Tulip Gums Elicit Bikaverin Production in the Isolates of Fusarium oxysporum Pathogenic for Tulip Bulbs

1. Introduction

Induction and formation of gums (gummosis) are found through the plant kingdom. Generally gums are induced by biotic and abiotic environmental factors such as bacterial and fungal infection, insect attack, mechanical and chemical injury, water stress, and other environmental stressors [1]. All these factors have been shown to promote also ethylene synthesis in plant tissues. It was reported that ethylene or ethylene-releasing compounds (i.e. ethephon; 2-chloroethylphosphonic acid) stimulate gum formation in stone-fruit trees and their fruits of the Rosaceae, apricot, cherry, peach, plum, almond [1,2], but also in tulip (Tulipa sp.) bulbs and other bulbous ornamental plants [3].

Tulip bulbs infected by Fusarium oxysporum f. sp. tulipae have been shown to produce considerable quantities of ethylene, causing gummosis in diseased and healthy bulbs stored in the same conditions (Figure 1) [3,4,5]. These results suggest that ethylene is a common factor involved in the induction of gummosis [1].

Figure 1. Gummosis in tulip bulbs cv. Apeldoorn induced by Fusarium oxysporum f. sp. tulipe (on left) and by ethephon (2-chloroethylphosphonic acid) – ethylene releasing compounds (on right).

Figure 1. Gummosis in tulip bulbs cv. Apeldoorn induced by Fusarium oxysporum f. sp. tulipe (on left) and by ethephon (2-chloroethylphosphonic acid) – ethylene releasing compounds (on right).

On the other hand it has been shown that jasmonates have a promoting effect on the induction and/or production of gums in tulip bulbs and in stone-fruit trees and their fruits of Rosaceae family, such as cherries, plums, peaches, apricots [6].

This was reported that a rapid increase in endogenous levels of jasmonates, mainly Jasmonic acid (JA), occurs in plants or their organs under stress conditions, e.g. after mechanical wounding, under osmotic stress conditions and after pathogens infection or insect attack [7]. These facts strongly suggest that jasmonates are important key compounds involved in induction and/or production of gums in plants together with or without endogenous ethylene. Jasmonates have also been well known to control ethylene production in plant tissues [7].

Gums are complexes of different substances, but most important constituents are polysaccharides of highly individual structure [1]. The composition of tulip gum polysaccharides has been determined and shown to be consisted of glucuronoarabinoxylan with an average molecular weight of ca 700 kDa [8]. It is well known that different kinds of polysaccharides function in plants as molecular signals (elicitors) that regulate growth, development and survival in the environment, through elicitation of various physiological and biochemical processes [9,10,11,12,13,14,15,16,17]. It is possible that a polysaccharides of tulip gums act as an elicitor, which regulates some processes connected with or responsible for mycelial growth and other physiological processes of Fusarium oxysporum f. sp. tulipae. Saniewska [18] studied the effect of gums induced by Fusarium oxysporum f. sp. tulipae in tulip bulbs on the mycelium growth and development of the pathogen in vitro.

It was previously demonstrated that incubation of Fusarium oxysporum f. sp. tulipae spores in the water suspension of tulip gums stimulated spore germination and greatly induced the appearance of red-colored hyphae and medium. It seems that the polysaccharide of tulip gums is the main agent responsible for the elicitation of the secondary metabolite(s) by the hyphae of the pathogen [19]. In the present study the red colored pigment occurring in Fusarium oxysporum f. sp. tulipae elicited by tulip gums was identified.

2. Materials and Methods

The spores of isolates of Fusarium oxysporum f. sp. tulipae were produced from 10 day old cultures grown on PDA medium. After that period the mycelium was washed with 20 cm³ of sterilized water. Then the mycelium was smoothed by glass stirrer for liberation of spores and after 30 minutes spores were separated from mycelium using filter paper. The spore density in 1 cm³ of filtered suspension estimated under a microscope using a Büker camera was 1.5 x 10⁶. Tulip gums, were dissolved in distilled and sterilized water. To a water solution of gum with a volume of 19 cm³, 1 cm³ of F. oxysporum f. sp. tulipae spores was added. The density of the pathogen was then checked again and determined to be 1.2 x 10⁶. The total concentration of tulip gums was 1.0% (w/w). As a control distilled water with F. oxysporum f. sp. tulipae spores was used. The solution was kept at room temperature for 15 days with occasional shaking, The mycelium was sampled after 7 and 15 days.

The lyophilized colored mycelium was extracted with 70% MeOH,. The extract was evaporated in vacuo, dissolved in MeOH and used for analysis. Thermo Finningan ICQ Advantage MAX mass spectrometer was used for quantitation and identification of compounds. An LC system consisting of Finnigan Surveyor pump equipped with a gradient controller, an automatic sample injector and Photodiode Array Detector (PDA) was used. The separation was performed on a 250 x 4 mm i.d., 5 μm Eurospher 100 C18 column (Knauer, Germany). A mobile phase consisted of 0.05% acetic acid in water (B) and 0.05% acetic acid in acetonitrile (A) was used for the separation. The flow-rate was kept constant at 0,5 mL/min for a total run time of 60 min. The system was run with a gradient program: 18% A to 36% A in 55 min, 36% A to 100% A in 20 min. The sample injection volume was 25 μL. Quantitation was based on external standardization by employing calibration curves for commercial standard in the range 0.0362 – 6.94 μg/mL. Standard was prepared in methanol prior to analysis. Structural analyses were performed with Thermo Finnigan LCQ Adventage Max ion-trap mass spectrometer with an electrospray ion source coupled to the HPLC system described above. The spray voltage was set to 4.2 kV and a capillary offset voltage –60 V. All spectra were acquired at a capillary temperature of 220 °C. The calibration of the mass range (400–2000 Da) was performed in negative ion mode. Nitrogen was used as sheath gas and the flow rate was 0.9 L/min. The structure of compounds was confirmed on the basis of pseudomolecular ion formation.

3. Results

Incubation of Fusarium oxysporum f. sp. tulipae in water solution of tulip gums resulted in the appearance or red color product in mycelium. Lyophilized mycelium extract was analyzed with liquid chromatography, which revealed two dominant peaks (1: R_t = 44.02 min. and 2: R_t = 47.15 min.) in the chromatogram (Figure 2). Based on the peak area these two peaks made over 90% of total. Their chemical structures were confirmed by comparing UV and MS data with those previously reported.

Figure 2. The UV chromatogram of the extract from mycelium of Fusarium oxysporum f. sp. tulipae grown in tulip gum solution.

Figure 2. The UV chromatogram of the extract from mycelium of Fusarium oxysporum f. sp. tulipae grown in tulip gum solution.

The UV spectrum taken from PDA detector of dominant compound 2 exhibited λ_max at 201.8, 227.6, 251.2, 274.9, 336sh and 507,8 nm (Figure 2), and m/z value of 381 in negative mode [M-H]^-. These values were similar to those obtained previously for bikaverin (6,11-dihydroxy-3-methoxy-1-methylbenzo[b]xantene-7,10,12-trione) the naphthoquinone pigment found in the wild Fusarium oxysporum LCP531 strain [20].

Compound 1 exhibited λ_max at 201.5, 227.5, 251.4, 275.2, 336sh and 508.2 nm and m/z value of 367 in negative mode [M-H]^-. These values were quite similar to compound 2 and were identical to those obtained previously for norbikaverin (6,10,11-trihydroxy-3-methoxy-1-methylbenzo[b]xantene-7,8,12-trione] [20].

The other minor peaks, the peak area of which made up less than 10% of total peak area were based on absorption spectra estimated as bikaverin related compounds, but their structures were not confirmed in this study. Presumably these were yellow naphthoquinone pigments (absorption maxima at 441 nm) reported previously [20].

Both bikaverin and norbikaverin content in the mycelium of Fusarium oxysporum f. sp. tulipe were determined after 7 and 15 days of incubation (Table 1). After 7 days of incubation intracellular concentration of bikaverin 2.94 mg/100 mg mycelial dry matter and was nearly 4.5 higher than that of norbicaverin. Incubation of the solution for the next 8 days resulted in nearly doubled concentration of both naphthoquinones.

Table 1. Bikaverin and norbikaverin content (mg/100 mg dry matter) in Fusarium oxysporum f. sp. tulipe mycelium grown with tulip gum solution.

Sample	Norbikaverin (Rt=44.02)	Bikaverin (Rt=47.15)	Total
Mycelium after 7 days
	0.64 ± 0.06	2.94 ± 0.26	3.58 ± 0.32
Mycelium after 15 days	1.02 ± 0.11	4.62 ± 0.55	5.64 ± 0.62

Table 1. Bikaverin and norbikaverin content (mg/100 mg dry matter) in Fusarium oxysporum f. sp. tulipe mycelium grown with tulip gum solution.

Sample	Norbikaverin (Rt=44.02)	Bikaverin (Rt=47.15)	Total
Mycelium after 7 days
	0.64 ± 0.06	2.94 ± 0.26	3.58 ± 0.32
Mycelium after 15 days	1.02 ± 0.11	4.62 ± 0.55	5.64 ± 0.62

4. Discussion

Fungal secondary metabolites display diverse biological activities. They may act as phytotoxins causing damages to plants, have important roles in other interactions with other plants or may exhibit antimicrobial activities and be involved in competition for survival with other microbes. Bikaverin, the reddish pigment, is produced by different fungal species, especially those belonging to the genus Fusarium. This compound shows antibiotic activity against certain fungi and protozoa [21,22], nematicidal activity [23] and anticancer activity [24,25,26], but its biological function in Fusarium remains unknown [26,27].

It was reported that the synthesis of bikaverin is regulated by the culture conditions, such as carbon and nitrogen sources, calcium content, pH and many other factors [28,29,30]. For example, in Fusarium fujikuroi fungus the synthesis of bikaverin was induced by nitrogen starvation and acidic pH, and it was favored by aeration, and sulfate and phosphate starvation [26].

Species of genus Fusarium produced also other red coloured naphthoquinone metabolites [20,31,32].

Poly- or oligosaccharides are the most well studied signal molecules in elicitors signal transduction. Many elicitors, such as chitin, chitosan, xyloglucans, β-glucans and their fragments, oligogalacturonides, laminarin, exhibit elicitor activity across different plant species and highly induce phytoalexins in plants, suggesting that different plants possess some common receptors to sense these signals [33]. One oligosaccharide elicitor can be recognized by several plants through their receptors with similar binding properties [34]. Dass and Ramawat [35] showed that gum arabic obtained from Acacia senegal (arabinogalactan-proteins) and mesquite gum obtained from Prosopis cineraria (arabinogalactan-proteins) evidently increased guggulsterone production in cell cultures of Commiphora weightii. Water-extracted mycelial polysaccharide prepared from the endophytic fungus Fusarium oxysporum Dzf17 isolated from the rhizomes of Discorea zingiberensis evidently increased diosgenin accumulation in cell suspension culture of D. zingiberensis [36]. Chitosan, pectin and alginate enhanced production of anthocyanin in Vitis vinifera cell suspension cultures [37]. In Cocos nucifera cell culture treatment with chitosan enhanced production of different secondary metabolites, e.g., p-hydroxbenzoic acid, p-coumaric acid and ferulic acid [38]. Chitosan/chitin and pectin were effective in inducing of anthraquinone synthesis in cultured cells of Morinda citrifolia [39]. Gum arabic significantly promoted accumulation of phenolic acids, particularly 3-0-glucosyl-resveratrol, in Vitis vinifera cultures, as well in the culture medium [37]. Letarte et al. [40] showed that arabinogalactan-protein from gum arabic increased microspore viability and induced embryogenesis in the microspore culture of wheat (Triticum aestivum), and finally obtained regenerated green plants. Arabinogalactan-protein from cashew-nut (Anacardium occidentale) gum greatly stimulated somatic embryogenesis in carrot culture and enhanced of the conversation rate of somatic embryos into plantlets [41].

The induction of red colour pigment in mycelium of Fusarium oxysporum f. sp. tulipae by tulip gums was previously reported, but its chemical structure was not confirmed [42]. In present study the red color pigment was identified to be a mixture of bikaverin and norbicaverin. Their concentration in mycelium was quite high, which may suggest that this process can be applied in pigment production for industrial purposes. However, it is interesting to mention that tulip gums induced pigment production exclusively by Fusarium oxysporum pathogenic for tulip bulbs. These gums did not elicit the red pigment formation through mycelium of F. oxysporum f. sp. callistephi, F. oxysporum f. sp. dianthi and F. oxysporum f. sp. narcissi [42]. These pathogens are not pathogenic for tulip bulbs and tulip gums did not stimulate linear mycelium growth of these pathogens on CzDA medium [43].