Fungi isolated from Sclerotia ofSclerotium cepivorum and from soil and their effects upon the pathogen

Summary Data are presented on the antagonistic effects of the fungi isolated from sclerotia ofSclerotium cepivorum and from nonrhizosphere soil taken from around the roots of infected onions upon mycelial growth and sclerotial germination ofS. cepivorum. Most of the isolated fungi especiallyPenicillium species were antagonistic to mycelial growth. Sclerotial germination was slightly inhibited by diffusates of these fungal isolates. Testing the antifungal effect of someAllium extracts against the fungal isolates by the inhibition zone method showed that garlic extract has the greatest antifungal effects and onion extract is the least potent. However, spore germination tests indicated that onion extract completely inhibits the spore germination of all test fungi. The role of host-plant extracts in stimulating sclerotial germination is discussed.     

The Effect of Orange Extract and Other Additives on Anthracnose of French Beans Caused by Colletotrichum lindemuthianum

Orange and carrot extract, pollen diffusate, peptone, and amixture of glucose and casamino acids greatly stimulated germinationof conidia of Colletotrichum lindemuthianum which was poor inwater. Urea and glucose and casamino acids separately had littleeffect. Orange extract and the glucose/casamino acids mixture alteredthe ways in which the pathogen grew after germination. Withincreasing concentration of these additives more spores germinatedwith appressoria at the ends of increasingly thicker germ tubesinstead of more directly with no or very thin germ tubes. Large-diametergerm tubes with no appressoria were also formed. These additives almost completely suppressed formation of lesionswhen added to spore suspensions placed on hypocotyls. Otheradditives that greatly stimulated spore germination did notaffect the development of lesions. Orange extract seems to control disease in the first place bychanging the form of growth of the parasite on the hypocotylsurface and not by altering the susceptibility of the host,though this may happen later. The implications of these results for the control of diseasescaused by this and similar fungi are discussed.     

The effect of pollen grains on infections caused by Botrytis cinerea Fr

The addition of pollen to spores of Botrytis cinerea Fr. in droplets of distilled water stimulates spore germination, growth of germ tubes and lesion development. Aqueous diffusate from pollen is as effective as pollen grains, and frozen pollen is more stimulatory than freshly collected pollen. The presence of pollen grains reduces considerably the number of spores needed to allow infection to occur. The lost germination ability and infectivity of old spores is restored by pollen. The stimulatory effect of the presence of pollen has been demonstrated both in vitro and on the surfaces of strawberry petals, strawberry fruits and broad bean leaves. Complete removal of the source of pollen, the anthers from strawberry fruits, markedly affected the speed and severity of infections of strawberry fruits. On broad bean leaves the addition of pollen grains to spores induced the development of spreading aggressive lesions. Preliminary work indicates that the effective principle in pollen is water-soluble, dialysable and heat-stable. Although glucose and fructose are important components of diffusate, neither glucose solution nor fructose solution nor a mixture of the two showed as marked effects as did pollen. Orange juice produces similar effects.     

Cooperativity between different nutrient receptors in germination of spores of Bacillus subtilis and reduction of this cooperativity by alterations in the GerB receptor

The GerA nutrient receptor alone triggers germination of Bacillus subtilis spores with L-alanine or L-valine, and these germinations were stimulated by glucose and K+ plus the GerK nutrient receptor. The GerB nutrient receptor alone did not trigger spore germination with any nutrients but required glucose, fructose, and K+ (GFK) (termed cogerminants) plus GerK for triggering of germination with a number of L-amino acids. GerB and GerA also triggered spore germination cooperatively with l-asparagine, fructose, and K+ and either L-alanine or L-valine. Two GerB variants (termed GerB*s) that were previously isolated by their ability to trigger spore germination in response to D-alanine do not respond to D-alanine but respond to the same L-amino acids that stimulate germination via GerB plus GerK and GFK. GerB*s alone triggered spore germination with these L-amino acids, although GerK plus GFK stimulated the rates of these germinations. In contrast to l-alanine germination via GerA, spore germination via L-alanine and GerB or GerB* was not inhibited by D-alanine. These data support the following conclusions. (i) Interaction with GerK, glucose, and K+ somehow stimulates spore germination via GerA. (ii) GerB can bind and respond to L-amino acids, although normally either the binding site is inaccessible or its occupation is not sufficient to trigger spore germination. (iii) Interaction of GerB with GerK and GFK allows GerB to bind or respond to amino acids. (iv) In addition to spore germination due to the interaction between GerA and GerK, and GerB and GerK, GerB can interact with GerA to trigger spore germination in response to appropriate nutrients. (v) The amino acid sequence changes in GerB*s reduce these receptor variants' requirement for GerK and cogerminants in their response to L-amino acids. (vi) GerK binds glucose, GerB interacts with fructose in addition to L-amino acids, and GerA interacts only with L-valine, L-alanine, and its analogs. (vii) The amino acid binding sites in GerA and GerB are different, even though both respond to L-alanine. These new conclusions are integrated into models for the signal transduction pathways that initiate spore germination.     

The effect of leaf exudates on the spore germination of phylloplane mycoflora of chilli (Capsicum annuum L.) cultivars

Summary Ninetten aminoacids, twelve sugars, eleven organic acids and ten phenols were detected in the leaf exudates of three cultivars of chilli. The number of aminoacids, sugars, organic acids and phenols increased as the plants grew older. More aminoacids and sugars were detected in the exudate from the susceptible cultivar (Malwa). More organic acids and phenols were detected from the resistant cultivar (Simla). The leaf exudate of the resistant cultivar (Simla) inhibited spore germination of the pathogen (Alternaria solani) while that of susceptible (Malwa) stimulated spore germination. The cultivar ‘Patna’ which is moderately resistant, occupied an intermediate position. Spore germination of the isolated fungi was enhanced in leaf exudate of susceptible cultivar (Malwa), while leaf exudates of the moderately resistant (Patna) and resistant (Simla) inhibited spore germination of the majority of fungi isolated. Most of the antagonistic fungi were not isolated from the susceptible cultivar and the percentage spore germination of these fungi was less in leaf exudate of the susceptible cultivar, while leaf exudates of resistant cultivars enhanced the percentage spore germination of antagonistic fungi,viz Aspergillus flavus, A. fumigatus, A. versicolor, Penicillium citrinum, P. restrictum andTrichoderma viride.     

Adhesion and development of the root rot fungus (Heterobasidion annosum) on conifer tissues: effects of spore and host surface constituents

The objective of this study was to correlate the occurrence of particular root and woody stump surface components with the ability of spores of the root rot fungus (Heterobasidion annosum) to adhere, germinate and establish on conifer tissues. With the aid of high performance liquid chromatography, several sugars (pinitol, xylitol, dulcitol, mannitol, D-glucose, mannose, fructose) were detected on both stump and fine root surfaces of Scots pine and Norway spruce. Of all the sugars observed, xylose and arabinose were poorly utilized for initiation of germ tube growth whereas spore germination was enhanced in the presence of D-glucose, mannose or fructose. Oxidation of these sugars by pretreatment of wood discs or roots with periodic acid abolished the ability of the spores to germinate. Non-sugar components such as long chain fatty acids on spores and root surfaces as detected with nuclear magnetic resonance were found to have a significant influence on adhesion and initiation of germ tube development. Removal of these aliphatic compounds from the root surface increased spore germination by 2-fold, whereas similar treatment on spores led to a 5-fold decrease in adhesiveness to root material. In vitro studies revealed that the di-ethyl ether extract from the roots had no long term adverse effect on spore germination which suggests that the fungus may possess the capability to detoxify this substance. Similarly, adhesion of spores was affected by low and freezing temperatures. The role of significant levels of mannitol and trehalose accumulated in spores and hyphae of the fungi on viability, survival and tolerance to adverse conditions such as oxidative stress, freezing and desiccation are discussed.     

Studies on the rhizosphere mycoflora of broad bean and cotton

Summary Seed and root exudates from three varieties of both broad bean and cotton grown aseptically were analyzed for amino acids and sugars. Broad bean varieties were found to excrete considerable amounts of amino compounds with 15 different amino acids, while cotton varieties excreted less consisting of 12 amino acids. Five sugars were identified in the seed and root exudates from broad bean varieties, while three were present in the seed and root exudates from cotton varieties. Seed and root exudates stimulated spore germination and growth of fungi isolated from the rhizosphere.     

Tarsal taste neurons of Helicoverpa assulta (Guenée) respond to sugars and amino acids, suggesting a role in feeding and oviposition

Helicoverpa assulta and Helicoverpa armigera are sibling species with different host-plant ranges. We have previously reported electrophysiological and behavioral responses of H.armigera to sugars and amino acids. Here we describe a parallel study performed on H. assulta and compare the results obtained with the two species. In females, fourteen gustatory chemosensilla, identified on one ventrolateral side of the fifth tarsomere were stimulated with sucrose, glucose, fructose, maltose, myo-inositol, and the twenty common amino acids, using the tip-recording technique. The taste receptor neurons in eight chemosensilla were identified sensitive to the sugars, myo-inositol, Lys, Glu, Arg, Trp, and Ser which all induced proboscis extension reflex (PER) when tarsi were stimulated. There was a positive correlation between electrophysiological activities and PER responses triggered by sucrose. No stimulatory effect on oviposition was observed with sugar or amino acid mixtures. In males, three chemosensilla showed responses to the four sugars, but generally weaker than in females. The major difference of the two species was the variety of amino acids triggering electrophysiological responses. The stimulatory effect of sugars and amino acids on H.assulta was also generally weaker than that on H. armigera.     

Some chemical and biological properties of culture filtrate ofNostochopsis lobatus

The blue-green algaNostochopsis lobatus released amino acids (glycine, serine, arginine and glutamic acid), sugars (glucose, fructose and sucrose), organic acids (oxaloacetic acid and oxalic acid) and protein in the culture medium. The quantity of extracellular amino acids and protein increased with age of culture from 30 to 120 d. All culture filtrates ofN. lobatus were highly toxic to spore formation and germination ofWestiellopsis prolifica and growth and conjugation ofSpirogyra decimina, while old-age culture filtrates inhibited total chlorophyll, heterocyst and akinete formation in the same alga and total chlorophyll and zoospore formation inChaetophora attenuata. The toxicity ofN. lobatus culture filtrate persists following high temperature treatment and at extremes of pH.     

Effect of root exudates on the spore germiantion of rhizosphere and rhizoplane mycoflora of chilli (Capsicum annuum L.) cultivars

Summary From root exudates of three cultivars of chilli (Capsicum annuum L.) 12 amino acids and 7 sugars were detected. Methionine, d-1- phenylalanine, citrulline and d-xylose were detected only from the root exudates of resdistant cultivars. The root exudates of resistant variety inhibited spore germination of the pathogen (Fusarium oxysporum f. sp.capsici), but that of susceptible variety enhanced spore germiantion of the same. Spore germiantion of antagonistic fungi (Trichderma viride andAspergillus sydowi) was also influenced by the root exudates of resistant and susceptible varieties, but the influence was different.Spore germiantion of a number of rhizosphere fungi was studied and in general root exudate of susceptible cultivar enhanced spore germiantion of majority of fungi, but spore germination of antagonistic fungi against the pathogen was inhibited. However, root exudate of resistant cultivar stimulated spore germination of antagonistic fungi.     

Metabolism of the germinating sclerotium of Claviceps purpurea.

The concentration of free amino acids, particularly alanine, altered as stromata developed on germinating sclerotia. Lipid reserves in the sclerotium provided the principal carbon and energy source, and the nutrients required for stomatal growth appeared to be provided by the sclerotial tissues immediately beneath the point of attachment of the stoma. Alkaloid did not disappear from the sclerotia during germination. Very high levels of polyol, particularly mannitol, were attained in the stromata as germination progressed; the synthesis of polyol was usually accompanied by that of soluble sugars, notably glucose. In an attempt to ascertain the role of mannitol in germination, the relationship between polyol and soluble sugars was investigated in excised stromata after their submergence in buffered media containing a variety of soluble carbohydrates. Mannitol was synthesized under these conditions by way of phosphorylated intermediates, and chiefly in the capitulum, after rapid, and probably passive, absorption of the exogenously supplied sugar. The tissues equilibrated rapidly with the supplied carbohydrate, whose concentration was maintained by further uptake whilst mannitol was being synthesized. The concentrations of assimilated glucose and synthesized mannitol tended toward the same value during incubation and the results suggested that the synthesis was compartmentalized within the stromata. Very little exogenously supplied mannitol was assimilated.     

Effect of individual amino acids and glucose on activation and germination of Rhizopus oligosporus sporangiospores in tempe starter

AIM: To understand the conditions promoting activation and germination of spores, and to contribute to the control of tempe starters. METHODS AND RESULTS: Using microscopic counts of fluorescent labelled spores, the following results were obtained: (1) L-alanine plays an important role (of the same order as that of peptone) in stimulation of germination of dormant spores. Alanine can satisfy the requirements of carbon as well as nitrogen for spore germination; (2) L-proline, on the other hand, inhibits alanine uptake presumably by blocking/congesting transporters of spore cells, resulting in apparent low viability on agar media; (3) L-leucine and L-isoleucine slightly favour spore germination while L-arginine and L-lysine do not have any stimulating effect; (4) The stimulatory role of glucose was only evident in the presence of phosphate (in minimal medium); when glucose is used in the absence of phosphate, either alone or in combination with single amino acids its role is hardly distinguishable; (5) Phosphate plays a facilitating role in spore germination. CONCLUSIONS: Glucose and amino acids play important roles in activation and germination of sporangiospores of Rhizopus oligosporus in tempe starter (stored for 12 months). The ability and rate of germination of dormant/old sporangiospores of R. oligosporus, depend on their ability for uptake of individual amino acids and/or glucose. SIGNIFICANCE AND IMPACT OF STUDY: New light was shed on the counteractive role of proline and the stimulating effect of phosphate. Soybeans subjected to traditional preparation for tempe making are heavily leached; germination of starter spores on such beans is sub-optimal, and bean processing could be optimized.     

Ingestion of sugar diets correlates with spike activity in labellar chemosensilla of the flesh-fly, Neobellieria (= Sarcophaga ) bullata

Abstract .Female 2-day-old Neobellieria (= Sarcophaga ) bullata (Parker) (Diptera: Sarcophagidae) were exposed to different concentrations of sucrose, glucose and fructose in a single-choice potometer, and the volume ingested in the first hour was measured. Nerve spike activity in response to the same sugars was recorded from medium labellar taste hairs of similar flies by tip-recording. Two classes of chemosensory cells responded to sucrose, glucose and fructose. Cell 1 showed an increasing spike activity with sugar concentration, whereas cell 2 did not; cell 1 was identified as the 'sugar cell'.
For both spike activity in cell 1 and feeding, sucrose was the most stimulatory sugar. The dose–response curves for glucose and fructose crossed over at about 200 m m . At higher concentrations, glucose was more stimulatory for both cell 1 and for feeding, and at lower concentrations, fructose. The pattern of spike activity supports a separate location on the sensory cells of receptors for pyranose and fructose forms of sugar. The strong correlation between volume ingested and spike activity indicates that sugar feeding is controlled by sensory input from the 'sugar' cells of labellar chemosensilla. Moreover, the results suggest that the flies do not distinguish between these sugars except by apparent 'sweetness'.     

Analysis of carbohydrates and amino acids in vegetable waste waters by ion chromatography

High-performance anion exchange chromatography coupled with pulsed amperometric detection was used for the quantitative determination of total and free sugars in olive oil mill waste waters (OMWW). Automated amino acid ion chromatography was employed to analyse total and free amino acids in the same OMWW. Sugars were analysed in samples pre-purified by means of a three-step purification procedure involving: (i) methanol precipitation of OMWW; (ii) dialysis of the obtained solid and liquid fractions; and (iii) chromatographic purification on RP18 phase followed by Amberlite resin. The amino acids were determined directly in samples obtained from the first two steps performed for sugar analysis. The analysis carried out with the reported methodologies allowed the quantitative determination of total sugars and amino acids and the differentiation between their free and bound forms. The sugars determined were arabinose, fructose, galactose, glucose, rhamnose, xylose, galacturonic and glucuronic acids, and the amino acids were Asp, Glu, Thr, Ser, Pro, Gly, Ala, Val, Met, Ile, Leu, Tyr, Phe, Lys, His, Arg and Cys. Asn, Gin, and Trp were not detected. The technological, biotechnological and environmental advantages arising from this analytical methodology applied to OMWW are briefly discussed.     

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.     

Identification of feeding stimulants in corn roots for western corn rootworm (Coleoptera: Chrysomelidae) larvae

Using a bioassay-driven approach, we have isolated and identified a blend of compounds from the roots of germinating corn, Zea mays L., that serve as feeding stimulants for neonate western corn rootworm larvae, Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae). The active blend is a combination of simple sugars (30:4:4 mg/ml glucose:fructose:sucrose in the corn root) plus at least one of the free fatty acids in germinating corn roots (2:5 mg/ml oleic acid:linoleic acid in the corn root). When an extract of germinating corn was partitioned into an ethyl acetate fraction and an aqueous fraction, full feeding occurred only when the two fractions were recombined, indicating that the phagostimulant was comprised of both polar and nonpolar components. Gas chromatography-mass spectrometry analysis of root extracts from germinating corn seedlings revealed a blend of 20 compounds from a variety of chemical classes, including small sugars, diacids, amino acids, inorganic compounds, and free fatty acids. When the major components were tested in feeding bioassays, the sugars and lipids were shown to be essential for feeding by larvae, but the two classes of compounds were only effective when combined. The sugars alone elicited feeding by only 40% of larvae, but the percentage of larvae feeding was increased significantly with the addition of linoleic acid (91.7% larvae feeding) or oleic acid (85.8% larvae feeding). The amino acids alone were not essential elements for feeding by western corn rootworm larvae.     

Oxidative Stress in Relation to Lipid Peroxidation, Sclerotial Development and Melanin Production by Sclerotium rolfsii

Melanin pigments constituted 13.9% of the chemical composition of the sclerotial walls of Sclerotium rolfsii and was associated with proteins, reducing sugars and amino acids. The lipid and ash contents in the sclerotial walls were double those in the hyphal walls of the fungus. Increasing age of the culture and maturation of the sclerotia were always accompanied by elevation of lipid peroxides and melanin pigments. Such behaviour may indicate that lipid peroxidation and melanin formation are operating in parallel during sclerotial biogenesis and maturation. These two processes depend on the theory of oxidative stress, as affected by growth conditions. Both processes could be stopped or sharply retarded when subjected to some antioxidant growth factors such as vitamins (ascorbic acid), micro-elements (selenium) and sulfhydryl compounds (glutathione). A clear relation between oxidative stress, myceliogenic germination and lytic activity via melanin production was observed. This finding appears promising in applying a new control measure against diseases caused by sclerotia-producing fungi without using traditional toxic fungicides.     

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.     

Reserve Mobilization During Germination of Tagetes minuta L.

Levels of albumins, globulins and starch were measured in dryTagetes minuta L. achenes (seeds) germinating at 25 °C andin those thermo-inhibited at 35 °C. In addition, the compositionof amino acids, fatty acids and free sugars was determined. In germinating seeds, albumins increased, while globulins decreased.At 35 °C, only a delayed decrease in globulins occurred.However, in both, the amino acids serine and glutamate increased.Lipid composition remained constant except for a slight declinein linoleic acid in the germinating seeds. At 25 °C, starchlevels increased with time while at 35 °C, there was onlya transient increase. Sucrose was the major free sugar foundin the seeds at all times, but its levels declined in the germinatingseeds. This was accompanied by an increase in the proportionsof glucose and fructose. No such changes in sugar compositionwere observed in the thermo-inhibited seeds. Tagetes minuta L., seed germination, reserve mobilization, thermo-inhibition     

Spatial and temporal distribution of solutes in the developing carrot taproot measured at single-cell resolution

The time-course and spatial distribution of sugars and ions in carrot (Daucus carota L.) was studied at fine resolution using single cell (SiCSA) and tissue analysis. Four phases of osmolyte accumulation in the taproot were identified: an amino acid (germination) phase, when internal sources of amino acids provide seedlings with osmotica; an ion phase, when inorganic and organic ions were the main solutes; a hexose phase, when concentrations of glucose and fructose sharply increased and reached their maximum; and a sucrose phase, when sucrose became the major solute. Spatial distribution of sugar in taproot cells showed a general trend of highest concentration on both sides of the vascular cambium (some 200 mM sucrose, 150 mM glucose) and a minimum in the pith (some 100 mM sucrose, 60 mM glucose) and in periderm. Electrolytes (e.g. potassium) followed a distribution generally reciprocal to that of sugars; minimum in the tissue adjacent to the cambium (some 10 mM) and maximum in the pith and periderm (some 60-100 mM). The cambial cells contained unexpectedly low concentrations of sugars and potassium. These spatial and temporal patterns indicate that amino acids, other electrolytes and sugars are interchangeable in the tissue osmotic balance. The nature of the solute is developmentally determined both temporally and spatially. During the accumulation of electrolytes following the initial amino acid phase, osmotic pressure to 420 mosmol kg-1 rises and then remains constant despite large changes in the concentration of individual solutes. This indicates that osmotic pressure is regulated independently of the individual concentrations of solutes.