Changes in the bacterial communities structure of the rhizosphere of four wild Lupinus species from flowering to fruiting

Abstract Microbial rhizosphere communities of four different Lupinus species were studied at two plant growth stages in order to determine the dominance of r/K strategists and the pattern of taxa and diversity (Shannon index). Five hundred and seventy-six strains were isolated, determined and classified into eleven different bacterial genera and the Enterobacteriaceae family. Aureobacterium dominated on L. hispanicus and L. angustifolius at both sampling times (GS1: Growth Stage 1 (flowering); GS2: Growth Stage 2 (fruiting)), whereas it only dominated in the rhizosphere of L. albus at GS1 (58.45%). Cellulomonas dominated at GS2 (42.93%). In L. luteus, Bacillus dominated at GS1 (41.21%) and Aureobacterium at GS2 (39.74%). Principal Component Analysis (PCAs) showed that the microbial composition of the rhizosphere of L. albus is the only one that changed considerably from GS1 to GS2. These changes were due to a proportional increase of r strategists at GS2. Sample size (18 to 54 colonies, depending on lupin species) was adequate according to the Shannon index. Despite the fact that diversity between GS1 and GS2 hardly varied in L. albus, L. luteus, and L. hispanicus, the PCAs revealed the largest differences in the microbial structure associated with L. albus. A tentative successional model for the lupin microbial population of the rhizosphere related to its biological cycle, is proposed. Key words:bacterial communities, biological diversity, Lupinus, rhizosphere, r/K strategies. Resumen. Cambios en la comunidad microbiana rizosfŽrica de cuatro especies salvajes de Lupinus desde la floraci—n hasta la fructificaci—n Se estudia la comunidad microbiana rizosfŽrica de cuatro especies de Lupinus en dos momentos de su ciclo biol—gico, con el objeto de determinar la dominancia de r y K estrategas, el patr—n de taxones y la diversidad biol—gica (’ndice de Shannon). Se aislaron 576 cepas, y se encontraron representantes de once gŽneros bacterianos y de la familia Enterobacteriaceae. Aureobacterium fue mayoritario en L. hispanicus y L. angustifolius en ambos momentos de muestreo (GS1: floraci—n; GS2: fructificaci—n), mientras que s—lo domin— en L. albus en GS1 (58.45%). Cellulomonas domin— en GS2 (42.93%). En L. luteus fue Bacillus el mayoritario en GS1 (41.21%) y Aureobacterium en GS2 (39.73%). Orsis 15, 2000 7-25 Changes in the bacterial communities structure of the rhizosphere of four wild Lupinus species from flowering to fruiting J.A. Lucas Garc’a, A. Probanza, B. Ramos, M.L. Barrientos, F.J. GutiŽrrez Ma–ero Universidad San Pablo CEU. Departamento de Biolog’a P.O. Box 67. 28668 Boadilla del Monte. Madrid (Spain) Fax 913 510 496 [email protected] Manuscript received in February 2000 Los An‡lisis de Componentes Principales (ACPs) indican que s—lo L. albus tiene cambios considerables en sus poblaciones microbianas rizosfŽricas de GS1 a GS2. Estos cambios se deben al incremento de estrategas de la r en GS2. El tama–o de la muestra (de 18 a 54 colonias, dependiendo de la especie de lupino) fue la adecuada, segœn indica el ’ndice de Shannon. A pesar de que la diversidad apenas var’a de GS1 a GS2 en L. albus, L. luteus y L. hispanicus, los ACPs reflejan cambios importantes en la estructura microbiana rizosfŽrica en L. albus. Se propone un modelo sucesional tentativo de las poblaciones microbianas de la rizosfera del lupino, relacion‡ndolo con su ciclo biol—gico. Palabras clave: comunidades bacterianas, diversidad biol—gica, estrategias de crecimiento r/K, Lupinus, rizosfera. Introduction Interactions plant-soil-microorganism have been known for a long time, however, little research has been done to understand microbial diversity, soil functioning, and ecosystem sustainability (Kennedy & Smith, 1995). The knowledge of microbial community structure, successional phenomena and growth strategies within the rhizosphere are essential for understanding of the Ecological Theory in this environment. This lack of knowledge of Ecological Theory (Margalef, 1968, 1973) is probably due to inconsistencies between evolutionary ecology (Kauffman, 1993, 1995), ecosystem ecology (Schulze & Mooney, 1993), and information theory (Rasmussen, 1988; Yockey, 1990, 1995). Previous cited inconsistences to be in other natural systems. The concept of r/K strategy (Luckinbill, 1978) is derived from evolutionary ecology and posits that there are genetic differences between organisms in their ability to exploit and survive in different kinds of environments (Luckinbill, 1978; Pianka, 1970). Knowledge of these interactions is also essential when considering the use of biological agents to improve crop yields. First, because the successful inoculation of biological agents depends on the bacterial ability of microorganisms to colonise the plant root (Kloepper et al., 1980; Michiels et al.,1989), and secondly, because the incidence and consequences of introducing foreign microorganisms in the rhizosphere must be known. Among the factors that determine the structure of microbial communities in the rhizosphere, the plant plays a major role (Marilley et al.,1998) by releasing organic compounds to soil, a process called rhizodeposition or exudation (Whipps & Lynch, 1985). The effect of specific compounds released by the plant root on the selection of the microbiota of the rhizosphere was first demonstrated by Gunner et al. (1966). The exudation pattern is affected by genetic factors of the plant (Bolton et al., 1993, Hedges & Messens, 1990), plant age, physiological status of the plant and environmental factors (Grayston et al.,1996). Chanway & Nelson (1991) suggested that rhizobacteria play a significant role in rhizodeposition, assuming that bacteria may modify the plant physiology. This hypothesis is supported by Wiehe & Hšflicht (1995) and others, who demonstrated that certain bacteria had different colonization patterns depending on the plant species. In view 8Orsis 15, 2000 J.A. Lucas; A. Probanza; B. Ramos; M.L. Barrientos; F.J. GutiŽrrez of this, rhizobacterial composition must be defined by an adaptive process based on the natural coexistence (Sumner, 1990) and metabolic compatibility (Chanway et al., 1989) between the plant and the bacteria. As proposed by Lynch (1990), soil also influences rhizobacterial composition to some extent. Humans have used the legume Lupinus for nutritional purposes because of the high protein content (36-52%) in seeds (Petterson & Mackintosh, 1994). Lupinus tolerates low temperatures, drought and poor soils, three qualities that make this crop quite suitable for marginal soils, where other crops cannot be cultivated (Mohamed & Rayas-Duarte, 1995). The aim of this study is to determine the influence of plant growth stage (flowering and fruiting) on i) pattern of bacterial taxa, ii) degree of diversity, and iii) community structure and natural succession of rhizosphere bacterial communities in the populations of four wild Lupinus species (Lupinus albus L., Lupinus luteus L., Lupinus hispanicus Boiss & Reuter, and Lupinus angustifolius L.). The two growth stages have been determined according to traditional agricultural cropping practices for Lupinus. Materials and Methods Field sites The four populations of Lupinus were located in different areas within the Comunidad de Madrid (Spain). Table 1 shows the geographical location, vegetation, type of soil, annual mean rainfall, and annual mean temperature. L. luteus grew in a soil with pH 6.3, 0.698% nitrogen, and 0.474 mg gÐ1 carbon and L. albus in a soil with pH 6.02, 1.077% nitrogen, and 0.741 mg gÐ1 carbon. Although these two populations probably originated from nearby cropping areas, they have been established in the area for at least four years. L. hispanicus was sampled in the Sierra de Ayll—n, in a soil with a pH, carbon, and nitrogen content of 5.71, 0.416 mg gÐ1, and 1.306%, respectively. L. angustifolius was sampled in the Valle del Lozoya, a soil with a pH, carbon, and nitrogen content of 6.03, 0.633 mg gÐ1, and 1.378%, respectively. These two species are indigenous to the Madrid area. Total nitrogen was determined colorimetrically, following the Smith method (1980), after a Kjeldhal digestion of 2 g of soil in a Prolabo Maxidigest-Mx350 microwave digestor. Organic C was determined as in Walkley & Black (1934). Root Sampling Three populations of each Lupinus species (50 x 50 m), separated by 500 to 1000 m, were marked on a map (1:10) and each population was divided into 1 x 1 m plots, numbered on the map. Ten plots were selected with a random number computer programme, sampling one plant from each of them. Roots from these 10 plants for each one population constituted a replicate (therefore, there were three replicates for each lupin species). Bacterial communities of Lupinus sp. Orsis 15, 2000 9 Samples were transported to the laboratory in sterile plastic bags at 4¼C. Roots from each replicate were cut into 2 cm pieces, mixed and 10 g were put into Erlenmeyer flasks filled with 100 ml of sterile distilled water. Roots were shaken at 2000 rpm with 4 mm-diameter glass beads for 10 minutes. Then, 10-fold dilutions were prepared in sterile distilled water and 1 mL of each was plated on a general solid media (23.5 g standard methods agar (Pronadisa), 10 mL soil extract, 50 mL Winogradsky saline solution, 1 mL oligoelement solution, 1000 mL distilled water) (Pochon & Tardieux, 1962) and incubated at 28¼C for 72 hours; cfus were isolated after 36 and 72h. Two sampling moments were determined at two Growth Stages (GS) of the plants, one at the beginning of flowering (GS1) and the other when fruiting was completed (GS2). Identification of isolates 12 isolates were randomly taken after 36 h of incubation at 28¼C from plates with approximately 100 cfus and another 12 in the following 36 h, that is, after 72 hours. Therefore, 24 colonies were isolated for each replicate (three populations of each Lupinus species), giving a total of 72 colonies for each lupin species (4). Two different growing stages were sampled, giving a total of 576 isolates. All isolates, except those that belonged to the Enterobacteriaceae family, were identified untill genus level according to the Acero et al. (1994) procedure modified by us. This methodological modification were done in order to a better identification of the Coryneforme genera (figure 1). Results are presented as frequencies (percentage) of total isolates. Diversity Analysis The Shannon index (Shannon & Weaver, 1949; H(e) = ÐS(pilog2 pi), where pi is the proportion of isolates belonging to each taxa) was applied to assess rhizobacterial diversity in each Lupinus species and growth stage (Bianchi & Bianchi, 1982). Statistical Analysis Three two-way ANOVAs with three replicates each were made (Sokal & Rohlf, 1969). The variation factors being the four Lupinus species and the frequencies of the isolated bacterial taxa in the two plant growth stages. ANOVA a was done with data after 72 h of incubation, ANOVA b with data after 36h of incubation, and ANOVA c with data from 36 to 72h of incubation. Three Principal Component Analysis (PCAs) were undertaken to determine main trends in microbiological data, previously log(%+1) transformed (Hartmann, 1967). PCAs nomenclature were the same as the ANOVAs and both were carried out with the same data. 10 Orsis 15, 2000 J.A. Lucas; A. Probanza; B. Ramos; M.L. Barrientos; F.J. GutiŽrrez Bacterial communities of Lupinus sp. Orsis 15, 2000 11 Isolates Filamentous Rods or cocci Heat-resistance yes no Catalase Gram –+ *+– Rods Cocci * Catalase Colonies pigment +– * +– –+ * –+ +– * +– * Semitransparent and white Hugh & Leifson's test Hugh & Leifson's test No acid Oxidative Fermentative Kovacs Kovacs Motility Yes No Yellow colonies Blue colonies No growth B-King McConkey D3 Yes No Rods Cocci Hugh & Leifson's test Oxidative No acid Fermentative B-King Kovacs Kovacs Agar D1 Catalase Yellow, pink and orange Oxidative No acid Fermentative X I XIII XIV XV II VII V VI IX XII III XI III VI XII IX VIII Figure 1. Modified Acero et al., (1994) protocol: I; Bacillus, II; Micrococcus, III; Pseudomonas, IV; Pseudomonas-Alcaligenes, V; Moraxella, VI; Xanthomonas, VII; Acinetobacter, VIII; Flavobacterium, IX; Enterobacteriaceae, X; Streptomyces, XI; Agrobacterium, XII; Erwinia, XIII; Aureobacterium, XIV; Cellulomonas, XV; Arthrobacter, asterisk; not found. 12 Orsis 15, 2000 J.A. Lucas; A. Probanza; B. Ramos; M.L. Barrientos; F.J. GutiŽrrez Table 1. Geographic coordinates, vegetation characteristics and type of soil, annual mean rainfall and annual mean temperature under each Lupinus species. Annual mean Annual mean Geographic coordinates Vegetation and type of soil rainfall temperature Lupinus albus 40¼24«N; 3¼50«W Junipero-oxycedri Quercetum rotundifoliae. Anfisol soil 464-508 mm 14-14.25 ¼C Lupinus luteus 40¼24«N; 3¼50«W Junipero-oxycedri Quercetum rotundifoliae. Anfisol soil 464-508 mm 14-14.25 ¼C Lupinus hispanicus 41¼3«30««N; 3¼31«30««W Luzulo forsteri Quercetum pyrenaicae. Brown forest soil 650-700 mm 10-10.3 ¼C on igneous rocks Lupinus angustifolius 40¼58«N; 3¼43«W Junipero-oxycedri Quercetum rotundifoliae. Brown forest 487-608 mm 10.8-11.4 ¼C soil on metamorphic rocks Table 2. Frequency of bacterial taxa isolated from the rhizosphere of Lupinus at the beginning of flowering (GS1) and fruiting (GS2) isolated at different times of incubation: 1a) after 72h, 1b) after 36h and 1c) from 36 to 72h. LA: Lupinus albus; LL:L. luteus; LH: L. hispanicus; LAN: L. angustifolius. a) 0 Þ72h LAGS1 LAGS2 LLGS1 LLGS2 LHGS1 LHGS2 LANGS1 LANGS2 Total Mean Bacillus 19.84 2.77 41.21 34.75 4.33 3.00 5.26 7.12 14.78 Aureobacterium 58.45 15.27 33.77 39.74 40.47 35.95 56.66 71.61 43.99 Cellulomonas 3.50 42.93 0 4.34 24.60 20.47 3.50 6.20 13.19 Pseudomonas 10.75 1.38 11.84 11.86 21.70 22.17 12.01 4.42 12.01 Arthrobacter 4.54 33.51 3.4 7.78 0 2.90 8.46 10.62 8.90 Micrococcus 0 0 6.73 0 0 0 0 0 0.84 Streptomyces 0 0 1.66 0 0 0 1.73 0 0.42 Erwinia 0 0 0 0 1.43 4.44 3.50 0 1.17 Acinetobacter 3.03 2.77 0 0 0 1.43 3.46 0 1.33 Flavobacterium 0 0 0 0 0 0 3.50 0 0.43 Enterobacteriaceae 1.51 1.56 0 1.43 2.86 0 1.73 0 1.13 Xanthomonas 0 0 0 0 1.43 5.66 0 0 0.88 Bacterial communities of Lupinus sp. Orsis 15, 2000 13 b) 0 Þ36h LAGS1 LAGS2 LLGS1 LLGS2 LHGS1 LHGS2 LANGS1 LANGS2 Total Mean Bacillus 37.11 0 58.33 49.99 11.44 6.11 12.50 13.88 23.67 Aureobacterium 47.13 16.66 22.22 25.15 19.69 26.11 61.43 36.11 31.43 Cellulomonas 7.40 47.22 0 6.06 32.31 38.33 6.66 11.61 18.69 Pseudomonas 0 2.77 11.10 15.75 16.66 14.99 3.03 2.77 8.38 Arthrobacter 2.77 27.77 0 0 0 2.77 6.36 0 4.95 Micrococcus 0 0 8.33 0 0 0 0 0 1.04 Streptomyces 0 0 0 0 0 0 0 0 0 Erwinia 0 0 0 0 2.77 6.11 3.33 0 1.52 Acinetobacter 2.77 2.77 0 0 0 0 0 0 0.69 Flavobacterium 0 0 0 0 0 0 3.33 0 0.41 Enterobacteriaceae 2.77 0 0 3.03 5.55 0 3.33 0 1.83 Xanthomonas 0 0 0 0 0 5.55 0 0 0.69 c) 36h Þ72h LAGS1 LAGS2 LLGS1 LLGS2 LHGS1 LHGS2 LANGS1 LANGS2 Total Mean Bacillus 3.33 2.77 8.33 15.58 2.77 5.80 0 0 4.82 Aureobacterium 68.18 13.88 52.38 58.40 53.02 48.73 51.51 71.62 52.21 Cellulomonas 0 38.38 0 2.77 17.17 3.03 0 0 7.66 Pseudomonas 18.48 0 21.42 8.33 23.98 28.02 20.87 9.09 16.27 Arthrobacter 6.66 39.14 8.94 14.89 0 3.03 10.43 22.30 13.17 Micrococcus 0 0 4.76 0 0 0 0 0 0.59 Streptomyces 0 0 4.16 0 0 0 3.03 0 0.89 Erwinia 0 0 0 0 0 2.77 3.70 0 0.81 Acinetobacter 3.33 2.77 0 0 0 3.03 6.73 0 1.98 Flavobacterium 0 0 0 0 0 0 3.70 0 0.46 Enterobacteriaceae 0 3.03 0 0 0 0 0 0 0.38 Xanthomonas 0 0 0 0 3.03 5.55 0 0 1.07 ANOVA a: Differ. between genera D.F.=46; LSD (0.05)=5.432; LSD (0.01)=7.420, differ. Interaction between genera and sampling moments D.F.=4; LSD (0.05)=21.101; LSD (0.01)= 34.973 ANOVA b: Differ. between genera D.F.=46; LSD (0.05)=7.427; LSD (0.01)=10.147, differ. Interaction between genera and sampling moments D.F.=4; LSD (0.05)=28.856; LSD (0.01)= 47.827 ANOVA c: Differ. between genera D.F.=46; LSD (0.05)=5.97; LSD (0.01)=7.97, differ. Interaction between genera and sampling moments D.F.=4; LSD (0.05)=23.196; LSD (0.01)= 38.445 Results Identification and frequency of isolates. The 576 isolates belonged to 11 different bacterial genera and to the Enterobacteriaceae family. Table 2 shows frequencies of each bacterial genera for each Lupinus species at the two growth stages determined (table 2a shows data of colonies grown from 0 to 72h of incubation; table 2b: data of colonies grown from 0 to 36h; table 2c: data from colonies that grew between 36 and 72h) . Considering total averages after 72 h (table 2a), Aureobacterium was the dominant genus (43.99%) followed by Bacillus (14.78%), and the least abundant were Streptomyces (0.42%) and Flavobacterium (0.43%). Aureobacterium showed the highest frequency (71.61%) when associated with L. angustifolius at GS2 and the lowest (15.27%) associated with L. albus at GS2. The corresponding ANOVA (ANOVA a, table 2a) shows significant differences between genera (p<0.01) and between genera and growth stages (p<0.01). The dominant genus was different depending on the plant species and growth stages. Aureobacterium dominated in L. hispanicus and L. angustifolius in both growth stages but it only dominated in L. albus in GS1 (58.45%), while Cellulomonas did in GS2 (42.93%). In L. luteus, Bacillus, and Aureobacterium dominated in GS1. Data from isolates that grew from 0 to 36h (table 2b) and that grew between 36 and 72h (table 2c) of incubation are treated separately in ANOVAs band c, respectively. These ANOVAs show significant differences between genera and between genera and growth stages (p<0.01 in all cases). Our results after 36h of incubation (table 2b) showed that Aureobacterium was the dominant genus (31.43%), followed by Bacillus and Cellulomonas (23.67% and 18.69%, respectively). Considering the dominant genera under each Lupinus species, Aureobacterium dominated only under L. angustifolius at GS1 and GS2, and under L. albus at GS. From 36 to 72h (table 2c), the frequency of Aureobacterium increased from 31.43% to 52.21%, while Bacillus and Cellulomonas frequencies decreased significantly (4.82 and 7.66%, respectively). In this case, Aureobacterium dominated in all the Lupinus species at both sampling moments, except for L. albus at GS2. Patterns of Taxa from Plant Species To establish differences between the Lupinus species at the two growth stages (GS) sampled, three PCAs were performed, based on the frequencies of bacterial genera. The loading factors of the variables (bacterial genera) determine the position of samples on to the corresponding diagram, defined by the two axes that account for the greatest variance percentages. PCA a was undertaken with data from 0 to 72h, PCA b with data from 0 to 36h, and PCA c with data from 36 to 72 h of incubation. Table 3 shows variance percentages for each PCA. 14 Orsis 15, 2000 J.A. Lucas; A. Probanza; B. Ramos; M.L. Barrientos; F.J. GutiŽrrez PCA a (figure 2) shows data from 0 to 72h. The first two axes account for 88.23% of the variation (table 3). The low frequencies of Bacillus (2.77%) and Pseudomonas (1.38%), and the high frequency of Cellulomonas (42.93%) separate L. albus GS2 from the other samples. L. luteus at GS1 and at GS2 are separated from the others because of the high percentage of Bacillus (41.21 and 34.75%,) and the absence of Cellulomonas in GS1. In PCA b (figure 3), all variables except for Aureobacterium, Arthrobacter, and the Enterobacteriaceae family have high load factors on axis I, accounting for 59.83% of the variation (table 3). L. albus at GS2 appears separated from all others due to the high Cellulomonas frequency. Once more this PCA evidences the differences between the two GS of L. albus because of the increase of Cellulomonas and the decrease of Aureobacterium at GS2. Bacterial communities of Lupinus sp. Orsis 15, 2000 15 1.0 0 –0.5 0.2 1.0 LA GS2 LH GS2 LH GS1 LAN GS1 LA GS1 LAN GS2 LL GS2 LL GS1 Axis I Axis II Figure. 2. PCA with bacterial genera isolated after 72h of incubation at two growth stages (GS1 and GS2) in the four species of Lupinus: L.albus, LA, L.luteus, LL, L.hispanicus, LH, and L.angustifolius LAN. root growth), and linked to another heterotrophic succession (root necrosis with the corresponding rhizospheric soil turning to bulk soil). 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