الفهرس 
Material and methodsOnly 14 pages are availabe for public view
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Abstract
Until perhaps 10 years, studies of environmental stress and the
legume-Rhizobium or Bradyrhizobium symbiosis were restricted to
defining the problem, and to using physical or chemical amendments to
overcome it, i.e., mulching to reduce soil temperature or liming to
ameliorate soil acidity. The identification of bacterial strains and in some cases host cultivC;lfs that are tolerant to these stresses opens the way for alternate, lower cost solutions to these problems. c’ Therefore, in the present study, the ability of2S local and foreign Rhizobium and Bradyrhizobium strains represent R. meliloti (5 strains),
R, leguminosarum bv. viceae (5 strains), R leg!lminosarum bv. trifolii
(2 strains), Bradyrhizobium sp. nodulating peanut (5 strains) and
.Bradyrhizobium . japonicum. ( 5 strains) to tolerate increased
concentrations of NaCI, high temperature, high levels of nitrogen, high pH values and desiccation was investigated. Rhizobial strains tolerant to the above mentioned stress conditions were selected and the symbiotic performance of the obtained strains was evaluated as well. Results are summarized as follows :
1- Effect of NaCI salinity on growth and survival of rhizobia:
1.AU tested strains were able to tolerate NaCl concentration up to O.1°/0 However, tolerance of NaCI salinization differed according to
rhizobial spp. and strains. In general, R. meliloti and K
leguminosarum bv. phseoli were found to be more sensitive to NaCl
salinity . 2.There was a great variation in the response of the tested strains tosodium chloride regimes. All strains were sensitive to higher
concentration of NaCI(S%). The growth of some strains was
completely inhi~ited at 20/0 NaCI. Some ,strains were sensitive to even
20g IL NaCl. 1. Two Rhizobium l~guminosa,.um bv. viceae ARC 200Fand ICARDA 441 were tolerant to 10 and 40 gIL NaCl, the two strains were
identified as low and high tolerance to NaCl salinity respectively ..
2- Growth and survival res onse of rhizobia to increasin tem erature:
I 1.The most of the rhizobial strains-tested showed optimum growth at 30-
350C , then higher temperature resulted in gradual decreases in
rhizobial numbers. Susceptibility to higher temperature of 50 ~Cis
common among the rhizobia! species and strains under investigation.
2. The decline in, the number of surviving strains was especially
noticeable between 40 and 45°C. This behavior depends on the
rhizobial sp. and ·strain.
3. In general, fourteen strains were found to tolerate temperature up to 45
0C. such as Rhizobium meliloii (ARC 1, ARC 2, and A 2), Rhizobium
leguminosarum bv. viceae (ARC 200 F and ARC 202F), Rhizobium
le~minosarum . bv. trifolii (TAL), Rhizobium lflEU_mirlOsaromby.
phaseoli tARC 302 and UMR), peanut Bradyrhizobium (619, 3339
and 601) and firadyrhizobium jqponicum (138, USDA 110 and ARC4. Three strains, however, were found to be susceptible to elevated temperature more than 3S °C such as Rhizobium le.,gpminosarufIl bv.viceae (ARC 206 F and ARC 207 F) and Rhizobium legumino.,arum
by. tYi/olii (ARC 103).5. Four rhizobia 1 strains tolerant to high temperature representing peanut Bradyrhizobium (3456 T 40°C and 619 T 45°C ) and
Bradyrhizobium jqponicIlm (3407 T 40°C and 138 T 45°C) were
selected for further competence study.
3- Effect of mineral nitrogen levels on survival of rhizobia:
1. All tested strains were able to grow and survive under stress of
200ppm N as KNO, (20 kg N I fed) in YEM medium 2. Strains representing Rhizobium le~”,i’lO.<arum bv. viceae, bv. trifolii,bv. pha.<eoliand Bradyrhizobium sp. (Peanut) were able to tolerate up
to 300ppm N111 liquid medium.
3.A11 tested strains representing R. meliloti were able to tolerate up to
400ppm N as KNO, (ARC 1, ARC 2, and ARC 3 ) . The other two
strains (ARC 6 and A 2) were superior in surviving up to 500 ppm N.
4. With increasing the dose of mineral nitrogen up to 600 ppm N
(KNO,), no growth was observed for any of the tested strains.
5.Two rhizobial strains Bradyrhizobium sp. nodulating peanut(619 T
300 ppm N .and 3456 T 500 ppm N) and two Bradyrhiz(lhi~;’l
japoniCllm strains (138 T 200 ppm Nand 3407 T 500 ppm N) were
selected for symbiotic perfonnance evaluation as compared to
sensitive original ones.4- Effect of acidity and alkalini~y on survival of rhizobia: 1.The different rhizobial strains under investigation varied widely in their growth tolerance to pH values in YEM medium. The pH value
most favourable for growth was 7.0 . A corresponding decrease in
growth of all tested strains was recorded with the increase or decrease
in pH values outside 7.0 .
1. Strains of R !cguminosarum bv. phaseoli ARC 302 and peanut
Bradyrhizobium 618, 3339 and 601 were more tolerant to lower pH
values up to 5. The growth rate ranged from 72-99 % as compared
with growth at pH 7 .
3. Regarding alkaline pH values tested only seven out of twenty - five
rhizobial strains were able to tolerate pH values up to \0.0 these
strains were R. meliloti (ARC 103), R le.guminosarnm bv. phaseoli
(UMR), peanut Bradyrhizobium (618) and .!3radyrhizohium
japonicum (138 and 3407).
4- Effect of desiccation on survival of rhizobia: 1. Growth and survival of representative five strains of R.
leguminosarum by. viceae and peanut Bradyrhizobium as affected by
desiccation for 24 ..96 hr were investigated. Drying. stress was
imposed on the legume seeds coated with the rhizobial strains as peat
_ based inoculant. kept in CaCh desiccator for 96 hr. Survival of
rhizobia was followed by determination of the viable rhizobial cells
number (log 10) per seed at intervals of 24 hr. 2. The growth rate of the rhizobial strains generally declined by
increasing the duration of desiccation. No strain from the two speciestested was able to survive desiccation up to 96 hr. However, one strain
(ICARD A 441) of R. leguminosarum bv. »tceae and two strains (619
and 3339) of Bradyrhizobium sp. retained viability after 72 hr. of
drying stress. The .viability rate recorded was 42.9,52.3 and 50.1%
respectively. 3. Comparing the viability responses as affected by desiccation after 2hr, R leguminosarum bv. viceae strains showed consistently lower growth rate than Bradyrhizobium sp. strains. The viability rate was
between 49.1 70.4% and between 93.5 - 98.7% for II.legum;nosarum bv. »tceae and Bradyrhizobium sp, respectively, as
compared to initial count After 48 hr of desiccation exposure, all
strains tested were able to tolerate drying except R. leguminosarum
bv. viceae ARC 200F which showed no growth. 4. The obtained results show the importance of drying as factor affects the viability and survival of the rhizobial strains in free living state. However, comparison of survival under drying conditions indicated
that Bradyrhizobium sp. (peanut) strains were more tolerant to
desiccation than strains of Rhizobium tested over short periOds (24 hr):
5. It appears that exposure to desiccation can be applied to select some rhizobia} strains (R. le.&JUminosarumby. vtceae, lCARDA 441) not seriously affected under stress of drying conditions.
5~Symbiotic performance of stress tolerant rhizobia strains: \ ,
In the present study , some rhizobial strains tolerant to different
soil~stress factors were selected and their competence with’ specific host
were evaluated as well 5.1. ’Y’iWbiUPJ ””wniAAsslUm by. vil<eM toltranl to djt!eranl corgnlraiions gf
NaCI with taba bean under soil salinity stress : 1- Inoculation of faba bean plants withR!Jizobium }egu.rn~nosarumbv.
viceae ARC 200 F’and~CARDA 441 (tolerant to 1 and 40/0 NaCl
respectively) resulted in significant increases in nodules number and
dry weight, dry matter and nitrogen content under stress ofNaCl
salinized soil up to 6% as compared to uninoculated plants. Strain
IcARDA 441 was superior in the above mentioned parameters
compared to ARC 200F
2- Strain ARC 200 F was able to induce few nodules (6 nodules I plant)
on faba bean plants under stress of 0.80/0 NaCI salinized soil.
However, strain ICARDA 441 was highly infective, the number of
nodules induced was 21 nodules / plant under the same level of soil
salinity.3- At high soil salinity (0.8% NaCI) which greatly depressed the growth
of uninoculated plants (2.88 g1plant, 75 DAP) , there was better
growth of 4.02 g/’plant due to inoculation with ICARDA 441. These
findings indicated that Rhizobium lezu.minosarum. by. viceae can
tolerate higher levels of soil salinity than the host plant.
5.2. Bradvrhizobium sp. strains tolerant to high temperature with peanut: 1- Inoculation with Bradyrhizobium sp. strains (peanut) tolerant to high
temperature (40 and 45°C) increased the number of nodules/plant.
up to 95 (3456 T 40 OC) and 106 (619 T 45°C) as compared with
inoculated plants with original sensitive ones (3456 and 619) , the
number of nodules formed ranged from 85 to 99 nodules/plant.2- The nodules formed on peanut plants inoculated with high temperature
tolerant strains were higher in dry weights (680-743 mg/plant).
However, Brad~rhizob;um peanut strain 3456 T 40°C was the
superior among the four strains tested in producing higher weight
nodules of 743 mg/plant. Peanut plants inoculated with original
strains induced lower nodules dry weight (503-523 mg/plant).
3- After 75 DAP, the influence of ill0culation with 3456 T 40°C and 619
T 450C was promotive and produced the highest dry matter yield of
19.9 and I 8.6 g/plant respectively, as compared to 10.1 and 1l.6 g J
plant for sensitive original strains.
4- Inoculation with the high temperature tolerant strains seemed to be superior in increasing the nitrogen content of shoots by 82.7-I 89%
(45 DAP) and 88.6-139.2% (75 DAP) over inoculated plants with
sensitive original ones.
5.3. Bradyrhizobiumja onicumstrains tolerant to hi h tem erature with so bean : 1- At 45 DAP no significant differences were obtained in nodules
number of soybean plants inoculated with original (3407 and 138)
and their high temperature tolerant derivatives (3407 T 40°C and
138 T 450C). where averages recorded were 105-109 and 99-11 5
nodules/plant resp~ctively. The same trend was noticed with older
plants, 75 OAP.2- At the plant age of75 days, an increase of 53.3 and 47.6°Atin nodules dry weight were attributed to inoculation with 3407 T 40°C. and 138
T 450C respectively over inoculation with strains 3407 and 138.3. The rate of increase in plant dry matter yield differed according to
rhizobial strain character as tolerant or sensitive to high remperaturPercentages of increase in plant dry biomass of 17.4 and 1.8 were
recorded for 3407 T 40·C and 138 T 45·C respectively.
4- For nitrogen content a’n increase of 154.7% was recorded for soybean
plants inoculated with 3407 T 40 ·C over that inoculated with
original strain 3407 .
5.4. Brad rtlizobium s. eanut strains tolerant to mineral nitro an under stresS of soil nitrogen
1_ At 45 DAP irrespective of .the rhizobia! strain, nodules number
decreased with increasing the level of nitrogen in the soil being lower
at 75 kg N/fed (7-27 nodules/plant) than at 20 kg N/fed (19-42
noduels/plant). However, peanut plants inoculated with nitrogen
tolerant rhizobial strains 619 T 300 ppm Nand 3456 T 500 ppm N
formed higher number of nodules 20 and 27/plant respectively, under
nitrogen fertilized soil with 75 kg N/fed compared to sensitive
original strains ( 7 _ 10 nodules I plant). The corresponding values
after 75 DAP were 22-23 for the original strains and 27 - 31
noduelslplant for their nitrogen tolerant derivatives ones.
2- Nodules dry weight drastically decreased by application of higher
doses of nitrogen (75 kg N/fed) at the two periods of plant sampling.
This finding was clear for inoculation with original Bradyrhizobium
peanut ones. 3- Competence of rhizobia! strains tested tolerant to mineral nitrogen was
more pronounced under stress of 50 kg N/fed than sensitive ones
regarding increased’ shoots dry weight and nitrogen content.1- At 75 DAP, the two Bra<Jyrhizobium japonicum selected strains tolerant to nitrogen showed different infectivity competence as
compared to the original sensitive ones. The higher infectivity of 94
nodules / plant of 3407 T 500 ppm N was demonstrated at 75 kg N/fed than the original one (25 nodules /plant). However, little
fluctuation of no significancy in nwuber of nodules was recorded
between strain 138 (104 nodules/plant) and strain 138 T 200 ppm N
( 114 nodules/plant) under stress of the same dose of nitrogen..
2- The same trend was observed regarding uodll1es dry weight and dry,
weight of plant shoots. In the majority of cases, inoculation with
Bradyrhizohium japonicum 3407 T 500 ppm resulted in higher dry
weight of both nodll1es and shoots as compared to 3407 at the two
periods of sampling. In contrast, there was no significant difference
between 138 and T 200 ppm N regarding the above mentioned parameters.
3- High significant quantities of nitrogen 781, 635 and 675 mg/plant
were accw,nulated in shoot tissues of soybean plants inoculated with
3407 T 500 ppm N under stress of 20, 50 and 75 kg N/fed
respectively. In contrast, the influence of inoculation with 138 T 200
ppm N on soybean shoots nitrogen content was nnpromotive (201,
284 and 251 mg I plant) as.comparedto Bradyrhizobium jCJponicum
138 original one (464 , 384 and 311 mg N/plant ) .5.6. Bradyrhizobium sQ. (oeanutyand BradWizQtl1!ml.§QnicY...fJJ tolerantto low and high pH values
1- One strain represent Bradyrhizobium sp. (peanut) was tolerant to low
pH 5 (618 T pH 5) and high pH 10 (618 T pH 10). Also, three strains
represent Bradl’yhizobium japonicum were loleranl.to high pH value
(3407 T pH 11 and 138 T pH 11) and low pH 5 (3407 T pH 5) were
selected. The symbiotic ·performance of the obtained strains were
investigated.2- Uninoculated peanut and soybean plants were free of nodIlles. At 45 OAP, however, inoculation of peanut or soybean plants with original
or tolerant rhizobial strains resulted in induction of high number of
nodules/plant for peanut (25-44) and soybean (54-92) . Nodules
number/plant increased up to 51-99 for peanut and 105-174 for
soybean by increasing the plant age (75 DAP) .3- On the other hand, tolerance of the rhizobial strains tested to high pH values of 10 and. 11 was parallel to increase the munber of nodules
induced for the two intervals of plant sampling of 4S and 7S DAP.
The corresponding numbers of nodules I plant recorded at 45 OAP
were 44~92 and 84 for 618 ,TpH 10,3407 T pH 11 and 138 T pH 11
respectively. Ho~ever, -at 75 DAP the number of nodules/planl
increased to 91, 174 and 123 in the same order.4- The dry weight of nodules was found to have the same trend as
nodulation, this fipding .was recorded for peanut and soybean
rhizobial strains tested.
5.- Inoculation with alkaline (pH 10 and 11) tolerant Bradyrhizobium sp.
(618 T pH 10yandBradyrhiznbium japonicum (3407 T pH 11 and
138 T pH 11) were superior in increasing peanut and soybean plant
’growth compared to original and acidity (pH 5) tolerant ones.
6- It was clear that inoculated peanut plants with 618 T pH 10 was
. ’ superior in increasing nitrogen content (396 mg I plant) as compared
with uninocu1ated (190 mglplant)~ inoculated with 618 T pH 5 (247
mg/plant) and original strain 618 (225 mg / plant).7_ The nitrogen content of .soybean plants showed the same trend as , peanut plants. the tolerant p” japonieum strains tolerant to pH II
(3407 T pH 11 and 138 T pH 11) were more effective in
accumulating more atmospheric nitrogen than the original ones.
5.7. Rhizobium leauminosarum by. viceae strains tolerant to desiccation
1- Two R. !eguminosarum bv viceae strains ARC 200 F and ICARD A
441 were selected as low and high desiccation tolerant, respectively.
The infectivity and effeciency of the two strains were evaluated with
faba bean under soil drought stress of75 ,60 ,45 and 30% WHC.
2- Uninoculated faba bean plants formed a considerable number of
nodules being 45 and 7 nodules / plant at 75 and 30% soil WHC
respectively. Inoculated plants with desiccation tolerant strains
beared higher number of nodules / plant being 114 and 13 ( ARC
200F) or 135 and 30 (ICARDA 441) at 75 and 300/0 WHC respecti vely.
3- Generally, the highest nodules number anddry weight were induced
on faba bean plants inoculated with ICARDA 441 which recorded
more desiccation tolerance up to 72 hrs.
4- The largest estimates of shoots dry weight were for inoculation with
ICARDA 441 being 9.7 and 8.18 g /plant at 45 and 30% WHC
respectively. The same trend was also observed for shoots nitrogen
content of95.1, 106.7 and 132.5 mg/plant for uninoculated , inoculated
with ARC 200 F and inoculated plants with ICARDA 441 respectively at
30%WHC.