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Biological control of root-knotnematode (Meloidogyne javanica)disease by Pseudomonas fluorescens(Chao)Maryem Tavakol Norabadia, Navazollah Sahebania & Hassan RezaEtebarianb

a Department of Plant Protection, University of Tehran, Tehran,Iran.b Department of Plant Protection, Islamic Azad University, Tehran,Iran.Published online: 12 Jul 2013.

To cite this article: Maryem Tavakol Norabadi, Navazollah Sahebani & Hassan Reza Etebarian(2014) Biological control of root-knot nematode (Meloidogyne javanica) disease by Pseudomonasfluorescens (Chao), Archives Of Phytopathology And Plant Protection, 47:5, 615-621, DOI:10.1080/03235408.2013.816102

To link to this article: http://dx.doi.org/10.1080/03235408.2013.816102

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Biological control of root-knot nematode (Meloidogyne javanica)disease by Pseudomonas fluorescens (Chao)

Maryem Tavakol Norabadia*, Navazollah Sahebania and Hassan Reza Etebarianb

aDepartment of Plant Protection, University of Tehran, Tehran, Iran; bDepartment of PlantProtection, Islamic Azad University, Tehran, Iran

(Received 6 June 2013; final version received 12 June 2013)

Plant growth-promoting Rhizobacteria is currently developed as an biocontrol agentagainst many plant pathogens. In this research, biological control of root-knotnematode (Meloidogyne javanica) by Pseudomonas fluorescens was investigated ingreenhouse and laboratory experiments. Results showed that 109 (CFU/ml) of P. fluo-rescens decreased nematode infection and other parameters significantly, comparedto the control. P. fluorescens was able to cause destruction of nematode egg massmatrix and significantly decreased nematode egg hatching level. Specific activities ofresistance-related enzymes, namely peroxidase (POX) and phenylalanine ammonialyase (PAL), increased significantly in P. fluorescens-inoculated plants. Maximumactivities of POX and PAL were observed at the 5 days after inoculation,respectively. Results suggested that the destruction of eggs and plant defence mecha-nisms leading to systemic resistance are two main suppression mechanisms used byP. fluorescens against nematode.

Keywords: biological control; Meloidogyne javanica; peroxidase; phenylalanineammonia lyase

Introduction

Tomato (Lycopersicon esculentum) is one of the most important vegetable crops in Iran.Most common tomato varieties are susceptible to the root-knot nematode Meloidogynejavanica. This nematode has been managed with soil fumigants, mainly methyl bro-mide. The mandated elimination of methyl bromide will make nematode control moredifficult and require alternative control methods (Noling & Becker 1994). Also, thetrend of reduced use of nematicides due to concerns for the environment and safe foodrequires environmentally friendly nematode control methods, including use of biologicalcontrol and resistant varieties. Unfortunately, no tomato varieties completely resistant tothe root-knot nematodes are available in Iran. So, scientists are looking for other waysto control disease agents. Rhizobacteria are a subset of total rhizosphere bacteria whichhave the capacity, upon reintroduction to seeds or vegetative plant parts (such as potatoseed pieces), to colonise the developing root system in the presence of competing soilmicroflora (Kloepper et al. 1999). One of the ways is the use of natural biocontrolbacterial such as Pseudomonas fluorescens (Chao) in which antagonist properties aregrowth enhancers. Certain root-associated strains of P. fluorescens produce and excrete

*Corresponding author. Email: sahebani@ut.ac.ir

Archives of Phytopathology and Plant Protection, 2014Vol. 47, No. 5, 615–621, http://dx.doi.org/10.1080/03235408.2013.816102

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metabolites that are inhibitory to soil-borne plant pathogens (Keel et al. 1992; Dowling& O’Gara 1994; Thomashow & Weller 1995). However, micro-organisms includingPGPR as biological control agents typically have a relatively narrow spectrum ofactivity compared with synthetic pesticides (Baker 1991; Raupach & Kloepper 1998)and often exhibit inconsistent performance in practical agriculture, resulting in limitedcommercial use of biocontrol approaches for suppression of plant pathogens (Backmanet al. 1997). Much of this variability has been attributed to differences in physical andchemical properties found in natural environments where biocontrol agents are applied(Thomashow & Weller 1996; Duffy et al. 1997). Understanding that environmentalfactors are important and how these influence disease suppression is widely recognisedas a key to improving the level and reliability of biocontrol. Previous studies haveshown that specific rhizosphere bacteria have an antagonistic activity against variousspecies of plant-parasitic nematodes. In a screening programme, 16 bacterial isolatesout of 179 isolated from root and cysts caused a significant (0.25%) reduction in Globo-dera pallida penetration of potato roots (Racke & Sikora 1992). A 68% reduction ofsugar beet cyst nematode root invasion was obtained by an application of the Rhizobac-terium P. fluorescens to beet seeds (Oostendorp & Sikora 1990). Latter studies indicatedthat Rhizobacteria can also induce systemic resistance in potato roots to infection bythe potato cyst nematode G. pallida (Reitz et al. 2000).

So with the introduction that mentioned above in the present study, we used green-house experiments to show the ability of P. fluorescens (Chao) to: (1) suppress the rootknot-nematode, M. javanica infection and nematode egg hatching on tomato plant; and(2) conduct laboratory experiments to monitor the assay of the effects of P. fluorescens(Chao) filtrate on the egg hatching and larve mortality of M. javanica.

Materials and methods

Nematode inoculum preparation

To obtain nematodes for the experiments, four-week-old tomato (L. esculentum)seedlings of cv. Early-Urbana were planted in soil and infested with 200–300 s-stagejuveniles of M. javanica (Infected sample was collected from a tomato field in Pakdasht(Tehran province, Iran) and single egg mass was used to establish a population). After2months in the greenhouse, the tomato roots were removed from the pots and eggswere extracted from infected tomato roots using 1% NaOCL. Extracted eggs weregently washed with tap water to remove NaOCL (Hussey & Barker 1973) and passedthrough 60–100 and 400 μm aperture sieves, from where they were washed into abeaker (Kim & Riggs 1995). The species of nematode was identified as M. javanicaaccording to morphological and morphometrical characters (Eisenback 1985).

Plant material preparation

Experiments were carried out with tomato (L. esculentum cv. Early-Urbana, susceptibleto M. javanica) grown in a controlled environment greenhouse. The air temperaturewas maintained at 27 ± 1 °C.

Bacterial inoculum preparation

P. fluorescens (Chao) was obtained from the plant pathology department, agriculturefaculty of Tehran University and was cultured on Nutrient agar. Two days after

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incubation (27 °C), the purified P. fluorescens was used to produce bacterial suspensionfor inoculation.

Soil treatment at planting (in vivo tests)

Tomato seeds were sown in pots (5 inches in diameter). Seedlings were then propagatedfor 45 days in the greenhouse in the pots containing sterilised mixture of field soil, leafcompost and sand at the rate of 1:2: 2:2. In this time, the seedlings (at four-leaf stage)were inoculated with P. fluorescens. Each seedling received a 20ml of liquid suspensionof the bacteria containing 109 (CFU/ml). The inoculum was injected 2 cm deep into therhizosphere using three holes made around the stem base with a plastic rod. Theabsolute controls were treated with distilled water. The pots were then immediatelyinoculated with a 5-ml sterile distilled water suspension containing 2000 J2 of M. java-nica. The inoculum was also injected into three holes roughly 2-cm deep around thestem base. The experiment consisted of four treatments: (1) P. fluorescens +M. javanica;(2) only P. fluorescens; (3) only M. javanica (positive control); and (4) non-inoculated(negative control). The treated pots were incubated in the greenhouse at 22 ± 5 °C with16 h of supplemental artificial light per day. The plants were watered and fertilised with2 g per litre water to insure proper plant growth. The experiment was terminated 45 daysafter fungal and nematode inoculation. Fresh root and shoot weight was measured(Al-Fattah et al. 2007). The roots were removed, washed free of soil and stained in0.015% Phloxine B for 20min to facilitate egg mass counting Shurtleff and Averre,(2000). The number of galls, egg masses, eggs and diameter of galls per plant was thendetermined. The experiment was done twice. Statistical analysis was done by employingcompletely randomised experimental design. ANOVA analysis was performed withcritical difference at 5% level of significance.

Resistance-related enzymes assays

Seeds of tomato were surface-sterilised and sown in pots containing sterile soil. Eachseedling received 20ml of a liquid suspension of the bacteria containing 109 (CFU/ml)of P. fluorescens at four-leaf stage and were irrigated with SD/water. Two days afterinoculation with bacteria, plant roots were inoculated with 2000 nematode J2/plant.Root sampling were done 7 days with 1-day intervals. Fresh tomato roots were washedand dried with filter paper after sampling and homogenised with liquid nitrogen in anice cold mortar and pestle. The homogenised tissue was rinsed with the same volumeof 10-mM sodium phosphate buffer (PH 6.0) at 4° C, and was filtered through a 0.2-mm nylon filter into a centrifuge tube. The tissue extracts were centrifuged at12,000 rpm for 20min at 4 °C. The supernatant was used for the enzymatic activityassay. The experiment was performed with five replications.

Results

Soil treatment at planting (in vivo tests)

The treatment of the soil at transplanting with P. fluorescens and M. javanicaresulted in a reduction of the number of galls, egg masses, eggs and diameter ofgalls. The significant reduction in gall formation was obtained with P. fluorescenswhen compared to the control. However, in the experiments, P. fluorescens gavehigher levels of control when compared to only M. javanica (Table 1). The

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suppressive effect of the P. fluorescens on egg mass production, which is a measureof nematode development over time, was confirmed for both treatments, when com-pared to the control (M. javanica). P. fluorescens reduces egg mass, eggs, gallsnumber and diameter of galls (Table 1). Root and shoot weight obviously increasedwhen the tomato plants were inoculated with P. fluorescens compared nematode con-trols (Table 2). The P. fluorescens exists on the surface of the root after 45 days andit was successfully reisolated from the rhizosphere.

PAL and PPO enzymes activities in roots inoculated with P. fluorescens andM. javanica

Inoculation of tomato roots with P. fluorescens significantly increased phenylalanineammonia lyase (PAL) and peroxidase (POX) enzyme activities compared to control(inoculated with nematode). Maximum (peak) level of the enzyme activities was at 5and 3 days after inoculation, respectively, and then slowly decreased (Figures 1 and 2).When the tomato roots were treated with P. fluorescens and nematode, PAL and POXactivity increased gradually in all the treated roots compared to activities from theinfected control plants with nematode. PAL and POX activities in the P. fluorescens-treated plants in the presence of pathogen inoculation were approximately 50% higherthan those in the inoculated control roots, at 5 days after inoculation. In all of the dayof sampling (except 1), there were a significant difference between P. fluorescens andother treatments (Figures 1 and 2).

Table 2. Influence of soil treatment with Pseudomonas fluorescens and Meloidogyne javanicaon fresh root and shoot weight 45 days after nematode inoculation at the time of transplantingtomato.

Treatment Fresh root Shoot weight

P. fluorescens 8.3 A 25 AM. javanica 4.9 D 19 DP. fluorescens +M. javanica 5.5 C 20.5 Cnon-inoculated 6.05 B 22 B

Note: Means with different capital letters in the collums are significantly different from each other based onduncan test (p6 0.05; n= 10).

Table 1. Influence of soil treatment with Pseudomonas fluorescens and Meloidogyne javanicaon gall, egg mass and egg number 45 days after nematode inoculation at the time of transplantingtomato.

TreatmentNo. Gall/plant

No. Eggmass/plant

N. Egg/individual eggmass

Diameter of Gall(mm)

P. fluorescens 0 C 0 C 0 C 0 CM. javanica 320 A 184 A 410 A 2.4 AP. fluorescens +

M. javanica112 B 100 B 215 B 1.6 B

non-inoculated 0 C 0 C 0 C 0 C

Note: Means with different capital letters in the collums are significantly different from each other based onduncan test (p6 0.05; n= 10).

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Discussion

Our results indicate that P. fluorescens has significant potential as biocontrol agentagainst the root-knot nematode M. javanica in greenhouse experiment. Inoculation oftomatoes with P. fluorescens can significantly reduce the population of this nematodeand disease severity. Suitable rate of P. fluorescens for suppressing nematode activitiessuch as nematode infection, egg mass production and number of eggs per egg masswas observed in 109 (CFU/ml) concentration. Results also showed that P. fluorescenssignificantly increased PAL and POX activities compared to control and this suggestthat P. fluorescens can induce such defence enzymes and probably other defence com-pounds leading to systemic resistance in plants. In addition to suppressing nematodedamage, treatments with the tested inoculants increased tomato root weight, which

Figure 1. Phenylalanine ammonia lyase specific activity in tomato roots (var. Roma VF) asrelease of trans-cinnamic acid from phenylalanine, inoculated with Pseudomonas fluorescens andMeloidogyne javanica. Each value represents the mean of five replicates from two paralleledexperiments. The bars correspond to the standard error.(B=Pseudomonas fluorescens – N=Meloidogyne javanica – N+B=P. fluorescens +M. javanica– P=Control.

Figure 2. Polyphenol oxidase specific activity in tomato roots (var. Roma VF) inoculated withPseudomonas fluorescens and Meloidogyne javanica. Each value represents the mean of 10replicates from two paralleled experiments.The bars correspond to the standard error. (B=Pseudomonas fluorescens – N=Meloidogynejavanica – N+B=P. fluorescens +M. javanica – P=Control.

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could also account for some of the observed suppression. Plants with larger rootsystems have been reported to tolerate a given population of nematodes (Gierth et al.2004; Kokalis-Burelle et al. 2006). The suppression of root-knot nematode by PGPRinoculants, as found in our study, agrees with previous reports with BioYield ingreenhouse and field trials (Kokalis-Burelle et al. 2002). Plant growth-promoting Rhizo-bacteria suppress plant parasitic nematode population through blockage of the receptorson roots and modification of root exudates of the host plant, thus hindering the attrac-tion, hatching and penetration behaviours of nematodes. P. fluorescens promotes thegrowth of plants and reduces the nematode multiplication, and has high potential asbiological control agents against wilt as well as root-knot disease. The results of ourstudy suggest that P. fluorescens can be used as a bioagent for the sustainable manage-ment of root-knot nematodes on selected crops and in particular circumstances. Futurestudies on nematode biocontrol will focus on the effect of biocontrol agent on otherplant defence mechanisms and extracellular enzymes.

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