Intracellular Hyphae Research Articles

Infection progress of arbuscular mycorrhizae on tissue-cultured plantlets of Pinellia ternata

To study the Arbuscular mycorrhizal (AM) formation progress and infection characteristics between tissue culture plantlets of Pinellia ternata and Glomus mosseae. The tissue culture plantlets of P. ternata were inoculated with G. mosseae, the formation of AM were sampled and observed with microscopy by staining. The hyphae of G. mosseae began to penetrate the root epidermis after 10 days of inoculation. Lots of intracellular hyphae formed in cortex cells at the 15th day. Arbuscules started to form and there were some hyphae on the root at the 20th day. At the 25th day, many arbuscules formed and most as Arum type. Some arbuscles started to disintegrate at the 30th day, and a few of vesicles occurred. Lots of spores formed after 35 days. At the 40th day, some vesicles began to decline. The hand section showed that the intercellular hyphae gradually formed in intercellular space, and the hyphae branched in cortex cells and occupied most cell lumen finally. It is expounded that P. ternata and G. mosseae could recognize each other quickly and form a symbiont system.

Arbuscular mycorrhizal fungal phylogeny-related interactions with a non-host

Arbuscular mycorrhizal fungi (AMF) form symbiotic relationships with most vascular plants including some gymnosperm species. Although species in the gymnosperm family Pinaceae normally develop ectomycorrhizal associations, AMF hyphae and vesicles, typical of members of the Suborder Glomineae, have been reported in the roots of some Pinaceae species. However, it is not known whether AMF belonging to various species and suborders are able to colonize roots of Pinaceae species and to what extent this influences the performance of Pinaceae seedlings. We tested in each of the Glomaceae, Acaulosporaceae and Gigasporineae AMF families two species for their ability to colonize and affect the growth of Pinus strobus (eastern white pine) in the presence or absence of an AMF host plant (Trifolium pretense—red clover). Glomus intraradices was the only AMF that colonized pine roots, predominantly in the presence of clover, forming intracellular hyphae and vesicles but not arbuscules. Colonization, however, did not relate to increased pine biomass and the overall presence of AMF, regardless of colonization abilities, resulted in a biomass reduction. This effect on pine seedling biomass was explained by the AMF family to which the AMF belonged, indicating that the effects of AMF on the non-host pine may be related to phylogeny. Acaulosporaceae species reduced pine biomass the most whereas, Gigasporineae species had the smallest effect on biomass. These preliminary results suggest that AMF may affect the soil microflora differently among AMF families in previously unsuspected ways with potential consequences for non-AMF host growth.

Identification of two conserved cis‐acting elements, MYCS and P1BS, involved in the regulation of mycorrhiza‐activated phosphate transporters in eudicot species

• In this study, six putative promoter regions of phosphate transporter Pht1;3, Pht1;4 and Pht1;5 genes were isolated from eggplant and tobacco using the inverse polymerase chain reaction (iPCR). The isolated sequences show evolutionary conservation and divergence within/between the two groups of Pht1;3 and Pht1;4/Pht1;5. • Histochemical analyses showed that all six promoter fragments were sufficient to drive β-glucuronidase (GUS) expression specifically in arbuscular mycorrhizal (AM) tobacco roots and were confined to distinct cells containing AM fungal structures (arbuscules or intracellular hyphae). • A series of promoter truncation and mutation analyses combined with phylogenetic footprinting of these promoters revealed that at least two cis-regulatory elements--the mycorrhiza transcription factor binding sequence (MYCS) first identified in this study and P1BS--mediated the transcriptional activation of the AM-mediated inorganic phosphate (Pi) transporter genes. Deletion or partial mutation of either of the two motifs in the promoters could cause a remarkable decrease, or even complete absence, of the promoter activity. • Our results propose that uptake of inorganic phosphate (Pi) by AM fungi is regulated, at least partially, in an MYCS- and P1BS-dependent manner in eudicot species. Our finding offers new insights into the molecular mechanisms underlying the coordination between the AM and the Pi signalling pathways.

The PAM1 gene of petunia, required for intracellular accommodation and morphogenesis of arbuscular mycorrhizal fungi, encodes a homologue of VAPYRIN

Most terrestrial plants engage into arbuscular mycorrhizal (AM) symbiosis with fungi of the phylum Glomeromycota. The initial recognition of the fungal symbiont results in the activation of a symbiosis signalling pathway that is shared with the root nodule symbiosis (common SYM pathway). The subsequent intracellular accommodation of the fungus, and the elaboration of its characteristic feeding structures, the arbuscules, depends on a genetic programme in the plant that has recently been shown to involve the VAPYRIN gene in Medicaco truncatula. We have previously identified a mutant in Petunia hybrida, penetration and arbuscule morphogenesis 1 (pam1), that is defective in the intracellular stages of AM development. Here, we report on the cloning of PAM1, which encodes a VAPYRIN homologue. PAM1 protein localizes to the cytosol and the nucleus, with a prominent affinity to mobile spherical structures that are associated with the tonoplast, and are therefore referred to as tonospheres. In mycorrhizal roots, tonospheres were observed in the vicinity of intracellular hyphae, where they may play an essential role in the accommodation and morphogenesis of the fungal endosymbiont.

Hudsonia ericoides and Hudsonia tomentosa: Anatomy of mycorrhizas of two members in the Cistaceae from Eastern Canada

Most species in the family Cistaceae are found in the Mediterranean basin. Several hosts are of special interest, owing to their associations with truffle species, while many are important as pioneer plants in disturbed areas and in soil stabilization. For these reasons, understanding their root systems and their associated fungal symbionts is important. Most studies of the structure of mycorrhizas in this family involve two genera, Cistus and Helianthemum . The present study examines structural features of mycorrhizas in two North American species, Hudsonia ericoides L. and Hudsonia tomentosa Nutt. Root systems of both species are highly branched with most fine roots colonized by mycorrhizal fungi. Based on morphological features, several mycorrhizal fungi were identified; structural details also provided evidence of more than one fungal symbiont for each host species. All mycorrhizas had a multi-layered fungal mantle and Hartig net hyphae confined to radially elongated epidermal cells; no intracellular hyphae were observed. Although the Hartig net was confined to the epidermis, the outer row of cortical cell walls lacked suberin, a known barrier to fungal penetration. Mycorrhizas in H. ericoides and H. tomentosa differed from those of Cistus and Helianthemum species that have a Hartig net that extends into the root cortex, as well as frequently present intracellular hyphae.

Rust fungi on Annonaceae: the genus Sphaerophragmium

Seven species of the rust genus Sphaerophragmium occur on members of the tropical plant family Annonaceae. Uropyxis gerstneri is recombined to S. gerstneri. A new species, S. xylopiae, is described from Xylopia acutiflora. The host plant of S. boanense is identified as Mitrella sp. Sphaerophragmium pulchrum is transferred to Dicheirinia. The anatomy of telia with teliospores and parasitizing mycelium is described and illustrated in detail. A new type of M-haustorium, which emanates laterally from intracellular hypha, is detected in S. monodorae. An identification key is given.

The infection biology of Fusarium graminearum: Defining the pathways of spikelet to spikelet colonisation in wheat ears

Fusarium graminearum is one of the main causal agents of Fusarium Ear Blight on wheat. How the pathogen colonises the entire ear is not known. There is controversy over whether this mycotoxin producing pathogenic fungus invades wheat floral tissue using a necrotrophic or another mode of nutrition. A detailed microscopic investigation has revealed how wild-type fungal hyphae, of the sequenced strain PH-1, colonised susceptible wheat ears and spread from spikelet to spikelet. At the advancing infection front, colonisation of the host cortex occurred ahead of any vascular colonisation and the hyphae adapted to the available intercellular space between host cells. Intercellular hyphae then became abundant and host cells lost their entire cellular contents just prior to intracellular colonisation. No host cells died ahead of the infection. However, while these deep cortex infections progressed, just below the surface the highly photosynthetic chlorenchyma cells were observed to have died prior to colonisation. Behind the infection front, hyphae were abundant in the vasculature and the cortex, often growing through the pit fields of thick walled cells. This high level of inter- and intracellular fungal colonisation resulted in the collapse of the non-lignified cell-types. In this middle zone of infection, hyphal diameters were considerably enlarged. Far behind the infection front inter- and intracellular hyphae were devoid of contents and had often collapsed. At later stages of infection, the pathogen switched from predominately vertical to lateral growth and accumulated below the surface of the rachis. Here the lignified host cell walls became heavily degraded and hyphae ruptured the epidermis and produced an aerial mycelium.

A glimpse into the past of land plants and of their mycorrhizal affairs: from fossils to evo‐devo

A glimpse into the past of land plants and of their mycorrhizal affairs: from fossils to evo‐devo

A locus conferring resistance to Colletotrichum higginsianum is shared by four geographically distinct Arabidopsis accessions

Colletotrichum higginsianum is a hemibiotrophic fungal pathogen that causes anthracnose disease on Arabidopsis and other crucifer hosts. By exploiting natural variation in Arabidopsis we identified a resistance locus that is shared by four geographically distinct accessions (Ws-0, Kondara, Gifu-2 and Can-0). A combination of quantitative trait loci (QTL) and Mendelian mapping positioned this locus within the major recognition gene complex MRC-J on chromosome 5 containing the Toll-interleukin-1 receptor/nucleotide-binding site/leucine-rich repeat (TIR-NB-LRR) genes RPS4 and RRS1 that confer dual resistance to C. higginsianum in Ws-0 (Narusaka et al., 2009). We find that the resistance shared by these diverse Arabidopsis accessions is expressed at an early stage of fungal invasion, at the level of appressorial penetration and establishment of intracellular biotrophic hyphae, and that this determines disease progression. Resistance is not associated with host hypersensitive cell death, an oxidative burst or callose deposition in epidermal cells but requires the defense regulator EDS1, highlighting new functions of TIR-NB-LRR genes and EDS1 in limiting early establishment of fungal biotrophy. While the Arabidopsis accession Ler-0 is fully susceptible to C. higginsianum infection, Col-0 displays intermediate resistance that also maps to MRC-J. By analysis of null mutants of RPS4 and RRS1 in Col-0 we show that these genes, individually, do not contribute strongly to C. higginsianum resistance but are both required for resistance to Pseudomonas syringae bacteria expressing the Type III effector, AvrRps4. We conclude that distinct allelic forms of RPS4 and RRS1 probably cooperate to confer resistance to different pathogens.

Basal stem rot of oil palm (Elaeis guineensis); mode of root infection and lower stem invasion by Ganoderma boninense

Reproducible infection of intact roots of oil palm (Elaeis guineensis) with Ganoderma boninense, the cause of basal stem rot, showed penetration followed by rapid longitudinal progression of hyphae and colonization of the lower stem (bole). Light and transmission electron microscopy showed invasion of the root cortex, with no evidence of selective progression through the vascular system or lacunae. In newly colonized tissue the fungus behaved as a hemibiotroph, with numerous, wide, intracellular hyphae occupying entire host cells that possessed intact cell walls and contained discernible cytoplasm and organelles. In the bole this phase coincided with a complete depletion of previously abundant starch grains in advance of invasion. Subsequently, in the roots and colonized stem base, widespread necrotrophic, enzymatic attack of all layers of the host cell walls occurred. Hyphae were intra‐ and intercellular and intramural and associated host cell wall degradation was often at a distance from hyphae, resulting in cavities within cell walls. A third developmental stage was the formation of an extensive, melanized, tough mycelium or pseudo‐sclerotium which surrounded roots and comprised many very thick‐walled cells encasing more typical thin‐walled hyphae. Macroscopic observation of and isolation from the bole of randomly felled, commercial palms provided confirmatory evidence that multiple infections originated in the roots before spreading into the base of long‐established palms.

CHARACTERIZATION OF INFECTION AND COLONIZATION OF STRAWBERRY CROWNS BY COLLETOTRICHUM ACUTATUM

Strawberry anthracnose, caused by Colletotrichum acutatum J.H. Simmonds, is one of the most important diseases of this crop in Huelva (southwestern Spain). Lesions can occur on all parts of the plant but anthracnose crown rot is specially severe leading to wilt and death of plants. In this work, the infection and colonization process of C. acutatum on strawberry plants was studied. 'Camarosa' strawberry plants (highly susceptible to C. acutatum) were inoculated by applying 100 μl droplets of a conidia suspension of the fungus on crowns, on buds and between second and third stipules. Plants were evaluated for response over a 30 days period. The severity of disease, estimated as percentage of plants showing symptoms on aerial parts, and the amount of infected crown tissues were determined at 2, 5,10, 15, 20, 25 and 30 days after inoculation. Depending on the inoculation site the severity of anthracnose crown rot varied between plants. Moreover, an ultrastructural study of infected plants exhibiting anthracnose crown rot was carried out by transmission electron microscopy. The colonization of the crown tissues by intracellular and intercellular hyphae in the cortex, medulla and vascular system was observed resulting in cell colapse and necrosis.

Flow cytometric purification ofColletotrichum higginsianumbiotrophic hyphae from Arabidopsis leaves for stage‐specific transcriptome analysis

Generation of stage-specific cDNA libraries is a powerful approach to identify pathogen genes that are differentially expressed during plant infection. Biotrophic pathogens develop specialized infection structures inside living plant cells, but sampling the transcriptome of these structures is problematic due to the low ratio of fungal to plant RNA, and the lack of efficient methods to isolate them from infected plants. Here we established a method, based on fluorescence-activated cell sorting (FACS), to purify the intracellular biotrophic hyphae of Colletotrichum higginsianum from homogenates of infected Arabidopsis leaves. Specific selection of viable hyphae using a fluorescent vital marker provided intact RNA for cDNA library construction. Pilot-scale sequencing showed that the library was enriched with plant-induced and pathogenicity-related fungal genes, including some encoding small, soluble secreted proteins that represent candidate fungal effectors. The high purity of the hyphae (94%) prevented contamination of the library by sequences derived from host cells or other fungal cell types. RT-PCR confirmed that genes identified in the FACS-purified hyphae were also expressed in planta. The method has wide applicability for isolating the infection structures of other plant pathogens, and will facilitate cell-specific transcriptome analysis via deep sequencing and microarray hybridization, as well as proteomic analyses.

Orchid mycorrhiza: implications of a mycophagous life style

Orchid mycorrhiza probably affects about 25 000 plant species and thus roughly one tenth of all higher plants. Histologically, this symbiosis resembles other kinds of endomycorrhiza, the fungal hyphae growing within living plant cells. Considerable evidence, however, suggests that it is not a two‐way exchange relationship and thus not potentially mutualistic, such as the wide‐spread endomycorrhiza between plants and Glomalean fungi, known as arbuscular mycorrhiza. During the achlorophyllous seedling stage orchids are obligately dependent on the fungi; some species remain so through life, while others establish photosynthesis but to varying degrees remain facultatively dependent of /responsive to fungal infection as adults. None of the fungi involved are so far known to depend on the symbiosis with orchids. Transfer of organic carbon compounds from hyphae to the orchid has been demonstrated repeatedly, but it is not clear to what extent this takes place during a biotrophic phase while the intracellular hyphae remain intact, or during the subsequent extensive degradation of the hyphal coils. The advantage of viewing orchid mycorrhiza basically as a unilateral mycophagous relationship, in spite of hypothetical beneficial spin‐offs to the mycobiont, is that it provides a conceptual framework similar to that of other parasitic or fungivore relationships; mechanisms known in such relationships could be searched for in future studies of the orchid–fungus symbiosis. These could include mechanisms for recognition, attraction and selection of fungi, physiological regulation of internal hyphal growth, breakdown, and material transfer, nutritional consequences of the plant's preference(s) and trophic changes, fungal avoidance mechanisms, and consequences at population and ecosystem levels. A whole range of possible life strategies becomes apparent that could support divergent evolution and lead to the proliferation of species that has indeed occurred in the orchid family. We outline some of the possible physiological mechanisms and ecological implications of this approach.

Ultrastructures of Colletotrichum orbiculare in the Leaves of Cucumber Plants Expressing Induced Systemic Resistance Mediated by Glomus intraradices BEG110

The colonization of an arbuscular mycorrhizal fungus Glomus intraradices BEG110 in the soil caused a decrease in disease severity in cucumber plants after fungal inoculation with Colletotrichum orbiculare. In order to illustrate the resistance mechanism mediated by G. intraradices BEG110, infection patterns caused by C. orbiculare in the leaves of cucumber plants and the host cellular responses were characterized. These properties were characterized using transmission electron microscopy on the leaves of cucumber plants grown in soil colonized with G. intraradices BEG110. In the untreated plants, inter- and intra-cellular fungal hyphae were observed throughout the leaf tissues during both the biotrophic and necrotrophic phases of infection. The cytoplasm of fungal hyphae appeared intact during the biotrophic phase, suggesting no defense response against the fungus. However, several typical resistance responses were observed in the plants when treated with G. intraradices BEG110 including the formation of sheaths around the intracellular hyphae or a thickening of host cell walls. These observations suggest that the resistance mediated by G. intraradices BEG110 most often occurs in the symplast of the host cells rather than in the apoplast. In addition, this resistance is similar to those mediated by biotic inducers such as plant growth promoting rhizobacteria.

Histopathology of spot blotch disease of wheat caused byBipolaris sorokiniana(Teleomorph:Cochliobolus sativus)

Bipolaris sorokiniana (Sacc.) Shoemaker (Teleomorph: Cochliobolus sativus (Ito and Kuribayashi) Drechs. Ex. Dastur) is a serious necrotrophic pathogen of wheat and barley in several countries. In this paper we report on mechanism of resistance and histopathological changes that occur on pathogen attack in resistant and susceptible genotypes. Callose deposition was observed in the epidermal cell wall of resistant genotype, Pusa T3336 at 2DAI. Lignin deposition around mesophyll tissues was also strongly observed by Phloroglucinol-HCl test in Pusa T3336. Four days after inoculation (4DAI), the pathogen colonized the leaf tissues showing inter and intracellular hyphae in susceptible genotype, Agralocal. In resistant genotype, no colonization was observed. The host tissue in susceptible genotype showed degeneration away from the site of colonization and also showed loss of structural integrity thereby indicating the role of toxin in pathogenesis. Separation of plasmalemma from the cell wall was observed due to loss of electrolytes and degradation of chloroplast at 5 DAI in the susceptible genotypes was prominently seen through light microscopy.

The histopathology of Phaeocryptopus gaeumannii on Douglas-fir needles

Germinating ascospores of Phaeocryptopus gaeumannii produce suprastomatal appressoria from which penetration pegs enter needles. Initial infection occurs between late May and early Jul and coincides with budbreak and shoot elongation. Colonization within needles is exclusively intercellular and increases continuously during Jul–May. No intracellular hyphae or haustoria were observed, but hyphae closely appressed to mesophyll and palisade cell walls are abundant by 3–5 mo after initial infection. Pseudothecial primordia begin to form in epistomatal chambers Oct–Apr, 4–9 mo after initial infection. Pseudothecial primordia developing in the epistomatal chamber are connected to the endophytic thallus by specialized cells in the substomatal chamber that have thickened apical walls and resemble phialides but are not involved in asexual reproduction. The apical wall thickenings instead appear to function as reinforcement against the turgor pressure of the guard cells, allowing cytoplasmic continuity to be maintained between the developing pseudothecium and vegetative hyphae within the needle. Concurrent with the formation of pseudothecial primorida, epiphytic hyphae emerge from the periphery of developing pseudothecia, grow across the needle surface, form numerous anastomoses and reenter the needle by producing appressoria above unoccupied stomata. Epiphytic hyphae and their associated appressoria gradually become more abundant during Oct–Jan.

Associations between microfungal endophytes and roots: do structural features indicate function?

Roots encounter a plethora of microorganisms in the soil environment that are either deleterious, neutral, or beneficial to plant growth. Root endophytic fungi are ubiquitous. These include dark septate endophytes whose role in plant growth and the maintenance of plant communities is largely unknown. The objectives of this review were to assess the structural features of the interactions between dark septate endophytic fungi and the roots of both angiopsperms and conifers, and to suggest avenues for further research. Several light microscopy studies of endophyte–root interactions have revealed a variety of structural features, depending on host species and plant growth conditions. In some cases, when fungal hyphae enter roots they cause cell breakdown, whereas in other situations there is little noticeable effect. In some tree species, associations with these endophytes may mimic ectomycorrhizas or ectendomycorrhizas. The few ultrastructural studies indicate that intracellular hyphae lack a host-derived perifungal membrane and interfacial matrix material, features typical of biotrophic fungus – plant cell interactions. This raises questions concerning nutrient exchange between these fungi and plant cells. Further research in this area is needed. New approaches that include molecular cytology and live-cell imaging are needed to determine early changes in plant cells when challenged with these fungi.

Ericoid mycorrhizal fungi are common root inhabitants of non-Ericaceae plants in a south-eastern Australian sclerophyll forest

Fungi were isolated from the roots of 17 plant species from the families Apiaceae, Cunoniaceae, Cyperaceae, Droseraceae, Fabaceae-Mimosoideae, Lomandraceae, Myrtaceae, Pittosporaceae, Proteaceae and Stylidiaceae at a sclerophyll forest site in New South Wales, Australia. Internal transcribed spacer (ITS) restriction fragment length polymorphism (RFLP) and sequence comparisons indicated that the isolated fungi had affinities to a range of ascomycetes, basidiomycetes and zygomycetes. Four RFLP types had closest affinities to previously identified Helotiales ericoid mycorrhizal (ERM) or Oidiodendron spp. Isolates representing six RFLP types, which were variously isolated from all 17 plant species, formed ERM coils in hair root epidermal cells of Woollsia pungens (Ericaceae) under gnotobiotic conditions. Three of these isolates formed intercellular hyphae, intracellular hyphae and/or microsclerotia, which are typical of dark septate endophyte infection, in roots of Stylidium productum (Stylidiaceae), indicating an ability to form different types of association with roots of different hosts. Overall the data indicate that a broad range of plant taxa may act as repositories for ERM fungi in sclerophyll forest soil.

Ultrastructural localization of acid phosphatase in arbusculate coils of mycorrhizal Phoenix canariensis roots

Acid phosphatase (ACP) activity has been detected in roots of mycorrhizal and non-mycorrhizal Phoenix canariensis. This enzyme was ultrastructurally localized in arbusculate coils for the first time. This localization was carried out using a cerium-based method, which minimizes non-specific precipitation. The ACP was localized in inter- and intracellular hyphae, in the fungal cytoplasm as well as at the interface and the fungal cell wall and the periarbuscular membrane limiting it. The novel localization of an ACP in the arbuscular mycorrhizal (AM) interface of arbusculate coils suggests that this enzyme may be involved in the phosphorus efflux from the mycorrhizal fungus to the host. The results presented in this article indicate that the role played by ACP in AM symbiosis may be more important than was previously thought and that arbusculate coils are highly relevant when considering nutrient transfer through AM symbiosis.

Interaction between an isolate of dark-septate fungi and its host plant Saussurea involucrata

A dark-septate endophytic (DSE) fungus EF-M was isolated from the roots of an alpine plant Saussurea involucrata Kar. et Kir. ex Maxim. The fungus was identified by sequencing the PCR-amplified rDNA 5.8S gene and ITS regions. The sequence was compared with similar sequences in the GenBank, and results showed that EF-M was congeneric to Leptodontidium. Resynthesis study was conducted to clarify the relationship between the root endophyte EF-M and the host plant S. involucrata using the material grown in sterile culture bottle. In roots recovered 6 weeks after inoculation, epidermal cells were colonized by intercellular and intracellular hyphae and "microsclerotia" formed within individual cells in the epidermis layers. However, hyphae did not invade the cortex and the stele. There were no profound effects of endophyte EF-M on plant root development, but significant differences were detected in plant height and shoot dry weight between the treatments. The present study is the first report hitherto on DSE fungi in S. involucrata.