Nikkomycin Z counteracts rylux BSU and Congo red inhibition ofSaccharomyces cerevisiae growth but does not prevent formation of aberrant cell walls

Rylux BSU and Congo red bind to chitin, interfere with proper cell-wall assembly, and stimulate chitin synthesis by increasing, most probably, chitin synthase 3 (ChS3) levels inSaccharomyces cerevisiae. On the other hand, the antibiotic nikkomycin Z inhibits chitin synthesis competitively. As ChS3 is the critical target of nikkomycin Z, its effect was tested in cells inhibited in growth by Rylux BSU or Congo red. Nikkomycin Z counteracted this inhibition but did not counteract aberrant cell-wall formation. These results indicate that chitin synthesis stimulation is the key step in Rylux BSU and Congo red inhibition and support the idea that increase in chitin synthesis represents a compensatory response to damaged cell-wall structure. As Rylux BSU and Congo red bind to newly synthesized chitin, further damage is caused in the wall and the response works in this case contrariwise. Nikkomycin Z breaks this vicious circle by counteracting the chitin synthesis stimulation.     

Staining of fungal cell walls with fluorescent brighteners: Flow-cytometric analysis

Spore walls ofBackusella lamprospora (Mucorales) were stained with ten fluorescent brighteners (FB) and the intensity of their fluorescence was determined. The fluorescence was most intense with Uvitex 2B (100%), other brighteners yielding lower fluorescence intensities: Blankophor BA 267% and BA 200% about 75%, Rylux BSU about 50%, other Rylux agents 10–30%. The agents most suitable for microscopic diagnostics of human and animal mycoses are Uvitex 2B, Blankophor BA 267% and BA 200%, Rylux BSU, and also Rylux BS and PRS. The regulation of excessive fluorescence of fungal cells during microscopic observation is discussed. For the purposes of microscopic diagnosis of human and animal mycosis Uvitex 2B, Blankophor BA 267% and BA 200%, Rylux BSU and, possibly, Rylux BS and PRS are recommended.     

Effect of the new fluorescent brightener Rylux BSU on morphology and biosynthesis of cell walls in Saccharomyces cerevisiae

Rylux BSU, a new fluorescent brightener from the family of 4,4-diaminostilbene-2,2disulfonic acid derivatives, inhibited growth and cytokinesis of the yeast Saccharomyces cerevisiae. In the presence of 0.1–1 mg/ml Rylux BSU the cells grew in clumps, had irregular shape and were larger than controls. They formed apparently normal primary septa but their secondary septa and lateral cell walls, especially those in older cells, were abnormally thick with large deposits of amorphous wall material in the periplasmic spaces all over the cell surface. Chitin content in the cell walls of cells grown in the presence of Rylux BSU was increased 2 to 5 times in comparison to that of the controls and glucan content was reduced by up to 30%. In the in vitro assays with particulate membrane fractions, Rylux BSU acted as a non-competitive inhibitor of -1,3-glucan synthase with inhibitory constant K _i=1.75 mg/ml whereas the chitin synthase was inhibited to a much lesser extent. From the difference of the effects of Rylux BSU on the synthesis of chitin in vivo and in vitro it is concluded that the brightener interacts with chitin synthase only indirectly, possibly by influencing the properties of integral plasma membrane.Abbreviations RBSU Rylux BSU, 1,4-benzenedisulfonic acid-2,2-[ethyleneidy]bis[(3-sulpho-4,1-phenylene)imino[6-bis(2-hydroxyethyl)amino]-1,3,5-triazine-4,2-diylamino]]bis-, hexasodium salt - FB fluorescent brightener     

The fluorescence brightener Rylux BSU induces dimorphism inBasidiobolus ranarum

The fluorescence brightener Rylux BSU (RBSU) showed an affinity for polysaccharide components of cell walls and accumulated in the extension zones of hyphal apices inBasidiobolus ranarum. It inhibited the polarized growth of mycelial hyphae and induced isotropic growth resulting in spherical thick-walled cells up to 456 μm in diameter. On the inner cell wall surface, massive protuberances were formed. The cell wall and protuberances were positive in PAS and the Grocott method and stained with fluorochromes Blankophor BA, Calcofluor, Uvitex 2B, Rylux BSU and FITC-labeled WGA- and ConA-lectins. The WGA-FITC fluorescence intensity of the wall’s outermost layer, if not connected with neighbouring cells, and the fluorescence intensity of the innermost layer and of some protuberances mainly in their apical parts were on the average twice higher than the fluorescence intensity of the remaining wall material. RBSU binding to the cell wall material was stable. The process of converting from polarized to isotropic growth was reversible, depending upon contact with RBSU-containing medium. Repeated transfers of cells from RBSU-containing medium to an RBSU-free medium resulted in the development of apical swollen dumbbell-shaped cells.     

Susceptibility of Larvae of Galleria mellonella to Infection by Aspergillus fumigatus is Dependent upon Stage of Conidial Germination

The ability of conidia of the human pathogenic fungus Aspergillus fumigatus to kill larvae of the insect Galleria mellonella was investigated. Conidia at different stages of the germination process displayed variations in their virulence as measured using the Galleria infection model. Non-germinating (‘resting’) conidia were avirulent except when an inoculation density of 1 × 10⁷ conidia per insect was used. Conidia that had been induced to commence the germination process by pre-culturing in growth medium for 3 h were capable of killing larvae at densities of 1 × 10⁶ and 1 × 10⁷ per insect. An inoculation density of 1 × 10⁵ conidia per insect remained avirulent. Conidia in the outgrowth phase of germination (characterised as the formation of a germ tube) were the most virulent and were capable of killing 100% of larvae after 5 or 24 h when 1 × 10⁷ or 1 × 10⁶ conidia, that had been allowed to germinate for 24 h, were used. Examination of the response of insect haemocytes to conidia at different stages of the germination process established that haemocytes could engulf non-germinating conidia and those in the early stages of the germination process but that conidia, which had reached the outgrowth stages of germination were not phagocytosed. The results presented here indicate that haemocytes of G. mellonella are capable of phagocytosing A. fumigatus conidia less than 3.0 μm in diameter but that conidia greater than this are too large to be engulfed. The virulence of A. fumigatus in G. mellonella larvae can be ascertained within 60–90 h if infection densities of 1 × 10⁶ or 1 × 10⁷ activated conidia (pre-incubated for 2–3 h) per insect are employed.     

Antifungal activity of berberine iodide, a constituent ofFumaria indica

Berberine iodide, an isoquinoline alkaloid was isolated fromFumaria indica and its efficacy was tested against spore germination of some plant parasitic as well as saprophytic fungi. The alkaloid significantly curbed spore germination ofCurvularia lunata, Erysiphe cichoracearum, E. pisi, Fusarium udum andPenicillium species. Complete inhibition (100%) of spore germination was observed inE. cichoracearum andPenicillium species at 1.5 g/L.Albugo candida andAlternaria species were not affected by the chemical.     

Studies on the phospholipase A of dermatophytes

The presence of phospholipase A activity was detected in three dermatophytes:Microsporum cookei, Trichophyton mentagrophytes, andEpidermophyton floccosum. The activity was always higher inT. mentagrophytes than inM. cookei andE. floccosum. All exhibited phospholipase A₁ and A₂ activities, but the activity was largely A₂ inM. cookei and A₁ inE. floccosum. T. mentagrophytes possessed almost equal activities of phospholipase A₁ and A₂.     

Spore Coat Protein Synergizes Bacillus thuringiensis Crystal Toxicity for the Indianmeal Moth (Plodia interpunctella)

Spores from Bacillus thuringiensis serovars kurstaki and entomocidus synergized crystal protein toxicity for larvae of the Indianmeal moth (Plodia interpunctella). Preparations of spore-crystal mixtures of either serovar were more toxic for the larvae than either purified spores or crystals alone (based on dry weight). Spores lost 53% of their toxicity for the Indianmeal moth after 2 h of UV-irradiation, but remained partially toxic (28%) even after 4 h of irradiation. Spore coat protein was toxic for the Indianmeal moth and was synergistic with B. thuringiensis serovar kurstaki HD-1 crystal protein. Enhanced toxicity of the combined spore-crystal preparation was attributed to a combination of crystal and spore coat protein, and included the effects of spore germination and resulting septicemia in the larval hemolymph. Ultraviolet irradiation of spores reduced the toxicity from septicemia but not the synergism caused by spore coat protein. The potencies of spore-crystal preparations must be carefully evaluated on the basis of contributions from all three factors. Received: 15 September 1997/Accepted: 21 October 1997     

Infection Process of Plectosporium tabacinum on False Cleavers (Galium spurium)

A native fungus, Plectosporium tabacinum (van Beyma) M. E. Palm, W. Gams et Nirenberg, has potential as a bioherbicide for the control of both herbicide-resistant and herbicide-susceptible false cleavers. Limited information is available on the infection process of P. tabacinum. P. tabacinum spore distribution pattern, germination, penetration, and colonization on false cleavers leaves were examined using confocal, light, and scanning electron microscopy. The results demonstrated that conidia were distributed over the entire surface of leaves and cotyledons. More than 90% of the conidia germinated on the leaf surface 6-8 h after inoculation. Penetration of the leaf epidermis by conidia started 8-10 h after inoculation. Histological observation showed that no appressoria were formed by P. tabacinum, but its hyphae produced appressed club-like structures that penetrated the cuticle and epidermal layers. No stomata or other natural openings were observed on the upper leaf surface of false cleavers seedlings. Penetration occurs directly on epidermal cells with more frequent intercellular penetrations. Hyphal penetration was visualized at a depth of 30 and 40 üm after 8 and 16 h of incubation, respectively. Secondary hyphae colonized mesophyll cells 16 h after inoculation. Even spore distribution, short spore germination time, club-like infection structure formation, direct penetration, quick colonization, and mucous secretion on false cleavers leaves may contribute to the kill of false cleavers by P. tabacinum. Slow spore germination and germ tube growth, low spore germination numbers, and no infection structure formation on Brassica napus leaves may be factors affecting the host selectivity of P. tabacinum.     

Nutritional requirements of keratinophilic fungi and dermatophytes for conidial germination

Germination of spores of Chrysosporium crassitunicatum, Nannizzia fulva (+), Nannizzia fulva (–) and Trichophyton equinum was studied in the presence of various carbon and nitrogen sources. Effect of different temperatures on spore germination was also determined. Maximum spore germination within 24 hours was recorded when glucose was used as a carbon source for all the test fungi. Except sodium nitrate all the inorganic nitrogen sources enhanced the spore germination at 0.05% concentration. Most of the organic nitrogen sources used were found to be stimulatory for the spore germination of test fungi. Optimum temperature i.e. 28 °C supported maximum spore germination of all the test fungi within 24 hours. C. Crassitunicatum, N. fulva(+), N. fulva(–) could germinate upto 35 °C but beyond that no spore germination was noticed in these fungi. T. equinum could germinate at a higher temperature of 40 °C but the percentage of germination was very low.     

Germination and physiological properties of Frankia spores

Spores from four Frankia strains were isolated and purified to homogeneity. The purified spores were biochemically and physiologically characterized and compared to vegetative cells. Frankia spores exhibited low levels of endogenous respiration that were at least ten-fold lower than the endogenous respiration rate of vegetative cells. The macromolecular content of purified spores and vegetative cells differed. One striking difference among the Frankia spores was their total DNA content. From DAPI staining experiments, only 9% of strain ACN1^AG spore population contained DNA. With strains DC12 and EuI1c, 92% and 67% of their spore population contained DNA. The efficiency of spore germination was correlated to the percentage of the spore population containing DNA. These results suggest that the majority of strain ACN1^AG spores were immature or nonviable. The presence of a solidifying agent inhibited the initial stages of spore germination, but had no effect once the process had been initiated. The optimal incubation temperature for spore germination was 25°C and 30°C for strains DC12 and EuI1c, respectively. A mild heat shock increased the efficiency of spore germination, while root extracts also stimulated spore germination. These results suggest that strains DC12 and EuI1c may be suitable strains for further germination and genetic studies.     

Control of Fusarium solani rot of potato tubers with fungicides

Tecnazene (up to 33 ppm) and dichloran (up to 500 ppm) had little effect on germination of spores or growth of Fusarium solani isolated from and causing a rot of potato tubers; they also did not decrease rotting when applied to wounds later inoculated with the pathogen. Benomyl and thiabendazole (up to 500 ppm) also had little effect on spore germination but did greatly decrease growth at 5 ppm. A pronounced pink coloration developed in cultures growing slowly in the presence of benomyl; a similar though less striking effect appeared in agar cultures containing thiabendazole. Benomyl suspended in water or diluted with Fuller's earth gave good control of rotting when applied to wounds inoculated later with F. solani. Still better control was obtained with thiabendazole; dusts containing 1% a.i. substantially decreased rots and those containing 10 % a.i. gave almost complete control when applied to wounds shortly before inoculation. Thiabendazole was also very effective when used 24 h after inoculation and a fair measure of control was obtained when it was applied 24 h later. Benomyl and thiabendazole placed on apparently intact surfaces of tubers caused tissue 5 mm deep to become toxic to F. solani 10 days later, and, unexpectedly, this tissue prevented spore germination. Fuller's earth alone substantially decreased rotting. The results obtained suggest that dusts containing thiabendazole have some promise for the control of Fusarium rots of potato tubers, especially of early crops.     

Enzyme make-up of trichophyton rubrum and t. mentagrophytes

Summary Aldolase activity in the cell-free extracts of two dermatophytes,T. mentagrophytes andT. rubrum, was investigated. The kinetics of the enzyme and the effects of metal ions and metal-binders are also reported. The enzyme was more active inT. mentagrophytes than inT. rubrum. The optimum pH for the enzyme action was 7.2 and it was completely inactivated at 60° C. Cobalt and magnesium ions and cysteine activated the enzyme. Inhibition caused by EDTA and o-phenanthroline was partially reversed by cobalt ions. The dermatophyte aldolase resembles bacterial aldolase in its properties.     

A mechanism of propagule release from unilocular reproductive structures in brown algae

Summary The structure of unilocular sporangia inP. littoralis was investigated along with several other species of brown algae in order to study the mechanism by which propagules are released from unilocular reproductive structures. Unilocular sporangia inP. littoralis are composed of a spherical cell wall of two distinct layers and contain a number of zoospores. The mass of spores is surrounded and permeated by mucilaginous carbohydrates. It is suggested that the production of these carbohydrates generates the necessary pressure to weaken the sporangial wall. In addition, ultrastructural observations indicate that further weakening seems to occur due to digestion of the inner wall layer. Walls of sporangia were mechanically broken just prior to normal spore release in order to investigate whether internal pressure exists, and if it can effect spore discharge. Results show that an internal pressure does exist prior to normal spore discharge and that this pressure is not generated by turgor pressure of the spores themselves or by a semi-permeable wall osmoticum system. The discharge of spores seems to occur when the carbohydrate around the spores swells. The adsorption of water when plants are immersed by the incoming tide thus seems a likely mechanism of spore discharge. The similarities of unilocular reproductive structures and spore release in several brown algal species suggests common mechanisms of propagule discharge for members of thePhaeophyta.     

Investigation of factors influencing spore germination of Paenibacillus polymyxa ACCC10252 and SQR-21

Bioorganic fertilizer containing Paenibacillus polymyxa SQR-21 showed very good antagonistic activity against Fusarium oxysporum. To optimize the role of P. polymyxa SQR-21 in bioorganic fertilizer, we conducted a study of spore germination under various conditions. In this study, l-asparagine, glucose, fructose and K⁺ (AGFK), and sugars (glucose, fructose, sucrose, and lactose) plus l-alanine were evaluated to determine their ability to induce spore germination of two strains; P. polymyxa ACCC10252 and SQR-21. Spore germination was measured as a decrease in optical density at 600 nm. The effect of heat activation and germination temperature were important for germination of spores of both strains on AGFK in Tris–HCl. l-Alanine alone showed a slight increase in spore germination; however, fructose plus l-alanine significantly induced spore germination, and the maximum spore germination rate was observed with 10 mmol l⁻¹ l-alanine in the presence of 1 mmol l⁻¹ fructose in phosphate-buffered saline (PBS). In contrast, fructose plus l-alanine hardly induced spore germination in Tris–HCl; however, in addition of 10 mmol l⁻¹ NaCl into Tris–HCl, the percentages of OD₆₀₀ fall were increased by 19.6% and 24.3% for ACCC10252 and SQR-21, respectively. AGFK-induced spore germination was much more strict to germination temperature than that induced by fructose plus l-alanine. For both strains, fructose plus l-alanine-induced spore germination was not sensitive to pH. The results in this study can help to predict the effect of environmental factors and nutrients on spore germination diversity, which will be beneficial for bioorganic fertilizer storage and transportation to improve the P. polymyxa efficacy as biological control agent.     

Antifungal potential of essential oil and various organic extracts of <Emphasis Type="Italic">Nandina domestica</Emphasis> Thunb. against skin infectious fungal pathogens

This study was undertaken to assess the in vitro antifungal potential of the essential oil and n-hexane, chloroform, ethyl acetate, and methanol extracts of Nandina domestica Thunb. against dermatophytes, the casual agents of superficial infections in animals and human beings. The oil (1,000 μg/disc) and extracts (1,500 μg/disc) revealed 31.1–68.6% and 19.2–55.1% antidermatophytic effect against Trichophyton rubrum KCTC 6345, T. rubrum KCTC 6375, T. rubrum KCTC 6352, Trichophyton mentagrophytes KCTC 6085, T. mentagrophytes KCTC 6077, T. mentagrophytes KCTC 6316, Microsporum canis KCTC 6591, M. canis KCTC 6348, and M. canis KCTC 6349, respectively, along with their respective minimum inhibitory concentration values ranging from 62.5 to 500 and 125 to 2,000 μg/ml. Also, the oil had strong detrimental effect on spore germination of all the tested dermatophytic fungi as well as concentration and time-dependent kinetic inhibition of T. rubrum KCTC 6375. The present results demonstrated that N. domestica mediated oil and extracts could be potential sources of natural fungicides to control certain important dermatophytic fungi.     

The physiology of spore-negative and spore-positive nodules ofMyrica gale

The physiology of spore-negative and spore-positive root nodules was investigated inMyrica gale L. grown in water culture in a growth chamber. Spore(–) nodules were induced withFrankia cultures and spore(+) nodules with crushed nodules. Gas exchange was measured in a flow-through system.The time course of acetylene reduction following addition of acetylene was essentially the same in both spore(–) and spore(+) nodules with a stable maximum between 2 and 4 minutes followed by a steep decline to a minimum (37% of the maximum) between 9 and 30 minutes depending on the plant. The minimum was followed by a partial recovery. Nodule CO₂ evolution showed a similar pattern but the minimum rate (83% of the maximum) was not nearly as low.Plants nodulated with one spore(–) and one spore(+) strain were compared at 6, 8 and 10 weeks after inoculation. At 6 weeks the spore(–) plants had 52% greater specific nitrogenase activity and 46% more biomass than the spore(+) plants. At 8 and 10 weeks, however, the differences between plants with spore(–) and spore(+) nodules became smaller.Plants nodulated with 4 spore(–) and 5 spore(+) strains were compared at 8 weeks after inoculation. Collectively the spore(–) plants exhibited a 32% greater specific nitrogenase activity, a 15% lower energy cost of nitrogenase activity (CO₂/C₂H₄), and invested 31% less biomass in nodules than the spore(+) plants. The spore(–) plants also produced 16% more biomass indicating that spore(–) strains are generally more desirable than spore(+) strains. However, two spore(+) strains were as effective as the spore(–) strains.     

Colonization capacity of the soil micro-organisms in relation to fungistasis

Summary Evans' tube and cellophane agar-disc techniques were used to evaluate the colonization capacity of the soil microbes and fungistasis, respectively. Spore germination ofAlternaria tenuis, Curvularia geniculata, Helminthosporium rostratum andPestalotia sp. was studied on a natural soil. Aspergillus fumigatus was the pioneer colonizer, followed in the order of decreasing colonizing capacity by,A. flavus, A. niger, A. terreus andPenicillium nigricans. Sensitivity of test spores to soil fungistasis declined in the order,C. geniculata → Pestalotia sp. →A. tenuis → H. rostratum. The fungistatic level of the soil samples decreased gradually from side arms 1 to 7 of the Evans tube, inoculated with unsterilized soil from one end. Studies conducted with an inoculum of four soil fungi (A. fumigatus, A. flavus, A. niger, andP. nigricans) as well as those involving fumigation of Evans tube by CS₂ also proved the greater colonizing capacity ofA. fumigatus. The level of fungistasis followed closely the colonization pattern of this fungus. When added individually to the soil, all fungi were able to inhibit spore germination of test fungi to some degree or other.     

Failure to decontaminate Glomus clarum NT4 spores is due to spore wall-associated bacteria

 Exposure of spores of Glomus clarum NT4 to solutions of chloramine-T (2.5–10% w/v) for 10–120 min failed to fully decontaminate all spores. Scanning electron microscopy did not show the presence of contaminants on treated spores, but transmission electron microscopy revealed bacterial cells embedded within the outer spore wall layer. Bacteria that remained protected within the spore walls were detected only when the spores were placed on appropriate media. Nutrient agar and tryptic soy agar supported relatively high levels of contaminant growth and were regarded as good media for assessing contamination, whereas the detection of contaminant growth on water agar required prolonged incubation. Contamination and germination of G. clarum NT4 spores following decontamination treatments were dependent on spore age. Generally, lower concentrations of chloramine-T and shorter incubation periods were required to reduce contamination of freshly harvested spores than of mature spores. Exposure to 10% chloramine-T for 120 min was required to reduce the levels of contamination of mature spores to ≤10%. Unfortunately, spore germination was compromised by rigorous decontamination treatments, thus the success of any decontamination procedure should be evaluated prior to its routine use. Moreover, if the interpretation of experimental results rests on the assumption of true surface sterility of VAMF spores, we suggest that the axenic condition of spores be confirmed prior to experimentation on a medium that encourages contaminant growth. Accepted: 12 July 1995     

In vitro evaluation of combination of Trichoderma harzianum and chitosan for the control of sapstain fungi

In vitro assays were undertaken to evaluate the control of two sapstain fungi, Leptographium procerum and Sphaeropsis sapinea by a combination of chitosan or chitosan oligomer and an albino strain of Trichoderma harzianum. Spore germination and hyphal growth of the test fungi were assessed on media amended with chitosan or chitosan oligomer with and without T. harzianum using either simultaneous inoculation with test fungus or inoculation 1, 2, or 3 days after pre-infection with test fungus.There was no mycelial growth of the test fungi regardless of chitosan concentrations used when either L. procerum or S. sapinea was simultaneously inoculated with T. harzianum. However, the dose–response of chitosan or chitosan oligomer on the test fungi was apparent when T. harzianum was not simultaneously inoculated with test fungus but introduced later. There was a greater growth reduction at higher concentrations (0.075–0.1% v/v) of chitosan, and overall chitosan oligomer was more effective than chitosan aqueous solution.Chitosan alone was able to restrict or delay the germination of spores but the combination of chitosan and T. harzianum inhibited spore germination and hence colony formation of test fungi regardless of time delay.