Mycol. Res. 102 (4) : 391–399 (1998) 391 Printed in the United Kingdom Scanning electron microscopy of early infection structure formation by Puccinia recondita f. sp. tritici on and in susceptible and resistant wheat lines G U A N G G A N H U* A N D F. H. J. R I J K E N B E R G Department of Microbiology and Plant Pathology, University of Natal, Private Bag X01, Scottsville 3209, Pietermaritzburg, South Africa The morphology of infection structure development of Puccinia recondita f. sp. tritici on and in susceptible and resistant wheat lines inoculated with urediospores was examined by SEM. The germ-tube extends over the leaf surface and elongates perpendicularly to the long axis of the leaf. When the germ-tube encounters the stomatal lip, an appressorium forms over the stoma and the pore is entered by an infection peg produced on the surface of the appressorium in contact with the host leaf. At 6 h post-inoculation (hpi), infection pegs develop terminally substomatal vesicles (SSVs) in the substomatal chambers of all wheat lines. A septum separates each SSV from its interconnective tube. A primary infection hypha forms terminally from the elongated SSV either parallel to the long axis of the stomatal slit or perpendicular to the leaf surface. When a primary infection hypha attaches to a host cell, a septum forms cutting off the tip of the hypha, delimiting a terminal haustorium mother cell (HMC) by 12 hpi. Secondary infection hyphae arise from a position proximal to, and in the proximity of, the HMC septum. Additional HMCs are formed when a secondary hypha or a tertiary hypha adheres to a plant cell. Infection sites with HMCs were observed at 24 hpi and at subsequent sampling stages. There were no significant differences between the infection processes on the three wheat lines examined in this study. Infection structure morphology may provide additional criteria valuable for the identification and classification of rust fungi on grasses and cereals (Niks, 1986 ; Niks & Dekens, 1991 ; Swertz, 1994). Many studies have been done on infection structure formation of rust fungi with light and transmission electron microscopy. However, until quite recently, scanning electron microscopy (SEM) of rust infections has been confined to describing infection structure differentiation from urediospores on artificial, host and non-host surfaces. Little SEM information is available on the initiation and formation of the substomatal vesicle and its subsequent development. Hughes & Rijkenberg (1985) developed a leaf-fracture technique in their studies on the early infection stages of Puccinia sorghi in Zea mays that enables the investigation of early infection structures in both host and non-host tissues. This technique has been applied to studies of several other rust–plant (host and non-host) interactions including those of Uromyces transversalis in Gladiolus and Zea mays (Ferreira & Rijkenberg, 1989), P. graminis f. sp. tritici in Triticum aestivum and several cereal species (Lennox & Rijkenberg, 1989) and Hemileia vastatrix in Coffea spp. and Phaseolus vulgaris (Coutinho, Rijkenberg & Van Asch, 1993 a). Davies & Butler (1986) used a similar method to describe infection structure formation by P. porri in leaves of leek (Allium porrum). The infection process of Puccinia recondita Roberge ex Desm. f. sp. tritici Erikss. & Henning (syn. P. triticina) has been investigated by light microscopy (Allen, 1926 ; Romig & * Current address : Department of Plant Pathology, University of Arizona, Tucson, AZ 85721, U.S.A. Caldwell, 1964 ; Niks, 1983, 1986 ; Lee & Shanner, 1984 ; Staples & Macko, 1984 ; Jacobs, 1989, 1990 ; Southerton & Deverall, 1989 ; Kloppers, 1994 ; Swertz, 1994). Details of early morphological development within the host, however, are few. The objective of this research was to describe by SEM the ontogeny and morphology of infection structure formation by P. recondita f. sp. tritici in susceptible and resistant near isogenic lines of wheat (Triticum aestivum L.). The leaf fracture method of Hughes & Rijkenberg (1985) was used. MATERIALS AND METHODS Plant materials, rust propagation and inoculation The near isogenic wheat lines used were RL 6040 and RL 6043, which have leaf rust resistance genes Lr19 and Lr21, respectively, and susceptible cv. Thatcher (kindly provided by Professor Z. A. Pretorius, Department of Plant Pathology, University of the Orange Free State, South Africa). RL 6040 is Thatcher*7}translocation 4 (derived from Agropyron elongatum) and is highly resistant to UVPrt 8 while RL 6043 is Thatcher*6}RL 5406 (Tetra Canthatch)}Aegilops squarrosa cv. Meyeri-RL 5289 and is intermediately resistant to UVPrt 8 at 20 °C. All seedlings were grown singly in 10 cm pots maintained at 18–20° in a leaf rust free growth chamber with a 12 h photoperiod. The South African pathotype UVPrt 8 of P. recondita f. sp. tritici (from Professor Z. A. Pretorius) was employed in this study. Race UVPrt 8 has an avirulence}virulence formula of 3a, 3bg, 3ka, 11, 16, 20, 26, 30}1, 2a, 2b, 2c, 10, 14a, 15, 17, 24. P. recondita f. sp. tritici infection structures 392 Figs 1–6. Fig. 1. Urediospore germ-tube on RL 6043 leaf at 6 hpi (bar, 40 µm). Fig. 2. Two appressoria over stoma. 6 hpi on RL 6040 (bar, 10 µm). Fig. 3. Appressorial lobes are present on periphery of appressorium. 6 hpi on RL 6043 (bar, 8 µm). Fig. 4. Mucilage produced by appressorium (Cryo-SEM). Appressorium contents have moved into the SSV. 24 hpi, Thatcher (bar, 12 µm). Fig. 5. A young appressorium detached from RL 6040 leaf showing rugose texture of lower surface of appressorium on which imprint of guard cells appears (bar, 8 µm). Fig. 6. Interconnective tube is visible after epidermis is stripped off. 24 hpi in Thatcher (bar, 2±4 µm). Abbreviations. Fungal structures : A, Appressorium ; B, Branches ; G, Germ-tube ; HM, Haustorial mother cell ; IT, Interconnective tube ; L, Lobe ; PH, Primary infection hypha ; PHi, Primary infection hypha initial ; S, Septum ; SH, Secondary hypha ; SHi, Secondary hypha initial ; SSV, Substomatal vesicle ; U, Urediospore. Host structures : M, Mesophyll cell ; Gi, Guard cell imprint. G. Hu and F. H. J. Rijkenberg 393 Figs 7–12. Fig. 7. Mature ellipsoidal SSV parallel to stomatal slit. 12 hpi in Thatcher (bar, 10 µm). Fig. 8. SSV oriented perpendicular to leaf surface. 24 hpi in RL 6040 (bar, 10 µm). Fig. 9. SSV positioned at right angle to stomatal opening. 12 hpi in RL 6043 (bar, 10 µm). Fig. 10. Two SSVs situated on same stomatal slit. 12 hpi in RL 6040 (bar, 17 µm). Fig. 11. Amorphous material on surface of SSV. 24 hpi in RL 6040 (bar, 7 µm). Fig. 12. Collapsed stub-shaped SSV, orientated parallel to stomatal slit. 24 hpi in RL 6040 (bar, 6 µm). P. recondita f. sp. tritici infection structures The respective reactions to this pathotype were : RL 6040 ¯ 0 ; RL 6043 ¯ 2, Thatcher ¯ 4. Freshly harvested urediospores of P. recondita f. sp. tritici produced on 15 d old plants of the susceptible wheat cv. Agent in a greenhouse (20–26°) were used to inoculate the adaxial surface of the first leaf of 10-d-old plants of wheat lines at an inoculum dose of 50 mg urediospores ml−" of Soltrol 130 (Phillips Chemical Co.). A modified Andres & Wilcoxson (1984) inoculator was used to inoculate the plants. To prevent Soltrol damage to leaves, inoculated seedlings were allowed to dry for approximately 1 h before placement in the dark in a dew chamber at 20° and 100 % r.h. for 24 h. Inoculated leaves of 10 seedlings of each wheat line were harvested at 6, 12, 24, 48, 72, 96, and 144 h post-inoculation (hpi). At 24 hpi, seedlings were removed from the dew chamber and placed in a growth chamber at 18–20°. Specimen preparation for SEM Harvested leaves were cut into about 3¬3 mm squares and fixed in 3 % glutaraldehyde in 0±05  sodium cacodylate buffer, pH 6±8–7±2, for 24 h, washed twice in the buffer, postfixed for 2 h in 2 % osmium tetroxide in the buffer, washed twice in the buffer, then dehydrated in a graded ethanol series. Specimens were critical-point dried with carbon dioxide as a transition fluid and were mounted on copper stubs. The leaf-fracture method of Hughes & Rijkenberg (1985) was used on specimens harvested at 12–96 hpi. Both stubs used in fracturing were processed for observation. Samples harvested at 6 and 144 hpi were left unfractured. All stripped epidermis and the tissue remaining on stubs were gold}palladium-coated in a Polaron sputter coater. Materials were examined with a Hitachi S-570 scanning electron microscope operating at 8 or 10 kV. Specimens harvested at 6 and 144 hpi were scanned only exteriorly. In some instances, after freeze-fracturing in an EM Scope SP 2000 cryo unit, or without such fracturing, freshly harvested materials were viewed at 8 kV. Counts of infection structures were made directly from the screen. RESULTS At germination, a germ-tube protrudes form a germ pore in the urediospore wall and then elongates closely appressed to the cuticular leaf surface. Along the germ-tube’s length, several exploratory branches are formed at anticlinal wall depressions (Fig. 1). Normally, germ-tubes extend perpendicularly to the leaf long axis (Fig. 1). When a stoma is encountered by a germ-tube, an appressorium is formed terminally (Fig. 1). Occasionally, germ-tubes fail to recognize the stoma. In this case, an appressorium is not formed and the germ-tube passes the stoma. Initially, the appressorium is approximately spherical. As it matures, it enlarges and becomes oval or oblong, with its long axis orientated parallel to the stomatal slit (Fig. 3). A mature appressorium is delimited from the germ-tube by a septum (Figs 2, 3). In many instances, two (Fig. 2), or even three appressoria are seen on a single stoma. Moreover, in many instances, a urediospore on or near a stoma produces 394 Table 1. Frequencies of long axis orientation of the stomatal vesicles of Puccinia recondita f. sp. tritici to the stomatal slits of the host Orientation Observed Expected Parallel Perpendicular Others 56 48 10 38 38 38 Note : The following χ# tests were performed : χ# (#) (parallel v. perpendicular v. others) ¯ 31±78, P ! 0±001 ; χ# (") (parallel v. perpendicular) ¯ 11±15, P ¯ 0±001 ; χ# (") (perpendicular v. others) ¯ 23±26, P ! 0±001 ; χ# (") (parallel v. others) ¯ 29±16, P ! 0±001. either a short germ-tube or a germ-tube appears to be absent from the spore}appressorium combination (Fig. 3). Some urediospores with long germ-tubes do not develop appressoria by 12 hpi. An appressorium at its periphery produces 4(–6) nearsymmetrically orientated lobes which appear to adhere closely to the guard cell surfaces (Fig. 3). The appressorium appears to secrete a mucilage that bonds it to the host cuticle (Fig. 4). At 6 hpi, mature appressoria typically are observed on leaves of all susceptible and resistant wheat lines. The lower surface of the appressorium, observable after it has been stripped from the stoma, has a rugose texture and carries an imprint of parts of the stoma to which it was attached (Fig. 5). Particles of host epicuticular wax appear to adhere to the appressorium surface (Fig. 5). The appressorium, albeit in collapsed form, remains on the leaf surface even 144 hpi. No significant difference is observed between the appearance of the appressorium on susceptible and that on resistant wheat near-isogenic lines. Originating from the lower surface of the appressorium, a single infection peg swells into a substomatal vesicle (SSV) in the substomatal chamber after entry through the stoma. The blade-like connection between the appressorium and the SSV has been termed the interconnective tube by Hughes & Rijkenberg (1985) and Lennox & Rijkenberg (1989). A septum delimits the SSV from the interconnective tube (Fig. 6). When the SSV is mature and the transfer of cytoplasm to this structure has taken place, the appressorium and the germ-tube on the exterior plant surface collapse. The SSV initial emerges from the stomatal slit as a round or near-spherical structure (approximately 8¬6 µm) and then increases in both dimensions to about 11¬7 µm. A subsequent increase in length is not associated with a further increase in width. The mature SSV is oblong or ellipsoid (Figs 7–9). At 12 hpi, the most mature SSVs measure about 22–25¬7–8 µm. In most cases, elongated SSVs lie parallel to the long axis of the stomatal slit (Fig. 7). However, other orientations of SSVs have also been observed (Figs 8, 9). Some are orientated perpendicular (or nearly so) to the stomatal opening, and develop further in this direction (Fig. 8). Few SSVs are orientated at right angles to the stomatal slit (Fig. 9). A statistical comparison between the types of SSV orientation (parallel, perpendicular, and at right angles) indicates that there is a nearly equal tendency for the SSVs to position their long axes perpendicular to the stomatal slit or parallel to it (Table 1). Occasionally, an amorphous material is associated with the collapsed SSV (Fig. 11). At 12 hpi, many mature SSVs G. Hu and F. H. J. Rijkenberg 395 Figs 13–18. Fig. 13. SSV parallel to leaf vein forming a primary hypha initial. 24 hpi in Thatcher (bar, 3 µm). Fig. 14. Primary hypha initial perpendicular to the stomatal slit on the chamber side of SSV which has formed at right angle to stomatal opening. 24 hpi in Thatcher (bar, 5 µm). Fig. 15. Primary hypha that has elongated between mesophyll cells. 24 hpi in Thatcher (bar, 5 µm). Fig. 16. Septum delimits HMC from primary hypha. 48 hpi in RL 6043 (bar, 3 µm). Fig. 17. HMC has formed on contact with host cell. Secondary infection hypha arises on the SSV side of HMC septum. 48 hpi in RL 6040 (Bar, 7 µm). Fig. 18. One HMC has formed where primary hypha has become attached to mesophyll cell and secondary hypha initial has formed in proximity of HMC. 48 hpi in Thatcher (bar, 7 µm). P. recondita f. sp. tritici infection structures 396 Table 2. Percentage of infection structures discovered at specific time intervals post-inoculation Hours post-inoculation Knott 12 Substomatal vesicle Collapsed SSVs Primary hypha Collapsed primary hypha Primary hypha with HMC Secondary hypha Intercellular mycelium and HMCs Total sites RL 6043 24 48 96 12 Thatcher 24 48 96 12 24 48 96 80 20 18 7 10 81 19 58 14 46 26 2 81 19 55 9 52 42 24 72 28 62 15 55 44 32 86 14 20 9 12 88 12 62 13 47 23 3 84 16 61 4 53 42 27 90 10 77 10 70 56 40 89 11 22 11 9 88 12 60 11 41 19 4 92 8 65 9 53 41 23 90 10 85 5 75 59 36 102 170 170 279 116 203 178 125 89 185 211 168 Data in table are based on the cumulative totals. Figs 19–22. Fig. 19. Abnormal formation of infection hyphae. SSV is perpendicular to stomatal slit. One primary hypha (arrowhead) has arisen from a location on SSV close to stomatal slit, while another (arrow) is located terminally at the end of the same SSV. HMCs and secondary hyphae have formed. 48 hpi in RL 6040 (bar, 10 µm). Fig. 20. Possibly collapsed mature HMC. 48 hpi in RL 6040 (bar, 2±4 µm). Fig. 21. Cryo-fractured leaf tissue. Haustorium in mesophyll cell. Note haustorial neck. 48 hpi in Thatcher (bar, 7 µm). Fig. 22. Overview of the infection by P. recondita f. sp. tritici. Appressorium on leaf surface has collapsed and intercellular hyphae ramify in the leaf tissue. 72 hpi in RL 6043 (bar, 24 µm). G. Hu and F. H. J. Rijkenberg are found in both susceptible and resistant wheat lines. A number of stomatal chambers are seen in which two SSVs have formed (Fig. 10). The near-spherical or stub-like SSVs (Fig. 12), often collapsed, observed at the later sampling stages, are assumed to be aborted structures. In instances where the SSV is parallel to the stomatal slit, the SSV progressively develops and elongates unilaterally, while remaining closely attached to the inner epidermal surface, in a direction parallel to the long axis of the stomatal slit, to form a primary hypha (Fig. 13). In instances where the SSV develops in a direction perpendicular to the long axis of the leaf, a primary hypha forms from one end of the SSV, generally in the proximity of a mesophyll cell, and extends further (Figs 14–17). In a few cases, the primary hypha arises from the surface of the SSV, away from the host epidermis, rather than from the end, in a direction perpendicular to the stomatal opening, and extends further into the mesophyll (Fig. 18). At 12 hpi, early stages of SSV development and the formation of primary infection hyphae are observed. A small number of collapsed SSVs is also found at this stage in all wheat lines investigated. More collapsed SSVs are counted at 24, 48, 72 and 96 hpi. Some SSVs (often of the near-spherical type) have formed a very elongate and slender primary infection hypha which apparently does not develop beyond this stage. Hence these are regarded as abortive primary hyphae and SSVs. Generally, relatively low numbers of the atypical primary hyphae are recorded at 48 hpi and the subsequent stages of harvest (Table 2). Once the primary infection hypha expands fully and}or becomes attached to a mesophyll cell or epidermal cell, a septum is produced which delimits a presumed haustorial mother cell (HMC) from the primary hypha at the hyphal tip (Fig. 16). This septum is seen at 24 hpi and thereafter. After the SSV and the primary infection hypha develop, a secondary infection hyphal initial may emerge at several sites on the primary hypha (Fig. 17). A secondary infection hypha mostly arises at a position adjacent to, or in the proximity of, the HMC on the SSV side of the HMC septum of the primary infection hypha (Figs 17, 18). By 48 hpi, many HMCs have formed from the secondary hyphae which form in this manner (Table 2). Abnormal patterns of secondary hypha formation are occasionally observed in leaves of both susceptible (Fig. 19) and resistant wheat lines. Some haustorial mother cells are collapsed (Fig. 20). The mature haustorial mother cell penetrates the host cell and a haustorium is formed (Fig. 21). Additional intercellular hyphae originate from the secondary infection hyphae with HMCs, and form the fungal thallus (Fig. 22). Relatively more collapsed SSVs and collapsed primary infection hyphae are observed in the tissues of the resistant line, i.e. RL 6040 (Lr19), than in the intermediately resistant RL 6043 (Lr21) and susceptible Thatcher lines (Table 2). However, there are no significant structural and statistically numerical differences in infection structure development between the wheat lines. At 144 hpi, uredia with a number of immature urediospores are found in Thatcher while fewer urediospores are found in RL 6043 and no sporulation is observed in RL 6040. 397 DISCUSSION Overall, infection structure formation of P. recondita f. sp. tritici on and in susceptible and resistant wheat lines in the present investigation is similar to that observed for other rust fungi (Hughes & Rijkenberg, 1985 ; Ferreira & Rijkenberg, 1989 ; Lennox & Rijkenberg, 1989 ; Coutinho et al., 1993 a). P. recondita f. sp. tritici germ-tubes grow and elongate perpendicular to the long axis of the leaf on susceptible, intermediately resistant and resistant wheat lines. Dickinson (1969) noted the directional growth of germ-tubes of P. recondita f. sp. tritici along the transverse axis of the plant surface. Dickinson (1970) suggested that the elongation toward the stoma was a curved thigmotropic stimulus. Directional growth of germ-tubes has also been observed to occur in other rust fungi, such as P. graminis f. sp. tritici (Johnson, 1934 ; Lewis & Day, 1972 ; Lennox & Rijkenberg, 1989), and Uromyces phaseoli var. typica (Wynn, 1976). Several investigators have proposed that the physical or chemical features of the leaf surface may influence the direction of growth. These may include cuticular ridges (Wynn, 1976) and patterns of epicuticular wax crystals (Lewis & Day, 1972), or pH gradients at the leaf surface (Edwards & Bowling, 1986). However, Hughes & Rijkenberg (1985) reported that P. sorghi germ-tubes grow towards maize stomata randomly as they traverse both axes of the leaf surface. Coutinho, Rijkenberg & Van Asch (1993 b) also noted that Hemileia vastatrix germtubes appeared to lack directional growth on both host and non-host leaf surfaces. Wynn (1976) proposed that random growth of germ-tubes may be due to the lack of close adhesion between the germ-tubes and the leaf surface. In the present investigation, some urediospores which landed on the stomatal pore directly or near the stoma develop appressoria without the apparent formation of germ-tubes, or form short germ-tubes, while a few urediospores with long germ-tubes fail to produce appressoria. Niks (1990) observed a negative correlation between the germ-tube length of P. hordei on Hordeum vulgare and establishment of a colony, and was of the opinion that the formation of a long germ-tube and exploratory branches reduced the amount of energy available to infect the plant. Ferreira & Rijkenberg (1989) showed that germ-tubes of Uromyces transversalis which failed to locate stomata often attained considerable length. The appressorium of P. recondita f. sp. tritici develops 4–6 lobes. The lobes appear to play a role in the adhesion of the appressorium to the stomatal apparatus. A putative mucilage, apparently formed by the appressorium, may play a role in cementing the appressorium to the host surface. More than one appressorium over a stoma has been reported in other host–pathogen interactions (Niks, 1981 ; Falahati-Rastegar, Manners & Smart, 1983 ; Ferreira & Rijkenberg, 1989 ; Lennox & Rijkenberg, 1989 ; Coutinho et al., 1993 b). In the present investigation, P. recondita f. sp. tritici urediospore germ-tubes occasionally form two or even three appressoria on one stoma which result in more than one functional substomatal vesicle occupying the same substomatal chamber. Torabi & Manners (1989) proposed that the proportion of appressoria of P. recondita resulting in successful P. recondita f. sp. tritici infection structures penetration was greater when two or more appressoria occurred over a stoma. Pole-Evans (1907), Niks (1983, 1986), Helfer (1987) and Swertz (1994) noted that the SSV of P. recondita f. sp. tritici tended to be orientated mostly parallel to the stomatal slit with the primary infection hypha them becoming oriented perpendicular to the leaf surface. In the present investigation, however, the orientation of SSVs of P. recondita f. sp. tritici in the substomatal chamber is found to be mainly either parallel or perpendicular to the stomatal opening. Swertz (1994) also observed that SSVs were oriented perpendicular to the leaf surface with primary hyphae parallel to the long axis of the leaf and proposed that the orientation of SSVs was highly influenced by temperature. Our experiment was carried out at 18–20° to enable Lr19 and Lr21 to express their resistance properly (Z. A. Pretorius, pers. comm.). Perpendicular orientation of the SSVs has also been described for P. porri on Allium porrum (Davies & Butler, 1986) and U. transversalis on Gladiolus (Ferreira & Rijkenberg, 1989). Ferreira & Rijkenberg (1989) postulated that SSV orientation has co-evolved with, or adapted to, substomatal chamber orientation. In the present study, there is no significant difference in SSV orientation between the susceptible, intermediately resistant and highly resistant wheat lines. The growth and extension of the primary infection hypha occurs at one side of the SSV. Similar to those of P. sorghi (Hughes & Rijkenberg, 1985) and P. porri (Davies & Butler, 1986), the primary hypha of P. recondita f. sp. tritici is twocelled. The terminal cell of the primary hypha is a haustorial mother cell (HMC). In most cases, the secondary infection hypha of P. recondita f. sp. tritici arises on the SSV side of the septum separating the primary hypha from the HMC, as reported in P. sorghi (Hughes & Rijkenberg, 1985), U. transversalis (Ferreira & Rijkenberg, 1989), P. graminis f. sp. tritici (Lennox & Rijkenberg, 1989) and H. vastatrix (Coutinho et al., 1993 a). 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