The Infection of Trifolium subterraneum Root Hairs by Rhizobium trifolii

Both host cultivar and Rhizobium strain influence the numberof infected root hairs of Trifolium subl-errctneum, seedlings;Yarloop had fewer infections than Cranmore, Mount Barker, orTallarook and Rhizobium trifolii strain 5 infected fewer hairsthan strain TA1. Hybrid lines bred for sparse or abundant nodulationhad similar numbers of infected hairs, but. as in the cultivars,these always greatly exceeded the number of nodules formed.More infection threads aborted early during growth in the roothairs of Cranmore than in other hosts and early abortion wasmore common with strain 5 than strain TA1 In all hosts and with both Rhizobium strains, infection beganon day 3 and was initially restricted to one or two zones alongthe root with later infections extending these zones or initiatingnew ones. The exponential rate of infection (least for Yarloop)slows sharply when nodules appear. Early nodules and lateral roots formed at different places indifferent hosts, and in most cultivars and hybrid lines nodulesand laterals occurred in mutually exclusive zones. Primordiaarising above the first nodule failed to develop.     

Rhizobium-stimulated Callose Formation in Clover Root Hairs and its Relation to Infection

Callose was detected in the cell walls of the tips of growingroot hairs of Trifolium species and the non-legume Phleum pratenseusing u.v. fluorescence of fresh material stained with 0·005%aniline blue. Inoculation of the roots with Rhizobium trifolii,R. leguminosarum, R. meliloti, and R. japonicum, or additionof 10^–7 and 10^–8 M indole-3-acetic acid (IAA) increasedtip callose formation. Most tip callose was formed at 12 °C, and amounts declinedprogressively at 18, 24, and 30 °C, with very little formedat 36 °C. Tip calloso usually became less and disappearedin individual root hairs as they aged. Callose which appeared prominently in the host cell walls atthe points of initiation of infection threads did not usuallydisappear as the hairs matured. There was little or no extensionof callose along the infection thread and none in the threadtip or in the cell nucleus. Presumptive regions of callose hadsimilar structure and electron density as root hair wall materialand were sometimes related to arrays of vesicles in the hostcytoplasm. The external surface of the hair wall bore smallpegs or papillae (0·1–0·2 µm) continuouswith the outer layer of the wall and possibly associated withattachment of bacteria. Bacteria were usually umboriate at thepoint of attachment and their polyphosphate granules were muchlarger near the root hair than at the distal end.     

Studies on the Characterization of Root Morphology of White Clover Infected by Transconjugant of Rhizobium leguminosarum

White clover plants were inoculated with transconjugant strain' 290 which was obtained from introduction of host specific nodulation genes of wild-type Rhizobium trifolii strain ANU 843 to Rhizobium leguminosarum strain 300. The characterization of root morphology of white clover induced by the transconjugant was observed and compared to the plants induced by the parent strains. White clover started tO form a typical root hair curling inoculated with transconjugant strain 290 24h after inoculation, at 48h a part of cell wall of root hair was degradated, infection thread was observed in the infected root hair cell, cortical cell divisions occurred extensively. All these characterizations were similar to that infected by strain ANU 843. Plant inoculation test indicated that no nodule was formed when inoculated by R. leguminosarum strain 300, while plants nodulated when inoculated with transconjugant strain 290 as well as R. trifolii ANU 843. This suggests that introduction of host specific nodulation genes of R. trifolii results in conferring the nodulation ability of R. leguminosarum on white clover.     

Study on Chemosensitive Hairs in Wolf Spiders (Araneae, Lycosidae) by Scanning Electron Microscopy

The occurrence of chemosensitive hairs in some lycosid species is examined. The distribution and shape of such hairs dorsally on the male subadult palpal tarsus and the cymbium are compared. In certain species (e.g. of the genera. Pardosa and Alopecosa) the cymbium has a dense covering of these hairs while there are rather few of these in the male subadult palpal tarsus. The cymbium of Aulonia atbimana has comparatively few chemosensitive hairs dorsally, a condition supposedly related to the web-building habit in this species. Indications are given that the cymbial chemosensitive hairs are involved in the detection of presumed contact sex pheromones produced by the female. Certain intergeneric differences in the shape of the apical part of the cymbial chemosensitive hairs are noted.     

Scanning Electron Microscopy of the Infection Process of Cercospora henningsii on Cassava Leaves

Germination, penetration and sporulation of Cercospora henningsii (Allesch.) on cassava leaves were studied by scanning electron microscopy. Conidia started to germinate 9 h postinoculation producing one to two germ tubes. The germ tubes entered the leaf tissue through the abaxial surface by direct penetration of the epidermis without forming appressoria. The cassava leaf is characterized by its papillose epidermis on the abaxial surface. The penetrations occurred at smooth areas of the leaf epidermis between the papillae. The germ tubes did not enter stomata even when they passed over stomatal openings. Leaf spots started to appear 9 days after the inoculation (dpi), and the emergence of conidia occurred 14 dpi. The symptoms appeared first on the abaxial leaf surface, followed 2 days later on the adaxial. Conidia emerged in clusters through ruptured epidermis on both sides of the leaves. Conidia emerged also through the epidermal papillae and the leaf veins. Even though small groups of conidia emerged through stomata also, emergence through stomata appeared to be random rather than a preferred route. Each conidium was born on a short conidiophore with a swollen base.     

Scanning Electron Microscopy of the Infection of Beans by Rhizoctonia solani Propagules

Germination, hyphal spread, and formation of infection cushionsand lesions on bean hypocotyls by infective large propagulesof Rhizoctonia solani were studied with the scanning electronmicroscope. The surface of old, but not of young, hyphae appearedtypically wrinkled and shrunken. Infection cushions appearedas a network of several layers of crossed anastomosing hyphae.These features could not be observed under the light microscope.No differences in the surfaces of infective and non-infectivesmall propagules could be observed. Non-infective propagulesgerminating on the hypocotyls gave rise to spreading hyphaebut did not develop infection cushions.     

Succinate-Induced Morphology of Rhizobium trifolii 0403 Resembles That of Bacteroids in Clover Nodules

Morphological changes which accompany nutrient enrichment of Rhizobium trifolii 0403 were studied. Assays of cell number and size coupled with scanning electron microscopy and immunofluorescence microscopy showed that succinate induces cells to stop dividing in vitro and to swell either in the cell center or at one cell pole. The extent and frequency of in vitro cell swelling were in direct relation to the concentration of succinate added to the enrichment medium. The in vitro swelling of cells in 16.6 mM succinate plus Casamino Acids, glucose, and yeast extract closely resembled that of bacteroids of R. trifolii 0403 in nitrogen-fixing nodules of white clover. We hypothesize that succinate may be involved in the transformation of vegetative bacteria into the bacteroid morphology found in nitrogen-fixing nodules.     

Mode of Infection of Phyllosticta maculata on Banana as Revealed by Scanning Electron Microscopy

The initial infection stages of Phyllosticta maculata on banana were studied using scanning electron microscopy. Conidial germination on the banana leaf surface commenced within 3 h postinoculation to produce a long and slender germ tube. The hyphae developed secondary branches and mostly grew randomly across the leaf surface. Appressoria were formed at the apex of the germ tubes within 18 h postinoculation and were variable in shape. A layer of an extracellular matrix surrounded the appressoria at the pathogen–host interface. On the fruit surface, conidia germinated to produce predominantly swollen germ tubes which functioned as lateral appressoria together with some slender ones. These germ tubes were formed within 3 h postinoculation. There was no stomatal penetration apparent on the leaf; instead, direct penetration through the cuticle with and without the formation of appressoria was observed. Cuticular degradation on the leaf surface was evident with a circular, darkened area around the point of penetration by hyphae or appressoria. The significant role of pycnidia and conidia in the epidemiology of the disease was further demonstrated in naturally infected leaf samples.     

Effects of Resistance in White Clover (Trifolium repens) on Heterodera trifolii

Clones of two partially resistant and two susceptible white clover, Trifolium repens, genotypes were exposed to eggs of Heterodera trifolii and nematode development in stained roots measured at 2, 4, 7, 11, 18, 23, and 37 days after inoculation. The differences in development between nematode populations in resistant and susceptible genotypes showed that resistance operated after infection during feeding and development. At 7 days after inoculation, counts of second-stage juveniles did not differ between genotypes, whereas at 37 days more adults had developed in the susceptible than in the resistant genotypes. In a separate experiment, cysts hosted by susceptible genotypes were larger and contained more eggs than those on resistant genotypes so that the product of the values for cysts per plant and for eggs per cyst resulted in a more sensitive measure of resistance than from using cysts per plant alone.     

Scanning Electron Microscopy of Budding and Metamorphosis in Discophrya collini (Root)*

SYNOPSIS. Budding and metamorphosis in the suctorian ciliate, Discophrya collini, have been investigated by scanning electron microscopy. The adult body form, tentacles, stalk, and attachment disk are described. A field of depressions or small pits was observed in the pellicle of adult suctorians in the early stages of bud formation. These pits deepen and coalesce until one large pore, the birth pore, remains. Cilia protrude through the pore, and as eversion of the bud proceeds the meridional arrangement of the larval ciliation is evident. After eversion is completed, a pronounced division furrow is found between the adult and soon-to-be-released swarmer. The stalk-forming region is seen on swarmers. Metamorphosing swarmers produce tentacles upon settling before any indication of ciliary resorption. Resorption of cilia and change in body form occur progressively with the production of the attachment disk and stalk.     

Scanning Electron Microscopy of Plant Roots

A glycol methacrylate infiltration and polymerization techniquewas used to prepare clover roots inoculated with Rhizobium forscanning reflection electron microscopy. Root hairs and epidermalcells were coated with many bacteria; some bacteria seemed tobe embedded in the wall surface. Root hair tips were often smoothbut some older root hair surfaces showed a fibrillar meshworkpattern. Small granules c. 0.18 µm diameter were presenton the root hair and epidermal cell walls. The root cap, someroot hairs, and some epidermal cells were covered by an amorphousfilm thought to be the mucigel.     

Scanning Electron Microscopy of Bacterial Colonies

A technique is described for observing bacterial colony growth. Bacillus cereus, B. subtilis, and B. cereus var. mycoides were grown on strips of dialysis membrane layered on nutrient agar. Microcolonies of the organisms on strips were fixed in Formalin vapor in situ; the strips then were removed from the agar and secured to scanning microscope specimen stubs without markedly disturbing the cellular arrangement. Scanning electron micrographs clearly depict morphology of individual cells, as well as the spatial orientation of cells within the colony. This technique is reproducible, adaptable, and simple.     

Scanning Electron Microscopy of Thermoplasma acidophilum

The scanning electron microscope was utilized to observe the morphology of the thermophilic, acidophilic mycoplasma, Thermoplasma acidophilum. Upon examination of the surface morphology, the size and shape of this unusual mycoplasma revealed its similarity to the other mycoplasmas that have been investigated.     

A Structural Comparison of the Acidic Extracellular Polysaccharides from Rhizobium trifolii Mutants Affected in Root Hair Infection

The structures of the acidic extracellular polysaccharides (EPSs) from several R. trifolii mutants were compared by examining their compositions and their sugar linkages as determined by methylation analysis. These mutant strains were derived from the wild-type R. trifolii ANU843 and were unable to induce normal root hair curling (Hac- phenotype) or nodulation response (Nod- phenotype) in clover plants. These strains included several transposon Tn5-induced Nod-mutants, strain ANU871, which possesses a 40 to 50 kilobase deletion of the resident Sym plasmid, and strain ANU845 which is missing the Sym plasmid (pSym-). Strains ANU845(pSym-) containing either plasmid pRt150 or pBR1AN were also used. The recombinant plasmid pRt150 restores only root hair curling capacity to ANU845 while plasmid pBR1AN (an R. trifolii pSym) restores both root hair curling and nodulation capacity to this strain. Our composition and methylation results show that the EPSs from all these strains have the same glycosyl and pyruvyl linkages. Thus we suggest that neither the nod genes involved in root hair curling nor the entire pSym encodes for the arrangement of glycosyl or pyruvyl residues in these EPSs. Whether or not the nod genes dictate the location of acetyl or β-hydroxybutyrate substituent groups remains to be determined.     

Root Hair Deformations Associated with Fractionated Extracts from Rhizobium trifolii

Components from culture fluid and whole cells of Rhizobium trifolii were examined for effects on root hair morphology of white clover seedlings (Trifolium repens var. Ladino). Cell-free culture fluid, exopolysaccharides, supernatant fluid from the precipitation of the exopolysaccharides, capsular polysaccharides, lipopolysaccharides, and a protein fraction from culture fluids were assayed for morphogenetic effects on the root hairs of axenically grown clover seedlings. Crude fractions were chromatographed on Bio Gel A-5m (Bio-Rad Laboratories), and fractions collected were similarly assayed. Hexose, uronic acid, and protein concentrations were determined for all fractions assayed. Gel chromatography indicated the materials with deforming ability to be of high molecular weight (>10,000). For all fractions except exopolysaccharide, deforming ability was associated with a protein component. This suggested that two components were associated with deformation; both contained polysaccharides and one contained protein. Crude fractions differed in their ability to cause deformations and indicated the following relative ability (in decreasing order) to deform root hairs: cell-free culture fluid, capsular polysaccharides, protein from culture fluids, exopolysaccharide, and cell envelope. Lipopolysaccharides had no effect.     

Transmission Electron Microscopy of Tissue Prepared for Scanning Electron Microscopy by Ethanol-Cryofracturing

Tissue processed for scanning electron microscopy by ethanol-cryofracturing combined with critical point drying was embedded and sectioned for transmission electron microscopy. Study of sections cut in a plane passing through the fracture edge indicated that preservation of cellular fine structure of fractured cells was excellent. Even at the most peripheral edge of the fracture there was no evidence that movement of cytoplasmic components occurred to distort the original structural organization of fractured cells. Lack of cytoplasmic detail in ethanol-cryofractographs has been due more to the nature of the fracturing of the tissue and to the obscuring effects of the metal coating than to structural deformation at the fracture edge or to limitations in resolving power of the scanning electron microscope used.