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The Effects of Some Biocontrol Agents and their Combination on Root-knot Disease on Tomato
Hadi Golzary, Masuod Ahmadzadeh, Naser Panjehkeh, Mohammad Salari and Ebrahim Sedaghati-khoravi
 
ABSTRACT:
The effects of pseudomonas sp., Glomus sp., their combination and Trichoderma harzianum on root-knot disease reduction were investigated. In this experiments, combination of bacterial strains UTPF86 (P2) and 7NSK2 (p1) with species G. mosseae (G1) and G. intraradices (G2) (G2P2M, G1P2M and G1P1M) gave the best results. These treatments reduced the gall number, gall diameter and nematode penetration, followed by 78, 70, 60, 79, 69 and 60%, respectively (p<0.05). Bacterial strains 7NSK2 and UTPF86 produced the maximum of salicylic acid (inducing resistance agent) and hydrogen cyanide (agent of neurosis and maim) in in vitro (p<0.01) and mycorrhizal fungi induces the resistance. Therefore, it was expected that the combination of mention two agents would give the best effect in enhancement of plant growth and nematode biocontrol. Among combined treatments, T2Mt1, P2M and G1M gave the best results. These treatments reduced the gall number, gall diameter and nematode penetration, followed by 59, 48, 32% and 71, 60, 49% and 58, 48, 32%, respectively so that their effects were not significant (p<0.05). T1Mt2 was the worst treatment. However, this treatment reduced the gall number, gall diameter and nematode penetration, followed by 16, 12 and 17%, respectively so that its effects against the contaminated control was significant (p<0.05). The present study demonstrated that AM fungus and bacteria can coexist without adversely affecting one another. In fact, suitable combinations of biocontrol agents can further increase the plant growth and resistance to pathogens.
 
    How to Cite:
Hadi Golzary, Masuod Ahmadzadeh, Naser Panjehkeh, Mohammad Salari and Ebrahim Sedaghati-khoravi , 2011. The Effects of Some Biocontrol Agents and their Combination on Root-knot Disease on Tomato. Insight Plant Disease, 1: 1-11
DOI: 10.5567/PLANTDIS-IK.2011.1.11
 

INTRODUCTION
Plant parasitic nematodes cause significant damage to agriculture within the EU and throughout the world (Sasser and Freckman, 1987).The root-knot nematode, Meloidogyne spp., is one of the most important Plant parasitic nematodes. Meloidogyne spp. is able to infect more than 2000 plant species (Jung and Wyss, 1999). The damage caused by the root-knot nematode is much higher in tropical and sub-tropical countries (Taylor and Sasser, 1978). Studies have shown that root-knot nematodes can cause suppression in yield of tomato as high as 85% (Sasser, 1979; Taylor and Sasser, 1978).

Beneficial pseudomonads can antagonize soil-borne pathogens through various mechanisms (Bakker et al., 1991). For example, bacterial siderophores inhibit plant pathogens through competition for iron, antibiotics suppress competing microorganisms or hydrogen cyanide (Ahl et al., 1986) and chitinases and glucanases lyse microbial cells; and these compounds have been implicated in the reduction of deleterious and pathogenic rhizosphere microorganisms, creating an environment more favorable for root growth (Leong, 1986). Recent studies have demonstrated that some rhizobacteria can also act indirectly by inducing systemic resistance in the plant towards soil-borne fungi and plant-parasitic nematodes (Siddiqui and Shaukat, 2002a, b).

The improvement and eventual commercialization of fluorescent pseudomonads as biocontrol agents depends in part on understanding and exploiting the mechanisms involved in these antagonistic interactions among bacteria, pathogens and their plant hosts.

The establishment of the AM in the roots of more than 80% of all land plants is the result of a complex exchange of signals between the host plant and AMF. Classically, four major groups of mycorrhizal mode of action mechanisms that mediated bioprotection have been considered: (1) direct competition, (2) mechanism mediated by alteration in plant growth, nutrition and morphology, (3) biochemical and molecular changes in mycorrhizal plants that induce pathogen resistance and (4) alterations in the soil microbiota and development of pathogen antagonism (Vierheilig et al., 2008). Arbuscular mycorrhizal fungi are of great value in promoting uptake of phosphorus, nitrogen, minor elements and water and also increase plant growth and yield of several crops (Hayman, 1982; Zambolim and Schenck, 1983; Allen, 1996).

Weindling (1932) over 75 years ago, demonstrated the antagonistic nature of Trichoderma fungal species from the genus, Trichoderma were demonstrated over 75 years ago. The genus, Trichoderma is common filamentous imperfect fungi (Deutromycetes, Dematiaceae), the most common saprophytic fungi in the rhizosphere and found in almost any soil. Trichoderma species are free-living fungi that are common in soil and root ecosystems. They are opportunistic, avirulent plant symbionts, as well as parasites of other fungi. Some strains establish robust and long-lasting colonizations (colonies) of (on) root surfaces and penetrate into the epidermis and a few cells below this level. Biocontrol activities of T. asperellum-203 and T. atroviride IMI 206040 (both fungi were previously defined as strains of T. harzianum) have been reported against M. javanica in soil (Sharon et al., 2001). Other Trichoderma species and isolates have also exhibited significant biocontrol activity against M. javanica in growth chamber experiments (Spiegel et al., 2006). Most studies on nematodes concurred that the promising fungal antagonists Trichoderma spp., had different and in fact multiple modes of action. For example, Trichoderma virens invaded, ramified, grooved and vacuolated the root-knot nematode eggs. Eapen et al. (2005) reported easy staining of eggs for microscopy due to the increased permeability of eggshell. The antagonistic action of Trichoderma spp. was chiefly attributed to chitinolytic activity of the fungi on cellular structure of nematodes which is rich in chitin. Early researchers identified key concepts and developed tactics for multiple option management of nematodes. Although, the emphasis on integrated pest management over the past three decades has promoted strategies and tactics for nematode management, comprehensive studies on the related soil biology ecology are relatively recent. Traditional management tactics include host resistance (where available), cultural tactics such as rotation with nonhosts, sanitation and avoidance and destruction of residual crop roots and the judicious use of nematicides. There have been advances in biological control of nematodes but field-scale exploitation of this tactic remains to be realized (Barker and Koenning, 1998).

This study is aimed on determination of effects of different antagonistic agents separately and their combination in decrease of the disease indices and increase of growth indices also decrease of damage due to Meloidogyne javanica nematode on tomato seedling to have a safe environment. With the increasing cost of inorganic fertilizers, the environmental and public health hazards associated with pesticides and pathogens resistant to chemical pesticides. AM fungi may provide a further suitable and environmental alternation for sustainable agriculture.


MATERIALS AND METHODS
Individuation, identification and reproduction of Nematode:
Mature females were dissected out from large galls on the roots of tomato plants. Perineal patterns slides (10-20) from each sample or locality were prepared and examined under microscope to study their characteristics Perennial patterns of mature females of root-knot nematode were prepared for identification. At least 10 perennial patterns of each sample of nematode species were examined to make more accurate identification (Jepson, 1987). For reproduction of nematode, single egg mass was separated from terminal of the females’ body and were inserted to the soil by making three holes around the tomato seedlings (Race of Early Urbana) in greenhouse conditions.

Nematode inoculums: Meloidogyne javanica nematode was obtained from pure cultures was maintained on roots of tomato. The entire root system was dipped in water and soil was removed gently without detaching egg sacs. Eggs were extracted by vigorous shaking of infested roots in a 1% sodium hypochlorite solution for 3 min. The resulting suspension was then passed through a range of different mesh-size sieves. The eggs were collected on a fine sieve (38 mm) and washed in tap water to remove all traces of sodium hypochlorite before use. Hatched juveniles of M. javanica were obtained by placing the eggs in sterile distilled water for 5 days at 28°C. The inoculum was used for the laboratory and glasshouse tests (Siddiqui et al., 2006).

Bacterial inoculums: The Fluorescent Pseudomonad bacterial strains (UTPF86, UTPF5, 7NSK2) that were used in this study were obtained from the Department of Plant Pathology, University of Tehran, in Iran. Bacterial Strains (1) were cultivated at 24°C for 24 h with shaking (150 rpm) in 250 mL Erlenmeyer flasks containing 100 mL of King’s B medium. The bacterial culture was centrifuged at 2800 rpm for 20 min, the supernatant discarded and the pallet resuspended in MgSO4 (0.1 M) (Siddiqui and Shahid, 2003).

Mycorrhizae inoculums: Two mycorrhizal species were used in this work. The air dried inoculum of Glomus intraradice and G. mosseae was obtained from the Soil and Water Research center in Iran, Tehran and prepared on Sudan grass (Sorghum bicolor L.) grown in sandy loam soil mixed with washed river sand in the ratio of 3:2 (v/v) respectively for four-month-old then spores of fungi were isolated by sieving and decanting and were counted under stereomicroscope to the help of a fine hair brush. There were 32 G. mosseae spores and 26 G. intraradices, spores per soil gram.

Trichoderma inoculums: Two Trichoderma harzianum strains (T1 and T2) were used in this work (Table 1). Mention strains were obtained from the Department of Plant Pathology, University of Tehran, in Iran. Two strains of T. harzianum that had been evaluated for their nematicide activity in vitro beforehand, was cultured in Agar water medium. Plates was incubated in 25°C for 5 days because the fungi growth as well as. A mixture of peat and wheat bran (1:1, v/v) has been used extensively as a medium for delivery of T. harzianum preparations (Sivan et al., 1984). In this modified preparation, the pH remains constant and low (5.5) during the entire growth period, thus preventing bacterial contamination. The mention mixture was autoclaved for 15 min, twice in two days continuously. The fungi suspension (10 mL, 108 CFU g-1 per mL) was inserted to the steriled mixture in the Erlen. The mention mixture was incubated for 18 days in 25°C because the fungi growth as well as (Spiegel and Chet, 1998).

Primary investigation of the effective metabolite production in bacterial antagonistic specifics: Some tests (hydrogen cyanide, protease, Salicylic acid and siderofor) was did to measure of the ability and secondary metabolite production meter in Fluorescent pseudomonad bacteria for primary screen of the thirty bacterial strains (Schaad et al., 2001). Eleven strains were chief in mention tests that selected for survey of the nematicide activity.

Biocontrol of root-knot nematode by Flourescent Pseudomonad bacteria (In greenhouse): Three-week-old tomato seedlings (Early Urbana) were planted in plastic pots (12 cm diameter) filled with 700 g unsterilized sandy-loam (pH 8.1; moisture retaining capacity 38%) and cultivated in a glasshouse (19-24 and 29-33°C day and night temperatures, respectively). After one week, the plants were treated with one of the following bacteria (three bacterial strains that had been evaluated for their nematicidal activity in vitro beforehand) by pipetting 30 mL of the bacterial suspension (diluted to OD600 = 0.5 which corresponds to 2.1x109 CFU mL-1) into soil around the root system. Each treatment was replicated 5 times. Control plants received 30 ml of one-fourth concentration Ringer solution. Two days after bacterial application, 2000 freshly hatched juveniles of M. javanica were inserted to the soil by making three holes around the seedlings (Siddiqui and Shaukat, 2002c). The plant samples were taken eight-week-old after nematode inoculation. Tomato roots were carefully rinsed in tap water, separated from the shoot, blotted dry and weighed. The numbers of galls produced on the entire root system were counted using a hand lens and their diameter was measured. Final larva mortality in percent and population densities of the nematode in the roots were estimated following the method outlined by Siddiqui and Shaukat (2002b).

Biocontrol of root-knot nematode by mycorrhizal (in greenhouse): Plants were prepared and cultivated similar to the experiment of eight. After one week, the plants were treated with one of the following fungi, 40 g soil (per gram 32 spores inclusive 1200 G. mosseae spores) and 50 g soil (per gram 26 spores inclusive 1200 G. intraradices spores) into soil around the root system for each treatment separately. Each treatment was replicated 5 times. Two-week after fungi application, 2000 freshly hatched juveniles of M. javanica were inserted to the soil by making three holes around the seedlings (Bhat and Mahmood, 2000). The plant samples were taken eight-week-old after nematode inoculation and shoot and root dry weight, number of galls per root system, gall diameter, nematode population in soil and root and percent root colonization of AM fungus were measured and recorded.

Biocontrol of root-knot nematode by Trichoderma (In greenhouse): Plants were prepared and cultivated similar to eight-experiment. After one week, the plants were treated with one of the following fungi. The trichoderma inoculum was mixtured (1% w/w) with soil in each pot (Sharon et al., 2001). Each treatment was replicated 5 times. In this section, three-time-factor was used in consist: In one-time-fictor (t1), Trichoderma fungi and 2000 j2 (juvenile larva) was inserted to the soil by making three holes around the seedlings and three-week-old tomato seedlings was transplanted after 18 days. In two-time factor (t2), seedlings was transplanted and inoculated with fungi and nematode simultaneity. In three-time factor (t3), seedlings was transplanted and inoculated with fungi then inoculated with nematode after 18 days (Spiegel and Chet, 1998). The plant samples were taken eight-week-old after nematode inoculation and shoot and root dry weight, number of galls per root system, gall diameter, nematode population in soil and root were measured and recorded.

Biocontrol of root-knot nematode to integrate flourescent pseudomonad bacteria and mycorrhzal fungi (In greenhouse): Plants were prepared and cultivated similar to mentioned experiment. After one week, the plants were treated with one of the following fungi, 40 g soil (per gram 32 spores inclusive 1200 G. mosseae spores) and 50 g soil (per gram 26 spores inclusive 1200 G. intraradices spores) into soil around the root system for each treatment separately (Shafi and Mahmood, 2000). Each treatment was replicated 5 times. Plants were treated with one of the following bacteria (three bacterial strains that had been evaluated for their nematicide activity in vitro beforehand) by pipetting 30 mL of the bacterial suspension (diluted to OD600 = 0.5 which corresponds to 2.1x109 CFU mL-1) into soil around the root system. Control plants received 30 mL of one-fourth concentration Ringer solution. Two-week after fungi and bacteria application, 2000 freshly hatched juveniles of M. javanica were inserted to the soil by making three holes around the seedlings. The plant samples were taken eight-week-old after nematode inoculation and shoot and root dry weight, number of galls per root system, gall diameter, nematode population in soil and root and percent root colonization of AM fungus were measured and recorded.

The evaluation of nematode population in root: The number of juveniles, eggs and females in the roots were also estimated. The roots were cut into small pieces and mixed.; gr root was macerated for 45s in blender to recover nematode eggs, females and larvae. The total root population of nematodes was determined with the number of larvae and females present in 1 g root and by multiplying it with total weight of root.

The Larva mortality of nematode in percent: The Larva mortality of nematode in percent was determined in infected roots against control roots via Abbott’s formula (Abbott, 1925):

Where:
Tn = Nematode population after inoculation
Cn = Nematode population in control
n = Nematode population
T = Treatment (fungi or bacteria)
C = Control (non treatment)

The evaluation of root colonization percentage by mycorrhizal fungi. The proportion of root colonized by G. mosseae was determined by the grid line intersecting method (Giovannetti and Mosse, 1980) after clearing the roots with KOH (Phillips and Hayman, 1970) and staining the roots in 0.05% trypan blue lactophenol.

Statistical analyses: Data were subjected to one-way analysis of variance (ANOVA) followed by the Complete Randomized Design (CRD) test using SAS software (ver. 9.1, USA). Meanwhile each treatment was replicated 5 times.


RESULTS
Primary investigation of the effective metabolite production in bacterial antagonistic specifics (In vitro): Its Information has been shown in under Table 1.

Investigation of nematicide activity (In vitro): Its Information has been shown in under Table 2 and 3.

Biocontrol of root-knot nematode by some antagonistic agents: The rate of growth and disease was different in plants of infected to nematode so that they were affected by antagonistic agents against the just nematode infected plants (as contaminated control). The differences were significant in all indices at level of five percent (Table 1, 2).

Growth indexes: Among all the treatments of infected to nematode and the antagonistic agents, root dry weight against contaminated control was in range of 29 to 76% and shoots dry weight was in range of 22 to 75%. The treatments of G2P2M and T1Mt2 in nematode infected plants had the best and worst effect in enhancement of the dry weight. Among the combined treatments, G2P2M and G2P3M increased the dry weight of the nematode infected plants, followed by 76 and 58 %, respectively so that they had the highest and lowest effects, respectively.

Table 1: Investigation of the effective metabolite production in bacterial antagonistic
P1 is the bacterial strain P. aeruginosa 7NSK2 and P3 (UTPF5), P2 (UTPF86) is the bacterial strain P. fluorescens

Table 2: Mean comparative of Root-Knot nematode control by three strains of bacterial (In vitro)
Means with the same letter are not significantly different at 1% level according to Dunkan. UTPa95 is the bacterial strain P. aeruginosa 7NSK2 and UTPF5, UTPF86 is the bacterial strain P. fluorescens that they were used from b collection, of University of Tehran, in Dep. Plan protection. Numbers is in percentage

Table 3: Mean comparative of Root-Knot nematode control by two strains of Trichoderma (In vitro)
Means with the same letter are not significantly different at 1 % level according to Dunkan. T1and T2 is two isolates of Trichoderma harzianum that they were used from mycology collection, of University of Tehran, in Dep. Plan protection. M is the plants inoculated by Meloidogyne javanica. Numbers is in percentage

Table 4: Mean’s comparison of the effects of some antagonistic agents on the plant growth
Means having common letters, of Duncan test at 5% level have not significant difference from each other treatment. M is the infected control that it has M. javanica nematode. P1 is the bacterial strain P. aeruginosa 7NSK2 and P3 (UTPF5), P2 (UTPF85) is the bacterial strain P. fluorescens. G is the Mycorrhizal species which include the G. mosseae (G1) and G. intraradices (G2). T is the T. harzianum isolate. t is the inoculated time factor. GPM is the integration of mycorrhizal species (G1 and G2), P. fluorescens strains and M. javanica.

The difference was significant in these two treatments (p<0.05). Among the Pseudomonas bacteria and nematode treatments, P2M and P3M increased the dry weight of the nematode infected plants, followed by 63 and 40%, respectively, so that they were the highest and lowest in treatments. The difference was significant in these two treatments (p<0.05). Between mycorrhizal and nematode treatments, G1M and G2M increased the dry weight of the nematode-infected plants, followed by 46 and 43%, respectively. The difference was not significant between these two treatments (p<0.05). Among the Trichoderma fungus and nematode treatments, T2Mt1 and T1Mt2 increased the dry weight of the nematode-infected plants, followed by 67 and 26%, respectively so that they were the highest and lowest in treatments, respectively. The difference was significant in these two treatments (p<0.05). Amount of damage to the root and shoot dry weight of infected plants against control were in range of 66 to 74%. Reduction of the dry weight was significant in nematode-infected plants against other treatments (p<0.05) (Table 4).

Disease indexes: Among all the treatments of nematode infected plant and antagonistic agents, reduction of the gall number against control was in range of 16 to 78% and reduction in the gall diameter was in range of 12 to 77%. The treatments of G2P2M and T1Mt2 caused the highest and lowest reduction in the number and gall diameter gall. Their differences was significant against other treatments (p<0.05). Among the combined treatments, G2P2M and G2P3M decreased the gall diameter and gall number against the nematode infected plants, followed by 78, 39, 77 and 54%, respectively. These treatments had the highest and lowest effect in reduction of the gall number and gall diameter, respectively. The difference was significant in these two treatments (p<0.05). Among the Pseudomonas bacteria and nematode treatments, P2M and P3M decreased the gall number and gall diameter against the nematode infected plants, followed by 48, 23, 60 and 37%, respectively. These treatments had the highest and lowest effect in reduction of the gall number and gall diameter, respectively. The difference was significant in these two treatments (p<0.05). Among the treatments of the plants infected to mycorrhizal fungi and nematode, G1M and G2M decreased the gall diameter and gall number against the just nematode infected plants, followed by 32, 29, 49 and 48%, respectively. The difference was not significant in these two treatments Among the Trichoderma fungus and nematode treatments, T2Mt1 and T1Mt2 decreased gall diameter and gall number, followed by 59, 16 and 71 and 12%, respectively. These treatments had the highest and lowest effect in reduction of the gall number and gall diameter. The difference was significant in these two treatments (p<0.05). In addition, gall number and gall diameter was significant in treatments against contaminated control at the level of five percent (Table 5). Number of eggs and second-stage juveniles (J2) per gram root were significantly lower in fungal treatments (p<0.05).

Table 5: Mean’s comparison of the effects of some antagonistic agent in reduction of nematode infection
Means having common letters, of Duncan test at 5% level have not significant difference from each other treatment. M is the infected control that it has M. javanica nematode. P1 is the bacterial strain P. aeruginosa 7NSK2 and P3 (UTPF5), P2 (UTPF85) is the bacterial strain P. fluorescens. G is the Mycorrhizal species which include the G. mosseae (G1) and G. intraradices (G2). T is the T. harzianum isolate. t is the inoculated time factor. GPM is the integration of mycorrhizal species (G1 and G2), P. fluorescens strains and M. javanica.

DISCUSSION
The nematode penetration to the root of plants so that treated with UTPF86 strain was more than P. aeruginosa 7NSK2 (P1) but total galls in inoculated roots with UTPF86 was lower than that may be caused by highest production of salicylic acid in UTPF86 strain (Table 1). Salicylic acid (SA) is known to play a critical signaling role in the activation of plant defense responses after attacking of pathogen (Klessig et al., 2000). This indicates that ISR by rhizobacteria is independent of in planet accumulation of SA. In recent literature, SA has been reported as an endogenous signal for the activation of certain plant defense responses, inclusion of expression of PR gene and enhanced resistance to pathogens (Conrath et al., 1995). In addition, the performance of UTPF86 strain in the biocontrol of root-knot nematode in greenhouses was better than the other strains. Also similar studies showed that production of salicylic acid by Pseudomonas sp. increases the defense mechanisms of host and the resistance will induce systemic or local defense and the pathogen indirectly dead. Application of SA is known to express resistance reaction in cowpea roots against M. incognita (Nandi et al., 2002) but the role of production of SA by rhizobacteria against M. javanica has not been investigated yet. Based on the presented results it is assumed that fluorescent pseudomonad mediate systemic resistance against root-knot nematode in tomato by pathway of SA-independent transduction (Siddiqui and Shaukat, 2004). The researches are shown that CAT extracts from leaves is found to be less sensitive to SA inhibition than root CAT SA (1 mM) completely inhibits root CAT, whilst the same concentration causes approx. 60% inhibition of CAT is in leaf (Molinari and Loffredo, 2006) Generally, SA treatment don’t seem to limit the degree of infestation of J2 significantly, although, it may have been an inhibited effect on the reproduction of nematode index (Molinari, 2005).

In this research, P. aeruginosa 7NSK2 (P1) strain was cultured, the presence of hydrogen cyanide UTPF86 was shown and Pykrete paper changed from yellow to reddish brown color. It seems, there is a positive relationship between hydrogen cyanide by bacteria and the rate of nematode mortality. Nematode population in soil and rhizosphere treated to 7NSK2M was lower than other treatments. It is probably due to the production of volatile compound of hydrogen cyanide that it is highest. Gallagher and Manoil (2001) founded that production of HCN by P. aeruginosa plays a key role in killing the juveniles of Meloidogyn sp., as inhabiting soil against nematode (Siddiqui et al. 2006). P. aeruginosa 7NSK2 (P1) strain UTPF86 strain produced hydrogen cyanide more than other strains in laboratory tests. On the other hand its nematicide also was higher in the laboratory. The penetration of nematode to the inoculated roots with P. aeruginosa 7NSK2 (P1) strain was lower than other strains. Siddiqui et al. (2006) investigated the role of cyanide production by Pseudomonas fluorescens CHA0 in the suppression of the root-knot nematode on tomato and suggested for effective control of M. javanica, there is a direct relation between production of HCN by CHA0 and mortality of nematode (Siddiqui et al., 2006). It cannot be stated with certainty whether HCN production by 7NSK2 (P1) strain is the only mechanism responsible for the suppression of root-knot nematode against other mechanisms (Bakker et al., 1991) such as, alteration of root exudates that reduce attraction of nematode, enhance host defense mechanism to systemic resistance and/or blockage of the potential entry sites for nematode following increase number of bacterial cells in the rhizosphere and roots can be involve in the reduction of invasion of nematode and development of ultimate root-knot (Siddiqui et al., 2003). Therefore, it is concluded DAPG, pyoluteorin and hydrogen cyanide play a critical role (but not primary) in nematode mortality through neuromuscular disorders (Siddiqui and Shaukat, 2004). Studies of human and animal cyanide poisoning indicate that the poison strongly affects neurological tissue (Way, 1984) and it is possible that this poison causes the nematode mortality also reflects hypersensitivity of neuromuscular tissues (Gallagher and Manoil, 2001).

7NSK2 (P1) strain in laboratory tests produces significant amounts of protease and it had the highest percentage of egg non-hatching. The AprA protease of CHA0 strain contributes in biocontrol of M. incognita directly or indirectly. These data support the involvement of AprA protease in the inhibition of egg hatching and juvenile’s mortality. However, AprA protease may not be the only antinematode factor of bacterial strain, antibiotic compounds produce under GacA control also have a role in biocontrol of nematode (Siddiqui et al., 2005). Briefly, this results demonstrate that protease enzyme in strain 7NSK2 (P1) is effective to biocontrol of M. javanica directly or indirectly.

In plants inoculated by nematodes and mycorrhiza indicated very small primary galls because the endomycorrhiza interfered with the development of nematodes. In endomycorrhizal root system was observed significant reduction in the number of M. incognita larvae that they develop into adult. Inoculation of tomato plants with G. mosseae and G. intraradises 14 days after nematode infection, significantly reduced the number and size galls. Our results are in agreement with those of Kellam and Schenck (1980), who reported a similar effect of G. mosseae on tomato plants infected by root-knot nematode.

The growth’s response of nematode-infected plant as a result of mycorrhiza assisted nutrition is commonly mentioned in the literature as the capacity of mycorrhiza to improve plant’s health, confer protection, prophylactic effect, or increase host tolerance against the nematode pest and etc. (Elsen et al., 2008). Increase in plant growth after colonization of root by AM fungi is due to improvement in the mineral nutrient status of host plant. During AMF colonization, there is little evidence that classic plant resistance responses occur at high levels. However, these responses are greatly stimulated when a subsequent challenge with a pathogen occurs (St-Arnaud and Vujanovic 2007; Gianinazzi-Pearson et al., 1996) but a good colonization of AMF is a prerequisite for this response (Cordier et al., 1998; Slezack et al., 2000). It seems that AMF colonization acts as a priming system, immunize the plant against a pathogen (Elsen et al., 2008).

In the present study, G. intraradices improved plant growth of nematode-infected plants by reduction in reproduction of nematode. This object has been shown for other AM fungus. We presumed inhibition of disease by G. intraradices that it might be related to the increase in content of phosphorus, because a significant increase in phosphorus and dry mass of roots was observed (data not shown). However, an increase in phosphorus may not be the sole cause of disease inhibition. In addition, change of nutrient uptake and root system, a mycorrhizosphere effect and activation of plant defense mechanisms are thought to be responsible for disease inhibition by AM fungi (Demir and Akkopru, 2005).

The inoculation of mycorrhiza and the number of spores was not significantly affected in infection of nematode. Inoculation of mycorrhizal before nematode intensifies and spores are formed more than other times. In this case, effective control of nematodes was done against inoculation of mycorrhizal and nematode simultaneity. In this research, the total spore of mycorrhiza was sufficient for inoculation. Based on references, 1200 spores inoculated for each plant. It is possible that higher initial inoculum density of nematode might affect on the initial colonization of the fungus. However, it is doubtful that enhancement of the fungus initial inoculum would exert such an effective agent on the number of penetrated nematodes to tomato seedling, because the nematode requires only a few hours to infection of root while the fungus requires at least 10 days to become established in the roots (Kellam and Schenck, 1980).

In this study, root colonization (%) by G. mosseae (G1) was 2% more than root colonization (%) by G. interaradices (G2).

If root colonization (%) is more each value the nematode penetration and gall number will reduce. Although, mycorrhizal plants have higher root systems than non-mycorrhizal plants, the total number of galls produces on each mycorrhizal plant is less than the number of produced galls on non-mycorrhizal plants. Fewer galls in mycorrhizal roots demonstrates reduction of ability of the nematode penetration or the presence of the fungus may influence the development of giant cells which can, in turn, interfere with development of nematode. Our results are in agreement with those of Kellam and Schenck (1980).

The time of inoculation is important in nematode biocontrol by Trichoderma sp. In this study, T2Mt1 was the best treatment to biocontrol of nematode. In this treatment, nematode responsed against Trichoderma sp. isolates for 18 days then transplanted three-week-seedling. The concentration of the acidic component(s) in the soil is very low and a certain period of time may therefore, was needed for its accumulation before its effect on J2 becomes significant. Thus in those experiments in which J2 were exposed to T. harzianum for 18 days prior planting, was observed maximum nematicidal efficacy; but when J2 were exposed to the fungus during planting. In treatments where T. harzianum had been assigned to the root-ball, was not recorded nematicidal activity. For all studied species, the use of wheat bran increased the production of spore. However, the enhancement was extremely high for T. harzianum and T. viride nutritional supplementation needed production of high spores’ (Cavalcante et al., 2008).

In this study, top wet weight in T2Mt1 and T2Mt3 treatments had not significant difference (in 0.05 levels) but its difference was significant in disease indexes (in 0.05 levels). Top wet weight had significant increase in pre-colonization plants by T. harzianum but they had not significant reduction in disease indexes (in 0.05 levels) also, Sharon et al. (2001) resulted that biocontrol of pre-colonization plants was not satisfactory but stem wet weight had significant increase in these plants (in 0.05 levels). Treatment of the soil by T. harzianum isolates at the time of transplanting caused the small reduction in nematode galling. However, total gall was reduced significantly when treatment was took place one week before transplanting-allowing to extend exposure of the nematode against the antagonist. Our results are in agreement with those Fattah and calleague (Al-Fattah et al., 2007).

In this research, larval mortality in percent and parasitism on egg’s nematode was great by T. harzianum isolates that it may be because of proteolytic or production of chitinolytic enzyme by the fungus. The germ has also been found to trigger proteolytic and production of chitinolytic enzyme by the fungus (Sharon et al., 2007). This combination of enzymes is required to disrupt the eggshell (Tikhonov et al., 2002; Khan et al., 2004), although, chitinolytic capacity is probably the most important activity on the eggshells (Morton et al., 2004). While T. atroviride presented the greatest efficiency for parasitism of J2s, probably because of its high proteolytic activities (Sharon et al., 2007).

However, secondary metabolites from fungi also contain compounds which are toxic to plant parasitic nematodes (Al-Fattah et al., 2007). Trichoderma may be effective as an egg pathogen; and a pre-plant treatment when eggs are present, not J2, may give better results (Al-Fattah et al., 2007). Additional studies are needed to clarify the interaction of Meloidogyne spp. with T. harzianum as a biocontrol agent in terms of the physiological roles of enzyme activities in response to attraction of nematode and fungal colonization.

In this research, the nematode population was very high in the soil of T1Mt3 and T2Mt3 treatments. The nematode penetration in root was less in the treatment of Trichoderma fungi and nematode simultaneity. This order may be because of pre-colonization of root by fungi. Our results are in agreement with those of Chet et al. (2006). Some Trichoderma rhizosphere-competent strains colonize also entire the surface of root with morphological features reminiscent of those seen during mycoparasitism. Penetration to the root tissue is usually limited to the first or second layers of cells and only in the intercellular spaces (Chet et al., 2006).

The strain UTPF86 (P2) produced the maximum of salicylic acid in vitro thus its role is important in induced resistance (Table 1). In other hand, induced resistance is one of the biocontrol mechanisms by mycorrhizal fungus. Therefore, it was not far that combined use of Glomus sp. and UTPF86 (P2) strain that it had high percentage of colonization, caused the greatest increase and the plant growth with adverse effects on gall number and gall diameter than other treatments. Also 7NSK2 (P1) strain produced cyanide hydrogen more than other strains in vitro (Table 1). This material has neurosis and maims effect on nematode larva and reduces the nematode penetration. Combined use of Glomus sp. and 7NSK2 (P1) sitted in secondary station after combined use of Glomus sp. and UTPF86 (P2) strain against other treatment. Combined use of Glomus sp. and Pseudomonas sp. caused the greater increase and plant growth coinciding by adverse effects on nematode multiplication and morphometric. When Pseudomonas sp. and Glomus sp. were applied together, the increase in tomato growth was greater than when either agent was applied alone.

Pseudomonas sp. was better in reduction of galls and reproduction of nematode against Glomus sp. whereas use of the two together was better than when either agent was applied alone. Namely, combined application of the Glomus sp. and the Pseudomonas sp. was better than when either agent was applied alone. Root colonization by Pseudomonas sp. was increased when inoculated by Glomus sp. more than inoculation of single. Also inoculation of Pseudomonas sp. increased the root colonization by the AM fungus. The present study demonstrated that AM fungus and plant-growth-promoting rhizobacterium can coexist without adversely affecting one another. In fact, suitable combinations of these biocontrol agents can further increase the plant growth and resistance to pathogens.

These results will help to define optimal conditions for better growth of tomato as well as and for management of root-knot nematode in infested field.


ACKNOWLEDGMENTS
This research has been done by supporting of pole biological control of plant pests and diseases, College of Agriculture and Natural Resources, University of Tehran in the laborator of biological control of plant diseases.


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