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.     

Chitosan-mediated changes in cell wall composition, morphology and ultrastructure in two wood-inhabiting fungi

The effect of chitosan on cell wall deposition was investigated in the two wood-inhabiting fungal species Trichoderma harzianum (CBS 597.91) and Sphaeropsis sapinea (NZFS 2725). The study used three independent analytical techniques to quantify chitin in the fungal mycelium. A colorimetric method for the detection of d-glucosamine was compared with two gas chromatography–mass spectroscopy (GC-MS) methods employing alditol acetates analysis and pyrolysis. The latter used a stable-isotope-labelled internal standard, d₃-N-acetyl glucosamine. At least in the case of S. sapinea, the study provided evidence of an increase in the chitin content in the mycelium due to chitosan treatment, indicating that chitosan treatment affected cell wall deposition. Electron microscopy techniques showed alteration in surface morphology and cell wall texture due to chitosan treatment. The implications of these results are discussed with a view to analysing possible mechanisms for growth inhibitory effects of chitosan on fungal hyphae.     

Bioactivity of intra and extracellular substances from cyanobacteria and lactic acid bacteria on “wood blue stain” fungi

Cyanobacteria (photoautotrophic prokariota) have potential for the control of pathogenic bacteria and fungi. The effect of intra and extracellular products from cyanobacterial strains on the growth of fungi isolated from “wood blue stain,” was tested. Extracellular products were obtained by concentration and sterilization of the culture medium where cyanobacteria were grown. Cyanobacterial substances promoted or inhibited fungal growth according to the fungal and cyanobacterial strains tested. Extracellular products from Nostoc muscorum 79a and the methanolic extract from Microchaete tenera 84b biomass inhibited growth of Sphaeropsis sapinea 2157 (64.7 and 775.6%, respectively). Extracellular products of Nostoc piscinale 59 and biomass methanolic extract from N. muscorum 79a produced the highest growth promotion of Trichoderma boningii 452 (105.0%) and T. viride 993 (136.7%). Extracellular products of the heterotrophic lactic acid bacterium Streptococcus termophilus were also tested and strongly inhibited (64–92%) all the fungal strains. The tested fungi have different sensitivity to the bioactive substances present in the biomass and/or the culture medium of the studied cyanobacteria and lactic acid bacterium. N. muscorum 79a, M. tenera 84b, and S. termophilus have potential to control the wood blue stain fungi by a friendly environmental alternative.     

Effect of inoculum density,nitrogen source and saprophytic fungi on Fusarium wilt of Mexican lime

Summary Mexican lime seedlings were inoculated with 0, 500, 1000, 2000, 4000 and 8000 microconidia ofFusarium oxysporum f. sp.citri per gram of potting media. The percent infection and mean disease severity rating increased with increasing inoculum density of the pathogen. In potting mix infested withAspergillus ochraceus, Penicillium funiculosum andTrichoderma harzianum at 5000 conidia per gram 2 weeks prior to infestation withF. oxysporum f. sp.citri at 0, 1000, 4000, and 8000 microconidia per gram,A. ochraceus reduced,P. funiculosum increased andT. harzianum had no effect on disease severity or pathogen population. OnlyP. funiculosum showed antagonistic activityin vitro against the pathogen. Disease severity and pathogen propagule densitites were greater and pH was lower in potting media fertilized with NH₄–N than in media fertilized with NO₃–N.Portion of M. S. thesis submitted by the senior author to the Graduate School, University of Florida, Gainesville. Florida Agricultural Experiment Stations Journal Series No. 4221.     

Metal loading and enzymatic degradation of fungal cell walls and chitin

The capacity of chitin (from crab shells) and of fungal cell walls from Trichoderma harzianum to accumulate zinc, cadmium and mercury was studied as well as the effects of adsorbed metals on the enzymatic hydrolysis by Novozym 234 of the two substrates. The total adsorbing capacity with respect to these metals was estimated to be at least 10 mmol kg^–1 chitin (dry weight) and 50 mmol kg^–1 fungal cell walls (dry weight), respectively, at pH 6.1. Enzymatic digestion of fungal cell walls preloaded with mercury and cadmium was significantly reduced, while zinc did not cause any significant inhibition. The effect of metal complexation by chitin on the enzymatic digestion was not as pronounced as for fungal cell walls. This could reflect the fact that chitin sorbed a lower total amount of metals. The inhibitory effect of metals on the enzymatic hydrolysis was caused by the association of the metals with the two substrates and not by the presence of free metals in solution.     

Comparative study on the delignification of Azadirachta indica (L) Del., wood by Chrysosporium asperatum and Trichoderma harzianum

Structural alterations induced in response to degradation by two white rot Basidiomycetes on the secondary xylem of Azadirachta indica (L) Del., was compared. In vitro decay test was employed to investigate the pattern of delignification of Azadirachta wood by Trichoderma harzianum and Chrysosporium asperatum. Wood samples inoculated with both the strains were analyzed for different periods viz. 30, 60, 90 and 120 days after fungal inoculation. Initially there was no appreciable percent weight loss of the wood blocks but later on (after 60 days) it increased rapidly and was found similar for both the strains (43-46% of wood mass). Samples inoculated with both the strains showed dual pattern of degradation i.e. selective delignification in the initial stage followed by simultaneous rot during advance stage of decay. Separation of the cells due to dissolution of middle lamella was the characteristic feature of both strains but in the advanced stage of decay, formation of erosion troughs were conspicuous in all the cell types. Other features such as cell wall thinning, rounded pit erosion, formation of erosion channels and bore holes were also observed frequently. Initially, fungal invasion started through the vessel lumen, followed by all the cell types of the xylem. From the vessels, mycelia entered into the adjacent rays and parenchyma cells through the pits. In advanced stage, degradation was so pronounced that rays were partially or even completely destroyed while many cells including vessels were either deformed or destroyed due to loss of rigidity of their walls. Structural alterations induced in response to C. asperatum and T. harzianum attack is described in details.     

Effect of anoxic conditions on wood-decay fungi treated with argon or nitrogen

The effects of low-oxygen conditions, achieved with either argon or nitrogen gas, on the viability of wood-decay fungi Coniophora puteana and Antrodia vaillantii, grown on artificial growth medium, were tested. Initial tests for viability were run after 1, 2, 3 and 4 weeks of exposure to low oxygen conditions, at oxygen levels in the vessels maintained below 10 ppm. Treatment was later extended to 10 and 16 weeks. Tests included measurements of CO₂ production and determination of the ability of fungal tissue to regenerate. The effect of anoxic conditions on the mycelia of treated fungal species was expressed as an increased time needed for regeneration or as a complete absence of growth of inocula taken from the exposed cultures. The cultures that were retarded by the low-oxygen environment consequently produced less CO₂ per hour. For C. puteana cultures, the effects of anoxic treatment became apparent in the second week of the treatment. The number of affected cultures rose steadily with the prolongation of anoxic treatment. By the 16th week of the experiment, 80% of the inocula of C. puteana did not regenerate. A. vaillantii inocula regeneration was not affected until after the fourth week of treatment. The influence of anoxic treatment on the cultures of this species was more pronounced in the test on the 10th and especially on the 16th week, when 67% of inocula did not regenerate. Argon or nitrogen gas caused the same degree of loss of viability in both the fungal species tested. In general, A. vaillantii mycelial cultures proved to be less sensitive to anoxic conditions, caused by either argon and nitrogen gas.     

Genome-wide identification,expression and chromosomal location of the genes encoding chitinolytic enzymes in <Emphasis Type="Italic">Zea mays</Emphasis>

Chitinolytic enzymes are important pathogenesis and stress related proteins. We identified 27 putative genes encoding endochitinases in the maize genome via in silico techniques and four exochitinases. Only seven of the endochitinases and segments of the exochitinases were heretofore known. The endochitinases included members of family 19 chitinases (classes I-IV of PR3, II of PR4) and members of family 18 chitinases (class III of PR8). Some similar enzymes were detected on adjacent regions of the same chromosome, and seem to result from duplication events. Most of the genes expressed were identified from EST libraries from plants exposed to biotic or abiotic stresses but also from libraries from tissues not exposed to stresses. We isolated proteins from seedlings of maize in the presence or absence of the symbiotic root colonizing fungus Trichoderma harzianum strain T22, and analyzed the activity of chitinolytic enzymes using an in-gel activity assay. The activity bands were identified by LC/MS/MS using the database from our in silico study. The identities of the enzymes changed depending on whether or not T22 was present. One activity band of about 95 kDa appeared to be a heterodimer between an exochitinase and any of several different endochitinases. The identity of the endochitinase component appeared to be dependent upon treatment.     

Biological and physiological characteristics of Neotyphodium gansuense symbiotic with Achnatherum inebrians

Biological and physiological characteristics of Neotyphodium gansuense were compared with Neotyphodium coenophialum and Epichlo? festucae at a range of temperatures and pH values, and on carbon and nitrogen amended media. N. gansuense was able to grow at 10-30 degrees C, but not at 5 degrees C, and slowly at 35 degrees C. The optimal temperature for both N. gansuense and N. coenophialum was 25 degrees C, but that of E. festucae was 20-25 degrees C. The optimal pH ranges for mycelial growth of N. gansuense, N. coenophialum and E. festucae were 5-9, 5-9 and 5-7, respectively. The Neotyphodium and Epichlo? endophytes varied in their ability to grow on media containing different carbon and nitrogen nutrients. The preference of N. gansuense for carbon source was sucrose>glucose, lactose, sorbitol, inulin, maltose, mannitol, starch, fructose>xylose. Growth of all three endophytes tested was significantly improved by peptone, tryptone, casein, yeast extract and l-proline. Yeast extract, peptone, casein, tryptone, l-proline, potassium nitrate, ammonium oxalic acid and l-leucine significantly improved growth of N. gansuense. However, ammonium nitrite was not utilized at all by any tested endophyte. N. gansuense grew significantly better on potato dextrose agar (PDA) and oat meal agar (OMA) than on corn meal agar (CMA) and drunken-horse-grass agar (DA), and most slowly on water agar (WA) and saltwater nutrient agar (SNA).     

Processing of Cry1Ab δ-endotoxin from Bacillus thuringiensis by Manduca sexta and Spodoptera frugiperda midgut proteases: role in protoxin activation and toxin inactivation

Activation of Cry protoxins is carried out by midgut proteases. This process is important for toxicity and in some cases for specificity. Commercial proteases have been used for in vitro protoxin activation. In the case of Cry1A protoxins, trypsin digestion generates a toxic fragment of 60–65 kDa. Here, we have analyzed the in vitro and in vivo activation of Cry1Ab. We found differences in the processing of Cry1Ab protoxin by Manduca sexta and Spodoptera frugiperda midgut proteases as compared to trypsin. Midgut juice proteases produced two additional nicks at the N-terminal end removing helices α1 and α2a to produce a 58 kDa protein. A further cleavage within domain II splits the toxin into two fragments of 30 kDa. The resulting fragments were not separated, but instead coeluted with the 58 kDa monomer, in size-exclusion chromatography. To examine if this processing was involved in the activation or degradation of Cry1Ab toxin, binding, pore formation, and toxicity assays were performed. Pore formation assays showed that midgut juice treatment produced a more active toxin than trypsin treatment. In addition, it was determined that the α1 helix is dispensable for Cry1Ab activity. In contrast, the appearance of the 30 kDa fragments correlates with a decrease in pore formation and insecticidal activities. Our results suggest that the cleavage in domain II may be involved in toxin inactivation, and that the 30 kDa fragments are stable intermediates in the degradation pathway.     

Comparative studies on the diversity of the edible mushroom Pleurotus eryngii: ITS sequence analysis, RAPD fingerprinting, and physiological characteristics

Verification of Pleurotus eryngii strains was assessed using ITS sequence analysis and RAPD fingerprinting. Sequence analysis of the ITS1–5.8S rDNA–ITS2 region of 24 strains of Pleurotus sp., which consisted of 22 strains of P. eryngii and the control strains P. ostreatus and P. ferulae, demonstrated that the DNA regions share mostly 99 % sequence identity, indicating that sequence-based analysis is not applicable for the verification of closely related mushroom strains. To verify the mushroom strains using RAPD, we amplified DNA fragments from the total cellular DNA of 24 mushroom strains with 18 different random primers, yielding 538 distinct DNA fragments ranging from 200–4000 bp. Analysis of the DNA fragment pattern showed that the 22 P. eryngii strains were clearly distinguished from the control strains P. ostreatus and P. ferulae, and could be categorized into five subgroups. Subsequent physiological studies on the development of fruiting bodies demonstrated the close correlation of the RAPD-based grouping with the phenotypical characteristics of mushroom fruiting bodies.     

Antagonistic potential of Gliocladium virens and Trichoderma longibrachiatum to phytopathogenic fungi

Three isolates of Gliocladium virens (G1, G2 and G3) and two of Trichoderma longibrachiatum (T1 and T2) were screened against isolates of three soilborne plant pathogens namely Rhizoctonia solani, Sclerotium rolfsii and Pythium aphanidermatum. G. virens exhibited stronger hyperparasitism and wider biological spectrum than T. longibrachiatum. Further, similarities as well as variation was observed in the ability of the various isolates to invade the test pathogens in dual culture. For the hyperparasites, acidic pH range (5.0 to 5.5) favoured both growth and spore germination. The hyperparasites made direct contact with the pathogens followed by varied modes of attack invariably leading to cell disruption. Antagonists, G1 and G3 revealed strong antibiosis while T2 showed moderate effect. All the isolates produced enhanced levels of lytic enzymes adaptively and there were marked differences among them. However, no correlation was observed between these attributes and the hyperparasitic potential of the various isolates in dual culture. The relevance and the role of enzymes and toxic metabolite(s) in the antagonism of G. virens and T. longibrachiatum to these pathogens are discussed.     

全氟辛烷磺酰胺降解菌的分离、鉴定和降解特性

【背景】全氟烷基和多氟烷基物质(per-and poly-fluoroalkyl substances,PFAS)是一类具有高表面活性、热稳定性、化学稳定性和疏水疏油性的难降解有机污染物。其长距离迁移性、极高的环境持久性和生物蓄积性给生态环境和生物体带来了严重的危害。【目的】从吉林石化公司污水处理厂水样中筛选获得以全氟辛烷磺酰胺(perfluorooctane sulfonamide,PFOSA)为唯一碳源生长的降解菌,并分析其降解特性及机理。【方法】以PFOSA为唯一碳源,通过富集、筛选、分离和纯化,从污水中筛选出PFOSA好氧降解菌,通过形态学观察、16S r RNA基因和全基因组测序分析对菌株进行鉴定,并采用三重四级杆液质联用仪分别对PFOSA的降解率和降解产物进行分析。【结果】筛选得到一株PFOSA好氧降解细菌C11,经形态学观察、16S r RNA基因序列分析和全基因组测序分析,初步鉴定该菌为睾丸酮丛毛单胞菌(Comamonas testosteroni)。经单因素优化试验发现,在培养温度为30℃、初始p H值为7.0、PFOSA初始浓度为30 mg/L的降解条件下,菌株C1...     

Phylogenetic relationships of Auriculoscypha based on ultrastructural and molecular studies

The phylogeny of Auriculoscypha anacardiicola, an associate of scale insects in India, is investigated using subcellular characters and MP and Bayesian analyses of combined nuLSU-rDNA, nuSSU-rDNA and 5.8S rDNA sequence data. It has simple septa with a pulley-wheel-shaped pore plug, which is diagnostic of phytoparasitic members of the Pucciniomycetes, and hyphal wall break on branching, a phenomenon unique to some simple septate heterobasidiomycetes. The septal ultrastructure of A. anacardiicola is similar to that of the genus Septobasidium. The close relationship to Septobasidium is also confirmed by rDNA sequence analyses. The polyphyletic nature of the order Platygloeales, noted in earlier studies, is evident from the present molecular analysis as well. The placement of Auriculoscypha in the Platygloeales can no longer be justified and both ultrastructural and molecular evidence strongly support the placement of Auriculoscypha in the Septobasidiales.     

降雪对荒漠地区藓类结皮中真藓生理生化的影响

生物土壤结皮的生存环境是地球上自养生物生存最为极端的生境之一,真藓是荒漠结皮中最为重要的组分之一。很少有研究涉及冬季降雪对结皮层生物体的影响。以宁夏沙坡头人工植被区内发育良好、长势均匀的真藓结皮为研究对象,系统研究降雪影响下荒漠地区藓类结皮层真藓的光合色素含量、可溶性糖含量、可溶性蛋白含量、丙二醛(MDA)含量以及脯氨酸含量的变化,并探讨其对降雪的生理生化响应。采用了4个降雪处理,分别是无降雪、0.5倍降雪、1倍降雪、2倍降雪。结果表明:随着降雪量的增加,其光合色素含量和可溶性蛋白含量显著增加;而可溶性糖含量、游离脯氨酸含量以及MDA含量均呈下降趋势。研究结果表明,作为生物土壤结皮重要水源之一的冬季降雪,能够为结皮层生物体提供适宜的水分条件以激发其生理生化活性,对维持荒漠生态系统的稳定性具有重要作用。     

Antifungal activity of Tagetes patula extracts on some phytopathogenic fungi: ultrastructural evidence on Pythium ultimum

Methanol extract, obtained from Tagetes patula plant, was assayed against three phytopathogenic fungi: Botrytis cinerea, Fusarium moniliforme and Pythium ultimum. The antifungal activity was tested both in the dark and in the light, using two different lighting systems. The data showed that the extract proved to have a dose-dependent activity on all the fungi with a marked difference between treatments in the light than in the dark. Good growth inhibition was observed in fungi only when these were treated with the highest dose of the extract and irradiated, whereas the same dose gave only a modest inhibition when the experiment was conducted in the dark. At 5 and 10 microg/ml in the dark, growth increased. The results indicated that the presence of a luminous source enhances the antifungal activity, with small differences between UV-A and solar spectrum light. SEM and TEM observations on Pythium ultimum revealed that the Tagetes patula extract induced alterations on cell fungal membranes with a photoactivation mechanism possibly involving the production of free radicals and leading to a premature aging of the mycelium.     

A comparative ultrastructural and physiological study on melanophores of wild-type and periodic albino mutants of Xenopus laevis

Summary The central and visceral nervous systems of the cockroach Periplaneta americana were studied by means of the peroxidase-antiperoxidase immunocytochemical method, with the use of antibody to bovine pancreatic polypeptide (PP). PP-like immunoreactive neuron somata are most numerous in the brain; at least 6 pairs of cell groups occur in clearly defined regions. Three pairs of cells each are also present in the suboesophageal ganglion and the thoracic ganglia, one pair of a single cell each in the first abdominal and the frontal ganglia, and 4 to 6 pairs of single cells in the terminal ganglion. No reactive cells were found in the retrocerebral complex and the second to the fifth abdominal ganglia. The axons containing PP-like immunoreactivity issue many branches that are distributed in the entire brain-retrocerebral complex, ventral cord, and visceral nervous system. PP-like immunoreactive material produced in the brain seems to be transported by three routes: protocerebrum to corpora cardiaca (-allata) through the nervi corporis cardiaci, tritocerebrum to visceral nervous system through frontal commissures, and to ventral cord through circumoesophageal connectives.A possible homology between the mammalian brain-GEP (gastro-enteropancreatic) system and the brain-midgut system of this insect is discussed.     

内生真菌对羽茅生长及光合特性的影响

通过温室栽培实验,以感染两种内生真菌(Neotyphodium sibiricum和Neotyphodium gansuence)和未感染内生真菌的羽茅(Achnatherum sibiricum)为实验材料,分析感染不同种内生真菌对宿主植物的生长及光合特性的影响。结果表明,感染两种内生真菌羽茅的株高和CO₂补偿点显著低于未染菌的羽茅,而染菌羽茅的蒸腾速率和气孔导度显著高于未染菌羽茅,但对于感染不同种内生真菌的羽茅,无论是分蘖数与生物量的积累还是光合生理值之间均无显著差异。     

Dyeing and antimicrobial characteristics of chitosan treated wool fabrics with henna dye

Chitosan, a naturally available biopolymer which is now increasingly being used as a functional finish on textile substrates to impart antimicrobial characteristics and increase dye uptake of fabrics was applied on wool fabrics. Henna a natural dye with proven bactericidal properties was applied on wool fabrics along with chitosan to impart antimicrobial characteristics. The effect of chitosan application on the dyeing properties of wool fabrics was studied by measuring the K/S values of the treated substrates at various concentrations of chitosan and the dye. The antimicrobial properties of chitosan and natural dyes both when applied independently and collectively on fabrics were assessed. The results proved that the chitosan treated wool fabrics showed increase dye uptake of fabrics. The treated fabrics were found to be antimicrobial and the chitosan treatment enhances the antimicrobial characteristics of the dyes. Fastness properties of the applied finish to washing, rubbing and perspiration have also been discussed.     

The effects of immobilization on the biochemical,physiological and morphological features of Anabaena azollae

Anabaena azollae, a presumptive isolate from Azolla filiculoides, was immobilized in polyurethane foam, hydrophilic polyvinyl foam and alginate. When viewed by low-temperature scanning electron microscopy a thick mucilage layer covered the surface of both cells and matrix; this closely resembles the mode of attachment of the symbiont Anabaena in the Azolla leaf cavity. The heterocyst frequency of the immobilized A. azollae doubled relative to free-living cells and reached a level of 14–17%. Immobilization induced increases in both hydrogen production via nitrogenase or hydrogenase and in the rates and stabilization of acetylene reduction (N₂-fixation). Ammonia production by immobilized cells with L-methionine-D,L-sulfoximine (MSX) is greater than that of freeliving cells. Immobilized cells without MSX were, however, able to excrete ammonium at lower rates thus emulating the characteristic of the symbiotic cyanobacteria (A. azollae) in the leaf cavity of Azolla.Abbreviations Chl chlorophyll - GS glutamine synthetase - MSX L-methionine-D,L-sulfoximine - SEM scanning electron microscopy - PU polyurethane - PV polyvinyl