Research Papers (Etiology: Mycology) :6t++...rr) rlj---.-r+

The Infection of Vascular Tissue of Crataegus monoglna by thle Monoka ryo tic Sta ge of Gy mno sp or angiu m c lav ariifor me

L. Larous and D.lU. Losel Institute de Biologie, Universite Ferhat Abbas, Setif 19000, Algerie

Abstract Larous, L. and D.M. Losel. 1996. The infection of vascular tissue ol Crdlaegus monogrra by the monokrryotic stage of Gymnosporangium clava iforme. Arab J, Pl. Prot. l4(l)t 57-62. The fne strurtwe of Crataeg$ monog,na mfeoted with the monokaryotio strugtwes of Gymnospomnghrm clavoiiforme was investigated. The distribrtion of fimgal thallus within host tissue, types of infection sfuctures and intersotions with host cells have been described with particular reference to vascular system infection. A large nucleus md the formation of8 septum.tthe level oforc p€netration site is Aequ€ntly observed. This stage of infection is characterized by the extensive growth offimgel structues in tlle htercellular spaces of leaves mesophyll, with low frequency of penetration of these cells, and the grester degre€ of invasion of all Spes of cells in the host plant vascular systeNn. Key words: Gyunosporangium clavaiilorme, ultastucture. vascular infection.

Introduction Results The ultrastructure of fungr has been studied Infection struclures extensively, particularly with respect to the uredinial stages The intercellular spaces of mesophyll become almost of infection (15, 21). It is generally known that obligate completely filled with the intercellular hyphae of the parasites, such as rust fungr form specialized type of monokaryotic stages of G. clavariiforme, but the fungal haustoria in host cells, during the uredinial-telial phases of penetration of these cells is less frequent than it is in the infection. This type of haustoria (Dikaryotic) is demonstrated vascular tissue region (Fig 1). to be morphologicatly difrerent from those produced by spermogonial-aecial stages of infection (l I , 12, 22,24). Most of these investigations have confirmed that the distribution of 's. fungal dikaryotic structures is confined to leaf mesophyll and t stem cortex cells. In contrast, recent reports have shown that L t the rust monokaryotic structures can invade different type of t) cells in the vascular tissues of host plant ( I , 5, 19 , 20). In an attempt to obtain a clearer understanding of physiologrcal and structural relationship of the monokaryotic infection of rust fungr, the distribution and nature of spermogonial-aecial ultrastructures of G. clavariiforme were y',tt investigated..

Material and Methods Light and electron microscopy Naturally infected leaves of Crataegus monogwa Jaq. bearing spermogonia and aecia of G. clavariiforme were .J collected from the field. Portions of infected leaves were processed for light and electron microscopy, by the procedure ( of Woods and Gay (26). The tissue was fixed in 2.5o/o * glutaraldehyde in 0.1 M. sodium cacodylate buffer, pH 7.4, \t post-fixed in l% osmium tetroxide in the same buffer, dehydrated in a series of ethanol concentrations and embedded in Araldite. Semi-thin sections for light microscopy were stained with toluidine blue. Ultrathin sections for electron microscopy were cut with a Reichart microtome using a diamond knife, post stained with 2o/o aqueous uranyl acetate, followed by lead citrate (23), and examined with Phillips 301 electron microscope. Figure 1. LM micrograph of C. monog/na leaf mesophytl inlixted with the monokaryotic stage of G. clavaidorme, showing the dense growth of intercellular hyryhae and the relatively ffiequent penetration ofthese cells. 40X.

(1996) I .r.rp ,14 .rl+r ,L,.ll clt+i$ L.EJ ilp 62 The intercellular hyphae were often observed partially the infected host cell. They arise relatively from a small or completely ernbedded in the host cell wall. Serial sections haustorial mother cell (HMC), which is always found from different specirnens showed that the nuclei has no appressed tolor embedded completely in host cell wall nucleoli and contain a variable amount of hetero- and material (Figs. 4, 5 and 6). The HMC uall appears of the euchronntin similar to that in the filamentous haustoria. szrme thickness as that of the filamentous haustoria and much Contrasting to the young hlphae. the older ones were more electron dense than the host wall. A septum separating vacuolated, with the cytoplasm confined to alayer lining the the HMC from developing haustoria is found repeatedly at cell wall (Figs. 2 & 3). the level of host cell wall penetration or proximal to it, which can be either complete or perforated. The pore apparatus of mature perforate septa are associated with numerous microbodies (Fig. 5). The filamentous haustoria are not constricted at the penetration site, and have no clearly differentiated neck region. The neck ban{ characteristic of dikaryotic haustoria, is absent, they possess one well developed large nucleus (Fig. 4). t There is no sign of break-down of the invaginated host plasmalemma which surrounds the haustorial body, and is separated from the haustorial wall by the extra-haustorial matrix @M) This matrix is relatively thin in young haustoria and larger in older ones and consists of two layers differing in chemical cornposition, the outer layer is more electron lucent than the inner and resembles host cell wall material. The inner contains fibrillar structures projecting outwards from the haustorial cell wall (Fig. 7).

Host response to infection The collars observed around the intracellular structures of the monokaryotic infections of G. clavariifurme are Frgure 2. Vacuolated intercellular hyphae (I) in leaf mesophyll. continuous with and indistinguishable from host cell wall some are partially embedded in host cell wall. Note the oval and material (Figs. 4, 5 and 6). The host endoplasmic reticulum elongated shape ofthe nuclei. 12400X (ER), chloroplasts, and mitochondria are in close association with the extrahaustorial membrane @HM) (Figs. 3 and l0).As the infection progress, the host cytoplasm becomes less dense in appearance with loss of ribosomes and mitochondria, and decreasing amount of ER.

r Yascular infection The spermogonial-aecial stages of G. clavariiforme showed extensive invasion of the conducting system of the infected leaves. Dense intercellular growth of hyphae is observed, not only in the mesophyll but also in the vascular tissue (Fig. l), and hyphae are frequently observed partially or completely embedded in host wall material (Figs. 8, 9 and l0). Intracellular structures of this rust occur within phloenr parenchyma and rylem vessels. where they are sometimes embedded in lignified bands of wall thickening and also in bundle sheath cells (Figs. 8, 9 and 10). The intracellular hyphae in rylem parenchyma, and phloem parenchym4 appear metabolically active (Fig. l0), although some are largely vacuolated. They contain t' mitochondria and dense cytoplasm, and are surrounded by the EM, bounded by the invaginated host plasmalemma. In this host cells, the rytoplasm generally remains well supplied Figure 3. Old monokaryotic haustorium in phloem region with organelles, including chloroplasts, mitochondria and surrounded by the extra-haustorial matrir (EM). The host ER. In contrast, the haustoria observed within xylem vissels mitochondria, ER, and vesicules are in close association with the haustorium. t8750X. are generally necrotic in appearance and lack an EHM (Figs 8 and 9). The spermogonial-aecial stages (monokaryotic) of this are characterized by the existence of the filamentous haustoria, which are irregular in shape, and terminate within

6l Arab J. Pl. Prot. Vol. 14, No. 1 (1996) Discussion The haustorial cell content are generally similar to those Tlre filamentous haustoria of G. clat,ariiforme, in most reported from other rust (15, 2l), except for that the respects, are sirnilar to those described earlier in the fungi nucleus is characterized large size containing a spennogonial-aecial stages of other rust ftrngi (6, I l), and by a and variable amount heteroand euchromatin. The absence of differ from dikaryotic haustoria in the absence of the of nucleoli intercellular hyphae and haustoria thickening of the HMC wall at the penetration site, and in in both correspond to previous reports (3, 11). having little or no constriction at the neck region and no The formation of collars were frequently linked to the neck-band. Howeve r, Gltmn osporang i u nr h ara e nu m infecting degree host-pathogen compatibility the age leaves of Japanese pear does not fornr haustoria in its of or to of haustorium (9,21). However, the filamentous haustoria of G. monokaryotic stage (18). The HMC wall is continuous and is clavariiforme partially encased in material of unifonn thickness with the wall of the haustorium. Harder is which appqrs as an extension to the host cell wall, having the same staining ( 14) considered thern as haustoria with a prirnitive level of properties host material found specialization, in that they lack some of the structural to it. Similar wall-like was around monokaryotic haustorium P. podophylli features of more specialized haustoria. However. the the of described Borland (6), poqrum (27), P. rnonokaryotic haustoria of Endctcronartium harknessi, (16), by and Mims P. (17). ('ronartium quercuum (13). and Endocronarlium pini (25) coronata Q$ and E. harlcnessi, The host wall-like rnaterial observed here seems to be different in cornposition are reported to have a constricted neck and expandedbody, from that in dikaryotic infection of rust fungr. although lacking a neck-band. The septal pore apparatus is sinrilar to that found in P. poarum (3) and P. punctiftrmis ( 5).

H S N

H

H/V\C

MC

Figure 5. Monokayotic haustorium (fD of G. clavariiforme showing a perforate septum (S) proximal to the panetration site, crystal containing microbodies are closely associated with the pore apparatus. 12400X.

The EM had previously been suggested to be of host origin (7, L4, 16), fungal origin (10) or an artifact of preparation (4) Recent cytochemical studies on HMC graminis (8) and on P. menthae (-arous and Losel, unpublished) support the point of view that it is of host origin. The continuity of EHM with the host plasma membrane, commonly accepted for both mono and dikaryotic haustoria. The extensive growth of the monokaryotic stages of G. Figure 4. Filamentous haustorium (tt) in mesophyll cell (MC), the clavariiforme in the intercellular spaces of the leaf furrg"l wall is densely stained and is of uniform thickness, there is mesophyll, accompanied by lowfrequency ofpenetration of no constriction at the penetration site, and the neck lacks a neck- cells in these tissues and the grater degree of invasion of the band. Note the localization ofthe septum and the oval nucleus (I.l-). t0000x. vascular system, correspond with the observation on the infection by P. poqrum of Tussulagofarfara (1). The above (1996) I r.e .14 $L. .L..fl €i+ill L,t33 iJy 60 observation, together with those of a series of ultrastructural studies by Harder (14) Al-Khesraji et al. (z),Baka, and Losel -(. (5) and Larous and Losel (19, 20),pointtotheconclusion that invasion of the vascular system is frequent in primary, or { spermogonial-aecial stages of rust infection but absent in the D secondary, uredinial-telial phases of infection. As in the systems described in the above studies, the cytoplasm in ?E' vascular tissue infected by G. clavariiforme remains of relatively healthy ultrastructural aprpearance. The seemingly gteater tolerance to infection by cells in the vascular region than in the mesophyll may be a consequence of their continuing supply of nutrients and would be consistent with the view that the adverse effect of infection on mesophyll cells result mainly from nutrient depletion by the pathogen. LI

\ cw Figure 8. Necrotic, filamentous haustorium GD of G. clavariiforme in xylem vessl (XV) of the Ci. monogtna le*rf. The haustorium --- mother cell ([IMC) is embedded in rylem vessel wall. 7500X

t* A -\, Figure 6. Haustorial mother cell (HMC) penetrating a mesophyll cell of C. monog/no Ieaf The host wall-like material (arrow heads) is surrormding the haustorium neck. 25000X.

IT L 7f- /

H .s:l \ Figure 7. A paft of a haustorium showingelectrondenseextra- Figure 9. The spermogonial-aecial stages of G. clavariiforme haustorial matrix (EM) surrorurding the haustorial body and is infecting C. monogtno leaf vascular tissue showing a necrotic composed oftwo different layers. The inner layer is more electron hatutorium (H) in xylem vessel (XV) contains vesicular bodies, dense than the outer, enclosed by the extra-haustorial membrane vacuoles and lipid droplets. Note also intercellular hyphae (I) (ErM) 32500X. embedded in xylem vessel wall with alarge nucleus (N). 7500X.

59 Arab J. Pl. Prot. Vol. 14, No. I (1996) ffi'/*t' *?yi.I t-'tl #i* t,,qB I tlil.-.t',t

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trigqr. I0. A h|ustorirm of G. clavoitfunae with exta-haustorial mstrix bounded by lhe invaginated host plssms membrang in xylem parenchyma c€ll O(P) adjscent to rylenr vessel (XV) of C. monogma. An'fracellular hyphae (D is €mHded completely in host well material. Note the olganelle-rich cyoplasm of both host rnd fimgal cells. 10000X

o.iLll ir-.ll *il il3r-ill 4rt-rl s)A4 Crataegus monogtna g*J .,'or-1ll C-^"lt iri'eJll iJ,Jyl .1996 .,JjrJ .p.r r ,i.yll .et! .62-37 t

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57 Arab J. Pl. Prot. Vol. 14, No. I (1996)