日 植 病 報 52: 577-584 (1986) Ann. Phytopath. Soc. Japan 52: 577-584 (1986)

Formae Speciales Differentiation of Phytophthora

vignae Isolates from Cowpea and Adzuki bean

Sadao TsucHIYA*, Mayumi YANAGAwA**, and Akira OGOSHI**

Abstract

The pathogenicity of 17 isolates of Phytophthora vignae Purss from adzuki beans and 4 isolates from cowpeas was compared. Isolates from adzuki beans were virulent to adzuki beans, but not to cowpeas, while isolates from cowpeas were virulent to cowpeas, but not to adzuki beans. The soluble proteins and isoenzymes patterns of these isolates, compared by zone electrophoresis, showed no qualitative differences between adzuki bean and cowpea iso lates. Although the pathogens of both adzuki beans and cowpeas have bean treated as P. vignae, two formae specials are proposed for these two fungi on the basis of their specific pathogenicity: P. vignae f. sp. adzukicola Tsuchiya, Yanagawa et Ogoshi for the former and P. vignae f. sp. vignae Tsuchiya, Yanagawa et Ogoshi for the latter. Furthermore, from differ ences in pathogenicity to six cultivars of adzuki bean, three races (1, 2 and 3) were recog nized within f. sp. adzukicola. (Received September 4, 1985)

Key words: Phytophthora vignae, forma specialis, race, adzuki bean, cowpea.

Introduction

The pathogen causing stem rot of adzuki bean (Phaseolus radiatus var. aurea Prain) was identified as Phytophthora vignae Purss in 1978 in Japan6). This fungus was first reported as the pathogen causing stem rot of cowpeas (Vigna sinensis Endl.); it had a narrow host range and did not attack leguminous plants other than cowpeas9). Race dif ferentiation (race 1-4) occurs among the cowpea isolates of P. vignae10). In contrast, isolates (adzuki bean isolates) from adzuki beans in Hokkaido, Japan were reported to be highly virulent only to adzuki beans6,12). In this paper, we report the results of experiments on the pathogenicity and electro phoresis of fungal proteins and enzymes of adzuki bean and cowpea isolates, to examine if there are formae speciales or races among these isolates.

Materials and Methods

Test plants. Ten cultivars of adzuki bean from Hokkaido and 7 cultivars of cow pea listed in Table 2 were used. Chinese red, Blackeye 5 and Bechuana white are the differential cultivars of cowpeas for races of P. vignae10). Seeds of these three differ

* Hokkaido Prefectural Kamikawa Agricultural Experiment Station , Nagayama, Asahikawa, Hok kaido 079, Japan 北 海 道 立 上 川 農 業 試 験 場 ** Faculty Of Agriculture , Hokkaido University, Sapporo 060, Japan 北 海 道 大 学 農 学 部 578 日本 植 物 病 理 学 会 報 第52巻 第4号 昭 和61年10月

ential cultivars and Poona were provided by Mr. K. Kitazawa, Hokkaido National Agri cultural Experiment Station, and these were originally from Dr. G. S. Purss, Qeensland Department of Primary Industries, Australia. Fungal Isolates. Fungal isolates from adzuki beans are listed in Table 1. Isolates from cowpeas, 20082 (race 1), 19915 (race 2), 19997 (race 3) and 20549 (race 4) were obtained from Dr. G. S. Purss, and also used in this study.

Table 1. Sources of isolates of Phytophthora uignae obtained from adzuki bean stem rot

a) All isolates were obtained in 1977 except for isolate Ph-61 in 1979.

Inoculation. Potato sucrose agar cultures in Petri dishes at 26-27 C for 20 days

were used as inocula for the test plants. Plants were inoculated with the following two

methods: (i) Soil inoculation: the fungal colony was cut lightly with a homogenizer and

resultant minced mycelia were mixed with the sterilized soil. This mixture was packed

in shallow plastic plates (30•~21•~5cm), then adzuki beans or cowpeas were sown. The

inoculum dose per plate was equivalent to half a colony of 9 cm diameter. Plants were

grown in a growth chamber at 27 C with supplementary light providing a 12 hr. daylight at approximately 2,300 lux. After germination of seeds, plants were flooded in water

almost as deep as 1.0 cm above from soil surface for 3 days. (ii) Hypocotyl inoculation:

Seeds were sown in sterilized soil in plates and seedlings were kept under the same

condition as described above. Two days after germination (6-7 days after sowing), a

toothpick infested with mycelia of pathogen was inserted into the seedling hypocotyl at 1.0 cm above soil surface, according to Kaufmann and Gerdemann5). Then the inoc

ulated seedlings were kept for 4 days at 27 C under the lighting conditions. Ann. Phytopath. Soc. Japan 52 (4). October, 1986 579

Observation of diseased plants. Development of disease was determined in 5 days after flooding in the soil inoculation, and in 5 days after hypocotyl inoculation. Eight to 10 seedlings of each cultivar were used for each inoculation test. The test was repeated at least 3 times.

Electrophoresis of proteins and isoenzymes. Adzuki bean and cowpea isolates were compared using polyacrylamide gel disk electrophoresis for their proteins and iso enzymes. Isolates were cultured in Asparagine medium (asparagine, 2.5 g; K2HPO4, 5 g;

MgSO4, 0.2 g; sucrose, 10 g; dist. water, 1,000 ml), GPY medium (NaNO,, 2.0 g; KCI,

0.5 g; FeSO4.7H20, 10 mg; yeast extract, 0.5 g; peptone, 7.0 g; glucose, 30.0 g; dist. water, 1,000 ml), or V-8 juice medium for 20 days at 27 C. The mycelial mats were separated by filtration, washed with sterilized water, then homogenized by a blender

(1,500 rpm) for 5 min in 2-fold volume of 1/15 M phosphate buffer (pH 7.2), and finally sonicated for 10 min. The homogenate was then centrifuged at 18,000 rpm for 60 min at 2 C and the resultant supernatant was used for electrophoresis. Using glass columns

(5 mm•~80 mm) with polyacrylamide gel (7.5%, pH 8.9) used as a supporting medium, the electrophoresis was carried out at 4 C for about 60 min with 3 mA/column current.

General proteins, esterase, peroxidase, lactate dehydrogenase, and glucose-6-phosphate dehydrogenase were detected by appropriate staining1-4,11).

Results

Pathogenicity of adzuki bean and cowpea isolates In pathogenicity test of adzuki bean isolates on adzuki bean and cowpea cultivars (Tables 2 and 3), all isolates were highly pathogenic to adzuki bean cultivars, irrespective of the inoculation methods. It was also revealed by the hypocotyl inoculation that iso late Ph-4 was virulent to Blackeye 5 and Poona, isolate IFO 30613 to Chinese red and Poona, and isolate Ph-39 to Bechuana white and Poona. With the soil inoculation meth od, IFO 30613. IFO 30614, Ph-13, Ph-24, Ph-34 and Ph-39 infected Poona with low frequency, but did not infect other 6 cowpea cultivars tested. In pathogenicity test of cowpea isolates on cowpea cultivars (Tables 4 and 5), isolate 20082 (race 1) did not infect all of the three cowpea differentials, but infected other 4 cowpea cultivars tested, and showed typical symptom on these, irrespective of the in oculation methods. This isolate was, therefore, recognized to be pathogenic on these cul tivars. Isolate 19915 (race-2) was pathogenic only on three cowpea cultivars, 19997 (race-3) on four cultivars, and 20549 (race-4) on all cultivars used, except Chinese red. Pathogenicity of cowpea isolates on adzuki bean cultivars is also shown in Tables 4 and 5. In case of the hypocotyl inoculation, isolate 20082 rarely infected 4 adzuki bean cul tivars. Isolate 19915 infected only one cultivar, and isolate 19997 infected four out of seven adzuki bean cultivars tested. However, isolate 20549 was not pathogenic on any adzuki bean cultivars used. Each isolate of 4 pathogenic races typically reacted to the differential cultivars of cowpeas, but did not show pathogenicity toward adzuki bean cul tivars. These results suggested that adzuki bean isolates are virulent to adzuki beans, but not to cowpeas, while cowpea isolates are virulent to cowpeas, but not to adzuki 580 日本 植 物 病 理学 会 報 第52巻 第4号 昭 和61年10月

Table 2. Pathogenicity of the adzuki bean isolates of Phytophthora vignae on cultivars of adzuki bean and cowpea by the soil inoculation method

a) -=healthy; •}=below 5% of plants diseased; +=6•`30% of plants diseased; ++=above 31% of plants diseased.

Table 3. Pathogenicity of the adzuki bean isolates of Phytophthora vignae on cultivars of adzuki bean and cowpea by the hypocotyl inoculation method

a) -=healthy; •}=below 5% of plants diseased; +=6•`30% of plants diseased; ++=above 31% of plants diseased. Ann. Phytopath. Soc. Japan 52 (4). October, 1986 581

Table 4. Pathogenicity of the cowpea isolates of Phytophthora vignae on cultivars of cowpea and adzuki bean by the soil inoculation method

a) -=healthy; •}=below 5% of plants diseased; +=6•`30% of plants diseased; ++=above 31% of plants diseased.

Table 5. Pathogenicity of the cowpea isolates of Phytophthora vignae on cultivars of cowpea and adzuki bean by the hypocotyl inoculation method

a) -=healthy; •}=below 5% of plants diseased; +=6•`30% of plants diseased; ++=above 31% of plants diseased. 582 日本 植 物 病 理 学 会 報 第52巻 第4号 昭 和61年10月 beans. Differentiation of pathogenicity of the adzuki bean isolates Seventeen isolates obtained from adzuki beans in various parts of Hokkaido district were tested for their pathogenicity on Notoshozu, Wasetairyu 1, Takarashozu and Cha garawase (selected as pure lines from local cultivars), and Hayateshozu and Kotobuki shozu (which were bred by crossing). The pathogenicity was compared by the symptoms on young seedlings of cultivars with the soil inoculation method. The result is shown in Table 6. Out of 17 isolates tested, 12 isolates including IFO 30613 and IFO 30614 intensely infected 6 cultivars of adzuki bean. Isolates Ph-18, Ph-32, Ph-39, and Ph-52 were virulent to Wasetairyu 1, Cha garawase, Takarashozu and Hayateshozu, but not to Notoshozu and Kotobukishozu. Isolate Ph-15 was virulent to all cultivars except Notoshozu. From these results, it is obvious that the pathogenicity of isolates of the adzuki bean pathogen differed in the reaction to cultivars of adzuki bean and the isolates can be divided into 3 groups on pathogenic differentiation.

Table 6. Pathogenic races of Phytophthora uignae f. sp. adzukicola causing stem rot of adzuki beans

a) R=Resistant; S=Susceptible.

Comparison of electrophoretic patterns of soluble proteins and isoenzymes of the adzuki bean and the cowpea isolates Electrophoretic patterns of soluble proteins, in which there were 10 distinctive stain ed bands, were not different between the adzuki bean and the cowpea isolates. Peroxi dase isoenzyme patterns (3 distinctive bands), were also not different between two groups of isolates, except that an isoenzyme of cowpea isolates at Ef 0.66-0.70 was less sharp than that of adzuki bean isolates. Non-specific esterase patterns, in which 2 bands were detected, were a little different quantitatively between two groups of iso lates. An isoenzyme of cowpea isolates was stained more distinctively than that of ad Ann. Phytopath. Soc. Japan 52 (4). October, 1986 583 zuki bean isolates. Isoenzyme patterns of lactate dehydrogenase were simple and only one band was detected. There was no difference between two groups of isolates. Iso enzyme patterns of glucose-6-phosphate dehydrogenase, in which 2 bands were detect able, had qualitatively no difference between two groups of isolates, although an isozyme of adzuki bean isolates was stained more distinctively than that of cowpea isolates.

Discussion

Phytophthora vignae, the pathogen of stem rot of cowpea, has a narrow host range and is pathogenic only on cowpeas9). However, isolates of P. vignae obtained from adzuki beans are pathogenic on adzuki beans6,12). In this paper we described that the patho genicity differed between the adzuki bean and the cowpea pathogen. The adzuki bean pathogen was virulent to adzuki beans, but not to cowpeas with both soil inoculation and hypocotyl inoculation methods, with the exception of slight infection of Poona, a cultivar of cowpea, using the soil inoculation method. The cowpea pathogen was almost not pathogenic to adzuki bean. These results show that there are pathogenic differentia tions between the adzuki bean and the cowpea pathogens. The results of electrophoresis of proteins and analysis of isoenzymes showed that there were no qualitative differences and only slight quantitative differences between two pathogens. The morphology and physiology of the adzuki bean pathogen is very similar to that of the cowpea pathogen6,7,12). They are different only in their pathogenicity and host range. Therefore, these pathogens should be treated as distinctive, pathogenic groups. Kuan and Erwin8) reported two formae speciales in P. megasperma, i.e., f. sp. glycinea for isolates that attack soybean and f. sp. medicaginis for isolates that attack alfalfa. As Kitazawa et al.7) suggested earlier, we also propose that adzuki bean and cowpea isolates of P. vignae be subdivided into two formae speciales, i.e., P. vignae Purss f. sp. adzukicola Tsuchiya, Yanagawa et Ogoshi for the former and P. vignae Purss f. sp. vignae Tsuchiya, Yanagawa et Ogoshi for the latter. It has already been reported that there are 4 races within the pathogen of stem rot of cowpea10). It is obvious in our experiments that there are also at least 3 races within the pathogen of stem rot of adzuki beans, P. vignae f. sp. adzukicola, namely, race 1 which is unable to attack Notoshozu and Kotobukishozu but virulent to all other culti vars tested, race 2 which is virulent to all cultivars except Notoshozu, and race 3 which is able to attack all six tested cultivars of adzuki bean.

We thank Dr. G. S. Purss, Queensland Department of Primary Industries, Australia, for sending isolates of Phytophthora vignae, Mr. K. Kitazawa, Hokkaido National Agricultural Experiment Station, for the differential cultivars of cowpea, and staff in Hokkaido Prefectural Tokachi Agricultural Experiment Station for cultivars of adzuki bean. We also thank Dr. G. D. Bowen, Division of Soils, CSIRO, Australia, for the critical reading of the manuscript. 584 日本 植 物病 理 学 会 報 第52巻 第4号 昭 和61年10月

Literature cited

1. Berry, J. A. and Franke, R. G. (1973). Amer. J. Bot. 60: 976-986. 2. Clare, B. G., Flentje, N. T. and Atkinson, M. R. (1968). Aust. J. Boil. Sci. 21: 275-279. 3. Gill, H. S. and Powell, D. (1968). Phytopath. Z. 63: 23-29. 4. Hall, R., Zentmyer, G. A. and Erwin, D. C. (1969). Phytopathology 59: 770-774. 5. Kaufmann, M. J. and Gerdemann, J. W. (1958). Ibid. 48: 201-208. 6. Kitazawa, K., Tsuchiya, S., Kodama, F., Kamjaipai, W., Ogoshi, A. and Yanagita, K. (1978) Ann. Phytopath. Soc. Japan 44: 528-531. 7. Kitazawa, K., Suzui, T. and Yanagita, K. (1979). Ibid. 45: 406-408. 8. Kuan, T. -L. and Erwin, D. C. (1980). Phytopathology 70: 333-338. 9. Purss, G. S. (1957). Qd. J. Agric. Sci. 14: 125-154. 10. Purss, G. S. (1972). Aust. J. Agric. Res. 23: 453-456. 11. Reddy, M. N. and Stahmann, M. A. (1972). Phytopath. Z. 74: 55-68. 12. Tsuchiya, S. and Kodama, F. (1978). Shokubutsu-boeki (Plant Protection) 32: 357-360.

和 文 摘 要

土 屋 貞 夫 ・梁 川 真 弓 ・生 越 明:ア ズ キ お よ び サ サ ゲ か ら分 離 さ れ たPhytophthora vignaeの 寄 生 性 の 分 化

Phytophthora vignaeの ア ズ キ か ら の17菌 株 お よ び サ サ ゲ か ら の4菌 株 を 用 い て,ア ズ キ お よ び サ サ ゲ に 対 す る 病 原 性 を 比 較 し た 。 ア ズ キ 菌 は ア ズ キ に 対 し て 病 原 性 を 示 し た が,サ サ ゲ に は ほ と ん ど 病 原 性 が な か っ た 。 一 方,サ サ ゲ 菌 は サ サ ゲ に 病 原 性 を 示 し た が,ア ズ キ に 対 し て は ほ と ん ど 病 原 性 が な か っ た 。 こ れ ら の 分 離 株 の た ん ぱ く質 と 酵 素 の 電 気 泳 動 パ タ ー ン を 比 較 し た 。 両 菌 間 に は ほ と ん ど 質 的 な 差 が な く,ま た 量 的 な 差 は ご くわ ず か で あ っ た 。 ア ズ キ 菌 も サ サ ゲ 菌 もPhytophthora vigraaeと さ れ て い る が,病 原 性 の 相 違 か ら,ア ズ キ 茎 疫 病 菌 に 対 し て はP. vignae Purss f. sp. adzukicola Tsuchiya, Yanagawa et Ogoshi,サ サ ゲ の 茎 腐 病 の 病 原 菌 に 対 して はP. vignae Purss f. sp. vignae Tsuchiya, Yanagawa et Ogoshiを 提 案 す る。 さ ら に ア ズ キ6品 種 に 対 す る 病 原 性 試 験 か らf. sp. adzukicolaの 中 に はrace 1, race 2, race3 が 存 在 す る こ と が 判 明 し た 。