Ultrastructure of the dikaryotic stage of anise rust , Puccinia pimpinellae ( F . Strauss ) Link

This study examined the ultrastructure of urediniospores, teliospores, intercellular hyphae and haustoria of the rust fungus: Puccinia pimpinellae on Pimpinella anisum. The morphology and structure of urediniospores and teliospores is similar to those of most other Puccinia species. The ultrastructure of intercellular hyphae and haustoria is mainly similar to those of other rust fungi. The intercellular hyphae are frequently containing two nuclei and an extramural substance is found between their walls and host cell wall. An electron-lucent collar is found around the haustorial neck and the proximal end of the haustorial body. In most cases, two nuclei are detected inside the haustorial body. A fibrillar extrahaustorial matrix is present around the old vacuolated haustoria. Necrotic haustoria are frequently detected inside host cell and encased by a wide sheath containing membranous structures and electron-dense deposits. The association of chloroplasts and nuclei with haustoria is also detected.


INTRODUCTION
Anise (Pimpinella anisum L.) is an annual plant belonging to the family Apiaceae.The plant is fragrant and widely used in medicine and as food flavorant.In Egypt, its cultivation has become more widespread in order to cover the increasing medicinal industries and exportation needs (Zaky et al., 2006).
Rust fungi are plant pathogens that can cause disease on a number of economically important crop species (Littlefield and Heath, 1979).The basidiospore derivedmonokaryotic stage usually penetrates plant epidermal cells directly, producing an intracellular hypha which subsequently branches out into an intercellular mycelium which develops intracellular M-haustoria, while the urediniospore-derived dikaryotic stage penetrates plant tissue through stomata openings, grows between plant cells and subsequently produces highly specialized Dhaustoria inside mesophyll cells (Littlefield and Heath, 1979).Therefore, intracellular structures (haustoria) are produced during both stages of growth.The haustoria remain enveloped by the invaginated plant plasma membrane so that the only plant structure penetrated is the cell wall.Nevertheless, fungal structures are in close contact with the plant protoplast in which they can cause considerable organelle rearrangements to be in close association with these structures for nutrient needs (Heath and Skalamera, 1997).
In Egypt, the rust disease caused by Puccinia pimpinellae (F.Strauss) Link. is prevalent and destructive disease for anise during the last few years.Since the disease was recorded by Ghoneem (2003), there are annually considerable losses in yields of fruits and its essential oil.Puccinia pimpinellae is an autoecious rust characterized by rust-colored pustules on the upper and lower leaf surfaces, being more predominant on the E-mail: zakariabaka@yahoo.com.
underside of the leaf.The infection extended to stem, flowering buds, inflorescence and seeds.Severe infection may cause leaves to curl upwards, desiccate, turn brown and drop prematurely (Ghoneem et al., 2009;Saber et al., 2009).Singh et al. (2011) reported that uredinia, telia and aecia were found on the same host, Osmorhiza longistylis (Apiaceae).
The present study investigates for the first time the ultrastructure of urediniospores, teliospores, intercellular hyphae and dikaryotic haustoria of P. pimpinellae using transmission and scanning electron microscopy.

Collection of plant materials
Infected leaves of anise were collected from the Agriculture College Farm at Damietta University, Egypt.The dried specimens were deposited in the Herbarium of Botany Department, College of Science, Damietta University.

Scanning electron microscopy (SEM)
Rust-infected leaves from fresh specimens were cut into 1 mm 2 pieces containing urediniosori or teliosori, and prefixed in 3% glutaraldehyde solution (Merkh) in 0.1 M cacodylate buffer (Sigma), and then postfixed in 1% osmium tetroxide in the same buffer.The leaf segments were dehydrated using a serial dilution of ethanol and finally by acetone.The processed samples were then dried using a critical point drier (EMS 850) and coated with gold using a sputter coater (EMS 550).The samples were examined using a JEOL T100 JSM scanning electron microscope.

Transmission electron microscopy (TEM)
Rust-infected leaves were processed according to the procedure described by Hayat (1989).Infected leaves were cut into 1 mm 2 pieces and fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer, and postfixed in 1% osmium tetroxide in the same buffer for 2 h.The fixed pieces were dehydrated in a graded series of ethanol and embedded in Spurr's resin.Ultrathin sections were cut with an ultramicrotome and glass knife, stained with 2% aqueous uranyl acetate followed by lead citrate, and examined using a JEOL 100-S TEM.

Urediniospores and teliospores
SEM observations revealed that uredinia developed under leaf epidermis and serial sections through uredinia confirmed the absence of paraphyses (Plate 1A).Short spines are scattered on the surface of the urediniospore Baka 889 walls (Plate 1B).The telia also developed under leaf epidermis and contain condensed bi-celled teliospores with pedicels (Plate 1C).The teliospore possesses a verrucose surface ornamentation and a transverse depression at the middle of the spore indicated the presence of the septum (Plate 1D).TEM observations indicated that the mature urediniospore exhibited dense cytoplasm with two nuclei, many lipid bodies and conical spines emerged from its wall (Plate 2A), while the mature teliospore consists of two cells each containing one nucleus, separated by a septum and dense cytoplasm with numerous lipid drops.The thickest parts of the teliospore wall occur at the germ pore positions (Plate 2B).

Intercellular hyphae and haustoria
Intercellular hyphae developed in the intercellular spaces of the mesophyll of anise leaves.These hyphal cells possess two nuclei, each with one nucleolus, mitochondria and glycogen appeared as electron-lucent areas (Plate 3A).An amorphous material (extramural substances as named by Harder et al., 1989) is present between the hyphae and host cell wall (Plate 3A).
Haustorium is produced by specialized intercellular hyphae, called haustorial mother cells (HMC) which adhered to the host cell wall.A very small penetration peg emerged from the HMC at the point of contact with the host cell wall (Plate 3B).Like D-haustoria of some rust fungi, the mature haustorium of P. pimpinellae, is clubshaped having two nuclei and a narrow cylindrical neck with a distinct electron-dense neckband in the wall midway along its length (Plate 3C, D).Transverse section in the haustorial body indicates the presence of two nuclei and other organelles similar to eukaryotic cells (Plate 4B).Old vacuolated haustorium possesses thicker extrahaustorial matrix with fibrillar material which are noticed (Plate 4A).

Host cell responses
Infected host cell contains spherical chloroplast with disorganized grana, absence of intergranal lamellae and increase of plastoglobuli with big starch grains.Host mitochondria, lipid body, Golgi body and crystal-containing microbodies are also observed near the penetration peg (Plate 3B).
The host plasma membrane is invaginated around an electron-lucent collar and the haustorium.This collar encloses the neck and the proximal part of haustorial body (Plate 3C, D).Close association of host rough ER with the invaginated host plasma membrane around haustoria is also observed (Plate 4B).Necrotic haustoria were always encased by a matrix containing membranous materials (Plate 4C).Host chloroplasts and nuclei are closely associated with the haustorium (Plate 4D).
This definition essentially describes the mode of uredi-niospore formation in most rust fungi, including Puccinia and Uromyecs ( (Harder, 1984).On the other hand, teliospores in P. pimpinellae, in most cases, resemble those found in other Puccinia species in that they are pedicellate, bi-celled and each cell contains one nucleus and one germ pore (Littlefield and Heath 1979;Baka and Lösel, 1992a;Baka, 1996).Littlefield and Heath (1979) pointed out that teliospores generally exhibited a greater variety of ornamentation types than do urediniospores and aeciospores.Teliospore ornamentation is an important character in the taxonomy of rust fungi (Baka and Gjaerum, 1996).SEM observations revealed that the teliospores of P. pimpinellae show ornamentation of the verrucose type, similar to P. nitida (Durrieu 1974) and P. punctiformis (Baka and Lösel, 1992a).The ultrastructure of intercellular hyphae of P. pimpinellae during dikaryotic stage of infection resembled Plate 2. TEM micrograph of urediniospre and teliospore. A. A longitudinal section of mature urediniospore (u) showing spines (arrowheads) emerging from thick wall (uw).The cytoplasm is very dense and contains large numbers of lipid bodies (L), some aggregated in rosettes and two nuclei (n) with nucleoli (nu).Note the thicker part of spore wall at the attachment of pedicel (pd) and the position of germ pore (gp).Bar = 2.0 μm.B. A mature bi-celled teliospore (t), each cell containing one nucleus (n) and numerous electron-lucent lipids drops.Note the thickening of spore wall at the position of germ pores (arrows).Note also a short terminal portion of pedicel (pd) still attached to the teliospore.Bar = 5.0 μm. that of most other rust fungi (Littlefield and Heath, 1979;Baka, 1992;Baka and Gjaerum, 1996;Baka and Lösel, 1992a).The extramural substances between intercellular hyphae and host cell wall may have adhesive function and related to the establishment of compatibility or incompatibility.Harder et al. (1989) concluded that these substances appear to be highly complex and variable according to the location of the hyphae within the rust colony.Components of this material of fungal origin may well be involved in adhesive functions, in conjunction with material of host origin and are thought to consist basically of glycoprotein (Harder et al., 1989).
The haustorium of P. pimpinellae showed the ultrastructural characteristics described by other investigators for other club-shaped dikaryotic haustoria with two nuclei in their bodies (Baka, 1992;Baka and Gjaerum, 1996;Baka, 2002;Baka and Lösel, 1992a;Classen et al., 2001;Mims et al., 2002).It is generally assumed that, at maturity, two nuclei are present in the haustorium body.
The neck ring is a specialized structure occurring in the haustorial neck of all rust fungi so far examined ultrastructurally.This ring, found in D-haustoria forms a seal between the host and fungal plasmalemma, preventing leakage, from the extrahaustorial matrix, of solutes undergoing transport from host to pathogen.Neck-ring function was first demonstrated by Heath (1976).Thus, the neck ring is instrumental in coupling the host and haustorial cytoplasm into a functional unit (Woods and Gay, 1987).The neck ring stains intensely after conventional preparation procedures for electron microscopy.Using the periodate-chromate-phosphotungstate (PACP) method for staining the plant plasmalemma, Littlefield and Bracker (1972) showed that the neck ring was unstained as compared to intense staining of remainder of the neck wall.This was attributed to the preferential extraction of the neck ring with periodic acid before the heavy metal stains were applied.In preliminary studies on haustorial development in Puccinia coronata f. sp.avenae, it was found that the neck ring was intensely electron-opaque in glutaraldehyde-fixed, but unosmicated, unstained tissue.This suggested that the neck ring contains mineral-based electron scattering substances (Littlefield and Bracker, 1972).Electron probe X-ray analysis of the haustorial neck ring of P. coronata avenae indicated the presence of silicon and phosphorus (Heath and Allen, 1985).
The haustorium is completely enclosed by host-wall like material, which is referred to as extrahaustorial matrix (Bushnell, 1972).This matrix becomes more fibrillar around the old vacuolated haustoria than around young haustoria.There has been considerable speculation regarding the nature of the extrahaustorial matrix.Evidence has been presented that it contains material of host and (or) fungal origin or that may in fact be an artifact of preparation method (Littlefield and Heath, 1979).Evidence has since been presented that the extrahaustorial matrix does contain identifiable substances (Coffey and Allen, 1983).Cytochemical investigation indicated that polysaccharides and glycoproteins are present in the extrahaustorial matrix (Chong et al., 1986).
In the present investigation, an electron-lucent collar enclosed the neck and the proximal part of haustorial body.Collar formation in rust infection is frequently linked to the degree of host-fungal compatibility (Heath, 1974).The chemical composition of the collar has been studied Plate 4. TEM micrograph showing the structure of haustorium and host responses.A. A fibrillar extrahaustorial matrix (Ex) of old vacuolated haustorium (H).Note invaginated (ip) and uninvaginated (Hp) host plasma membrane.Note also electron-dense haustorial wall (arrow) and host cell wall (w).Bar = 0.5 μm.B. A transverse section of mature dikaryotic haustorium body is showing two nuclei (n) with nucleoli (nu) and mitochondria (m).Note small patches of heterochromatin (arrowheads) and large amounts of euchromatin (eu).Note also vesicle-like structure (arrows) and what seems to be endoplasmic reticulum is associated with invaginated host plasma membrane (ip) and host vacuole (v).Close association of host mitochondria (M), Golgi body (g), rough endoplasmic reticulum (ER) and lipid drop (L) to the haustorium can also be seen.Bar = 2.0 μm.C. A necrotic haustorium (H).Note that the haustorium is encased by a matrix containing membranous materials (F) as a result of host response.Bar = 0.5 μm.D. An infected host cell showing its response for infection.Note the close association of nucleus (N) and chloroplasts (c) with haustorium (H).Note also small clumps of heterochromatin (arrows), large amounts of euchromatin (Eu) and disintegration of nuclear envelope (arrowhead).Host cell vacuole (v) can also be seen.Bar = 0.5 μm. by many workers.Heath (1974) suggested that the electron-lucent type is formed of callose-like materials.Cytochemically, Chong and Harder (1982) showed that several types of polysaccharides are the main constituents of the collar of P. coronata avenea and it was unaffected by protease, cellulose, or lipid-solvent treatments but stained heavily with periodic acid-thiocarbohydrazidesilver proteinate.
Necrotic haustoria were encased by a wide sheath containing membranous structures and electron-dense deposits.The characteristically wide encasement, frequently detected around necrotic haustoria may be a defense of the host cell to toxins produced after the death of haustoria as indicated by Ehrlich and Ehrlich (1971).
The close association of host chloroplasts and nuclei with haustoria, in the present investigation, was in agreement with studies by many workers.Littlefield and Heath (1979) suggested that this close association may be a characteristic response of the host to invasion and the interaction between host and haustorium is generally assumed to be mainly concerned with nutritional requirements of the parasite.Cytochemical detection of carbohy-drates by means of gold-lectin complex may shed further light on the structure of this fungus.

Plate 3 .
TEM micrograph of intercellular hypha and haustorium. A. An intercellular hypha (Ih) is showing close adherence of this hypha to host cell wall.The hypha contains two nuclei (n) with nucleoli (nu), mitochondria (m) and electron-lucent areas suggesting glycogen (arrowheads).Note amorphous material (arrow) between the hypha and host cell wall.Note also the host chloroplast (c).Bar = 2.0 μm.B. A penetration peg (big arrow) emerging from haustorial mother cell (HMC).Amorphous material (arrowhead) between haustorial mother cell wall and host cell wall (w) can be seen.Note the affected spherical chloroplast (c) with disorganized grana, absence of intergranal lamellae, increas e of plastoglobuli (os) and big starch grains (st).Note also host mitochondria (M), lipid body (L), vacuole (v), Golgi body (small arrow) and microbody (Mb) containing crystal (cr).Bar = 0.5 μm.C. A median section through haustorium mother cell (HMC) and mature dikaryotic haustorium (H), containing two nuclei (n) and lipid drop (L), inside host cell.Note the extensive collar (CO) surrounding the haustorial neck and also the continuity of the invaginated host plasmalemma (arrowhead) around the collar.Bar = 1.0 μm.D. A magnified part showing haustorium neck (HN) with neckband (arrowheads).Note thickening (arrows) of the haustorial mother cell (HMC) wall at the site of penetration and the collar (CO).Bar = 1.0 μm.