Interaction of Plant Epicuticular Waxes and Extracellular Esterases of Curvularia eragrostidis during Infection of Digitaria sanguinalis and Festuca arundinacea by the Fungus

Curvularia eragrostidis, a causal agent of head blight on the weed (Digitariasanguinalis), did not cause disease on the turfgrass Festuca arundinacea. Differentextracellular esterase isoenzymes were detected in saprophytic and parasitic phases duringthe fungal germination. The epicuticular waxes of D. sanguinalis were more efficient toinduce the secretion of esterases from the fungus than that of F. arundinacea, but were morerapidly degraded by the fungal enzymes. Component analysis indicated that the epicuticularwaxes from D. sanguinalis were mostly composed of alcohols, with 54.3% being 9,12-Octadecadien-1-ol. The main component of F arundinacea waxes was alkyl compounds,with 49.8% being olefin, 9-Tricosence. More long-chained esters were found in D.sanguinalis waxes, which were easier to be digested than those in F. arundinacea waxes byextreacellular esterases of the fungus. Epicuticular waxes play a role in varyingpathogenicity of C. eragrostidis on D. sanguinalis and F arundinacea.


Introduction
Curvularia eragrostidis, a causal organism of head blight disease of Digitaria sanguinalis, has potential to be developed into a bio-herbicide for removal of the weed [1].Our previous studies showed that the fungus could cause typical disease symptoms on D. sanguinalis, but was safe to many crops and lawn grasses including F. arundinacea, a turf grass of family Poaceae and widely-growing in China [1,2].C. eragrostidis adheres to and breaches the cuticle of leaf, then penetrate into epidermis and mesophyll cell directly.It is known, during the fungus infection, cuticle is the first barrier, which comprises waxes and cutin.Formation of penetration peg and differentiation of appressorium by fungus on cuticle are associated with secretion of esterases.But the role of esterases in direct cuticle penetration by fungus is not well understood.[3][4][5].
Function of cutin and cutinase in pathogenesis has been reported in many studies [6][7][8].In plants, epicuticular waxes are located on the outermost part of cuticle [9].When microbes adhered to the epicuticular waxes, the waxes showed some important changes [10].It has been shown that wax components acted as allelochemicals by influencing fungal development [11][12][13].Changes in components of waxes depend on spatial, temporal variety and species [14][15][16][17][18].But more evidences are required to elucidate the function of waxes during the plant-pathogen interactions.
Previous reports suggest that the appressorium differentiation of barley powdery mildew fungus was possibly controlled by composition of the plant waxes, which might potentially be an important element determining host or non-host relationship [19].The main purpose of the present study is to investigate the corresponding relationship between the epicuticular waxes of two plant species and extracellular esterase of fungal conidia, and understand the role of plant epicuticular waxes and fungal extracellular esterases in host selectivity and pathogenesis.

Localization of esterase secreted by conidia
Much mucilage secreted by conidia was visualized with the fixation (Figure 1a) and invisible without fixation process (Figure 1b).Only new hyphae, germ tubes and appressoria of germinating conidia were stained black by esterase staining reagent on leaf surface (Figure 1b) and on glass slide, however, old hypha could not be stained (Figure 1c, d).

Conidia adhesion
The count of conidia distributed by copper-grid inoculation showed no difference in the four surfaces of leaves and glass slides after inoculation (data not shown).More conidia adhered to the wax layer of D. sanguinalis than to that of F. arundinacea on leaves and wax-coated slides at both incubation times.However the numbers of adhered conidia incubated for 8 h in all treatments were less, but not significantly, than that for 4 h.The conidia with longer germ tubes are rinsed out more easily (Table 1).

Extracellular esterase isoenzymes from germinating spore
Microscopic observation demonstrated that the wash procedure neither damaged conidia nor reduced their germinating ability.There were obvious different profiles of esterases in the four extraction solutions (Figure 2).More esterase activities were observed in wash liquid of phosphate buffer supplemented with 0.1% Tween-20.Esterase isoenzyme activities were visualized by esterase staining reagent.Three esterase isoenzymes, E2, E3 and E4, were localized on the surface of ungerminated conidia of C. eragrostidis.Each value was the average of conidial number appeared in 20 random fields without overlap.After washing by 5 drops of water from a height of 5 cm.Values with letter in common within a column are not statistically significant (p < 0.05), LSD test.
Different esterase secretion was checked 6 h after conidia germination on glass and the leaf surfaces of D. sanguinalis.E5, an esterase isoenzyme mainly appeared in the intracellular (Figure 2, lane 7), was secreted from conidia germinated on glass slides (Figure 2, lane 5).When the conidia a b d c germinated on D. sanguinalis leaves, E4 and E5 were not secreted, but E1 and E2 were released and showed strong activities.Meanwhile, E6 in lane 6 indicated that the leaves of C. eragrostidis were damaged when inoculated by the conidia for 6 h (Figure 2).

Induction of esterase activity by waxes
Each culture of C. eragrostidis contained an extracellular p-nitrophenyl butyrate (PNB)hydrolysing activity after the conidia of the fungus were incubated for 24 h.And esterase secretion was induced by epicuticular waxes of both plants (Figure 3).In the absence of waxes, the esterase activity was much lower.The waxes of D. sanguinalis showed more efficiency on inducing esterase of the fungus than that of F. arundinacea.In the culture with waxes of D. sanguinalis, esterase activity reached the highest level when the conidia were incubated for 72 h, but showed obvious decrease on the fourth and fifth days.In the culture with waxes of F. arundinacea, esterase activity was less than half of that in culture with waxes of D. sanguinalis on the third and fourth days (Figure 3).As to the two aliphatic acids, 16-hydroxyhexadecanoic acid was more efficient to induce secretion of esterases from C. eragrostidis than palmitic acid.

Degradation of two plants waxes by extracellular esterase of C. eragrostidis conidia
Components of waxes from both plants were mainly aliphatic in nature.The most abundant component found was alcohol (9,12-Octadecadien-1-ol, (Z, Z)-), which contained 54.3% in the epicuticular waxes of D. sanguinalis, and olefin (9-Tricosene, (Z)-), contained 49.8% in that from F. arundinacea.A few esters with phenyl were directly detected in the process and performed nearly invariable in waxes of the two plants.After the treatments by crude esterase solution from extracellular conidia of C. eragrostidis, 9,12-Octadecadien-1-ol, (Z, Z)-was disappeared in waxes of D. sanguinalis, but 9-Tricosene, (Z)-was stable in waxes of F. arundinacea.In waxes of D. sanguinalis, other alcohols such as 1-Dodecanol, 1-Tridecanol et al showed much variety.The stable hydrophobic alkane contained only 9.6% of total waxes.There was a 68% decrease in the amount of waxes of D. sanguinalis after the hydrolysis by crude esterase solution.As for waxes of F. arundinacea, longchain hydrocarbons (alkane) accounted for 67% of the total epicuticular waxes.Alcohols and the esters with phenyl in the waxes of F. arundinacea were kept stable.There was only 3.4% decrease in the amount after the hydrolysis (Table 2).

Discussion
Possible components of epicuticular waxes could be alkane, alcohol, aldehyde, carboxylic acid ester (predominantly acetate) or triterpenoid [15].It was also found that there were such components in the epicuticular waxes of the two plants.But the variety and content were greatly different in the waxes of D. sanguinalis and F. arundinacea according to gas chromatography-mass spectrometry (GC-MS) analysis (Table 2).Previous reports indicated that the rates of esters including straight-chain n-alkyl esters, triterpenoid esters and triacylglycerols in waxes were 7-79.5% [20][21].In this study, only a few phenyl esters in waxes of the two plants were detected.Possibly, many esters had been splited under high temperature condition except phenyl esters.In order to know the original hydrophobic components, the waxes without derivatization were also applied to GC-MS, but there are no any acids detected may due to the difficult gasification.
Adhesion to the surface of host plant is the first step of infection, so the epicuticular waxes played a barrier role in defending potential pathogens infection [22,23].The barrier efficiency of plant waxes may determine the possibility of a microbe to invade plants.Previous report illustrated that the surface hydrophilicity was related to attachment of fungi to leaves [24], the adhesion assay in this project also indicated the epicuticular wax layer of F. arundinacea was much less hydrophilic, so it could be more efficient as a barrier to the conidia of C. eragrostidis than that of the host plant of D. sanguinalis.This barrier efficiency in wax of F. arundinacea may be partly due to main hydrophobic component of 9-Tricosene, (Z)-.
The extracellular esterases of pathogen conidia, including cutinase, were also important factors for the pathogen adhesion to the host plant.These enzymes help germinating conidia get food supply from leaf cuticle and make successful infection at last [4,7,8,25,26,27].But there was no previous reports about the epicuticular esterase from Curvularia spp.. Our study obviously showed that the esterases were formed as constitutive and induced enzymes on surface of the fungal conidia and invasive structures at the early phase of infection.
This study showed that different esterase isoenzymes were secreted when the conidia germinated on surface of glass and host leaves, and the activities of esterase were also different in the culture mediums containing waxes of the two plants or two long-chain fatty acids.These results indicated that the secretion of extracellular esterase of the fungus was affected by the waxes of plant.Actually it was greatly affected by different substrates.16-hydroxyhexadecanoic acid was used as substrate of esterase in previous study [28], which showed more efficiency to induce the secretion of esterases from C. eragrostidis than palmitic acid.So esterase inducers for the fungus should be some specific components.16-hydroxyhexadecanoic acid always presents in the epicuticular waxes of several plants [21,29,30], but it was not detected in the waxes of D. sanguinalis.However, D. sanguinalis waxes were obviously more efficient on esterase induction of the fungus than that of F. arundinacea.The results above suggested that the inducer of esterases could be the other specific components, which might be 9,12-Octadecadien-1-ol, (Z,Z)-or its ester form in the epicuticular waxes of D. sanguinalis.
So the different results would also be appeared when two plant waxes met the esterases of conidia.The esterases degraded a majority of the waxes of D. sanguinalis.Especially 9,12-Octadecadien-1-ol, (Z,Z)-, which might present as its ester form in the waxes, was disappeared after the treatment.But the hydrophobic alkyl (9-Tricosene, (Z)-) in waxes of F. arundinacea could not be hydrolyzed by the esterases.As a result, the hydrophobic components present in F. arundinacea efficiently prevented from both adhesion of conidia and the hydrolysis of esterases, and the waxes layer could form an efficient barrier against the C. eragrostidis's infection.Hoverver, the waxes present in D. sanguinalis could not efficiently prevent from adhesion of conidia, but stimulate the invasion of C. eragrostidis.So different plant epicuticular waxes and extracellular esterases of C. eragrostidis lead to the different results of interaction during infection of D. sanguinalis and F. arundinacea by the fungus.

Conclusion
When conidia of a potential fungal pathogen land on a plant surface, plant waxes may induce the production of extracellular esterases of germinating spores that digest hydrophobic esters in the wax layer [31,32].On host plants, degradation of epicuticular waxes facilitates penetration of cell walls by the pathogen.On non-host plants, however, the waxes are hardly degraded and the efficiency barrier to fungal pathogen.So it's the differences of interaction between epicuticular waxes of two plant and extracellular esterases of fungus, which determined the success infection of C. eragrostidis to D. sanguinalis and the failed infection to F. arundinacea.

Plants and Fungal Material
After sporulation induced by providing 48 h black light and at 25 °C, the conidia of C. eragrostidis were harvested from potato dextrose agar.Pre-chilled autoclaved water (4 °C) was used to avoid germination.The conidia were washed twice and stored at -70 °C.
Seeds of D. sanguinalis and F. arundinacea were sown in pots in greenhouse maintained at 25±2 °C and 14-h Iight/10-h dark photoperiod.All seedlings for the following experiments were harvested at four-leaf stage.

Assay of surface esterase activity
Conidia of C. eragrostidis were diluted to the suspension with about 1000 conidia per 100 µL.200 µL of conidia suspension and 2 ml of 1% agar were mixed at about 40 °C, then the mixtures was laid quickly on a thin glass and spread even film on a glass slide prior to culture at 28 °C in a humid chamber.After 6 h and 12 h of incubation, presence of esterase activity on the surface of conidia and germ tubes was assessed by esterase staining reagent (0.03 g αand β-napthyl acetate, 0.06 g Fast Blue RR were dissolved in 2 ml of acetone, and then mixed with 200 ml of 0.5 M sodium phosphate buffer, pH 7.5).The germinating conidia were treated with esterase staining reagent for 2 h with constant gentle shaking at 28 °C, and then observed with the aid of light microscope [33].

Isolation of epicuticular waxes
Epicuticular waxes of leaves were isolated by dipping intact leaves in chloroform for 30 s with gentle agitation.The solvent was evaporated under reduced pressure in a rotary evaporator as suggested by Podila et al. [12].The residues were waxes stored in dry chamber at -20 °C.

Adhesion assay
Chloroform solutions with 1µg waxes from the intact leaves of D. sanguinalis and F. arundinacea were respectively dried within 1×1 cm 2 area on glass slides surface.After the chloroform evaporated, the glasses with wax layer were kept in a humid chamber together with freshly prepared leaves of D. sanguinalis and F. arundinacea (with adaxial surface upside).Dry conidia were dusted onto the surface of leaves and wax layer on the glass slides through a copper grid of 0.8 mm pore size.Incubated for 4 h and 8 h at 25 °C in darkness, the leaves and glass slides were uniformly washed by 5 drops of water from a height of 5 cm.Immediately after washing, spores remaining on the leaf surface and wax layer on glass slides were counted under light microscope.Four repeats were carried for every treatment, and the conidial number in 5 random fields without overlap in every repeat was recorded [34].

Extraction of extracellular esterases of conidia
Extracellular esterase from 0.5 g of wet conidia was extracted respectively in four 20 ml of extracting solutions [Water, 0.1 M sodium phosphate buffer (pH 7.5), the buffer supplied with Tween-20 (0.1%, w/v), the buffer supplied with SDS (0.2%) and Phenylmethanesulfonyl fluoride (PMSF) (1mM)].After being gently shaken for 8 h at 4 °C in 50 ml sterile centrifugal tubes, conidia were centrifuged (1000 rpm, 2 °C, 10 min), and supernatant was collected to centrifuge once again.The final supernatant was filtered to ensure the absence of conidium, and the crude esterases solution was stored at -70 °C [4].

Esterases from germinating conidia induced by waxes and organic acids
Wet conidia (0.05 g) were suspended in 1.5 ml autoclaved water and divided into two equal portions, then one portion was distributed on surface of intact leaf (washed by autoclaved water) of D. sanguinalis and the other portion was distributed on glass.Incubated for 6 h at 25 in humid chambers, then the extracellular esterase solutions secreted by germinating fungi were collected by 0.05 M sodium phosphate buffer (pH 7.5).The solutions were centrifuged at 10000 rpm for 10 min, and the supernatants were treated with 4-time pre-cold acetone.The deposits were collected by centrifuging at 5000 rpm for 5 min, re-dissolved in 100 µl of 0.1 M sodium phosphate buffer (pH 7.5), the solution was purified by centrifugation at 10000 rpm for 10 min, the supernatants were stored at -70 °C for esterase assay.Intracellular protein from the washed germinating conidia and 0.5 g of the leaf of D. sanguinalis (had been washed out the inoculated conidia) were extracted to use as two controls.
Wax effects on esterase induction from the germinating conidia were also investigated.20 mg of epicuticular waxes from both plants were re-dissolved in 2 ml of acetone, than added to 200 ml of liquid inorganic Czapek medium (0.6 g NaNO 3 , 0.6 g K 2 HPO 4 , 0.2 g MgSO 4 , 0.2 g KCI, 0.01 g FeSO 4 •7H 2 O per litre) in 500 ml conical flacks, respectively.The mediums were inoculated with 1 ml of prepared fungal spore solution, at the cultured conditions of darkness, 25 °C and 190 rpm.The control mediums were supplemented with 2 ml of acetone solution of 16-hydroxyhexadecanoic acid and palmitic acid, respectively.Three repeats were carried out for every treatment.5 ml of conidium suspension (10 6 conidia/ml) was added to every medium and cultured for 5 d.20 ml of culture solution was sampled for enzyme assay every 24 h [35].

Esterase assay
Separation of esterase was carried out by running a gel of 7.5% polyacrylamide with a 4% stacking gel.The esterase activity was assayed by esterase staining reagent.
Esterase activity released from conidia to the medium was determined by the modified spectrophotometrical method of Fett et al. [36] using PNB as the substrate as described previously [37].Reaction mixtures consisted of 980 µl of Tris-HCI buffer (0.1 M, pH 8.0), 10 µl of crude esterase solution and 10 µl of a stock solution (16 µl p-nitrophenyl butyrate resolved in 1ml of acetonitrile).Absorbance change due to the appearance of p-nitrophenyl at room temperature was monitored at 405 nm in 0.5 cm colorimetric cup.The reaction mixtures with heat denatured esterase solution were used as controls in this trial.

Degradation of epicuticular waxes by the extracellular esterase of conidia
Prepared waxes (1mg) from leaves surface of the two plants were resolved in 100 µL of acetone respectively, then mixed with 500 µL of 0.05 M sodium phosphate buffer (pH 7.0) in assay tubes, then 10 µL of extracellular esterase solution extracted from spore surface was added, the tubes with heatdenatured enzyme added were used as control.After incubation at 28 °C for 4 h with gentle shaking, 2 ml of acetone was added to each tube and swirled vigorously, subsequently adequate anhydrous MgSO 4 was also added.Tubes were kept in constant state without any shaking for 5 h at 4°C to allow protein precipitation.After 5 hours, centrifuged at 12000 rpm for 20 min, then the supernatant of acetone solutions were collected and concentrated to 1 ml under vacuum, 1 µL of this solution was applied to gas chromatography with a mass detector (Agilent5973N, capillary column 30 m×0.25 mm×0.25 µm , 70 eV, m/z 50-500) to identify the components and the variety of components among the waxes changed by extracellular esterase.The temperature chromatograph was set with an initial temperature of 100 °C for 1 min, and then increased at 15 °C per min to 300 °C, held for 5 min at 300 °C.Peaks were identified with the aid of the NIST mass spectra library [38,39].

Figure 1 .
Figure 1.Esterases around the surface of germinating conidia a. Conididium germinated on D. sanguinalis for 6 h, fixed with acetic acid and ethanol (1:1), then stained with aniline blue.b.Appressoria from the germination conidia were stained by esterase stain reagent, indicated by the arrowhead.c. and d.After incubation for 4 h (c) and 8 h (d) Conidia buried in 1% agar on glass slide were stained by esterase stain reagent.Old hypha was indicated by the white arrowhead.

Table 1 .
Conidial number of C. eragrostidis adhered to the surface of leaves of D. sanguinalis and F. arundinacea and wax layer from the two plants on glass slides

Table 2 .
The components analysis of epicuticular waxes incubated with crude esterase solution from extracellular conidia of C. eragrostidis