S.AfrJ.Bot., 1994, 60(5): 257 - 260 257

Phytophthora capsici (Oomycota: Fungi), a first report from South Africa

A.H. Thompson: W.J . Bothat and M.D.R. Uys Agricultural Research Council, Vegetable and Ornamental Plant Institute, Private Bag X293, Pretoria, 0001, Republic of South Africa

tPlant Protection Research Institute, Private Bag X134, Pretoria, 0001, Republic of South Africa

Received 28 February 1994; revised 28 June 1994

Phytophthora capsici Leonian is reported for the first time from South Africa, causing a root rot of paprika (Capsicum annuum L.). A morphological description of the fungus is given, supplemented by restriction enzyme digest analysis to distinguish it from the closely related species P. nicotianae. Pathogenicity to paprika was confirmed.

Phytophthora capsici Leonian word vir die eerste keer in Suid-Afrika aangemeld as 'n wortel-vrot van paprika (Capsicum annuum L.). Die swam word morfologies beskryf met behulp van die analise van beperkingsensiem­vertering om dit te onderskei van die verwante spesie P. nicotianae. Patogenisiteit op paprika is bevestig.

Keywords: Morphology, pathogenicity, paprika, Phytophthora capsici, RFLP .

• To whom correspondence should be addressed.

Introduction Phytophthora capsici Leonian is a worldwide pathogen of pepper (Capsicum spp.), tomato, eggplant, macadamia, lima bean and various members of the Cucurbitaceae (watermelon, squash, pumpkin, cucumber and honeydew melon), causing root, stem and fruit rots (Ribeiro 1978; Stamps 1985).

A species of Phytophthora was consistently isolated from diseased roots of paprika (Capsicum annuum L. cv. Papri Queen) from a producer near Brits, Transvaal, who reported severe losses as a result of root rot. To select for pythiaceous fungi, isolations were made on com meal agar plates amended with 340 ~g ml-1 ampicillin, 20 ~g ml-1 rifampicin, 20 ~g ml-l pimaricin and 200 ~g mrl PCNB (modified from Kann­wischer & Mitchell 1978).

Representative single zoospore isolates were identified as P. capsici using the keys and descriptions of Ho (1981), Krober (1985), Stamps (1985) and Stamps et al. (1990). Two isolates were deposited at the National Collection of Fungi, Plant Protection Research Institute (PPRI), Pretoria, and one of these (PPRI 4845) was sent to the Centraalbureau voor Schimmel­cultures (CBS), Baarn, Netherlands, for confirmation of identi­fication.

This is the first report of P. capsici from South Africa.

Description

Phytophthora capsici Leonian in Phytopathology 13: 401 - 408 (1922). (Figures 1 - 6)

= P. hydrophilaCurzi Riv. Patol. veg. Padov 17: 1 - 19 (1927)

Colonies on V -8 juice agar uniform in appearance, felty to downy; optimal and maximum growth temperatures were 30 and 36 - 37°C, respectively. Hyphal swellings sparsely formed on solid media, abundantly in water; usually single, terminal or intercalary, but sometimes forming a small 'net'; central vacu­ole occasionally present; shape varying from globose, ovoid, angular to irregular; 28.0 - (37.6) - 48.0 ~m in diameter. Chlamydospores not formed. Sporangiophores unbranched or irregularly branched. Sporangia clearly papillate, apical thickening deep (average 3.6 ~m), exit pore narrow (average 4.9 ~m); usually one apex, sometimes two; base usually distinctly tapered, but often rounded; basal plug and septum usually distinct; position terminal, occasionally intercalary; attachment to sporangiophore basal, sometimes slightly lateral;

caducous in water only, with pedicel length very variable, 2.0 - (38.2) - 132.0 ~m; no internal proliferation; central vacuole sometimes present; shape very variable, usually oblong/ellip­soid (often elongated), ovoid or obovoid, occasionally ovoid/ ellipsoid or ovoid/navicular, in water tending to be ovoid to subglobose; distorted shapes present in water; 34.0 - (58.2) -76.0 X 20.0 - (25.4) - 34.0 ~m; average L:B ratio 1.5:1; spor­angia produced abundantly in light on solid media (formed peripherally on dishes) and in water; optimum temperature for production 25°C. Sex organs of both isolates were heterothallic with no sex organs produced either in culture or in host root tissues; intraspecific mating of our isolates with a confirmed isolate of P. capsici (ATCC 66768) on polycarbonate membranes (Ko 1978) produced no sex organs.

For assessing production of sex organs, sporangia, hyphal swellings and chlamydospores on solid media and in water, isolates were incubated at 15 - 30°C for 2 - 3 weeks in the dark. Sporangia used for descriptions were from Petri dish cultures incubated at room temperature (±25°C) in daylight. All dimensions were based on 50 measurements.

Specimens examined

-389 South Africa; Transvaal: Rooikoppies area, near Brits, dis­eased roots, Thompson 389, Feb. 1992, CBS 254.93, PPRI 4845. -390 As above, Thompson 390, PPRI 4846.

Two isolates from the same locality were examined, as both showed considerable variation in culture morphology. Our identification was confirmed by the Centraalbureau voor SchimmeIcultures, Baam, Netherlands (CBS 254.93).

In the Transvaal Province, P. nicotianae van Breda de Haan is a widespread pathogen on citrus, tobacco and ornamentals (Gorter 1977; Thompson & Naude 1992). Although unreported from pepper in South Africa, it was reported as a fruit rot of Capsicum from Zimbabwe (Griffith 1968). Borzini (1956) reported P. nicotianae (= P. parasitica) as the cause of a foot rot of pepper from Italy.

P. capsici and P. nicotianae are morphologically very similar, and can be easily confused. Only sporangia produced in light show the basic difference between the two species, viz. the tapered base (P. capsici) versus rounded base (P. nicotianae). However, in water culture, P. capsici can show a predominantly rounded base, while P. nicotianae can also

258 S.-Afr.Tydskr.Plantk., 1994, 60(5)

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Figures 1 - 6 Phytophlhora capsici. 1. Elongated oblong/ellipsoid sporangia typically produced in light. Papillate (arrow) with short pedicels (p). 2. Ovoid papillate sporangium with tapered base (arrow) and long pedicel (p) . 3. Ovoid papillate sporangium with tapered base and short pedicel (p). 4. Ovoid papillate sporangium with rounded base (arrow) and short pedicel. S. Sporangium with two apices (arrows) . 6. Single hyphal swellings. Scale bar: 30 IJ.-m.

show a high percentage of caducity, making species separation difficult. To further confirm that our isolates were P. capsici and not P. nicotianae, molecular methods were used to compare them with confirmed isolates of P. capsici (ATCC 66768) and P. nicotianae (IMI 189725). ATCC 66768 was isolated from Capsicum sp. (hot pepper, M.D. Coffey, Bulgaria), while IMI 189725 was isolated from Citrus sp. (P.H. Tsao and W. Kanjaipai, Thailand).

For DNA restriction enzyme analysis, five mycelial plugs from the margin of young cultures were inocula~ed into flasks of GYP medium (Gill & Zentmyer 1978) containing 200 ml per flask. Liquid cultures were incubated at 25°C in the dark for 16 h in the stationary flasks which were then shaken for 10 - 14 days at 100 r/rnin under the same conditions. Mycelium was collected by vacuum filtration, rinsed with sterile distilled

water, then frozen at -170°C and lyophilized. The method of Panabieres et al. (1989) was used to extract and purify total DNA. Mini-preparation DNA (2 - 4 ILg) was digested over­night with Hind III, Kpn I, Sal I, Xba I, Pst I, Bel I, Apa I, Msp I, Hae III and Eco RI following the manufacturer's recommen­dations (Boehringer Mannheim). Restricted DNA was sepa­rated using 0.7% agarose gels in 50% TBE buffer (90 mM Tris borate; pH 8.0) and electrophoresed at 60 V for 4 h. Gels were stained with ethidium bromide and photographed under UV light.

Restriction fragment length polymorphism (RFLP) patterns of isolates PPRI 4845 and PPRI 4846 were identical to those of P. capsici (ATCC 66768), confirming the identification. All three isolates were distinctly different from that of P. nicotia­nae (IMI 189725) (Figure 7). Hae III and Msp I showed poly-

S.AfrJ.Bot.,1994,60(5) 259

Hind III Sal I Pst I

Figure 7 Restriction fragment patterns of total DNA from Phytophthora capsici and P. nicotianae restricted with the enzymes Hind Ill, Sal I and Pst I. Lane I, P. nicotianae (IMI 189725); lane 2, P. capsici (PPRI 4845); lane 3, P. capsici (PPRI 4846); lane 4, P. capsici (ATCC 66768); M, molecular marker (Boehringer Mannheim marker 3); lambda DNA digested with Eco RI and Hind Ill; range size 21.2,5.1,4.9,4.2,3.5,2.0, 1.9, 1.5, 1.3, 0.9, 0.8 and 0.5 kb.

morphisms in the mitochondrial DNA of the P. capsici isolates and could not be used to confirm identification.

Proof of pathogenicity Pathogenicity was tested on Papri King and Papri Queen, the major paprika cultivars grown in South Africa. Seeds were sown in trays containing a steam-pasteurized vermiculite/ peatrnoss/sand mixture. Seedlings were then transplanted after 6 weeks into pots containing a steam-pasteurized sand/soil mix, and inoculated after 15 weeks using the methods described by Thompson and Phillips (1988). There were 12 inoculated and 12 uninoculated control plants for each variety. Pots were watered with 500 ml of distilled water twice daily for 2 days, followed by once daily for 3 weeks. Plants were then harvested and examined for root rot.

Inoculated plants of both paprika cultivars tested were severely wilted by P. capsici (Figure 8) while controls remained healthy. Extensive necrosis of the roots and a basal stem rot were present. The rot started at soil level, extending down into the roots and up into the stem. The taproot, secon­dary and feeder roots were brown, often with the tips still healthy (white). The stem-base necrosis extended 3 - 6 cm above the soil line, with sloughing off of bark, and with a light brown internal discolouration of tissues. P. capsici was re­isolated from all diseased roots.

The identity of our isolates was clearly P. capsici and not P. nicotianae, although some morphological variability existed. The obovoid shape of sporangia, their minimum length of <45 jJ.m (34 jJ.m), occasional intercalary position and conspicuous basal plug, were atypical of the description given by Stamps et at. (1990). Average lengthibreadth ratio was less than that reported by Ho (1981) and Krober (1985). Although Stamps et at. (1990) reported no hypha I swellings on agar, our isolates formed hyphal swellings sparsely on agar. Ho (1981) reported swellings to be under 25 jJ.m in diameter, while our isolates had very large swellings (up to 48 jJ.m). However, sporangial shape and size, and production of hyphal swellings, are known to be variable within the genus (Waterhouse et al. 1983; Ho & Jong 1987).

Figure 8 Paprika plants showing wilted plant (1) inoculated with P. capsici, and healthy control plant (C).

RFLP patterns of total DNA can be used to distinguish species of Phytophthora (Panabieres et at. 1989; Blaha 1990). Although unsuitable for determining interspecific similarities, they can supplement morphological studies. In our study, RFLP patterns supported our morphological identification. It is probably coincidental that restriction patterns of the P. capsici isolates used in this study were identical. Forster et al. (1990) reported that some isolates of P. capsici had identical RFLP patterns, but most isolates showed a high degree of mitochon­drial and nuclear DNA diversity. Hwang et at. (1991) also distinguished four homogeneous groups within this species,

260

with isolates from each of four groups having identical restric­tion patterns.

Acknowledgements We thank: the Agricultural Research Council for funding this research.

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