Search for Causative Agents of the
Figure 1.—Sugarbeet field in early September at the La Platina Experi- mental Station; 11 months old. More than 95% of the plants showed yellow- wilt symptoms.
Figure 2.—Diseased plants showing advanced stages of the typical yellowing phase, including witches' broom clusters, dwarfing, and leaf necrosis.
t h e o t h e r was a n e l o n g a t e d , f l e x u o u s v i r u s l i k e p a r t i c l e a s s o c i a t e d with c h a r a c t e r i s t i c i n c l u s i o n s .
Mycoplasmalike organism (MLO)
P l e o m o r p h i c b o d i e s w h o s e m o r p h o l o g y r e s e m b l e d t h a t o f m i c r o - o r g a n i s m s b e l o n g i n g t o M y c o p l a s m a i a l e s w h i c h h a v e b e e n isolated f r o m a n i m a l s ( 1 , 1 3 ) , a n d M I . O w h i c h h a v e b e e n o b s e r v e d i n m o r e t h a n
Vol.. 18. No. 2, O C T O B E R 1974 145
50 yellows-type diseased plants (16-18, 25, 33), were detected in the phloem tissues of the yellow wilt-affected plants. The MLO were ob- served either in mature sieve tube elements, devoid of cytoplasm and filled with sieve tube sap (Fig. 3), or in the cytoplasm of phloem paren- chyma cells (Fig. 4).
These bodies, bounded by a trilaminar membrane (unit mem- brane), contained ribosomes and deoxyribonucleic acid (DNA)-like strands and lacked a nuclear envelope and a cell wall (Figs. 3 to 6). They varied considerably in size and electron opacity. Wide variations of the morphological profiles, such as spherical, ovoidal, amoeboidal, long elliptical, horseshoe-shaped, and doughnut-shaped forms, were ob- served (Figs. 3 to 6). T h e majority of MLO varied in size from approxi- mately 100 nm to 800 nm (Figs. 3 and 4). However, some of them, parti- cularly those in the phloem parenchyma cells, had fine, long cyto- plasmic protrusions containing electron-lucent cytoplasm (Fig. 6).
T h e n u m b e r of MLO in each host cell varied considerably (Figs. 3 and 4). Some sieve tube elements, as well as phloem parenchyma cells, were tightly packed with numerous MLO, while others contained a small number of MLO scattered in the elements. In the cytoplasm of phloem parenchyma cells, "colony-type" accumulations (17) of the MLO were often seen (Fig. 4). In general, MLO around the sieve plates were more crowded on one side of the plate than on the other (Fig. 3).
MLO, partially squeezed through the sieve pores, were observed often.
This may indicate that the MLO move from one sieve element to a neighboring one via the sieve pores.
Morphological configuration indicative of binary fission, budding, extrusion of the protoplasm and tightly lined bodies, which were similar to those considered earlier to be undergoing reproduction in sieve tube elements (16) or in phloem parenchyma cells (17), were also seen in the phloem of vellow wilt-affected sugarbeet plants.
Elongated viruslike particles
Elongated, flexuous viruslike particles were observed in the cyto- plasm of mesophyll (ells and phloem parenchyma cells of leaves ob- tained from the diseased plants, but. not in the normal-appearing materials. Morphological characteristics of the particles differed from that of microtubules and phloem protein (P-protein) fibrils, which are normal cytoplasmic constituents (Fig. 7). Although measurements of elongated, flexuous particles in thin sections did not reveal accurately their length, thev appeared to be more than 650 nm in length and 10-12 nm in diameter. T h e particles often appeared in more or less parallel arrangements, oriented perpendicularly to cell walls (Fig. 7 to 9).
VoL. I8. No. 2. OCTOBER 1974 151 The particles were closely associated with various t y p e s of cyto-
p l a s m i c i n c l u s i o n s , s u c h as bundles (fig 8), l a m i n a t e d aggregates (Fig 9).and p i n w h e e l s (Fig. 10). C o m p l e x inclusions consisting of p i n w h e e l arms, bundles, and or laminated aggregates were also s e e n The m o r p h o l o g i c a l p r o f i l e s these i n c l u s i o n s s t r i k i n g l y r e s e m b l e d those observed by o t h e r s in diseased p l a n t s infected with certain t y p e s of elongated viruses (2,6,8,10,14,15,20-24,26,28-32,34)
Single p a r t i c l e s , a s w e l l a s i n c l u s i o n s . o f t e n a p p e a r e d i n t h e cytoplasm close to t h e plasmodesmata b e t w e e n parenchyma cells (Fig 11) i n d i c a t i n g that they may m o v e from cell to cell t h r o u g h t h e plasmodesmata as pointed o u t recently by Weintraub et al (32) Occas- i o n a l l y t h e v i r u s l i k e p a r t i c l e s w e r e a l i g n e d a l o n g t h e l a m i n a t e d a g g r e - g a t e s (Fig 12). At certain a n g l e s of s e c t i o n i n g , very fine s p i k e s were seen on the surface of the aggregates.
I n s o m e i n s t a n c e s , . t h e i n c l u s i o n s w e r e o b s e r v e d i n t h e p h l o e m parenchyma cell a d j a c e n t to the sieve elements in which M L O were s e e n (Fig 12). Both MLO) a n d the i n c l u s i o n s occaisonally c o e x i s t e d within the s a m e p h l o e m parenchma cell (Fig. 13).
F i g u r e 10.—Characteristic pin wheel i n c l u s i o n s (PW) in m e s o p h y l l cells of a yellow wilt-diseased plant. C o m p l e x inclusions consist of pin- wheel a r m s and a laminated aggregate (LA). X 54,600.
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Figure 11.- Elongated viruslike particles (VLP) apparently passing
t h r o u g h the plasmodesmata (PD) of p a r e n c h y m a cells of a yellow wilt- diseased plant. CW: Cell wall. X 57,400.
Long filamentous particles
Masses of long filamentous particles were frequently observed
in the cytoplasm of mesophyll, as well as phloem parenchyma, cells of both yellow wilt-infected and normal-appearing plants (Figure 14) Morphological features, size (approximately 1200-1300 nm in length) and appearance of the particles in the host cells were very similar to those of the presumed beet yellows virus particles in sugarbeet yellows- infected Beta vulgaris, described by Esau et al (12).
T h e filamentous particles that often formed large spindle-shaped aggregates are shown in Figures 1 5 and 1 6 at high magnifications the particles also appear in irregularly aligned masses of various sizes (Fig. 14). n some instances, the particles formed highly ordered a b l e - gates. A longitudinal section of the ordered aggregate is shown in
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Figure 12.—Portion of phloem tissue from a yellow wilt-infected plant.
Note MLO in the sieve tube element (S) and elongated viruslike particles (VLP), associated with the laminated aggregates (LA), in a p h l o e m p a r e n - c h y m a cell (PP). CW: Cell wall; SPK: Fine spikes. X 68,250.
Figure 13.—Coexistence of mycoplasmalike o r g a n i s m s (MLO) and the laminated aggregates (LA) in the phloem p a r e n c h y m a cell of a yellow wilt- infected plant. X 41,750.
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Figure 14.—Portion of mesophyll cell obtained from a n o r m a l - a p p e a r - ing sugarbeet plant. Note a large spindle-shaped aggregate, as well as small i r r e g u l a r m a s s e s , of l o n g filamentous p a r t i c l e s (FP). CW: Cell wall X 18,750.
V o L 1 8 . N o . 2 . O C T O B E R 1 9 7 4
Figures 15 and 16.—Portion of large s p i n d l e aggregate of the long fila- m e n t o u s particles (FP) in the p h l o e m p a r e n c h y m a cells of a yellow wilt- d i s e a s e d p l a n t . N o t e loosely aligned p a r t i c l e s in l o n g i t u d i n a l section (Figure 15, X 49,000) a n d transverse section (Figure 16, X 49,000).
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in F i g u r e 17 a n d t h e t r a n s v e r s e section in F i g u r e 18. No e n c l o s i n g m e m b r a n e was s e e n a r o u n d e i t h e r l a r g e o r small f i l a m e n t o u s m a s s e s ( F i g u r e s 14. 15, 17, a n d 18).
I n t h e yellow wilt-infected p l a n t s , f i l a m e n t o u s a g g r e g a t e s w e r e o f t e n o b s e r v e d i n p h l o e m p a r e n c h y m a cells w h i c h c o n t a i n e d i n t r a - c y t o p l a s m i c M L O ) ( F i g u r e 19). Occasionally t h e f i l a m e n t o u s p a r t i c l e s also w e r e p r e s e n t in h o s t cells, closely a s s o c i a t e d with t h e l o n g cyto- p l a s m i c p r o t r u s i o n s o f t h e M L O ( F i g u r e 6 ) o r with s p h e r i c a l M L O .
D i s c u s s i o n
B e f o r e t h e e x i s t e n c e o f m y c o p l a s m a l i k e o r g a n i s m s a s p a t h o g e n i c a g e n t s i n p l a n t s b e c a m e k n o w n , B e n n e t t a n d c o - w o r k e r s (3) m e n - t i o n e d t h a t " t h e d i v e r s i t y o f s y m p t o m s o f t h e yellow wilt o f s u g a r b e e t h a s led t o s p e c u l a t i o n a s t o w h e t h e r m o r e t h a n o n e c a u s a l a g e n t i s i n v o l v e d . " H o w e v e r , t h e y c o n c l u d e d t h a t " r e s u l t s o f o b s e r v a t i o n s a n d e x p e r i m e n t s s t r o n g l y s u p p o r t t h e c o n c e p t t h a t all t h e d i f f e r e n t t y p e s o f s y m p t o m s o b s e r v e d a r e t h e r e s u l t o f t h e a c t i o n o f a single a g e n t a n d t h a t this a g e n t i s a v i r u s . " T h e y also c o n c l u d e d that t h e t y p e o f s y m p - t o m s p r o d u c e d was d e t e r m i n e d largely b y e n v i r o n m e n t a l c o n d i t i o n s : t h e w i l t i n g p h a s e o f t h e d i s e a s e a p p e a r e d u n d e r c o n d i t i o n s o f h i g h t e m p e r a t u r e a n d low h u m i d i t y , w h e r e a s t h e y e l l o w i n g p h a s e a p p e a r e d u n d e r c o n d i t i o n s o f l o w e r t e m p e r a t u r e a n d h i g h e r h u m i d i t y .
T h e c h a r a c t e r i s t i c s y m p t o m e x p r e s s i o n s o f t h e y e l l o w i n g p h a s e d e s c r i b e d by B e n n e t t et, al ( 3 , 4) s t r i k i n g l y r e s e m b l e t h o s e of yellows t y p e d i s e a s e s , n o w k n o w n t o b e a s s o c i a t e d with o r c a u s e d b y M L O ( 1 6 - 1 8 , 2 5 , 3 3 ) . F u r t h e r m o r e , successful s u p p r e s s i o n o f t h e d i s e a s e with t e t r a c y c l i n e t r e a t m e n t s (11) a n d t h e p r e s e n t f i n d i n g s o f M L O i n yellow w i l t - d i s e a s e d Beta vulgaris p l a n t s s t r o n g l y s u p p o r t a n e w c o n - c e p t t h a t a t least s y m p t o m e x p r e s s i o n s o f t h e y e l l o w i n g p h a s e a r e c a u s e d b y M L O a g e n t s . H o w e v e r , n o M L O a g e n t s a s s o c i a t e d w i t h a n y w i l t i n g d i s e a s e s a r e k n o w n t o d a t e d e s p i t e t h e fact t h a t m o r e t h a n 5 0 d i f f e r e n t M L O - i n d u c e d d i s e a s e s h a v e b e e n r e p o r t e d . I f t h e M L O a r e i n d e e d t h e c a u s a t i v e a g e n t s o f t h e y e l l o w i n g p h a s e , a c o m p l e x e t i o l o g y , m o s t likely M L O a n d a v i r u s (or viruses), of t h e s u g a r b e e t yellow wilt i s m o r e a c c e p t a b l e t h a n t h e single viral etiology (3).
T h e r e exists a c o n t r o v e r s y a s t o w h e t h e r t h e c h a r a c t e r i s t i c i n - c l u s i o n s , s u c h a s p i n w h e e l s , b u n d l e s , a n d l a m i n a t e d a g g r e g a t e s , c o n - sist o f c e r t a i n e l o n g a t e d v i r u s p a r t i c l e s (6, 15, 2 4 , 2 6 , 34) o r w h e t h e r t h e y a r e c o m p l e x o r g a n i z a t i o n s o f v i r u s p a r t i c l e s a n d / o r s o m e s u b - s t a n c e s o f u n k n o w n n a t u r e (2, 10, 21). N e v e r t h e l e s s , b a s e d o n t h e i n t i m a t e association b e t w e e n t h e v i r u s e s a n d t h e c h a r a c t e r i s t i c i n - c l u s i o n s , E d w a r d s o n (9) p r o p o s e d t h a t t h e i n c l u s i o n s a r e d i a g n o s t i c for i n f e c t i o n b y v i r u s e s o f t h e p o t a t o v i r u s Y g r o u p (5), w h o s e l e n g t h s vary f r o m 7 3 0 n m t o 7 5 0 n m . L a t e r s i m i l a r i n c l u s i o n s i n d u c e d b y s h o r t e r (31), as well as l o n g e r (14), v i r u s e s w e r e also r e p o r t e d . It is
VOL. 18, No. 2, O C T O B I R 1971
Figures 17 and 18.—Highly o r d e r e d aggregates of the long filamentous particles (FP) in the mesophyll cells obtained from a yellow wilt-infected plant. Note highly aligned particles in longitudinal section (Figure 17, X 49,100) and transverse section (Figure 18, X 49,100).
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Figure 19.—Coexistence of the long filamentous particles (FP) and mycoplasma-like organisms (MLO) in a phloem parenchyma cell obtained trom a yellow wilt-infected plant. MT: Microtubules. X 32,630.
generally accepted that the presence of the characteristic inclusions indicates the occurrence of infection with certain elongated viruses.
Thus, the finding of the elongated viruslike particles with the charac- teristic inclusions suggests that the diseased sugarbeet plants examined may be infected with an inclusion-inducing virus. T h e inclusion- inducing elongated viruses are known to be causative agents of a number of plant diseases (2, 6, 8-10, 14, 15, 19, 20, 22-24,26, 28-31.
34). Although no elongated viruses which induce wilting have been reported, one of the potato virus Y group is known to be associated with the sugarbeet wilting in Brazil (personal communication: A. S.
Costa, Virus Section, T h e Agricultural Institute, C.P. 28, 13100 Cam- pinas, Sao Paulo). At present, it is uncertain whether elongated virus- like particles are responsible in part for causing symptoms of the sugarbeet yellow wilt disease (e.g. in the wilting phase). Further elec- tron microscopic examination of diseased plants representing the two distinct types (i.e. the yellowing phase and the wilting phase) may provide more critical information on this problem. Recently the co- existence of MLO and an elongated virus, russet crack virus, asso- ciated with pinwheel inclusions in sweet potato plants was reported
V O L . 18, No. 2, OCTOBER 1974 159
T h e nature of the long filamentous particles observed in both yellow wilt-infected and apparently noninfected sugarbeet plants remains unknown. However, since those particles resembled the beet yellows virus (12), and since one or more types of virus yellows usually is quite prevalent in the sugarbeet crop in Chile, including La Platina (personal communication: J. O. Gaskill, Beet Sugar Development Foundation, Fort Collins, Colo.), it seems probable that the long fila- mentous particles observed in this study resulted from infection by at least one of the viruses of the sugarbeet virus yellows complex. Since the effects apparently were mild, as evidenced by the appearance of plants without yellow wilt symptoms, it is assumed that a mild type of virus was involved, such as a mild strain of beet yellows virus.
If the above assumption of a yellows-virus involvement is correct the findings indicate that the yellow wilt-diseased plants examined were super infected with the yellows virus. T h e role, if any, of that virus in the development of symptoms of yellow7 wilt is not known.
However, it is apparent that controlled conditions of plant growth and inoculation are needed in future electron microscopy studies of the causal agent or agents of yellow wilt.
Summary
An electron microscopic search for the causative agents of the sugarbeet yellow wilt disease in Chile was carried out. Mycoplasmalike organisms (MLO), as well as elongated viruslike particles associated with characteristic inclusions (such as pinwheels, bundles, and laminated aggregates), were observed in the diseased plants, but were absent from normal-appearing plants. T h e presence of the MLO strongly suggests that they are largely responsible for causing the yellowing phase of the disease.
T h e presence of the elongated viruslike particles associated with characteristic inclusions suggests that the diseased plants were infected with a virus. Although the elongated particles may be responsible in part for yellow wilt symptom expression, further studies are needed to clarify their etiologic role.
These findings suggest the possibility of a complex disease etiology, presumably with the MLO and a viral agent rather than the single MLO or viral etiology. ,
Long filamentous particles, morphologically resembling the beet yellows virus, were observed in both the yellow wilt diseased and the normal-appearing plants grown in the same field. This suggests that the yellow wilt-diseased sugarbeet plants may also have been infected with a type of beet yellows virus.
1 6 0 JOURNAL OF THE A. S. S. B. T . A c k n o w l e d g m e n t s
T h e a u t h o r s t h a n k J . O . Gaskill, P r o j e c t C o n s u l t a n t , B e e t S u g a r D e v e l o p m e n t F o u n d a t i o n , F o r t C o l l i n s , C o l o r a d o , f o r his v a l u a b l e s u g g e s t i o n s a n d critical r e v i e w o f t h e m a n u s c r i p t . T h e a u t h o r s also t h a n k G . L . M c N e w , M a n a g i n g D i r e c t o r , B o y c e T h o m p s o n I n s t i t u t e for P l a n t R e s e a r c h , Y o n k e r s , N . Y . , a n d t h e U . S . A g e n c y f o r I n t e r n a - t i o n a l D e v e l o p m e n t for t h e i r p a r t i c i p a t i o n i n m a k i n g t h e a r r a n g e - m e n t s for t h e s e n i o r a u t h o r ' s visit t o B o y c e T h o m p s o n I n s t i t u t e t o c a r r y o u t this r e s e a r c h .
T h a n k s also t o K a z u k o H i r u m i o f B o y c e T h o m p s o n I n s t i t u t e for h e r v a l u a b l e a s s i s t a n c e i n e l e c t r o n m i c r o s c o p y , a n d t o R o b e r t o E h r e n f e l d K . o f I n d u s t r i a A z u c a r e r a N a t i o n a l S.A., S a n t i a g o , C h i l e , for c o n d u c t i n g t h e f i e l d w o r k a t L a Platina.
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