5. Sri Wilarso.cdr BIOTROPIA Vol. 20 No. 1, 2013: 38 - 49 BACTERIA FROM ARBUSCULAR MYCORRHIZAL FUNGI SPORES sp. AND sp. : THEIR ANTAGONISTIC EFFECTS TOWARDS SOILBORNE FUNGAL PATHOGENS AND GROWTH STIMULATION OF sp. Gigaspora Glomus Gigaspora IN VITRO SRI WILARSO BUDI * and NUNANG LAMAEK MAY Recipient of Biotrop Research Grant 2010/Accepted 21 May 2012 This research was aimed at obtaining bacterial isolate from arbuscular mycorrhizal fungi (AMF) spores showing antagonistic effects against fungal pathogens and yet compatible with AMF. Seven isolates of bacteria were isolated from surface-sterilized AMF spores of sp. (GG) and five isolates of bacteria isolated from sp. (GL). All bacterial isolates were identified based on its morphological methods and biochemical reaction and revealed that 9 isolates belong to genus , while the other 3 isolates belong to genus , and respectively. The tests to the antagonists against fungal pathogens and stimulation of AMF hyphal development of sp. showed that there were 3 isolates of bacteria ( GG1, GG5 and GL3) had the ability to inhibit the growth of pathogens and to enhance the development of AMF hyphae sp . These three bacteria isolates potentially can be used to enhance the quality of AMF inoculants as biocontrol and biofertilizers. Enzymatic activity test showed that there were 7 isolates of bacteria that produced cellulase and protease activities, i.e. GG1, GG3, GG6, GG7, GL2, GL4 and GL5. bacteria, Arbuscular Mycorrhizal Fungi Spores, antagonistic effects, stimulation effects, fungal pathogens 1 2 1 2 Department of Silviculture, Faculty of Forestry, Bogor Agricultural University, Bogor 16680, Indonesia Faculty of Forestry, The State University of Papua, West Papua, Indonesia Gigaspora Glomus Bacillus Pseudomonas Proteous Enterobacter Gigaspora in vitro Bacillus subtilis Pseudomonas diminuta Enterobacter hormaechei Gigaspora in vitro Bacillus subtilis Bacillus cereus Bacillus laterosporus Bacillus pasteurii Proteus penneri Bacillus firmus Bacillus cereus ABSTRACT Key words: * Corresponding author : Wilarso62@yahoo.com 38 INTRODUCTION Many kind of microorganisms have been observed to be associated with mycorrhizosphere of different host plants. The functional significance of the microbial associate in the ecosystem has been well documented (Budi 2012) including associative N -fixing bacteria (Cruz & Ishii 2011), plant growth-promoting rhizobacteria (Siddikee . 2010), phosphate solubilizing bacteria (Khan . 2009), and antagonists of plant pathogens (Artursson . 2006, Bakhtiar 2010). In addition, the bacteria associated with spores of AMF and (Roesti . 2005), (Cruz . 2008, Cruz & Ishii 2011, Horii & Ishii 2006, Budi 2012), and (Bharadwaj . 2008) have been reported. These AMF spores associated bacteria has been reported significantly increased AMF hyphal growth and suppress fungal pathogens (Cruz & Ishii 2011). Associated microorganisms may complement mycorrhizal activities, particularly in biological control and biofertilizer in agricultural systems. Nowadays, the awareness of international community to food safety has increased due to negative impacts of the use of chemical substances in crop production. The movement of “back to nature” of international community increases and organic food has become a new trend for their life style. Intensive agriculture in many countries including Indonesia have led to excessive inputs of agrochemicals (fertilizers, pesticides) but agricultural policies are now tending towards more organic for sustainable agricultural systems. Sustainability in agriculture has been defined as the successful management of resources for agriculture to satisfy enhancing the quality of the environment and conserving resources (Ladha 1992). In this respect, the Government of Indonesia c.q Ministry of Agriculture has declared “Go Organic in 2010”. It implies better soil management to avoid modifications that have adverse effects on chemical and biological processes supporting plant growth. One approach is the decrease in chemical inputs to rational (economic but non-polluting) levels through the development of alternative strategies to ensure acceptable yield. An increasing demand for low-input of chemical substance in agriculture has resulted in greater interest in soil microorganisms that increase soil fertility or improve plant nutrition and health including the use of AMF as biofertilizer and biocontrol agents. The use of AMF for sustainable agriculture has been widely used and reported significantly increased plant productivity (Wu & Zou 2009; Siddikee . 2011), but the avaibility of high quality inoculants is very limited due to the production of this fungi as biofertilizer. Up to now, biocontrol is carried out in open pot culture which is sensitive to contamination by other organism or soil borne plant pathogen. Therefore, the development of AMF inoculum production is needed in order to obtain high quality and purity of AMF inoculum. One approach to produce a high quality and purity of AMF inoculum is by using AMF spore associated with bacteria. The objective of this study was to acquire bacteria from surface-sterilized spores of AMF that showed antagonistic effects against fungal pathogens and yet compatible with AMF which potential to improve inoculum quality. et al. et al et al et al et al. Glomus constrictum Glomus geosporum et al Gigaspora margarita et al et al. Glomus intraradices Glomus mosseae et al et al 2 39 Bacteria from Arbuscular Mycorrhizal Fungi Spores sp. and sp. Sri Wilarso BudiGigaspora Glomus – et al. MATERIALS AND METHODS Isolation and identification of bacteria from the AMF spores Antagonistic effects of isolated bacteria towards soil borne plant pathogen Stimulation effect of bacteria on hyphal growth of AMF spores The spores of AMF sp. and sp. originally from the collection of Silviculture Laboratory, Faculty of Forestry IPB were sieved by wet sieving according to method of Gardeman and Nicholson (1963). Hundred spores of each species were surface sterilized according to the method described by Budi . (1999a). The surface sterilized spores were crushed using sterile needles and transferred to Petri dishes containing sterile agar base (Sigma-Aldrich) and Nutrient agar (Oxoid) and then incubated in the dark at 30 C for 24 h. The bacteria that grew were transferred to another Petri dishes containing the same media until single colonies were obtained. A single colony was then identified based on morphological methods using biochemical reaction developed by bioMerieux and carried out at Laboratory of Identification and Determination, School of Life Science and Technology, Bandung Institute of Technology, Bandung, Indonesia. Isolated bacteria were tested toward soil borne plant pathogen . The common soil borne plant pathogens like sp., sp. and sp. originally from the Laboratory of Pest and Diseases, Faculty of Forestry IPB, were used in this study. The test was carried out according to the method of Varese . (1996). The pathogens were grown on Petri dishes containing Potato Dextrose Agar (Difco) Media. Colony of bacteria were placed about 1.5 cm from the pathogen. The Petri dishes were then sealed with parafilm, incubated in the dark at 25 C for 7 days. The radial growth of mycelium were measured and compared to the control treatment. The experiment was carried out in a completely randomized design composed of 12 bacterial treatments and one control in triplicates for each pathogenic fungi. Data were analysed by one-way ANOVA. The AMF sp. spores were collected and surface sterilized according to the method of Budi . (1999a). The spores were then placed in Petri dishes containing sterile zeolit medium supplemented with nutrient. Ten spores were put on sterile filter paper saturated with 50 l suspension of bacteria and were then placed on each Petri dish. The control sterile filter paper was saturated with sterile water. Petri dishes were then sealed with parafilm, incubated in the dark at 25 C for 21 days. The hyphal growth were recorded starting from germ tube and only the main hyphae was measured (Fig. 1) and compared to the control treatment. The experiment was performed in a completely randomized design and composed of 12 bacterial treatments and one control in triplicates. Data were analysed by one-way ANOVA. Gigaspora Glomus et al Pseudomonas in vitro Slerotium Rhizoctonia Ganoderma et al Gigaspora et al o o o in vitro in vitro BIOTROPIA Vol. 20 No. 1, 2013 40 Plate assay for enzymatic activities test Isolated bacteria from AMF spores Bacterial isolates were grown in Erlenmeyer flasks for 24 h on a culture shaker at 30 C in Nutrient Broth buffered medium. Ten micro liters of each bacterial suspension ±10 CFU/m/l was spotted onto plates containing the substrate of the enzyme to be tested and grown at 25 C for 48 h. Cellulolytic activity was assessed as described by Teather and Wood (1982) using a solid medium, containing MgSO 7H O (0.1 g/l), CaCl 2H O (0.2 g/l), FeSO 7H O (0.04 g/l), NaCl (0.2 g/l), KH PO4 (0.3 g/l), K HPO4 (0.5 g/l), CMC (carboxymethylcellulose) (Sigma) (5 g/l), yeast extract (0.1 g/l) and Bacto agar (15 g/l). For visualization of -D-glucan hydrolysis, the agar medium containing CMC was flooded with an aqueous solution of congo red (1 mg/ml) for 15 min (Budi . 2000). The clear zones was measured after 48 h incubation. The proteolytic activity test was performed according to the method described by Dunne . (1997). The experiment was performed in a completely randomized design and composed of 12 bacterial treatments and one control with triplicates. Data were analysed by one-way ANOVA. A total of 12 bacteria isolates were isolated and identified from surface sterilized spores of AM fungi. There were 7 isolates of bacteria isolated from sp. and 5 isolates isolated from sp. The morphological characteristics of each isolate of bacterial colony and its identification are shown in Table 1. 0 8 0 4 2 2 2 4 2 2 2 β et al et al Gigaspora Glomus RESULTS AND DISCUSSION Figure 1. Hyphal growth measurement 41 Bacteria from Arbuscular Mycorrhizal Fungi Spores sp. and sp. Sri Wilarso BudiGigaspora Glomus – et al. Source of isolate Isolates Code Morphological characteristics of bacterial colony (colour, colony surface and type) Gram Species name Gigaspora sp. GG1 Positive Bacillus subtilis GG2 Positive Bacillus licheniformis GG3 Positive Bacillus cereus GG4 Positive Bacillus brevis GG5 Negative Pseudomonas diminuta GG6 Positive Bacillus laterosporus GG7 Circular, entire, umbonate, cream, opaque Circular, undulated, umbonate, cream, opaque Circular, serrate, raised, opaque Circular, undulated, raised, surface dried, opaque Circular, entire, convex, transparent, yellowish Circular, filamentous, raised, opaque Circular, undulate, filamentous, raised, opaque Positive Bacillus pasteurii Glomus sp. GL1 Negative Bacillus circulans GL2 Negative Proteus penneri GL3 Negative Enterobacter hormaechei GL4 Positive Bacillus firmus GL5 Circular, entire, umbonate, translucent Circular, entire, convex, translucent, yellow Circular, entire, convex, transparent Irregular, undulate, raised, transluent Circular, serrate, raised, opaque, Positive Bacillus cereus Table 1. Species identification and morphology characteristics of isolates of bacteria isolated from AMF spores of sp. and sp.Gigaspora Glomus Antagonistic effects of isolated bacteria against fungal pathogen. The results indicated that there are 2 bacterial isolates originated from sp. spores ( GG1 and GG5) and 2 bacterial isolates from sp. spores ( GL2 and GL3) have the ability to inhibit the growth of three pathogens tested. The percentages of inhibition Gigaspora Bacillus subtilis Pseudomonas diminuta Glomus Proteus penneri Enterobacter hormaechei BIOTROPIA Vol. 20 No. 1, 2013 42 varied among pathogens tested. The bacterial isolates of GG1 and GG5 inhibited the growth of mycelium of sp. by 79- 80%. While four isolates of bacteria ( GG1, GG5, GL2, and GL3) showed inhibitory effects to by 46-54%. Testing against sp. showed that bacterial isolates GG1, GG5, GL2 and GL3 gave inhibitory effects of 59-90 %. (Table 2). Among the 12 isolates of bacteria tested, one isolate ( GG5) had a significantly higher effect than the control, while 6 other isolates ( GG1, GG2, GG3, GG4, GG6 and GL3) increased AMF hyphal development higher than the control treatment but were statistically not different. Of the remaining 5 isolates, 2 isolates significantly inhibited the AMF hyphal growth, i.e. GG7 and GL5 while the other 3 isolates inhibited the AMF hyphal growth but were not significally different from the control treatment (Table 3). Ten out of the 12 isolates of bacteria produced cellulase activities, i.e. GG1, GG2, GG3, GG5, GG6, GG7, GL2, Bacillus subtilis Pseudomonas diminuta Sclerotium Bacillus subtilis Pseudomonas diminuta Proteus penneri Enterobacter hormaechei Rhizoctonia Ganoderma Bacillus subtilis Pseudomonas diminuta Proteus penneri Enterobacter hormaechei Pseudomonas diminuta Bacillus subtilis Bacillus licheniformis Bacillus cereus Bacillus brevis Bacillus laterosporus Enterobacter hormaechei Bacillus pasteurii Bacillus cereus Bacillus subtilis Bacillus licheniformis Bacillus cereus Pseudomonas diminuta Bacillus laterosporus Bacillus pasteurii Proteus penneri Enterobacter Stimulation effect of isolated bacteria on hyphal growth of AMF spores sp. Enzymatic activity characterization of Bacteria Gigaspora in vitro Table 2. yRadial growth of pathogenic fungal m celium in vitro No Isolates Radial growth of mycelium (cm)* and % Inhibition Sclerotium sp. % Rhizoctonia sp. % Ganoderma sp. % 1 Control 9.00 a 0 9.00 a 0 9.00 a 0 2 GG1 1.70 d 81.11 4.17 d 53.66 1.04 c 88.44 3 GG2 9.00 a 0 9.00 a 0 9.00 a 0 4 GG3 9.00 a 0 9.00 a 0 9.00 a 0 5 GG4 9.00 a 0 9.00 a 0 9.00 a 0 6 GG5 1.88 d 79.11 4.35 d 51.66 1.12 c 87.55 7 GG6 9.00 a 0 9.00 a 0 9.00 a 0 8 GG7 9.00 a 0 9.00 a 0 9.00 a 0 9 GL1 9.00 a 0 4.65 b 48.33 9.00 a 0 10 GL2 2.99 c 66.77 4.16 d 53.77 0.92 d 89.77 11 GL3 3.86 b 57.11 4.86 c 46 3.68 b 59.11 12 GL4 9.00 a 0 9.00 a 0 9.00 a 0 13 GL5 9.00 a 0 9.00 a 0 9.00 a 0 *Values in a column followed by the same letter do not differ significantly from each other at value of 0.05.P 43 Bacteria from Arbuscular Mycorrhizal Fungi Spores sp. and sp. Sri Wilarso BudiGigaspora Glomus – et al. Table 3. Stimulation of AMF hyphae of sp. growth after 21 days incubation by bacteria Gigaspora No Isolates Hyphal length (μm)* % Increased/ decreased (-) 1 Control 202.44 bc - 2 Bacillus subtilis GG1 286.31 ba 41.43 3 Bacillus licheniformis GG2 369.84 ba 82.69 4 Bacillus cereus GG3 231.79 bc 14.50 5 Bacillus brevis GG4 287.37 ba 41.95 6 Pseudomonas diminuta GG5 499.19 a 146.59 7 Bacillus laterosporus GG6 363.89 ba 79.75 8 Bacillus pasteurii GG7 19.62 e -90.31 9 Bacillus circulans GL1 139.46 bcd -130 10 Proteus penneri GL2 160.68 bcd -20.62 11 Enterobacter hormaechei GL3 324.47 ba 60.28 12 Bacillus firmus GL4 181.46 bc -11.56 13 Bacillus cereus GL5 156.38 bcd -22.75 Note : * Values in a column followed by the same letter do not differ significantly from each other at value ofP hormaechei Bacillus firmus Bacillus cereus Bacillus subtilis Bacillus cereus Bacillus brevis Bacillus laterosporus Bacillus pasteurii Proteus penneri Bacillus firmus Bacillus cereus Bacillus subtilis Bacillus cereus Bacillus laterosporus Bacillus pasteurii Proteus penneri Bacillus firmus Bacillus cereus et al. et al. et al. et al. GL3, GL4, and GL5, while 8 isolates produced protease activities, i.e. GG1, GG3, GG4, GG6, GG7, GL2, GL4, and GL5. There were 7 isolates that produced both cellulase and protease activities, i.e. GG1, GG3, GG6, GG7, GL2, GL4 and GL5, as indicated by clear zone (halo) around the colony (Table 3, Fig. 2). Bacteria have been observed to live in close association with AMF (Gopal 2012), and have been isolated from different AMF spores (Xavier & Germida 2003; Roesti 2005; Horii & Ishii 2006; Cruz 2008; Bharadwaj 2008;, Figure 2. Celullase activity (A) and Protease activity (B) as indicated by clear zones (halo) BIOTROPIA Vol. 20 No. 1, 2013 44 Cruz & Ishii 2011). Cruz and Ishii (2011) found 2 bacteria sp. and isolated from liquid present inside AMF spore of , while Budi (2012) found four bacteria sp. and KSC_SF9c isolated from surface sterilized AMF spores . Xavier and Germida (2003) found 5 bacteria, , sp. , and sp. from surface sterilized AMF spores of . This study demonstrated different bacteria species isolated from surface sterilized AMF spores of sp. and sp. We found 7 bacteria from sp. and 5 bacteria from sp. as shown in Table 1. As reported by Roesti . (2005), the bacterial community associated with the AMF spores was more inf luenced by the AMF identity (AMF species) than by the host plant. The difference in composition of the spore walls or exudates of AMF species may have played a major role in the selection of bacterial populations living on the spore (Roesti . 2005). Bacteria associated with AMF spores have been reported to play an important role in antagonistic effect of soil borne plant pathogens, AMF spore germination and hyphal growth (Horii & Ishii 2006; Cruz & Ishii 2011). The interesting thing from this study was that among the 12 isolate bacteria tested, 3 bacterial isolates ( GG1, GG5 and GL3) have the ability to inhibit the growth of three pathogens and stimulate hyphal growth of sp. (Table 2 and 3). This finding was in line with Cruz and Ishii (2011), who found that sp., and isolated from surface sterilized Bacillus B. thuringiensis Gigaspora margarita et al. Bacillus Bacillus megaterium, Bacillus subtilis Bacillus flexus strain G. margarita Alcaligenes Bacillus Burkholderia Flavobacterium Pseudomonas Glomus clarum Gigaspora Glomus Gigaspora Glomus et al et al Bacillus subtilis Pseudomonas diminuta Enterobacter hormaechei Gigaspora Bacillus B. thuringiensis Paenibacillus rhizosphaerae Table 4. Enzymatic activities of bacteria isolated from sp. and sp. as indicated by clear zones diameter after 48 h incubation Gigaspora Glomus No Bacteria Isolates Enzymatic activities ( Ø clear zones, cm) Celluase Protease 1 Bacillus subtilis GG1 4.80 cd 4.80 ba 2 Bacillus licheniformis GG2 2.83 e 0.00 c 3 Bacillus cereus GG3 5.70 b 6.50 a 4 Bacillus brevis GG4 0.00 f 5.77 a 5 Pseudomonas diminuta GG5 5.10 cb 0.00 c 6 Bacillus laterosporus GG6 5.87 b 6.20 a 7 Bacillus pasteurii GG7 7.17 a 5.83 a 8 Bacillus circulans GL1 0.00 f 0.00 c 9 Proteus penneri GL2 5.07 cb 4.73 ba 10 Enterobacter hormaechei GL3 2.97 e 0.00 c 11 Bacillus firmus GL4 3.23 e 3.73 b 12 Bacillus cereus GL5 4.10 d 4.73 ba * Values in a column followed by the same letter do not differ significantly from each other at value of ,0.05P 45 Bacteria from Arbuscular Mycorrhizal Fungi Spores sp. and sp. Sri Wilarso BudiGigaspora Glomus – et al. AMF spores had antagonistic effect to the pathogenic fungi and and stimulate hyphal growth of . Many studies have reported that has the potential to be used as biocontrol agent against soil borne plant pathogens (Nalisha . 2006; Velmuragan . 2009). Phae . (1990) found that produced , a bioactive compound that are active as antifungal agents against several plants pathogenic fungi as well as antibacterial. The production of hydrolytic enzyme protease by sp. isolated from mycorrhizosphere that caused cell wall degradation of pathogenic fungi have also been reported (Budi 2000). In this experiment, also produced hydrolitic protease enzyme. We assume that combination of protease enzyme with the other active compounds produced by could inhibit pathogenic fungal growth. Other bacteria such as and have been reported effectively control diseases caused by soil borne plant pathogens (Thomashow . 1990; Chermin . 1995, Barea . 1998). These bacteria produced bioactive compound Phenazine-1-Carboxylic Acid responsible for suppression of soil borne plant pathogenic fungi (Thomashow . 1990). In this study only GG5 produced hydrolytic protease enzyme but not GL3, indicating that the role of these enzymes in controlling the growth of soil borne plant fungal pathogens is not clear and it seems that the inhibitory effect of these bacteria to soil borne plant fungal pathogen development is probably derived from more than one mechanism (Budi . 2000). Further investigation is necessary to isolate bioactive compound and its mode of action from these 3 bacteria isolates. The stimulation of AMF hyphal growth by AMF spores associated with bacteria have been reported by several researchers (Budi 1999b; Cruz & Ishii, 2011). In this study GG1, GG5 and GL3 have the ability to stimulate hyphal growth of sp. and supressed several soil borne fungal pathogens . Four others isolates only have the ability to stimulate hyphal growth of sp. (Table 3). The exact mechanism of hyphal growth stimulation by spores associoated with bacteria is not known, but Cruz and Ishii (2011) reported that bacteria released volatile compounds and exudates involved in stimulating hyphal growth. Futher study is needed to investigate the possible isolate volatile compounds and exudate released by these 3 bacteria isolates. Certain Arbuscular Mycorrhizal Fungi hyphae produce hydrolytic enzymes which hydrolyses the biopolymers such as protein, chitin and cellulose that will help the AMF to degrade and infect the plant cell walls (Gopal . 2012). Furthermore, the hyphae and spores of AMF also harbour associated bacteria that produce hydrolytic enzyme (Budi . 1999b). In this experiment, 7 isolates produced both cellulase and protease activities, i.e. GG1, GG3, GG6, GG7, GL2, GL4 and GL5, as indicated by clear zone (halo) around the colony (Table 3, Fig. 2). As reported by Budi (2012) isolated from surface sterilized AMF spores have shown to increase mycorrhizal root colonization of neem seedling in the nursery and also produced cellulase and protease activities. Gigaspora margarita Rosellimia necatrix, Phytium ultimum, Fusarium oxysporum Rhizoctonia solani G. margarita B. subtilis et al et al et al B subtilis iturin Paenibacillus et al. B. subtilis B. subtilis Pseudomonas Enterobacter et al et al et al et al Pseudomonas diminuta Enterobacter hormaechei et al et al. Bacillus subtilis Pseudomonas diminuta Enterobacter hormaechei Gigaspora in vitro Gigaspora in vitro et al et al Bacillus subtilis Bacillus cereus Bacillus laterosporus Bacillus pasteurii Proteus penneri Bacillus firmus Bacillus cereus et al. Bacillus subtilis G. margarita BIOTROPIA Vol. 20 No. 1, 2013 46 Production of volatile compounds that can positively influence germination of AMF spores, provision of nitrogen through nitrogen fixation, solubilization of soil phosphate sources, detoxification of the fungal microhabitat, change of pH level of siderophores are also some of the proposed mechanisms underlying stimulation of mycorrhization (Bharadwaj 2012). Further study is needed to test the consistency effects in the field, and to investigate the mechanisms involved in the antagonism towards fungal pathogens as well as stimulation effect to the hyphal growth of AM fungi. There is a possibility that 3 isolate bacteria ( GG1, GG5 and GL3) isolated from surface sterilized spores of AM fungi sp. and sp. have the ability to inhibit the growth of 3 pathogens and stimulate hyphal growth of sp. . These bacteria have a great opportunity to be used as biocontrol agents for soil borne plant pathogens as well as for improving the AMF inoculum quality, since up to now the production of these fungi for biofertilizers and biocontrol is carried out in open pot culture sensitive to contamination by other organisms or pathogens. This research was supported by BIOTROP through the competitive research grant No 050.7/PSRP/SPK-PNLT/III/10. The authors would like to thank the Department of National Education of the Republic Indonesia for the financial support to conduct this research. et al. Bacillus subtilis Pseudomonas diminuta Enterobacter hormaechei Gigaspora Glomus Gigaspora in vitro CONCLUSIONS ACKNOWLEDGMENTS REFERENCES : Artursson V, Finlay RD, Jansson JK. 2006. Interactions between arbuscular mycorrhizal fungi and bacteria and their potential for stimulating plant growth. 8: 1-10. Bakhtiar Y, Yahya S, Sumaryono W, Sinaga MS, Budi SW, Tajudin T. 2010. 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