Survival and Symbiotic Effectivity of Lyophilized Root Nodule Bacteria
The study has been carried out under the greenhouse
and laboratory conditions, using the lyophilization technique to determine
the preserving possibilities of 3 native strain isolated from bean root
nodules that been widely cultivated in Turkey and was defined as Rhizobium
bacteria. The strains been developed separately in the Petri dishes
with YMA (Yeast Extract Mannitol Agar) sown into lyophilization tubes
containing skim milk, frozen at -85 °C and dehydrated at -55 °C
with vacuum. According to the cultural counting results continued for
35 months after lyophilization, while the living cells in R11 strains
and R12 strains found as 41x109 and 35.5x109 cell
mL-1, respectively, the best result was determined in the R13
strain as 51.5x109 cell mL-1. In the greenhouse
trials, the dry weight of plant`s green parts, the number of nodules and
total nitrogen amounts were evaluated. The tested strains were inoculated
with bean varieties peculiar to themselves and the lyophilizated cultures
gave similar results with the main cultures kept in tubes with YMA.
Rhizobium bacteria are preserved under various conditions in order
not to lose their nitrogen fixation and genetic change (Antheuniss, 1973;
Miyamato-Shinogara et al., 2006). Since, the cultures used continuously
in the production are preserved best in the leaning tubes of agar, it
facilitates its reproduction (Antheuniss et al., 1981). However,
when preserving cultures in the agar medium, it takes a lot of efforts
to transfer them repeatedly into a new medium every other month (Abadias
et al., 2001; Miyamato-Shinora et al., 2006). In the recent
studies, performing the control of cultures` activeness and effectively
nodulation has been reported to be essential (Safronova and Novikona,
1996; Willems et al., 2001). When compared to the other preservation
methods, the lyophilization method (frozen and dehydrated) has been determined
to be used successfully with a widely range (Abadias et al., 2001;
Miyamato-Shinogara et al., 2000).
Although, there are different views on the preservation temperatures
of frozen and dehydrated cultures, preservation at low temperatures is
recommended by Miyamato-Shinogara et al. (2006), Malik (1990) and
Revellin et al. (2000). Willems et al. (2001) has suggested
that Rhizobium cultures are preserved at either 15 °C in a
dry place or at 4 °C in the fridge. The vigor tests carried out after
lyophilization are usually done in the cultural counting technique and
the results are presented as the number of living cells per milligram
(Peter and Reichart, 2001; Ekdawi-Sever et al., 2003). In the preparation
of culture collections of Rhizobium, lyophilized cultures are reported
to be preferred in the greenhouse and field trials (Miyamato-Shinogara
et al., 2006).
This study has been carried out to constitute the long time preservation
of native Rhizobium strains, whose activeness was determined in
the earlier study (Küçük, 2004), isolated from the root
nodules of bean varieties that are cultivated widely in Turkey, providing
the supply of the essential materials of Turkey`s developing microbial
inoculant production and the formation of Rhizobium cultural collections.
MATERIALS AND METHODS
The strains: In this study, Rhizobium R11, R12 and R13
strains, whose activeness was determined from earlier study (Küçük,
2004), were used.
The preparation of lyophilization process: The cultures been purified
in Petri dishes with Yeast Extract Mannitol Agar (YMA) then transferred
into medium containing 20% skim milk in lyophilization tubes (2 mL) (Malik,
1990). After the tubes having been frozen at -85 °C, they were dehydrated
at -55 °C under vacuum (Jenning, 1999). The samples were accumulated
at +4 °C.
The vigor tests: During the preservation, a vigor test was performed
for the cultures every other month. The cultures were dissolved in the
yeast extract mannitol broth at the time of counting (Ekdawi-Sever et
al., 2003). At the end of three days development, the number of living
cells after lyophilization was obtained by cultural counting technique
(Schoung et al., 2006).
Greenhouse tests: The test subjects include lyophilizated R11,
R12, R13 strains, inoculated and noninoculated subjects. The Rhizobium
strains that defined as Rhizobium bacteria in the earlier study
(Küçük, 2004) were inoculated with bean varieties having
high nodulation and nitrogen fixation peculiar to themselves. (R11, R12,
R13 strains inoculated with Akman 98, Göynük 98 and Şehirali
90 bean varieties, respectively). The bean seeds (Phaseolus vulgaris
L.) were provided from Anatolian Agricultural Research Institute in Eskişehir,
Turkey.The seeds were soaked into 95% sodium hypochloride solution for
5 min and their surface sterilization were made by washing using sterile
distilled water (Daba and Haile, 2002).
Afterwards, the seeds were sown into pots containing sterile peat (3
g). After the lyophilization strains having been developed in yeast extract
mannitol broth 10 mL of culture (109 cell mL-1)
was added to developing roots. At the end of 35 mount development period,
the roots sections of plants were cut and the numbers of nodules were
examined by Mostasso et al. (2002) and Daba and Haile (2002).
The green part sections of the plants were dehydrated at 65 °C in
the oven until they were with stable weight, then weighted and their nitrogen
amounts were determined with Kjeldahl method. All parameters were statistically
analyzed using an analysis of variance (Yurtsever, 1984).
RESULTS AND DISCUSSION
In the recent study, while lyophilizated bacteria cultures were preserved
at +4 °C, the vigor test was carried out every other month for 35
months. At the end of 35th month, nodulation and nitrogen fixation activity
in the tested bean varieties were determined. The living Rhizobium
numbers obtained from culture counting after the lyophilization process
performed every other month are shown in Table 1. The
starter values obtained after lyophilization varied between 263.5x109
and 35.5x109 cell number mL-1 (Table
1). The results in Table 1 show that, the existence
of change on the living Rhizobium cell numbers performed every
other month after the lyophilization process was determined. An analysis
of variance of data indicated that strain, time, strainxtime (SxT) were
significantly (0.01 level) (Table 1). Since the lyophilization
is a method preventing the biological originated material from being spoiled,
it is preferred in the Rhizobium microbiology to preserve the cultures
for long time (Antheuniss, 1973; Miyamato-Shinogara et al., 2000,
2006; Safronova and Novikona, 1996; Willems et al., 2001; Abadias
et al., 2001; Jenning, 1999).
|| Colony forming units after freeze-drying (109
|Significant at the **0.01 levels of probability, respectively
Miyamato-Shinogara et al. (2006), Willems et al. (2001)
and Peter and Reichart (2001) showed that after lyophilization, the starter
values being high like 109 cell mL-1 originated
from their using intensive cell numbers at the start of lyophilization,
taking possible bacteria loss number into account during the lyophilization
process. In addition, in the performed culture counting after lyophilization
every other month continued for 35 months, the decreasing vigor of Rhizobium
strains when compared to their starter values, as Safronova and Novikona
(1996) observed that the deaths occurred in time are thought to be originated
from Rhizobium bacteria`s being quite sensitive to the dry environment.
In the studies carried by Safronova and Novikona (1996), in order to
preserve Rhizobium bacteria, when they compared the methods of
lyophilization with the method of keeping in the liquid nitrogen, they
determined the lyophilization as the best preserving method for Rhizobium
bacteria and also stated that the symbiotic characteristics of strains
do not change at the end of the study.
The number of counted nodules of tested plants in their flowering period,
the dry substance weight dehydrated at 65 °C and their total nitrogen
extent are shown on Table 2. The highest nodule number
which is R12 was obtained from inoculated Göynük 98 bean variety.
Nodulation was not detected in the uninoculated beans. While the highest
dry weight 2.90 g pot-1 was given by the inoculated with R12,
the least dry weight 1.21 g pot-1 was given by the control
(uninoculated) application. An analysis of variance of data indicated
that, combination and treatment were significantly on nodule number (Table
2) and combination, strain, strainxtime were significantly on shoot
dry weight (Table 2), combination, treatment and strain
were significantly on N% (Table 2). The results in Table
2 show that, the total nitrogen varied between 83.43 and 130.5 mg
plant-1. In the greenhouse trials carried out by Mostasso et
al. (2002) and Daba and Haile (2002), the variation of the dry weights
of bean plants being between 2.75 and 2.68 g pot-1 is harmonious
with the our trial`s results.
|| Effects of strains on bean varieties
|*Inc.: Increase, Significant at the **0.01 levels of
Consequently, according to the cultural counting performed after lyophilization,
the starter values of lyophilizated cultures varied between 35.5x109
to 263.5x109 cell mL--1. Between three tested strains,
the highest vigor value was obtained from R13. The values belong to lyophilizated
cultures and the main cultures kept in tubes with YMA were found similar
to each other. During the preservation of isolates, when taking some factors
into consideration such as absence of contamination, having no mutations
due to the continuous inoculated that happening continuously, having economically
low cost, the ability of being preserved securely for a long time, using
the lyophilization technique can be recommended for preservation of Turkish
This study supported by the Scientific and Technological Research Council
of Turkey (TUBITAK-BAYG).
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