Phenol oxidase in crayfish blood: Activation by and attachment on cells of other organisms

Cell walls from the crayfish parasite Aphanomyces astaci strongly enhanced phenol oxidase activity in crayfish blood or cell-free serum. The activation was not very specific since bacteria, cells, and cell walls of some algae, fungi, and higher plants also activated the enzyme strongly. Only cell walls from one fungus lacked this property. Laminaran, a purified glucan found in many plant cell walls, activated the enzyme as well, but cellulose, chitin, or nylon did not. On the other hand, attachment of the enzyme to the wall surfaces and subsequent strong local melanization was much more specific and occurred only on a few fungi but not on other plant cell walls, bacteria, or other solid, enzyme-activating or nonactivating material. The mechanism of activation and attachment is discussed.     

Attachment of phenoloxidase to fungal cell walls in arthropod immunity

In crayfish, phenoloxidase was located in the hemocytes. The plasma had infinitesimal enzyme activity. A phenoloxidase preparation from hemocytes precipitated spontaneously after approximately 1.5 hr at 22°C, which became attached spontaneously to glass, Plexiglas, and polystyrene plastic. The enzyme preparation could also become attached to Saccharomyces cerevisiae cell walls. Attachment was mediated by a proteinaceous substance, since trypsin significantly decreased the degree of attachment. Calcium ions were also necessary for attachment. A β-1,3-glucan, laminaran, partially prevented attachment to the fungal cell walls. Heparin caused precipitation of the phenoloxidase preparation from hemocytes. In crayfish cuticle, proteins with associated phenoloxidase activity were attached to cell walls of Aphanomyces astaci as well as to those of S. cerevisiae.     

Wound reactions and Aphanomyces astaci growth in crayfish cuticle

Superficial wounding of crayfish soft cuticle by removal of the epicuticle caused hemocyte aggregation beneath the wound. The aggregation seemed to be induced by osmotic factors. Subsequently, melanin was only formed in parts of the wound region. Swimming zoospores of the fungal parasite Aphanomyces astaci preferentially encysted in the near vicinity of fresh superficial wounds. Cysts attached onto the naked endocuticle became encapsulated by melanin and were mostly killed during the process. The induced melanization reactions may have antifungal effects. The intermolt cuticle of a highly resistant crayfish species, Pacifastacus leniusculus, was easily penetrated by the fungus only via wounds. The cell walls of the penetrating hyphae became heavily melanized, growth was visibly disturbed, and they grew sparsely in the wound region. As soon as they reached outside the wound region they grew profusely. In susceptible crayfish Astacus astacus melanization was slower and the wound reactions prevented fungal growth much less as evidenced by microscopy. The preferential direction of hyphal growth is parallel to the chitin fibrils in both the resistant crayfish P. leniusculus and the susceptible A. astacus.     

Properties of Extracellular Enzymes from Aphanomyces astaci and Their Relevance in the Penetration Process of Crayfish Cuticle

In culture filtrates from the crayfish plague parasite, Aphanomyces astaci, protease and a low level of hyaluronidase activity were found. The hyaluronidase activity was highest at pH 6.5 or above and at about 23°C. The protease activity had a broad pH-optimum, between pH 7 and at least pH 10, and was partially denatured at 30°C. However, when incubated for 30 min with the substrate, casein, the activity increased logarithmically up to about 35–40°C and had an apparent optimum at 45–50°C. The proteases from the parasitic as well as from two less proteolytic, saprophytic Aphanomyces species were predominantly constitutive and were excreted mainly by the older mycelia. Proteases from the parasite and a saprophyte did not reach full activity until 10–30 min after substrate addition. No lipase activity was found in the case of the mycelium of the parasitic species. However, esterase was apparently present inside germinating zoospores. The native enzymes of A. astaci could degrade freeze-dried soft cuticle from crayfish. The relevance of the different enzymes of A. astaci for the penetration process within the cuticle of crayfish is discussed.     

Blood reactions in vitro in crayfish against a fungal parasite, Aphanomyces astaci

The immediate and subsequent reactions in crayfish blood upon making contact with hyphae of the crayfish plague fungus were observed in vitro. Blood cells agglomerated and clumped rapidly on the hyphae in streaming blood. Very brief contact by chance with a hypha, or with cell extensions from cells or a cell clump on the hypha, caused flowing cells to stop and adhere. Chemotaxis was not believed to be of any importance. Particles originating from hemocyte granules were specifically attached to, and “encapsulated” the distal portion of, the hypha (even without the presence of the cells themselves), where their content of polyphenol oxidase became activated. These processes may be connected with the defense mechanism in crayfish. Within a few hours the process of melanization could be observed on the hyphal surface. Both the enzyme(s) and the substrate(s) for the melanization reaction seem to originate from the blood cells. Dihydroxyphenylalanine (DOPA) could replace the natural substrate and greatly enhanced the process. The melanization in whole blood was much heavier in a crayfish resistant to the disease than in a susceptible one.     

Histological and Ultrastructural Changes in Leaves and Stems of Resistant and Susceptible Chickpea Cultivars to Ascochyta rabiei

The histo- and cytopathological effects in resistant (ILC-195) and susceptible (Canitez-87) chickpea cultivars were examined by light, transmission and scanning electron microscopy 3, 5 and 7 days after inoculation (d.a.i) of seedlings with Ascochyta rabiei. The fungus produced typical appressoria that penetrated both cuticle and stomata. The resistant plants had physical barriers and a cuticle layer against fungal penetration 3 d.a.i. The fungus spread intercellularly and subepidermally in the leaves and stems of susceptible plants 3 d.a.i., and was followed 5 d.a.i. by cell plasmolysis, degeneration of organelles and of cellulose, but not lignified, walls. Pycnidia formation occurred between 5 and 7 d.a.i. 7 d.a.i., organelle degeneration, pycnidia formation and symptom severity increased. Tracheidal elements, including lignified elements, were almost intact in both resistant and susceptible cultivars. In the susceptible plants, lignin cell walls were slightly degraded after 7 days. There was less cell degeneration and pycnidia formation in resistant plants. Some electron-dense large bodies and lipid granules were observed within intracellular fungal hyphae in infected cells of resistant plants 7 d.a.i.     

Heterologous chitinase gene expression to improve plant defense against phytopathogenic fungi

Agricultural crops worldwide suffer from a vast array of fungal diseases which cause severe yield losses. Upon interaction with a pathogen, plants initiate a complex network of defense mechanisms, among which is a dramatic increase in chitinase activity. Chitinases are capable of hydrolyzing chitin-containing fungal cell walls and are therefore thought to play a major role in the plant’s response. One of the strategies to increase plant tolerance to fungal pathogens is the constitutive overexpression of proteins involved in plant-defense mechanisms. The level of protection observed in transgenic plants harboring heterologous chitinase genes varies, depending on the particular combination of enzyme, plant and pathogen tested. Nevertheless, most of these transgenic plants exhibit increased tolerance to fungal diseases relative to their non-transgenic counterparts. The combined expression of chitinases with other plant-defense proteins such as glucanases and ribosome-inactivating proteins further enhances the plant’s resistance to fungal attack. Received 29 January 1997/ Accepted in revised form 01 July 1997     

Fungal entomopathogens in a tritrophic context

Variation in plant quality has an important impact on insect growth and development and there is considerable evidence that plants can also influence an insect’s natural enemies. Here we discuss the potential for plant-mediated effects on fungal entomopathogens. Fungi differ from other insect pathogens in that they infect an insect directly through its cuticle. This means that they are particularly vulnerable to changes in microclimate and properties of the insect cuticle. Potential direct and indirect mechanisms for plant-mediated effects on fungal entomopathogens are discussed. It is clear from these studies that fungal entomopathogens could be affected by plant volatiles and plant surface chemistry. Plant secondary chemicals can also inhibit fungal growth, potentially protecting the insect herbivore. However, the site of action and the mechanism behind these effects in plant-based studies is not always clear. The implications for biocontrol using fungal entomopathogens are discussed.     

硅在植物体内的分布和吸收及其在病害逆境胁迫中的抗性作用

硅在地壳中含量位居第二位,尽管还没有被列为植物生长的必需营养元素,但它在促进植物生长发育和营养吸收、提高植物对非生物逆境胁迫和生物逆境胁迫的抗性等方面都具有重要作用。综述了近些年来国内外关于硅在植物体内的分布、吸收及其生理效应,重点介绍了硅在病害逆境胁迫中的抗性作用机理。高等植物以单硅酸[Si(OH)4]的形式吸收硅,存在硅的主动吸收和被动吸收机制。硅主要沉积在叶片及叶鞘表皮细胞,形成硅化细胞和角质-硅双层结构,能增强寄主植物细胞壁的机械强度和稳固性,从而延缓和抵御病菌的侵入和扩展。更多的证据表明,硅处理能增加植物叶片保护酶(过氧化物酶、多酚氧化酶、苯丙氨酸解氨酶等)活性和诱导寄主产生次生代谢抗性物质(如植保素、多酚类化合物、木质素),从而激活植物的防御系统,增强对病原菌的抵抗能力。分子水平上的研究显示,硅能诱导与植物防御机制相关的基因表达,参与抗病信号分子(如水杨酸、茉莉酸和乙烯)在信号传导中的作用。     

Polyphenol oxidase activity in the roots of seedlings of Bromus (Poaceae) and other grass genera

? Premise of the study: Phenolic compounds exuded by roots have been implicated in allelopathic interactions among plants. Root enzymes that destroy phenolics may protect plants against allelopathic inhibition and thus may aid in invasiveness. Phenolic-degrading enzymes are chiefly found in aboveground plant parts, but have also been previously reported in root tissues where the enzyme's function is unknown. We explored phenolic oxidase activity in emerging roots of grasses in a survey across different grass genera; in particular, we aimed to test whether grasses of the genus Bromus, known for their large invasion potential, differ in this respect from other grass taxa. ? Methods: We assayed a range of grass genera commonly found in the United States for root enzyme activity with spectrophotometric assays of phenol oxidase activity using l-DOPA as the main substrate. ? Key results: In the survey of a grass genera, we discovered that roots of the genus Bromus contain large amounts of polyphenol oxidase (PPO) activity, while all other tested grass genera, even ones closely related to Bromus, did not. PPO was found to be present at germination and remained active throughout the life of the plant. Compared to other PPOs, the enzyme present in Bromus appears to have a narrow substrate range. ? Conclusions: The specific functions of the root PPO and the ecological ramifications of the special status of Bromus are not yet clear. The possibility that the enzyme plays a role in plant species interaction for bromes, a genus of grasses known to have high invasive potential, is raised.     

The role of fungi in the diet of the amphipod Gammarus pulex (L.): an enzymatic study

SUMMARY. 1. Sets of ten Gammarus pulex fed on controlled diets of sterile alder leaves, or fungal mycelium, or alder leaves incubated for 10 days with an aquatic hyphomycete, were assayed for cellulase, β-1,3-glucanase an d chiitinase activity and compared with (a) animals taken directly from the stream, (b) animals starved for 2 days, and (c) enzyme activity in fungal mycelium.
2. Gut enzyme activity was compared on natural substrates of sterile leaves, mycelium and inoculated leaves as well as on model substrates.
3. G. pulex secretes an endogenous coupled cellulase system capable of degrading native cellulose in plant cell walls. It also secretes β-1,3-glucanase and chitinase capable of degrading fungal cell walls thus affording access for gut enzymes to cell contents.
4. Secretion of enzymes active on native cellulose is enhanced on a diet of leaves already partially degraded by fungal enzymes. Gut enzymes extract more reducing sugar from this substrate than from sterile leaves. Specific enzyme secretion is enhanced by the presence in the diet of exposed, accessible substrates. Fungal enzymes do not appear to contribute to the digestive processes of G. pulex.     

Identification and characterization of an alternative oxidase in the entomopathogenic fungus Metarhizium anisopliae

Mitochondria of Metarhizium anisopliae contain an alternative oxidase (AOX), which reduces oxygen to water by accepting electrons directly from ubiquinol. AOX activity is demonstrated in situ as a constitutive enzyme. Greatest activity of AOX appears at the beginning and at the end of the fungal developmental cycle, germination of aerial conidia and the formation of submerged conidia, respectively. Changes in nutritional conditions, e.g., the presence of host insect cuticle or nutrient starvation had no effect on the induction of AOX activity. Antimycin A, an electron transport chain inhibitor, induced AOX activity. Cloning of the AOX DNA and the alignment of the deduced amino acid sequence of a segment of the AOX gene from M. anisopliae shows structural similarities with other AOX sequences with differing levels of variation when compared with homologous sequences from plants, yeasts, and filamentous fungi. Alternative oxidase in entomopathogenic fungi may have a positive contribution to ecological fitness.     

盐胁迫下接种AM真菌对玉米耐盐性的影响

以玉米品种陕单16号幼苗为材料,用盆栽法研究了不同含盐量(0、0.5、1.0、1.5和2.0 NaCl g/kg)土壤接种AM真菌(Glomus mosseae)对玉米幼苗生物量、盐害级数,以及叶片中电解质透出率、丙二醛、O·2-、H2O2含量和保护酶活性的影响.结果表明:在盐胁迫下,接种AM真菌增加了玉米植株生物量,降低了玉米的盐害级数;菌根植株叶片中过氧化氢酶的活性高于非菌根植株,而过氧化物酶、抗坏血酸氧化酶和多酚氧化酶的活性则为非菌根植株高于菌根植株;超氧化物歧化酶的活性在NaCl浓度为0、0.5和1.0 g/kg时为非菌根植株高于菌根植株,而在NaCl浓度为1.5和2.0 g/kg时则为菌根植株高于非菌根植株;菌根植株叶片中电解质透出率、丙二醛、O·2-和H2O2的含量低于非菌根植株.可见,AM真菌的侵染提高了玉米的耐盐性,缓解了由盐胁迫引起的过氧化胁迫对玉米植株的伤害,但这一缓解作用并不只是通过提高保护酶活性来实现的,可能还存在一些非酶促的调节机制.     

Purification and characterization of an endo-beta-1,6-glucanase from Trichoderma harzianum that is related to its mycoparasitism.

The enzymes from Trichoderma species that degrade fungal cell walls have been suggested to play an important role in mycoparasitic action against fungal plant pathogens. The mycoparasite Trichoderma harzianum produces at least two extracellular beta-1,6-glucanases, among other hydrolases, when it is grown on chitin as the sole carbon source. One of these extracellular enzymes was purified to homogeneity after adsorption to its substrate, pustulan, chromatofocusing, and, finally, gel filtration. The apparent molecular mass was 43,000, and the isoelectric point was 5.8. The first 15 amino acids from the N terminus of the purified protein have been sequenced. The enzyme was specific for beta-1,6 linkages and showed an endolytic mode of action on pustulan. Further characterization indicated that the enzyme by itself releases soluble sugars and produces hydrolytic halli on yeast cell walls. When combined with other T. harzianum cell wall-degrading enzymes such as beta-1,3-glucanases and chitinases, it hydrolyzes filamentous fungal cell walls. The enzyme acts cooperatively with the latter enzymes, inhibiting the growth of the fungi tested. Antibodies against the purified protein also indicated that the two identified beta-1,6-glucanases are not immunologically related and are probably encoded by two different genes.     

Norepinephrine, monoamine oxidase, and acetylcholinesterase in the rat jugular vein compared with other blood vessels

The observation that the rat jugular vein relaxed in response to norepinephrine but not to field stimulation prompted us to evaluate the extent of innervation in this tissue. The norepinephrine concentration in the jugular vein was about 10% of that in the mesenteric artery and vein. The low levels of norepinephrine were not due to higher monoamine oxidase activity relative to the enzyme activity in other blood vessels. In the jugular vein, as in heart and brain, serotonin was preferred substrate for monoamine oxidase whereas in the femoral vein, mesenteric vein, and mesenteric artery, phenylethylamine oxidation was greater. Based on kinetic and inhibitory studies with LY51641, a selective type A inhibitor, monoamine oxidase activity was not found to be uniform throughout the cardiovascular system. In addition to low levels of norepinephrine, acetylcholinesterase activity in the jugular vein was only 5 and 13% of the activity in the portal vein and mesenteric artery, respectively. Thus, we provide strong evidence that our inability to generate a response to field stimulation in the rat jugular vein results from the lack of functional innervation in this tissue. This information adds to the usefulness of this preparation for comparative studies of agents acting on the smooth muscle without the added complication of neuronal uptake mechanisms.     

Host-Pathogen Interactions: I. A Correlation Between alpha-Galactosidase Production and Virulence

Resistance or susceptibility of Red Kidney, Pinto and Small White beans (Phaseolus vulgaris) to the alpha, beta, and gamma strains of Colletotrichum lindemuthianum was either confirmed or established. These fungal strains secrete α-galactosidase, β-galactosidase and β-xylosidase when grown on cell walls isolated from the hypocotyls of any of the above bean varieties. These enzymes effectively degrade cell walls isolated from susceptible 5-day old hypocotyls but degrade only slightly the walls isolated from resistant 18-day old hypocotyls. The amounts of the β-galactosidase and β-xylosidase secreted by the 3 fungal strains are relatively low and are approximately equivalent. The secretion of these 2 enzymes is not dependent upon the bean variety from which the hypocotyl cell walls used as a carbon source were isolated. However, the fungal strains secrete greater amounts of α-galactosidase when grown on hypocotyl cell walls isolated from susceptible plants than when grown on walls from resistant plants. Virulent isolates of the fungus, when grown on hypocotyl cell walls isolated from a susceptible plant, secrete more α-galactosidase than do attenuated (avirulent) isolates of the same fungal strain grown under the same conditions. The α-galactosidase secreted by each of the fungal strains is capable of removing galactose from the hypocotyl cell walls of each bean variety tested. Galactose is removed from the cell walls of each variety at the same rate regardless of whether the cell walls were isolated from a susceptible or resistant plant.     

Detection of Aphanomyces astaci in North American crayfish by polymerase chain reaction

We present a PCR based method to detect Aphanomyces astaci in North American crayfish. Primers were designed to specifically amplify parts of the internal transcribed spacer (ITS) regions and the 5.8 rRNA gene of A. astaci. A single round and a semi-nested assay were tested for their sensitivity and specificity. Specificity of the PCR assays was tested against several closely related Aphanomyces species, other Oomycetes and some non-A. astaci DNA that might be found in or on crayfish. The single round assay was fully specific against all DNA tested. In the semi-nested assay, cross-reaction was seen when the equivalent of 40,000 or more genomic units of A. invadans or A. frigidophilus were entered into the PCR reaction. The lower detection limit of both assays lies around 1 genomic unit of A. astaci. Investigation of various parts of the exoskeleton of 3 North American crayfish species revealed that for O. limosus and P. leniusculus the telson and soft abdominal cuticle yielded a positive PCR reaction most frequently. For the third species, Procambarus clarkii, only 1 individual tested positive, so no conclusion as to preferred infestation site(s) could be drawn.     

Ruminococcus flavefaciens Cell Coat and Adhesion to Cotton Cellulose and to Cell Walls in Leaves of Perennial Ryegrass (Lolium perenne)

Ruminococcus flavefaciens was shown to possess a prominent glycoprotein coat, which contained rhamnose, glucose, and galactose as its principal carbohydrates. Periodate-reactive carbohydrate occurred as a surface layer of the coat. The ruminococci adhered strongly by means of this coat to cotton cellulose and to cell walls in leaf sections of Lolium perenne L. (perennial ryegrass). The coat was diffuse at the point of contact so that the bacterial cell wall was in close contact with the substrate. Adhesion was influenced by the availability of damaged plant cell walls and by the cell wall type and occurred most rapidly to cell walls of the epidermis and sclerenchyma, followed by the phloem and mesophyll. Plaques of bacteria with filamentous coat extensions developed on all these tissues. The bacteria did not readily adhere to the walls of the bundle sheath cells or metaxylem or protoxylem vessels and did not adhere to the cuticle or chloroplasts. The epidermal and phloem cell walls were more rapidly digested than the walls of other cell types.     

Reactive oxygen species play a role in regulating a fungus-perennial ryegrass mutualistic interaction

Although much is known about the signals and mechanisms that lead to pathogenic interactions between plants and fungi, comparatively little is known about fungus-plant mutualistic symbioses. We describe a novel role for reactive oxygen species (ROS) in regulating the mutualistic interaction between a clavicipitaceous fungal endophyte, Epichlo? festucae, and its grass host, Lolium perenne. In wild-type associations, E. festucae grows systemically in intercellular spaces of leaves as infrequently branched hyphae parallel to the leaf axis. A screen to identify symbiotic genes isolated a fungal mutant that altered the interaction from mutualistic to antagonistic. This mutant has a single-copy plasmid insertion in the coding region of a NADPH oxidase gene, noxA. Plants infected with the noxA mutant lose apical dominance, become severely stunted, show precocious senescence, and eventually die. The fungal biomass in these associations is increased dramatically, with hyphae showing increased vacuolation. Deletion of a second NADPH oxidase gene, noxB, had no effect on the E. festucae-perennial ryegrass symbiosis. ROS accumulation was detected cytochemically in the endophyte extracellular matrix and at the interface between the extracellular matrix and host cell walls of meristematic tissue in wild-type but not in noxA mutant associations. These results demonstrate that fungal ROS production is critical in maintaining a mutualistic fungus-plant interaction.