Quantification of Wautersia [Ralstonia] basilensis in the mycorrhizosphere of Pinus thunbergii Parl. and its effect on mycorrhizal formation

The bacterium Wautersia [Ralstonia] basilensis has been shown to enhance the mycorrhizal symbiosis between Suillus granulatus and Pinus thunbergii (Japanese black pine). However, no information is available about this bacterium under field conditions. The objectives of this study were to detect W. basilensis in bulk and mycorhizosphere soils in a Japanese pine plantation in the Tottori Sand Dunes, determine the density of W. basilensis in soil, and determine the optimal cell density of W. basilensis for mycorrhizal formation in pine seedlings. We designed and validated 16S rRNA gene-targeted specific primers for detection and quantification of W. basilensis. SYBR Green I real-time PCR assay was used. A standard curve relating cultured W. basilensis cell density (10³-10⁸ cells ml⁻¹) to amplification of DNA showed a strong linear relationship (R = 0.9968). The specificity of the reaction was confirmed by analyzing DNA melting curves and sequencing of the amplicon. The average cell density of W. basilensis was >4.8 × 10⁷ cells g⁻¹ of soil in the mycorrhizosphere and 7.0 × 10⁶ cells g⁻¹ in the bulk soil. We evaluated the W. basilensis cell density required for mycorrhizal formation using an in vitro microcosm with various inoculum densities ranging from 10² to 10⁷ cells g⁻¹ soil (10⁴-10⁹ cells ml⁻¹). Cell densities of W. basilensis of >10⁶ cells g⁻¹ of soil were required to stimulate mycorrhizal formation. In vivo and in vitro experiments showed that W. basilensis was sufficiently abundant to enhance mycorrhizal formation in the mycorrhizosphere of Japanese black pine sampled from the Tottori Sand Dunes.     

Trophic transfer of fatty acids from gut microbiota to the earthworm Lumbricus terrestris L

The diet of earthworms includes soil organic matter, soil microbes and other microfauna, but the relative contribution of these dietary components to earthworm nutrition is not well known. Analysis of fatty acid (FA) profiles can reveal trophic relationships in soil food webs, leading to a better understanding of the energy and nutrient flows from microbiota to earthworms. The objective of this study was to determine the origin of FAs assimilated by the earthworm Lumbricus terrestris L. We analysed the pattern of FAs in: (i) the bulk soil, (ii) soil in the earthworm gut, (iii) the absorptive tissue of the earthworm gut wall, and (iv) the muscular layers of the earthworm body wall. Multivariate analyses performed on the FA profiles suggest that the microbial community in the earthworm gut differs from that in bulk soil. Diverse bacterial and fungal derived FAs, which earthworms cannot synthesize, were found in the earthworm gut wall and body wall, and in the neutral lipids (storage lipids) of the gut wall. The major compounds isolated were 20:4ω6, 20:5ω3 and 18:2ω6, followed by the monoenoic 18:1ω7 and 18:1ω9c, and the saturated 18:0. The microbial FA assemblage in the gut wall resembled the gut soil more than the bulk soil, and the body wall of L. terrestris showed the same microbial derived FA pattern as the gut wall, although at reduced concentrations. We propose the existence of a specific microbial community in the earthworm gut that provides FAs to the earthworm. It appears that L. terrestris may derive more of its energy and nutrients from gut specific microbiota than from microbiota already present in the ingested soil, based on the trophic relationships revealed through FA analysis.     

Development of an immunological technique for estimating mycelial biomass of Mycena galopus in leaf litter

Antisera were produced in rabbits against cell wall and cytoplasmic components of the mycelium of a litter-decomposing basidiomycete, Mycena galopus (Pers. ex Fr.) Kummer. The antisera were conjugated with fluorescein isothiocyanate, and positive reactions were obtained in direct and indirect fluorescent antibody (FA) tests with homologous antigens and with heterologous antigens of M. galopus from pure cultures and field material. Cross reactions occurred with fungal associates of M. galopus on Quercus litter from a deciduous woodland, but these were mostly removed by absorption with a preparation of Cladosporium herbarum Link ex Fr. Membrane filtration combined with FA staining and rhodamine-gelatin counter-staining was found to be a feasible means of estimating the biomass of FA-reactive mycelium of M. galopus in leaf litter.     

Arbuscular mycorrhizal colonisation increases copper binding capacity of root cell walls of Oryza sativa L. and reduces copper uptake

There is evidence that colonisation by mycorrhizal fungi can protect host plants from toxic concentrations of heavy metals. The mechanism by which protection is provided by the fungus for any particular metal is poorly understood. Rice (Oryza sativa L.) plants were inoculated with Glomus mosseae and grown for 4 weeks to ensure strong colonisation. The plants were then exposed to low to toxic concentrations of copper (Cu) and the uptake and distribution were examined. The effect of mycorrhizal colonisation on the cell wall composition and Cu binding capacity of roots was also investigated. Mycorrhizal plants showed moderate reductions in Cu concentrations in roots but large reductions in shoots. In roots, mycorrhizal plants accumulated more Cu in cell walls but much less in the symplasm compared to non-mycorrhizal plants. The differences in cell wall binding of Cu could be partly explained by changes in the composition of the cell wall. The mechanistic basis for the reduced Cu accumulation and the potential beneficial consequences of mycorrhizal associations on plant growth in Cu toxic soil are discussed.     

Potential gene exchange between Bacillus thuringiensis subsp. kurstaki and Bacillus spp. in soil in situ

The possible transfer of genes from Bacillus thuringiensis subsp. kurstaki (Btk) to indigenous Bacillus spp. was investigated in soil samples from stands of cork oak in Orotelli (Sardinia, Italy) collected 5 years after spraying of the stands with a commercial insecticidal preparation (FORAY 48B) of Btk. Two colonies with a morphology different from that of Btk were isolated and identified as Bacillus mycoides by morphological and physiological characteristics and by 16S rDNA analysis. Amplification by the polymerase chain reaction (PCR) of the DNA of the two isolated B. mycoides colonies with primers used for the identification of the Btk cry genes showed the presence of a fragment of 238 bp of the cry1Ab9 gene that had a similarity of 100% with the sequence of the cry1Ab9 gene present in GenBank, indicating that the isolates of B. mycoides acquired part of the sequence of this gene from Btk. No cells of Btk or B. mycoides carrying the 238-bp fragment of the cry1Ab9 gene were isolated from samples of unsprayed control soil. However, the isolates of B. mycoides were not able to express the partial Cry1Ab protein. Hybridization with probes for IS231 and the cry1Ab9 gene suggested that the inverted repeated sequence, IS231, was probably involved in the transfer of the 238-bp fragment from Btk to B. mycoides. These results indicate that transfer of genes between introduced Btk and indigenous Bacillus spp. can occur in soil under field conditions.     

Parasitism of sclerotia of Sclerotium rolfsii by trichoderma harzianum

The ability of Trichoderma harzianum isolate 203 to attack the soil-borne plant pathogen Sclerotium rolfsii is apparently connected with the production by the isolates of chitinase and β-(1,3)-glucanase inside the attacked sclerotia during parasitism.SEM and TEM micrographs show that the mycoparasite degraded walls of sclerotial cells and the attacked cells lost their cytoplasmic contents. It is assumed that T. harzianum utilizes sclerotial cell contents thus enabling it to sporulate intensively on the sclerotial surface and inside the digested cells.     

Electron microscopical studies of chlamydospores of Fusarium solani F. cucurbitae formed in natural soil

Macroconidia of Fusarium solani f. cucurbitae were placed into natural soil, incubated for various times, recovered and examined by light and transmission electron microscopy. Lysis of macroconidia and formation of chlamydospores were studied and the fine structure of these propagules and their associated microflora was investigated. The two most obvious features of chlamydospore morphology were the sloughing of outer wall layers and the accretion of micro-fibrillar elements adjacent to the plasmalemma. Chlamydospore formation was partially suppressed by addition of nitrogenous compounds to the soil.     

Micromonospora: An important microbe for biomedicine and potentially for biocontrol and biofuels

Micromonospora species have long been recognized as important sources of antibiotics and also for their unusual spores. However, their involvement in plant-microbe associations is poorly understood although several studies demonstrate that Micromonospora species function in biocontrol, plant growth promotion, root ecology, and in the breakdown of plant cell wall material. Our knowledge of this generally understudied group of actinomycetes has been greatly advanced by the increasing number of reports of their associations with plants, by the deployment of DNA cloning and molecular systematics techniques, and by the recent application of whole genome sequencing. Efforts to annotate the genomes of several Micromonospora species are underway. This information will greatly augment our knowledge of these versatile microorganisms.     

Fine structure of a new mycophagous amoeba and its feeding on Cochliobolus sativus

An unidentified mycophagous soil amoeba is described. The pigmented soil-borne fungus Cochliobolus sativus and four other fungal species, both pigmented and hyaline, were utilized as food. Spores were ingested and lysed within digestive vacuoles by general wall erosion. This contrasts with the wall perforation mechanism described for other mycophagous amoebae. Ultrastructural studies of trophozoites showed that large quantities of electron dense granules were released into the digestive vacuoles during fungal cell lysis. These were incorporated into the amoebal protoplast. Bacteria were commonly present in the amoebal protoplasts and within digestive vacuoles. Their possible role as endosymbionts is discussed.     

Genetic diversity among Elaeagnus compatible Frankia strains and sympatric-related nitrogen-fixing actinobacteria revealed by nifH sequence analysis

Elaeagnus compatible Frankia isolates from Tunisian soil have been previously clustered with Frankia, colonizing Elaeagnaceae and Rhamnaceae in two different phylogenetic subgroups, while strain BMG5.6 was described as a new lineage closely related to Frankia and Micromonospora genera. In this study we further assess the diversity of captured Frankia and the relationship with BMG5.6-like actinobacteria, by using nifH gene sequences. Using PCR-RFLP screening on DNA extracted from lobe nodules, additional microsymbionts sharing BMG5.6 features have been detected proving a widespread occurrence of these actinobacteria in Elaeagnus root nodules. Neighbour-Joining trees of Frankia nifH sequences were consistent with previously published 16S rRNA and GlnII phylogenetic trees. Although four main clades could be discerned, actinobacterial strain BMG5.6 was clustered with Frankia strains isolated from Elaeagnus. The present study underscored the emanation of new diazotrophic taxon isolated from actinorhizal nodules occupying intermediate taxonomic position between Frankia and Micromonospora. Moreover, its aberrant position in nifH phylogeny should open network investigations on the natural history of nitrogen-fixing gene among actinobacteria.     

Mycoparasitism by Pythium oligandrum and P. acanthicum

Isolates of the reported mycoparasites Pythium oligandrum, P. acanthicum and P. periplocum markedly reduced growth and cellulolysis by Botryotrichum piluliferum, grew rapidly across agar plates precolonized by Phialophora radicicola var radicicola (sensu Deacon) and, where tested (not P. periplocum), were non-pathogenic towards higher plants. Isolates of P. echinulatum, P. mamillatum. P. megalacanthum, P. spinosum, P. ultimum and one isolate of P. acanthicum behaved differently from the mycoparasites and could, themselves, be placed in two groupings in these tests. It is suggested that the ability or otherwise to grow on Phialophora-precolonized agar plates may help to distinguish broad biological groupings within the genus Pythium, but these groupings may cut across conventional taxonomic ones.One isolate of P. acanthicum was tested for its effects on a range of cellulolytic fungi: it reduced their growth to different extents, as did P. oligandrum.Plates of potato-dextrose agar precolonized by Phialophora radicicola were used to isolate selectively P. oligandrum and similar fungi from soils, but the use of hemp seed baits in conjunction with precolonized plates was less selective for these fungi.Straw pieces precolonized by P. oligandrum and buried in soil decomposed at the same rate as virgin straws or those precolonized by P. ultimum or Mucor hiemalis. Subsequently, Stachyholrys atra appeared to colonize straws more frequently from soil, and Fusarium spp. less so, in the presence of P. oligandrum than in its absence. In the laboratory, P. oligandrum was antagonized by Slachyholrys, whereas Fusarium spp. were frequently overgrown by the Pythium.     

In vitro effects of Fusarium blight-controlling fungicides on pathogens of Poa pratensis

An experimental iprodione fungicide,3-(3,5dichlorophenyl)-N-(1-methylethyl)-2,4-dioxo-1-imidazolidinecarboxamide, controls Fusarium blight of Kentucky bluegrass (Poa pratensis L.), but it also amplifies the proportion of crowns colonized by Fusarium and the number of its propagules in soils. In contrast, the disease, the proportion of infected crowns, and the numbers of propagules in soil are generally suppressed by benomyl, methyl 1-(butylcarbamoyl)-2-benzimidazolecarbamate. Triadimefon, 1-(4-chlorophenoxy)-3,3-dimethyl-1-(1H-1,2,4-triazol-1-yl)-2-butanone, also controls the disease but is not stimulatory or inhibitory of fusaria. Iprodione and benomyl were studied for their effects on growth and sporulation of Fusarium acuminatum isolated from diseased crowns; iprodione had no or slightly stimulatory effects, and benomyl greatly suppressed these processes, except in a benomyl-tolerant strain.Toxicities of iprodione and benomyl to 1555 identified Fusarium isolates from Kentucky bluegrass turf were determined, as were the toxicities of iprodione to 23 turfgrass pathogens. Of the Fusarium spp, only F. solani was significantly inhibited by iprodione, whereas all were inhibited by benomyl. Iprodione-sensitive fungi included species of Bipolaris, Corticium, Curvularia, Drechslera, Rhizoctonia, Sclerotinia, and Typhula. Insensitive fungi included Colletotrichum, Fusarium, Gaeumannomyces, and Pythium.Investigations with selective fungicides indicate that the primary causal agent of Fusarium blight is not among the fusaria, and that re-interpretation of the disease and its etiology is necessary.     

Hyperparasitism of oospores of Phytophthora megasperma var. sojae

Hyperparasites of oospores of Phytophthora megasperma Drechs. var. sojae Hildb. were present in each of 15 field soils tested. Maximum numbers of oospores parasitized ranged from 42.5 to 87.5% for flooded soils, and from 25.5 to 73.0% for soils adjusted to 50% water holding capacity; the mean for all soils was 51.5%. The frequency of hyperparasitism was not correlated with the disease potential soils for Phytophthora root-rot of soybean as determined in seedling tests on flooded soil samples. Of eight isolated hyperparasitic fungi tested in steamed soil, the most efficient parasites were Hyphochytrium catenoides, Humicola fuscoatra, and Pythium monospermum, each of which parasitized at least 76% of oospores during 3 weeks. Hyphae were not parasitized by any of the eight fungi. Parasitism by H. catenoides in sterilized soil increased as soil temperature increased from 16° to 28°C. Parasitism by P. monospermum was maximum at 20°–24°C. Oospores of P. meyasperma var. sojae race 7 were more resistant to infection by hyperparasites than were oospores of races 1 and 3. Oospores produced in culture were slightly more susceptible to hyperparasitism in soils than were oospores produced in soybean seedlings.     

Non-pathogenic Fusarium strains protect the seedlings of Lepidium sativum from Pythium ultimum

Two root-colonizing Fusarium strains, Ls-F-in-4-1 and Rs-F-in-11, isolated from roots of Brassicaceae plants, induced the resistance in Lepidium sativum seedlings against Pythium ultimum. These strains caused an increase in the content of benzyl isothiocyanate, and of its precursor glucotropaeolin, in the roots of the host plants. The increased isothiocyanate content is one of the factors contributing to the resistance of L. sativum against P. ultimum. To be transformed into the fungitoxic compound benzyl isothiocyanate, glucotropaeolin has to be hydrolyzed by myrosinase, which can be produced either by plants or microorganisms. The Fusarium strain Ls-F-in-4-1 has a myrosinase activity but the strain Rs-F-in-11 has not. These results suggest that both strains are able to trigger the metabolic pathway leading to benzyl isothiocyanate production in the plant. In the case of the myrosinase-negative strain Rs-F-in-11, hydrolyzation into isothiocyanate is only due to the myrosinase activity of the plant, and in the other case, the myrosinase produced by the strain Ls-F-in-11 also would contribute to the production of isothiocyanate. This paper reports a new mode of action of non-pathogenic Fusarium strains in controlling P. ultimum.     

Competition between species of Rhizobium for nodulation of Glycine max

Glycine max cv. Malayan is a promiscuously nodulating cultivar which formed nodules with 6 out of 9 strains of Rhizobium spp of diverse origin and all strains of R. japonicum tested. No generalizations can be made as to the probability of strains isolated from a particular host being infective on Malayan as only some isolated from Centrosema pubescens, and Cajanus cajan were able to form nodules. In competition with R. japonicum at 30°C all 20 strains of Rhizobium spp isolated from Malayan grown in Nigeria formed fewer than 50% of the nodules and 14 strains fewer than 25%. Competition was influenced by root temperature. Three strains of Rhizobium spp were poor competitors with R. japonicum between 24° and 33°C but at 36°C they formed more nodules (74–88%) than R. japonicum. Another strain of Rhizobium spp formed the majority of the nodules between 27° and 36°C whereas R. japonicum formed the most at 24°C.     

Density loss and respiration rates in coarse woody debris of Pinus radiata, Eucalyptus regnans and Eucalyptus maculata

This study compared field and laboratory decomposition rates of coarse woody debris (CWD) (>10 cm diameter) from three tree species: Pinus radiata, Eucalyptus regnans, and Eucalyptus maculata. For this purpose, the density loss of logs on the ground sampled from chronosequences of sites following harvesting was determined using the water replacement technique. P. radiata logs were sampled 1, 2.5, 6, and 9 years following harvesting, and logs of E. regnans and E. maculata were collected from sites that were harvested 1, 3.5, 6.5, and 12 and 1.5, 6.5, and 11.5 years ago, respectively. In addition, the C/N ratio of wood was determined and current respiration rates of logs from these different age classes were measured through laboratory incubation. The times for loss of 95% of material (t_0.95) determined from density loss for these species were 24 years for P. radiata, 43 years for E. regnans, and 62 years for E. maculata. The decomposition rates of CWD derived from laboratory respiration were 6.1, 5.9 and 11.9 times higher than the decay rates from density loss in P. radiata, E. regnans, and E. maculata, respectively. This points to severe constraints of decomposition through adverse conditions in the field. The changes in respiration rates and C/N ratio with age of decaying logs indicated that the single component, negative exponential decay model could be applied satisfactorily only to P. radiata. In the case of the eucalypt species, substrate quality (expressed through respiration rates) declined in the oldest samples. This may be explained by the loss of rapidly decomposing sapwood and the retention of more decay-resistant heartwood. In these cases, a two-component model will be more suitable to describe the density loss of decaying wood.     

Reduction of sclerotial inoculum of Sclerotinia sclerotiorum with Coniothyrium minitans

Aspects of the biology of C. minitans and its potential for control of S. sclerotiorum were investigated.Temperatures below 7°C resulted in comparatively slow rates of germination and infection of sclerotia by C. minitans. The optimum temperature for germination, growth, infection of sclerotia, and destructive parasitism by C. minitans was 20°C. The optimum relative humidity for germination, growth and infection by C. minitans was above 95%.Autumn inoculations with suspensions of conidia, pycnidia and mycelium of C. minitans in the field resulted in negligible numbers of sclerotia remaining viable after 1 month. With culture-grown sclerotia 2 months were required for a similar reduction of sclerotial viability. In the absence of C. minitans mulching had no significant effect on sclerotial viability. In the presence of C. minitans mulching did, however, influence the viability and infection by C. minitans of culture-grown sclerotia. Populations of field sclerotia also differed from culture-grown sclerotia in that they harboured an internal population of microorganisms, which included C. minitans, and had a lower level of viability at the commencement of the treatments.A winter application of C. minitans did not result in significant infection of sclerotia nor in a reduction in viability of sclerotia. This failure is believed to have resulted from low temperatures and dry conditions.     

Distribution of Pseudomonas populations harboring phlD or hcnAB biocontrol genes is related to depth in vineyard soils

The abundance and population structure of pseudomonads in soils collected from long-(1006 years) and short-(54 years) term grapevine monocultures in Switzerland were examined across five soil horizons within the 1.20-1.35 m range. Soil samples were baited with grapevine, and rhizosphere pseudomonads containing the biocontrol genes phlD (2,4-diacetylphloroglucinol synthesis) and/or hcnAB (hydrogen cyanide synthesis) were analyzed by MPN-PCR. The numbers of total, phlD⁺ and hcnAB⁺ pseudomonads decreased with depth by 1.5-2 log (short-term monoculture) and 3-3.5 log (long-term monoculture). In addition, the percentages of phlD⁺ (except in short-term monoculture) and hcnAB⁺ pseudomonads were also lower in deeper horizons. RFLP-profiling of phlD⁺ and hcnAB⁺ pseudomonads revealed three phlD and twelve hcnAB alleles overall, but the number of alleles for both decreased in relation to depth. The only phlD allele found in deeper horizons was also found in topsoil, whereas one hcnAB allele (k) found in deeper horizons in long-term monoculture was absent in the topsoil. This suggests that certain Pseudomonas ecotypes are adapted to specific depths. Four hcnAB alleles enabled discrimination between monocultures. We conclude that soil depth is a factor selecting phlD and hcnAB genotypes, and that the allelic diversity of the two biocontrol genes decreases with depth.     

Transgenic Bt plants decompose less in soil than non-Bt plants

Bt plants are plants that have been genetically modified to express the insecticidal proteins (e.g. Cry1Ab, Cry1Ac, Cry3A) from subspecies of the bacterium, Bacillus thuringiensis (Bt), to kill lepidopteran pests that feed on corn, rice, tobacco, canola, and cotton and coleopteran pests that feed on potato. The biomass of these transgenic Bt plants (Bt+) was decomposed less in soil than the biomass of their near-isogenic non-Bt plant counterparts (Bt−). Soil was amended with 0.5, 1, or 2% (wt wt⁻¹) ground, dried (50 °C) leaves or stems of Bt corn plants; with 0.5% (wt wt⁻¹) ground, dried biomass of Bt rice, tobacco, canola, cotton, and potato plants; with biomass of the near-isogenic plants without the respective cry genes; or not amended. The gross metabolic activity of the soil was determined by CO₂ evolution. The amounts of C evolved as CO₂ were significantly lower from soil microcosms amended with biomass of Bt plants than of non-Bt plants. This difference occurred with stems and leaves from two hybrids of Bt corn, one of which had a higher C:N ratio than its near-isogenic non-Bt counterpart and the other which had essentially the same C:N ratio, even when glucose, nitrogen (NH₄NO₃), or glucose plus nitrogen were added with the biomass. The C:N ratios of the other Bt plants (including two other hybrids of Bt corn) and their near-isogenic non-Bt counterparts were also not related to their relative biodegradation. Bt corn had a significantly higher lignin content than near-isogenic non-Bt corn. However, the lignin content of the other Bt plants, which was significantly lower than that of both Bt and non-Bt corn, was generally not statistically significantly different, although 10-66% higher, from that of their respective non-Bt near-isolines. The numbers of culturable bacteria and fungi and the activity of representative enzymes involved in the degradation of plant biomass were not significantly different between soil amended with biomass of Bt or non-Bt corn. The degradation of the biomass of all Bt plants in the absence of soil but inoculated with a microbial suspension from the same soil was also significantly less than that of their respective inoculated non-Bt plants. The addition of streptomycin, cycloheximide, or both to the soil suspension did not alter the relative degradation of Bt+ and Bt− biomass, suggesting that differences in the soil microbiota were not responsible for the differential decomposition of Bt+ and Bt− biomass. All samples of soil amended with biomass of Bt plants were immunologically positive for the respective Cry proteins and toxic to the larvae of the tobacco hornworm (Manduca sexta), which was used as a representative lepidopteran in insect bioassays (no insecticidal assay was done for the Cry3A protein from potato). The ecological and environmental relevance of these findings is not clear.     

Infection of wheat seminal roots by varieties of Phialophora radicicola and Gaeumannomyces graminis

The growth of isolates of Phialophora radicicola var. radicicola, P. radicicola var. graminicola, Gaeumannomyces graminis var. graminis, G. graminis var. tritici and Leptosphaeria narmari was compared on the coleoptiles and roots of wheat seedlings. Fungal growth was measured as the extent and density of dark runner hyphae. All except P. radicicola var. graminicola grew on coleoptiles and all grew on roots although only G. graminis var. tritici extensively colonized the root stele. Growth rate on roots was positively correlated with that on agar, P. radicicola var. graminicola and L. narmari growing at about half the rate of the other fungi; hyphal density was high for P. radicicola var. graminicola but relatively low for the other fungi. For P. radicicola var. radicicola, P. radicicola var. graminicola and G. graminis var. tritici growing from buried inocula, the extent and density of hyphae up roots towards the seed was similar to that down, but G. graminis var. tritici caused chocolate-brown stelar discoloration up roots only.Root invasion by P. radicicola var. radicicola, P. radicicola var. graminicola and G. graminis var. tritici was described from sections. Each gave a different pattern of hyphae and host response within an inoculum layer, and progressive changes occurred away from the inoculum. Studies of the rate of penetration by each fungus and the rate and pattern of death of cortical cells explained the differences between fungi. G. graminis var. tritici penetrated living cells in advance of other soil micro-organisms, and hence by hyaline hyphae inducing much lignituber formation as a host resistance reaction. P. radicicola var. graminicola penetrated only senescent or dead cells in association with other soil microorganisms, and hence by dark hyphae, inducing little lignituber formation. P. radicicola var. radicicola was intermediate in all these respects. The high hyphal density of P. radicicola var. graminicola was due to the colonization of cortical cells and spaces by dark, clearly visible, rather than hyaline hyphae, which are invisible in unstained roots. Cell death in the outer cortex explained the observed progressive restriction of growth by all fungi to the inner cortex with increasing distance from the inoculum. Spread by G. graminis var. tritici up roots was ectotrophic relative to the stele but down roots hyphae spread rapidly within the stele. Stelar reactions suggested as resistance mechanisms occurred up roots only. Their absence down roots is attributed to infection disrupting stelar transport.