Morphological and Physiological Changes on Rhizopus stolonifer by Effect of Chitosan,Oligochitosan or Essential Oils

Effects of chitosan, oligochitosan and the essential oils of clove and cinnamon were evaluated on hyphal morphology, cell wall thickness, minimum medium pH changes and respiration of Rhizopus stolonifer. Changes in hyphal morphology were observed due to chitosan or oligochitosan treatment in this fungus. Mycelial branching, abnormal shapes and swelling were showed on hyphae of R. stolonifer treated with chitosan, whereas the development of hyphae was markedly inhibited by the effect of oligochitosan. Clove and cinnamon oils caused few morphological changes in the hyphae of R. stolonifer. Cell wall thickness was increased approximately 2‐ to 3‐fold by effect of chitosan, oligochitosan and the essential oil of clove. R. stolonifer grown in minimum medium generated a decrease in the medium's pH. However, the addition of chitosan or oligochitosan caused increases in pH of medium culture. The highest pH value (5.4) was observed in the presence of chitosan. The respiration of R. stolonifer was stimulated at low concentrations of chitosan, oligochitosan or essential oils. Significant changes in morphology and physiology of this fungus were demonstrated by the effect of all evaluated compounds. The most important changes were induced on cells of R. stolonifer treated with chitosan and oligochitosan.     

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.     

Non-Specific Induction of Pisatin and Local Resistance in Pea Leaves by Elicitors from Mycosphaerella pinodes, M. melonis and M. ligulicola and the Effect of Suppressor from M. pinodes

The accumulation of pisatin was induced non-specifically by elicitors prepared from the high molecular weight fraction (molecular weight: more than 10,000 daltons) of the spore germination fluid of three species of Mycosphaerella-plant pathogens in pea leaves with epidermis removed, regardless of the pathogenicity of the fungi to pea. Before the elicitation of pisatin synthesis, local resistance to infection by Mycosphaerella pinodes was induced by elicitors again non-specifically inpea leaves in which wax had been removed from the leaf surface. The substance responsible for local resistance could be extracted with ethylacetate from the elicitor-containing drop diffusate which was placed on pea leaves. The substance prevented the penetration of M. pinodes through heat-killed pea epidermis, but did not affect spore germination. The suppressor prepared from the low molecular weight fraction (molecular weight: less than 10,000 daltons) of the spore germination fluid of M.pinodes counteracted the ability of elicitors to induce both phases of resistance mechanisms.     

Structural and developmental studies of Chlamydomyzium oviparasiticum from Rhabditis nematodes and in culture

The oomycete (Peronosporomycete) Chlamydomyzium oviparasiticum, previously recorded as a parasite of rotifer eggs, was found infecting Rhabditis nematodes in a sample of rotting garden compost. For the first time C. oviparasiticum was cultured in liquid media, which enabled more detailed studies of zoospore behaviour and facilitated the use of confocal microscopy. Rhabditis nematodes were successfully re-infected from liquid-cultured inoculum. Light (including video) microscopy and transmission electron microscopy were used to document details of thallus development, zoospore release and resting spore morphology to enable comparison with other oomycete species. This species showed several significant saprolegnialian characters such as the ‘achlyoid’ pattern of spore formation, centrifugal cleavage and structured encystment vesicles. In contrast, spore release into a transient vesicle was a peronosporalean characteristic. The thick-walled resting spores showed relatively poor cytoplasmic preservation and had a thick multi-layered wall. It was still not possible to unequivocably decide whether these were chlamydospores or parthenogenically formed oospores. The phylogenetic significance of these observations is discussed.     

Intradiurnal periodicity of fungal spore concentrations (Alternaria, Botrytis, Cladosporium, Didymella, Ganoderma) in Cracow, Poland

Aerobiological monitoring enables the definition of seasonal fungal spore concentrations and also intradiurnal time when the highest concentrations of spores could cause or increase allergy symptoms. These data are useful to estimate symptoms of disease, duration of infection and how advanced the illness is in people suffering from fungal allergens. The aim of the study was to compare the concentrations of fungal spores (Alternaria, Botrytis, Cladosporium, Didymella, Ganoderma) during dry and rainy periods and to analyse their intradiurnal changes. Average daily spore concentrations in dry and rainy periods were compared, using z test, separately for each taxon, season and for a combined 3-year period. Intradiurnal periodicity of fungal spore concentrations was analysed on the basis of three complementary diagrams. These spore concentrations were presented using three curves for all, dry and rainy days in 1997–1999 (April–November). The spore percentage in particular hours was normalized in relation to the daily spore sum accepted as 100%. Two further diagrams enabled the more precise analysis of the highest concentrations in dry days. Daily Botrytis and Cladosporium spore concentrations did not show significant differences between dry and rainy periods. In the case of Didymella and Ganoderma spore concentrations, there were no significant differences between both weather types in the single years, although there was a significant difference when a 3-year period was considered. The differences between daily concentrations of Alternaria spores in dry and rainy periods occurred in 1997 and in a 3-year period. Intradiurnal periodicity of spore concentrations was different for ‘dry’ and ‘wet’ fungal spores. Dry spores are released from the spore-producing parts of the fungus under conditions of decreasing humidity and increasing airflow. Examples of dry spores are those from Alternaria, Cladosporium and Botrytis. Wet spores, such as those from many Ascomycetes (Didymella) and Basidiomycetes (Ganoderma), are released into the atmosphere by processes related to humidity conditions or rain. The highest concentrations of ‘dry’ spores were observed early in the afternoon, while highest values of ‘wet’ spore concentrations occurred in the predawn hours. Statistically non-significant differences between daily spore concentrations in dry and rainy periods of single seasons were found except for Alternaria. Statistically significant differences could occur when the studied period was longer than one season (Alternaria, Didymella, Ganoderma). The highest concentrations of Alternaria, Botrytis and Cladosporium spores were recorded at noon and early in the afternoon. Concentrations of Didymella and Ganoderma spores were highest in the predawn hours.     

Molybdenum hydroxylases in Drosophila

Summary The molybdenum hydroxylases are a ubiquitous class of enzymes which contain molybdenum in association with a low molecular weight cofactor. Genetic evidence suggests that the Drosophila loci, ma-1, cin and lxd are concerned with this cofactor because mutants for any one of these loci simultaneously interrupt activity for two molybdenum hydroxylases, XDH and A0. A third enzyme activity, P0, is also absent in each of the three mutants but evidence classifying P0 as a molybdoenzyme has been lacking. This study utilizes the known tungsten sensitivity of molybdoenzymes to demonstrate directly that pyridoxal oxidase is also a molybdoenzyme. The low molecular weight molybdenum cofactor is found to be severely reduced in extracts of the lxd and cin mutants but ma-1 mutants have high levels of cofactor. A partially purified preparation of XDH crossreacting material from ma-1 was also shown to contain the molybdenum cofactor. These results, considered with data from other workers are taken to indicate that the functions of all three of the loci examined could be concerned with some aspect of cofactor biosynthesis.This work was supported by PHS grant GM 23736 to V. Finnerty     

中国产石韦属植物孢子形态特征及分类学意义

利用光镜和扫描电镜,对石韦属(Pyrrosia)19种植物的孢子纹饰进行了研究。结果表明:19种石韦属孢子都为黄色,形态均为肾形,两侧对称,单裂缝,说明该属植物是一个自然类群。表面纹饰类型有3种,即瘤状、瘤状—疣状和瘤状—网脊状。孢子表面纹饰特征性状稳定,在种间存在较大差异。该研究结果可以为形态近似种的分类提供参考依据,同时也为石韦属属下分类系统的建立提供了重要证据。     

Kinetic study of acid depolymerization of chitosan and effects of low molecular weight chitosan on erythrocyte rouleaux formation

In this study, the depolymerization of chitosan was carried out in an acetic acid aqueous solution and was followed by viscometry for molecular weight determination. It was found that the depolymerization rate increased with elevated temperatures and with high acid concentrations. Based on FTIR analysis, the chitosan was depolymerized randomly along the backbone; no other structural change was observed during the acid depolymerization process. Revealed in the TGA study, the degradation temperature and char yield of LMWCs (low molecular weight chitosan) were molecular weight dependent. The blood compatibility of LMWCs was also investigated: rouleaux formation was observed when erythrocyte contacted with LMWCs, which showed that LMWCs are able to interfere with the negatively charged cell membrane through its polycationic properties. Furthermore, as regards a kinetics investigation, the values of M_n (number-average molecular weight) were obtained from an experimentally determined relationship. The kinetics study showed that the complex salt, formed by amine on chitosan and acetic acid, acted as catalyst. Finally, the activation energy for the hydrolysis of the glycosidic linkage on chitosan was calculated to be 40 kJ/mol; the mechanism of acid depolymerization is proposed. In summary, LMWCs could be easily and numerously generated with acid depolymerization for further biological applications.     

Biosynthesis of scopoletin in Hevea brasiliensis leaves inoculated with Phytophthora palmivora

Inoculation of resistant (R) and susceptible (S) Hevea brasiliensisleaves with Phytophthora palmivorainduced foliar necrosis and biosynthesis of scopoletin (Scp), considered as a Heveaphytoalexin. The degree of resistance of four clones, as classified by the necrotic lesions, was related to the rapidity and intensity of Scp production. The resistant BPM-24, and marked partially resistant clone PB-235, displayed an early secretion of scopoletin that intensified and lasted longer than the weak partially resistant RRIT251, and susceptible clone RRIM600. The lesion size and amount of Scp after infection were positively correlated to the concentration of spores applied to Hevealeaves. In addition, in leaflets inoculated with high spore concentration, Scp reached the highest level earlier than those with low spore concentration. A fungitoxic effect of Scp on mycelium growth was shown in bioassays; the I₅₀ value tested on Phytophthora palmivora was relatively the same as on Phytophthora botryosa, but much lower than those found on other leaf pathogens of rubber tree, Corynespora cassiicolaand Colletotrichum gloeosporioides. The lesion size and level of Scp upon spore inoculation may be appropriate for classifying Heveaclones according to their R/S with respect to P. palmivora     

三株海洋木霉的应用潜力

【目的】探索3株海洋生境木霉的应用潜力。【方法】经过筛选和诱变,获得高抑菌活性及产孢量的木霉突变株;通过优化培养基、温度、初始p H考察其产孢量及最适培养条件;综合抑菌谱、重寄生及抑菌相关基因考察其抑菌活性;采用特殊培养基法考察其产纤维素酶、植酸酶、铁载体以及降解磷钾的能力,高效液相色谱法测定其产吲哚乙酸能力。【结果】3株木霉菌的产孢量分别为3.45×108、3.10×108和2.55×108 CFU/cm2,与野生型相比分别提高了88.52%、63.16%和180.22%;且均可产生厚垣孢子,其中XG20-1厚垣孢子产量最高,达到3.56×108 CFU/m L。3株木霉菌具有较广抑菌谱及对番茄早疫病菌的重寄生作用,同时扩增得到Tex1、Nag1、Eg1基因,生物学测试显示其均具有产纤维素酶、几丁质酶以及铁载体的能力,证明其抑菌活性是多种机制共同作用的结果;菌株可以降解磷钾,且吲哚乙酸产量分别为2.61、1.57和1.92 mg/L,具有促进植物生长的潜力。【结论】本文中3株木霉菌在开发为生防菌与生物肥料方面展现出良好的应用潜力。     

Extrachromosomal inheritance in Schizosaccharomyces pombe

Summary Primary and secondary spore clones were analyzed from two- and three-factor crosses involving the mitochondrial markers conferring resistance to antimycin (A ^R ), chloramphenicol (C ^R ), and erythromycin (E ^R ). As in zygote clones (Seitz-Mayr et al., 1978), transmission of markers is higher in two-factor trans-crosses than in cis-crosses. Except transmission of C ^R in the cross A ^R C ^R E ^R xA ^S C ^{S E S} , no significant differences between cis- and trans-configuration were observed in three-factor crosses. In contrast to zygote clones, in spore clones transmission rates of the two or three markers in a given cross are roughly equal. 18 out of 20 secondary spore clones of different mitochondrial phenotypes appeared to be homoplasmic, whereas 2 still continued to segregate. One of these spore isolates was analyzed, and segregation was found to continue for more than 150 generations after spore germination. Whereas up to more than 80% of zygote clones in certain crosses were uniform, only 2 out of 91 tetrads were uniform, i.e. all four spores were homoplasmic for the same mitochondrial genotype. Presence or absence of recombinant mitochondrial phenytypes among secondary spore clones from tetrads indicated, whether, cytoplasmic mixing had occurred in the original zygote or not. Within an ascus, the number of spores containing recombinant genotype(s) is a direct measure for the extent of cytoplasmic mixing in the zygote. In 82 tetrads analyzed, the number of tetrads with 0, 1, 2, 3, and 4 spores containing recombinant genotype(s) were 25, 37, 14, 5, and 1, respectively. In conclusion, the extent of cytoplasmic mixing at the cell stage before forespore membrane formation is highly variable.     

Autotropism in Fungal Spores

Autotropism was examined in germinating spore pairs of Rhizopusstolonifer, Mucor plumbeus, Trichoderma viride, and Botrytiscinerea. When germinated on agar surfaces the first three speciesexhibited negative autotropism, B. cinerea being neutral inits autotropic behaviour. More pronounced negative autotropismwas shown by the first three species when germinated on a filmof Cellophane applied to an agar surface. Under these conditionsB. cinerea displayed positive autotropism. Spore pairs of R. stolonifer germinated on agar containing cellulosepowder or charcoal showed less negative autotropism than onagar alone. Touching spore pairs of each species showed a markedtendency towards cis-ness, i.e. germ-tubes beginning on thesame side of a line joining the two spore centres, under theculture conditions described, the one exception being the reductionin cis-ness recorded when R. stolonifer was germinated on agarcontaining charcoal. Time courses of germination were determined for single sporesand touching spore pairs of R. stolonifer and M. plumbeus anda significant promotion was obtained in the spore pairs as comparedwith the single spores. Although both these species exhibitmarked negative autotropism there was a strong tendency forthe positive germ-tube, i.e. one beginning more nearly towardsits neighbour, to emerge before the negative germ-tube in thosespore pairs having one germtube positive and the other negativein orientation. Also, in R. stolonifer, the replacement of germination-promotersby germination-inhibitors in filtrates from spore suspensionsas they age is correlated with a change from positive to negativeautotropism in germinating members of (+ –) spore pairs. Possible mechanisms are discussed to account for the observedeffects.     

Biological control of Pythium aphanidermatum in cucumber with a combined application of Lysobacter enzymogenes strain 3.1T8 and chitosan

Pythium aphanidermatum (Edson) Fitzp., causing root and crown rot in cucumber, was successfully managed by Lysobacter enzymogenes strain 3.1T8. Greenhouse experiments were performed with cucumber plants grown in rockwool blocks up to 5 weeks with a recirculated nutrient solution. Application of L. enzymogenes 3.1T8 in combination with chitosan (the deacetylated derivative of chitin) reduced the number of diseased plants by 50–100% in four independent experiments relative to the Pythium control. Application of chitosan or the bacterial inoculant alone was not effective. Washed bacterial cells plus chitosan inhibited Pythium-induced disease, but the supernatant without bacterial cells combined with chitosan was not effective. The most effective and convenient type of commercially available chitosan was selected. Chitosan disappeared from the hydroponic system within 24 h after application, which we attribute to enzyme expression of L. enzymogenes 3.1T8 induced by the exposure to chitosan. Plate counts of the nutrient solution on a general bacterial medium showed the dominance of the inoculated strain, and an increased bacterial population growing on chitin and chitosan as single carbon source. The population density of L. enzymogenes 3.1T8 on the cucumber roots was investigated with a strain specific real-time TaqMan PCR. Highest chitosan concentrations applied (0.1 and 0.03 g/plant) resulted in the highest numbers of L. enzymogenes 3.1T8 present on roots; i.e. 10⁸–10⁹ cells/g root. Substantially higher numbers of bacterial cells were observed by scanning electron microscopy after application of chitosan; no morphological or other qualitative differences were found. The results indicate that addition of chitosan enhanced the biocontrol efficacy of L. enzymogenes 3.1T8; either chitosan serves as C- and N-source for the antagonist, induces antagonistic gene expression, or both.     

Physical properties of fungal chitosan

Fungi are promising alternative sources of chitosan. This study evaluated the physical properties of fungal chitosan from Absidia coerulea (AF 93105), Mucor rouxii (Ag 92033), and Rhizopus oryzae (Ag 92033). FT-IR and X-ray diffraction of the extracted products showed typical chitosan peak distributions which confirmed the extracted products to be chitosan. All of their glucosamine contents and degrees of deacetylation (DD) were over 80%, not showing obvious differences respectively. However, differences had been observed in their molecular weight (M_w), ranging from 6.6  to 560 kDa. The results of this study demonstrated that different fungi could produce different M_w chitosan with high DD and high purity.     

蛋白和酯酶电泳在根霉鉴定上的应用

对根霉(Rhizopus)属的十个种或变种共二十四株菌的菌体可溶性蛋白和酯酶同工酶进行了电泳的研究得到对这个属分类上更多的依据.在严格控制培养,提取和电泳条件的情况下,同一株菌不同批次所得菌体蛋白电泳图谱有较好的重复性.在相同的条件下,每个种的根霉有各自特征性蛋白图谱,种内不同菌株的蛋白图谱和酯酶酶谱基本相同.特别是形态特征明显、分类地位明确的种,种内各株的图谱也较一致,如R. stolonifer;与R.circinans.在确定新变种R. delemar var. latoapicalis时,电泳图谱与R.delemar var.delema:有明显不同,起到了佐证作用.因此认为,蛋白图谱与酯酶酶谱相辅相成,在根霉种的分类中是一有效的辅助手段.     

Evaluation of chitosans and Pichia guillermondii as growth inhibitors of Penicillium digitatum

Chitosans were obtained by room-temperature-homogeneous-deacetylation (RTHD) and freeze-pump-out-thaw-heterogeneous-deacetylation (FPT) from chitins purified from fermentations. Commercial chitosan was deacetylated by three-FPT-cycles. Chitosans and Pichia guillermondii were evaluated on the growth of Penicillium digitatum. Medium molecular weight (M(W)) chitosans displayed higher inhibitory activity against the yeast than low M(W) biopolymers. Chitosans with low degree of acetylation (DA) were inhibitory for yeast and mould. Therefore, a low M(W) and high DA chitosan was selected for use against moulds combined with yeasts. Biopolymer and yeasts presented an additive effect, since chitosans were effective to delay spore germination, whereas yeast decreased apical fungal growth.     

Spore control (Sco) mutations in Bacillus subtilis

Summary Morphogenesis of spore control (Sco) mutants of Bacillus subtilis was followed by quantitative electron microscopy. In wild type cultures the morphological stages II, III, IV and V attain their peaks of frequency at 1.5, 3.5, 4.5 and 5 h after t ₀, respectively. Stages II and IV are short, stages III and V occupy the main part of the process. Morphogenesis is slowed down in Sco A1, Sco B2 and Sco12 strains: all the stages are delayed and prolonged. Stage III cells are predominant for a long time, until t ₉ or later. It is suggested that the mechanism which switches off sporulation genes is affected and this leads to overproduction of sporulation-associated products. In other Sco mutants, such as Sco C3, part of the population sporulates normally but a fraction of cells persists for a long time at stages III and V. The pleiotropy of the spore control mutations is discussed.     

Chitosan topical gel formulation in the management of burn wounds

Wound healing properties of chitosan with different molecular weight and degree of deacetylation ranges have been examined. The macroscopic image and histopathology were examined using chitosan, Fucidin^® ointment and to blank. The rate of contraction was evaluated by determination of the unclosed area as a function of time. The treated wounds were found to contract at the highest rate with high molecular weight–high degree of deacetylation chitosan-treated rats as compared to untreated, treated, and Fucidin^® ointment-treated rats. Wounds treated with high molecular weight chitosan had significantly more epithelial tissue (p < 0.05) than wounds with any other treatment and the best re-epithelization and fastest wounds closure were found with the high molecular weight chitosan treatment group. Histological examination and collagenase activity studies revealed advanced granulation tissue formation and epithelialization in wounds treated with high molecular weight chitosan (p < 0.05). High molecular weight with high degree of deacetylation chitosan samples therefore demonstrates potential for use as a treatment system for dermal burns.     

Crystallinity of Partially N-Acetylated Chitosans

In order to search for a chitosan with low crystallinity, partially N-deacetylated chitins (PDC) and partially N-acetylated chitosans (PAC) with a low degree of N-acetylation (DAc) were examined by X-ray powder diffraction measurements. Three PDC samples, having less than 30% DAc and prepared by solid-state deacetylation, gave X-ray powder patterns showing the presence of α-chitin, a hydrated crystal of chitosan, or their mixture, respectively. When these PDC samples were treated with an acid-alkali, however, reduced crystallinity was observed. By annealing in water at 160 or 200°C, the latter PDC having DAc less than approx. 22% gave powder patterns indicating the presence of an anhydrous crystal which may spoil the chitosan’s functionality. In contrast, PAC prepared by N-acetylating pure chitosan (DAc=0%) in a swollen state, which can be expected to have random copolymers in the chain, was always less crystallized than PDC, this crystallinity depending on the molecular weight. In the case of high-molecular-weight PAC samples, whose DAc was in the range of 5–21%, the effect of high molecular weight on reducing crystallinity was larger than that of the degree of N-acetylation.     

Lyophilized Sustained Release Mucoadhesive Chitosan Sponges for Buccal Buspirone Hydrochloride Delivery: Formulation and In Vitro Evaluation

This work aims to prepare sustained release buccal mucoadhesive lyophilized chitosan sponges of buspirone hydrochloride (BH) to improve its systemic bioavailability. Chitosan sponges were prepared using simple casting/freeze-drying technique according to 3² factorial design where chitosan grade was set at three levels (low, medium, and high molecular weight), and concentration of chitosan solution at three levels (0.5, 1, and 2%). Mucoadhesion force, ex vivo mucoadhesion time, percent BH released after 8 h (Q8h), and time for release of 50% BH (T_50%) were chosen as dependent variables. Additional BH cup and core buccal chitosan sponge were prepared to achieve uni-directional BH release toward the buccal mucosa. Sponges were evaluated in terms of drug content, surface pH, scanning electron microscopy, swelling index, mucoadhesion strength, ex vivo mucoadhesion time, and in vitro drug release. Cup and core sponge (HCH 0.5E) were able to adhere to the buccal mucosa for 8 h. It showed Q8h of 68.89% and exhibited a uni-directional drug release profile following Higuchi diffusion model.KEY WORDS: buspirone HCL, casting/freeze-drying technique, chitosan, cup and core sponge, mucoadhesive buccal sponges