Pathogenic potential of bacteria isolated from commercial biostimulants.

Microbial-based products are a promising alternative to agrochemicals in sustainable agriculture. However, little is known about their impact on human health even if some of them, i.e., Bacillus and Paenibacillus species, have been increasingly implicated in different human diseases. In this study, 18 bacteria were isolated from 2 commercial biostimulants, and they were genotypically and phenotypically characterized to highlight specific virulence properties. Some isolated bacteria were identified as belonging to the genus Bacillus by BLAST and RDP analyses, a genus in-depth studied for plant growth-promoting ability. Moreover, 16S rRNA phylogenetic analysis showed that seven isolates grouped with Bacillus species while two and four clustered, respectively, with Neobacillus and Peribacillus. Unusually, bacterial strains belonging to Franconibacter and Stenotrophomonas were isolated from biostimulants. Although Bacillus species are generally considered nonpathogenic, most of the species have shown to swim, swarm, and produced biofilms, that can be related to bacterial virulence. The evaluation of toxins encoding genes revealed that five isolates had the potential ability to produce the enterotoxin T. In conclusion, the pathogenic potential of microorganisms included in commercial products should be deeply verified, in our opinion. The approach proposed in this study could help in this crucial step.

GUN1 influences the accumulation of NEP-dependent transcripts and chloroplast protein import in Arabidopsis cotyledons upon perturbation of chloroplast protein homeostasis.

Correct chloroplast development and function require co-ordinated expression of chloroplast and nuclear genes. This is achieved through chloroplast signals that modulate nuclear gene expression in accordance with the chloroplast's needs. Genetic evidence indicates that GUN1, a chloroplast-localized pentatricopeptide repeat (PPR) protein with a C-terminal Small MutS-Related (SMR) domain, is involved in integrating multiple developmental and stress-related signals in both young seedlings and adult leaves. Recently, GUN1 was found to interact physically with factors involved in chloroplast protein homeostasis, and with enzymes of tetrapyrrole biosynthesis in adult leaves that function in various retrograde signalling pathways. Here we show that following perturbation of chloroplast protein homeostasis: (i) by growth in lincomycin-containing medium; or (ii) in mutants defective in either the FtsH protease complex (ftsh), plastid ribosome activity (prps21-1 and prpl11-1) or plastid protein import and folding (cphsc70-1), GUN1 influences NEP-dependent transcript accumulation during cotyledon greening and also intervenes in chloroplast protein import.

The Fusarium graminearum FGSG_03624 Xylanase Enhances Plant Immunity and Increases Resistance against Bacterial and Fungal Pathogens.

Fungal enzymes degrading the plant cell wall, such as xylanases, can activate plant immune responses. The Fusarium graminearum FGSG_03624 xylanase, previously shown to elicit necrosis and hydrogen peroxide accumulation in wheat, was investigated for its ability to induce disease resistance. To this aim, we transiently and constitutively expressed an enzymatically inactive form of FGSG_03624 in tobacco and Arabidopsis, respectively. The plants were challenged with Pseudomonas syringae pv. tabaci or pv. maculicola and Botrytis cinerea. Symptom reduction by the bacterium was evident, while no reduction was observed after B. cinerea inoculation. Compared to the control, the presence of the xylanase gene in transgenic Arabidopsis plants did not alter the basal expression of a set of defense-related genes, and, after the P. syringae inoculation, a prolonged PR1 expression was detected. F. graminearum inoculation experiments of durum wheat spikes exogenously treated with the FGSG_03624 xylanase highlighted a reduction of symptoms in the early phases of infection and a lower fungal biomass accumulation than in the control. Besides, callose deposition was detected in infected spikes previously treated with the xylanase and not in infected control plants. In conclusion, our results highlight the ability of FGSG_03624 to enhance plant immunity, thus decreasing disease severity.

The Structural Characterization and Antipathogenic Activities of Quinoin, a Type 1 Ribosome-Inactivating Protein from Quinoa Seeds.

Quinoin is a type 1 ribosome-inactivating protein (RIP) we previously isolated from the seeds of pseudocereal quinoa (Chenopodium quinoa) and is known as a functional food for its beneficial effects on human health. As the presence of RIPs in edible plants could be potentially risky, here we further characterised biochemically the protein (complete amino acid sequence, homologies/differences with other RIPs and three-dimensional homology modeling) and explored its possible defensive role against pathogens. Quinoin consists of 254 amino acid residues, without cysteinyl residues. As demonstrated by similarities and homology modeling, quinoin preserves the amino acid residues of the active site (Tyr75, Tyr122, Glu177, Arg180, Phe181 and Trp206; quinoin numbering) and the RIP-fold characteristic of RIPs. The polypeptide chain of quinoin contains two N-glycosylation sites at Asn115 and Asp231, the second of which appears to be linked to sugars. Moreover, by comparative MALDI-TOF tryptic peptide mapping, two differently glycosylated forms of quinoin, named pre-quinoin-1 and pre-quinoin-2 (~0.11 mg/100 g and ~0.85 mg/100 g of seeds, respectively) were characterised. Finally, quinoin possesses: (i) strong antiviral activity, both in vitro and in vivo towards Tobacco Necrosis Virus (TNV); (ii) a growth inhibition effect on the bacterial pathogens of plants; and (iii) a slight antifungal effect against two Cryphonectria parasitica strains.

New Insight into Justicidin B Pathway and Production in Linum austriacum.

Lignans are the main secondary metabolites synthetized by Linum species as plant defense compounds but they are also valuable for human health, in particular, for novel therapeutics. In this work, Linum austriacum in vitro cultures, cells (Cc), adventitious roots (ARc) and hairy roots (HRc) were developed for the production of justicidin B through elicitation with methyl jasmonate (MeJA) and coronatine (COR). The performances of the cultures were evaluated for their stability, total phenols content and antioxidant ability. NMR was used to identify justicidin B and isojusticidin B and HPLC to quantify the production, highlighting ARc and HRc as the highest productive tissues. MeJA and COR treatments induced the synthesis of justicidin B more than three times and the synthesis of other compounds. RNA-sequencing and a de novo assembly of L. austriacum ARc transcriptome was generated to identify the genes activated by MeJA. Furthermore, for the first time, the intracellular localization of justicidin B in ARc was investigated through microscopic analysis. Then, HRc was chosen for small-scale production in a bioreactor. Altogether, our results improve knowledge on justicidin B pathway and cellular localization in L. austriacum for future scale-up processes.

Gene Organization, Expression, and Localization of Ribotoxin-Like Protein Ageritin in Fruiting Body and Mycelium of Agrocybe aegerita.

The edible mushroom Agrocybe aegerita produces a ribotoxin-like protein known as Ageritin. In this work, the gene encoding Ageritin was characterized by sequence analysis. It contains several typical features of fungal genes such as three short introns (60, 55 and 69 bp) located at the 5' region of the coding sequence and typical splice junctions. This sequence codes for a precursor of 156 amino acids (~17-kDa) containing an additional N-terminal peptide of 21 amino acid residues, absent in the purified toxin (135 amino acid residues; ~15-kDa). The presence of 17-kDa and 15-kDa forms was investigated by Western blot in specific parts of fruiting body and in mycelia of A. aegerita. Data show that the 15-kDa Ageritin is the only form retrieved in the fruiting body and the principal form in mycelium. The immunolocalization by confocal laser scanning microscopy and transmission electron microscopy proves that Ageritin has vacuolar localization in hyphae. Coupling these data with a bioinformatics approach, we suggest that the N-terminal peptide of Ageritin (not found in the purified toxin) is a new signal peptide in fungi involved in intracellular routing from endoplasmic reticulum to vacuole, necessary for self-defense of A. aegerita ribosomes from Ageritin toxicity.

Effect of sprouting on nutritional quality of pulses.

In order to investigate the nutritional quality of industrial-scale sprouted versus unsprouted chickpeas and green peas, before and after cooking, the ultrastructure, chemical composition, antioxidant capacity, starch digestibility, mineral content and accessibility were analysed. Sprouting did not deeply affect raw seed structure, although after cooking starch granules appeared more porous and swelled. Chemical composition of raw sprouted seeds was not strongly affected, excepting an increase in protein (both pulses), and in free sugars (in peas; +10% and +80%, respectively, p < .05). The industrial sprouting favoured phytic acid leaching in cooking water (-35% in seeds, compared to unsprouted cooked ones, p < .05), and promoted antioxidant capacity reductions in raw and cooked seeds (-10% and -37%, respectively, p < .05). In conclusion, sprouting on an industrial-scale induced mild structural modifications in chickpeas and peas, sufficient to reduce antinutritional factors, without strongly influencing their nutritional quality. These products could represent nutritionally interesting ingredients for different dietary patterns as well as for enriched cereal-based foods.

Maize 16-kD γ-zein forms very unusual disulfide-bonded polymers in the endoplasmic reticulum: implications for prolamin evolution.

In the lumen of the endoplasmic reticulum (ER), prolamin storage proteins of cereal seeds form very large, ordered heteropolymers termed protein bodies (PBs), which are insoluble unless treated with alcohol or reducing agents. In maize PBs, 16-kD γ-zein locates at the interface between a core of alcohol-soluble α-zeins and the outermost layer mainly composed of the reduced-soluble 27-kD γ-zein. 16-kD γ-zein originates from 27-kD γ-zein upon whole-genome duplication and is mainly characterized by deletions in the N-terminal domain that eliminate most Pro-rich repeats and part of the Cys residues involved in inter-chain bonds. 27-kD γ-zein also forms insoluble PBs when expressed in transgenic vegetative tissues. We show that in Arabidopsis leaves, 16-kD γ-zein assembles into disulfide-linked polymers that fail to efficiently become insoluble. Instead of forming PBs, these polymers accumulate as very unusual threads that markedly enlarge the ER lumen, resembling amyloid-like fibers. Domain-swapping between the two γ-zeins indicates that the N-terminal region of 16-kD γ-zein has a dominant effect in preventing full insolubilization. Therefore, a newly evolved prolamin has lost the ability to form homotypic PBs, and has acquired a new function in the assembly of natural, heteropolymeric PBs.

Evidence of a bacterial core in the stored products pest Plodia interpunctella: the influence of different diets.

The potential influence of insects' feeding behaviour on their associated bacterial communities is currently a matter of debate. Using the major pest of commodities, Plodia interpunctella, as a model and adopting a culture-independent approach, the impact of different diets on the host-associated microbiota was evaluated. An analysis of similarity showed differences among the microbiotas of moths fed with five substrates and provided evidence that diet represents the only tested factor that explains this dissimilarity. Bacteria shared between food and insects provide evidence for a limited conveyance to the host of the bacteria derived from the diet; more likely, the content of carbohydrates and proteins in the diets promotes changes in the insect's microbiota. Moth microbiotas were characterized by two robust entomotypes, respectively, associated with a carbohydrate-rich diet and a protein-rich diet. These results were also confirmed by the predicted metagenome functional potential. A core microbiota, composed of six taxa, was shared between eggs and adults, regardless of the origin of the population. Finally, the identification of possible human and animal pathogens on chili and associated with the moths that feed on it highlights the possibility that these bacteria may be conveyed by moth frass.

Construction of a synthetic infectious cDNA clone of Grapevine Algerian latent virus (GALV-Nf) and its biological activity in Nicotiana benthamiana and grapevine plants.

BACKGROUND: Grapevine Algerian latent virus (GALV) is a tombusvirus first isolated in 1989 from an Algerian grapevine (Vitis spp.) plant and more recently from water samples and commercial nipplefruit and statice plants. No further reports of natural GALV infections in grapevine have been published in the last two decades, and artificial inoculations of grapevine plants have not been reported. We developed and tested a synthetic GALV construct for the inoculation of Nicotiana benthamiana plants and different grapevine genotypes to investigate the ability of this virus to infect and spread systemically in different hosts. METHODS: We carried out a phylogenetic analysis of all known GALV sequences and an epidemiological survey of grapevine samples to detect the virus. A GALV-Nf clone under the control of the T7 promoter was chemically synthesized based on the full-length sequence of the nipplefruit isolate GALV-Nf, the only available sequence at the time the project was conceived, and the infectious transcripts were tested in N. benthamiana plants. A GALV-Nf-based binary vector was then developed for the agroinoculation of N. benthamiana and grapevine plants. Infections were confirmed by serological and molecular analysis and the resulting ultrastructural changes were investigated in both species. RESULTS: Sequence analysis showed that the GALV coat protein is highly conserved among diverse isolates. The first epidemiological survey of cDNAs collected from 152 grapevine plants with virus-like symptoms did not reveal the presence of GALV in any of the samples. The agroinoculation of N. benthamiana and grapevine plants with the GALV-Nf binary vector promoted efficient infections, as revealed by serological and molecular analysis. The GALV-Nf infection of grapevine plants was characterized in more detail by inoculating different cultivars, revealing distinct patterns of symptom development. Ultrastructural changes induced by GALV-Nf in N. benthamiana were similar to those induced by tombusviruses in other hosts, but the cytopathological alterations in grapevine plants were less severe. CONCLUSIONS: This is the first report describing the development of a synthetic GALV-Nf cDNA clone, its artificial transmission to grapevine plants and the resulting symptoms and cytopathological alterations.

Phytotoxic Ozone Dose-Response Relationships for Durum Wheat (Triticum durum, Desf.).

Ozone (O3) pollution poses a significant threat to global crop productivity, particularly for wheat, one of the most important staple foods. While bread wheat (Triticum aestivum) is unequivocally considered highly sensitive to O3, durum wheat (Triticum durum) was often found to be more tolerant. This study investigated the O3 dose-response relationships for durum wheat in the Mediterranean region, focusing mainly on grain yield losses, and utilizing the phytotoxic ozone dose (POD) metric to describe the intensity of the stressor. The results from two experiments with Open-Top Chambers performed in 2013 and 2014 on two relatively sensitive durum wheat cultivars confirmed that this wheat species is far more tolerant than bread wheat. The use of a local parameterization of a stomatal conductance model based on field measurements did not significantly improve the dose-response relationships obtained in comparison to the generic parameterization suggested by the Mapping Manual of the United Nations Economic Commission for Europe (UNECE). The POD6 critical level of 5 mmolO3 m-2 for 5% grain yield loss was remarkably higher than the one established for bread wheat with analogous experiments, highlighting that O3 risk assessments based on bread wheat may largely overestimate the damage in the Mediterranean region where durum wheat cultivation prevails.

Internalisation and multiple phosphorylation of γ-Conglutin, the lupin seed glycaemia-lowering protein, in HepG2 cells.

Lupin seed γ-Conglutin is a protein capable of reducing glycaemia in mammalians and increasing glucose uptake by model cells. This work investigated whether γ-Conglutin is internalised into the target cells and undergoes any covalent change during the process, as a first step to understanding its mechanism of action. To this purpose, γ-Conglutin-treated and untreated HepG2 cells were submitted to confocal and transmission electron microscopy. Immune-revelation of γ-Conglutin at various intervals revealed its accumulation inside the cytosol. In parallel, 2D-electrophoresis of the cell lysates and antibody reaction of the blotted maps showed the presence of the protein intact subunits inside the treated cells, whilest no trace of the protein was found in the control cells. However, γ-Conglutin-related spots with an unexpectedly low pI were also observed in the maps. These spots were excised, trypsin-treated and submitted to MS/MS spectrometric analysis. The presence of phosphorylated amino acids was detected. These findings, by showing that γ-Conglutin is internalised by HepG2 cells in an intact form and is modified by multiple phosphorylation, open the way to the understanding of the lupin γ-Conglutin insulin-mimetic activity.

Differential timing of defense-related responses induced by cerato-platanin and cerato-populin, two non-catalytic fungal elicitors.

The cerato-platanin (CP) family consists of fungal-secreted proteins involved in various stages of the host-fungus interaction and acting as phytotoxins and elicitors of defense responses. The founder member of this family is CP, a non-catalytic protein with a six-stranded double-ψß-barrel fold. Cerato-populin (Pop1) is an ortholog showing low sequence identity with CP. CP is secreted by Ceratocystis platani, the causal agent of the canker stain of plane. Pop1 is secreted by Ceratocystis populicola, a pathogen of poplar. CP and Pop1 have been suggested to act as PAMPs (pathogen-associated molecular patterns) because they induce phytoalexin synthesis, transcription of defense-related genes, restriction of conidia growth and cell death in various plants. Here, we treated plane leaves with CP or Pop1, and monitored defense responses to define the role of these elicitors in the plant interactions. Both CP and Pop1 were able to induce mitogen-activated protein kinases (MAPKs) phosphorylation, production of reactive oxygen species and nitric oxide, and overexpression of defense related genes. The characteristic DNA fragmentation and the cytological features indicate that CP and Pop1 induce cell death by a mechanism of programmed cell death. Therefore, CP and Pop1 can be considered as two novel, non-catalytic fungal PAMPs able to enhance primary defense. Of particular interest is the observation that CP showed faster activity compared to Pop1. The different timing in defense activation could potentially be due to the structural differences between CP and Pop1 (i.e. different hydrophobic index and different helix content) therefore constituting a starting point in unraveling their structure-function relationships.

Effect of resistant starch on the cooking quality of yam (Dioscorea spp.) and cassava (Manihot esculenta) based paste products.

Total starch (TS) and resistant starch (RS) contents in pasty edible product of mealy and hard cooking tubers of three yam varieties and four cassava varieties were determined to evaluate their contribution in their cooking quality. TS and RS contents appeared as the main components in determining yam cooking quality. Mealy cooking yam varieties were characterized by a significant higher TS content (75.2 ± 7.7 g/100 g d.m.) and lower RS content (13.8 ± 3.4 g/100 g d.m.) than hard cooking yam varieties, which, in contrast, contained less TS (61.7 ± 12.1 g/100 g d.m.) and particularly high RS (21.8 ± 9.9 g/100 g d.m.), possibly as a consequence of the prevalence of large granules (35-40 µm) observed by light microscope. Conversely, TS and RS contents appeared not determinant on the cooking quality of cassava. Moreover, higher amylose contents were associated with substantially elevated percentages of RS in yam and cassava, and high RS content in samples modulates their pasting properties by reducing the peak viscosity and the breakdown and requiring higher temperature and longer time to the peak.

Stocky1, a Novel Gene Involved in Maize Seedling Development and Cuticle Integrity.

The cuticle is the plant's outermost layer that covers the surfaces of aerial parts. This structure is composed of a variety of aliphatic molecules and is well-known for its protective role against biotic and abiotic stresses in plants. Mutants with a permeable cuticle show developmental defects such as organ fusions and altered seed germination and viability. In this study, we identified a novel maize mutant, stocky1, with unique features: lethal at the seedling stage, and showing a severely dwarfed phenotype, due to a defective cuticle. For the first time, the mutant was tentatively mapped to chromosome 5, bin 5.04. The mutant phenotype investigated in this work has the potential to contribute to the elucidation of the role of the cuticle during plant development. The possibility of controlling this trait is of relevance in the context of climate change, as it may contribute to tolerance to abiotic stresses.

Oxidative Stress Mitigation by Chitosan Nanoparticles in Durum Wheat Also Affects Phytochemicals and Technological Quality of Bran and Semolina.

In our previous work, durum wheat cv. Fabulis was grown over two consecutive seasons (2016-2017 and 2017-2018) in an experimental field in the north of Italy. With the aim of mitigating oxidative stress, plants were subjected to four treatments (deionized water, CHT 0.05 mg/mL, CHT-NPs, and CHT-NPs-NAC) three times during the experiment. Chitosan nanoparticles (CHT-NPs) reduced symptom severity on wheat leaves and positively influenced the final grain yield. The present work aimed at investigating whether CHT treatments and particularly N-acetyl cysteine (NAC)-loaded or -unloaded CHT-NPs, while triggering plant defense mechanisms, might also vary the nutritional and technological quality of grains. For this purpose, the grains harvested from the previous experiment were analyzed for their content in phytochemicals and for their technological properties. The results showed that CHT increased the polyphenol and tocopherol content and the reducing capacity of bran and semolina, even if the positive effect of the nano-formulation remained still unclear and slightly varied between the two years of cultivation. The positive effect against oxidative stress induced by the chitosan treatments was more evident in the preservation of both the starch pasting properties and gluten aggregation capacity, indicating that the overall technological quality of semolina was maintained. Our data confirm the role of chitosan as an elicitor of the antioxidant defense system in wheat also at the grain level.

The presence of melatonin in grapevine (Vitis vinifera L.) berry tissues.

Melatonin has been reported in a variety of food plants and, consequently, in a number of plant-derived foodstuffs. In grapevine (Vitis vinifera L.) products, it was found in berry exocarp (skin) of different cultivars and monovarietal wines. Herein, we assessed, by means of mass spectrometry, the occurrence of melatonin in all berry tissues (skin, flesh, and seed) at two different phenological stages, pre-véraison and véraison. We detected the highest melatonin content in skin, at pre-véraison, whereas, at véraison, the highest levels were reported in the seed. Furthermore, during ripening, melatonin decreased in skin, while increasing in both seed and flesh. The relative concentrations of melatonin in diverse berry tissues were somewhat different from those of total polyphenols (TP), the latter measured by the Folin-Ciocalteau assay, and more abundant in seed at pre-véraison and in exocarp at véraison. The highest antiradical activity, determined by both DPPH (2,2-diphenyl-1-pycryl hydrazyl) and ABTS [(2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid)] radical-scavenging assay, was reported at pre-veráison in seed. To the best of our knowledge, we reported, for the first time, the occurrence of melatonin in grape seeds.

From vineyard to glass: agrochemicals enhance the melatonin and total polyphenol contents and antiradical activity of red wines.

Resistance inducers are a class of agrochemicals, including benzothiadiazole and chitosan, which activate the plant own defence mechanisms. In this work, open-field treatments with plant activators were performed on two red grape (Vitis vinifera L.) varieties cultivated in different localities, Groppello (Brescia, Lombardia, Italy) and Merlot (Treviso, Veneto, Italy). Treatments were carried out every 10 days until the véraison and, after harvesting, experimental wines (microvinificates) were prepared. In general, both melatonin and total polyphenol content, determined by mass spectrometry and Folin-Ciocalteu assay, respectively, were higher in wines produced from grapes treated with resistance inducers than in those obtained from untreated control and conventional fungicide-treated grapes. Accordingly, antiradical power of wines derived from plant activator-treated grapes, measured by both DPPH (2,2-diphenyl-1-picrylhydrazyl) and the ABTS [(2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)] radical-scavenging assay, was higher than in their counterparts. To the best of our knowledge, this is the first report on the effects of agrochemicals on the melatonin content of red wine.

Benzothiadiazole (BTH) activates sterol pathway and affects vitamin D3 metabolism in Solanum malacoxylon cell cultures.

Benzo-(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester (BTH), a particularly efficient inducer of systemic acquired resistance (SAR), was developed as an immunizing agent to sensitize various crop species against pathogen infections. Recent works highlighted its activating effect on different metabolic pathways, concerning both primary and secondary metabolites. In this study, we investigated the effect of BTH treatment on sterol levels and vitamin D(3) metabolism in Solanum malacoxylon cultures. Calli of S. malacoxylon were incubated in Gamborg B5 liquid medium alone or added with 50 µM BTH for different times (one, two or three cycles of light). Histocytochemical investigations performed on our experimental system using 3,3'-diaminobenzidine (DAB) for hydrogen peroxide (H(2)O(2)) detection and phloroglucinol for lignin staining showed that BTH causes H(2)O(2) accumulation and lignin deposition in treated calli. Gas chromatographic analysis of principal cell membrane sterols (ß-sitosterol, campesterol, stigmasterol) showed that BTH transiently increases their cellular levels. Callus cultures were found to contain also cholesterol, 7-dehydrocholesterol, the putative precursor of vitamin D(3), and the hydroxylated metabolites 25-hydroxyvitamin D(3) [25(OH)D(3)] and 1α,25-dihydroxyvitamin D(3) [1α,25(OH)(2)D(3)]. BTH treatment enhanced 7-dehydrocholesterol while reduced cholesterol. HPLC analysis of sample extracts showed that BTH does not affect the cell content of vitamin D(3), though results of ELISA tests highlighted that this elicitor moderately enhances the levels of 25(OH)D(3) and 1α,25(OH)(2)D(3) metabolites. In conclusion, BTH treatment not only causes cell wall strengthening, a typical plant defence response, as just described in other experimental models, but in the same time increases the cellular level of the main sterols and 7-dehydrocholesterol.

Uromyces appendiculatus infection in BTH-treated bean plants: ultrastructural details of a lost fight.

The mechanisms of BTH [benzo-(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester]-induced resistance against bean rust caused by Uromyces appendiculatus have been explored in Phaseolus vulgaris by light and transmission electron microscopy, following the infection progression in plants challenged 7 days after treatment. While BTH did not affect uredospore germination and fungal penetration in the substomatal cavity, a first impairment to the colonization appeared evident about 48-96 h after inoculation, with alterations of infection hypha structure and reduction in mycelium expansion. No differences were found in this phase regarding the formation and ultrastructure of haustoria in untreated and BTH-treated plants, except for the deposition of electron-opaque material in the extrahaustorial matrix of the latter. A second and decisive impairment in fungal progression was observed at 7-10 days after inoculation when host cell penetrated, or in close contact with the fungal hyphae, were impregnated by phenolic compounds. The same was observed in fungal walls, particularly around haustoria, thus hampering the biotrophic habitus of the fungus and further mycelium spreading. This, in turn, prevented the evasion of fungal reproductive structures, the uredinia, and the appearance of visible symptoms. No particular ultrastructural alterations were observed in most of the penetrated cells, even at late stages of infection, indicating that BTH treatment does not induce host cells to respond with a hypersensitive reaction (HR). A parallel time course of the expression of phenylalanine ammonia lyase (PAL) gene, the key enzyme for the synthesis of phenylpropanoidic phytoalexins and many other phenolics, has shown that PAL mRNA is strongly and persistently transcripted in BTH-treated plants since the 6th h after treatment, though no apparent ultrastructural alterations were detectable up to some days after pathogen challenging. This indicates that BTH, at the employed concentration of 0.3 mM, directly activates the plant's own defences, thus accounting for the observed full protection against bean rust.