Developments in using fatty acids in fungal chemotaxonomy

The cellular fatty acid composition of nine species of Fusarium; namely, Fusarium anthophilum, F. avenaceum, F. cerealis, F. graminearum, F. graminum, F. oxysporum f. sp. conglutinans, F. pseudograminearum, F. roseum and F. sacchari var. elongatum growing on malt extract medium were determined. The fatty acid profiles of the investigated fungi showed very little variation and could only differentiate between few species. However, by adding certain chemical compounds including aspartic acid, glutamic acid, methionine, selenium and urea to the growth medium, the variation of the fatty acid profile was greatly increased and differentiated between all the investigated fungi. For example, pentadecanoic acid was not produced by F. anthophilum on malt extract broth (MEB) but only produced on MEB supplemented with aspartic acid. On the other hand, linolenic fatty acid was neither produced by F. anthophilum nor F. roseum grown on MEB, but it was produced by F. anthophilum in presence of aspartic acid and by F. roseum in the presence of glutamic acid. The fatty acid profiles could be useful for characterization and identification of fungi if determined under different conditions.


INTRODUCTION
The Fusarium species are widely distributed in soil and on subterranean and aerial plant parts, plant debris, and other organic substrates.They are common in tropical and temperate regions.They are found in desert, alpine, and arctic areas, where harsh climatic conditions prevail (Nelson et al., 1994).Many Fusarium species are abundant in fertile cultivated and rangeland soils but are relatively uncommon in forest soils (Jeschke et al., 1990).Fusarium species are often regarded as soil-borne fungi because of their abundance in soil and their frequent association with plant roots, as either parasites or saprophytes.
The taxonomy of Fusarium spp. is confusing and various classification systems have been proposed (Nelson, 1991).Species identification by morphological traits is problematic because characteristics like mycelial pigmentation, formation, shape and size of conidia are unstable and highly dependent on composition of media and environmental conditions.Phenotypic variation is abundant and expertise is required to distinguish between closely related species and to recognize variation within species (Nelson et al., 1983).Chemotaxonomy seems to mean different things to different people depending on their subject area.To a bacteriologist, chemotaxonomy has meant nucleotide, amino acid, carbohydrate or lipid based taxonomy (Fox et al., 1990), and to a mycologist working with yeasts, it often means a carbohydrate or lipid-based taxonomy (Tosch et al., 2006;Velazques et al., 2006).
Fatty acid (FA) profiles are used increasingly as a chemotaxonomic tool for the identification and classification of bacteria (O 'Donnell, 1994).There are still few publications on the use of this tool for fungal taxonomy, although it has proved to be useful with fungi that can be grown in carefully standardized axenic culture conditions (Stevens andJones 1993, 1994;Koppova et al., 2008;Zain, 2009;Dembitsky et al., 2010), as well as for lichens (Sassaki et al., 2001), arbuscular-mycorrhizal fungi (Madan et al., 2002), mushrooms, basidiomycetes (Ben-Ze'ev et al., 2005), and Oomycetes (Larkin and Groves, 2003).
The objectives of this study was: (i) to analyze the cellular fatty acid compositions of some species of the genus Fusarium, (ii) to evaluate the usage of FA composition to differentiate each species (iii) to determine the susceptibility of the FA composition to the environmental conditions and (iv) its impact on chemotaxonomy of the fungi.

Fungal strains
The fungal strains were obtained from different culture collections; Fusarium oxysporum Schlechtendahl : Fries f. sp.

Media
For growing and maintenance of stock cultures, the malt extract agar (MEA) (malt extract, 20 g; peptone, 1 g; glucose, 20; agar, 20 g; and distilled water, 1 L) was used.To determine the effect of chemical compounds, the fungal strains were grown on malt extract broth (MEB) medium separately supplemented with different chemical compound with the concentration of 0.1% (1 gm/L) (aspartic acid, glutamic acid, methionine, and urea) and 0.01% (0.1 gm/L) for selenium selenite and incubated at 25°C for 10 days.Discs, 9 mm in diameter, of agar media containing the fungal materials were picked up from the margin of actively growing colonies, using sterile cork borer and each disc was transferred into 100 ml liquid medium; in 500 ml conical flasks.The flasks were then incubated at 25°C for 10 days.

Determination of the fatty acids
Fungal mycelium was harvested from broth medium by vacuum filtration.The harvested biomass was rinsed with nano-pure water while still in the funnel and then placed on a lipid-free paper towel for several minutes to remove excess moisture.The lipids were extracted from the dried biomass using chloroform/methanol (2:1 v/v) according to the method described by Folch et al. (1957). 1 g (wet weight) samples of fungal tissue were then placed into 4.0 ml of a saponification reagent and homogenized with a tissue grinder.
The extract of each sample was dried under a stream of nitrogen gas, after which the lipid was dissolved in chloroform and methylated.To methylate the liberated fatty acids, 2.0 ml of 6 N HCl in methanol was added to each tube.Subsamples were placed in water bath at 80°C for 10 min and immediately cooled to room temperature.Prior to fatty acid methyl ester (FAMEs) analyses, the samples were evaporated under nitrogen and resuspended in 50 µl of hexane.The fatty acid methyl esters were analysed at the Regional Center for Mycology and Biotechnology, Al-Azhar University, Cairo, Egypt using a SHIMADZU 5050 type gas chromatograph equipped with mass detector using a 30 x 0.32 mm, 0.53 µm internal diameters, DB1 fused silica capillary column.The carrier gas was helium at a flow rate of 10 ml/min.
The temperature of the injector was 250°C and that of the detector was 280°C.The oven temperature after sample injection (2 µl) was 1 min at 115°C, increasing to 200°C at 7.5°C/min and then raised at a rate of 5°C/min to 240°C held at this temperature for 2 min and then raised at a rate of 3.5°C/min to 260°C and held at this temperature for 2 min.Peaks were identified by reference to authentic standards and verified using mass selective detector.

RESULTS
Nine species of the genus Fusarium were grown on malt extract broth medium and media separately supplemented with chemical compounds, incubated at 25°C for 10 days, and the fatty acid profiles were determined (Tables 1 to 9).
The fatty acid profile of F. anthophilum revealed that there were some fatty acids produced only in the presence of one or more chemical compound in the growth medium, such as capric fatty acid which is produced in the presence of all the investigated chemical compounds used in our study, while tridecanoic was induced in the presence of the glutamic acid and methionine.On the other hand, pentadecanoic was only induced in the presence of the aspartic acid, while linolenic was induced in the presence of aspartic acid, urea and selenium.Caprylic was not produced in the presence of urea, while linoleic was not produced in the presence of glutamic acid (Table 1).
The fatty acid profile of F. avenaceum was affected by the chemical constituents of the growth medium.Eight out of 15 fatty acids were affected by various aspects.Caprylic was not produced in the presence of aspartic acid, glutamic acid, methionine, urea and selenium, while capric was not produced in presence of glutamic acid, methionine, and selenium.Tridecanoic was not produced in the presence of glutamic acid and methionine, whereas palmitoleic was not produced in the presence of urea.Heptadecanoic was not produced in the presence of methionine, while stearic and linolenic were not produced in the presence of aspartic acid, methionine and urea.Seven out of 15 fatty acids produced by F. avenaceum including myristic, pentadecanoic, palmitic, oleic, linoleic, arachidic, and behenic were not affected by the addition of certain chemical compounds such as aspartic acid, glutamic acid, methionine, urea, and selenium (Table 2).The caprylic and capric fatty acids were not produced by F. cerealis growing on malt extract medium (control medium) or in presence of the different chemical compounds used in this study while tridecanoic fatty acid was not produced in presence of only urea (Table 3).Most of the fatty acids produced by F. cerealis including lauric, myristic, pentadecanoic, palmitoleic, palmitic, heptadecanoic, oleic, stearic, linoleic, linolenic, arachidic, and behenic were not affected by the addition of certain chemical compounds such as aspartic acid, glutamic acid, methionine, urea, and selenium (Table 3).The fatty acid profile of F. graminearum showed that the fatty acid was not produced by F. graminearum growing on malt extract medium (control medium), or any of the treatments used in this study (4).Caprylic, capric, pentadecanoic and behenic were not produced in the presence of aspartic acid which is considered the most influential negative impact on F. graminearum, treatment by methionine prevented formation of both caprylic and pentadecanoic.However, only capric fatty acid was not produced in presence of glutamic acid.However, the caprylic and pentadecanoic were not produced in the presence of methionine.Caprylic was not produced in the presence of urea; only four fatty acids acids produced by F. graminearum were affected by the addition of certain chemical compounds (Table 4).On the other hand, fatty acid profile of F. graminum was not affected by the chemical constituents of the growth medium.However, only caprylic was affected; not produced in presence of urea (Table 5).
The fatty acid profile of F. oxysporum f. sp.conglutinans was affected by the addition of certain compounds to growth medium (Table 6).Caprylic and capric were not produced when urea or selenium were added to the growth medium.However, both fatty acids in addition to tridecanoic were not produced in the presence of glutamic acid.The stearic was not produced in the presence of methionine.Most of the fatty acids produced by F. oxysporum f. sp.conglutinans including lauric, myristic, pentadecanoic, palmitoleic, palmitic, heptadecanoic, oleic, linoleic, linolenic, arachidic, and behenic were not affected by the addition of aspartic acid, glutamic acid, methionine, urea, and selenium.On the other hand, the production of caprylic and capric was strongly affected and coined to the chemical constituents of growth medium.However, tridecanoic and stearic were slightly susceptible to the constituents of the growth medium (Table 6).
The fatty acid profile of F. pseudograminearum was affected by the chemical constituents of the growth medium (Table 7).Caprylic, tridecanoic, stearic, in addition to linolenic were not produced in the presence of aspartic acid.Tridecanoic was not produced in the presence of urea, and selenium as well.Linolenic was not produced in presence of aspartic acid and glutamic acid.However, pentadecanoic, heptadecanoic in addition to linolenic fatty acids were not produced in the presence of glutamic acid (Table 7).The fatty acid profile of Fusarium roseum was slightly affected by the chemical constituents of the growth medium (Table 8).However, linolenic was produced only in the presence of glutamic acid, urea, and selenium (Table 8).
Most of the fatty acids produced by Fusariumsacchari var.elongatum including capric, lauric, myristic, palmitoleic, palmitic, heptadecanoic, oleic, stearic, linoleic, arachidic, and behenic were not affected by the addition of certain chemical compounds such as aspartic acid, glutamic acid, methionine, urea, and selenium.On the other hand, the production of tridecanoic was strongly affected and coined to the chemical constituents of growth medium.However, caprylic, pentadecanoic and linolenic were slightly susceptible to the constituents of the growth medium (Table 9).

DISCUSSION
The results of the current study reveal that the fatty acid profiles of the investigated Fusarium species were varied.Most of the Fusarium species, seven out of nine, contained the same fatty acids but differed in the relative amounts of each, however, F. anthophilium and F. roseum differed in both the type and amount of fatty acids produced .The cellular fatty acid composition is now routinely used for the identification and differentiation of microorganisms (Lechevalier and Lechevalier, 1988;Tuner et al., 1992;Graham et al., 1995;Stahl and Klug, 1996;Bentivenga and Morton, 1996;Tighe et al., 2000;Whittaker et al., 2005Whittaker et al., , 2007;;Devi et al., 2006;Koppova et al., 2008;Zain, 2009;Dembitsky et al., 2010).
However, addition of certain chemical compounds to the growth medium affected both the kinds and amounts of the fatty acid composition.It is strongly believed that when employing fatty acid profiles to differentiate or compare fungi, it is important to minimize sources of variation in fatty acid composition from culture conditions (Stahl and Klug, 1996;Zain et al., 2009).The results of the present study confirmed this postulation since the fatty acid profiles changed when certain chemical compounds were added to the growth medium.However, the addition of chemical compounds to the growth medium revealed significant disparities in both the amounts and type of fatty acid composition for all the investigated Fusarium species.
The results of this study strongly demonstrate that the use of fatty acid composition in characterization and iden-tification of fungi could be significantly improved by using more than two different type of growth medium.

Table 1 .
The fatty acid profile of Fusarium anthophilumgrown on MEB medium amended with different chemical compounds.

Table 2 .
The fatty acid profile of Fusarium avenaceum grown on MEB medium amended with different chemical compounds.

Table 3 .
The fatty acid profile of Fusarium cerealis grown on MEB medium amended with different chemical compounds.

Table 4 .
The fatty acid profile of Fusarium graminearum grown on MEB medium amended with different chemical compounds.

Table 5 .
The fatty acid profile of Fusarium graminum grown on MEB medium amended with different chemical compounds.

Table 6 .
The fatty acid profile of Fusarium oxysporumf.sp.conglutinans grown on MEB medium amended with different chemical compounds.

Table 7 .
The fatty acid profile of Fusarium pseudograminearum grown on MEB medium amended with different chemical compounds.

Table 8 .
The fatty acid profile of Fusarium roseum grown on MEB medium amended with different chemical compounds.

Table 9 .
The fatty acid profile of Fusarium sacchari var.elongatum grown on MEB medium amended with different chemical compounds.