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Azospírillum VI and Related Microorganisms Genetics - Physiology - Ecology Edited by István Fendrik Maddalena del Gallo Jos Vanderleyden Miklos de Zamaroczy NATO ASI Series Series G: Ecological Sciences, Vol. 37 Yoav Bashan", M. Esther Puente", M. Nieves Rodri Toledo*, Ronald Ferrera-Cerrato*. and Sergio Pedrin "Department of Microbiology, and ^Department of The Center for Biological Research (CIB), A.P. 128 Abstract The survival of Azospirillum brasilense strains rhizosphere of wheat and tómalo plañís and in 23 from a wide range of environmental conditions in brasilense was analyzed for 15 soil parameters rhizospheres tested, regardless of soil type, bact amount of rainfall each soil received prior to sam survival characteristics of A. brasilense differed an origin of the soil and not to prevailíng environm brasilense is a rhizosphere colonizer which surv periods. (ii) Some major physical and chemical s bacteria in plantless soils. Introduction There is no doubt that Azospirillum species surviv the rhizosphere of numerous plant species (Basha in the soil per se is controversial. On one hand, se Dobereiner and Baldani, 1979; Dobereiner et al. 1 but not exclusively (Germida, 1986) from tropical found in nearly every sampling of soil, indicating hand, studies mainly from températe and semi-a (Nayak et al. 1986) found that Azospirillum surv 1983; Bashan, 1986 a; Bashan et al. 1987; De Vandenhove et al. 1993) and hardly lasted from o '*Institute of Natural Resources, Colegio de Po 56230, México. Organisms For all experiments we used Azospiríllum brasilense strains Cd (ATCC 29710) and Sp-24 (Baldani et al. 1986) with the plants wheat (Tríticum aestivum) cvs. "Deganit" (Israel) an "Morelos" (Central México) and tomato (Lycopersicon esculentum) cv. "UC-82-L" (Baj California Sur, México). Bacterial inoculation Bacteria were grown in one of the following growth media: Nutrient Broth (ín Baj California, México and Israel) or N-free médium (NFb) (in mainland México) and prepare for inoculation at various concentraüons as previously described (Bashan, 1986 b, Bashan e al. 1993). The final bacterial concentrations were: IxlO 6 cfu/ml soil either in the presence o absence of plants in Israel, 1.77xl07 cfu/ml soil for both soil or plant inoculation in Baj California, México, and 4.2xl04 (to inocúlate plants) or 1.44xl08 cfu/ml (to inocúlate soil) i Central México. Soil was directly inoculated by applying double washed bacterial suspensió to each pot. Plants were inoculated at sowing as previously described (Puente and Bashan 1993). hermetically sealed plástic containers at 4±1° :s used in the studies. No two commercial core samplers from the soil layer 20-3 :re not compared with those of attempt was made to preserve the soil intact and ntroversy in the soü survival of disturbed. Several soils were from cultivated áre ¡süons of Azospiríllum research land. Soils were collected from arid (< 200 mm r p, 1991, Germany). mm rainfall /year) (5 soils), mountains (500-800 >rrelating the soil parameters of (1800+ mm rainfall /year) (one soil). itain soils from Israel, semi-arid The physical and chemical characteristics of eac ya California, México) with the standard methods: texture and organic matter (Ro 1 and Sp-245. electric conducüvity (Chapman and Pratt, 1984 CaCo, content (Jackson, 1976). Soil sterilization To avoid competition with native microorganism analysis, all soils were sterilized by a standard ty autoclave. Later, the soil was incubated for 24 Cd (ATCC 29710) and Sp-245 Preliminary comparison between sterile and non- ím) cvs. "Deganit" (Israel) and in the level of survival for both strains Cd and Sp- ilentum) cv. "UC-82-L" (Baja Plant growth conditions and inoculation Plants were grown in 500 mi plástic pots contain with 10% NaOCl and thoroughly washed with s :día: Nutrient Broth (in Baja soils without plants was carried out in identical 2 nainland México) and prepared chamber at 25±1°C, 100 mole/m2/sec and 60±2 >ed (Bashan, 1986 b, Bashan et every week with 5-15 mi sterile, distilled wat ni soil either in the presence or different size of the growing plants. Plants w il or plant inoculation in Baja half-strength Hoagland's solution. 10* cfu/ml (to inocúlate soil) in Sampling and bacterial countsfrom solí and root ile washed bacterial suspensión Samples (2 g soil or approx 500-1000 mg (fresh w described (Puente and Bashan, were taken at each sampling. Rhizosphere bac colonize the roots and the adhering soil parücles lightly sonicated al 25 W for 5 min (Colé Parm procedure. Resulte and discussion The reléase of Azospiriüum and other PGPR into soils has a long history of unpredictable and often disappointing results. One of the main obstacles has been the often poor establishmen and survival of the introduced bacteria in the soil prior to root colonization (Bashan and Levanony, 1990, Michiels et al. 1989, Jagnow, 1987). Survival of PGPR in the soil is crucial issue for both ínoculant users and manufacturen, stemming from the commo agrotechnical belief that seed inoculaüon is impracücal in many cases (perennial plants vegetativo propagated plants, trees, or where more than one inoculaüon per season i required, etc). When the PGPR is applied to the soil, it is hoped that it will survive lon enough to fmd its target plant. Thus, before one considers an expensive field inoculaüon, th prospect of bacterial survival in the soil must be considered. The aim of this study was to address the soil phase survival of Azospirillum by creatin sufficient data which will allow future modeling and prediction of bacterial behavior in an gíven soil without laborious studies of bacterial survival in every field. To this end, w collected 23 soil types representing different climatological conditions, from tropical to ari ar (soils from Baja California, available stains of A. brasilense. >ils from Central México), by One fact emerged immediately and was clear even 1) or by the time-limited liquid A. brasilense proved to be a rhizosphere bacteria. I bacteria fell below the level of characteristics as long as plants were growing in survival picture differed significantly. The g geographical than climatological, i.e., in the arid iL The rate of growth or death while it proliferated in the arid soils of Baja Califo ;' Logistic Equation of Growth in Fig. 1 in which the survival of A. brasile representative soil types is demonstrated. e soils were correlated with the With the aid of several statistical analyses perfor . type of soils using Linear and the survival data from all the Principie Componen! Analysis soil parameters data, we were able to sort out the A. brasilense in these soils. Two factors, the levéi and significantly correlated with bacterial surviva d c s- ttí ng history of unpredictable and -O "E 6 :n the often poor establishment root colonization (Bashan and • O • n » ival of PGPR in the soil is a stemming from the common o Mexican soil many cases (perennial plants, me inoculation per season is Israeli rhizosphere Israeli soil Baja California rh Baja C a l i f o r n i a soi Mexican rhizosphe tjfl O loped that it will survive long O ;xpensive fíeld inoculation, the Days a f t e r in il of Azospirillum by creating Fig. 1. Survival of Azospirillum brasilense in the from Israel, Baja California Sur, México a Each point represents the mean of 3-5 conducted in triplicates. For simplicity, t are: Israeli soil; rhizosphere (R) ±1.4851 ±1.39024, S ±1.5149. Central México soil; :m of bacterial behavior in any every fíeld. To this end, we Dnditions, from tropical to arid 10 20 T—• 1 —-1 1 T y=-0.03644x-0.08098 r=0.86* o - y=-0.02354x-0.46746 r=0.82* -0.5 -1.0 O 10 20 30 CaCO,, (%) 40 50 O 10 20 30 40 Rou»h Sand (%) Fig. 2. Linear regression analyses between percentage of CaCO3 (A) and rough sand (B) and the survival rate of A. brasilense in 23 and 13 soil types, respectively. Missing points in each sub-figure are overlapped by other printed points. * - Significance of the regression at P<0.05. iüvely affected survival. On the parameters may affect the survival of this bacteria in level of fine sand in the soil had :ts of single soil parameters on L are known (Foster, 1988; van Acknowledgements y of this study is that only the and positively determined the This study was written for the memory of the l ameters are unimportant when encouraged agricultural research. We thank Ms. Lan unknown are the proportional for excellent technical assistance, Mr. Gustavo Pa her manipulation of the soil discussions on soil analyses and soil classificat clarifying the English and Dr. J. Dübereiner, EMB This study was partially supported by Conse (CONACyT), México grant # 225130-5-3541 A. References 02354X-0.46746 2- ; 20 30 40 Rough Sand (%) D3 (A) and rough sand (B) and :s, respectively. Missing points oints. * - Significance of the Albrecht SL, Gaskins MH, Milam JR, Schank S affecting survival and activity of Azospirillum Genetics, physiology, ecology. Edited by W. K 138-148. Baldani VLD, Alvarez MA de B, Baldani JI, Dober Azospirillum spp. in the rhizosphere and in roo Soil 90:35-46. Baldani VLD, Baldani JI, Dobereiner J (1987) In aestivum) with Azospirillum spp. in Brazil. Biol Bashan Y (1986 a) Enhancement of wheat roots Azospirillum brasilense Cd. following tem mícroflora. Appl. Environ. 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