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Studies on Nodulation of Two Lima Bean and Six Cowpea Cultivars
Zentralbl. Mikrobiol. 141 (1986), I07-113 VEB Gustav Fischer Verlag Jena [From the Department of Agric. Microbiology, Faculty of Agriculture, 3'Iinia University, Minia, Egypt]
Studies on Nodulation of Two Lima Bean and Six Cowpea Cultivars II. Effect of Seed Treatments F. S. ALI and A. A. ABDEL-l\10NEIl\1 With 3 Figures
Summary The inhibitive effect of seed diffusates of two Lima bean and six cowpea cultivars on three strains of Rhizobium sp. in Petri dishes was investigated. This effect could be eliminated by seed germination for several hours, differing, however, according to the seed's cultivar. The cowpea seeds Magnolia Blackeye, Cream 7, and Cream 12 still inhibited the three strains of Rhiz. sp. until 42 h of germination. However, in pot experiment, germinating 1Iagnolia Blackeye seeds (six hours before inoculation and sowing) was the best treatment in number of nodules, fresh and dry mass of nodules, dry mass of shoots and whole plants as well a" phosphorus and total nitrogen content in whole plants per pot on the 60th day after sowing.
Zusammenfassung Es wurde die inhibierende Wirkung der Diffusate keimender Samen von 2 Lima-Bohnen- u nd 6 cowpea-Sorten auf :3 Rhizobium-Stamme getestet. Dabei zeigten sich Sortenunterschiede. Die cowpea-Sorten Magnolia Blackeyo, Cream 7 und Cream 12 hemmtcn die Rhizobien 42 Stunden lang. In einem GefaEversuch ergab eine Gstundige Vorkcimung der }Iagnolia-Blackeye-Samen vor der Beimpfung mit Rhizobium und Aussaat die hochst.e Knollchenzahl, -frisch- und -trockenmasse, die hochste Trockenmasse und den hochst en Phosphor- und Stickstoffgehalt dor Pflanzen pro GefaE naeh 60tagiger Vegetationszeit.
Several investigators had referred to the presence of inhibitory substances (especially rhizobia) in seeds of soybeans, beans, as well as in other leguminous plants (IswARAN 1970; KAREAMAN et al. 1970; DADARWAL and SEN 1971 and 197B; ELl\1ALLAH 1974; JAIN and REWARI 1976; ABDEL-GHAFFAR 1977; ABDEL-1\ASSER et al. 1979). Recently, ALI and ABDEL-MoNEIM (1986) could detect antirhizobial substances present in the seeds (cotyledon, dicotyledon, and seed coat) of two Lima bean cultivars and six cowpea cultivars, which in laboratory medium inhibited the growth of Rhizobium sp, The antirhizobial substances were water-soluble or partially insoluble. Therefore in this investigation, several treatments were done in trial to eliminate or decrease the observed inhibitive effects of the seed diffusates in order to obtain successful nodulation.
108
F. S. ALI and A. A. ABDEL·:\IoNEDI
Fig. 1. Effect of seed germination time 0 (1), 2 (2), and 4 h (3) for six cowpea cultivars on three strains of Rhizobium sp. A - Blackeye, B - Cream 12, C - Magnolia Blackeye, D - Mungbean line 1, E - Mungbean line 2, and F - Cream 7.
Studies on Nodulation. II. Effect of Seed Treatments
109
no
F. S. ALI and A. A. ABDEL·)IoNEIM
Materials and Methods L ab or a t or y e x p e ri me nt H eavy in oculum of Rh i zobium sp . st ra in 1 or 2 and/or 3 wa s a dded to a m elted (45-50 °C) y east ex t ract m annitol agar " Med iu m 79" (ALLEN 1959) a nd po ured in ste r ilized P etri di sh es. Aft er so lid ifica t ion the st eri lized a n d t r ea t ed se eds were put eac h in di sh es in triplicates. Sur fa cest e r ilized se eds (ALLEN 1959, m odi fied by EL ·l\IALLAH 1974) were aseptically pl a ced in ster ilize d di still ed wa ter in P etri di sh es for differ ent periods (0 to 42 h) a n d then asepti call y transferred t o P et ri di sh es, co n tain ing the yeast ex t ract m annitol agar m ed ium , see ded with each of the R. sp . s train. Rh izobium sp . st ra in No . I a n d 2 were obtained fr om Sa k ha Agri cul ture R esearch Station, K afer El-Sheikh, a n d strain No. 3 fr om So ill\Iicrobiology , Ins t itute of So il a n d Wa t er R esearch , Agri cul ture Research Center, Giza, Egypt. P ot e x p e r i m e n t Soil :
Three kg portion s of loa m y so il were put in pl ast ic pots, 20 em in diameter and 25 cm in h eight. Seed s : Lima bean and cowpea cult ivars were ob taine d from Hortieulture Depar t me nt, Faculty of Agriculture, Minia Univ. , Mini a . The effec t of seed germination, wash ed with tap. water for fiv e minutes every ho ur during 6, 12, and 24 h a n d removal of excess wat er from the seed con tainer (MAHMOUD et al , 1981), prior t o inoculation with mixed ino culum of R. sp . (the three st rains) , was done for cowpea (Magnolia Bl ackeye) . Seeds withou t wash ing a nd ino cul ated with the m entioned inoculum immediately before sowing and con t rol (uninocul ated seeds) were al so used . The ex pe rim en t was ca rr ied out in com p lete randomized bl ock design , using five seed trea tmen t s wi th three r ep licates, ea ch p ot eont a ining four p lants . The p ots wer e k ep t in t he green h ouse until 60th day aft er sowing . Che m ical m ethods: To t al nitrogen was determined u sing t he m odified micro-Kjehld ahl m ethod as describ ed by \YOLDENDORP (I 96 3). Phosphorus wa s det ermined color imotrically by r eduction of t h e pho spho m oly bda te co m p lex w ith stann ous ch lor ide in the sulph uric acid syst em ac cor d ing t o JA CKSON (l958). Th e ob t a in ed dat a wer e st a t ist ica lly a n aly zed .
Resu lts an d Discussion Pre-germination of t he see ds of two Lima bean (Dixie Butterpea white and H en ders on) and six Cowp ea cultivars (Blackeye, Cream 12, Magnolia Blackeye, Mungbean line 1, Mungbean line 2, a nd Cream 7), before t esting on the cult ure surface of t he H Rhizo bium st ra ins in P etri dishes, showed that the inhibit ion zone decreased with the inc re asing t ime of seed germinati on . Fi g. 1 sh ows that germinat ion for 4 h was not sufficient for elimination of the in hibit ing substances from t he seed s. Many experiments were ca rr ied out to eliminate the in hibiting effect of the seed s by germinat ion for 14, 18, a nd 42 h. The germination of Mun gbe an line 1 for 14 h did not result in an inhibition effect on the three strains of R. sp . Such substances m ay be diffusated orland utilized by t he embryo during germination (ABDEL-NASSER et al. 1979). . Fig. 2 shows the inhibition effect of t he seeds of Dixie Bu t t erpea white, Henderson , Bla ck eye, Cream 12, Magnolia B la ck eye, l\Iun gb ean line 2, and Cream 7 cult iva rs aft er germinat ion for 18 h. Th e diffu sates of the seed" d iffered a ccording to t he seed's cult ivar and R. sp . strain (ALI and ABDEL-l\ImmIi\I 1986). Th e seed germination up t o 42 h eliminat ed the inhibi ti on effe ct of Dixie Bu tter pea white, H enderson , Blackeye, a nd Mungbean line 2 cult ivars, however , cult iva r seeds of Magno lia Blackeye, Cream 7, and Cream 12 st ill inhibit ed t he three st rai n!'! of R. sp .
Studies on Nodulation. II. Effect of Se ed Treatments
111
Fi g. 2. Effect of seed germinat ion (18 h) for 2 Lima bean ancl 5 co wpea cult ivars on thre e strains of R. sp . I - Dixie But.terpea wh it e, 2 - H on rlorcon, 3 - Bl a ek eyo, 4 - - Cream 12,5 - Magnolia Bl a ck ey o, 'I - Mungbea n line 2, an d 8 - Cream 7,
From t he previou s resul t s it is evide nt t hat the seeds differ in ger mination time, needed for eliminating t he inh ibiting substan ces, which agrees with A HD E L- K.-\.SSE R et al. (1979), who could elimina te t he inhibition effect of H ar osoy soy bean seeds on R. jap onic11m by ger mina t ion up to 12 h or more. Th e pot experiment (Fig. ;{ a nd Ta ble 1) shows t ha t gern tinatioll of cowpea (~Iag nolia Bla ckeye) for () h before inoculation and sowing was t he best treatment for in creasing the number of nodul es, fresh a nd dry mass of nodul es, dr y Blass of shoots
F ig. 3, E ffect of sor« l g c r m ;Il.,r! ;O lt \. " Il<' (I ( \) , fi (2), 12 (:1) , a n d 24 It inoc ula ted co ut.ro l] 0" u orlul a t ion of ('()\\,fea p h"gno !;;. B i,w kc.\',' ).
( ~)
.u id ;" .-",,,I,,t;,"l (C -
un -
24
12
0
0
..c:
Q)
eo
'S...
I':
I':
0 +' al
L.S.D.
5% 1%
Uninoculated (control)
I-i
I':
0
"2o
+' al
-e Q)
et:al
Q)
...
Treatments
3.55 5.05
32.20
30.37
30.4()
36.14
36.50
Height of plants (crn)
NSD
31.32
12.08
27.48
29.44
29.00
24.20
28.34 40.31
Whole plants
NSD
7.92
4.96
e.os
4.88
4.32
0.079 0.112
0.064
0.064
0.048
0.160
0.152
NSD
39.304
29.224
33.()08
34.480
32.472
1.04 1.48
5.5fl8
4.900
4.128
7.072
4.672
NSD
1.300
0.840
1.044
0.880
0.768
Roots
Shoots
Nodules
Shoots Roots
Dry mass (g/pot) of
Fresh mass (g/pot) of
41.00
30.32
64.00
44.44
Number of nodules per pot
0.031 0.044
0.016
0.012
0.008
0.000
0.052
Nodules
1.512 2.152
0.884
5.770
5.180
8.012
5.492
Whole plants
Total N
NSD
88.24
82.00
67.64
103.88
74.72
84.05 119.56
323.16
462.56
441.40
590.24
498.04
in whole plants (mg/pot)
P 20S
Table 1. Effect of germination and inoculation on means of height of plants, number of nodules, fresh and dry mass of whole plants, P20S' and total nitrogen of cowpea (Magnolia Blackeye)
;;;:
o-l
l'J
':2i
0
~
t"'
l'J
~
?> > to
~
= c,
o-l
t"'
>
~ U1
l>:l
l-' l-'
Studies on Nodulation. II. Effect of Seed Treatments
113
and whole plants, and phosphorus and total nitrogen contents in whole plants per pot on the 60th day after sowing, which agrees with MAHMOUD et al. (1981), who recommended the germination of Harosoy soybean seeds for 3 to 12 h and then inoculation just before sowing to improve nodulation and yield. Inoculation with R. sp. increased the number of nodules and total nitrogen in whole plants (ALI 1977 and MAHMOUD et al. 1981). Therefore, it is recommended to apply germination to these seeds before inoculating them with the specific active strains.
References ABDEL-GHAFFAR, S. A. 1\1.: Some factors affecting nodulation and nitrogen fixation of soybean (Glycine max). 1\l.Sc. Thesis, Fac. Agric., Univ, Alexandria, Egypt (1977). ABDEL-NAssER, M., MAHM01:D, S. A. Z., MAKAWI, A., and ALI, F. S.: Studies on nodulation of soybeans in Egypt. II. Effect of seed-diffusates on Rhizobium japonicum. Zbl. Bakt, II 139 (1979),600. ALI, F. S.: Studies on nodulation of soyabeans in Egypt. 1\1.Sc. Thesis, Fac. Agric. Minia Univ., Egypt (1977). - and ABDEL-MmmI1VI, A. A.: Studies on nodulation of two Lima bean and six Cowpea cultivars. 1. Effect of seed's diffusates on Rhizobium sp. Zbl. Mikrobiol. 141 (1986), 25-30. DADARwAL, K. R., and SEX, A. N.: Survival of R. japonicum on soybean seeds. Indian J. Agric. Sci. 41 (1971), 564. DADARWAL, K. R., and SEN, A. N.: Inhibitory effect of seed diffusates of some legumes on rhizobia and other bacteria. Indian. J. Agric. 43 (197:3),82. EL-MALLAH, M. 1.: Studies on soybean. M.Sc. Thesis, Fac. Agric. AI-Azhar Univ., Cairo, Egypt (1974). ISWARAN, V.: Soaking of soybean seed and its effect on nodulation and yield. Mysore J. Agric. Sci. 4 (1970), 364. JACKSON, 1\1. L.: Soil Chemical Analysis. London 1958. JAIN, 1\1. K., and REWARI, R. B.: Studies on seed coat toxicity to rhizobia of urid (Phaseolus mungo), mung (Phascolus aureus) and soybean (Glycine max). Zbl. Bakt. II 131 (1976), 193. KAREAMAN, M. F., ABDEL-GHAFFAR, A. S., and EL-GABALY, 1\1. M.: Inhibition effect of bean seeds on Rhizobia. Agrokemia es Talajtan 19 (1970), 546. MAHMOUD, S. A. Z., ABDEL-NASSER, M., and ALI, F. S.: Effect of seed treatments and time of inoculation on nodulation and yield of soybean. Pesq. agropec. bras. Brasilia 16 (1981),617. 'VOLDENDORP, J. 'V.: The influence of living plants on denitrifieation. :Ucded. Landb. Hogesch. Wagcningen 63 (196:1),1. Authors' address: Dr. F. S. ALI and Dr. A. A. ABDEL-MoXEIM, Department of Agrie. Mierobiology, Fac, of Agrieulture, Minia Univ., ::\finia, Egypt.
8
Zhl. 'Ilikrohiol., Bd. 141
Studies on Nodulation of Two Lima Bean and Six Cowpea Cultivars II. Effect of Seed Treatments F. S. ALI and A. A. ABDEL-l\10NEIl\1 With 3 Figures
Summary The inhibitive effect of seed diffusates of two Lima bean and six cowpea cultivars on three strains of Rhizobium sp. in Petri dishes was investigated. This effect could be eliminated by seed germination for several hours, differing, however, according to the seed's cultivar. The cowpea seeds Magnolia Blackeye, Cream 7, and Cream 12 still inhibited the three strains of Rhiz. sp. until 42 h of germination. However, in pot experiment, germinating 1Iagnolia Blackeye seeds (six hours before inoculation and sowing) was the best treatment in number of nodules, fresh and dry mass of nodules, dry mass of shoots and whole plants as well a" phosphorus and total nitrogen content in whole plants per pot on the 60th day after sowing.
Zusammenfassung Es wurde die inhibierende Wirkung der Diffusate keimender Samen von 2 Lima-Bohnen- u nd 6 cowpea-Sorten auf :3 Rhizobium-Stamme getestet. Dabei zeigten sich Sortenunterschiede. Die cowpea-Sorten Magnolia Blackeyo, Cream 7 und Cream 12 hemmtcn die Rhizobien 42 Stunden lang. In einem GefaEversuch ergab eine Gstundige Vorkcimung der }Iagnolia-Blackeye-Samen vor der Beimpfung mit Rhizobium und Aussaat die hochst.e Knollchenzahl, -frisch- und -trockenmasse, die hochste Trockenmasse und den hochst en Phosphor- und Stickstoffgehalt dor Pflanzen pro GefaE naeh 60tagiger Vegetationszeit.
Several investigators had referred to the presence of inhibitory substances (especially rhizobia) in seeds of soybeans, beans, as well as in other leguminous plants (IswARAN 1970; KAREAMAN et al. 1970; DADARWAL and SEN 1971 and 197B; ELl\1ALLAH 1974; JAIN and REWARI 1976; ABDEL-GHAFFAR 1977; ABDEL-1\ASSER et al. 1979). Recently, ALI and ABDEL-MoNEIM (1986) could detect antirhizobial substances present in the seeds (cotyledon, dicotyledon, and seed coat) of two Lima bean cultivars and six cowpea cultivars, which in laboratory medium inhibited the growth of Rhizobium sp, The antirhizobial substances were water-soluble or partially insoluble. Therefore in this investigation, several treatments were done in trial to eliminate or decrease the observed inhibitive effects of the seed diffusates in order to obtain successful nodulation.
108
F. S. ALI and A. A. ABDEL·:\IoNEDI
Fig. 1. Effect of seed germination time 0 (1), 2 (2), and 4 h (3) for six cowpea cultivars on three strains of Rhizobium sp. A - Blackeye, B - Cream 12, C - Magnolia Blackeye, D - Mungbean line 1, E - Mungbean line 2, and F - Cream 7.
Studies on Nodulation. II. Effect of Seed Treatments
109
no
F. S. ALI and A. A. ABDEL·)IoNEIM
Materials and Methods L ab or a t or y e x p e ri me nt H eavy in oculum of Rh i zobium sp . st ra in 1 or 2 and/or 3 wa s a dded to a m elted (45-50 °C) y east ex t ract m annitol agar " Med iu m 79" (ALLEN 1959) a nd po ured in ste r ilized P etri di sh es. Aft er so lid ifica t ion the st eri lized a n d t r ea t ed se eds were put eac h in di sh es in triplicates. Sur fa cest e r ilized se eds (ALLEN 1959, m odi fied by EL ·l\IALLAH 1974) were aseptically pl a ced in ster ilize d di still ed wa ter in P etri di sh es for differ ent periods (0 to 42 h) a n d then asepti call y transferred t o P et ri di sh es, co n tain ing the yeast ex t ract m annitol agar m ed ium , see ded with each of the R. sp . s train. Rh izobium sp . st ra in No . I a n d 2 were obtained fr om Sa k ha Agri cul ture R esearch Station, K afer El-Sheikh, a n d strain No. 3 fr om So ill\Iicrobiology , Ins t itute of So il a n d Wa t er R esearch , Agri cul ture Research Center, Giza, Egypt. P ot e x p e r i m e n t Soil :
Three kg portion s of loa m y so il were put in pl ast ic pots, 20 em in diameter and 25 cm in h eight. Seed s : Lima bean and cowpea cult ivars were ob taine d from Hortieulture Depar t me nt, Faculty of Agriculture, Minia Univ. , Mini a . The effec t of seed germination, wash ed with tap. water for fiv e minutes every ho ur during 6, 12, and 24 h a n d removal of excess wat er from the seed con tainer (MAHMOUD et al , 1981), prior t o inoculation with mixed ino culum of R. sp . (the three st rains) , was done for cowpea (Magnolia Bl ackeye) . Seeds withou t wash ing a nd ino cul ated with the m entioned inoculum immediately before sowing and con t rol (uninocul ated seeds) were al so used . The ex pe rim en t was ca rr ied out in com p lete randomized bl ock design , using five seed trea tmen t s wi th three r ep licates, ea ch p ot eont a ining four p lants . The p ots wer e k ep t in t he green h ouse until 60th day aft er sowing . Che m ical m ethods: To t al nitrogen was determined u sing t he m odified micro-Kjehld ahl m ethod as describ ed by \YOLDENDORP (I 96 3). Phosphorus wa s det ermined color imotrically by r eduction of t h e pho spho m oly bda te co m p lex w ith stann ous ch lor ide in the sulph uric acid syst em ac cor d ing t o JA CKSON (l958). Th e ob t a in ed dat a wer e st a t ist ica lly a n aly zed .
Resu lts an d Discussion Pre-germination of t he see ds of two Lima bean (Dixie Butterpea white and H en ders on) and six Cowp ea cultivars (Blackeye, Cream 12, Magnolia Blackeye, Mungbean line 1, Mungbean line 2, a nd Cream 7), before t esting on the cult ure surface of t he H Rhizo bium st ra ins in P etri dishes, showed that the inhibit ion zone decreased with the inc re asing t ime of seed germinati on . Fi g. 1 sh ows that germinat ion for 4 h was not sufficient for elimination of the in hibit ing substances from t he seed s. Many experiments were ca rr ied out to eliminate the in hibiting effect of the seed s by germinat ion for 14, 18, a nd 42 h. The germination of Mun gbe an line 1 for 14 h did not result in an inhibition effect on the three strains of R. sp . Such substances m ay be diffusated orland utilized by t he embryo during germination (ABDEL-NASSER et al. 1979). . Fig. 2 shows the inhibition effect of t he seeds of Dixie Bu t t erpea white, Henderson , Bla ck eye, Cream 12, Magnolia B la ck eye, l\Iun gb ean line 2, and Cream 7 cult iva rs aft er germinat ion for 18 h. Th e diffu sates of the seed" d iffered a ccording to t he seed's cult ivar and R. sp . strain (ALI and ABDEL-l\ImmIi\I 1986). Th e seed germination up t o 42 h eliminat ed the inhibi ti on effe ct of Dixie Bu tter pea white, H enderson , Blackeye, a nd Mungbean line 2 cult ivars, however , cult iva r seeds of Magno lia Blackeye, Cream 7, and Cream 12 st ill inhibit ed t he three st rai n!'! of R. sp .
Studies on Nodulation. II. Effect of Se ed Treatments
111
Fi g. 2. Effect of seed germinat ion (18 h) for 2 Lima bean ancl 5 co wpea cult ivars on thre e strains of R. sp . I - Dixie But.terpea wh it e, 2 - H on rlorcon, 3 - Bl a ek eyo, 4 - - Cream 12,5 - Magnolia Bl a ck ey o, 'I - Mungbea n line 2, an d 8 - Cream 7,
From t he previou s resul t s it is evide nt t hat the seeds differ in ger mination time, needed for eliminating t he inh ibiting substan ces, which agrees with A HD E L- K.-\.SSE R et al. (1979), who could elimina te t he inhibition effect of H ar osoy soy bean seeds on R. jap onic11m by ger mina t ion up to 12 h or more. Th e pot experiment (Fig. ;{ a nd Ta ble 1) shows t ha t gern tinatioll of cowpea (~Iag nolia Bla ckeye) for () h before inoculation and sowing was t he best treatment for in creasing the number of nodul es, fresh a nd dry mass of nodul es, dr y Blass of shoots
F ig. 3, E ffect of sor« l g c r m ;Il.,r! ;O lt \. " Il<' (I ( \) , fi (2), 12 (:1) , a n d 24 It inoc ula ted co ut.ro l] 0" u orlul a t ion of ('()\\,fea p h"gno !;;. B i,w kc.\',' ).
( ~)
.u id ;" .-",,,I,,t;,"l (C -
un -
24
12
0
0
..c:
Q)
eo
'S...
I':
I':
0 +' al
L.S.D.
5% 1%
Uninoculated (control)
I-i
I':
0
"2o
+' al
-e Q)
et:al
Q)
...
Treatments
3.55 5.05
32.20
30.37
30.4()
36.14
36.50
Height of plants (crn)
NSD
31.32
12.08
27.48
29.44
29.00
24.20
28.34 40.31
Whole plants
NSD
7.92
4.96
e.os
4.88
4.32
0.079 0.112
0.064
0.064
0.048
0.160
0.152
NSD
39.304
29.224
33.()08
34.480
32.472
1.04 1.48
5.5fl8
4.900
4.128
7.072
4.672
NSD
1.300
0.840
1.044
0.880
0.768
Roots
Shoots
Nodules
Shoots Roots
Dry mass (g/pot) of
Fresh mass (g/pot) of
41.00
30.32
64.00
44.44
Number of nodules per pot
0.031 0.044
0.016
0.012
0.008
0.000
0.052
Nodules
1.512 2.152
0.884
5.770
5.180
8.012
5.492
Whole plants
Total N
NSD
88.24
82.00
67.64
103.88
74.72
84.05 119.56
323.16
462.56
441.40
590.24
498.04
in whole plants (mg/pot)
P 20S
Table 1. Effect of germination and inoculation on means of height of plants, number of nodules, fresh and dry mass of whole plants, P20S' and total nitrogen of cowpea (Magnolia Blackeye)
;;;:
o-l
l'J
':2i
0
~
t"'
l'J
~
?> > to
~
= c,
o-l
t"'
>
~ U1
l>:l
l-' l-'
Studies on Nodulation. II. Effect of Seed Treatments
113
and whole plants, and phosphorus and total nitrogen contents in whole plants per pot on the 60th day after sowing, which agrees with MAHMOUD et al. (1981), who recommended the germination of Harosoy soybean seeds for 3 to 12 h and then inoculation just before sowing to improve nodulation and yield. Inoculation with R. sp. increased the number of nodules and total nitrogen in whole plants (ALI 1977 and MAHMOUD et al. 1981). Therefore, it is recommended to apply germination to these seeds before inoculating them with the specific active strains.
References ABDEL-GHAFFAR, S. A. 1\1.: Some factors affecting nodulation and nitrogen fixation of soybean (Glycine max). 1\l.Sc. Thesis, Fac. Agric., Univ, Alexandria, Egypt (1977). ABDEL-NAssER, M., MAHM01:D, S. A. Z., MAKAWI, A., and ALI, F. S.: Studies on nodulation of soybeans in Egypt. II. Effect of seed-diffusates on Rhizobium japonicum. Zbl. Bakt, II 139 (1979),600. ALI, F. S.: Studies on nodulation of soyabeans in Egypt. 1\1.Sc. Thesis, Fac. Agric. Minia Univ., Egypt (1977). - and ABDEL-MmmI1VI, A. A.: Studies on nodulation of two Lima bean and six Cowpea cultivars. 1. Effect of seed's diffusates on Rhizobium sp. Zbl. Mikrobiol. 141 (1986), 25-30. DADARwAL, K. R., and SEX, A. N.: Survival of R. japonicum on soybean seeds. Indian J. Agric. Sci. 41 (1971), 564. DADARWAL, K. R., and SEN, A. N.: Inhibitory effect of seed diffusates of some legumes on rhizobia and other bacteria. Indian. J. Agric. 43 (197:3),82. EL-MALLAH, M. 1.: Studies on soybean. M.Sc. Thesis, Fac. Agric. AI-Azhar Univ., Cairo, Egypt (1974). ISWARAN, V.: Soaking of soybean seed and its effect on nodulation and yield. Mysore J. Agric. Sci. 4 (1970), 364. JACKSON, 1\1. L.: Soil Chemical Analysis. London 1958. JAIN, 1\1. K., and REWARI, R. B.: Studies on seed coat toxicity to rhizobia of urid (Phaseolus mungo), mung (Phascolus aureus) and soybean (Glycine max). Zbl. Bakt. II 131 (1976), 193. KAREAMAN, M. F., ABDEL-GHAFFAR, A. S., and EL-GABALY, 1\1. M.: Inhibition effect of bean seeds on Rhizobia. Agrokemia es Talajtan 19 (1970), 546. MAHMOUD, S. A. Z., ABDEL-NASSER, M., and ALI, F. S.: Effect of seed treatments and time of inoculation on nodulation and yield of soybean. Pesq. agropec. bras. Brasilia 16 (1981),617. 'VOLDENDORP, J. 'V.: The influence of living plants on denitrifieation. :Ucded. Landb. Hogesch. Wagcningen 63 (196:1),1. Authors' address: Dr. F. S. ALI and Dr. A. A. ABDEL-MoXEIM, Department of Agrie. Mierobiology, Fac, of Agrieulture, Minia Univ., ::\finia, Egypt.
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Zhl. 'Ilikrohiol., Bd. 141