Bloodmeal digestion in the midgut of Phlebotomus papatasi and Phlebotomus langeroni

Abstract. Bloodmeal digestion in midguts of the sandflies Phlebotomus papatasi and Phlebotomus langeroni (Diptera: Psychodidae) was investigated in optimized assays to detect general protease, trypsin and aminopeptidase activities using synthetic substrates. Optimal activity occurred at pH 8-9 for all enzymes examined in both species. Protease activity peaked at 24-34h post human bloodmeal in midguts of P.papatasi and 34-48h in P'.langeroni; all endo- and exoprotease activities were completed by 50 h in P.papatasi compared to 72 h in P. langeroni. Hydrolysis of two chymotrypsin substrates was <2% of trypsin activity in both species. Aminopeptidase activity was associated mainly with the midgut wall, whereas trypsin activity was confined to the midgut lumen. A feature of digestion in P.langeroni was the high level of aminopeptidase recorded within 10h of the bloodmeal.     

Structural features of mycorrhizal associations in two members of the Monotropoideae, Monotropa uniflora and Pterospora andromedea

Species in the subfamily Monotropoideae (family Ericaceae) are achlorophyllous and myco-heterotrophic. They have become highly specialized in that each plant species is associated with a limited number of fungal species which in turn are linked to autotrophic plants. This study provides an updated and comprehensive examination of the anatomical features of two species that have recently received attention with respect to their host-fungal specificity. Root systems of Monotropa uniflora and Pterospora andromedea collected from the field were characterized by light microscopy and scanning electron microscopy. All roots of both species were associated with fungi, each root having a well-developed mantle, paraepidermal Hartig net, and intracellular fungal pegs within epidermal cells. The mantle of M. uniflora was multi-layered and numerous outer mantle hyphae developed into cystidia of two distinct morphologies. Large calcium oxalate crystals were present, primarily on the mantle surface. The outer mantle of P. andromedea was more loosely organized, lacked cystidia, and had smaller plate-like as well as cylindrical crystals on the surface and between outer mantle hyphae. Fungal pegs in M. uniflora originated from inner mantle hyphae that penetrated the outer tangential wall of epidermal cells; in P. andromedea, these structures were initiated either from inner mantle hyphae or Hartig net hyphae and penetrated radial walls of epidermal cells. With respect to function, fungal pegs occurred frequently in both host species and, although presumed to be the sites of active nutrient exchange, no direct evidence exists to support this. Differences between these two monotropoid hosts, resulting from the mycorrhizal fungi with which each associates, are discussed.     

Nochascypha jacksonii comb. nov. and N. paraensis comb. nov., additional members of the cyphellaceous genus Nochascypha formerly placed in Maireina

Nochascypha jacksonii and N. paraensis proposed for combination were previously placed in the cyphellaceous genus Maireina. Unlike the typical representatives of the genus Maireina, both taxa have only pale coloured surface hyphae. The morphological and anatomical re-examination of type material has shown both species belong to the genus Nochascypha. Their inclusion extends the former range of Nochascypha species whose surface hyphae are colourless by two distinct members with yellowish surface hyphae. Nochascypha jacksonii and N. paraensis are described and illustrated in detail. An updated key for Nochascypha is presented.     

Anatomical Structures of the VA Mycorrhiza in the Apocynaceae (Gentianales)

Roots of 19 Apocynaceae species were studied anatomically with respect to their symbiosis with VAM-fungi. In plants collected from the field, VAM-fungi were established in the root cortex. Also, inoculations with different Glomus species on the cultured plants are very successful in the infection and colonization of the root cortex. After penetration of the rhizodermis, the special exodermal short cells become colonized by winding hyphae. Then, in the root cortex of many Apocynaceae species, the VAM-fungi produce intercellular running hyphae which leads to extensive colonization of the root. Arbuscules develop on intracellular running hyphae, whereas vesicles develop mainly on intercellular hyphae. Except for some special details, this is the most common type of colonization of VAM fungi in flowering plants. But in Amsonia tabernaemontana, Nerium oleander, and Thevetia peruviana, another type of colonization could be observed. In these species, the colonization of the hyphae within the root cortex is only possible by intracellular growth. Intercellular running hyphae in the root are lacking. Therefore, after penetration the colonization in the cortex is cluster-like and strictly limited. Only by additional penetrations from hyphae in the soil, will roots show heavy infestations. This type of growth of the VAM fungi in the root is well known from the Gentianaceae and was explained as a structural incompatibility. In Catharanthus roseus, Pachypodium lamerei, and Trachelospermum jasminoides, intermediate stages of both types of colonization could be described. The results are discussed in the search for possible stimulants for structural incompatibility.     

<Emphasis Type="Italic">Maireina afibulata</Emphasis> and <Emphasis Type="Italic">M. attenuatipilis</Emphasis>, new members of the cyphelloid genus <Emphasis Type="Italic">Maireina</Emphasis> (Basidiomycota,Agaricomycetes)

Maireina afibulata and M. attenuatipilis are proposed as new members of the recently revised genus Maireina, one of the few anatomically well-studied cyphelloid groups in the Basidiomycota. Maireina species have brownish basidiomes, smooth, parietally unpigmented spores, and are characterized by non-ramified external hyphae with more or less thick, yellow to brown cell walls and distinct crystalline incrustations that always include their distal ends. A key to the species of the genus Maireina is presented, and M. afibulata as well as M. attenuatipilis are described and discussed in detail. Taxonomic novelties: Maireina afibulata Bodensteiner, M. attenuatipilis Bodensteiner     

5种虾脊兰菌根显微结构观察

采用石蜡切片法观察5种虾脊兰菌根的显微结构、菌根真菌的侵入途径与分布特征,为更好地保护和开发利用虾脊兰属植物资源提供理论依据.结果表明:(1)5种虾脊兰菌根的显微结构由根被、皮层和中柱组成,根被细胞3～6层,皮层由9～13层薄壁细胞组成,韧皮部与木质部8～12束,呈辐射状相间排列,中柱中央为薄壁细胞组成的髓.(2)菌丝...     

Tropic failure of Phyllactinia corylea contributes to the mildew resistance of mulberry genotypes

Different mulberry genotypes show great variation in their resistance to the powdery mildew Phyllactinia corylea. Conidial germination and hyphal growth of P. corylea on the leaf surface of two susceptible mulberry genotypes, viz., Kanva 2 (K2) and Victory 1 (V1) varieties of Morus indica, and on two resistant species, viz., M. laevigata and M. serrata were studied by scanning electron microscopy. Conidial germination and growth of germ tubes were normal on all the leaves. The hyphae of P. coryleaidentify stomata on host leaves by their topographical features to produce the stomatopodia precisely over them. The holes and/or the grooves of stomata appear to provide the signals for the initiation of stomatopodia and similar structures are erratically developed over many local depressions or grooves on leaf surface. The abaxial surface of K2 leaf is smooth without prominent undulations of epidermal cell surface, and the stomata are flush with the leaf surface. Although successful penetration is also achieved on V1 leaf, its slightly undulated surface occasionally provides inaccurate tropic signals to the hyphae, inducing the development of stomatopodia away from the stomata. The leaf surfaces of M. laevigata and M. serrata are very rough with highly sculptured cuticle and abundant epidermal outgrowths. Stomata mostly remain sunken or hidden amidst the cuticular ornamentations and the hyphae fail to recognise the precise signals from them. As the surface architecture of the leaves provides many immense sources of tropic signals, stomatopodia are often produced over local depressions or grooves. In these cases the fungus fails to penetrate the leaf, does not develop beyond 24 h and penetration is rarely achieved on the leaves of the resistant plants. The study indicates that the stimulatory effect of the leaf surface topography of resistant varieties misleads the pathogen from successful penetration, thus contributing to the plant's resistance.This revised version was published online in October 2005 with corrections to the Cover Date.     

The ectomycorrhiza <Emphasis Type="Italic">Piceirhiza internicrassihyphis</Emphasis>: A weak competitor of <Emphasis Type="Italic">Cortinarius obtusus</Emphasis>?

The ectomycorrhiza (ECM) Piceirhiza internicrassihyphis on Picea abies is described in detail. It is yellowish brown to brown with a mantle of a transitional type between plectenchymatous and pseudoparenchymatous. The inner mantle layers show some thick-walled hyphae, often with a few of them growing in parallel. These thick-walled hyphae contain particles which turn brownish in Melzer’s reagent, and their septa and walls are partially amyloid. Rhizomorphs occur infrequently and are undifferentiated, and are composed of rather loosely woven hyphae of uniform diameter. This combination of characteristics has not yet been identified in any other ECM. The lack of cystidia and the presence of amyloidy suggest that this ECM is formed by a species of the family Thelephoraceae. Piceirhiza internicrassihyphis can be found associated with ECM of Cortinarius obtusus, and the hyphae of the latter species often grow on the surface of P. internicrassihyphis and can even cover the whole tip. Although there is a close association and possibly a penetration between these two fungi, it can be excluded that the thick-walled hyphae of the inner mantle layers originate from C. obtusus. Some emanating hyphae of C. obtusus ECM seem to interact with those of P. internicrassihyphis, as indicated by short, hyphal outgrowths which were found attached to the foreign hyphae.     

Arbuscular mycorrhizal structure and fungi associated with mosses

We investigated the colonization and diversity of arbuscular mycorrhizal (AM) fungi associated with 24 moss species belonging to 16 families in China. AM fungal structures, i.e. spores, vesicles, hyphal coils (including intracellular hyphae), or intercellular nonseptate hyphae, were found in 21 moss species. AM fungal structures (vesicles, hyphal coils, and intercellular nonseptate hyphae) were present in tissues of 14 moss species, and spores and nonseptate hyphae on the surface of gametophytes occurred in 15 species. AM fungal structures were present in 11 of the 12 saxicolous moss species and in six of the ten terricolous moss species, but absent in two epixylous moss species. AM fungal structures were only observed in moss stem and leaf tissues, but not in rhizoids. A total of 15 AM fungal taxa were isolated based on trap culture with clover, using 13 moss species as inocula. Of these AM fungi, 11 belonged to Glomus, two to Acaulospora, one to Gigaspora, and one to Paraglomus. Our results suggest that AM fungal structures commonly occur in most mosses and that diverse AM fungi, particularly Glomus species, are associated with mosses.     

Production of Polyclonal and Monoclonal Antibodies Against Hyphae from Arbuscular Mycorrhizal Fungi

Abstract

Polyclonal and monoclonal antibodies were produced against hyphae of the arbuscular mycorrhizal fungus Glomus monosporum. The polyclonal antibodies (pAbs) were raised in a rabbit by immunizing with hyphae. They were tested for their specificity by a dot-immunoblot assay (DIBA). After the third immunization, a distinct difference in the signal strength was observed between the antisera and the preimmune serum. The pAbs showed cross-reactions to a number of fungal species, both mycorrhizal and other. For the production of monoclonal antibodies (mAbs), mice were immunized intraperitoneally with hyphae. The resulting hybridoma cell culture supernatants were tested by an indirect immunolabeling procedure. For this purpose the hyphae were immobilized on silane-coated microscopic slides. The mAb 8A7 reacted with hyphae from all Glomus isolates tested so far. Cross-reactivities were not observed with hyphae from fungi of the family Acaulosporaceae, phytopathogenic fungi tested so far, or from spores from Glomus species.     

Systematics of <Emphasis Type="Italic">Mycosphaerella</Emphasis> species associated with the invasive weed <Emphasis Type="Italic">Fallopia japonica</Emphasis>, including the potential biological control agent <Emphasis Type="Italic">M. polygoni-cuspidati</Emphasis>

Native to Japan, Fallopia japonica, most frequently referred to as Japanese knotweed, is a highly problematic invasive weed, particularly in the UK and North America. During surveys for natural enemies of this plant in Japan, two species of Mycosphaerella were collected. One of these was identified as M. polygoni-cuspidati, and is redescribed and neotypified. Causing a damaging leaf spot disease of F. japonica throughout its natural range in Japan, it is absent from the host’s exotic range. The restriction of M. polygoni-cuspidati to F. japonica in its center of origin, together with its severe impact on host fitness, indicates that this is a coevolved natural enemy with high potential as a classical biological control agent for the long-term management of this ecologically and economically important weed. In the field, the fungus has a reduced life cycle, with only spermogonia and pseudothecia (ascomata) being formed. Ascospores are the primary source of infection, and studies show that the mycelium from in vitro cultures is also infective and hyphae penetrate mainly via the stomata. A further, undescribed species of Mycosphaerella co-occurs with M. polygoni-cuspidati, here proposed as the new species M. shimabarensis. Both species have been studied using cultural, morphological and molecular phylogenetic methods.     

Studies in Heterobasidiomycetes, Part 31*). On the Anther Smuts of Silene vuigaris (Moench) Garcke

Microbotryum silenes-inflatae (DC) G. Deml & Oberw. and Microbotryum violaceoirregulare (Brandenburger & Schwinn) G. Deml & Oberw., both parasitic in the anthers of Silene vulgaris (Moench) Garcke, are compared by their morphology, karyology, and some cultural characteristics. The infection of their hosts is similar in both species, but causes different sizes of the flowers and different color of the spore mass. The teliospore initials of that smuts are produced in dikaryotic sporogenous hyphae. During gelatinization of the hyphal coat, surface ornamentations on the teliospores are formed. Mature teliospores are monokaryotic, and presumably diploid, reticulate in Microbotryum silenes-inflatae and echinulate in M. violaceo-irregulare. Teliospores of both species germinate without a resting periode. Germination results in a commonly three-celled promycelium, which in M. silenes-inflatae separates from the producing teliospore, while in M. violaceo-irregulare it remains on the teliospore during basidiospore formation. The basidiospores are monokaryotic and propagate by budding. Monokaryotic strains were isolated and characterized by standard yeast identification tests and by some enzymatic activities on solid media. While no differences in the enzymatic activities can be found, the species differ in their utilization of carbon sources.     

A new species of petalosporus

Summary A new species ofPetalosporus, P. anodosus, is described and illustrated. Two isolates representing this species were isolated from dung in California. The new species differs from the only other species of the genus,P. nodulosus, by the lack of thick-walled peridial hyphae with enlargements at the septa and by the absence of disarticulation of peridial elements. Peridial hyphae inP. anodosus have walls which are somewhat thickened and the cells are often variable in diameter. Conglomerate ascospores usually assume the petaloid arrangement characteristic of the genus.     

Ustilago maydisMating Hyphae Orient Their Growth toward Pheromone Sources

Snetselaar, K. M., Bölker, M., and Kahmann, R. 1996.Ustilago maydismating hyphae orient their growth toward pheromone sources.Fungal Genetics and Biology20,299–312. When small drops ofUstilago maydissporidia were placed 100–200 μm apart on agar surfaces and covered with paraffin oil, sporidia from one drop formed thin hyphae that grew in a zig-zag fashion toward the other drop if it contained sporidia making the appropriate pheromone. For example,a2b2mating hyphae grew towarda1b1anda1b2mating hyphae, and the filaments eventually fused tip to tip. Time-lapse photography indicated that the mating hyphae can rapidly change orientation in response to nearby compatible sporidia. When exposed to pheromone produced by cells in an adjacent drop, haploid sporidia with thea2allele began elongating before sporidia with thea1allele. Sporidia without functional pheromone genes responded to pheromone although they did not induce a response, and sporidia without pheromone receptors induced formation of mating hyphae although they did not form mating hyphae. Diploid sporidia heterozygous atbbut not ataformed straight, rigid, aerial filaments when exposed to pheromone produced by the appropriate haploid sporidia. Again, thea2a2b1b2strain formed filaments more quickly than thea1a1b1b2strain. Taken together, these results suggest that thea2pheromone diffuses less readily or is degraded more quickly than thea1pheromone.     

Antifungal action of human cathelicidin fragment (LL13-37) on Candida albicans

Human cathelicidin LL37 and its fragments LL13–37 and LL17–32 exhibited similar potencies in inhibiting growth of the yeast Candida albicans. After treatment with 0.5 μM and 5 μM LL13–37, the hyphae changed from a uniformly thick to an increasingly slender appearance, with budding becoming less normal in appearance and cell death could be detected. Only the yeast form and no hyphal form could be observed following exposure to 50 μM LL13–37. LL13–37 at a concentration of 5 μM was able to permeabilize the membrane of yeast form as well as hyphal form of C. albicans since the nuclear stain SYTOX Green was localized in both forms. Mycelia treated with LL13–37 stained with SYTOX Green, but did not stain with MitoTracker deep red, indicating that the mitochondria were adversely affected by LL13–37. Bimane-labeled LL13–37 was able to enter some of the hyphae, but not all hyphae were affected, suggesting that LL37impaired membrane permeability characteristics in some of the hyphae. Reactive oxygen species was detectable in the yeast form of C. albicans cells after treatment with LL13–37 but not in the untreated cells. The results suggest that the increased membrane permeability caused by LL13–37 might not be the sole cause of cell death. It might lead to the uptake of the peptide, which might have some intracellular targets.     

Interactions of soil fungi with Macrophomina phaseoli (Maubl.) ashby,the cause of root rot of cotton

Summary Interactions of 46 isolates of fungi with an isolate ofM. phaseoli from cotton, was studied in agar culture. They were grouped into 4 different types of reactions.T. viride was found to inhibit the growth ofM. phaseoli and grow over its colony. The hyphae ofT. viride were observed to coil around the hyphae ofM. phaseoli. A majority of the isolates tested had no effect on the growth ofM. phaseoli. M. phaseoli, however, was found to overgrow the test fungi.     

Peptidase activities during morphogenesis of Mucor racemosus

Multiple peptidase activities are expressed in the dimorphic fungus Mucor racemosus. Peptide hydrolysis was measured using an enzyme-coupled colorimetric assay. Aminopeptidase as well as carboxypeptidase activities increased during spore, swollen spore, and budding yeast-to-hyphae conversions, and activities achieved a maximum level prior to the period of rapid germ tube formation. These increases in peptidase activity were prevented by cycloheximide. Three distinct aminopeptidases (AP) and three distinct carboxypeptidases (CP) were partially purified by gel filtration column chromatography. AP1 (235 kDa), AP2 (112 kDa), and AP3 (70 kDa) were all expressed in spore, yeast, and hyphae. The activity levels of AP2 and AP3 decreased in hyphae entering stationary growth. CP1 (250 kDa) and CP3 (50 kDa) activities were expressed exclusively in hyphae, whereas CP2 (77 kDa) was expressed in spore, yeast, and hyphal forms. CP1 activity was most pronounced in hyphae entering stationary growth. We concluded that M. racemosus expresses a multiplicity of peptidases and that CP1 and CP3 are morphology-specific carboxypeptidases.     

Comparison of phosphatase localization in the intraradical hyphae of arbuscular mycorrhizal fungi,Glomus spp. and Gigaspora spp.

The localization of acid and alkaline phosphatases in the intraradical hyphae of the arbuscular mycorrhizal fungi, Glomus mosseae (Nicol. and Gerd.) Gerd. and Trappe (Gm), Gl. etunicatum Becker and Gerd. (Ge) and Gigaspora rosea Nicol. and Schenck (Gir) were compared. Marigold (Tagetes patula L.) and leek (Allium porrum L.) were inoculated with each of the three fungi. The mycorrhizal roots were harvested at 3, 4, 5 and 6 weeks after sowing (WAS), treated with a digestion solution containing cellulase and pectinase, and then stained for phosphatase activities at pH 5.0 and pH 8.5. The development of fungal structures in the host root was also examined. Gm formed fine-branched (mature) arbuscules only at the early phase of infection (3 to 4 WAS). Mature arbuscules of Ge and Gir were observed from the early phase (4 WAS) up to the end of experiment. At pH 5.0, the localization of the phosphatase activities of the three fungi were similar irrespective to host plant species. The activity appeared in mature arbuscules and intercellular hyphae, whereas the collapsed arbuscules were inactive. Ten millimolar NaF, an acid phosphatase inhibitor, inhibited the phosphatase activities of Gm and Ge but did not affect that of Gir. At pH 8.5, a difference among the fungal species was found in the localization of phosphatase activity while that between host species was not. The mature arbuscules were also the active sites in all three species. Only Gir showed the activity in the intercellular hyphae while the two Glomus spp. did not. Five millimolar EDTA inhibited the activity of Gir at pH 8.5 while the activities of Ge and Gm were not affected by either 5 mM EDTA or 10 mM KCN (both are alkaline phosphatase inhibitors).     

The trapping fungus Dactylaria Brochopaga: induction of trap formation,attraction, trapping and the development in some phytonematodes

All the six species of the phytonematodes induced traps directly on the spores of all the five isolates of Dactylaria brochopaga. The maximum rings induction was observed in the presence of second-stage juveniles of Meloidogyne incognita followed by Meloidogyne graminicola (J₂), Xiphinema basiri, Helicotylenchus dihystera, Tylenchorynchus brassicae and Hoplolaimus indicus. Maximum number of induced traps was recorded at temperature 28°C (98.3%) and pH 7 (96.7%) in the presence of H. indicus. In attraction assay, irrespective of the isolates of the fungus, all the six phytonematodes were attracted towards the fungal discs of the different isolates of D. brochopaga. Among all the five isolates, Isolate D attracted maximum (34.7%) number of nematodes closely followed by Isolate C (31.0%), whereas, Isolate A, B and E did not differ significantly. Maximum in vitro trapping was recorded in the case of M. incognita (J₂) followed by M. graminicola (J₂), H. dihystera and T. brassicae. The maximum ringing (pre-capturing), trapping and killing was recorded for the second-stage juveniles of M. incognita followed by M. graminicola, T. brassicae and H. dihystera. The inflation of ring cells did not occur immediately after ringing; however, the time between ringing and inflation varied with different nematodes species. Clear hyphae were observed in the invaded nematodes body after the content of nematode body was consumed. External growth of hyphae occurred only from the inflated cells of the constricting rings, which further developed several rings. Higher number of rings was observed in mycelia developed from bigger nematodes.