Substomatal Vesicles Research Articles

Histological and molecular responses of Vigna angularis to Uromyces vignae infection

BackgroundTo advance the understanding of adzuki bean (Vigna angularis) resistance to infection with the rust-causing fungus Uromyces vignae (Uv), we comprehensively analyzed histological events and the transcriptome of Uv-infected adzuki bean.ResultsCompared with the susceptible cv. Baoqinghong (BQH), the resistant cv. QH1 showed inhibition of uredospore germination and substomatal vesicle development, intense autofluorescence of cells around the infection site, and cell wall deposit formation in response to Uv infection. In cv. QH1, gene set enrichment analysis (GSEA) showed enrichment of chitin catabolic processes and responses to biotic stimuli at 24 h post-inoculation (hpi) and cell wall modification and structural constituent of cytoskeleton at 48 hpi. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis indicated enrichment of WRKY transcription factors (TFs), the calcium binding protein cml, and hydroquinone glucosyltransferase at both 24 and 48 hpi. In total, 1992 and 557 differentially expressed genes (DEGs) were identified at 24 and 48 hpi, respectively. Cell surface pattern-recognition receptors (PRRs), WRKY TFs, defense-associated pathogenesis-related (PR) proteins, and lignin and antimicrobial phenolic compound biosynthesis were significantly induced. Finally, we detected the chitinase (CHI) and phenylalanine ammonia-lyase (PAL) activity were higher in QH1 and increased much earlier than in BQH.ConclusionIn cv. QH1, cell-surface PRRs rapidly recognize Uv invasion and activate the corresponding TFs to increase the transcription of defense-related genes and corresponding enzymatic activities to prevent fungal development and spread in host tissues.

Pathogenesis of Plasmopara viticola Depending on Resistance Mediated by Rpv3_1, and Rpv10 and Rpv3_3, and by the Vitality of Leaf Tissue.

Grapevine cultivars vary in their resistance to Plasmopara viticola, causal agent of downy mildew. Genes from various Vitis species confer pathogen resistance (Rpv), resulting in reduced compatibility of the host-pathogen interaction and partial disease resistance that may become apparent at different stages of pathogenesis. This study describes the pathogenesis of P. viticola on the partially resistant cultivars Regent (Rpv3-1) and Solaris (Rpv3-3, Rpv10) as compared with the susceptible cultivar Mueller-Thurgau using various microscopic techniques, visual disease rating as well as qPCR. Host plant resistance had no effect on the initial steps of pathogenesis outside the host plant cells (zoospore attachment, formation of substomatal vesicle) and became detectable only after the formation of primary haustoria. The restricted compatibility resulted in reductions in haustorium size and in the number of secondary haustoria and was associated with callose depositions around haustoria and stomatal guard cells, collapsed mesophyll cells (hypersensitive reaction), and additional production of an amorphous substance in the intercellular space of cultivar Solaris. Resistance mechanisms reduced the efficiency of P. viticola haustoria and largely restricted tissue colonization to the spongy parenchyma; resistance of cultivar Solaris having thicker leaves was more effective than that of cultivar Regent. Despite of the effects of resistance genes, P. viticola was able to complete its life cycle by forming sporangiophores with sporangia through the stomata on both resistant cultivars indicating partial resistance. Differences in the pathogenesis on detached and attached grapevine leaves highlighted the impact of host tissue vitality on both resistance and susceptibility to the biotrophic pathogen.

Comparing infection and colonisation of Puccinia graminis in barley and wheat

Barley, host to the wheat and rye attacking forms of the stem rust pathogen, Puccinia graminis (Pg), is considered inherently more resistant to Pg than wheat. To investigate whether this enhanced basal defence is associated with early infection or colonisation processes, adult plants of selected barley and wheat entries were inoculated with Pg f. sp. tritici (Pgt) races BNGSC and PTKST, and Pg f. sp. secalis (Pgs) isolate UVPgs1. Flag leaf sheaths on the last stem internode were sampled for analysis. Using scanning electron microscopy and epidermal stripping, early Pg infection structure development was compared between barley and wheat. Sub-stomatal vesicle appearance and production of haustorial mother cells were similar at 24 and 48 h post inoculation (hpi), respectively. Significant variation occurred among host entries (P ≤ 0.01) for colony size and fungal biomass at 120 hpi. Differences between the Pgt races and the Pgs isolate as well as the host entry-Pg isolate interaction proved insignificant (P ≥ 0.05). The period between 120 and 240 hpi may hold some explanation for the perceived lower stem rust receptivity in barley, since a steep increase in accumulated fungal biomass observed in the susceptible wheat Line 37 − 07 was not seen in the susceptible barley entries at the same time interval.

Resistance mechanisms involved in complex immunity of wheat against rust diseases

The review is devoted to the disclosure of the modern concept of plant immunity as a hierarchical system of plant host protection, controlled by combinations of major and minor resistance genes (loci). The “zigzag” model is described in detail for discussing the molecular bases of plant immunity with key concepts: pathogen-associated molecular patterns triggering innate immunity, ambivalent effectors causing susceptibility, but when interacting with resistance genes, a hypersensitive reaction or alternative defense mechanisms. There are three types of resistance in cereals: (1) basal resistance provided by plasma membrane-localized receptors proteins; (2) racespecific resistance provided by intracellular immune R-receptors; (3) partial resistance conferred by quantitative gene loci. The system ‘wheat (Triticum aestivum) – the fungus causing leaf rust (Puccinia triticina)’ is an interesting model for observing all the resistance mechanisms listed above, since the strategy of this pathogen is aimed at the constitutive use of host resources. The review focuses on known wheat genes responsible for various types of resistance to leaf rust: race-specific genes Lr1, Lr10, Lr19, and Lr21; adult resistance genes which are hypersensitive Lr12, Lr13, Lr22a, Lr22b, Lr35, Lr48, and Lr49; nonhypersensitive genes conferring partial resistance Lr34, Lr46, Lr67, and Lr77. The involvement of some wheat R-genes in pre-haustorial resistance to leaf rust has been discovered recently: Lr1, Lr3a, Lr9, LrB, Lr19, Lr21, Lr38. The presence of these genes in the genotype ensures the interruption of early pathogenesis through the following mechanisms: disorientation and branching of the germ tube; formation of aberrant fungal penetration structures (appressorium, substomatal vesicle); accumulation of callose in mesophyll cell walls. Breeding for immunity is accelerated by implementation of data on various mechanisms of wheat resistance to rust diseases, which are summarized in this review.

Autophagy is induced in haustorial mother cells of Puccinia triticina and is necessary for plant infection

Autophagy is a highly conserved pathway that mediates bulk degradation of cellular components in lysosomes/vacuoles and plays an important role in filamentous fungi by impacting growth, morphology and development. However, in leaf rust, the role of autophagy is poorly understood. In this study, we identified the structure of autophagy in leaf rust infection structures by using transmission electron microscopy (TEM). The results suggest that autophagy can be strongly induced in the haustorium mother cell (HMC) but did not occur in other special infection structures, such as the appressorium, substomatal vesicle and haustorial. Applying the autophagy inhibitor 3-mehyladenine (3-MA) to leaf rust resulted in significant arrest of mycelial growth and delays in leaf rust infection. These results suggest that autophagy can be induced in HMC and is important for fungal infection of wheat. This research will help us to expand our understanding of the physiological functions of autophagy in filamentous fungi.

TaMDAR6 acts as a negative regulator of plant cell death and participates indirectly in stomatal regulation during the wheat stripe rust-fungus interaction.

We identified a new monodehydroascorbate reductase (MDAR) gene from wheat, designated TaMDAR6, which is differentially affected by wheat-Puccinia striiformis f. sp. tritici (Pst) interactions. TaMDAR6 is a negative regulator of plant cell death (PCD) triggered by the Bax gene and Pst. Transcript levels of TaMDAR6 are significantly upregulated during a compatible wheat-Pst interaction, indicating that TaMDAR6 may contribute to plant susceptibility. In addition, H2 O2 production and PCD are significantly induced and initial pathogen development is significantly reduced in the TaMDAR6 knocked-down plants upon Pst infection. Thus, the suppression of TaMDAR6 enhances wheat resistance to Pst. Besides, the suppression of TaMDAR6 during an incompatible interaction induces a change in the morphology of stomata, which leads to poor stoma recognition and as a consequence to reduced infection efficiency. The percentage of infection sites that develop substomatal vesicles decreases in the TaMDAR6 knocked-down plants during the incompatible interaction presumably due to the increase in ROS accumulation, which is likely to activate other resistance mechanisms that have a negative effect on substomatal vesicle formation. TaMDAR6 can therefore be considered a negative regulator of PCD and of wheat defense to Pst.

Different non-host resistance responses of two rice subspecies, japonica and indica, to Puccinia striiformis f. sp. tritici

Japonica and indica have different non-host resistance (NHR) abilities to Puccinia striiformis f. sp. tritici ( Pst ), and hydrogen peroxide (H 2 O 2 ) has a positive function in NHR to japonica against Pst. Non-host interactions between Puccinia striiformis f. sp. tritici (Pst) and two rice subspecies were characterized using 23 rice varieties, including 11 japonica and 12 indica. Results showed that the infected fungal structures were easily produced in the leaves of indica, whereas only several substomatal vesicles and primary infection hyphae were observed in the leaves of japonica. This result indicated that indica is less resistant or more susceptible to Pst than japonica. Hydrogen peroxide accumulated in the initial phase of japonica-Pst interaction but not in indica-Pst interaction. A set of reactive oxygen species (ROS)-related genes was also induced in response to Pst infection, suggesting that ROS activation is one of the major mechanisms of non-host resistance of rice to Pst.

HISTOCHEMICAL RESPONSE OF NONHOST RESISTANCE IN PEPPER TO THE STRIPE RUST FUNGUS (PUCCINIA STRIIFORMIS f. sp. TRITICI)

Nonhost resistance (NHR) confers plants immunity to most microbial pathogens. Resistance responses in nonhost pepper cv. A11 and the incompatible host wheat cv. Suwon 11 against the stripe rust fungus Puccinia striiformis f. sp. tritici (Pst) were studied histochemically. Similarly high germination rates of Pst were observed on both nonhost and resistant host. However, the majority of germ tubes failed to form substomatal vesicles and fewer haustoria formed in the nonhost interaction, while the ratio of H2O2 accumulation was considerably high in pepper. During a further incubation period, no secondary hyphae were found in nonhost pepper-Pst. In contrast, significantly higher proportion of haustoria and a much lower production of H2O2 and secondary hyphae were detectable in the incompatible host resistance deployed by wheat. The incidence of hypersensitive cell death was found to be slightly higher in the nonhost interaction. These results indicate that H2O2 and hypersensitive cell death play important roles in the NHR of pepper to Pst, and nonhost and host resistance may share similar mechanisms.

Infection of Brachypodium distachyon by formae speciales of Puccinia graminis: early infection events and host-pathogen incompatibility.

Puccinia graminis causes stem rust, a serious disease of cereals and forage grasses. Important formae speciales of P. graminis and their typical hosts are P. graminis f. sp. tritici (Pg-tr) in wheat and barley, P. graminis f. sp. lolii (Pg-lo) in perennial ryegrass and tall fescue, and P. graminis f. sp. phlei-pratensis (Pg-pp) in timothy grass. Brachypodium distachyon is an emerging genetic model to study fungal disease resistance in cereals and temperate grasses. We characterized the P. graminis-Brachypodium pathosystem to evaluate its potential for investigating incompatibility and non-host resistance to P. graminis. Inoculation of eight Brachypodium inbred lines with Pg-tr, Pg-lo or Pg-pp resulted in sporulating lesions later accompanied by necrosis. Histological analysis of early infection events in one Brachypodium inbred line (Bd1-1) indicated that Pg-lo and Pg-pp were markedly more efficient than Pg-tr at establishing a biotrophic interaction. Formation of appressoria was completed (60–70% of germinated spores) by 12 h post-inoculation (hpi) under dark and wet conditions, and after 4 h of subsequent light exposure fungal penetration structures (penetration peg, substomatal vesicle and primary infection hyphae) had developed. Brachypodium Bd1-1 exhibited pre-haustorial resistance to Pg-tr, i.e. infection usually stopped at appressorial formation. By 68 hpi, only 0.3% and 0.7% of the Pg-tr urediniospores developed haustoria and colonies, respectively. In contrast, development of advanced infection structures by Pg-lo and Pg-pp was significantly more common; however, Brachypodium displayed post-haustorial resistance to these isolates. By 68 hpi the percentage of urediniospores that only develop a haustorium mother cell or haustorium in Pg-lo and Pg-pp reached 8% and 5%, respectively. The formation of colonies reached 14% and 13%, respectively. We conclude that Brachypodium is an apt grass model to study the molecular and genetic components of incompatiblity and non-host resistance to P. graminis.

Microscopy and proteomic analysis of the non-host resistance of Oryza sativa to the wheat leaf rust fungus, Puccinia triticina f. sp. tritici

Rice (Oryza sativa) cv. Nipponbare expresses non-host resistance (NHR) to the wheat leaf rust fungus, Puccinia triticina f. sp. tritici (Ptt). When the leaves of cv. Nipponbare were inoculated with Ptt, approx 93% of the urediniospores germinated on the leaf surface, but only 10% of the germinated spores formed appressoria over the stomata at one day post inoculation (1 dpi). Hydrogen peroxide (H(2)O(2)) accumulated in host cells around the appressoria at 3 dpi. Approx. 3% of the appressoria produced short hyphae inside the leaf, and fluorescence was observed in tissue invaded by the hyphae by 7 dpi. At 22 dpi, 0.2% of the sites with appressoria formed branching infection hypha in mesophyll cells, but no substomatal vesicles, haustorial mother cells or haustoria were observed. Proteins were extracted from leaves 3 dpi and analyzed by two-dimensional gel electrophoresis (2-DE). A total 33 spots were reproducibly up-regulated and 9 were down-regulated by infection compared to the water inoculated control. Of these, 30 were identified by MALDI-TOF Mass Spectrometry. The identified proteins participate in defense/stress responses, energy/carbohydrate metabolism, oxidation-reduction processes, protein folding/turnover/cleavage/degradation, signal transduction and cell death regulation. The results indicates that NHR of rice to Ptt is consistent with a shift in protein and energy metabolism, increased antimicrobial activities, possibly including phytoalexin accumulation and cell wall reinforcement, increased cell repair, antioxidive and detoxification reactions, and enhanced prevention of plant cell death. Nearly half of the up-regulated identified proteins were associated with chloroplast and mitochondrial physiology suggesting important roles for these organelles during NHR.

Identification of loci controlling non‐host disease resistance in Arabidopsis against the leaf rust pathogen Puccinia triticina

SUMMARY Plant immunity against the majority of microbial pathogens is conveyed by a phenomenon termed non-host resistance (NHR). This multifactorial trait provides durable protection against a given pathogen species. We investigated the molecular basis of NHR in Arabidopsis against the wheat leaf rust pathogen, Puccinia triticina (Ptr). Urediospores germinated with high efficiency and grew randomly over the Arabidopsis leaf surface. However, only 12% of urediospores produced a germ tube that successfully located a stoma and just 0.2% of urediospores went on to produce a haustorium within a penetrated mesophyll cell. Attempted Ptr infection induced the production of reactive oxygen intermediates (ROIs), nitric oxide (NO), salicylic acid (SA) and camalexin. The expression of SA, jasmonic acid (JA) and ROI-dependent genes was also detected. A series of well-characterized defence-related mutants were challenged with Ptr, but none of these lines exhibited significantly increased susceptibility to this fungus. Our findings also suggest that attempted Ptr infection triggers transient stomatal closure in Arabidopsis. We assessed the response of a collection of 79 geographically diverse Arabidopsis accessions to Ptr. Wa-1 plants supported a striking increase in Ptr substomatal vesicle frequency relative to all other tested accessions. Furthermore, SA and camalexin levels became elevated in Wa-1 plants relative to the Col reference line, in response to attempted Ptr infection. Additionally, the kinetics of SA-dependent gene expression was expedited in this accession relative to Col plants. To uncover the genetic architecture of NHR against Ptr, we assayed the response of the Arabidopsis Landsberg erecta (Ler) x Columbia (Col) recombinant inbred population to this fungus. Multiple small-to-medium effect quantitative trait loci were identified that govern the expression of NHR in Arabidopsis against Ptr.

Histological investigation of stripe rust (Puccinia striiformis f.sp. tritici) development in resistant and susceptible wheat cultivars

The wheat cultivar Kariega expresses complete adult plant resistance against stripe rust, whereas cv. Avocet S is susceptible. Using confocal laser scanning microscopy, initial fungal penetration into flag leaves was identical in both cultivars, with directional germ‐tube growth towards stomata that were penetrated without the formation of an appressorium, followed by differentiation of a substomatal vesicle, infection hyphae, haustorial mother cells and haustoria. During the following 4 days, further fungal development occurred more quickly in the resistant than in the susceptible cultivar. However, by 7 days postinoculation (dpi) the situation changed, with exponential growth of the pathogen occurring only in the susceptible line. Induced cellular lignification, a typical defence reaction of cereals, was observed at 4 dpi in the resistant cultivar, and 2 days later lignified tissue completely surrounded the fungal colonies. In the susceptible cultivar, isolated lignified host cells occurred at 6 dpi, and long, unbranched fungal hyphae outgrowing the resistance reaction were observed.

The C28 aldehyde octacosanal is a morphogenetically active component involved in host plant recognition and infection structure differentiation in the wheat stem rust fungus

Using Langmuir–Schäfer technology, a very thin, homogeneous coating of glass surfaces with leaf epicuticular waxes was achieved, allowing the establishment of a bio-assay for morphogenetically active, hydrophobic surface components triggering infection structure differentiation in a rust fungus. A chloroform extract of wheat leaf epicuticular wax coated onto a flat glass surface induced the formation of appressoria, substomatal vesicles, and infection hyphae in about 50% of the germ tubes of Puccinia graminis f.sp. tritici. No induction was seen on a glass surface coated with an inert wax which confered the same hydrophobicity to the surface as the leaf wax. The chemical composition of the leaf epicuticular wax extract was analysed, and the active ingredient was shown to be the C28-aldehyde, octacosanal. The importance of multiple recognition of a ‘host associated surface pattern’ (HASP) integrating both physical and chemical signaling cues for the crucial differentiation of an appressorium upon recognition of a host stoma is discussed.

Infection of radiata and bishop pine by Mycosphaerella pini in California

Infection of radiata pine (Pinus radiata D. Don) and bishop pine (Pinus muricata D. Don) by Myco sphaerella pini Rostr. in Munk was determined on needles of infected forest trees in central to northern coastal California. Conidia from infected trees were used to inoculate radiata pine seedlings maintained in constant and (or) fluctuating moisture and temperature regimes. On needles of radiata pine but not bishop pine plantation trees, most conidial germ tubes grew directly towards and entered the nearest epistomatal opening. On inoculated radiata pine seedlings in infection chambers, germ tubes grew haphazardly and few entered openings. Germ tubes entered epistomatal openings more often on abaxial than adaxial needle surfaces of both plantation trees and inoculated seedlings, and more lesions developed on the abaxial surface. Simple hyphae penetrated through epistomatal chambers, between guard cells, and into substomatal chambers. On radiata pine plantation trees, germ tubes that penetrated below guard cells occasionally formed substomatal vesicles. On needles of northern race bishop pine that had few disease lesions per needle, substomatal vesicles were common and frequently partially disintegrated. On seedlings, "water-soaked" spots formed 5 days after hyphae of the fungus penetrated slightly below guard cells. Development of typical lesions was delayed when seedlings were initially exposed to up to 16 days of continuous mist spray and then kept dry for 8 weeks. Penetration on inoculated seedlings was significantly greater in a variable than in a constant air temperature regime on abaxial needle surfaces, and greater in 24 h/day than in a 16 h/day exposure of seedlings to mist spray. In both temperature regimes more needles were infected on seedlings exposed to 24 h/day mist spray and fewest were infected in the variable temperature, 16 h/day moisture regime.

Infection Structures of Host-Specialized Isolates of Uromyces viciae-fabae and of Other Species of Uromyces Infecting Leguminous Crops

A study was made of the morphology of urediniospores and primary infection structures of 12 isolates of six legume-infecting species of Uromyces. Infection structures were sufficient to distinguish among species. Isolates of Uromyces viciae-fabae proved to be specialized with respect to host, because each isolate infected only cultivars of the species from which it was collected. Host-specialized isolates of U. viciae-fabae also were morphologically distinct, differing in both spore dimensions and infection structure morphology. In particular, the shape and dimensions of the substomatal vesicle were distinctive. These results support the view that U. viciae-fabae sensu lato is a species complex.

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 microm 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.

Morphology, Life Cycle Biology, and DNA Sequence Analysis of Rust Fungi on Garlic and Chives from California

ABSTRACT In the late 1990s, commercial garlic fields in California (CA) were devastated by an outbreak of rust caused by Puccinia allii. We compared collections of the pathogen from garlic (Allium sativum) and chives (A. schoenoprasum) in central CA and Oregon (OR) to collections from garlic and leek (A. porrum and A. ampeloprasum) in the Middle East. Teliospores from the CA and OR collections were smaller in length, width, and projected cross-sectional area compared with collections from the Middle East. CA and OR collections had a shortened life cycle, in which pycnia and aecia were not formed. Germinating teliospores produced a two-celled promycelium, resulting in two basidiospores, each initially with two nuclei, indicating that this rust was homothallic. In addition, the morphology of the substomatal vesicles was different between the CA-OR (fusiform) and the Middle Eastern (bulbous) collections. DNA sequence analysis of the nuclear ribosomal internal transcribed spacer region showed that the CA and OR rust collections formed a well-supported cluster distinct from the Middle Eastern and European samples. These results suggest that the rust on garlic and chives in CA and OR is a different species than the rust fungus on garlic and leek in the Middle East.

Leaf Rust onAegilops speltoidesCaused by a New Forma Specialis ofPuccinia triticina

ABSTRACT A leaf rust attacking Aegilops speltoides in its natural habitat is reported for the first time. It was found in two locations in northern and central Israel. The two collections from A. speltoides resemble wheat leaf rust, Puccinia triticina, in most spore dimensions, in the morphology of the substomatal vesicle of the urediniospore, and in DNA content in pycniospore nuclei. Similarly to P. triticina isolates from wheat, isolates taken from A. speltoides are compatible with Thalictrum speciosissimum as an aecial host and they are crossed easily with wheat leaf rust isolates. However, isolates from A. speltoides differ from wheat leaf rust in their telial host range. They are avirulent to cultivated wheat cultivars, but attack hundreds of A. speltoides accessions that were immune to wheat leaf rust. This distinct host preference justifies delineation of the newly found leaf rust as a forma specialis (f. sp. speltoides) within P. triticina.

In vitro differentiation of haustorial mother cells of the wheat stem rust fungus, Puccinia graminis f. sp. tritici, triggered by the synergistic action of chemical and physical signals

Biotrophic plant pathogenic fungi often develop a sophisticated series of infection structures for non-destructive host tissue penetration. In vitro, early infection structures of rust fungi—germ tube, appressorium, substomatal vesicle, infection hyphae—can easily be induced, but in vitro differentiation rates of late infection structures—haustorial mother cells (hmc), haustoria—are low at best. Under appropriate conditions (humid atmosphere), a combination of physical (mild heat shock) and chemical signals ( trans-2-hexen-1-ol) induced the in vitro differentiation of hmc in the wheat stem rust fungus, Puccinia graminis f. sp. tritici. Around two thirds of the in vitro differentiated germlings developed up to three hmc which were cytologically identical to hmc formed in planta. Efficient in vitro differentiation of hmc will allow us to analyse in molecular detail the processes involved in the induction and differentiation of this critically important developmental stage of the economically important plant pathogenic rust fungi.

Surface ultrastructural studies on the infection process of Pseudocercospora mori causing grey leaf spot disease in mulberry

Pseudocercospora mori produced three kinds of hyphae — primary infection hyphae, internal hyphae and secondary infection hyphae. The primary infection hyphae were developed from conidia and they penetrated the leaf through stomata directly or by lateral penetration branches — the stomatopodia. Inside the stomatal chambers the stomatopodia swelled to become vesicle-like structures. The internal hyphae produced compact stromata in the sub-stomatal chambers. Each stromata carried 1–5 conidia at a time on short unbranched conidiophores. The secondary infection hyphae were developed from internal hyphae directly or from stromata. The secondary infection hyphae emerged through stomata, branched profusely on the leaf surface and produced conidia singly on short conidiophores. The secondary infection hyphae re-entered the leaf directly or by stomatopodia. Conidia were developed also from stomatopodia and from their substomatal vesicles. The conidia developed from substomatal stromata, and those produced from the secondary infection hyphae on leaf surface were similar in their morphology and size of conidiophores. The conidia were obclavate to cylindrical, 3–17 celled and measured 4–35 x 1.5–2 μm. Germ tubes were often found to emerge from the conidial cell nearer to stomata. The primary infection hyphae and secondary infection hyphae produced stomatopodia above stomata.