Germination of Melampsora larici-populina uredosporeson poplar leaves'

By F. MLODZIANOWSKI, A. WERNER and R. SiwiccKi

Receipt of Ms. 26. 6. 1977

Abstract

Using an electron scanning microscope the germination of Melampsora larici-populina ure-dospores was observed on the lower leaf surfaces of a susceptible poplar clone Populus tricho-carpa and a resistant one P. X 'Serotina de Poitou'. The observations were made from a fewhours to more then a day after placing the uredospores on the leaf surfaces.

In the life cycle of Melampsora lariei-populina the uredial stage on the leaves of pop-lars constitutes the most important phase being of considerable pathogenic significance.Under favourable conditions the rapidly forming ureciospores infect le.ives on thesame or neighbouring trees. In this manner the disease spreads rapidly and reduces theassimilation surfaces of affected plants.

The uredial stage offers an easy opportunity to select resistant poplar clones. Com-parative studies in the Institute of Dendrology in Kornik on the resistant clone P. X'Serotina de Poitou' and a clone of the very susceptible /'. triehoearpa have demon-strated differences in the degree of chloroplast degradation in these poplars by the rust(MLODZIANOWSKI and SIWECKI 1976). Also the protein spectra of chloroplasts fromthese two poplars differ quantitatively and qualitatively (SIWECKI 1976). The leavesof P. triehoearpa have much larger stomata than P. X 'Serotina de Poitou' and thereis a difference also in their intensity of stomatal transpiration (STWECKI 1975, 1976).The morphology and ultrastrueture of uredospores collected on P. trichocarpa and onP. nigra was also compared (MLODZIANOWSKI and SIWECKI 1978).

The purpose of the present study was to follow the germination of uredospores onthe leaf surface of two poplar clones of different susceptibility. The studies were con-ducted using a scanning electron microscope.

Materials and methods

Uredospores of M. larici-populina were collected in July 1975 from natur.-\lly infectedleaves of Populus nigra in a stool bed. Immediately after collection under controlledconditions of moisture and temperature, the uredospores were brushed on to the lowerleaf surfaces of the poplar clones P. X 'Serotina de Poitou' and P. triehoearpa. Plantsof these poplars with well formed leaves had been raised earlier from cuttings in agreenhouse.

' This work has been partially supported by grant FG-Po-298 from the OS Department ofAgriculture under PI,-480 and partially by gram 10.2.10 from ihe Polish Academy of Sciences.

Eur. J. For. Path. 8 (1978) 119-125© 1978 Verlai- Paul Parey, HaniburR uiul licrliii

ISSN 0300-1237 / ASTM-Coilcn: EJ l 'PA 9

1 20 P- Mlodzianowski, A. Werner and R. Siwecki

The material was collected for examination after 4, 6, 12, 24, 48 and 72 hours fromthe time of inoculation. Small leaf fragments were cut out and fixed in 5 "/o glutaral-dehyde in a 0.1 M cacodylate buffer. After fixation the samples were dehydratedthrough an ethanol series and acetone. The air-dried material was covered with pla-tinum and gold. The observations were conducted using 1SM-V3 scanning electronmicroscope.

Results

As well as observations on the germination of uredospores the work permitted adetailed description and comparison of the morphology of leaves of the two poplars.Differences were observed in their lower epidermal surfaces which might affect thegrowth of uredospore hyphae.

The epidermis of the lower leaf surface of /'. trichocarpa was strongly folded (fig.1 and 2). On such a plicate surface it was often difficult to identify the fungal hyphae.Small, cylindrical surface structures were arranged particularily densely around thestomata forming characteristic radially arranged patterns (fig. 3).

In P. X 'Serotina de Poitou' the lower epidermis was also folded, but the hollowswere not as deep as in P. trichocarpa, and the crests of the pleats were not so denslypacked (fig. 4).

From earlier studies by WERNER (SIWECKI 1975) it appears that the mean lengthand width and the frequency of occurrence of the stomata on the lower epidermalsurface of the susceptible clone P. triehoearpa were much greater than on the resistantclone P. X 'Serotina de Poitou' (table 1). Similar conclusions may be drawn from thephotograms in figs. 2 and 4 made with the same magnification.

24 hours after placement of uredospores on the lower epidermis of P. triehoearpavery strongly ramified hyphae (fig. 5) were already visible. They appeared some-times to distinctly point towards the stomata, growing into them (fig. 6). After en-tering the leaf the hyphae spread in the intercellular spaces, and their walls were closelyappressed to the walls of the mesophyll cells of the host (fig. 7). This was earlierdescribed in greater detail (MLODZIANOWSKI and SIWECKI 1976).

On leaves of the resistant clone P. X 'Serotina de Poitou' germination had pro-gressed much more slowly, 24 hours after placement of uredospores lorming shortramified hyphae (fig. 8). After 24 and 48 hours since uredospores placement on P. X '.S'e-rotina de Poitou', no penetration by hyphae through stomata was observed, regardlessof whether they were open (fig. 9) or closed (fig. 10).

The results show clear differences in the degree of uredospore germination and inthe growth and development of the rust hyphae on the two clones. Further observa-

1

Fig. I. Surface of the lower epidermis of a P. trichocarpaleaf (X 660)

Germination of Melampsora larici-populina uredospores on poplar leaves 121

Fig. 2. Closed stomata on the lower epidermis of a /'. triehoearpa leaf (X 920). - Fig. 3.An open stomata in P. triehoearpa with the characteristic sculpturing of the epidermal surface(X 1150). - Fig. 4. Surface of the lower epidermi.s with a visihie stoma in P. X 'Serotina dePoitou' (X 920). - Fig.y The developinj; mycelium from several uredospores on the lowerepidermal surface of a /'. trichocarpa leaf 24 hours .ifter placing the uredo.spores on it (X 6S0)

Fig. 6. The rust hyphae on the lower epidermal surface of a susceptiblepoplar clone, P. trichocarpa, in most cases i;row in the direction of thestomata. The material was collected 24 hours since placing the uredosporeson the leaves (X 695). - Fig. 7. Cross section through a leaf of P. tricbo-carpa with visible uredospores on the surface and with hyphae in theintracellular spaces 48 hours after placing the uredospores on the leaves

(X 910)

122 F. Mlod'/.ianowski, A. Werner and R. Siwecki

tjons were then made m uredospore germination alter several hours on the lower leafsurface.

After 3—4 hours since placement on P. triehoearpa uredospores had germinatedabundantly and some hyphae were clearly growing in the direction of stomata (figs.11 and 12). In most cases germination tubes emerged from the poles of the uredospores(fig. 12). After 6 hours numerous ramifications of rapidly growing hyphae were seen.Some long hyphae grew directly towards the stomata (fig. 13). One (fig. 14) or some-times two, hyphae (fig. 15) were seen entering stomata.

In similar experiments with the resistant clone P. X 'Serotina de Poitou' no ger-mination tubes were observed. As mentioned above, this process was observed onlyafter 12 or 24 hours and then in a much inhibited form.

Discussion

The results might have been affected by the methods used to fix the material andprepare the slides. For example the greater pleating of the lower epidermal surfacein P. trichocarpa could have caused the retention of a greater number of germinatinguredospores than in P. X 'Serotina de Poitou'. Only two clones were studied comparedto the study made by one of us (A. W.) under light and fluorescent microscopy, whereseveral resistant and several susceptible clones were looked at. However, the resultspresented here help to interpret results obtained by other microscopic techniques.

Distinct differences were sh.own to exist in the surface morphology of the epidermisof the two clones. Size and frequency of stomata also differed (table 1). However itis not possible to say that these determine the relative resistance of the two clones.COIFEY'S (1975) work showed that the development of infective structures is deter-mined by the host cuticle. It acts however non specifically on the initiation of thesestructures. It is possible that the mechanism responsible for the development of thepathogen and its penetration through the stomata is to be sought in the cuticle of thepoplar leaf. However, this may depend on its chemistry rather than its morphology.It is also possible that the whole mesophyll participates m the infection or defenceprocess.

fig. 8. A germinating uredospore with short ramifications of ihe hyphae from the lowerepidermal surface of a resistant poplar clone P. X 'Serotina de Poitou' 24 hours after plaeinj;the uredospores on the leaf (X 695). - Fig. 9. Surface of the lower epidermis of a leaf of

/•". X 'Serotina de Poitou' 48 liours after placing uredospores on it (X 230)

Germination of Melampsora lariei-populina itredospores on poplar leaves I 23

This is supported by observed inhibition ol germination ot uredospores on theepidermis of the resistant clone. On this poplar, uredospores germinated to a muchlesser extent and only 12 hours after being placed on the leaf surface. On the suscep-tible clone this process had started after 3-4 hours.

Similar conclusions were drawn by Horn, and SUTI.R (1976) who used transmissionand scanning microscopes to study germination of spores and the penetration by Phy-

15

Fig. 10. A closed stcmata of /'. X 'Serotina de Poitou' with a visible hypha above it (X 1450).— Fig. 11 and 12. Cerminating uredospores on the surface of the lower epidermis of a P.trichocarpa leaf, 4 hours after being placed here. The growth hypha points directly to thestomata (X 970). - In Fig. 12 the growth hyphae originate from opposite poles of the ure-dospore (X 915). - Fig. 13. A long fungal hypha growing along the characteristic cylindricalsurface struetures and penetrates through a stoma into the leaf of /'. triehoearpa 6 hours afterthe uredospores were placed on it (X 930). - Tig. 14. and /J. Stomata in P. triehoearpa beingpenetrated hy hyphae of the fungus 6 hours after placing uredospores on the leaf surface-

(X HOO)

124 F. Mlodzianowski, A. Werner and R. Siweeki

Differences in size and frequency of stomat.i on the lower surfaces of poplar UMve.*

Poplar clones ut stnitiaia in innl d i h Mean fit-quciicy of htoin.ita ̂

per cm •'

P. trichocarpa 37.0X32.1 19,030 ± 3 8 0Susceptible

P. X 'Serotina de Poitou' 32.6 X 20.1 14,150 ± 340Resistant

tophtora infestans of leaves of a resistant and susceptible variety of Solarium tubero-sum. However, in that case penetration into the mesophyll tissues of the resistant andthe susceptible variety took place both through the stomata and directly through theepidermis. In poplar leaves, however, the infection of the mesophyll took place exclu-sively through the stomata.

SuiTim.iry

Uredospores of Melampsora lariei-populina germinated more intensely and more rapidly onthe lower epidermis of /'. trieboearpa, a susceptible clone, than on P. X'Serotina de Poitou',a resistant clone. 3-4 hours after uredospore placement on leaves of P. trichocarpa, hyphaefrom germinating spores had frequently penetrated the stomata. This process in P. X 'Serotinade Poitou' took place much later in samples collected more hours after placing the uredosporeson the leaves and this to a limited extent.

Differences were also observed in the sculpturing of the lower epidermal surface of thetwo poplars. To what extent this factor determines resistance to infection is not known.

Earlier observations that stomata were larger and more numerous" on the lower epidermisof P. trichocarpa than on P. X 'Serotina de Poitou' were confirmed.

Resume

Germination des urediospores de Melampsora larici-populina sur les feuilles de Peuplier

Les urddiospores de Melampsora larici-populina germaient plus intenscment et plus rapidementsur l'dpiderme de la face inferieure des feuilles de Populus trichocarpa, un clone sensible, quesur P. X 'Serotina du Poitou', un clone resistant. Trois a quatre heures apres mise en placedes urediospores sur les feuilles de P. trichocarpa, les hyphes provenant des spores en germi-nation avaient frc'quemment investi les stomates. Ce processus chez P. X 'Serotina du Poitou'se produisait beaucoup plus tard sur des c'chantillons preleves apres un temps plus long depresenee des urediospores sur les feuilles et ceci dc fafon limitce.

Des differences ont etc cgalement notees sur la structure des surfaces folaires inferieuresde l'epiderme des deux peupliers. La mesure dans laquelie ce facteur determine la resistance aI'infection est inconnue.

Des observations anterieures portant sur la largeur supcrieure et le nombre plus eleve desstomates a la face inferieure des feuilles de /-". trichocarpa par rapport a celles de P. X 'Serotinadu Poitou' sont confirmees.

Zusammenfassung

Die Keimung der Uredosporen von Melampsora larici-populina auf Pappelblcittern

Uredosporen von Melampsora larici-populina keimten besser und schneller auf der Blattunter-seite eines anfalligen P. trichocarpa-K.]ons als auf P. X 'Serotina de poitou', einem resistentenKlon. 3 bis 4 Studen nach der Applikation der Uredosporen auf P. trichocarpa waren dieKeimhyphen sehon haufig durch die Stomata eingewachsen. Dieser Vorgang fand bei P. X'Serotina de Poitou' viel spater und in begrenztem Umfang statt.

Untersdiiede waren auch in der Oberflachenskulptur der unteren Epidermis zwischen denBlattern der beiden Pappelsorten zu erkennen. Es ist jedoch nicht bekannt, in welehem Umfangdieses Merkmal die Infektionsresistenz bestimmt. Friihere Beobachtungen, wonach die Stomataan der unteren Blattepidermis von P. trichocarpa grofk-r und zahlreidier ausfallen als bei/'. X 'Serotina de Poitou', wurden bestatigt.

Mycoplasma like organisms assoeiated with yellow leaf disease of Areca catechu 125

References

CorrriY, M. D., 1975: Obligate parasites of higher plants, particularly rust fungi. Symposiaof the Society for Experimental Biology, Cambridge, No. XXIX; 297-323.

L, H. R.; SuTKR, E., 1976: Host-parasite interfaces in resistant and suseeptible cultivarsof Solatium tuberosum inoculated with Pbytopbtora infestans leaf tissue. Can. ]. Bot. 54,1956-1970.

MtODZiANOWSKi, F.; SiwiiCKi, R., 1976: Ultrastrueture of poplar leaves naturally infected byrust Melampsora lariei-populina Kleb. Arboretum Kornickie 21, 375-400.

— — 1978: Morphology and ultrastrueture of uredospores of Melampsora larici-populinaKleb. Eur. J. For. Path. 8, 43-48.

SIWECKI, R., 1975: The mechanism of poplar leaf resistance to fungal infection. 2. Ann. Rep.,Inst. Dendrology, Kornik, Poland.

— 1976: The mechanism of poplar leaf resistance to fungal infection. 3. An., Rep., Inst.Dendrology, Kornik, Poland.

yluthors' addresses: Dr. F. MLODZIANOWSKI, Department of Ceneral Botany, Institute ofBiology, A. Mickiewicz University, Stalingradzka 14, 61-713 Poznan,Poland; Dr. R. SIWECKI and Mr. A. WEKNKU, Polish Academy of Sciences,Institute of Dendrology, 63-120 Kornik, Poland

WISSENSCHAFTLICHE KURZMITTEILUNG

Forest Research Laboratory, Bangalore, India, and NortbeasternExperiment Station, Delaware, Obio, USA

Mycoplasma like organisms associated with yellow leaf diseaseof Areca catechu L.

By R. NAYAR and C. E. SELISKAR

Reeeipt of Ms. 28. 11. 1977

Abstract

Mycoplasma like organisms (MLO) were found in the young sieve elements of arecanut palmsdeclining with yellow leaf disease in Kerala and Karnataka States of India. The organismsresembled those MLO found in coconut palms affected with lethal yellowing in Florida andAfrica.

Yellow leaf disease of arecanut palm {Areca catechu) remains the most serious problemof betelnut palm growers of Kerala and Karnataka States. MHNON (1960, 1963, 1968)has described the symptoms, responses to micro and macro nutrients, to mites, and tofungi isolated from diseased palms. MENON-NAYAR (1971, 1976) has indicated thatthe causal organism is possibly mycoplasma like organisms (MLO). This paper pre-sents the results of electron microscopic investigations of the affected palms fromboth states.

Eur. J. For. Path. 8 (1978) 125-128© 1978 Vcrlag Paul Parey, Hamburp uiij licrlinISSN 0300-1237 / ASTM-CodcMi; EIFPA 9