Degenerative changes in the epidermal cell wall of Allium cepa caused by Botrytis allii: Loss of dry mass,pectin, and cellulose in halos

Halos detected using interference microscopy (even- and fringle-field modes with monoand poly-chromatic light) around penetration sites of Botrytis allii in cell walls of normal and protoplast-free outer epidermal tissue of white, yellow, and red onions were alike. Halos in protoplast-free cell walls contained 33% less dry mass than areas of these walls adjacent to halos (quantitative interference miscroscopy with 546 nm light in the even-field mode). Halos were significantly larger in the white onion than in the yellow and red varieties. The loss of cell wall dry mass during the production of halos involved the loss of pectin and cellulose. We infer that this is caused by enzymes released from the pathogen. Cuticle degradation at penetration sites was not observed.     

Involvement of carbohydrates and cytokinins in pathogenicity ofHelminthosporium carbonum

Significant stimulation of the number of appressoria, penetration and colonization by conidia ofHelminthosporium carbonum occurred on decolorized maize leaves when exogenous carbohydrates and leaf leachates were added. Germination and germ tube length, however, did not exhibit appreciable differences on decolorized or non-decolorized maize leaves. Lower germination was recorded by leached conidia on decolorized leaves; while appressoria, penetration and colonization were absent. Addition of exogenous nutrients (sucrose>leaf leachates>yeast extract>glucose) enabled conidia to accomplish appressoria, penetration and colonization. Optimum levels for various nutrients observed were 2% (w/v) sucrose/glucose or 0.1% (w/v) yeast extract. Higher concentrations inhibited the infection stages of the pathogen. Depletion of host carbohydrates from green islands/infection sites adversely affect appressoria formation, penetration and colonization; and the loss of carbohydrates from the spore affects germination. Cytokinin-like activity at the infection site/green islands increased with the period of incubation of the host as compared to the surrounding tissue or tissue under water drops. The culture filtrate extracts ofH. carbonum recorded cytokinin-like activity which increased with growth of the fungus. TLC (thin layer chromatography) of cytokinin-like substances (tissue extract and culture filtrate) revealed major activity was confined to Rf zones 0.6 to 0.8 which co-chromatographed with zeatin and zeatin riboside. These substances increase at infection sites by virtue of which carbohydrates accumulate at these sites ensuring a continuous supply to the growing pathogen.     

Ultrastructure of infection-thread development during the infection of soybean by Rhizobium japonicum

The location and topography of infection sites in soybean (Glycine max (L.) Merr.) root hairs spot-inoculated with Rhizobium japonicum have been studied at the ultrastructural level. Infections commonly developed at sites created when the induced deformation of an emerging root hair caused a portion of the root-hair cell wall to press against an adjacent epidermal cell, entrapping rhizobia within the pocket between the two host cells. Infections were initiated by bacteria which became embedded in the mucigel in the enclosed groove. Infection-thread formation in soybean appears to involve degradation of mucigel material and localized disruption of the outer layer of the folded hair cell wall by one or more entrapped rhizobia. Rhizobia at the site of penetration are separated from the host cytoplasm by the host plasmalemma and by a layer of wall material that appears similar or identical to the normal inner layer of the hair cell wall. Proliferation of the bacteria results in an irregular, wall-bound sac near the site of penetration. Tubular infection threads, bounded by wall material of the same appearance as that surrounding the sac, emerge from the sac to carry rhizobia roughly single-file into the hair cell. Growing regions of the infection sac or thread are surrounded by host cytoplasm with high concentrations of organelles associated with synthesis and deposition of membrane and cell-wall material. The threads follow a highly irregular path toward the base of the hair cell. Threads commonly run along the base of the hair cell for some distance, and may branch and penetrate into subjacent cortical cells at several points in a manner analagous to the initial penetration of the root hair.     

Photoreactivation of UV-irradiated blue-green algae and algal virus LPP-1

Ultraviolet (UV) sensitivity and photoreactivation of blue-green algae Cylindrospermum sp., Plectonema boryanum, spores of Fischerella muscicola and algal virus (cyanophage) LPP-1 were studied. The survival value after UV irradiation of filaments of Cylindrospermum sp. and Virus LPP-1 showed exponential trend and these were comparatively sensitive towards UV than F. muscicola and P. boryanum. Photoreactivation of UV-induced damage occurred in black, blue, green, yellow, red and white light in Cylindrospermum sp., however only black, blue and white light were capable of photorepair of UV-induced damage in P. boryanum, spores of F. muscicola and virus LPP-1 in infected host alga. Pre-exposure to yellow and black light did not show photoprotection. The non-heterocystous and nitrogen fixation-less mutants of Cylindrospermum sp. were not induced by UV and their spontaneous mutation frequency was not affected after photoreactivation. The short trichome mutants of P.boryanum were more resistant towards UV.The occurrence of photoreactivation of UV-induced killing in wide range of light in Cylindrospermum sp. is the first report in organisms.     

The role of bacterial attachment in the transformation of cell-wall-regenerating tobacco protoplasts by Agrobacterium tumefaciens

The presence of a newly formed primary cell wall was shown to be required for attachment and subsequent transformation of tobacco leaf protoplasts by Agrobacterium tumefaciens in cocultivation experiments. In these experiments both protoplasts at different stages after their isolation and cell-wall inhibitors were used. The specificity of Agrobacterium attachment was shown by using other kinds of bacteria that did not attach. By diminishing the concentration of divalent cations using ethylenediaminetetraacetic acid, neither attachment nor transformation was found; however, when more specifically the Ca²⁺concentration was lowered by ethylene glycol-bis (-aminoethyl ether)-N,N,N,N-tetraacetic acid, both phenomena occurred. Commercial lectins had no effect on binding, but this observation does not exclude the involvement of other lectins. Protoplasts isolated from various crown-gall callus tissues also developed binding sites, but when they were at the stage of dividing cells, attachment of agrobacteria was no longer observed. In this respect, cells from protoplasts of normal tobacco leaves behaved differently. Even 16 d after protoplast isolation, the dividing cells were still able to bind A. tumefaciens, while transformation was not detected. For transformation of 3-d-old tobacco protoplasts, a minimal co-cultivation period of 24 h was required, while optimal attachment took place within 5 h. It is concluded that the primary cell wall was sufficiently well formed that certain functional receptor molecules were available for attachment of Agrobacterium as the first step of a multistep process leading to the transformation of cells. The expression of bacterial functions required for attachment, moreover, was independent of the presence of Ti-plasmid.Abbreviations ConA concanavalin A - CW calcofluor white - EDTA ethylenediaminetetraacetic acid - EGTA ethylene glycol-bis (-aminoethyl ether)-N,N,N,N-tetraacetic acid - -Man -methyl-d-mannoside     

The combination of electronic monitoring and video-assisted observations of plant penetration by aphids and behavioural effects of polygodial

Simultaneous electronic and close-up video recordings were made of behaviour during the initial 15 min of plant contact by adult, apterous Aphis fabae Scopoli on tick bean seedlings (Vicia faba Moench). Electronic techniques accurately determined stylet penetration of plant tissue and there was a close correlation between penetration and periods during which the insect antennae and body were immobile (r=0.994, n=60). Video techniques were then used alone to infer stylet penetration and the behaviour of aphids after various treatments was monitored. In particular, the time to first penetration, the number of penetrations, the mean duration of penetrations and the total time of penetration were observed. Behavioural differences were recorded between tethered (as required for electronic recording) and freelymoving insects as well as between fed and starved insects. The behaviour of starved aphids placed on beans treated with the plant-derived antifeedant, polygodial could not be distinguished from aphids on solvent-treated control beans. However, there were significant differences in behaviour of aphids which had previously been exposed to polygodial on plant or green/yellow paper surfaces for 24 h when compared with insects exposed to solvent alone. The possible modes of action of polygodial are discussed.     

古林箐秋海棠叶斑结构对叶色的影响

该研究以古林箐秋海棠(Begonia gulinqingensis)为材料,通过分析叶片形态特征、上表皮光学特性、组织结构、叶绿素含量及叶绿素荧光参数(F_v/F_m),探讨了叶片色斑的形成原因。结果表明:(1)古林箐秋海棠叶斑发生频率和数量无明显规律,但发生部位相对稳定,叶斑主要发生在正对叶柄的两条主脉之间。(2)斑区有两种光反射模式,点状反射和多角形反射,栅栏组织细胞呈近等轴的圆形,排列疏松,与上表皮细胞间存在空隙;非斑区只有点状反射模式,栅栏组织细胞为漏斗型,排列紧密,与上表皮细胞间不存在空隙。(3)斑区和非斑区叶绿体均有密集的堆积基粒和丰富的类囊体膜,斑区叶绿素a、b及总叶绿素含量仅比非斑区分别低24.9%、25.2%、25.1%。(4)叶绿素荧光参数(F_v/F_m)值斑区为0.793,非斑区为0.790。虽然斑区叶绿素含量比非斑区略低,但叶绿体结构完整,且叶绿素荧光参数与非斑区无显著差异。斑区上表皮与栅栏组织细胞间的空隙可使光线到达绿色组织时发生二次反射,在叶片表皮细胞边缘形成白色多边形光反射使该区域相对周围正常叶片区域偏白,基于上述结果可推测古林箐秋海棠的淡绿色块斑形成与特殊的叶片结构有关。     

Isolation and culture of protoplasts from callus tissue of Hibiscus syriacus L.

Protoplasts were isolated from callus tissue of Hibiscus syriacus L. using a solution of 3% Onozuka cellulase, 1% Onozuka macerozyme, and 0.5% hemicellulase. Highest yields of viable protoplasts were obtained from friable, white or yellow callus 8–9 days after subculture on Murashige & Skoog medium with 0.5 mg l^-1 2,4-dichlorophenoxyacetic acid and 0.1 mg l^-1 kinetin. Protoplasts cultured in thin liquid layers of this medium with mannitol continued dividing for longer than those cultured in droplets or in an agar medium. Cultures were maintained until protoplasts had divided to form groups of more than ten cells. Cell groups developed into callus and continued to grow on an agar medium, but failed to differentiate on a regeneration medium with 2 mg l^-1 naphthalene acetic acid and 1 mg l^-1 benzylaminopurine.     

Effect of shape, size and color on selection of oviposition sites by Chaetorellia australis

The effect of shape, size and color on the selection of oviposition sites by females of Chaetorellia australis Hering (Diptera, Tephritidae) originating from infested yellow starthistle (YST) Centaurea solstitialis L. (Asteraceae, Cardueae) heads, was studied in the laboratory by direct observation of females sitting on or attempting to oviposit into different objects. Artificial substrates made of yellow-colored parafin was mimicking the natural oviposition sites (floral buds of YST), were used to study the effect of shape and size. The results revealed that spherical and conical objects of a total surface area of 310 mm², were visited more frequently than cylindrical and cubical ones of the same surface area. Also more females were observed and more oviposition attempts were recorded on spheres of 5 and 10 mm diameter than of 15 and 20 mm diameter. Yellow and orange dry YST heads, colored by dipping in molten colored ceresin wax, were preferred for oviposition to black, green, blue, red and white ones. The observed preference for certain colors depended primarily on the color hue and not on the intensity of the total reflected light. The females responded positively to hues reflecting maximally between 560 and 610 nm, optimum at 590 nm, while hues reflecting below 540 nm and especially between 400 and 500 nm appeared to have no positive effect.     

Coral disease physiology: the impact of Acroporid white syndrome on <Emphasis Type="Italic">Symbiodinium</Emphasis>

Acroporid white syndrome, a disease-like syndrome from the Great Barrier Reef, results from degenerative host tissue at lesion borders. Tissue preceding lesion borders appears visually healthy, but it is currently unclear whether the endosymbiotic zooxanthellae (Symbiodinium) are physiologically impacted. Compared to healthy colonies, this study found no significant differences in symbiont density, mitotic index or chlorophyll a content in tissue bordering (0 cm), and 8 cm away from white syndrome lesions. Using chlorophyll a fluorescence techniques, the border tissue did not appear to be photosynthetically compromised, and Symbiodinium extracted from this area were photosynthetically competent. Transmission electron microscopy revealed extensive degeneration of host tissues surrounding symbionts in affected areas, however, Symbiodinium cells were structurally intact with no sign of in situ degradation. Collectively, these results suggest that Symbiodinium at white syndrome lesion borders exist in a dynamic intra-cellular state during active host tissue loss, yet remain physiologically uncompromised.     

Microtubules and coated vesicles in guard-cell protoplasts ofAllium cepa L.

Protoplasts were prepared from the guard cells ofA. cepa. Epidermal peels taken from expanding green leaves and largely free of mesophyll were treated with Cellulysin, and protoplasts were harvested after 18 h of digestion. That the protoplasts were derived from guard cells was ascertained from their characteristic vacuolar autofluorescence and from observations showing that all other epidermal cells are killed in the peeling procedure. The protoplasts proved to be a good system with which to view the cell cortex and inner surface of the plasmalemma. The lysis of cells adhering to polylysine-treated, Formvar-coated grids, followed by negative staining in uranyl acetate, showed that many microtubules normally present in ordered arrays in situ remain closely applied to the inner surface of the plasmalemma in protoplasts. In addition, numerous vesiculate elements including coated vesicles and/or pits are present amongst the microtubules. Similar vesicles are evident in thin sections of fixed, embedded guard cells and protoplasts. The significance of these structures in the cell cortex is discussed.     

Colonisation patterns of root tissues byPhytophthora nicotianae var.parasitica related to reduced disease in mycorrhizal tomato

Tomato plants pre-colonised by the arbuscular mycorrhizal fungusGlomus mosseae showed decreased root damage by the pathogenPhytophthora nicotianae var.parasitica. In analyses of the cellular bases of their bioprotective effect, a prerequisite for cytological investigations of tissue interactions betweenG. mosseae andP. nicotianae v.parasitica was to discriminate between the hyphae of the two fungi within root tissues. We report the use of antibodies as useful tools, in the absence of an appropriate stain for distinguishing hyphae ofP. nicotianae v.parasitica from those ofG. mosseae inside roots, and present observations on the colonisation patterns by the pathogenic fungus alone or during interactions in mycorrhizal roots. Infection intensity of the pathogen, estimated using an immunoenzyme labelling technique on whole root fragments, was lower in mycorrhizal roots. Immunogold labelling ofP. nicotianae v.parasitica on cross-sections of infected tomato roots showed that inter or intracellular hyphae developed mainly in the cortex, and their presence induced necrosis of host cells, the wall and contents of which showed a strong autofluorescence in reaction to the pathogen. In dual fungal infections of tomato root systems, hyphae of the symbiont and the pathogen were in most cases in different root regions, but they could also be observed in the same root tissues. The number ofP. nicotianae v.parasitica hyphae growing in the root cortex was greatly reduced in mycorrhizal root systems, and in mycorrhizal tissues infected by the pathogen, arbuscule-containing cells surrounded by intercellularP. nicotianae v.parasitica hyphae did not necrose and only a weak autofluorescence was associated with the host cells. Results are discussed in relation to possible processes involved in the phenomenon of bioprotection in arbuscular mycorrhizal plants.     

Pathogenesis of barley leaves by Helminthosporium teres I: Green island formation and the possible involvement of cytokinins

Infection sites/green islands were formed in host leaf tissue infected with drops of H. teres. They exhibited higher cytokinin-like activity, sugar and starch than their surrounding tissue and tissue under water drops. The cytokinin-like activity at the infection sites increased from 24 to 72 h of incubation. However, the cytokinin-like activity of the tissue surrounding the infection drops and the tissue under water drops fell from 24 to 72 h incubation. The culture filtrate extracts of the fungus also produced cytokinin-like activity which increased from 1 to 10 days incubation. Application of this culture filtrate extract evoked green island formation. Application of kinetin to host leaves duplicated the green island effect. Thin-layer chromatographic fractions of the tissue extracts and the culture filtrate extracts revealed that a major portion of cytokinin-like activity corresponded to zeatin and zeatin riboside. The presence of zeatin and zeatin riboside (both in tissue and culture filtrate extracts) was confirmed by high performance liquid chromatography. Increases in the amounts of cytokinin-like substances, particularly zeatin and zeatin riboside, attributed to pathogen influence are suggested to be involved in infection and pathogenicity of H. teres.     

Protoplasts of Gelidium robustum (Rhodophyta)

Viable protoplasts were isolated from apices of the agarophyte Gelidium robustum (Gardn.) Hollenb. & Abb. using a combination of commercial cell-wall degrading enzymes and extracellular wall-degrading enzymes isolated from a marine bacterium. The protoplasts were approximately 8–15 µm in diameter, liberated mainly from the surface cell layers and from cells at the distal ends of medullary filaments. The bacterial enzyme alone was not sufficient to liberate significant numbers of protoplasts. Maximum yield was 9 × 10⁵ protoplasts/g tissue (wet wt.). Optimum osmolality occurred between 1750–1950 mOs kg^–1; yield and viability were severely diminished at osmolalities less than 1350 mOs kg^–1. Viability, as determined by flurorescein diacetate staining and Evans Blue exclusion 1 hr after removal from the enzyme solution, was approximately 80–95%. Roughly 80% of the cells did not show Calcofluor fluorescence, while 40% stained positively for the presence of sulfated polysaccharides. Cell wall regeneration was observed with inconsistent reproducibility, and no cell division was observed when the protoplasts were placed in culture medium.Dedicated to the memory of Professor Michael Neushul.     

Autofluorescence of the fungus <Emphasis Type="Italic">Morchella conica</Emphasis> var. <Emphasis Type="Italic">rigida</Emphasis>

Autofluorescence (primary fluorescence (AF)) of fruiting bodies and stems of the fungus Morchella conica var. rigida was studied by fluorescence microscopy including sporangia and ascospores. The ascospores were characterized by a weak green–yellow AF at blue excitation. Using a green excitation, no AF was observed. The hyphae located under the layer of asci with ascospores exhibited a higher primary fluorescence, namely their walls that had green-yellow color at blue excitation. Also, their red AF observed when a green excitation was used was significant. Similarly, the hyphae located in the fungal stem exhibited a significant AF, especially their walls when the blue light was used for excitation. In addition, large, yellow-to-yellow/green, oval-to-round bodies with strong fluorescence were detected whose morphological equivalents were not clearly visible in the white halogen light. The AF of the fungus M. conica var. rigida was lower compared with the other higher fungi studied so far.     

Field studies on colour preferences of Ctenarytaina thysanura in Tasmanian boronia farms

Field tests on attraction of Ctenarytaina thysanura (Hemiptera: Psyllidae) adults to different coloured 30×30 cm sticky traps revealed a preference for yellow. Among the enamel colours tested, more psyllids were captured on yellow traps followed by green, then blue and least on red, cyan and magenta. Dilution of yellow enamel with 50% white (1Y: 1W) and 75% white (1Y: 3W) to produce yellow-white hues resulted in a significant decrease in psyllid capture indicating that the psyllids response to yellow was one of positive attraction and could suggest true colour discrimination. Reflectance spectra of painted surfaces of the enamel colours and also yellow to white hues indicated that psyllid capture rates were directly related to the proportion of light reflected in the 500–560 nm region. The biological basis of the observed C. thysanura response may be that yellow is the most intensely reflective colour in the general part of the spectrum for leaves which reflect most light in the 500–600 nm (peak 550 nm) range.     

Restriction fragment length polymorphism (RFLP) analyses of plants produced by in vitro anther culture of Solanum chacoense Bitt

Summary Green mesophyll protoplasts of the dihaploid potato line 1982 (Solanum tuberosum L.) were fused with herbicide-bleached mesophyll protoplasts of the dihaploid potato line 679 using a polyethylene glycol protocol. Heterokaryons were identified under a fluorescence microscope using the dual fluorescence of carboxyfluorescein-stained, herbicide-bleached protoplasts and the autofluorescence of green mesophyll protoplasts. About 20% of the protoplasts survived the fusion treatment, and the fusion frequency was 3%–4%. Unfused and fused protoplasts were mass cultured for 6 weeks after which vigorously growing calli were selected and transferred to shoot regeneration medium. Somatic hybrids were identified by a combination of five isozyme markers, and the ploidy level was determined by flow cytometry. Out of 15 calli that regenerated shoots, 6 plants derived from 2 different calli were identified as hexaploid somatic hybrids, while one morphologically deviant plant from a third callus was identified as a mixoploid that had lost some enzyme markers after 4 months of culturing.     

Ultrastructural characterisation of the host–pathogen interface in white blister-infected Arabidopsis leaves

In this study transmission electron microscopy (TEM) was used to examine details of the host–pathogen interface in Arabidopsis thaliana cotyledons infected by Albugo candida, causal agent of white blister. After successful entry through stomatal pores, the pathogen developed a substomatal vesicle and subsequently produced intercellular hyphae. TEM observations revealed that coenocytic intercellular hyphae ramified and spread intercellularly throughout the host tissue forming several haustoria in host mesophyll cells. Intracellular haustoria were spherical and 4.5 μm in diameter. Each haustorium was connected to intercellular hyphae by a narrow, slender haustorium neck. The cytoplasm of the haustorium included the organelles characteristic of the pathogen. No obvious response was observed in host cells following formation of haustoria. Most of the mesophyll cells contained normal haustoria and the host cytoplasm displayed a high degree of structural integrity. Absence of host cell wall alteration and cell death in penetrated host cells suggest that the pathogen exerts considerable control over basic cellular processes and in this respect, response to this biotrophic Oomycete differs considerably from responses to other pathogens such as necrotrophs. Modification of the host plasma membrane (PM) along the cell wall and around the haustoria, was detected by applying the periodic acid-chromic acid-phosphotungstic acid (PACP) staining technique. After staining with PACP, the host PM was found to be intensely electron dense where it was adjacent to the host cell wall and the distal region of the haustorial neck. By contrast, the extrahaustorial membrane, where the host PM surrounded the haustorium, was consistently very lightly stained.     

Influence ofRhizobium leguminosarum biovartrifolii host specific nodulation genes on the ontogeny of clover nodulation

Summary The infection of white clover seedlings byRhizobium strains with different host range properties was assessed using various microscopic techniques. Several wild-type andRhizobium leguminosarum biovarvicias hybrid strains containing definedR. l. bv.trifolii host range genes were used. The morphological changes in the root tissue of uninoculated and rhizobia inoculated white clovers were identified and compared. In particular, changes were observed in the induction of inner cortical cell division, alterations to nodule development and lateral root formation. The responses of the infected roots and the types of structures formed support the hypothesis that lateral roots and nodules may be physiologically homologous structures. To establish a normal pattern of nodulation on white clover roots, both sets of known host specific nodulation genes (operonsnod FERL andnod MNX) ofR. l. bv.trifolii were required. However, some nodule development occurred when only thenod FERL genes were present in the hybrid strain.     

Recognition of N-acetylchitooligosaccharide elicitor by rice protoplasts

The response by rice protoplasts to N-acetylchitooligosaccharide elicitor was examined by monitoring the production of reactive oxygen species (ROS), and the expression of the two early-responsive genes, EL2 and EL3. Freshly prepared rice protoplasts produced a high level of ROS in the absence of the elicitor, and did not show further increase of the ROS generation in response to N-acetylchitooligosaccharide elicitor. By incubating protoplasts for 1 d, the background level decreased and the induction of ROS production and the induction of mRNAs for the two genes were observed. The structural requirements of N-acetylchitooligosaccharides for elicitor-activity, as well as the effects of inhibitors of protein kinase (K-252a), protein phosphatase (calyculin A) and protein synthesis (cycloheximide) on the ROS production and gene expression were very similar to those observed in suspension-cultured rice cells, indicating that rice protoplasts retain the machinery for the recognition of, and initial signaling from, N-acetylchitooligosaccharide elicitor.