Penicillium chrysogenum Fermentation and Analysis of Benzylpenicillin by Bioassay and HPLC.

Penicillium chrysogenum, recently re-identified as Penicillium rubens, is the microorganism used for the industrial production of penicillin. This filamentous fungus (mold) probably represents the best example of adaptation of a microorganism to industrial production conditions and therefore, it can be considered as a model organism for the study of primary and secondary metabolism under a highly stressful environment. In this regard, biosynthesis and production of benzylpenicillin can be used as an interesting phenotypic trait for those studies. In this chapter, we describe P. chrysogenum culture procedures for the production of benzylpenicillin and the process of antibiotic quantitation either by bioassay or by high-performance liquid chromatography (HPLC).

Competitiveness of three biocontrol candidates against ochratoxigenic Penicillium nordicum under dry-cured meat environmental and nutritional conditions.

The environmental conditions during the ripening of dry-cured meats and their nutritional composition promote the colonisation of their surface by Penicillium spp., including P. nordicum producer of ochratoxin A (OTA). The objective of this work was to study the competitiveness of three potential biocontrol candidates (Debaryomyces hansenii FHSCC 253H, Enterococcus faecium SE920 and Penicillium chrysogenum CECT, 20922) against the ochratoxigenic P. nordicum FHSCC4 under environmental and nutritional conditions simulating the ripening of dry-cured meat products. For this, the nutritional utilisation pattern, niche overlap index (NOI), interactions by dual-culture assays and OTA production were determined. The number of carbon sources (CSs) metabolised depended on the microorganism and the interacting water activity (aw) x temperature conditions. The number of CSs utilised by both filamentous fungi was quite similar and higher than those utilised by D. hansenii and E. faecium. The yeast isolate metabolised a number of CSs much larger than the bacterium. The NOI values showed that, in general, P. nordicum nutritionally dominated E. faecium and D. hansenii regardless of the environmental conditions evaluated. The relationship between the toxigenic and non-toxigenic fungal isolates depended on the aw x temperature combinations, although in none of the conditions a dominance of P. nordicum was observed. According to the interaction assays, both D. hansenii and P. chrysogenum decreased the growth of P. nordicum. The effect of D. hansenii could be attributed to the production of some extra-cellular compounds, while the action of P. chrysogenum is likely related to nutritional competition. In addition, both P. chrysogenum and D. hansenii reduced the OTA levels produced by P. nordicum. The effect of the yeast was more pronounced decreasing the concentration of OTA at quantities lower than the limit established by the Italian legislation. Therefore, P. chrysogenum and D. hansenii can be suggested as biocontrol candidates in the manufacture of dry-cured meat products.

Antarctic root endophytes improve physiological performance and yield in crops under salt stress by enhanced energy production and Na+ sequestration.

Climatic change is pointed as one of the major challenges for global food security. Based on current models of climate change, reduction in precipitations and in turn, increase in the soil salinity will be a sharp constraint for crops productivity worldwide. In this context, root fungi appear as a new strategy to improve plant ecophysiological performance and crop yield under abiotic stress. In this study, we evaluated the impact of the two fungal endophytes Penicillium brevicompactum and P. chrysogenum isolated from Antarctic plants on nutrients and Na+ contents, net photosynthesis, water use efficiency, yield and survival in tomato and lettuce, facing salinity stress conditions. Inoculation of plant roots with fungal endophytes resulted in greater fresh and dry biomass production, and an enhanced survival rate under salt conditions. Inoculation of plants with the fungal endophytes was related with a higher up/down-regulation of ion homeostasis by enhanced expression of the NHX1 gene. The two endophytes diminished the effects of salt stress in tomato and lettuce, provoked a higher efficiency in photosynthetic energy production and an improved sequestration of Na+ in vacuoles is suggested by the upregulating of the expression of vacuolar NHX1 Na+/H+ antiporters. Promoting plant-beneficial interactions with root symbionts appears to be an environmentally friendly strategy to mitigate the impact of climate change variables on crop production.

Drought-Induced Stress Priming in Two Distinct Filamentous Saprotrophic Fungi.

Sessile organisms constantly face environmental fluctuations and especially drought is a common stressor. One adaptive mechanism is "stress priming," the ability to cope with a severe stress ("triggering") by retaining information from a previous mild stress event ("priming"). While plants have been extensively investigated for drought-induced stress priming, no information is available for saprotrophic filamentous fungi, which are highly important for nutrient cycles. Here, we investigated the potential for drought-induced stress priming of one strain each of two ubiquitous species, Neurospora crassa and Penicillium chrysogenum. A batch experiment with 4 treatments was conducted on a sandy soil: exposure to priming and/or triggering as well as non-stressed controls. A priming stress was caused by desiccation to pF 4. The samples were then rewetted and after 1-, 7-, or 14-days of recovery triggered (pF 6). After triggering, fungal biomass, respiration, and ß-glucosidase activity were quantified. P. chrysogenum showed positive stress priming effects. After 1 day of recovery, biomass as well as ß-glucosidase activity and respiration were 0.5 to 5 times higher during triggering. Effects on biomass and activity decreased with prolonged recovery but lasted for 7 days and minor effects were still detectable after 14 days. Without triggering, stress priming had a temporary negative impact on biomass but this reversed after 14 days. For N. crassa, no stress priming effect was observed on the tested variables. The potential for drought-induced stress priming seems to be species specific with potentially high impact on composition and activity of fungal communities considering the expected increase of drought events.

The filamentous fungus Penicillium chrysogenum analysed via flow cytometry-a fast and statistically sound insight into morphology and viability.

Filamentous fungi serve as production host for a number of highly relevant biotechnological products, like penicillin. In submerged culture, morphology can be exceptionally diverse and is influenced by several process parameters, like aeration, agitation, medium composition or growth rate. Fungal growth leads to several morphological classes encompassing homogeneously dispersed hyphae and various forms of hyphal agglomerates and/or clump structures. Eventually, the so-called pellet structure can be formed, which represents a hyphal agglomerate with a dense core. Pellet structures can hinder oxygen and substrate transport, resulting in different states of viability, which in turn affects productivity and process control. Over the years, several publications have dealt with methods to either gain morphological insight into pellet structure or determine biomass viability. Within this contribution, we present a way to combine both in a flow cytometry-based method employing fluorescent staining. Thereby, we can assess filamentous biomass in a statistically sound way according to (i) morphology and (ii) viability of each detected morphological form. We are confident that this method can shed light on the complex relationship between fungal morphology, viability and productivity-in both process development and routine manufacturing processes.

A 9-pool metabolic structured kinetic model describing days to seconds dynamics of growth and product formation by Penicillium chrysogenum.

A powerful approach for the optimization of industrial bioprocesses is to perform detailed simulations integrating large-scale computational fluid dynamics (CFD) and cellular reaction dynamics (CRD). However, complex metabolic kinetic models containing a large number of equations pose formidable challenges in CFD-CRD coupling and computation time afterward. This necessitates to formulate a relatively simple but yet representative model structure. Such a kinetic model should be able to reproduce metabolic responses for short-term (mixing time scale of tens of seconds) and long-term (fed-batch cultivation of hours/days) dynamics in industrial bioprocesses. In this paper, we used Penicillium chrysogenum as a model system and developed a metabolically structured kinetic model for growth and production. By lumping the most important intracellular metabolites in 5 pools and 4 intracellular enzyme pools, linked by 10 reactions, we succeeded in maintaining the model structure relatively simple, while providing informative insight into the state of the organism. The performance of this 9-pool model was validated with a periodic glucose feast-famine cycle experiment at the minute time scale. Comparison of this model and a reported black box model for this strain shows the necessity of employing a structured model under feast-famine conditions. This proposed model provides deeper insight into the in vivo kinetics and, most importantly, can be straightforwardly integrated into a computational fluid dynamic framework for simulating complete fermentation performance and cell population dynamics in large scale and small scale fermentors. Biotechnol. Bioeng. 2017;114: 1733-1743. © 2017 Wiley Periodicals, Inc.

Penicillin production in industrial strain Penicillium chrysogenum P2niaD18 is not dependent on the copy number of biosynthesis genes.

BACKGROUND: Multi-copy gene integration into microbial genomes is a conventional tool for obtaining improved gene expression. For Penicillium chrysogenum, the fungal producer of the beta-lactam antibiotic penicillin, many production strains carry multiple copies of the penicillin biosynthesis gene cluster. This discovery led to the generally accepted view that high penicillin titers are the result of multiple copies of penicillin genes. Here we investigated strain P2niaD18, a production line that carries only two copies of the penicillin gene cluster. RESULTS: We performed pulsed-field gel electrophoresis (PFGE), quantitative qRT-PCR, and penicillin bioassays to investigate production, deletion and overexpression strains generated in the P. chrysogenum P2niaD18 background, in order to determine the copy number of the penicillin biosynthesis gene cluster, and study the expression of one penicillin biosynthesis gene, and the penicillin titer. Analysis of production and recombinant strain showed that the enhanced penicillin titer did not depend on the copy number of the penicillin gene cluster. Our assumption was strengthened by results with a penicillin null strain lacking pcbC encoding isopenicillin N synthase. Reintroduction of one or two copies of the cluster into the pcbC deletion strain restored transcriptional high expression of the pcbC gene, but recombinant strains showed no significantly different penicillin titer compared to parental strains. CONCLUSIONS: Here we present a molecular genetic analysis of production and recombinant strains in the P2niaD18 background carrying different copy numbers of the penicillin biosynthesis gene cluster. Our analysis shows that the enhanced penicillin titer does not strictly depend on the copy number of the cluster. Based on these overall findings, we hypothesize that instead, complex regulatory mechanisms are prominently implicated in increased penicillin biosynthesis in production strains.

Production of 8,11-dihydroxy and 8-hydroxy unsaturated fatty acids from unsaturated fatty acids by recombinant Escherichia coli expressing 8,11-linoleate diol synthase from Penicillium chrysogenum.

Hydroxy unsaturated fatty acids can be used as antimicrobial surfactants. 8,11-Linoleate diol synthase (8,11-LDS) catalyzes the conversion of unsaturated fatty acid to 8-hydroperoxy unsaturated fatty acid, and it is subsequently isomerized to 8,11-dihydroxy unsaturated fatty acid by the enzyme. The optimal reaction conditions of recombinant Escherichia coli expressing Penicillium chrysogenum 8,11-LDS for the production of 8,11-dihydroxy-9,12(Z,Z)-octadecadienoic acid (8,11-DiHODE), 8,11-dihydroxy-9,12,15(Z,Z,Z)-octadecatrienoic acid (8,11-DiHOTrE), 8-hydroxy-9(Z)-hexadecenoic acid (8-HHME), and 8-hydroxy-9(Z)-octadecenoic acid (8-HOME) were pH 7.0, 25°C, 10 g/L linoleic acid, and 20 g/L cells; pH 6.0, 25°C, 6 g/L α-linolenic acid, and 60 g/L cells; pH 7.0, 25°C, 8 g/L palmitoleic acid, and 25 g/L cells; and pH 8.5, 30°C, 6 g/L oleic acid, and 25 g/L cells, respectively. Under these optimized conditions, the recombinant cells produced 6.0 g/L 8,11-DiHODE for 60 min, with a conversion of 60% (w/w) and a productivity of 6.0 g/L/h; 4.3 g/L 8,11-DiHOTrE for 60 min, with a conversion of 72% (w/w) and a productivity of 4.3 g/L/h; 4.3 g/L 8-HHME acid for 60 min, with a conversion of 54% (w/w) and a productivity of 4.3 g/L/h; and 0.9 g/L 8-HOME for 30 min, with a conversion of 15% (w/w) and a productivity of 1.8 g/L/h. To best of our knowledge, this is the first report on the biotechnological production of 8,11-DiHODE, 8,11-DiHOTrE, 8-HHME, and 8-HOME. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:390-396, 2017.

At-line determining spore germination of Penicillium chrysogenum bioprocesses in complex media.

Spore inoculum quality in filamentous bioprocesses is a critical parameter associated with viable spore concentration (1) and spore germination (2). It influences pellet morphology and, consequently, process performance. The state-of-the-art method to measure viable spore concentration is tedious, associated with significant inherent bias, and not applicable in real-time. Therefore, it is not usable as process analytical technology (PAT). Spore germination has so far been monitored using image analysis, which is hampered by complex medium background often observed in filamentous bioprocesses. The method presented here is based on the combination of viability staining and large-particle flow cytometry which enables measurements in real-time and hence aims to be applicable as a PAT tool. It is compatible with the complex media background and allows the quantification of metabolically active spores and the monitoring of spore germination. A distinction of germinated spores and not germinated spores was based on logistic regression, using multiparameteric data from flow cytometry. In a first step, a significant correlation between colony-forming unit (CFU) counts and viable spore concentration (1) in an industrially relevant model bioprocess was found. Spore germination (2) was followed over the initial process phase with close temporal resolution. The validation of the method showed an error below 5 %. Differences in spore germination for various spore inocula ages and spore inoculum concentrations were monitored. The real-time applicability of the method suggests the implementation as a PAT tool in filamentous bioprocesses.

Identification and control of moulds responsible for black spot spoilage in dry-cured ham.

The aims of this work were to identify moulds responsible for black spot spoilage in the drying and cellar stages of dry-cured ham processing and evaluate the effectiveness of preventive actions for controlling this alteration. Four mould strains isolated from spoiled hams were identified by morphological characteristics and the ITS and ß-tubulin sequencing. Two of them were Cladosporium oxysporum, one was C. cladosporioides and the remaining one was C. herbarum. These spoiling strains reproduced the black spots on dry-cured ham-based media and ham slices. Additionally, the effect of water activity (aw) conditions reached throughout dry-cured ham ripening and the activity of the protective culture Penicillium chrysogenum CECT 20922 against the spoiling moulds were evaluated. In the dry-cured ham model system the growth of the Cladosporium strains was minimised when the aw approaches 0.84 or in P. chrysogenum CECT 20922 inoculated dry-cured ham slices. Therefore such combination could be used to avoid the black spot formation in dry-cured ham.

At-line determination of spore inoculum quality in Penicillium chrysogenum bioprocesses.

Spore inoculum quality in filamentous bioprocesses is a critical parameter influencing pellet morphology and, consequently, process performance. It is essential to determine the concentration of viable spores before inoculation, to implement quality control and decrease batch-to-batch variability. The ability to assess the spore physiologic status with close-to-real time resolution would offer interesting perspectives enhanced process analytical technology (PAT) and quality by design (QbD) strategies. Up to now, the parameters contributing to spore inoculum quality are not clearly defined. The state-of-the-art method to investigate this variable is colony-forming unit (CFU) determination, which assesses the number of growing spores. This procedure is tedious, associated with significant inherent bias, and not applicable in real time.Here, a novel method is presented, based on the combination of viability staining (propidium iodide and fluorescein diacetate) and large-particle flow cytometry. It is compatible with the complex medium background often observed in filamentous bioprocesses and allows for a classification of the spores into different subpopulations. Next to viable spores with intact growth potential, dormant or inactive as well as physiologically compromised cells are accurately determined. Hence, a more holistic few on spore inoculum quality and early-phase biomass composition is provided, offering enhanced information content.In an industrially relevant model bioprocess, good correlation to CFU counts was found. Morphological parameters (e.g. spore swelling) that are not accessible via standard monitoring tools were followed over the initial process phase with close temporal resolution.

Penicillium chrysogenum DSOA associated with marine sponge (Tedania anhelans) exhibit antimycobacterial activity.

A strain of Penicillium chrysogenum was isolated from Tedania anhelans (marine sponge) collected from Indian Ocean (8°22'30″N latitude and 76°59'16″ longitude) and deposited in culture collection centers. The strain subjected to different culture conditions for production of extrolites were extracted using ethyl acetate and chloroform. When both extracts were subjected for antibacterial activity, latter had high activity. Minimum inhibitory concentration of chloroform extract ranged from 31.25-1000 µg/mL in tested microbes such as, Mycobacterium tuberculosis H37Ra, Mycobacterium avium, Mycobacterium fortuitum, Mycobacterium smegmatis, Mycobacterium vaccae, Staphylococcus aureus, Aeromonas hydrophila, Pseudomonas aeruginosa and Vibrio cholerae. No cytotoxicity was observed in Vero cell line up to 399.10 µg/mL. Antibacterial activity previously reported by Parameswaran et al. in 1997 from ethyl acetate extract of T. anhelans might be due to the diketopiperazines, Cyclo-(L-Pro-L-Phe) and Cyclo-(L-Leu-L-Pro) produced by the associated fungi-P. chrysogenum DSOA. It is producing a metabolites having antimycobacterial activity, a first report.

Characterization of a novel 8R,11S-linoleate diol synthase from Penicillium chrysogenum by identification of its enzymatic products.

To identify novel fatty acid diol synthases, putative candidate sequences from Penicillium species were analyzed, and hydroxy fatty acid production by crude Penicillium enzyme extracts was assessed. Penicillium chrysogenum was found to produce an unknown dihydroxy fatty acid, a candidate gene implicated in this production was cloned and expressed, and the expressed enzyme was purified. The product obtained by the reaction of the purified enzyme with linoleic acid was identified as 8R,11S-dihydroxy-9,12(Z,Z)-octadecadienoic acid (8R,11S-DiHODE). The catalytic efficiency of this enzyme toward linoleic acid was the highest among the unsaturated fatty acids tested, indicating that this enzyme was a novel 8R,11S-linoleate diol synthase (8R,11S-LDS). A sexual stage in the life cycle of P. chrysogenum has recently been discovered, and 8R,11S-DiHODE produced by 8R,11S-LDS may constitute a precocious sexual inducer factor, responsible for regulating the sexual and asexual cycles of this fungus.

Common airborne fungi induce species-specific effects on upper airway inflammatory and remodelling responses.

OBJECTIVE: Whilst the exact cause of chronic rhinosinusitis (CRS) remains elusive, it is clear that both inflammation and remodelling are key disease processes. Environmental fungi have been linked to airway inflammation in CRS; however, their role in the pathogenesis of this condition remains controversial. The current consensus suggests that whilst fungi may not be directly causative, it is likely that CRS patients have deficits in their innate and potentially acquired immunity, which in turn may modify their ability to react to fungi. This study used a nasal polyp explant tissue stimulation model to study the inflammatory and remodelling responses related to challenge with common airborne fungal species. METHODS: Ex vivo nasal polyp tissue from six well phenotyped CRSwNP patients undergoing functional endoscopic sinus surgery was stimulated with 1, 10 and 100 µg/ml of Alternaria alternata, Aspergillus niger, Cladosporium sphaerospermum and Penicillium notatum and compared with unchallenged polyp tissue as control. Enzyme-linked immunosorbent assay (ELISA) was used to measure the levels of pro-inflammatory cytokines interleukin-6 (IL-6), granulocyte macrophage colony stimulating factor (GM-CSF) and tumour necrosis factor-α (TNF-α); and pro-remodelling cytokines transforming growth factor-b1 (TGF-b1), and basic fibroblast growth factor (bFGF) in the polyp supernatant. RESULTS: Aspergillus niger stimulation increased pro-inflammatory cytokines TNF-α, GM-CSF and IL-6 whilst having little effect on the remodelling cytokines bFGF and TGF-b1. In contrast, stimulation with Cladosporium sphaerospermum, Alternaria alternata and Penicillium notatum reduced pro-inflammatory cytokines TNF-α and IL-6, but induced a dose-dependent increase in remodelling cytokines TGF-b1 and bFGF. CONCLUSIONS: This study shows that common airborne fungi induce species-specific effects on the upper airway inflammatory and remodelling responses. These findings provide further immunological evidence of a disease-modifying role for fungi in CRS.

Microarray hybridization analysis of light-dependent gene expression in Penicillium chrysogenum identifies bZIP transcription factor PcAtfA.

The fungal velvet complex is a light-dependent master regulator of secondary metabolism and development in the major penicillin producer, Penicillium chrysogenum. However, the light-dependent mechanism is unclear. To identify velvet-dependent transcriptional regulators that show light-regulated expression, we performed microarray hybridizations with RNA isolated from P. chrysogenum ΔPcku70 cultures grown under 13 different long-term, light-dependent growth conditions. We compared these expression data to data from two velvet complex deletion mutants; one lacked a subunit of the velvet complex (ΔPcvelA), and the other lacked a velvet-associated protein (ΔPclaeA). We sought to identify genes that were up-regulated in light, but down-regulated in ΔPcvelA and ΔPclaeA. We identified 148 co-regulated genes that displayed this regulatory pattern. In silico analyses of the co-regulated genes identified six proteins with fungal-specific transcription factor domains. Among these, we selected the bZIP transcription factor, PcAtfA, for functional characterization in deletion and complementation strains. Our data clearly indicates that PcAtfA governs spore germination. This comparative analysis of different microarray hybridization data sets provided results that may be useful for identifying genes for future functional analyses.

Effect of low shear modeled microgravity on phenotypic and central chitin metabolism in the filamentous fungi Aspergillus niger and Penicillium chrysogenum.

Phenotypic and genotypic changes in Aspergillus niger and Penicillium chrysogenum, spore forming filamentous fungi, with respect to central chitin metabolism were studied under low shear modeled microgravity, normal gravity and static conditions. Low shear modeled microgravity (LSMMG) response showed a similar spore germination rate with normal gravity and static conditions. Interestingly, high ratio of multiple germ tube formation of A. niger in LSMMG condition was observed. Confocal laser scanning microscopy images of calcofluor flurophore stained A. niger and P. chrysogenum showed no significant variations between different conditions tested. Transmission electron microscopy images revealed number of mitochondria increased in P. chrysogenum in low shear modeled microgravity condition but no stress related-woronin bodies in fungal hyphae were observed. To gain additional insight into the cell wall integrity under different conditions, transcription level of a key gene involved in cell wall integrity gfaA, encoding the glutamine: fructose-6-phosphate amidotransferase enzyme, was evaluated using qRT-PCR. The transcription level showed no variation among different conditions. Overall, the results collectively indicate that the LSMMG has shown no significant stress on spore germination, mycelial growth, cell wall integrity of potentially pathogenic fungi, A. niger and P. chrysogenum.

Effect of fungi and light on seed germination of three Opuntia species from semiarid lands of central Mexico.

Fungal attack under light reduces mechanical resistance of the testa of Opuntia seeds, making it easier for the embryo to emerge. However, the effect of fungi on Opuntia seed germination in darkness is unknown. We evaluated the combined effects of light and inoculation with Phoma medicaginis, Trichoderma harzianum, Trichoderma koningii, and Penicillium chrysogenum on germination of O. streptacantha, O. leucotricha, and O. robusta seeds, from central Mexico. We also evaluated the combined effects of seed age (2-, 3-, and 12-year-old seeds) and presence of fungi on the testa on O. streptacantha germination. All fungal species eroded the funicular envelope and promoted seed germination for O. leucotricha and O. streptacantha, but did more so in light than in darkness. For the latter species, younger seeds inoculated with fungi had lower germination than older ones. For O. robusta, we found that seeds inoculated with P. medicaginis and T. harzianum had similar germination in light and in darkness. Our results strongly indicate that deterioration of the testa by fungi is higher in light than in darkness.

Sexual reproduction and mating-type-mediated strain development in the penicillin-producing fungus Penicillium chrysogenum.

Penicillium chrysogenum is a filamentous fungus of major medical and historical importance, being the original and present-day industrial source of the antibiotic penicillin. The species has been considered asexual for more than 100 y, and despite concerted efforts, it has not been possible to induce sexual reproduction, which has prevented sexual crosses being used for strain improvement. However, using knowledge of mating-type (MAT) gene organization, we now describe conditions under which a sexual cycle can be induced leading to production of meiotic ascospores. Evidence of recombination was obtained using both molecular and phenotypic markers. The identified heterothallic sexual cycle was used for strain development purposes, generating offspring with novel combinations of traits relevant to penicillin production. Furthermore, the MAT1-1-1 mating-type gene, known primarily for a role in governing sexual identity, was also found to control transcription of a wide range of genes with biotechnological relevance including those regulating penicillin production, hyphal morphology, and conidial formation. These discoveries of a sexual cycle and MAT gene function are likely to be of broad relevance for manipulation of other asexual fungi of economic importance.

Rhizosphere fungus Penicillium chrysogenum promotes growth and induces defence-related genes and downy mildew disease resistance in pearl millet.

Susceptible pearl millet seeds (cv 7042S) were treated with the plant growth promoting fungus Penicillium chrysogenum (PenC-JSB9) at 1 × 10(8) spores·ml(-1) to examine mRNA expression profiles of five defence responsive genes and test its ability to induce resistance to downy mildew caused by Sclerospora graminicola. PenC-JSB9 treatment at 1 × 10(8) CFU·ml(-1) for 6 h significantly enhanced seed germination (9.8- 89%), root length (4.08% to 5.1 cm), shoot length (18.9% to 7.77 cm) and reduced disease incidence (28%) in comparison with untreated controls. In planta colonisation of PenC-JSB9 showed that all three root segments (0-6 cm) and soil dilutions incubated on PDA produced extensive mycelial growth, however colonisation frequency of PenC-JSB9 was significantly higher in soil than in root segments. Spatiotemporal studies revealed that induction of resistance was triggered as early as 24 h and a minimum 2-3 days was optimal for total resistance to build up between inducer treatment and challenge inoculation in both experiments. In Northern blot analysis, transcript accumulation of resistant and PenC-JSB9 induced susceptible cultivars showed higher basal levels of defence gene expression than non-pretreated susceptible controls. Transcript accumulation in resistant seedlings challenge-inoculated with the pathogen showed maximum expression of CHS (3.5-fold increase) and Pr-1a (threefold increase) at 24 and 12 h, respectively. While PenC-JSB9 pretreated susceptible seedlings challenge-inoculated showed rapid and enhanced expression of LOX and POX at 48 h and for CHT at 24 h, whereas non-pretreated susceptible seedlings after pathogen inoculation showed weak expression of hybridised defence genes. Enhanced activation of defence genes by PenC-JSB9 suggests its role in elevated resistance against S. graminicola.