verticillium wilt of chrysanthemum: colonization of leaves in relation to symptom...
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Canadian Journa of Botanv Published by THE NATIONAL RESEARCH COUNCIL OF CANADA
VOLUME 49 FEBRUARY 1971 NUMBER 2
Vevticillizlm wilt of chrysanthemum: coIonization of leaves in relation to symptom development
R. HALL AND L. V. BUSCH Department of Botany, University of Glrelph, Guelph,'Ot~tario
Received September 30, 1970
HALL, R., and L. V. BUSCH. 1971. Verticillirun wilt of chrysanthemum: colonization of leaves in relation to symptom development. Can. J. Bot. 19: 181-185.
The vascular system of leaves of chrysanthemum plants inoculated with Verticillirrtn dahliae was in- vaded by mycelium before the appearance of visible wilt symptoms. After flower buds appeared a rapid rise in the number of propagules of fungus in the leaf was followed by a rise in severity of visible wilt symp- tom expression and conductivity of leaf exudate. Those portions of the leaf with the most advanced stages of wilt contained the greatest amounts of fungus. It is suggested that the mycelium within the leaf makes a significant contribution to the development of symptoms of wilt.
Introduction relation to the onset and development of foliar I
The two general theories concerning the Symptoms.
mechanism of wilting by vascular pathogens both attempt to explain a basic observation
, (Beckman 1964) that cells of wilted leaves are deficient in water. The "plugging" theory as-
I serts that insufficient water reaches the leaf parenchyma because of malfunction of the vascular system. The "toxin" theory claims that there is excessive loss of water from leaf paren- chyma cells due, in part, to damage to semi- permeable membranes (Dimond 1967; Sadasivan 1961 ; Talboys 1968). However, it has been sug- gested (Talboys 1968) that these two theories do not cover all observations and oversimplify the complexities of several types of pathogenesis and symptom expression which have been collectively referred to as wilt diseases.
Implicit in both theories is a concept of "action at a distance." Damage, obstruction, or production of toxin in the vascular system is considered to lead to wilt symptoms in the leaves. Usually emphasis has been placed on the vascular system of root, stem, and petiole. Since symptoms of Verticilliunz wilt of herbaceous plants occur most noticeably in the leaves and little attention has been given to these organs, a study was made of colonization of chrysanthe- mum leaves by Verticillium dal~liae Kleb. in
Materials and Methods Chrysanthemums (Chrysatlthernrrm morifoliwn Ramat)
of the cultivar Princess Ann were used for all experiments. The plants were grown in pots, 4 in. in diameter, in a growth room kept at 21°C t 1°C, lighted by fluorescent cool white lamps augmented by incandescent lamps at a photoperiod cycle of 10 h light - 14 h dark. Inoculation took place at potting by mixing with the steamed potting mix about 4 g of a wheat grain culture of V. clahliae which had been obtained froin an infected Princess Ann chrysanthemum.
An effort was made to measure the effect of the fungus in the leaf on cell membrane integrity. Leaves were r s moved from plants which were being sampled, from the bottom to the top, numbered in consecutive order and weighed, and the wilt symptoms rated on a scale of 0 (no symptoms), 1 (wilt perceptible at leaf margin), 2 (wilt involving half of leaf), and 3 (wilt involving most or all of leaf). The leaves were then placed in glass-distilled water containing 0.1% Tween 20 (polyoxyethylene sorbi- tan monolaurate) for 30 min to remove surface contami- nants. The solution was then discarded and replaced with fresh solution, each leaf being immersed in 40 times its weight for 3 h. The conductivity of the solution was then measured with a Radiometer conductivity meter type CDM2e using a CDC114 electrode.
To determine the amount of fungus in the leaf, each leaf was then sterilized for 1 min in a 10% commercial Javex solution (0.5% sodium hypochlorite), rinsed in sterile water, comminuted for 1 min in an Eberback semimicro blendor, and plated on water-agar using a standard dilution series. Plates were incubated until the
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182 CANADIAN JOURNAL OF BOTANY. VOL. 49, 1971
Verricillin~n colonies developed microsclerotia. Consider- (day 36) there was no change in conductivity able care was taken to ensure that the colonies counted values to those for symptomless leaves. were not verticillate stages of fungi such as Gliocladium. The colonies of Verticillium were counted at the most began show a auurouriate dilution and an estimate made of the number significant (P = 0.05) increase when wilting in- ofcolbnies per gram of fresh weight of leaf. volved 50% or more of the leaf.
Results 1. Colonization and Symptom Developnzent
Rooted cuttings were inoculated and incubated as described above. At intervals after inocula- tion, the leaves were stripped from a single plant and the number of propagules per gram, con- ductivity of leaf exudate, and symptom rating of each leaf were determined as above. The mean values per plant are plotted against days after inoculation in Fig. 1. V. dahliae was recovered from leaves at day 24, 12 days before symptoms appeared. Symptomless leaves contained up to 11 000 propagules per gram. Symptoms of wilt began to appear soon after flower buds were apparent to the naked eye (day 28). As flowering progressed, there was a rapid rise in colonization of the leaves, accompanied by a rapid rise in the mean symptom rating.
The conductivity of 0.1% Tween 20 was 3.6 pmho. Throughout the sampling period washings from healthy leaves produced readings ranging from 3.6 to 16.6 pmho. At an early stage of wilt
I conductivity 3 VI VI
colonies
0 U .
z 3
- 8 0 -0
DAYS AFTER INOCULATION
FIG. 1. Mean conductivity (e), colonies of V. dahliaelg leaf tissue (O), and wilt rating (X) per leaf of chrysan- themum plants in relation to time after inoculation. Arrow indicates time at which flower buds (FB) first observed. Each point is a mean der~ved from leaves taken from one plant.
2. Form and Location of Fungus The form and location of the fungus within the
leaf was determined by microscopic examination of comminuted and sectioned leaf tissue and of strips of vascular tissue dissected directly from leaves.
Mycelium was readily observed in unstained preparations of vascular tissue at a magnification of 1250 diameters (Fig. 2a). It occurred exten- sively in vascular elements of stems, petioles, and leaves before and during the period of symptom expression in leaves. The vascular system was not, however, intensively invaded by mycelium.
In leaves attached to the plant the mycelium was invariably confined to the vascular system at all stages of symptom expression. However, it rapidly grew out from the vascular system in comminuted leaf preparations (Fig. 2b) or in detached leaves placed in petri dishes lined with moist filter paper. No conidia were seen in sections or in comminuted preparations. Co- nidia added to comminuted preparations ger- minated within 6 h and germ- tubes continued to elongate. No germinated conidia were ob- served in comminuted preparations of diseased leaves after 24 h.
3. Symptoms, Colonization, and Recovery from Wilt
To relate symptom rating of the leaf to reversi- bility of wilt, leaves were removed from plants 47 days after inoculation and divided into three groups according to the overall severity of symptoms expressed by the leaf (Table 1). Petioles were removed from the leaves, the cut end of the midrib was immersed in water, and each leaf lobe was tested for wilting. This was done by lifting or depressing the lobe with the forefinger. If the lobe moved freely without moving the rest of the leaf then the lobe was rated as wilted. The leaves were left in the water for 4 days, then tested again in the same way. As the symptom rating of the leaves rose from 1 to 3 the percentage of wilted lobes that re- covered from wilt declined from 90% to 20% (Table 1).
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FIG. 2. (a, upper photograph) Myceliilm (M) in vascular tissue of chrysantliemi~~ii leaf. x 1250. (b, lower photograph) Growth of ~ i i y c e l i i ~ ~ i ~ 6 h after removal of vascular tissue from chrysanthcmurn leaf. Phase con- trast, X 500.
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HALL AND BUSCH: VERTICILLIU rM WILT O F CHRYSANTHEMUM 183
TABLE 1
Recovery of chrysanthemum leaf lobes from wilt in relation to symptom rating of leaf
-- Symptom rating Percentage of wilted lobes
of leaf with reversible wilt
*Ten wilted lobes in each sample.
TABLE 2
Colonies of Verficillilr~n rlahline per gram of chrysanthemum leaf lobe tissue in relation to
symptom expression -
Wilt Other Colonies/g
Reversible None 7 333 Irreversible None 158 860 Irreversible Chlorosis and necrosis 221 140
To relate the symptoms expressed by the lobes to the amount of fungus in them lobes were cut from the leaves and grouped according to their symptoms, viz. reversible wilt, irreversible wilt but no other marked symptoms, and irreversible wilt with extensive chlorosis or necrosis (Table 2). Lobes with reversible wilt contained relatively little fungus (7330 propagules/g). The irrever- sible stage of wilt was associated with 20 to 30 times as much fungus.
The levels of colonization in the leaf tissue in Table 2 are higher than those recorded in Fig. 1 because only the area of the leaf expressing symp- toms was sampled. Further, some development of the fungus probably occurred during the 4 days the leaves were detached from the plant.
Discussion
Busch and Schooley (1970) showed that in chrysanthemum plants grown under short-day conditions visual symptoms of Verticillium wilt did not appear until flower buds were apparent on the plants. The number of propagules of V. dahliae isolated from stems increased rapidly as flowering progressed. The present results show a similar pattern of development of the fungus in the leaves.
Since the aim of this investigation was to relate symptom development to leaf coloniza- tion, an objective measure of symptom ex- pression was sought. Conductivity readings
were objective and gave a measure of tissue damage but were not significantly greater than those of healthy leaves until the wilt entered the irreversible stage. This can be interpreted in two ways. Since permeability changes did not pre- cede colonization it could be concluded that the permeability changes result, not from desicca- tion or a highly active toxin moving well in ad- vance of the mycelium, but from extensive colonization of the leaf and localized production of materials that damage membranes. Such an interpretation complements our data relating visual symptom expression to colonization.
On the other hand, permeability changes often occur very early in the disease process, before visual symptoms develop (Wheeler and Hanchey 1968). It is therefore possible that permeability changes occurred in infected chrysanthemum leaves earlier than they could be detected by the method of floating whole leaves. Emphasis is therefore placed in this discussion on visual ratings of symptom development.
Three stages in the development of wilt symp- toms are recognized. In the first stage of disease the leaf is colonized by the fungus but expresses no visible wilt symptoms. The mean level of colonization of symptomless leaves containing fungus was 5000 propagules per gram.
In the second stage, symptoms of wilt appear and colony counts increase. Leaves given a wilt rating of 1 contained, on the average, 11 000 propagules per gram. Values for individual leaves ranged from 7500 to 16 500. This early stage of wilt is reversible. Wilted leaves removed from the plant and placed in water regain turgor if the petiole is removed.
Reversibility of wilt has been interpreted by others in other hosts as evidence that the leaf wilts because it is not receiving sufficient water from the stem (Dimond 1967). Our data do not conflict with the possibility that the onset of the first visible and reversible wilt symptoms in the chrysanthemum leaf are triggered by events in the roots, stem, and petiole. However, since at this time the fungus is present as active mycelium in the leaf, we believe it is likely to be contributing to symptom development. There are indications in other hosts of Verticillium that early symp- toms of wilt are not entirely due to shortage of water. Schnathorst (1969) found that there was little disruption of vascular flow in stems of cotton plants infected with V. dalzliae until
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184 CANADIAN JOURNAL OF
symptoms had reached a rating of 3 on a scale of 0 (healthy) to 4 (dead). This suggested that early symptoms of wilt in cotton were not due to disruption of flow of water through the stem.
In the third stage of wilt of chrysanthemum the amount of mycelium in the leaf continues to increase and the wilt becomes irreversible and the leaf becomes increasingly chlorotic and necrotic. It is not possible at this time to say whether the increasing amount of mycelium leads to increasing amounts of tissue damage or vice versa. We suggest that the fungus in the leaf makes a major contribution to damage to the leaf at the irreversible stage of wilt and that its development and pathogenic activities increase as host tissue damage increases. In this respect Verticillium wilt of chrysanthemum leaves is analogous to diseases caused by foliar pathogens which invade through the epidermis. Verticillium will invade and cause chlorosis and necrosis of leaves of red clover (Sackston 1959), tomato and eggplant (Provvidenti and Schroeder 1959), and potato (Thanassoulopoulos and Hooker 1970) when applied as conidia to the surface. In the case of the wilt syndrome access of the fungus or itsmetabolic products to the leaf parenchyma is provided by the vascular system, although it is important to note that in this case the fungus in the leaf is confined to the vascular system during the wilt phase.
It has been claimed (Dimond 1967) that "in the wilt diseases we are always speaking of re- versible wilting." Irreversible wilting is con- sidered to be a passive manifestation of damage caused by desiccation. In contrast, the following observations suggest that the fungus in the chrysanthemum leaf actively contributes to symptom expression at both the reversible and irreversible stages of wilt. The fungus is present in the leaf as active mycelium before symptoms are visible. Secondly, the severity of wilt symp- toms increases as the amount of fungus in the leaf increases. Thirdly, those portions of the leaf with the greatest amounts of fungus show the most advanced stages of wilt.
The normal wilt syndrome caused by the pathogenic species of Verticillium is expressed in the leaves yet a search of the literature reveals few attempts to examine this organ for coloniza- tion. Wood (1967) in fact states that "many of the most typical (symptoms of wilt) appear in parts of the plants not yet colonized by the
BOTANY. VOL. 49, 1971
parasite." But there are some indications in the literature that, in some hosts, mycelium in the leaf contributes to symptoms.
In cotton, chloroplasts begin to disintegrate soon after Verticillium invades the leaf (Leyen- decker 1950), and the fungus can be isolated from all portions of the leaf and petiole when the first (epinastic) symptoms appear (Mathre 1968).
Colonies of V. dahliae may develop from discs taken from symptomless or diseased leaves of tobacco plants invaded by the fungus (Wright 1969). Using the size of the colony that developed as a measure of the amount of fungus in the leaf disc Wright found that necrotic areas of the leaf contained the highest concentrations of V. dahliae, yellow areas contained less fungus, and green areas contained little or no fungus. In this host Wright suggested that development of the fungus is stimulated by changes associated with senescence of the host cells.
In his discussion of the "tiger-stripe" syn- drome associated with the progressive wilt of hop Talboys (1968) suggests that "as a result of vascular occlusions the fungal metabolites are exerting a toxic action close to the site of pro- duction rather than at a distance." Since the "tiger-stripe" symptoms are expressed in the leaves, "close to the site" presumably refers to the vascular system of the leaf itself or of the petiole.
Pomerleau (1968) showed that the vascular pathogen Ceratocystis ulini often invades the leaves of American elm before symptoms are expressed, although infected leaves do not nec- essarily wilt, and wilted leaves are not always infected. In this case Pomerleau believes that the necrotic processes initiating wilting take place in the young shoots.
We propose that the activities and develop- ment of the mycelium of V. dahliae in chrysan- themum leaves make a significant contribution to the development of visible symptoms and that the concept of the influence of development of the fungus in the leaf on symptom expression may apply to Verticillium wilt of other herba- ceous plants.
We thank Mrs. Donna Sanford for technical assistance. The investigation was supported in part by the National Research Council of Canada.
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HALL AND BUSCH: VERTICILLIUM WILT OF CHRYSANTHEMUM 185
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