Nematicidal Effects of Bacillus subtilis and Bacillus pumilus Against Meloidogyne incognita infecting Pea

Volume06-2018
Advances in Agricultural Science 06 (2018), 04: 52-59

Nematicidal Effects of Bacillus subtilis and Bacillus pumilus Against Meloidogyne incognita infecting Pea

Wafaa M. A. El-Nagdi 1*, H. Abd-El-Khair 2 and Mona G. Dawood 3

Nematology Laboratory, National Research Centre, Giza, Egypt.
Plant Pathology Department, National Research Centre, Giza, Egypt.
Botany Department , National Research Centre, Giza, Egypt.

ABSTRACT

Bacillus subtilis (BS) and B. pumilus (BP1 and BP2) were applied alone as well as in combination for controlling Meloidogyne incognita infecting pea in pot experiment. Single treatments of bacteria significantly reduced the numbers of juvenile (J2) in soil (50-80%) ,J2 in roots (57-78%), females (55-74%), galls  (65-74%) and egg-masses (56-70%) , while the combination treatments of them significantly reduced the same parameters in the ranges of 29-76% , 61-77% , 46-69% , 47-67% and 40-61% , compared to 71 , 70 , 70 ,68 and 65% , with Carbofuran 10%, respectively.  Results showed that BP2 highly reduced the J2 in soil , BS highly reduced J2 in roots BS and BP1 highly reduced the numbers of females, galls and egg-masses in roots. On the other hand, BP1 + BP2 highly reduced the numbers of J2 in soil, J2, females and egg-masses in roots, while BS+BP1+BP2 treatment highly reduced the galls number.  B. subtilis and B. pumilus, as single treatment, in some parameters, had more nematicidal activity against M. incognita, than in combination treatments. Treatments also significantly increased the growth parameters of pea plants shoot length, shoot fresh and dry weight, leaves numbers and pod fresh and dry weight. The treatments improved the soluble protein and total phenolic compounds in treated pea plants.

KeywordsBacillus subtilisBacillus pumilusMeloidogyne incognitaPeaBiological control


How to Cite: El-Nagdi, W. M., Abd-El-Khair, H., & Dawood, M. G. (2018). Nematicidal Effects of Bacillus subtilis and Bacillus pumilus Against Meloidogyne incognita infecting Pea. Advances in Agricultural Science6(4), 52-59.  

Introduction

Meloidogyne incognita  Kofoid and White Chitwood (root knot nematode) attacked  pea plants (Pisum sativum L.) and cause severe damage (Samaliev and Stoyanov2008 and Satyandra et al., 2011). Chemical treatments can be managed of root-knot nematodes but application of the nematicides are expensive, non-eco-friendly and causing risk to human and environmental (Abd-Elgawad, 2008). Therefore, biological control is safe and cheap alternative method to chemical controls and its ability to antagonize the nematodes by different mode of action (Gowen and Ahmad,1990 and Choudhary and Johri, 2009).  

The colonization of plant roots is useful step for beneficial rhizobacteria act , as biocontrol agents as well as growth-promoting bacteria, depended on microcolonies or biofilms of root exudation (Choudhary and Johri. 2009). Therefore, Bacillus spp. considered one of the important bacterial genera plays an important role in suppressive nematode invasion (Kloepper and Ryu. 2006). Bacillus spp. is effective in root colonization, versatile activity against multiple nematodes as well as ability to produce endospores when environmental conditions are stressful and the most species of Bacillus are harmless saprophytes (Siddiqui and Mahmood. 1999 and Dawar et al., 2008). B. subtilis and B. pumilus exhibited larvicidal activity against the second stage juveniles (J2) of M. incognita in vitro (Gokte and Swarup. 1988). Application of Bacillus spp. significantly reduced hatching of larvae of Meloidogyne javanica, whereas mortality of larvae was significantly increased with the increase in time. The growth parameters cowpea and mash bean viz. shoot length, root length, shoot weight and root weight significantly increased in treated plants, compared to control (Dawar et al., 2008). B. subtilis had highest efficacy against M. arenaria in potato, where nematode development in treated plant roots was lower than the untreated control (Mohamedova and Samaliev. 2011). Application of the two isolates of B. pumilus against M. javanica reduced the number of galls and eggs in tomato production. Significant enhancement in root and shoot length and dry root and shoot weight was recorded (Moghaddam et al., 2014). In previous study, Abd-El-Khair et al. (2016) mentioned that Bacillus subtilis and Bacillus pumilus showed the highest net mortality of M. incognita about ≥ 95% in bioassay test.

The objective of this research was to study the nematicidal activity of Bacillus subtilis and  B. pumilus  in pea under pots experiment.

 

Materials and Methods

Identification, Culturing and Extraction of M. incognita

Adult females were isolated from galled roots of pea plants and identified as M. incognita by examination of their cuticular perineal patterns and morphological characteristics according to method described by Taylor and Sasser (1978). The nematode was reared on eggplant in greenhouse at 30 ± 5 ºC. Second stage juveniles (J2) of M. incognita were extracted from these cultures by incubating infected roots in water for three days at 30 ± 5 ºC and then hatched J2 were collected and counted.

 

Preparation of Bacillus species inoculums

Cultures of Bacillus species viz., B. subtilis (BS isolate) and B. pumilus (BP1 and BP2 isolates) were obtained from Department of Plant Pathology, National Research Centre, Egypt. For the preparation of Bacillus species inoculums, pure culture tubes of BS, BP1 and BP2 were incubated for 48 h at (30±2)°C for the multiplication of bacteria. For mass production, one-liter Conical flasks containing 500 ml of broth nutrient glucose (2%) medium (3 g beef extract; 5 g peptone, 20 g glucose, in 1000 ml distilled water and pH at 7.2) were autoclaved .Then, each flask was separately inoculated with 1.0 ml of tested bacteria suspension. The flasks were kept at (30±2) °C in for 48 h and were shaken two times a day. Inoculums culture of bacteria was mixed and adjusted to 107-109 colony forming unit (CFU)/ml using dilution method. as previously mentioned.

 

Pots experiment

The experiment was conducted to assess the nematicidal effects of BS, BP1 and BP2 against M. incognita under glasshouse conditions in a greenhouse of Department of Plant Pathology, NRC. The plastic pots (20 cm diameter), containing 2 kg of a sterilized mixture of sandy and loamy soil (1:1, w/w), were arranged according to a completely randomized design on a bench in the glasshouse. Seeds were sown in pot in April at 2017. After seed germination, each pot was thinned to two plants. Then, the pots were inoculated (in four holes made by wooden stalk around the plant roots in the soil) with 1000 newly hatched J2 of M. incognita. At the same time of nematode inoculation, Bacillus spp. viz. BS, BP1 and BP2 was applied, as soil drenching, at 30 ml of bacterial suspension [107-9 colony forming unit (CFU)/ml]. The  sterilized soil was separately mixed with bacterial suspension and then the pots watered. Pots without bacterial suspension served as control. These treatments were compared Furadan (Carbofuran 10% ) as a nematicide, at the rate of 0.02g/pot (equivalent to 10kg Fed.). Six replicated pots were used per treatment as well as for the control.

After 60 days of growth, the nematicidal effects of tested Bacillus species against M. incognita parameters viz. numbers of J2 in soil as well as J2 , galls , females and egg-masses in roots of pea plants (six plant roots per treatment) and the percentages of reduction were determined. Reduction of nematode parameters was calculated according to the following formula:

 

The growth parameters of pea plants, viz. shoot length, shoot fresh and dry weight, leaves numbers and pod fresh and dry weight were recorded. Increase in growth parameters was calculated according to the following formula:

 

Total protein content in dry pea seeds was determined according to Bradford method (1976), using a bovine serum albumin as a standard. Two grams of each sample were separately grinded in mortar . Then, 5ml of phosphate buffer (pH 7.6) was added and then transformed to the centrifuge tubes. The sample was centrifuged at 8000 rpm for 20 minutes After extraction, 30μl of each sample was separately mixed with 70μl of distilled water in separate tube. Coosmassic Brillaint Blue solution was added .A 3 ml as total volume was made and then the tubes were incubated for 5 minutes at room temperature. The total protein content was measured as the absorbance by spectrophotometer  against the control at 600 nm.

Soluble sugar content was determined in the dry seeds using the colorimetric method described by Dubois et al. (1956). One dried seeds was weighed and then 10 ml ethanol 70% was added . Then, the mixtures were kept in refrigerator for 1 week. One ml of the supernatant was mixed with 1 ml distilled water. Then, 1 ml of phenol 5% and 5 ml pure sulfuric acid were added to the solution. Soluble sugar was measured as the absorbance by spectrophotometer against the control at 490 nm. To obtain the standard curve of soluble sugars using, different concentrations of glucose were used.

Total phenolic compounds were extracted from dry seeds and determined calorimetrically according to the method defined by Snell and Snell (1953) using Folin Ciocalteu phenol reagent.

One gram of pea seeds was macerated in 5-10 ml  ethanol (80%) for at least 24 h at zero °C, the alcohol was clarified.

The remained residue was re-extracted for 3 times with 5-10 ml of ethanol. At the end, the clarified extract was completed to 50 ml using  ethanol .

 

Statistical analysis

Nematode data were normalized before analysis by log transformation. The obtained data were analyzed using Computer Statistical Package (CO-STATE) User Manual Version 3.03, Barkley Co.USA and means compared with the Duncan’s Multiple Range Test at P=0.05(Snedecor and Cochran,1980).

 

Results and Discussion

Application of BS, BP1 and BP2, as single or in combined as soil treatment with M. incognita  inoculations, significantly reduced the M. incognita parameters viz. numbers of J2 in soil , J2 in roots , females , galls and egg-masses , where the reduction percentages were in the ranges of 60-80% , 57-78% , 55-74% , 65-74% and 56-70% , compared to nematode only (Untreated control). Carbofuran 10% reduced the above nematode parameters to 71, 70, 70, 68 and 65%, respectively. Results showed that BP2 highly reduced the J2 number in soil, followed by BS and BP1, respectively. BS highly reduced the J2 number in roots, followed by BP2 and BP1, respectively. BP1 highly reduced the numbers of females, galls and egg-masses, followed by BS and BP2, respectively.. In combination treatments, BS+BP1, BS+BP2, BP1+BP2 and BS+BP1+BP2significantly reduced the above nematode parameters in the ranges of 29-76%, 61-77%, 46-69%, 47-67% and 40-61%. BP1 + BP2 highly reduced the numbers of J2 in soil and roots, followed by BS+BP1, BS+BP1 +BP2 and BS+BP1, respectively. BP1 + BP2 highly reduced the numbers of females and egg-masses, followed by BS+BP1 +BP2, BS+BP1 and BS+BP1, respectively. BS+BP1+BP2 highly reduced the galls number, followed by BP1+BP2, BS+BP1 and BS+BP2, respectively (Table 1)..

It is cleared that as single treatment , BP2 highly reduced the J2 in soil , BS highly reduced J2 in roots BS and BP1 highly reduced females, galls and egg-masses in roots. On the other hand , in combination treatment, BP1 + BP2  highly reduced the  tested nematode parameters. Our results are agreement with those recorded by Priest (1999) and Choudhary and Johri (2009) . They mentioned that the mechanisms of biological agents affecting root gall development, egg hatching or nematode survival were either directly through the production of toxic metabolites or indirectly by induction of systematic resistant. Sarangi and Ramakrishnan, (2016) also that Bacillus spp. had the nematicidal activity , may be due to the bacteria able to produce a wide variety of secondary metabolites viz. lipopeptide antibiotics of Surfactin and iturin A. Bacillus species also can kill nematodes by Bacilli that affect nematode development, fecundity and survival (Zheng et al., 2016). B. pumilus able to produce the protease and chitinase that demonstrated their ability as a potential biocontrol agent against root-knot nematode (Ahmadian et al., 2007 and Lee and Kim, 2016). B. subtilis and B. pumilus had antibiotic activity (Leifert et al., 1995) as well as application of Bacillus spp. significantly reduced hatching of larvae of root- knot (Dawar et al., 2008) . In pot experiment , El-Nagdi and Abd-El-Khair (2017) reported that B.subtilis and B. megaterium had nematicidal activity against M. incognita in Cowpea . In field application the same bacterial bio-agents controlled M. incognita in bean plants under natural infection conditions (El-Nagdi and Abd-El-Khair, 2014). Application of BS, BP1 and BP2, as single soil treatment together with M. incognita  inoculations, significantly increased the growth parameters of pea plants viz. shoot length , shoot fresh weight , shoot dry weight, leaves numbers, pod fresh weight and pod dry weight in the ranges of 60-102%, 71-129% , 30-60% , 31-39%, 21-86% and 21-43%  as well as the growth parameters of 48, 40, 25, 14, 18 and 14% with Carbofuran 10%  comparing to the control, respectively. BP2 highly increased the growth parameters viz. shoot fresh and dry weight and pod fresh and pod dry weight, followed by BP1 and BS, respectively. BP1 highly increased the shoot length and leaves numbers, followed by BP2 and BS, respectively.  In the combined treatments, BS+BP1, BS+BP2, BP1+BP2 and BS+BP1+BP2 significantly increased the above growth parameters in the ranges of 63-86%, 101-172%, 22-70%, 37-43%, 24-96% and 21-50%, respectively. BS + BP1 + BP2 highly increased the shoot fresh weight , leaves numbers and pod fresh

      Table 1. Nematicidal activity of Bacillus subtilis and Bacillus pumilus as single or combined treatments on Meloidogyne incognita parameters as log10 and reduction (%) in pea plants in pots.

 

 

Treatments

log 10 of numbers and reduction  (Red.%) of M. incognita parameters
J2 no. /200g

soil

 

J2 no. /5g

roots

 

 Females no./5g roots Galls no. /5g roots

 

Egg-masses  no./5g roots
log 10 Red. % log 10 Red. % log 10 Red. % log 10 Red. % log 10 Red. %
Bacillus subtilis (BS) 2.28bc 77 2.60c 88 1.08cd 64 1.21bcd 65 1.05c 64
Bacillus pumilus  (BP1) 2.53b 60 2.89b 57 0.95d 74 1.07d 74 0.89d 74
Bacillus pumilus  (BP2) 2.24c 80 2.70c 72 1.09c 55 1.24bc 65 1.14c 55
BS + BP1 2.52b 60 2.62c 77 1.10c 56 1.24bc 63 1.14c 56
BS + BP2 2.78a 29 2.85b 61 1.27b 40 1.32b 46 1.27b 40
BP1+ BP2 2.31bc 76 2.25d 90 1.09c 61 1.12cd 65 1.07c 61
BS + BP1+ BP2 2.46bc 49 2.68c 73 1.02cd 58 1.19bcd 69 1.11c 58
Carbofuran 10% 2.39bc 71 2.25d 90 1.01cd 64 1.10cd 70 1.04c 65
Nematode only 2.93a 3.26a 1.54a 1.59a 1.29a

Means are averages of six replicates. Means followed by different letter(s)are significantly different according to Duncans Multiple Range Test at p≤0.05.

 

Table 2. Effects of Bacillus subtilis and Bacillus pumilus as single or combined treatments on growth characteristics in pea plants infected by the root- knot nematode, Meloidogyne incognita.

 

 

Treatments

Growth parameters Pod parameters
Shoot Leaves

 

Fresh weight Dry weight
Length Fresh weight Dry weight
cm. Increase

%

g Increase

%

g Increase

%

No. Increase

%

g Increase

%

g Increase

%

Bacillus subtilis (BS) 27.67bc 60 2.50bcd 71 1.31f 30 17.83ab 35 1.02g 21 0.17de 21
Bacillus pumilus  (BP1) 35.00a 102 2.79abc 91 1.35e 34 19.50ab 39 1.45b 73 0.20ab 43
Bacillus pumilus  (BP2) 32.33ab 87 3.34ab 129 1.62b 60 18.33ab 31 1.56a 86 0.20ab 43
BS + BP1 28.17bc 63 2.94abc 101 1.51c 50 19.33ab 38 1.22c 45 0.18cd 29
BS + BP2 32.17ab 86 3.40ab 133 1.23h 22 19.17ab 37 1.04f 24 0.19bc 36
BP1 + BP2 31.67ab 83 3.65ab 150 1.72a 70 19.33ab 38 1.13d 35 0.17de 21
BS + BP1+ BP2 30.67abc 77 3.97a 172 1. 41d 40 20.00a 43 1.05e 96 0.21a 50
Carbofuran 10% 25.82c 48 2.05cd 40 1.26g 25 16.00bc 14 0.99h 18 0.16e 14
Nematode only 17.33d 1.07d 1.01i 14.00c 0.74i 0.14f

Means are averages of six replicates. Means followed by different letter(s)are significantly different according to Duncans Multiple Range Test at p≤0.05.

 

 

Table 3. Effect of Bacillus subtilis and Bacillus pumilus on biochemical compounds of pea plants infected by the root- knot nematode, Meloidogyne incognita.

 

 

Treatments

Biochemical compounds (mg/g)
Soluble protein Soluble sugar Total phenolic compound
Bacillus subtilis (BS) 10.45a 65.53c 26.21a
Bacillus pumilus  (BP1) 8.45cde 61.68de 23.31b
Bacillus pumilus  (BP2) 8.73b 76.76a 21.65c
BS + BP1 8.65cd 73.50b 20.35d
BS + BP2 8.33de 51.49f 17.44ef
BP1 + BP2 9.05b 60.35e 18.44e
BS + BP1+ BP2 8.30e 48.80f 17.28ef
Carbofuran 10% 8.14ef 51.75f 17.62ef
Nematode only 7.95f 64.64cd 16.54f

Means are averages of six replicates. Means followed by different letter(s)are significantly different according to Duncans Multiple Range Test at p≤0.05.

 

and dry weight averages, while BS + BP2 and BP1 + BP2 Highly increased shoot length and shoot dry weight, respectively (Table 2). A group of rhizosphere bacteria (rhizobacteria)  play an important role a beneficial effect on plant growth such as Bacillus spp. by several mechanisms of growth promotion include production of growth stimulating phytohormones, solubilization and mobilization of phosphate, siderophore production, antibiotics production, ethylene synthesis inhibition and induction of plant systemic resistance to pathogens (Richardson et al., 2009). Significant enhancement in root and shoot length and dry root and shoot weight in potato plants was recorded by Moghaddam et al. (2014).

Effects of Bacillus spp. viz. BS, BP1 and BP2, as single soil treatment together with M. incognita inoculations, on soluble protein, soluble sugar and total phenolic compounds as mg/g in pea seeds are listed in Table (3). The above biochemical compounds ranged from 8.45-10.45 mg/g; 61.68-76.76 mg/g and 21.65-26.21 mg/g when BS, BP1 and BP2 was applied alone, respectively. BS significantly increased the soluble protein and total phenolic compounds, while BP2 highly increased the soluble sugar, respectively. The same compounds ranged from 8.30-9.05 mg/g: 48.80-73.50 mg/g and 16.85-20.35 mg/g in combined treatments of BS+BP1, BS+BP2, BP1+BP2 and BS+BP1+BP2, respectively. BS + BP1 highly increased the soluble protein and total phenolic compounds, while BP1+BP2 highly increased the soluble sugar, respectively. The biochemical compounds were 8.14, 51.75 and 16.64 mg/g with Carbofuran 10%  the , compared to 7.95 , 64.64 and 16.64 mg/g in nematode only (Table 3).  These results are agreement with those recorded by Shama et al. (2010). They indicated that the biocontrol agents viz. Trichoderma harzianum, Pseudomonas fluorescens  and B. subtilis enhanced the contents of phenol,  soluble sugars, and total protein Brassica juncea  . The sugar beet plant residues also increased the soluble carbohydrates, total carbohydrates, phenols and soluble proteins in cowpea seeds (Youssef el al., 2018).

 

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