Elemental Sulfur and Thiol Accumulation in Tomato and Defense against a Fungal Vascular Pathogen

The occurrence of fungicidal, elemental S is well documented in certain specialized prokaryotes, but has rarely been detected in eukaryotes. Elemental S was first identified in this laboratory as a novel phytoalexin in the xylem of resistant genotypes of Theobroma cacao, after infection by the vascular, fungal pathogen Verticillium dahliae. In the current work, this phenomenon is demonstrated in a resistant line of tomato, Lycopersicon esculentum, in response to V. dahliae. A novel gas chromatography-mass spectroscopy method using isotope dilution analysis with 34S internal standard was developed to identify unambiguously and quantify 32S in samples of excised xylem. Accumulation of S in vascular tissue was more rapid and much greater in the disease-resistant than in the disease-susceptible line. Levels of S detected in the resistant variety (approximately 10 microg g-1 fresh weight excised xylem) were fungitoxic to V. dahliae (spore germination was inhibited >90% at approximately 3 microg mL-1). Scanning electron microscopy-energy dispersive x-ray microanalysis confirmed accumulation of S in vascular but not in pith cells and in greater amounts and frequency in the Verticillium spp.-resistant genotype. More intensive localizations of S were occasionally detected in xylem parenchyma cells, vessel walls, vascular gels, and tyloses, structures in potential contact with and linked with defense to V. dahliae. Transient increases in concentrations of sulfate, glutathione, and Cys of vascular tissues from resistant but not susceptible lines after infection may indicate a perturbation of S metabolism induced by elemental S formation; this is discussed in terms of possible S biogenesis.     

Accumulation of Heavy Metals in Cell Walls of Polygonum cuspidatum Roots from Metalliferous Habitats

The distribution and accumulation of Cu²⁺, Zn²⁺ and Cd²⁺ ionsin the roots of Polygonum cuspidatum, collected from metalliferousand non-metalliferous habitats, were examined. About 90% ofthe metal ions was located in the cell wall fraction of rootsof plants grown in either type of habitat. The metal-ion exchangecapacity of the cell walls is not specific to the plants frommetalliferous habitats, and there were no significant differencesamong plants from the various habitats. The capacity for Cu²⁺ions was greater than that for Zn²⁺ or Cd²⁺ ions. Cu²⁺ ionshave a high affinity for the cell wall and, thus, it appearsthat the cell wall is a barrier for the transportation of Cu²⁺into the cytoplasm. (Received March 10, 1988; Accepted March 17, 1989)     

Activation of Glycosidases as a Consequence of Infection Stress in Fusarium Wilt of Tomato

Changes of β-1,3-glucanase, chitinase, β-1,4-glucosidase and N-acetylglucosaminidase activity have been investigated in relation to the development of symptoms and colonization by the pathogen in roots, stems and leaves of susceptible (‘Improved, Pearson’) and resistant (‘Improved Pearson VF11’) tomato plants infected by Fusarium oxysporum f. sp. lycopersici. Glycosidase activities increased after inoculation to different extents depending on the plant part and cultivar. Increases were always higher in susceptible than in resistant plants. Changes in the β-1,3-glucanase activity after inoculation were particularly large in stems of infected plants. In contrast, chitinase activity increased more in roots than in stems. The β-1,3-glucosidase and chitinase activity decreased slightly from the basal to the apical third of stems. The trend of changes of the glycosidase activity generally were well related with the severity of disease symptoms and the fungal colonization of basal stem segments. There was no evidence that the increase of glycosidase activity after the infection was directly related with the resistance to Fusarium wilt in tomato.     

Mitigation of Tomato Spotted Wilt Virus Infection and Symptom Expression by Water Stress

The effects of water stress on the infection and systemic movement of tomato spotted wilt virus (TSWV) in vegetative tomato plants are examined. Two groups of plants: water stressed (water potential ?1.31 MPa) and well-watered ones (water potential ?0.65 MPa), were mechanically inoculated on the third expanded leaf from the top. Each group was then divided into a well-watered and a water stressed one. TSWV inoculation was effective both on water-stressed and well-watered plants. In all groups, infection was first detected in roots, then in shoot apices and subsequently in the rest of the leaves. Systemic virus infection was somewhat slower in the water stressed plants. Post inoculation stress treatments had a significant effect in reducing infection as well as in attenuating TSWV symptoms. Results from the present work suggest that water stress may be effective in controlling virus concentration and systemic infection symptoms in tomato plants and could thus be used as a tool to study the regulation of development of TSWV infection in this system.     

Ion-Exchange Properties of Cell Walls Isolated from Lupine Roots

Acid–base properties of cell walls isolated from various root tissues of 7-day-old lupine seedlings and 14-day-old lupine plants grown in various media were studied. The ion-exchange capacity of root cell walls was estimated at various pH values (from 2 to 12) and constant ionic strength (10 mM). The parameters determining the qualitative and quantitative composition of cell wall ionogenic groups along the root length and in its radial direction were estimated using Gregor's model. This model fits the experimental data reasonably well. Four types of ionogenic groups were found in the cell walls: an amino group (pK _a 3), two types of carboxylic groups (pK _a 5 and 7.3, the first being the carboxylic group of galacturonic acid), and a phenolic group (pK _a 10). The number of functional groups of each type was estimated, and the corresponding ionization constant values were calculated. It is shown that the chemical composition of the ionogenic groups was constant along the root length as well as in its radial direction and did not depend on either physiological state or root nutrition, while the number of different groups varied. The content of carboxylic groups of -D-polygalacturonic acid in the root cell walls of 14-day-old plants was shown to depend on the distance from the root tip, being maximal in the zone of lateral roots. The number of these groups was 10- and 2-fold less in the central cylinder compared to that of cortex for 14-day-old plants and 7-day-old seedlings, respectively.     

Modifications of Cortical Cell Walls in Roots of Seedless Vascular Plants

Forty-three species of seedless vascular plants were assessed for modifications to root cortical cell walls. All species except Lycopodium had an endodermis with distinct Casparian bands. Experiments with the apoplastic tracer berberine hemisulfate showed that walls of all root cortical cells in the two Lycopodium species tested were permeable to this tracer. Although most species examined lacked a hypodermis several Equisetum species had a hypodermis with modified walls. Three Selaginella species had distinct Casparian bands in this cortical cell layer. This layer, therefore, is an exodermis in Selaginella and its presence limited the inward diffusion of the apoplastic tracer berberine hemisulfate.     

Formation of Di-Isodityrosine and Loss of Isodityrosine in the Cell Walls of Tomato Cell-Suspension Cultures Treated with Fungal Elicitors or H2O2

About 84% of the hydroxyproline residues in a cell culture of tomato (Lycopersicon esculentum x Lycopersicon peruvianum) were present in phenol-inextractable (i.e. covalently wall-bound) material. Treatment of the cells with any of three fungal elicitors (wall fragments from Phytophthora megasperma and Pythium aphanidermatum and xylanase from Aureobasidium pullulans) or with 1 mM H2O2 had little effect on the quantity of phenolinextractable hydroxyproline per milligram of freeze-dried cells. However, each treatment induced a decrease in the content of phenol-inextractable isodityrosine (Idt) residues. Each treatment, except with the P. megasperma fragments, also induced an increase in phenol-inextractable di- (Di-Idt). The increase in Di-Idt partly accounted for the loss of Idt. We conclude that the elicitors and H2O2 acted to reinforce the existing cross-linking of cell wall (glyco)proteins by evoking oxidative coupling reactions to convert Idt to Di-Idt plus unidentified products. The promotion of cross-linking by elicitor treatment is proposed to be a defensive response that restricts the penetration of pathogens.     

miR482 Regulation of NBS-LRR Defense Genes during Fungal Pathogen Infection in Cotton

In this study, we characterized the miR482 family in cotton using existing small RNA datasets and the recently released draft genome sequence of Gossypium raimondii, a diploid cotton species whose progenitor is the putative contributor of the D_t (representing the D genome of tetraploid) genome of the cultivated tetraploid cotton species G. hirsutum and G. barbadense. Of the three ghr-miR482 members reported in G. hirsutum, ghr-miR482a has no homolog in G. raimondii, ghr-miR482b and ghr-miR482c each has a single homolog in G. raimondii. Gra-miR482d has five homologous loci (gra-miR482d, f-i) in G. raimondii and also exists in G. hirsutum (ghr-miR482d). A variant, miR482.2 that is a homolog of miR2118 in other species, is produced from several GHR-MIR482 loci in G. hirsutum. Approximately 12% of the G. raimondii NBS-LRR genes were predicted targets of various members of the gra-miR482 family. Based on the rationale that the regulatory relationship between miR482 and NBS-LRR genes will be conserved in G. raimondii and G. hirsutum, we investigated this relationship using G. hirsutum miR482 and G. raimondii NBS-LRR genes, which are not currently available in G. hirsutum. Ghr-miR482/miR482.2-mediated cleavage was confirmed for three of the four NBS-LRR genes analysed. As in tomato, miR482-mediated cleavage of NBS-LRR genes triggered production of phased secondary small RNAs in cotton. In seedlings of the susceptible cultivar Sicot71 (G. hirsutum) infected with the fungal pathogen Verticillium dahliae, the expression levels of ghr-miR482b/miR482b.2, ghr-miR482c and ghr-miR482d.2 were down-regulated, and several NBS-LRR targets of ghr-miR482c and ghr-miR482d were up-regulated. These results imply that, like tomato plants infected with viruses or bacteria, cotton plants are able to induce expression of NBS-LRR defence genes by suppression of the miRNA-mediated gene silencing pathway upon fungal pathogen attack.     

Environmentally Induced Changes in the Cell Walls of Tomato Leaves in Relation to Cell and Protoplast Release

Factors involved in the isolation of protoplasts from the leaves of tomato plants grown over a wide range of environmental conditions have been studied. Increases in calcium pectate in summer grown (“hard”) plants are suggested as a barrier to cell wall degradation. A one-step method involving the addition of sodium citrate to pectinase plus cellulase gives high yield of protoplasts from hard plants. Attempts to convert isolated palisade cells to protoplasts have failed. The plant culture conditions are described such that protoplasts may be isolated throughout the year using low enzyme concentrations.     

Reduction of Feeding by the Variegated Grasshopper, Zonocerus variegatus , Following Infection by the Fungal Pathogen, Metarhizium flavoviride

The effect of infection by the fungal entomopathogen , Metarhizium flavoviride, on feeding by the tropical grasshopper pest , Zonocerus variegatus, was investigated in field - cage studies . A significant reduction in feeding , as indicated by faecal production , was recorded 2 - 3 days after inoculation for a range of spore doses (104 , 105 and 5 105 spores per insect) . This was before any mortality was recorded due to infection . All infected individuals died by day 7 . At this time , faecal production of the treated grasshoppers was equivalent to less than 2 days faecal production by grasshoppers untreated with spores . This reduction in feeding (69 , 71 and 74% total reduction by day 7 in the 104 , 105 and 5 105 doses respectively in comparison to controls) is a substantial contribution to the overall effects of the slow acting pathogen . Furthermore , the rapid reduction in feeding indicated that this effect was not simply due to invasion of the host tissues by the pathogen or production of secondary metabolites . The possibility that reduction in feeding is associated with a behavioural response in which there is a trade - off between host defence and feeding during early stages of infection is discussed     

Effect of Short-Term Salinity on Lipid Metabolism and Ion Accumulation in Tomato Roots

To examine the ion accumulation and membrane lipid metabolism in response to salinity we compared two tomato cvs. Pera and Hellfrucht Fruhstamm (HF), considered to be salt-tolerant and sensitive respectively. Na⁺ and K⁺ accumulation was significantly higher in roots of cv. Pera after 24 h and 72 h of 100 mM NaCl. While in cv. HF, a temporary increase in K⁺ accumulation at 24 h was accompanied by a sustained increase in Na⁺ content. Both cultivars enhanced incorporation of [³²P]orthophosphate into phosphatidylinositol 4,5-bisphosphate at 24 h and 72 h of NaCl. In parallel to the increase of phosphatidylinositol 4,5-bisphosphate a decrease in phosphorylation of phosphatidic acid and phosphatidylcholine were observed in the sensitive cv. HF. Structural and signal lipid changes in response to salinity were more evident in the sensitive cv. HF. Salt tolerant cv. Pera accumulated Na⁺ ions in the roots without considerable modifications in lipid metabolism. This revised version was published online in July 2006 with corrections to the Cover Date.     

Salicylate Degradation by the Fungal Plant Pathogen Sclerotinia sclerotiorum

The fungal plant pathogen Sclerotinia sclerotiorum was studied to determine its ability to degrade salicylate, an important defense-signaling molecule in plants. S. sclerotiorum D-E7 was grown at 25 °C in an undefined medium (50 ml) containing minerals, 0.1 % soytone, 50 mM MES buffer (pH 6.5), 25 mM glucose, and 1 mM salicylate. Glucose, oxalate, and salicylate concentrations were monitored by HPLC. S. sclerotiorum D-E7 was found to be active in salicylate degradation. However, salicylate alone was not growth supportive and, at higher levels (10 mM), inhibited glucose-dependent growth. Biomass formation (130 mg [dry wt] of mycelium per 50 ml of undefined medium), oxalate concentrations (~10 mM), and culture acidification (final culture pH approximated 5) were essentially the same in cultures grown with or without salicylate (1 mM). Time-course analyses revealed that salicylate degradation and glucose consumption were complete after 7 days of incubation and was concomitant with growth. Trace amounts of catechol, a known intermediate of salicylate metabolism, were detected during salicylate degradation. Overall, these results indicated that S. sclerotiorum has the ability to degrade salicylate and that the presence of low levels of salicylate did not affect growth or oxalate production by S. sclerotiorum.     

External Reinfection of a Fungal Pathogen Does not Contribute to Pathogen Growth

Chytridiomycosis is an emerging infectious disease of amphibians caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd), which has led to devastating declines in amphibian populations worldwide. Current theory predicts that Bd infections are maintained through both reproduction on the host’s skin and reinfection from sources outside of the host. To investigate the importance of external reinfection on pathogen burden, we infected captive-bred individuals of the highly susceptible Panamanian Golden Frog, Atelopus glyphus, and wild-caught glass frogs, Espadarana prosoblepon, with Bd. We housed the animals in one of three treatments: individually, in heterospecific pairs, and in conspecific pairs. For 8 weeks, we measured the Bd load and shedding rate of all frogs. We found that Atelopus had high rates of increase in both Bd load and shedding rate, but pathogen growth rates did not differ among treatments. The infection intensity of Espadarana co-housed with Atelopus was indistinguishable from those housed singly and those in conspecific pairs, despite being exposed to a large external source of Bd zoospores. Our results indicate that Bd load in both species is driven by pathogen replication within an individual, with reinfection from outside the host contributing little to the amplification of host fungal load.