Effect of culture conditions on the lipid composition of Phytophthora infestans

Authors


Author for correspondence: John L. Harwood Tel: +44 (0)29 20874108 Fax: +44 (0)29 20874116 Email: harwood@cardiff.ac.uk

Summary

  • • Phytophthora infestans is one of the most important of all plant pathogens and there is a demand for its more effective control. One target area for pesticides is lipid metabolism. To provide background information for studies with pesticides, we examined the lipids of P. infestans and how their composition is altered by culture conditions.
  • • Phytophthora infestans was grown in a pea-broth and three different minimal media. Acyl lipids were extracted, separated by thin-layer chromatography, identified and quantified. Total fatty acid patterns and those in individual lipid classes were measured by gas–liquid chromatography.
  • • Major polar lipids were the phosphoglycerides, phosphatidylcholine > phosphatidylethanolamine > phosphatidylinositol. The proportion of total polar lipids was reduced in minimal media. The similar fatty acid patterns of phosphatidylcholine and phosphatidylethanolamine were consistent with their metabolic relationship. Phytophthora infestans contained significant amounts of 22C unsaturated fatty acids, in addition to common fungal or oomycete fatty acids. Growth on minimal media caused changes in fatty acid patterns with increases in palmitate and linoleate and decreases in oleate being most obvious. Particular changes were also associated with the specific fatty acid patterns of individual lipids.
  • • The changes found in total fatty acids and in the acyl profiles of individual lipids suggest that desaturase activities are altered by growth conditions. These results and the greater proportions of storage triacylglycerol in minimal media agree with data for some other lower plants or fungi.

Introduction

Phytophthora infestans is probably the most notorious of all plant pathogens, being responsible for the Irish potato famine in the mid-nineteenth century (Griffith et al., 1992). Although the host range of P. infestans is limited mainly to potato and tomato, the number of other Phytophthora species stands at more than 50 and some, such as Phytophthora cinnamomi (Erwin & Ribeiro, 1996), parasitize hundreds of plant species. The blight disease caused by P. infestans can affect not only the stem and leaves but also the tuber/fruit or both (Parry, 1990; Erwin & Ribeiro, 1996).

The control of Oomycetes, such as P. infestans, has always been a major aim in chemical plant protection. Indeed, a significant proportion of the world-wide pesticide expense has been devoted to the control of Oomycetes, of which the specific control of P. infestans represents about one-quarter (Schwinn & Staub, 1995). Foliar disease can be controlled by a number of pesticide classes (e.g. dithiocarbamates and thalimides) but these are not very effective for systemic infections and soil-borne disease (Staub & Hubele, 1981; Schwinn, 1983; Cochwinn & Staub, 1995). Moreover, the Oomycetes are often very persistent because of the varieties of structures which can survive various environmental conditions. Furthermore, because the biochemistry of Oomycetes is different from that of true fungi, they are often tolerant of fungicides that can control the latter (Griffith et al., 1992; Erwin & Ribeiro, 1996).

The fatty acids of Oomycetes tend to differ from these of the true fungi. In fact, Oomycetes generally have a greater diversity of fatty acids, including a greater preponderance of 12C and 14C chain lengths, odd-numbered chains and polyunsaturated acids of chains greater than 18C. Typically, they produce γ-linolenic rather than α-linolenic acid, which is found in true fungi (Weete, 1974; Lösel, 1988).

There is not a great deal of information about the phospholipids of fungi apart from yeasts such as Saccharomyces cerevisiae. The phospholipid content of the few filamentous fungi analysed tends to be more variable than for yeasts (Weete, 1980). Moreover, the composition varies considerably with the developmental stage and the environmental conditions. In general, the main phospholipids of filamentous fungi are phosphatidylcholine (PtdCho) and phosphatidylethanolamine (PtdEtn) with smaller amounts of phosphatidylinositol (PtdIns), diphosphatidylglycerol (DiPtdGly) and the methyl derivatives of PtdEtn (Letters, 1966; Weete, 1974; Lösel, 1988).

Sphingolipids are often overlooked in fungal extracts partly because they can be lost during extraction procedures (Lösel, 1988). However, an unusual phosphosphingolipid, ceramide aminoethylphosphonate, has been isolated from P. infestans (Weete, 1980; Creamer & Bostock, 1986). Triacylglycerols (TAGs) are the main nonpolar lipids in most fungi and, in some fungi, may account for the majority of their lipids. However, the TAG content is particularly influenced by developmental and environmental factors (Weete, 1974; Wassef, 1977; Lösel, 1988).

Members of the Phycomycetes, particularly Oomycete species of Saprolegniales and Heptomitales, unlike true fungi, do not produce ergosterol as their main sterol. In addition, members of the Oomycetes, the Pythiaceae, cannot produce sterols and need them exogenously for growth and reproduction (Nes et al., 1979; Warner et al., 1983). Moreover, certain long-chain fatty alcohols, the ‘phytophthorols’, have been detected in Phytophthora species, where they have been suggested to fulfil the role of sterols in membranes in the absence of an exogenous supply of sterols (Nes, 1988).

Because of the continuing agricultural problems caused by P. infestans, there is a demand for more effective control. Thus, efforts are continually being made to develop new types of pesticides. One area to target is that of lipid metabolism and this area has been shown to be a good target for pesticides against other organisms (Harwood, 1991). Indeed, some compounds that show activity against Phytophthora species have been reported to affect lipids (Burden et al., 1988; Reiter et al., 1996) As part of a study to examine the possible mode of action of such pesticides against primary pathways of lipid metabolism (which are essential for normal growth and metabolism), we first made an examination of the lipids of P. infestans. Because environmental conditions are known to be important determinants of lipid composition (Lösel, 1988), we included a consideration of different culture media in our studies.

Materials and Methods

Culture growth

Cultures of P. infestans were maintained on pea broth medium. The latter was made by boiling 300 g of frozen peas in 1 l of distilled water for 10 min, filtering through four layers of muslin and autoclaving. For agar plates, the medium contained an addition of 2% agar (w : v) and growth was maintained at 20°C in a 12-h light/dark cycle. Subculturing was every 4–5 wk. Minimal media were made up as described by Henniger (1959), by Merz et al. (1969) and by Reich (1994) for the CaCl2-free version of Henniger medium.

For liquid cultures of P. infestans, cultures were grown on pea growth agar plates for 14 d. Ten millilitres of sterile medium was used to flood the plate and the surface of the plate was gently scraped to create a sporangial suspension and 1 ml of the latter was then used to inoculate Petri dishes containing appropriate media to a final volume of 10 ml. Incubation continued at 20°C in a 12-h light/dark cycle for 7–14 d, depending on the culture medium used.

Lipid extraction and analysis

A comparison of lipid extraction methods was made initially to determine the most suitable for P. infestans. The methods compared were those of Garbus et al. (1963), a modification by Smith et al. (1982), the method of Kates (1986) which was based on that of Kates & Eberhardt (1957) and the method of Crescenzi (1984). All methods were found to give comparable quantitative and qualitative extraction of lipids and the method of Kates (1986) was used routinely. This method included an immediate treatment of tissue with hot 2-propanol (70°C) for 15 min to inactivate any endogenous lipid-degrading enzymes.

The total lipid extracts were separated into lipid classes by thin-layer chromatography (TLC) on silica gel G60 plates (E. Merck, Darmstadt, Germany). Polar lipids were separated using chloroform–methanol–acetic acid–water (170 : 30 : 20 : 7, by volume) (solvent 1) or using chloroform–methanol–ammonium hydroxide (60 : 25 : 4, by volume) (solvent 2) which allowed phosphatidic acid (PtdOH) to be resolved from other phosphoglycerides. Neutral lipids were separated using petroleum ether (60–80°C b.p.)–diethyl ether–acetic acid (80 : 20 : 1, by volume) (solvent 3). An alternative solvent was used to better resolve diacylglycerol (DAG) from other lipids. This consisted of an initial development using diethyl ether–toluene–ethanol–acetic acid (40 : 50 : 2 : 0.2, by volume) run three-quarters up the plate, followed (after drying under nitrogen) by diethyl ether–hexane (6 : 94, by volume) to the top of the plate (solvent 4).

Lipid bands separated by TLC were routinely visualized by spraying the plates with 8-anilino-1-napthalenesulphonic acid (ANSA) in anhydrous methanol (0.2%, w : v), and viewing under UV light. Routine identification was by comparison with standards (Sigma, Poole, Dorset, UK) but further identification was made by using colour stains and destructive reagents as described in Christie (1982) and Kates (1986).

Fatty acid methyl esters (FAMEs) for gas–liquid chromatography (GLC) were prepared by acid-catalysed methanolysis using 2.5% H2SO4 in anhydrous methanol and heating at 70°C for 120 min The FAMEs were separated in a glass (1.5 m × 3 mm internal diameter) column packed with 10% SP-2330 on 100/120 mesh Supelcoport (Supelco UK, Poole, Dorset, UK) at 180°C. Routine identification was by comparison of retention times with standards (Nu-Chek Prep. Inc., Elysian, MN, USA) but additional confirmation was by AgNO3-TLC (Christie, 1982) or by relative retention times in different GLC columns (Kates, 1986). Quantification of FAMEs and of acyl lipids after FAME production was made using an internal standard of pentadecanoic acid.

Results

Fatty acid content and composition in different media

The total fatty acid contents of P. infestans varied somewhat, depending on the growth media used. Material was harvested during the rapid phase of growth to minimize variations caused by exhausting the medium of growth factors. However, different lengths of time were needed to reach the equivalent amounts of mycelial development and, hence, the same tissue density (20–30 mg dry wt per 10 ml). Average total fatty acid contents were 30.3 mg g−1 dry wt in pea medium, 11.4 mg g−1 dry wt in Merz and 55.4 mg g−1 dry wt in CaCl2-free Henniger. Standard growth conditions were for 7 d in pea medium and 14 d in the minimal media. It has been suggested that sometimes the actual growth time is more important than mycelial development in determining aspects of metabolism. We checked the lipid profiles for growth in pea media at longer times of up to 12 d. Beyond this, growth rates diminished, perhaps as a result of exhaustion of components in the media. For CaCl2-free Henniger media, we examined the lipid profiles at 7 d, 10 d and 14 d. No statistically significant effect was found on the lipid class or fatty acid patterns within these growth times (data not shown). Therefore, we are confident that our decision to choose to examine P. infestans after it developed to the same extent in the various media did not influence the results.

In pea medium, P. infestans had oleate (33%), docosadienoate (17%), palmitate (15%) and myristate (10%) as its main fatty acid components. Docosenoate, linoleate, arachidonate, palmitoleate, eicosenoate and stearate were also found in significant amounts and a host of other acids, including α-linolenate, were found in trace amounts (Table 1).

Table 1.  Effect of growth media on the fatty acid composition of Phytophthora infestans
Fatty acidFatty acid composition (% total)
Pea mediumMerz mediumHennigerHenniger minus CaCl2
  1. Results are means ± SD for three independent experiments. Fatty acids are abbreviated with the figure before the colon indicating the number of carbons and the number after the colon showing the number of double bonds (see text for further details); tr, trace (< 0.05%).

12 : 0 0.6 ± 0.4trtrtr
14 : 0 9.6 ± 0.3 6.1 ± 1.4 5.1 ± 1.6 7.6 ± 0.6
14 : 1tr. 2.1 ± 0.1tr. 2.1 ± 0.2
16 : 014.8 ± 1.517.9 ± 1.219.8 ± 2.521.5 ± 0.2
16 : 1 2.8 ± 0.3 1.8 ± 0.3 3.1 ± 0.4 3.5 ± 0.3
18 : 0 1.6 ± 0.2 6.7 ± 0.3 2.0 ± 0.4 1.8 ± 0.3
18 : 133.2 ± 2.724.2 ± 1.514.4 ± 0.713.3 ± 0.6
18 : 2 6.5 ± 0.412.2 ± 0.917.6 ± 1.013.5 ± 0.9
γ18 : 3/20 : 0 0.6 ± 0.5 2.3 ± 0.3 2.0 ± 0.0 1.9 ± 0.3
20 : 1 2.5 ± 0.4 2.7 ± 0.2 1.9 ± 0.5 1.8 ± 0.6
20 : 2trtrtr 0.9 ± 0.5
20 : 3tr 2.0 ± 0.3 2.2 ± 0.4 1.9 ± 0.6
20 : 4 3.4 ± 0.3 3.4 ± 0.4 2.3 ± 0.310.1 ± 0.4
22 : 1 7.1 ± 1.512.2 ± 1.110.1 ± 3.4 8.4 ± 1.8
22 : 216.8 ± 1.9 6.5 ± 1.018.6 ± 1.711.7 ± 2.0

Growth in Merz medium resulted in a significant difference in the pattern of fatty acids accumulated. Thus, while oleate (24%) was still the main component, palmitate was 18%, docosenoate and linoleate had both increased to 12% while docosadienoate was now 7%. Stearate and myristate were still found in small amounts (6–7%). A number of other acids, including palmitoleate each accounted for 2–3% of the total (Table 1).

Two variations of Henniger medium were tested. For growth in both, palmitate was now the most abundant fatty acid (20–21%). For CaCl2-free Henniger, the dienoic acids, linoleate and docosadienoic acids were the next most abundant, at 18% and 19%, respectively, while oleate was reduced to 14%. The next most abundant acids were docosenoate (10%) myristate (5%) and palmitoleate (3%). Various 20C unsaturated acids, α-linolenate and stearate were also significant but minor components (Table 1).

By contrast, P. infestans grown in the complete Henniger medium contained oleate and linoleate, each at 13%, with docosadienoate (12%), arachidonate (10%) docosenoate (8%) and myristate (8%) as the next most abundant. Palmitoleate was present at 4% but other fatty acids each only made up 2% or less (Table 1). When comparing the two variants of the Henniger media, the most obvious difference was that, in the absence of CaCl2, docosadienoate accumulated at the expense of arachidonate.

Acyl lipids present in P. infestans

We isolated total lipid extracts from Phytophthora infestans and, by using a combination of different TLC separations and identification methods, examined the extracts for individual polar and neutral lipid classes. The major polar lipid component was clearly PtdCho (nearly 40%), with PtdEtn and PtdIns also identified as significant constituents (Table 2). Minor phosphoglycerides also identified were PtdGly, DiPtdGly (cardiolipin) and, interestingly, methyl derivatives of PtdEtn. A significant, unidentified, component was observed which migrated with a low Rf. It did not stain positively with any of the selective sprays (Kates, 1986) used and, although it migrated similarly to lysoPtdCho, was obviously not that compound. Neither did it appear to be ceramide aminoethylphosphonate (Weete, 1980). It represented about 5% of the total polar lipids on a fatty acid basis. Although glycosyldiacylglycerols have been reported as minor lipids in lower plants, we could not detect them in P. infestans under the culture conditions used.

Table 2.  Comparison of the major lipid class composition for Phytophthora infestans grown in pea or CaCl2-free Henniger media
MediumPolar lipids (% total)
PtdOH/DiPtdGlyPtdEtnPtdChoPtdIns/PtdSerUnknown
Pea2.2 ± 0.510.5 ± 1.829.4 ± 3.6 4.0 ± 0.61.3 ± 0.4
Henniger minus CaCl23.1 ± 1.0 9.3 ± 1.010.4 ± 1.8 2.5 ± 0.11.6 ± 0.6
  1. Results are mean ± SD (three independent experiments). Abbreviations: DAG, diacylglycerol; DiPtdGly, diphosphatidylglycerol (cardiolipin); MAG, monoacylglycerol; NEFA, nonesterified fatty acid; PtdCho, phosphatidylcholine; PtdEtn, phosphatidylethanolamine; PtdIns, phosphatidylinositol; PtdOH, phosphatidic acid; PtdSer, phosphatidylserine; PUFA, polyunsaturated fatty acid; TAG, triacylglycerol. For further discussion see text.

 Nonpolar lipids (% total)
MAGDAGNEFATAG
Pea2.0 ± 0.84.5 ± 0.72.9 ± 2.041.1 ± 4.3
Henniger minus CaCl22.6 ± 0.96.9 ± 1.47.1 ± 2.854.7 ± 4.0

The proportion of the total lipids which were represented by polar constituents was very much dependent on the culture medium. In Fig. 1 a comparison is given of the lipid classes of P. infestans grown in pea or CaCl2-deficient Henniger media. Whereas, polar and, hence, membrane lipids represented just under half of the total lipids when P. infestans was grown in pea medium, they were only 27% of the total for growth in CaCl2-deficient Henniger medium (Fig. 1). The decrease in polar lipid proportions was compensated by an increase in nonpolar acyl glycerols (especially DAG and TAG) and in nonesterified fatty acids (NEFA). Bearing in mind that the total lipid content of P. infestans grown in CaCl2-deficient Henniger medium had increased to 55.4 mg g−1 dry wt from 30.3 mg g−1 dry wt in the pea medium, the increased proportion of TAG meant that this storage lipid had approximately doubled in amount.

Figure 1.

The lipid content of Phytophthora infestans is influenced by the culture medium. The proportions of the main nonpolar lipid classes for P. infestans (a) grown in pea medium or (b) grown in CaCl2-deficient Henniger medium. Data are mean ± SD (n = 3). Lipid abbreviations: DAG, diacylglycerol; MAG, monoacylglycerol; NEFA, nonesterified fatty acid; TAG, triacylglycerol.

The fatty acid compositions for the main lipid classes were determined. Table 3a shows the data for polar lipids of P. infestans grown in pea medium. The main phosphoglycerides contained palmitate and oleate as major constituents but, whereas PtdEtn and PtdCho had oleate as their dominant acyl constituent, in PtdIns palmitate was the major fatty acid. As expected from their common pathway of synthesis, the fatty acid compositions of PtdCho and PtdEtn were similar except for some polyunsaturated components. The unknown polar lipid contained a particularly large amount of palmitate and the total neutrals had a significant proportion of myristate (Table 3a).

Table 3.  Fatty acid patterns of major polar lipid classes separated from Phytophthora infestans grown in pea or CaCl2-deficient Henniger media
Fatty acidsFatty acid composition (% total)
UnknownPtdInsPtdChoPtdEtnDiPtdGly1Nonpolars
  • Data as means ± SD (three experiments). Fatty acids are abbreviated with the figure before the colon indicating the number of carbons and the number after the colon showing the number of double bonds (see text for further details). Abbreviations: DAG, diacylglycerol; DiPtdGly, diphosphatidylglycerol (cardiolipin); MAG, monoacylglycerol; NEFA, nonesterified fatty acid; PtdCho, phosphatidylcholine; PtdEtn, phosphatidylethanolamine; PtdIns, phosphatidylinositol; PtdOH, phosphatidic acid; PtdSer, phosphatidylserine; PUFA, polyunsaturated fatty acid; TAG, triacylglycerol; nd, none detected.

  • 1

    Contained PtdOH.

(a) Pea medium
12 : 0 7.3 ± 6.9nd 0.2 ± 0.4 0.4 ± 0.7 1.6 ± 2.7 0.8 ± 0.3
14 : 0 7.5 ± 2.9 4.6 ± 1.6 4.5 ± 1.9 4.9 ± 2.4 8.7 ± 11.417.1 ± 3.3
16 : 055.2 ± 22.437.7 ± 4.217.0 ± 2.419.9 ± 0.915.2 ± 2.616.4 ± 2.6
16 : 1nd 1.1 ± 1.2 2.2 ± 0.4 1.1 ± 0.6nd 4.1 ± 0.1
16 : 2 4.6 ± 4.0 4.4 ± 3.9 0.9 ± 0.9 2.0 ± 1.7 5.2 ± 6.8nd
18 : 0 1.6 ± 2.8 2.9 ± 1.4 2.5 ± 0.6 2.2 ± 1.4 2.1 ± 1.9 2.1 ± 0.3
18 : 1 3.0 ± 5.233.3 ± 3.142.0 ± 4.439.2 ± 6.033.6 ± 2.833.2 ± 4.8
18 : 2nd10.0 ± 3.3 6.5 ± 2.8 6.5 ± 2.6 6.1 ± 5.4 5.7 ± 1.2
20 : 0/γ18 : 3ndnd 0.2 ± 0.3 0.5 ± 0.8nd 0.3 ± 0.3
20 : 1ndnd 2.8 ± 0.6ndnd 2.2 ± 0.9
20 : 2ndnd 0.4 ± 0.7 0.6 ± 1.0ndnd
20 : 3ndndnd 3.9 ± 0.1nd 0.4 ± 0.6
20 : 4 5.2 ± 9.1 3.2 ± 3.6 2.0 ± 2.4 7.3 ± 2.2nd 0.8 ± 0.7
22 : 115.5 ± 14.6nd11.5 ± 2.0 2.6 ± 2.5nd 8.5 ± 1.4
22 : 2ndnd 7.4 ± 2.8 9.0 ± 5.525.5 ± 23.7 8.4 ± 2.0
(b) CaCl2-dependent Henniger medium
12 : 0 1.7 ± 2.9 2.2 ± 3.2 0.1 ± 0.2 0.6 ± 0.6 1.8 ± 2.1 0.6 ± 0.4
14 : 0 2.1 ± 0.7 2.0 ± 0.1 1.4 ± 0.2 2.3 ± 0.5 5.8 ± 7.9 9.5 ± 0.9
16 : 025.4 ± 0.745.9 ± 1.929.8 ± 3.627.9 ± 3.317.2 ± 3.721.2 ± 3.1
16 : 1 2.8 ± 4.8 9.0 ± 10.3 2.2 ± 0.4 2.1 ± 0.3 1.9 ± 1.7 3.8 ± 0.3
16 : 2nd 0.9 ± 1.2 1.2 ± 0.2 0.7 ± 0.5nd 0.6 ± 0.1
18 : 0 2.1 ± 2.1 4.4 ± 2.2 3.5 ± 0.5 1.3 ± 0.8 3.7 ± 2.9 1.5 ± 0.3
18 : 1 2.9 ± 2.7 8.7 ± 1.811.6 ± 1.010.5 ± 1.8 9.9 ± 1.516.9 ± 0.4
18 : 2 3.6 ± 3.414.0 ± 4.215.3 ± 2.920.4 ± 3.313.0 ± 11.317.3 ± 0.1
20 : 0/γ18 : 3ndnd 0.9 ± 1.0 1.8 ± 0.2 1.7 ± 1.6 2.0 ± 0.2
20 : 1ndnd 1.4 ± 0.6 0.4 ± 0.7nd 2.1 ± 0.4
20 : 3ndnd 0.2 ± 0.3 3.4 ± 2.9 6.8 ± 4.3 1.7 ± 0.4
20 : 416.9 ± 15.413.4 ± 11.6 6.6 ± 2.715.9 ± 0.210.6 ± 6.0 1.0 ± 0.9
22 : 142.6 ± 6.1nd20.8 ± 6.5nd 1.4 ± 2.410.2 ± 2.7
22 : 2ndnd 4.7 ± 6.012.7 ± 2.926.2 ± 7.911.5 ± 1.8

As for growth in pea medium, the fatty acid composition of individual polar lipids for P. infestans grown in CaCl2-deficient Henniger medium, showed several distinctive features. As before, the fatty acid compositions of PtdCho and PtdEtn were similar except for very long-chain unsaturated components. Also, PtdIns was enriched in palmitate compared with the other phosphoglycerides (Table 3b). However, there were two major differences between growth in the two media. These were that growth in CaCl2-deficient Henniger medium led to a notable decrease in medium-chain saturated acids (laurate, myristate) and a reciprocal change in the proportions of oleate and linoleate. For example, in the total nonpolars oleate decreased from 33% to 17% while linoleate rose from 6% to 17% in the CaCl2-deficient Henniger medium (Table 3).

The fatty acid composition of TAG (Table 4a) was similar to that of the total nonpolar lipids (Table 3a) for growth in pea medium, reflecting the fact that TAG was the major component of this fraction. All of the nonpolar acyl glycerols contained more saturated and less unsaturated acids than the polar lipid fraction. TAG contained a significant proportion of myristate while MAG and DAG contained increased levels of laurate and stearate. The NEFA fraction contained over half of its acyl chains as saturated acids with stearate notably increased (Table 4a).

Table 4.  Fatty acid patterns of major lipid classes separated from Phytophthora infestans grown in pea or CaCl2-deficient Henniger medium
Fatty acidFatty acid composition (% total)
PolarsMAGDAGNEFATAG
  1. Data are means ± SD (three experiments). Abbreviations: DAG, diacylglycerol; MAG, monoacylglycerol; NEFA, nonesterified fatty acid; TAG, triacylglycerol; nd, none detected.

(a) Pea medium
12 : 0 0.3 ± 0.2 4.7 ± 2.3 3.4 ± 1.6 4.1 ± 3.9 0.2 ± 0.3
14 : 0 3.5 ± 0.9 2.1 ± 1.0 8.5 ± 1.5 4.1 ± 1.319.4 ± 3.1
16 : 015.8 ± 1.027.5 ± 9.221.5 ± 1.734.1 ± 5.715.1 ± 2.2
16 : 1 1.9 ± 0.5 6.1 ± 5.6 2.7 ± 0.4 0.7 ± 1.2 4.4 ± 0.9
16 : 2 0.2 ± 0.2 0.4 ± 0.7 2.5 ± 0.7 2.4 ± 4.2 0.2 ± 0.2
18 : 0 1.3 ± 0.4 4.8 ± 1.9 3.0 ± 1.310.9 ± 7.0 1.4 ± 0.4
18 : 140.6 ± 2.0 6.1 ± 1.431.0 ± 2.222.1 ± 3.533.8 ± 1.9
18 : 2 6.1 ± 1.0 4.0 ± 3.6 7.7 ± 2.0 9.0 ± 1.0 5.8 ± 1.1
20 : 0/γ18 : 3 1.1 ± 0.3ndndndnd
20 : 1 1.6 ± 0.1nd 0.7 ± 1.2 1.3 ± 2.2 2.7 ± 0.3
20 : 3 0.2 ± 0.3 2.7 ± 4.8 3.0 ± 3.2 1.8 ± 3.1nd
20 : 4 2.2 ± 0.411.7 ± 3.4 6.4 ± 0.6 5.0 ± 4.5 0.6 ± 0.5
22 : 1 8.1 ± 0.929.9 ± 5.5 4.5 ± 3.9 4.5 ± 5.2 7.0 ± 2.6
22 : 217.3 ± 0.7nd 5.3 ± 4.6nd 8.8 ± 0.9
(b) CaCl2-dependent Henniger medium
12 : 0 0.3 ± 0.5 2.5 ± 3.7nd 0.4 ± 0.6 0.4 ± 0.7
14 : 0 1.3 ± 0.7 3.6 ± 1.1 6.9 ± 1.2 4.5 ± 2.2 7.7 ± 2.8
14 : 1 0.9 ± 1.6 8.8 ± 9.8 0.8 ± 1.4 2.2 ± 3.0nd
16 : 025.4 ± 0.919.6 ± 2.222.8 ± 6.333.7 ± 7.517.3 ± 3.7
16 : 1 1.8 ± 0.7 4.8 ± 0.9 4.2 ± 0.9 3.0 ± 0.8 3.7 ± 0.3
16 : 2 0.6 ± 0.3 3.3 ± 0.6 2.0 ± 1.7 1.0 ± 0.9 0.6 ± 0.1
18 : 0 2.2 ± 0.4 5.9 ± 2.5 3.2 ± 0.7 5.5 ± 0.3 1.4 ± 0.3
18 : 110.5 ± 0.5 7.4 ± 1.516.2 ± 0.113.3 ± 1.117.7 ± 0.7
18 : 219.5 ± 0.8 5.0 ± 0.716.4 ± 1.318.8 ± 2.918.2 ± 0.6
20 : 0/γ18 : 3 2.2 ± 0.1nd 1.1 ± 0.2 0.9 ± 0.8 2.3 ± 0.2
20 : 1 1.8 ± 0.2 3.7 ± 1.0 2.9 ± 0.9 2.0 ± 2.0 2.1 ± 0.6
20 : 2nd 1.0 ± 1.6 0.4 ± 0.8 0.4 ± 0.6nd
20 : 3 2.4 ± 0.4 2.2 ± 2.0 2.0 ± 0.4 1.5 ± 1.4 2.1 ± 0.4
204 2.7 ± 0.4 6.0 ± 4.0 1.8 ± 1.6 2.5 ± 0.4 1.0 ± 0.9
22 : 110.8 ± 2.425.9 ± 7.0 8.3 ± 2.1 6.2 ± 3.3 8.3 ± 2.8
22 : 217.8 ± 0.4nd11.0 ± 0.9 4.3 ± 2.417.3 ± 2.8

The changes for growth in the two media which were reflected in the total nonpolars lipids (Table 3) can also be seen clearly in the individual nonpolar lipid classes (Table 4). Thus, the nonpolar acyl glycerols (MAG, DAG, NEFA) after growth in ClCl2-deficient Henniger medium contained much less laurate and myristate had decreased from 19% to 8% in TAG. In most nonpolar lipid classes, linoleate was now the most abundant unsaturated fatty acid (Table 4b) compared with when P. infestans was grown in pea medium (Table 4a).

Discussion

The lipid content for P. infestans reported here is of the same order as that published for Phytophthora capsici (Souliéet al. 1995) and for P. infestans and other Phytophthora species by Lösel (1988). Moreover, variations in total lipid content due to different growth conditions have been observed for similar organisms and reported for a related pythiaceous organism, Pythium ultimum (Bowman & Mumma, 1967).

The total fatty acid compositions of P. infestans grown in various media agreed generally with these reported for the same organism or for other Phytophthora or Pythium species (Bowman & Mumma, 1967; Tyrrell, 1967; Brushaber et al., 1972; Lösel, 1988; Kerwin & Duddles, 1989; Gandhi & Weete, 1991). However, some investigators detected eicosapentaenoate instead of docosenoate or docosadienoate (Brushaber et al., 1972; Kerwin & Duddles, 1989; Gandhi & Weete, 1991) whereas Bowman & Mumma (1967) found docosenoate and docasadienoate but no eicosapentaenoate. These variations are most likely caused by species and strain differences although different growth conditions may have played a part.

Phytophthora infestans contained the same fatty acids in each of the four growth media. However, their proportions were different. Cultures grown in the pea medium contained the highest proportion of oleate. In Merz medium there was less oleate and more linoleate and more docosenoate and less dodecadienoate. We did not analyse the double bond positions of the very long-chain fatty acids so cannot determine whether the conversion of both monoenoates into dienoate points to different fatty acid desaturases responding to growth in the Merz medium. Transfer to the Henniger media gave an increased conversion of oleate to linoleate as well as greater elongation to very long-chain acids. Taken together, these results add to the already substantial evidence (Creamer & Bostock, 1986; Gandhi & Weete, 1991) that lipid composition is very much influenced by environment. The presence of polyunsaturated fatty acids (PUFAs) with a range of chain lengths and unsaturation is thought to be a feature that allows lower plants such as the Oomycetes to tolerate a variety of cold aquatic and soil environments (Lösel, 1988).

One other factor that could have influenced fatty acid composition and lipid quality could have been the developmental stage of mycelial growth. We tried to guard against this by only examining growing cultures and ones that had developed their mycelia to the same extent. Nevertheless, because P. infestans grew faster in pea medium than in the minimal media, the cultures were of different ages and, although none had yet reached a stationary phase, it was always possible that the variation in growth period might cause changes in lipid composition. However, as discussed in the Results, examination of the lipid composition at other time-points during the rapid phase of mycelial development in pea and in CaCl2-deficient Henniger media did not reveal any changes due to growth period.

Although the overall patterns of polar lipids in P. infestans grown in pea or CaCl2-deficient Henniger media were similar, growth in the latter produced a higher proportion of nonpolar lipids (Fig. 1). Taken together with the higher lipid content of the latter this meant that the TAG accumulated to about double the amount (on a dry weight basis) compared with growth in pea medium. This greater accumulation of storage lipid in CaCl2-deficient Henniger medium is likely to reflect the poorer growth conditions in the minimal medium (Weete, 1980).

Overall, the distribution of polar lipids reflected well the profile reported by Brushaber et al. (1972) except for the higher proportion of PtdSer observed in their investigation. The proportions of phosphoglycerides (with PtdCho > PtdEtn > PtdIns as the major components) is in good agreement with other Phytophthora species (Bowman & Mumma, 1967; Souliéet al., 1995). We took great care to prevent lipid degradation occurring during extraction and analysis (see the Materials and Methods) and such phenomena may explain the variable amounts of PtdOH reported in some earlier studies (discussed by Lösel, 1988). We found very little of this key metabolic intermediate, which was as expected if the fungi were in a healthy condition. The small amounts of NEFA also confirms that unwanted degradation had not occurred during analysis (Fig. 1).

The fatty acid profiles of different lipid classes showed subtle variations. The generally similar compositions for PtdCho and PtdEtn is in keeping with the fact that they both derive from the same DAG pool during synthesis by the CDP-base pathway (Gurr et al., 2002). Moreover, the presence of significant quantities of methylated derivatives of PtdEtn implied that conversion of PtdEtn into PtdCho by successive methylation was also important in P. infestans, and we showed this to be true by radiolabelling studies (Griffiths et al., 2002). Such a metabolic relationship would also contribute to the similar fatty acyl patterns of the two phosphoglycerides. The increased percentage of saturated (especially palmitate) fatty acids in PtdIns compared with other phosphoglycerides is a well-known observation in many organisms (Harwood, 1980).

Although there have been relatively few studies of the fatty acid profiles of individual lipids in other lower plants such as true fungi, Souliēet al. (1995) reported data for TAG, PtdCho and PtdEtn in P. capsici which showed broadly similar characteristics to our results. In true fungi, polar lipids tend to have a greater proportion of longer-chain polyunsaturated fatty acids while neutral lipids tend to possess more shorter-chain saturated fatty acids (see Lösel, 1988), as found in our study (Tables 3 and 4).

The changes found in total fatty acids and in the acyl profiles of individual lipid classes, point to alterations in relative activity of fatty acid desaturases caused by growth conditions. These results and the greater accumulation of storage TAG in minimal media agree with data for fungi (see Weete, 1980; Lösel, 1988). The fatty acid synthase (FAS) of S. cerevisiae is a Type I enzyme (Gurr et al., 2002) where the chain length of product is controlled by the relative efficiencies of transfer of the growing acyl chain to the FAS-condensing enzyme or to the acyl-ACP: CoA transacylase. Moreover, Wakil et al. (1983) have shown that growth conditions can cause significant changes in the chain length of yeast FAS products. Indeed, if P. infestans contains a similar FAS, then it is possible that growth-induced changes in the relative proportion of laurate and myristate products (Tables 1, 3 and 4) are due to an alteration in the transacylase activity. Clearly, with increased knowledge of the molecular biology of both FAS and fatty acid desaturases (Harwood, 1996) a detailed study of the mechanisms underlying the fatty acid changes induced by growth conditions is a feasible topic for future research.

Acknowledgements

We are grateful to the BBSRC for a CASE studentship to support R.G.G.

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