Physiological Plant Pathology (1976) 9,95-100

Xylem morphology of Pierce’s disease-infected grapevines

with different levels of tolerance
HILTON H. MOLLENHAUER
Vetzrinq Toxicology aad Entomology ResearchLaboratory, Agricultural ResearchService,
U.S. Department of Agriculture, P.O. Drawer GE, College Station, Texas 77840, U.S.A.

DONALD L. HOPKINS
Agricultural ResearchCenter, University of Florida,
Leesburg, Florida 32748, U.S.A.
(Acceptedfw publication April 1976)

Pierce’s disease bacteria formed aggregates in the xylem of susceptible grapevines that
appeared to hinder the flow of water and mineral nutrients through the plant. Similar
aggregates were present in tolerant grapevines, but in substantially lower numbers. Pierce’s
disease induced the formation of tyloses and gums in the tracbeary elements of infected
grapevines. The frequencies of tyloses and gums in tolerant muscadine and wild grapevines
were 7 or 8 times those in susceptible bunch grapevines. These tyloses and gums often
encapsulated the bacteria and occasionally filled the infected elements of the xylem. The
results suggest that gums and tyloses restrict the spread of infecting bacteria, thus imparting
tolerance to Pierce’s disease in certain types of grapevines.

INTRODUCTION
Pierce’s disease is a limiting factor in the production of bunch grapes in the Gulf
Coast states of the United States of America and also has resulted in large losses of
vines in California [IO, II, 13, 191. The causal agent appears to be a bacterium that
is confined to the xylem [I, 9, 12, 161. The bacteria multiply and form aggregates
which plug xylem vessels and restrict the flow of water and mineral nutrients through
the plant [ 151.
Vitis species native to the Gulf Coastal plains of the United States are the only
grapevines known to be resistant, or tolerant, to Pierce’s disease [IO, II]. A thriving
and rapidly expanding muscadine grape (Vitis rotunda$Xa Michx.) industry in the
south-eastern states has developed as a result of this resistance; however, muscadines
are not immune to Pierce’s disease. Symptoms of the disease may be observed in
some muscadine cultivars, and bacteria have been found in the xylem of these plants
l-141*
In an earlier ultrastructural study of the Pierce’s disease bacterium, we reported
two general patterns of bacterial distribution in xylem vessels. Bacteria were found
evenly distributed throughout the lumen of the vessel and as aggregates appressed
along the inner surfaces of the xylem vessel wall [l/F& The concentration of bacteria
in the lumen was quite variable in both distribution patterns. This study was
undertaken to determine whether differences in bacterial distribution patterns are
96 H. H. Mollenhauer and D. L. Hopkins
related to susceptibility and tolerance of grape cultivars, and to characterize morpho-
logical changes in the xylem of grapevines infected with Pierce’s disease bacteria.
The possible relationship between these morphological changes and resistance to
Pierce’s disease is discussed.

MATERIALS AND METHODS

Samples of Vitis vinijka L. cv. Thompson Seedless (bunch grape), Vitis rotundz$Xa
Michx. cvs Scuppernong and Pride (muscadine grape) and Vitis munsoniana Simpson
(wild grape) were taken from mid to late summer. Samples consisted of thin,
transverse slices of leaf veins and petioles of leaves taken from both grapevines
showing disease symptoms and from symptomless ones. Tissue samples were never
taken from necrotic areas of the leaf.
The leaf and petiole slices were fixed in 2% glutaraldehyde-2% paraformaldehyde
in 0.05 M-sym-collidine buffer (2,4,6&methylpyridine), pH 7.3, at 2 to 4 “C for
1 to 2 h; rinsed 5 to 6 times in 0.1 M-sym-collidine buffer over a period of 1 to 2 h at
2 to 4 “C; and post-fixed overnight at 2 to 4 “C in 1 y0 0~0, buffered in 0.05 M-sym-
collidine as above. Dehydration was carried out in an ethanol and acetone series,
and embedding was carried out in a mixture of Spurr [18].
The cell surveys were made from large, transverse sections of grape tissues
(approximately O-2 x 1.0 mm) aligned parallel to the grid bars of a 300-mesh grid.
A low magnification electron micrograph was made of each grid opening containing
a portion of a section (1 picture per 34 x 4 in. plate). This photographed portion
was subsequently enlarged into a 8 x 10 inch print that provided enough magnification
to count individual bacteria.
Determination of tyloses was simple because all but one or two tyloses examined
were fully expanded into the vessels. Double and even triple tyloses in a single
cross-section through a vessel were common, but were classified in the table as a
single tylosis. The determination of cells containing gums was a subjective process
because many forms and distribution patterns of gums were present. Any xylem cell
containing enough gum to coat heavily the cell walls and the debris in the xylem
was classified as a cell containing gum.

RESULTS
Susceptible bunch grape
The tracheary cells of the xylem were easily recognized by the secondary thickenings
of the cell walls (Plate 1). These cells were generally devoid of cytoplasm, but
sometimes contained dense-staining substances (debris) along the cell walls or free
within the intercellular spaces (Plate 1).
Bacteria were found in approximately 40% of the tracheary elements from
Thompson Seedless grapevines with symptoms of Pierce’s disease (Table l), and none
were found in sections of leaf tissue from symptomless grapevines. The bacteria were
rod-shaped organisms about 0.25 to O-5 pm in diameter by 1.0 to 4.0 pm long, bounded
by a relatively thick complex of membranes and wall materials as described earlier
[15J. In all cases the bacteria were confined to the xylem and, most frequently, to
the smaller vessels of the xylem (Plate 1). No intracytoplasmic bacteria were visible.
 PLATE 1. Transverse section through a leaf petiole of a Pierce’s disease-infected Thompson
seedless grape showing several infected xylem cells. The bacteria are about 0.25 to 0.5 pm in
diameter by 1.0 to 4.0 pm long and may appear singly or in sizable aggregates. Tyloses and
heavy gum deposits are seldom present in the tracheary elements of the xylem of these leaves.
x 4000.

[facing Page 961

 PLATE 2. Muscadine grapes with Pierce’s disease differ from Thompson seedless grapes in
the increased presence of tyloses and accumulations of dense-staining gums. A full tylosis
(large arrows) is shown at the lower left and a partial tylosis (small arrow) at the upper part of
the micrograph. x 8000.
 PLATE 3. Tyloses often trap debris and Pierce’s disease bacteria as they invade the intraxylar
cavity. From muscadine grape. x 20 000.
PLATES 4 and 5. Gums often coat the internal surfaces of the xylem walls and encapsulate
Pierce’s disease bacteria. In extreme cases, the gums and entrapped bacteria completely fill
the cell cavity. From muscadine grapes. Plate 4 x 24 000; Plate 5 x 9500.
Pierce’s disease of grapevines 97
TABLE 1
Degree of bacterial infection of the xylem vesselsof grapevines with Pierce’s disease

Average number of
Sources of Number of o/o of vessels bacteria per section
samplesa vessels infected of infected vessel

Bunch grape 173 38 41

Muscadine grape 256 22 27
Wild grape 46 21 45

s Results for bunch grape were derived from five samples of Vitis vinifera cv. Thompson
Seedless; results for muscadine were obtained from four samples of Vih rotundzjblia cvs
Scuppernong and Pride; and results for wild grape came from two samples of L’i’itis
munsoniana.

The morphology of most xylem vessels from Thompson Seedless leaves was normal
but many of them contained bacteria. Only 5% of the xylem vessels of the infected
leaves had tyloses, and less than 1 y0 contained gums (Table 2). Tyloses and gums
were not found in the xylem of healthy grapevines.
Thompson Seedless grapevines usually die of Pierce’s disease within 2 to 3 years in
Florida. Two of the vines used in this study were dead 6 months after sampling.

Tolerant muscadinegrape
Bacteria were found only in about 58% as many tracheary elements of Pierce’s
disease-infected muscadines as in the bunch grapes (Table 1). The average number
of bacteria per infected xylem vessel was lower. The distribution patterns and the
form of bacteria were similar to those of the susceptible bunch grape.
There were significant differences in the morphology of the tracheary cells of the
xylem of infected muscadines when compared to those cells of infected bunch grapes.
Pierce’s disease apparently induced the formation of numerous tyloses and accumu-
lations of dense-staining substances thought to be gums (Table 2, Plate 2). Most of
the tyloses completely filled the vessels (Plate 2), although occasionally a forming
tylosis was found (Plate 2). The tyloses tended to trap intracellular debris and gum
in the space between the xylem vessels and tylosis walls (Plate 3). In many of the
tyloses, this trapped material included bacteria (Plate 3). Dense-staining gums lined
the intracellular walls of many xylem vessels (Plates 4 and 5), and occasionally
completely (or nearly) filled the lumina of the vessels. These gums often covered
bacteria and seemed to trap them (Plates 4 and 5). Tyloses and gums were rarely
found ( < 4%) in vessels of leaves from symptomless muscadine grapes.

Highly tolerant wild grape

Bacteria were found in approximately the same percentage of the tracheary elements
in wild grapes as in muscadines (Table 1). The pattern of distribution and number
of bacteria per cell were similar to those of the bunch grapes.
The morphology of the xylem vessels of infected wild grapes was similar to that
of the muscadine grapes. Tyloses or gums were present in almost half of the xylem
IO
98 H. H. Mollenhauer and D. L. Hopkins
TABLE 2
Occurrenceof &dosesand gum in tolerant and susceptible
grape&es infected with Pierce’s disease

Sources of Number of o/O of vessels o/o of vessels “/Q tyloses

SZiI@?S” vessels with tylose@ with gums +gums
Bunch grape 173 5 0.6 5.6
Muscadine grape 256 27 34.4
Wild grape 46 14 49.2

a Results for bunch grape were
Seedless; results for muscadine
Scuppernong and Pride; and
munsoniana.
8 Most vessels with tyloses also
“vessels with tyloses”.
derived from five samples of Pink oitlifer cv. Thompson
were obtained from four samples of Vitis rotundifolia cvs
results for wild grape came from two samples of Vitis
contained gums. However, they were classified only as

cells examined (Table 2). However, the wild grapes had much more gum and
fewer tyloses than did the muscadines.

DISCUSSION
The symptoms of Pierce’s disease were correlated with the presence of intraxylar
bacteria. Grapes native to the Gulf Coastal plain had fewer bacteria than did the
Vitis vinifera. However, even the tolerant grape cultivars contained aggregates of
bacteria in the xylem in parts of the plant showing symptoms of Pierce’s disease.
Most interesting to us were the morphological differences in the xylem vessels of
tolerant and susceptible cultivars after infection with Pierce’s disease bacteria. Large
quantities of electron-dense gums and numerous tyloses formed after infection in the
tolerant grapevines, whereas gums and tyloses were not common in the very suscep-
tible bunch grapes. Esau [S] described gums and tyloses in susceptible grapevines.
Gums and tyloses appear to have divergent roles in Pierce’s disease. That is,
they may themselves contribute to the development of disease symptoms by reducing
water movement in the vessels while simultaneously restricting the internal spread
of the bacteria and localizing the infection [3, 61. The speed and extent of the for-
mation of gum deposits and tyloses have been related to resistance to vascular
diseases in many plants [Z-.5, 7, 171. Beckman [Z, 31 has proposed that localization
of infection by vascular occlusion is a general mechanism of resistance to vascular
infections. The observed differences between susceptible and tolerant grapevines in
the production of gums and tyloses in this study indicate that this resistance
mechanism may be operative in Pierce’s disease of grape. Gums were frequently
deposited with the tyloses and, together, they often sealed the vessels of the more
tolerant muscadines and wild grapes. Bacteria were often encapsulated in the gum.
The hypothesis of resistance by the localization of infection is compatible with the
results of this study.
The purpose of this work was to study the morphology of the xylem in grapes
infected with Pierce’s disease. It is important in interpreting the results to remember
that the samples from the tolerant muscadines and wild grapes used were taken from
individual grapevines in which the tolerance, or resistance, was failing. The majority
Pierce’s disease of grapevines 99
of the muscadines and wild grapes do not develop any Pierce’s disease symptoms.
If localization of infection by vascular occlusions were effective, only the vessels
around the site of primary infection would be blocked. Perhaps this is the case with
most of the tolerant grapevines. Where the tolerance fails, as in the muscadine and
wild grapes that were sampled, one would expect to see a much higher number of
plugged vessels as the localization mechanism is repeatedly activated.
Comparisons of the rate and extent of tylosis formation, as well as bacterial
movement from single local inoculations in resistant and susceptible vines, are
needed to elucidate the role of tyloses and gums in Pierce’s disease. The possibility
of the gums having antibacterial properties and functioning as chemical resistance
mechanisms are also considered important for future studies. At the present time,
resistance is the only means of controlling Pierce’s disease. Therefore, an under-
standing of the mechanisms of resistance would be extremely valuable in the develop-
ment of high quality resistant grape cultivars for the southern states.

REFERENCES
1. AUGER,J. G., SHALLA, T. A. & I(ADo, C. I. (1974). Pierce’s disease of grapevines: Evidence for a
bacterial etiology. Science184, 13751377.
2. BECKMAN,C. H. (1964). Host responses to vascular infection. Annual Review of Phytopathologv 2,
231-252.
3. BECKM.W, C. H. (1966). Cell irritability and localization of vascular infections in plants. Phyto-
pathology 56, 82 l-824.
4. BECKMAN,C. H., ELGERSMA,D. M. & MIACHARDY, W. E. (1972). The localization of fusarial
infections in the vascular tissue of single-Dominant-gene resistant tomatoes. Phytopathologv62,
1256-1260.
5. BELL, A. A. (1974). Biochemical bases of resistance of plants to pathogens. In Publication and
Proceedingsof the SummerInstitute on Biological Control of Plants, Inreck, and Diseases, Ed. by F. G.
Maxwell & F. A. Harris. p. 647. The University Press of Mississippi, Jackson.
6. DIMOMD,A. E. (1970). Biophysics and biochemistry of the vascular wilt syndrome. Annual Review
of Phytofiathology8, 301-322.
7. ELGERSMA,D. M. (1973). Tylose formation in elms after inoculation with Ceratocystis ulmi, a
possible resistance mechanism. Netherlande Journal of Plant Pathology 79, 218-220.
8. ESAU, K. (1948). Anatomic effects of the viruses of Pierce’s disease and phony peach. Hilgardiu
18,423-482.
9. GOHEEN, A. C., NYLAND, G. & Lawn, S. K. (1973). Association of a rickettsia-like organism with
Pierce’s disease of grapevines and alfalfa dwarf and heat therapy of the disease in grapevines.
Phytopathology63,341345.
10. H~wrrr, W. B. (1958). The probable home of Pierce’s disease virus. Plant Disease Reporter 42,
211-215.
11. HEWITT, W. B., Looms, N. H., OVERCASE, J. P. & PARRIS,G. K. (1958). Pierce’s disease virus in
Mississippi and other southern states. Plant Disease Reporter 42, 207-210.
12. HOPKINS, D. L. & MOLLENHAUER, H. H. (1973). Rickettsia-like bacterium associated with
Pierce’s disease of grapes. Science179, 298-300.
13. HOPKINS, D. L. & MOLLENHAUER, H. H. (1974). Grape culture. In McGraw-Hill Earbook of
Scienceand Technology(In press).
14. HOPKINS,D. L., MOLLENHAUER,H. H. & MORTENSEN,J. A. (1974). Tolerance to Pierce’s disease
and the associated rickettsia-lie bacterium in muscadine -grape - cultivars. .~ournal of the
American Societyof Horticultural Science99, 436439.
15. MOLLENHAUER.H. H. & HOPKINS.D. L. (1974). Ultrastructural studv of Pierce’s disease bacterium
in grape xykm tissue. Journal’of Bacteriolo~ 119,612-618. ’
16. PERRY, R., MOLLENHAUER, H. H. & BOWEN, H. H. (1974). Electron micrograph studies of
Pierce’s disease in Texas. Plant DieeaeeReporter 58, 780-782.
17. SINCLAIR,W. A., &HAND, J. P. & MELCHING,J. B. (1975). Localization of infection in American
elms resistant to Cerato&e ulmi. Phytopathology65, 129-133.
100 H. H. Mollenhauer and D. L. Hopkins
18. SPURR,A. R. (1969). A low-viscosity epoxy resin embedding medium for electron microscopy.
3ournalqf Ultrastructural
Research26, 31-43.
19. STONER,W. N., STOVER, L. H. & PARRIS,G. K. (1951). Field and laboratory investigations
indicate grape degeneration in Florida due to Pierce’s disease virus infection. Plant Disease
Reporter 35, 341-344.