Botanical Studies (2010) 51: 203-207.
MICROBIOLOGY
Reevaluation of the report of the A2 mating type of Phytophthom infestans on tomato in Taiwan
Pao-Jen ANN1, Jyh-Nong TSAI1, Tien-Cheng WANG2, Chien-Hua CHEN2, Mei-Ju LIN3, and Wen-
Hsiung KO3 *
1Division of Plant Pathology, Taiwan Agricultural Research Institute, Wufeng, Taichung, Taiwan
2Asian Vegetable Research and Development Center, The World Vegetable Center, Shanhua, Tainan, Taiwan
3Department of Plant Pathology, National Chung Hsing University, Taichung, Taiwan
(Received November 27, 2009; Accepted February 8, 2010)
ABSTRACT. In June 2008, Deahl et al. reported the first detection of two isolates of the A2 mating type of Phytophthom infestans on tomato from two locations in Taiwan based on the tests performed at USDA, Belts-ville, using A1 and A2 mating types of P. infestans as testers. However, the third and fourth authors of the paper showed that these two isolates (Pi 214 and Pi 566) behaved as A1 mating type when paired with A1 and A2 testers of P. nicotianae (= P. parasitica) at Asian Vegetable Research and Development Center, Shanhua, Taiwan. This information was not included in the report. These two isolates along with two other isolates of P. infestans (Pi 215 and Pi 564) isolated from the same locations on the same dates were re-tested independently in three laboratories using A1 and A2 mating types of P. infestans, P. nicotianae and P. capsici as testers. All four isolates displayed oospore formation when paired with A2 but not A1 mating type regardless of species used as the testers, indicating that all of them are of the A1 mating type. New isolation of P. infestans from diseased tomato plants from the same locations also showed the presence of only the A1 mating type. These results refute the claim by Deahl et al. of the discovery of the A2 mating type of P. infestans from Taiwan.
Keywords: Late blight; Mating type; Phytophthora infestans; Tomato.
INTRODUCTION
Late blight of potato and tomato caused by Phytoph­thora infestans (Mont.) de Bary is one of the most devas­tating plant diseases worldwide. The disease was reported from Taiwan in 1908 by Kawakami and Suzuki and in 1919 by Sawada. It had not received much attention until 1995 when Hartman and Huang reported the isolation of 13 isolates of P. infestans from tomato and the discovery that all isolates were of the A1 mating type. The presence of only the A1 mating type of P. infestans in Taiwan was confirmed in 1998 by Ann et al. (1998) who reported that 68 isolates from tomato and 2 isolates from potato, were all of the A1 mating type. In 2004, Jyan et al. reported collection of 94 tomato and potato isolates of P. infestans throughout Taiwan from 1992 to 2002 and found that all were of the A1 mating type. Since 1998, the senior author has been surveying the mating type distribution of P. infes-tans in Taiwan yearly. As of 2008, 409 potato isolates and 1172 tomato isolates were collected and all have been of the A1 mating type (Ann, unpublished data).
In 2008, Deahl et al. reported the detection in Taiwan of one A2 isolate of P. infestans (Pi 214) out of 200 iso­lates tested in 2004 and another A2 isolate (Pi 566) out
*Corresponding author: E-mail: kowh@dragon.nchu.edu.tw;
Tel: +886-4-2284-0780 ext 371; Fax: +886-4-2287-7585.
of 102 isolates tested in 2006. Due to the importance of the A2 mating type of P. infestans for quarantine status in Taiwan, it is essential to confirm this purported A2 mating type. We, therefore, wrote to Deahl to request cultures of these two A2 isolates (Pi 214 and Pi 566). He replied that the quarantine permit only allowed the receipt of cultures but no redistribution to others, and suggested that Wang the fourth author be contacted for cultures (W. H. Ko, personal communication). Wang shared the two putative A2 cultures (Pi 214 and Pi 566) along with two A1 cul­tures (Pi 215 and Pi 564) obtained from the same locations in 2004 and 2006 (Table 1). He mentioned that he and Black, the third author, informed Deahl that Pi 214 and Pi 566 were A1 based on the result of pairing with the A1 and A2 testers of Phytophthora nicotianae van Breda de Haan (= Phytophthora parasitica Dastur) and requested that this result be added to the article. However, the request was ignored because it was not based on the pairing with the A1 and A2 testers of P. infestans, a procedure used at USDA, Beltsville (T. C. Wang, personal communication). This rejection is not logical because sexual reproduction of heterothallic Phytophthora is unique with normal oo-spores produced readily when opposite mating types of different species are paired (Shen et al., 1983; Ko, 2007). Moreover, all 302 P. infestans isolates were tested using A1 and A2 testers of P. nicotianae in Taiwan and all were found to be of the A1 mating type. Therefore, the mating
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type of the four isolates of P. infestans mentioned above were reevaluated by performing independent experiments in three different laboratories using A1 and A2 testers of P. infestans, P. nicotianae and P. capsici Leonian and by obtaining quantitative data from each test. In addition, new isolates were collected from diseased leaves, fruit and stems of infected tomato plants from Nantou and Hualien counties where the two purported A2 isolates were collected. Details of these studies are reported herein.
MATERIALS AND METHODS
Sources of isolates
The origins of P. infestans isolates included in the report of Deahl et al. (2008) and used in this study for reevaluation of mating type are described in Table 1. New P. infestans from diseased tomato leaves, stems and fruit were collected from Hsinyi of Nantou county in June and August 2008, and from Shoufeng of Hualein county in February and April 2009. These are the sites for the isolation of Pi 214 and Pi 566, the purported A2 isolates. Pieces of diseased leaves (10 x 10 mm), stems (5 x 5 mm) and fruit (5 x 10 x 10 mm) each with a single lesion were surface-sterilized with 0.5% sodium hypochlorite for 1 min and placed on rye A agar (Caten and Jinks, 1968) supplemented with 100 ppm ampicillin, 50 ppm nystatin and 10 ppm pentachloronitrobenzene to inhibit bacteria and non-pythiaceous fungi (Ko et al., 2006). Plates were incubated at 20°C and observed daily. Hyphae of P. in-festans emerging from a tissue were transferred to a plate of rye A agar. Cultures were maintained on rye agar and stored in culture slants covered with mineral oil in test tubes at 16°C.
Determination of mating type
The sources of mating type testers of Phytophthora used in this study are listed in Table 2. At the Taiwan Agricultural Research Institute (TARI), rye broth used to prepared rye agar was prepared as described by Caten and Jinks (1968), while at the Asian Vegetable Research and Development Center (AVRDC) and the National Chung Hsing University (NCHU), it was modified by grinding the rye grain preparation in an Omni mixes at 4,000 rpm for 1 min before filtration through three layers of cheese­cloth. The modified rye agar was used for pairing with P. nicotianae testers, and rye agar supplemented with 10% V-8 juice (V-8 rye agar) (Ho and Ko, 1999) was used for pairing with P. capsici or P. infestans.
At AVRDC, 5-mm culture discs of P. infestans and the A1 or A2 tester of P. nicotianae were paired 5 mm apart on the center of a rye agar plate (9 cm), and incubated at 20°C in darkness. After 12 days, the paired culture was mixed with 100 ml water in a blender at high speed for 1 min and oospore concentration was determined with a haemato-cytometer. At TARI and NCHU, a small piece (3 x 3 x 3 mm) of P. infestans culture was paired with the same size culture of a tester on a rye or V-8 rye agar block (20 x 15 x 3 mm). Ten blocks were placed in a 9-cm plate at equal distance from each other. After incubation at 20°C for 21 days at TARI or 16°C for 21 days at NCHU, all the blocks were examined under a microscope for oospore formation. The total number of oospores was counted for the blocks producing less than 500 oospores each. For those forming large amount of oospores, each block was mixed with 100 ml water in an Omni mixer at 4,500 rpm for 1 min, and oospore concentration was determined with a Pipetman microliter pipette (West Coast Scientific Inc., Oakland,
Table 1. Sources of isolates of Phytophthora infestans from tomato in Taiwan used in the reevaluation of mating type.
Isolate No. Location
Reported mating type
Source
Reference
Pi 214 Shoufeng, Hualien
A2
T. C. Wang
Deahl et al., 2008
Pi 566 Hsinyi, Nantou
A2
T. C. Wang
Deahl et al., 2008
Pi 215 Shoufeng, Hualien
A1
T. C. Wang
Deahl et al., 2008
Pi 564 Hsinyi, Nantou
A1
T. C. Wang
Deahl et al., 2008
Table 2. Sources and habitats of mating type testers of Phytophthora used in the study.
Species and isolate
Mating type
Habitat
Origin
Source
P. nicotianae
P 991(ATCC 38607)
A1
Soil
U.S.A.
G. A. Zentmyer
P 731(ATCC 38606)
A2
Soil
U.S.A.
G. A. Zentmyer
P. infestans
P 95039
A1
Potato
Taiwan
P. J. Ann
DN 103
A2
Potato
Japan
A. Ogoshi
P. capsici
P 28120
A1
Tomato
Taiwan
P. J. Ann
P 28117
A2
Tomato
Taiwan
P. J. Ann
ANN et al. 一 Phytophthora infestans in Taiwan
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California) (Ko et al., 1973). Three replicates were used and all the experiments were repeated at least once.
RESULTS
When the four isolates of P. infestans used in the re­port of Deahl et al. (2008) including the two purported A2 isolates (Table 1) were paired with the A1 tester of P. nicotianae at AVRDC, oospores were not produced in any pairing test. However, when paired with the A2 tester of P. nicotianae abundant oospores ranging from 14,200 to 85,600/cm3 were produced by every pair (Table 3). Similar result was obtained at NCHU when P. nicotianae testers were used. Pairing of the same P. infestans isolates. with the A1 tester did not result in oospore formation, but oo-spores ranging from 7,200 to 23,400/cm3 were produced when paired with the A2 tester (Table 4).
When Deahl's A2 isolates of P. infestans were paired with P. capsici testers at NCHU, oospores were not pro­duced in the pairings with the A1 tester, but oospores rang­ing from 13,900 to 42,400/cm3 were produced with the A2 tester (Table 4). Similar result was obtained at TARI where oospores ranging from 6,300 to 22,800/cm3 were produced when paired with the A2 P. capsici tester (Table 5).
Table 5. Oospore formation resulting from pairings at TARI between Phytophthora infestans isolates from Taiwan and A1 or A2 tester of P. capsici. or P. infestans.
P. infestans isolates
Oospores produced (no. ± SD/cm3)a
P. capsici tester
P. infestans tester
A1
A2
A1 A2
Pi 214
0
6,300 ± 1,200
0 38,500 ± 3,200
Pi 566
0
22,800 ± 900
0 40,100 ± 13,700
Pi 215
0
12,900 ± 1,500
0 29,000 ± 3,900
Pi 564
0
19,700 ± 2,900
0 53,400 ± 12,200
aData were subjected to ANOVA. Numbers are the mean ± standard deviation.
The four isolates of P. infestans behaved similarly when paired with P. infestans testers at TARI. No oospores were produced when Deahl's purported A2 isolates were paired with the A1 tester, but oospores ranging from 38,500 to 40,100/cm3 were produced when paired with the A2 P. infestans tester (Table 5).
At TARI, 41 and 40 new isolates of P. infestans were obtained from Nantou and Hualien, respectively. Phytoph-thora infestans testers were used for mating type determi­nation and all new isolates were of the A1 mating type.
DISCUSSION
Data from tests performed in the three laboratories are consistent in showing that all the four isolates of P. infes-tans including the two purported A2 isolates from Deahl et al. (2008) displayed oospore formation only when paired with A2 but not A1 testers. This indicates that all of them are of the A1 mating type. Our results unequivocally refute the claim by Deahl et al. (2008) of the discovery of A2 mating type in Taiwan. This study also shows that the ini­tial designation of Pi 214 and Pi 566 as A1 mating type in Taiwan based on results using A1 and A2 mating types of P. nicotianae as testers was sound. These two isolates be­haved as A1 type regardless of pairing with P. nicotianae (Tables 3 and 4), P. capsici (Tables 4 and 5) or P. infestans (Table 5). This is consistent with the previous reports that in heterothallic Phytophthora oospore production oc­curs when opposite mating types are paired regardless of whether they are the same or different species (Ko, 2007). Our recent collection of P. infestans isolates also show the presence of only the A1 mating type of this species in the Nantou area in 2008 and in the Hualien area in 2009.
The reason for Pi 214 and Pi 566 to behave as the A2 mating type when tested previously is not known. Mating type change in Phytophthora has been reported to oc­cur as a result of aging (Ko, 1981), germination of selfed oospores (Ann and Ko, 1988; Ko, 1994) or exposure to fungicides (Ko, 1981; Chang and Ko, 1990; Groves and Ristaino, 2000). It is not known if any of these events oc­curred. In this study, not unexpectantly, environmental
Table 3. Oospore formation resulting from pairings at AVRDC between Phytophthora infestans isolates from Taiwan and the A1 or A2 tester of P. nicotianae.
Oospores produced (no. ± SD/cm3)a
P. infestans isolates
P. nicotianae tester
A1
A2
Pi 214
0
15,000 ± 3,300
Pi 566
0
14,200 ± 9,100
Pi 215
0
85,600 ± 16,500
Pi 564
0
60,700 ± 6,900
aData were subjected to ANOVA. Numbers are the mean ± standard deviation.
Table 4. Oospore formation resulting from pairings at NCHU between Phytophthora infestans isolates from Taiwan and the A1 or A2 tester of P. nicotianae or P. capsici.
Oospores produced (no. ± SD/cm3 )a
P. infestans isolates
P. nicotianae tester
P. capsici tester
A1
A2
A1 A2
Pi 214
0
8,300 ± 2,200
0 42,400 ± 5,300
Pi 566
0
7,200 ± 3,500
0 37,500 ± 10,400
Pi 215
0
10,200 ± 200
0 13,900 ± 5,600
Pi 564
0
23,400 ± 17,300
0 16,000 ± 5,000
aData were subjected to ANOVA. Numbers are the mean ± standard deviation.
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conditions such as temperature affect oospore production. When Pi 215 was paired with P. nicotianae A2 tester on rye agar plate at 20°C at AVRDC, 85,600 oospores/cm3 were produced (Table 3), but only 10,200 oospores/cm3 were produced when the same two isolates were paired on the same rye agar block at 16°C at NCHU (Table 4). When Pi 215 was paired with P. infestans A2 tester on V-8 rye agar block at 20°C at TARI, 29,000 oospores/cm3 were produced and other P. infestans isolates formed between 38,500 and 53,400 oospore/cm3. This indicates that pair­ing of A1 and A2 mating types between the same species is not necessary better than that between different species. Similar results were obtained when different combination of mating types and species of Phytophthora were paired on the opposite sides of polycarbonate membranes (Ko, 1978; Ko and Kunimoto, 1981). This is because sexual reproduction in Phytophthora is regulated by mating type specific a hormones (Ko, 2007), and different isolates of the same or different species of Phytophthora vary in abil­ity to produce and receive hormones (Ko and Kunimoto,
1981).
In conclusion, research at TARI, AVRDC and NCHU
all indicates that there is no evidence of the presence of the A2 mating type of P. infestans in Taiwan. It is, therefore, recommended that the quarantine regulations are enforced continuously in Taiwan to prevent the introduction of the A2 P. infestans from foreign countries.
Acnowledgments. We thank A. Ogoshi for supplying A2 mating type tester of P. infestans to the senior author. This study was supported in part by grants from National Sci­ence Council and Council of Agriculture of Taiwan.
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Groves, C.T. and J.B. Ristaino. 2000. Commercial fungicide for­mulations induce in vitro oospore formation and phenotypic change in mating type in Phytophthora infestans. Phytopa­thology 90: 1201-1208.
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重新評審在台灣番茄上發現Phytophthora infestans A2
配對型的報告
安寶貞1 蔡志濃1 王添成2 陳堅華2 林玫珠3 柯文雄3
1行政院農委會農業試驗所植物病理組
2亞洲蔬菜研究中心
3國立中興大學植物病理學系
               Deahl等於2008年6月報告說，他們在美國農部，以Phytophthora infestans A1及A2配對型當測試
菌株，第一次在台灣二個地方的番茄上，各測到一株P. infestans的A2配對型。但是該論文的第三及第
四作者，在台灣亞洲蔬菜研究中心，以P. nicotianae的A1及A2配對型當測試菌株時，那二株菌是A1
配對型，他們的試驗結果，在報告裡卻沒有被提及。我們將這兩株菌同另外二株同時在同地點分離到
的，以P. nicotianae, P. capsici及P. infestans之A1同A2配對型當測試株，重新在三個不同試驗室，進
行配對型的測試。這四株菌同A2配對型，不管是那一個種，配對時均會產生卵孢子，但同A1配對型
配對時則不會，顯示這四株菌全屬A1配對型。從那二個地方採番茄病組織回來分離的結果，所得的菌
株也全屬於A1配對型。我們的結果顯示Deahl等的報告說在台灣發現P. infestans A2配對型是錯誤的。
關鍵詞：晚疫病；配對型；Phytophthora infestans ；番茄。