NATURAL OCCURRENCE OF Pasteuria nishizawae IN SOYBEAN AREAS OCORRÊNCIA NATURAL DE Pasteuria nishizawae EM ÁREAS DE SOJA

: Soybean ( Glycine max (L.) Merrill) is a widely grown crop of economic prominence. Brazil is considered as the second largest worldwide producer and exporter. Soybean cyst nematode, Heterodera glycines Ichinohe, in one of the most serious threats for this crop, considered as its most destructive parasite. The first report of this disease in Brazil was recorded during the 1991/1992 harvest. Control of nematodes is more challenging if compared with other plant diseases control. Thus, there is a growing demand to search for alternative control practices that will not harm the environment nor the human being. Therefore, the highly specific bacteria Pasteuria spp. Metchnikoff, represents an auspicious biological control agent against nematodes. The biological control of the soybean cyst nematode with the bacterium Pasteuria nishizawae Sayre has proven an excellent choice and has been studied by different scientists. The objective of this work was to determine the natural occurrence of Pasteuria nishizawae in Brazilian soils. The experiment was performed under greenhouse conditions at the Research unit from Syngenta® in Uberlandia-MG, Brazil, with soil samples originated from soybean planted areas from the Brazilian states of Bahia, Goiás, Mato Grosso and Paraná. Fractions of 150 cm 3 of soil were withdrawn to be processed by the centrifugal flotation technique in sucrose solution. Aliquots of 1 mL from the obtained suspension were observed in Peters’ chamber with the aid of an inverted light microscope, in order to verify the absence or presence of bacterial endospores attached to the cuticle of the nematodes extracted from the soil samples. The frequency of Pasteuria nishizawae incidence was of 100% within the samples analyzed.


INTRODUCTION
Soybean is a crop of economic importance, being the most planted oilseed in the world. Brazil is the second largest producer and exporter of soybean in the world, with a planted area of approximately 26,4 to 27,3 million hectares and a production of about 80,08 to 82,99 million tons in 2012/2013 (CONAB, 2013).
The soybean cysts nematode (SCN), Heterodera glycines, is one of the most severe plant disease problems of this crop, considered as the most destructive parasite of soybean plants (NOEL, 1992).
In order to control these nematodes the primary common methods used are chemical control (nematicides), the use of resistant varieties and crop rotation. Chemical control by means of nematicide application has proven expensive as well as detrimental to the environment, to the human being, to the wild fauna and to the beneficial organism within the soil. The use of resistant varieties is a natural approach, highly recommended to control plant pests and diseases. However, in the case of this specific nematode, the availability of resistant varieties for the farmer is scarce, due to the great number of races or HG types of SCN occurring in Brazil (FERRAZ;FREITAS, 2004).
In tropical and subtropical countries nematodes find ideal humidity and temperature conditions for reproduction and feeding. Thus, worsening the efficiency of control of these pathogens, that are very difficult to eradicate once they are established within an area (TORRES et al., 2008).
Biological control is an alternative method to control plant parasites, as nematodes. Biological control of nematodes can be achieved by the use of appropriate fungi or bacteria. Predators and egg parasites are certainly the most studied organism and the best fitted as nematode's biological control agents (JATALA, 1986;NORDBRING-HERTZ et al., 2002).
The first observation of Pasteuria in a plant parasite nematode, Pratylenchus pratensis de Man, was made by Thorne (1940) whom classified the organism as a Protozoa and named it as Dubosquia penetrans. However, studies performed by Mankau (1975), re-classified the organism as a prokaryote of the Genus Bacillus Cohn. The re-discovery of Pasteuria ramose by Sayre et al. (1977) and its morphological similarities with Bacillus penetrans Mankau suggested that these two bacteria had a common generic relation. In 1985, Sayre and Starr named the organism that parasite Meloidogyne incognita as Pasteuria penetrans (Thorne) Sayre & Starr, as well as Pasteuria nishizawae is specific to the soybean cysts nematode (Heterodera glycines), Pasteuria thornei Sayre & Starr is specific to the nematode of the root lesions according to Stirling & Wachtel (1980), while Pasteuria usgae is specific to the nematode Belonolaimus longicaudatus Rau according to Rau Giblin-Davis et al. (2003).
Pasteuria is a gram-positive bacterium that forms an endospore. These bacteria were found as parasites of many economically important nematodes (SAYRE; STARR 1988). The Genus Pasteuria spp. was structured from 323 nematode species belonging to 116 Genera, including plant parasitic nematodes, entomopathogenic nematodes, predator nematodes and free living nematodes (CHEN;DICKSON 1998).
The analysis of the 16S rRNA gene sequences is a sensible and well-established tool for the detection and phylogenetic analysis of bacteria (STAHL, 1997). The use of sequences of the gene 16S rRNA may lead to identification of species of Pasteuria and to evaluation of the diversity of these microorganisms within a population of nematodes' samples, thus being a tool of a great utility for the comprehension of the parasite-nematode interactions and the ecology of Pasteuria (EBERT et al., 1996;ANDERSON et al., 1999;ATIBALENTJA et al., 2000;BEKAL et al., 2001).
For many years attempts to obtain an axenic culture of Pasteuria spp. resulted in failure (WILLIAMS et al., 1989;BISHOP;ELLAR, 1991). However, an advance towards the success to obtain an in vitro culture of P. penetrans was announced by Hewlett et al. (2002).
A life cycle study of the bacterium that parasites the nematode Heterodera glycines was performed by means of germination of the endospore that infects J2's for production of a next generation endospores in adult females and cysts. Descriptions were based in microscopic examination of successive juvenile stages of H. glycines excised from soybean roots, unlike P. nishizawae, that were based exclusively on the examination of diseased cysts (SAYRE et al., 1991).
According to Atibalentja et al. (2004), bacterium development, germination of the endospore and penetration of the germ tube inside the nematode begin soon after penetration of the J2's in the radicular system. Otherwise, the endospore does not germinate and consequently no infection by Pasteuria will arise. For this reason, observations based only on diseased cysts will result in an incomplete analysis of Pasteuria's life cycle, which would explain why germination of P. nishizawae is not observed (SAYRE et al., 1991).
After the endospore's germination, primary cauliflower-like colonies are formed within the J3. Development of the bacterium was observed only in female adults but not in males. Later, the bacterial sporulation can be observed with the development of a structure similar to a chunk of grapes, which can be observed also in immature females of fourth stage and cysts. Finally, the presence of mature sporangia and endospores can be observed, varying in number (30.000 to 820.000 with mean value and standard deviation of 314.00 and 234.000, respectively) as a function of the size of the cyst or female nematode (ATIBALENTJA et. al., 2004).
Until now, P. penetrans is the most studied species due to its potential as a biological control agent. However, technical hitches for mass production in vitro had made its commercial production problematic. In order to introduce bacteria in the soil environment for field experiments, dry roots' powder infected by the bacteria was used, as proposed by Stirling and Wachtel (1980). Thus, the objective of the present work was to verify the natural occurrence of Pasteuria nishizawae Sayre in soils under soybean cultivation.

MATERIAL AND METHODS
Soil samples with cysts coming from different established soybean growing areas from Brazil (Table 1), were processed by the centrifugal flotation in sucrose solution technique (Jenkins, 1964).
Part of those ten processed soil samples were added, individually, to ceramic pots and preserved under greenhouse at Syngenta's experimental station in Uberlândia, MG -Brazil. These pots were planted with the susceptible soybean cultivar 'Lee 74E', in order to promote multiplication of Heterodera glycines, as shown in Figure 1.   A soil aliquot of 150 cm 3 was obtained 28 days after sowing and then processed by the centrifugal flotation in sucrose solution technique, according Jenkins (1964).
From the obtained suspension solution an aliquot of 1 mL was observed in Peters chamber under an inverted light microscope, verifying the presence or absence of endospores of the bacterium attached to the nematode's cuticle ( Figure 2). Subsequently, the percentage of nematodes showing bacteria attached to its body in relation to the total number of nematodes observed in the suspension was calculated.

RESULTS AND DISCUSSION
Within all ten samples the frequency of occurrence of the bacterium Pasteuria nishizawae was of 100%, that is to say, there were observed bacterial endospores attached to the cuticle of all the nematodes Heterodera glycines extracted from the soil 28 days after soybean sowing (Table 2), as showed in Figure 3.  In Brazil, studies regarding Pasteuria spp. are still developing, especially when concerning to its employment to control species of Pratylenchus and Heterodera. The vast majority research about the subject is developed in the country focusing the root-knot nematode (Meloidogyne spp.).
According to Tzortzakakis and Gowen (1994), a great number of endospores attached to the J2 nematodes, does not ensure reduction in the number of eggs produced, if a high variance of attachment and infectivity of endospores occurs. These remain viable in the soil by many years, are resistant to desiccation (STIRLING, 1984) and relatively resistant to high temperatures. Lordello (1966) reported for the first time in Brazil, the occurrence of Pasteuria spp. in females of Meloidogyne javanica, infecting plants of tomato coming from Vargina in Minas Gerais. Santos (1981), observed the bacterium in juveniles of the second stage of M. javanica extracted from soil collected from the rhizosphere of bean plants intensively infected by the nematode in Petrolina, Pernambuco. Subsequently, several works were performed on the parasitism of Pasteuria spp. in nematodes, verifying in the average, activity of the bacterium over Meloidogyne species (PIMENTA; CARNEIRO, 2005).
According to Souza and Campos (1996), Pasteuria spp. was observed in 29,69% of the samples collected in 128 locations and 28 vegetal species in Minas Gerais, similar results were also verified in other countries: South Africa (Spaull, 1984), Australia (STIRLING; WHITE, 1982; BIRD; Natural occurrence of Pasteuria nishizawae… VICENTE, C. B.;SANTOS, M. A. BRISBANE, 1988), USA (WALTER; KAPLAN, 1990;HEWLETT et al., 1994) and Spain (VERDEJO;LUCAS, 1992 There is no record of the occurrence of Pasteuria spp. in regions with annual mean temperature under 10ºC,with the higher occurrence being observed under annual mean temperatures above 21ºC (CHEN; DICKSON, 1998).

CONCLUSIONS
Pasteuria nishizawae has natural occurrence in Brazilian soils under soybean cultivation, specifically in the States of Bahia, Goiás, Mato Grosso and Paraná.
The frequency of Pasteuria nishizawae occurence within the analyzed soils was of 100%.

ACKNOWLEDGMENTS
The author would like to thank Syngenta's experimental station staff at Uberlândia -MG, where this research was performed.