Introduction

The collections of microorganisms have academic or reference purposes (^{Borman et al., 2006}). And is the maintenance and viability of these collections, one of the most common problems in the laboratory diagnosis of plant diseases. Conservation methods of the causative organisms are presented as a critical step in meeting these goals. Due to their physiological characteristics, species of Phytophthora and Pythium genera present the biggest problems in their survival, and even if they do not have the necessary infrastructure. Fungi and oomycetes lose viability in different conservation methods and otherwise lose virulence and pathogenicity, as in the method of periodic reseeding (^{Dhingra and Sinclair, 1987}; ^{Richter and Bruhn, 1989}; ^{Humber, 1997}).

The oomycetes can be preserved through short-term methods such as planting on slopes with potato-dextrose agar or V8-agar covered with lugol or mineral oil tubes, where it is necessary to replant every two years (^{Humber, 1997}; ^{Heckely, 1978}). Large collections, such as the World Phytophthora Collection, are in cryopreservation at -120 °C in deep freezers (^{Coffey, 2008}) and even reported immersion in liquid nitrogen at -190 °C, where they remain viable for more than 9 years (^{Smith, 1982}; ^{Dahmen et al., 1983}). Although cryopreservation is able to keep the body in its original genetic state, it requires expensive equipment, being its main limitation.

One of the most practical and reliable store Phytophthora forms is in sterile distilled water (^{Dhingra and Sinclair, 1987}; ^{Simpfendorfer et al., 1996}; ^{Ryan et al., 2000}; ^{Ko, 2003}), especially in places where no infrastructure are necessary for cryopreservation. ^{Boesewinkel (1976)} reports have kept Phytophthora cactorum, P. cinnamomi and P. megasperma var. sojae by 6 to 7 years in sterile distilled water at room temperature; Moreover, ^{Ko (2003)} retained viable isolates of de P. cinnamomi, P. parasitica and P. palmivora in sterile distilled water at room temperature for periods of 6-23 years. In the case of conservation in Pythium and Phytophthora floor, this has been poorly documented.

Commonly, this method is considered conservation of short term and its success largely depends on the soil used and the amount of water that is able to retain (^{Dhingra and Sinclair, 1987}) since the formation of structures propagation Phytophthora requires high humidity, high and low oxygen concentration of carbon dioxide (^{Ribeiro, 1983}). With the intention to protect isolates of Phytophthora and Pythium by a method that would prevent loss of virulence and pathogenicity, besides being economically accessible, they have been stored in test tubes containing sterile distilled water and sterile soil in a 2 to 19 years . Recently, was determined its viability in order to know what was the best method for the conservation of these two genders.

Materials and methods

For the conservation of different isolates of Phytophthora and Pythium in sterile soil was used the following protocol Dr. García-Espinosa (Com. Per.): 2 circular segments (9 mm diameter and of varying thickness) of placed different isolates growing medium cornmeal-agar (100%), agar (1.9%) and antibiotics (250 ampicillin ppm, 10 ppm rifampicin and 10 ppm pimaricin) in test tubes of 30 ml capacity containing soil previously sieved (mesh # 20) and autoclaved at 15 lbs pressure for three hours for two consecutive days. After planting, the tubes were closed with its stopper Bakelite, were incubated at 28 °C for 48 h, and stored in dark at room temperature.

In addition, it was stored in tubes with sterile water Dr. García-Espinosa (Com. Per.). A test tubes, about 20 ml capacity, were added 12 ml of distilled water and sterilized for 30 minutes at 15 lbs pressure; subsequently, they were placed in each tube, four circular segments (9 mm diameter and of varying thickness) of different isolates growing medium cornmeal-agar (100%), agar (1.9%) and antibiotics (250 ampicillin ppm, 10 rifampin ppm and 10 ppm pimaricine). The tubes were stored in the dark at room temperature.

At least, there were five tubes in sterile soil conservation for each isolate. To determine the viability of cultures preserved in ground floor of each test tube was subtracted, making prior homogenization, and placed (in duplicate) in Petri dishes with medium cornmeal-agar (100%), agar (1.9% ) and antibiotics (ampicillin 250 ppm, 10 ppm and 10 ppm natamycin rifampicin (Delvocid®), was incubated at 28 °C in dark. in the case of the method in sterile water conservation, the circular portions of each tube were subtracted and they placed (in duplicate) in petri dishes with the means described above.

Isolates were kept in sterile soil: Phytophthora cinnamomi, Phytophthora sp., Pythium vexans and Pythium aphanidermatum. Conserved in sterile distilled water were Phytophthora citricola, Phytophthora sp., Phytophthora cinnamomi, Phytophthora capsici, Pythium sp. and Pythium aphanidermatum (Table 1).

Table 1 Survival of Phytophthora and Pythium species in sterile distilled water and sterile soil. 

Results and discussion

In either replicas made from each tube conservation, he survived the shelf in sterile soil and there was no growth of the pathogen in culture medium. In the case of sterile distilled water conservation in both or one of the aftershocks, there was positive growth response, so they remained viable for the time were preserved. The two isolates of Phytophthora citricola remained viable after 18 and 19 years, while P. cinnamomi was grown successfully after 10 years. P. capsici could be reactivated after 10 and 21 years, like Pythium aphanidermatum reactivated 7 years later. The P. citricola, P. capsici and Pythium aphanidermatum is the first report in sterile water conservation and P. cinnamomi had been reported survival period of between 9-18 years in sterile water (^{Ko, 2003}).

All isolates showed a characteristic mycelial growth in Petri dish, and only produced Phytophthora citricola propagation structures (oospores) six days after being reactivated.

Commonly Pythium and Phytophthora grown-free medium prior to being preserved under various methods (^{Dhingra and Sinclair, 1987}; ^{Ko, 2003}) antibiotics. In this case, the different isolates grew in medium containing pimaricine prior to being preserved, and this condition did not affect its viability as it was possible cultivation after periods of 3-19 years. ^{Tsao and Ocana (1969)} comment that 100 ppm of pimaricin in medium inhibits the germination of chlamydospores, sporangia and zoospores of many species, but also comment that there is a partial inhibition at 25 ppm and ceases inhibition below 12 ppm. Half segments stored in sterile distilled water and sterile soil did not exceed 10 ppm pimaricine.

Success in soil conservation depends on the development of isolation in the early days of conservation. In soil conservation in the mycelium and propagative units of the second generation are those that are preserved (^{Dhingra and Sinclair, 1987}) and this event depends on the success and the time can be preserved.

Unlike the above, the conservation in sterile distilled water offers a longer preservation. This study confirms that species of Phytophthora and Pythium gender successfully conserved in sterile water. The structures of Pythium and Phytophthora resistance can be oospores, chlamydospores, and sporangia (^{Ribeiro, 1983}), although the latter are highly sensitive to desiccation (^{Judelson and White, 2005}). Either in the case of homothallic species (P citricola and Pythium aphanidermatum) where oospores, chlamydospores and sporangia act as survival mechanisms, or in the case of heterothallic species (P. capsici and P. cinnamomi), considering only one group supported in the middle, where sporangia and chlamydospores act as resistance structures (Ribeiro, 1983; ^{Elliot, 1983}), the water conservation prevents desiccation of propagation structures. Sterile water conservation of Phytophthora and Pythium species is simple, reliable and cost, especially compared to methods where reseeding or ultrafreezers required as in the case of cryopreservation.

Conclusions

Viability was confirmed by plating on medium cornmeal-agar culture medium supplemented with antibiotics. None of the oomicetos survived preserved in soil conservation during periods while in sterile water conservation remained viable Phytophthora species for up to 21 years and Pythium for 7 years.