Abstract: An attempt was performed to achieve four granular formulations from two isolates of Fusarium oxysporum (Foxy I and Foxy II) for biocontrol of Orobanche ramosa and Orobanche crenata in tomato and faba bean; two formulations from microconidia (PM I and PM II) and two from chlamydospores (PC I and PC II). Doses of all formulations (0.5, 0.75, 1.0 and 1.25 g kg-1 soil) differentially reduced number of emerged shoots, shoot height and shoot dry weight of both Orobanche species with a remarkable increase in disease incidence on the parasitic plant, the highest dose giving the most severe symptoms. On the other hand, Orobanche-infected tomato and faba bean plants showed significant reductions in shoot height and root length as well as in shoot and root dry weights concomitant with significant increases in H2O2 content and superoxide dismutase (SOD) activity and significant decreases in ascorbic acid (AsA) content and activities of catalase (CAT), guaiacol peroxidase (GPX) and glutathione-S-transferase (GST). However, application of the highest dose of Pesta fungi overcame, to a great extent, the changes in host growthand oxidative stress parameters so that levels were similar to those of normally grown plants, the preparation PC II had the most pronounced effect. These findings showed that considerable control of the two species of Orobanche could be obtained by the formulations of the biocontrol agents, particularly at the highest dose (1.25 g kg-1 soil).
INTRODUCTION
Parasitic angiosperms are a taxonomically diverse group of plants that invade host plant tissues and remove resources via a specialized structure known as the haustorium. Through the haustorium, carbon, water and mineral nutrients are withdrawn, often at the expense of host growth and vigour (Watling and Press, 2001). The host selectivity of these plants is mediated by chemical signals, including germination stimulants and haustorial inducing factors (Shen et al., 2006). Parasitic weeds of the genus Orobanche represents a serious threat to a wide range of economically important crops. Orobanche crenata parasitizes major legume crops while Orobanche ramosa, which is closely related to aegyptiaca, attacks mainly Solanaceae (Rubiales et al., 2004). Due to infection with biotic agents such as Orobanche, oxidative stress could be arisen in host plants with a consequent production of Reactive Oxygen Species (ROS). Moreover, during O2 reduction, cells continuously produce ROS. They have been implicated in damaging cells. To protect cellular membranes against the harmful ROS levels, plants developed defense antioxidants (Aravind and Prasad, 2005). Antioxidants are crucial for plant defense against oxidative stress (Gomez et al., 2004). However, ROS accumulation is crucial to plant development as well as defense (Pavet et al., 2005). Because of the intimate host-parasite relationship and the anatomical-physiological connections, Orobanche is a particularly difficult target for selective chemical control (Kleifeld et al., 1998). Parasitic weeds are one of the major intractable regional problems and require biotechnological solutions (Gressel et al., 2004). Biological control using fungal pathogens could provide a possible solution because of the high specificity of the fungal pathogens used as biocontrol agents (Boari and Vurro, 2004; Elzein et al., 2006). Indigenous, weed-specific fungal pathogens can be developed and used as safe and effective bioherbicides (Charudattan, 2001). This study was an attempt to formulate granular Pesta from Fusarium oxysporum and to evaluate their role in both Orobanche biocontrol and mitigation of Orobanche-induced growth reduction and oxidative stress of host plants, tomato and faba bean.
MATERIALS AND METHODS
Plant Materials and Growth Conditions
This study was conducted in Botany Department, Faculty of Science at Damietta,
Mansoura University, Egypt during the year 2006. Surface-disinfected seeds of
tomato (Supper strain B) and faba bean (Giza 3) were used. Root exudate was
produced by sowing ten seeds in plastic pots filled with 5 kg of sand. After
14 days, the root system was washed, grouped together and completely immersed
in 200 mL water in a 250 mL glass flask. The flasks were then wrapped in black
polyethylene. Plants were incubated for three days while water content of the
flasks was held constant. Afterwards, plants were removed and the liquid content
of the flasks was stored at -20°C until use.
Seeds of O. aegyptiaca/ramosa and O. crenata were
germinated in vitro in order to determine the seed amounts needed to
achieve a concentration of approximately 10,000 germinating seeds per pot (2
kg of soil). For the in vitro germination, surface-sterilized Orobanche
seeds were placed on small glass fiber filter paper discs in petri dishes containing
one layer of witted filter paper, sealed with parafilm and incubated for seven
days at 20°C in the dark. The small discs were then transferred to other
petri dishes with exudate. After ten days, the percentage of germinated seeds
was determined using a binocular microscope.
For pot experiments, Orobanche seeds (60 mg to give 10000 seeds) were
sprinkled onto the soil surface of plastic pots (25 cm diameter x 20 cm height)
filled up to 2/3 of their height with 1:1 (v:v) sand:clay soil. Inoculum was
added and mixed into the soil together with the seeds. The pots were then filled
with soil and host plant seeds were sown. Fourteen days after sowing, plants
were thinned out to three per pot. The pots were irrigated as required.
Granular Formulation of Mycoherbicides
About 300 infected Orobanche plants were collected from faba bean
and tomato fields. Shoots were carefully collected and stored in a refrigerator.
Small pieces of infected shoots were surface-disinfected, rinsed with sterile
water, blotted dry and placed on Potato Dextrose Agar (PDA) plates supplemented
with 200 ppm chloramphenicol and 100 ppm streptomycin sulfate. The plates were
incubated at room temperature until fungal mycelium grew out of the plant pieces.
The mycelium was transferred to fresh PDA plates to obtain pure cultures. One
hundred and 69 fungal isolates obtained from infected shoots from infested fields
were tested for their pathogenicity to Orobanche. Only two fungi isolated
from diseased Orobanche shoots, classified as F. oxysporum isolate
I (Foxy I) and isolate II (Foxy II), inhibited Orobanche germination.
Microconidia and chlamydospores of both isolates were used to achieve prepare
granular formulations for biocontrol of Orobanche. The most suitable
formulations of Fusarial species for biocontrol of Orobanche are those
that contain microconidia and chlamydospores as these remain viable in the soil
for long periods and can re-germinate. Foxy isolates were grown on Potato Dextrose
Broth (PDB) for six days on a reciprocating shaker at 125 strokes per min (spm)
at room temperature to produce microconidia or for 20 days at 100 spm to produce
chlamydospores. The contents of culture flasks were blended and the number of
spores was determined using a haemocytometer. The resulting suspensions were
centrifuged at 4,000 x g for 10 min and adjusted to 3.5x108 and 5x108
microconidia mL-1 for Foxy I and Foxy II, respectively and
to 2.5x107 and 2.7x107 chlamydospores mL-1.
The two types of spores were used as active ingredients for the formulations along with three adjuvants (sucrose, yeast extract and glycerol). Dough was prepared by blending 38 g semolina, 4 g kaolin, 6 g yeast extract, 2 g sucrose, 20 mL spore suspension and 2 mL glycerol. The dough was then rolled through a pasta machine, folded and extruded several times. A 1 mm thick sheets were produced, air-dried at room conditions, ground and sieved. Four formulations resulted from both spore types of both isolates. Microconidia of Foxy I and Foxy II gave rise to PM I and PM II, chlamydospores gave rise to PC I and PC II.
Evaluation of Mycoherbicides Formulations
Plastic pots were filled up to 2/3 the height with sand/clay (1/1, v/v)
soil and divided into groups. Sixty milligram of O. crenata or O.
aegyptiaca/ramosa seeds together with the different fungal preparations
at 0.0, 0.5, 0.75, 1.0 and 1.25 g kg-1 soil were well mixed with
the sub layer. Five faba bean seeds were placed in O. crenata infested
pots and covered with an additional 3 cm thick layer of soil. Faba bean seedlings
were thinned to 3 per pot 14 days after sowing. The O. aegyptiaca/ramosa
infested pots were planted with 3 tomato seedlings. Disease incidence on Orobanche
and the number of emerged shoots were determined two months after planting.
In addition, the Orobanche shoot height and shoot dry weight were determined.
Meanwhile, an evaluation of host plants growth and oxidative stress signals
was performed.
Determination of H2O2
Extraction was carried out in 200 mM perchloric acid and centrifuged at
5000 x g for 10 min. The supernatant was neutralized with 4 M KOH and centrifuged
at 3000 x g for 5 min. An aliquot (0.2 mL) of the supernatant was loaded on
1 mL column of anion exchange resin and eluted with 0.8 mL of distilled water.
H2O2 was assayed in 12.5 mM 3-dimethylaminobenzoic acid
in 375 mM phosphate buffer pH 6.5, 1.3 mM 3-methyl-2-benzothiazolinone hydrazone
and 0.25 units horseradish peroxidase (Okuda et al., 1991). The reaction
was initiated by the addition of peroxidase and the increase in absorbance at
590 nm was monitored for 3 min.
Determination of Ascorbate (AsA)
Extraction was performed in 62.5 mM phosphoric acid, centrifuged at 12000x
g for 20 min and filtered through a 0.5 μm Millipore filter. The filtrate
was loaded onto an ion exclusion column (300x7.8 mm) connected to analytical
HPLC system and eluted with 4.5 mM H2SO4 at a flow rate
of 0.5 mL min-1. The elution of AsA was detected at 245 nm (Ahn
et al., 1999).
Assays of Superoxide Dismutase (SOD), Catalase (CAT) Guaiacol Peroxidase
(GPX) and Glutathione-S-Transferase (GST) Activities
All extraction steps were carried out at 4°C. SOD (EC 1.15.1.1) was
extracted in 50 mM phosphate, pH 7.8, 0.1% (w/v) bovine serum albumin, 5.5 mM
AsA and 8 mM β-mercaptoethanol. SOD was assayed in 50 mM phosphate, pH
7.8, 9.9 mM L-methionine, 0.057 mM nitroblue tetrazolium (NBT), 0.025% (w/v)
Triton X-100 and 0.1 mM riboflavin by using the photochemical NBT method in
terms of SOD’s ability to inhibit reduction of NBT to form formazan by
superoxide (Beyer and Fridovich, 1987). The photoreduction of NBT was measured
at 560 nm.
CAT (EC 1.11.1.6) was extracted in 50 mM phosphate buffer, pH 7 and 1 mM dithiothreitol. CAT was evaluated spectrophotometrically by determining the consumption of H2O2 at 240 nm in 50 mM phosphate buffer, pH 7.5 and 200 mM H2O2 (Aebi, 1984).
GPX (EC 1.11.1.7) was extracted in 220 mM Tris-HCl, pH 7.4, 250 mM sucrose, 50 mM KCl, 1 mM MgCl2, 160 mM β-mercaptoethanol and 0.57 mM phenyl methyl sulphonyl fluoride. GPX was assayed in 20 mM acetate, pH 5, 30 mM H2O2 and 2 mM guaiacol. The absorption at 470 nm was recorded and the activity was calculated using the extinction coefficient of 26.6 mM-1 cm-1 (Ranieri et al., 1997).
GST (EC 2.5.1.18) was extracted in 100 mM Tris-HCl, pH 7.5, 2 mM EDTA, 14 mM β-mercaptoethanol and 7.5% (w/v) polyvinylpolyprollidone, centrifuged at 15000 x g for 15 min, ammonium sulfate was added to 80% saturation and the protein pellets were collected (Dixon et al., 1995). GST was assayed in 100 mM phosphate, pH 6.5, 5 mM GSH and 1 mM CDNB. The absorbance at 340 nm was measured and the activity was calculated by the extinction coefficient E = 9.6 mM-1cm-1 (Ando et al., 1988).
Protein content was determined spectrophotometrically by reaction with Commassie Brilliant Blue G according to Bradford (1976). All values reported herein are means of at least six replications from two independent experiments. The full data were statistically analyzed using the Least Significant Difference (LSD) test at 5% level (Snedecor and Cochran, 1980).
RESULTS
Only two fungi isolated from diseased Orobanche shoots, classified as F. oxysporum isolate I (Foxy I) and isolate II (Foxy II), inhibited Orobanche germination. Microconidia and chlamydospores of both isolates were used for achieving formulations as Pesta (PM I and PM II from microconidia while PC I and PC II from chlamydospores). As shown in Table 1, the number of emerged and the total number of Orobanche shoots were decreased after the application of Pesta formulations.
| Table 1: | Effect of granular Pesta formulations from microconidia and chlamydospore (PM and PC) of two Fusarium oxysporum isolates (I and II) on emerged shoot number, shoots height, shoot dry weight and disease incidence in Orobanche ramosa and Orobanche crenata |
Values are mean of at least six determinations from two independent experiments. *: Values are significantly different at 5% level with respect to untreated control |
|
However, low doses had, in general, no significant effects on the number of emerged shoots of both Orobanche species. Increasing Pesta dose retarded Orobanche emergences so that high doses resulted in much diminutions in emerged shoots. Moreover, Pesta significantly reduced shoot height of both Orobanche species compared with their respective controls. The magnitude of reduction increased with increasing doses. In addition, Orobanche shoot dry weight was significantly decreased by all doses of the applied Pesta formulations, the magnitude of decrease was greatest with the highest dose. Nevertheless, PC II seemed to be the most effective formulation. The trend of response to the formulations was most likely similar in both Orobanche species. The emerged shoots of the Pesta-treated Orobanche exhibited disease symptoms at emergence, which continued up to the flowering stage. Disease symptoms consists of inhibition of Orobanche germination, wilting, necrosis and shoot curvature of those plants that did emerge. High doses mostly prevented flowering and fruit production of Orobanche. Treatments with either of the different formulations highly enhanced Disease Incidence (DI) of the emerged Orobanche shoots either with faba bean or with tomato. DI appeared to be mostly related to the doses of formulations. Treatments with either of the different formulations highly enhanced Disease Incidence (DI) of the emerged Orobanche shoots either with faba bean or with tomato. DI appeared to be mostly related to the formulation doses.
Regarding growth parameters of host plants, Table 2 shows that Orobanche reduced shoot height, root length as well as shoot and root dry weights of tomato and faba bean as compared with normally grown plants. However, the Orobanche-induced reduction in host growth was ameliorated by application of Foxy Pesta formulations. Shoot height and root length of Orobanche-infected tomato and faba bean seemed to be mitigated by all types of formulations to become comparable to control levels.
| Table 2: | Effect of granular Pesta formulations from microconidia and chlamydospore (PM and PC) of two Fusarium oxysporum isolates (I and II) on shoot height and root length as well as shoot and root dry weight of tomato and faba bean infected with Orobanche ramosa and Orobanche crenata, respectively |
Values are mean of at least six determinations from two independent experiments. *: Values are significantly different at 5% level with respect to untreated control |
|
| Table 3: | Effect of granular Pesta formulations from microconidia and chlamydospore (PM and PC) of two Fusarium oxysporum isolates (I and II) on ascorbate (AsA) and H2O2 contents in shoots and roots of tomato and faba bean infected with Orobanche ramosa and Orobanche crenata, respectively |
Values are mean of at least six determinations from two independent experiments. *: Values are significantly different at 5% level with respect to untreated control |
|
| Table 4: | Effect of granular Pesta formulations from microconidia and chlamydospore (PM and PC) of two Fusarium oxysporum isolates (I and II) on activity of superoxide dismutase (SOD) in shoots and roots of tomato and faba bean infected with Orobanche ramosa and Orobanche crenata, respectively |
Values are mean of at least six determinations from two independent experiments. *: Values are significantly different at 5% level with respect to untreated control |
|
The effect of PC II was greater compared to the other formulations. Similar increases were also detected in shoot and root dry weights. Such reduction in symptoms became more pronounced at higher doses of biocontrol formulations and plants treated with the highest doses were indistinguishable from controls.
The H2O2 content in shoots and roots of the Orobanche-infected tomato and faba bean plants were significantly higher than in the control (Table 3). The application of Pesta formulations reduced the magnitude of the Orobanche-induced accumulation. The reduction in H2O2 accumulation was greater with higher doses of the formulations and the PC II formulation was the most effective.
In contrast to H2O2, there was a significant drop in the contents of AsA in tomato and faba bean plants due to Orobanche infection. The application of all formulations resulted in significant increases in AsA content of the infected plants to reach mostly the contents of the healthy non-infected plants. The magnitude of increase in AsA content was proportional to the increase in Pesta doses; the higher the Pesta dose was, the greater was the enhancement of AsA content.
| Table 5: | Effect of granular Pesta formulations from microconidia and chlamydospore (PM and PC) of two Fusarium oxysporum isolates (I and II) on activity of catalase (CAT), guaiacol peroxidase (GPX) and glutathione-S-transferase (GST) in shoots and roots of tomato and faba bean infected with Orobanche ramosa and Orobanche crenata, respectively |
Values are mean of at least six determinations from two independent experiments. *: Values are significantly different at 5% level with respect to untreated control |
|
SOD activity was significantly higher in shoots and roots of the Orobanche infected tomato and faba bean plants than in the control plants (Table 4). The application of Pesta formulations significantly decreased the enhancement of SOD activity. The reduction of theinduced increases in SOD activity was related to dosage of the formulations and at the higher doses values were not significantly different from uninfected control plants.
Infection with Orobanche led to decreases in activities of CAT, GPX and GST in shoots and roots of tomato and faba bean plants as compared to the control plants (Table 5). Pesta application overcame the infection-induced inhibition of the enzyme activity. The release of the enzymes activities from inhibition was related to the dose of the Pesta formulations and became comparable with those of controls at the higher levels.
DISCUSSION
In the present study, two isolates of F. oxysporum showed pathogenicity to O. crenata and O. aegyptiaca/ramosa. Microconidia and chlamydospores of both isolates were used to achieve granular mycoherbicide formulations for biocontrol of Orobanche. Severe reductions were obtained in shoot emergence of O. crenata and O. ramosa, respectively by application of all Foxy Pesta formulations particularly at the highest dose. Similar reductions were observed in shoot height and shoot dry matter. On the contrary, disease incidence of Orobanche was highly accelerated upon application of Pesta. In this connection, Shabana et al. (2003) reported that application of Pesta formulations containing microconidia and chlamydospores of F. oxysporum f. sp. orthoceras (FOO) as a bioherbicide for O. cumana resulted in a reduction in Orobanche biomass and increase in disease severity. Moreover, Pesta granules reduced the emergence of O. cumana shoots (Müller-Stöver et al., 2004). Similar results were also observed by Müller-Stöver and Kroschel (2005) on O. crenata by inoculation with Ulocladium botrytis. The reduction of Orobanche growth by Foxy isolates may be due to the production of toxic metabolites (Zonno and Vurro, 2002; Müller-Stöver and Kroschel, 2005).
Growth of the host species (faba bean and tomato) was significantly reduced by Orobanche infection. The Orobanche-induced growth inhibition of tomato and faba bean due to parasitism might result from depletion of host nutrition. However, these reductions appeared to be counterbalanced following the application of the formulated Pesta. The biocontrol agents did not only prevent germination but also attacked developed shoots overcoming, therefore, any malfunction in host metabolism probably caused by Orobanche infection. Consequently a repair in host metabolism might support new syntheses and consequently normal and even vigor growth. In this context, Müller-Stöver and Kroschel (2005) found that dry matter accumulation of faba bean was significantly increased by treatment with U. botrytis. Also, Shabana et al. (2003) observed an increase in sunflower dry weight as a result of treatments with FOO. Therefore, Pesta not only overcame the Orobanche-induced reductions in host growth but also seemed to serve as amendments, to some extent, supplying host species with good conditions for growth. The inhibition in Orobanche growth following Pesta application accompanied with recoveries in host growth could point to the suitability of the formulations as mycoherbicides for Orobanche biocontrol with no negative side-effects on host growth.
The stress imposed in tomato and faba bean by Orobanche could generate oxidative stress resulted from both infection and normal growth, a state that could be indicated by the accumulation of H2O2 and the decline of AsA. Aerobic organisms depend on O2 as electron acceptor in electron transfer reactions. During O2 reduction, cells continuously produce ROS (Mittler, 2002). ROS are involved in molecular damage in plants (Noctor and Foyer, 1998). Aerobic organisms evolve numerous antioxidants to minimize the adverse effects of ROS (Durmus and Kadioglu, 2005; Hassan and Nemat Alla, 2005; Nemat Alla et al., 2007). The accumulation of H2O2 and the decline of AsA could be considered as indices of oxidative stress. However, H2O2 accumulation is also very important to trigger defense mechanisms against pathogens. ROS accumulation is crucial to plant development as well as defense (Foyer and Noctor, 2005; Pavet et al., 2005). Signals of oxidative stress could be evidenced from the abnormal changes in activities of antioxidative stress enzymes (Nemat Alla and Hassan, 2007; Nemat Alla et al., 2007). Within a cell, SOD constitutes the first line of defense against ROS (Alscher et al., 2002). SOD dismutates the superoxide radicals into H2O2 (Noctor and Foyer, 1998). H2O2 is detoxified by GPX or CAT and sometimes by GST which may act as peroxidase. However, under unstressed conditions, the formation and removal of ROS are in balance. Therefore, the defense system, with increased ROS formation under stress conditions, can be overwhelmed. Detoxification reactions must involve right balance between the formation and detoxification of ROS.
The increases in SOD activity of the Orobanche infected plants concomitant with the inhibited activities of CAT, GPX and GST could explicate the great accumulation of H2O2. Thus, the accumulated H2O2 might arise from dismutation of superoxide radical with no enhanced detoxification routes. Under these conditions where CAT, GPX and GST were inhibited, the cell is not competent to scavenge H2O2. On the other hand, application of Pesta formulation overcame the stress inducers and consequently could terminate and moreover eliminate the stress circumstances. Therefore, AsA content and activities of CAT, GPX and GST showed enhancements supporting more detoxification of H2O2 with a subsequent decline in its accumulation. AsA increases H2O2 detoxification through a played role by the AsA-glutathione (AsA-GSH) cycle. This cycle is an important and powerful detoxifying mechanism in the plant cells (Ma and Cheng, 2003) in which H2O2 is reduced to water by peroxidase on the expense of AsA oxidation (Mittler, 2002; Aravind and Passad, 2005; Nemat Alla et al., 2007). AsA is regenerated again by monodehydroascorbate reductase and GSH-dependent dehydroascorbate reductase coupled with glutathione reductase to maintain GSH levels for elimination of ROS (Nagalakshmi and Prasad, 2001). These antioxidants are crucial for plant defense against oxidative stress (Gomez et al., 2004; Nemat Alla and Hassan, 2007). In addition, CAT, GPX and GST help in H2O2 scavenge by catalyzing its reduction to water (Nagalakshmi and Prasad, 2001; Nemat Alla and Hassan, 2006; Nemat Alla et al., 2007).
The present results clearly revealed that application of Pesta formulations particularly at 1.25 g kg-1 soil to Orobanche infected plants retarded Orobanche growth and simultaneously retracted the Orobanche-induced reductions in host plants and moreover delayed oxidative stress. These findings could suggest that an increase in the defense of host plants was developed by Pesta application against the stress elicited by the infection. Antioxidative mechanism seemed to be modified as activities of SOD, CAT, GPX and GST as well as contents of AsA were greatly improved following the application of Pesta formulations. As a result, H2O2 and certainly other ROS could be eliminated. Therefore, it could be suggested that these formulations have positive influences not only because of overcoming the infection effect but also of improving plant metabolites and antioxidative defense mechanism.
The present findings, moreover, could suggest a high virulence of chlamydospores of isolate II in performing the Pesta against Orobanche. Anyway, H2O2 levels and SOD activity were decreased by Pesta to reach those of normally grown non-infected plants. In the mean time, AsA content and activities of CAT, GPX and GST raised. The retractions in oxidative stress of host plants by Foxy Pesta in addition to the recoveries of growth reductions could suggest that plants seemed more healthy to withstand even with the presence of Orobanche. These findings conclude that the application of Foxy formulations particularly PC II at 1.25 g kg-1 soil seemed to scavenge the negative effects of parasitism and appeared efficient to mitigate oxidative stress and growth reduction in tomato and faba bean resulted from Orobanche infection.