Evidence for the Importance of Enzymatic Digestion of Epidermal Walls During Subepidermal Sporulation and Pustule Opening in White Blister Rusts (Albuginaceae)
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mycological research 113 (2009) 657–667 journal homepage: www.elsevier.com/locate/mycres Evidence for the importance of enzymatic digestion of epidermal walls during subepidermal sporulation and pustule opening in white blister rusts (Albuginaceae) Annerose HELLER*, Marco THINES Institute of Botany 210, University of Hohenheim, Garbenstrasse 30, D-70599 Stuttgart, Germany article info abstract Article history: Albugo candida, A. ipomoeae-panduratae, Pustula tragopogonis, Wilsoniana bliti and W. portulacae are Received 1 July 2008 widespread obligate biotrophic plant pathogens causing white blister diseases on a variety Received in revised form of flowering plants. Their subepidermal mode of sporulation is unique amongst Oomycetes 22 December 2008 and leads to blister-like structures on their hosts similar to those produced by true rusts Accepted 30 January 2009 (Uredinales). Unlike in true rusts, sporangia are colourless and produced in chains; the first Published online 7 February 2009 formed, primary sporangium, differing in size and morphology from subsequent secondary Corresponding Editor: sporangia. According to current interpretations of pustule development the rising pressure Nicholas P. Money of the growing chains of sporangia tear off the epidermal layer from the mesophyll and, in the end, ruptures the epidermis to release the sporangia. This is not convincing consider- Keywords: ing the rigidity of the epidermal layer and the fact that thin-walled mesophyll cells show Albuginales no signs of pressure endurance. Our detailed light-, scanning electron-, and transmission Albugo candida electron microscopic observations provide evidence that pustule development and opening Albugo ipomoeae-panduratae are regulated and delicate processes that involve directed enzymatic dissection of host tis- Electron microscopy sue cell walls. The process starts when intercellular hyphae separate the epidermal layer Light microscopy from the parenchyma, forming a cavity in which sporulation takes place. Then thick- Oomycetes walled sporogenous hyphae with club-shaped but thin-walled tips develop and produce Pustula tragopogonis sporangia in basipetal succession from the apices of the sporogenous hyphae. The short- Pustule opening living primary sporangia attach tightly to the inner cell walls of the epidermal layer and un- Sporulation dergo dramatic cytological changes during pustule maturation, including vacuolisation and Ultrastructure development of numerous electron-dense vesicles that might deliver cell wall degrading Wilsoniana bliti enzymes. In ripe pustules, the disintegration of areas of epidermal cells leads to the open- Wilsoniana portulacae ing of the pustules and to the release of the secondary sporangia. Also the comparison of samples prepared for scanning electron microscopy with fresh pustules, as well as the comparison of the inner epidermal layers detached by the pathogens and detached by force supports our conclusion that delicate enzymatic activity and not force are involved in pustule development and opening by these highly sophisticated pathogens. ª 2009 The British Mycological Society. Published by Elsevier Ltd. All rights reserved. Introduction relatives of the downy mildews (Peronosporaceae), until it was revealed through the molecular phylogenetic investigations The obligate biotrophic plant parasites causing white blister of Riethmu¨ ller et al. (2002) and Hudspeth et al. (2003) that these disease (Albuginaceae) were previously considered to be pathogens are largely unrelated and, therefore, were placed in * Corresponding author. Tel.: þ49 711 459 22180, fax: þ49 711 459 23355. E-mail address: [email protected] 0953-7562/$ – see front matter ª 2009 The British Mycological Society. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.mycres.2009.01.009 658 A. Heller, M. Thines an order of their own by Thines & Spring (2005). Their unique mode of sporulation and oospore formation (Ruhland 1904; Materials and methods Stevens 1904) and their basal position in the ‘‘Peronosporalean galaxy’’ (Sparrow 1976) as inferred in Thines et al. (2008) may All plants were collected from sites with a natural occurrences even justify considering them as a subclass of their own to of the host plants: Capsella bursa-pastoris infected by A. candida acknowledge their distinctiveness. The three genera of the near Undine, Italy, Ipomoea hederacea infected by Albugo Albuginaceae hitherto described, Albugo, Pustula, and Wilsoni- ipomoeae-panduratae in the Great Smoky Mountains National ana contain numerous species which still await scientific Park, near Gatlinburg, TN, U.S.A., Tragopogon pratensis infected description (Choi et al. 2008). They often have a narrow host by Pustula tragopogonis s.l. near Ostfildern-Nellingen, range, although Albugo candida is apparently capable of para- Germany, Amaranthus lividus infected by Wilsoniana bliti in sitizing a great variety of Brassicaceae (Choi et al. 2006, 2007). the Botanical Garden of the University of Hohenheim, Stutt- The outbreak of the disease is characterized by host plants de- gart, Germany and Portulaca oleracea infected by Wilsoniana veloping white blister-like pustules on the lower side of leaves portulacae in Moos, Lake Constance, Germany. and on stems, but sometimes also on the upper leaf surface. In Photos of fresh leaves with developing pustules were taken all Albuginaceae, colonization of hosts and sporulation take with a Photo Macroscope M 400 (Wild, Heerbrugg, Switzer- place in a similar manner. Coenocytic hyphae ramify and land) coupled to a Canon Powershot A95 digital camera spread within the intercellular space of the host plants. The (Canon, Tokyo, Japan). Albuginaceae penetrate host cells only to establish their Samples for transmission electron and light microscopy globose haustoria. Except for their stalks, haustoria of the were prepared by using a razor blade to excise approximately Albuginaceae are similar to those found in some downy mildew 1-mm2 pieces of leaf tissue bearing sori in different develop- genera (e.g. Plasmopara (Fraymouth 1956)). In contrast to the mental stages; only for Capsella bursa-pastoris also stem tissue downy mildews, however, which sporulate by producing spo- was used. The samples were fixed in 5 % (v/v) buffered glutar- rangiophores protruding through host stomata, white blister aldehyde (0.1 M phosphate buffer, pH 7.2), and stored at þ 4 C rusts produce chains of sporangia in sori between epidermis for 1 d up to 29 d for samples of I. hederacea. Sample prepara- and mesophyll in leaves or between epidermis and cortical tion continued at room temperature by three washing steps parenchyma in stems. in 0.1 M phosphate buffer, pH 7.2, followed by a second The first-formed sporangia are always distinct from others fixation step in 1 % (w/v) buffered osmium tetroxide (0.1 M in the chains, a fact which has already been noted in early phosphate buffer, pH 7.2) for 1 h. After rinsing the samples mycological works (De Bary 1863; Tulasne 1854), but is often three times in distilled water, they were dehydrated in an ignored in more recent investigations and monographs ethanol series [30, 50, 70, 100, 100 % (v/v)], infiltrated and (Ananthanarayanan 1964; Jørstad 1964; Zhang & Wang 1998). embedded in LR-White resin, hard grade (Science Services, Recently, Constantinescu & Thines (2006) demonstrated that Munich, Germany) in gelatine capsules and polymerized in the sporangium dimorphism is present in all species of the an oven at 60 C for 24 h. Albuginaceae. The function of the primary sporangia remained All sectioning was carried out with an Ultracut UCT (Leica, unclear, however. Ga¨umann & Dodge (1928) and Ga¨umann Wetzlar, Germany) using glass and diamond knives. Serial sec- (1949) proposed the term ‘‘buffer cells’’ for these sporangia, tions of the pustules were taken in 50 mm intervals. Semi-thin which are usually sterile and often exhibit a massive wall sections of 1.5 mm were used to locate the pustules and to show thickening compared to the secondary sporangia. The first different developmental stages of the pustules. Sections of formed sporangia are not released from the pustules, but ad- 80–90 nm were used for investigations of the ultrastructure. here to the epidermal layer. They concluded that the function Ultrathin sections were mounted on pioloform-coated and of these cells is to detach and to lift the host epidermis. carbon-coated copper grids. These were post-stained for After the first sporangium is formed, multiple secondary 10 min each with a saturated aqueous solution of uranyl ace- sporangia will develop leading to dome-like swellings filled tate followed by lead citrate, 90 sec each (Reynolds 1963), and with sporangia, which finally open and expose a powdery examined with an EM 10 transmission electron microscope mass of spores, which are disseminated by wind, rain or in- (Zeiss, Oberkochen, Germany) operating at 60 kV. Semithin sects. It is generally assumed that in the Albuginaceae – and sections were mounted on glass slides, stained in 0.05 % aque- this is also found in recent text books (Webster & Weber ous toluidine blue O (Merck, Germany) for 30 min and exam- 2007) – the increasing pressure caused by the developing ined with an Axioplan light microscope (Zeiss, Go¨ ttingen, chains of sporangia, massed side by side between parenchyma Germany) coupled to a Canon Powershot A640 digital camera and epidermis, leads to a rupturing of the epidermal layer. (Canon, Germany). TEM negatives were scanned with an Epson However, this