Mycelial Walls Research Articles

Roles of Small Subunits of Laccase (ssPOXA3a/b) in Laccase Production by Pleurotus eryngii var. ferulae.

The small subunit, ssPOXA3a/b, and the large subunit, POXA3, are indispensable components of typical heterodimeric laccase (Lacc2) in white rot fungi. However, the enzymatic and biological functions of ssPOXA3a/b remain unclear. The present study revealed that neither ssPOXA3a nor ssPOXA3b per se has a catalytic ability, whereas their combination with POXA3 (and especially ssPOXA3b) enhances the activity, thermostability, and pH stability of POXA3. In Pleurotus eryngii var. ferulae, there was no regulatory relationship between ssPOXA3a/b and POXA3 at the transcriptional level. However, sspoxa3a/b overexpression had a negative feedback effect on lacc6 transcription. By contrast, poxa3 transcripts had no effect on any other laccase isoenzyme. Overexpression of sspoxa3a/b resulted in small fungal pellets, thin mycelial walls, and facilitated laccase secretion. However, poxa3 overexpression had no influence on pellet morphology. Collectively, this work elucidated the functions of ssPOXA3a/b and laid an empirical foundation for the development of high-yield laccase.

Effects of β-Glucans from Aureobasidium pullulans on Cucumber Mosaic Virus Infection in Chili Pepper

Cucumber mosaic virus (CMV), the most prevalent virus in chili pepper (Capsicum annuum L.), negatively affects chili pepper production in South Korea. In this study, foliar spraying with β-glucans obtained from the mycelial walls of the yeast-like fungus Aureobasidium pullulans inhibited CMV infection of chili pepper if applied before virus inoculation. At three concentrations, β-glucans from A. pullulans significantly ameliorated CMV symptoms in treated chili pepper; the effect was greater in plants treated with 0.01% β-glucans than 0.005% or 0.001% β-glucans. Double antibody sandwich enzyme-linked immunosorbent assay showed that these β-glucans treatments resulted in 1.7- to 10-fold reductions in CMV accumulation in the treated chili pepper. The glucans did not act directly on the virus and did not interfere with virus disassembly or replication. Foliar spraying with 0.01% β-glucans from A. pullulans at 24 hr intervals for 3 days significantly increased plant height, the total number of fruit, and the fresh weight of chili pepper fruit. However, the stem diameter of chili pepper treated with β-glucans did not increase significantly. These results indicate that foliar spraying with β-glucans from A. pullulans acts an antiviral agent against CMV infection and stimulates chili pepper growth.

Sodium pheophorbide a controls cherry tomato gray mold (Botrytis cinerea) by destroying fungal cell structure and enhancing disease resistance-related enzyme activities in fruit

Sodium pheophorbide a (SPA) is a natural photosensitizer. The present study investigated the antifungal activity and mechanism of SPA against Botrytis cinerea in vitro and in vivo. Its inhibitory effect was studied on the spore germination and mycelial growth of B. cinerea. The effects of SPA on cell wall integrity, cell membrane permeability, and mycelial morphology of B. cinerea were also determined. Additionally, how SPA effected B. cinerea in vivo was evaluated using cherry tomato fruit. The results showed that SPA effectively inhibited the spore germination and mycelial growth of B. cinerea under light conditions (4000 lx). SPA significantly affected both cell wall integrity and cell membrane permeability (P < .05). In addition, SEM analysis suggested that B. cinerea treated with SPA (12.134 mg/mL) showed abnormal mycelial morphology, including atrophy, collapse, flattening, and mycelial wall dissolution. In vivo tests showed that SPA could increase the activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) significantly (P < .05); however, SPA had no significant effect on phenylalanine ammonia lyase (PAL) activity. In short, SPA could destroy the fungal cell structure and enhance disease resistance-related enzyme activity in cherry tomatoes, thereby controlling cherry tomato gray mold.

Calcineurin signaling pathway influences Aspergillus niger biofilm formation by affecting hydrophobicity and cell wall integrity

BackgroundBiofilms, as a kind of fixed-cell community, can greatly improve industrial fermentation efficiency in immobilized fermentation, but the regulation process is still unclear, which restricts their application. Ca2+ was reported to be a key factor affecting biofilm formation. However, the effect of Ca2+ on biofilm structure and microbiology was yet only studied in bacteria. How Ca2+-mediated calcineurin signaling pathway (CSP) alters biofilm formation in bacteria and fungi has rarely been reported. On this basis, we investigated the regulation of CSP on the formation of biofilm in Aspergillus niger.ResultsDeletion of the key genes MidA, CchA, CrzA or CnaA in the CSP lowered the Ca2+ concentration in the mycelium to a different extent, inhibited the formation of A. niger biofilm, reduced the hydrophobicity and adhesion of spores, destroyed the cell wall integrity of hyphae, and reduced the flocculation ability of hyphae. qRT-PCR results showed that the expression of spore hydrophobic protein RodA, galactosaminogalactan (GAG) biosynthesis genes (uge3, uge5, agd3, gtb3), and α-1,3-glucan biosynthesis genes (ags1, ags3) in the ∆MidA, ∆CchA, ∆CrzA, ∆CnaA strains were significantly down-regulated compared with those of the wild type (WT). In addition, the transcription levels of the chitin synthesis gene (chsB, chsD) and β-1,3-glucan synthesis gene (FksA) were consistent with the change in chitin and β-1,3-glucan contents in mutant strains.ConclusionThese results indicated that CSP affected the hydrophobicity and adhesion of spores, the integrity of mycelial cell walls and flocculation by affecting Ca2+ levels in mycelium, which in turn affected biofilm formation. This work provides a possible explanation for how CSP changes the formation of A. niger biofilm, and reveals a pathway for controlling biofilm formation in industrial immobilized fermentation.

Bioimaging structural signatures of the oomycete pathogen Sclerospora graminicola in pearl millet using different microscopic techniques

In this case study, the mycelium growth of Sclerospora graminicola in the infected tissues of pearl millet and the process of sporulation and liberation of sporangia and zoospores were observed using four different microscopic techniques. The cotton blue-stained samples observed under light microscope revealed the formation of zoospores with germ tubes, appressoria and initiation of haustorium into the host cells, while the environmental scanning electron microscopy showed the rapid emergence of sporangiophores with dispersed sporangia around the stomata. For fluorescence microscopy, the infected leaf samples were stained with Fluorescent Brightener 28 and Calcofluor White, which react with β-glucans present in the mycelial walls, sporangiophores and sporangia. Calcoflour White was found to be the most suitable for studying the structural morphology of the pathogen. Therefore, samples observed by confocal laser scanning microscopy (CLSM) were pre-treated with Calcofluor White, as well as with Syto-13 that can stain the cell nuclei. Among the four microscopic techniques, CLSM is ideal for observing live host-pathogen interaction and studying the developmental processes of the pathogen in the host tissues. The use of different microscopic bioimaging techniques to study pathogenesis will enhance our understanding of the morphological features and development of the infectious propagules in the host.

The metabolic response of suspension-cultured cells from blast-resistant and -susceptible rice (Oryza sativa L.) genotypes to a Pyricularia oryzae elicitor

Rice blast, incited by Pyricularia oryzae Cavara, is an important fungal disease of rice (Oryza sativa L.) worldwide. To understand the resistance mechanisms of rice to P. oryzae, we studied metabolic responses of rice suspension-cultured cells to treatment with an elicitor prepared from the mycelial walls of P. oryzae. Suspension-cultured cells of blast- resistant (Usen) and -susceptible (CO39) rice genotypes were profiled for differential metabolic responses at 1, 3, 5 and 7 days after elicitor treatment using gas chromatography coupled to electron impact ionization-time of flight-mass spectroscopy (GC/EI–TOF–MS). A total of 99 metabolites were identified by mass spectral and retention index match to the reference data of the Golm Metabolome Database (GMD). Of the 99 metabolites, 66 metabolites were classified into different chemical classes representing mostly primary metabolites. The remaining 33 metabolites are yet non-identified but are frequently observed metabolites that are part of the GMD reference data and are designated with a unique GMD identifier. Multivariate analysis revealed that the suspension-cultured cells of susceptible and resistant genotypes differ in regard to primary metabolism. Differential responses to the P. oryzae elicitor included significant changes among aromatic amino acids, phenylpropanoids, polyamines and sugars of major carbohydrate metabolism.

Assessment of bioactivity of endophytic actinomycetes from some medicinal plants

Out of 70 isolates of actinomycetes obtained from medicinal plants, 21 displayed antagonistic activity against Alternaria alternata, Fusarium oxysporum and Rhizoctonia solani. All the isolates were screened for hydrolytic enzymes production. Among these, 47 isolates produced amylase, 25 protease and 16 were having ability to produce chitinase. The extracellular chitinase activity of two selected isolates AR3 and O9 was evaluated (0.083 and 0.080 U/ml) with 0.6% colloidal chitin, which was found better as compared to 1% colloidal chitin. Scanning electron microscopy revealed rupture of F. oxysporum mycelial cell wall at the area of interaction with O9. The endophytic actinomycete isolate O9 was identified as Streptomyces rochei strain KMB-1 by 16S rDNA sequencing. This isolate was found to be promising in terms of seed germination and Fusarium wilt control in muskmelon under green house conditions.

A toolbox to measure changes in the cell wall glycopolymer composition during differentiation of Streptomyces coelicolor A3(2)

Cell wall glycopolymers (CWG) represent an important component of the Gram-positive cell envelope with many biological functions. The mycelial soil bacterium Streptomyces coelicolor A3(2) incorporates two distinct CWGs, polydiglycosylphosphate (PDP) and teichulosonic acid, into the cell wall of its vegetative mycelium but only little is known about their role in the complex life cycle of this microorganism. In this study we established assays to measure the total amount of CWGs in mycelial cell walls and spore walls, to quantify the individual CWGs and to determine the length of PDP. By applying these assays, we discovered that the relative amount of CWGs, especially of PDP, is reduced in spores compared to vegetative mycelium. Furthermore we found that PDP extracted from mycelial cell walls consisted of at least 19 repeating units, whereas spore walls contained substantially longer PDP polymers.

Purification of a novel hydrophobin PN1 involved in antibacterial activity from an edible mushroom Pleurotus nebrodensis

Due to their hydrophobic characteristics, hydrophobins function in a broad range of processes in the growth and development of filamentous fungi. In this study, a novel hydrophobin, PN1, was purified from the fruiting bodies of Pleurotus nebrodensis by first removing other proteins from mycelial cell walls with hot SDS solution. The molecular mass of PN1 was approximately 10.7 kDa. The N-terminal AA sequence was QESPVNQCNTGSIEECKTVQ, which did not match that of any other mushroom hydrophobin described thus far. Levels of hydrophobin PN1 protein in the tissue of fruiting bodies was decreased by fruiting body development under low temperatures. Low levels of hydrophobin PN1 was correlated to the formation of water-soaked blotches on fruiting body tissues that appeared dark and translucent, and were usually sunken. In addition, hydrophobin PN1 levels were correlated with protecting fruiting bodies against surface bacterial colonization and multiplication. Low levels of hydrophobin PN1 resulted in water-soaked blotches that had high levels of surface bacteria belonging to multiple species when compared with nonsymptomatic tissue of fruiting bodies with higher hydrophobin PN1. These results show a direct link between mushroom hydrophobins and resistance of mushrooms to bacterial attack.

A Multifaceted Study of Scedosporium boydii Cell Wall Changes during Germination and Identification of GPI-Anchored Proteins.

Scedosporium boydii is a pathogenic filamentous fungus that causes a wide range of human infections, notably respiratory infections in patients with cystic fibrosis. The development of new therapeutic strategies targeting S. boydii necessitates a better understanding of the physiology of this fungus and the identification of new molecular targets. In this work, we studied the conidium-to-germ tube transition using a variety of techniques including scanning and transmission electron microscopy, atomic force microscopy, two-phase partitioning, microelectrophoresis and cationized ferritin labeling, chemical force spectroscopy, lectin labeling, and nanoLC-MS/MS for cell wall GPI-anchored protein analysis. We demonstrated that the cell wall undergoes structural changes with germination accompanied with a lower hydrophobicity, electrostatic charge and binding capacity to cationized ferritin. Changes during germination also included a higher accessibility of some cell wall polysaccharides to lectins and less CH3/CH3 interactions (hydrophobic adhesion forces mainly due to glycoproteins). We also extracted and identified 20 GPI-anchored proteins from the cell wall of S. boydii, among which one was detected only in the conidial wall extract and 12 only in the mycelial wall extract. The identified sequences belonged to protein families involved in virulence in other fungi like Gelp/Gasp, Crhp, Bglp/Bgtp families and a superoxide dismutase. These results highlighted the cell wall remodeling during germination in S. boydii with the identification of a substantial number of cell wall GPI-anchored conidial or hyphal specific proteins, which provides a basis to investigate the role of these molecules in the host-pathogen interaction and fungal virulence.

Differential induction of peroxidase and polyphenol oxidase isozymes in suspension-cultured cells of blast resistant and susceptible genotypes of rice in response to treatment with Magnaporthe grisea elicitor and toxin

The effect of elicitor from mycelial walls of Magnaporthe grisea, the rice blast fungus and α-picolinic acid, one of the toxins produced by M. grisea on induction of peroxidase (PO), polyphenol oxidase (PPO) in suspension-cultured rice (Oryza sativa L.) cells was studied. Cultured cells of blast resistant (Usen) and susceptible (CO39) rice genotypes were treated with elicitor (50 μg of glucose equivalents per ml) or α-picolinic acid (400 ppm). The cells were harvested at different time intervals and analysed for the induction of PO and PPO. PO isozyme analysis indicated that the elicitor strongly induced the activities of PO-2 and PO-3 in cultured cells of Usen 3 days after treatment. In Usen, toxin also induced the activities of PO-3 and PO-4. However, similar levels of activities corresponding to these isozymes were recorded 7 days after treatment. In CO39, the activities of PO-1 and PO-2 were induced 3 days after elicitor treatment. In contrast, the toxin suppressed the activity of PO-2. The elicitor induced the activities of PPO-1, PPO-2 and PPO-3 in both Usen and CO39. In Usen, steady increase of PPO-3 was observed and higher level of activity was recorded 5 days after treatment. In CO39, higher level of PPO-3 was observed 1 day after treatment and declined thereafter. However, the activities of PPO-1 and PPO-2 increased 3 days after treatment in CO39. In the toxin-treated cells of Usen, higher level of activity of PPO-3 was observed 3 days after treatment.

Immunostimulatory properties and antitumor activities of glucans

New foods and natural biological modulators have recently become of scientific interest in the investigation of the value of traditional medical therapeutics. Glucans have an important part in this renewed interest. These fungal wall components are claimed to be useful for various medical purposes and they are obtained from medicinal mushrooms commonly used in traditional Oriental medicine. The immunotherapeutic properties of fungi extracts have been reported, including the enhancement of anticancer immunity responses. These properties are principally related to the stimulation of cells of the innate immune system. The discovery of specific receptors for glucans on dendritic cells (dectin-1), as well as interactions with other receptors, mainly expressed by innate immune cells (e.g., Toll-like receptors, complement receptor-3), have raised new attention toward these products as suitable therapeutic agents. We briefly review the characteristics of the glucans from mycelial walls as modulators of the immunity and their possible use as antitumor treatments.

温郁金内生真菌Chaetomium globosumL18对植物病原菌的抑菌谱及拮抗机理

PDF HTML阅读 XML下载 导出引用 引用提醒 温郁金内生真菌Chaetomium globosum L18对植物病原菌的抑菌谱及拮抗机理 DOI: 10.5846/stxb201110081465 作者: 作者单位: 吉林农业大学农业部参茸产品质量监督检验测试中心,吉林农业大学附属医院,吉林农业大学农业部参茸产品质量监督检验测试中心,,吉林农业大学农业部参茸产品质量监督检验测试中心,吉林农业大学农业部参茸产品质量监督检验测试中心,吉林农业大学农学院 作者简介: 通讯作者: 中图分类号: 基金项目: 吉林农业大学博士启动基金(201110);浙江省重大科技专项资助项目(2005C13019) Inhibition effects and mechanisms of the endophytic fungus Chaetomium globosum L18 from Curcuma wenyujin Author: Affiliation: ．Jilin Agriculture University，Ginseng and Cartialgenous testing center of Ministry of Agriculture PRC,,,,, Fund Project: 摘要 | 图/表 | 访问统计 | 参考文献 | 相似文献 | 引证文献 | 资源附件 | 文章评论 摘要:通过体外培养法,研究了药用植物温郁金内生真菌Chaetomium globosum L18对几种常见的植物病原菌的抑菌谱及其拮抗机理。结果表明,Chaetomium globosum L18对多种植物病原真菌和细菌均有不同程度的抑制作用,具有较广的抑菌谱,但对不同植物病原菌的抑制作用具有显著性差异(P＜0.05),抑制率最高可达到92.9%;抑菌机制结果显示,竞争作用和重寄生作用是其主要的拮抗机制之一;发酵产物抑制作用测定发现, 内生真菌Chaetomium globosum L18能够分泌产生抗菌物质抑制病原菌菌丝的生长和孢子萌发,可引起病原菌菌丝菌丝膨大成串珠状,分枝增多,分枝顶端膨胀后细胞壁破裂,原生质外溢,产生溶菌作用;使分生孢子萌发畸形,萌发率降低。 Abstract:Fungal endophytes are microorganisms that colonize living, internal tissues of plants without causing any immediate, overtly negative effects. Many fungal endophytes produce secondary metabolites and some of these compounds exhibit antifungal and antibacterial activity that strongly inhibits the growth of other microorganisms. Antimicrobial strains are widely distributed among endophytic fungi and do not cause any obvious symptoms on host plants. Infected hosts usually grow quickly, have strong resistance to adverse conditions and diseases, and are immune to herbivory in comparison with uninfected plants. Endophyte-infected plants thus have competitive advantage. Endophytes act as biological control agents by inhibiting pathogen growth by the production of antibiotics, hydrolases, plant growth regulators and alkaloids. They also compete with pathogens for nutrition, enhancing resistance of host plants to diseases and inducing systemic resistance. Endophytic fungi are therefore biological control agents with great potential for applied use. We investigated the antimicrobial properties of an endophytic fungus Chaetomium globosum L18 from a traditional Chinese medicinal plant Curcuma wenyujin Y. H. Chen et C. Ling (Zingiberaceae), on several conventional plant pathogens, using in vitro culturing methods. We thus present a preliminary discussion of the antimicrobial spectrum and antagonistic mechanisms of Chaetomium globosum L18. This fungus had different inhibitory actions on plant pathogenic fungi and bacteria, and wide-ranging inhibitory effects. There was significant difference (P<0.05) on inhibition of different plant pathogenic bacteria; and the inhibition rate was up to 92.9%. Our inhibition experiment showed that competition and mycoparasitism were the main antagonistic mechanisms. When two strains intersected with pathogenic fungi on co-culturing plates, plant pathogens were surrounded, and the margins of colonies gradually collapsed and shriveled. The pathogens stopped growing and began to die; and Chaetomium globosum L18 colonized the nutritious space. Chaetomium globosum L18 parasitized pathogens by parallel intergrowth, intertwining, and even penetrating their mycelia. Our fermented product test showed that Chaetomium globosum L18 was able to strongly inhibit mycelial growth and spore germination. Its presence resulted in mycelia that were twisted and inflated, with increased abnormal branching, broken mycelial walls, and cell contents released. Spores were reduced in number and deformed; germination rate and germination tube length were decreased. 参考文献 相似文献 引证文献

A study of the Candida albicans cell wall proteome

Considering the importance of proteins in the structure and function of the cell wall of Candida albicans, we analyzed the cell wall subproteome of this important human pathogen by LC coupled to MS (LC-MS) using different protein extraction procedures. The analyzed samples included material extracted by hydrogen fluoride-pyridine (HF-pyridine), and whole SDS-extracted cell walls. The use of this latter innovative procedure gave similar data as compared to the analysis of HF-pyridine extracted proteins. A total of 21 cell wall proteins predicted to contain a signal peptide were identified, together with a high content of potentially glycosylated Ser/Thr residues, and the presence of a GPI motif in 19 of them. We also identified 66 "atypical" cell wall proteins that lack the above-mentioned characteristics. After tryptic removal of the most accessible proteins in the cell wall, several of the same expected GPI proteins and the most commonly found "atypical" wall proteins were identified. This result suggests that proteins are located not only at the cell wall surface, but are embedded within the cell wall itself. These results, which include new identified cell wall proteins, and comparison of proteins in blastospore and mycelial walls, will help to elucidate the C. albicans cell wall architecture.

Disease resistance in tomato induced by an elicitor derived from cell walls of the fungal pathogen,Botrytis cinerea

Cell walls of the phytopathogenic fungus, Botrytis cinerea were identified for the presence of biotic elicitors. The isolation of carbohydrates from purified mycelial cell walls of B. cinerea (CCW) and the effect of CCW on induction of disease resistance of Lycopersicon esculentum were carried out. CCW, Immunocytophyt and a fungicide, Ridomil Gold, were evaluated for the control of late blight, early blight and nailhead spot, the most damaging diseases of tomato in Ukraine. The results of 2-year greenhouse experiments and field trials demonstrated that foliar spraying of tomato plants with sensitizing concentration of CCW (0.01%) reduced development of infection during the vegetation period and crop losses. Our results indicate that the elicitoractive carbohydrates from cell walls of the fungal pathogen B. cinerea are effective to induce systemic resistance against tomato foliar diseases under greenhouse and field conditions.

Effects of elevated CO 2 on the phytoalexin production of two soybean cultivars differing in the resistance to stem canker disease

The excess of carbon leads to changes in the production of plant defensive secondary chemicals, which in turn may modify the plant responses to herbivores and pathogens. Age and/or developmental stage as well as environmental conditions influence race-specific accumulation of phytoalexins in soybean. Thus, we hypothesized that elevated CO 2 atmospheres could lead to distinct phytoalexin production in soybean cultivars differing in their resistance to fungal attack. Two cultivars of soybean ( Glycine max, IAC-14 and IAC-18) differing in the resistance to the causal agent of the stem canker disease were subjected to 320 and 720 μmol mol −1 of CO 2 in open top chambers. Plants were kept under both CO 2 atmospheres from germination up to 9 days (VC stage), when CO 2 assimilation rates, chlorophyll a fluorescence, leaf and cotyledon area, dry mass and root: shoot ratio were measured. Phytoalexin content were evaluated in detached cotyledon diffusates of both cultivars elicited with β-glucan elicitor from mycelial walls of Phythophytora sojae. Exposure to elevated CO 2 stimulated photosynthesis, leaf and cotyledon areas and the enhancement of glyceollin production by the elicitor in the resistant cultivar (IAC-18). No changes were observed in growth parameters (biomass and area of photosynthetic tissues) and phytoalexin response in susceptible cultivar (IAC-14), despite the stimulation of photosynthetic assimilation in cotyledons under high CO 2. These findings corroborate the assumption that exposure to enriched CO 2 atmospheres can change inducible defensive responses in soybean against pathogens. These changes occurred in individual metabolites and were dependent on the cultivar resistance patterns.

Cell-wall structure and composition of Penicillium janczewskii as affected by inulin

Penicillium janczewskii, a filamentous fungus isolated from the rhizosphere of Vernonia herbacea (Asteraceae), grows rapidly on media containing either sucrose or inulin as carbon sources. Maintenance of P. janczewskii on inulin medium induces secretion of proteins with high inulinase activity but results in a mycelium that easily collapses and breaks. We evaluated the influence of inulin on fungal growth and colony morphology and on cell-wall structure and composition in comparison with growth and wall characteristics on sucrose-containing medium. P. janczewskii grown on Czapek medium with agar containing 1% (w/v) sucrose or inulin showed differences in the color and morphology of the colonies, although growth rates were similar on both carbon sources. Scanning-electron microscopy revealed that the hyphae from fungus grown on inulin-containing medium are much thinner than those from fungus cultivated on sucrose. Ultrastructural analysis of 5 d old cultures using transmission-electron microscopy indicated significant differences in the cell-wall thickness between hyphae grown on inulin or sucrose media. No differences were detected in the overall carbohydrate and protein contents of cell walls isolated from cultures grown on the two carbon sources. Glycosyl composition analyses showed glucose and galactose as the predominant neutral monosaccharides in the walls but showed no differences attributable to the carbon source. Glycosyl linkage composition analyses indicated a predominance of 3-linked glucopyranosyl in the hyphal walls when P. janczewskii was grown on inulin-containing medium. Our results suggest that growth on inulin as the sole carbon source results in structural changes in the mycelia of P. janczewskii that lead to mycelial walls with altered physical and biological properties.

Cell-wall structure and composition of Penicillium janczewskii as affected by inulin

Penicillium janczewskii, a filamentous fungus isolated from the rhizosphere of Vernonia herbacea (Asteraceae), grows rapidly on media containing either sucrose or inulin as carbon sources. Maintenance of P. janczewskii on inulin medium induces secretion of proteins with high inulinase activity but results in a mycelium that easily collapses and breaks. We evaluated the influence of inulin on fungal growth and colony morphology and on cell-wall structure and composition in comparison with growth and wall characteristics on sucrose-containing medium. P. janczewskii grown on Czapek medium with agar containing 1% (w/v) sucrose or inulin showed differences in the color and morphology of the colonies, although growth rates were similar on both carbon sources. Scanning-electron microscopy revealed that the hyphae from fungus grown on inulin-containing medium are much thinner than those from fungus cultivated on sucrose. Ultrastructural analysis of 5 d old cultures using transmission-electron microscopy indicated significant differences in the cell-wall thickness between hyphae grown on inulin or sucrose media. No differences were detected in the overall carbohydrate and protein contents of cell walls isolated from cultures grown on the two carbon sources. Glycosyl composition analyses showed glucose and galactose as the predominant neutral monosaccharides in the walls but showed no differences attributable to the carbon source. Glycosyl linkage composition analyses indicated a predominance of 3-linked glucopyranosyl in the hyphal walls when P. janczewskii was grown on inulin-containing medium. Our results suggest that growth on inulin as the sole carbon source results in structural changes in the mycelia of P. janczewskii that lead to mycelial walls with altered physical and biological properties.

Multiple Forms of Calmodulin Genes in Carrot Treated with Fungal Mycelial Walls

Eleven cDNA clones encoding the open-reading frame of calmodulin and calmodulin-like proteins were isolated from carrot seedlings treated with fungal mycelial walls. These calmodulin genes were mainly expressed in the stems and leaves of carrot, although the transcriptional level was almost negligible in the seeds and root tissues. Based on nucleotide homology, these genes were divided into two classes, class I and class II, and reverse transcription-polymerase chain reaction analyses revealed that the expression level of class II genes transiently increased when carrot seedlings were treated with the mycelial walls. In contrast, the level of class I genes apparently did not show any significant change even after treatment with the fungal components. These results suggest that the defense responses induced in the fungi-infected carrot accompany the increase in the transcriptional levels of a certain calmodulin gene(s).

Cloning and expression of calmodulin genes regulating phytoalexin production in carrot cells.

A homology-based cloning strategy yielded four cDNA clones encoding the open reading frame of carrot calmodulin, designated cam-4 and cam-8 from an oligogalacturonide elicitor-treated cell culture and cam-11 and cam-16 from cells exposed to ethylene, respectively. Reverse-transcription polymerase chain reaction analyses revealed that the expression of the cam-4 gene increased transiently when carrot seedlings were treated with oligogalacturonides, although, the cells incubated with fungal mycelial walls or ethylene did not show a significant change in the expression level. In contrast, marked and transient expression was observed for either cam-11 or cam-16 only when carrot cells were treated with ethylene. These results suggest that two classes of stimuli which are capable of triggering phytoalexin production in carrot cells, oligogalacturonides and ethylene, evoke the activation of the Ca2+ cascade in the cells by expressing distinct calmodulin genes to initiate the biosynthesis of the compound.