Sexual reproduction of Zymoseptoria tritici on durum wheat in Tunisia revealed by presence of airborne inoculum, fruiting bodies and high levels of genetic diversity.

Septoria tritici blotch (STB) caused by the heterothallic ascomycete Zymoseptoria tritici is currently one of the most devastating diseases of wheat worldwide. The extent of sexual reproduction of this pathogen is well documented on bread wheat, but not on durum wheat. The objective of the present study was to quantify the occurrence of Z. tritici sexual reproduction on durum wheat in the Tunisian environment. The assessment was undertaken using a triple approach combining fruiting body assessment, ascospore trapping and population genetic analyses. The results highlighted the formation of pseudothecia on leaves and stubble from the autumn until the end of the growing season. Likewise, qPCR monitoring highlighted a constant release of Z. tritici airborne inoculum during the wheat-growing season, with a peak of production at the end of the season. Genetic investigations using microsatellites revealed high levels of gene and genotypic diversities, an equal distribution of mating types, and a lack of genetic clustering within and between growing seasons. Taken together, these findings indicate that Z. tritici undergoes sexual reproduction on durum wheat in Tunisia at least to the same extent than on bread wheat in Western Europe, and that the dry and warm climate does not affect the mating process of the fungus. Frequent occurrence of sexual reproduction is a valuable knowledge to take into account in STB control strategies on durum wheat.

Thyme essential oil as an alternative mechanism: biofungicide-causing sensitivity of Mycosphaerella graminicola.

AIMS: To understand the mode of action of thyme essential oil as an alternative biofungicide. METHODS AND RESULTS: The chemical composition of thyme essential oil isolated by hydrodistillation from the aerial parts of Thymus vulgaris was analyzed. The main constituents of thyme essential oil were thymol (76·96%), ρ-cymene (9·89%), γ-terpinene (1·92%) and caryophyllene oxide (1·69%). The antifungal activity of the oil and its pure major component (thymol) was assessed by the in vitro assay against Mycosphaerella graminicola. Thyme oil exhibited higher antifungal activity than thymol. The expression pattern of genes involved in fungal development and detoxification acting in M. graminicola under thyme oil and thymol treatment was analyzed. Thyme oil overexpressed, more than thymol, the genes encoding for the efflux pump (MgMfs1, MgAtr4), the regulatory subunit of protein kinase A (PKA) (MgBcy1) and the MAPK MgHog1. Thyme oil repressed the expression of the genes encoding for the efflux pump MgAtr4, the MAPK (MgSlt2) and the regulatory subunit of PKA (MgBcy1). However, thymol repressed only MgAtr4 and MgSlt2 expression. CONCLUSIONS: These data highlight the ability of thyme oil to target genes involved in fungal development and virulence of the yeast-like fungi M. graminicola, which explain its higher antifungal activity. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings will probably be useful to design an alternative biofungicide which will not lead to pathogen multidrug resistance.

PLANT OLIGOSACCHARIDES ENHANCE WHEAT DEFENCE RESPONSE AGAINST SEPTORIA LEAF BLOTCH.

Our work provides the first evidence for elicitation and protection effects of preventive treatments with oligosaccharides (20%)-based new formulation (Oligos) against Mycosphaerella graminicola, a major pathogen of bread wheat (BW) and durum wheat (DW). In planta Oligos treatment led to strongly reduced hyphal growth, penetration, mesophyll colonization and fructification. During the necrotrophic phase, Oligos also drastically decreased the production of M. graminicola CWDE activities, such as xylanase and glucanase as well as protease activity in both wheat species, suggesting their correlation with disease severity. Concerning plant defence markers, PR2, Chi 4 precursor-, Per- and LOX-1-encoding genes were up-regulated, while glucanase (GLUC), catalase (CAT) and lipoxygenase (LOX) activities and total phenolic compound (PC) accumulation were induced in both (non-inoculated and inoculated contexts. In inoculated context, a localized accumulation of H2O2 and PC at fungal penetration sites and a specific induction of phenylalanine ammonia-Lyase (PAL) enzymatic activity were observed. Moreover, our experiment exhibited some similarities and differences in both wheat species responses. GLUC and CAT activities and H2O2 accumulation were more responsive in DW leaves, while LOX and PAL activities and PC accumulation occurred earlier and to a stronger extent in BW leaves. The tested Oligos formulation showed an interesting resistance induction activity characterized by a high and stable efficiency whatever the wheat species, suggesting it integration in common control strategies against STB on both DW and BW.

ASCORBIC ACID CONTROLS MYCOSPHAERELLA GRAMINICOLA IN BREAD AND DURUM WHEAT THROUGH DIRECT EFFECT ON THE PATHOGEN AND INDIRECT ACTION VIA PLANT DEFENCE.

Septoria tritici blotch (STB) caused by Mycosphaerella graminicola is one of the most devastating foliar diseases on wheat. Due to the emergence of fungicide-resistant M. graminicola strains and in an effort to reduce the impact of pesticides on the environment, considerable interest has been devoted to alternative control strategies. The use of natural products, especially through a defense-activating effect on the host, could be considered. Acid ascorbic (AA) is synthesized by plants and most animal cells with antioxidant properties. This study aimed at: (i) assessing the protective effect of an AA-based product on bread (BW) and durum (DW) wheat (Triticum aestivum and T. durum, respectively) susceptible cultivars against M. graminicola and (ii) investigating the mechanisms involved in wheat protection. Therefore, the foliar application of a formulated AA-based product (50 mg L-) on 3-week-old wheat plants reduced the infection level by more than 75% for both BW and DW. In vitro experiments revealed that AA induced a strong inhibition of spore germination (at 50 mg L.(-1)) and hyphal growth (at 16 mg L.(-1)) for both M. graminicola strains, infecting either BW or DW. Used as a preventive foliar spray on wheat leaves, microscopic observations revealed that AA inhibits in planta spore germination, hyphal growth, leaf penetration, substomatal colonization and eventually sporulation. Moreover, AA treatment also decreased fungal protease and cell wall degrading enzyme activities, putative pathogenicity determinants of M. graminicola. In addition to these effects on the fungus, AA induced defence reactions in both BW and DW. Indeed, in non-inoculated context, eliciting effect was observed on (i) stimulation of enzymatic activities such as lipoxygenase, peroxydase and catalase and (ii) transcript accumulation of genes encoding for pathogenesis-related (PR) proteins (chitinase class IV, peroxidase). In inoculated condition, accumulation of H2O2 and phenolic compounds increased at the penetration site in AA-treated leaves. In addition, AA treatment impacted the phenylpropanoid pathway through the induction of phenylalanine ammonia lyase activity. These results show that, in our conditions, AA both presents an antifungal activity and triggers several plant defences in wheat and suggest its use to control M. graminicola on both DW and BW.

EFFICACY AND MODES OF ACTION OF RESISTANCE INDUCERS ON TWO WHEAT SPECIES AGAINST MYCOSPHAERELLA GRAMINICOLA.

Plant resistance inducers could be an alternative to conventional fungicides to control in a more durable and environmentally friendly manner fungal pathogens. Here, we tested the protection efficacy and the modes of action of four resistance inducers (R1, R2, R3 and R4) against the causal agent of Septoria tritici blotch, Mycosphaerella graminicola, the most frequently occurring pathogen on wheat crops worldwide. The four inducers were tested on two wheat cultivars, Premio (a French bread wheat cultivar) and Karim (a Tunisian durum wheat cultivar), each inoculated with a bread-wheat or a durum-wheat adapted isolate; respectively. All inducers exhibited in the greenhouse a significant protection level on both cultivars regarding disease symptoms (necrosis and chlorosis) and sporulation (pycnidium density). The most efficient inducer was R3 which showed 84% symptom reduction, while the less efficient one was R2 with only a 39% reduction. None of the studied inducers showed direct biocide effect against the fungus, except R4 which displayed a significant in planta inhibition of spore germination. Further investigations revealed that all inducers elicited the plant defence enzymes peroxidase and lipoxygenase, but the activity levels varied depending on the considered inducer. In addition, the effect of resistance inducers on the infection process and the fungal cell-wall degrading enzymes xylanases and glucanases was also investigated. Our study allowed us to find out four efficient resistance inducers on wheat against M. graminicola and to establish data about the modes of action of these inducers.

Isolation and characterisation of five different hydrophobin-encoding cDNAs from the fungal tomato pathogen Cladosporium fulvum.

Five different hydrophobin-encoding cDNA clones from Cladosporium fulvum were isolated from cDNA libraries, made from nutrient-depleted mycelium. One cDNA clone was identical to the previously isolated hydrophobin HCf-1. The other clones were named HCf-2, -3, -4 and -5. HCf-1, -2, -3 and -4 show a high degree of identity, and are predicted to encode class I hydrophobins. HCf-5 encodes a class II hydrophobin. The expression patterns of these hydrophobins at various stages of development, and in liquid media lacking either carbon or nitrogen, or both, showed clear differences. All hydrophobins were more strongly expressed during sporulation than before, with HCf-4 and -5 showing the highest increase. Expression of HCf genes in infected plants was also higher at 16 days than at 10 days after infection. The expression of HCf-5 in sporulating mycelium was much lower in planta than in vitro. All HCf genes were upregulated under conditions of nutrient deprivation. HCf-1, -2, -3 and -4 showed highest levels of transcription in medium lacking both carbon and nitrogen. Expression of HCf-5 was highest in medium lacking nitrogen but containing carbon. HCf-1 was generally the most abundant hydrophobin. The introduction of multiple copies of HCf-1, which caused co-suppression of the endogenous HCf-1 gene, was shown to affect the expression of HCf-2, -3 and -4 also. Expression of HCf-4 was suppressed, but expression of HCf-2 and -3 was upregulated. Expression of HCf-5 was not changed.

[Rapid ACTH test to judge recovery of adrenal function in patients with Cushing's syndrome].

BACKGROUND: Steroid hormone replacement and recovery of adrenal function after unilateral adrenalectomy were studied in 12 patients with Cushing's syndrome due to adenocortical adenoma. PATIENTS AND METHOD: The patients were 3 males and 9 females aged 19 to 61 years old (mean 34.3 years old). Recovery of adrenal function was judged by plasma cortisol level and rapid ACTH test peridocally. RESULTS: Plasma cortisol level was 13.7 to 20.8 micrograms/dl without diurnal rythme before adrenalectomy. Average replacement period with low dose hydrocortisone was 15 +/- 9.2 months after adrenalectomy. Plasma ACTH and cortisol concentration was recovered to normal range after 3.4 +/- 2.9 months and 12.2 +/- 8.2 after adrenalectomy, respectively. Plasma ACTH concentration was recovered to normal range earlier than plasma cortisol level in all patients. Sufficient response of plasma cortisol concentration in rapid ACTH test was seen a little later than recovery of plasma cortisol level. CONCLUSION: Present data suggest that it is insufficient to judge recovery of adrenal function only by the rise of plasma ACTH and/or cortisol concentrations. Therefore rapid ACTH test is useful to judge sufficient recovery of adrenal function during and after steroid hormone replacement.

Co-suppression of the hydrophobin gene HCf-1 is correlated with antisense RNA biosynthesis in Cladosporium fulvum.

Transformation of Cladosporium fulvum with DNA containing a truncated copy of the hydrophobin gene HCf-1 causes co-suppression of hydrophobin synthesis in 30% of the transformants. The co-suppressed isolates have a hydrophilic phenotype, lower levels of HCf-1 mRNA than wild type and contain multiple copies of the plasmid integrated as tandem repeats at ectopic sites in the genome. Gene silencing is not associated with DNA cytosine methylation. Nuclear run-off experiments reveal that transcription rate of HCf-1 in the co-suppressed isolates is higher than in the untransformed strains, suggesting that silencing acts at the post-transcriptional level. We show, for the first time in fungi, that co-suppression is correlated with the presence of antisense RNA, and that this is synthesised on a DNA template. Derivatives showing reversion to the wild-type phenotype and restoration of HCf-1 gene expression were also observed. Reversion is associated with loss of some copies of the transgene. We propose that co-suppression is due to ectopic integration of the transgene next to promoters which initiate transcription to form antisense RNA and that this in turn determines down-regulation of HCf-1.

Stability and modulated expression of a hygromycin resistance gene integrated in Botrytis cinerea transformants.

When transformation of Botrytis cinerea occurred in mononucleated protoplasts the hygromycin resistance phenotype was stable and integrated plasmid DNA although rearranged was transmitted through meiosis. We observed that transformants were often heterokaryotic and using serial conidial transfer, we showed failure of expression of the entire copies of integrated plasmids in some conidial isolates. A non-Mendelian segregation of the hygromycin resistance phenotype was observed in most crosses between these transformants and sensitive strains. However, a 1:1 segregation ratio of plasmid DNA hybridisation was observed. Mechanisms of gene silencing in B. cinerea, in both the asexual and the sexual cycle, are discussed.

Transformation of Botrytis cinerea with the hygromycin B resistance gene, hph.

A transformation method has been developed for the phytopathogenic fungus Botrytis cinerea. Protoplasts were transformed with pAN7-1 plasmid carrying the Escherichia coli hygromycin phosphotransferase gene (hph), conferring hygromycin B resistance, downstream from an Aspergillus nidulans promoter. Molecular analysis, showed that transformation resulted in an integration of the plasmid into different regions of the B. cinerea genome and occurred through non-homologous recombination. The frequency was 2-10 transformants per micrograms of DNA. Transformants expressed phosphotransferase activity confirming that the hph gene conferred the hygromycin-resistance phenotype. All transformants analysed so far proved to be stable after several subcultures without any selective pressure.

[Clinical study of cefotetan in complicated urinary tract infections].

Clinical effectiveness and safety of Cefotetan were evaluated in 28 patients with complicated urinary tract infections. The results were excellent in 12 patients (42.9%), moderate in 10 patients (35.7%) and poor in 6 patients (21.6%), and the effectiveness rate was 78.6%. Bacteriologically, 24 (75%) out of 32 strains were eradicated. Subjective side effects, nausea and abdominal discomfort, were observed in one patient. Abnormal laboratory findings were observed in 6 patients, eosinophilia in 3 patients and slight elevation of transaminase in 3 patients.

[Clinical evaluation of cefmenoxime in chronic complicated urinary tract infection].

The efficacy of cefmenoxime (CMX), which is a third generation, beta-lactamase-resistant cephem with a broad antibacterial spectrum, was examined in 43 patients with chronic complicated urinary tract infections. The usual dosage regimen was given 2 approximately 4 g/day of CMX by intravenous drip infusion over 1 hour. The duration of treatment was 5 days. Fifteen patients were cured and 21 improved, and the effective rate was 83.7%. Bacterial eradication rate in these cases was 88.2%, especially eradication of the original pathogens such as Serratia marcescens, Proteus species and Klebsiella species, occurred in high frequency. Laboratory abnormalities were slight elevation of serum GOT and GPT in 2 cases. From these findings, CMX was considered to be very effective in complicated urinary tract infections.

Effects of benzothiazepine derivative (CRD-401) on blood pressure, excretion of electrolytes, and plasma renin activity.

A benzothiazepine derivative, CRD-401, was administered orally in a dosage of 60 to 120 mg/day to 14 patients with various renal diseases. The systolic pressure was lowered slightly in some cases after administration of CRD-401. Urine volume and urinary excretion of electrolytes were increased by the drug in most patients except those with severe renal dysfunction. The plasma renin activity was increased to about twice the premedication value in most patients. Although the mechanism of increasing action of CRD-401 on plasma renin activity was assumed to be related to the reabsorption capacity of renal tubules for electrolytes, some questions still remain unanswered. The mechanism of this drug action, therefore, could not be elucidated completely in this study.