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International Journal of PharmTech Research CODEN (USA): IJPRIF, ISSN: 0974-4304, ISSN(Online): 2455-9563 Vol.9, No.10, pp 097-107, 2016
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Antagonistic effects of rhizobacteria isolates against Meloidogyne incognita infecting tomato plants under greenhouse conditions
Hassan Abd-El-Khair1*, Wafaa, M.A. El-Nagdi2 and Hoda, H. Ameen2
1Plant Pathology Department, National Research Centre, Giza, Egypt.
Abstract : Thirty rhizobacteria (RB) isolates isolated from rhizospheres of healthy plants - free from nematode infection viz. 13 isolates (RBba1-RBba13) from banana; 6 isolates (Rbbe1-RBbe6) from bean ; and 11 isolates (RBcu1 - RBcu11) from cucumber. All the thirty RB isolates were primarily identified according to cultural characters using standard bacteriological method and their nematicidal activity were evaluated against Meloidogyne incognita at second stage juveniles (J2) in vitro. Results of primary bioassay test of the thirty RB isolates against Meloidogyne incognita J2 showed that the percentages of mortality ranged from 81 – 97%. RB isolates of banana, bean and cucumber reduced the mortality of M. incognita J2 in the ranges of 81-97%, 85-96% and 84-95%, respectively. Isolates of RBba9, RBba10, RBba12, RBba13, RBbe5, RBbe6 RBcu1and RBcu6 showing the highest net mortality of nematode about ≥ 95% were selected and identified as Bacillus sp.ba9, Bacillus sp.ba10, Bacillus sp.ba12, Bacillus sp.ba13, Bacillus sp.be5, Bacillus sp.be6, Bacillus sp.cu1 and Bacillus sp.cu6 according to morphological, cultural and biochemical characters. Under greenhouse conditions, the eight select Bacillus spp. significantly reduced the root-knot nematode parameters, i.e. numbers of J2 in soil (82.7 – 97.6%); J2 in roots (91.7 – 95.8%) ; Galls (61.1- 85.3%) and egg-masses (63.8 - 87.0%), compared to untreated controls. The treatments also improved tomato plant growth parameters such as shoot length, shoot and root dry weight, compared to untreated controls.
Key words: Bacillus spp., Nematicidal activity, Meloidogyne incognita, Rhizo-bacteria, Tomato.
Plant growth promoting rhizo-bacteria (PGPR) are the soil bacteria inhabiting around/on the root surface, they affect plant growth directly through synthesized compounds i.e. phytohormones, auxin, facilitating the uptake of certain nutrients from the environment or indirectly when they prevent the deleterious pathogens to came in contact with the plant 1. Rhizo-bacteria affect nematode by more than one mechanisms: direct effects on egg hatch and nematode mobility through the production of toxins; interference with plant-nematode recognition ; competition for nutrients; plant growth promotion; synthesize enzymes; production of metabolites such as protease, chitinase, glucanase, antibiotics, alteration of root exudates which make roots less attractive to nematodes and induced systemic resistance 2,3,4 . Out of several PGPR genera; Bacillus spp. have considerable potential effect in the biocontrol of plant parasitic nematodes by their effective root colonization, multiple modes of action and promising ability to sporulate under stressed conditions 5. Moussa and Zawam6 reported that Bacillus amyloliquefaciens, Brevibacterium otitidis and Sanguibacter inulinus inhibited the egg-masses hatching of M. incognita in vitro and exhibited strong nematicidal activity by killing the second stage juveniles
(J2) of nematode in tomato plants under greenhouse conditions. El-Hadad et al.7found that application of Bacillus polymyxa NFB7, B. megaterium PSB2 and B. circulans KSB2 gave the highest reduction in root-knot nematode population on tomato plants under greenhouse conditions, comparing with the un-inoculated nematode-infested control.
Xiao et al.8 noted that Bacillus cereus X5-gfp can kill J2 of root -knot nematodes and reduce egg hatching rates in pot experiment by colonized the rhizosphere soil and the root surfaces of tomato plants. Khalil et al.9 reported that Bacillus subtilis, Pseudomonas fluorescens and Paecilomyces lilacinus reduced root galls of M. incognita and egg masses on root system as well as J2 numbers in the soil on the tomato plants under greenhouse conditions. Their data obvious that, B. subtilis recorded the highest increase in fresh root weight, followed by P. fluorescens with values of 125.75 and 86.57%, consecutively, while the great increase in root dry weight 68.14% resulted from P. fluorescens followed by B. subtilis which recorded 35.40%. Khalil et al.9 found that application of B. subtilis and B. thuringiensis suppressed populations of M. incognita infesting tomato plants in the soil with 82.6 and 80.5%, respectively as compared to control. They mentioned that B. thuringiensis also increased shoot weight and root length than B. subtilis. Almaghrabi et al.10 noted that B. amyloliquefaciens, B. subtilis and B. cereus were effective in reducing numbers of J2 in soil, galls and egg masses/root of root-knot nematode in tomato plant after 45 days from nematode infection. They mentioned that treatments increased shoot dry weight, plant height, fruit/plant and weight of plant yield, compared to infected untreated plants. Amin et al.11 reported that Bacillus brevis, B. cereus and Bacillus firmus showed significant reduction in M. incognita development and reproduction in tomato under greenhouse condition. El-Nagdi and Abd-El-Khair12 mentioned that B. subtilis (as commercial product of Rhizo-N®) highly reduced the number of rook knot nematode J2 in soil.
This work is aimed to 1) Isolate of common rhizo-bacteria from the rhizospheres of banana , bean and cucumber healthy plants-free from root knot nematode infection ; 2) Study the nematicidal activity of the isolated rhizo-bacteria against M. incognita second stage juveniles (J2) in vitro ;and 3) Determine their antagonistic activity on nematodes parameters viz. numbers of J2 in soil and roots and number of galls and egg-masses on tomato roots in pot experiment.
Materials and methods
1. Sampling
Thirty rhizosphere soil samples were collected from healthy plants -free from root-knot nematode infection- from banana (13 samples), bean (6 samples) and cucumber (11 samples) from Gazeret El-Dahab, Giza Governorate, Egypt. About 200 g soil as collective sample were collected around each plant root at a depth of 20–30 cm. The samples were kept in polyethylene bags and immediately transferred to the Plant Pathology Department, National Research Centre for isolation of common rhizo-bacteria.
2. Isolation and identification of common rhizo-bacteria (RB)
The isolation of RB was made according to total plate count technique and dilution method13. Ten grams of each collective sample were transferred into 250 ml conical flask containing 90 ml of sterile distilled water to give dilution of 10-1. Serial dilutions from 10 -2 to 10-7 were prepared. One ml from each dilution was pipette into sterile Petri-dish (9 cm diam.) containing Nutrient Glucose 1% Agar (NGA) medium [ Beef extract ,3.0g ; Peptone ,5.0g ; Glucose , 10.0g ; Agar 15.0g ; in 1.0 liter of distilled water and pH ,7.4 ± 0.2 ]14. Each dilution was replicated three times and incubated at 28°C for 48h. The thirty RB isolates were firstly identified according to the growth cultural characters on NGA medium using standard bacteriological methods15. Then, the eight RB isolates named RBba9, RBba10, RBba12, RBba13, RBbe5, RBbe6, RBcu1, and RBcu6 which showed the net mortality of M. incognita J2 about ≥ 95% were selected to identify to genera level using cultural, morphological and biochemical characters as Bacillus sp.ba9, Bacillus sp.ba10, Bacillus sp. ba12, Bacillus sp. ba13, Bacillus sp. be5, Bacillus sp.be6, Bacillus sp.cu1 and Bacillus sp.cu6. 15, 16
3. In vitro experiments
3.1. Preparation of RB suspension
For the preparation of RB suspension, each bacterial isolate was separately grown in Nutrient Glucose 1% Broth (NGB) medium for four days at 28°C and then adjusted to 107- 109 colony forming unit (CFU)/ml using turbidity method for in vitro studies17 and considering as S concentration.
3.2. Root knot nematode inoculums
The second stage juveniles (J2) of root-knot nematode used throughout the study were extracted according to Hussey and Barker18 from a pure culture maintained in eggplant roots in the greenhouse of the National Research Centre and previously identified as Meloidogyne incognita according to Taylor and Sasser 19.
3.2.1. Primary bioassay test
Twenty five ml plastic capsule supplied with nine ml from each of the thirty RB isolates suspension plus one ml of nematode suspension containing 300 M. incognita J2. Sterile distilled water and NGB medium without bacteria were served as control. The numbers of viable and dead nematodes were counted under a light microscope after 24, 48 and 72 h of exposure at 25°C. Nematodes were considered alive if they moved or assumed a winding shape and dead if they were straight and immobile. After the exposure periods the nematodes in each treatment were transferred to distilled water and left for 24 h to see whether immobile nematodes resumed activity or not. The corrected percentages of nematode mortality were calculated according to Abbott’s20 formula:
Mortality (%) = (m – n)/ (100 – n) × 100
Where: m and n indicate the percentages of mortality in treatments and control, respectively.
3.2.2. Secondary bioassay test
The nematicidal activity of the eight RB isolates viz. Bacillus sp.ba9, Bacillus sp.ba10, Bacillus sp.ba12, Bacillus sp.ba13, Bacillus sp.be5, Bacillus sp.be6, Bacillus sp.cu1and Bacillus sp.cu6, which gave ≥ 95%, mortality in the primary bioassay test , were re-evaluated for their nematicidal activity against M. incognita J2 mortality in vitro as previously described.
4. Greenhouse study
A pot experiment was conducted to assess the nematicidal effects of the eight selected Bacillus spp. isolates viz. Bacillus sp.ba9, Bacillus sp.ba10, Bacillus sp. ba12, Bacillus sp.ba13, Bacillus sp.be5, Bacillus sp.be6, Bacillus sp.cu1 and Bacillus sp.cu6 against M. incognita reproduction infecting tomato plants as well as plant growth parameters in comparing with Micronema® as commercial bio-product (containing109 CFU/ml of Serratia sp., Pseudomonas sp., Azotobacter sp., Bacillus circulans and Bacillus thuringiensis) and NGB medium and untreated plant as control under greenhouse conditions at Plant Pathology Department, National Research Centre (NRC) . Plastic pots (20 cm in diam.) containing 2 kg autoclaved sandy loamy soil (1:1 v/v) were arranged in randomized completely design on a bench in the greenhouse at 25 ± 5 °C. Soil of each pot was separately mixed with 20 ml from each of the previous RB suspension and NGB medium without bacteria then transplanted with two -one month old- tomato seedlings. One week later, each pot received 1,000 newly hatched M. incognita J2 (in four holes around plants). Pots received only 1,000 newly hatched M. incognita J2 served as control. The plants were watered after inoculation and thereafter, whenever required. Each treatment replicated four times as well as the control. After 6 months from nematode infestation, the following data were collected on nematode reproduction [No. of J2 in soil and roots, No. of root galls, and egg-masses / root system (eight plant roots per treatment)] as well as dry shoot length and weight. All collected data were subjected to analysis of variance ANOVA procedures which reported by Snedecor and Cochran21 and means of treatments were compared by the least significant difference test “LSD” at 5% level of probability using Computer Statistical Package (CO-STATE) User Manual Version 3.03, Barkley Co.
Results
1. Rhizo-bacteria (RB) isolates
The thirty RB isolated from healthy plant were classified into thirteen isolates from banana rhizosphere (RBba1 to RBba13); six isolates from bean rhizosphere (RBbe1 to RBbe6) and eleven isolates from cucumber rhizosphere (RBcu1 to RBcu11), respectively. Their cultural characters details according to standard bacteriological methods are listed in Table (1).
Table 1. Cultural characters of rhizo-bacteria isolates (RB) isolated from the rhizosphere of some plant species on nutrient glucose 1% agar medium.
No. |
Rhizo-Bacteria isolates |
Plant species |
Cultural characters* |
1 |
RBba1 |
Banana
|
Irregular , Raised , Undulate , Smooth , Transluscent , Butyrous , Creamy white |
2 |
RBba2 |
Irregular , Flat , Erose , Smooth , Transluscent , Butyrous , Creamy white |
|
3 |
RBba3 |
Circular , Flat , Entire , Smooth , Translucent , Botyrous , Creamy white |
|
4 |
RBba4 |
Circular , Convex , Entire , Smooth , Translucent , Botyrous , Creamy white |
|
5 |
RBba5 |
Circular , Raised , Entire , Smooth , Opaque , Butyrous , White |
|
6 |
RBba6 |
Circular , Raised , Entre , Smooth , Opaque , Butyrous , White |
|
7 |
RBba7 |
Circular , Convex, Entire , Smooth , Opaque , Butyrous , creamy white |
|
8 |
RBba8 |
Circular , Convex , Entire , Smooth , Opaque , Viscoud , White |
|
9 |
RBba9 |
Rhizoid , Flat , Filamentous , Rough , Translucent , Membranous ,Creamy white |
|
10 |
RBba10 |
Irregular , Raised , Undulate , Rough , Opaque , Brittle, White |
|
11 |
RBba11 |
Irregular , Flat , Lobate , Rough , Opaque , Brittle , White |
|
12 |
RBba12 |
Irregular , Flat , Lobate , Rough , Opaque , Brtittle , White |
|
13 |
RBba13 |
Filamentous , Flat , Filamentous , Rough , Translucent , Brtittle , white |
|
14 |
RBbe1 |
Bean |
Circular , Convex , Entre , Smooth , Opaque , Viscoud , Creamy white |
15 |
RBbe2 |
Rhizoid , Flat , Filamentous , Rough , Translucent , Membranous, White |
|
16 |
RBbe3 |
Circular , Convex , Entire , Smooth , Opaque, butyrous , Creamy white |
|
17 |
RBbe4 |
Irregular , Flat , Lobate , Rough , Translucent , Brittle , Creamy white |
|
18 |
RBbe5 |
Irregular , Flat , Undulate , Rough , Translucent , Membranous , Creamy white |
|
19 |
RBbe6 |
Circular , Convex, Entire , Smooth , Opaque , Butyrous , creamy white |
|
20 |
RBcu1 |
Cucumber |
Irregular, Raised, Undulate, Rough, Opaque, Butyrous, Creamy white. |
21 |
RBcu2 |
Irregular , Raised , Undulate , Smooth , Translucent , Butyrous , Creamy while |
|
22 |
RBcu3 |
Circular , Raised , Entire , Smooth , Opaque , Butyrous , White |
|
23 |
RBcu4 |
Circular , Raised , Entire , smooth, Opaque , Butyrous , Creamy white |
|
24 |
RBcu5 |
Rhizoid , Flat , Filamentous , Rough , Translucent , Membranous, White |
|
25 |
RBcu6 |
Circular , Convex , Entire , Smooth , Opaque, Viscoud , Creamy white |
|
26 |
RBcu7 |
Circular , Convex , Entire , Smooth , Opaque, butyrous , Creamy white |
|
27 |
RBcu8 |
Irregular , Flat , Lobate , Rough , Opaque , Membranous , Creamy white |
|
28 |
RBcu9 |
Circular , Convex , Entire , Smooth , Opaque , Butyrous , Creamy white |
|
29 |
RBcu10 |
Circular , Raised , Entire ,Smooth , Opaque, Butyrous , Creamy white |
|
30 |
RBcu11 |
Circular , Convex , Entire , Smooth , Opaque , Butyrous , White |
*Cultural of signal bacteria colony as Form, Elevation, Margin of edge, Surface, Optical features, and Consistency
2. In vitro bioassay
2.1. Primary bioassay test
As shown in (Table 2), all the 30 RB isolates at concentration (S) could kill M. incognita J2 and the percentage mortality increased by increasing the exposure time. The percentages of mortality after 24 h of exposure were in the ranges of 81-92%, while it's were in the range of 86-94% and 90- 97% after 48 and 72 h, respectively. The net mortality was in the ranges of 90 - 97%. No recovery % was occurred. The RB isolates of banana; bean and cucumber reduced the J2 of nematode in the ranges of 81-97%; 85-96% and 84-95% after the different exposure periods, respectively. The net mortality of J2 was in the ranges of 90-97%, 92-96% and 92-95% with RB isolates of banana, bean and cucumber rhizospheres, respectively. Results obvious that the eight of RB isolates viz. RBba9, RBba10, RBba12, RBba13, RBbe5, RBbe6, RBcu1 and RBcu6 which showing mortality of M. incognita J2 by ≥ 95 % were identified as Bacillus spp.
Table 2. In vitro nematicidal activities of 30 rhizosphere bacteria from banana bean and cucumber plants against Meloidogyne incognita based on juvenile mortality.
No. |
Rhizo-bacteria isolates |
Mortality % |
Net Mortality % |
||
24h |
48h |
72h |
|||
1 |
RBba1 |
81 |
86 |
90 |
90 |
2 |
RBba2 |
83 |
88 |
91 |
91 |
3 |
RBba3 |
89 |
91 |
94 |
94 |
4 |
RBba4 |
86 |
90 |
93 |
93 |
5 |
RBba5 |
87 |
89 |
91 |
91 |
6 |
RBba6 |
90 |
92 |
94 |
94 |
7 |
RBba7 |
89 |
91 |
94 |
94 |
8 |
RBba8 |
87 |
89 |
92 |
92 |
9 |
RBba9 |
88 |
90 |
95 |
95 |
10 |
RBba10 |
90 |
93 |
97 |
97 |
11 |
RBba11 |
89 |
91 |
94 |
94 |
12 |
RBba12 |
91 |
94 |
96 |
96 |
13 |
RBba13 |
92 |
93 |
95 |
95 |
14 |
RBbe1 |
85 |
90 |
93 |
93 |
15 |
RBbe2 |
86 |
90 |
93 |
93 |
16 |
RBbe3 |
87 |
90 |
92 |
92 |
17 |
RBbe4 |
85 |
89 |
93 |
93 |
18 |
RBbe5 |
90 |
93 |
96 |
96 |
19 |
RBbe6 |
88 |
92 |
95 |
95 |
20 |
RBcu1 |
89 |
93 |
95 |
95 |
21 |
RBcu2 |
88 |
92 |
93 |
93 |
22 |
RBcu3 |
84 |
88 |
92 |
92 |
23 |
RBcu4 |
86 |
89 |
92 |
92 |
24 |
RBcu5 |
89 |
92 |
93 |
93 |
25 |
RBcu6 |
90 |
94 |
95 |
95 |
26 |
RBcu7 |
88 |
91 |
94 |
94 |
27 |
RBcu8 |
87 |
90 |
92 |
92 |
28 |
RBcu9 |
88 |
90 |
93 |
93 |
29 |
RBcu10 |
87 |
92 |
93 |
93 |
30 |
RBcu11 |
89 |
90 |
94 |
94 |
Medium |
6 |
8 |
10 |
10 |
|
Water only |
0 |
0 |
0 |
0 |
|
L.S.D. 0.05 |
Bacterial isolates (B) |
Period (P) |
B x P |
||
0.7 |
0.2 |
1.1 |
2.2. Second bioassay test
Data in Table (3) showed that the eight Bacillus spp. isolates viz. Bacillus sp.ba9, Bacillus sp. ba10, Bacillus sp.ba12, Bacillus sp.ba13, Bacillus sp.be5, Bacillus sp.be6, Bacillus sp.cu1 and Bacillus sp.cu6 at concentration (S) could kill M. incognita J2. The percentages of mortality after 24 h of exposure were in the ranges of 88-95%, while it's were in the ranges of 90 – 96% after 48 and 72 h of exposure period, respectively. The net mortality was in the ranges of 90 – 96%. Bacillus sp.ba10 gave the highly percentage of mortality in M .incognita J2 , followed by Bacillus sp.ba12, Bacillus sp.be6 , Bacillus sp.cu6 , Bacillus sp.be5 , Bacillus sp.ba9 and Bacillus sp.cu1, where the net mortalities were 96 , 95 , 95 , 95 , 95 ,93 , 90 and 90% , respectively. While Micronema® resulted in 93, 95 and 95% mortality for 24, 48 and 72 h of exposure. The NGB medium induced only 6, 8 and 8 % mortality during the previous exposure time, respectively. No recovery % was occurred. As observed in Table (3), the nematicidal effect of the selected bacterial isolates was increased by increasing the exposure periods.
Table 3. Effects of Bacillus spp. isolates on the mortality of second-stage juveniles of Meloidogyne incognita in vitro test (Second bioassay).
Rhizo-bacteria treatments |
Mortality % |
Net Mortality % |
||
24h |
48h |
72h |
||
M. incognita + water only |
0 |
0 |
0 |
0 |
M. incognita + NGB Medium only |
6 |
8 |
8 |
8 |
M. incognita + Micronema® |
93 |
95 |
95 |
95 |
M. incognita + Bacillus sp.ba9 |
88 |
90 |
90 |
90 |
M. incognita + Bacillus sp.ba10 |
96 |
96 |
96 |
96 |
M. incognita + Bacillus sp.ba12 |
93 |
95 |
95 |
95 |
M. incognita + Bacillus sp.ba13 |
94 |
95 |
95 |
95 |
M. incognita + Bacillus sp.be5 |
91 |
93 |
93 |
93 |
M. incognita + Bacillus sp.be6 |
95 |
95 |
95 |
95 |
M. incognita + Bacillus sp.cu1 |
89 |
90 |
90 |
90 |
M. incognita + Bacillus sp.cu6 |
92 |
94 |
95 |
95 |
3. Greenhouse experiment
3.1. Effect on root-knot nematode parameters
Data in Table (4) showed that the entire eight selected Bacillus spp. isolates as well as the commercial product Micronema® significantly reduced numbers of M. incognita J2 in soil and roots as well as root galls and egg-masses counts in roots, comparing with untreated control after 6 months from nematode infestation. The percentages of reduction in M. incognita J2 ∕ 200 g soil due to the application of the Bacillus spp. isolates ranged between 82.7 - 97.6%, while Micronema® and the NGB medium resulted in 93.1 and 79.5% reduction ,respectively, as compared to untreated control. Data showed that Bacillus sp. be5 gave the highest reduction in M. incognita J2 in soil , followed by Bacillus sp. ba12, Bacillus sp.cu6 , Bacillus sp.be6, Bacillus sp.ba9 , Bacillus sp.ba10 , Bacillus sp.cu1 and Bacillus sp.cu13 , where the percentages of reduction recorded were 97.6 , 95.2 , 95.2 , 93.7 , 92.2 , 91.3 , 89.4 and 82.7% , respectively (Table,4). While the percentages of reduction recorded in M. incognita J2 in roots ranged from 91.7 to 95.8%, while those resulted from the application of Micronema® and NGB medium were 94.8 and 75.5 %, respectively, compared to untreated control. Bacillus sp. be5 also resulted in the highly reduction in M. incognita J2 in roots , followed by Bacillus sp.ba13 , Bacillus sp.ba9 , Bacillus sp.ba10 , Bacillus sp.cu6 , Bacillus sp.ba12 , Bacillus sp.cu1and Bacillus sp.be6 , where the percentages of reduction were 95.8 , 95.8 , 95.7 , 95.2 , 94.3 , 93.4 , 92.9 and 91.7% , respectively (Table, 4).
Table 4. Nematicidal effects of Bacillus spp. isolates on Meloidogyne incognita reproduction infecting tomato plants.
Rhizo-bacteria treatments |
M. incognita parameters |
|||||||
J2 in /200g soil
|
J2 in /5g roots
|
Root galls /5g roots
|
egg-masses/5g roots
|
|||||
Count |
Red. % |
Count |
Red. % |
Count |
Red. % |
Count |
Red. % |
|
Nematode only |
2600 |
- |
2100 |
- |
95 |
- |
69 |
- |
M.incognita+NGB Medium only |
533 |
79.5 |
515 |
75.5 |
66 |
30.5 |
45 |
34.8 |
M. incognita + Micronema® |
180 |
93.1 |
110 |
94.8 |
18 |
81.1 |
14 |
79.7 |
M. incognita + Bacillus sp.ba9 |
203 |
92.2 |
90 |
95.7 |
27 |
71.6 |
20 |
71.0 |
M. incognita + Bacillus sp.ba10 |
225 |
91.3 |
100 |
95.2 |
37 |
61.1 |
32 |
53.6 |
M. incognita + Bacillus sp.ba12 |
125 |
95.2 |
138 |
93.4 |
22 |
76.8 |
18 |
73.9 |
M. incognita + Bacillus sp.ba13 |
450 |
82.7 |
88 |
95.8 |
15 |
4.2 |
11 |
84.1 |
M. incognita + Bacillus sp.be5 |
63 |
97.6 |
88 |
95.8 |
18 |
81.1 |
13 |
81.2 |
M. incognita + Bacillus sp.be6 |
165 |
93.7 |
175 |
91.7 |
35 |
63.2 |
25 |
63.8 |
M. incognita + Bacillus sp.cu1 |
275 |
89.4 |
150 |
92.9 |
14 |
85.3 |
9 |
87.0 |
M. incognita + Bacillus sp.cu6 |
125 |
95.2 |
120 |
94.3 |
17 |
82.1 |
12 |
82.6 |
L.S.D. 0.05 |
144.4 |
- |
76.4 |
- |
7.6 |
- |
6.0 |
- |
Also the numbers of root galls were affected by the application of the eight of Bacillus spp. isolates, the detected reduction percentages ranged between 61.1-85.3%, while the application of Micronema® and NGB medium resulted in 81.1 and 30.5% reduction, respectively, as compared to untreated control. As shown in (Table, 4) , Bacillus sp.cu1 showed the highly reduction in root galls followed by Bacillus sp.ba13 , Bacillus sp.cu6, Bacillus sp.be5, Bacillus sp.ba12, Bacillus sp.ba9, Bacillus sp.be6 and Bacillus sp.ba10, where the percentages of reduction were 85.3, 84.2, 82.1, 81.1, 76.8, 71.6, 63.2 and 61.1% , respectively. All Bacillus spp. isolates suppressed number of egg-masses in tomato roots, as compared to control. The percentages of reduction ranged between 53.6 – 87.0% and for Micronema® and NGB medium were 79.7 and 34.8% reduction respectively as compared to untreated control. Bacillus sp.cu1 induced the highly reduction in egg-masses numbers, followed by Bacillus sp.ba13, Bacillus sp.cu6, Bacillus sp.be5, Bacillus sp.ba12, Bacillus sp.ba9, Bacillus sp.be6 and Bacillus sp.ba10, where the percentages of reduction were 87.0 , 84.1 , 82.6 , 81.2 , 73.9 , 71.0 , 63.8 and 53.6% , respectively (Table, 4).
3.2. Effect on Growth parameters of tomato plants
All the eight of Bacillus spp. isolates increased the growth parameters of tomato plants viz. plant length (cm), shoot and root dry weight (g) , compared to untreated control. Results showed that the percentage increase in plant length due to Bacillus spp. isolates ranged between 12.5-31.0%, while Micronema® and NGB medium showed 5.2 and 20.3% increase, respectively as compared to control. Bacillus sp.ba12 gave the highly increase in plant length, followed by Bacillus sp.cu1, Bacillus sp.ba13 , Bacillus sp.cu6 , Bacillus sp.ba9 , Bacillus sp.be6 , Bacillus sp.be5 and Bacillus sp.ba10 , where the percentages increase were 31.0 , 29.0 , 27.0 , 21.7 , 16.8 , 15.9 , 15.4 and 12.5% , respectively , as compared to untreated control .Also, all Bacillus spp. isolates increased shoot dry weight as compared to control. The increase was in the ranges of 9.2-107.7%, while it was 7.7 and 35.4 % with Micronema® and NGB medium, respectively (Table, 5).
Table 5. Effects of Bacillus spp. isolates on growth parameters of tomato plants infected with Meloidogyne incognita.
Rhizo-bacteria treatments |
Growth parameters |
|||||
Shoot length |
Shoot dry weight |
Root dry weight |
||||
Length (cm) |
% Inc. |
Weight (g) |
% Inc. |
Weight (g) |
% Inc. |
|
Nematode only |
34.5 |
- |
6.5 |
- |
2.5 |
- |
M. incognita +NGB medium only |
41.5 |
20.3 |
8.8 |
35.4 |
3.3 |
32.0 |
M. incognita + Micronema® |
36.3 |
5.2 |
7.0 |
7.7 |
3.1 |
24.0 |
M. incognita + Bacillus sp.ba9 |
40.3 |
16.8 |
9.2 |
41.5 |
3.2 |
28.0 |
M. incognita + Bacillus sp.ba10 |
38.8 |
12.5 |
7.1 |
9.2 |
3.7 |
48.0 |
M. incognita + Bacillus sp.ba12 |
45.3 |
31.0 |
13.5 |
107.7 |
4.6 |
84.0 |
M. incognita + Bacillus sp.ba13 |
43.8 |
27.o |
8.8 |
35.4 |
5.6 |
124.0 |
M. incognita + Bacillus sp.be5 |
39.8 |
15.4 |
9.8 |
50.8 |
4.4 |
76.0 |
M. incognita + Bacillus sp.be6 |
40.0 |
15.9 |
8.4 |
29.3 |
3.2 |
28.0 |
M. incognita + Bacillus sp. cu1 |
44.5 |
29.0 |
10.4 |
60.0 |
5.1 |
104.0 |
M. incognita + Bacillus sp. cu6 |
42.0 |
21.7 |
8.8 |
35.4 |
3.0 |
20.0 |
L.S.D. 0.05 |
9.5 |
- |
3.9 |
- |
2.0 |
- |
Bacillus sp.ba12 induced the highly increased in shoot dry weight of treated plants, followed by Bacillus sp.cu1, Bacillus sp.be5 , Bacillus sp.ba9, Bacillus sp.ba13 , Bacillus sp.be6 , Bacillus sp.be6 and Bacillus sp.ba10 , where the percentages of increase were 107.7 , 60.0 , 50.8 , 41.5 , 35.4 , 35.4 , 29.3 and 9.2% , respectively (Table,5). As well as the root dry weight increased in all treated plants as compared to control. The percentages of increase ranged between 20.0-124.0%, while Micronema® and NGB medium increased root dry weight by 24.0 and 32.0%, respectively. Bacillus sp.ba13 resulted in the highly increase in root dry weight, followed by Bacillus sp.cu1, Bacillus sp.ba12, Bacillus sp.be5, Bacillus sp.ba10, Bacillus sp.ba9, Bacillus sp.be6 and Bacillus sp.cu6, where the percentages of increase were 124.0 , 104.0 , 84.0 , 76.0 ; 48.0 , 28.0 , 28.0 and 20.0% ,respectively (Table, 5).
Discussion
The use of pesticides to control plant diseases became limited because of the environmental concerns, health conscious attitude of human beings and other hazards associated with the use of chemicals. It is clear that using PGPR to suppress the plant pathogens is gaining importance. They exhibit diverse modes of action against plant parasitic nematodes include antibiosis, competition, myco-parasitism, cell wall degradation, induced resistance, plant growth promotion and rhizosphere colonization capability22-28. This work is aimed to isolate the common rhizo-bacteria from rhizosphere of some plants to control root-knot nematode, M. incognita in vitro and in pots. Our results clearly illustrated the potential of the thirty isolated rhizo-bacteria from the rhizosphere of healthy banana, bean and cucumber plants to kill and immobilized M. incognita second stage juveniles under laboratory conditions with variation in their potentialities to kill. These results are in agreement with that cited by Becker et al.29 who reported that bacteria isolated from rhizosphere of different plants affected the vitality of second-stage juveniles of M. incognita in vitro test, also ElSayed and Edrees30found that microorganisms that can grow in the rhizosphere are ideal for use as bio-control agents where the rhizosphere provides the front line defense for roots against attack by pathogens. Present results showed that the most potent rhizo-bacteria for killing M. incognita J2 are identified as Bacillus spp. these are in accordance with Daward et al.31 who found that application of Bacillus spp., significantly reduced hatching of M. javanica eggs, whereas mortality of larvae was significantly increased with the increase in time. El-Nagdi and Abd-El-Khair32, Ruiz et al.33 and Wei et al.34found that cell-free culture filtrate of B. subtilis increased M. incognita J2 mortality and inhibited egg hatch.
The greenhouse evaluation confirm the results observed in vitro bioassay where the selected Bacillus spp. significantly reduced the numbers of J2 in soil and roots as well as galls and egg-masses/root system infected with M. incognita this documented the findings of Mannanov and Sattarova35 who reported that Bacillus species are effective in the management of plant pathogens owing to their ability to produce
antimicrobial compounds and the wide distribution of the cuticle-degrading proteases with nematicidal activity which play an important role in bacteria–nematode interactions and serve as important nematicidal factors in balancing nematode populations in the soil. Ahmad et al.36noted that the nematicidal activity of Bacillus spp. may due to their capabilities to produce indole acetic acid and hydrogen cyanide. While Oliveira et al.37 refer the nematicidal of B. megaterium to the production of threonine, glutamic acid, glycine, alanine, valine, methionine, isoleucine, leucine, tyrosine, phenylalanine and histidine that cause significantly higher J2 mortalities of M. exigua than control (water). Kavitha et al.5 mentioned that B. subtilis with high surfactin and iturin activity suppressed hatching of eggs and killed second stage juveniles of the nematode under in vitro condition. Abdel –Aziz et al.38noted that the potentiality of B. alvei against nematode’s eggs and larvae due to the hydrolytic enzymes which directly hydrolyze nematode’s eggs and larvae .The strain also produced lytic enzymes viz. chitinase, chitosanase, proteases, as well as other potential bioactive metabolite.
Obtained results demonstrated the effect of the selected Bacillus spp. on tomato growth parameters by enhancing plant length and shoot and root dry weight over control. This is in agreement with Siddiqui and Mahmood39 who reported that the rhizosphere supports large and active microbial populations capable of exerting beneficial, neutral or detrimental effects on plant growth and confirm the previous findings of Antoun and Prevot3 who found that PGPR may induce plant growth promotion directly by invade plant roots and produce different metabolites like phyto-hormones , auxin derivatives and gibberellin-like substances which have been reported to elongate stem, enlarge cells and leaves, expand the root system, induce cell-division and cause early flowering and fruiting, as well as, the metabolites of Bacillus spp. increase the mineral availability to plants by solubilization of inorganic phosphate and mineralization of organic phosphate or indirect action by stimulation of disease-resistance mechanisms40.
It is clear that PGPR are the most abundant in plant rhizosphere and exhibit diverse modes of action against nematodes includes antibiotics , enzymes and toxins production; parasitizing; competing for nutrients; inducing systemic resistance of plants and improve of plant health23,24 . Rhizo-bacteria are capable of stimulating plant growth through a variety of mechanisms that include improvement of plant nutrition, production and regulation of phyto-hormones, and suppression of disease causing organisms40. PGPR are naturally occurring soil bacteria that aggressively colonize plant roots and benefit plants by providing growth promotion. These results demonstrated that inoculation of crop plants with certain strains of PGPR at an early stage of development improves biomass production through direct effects on root and shoots growth and controlling of nematode infection.
Acknowledgement
This research work was supported in part by the In-house Project No. 10120603 entitled “Improving Activity of some Microorganisms through Biotechnological Approaches to Control plant Parasitic Nematodes” funded by National Research Centre, Egypt.
References
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