J Gen Plant Pathol (2009) 75:144–153 DOI 10.1007/s10327-009-0154-4

DISEASE CONTROL

Effects of antagonistic fungi, plant growth-promoting rhizobacteria, and arbuscular mycorrhizal fungi alone and in combination on the reproduction of Meloidogyne incognita and growth of tomato Zaki A. Siddiqui Æ M. Sayeed Akhtar

Received: 24 February 2008 / Accepted: 17 December 2008 / Published online: 24 February 2009 Ó The Phytopathological Society of Japan and Springer 2009

Abstract Antagonistic fungi (Aspergillus niger CA and Penicillium chrysogenum CA1), plant growth-promoting rhizobacteria (PGPR) (Burkholderia cepacia 4684 and Bacillus subtilis 7612) and AM fungi (Glomus intraradices KA and Gigaspora margarita AA) were assessed alone and in combination for their effects on the growth of tomato and on the reproduction of Meloidogyne incognita in glasshouse experiments. Application of antagonistic fungus, PGPR, or AM fungus alone or in combination significantly increased the length and shoot dry mass of plants both with and without nematodes. The increase in shoot dry mass caused by Gl. intraradices KA in plants without nematodes was greater than that caused by PGPR or antagonistic fungi. Similarly, the increase in shoot dry mass caused by Bu. cepacia 4684 in plants with nematodes was greater than that caused by P. chrysogenum CA1. Application of Bu. cepacia 4684 caused a 36.1% increase in shoot dry mass of nematode-inoculated plants followed by Ba. subtilis 7612 (32.4%), A. niger CA (31.7%), Gl. intraradices KA (30.9%), Gi. margarita AA (29.9%) and P. chrysogenum CA1 (28.8%). Use of Bu. cepacia 4684 with A. niger CA caused a highest (65.7%) increase in shoot dry mass in nematode-inoculated plants followed by Ba. subtilis 7612 plus A. niger CA (60.9%). Burkholderia cepacia 4684 greatly reduced (39%) galling and nematode multiplication, and the reduction was even greater (73%) when applied with A. niger CA. Antagonistic fungi had no significant effect on root colonization caused by AM fungi. Applying Bu. cepacia 4684 with A. niger CA may be useful in the biocontrol of M. incognita on tomato. Z. A. Siddiqui (&)
M. Sayeed Akhtar Department of Botany, Aligarh Muslim University, Aligarh 202002, India e-mail: [email protected]

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Keywords AM fungi
Antagonistic fungi
Biocontrol
Meloidogyne incognita
Nematode
PGPR
Solanum lycopersicum

Introduction Tomato, Solanum lycopersicum L., is an important vegetable crop, cultivated worldwide. Yield loss due to root-knot nematodes (Meloidogyne spp.) on tomato can reach 40–46% in India (Bhatti and Jain 1977; Reddy 1985). Plants infected with Meloidogyne spp. have typical root galling. Some infected plants also express nutrient deficiency symptoms, particularly for nitrogen (Good 1968). This disease has become a major constraint to the successful cultivation of tomato in India (Siddiqui et al. 2001b). Rhizosphere microorganisms provide an initial barrier against pathogen attack to the root (Weller 1988). Of the rhizosphere organisms, antagonistic fungi have great potential against plant pathogens (Kiewnick and Sikora 2006; Papavizas 1985). Though hundreds of organisms have been reported to parasitize or prey on nematodes, fungal antagonists have been predominantly used for the biological suppression of nematodes. Several reviews have been published exclusively on fungal antagonists of nematodes (Barron 1977; Kerry 1984; Morgan-Jones and Rodrı´guez-Ka´bana 1988). Of the antagonistic fungi, Aspergillus and Penicillium are common genera in most agricultural fields of India. Aspergillus species are known to produce a variety of secondary metabolites and are useful in the biocontrol of nematodes (Siddiqui et al. 2004). Similarly, Penicillium spp. are also useful as antagonists of nematodes (Eapen et al. 2005). Plant growth-promoting rhizobacteria (PGPR) are free living and may impart beneficial effects on plants. PGPR

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enhance emergence and stimulate overall plant growth. PGPR also improve seed germination, root development, mineral nutrition, water utilization and disease suppression (Siddiqui 2006). The manipulation of crop rhizosphere with PGPR for biocontrol of plant pathogens has shown considerable promise (Nelson 2004; Siddiqui 2006; Tian et al. 2007). Arbuscular mycorrhizal (AM) fungi colonize the roots of many crop plants (Ozgonen et al. 1999; Smith and Read 1997) and are of great value in promoting the uptake of phosphorus, minor elements and water (Allen 1996; Siddiqui and Mahmood 1995). They also reduce the severity of several plant diseases (Akkopru and Demir 2005; Barea et al. 2002; Masadeh et al. 2004; Siddiqui and Mahmood 1995). Biocontrol agents are affected by biotic and abiotic conditions. Because different mechanisms of control may be dissimilarly influenced by environmental conditions, it is possible that if multiple mechanisms are involved, under a certain set of conditions, one mechanism may compensate for the other (Guetsky et al. 2002). Therefore, the control achieved by biocontrol agents with several distinct mechanisms of control may be additive or synergistic. Biocontrol agents that have different traits in the soil and rhizosphere may be used together to control plant pathogens via different mechanisms of disease suppression. Siddiqui et al. (2001b) observed that Pseudomonas fluorescens GRP3 applied with organic manure was effective for the management of the root-knot nematode M. incognita on tomato. In addition, the intensity of the chickpea root-rot disease complex was reduced after the combined application of Glomus intraradices with either Pseudomonas straita or Rhizobium than when they were applied separately (Akhtar and Siddiqui 2008a). Similarly, the greatest reduction in the chickpea root-rot disease complex was observed when Pseudomonas alcaligenes, Bacillus pumilus and Glomus intraradices were used together (Akhtar and Siddiqui 2008b). Application of Paecilomyces lilacinus with composted cow manure on tomato also gave better control of M. incognita than either did individually (Siddiqui and Akhtar 2008). Antagonistic fungi, PGPR, and AM fungi have been used extensively to manage nematode diseases. They have also been used with organic manures and root-nodule bacteria to control nematode diseases. The aim of the present study was to use these three groups of microorganisms in combinations of two to test which two groups may achieve the best control of root-knot nematodes. Two antagonistic fungi, two PGPR, and two AM fungi were tested alone and in various combinations for their effect on tomato growth and on the reproduction of the root-knot nematode Meloidogyne incognita.

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Materials and methods Two antagonistic fungi (Aspergillus niger CA and Penicillium chrysogenum CA1), PGPR (Burkholderia cepacia MTCC No. 4684 and Bacillus subtilis MTCC No. 7612) and AM fungi (Glomus intraradices KA and Gigaspora margarita AA) were tested alone and in groups of two for their effect on tomato growth and on the reproduction of root-knot nematode Meloidogyne incognita. Preparation and sterilization of soil mixture Sandy loam soil (pH 7.2) collected from the field of the Department of Botany, A.M.U. Aligarh, India was added to jute bags. Water was poured into each bag to wet the soil before transferring them to an autoclave for sterilization at 137.9 kPa for 20 min. Sterilized soil was allowed to cool to room temperature (20°C) before filling 15-cm-diameter clay pots with 1 kg of sterilized soil. Growth and maintenance of test plants Seeds of tomato cultivar K-25 were surface sterilized in 0.1% sodium hypochlorite for 2 min and then washed three times with distilled water. Our previous study showed that cultivar K-25 is susceptible to M. incognita (Singh and Siddiqui 2008). Seeds were sown in a seedling tray, and each germinated seedling was transplanted to its own clay pot. Seedlings were placed in a glasshouse at 20 ± 3°C and watered as needed but without any added nutrients. Two days after transplantation, seedlings were inoculated with biocontrol agents; noninoculated plants served as controls. Nematode inoculum Meloidogyne incognita was collected from tomato field soil and multiplied on eggplant (Solanum melongena L.) using a single egg mass. Egg masses were hand picked using sterilized forceps and placed in 9-cm-diameter sieves of 1-mm pore size, which had been lined with cross-layered tissue paper. The sieves were placed in Petri dishes with distilled water for hatching and incubated at 27°C. Two thousand hatched J2s were applied to each plant. Selection of biocontrol agents and inoculum preparation Glomus intraradices KA and Gigaspora margarita AA, isolated from the soil of chickpea fields of Kasimpur and Atrauli, respectively, where the intensity of root-knot disease was lower than in the surrounding fields, were used as AM fungi. For inoculum, these fungi were produced

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separately on Chloris gayana Kunth (Rhodes grass) grown in sandy loam soil collected from the field of the Botany Department, A. M. U. Aligarh, without addition of nutrients. The population of AM fungi in the inoculum was assessed by the most probable number method (Porter 1979). Fifty grams of inoculum with soil was added around the seed to provide 500 infective propagules of AM fungi per pot (1 g inoculum contains 10 infective propagules). The crude inoculum consisted of soil, extramatrical spores and sporocarps, hyphal fragments and infected Rhodes grass fragments. PGPR (Bu. cepacia MTCC No. 4684 and Ba. subtilis MTCC No. 7612) were obtained from Microbial Type Culture Collection and Gene Bank, Institute of Microbial Technology, Chandigarh, India (Table 1). In separate experiments, both isolates were inhibitory to the penetration of tomato by M. incognita and promoted plant growth. Both PGPR species were grown separately in nutrient broth (HiMedia Laboratories, Mumbai, India) at 37 ± 2°C for 72 h. Ten milliliters of the suspension (1.5 9 107 cells/ml) was used as inoculum. Two fungi, A. niger CA and P. chrysogenum CA1, were isolated from rhizosphere soil tomato field at Chherat, Aligarh, India (Table 1). Pure cultures of these fungi were stored at 5°C in the Mycology Laboratory, Department of Botany, Aligarh Muslim University, Aligarh, India. Although M. incognita penetrated tomato roots at 25°C 10 days after inoculation with the antagonistic fungus and in the noninoculated controls in separate experiments, both fungal species had an adverse effect on nematode penetration. These fungi were separately cultured on Richard’s liquid medium for 15 days at 25°C (Riker and Riker 1936). Mycelial mats were collected separately on blotting paper to absorb excess of water and nutrients, then macerated in distilled water to make a mycelial suspension that contained 10% (w/v) fresh mass of mycelium. Ten milliliters of the suspension was used as the inoculum in treatments. The inoculum concentrations for these biocontrol agents were selected after preliminary studies. Moreover, similar

concentrations of these biocontrol agents were also used in our earlier studies (Akhtar and Siddiqui 2008a; Siddiqui and Mahmood 1996). Biocontrol inoculum of PGPR and AM fungi contained culture media. PGPR were grown in nutrient broth medium for 72 h at 37°C before their use as biocontrol agents; hence, no significant nutrients remained in the medium (Siddiqui and Akhtar 2008). Similarly, the AM fungal inoculum included a few Rhodes grass segments and some soil particles, but these additions did not significantly affect plant growth (Akhtar and Siddiqui 2008b). Inoculation techniques For inoculation with M. incognita, AM fungi, PGPR and antagonistic fungi, the layer of soil within 6 cm of the entire root system was carefully removed without damaging the roots. A prepared inoculum suspension was poured into the removed soil, and the AM fungal inoculum was placed around the roots, and the soil was then replaced. An equal volume of sterile water was added to control treatments. All the inoculations were performed 2 days after transplantation when seedlings were 1-week-old. Experimental design The experiment was set up in a completely randomized blocked design with 19 treatments: (1) control; (2) Bu. cepacia (Bc); (3) Ba. subtilis (Bs); (4) A. niger (An); (5) P. chrysogenum (Pc); (6) Gl. intraradices (Gi); (7) Gi. margarita (Gm); (8) Bc ? An; (9) Bc ? Pc; (10) Bc ? Gi; (11) Bc ? Gm; (12) Bs ? An; (13) Bs ? Pc; (14) Bs ? Gi; (15) Bs ? Gm; (16) An ? Gi; (17) An ? Gm; (18) Pc ? Gi; (19) Pc ? Gm. These 19 treatments were tested in the presence and absence of M. incognita (19 9 2 = 38 treatments). Each treatment was replicated five times (38 9 5 = 190 pots). The experiment was performed in a glasshouse at 20 ± 3°C and repeated once. Data for both experiments were almost identical, so we pooled the data.

Table 1 Character and origins of the isolates tested in this study Species, character

Isolate

Geographic origin; year isolated, host or origin

Glomus intraradices, AM

KA

Kasimpur, Aligarh; 2004, chickpea field soil

Gigaspora margarita, AM

AA

Atrauli, Aligarh; 2004, chickpea field soil

Burkholderia cepacia, PGPR

4684

MTCC, Chandigarh; 2005, PGPR

Bacillus subtilis, PGPR

7612

MTCC, Chandigarh; 2005, PGPR

Aspergillus niger, SA

CA

Chherat, Aligarh; 2005, Tomato field soil

Penicillium chrysogenum, SA

CA1

Chherat, Aligarh; 2005, Tomato field soil

For details on MTCC isolates, see website http://www.imtech.res.in/mtcc AM arbuscular mycorrhizal fungus; PGPR plant growth promoting rhizobacterium; SA fungal soil antagonist

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Observations

Results

The plants were harvested 90 days after inoculation. Data were recorded on plant length (root tip to shoot tip), shoot dry mass, number of galls, percentage root colonization, and size of nematode population. A 250-g subsample of well-mixed soil from each treatment was processed by Cobb’s sieving and decanting method, followed by Baermann’s funnel extraction to extract the nematode population (Southey 1986). For estimating the number of juveniles, eggs, and females inside the roots, a 1-g subsample of roots was macerated in a Waring blender, and nematodes in the resulting suspension were counted. The number of nematodes in roots was calculated by multiplying the number of nematodes in 1 g of root by the total root mass. The proportion of roots colonized by Gl. intraradices and by Gi. margarita was determined using a grid intersection method (Giovannetti and Mosse 1980) after clearing the root with KOH in 0.05% (w/v) trypan blue in lactophenol. Tomato roots inoculated with PGPR were collected 1 month after sowing. One gram of surface-sterilized roots was crushed in sterile normal saline solution (NSS), and 0.1 ml serially diluted extracts were plated on nutrient agar plates and incubated at 37°C for 24 h. Five samples (1 g root) from each replicate were used to determine root colonization by PGPR. Recovered bacteria were roughly identified based on their colony shape, morphological characteristics and Gram staining. The plates were placed on a Quebec colony counter for counting the bacterial colonies. The number of colonies on plates with 30–300 colonies was multiplied by the reciprocal dilution factor to obtain the size of the bacterial population (Sharma 2001) and expressed as colony forming units (CFU) per gram root.

Application of antagonistic fungi, PGPR and AM fungi alone or in combination against plants without nematodes caused a significant increase in the growth of tomato (based on plant length and shoot dry mass) over the control (Table 2). Application of AM fungus Gl. intraradices KA was better at improving shoot dry mass of plants without M. incognita than was the antagonistic fungus or the PGPR. Application of Gl. intraradices KA to plants without nematodes caused a 17.6% increase in shoot dry mass, which was significantly higher than that caused by Gi. margarita AA (13.8%), A. niger CA (13.2%), Bu. cepacia MTCC No. 4684 (12.2%), P. chrysogenum CA1 (10.2%) and Ba. subtilis MTCC No. 7612 (9.7%). When nematodes were absent, combined use of Gl. intraradices KA with A. niger CA increased shoot dry mass the most (26.6%, followed by Gi. margarita AA plus A. niger CA (25.5%) and Bu. cepacia MTCC No. 4684 plus AM fungus (24.5, 23.1%), none of which differed statistically from each other (Table 2). Application of antagonistic fungi, PGPR, and AM fungi alone or in combination significantly increased the growth of plants inoculated with nematodes over the control (Table 3). Application of an antagonistic fungus, PGPR, or AM fungus alone was almost equally effective in improving growth of plants with M. incognita. Use of Bu. cepacia MTCC No. 4684 caused a 36.1% increase in shoot dry mass of nematode-inoculated plants, followed by Ba. subtilis MTCC No. 7612 (32.4%), A. niger CA (31.7%) and Gl. intraradices KA (30.9%), Gi. margarita AA (29.9%), and P. chrysogenum CA1 (28.8%). Moreover, integration of Bu. cepacia MTCC No. 4684 with A. niger CA or Ba. subtilis MTCC No. 7612 with A. niger CA caused the greatest (65.7 and 60.9%, respectively) increase in shoot dry mass of nematode-treated plants. Use of P. chrysogenum CA1 with Gi. margarita AA or Gl. intraradices KA were least effective in increasing shoot dry mass (36.3 and 37.4%, respectively) of nematode-treated plants among combined treatments. Use of any PGPR (Bu. cepacia MTCC No. 4684 or Ba. subtilis MTCC No. 7612) plus AM fungi (Gl. intraradices KA or Gi. margarita AA) in different combinations caused a statistically similar increase in the growth of nematode-inoculated plants. Similarly, P. chrysogenum CA1 with Bu. cepacia MTCC No. 4684 or with Ba. subtilis MTCC No. 7612 resulted statistically no different increase in the growth of nematode-inoculated plants (Table 3). Treatment with Bu. cepacia MTCC No. 4684 significantly increased colonization of tomato roots by the PGPR more than did Ba. subtilis MTCC No. 7612 with (Table 4) and without nematode (Table 5) treatment. However,

Statistical analysis The experiments had two-way combinations, i.e., with/ without M. incognita and one or two of the following groups: PGPR, AM fungi, soil antagonistic fungi. Data without and with nematodes were analysed separately to observe the growth-promoting effect of inoculated microorganisms in the absence of nematodes and the biocontrol effect in the presence of nematodes. Data were analyzed statistically by analysis of variance (P = 0.05). Tukey’s tests (P = 0.05) were then used to distinguish differences between treatments. All these analyses were performed by program Stat View 5.0 (SAS Institute, Cary, NC, USA). The graph for galling and nematode population was prepared using the program Sigma plot with error bars showing standard error.

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Table 2 Effects of plant growth-promoting rhizobacteria (PGPR), soil antagonistic fungi (SA), and arbuscular mycorrhizal fungi (AM) on mean growth of tomato plants without nematodes Plant length (±SD) (cm)a

Treatment

Shoot dry mass (±SD) (g)a

% Increase over control Plant length

Shoot dry mass

Nil

Control

65.7 ± 1.86 j

17.89 ± 0.37 l

–

–

PGPR

Bu. cepacia 4684

72.9 ± 2.14 hi

20.08 ± 0.37 jk

10.9

12.2

SA

Ba. subtilis 7612 A. niger CA

71.4 ± 1.47 i 73.6 ± 1.87 ghi

19.63 ± 0.34 k 20.26 ± 0.36 ijk

8.7 12.0

9.7 13.2

P. chrysogenum CA1

71.6 ± 1.44 i

19.72 ± 0.41 k

8.9

10.2

AM PGPR 9 SA

PGPR 9 AM

SA 9 AM

Gl. intraradices KA

76.6 ± 1.41 defgh

21.04 ± 0.34 fgh

16.6

17.6

Gi. margarita AA

73.8 ± 1.70 fghi

20.35 ± 0.27 hijk

12.3

13.8

Bc ? An

77.7 ± 1.30 cdef

21.29 ± 0.32 defg

18.3

19.0

Bc ? Pc

76.5 ± 1.36 defgh

20.94 ± 0.18 fghi

16.4

17.0

Bs ? An

77.2 ± 2.43 cdefg

21.16 ± 0.38 efg

17.5

18.3

Bs ? Pc

75.8 ± 1.45 efgh

20.77 ± 0.31 ghij

15.4

16.1

Bc ? Gi

81.2 ± 1.34 abc

22.27 ± 0.32 abc

23.6

24.5

Bc ? Gm

80.4 ± 2.61 abcd

22.02 ± 0.30 abcd

22.4

23.1 20.5

Bs ? Gi

78.6 ± 1.67 abcde

21.55 ± 0.31 cdef

19.6

Bs ? Gm

78.2 ± 1.82 bcde

21.36 ± 0.38 defg

19.0

19.4

An ? Gi

82.6 ± 1.72 a

22.64 ± 0.29 a

25.7

26.6

An ? Gm

81.9 ± 1.45 ab

22.45 ± 0.34 ab

24.7

25.5

Pc ? Gi Pc ? Gm

79.8 ± 1.20 abcde 79.1 ± 2.36 abcde

21.84 ± 0.31 bcde 21.62 ± 0.24 cdef

21.5 20.4

22.1 20.8

Bc = Burkholderia cepacia MTCC No. 4684; Bs = Bacillus subtilis MTCC No. 7612; An = Aspergillus niger CA; Pc = Penicillium chrysogenum CA1; Gi = Glomus intraradices KA; Gm = Gigaspora margarita AA a

Values within each column followed by same letters are not significantly different (Tukey’s test, P \ 0.05)

adding A. niger CA with Ba. subtilis MTCC No. 7612 reduced root colonization by the rhizobacteria compared to Ba. subtilis MTCC No. 7612 alone in plants without nematodes (Table 4). Combined inoculation of Gl. intraradices KA with Ba. subtilis MTCC No. 7612 increased root colonization caused by Ba. subtilis MTCC No. 7612 compared to use of Ba. subtilis MTCC No. 7612 alone. Glomus intraradices KA caused colonization of roots more than did Gi. margarita AA. Antagonistic fungi had no significant effect on root colonization by AM fungi. Use of Bu. cepacia MTCC No. 4684 with AM fungi resulted in a level of colonization by AM fungi that did not differ statistically from that after treatment with an AM fungus alone and without nematodes (Table 4). Inoculation with antagonistic fungi, PGPR, and AM fungi significantly reduced galling and nematode population over the control (Table 6; Fig. 1). In treatments with one microorganism, reductions in galling ranged from 39% (Bu. cepacia MTCC No. 4684) to 26% (Gi. margarita AA). In combined treatments, reductions in galling ranged from 41 to 73%. When PGPR were used with an antagonistic fungus, galling was reduced by 54–73%. Similarly, galling was reduced by 60–68% when PGPR were used with an AM fungus, while adding an antagonistic fungus with the

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AM fungus resulted in decreases of 41–51%. Galling was reduced the most (73%) when Bu. cepacia MTCC No. 4684 was used with A. niger CA; reduction was least when P. chrysogenum CA1 was used with Gi. margarita AA (41%). Nearly the same trends were observed for nematode population (Table 6; Fig. 1).

Discussion Aspergillus species are common, occurring in habitats such as soils in warmer climates, compost, decaying plant material, and stored grains, and many are known to produce a variety of secondary metabolites (Domsch et al. 1980). Some Aspergillus species have also been reported for their biocontrol potential against root knot nematodes (Siddiqui et al. 2001a). Aspergillus species isolated from the rhizosphere of crop plants produced a number of secondary metabolites that are soluble in ethyl acetate and can potentially influence the efficacy of the biocontrol strains of PGPR (Siddiqui et al. 2004). Thus, biocontrol by A. niger CA may be attributed to its production of secondary metabolites. Moreover, Aspergillus and Penicillium inhibit egg hatch, indicating the involvement of mechanisms other

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Table 3 Effects of plant growth-promoting rhizobacteria (PGPR), soil antagonistic fungi (SA), and arbuscular mycorrhizal AM fungi (AM) on mean (±SD) growth of tomato in plants inoculated with root-knot nematode Meloidogyne incognita Plant length (cm)a

Treatment

Shoot dry mass (g)a

% Increase over control Plant length

Shoot dry mass –

Nil

Control

42.7 ± 1.73 k

11.62 ± 0.43 k

–

PGPR

Bu. cepacia 4684

57.8 ± 2.32 ghi

15.81 ± 0.29 ghi

35.4

36.1

SA

Ba. subtilis 7612 A. niger CA

56.3 ± 0.97 ghij 56.2 ± 0.72 hij

15.39 ± 0.20 hij 15.30 ± 0.27 hij

31.9 31.6

32.4 31.7

P. chrysogenum CA1

54.8 ± 1.32 ij

14.97 ± 0.23 j

28.3

28.8

AM PGPR 9 SA

PGPR 9 AM

SA 9 AM

Gl. intraradices KA

55.6 ± 1.53 hij

15.21 ± 0.25 ij

30.2

30.9

Gi. margarita AA

53.7 ± 1.29 j

15.09 ± 0.39 j

25.8

29.9

Bc ? An

70.4 ± 1.73 a

19.26 ± 0.26 a

64.9

65.7

Bc ? Pc

64.8 ± 1.25 cde

17.72 ± 0.32 cd

51.8

52.5

Bs ? An

68.5 ± 1.76 ab

18.70 ± 0.20 ab

60.4

60.9

Bs ? Pc

63.5 ± 1.27 de

17.36 ± 0.31 de

48.7

49.4

Bc ? Gi

67.2 ± 2.10 abc

18.37 ± 0.23 bc

57.4

58.1

Bc ? Gm

66.7 ± 1.55 bcd

18.25 ± 0.30 bc

56.2

57.1 56.1

Bs ? Gi

66.3 ± 1.60 bcd

18.14 ± 0.18 bc

55.3

Bs ? Gm

65.4 ± 1.83 bcd

17.88 ± 0.28 cd

53.2

53.9

An ? Gi

61.7 ± 1.36 ef

16.86 ± 0.27 ef

44.5

45.1

An ? Gm

59.8 ± 1.38 fg

16.35 ± 0.38 fg

40.0

40.7

Pc ? Gi Pc ? Gm

58.4 ± 0.99 fgh 57.9 ± 1.54 ghi

15.97 ± 0.49 gh 15.84 ± 0.15 ghi

36.8 35.6

37.4 36.3

Bc = Burkholderia cepacia MTCC No. 4684; Bs = Bacillus subtilis MTCC No. 7612; An = Aspergillus niger CA; Pc = Penicillium chrysogenum CA1; Gi = Glomus intraradices KA; Gm = Gigaspora margarita AA a

Values within each column followed by the same letter are not significantly different (Tukey’s test, P \ 0.05)

than parasitism (Eapen et al. 2005). Neither of these species were isolated from eggs or females of nematodes, so they apparently do not parasitize nematodes; hence their effect is exogenous. Moreover, enzymatic disintegration of the vitelline and chitin layers of the nematode eggshell might have increased the permeability of the eggshell and enhanced mycelial penetration, leading to total disintegration of the egg contents (Eapen et al. 2005). In the present study, AM fungi improved plant growth of nematode-infected plants by reducing nematode multiplication as shown earlier (Bagyaraj et al. 1979). In our study, we presumed that disease inhibition by Gl. intraradices KA or Gi. margarita AA might not be completely related to an increase in phosphorus content despite a significant increase in phosphorus and dry mass of roots (data not shown). In addition to changes in nutrient uptake by the 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). Treatment with AM fungi is also reported to increase phenylalanine and serine in tomato roots (Suresh 1980); these amino acids have an inhibitory effect on nematodes (Reddy 1974). Similarly, Bacillus isolates have been reported to promote the growth of a wide range of plants (De Freitas et al.

1997; Kokalis-Burelle et al. 2002). Bacillus subtilis reduced nematode galling and multiplication, resulting in improved growth of nematode-inoculated plants. Improvement in growth can be attributed to an inhibitory effect of Ba. subtilis MTCC No. 7612 against plant pathogens (Siddiqui and Mahmood 1995; Yuen et al. 1985). Previous studies indicate that treatments with Ba. subtilis increased yields of several crops (Merriman et al. 1974; Turner and Backman 1986). Additionally, this bacterium improved plant growth by inhibiting root pathogens, producing biologically active substances, or by transforming unavailable mineral and organic compounds into form available to plants (Broadbent et al. 1977). Moreover, a noncellular extract of Ba. subtilis is also reported to have a high degree of larvicidal properties against root-knot and cyst nematodes (Gokte and Swarup 1988). Burkholderia cepacia is recognized for its abilities to promote plant growth (Bevivino et al. 1998) and enhance crop yields (Chiarini et al. 1998; Tabacchioni et al. 1993). It also suppresses many soilborne plant pathogens (Daubaras et al. 1996; Mueller et al. 1997). The increase in plant growth and reduction in nematodes multiplication that we observed agrees with earlier studies (Meyer et al. 2000). Moreover, economically important crop diseases

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Table 4 Root colonization by plant growth-promoting rhizobacteria (PGPR), and arbuscular mycorrhizal fungi (AM) alone and in combination with soil antagonistic fungi (SA) in plants without nematodes Treatment

Root colonization by PGPR (cfu/g soil)a AM (%)a

Table 5 Root colonization by plant growth-promoting rhizobacteria (PGPR) and arbuscular mycorrhizal fungi (AM) alone and in combination with soil antagonistic fungi (SA) in plants inoculated with root-knot nematode Meloidogyne incognita Treatment

Root colonization by PGPR (cfu/g soil)a AM (%)a

Nil

Control

2.3 9 103 g

–

PGPR

Bu. cepacia 4684

2.9 9 105 abc

–

Nil

Control

2.2 9 103 e

–

Ba. subtilis 7612

2.5 9 105 de

–

PGPR

Bu. cepacia 4684

2.6 9 105 ab

–

Ba. subtilis 7612

2.1 9 105 cd

–

A. niger CA

–

–

P. chrysogenum CA1 –

–

Gl. intraradices KA

–

51 b

Gi. margarita AA

–

44 d

Bc ? An

2.4 9 105 bc

–

SA

A. niger CA

–

–

P. chrysogenum CA1 – AM PGPR 9 SA

Gl. intraradices KA

–

54 ab

Gi. margarita AA

–

48 cd

Bc ? An

2.6 9 105 cd

–

5

PGPR 9 SA

Bc ? Pc

2.7 9 10 bcd

–

2.0 9 105 f

–

Bc ? Pc

2.5 9 105 b

–

5

–

5

3.2 9 10 a

59 a

Bs ? An Bs ? Pc

1.8 9 105 d 1.9 9 105 d

– –

2.8 9 105 bcd

53 bc

PGPR 9 AM Bc ? Gi

2.9 9 105 a

57 a

54 ab 51 bcd

Bc ? Gm

2.5 9 105 b

51 b

Bs ? Gi

2.7 9 105 ab

51 b

Bs ? Gm

2.5 9 105 b

49 bc

An ? Gi

–

49 bc

PGPR 9 AM Bc ? Gi Bc ? Gm Bs ? Gi Bs ? Gm

2.2 9 10 ef

5

3.0 9 10 ab 2.8 9 105 bcd

An ? Gi

–

50 bcd

An ? Gm

–

47 d

Pc ? Gi

–

52 bcd

An ? Gm

–

45 d

Pc ? Gm

–

50 bcd

Pc ? Gi

–

49 bc

Pc ? Gm

–

46 cd

Bc = Burkholderia cepacia MTCC No. 4684; Bs = Bacillus subtilis MTCC No. 7612; An = Aspergillus niger CA; Pc = Penicillium chrysogenum CA1; Gi = Glomus intraradices KA; Gm = Gigaspora margarita AA a

Values within each column followed by the same letter are not significantly different (Tukey’s test, P \ 0.05)

such as blight caused by Alternaria solani or the blight of oil-producing plants rape and canola by A. brassicae and A. brassicola, have been controlled by Bu. cepacia. The organism is also being used to prevent the blight of ginseng plants by Alternaria panax (Joy and Parke 1995). The Bu. cepacia complex (Bcc) is a group of closely related, remarkably versatile bacteria found naturally in soil, water, and the rhizosphere of plants (Parke and Gurian-Sherman 2001). Individual strains within this complex have the ability to cause plant disease, protect plants from disease, cause nosocomial infections, and degrade environmental pollutants. In the last two decades, the Bu. cepacia complex has also emerged as opportunistic pathogens of humans with cystic fibrosis. The dominance of the Bu. cepacia complex in the rhizosphere probably results from a suite of physiological and genetic traits that confer its competitive saprophytic survival and may contribute to root colonization, biocontrol, and plant growth promotion. This is probably true even if introduction of the biocontrol strain does not increase the level of Bcc above the indigenous population already present, and thus risk to

123

AM

Bs ? An Bs ? Pc

SA 9 AM

–

SA

SA 9 AM

Bc = Burkholderia cepacia MTCC No. 4684; Bs = Bacillus subtilis MTCC No. 7612; An = Aspergillus niger CA; Pc = Penicillium chrysogenum CA1; Gi = Glomus intraradices KA; Gm = Gigaspora margarita AA a

Values within each column followed by the same letter are not significantly different (Tukey’s test, P \ 0.05)

the cystic fibrosis population is not increased (Parke and Gurian-Sherman 2001). The PGPR, AM fungi, and antagonistic fungi were used individually and in combination to determine which two groups together are most suitable to control root-knot nematodes. Dual inoculation with biocontrol agents having different mechanisms of action is known to provide greater biocontrol against plant pathogens on different crops than an inoculation with a single agent (Akhtar and Siddiqui 2008a, b; Guetsky et al. 2002; Siddiqui and Akhtar 2008). The effectiveness of the PGPR, AM fungi and antagonistic fungi was found to be species dependent. Use of PGPR with either AM fungi or with antagonistic fungi caused a greater decrease in galling and the nematode populations than with an antagonistic fungus plus AM fungus. Of the antagonistic fungi tested with PGPR, A. niger CA was better than P. chrysogenum CA1 at reducing galling and nematode populations. Antagonistic fungi had no adverse effect on the PGPR as evident from the enhanced plant growth and similarity in the level of root colonization by

J Gen Plant Pathol (2009) 75:144–153

Treatment

138 a

14,760 a

Control

PGPR

Bu. cepacia 4684

84 e

8,920 e

Ba. subtilis 7612

90 d

9,170 de

A. niger CA

94 cd

9,570 cd

P. chrysogenum CA1

98 bc

9,960 c

AM

Gl. intraradices KA

97 c

9,840 c

102 b

11,130 b

PGPR 9 SA

Bc ? An

37 o

3,940 m

Bc ? Pc

59 ij

6,120 ij

Bs ? An

40 no

4,120 m

Bs ? Pc Bc ? Gi

63 i 44 mn

6,480 i 4,570 l

Bc ? Gm

47 lm

4,890 kl

SA

PGPR 9 AM

SA 9 AM

a

Values within each column followed by the same letter are not significantly different (Tukey’s test, P \ 0.05)

Bs ? Gi

51 kl

5,260 k

Bs ? Gm

55 jk

5,710 j

An ? Gi

68 h

6,940 h

An ? Gm

73 g

7,480 g

Pc ? Gi

77 fg

7,890 fg

Pc ? Gm

81 ef

8,270 f

16000

Total number of juveniles, eggs and females in root and soil

Fig. 1 Effects of plant growthpromoting rhizobacteria, soil antagonistic fungi, and arbuscular mycorrhizal (AM) fungi on the galling of tomato and nematode multiplication. Error bars represent standard error. Bc = Burkholderia cepacia MTCC No. 4684; Bs = Bacillus subtilis MTCC No. 7612; An = Aspergillus niger CA; Pc = Penicillium chrysogenum CA1; Gi = Glomus intraradices KA; Gm = Gigaspora margarita AA

No. nematodes (root ? 1 kg soil)a

Nil

Gi. margarita AA

Bc = Burkholderia cepacia MTCC No. 4684; Bs = Bacillus subtilis MTCC No. 7612; An = Aspergillus niger CA; Pc = Penicillium chrysogenum CA1; Gi = Glomus intraradices KA; Gm = Gigaspora margarita AA

No. of galls/root systema

Total no. of juveniles, eggs and females in root and soil No. of galls / root system

160

14000

140

12000

120

10000

100

8000

80

6000

60

4000

40

2000

20

G i G m Bc +A Bc n +P Bs c +A Bs n +P c Bc +G Bc i +G m Bs +G Bs i +G m An +G An i +G m Pc +G Pc i +G m

Pc

Bs

An

C

0 Bc

0

No. of galls per root system

Table 6 Effects of plant growth-promoting rhizobacteria (PGPR), soil antagonistic fungi (SA), and arbuscular mycorhizal AM fungi (AM) on mean number of root galls and population of root-knot nematode Meloidogyne incognita

151

Treatments

PGPR. Because Bu. cepacia MTCC No. 4684 colonized host roots, the effects on nematodes may be plant mediated because PGPR strains were shown to trigger a plant-mediated resistance in different plant parts (Van Peer et al. 1991; Wei et al. 1991). Bu. cepacia MTCC No. 4684 also was antagonistic to nematodes hatching in a preliminary study (data not shown). On the other hand, A. niger acts

directly through enzymatic disintegration of the nematodes egg contents (Eapen et al. 2005), which is the probable reason for A. niger CA with Bu. cepacia MTCC No. 4684 causing the greatest reduction in galling and nematode populations in this study. Different modes of action by these organisms probably resulted in synergistic effects in increasing plant growth (Guetsky et al. 2002). Similarly,

123

152

antagonistic fungi with AM fungi also had an additive effect in reducing galling and nematode population, but less than when either was used with PGPR because PGPR is better than either AM fungi or antagonistic fungi at reducing galling and nematode population. Moreover, PGPR are also known to synthesize antibiotics that prevent proliferation of phytopathogens (Siddiqui 2006). The experiments described here were done in pots with sterilized soil. When these microorganisms are added in the field, they will have to compete with other soil microorganisms and survive environmental conditions that will influence their efficacy as biocontrol agents. Although the present study indicates that the antagonistic fungus A. niger CA may be applied with Bu. cepacia MTCC No. 4684 to control M. incognita on tomato, studies under different field conditions are required to confirm these results.

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