Breeding Soybean Cyst Nematode Resistant Azuki Beanusing Wild Vigna Germplasm
Aoyama Satoshi1) and Atsuhiko Kushida2)
1) Hokkaido Prefectural Tokachi Agricultural Experiment Station (Research Conducted by Special Assignment of

The Ministry of Agriculture, Forestry & Fisheries of Japan ),Present address: Hokkaido Prefectural Kamikawa Agricultural Experiment Station .
2) National Agricultural Research Center for Hokkaido Region ［Memuro Upland Farming Research Station ］

Abstract
Wild accessions of Vigna riukiuensis have been crossed to azuki bean (V. angularis ) cultivars in aseries of breeding programs at Hokkaido Prefectural Tokachi Agricultural Experiment Station aiming
to introduce resistance of soil-borne diseases and abiotic stresses. In one of the characterization
process, we have found an accession “Acc2482 (JP235878)” of V. riukiuensis which was collectedfrom Iriomote island of Okinawa prefecture, Japan showed resistance to Soybean Cyst Nematode(SCN), Heterodera glycines . In an evaluation experiment, SCN could produce significantly lessnumber of cysts on the roots of “Acc2482”. To confirm SCN resistance of “Acc2482”, we reinvestigated the level of SCN resistance of this accession and the hybrid progenies. The results
showed that the SCN resistance of “Acc2482” was stable and genetically inherited in the hybrid
progenies produced from a hybrid using a susceptible azuki bean breeding line as a female parent.
This suggested a possibility of introducing SCN resistance gene(s) from V. riukiuensis to azuki bean.

Introduction

Hokkaido is the largest beans producing area
in Japan. It is located in the northern part of Japan,
as illustrated in Fig.1. Soybean Cyst Nematode(SCN), Heterodera glycines , is one of the most serious pests that cause yield loss of bean crops suchas soybean (Glycine max ), azuki bean (Vigna angularis ), common bean (Phaseolus vulgaris ) (Ichinohe1953). Until recently, breeding of SCN resistantazuki bean cultivar has not been considered as ahigh priority objective, since azuki bean is relatively tolerant to the infestation of SCN compared withsoybean and yield loss is less prominent. However,
the relatively high level of tolerance in azuki bean
revealed to cause an adverse effect on the subsequent susceptible crops such as soybean by increasing population density of SCN in the field. In addition, since the population density of SCN in thefield of short-term rotation of beans becomes very
high recently, yield loss of azuki bean itself has
been becoming a serious problem too. At present,
use of resistant varieties is the most effective wayof controlling SCN damage.
In 1954, screening of SCN resistant varieties in azuki bean germplasm was carried out inTokachi Agricultural Experiment Station. However,no promising SCN resistant azuki bean variety was
found at that time. Shimizu and Mitsui (1987) reported a phenotypic difference in resistance levelto SCN among azuki bean varieties. However, thelevel of resistance detected was insufficient for thebreeding of SCN resistant cultivar. On the otherhand, Sawa (1973) reported that green gram (V.
radiata ) showed higher level of resistance to SCN.
Interspecific hybridization was tried, however tothe authors' knowledge, there have been no experimental data on SCN resistance in the hybrid progenies.
Collection, conservation and classification ofwild relatives of azuki bean have been conducted inthe NIAS genebank, Tsukuba, Japan (Tomooka etal ., 2002). In addition to the germplasm collection,molecular markers and molecular linkage maps ofazuki bean have been developed (Han et al . 2005,Isemura et al ., 2007, Kaga et al ., 2008, Tomoka etFig. 1. Hokkaido (shaded) located in northern Japan- 103 -Fig. 2. Gene pool classification of azuki bean (moodified from Tomooka et al., 2000)

al . 2006, Xu et al . 2008).
Availability of the wild germplasm collection
and molecular tools has encouraged us to use wild
genetic resources for the azuki bean breeding. For
example, a powdery mildew-resistant accession ofV. hirtella was found and introduction of resistancegene into azuki bean was attempted (Egawa et al .,2003).
Egawa et al . (1999) have reported that accessions of V. riukiuensis , which belongs to the primary

genepool of azuki bean (see Fig. 2, Vaughan et al .,2005), showed significantly higher levels of heat tolerance compared with azuki bean. Previous researchin Hokkaido Prefecture Tokachi Agricultural Experiment Station (1997) showed that V. riukiuensis has
resistance to brown stem rot, phytophthora stem
rot and fusarium wilt. Therefore, we have crossed
V. riukiuensis with azuki bean in order to introduce
these resistance genes of V. riukiuensis . In one of thecharacterization process, we accidentally found theTable 1. The number of cysts in breeding line in F5 generation
LineSpecies
orCross

Evaluationof resistance1)

Root
weightAverage (Fwt/plant)

No. of cysts (/plant)

F3 or F4F512

34

Acc2482

V.riukiuensis

RR

26

2143

1727

6.6

Toiku No.150

V.angularisSS

219231197224

218

8.30321- ② 83-2

Toiku No.150

SS

256

255

241

260

253

21.1

0321- ② 85-1-9

×R

S

229

216--223

11.40321- ② 85-1-13

Acc2482

RS

97

153

159

220

157

4.80321- ② 85-2-1

R

R

45

34

34-

38

16.5

0321- ② 85-3-5

RSeg

44

130

41

98

78

16.8

0321- ② 85-4-2

R

S

190

87

149

102

132

5.6

0321- ② 85-6-2

SS

267

216

93

254

208

12.41) R : resistance, S : susceptible, Seg : segregating.

- 104 -Fig. 3. Procedure used to obtain lines for resistance to SCNSCN resistance in an accession “Acc2482 (JP235878,NIAS genebank passport number)” of V. riukiuensis
The objectives of this study are to confirm
SCN resistance found in an accession “Acc2482”
of V. riukiuensis and to examine the possibility of
introducing SCN resistance to azuki bean. Additionally, we attempted to find novel germplasm of SCN
resistance by screening larger number of wild Vigna
germplasm.

Materials and Methods

The procedure to develop azuki bean lines
harboring resistance to SCN is shown in Fig. 3. In
2003, an accession “Acc2482” of V. riukiuensis wascrossed with an azuki bean breeding line “ToikuNo.150” (the cross number is “0321”). Selection
based on the resistance level to SCN has been performed from F2 to F4 generation. In F5 generation,line test for the SCN resistance was performed using
lines which have enough number of seeds.
Searching of novel source of SCN resistance
germplasm from wild relatives of azuki bean has
been conducted using 9 accessions consist of 4 species (Table 2). Seeds were directly sown in a plastic
pot filled with autoclaved soil and maintained in agreenhouse. About 1,000 larvae of SCN were inoculated into each pot about one month after sowing.
At 35 days after SCN inoculation, cysts on the roots

Table 2. The number of cysts in wild relatives
Acc No.
-Species

V.angularis

1)

OriginNo. of cysts (/pot)
Average

S.E.

Hokkaido Pref.

198

142463

V.angularis var.nipponensis

Miyagi Pref.

278

102464

V.angularis var.nipponensis

Ibaraki Pref.

246

212477

V.riukiuensis

Okinawa Pref.

124132482

V.riukiuensis

Okinawa Pref.

15

32513

V.nepalensisNepal

249

102243

V.nepalensis

Nepal

318

222502

V.umbellata

Thailand(Phrae)

144132503

V.umbellata

Thailand(Phrae)

117131) Erimoshouzu is a leading variety in Hokkaido Prefecture.
- 105 -were collected from each pot (or plant) and numberof cysts was counted.

Results and Discussion

SCN resistance of F5 hybrid breeding lines
and their parents
Number of cysts on the roots of parents isshown (upper 2 rows, Table 1). Average number ofcysts detected on the roots of “Toiku No.150” (azukibean breeding line) was 218 cysts / plant, while thatdetected on the roots of “Acc2482” (a resistant wild
accession of V. riukiuensis ) was 27 cysts/plant. Although the root mass of “Acc2428” was about 80%of “Toiku No.150”, the number of cysts of “Acc2482”
was only 12 % of “Toiku No.150”. Since the tested
4 individuals of “Acc2482” and “Toiku No.150”
showed stable response to SCN infestation, resistance of “Acc2482” was confirmed to be geneticallystable.
Number of cysts on the roots of F5 breedinglines is shown (lower 7 rows, Table 1). Among 7lines tested, 5 lines were evaluated as susceptible (S),
1 as resistant (R) and 1 as segregating (Seg). Threelines formerly evaluated as R in F3 or F4 generation
were evaluated as S in F5 generation. One line evaluated as R in F3 or F4 generation (0321- ② 85-3-5)
was evaluated as segregating and another line evaluated as R in F3 or F4 generation (0321- ② 85-2-1)
was evaluated as R. These results suggest that SCN
resistance of V. riukiuensis accession is incorporated,
genetically inherited between generations and segregating in the hybrid lines. Judging from the root
weight of hybrid lines, level of SCN resistance is notdirectly related to the amount of roots (Photo 1).
Searching novel source of SCN resistance
Since promising SCN resistance source wasfound in an accession of cross compatible wild relative of azuki bean (V. riukiuensis ), we have started
a screening using comprehensive collection of wild
Vigna species which can cross with cultivated azuki
bean. Preliminary result was shown in Table 2. A
leading azuki bean variety “Erimoshouzu” showing susceptibility (198 cysts / plant) was used as acontrol (uppermost row in Table 2). Two accessionsof wild ancestor of azuki bean (V. angularis var. nipponensis ) collected from Japan showed higher levelsof susceptibility. Higher levels of susceptibility werealso recorded for V. nepalensis accessions collectedfrom Nepal. Two cultivated accessions of rice bean(V. umbellata ) from Thailand showed similar levelsof susceptibility to “Erimoshouzu”.
“ACC2477” of V. riukiuensis collected fromthe same prefecture as “ACC2482” did not show resistance to SCN. It was found that intraspecific variation of SCN resistance exists in V. riukiuensis .
Further screening program using larger
number of wild accessions is in progress.

Acknowledgments

We thank Dr. N. Tomooka and Dr. Y. Egawa
for suggesting the importance of wild genetic recourse in the genus Vigna . Technical help from Mrs.
C. Suzuki is greatly appreciated. The authors express
their sincere thanks to Mr. H. Shimada for criticizing the manuscript. This research was conducted in
Hokkaido Prefectural Tokachi Agricultural Experiment Station.

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- 107 -Maturing

V. riukiuensis

Seedling

F2 (0321)

V. angularis

V. riukiuensis

Testing of SCN resistanceV. riukiuensis

F3(0321- ② 85)

F3 (0321)

V. angularisRoot

V. angularis

V. riukiuensis

Photo 1. Comparison of morphology- 108 -

F3 (0321- ② 85)

V. angularis