Biology and Fertility of Soils

, Volume 41, Issue 5, pp 350–358

Pink-pigmented facultative methylotrophic bacteria accelerate germination, growth and yield of sugarcane clone Co86032 (Saccharum officinarum L.)

Authors

    • Department of Agricultural ChemistryChungbuk National University
  • S. Poonguzhali
    • Department of Agricultural ChemistryChungbuk National University
  • H. S. Lee
    • Department of Agricultural ChemistryChungbuk National University
  • K. Hari
    • Division of Crop Production, Sugarcane Breeding InstituteICAR
  • S. P. Sundaram
    • Department of Agricultural MicrobiologyTamilnadu Agricultural University
  • T. M. Sa
    • Department of Agricultural ChemistryChungbuk National University
Original Paper

DOI: 10.1007/s00374-005-0838-7

Cite this article as:
Madhaiyan, M., Poonguzhali, S., Lee, H.S. et al. Biol Fertil Soils (2005) 41: 350. doi:10.1007/s00374-005-0838-7

Abstract

The existence of Methylobacterium as a symbiont with sugarcane and its influence on crop growth at various stages was examined. Pink-pigmented facultative methylotrophic bacteria (PPFMs) strains isolated from different parts of the sugarcane clone Co86032 showed growth on methanol, and were further confirmed based on the mxaF gene encoding the α-subunit of the methanol dehydrogenase by polymerase chain reaction amplification using specific primers. True seeds inoculated with PPFMs had a higher germination percent and rate of germination than the control. A combined treatment of seed imbibition, soil application and phyllosphere spray increased specific leaf area, plant height, number of internodes, and cane yield. Immunological determination of cytokinin in young and mature leaves significantly increased when the epiphytic population on the leaf surface increased. Trends in sugar qualities in the form of Pol (sucrose) % in cane, Brix % in cane, and commercial cane sugar were similar to that of cane yield. These effects might be mediated by the production or synthesis of plant hormones.

Keywords

Pink-pigmented facultative methylotrophic bacteriaMethylobacterium spp.Plant hormonesTrue seed germinationSpecific leaf area

Introduction

Pink-pigmented facultative methylotrophic bacteria (PPFMs), ubiquitous in nature and frequently reported on various plant species, are a substantial part of the aerobic, heterotrophic microflora of the surfaces of young leaves. They were first isolated as covert contaminants from the tissue cultures of liverwort, Scapania nemorosa (Basile et al. 1969), but later identified as belonging to the genus Methylobacterium. PPFMs are bacteria capable of growing on C1 compounds like methanol and methylamine and also on a variety of C2, C3, and C4 compounds, and are classified as Alphaproteobacteria (Patt et al. 1976; Lidstorm 1992). These bacteria are commonly found in soils, as well as on the surfaces of leaves, seeds and in the rhizosphere of a wide variety of plants, with highest numbers present on actively growing and meristamatic tissue, sometimes averaging 104–106 colony-forming units (cfu) per leaflet (Dunleavy 1988; Corpe 1985; Corpe and Rheem 1989; Hirano and Upper 1992; Holland and Polacco 1992, 1994; Chanprame et al. 1996; Holland 1997; Shepelyakovskaya et al. 1999). They have been reported to influence seed germination and seedling growth by producing plant growth regulators like zeatin and related cytokinins and auxins (Dileepkumar and Dube 1992; Holland and Polacco 1992, 1994; Holland 1997; Ivanova et al. 2001; Omer et al. 2004) and to alter agronomic traits like branching, seedling vigour, rooting and heat/cold tolerance (Holland 1997; Freyermuth et al. 1996). Methylotrophs indirectly reduce or prevent the deleterious effects of pathogenic microorganisms, through induced systemic resistance (Madhaiyan et al. 2004), and their inoculation was found to increase the photosynthetic activity by enhancing the number of stomata, chlorophyll concentration and malic acid content of crops (Cervantes-Martinez et al. 2004).

The sugarcane (Saccharum officinarum L.) crop, covering an area of >4.0 million ha worldwide with an average productivity of about 67.8 t ha −1offers definite scope for improvement. Its fertilizer consumption being higher than that of other crops might negatively affect soil health in the long term. The bioinoculants used as alternatives to fertilizer usually target only the rhizosphere, and less attention has been paid to the phyllosphere. Although there are no prior reports on the beneficial effects of methylotrophic bacteria on the sugarcane phyllosphere, Holland and Polacco (1994) and Holland (1997) reported the beneficial effects of methylotrophic bacteria on various crop plants. In the present study, we report the beneficial effect of methylotrophic bacterial inoculation on true seed germination, early growth of sugarcane, plant colonization, cytokinin contents, cane yield and sugar quality.

Materials and methods

Bacterial isolates and cultural conditions

We employed five methylobacterial strains, PPFM-So77, PPFM-So78, PPFM-So79, PPFM-So80, and PPFM-So81, isolated from leaf, germinated buds, stalk, root, and rhizosphere of sugarcane clone Co86032, respectively (Madhaiyan 2003) and two reference strains, Methylobacterium extorquens AM1 and M. extorquens miaA mutant. All the methylotrophic strains were polymerase chain reaction (PCR) amplified to detect the presence of methanol oxidation genes using the nondegenerate primers mxa f1003 (5′-GCG GCA CCA ACT GGG GCT GGT-3′) and mxa r1561 (5′-GGG CAG CAT GAA GGG CTC CC-3′) defined from conserved parts of mxaF genes (McDonald and Murrell 1997). An amplification product of the expected size, 560 bp, for all the isolates from sugarcane similar to reference strain M. extorquens AM1 were noted (Fig. 1). These bacteria were grown on ammonium mineral salt (AMS) medium (Whittenbury et al. 1970), supplemented with 0.5% (v/v) methanol as a sole C source and cycloheximide (20 μg ml−1) (Corpe 1985; Corpe and Basile 1982) and incubated at 28°C on a shaker at 120 r.p.m. under a 16-h photoperiod. M. extorquens miaA mutant was grown in the medium containing 15 μg tetracycline ml−1 (Sy et al. 2001). Bacterial cells harvested at the end of the exponential growth phase with a suspension density of 1.6–1.8 optical units at 600 nm and a concentration of 109 bacterial cell ml−1 were used for colonizing plants.
Fig. 1

Polymerase chain reaction amplification products of methanol dehydrogenase (mxaF gene) obtained by using mxa f1003 and mxa r1561 primers. Lane 1 Negative control, lane 2 Methylobacterium extorquens (Me) AM1, lane 3 sugarcane isolated pink-pigmented facultative methylotrophic bacteria (PPFM)-So77, lane 4Me PPFM-So78, lane 5 PPFM-So79, lane 6 PPFM-So80, lane 7 PPFM-So81, M molecular marker of 500-bp DNA ladder

True seed germination of sugarcane

To determine whether stimulation of germination was due to PPFM inoculation, we tested the effects of methylotrophic bacteria on true seed germination under in vitro conditions. Seed germination studies were carried out as described by M. A. Holland (personal communication). A total of 0.1 g true seeds (fuzz) was immersed in 0.525% NaOCl solution for 30 min followed by heat treatment at 50°C for 48 h for removal of seedborne methylotrophic bacteria (Holland and Polacco 1992). The heat-treated seeds were sparsely placed (approximately 200 seeds/plate) in a Petri dish containing a thin layer of sterile absorbent cotton and were imbibed in liquid culture or spent media (stationary phase culture) for 5 h at room temperature. Plates with fresh AMS medium were used for the control. The Petri dishes were incubated at 25°C for 7 days with 16-h light and 8-h dark periods. The seeds germinated over wet absorbant cotton (Fig. 2), and the germinated seedlings were counted; the results are expressed in percentages.
Fig. 2a–d

Effect of PPFMs on true seed germination of sugarcane. a Untreated true seeds, bMe AM1, cMe miaA mutant, dMe strain PPFM-So78. For abbreviations, see Fig. 1

Pot culture study

Experiments were conducted at Tamilnadu Agricultural University Experimental Station, Department of Agricultural Microbiology, Coimbatore, India, situated in the northwestern agroclimatic zone of Tamilnadu (11°N, 77°E; 426.72 m a.s.l.). Pot experiments were conducted to study the response of sugarcane to different methods of methylotroph application. The various treatments and the methods used are given below. The experimental design was a completely randomized block with three replications. The soil used had the following physiochemical properties: pH 7.7, CEC 26.67 cmol(p+) kg−1, EC 0.42 dSm−1, organic C 0.57%, available N 228 kg ha−1, available P2O5 11 kg ha−1, available K2O 267 kg ha−1 and available Ca 11.5 cmol kg−1, Zn 2.83 ppm, Fe 29.81 ppm, Mn 4.12 ppm and Cu 2.42 ppm. The soil samples were collected from an experimental field, dried and passed through a 4-mm sieve, and mixed with farmyard manure (2:1, w/w); 60-l pots were filled with 50 kg of the mixture. Sugarcane setts (clone Co86032) were collected from the sugarcane experimental field (Tamilnadu Agricultural University, Coimbatore) at the time of planting. The setts were dipped in 10 l bacterial suspension for 5 h, and planted in pots (sett treatment). For the soil application (SA), the bacterial suspension (25 ml pot−1) was poured after planting the untreated setts, and for the foliage application 25 ml of bacterial suspension per pot was sprayed over the foliage on 30, 60, and 90 days after planting (DAP) using a hand sprayer. For the control, the foliage of non-foliage treated plants was sprayed with distilled water. All the treatments were replicated 6 times in a factorial randomized complete block design. Filtered tap water was used to irrigate pots. Plants were harvested 3, 6 and 10 months after planting and subjected to biometric analysis.

Specific leaf area

Forty-five and 90 days after emergence (DAE), the treated and untreated plants were uprooted and used to measure leaf area and specific leaf area (SLA) (Terauchi et al. 2001). The leaf area was measured using a LI-3000 portable area meter (LICOR, Lincoln, Neb.) and the SLA, calculated after drying the leaf samples to a constant mass at 70°C, is expressed in cm2 g−1.

Measurement of leaf PPFMs population sizes

PPFMs populations were enumerated on both abaxial and adaxial surfaces of leaves by a leaf imprinting method (Corpe 1985; Holland et al. 2002) and expressed in cfu cm−2. Fully expanded leaves were collected and the samples were handled aseptically. Representative samples of a large number of leaves (three leaves per treatment) were cut into small pieces to fit them into 10-cm-diameter Petri dishes and were then pressed firmly onto the surface of AMS solid medium (Corpe and Basile 1982). A serial dilution method was employed to analyse population size in young and matured fully expanded leaves at different stages of crop growth. Leaf homogenates prepared by homogenizing the leaves separately in 5 ml sterile distilled water in an autoclaved glass teflon homogenizer with the pestle driven by a drill press, were serially diluted and 1.0-ml aliquots were plated and incubated at 28°C under a 16-h photoperiod. PPFMs producing visible pink colonies were counted after 7 days and expressed as cfu g−1 fresh weight leaves (Chanprame et al. 1996).

Hormone analysis

Cytokinins from freeze-dried material of young and matured leaves were extracted using 80% methanol (Matsuchke and Machackova 2002), and the water phase of the extract was purified on P-cellulose in dilute acetic acid (pH 3.0), DEAE-cellulose in 0.04 M ammonium acetate buffer pH 6.5 and C-18 Sep-Pak cartridges, from which cytokinins were eluted by 80% methanol. The individual cytokinins, dihydrozeatin riboside, trans-Zeatin riboside and isopentenyladenosine were separated by HPLC on a C18 reverse phase column (Machackova et al. 1993). The amount of cytokinins in individual HPLC fractions was determined by ELISA performed with antibodies and conjugates with an alkaline phosphatase phytodetek kit (AGDIA, Ind.) and was compared with cytokinin standards (Sigma, USA).

Sugar quality

The Brix–Pol analysis was carried out immediately after the cane juice samples were extracted from three canes cut at random from each treatment. Brix % in cane was measured as follows: \({\text{Brix \% = Brix in first expressed juice}} \times {\left[ {{\text{100 - (\% fibre + 3)}}} \right]}{\text{/100}}\). Sucrose content was determined as percent Pol, where Pol was read from a saccharimeter (Meade 1963; Meade and Chen 1977). Pol % in cane was measured as follows: \({\text{Pol \% = Pol in first expressed juice}} \times {\left[ {{\text{100 - }}{\left( {{\text{\% fibre + 5}}} \right)}{\text{/100}}} \right]}\). Commercial cane sugar (CCS) provides an estimate of the recoverable sucrose from sugarcane. CCS can be calculated when both Brix and Pol in cane are known: CCS=pol in cane−0.75 (impurities in cane×40/60) (Mathur 1961; Engelke 2002).

Statistical methods

A completely randomized design was used in all experiments. The data were subjected to statistical analysis and significant difference was calculated at P=0.05 using SAS version 8.2 (SAS 2001). Appropriate arcsine transformation was used to normalize the data. Bacterial population data were log10 transformed prior to calculations.

Results

Effect of bacterial inoculation on true seed germination

PPFMs had significant and consistent stimulating effects on the germination of treated true seeds of sugarcane. To determine whether methylotrophic bacteria can stimulate seed germination, we tested seven strains of M. extorquens, M. extorquens AM1, M. extorquens miaA mutant, PPFM-So77, M. extorquens strain PPFM-So78, PPFM-So79, PPFM-So80, and PPFM-So81, in their spent media (Table 1). The degree of stimulation obtained among sugarcane differed according to the strain employed. M. extorquens PPFM-So78 stimulated better seed germination of sugarcane true seeds than the other strains. In the case of nonheated seeds, the bacterial inoculation was found to be more effective than clarified spent media. Inoculation with M. extorquens PPFM-So78 strain produced the highest germination (24.41% increase over control), followed by strain PPFM-So80 (18.79% increase over control).
Table 1

Effect of pink-pigmented facultative methylotrophic bacteria (PPFMs) inoculation on quality of sugarcane true seed under in vitro conditions. Values represent all treatment with an average of three replicates of 200 seeds each. Values in parentheses are arcsine-transformed values. In the same column, significant differences according to LSD at P=0.05 are indicated by different letters. Me Methylobacterium extorquens

Strains

Sugarcane true seed treatment

Rate of germination

Nonheated

Heated (50°C, 48 h)

Culture

Spent media

Culture

Spent media

Me AMI

61.04 (33.52) b

56.88 (32.21) b

64.20 (34.50) b

63.66 (34.34) c

5.31 c

Me miaA mutant

60.96 (33.50) b

56.28 (32.03) b

62.84 (34.08) b

60.96 (33.50) d

5.42 b

PPFM-So77

51.52 (30.48) c

48.52 (29.48) c

56.64 (32.15) c

52.98 (30.96) e

6.11 c

Me PPFM-So78

70.56 (36.42) a

68.50 (35.80) a

76.24 (38.13) a

74.46 (37.60) a

7.18 a

PPFM-So79

46.76 (28.91) d

46.66 (28.85) d

50.24 (30.06) d

48.30 (29.42) f

5.14 c

PPFM-So80

62.20 (33.90) b

49.44 (29.79) c

72.64 (37.05) a

70.96 (36.56) b

5.49 bc

PPFM-So81

44.86 (28.26) ed

45.72 (28.56) d

46.36 (28.76) e

45.44 (28.45) g

5.63 bc

Control

42.80 (27.53) e

43.20 (27.68) e

38.24 (25.85) f

37.00 (25.46) h

5.10 c

LSD (P=0.05)

0.985

2.145

2.145

0.463

0.628

The rate of germination (RG) of sugarcane seeds was calculated based on the germination counts recorded over time. The inoculated seeds had increasing RG over the control. The RG of PPFM-treated seeds ranged from 5.14 to 7.18 and in the control it was 5.10 (Table 1). The difference in RG between PPFM strains could be related to the ability of individual strains to promote early germination. From the results, it is evident that M. extorquens PPFM-So78 strain induced growth earlier than the control.

Growth and yield parameters

Pot experiments established the efficacy of M. extorquens PPFM-So78 in improving plant growth as determined by plant height, number of internodes and cane yield (Table 2). Though maximum plant growth was recorded in the treatments where the strain was applied by sett imbibition (SI) plus SA plus phyllosphere spray (PS), and by SI+PS, during all stages of crop growth, plant growth in response to individual methods of strain application differed. Application of the M. extorquens strain PPFM-So-78 by all three methods viz, SI+SA+PS, led to higher biometric measurements when compared to the control and other treatments at P=0.05. Treatments which included SI+PS gave the next highest biometric results.
Table 2

Effect of different methods of application of Me PPFM-So78 on sugarcane clone Co86032. Each value represents mean±SE of three replicates per treatment. The data were statistically analysed using DMRT. In the same column, significant differences according to LSD at P=0.05 are indicated by different letters

Sugarcane strain Me PPFM-So-78

Germination (%)

Plant height (cm)

Number of internodes

Cane yield (kg per cane)

After 3 months

After 6 months

After 10 months

Sett imbibition (SI)

89.29±3.08 a

95.91±3.40 c

175.40±6.32 e

221.92±5.10 c

21.12±0.25 abc

1.16±0.07 b

Phyllosphere spray (PS)

70.62±2.68 b

96.32±3.45 c

180.60±5.00 d

237.24±8.13 b

21.23±0.23 abc

1.17±0.07 b

Soil application (SA)

75.35±3.45 b

89.36±4.34 d

165.23±3.55 g

217.63 cd±3.60 cd

20.56±0.22 bc

1.00±0.04 c

SI+PS

89.35±3.84 a

104.40±4.43 b

195.20±6.22 b

240.24±7.18 ab

21.67±0.22 ab

1.24±0.09 ab

SI+SA

89.14±2.94 a

96.56±2.95 c

170.31±3.54 f

222.42±4.91 c

21.44±0.21 abc

1.18±0.02 b

PS+SA

75.74±2.47 b

96.82±3.40 c

182.62±5.90 c

237.55±9.69 b

21.42±0.22 abc

1.19±0.05 b

SI+PS+SA

89.36±3.94 a

107.32±4.40 a

198.31±5.90 a

247.87±7.48 a

22.30±0.21 a

1.31±0.02 a

Control

70.69±1.75 b

79.21±4.34 e

161.20±3.58 h

210.14±4.73 d

20.32±0.21 c

0.90±0.07 d

LSD (P=0.05)

7.49

2.22

4.67

7.70

1.31

0.09

Specific leaf area

At 45 and 90 DAE, both SI and PS treatments had increased SLA compared to other treatments. The SI+SA+PS treatment gave the highest SLA followed by SI+PS, SI+SA, PS+SA, SI, PS, and SA treatments at 45 DAE. The trend differed slightly at 90 DAE, where the maximum SLA was recorded in the SI+SA+PS treatment followed by SI+PS, PS+SA, PS, SI+SA, SI, and SA (Fig. 3).
Fig. 3

Variation in the specific leaf area [leaf area (cm2 plant−1)/dry weight (g plant−1)] of sugarcane clone Co86032 during the early growth stage due to different methods of application of methylotrophic bacteria strain Me PPFM-So78. Error bars indicate ±SE. SI Sett imbibition, SA soil application, PS phyllosphere spray, DAE days after emergence; for other abbreviations, see Fig. 1

Determination of sugar quality

Cane yield increased significantly with the combined application of Methylobacterium by SI+SA+PS (Table 3). The measurements of Pol (%), Brix (%) and CCS contribute to an assessment of the quality of sugarcane. Methylobacterium-treated plants had significantly higher values of Pol (%), Brix (%) and CCS compared to the control. The trends were similar to that of cane yield. The treatments which combined the three methods (SI, SA and PS) gave a higher percentage of Pol (%) and Brix (%), 15.03 and 18.02, respectively, and a CCS of 14.85. For the control 13.32, 15.69 and 13.14 were recorded for Pol (%), Brix (%) and CCS, respectively. The other treatments followed the order: SI+PS, SI+SA, PS+SA, PS+SA, PS, SI and SA.
Table 3

Sugar compositions of clone Co86032. Each value represents mean±SE of three replicates per treatment. In the same column, significant differences according to LSD at P=0.05 are indicated by different letters. CCS Commercial cane sugar, Brix (%) % Brix of sugarcane, Pol (%) % Pol of sugarcane

Sugarcane strain Me PPFM-So-78

Brix (%)

Pol (%)

CCS

Sett imbibition (SI)

16.50±0.38 d

14.17±0.17 cd

14.01±0.17 ab

Phyllosphere spray (PS)

17.00±0.24 c

14.25±0.49 cd

13.88±0.64 b

Soil application (SA)

16.32±0.34 d

14.07±0.32 d

13.95±0.62 ab

SI+PS

17.56±0.36 b

14.73±0.53 ab

14.32±0.45 ab

SI+SA

17.27±0.34 bc

14.30±0.42 cd

13.82±0.59 b

PS+SA

17.41±0.57 b

14.52±0.41 bc

13.93±0.48 ab

SI+PS+SA

18.02±0.43 a

15.03±0.37 a

14.85±0.55 a

Control

15.69±0.51 e

13.32±0.33 e

13.14±0.34 c

LSD (P=0.05)

0.395

0.419

0.594

PPFMs population size

The population of PPFMs in general was higher on the abaxial surface than the adaxial surface of leaves. The PPFM density ranged from 33 to 52 cfu/cm2 in treated plants, while for untreated plants the density was only 18 cfu cm-2 3 months after planting (Fig. 4a, b). The population size increased significantly after 6 months. Among the treatments the combined application using SI+SA+PS gave a maximum population (55 cfu/cm2) followed by SI- and PS-treated plants. The population declined after 10 months in both treated and untreated plants. While PPFMs covered the entire leaf surface of treated plants, in untreated plants, the distribution of PPFMs was more restricted or the population size was low, and these bacteria generally were abundant near the margins of the abaxial surfaces of the sugarcane leaves.
Fig. 4

PPFMs population [colony-forming units (cfu)/cm2] of young (a) and mature (b) leaves of sugarcane at different stages of plant growth. Error bars indicate ±SE. For other abbreviations, see Figs. 1 and 3

The maximum PPFM population was recorded in young leaves at all three sampling times. The population size reached the maximum at 6 months after planting, and ranged from 5.3 to 7.32 log cfu g−1 in young leaves and 4.1–5.04 log cfu g−1 in mature leaves (Fig. 5a, b) which was higher than in untreated plants, where it ranged from 5 and 3 log cfu g−1 in young and mature leaves, respectively.
Fig. 5

PPFMs population of young (a) and mature (b) leaves of fresh tissue of sugarcane at different stages of plant growth. Error bars indicate ±SE. For abbreviations, see Figs.  1, 3 and 4

Hormone analysis

The cytokinin contents significantly increased when the total epiphytic population increased in leaves. Young leaves had higher cytokinin contents than older leaves, mediated by the methylotrophic population. Among the treatments, sugarcane plants that received a combined application of SI+SA+PS showed maximum cytokinin contents followed by SI and PS treatments. The cytokinin contents increased during early stages of plant growth, reached the maximum at 6 months, and declined at 10 months (Fig. 6a, b).
Fig. 6

Effect of PPFMs inoculation on cytokinin contents of young (a) and mature (b) leaves of sugarcane clone Co86032. Error bars indicate ±SE. FW Fresh weight; for abbreviations, see Figs.  1 and 3

Discussion

It is well established that PPFMs are colonizers of a wide array of crops and are capable of growing on C1 compounds as well as on a wide range of multicarbon substrates (Green and Bousifield 1983; Green 1992). All isolates used in the study were Gram-negative rods and grew oxidatively on methanol-amended medium. Based on cultural, biochemical and C utilization tests the isolates were identified as Methylobacterium spp. (Green and Bousifield 1982; Oppong et al. 2000). The presence of a methanol dehydrogenase gene encoded by mxaF, is well conserved among methylotrophic bacteria (Machlin and Hanson 1988), and confirms the above results. The mxaF gene has been successfully amplified and sequenced using nondegenerate primers f1003 and r1561, and produced an amplified product of 560 bp (McDonald et al. 1995; McDonald and Murrell 1997).

Studies showing that the imbibition of heat-treated seeds with PPFMs restores germination frequency of soybean seeds indicate their positive role (Holland and Polacco 1994). In our study, experiments on true seed germination of sugarcane showed significantly higher results when seeds were treated with PPFMs. This corroborates results obtained in soybean and maize when their seeds were imbibed with PPFMs (Holland and Polacco 1994; Madhaiyan et al. 2002).

The pot culture study revealed that all the treatments viz, SI, SA and PS, resulted in a significant increase in sugarcane plant growth, cane yield and sugar quality. Earlier studies also report the beneficial effects of application of methylotrophic bacteria through SI (Holland and Polacco 1994) and PS (Holland 1997) in increasing soybean plant growth and productivity. SLA can be employed as a breeding index when seeking to improve the early growth of sugarcane, and a smaller SLA is considered as one of the main reasons for the slow growth of sugarcane at the early growth stage (Terauchi and Matsuoka 2001) that leads to low productivity. Rapid expansion of leaf area at the early growth stage of sugarcane plants as well as crop growth, influenced by methylotroph inoculation, could be related to beneficial mutualistic interactions.

The abaxial surface of leaves always registered a higher PPFM population than the adaxial surface (Madhaiyan et al. 2004). This could be directly related to the emission of methanol through stomatal openings during leaf expansion by pectin demethylation (Nemecek-Marshall et al. 1995), which may be utilized by PPFMs to survive on the surface of leaves. Conversely, a smaller population on the adaxial surface could be attributed to several biotic and abiotic factors viz, rainfall, wind, irradiation, temperature, and humidity and competition for nutrients with other epiphytic bacteria and fungi (Hirano et al. 1996; Lilley et al. 1997; Lindemann and Upper 1985; Walker and Patel 1964; Beattie and Lindow 1995; Hirano and Upper 2000). The variation in the population of PPFM on the surfaces could also be attributed to the foliar spray applied at 30, 60, and 90 DAP. The population density of PPFM was higher in young leaves compared to mature leaves, shown by the proportional total cytokinin contents due to their activity.

There are a number of measurements that contribute to an assessment of the quality of sugarcane viz, Pol (sucrose) % in cane; Brix % in cane; fibre %; CCS; and purity (Meade 1963; Meade and Chen 1977). In the present study, the sugar quality parameter was significantly increased in all Methylobacterium treatments. These effects were generally attributed to the synthesis of biologically active compounds, such as cytokinins and auxins, pyrroloquinoline quinone synthase, urease, and polyhydroxybutyrates, and the ability of methylotrophs to efficiently survive on leaves by using methanol, a product of pectin demethylation that is released through stomata (Holland and Polacco 1994; Ivanova et al. 2000, 2001; Freyermuth et al. 1996; Avezoux et al. 1995; Knani et al. 1994; Breuer et al. 1995). The sugarcane growth also significantly increased as a result of PPFM inoculation through SI and SA initially, and by PA given at 30, 60 and 90 DAP, the latter being the treatment which provides a larger contact area for PPFMs allowing them to adhere for a longer period thus facilitating their establishment on the surface.

Traditionally, the study of cytokinin production by plant-associated bacteria has been associated with microbes known to cause plant disease or to enter into an intimate symbiosis with a plant host. Koenig et al. (2002) sought to rectify the omission of plant commensal bacteria from this field of study by making a detailed examination of cytokinin production by PPFMs. Circumstantial evidence indicates that plant hormones might be produced by these bacteria (Corpe and Basile 1982). Methylotrophic strains representing leaf isolates are reported not only to produce cytokinin in pure culture but also to excrete it into the culture medium (Long et al. 1997; McDonald and Murrell 1997; Ivanova et al. 2000; Koenig et al. 2002). The first report on the production of indole acetic acid in significant amounts by four different methylotrophs was by Ivanova et al. (2001). As mentioned earlier, the mechanism behind cytokinin production during symbiosis requires further investigation to determine whether it could be attributed to its induction in plants or to the production of cytokinins compounds by PPFMs (Holland 1997).

Sugarcane breeding programs typically commence by exploiting a large number of seedlings derived from true seed, whose storage will preserve the genetic resources of the wild canes, needed to enhance the improvement of cultivars (Peter et al. 1998). Methylotrophic bacteria used to treat seeds to prolong storage (Holland and Polacco 1994) and improve germination rates, as observed in this study, will be helpful in supporting the germplasm conservation of wild canes through true seed storage. Most of the earlier reports on methylotrophs concentrated on soybean (Long et al. 1997), maize (Madhaiyan et al. 2002), Arabidopsis and safflower (Holland and Polacco 1994) while this study forms the first of its kind on sugarcane, and potentially opens a new area of economic development. Because PPFMs are able to colonize plant tissue cultures, an examination of cytokinins produced by PPFMs may shed some light on the importance of bacterially produced cytokinins for normal plant growth and development. The potential use of PPFMs in cultured tissues remains unproven but may prove valuable.

Acknowledgements

We thank M. A. Holland (Department of Biology, Salisbury University, Salisbury, Maryland) and Joe C. Polacco (Department of Biochemistry, University of Missouri–Columbia, Columbia, Missouri), for their valuable suggestions and provision of strains. The authors thank the Korea Science and Engineering Foundation for financial assistance through the Research Centre for the Development of Advanced Horticultural Technology at Chungbuk National University.

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© Springer-Verlag 2005