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Isolation of potential fluorescent pseudomonads from Kuini (Mangifera odorata) planted soil and their potential as biofertilizer

Pseudomonas sp. are known to be good Plant Growth Promoting Rhizobacteria (PGPR). In this study, Pseudomonas sp. were isolated from soil planted with kuini (Mangifera odorata) using soil dilution method and spread onto King’s B media. Five isolates of Pseudomonas sp. were observed to give promising results in the phytohormone and antimicrobial test conducted. These isolates are Pseudomonas sp. isolate K24pf, K29pf, K32pf, K33pf and K37pf. From the 5 potential isolates, Pseudomonas sp. isolate K29pf was chosen because it showed potential activity in producing marked amounts of Indole-3-Acetic Acid and gibberellic acid. Pseudomonas sp. isolate K29pf also produced antimicrobial activities towards Ralstonia solanacearum, Erwinia caratovora, Erwinia mallotivora and Colletotrichum gloeosporioides. Seed germination test showed that Pseudomonas sp. isolate K29pf was able to promote approximately 90% growth of Brassica chinensis seeds. Pot trial conducted showed that Treatment 3 (+OF+PGPR) was able to increase Brassica chinensis root by 36.5% and 28.4% of its biomass compared to treatment using Treatment 1 (+OF).


Introduction
Soil is the most complex and heterogeneous of natural system composed of solid, liquid and gaseous phase. Soil is the habitat for many beneficial and pathogenic microorganisms. Bacteria inhabiting the rhizosphere can influence plant growth by producing certain bioactive compounds beneficial to the plants such as phytohormones and antibiotics. 1,2 These bacterial groups are known as Plant Growth Promoting Rhizobacteria (PGPR). 3 Plant Growth Promoting Rhizobacteria include the species in the genera of Bacillus, Pseudomonas, Azotobacter and Rhizobium. An effective PGPR should have these characteristics; ability of root colonization, phytostimulator and biocontrol agent against certain phytopathogens. 3 Pseudomonas sp. has been known as one of the most important members of PGPRs that showed all these three characteristics of PGPR. 3 Wahyudi et al. 4 and Vasanthakumar and McManus, 5 showed that some Pseudomonas sp. such as Pseudomonas gingeri and Peudomonas alcaligenes has the ability to produce Indole Acetic Acid (IAA), which is important for the root growth. 5 Studies by Pandey and Desai 6 showed that Pseudomonas sp. also has the ability to produce gibberellic acid which is important to promote the growth and of yield of plants. Dey et al. 7 showed that Pseudomonas sp. isolated were effective to produce phyto-activities toward phytopathogens such as Aspergillus niger and A. flavus. In another study, Wahyudi et al. 4 showed that Pseudomonas sp. isolated form rhizosphere of soybean field have the ability to inhibit the growth of Fusarium oxysporum, Rhizoctonia solani and Sclerotium rolfsii, the causal agent of root rot disease.
In this study, our objectives are to isolate pseudomonas from kuini (Mangifera ordorata) planted soil, characterize their potential bioactivities and analyzed their efficacy on a pot trial. As this is the pioneer work regarding isolation of PGPR from Mangifera ordorata in Malaysia, we will focus on the best known PGPR which is the Pseudomonas sp.

Isolation of soil microbes
Pseudomonas sp. were isolated from kuini (Mangifera ordorata) planted soil collected from MARDI Sintok, Kedah, Malaysia (Latitude: 6°29'6.98" Longitude: 100°29'2.27"). Soil samples were collected at the depth of 15 cm from the rhizosphere. These soils were from the soil type of Siri Napai a type of laterite soil. The collected soil samples were kept in ice box when transporting back to MARDI Headquater located at Serdang, Selangor, Malaysia. The samples were crushed into smaller pieces and were processed by diluting 10 g of the soil sample in 100 ml of sterile distilled water (sH 2 O). The soil suspension was vigorously agitated at 200 rpm for 1 h. After 1 h, 150 µl of the soil suspension was pipetted onto King's B agar. 8 Agar plates were then incubated at 28 ± 2 o C for 48 h. After that, the agar plates were exposed to UV light at 365 nm for selection of any fluorescent colonies. The emerging colonies were selected and streaked onto a fresh new agar plate for further usage.

Screening of Indole-3-Acetic Acid
For Indole-3-Acetic Acid (IAA) estimation, method used by Iqbal and Hasnain, 9 was followed with a little modification. Fluorescent pseudomonads isolated were incubated at 30 ± 2 o C for 48 hrs. The density of the culture broths were then adjusted to 10 6 cfu/mL for each test isolates. Two mililiter of the culture broth was pipetted into a new Eppendorf tube and centrifuged at 10,000 rpm for 30 min. After centrifugation, 1 ml of the supernatant was mixed with 2 mL of Salkowski's reagent in a test tube and allowed to stand in dark for 30 min. Development of pink color indicated IAA production and the amount of IAA was measured using Nanodrop spectrophotometer at wavelength of 530 nm. A standard curve was constructed using synthetic IAA (Sigma, Seelze, Germany) with purity of 99.3% to determine the amount of IAA secreted by each fluorescent pseudomonads.

Screening of gibberellic acid
Screening of gibberellic acid (GA 3 ) producing fluorescent pseudomonad was done according to method proposed by Panday and Desai. 6 Standard curve was constructed using synthetic GA 3 (Sigma) and was measured at 254 nm using Nanodrop spectrophotometer.

Screening for nitrogen fixer and phosphate solubilizer activity
For the screening of nitrogen fixer from fluorescent pseudomonads, Burk's Nitrogen free medium was used (10.0 g glucose, 0.41 g KH 2 PO 4 , 0.52 g K 2 HPO 4 , 0.05 g Na 2 SO 4 , 0.2 g CaCl 2 , 0.1 g MgSO 4 7H2O, 0.005 g FeSO 4 7H 2 O, 0.0025 g Na 2 MoO 4 2H 2 O and 15 g agar-agar in 1 L of sdH 2 O). 10 The pH of the medium was adjusted to 7.0±0.2 prior autoclaving at 121 o C for 15 min. The fluorescent pseudomonads were screened for their phosphate solubilizing ability using Pikovskaya's agar (10.0 g glucose, 5 g Ca 3 (PO 4 ) 2 , 0.5 g yeast extract, 0.5 g (NH 4 ) 2 SO 4 ; 0.2 g NaCl; 0.2 g KCl; 0.1 g MgSO 4 , 0.005 g FeSO 4 7H 2 O MnSO 4 H2O 0.001 g and 15.0 g agar-agar in 1 L of sdH 2 O). 11 The pH was adjusted to 7.0±0.2 autoclaving at 121 o C for 15 min. Isolates of fluorescent pseudomonads were then grown on the each of the plates in triplicates and halo zones formed indicate the positive activity for the tested bacteria.

Screening of antimicrobial activities
Screening of anitimicrobial activities were conducted based on the culture overlay method. The phyto-pathogens used were Ralstonia solanacearum (MMCC10019), Erwinia caratovora (MMCC10018), Erwinia mallotivora (MMCC10032), Colletotrichum capsicii (MMCC20012) and Colletotrichum gloeosporioides (MMCC20013). Anti-bacterial test was conducted by lawning the test pathogenic bacteria onto the media plate and the agar block with pseudomonas was overlayed on top of the phytopathogens. For anti-fungal test, a dual cultures agar test was performed, where the pathogenic fungi was inoculated at the center of the agar plate and flanked by tested fluorescent pseudomonads on left and right side of the agar. Clear zone produced indicates the ability to secrete antimicrobial compound against the tested pathogens. Antimicrobial activity was evaluated after 24 h of incubating and the clear zones were rated using the rating scheme suggested by Baniasadi et al. 12 from the modification of rating by Lee and Hwang, 13 whereby; 0-4 mm= no inhibition, 5-9 mm= weak inhibition, 10-19 mm= moderate inhibition and ≥20 mm= strong inhibition.

Identification of beneficial microbes
Isolation of DNA was conducted using Qiagen DNeasy DNA extraction protocol as suggested by the manufacturer. PCR parameters and conditions used for this study followed the optimization made by Jeffrey 14 with the modification on the primers used. The 16S rRNA gene region was amplified using universal primers F8 (AGA GTT TGA TCM TGG CTC) and rP2 (ACG GCT ACC TTG TTA CGA CTT). 15 PCR products obtained were subjected to purification using Vivantis GF-1 Gel DNA recovery kit following the protocol provided by the manufacturer. The purified PCR products were sent for sequencing at First Base Laboratories Sdn. Bhd., Selangor. Results obtained from the sequencing were then compared with the databases from National Center for Biotechnology Information.

Germination test
Germination test was conducted using 20 seeds of Brassica chinensis var parachinens for each treatment. Mean of 20 seeds weighted at 0.0464±0.002 g. The seeds were pre-soaked with 10 ml of selected fluorescent pseudomonads culture broths for 30 mins while control seeds were soaked with 10 ml of water for the same duration. After 30 min, seeds from each treatment were dried in laminar flow for 2 hrs before being transferred onto filter paper placed in the 90×15 mm petri dishes. The filter papers were then wetted with 5 ml of fluorescent pseudomonads broths while sterile distilled water of 5 ml was used as control for 5 days. Number of seed germinated in 5 days were counted and represented as percentage of germination. Tests were conducted in room temperature 28±2 o C and all tests were conducted in triplicates.

Pot trial
Soil used for the planting of Brassica chinensis var parachinens was cocoa peat obtained from local market. Three seeds of Brassica chinensis var parachinens were planted in each pot. A total 50 ml broth containing Pseudonomas sp. strain K29pf with the concentration of 10 6 were inoculated onto the soil for each pot in treatment 2 and 3, while 3g of organic fertilizer was added around the plant in each pot in treatment 1 and 3 (Table 1). No artificial lighting were provided to induce the plant growth. The plants were watered manually every morning and evening during the experiment. Temperature was recorded twice a day in the morning (around time 0900) and in the evening (around time 1700). The treatments used are shown in Table 1.
Treatments were applied twice throughout the experiment on 1 st and 15 th day after planting. B. chinensis var parachinens was harvested on day 30 after planting. The fresh weight, dry weight and root length of the plants for each treatment were recorded. The tests were conducted with 5 replicates for each treatment and in a randomized complete block design method ( Table 2).   Fresh weight of the plants were taken immediately after harvesting. While the dry weight of the plants were taken after oven drying at 55 o C overnight. The dried plants were place in a ziplock bag once it is removed from the oven to prevent the plant from contacting with the surrounding moisture. The dry weight was determine by subtracting the weight of the dry plant with the ziplock bag with the emptied ziplock bag.

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Dry weight (g) = (weight of dried plant in ziplock bag) -(weight of emptied ziplock bag).

Statistical data analysis
All data for fresh plant weight, dry plant weight and root elongation was done using one way analysis of variance was done using SAS 9.3 software. The Tukey's honest significant difference at P=0.05 was used to determine the significance among the samples.

Isolation of soil fluorescent pseudomonads and screening of IAA and GA 3 activities
A total of 25 Pseudomonas sp. was isolated soil of kuini plot from MARDI Sintok. The average colony forming unit per gram soil (cfu/g) for Pseudomonas sp. isolated was 7.3×10 5 . We observed that 100% of the fluorescent pseudomonads presented the ability to produce IAA but only 20% showed the ability to produce GA 3 . The results are shown in Table 3.

Screening of nitrogen fixation, phosphate solubilization, antimicrobial activities and identification of fluorescent pseudomonads
Secretion of bioactivities by microbes was known to be an important characteristic for the microbes to stay viable in their ecosystem. From the total of 25 fluorescent pseudomonads isolated, 60% of the Pseudomonas sp. showed antimicrobial activity towards Ralstonia solanacearum (MMCC10019), 20% for Erwinia caratovora (MMCC10018), 18% towards Erwinia mallotivora (MMCC10032), 4% Colletotrichum capsicii (MMCC20012) and 8% for Colletotrichum gloeosporioides (MMCC20013). Antimicrobial activity of selected fluorescent pseudomonads were tabulated in Table 3.
Screening for phosphate solubilizing and nitrogen fixation ability of 25 isolates of fluorescent pseudomonads isolated showed that only 8% of the fluorescent pseudomonads isolates gave positive reaction to the phosphate solubilizing media used, while no nitrogen fixation activities was observed for all 25 isolates of fluorescent pseudomonads ( Table 3). The 5 poten-   Table 4.

Seed germination test
Microbial benefit of seed germination can be an index of its potential benefit for agricultural, including propagation. Here, Pseudomonas sp. strain K29pf allowed a 90% germination of the seeds while Pseudomonas putida strain K33pf and Pseudomonas sp. K37pf had the lowest seeds germination rate of 70% ( Table 5).

Efficacy of Pseudomonas sp. strain K29pf on pot trial
The mean temperature recorded of the glass house was 27 o C during the morning and 34 o C during the evening throughout the experiment. From all the test conducted, Pseudomonas sp. strain K29pf was chosen for the pot trial. It was observed that Treatment 3 gave the highest wet weight with the average wet weight of 75.6 g whereas Treatment 4 gave the lowest average wet weight of 28.5 g ( Table 6). The observation on the root length indicated that Treatment 3 stimulate the longest roots elongation with the average of 13.1 cm. The results also indicated that Treatment 2 stimulated the elongation of roots, however does not gave significant increase in the weight of Brassica chinensis var parachinens compared to Treatment 1 ( Table 6).

Discussion
Indole-3-Acetic Acid which is an auxin, can stimulate cell elongation and division and also promote plant growth and development. However, high concentrations of IAA will cause toxicity to the plant such prevention of shoot and root growth. 16 Study done by Rodrigues et al. 17 indicated that 80% of the Pseudomonas sp. isolated from sugarcane showed the ability to produce IAA. In our study, all the 25 isolates of fluorescent pseudomonads isolated were observed to produce IAA. From this, we observed that the highest IAA produced was only 8.52 µg/ml which was considered low compared to IAA produced by Pseudomonas putida UB1 (591.8 µg/ml) is a study conducted by Bharucha et al. 18 This observation indicated that the IAA reading obtained in this study were significantly low. However, study done by Dagnaw et al. 19 stated that it is normal to obtained plant growth promoting rhizobacteria with low production of IAA due to the differences in species, strains and also the influence of different culturing condition.
Gibberellin acid is an endogenous hormone that function as plant growth regulators and influencing the developmental process of plants such as stem elongation, seed germination and sex expression. In this study, we observed that Pseudomonas aeruginosa strain K32pf produced the highest GA 3 (91 µg/ml). This was higher compare to GA 3 produced by Pseudomonas isolate K8 (70 µg/ml) isolated by Desai. 20 However comparison of GA 3 production from Pseudomonas sp. done by Sharma et al., 21 GA 3 production by Pseudomonas sp. isolated in this study were very low. Bottini et al., 22 noted that production of GA 3 might be influenced by factors such as the media used, the supply of O 2 and N.
In this study, Pseudomonas sp. isolate K29pf showed the most potential with the 15.0, 20.3 and 21.5 mm respectively of clear zones produced towards Ralstonia solanacearum, Erwinia caratovora and Erwinia mallotivora respectively. In a study conducted by Zhou et al., 23 Pseudomonas brassicacearum J12 was observed to inhibit the growth of Ralstonia solanacearum. Pseudomonas putida ICCF 391 on the other hand showed antagonistic activity against E. carotovora. 24 Soare et al., 24 mention that E. caratovora have the ability to produce bacteriocine, a compound that enable them to grow and inhibit other microbial growth. With the compound, E. carotovora can be seen as a microbes that are resistant to many other microbes. The formation of clear zone by only Pseudomonas sp. strain K29pf showed that this Pseudomonas sp. strain K29pf might have the ability to neutralize the bacteriocine produced by E. carotovora. 24 Apart from producing antibacterial activity, Pseudomonas sp. strain K29pf also showed its ability to inhibit the growth of Colletotrichum gloeosporioides. These findings suggested that Pseudomonas sp. isolate K29pf has the potential to be develop into a beneficial biocontrol agent due to its effectiveness and board range of antimicrobial spectrum.
The ability of Pseudomonas sp. to fix nitrogen are considered to be rare. 25 Indeed, in the study conducted by Lin et al., 25 Pseudomonas stutzeri was the only known Pseudomonas sp. that fixed nitrogen. Unlike its rareness in fixing nitrogen, Pseudomonas sp. was well known for its ability to solubilize phosphate. In a study conducted by Dipak and Sankar, 26 Pseudomonas sp. was noted as a good phosphate solubilizer and most Pseudomonas sp. have the ability to solubilize phosphate [26][27][28] from the environment. However, in the present study we only isolated 2 pseudomonas Pseudomonas putida 98 K37pf

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Pseudomonas sp. 99   with phosphate solubilizing properties. This could be due to the fact that fluorescent pseudomonads in this study were isolated form laterite soil which is well known for its low content of phosphorous. A study by Tiwari and Singh 29 indicated that Triticum aestivum (L) and Zea mays (L) seeds were inhibited by Pseudomonas aeruginosa. The infected seeds shown the character of soft and was covered by biofilm of the bacteria. However, in this study the seeds that did not germinate showed none of these attributes. However, Tabatabaei et al., 30 stated that IAA levels, unknown metabolites produced by the bacteria and stress induced by the bacteria might also be one of the reason that seeds germination was inhibited.
Application of the potential microbes to the real world is away to test it functionality after completion of lab tests. From the study, +PGPR increased the weight of Brassica chinensis var parachinens but did not surpassed the wet weight of +OF. This showed that by adding the Pseudomonas sp. alone, the microbes might not be able to provide the plants with the required nutrients from the soil for its growth. In +OF+PGPR, we saw an increased of 20% in the plant biomass compared to +OF only. This showed that combination of organic fertilizer and Pseudomonas sp. strain K29pf provide the plant with the needed nutrients and thus boosting the weight increment. This argument above is supported by finding from Antonius and Agustiyani, 31 where they noted that application of beneficial microbes to the compost and application thereafter is the effective method to increase the biomass of the plants. Previous studies show that a biofertilizer prepared by combining PGPR with composts enhance growth-promoting effects and bio-control of the microbes towards the plants. 32 Saia et al. 33 also observed that soil under fertilized condition with addition of PGPR increased the density of bacteria on the rhizoplane and also the N concentration of the plant.

Conclusions
Pseudomonas sp. are well known for their PGPR ability. In this study, Pseudomonas sp. K29pf isolated from kuini (Mangifera odorata) soil had showed its potential to produce phytohormones such as Indole-3-Acetic Acid and gibberellic acid, antimicrobial activities against Ralstonia solanacearum, Erwinia caratovora and Erwinia mallotivora and Colletotrichum gloeosporioides and also the ability to solubilize phosphate. This shows that Pseudomonas sp. K29pf can act as a good source of PGPR; further studies are needed to fully understand the efficacy of this bacteria on field.