Nicole Benhamou

Accumulation of an acidic dehydrin in the vicinity of the plasma membrane during cold acclimation of wheat

Expression of the acidic dehydrin gene wcor410 was found to be associated with the development of freezing tolerance in several Gramineae species. This gene is part of a family of three homologous members, wcor410, wcor410b, and wcor410c, that have been mapped to the long arms of the homologous group 6 chromosomes of hexaploid wheat. To gain insight into the function of this gene family, antibodies were raised against the WCOR410 protein and affinity purified to eliminate cross-reactivity with the WCS120 dehydrin-like protein of wheat. Protein gel blot analyses showed that the accumulation of WCOR410 proteins correlates well with the capacity of each cultivar to cold acclimate and develop freezing tolerance. Immunoelectron microscope analyses revealed that these proteins accumulate in the vicinity of the plasma membrane of cells in the sensitive vascular transition area where freeze-induced dehydration is likely to be more severe. Biochemical fractionation experiments indicated that WCOR410 is a peripheral protein and not an integral membrane protein. These results provide direct evidence that a subtype of the dehydrin family accumulates near the plasma membrane. The properties, abundance, and localization of these proteins suggest that they are involved in the cryoprotection of the plasma membrane against freezing or dehydration stress. We propose that WCOR410 plays a role in preventing the destabilization of the plasma membrane that occurs during dehydrative conditions.

Induction of Defense-Related Ultrastructural Modifications in Pea Root Tissues Inoculated with Endophytic Bacteria

The stimulation exerted by the endophytic bacterium Bacillus pumilus strain SE34 in plant defense reactions was investigated at the ultrastructural level using an in vitro system in which root-inducing T-DNA pea (Pisum sativum L.) roots were infected with the pea root-rotting fungus Fusarium oxysporum f. sp. pisi. In nonbacterized roots, the pathogen multiplied abundantly through much of the tissue including the vascular stele, whereas in prebacterized roots, pathogen growth was restricted to the epidermis and the outer cortex In these prebacterized roots, typical host reactions included strengthening the epidermal and cortical cell walls and deposition of newly formed barriers beyond the infection sites. Wall appositions were found to contain large amounts of callose in addition to being infiltrated with phenolic compounds. The labeling pattern obtained with the gold-complexed laccase showed that phenolics were widely distributed in Fusarium-challenged, bacterized roots. Such compounds accumulated in the host cell walls and the intercellular spaces as well as at the surface or even inside of the invading hyphae of the pathogen. The wall-bound chitin component in Fusarium hyphae colonizing bacterized roots was preserved even when hyphae had undergone substantial degradation. These observations confirm that endophytic bacteria may function as potential inducers of plant disease resistance.

Concomitant Induction of Systemic Resistance to Pseudomonas syringae pv. lachrymans in Cucumber by Trichoderma asperellum (T-203) and Accumulation of Phytoalexins

ABSTRACT Most studies on the reduction of disease incidence in soil treated with Trichoderma asperellum have focused on microbial interactions rather than on plant responses. This study presents conclusive evidence for the induction of a systemic response against angular leaf spot of cucumber (Pseudomonas syringae pv. lachrymans) following application of T. asperellum to the root system. To ascertain that T. asperellum was the only microorganism present in the root milieu, plants were grown in an aseptic hydroponic growth system. Disease symptoms were reduced by as much as 80%, corresponding to a reduction of 2 orders of magnitude in bacterial cell densities in leaves of plants pretreated with T. asperellum. As revealed by electron microscopy, bacterial cell proliferation in these plants was halted. The protection afforded by the biocontrol agent was associated with the accumulation of mRNA of two defense genes: the phenylpropanoid pathway gene encoding phenylalanine ammonia lyase (PAL) and the lipoxygenase pathway gene encoding hydroxyperoxide lyase (HPL). This was further supported by the accumulation of secondary metabolites of a phenolic nature that showed an increase of up to sixfold in inhibition capacity of bacterial growth in vitro. The bulk of the antimicrobial activity was found in the acid-hydrolyzed extract containing the phenolics in their aglycone form. High-performance liquid chromatography analysis of phenolic compounds showed a marked change in their profile in the challenged, preelicited plants relative to that in challenged controls. The results suggest that similar to beneficial rhizobacteria, T. asperellum may activate separate metabolic pathways in cucumber that are involved in plant signaling and biosynthesis, eventually leading to the systemic accumulation of phytoalexins.

Antifungal activity of chitosan on post-harvest pathogens: induction of morphological and cytological alterations in Rhizopus stolonifer

The effect of chitosan preparations with different levels of deacetylation and other polyanions on the growth of post-harvest pathogens was investigated. Chitosan markedly reduced the radial growth of all the fungi tested, with a greater effect at higher concentration. Chitosan was more effective than N,O-carboxymethylchitosan, polygalacturonate or d -glucosamine, and its inhibitory activity appeared to increase with the level of deacetylation. In addition to inducing cellular leakage of amino acids and proteins in Botrytis cinerea and Rhizopus stolonifer, chitosan also caused morphological changes in R. stolonifer. The ultrastructural study showed that chitosan caused deep erosion of the cell wall as well as increasing the cell-wall thickness. Although chitosan treatment did not affect chitin distribution in R. stolonifer wall, it stimulated the activities of chitin deacetylase, an enzyme involved in the biosynthesis of chitosan. This may well upset the balance between biosynthesis turnover of chitin, thereby rendering the cell wall more viscoelastic.

Elicitor-induced plant defence pathways

During the course of their coevolution, plants and pathogens have evolved an intricate relationship, resulting from a continuous exchange of molecular information. Pathogens have developed an array of offensive strategies to parasitize plants and, in turn, plants have deployed a wide range of defence mechanisms. In recent years, major advances have been made in understanding the sequential events taking place during the induction and expression of plant defence proteins. As the signal transduction pathways responsible for triggering plant defence responses become clearer, the possibility of sensitizing a plant by prior application of elicitors has become a promising option for effective management of plant diseases.

Cytological Evidence of an Active Role of Silicon in Wheat Resistance to Powdery Mildew (Blumeria graminis f. sp. tritici).

ABSTRACT Silicon (Si) amendments in the form of exogenously supplied nutrient solution or calcium silicate slag protect wheat plants from powdery mildew disease caused by the fungus Blumeria graminis f. sp. tritici. The most striking difference between Si- and Si+ plants challenged with B. graminis f. sp. tritici was the extent of epidermal cell infection and colonization by B. graminis f. sp. tritici. Histological and ultrastructural analyses revealed that epidermal cells of Si+ plants reacted to B. graminis f. sp. tritici attack with specific defense reactions including papilla formation, production of callose, and release of electron-dense osmiophilic material identified by cytochemical labeling as glycosilated phenolics. Phenolic material not only accumulated along the cell wall but also was associated with altered integrity of haustoria in a manner similar to localized phytoalexins as reported from other pathosystems. These results strongly suggest that Si mediates active localized cell defenses against B. graminis f. sp. tritici attack.

Silicon Enhances the Accumulation of Diterpenoid Phytoalexins in Rice: A Potential Mechanism for Blast Resistance

ABSTRACT Although several reports underscore the importance of silicon (Si) in controlling Magnaporthe grisea on rice, no study has associated this beneficial effect with specific mechanisms of host defense responses against this fungal attack. In this study, however, we provide evidence that higher levels of momilactone phytoalexins were found in leaf extracts from plants inoculated with M. grisea and amended with silicon (Si(+)) than in leaf extracts from inoculated plants not amended with silicon (Si(-) ) or noninoculated Si(+) and Si(-) plants. On this basis, the more efficient stimulation of the terpenoid pathway in Si(+) plants and, consequently, the increase in the levels of momilactones appears to be a factor contributing to enhanced rice resistance to blast. This may explain the lower level of blast severity observed on leaves of Si(+) plants at 96 h after inoculation with M. grisea. The results of this study strongly suggest that Si plays an active role in the resistance of rice to blast rather than the formation of a physical barrier to penetration by M. grisea.

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

Abstract. 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.

Silicon induced resistance in cucumber plants against Pythium ultimum

Abstract The effect of the amendment of nutrient solutions with soluble potassium silicate on the response of cucumber (cv. Corona) root and hypocotyl tissues infected by Pythium ultimum was examined by light and electron microscopy, and by energy dispersive X-ray analysis (EDX). Plants were grown in 0 or 1·7 m m Si-amended nutrient solutions, and root and hypocotyl samples were collected at different times after inoculation with P. ultimum . By 48 h after infection, striking differences in the expression of defence reactions were observed between Si-amended and Si-free cucumber plants. Treatment of plants with Si markedly stimulated the accumulation of an electron-dense, phenolic-like material in infected host tissues, and significantly increased the percentage of cells filled with this material. Fungal hyphae colonizing occluded host cells were seriously damaged, and were often reduced to empty hyphal shells. Additionally, Si-treated cucumber plants responded to P. ultimum infection by forming electron-dense layers along primary and secondary cell walls, as well as over pit membranes of xylem vessels. EDX analysis failed to reveal the presence of silica deposits in P. ultimum -infected plants grown in Si-supplemented media. Our results suggest that a relationship exists between Si treatment, resistance to P. ultimum attack, and expression of plant defence mechanisms.

Treatment with chitosan enhances resistance of tomato plants to the crown and root rot pathogen Fusarium oxysporum f. sp. radicis-lycopersici☆

Abstract Chitosan, a polymer of β-1,4-D-glucosamine derived from crab-shell chitin was applied to tomato plants prior to inoculation with the root pathogen, Fusarium oxysporum f. sp. radicis-lycopersici. Whether chitosan was applied by leaf spraying or root coating, it was found to markedly reduce the number of root lesions caused by the fungus, and to drastically increase the formation of putative physical barriers in infected root tissues. The effect of chitosan on the induction of host cell reactions was observed at concentrations ranging from 0·5 to 2 mg ml−1 with an optimal effect at 2 mg ml−1. The enhanced protection of tomato roots to fungal attack upon application of chitosan to leaves suggests that chitosan-induced resistance is systemic. Formation of wall oppositions such as papillae and occlusion of xylem vessels with either a network of bubble-like structures or a coating material were among the most typical features of host reactions. In addition, the accumulation of amorphous deposits, probably infused with phenolics from their electron-density, was observed in most intercellular spaces and some host cells. These deposits were often found to interfere with the walls of invading hyphae causing severe alterations. The application of wheat germ agglutinin, a lectin with N-acetylglucosamine-binding specificity, in conjunction with gold-complexed ovomucoid, to tissue sections showed that the walls of severely altered hyphal cells were labelled except in the area closely appressed to host cell walls. This suggests that extracellular chitinases accumulate in the hosts cell walls but are not the primary determinants of fungal damage. The possibility that toxic compounds such as phenols and chitosan-induced phytoalexins may be responsible for the observed damage of invading hyphae is discussed.