella, Phoma, Sclerotium, and non-sporing mycelia.The study indicated a marked succession of fungal species during the decomposition of the root debris in newly cleared soils, and also that some early members of this changing population might exert an adverse effect on the establishment of clover.     

Endophytic microorganisms as potential growth promoters of banana

The potential of endophytic microorganisms in promoting the growth of their host plant was determined by artificially introducing five isolates (bacterial and fungal strains: UPM31F4, UPM31P1, UPM14B1, UPM13B8, UPM39B3) isolated from the roots of wild bananas into both healthy and diseased banana plantlets (Berangan cv. Intan). The response of the host plants to endophytic infection was assessed by measuring the change in four growth parameters: plant height, pseudostem diameter, root mass and total number of leaves. The endophytes tested as growth promoters were found to have a significant effect in both healthy and Fusarium-infected (diseased) plantlets. In both experimental systems, the bacterial isolate UPM39B3 (Serratia) and fungal isolate UPM31P1 (Fusarium oxysporum) showed promising growth-promoting properties. Isolate UPM39B3 (Serratia) induced the largest increases in all four growth parameters in healthy plantlets – 3.14 cm (height), 1.12 cm (pseudostem diameter), 2.12 g (root mass) and 1.12 (total number of leaves plant⁻¹) – followed by isolate UPM31P1 (Fusarium oxysporum). The beneficial effect of UPM39B3 (Serratia) and UPM31P1 (Fusarium oxysporum) was also reflected in the diseased plantlets, where pre-treatments with the isolates either singly (T6: UPM31P1; T8: UPM39B3) or in a mixture (T7: UPM31P1 + UPM39B3; T9: UPM14B1 + UPM13B8 + UPM39B3) were able to sustain the growth of plantlets, with significantly higher growth values than those in diseased plantlets that were not infected with endophytes (T10: FocR4). These results demonstrate the economic significance of these endophytic isolates, particularly UPM39B3 (Serratia) and UPM31P1 (Fusarium oxysporum), both as potential growth promoters of banana and as agents rendering tolerance towards Fusarium wilt as a strategy in the management of Fusarium wilt of banana via improved vegetative growth.     

Accumulation of secondary compounds in barley and wheat roots in response to inoculation with an arbuscular mycorrhizal fungus and co-inoculation with rhizosphere bacteria

 Colonization of Hordeum vulgare L. cv. Salome (barley)and Triticum aestivum L. cv. Caprimus (wheat) roots by the arbuscular mycorrhizal fungus Glomus intraradices Schenck & Smith leads to de novo synthesis of isoprenoid cyclohexenone derivatives with blumenin [9-O-(2′-O-β-glucuronosyl)-β-glucopyranoside of 6-(3-hydroxybutyl)-1,1,5-trimethyl-4-cyclohexen-3-one] as the major constituent and to transient accumulation of hydroxycinnamate amides (4-coumaroylagmatine and -putrescine). Accumulation of these compounds in mycorrhizal wheat roots started 2 weeks after sowing together with the onset of arbuscule formation and proceeded with mycorrhizal progression. Highest levels were reached in 3- to 4-week-old secondary roots (root branches of first and higher order) characterized by the formation of vesicles. In the final developmental stages, the fungus produced massive amounts of spores, enclosing the stele of older root parts (older than 5 weeks) characterized by cortical death. In these root parts, the secondary compounds were detected in trace amounts only, indicating that they were located in the cortical tissues. Some rhizosphere bacteria tested, i.e. Agrobacterium rhizogenes, Pseudomonas fluorescens, and Rhizobium leguminosarum, markedly stimulated both fungal root colonization and blumenin accumulation, thus, acting as mycorrhiza-helper bacteria (MHB). Application of blumenin itself strongly inhibited fungal colonization and arbuscule formation at early stages of mycorrhiza development. This was associated with a markedly reduced accumulation of the hydroxycinnamate amides 4-coumaroylputrescine and -agmatine. The results suggest that both the isoprenoid and the phenylpropanoid metabolism are closely linked to the developmental stage and the extent of fungal colonization. Their possible involvement in the regulation of mycorrhiza development is discussed. Accepted: 18 September 1998     

Induction of resistance against Fusarium wilt of tomato by combination of chitosan with an endophytic bacterial strain: ultrastructure and cytochemistry of the host response

The potential of Bacillus pumilus (PGPR strain SE 34), either alone or in combination with chitosan, for inducing defense reactions in tomato (Lycopersicon esculentum Mill.) plants inoculated with the vascular fungus, Fusarium oxysporum f. sp. radicis-lycopersici, was studied by light and transmission electron microscopy and further investigated by gold cytochemistry. The key importance of fungal challenge in the elaboration of defense mechanisms is discussed in relation to the possibility that an alarm signal provided by the pathogen itself is required for the expression of resistance in plants previously sensitized by biotic agents. Ultrastructural investigations of the infected root tissues from water-treated (control) plants showed a rapid colonization of all tissues including the vascular stele. In root tissues from bacterized tomato plants grown in the absence of chitosan, the limited fungal development coincided with marked changes in the host physiology. The main facets of the altered host metabolism concerned the induction of a structural response at sites of fungal entry and the abnormal accumulation of electron-dense substances in the colonized areas. A substantial increase in the extent and magnitude of the cellular changes induced by B. pumilus was observed when chitosan was supplied to bacterized tomato plants. These changes were characterized by a considerable enlargement of the callose-enriched wall appositions deposited onto the inner cell wall surface in the epidermis and the outer cortex. The use of the wheat germ agglutinin-ovomucoid-gold complex provided evidence that the wall-bound chitin component in Fusarium cells colonizing bacterized tomato roots was not substantially altered. One of the most-typical fungal cell reactions, observed only when bacterized tomato plants were grown in the presence of chitosan, was the formation of abnormal chitin-enriched deposits between the retracted plasma membrane and the cell wall. Results of the present study provide the first evidence that combination of biocontrol approaches is a promising step towards elaborating integrated pest management programmes. Received: 6 June 1997 / Accepted: 8 July 1997     

Developmental Regulation of Susceptibility and Tolerance to Fusarium Root Rot in Beans

The soil-borne fungus, Fusarium solani f. sp. phaseoli, attacks roots and hypocotyls of bean (Phaseolus vulgaris) plants causing a devastating disease called root and foot rot. In a study of the host-pathogen relationship it was found that young bean roots, with the radicle just emerging, were highly tolerant to the pathogen, whereas older bean seedlings, with a fully developed root system, were completely susceptible. Investigations by low-temperature scanning electron microscopy demonstrated that significantly fewer spores and hyphae were present on the root surface of young bean seedlings as compared to older ones. A similar pattern of attachment was found when bean roots were inoculated with spores of F. solani f. sp. pisi, a related pathogen causing disease on peas but not on beans. Light microscopic studies showed that F. solani f. sp. pisi did not penetrate the root but rapidly formed thick-walled resting spores on the root surface. F. solani f. sp. phaseoli on the other hand quickly penetrated the root and formed an extensive network of fungal hyphae. These results demonstrate that the ability of fungal propagules to adhere to and to penetrate host tissues are two distinct processes. Furthermore, the data indicate that young bean roots lack a surface component necessary for attachment of fungal spores which may help explain their tolerance to Fusarium root rot.