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Effect of Carrier and Temperature on the Viability of Burkholderia sp. (UPMB3) and Pseudomonas sp. (UPMP3) during Storage.

Byline: A.B. ILI BAZILAH, M. SARIAH, M.A. ZAINAL ABIDIN AND S. YASMEEN

ABSTRACT

This study was aimed at to determine the ability of different carriers to sustain the viability and efficacy of endophytic bacteria: Burkholderia sp (UPMB3) and Pseudomonas sp (UPMP3) during storage. UPMB3 and UPMP3 were formulated as dry formulation using vermiculite and coir dust as carriers and liquid formulation with Luria broth (LB) as the culture substrate.

These bacterial formulations developed were assessed for the viability and efficacy as fresh preparations and after nine months stored at 10, 20 and 30degC. Formulations stored at 10 and 20degC provided a longer shelf-life than those stored at 30degC based on viability at monthly intervals over a 9-month storage period. At 10 and 20degC, the LB-based and vermiculite-based formulations were found to be the most stable by sustaining 86% of viable bacteria cells after 6 months of storage. However, at the end of 9 months, the number of viable bacteria cells in both formulations declined to 71 and 57%, respectively. Coir dust-based formulation was the least stable at 10 and 20degC storage, when only 43 and 29% viable cells were detected at the end of 9-months storage. (c) 2011 Friends Science Publishers

Key Words: Burkholderia; Pseudomonas; Formulation; Vermiculite; Coir dust; Luria broth

INTRODUCTION

Studies on plant disease control by using endophytic bacteria have gained importance due to their ability to colonize intercellular and intracellular tissues of plants to restrain the invasion of pathogen and minimize the chances of such pathogen to colonize the area (Kloepper et al., 1999; Hallmann et al., 2001). Introduction of endophytic bacteria including Rhizobium etli against root-knot nematode

Meloidogyne incognita (Hallmann et al., 2001), Bacillus pumilus and Corynebacterium flavescens against Azospirillum brasilense (Bacilio-Jimenez et al., 2001), Bacillus thuringiensis and Serratia sp. against wilt of tomato caused by Ralstonia solanacearum (Jian et al., 2004), Pseudomonas fluorescens against Fusarium oxysporum wilt of tomato (Dieter and Genevieve, 2005), Pseudomonas and Burkholderia spp. against Basal stem rot in oil palm by G. boninense (Zaiton et al., 2008) have shown significant result in plant disease control.

However, the feasibility of using the endophytic biological control agent (BCA) in greenhouses or the field is determined largely by its formulation, shelf life and delivery techniques (Stephens and Rask, 2000; Albareda et al., 2008; Khavazi et al., 2007). The development of cost effective, user friendly and readily available commercial formulations for beneficial microbes has always been a constraint in sustaining the performance of the BCAs. Formulation of biological control agents for commercial use generally involves the mixing of viable BCAs cells with carrier-based materials in liquid or dry form and nutritional supplements such as glucose to develop fine formulations that not only can stabilize and enhance the growth of BCAs but also convenient for storage and user friendly for field and glasshouse application. Thus, the nature of BCAs, target pathogen and site of action and delivery requirements are the major deciding factors in determining the type of formulation.

Many studies on formulated endophytes for plant protection purpose have been reported including

Pseudomonas fluorescens and Bacillus megaterium for sheath blight of rice (Vidhyasekaran et al., 1997; Wiwattanapatapee et al., 2004 and 2007), B. subtilis and Rhodotorula minuta for mango anthracnose (Patino-Vera et al., 2005; Chung et al., 2007), Streptomyces for damping-off in tomato (Sabaratnam and Traquair, 2002) and plant growth promoting rhizobacteria (PGPR) (Albareda et al., 2008).

However, no formulated BCAs against Ganoderma boninense of oil palm (Elaeis guineensis) have been reported. Burkholderia cepacia (UPMB3) and Pseudomonas aeruginosa (UPMP3), two species of endophytic bacteria previously isolated from roots of healthy oil palm samples were reported to suppress BSR disease of oil palm caused by Ganoderma boninense under controlled environmental conditions (Zaiton et al., 2008). High abundance of such bacteria appeared to influence the incidence of BSR disease by preventing the establishment of G. boninense population in oil palm tissues.

The objectives of this study were to formulate the vegetative biomass of such endophytic bacteria, B. cepacia (UPMB3), P. aeruginosa (UPMP3) and mix culture of UPMB3 and UPMP3 as liquid formulation in LB-based carrier and also as dry formulation in vermiculite and coir dust-based carriers and test the viability of the bacterial formulations in storage.

MATERIALS AND METHODS

Preparation of endophytic bacteria:

Two species of endophytic bacteria, Burkholderia cepacia (UPMB3) and Pseudomonas aeruginosa (UPMP3) that were previously isolated from roots of healthy oil palm samples (Zaiton et al., 2008) were used in this study. To obtain viable cells for formulation, UPMB3 and UPMP3 were grown in liquid culture on Luria broth (LB; g liter-1 distilled water in the following proportions: 10 tryptone, 5 yeast extract, 10 sodium chloride) and harvested after 48 h incubation at 35 and 40oC, respectively on rotary incubator shaker operating at 150 rpm. The 48-h culture of UPMB3 and UPMP3 in LB medium was centrifuged at 5000 rpm for 10 min to obtain the bacterial pellet.

Formulation of endophytic bacteria UPMB3, UPMP3 and mix culture of UPMB3 and UPMP3:

Two types of bacterial formulations were developed, liquid and dry formulation. Endophytic bacterial inoculants that were formulated as liquid form in LB-based carrier was prepared based on Walker et al. (2004) with modifications. 10 g pellet of UPMB3 and UPMP3 was re-suspended separately in 1 L sterile LB as liquid formulation and for the mixture (UPMB3 + UPMP3) equal volume of UPMB3 and UPMP3 suspensions were mixed. The concentration of liquid formulated bacterial products (single and mixture) were adjusted to log 107 cfu mL-1 before storing in bottles and maintained at, 10, 20 and 30oC.

Endophytic bacterial inoculants that were formulated as dry form in vermiculite and coir dust-based carriers were prepared by re-suspending bacterial pellet of UPMB3 and UPMP3 in 1 L sterile LB medium and mixed thoroughly with either 100 g of vermiculite or coir dust carriers. For mixed culture of UPMB3 and UPMP3, equal volume of each bacterial suspension was used. The moisture content of the formulated bacterial products (single and mixture) was adjusted to 50% (w/v) by adding a known volume of sterilized distill water calculated from the moisture content of the carrier before packed into separate packages and stored at three different temperatures, 10, 20 and 30oC.

Stability of endophytic bacteria in various formulations and storage temperatures:

The stability of the bacterial inoculants was determined monthly during storage by suspending 1 g of the formulations in 9 mL of sterile distill water. Serial dilutions were made and 0.1 aliquot plated on selective agar (Burkholderia cepacia selective agar for UPMB3 and King's B agar for UPMP3) and incubated at 28+-2degC. Bacterial colonies that were formed after 48 h incubation, was counted as colony form units (c.f.u) mL-1 and expressed as log cfu g/substrate. The plate count was carried out in triplicates and the final value of c.f.u bacteria was average of three readings. pH of each formulation was also measured at monthly intervals throughout the nine months storage period.

Efficacy of the viable bacterial cells surviving in each formulation during storage was conducted as described by Sabaratnam and Traquair (2002) with modifications. The efficacy of the bacterial cells surviving in each formulation during storage was assessed in vitro at 3-months intervals over a 9-month storage period. Efficacy was measured as the percentage of endophytic bacteria colonies capable of reducing the mycelial growth of G. boninense based on dual culture test. For each formulation, 10-fold serial dilutions were plated onto bacteria selective media (Burkholderia cepacia selective medium and King's B) and incubated at 28degC for 48 h. After 48 h, a mycelial plug cut from 7-day old G. boninense, were placed on the center of agar plate that widely separated with 48-h endophytic bacterial colonies. Plate was sealed and reduction of Ganoderma's mycelial growth was measured after seven days.

The data were later transformed into percentage inhibition of radial growth (PIRG) in relation to the radial growth of G. boninense in the control plate (R1) using the formula:

R1 - R2

PIRG = x 100%

R1

Where by, R1 - Radius of the G. boninense colony in the control plate.

R2 - Radius of the G. boninense colony in the dual culture plate.

The PIRG of the mycelia represents the efficacy of each bacteria and expressed as percentage efficacy of the viable endophytic bacteria.

Experimental design and statistical analysis:

Experiment was repeated twice. Each formulation was replicated five times with four assessment units per replicate. All treatments were arranged in Randomize Complete Block Design (RCBD) with each formulations being blocked with three different incubation temperatures. Data expressed in percentages were transformed by arcsine and analyzed by Analysis of Variance (ANOVA). Treatment means were compared by using Least Significant Difference (LSD) and significant differences were detected at P less than 0.05.

RESULTS AND DISCUSSION

Effect of storage temperature on viability of endophytic bacteria in various formulations:

The stability test showed that UPMB3 and UPMP3 formulated singly or as mixture in dry and liquid formulations and stored at 10 and 20degC had longer shelf-life than those at 30degC. At 10 and 20degC, Luria broth and vermiculite carriers were capable in sustaining the viable cells of endophytic bacteria up to log 106 cfu g-1 for at least 5 months (Fig. 1). The only exception was the coir dust carrier which sustained less number of cells with values of log 104 cfu g-1. There was a slight reduction of viable cells in Luria broth carrier during the sixth month towards the end of assessment with values of log 106 cfu g-1 to log 105 cfu g-1. Vermiculite carrier sustained less number of viable cells compared to Luria broth with values of log 105 cfu g-1 during the sixth month to log 104 cfu g-1 during the ninth month storage.

The viable cells in coir dust carrier suffered the highest reduction with number of viable cells of log 102 cfu g-1 during the final month. The viable cells of UPMB3, UPMP3 and mixture inoculum of both isolates were highly survived in Luria broth and vermiculite at 10 and 20degC probably attributable to the moisture availability surrounding the cells (Beatrice et al., 1991). This was confirmed by other studies when many species of beneficial microbes have longer shelf-life after stored at lower temperatures (Cigdem and Merih, 2005; Walker et al., 2004; Daza et al., 2000).

High water holding capacity (WHC) property of vermiculite could also be the essential characteristic of good carrier (Nakkeeran et al., 2005). Other studies have also shown liquid carrier and vermiculite to be good carrier for several biological control agents including Rhizobium spp. (Weiss et al., 1987), Azospirillum brasilense (Elazar and Yaakov, 1996), Pseudomonas fluorescens (Loccoz et al., 1999) and Pichia anomala (Melin et al., 2006).

At 30degC the number of viable cells in all three formulations declined sharply throughout the experiment (Fig. 2). The number of viable cells detected in vermiculite carrier was slightly higher than in Luria broth and coir dust with values of log 103 cfu g-1 after five months of storage. Only 14% of viable cells were detected in vermiculite, while almost no viable cells were detected in Luria broth and coir dust after nine months. Greater loss of viable cells stored at 30degC could be explained by the desiccation process that happen as a result of prolong exposure to high temperature (Cigdem and Merih, 2005).

Higher temperature promotes bacterial growth that consequently causes the bacteria to produce more wastes. These wastes are toxic and lead to pH changes to the environment surrounding the bacteria (Dearmon et al., 1962). This could explain the high reduction of bacterial viability within coir dust and LB at 30degC. At this temperature, coir dust and LB contributed to the lowest pH, which is highly acidic for UPMB3 and UPMP3 (Table I). Vermiculite managed to stabilize the pH of the micro-environment within range of 4.7 to 6.2 at all temperatures during the nine months of storage period, which is close to the range of optimum pH for bacterial growth (Nakkeeran et al., 2005).

Table I: Effect of various storage temperatures and carriers on pH changes in bacterial formulations

Carrier###Average pH###

###UPMB3###UPMP3###UPMB3 + UPMP3###

Vermiculite at 10degC###5.7###6.2###5.8###

Vermiculite at 20degC###5###6###5###

Vermiculite at 30degC###4.7###5###4.7###

Luria broth at 10degC###4.8###5.3###4.8###

Luria broth at 20degC###4.5###4.4###4.5###

Luria broth at 30degC###3###3###3.4###

Coir dust at 10degC###3.5###3###3.8###

Coir dust at 20degC###3.1###3###3.2###

Coir dust at 30degC###3###3###3.1

Initial pH was 7

Efficacy of viable endophytic bacteria in each formulation during storage:

The efficacy of viable UPMB3 and UPMP3 in all carriers reduced through time and it varied depending on temperatures, carriers and the bacteria itself. At the time of bagging, viable cells of endophytic bacteria extracted from all carriers were effective by causing 100% inhibition to the growth of G. boninense. Generally, formulations stored at 10 and 20degC showed higher percentage of efficacy against G. boninense compared to those at 30degC. According to Zaiton et al. (2008), oil palm seedlings treated with UPMP3 showed the lowest percentage of disease severity of BSR disease assuming this isolate was also effective in inhibiting the growth of G. boninense on culture plate. As expected, UPMP3 was more effective in inhibiting the growth of G. boninense than mix inoculum and UPMB3 during 9-month assessment.

Generally, UPMP3 was more effective in inhibiting the growth of G. boninense followed by mix inoculum and UPMB3 along 9-month assessment (Table II). After three months in storage, UPMP3 incorporated into Luria broth that stored at 10 and 20degC showed the highest percentage of efficacy against G. boninense with respective values of 88 and 80%. UPMP3 incorporated into vermiculite that stored at similar temperatures also highly effective in inhibiting the growth of G. boninense with percentage of efficacy of 85 and 78%, respectively. Mix inoculum extracted from similar carriers and stored at 10 and 20degC showed a slight difference from UPMP3 by causing 80, 78, 78 and 76% inhibition to the growth of Ganoderma. Lower efficacy was showed by UPMB3 in Luria broth and vermiculite stored at similar temperatures with percentage of 80, 75, 74 and 72%, respectively. Overall, coir dust failed to sustain the efficacy of bacterial cells although stored at lower temperatures through out the 9-month storage period.

Lower storage temperatures minimized water loss within the formulations and maintained the efficacy of the bacteria. At 10 and 20degC, UPMP3 in Luria broth and vermiculite carriers showed a slight reduction in efficacy on Ganoderma with respective percentage 70, 69, 69 and 65% although after nine months of storage. The efficacy of mix inoculum in similar carriers reduced to 70, 65, 65 and 63%, respectively. UPMB3 extracted from Luria broth and vermiculite carriers caused lower inhibition to the growth of G. boninense with respective percentage of efficacy of 66, 55, 63 and 60%. As in viability test, the efficacy of UPMB3, UPMP3 and mix inoculum in coir dust was very low even when stored at lower storage temperature of 10 and 20degC.

UPMB3 and UPMP3 formulated singly or as mixture in all carriers and stored at 30degC were the least effective in retarding the mycelial growth of G. boninense concluding this temperature may not be suitable to sustain the effectiveness of endophytic bacteria, while in storage. Result also suggested LB and vermiculite carriers are not only stable to sustain the viability but also efficacy of bacterial viable cells against G. boninense compared to coir dust.

Table II: Efficacy of endophytic bacteria (UPMB3, UPMP3, UPMB3 + UPMP3) in various carriers and storage temperatures

PIRG (%) a###

Storage period (month) b###

Carrier###UPM B3###UPM P3###UPM B3 + UPM P3###

###0###3###6###9###0###3###6###9###0###3###6###9###

Luria broth at 10degC###100###80a###72a###66a###100###88a###81a###70a###100###80a###78a###70a###

Luria broth at 20degC###100###74b###68a###55b###100###80a###78a###69a###100###78a###70a###65a###

Luria broth at 30degC###100###40c###30c###18d###100###50c###40b###25c###100###50b###40c###25c###

Vermiculite at 10degC###100###75b###70a###63a###100###85a###79a###69a###100###78a###75a###65a###

Vermiculite at 20degC###100###72b###68a###60a###100###78a###76a###65a###100###76a###70a###63a###

Vermiculite at 30degC###100###45c###40b###32c###100###50c###43b###40b###100###55b###48b###40b###

Coir dust at 10degC###100###48c###35b###22d###100###65b###45b###32b###100###55b###35c###22c###

Coir dust at 20degC###100###50c###33c###19d###100###51c###30c###15d###100###50b###33cd###19c###

Coir dust at 30degC###100###20d###17d###9e###100###30d###20d###9d###100###30c###20d###9d###

The efficacy is expressed as a percentage of inhibition in radial growth of G. boninense and was recorded in every 3-months intervals over a 9-month period

The data were transformed by arcsine and values with similar letter within a column are not significantly different at P less than 0.05 according to Least Significant Difference (LSD)

growth of G. boninense on culture plate. As expected, UPMP3 was more effective in inhibiting the growth of G. boninense than mix inoculum and UPMB3 during 9-month assessment.

Generally, UPMP3 was more effective in inhibiting the growth of G. boninense followed by mix inoculum and UPMB3 along 9-month assessment (Table II). After three months in storage, UPMP3 incorporated into Luria broth that stored at 10 and 20degC showed the highest percentage of efficacy against G. boninense with respective values of 88 and 80%. UPMP3 incorporated into vermiculite that stored at similar temperatures also highly effective in inhibiting the growth of G. boninense with percentage of efficacy of 85 and 78%, respectively. Mix inoculum extracted from similar carriers and stored at 10 and 20degC showed a slight difference from UPMP3 by causing 80, 78, 78 and 76% inhibition to the growth of Ganoderma. Lower efficacy was showed by UPMB3 in Luria broth and vermiculite stored at similar temperatures with percentage of 80, 75, 74 and 72%, respectively.

Overall, coir dust failed to sustain the efficacy of bacterial cells although stored at lower temperatres through out the 9-month storage period.

Lower storage temperatures minimized water loss within the formulations and maintained the efficacy of the bacteria. At 10 and 20degC, UPMP3 in Luria broth and vermiculite carriers showed a slight reduction in efficacy on Ganoderma with respective percentage 70, 69, 69 and 65% although after nine months of storage. The efficacy of mix inoculum in similar carriers reduced to 70, 65, 65 and 63%, respectively. UPMB3 extracted from Luria broth and vermiculite carriers caused lower inhibition to the growth of G. boninense with respective percentage of efficacy of 66, 55, 63 and 60%. As in viability test, the efficacy of UPMB3, UPMP3 and mix inoculum in coir dust was very low even when stored at lower storage temperature of 10 and 20degC.

UPMB3 and UPMP3 formulated singly or as mixture in all carriers and stored at 30degC were the least effective in retarding the mycelial growth of G. boninense concluding this temperature may not be suitable to sustain the etiveness of endophytic bacteria, while in storage. Result also suggested LB and vermiculite carriers are not only stable to sustain the viability but also efficacy of bacterial viable cells against G. boninense compared to coir dust.

CONCLUSION

There were differences in the shelf-life and efficacy of the bacteria for each formulation. Formulations of UPMB3 and UPMP3 either singly or as mixture stored at 10 and 20degC showed better shelf-life and percentage of efficacy than those stored at 30degC. Least reduction of viability and efficacy of the viable cells in Luria broth and vermiculite-based formulations suggested that both carriers and storage temperatures were excellent for extending shelf-life of UPMB3 and UPMP3 during storage. Least reduction of bacteria viable cells could be attributed to the properties of the carrier materials.

Luria broth and vermiculite managed to prolonged the shelf-life and sustain the efficacy of the bacteria suggesting both carriers were stable for the bacteria. Coir dust failed to sustain the viability and efficacy of the bacteria even at lower storage temperatures showing that the carrier may not be stable to maintain the performance of the bacteria in the field. Although Luria broth was an excellent formulation for endophytic bacteria, it is probably not practical for large scale use in the field considering simpler and cost effective preparation technology by using vermiculite-based formulation.

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Institute of Tropical Agriculture, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, D.E., Malaysia Corresponding E-mail: sariahm@putra.upm.edu.my
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Author:Bazilah, A.B. Ili; Sariah, M.; Abidin, M.A. Zainal; Yasmeen , S.
Publication:International Journal of Agriculture and Biology
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Geographic Code:9MALA
Date:Apr 30, 2011
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