ARTICLE IN PRESS

Biological control potential and modes of action of Bacillus megaterium against Meloidogyne graminicola on rice
J.L. PadghamÃ, R.A. Sikora
Institute of Crop Sciences and Resource Conservation, Department of Plant Health, Nematology and Soil Ecosystems Laboratory, University of Bonn, 9 Nussallee, D-53115 Bonn, Germany

Abstract This paper describes the development of a biological control system for Meloidogyne graminicola using rice root inoculations of the bacterium, Bacillus megaterium, which was isolated from a rice-growing region of Taiwan. Treatment with B. megaterium resulted in a greater than 40% reduction in nematode penetration and gall formation compared with non-treated rice roots, and, in a separate study, colonization of rice roots with B. megaterium decreased migration of M. graminicola to the root zone by nearly 60% compared with that of non-treated roots. Exposure of M. graminicola eggs to secondary metabolites of B. megaterium reduced hatching by over 60% compared with eggs not exposed to the bacteria. This paper will present modes of action through which B. megaterium reduces M. graminicola damage, and it will discuss challenges in developing nematode biological control systems for rice cultivation in intermittently anoxic and oxic soil environments. r 2006 Elsevier Ltd. All rights reserved.
Keywords: Bacteria; Nematode; Biological control; Rice

1. Introduction The rice root-knot nematode (Meloidogyne graminicola) is an important pest in several major rice growing areas of South and South-east Asia, causing estimated yield losses of between 20% and 80% (Arayarungsarit, 1987; Bridge and Page, 1982; Netscher and Erlan, 1993; Padgham et al., 2004; Soriano and Reversat, 2003). While M. graminicola is unable to penetrate rice roots in flooded soils, second-stage juveniles have been reported to remain viable under a continuously anoxic soil environment for at least 5 months (Bridge and Page, 1982), and egg masses for as long as 14 months (Roy, 1982). The ability of M. graminicola to survive long periods under anoxic conditions and rapidly re-infect roots at the subsidence of flooded conditions, has led to its widespread distribution in upland, lowland
ÃCorresponding author. Current address: AAAS Fellow, American Association for the Advancement of Science, 1200 New York Ave. NW, Washington, DC 20005, USA. Tel.: +1 202 712 0375; fax:+1 202 216 3174. E-mail address: jonathanpadgham@yahoo.com (J.L. Padgham).

rainfed, deepwater, and lowland irrigated rice production systems (Bridge et al., 2005). The greatest damage from M. graminicola occurs in the absence of flooded conditions, such as in aerobic upland systems (Soriano and Reversat, 2003); however, substantial yield loss also occurs in drought-prone lowland rainfed systems, where the timing and duration of early season soil flooding is often inadequate to prevent nematode attack (Padgham et al., 2004). The lack of nematode-resistant rice cultivars, and the economic unviability of nematicide application, severely limit options for effective nematode management. Alternative strategies therefore need to be developed so that rice producers do not have to simply cope with the inevitable crop loss caused by this parasite. One such strategy would be to deploy a biological control system that could be used during rice nursery seedbed establishment and/or at transplanting to protect the host plant during early plant growth stages when it is most vulnerable to damage caused by nematodes. The development of a biological control system for rice nematodes is best suited to transplanted lowland rainfed rice zones that experience fluctuating anaerobic–aerobic

0261-2194/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.cropro.2006.09.004

ARTICLE IN PRESS
periods during the cropping cycle. Biocontrol could be utilized to protect the root during brief but critical periods when the soil is aerobic, such as during seedling establishment in nematode-infested nursery beds, or directly after transplanting when the root system is becoming reestablished and is again vulnerable to nematode attack. In such a system, the nematode antagonist(s) could be introduced into the plant as a seed coat on pre-germinated seeds or as a root dip to the mature seedling at transplanting. Despite M. graminicola’s widespread occurrence in Asian rice production systems, only limited research has been done on its biological control, and these have primarily focused on soil applications of biocontrol organisms (Ahmed and Gowen, 1991; Duponnois et al., 1997). No investigations have been conducted on ways of directly targeting the rice seed or seedling with the biocontrol organism prior to invasion by the parasite. Thus, a project was recently undertaken to investigate the antagonistic potential of rice-associated endophytic bacteria for biological control of M. graminicola. Bacterial and fungal endophytes have been utilized for biocontrol of both sedentary and migratory endoparasitic nematodes that attack banana and tomato (Hallmann and Sikora, 1994; Hallmann et al., 2001; Munif et al., 2000; Pocasangre et al., 2000). Detailed mode of action data in these studies revealed that root endophytes are capable of targeting multiple points of vulnerability in the nematode’s lifecycle through inhibiting penetration, reducing reproductive capacity, and retarding nematode mobility and egg hatch. This paper describes research conducted on isolating and testing endophytic bacteria with antagonistic potential towards M. graminicola, and on possible modes of action of the bacteria against nematode activity related to host finding and penetration activities, and nematode egg development. 2. Materials and methods 2.1. Bacteria isolation and identification In 2003, endophytic bacteria were isolated from rice (Oryza sativa, L.) seedling roots grown in soil collected from several upland and lowland rice fields in Myanmar and Taiwan. All bacterial isolations were done at the Department of Plant Health and Nematology, University of Bonn, Bonn, Germany. Surface sterilized seeds (95% ethanol, 30 s and 2.5% NaOCl, 10 min) were planted in 750 cm3 pots containing natural rice paddy soil, and maintained in a greenhouse at 27 1C. After 5 weeks, the roots were washed free of soil, and surface sterilized in 1.5% NaOCl for 3 min followed by several rinses with sterile phosphate buffer (PB) to remove hypochlorite solution. Roots were imprinted on 5% trypic soy agar (TSA) for a sterility check after the final rinse with PB. The roots were then macerated in PB with a mortar and pestle under aseptic conditions and the solution was plated on 5% TSA using a spiral platter and incubated at 28 1C for 2 d. Individual colonies were then transferred using a sterile loop to 100% TSA and placed in an incubator. The resulting isolates were identified using FAME-GC analysis (Sasser, 1990). After identification, isolates were transferred to vials containing a 50% mixture of TSB and glycerol, and were stored at À18 1C. Surface sterilized rice seeds were also grown in sterile soil as a control to distinguish bacteria originating from the seed source from those that colonized the root in the field soils. Bacterial endophytes were also isolated from seeds of five rice cultivars commonly cultivated in lowland rice areas of Bangladesh. Surface sterilized seeds of each cultivar were placed on wet sterile filter paper in a sterile petri dish. The seeds were then germinated in an incubator at 28 1C. Six days later, the emerging roots were washed with 12 ml of sterile phosphate buffer and placed on a horizontal shaker table for 4 h at 25 1C. The solution was transferred to a sterile glass vial and heated to 80 1C for 10 min in order to kill all but the spore-forming bacteria. After cooling, the solution was plated on 5% TSA and incubated at 28 1C. Single colonies were transferred to 100% TSA and identified using FAME-GC analysis. The isolates were stored as described above. 2.2. Screening tests Only bacteria isolated from the inside of rice roots and not recovered in the sterility check were used for testing against M. graminicola. We consider these as putative endophytes. One-day-old cultures of each bacteria were scraped from the TSA plate surface and collected at the bottom of the plate. Each of the harvested bacteria were then mixed with 1 ml of sterile 2% methyl cellulose, and nine surface sterilized rice seeds were soaked in this solution for 30 min in order to coat the seed with the bacteria. Non-treated (control) seeds were coated with sterile methyl cellulose alone. After coating, the seeds were placed in a sterile petri dish and dried for several hours under a laminar flow hood. Three of the coated, dried seeds were placed in 9 ml of sterile quarter-strength Ringer’s solution and vortexed every 15 min for 1 h. The suspension was then serially diluted from 10À1 to 10À4 and plated out on TSA using a spiral platter. The plates were incubated at 28 1C for 24 h and the number of CFU per seed was determined as being between 107 and 108. The six remaining bacteria-coated seeds were individually planted in 100 cm3 pots filled with a sterile 2:1 sand–soil mixture. The seedlings were watered daily after emergence and maintained in a greenhouse at 27 1C with 15 h of supplemental light. Two weeks after seeding, approximately 500 J2 of M. graminicola were introduced around the roots. Ten to 12 d after nematode infestation, roots were washed, weighed, and the number of root-knot nematode galls per root system counted. A total of 31 isolates were tested in this manner, over a course of four tests. All treatments were replicated six times

ARTICLE IN PRESS
and a non-treated control and a nematicide control, consisting of 3 mg gÀ1 a.i. of Nemathorin (fosthiazate) were included for comparison. Tests were repeated for six of the 31 isolates that were found to reduce root-knot galling severity by approximately 20% or more compared with the non-treated control. Repeat tests were done on those isolates that met these criteria and that were identifiable to species level in the FAME analysis. 2.3. Root and soil inoculation test An isolate of Bacillus megaterium demonstrated consistently good nematode suppression in repeated screening tests, and was thus selected for further testing against M. graminicola. A combined root dip and soil drench method was used to inoculate the plant with the test bacterium because the presence of the growing root, as opposed to the germinating seed, reduced the time needed between bacteria inoculation and nematode infestation. B. megaterium inoculum was produced by culturing the bacteria in TSB on a rotary shaker (100 rpm, 28 1C) 1 d prior to inoculating the roots. The bacteria were then centrifuged at 5000 rpm for 20 min and the bacterial pellet re-suspended in quarter-strength Ringer’s solution. Bacterial density was adjusted to an optic density 560 nm (OD) of 2.0, and the bacteria was then diluted 1:10 with Ringer’s solution. The final concentration of B. megaterium was approximately 6 Â 106 cfu mlÀ1, as determined by dilution plating. Immediately following preparation of the bacteria, 3-week-old rice seedlings were soaked in either the B. megaterium suspension or in Ringer’s solution (control) for 30 min. After inoculation, rice seedlings were transplanted into 250 cm3 pots containing sterile 2:1 sand–soil mix, and the soil around the roots drenched with 5 ml of the bacteria or of Ringer’s. One week later, approximately 900 second-stage juveniles (J2) of M. graminicola were introduced around the roots, and 20 d after nematode infestation the roots were washed and root-knot galls were counted. The treatments were replicated five times in the initial experiment and six times in the repeat experiment. 2.4. Nematode penetration test The ability of B. megaterium to reduce M. graminicola penetration of the rice root was evaluated in a pot study using the same root and soil inoculation method and same number of nematode inocula as described in the previous section. For the penetration test, rice seedlings were transplanted into 100 cm3 pots to ensure a sufficiently high rate of root infection by the nematode, as roots were evaluated for penetration at only 7 d after M. graminicola infestation. The M. graminicola-infected roots were stained with acid fuchsin (Hooper et al., 2005) overnight and then macerated in water using a Torrax blender (IKA Labortechnik, Staufen, Germany). The number of invading juveniles was counted under a stereomicroscope. Treatments were replicated eight times, and the experiment was conducted twice. 2.5. Attraction test The effect of B. megaterium colonization of the rice root on M. graminicola host finding activity was evaluated using a 12 Â 2 Â 2 cm plastic chamber. The chamber was filled with fine quartz sand sieved through a 630 mm aperture, and two 5-d-old rice seedlings, soaked in either a B. megaterium suspension (107 cfu mlÀ1) or in blank Ringer’s solution for 30 min, were planted at opposite ends of the chamber. Directly after planting, approximately 1000 J2 of M. graminicola contained in 1 ml of water were pipetted in a small trough in the middle of the chamber. The trough was covered with additional soil, and the length of the chamber, up to the seedlings, was then covered with a plastic shell and sealed with Parafilm to prevent moisture loss. A treatment containing nematodes but no plants was included as a control. The chambers were placed in a greenhouse, and left undisturbed. After 1 week, the chambers were disassembled and the wet sand was removed at 1.5 cm intervals, starting at each end and moving towards the centre, and transferred into individual vials. The nematodes were separated by wet sieving through a 250 mm sieve and collected on a 5 mm sieve, and counted using a stereomicroscope. Infected rice roots were stained and macerated as described above, and the number of invading J2 counted. The treatments were replicated six times and the experiment was conducted twice. 2.6. Secondary metabolite test An in vitro test was conducted to determine whether secondary metabolites of B. megaterium affect M. graminicola egg hatching. M. graminicola eggs for this experiment were obtained by macerating M. graminicola-infected rice roots for 2 min in a Waring blender with 1% NaOCl. Eggs were collected on a surface-sterilized 0.25 mm sieve and rinsed several times with sterile tap water to remove excess hypochlorite solution. The eggs were stored in sterile water, and were used within 2 h of extraction from the root. B. megaterium was produced by multiplying the bacteria in Nutrient Broth (NB) on a rotary shaker (100 rpm, 28 1C) 1 day prior to nematode egg extraction. After shaking, the bacteria were centrifuged at 5000 rpm for 20 min to separate bacterial cells from cultural filtrate. After centrifuging, sterile filtration was done on the B. megaterium cultural filtrate, and on sterile tap water and NB control treatments by passing 20 ml of each suspension through 5.0 and 0.22 mm sterile filter packs, consecutively. The tests were conducted in 6-well plates which contained 2 ml of cultural filtrate or control, 1 ml of 150 mg mlÀ1 solution of streptomycin sulfate and penicillium, and approximately 2000 eggs of M. graminicola suspended in approximately 1 ml of sterile water. Lastly, sterile tap water was added to

ARTICLE IN PRESS
974 J.L. Padgham, R.A. Sikora / Crop Protection 26 (2007) 971–977

dilute the final concentration of the cultural filtrate and the NB control to 25% of full strength. The plates were then placed in a 25 1C incubator. After 4 d, half of the plates were removed and the number of J2 counted to determine the percentage egg hatch. A determination of egg hatch percentage was repeated again 11 d after exposing the eggs to the cultural filtrate. The experiment was conducted twice. A test for the production of protease enzymes was evaluated through streaking B. megaterium on skim milk agar plates (Berg et al., 2005). The plates were incubated at 28 1C, and periodically evaluated for the detection of clearing zones. Chitinase production was similarly tested by streaking B. megaterium on 10% TSA amended with 0.2% chitin colloid. Analysis of variance tests were performed for all of the experiments, and mean treatment differences were estimated using a t-test for all experiments, except that a Tukey’s test was used for separating mean differences in the secondary metabolite experiment, as there were more than two treatments.

3. Results Isolates belonging to the genus Bacillus represented the largest percentage of bacteria recovered from the rice roots and seeds; accordingly the majority of the isolates selected for the screening test against M. graminicola belonged to this genus. In repeated tests, four Bacillus isolates, among the 31 isolates originally screened, reduced nematode galling severity by 25–30%, though the combined result of the repeated screening tests was not significant (Table 1). The root and soil inoculation treatment used in the subsequent test with B. megaterium (isolate Ni5SO11) further reduced the severity of root-knot galling over the previous seed coating inoculation methods, resulting in a mean reduction in galling severity of 40% compared with the non-inoculated control (Fig. 1). Similar results were obtained in the penetration test, where a root and soil treatment with B. megaterium suspension reduced M. graminicola penetration of the rice root by 47% compared with non-treated control roots (Fig. 1). Inoculation of 5d-old seedlings with B. megaterium reduced the attraction

Table 1 Screening test to assess the effect of coating seed with bacteria on galling severity of rice roots caused by M. graminicola Isolate Species % change in root-knot gallinga Initial test 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31
a b

Source of isolate Mean À17 À25 À26
b

Repeat test À10 À28 À31

Ni-1-KA-4 Ni-6-PE-9 Ni-9-MO-3 UPLI-b48 Ni-5-SO-7 Ni-5-SO-10 Ni-5-SO-11 Ni-9-MO-7 Ni-5-SO-2 Ni-6-PE-1 LOWLIV-6 UPLI-1 UPLII-3 LOWLIII-5 Ni-9-MO-12 UPLII-a-28 UPLII-a-16 UPLI-b-43 11 PA 11 PB 29 PB 29 PD 31 PA 32 PA 32 PB 39 PB LOWLIII 90 LOWLIV 120 Ni-9-MO-15 Ni-3-TO-3 Ni-3-TO-4

Arthrobacter oxydans Bacillus circulans B. laterosporus B. laterosporus B. laterosporus B. laterosporus B. megaterium B. megaterium B. megaterium B. megaterium B. mycoides B. pumilus B. pumilus B. pumilus B. pumilus B. subtilis B. subtilis B. subtilis Bacillus sp. Bacillus sp. Bacillus sp. Bacillus sp. Bacillus sp. Bacillus sp. Bacillus sp. Bacillus sp. Burkholderia pickettii Burkholderia cepacia Cellulomonas biazatea Methylobacterium organophilu Micrococcus luteus

À24 À19 À15 À29 À10 0 À19 +19 À1 À3 60 À6 14 27 À44 17 5 3 À23 À9 À23 À12 À15 À25 À19 8 9 27 À17 À19 À4

À29

À26

À25

À30

10

À15

Taiwan Taiwan Taiwan Myanmar Taiwan Taiwan Taiwan Taiwan Taiwan Taiwan Myanmar Myanmar Myanmar Myanmar Taiwan Myanmar Myanmar Myanmar Bangladesh Bangladesh Bangladesh Bangladesh Bangladesh Bangladesh Bangladesh Bangladesh Myanmar Myanmar Taiwan Taiwan Taiwan

Root damage was expressed as a reduction in the number of nematode galls per g fresh weight of root compared to a non-inoculated control. There were no statistically significant differences, at 95% confidence intervals, between the test isolates and the control for the mean of the two tests.

ARTICLE IN PRESS
of the root to M. graminicola, resulting in a 53% and 62% reduction in J2 migration to the 0–1.5 and 1.5–3 cm soil zones, respectively, closest to the seedlings, and a 55% reduction in J2 penetration of the root (Fig. 2). No significant movement of J2 from the centre of the soil chamber was detected in the control treatment that contained no plants (data not shown). Secondary metabolites of B. megaterium significantly reduced nematode egg hatch compared with the NB control at both 4 and 11 d incubation (Fig. 3). The percentage egg hatch in the B. megaterium treatment was 4% and 14% after 4 and 11 d incubation, respectively, whereas in the NB treatment, the percentage egg hatch was 16% and 33%, respectively, over the same two time periods. Egg hatch increased for all treatments between the 4 and 11 d intervals. In the enzyme tests, strong clearing zones were detected in the skim milk agar after 3 d incubation (data not shown), indicating the presence of protease production by B. megaterium. No clearing zones were detected in the chitinase-amended agar plates after 10 d, indicating that chitinase was not produced by B. megaterium. 4. Discussion Thirteen per cent of the isolates tested in the screening tests showed a greater than 25% reduction in root galling demonstrating that root-associated bacteria of rice have reasonable biocontrol potential against the economically important M. graminicola nematode, and that more research with this group of organisms would be justified in order to try to improve their biocontrol performance, as was done with B. megaterium (isolate Ni5SO11) in our subsequent experiments. Bacillus sp. are particularly wellsuited for further development of a biocontrol system
Percentage M. graminicola hatch 35 30 25 20 a 15 10 5 0 4 days Root galling J2 penetration
Fig. 3. Effect of quarter strength B. megaterium cultural filtrate on egg hatch of M. graminicola after 4 and 11 d, as compared with water and nutrient broth controls. Columns having the same letter are not significantly different (po0:05) for comparisons with lower case and upper case groupings.

100

∗ Control B. megaterium

Percent reduction from the control

80

60

water Nutrient Broth B. megaterium metabolite

B

40

A A

a

20

0
Fig. 1. Effect of B. megaterium treatment of rice roots as a root dip on root-knot galling severity of and penetration by M. graminicola. *Indicates a significant difference between the bacteria treatment and the control at po0:05.

11 days

120 100 80 60 40 20 0 root 0 - 1.5 ∗ ∗

control B. megaterium

1.5 - 3.0

3.0 - 4.5

4.5 - 6.0

Distance from plant root (cm)
Fig. 2. Effect of B. megaterium application to rice roots on M. graminicola migration towards, and penetration of, the root. *Indicates a significant difference between the bacteria treatment and the control at po0:05.

ARTICLE IN PRESS
because their endospore-forming capacity provides favourable characteristics for endurance of biological control function under natural field conditions. Dry spores of Bacillus can be applied directly to the seed as a commercial seed coating. Since these bacteria rapidly germinate when introduced into soil, they should be able to colonize the root quickly with out impregnation of the seed. The recovery and isolation of predominately Bacillus sp. from rice roots grown in Taiwan and Myanmar rice paddy soils, and from the germinated seed of Bangladesh rice cultivars is consistent with previous reports that Bacillus was among the dominant bacterial genera recovered from the rice rhizosphere (Watanabe and Hayano, 1993), endorhiza (Xia et al., 1999) and seed (Cottyn et al., 1991) in long-term rice-producing areas of Asia. The reductions in penetration achieved with B. megaterium in this study confirm previous work showing that B. megaterium is a promising candidate for nematode biocontrol. For instance, Neipp and Becker (1999) reported that several isolates of B. megaterium were effective against Heterodora schachtii, reducing J2 penetration of sugar beet by 38–59%. Similarly, B. megaterium was reported to reduce by 50% penetration of both M. chitwoodi and Pratylenchus penetrans in potato (Al-Rehiayani et al., 1999). The reduction in migration of M. graminicola J2 towards the bacteria-treated root compared with the non-treated root, suggests that impairment of the nematode’s host finding abilities is an important means by which B. megaterium colonization reduces the rate of nematode invasion. On the other hand, Oostendorp and Sikora (1990) reported no effect of Pseudomonas fluorescens colonization of sugar beet roots on the movement of H. schachtii juveniles. The reduction in egg hatch that we measured from bacterial secondary metabolites at 25% strength is considerably less than that of Neipp and Becker (1999), who reported near complete inhibition of H. schachtii hatching with B. megaterium at 50% strength. In a separate test, we found that secondary metabolite concentrations below 25% did not result in a significant reduction in egg hatch compared with the NB or water controls (data not shown). Additional research is needed in order to better understand the parameters of this biocontrol system, in preparation for possible field testing of the organism against M. graminocola. For instance, one area of further investigation would be to evaluate the effectiveness of the biocontrol system when the bacteria and nematode are simultaneously introduced into the rhizosphere. In the root dip and soil drench experiments for penetration and galling severity, M. graminocola was added at 4 d after inoculation with the bacteria. This interval was chosen to allow for adequate colonization by B. megaterium in advance of nematode invasion. On the other hand, the nematode and bacteria were introduced simultaneously, though 6 cm apart, in the attraction experiment with similar reductions in root penetration observed in this experiment as compared to the penetration experiment. Even at this distance, the nematode is able to rapidly find its host, as indicated by our results, and as supported by Prot and Van Gundy (1981) who measured M. incognita migrations of 5 cm over a 48 h period in the presence of host exudates. Thus, the results of the attraction experiment indicate that the interval between bacteria inoculation and nematode infestation can be further reduced for experimental purposes. Our study demonstrated that the Ni5SO11 strain of B. megaterium significantly repelled invasion by the nematode. However, the ability of a microbial antagonist to disrupt the developing nematode inside the root is important as well for achieving successful biological control in the field. This is particularly the case for M. graminicola because the female’s egg sac is completely embedded inside the root cortex, rather than residing on the rhizoplane (as occurs with many root-knot nematode species) where the egg sac is vulnerable to attack from antagonists that colonize the root surface. Thus further research on the range of possible modes of action in the endorrhiza for biologically controlling M. graminicola is needed. A further challenge in designing endophytic biocontrol systems for M. graminicola and other rice root parasites is to understand whether the durability and efficacy of the biocontrol organism inside the root cortex is affected by anoxic soil conditions that exists in a flooded rice paddy. Endorrhizal microbial communities exist in a continuum with the rhizosphere and bulk soil, and can, in upland agriculture, change in response to shifts in soil microbial composition (Hallmann et al., 1999). A parallel issue in a flooded rice system becomes one of whether the shift in soil microbial communities that occurs with the evolution of anoxic conditions has any influence on the introduced endophyte and key functional groups inside the root. External leakage of oxygen from the rice aerenchyma generates an oxic layer in the rhizosphere that is sufficient to support a number of aerobic microbial communities and processes on the root surface in flooded soils (Briones et al., 2002; Liesack et al., 2000; Reichardt et al., 1997; Scheid et al., 2004); thus it is conceivable that the aerenchymal function may buffer against structural changes in endorrhizal communities. Soil flooding appears to enhance the functional efficiency of some root-associated bacteria. For instance, Engelhard et al. (2000) reported greater N-fixing efficiency of Azoarcus sp. in rice under oxygen-stressed environments. Similarly, low oxygen conditions in soil were reported to stimulate greater Pseudomonas colonization in the rhizosphere of tomato, resulting in enhanced fungal pathogen (Kim et al., 1996) and nematode (Siddiqui et al., 2003) biocontrol relative to that achieved in oxic soil environments. These studies suggest that there is considerable potential to exploit the conditions created by oxygen-limited soil environments for biocontrol of rice nematodes. We observed that the Ni5SO11 strain of B. megaterium used

ARTICLE IN PRESS
in this study grew abundantly on TSA plates kept under anaerobic conditions for 48 h (data not shown); thus this isolate could function over a wide range of oxygen environments making it a suitable candidate for further investigations on its biocontrol potential under flooded and non-flooded soil environments. Our study is the first to demonstrate nematode biological control in rice using bacterial antagonists introduced on the plant rather than added to the soil, and it is the first study to describe specific modes of action through which decreased nematode damage occurs in rice. Acknowledgements The authors wish to thank the US National Science Foundation for funding this research through an NSF International Research Post-Doctoral Fellowship grant. References
Ahmed, R., Gowen, S., 1991. Studies on the infection of Meloidogyne spp. with isolates of Pasteuria penetrans. Nemat. Medit. 19, 229–233. Al-Rehiayani, S., Hafez, S.L., Thorton, M., Sundararaj, P., 1999. Effects of Pratylenchus neglectus, Bacillus megaterium, and oil radish or rapeseed green manure on reproductive potential of Meloidogyne chitwoodi on potato. Nematropica 29, 37–49. Arayarungsarit, L., 1987. Yield ability of rice varieties in fields infested with root-knot nematode. Int. Rice Res. Notes 12, 14. Berg, G., Krechel, A., Ditz, M., Sikora, R.A., Ulrich, A., Hallmann, J., 2005. Endophytic and ectophytic potato-associated bacterial communities differ in structure and antagonistic function against plant pathogenic fungi. FEMS Mic. Ecol. 51, 215–229. Bridge, J., Page, S., 1982. The rice root-knot nematode, Meloidogyne graminicola, on deep water rice (Oryza sativa subsp. indica). Rev. Nemat. 5, 225–232. Bridge, J., Plowright, R.A., Peng, D., 2005. Nematode parasites of rice. In: Luc, M., Sikora, R.A., Bridge, J. (Eds.), Plant Parasitic Nematodes in Subtropical and Tropical Agriculture. CAB International, London, UK, pp. 87–130. Briones, A.M., Okabe, S., Umemiya, Y., Ramsing, N.B., Reichardt, W., Okuyama, H., 2002. Influence of different cultivars on populations of ammonia-oxidizing bacteria in the root environment of rice. Appl. Environ. Microbiol. 68, 3067–3075. Cottyn, B., Regalado, E., Lanoot, B., De Cleene, M., Mew, T.W., Swings, J., 1991. Bacterial populations associated with rice seed in the tropical environment. Phytopathology 3, 282–292. Duponnois, R., Netscher, C., Mateille, T., 1997. Effect of the rhizosphere microflora on Pasteuria penetrans parasitizing Meloidogyne graminicola. Nemat. Medit. 25, 99–103. Engelhard, M., Hurek, T., Reinhold-Hurek, B., 2000. Preferential occurrence of diazotrophic endophytes, Azoarcus spp., in wild rice species and land races of Oryza sativa in comparison with modern races. Environ. Microbiol. 2, 131–141. Hallmann, J., Sikora, R.A., 1994. Influence of Fusarium oxysporum, a mutualistic fungal endophyte, on Meloidogyne incognita infection of tomato. Zeit. Pflanzenkrank. Pflanzenschutz. 101, 475–481. Hallmann, J., Rodriguez, K.R., Kloepper, J., 1999. Chitin-mediated changes in bacterial communities of the soil, rhizosphere and within roots of cotton in relation to nematode control. Soil Biol. Biochem. 31, 551–560. Hallmann, J., Quadt, H.A., Miller, W.G., Sikora, R.A., Lindow, S.E., 2001. Endophytic colonization of plants by the biocontrol agent Rhizobium etli G12 in relation to Meloidogyne incognita infection. Phytopathology 91, 415–422. Hooper, D.J., Hallmann, J., Subbotin, S.A., 2005. Methods for extraction, processing and detection of plant and soil nematodes. In: Luc, M., Sikora, R.A., Bridge, J. (Eds.), Plant Parasitic Nematodes in Subtropical and Tropical Agriculture. CAB International, London, UK, pp. 53–86. Kim, D.H., Misaghi, I.J., Pierson, E.A., 1996. Fluorescent pseudomonad populations in modified rhizosphere atmospheres. Soil Biol. Biochem. 28, 497–501. Liesack, W., Schnell, S., Revsbech, N.P., 2000. Microbiology of flooded rice paddies. FEMS Microbiol. Rev. 24, 625–645. Munif, A., Hallmann, J., Sikora, R.A., 2000. Evaluation of the biocontrol activity of endophytic bacteria from tomato against Meloidogyne incognita. Mededelingen Faculteit Landbouwkundige en Toegepaste Biologische Wetenschappen Universiteit Gent 65, 471–480. Neipp, P.W., Becker, J.O., 1999. Evaluation of biocontrol activity of rhizobacteria from Beta vulgaris against Heterodora schachtii. J. Nemat. 31, 54–61. Netscher, C., Erlan, X., 1993. A root-knot nematode, Meloidogyne graminicola, parasitic on rice in Indonesia. Afro-Asian J. Nemat. 3, 90–95. Oostendorp, M., Sikora, R.A., 1990. In-Vitro interrelationships between rhizosphere bacteria and Heterodera schachtii. Rev. Nemat. 13, 269–274. Padgham, J.L., Mazid, M.A., Duxbury, J.M., Abawi, G.S., Hossain, M., 2004. Yield loss caused by Meloidogyne graminicola on lowland rainfed rice in Bangladesh. J. Nemat. 36, 42–48. Pocasangre, L., Sikora, R.A., Vilich, V., Schuster, R.P., 2000. Survey of banana endophytic fungi from Central America and screening for biological control of Radopholus similis. Act. Hort. 531, 283–289. Prot, J.C., Van Gundy, S.D., 1981. Effect of soil texture and clay component on migration of Meloidogyne incognita second stage juveniles. J. Nemat. 13, 213–219. Reichardt, W., Mascarina, G., Padre, B., Doll, J., 1997. Microbial communities of continuously cropped, irrigated rice fields. Appl. Environ. Microbiol. 63, 233–238. Roy, A.K., 1982. Survival of Meloidogyne graminicola eggs under different moisture conditions in vitro. Nemat. Medit. 10, 221–222. Sasser, M., 1990. Identification of bacteria through fatty acid analysis. In: Klement, Z., Rudolph, K., Sands, D.C. (Eds.), Methods in Phytobacteriology. Akademiai Kiado, Budapest, Hungary, pp. 199–204. Scheid, D., Stubner, S., Conrad, R., 2004. Identification of rice root associated nitrate, sulfate and ferric iron reducing bacteria during root decomposition. FEMS Microbiol. Ecol. 50, 101–111. Siddiqui, I.A., Shaukat, S.S., Ghazala, H.K., Ali, N.I., 2003. Suppression of Meloidogyne javanica by Pseudomonas aeruginosa IE-6S in tomato: the influence of NaCl, oxygen and iron levels. Soil Biol. Biochem. 35, 1625–1634. Soriano, I.R., Reversat, G., 2003. Management of Meloidogyne graminicola and yield of upland rice in South-Luzon, Philippines. Nematology 5, 879–884. Watanabe, K., Hayano, K., 1993. Source of soil protease in paddy fields. Can. J. Microbiol. 39, 1035–1040. Xia, Y.L., Wen, Q.Z., Zang, M.Z., 1999. Population dynamics of endophytic bacteria in symptom-free rice plants. Chin. J. Appl. Ecol. 10, 735–738.