Phlgaie sarel of Oop Scere PICS se fg 08 33): 0224 aprile 2008, Cr Skee ace of te Php. eee? gat 2008 GROWTH ENHANCEMENT AND ROOT COLONIZATION OF SUGARCANE By PLANT GROWTH-PROMOTING BACTERIA. LUCILLE C VILLEGAS' & ERLINDA S PATERNO™ ‘Microbiology Division, Institute of Biological Sciences, College of Arts and Sciences, UP Los Baos, College, Laguna 4031. "crop Science Chater, Collegeof Agriculture, UP Les Bats, Coleg, Laguna 4031 lant growth promoting bacteria were isolated from the sugarcane rhizosphere. They were found to possess growth ‘enhancing activities which included indole aceti acid production, phosphate solubilization, siderophore produetion and rivogen fication. Physiological, biochemical and genomic characterizations indicated that the two selected isolates, designated as LV7 and LV10, belong to Pseudomonas genus, Plant growth promotion due to bacterial inoculation was evaluated atthe in-vitro shoot multiplication and root development stages of sugarcane. LV10 significantly increased the ‘number of tlers by 31% and the fresh shoot weight by 26% above the uninoculated control. LV? significantly increased the fresh shoot weight by 1796 and promoted the in-vitro rooting ofthe plantlets by 55% above the uninoculated control Under nursery conditions, both isolates enhanced the growth of sugarcane. Inoculated plants were significantly taller are accumulated significant amounts of biomass. Shoot dry weight Increased up to 61% and root dry welght up 0 67% Microbiological analyses revealed that both bacteria survived and persisted in the rhizosphere and within sugarcane tissues. The mode of invasion and extent of colonization of sugarcane roots was studied using LV7-tagged with gus gene. Microscopic examination of the x-gluc siined plants revealed that LV7 was an imasive colonizer capable of Iimading sugarcane root via root tips and fissures created by the emerging lateral roots. LV7 was found to colonize the ‘root cortex and vascular bundles. Due 10 the growth promoting activities and invasive colonization of LV7 and LVI0, these strains may be most suited as inoculants for sugarcane ‘gusA gene, microbial inoculant, plant growth promoting bacteria, Pseudomonas, root colonization, sugarcane, ‘issue culture INTRODUCTION Sugarcane is one of the Philippines’ major crops and is grown mainly for sugar. Cane sugar isa highly prized commodity and has become one of the country’s major traditional exports. The sugar industry is a major employer both in the agricultural and industrial sectors (PSTC 2001), Sugarcane requires large inputs of fertilizers Several bags to more than a ton of fertilizers are applied to a hectare annually (Ledesma 1997). In February 2006, the price of commercial inorganic fertilizer increased up 10 4.5%, discouraging some farmers from planting sugarcane (Trinidad 2006), Derived from nonrenewable energy resources such as petroleum, fertilizers and have been implicated in the pollution of air and groundwater. The application of microbial inoculants promising tool by which synthetic fertilizer use ‘might be decreased in the cultivation of sugarcane. Plant growth promoting bacteria (PGPB) are being tapped as microbial inoculants, they being, reported to influence the growth and development of associated crop by several mechanisms (Glick 1995). They are capable of fixing. atmospheric nitrogen and supplying the fixed nitrogen to the offers aplant, They synthesize several phytohormones that enhance the various stages of plant growth. They have mechanisms for the solubilization of minerals such as phosphate that then become more readily available for plant growth, and they synthesize some of the well-characterized low molecular weight compounds or enzymes that can modulate plant growth and development (Glick 1995, Cleyet-Marel fal 2000), Indirectly, they promote plant growth by Preventing the deleterious effects of phyto- pathogenic organisms through the synthesis of antibiotics (Schinder et al 1994) and secretion of siderophores (Castignetti_ & Smarrell 1986, O'Sullivan & O’Gara 1992). Several commercial inoculants are marketed in the Philippines for specific crops such as legumes, rice, com, fruit trees and forestry crops, which were developed by the National Institute of Molecular Biology and Biotechnology (Biotech), UP Los Baitos, Laguna, Philippines. None has been specifically developed for sugarcane, and this is ‘necessary, considering that sugarcane is one of the heavy users of chemical fertilizers. (One essential approach in developing microbial inoculant for sugarcane is the identification of the mest effective microorganisms. To fully exploit the microbes’ beneficial effects, a better understanding, of the mechanisms of growth promotion, improved strategies to enhance inoculation and detivery ‘methods, long-term persistence and survival, and the stability of growth promotion effects, is necessary. This study was undertaken to identify PGPB that ‘can be utilized for sugarcane production, We report here the identification of two promising PGPB that are capable of enhancing sugarcane growth, the mode of invasion and extent of colonization of sugarcane roots using the Gus {gene biomarker, and the persistence and survival of the introduced PGPB in the rhizosphere and inside the plant. A successful method of introducing and ‘maintaining the PGPB population in the sugarcane plant is presented. MATERIALS AND METHODS Isolation of Bacteria and Determination of Growth Promoting Activities Soil samples fom 0-15 em of Silay series (Typic Duraqualf) and Guimbataon series (Vitrandic Dystrustept) were collected from sugarcane fields in Negros Occidental. The bacterial species of interest were selectively isolated from these samples. Non-soil materials were discarded from the soil samples. About 10 g of soil was pulverized and then shaken in 90 mL saline solution for 15 minutes. Several dilutions were prepared from this stock and 100 tL from each dilution (10°, 10°, 10 and 10%) were plated onto appropriate media, The culture media used included King’s Medium B (KMB), Congo red NFb medium (CNF) (Dobereiner and Day 1976), nitrogen free medium (NFM) (Kole et al 1988) and tryptic soy agar medium (TSA) (Tipping etal 1989). ‘The phosphate solubilizing activity ofthe isolates was determined following the procedure of Goldstein (1986). Indole acetic acid (IAA) production was determined by growing the bacteria in Minimal Salts (MS) medium (Frankenberger & Poth 1987) supplemented with tryptophan. After 72 hr incubation, the cultures were centrifuged and the IAA in the supernatant was detected colori ‘metrically (Gordon & Weber 1951). ‘The siderophore secretion was determined following the’ procedure of Alexander & Zuberer (1991). Diazotrophy was detected in isolates that gave positive results in the above growth promoting properties through acetylene reduction assay (Rennie 1981) and Polymerase Chain Reaction (PCR) method (nif PCR) developed by de Bruijn et al (1995). Growth Enhancement Of Sugarcane(Characterization and Identification ofthe Bacteria Morphological characteristics of the bacteria ‘were investigated microscopically. The physio- logical and biochemical properties were screened using automated microbiological identification systems including Biolog (Biolog Inc, California, USA) and APL 20 NE (bioMerieux, France) according to the manufacturer’s instructions. The Biolog GN2 MicroPlate was used, which contained 95 discrete carbon-source utilization tests and gives 2 characteristic reaction patter called a ‘metabolic fingerprint”. The metabolic fingerprint pattems or the biochemical results of the isolates were automatically read and recorded and then compared to the extensive MicroLog database software for final identification. In addition, biochemical characters were recorded as ‘I’ (indicating growth) or *-" (no growth) and clustered by UPGMA method (Unweighted Pair Group Method using arithmetic average (Sneath & Sokal 1973). Reference strains, Pseudomonas fluorescens NRRL 24709 Biotech ‘Accession Number 1123 and Pseudomonas putida NRRL B13 Biotech Accession Number 1337, were included in the tests. API 20 NE on the other hand is a standard system consisting of 8 conventional tests, 12 assimilation tests and a database. The reactions were read according to the Reading Table and the identification was obtained by referring to the ‘Analytical Profile Index and confirmed through the APL identification software. ‘The genomic fingerprints of the isolates were obtained using three sets of amplification oligo- nucleotide primers. The bacterial DNA extraction ‘was done following standard techniques (Sambrook ef al 1989). For ribosomal gene spacer sequence (RS}PCR, a pair of 15-mer primers (L1, 5-CAA GGC ATC CAC CGT-3', and Gl, 5’-GAA GTC GTA ACA AGG-3’) (Jensen et al 1993) was used. LC Villegas & ES Paterno For repetitive extragenic palindromic sequence (REP}PCR, the pairs of 18-mer primers used were: REP-ID, 5'-NNN RCG YCG NCA TCM GGC:3", and REP-2D, 5"-RCG YCT TAT CMG GCC TAC- 3°, where Mis A or C, Ris A or G, Y is Cor T, and N is any nucleotide (Stem et al 1984). For enterobacterial repetitive intergenic consensus (ERIO)-PCR, a pair of 22-mer primers (ERIC IR, S'-ATG TAA GCT CCT GGG GAT TCA C3, and ERIC2, 5'-AAG TAA GTG ACT GGG GTG AGC G-3') was used (Versalovic et al 1991). Polymerase chain reaction was carried out in a volume of 25 ul. containing the following: 25 uL of 10x PCR buffer, 20mM of MgSO4,; 10mM of NTP mix (Invitrogen life technologies); 5 U/l of. DNA taq polymerase, 10 mM of oligonucleotide primer and 2Sng/u of DNA template, The DNA ‘was frst denatured for 7 min at 95°C, followed by 35 cycles of 30 sec denaturation at 90°C, 1 min annealing at 52°C and 5 min elongation at 70°C, with a final elongation at 70°C for 10 min. The soaking temperature was 4°C. Amplification products were resolved on 2% (w/) agarose, visualized following ethidium bromide staining and photographed under UV light. The bands were recorded as ‘I’ (indicating presence of DNA band) or ‘ (absence of DNA band) and clustered by UPGMA method (Unweighted Pair Group Method using arithmetic average (Sneath & Sokal 1973). Reference strains were included in the analysis, Sugarcane Tissue Culture Bioassay's Variety VMC 86-550 was used throughout this study, Plantlets at the shoot multiplication and root development stages were aseptically separated and three plantlets were transferred to tissue culture bottles containing modified Murashige-Skoog medium (Morales et al 1991). The plantlets were allowed to establish for 5 days before bacterial inoculation.Inoculation was done by first growing the bacterial isolates ovemight in King’s medium B. ‘The bacterial cells were harvested by centrifugation at $000 rpm for 5 min, The pelleted cells were washed and resuspended in 0.85% NaCl. Cell population was adjusted to 10° CFU/mL by spectrophotometry or using McFarland standards. (One ml of this cell suspension was inoculated onto ach bottle containing plantlets, The inoculated plantlets were maintained in a environment with 1000-3000 lux light intensity at 26-28°C. Number of tillers, total biomass accumulated and root weights were determined 21 days after inoculation (DAL) controlled Growth Promotion and Stavival of Isolate Rooted plantlets (je, 6 days at the rooting period) were individually separated and transplanted onto big tubes containing rooting medium without naphthalene acetic acid (NAA). Three days after transfer, 20 plantlets were inoculated with 10* cells ‘These were maintained at the growth chamber at controlled conditions as above. Fourteen DA, five plant samples were obtained and the bacterial populations inside the root and culm were estimated by plate count. The remaining 15 plantlets were transplanted individually onto cups disinfected with 15% hypochlorite solution and sterile mixture of sand, vermiculite and peat (2:1:1/2) and maintained in the growth room at controlled conditions for 2 ‘weeks and then transferred to the nursery. Thirty days after transplanting (DAT), plant height, shoot weight, root length and root weight were determined. The bacterial populations in the suistrate, rhizosphere, root and culm were also determined. The procedure of Stoltzfus et al (1997) for surface sterilization was followed. Dilutions ‘were prepared fom the macerate and SOL of the appropriate dilutions were spread onto plates containing tryptic soy agar medium supplemented with at least two antibiotics. Plates were incubated ovemight at 30°C. Number of colonies were counted and the colony forming units per rmL(ofu/mL) was calculated. GusA Gene Labeling and Microscopy Plasmid pKW107, a suicide plasmid carrying ‘gus inserted in a transposon (mTNSSSsgus421) bom in E.coli $17-2-pir, was introduced to LV7 by Conjugation following the protocol of Barraquio et al (1997) with slight modifications. Selection of transconjugants was based on spectinomycin and ritrofurantoin antibiotic resistance instead of the chloramphenicol used by Barraquio etal (cited), and development of blue colored colonies in the presence of Gus substrate 5-bromo-4chloro-3indolyl B-D-glucoronide (x-gluc) (Wilson 1996). Individualized —micropropagated sugarcane plantlets were aseptically inoculated with the gus marked LV7 to determine its endophytic. characteristic, mode of invasion and extent of colonization, Plantlets inoculated with the gus+ LV7 ‘were sampled at predetermined intervals. Histo- chemical staining ofthe root and whole plant tissues with x-gluc allowed localization of the bacteria Bacterial Gus activity was indicated by the formation of blue precipitate. The staining was observed with naked eye as well as under the ‘microscope. To further confirm the localization of bacteria, semithin sections (30 pm thick; Microcut H11250; Energy Beam Sciences) were prepared from agar embedded roots and were analyzed micro- scopically. RESULTS & DISCUSSION Isolation of Bacteria and Growth-Promoting Activities A total of 135 isolates were obtained from different sugarcane thizospheres; 99 isolates were Growth Enhancement Of Sugarcaneobtained using KMB, eight isolates using CNF, 20 isolates using NFM based on soil extract and ‘mannitol, and eight isolates using TSA. Of these, 44 were found to produce LAA (Table 1). Positive test for IAA was pink to red color when the bacterial supernatant was treated with Fe-H,SO,, Isolates with no capacity to synthesize IAA showed yellow reaction. IAA secretion was reported as a mechanism by which beneficial bacteria stimulate plant growth. Specific examples include two-three fold increased in root length of canola due to [AA produced by P. putida (Glick 1995); increased in the number and length of lateral roots of wheat seedlings and increased root hairs in Arabidopsis thaliana due to 1AA produced 4. brasilense (Omay etal 1993), and improved growth of tomato, lettuce, Centrosema pubescens and Paspalum norarum due to IAA secreted by 4. paspali in culture media (Barea & Brown 1974). Twenty eight isolates were found to solubilize Phosphate (P) (Table 1). In the plate assay for P solubilizing activity, observation of clearing zone around the bacterial growth was considered as positive reaction, P solubilization is one trait being exploited in selecting microorganisms as inoculants. While only 10-20% of fertilizer P can be utilized by plants, the major part is deposited in the soil and as such P becomes limiting (Hoflich et al 1995). Microorganisms solubilize inorganic P compounds by excreting organic acids and mineralizing organic P by phosphatases, thus making the P compounds utlizable for plants (Lifshitz etal 1987). ‘The capacity of the different bacterial isolates to ‘produce siderophores was determined using chrome anol S (CAS) reagents. Orange halos developed around colonies of siderophore-producing bacteria 1s the siderophore removes the Fe from the Fe-CAS, dye complex which gives the CAS medium its characteristic color. Twelve isolates were found capable of producing siderophores (Table 1). £C Villegas & ES Paterno Siderophores produced by bacteria may enhance ant growth by increasing the availability of Fe near the root o by inhibiting the colonization of roots by plant pathogens or other harmful bacteria, Kloepper etal (1980) suggested that root colonizing, beneficial fluorescent pseudomonads enhance plant growth in part by producing siderophores. which efficiently sequester iron in the root zone, making it unavailable to certain rhizoplane microorganisms, including microorganisms deleterious to plants. The Populations of the deleterious microorganisms are thereby reduced and the roots are healthier. Fourteen isolates gave positive reactions in all the assays performed, indicating that the isolates have the potential of promoting plant growth. These isolates were further screened for nitrogenase activity through acetylene reduction assay. Six isolates were found positive for acetylene reduction and were tested for possible pathogenicity. Due to the absence of pathogenic reactions in tissue cultured sugarcane, LV7 and LV10 were selected as the best isolates. Theit diazotrophic nature was positively confirmed through nif PCR. Characterization and Identification of Bacteria ‘The selected isolates designated as LV7 and LV10 were both Gram-negative aerobic rods, LV7 forms circular, opaque white to off-white, colonies with entire margin and convex elevation on agar plate while LV10 forms iregular, flat, beige colonies with undulate to lobate margin. AS both cultures age, fluorescent yellow green/yellow diffusible pigment is produced, Both LV7 and LV10 are capable of growing on potato agar with 10% and 30% cane sugar. ‘A total of 115 physiological and biochemical tests checked for identities of the isolates. The properties obtained using the API 20 NE galleries idemified LV7 to be Pseudomonas fluorescens and LV10 to be P. putida (Table 2a). Further bio-‘Table 1. Phosphate solubilizing activity, indole acetic acid production and si bacterial isolates obtained from the sugarcane rhizosphere 1 n GB “4 8 6 7 8 a Q 3 a G o a MI Me Lvio Ma MS M6 MT MB. M9 mio Mu Miz M3 Mit Mis Mis M7 Mis Mig ‘20 KI K 3 Ké KS Ks KI Kg ko Ki0 "sapien sc amagone reaction Toole Phosphate Solubilization “TAA Production Growth Enhancement Of SugarcaneTable 1. Phosphate solubilizing activity(cont’d) sols Phosphate Solubilization TAA Production ‘Siderophore Production KI 5 : + Ki z : : KB : Kis Lr Kis KV? Kis Kia : : : 20 I : 5 Kn : : Kt : : : 2s : 26 : kar : + 19 30 I : 5 Kx : + 133 : + 134 : + Kas : 136 : : 37 : x38, : 39 : Kao : Kal z Ka z : : Ka : kat : 7 : Kas : : kay : : : Kas. z : : Ka. : + A Ks : : : Kst : : : Ks + + 5 Ks3 : 7 + Ks : : Kss : : KS : : Ko : + : Kot . : + Keo : - + Wapatine reaction isa mega eon IC Villegas & ES PaternoTable 1, Phosphate solubilizing activity(cont’d) Soles K63 K64 : 6s : 66 5 6 : Ket : Koo : Kn + Km K76 Kn Kp 80 Kal Kg : KB 5 Ket + Kas Kes Kas Keo KM Kot KM K93 ko Kos K96 K97 K98 koe Phosphate Solubilization TAA Production Siderophere Production Tapeume racion ir anegathe reco chemical characterizations were done using the Biolog GN2 MicroPlate that contained 95 discrete carbon-source utilization tests, These 95 characters (Table 2b) were used for cluster analysis. At 90% similarity level, LV7 grouped together with Pseudomonas fluorescens NRRL 24709 Biotech ‘Accession Number 1123 while LV10_ grouped together with Pseudomonas putida NRRL B13 Biotech Accession Number 1337 at 85% similarity level. ‘The isolates were further investigated by PCR- based genomic fingerprinting using three sets of amplification oligonucleotide primers. Fig 1 shows 10 the pattem of the PCR products separated by agarose gel electrophoresis. After analyzing the data using UPGMA method, RS-PCR resulted in clustering of LV7 with P. fluorescens reference strain and LV10 with P. putida reference strain, both at the 83% similarity level. This primer was based on the spacer sequences of 16S and 238 ribosomal DNAs (Jensen et al 1993). Using ERIC PCR, LV10 clustered with P. putida reference strain at 70% similarity level while LV7 was only 28% similar to the reference strain. This may indicate that ERIC-PCR discriminate LV7 and LV10 as strains possibly different from the reference strains. ERIC Growth Enhancement Of Sugarcane‘Table 2a, Physiological and biochemical characteristis ofthe isolates as determined using API20 NE, = ‘Test ‘itatereduction| Indole production Acidification of glucose Arginine dihydrolase Urease Esculin hydrolysis Gelatin hydrolysis galactosidase Assimilation of Glucose Arabinose Mannose Mannitol Neacety-glucosamine Maltose Gluconate Caprate Adipate Malate Citrate Phenylacetate ‘dentifcation primers are oligonucleotides targeting short repetitive sequences dispersed throughout various bacterial genomes. Their location in bacterial ‘genomes allows discrimination atthe genus, species and strain level based on electrophoretic pattems of amplification products (Versalovic et al 1991). REP PCR confirmed the ERIC-PCR results of LV7 wherein it clustered with P. fluorescens reference strain at 60% similarity level. LVIO clustered with P. putida reference strain at 58% similarity level Like ERIC PCR, REP-PCR is based on the presence of repetitive conserved sequences in bacteria. The primers contain multiple nucleotides at ambiguous positions in the consensus REP (Stem etal 1984). Inoculation Responses of Sugarcane ‘Sugarcane tissue culture involves three stages, LC Villegas & ES Paterno Pseudomonas fluorescens LvI0 Pseudomonas putida which include culture establishment, shoot ‘muttipfcation and root development (Morales et al 1991), In all these stages, growth factors such as vitamins and hormones are incorporated into the ‘media to stimulate the growth of the plantlets In this, study, the multiplication and root development stages were utilized to demonstrate that the selected bacterial isolates by themselves can stimulate sugarcane growth. Bacterial inoculations were performed with the plantlets growing in media missing with atleast one important growth factor such as _kinetin, benzylaminopurine, and naphthalene acetic acid, In plant-bacteria co-culture, growth effects reported include increases in plant height, root and shoot biomass and root formation (Muthukumarasamy et WWTable 2b. Physiological and biochemical characteristics ofthe isolates as determined using BIOLOG™ GN? MicroPlate Tea ‘acyclodextrin Dextrin Glycogen ‘Tween 40 ‘Tween 80 ‘N-acetyl-D- galactosamine Neacety-D- glucosamine Adonitol L-arabinose Danbitol Decellobiose erythritol Detructose Lefucose Degalactose Gentiobiose a D-glucose Meinasitl a -Dlactose Lactulose Maltose D-mannitol D-mannose D-melibiose Brmethy/D -glucoside Depsicose Deaifinase L+thamnase Desorbitol Sucrose Detchalose Turanose Xylitol Methyl pyruvate Mano -Methy! Succinate Acetic acid Cis-aconitic acid Citric acid Formic acid D-galactonic acid lactone D- galacturonic acid Degluconic acid D-glucosaminic acid Deglucoronic acid artiydroxy butyric acid B- hydroxy butyric ac ‘y— Hydroxy butyrie acid Brhydroxy phenylacetic acid * sposneraacon - i pega eatin 2 LT eeette Peete et eee eee ttaeeetee v0 Growth Enhancement Of Sugarcane‘Table 2b, Physiological and biochemical (cont'd) Tet Taconic al a-Keto butyric acid ‘a Keto glutaric acid ‘a-Keto valeric acid Dil actic acid Malonic acid Propionic acid Quinic acid Dsaccharie acid Sebucic acid Succinicacid Bromo Suetinic acid Succinamio acid ‘Glucuremomide Lalaninamide Dalani Lealanine Lealanyl-slycine asparagine aspartic acid Leglutamic acid ‘Glyoyl-L -aspartic acid Glycylt, -glutamie acid histidine Hydroxy+L proline Leleucine L-omithine L-phenylalanine L-protine L-pyroglutamic D-serine Leserine threonine DjL-carithine ‘y-Amino Butyric acid Uroeanio acid Inasine Uridine ‘Taymidine Pheny-ethylamine Purescine 2-Amino ethanol 23 butanediol | Glycerol D-L-a~ glycerol phosphate Glucose-- phosphate Glucose -6 phosphate Identification spine easton sme racton LC Villegas & ES Paterno eeeeteeeeee Pseudomonas frescens beeeeeteeeeaee Pseuetomonas purida BElectropherogeam of PCR products obtained from the amplification of LV7 and LV10 DNA samples in the presence of primers FRIC, REP and RS. Lane M1 kb plus marker. Lane 7~LV7. Lane 8~ LV10. Lane 9 P. putida reference strain. Lane 10 ~P. fluorescens reference strain. Lane W negative control al 2006, Conn et al 1997, Pillay & Nowak 1997, Fromme! et al 1991), Growth responses due to inoculation were ‘observed when plantlets were grown in. medium lacking in kinetin (Table 3). LV10_ significantly increased the number of tillers by 31% above the uninoculated control. In terms of shoot biomass, significant increases of 17% in LV-inoculated plants, and 26% in LV10-inoculated plants were observed. ‘These significant increases in shoot biomass and number of tillers may indicate that the introduced bacteria provided the missing growth factor which can be a cytokinin-like or kinetin-like ‘growth factor. In tissue culture, cytokinins such as 4 kinetin are incorporated in the medium to promote shoot proliferation (Bhojwani & Razdan 1983). In ‘cases where bioassays were done in the absence of BAP and in complete medium, no significant differences in number of tillers and shoot biomass were observed between inoculated and uninoculated plants. This observation confirmed the study of Chanway & Nelson (1991) wherein growth promotion due to bacterial inoculation was not detected when bioassays were performed using full nutrient medium, Results on the root development assays showed that in the absence of the auxin NAA, no significant differences in root weights were observed between Growth Enhancement Of SugarcaneTable3. Growth responses ofsugarcane to bacterial inoculation atthe in-vitro shoot multiplication and root development stages 21 days after inoculation Inoculation Gromeh responses Treatment ‘Shoot multiplication (medum wo oot development (edium wo NAA) kinetin) Number of Tillers Biomass (g) Root Weight) Number of Tilers ‘Uninoculated contro! 2 288 os" a Lr 28° 6si* oss or Lvi0 36 802 ois, 28 “Man ce co lowed bythe same ar rem scaly fren a 5% ee by DMT. the inoculated and uninoculated plants (Table 3). While in-vitro rooting was insignificant, significant differences were noted in the number of tillers, Plantlets inoculated with LV7 developed tillers 15% ‘more than the uninoculated control and LV10 had 18% more than the uninoculated control. These observations confirmed the results obtained in the bioassay on shoot multiplication where LV7 was found to provide a cytokinin-like growth factor that promoted shoot multiplication. ‘The rooting medium without NAA is similar to the multiplication medium used in the previous bioassay but with a higher level of sucrose and lacking the growth hormones kinetin and benzyl ‘aminopurine. In the complete rooting medium, LV7 significantly increased the root weight of inoculated plants by 55% above the uninoculated control. Based on the results, the significant increase in root ‘weight of LV7-inoculated plants suggests that LV7 may be producing auxin-like substances as well. Root development is reported to be influenced by the phytohormone auxin (Rolfe et al 1997). In nature, auxins are involved in a number of plant functions such as promotion of cell elongation and call division, apical dominance, root initiation, differentiation of vascular tissue, ethylene biosynthesis, mediation of tropistic responses and the alteration of the expression of specific genes (Bhojwani & Razdan 1983, Rolfe et al 1997), In LC Villegas & ES Paterno tissue culture, auxins such as NAA are incorporated inthe medium to promote root initiation. ‘The use of mixed substrates with low nutrient concentrations would induce the plant to respond to inoculation (Reis et al 1999). In the current study, a presterilized low nutrient mixture of sand- vermiculite-peat (2:1:1/2) was used to evaluate growth promotion. The plants depended on the nutrients irrigated once a week and sterile distilled ‘water supplied the water requirement of the plants. Results showed that the LV7-inoculated plants were significantly taller by 20% and LVIO- inoculated were 19% taller than the uninoculated plants (Table 4). Similarly, the accumulated shoot and root dry weights were significantly greater than the uninoculated plants, LV7 and LV10-inoculated plants accumulated shoot dry weight by 55 and 52% above the uninoculated control while root dry ‘weight were 67 and 65% above the uninoculated ‘controls. LV7 significantly increased root weight of the inoculated plants. LV7 and LV10 clearly enhanced the growth of sugarcane when grown in mixed substrate with low nutrient concentration under nursery condition. Since during this period, there was no carbon substrate, it is possible that the absence of a carbon source induced photosynthesis. In this carbon-limited condition, biomass could only be derived from CO; lated through Photosynthesis (Zelitch 1982). Since plants 15Table 4 substrate 30 days after inoculation. Growth responses of sugarcane to bacterial inoculation when grow in low nutrient pre-steriized Inoculation teatment Growth Responses Pant Height em) Shoot Weight (g) Uninoculated control 39.61" 038s" Lv? 49.36 o.gss* Lv10 4371" 0.798" Root Length (cm) __ Root Weight sor” 0.127 675" oss" 631% 0.360" ‘Ms sae oar lowe by th sre ere nisi fern a 5% eel ONT inoculated with LV7 and LV10 were significantly taller and accumulated a significant amount of biomass compared with the uninoculated plants, it is suggested that bacterial inoculation promoted sugarcane growth through @ mechanism that improves the photosynthetic rate, Increased Photosynthetic rates were reported following Thizobial inoculation (Peng 2000; Biswas et al 2000). They showed that certain strains of rhizobia ‘promote rice growth and yield through mechanism that improves single-teaf net photosynthetic rate rather than biological nitrogen fixation. Also, significant increase in root weight and root length (for LV7 only) compared to the uninoculated control suggests bacterial inoculation favored the quick growth of the root system considering that the plants needed to take up nutrients from the solution. Based on these observations, the mechanism of plant growth promotion is probably through the enhancement of root development, which resulted in increase uptake of nutrients fom the solution. We cannot discount, however, that other mechanisms of ‘growth promotion may be involved. Colonization, Swavival and Persistence of the Introduced Bacteria Plants inoculated with the GusA marked LV7 showed the characteristic blue color when stained with x-gluc, whereas uninoculated plants and those inoculated with the wild type did not show any blue 16 staining (Fig 2). Microscopic examination of the roots 2 DAI revealed slight blue staining on the root. tips. Six DAI, blue staining on the root tips and emerging lateral roots became evident (Fig 2C & D). The blue staining became intense 14 DAI wherein all of the emerging lateral roots were stained (Fig 3A), Further microscopic analysis indicated that the bacteria had entered the root tips and wounds caused by the splitting ofthe epidermis at the emergence of ‘young lateral roots (Fig 3B & C). The root tips, lateral roots as well as the vascular bundles are intensely stained with x-gluc 14 DAI (Fig 3 & 4). These results clearly indicate that the bacteria successfully colonized the sugarcane root. As early as 2 DAI, it had gained entry into the plant through the root tips. By 6 DAI, points of tateral root cemergence had been invaded such that the bacteria had reached the inside of root. Root tips and the lateral root emergence were the entry points of the invading microorganism. Semithin section (30 um) preparations analyzed under the microscope ‘confirmed that the bacteria colonized the epidermis and the cortex within the first 6 DAI (Fig 4D). Examination of root hairs 14 DAI showed no Gus staining, which would mean thatthe bacteria did not colonize root hairs. Cross sections (30 um) prepared from the roots sampled at 14 DAI confirmed as well the invasion of the vascular bundles (Fig 4C & 4B). Cross Growth enhancement of sugarcaneCross sections (30 um) prepared from the roots sampled at 14 DAI confirmed as well the invasion of the vascular bundles (Fig 4C & 4B). Cross sections of the lateral root emergence showed heavy ‘colonization by the bacteria (Fig 3C). From this study, the following conclusions can needed as they may be the predominant Np fixing heterotrophs in the rhizosphere, specifically of rice (Barraquio et al 1983; Watanabe et al 1987), and ‘most probably in sugarcane as well. By inhabiting the interior of the plants, they avoid competition phere bacteria and derived nutrients Fig? Gus staining ofthe sugarcane roots six days after inoculation. Plantets were inoculated with LV7 ‘expressing the Gus biomarker. Note the colonization of the root tips as indicated by blue color. A. Uninoculated control (30X) B. magnified view of the root tip ofthe uninoculated control (100X) C. inoculated plantiet (30X) D. magnified view of the root tip ofthe inoculated plantlt (100X) bbe made: That LV7 is an invasive colonizer capable of invading sugarcane root via root tips and fissures created by the emerging lateral root, It is able t0 colonize the root epidermis, cortex and the vascular bundles but not the root hairs. LV7 was identified to be Pseudomonas fluorescens. Acetylene reduction assay and nif PCR showed that it has nitrogenase activity, which indicates its diazotrophic nature. ‘According to James et al (1994), the endophytic nature of diazotrophie Pseudomonas spp. is clearly LC Villegas & ES Paterno directly from the host plants. It was also observed in this study that inoculation of the plants with LV7gus+ caused the formation of lateral roots different from the uninoculated plants ‘The inoculated plants developed short and thick lateral roots while the uninoculated were long and thin, This was similar to the report of Reddy et al (1997) wherein rhizobial inoculation promoted the formation of thick short lateral roots in rice. They buted this morphological response to the 0Fig3. Gus staining of the sugareane roots 14 days after inoculation. Note the concentration of bacteria on the lateral root emergence points as indicated by blue color. 4: Stained whole root system of the inoculated plantlet withthe emerging lateral roots (30X) B: magnified view of the lateral root emergence points (100X) C: crass section of the main root at the site of lateral root emergence (400X) AL & A2: Stained whole roots (30X) B: vascular bundle of the uninoculated control (100X)- C: Vascular bundle of the plantlet inoculated with the bacteria expressing the Gus biomarker (200X). D: Cross section of the main root as indicated in A2 showing bacterial colonization of the cortex. (400X). £: Cross ection of the main root as indicated in AI showing bacterial colonization ofthe epidermis, cortex and vaseular bundle (400X) production of IAA of the inoculant as well as cytokinins. The formation of short lateral roots in sugarcane as observed here may probably be due to the IAA or cytokinin-like substances produced by LVI. ‘The population estimates ofthe different strains prior to transplanting were 10° cfug fresh weight for 18 the roots and 0 to 10* efuvg fresh weight for the ‘culm (Table 5). LV7 and LV10 were detected both in surface sterilized roots and culms, which would imply that they are capable of colonizing other plant parts beside the roots. Therefore, the inoculation system favored the infection of the plant tissues by the bacteria, Growth Entancement Of Sugarcan’‘At 30 DAT, the introduced bacteria could be detected in the substrate and root surface. Population estimates ranged from 10° to 107.cfwmL. in the substrate and 10° to 10° efiwimL. in the rhizosphere (Table 5). Both strains were found inside the roots, as was observed at 14 DAI, but this time the population increased to 10° cfwmL which would imply that the inoculated bacteria did not only survive but able to establish themselves within the plant tissues. While LV7 was detected in the culm, LV10 was absent Population estimates ofthe introduced bacteria 14 days after inoculation accounted for both dead and viable cells. it could have been better if the initial population was ‘obtained from the medium by plate count right after inoculation. In that way, the viable bacteria could have been correctly accounted for and then compared with the final population estimates 30 DAT. Nevertheless, from the point of view of their persistence in the mixed substrate and in gnotobiotic culture in association with the plant, the selected isolates may be well suited for use as inoculant. To function asa biofertilizer or phytostimulator, nd 30 days after Table’. transplanting Inoculation Treatment Sample Plated Lv? Substrate Rhizosphere Root * Culm* Lvi0 Substrate Bacterial Population (cfulgfesh weight) MDAL 30DaT : 253% 10" : 35x10 8.45 x 10° 918K 1 503x10° Larx ot - Axe : 192x107 525x108 1.07 10° 157x 10" None The results obiained indicate that the inoculated bacteria persisted inside and outside the plans However, the population is relatively lower than in a large soybean nodule which can contain 10° bacteria. However, if the bacteria are distributed evenly throughout the large plant like sugarcane then these population estimates may be sufficient to exert positive effect on plant growth. Bacteria within oF outside plants should be sufficient in number to be of significance in plant growth promotion. The initial population of 10% cellsiml. was estimated only through turbidimetric method, which LC Villegas & ES Paterno a PGPB must be present at the right site and at the right time atthe place of action (Okon et al 1994). Since colonization can be considered as the delivery system of the microbe’s beneficial factors, the identified PGPB should be able to colonize sugarcane tissues and this was so with LV7 and LV10. This would allow the direct supply of benefits to the host plant. To the organism, there ‘would be reduced competition from other bacteria and a reliable supply of metabolic substrate. It is reported that the effects of PGPB inoculation are difficult to reproduce and as such only a few PGPB 19have been utilized in agricultural production (Reddy €t al 1991), Inconsistent results in the agronomic uses of PGPB are frequently attributed to poor rhizosphere colonization resulting from the adverse edaphic and environmental conditions. The ability to establish high population densities in the thizosphere is a characteristic suggested to be an essential factor forthe production of consistent plant growth responses (Bakker & Schippers 1987, Klein etal 1990, Kloepper et al 1991, Parke 1991). CONCLUSION & IMPLICATIONS Pseudomonas fluorescens LV7 and P. putida LV10 were identified as the most promising PGPB for sugarcane. LV7 and LV10 can be introduced back to sugarcane tissues to enhance their population in order to exert positive effects on sugarcane growth. To our knowledge, there is no report yet of a Pseudomonas species being applied ‘as microbial inoculant specific for sugarcane and Pseudomonas being incorporated into sugarcane through tissue culture inthe Philippines. ‘The information generated in this study has important implications in our ultimate goal: to identify an inoculant specific for sugarcane. Since ‘micropropagated plantlets are currently being used as planting materials in addition to sugarcane seedpieces and that the Philippine sugar industry has ‘well established micropropagation laboratories that are currently being used in the rapid propagation of new high yielding varieties, we can therefore capitalize on this system in devetoping our inoculant. We recognize the fact that ‘micropropagated plantlets are produced free of microbial contaminants and therefore are ideal for inoculation prior to transplanting in the field. With this, all the crucial and dificult aspects of producing viable, cost-effective and user-friendly inoculant formulations are eliminated, Instead of developing a formulated inoculant in powder, granule or liquid form, the microbial directly incorporated into the host plant through the micropropagation system such that a biotized plant is produced, Since colonization is the delivery system of the ‘microbe’s beneficial factors, what is essential is that inoculum is the bacteria should be able to colonize plant tissues Internal colonization would allow the direct supply of benefits to the host plant; and for the introduced organism, there would be reduced competition from other bacteria and a reliable supply of metabolic. substrate Since the inoculant is incorporated into the plantlets, the ultimate product then is a biotized sugarcane plant which can then be made available for farmers. There is no need for reinoculation because the introduced bacteria persist and grow along with the plants and therefore should exert the beneficial effects throughout the life cycle of the plant, Since sugnreane is propagated using cutings, the original bacterial population is carried in the subsequent plants, With that, the seedpieces derived from the inoculated plantlets need not be inoculated again but can be directly used as planting materials, In this way, a significant economic advantage can be realized. And far and beyond this economic advantage will be the environmental benefits Acknowledgment This study was funded by the Philppine Sugar Research Institute Foundation Inc, for which the euthors are grate 20 Growth Enhancement Of SugarcaneLiTeRATURE CITED Alexander DB & DA Zuberer. 1991. Use of chrome azurol $ reagents to evaluate siderophore production by rhizosphere bacteria, Biology and Fertility of Soils 12: 39-45 Bakker AW & B Schippers. 1987. 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