Impact of the exodermis on infection of roots byFusarium culmorum

Patterns of infection withFusarium culmorum (W G Smith) Saccardo were observed in seedling roots of barley (Hordeum vulgare L.), wheat (Triticum aestivum L.), maize (Zea mays L.) and asparagus (Asparagus officinalis L). Apical regions of the main roots were not infected. Since penetration into the root occurred several days after inoculation and the roots were growing during the experiment, these regions had apparently not been in existence long enough to be infected. In older regions of barley, wheat and asparagus, hyphae entered through the tips of lateral roots. In barley and wheat, which had not developed any suberin lamellae in their subepidermal layer, infection occurred randomly over the remainder of the root. In maize, the fungus penetrated the epidermis at many sites but did not breach the exodermis in which all cells possessed both Casparian bands and suberin lamellae. Maize roots, therefore, sustained only minimal infections. In asparagus, the fungus grew through the short (passage) cells but never the long cells of the exodermis. In doing so, it penetrated cells possessing Casparian bands but lacking suberin lamellae. The results support the hypothesis that suberin lamellae provide effective barriers to the growth ofF. culmorum hyphae.     

Surface interactions of Fusarium graminearum on barley

The filamentous fungus Fusarium graminearum, a devastating pathogen of barley (Hordeum vulgare L.), produces mycotoxins that pose a health hazard. To investigate the surface interactions of F. graminearum on barley, we focused on barley florets, as the most important infection site leading to grain contamination. The fungus interacted with silica‐accumulating cells (trichomes and silica/cork cell pairs) on the host surface. We identified variation in trichome‐type cells between two‐row and six‐row barley, and in the role of specific epidermal cells in the ingress of F. graminearum into barley florets. Prickle‐type trichomes functioned to trap conidia and were sites of fungal penetration. Infections of more mature florets supported the spread of hyphae into the vascular bundles, whereas younger florets did not show this spread. These differences related directly to the timing and location of increases in silica content during maturation. Focal accumulation of cellulose in infected paleae of two‐row and six‐row barley indicated that the response is in part linked to trichome type. Overall, silica‐accumulating epidermal cells had an expanded role in barley, serving to trap conidia, provide sites for fungal ingress and initiate resistance responses, suggesting a role for silica in pathogen establishment.     

Enhanced production of valerenic acid in hairy root culture of Valeriana officinalis by elicitation

Valerenic acid (VA) is a pharmacologically-active sesquiterpene found in valerian (Valeriana officinalis L., Valerianaceae) roots and rhizomes. The plant produces only small amounts of this metabolite naturally. So, induction of hairy roots as well as elicitation can be useful to increase its commercial production. In this study, Wild-type strain ‘A13’ of Agrobacterium rhizogenes was used to induce hairy roots in valerian. The influence of three different elicitors including Fusarium graminearum extract (FE), methyl jasmonate (MJ) and salicylic acid (SA) on VA production in the selected hairy root line ‘LeVa-C4’ was also investigated. The 23-day-old cultures were treated with different concentrations of the elicitors at exposure time of 3 and 7 days. FE (1%) and MJ (100 µM L^?1) highly promoted VA production at 7 days after elicitation, to a level of 12.31- and 6-fold higher than that of non-elicited controls, respectively, and FE did not exert any negative effects on biomass yield of hairy root. SA did not significantly increase the production of VA. This is the first time study to assess the elicitation of hairy root cultures to promote VA biosynthesis in valerian and the resulting experiments demonstrated that F. graminearum extract and MJ were indeed a potent inducer of VA biosynthesis.     

Phytopathogenic fungus Fusarium cerealis in Russia

The fungus Fusarium cerealis is described, which had not been previously identified in Russia. F. cerealis was isolated from wheat and barley roots and grains, as well as from the leaves of thistle collected in the Far Eastern, North Caucasus, and Central regions. F. cerealis strains may be misidentified as F. culmorum or F. graminearum, since the morphological characteristics of these fungi are similar.     

Regulation of a novel Fusarium cytokinin in Fusarium pseudograminearum

Fusarium pseudograminearum is an agronomically important fungus, which infects many crop plants, including wheat, where it causes Fusarium crown rot. Like many other fungi, the Fusarium genus produces a wide range of secondary metabolites of which only few have been characterized. Recently a novel gene cluster was discovered in F. pseudograminearum, which encodes production of cytokinin-like metabolites collectively named Fusarium cytokinins. They are structurally similar to plant cytokinins and can activate cytokinin signalling in vitro and in planta. Here, the regulation of Fusarium cytokinin production was analysed in vitro. This revealed that, similar to deoxynivalenol (DON) production in Fusarium graminearum, cytokinin production can be induced in vitro by specific nitrogen sources in a pH-dependent manner. DON production was also induced in both F. graminearum and F. pseudograminearum in cytokinin-inducing conditions. In addition, microscopic analyses of wheat seedlings infected with a F. pseudograminearum cytokinin reporter strain showed that the fungus specifically induces its cytokinin production in hyphae, which are in close association with the plant, suggestive of a function of Fusarium cytokinins during infection.     

Toxicity,of extracts of barley kernels inoculated withFusarium spp. toArtemia salina

Toxicity toA. salina, of the Fusarium metabolites: deoxynivalenol (DON), its acetylated derivatives (3- and 15-AcDON), zearalenone (ZON), neosolaniol (NEO), nivalenol (NIV), T-2, HT-2 toxins, has been examined and compared with toxicity of extracts of barley kernels (8 cultivars and 4 lines) inoculated withFusarium culmorum, F. graminearum andF. sporotrichioides respectively. Estimated LC₅₀ values were expressed as relative toxicity (RT) in mg DON/kg for samples inoculated withF. culmorum, F. graminearum or in mg T-2/kg forF. sporotrichioides inoculations. Toxicity of extracts of the same genotype/line kernels was compared among different pathogens used for inoculation and differences in Fusarium head blight susceptibility of different genotypes/lines inoculated with the sameFusarium strain were found. Significant correlation between toxicity of extracts (LC₅₀, RT) and toxic metabolites concentration was found ( $\bar r = 0.82$ ; P = 0.01). Bioassays withA. Salina offer a fast, easy and inexpensive method to examine cereal genotypes susceptibility to Fusarium head blight and mycotoxins accumulation in kernels.     

Characterization of Fusarium secorum,a new species causing Fusarium yellowing decline of sugar beet in north central USA

This study characterized a novel sugar beet (Beta vulgaris L.) pathogen from the Red River Valley in north central USA, which was formally named Fusarium secorum. Molecular phylogenetic analyses of three loci (translation elongation factor1α, calmodulin, mitochondrial small subunit) and phenotypic data strongly supported the inclusion of F. secorum in the Fusarium fujikuroi species complex (FFSC). Phylogenetic analyses identified F. secorum as a sister taxon of F. acutatum and a member of the African subclade of the FFSC. Fusarium secorum produced circinate hyphae sometimes bearing microconidia and abundant corkscrew-shaped hyphae in culture. To assess mycotoxin production potential, 45 typical secondary metabolites were tested in F. secorum rice cultures, but only beauvericin was produced in detectable amounts by each isolate. Results of pathogenicity experiments revealed that F. secorum isolates are able to induce half- and full-leaf yellowing foliar symptoms and vascular necrosis in roots and petioles of sugar beet. Inoculation with F. acutatum did not result in any disease symptoms. The sugar beet disease caused by F. secorum is named Fusarium yellowing decline. Since Fusarium yellowing decline incidence has been increasing in the Red River Valley, disease management options are discussed.     

Weed species within cereal crop rotations can serve as alternative hosts for Fusarium graminearum causing Fusarium head blight of wheat

Fusarium graminearum is the main causal agent of Fusarium head blight (FHB) of small grain cereals, but the importance of weeds in the FHB disease cycle and the establishment of F. graminearum in agroecosystems are still not fully understood. The objective of this study was to determine the potential role of weeds present within cereal crop rotations as alternative hosts. F. graminearum was isolated from different organs of asymptomatic weeds sampled from six fields with cereal-crop rotations in Lithuania for two consecutive years (2015 and 2016). The fungi were identified using morphological and molecular methods. Out of 57 weed species that were investigated, 41 (71.9%) harboured F. graminearum isolates. Twenty five weed species were identified as new, previously undocumented, hosts. The majority (73.3%) of the isolates of F. graminearum from this study belonged to the 15ADON genotype while a smaller proportion (23.4%) belonged to the 3ADON genotype. All F. graminearum isolates that were assessed induced FHB symptoms on artificially inoculated spring wheat tested in the field.     

Recent advances in genes involved in secondary metabolite synthesis,hyphal development,energy metabolism and pathogenicity in Fusarium graminearum (teleomorph Gibberella zeae)

The ascomycete fungus, Fusarium graminearum (teleomorph Gibberella zeae), is the most common causal agent of Fusarium head blight (FHB), a devastating disease for cereal crops worldwide. F. graminearum produces ascospores (sexual spores) and conidia (asexual spores), which can serve as disease inocula of FHB. Meanwhile, Fusarium-infected grains are often contaminated with mycotoxins such as trichothecenes (TRIs), fumonisins, and zearalenones, among which TRIs are related to the pathogenicity of F. graminearum, and these toxins are hazardous to humans and livestock. In recent years, with the complete genome sequencing of F. graminearum, an increasing number of functional genes involved in the production of secondary metabolites, hyphal differentiation, sexual and asexual reproduction, virulence and pathogenicity have been identified from F. graminearum. In this review, the secondary metabolite synthesis, hyphal development and pathogenicity related genes in F. graminearum were thoroughly summarized, and the genes associated with secondary metabolites, sexual reproduction, energy metabolism, and pathogenicity were highlighted.     

Screening of certain barley lines for resistance to root rot disease caused by Fusarium graminearum

This investigation was conducted in 2005/2006 and 2006/2007 to test 235 barley lines plus two varieties Giza 127 and Giza 128 for resistance and susceptibility to Fusarium graminearum. All screened barley lines showed varied significant degrees of infestation to root rot pathogen. A screening system is described for identifying barley lines which are effective in controlling resistant or susceptible lines. By detecting small but consistent differences in root rot severity, the bioassay proved effective in large-scale screening for partial resistance: already 335 barley lines and two varieties have been screened. We found five groups (7.12%), 22 barley lines and both varieties are resistant (R) (8.31%); 28 barley lines are moderately resistant (MR) (19.29%); 65 barley lines are moderately susceptible (MS) (27.89%); 94 barley lines are susceptible (S) and (37.39%) 126 barley lines are highly susceptible (HS). The high degree of precision makes this an invaluable tool in the understanding of pathogen aggressiveness, host specialisation and parasitic fitness. Disease scale was strongly negative and had moderate correlation with germination (?0.309?? and ?0.649??) under normal and disease treatment. The correlation between yield and normal and disease treatment during two seasons was strong and negative (?0.834?? and ?0.847??, respectively were detected).     

Accumulation of reactive oxygen species in arbuscular mycorrhizal roots

We investigated the accumulation of reactive oxygen species (ROS) in arbuscular mycorrhizal (AM) roots from Medicago truncatula, Zea mays and Nicotiana tabacum using three independent staining techniques. Colonized root cortical cells and the symbiotic fungal partner were observed to be involved in the production of ROS. Extraradical hyphae and spores from Glomus intraradices accumulated small levels of ROS within their cell wall and produced ROS within the cytoplasm in response to stress. Within AM roots, we observed a certain correlation of arbuscular senescence and H₂O₂ accumulation after staining by diaminobenzidine (DAB) and a more general accumulation of ROS close to fungal structures when using dihydrorhodamine 123 (DHR 123) for staining. According to electron microscopical analysis of AM roots from Z. mays after staining by CeCl₃, intracellular accumulation of H₂O₂ was observed in the plant cytoplasm close to intact and collapsing fungal structures, whereas intercellular H₂O₂ was located on the surface of fungal hyphae. These characteristics of ROS accumulation in AM roots suggest similarities to ROS accumulation during the senescence of legume root nodules.     

Significance of the class II hydrophobin FgHyd5p for the life cycle of Fusarium graminearum

Hydrophobins are small secreted proteins ubiquitously found in filamentous fungi. Some hydrophobins were shown to have functions in fungal development, while others lack known function. Class II hydrophobins from Fusarium graminearum and Fusarium culmorum are characterized by formation of low stability aggregates and their solubility in organic solvents. They are economically relevant to the brewing industry because they can induce beer gushing. Since cellular functions of Hyd5p's are still unknown, we analyzed the influence of FgHyd5p on growth and morphology of F. graminearum using FgΔhyd5 knock-out mutants expressing sGFP under the control of the hyd5 promoter and compared them with the performance of the parent wild type strain. Results demonstrate that FgHyd5p does not affect the colony and hyphal morphology. FgHyd5p affects the hydrophobicity of aerial mycelia but had no obvious function in penetration of hyphae through the water air interface. The hydrophobin affects the morphology of conidia, but not their fitness. Different sources of carbon and nitrogen as well as different pH have no effect on the expression of the hyd5 gene, which was demonstrated to be expressed upon growth of F. graminearum on hydrophobic surfaces.     

Colonization of barley roots by Fusarium culmorum and influence of Pseudomonas fluorescens on the process

The stages of barley root colonization by Fusarium culmorum were studied in sterile vermiculite by the method of fluorescent antibodies. The influence of the antagonistic bacterium Pseudomonas fluorescens on the process of root colonization by F. culmorum was demonstrated. In vermiculite inoculated with F. culmorum, the fungus density on the roots increased gradually. In the case of joint inoculation of vermiculite with the fungus and the bacterium, the F. culmorum density on the roots changed abruptly. It was shown that the site of primary colonization of the roots by the fungus was mainly the zone of root hairs. When Pseudomonas fluorescens was present on the roots, F. culmorum colonized not only root hairs, but also the elongation zone, during the first two days. Introduction of Pseudomonas fluorescens into vermiculite resulted in lower intensity of barley root rot.     

Shotgun Analysis of the Secretome of Fusarium graminearum

Fusarium head blight, caused predominately by Fusarium graminearum, is one of the most destructive diseases of wheat (Triticum aestivum L.) worldwide. To characterize the profile of proteins secreted by F. graminearum, the extracellular proteins were collectively obtained from F. graminearum culture supernatants and evaluated using one-dimensional SDS-PAGE and liquid chromatography-tandem mass spectrometry. A total of 87 proteins have been identified, of which 63 were predicted as secretory proteins including those with known functions. Meanwhile, 20 proteins that are not homologous to genomic sequences with known functions have also been detected. Some of the identified proteins are possible virulence factors and may play extracellular roles during F. graminearum infection. This study provides a valuable dataset of F. graminearum extracellular proteins, and a better understanding of the virulence mechanisms of the pathogen.     

Early generation of nitric oxide contributes to copper tolerance through reducing oxidative stress and cell death in hulless barley roots

The objective of this study was to investigate the specific role of nitric oxide (NO) in the early response of hulless barley roots to copper (Cu) stress. We used the fluorescent probe diaminofluorescein-FM diacetate to establish NO localization, and hydrogen peroxide (H₂O₂)-special labeling and histochemical procedures for the detection of reactive oxygen species (ROS) in the root apex. An early production of NO was observed in Cu-treated root tips of hulless barley, but the detection of NO levels was decreased by supplementation with a NO scavenger, 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO). Application of sodium nitroprusside (a NO donor) relieved Cu-induced root inhibition, ROS accumulation and oxidative damage, while c-PTIO treatment had a synergistic effect with Cu and further enhanced ROS levels and oxidative stress. In addition, the Cu-dependent increase in activities of superoxide dismutase, peroxidase and ascorbate peroxidase were further enhanced by exogenous NO, but application of c-PTIO decreased the activities of catalase and ascorbate peroxidase in Cu-treated roots. Subsequently, cell death was observed in root tips and was identified as a type of programed cell death (PCD) by terminal deoxynucleotidyl transferase dUTP nick end labeling assay. The addition of NO prevented the increase of cell death in root tips, whereas inhibiting NO accumulation further increased the number of cells undergoing PCD. These results revealed that NO production is an early response of hulless barley roots to Cu stress and that NO contributes to Cu tolerance in hulless barley possibly by modulating antioxidant defense, subsequently reducing oxidative stress and PCD in root tips.     

Pathogenic Variation of Fusarium Isolates Associated with Head Blight of Wheat in Australia

This paper examines the level of pathogenic diversity in Australian Fusarium pseudograminearum and Fusarium graminearum isolates for head blight from the assessment of 51 wheat germplasm lines, barley, triticale, rye, maize and sorghum plants. A set of nine putative wheat differentials were selected and assessed with 10 F. graminearum and 12 F. pseudograminearum isolates. Isolates of both species were pathogenic on all the wheat germplasm lines, barley triticale and rye. The isolates differed largely in a quantitative way with only small differential effects and were statistically demarcated into three pathogenicity groups: low, intermediate and high. Such distribution patterns suggest that wheat germplasm lines employ different resistance mechanisms to each group of isolates and the three pathogenicity groups may have different mechanisms controlling pathogenicity. The aggressiveness of F. graminearum and F. pseudograminearum isolates on the wheat germplasm lines were marginally correlated (r = 0.40). Durum wheats were ranked as the most susceptible while Sumai 3, Ituo Komugi, Sotome A, Sotome and Nobeokabouzu komugi were consistently grouped as resistant by both species. These findings reiterate the need to consider pathogen variability in the screening, selection and improvement of resistance to head blight in wheat.     

Induced root-secreted phenolic compounds as a belowground plant defense

Rhizosphere is the complex place of numerous interactions between plant roots, microbes and soil fauna. Whereas plant interactions with aboveground organisms are largely described, unravelling plant belowground interactions remains challenging. Plant root chemical communication can lead to positive interactions with nodulating bacteria, mycorriza or biocontrol agents or to negative interactions with pathogens or root herbivores. A recent study¹ suggested that root exudates contribute to plant pathogen resistance via secretion of antimicrobial compounds. These findings point to the importance of plant root exudates as belowground signalling molecules, particularly in defense responses. In our report,² we showed that under Fusarium attack the barley root system launched secretion of phenolic compounds with antimicrobial activity. The secretion of de novo biosynthesized t-cinnamic acid induced within 2 days illustrates the dynamic of plant defense mechanisms at the root level. We discuss the costs and benefits of induced defense responses in the rhizosphere. We suggest that plant defense through root exudation may be cultivar dependent and higher in wild or less domesticated varieties.Key words: root exudates, plant defense, t-cinnamic acid, fusarium, induced defensePlants grow and live in very complex and changing ecosystems. Because plants lack the mobility to escape from attack by pathogens or herbivores, they have developed constitutive and in addition inducible defenses that are triggered by spatiotemporally dynamic signaling mechanisms. These defenses counteract the aggressor directly via toxins or defense plant structures or indirectly by recruitment of antagonists of aggressors. Whereas induced defenses are well described in aboveground interactions, evidence of the occurrence of such mechanisms in belowground interactions remains limited. The biosynthesis of a defensive molecule could be both constitutive and inducible with a low level of a preformed pool (Fig. 1). In addition, upon encounter of an attacking organism, those levels could be induced to rise locally to a high level of active compound that is able to disarm the pathogen.^2,3 Only a few examples show that root exudates play a role in induced plant defense. Hairy roots of Ocimum basilicum secrete rosmarinic acid only when challenged by the pathogenic fungus Pythium ultimum.⁴ Wurst et al.⁵ reported on the induction of irridoid glycosides in root exudates of Plantago lanceolata in presence of nematodes. In vivo labelling experiments² with ¹³CO₂ showed the induction of phenolic compounds secreted by barley roots after Fusarium graminearum infection and the de novo biosynthesis of root secreted t-cinnamic acid within 2 days. These results show that the pool of induced t-cinnamic acid originated from both pre-formed and newly formed carbon pools (Fig. 1), highlighting a case of belowground induced defense inside and outside the root system.Open in a separate windowFigure 1Suggested mechanisms for the induction of root defense exudates in barley in response to Fusarium attack. Upon pathogen attack by Fusarium, the initial preformed pool of phenolic compounds is increased by the addition of inducible, de novo biosynthesized t-cinnamic acid. Both, the preformed pool and the de novo biosynthesized pool fuel the exudation of defense compounds from infected roots.The concept of fitness costs is frequently presented to explain the coexistence of both constitutive and induced defense.⁶ In the case of induced defense, resources are invested in defenses only when the plant is under attack. In the absence of an infection, plants can optimize allocation of their resources to reproduction and growth to compete with neighbours.⁷ Constitutive defenses are thought to be more beneficial when the probability of attack is high, whereas adjustable, induced defenses are more valuable to fight against an unpredictable pathogen. Non disturbed soil is a heterogeneous matrix where biodiversity is very high and patchy^8,9 and organism motility is rather restricted.¹⁰ As a consequence of the patchiness, belowground environment is expected to be favourable to selection for induced responses.¹¹ The absence of defense root exudates between two infections may form an unpredictable environment for soil pathogens and reduce the chance for adaptation of root attackers. Plants may also use escape strategies to reduce the effect of belowground pathogens. Henkes et al. (unpublished) showed that Fusarium-infected barley plants reduced carbon allocation towards infected roots within a day and increased allocation carbon to uninfected roots. These results illustrate how reallocation of carbon toward non infected root parts represents a way to limit the negative impact of root infection.We have demonstrated the potential of barley plants to defend themselves against soil pathogen by root exudation.² Even the barley cultivar ‘Barke’ used in our study, a modern cultivated variety, was able to launch defense machinery via exudation of antimicrobial compounds when infected by F. graminearum. We suggest that plant defense through root exudation might be cultivar dependent and perhaps higher in wild or less domesticated varieties. Taddei et al.¹² reported that constitutivelyproduced root exudates from a resistant Gladiolus cultivar inhibit spore germination of Fusarium oxysporum whereas root exudates from a susceptible cultivar do not affect F. oxysporum germination. Root exudates from the resistant cultivar contained higher amounts of aromaticphenolic compounds compared to the susceptible cultivar and these compounds may be responsible for the inhibition of spore germination. Metabolic profiling of wheat cultivars, ‘Roblin’ and ‘Sumai3’, respectively, susceptible and resistant to Fusarium Head Blight, showed that t-cinnamic acid was a discriminating factor responsible for resistance/defense function.¹³ Therefore it is likely that wild barley varieties hold higher defense capacities compare to cultivated varieties selected for high yield. In the future, plant breeders in organic and low-input farming could use root-system defense ability as new trait in varietal variation.