J. Phytopathology 121, 33—39 (1988)© 1988 Paul Parey Scientific Publishers, Berlin and HamburgISSN 0931-1785

Biologische Bundesanstalt fur Land- und Forstwirtschaft,Institut fur Pflanzenschutz im Ohsthau, Dossenheim,

Federal Republic of Germany

MLO-Associated Decline of Alnus glutinosa,Populus tremula and Crataegus monogyna

E. SEEMULLER and W. LEDERER

Authors' address: E. SEEMOLLER and W. LEDERER, Biologische Bundesanstalt fur Land- undForstwirtschaft, Postfach 73, D-6915 Dossenheim (F.R.G.).

With 12 figures

Reeeived January 28, 1987; aecepted March 10, 1987

Abstract

In the Heidelberg area of southwestern Germany declining trees oi Alnus glutinosa (L.) Gaertn.and Populus tremula L. as well as stunting shrubs of Crataegus monogyna Jacq. were observed. In all 3species mycoplasmalike organisms, which appear to be the causal agents of the disorders, weredetected by fluorescence and electron microscopy. The diseases of alder and aspen seem to be lethalwhile the hawthorn apparently is less affected.

Zusammenfassung

MLO-assoziierter Verfall bei Alnus glutinosa, Populus tremulaund Crataegus monogyna

In der weiteren Umgebung von Heidelberg wurden verfallskranke Baume von Alnus glutinosa(L.) Gaertn. und Populus tremula L. sowie kiimmernde Straucher von Crataegus monogyna Jacq.beobachtet. Durch fluoreszenz- und elektronenmikroskopische Untersuchungen konnten in krankenPflanzen aller 3 Arten mycoplasmaahnliche Organismen beobachtet werden, die allem Anschein nachfiir die Krankheiten verantwortlich sind. Die Krankheiten bei Erie und Pappel seheinen letal zu sein,wahrend der Weifidom offenbar weniger in Mitleidenschaft gezogen wird.

Mycoplasma-like organisms (MLOs) induce many diseases of trees andshrubs. In general MLO-diseases are most serious in woody hosts because oftheir long-lasting effect and the importance of the individual plant. The disorders

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34 SEEMULLER and LEDERER

are most often lethal, although death may be slow. Less common is the exhibitionof chronic symptoms over a more or less normal live span or the partial recoveryof infected plants.

The majority of MLO-diseases of woody hosts were found in the USA(MARAMAROSGH and RAYGHAUDHURI 1981), In Europe they are mainly known tooccur on several fruit crops but MLOs were also associated with the witches'-broom diseases of Rohinia pseudoacacia L. (BLATTNY 1959, CIFERRI and CORTE

1960) and of Ulmus carpinifolia Cled. (BOJNANSKY 1969) and U. "campestris" (Pisiet al. 1981). In the genus Populus witches'-broom diseases presumably induced byMLOs were observed on P. nigra L, 'Italica' and P. X canadensis Moench(ATANASOFF 1973), P. x canescens (Ait.) Sm. (VAN DER MEER 1980) and P. alba L.(SHARMA and COUSIN 1986). — In this paper the demonstration of MLOs indeclining trees of alder {Alnus glutinosa [L.] Caertn.) and aspen {Populus tremulaL.) and in hawthorn shrubs {Crataegus monogyna Jacq.) is reported.

Material and Methods

Samples were taken from branches, shoots and roots of healthy and diseased alder and aspentrees and hawthorn shrubs in September and October 1986. The inner bark was cut into small bits andfixed in a cacodylate-buffered mixture of paraformaldehyde and glutaraldehyde (PGA), pH 7.3, afterKARNOVSKY (1965). All samples were screened for the presence of MLOs with the DAPI method(SEEMULLER 1976). In this procedure the tissue is sectioned with a freezing microtome, stained withthe DNA indicator 4'-6-diamidino-2-phenyHndole (Serva, Heidelberg) and examined by fluorescencemicroscopy. A number of DAPI-positive samples of each host were also examined by electronmicroscopy for verification of the DAPI results. In this case, the PGA-fixed material was postfixed inbuffered 1 % osmium tetroxide and infiltrated with Epon-Araldit. Thin sections were cut with adiamond knife and stained with uranyl acetate and lead citrate.

Results

Alnus glutinosa

In summer and fall of 1986 declining alder trees were observed m theHeidelberg region of southwestern Cermany. At several sites of this area themajority of the trees were diseased or dead. In other places only a few treesshowed symptoms of the disease or the disease seemed not occur at all.

The first symptoms of the disease are light green leaves. Later, the leavesbecome yellowish-green to yellow and often necrotic from the margin (Fig. 1).They are usually smaller than normal and drop prematurely. As the diseaseprogresses, the foliage becomes sparse and the vegetativ growth decreases stead-ily. Then symptoms of die-back appear, first on a few branches and thenspreading rapidly until the death of the tree (Fig. 2). The disease seems to belethal in all cases, Witches'-brooms were never observed in the crown butbroomed shoots were sometimes found among the stump and root sprouts whichoccur often on diseased trees (Fig, 3),

Typical MLO fluorescence was detected in sieve tubes of all root samplesand in nearly all of the stem samples of the diseased trees examined. The MLO

liP^-:>:^'^i.-V;

! • •

4

"iMM-^^^'n. ^';3^

Fig. 1—6. Alnus glutinosa: Fig. 1. Yellowing and leaf necrosis; left healthy leaf. Fig. 2. Dying tree.Fig. 3. Small witches'-brooms of stump sprouts. Fig. 4. Various MLO forms. Fig. 5. DividingMLOs. Fig. 6. MLOs showing DNA strands (arrowheads) and the trilaminar unit membrane

(arrows). All bars 200 nm

36 SEEMULLER and LEDERER

population was always higher in the roots than in the stem. The occurrence ofMLOs was confirmed by electron microscopy. The organisms varied in size andshape. They were mainly spherical or elliptical in form, but filamentous anddividing forms were also observed (Fig. 4 and 5). The diameter usually rangedfrom 200 to 400 nm, that of filaments was sometimes smaller. The organismswere bounded by a trilaminar unit membrane and contained peripheral ribosomesand a central DNA molecule (Fig. 6). As typical for diseased trees the number ofthe organisms was usually low. They occurred only in the functional sieve tubesof a few cell layers adjacent to the cambial zone. The sieve tubes in the olderphloem were all necrotic due to the disease. No sieve tube necrosis and no MLOswere found in healthy looking trees.

Populus tremula

At the same time and in the same area as described for alder, declining aspentrees were observed. The symptoms of the disease vary. The first indication is thereddening of the foliage in late summer and fall. The discolored foliage oftendrops prematurely. In a few cases, witches'-brooms were observed, mostly onvigorously growing shoots. The branching of the brooms is either inconspicuousor pronounced but in both cases the lateral shoots are thin (Fig. 7 and 8). Theleaves of the brooms are often extremely large at the shoot base. They increase innumber and become dwarfed towards the tip. On non-broomed shoots the leavesmay be reduced in size and have short petiols which give the shoot a bushyappearance. As the disease progresses the foliage become sparse and die-back,which usually proceeds rapidly, occurs. The disease was found in all areas whereaspen grow and trees at all stages of growth were affected. In many cases a greatnumber of trees was diseased or dead.

In nearly all root and stem samples of diseased trees, MLOs were detectedby fluorescence microscopy. The presence of the organisms could be confirmedby electron microscopy. In general, the shape of the organisms was similar to thatof the alder decline agent, although filaments were more prevalent and relativelylong chains of MLOs occurred. The diameter of the organisms ranged from 100to 400 nm and was as small as 60 nm for tender filaments (Fig. 9—11). Theaverage size was significantly smaller than that of the alder decline agent, but theMLO population in the sieve tubes was higher. Usually, intact sieve tubes inwhich the organisms were observed occurred only in the youngest portion of thecurrent season's phloem while the older sieve tubes were necrotic. No sieve tubenecrosis and no MLOs were found in healthy looking trees.

Crataegus monogyna

Diseased hawthorns were also observed in the Heidelberg area. The symp-toms of the disease are neither very conspicuous nor very specific for a MLO

Fig. 7—11. Populus tremula. Fig. 7. Witches'-broom with decreasing leaf size towards the top.Fig. 8. Witches'-broom with short laterals and dwarfed leaves; left healthy shoot. Fig. 9. SmallMLOs in a chain and dividing forms. Fig. 10. Medium-sized MLOs, some are dividing. Arrows =

trilaminar unit membrane.' Fig. 11. Large MLO. Bar 200 nm (same scale for all MLC) pictures)

• - ' ' v ' .

'i •

/ I . ' - • 1. •' -*

38 SEEMULLER and LEDERER

Fig. 12. MLOs in a root sieve tube of Crataegus monogyna. Bar 200 nm

infection. Affected shrubs show light green to yellowish-green leaves and areddening of the foliage in late summer and fall. Also, the leaves may be smallerthan normal and may be rolled downward at the margin. Furthermore, the foliageis often sparse and the vigor is reduced. The decline of the shrubs appears to beslow and seems to result in a more or less pronounced stunting. Dead plants werenot observed and the incidence of the disease seems to be lower than in the case ofalder and aspen.

The nonspecific symptoms make the diagnosis of the disease difficult. MLOswere detected by fluorescence microscopy in only 10 % of the stem samples butin 60 % of the root samples examined. The presence of the organisms in theDAPI-positive samples was confirmed by electron microscopy. They weresimilar in shape and size to the organisms found in alder (Fig. 12),

Discussion

This is the first time that MLO diseases are reported within the genera Alnusand Crataegus and in Populus tremula. Because the organisms were always foundin diseased plants and never in healthy plants there is no doubt that they are thecausal agents of the diseases although no transmission experiments have beenperformed yet. The diseases of alder and aspen are widespread in the Heidelbergarea and both lead to a lethal decline. The disease of hawthorn seems to be lesssevere and less prevalent, but because of their nonspecific symptoms and the weakand irregular colonization of the stem, evaluation is more difficult.

Since the survey was confined to the Heidelberg area, further investigationsare necessary to evaluate the importance of the three diseases in other regions of

MLO-Associated Decline of Alnus glutinosa 39

Germany. The description of the diseases given in this paper is based onobservations of one growing season only. For better description of the diseasesmore work is required.

The work was supported by a grant from the Bundesminister fiir Forschung und Technologie.We thank Prof. Dr. H. BuTIN for his help in the taxonomy of Populus tremula.

Literature

ATANASOFF, D. , 1973: Witches' brooms on stems of Elms and other trees. Archiv PhytopathologiePflanzenschutz 9, 241—243.

BLATTNY, G., 1959: Viroza a z virozy podezrela nemoc Trnovniku Akatu (Robinia pseudoacacia L.).Ann. Acad. Tchecosl. Agr. 5, 291—294.

BojNANSKY, V., 1969: Elm witches' broom a new virus disease in Gzechoslovakia. Proc. 6thGzechoslov. PL Virol. Olomouc 1967, 211—215.

GiFERRi, R., e A. GORTE, 1960: Gli ,,scopazzi virosici" della Robinia pseudoacacia, malattia nuova perritalia. Atti. Ist. Bot. Univ. Pavia Ser. 5, 17, 122—128.

KARNOVSKY, J. M., 1965: A formaldehyde-glutaraldehyde fixative of high osmolality for use inelectron microscopy. J. Gell. Biol. 27, 137a—138a.

MARAMAROSCH, K., and S. P. RAYCHAUDHURI, 1981: Mycoplasma diseases of trees and shrubs.Acad. Press, 362 pp.

PISI, A., F. MARANI, and A. BERTACCINI, 1981: Mycoplasmalike organisms associated with elmwitches' broom symptoms. Phytopath. medit. 20, 189—191.

SEEMULLER, E., 1976: Investigations to demonstrate mycoplasmalike organisms in diseased plants byfluorescence microscopy. Acta Hort. 67, 109—-112.

SHARMA, A. K., and M.-T. GOUSIN, 1986: Mycoplasmalike organisms (MLOs) associated with thewitches' broom disease of poplar. J. Phytopathology 117, 349^356.

VANDERMEER, F . A. , 1980: Witches' broom in poplars. Populier 17, 42—43.