Occurrence and Host Specificity of Indigenous Rhizobia from Soils of São Paulo State, Brazil
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Indigenous rhizobia from Brazilian soils 543 Note OCCURRENCE AND HOST SPECIFICITY OF INDIGENOUS RHIZOBIA FROM SOILS OF SÃO PAULO STATE, BRAZIL Maria Luiza Colognesi de Oliveira Lombardi1; Milene Moreira2*; Luís Alberto Ambrósio1; Elke Jurandy Bran Nogueira Cardoso3 1 IAC - Centro de Pesquisa e Desenvolvimento de Solos e Recursos Ambientais, C.P. 28 - 13012-970 - Campinas, SP - Brasil. 2 APTA - Pólo Médio Paranapanema, C.P. 263 - 19800-970 - Assis, SP - Brasil. 3 USP/ESALQ - Depto. de Ciência do Solo, C.P. 9 -13418-900 - Piracicaba, SP - Brasil. *Corresponding author <[email protected]> ABSTRACT: The occurrence of rhizobial communities at four sites under natural vegetation and one site under pasture were examined. Isolates of rhizobia originating from crotalaria (C. junceae), common bean (Phaseolus vulgaris) and pigeon pea (Cajanus cajan) were studied in relation to population density, host specificity and the interaction between rhizobial occurrence, climatic conditions and soil properties. pH values and potential acidity were the soil properties that most affected rhizobial occurrence. Rhizobia from crotalaria and common bean were evaluated at four sites, and from pigeon pea, at five sites. Common bean was the most specific legume, followed by peanuts, crotalaria and pigeon pea. Key words: Rhizobium, Bradyrhizobium, crotalaria, pigeon pea, common bean OCORRÊNCIA DE RIZÓBIOS NATIVOS EM PLANTAS HOSPEDEIRAS DE SOLOS DO ESTADO DE SÃO PAULO, BRASIL RESUMO: Foi examinada a ocorrência de comunidades de rizóbios em quatro locais de vegetação natural e um local de pastagem. Isolados de rizóbio originados de crotalária (C. junceae), feijão (Phaseolus vulgaris) e guandu (Cajanus cajan) foram estudados em relação à densidade populacional, planta hospedeira e interação entre ocorrência de rizóbio, condições climáticas e propriedades do solo. Os valores de pH e potencial de acidez foram as propriedades do solo que mais contribuíram para a ocorrência de rizóbio. A ocorrência de rizóbio em crotalária e feijão foi estudada em quatro locais, e em guandu em cinco locais. O feijão foi mais específico, seguido por crotalária e guandu. Palavras-chave: Rhizobium, Bradyrhizobium, crotalária, guandu e feijão INTRODUCTION dicated that the rhizobial communities can be affected by the properties of the soil and by the host plants Biological nitrogen fixation is a vital component of ag- (Palmer & Young, 2000, Zhang et al., 2001; Laguerre ricultural sustainability, and the understanding of rhizobial et al., 2003), as well as by soil tillage (Cattelan & Vidor, diversity in different ecosystems is therefore fundamen- 1990; Castro et al., 1993, Carvalho et al., 2004), and tal. It is necessary to consider the complexity of the in- crop history (Venkatesvarlu et al., 1997). teractions among rhizobia and the soil characteristics that The natural vegetation in the State of São Paulo is influence the occurrence and diversity of these microor- composed of forest, savanna (cerrado) and “cerradão”, ganisms in natural ecosystems. Also, the discovery of new but little is known about communities of rhizobia in genetic materials that can be used in biotechnological ap- these areas. Although the presence of the leguminous plications is essential for sustainable agriculture. host is one of the determinants of the size of the Cultivated areas containing Neonotonia wightii rhizobial population, other factors, such as environmen- (Lopes et al., 1972), peanut (Giardini et al., 1985; tal stresses, soil acidity and the presence of toxic ele- Lombardi et al., 1992) or green manure legumes ments in the soil, may play an important role in the (Chada & De Polli, 1988; Rodrigues et al., 1994, Zilli establishment of rhizobial communities (Kahindy et al., et al., 2004) were capable of maintaining rhizobial com- 1997). Brazilian soils are in general acidic and can munities that were highly infective for their host plants present high exchangeable aluminum concentrations, and well adapted to soil conditions. Several studies in- a factor that can limit rhizobial survival. Sci. Agric. (Piracicaba, Braz.), v.66, n.4, p.543-548, July/August 2009 544 Lombardi et al. A survey of the presence of rhizobial strains was Soil samples were collected at the top layer (0-20 conducted at four sites under natural vegetation and cm depth) and consisted of 20 sub-samples taken at one site under pasture in the State of São Paulo, Bra- a distance of at least 15 m between the sampling zil. Rhizobia and bradyrhizobia were isolated from soil points. The sub-samples were mixed and submitted to using Phaseolus vulgaris, Crotalaria juncea and chemical analysis (Table 2). Cajanus cajan as trap hosts. The population density of rhizobia nodulating each host plant was evaluated. Rhizobial counts in soil The objective of this study was to examine the oc- A greenhouse experiment was carried out in plas- tic pots (300 cm3) filled with sterilized sand. Seeds of currence and diversity of the rhizobial communities at crotalaria were sterilized with concentrated sulphuric the sites and to determine whether the presence of the acid for three minutes and washed with sterilized wa- rhizobial strains is dependent on soil chemical proper- ter. Seeds of common bean and pigeon pea were dis- ties. infected with a 5% solution of sodium hypochlorite for MATERIAL AND METHODS five minutes and washed with sterilized water. Three seeds of each legume were planted per pot and, after Soil samples 5 days, seedlings were thinned to one per pot. A four- –4 –7 Soil samples were collected at five sites with dif- fold soil dilution series (4 to 4 ) with three replicates ferent plant covers: (i) cerradão (Morro Agudo - for each soil type and four replicates for each dilution 20º43’S and 48º55’ W); (ii) savanna (cerrado) was used to determine the Rhizobium most probable (Campinas - 22º53’ S and 47º04’ W); (iii) Paspalum number (Somasegaran & Hoben, 1985). Each pot re- notatum pasture (Anhembi - 24º48’ S and 48º07 W), ceived 1 mL of each soil dilution. Pots were distrib- and (iv) forest (Pariquera-Açu - 24º43’ S and 47º52’ uted in a completely randomized design in a green- W, and Pindorama - 21º13’ S and 48º55’ W). Some house, and plants were supplied weekly with minus native legumes mixed under the native vegetation were N nutrient solution (Norris, 1965). The pots were ir- observed at Anhembi, Campinas and Morro Agudo, rigated with sterilized water, whenever necessary. while no leguminous plants were found at the two other Plants were harvested 40 days after planting. Non-in- sites. The soil types corresponding to these areas are: oculated control plants were sown in sterilized sand, Kandiudult, under pasture in Anhembi, Hapludox un- to verify the aseptic conditions of the experiment, in der savanna in Campinas (Oliveira et al., 1976), addition to the plants grown in this experiment. Eutrudox under forest, in Pariquera-Açu (Sakai & Nodule formation and rhizobia isolation Lepsch, 1984), and Kanhapludult in Pindorama Crotalaria, common bean and pigeon pea were cul- (Lepsch & Valadares, 1976). The temperature and pre- tivated in plastic pots (500 cm3) with washed and ster- cipitation regimes of the different regions were quite ilized sand plus soil, in a proportion of two parts of variable (Figure 1). sand (300 cm3) to one part of soil (150 cm3). Con- 30 trols were set up in pots that contained only sterilized A) 25 sand. Seeds of each legume were prepared as in the 20 first experiment. Six seeds for each host plant were 15 planted per pot and, after five days, seedlings were 10 thinned to two per pot. The pots were distributed in a Anhembi Campinas Morro Agudo greenhouse, in a completely randomized design. Plants 5 Pariquera-Açu Pindorama Mean temperature (°C) 0 were harvested 40 days after planting. Jan Feb Mar Apr May Jun Jul Aug Sept Oct Nov Dec Nodules of the five replicates of each legume and of each soil were removed, mixed, counted and six nodules were chosen at random for isolation of rhizo- 350 B) Anhembi Campinas Morro Agudo 300 bia, according to Vincent (1970). 250 Pariquera-Açu Pindorama 200 Statistical analysis 150 100 Analyses of variance were conducted to test the Total rainfall(mm) 50 effects of the treatments corresponding to different 0 sites and host plants, on log transformed data for Jan Feb Mar Apr May Jun Jul Aug Sept Oct Nov Dec 10 rhizobial number [log10(CFU+1)], with the F test for Figure 1 - Monthly distribution of (a) mean temperature and (b) treatments at the 5% level of significance. For mul- total rainfall at sites from which soils were sampled. tiple comparisons of means of the five sites (Anhembi, Means were for 1961-1990. Campinas, Morro Agudo, Pariquera-Açu, and Sci. Agric. (Piracicaba, Braz.), v.66, n.4, p.543-548, July/August 2009 Indigenous rhizobia from Brazilian soils 545 Pindorama) and three host plants (Crotalaria, Common soils, with a significance level of 5%. Common bean bean, and Pigeon pea) the Tukey test at the 5% level rhizobia were more specific than those of other le- of significance was used. gumes (Table 6). Peanut and crotalaria did not nodu- To identify a useful subset of predictors of rhizobial late with isolates originated from common bean. number (CFU) in soil, a stepwise regression was used The F test of the ANOVA showed that the main ef- which removes or adds variables to the regression fects of sites and host plants and their interactions upon model (Yamane, 1973). These variables are removed the variable log (cfu+1) were highly significant (p < or added if their p - values are greater than the alpha 0.0001). Thus, we compared the means of log (cfu+1) value, alpha here considered as 0.25 for the F-to-en- for each host plant at each site, using the Tukey test ter and F-to-remove test. The high value of 0.25 for (p < 0.05) (Table 7).