Allelopathic potential of macrofungi on germinating maize (Zea mays L.) grain

The effect of methanol extracts of 10 macrofungi was evaluated on grain germination of maize (Zea mays L.). Germination percentage, radicle and plumule length and the level of carbohydrates and fatty acids were measured. Fungal metabolites inhibited germination up to 90.96%, plumule (97.77%) and radicle (92.83%) development. Plumule and radicle lengths were stimulated 35.26 and 10.60% in some assays, respectively. The fungal metabolites decreased the glucose (97.60%), sucrose (90.34%), fructose (96.85%), maltose (95.64%), oleic acid (97.50%) and linoleic acid (98.25%) levels, whereas increasing levels of the glucose (165.14%), sucrose (166.53%), fructose (83.18%), maltose (124.73%), oleic acid (6975.00%) and linoleic acid (5233.33%) were detected in some assays. It is concluded that macrofungi metabolites have commonly inhibitory effects on physiological and morphological processes of germinating maize grain except for considerable increases in the some parameters investigated.


Abbreviations:
HPLC, High performance liquid chromatography; MF, macrofungi; MFE, macrofungi extracts; LA, linoleic acid; OA, oleic acid; ANOVA, analysis of variance.barnyardgrass and stimulated rice shoot height (Mo et al., 2005).The activity of metabolites from macrofungi may be also used together to synthetic biocides.The germination and development of weeds were suppressed by bioherbicidal and soil-born fungi at different inoculum concentrations alone and in combination with glyphosate (Molish, 1937;Boyette et al., 2008).
Allelopathy is a useful tool in sustainable agriculture (Rizvi et al., 1999).Most allelopathy research is focused on weed suppression (Narwal, 1994;Rice, 1995;Kastanias and Chrysayi-Tokousbalides, 2000;Kadio lu and Yanar, 2004).Nevertheless, physiological effects and action mechanisms of allelopathic chemicals are sometimes not well understood.The aim of this work was to evaluate the effects of macrofungi extracts on physiology of grain germination and seedling growth using maize as a model system.
Although maize plant does not share the same living place with macrofungi tested, it was used as a model plant to understand the effects of alleochemicals in macrofungi on physiological and developmental processes in plants.Some allelochemicals in macrofungi tested may be useful as alternative to synthetic compounds used for producing more crops.

Preparation of the methanol extracts
Twenty grams of fruiting bodies of dried macrofungi were powdered in liquid nitrogen.Each powder was shaken with 200 ml of methanol for 24 h at room temperature in the dark.Then, the extract was filtered through Whatman filter paper (No.1) and methanol was removed under vacuum at 40°C using a rotary evaporator.The residues obtained were stored at -80°C until use.

Bioassay of macrofungi extracts on maize grain
Grains of Zea mays with uniform size were immersed in 2.5% (w/v) NaOCl for 3 min.After sterilization, grains were washed with sterile distilled water and dried with sterile filter paper.Grains were soaked in sterile distilled water for 1 h and 15 grains were germinated in Petri dishes on four layers of sterile Whatman No.1 filter paper.Concentration of macrofungi extracts was adjusted to 3.3, 5.0 and 10.0% (w/v) with deionized water.Then, 20 ml of each extract were added to each Petri dish containing 15 grains and allowed to germinate at 25°C in dark for 7 days.Seedlings were harvested with root lengths of 1.5 -3.0 cm.Plumule and radicle length were measured and germination percentage was calculated and then they were maintained until analysis at -80°C.The macrofungi extracts were tested in triplicate for each parameter.Sterile distilled water was added to a Petri dish containing 15 grains as control.For biochemical and physiological analysis the germinated grains were used.

Determination of fatty acids
Quantitative determination of oleic and linoleic acids was performed according to the methods of Christie (1997) and Hamrouni et al. (2001).Two grams of sample were ground to powder in liquid nitrogen and homogenized in 20 ml of a chloroform : methanol mixture (2:1).Then, the mixture was diluted 1/5 by adding 4 ml of double distilled water.The mixture was separated in two phases.The upper layer was removed and the lower layer containing the fatty acids was evaporated under vacuum.The residue was dissolved by methanol and injected into HPLC equipped with a µBondapak C18 column (Waters, Hicrom Ltd.UK); Waters 6000 A pump (Waters, Hicrom Ltd.UK); Refractive Index detector (Waters Uzun et al. 1017Uzun et al. 2414)).Acetonitrile: water (78:22) was used as mobile phase at a flow rate of 1.5 ml/min.The absorption wavelength was chosen at 412 nm.The retention times for linoleic and oleic acid standards were 6.25 and 9.04 min, respectively.

Determination of free sugars
Free sugars were analyzed by the methods of Torije et al. (1998) and Karkacier et al. (2003).Five grams of sample were powdered in liquid nitrogen and 40 ml of methanol was added.The mixture was incubated on a magnetic stirrer at 65°C for 30 min.Then, it was centrifuged at 4°C, 1300 rpm for 40 min.The supernatant was transferred in a clean tube and made up to 50 ml with methanol.
Then, methanol was removed in a rotary evaporator and the residue was dissolved in 25 ml of double distilled water.The extract was passed through Sep-Pak C18 cartridge and 2.5 ml of filtrate was mixed with 7.5 ml acetonitrile.Then, it was filtrated with 0.45 µm membrane filter and injected into HPLC equipped with Spherisorb 5 µm NH2 column (250 × 4.6 mm; Waters, Ireland); Waters 6000 A pump (Waters, Hicrom Ltd.Uk); Refractive Index detector (Waters 2414).Acetonitrile : water (80:20) was used as the mobile phase at a 1.5 ml/min flow rate.Eluted compounds were detected at 412 nm.The retention times were determined for fructose (5.12 min), glucose (5.65 min), sucrose (7.87 min) and maltose (9.25 min).

Statistical analysis
All data were expressed as mean ± standard deviation (SD).The statistical analyses were made using the Minitab 13 for windows packet program.Means and Standard deviations were calculated according to the standard methods for all parameters.One way ANOVA statistical test was used to determine the differences between means of the treatments and the control group accepting the significance level at p < 0.05.The statistically significant changes were indicated on the figures and table by asterisks.

Effect of macrofungi extracts on grain germination
Several macrofungi extracts significantly reduced percentage of grain germination.The highest inhibition of grain germination was observed for extracts of V. gloiocephala and P. aurivella, M. vulgaris and M. procera var.procera at concentration of 5% and S. luteus at 10% (Figure 1).

Effect of macrofungi extracts on seedling growth
The plumule growth of germinating maize was inhibited by some extracts and stimulated by others.Plumule growth was stimulated by extracts of Pleurotus ostreatus at 3.3 and 10%, A. muscaria var.muscaria at 10%, P. squamosus and Volvaria gloicephala at 5% and L. leucothites at 3.3% (Figure 2).The most suppressive effect on plumule length was determined for V. gloiocephala and S. luteus at 10% and M. vulgaris at 5%.Macrofungi extracts inhibited radicle length of maize except for 10% of A. muscaria var.muscaria and 3.3% of L. leucothites where radicle length was higher than that of the control.The most suppressive effect of macrofungi extract on radicle development was determined in the applications of all P. ostreatus, 10% of V. gloiocephala and S. luteus and 5% of M. vulgaris extracts (Figure 3).

Physiological parameters
The decreased glucose levels were also increased with changing dilution amount of macrofungi extracts (MFE).

M . v u l g a r i s P . o s t r e a t u s L . l e u c o t h i t e s C . a t r a m e n t a r i a P . a u r i v e l l a A . m u s c a r i a P . s q u a m o s u s V . g l o i o c e p h a l a M . p r o c e r a S . l u t e u s c o n t r o l
Macrofungi Glucose levels (mg/g FW) .All P. ostreatus and P. aurivella applications and 5% of S. luteus and C. atramentaria, 3.3% of P. squamosus, A. muscaria var.muscaria and 10 and 3.3% of L. leucothites extracts increased glucose levels whereas, all M. procera.var.procera and M. vulgaris extracts and 10% of C. atramentaria, 5% of V. gloiocephala, 10 and 3.3% of S. luteus, 5% of L. leucothites, A. muscaria var.muscaria, 10 and 5% of P. squamosus extracts decreased glucose levels (Figure 4).The lowest glucose level of germinating maize grains was determined in the applications of M. procera var.procera extract.
The 10% of M. vulgaris, P. ostreatus, L. leucothides, P. aurivella and V. gloiocephala and 3.3% of V. gloicephala, P. squamosus and A. muscaria var.muscaria and 5% of P. aurivella extracts increased the sucrose level compared to that of the control.The highest sucrose levels were seen in the application of 3.3% of A. muscaria var.muscaria and P. squamosus extracts.M. procera var.procera, S. luteus extracts decreased sucrose levels.The lowest sucrose levels were determined in 5% of M. vulgaris, 5 and 3.3% of P. ostreatus, 3.3% of C. atramentaria, 10 and 5% of A. muscaria var.muscaria extract applications (Figure 5).
In general, fructose level was seriously inhibited by macrofungi extracts, whereas 10% of P. squamosus and 5% of P. aurivella and M. procera var.procera extracts increased fructose level compared to that of the control.The lowest fructose levels were determined in the application of 10% of M. procera var.procera, P. aurivella and C. atramentaria, 5 and 3.3% of P. squamosus, C. atramentaria, M. vulgaris and S. luteus extracts.The most inhibited results on fructose concentration were observed in L. leucothites, P. ostreatus and V. gloiocephala extract applications where any fructose amount was not detected by HPLC (Figure 6).
Maltose concentrations decreased in changing levels with different dilution of macrofungi extract applications except for all P. squamosus, 5% of A. muscaria var.muscaria and 3.3% of P. aurivella, L. leucothites extract where maltose levels were found to be higher than that of the control.The significantly lowest maltose levels were observed in the applications of M. vulgaris and P. ostreatus extracts, 10 and 3.3% of S. luteus, V. gloiocephala, 10 and 5% of L. leucothites, C. atramentaria extracts (Figure 7).
The oleic acid (OA) levels in general were decreased by MFE applications.All M. vulgaris, A. muscaria, M. procera var.procera, S. luteus and 10 and 3.3% of P. squamosus, 5 and 3.3% of P. aurivella, V. gloiocephala extract applications did not reveal any OA level in germinating maize grains.The OA levels were found to be higher in all P. ostreatus, C. atramentaria and 5% of P. squamosus extract applications than that of control (Table 1).

DISCUSSION
The extracts from macrofungi (MF) species assayed showed a significant inhibitory effect on grain germination and plumule and radicle growth.This inhibition may be due to the alteration of enzyme activity, which affects the mobilization of storage compounds during germination (Einhellig, 1995).The data are in accordance with Mo et al. (2005) findings indicating that extract of L. hatsudake inhibited the root development of plants.The most important effect of allelopathy was reported to be on seed germination (Rizvi et al., 1999).The increasing dilution rate of extracts is expected to cause decrease in the inhibition level of maize grain germination.The results presented (Figure 1) are in contrast to the previous findings in the literature suggesting that higher concentration of allelopatic chemicals may cause higher level of phytotoxicity in plant germination (Kadio lu and Yanar, 2004).Fungi are well recognized for their ability to produce diverse biologically active metabolites (Saxena and Pandey, 2001).Some fungal metabolites are known to have allelopathic effects on cell division, germination and specific enzyme function (Magan, 2001).The provided data showed that the effects of MFE on germination depend on the MF species and concentration of the extract.Plant grains metabolize starch, proteins and fatty acids to provide energy and synthesize the enzyme as well as The higher level of the compounds might be due to the result of metabolic pathway suppression which does not allow metabolizing synthesized compounds; or the allelochemicals in macrofungi extract may stimulate the synthesis of the compounds of which levels were found to be higher in tested grains than that of the control.Besides, the higher carbohydrate level may be attributed to cell wall metabolizing enzymes which degrade oligosaccharide to release the small sugars (Grant Reid, 1997).The most stimulative effect of MF extract was observed in LA synthesis whereas the OA levels were tremendously inhibited by MFE.It was reported that the changing level of fatty acids affects the turnover of membrane lipid composition, the fluidity, elasticity and mobility of bilayer as plant answer to stress (Svenningsson et al., 1990;Surjus and Durand, 1996).Unfortunately we do not have the chance to compare our results with the previous studies, because of the limited reports on the allelopathic effects of macrofungi on plants.
In general, it might be stated that the macrofungi extract inhibited the germination and physiological processes of maize grain.However, some stimulating effects on these parameters were also determined.It could be concluded that although some of the macrofungi allelochemicals have stimulating effects, others are inhibitive.The allelochemicals are needed to be characterized and tested on plants in order to determine inhibitive and stimulative substances which might be used as plant growth regulators, herbicide or insecticide an alternative to synthetic compounds.
When germination, plumule and radicle length and sugar and fatty acids concentrations are considered together, the effects of MFE showed similar pattern on the parameters.The MFE may be tested on economically important plant seeds using this model system and has great potential to be used for controlling dormancy, senescence, cell division and physiological processes in organic and sustainable agricultural systems where synthetic herbicides are not allowed.

Figure 1 .Figure 2 .
Figure 1.The effect of macrofungi extracts on germination of maize plant.

Figure 3 .
Figure 3.The effect of macrofungi extracts on radicle length of maize plant.

Figure 4 .
Figure 4.The effect of macrofungi extracts on glucose levels in maize plant.

Figure 5 .
Figure 5.The effect of macrofungi extracts on sucrose levels in maize plant.

Figure 6 .Figure 7 .
Figure 6.The effect of macrofungi extracts on fructose levels in maize plant.

Table 1 .
The effect of macrofungi extracts on linoleic and oleic acid levels in maize plant.