Potential of Bacillus species against Meloidogyne javanica parasitizing eggplant (Solanum melongena...

REGULAR ARTICLE

Potential of Bacillus species against Meloidogyne javanicaparasitizing eggplant (Solanum melongena L.) and inducedbiochemical changes

Muhammad Waseem Abbasi & Naeem Ahmed &

Muhammad Javed Zaki & S. Shahid Shuakat & D. Khan

Received: 13 March 2013 /Accepted: 3 October 2013 /Published online: 28 October 2013# Springer Science+Business Media Dordrecht 2013

AbstractAims The biocontrol potential of three Bacillus species,namely Bacillus subtilis (BS), Bacillus firmus (BF),and Bacillus coagulans (BC) was tested against theroot-knot nematode Meloidogyne javanica (Treub)Chitwood in eggplants (Solanum melongena L.). Plantgrowth and biochemical effects were also measuredin these interactions.Methods Bacillus species were inoculated in soil aroundthe seedlings of eggplants (Solanum melongena L.)with andwithout nematodes in a greenhouse experiment.Plant growth, biochemical changes, and nematodeparasitism were observed at 15 and 45 days afterinoculation (DAI).Results BC significantly enhanced plant growth,chlorophyll “b” and total chlorophyll contents, andpolyphenol oxidase (PPO) activity in the leavesof eggplants, while BS showed greatest reductionin root-knot nematode parasitism. Non-infected anduntreated control (C−) plants showed lesser chlorophyll“b,” carotenoids, soluble protein contents, and guaiacolperoxidase but higher catalase and PPO activitiescompared to infected and untreated controls (C+) at 15and 45 DAI. Superoxide dismutase activity declined inmost of the treated plants at 45 DAI following rise at 15DAI. Ascorbate peroxidase activity increased at 45 DAI

compared to 15 DAI in C− and C+ plants. PAL activitywas greatly enhanced at 45 DAI in all treatments andcontrols over that at 15 DAI.Conclusions BC is a potentially plant growth-promotingbacteria although it was less effective against nematodeinfection compared to BS. Enzymes activities variedwith infection and DAI. BC at 15 DAI in generalincreased the activity of most of the stress enzymesand thereby overcoming the effect of nematodeparasitism.

Keywords Bacteria . Biocontrol . Nematode .

Photosynthetic pigments . Enzymes activities

Introduction

Synthetic pesticides usually control plant diseasesefficiently, but many of them exhibit non-targeteffects. Besides, pathogens are constantly developingresistance against synthetic pesticides. Some of thesechemicals are equally hazardous to livestock, plants,and also to the beneficial fauna and flora of thesoil. This situation stimulated the development ofnew strategies for better disease control. Beneficialrhizobacteria have been used to control plant pathogensand promote plant growth as well. Various speciesof Bacillus have been tested against plant pathogenicfungi. But, there is a need to seek suitable Bacillusisolates against plant parasitic nematodes. Bacillusspecies are effective in the management of plantpathogens owing to their ability to produce antimicrobial

Plant Soil (2014) 375:159–173DOI 10.1007/s11104-013-1931-6

Responsible Editor: Jesus Mercado-Blanco.

M.W. Abbasi (*) :N. Ahmed :M. J. Zaki : S. S. Shuakat :D. KhanDepartment of Botany, University of Karachi,Karachi 75270, Pakistane-mail: [email protected]

compounds (Mannanov and Sattarova 2001) andplant growth promoting potential, especially byincreasing the performance of plant roots (Compantet al. 2005; Zhang et al. 2011). Metabolites of Bacillusspp. increase the mineral availability (Ongena andJacques 2007) to plants by solubilization of inorganicphosphate and mineralization of organic phosphate.Strains of Bacillus subtilis, Bacillus pasteurii, Bacilluscereus, and Bacillus pumilus are involved in theinduction of systemic resistance in plants against avariety of pathogens (Kloepper et al. 2004). SomeBacillus species can also invade plant roots and producedifferent metabolites like phytohormones and enzymes(Selvadurai et al. 1991).

Root-knot nematodes (RKN) (Meloidogyne spp.)are obligate parasites and cause root cells to becomegiant and multinucleated. The giant cells are specializedfeeding sites with dense cytoplasm containing manyorganelles. RKN feed through feeding tubes intothe cytoplasm of giant cells acting as molecularsieves (Bockenhoff and Grundler 1994). Parasitismchanges the anatomy and efficiency of roots bydisrupting the vascular system resulting in reductionof water uptake and nutrients transport (Abad et al.2003) and ultimately inhibiting photosynthesis. Thesecretions produced by nematodes play an importantrole affecting many biochemical and molecular eventsin plant roots.

Plants adopt various changes at physiological andbiochemical levels against pathogens. The changesinitiated by the host plant after the pathogen entry areboth rapid as well as slow (Strange 2003). Slowresponses comprise of production of phytoalexins,hypersensitive response in host cells that die rapidly,inducing a limited necrosis associated with resistanceof the plant as a whole. The rapid response afterpathogen attack involves the generation of reactiveoxygen intermediates (ROIs) and nitric oxide (NO)(Dat et al. 2000). ROIs include superoxide (O2

−),hydrogen peroxide (H2O2), and hydroxyl radical(OH). The generation of ROIs as a defense responseoccurs most dramatically in localized infections andalso in general and systemic infections, as well as inplants treated with chemicals that induce systemicacquired resistance. ROIs along with NO act asantimicrobial agents that are involved in signaltransduction and induction as well as expression ofprotective genes. ROIs also behave as substrate forthe rapid oxidative cross-linking of cell-wall proteins

and lignification, making cell walls more difficult topenetrate (Shetty et al. 2008).

The purpose of the present study was to investigatethe in vivo nematicidal potential of three Bacillusspecies including B. subtilis (BS), Bacillus firmus(BF), and Bacillus coagulans (BC) againstMeloidogynejavanica (Treub) Chitwood in order to determinetheir effect on plant growth and the biochemical changesinduced in eggplant following application. Particularattention has been paid on six stress enzymesincluding superoxide dismutase (SOD), catalase (CAT),ascorbate peroxidase (APX), guaiacol peroxidase(GPX), polyphenol oxidase (PPO), and phenylalanineammonia lyase (PAL).

Materials and methods

Nematode and bacterial cultures

Roots of infested eggplants were collected from Malir(near Karachi), and RKN was identified asM. javanica(Treub) Chitwood in the laboratory by observingperennial pattern of adult females as described byTaylor and Netscher (1974). Nematode was culturedon tomato seedlings in a greenhouse from a single eggmass in earthen pots. Infested tomato roots werecut into small pieces and transferred in a sodiumhypochlorite solution (1 %). After vigorous shakingfor 4 min., the suspension was passed through 75and 37-μm sieves. Eggs were collected on 37-μmsieve and transferred into 250-ml beaker. Nematodeeggs were incubated for 72 h at 28±2 °C forhatching of juveniles (J2). Subsequently, the J2 werecollected and suspended in distilled water. The numberof J2 per milliliter was counted using a countingchamber (Hussey and Barker 1973).

Bacillus species were isolated from the rhizosphereof different cultivated crops around southern Sindh,Pakistan by serial dilution technique. Bacillus specieswere identified according to Bergey's manual ofdeterminative bacteriology (Holt et al. 1994) usingphenotypic characteristics. B. subtilis (collection codenumber KUB-15) isolated from the tomato field(locality, Thatta), B. firmus (KUB-27) from cottonfield (Tandojam), and B. coagulans (KUB-20) fromsugarcane field (Hyderabad). Bacterial cell suspensionwas prepared by inoculating bacteria on Luria Bertanibroth for 48 h at 37 °C in a shaking incubator at

160 Plant Soil (2014) 375:159–173

140 rpm in dark. Bacterial cultures were centrifuged at4,000×g for 15 min in 15-ml sterilized centrifugetubes. The cell pellets were washed once and resuspendedin sterilized distilled water and used for inoculationimmediately.

Treatments and experimental design

Seedlings of eggplant (Solanum melongena L.) var.Black Beauty were raised in large earthen pots(diameter, 24 cm) containing 5 kg sandy clay soil (sand58 %, silt 4 % and clay 38 %, bulk density 1.6 g cm−3,porosity 40 %, water holding capacity 32 %). One-month-old seedlings were transplanted in plastic pots(12 cm diameter), each containing 1 kg sterilized soil(soil was autoclaved twice at 121 °C for 15 min insmall woven cloth bags). Two plants were maintainedin each pot with four pots for each treatment.Experiment was divided into two sets. One set withRKN and treated with bacterial cell suspension (BC+,BF+, and BS+). The second set was treated withbacterial cell suspension without nematodes (BC−,BF−, and BS−). Two types of controls were kept,positive control (C+) containing nematodes andnegative control (C−) without nematodes. Twomilliliters of bacterial cell suspension {BS (11·109

colony forming units (CFU)/ml), BF (55·109 CFU/ml),and BC (78·109 CFU/ml)} was applied near the rootsin soil 4 days after transplantation. Three days afterbacterial application, 2,000 freshly hatched M.javanica J2 were inoculated in four holes around theroots of each plant. Plants were uprooted 15 and45 days after nematode inoculation. To determinenematode penetration, the roots were stained in boiling(80 °C) acid fuchsin (0.25 %) and macerated in anelectric blender. The released nematodes were countedand expressed as the number of nematodes per gram ofroots (Bridge et al. 1982). Bacterial population wascounted in the rhizosphere soil and roots of eggplantby serial dilution technique at 45 days after inoculation(DAI) as reported by Shishido et al. (1996). Forbiochemical analysis in plants, leaf samples (1.5 g perplant) were collected, quickly frozen into liquidnitrogen and stored at −20 °C.

Photosynthetic pigments

For the determination of photosynthetic pigments, 0.2-g fresh leaf samples were crushed in 5 ml of 80 % cold

acetone and centrifuged at 4,000×g for 10 min.Supernatants were separated, and absorbance wasrecorded on a Janway 6305 spectrophotometer (BibbyScientific, Essex, UK) at 445 and 663 nm forchlorophylls and 480 and 510 nm for carotenoids.Chlorophylls and carotenoids were estimated inaccordance with Arnon (1949) and Duxbury and Yentsch(1956), respectively, and expressed as micrograms pergram of fresh weight.

Enzyme assays

Leaf samples (1 g) were ground with liquid nitrogenand homogenized in chilled potassium phosphatebuffer (0.1 M, pH 7), 0.1 mM EDTA (Sigma-Aldrich),and 1 % PVP (Sigma-Aldrich), filtered by muslin clothand centrifuged at 21,000×g at 4 °C for 20 min. Thesupernatant was separated and stored at −20 °C.

Protein contents

The protein contents were determined for the extractused in the enzyme assay by the method of Bradford(1976) using bovine serum albumin as standard.

Enzyme activities

Superoxide dismutase

SOD (EC 1.15.1.1) activity was ascertained by themethod of Beauchamp and Fridovich (1971) basedon the inhibition of the reduction of nitrobluetetrazolium (NBT) (Sigma-Aldrich) by ·O2

− producedvia riboflavin photoreduction. The reaction mixture(3 mL) contained 50 mM phosphate buffer (pH 7.8),0.1 mM EDTA, 13 mM methionine (Sigma-Aldrich),75 μM NBT, 2 μM riboflavin (Sigma-Aldrich), and100 μL of enzyme extract. Riboflavin was added atthe end, and the reaction was initiated by placingthe tubes under fluorescent lamps. The reaction wasterminated after 10 min by removing the tubes fromlight. Non-illuminated and illuminated reactions withoutthe extract served as calibration standards. Reactionproducts were measured at 560 nm. One unit of SODactivity was defined as the amount of enzyme requiredfor 50 % inhibition of NBT conversion.

Plant Soil (2014) 375:159–173 161

Catalase

CAT (E.C. 1.11.1.6) activity was determined inaccordance with Aebi (1984). The reaction mixture(3 mL) contained potassium phosphate buffer(100 mM, pH 7.0), 30 mM H2O2, and 50 μL of dilutedenzyme extract, and the activity was estimated bymonitoring the decline in absorbance due to H2O2

reduction at 240 nm for 30 s (extinction coefficient=36 M−1 cm−1). One unit of CAT decomposes 1 μmol ofH2O2 per minute at 25 °C.

Ascorbate peroxidase

APX (EC 1.11.1.11) activity was determined by themethod of Nakano and Asada (1981). The reactionmixture (3 mL) contained potassium phosphate buffer(50 mM, pH 7.0), 0.1 mM H2O2, 0.50 mM ascorbate(Merck), and 100 μL of enzyme extract. The activitywas determined from the decrease in A290 for 1 min dueto ascorbate oxidation by H2O2 at 25 °C (extinctioncoefficient, 2.8 mM−1 cm−1). The reaction was startedby adding enzyme extract. Correction was done for thelow, non-enzymatic oxidation of ascorbate by H2O2.

Guaiacol peroxidase

GPX (EC 1.11.1.7) activity was determined by themethod of Anderson et al. (1995). The reaction mixture(3 mL) contained potassium phosphate buffer (50 mM,pH 7.0) guaiacol (Scharlau Chemie) 75 mM, H2O2

10 mM, and 20 μL of enzyme extract. The activitywas determined from the increase in absorbance due tothe formation of tetraguaiacol at 470 nm for 2 min(extinction coefficient, 26.6 mM−1 cm−1).

Polyphenol oxidase

PPO (EC 1.14.18.1 or EC 1.10.3.2) activity wasassessed by the method of Şakiroğlu et al. (2008).The reaction mixture (3 mL) contained potassiumphosphate buffer (50 mM, pH 6.5), catechol (Sigma-Aldrich) 50 mM, ascorbic acid 2 mM, and 50 μL ofenzyme extract. One unit of PPO activity was definedas the amount of enzyme that causes an increase inabsorbance of 0.001 min−1 (Fenoll et al. 2002).

Phenylalanine ammonia lyase

PAL (EC 4.3.1.5) activity was assayed by the methodof Reichert et al. (2009) with minor modifications. Thereaction mixture (3 mL) contained Tris–HCl buffer(150 mM, pH 8.5), L-phenylalanine 2 mM, and50 μL of enzyme extract. The activity was determinedby the increase in absorbance at 270 nm for 1 min.Reaction mixture without enzyme extract served asblank. One unit of PAL activity was defined as theamount of enzyme that deaminated 1 μmol of L-phenylalanine to trans-cinnamate per minute at 30 °C.

Statistical analysis

Data were subjected to either two-factor or three-factoranalysis of variance (ANOVA), followed by Fisher'sleast significant test (Zar 2009) using the softwareSTATISTICA ver. 5.0 (Statsoft Inc., Tulsa, Oklahoma,USA). Prior to analysis of variance, the homogeneityof variances was checked using Bartlett's chi-squaredstatistic (Zar 2009). In addition, Scheffe's multiplecontrast post-hoc test was employed to compareselected groups of means. Scheffe's method is moreflexible compared to other available post-hocprocedures and permits a variety of comparisons(Rosner 2006). Basically, six types of comparisons wereperformed as outlined in Table 3. In addition, some othercomparisons of interest were also performed. ForScheffe's multiple contrast, a computer program waswritten by one of us (SSS) in C++ and is available onrequest. Bacterial population was transformed to log10(x+1) prior to analysis to achieve the normalityand homoscedasticity of the data. Part of the experimentwas repeated for plant growth and nematodedevelopment using similar protocol as mentionedearlier. Data were reanalyzed using pooled replicates(four new replicates and four replicates from the firstexperiment). The error mean squares for this part ofANOVA for the two trials were similar, and therefore,combined analysis was validly performed.

Results

Plant growth

Nematode parasitized eggplants showed lower heightcompared to non-inoculated plants at 15 and 45 DAI

162 Plant Soil (2014) 375:159–173

(P<0.001, P<0.05; Tables 1, 2, and 3). Soil inoculationwith Bacillus species significantly (P<0.001) increasedtheir height compared to controls at 45 DAI exceptBS-treated plants. Maximum plant heights wererecordedwhenBacillus species (BC andBF)were appliedalone without nematode inoculation. Interestingly,BS and BC when applied along with RKN significantly(P<0.001) increased plant height at 15 DAI (Table 1).The plants with RKN had fresh weights relativelylesser than those without RKN (P<0.001). Maximumplant fresh weight was recorded in plants appliedwith BC− at 45 DAI (P<0.001). Comparison ofmean fresh weights of controls (C+, C−) and alltreatments (B+ and B−) showed a nonsignificantdifference (Table 3).

Root-knot nematode development and bacterialcolonization of roots

RKN parasitism was measured in terms of number ofpenetrated nematodes per gram of roots recorded at 15and 45 DAI, while galls were observed at 45 DAI. Alltested Bacillus isolates significantly reduced the rootpenetration of nematodes at both 15 and 45 DAI andgalling intensity at 45 DAI (P<0.001). The maximumreduction of nematode penetration was observedin plants treated with BS particularly at 45 DAI.Likewise, BS reduced the galling intensity,particularly at 45 DAI. Bacterial populationswere also recorded in roots and the rhizosphere soilsat 45 DAI. BF plants roots without RKN showedmaximum population (8.02 log CFU g−1 roots).However, in the plants roots treated with RKNand BF, bacterial population declined. A similartrend was observed in BC-treated plants, but in BStreated plants, roots without RKN gave lesser bacterialcount compared to roots with RKN. Maximumcell count in the rhizosphere soil was observedin BC-treated experimental units, followed by soilstreated with BF along RKN and BS without RKN(Table 1).

Photosynthetic pigments and protein concentration

Chlorophyll “a” concentration in Bacillus inoculatedplants declined dramatically during early growthperiod in nematode inoculated plants compared tonon-inoculated plants (P<0.05, Table 3). BF withRKN gave maximum decrease in chlorophyll “a”

contents followed by BS and BC. However, at 45DAI, chlorophyll “a” concentration was recoveredand attained a higher level (P<0.05) in nematode-parasitized plants and maximum concentration wasobserved at 45 DAI in plants treated with BS andRKN. Treatments, inoculation, time, and their interactionsshowed significant effect on chlorophyll “a” concentration(P<0.001) (Fig. 1a). The plants with RKN comparedto healthy plants showed higher concentration ofchlorophyll “b” (P<0.05, Table 3). Chlorophyll “b”concentration was increased in pants treated withbacteria particularly the inoculated plants at 15 DAIcompared to controls with and without RKN (P<0.05,Table 3), although a decline in chlorophyll “b” wasnoticed at 45 DAI. On the other hand, both controls(C− and C+), BF and BS without RKN plants showednon-significant changes in chlorophyll “b” over plantswith bacteria at both the observation periods (P<0.05)(Fig. 1b). Total chlorophyll concentration in negativeand positive controls (C− and C+) differed significantlyat 15 DAI but not at 45 DAI (P<0.05). Surprisingly,BC and BS with RKN showed significant elevationof total chlorophyll content compared to BC andBS without RKN, respectively (P<0.05) (Fig. 1c).Maximum total chlorophyll concentration was observedin BC with RKN-treated plants at both samplingtimes (P<0.001). RKN-parasitized control (C+) plantsshowed higher carotenoid concentration than didhealthy control (C−) plants (P<0.05, Table 3). Maximumcarotenoid concentration was observed in plants treatedwith BS along RKN followed by BC without RKNat 45 DAI and BC with RKN at both 15 and 45 DAI(P<0.001) (Fig. 1d). All treatments showed increasein carotenoid concentration at 45 DAI compared to 15DAI (P<0.05, Table 3).

Higher protein concentration was recorded in RKN-parasitized plants in comparison to healthy plants(Table 3). Maximum protein content was, however,observed in plants treated with BC along RKN at 45DAI followed by positive controls (C+) at 15 DAI.Generally, protein concentration was found to be lowerat 45 DAI compared to that at 15 DAI (P<0.001)(Fig. 2).

Enzymes activities

SOD activity was significantly (P<0.001, P<0.05,Tables 2 and 3) higher in all plants at 15 DAI comparedto C− plants. At 15 DAI, the maximum SOD activity

Plant Soil (2014) 375:159–173 163

was observed in BS without RKN plants, followed byBC without RKN. However, greatest decline in theactivity was recorded in C+ plants at 45 DAI(P<0.05, Table 3) (Fig. 3a). Nematode parasitized andhealthy controls (C+, C−) showed significant differencein CAT activity (P<0.05). CAT activity was retardedin RKN-treated plants, while healthy plants showedhigher activity (P<0.05, Table 3). Bacillus-treatedplants showed greater CATactivity at 15 DAI comparedto those at 45 DAI. The maximum CAT activitywas recorded in C− plants at 45 DAI, followedby the BF without RKN plants at both 15 and45 days of incubation and BC with RKN at 15 DAI(P<0.001) (Fig. 3b). A significant increase was obtained

in APX activity in C+ plants at 45 DAI compared to C−(P<0.001). Bacillus treatment along with nematodesincreased APX activity relative to non-inoculated andBacillus-treated plants at 15 DAI. Healthy plants (C−)and controls with RKN (C+) showed higher activity at45 DAI compared to plant at 15 DAI (Fig. 3c). GPXactivity was enhanced in controls with RKN (C+)-treated plants compared to healthy (C−) plants (P<0.001,P<0.05; Tables 2 and 3). Maximum GPX activitywas recorded in plants treated with BC along RKNat 45 DAI followed by BF with RKN at 15 DAI.In most of the treated plants, higher GPX activity wasnoticed at 45 DAI than at 15 DAI (P<0.001, P<0.05;Tables 2 and 3, Fig. 3d).

Table 1 Effect of different Bacillus species on growth and parasitism of root-knot nematodes in eggplant after 15 and 45 days

Treatments Shoot length(cm)

Shoot freshweight (g)

Galls per rootsystem

Nematodes (g−1

fresh roots)Bacterial populationa

(g−1 roots)Bacterial population(g−1 soil)

15 DAI

C− 15.06±0.27 2.82±0.07 – – – –

C+ 12.81±0.38 2.30±0.09 – 138±5.9 – –

BF− 15.44±0.29 2.82±0.06 – – – –

BF+ 13.94±0.33 2.76±0.15 – 101±9.2 – –

BS− 14.62±0.42 2.63±0.07 – – – –

BS+ 15.93±0.64 2.99±0.30 – 59±5.2 – –

BC− 16.18±0.76 3.16±0.06 – – – –

BC+ 16.12±0.44 3.45±0.30 – 124±4.4 – –

45 DAI

C− 16.94±0.49 3.31±0.07 – – – –

C+ 13.31±0.37 2.61±0.07 121±8.5 2540±144 – –

BF− 19.25±0.65 3.46±0.06 – – 8.0247±0.038 7.5604±0.139

BF+ 14.75±0.84 2.52±0.05 92±8.7 1738±110 5.1590±0.160 7.9904±0.063

BS− 15.38±1.03 2.85±0.13 – – 5.0528±0.077 7.9075±0.076

BS+ 13.50±0.73 2.65±0.06 43±3.0 748±81 7.3111±0.046 7.1563±0.224

BC− 20.63±0.75 3.55±0.04 – – 7.5528±0.320 8.0302±0.104

BC+ 17.50±0.50 3.30±0.07 69±6.1 1044±79 6.0473±0.055 8.2617±0.275

BacteriaLSD0.05

b0.84 0.18 0.333 0.362

NematodesLSD0.05

0.59 0.13 0.272 0.295

Time LSD0.05 0.59 0.13

Data of plant growth and nematode development comprises of means of eight replicates ± standard error. The part of experimentinvolving shoot length, shoot fresh weight, number of galls, and penetrated nematodes was repeated with four replications; since the errormean square for the two experiments were similar, the data were pooled. The data on bacterial populations comprised of means of fourreplicates ± standard error

C− control (without root-knot nematodes and bacteria), C+ control with nematode only, BF B. firmus, BS B. subtilis, BC B. coagulansa Data of colony forming units was transformed into log 10b Fisher's least significant difference

164 Plant Soil (2014) 375:159–173

Table 2 F values of three-way ANOVA for various growth parameters against Bacillus species, nematode presence, and time afterinoculation and their interactions as observed in S. melongena L.

Parameters Bacteria Nematodes Time B × N B × T N × T B × N × T(B) df=3 (N) df=1 (T) df=1 df=3 df=3 df=1 df=3

Shoot length 21.49*** 42.86*** 21.73*** 4.67** 7.66*** 19.82*** 0.16 ns

Shoot weight 19.80*** 14.41*** 6.86* 7.60*** 2.08 ns 16.10*** 1.24 ns

Chlorophyll “a” 22.76*** 121.93*** 554.31*** 15.31*** 56.58*** 108.38*** 42.23***

Chlorophyll “b” 336.62*** 402.84*** 372.22*** 32.18*** 83.11*** 67.59*** 66.33***

Total chlorophyll 78.23*** 28.75*** 4.40* 16.82*** 2.61ns 1.22ns 7.21***

Carotenoids 18.10*** 0.16 ns 46.27*** 6.94*** 4.46*** 1.57 ns 9.70***

Proteins 16.70*** 32.96*** 38.31*** 13.50*** 8.60*** 1.68 ns 9.90***

Catalase 12.12*** 4.00 ns 12.87*** 50.06*** 25.33*** 0.96 ns 2.97*

Ascorbate peroxidase 6.29** 55.45*** 43.12*** 2.43 ns 73.53*** 28.22*** 20.59***

Superoxide dismutase 84.77*** 0.03 ns 266.00*** 28.03*** 4.21* 6.29* 73.73***

Guaicol peroxidase 7.36*** 45.35*** 59.71*** 31.11*** 7.12*** 2.01 ns 33.97***

Polyphenol oxidase 21.27*** 6.39* 20.53*** 10.92*** 13.00*** 39.57*** 3.49*

Phenylalanine ammonia lyase 60.40*** 45.02*** 2872.89*** 60.24*** 52.60*** 27.88*** 68.49***

Bacteria population (roots) 8.45** 31.80*** – 150.70*** – – –

Bacteria population (soil) 6.92* 0.05 ns – 7.24** – – –

ns non-significant, df degrees of freedom

*P<0.05; **P<0.01; ***P<0.001

Table 3 Scheffe′s multiple contrast tests between different groups of means

Parameters Comparisons

A B C D E F

Shoot length ns ns ns Ns ns –*

Shoot weight ns ns ns Ns ns ns

Chlorophyll “a” ns –* –* –* ns –*

Chlorophyll “b” –* –* –* –* –* –*

Total chlorophyll ns –* –* –* –* –*

Carotenoids –* ns –* –* –* –*

Proteins –* ns ns ns ns ns

Catalase –* –* ns –* –* ns

Ascorbate peroxidase ns ns ns ns ns –*

Superoxide dismutase –* –* –* –* –* –*

Guaicol peroxidase –* –* –* –* –* –*

Polyphenol oxidase –* –* –* –* –* –*

Phenylalanine ammonia lyase ns –* –* –* –* –*

Bacteria population (roots) – – – – – –*

Bacteria population (soil) – – – – – ns

A negative and positive control, B controls against bacteria, C between times (15 and 45 DAI), D negative control against bacteriainoculated (non-infected), E positive control against bacteria inoculated (infected), F all infected (including bacteria inoculated) againstall non-infected (without nematodes having bacteria inoculation), ns non-significant difference between the comparison

*P<0.05, Significant difference between the comparison at this level

Plant Soil (2014) 375:159–173 165

No nematodes with nematodes

0

200

400

600

800

1000

1200

C- BF- BS- BC- C+ BF+ BS+ BC+

TreatmentsLSD0.05 for Bacteria: 48.36, Nematodes:34.19, Time: 34.19

Ch

loro

ph

yll "

a" (µ

g.g-1

)

15 days 45 days


0

200

400

600

800

1000

1200

1400

1600

1800

C- BF- BS- BC- C+ BF+ BS+ BC+Treatments

LSD0.05 for Bacteria: 49.64, Nematodes:35.1, Time: 35.1

Ch

loro

ph

yll "

b" (

µg.g

-1)

15 days 45days

No nematode with nematodes

0

500

1000

1500

2000

2500



To

tal C

hlo

rop

hyl

ls (

µg.g

-1)

15days 45days


0

100

200

300

400

500

600



Car

ote

no

ids

(µg.

g-1)

15days 45days

a

b

c

d

Fig. 1 Effect of Bacillusspecies on photosyntheticpigments in eggplant withand without root-knotnematodes parasitism after15 and 45 days. C− control(without nematodes andbacteria), C+ control withnematode only, BF B.firmus, BS B. subtilis, BC B.coagulans, LSD Fisher’sleast significant difference.Figure comprising means offour replicates, error barspresenting standard error ofmean

166 Plant Soil (2014) 375:159–173

PPO activity was suppressed in C+ compared to C−plants at both 15 and 45 DAI (P<0.05 Table 3). Themaximum PPO activity was recorded in plants treatedwith BC along RKN at 15 DAI followed by BCwithout RKN at 15 and 45 DAI. However, BC withRKN at 45 DAI showed minimal activity (P<0.001).Healthy plants exhibited greater PPO activity at 45DAI compared to 15 DAI except BC without RKNtreated plants. Though in the RKN-treated plants, anopposite trend was observed (Fig. 4a). Both controls(C−, C+) did not show a significant difference inPAL activity. However, plants treated with bacteria(B−, B+) and the controls (C−, C+) gave asignificant difference in PAL activity (P<0.05).PAL activity increased with time in treatments aswell as controls (Fig. 4b) and was greatlyenhanced at 45 DAI in all treatments over that at15 DAI (P<0.05, Table 3) (Fig. 5). The maximumPAL activity at 15 DAI was recorded in plantstreated with BC along RKN, followed by positivecontrols (C+) with minimum activity noticed in C−plants. At 45 DAI C− plants showed maximumPAL activity. The maximum promotion in activityat 45 DAI over 15 DAI was observed in C− plantsand the minimal in BS plants without RKN.

Discussion

Plant growth varied with the bacterial strains; BCshowed stimulatory effect on plant growth while theresponse to BS was somewhat inhibitory to neutral.Surette et al. (2003) tested a number of bacterial strainson a variety of crops and demonstrated that 10–38 % ofstrains were plant growth neutral while 7–29 % were

plant growth inhibitory. Won-Chan and Rhee (2012)showed that a strain of B. subtilis produces someallelochemicals that suppress growth hormones likegibberellins and thereby inhibit plant growth. Thecriteria for plant growth in this study were plant freshweight and height. Bashan and de Bashan (2005)advocated that plant fresh weight as a criterion forgrowth is dubious as the fresh weight can depend upona multitude of factors including air temperature,relative humidity, air current, light intensity duringplant harvest, time taken during harvest, and type ofmaterial used for absorption of excess moisture.However, the environmental factors during harvest ofpots were kept more or less constant, and time toharvest for each treatment was also almost the same.Thus, it is reasonably believed that the extrinsic factorsbesides the intended treatments did not alter themagnitude of fresh weight or height.

Bacillus species have the ability to enter root tissueswhen applied in soil and ultimately colonize stemtissues without causing any disease symptoms(Shishido et al. 1999). Reports exist regarding theinduced protection of plant root cells elicited by B.pumilus strain SE34 due to cell wall deposition inepidermal and cortical cells (Benhamou et al. 1996).Bacillus inoculated plants showed reduced penetrationof RKN compared to infected control plants at both thetime periods. Bacillus species are known to producedifferent exotoxins and antibiotics some of which arereported to be nematicidal (Araújo and Marchesi 2009;Cawoy et al. 2011). Some saprotrophic species areknown to penetrate the cuticle of nematodes, propagaterapidly, and cause degradation and digestion ofnematode tissues by the involvement of hydrolyticenzymes (Huang et al. 2005; Niu et al. 2006). The


0

0.5

1

1.5

2

2.5

3



Pro

tein

s (m

g.g-1

)

15 days 45 days

Fig. 2 Effect of Bacillus species on soluble protein contents in eggplant with and without root-knot nematodes parasitism after 15 and45 days

Plant Soil (2014) 375:159–173 167


0

5

10

15

20

25

30



SO

D (

Un

its.m

g-1

pro

tein

s. m

in-1

)

15 days 45 days


0

1

2

3

4

5

6



CA

T (U

nits

.mg

-1 p

rote

ins.

min

-1)

15 days 45 days


0

0.05

0.1

0.15

0.2

0.25

0.3



GP

X (U

nits

.mg

-1 P

rote

ins.

min

-1)

15 days 45 days

a

b

c

d


0

5

10

15

20

25



AP

X (U

nits

.mg

-1 p

rote

ins.

min

-1)

15 days 45 days

Fig. 3 Effect of Bacillusspecies on antioxidantenzymes in eggplant withand without root-knotnematodes parasitism after15 and 45 days

168 Plant Soil (2014) 375:159–173

ability of some rhizobacteria to induce systemicresistance against nematodes has also been reported(Siddiqui and Shaukat 2004). The decrease innematode penetration may be ascribed to the abilityof rhizobacteria to provide mechanical and physicalsupport to plant cell wall, accumulation of phenoliccompounds, and production of lipopeptides. In ourstudy, BS showed maximum inhibition of RKNparasitism. However, plant growth in term of heightand fresh weight were found insignificant compared toRKN treated controls. It seems that BS produced somekind of metabolites which were toxic to RKN as wellas for the eggplant.

The concentration of chlorophyll pigments variedfollowing different treatments applied to the plants.Concentration of chlorophyll “a”was remarkably reducedin parasitized plants at 15 DAI compared to non-parasitized plants. However, at 45 DAI, its concentrationincreased over that recorded at 15 DAI in all treatments.The decrease in chlorophyll “a” concentration in Bacillus+ nematode-treated plants suggests that some biochemicalchanges occurred in plants at an early stage of combinedpathogen and bacterial inoculation. The same treatmentsshowed higher levels of chlorophyll “b” concentrationssuggesting degradation or conversion of chlorophylls. It isreported that chlorophyll “a” is first converted into

chlorophyll “b” before degradation (Ahmed et al. 2009).Early decrease in chlorophyll “a” concentration wasrecovered at 45 DAI. This improvement seems to indicatea possible reaction of plants to cope with the nematodestress. However, data on total chlorophyll concentrationsindicated a significant decline compared to healthy plants,providing evidence that chlorophylls are sensitive tostress. Downregulation of photosynthesis-related geneswas found to be part of a defense or adaptive responseto stress (Bilgin et al. 2010).

Carotenoids concentration increased in nematode-parasitized plants compared to healthy ones. Carotenoidsperform many important functions in plants suchas dissipating excess light energy, scavenging ROI,accessory light harvesting, behaving as antioxidantsduring oxidative stress, and stabilizing the thylakoidmembrane. During stress conditions such as RKNparasitism, chlorophyll concentration and ultimatelyphotosynthetic rate are decreased due to pooravailability or unavailability of water and nutrients.A variety of stresses have been shown to affectalso the levels of carotenoids (Demmig-Adams andAdams 1996).

Higher levels of soluble protein concentration werenoticed in infected plants compared to non-infectedones at 15 DAI. Accumulation of soluble proteins for


0

12

34

56

78

9



PP

O (

Un

its.m

g-1

Pro

tein

s. m

in-1

)

15 days 45 days


0

0.2

0.4

0.6

0.8

1

1.2

1.4


TreatmentsLSD0.05 for Bacteria: 0.042, Nematodes:0.0.029, Time: 0.0.029

PA

L (u

nits

.mg

-1 p

rote

ins.

Min

-1)

15 days 45days

a

b

Fig. 4 Effect of Bacillusspecies on polyphenoloxidase and phenylalanineammonia lyase activity ineggplant with and withoutroot-knot nematodesparasitism after 15 and45 days

Plant Soil (2014) 375:159–173 169

survival is reported to protect cells from stress (Wanget al. 2003).Many stress-related proteins are synthesizedin response to various forms of stress shortly followingits onset (Astorga and Meléndez 2010). Potato leavesshowed increased levels of pathogenesis-related proteinsafter being infected with the potato cyst nematode,Globodera species (Rahimi et al. 1996). The increasedconcentration of proteins at 15 DAI may be due to highrate of metabolic activities during early days of plantgrowth and development. The decline at 45 DAIin protein may be ascribed to prolonged effect ofnematodes on plants, as well as increase in parasitismseverity with time. Another reason for reduced proteinconcentration may be the formation of ROI that

target proteins whose carbonylation may take placedue to oxidation of amino acid chains (Gill andTuteja 2010).

The enhanced production of ROI is considered asthe most important metabolic response of plants to apathogen attack. In order to avoid damage caused byROI, synthesis of antioxidant enzymes is required.Biotic and abiotic environmental stresses enhance theactivity of SOD in plant tissues. Bacterial applicationin the rhizosphere may promote SOD activity.Plant growth promoting strain of Pseudomonasafter inoculation increased the activity of SOD in leavesof eggplant (Fu et al. 2010). Pseudomonas fluorescensCHA0 was also reported to significantly stimulate SODactivity in tomato plants (Ardebili et al. 2011).

Reduced CAT activity was observed in RKN-parasitized controls compared to healthy plants.Cavalcanti et al. (2006) observed that oxidativeresponses in roots and leaves exhibit contrastingeffects and decreased level of CAT in leaves of cowpeafollowing abiotic stress. RKN parasitic activitydisrupts the water supply to foliar parts inducingdrought conditions. The combined effect of salt anddrought decreased CAT activity in Glycyrrhizauralensis Fisch. seedlings (Pan et al. 2006). Previousstudies indicate increased CAT activity in roots ofRKN-parasitized plants (Niebel et al. 1995). Gallformation requires high level of CAT activity duringnematode development in roots. The peroxisomalcatalase protein is very sensitive to salt and hightemperatures stress (Foyer and Noctor 2000). Also,CAT is a light-sensitive protein and nematodes inducedchlorosis, and high light intensity can lower thetotal protein through accelerated inactivation ordecreased replacement capacity (Streb and Feierabend1996). Furthermore, CAT is typically found only inperoxisomes, while peroxidases (APX and GPX)are found throughout the cell including cytosol,mitochondria, and chloroplast, playing a vital rolein H2O2 destruction. APX has higher affinity forH2O2 than CAT (Asada 1992). Increased level of APXreduces the level of H2O2 and thus protects cells fromcell death as demonstrated during different stressconditions (Gill and Tuteja 2010). Under drought andheat stress, increased activity of cytosolic APX protectsplant from cellular damage. Different types of biotic andabiotic stresses are known to upregulate the APX genes(Sarowar et al. 2005; Dabrowskai et al. 2007). APX alsoincreases the GPX activity to increase ROI scavenging

1748 %

1676 %

928 %

1612 %

736 %

4368 %

5851 %

1897 %

DAYS AFTER NEMATODE INOCULATION

Fig. 5 Activity of phenylalanine ammonia lyase in egg plant on15th and 45th day of root-knot nematode inoculation for varioustreatments as outlined in the legend. Figures beside curvesindicate percent promotion of activity over the 15th day activity.C control, CN control with nematode only, Bf B. firmus, Bs B.subtilis, Bc B. coagulans, N root-knot nematode (M. javanica)

170 Plant Soil (2014) 375:159–173

system and leads to oxidative stress tolerance andpathogen resistance (Sarowar et al. 2005). Peroxidasedecomposes H2O2 by oxidation of co-substratessuch as phenolic compounds and/or antioxidants.Kovács et al. (2011) reported increased APX and GPXactivities 7 days after powdery mildew infection inwheat plant. GPX in plants have also been suggestedto be involved in defense against other pathogens(Kristensen et al. 1997).

PPO oxidizes phenolic compounds into quinonesduring pathogen invasion in plants. Quinines are toxicto pathogens. Generally, increase in PPO activity isreported following infections in plants (Luthra et al.1988) and cause browning at wounded parts of plants.In our studies, reduction in PPO activity was recordedafter 15 and 45 DAI in leaves compared to healthycontrols. RKN injuries in roots may trigger localizedaccumulation of PPO or its mobility from leaves toroots, thus reducing its activity in leaf tissues. PPOactivity was found to decline during the progressionof drought stress (Sofo et al. 2004).

PAL activity provides precursor in plants forbiosynthesis of phenolic compounds like lignin andanthocyanins in response to various stresses includinga pathogen attack and frequently correlated withresistance. In Linum usitatissimum L., increased PALactivity was observed after 4 days of fungal infection(Hano et al. 2006). In roots of potato, increased PALactivity was found in association with cyst nematodes(Heterodera rostochiensis Woll.) parasitism (Giebel1973).

Conclusions

BC appears as a possible plant growth-promotingbacterium. Although it was less effective against root-knot nematode parasitism compared to BS, it exhibitedenhanced APX, GPX, PPO, and PAL activities. BSshowed maximum inhibition of RKN parasitism. Thiseffect was correlated with increased APX, SOD, andPAL (at 45 DAI) activities. BF enhanced CAT, SOD,and GPX activities in nematode-exposed plants. Allstrains when applied with nematodes reducedchlorophyll “a” contents and elevated chlorophyll “b”contents. Soluble proteins increased in controls withRKN compared to healthy control plants. Enzymeactivities exhibited varied responses with treatmentsand time.

Acknowledgments Financial support provided by The Dean,Faculty of Science, University of Karachi, is gratefullyacknowledged.

References

Abad P, Favery B, Rosso M, Castagnone-Sereno P (2003) Root-knot nematode parasitism and host response: molecularbasis of a sophisticated interaction. Mol Plant Pathol 4:217–224

Aebi H (1984) Catalase in vitro. Methods Enzymol 105:121–126Ahmed N, Abbasi MW, Shaukat SS, Zaki MJ (2009)

Physiological changes in leaves of mung bean plantinfected with Meloidogyne javanica. Phytopathol Mediterr48:262–268

Anderson MD, Prasad TK, Stewart CR (1995) Changes inisozymes profile of catalase, peroxidas and glutathionereductase during acclimation of chilling in mesocotyls ofmaize seedlinhg. Plant Physiol 109:1247–1257

Araújo FF, Marchesi GVP (2009) Uso de Bacillus subtilis nocontrole da meloidoginose e na promoção do crescimentodo tomateiro. Ciênc Rural 39:1558–1561

Ardebili ZO, Ardebili NO, Hamdi SMM (2011) Physiologicaleffects of Pseudomonas fluorescens CHA0 on tomato(Lycopersicon esculentum Mill.) plants and its possibleimpact on Fusarium oxysporum f. sp. Lycopersici. Aust JCrop Sci 5:1631–1638

Arnon DI (1949) Copper enzyme in isolated chloroplastpolyphenol oxidase in Beta vulgaris. Plant Physiol 24:1–15

Asada K (1992) Ascorbate peroxidase. A hydrogen peroxidescavenging enzyme in plants. Physiol Plant 85:235–241

Astorga GIA, Meléndez LA (2010) Salinity effects on proteincontent, lipid peroxidation, pigments, and proline inPaulownia imperialis (Siebold & Zuccarini) andPaulownia fortunei (Seemann & Hemsley) grown in vitro.Electron J Biotechnol 13:1–15

Bashan Y, de-Bashan LE (2005) Fresh-weight measurements ofroots provide inaccurate estimates of the effects of plantgrowth-promoting bacteria on root growth: a criticalexamination. Soil Biol Biochem 37:1795–1804

Beauchamp C, Fridovich I (1971) Superoxide dismutase:improved assays and an assay applicable to acrylamidegels. Anal Biochem 44:276–287

Benhamou N, Kloepper JW, Quadt-Hallmann A, Tuzun S (1996)Induction of defense-related ultrastructural modifications inpea root tissues inoculated with endophytic bacteria. PlantPhysiol 112:919–929

Bilgin DD, Zavala JA, Zhu J, Clough SJ, Ort DR, DeLucia EH(2010) Biotic stress globally downregulates photosynthesisgenes. Plant Cell Environ 33:1597–1613

Bockenhoff A, Grundler FMW (1994) Studies on the nutrientuptake by the beet cyst nematode Heterodera schachtii byin situt microinjection of fluorescent probes into the feedingstructures in Arabidopsis thaliana. Parasitol 109:249–254

Bradford MM (1976) A rapid and sensitive method for thequantitation of microgram quantities of protein utilizing

Plant Soil (2014) 375:159–173 171

the principle of protein-dye binding. Anal Biochem 72:248–254

Bridge J, Page SLJ, Jordan SM (1982) An improved method forstaining nematodes in roots. In: Rothamsted ExperimentalStation Annual Report 1981. Rothamsted ExperimentalStation, Hertfordshire

Cavalcanti FR, Resende MLV, Lima JPS, Silveira JAG, OliveiraJTA (2006) Activities of antioxidant enzymes andphotosynthetic responses in tomato pre-treated by plantactivators and inoculated by Xanthomonas vesicatoria.Physiol Mol Plant Pathol 68:198–208

Cawoy H, Bettiol W, Fickers P, OngenaM (2011) Bacillus-basedbiological control of plant diseases. In: Stoytcheva M (ed)Pesticides in the modern world—pesticides use andmanagement. InTech, Rijeka, pp 273–302

Compant S, Duffy B, Nowak J, Clément C, Barka EA (2005)Use of plant growth-promoting bacteria for biocontrol ofplant diseases: principles, mechanisms of action, and futureprospects. Appl Environ Microbiol 71:4951–4959

Dabrowskai G, Kata A, Goc A, Szechyńska-Hebda M, SkrzypekE (2007) Characteristics of the plant ascorbate peroxidase.Familacta Biol Cracov Ser Bot 49:7–17

Dat J, Vandenabeele S, Vranova E, Montagu MV, Inze D,Breusegem FV (2000) Dual action of the active oxygenspecies during plant stress responses. Cell Mol Life Sci57:779–795

Demmig-Adams B, Adams WW (1996) The role of xanthophyllcycle carotenoids in the protection of photosynthesis.Trends Plant Sci 1:21–26

Duxbury AC, Yentsch CS (1956) Plankton pigment monograph.J Mar Res 15:93–101

Fenoll LG, Rodriguez-Lopez JN, Garcia-Molina F, Garcia-Conovas F, Tudila J (2002) Unification for the expressionof the monophenolase and diphenolase activities oftyrosinase. IUBMB Life 54:137–141

Foyer CH, Noctor G (2000) Oxygen processing inphotosynthesis: regulation and signaling. New Phytol 146:359–388

Fu Q, Liu C, Ding N, Lin Y, Guo B (2010) Ameliorative effectsof inoculation with the plant growth-promotingrhizobacterium Pseudomonas sp. DW1 on growth ofeggplant (Solanum melongena L.) seedlings under saltstress. Agric Water Manag 97:994–2000

Giebel J (1973) Phenylalanine and tyrosine ammonia lyaseactivities in potato roots and their significance in potatoresistance to Heterodera rostochiensis. Nematologica 19:3–6

Gill SS, Tuteja N (2010) Reactive oxygen species andantioxidant machinery in abiotic stress tolerance in cropplants. Plant Phys Biochem 48:909–930

Hano C, Addi M, Bensaddek L, Cronier D, Baltora-Rosset S,Doussot J, Maury S, Mesnard F, Chabbert B, Hawkins S,Laine E, Lamblin F (2006) Differential accumulation ofmonolignol-derived compounds in elicited flax (Linumusitatissimum) cell suspension cultures. Planta 223:975–989

Holt JG, Krieg NR, Sneath PHA, Staaley JT, Williams ST (1994)Bergey’s manual of determinative bacteriology, 9th edn.The Williams and Wilkins Company, Baltimore

Huang XW, Tian BY, Niu QH, Yang JK, Zhang LM, Zhang KQ(2005) An extracellular protease from Brevibacillus

laterosporus G4 without parasporal crystal can serve as apathogenic factor in infection of nematodes. Res Microbiol156:719–727

Hussey RS, Barker KR (1973) A comparison of methods ofcollecting inocula of Meloidogyne spp., including a newtechnique. Plant Dis Rep 57:1025–1028

Kloepper JW, Ryu CM, Zhang S (2004) Induced systemicresistance and promotion of plant growth by Bacillus spp.Phytopathol 94:1259–1266

Kovács V, Pál M, Vida G, Szalai G, Janda T (2011) Effect ofpowdery mildew infection on the antioxidant enzymeactivities in different lines of Thatcher-based wheat. ActaBiol Szeged 55:99–100

Kristensen BK, Brandt J, Bojsen K, Thordal-Christensen H,Kerby KB, Collinge DB (1997) Expression of a defence-related intercellular barley peroxidase in transgenictobacco. Plant Sci 122:173–182

Luthra YP, Ghandi SK, Joshi UN, Arora SK (1988) Totalphenols and their oxidative enzymes in sorghum leavesresistant and susceptible to Ramulispora soghicola Harris.Acta Phytopathol Entomol Hung 23:393–400

Mannanov RN, Sattarova RK (2001) Antibiotics produced byBacillus bacteria. Chem Nat Comp 37:117–123

Nakano Y, Asada K (1981) Hydrogen peroxide is scavenged byascorbate-specific peroxidase in spinach chloroplasts. PlantCell Physiol 22:867–880

Niebel A, Heungens K, Barthels N, Inze D, Montagu MV, GheysenG (1995) Characterization of a pathogen-induced potatocatalase and its systemic expression upon nematode andbacterial infection. Mol Plant Microbe Interact 8:371–378

Niu Q, Huang X, Zhang L, Li Y, Li J, Yang J, Zhang K (2006) Aneutral protease from Bacillus nematocida, anotherpotential virulence factor in the infection againstnematodes. Arch Microbiol 185:439–448

Ongena M, Jacques P (2007) Bacillus lipopeptides: versatileweapons for plant disease biocontrol. Trends Microbiol16:115–125

Pan Y, Wu LJ, Yu ZL (2006) Effect of salt and drought stress onantioxidant enzymes activities and SOD isoenzymes ofliquorice (Glycyrrhiza uralensis Fisch). Plant GrowthRegul 49:157–165

Rahimi S, Perry RN, Wright DJ (1996) Identification ofpathogenesis-related proteins induced in leaves of potatoplants infected with potato cyst nematodes, Globoderaspecies. Physiol Mol Plant Pathol 49:49–59

Reichert AI, Xian-Zhi HE, Dixon RA (2009) Phenylalanineammonia-lyase (PAL) from tobacco (Nicotiana tabacum):characterization of the four tobacco PAL genes and activeheterotetrameric enzymes. Biochem J 424:233–242

Rosner B (2006) Fundamentals of biostatistics, 6th edn.Thomson Books/Cole, Duxbury

Sakiroglu H, Ozoturk AE, Pepe AE, Erat M (2008) Some kineticproperties of polyphenol Oxidase obtained from dill(Anethum graveolens). J Enzym Inhib Med Chem 23:380–385

Sarowar S, Kim EN, KimYJ, Ok SH, KimKD, Hwang BK, ShinJS (2005) Overexpression of a pepper ascorbate peroxidase-like 1 gene in tobacco plants enhances tolerance tooxidative stress and pathogens. Plant Sci 169:55–63

Selvadurai EL, Brown AE, Hamilton JTG (1991) Production ofindole-3-acetic acid analogues by strains of Bacillus cereus

172 Plant Soil (2014) 375:159–173

in relation to their influence on seedling development. SoilBiol Biochem 23:401–403

Shetty NP, Hans J, Jørgensen L, Jensen JD, Collinge DB, ShettyHS (2008) Roles of reactive oxygen species in interactionsbetween plants and pathogens. Eur J Plant Pathol 121:267–280

Shishido M, Massicotte HB, Chanway CP (1996) Effect of plantpromoting Bacillus strains on pine and spruce seedlinggrowth and mycorrhizal infection. Ann Bot 77:433–441

Shishido M, Breuil C, Chanway CP (1999) Endophyticcolonization of spruce by plant-promoting rhizobacteria.FEMS Microbiol Ecol 29:191–196

Siddiqui IA, Shaukat SS (2004) Systemic resistance in tomatoinduced by biocontrol bacteria against root-knot nematodeMeloidogyn javanica is independent by salicylic acid. JGeophys Res 152:48–54

Sofo A, Dichio B, Xiloyannis C, Masia A (2004) Effects ofdifferent irradiance levels on some antioxidant enzymesand on malondialdehyde content during rewatering in olivetree. Plant Sci 166:293–302

Strange, RN (2003) Introduction to plant pathology. Wiley, NewYork

Streb P, Feierabend J (1996) Oxidative stress responsesaccompanying photoinactivation of catalase in NaCl-treated rye leaves. Bot Acta 109:125–132

Surette MA, Sturz AV, Lada RR, Nowak J (2003) Bacterialendophytes in processing carrots (Daucus carota L. var.sativus): their localization, population density, biodiversityand their effects on plant growth. Plant Soil 253:381–390

Taylor DP, Netscher C (1974) An improved technique forpreparing perennial pattern of Meloidogyne spp. Nematol20:268

Wang W, Vinocur B, Altman A (2003) Plant responses todrought, salinity and extreme temperatures: towards geneticengineering for stress tolerance. Planta 218:1–14

Won-Chan K, Rhee IK (2012) Functional mechanism ofplant growth retardation by Bacillus subtilis IJ-31 and itsallelochemicals. J Microbiol Biotechnol 22:1375–1380

Zar JH (2009) Biostatistical analysis, 5th edn. Prentice-Hall,Englewoods Cliff

Zhang N,WuK, He X, Li S, Zhang Z, Shen B, Yang X, Zhang R,Huang Q, Shen Q (2011) A new bioorganic fertilizer caneffectively control banana wilt by strong colonization withBacillus subtilis N11. Plant Soil 344:87–97

Plant Soil (2014) 375:159–173 173

Potential of Bacillus species against Meloidogyne javanica parasitizing eggplant (Solanum melongena...

Documents

Transcript of Potential of Bacillus species against Meloidogyne javanica parasitizing eggplant (Solanum melongena...