Toward alternative sources of milk coagulants for cheese manufacturing: establishment of hairy roots culture and protease characterization from Cynara cardunculus L.

KEY MESSAGE: Extracts from hairy root cultures of Cynara cardunculus L. contain proteases and show milk-clotting activity. Cynara cardunculus L. or cardoon is often used as rennet in traditional cheese manufacturing, due to the presence of specific proteases in the flower. However, the flower extracts are variable depending on the provenance and quality of the flowers as well as high genetic variability among cardoon populations, and this affects the quality of the final product. In search for alternative sources of milk-clotting enzymes, hairy root cultures from cardoon were obtained and characterized regarding their protease content and proteolytic activity toward milk proteins. Aspartic, serine and cysteine proteases were identified in hairy roots by mass spectrometry analysis and an azocasein assay combined with specific inhibitors. RT-PCR analysis revealed the expression of cardosin A and D, and immunoblotting analysis suggested the presence of cardosin A or cardosin A-like enzyme in its mature form, supporting this system as an alternative source of cardosins. Hairy root protein extracts showed activity over caseins, supporting its use as milk coagulant, which was further tested by milk-clotting assays. This is also the first report on the establishment of hairy root cultures from cardoon, which paves the way for future work on controlled platforms for production of valuable metabolites which are known to be present in this species.

A first insight into the involvement of phytohormones pathways in coffee resistance and susceptibility to Colletotrichum kahawae.

Understanding the molecular mechanisms underlying coffee-pathogen interactions are of key importance to aid disease resistance breeding efforts. In this work the expression of genes involved in salicylic acid (SA), jasmonic acid (JA) and ethylene (ET) pathways were studied in hypocotyls of two coffee varieties challenged with the hemibiotrophic fungus Colletotrichum kahawae, the causal agent of Coffee Berry Disease. Based on a cytological analysis, key time-points of the infection process were selected and qPCR was used to evaluate the expression of phytohormones biosynthesis, reception and responsive-related genes. The resistance to C. kahawae was characterized by restricted fungal growth associated with early accumulation of phenolic compounds in the cell walls and cytoplasmic contents, and deployment of hypersensitive reaction. Similar responses were detected in the susceptible variety, but in a significantly lower percentage of infection sites and with no apparent effect on disease development. Gene expression analysis suggests a more relevant involvement of JA and ET phytohormones than SA in this pathosystem. An earlier and stronger activation of the JA pathway observed in the resistant variety, when compared with the susceptible one, seems to be responsible for the successful activation of defense responses and inhibition of fungal growth. For the ET pathway, the down or non-regulation of ET receptors in the resistant variety, together with a moderate expression of the responsive-related gene ERF1, indicates that this phytohormone may be related with other functions besides the resistance response. However, in the susceptible variety, the stronger activation of ERF1 gene at the beginning of the necrotrophic phase, suggests the involvement of ET in tissue senescence. As far as we know, this is the first attempt to unveil the role of phytohormones in coffee-C. kahawae interactions, thus contributing to deepen our understanding on the complex mechanisms of plant signaling and defense.

A transposable element insertion in the susceptibility gene CsaMLO8 results in hypocotyl resistance to powdery mildew in cucumber.

BACKGROUND: Powdery mildew (PM) is an important disease of cucumber (Cucumis sativus L.). CsaMLO8 was previously identified as a candidate susceptibility gene for PM in cucumber, for two reasons: 1) This gene clusters phylogenetically in clade V, which has previously been shown to harbour all known MLO-like susceptibility genes for PM identified in dicot species; 2) This gene co-localizes with a QTL on chromosome 5 for hypocotyl-specific resistance to PM. METHODS: CsaMLO8 alleles from susceptible and resistant cucumber were cloned and transformed to mlo-mutant tomato. Cucumber seedlings were inoculated with Podosphaera xanthii, tissues were studied for CsaMLO8 expression at several timepoints post inoculation using qRT-PCR. The occurrence of the observed loss-of-function allele of CsaMLO8 in resequenced cucumber accessions was studied in silico. RESULTS: We cloned CsaMLO8 alleles from susceptible and resistant cucumber genotypes, the latter carrying the QTL for hypocotyl resistance. We found that insertion of a non-autonomous Class LTR retrotransposable element in the resistant genotype leads to aberrant splicing of CsaMLO8 mRNA. Heterologous expression of the wild-type allele of CsaMLO8 in a tomato mlo-mutant restored PM susceptibility. However, heterologous expression of the CsaMLO8 allele cloned from the resistant cucumber genotype failed to restore PM susceptibility. Furthermore we showed that inoculation of susceptible cucumber with the PM pathogen Podosphaera xanthii induced transcriptional upregulation of CsaMLO8 in hypocotyl tissue, but not in cotyledon or leaf tissue. This coincides with the observation that the QTL at the CsaMLO8-locus causes full resistance in hypocotyl tissue, but only partial resistance in cotyledons and true leafs. We studied the occurrence of the loss-of-function allele of CsaMLO8 in cucumber germplasm by an in silico approach using resequencing data of a collection of 115 cucumber accessions, and found that this allele was present in 31 out of 115 accessions. CONCLUSIONS: CsaMLO8 was characterised as a functional susceptibility gene to PM, particularly in the hypocotyl where it was transcriptionally upregulated upon inoculation with the PM pathogen P. xanthii. A loss-of-function mutation in CsaMLO8 due to the insertion of a transposable element was found to be the cause of hypocotyl resistance to PM. This particular allele of CsaMLO8 was found to occur in 27 % of the resequenced cucumber accessions.

Optimization of in vitro regeneration and Agrobacterium tumefaciens-mediated transformation with heat-resistant cDNA in Brassica oleracea subsp. italica cv. Green Marvel.

An efficient system for shoot regeneration and Agrobacterium tumefaciens-mediated transformation of Brassica oleracea cv. Green Marvel cultivar is described. This study focuses on developing shoot regeneration from hypocotyl explants of broccoli cv. Green Marvel using thidiazuron (TDZ), zeatin, and kinetin, the optimization of factors affecting Agrobacterium-mediated transformation of the hypocotyl explants with heat-resistant cDNA, followed by the confirmation of transgenicity of the regenerants. High shoot regeneration was observed in 0.05-0.1 mg dm(-3) TDZ. TDZ at 0.1 mg dm(-3) produced among the highest percentage of shoot regeneration (96.67 %) and mean number of shoot formation (6.17). The highest percentage (13.33 %) and mean number (0.17) of putative transformant production were on hypocotyl explants subjected to preculture on shoot regeneration medium (SRM) with 200 µM acetosyringone. On optimization of bacterial density and inoculation time, the highest percentage and mean number of putative transformant production were on hypocotyl explants inoculated with a bacterial dilution of 1:5 for 30 min. Polymerase chain reaction (PCR) assay indicated a transformation efficiency of 8.33 %. The luciferase assay showed stable integration of the Arabidopsis thaliana HSP101 (AtHSP101) cDNA in the transgenic broccoli regenerants. Three out of five transgenic lines confirmed through PCR showed positive hybridization bands of the AtHSP101 cDNA through Southern blot analysis. The presence of AtHSP101 transcripts in the three transgenic broccoli lines indicated by reverse transcription-PCR (RT-PCR) confirmed the expression of the gene. In conclusion, an improved regeneration system has been established from hypocotyl explants of broccoli followed by successful transformation with AtHSP101 for resistance to high temperature.

Phenotypic expression of blast resistance gene Pi54 is not affected by its chromosomal position.

KEY MESSAGE: This is a novel report in which chromosomal position of the rice blast resistance gene Pi54 was not found to affect significantly the resistance phenotype or morphological traits. Blast disease caused by Magnaporthe oryzae is a serious constraint in rice production at global level. Pi54 gene imparts resistance against M. oryzae. Three different transgenic lines containing Pi54 and its orthologue Pi54rh were shown to be resistant to different races of M. oryzae. To determine the chromosomal location of Pi54 gene in transgenic lines, inverse PCR was performed. Our analysis showed that in two transgenic lines, Pi54 gene was integrated on chromosomes 6 and 10 at 12.94 and 22.30 Mb, respectively. Similarly, Pi54rh allele was integrated on chromosome 1 at 16.25 Mb. The Pi54 gene present on chromosome 6 was located in a non-coding region whereas in the other TP-Pi54 line, the gene was introgressed on chromosome 10 in between the coding region of SAP domain gene. The Pi54rh was also located in the non coding region flanked by the retrotransposon genes. These rice lines were evaluated for eight different traits related to seed and plant morphology and agronomic features for two consecutive years. The transgenic lines containing Pi54 gene have higher tiller number, grain weight, epicotyl length, and yield compared to the non-transgenic control. Multivariate correlation analysis shows that blast resistance was positively correlated with the number of tillers; thousand grain weight and epicotyl length. These results will facilitate precise utilization of Pi54 gene and its orthologue in breeding programs for the development of rice cultivars with broad spectrum and durable resistance to M. oryzae.

An efficient plant regeneration and Agrobacterium-mediated genetic transformation of Tagetes erecta.

Tagetes erecta, L. an asteraceous plant of industrial and medicinal value, contains important compounds like pyrethrins, thiophenes and lutein, possessing immense potential for insecticidal, nematicidal and nutraceutical activities. Considering the importance and demand for these natural compounds, genetic manipulation of this crop for better productivity of secondary metabolites holds great significance. A rapid and reproducible direct regeneration and genetic transformation system is the prerequisite for genetic manipulation of any crop. This paper elucidates the establishment of an efficient direct regeneration and transformation protocol of T. erecta using Agrobacterium tumefaciens. Investigation of the effects of different types of explants (Hypocotyls, cotyledonary leaves, rachis and leaf sections) and different BAP and GA3 combinations on the regeneration frequency of T. erecta suggested that the best regeneration frequency (66 %) with an average of 5.08 ± 0.09 shoot buds/explant was observed from hypocotyl explants cultured on media containing 1.5 mg/l BAP and 5 mg/l GA3. The transformation protocol was established using A. tumefaciens strain LBA4404, containing the binary vector pBI121, along with the gusA reporter gene with intron under the transcriptional control of the Cauliflower Mosaic Virus (CaMV) 35S promoter and the neomycin phosphotransferase II (nptII) gene as a kanamycin-resistant plant-selectable marker. Various parameters like optimization of kanamycin concentration (200 mg/l) for selection, standardization of cocultivation time (45 min) and acetosyringone concentration (150 µM) for obtaining higher transformation frequency were established using hypocotyl explants. The selected putative transgenic shoots were subsequently rooted on the Murashige and Skoog medium and transferred to the green house successfully. The plants were characterised by analysing the gus expression, amplification of 600 bp npt II fragment and Southern blot hybridization using the PCR-amplified gusA fragment as probe. The standardised protocol established during the study will open new vistas for genetic manipulation and introduction of desired genes for genetic improvement of T. erecta.

Induction of hairy roots by various strains of Agrobacterium rhizogenes in different types of Capsicum species explants.

BACKGROUND: Capsicum annuum and Capsicum frutescens, also known as "chilies", belong to the Solanaceae family and have tremendous beneficial properties. The application of hairy root culture may become an alternative method for future development of these species by adding value, such as by increasing secondary metabolites and improving genetic and biochemical stability compared with normal Capsicum plants. Therefore, in this research, different types of explants of both species were infected with various Agrobacterium rhizogenes strains to provide more information about the morphology and induction efficiency of hairy roots. After 2 weeks of in vitro seed germination, young seedling explants were cut into three segments; the cotyledon, hypocotyl, and radical. Then, the explants were co-cultured with four isolated A. rhizogenes strains in Murashige & Skoog culture media (MS) containing decreasing carbenicillin disodium concentrations for one month. RESULTS: In this experiment, thick and short hairy roots were induced at all induction sites of C. annuum while thin, elongated hairy roots appeared mostly at wound sites of C. frutescens. Overall, the hairy root induction percentages of C. frutescens were higher compared with C. annuum. Hairy root initiation was observed earliest using radicles (1st week), followed by cotyledons (2nd week), and hypocotyls (3rd week). Cotyledon explants of both species had the highest induction frequency with all strains compared with the other explants types. Strains ATCC 13333 and ATCC 15834 were the most favourable for C. frutescens while ATCC 43056 and ATCC 43057 were the most favourable for C. annuum. The interactions between the different explants and strains showed significant differences with p-values < 0.0001 in both Capsicum species. CONCLUSIONS: Both Capsicum species were amenable to A. rhizogenes infection and hairy root induction is recommended for use as an alternative explants in future plant-based studies.

The endophyte Verticillium Vt305 protects cauliflower against Verticillium wilt.

AIMS: To investigate the interaction between cauliflower and the isolate VerticilliumVt305, obtained from a field suppressive to Verticillium wilt of cauliflower, and to evaluate the ability of VerticilliumVt305 to control Verticillium wilt of cauliflower caused by V. longisporum. METHODS AND RESULTS: Single and combined inoculations of VerticilliumVt305 and V. longisporum were performed on cauliflower seedlings. Symptom development was evaluated, and fungal colonization was measured in the roots, hypocotyl and stem with real-time PCR. No symptoms were observed after single inoculation of VerticilliumVt305, although it colonized the plant tissues. Pre-inoculation of VerticilliumVt305 reduced symptom development and colonization of plant tissues by V. longisporum. CONCLUSIONS: VerticilliumVt305 is an endophyte on cauliflower plants and showed effective biological control of V. longisporum in controlled conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: This work can contribute to the development of a sustainable control measure of V. longisporum in Brassicaceae hosts, which is currently not available. Additionally, this study provides evidence for the different roles of Verticillium species present in the agro-ecosystem.

Validation of reference genes for normalization of qPCR gene expression data from Coffea spp. hypocotyls inoculated with Colletotrichum kahawae.

BACKGROUND: Coffee production in Africa represents a significant share of the total export revenues and influences the lives of millions of people, yet severe socio-economic repercussions are annually felt in result of the overall losses caused by the coffee berry disease (CBD). This quarantine disease is caused by the fungus Colletotrichum kahawae Waller and Bridge, which remains one of the most devastating threats to Coffea arabica production in Africa at high altitude, and its dispersal to Latin America and Asia represents a serious concern. Understanding the molecular genetic basis of coffee resistance to this disease is of high priority to support breeding strategies. Selection and validation of suitable reference genes presenting stable expression in the system studied is the first step to engage studies of gene expression profiling. RESULTS: In this study, a set of ten genes (S24, 14-3-3, RPL7, GAPDH, UBQ9, VATP16, SAND, UQCC, IDE and ß-Tub9) was evaluated to identify reference genes during the first hours of interaction (12, 48 and 72 hpi) between resistant and susceptible coffee genotypes and C. kahawae. Three analyses were done for the selection of these genes considering the entire dataset and the two genotypes (resistant and susceptible), separately. The three statistical methods applied GeNorm, NormFinder, and BestKeeper, allowed identifying IDE as one of the most stable genes for all datasets analysed, and in contrast GADPH and UBQ9 as the least stable ones. In addition, the expression of two defense-related transcripts, encoding for a receptor like kinase and a pathogenesis related protein 10, were used to validate the reference genes selected. CONCLUSION: Taken together, our results provide guidelines for reference gene(s) selection towards a more accurate and widespread use of qPCR to study the interaction between Coffea spp. and C. kahawae.

MtQRRS1, an R-locus required for Medicago truncatula quantitative resistance to Ralstonia solanacearum.

Ralstonia solanacearum is a major soilborne pathogen that attacks > 200 plant species, including major crops. To characterize MtQRRS1, a major quantitative trait locus (QTL) for resistance towards this bacterium in the model legume Medicago truncatula, genetic and functional approaches were combined. QTL analyses together with disease scoring of heterogeneous inbred families were used to define the locus. The candidate region was studied by physical mapping using a bacterial artificial chromosome (BAC) library of the resistant line, and sequencing. In planta bacterial growth measurements, grafting experiments and gene expression analysis were performed to investigate the mechanisms by which this locus confers resistance to R. solanacearum. The MtQRRS1 locus was localized to the same position in two recombinant inbred line populations and was narrowed down to a 64 kb region. Comparison of parental line sequences revealed 15 candidate genes with sequence polymorphisms, but no evidence of differential gene expression upon infection. A role for the hypocotyl in resistance establishment was shown. These data indicate that the quantitative resistance to bacterial wilt conferred by MtQRRS1, which contains a cluster of seven R genes, is shared by different accessions and may act through intralocus interactions to promote resistance.

The KEEP ON GOING protein of Arabidopsis regulates intracellular protein trafficking and is degraded during fungal infection.

In plants, the trans-Golgi network and early endosomes (TGN/EE) function as the central junction for major endomembrane trafficking events, including endocytosis and secretion. Here, we demonstrate that the KEEP ON GOING (KEG) protein of Arabidopsis thaliana localizes to the TGN/EE and plays an essential role in multiple intracellular trafficking processes. Loss-of-function keg mutants exhibited severe defects in cell expansion, which correlated with defects in vacuole morphology. Confocal microscopy revealed that KEG is required for targeting of plasma membrane proteins to the vacuole. This targeting process appeared to be blocked at the step of multivesicular body (MVB) fusion with the vacuolar membrane as the MVB-associated small GTPase ARA6 was also blocked in vacuolar delivery. In addition, loss of KEG function blocked secretion of apoplastic defense proteins, indicating that KEG plays a role in plant immunity. Significantly, KEG was degraded specifically in cells infected by the fungus Golovinomyces cichoracearum, suggesting that this pathogen may target KEG to manipulate the host secretory system as a virulence strategy. Taking these results together, we conclude that KEG is a key component of TGN/EE that regulates multiple post-Golgi trafficking events in plants, including vacuole biogenesis, targeting of membrane-associated proteins to the vacuole, and secretion of apoplastic proteins.

Agrobacterium rhizogenes-dependent production of transformed roots from foliar explants of pepper (Capsicum annuum): a new and efficient tool for functional analysis of genes.

Pepper is known to be a recalcitrant species to genetic transformation via Agrobacterium tumefaciens. A. rhizogenes-mediated transformation offers an alternative and rapid possibility to study gene functions in roots. In our study, we developed a new and efficient system for A. rhizogenes transformation of the cultivated species Capsicum annuum. Hypocotyls and foliar organs (true leaves and cotyledons) of Yolo Wonder (YW) and Criollo de Morelos 334 (CM334) pepper cultivars were inoculated with the two constructs pBIN-gus and pHKN29-gfp of A. rhizogenes strain A4RS. Foliar explants of both pepper genotypes infected by A4RS-pBIN-gus or A4RS-pHKN29-gfp produced transformed roots. Optimal results were obtained using the combination of the foliar explants with A4RS-pHKN29-gfp. 20.5% of YW foliar explants and 14.6% of CM334 foliar explants inoculated with A4RS-pHKN29-gfp produced at least one root expressing uniform green fluorescent protein. We confirmed by polymerase chain reaction the presence of the rolB and gfp genes in the co-transformed roots ensuring that they integrated both the T-DNA from the Ri plasmid and the reporter gene. We also demonstrated that co-transformed roots of YW and CM334 displayed the same resistance response to Phytophthora capsici than the corresponding untransformed roots. Our novel procedure to produce C. annuum hairy roots will thus support the functional analysis of potential resistance genes involved in pepper P. capsici interaction.

Agrobacterium rhizogenes-mediated transformation of the parasitic plant Phtheirospermum japonicum.

BACKGROUND: Plants within the Orobanchaceae are an agriculturally important group of parasites that attack economically important crops to obtain water and nutrients from their hosts. Despite their agricultural importance, molecular mechanisms of the parasitism are poorly understood. METHODOLOGY/PRINCIPAL FINDINGS: We developed transient and stable transformation systems for Phtheirospermum japonicum, a facultative parasitic plant in the Orobanchaceae. The transformation protocol was established by a combination of sonication and acetosyringone treatments using the hairy-root-inducing bacterium, Agrobacterium rhizogenes and young seedlings. Transgenic hairy roots of P. japonicum were obtained from cotyledons 2 to 3 weeks after A. rhizogenes inoculation. The presence and the expression of transgenes in P. japonicum were verified by genomic PCR, Southern blot and RT-PCR methods. Transgenic roots derived from A. rhizogenes-mediated transformation were able to develop haustoria on rice and maize roots. Transgenic roots also formed apparently competent haustoria in response to 2,6-dimethoxy-1,4-benzoquinone (DMBQ), a haustorium-inducing chemical. Using this system, we introduced a reporter gene with a Cyclin B1 promoter into P. japonicum, and visualized cell division during haustorium formation. CONCLUSIONS: We provide an easy and efficient method for hairy-root transformation of P. japonicum. Transgenic marker analysis revealed that cell divisions during haustorium development occur 24 h after DMBQ treatment. The protocols described here will allow functional analysis of genes involved in plant parasitism.

Organ identity and environmental conditions determine the effectiveness of nonhost resistance in the interaction between Arabidopsis thaliana and Magnaporthe oryzae.

Mechanisms leading to nonhost resistance of plants against nonadapted pathogens are thought to have great potential for the future management of agriculturally important diseases. In this article, we report an investigation of nonhost resistance motivated by the advantages of studying an interaction between two model organisms, namely Arabidopsis thaliana and Magnaporthe oryzae. During the course of our studies, however, we discovered an unexpected plasticity in the responses of Arabidopsis against this ostensibly nonhost pathogen. Thus, we elucidated that certain experimental conditions, such as the growth of plants under long days at constantly high humidity and the use of high inoculum concentrations of M. oryzae conidia, forced the interaction in leaves of some Arabidopsis ecotypes towards increased compatibility. However, sporulation was never observed. Furthermore, we observed that roots were generally susceptible to M. oryzae, whereas leaves, stems and hypocotyls were not infected. It must be concluded, therefore, that Arabidopsis roots lack an effective defence repertoire against M. oryzae, whereas its leaves possess such nonhost defence mechanisms. In summary, our findings point to organ-specific determinants and environmental conditions influencing the effectiveness of nonhost resistance in plants.

Development of the molecular methods for rapid detection and differentiation of Fusarium oxysporum and F. oxysporum f. sp. niveum in Taiwan.

Fusarium wilt, caused by Fusarium oxysporum (Fo), is one of the most important fungal diseases worldwide. Like other plant pathogens, Fo displays specialized forms in association with its hosts. For example, F. oxysporum f. sp. niveum (Fon) is the damaging pathogen causing Fusarium wilt disease on watermelon, whereas F. oxysporum f. sp. cubense is the pathogen that infects banana. A rapid and reliable pathogen identification or disease diagnosis is essential for the integrated disease management practices in many crops. In this study, two new primer sets, Fon-1/Fon-2 and FnSc-1/FnSc-2, were developed to differentiate Fon and Fo, respectively. The PCR method using the novel primer sets has high sensitivity to detect Fon when the DNA concentration was as low as 0.01 pg or when the conidia number was as few as 5. In comparison with the published primer set, the Fon-1/Fon-2 primer set, derived from the sequence of OP-M12 random primer-amplified fragment, produced a 174 bp DNA fragment, and was more specific to Fon in Taiwan. In addition, with optimized PCR parameters, the molecular method using the Fon-1/Fon-2 primer set could directly detect Fon even when watermelon samples were collected in its early stage of disease development.

Monitoring growth and movement of Ralstonia solanacearum cells harboring plasmid pRSS12 derived from bacteriophage phiRSS1.

We monitored growth and movement of Ralstonia solanacearum harboring the plasmid pRSS12 in tomato seedlings. The plasmid contains a gene for green fluorescent protein (GFP) and is stably maintained in R. solanacearum cells without selection pressure. Bacteria harboring the plasmid can be tracked in planta by visualizing GFP fluorescence. Stems of seedlings were infected with R. solanacearum cells transformed with pRSS12, and bacterial growth and movement, particularly around the vascular bundles, were monitored for more than 7 days. Our results showed that vascular bundles are independent of each other within the stem, and that it takes a long time for R. solanacearum cells to migrate from one vascular bundle to another. For real-time monitoring of bacteria in planta, tomato seedlings were grown on agar medium and bacterial suspension was applied to the root apex. The bacterial invasion process was monitored by fluorescent microscopy. Bacteria invaded taproots within 6 h, and movement of the bacteria was observed until 144 h after inoculation. In susceptible tomato cultivars, strong GFP fluorescence was observed in hypocotyls and lateral roots as well as the taproot. In resistant cultivars, however, GFP fluorescence was rarely observed on lateral roots. Our results show that this monitoring system can be used to assess bacterial pathogenicity efficiently.

Changes in cucumber hypocotyl cell wall dynamics caused by Azospirillum brasilense inoculation.

We previously reported that Azospirillum brasilense induced a more elastic cell wall and a higher apoplastic water fraction in both wheat coleoptile and flag leaf. These biophysical characteristics could permit increased growth. Knowledge of the biochemical effects the bacteria could elicit in plant cell walls and how these responses change plant physiology is still scarce. The objective of this work was to analyze whether A. brasilense Sp245 inoculation affected elongation and extensibility of growing cucumber (Cucumis sativus) hypocotyls and ionically bound cell wall peroxidase activities. Hypocotyl tip and basal segments were excised from A. brasilense Sp245-inoculated cucumber seedlings growing in darkness under hydroponic conditions. Elongation, cell wall extensibility, cell wall peroxidase activities against ferulic acid and guaiacol and NADH oxidase activities were analyzed. Azospirillum-inoculated cucumber seedlings grew bigger than non-inoculated ones. Dynamic cell wall differences were detected between inoculated and non-inoculated hypocotyls. They included greater acid-induced cell wall extension and in vivo elongation when incubated in distilled water. Although there was no difference between treatments in either region of the hypocotyl NADH oxidase and ferulic acid peroxidase activities were lower in both regions in inoculated seedlings. These lesser activities could be delaying the stiffening of cell wall in inoculated seedlings. These results showed that the cell wall is a target for A. brasilense growth promotion.

Effect of light conditions on the resistance of current-year Fagus Crenata seedlings against fungal pathogens causing damping-off in a natural beech forest: fungus isolation and histological and chemical resistance.

Forest gap dynamics affects light intensity on the forest floor, which in turn may influence defense and survival of tree seedlings. Current-year Fagus crenata seedlings show high mortality under the canopy caused by damping-off. In contrast, they survive pathogen attacks in gaps. However, defense mechanisms against damping-off have not been fully understood. In order to determine the resistance factors that affect mortality in current-year seedlings, we compared seedling survival and chemical and histological characteristics of the hypocotyls of seedlings from closed-stand and forest-edge plots. Damping-off occurred in the current-year seedlings mainly from the end of June to July; survival rate of the seedlings was higher in the forest-edge plot than in the closed-stand plot. By performing an inoculation test on the seedling hypocotyls, we identified Colletotrichum dematium and Cylindrocarpon sp. as the causative pathogens under low illumination only. In the beginning of July, only seedling hypocotyls from the forest-edge plot exhibited periderm formation. From mid-June to July, seedling hypocotyls from the forest-edge plot accumulated approximately twice the amount of total phenols as those accumulated by seedling hypocotyls from the closed-stand plot. The ethyl acetate phase of methanol extracts of hypocotyls showed antifungal activity. We conclude that seedlings from the forest-edge plot may resist pathogenic attack via periderm formation and increased phenol synthesis. Plant defense mechanisms that are controlled by light intensity may be important for promoting seedling regeneration in forest gap dynamics.

Internal resistance in winter oilseed rape inhibits systemic spread of the vascular pathogen Verticillium longisporum.

Verticillium longisporum is a vascular fungal pathogen presently threatening oilseed rape production in Europe. Systemic spread and vascular responses were studied in a susceptible ('Falcon') and a resistant genotype (SEM 05-500256) of Brassica napus. Colonization of both genotypes after dip-inoculation of the roots followed by quantitative polymerase chain reaction revealed similarities only in the initial stages of root penetration and colonization of the hypocotyl, while a substantial invasion of the shoot was only recorded in 'Falcon'. It is concluded that the type of resistance represented in SEM 05-500256 does not prevent the plant base from being invaded as it is internally expressed well after root penetration and colonization of the plant base. The morphological and biochemical nature of barriers induced in the hypocotyl tissue upon infection was studied with histochemical methods accompanied by biochemical analyses. Histochemical studies revealed the build-up of vascular occlusions and the reinforcement of tracheary elements through the deposition of cell wall-bound phenolics and lignin. Furthermore, the accumulation of soluble phenolics was observed. Although these responses were found in vascular tissues of both genotypes, they occurred with a significantly higher intensity in the resistant genotype and corresponded with the disease phenotype. In the resistant genotype phenols were differentially expressed in a time-dependent manner with preformed soluble and cell wall-bound phenolics at earlier time points and de novo formation of lignin and lignin-like polymers at later stages of infection. This is the first study identifying a crucial role of phenol metabolism in internal defense of B. napus against V. longisporum and locating the crucial defense responses in the plant hypocotyl.

White mould of common bean incited by Sclerotinia sclerotiorum Lib. de Bary in Egypt.

White mould, caused by Sclerotinia sclerotiorum (Lib.) de Bary, is a destructive yield-limiting disease of common bean (Phaseolus vulgaris L.) in Egypt. Forty eight isolate of S. sclerotiorum were isolated from diseased bean tissues taken from 9 geographical regions (Al-Behaira, Alexandria and Assiut governorates) during winter season in 2008. The pathogenicity studies showed that the tested bean cultivars (Bronco, Contender, Giza 6 and Nebraska) varied in disease incidence. Contender bean cultivar was more resistant than other cultivars. Whereas, the more virulent isolates were S5 and S6. Histology investigation of seedlings bean hypocotyls inoculated with S. sclerotiorum after 24, 48, 72 and 96 hours after inoculation indicated that penetration of bean seedlings occurred, during the first 48 hours after inoculation, through the epidermis and the outer layer of the cortex. 72 hours after inoculation, damage extended deeper into the cortical cells. Infection took place inter-and interacellularly after 96 hours more damage occurred. In addition, the invasion of the fungal hyphae through the cortical cells occurred both inter-, and intracellularly. Moreover, the observed of electron microscope both transmission and scanning investigations concluded that penetrating hyphae progressed through bean seedlings tissues leading to complete destruction of epidermis, fully colonization and death of cortical cells, partial invasion of vascular tissues. However, presence of the fungal structures in pith cells was observed.