Effects of rhizobacteria on Meloidogyne javanica infection on eggplants

The effect of two rhizobacteria, Pantoea agglomerans and Bacillus subtilis on parasitism of the rootknot nematodes, Meloidogyne javanica on eggplants was evaluated in laboratory and greenhouse experiments. The impacts of the bacteria application as seed treatment, root dipping, and soil drench on early nematode penetration into plant roots, as well as, on eggplant growth were tested. The number of penetrated second stage juveniles of M. javanica was significantly reduced after P. agglomerans and B. subtilis application at concentration of 10 8 CFU ml -1 . P. agglomerans increased the eggplant fresh shoot and root weight following seed treatment. In the greenhouse experiment the bacteria were applied alone and in combination with a carbamate nematicide, Oxamyl (Vydate®). P. agglomerans was able to suppress M. javanica development into plant roots through a first half of eggplant cropping season. At the end of cropping season B. subtilis significantly reduced root gall index and number of nematode juveniles in soil and roots. This bacterium applied in combination with Oxamyl was the most efficient against M. javanica reproduction. Thereby, B. subtilis might be considered as a good candidate for biological or integrated control of the root knot nematodes.


INTRODUCTION
Root-knot nematodes are one of the main problems for protected crops, especially in south-eastern countries of Europe where climatic conditions favour their development.Species of Meloidogyne have a wide range of plant hosts and cause severe damage to vegetable crops.Crop losses due to Meloidogyne exceed 32% on tomato, 30% on melon and 20% on eggplant (Netscher and Sikora, 1990).Eggplant (Solanum melongena L.) is a traditional vegetable crop grown in Bulgaria.One of the most frequently found root-knot nematode species on eggplant is Meloidogyne javanica, present alone or in combination with other Meloidogyne species.In Bulgaria annual crop losses due to M. javanica largely depend on vegetable crops, their resistance to the nematode and the methods of control that have been used (Samaliev and Baicheva, 2010;Masheva et al., 2016).Present strategies for nematode management involved some cultural practices such as crop rotations and resistant *Corresponding author.E-mail: m.mohamedova@au-plovdiv.bg.
Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License varieties.Crop rotation is widely use but is not effective when applied for control of polyphagous pests.Due to the lack of resistance genes in many vegetables, resistant varieties are not readily available (Dias-Arieira et al., 2012).During the last ten years, a series of field studies employed throughout the world demonstrate that fumigant and non-fumigant nematicides are still efficient for the control of the root-knot nematodes (Giannakou and Anastasiadis, 2005;Choudhary et al., 2006;Hadian et al., 2011).However, the nematicides are usually toxic to highly toxic and there is much concern about their residual effects on the environment and human health.For this reason the interest in biological control as an alternative to chemical control of Meloidogyne has increased.
Bacteria associated with roots and rhizosphere of many plant species have been extensively tested for the control of various soil borne pathogens (Haseeb et al., 2006;Vasebi et al., 2015) including plant parasitic nematodes (Kluepfel et al., 1993;Insunza et al., 2002;Aballay et al., 2013).Currently, Bacillus spp.and Pseudomonas spp.are the most studied bacteria as biological control agents of root-knot nematodes (Hallmann et al., 2009).Other rhizosphere bacteria expressing antagonistic potential against Meloidogyne include, Burkholderia, Paenibacillus, Pantoea and Seratia (Duponnois et al., 1999;Oliveira et al., 2007;Abd-Elgawad and Kabeil, 2010).Control of nematodes by rhizosphere bacteria is achieved by mechanisms such as: direct antagonism through the production of secondary metabolites; interference with plant-nematode recognition; competition for nutrients; plant growth promotion; and induced systemic resistance (Tian et al., 2007).The low level in consistency of control of nematodes by many bacteria under field conditions is most likely due to the variability of the soil properties (physical and chemical), microbial activity in the soil as well as due to other environmental factors (Kerry, 2000).Thus, achieving efficient and consistent performance of biocontrol agents requires a more knowledge of effective screening techniques, and development of appropriate formulation and application techniques.The objectives of the present studies were: (i) to determine the effect of two rhizobacteria on early root penetration and gall formation of M. javanica into eggplant roots; (ii) to observe the influence of the bacteria on the plant growth; to examine the effect of bacteria alone or in combination with an organophosphate nematicide under greenhouse condition.

Strains of rhizobacteria
Rhizobacteria were isolated in Bulgaria from rhizosphere of tomato plants.Two selected strains of the rhizobacteria, Pantoea agglomerans and Bacillus subtilis (collection of LMBT, Agricultural University-Plovdiv) which can significantly reduce incidence of galls of nematodes on tomato were used in this study.

Bacterium culture and identification
The strains of P. agglomerans and B. subtilis were grown in the dark for 48 h at 24°C on tryptic soy broth agar (TSBA).For inoculum production a loop of the bacteria was transferred into 100 ml of TSB and allowed to multiply for 48 h on a rotary shaker (160 rpm) at the same temperature.Bacterial suspensions were centrifuged at 4000 rpm for 20 min, and the bacterial pellet was resuspended in sterile ¼ strength Ringer's solution (Merck).The bacterial suspension was adjusted to a final concentration of 10 8 CFU ml -1 by dilution with Ringer's solution.The bacterial strains identifications were determined by FAME Analysis, following by BIOLOG Analysis for P. agglomerans.

Nematode culture and sterilization
M. javanica was obtained from cultures derived from single egg masses maintained on tomato (Solanum lycopersicum L., cv.Ideal) in a greenhouse at 24 to 26°C.Mature egg masses of M. javanica were hand-picked, using a sterilized needle and forceps from heavily infested tomato roots.These egg masses were sterilized in streptomycin sulphate (0.1%) for 45 min and rinsed in sterile distilled water (SDW) before being used as inoculum in experiments.Second stage juveniles (J2) of M. javanica were extracted from infested tomato roots following the procedure described by Stetina et al. (1997).Galled roots with egg masses were washed free of soil, cut into small pieces, placed in 1.5% NaOCl and homogenized at 8000 rpm (T 18 digit al ULTRA-TURRAX) (Hussey and Barker, 1973).The suspension was poured onto cotton-wool filter, incubated at 24 to 26°C and hatched J2 were collected every 24 h.The second stage juveniles were also sterilized in streptomycin sulphate (0.1%) for 15 min and rinsed in SDW before being used in experiments.

Effect of rhizobacteria on the penetration rate of M. javanica juveniles
The bacteria were applied as follows: 1. Seed treatment: The eggplant seeds, cv.Lych were soaked in the suspensions of P. agglomerans and B. subtilis for 20 min and then planted into pots containing 500 ml soil/sand mixture (1:1, v/v).Each pot received 1 seed.After four weeks, the plants were inoculated with 2500 J2 of M. javanica per pot.The inoculation of nematodes was carried out by drenching 5 ml suspension of J2 in distilled water into the potted soil arround the roots.The control treatments received 5 ml distilled water; 2. Root dipping: Roots of three-week-old eggplants were dipped for 20 min into the bacterial suspensions and then planted into pots containing 500 ml soil/sand mixture (1:1, v/v).After a week, the plants were inoculated with 2500 J2 of M. javanica per pot.3. Soil drench: Ten milliliter of bacterial suspensions was pipetted onto the soil surface around three-week-old eggplants.Plants were inoculated with 2500 J2 of M. javanica one week after bacteria application.The inoculation of nematodes was carried out by drenching 5 ml suspension of J2 in distilled water into the potted soil arround the roots.The control treatments received 5 ml distilled water.Plants were kept in a growth room with following conditions, 28±1°C temperature, 70±5% relative humidity (RH) and 16:8 (L:D) photoperiod.All the experiments was replicated eight times, end terminated two weeks after nematode inoculation.
At the end of the experiments eggplant fresh shoot and root weight was measured and the penetration rate of M. javanica was recorded.Penetrated J2 of M. javanica into eggplant roots was determined after staining the roots with red food color (33,3% (v/v) solution of acid fuchsine available in ROTH®) following the method described by Thies et al. (2002).Stained roots were washed with tap water and homogenized at 8000 rpm.The number of J2 extracted from the root tissue was counted under a stereomicroscope.

Greenhouse efficacy experiment in eggplant production system
The experiment was conducted in a commercial greenhouse near Svilengrad, Southern Bulgaria.No nematicides had been applied in the greenhouse for at least 2 years prior to the experiment.Plots were 20 m -2 (2 m × 10 m) each.The treatments were arranged in randomized complete block design with four replications.One week before planting of the eggplant seedlings all plots were irrigated with 13 L of water m -2 and ploughed with rotary cultivator to ensure moisture uniformity.The distances between and within rows were respectively 0.64 and 100 cm.The greenhouse temperature during the cropping period was 18 to 43°C (March-June).Plants were irrigated every three days with a drip system and fertilized every week with compound fertilizer (N: 15 %, P: 15%, K: 15%).
Two weeks eggplant seedlings, cv.Lych were transplanted in the greenhouse on 15 March, 2014.Before planting, the soil into the band had been infested with M. javanica at a rate of 5000 eggs and J2 per plant.The experiments included the following treatments: 1.Control with nematodes; 2. Susspension of P. agglomerans at dose 10 ml per plant (per treatment) on 1 th , 8 th and 15 th day after transplanting; 3. Susspension of B. subtilis at dose 10 ml per plant (per treatment) on 1 th , 8 th and 15 th day after transplanting; 4. Oxamyl (Vydate® 10L) at dose 1 ml plus P. agglomerans at dose 10 ml per plant (per treatment) on 8 th and 15 th day after transplanting; 5. Oxamyl (Vydate® 10L) at dose 1 ml plus B. subtilis at dose 10 ml per plant (per treatment) on 8 th and 15 th after transplanting; 6. Oxamyl (Vydate® 10 L) at dose 1 ml per plant (per treatment) on 1 th , 8 th and 15 th day after transplanting.
The following observations were made: 1.During the cropping season -numbers of females of M. javanica (45 th day after transplanting the eggplants); 2. At the end of cropping season (94 th day after transplanting) -root gall index, numbers of eggs and J2 in the soil (g), and numbers of eggs and J2 in the roots (g).The soil samples on 45 th day were taken by means of an auger (2 cm/d) at a distance 10 cm from the plant stem, upon which the roots were separated, washed, stained in acid fuchsine and the numbers of females were counted by direct examination of the roots using a stereomicroscope and determined per gram of fresh roots.At the end of cropping season, the nematodes were extracted from soil samples using a modified Baermann funnel technique (Southey, 1986).Eggs were extracted from roots samples according to hypochlorite procedure (Hussey and Barker, 1973).Root gall index of eggplants was assessed according to a 0 to 10 scale (Bridge and Page, 1980).

Statistical analysis
Obtained data were subjected to one way analysis of variance (ANOVA) and the treatment means were compared with the control plants, according to the Dunnet's test (P<0.05).

Effect of rhizobacteria on the penetration rate of M. javanica second stage juveniles
The experiments showed that both tested rhizobacteria Mohamedova et al. 4143 influenced the penetration of J2 of M. javanica into the eggplant roots (

Greenhouse efficacy experiment in eggplant production system
At the mid-season ( 45th day) plants treated with the rhizobacteria alone or in combination with Oxamyl had significantly lower number of females present in the roots than the plants in the control plots (Table 3, P<0.05).The lowest number of M. javanica females was observed in the treatment B. subtilis plus Oxamyl (52).Pantoea agglomerans plus Oxamyl, as well as Oxamyl applied separately reduced the number of females (78 and 67, respectively) compared with the control (168).Both, P. agglomerans and B. subtilis affected the development of M. javanica J2 and the number of reproductive females was 128 and 111, respectively.At the end of the cropping season, the gall index varied from 5.2 for the plants in the control plots to 1.9 for the plants treated with Oxamyl -B.subtilis combination.Significantly fewer galls were also observed on the plant roots in the plots treated with P. agglomerans plus Oxamyl (3.0).Bacillus subtilis, as well as Oxamyl decreased the number of eggs and J2 in the soil and in the roots, but there was no statistical difference between the treatments.Further, there was no significant difference between P. agglomerans and the control in all three parameters of assessment -gall index, eggs and J2 in the soil and eggs and J2 in the plant roots (Table 3, P<0.05).

DISCUSSION
During the last ten years numerous scientific articles have acquainted with achievements of successful Meloidogyne control using rhizobacteria (Mendoza et al., 2008;Vetrivelkalai et al., 2010;Majzoob et al., 2012;Bonaterra et al., 2014).In general, our study has also  demonstrated the efficacy of the rhizobacteria as biological control agents of M. javanica on eggplants.In laboratory pot experiment, P. agglomerans and B. subtilis caused a reduction of penetration of J2 into the roots up to 44.6 and 66.8%, respectively, following soil drench application.In the same experiment reduction of juvenile penetration was also observed when the bacteria applied as a root dipping.However, the number of J2 in the eggplant roots at this treatment was higher than the number of J2 at the soil drench treatment.Application of B. subtilis as a seed treatment resulted in significantly lower number of juveniles in the root systems compared with the control and P. agglomerans treatment.Although, the fresh root and shoot weight of eggplants treated with P. agglomerans and B. subtilis were higher compared with the control the differences were not significant.Amellal et al. (1999) reported that P. agglomerans is a competent colonizers of the rhizosphere and therefore a promising candidate for biological control.Plant-beneficial effect of P. agglomerans was observed by Majzoob et al. (2012) and Bonaterra et al. (2014) on cucumber and tomato, both infested with M. javanica.Several Bacillus strains have been assessed for their potential as a biological control agent of Meloidogyne species.Mendoza et al. (2008) reported in vitro activity of a Bacillus firmus strain against eggs and J2 of M. incognita.

Strains of Bacillus megaterium, Bacillis pumilus and
Bacillus mycoides reduced the number of egg masses of M. incognita on tomato by 31, 30% and 39% respectively (Mekete et al., 2009).The results obtained in the current experiments indicate that the effects of rhizobacteria treatments were similar to those described above.Our results also support the finding of Munif et al. (2013), who reported that P. agglomerans reduced M. incognita J2 root penetration by 31% following soil drench application.
In addition, the authors discussed that application of endophytic bacteria with root dipping and soil drench is more effective for suppressing the penetration of nematode juveniles compared with seed treatment application of (Munif et al., 2013).
According to Thies et al. (1992) biological control of root-knot nematodes usually has not been a stand-alone practice in control of these nematodes.Our results on the other hand, showed that B. subtilis can potentially reduce M. javanica population in a greenhouse up to the end of the cropping season.The suppressive effect was observed within 45 days after transplanting eggplants and continued through the end of the greenhouse experiment.In addition, the obtained data from this experiment indicate that the chemical nematicides such as carbamates are more effective at the beginning of cropping season, and their efficiency is higher in combinations with the tested bacteria.Furthermore, P. agglomerans applied separately does not provide longlasting protection of plants.However, in the plots treated with P. agglomerans plus Oxamyl, final M. javanica population was significantly lower than in the control plots.Suppression of the M. javanica populations throughout the plant growth is probably due to the successful colonization of the plant rhizosphere and good multiplication rates of the B. subtilis and P. agglomerans.This is consistent with previous findings reporting successful control of root-knot nematodes using bacteria under greenhouse condition (Giannakou et al., 2004).These authors reported that B. firmus significantly reduced the root-knot index on cucumber plants in a greenhouse in Greece.Similarly, Majzoob et al. (2012) stated suppression of M. javanica reproduction levels on greenhouse cucumbers in Iran.
It is important to note that although the rhizobacteria reduced the gall index the effect was not uniform, and eggplants within individual plots showed certain variation in the gall index.Moreover, the heavily galled plants suffered from secondary infection by soil-borne fungi (data not presented).This might be the reason for a few dead plants observed at the end of cropping season.Hence, the application of bacteria and chemicals, whose efficacy is limited to nematodes, should be accompanied by additions of fungicides to prevent infestation by soilborne fungi, such as Fusarium sp. and Verticillium sp.

Conclusion
The rhizobacterium B. subtilis showed promising results in reduction of M. javanica reproduction in eggplant and could be consider as a good non-chemical alternative for the control of root-knot nematodes.The superior performance of the B. subtilis in combination with an organophosphate nematicide (Oxamyl) in comparison with their separate application, suggests that this bacteria may be successfully included in an integrated scheme for the control of root-knot nematodes.
different letters in the column differ by Dunnet's test (P<0.05).

Table 1
P. agglomerans and B. subtilis slightly enhanced the eggplants growth.Statistically, differences in both shoot and root weight were not significant neither for P. agglomerans nor for B. subtilis when applied as a seed treatment, root dipping and soil drench (Table2, P<0.05).Overall, the eggplants with P. agglomerans application showed a better performance in plant growth parameters compared with those treated with B. subtilis.

Table 1 .
Effect of the rhizobacteria Bacillus subtilis and Pantoea aggromelans on root penetration of Meloidogyne javanica second stage juveniles

Table 2 .
Effect of the rhizobacteria B. subtilis and P. aggromelans on root and shoot fresh weight of eggplants.
a Means followed by different letters in the column differ by Dunnet's test (P<0.05).

Table 3 .
Effect of the rhizobacteria B. subtilis and P. aggromelans alone and in combination with Oxamyl on numbers of M. javanica females, root-gall index, numbers of J2 soil and root in greenhouse experiment.