29/05/13 Studies in Mycology Studies in Mycology. No. 21 22 Dec. 1981 Monograph of the genus Pythium J. VAN DER PLAATS-NITERINK Centraalbureau voor Schimmelcultures, Baarn Summary This revision of the species of Pythium Pringsh. is mainly based on living cultures preserved at the Centraalbureau voor Schimmelcultures. Eighty five species are recognized and described in alphabetical sequence; sixty four of these are available as living strains. In addition, two highly specialized cellulolytic species are treated in an appendix. Pythium buismaniae and P. macrosporum are described as new species. These species and some incompletely known ones are keyed out dichotomously. Seven species are heterothallic and produce sexual organs only in combinations of compatible isolates, whilst others that fail to produce oogonia can generally not be identified at species level. Sixty five taxa are listed as excluded, incompletely known or doubtful. The species are illustrated by line drawings, a few light micrographs and scanning electron micrographs of omamented oogonia. For each species a compilation of literature data on occurrence and pathogenicity is given, reference being made to 1133 pubfications. I. Introduction Butler’s (1907), Matthews’s (1931), Sideris’s (1931, 1931) and Frezzi’s (1956) treatments of Pythium dealt with limited numbers of species. Middleton’s work (1943) comprised all the species known at that time. Waterhouse (1967, 1968a) compiled the diagnoses and descriptions of more than 180 species of Pythium and provided a key to 89 recognized species. As several new species were subsequently described, a new monograph was required to cover all present-day knowledge of the genus and to evaluate the available taxonomic criteria. Most of the recognized species are represented by living strains preserved at the Centraalbureau voor Schimmelcultures (CBS). Pythium species are often pathogenic to many plants and to animals such as fishes or Crustacea. They can cause severe losses in cereals and other crops, as well as ornamental plants. For this reason, condensed data about the geographic distribution and the pathogenicity have been included after the taxonomic treatment of each species. The books by Rangaswami (1962) and Tompkins (1975) proved particularly useful as sourcebooks for the compilation work. In the compilation of literature data concerning each species, no critical judgement of correct identification could be made. For the sake of convenience, literature given in ecological-phytopathological context is referred to numerically, in taxonomical context with author and year. [p. 2] II. Material and methods A thorough study was made of the isolates of Pythium preserved in the CBS collection, which had been obtained from other investigators and institutes or isolated by the author. Many isolates were examined in addition to those in the collection. Cultures were kept at 16°C and transferred every 10 weeks on two alternating media, viz. cornmeal agar and potato-carrot agar, as well as being stored on cornmeal agar covered with mineral oil for up to 10 years. The media used for Petri dish cultures were cornmeal, potato-carrot and hempseed agars, and rarely Schmitthenner’s (1962b) agar. These media were prepared in the following way: Cornmeal agar: 60 g freshly ground maize kernels are boiled in 1 l water and allowed to simmer for 1 h. The suspension is then strained through a cloth and filled up with water to 1 1. Potato-carrot agar: 20 g carrots and 20 g potatoes are chopped and boiled for 10 min in 1 1 water; after filtration through a cloth the liquid is filled up with water to 1 l. Hempseed agar: 20 g non-denaturated hempseed is washed in distilled water, boiled for 30 min in 1 l distilled water, and filtered through cotton-wool; the liquid is filled up with distilled water to 1 l. The pH should be between 6 and 7. All media are made up with 15 g agar per litre and autoclaved at 120°C for 20 min. The development of sporangia generally requires water. To induce their formation a small piece of a culture on cornmeal or hempseed agar is placed in a Petri dish in a shallow layer of water, to which a 1-2 cm piece of grass leaf is added. The water used consisted of one part of sterilized pond water and one part of distilled water. The grass leaf was boiled for 10 min. The pond should not be polluted with chemicals or eutrophicated. Changing the water www.cbs.knaw.nl/publications/1021/content_files/content.htm 1/149 29/05/13 Studies in Mycology additionally favours the production of sporangia and discharge of zoospores. After a few hours to a number of days, the Pythium colonizes the grass leaf and develops zoosporangia along its margin (Emerson, 1958; Webster and Dennis, 1967; San and Srivastava, 1968). Oogonia mostly show optimal development on cornmeal agar. The measurements were made on slides in lactophenol-cotton blue. Antheridia often fade away when empty and must be studied in a young stage of development. Maintenance at an appropriate temperature is important, as many species originating from the temperate zones will not produce zoospores above 20°C and sometimes only at about 5°C. On the other hand, tropical species produce zoospores at 20-30°C. In the case of heterothallic species, crossings were made on cornmeal or potato-carrot agar, or in the case of P. intermedium, on a mixture of equal parts of both media. The compatible partners are inoculated at opposite sides of a Petri dish. Oogonia are formed in a dense zone of contact of the mycelia. The shape of these contact lines is characteristic of the species (Fig. 1). This zone is usually sharply delimited towards the male isolate and more diffuse on the female side. Fig. l. - Contact lines between compatible colonies of heterothallic species: a. P. sylvaticum (male strain CBS 452.67 left), b. P. heterothallicum (male strain CBS 450.67 left), c. P. catenulatum (CBS 842.68 x 843.68), d. P. intermedium (CBS 221.68 x 267.38), e. P. macrosporum (CBS 574.80 x 575.80), f. P. splendens (CBS 269.69 x 266.69). Isolates which showed no sexual reproduction are transferred into a Petri dish together with two compatible strains of possibly identical heterothallic species. The reaction on the contact lines of the mycelium with either tester strain gives information about the identity of the isolate. To distinguish oogonial and antheridial strains, transfers of compatible isolates were made to opposite places of a microscope slide covered with a thin layer of a [p. 4] suitable agar medium. These slides were supported on glass rods in a sterile Petri dish containing some sterile water. In certain species the origin of oogonia and antheridia could be traced to the respective strain, in other trials they were complicated or surrounded by mycelium so that male and female strains could not be distinguished, but only an arbitrary designation of + and - strains was made (Campbell and Hendrix, 1967c; Hendrix and Campbell, 1968b, 1969b; Van der Plaats-Niterink, 1968, 1969, 1972). The temperature relationships are characteristic for the various species. In routine examinations cultures were maintained at room temperature (20±2°C). Isolation Most Pythium species are easily isolated by using one of the following methods: 1. Particles of the diseased material or soil or some drops of water are placed in Petri dishes containing 2% water agar (Meredith, 1940). As the Pythium species usually grow more rapidly than other fungi, hyphal tips can be transferred to another medium after 2 or 3 days. 2. The same procedure can be followed using a medium containing an antibiotic substance to suppress the development of bacteria and/or benlate or a polyene antibiotic to suppress the growth of other fungi (see below). This may be especially useful when soil samples are investiged. 3. Small particles of the material are placed in Petri dishes containing water (as described on p. 2) and a few sterilized hemp-seeds. Within a number of days swarming zoospores colonize the hemp-seeds, which are then transferred to a medium containing antibiotics, mostly 415 I U. ml-¹ penicillin and 50 µg ml-¹ streptomycin. 4. One part of a soil sample can be mixed with about 15 parts of sterile sand and moistened. Seeds of corn, hemp or other plants are buried in the soil mixture as baits. After some time they are treated as the hemp-seeds in method 3. 5. Baits consisting of different kinds of fruit (apple, pear, tomato, etc.) can be buried in the soil or in a nylon net placed under water in a ditch, stream, pond etc. After some weeks, depending on the temperature, they are removed and treated as diseased plant material. Generally basic media were used with the addition of antibiotics to suppress the growth of other fungi and bacteria. Basic media include cornmeal, potato-carrot or Schmitthenner’s (1962b) synthetic medium. Pimaricin (50-100 µg ml-¹) or benomyl (2 µg ml-¹ or more) are particularly helpful to suppress the development or other fungi (Singh and Mitchell, 1961; Eckert and Tsao, 1962; Hine and Luna, 1963; Ocana and Tsao, 1966; Vaartaja, 1968; Van der Plaats- www.cbs.knaw.nl/publications/1021/content_files/content.htm 2/149 29/05/13 Studies in Mycology Niterink, 1968, 1975; Tsao and Ocana, 1969; Mircetich and Fogle, 1969; Tsao, 1970; Bollen and Fuchs, 1970; Burr, 1973; Ricci and Messiaen, 1973; Lumsden et al., 1975; Robertson, 1975). A variety of antibiotics have been used in the course of the years to purify the cultures from bacteria. Combinations of different antibiotics often gave good results: penicillin and streptomycin (Klemmer and Nakano, 1962; Van der Plaats- Niterink, 1968, 1975) or rose bengal in combination with streptomycin, pentachloronitrobenzene (PCNB), penicillin, vancomycin, chlortetracyclin, neomycin or agrimycin (Martin, 1950; Singh and Mitchell, 1961; Kerr, 1963; Vaartaja and Bumbieris, 1964b; Takahashi and Ozaki, 1965; Vaartaja, 1967a; Flowers and Hendrix, 1969; Burr, 1973; Mircetich and Kraft, 1973; Lumsden et al., 1975).