Brown Stem rot and Phytophthora Stem Rot:
Limiting Factors in Azuki Bean Production
Kondo Norio
Laboratory of Plant Pathology, Research Faculty of Agriculture , Hokkaido University
Sapporo, Japan

Summary
Information on the distribution of races of Cadophora gregata f. sp. adzukicola and Phytophthora vignae
f. sp. adzukicola is important for the management of brown stem rot and Phytophthora stem rot of azuki beans,respectively. Potential lines or cultivars with multiple resistances are developed using information on virulence ofraces and infection methods.

Introduction

Brown stem rot (BSR) of azuki bean [Vignaangularis (Willd.) Ohwi et Ohashi] caused by
Cadophora gregata (syn. Phialophora gregata )Harrington & McNew f. sp. adzukicola Kobayashi,Yamamoto, Negishi, and Ogoshi (Harrington andMcNew, 2003; Kobayashi et al . 1991), a soilborne fungus, is a serious problem in Hokkaido,
Japan (Fujita et al ., 2007). BSR is characterizedby wilting, and a reddish brown discoloration ofvascular and pith tissues of the stem and the petiole
that often occur in conjunction with leaf chlorosis,necrosis or defoliation. BSR was first observed inlate 1960’s in Tokachi district, the eastern part of
Hokkaido, and then the infested area extended to
Kamikawa and Shiribeshi, the central and westernparts during 1970’s. In 1977 and 1981 about
75% of the commercial fields in Tokachi district,accounting for about 40% of the total production
area of Hokkaido, were infected with the BSR
pathogen. Even in recent years the proportion ofseverely diseased fields has remained about 10 to15% (Fujita, 2004). BSR is estimated to reduce theactual seed weight per plant by up to 77%.
Phytophthora stem rot (PSR) caused by
Phytophthora vignae Purss f. sp. adzukicola
Tsuchiya, Yanagawa et Ogoshi (Tsuchiya et al .
1986) is also an economically important constraint
to azuki bean production (Kondo et al . 2004).
The PSR pathogen, an oomycete that is classified
in the kingdom Stramenopiles, infects the roots,
epicotyls, and stems of young seedlings andmature azuki plants, eventually causing wilt orleaf blight. Leaves can be infected by deliberate
inoculation in the laboratory (Harada and Kondo,2008). Beginning in the 1960’s overproductionof rice became a problem in Japan leading to
the national government establishing an act toequilibrate supply with demand for rice. Hence,
during the 1970’s farmers were obliged to convert
rice paddies into upland, non-flooded, fields.
Azuki bean was grown widely in the converted
fields in the central and western parts of Hokkaido.
Those poorly drained fields provide the moist
environmental conditions that favor infection by P.
vignae and disease development. This disease wasfirst reported in Hokkaido in 1967 and became themain constraint in the production of azuki beans in
Hokkaido including Tokachi district.
Generally, practical methods of controllingthese diseases are crop rotations and the use ofresistant cultivars. With these disease pressures,producers hoped for the development of cultivars
with resistance to both pathogens. Hence, thedevelopment of multiple resistant cultivars withgood agronomic qualities has been continued since
1981 in collaboration with the Tokachi and Central
Agricultural Experiment Stations in Hokkaido,
and recently resulted in the release of somepromising cultivars. In this paper it is shown how
to identify physiological races of the BSR and PSR
pathogens. It is important to know the frequency
and distribution of races of these pathogens for
managing the damage to azuki bean production by
new races in the future.

Methods for inoculation of C. gregata

Inoculum is grown in V-8 juice broth (200ml V-8 juice and 2 g CaCO3 centrifuged at 5,000rpm for 15 min, and the supernatant is diluted to- 99 -1 L with distilled water) at 25°C on a reciprocal
shaker at 120 oscillations/ min. After 2 to 3 weeks
of incubation, mycelia and spores are collected byfiltration through Whatman No.1 filter paper, andwashed with distilled water. The fungal pellets
are homogenized with distilled water (10,000 rpm
for 3 min) and diluted to 107 spores and mycelial
fragments per milliliter. Seedlings grown in sterilizedvermiculite (about 10 to 14 days) are removed,
washed gently, dipped into each inoculum suspension
for 12 hr, then transplanted into a disinfested soil /vermiculite mixture (1:1) in 12-cm diameter plastic
pots. The response to the pathogen was evaluatedafter 8 weeks growth in the greenhouse. The severityof symptoms is assessed in terms of the mean diseaseseverity index (DSI) of plants with a scale where 0= no symptoms, 1 = a slight vascular discoloration
without foliar symptoms, 2 = foliar symptoms or
severe vascular discolorations (above the first nodeor in petioles) , and 3 = death. Plants with a DSI <0.5 are rated resistant (R), plants with 0.5 ≤ a DSI <1.0 are regarded as intermediate (I) and plants with aDSI ≥ 1.0 were considered susceptible (S).

Races of C. gregata and their distribution
There are three races of C. gregata inHokkaido so far; race 1, virulent on Erimo-shozu
and avirulent on Kita-no-otome and Acc259; race
2, virulent on Erimo-shozu and Kita-no-otome but
avirulent on Acc259; and race 3, virulent on Erimoshozu and Acc259 but avirulent on Kita-no-otome(Table 1). Race 1 is predominant in the commercial
fields in Hokkaido. Although the frequency ofisolation is lower than that of race 1, race 2 waswidely distributed in most of the production areas(Kondo et al ., 2002). Meanwhile, race 3 was actually
restricted to certain fields only (Kondo et al ., 2005).
Methods for Inoculation of P. vignae
Root dipping
One gram (fresh weight) of mycelia from

2-week-old cultures of each isolate, grown in pea
broth at 25°C, is homogenized (Nissei AM-5, 5,000rpm, 3 min.) with 100 ml of sterilized distilled water.
Seven- to ten-day-old seedlings, grown in sterilizedvermiculite, are washed gently with running tap
water before inoculation. Seedlings of each cultivar
grown in sterilized vermiculite are removed andsoaked for 12 hr in inoculum suspensions Then, theseedlings are transplanted into a mixed soil (vermiculite/ Pot Ace, Katakura Chikkarin K. K., Tokyo,
Japan (1/1) v/v mixture) in plastic pots (18 cm indiameter). Three weeks after transplanting undergreenhouse conditions (12 to 25°C), the incidence ofdisease (dead or alive) is evaluated.
Epicotyl inoculation
Fourteen-day-old seedlings (planted in vermiculite and grown in growth chamber at 25℃ underfluorescent light for 15 h and 18℃ in the dark for 9h) are removed from the vermiculite and placed horizontally in plastic containers (15 × 20 × 5 cm) with
their roots covered with moistened paper towels. An
approximately 1.5 cm long slit wound is made on
the upper part of the epicotyl (about 1 cm below theprimary node) with a sterilized razor blade. Then,
200 μl drops of minced mycelial (10 mg/ml distilledwater) suspension from 3-day-old cultures grown in
V8 juice medium (200 ml V8 juice and 2.6g CaCO3
centrifuged at 5000 rpm for 15 min, and the supernatant diluted to 1 L with distilled water) are inoculated
into the wounded site. After inoculation, the containers are kept closed with plastic (poly vinylidenechloride) cling film (Asahi kasei chemicals, Tokyo)
to maintain high humidity and incubated at 25°C
in the dark. The severity is evaluated as describedabove. The disease severity is assessed two days after inoculation using the mean disease severity index(DSI) on a scale of 0 to 3, where 0 = no symptoms, 1= a slight or restricted lesion on the epicotyl, 2 = an
expanding lesion on the epicotyl, and 3 = death.

Table 1. Races of Cadophora gregata f. sp. adzukicola and differential cultivars/line
Erimo-shozuKita-no-otome

Acc259Race 1S

RR

Race 2SSR

Race 3S

RS

S: susceptible, R: resistant- 100 -Races of P. vignae and their distribution

The extensive surveys of the races distribution of P. vignae f. sp. adzukicola during 1977 to1979 and 1994 to 1995, showed that race 1 (virulenton cv. Erimo-shozu, but avirulent on cv. Kotobukishozu and cv. Noto-shozu) and race 3 (virulent tocvs. Erimo-shozu, Kotobuki-shozu and Noto-shozu)
are predominant in Hokkaido, while race 2 (virulenton cvs. Erimo-shozu and Kotobuki-shozu, but not
cv. Noto-shozu) is present at a low frequency or not
observed. Race designation tests in the latter survey
used cv. Urasa-Shimane, which is one of the parentsof cv. Syumari, as a resistant host to all races. No isolates were virulent on cv. Urasa-Shimane. However,in 1999 when cv. Syumari was first grown in several
experimental fields, PSR was observed on it (Notsuet al ., 2003). The presence of a new race, designatedrace 4, was confirmed. The differential cultivars forthe races were proposed, i.e. Erimo-shozu, Kotobukishozu, Noto-shozu, Urasa-shimane or Syumari (Table2). Race 4 isolates had the broadest host range andwere widely distributed in the azuki bean-producing
regions, especially in the central and western partsof Hokkaido (Kondo et al ., 2004). The appearance ofrace 4 immediately after the release of cv. Syumari
has raised concerns about promoting this new cultivar, and the effectiveness and durability of its resistance.

Conclusion

At Tokachi agricultural experiment station in
Hokkaido, breeding for BSR and PSR resistance has
been carried out in the infested fields since the late1970's. Field screening for BSR and PSR resistancecan be difficult as infection and subsequent diseasedevelopment are highly influenced by the environmental conditions. Factors such as inoculum levels
and isolate aggressiveness, along with air temperature, soil fertility, and soil moisture all impact disease
severity. At least race frequency in field soils should
be clarified before screening in the field. Screening
in the greenhouse allows for control of these factors,
thereby improving the chance of discovering truly
resistant lines. After identifying races of the BSR andPSR pathogens, new sources of BSR and PSR resistance have been sought(Fujita et al ., 2007; Kondo etal . 2009). Azuki bean or wild azuki bean germplasm
from some Asian countries including Japan havebeen evaluated so far for resistance genes and somepotential candidates have been identified. Thus,
multiple resistant lines are developed from the crosswith new resistant gene sources. In addition, now wefocus on using partial resistance (also termed fieldresistance) against PSR as well as Phytophthora stem
and root rot of soybean caused by P. sojae .

Table 2. Races of Phytophthora vignae f. sp. adzukicola and differential cultivars
Erimo-shozu

Kotobuki-shozu

Noto-shozu

SyumariRace 1

SRRR

Race 2

SSR

R

Race 3SSS

R

Race 4SS

S

S

S: susceptible, R: resistant- 101 -

References
Fujita S (2004) Breeding of adzuki bean cultivars
resistant to soilborne diseases. PSJ Soilborne
Disease Workshop Report 22: 88-99 (in Japanese).
Fujita S, Kondo N, Shimada H, Murata K, Naito
S (2007) Re-evaluation and selection of adzukibeans to breed cultivars resistant to a new race ofPhialophora gregata f. sp. adzukicola , the causal
agent of adzuki bean brown stem rot (BSR).
Breeding Research 9: 87-95 (in Japanese withEnglish summary).
Harada G, Kondo N (2009) Adzuki bean leaf infection by Phytophthora vignae f. sp. adzukicola
and resistance evaluation using detached leaves
inoculated with zoospores. Journal of General
Plant Pathology 75: 52-55.
Harrington TC, McNew DL (2003) Phylogenetic
analysis places the Phialophora -like anamorph
genus Cadophora in the Helotiales. Mycotaxon
87: 141-151.
Kobayashi K, Yamamoto H, Negishi H, Ogoshi A(1991) Formae speciales differentiation of Phialophora gregata from adzuki bean and soybean
in Japan. Annals of the Phytopathol Society ofJapan 57: 225-231.
Kondo N, Kobayashi Y, Sakuma F, Fujita S, Murata
K (2002) Regional distribution of two races ofPhialophora gregata f. sp. adzukicola , the causal

agent of adzuki bean brown stem rot, and their
genetic diversity in Hokkaido, the northernmost
island of Japan. Journal of General Plant Pathology 68: 284-291．
Kondo N, Notsu A, Naito S, Fujita S, Shimada H.
(2004) Distribution of Phytophthora vignae f. sp.
adzukicola races in adzuki bean fields in Hokkaido, Japan. Plant Disease 88: 875-877.
Kondo N, Nakazawa K, Fujita S, Shimada H, Naito
S (2005) New virulent race of Phialophora gregata f. sp. adzukicola associated with continuous
cultivation of adzuki bean cultivar Acc259. Journal of General Plant Pathology 71: 360-363.
Kondo N, Shimada H, Fujita S (2009) Screeningof cultivated and wild adzuki bean for resistanceto race 3 of Cadophora gregata f. sp. adzukicola ,cause of brown stem rot. Journal of General
Plant Pathology 75:181-187.
Notsu A, Kondo N, Fujita S, Murata K, Naito S(2003) New race of Phytophthora vignae f. sp.
adzukicola , the causal agent of Phytophthora
stem rot of the adzuki bean. Journal of General
Plant Pathology 69: 39-41.
Tsuchiya S, Yanagawa M, Ogoshi A (1986) Formae
speciales differentiation of Phytophthora vignae
isolates from cowpea and adzuki bean. Annals of
The Phytopathological Society of Japan 52: 577584.

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