The Effect of Serratia marcescens and Genetically Improved Pseudomonas fluorescens on Meloidogyne incognita

Egyptian Academic Journal of Biological Sciences is the official English language journal of the Egyptian Society of Biological Sciences, Department of Entomology, Faculty of Sciences Ain Shams University. The Journal publishes original research papers and reviews from any zoological discipline or from directly allied fields in ecology, behavioral biology, physiology, biochemistry, www.eajbs.eg.net Provided for non-commercial research and education use. Not for reproduction, distribution or commercial use.


INTRODUCTION
Root-knot nematodes is one of the critical pests that causes fatal damage for wide range of important crops in tropical and subtropical regions (Zeinat et al. 2010).Endoparasites nematodes (Meloidogyne spp.) is the most common parasite nematodes in all over the world.Some of rhizosphere microorganisms are able to grow in root area and stimulate systemic defense against parasites including nematodes, this prevent pathogen to attach the plant and considering ideal tools for biocontrol agents.Both eukaryotic and prokaryotic microorganisms are widely present in the soil.Some of these have a potential for biocontrol of nematodes (Zaied et al., 2009, andRehanadeh et al., 2013).The ability of P. fluorescens and S. marcescens to produce biological active ingredients have been reported in several studies (Shanthi, 1998;and Ramakrishnan, 1999) who find that, Pseudomona fluorescens as a biocontrol agent of M. incognita, and have a suppressing effect on nematodes multiplication and increase host plant growth.Microorganisms such as Serratia marcescens has been considered as a more natural and environmentally acceptable alternative to chemical control (Abd-Elgawad, 2006;and Zeinat et al. 2010).
Serratia sp. are short straight rods gram-negative bacteria, it found in soil, water and air.Their colonies are most often opaque.Somewhat iridescent and white, pink or red in color (Richared et al., 1995).Chitenase has a greatest effect in biocontrol activity, (Gomaa, 2012).Chitenase could be suppressed egg hatching via deformed and destroyed the eggshell of Meloidgyne (Jung et al., 2002).In addition, more efficient bacteria that may able to control nematodes i.e have nematicidal activity, as well as maintaining the soil fertility via protoplast fusion between different species of Peseudomones and Serratia.Fusents were produced high mortality levels against nematodes (Zaied et al., 2009) This study aim to 1-cloning of S. marcescens genes encoding the most abundant S. marcescens chitinase into both E. coli and P. fluorescens.2-investigation of chitinase expression from various constructs of both E. coli and P. fluorescens.3improvement the transformed P. fluorescens as a biocontrol against plant parasitic nematodes, M incognita.

MATERIALS AND METHODS
The present study was carried out in plant protection department and Ain Shams Center for Genetic Engineering and Biotechnology (ACGEB), Faculty of Agriculture Ain Shams university.

A-Materials 1-Bacterial strains
The following bacterial strains were used:

4-Preparation of colloidal chitin
Colloidal chitin was prepared by partial hydrolysis of chitin (Sigma) with HCl 1M for 2 h at room temperature.The colloidal chitin was washed several times with large volumes of distilled water to eliminate all HCl and the pH was adjusted to 7.0.

7-Root-knot nematode culture:
Culture of Meloidogyne incognita (Mi) was maintained on tomato plants (Lycopersicum esculentum L.) growing in 20-cm pots in sterile sandy clay soil until needed, galled cucumber roots were washed with tap water, cutting into small pieces and placed in the mist chamber for egg hatching.The catch of the first day was excluded, then the following hatched IJs were collected daily and refrigerated at 5 o C for using.B-Experimental methods: 1-Evaluation of bacterial activity on Meloidogyne incognita in greenhouse: Pots filled with sterilized soil were divided into four groups (G1, G2, G3 and G4).The first group used as control without microbes (halve pots received 1000Mi as a positive control, the rest pots without Mi, were used as a negative control) All the other groups received microbes (one microbe/group) 10 ml/pot (2.1x10 8 cfu/ml).Half of the pots of each group was infected with Mi (1000 IJs/pot) and the other half was left Mi-free.All the tretments were replicated four times, each replicate was cultivated with three weeks seedling of cucumber (Cucumis sativus L.).
After 60 days screenhouse experiments were ended, the height of the plants was measured, fresh weight of roots and shoots was measured, and shoot/root ratio was calculated.IJs were extracted from roots after washing by mist chamber for 7 days, and from soil using modified Burmman funnel.All IJs which extracted were counted using light microscop.After that, roots were removed from the mist chamber and stained with acid fuchsin in cold lacto-phenol (McBeth et al., 1941).Galls were counted in the stained roots.
Survival bacteria in the soil was estimated after plant harvest by mixing 10g samples of soil from each pot of the bacterial treatment or control and estimating CFU per gram of soil by serial dilution plating on selective medium.

2-Chitinolytic activity; Agar medium screening test
Plates containing 0.5% acid swollen chitin was prepared.Two µl from each bacterial isolates was pipetted.After 2 days from incubation, the diameters in cm of the chitin hydrolysis zones were measured.

3-Chitinolytic activity
Chitinase activity was determined colorimetrically by detecting the amount of N-acetyl glucosamine (GlcNAc) released from a colloidal chitin substrate (Reissig et al. 1955).

4-Bacterial Transformation Genomic library construction and screening
Chromosomal DNA was prepared from S. marcescens (ACGEB Ser1) according to the method described by Sambrook et al. (1989).

5-Preparation of Competent Bacterial Cells
Two hundred and fifty µl of 50 mM CaCl 2 was added into 2 tubes.The tubes was placed on ice, a sterile inoculating loop was used to aseptically transfer a single colony of bacteria from the starter plate to each tube.Ten µl of the ligation solution was added directly into the cell suspension in one of tubes.Ten µl (50 ng) of the control was added, linear, unligated pGEM T-Easy vector into the cell suspension of the second tube and returned to ice and incubated for 15 minutes.

6-Heat Shock of Bacterial Cells
Tubes was carried on ice to the water bath and transferred from ice and immediately immersed them into 42°C water bath for 90 seconds.It is critical that cells receive a sharp and distinct shock, immediately returned both tubes to ice and was leted them stand on ice for one additional minute and was placed in a test tube rack at room temperature.After that 250 µl of sterile liquid LB medium was added to each tube.Gently the tubes was taped to mix to allow for the bacteria to recover from the heat shock at room temperature for about 30 minutes.

7-Plating the Transformation
Forty µl of 20-mg/ml X-Gal were spreaded onto each agar plate about 60 minutes before use, 100 µl of cells were pipetted onto each plate after that 4 µl of 1 M IPTG was added to the 100 µl of bacterial cells on the agar.Both together were spreaded onto the plate.A sterile cell spreader spread the bacteria was used over the surface of the agar in each plate.The spreader was moved back and forth on the agar while turning the plate.This will spread the bacteria evenly across the agar surface and placed the plates upside down in a 37°C incubator, and allowed them to grow for 24 to 48 hours.Alternatively, plates may be incubated at room temperature for 2 to 3 days, finally the number of blue colonies and white colonies on each plate was counted and recorded.

8-Plasmid Isolation
Single bacterial colony was transferred into 5 ml of LB according to Diab et al., (2002).

Chitinolytic activity; Agar medium screening test
The Gram-negative bacterium secretes a variety of extracellular enzymes including chitinases; S. marcescens is one of the most effective bacteria for degradation of chitin (Brurberg et al., 2000).Serratia was subjected to test for their chitinase productivities as clear zone test for determination of the enzyme activity,

Chitinolytic activity spectro-photometric assay
Chitinase activity was determined colorimetrically by detecting the amount of GlcNAc released from a colloidal chitin substrate (Reissig et al. 1955).Serratia strain gave the highest enzyme activity (1744.5 µg/m).Refaat et al, (1982) and Bechina et al, (1982) noted that, a correlation was not always observed between the largest zone with respect to activity on solid medium and the activity of the cultures on liquid medium (Table 1).
These data confirmed that, E. coli (pGEM 1) produces higher chitinase activity than the same strain containing pGEM2.Chitinase activities of the P. fluorescens ACGEB ps111 without transformed plasmid constructs were below the level of detection for this assay.
The genes encoding ChiB and CPB21 are linked, Gal et al., (1997) but the DNA sequence suggests that transcription of the two genes is not coupled.ChiB are found in the periplasm and/or culture medium of S. marcescens but this protein does not contain typical N-terminal signal peptides.There are no indications of any proteolytic processing of ChiB Brurberg et al., (1995).The chiB genes from S. marcescens have been transformed into other bacterial species like P. fluorescens and E. coli in an attempt to improve their ability to control fungal plant pathogens Sundheim et al, (1988) or to create new biocontrol agents.

Chitinolytic activity of transformed E. coli and P. fluorescens
The amount of N-acetyl glucosamine were determined colorimetrically by detecting released from a colloidal chitin substrate as described by Reissig et al. (1955).Results are shown in the previous table demonstrated that, the transformed P. fluorescens ACGEB ps111 (PGEM1) gave the highest enzyme activity (1067.9µg/m) but P. fluorescens ACGEB ps111 (pGEM3) had no activity.The level of chitinase in Transformed P. fluorescens ACGEB ps111 (pGEM1) indicates that chitinase synthesis in these constructs is driven by S. marcescenis promoter that contained on the fragment insert, perhaps the chitinase promoter itself, and not from vector promoters.Clones producing significantly lower enzyme levels may therefore be undetectable and require a more sensitive screen.This conclusion is supported by Horwitz et al. (1984).

Cloning of a S. marcescens gene encoding chitinase
S. marcescens secretes chitinses of five distinct molecular masses of 58, 54, 52, 35, 22 kDa into the culture broth.S. marces-cens plasmid library was constructed using E. coli Top 10 by pGEM-T-Easy with S. marcescens ACGEB Ser1 chromosomal DNA.Overlay plates were used to identify chitinase producing clones.S .marcescensACGEB Ser1 produced chitinolytic activity.Out of 320 clones screened, only four chitinase positive clones were identified by the production of clear zones, visible against the trans-lucent background of colloidal chitin.
A total of four independent clones (pGEM 1, 2, 3 and 4) express chitinase activity in E. coli Top10 on chitin overlay plates incubated at 30°C.E. coli Top 10 containing produces pGEM 1 (the largest zone of clearing), whereas the same strain containing pGEM 4 produces the smallest.
All four plasmid were mobilized into P. fluorescens ACGEB ps111, and the resultant isolate were analyzed for the rate of clearing on chitin overlay plates.In contrast to the results with E. coli, Out of 80 colonies screened on LB agar plates supplemented with 1% swollen chitin, three of them were found surround by chitin clear zone (Fig. 2).
The pGEM 1 construct produces the largest zone of clearing in P. fluorescens ACGEB Ps111 (Fig. 3), and the PGEM 3 construct produces the smallest However, P. fluor-escens ACGEB Ps111 (pGEM 2) required considerably longer time (>10 days) than E. coli (pGEM 1) 4 to 6 days to produce a detectable zone of clearing.This data was conformed with Fuchs et al., (1986).In vitro chitinase activities were determined for cultures of E. coli Top 10 and P. fluorescens ACGEB ps111 containing either no plasmid to quantitative the levels of chitinase produced.

Plasmid transfer to E. coli clone selection and expression
The vector containing the DNA fragment was transformed to E. coli top10, after transformation the bacteria was screened on LB plates containing Ampicillin, IPTG and XGAl and after 2 days only the white colonies were selected.Approximately 320 clonies were screened by transfer to LB agar plates supplemented with 1% swollen chitin.Out of 320 clonies screened, 4 clonies produced clear chitinolytic zones after 3 days of growth at 37 Ο C and were isolated.All clonies were chitinase positive, as judged by a clear zone around the colonies.The size of DNA insert was determined to be 6.5 kb.

Selection of transformed P. fluorescens
There is a narrow range of pH (about 12.0 -12.5) which denatu-ration of linear DNA but not CCC-DNA occurs and that this property can be used for purifying CCC-DNA (Sharp et al., 1972 andCurrier andNester 1976).The high concentration of sodium acetate causes precipitation of protein-SDS complexes (Kay et al., 1952).Most of the three major contaminating macromolecules are co-precipitated and may be remo-ved by a single centrifugation in a bench top centrifuge.Plasmid DNA (and residual low molecular weight RNA) is recovered from the supernatant by ethanol precipita-tion.Plasmid DNA may be ana1yzed by gel electrophoresis either intact in the CCC form or after digestion with a restriction enzyme.After isolation of pGEM vector that contain ChiB insert, it retransformed to P. fluorescens ACGEB ps111.
The result of electrophoretic plasmid separation using 1.5% agarose gel electrophoresis is photographed (fig.4).As shown from photo, (A) the transformed E. coli pGEM contains the pGEM vector with Chi B fragment, (B) The Transformed P. fluorescens ACGEB ps111-PGEM with the same vector.Nematode parameters (number of galls, egg masses, and final population) were higher in unbacterized plants than on bacterized ones.Moreover, When the plants were inoculated with Mi only, significant suppression in the plant parameters (Shoot length, Shoot and Root Fresh weight, and shoot/root ratio) were recorded.Improving in these parameters was observed when adding different microbes (Serratia marcescens, Pseudomonas fluorescens, and transformed P. fluorescens) these data agreed with (Zaied et al., 2009).
Table (2) showed that: Maximum significant increasing in all the growth and yield parameters was found in treated plants by transformed P. fluorescens , P. fluorescens and Serratia as compared to untreated inoculated or other treatments.Numbers followed by the same letters did not differ significantly in their effects while, different letters had a statistically significant differences.
The obtained results indicated that, transformed P. is the most effective among all treatments in improving plant growth and reducing Mi population densities in soil.
In Table (3) results showed significant suppression of Mi., Maximum reduction in nematodes parameters was recorded with the transformed P. fluorescens.
Chitinases may have direct effect in nematode suppression (Elad et al., 1982).In the present study, the microbes which adding closed to the plant roots in large numbers in their media, that may contained toxins or by product of metabolites that may toxic to plant-parasitic nematodes and decrease other deleterious microorganisms (Khan and Saxena, 1997;Padgham and Sikora, 2007).Chitinase has a greatest effect in biocontrol activity (Gomaa, 2012), and it could be suppressed egg hatching via deformed and destroyed the eggshell of Meloidgyne (Duponnois, and Mateille, 1999;4. Ali, 1996;and Mercer, et al., 1992).Moreover, it may be cause immature hatch of nematode eggs (Jung et al., 2002).
The use of Serratia may be able to control nematodes.This is in agreement with results reported by other researchers (Kurz et al., 2003, El-Nagdi and Youssef, 2004, Darby.2005, Zaied et al., 2009and Zeinat et al., 2009).548 Numbers followed by the same letters did not differ significantly in their effects while, different letters had a statistically significant differences.

CONCLUSION
Each of the microbes: S. marcescenis , P. fluorescens and transformed P. fluorescens Serattia has a reducing effect on M. incognita but, the last one has the great effect in reduction followed by P. fluorescens and S. marcescenis.
All of these microbes recorded highly improving in plant growth.So, we can use transformed P. as a bio control agent to control plant parasitic nematodes, and improving plant growth.

Fig. 1 :
Fig. 1: Chitinase activity of Serratia strain as clear zone on chitin over lay plates.

Table 1 :
Concentrations of Chitinolytic activity of transformation measured by spectrophotometer

Table 2 :
Effect of different bacterial strains (with and without Mi) on Cucumber growth parameters under greenhouse conditions.

Table 3 :
Effect of different bacterial strains on the root-knot nematodes Meloidogyne incognita infecting Cucumber under greenhouse conditions.