Philippine Journal of Systematic Biology Vol. III (June 2009) ISOLATION AND CHARACTERIZATION OF PINK- PIGMENTED, FACULTATIVE METHYLOTROPHIC (PPFM) BACTERIA FROM LEAVES OF NEEM, Azadirachta indica A. Juss. RINKI KUMAR AND ANTHONY C. LEE Biology Department/Center for Natural Sciences and Environmental Research, De La Salle University-Manila 2401 Taft Ave., Manila, Philippines ABSTRACT A total of twenty isolates of pink-pigmented, facultative methylotrophic bacteria were obtained from the leaves of neem. All isolates exhibited pink to orange-pink pigmentation, entire margin, round colonies with a smooth glistening surface, and convex elevation. Most of the colonies were opaque with butyrous consistency. Staining revealed rod to coccobacilli shaped, Gram negative cells, containing poly-β-hydroxybutyrate granules. Biochemical analyses showed that all were catalase positive; majority of them were positive for citrate utilization, urease and oxidase activities but were negative for amylase activity. They can be cultivated on ammonium mineral salt (AMS) agar with methanol, glycerol peptone agar (GPA) and tryptic soy agar (TSA) with variations in colonial morphology. Based on the observed characteristics, the isolates obtained belong to the genus Methylobacterium. Keywords: leaf bacteria, PPFM bacteria, Methylobacterium INTRODUCTION Pink-pigmented, facultative methylotrophic (PPFM) bacteria are an interesting group of prokaryotic eubacteria. Their ability to metabolize C-1 compounds like methanol and a variety of organic compounds makes them highly ubiquitous in distribution. Moreover, their distinctive pink pigmentation due to carotenoids; render them to be tolerant to extreme light conditions and radiations. These features could explain their occurrence in diverse ecological systems such as soil, plants, air, water and even humans. (Anesti et al., 2005; Rice et al., 2000; Barbeau, 1996). PPFM bacteria have been isolated from a variety of plants, ranging from mosses and ferns to seed producing plants (Lee, 2007). However, Hirano and Upper (2000) reported that some plants like olive trees may not harbor PPFM bacteria. It is worthwhile to note that the relationship of PPFM bacteria to plants is not completely understood. However, there are studies to show that Philippine Journal of Systematic Biology Vol. III, No.1 (June 2009) PPFM bacteria promote plant growth and development by generating vitamins, phytohormones as well as supply nitrogen to the plant through diazotrophy (Madhaiyan et al., 2005; Van Aken et al., 2004; Koenig et al., 2002; Basile et al.,1985) Neem (Azadirachta indica A.Juss.), a relative of the mahogany, is a native of South East Asian countries. Various parts of the tree have been used to effectively treat or control a wide range of diseases, thus called a “wonder plant”. The antibacterial properties are attributed to the presence of substances like azadirachtin, salannin, meliantriol, and nimbin. As such, neem extracts were found effective against the multi-drug-resistant Vibrio cholerae (Thakurta et al., 2007), pathogenic bacteria of fish (Das et al.,1999), Propionibacterium acnes (Jain and Basal, 2003) and as a molluscicide for different kinds of snails (Ebenso, 2004). Its biodegradable nature makes azadirachtin a potential eco-friendly biopesticide. Various studies done on neem prove its economic value for potential drugs, pesticides and remedies. However, no studies have been done to explore the presence of PPFM bacteria in the neem tree (Azadirachta indica A.Juss.). While neem is well known for its antimicrobial and insecticidal properties, it is worthwhile to investigate whether this plant harbors PPFM bacteria. Hence, this study determined the presence of PPFM bacteria in the leaves of neem. METHODOLOGY PPFM Bacterial Isolation A neem tree within a household in Dasmariñas Village, Makati City, Philippines, served as source of leaf samples. The imprint method of Holland et al. (2000), using ammonium mineral salt (AMS) agar with 0.5% methanol, was employed to isolate PPFM bacteria from the neem leaves. Leaf samples were washed with sterile water to remove dirt and soil that may adhere to the surface. A total of fifteen leaf imprints were done. Inoculated plates were incubated at 25°C for one week. Observation was done daily from the time of inoculation. Pink colonies that appeared on the plates were then picked and re-streaked on fresh media, until pure cultures were obtained. Pure isolates were then maintained in slants at 4oC. These isolates are currently kept at the DLSU-Microbiology Laboratory. Characterization of the Bacterial Isolates Colonial morphology of the isolates was described after growing them on glycerol peptone agar (GPA) after one week of incubation at 25oC. Isolates were also streaked on Tryptic Soy Agar (TSA) to check their ability to grow in the said medium. Microscopic morphology ws described after performing Gram, background and polyβ-hydroxybutyrate (PHB) staining. All of the isolates were subjected to the following biochemical tests: oxidase, catalase, urease, citrate utilization and starch hydrolysis tests. 9 Philippine Journal of Systematic Biology Vol. III, No.1 (June 2009) RESULTS AND DISCUSSION Out of the fifteen leaf imprints, twenty (20) PPFM bacterial isolates were obtained in this study. Pink colonies were observed by the 6th to 7th day of incubation. Other bacterial and fungal colonies also appeared during the one week observation period. This suggests that a plethora of microorganisms may reside in the leaves of neem. The twenty isolates were categorized into seven groups based on their morphological and biochemical characteristics. Table 1 summarizes the phenotypic characteristics of the PPFM bacterial isolates. The colonial morphology of the isolates in GPA was similar in terms of shape, margin, elevation and optical density. They possessed round, raised, smooth margined and transluscent colonies. Variations were observed in terms of intensity of pigmentation, size and consistency of the colonies as shown in Figure 1. Based on these morphological differences, the isolates were classified into seven colonial types (A-G). Variations in the colonial morphology among PPFM bacteria isolated from various sources were also observed in previous studies (Jang and Lee 2008; Lo and Lee, 2007; Carvajal et al.,2006). Among the media tested, GPA seemed to be a better culture medium compared to AMS and TSA. It was observed that colonies of PPFM bacteria start to appear around three days after inoculation in GPA while it took a week before growth was observed in AMS. This could be due to the fact that AMS is a minimal medium, while GPA is an enriched medium. Faster and more luxuriant growth is expected when bacteria are grown in enriched medium. Nonetheless, there are isolates that showed little or no growth in TSA. This observation was consistent with the report of Carvajal et al. (2006). Examination of microscopic morphology revealed that all isolates yielded Gram negative rods to cocco-bacilli. Figure 2 shows the microscopic appearance of representative PPFM bacterial isolates. The observed microscopic appearance is similar with the description of PPFM bacteria by Green (2001). Moreover, it was observed that all isolates showed PHB granules. Studies by Corpel et al. (1986) indicate occurrence of poly-β- hydroxybutyrate and polyphosphate bodies in all the strains of PPFM they isolated from plant surfaces. The different carbon sources, such as glycerol, methanol and formate are used to form these inclusion bodies, which may serve as nutrient reserves. Biochemical analyses of the bacterial isolates revealed that all of them were catalase and urease positive. Majority of them were positive for oxidase and amylase activities and have the ability to utilize citrate as alternative carbon source. Variations however, were observed in terms of intensity of these activities as indicated by differences in the color change of chemical indicators of the test media used. Extension of the incubation period for one to 10 Philippine Journal of Systematic Biology Vol. III, No.1 (June 2009) two weeks was done in order to observe the enzymatic activities in some isolates. Previous reports (Jang and Lee, 2008; Idris et al. 2006; Gallego et al., 2005; Madhaiyan et al, 2005; Jourand et al., 2004) on the biochemical properties of PPFM bacteria support these findings. The biochemical features of these isolates may help PPFM bacteria thrive in the leaves of plant (Holland and Pollaco, 1994). During the course of the study, it was noted that some isolates were able to grow at low temperatures, while incubating the isolates at higher temperature tend to slow down their growth. This observation was similar to the previous studies by Jang and Lee (2008) and Carvajal et al.,(2006). They noted that some PPFM bacterial strains are psychrotropic. It is worthwhile to note that some isolates, although were derived from the same leaf imprint, vary in terms of phenotypic characteristics. This suggests that there is diversity of PPFM bacterial strains residing within a leaf of the plant. CONCLUSION AND RECOMMENDATIONS The study demonstrated the occurrence of PPFM in the leaves of the neem tree. Based on the phenotypic characteristics of the isolates, they can be assigned to the genus Methylobacterium based on the minimum criteria set by Green (2001). Molecular techniques, such as, 16S rDNA sequence analysis and multi-locus sequence typing (MLST), may be employed to further identify the isolates up to the species level. Variations on the morphological and biochemical properties of the different isolates underscore the diversity of PPFM bacterial strains residing the leaves of the plant. As neem is known to have antimicrobial properties, it is interesting to note that this plant harbor PPFM bacteria. The ability of these bacteria to thrive in this plant therefore necessitates further investigation. More studies are required to explain the association of PPFM bacteria with the neem leaves. Furthermore, it is recommended that that other plant parts be explored for the presence of these ubiquitous bacteria. ACKNOWLEDGEMENT The authors are sincerely thankful to the Biology Department of De La Salle University, for providing the necessary supplies and equipment for the experimentation; Mr. Ramonito Oroceo and Mr. Giovanni Perez, the lab technicians in the DLSU, Biology Department, for their kind assistance during the study; Dr. Emelina Mandia and Dr. Maribel Esperanza Agoo for their technical advise and to Mr. Raj Kumar, Ms.Neha Kumar and the Kumar Family for their unwavering support. Accomplishment of this research was possible due to the immense blessings of God, Family and Friends. 11 Philippine Journal of Systematic Biology Vol. III, No.1 (June 2009) LITERATURE CITED Anesti V., McDonald I.R., Ramaswamy M., William G. W, Donovan P. K. and, A P. Wood. 2005. Isolation and molecular detection of methylotrophic bacteria occurring in the human mouth. 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Molluscicidal effects of neem (Azadirachta indica) extracts on edible tropical land snails. Dept of Animal Science, University of Uyo, PMB 1017, Uyo, Nigeria. Pest Management Science.60 (2): 178-182. Gallego V. Ventosa, M.T., Antonio G. 2006. Methylobacterium adhavesium sp.nov., a methylotrophic bacterium isolated from drinking water, Int J Syst Evol Microbiol. 56: 339-342. Green, P.N. 2001. The genus Methylobacterium, In The Prokaryotes ed. by M. Dwokin. Release 3.5, 1994-2004, Springer –Verlag, New York, LLC. Available at: http://141.150.157.117:8080/prokPUB/chaprender/jsp /showchap.jsp?chapnum=302 Hirano S. S. and C.D. Upper. 2000. Bacteria in the leaf ecosystem with emphasis on Pseudomonas syringae a pathogen, ice nucleus, and epiphyte. Microbiology and Molecular Biology Reviews 3: 624-653. Holland M.A., Davis R., Moffitt S., O’ Laughlin K., , Peach D. Susan S., Wimbrow L. and B. Tayman. 2000. Using “leaf prints” to investigate a common bacterium. American Biology Teacher 62(2):128-131. Holland, M.A. and J.C. Polacco. 1994. PPFM’s and other covert contaminants: Is there more to plant physiology than just plant? Annual Rev. of Plant Physiology and Plant Molecular Biology 45:197-209. 12 Philippine Journal of Systematic Biology Vol. III, No.1 (June 2009) Jain A. and E. Basal. 2003. Inhibition of Propionibacterium acnes-induced mediators of inflammation by Indian herbs. Phytomedicine; 10: P34-38. Jang S.B. and Lee A.C. 2008. Phentoypic characterization of pink pigmented facultative methylotrophic bacteria from soil exposed to vehicular soot. Philippine Journal of Systematic Biology, 2(1): 32-39. Jourand, P., Giraud E., Bena G., Sy A., Willems A., Gillis M., Dreyfus B. and P. de Lajudie. 2004. Methylobacterium nodulans sp. nov., for a group of aerobic, facultatively methylotrophic, legume root-nodule forming and nitrogen fixing bacteria. Int. J. Syst. Evol Microbiol 54: 2269-2273. Lee, A.C. 2007. Pink pigmented facultative methylotrophic bacteria: common yet unexplored locally. Philippine Journal of Systematic Biology; 1(1): 61-72. Lo, J.M. and A.C. Lee. 2007. Phenotypic characterization of air-borne pink pigmented facultative methylotrophic bacteria from a high vehicular traffic density environment in Manila, Philippines. The Philippine Scientist 44:25-34. Koenig RL, Morris RO, and J.C. Polacco. 2002. tRNA is the source of low- level trans-zeatin production in Methylobacterium spp. Journal of Bacteriology; 184(7): 1832-1842. Madhaiyan M., Poonguzhali S., Lee HS., Hari K., Sundaram S.P., and Sal T.M. 2005. Pink-pigmented facultative methylotrophic bacteria accelerate germination, growth and yield of sugarcane clone Co86032 (Saccharum officinarum L.). Biology and Fertility of Soils; 41(5): 350-358. Maliti, Charles M., Basile, Dominick V., Corpe, William A, 2005. Effects of Methylobacterium spp. strains on rice Oryza sativa L. callus induction, plantlet regeneration, and seedlings growth in vitro. Journal of the Torrey Botanical Society 132(2): 355-367. Thakurta P., Bowmik P., Mukherjee S., Hajra TK., Patra A. and P.K. Bag. 2007. Antibacterial, antisecretory and antihemorragic activity of Azadirachta indica used to treat cholera and diarrhea in India. J. of Ethnopharmacol. 111(3):607-611. Trotsenko Y.A., Ivanova E. G., and N.V. Doronina .N. V. 2004. Aerobic Methylotrophic Bacteria as Phytosymbionts. Journal of Microbiology; 70 (6): 623-632. Van Aken B., Peres C.M., Doty S L., Yoon J. M., Schnoor J. L. 2004. Methylobacterium populi sp. nov., a novel aerobic, pink-pigmented, facultatively methylotrophic, methane-utilizing bacterium isolated from poplar trees (Populus deltoidesxnigra DN34). Int J Syst Evol Microbiol; 54: 1191-1196 13 Philippine Journal of Systematic Biology Vol. III, No.1 (June 2009) Table 1. PPFM isolates grouped based on similarities in biochemical and morphological characteristics. * Group I II III IV V VI VII number Isolate 1b,1c,2a, 2b, 7c, 3c,4a, 5a,5b, 11a, 7b, 3a,3b Designation 9b, 14a 8b 8a 12a 11b 13a Number of 5 3 2 3 3 2 2 isolates Colonial A B C D E F G Type in GPA GROWTH in: AMS + + + + + + + GPA + + + + + + + TSA + LG + NG + + + BIOCHEMICAL +/ Urease + w+ w+ + w+ + w+ – / Amylase – / w+ – – – – – w+ w+ / – / Citrate + –/+ + + + – w+ Catalase + + + + + + + +/ Oxidase + + + - – w+ w+/ – STAINING Gram’s stain – – – – – – – PHB + + + + + + + 14 Philippine Journal of Systematic Biology Vol. III, No.1 (June 2009) Legend * Isolate designation with same number indicate isolates obtained from the same leaf print. The letter in the isolate designation indicates that it is a different colony picked from the leaf print of the designated number. A- Pink, medium to large size, entire margin, convex elevation, butyrous to brittle consistency, opaque density, smooth and glistening to dull appearance. B- Light pink to pink, medium to small size, entire margin, convex elevation, butyrous consistency, opaque density, smooth and glistening to dull appearance. C- Pink, small sizes, entire margin, convex elevation, butyrous consistency, opaque density, smooth and glistening appearance. D- Pink, large to small size, entire margin, convex elevation, butyrous to brittle consistency, opaque density, smooth and dull to glistening appearance. E- Light pink, medium to small size, entire margin, convex elevation, butyrous to sticky and watery consistency and transluscent density, smooth and glistening appearance F- Light pink, medium to small size, entire margin, convex elevation, watery consistency, opaque density, smooth and glistening appearance G- Light pink, medium to very small size, entire margin, convex elevation, butyrous consistency, opaque density, smooth and glistening appearance + = Positive – = Negative w+ = Weakly positive NG = No growth LG = Limited growth 15 Philippine Journal of Systematic Biology Vol. III, No.1 (June 2009) a. Isolate 2b:light pink pigmentation b. Isolate 3a: pink pigmentation c. Isolate 11a: orange/pink pigmentation d. Isolate 3a: small size colonies e. Isolate 3c: medium size colonies f. Isolate 9b: large size colonies Figure 1. Variations in terms of colonial morphology of the PPFM bacterial isolates grown in GPA after one week of one week incubation at 25oC. Images a-c, show the differences in the intensity of pigmentation, while images d-f shows the range of colony size, at the quaternary streak of the inoculated plate. Description on the size of the colonies was based on the following: diameter of colony; small (<1mm), medium (~1mm) and large (>1mm) colonies. a. gram staining b. background staining c. PHB staining Figure 2. Microscopic morphology of the PPFM bacterial isolates. a. Gram staining revealed gram negative rod shaped cells, b. Background staining showed the isolates are rod to cocco-bacilli in shape c. Cells showing PHB granules (black dots). 16