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Rhizosphere microbial community structure at different maize plant growth stages and root locations
ARTICLE IN PRESS Microbiological Research 164 (2009) 391—399
www.elsevier.de/micres
Rhizosphere microbial community structure at different maize plant growth stages and root locations Lilia Cavaglieria,b,, Julieta Orlandoa, Miriam Etcheverrya,b a
Departamento de Microbiologı´a e Inmunologı´a. Universidad Nacional de Rı´o Cuarto. Ruta 36 km 601. 5800. Rı´o Cuarto. Co ´rdoba. Argentina b Member of Consejo Nacional de Investigaciones Cientı´ficas y Te´cnicas (CONICET), Argentina Accepted 29 March 2007
KEYWORDS Culturable microflora; Maize plant growth stages; Rhizosphere
Summary The aims of the present work were (1) to determine the influence of plant growth stages on the population size of culturable bacteria and fungi associated with rhizoplane and endo-rhizosphere of maize grown in field and (2) to establish the community structure of total culturable bacteria and fungi. Density, diversity and community structure of culturable rhizoplane and endo-rhizosphere populations at different maize plant growth stages were estimated. Plant development did not have influence on total culturable microflora density but it selectively influenced some bacterial and fungal groups present in the rhizosphere. However, the microbial community structure changed markedly over time. This knowledge is relevant for exploring endophytic rhizobacteria potential in the promotion of plant-growth, protection against pathogens and to detect perturbations in natural agro ecosystems. & 2007 Elsevier GmbH. All rights reserved.
Introduction The study of microbial communities associated with plants is important for understanding their ecological role in nature environments. Microorganisms, which establish positive interactions with Corresponding author.
E-mail address: [email protected] (L. Cavaglieri).
plant roots, play a key role in agricultural environments and are promising for their potential use in sustainable agriculture (Di Cello et al., 1997). Microbial populations can undergo temporary variations in its structure due to the selective pressure exerted by the environment that causes exchanges within a local population and migrations between distinct populations. Knowledge of such process in the rhizosphere can help in relating microbial
0944-5013/$ - see front matter & 2007 Elsevier GmbH. All rights reserved. doi:10.1016/j.micres.2007.03.006
ARTICLE IN PRESS 392 community changes to environmental variations over time (Wise et al., 1996). For example, it is well known that production and diffusion of root exudates are affected by plant development (Hamlen et al., 1972). These exudates exert a selective microbial stimulation (Buir and Caesar, 1984, Miller et al., 1989) that varies in function of time due to plant age. Consequently, the development of more adapted microorganisms at different plant growth stages may be favored. Di Cello et al. (1997) found that Burkholderia cepacia populations associated with maize roots decreased significantly during plant development. Moreover, several studies on the rhizosphere microbial community structure of wheat and maize revealed that fastgrowing bacteria (r-strategist) were predominant on young immature roots, whereas slow growing bacteria (k-strategist) became predominant on mature roots (De Leij et al., 1995, Nacamulli et al., 1997). Young and Kucharek (1977) reported the increase Trichoderma viride and Fusarium moniliforme counts in roots from the silking stage through subsequent growth stages. On the other hand, Windham (1983) found that fungi generally were isolated more frequently from roots of seedling stages than from roots of plants at silking stage. The capacity of bacteria and fungi to colonize internal tissues of plants could confer an ecological advantage over others that can only colonize plants epiphytically (Hallmann et al., 1997). Rhizosphere is widely accepted to be an important source of root endophytes, and most endophytic species are present in the rhizosphere (Germida et al., 1998, Sessitsch et al., 2002). This knowledge is relevant for exploring endophytic rhizobacteria potentially promoting plant-growth and protecting the plant against pathogens (Benhamou et al., 2000, Stirz and Nowak, 2000). Frommel et al. (1993) recorded a positive correlation of potato growth stimulation and yield increase with the occurrence of high population densities of Pseudomonas sp. on and in potato roots. Hinton and Bacon (1995) found one isolate of Enterobacter cloacae, endophytically associated with corn enhanced the biological control of systemic pathogens of corn. Among the microorganisms inhabiting the maize rhizosphere, Fusarium Section Liseola are the major soil-borne fungal pathogens (Marasas et al., 1984). Pathologists have special interest to control these pathogens. Therefore, it is important to characterize the indigenous microbial communities naturally associated with maize root systems to identify potential biocontrol agents. Although it has long been known that indigenous microorganisms are naturally associated with maize
L. Cavaglieri et al. roots, no experimental work has been reported for Cordoba, Argentina, maize production area to study the structure of native rhizosphere microbial populations at all growing stages. The aims of the present work were to: (i) determine the influence of plant growth stages on the population size of culturable bacteria and fungi associated with rhizoplane and endorhizosphere of maize grown in the field. (ii) establish the community structure of culturable bacteria and fungi.
Materials and methods Sampling Root samples (Zea mays L.) from an experimental maize field of Universidad Nacional de Rio Cuarto, Rio Cuarto, Cordoba, Argentina, were collected. The soil was loamy sand. Plants were collected throughout the 2003 growing season at the five more significant stages of maize development (Table 1). At each sampling, eight plants were randomly harvested.
Estimation of rhizosphere microbial populations To quantify culturable rhizoplane populations, roots were excised and loosely adhering soil was removed. Each root was weighted, blended and resuspended in phosphate-buffered saline (PBS, Oxoid Ltd., London, UK). Serial dilutions of these suspensions were plated onto the following media: (i) tryptic soy broth plus 2% agar (TSBA) in order to estimate total culturable bacteria; (ii) selective King0 s medium B (King et al. 1954) for pseudomonads; (iii) dichloran rose bengal-chloramphenicol agar (DRBC) (Abarca et al. 1994) to estimate total culturable micoflora; (iv) Aspergillus flavus and parasiticus agar (AFPA) for potentially aflatoxigenic fungi; (v) Nash-Snyder medium to enumerate Fusarium species (Nelson et al. 1983). Table 1. Microbial isolation from maize roots during plant development Sampling
Time (days)
Maize growth stage
I II III IV V
20 35 60 80 130
Germination Elongation Tassel appearance End of flowering Physiological maturation
ARTICLE IN PRESS Rhizosphere microbial community structure at different maize plant growth stages To quantify endo-rhizosphere microflora, roots were washed and dried between sheets of tissue, weighed and surface sterilized by gently shaking in 70% ethanol (1 min), 20% household bleach (5 min) and thiosulphate Ringer solution (5 min) (Oxoid Ltd., London, UK). Roots were macerated in 90 ml of PBS with a mortar and pestle. Serial dilutions of the homogenates were plated onto the media previously described. TSBA, King0 s medium B, DRBC and AFPA plates were incubated at 25 1C for 1-7 days. The Nash-Snyder medium plates were incubated at 24 1C for 7 days under 12/12 h photoperiod cold white and black fluorescent lamps. Only the plates containing 10–100 CFU were used for counting and the results were expressed as CFU per gram. Bacterial identification was performed according to Bergey0 s Manual of Systematic Bacteriology (Holt 1993, Krieg 1984, Sneath 1986). Fungal identification was performed according to Nelson et al. (1983), Pitt and Hocking (1997) and Samson et al. (2000).
Diversity index Diversity indices represent a useful means for quantifying community diversity and have been instrumental in the knowledge on resident populations (Natsch et al., 1997). The general diversity of total microorganisms, bacterial and fungal communities was measured by the generally accepted Shannon-Wiener information theory function: H0 :
C ðN log N ni log ni Þ N
where H0 is the general diversity index, C is 2.3, N is the number of CFU for total microorganisms, bacteria and fungi, ni is the number of CFU for the i genera (Shannon and Wiener, 1963).
Statistical Analysis Microbial rhizosphere populations CFU counts were transformed to log10 (x+1) to obtain the homogeneity of variance. Data were analyzed using one-way ANOVA. Least Significant Difference Test (LSD) was used to determine the differences between microbial total counts and genera counts at different maize growth stages (Quinn and Keough 2002). The analysis was conducted using PROC GLM in SAS (SAS Institute, Cary, NC).
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Results Colonisation of maize roots by total culturable microflora Bacterial population At the first sampling (20th days of plant growth) 1 1013 and 3.16 109 CFU [g of fresh weight (wt.) of roots]1 of total culturable bacteria were recovered from the rhizoplane and endorhizosphere, respectively (Fig. 1(a)). After 35th days of plant growth, a significant drop of population density occurred on the rhizoplane (po0.001). No statistically significant effect was visible for the endorhizosphere culturable bacterial population. The rhizoplane culturable populations, on the other hand, significantly decreased throughout plant growth and approached 1.99 108 CFU [g of fresh wt. of roots]1 after 130 days. Fungal population At the first sampling, fungal populations in the endorhizosphere were recorded with 1 105 CFU [g of fresh wt. of roots]1, which was significantly higher than on the rhizoplane [1.58 103 CFU (g of fresh wt. of roots)1] (Fig. 1(b)). After time, an increase of population density occurred for both rhizoplane and endorhizosphere fungal populations. However, at the third sampling (60th days of plant growth), the populations decreased at the two maize root levels tested. The fungal populations densities at 60, 80 and 130 days did not vary significantly when compared between them. The oscillations from the second sampling to the next successive samplings were not statistically different on both rhizoplane and endorhizosphere levels.
Rhizosphere microbial diversity Table 2 shows the general diversity index for total microbial communities, bacteria and fungi at different maize plant growth stages and root levels. General diversity increased from growth stages I to III for total microorganisms, bacteria and fungi at rhizoplane whereas there was an increment from growth stages I to II at the endo-rhizosphere. The general diversity index decreased at stages IV and V at both rhizoplane and endo-rhizosphere, respectively. Bacterial diversity was greater than fungal diversity at all maize growth stages studied.
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Fig. 1. Total culturable bacterial (a) and total fungal (b) populations from rhizoplane and endo-rhizosphere maize roots followed over time. Populations at each time represent mean values of eight replicates. Bars represent standard deviation. Letters in common are not significantly different according to Fisher0 s protected LSD test.
Community structure of total culturable bacteria The structure of bacterial community during plant growth at two maize root levels was investigated (Fig. 2). At the first sampling, Bacillus spp., Azotobacter spp. and Arthrobacter spp. were the dominant genera in the rhizoplane and endorhizospheric communities, with 1 1010, 3 108 and 3.16 1013 CFU (g of fresh wt. of roots)1 respectively. At
the second sampling, the bacterial genera counts ranged between 1 108 and 3.16 109 CFU (g of fresh wt. of roots)1 at the two maize levels studied. In the rhizoplane, Bacillus, Arthrobacter, Listeria and Sporolactobacillus were the predominant genera followed by Azotobacter, Micrococcus and Pseudomonas genera. When the endorhizospheric counts were determined, lower oscillations between bacterial genera were observed, except for Sporolactobacillus that significantly decreased its CFU counts. At the third sampling, Bacillus,
ARTICLE IN PRESS Rhizosphere microbial community structure at different maize plant growth stages Table 2. General diversity (H0 ) of total microbial communities, bacteria and fungi at different maize plant growth stages and root levels Root level
Rhizoplane
Growth General diversity (H0 ) stages Total Bacteria Fungi microorganisms I II III IV V
Endorhizosphere I II III IV V
0.04 1.39 3.8 3.76 0.007
0 1.27 1.56 0.5 0.007
0.64 0.87 1.23 0.39 0.8
0.72 1.35 1.21 0.08 0.02
0.33 1.28 1.21 0.35 0.03
0.11 0.74 0.07 0.04 0.04
Arthrobacter, Listeria and Agromyces genera maintained the CFU counts previously obtained at rhizoplane and endo-rhizosphere. A significant decrease on Azotobacter spp. CFU counts was observed at the two root levels, while an important recover of Micrococcus spp. [5.01 108 CFU (g of fresh wt. of roots)1] at rhizoplane was obtained. At the next sampling days (80th and 130th days of plant growth), except for Bacillus and Arthrobacter genera in the rhizoplane and for Bacillus, Arthrobacter and Azotobacter genera in the endo-rhizosphere all bacterial CFU counts significantly decreased.
Community structure of total culturable fungi Fig. 3 shows the structure of fungal communities during maize plant growth at rhizoplane and endorhizosphere root levels. Aspergillus and Fusarium were, in general, the predominant genera found at all stages of plant growth at the two root levels tested. At the initial stages (0–35 days) they were able to reach between 1 105 and 1 106 and between 1 103 and 1 106 CFU (g of fresh wt. of roots)1 in the rhizosphere and endo-rhizosphere, respectively. In the rhizosphere, Cladosporium and Eurotium genera did not change the CFU counts at all sampling stages. At the first sampling (20 days), Ulocladium, Alternaria and Trichoderma genera reached the highest CFU counts. They varied between 1 101 and 1 103 CFU (g of fresh wt. of roots)1 through the sampling days. In the endo-rhizhosphere, Cladosporium, Eurotium, Ulocladium, Alternaria
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and Trichoderma genera had the lowest CFU counts and did not vary significantly through stages of plant growth determined.
Colonisation of maize roots by Fusarium species over time At 20th days of plant growth, 1 104 and 4.3 103 CFU g1 of fresh wt. of roots of F. verticillioides were recovered from endo-rhizosphere and rhizoplane respectively (Fig. 4). At the next two samplings (30th and 60th days), F. verticillioides did not appear. At 80th and 130th days of plant growth, F. verticillioides gradually increased the CFU counts from 1 102 to 2.6 103 in the rhizoplane. F. proliferatum appeared only at the first sampling period with 7 102 CFU g1 of fresh wt. of roots in the rhizoplane. Other Fusarium species did not appear at the first sampling period. They predominated, except at 130 days, over F. verticillioides and F. proliferatum at the next sampling periods, at the two maize root levels studied and gradually decreased over time.
Discussion This is the first study for Argentina in which plant growth and total culturable rhizosphere microflora density and community structure the rhizoplane and endo-rhizosphere microbial populations at different root levels. The results indicate that during maize growth, except at the first sampling, the microbial density did not vary significantly at rhizoplane and endorhizosphere. Plant development did not have detectable influence on the total culturable microflora density, but it selectively influenced some bacterial and fungal groups present in the rhizosphere. Previuos works on the composition of microbial population in the maize rhizosphere pointed out the large numbers of diverse types of bacteria and fungi associated with root surfaces. Siciliano et al. (1998) established the diversity of root-associated microorganisms by utilizing fatty acid methyl ester (FAME) patterns. Buyer et al (1999) assessed the microbial diversity by determining the composition of fatty acids present in the microbial community. Chiarini et al. (1998) evaluated the influence of plant growth on the total rhizospheric microflora density and community structure by determining the percentage of total microbial colonies. Other researchers (Stirz and Nowak 2000, Soonthornpoct et al. 2000) determined the isolation frequency and occurrence of
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Fig. 2. Log10 of bacterial genera counts. Populations were extracted from rhizoplane (a) and endorhizosphere (b) at different growth stages. n ¼ 8. Letters in common are not significantly different according to Fisher0 s protected LSD test.
each fungal genus colonizing maize at the seedling or/and silking period. More recently, Seghers et al. (2004) used group-specific DGGE to fingerprint the endophytic community in the roots and kernels of Zea mays L. to evaluate the impact of agricultural practices. None of the works have determined the influence by plant growth over the culturable microbial size and composition at rhizoplane and endorhizosphere levels of agricultural plants like maize in the subtropics. In this study, the general diversity of total microorganisms remained more or less similar when
the two root levels, at the same maize plant growing period, were compared. The same occurred when bacterial diversity was studied. A detectable difference between rhizoplane and endo-rhizosphere fungal diversity was observed. Other researchers (Siddiqqi and Shaukat, 2003) found a decreasement on general diversity of Pseudomonas aeruginosa when the first and fourth growing periods of tomato were compared. However, the last period coincides with our second so these results are not in agreement with ours.
ARTICLE IN PRESS Rhizosphere microbial community structure at different maize plant growth stages Aspergillus spp. Penicillium spp. Fusarium spp. Cladosporium spp. Eurotium spp. Ulocladium spp. Alternaria spp. Trichoderma spp.
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Fig. 3. Log10 fungal genera counts. Populations extracted from rhizoplane (a) and endorhizosphere (b) at different growth stages. n ¼ 8. Letters in common are not significantly different according to Fisher0 s protected LSD test.
In the present work, the microbial community structure changed markedly at all stages of plant growth. These results are in agreement to those of Chiarini et al. (1998) Strains belonging to Bacillus, Arthrobacter and Azotobacter appeared at all plant growth stages at rhizoplane, but only Arthrobacter and Azotobacter genera appeared at the two maize root levels over time. On the other hand, strains belonging to Aspergillus and Fusarium genera predominated at the same growth conditions and root levels tested. Fusarium verticillioides, was the most prevalent Fusarium sp. at rhizoplane and endo-rhizosphere only at the first growing period. Arthrobacter
and Azotobacter strains could have a potential in the control of this soil-borne pathogen at root level. This work could be important in the future in order to detect possible perturbations in the maize agroecosystem, such as agronomic practices, pesticide applications and/or introduction of biological control agents.
Acknowledgements This work was carried out thanks to grants from Secretarı´a de Ciencia y Te ´cnica de la Universidad
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Fig. 4. Fusarium spp. log10 CFU counts from endorhizosphere and rhizoplane maize roots followed over time. CFU counts represent mean values of eight replicates.
Nacional de Rı´o Cuarto and Agencia Nacional de Promocio ´n Cientı´fica y Tecnolo ´gica (PICT 03/0814551) throughout the years 2004–2007.
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Plant growth responses and bacterium distribution in the rhizosphere. Plant Soil 1993;1(50):51–60. Germida JJ, Siciliano SD, de Freitas JR, Seib AM. Diversity of root-associated bacteria associated with heldgrown canola (Brassica napus L.) and wheat (Triticum aestivum L.) FEMS Microbiol. Ecol 1998;26:43–50. Hallmann J, Quadt-Hallmann A, Mahaffee WF, Kloepper JW. Bacterial endophytes in agricultural crops. Can J Microbiol 1997;43:895–914. Hamlen RA, Lukezie FL, Bloom JR. Influence of age and state of development of the neutral carbohydrates components in root exudates from alfalfa plants grown in a gnotobiotic environment. Can J Plant Sci 1972;52:633–42. Hinton DM, Bacon CW. Enterobacter cloacae is an endophytic symbiont of corn. Mycopathologia 1995;129:117–25. Holt JG., editors. Bergey0 s Manual of Systematic Bacteriology 9th ed, Baltimore, 1993. King EO, Ward MK, Ranney DE. Two simple media for the demonstration of pyocianin and fluorescin. J Lab Clin Med 1954;44:301–7. Krieg NR, editor. Bergey0 s Manual of Systematic Bacteriology, vol. 1. Baltimore: Williams and Wilkins; 1984. Marasas WFO, Nelson PE, Tousson TA, editors. Toxigenic Fusarium species. University Park: The Pennsylvania State University Press; 1984. Miller HJ, Heken G, van Veen JA. Variation and composition of bacterial populations in the rhizosphere of maize, wheat and grass cultivars. Can J Microbiol 1989;35:656–60. Nacamulli C, Bevivino A, Dalmastri C, Tabacchioni S, Chiarini L. Perturbation of maize rhizosphere microflora following seed bacterization with Burkholderia cepacia. MCI 7. FEMS Microbiol Ecol 1997;23:183–93. Natsch A, Keel C, Hebecker N, Laasik E, De´fago G. Influence of biocontrol strain Pseudomona fluorescens
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Rhizosphere microbial community structure at different maize plant growth stages and root locations Lilia Cavaglieria,b,, Julieta Orlandoa, Miriam Etcheverrya,b a
Departamento de Microbiologı´a e Inmunologı´a. Universidad Nacional de Rı´o Cuarto. Ruta 36 km 601. 5800. Rı´o Cuarto. Co ´rdoba. Argentina b Member of Consejo Nacional de Investigaciones Cientı´ficas y Te´cnicas (CONICET), Argentina Accepted 29 March 2007
KEYWORDS Culturable microflora; Maize plant growth stages; Rhizosphere
Summary The aims of the present work were (1) to determine the influence of plant growth stages on the population size of culturable bacteria and fungi associated with rhizoplane and endo-rhizosphere of maize grown in field and (2) to establish the community structure of total culturable bacteria and fungi. Density, diversity and community structure of culturable rhizoplane and endo-rhizosphere populations at different maize plant growth stages were estimated. Plant development did not have influence on total culturable microflora density but it selectively influenced some bacterial and fungal groups present in the rhizosphere. However, the microbial community structure changed markedly over time. This knowledge is relevant for exploring endophytic rhizobacteria potential in the promotion of plant-growth, protection against pathogens and to detect perturbations in natural agro ecosystems. & 2007 Elsevier GmbH. All rights reserved.
Introduction The study of microbial communities associated with plants is important for understanding their ecological role in nature environments. Microorganisms, which establish positive interactions with Corresponding author.
E-mail address: [email protected] (L. Cavaglieri).
plant roots, play a key role in agricultural environments and are promising for their potential use in sustainable agriculture (Di Cello et al., 1997). Microbial populations can undergo temporary variations in its structure due to the selective pressure exerted by the environment that causes exchanges within a local population and migrations between distinct populations. Knowledge of such process in the rhizosphere can help in relating microbial
0944-5013/$ - see front matter & 2007 Elsevier GmbH. All rights reserved. doi:10.1016/j.micres.2007.03.006
ARTICLE IN PRESS 392 community changes to environmental variations over time (Wise et al., 1996). For example, it is well known that production and diffusion of root exudates are affected by plant development (Hamlen et al., 1972). These exudates exert a selective microbial stimulation (Buir and Caesar, 1984, Miller et al., 1989) that varies in function of time due to plant age. Consequently, the development of more adapted microorganisms at different plant growth stages may be favored. Di Cello et al. (1997) found that Burkholderia cepacia populations associated with maize roots decreased significantly during plant development. Moreover, several studies on the rhizosphere microbial community structure of wheat and maize revealed that fastgrowing bacteria (r-strategist) were predominant on young immature roots, whereas slow growing bacteria (k-strategist) became predominant on mature roots (De Leij et al., 1995, Nacamulli et al., 1997). Young and Kucharek (1977) reported the increase Trichoderma viride and Fusarium moniliforme counts in roots from the silking stage through subsequent growth stages. On the other hand, Windham (1983) found that fungi generally were isolated more frequently from roots of seedling stages than from roots of plants at silking stage. The capacity of bacteria and fungi to colonize internal tissues of plants could confer an ecological advantage over others that can only colonize plants epiphytically (Hallmann et al., 1997). Rhizosphere is widely accepted to be an important source of root endophytes, and most endophytic species are present in the rhizosphere (Germida et al., 1998, Sessitsch et al., 2002). This knowledge is relevant for exploring endophytic rhizobacteria potentially promoting plant-growth and protecting the plant against pathogens (Benhamou et al., 2000, Stirz and Nowak, 2000). Frommel et al. (1993) recorded a positive correlation of potato growth stimulation and yield increase with the occurrence of high population densities of Pseudomonas sp. on and in potato roots. Hinton and Bacon (1995) found one isolate of Enterobacter cloacae, endophytically associated with corn enhanced the biological control of systemic pathogens of corn. Among the microorganisms inhabiting the maize rhizosphere, Fusarium Section Liseola are the major soil-borne fungal pathogens (Marasas et al., 1984). Pathologists have special interest to control these pathogens. Therefore, it is important to characterize the indigenous microbial communities naturally associated with maize root systems to identify potential biocontrol agents. Although it has long been known that indigenous microorganisms are naturally associated with maize
L. Cavaglieri et al. roots, no experimental work has been reported for Cordoba, Argentina, maize production area to study the structure of native rhizosphere microbial populations at all growing stages. The aims of the present work were to: (i) determine the influence of plant growth stages on the population size of culturable bacteria and fungi associated with rhizoplane and endorhizosphere of maize grown in the field. (ii) establish the community structure of culturable bacteria and fungi.
Materials and methods Sampling Root samples (Zea mays L.) from an experimental maize field of Universidad Nacional de Rio Cuarto, Rio Cuarto, Cordoba, Argentina, were collected. The soil was loamy sand. Plants were collected throughout the 2003 growing season at the five more significant stages of maize development (Table 1). At each sampling, eight plants were randomly harvested.
Estimation of rhizosphere microbial populations To quantify culturable rhizoplane populations, roots were excised and loosely adhering soil was removed. Each root was weighted, blended and resuspended in phosphate-buffered saline (PBS, Oxoid Ltd., London, UK). Serial dilutions of these suspensions were plated onto the following media: (i) tryptic soy broth plus 2% agar (TSBA) in order to estimate total culturable bacteria; (ii) selective King0 s medium B (King et al. 1954) for pseudomonads; (iii) dichloran rose bengal-chloramphenicol agar (DRBC) (Abarca et al. 1994) to estimate total culturable micoflora; (iv) Aspergillus flavus and parasiticus agar (AFPA) for potentially aflatoxigenic fungi; (v) Nash-Snyder medium to enumerate Fusarium species (Nelson et al. 1983). Table 1. Microbial isolation from maize roots during plant development Sampling
Time (days)
Maize growth stage
I II III IV V
20 35 60 80 130
Germination Elongation Tassel appearance End of flowering Physiological maturation
ARTICLE IN PRESS Rhizosphere microbial community structure at different maize plant growth stages To quantify endo-rhizosphere microflora, roots were washed and dried between sheets of tissue, weighed and surface sterilized by gently shaking in 70% ethanol (1 min), 20% household bleach (5 min) and thiosulphate Ringer solution (5 min) (Oxoid Ltd., London, UK). Roots were macerated in 90 ml of PBS with a mortar and pestle. Serial dilutions of the homogenates were plated onto the media previously described. TSBA, King0 s medium B, DRBC and AFPA plates were incubated at 25 1C for 1-7 days. The Nash-Snyder medium plates were incubated at 24 1C for 7 days under 12/12 h photoperiod cold white and black fluorescent lamps. Only the plates containing 10–100 CFU were used for counting and the results were expressed as CFU per gram. Bacterial identification was performed according to Bergey0 s Manual of Systematic Bacteriology (Holt 1993, Krieg 1984, Sneath 1986). Fungal identification was performed according to Nelson et al. (1983), Pitt and Hocking (1997) and Samson et al. (2000).
Diversity index Diversity indices represent a useful means for quantifying community diversity and have been instrumental in the knowledge on resident populations (Natsch et al., 1997). The general diversity of total microorganisms, bacterial and fungal communities was measured by the generally accepted Shannon-Wiener information theory function: H0 :
C ðN log N ni log ni Þ N
where H0 is the general diversity index, C is 2.3, N is the number of CFU for total microorganisms, bacteria and fungi, ni is the number of CFU for the i genera (Shannon and Wiener, 1963).
Statistical Analysis Microbial rhizosphere populations CFU counts were transformed to log10 (x+1) to obtain the homogeneity of variance. Data were analyzed using one-way ANOVA. Least Significant Difference Test (LSD) was used to determine the differences between microbial total counts and genera counts at different maize growth stages (Quinn and Keough 2002). The analysis was conducted using PROC GLM in SAS (SAS Institute, Cary, NC).
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Results Colonisation of maize roots by total culturable microflora Bacterial population At the first sampling (20th days of plant growth) 1 1013 and 3.16 109 CFU [g of fresh weight (wt.) of roots]1 of total culturable bacteria were recovered from the rhizoplane and endorhizosphere, respectively (Fig. 1(a)). After 35th days of plant growth, a significant drop of population density occurred on the rhizoplane (po0.001). No statistically significant effect was visible for the endorhizosphere culturable bacterial population. The rhizoplane culturable populations, on the other hand, significantly decreased throughout plant growth and approached 1.99 108 CFU [g of fresh wt. of roots]1 after 130 days. Fungal population At the first sampling, fungal populations in the endorhizosphere were recorded with 1 105 CFU [g of fresh wt. of roots]1, which was significantly higher than on the rhizoplane [1.58 103 CFU (g of fresh wt. of roots)1] (Fig. 1(b)). After time, an increase of population density occurred for both rhizoplane and endorhizosphere fungal populations. However, at the third sampling (60th days of plant growth), the populations decreased at the two maize root levels tested. The fungal populations densities at 60, 80 and 130 days did not vary significantly when compared between them. The oscillations from the second sampling to the next successive samplings were not statistically different on both rhizoplane and endorhizosphere levels.
Rhizosphere microbial diversity Table 2 shows the general diversity index for total microbial communities, bacteria and fungi at different maize plant growth stages and root levels. General diversity increased from growth stages I to III for total microorganisms, bacteria and fungi at rhizoplane whereas there was an increment from growth stages I to II at the endo-rhizosphere. The general diversity index decreased at stages IV and V at both rhizoplane and endo-rhizosphere, respectively. Bacterial diversity was greater than fungal diversity at all maize growth stages studied.
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Fig. 1. Total culturable bacterial (a) and total fungal (b) populations from rhizoplane and endo-rhizosphere maize roots followed over time. Populations at each time represent mean values of eight replicates. Bars represent standard deviation. Letters in common are not significantly different according to Fisher0 s protected LSD test.
Community structure of total culturable bacteria The structure of bacterial community during plant growth at two maize root levels was investigated (Fig. 2). At the first sampling, Bacillus spp., Azotobacter spp. and Arthrobacter spp. were the dominant genera in the rhizoplane and endorhizospheric communities, with 1 1010, 3 108 and 3.16 1013 CFU (g of fresh wt. of roots)1 respectively. At
the second sampling, the bacterial genera counts ranged between 1 108 and 3.16 109 CFU (g of fresh wt. of roots)1 at the two maize levels studied. In the rhizoplane, Bacillus, Arthrobacter, Listeria and Sporolactobacillus were the predominant genera followed by Azotobacter, Micrococcus and Pseudomonas genera. When the endorhizospheric counts were determined, lower oscillations between bacterial genera were observed, except for Sporolactobacillus that significantly decreased its CFU counts. At the third sampling, Bacillus,
ARTICLE IN PRESS Rhizosphere microbial community structure at different maize plant growth stages Table 2. General diversity (H0 ) of total microbial communities, bacteria and fungi at different maize plant growth stages and root levels Root level
Rhizoplane
Growth General diversity (H0 ) stages Total Bacteria Fungi microorganisms I II III IV V
Endorhizosphere I II III IV V
0.04 1.39 3.8 3.76 0.007
0 1.27 1.56 0.5 0.007
0.64 0.87 1.23 0.39 0.8
0.72 1.35 1.21 0.08 0.02
0.33 1.28 1.21 0.35 0.03
0.11 0.74 0.07 0.04 0.04
Arthrobacter, Listeria and Agromyces genera maintained the CFU counts previously obtained at rhizoplane and endo-rhizosphere. A significant decrease on Azotobacter spp. CFU counts was observed at the two root levels, while an important recover of Micrococcus spp. [5.01 108 CFU (g of fresh wt. of roots)1] at rhizoplane was obtained. At the next sampling days (80th and 130th days of plant growth), except for Bacillus and Arthrobacter genera in the rhizoplane and for Bacillus, Arthrobacter and Azotobacter genera in the endo-rhizosphere all bacterial CFU counts significantly decreased.
Community structure of total culturable fungi Fig. 3 shows the structure of fungal communities during maize plant growth at rhizoplane and endorhizosphere root levels. Aspergillus and Fusarium were, in general, the predominant genera found at all stages of plant growth at the two root levels tested. At the initial stages (0–35 days) they were able to reach between 1 105 and 1 106 and between 1 103 and 1 106 CFU (g of fresh wt. of roots)1 in the rhizosphere and endo-rhizosphere, respectively. In the rhizosphere, Cladosporium and Eurotium genera did not change the CFU counts at all sampling stages. At the first sampling (20 days), Ulocladium, Alternaria and Trichoderma genera reached the highest CFU counts. They varied between 1 101 and 1 103 CFU (g of fresh wt. of roots)1 through the sampling days. In the endo-rhizhosphere, Cladosporium, Eurotium, Ulocladium, Alternaria
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and Trichoderma genera had the lowest CFU counts and did not vary significantly through stages of plant growth determined.
Colonisation of maize roots by Fusarium species over time At 20th days of plant growth, 1 104 and 4.3 103 CFU g1 of fresh wt. of roots of F. verticillioides were recovered from endo-rhizosphere and rhizoplane respectively (Fig. 4). At the next two samplings (30th and 60th days), F. verticillioides did not appear. At 80th and 130th days of plant growth, F. verticillioides gradually increased the CFU counts from 1 102 to 2.6 103 in the rhizoplane. F. proliferatum appeared only at the first sampling period with 7 102 CFU g1 of fresh wt. of roots in the rhizoplane. Other Fusarium species did not appear at the first sampling period. They predominated, except at 130 days, over F. verticillioides and F. proliferatum at the next sampling periods, at the two maize root levels studied and gradually decreased over time.
Discussion This is the first study for Argentina in which plant growth and total culturable rhizosphere microflora density and community structure the rhizoplane and endo-rhizosphere microbial populations at different root levels. The results indicate that during maize growth, except at the first sampling, the microbial density did not vary significantly at rhizoplane and endorhizosphere. Plant development did not have detectable influence on the total culturable microflora density, but it selectively influenced some bacterial and fungal groups present in the rhizosphere. Previuos works on the composition of microbial population in the maize rhizosphere pointed out the large numbers of diverse types of bacteria and fungi associated with root surfaces. Siciliano et al. (1998) established the diversity of root-associated microorganisms by utilizing fatty acid methyl ester (FAME) patterns. Buyer et al (1999) assessed the microbial diversity by determining the composition of fatty acids present in the microbial community. Chiarini et al. (1998) evaluated the influence of plant growth on the total rhizospheric microflora density and community structure by determining the percentage of total microbial colonies. Other researchers (Stirz and Nowak 2000, Soonthornpoct et al. 2000) determined the isolation frequency and occurrence of
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Fig. 2. Log10 of bacterial genera counts. Populations were extracted from rhizoplane (a) and endorhizosphere (b) at different growth stages. n ¼ 8. Letters in common are not significantly different according to Fisher0 s protected LSD test.
each fungal genus colonizing maize at the seedling or/and silking period. More recently, Seghers et al. (2004) used group-specific DGGE to fingerprint the endophytic community in the roots and kernels of Zea mays L. to evaluate the impact of agricultural practices. None of the works have determined the influence by plant growth over the culturable microbial size and composition at rhizoplane and endorhizosphere levels of agricultural plants like maize in the subtropics. In this study, the general diversity of total microorganisms remained more or less similar when
the two root levels, at the same maize plant growing period, were compared. The same occurred when bacterial diversity was studied. A detectable difference between rhizoplane and endo-rhizosphere fungal diversity was observed. Other researchers (Siddiqqi and Shaukat, 2003) found a decreasement on general diversity of Pseudomonas aeruginosa when the first and fourth growing periods of tomato were compared. However, the last period coincides with our second so these results are not in agreement with ours.
ARTICLE IN PRESS Rhizosphere microbial community structure at different maize plant growth stages Aspergillus spp. Penicillium spp. Fusarium spp. Cladosporium spp. Eurotium spp. Ulocladium spp. Alternaria spp. Trichoderma spp.
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Fig. 3. Log10 fungal genera counts. Populations extracted from rhizoplane (a) and endorhizosphere (b) at different growth stages. n ¼ 8. Letters in common are not significantly different according to Fisher0 s protected LSD test.
In the present work, the microbial community structure changed markedly at all stages of plant growth. These results are in agreement to those of Chiarini et al. (1998) Strains belonging to Bacillus, Arthrobacter and Azotobacter appeared at all plant growth stages at rhizoplane, but only Arthrobacter and Azotobacter genera appeared at the two maize root levels over time. On the other hand, strains belonging to Aspergillus and Fusarium genera predominated at the same growth conditions and root levels tested. Fusarium verticillioides, was the most prevalent Fusarium sp. at rhizoplane and endo-rhizosphere only at the first growing period. Arthrobacter
and Azotobacter strains could have a potential in the control of this soil-borne pathogen at root level. This work could be important in the future in order to detect possible perturbations in the maize agroecosystem, such as agronomic practices, pesticide applications and/or introduction of biological control agents.
Acknowledgements This work was carried out thanks to grants from Secretarı´a de Ciencia y Te ´cnica de la Universidad
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Fig. 4. Fusarium spp. log10 CFU counts from endorhizosphere and rhizoplane maize roots followed over time. CFU counts represent mean values of eight replicates.
Nacional de Rı´o Cuarto and Agencia Nacional de Promocio ´n Cientı´fica y Tecnolo ´gica (PICT 03/0814551) throughout the years 2004–2007.
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