Resistance of eggplant, its wild relatives and their hybrids to different strains of Pseudomonas solanacearum

Scientia Horticulturae, 45 (1990) 1-9

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Elsevier Science Publishers B.V., A m s t e r d a m

Resistance of eggplant, its wild relatives and their hybrids to different strains of Pseudomonas solanacearum Mohammad Ali, Mohammed Abdul Quadir*, Hiroshi Okubo and Kunimitsu Fujieda Faculty of Agriculture, Kyushu University, Hakozaki, Higashi-ku, Fukuoka 812 (Japan) (Accepted for publication 17 April 1990)

ABSTRACT Ali, M., Quadir, M.A., Okubo, H. and Fujieda, K., 1990. Resistance of eggplant, its wild relatives and their hybrids to different strains ofPseudomonas solanacearum. Scientia Hortic., 45: 1-9. Resistance of eggplant (Solanum melongena L. ), its wild relatives and six intervarietal/interspecific hybrids of Solanum was evaluated with respect to five specialized plant pathogenic strains of Pseudomonas solanacearum. Eggplant was highly susceptible to all the strains except one to which it showed moderate reaction. Solanumferox and Solanum toxicarium were found to be immune to four and five strains, respectively. Moderate susceptible reaction was evident in Solanurn sisymbriifolium against most of the strains. Solanum integrifolium and its hybrids with eggplants were found to be highly tolerant to two to three strains. The poorest level of resistance was observed in S. melongena var. insanum, Solanum gilo and their hybrids with eggplants. In general, symptom severity decreased with age. Differential pathogenicity among the strains was also recorded. Keywords: bacterial wilt; eggplant; interspecific hybrids; Pseudomonas solanacearum; resistance; wild relatives. Abbreviations: PS = potato-sucrose; TTC = triphenyl tetrazolium chloride.

INTRODUCTION

Bacterial wilt of eggplant, caused by the soilborne bacterium Pseudomonas solanacearum E.F. Smith, is a serious disease, which limits eggplant (Solanum melongena L. ) production. Very little success has been attained using chemical control measures. The only effective means to control bacterial wilt is to *Present address: Institute o f Postgraduate Studies in Agriculture, Salna, Gazipur, Bangladesh.

0304-4238/90/$03.50

© 1990 - - Elsevier Science Publishers B.V.

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M. ALI ET AL.

develop resistant cultivars. Reports on complete resistance to bacterial wilt of eggplant are very limited. Recently Goth et al. (1986) reported some cultivars from India, Turkey and New Guinea with high resistance. Cultivars found to be resistant in the subtropics seem to be susceptible in the humid tropics (Mew and Ho, 1977). There is evidence (Mochizuki and Yamakawa, 1979a,b; Ozaki, 1985 ) that close relatives of eggplant possess very high to complete resistance to bacterial wilt, but most of the previous reports on resistance are against a single undefined strain (Mochizuki and Yamakawa, 1979a,b). Only a few Solanum species have been tested against defined strains (Ozaki, 1985). Moreover, bacterial wilt disease reaction of interspeeific hybrids and some Solanum species have not yet been reported. The present study was undertaken with the objective of confirming the precise reaction of the selected commercial cultivars, wild relatives and their hybrids against all available well-defined virulent strains of P. solanacearum. This study will provide information useful for undertaking interspecific sexual and somatic hybridization to develop resistant eggplant cultivars and to exploit suitable root stock for grafting which possesses a high degree of resistance to the disease along with other desirable characteristics. MATERIALS AND METHODS

Two cultivars of eggplant (Solanum melongena L.), 'Senryo 2 gou' and 'Uttara', five wild relatives of eggplant, namely: S. melongena L. var. insanum, Solanum ferox L., Solanum gilo Raddi, Solanum integrifolium Poir, Solanum sisymbriifolium Lam and Solanurn toxicarium Lam, and six intervarietal/interspecific hybrids of eggplant and wild relatives were evaluated for their resistance to five specialized pathogenic strains of P. solanacearum which were defined and grouped by Ozaki (1985). Pure cultures of the five strains: 8107, 8109, 8215, 8224 and 8503 - Groups, I, V, II, IV and III, respectively (obtained from Mr. Ozaki, National Research Institute of Vegetable, Ornamental Plants and Tea, Morioka Branch, Japan) were kept at 4°C until the preparation of inocula. Experiments were carried out during the summer of 1989. Preparation of inoculum. - Inocula of different strains were prepared from virulent isolates selected with tetrazolium (TTC) medium (Kelman, 1954) and multiplied in potato-sucrose (PS) medium (Wakimoto and Yoshii, 1955 ). Bacteria were harvested and resuspended in sterilized distilled water maintaining inoculum levels of ca. 108 cfu ml -~ (Mew and Ho, 1977; Ozaki and Kimura, 1989)for all five strains. Inoculation. - Seedlings, grown in plastic pots (9 cm diameter) containing heat sterilized sand, were inoculated by the injured-root-dipping method (Winstead and Kelman, 1960) at the three to four leaf stage (33 days old). After dipping in the bacterial suspension for 15 min seedlings were trans-

RESISTANCE OF EGGPLANT TO PSEUDOMONAS

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TABLE 1 Symptom classification of wilting and vascular bundle browning ofSolanum genotypes due to inoculation of Pseudornonas solanacearum Symptom grade

0 1 2 3 4 5

Symptom Leaf wilting

Vascular bundle browning

No wilting One leaf wilted Two leaves wilted Three leaves wilted More than three leaves wilted Dead

Unaffected < 1/6 brown 1/6 to < 1/2 brown 1/2 to < 2 / 3 brown 2 / 3 to totally brown Totally brown

planted back into the same pots. Twenty seedlings of each entry (except in the case of S. f e r o x and S. sisymbriifolium, where, because of lower emergence only 10 seedlings were used) were inoculated with each strain. A similar experiment was repeated with another batch of plants at the four to five leaf stage (40-44 days old). A control treatment was given for each entry where seedlings were dipped only in sterilized distilled water. To avoid contamination among strains, inoculated plants of each strain were allowed to grow in separate trays avoiding any water flow from one to another. The average maxi m u m and m i n i m u m temperatures of the glasshouse during the study were 40 and 24 ° C, respectively. Data collection. - Data on the degree of foliar wilting were recorded at 9 and 15 days and on vascular bundle browning at 20 days after inoculation following the scale m e n t i o n e d in Table 1. Readings were converted to disease indices according to Winstead and Kelman (1952) as follows: the n u m b e r of plants in each s y m p t o m category was multiplied by the corresponding numerical grade and the products added. The s u m m a t i o n was converted to disease index value by dividing by the total n u m b e r of plants and multiplying by 100. Rate of disease occurrence was calculated as the percentage of diseased plants recorded after 15 days of inoculation. RESULTS

The disease severity in respect of wilting and vascular bundle browning are presented in Figs. 1 and 2. In general, disease resistance was observed to increase with increasing seedling age, as demonstrated by the lower disease se~ verity index as recorded in the second experiment (four to five leaf stage ). N o n e of the commercial cultivars of eggplant tested were resistant to any

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Zig. 1. Severity of foliar wilting resulting from inoculation by five strains (8107, 8109, 8215, 8224 and 8503, followed by corresponding group mmber) ofP. solanacearum in different entries of Solanum. Cumulative disease index after 9 ( [ ] ) and 15 ( [ ] ) days of the inoculation at three o four leaf stage and after 9 ( [ ] ) and 15 ( [ ] ) days of the inoculation at four to five leaf stage.

S. inteQrifoli~ x 'Uttara'

~. gito x 'Uttara'

car..insanum x 'Uttara'

'Senryo 2 9ou'

'Senryo 2 gou' ~. inte~rifoliumx

'Senryo 2 gou'

car. jnsanum x

~. to×icarium

i. sisymbriifolium

i. ~tegr ifolium

i. ferox

vat. insanum

cv. Uttara

i. metongena cv. Senryo 2 g0u

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:ig. 2. Severity of vascular bundle browning following inoculation by 5 strains (8107, 8109, 8215, 8224 and 8503 followed by corresponding ;roup number) ofP. solanacearum in different entries ofSolanum. [~, three to four leaf stage; [], four to five leaf stage. Data recorded after 20 lays of the inoculation.

i. integrifolium x 'Uttara'

~. ~..gx 'Uttara'

tar. insanum x 'Uttara'

mr. insanum x 'Senryo 2 ~ou' ~. ~tto x 'Senryo 2 9ou' i. ,integrifolium x 'Senryo 2 9ou'

~. toxicarium

~. sisymbriifolium

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var. insanum

cv. Ottara

S. metongena cv. Senryo 2 9ou

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6

M.ALIETAL.

of the five strains. However, strain 8215 (Group II) seems to be the most damaging for both cultivars whereas there was moderate susceptibility against strain 8224 (Group IV). Among the wild relatives, S. toxicarium was completely resistant as not a single plant was affected by any of the strains. Solanum ferox was similar in this respect except that strain 8109 (Group V) resulted in high degrees of infection. Solanum sisymbriifolium showed greater susceptibility towards 8215 followed by 8109 and 8107 (Group I). With strain 8224 very few plants wilted but moderate vascular browning was recorded (Fig. 2), and in the case of 8503 (Group III) younger plants were susceptible, whereas older plants showed no symptoms at all. Solanum integrifolium was highly susceptible to 8109, 8224 and 8503; whereas with 8107 and 8215 little wilting or vascular browning was observed only on older plants. Solanum rnelongena var. insanum was one of the most susceptible among the wild relatives and S. gilo exhibited similar disease severity. Among the hybrids, F1 combinations ofS. integrifolium with 'Senryo 2 gou' and 'Uttara' showed moderate to low susceptibility against strains 8107, 8215 and 8224. For the other two strains, plants were moderately to completely susceptible. Against strains 8107 and 8215 the hybrid showed slight or no symptoms of infection with younger seedlings, while older seedlings developed few disease symptoms (Figs. 1 and 2). The F1 combinations of both cultivars with S. melongena var. insanum were completely susceptible, while with S. gilo showed moderate to high susceptibility against all strains except 8224. In all cases, disease advanced vigorously with time. In some cases, although a low level of symptoms was observed on the 9th day, the plants succumbed rapidly thereafter (see severity ratings for day 15 ). Regarding the rate of disease occurrence both cultivars of eggplant showed a similar response to all the strains (Table 2). With strains 8107, 8109, 8215 and 8503 they showed a high rate of disease occurrence, whereas with strain 8224 they were moderately susceptible. Solanumferox followed S. toxicarium with respect to infection rate and was resistant to all strains except 8109. Solanum integrifolium was little affected by strains 8107 and 8215 but was moderately to highly susceptible to other strains. Solanum sisymbriifolium had a low rate of infection with strain 8224, a moderate rate with 8503 but high susceptibility towards the other three strains. Solanum melongena var. insanum and S. gilo showed the greatest susceptibility to all of the strains. The F1 hybrids of both commercial cultivars of eggplant and S. integrifolium showed no disease symptoms after inoculation in the three to four leaf stage with 8107, mild symptoms with 8215, moderate with 8224 but severe symptoms with the other two strains. Hybrids having parentage from var. insanum and S. gilo showed higher infection rates. Concerning the pathogenicity of different strains, strain 8503 caused the

RESISTANCEOF EGGPLANTTOPSEUDOMONAS

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TABLE 2 Percentage of diseased plants after 15 days of inoculation by five bacterial strains in Solanum genotypes. Values in parentheses are the results of the second experiment (four to five leaf stage) Genotypes

S. melongena cv. 'Senryo 2 gou' cv. 'Uttara' var. insanum S.ferox S. gilo S. integrifolium S. sisymbriifolium S. toxicarium var. insanum×cv. 'Senryo 2 gou' S. gilo×cv.'Senryo2 gou' S. integrifoliumXcv. 'Senryo 2 gou var. insanurnxcv. 'Uttara' S. giloxcv.'Uttara' S. integrifoliumXcv. 'Uttara'

Strains of bacteria

Control

8107

8109

8215

8224

8503

95(100) 100(100) 100(100) 0 (0) 100(100) 0 (50) 100 (60) 0 (0) 100(100)

100(100) 90(100) 95(100) 85(100) 100 (60) 85(100) 100(100) 0 (0) 100(100)

100(100) 100(100) 75(100) 0 (0) 95(100) 0 (60) 100 (80) 0 (0) 100(100)

90 (80) 35 (20) 75(100) 0 (0) 100 (90) 85 (90) 10 (20) 0 (0) 80(100)

100(100) 100(100) 100(100) 10 (0) 100(100) 100(100) 50 (0) 0 (0) 100(100)

0 0 0 0 0 0 0 0 0

100 (70)

95 (90)

95 (80)

47 (60)

90(100

0

0 (30)

100 (70)

10 (40)

55 (40)

100(100)

0

100(100)

100(100)

100(100)

95(100)

100(J00)

0

100(100) 0 (20)

100 (90) 100(100)

100(100) 20 (50)

100(100) 38 (40)

100(100) 100(100)

0 0

greatest disease severity and infection rate. Strain 8224, although quite damaging, seems to be relatively weak compared with the other 4 strains. DISCUSSION

In the present study the symptoms of bacterial wilt were well expressed as the temperature of the greenhouse was maintained above 21 oC, which is a prerequisite condition for symptom appearance (Gallegly and Walker, 1949 ). Variable resistance or susceptibility observed in eggplant cultivars, wild relatives and their hybrids against different strains has also been reported by several other workers (Winstead and Kelman, 1960; Kelman and Person, 1961; Gleddie et al., 1986). This difference was attributed to differences in genotypes of the hosts as well as the pathogenic strain concerned. Plants after inoculation passed through an incubation period, an the early symptoms were expressed after 6 days. At 9 days the plants (Fig. 1 ) showed clearcut symptoms with many host genotypes. Vascular bundle browning (Fig. 2 ) recorded at the 20th day showed the extent of internal damage due to the death of vascular tissues. Extensive vascular browning at later stages of symptom development was also reported by Winstead and Kelman (1960).

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M, ALl ET AL.

The highest resistance level of S. toxicarium and moderate resistance of S. integrifolium and S. sisymbriifolium observed in the present study confirm previous reports (Mochizuki and Yamakawa, 1979a; Ozaki, 1985; Ozaki and Kimura, 1989). However, this study revealed the complete resistance of S. toxicarium against all available well-defined virulent strains. The resistance of S. ferox to most strains and the relatively high resistance of eggplant hybrids with S. integrifolium are noteworthy as this information has not been reported before. The resistance of S. integrifolium was also expressed in the F1 hybrids. Partial dominance of genes for bacterial wilt resistance has been shown by Acosta et al. (1964) in tomato. The lower symptom appearance observed in the second experiment may be related to the greater age of seedlings at inoculation. Older plants developed resistance against wilting organisms as observed by Mew and Ho (1976) and Winstead and Kelman ( 1952 ) in tomato - a host similar to eggplant. Relative susceptibility of the two commercial cultivars of eggplants showed that they could probably be improved by a conventional breeding procedure, or through use of tolerant rootstocks. Although the wild species such as S. toxicarium and S. ferox were found to be resistant, the transfer of genes conferring resistance to eggplants from these species may not be possible because of sexual incompatibility a n d / o r hybrid sterility. Direct use of these species as rootstock also faces commercial difficulty because of the slow growth rate of their seedlings. Moreover, the vigor, fruit yield and fruit quality of the scion were poorer than those on the commercial rootstock (Mochizuki and Yamakawa, 1979b; M. Ali et al., unpublished data, 1989 ). Protoplast fusion would be a proper tool for the utilization of the available resistance from the wild species. The possible somatic hybrid o u t o f protoplast fusion might offer a better stock plant with better grafting quality. Reports of successful fusion of Solanum species (Gleddie et al., 1986; Sihachakr, 1989) may show the way for this type of work. The present investigation has provided information for exploitation of bacterial wilt resistance with a view to developing resistant or highly tolerant stock or cultivated type of eggplant. ACKNOWLEDGEMENT We are grateful to K. Ozaki for providing the bacterial strains used in this study and we also wish to thank N. Furuya for his technical assistance during inoculum preparation. REFERENCES Acosta, J.C., Gilbert, J.C. and Quinon, V.L., 1964. Heritability of bacterial wilt resistance in tomato. Proc. Am. Soc. Hortic. Sci., 84: 455-462.

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Gallegly, M.E., Jr. and Walker, J.C., 1949. Relation of environmental factors to bacterial wilt of tomato. Phytopathology, 39: 936-946. Gleddie, S., Keller, W.A. and Setteriield, G., 1986. Production and characterization of somatic hybrids between Solanum melongena L. and Solanum sisymbriifolium Lam. Theor. Appl. Genet., 71: 613-621. Goth, R.W., Madalageri, B.B., Barksdale, T.H. and Webb, R.E., 1986. Screening world eggplant germplasm collection for resistance to Pseudomonas solanacaerum (abstract). Phytopathology, 76: 563. Kelman, A., 1954. The relationship of pathogenicity in Pseudomonas solanacearum to colony appearance on a tetrazolium medium. Phytopathology, 44: 693-695. Kelman, A. and Person, L.H., 1961. Strains of Pseudomonas solanacearum differing in pathogenicity to tobacco and peanut. Phytopathology, 51: 158-161. Mew, T.W. and Ho, W.C., 1976. Varietal resistance to bacterial wilt in tomato. Plant Dis. Rep., 60: 264-268. Mew, T.W. and Ho, W.C., 1977. Effect of soil temperature on resistance of tomato cultivars to bacterial wilt. Phytopathology, 67:909-911. Moehizuki, H. and Yamakawa, K., 1979a. Resistance of selected eggplant cultivars and related wild Solanum species to bacterial wilt (Pseudomonas solanacearum). Bull. Veg. Ornam. Crops Res. Stn. Ser. A, No. 6:1-10 (in Japanese, with English summary). Mochizuki, H. and Yamakawa, K., 1979b. Potential utilization of bacterial wiltresistant Solanum species as rootstock for commercial eggplant production. Bull. Veg. Ornam. Crops Res. Stn. Ser. A, No. 6:11-18 (in Japanese, with English summary). Ozaki, K., 1985. Pathogenic specialization ofPseudomonas solanacearum in solanaceous plants. Abstract of the 13th Meeting of Plant Bacterial Disease. Phytopath. Soc. Jpn., pp. 1-6 (in Japanese). Ozaki, K. and Kimura, T., 1989. Method for evaluating the resistance of Solanum plants to bacterial wilt caused by Pseudomonas solanacearum. Bull. Chugoku Natl. Agric Exp. Stn. No. 4:103-117 (in Japanese, with English summary). Sihachakr, D., Haicour, R., Chaput, M.H., Barrientos, E., Ducreux, G. and Rossignol, L., 1989. Somatic hybrid plants produced by electrofusion between Solanum melongena L. and Solanum torvum Sw. Theor. Appl. Genet., 77: 1-6. Wakimoto, S. and Yoshii, H., 1955. Quantitative determination of the population of bacteria by the phage technique. Sci. Bull. Fac. Agric. Kyushu Univ., 15: 161-169 (in Japanese, with English summary), Winstead, N.N. and Kelman, A., 1952. Inoculation techniques for evaluating resistance to Pseudomonas solanacearum. Phytopathology, 42: 628-634. Winstead, N.N. and Kelman, A., 1960. Resistance to bacterial wilt in eggplant in North Carolina. Plant Dis. Rep., 44: 432-434.