Selection of mutants resistant to Alternaria blotch from in vitro -cultured apple shoots irradiated with X- and γ-rays

J. Plant Physiol. 158. 391 – 400 (2001)  Urban & Fischer Verlag http://www.urbanfischer.de/journals/jpp

Selection of mutants resistant to Alternaria blotch from in vitrocultured apple shoots irradiated with X- and γ-rays Akira Saito, Norio Nakazawa, Masahiko Suzuki* Aomori Green BioCenter, 221-10, Yamaguchi, Nogi, Aomori, 030-0142, Japan

Received August 17, 2000 · Accepted October 15, 2000

Summary We produced mutants resistant to Alternaria blotch disease in several cultivars of apple (Malus × domestica Borkh.) by irradiation with X- or γ-rays. An efficient in vitro assay method was established using chemically-synthesized AM-toxin I of Alternaria alternata (Fr.) Keissler to screen for mutants resistant to Alternaria blotch disease. The frequency of necrotic lesions was investigated by applying various concentrations of AM-toxin I to leaf discs of the first, third, and fifth leaves from the shoot apex of several apple cultivars, including Jonathan, Fuji, Oorin, and Indo. In vitro-grown apple shoots of susceptible cultivars were then treated with various doses of X- or γ-ray irradiation. Several mutants resistant to AM-toxin I were obtained by combining the techniques for tissue culture of apple shoots with the AM-toxin I screening method. Following a repeat second screening test with AMtoxin I, mutant plants were sprayed with a spore suspension of A. alternata and found resistant to be the fungal pathogen. These mutants showed normal phenotypic appearance, and so far, no difference has been observed between the original plants and mutants except for the susceptibility to Alternaria blotch. Key words: Alternaria blotch – apple – mutant selection – γ-ray irradiation – X-ray irradiation Abbreviations: BA 6-benzyladenine. – IBA indole-butyric acid. – MS Murashige and Skoog medium

Introduction Alternaria blotch disease of apple (Malus × domestica Borkh.), caused by the apple pathotype of Alternaria alternata (Fr.) Keissler, is one of the most serious fungal diseases affecting apples in Japan. Leaves infected by this fungus show necrotic lesions and leaf-drop, following progression of the disease, that result in a decrease in fruit yield. The disease can usually * E-mail corresponding author: [email protected]

be controlled by spraying trees with several fungicide applications during the growing season. Control is difficult, however, if primary leaf infections are already abundant before the first spray application. In addition, chemical sprays are costly, laborious, and cause health hazards to farmers. Disease susceptibility is controlled by a single dominant gene (Saito and Takeda 1984), and most susceptible cultivars are heterozygous. Cultivars containing homozygous recessive genes show resistance to this disease. Therefore, irradiation with X- or γ-rays is an efficient tool for production of 0176-1617/01/158/03-391 $ 15.00/0

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Akira Saito, Norio Nakazawa, Masahiko Suzuki

resistant mutants, as irradiation usually induces recessive mutants. Irradiation has already been used to develop disease-resistant cultivars in both pears and apples. The Japanese pear cultivars Nijisseiki and Osanijisseiki were selected for resistance to black spot disease caused by the Japanese pear pathotype of Alternaria alternata (Fr.) Keissler, following chronic irradiation of somatic tissues with γ-rays (Sanada et al. 1988, Masuda et al. 1997). The homozygous recessive genes in these cultivars also result in resistance to Alternaria blotch disease as well. Attempts to select for mutants resistant to Alternaria blotch in the susceptible apple cultivar Indo have produced only moderately resistant mutants (Tabira et al. 1993 a, Sanada et al. 1994, Masuda and Yoshioka 1996). The pathogen produces host-specific toxins at the time of spore germination, which causes damage to the plant cytoplast and to chloroplasts (Park et al. 1981). Ueno et al. (1975) purified these toxins and classified them into 3 groups, namely AM-toxin I, II, and III. AM-toxin I was demonstrated to be the same as the compound Alternariolide, corresponding to C23 H31 N3 O6, which was previously reported by Okuno et al. (1974). Hashimoto et al. (1996) reported an efficient synthesis of AM-toxin I, and the synthetic AM-toxin I was identical to a natural sample from both spectral and biological aspects. In the present study, we developed an efficient in vitro-assay system to select for resistantce to Alternaria blotch using a chemically-synthesized toxin, and then produced apples resistant to Alternaria blotch disease through a highly efficient shoot regeneration procedure for apple shoots (Saito and Suzuki 1999) following X- and γ-ray irradiation of several susceptible cultivars.

Materials and Methods Method for assaying for susceptibility to pathogenic toxins using leaf discs Four apple cultivars with known field resistance or susceptibility to Alternaria blotch disease, including Jonathan (resistant), Fuji (intermediately resistant), Oorin, and Indo (both susceptible) were used to evaluate the reaction to inoculation with a chemically-synthesized AM-toxin I. The upper 10 leaves of the first-, third-, and fifth-leaves from the shoot apex of 10 plants were collected from each of the cultivars grown in the greenhouse. Leaf discs (8 mm diameter) were cut out with a corkborer from the central section of the leaf. The chemically-synthesized AM-toxin I was dissolved in 1 mL of DMSO (dimethylsulfoxide), and suspended with sterile water to a final concentration of 100 µmol/L. This solution was diluted to 10, 1, and 0.1 µmol/L sequentially, and sterilized by filtration through a 0.45 µm membrane filter. Each 0.5 mL of the diluted toxin was placed on a 24-titer plate, and leaf discs were placed in each well and immersed for 48 h in the dark at 28 ˚C. Thereafter, the degree of necrotic lesions was evaluated. Lesions were graded by the degree of necrosis, and classified into 4 groups as follows: grade 0, no necrotic lesion; grade 1, necrotic lesions on the periphery of the leaf disc; grade 2, necrotic lesions on less than half of a leaf disc; grade 3, necrotic lesions on half to 3⁄4 of a leaf disc; and grade 4, necrotic lesions on 3⁄4 to whole leaf disc.

Irradiation of shoots with various doses Apical shoots of in vitro-cultured of the susceptible cultivars Hokuto and Aori 10 were cut into 5 mm pieces, and 10 cut-up shoots of each were incubated on a shoot proliferation medium consisting of MS (Murashige and Skoog 1962) medium (pH 5.8) containing 1.0 mg/L BA, 30 g/L sucrose, and 0.8 % Difco Bact-agar. To evaluate the effect of X-ray irradiation on survival of in vitro-cultured shoots, Petri dishes containing these 10 shoots were irradiated with various doses of X-ray in soft X-ray irradiation equipment (Ohmic Ltd. Soft X-ray systems, OM-100 RAL). As for γ-ray irradiation, irradiation with doses of 60, 80, or 120 Gy at a dose rate of 5 Gy h –1 was carried out in the γ-room (44.4 TBq 60Co source) of the Institute of Radiation Breeding, NIAR, MAFF. After irradiation, 10 Petri dishes containing shoots were cultured at 25 ˚C with a 16 h photoperiod. The numbers of surviving shoots and morphological properties were investigated at 30 days after irradiation.

The first and secondary screening test of susceptibility in irradiated shoots To produce resistant mutants from susceptible cultivars, including Hokuto, Oorin, Fuji, and Aori 10, X-ray irradiation was carried out with various dose rates and radiation doses. In standard experiments, 10 Petri dishes each containing 10 cut-up shoots were used for irradiation. After 30-days culture, shoots of surviving plants elongated, and each of 10 segments of the elongated shoots was cultured into a shoot proliferation medium. Shoots were subcultured three times, cut off, and transferred to a rooting medium composed of MS medium (pH 5.8) containing 1.0 mg/L IBA, 30 g/L sucrose, and 0.8 % agar. Rooted plantlets were acclimatized and transferred to pots. Thereafter, these plants (ca. 15 cm) were screened with toxin to evaluate the degree of susceptibility. Six mm diameter discs were cut from the third-leaf of each plant with a corkborer. Two discs from each leaf were immersed with the toxin solution and incubated at 28 ˚C in darkness for two days to see development of necrotic lesions. We selected for resistant mutants, and they were evaluated grade 0 after immersion of 10 µmol/L toxin in this screening test. Resistant mutants selected by the first screening test were grown in a greenhouse, and the third- leaves from the tips of shoots were collected. Leaf discs (8 mm diameter) were cut out with a corkborer from the central region of those leaves. To select resistant mutants, the leaf discs were immersed in the toxin solution at high concentration (100 µmol/L) used for the titer plates, and incubated for 2 days at 28 ˚C in darkness. We selected for resistant mutants, and they were evaluated grade 0 to 1 after immersion of 100 µmol/L toxin in this screening test.

Inoculation of a spore suspension in selected resistant mutants by secondary screening test After selection by the secondary screening test, a suspension containing spores of the pathogen (3.3 × 104 mL –1) was sprayed onto the leaves of selected mutant plants of Hokuto, Fuji, Oorin, and several other cultivars of different susceptibility to the pathogen. Leaves inoculated with the pathogen were kept for 2 days at 25 ˚C in the inoculation box (relative humidity: 90 to 95 %). Thereafter, plants were transferred to a greenhouse, and disease symptoms and number of necrotic le-

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sions were determined for each of the upper 10 leaves of the shoot tips 7 days after inoculation. Among them, severity of symptoms was calculated following the conversion formula: Severity of symptoms = Σ(G × n) × 100/(N × 6). Where n is number of leaf corresponding to Grade (G) and N is all leaves used in test (10 leaves per 1 plant). Grade of necrosis was classified into 7 groups as follows: grade 0, no necrosis spots per leaf; grade 1, 1 to 5 necrosis spots per leaf; grade 2, 6 to 10 necrosis spots per leaf; grade 3, 11 to 30 necrosis spots per leaf; grade 4, 31 to 50 necrosis spots per leaf; grade 5, over 51 necrosis spots per leaf; grade 6, leaf fall.

Results Establishment of an assay method for evaluating the degree of susceptibility In the beginning, we investigated the concentration of toxin and position of leaves for screening. At a concentration of 100 µmol/L toxin, leaf discs of all 3 cultivars tested here, except for Jonathan, showed necrotic lesions with grades ranging from 3 to 4. For Jonathan, they were from 2.1 to 4.0 (Table 1). Thus, except for Jonathan, it was difficult to classify the other 3 cultivars based on the degree of susceptibility at this concentration. At toxin concentrations from 0.1 to 1 µmol/L, however, the grade of necrotic lesions of leaf discs properly corresponded to the field susceptibility previously known. Namely, the grades of the resistant cultivar (Jonathan) were from 0 to 0.5, the intermediate cultivar (Fuji) was from 0.5 to 3.5, and the susceptible cultivars (Oorin and Indo) were from 2.0 to 4. Thus, the degree of susceptibility to Alternaria blotch was successfully evaluated by measuring necrotic lesions of leaf discs exposed to chemically synthesized toxin. The position of leaves used for the screening test was another important factor, as was the concentration of toxin. The first leaves from the tip of the shoot were very susceptible to toxin; however, it was difficult to distinguish the degree of susceptibility among cultivars at 0.1 µmol/L of toxin. First leaves of all cultivars tested, except Jonathan, showed grades from 3.3 to 4.0 at this concentration. The third and fifth leaves from the tip of shoots were appropriate materials for the evaluation of the degree of susceptibility. In this study, the third leaves were used for the evaluation with toxin at the concentrations of 1 and 10 µmol/L.

Survival of irradiated shoots with various dose rates of irradiation Percentage of survival was investigated in cvs. Hokuto and Aori 10 for various dose rates of irradiation (Fig. 1). For a dose rate of 0.01 KR min –1, no plant died following radiation doses of up to 12 KR. However, the survival rate decreased gradually as the dose rate increased above 0.1 KR min –1. In cv. Hokuto, almost no plant died at a dose rate of 0.01 KR min –1 or at doses of up to 12 KR, whereas the survival rate

Figure 1. Survival rate of X- and γ-ray irradiated shoots of cvs. Hokuto and Aori 10. (A) In vitro cultured shoots of cv. Hokuto were irradiated with increasing doses of X-ray irradiation. (B) In vitro cultured shoots of cv. Aori 10 were irradiated with increasing doses of X-ray irradiation. (C) In vitro cultured shoots of cvs. Hokuto and Aori 10 were irradiated with increasing doses of γ-ray irradiation.

decreased to 50 % even at the radiation dose of 13 KR with the dose rate of 0.1 KR min –1 (Fig. 1 A). The extent of irradiation damage varied between the two cultivars. The survival rate was always higher in cv. Hokuto than in cv. Aori 10 for the dose range tested here, both with X-ray (Figs. 1 A, 1 B) and γ-ray irradiation (Fig. 1 C). At the dose rate of 0.01 KR

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Table 1. Effect of AM-toxin I on the necrotic lesions of leaf discs among cultivars with various degrees of susceptibility to Alternaria blotch. *The grade of necrotic lesions of leaf discs was classified into 4 groups as follows; 0, no necrotic lesion; 1, a few necrotic lesions at the cut end of leaf discs; 2, necrotic lesions on less than half of a leaf disc; 3, necrotic lesions on half to 3⁄4 of leaf dics; 4, necrotic lesions on 3⁄4 to whole leaf disc. Each grade was a mean of 10 leaves. Values are means ± SE (n = 10 leaves). Means followed by the same letter are not significantly different (P < 0.05). Cultivar

Degree of

Position of leaves

Grade of necrotic lesions*

susceptibility

from top of shoots

Concentration of toxin 100 µmol/L

10 µmol/L

1 µmol/L

0.1 µmol/L

Indo

highly susceptible

1st 3rd 5th

4.0 ± 0.00 a 4.0 ± 0.00 a 4.0 ± 0.00 a

4.0 ± 0.00 a 4.0 ± 0.00 a 4.0 ± 0.00 a

4.0 ± 0.00 a 4.0 ± 0.00 a 4.0 ± 0.00 a

4.0 ± 0.00 a 3.4 ± 0.48 b 3.2 ± 0.32 b

Oorin

susceptible

1st 3rd 5th

4.0 ± 0.00 a 4.0 ± 0.00 a 3.8 ± 0.32 a

4.0 ± 0.00 a 4.0 ± 0.00 a 3.8 ± 0.32 a

4.0 ± 0.00 a 3.6 ± 0.48 a 3.2 ± 0.48 b

3.6 ± 0.48 a 2.7 ± 0.42 d 2.0 ± 0.20 e

Fuji

intermediate

1st 3rd 5th

4.0 ± 0.00 a 4.0 ± 0.00 a 3.2 ± 0.32 b

4.0 ± 0.00 a 3.4 ± 0.48 b 2.1 ± 0.18 e

3.5 ± 0.50 b 2.6 ± 0.48 d 1.1 ± 0.36 g

3.3 ± 0.42 b 1.2 ± 0.32 g 0.5 ± 0.50 i

Jonathan

resistant

1st 3rd 5th

4.0 ± 0.00 a 2.8 ± 0.48 c 2.1 ± 0.36 e

3.2 ± 0.64 b 0.4 ± 0.48 i 0.2 ± 0.32 i

0.5 ± 0.50 i 0.0 ± 0.00 j 0.0 ± 0.00 j

0.0 ± 0.00 j 0.0 ± 0.00 j 0.0 ± 0.00 j

min –1 for cv. Aori 10, no plant died; however, only 50 % survived the radiation dose of 6 KR with a dose rate of 0.1 KR min –1 (Fig. 1 B). Moreover, under the latter conditions, many of the surviving plants showed rosette type of shoots or abnormal leaf morphology (Figs. 2 D, 2 F). Survival rate increased when chronic radiation was given at low dose rates compared with those at high dose rates for the same radiation dose (data not shown). Survival rate also decreased with the increase of total dose.

Efficient radiation dose and dose rate for production of mutants As the degree of susceptibility could be evaluated by screening leaf-discs against the toxin, the production of resistant mutants from susceptible cultivars was attempted by X-ray irradiation. After leaf-disc screening, selected resistant shoots were rooted and acclimatized. In cv. Hokuto, non-irradiated plants showed severe necrotic lesions (more than grade 3) at toxin concentrations of 1 (data not shown) and 10 µmol/L (Table 2). When radiation dose was lower than 4 KR, almost all irradiated plants showed the same necrotic lesions as those of non-irradiated plants at 0.01 KR min –1 (data not shown) and 0.1 KR min –1 (Table 2). Therefore, 4 KR irradiation was insufficient for production of resistant mutants. At a radiation dose of 6 KR, necrotic lesions decreased among some irradiated plants and some showed no necrotic lesions, as in the case of the resistant cultivar Jonathan, which was graded as 0. Irradiation with a 6 KR dose was sufficient for mutant produc-

tion. Thus, the first screening of resistant mutants was carried out in plants irradiated with radiation doses higher ranging from 6 KR to 12 KR. After the first screening, those plants whose leaf discs remained green in the toxin screening solution (Fig. 3 A), were grown in a greenhouse and used for the second screening test. Leaf discs were prepared from the third leaves from the top of the shoots of juvenile plants selected by the first screening. These leaf discs were selected by immersion in a solution containing 100 µmol/L toxin, and yielded highly resistant mutants showing grade 0 or 1 in plants irradiated with 8 KR and 10 KR. These mutants were more resistant than the resistant cultivar, Jonathan. Besides the susceptible cultivar Hokuto, 3 other susceptible cvs. Fuji, Oorin, and Aori 10, were also treated, and resistant mutants were selected from among them (Table 3). In the first screening of cv. Fuji, more resistant mutants were obtained in plants irradiated with a 0.1 KR min –1 dose rate than with a 0.01 KR min –1 dose rate. The frequency of occurrence of resistant mutants did not differ greatly among irradiated plants at radiation doses between 8 and 10 KR. For the cv. Oorin, resistant mutants were obtained in plants irradiated with 8 and 10 KR, whereas only a few resistant plants were obtained with the radiation dose of 6 KR. No resistant mutants were obtained for cv. Aori 10 at any dose of irradiation. Resistant mutants of cvs. Fuji and Oorin identified in the first screening were grown in a greenhouse, and the second screening was carried out. After the second screening, resistant mutants showing the highly resistant grade 0 and grade 1 appeared among the cv. Fuji plants irradiated with 6, 8, and 10 KR at the dose rate of 0.1 KR min –1, whereas no re-

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395

Figure 2. Appearance of in vitro cultured shoots after γ-ray irradiation on cvs. Hokuto and Aori 10. (A) In vitro cultured shoots of cv. Oorin (after cultured 30 days on shoot proliferation medium). Scale bar represents 2 cm. (B) Non-irradiated in vitro cultured shoots of cv. Hokuto. Scale bar represents 2 cm. (C) Appearance of in vitro cultured shoots 1 month after irradiation on cv. Hokuto (radiation doses of 60, 80, and 120 Gy). Shoots appeared as normal as non-irradiated one. Scale bar represents 3 cm. (D) Appearance of in vitro cultured shoots 1 month after irradiation on cv. Aori 10 (radiation doses of 60, 80, and 120 Gy). Callus formation from shoots appeared in parts of the irradiated shoots. Scale bar represents 3 cm. (E) Magnification of a part of (C). Normal phenotypes were shown in 120 Gy-irradiated cv. Hokuto. Scale bar represents 2 cm. (F) Magnification of a part of (D). Rosette-type shoots and abnormal leaf morphology appeared 1 month after γ-ray irradiation on cv. Aori 10 (radiation doses of 120 Gy). Scale bar represents 2 cm.

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Table 2. Production of resistant mutants in cv. Hokuto by X-ray irradiation and selection by toxin of Alternaria alternata. a Evaluation of susceptibility marked with grades from 0 to 4 was the same as described in Table 1. b Plants evaluated grade 0 after immersion of 10 µmol/L toxin in the first leaf-disc screening test. c Plants evaluated grade 0 or 1 after immersion of 100 µmol/L toxin in the second leaf-disc screening test. Cultivar

Hokuto

Dose rate

Radiation Toxin Number of Number of each group corresponding to the dose concentration plants for degree of susceptibility by leaf disc testa

Selected plantsb Selected plantsc of the first of the second

(KR min – 1)

(KR)

(µmol/L)

screening

0

1

2

3

4

screening test

screening test

Nonirradiated 0.01

-

10

49

0

0

0

6

43

-

-

6 8 12

10 10 10

263 223 232

1 3 5

33 25 48

86 58 51

63 52 63

80 85 65

1 3 5

0 0 0

1 2 4 6 8 10 -

10 10 10 10 10 10 10

92 88 82 276 312 303 10

0 0 0 4 5 8 8

0 0 1 55 30 38 2

0 8 17 54 87 116 0

3 21 29 79 103 78 0

89 59 35 84 87 63 0

0 0 0 4 5 8 -

0 1 4 -

0.1

Jonathan Non(resistant) irradiated

Table 3. Production of resistant mutants in cvs. Fuji, Oorin and Aori 10 by X-ray irradiation and selection by toxin of Alternaria alternata. a Evaluation of susceptibility marked with grades from 0 to 4 was the same as described in Table 1. b Plants evaluated grade 0 after immersion of 10 µmol/L toxin in the first leaf-disc sceening test. c Plants evaluated grade 0 or 1 after immersion of 100 µmol/L toxin in the second leaf-disc screening test. Cultivar

Fuji

Aori 10

screening test

sreening test

25 25 31 11 52 97 115

2 1 2 22 53 44

0 0 0 6 12 9

1 52 10 11

19 18 14 6

12 27 21

10 18 11

0 0 0 0 0 0

0 0 0 0 1 0

40 123 125 121 109 98

0 0 0 0 0

-

0

0

0

-

-

Number of plants for

(KR min – 1)

(KR)

screening

0

1

2

3

4

6 8 10 6 8 10

38 99 111 102 280 527 533

0 2 1 2 22 53 44

0 11 32 33 67 164 125

0 33 29 29 73 132 146

13 28 18 27 66 81 103

6 8 10

20 189 185 156

0 12 27 21

0 41 70 61

0 66 64 57

6 12 6 8 10

40 123 125 121 110 98

0 0 0 0 0 0

0 0 0 0 0 0

10

8

2

Non-irradiated 0.01

Non-irradiated 0.1

Non-irradiated 0.01 0.1

Jonathan

Selected plantsc of the second

Radiation dose

0.1

Oorin

Selected plantsb of the first

Dose rate

Non-irradiated

Number of each group corresponding to the degree of susceptibility by leaf disc testa

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397

Figure 3. Screening test by AM-toxin I and mutant resistant to Alternaria blotch selected by AM-toxin I. (A) Screening test for selecting cv. Fuji mutants resistant to Alternaria blotch by AM-toxin I. Leaf-discs of resistant mutants remained green, whereas necrotic lesions appeared in susceptible ones. (B) A regenerated plant of a mutant resistant to Alternaria blotch (right) and the original plant of cv. Fuji (left), which showed necrotic lesions 10 days after inoculation with a spore suspension of the apple pathotype of Alternaria alternata (Fr.) Keissler.

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Table 4. Production of resistant mutants in cvs. Hokuto, Fuji and Aori 10 by γ-ray irradiation and selection by toxin of Alternaria alternata. a Evaluation of susceptibility marked with grades from 0 to 4 was the same as described in Table 1. b Plants evaluated grade 0 after immersion of 10 µmol/L toxin in the first leaf-disc sceening test. c Plants evaluated grade 0 or 1 after immersion of 100 µmol/L toxin in the second leaf-disc screening test. Cultivar

Selected plantsb of the first

Selected plantsc of the second

screening test

sreening test

43 109 71 110

3 5 4

0 0 0

13 38 81 71

25 25 86 45

5 35 46

1 12 20

0 0 0 0

0 0 1 0

40 96 86 45

-

-

0

0

0

-

-

Dose rate

Radiation dose

Number of plants for

Number of each group corresponding to the degree of susceptibility by leaf disc testa

(Gy hr – 1)

(Gy)

screening

0

1

2

3

4

Hokuto

Non-irradiated 5

60 80 120

49 181 168 203

0 3 5 4

0 14 19 14

0 26 38 35

6 29 35 40

Fuji

Non-irradiated 5

60 80 120

38 116 410 491

0 5 35 46

0 15 115 164

0 33 93 165

Aori 10

Non-irradiated 5

60 80 120

40 96 87 45

0 0 0 0

0 0 0 0

Jonathan (resistant)

Non-irradiated

-

10

8

2

sistant mutants were obtained at 0.01 KR min –1. For cv. Oorin, the same tendency was observed as for cv. Fuji, although a higher number of resistant mutants was obtained than for cv. Fuji. As for the γ-ray irradiated plants (Table 4), more resistant mutants were selected in the first screening with 80 and 120 Gy than with 60 Gy. Among resistant mutants selected in the second screening, frequency of disease symptoms was tested by inoculation with a spore suspension of the pathogenic fungus (Table 5). Many of these mutants were more resistant than the original plants (Fig. 3 B), and showed a degree of resistance similar to that of the resistant cultivar Jonagold or Jonathan and the highly resistant cultivar Sansa. All had evaluation grades of 0. Altogether, we have obtained 4 resistant mutants in cv. Hokuto by X-ray irradiation (dose rate 0.1 KR min –1, radiation dose 10 KR), 13 resistant mutants by X-ray irradiation and 20 resistant mutants by acute γ-ray irradiation in cv. Fuji, and, in cv. Oorin, 12 resistant mutants by acute X-ray irradiation. The resistant mutants are being cultivated in a greenhouse and thus far have shown no morphological abnormality.

Discussion Saito et al. (1989) suggested that screening of the resistant mutants by the toxin might be an alternative to screening by the pathogen culture filtrate. They compared the results of screening obtained by crude AM-toxin with that by culture filtrate and showed they were almost consistent. Previously, fungal-culture filtrates or partially purified crude toxin from

culture filtrates was used for evaluating plant susceptibility to Alternaria blotch. However, when using culture filtrate or crude toxin, intermediate types of resistance were not completely distinguished (Tabira et al. 1993 a). Moreover, the toxin or culture filtrate titers varied with each culture. Therefore, to eliminate variability, we used chemically synthesized toxin (AM-toxin I) in this study. The age of leaves used for the screening test as well as the concentration of toxin, was another important factor. Generally, young leaves were more susceptible to toxin or fungal culture filtrate than were older leaves (Saito and Takeda 1984, Sawamura and Yanase 1963). In this study, the third leaves from the tip of the shoot were appropriate materials for evaluation for reaction to toxin treatments. Tabira et al. (1993 a) and Masuda (1995) observed that susceptibility to pathogenic toxin was correlated with the field susceptibility of both apple and pear cultivars. However, they observed no correlation for those cultivars with susceptibility to intermediate level of resistance when testing was done with in vitro-cultured shoots. In vitro-cultured shoots could always be prepared throughout the year and were easy to handle compared with shoots grown outside. Therefore, it seemed important for the selection of resistant mutants to establish an assay method for determining the degree of susceptibility by using in vitro-cultured shoots. We previously observed that in vitro-cultured shoots were not as responsive to toxin as leaves grown outdoors. They required higher concentrations of toxin (10 µmol/L) to evaluate the degree of susceptibility than did the leaves grown outdoors (1 µmol/L). Generally, the frequency of mutation generation by acute irradiation with X- or γ-rays increases exponentially compared

Selection of mutants resistant to Alternaria blotch in apples

399

Table 5. Appearance of symptoms after inoculation of a spore suspension to control plants and selected resistant mutant plants by toxin. a Rate of symptoms were appeared within group after the inoculation of spore suspension. b Severity of symptoms were appeared within group after the inoculation of spore suspension. Severity of symptoms calculated as described in Materials and Methods. c In cvs. Hokuto and Oorin we selected resistant mutants under 40 % rate of infected leaf symptoms and severity of symptoms was under 15. In cv. Fuji, we selected resistant mutants under 40 % rate of infected leaf symptoms and severity of symptoms was under 10. Values are means ± SE (n = 1–15 plants). Means followed by the same letter are not significantly different (P < 0.05). Cultivar

Susceptibility of Source of Alternalia blotch disease radiation

Hokuto

Susceptible control Selected mutant

Non-irradiated X-rays

Oorin

Susceptible control Selected mutant

Fuji

Susceptible control Selected mutant

No. of used plants

Rate of leaf (96)a Severity of b No. of selectedc appeared symptoms symptoms resistant mutants

10 KR

5 4

84.0 ± 4.8 a 20.0 ± 5.0 e

45.5 ± 5.3 a 6.7 ± 4.2 g

4

Non-irradiated X-rays 0.1 KR min – 1

6 KR 8 KR 10 KR

2 5 9 4

75.0 ± 5.0 a 56.0 ± 12.8 c 37.8 ± 30.9 d 20.0 ± 25.0 e

49.2 ± 0.9 a 15.3 ± 7.7 f 10.7 ± 10.1 g 3.4 ± 4.2 h

3 6 3

Non-irradiated X-rays 0.1 KR min – 1

6 KR 8 KR 10 KR 60 Gy 80 Gy 120 Gy -

5 2 8 4 1 8 15 4 3 3

54.0 ± 4.8 c 40.0 ± 0.0 d 25.0 ± 22.5 e 5.0 ± 5.0 f 10.0 ± 0.0 f 16.3 ± 17.8 f 20.0 ± 12.9 e 15.0 ± 10.0 f 3.3 ± 4.4 f 0.0 ± 0.0 g

19.3 ± 6.7 ± 4.8 ± 0.9 ± 1.7 ± 4.2 ± 4.1 ± 3,3 ± 0.6 ± 0.0 ±

γ-rays

Jonagold Resistant controll Jonathan Sansa

Dose rate

Non-irradiated

-

5 Gy h – 1

-

to that by chronic irradiation (Sparrow et al. 1961). As for dose rate, irradiation at high dose rates tended to cause chromosomal rearrangements and resulted in the occurrence of inferior phenotypes. In the breeding of Japanese pears, mutants resistant to black spot disease were obtained for cultivars Nijisseiki (Sanada 1986, Kotobuki et al. 1992) and Osanijisseiki (Masuda et al. 1997) by chronic irradiation (6.25 and 13.9 mGy h –1), and for cvs. Shinsui and Osanijisseiki (Murata et al. 1994) by acute irradiation (dose rate 2.5 Gy h –1, total dose 80 Gy). One resistant mutant was obtained from among 2,168 dormant shoots of Shinsui and 1 mutant from 2,335 dormant shoots of cv. Osanijisseiki by acute irradiation. Tabira et al. (1993 b) also produced one resistant mutant from 671 in vitro-cultured shoots of cv. Osanijisseiki by acute irradiation (dose rate 5 Gy h –1, total dose 80 Gy). The frequency of resistant mutants resulting from acute irradiation is estimated at 10 – 3, which is almost the same as that for chronic irradiation (Sanada et al. 1988). However, all mutants were intermediate-resistant types. As one of the reasons for the difficulty of producing highly resistant mutants, only the L-2 layer of the apical dome of shoots was genetically altered by irradiation, whereas L-1 remained susceptible; the L-1 and L-2 cells formed a chimera. These chimeras were passed on genetically to progenies produced by crossing. To avoid these chimeras and produce stable mutants, the procedure called ‹cutting back› has been used in orchard trees; however, this process requires a long period of time. Thus, plant tissue culture techniques were utilized to avoid

3.5 e 0.0 g 4.4 g 0.9 h 0.0 h 4.8 h 3.1 h 1.7 h 0.8 h 0.0 h

2 7 4 1 5 14 -

the formation of chimeras in combination with X- or γ-ray irradiation (Daub 1986, Novak 1991). In apple, Tabira et al. (1993 a) produced one resistant mutant from 453 in vitro-cultured shoots of cv. Indo by acute γ-ray irradiation (dose rate 5 Gy h –1, total dose 120 Gy), and this resistant mutant showed a 10,000-fold higher level of resistance than the original plant. Using a similar procedure, Masuda and Yoshioka (1996) selected one resistant mutant from among 3,602 in vitro-cultured shoots by screening shoots with a crude pathogenictoxin and acute γ-ray irradiation (dose rate 5 Gy h –1, total dose 80 Gy); this resistance was greater than that of cv. Fuji. The frequency of occurrence of resistant mutants was on the order of 10 – 2 to 10 – 3 in this study, although the general frequency of mutation is reportedly, 10 – 3 to 10 – 4. In this study, X-ray irradiation was shown to be as effective as γ-ray, which has mostly been used in mutation-based breeding. RAPD markers linked to susceptibility to Alternaria blotch were recently found by bulked- segregation analysis of the F1 population of a cross between a resistant and a susceptible cultivar (Fukasawa-Akada et al. 1999). The RAPD markers were found in the original Hokuto cultivar, whereas some were absent in resistant mutants of irradiated cv. Hokuto (FukasawaAkada et al. unpublished results). The data presented in this paper showed that the gene responsible for susceptibility to Alternaria blotch was eliminated by X-ray irradiation, which resulted in the production of resistant mutants.

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Akira Saito, Norio Nakazawa, Masahiko Suzuki

Acknowledgements. The authors thank Prof. T. Okuno of Hirosaki University for his generous gift of AM-toxin I.

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