Control Efficiency Enhancement by Using Biocontrol Agents, Bacillus thuringiensis, B.

megaterium and Trichoderma album As Single and Combined Treatments Against Meloidogyne incognita of Tomato A.A. Osman1, A.H. Hussein1, and Esraa.O. Abdel-raouf1 Zoology and Agricultural Nematology Dept., Faculty of Agriculture, Cairo University, Giza 12613Egypt. Three local commercially available bio-based pesticides; Agerin (Bacillus thuringiensis), BioArc (Bacillus megaterium) and BioZeid (Trichoderma album); applied as single and combined treatments; were tested under greenhouse conditions for their suppressive influence on the development of root-knot nematode, M. incognita on tomato plants. In the single treatments, B. thuringiensis had the highest reducing effect on the number of galls, females, egg-masses and eggs/egg-mass, followed by T. album, while B. megaterium had a negligible impact; compared to the check. In the combined (double) treatments, B. thuringiensis+ T. album recorded the highest percentages reduction in each of the number of galls (65.69%), females(68.96%), egg-masses (68.89%) and eggs/egg-mass (34.98%), followed by B. thuringiensis+ B. megaterium, then T. album+ B. megaterium which had the least reduction, compared to the check. B. thuringiensis+ B. megaterium+ T. album combined together in the same treatment attained significant reduction in all the counted nematode criteria, except for the number of eggs/egg-mass in comparison to the check. These results manifested that the effect of each of B. thuringiensis and T. album was utmost when they were applied together, rather than singly applied. It was also signified that B. megaterium lessened the effect of both B. thuringiensis and T. album in the coupled treatments. Plant growth parameters as indicated by shoot length, shoot and root fresh weights positively responded to the applied treatments, with exception to the single treatment of B. thuringiensis which did not improve shoot length and root weight, similarly the combined (double) treatment, B. thuringiensis+ T. album had resulted in a little non-significant increase in root weight. Keywords: Meloidogyne incognita, combination, biological control, Bio-based pesticides, Bacillus thuringiensis, B. megaterium, Trichoderma album, Tomato.

Introduction Plant parasitic nematodes strongly reduce the performance of their host plants when abundance is not controlled. Conventional chemicals may be deleterious to the environment and to non-target organisms, so alternatives are being investigated for use against plant-parasitic nematodes. Additionally, the restrictions on the use of conventional nematicides have increased the need for new methods of managing plant parasitic nematodes. Moreover, due to environmental and health concerns, the use of several widely applied nematicides have been now restricted to control nematodes. Therefore, there is a need to develop alternative strategies and tactics, including biological control to manage plant parasitic nematodes. A plenty of bioagents are antagonistic to pathogenic nematodes and fungi. A wide range of microbes antagonistic to soil borne diseases including nematodes are extensively documented by many researchers. Biological control agents have been extensively studied to manage plant parasitic nematodes. Root-knot nematodes are a top priority to deserve the majority of this concern. Application of bioagents individually to control nematodes is one of the many factors limiting its success in addition to the inconsistent performance in field. Numerous bioagents were subjected to studies concerned with their efficiency in controlling parasitic nematodes. Numerous microbes are antagonistic to soil borne plant pathogens and plant parasitic nematodes. Relatively few of microbes antagonistic to soil borne and nematode diseases have been commercialized as biocontrol agents due to many problems such as an inconsistent performance in the field or slower or less complete suppression when compared with chemical pesticides (Larkin et al., 1998, Meyer and Roberts, 2002). Inconsistent performance by microbial antagonists has been attributed to biotic and abiotic factors (environmental conditions). The majority of strategies for biocontrol of soil borne plant pathogens and plant parasitic nematodes rely on a single microbial agent for pathogen or nematode suppression. Considerable information available in the literature has documented the effectiveness of several biological control agents to manage plant-parasitic nematodes (Meyer and Meyer, 1996; Siddiqui and Mahmood, 1997; Walia, 1996; Chen and Dickson, 1998; Kerry, 1998; Siddiqui and Mahmood, 1996a, 1999; Sikora and Hoffmann-Hergarten, 1993). Unfortunately, biocontrol agents applied individually are not likely to perform consistently against all parasitic nematodes under soil environmental conditions. Because different mechanisms of control may be dissimilarly influenced by environmental conditions, it is possible that if multiple mechanisms are involved under a certain set of conditions, one mechanism may compensate for the other (Guetsky et al., 2002). Therefore, the control achieved by biocontrol agents with several distinct mechanisms of control may be additive or synergistic.

The approach of combining biocontrol agents to manage various soilborne pathogens including plant parasitic nematodes has been studied extensively e.g. (Duffy et al., 1996; Duponnis et al., 1999; Hojat jalali et al., 1998; Lemanceau and Alabouvette, 1991; Mao et al., 1998a,b;, Pierson and Weller, 1994; Siddiqui and Mahmood, 1993). Recently, few studies concerning this attitude have been published against root-knot nematodes, Meloidogyne spp. The use of Burkholderia cepacia plus Trichoderma virens against M. incognita on Bell pepper (Meyer et al., 2001), T. virens plus B. cepacia, T. virens plus B.ambifaria, T. virens plus S. marcescens (Roberts et al., 2005); combinations of Aspergillus niger CA, Penicillium chrysogenum, Burkholderia cepacia, Bacillus subtilis, Glomus intraradices, Gigaspora margarita were assessed by Siddiqui and Akhtar,2009; Bacillus thuringiensis plus Streptomyces costaricanus against M. hapla infecting lettuce by Chen et al., 2000; Serratia marcescens plus Pseudomonas fluorescens and Rhizobium leguminosarium against Meloidogyne incognita of faba bean by Mohammed et al., 2009. A number of research investigations indicate that biocontrol combinations may have a future for management of plant parasitic nematodes. In these studies, certain microbe combinations resulted in increased plant vigor or yield, and (or) reduction of nematode populations or penetration on roots, compared with individual applications of the biocontrol agents ( Duponnis et al., 1998; Gautam et al., 1995; Hojat Jalali et al., 1998; Khan et al., 1997; Perveen et al., 1998; Siddiqui et al., 1999; Siddiqui and Mahmood, 1993, 1995b, 1996b; Vidya and Reddy, 1998; Youssef and Ali, 1998; Raupach and kloepper, 1998; Meyer and Roberts, 2002). The effects on nematode populations included suppression in numbers of females, eggs, eggmasses, juveniles, or galls. The goal of this research was to compare the efficacy of three commercial bioagents applied individually and in combinations to act as biocontrol agents for root-knot nematode, Meloidogyne incognita on tomato plants under greenhouse conditions.

Materials and Methods Source of nematodes inoculum: Newly hatched second stage juveniles (j2) of the root-knot nematode, Meloidogyne incognita, were obtained by sieving and modified Baermann technique (Goodey, 1957) from a pure culture that was initiated using a single egg mass and propagated on eggplant transplants in a greenhouse located at Nematology Research Center, Department of Zoology and Agricultural Nematology, Nematodes Branch, Faculty of Agriculture, Cairo University, Egypt. Source of the microbial antagonists: This study included three commercial microbial formulations that are registered and sold as biopesticides in the Egyptian market. The first one is Agerin which contains 32,000 IU/mg of the bacterium Bacillus thuringiensis sub sp. aegypti with registration no. 580 and recommended to be applied at rate of 250 g/ feddan, and it was brought from BIOGRO international Egypt company. BioArc 6% powder is the second with registration no.1087 and it contains 25 million spores/g of B. megaterium and the third one is BioZeid containing 10 million spores/g of Trichoderma album with registration no. 1088, both were recommended to be applied for root-knot nematodes at rate of 40 kg/feddan and were brought from Plant Pathology Research Institute, Agricultural Research Center, Giza, Egypt. The effect of using the microbial antagonists singularly and in combinations on the suppression of Meloidogyne incognita infecting tomato, Solanum lycopersicum : A greenhouse experiment was conducted to test the efficacy of single and combined application of bioagents in controlling the rootknot nematode, Meloidogyne incognita infecting tomato plants cv. Super strain-b. For executing the experiment, 10-cm diameter conical shaped earthen pots were filled with 1:1, sand: clay, natural soil. All the bioagents in all treatments were incorporated into the top 5-cm of each pot concomitantly with nematode inoculation, where each pot received 2000 newly hatched juveniles of M. incognita. The treatments were as follows: 1- nematodes + B. thuringiensis (1g), 2- nematodes + B. megaterium (1g), 3- nematodes + T. album (1g), 4- nematodes + B. thuringiensis + B. megaterium (0.5g each), 5- nematodes + B. thuringiensis + T. album (0.5g each), 6- nematodes + B. megaterium + T. album (0.5g each), 7- nematodes + B. thuringiensis + B. megaterium + T. album (0.33g each) and 8- nematodes alone. Pots were arranged in a completely randomized design under greenhouse conditions, and each treatment was replicated five times. Tomato seedlings were immediately transplanted into the untreated pots and six days later into the treated pots. The experiment was terminated 52 days after nematode inoculation. Vegetative growth parameters (shoot length, shoot and root fresh weights) were recorded as measured, while the roots were washed with a gentle stream of tap water to free from soil particles. The most homogenous four plants were selected, stained with acid- fuchsine and examined under stereo microscope for counting the numbers of galls, females, egg masses and eggs per egg mass. Differences between treatments were determined according to Analysis of Variance (ANOVA), using MSTAT-C statistical package (MSTATC, Michigan state University, 1989) and whenever differences were detected, means were separated using Least Significant Difference test (LSD);in the same package; at the 5% level of significance.

Results A greenhouse pot-experiment was set up to determine the effect of applying three bioagents Bacillus thuringiensis (Agerin), Bacillus megaterium (Bio Arc), and Trichoderma album (Bio Zeid), as single and combined treatments for controlling root-knot nematode, Meloidogyne incognita on tomato plants, figures presented in Table.1 , Figure.1,and Figure .2, displayed the obtained results concerning nematode development and plant growth as follows: Number of galls: The three bioagents, Bacillus thuringiensis (Bt), Bacillus megaterium (Bm), and Trichoderma album (Ta),when applied singly reduced gall numbers by 55.73, 9.75 & 34.79%, respectively, compared to the untreated check. When the three bioagents were applied as combined treatments of two bioagents, B. thuringiensis+T. album was found to maximize the galling percentage reduction with 65.69% compared with the check, but B. megaterium+T. album resulted in minimizing galling percentage reduction 24.3%. A similar achievement was resulted from the triple applied bioagents, B.thuringiensis+B.megaterium+T.album, 32.16%. It was remarkably noticed that the presence of B. thuringiensis in the combined treatments with the other bioagents resulted in high suppressive effect on gall numbers, compared to the other combinations. B.megaterium, mixed with the other bioagents led to a feeble effect in reducing the number of galls. T.album mixed with B.thuringiensis; in the two bioagents combined treatment; was the superior in reducing gall numbers. Number of females: In the single treatments, only B.thuringiensis and T. album showed significant reduction in the number of females per plant, with percentage reduction of 59.39 & 41.01 %, respectively, in comparison to the untreated check, while B.megaterium was the least effective in reducing females number per plant, 15.32%. As for the two bioagents treatments, all the treatments proved to attain significant reduction in number of females per plant, compared to the check but not to each other. Moreover, B.thuringiensis + T. album was the most efficient among all the double applied treatments, recording females reduction percentage of 68.96%. The treatment B.thuringiensis + B.megaterium came next with a reduction percentage 39.41%, but B.megaterium + T. album was found to be the least among the two-applied bioagents treatments, recording 30.8 percentage reduction in females number per plant. The treatment that contained the three bioagents together in comparison to the check, succeeded in achieving a significant reduction percentage of 38.15%, which didnt differ significantly from the other treatments. Number of egg-masses: Compared to the untreated check, the single application of B.thuringiensis and T. album significantly reduced egg-mass formation by 63.79 and 48.16%, respectively, while B.megaterium did not where it recorded 17.57%. The highest percentage reduction in egg-mass numbers, in comparison to the check, was attained by the two-applied bioagents treatment B.thuringiensis + T. album (68.89%), almost half of this value was obtained by the combination of B.megaterium with each of B.thuringiensis

and T. album, which was 36.84 and 35.08%, respectively. Although the triple bioagents application didnt significantly differ from the other applied treatments, it significantly differed from the control; recorded 37.06% reduction in egg-mass numbers. Number of eggs/egg-mass: The only treatment that significantly reduced the no of eggs/egg-mass in comparison to the check was the combined treatment of B.thuringiensis + T. album (34.98%), however, it did not differ significantly from the other applied treatments either singly or combined. B.thuringiensis applied singly came in the second rank with a eggs/egg-mass reduction percentage of 26.14%, while the least effective bioagent treatment among all the applied treatments was the single treatment of B.megaterium where the number of eggs/egg-mass increased with a percentage of +13.94% compared to the check. Shoot length: results as presented in table (2), displayed that all the applied single and combined treatments significantly improved the shoot length compared with the untreated check, except for the single treatment of B. thuringiensis. When B. Thuringiensis was mixed with B. Megaterium, it recorded the highest increase in shoot length (37.03%). No significant differences were observed among all the treatments compared to each other. Shoot fresh weight: All the applied treatments caused significant improvement in the shoot fresh weight; which ranged from 45.73 to 59.27 %; compared with the untreated check, although they were not significantly different compared to each other. Root fresh weight: Highest root weight was recorded by the single treatment of B. megaterium (14.98 g) which was highly significant compared with the untreated check, but not with the other treatments. B. thuringiensis applied singularly and combined with T. album didn't succeed in attaining significant improvement of root fresh weight compared with the untreated check. Additionally, when compared with each other, no significant differences were noticed among all the treatments, except for B. thuringiensis applied singularly, which achieved the lowest root weight which was 7.625 g.

Discussion In our study, three biocontrol agents, Bacillus thuringiensis, Bacillus megaterium and Trichoderma album were evaluated for their effectiveness in controlling root knot nematode, Meloidogyne incognita infecting tomato plants under greenhouse conditions. The three mentioned bioagents were applied individually and concomitantly to study the interaction among each others. The individual treatment of B. thuringiensis and T. album were highly effective in reducing number of galls, females, egg-masses formation in comparison to the other treatments and non-treated plants. The dual application of B. thuringiensis and T. album had resulted in the maximum effect on nematode suppressiveness followed by joint application of B. thuringiensis+B. megaterium. Incorporation of B. thuringiensis with the other bioagents in one treatment led to achieve highly significantly results of nematode suppression which could be considered as efficacy enhancer. T. album was the second potent bioagent following B. thuringiensis in suppressing nematode developmental stages in this trial. The coupling of B. thuringiensis and T. album in one concomitant treatment means coupling of two potent bioagents capable of influencing nematode activity without antagonistic reaction. Many investigations have documented the potentiality of Trichoderma spp. as a good candidate for biological control of many soil-borne diseases including plant parasitic nematodes. The successfulness of Trichoderma to suppress the incidence of diseases depending on many factors, the easy technique to isolate and culture, grow rapidly on many substrates, affect a wide range of plant pathogens, and rarely pathogenic on higher plants, act as mycoparasites, compete well for food and site, produce antibiotics and have an enzyme system capable of attacking a wide range of plant pathogens and are effective bioagents against root-knot nematodes(Khan and Saxena, 1997; Hafeez et al., 2000; Meyer et al., 2000; Rangaswamy et al., 2000; and Sharon et al., 1 . u re et al., 2005, reported that Trichoderma mycoparasitic activity depends on the secretion of complex mixtures of hydrolytic enzymes able to degrade the host cell wall. The incorporation of Trichoderma with B. thuringiensis in our study indicates the increased suppressive effect of this treatment on the number of galls more than the individual treatment by each of them. This typically happened with M. incognita on lettuce when T. harzianum was applied with Paecilomyces lilacinus in combined treatment and results were significantly different from the other treatments and control (Songsak, 1999). Maximum reduction of root-knot nematode, M. incognita in chick pea was recorded with a triple combined treatment of (P. lilacinus+Aspergillus niger and T. harzianum). The same success was achieved in our study with the triple treatment including T. album+B. thuringiensis and B. megaterium, Hemlata Pant et al., 2001. The remarkable effect of the mixture of two bioagents (B. thuringiensis+T. album) in our study in reducing nematode activity, was documented by Siddiqui and Shaukat, 2004. They found that P. fluorescens and T. harzianum applied together in unsterilized sandy loam soil caused greater reduction in

nematode population densities on tomato roots. Our study displayed the effects of the single and combined treatments on nematode developmental stages, egg mass formation and fecundity. Similar to our results, Goswami et al., 2006, found that P. lilacinus and T. viride in a combined treatment promoted plant growth, reduced number of galls/plant, egg masses/root system and eggs/egg mass. In another study by Oyekanmi et al., 2007, it was found that the application of three bioagents included T. pseudokoningii to control M. incognita on soybean suppressed nematode reproduction and reduced galling comparable to the nematicide Furadan treatment. Outstanding results were obtained when T. album and B. thuringiensis were combined in one mixture. Microorganisms with broad spectrum activity showed promise for increasing the level of suppression of nematodes on tomato. Fungal species belonging to the genus Trichoderma are worldwide in occurrence and easily isolated from soil. The good role played by Trichoderma spp. in reducing nematode activities and suppression on nematode infection and parasitism relies on many and varied mechanisms. T. virens produced Gliotoxin which is a lethal principle to disease (Rehner and Samuels, 1994). Gliovirin was also reported as a toxin produced by T. virins and this is a strong microorganisms inhibitor. (Howell and Stipanovic, 1983). Fortunately, Gliovirin was not inhibitory to the bacteria B. thuringiensis, was a very potent inhibitor to many microbes. Results of Howell, 1987, indicated that mycoparasitism was not a major mechanism in the biocontrol by this fungus. Protease production by T. harzianum has been associated with biocontrol of the root-knot nematode, M. javanica on tomato plants. Sharon et al., 2001, showed that tomato plants treated with biocontrol agent (T-203) and grown in nematode infested soil, exhibited a drastic reduction in root galling when compared with the control. It could be said that the combination of enzymes and antibiotics produced by Trichoderma spp. in one treatment against soil-borne pathogens resulted in a great control than the single treatment by antibiotics or lytic enzymes and was found more effective. Yedida et al., 2000, showed that inoculation of cucumber roots with T. harzianum (T-203) induced an array of pathogenesis-related proteins, including a number of hydrolytic enzymes. In another study, Howell et al., 2000, demonstrated that seed treatment of cotton with biocontrol preparations of T. virins (G-6, G-11, G-6-5) or application of T. virins culture filtrate to cotton seedling radicles induced synthesis of much higher concentrations of the terpenoids desoxyhemigossypol (dHG), Hemigossypol (HG) and gossypol (G) in developing roots than those found in untreated controls. Gossypol was toxic only at high levels, but the pathway intermediates (dHG) and (HG) were strongly inhibitory to the cotton seedling pathogen. In addition to terpenoid synthesis, treatment of cotton roots

with T. virens also induced significantly higher levels of peroxidase activity than that found in control roots. The reduced number of galls as resulted from T. album application wheather in single or combined treatments was similar to what was reported by Spigel and Chet, 1998. Our results are highly satisfactory. So, it can be said that the bioagent effectiveness can be improved when combined with either of the bacteria or fungi. This research demonstrated that the tested bioagent combinations are active against the root-knot nematode, M. incognita and suppressiveness of nematode stages needs to be taken into account at different concentrations and time of application. Combining biocontrol agents resulted in beneficial synergistic interactions with some exceptions. In our test, combinations of microorganisms were more effective than individual agents for nematode control on tomato for improving plant vigor and suppressing disease. It is not clear why some combinations (including B. megaterium) did not reduce root-knot nematode effectively on tomato in our study. It is possible that the tested organisms were antagonistic to each other under the conditions used for this experiment, or that the combinations tested here suppressed production of nematode antagonistic compounds. Incompatibility among biocontrol organisms (B. thuringiensis plus B. megaterium) has been observed clearly in this study. Similar behavior was realized by Meyer et al., 2001, when Burkholderia cepacia and T. virins were applied together in combination. Many strains of Bacillus have been tested for nematicidal activity against Meloidogyne spp. on many vegetables crops. These strains were Bacillus thuringiensis aizawi, B. thuringiensis morrisoni, B. brasiliensis, B. laterosporus and B. circulans (Carneiro et al., 1998). Their studies manifested that B. thuringiensis sub sp. Brasiliensis and B. laterosporus caused high mortality to M. javanica larvae, whereas B. thuringiensis sub sp. aizawi and B. circulans caused larval immobilization and B. thuringiensis sub sp. morrisoni produced only mobility reduction of M. javanica J2 . The remarkable effect of B. thuringiensis was attributed to the production of a delta-endotoxin in cells as well as several exotoxins in the culture media (Oka et al., 1993). Accordingly, Sharma and Gomes, (1996 a,b) found that the delta endotoxin from B. thuringiensis and B. sphaericus inhibited the hatch of H. glycines J2 in one case, but in another the hatch was stimulated. Strains of B. thuringiensis produce toxic compounds of various chemical structures and properties. Most studies mention -endotoxin, a crystal protein that is produced by the bacilli during the sporulation process. Some strains of B. thuringiensis can produce both and toxins. Additionally, entomopathogenic bacilli also produce various exoenzymes, (e.g. Lecitinases, Proteases, Chitinases, Collagenases) (Sela et al., 1998).

The successfulness of B. thuringiensis in reducing nematode activity on many host plants was reported by Zuckerman et al., 1993, who reported that tomato and pepper treated with B. thuringiensis isolate CR-371 had significantly fewer galls caused by M. incognita than the untreated control. Similarly, Sharma, 1993, found that B. thuringiensis var. thuringiensis (Btt) and B. thuringiensis var. israelensis (Bti), significantly controlled M. incognita on Barley under greenhouse conditions. Because different mechanisms of control may be dissimilarly influenced by environmental conditions, it is possible that if multiple mechanisms are involved under a certain set of conditions, where one mechanism may compensate for the other (Guetsky et al., 2002). Therefore, the control achieved by biocontrol agents with several distinct mechanisms of control may be additive or synergistic. The intensity of root disease was reduced after the combined application of two biocontrol agents than when applied separately (Akhtar and Siddiqui, 2008a). Co-inoculation of Bacillus thuringiensis and Trichoderma album showed better results in the control of Meloidogyne incognita and produced greater increase and enhancement in plant growth (Biomass) as compared to check and the single treatments. The highest reduction in numbers of galls, females, egg masses/plant was only confined with B. thuringiensis when applied concomitantly with T. album. This research demonstrated that the tested bioagent combinations are highly active against the rootknot nematode, Meloidogyne incognita and could be good control enhancers, as the rates of reduction in numbers of galls, females, egg masses/plant were outstanding. The failure of B. megaterium in achieving acceptable rates of nematode reduction and fecundity was disappointing. Combining different microorganisms with broad spectrum activity showed promise for increased consistency of suppression of pathogenic nematodes. This broad spectrum activity is likely due to inhibitory metabolites produced by these organisms. Some of these metabolites produced in the rhizosphere in soil such as Pyrolnctrin, Oocydin A., Carbapenem, Prodigiosin, and Serrawettin as well as chitinase and others (McGowan et al., 1999; Strobel et al., 1999, Kamensky et al., 2003). Additionally, some microbes have been shown to produce altericidins and other compounds with antibiotic activity (Kang et al., 1998). Finally, it could be said that the compatibility between particular strain or bioagent pairs is determined with regard to suppression of nematode infection. Compatibility between particular isolate pairs varied with the assay and possibly the method of application. This suggests that, compatibility between particular isolates in strain combinations needs to be carefully determined for all applications of these combinations treatments (Roberts et al., 2005). The impact of this research is that considering the effect of biocontrol combined treatments will lead to more reliable implementation of biocontrol agents as a management strategy.

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 Meloidogyne incognita Trichoderma album Bacillus thuringiensis, B. megaterium . - - .

Bacillus thuringiensis B. megaterium Trichoderma album Meloidogyne incognita . B. thuringiensis / . T. album B. megaterium . B. thuringiensis + T. album %56.56 %59.65 %59.96 / %89.69 B. thuringiensis +B. megaterium T. album+ B. megaterium . / . B. thuringiensis T. album . B. megaterium B. thuringiensis T. album . . B. thuringiensis B. thuringiensis + T. album .