The nematicidal effect of some bacterial biofertilizers on Meloidogyne incognita in sandy soil.

In a greenhouse experiment, the nematicidal effect of some bacterial biofertilizers including the nitrogen fixing bacteria (NFB) Paenibacillus polymyxa (four strains), the phosphate solubilizing bacteria (PSB) Bacillus megaterium (three strains) and the potassium solubilizing bacteria (KSB) B. circulans (three strains) were evaluated individually on tomato plants infested with the root-knot nematode Meloidogyne incognita in potted sandy soil. Comparing with the uninoculated nematode-infested control, the inoculation with P. polymyxa NFB7, B. megaterium PSB2 and B. circulans KSB2, increased the counts of total bacteria and total bacterial spores in plants potted soil from 1.2 to 2.6 folds estimated 60 days post-inoculation. Consequently, the inoculation with P. polymyxa NFB7 increased significantly the shoot length (cm), number of leaves / plant, shoot dry weight (g) / plant and root dry weight (g) / plant by 32.6 %, 30.8 %, 70.3 % and 14.2 %, respectively. Generally, the majority treatments significantly reduced the nematode multiplication which was more obvious after 60 days of inoculation. Among the applied strains, P. polymyxa NFB7, B. megaterium PSB2 and B. circulans KSB2 inoculations resulted in the highest reduction in nematode population comparing with the uninoculated nematode-infested control. They recorded the highest reduction in numbers of hatched juveniles/root by 95.8 %, females/root by 63.75 % and juveniles/1kg soil by 57.8 %. These results indicated that these bacterial biofertilizers are promising double purpose microorganisms for mobilizing of soil nutrients (nitrogen, phosphate and potassium) and for the biological control of M. incognita.

INTRODUCTION

Root-knot nematodes Meloidogyne spp., are obligate endoparasites of great economic importance, being among the major limiting factors in the production of field and plantation crops, predominantly in the tropics but also in Europe and North America (17).

A number of methods for the management of root-knot nematode such as chemical control, organic amendments, and biological control have been tried with different levels of successes for the protection of tomato plants (22, 24). Chemical management is effective, but expensive and may lead to soil pollution problems (32). Over the last decades, researchers all over the world are engaged in standardizing the nematode management strategies by following non-chemical and ecofriendly approaches (3, 21, 31). A great diversity of rhizospheric microorganisms has been described, characterized, and tested for activity as biocontrol agents against soil nematodes. These microorganisms possess some mechanisms to promote plant growth and control pathogens. Explanation among ecological interactions that occur between nematodes and bacteria, amensalism and parasitism happens in soil largely as the result of the stepwise bacterial degradation of plant and animal residues. The widely recognized mechanisms of biocontrol mediated by plant growth promoting bacteria (PGPB) are competition for an ecological niche or a substrate, production of inhibitory substances, and induction of systemic resistance in host plants to a broad spectrum of pathogens and/or abiotic stresses (7, 13). Paenibacillus polymyxa, B. megaterium and B. circulans are common soil bacterial biofertilizers belonging to plant growth promoting bacteria (PGPB). Activities associated with these bacteria include nitrogen fixation (6), soil phosphorus solubilization (9) and solubilizing insoluble potassium (10, 26). Research into the impact of PGPB has provided a greater understanding of the multiple facets of disease suppression by these biocontrol agents. Much remains to be learned from these bacteria that have unique associations and a more pronounced growth-enhancing effect on host plants. Moreover, additional environmentally safe and economically feasible root-knot nematode control practices needs to be available (7, 14, 27, 32).

The main focus of this work was to evaluate the dual effect of some bacterial strains as biofertilizers and biocontrol agents against the root-knot nematode M. incognita infestation in sandy soil cultivated with tomato.

MATERIALS

Bacterial biofertilizers and nematode used

Strains of bacterial biofertilizers including nitrogen fixing bacteria (Paenibacillus polymyxa NFB2, NFB5, NFB6, NFB7), phosphate solubilizing bacteria (Bacillus megaterium PSB2, PSB4, PSB5), potassium solubilizing bacteria (B. circulans KSB2, KSB4, KSB7) and the root-knot nematode Meloidogyne incognita were isolated and identified by the authors in a previous work (8). Bacterial strains were maintained on nutrient agar slants at 4ºC, while pure culture of M. incognita was maintained on roots of tomato plants (Lycopersicon esculantium Mill cv. Castel Rock) growing in 20-cm diameter pots in sterile sandy-loam soil in greenhouse.

Microbiological Media used

a- Nutrient agar medium (12) was used to determine the total bacterial count. b- Soil extract agar medium (5) was used to determine the densities of spore forming bacteria. c- Modified Buntt and Rovira medium (16) was used for counting of phosphate solubilizing bacteria. d- Modified Aleksandrov’s medium (34) was used for counting of potassium solubilizing bacteria. e- MBS medium (18) was used for counting of nitrogen fixing bacteria. On all media, counting was done by using the plate count method (12).

Plant and soil used

Thirty days old seedlings of tomato (Lycopersicon esculantium Mill cv. Castel Rock) were used as the host plant for M. incognita. They were kindly provided from Central Laboratory of Agricultural Climate, Agricultural Research Center, Dokki, Giza, Egypt. Seedlings were transplanted into 25-cm diameter plastic pots (one seedling/pot) filled with 5 kg of sterilized sandy soil. They were watered daily with tap water and with a nutrient solution once a week.

Chemical fertilizers

Ammonium sulfate (20.5% N), Potassium sulfate (48% K2O) and phosphoric acid (80% P2O5) were used for nitrogen, potassium and phosphorus fertilization, respectively. Rock phosphate (16.7% P2O5) and Feldspar (Potassium ore 11% K2O) were used as cheap sources for phosphorus and potassium fertilizers. All chemical fertilizers were provided from Al-Ahram Company for Mining and Natural Fertilizers, Giza, Egypt.

METHODS

Preparation of bacterial inocula

For each bacterial strain, a conical flask (250 ml) containing 100 ml of nutrient broth medium consisted of 5.0 g peptone, 3.0 g beef extract, 1000 ml tap water, (pH was adjusted to 7.0) was inoculated and incubated at 28-30oC with shaking at 150 rpm for two days prior to application.

Preparation of M. incognita inoculum

When nematode inoculum as the second stage juveniles (J2s) was needed, galled tomato roots were washed thoroughly with tap water, cut into pieces then placed in mist chamber for egg hatching (23). The first catch was discarded, and the following emerged J2s were collected daily and refrigerated at 6ºC for the experimental use. J2s were placed in 0.5% sodium hypochlorite, agitated and rinsed with sterile water immediately before infestation (15).

Greenhouse pot experiment

Four days after seedlings transplantation, seedlings were infested with 1000 fresh J2s/plant in 10 ml suspension. Two days later, each pot was inoculated with a single bacterial culture at the rate of 10 ml/pot (containing 2x107 cfu/ml). There were also two uninoculated controls: In uninoculated control 1, the nematode was applied but the bacteria were not, whilst in uninoculated control 2, neither the nematode nor the bacteria were applied. The inoculated plants were maintained in greenhouse for 60 days at 28 +mn; 2oC. Throughout 60 days after bacterial inoculation, the plants were carefully removed from pots and the rhizosphere and soil of each pot were thoroughly mixed to compose representative samples then collected in labeled plastic bags for the determination of bacterial populations, CO2 evolution and second stage juveniles (J2s). Roots of plants taken 30 and 60 days post-bacterial inoculation were gently washed to remove soil particles for the determination of nematode population in roots. Plant growth was determined 60 days post-bacterial inoculation. The shoot length was measured from the soil line to the tip of the stem and expressed in cm. The dry weight of shoots and roots was recorded after drying in oven at 70oC. Nitrogen, phosphorus and potassium contents of tomato plants were determined according to Jackson (11). The rate of CO2 evolution was determined according to the method described by Shehata (25) and calculated according to Alef and Nannipieri (1). Ten replicates of each treatment were done and average response was taken. The obtained data were subjected to an analysis by using Statistical Analysis System (SAS) (version 6.12, SAS, Institute Inc., Cary, NC, USA). Duncan`s Multiple Range Test was used to test significance of means according to Snedecor and Cochran (29). Application treatments of this experiment are illustrated in Table (1).

M. incognita measurements

For counting of J2s, the soil of each pot was mixed thoroughly and about 250 g of soil was used for nematode extraction by using the Baermann funnel technique (30). Extracted J2s were counted in 1 ml suspension using counting slides. Number of J2s was attributed to 1 kg soil. Washed root was placed in a mist chamber for egg hatching (23). The hatched J2s were collected daily and refrigerated at 6oC for 5 days after which were counted. In one ml of extracted nematode suspension, J2s were counted using nematodes counting slides. Roots were removed from the mist chamber and stained with acid fuchsin in cold lactophenol for more than 24 h. The stained roots were rinsed with tap water and cut into pieces to facilitate counting of galls, females and egg masses using a stereomicroscope. The reduction of nematode population due to different bacterial inoculations was calculated compared with the uninoculated infested tomato control.

RESULTS AND DISCUSSION

The benefits of different bacterial inoculations on the growth of infested tomato plants with the root-knot nematode M. incognita, and their impact as biocontrol agents were studied. Within 60 days after tomato seedlings inoculation, the counts of bacterial strains were increasing (Table 2). The inoculation with P. polymyxa NFB7 exhibited the highest total nitrogen fixing bacterial (NFB) counts comparing with other P. polymyxa strains (131 x 104 and 172 x 104 cells / g dry soil after 30 and 60 days of inoculation, respectively). Also, this treatment achieved the highest total bacterial count and total bacterial spores count after 60 days of inoculation (221 x 106 and 195 x 105 / g dry soil, respectively). Comparing with control 1, this treatment enhanced the growth of total bacteria and total bacterial spores populations after 60 days of inoculation by 2.6 and 1.89 folds, respectively. The CO2 evolution induced under the effect of P. polymyxa NFB7 inoculation were 13 and 19.6 mg / g dry soil / h, after 30 and 60 days, respectively.

Table 2

Bacterial activity and CO2 evolution per gram of dry potted soil of tomato plants infested with M. incognita and inoculated with different P. polymyxa, B. megaterium and B. circulans strains in sandy soil.

Treatments	Time after bacterial inoculation(days)	Total bacterial count / g dry soil (x106)	Total sporescount / g dry soil (x105)	Nitrogen fixing bacterial count / g dry soil (x104)	Phosphate solubilizing bacterial count / g dry soil (x104)	Potassium solubilizing bacterial count / g dry soil (x104)	CO2 evolution(mg/g dry soil/h)
	0	55d	55d	28e	35d	45d	4.13d
Control(1)*	30	73f	71i	54e	54d	92d	8.2d
	60	85hi	103f	133c	73c	132d	9.2e
P. polymyxaNFB2	0	28g	73b	42d	-	-	2.8e
	30	82e	104fg	82d	-	-	8.2d
	60	123d	132e	122d	-	-	12.6d
NFB5	0	63c	63c	56c	-	-	6.3c
	30	102c	98g	110c	-	-	10c
	60	173c	153d	146b	-	-	17.7ab
NFB6	0	73b	82a	76b	-	-	7.3bc
	30	114b	106ef	115b	-	-	11.5c
	60	198b	165c	169a	-	-	15.5c
NFB7	0	85a	85a	84a	-	-	8.5b
	30	129a	116d	131a	-	-	13b
	60	221a	195a	172a	-	-	19.6a
B. megateriumPSB2	0	54d	87a	-	61b	-	10.48a
	30	81e	162a	-	130a	-	15.01a
	60	104g	193a	-	190a	-	18.3a
PSB4	0	36f	73b	-	51c	-	8.17b
	30	63g	142b	-	115b	-	13.48b
	60	91h	174b	-	163b	-	16.1b
PSB5	0	46e	61c	-	72a	-	10.48a
	30	75f	90h	-	94c	-	8.23d
	60	83i	131e	-	166b	-	11.9d
B. circulansKSB2	0	89a	82a	-	-	100a	8.14b
	30	132a	134c	-	-	146a	15.41a
	60	198b	189a	-	-	190a	19.4a
KSB4	0	65c	63c	-	-	76b	5.62cd
	30	74f	112e	-	-	105c	12.45bc
	60	124e	153d	-	-	165b	17.74ab
KSB7	0	44e	73b	-	-	63c	7.01bc
	30	88d	123d	-	-	124b	13.21b
	60	116f	174b	-	-	153c	15.96bc

Uninoculated tomato plants infested with M. incognita were supplemented with full-dose of chemical NPK fertilizers before transplantation.

Among B. megaterium and B. circulans strains inoculations, B. megaterium PSB2 and B. circulans KSB2 recorded the highest PSB and KSB counts (130 x 104, 190 x 104 and 146 x 104, 190 x 104 / g dry soil after 30 and 60 days of inoculation, respectively (Table 2). The equivalent CO2 evolutions due to the inoculation with the two strains were 15.01, 18.3 and 15.41, 19.4 mg / g dry soil / h, respectively. B. megaterium PSB2 and B. circulans KSB2 inoculations recorded the highest total bacterial count and total bacterial spores count after 60 days of inoculation (104 x 106, 193 x 105 and 198 x 106, 189 x 105 / g dry soil, respectively), which represented 1.2, 1.87 and 1.5, 1.83 folds comparing with control 1. These results indicated that these bacterial biofertilizers persisted in soil up to 60 days after inoculation despite of the lack of organic matter and depending on the inorganic compounds used for plants fertilization.

By comparing the plant growth of uninoculated infested plants (control 1) with those of uninoculated uninfested plants (control 2), a great reduction was achieved (Table 3). The reduction in shoot length (cm), number of leaves / plant, shoot dry weight / plant and root dry weight / plant were 21.2 %, 13.3 %, 23.7 % and 17.4 %, respectively. The majority of bacterial strains inoculations resulted in prevention or even reduction of the consequences of nematode infection which reflected on increasing the plants growth comparing with control 1 (Table 3). The inoculation with P. polymyxa NFB7 achieved the most promising results. This treatment increased the shoot length (cm), number of leaves / plant, shoot dry weight (g) / plant and root dry weight (g) / plant by 32.6 %, 30.8 %, 70.3 % and 14.2 %, respectively. Lower percentages could be obtained if results were compared with those of uninfested plants (control 2) (4.4 %, 13.3 %, 29.9 % and -; 5.6 %, respectively) (Table 3). B. megaterium PSB2 and B. circulans KSB2 inoculations were the most efficient among the other PSB and KSB strains. They increased the shoot length (cm), number of leaves / plant, shoot dry weight (g) / plant, root dry weight (g) / plant comparing with uninoculated infested plants (control 1) by 25.6 %, 15.4 %, 29.2 %, 3.8 % and 2.3 %, 7.7 %, 66.8 %, 9.7 %, respectively. The root weight was not relevant to the weight of the shoot. This could be due to root galling on infested plants and induced systemic resistance or multiple potential defense mechanisms due to the interaction between the host, the bacteria, and the nematode (33).

Table 3

Effect of various bacterial biofertilizers treatments on growth of tomato plants infested with M. incognita 60 days post-inoculation.

Treatments	Growth parameters
	Shoot length(cm)	No. of leaves / plant	Shoot dry weight(g/ plant)	Root dry weight(g/ plant)	Shoot content (%)	Root content (%)
						N	P	K	N	P	K
P. polymyxa
NFB2	44.1e	11e	3.12h	2.21c	1.23e	0.62ab	1.46c	0.26e	0.43b	0.30c
NFB5	46.6d	15bc	6.11f	3.18b	2.2a	0.61b	1.45cd	0.52b	0.42b	0.18f
NFB6	54b	15b	10.25b	4.01a	1.43d	0.53c	1.03e	0.26e	0.38d	0.37b
NFB7	57a	17a	11.63a	4.01a	2.32a	0.63a	1.53b	0.58a	0.47a	0.40a
B. megaterium
PSB2	54bc	15b	8.82c	3.64b	2.2a	0.61b	1.46c	0.48c	0.43b	0.36b
PSB4	52c	13cd	5.14g	2.04c	1.82c	0.51a	1.64a	0.46c	0.41bc	0.23e
PSB5	53c	12d	6.02f	2.5c	2.20a	0.55c	1.43cd	0.32d	0.41bc	0.23e
B. circulans
KSB2	44e	14bc	11.39a	3.85a	2.38a	0.65a	1.44cd	0.54b	0.44b	0.39a
KSB4	38g	13cd	7.02ed	1.06d	2.22a	0.52c	1. 39d	0.51b	0.43b	0.28d
KSB7	42f	13cd	6.14fe	2.21c	1.89c	0.56c	1.09e	0.46c	0.42b	0.18f
Control(1)	43e	13cd	6.83d	3.51b	2.17b	0.53c	1.46c	0.52b	0.42b	0.35b
Control(2)	54.6b	15b	8.95c	4.25d	2.24a	0.61b	1.48c	0.54b	0.43b	0.39a

N P K contents of inoculated plants by any bacterial strain gave close or in some cases slightly higher determinations comparing with control 1 (Table 3). P. polymyxa NFB7, B. megaterium PSB2 and B. circulans KSB2 inoculations achieved the highest N P K contents. P. polymyxa NFB7 increased the N P K contents of shoots by 6.91 %, 65.86 %, 41.20 % and roots by 11.5 %, 11.9 %, 14.2 %, respectively. Bacterial inoculations induced tomato plants to endure the nematode infection, N P K contents of endured plants were in general close to those of uninfested plants (control 2).

Regarding to the effectiveness of the strains of bacterial biofertilizers in suppressing M. incognita reproduction, it could be observed that, most of bacterial inoculations had significantly reduced nematode population by comparing with the uninoculated infested plants (control 1), especially for the counts of hatched J2s/root, females/root and J2s/1kg soil (Table 4). The reduction in nematode population was more obvious after 60 days of inoculation by most inoculated treatments. However, inoculating P. polymyxa NFB7, B. megaterium PSB2 and B. circulans KSB2 resulted in the highest control effect comparing with control 1. After 30 days of inoculation, they recorded the highest reduction in numbers of females/root by 14.3%, hatched J2s/root by 87.2% and in J2s/1kg soil by 79.3%, respectively (Table 5). While, after sixty days of inoculation, the highest reduction by the three biofertilizers were obtained on numbers of hatched J2s/root (95.8 %), females/root (63.75%) and J2s/1kg soil (57.8%), respectively (Table 5).

Table 4

Effect of different biofertilizers strains inoculations on M. incognita infestation throughout 60 days post-inoculation in sandy soil.

Treatments	Time after bacterial inoculation(days)	M. incognita measurements
No. of galls / root	No. of egg masses / root	No. of hatched J2s / root	No. of females / root	No. of J2s /1kg soil
P. polymyxa NFB2	30	135f	79e	8790h	64d	16200i
	60	56b	31a	990c	18a	4800b
NFB5	30	76bc	49bc	6060g	35b	11880g
	60	60b	36a	2580d	22a	9240d
NFB6	30	89d	62d	6960g	49c	12240h
	60	66bc	35a	3300e	25ab	5760c
NFB7	30	63b	40b	4050f	30b	10560f
	60	41a	28a	390b	12a	1452a
B. megateriumPSB2	30	50a	26a	360a	22a	1560a
	60	70c	44b	4980f	29b	11000e
PSB4	30	67b	41b	690b	50c	4670d
	60	89d	59bc	7980i	52c	17450h
PSB5	30	61b	35b	660b	40bc	3480c
	60	82d	56b	5970h	33b	14660g
B. circulansKSB2	30	68b	44b	1320c	15a	2520b
	60	88d	49b	5490g	47c	12650f
KSB4	30	92d	60d	2280d	22a	8760e
	60	112e	80d	296a	55cd	19230i
KSB7	30	102e	65d	2970e	26ab	22600j
	60	123f	83d	9900k	70e	26400j
Control(1)*	30	71b	46b	4050f	35b	12180h
	60	133g	86d	9360j	80f	30000k

Uninoculated tomato plants infested with Meloidogyne incognita were supplemented with full-dose of chemical NPK fertilizers before transplantation.

Table 5

Effect of the most efficient biofertilizer strains treatments on nematode population after 30 and 60 days of inoculation comparing with control 1*.

Treatments	Time after bacterial inoculation(days)	Reduction of nematode population measurements
No. of galls / root	No. of egg masses / root	No. of hatched J2s / root	No. of females / root	No. of J2s /1kg soil
P. polymyxa NFB7	30	11.3	13	0	14.3	13.3
	60	69.2	67.4	95.8	85	95.16
B. megateriumPSB2	30	1.4	4.3	23	17.1	87.2
	60	47.4	48.8	46.8	63.75	63.3
B. circulansKSB2	30	4.2	4.3	67.4	57.1	79.3
	60	33.8	43	41.3	41.25	57.8

Control 1: Uninoculated tomato plants infested with M. incognita were supplemented with full-dose of chemical NPK fertilizers before transplantation.

The present pot experiment indicated that bacterial biofertilizers reduced significantly the ability of M. incognita to reproduce in soil which was a strain dependant. Several reports were focused on the benefits of rhizosphere colonizing bacteria especially nitrogen fixing bacteria (NFB), phosphate solubilizing bacteria (PSB) and potassium solubilizing bacteria (KSB) as biocontrol agents, some of important genera include Agrobacterium, Alcaligenes, Bacillus, Clostridium, Desulfovibrio, Pseudomonas, Serratia, Paenibacillus and Streptomyces. Application of these bacteria has given very promising results (28, 13, 14). Whole cultures and supernatants (exotoxins) of Bacillus thuringiensis subsp. Brasiliensis and B. laterosporus caused high mortality of Meloidogyne javanica in in-vitro bioassays and greenhouse tests on tomato (4). Neipp and Becker (19) reported that several strains of B. megaterium were found to be effective against Heterodera schachtii, they reduced J2s penetration of sugar beet by 38%-59%. Similarly, B. megaterium was reported to reduce by 50% the penetration of both M. chitwoodi and Pratylenchus penetrans in potato (2). Padgham and Sikora (20) found that the inoculation with B. megaterium reduced more than 40% of nematode penetration and gall formation compared with non-;treated rice root. 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 nontreated roots. Exposure of M. graminicola eggs to secondary metabolites of B. megaterium reduced hatching eggs by over 60% compared with eggs not exposed to the bacteria.

CONCLUSIONS

The present greenhouse pot experiment indicated that the individual inoculations of the bacterial biofertilizers P. polymyxa NFB7, B. megaterium PSB2 and B. circulans KSB2, enhanced significantly the growth of tomato plants, and suppressed the nematode population in infested plants roots and soil. Such effect was prolonged and increased through the 60 days after plant transplantation. Depending on these results, the combined addition of these bacterial cultures may be more effective to increase the nematode suppression in infested plants. The reliance on bacterial biofertilizers that have the ability to fix nitrogen or solubilize either insoluble phosphate or potassium, in the biological control of root-knot nematode helps in the same way to minimize the quantities of chemical nitrogen fertilizers used and may open the door for a wide application of cheap and more available crude sources of insoluble phosphate and potassium in agriculture.

Table 1

Application treatments of tomato plants infested with M. incognita and inoculated with different P. polymyxa, B. megaterium and B. circulans strains in potted sandy soil.

	Treatments	Chemical fertilizations			Bacterial*inoculation	Infestation with M. incognita
	N	P**	K**
1(control)	Full dose of (NH4)2SO4	Full dose of H3PO4	Full dose of K2SO4	-	+
2(control)	Full dose of (NH4)2SO4	Full dose of H3PO4	Full dose of K2SO4	-	-
3	75% of full dose of (NH4)2SO4	Full dose of H3PO4	Full dose of K2SO4	Paenibacillus polymyxa (4 strains)	+
4	Full dose of (NH4)2SO4	Rock phosphate	Full dose of K2SO4	Bacillus megaterium (3 strains)	+
5	Full dose of (NH4)2SO4	Full dose of H3PO4	Feldspar	B. circulans (3 strains)	+

Each strain was tested alone.

Rock phosphate and feldspar were added in equivalent to (P%, K %) in full recommended dose of H3PO4 and K2SO4.