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Verticillium wilt of tobaccoL.N. Gibbins a b & D.S.C. Wright c da Cawthron Institute , P.O. Box 175 , Nelson , NewZealandb Department of Microbiology , University ofGuelph , Ontario , Canadac Tobacco Research Station, D.S.I.R. , R.D. 3,Motueka , New Zealandd Crop Research Division, D.S.I.R. , P.B.,Christchurch , New ZealandPublished online: 05 Jan 2012.

To cite this article: L.N. Gibbins & D.S.C. Wright (1968) Verticillium wilt oftobacco, New Zealand Journal of Agricultural Research, 11:4, 789-796, DOI:10.1080/00288233.1968.10422411

To link to this article: http://dx.doi.org/10.1080/00288233.1968.10422411

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789

VERTICILLIUM WILT OF TOBACCO

VI. The influence of roots and stems on Verticillium wilt symptoms in tobacco

By L. N. GIBBINS'" AND D. S. C. WRIGHT**

(Received 5 June 1968)

ABSTRACT

Tobacco root and stem systems were studied to determine their relative importance in resistance to Verticillium dahliae Kleb.

Three cultivars, the most susceptible and resistant of which differed widely in resistance to the disease, were grafted so that the stems and roots were united in all possible combinations.

The act of grafting lowered resistance in the more susceptible cultivars. The rootstocks of the most resistant cultivar marked Iv reduced symptoms in the susceptible scions grafted to them, and the stems of the resistant cultivar had more resistance than the stems of the susceptible ones.

A method of estimating the amount of susceptibility of the different graft combinations at different periods of growth is described.

The data most closely fitted a hypothetical model in which 74 percent of the resistance in all varieties at all stages of growth was contributed by the root system, the remainder corning from the stem.

INTRODUCTION

It has long been known that different tissues from the same plant differ in level of resistance to pathogenic organisms. Several workers have used grafting techniques to determine the relative importance of the root and shoot systems on vascular wilt syndromes. Heinze and Andrus (1954) found that a resistant tomato rootstock inoculated with Fusarium oxysporumgave resistance to a normally susceptible shoot. Keyworth (1953) obtained similar results in a study of Verticillium wilt in hops. But when Blackhurst and Wood (1963) infected cross­grafted resistant and susceptible, tomato plants with Verticillium albo­atrum Rke. and Berth. by root inoculation they found that resistance was not localised in the root, and they conrlrmed this finding by stem inoculation. Sewell and Wilson (1964) concluded that the stems of wilt-tolerant hops were slightly more resistant than those of wilt­sensitive ones, but the root was the determinative factor in the hop-wilt

• Cawthron Institute, P.O. Box 175, Nelson. Present address: Department of Microbiology, University of Guelph, Ontario, Canada .

.. Tobacco Research Station, D.S.I.R., R.D. 3, Motueka. Present address: Crop Research Division, D.S.I.R., P.B., Christchurch.

N.Z. Jl agric. Res. (1'968), 11: 789-96

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790 Verticillium wilt of tobacco. VI

syndrome, as previously suggested by Keyworth (1953). Likewise Fronek (1965) grafted potato stems to show that resistance to Verticil­lium was present in the tops as well as the roots. Berry and Thomas ( 1961), using different inoculation methods, found that resistance to V. albo-atrum (microsclerotial form = V. dahliae; in mint was common to both root and stem, but that the level was greater in the stem.

The grafting process reduced the growth of leaves, stems, and roots relative to intact plants in Nicotiana tabacum L. and N. rustica L. (Seltmann, Mann, and Waybrew 1962). The same authors concluded that the scion influenced the formation of alkaloids in the rootstock.

Preliminary work was carried out on cross-grafted plants of the tobacco cuItivars Virginia Gold and Coker 187 Hicks. Under glasshouse conditions these varieties showed very similar susceptibilities to the disease, a result confirmed in the work reported here. The purpose of the present investigation was to extend the study to field conditions, and to incorporate into the trial a variety with more marked resistance to the disease than Coker 187 Hicks. Information was sought on the relative contributions of the roots and aerial parts of the tobacco plant to resistance to Verticillium dahliae. The progression of the disease, as shown by external symptoms, was studied throughout the 1966-67 growing season. No growth measurements were made, but there were no obvious differences in size between the grafted and ungrafted plants.

MATERIALS AND METHODS The three cuItivars of N. tabacum used in this trial (Virginia

Gold, Coker 187 Hicks, and a biotype of McNairs 121) were respectively very susceptible, moderately field resistant, and resistant to Verticillium

TABLE l-Combinations of the Three Cultivars of Nicotiana tabacum Used in the Experiment

VG ungrafted (S)

CH ungrafted (M)

MN ungrafted (R)

VG/VG from same plant (S/S)

CHICH from same plant (M/M)

MN/MN from same plant (R/R)

VG/VG from different plants (SIS)

CHjCH from different plants (M/M)

Abbreviations used:

MN/MN from different plants (RjR)

VGjCH (SjM)

VGjMN (SIR)

CH/MN (MjR)

CHjVG (MjS)

MNjVG (RjS)

MNjCH (RjM)

VG, cultivar Virginia Gold; CH, cuItivar Coker 187 Hicks; MN bio~ype of cultivar McNairs 121; (R) resistant; (M) moderate field resistance; (S) susceptible.

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L. N. GIBBINS AND D. S. C. WRIGHT 791

wilt (Wright 1968a). Seedlings were grown in sterilised soil and single plants transferred to polythene sleeves (3t in. by 5 in. deep) when the largest leaves were about 2 in. long.

Razor blades were used to prepare cleft grafts at approximately 1 in. above soil level, when the stems were 3 in. to 4 in. long and approximately t in. thick. One leaf was left on each rootstock. The scion was trimmed of all leaves except one expanding leaf just below the apex. The graft tongue was cut so that about 1 in. of stem was left between the top of the graft and the apex. The graft union was held in place by at in. wide strip of "Parafilm", a self-sealing, water­proof plastic tape, which was stretched and wrapped firmly around the stem. To hold the graft together a stainless steel pin was inserted through the "Parafilm" and the graft.

The steel pins were removed 5 days after grafting, and 15 days later the "Parafilm" was stripped off. Only one graft was unsuccessful, and the plants grew well once the union was complete.

Inoculum was prepared from four pathogenic isolates of V. dahliae (Wright and Biss 1968) grown for 7 days at 24°c on potato glucose agar. The contents of 24 Petri dishes, six of each isolate, were mixed with 2.5 1. of water in a Waring blender. Three weeks after grafting, four holes t in. deep were made in the soil around each plant with a t in. diameter cork borer, and 1 ml of the inoculum was pipetted into each hole.

After inoculation, the polythene sleeves were removed, and the plants, with the inoculated soil intact, were planted at the Tobacco Research Station, D.S.I.R., Motueka, Nelson, in an area which was heavily infected with Verticillium dahliae and V. alba-atrum. The plants were fertilised and cultivated in the same manner as a commercial tobacco crop.

The 15 combinations used III this experiment are listed in Table 1. One plant of each type was planted at random in each of 20 rows, making a total of 300 plants. The plants were topped 64 days after planting in the field, and lateral growth was removed fortnightly.

Wilt symptoms first appeared in the plants early in January (7 weeks after inoculation), and the plants were examined fortnightly thereafter for 2 months, by which time 90% of the leaves on the Virginia Gold ungrafted controls showed symptoms.

At the end of the experiment sample isolations were made from the stems of five plants of each treatment, using the method described by Wright (1966) to confirm the presence of the fungus.

RESULTS AND DISCUSSION

Significant differences between the various combinations for the per­centage of leaves with symptoms were present at the first scoring, whereas the percentages of plants with symptoms did not show significant variation until the assessment at 11 weeks (Table 2).

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L. N. GIBBINS AND D. S. C. WRIGHT 793

The process of grafting per se significantly (P = 0.05) lowered the resistance of the more susceptible varieties Coker 187 Hicks and Virginia Gold, but not of McNairs 121. However, the very low score recorded 7 weeks after inoculation for the ungrafted McNairs 121, compared with the score for the grafted plants of the same variety, showed that disease expression was increased for a time in this variety also.

The assumption that grafting to a different plant of the same cultivar gave a different susceptibility to grafting back on to the same plant was tested. In the two more susceptible cultivars grafting on to the same plant appeared to increase susceptibility slightly compared with grafting on to different plants, but this difference was not significant.

Comparison of the two sets of figures, percentage of plants with some symptoms and percen.tage of leaves affected (Table 2), indicated the severity of the disease on the various plant groups. When McNairs 121 M:ions were grown on Coker 187 Hicks or Virginia Gold rootstocks, the number of plants showing symptoms was not altered, but the severity of the disease expression was reduced.

Rootstocks of McNairs 121 increased the resistance of the scions of both Coker 187 Hicks and Virginia Gold. Similarly the McNairs 121 scions showed some resistance, but this was not as marked as that contributed by the rootstock of this cultivar.

To arrive at a model to describe the results obtained it was assumed that for any graft combination, at any time, a certain proportion of the resistance was contributed by the roots, the remaining resistance coming from the stem. The following hypotheses may then be tested: (a) that the proportion contributed by the stock was independent of the variety grafted on to it; (b) that the proportion contributed by the stock was the same for all cultivars; and (c) that the proportion contributed by the stock was the same at all stages of growth observed. All these hypotheses were tested on the data, and none was shown to be invalid.

The mean proportion of leaves with symptoms in a graft com­bination at any stage was regarded as the total of a stock contribution and a scion contribution. A completely susceptible stem grafted to a fully resistant stock would give a fairly resistant plant, and the reciprocal graft a fairly susceptible plant. Values for the relative contributions to susceptibility of the graft combinations of the three cultivars under study were calculated. The range in susceptibility was from zero (complete resistance) to one (complete susceptibility). These values have been termed the "varietal susceptibility factors" and are presented in Table 3.

The transformed data derived from the angular transformation fitted closely a hypothetical model in which 74 percent of the resistance of graft combinations was contributed by the rootstock and the remaining 26 percent by the scion.

With the aid of the varietal susceptibility factors and a knowledge of the proportions contributed by the root and the stem, relative susceptibility values could be predicted, and these have been calculated for each pultivar combination for the five times of observation (Table 4).

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794 Verticillium wilt of tobacco. VI

For example, for a Virginia Gold scion grafted on to a McNairs 121 stock, the susceptibility value at 13 weeks after inoculation was derived as follows:

Contribution from Virginia Gold scion: = scion factor (0.26) X varietal susceptibility factor at 13 weeks

for Virginia Gold (0.72) = 0.187.

Contribution from McNairs 121 stock: = stock factor (0.74) X varietal susceptibility factor at 13 weeks

for McNairs 121 (0.01) = 0.007.

Cultivar

Virginia Gold

Coker 187 Hicks

McNairs 121

TABLE 3-Re1ative Contributions to Susceptibility by Varietal Susceptibility Factors

Weeks after Inoculation

7 9 II 13

I I

0.09 0.13 0.38 0.72 I

I

I 0.09 I 0.14 0.30 0.61

I

I 0.04

I 0.04 0.02 0.01

TABLE 4-Re1ative Susceptibility to Verticillium Wilt of the Inter-Variety Graft Combinations

Weeks after Inoculation

Combination

I I

I

I 7 9 11 ! 13

I

VGjCl-l 0.090 I 0.138 I

0.321 0.638 I

VGjMN 0.053 0.064 0.114 0.194

CHjMN 0.053 0.066 0.093 0.166

CH/VG 0.090 0.140 I

0.359 0.692 I

MN/VG 0.077 0.114 I 0.333 0.536

MNjCH 0.077 0.114

I 0.274

I 0.454

15

0.92

0.88

0.07

I 15

0.890

0.291

0.281

0.910

0.699

0.569

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L. N. GIBBINS AND D. S. C. WRIGHT

Therefore total susceptibility value of the combination 0.007 = 0.194.

795

0.187 +

This figure is an "equivalent angle"; the corresponding percentage would be 11.0 compared with the figure actually obtained in the trial of 7.8 -+- 3.1 percent (Table 2).

The results confirmed the report of Wright (1968a) that McNairs 121 was much more resistant to Verticillium wilt than the other two cuItivars. Coker 187 Hicks was not significantly more resistant than Virginia Gold until towards the end of the growing season.

In Coker 187 Hicks and Virginia Gold the number of leaves with symptoms increased in a linear manner over the period studied; but in McNairs 121 the symptoms observed early in the season dis­appeared as the plants grew and as leaves which showed early symptoms dropped from the plants after senescence. The control of Verticillium dahliae within the tissues of plants of this cuItivar during the logarithmic stage of growth has been demonstrated (Wright 1968b).

There was a marked increase in symptom expression in McNairs 121 at the 15th week, but this was small compared to that shown at the same stage by other cuItivars.

Close agreement was found between the disease symptoms observed and the amount of Verticillium in the stems at the end of the growing season.

CONCLUSIONS

Grafting significantly increased susceptibility to Verticillium wilt in the two susceptible cultivars. Disease symptoms increased linearly over the 2 months of the trial in the cuItivars Virginia Gold and Coker 187 Hicks, whereas symptoms decreased (and in autografts disappeared for a time) in the resistant cultivar McNairs 121. At the end of the trial there was a slight but significant increase in disease symptoms in the resistant variety. The percentage of leaves with symptoms in the susceptible cuItivars was greater by more than a factor of 10 than in McNairs 121.

The rootstock of resistant McNairs 121 markedly increased the resistance of the susceptible scions. Stems of McNairs 121 also showed some resistance to the disease when grown on susceptible rootstocks.

In all varieties, at all the stages of growth observed in the trial, the root system contributed 74 percent of the total resistance.

Acknowledgments

Dr M. P. Griffin, Appied Mathematics Division, D.S.I.R., Wellington, for statistical analyses; Mr R. W. James, Superintendent, Tobacco Research Station, D.S.I.R., Riwaka, for help in designing the experiment; Mr D. J. Stanton, Cawthron Institute, Nelson, for co-operation in developing a satisfactory grafting technique; Mr J. M. Biss, Miss A. L. Tindale, and Miss L. M. Buckland for technical assistance.

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796 Verticillium wilt of tobacco. VI

REFERENCES

BERRY, S. Z.; THOMAS, C. A. 1961: Influence of soil temperature, isolates, and method of inoculation on resistance of mint to Verticillium wilt. Phytopathology 5]: 169-73.

BLACKHURST, F. M.; WOOD, R. K. S. 1963: Resistance of tomato plants to Verticillium albo-atrum. Trans. Br. mycol. Soc. 46: 385-92.

FRONEK, F. R. 1965: Verticillium wilt resistance in potato. Diss. Abstr. XXVI: 607.

HEINZE, P. H.; ANDRUS, C. F. 1945: Apparent localization of Fusarium wilt resistance in the Pan America tomato. Am. 1. Bot. 32: 62-6.

KEYWORTH, W. O. 1953: Verticillium wilt of the hop VI. The relative roles of root and stem in the determination of wilt severity. Ann. appl. Bioi. 40: 344-61.

SEl.TMANN, H.; MANN, T. J.; WAYBREW, J. A. 1962: Dry weights a:ld alkaloid content of reciprocal grafts between Nicotiana tabacllm and N. rustica. Tobacco Science VI: 37.

SEWELL, O. W. F., WILSON, J. F. 1964: Occurrence and dispersal of Verticil/ium conidia in xylem sap of hop (Humulus lupulus LJ. Nature, Lond. 204: 901.

WRIGHT, D. S. C. 1966: Verticillium wilt of tobacco (a note). N.Z. Jl agric. Res. 9: 448-51.

-----1968a: Verticillium wilt of tobacco. V. Varietal screening for resistance to Verticillium dahliae Kleb., and mode of inheritance of resistance. N.Z. Jl agric. Res. ]]: 655-64.

----- 1968b: Verticillium wilt of tobacco. VIII. Movement of Verticillium dahliae conidia in tobacco plants. N.Z. Jl agric. Res. 11: 803-11.

BISS, J. M. 1968: Verticillium wilt of tobacco. VII. Pathogenicity of Verticilliwn dahliae isolates. N.Z. II agric. Res. II: 797-802.

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