Indigenous rhizobia from Brazilian soils 543

Note OCCURRENCE AND HOST SPECIFICITY OF INDIGENOUS RHIZOBIA FROM SOILS OF STATE,

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 - , SP - Brasil. 2 APTA - Pólo Médio , C.P. 263 - 19800-970 - Assis, SP - Brasil. 3 USP/ESALQ - Depto. de Ciência do Solo, C.P. 9 -13418-900 - , SP - Brasil. *Corresponding author

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 () 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.

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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 ( - 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 ( - 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 - 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

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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). alpha that increases the rejection zone of the null hy- pothesis (H0) is due to the exploratory character of DISCUSSION this investigation. Therefore, all potential predictor vari- ables (even those with low values) are included in this Temperature and rainfall distribution (Figure 1) model to explain the variations of the dependent vari- showed a certain variation form site to site, but nor- mally followed the expected pattern of a warn and rainy able (Conagin et al., 2006). The log10 (CFU+1) values were considered as the predicted variable and the pre- summer, followed by a colder and drier winter with dictor variables were the soil properties: pH, Al+3, or- low temperatures and a very low soil pH. Morro Agudo ganic matter, H+Al, sum of bases, bases saturation and under savanna vegetation and Anhembi had the high- phosphorus level. The winter temperatures and pre- est pH values and there, all three legumes produced cipitation means for May, June, July and August were nodules. However, in Campinas, with a much lower also considered as predictor variables. pH value and a medium clayey texture soil, there was also generalized nodulation, while in Pindorama, with RESULTS a much higher pH value and a very sandy texture, crotalaria failed to nodulate. Finally, in the forest soil All soil samples contained either slow growing or from Pariquera-Açu, highly acidic and slightly clayey, fast growing Rhizobium or Bradyrhizobium species, most legumes did not nodulate. Only pigeon pea pre- generally in low numbers, that varied from 12.7 cfu sented some nodules. g–1 soil for pigeon pea in Pariquera-Açu to 783 cfu g–1 For the increase of each pH unit, there was an in- soil for bean plants in Morro Agudo (Table 3). In crease of 36.6 cfu (antilog =1.5) and for an increase Pariquera-Açu, with the most acidic soil, the lowest of each H + Al unit there was an increase of 1.02 cfu rhizobial numbers were found, while Morro Agudo (antilog 0.0076) (Table 1). pH and H + Al are soil prop- presented the highest total rhizobial numbers. Table 4 erties that affect rhizobial growth and can influence shows the descriptive statistics for the observations the availability of some important nutrients for rhizo- of the sites. Campinas was not considered because the bia, like phosphorus and calcium. All soils contained high rhizobial numbers (mean = 65.7) in combination indigenous rhizobia capable of inducing nodulation in with the low base saturation value (mean = 3.0) in this at least one or more of the host legumes, which agrees location differed totally from those found at the other with Bala et al. (2003). Numbers as low as 6 × cfu g–1 sites. However, no special attempt was made to really soil in four Hawaiian soils cultivated with six legume classify the bacteria at the species or even at the ge- species were found by Singleton & Tavares (1986). nus level. Woomer et al. (1988) examined fourteen sites with four

The model fitted by stepwise regression analysis soils and five legumes and found 1.1 to 4.8 log 10 cells was: g–1 soil. In two sandy soils classified as Kandiudult (pas- Log (cfu+1) = a + b pH + b H+Al 10 1 2 ture and forest) all plants had fewer nodules compared 2 with legumes cultivated in soils with high clay con- The adjusted determination coefficient (R adj.) tents such as Hapludox under savanna and Eutrudox shows that 64.16% of the variability of log10 (cfu+1) accounted for the model. Results referring to legume nodulation in each soil are presented in Table 5, and Table 1 - Values of the terms used in the regression analysis. the same tendency was observed: common bean cul- S)scitsitat CHa(mreTtnatsno plA+H tivated in Morro Agudo presented the highest nodule stneiciffeoC 295.2- 05.1 6700.0 number, and pigeon pea cultivated in Pariquera-Açu the lowest. The pH and (H + Al) were the soil properties t6eulav- 575. 6.2 that contributed most to the rhizobial occurrence in eulav-p 00.0 210.0

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Table 2 - Chemical properties of soil samples.

)1( )2( Sleti CaCHp 2 OPM KCg aMHl+BA S A%l V gkg –1 gkgm –1 -g------kclomm –1 ------ibmehnA 0.5 6.1 5 3.4 21 5 52 3.12 02.0 64 C9sanipma 33. 32. 0119. 9 9920. 134. odugAorroM 4.5 0.6 57 6.6 86 82 34 6.201 52.0 07 P3uçA-areuqira 34. 42. 181435. 280 80. 244. amarodniP 6.4 7.2 8 5.3 91 8 23 5.03 10.0 84 1SB = sum of bases; 2V = base saturation

Table 3 - Rhizobia means as log10 (cfu+1), found in roots of three legumes planted in soils sampled at sites from the State of São Paulo, Brazil.

Sneti Osoitategevlanigir Naemugellaruta Cniralator Caaebnommo Plepnoegi atoT ibmehnA erutsaP 54.1 54.1 67.1 14.1 50.2 Casanipma S6nnava 167. 117. 270. 195. 2.2 odugAorroM oãdarreC 72.2 72.2 98.2 06.1 00.3 PtuçA-areuqira F---0sero 101. 1.1 amarodniP tseroF - - 13.1 65.1 57.1

Table 4 - Descriptive statistical data of four sites. P*retemara Mrnae Smorredradnat Mmumini umixaM **UFC 42.99 80.722 00.0 00.0011 A2munimul 097. 019. 000. 4.2 Hp 85.4 08.0 03.3 04.5 O8rettamcinagr 306. 107. 106. 0.6 sesabfomuS 08.04 20.73 08.8 06.201 B2noitarutasesa 412402.4 7 surohpsohP 52 93.92 5 57 H5lA+ 772.6 706.5 200.5 0.502 erutarepmeT 98.81 59.0 79.71 01.02 P5noitatipicer 450.8 15.6 357.4 9.47 *Means of four sites. Samples from Campinas were not considered (outlier data). CFU** = colony forming units.

Table 5 - Number of nodules per plant when crotalaria, common bean, and pigeon pea were inoculated with Rhizobium isolates from different plants.

Sreti Paevoctnal Cniralator Caaebnommo Plepnoegi atoT ibmehnA erutsaP 00.5 9.61 7.3 8.52 Casanipma S0nnava 200.3 30.1 30. .75 odugAorroM oãdarreC 00.13 7.78 2.81 8.731 PtuçA-areuqira F*sero n*.d. n0.d. 80. .8 amarodniP tseroF *.d.n 2.3 9.4 1.8 *n.d. = not detected under “cerradão”. In the clayey Hapludox from soil compared to a very clayey soil. Similar results Pariquera-Açu, the acidity seems to have affected the were attained by Rupela et al. (1982), who studied rhizobia more than soil texture. Few studies have rhizobial communities capable of nodulating Cicer shown the influence of soil texture on rhizobial com- arietinum. munities, but Danso & Owiredu (1988) observed The presence of a particular Rhizobium or fewer Vigna unguiculata rhizobia in a sandy or acidic Bradyrhizobium was correlated with the occurrence

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Table 6 - Host specificity of rhizobia isolates from crotalaria, common bean and pigeon pea: number and percent of nodules from one legume that nodulates the other legumes. Oeemugellanigir Tatalosilato Cniralator Caaebnommo epnoegiP n%.o n%.o n%.o .on airalatorC 81 00181 00 1611 C4naebnommo 200020014 0 aepnoegiP 03 7.6732 00 00103

Table 7 - Means as log(cfu +1)for the different plants at each site. HitnalPtso Asbmehn Coanipma MudugAorro PaçA-areuqira marodniP airalatorC ba64.1 ba67.1 b52.2 b00.0 b00.0 Canaebnommo 1a67. 2a00. 2b78. 0a00. 23.1 aepnoegiP b14.1 b65.1 c16.1 a50.1 a65.1 *Values in the columns followed by different letters indicate differences, among the means (Tukey, p < 0.05). of its appropriate host legume in soils from Hawaii to be a highly promiscuous legume and is capable of (Woomer et al., 1988). In our study, the highest nodulating with slow and fast growing rhizobia rhizobial number was observed in common bean in (Bromfield & Kuma Rao, 1983; Anand & Droga, four of five sites examined, and pigeon pea consis- 1991). Pigeon pea isolates could be either slow or tently had the fewest nodules (Tables 5). However, fast growing. This may explain the poor nodulation pigeon pea was nodulated in all soils. Therefore, demonstrated when peanut was inoculated with iso- these rhizobia were widely distributed, although the lates from pigeon pea, since peanut is nodulated pref- populations of pigeon pea rhizobia were not high in erentially by slow-growing rhizobia. Crotalaria did not any of these soils. Crotalaria did not nodulate in two nodulate with all the isolates from common bean be- soils and the lack of nodules in the soil from cause this legume nodulates only with slow-growing Pindorama had not been expected because this le- rhizobia and common bean is nodulated only by fast- gume is commonly nodulated in tropical soils (Tables growing rhizobia. Also, the fast-growing isolates from 5 and 6). pigeon pea were not capable of nodulating crotalaria, All sampling sites, except the Kandiudult under probably for the same reason (Table 7). To know the pasture in Anhembi, were under native vegetation: number of indigenous rhizobia in soils is important forest, savanna or “cerradão”. At all these sites, in- when soils are to be utilized for agricultural practices. digenous legumes were found. In an area close to The response to inoculation was affected by the soil Campinas, only few alterations in the native floristic population when the number of counted rhizobia was composition were found, and Leguminosae prevailed at or above 20 cells g–1 soil (Singleton & Tavares, over other plant families and occurred as native her- 1986). baceous and woody components (Matthes et al., A better knowledge of these communities can per- 1988). This fact may have contributed to the occur- haps answer questions about the potential efficacy of rence of rhizobia in all areas where the soils were legume inoculation in soils of the São Paulo State. In- sampled. The Kandiudult from Anhembi has been cul- oculation has not always produced consistent results, tivated with Paspalum notatum for ten years, and pre- especially in areas recently brought into agricultural viously had been cultivated with corn. No legumes productivity. In this study the nitrogen fixation effi- were observed in this area, but this site was next to ciency of the isolates was not evaluated, although it areas with native forest which contained shrubs, her- is necessary to obtain information about it and also, baceous and woody legumes. The legumes used in to obtain new strains for studies. this study became nodulated by the native commu- nities of rhizobia that were present, and, if we con- ACKNOWLEDGEMENTS sider the presence of wild legumes in all soils, this nodulation had been expected. The authors thank Dr. Dilmar Baretta for critical Only bean did not nodulate with the rhizobia from review of the text and Dr. Ricardo M. Coelho for soil crotalaria and pigeon pea (Table 6). Most isolates classification. Thanks also go to Rosana Gierts for from crotalaria (83%) nodulated peanut and 61% technical assistance. E.J.B.N.C. thanks CNPq for a formed nodules in pigeon pea. Pigeon pea is known research fellowship.

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