VERTICILLIUM WILT OF TOBACCO

V. Varietal screening for resistance to Verticillium dahliae Kleb., and mode of inheritance of resistance

By D. S. C. WRIGHT*

(Received 5 June 1968)

ABSTRACT

Eight newly introduced tobacco cultivars were screened for resistance to Verticillium dahliae Kleb. in 1965-6 season, and the following year 10 cultivars and four advanced breeding lines were tested for resistance.

The mode of inheritance of resistance found in four of the cultivars used in the breeding programme was studied in a set of diallel crosses. Most of the genetic variance appeared to be controlled by additive factors, with the high level of resistance found in a biotype of McNairs 121 conditioned by polygenes. However, in the field-tolerant cultivar Coker 187 Hicks, most of the resistance was the result of epistatic effects.

INTRODUCTION

655

Thomson and McLeod (1959) and McLeod and Thomson (1959) described the disease caused by Verticillium dahliae Kleb. in New Zealand tobacco and outlined the results of their varietal screening and breeding for resistance. More recent work is reported in the present paper.

Literature related to Verticillium alba-atrum Rke. and Berth. and V. dahliae as pathogens is detailed and extensive (Rudolf 1931; Panton 1964), and marked similarities exist between the Verticillium-tobacco syndrome and the expression of this disease in other crops. Variety and species differences in resistance have been noted in tomato (Bewley 1922), cotton (Jameson 1946), bramble fruits (Wilhelm and Thomas 1950). and many others. Since McLeod and Thomson first evaluated the resistance of Nicatiana species and N. tabacum L. varieties. several flue­cured cultivars with tolerance to the disease have been introduced into New Zealand. These had inferior leaf quality and were susceptible to black root rot (Thielaviopsis basicala (Berk. and Br.) Ferraris), known to be widespread in local tobacco fields. A breeding programme was under­taken to improve quality and incorporate multiple disease resistance (Wright 1965).

• Crop Research Division, Lincoln, Department of Scientific and Industrial Research, Private Bag, Christchurch. Previous address: Tobacco Research Station, Department of Scientific and Industrial Research, Riwaka, R.D. 3, Motueka.

N.Z. Jl agric. Res. (1968), 11: 655--64

656 Verticillium wilt of tobacco. V.

McLeod suspected that the high levels of tolerance found in his material were quantitatively inherited. Putt (1964), however, noted that breeding behaviour was not consistent among the various host species which had been studied. Recessive, dominant. and additive factors were found to be responsible for field resistance. and in some plants at least two types of inheritance were present (Putt 1964; Bowen 1965). Gopinath, Lakshminarayana, and Narayana (1967) found significant epistatic effects in the inheritance of some traits in N. tabacum. Although epistasis caused complications in a diallel, Hayman (1 954b ) stated that in diallel studies duplicate epistasis left the ratios of some components of genetic variation and the Vr.Wr graph almost unaltered. He pointed out later (Hayman 1957) that pronounced deviations from the simple additive-dominance genetic model made the model unreliable.

MATERIAL AND METHODS

In the 1965-6 season, eight cultivars, with Golden Wilt (resistant control) and Virginia Gold (susceptible control), were planted in three randomised blocks. The method of inoculating seedlings as they were transplanted from seedbed to field (Wright 1966) lessened maturity differences which influence symptom expression. and "escapes" which troubled McLeod were virtually eliminated. Inoculation was known to give approximately a twofold increase in symptoms over natural infection when tobacco was grown in heavily infested soil (Wright and Biss in press).

Individual plants (28 per plot) were scored when disease expression was severest. at the end of the growing season. by counting the numbers of leaves with and without symptoms.

Included among the 16 cultivars in the 1966-7 trial were the eight cultivars grown in the previous season and four F8 breeding lines. In these lines single plants had been selected and selfed in each generation except the third, in which six selected plants were inter-pollinated and bulked to give the fourth generation seed (Palmer 1953). Also included were Coker 187 Hicks, which had been grown in New Zealand on a commercial scale, and another introduced line (NC2514). The resistant control in this trial was a biotype of McNairs 121.

The design was a triple lattice with 20 plants. per plot. Methods of inoculation ,and scoring were the same as in previous years.

A complete diallel cross was made using four of the cultivars which had been used in the Verticillium wilt breeding programme. These had all been inbred for at least 10 generations. The varieties used were the susceptible Virginia Gold (VG), Virginia 724 (V7), and Coker 187 Hicks (CH) with some field resistance, and McNairs 121 selected bio­type (MN) resistant.

No back-crosses were available and therefore maternal influences and discrepancy in reciprocals were not estimated. The four cultivars (selfed seed from the plants used as parents) and their six FI, F2, and

D. S. C. WRIGHT 657

F3 families were inoculated and scored as described previously. The p

raw data were transformed using the formula log -- and analysed as 1-p

six incomplete blocks with 10 plants per plot.

The additive-dominance model used was that described by Hayman (1957) and extended by M. P. Griffin (pers. comm.) to include F3 families. Derivation of the genetic model from various parameters was therefore as follows:-

P F1

. F2

F3'

Pr =m+dr frs =m+Hdr+ds) +hrs grs=m+Hdr+ds ) +ihrs ers=m+i(dr+ds) +thrs'

where dr was the sum of positive and negative additive contributions of genes in the rth parent, hrs was the sum of positive and negative domin­ance deviations of genes by which Pr and Ps differ, and m was the mean, where ~ d r = O. The analysis of the diallel cross and notations used followed Hayman (1954a, 1954b, 1957).

RESULTS Varietal screening

The results of the trials in the two seasons are presented in Table 1. Marked variation was found among the cultivars tested, ranging from very high resistance to almost complete susceptibility. Most of the cultivars grown in both seasons gave similar results. and small differences may be the result of environmental influence (McLeod and Thomson 1959). The marked change in resistance of NC2512 may be explained by the fact that seed sown in 1966-7 came from plants with a high level of tolerance in the previous season. and therefore the more susceptible biotypes would have been discarded. Prior to this no selection for resistance to Verticillium had been made within this cultivar. The author has evidence that wide biotypic variation exists in some of the cultivars and selection within these may be expected to improve resistance.

The standard deviations shown in Table 1 illustrate that cultivars intermediate for resistance were the most variable.

Genetics

From the diallel cross data a Vr.Wr graph was prepared (Fig. 1). This indicated that resistance was controlled mainly by additive factors in 'the case of Virginia Gold and McNairs 121 and by dominant factors in the case of Coker 187 Hicks. The graph was drawn on the assumption that the additive dominance model was correct. so that H / D was constant and therefore the line drawn from A was of unit slope. The line of best fit through the plotted points of the parental arrays had b=0.823. Although only four parents were included in the diallel, the points were well dispersed along the regression line and a reasonably reliable estimate of,slope could be obtained.

658 Verticillium wilt of tobacco. V.

TABLE 1. Screening cultivars for resistance to verticillium wilt

I % of leaves per :

plant with symptoms I *Statistical significance (± S.D. %)

Cultivar

--------------------

1965·6 Golden Wilt 24 ± 3.4 la NC95 30 ± 3.6 la NC2512 50 ± 4.1 lb NC2326 55 ± 4.1 Ib Va115 58 ± 4.0 Ibl~ Va218 63 ± 3.9 Virginia Gold 67 ± 3.8 Ic McNairs 30 76 ± 3.3

I~ Kutsaga 51 77 ± 3.3 McNairs 20 82 ± 3.1

SE's of means ± 4.0 Coefficient of variation = 7% 1966-7

McNairs 121 I ± 0.8 a BLI054 14 ± 3.3 b NC2512 15 ± 3.4 b BLI058 19 ± 3.8 b NC2514 20 ± 3.8 b BL801 34 ± 4.6 I~ NC95 40 ± 4.7 BLI037 44 ± 4.7 IC NC2326 55 ± 4.8

Iii: Va1l5 60 ± 4.7 Vall 8 65 ± 4.6 Coker 187 Hicks 72 ± 4.3 McNairs 30 79 ± 3.9 lel~ Virginia Gold 83 ± 3.6 McNairs 20 90 ± 2.9

fl: Kutsaga 51 97 ± 1.6

SE's of means = ± 3.5 Coefficient of variation = 5%

• Bars mark non-significant groups, using t - test at the 5% level of probability.

(4 X 4)

(3 X 3)

(4 X 4)

(3 X 3)

(4 X 4)

TABLE 2. Parameters in diallel analysis Mean including CH (4 X 4) = 0.362 Mean excluding CH (3 X 3) = 0.385

Contribution to additivity (d,)

VG V7 CH

2.12

2.32

0.80

1.01

. 0.42

Sum of dominance deviations (h,,)

VG X V7 VG X MN V7 X MN

0.41 --0.28 --0.39

0.13 --1.45 0.34

VG X CH V7 X CH MN X CH

-1.75 --1.10 --0.49

MN

-3.35

-3.33

D. S. C. WRIGHT 659

The parameters m, dr. and hrs were estimated (Table 2) and used to determine expected mean scores for each generation using the additive­dominance model; these estimated expected scores, with those actually observed in the trial, are presented in Table 3. Wide deviations were found in each generation, the largest being from filial generations where Coker 187 Hicks was the parent.

Estimated components of variation obtained from the analysis of variance of the diallel are shown in Table 4. Variance due to environment (E) was negligible, most of the variance was related to additive effects

Wr2'S'S2Vr

o 2

v.

Fig I.-Graph of dominance relations for best estimate of hr. using P, FI, F2, and F3.

TA

BL

E

3.

Obs

erve

d m

ean

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es

and

expe

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mea

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us

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aren

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I

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on

b

on

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on

_ _

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erve

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2.8

5 +

1.6

1 +

1.4

1 -2

.88

I

Exp

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d (w

ith

CH

) +

2.4

9

+0

.36

+

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7 +

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I

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itho

ut C

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0.06

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ial

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ons

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I V

G

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tion

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erve

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2.5

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--0.

63

+0.

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---0

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-1.6

1

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4

+0

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--

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--

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9

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9

---0

.02

No

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+

2.1

8

+0

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--

-0.5

7 --

0.0

6

---0

.43

+0

.05

F2

O

bser

ved

+ 1

.55

+0

.08

--

0.14

--

-O.6

R --

0.4

6

--0.

86

CH

+

2.0

3

----{

l.48

+0

.76

--

-0.6

8 --

0.39

+

0.2

5

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-1

.10

-0

.65

+-

0.19

-1

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+-

0.49

N

o C

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+ 2

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4 +

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+

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I -

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erve

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7

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9 -1

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6 N

o C

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+2

.08

--

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6 --

0.23

--

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-0.6

9 --

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D. S. C. WRIGHT 661

CD), and this was supported by the estimated value of general combining ability (a=D-F+HI-H2) 4.86, compared with a value of 1.86 for specific combining ability (H2). The genetic variance due to intra­allelic interaction (D-Hl) was relatively high. Other ratios derived from the components of variation provided additional information regard­ing the inheritance of resistance to this disease. The ratio (Hl jD)!i, an overall measure of the degree of dominance, was 0.66. which suggested partial or incomplete dominance. Hayman (1954b) stated that h2 j He, which estimated the number of gene groups controlling a character, was depressed by the presence of epistasis; however, the figure of 0.2 obtained here suggested that no major genes were involved.

The estimated relative dominance (Fr) of each parent is shown in Table 4, these figures supporting the Vr,Wr graph.

Using the diallel results, a chi-squared test for epistasis gave a value of 85.7, which was highly significant (P=O.l%). The data were then analysed omitting CH and its families, giving a chi-squared figure of 12.76 with six degrees of freedom (p=5%). When the susceptible VG and the resistant MN and their filial generations alone were analysed the chi-squared test for epistasis was not significant.

The results suggest that the inheritance of resistance in Coker 187 Hicks was conditioned mainly by epistatic effects and that this was also the case, but to a lesser extent, in Virginia 724. When the data were

TABLE 4. Diallel cross components of variation

D F HI H2 h2 E D-Hl

(.~ X 4) 5.45 1.31 2 .. 37 1.86 0.40 0.07 3.08

(.1 X 3) 7.93 1.40 3.21 2."(2 0.39 0.07 ·k72

Relative dominance of eac!'t parent (F,)

VG V7 ,I

CH MN

(4 X 4) -1.35 1.27 3.95 1.09 .' (J x 3) -1.38 J.34 II 2.62

D - additive component; F - co-variance of additive and dominance effects; HI - dominance component; H2 - dominance indicating assymetry; E - environ­mental component; h2 - dominance effect over all loci.

662

VG X CH fl

Verticillium wilt of tobacco. V.

V7 X CH f fl

MN X CH f2

VG X MN ~7XMN f1

f2

Yrs.

VG X V7 FI

hrs _-+ ______ + ______ -t----f--'f-'-'I 1--'-'f3=___ ---+------i--hrs.

f2

f3

f3

f2

fl

L

FI

f3

Yrs.

Fig. 2.-Graph of apparent dominance vs epistatic deviation.

f3 f2

examined for the type of epistasis present, as suggested by Hayman (1957), the crosses VG X CH and V7 X CH showed significant levels of duplicate epistasis (Fig. 2).

Because of the pronounced epistasis present no standard errors could be calculated for the components of variation presented in Table 4. The data indicated. however, that a large part of the genetic variance was the result of additive gene action, especially in the susceptible Virginia Gold and the resistant biotype of McNairs 121.

DISCUSSION

All the introduced cultivars with field resistance to Verticillium have been bred in the U.S.A. for multiple resistance to other diseases, namely Pseudomona.\ solanacearum (Smith) Dowson. Fusarium oxy­sporum Schlecht var. nicotianae (Johnson) Snyder and Hansen. and Phytophthora parasitica Dast. var. nicotianae (Breda de Haan) Tucker. In almost all of these cultivars the resistance may be traced back to T1448A, a Colombian variety resistant to Pseudomonas and Fusarium wilts, and to Florida 301, a cigar variety with Phytophthora canker resis­tance. Lucas (1965) commented on the similarity of wilt diseases in tobacco. and resistance to Verticillium has been associated with resis­tance to other diseases in several host species (Wilhelm 1954; Sappenfeld 1963; and others). According to Lucas (loc. cit.) the resistance of tobacco to wilt diseases has been described in each case as additively inherited.

D. S. C. WRIGHT 663

The development of the breeding lines included in the screening trial has already been described (Wright 1965). In this programme selection pressure for resistance to several diseases could be applied to the same plants, as each pathogen could be made to attack the tobacco at a different stage of growth. Small seedlings (up to 4 in. high) were assessed for black root rot resistance and those with an acceptable level were then inoculated with Phytophthora. Finally, at the stage seedlings were normally planted out in the field (6-8 in. high), selected plants were inoculated with Verticillium for evaluation towards the end of the season, some five months later. Evaluations of yield and quality (both physical and chemical) were made on cured leaf in each alternate generation.

Driver ( 1962) 'stressed the importance of knowing as much as possible about the plant-pathogen complex, including the inheritance of resistance, before beginning a breeding programme. Not only was this important in deciding the best breeding technique to use, but also an understanding of the type of resistance gave an indication of its probable permanence. The breeding of cultivars with tolerance derived from the additive factors available in the material described above may offer the best prospect for obtaining a permanent method of controlling this serious disease in New Zealand tobacco. The high level of tolerance in the selected biotype of McNairs 121 should be fully exploited, whereas the use of Coker 187 Hicks as a parent should be avoided because of epistasis. Although epistatic effects are useful where heterosis, especially complementary heterosis, may be exploited, epistatic effects are not fix­able (Lush 1943); furthermore, Hayman (1957), analysing observations made on N. rustica L. by links, found epistatic effects were inconsistent from year to year. From this it must be concluded that resistance based on epistatic gene action, if it behaved in the same way as differences in height and time of flowering in N. rustica, may be unreliable.

It was of interest to note, however, that the promising breeding line BL801 was derived from a cross between McNairs 121 and Coker 187 Hicks. In the work reported here the F3 of this cross had the very good score for wilt resistance of -1.99 (Table 3), and the deviation of this from the expected score was large and negative, the opposite of that which would have been predicted (Fig. 2).

In work reported elsewhere (Wright in press) it has been established that in the cultivars Virginia Gold, Coker 187 Hicks, and McNairs 121, although the fungus may be present throughout the plant, it is unable to become dominant until after the plant has flowered. At this stage in the susceptible cultivars the fungus overcame the plant's defence mechanism in the leaves and symptoms appeared. In tolerant plants the fungus was checked even after flowering and even when present in the leaves. All cultivars have this defence mechanism during the logarithmic stages of growth and to varying degrees after this. In Coker 187 Hicks seedlings the movement of the fungal conidia in the vascular system was restricted to a greater degree than in Virginia Gold and McNairs 121 (Wright in press). The different resistance mechanism found in Coker 187 Hicks may be conditioned by epistasis and the underlying resistance found in

664 Verticillium wilt of tobacco. V.

all cultivars may be the system which is additively inherited. This appears to be common to all tobacco plants and may be the basic natural defence against many disease organisms.

ACKNOWLEDGMENTS Thanks are expressed to Dr M. P. Griffin, Applied Mathematics

Division, D.S.I.R., Wellington, for statistical analysis of the data; to Mr R. W. James, Superintendent, Tobacco Research Station, D.S.I.R., Motueka, for help in planning the work; and to Mr J. M. Biss for tech-

nical assistance in the field and laboratory.

REFERENCES BEWLEY, W. F. 1922: Sleepy disease of tomato. Ann. appl. Bioi. 9: 116-34. BOWEN, H. H. 1965; Breeding studies of Verticillium wilt resistance in

Fragaria ananassa Duch. Diss. Abstr. XXVI: 5664. DRIVER, C. M. 1962: Breeding for disease resistc.Ilce. Scott. Pl. Breed. Stn

Rec.: 28-38. GOPINATH, D. M.; LAKSHMINARAYANA, R.; NARAYANA, C. L. 1967: The

mode of gene actlOn in flue-cured tobacco. Eupilytica i6: 293-9. HAYMAN, B. 1. 1954a: The analysis of variance of dialle! tables. Bio­

metrics 10: 235-44. ----1954b: The theory and analysis of diallel crosses. Genetics,

Princeton 39: 789-809. -----1957: Interaction, heterosis and diallel crosses. ibid. 42: 336-

55. JAMESON, J. D. 1946: A. Rep. Dep. Agric. Uganda Protectorate. Part II.

pp. 32-3. LUCAS, G. B. 1965: "Diseases of tobacco". 2nd ed. Scarecrow Press,

New York and London. 778 pp. LUSH, J. L. 1943: "Animal breeding plans," 2nd ed. pp. 127-30. Iowa

State College Press, New York and London, 437 pp. McLEO;), A. G.; THOMSON, R. 1959: Verticillium wilt or tobacco. II.

Field and glasshouse trials to determine resistance of varieties and species to Verticil/hun dahliae Kleb. N.Z. JI agric. Res. 2: 792-6.

PALMER, T. P. 1953: Progressive Improvement III self-fertilised crops. Heredity, Lond. 7: 127-9.

PANTON, C. A. 1964: A review of some aspects of the wilt pathogen Verticillium albo-atrum. Acta Agric. scand. 14: 97-112.

PUTT, E. D. 1964: Breeding behaviour of resistance to leaf mottle or Verticillillm in sunflowers. Crop. Sci. 4: 177-9.

RUOOLF, 8. A. 1931: Verticillium hadromycosis. Hilgardia 5: 201-353. SAPPENFELD, W. P. 1963: Fusarium wilt-root knot nematode and Vertic. 1-

lillm wilt resistance in cotton: possible relationships and influence on cotton breeding method3. Crop. Sci. 3: 133-5.

THOMSON, R.; McLEOD, A. G. 1959: Verticillium wilt of tobacco. I. A new disease of tobacco caused by Verticillillm dahliae Kleb. N.z. Jl agric. Res. 2: 785-91.

WILHELM, S. 1954: Verticillillm wilt resistance in strawberry. Phyto­pathology 44: 510 (abstract).

WILHELM, S.; THOMAS, H. E. 1950: Verticillium wilt of bramble fruits with special reference to Rubus llrsillUS derivatives. Ibid. 40: 1103-10.

WRIGHT, D. S. C. 1965: Breeding better tobacco. N.Z. Tab. Grow. J. (July): 10-12.

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

-----(in press): Verticillium wilt of tobacco. VIII. Movement of Verticillillm dahliae Kleb. conidia in tobacco plants. I bid.

WRIGHT, D. S. C.; BISS, J. M. (in press): VerticilJium wilt of tobacco. VII. Pathogenicity of Verticillillm dahliae isolates (a note). Ibid.