5 . IR e S3 xj. 1 t s St r'l cii D ± s c; xj. s s :i- c> ra
5.1 Collection of leguminous samples
A total number of 87 nodulated leguminous species, collected during the course of investigation were identified to species level and a lis t was prepared. When the li s t was checked against the global listin g of nodulated and non-nodulated leguminous species (Allen and Allen 1981 and personal communication) 19 species were found to be new reports to the global list and thus were selected for detailed studies. The details regarding their taxonomic position, habit and place of their collection are given in Table 2.
It could be seen from the table that the species comprised one from Caesalpinioideae, seventeen from Papilionoideae and one from Mimosoideae. They included 13 shrubs, 5 arboreal trees and 1 climber. It may be mentioned here that of the 19 species studied, 11 can be considered as rare plants because of the enormous d iffic u ltie s encountered during their collection.
The 19 species were studied in detail for their nodule morphology. A special mention may be made here of the nodulation of 5 tree legumes wherein examination for the presence of nodules on the roots becomes normally d iffic u lt. The details regarding occurrance and position of nodules on the roots and their description v i z size, shape, interio r and exterior colour and surface are given in Table 3. Table 2 : New reports of leguminous species for nodulation, along with their taxonomic position, habit, and place of collection.
Sr. Plant Species Subfamily Tribe Habit Place of No. Subtribe of the Collection plant
1. Cassia kleinii W. 5 A. Caesalpinioideae Cassieae Shrub Kam ala h i l l Dist. Raigad.
2. Alysicarpus pubescens Papilionoideae Headysareae Shrub Maishej Ghat Law . Desmodiinae 3. Atylosia sericea Phaseoleae Shrub Varandha Ghat Benth. Cajaninae 4. Butea monospemia (Lam) Phasealeae Tree Kamala hill Taub. Erythrininae Dist. Raigad. 5. Crotalaria albida Genisteae Shrub Mulshi Heyne. Crotalariinae Dist. Pune 6. Crotalaria calycina Genisteae Shrub Sinhagad Schranko Cotalariinae Dist. Pune 7. Crotalaria leptostachya Genisteae Shrub Bhimashankar Benth. Crotalariinae Dist. Pune Crotalaria triquetra Genisteae Shrub Shivneri h i l l Dalz. Crotalriinae Dist. Pune. 9. Dalbergia lanceolaria Dalbergieae Tree Katraj Ghat L. Lonchocarpinae Dist. Pune
10. Erythrina stricta Phaseoleae Tree Pune Roxbo phaseolinae 11. Erythrina suberosa Phaseoleae Tree Parvati hill Roxb. phaseolinae Pune
12. Indigofera cassioides Galgeae Shrub Mahabaleshwar Rottler ex. Dc. Indigoferinae Dist. Satara
13. Mucuna monosperma Phasealeae Climber Malkapur Erythrininae Dist. Kolhapur
14. Priotropsis cystisoides Genisteae Slirub Pune ITFX Crotalariinae
15. Rhynchosia rothii Phaseoleae Shrub Kliandala BenthoEx.Ait. Cajaninae Dist. Pune Sr. Plant Species Subfamily Tribe § Habit Place of NOo Subtribe of the Collection plant
16. Smithia salsuginea Papilionoideae Headysareae Shrub Amboli Ghat Hance Aeschynominae Dist. Kolhapur
17. Tephrosia pauciflora Galegeae Shrub Dapoli Grab, Tephrosiinae Dist. R a tn a g iri.
18. Tephrosia tinctoria Galegeae Shrub Bhimashankar Pers. Tephrosiinae Dist. Pune
19. Acacia tomentosa Mimosoideae Acacieae Tree Paud WTT3 Dist. Pune. X >< X r : EEE X X E (D EEEE n E E E E N t o t^) E E E E E E E E E E LO g ^ e E E CO r-H E E (N1 1 n r - j t o t^') -=f fNl r-l .o to CM to rH •-.-I fU o O tiO to 1 O') I V ) CJ U 0 ) cd ^ 03 ^ r H r : r H + J ^ QJ ;> H 0 ) •M cu D p : 0 ) ,Q 5 CD C c n 0 03 -C J E Mh P u S c u 03 T—1 w 0 0 0 p • H (D 'J o ::3 • H It could be seen from the table that position of nodules was on tap root in 4 species, on lateral roots in 11 species and on both tap and lateral roots in 4 species. The root nodules of the 19 species exhibited different shapes, sizes and colours. As fcr the out side colour, all the nodules showed a variety of colours ranging from light brown to dark brown except for flucuna monosperma which had black coloured nodules. It may be mentioned here that Dolichos lablab, Centrosema pubescens and Macroptilium atropurpureum have been reported to be having black coloured nodules (Cloonan 1963, Brockwell 1980}. When the nodules were cut, following 13 species viz. A3ysicarpus~ pubescens, Atylosia sericea, Crotalaria albida, C.calycina, C.leptostachya, C.triqetra, Dalbergia lanceolaria, Erythrina suberosa, Indigofera cassioides, Priotropsis cystisoides, Rhynchosia ro th ii, Smithia salsuginea and Tephrosia pauciflora had pink coloured interior. The remaining six species v iz . Cassia k le in ii, Butea monosperma, Erythrina stricta, Mucuna monosperma, Tephrosia tinetoria and Acacia tbmentosa, on the other hand, had reddish to brown colouration of the in te rio r. As for the shape of the nodules, Cassia kle in ii, Alysicarpus pubescens, Atylosia sericea, Butea monosperma, Pelbergia lanceolaria, Erythrina stricta, F.suberosa, Indigofera 48 cassioides and Acacia tomentosa had globose shape (F ig .2) Crotalaria albida, Rhynchosia rothii, Tephrosia pauciflora and T .tinctoria had elongate shape (Fig. 3), Crotalaria leptostachya , C . tr iquetra and Priotropsis cys 11 soi,des had bifurcate shape (Fig.4) C.albida, C.calycina and C.triquetra had fan shaped nodules (F ig .5) C.leptostachya and P.cystiso ides had Coralloid nodules (F ig .6] and Smith1 a salsucinea had siamese nodules (F ig .7). Tlie nodules o ^ Mucuna monosperma were irregular in shape (F ig .8). According to Corby (1971) and Lim and Ng (1977) nodule shapes are characteristics of legume species and independent of Rhi zobium strain s. As for the size of the nodules, it ranged between 2 mm to 18 mm, the smallest being shown by Smithia salsuginea and largest by Crotalaria leptostachya. As for the surface of the nodules, i: varied from smooth to hard and woody. In case of Frythrina suberosa the hard surface of nodule also showed whitish streaks. The data thus show that nodulation on 19 wild leguminous species is a first report. 5.2 Isolation and Identification of root nodule bacteria The details of the growth of the isolates from root nodules of 19 leguminous spp. on YB>! agar are given in Table 4, together with the results of Ketolactose test, growth in 0) vj- u.D’ U'J u (/} rC Cj • H rt X X rC ■fj 4-> rC 4-> rC rC 4-> u-» X J-J a) ciO":3 tO 'O o o t O cj)nd CO O •#-> 'H D o :3 ^ o o o ;n. o ::3 -*--> o o o rt e G c E O CTj o E cy^ 1/1 c^: a:: CO lO q : CO Cr: cx in rC q; tn (/) T3 ■M P •H •H •H c •H rP ^ 'T^ in t/) o P r: r:J C C q P •T3 o P •ri f~. •t-i •H G) ' H •H •H •H (D hH P. CIh a . pH Ch C< fO D o t-H o o i/-> rc: u •x) to •H tn O') if) •H r: •M P •r-i P •H +-) c: •H p •H P 1/1 rP ^ O ' :>, ^ ;s +-* :s- +-> ^ . 4-> o n^o o -J O Cj P O Ci.'. 3 bO O •H o •rH O P •H >H 0 ^ f—( *H ^-i •H w •rs rO M C3 o « Pi ^ fQ .a :==' iTj Q r-; X >< E X E CD E; e E n e EE N o LD oo E; E e E f-( 0) (d +-> » 'H I (D o CD I—<• o ■1- o 1 O fJ ■M (D a> ;i3 cd c/) s u O r-H c to in 0) W Oj to (D O o O w) tio: J-* i-H t 0> tiO o »H bO DO O P. X) 0 ). c P c3 p E p : ^ p p: P u ::) rj o o O :3 t ■P C 03 P o +-> o O o r—< ?-l 03 O r - • iH O r H ct3 I—( t—i f-H oo CJ cc ^ O P. OO >- a W w O + j o T} f-H 'Td r-( 'TJ f—( «—( rH «r-^ o r-H !—< 1-H •H o c Cd f: rt p rt rt rt C' o 03 cd cd 4-J x : cd 10 rt CO cd J-t (/> CO J 1/) u W c/> If) u to • rH •M +-> (D ■M (D 4-> d) •M (D +-> • H rj o o Oj «r.- O rH T-( •r-' P *4-‘ 03 10 E •r-( *rH to I/; o 4-^ fc/j u O r3 p< C :3 +-• Indiqofera cassioides. F ig .3; Elongate nodules of Rhynchosia rot:h i i . F ig .4; Bifurcate nodules of Crotalaria triguetra F ig .5: Fan shaped nodules of Crotalaria calycina. Fig.6: Corailoid nodules of Crotalaria leptostachya F ig .7: Siamese nodules of Smithia salsuginea. F ig .8; Black coloured nodules of Mucuna monosperma. "J 1—H r^. d C' -M vO CO r o hO 'JD CO CO O t-o O H '-M 4-> '/) X O O I—( ^ i-H cn in i-> -*-> O D o o cni rv] C'j coocnojrgcovovooo C C ■M O to ci u '-M o i-H O f-H O O o JZ SI 4-> {H. O ro CO cn rg r-- r - j CO ro S c O o H Jh ^ 4-J (IL,< o r s ] in o > . C 03 w O •H ^ ot/) •M I 0 c3 o ^ O& C o bD 4-1D Oj O C in Xi -H O P.o < -MO X o o DC 4-1 O r—i t 3 O in O 1—1 o •r-* o PQ S-< 4-J 4-> T 3 c tn nj -C - g 4-< Jh 5 o •H ■P O ^4-1 •n) in S-. C o o c : • t-i _u e 4-J rH (D O o OC3 OC3 o ■ r-l d If. 0 o •H o o d •M c ir. -C d in 0 u •H o C u d o d C3 P. • f—1 O d d X 5 o P d • H r-H I—) U c +-J 4- > d d •H d 0 • rH 0 03 c i »-H W d •H t/^ CJ —4 d i/i o d • rH • iH 0 rH P CO x: O d ct ' ’J u o r.j o 4-J o •H c 4-> • H P Mh 0 0 d u o r- o 4-J ' y o U t/) p t/) X, •H •r-i 4-J p CO C3 •H u JD r-H P-« ••H u • r-l o 10 O 4-> bO u U ■ H 0 U •■H P- •H o I—' d o '. H d CL U o p P 4-J c 5m p - d u ?-H 4-> d 4-> u c/)^ P CO d • rH P «. CO O' ir. tn o CO rH P- 4-* 0 o o (y*, O d d d •3 d p •r- d d s Si >v I—< > . C •r-^. . - j ■ f-^ H d d d P o co • <-> CO d d 0 t—t 1—< & rt O 5-^ • r—t •—' c o g p . • H • H rH 4H •H ■ r-< d • H £ d d d d •rH • rH <+4 o O d if) IT. • rH a ' G u to r —I rH rH r H P O d p X. • H 0 0 s d • rH ' « •H o d d d d d o rC 4=: Oi} C 4-J u X PP d • H O'; r-H o 4-* +J • 4-J , j JIj 4-J +-> ■ H P O p 4-> -C , r~, t+H u t/i r £ +-) o o o .) rH X) u • H •H p - P. X d a 03 r-^ 4-* ' J-. J-< d P c 0 y-> 4:: 0> 0 P u i n U (7 ) < < : CQ u c_; UJUJ t-H p - c:: H in < c: }- o ..... • .,.....,, CO X rH rH OJ hO r-- LO vD t - CO c% o rH ro LO \C r - t-H t-H ?-H rH t-H t-H ^—t t-H t-H 50 Kofer's alkaline medium, Nile blue reduction test, growth on Congo red medium and Koch's postulates. It could be seen fi'om the table that 19 isolates could be divided into two groups v iz fa s t growing and slow growing on the basis of their growth rate. Colonies of fast growing isolates could be detected betw'een 2nd and 3rd day, their diameter ranged inbetween 2 to 4 mm on 4th day and 8 to 11 mm on 8th day. Colonies of slow growing iso lates, on the other liand, could only he detected after 5 to 6 days of incubation and their diameter was 1 mm or even less after 8 days of incubation. The fast growing group included isolates from Cassia kleinii , Erythrina stricta, F.suberosa, Priotropsi s cystisoides and Tephrosia pauciflora while the slow growing group included isolates from Alysicarpus pubescens, Atylosia sericea, Butea monosperma, Crotalaria albida, C.calycina, C. leptostachya, C.triquetra, Dalbergia lanceolaria, Indigofera cassioides, Mucuna monosperma, Rhynchosia ro th ii, Smithia salsuginea, Tephrosia tinctoria and Acacia tomentosa. The sole isolate from Mimosoideae (Acacia tomentosa) belonged to slow growing group while the isolate from C aesaljinioid eae (Cassia k le in ii) belonged to fast growing group. Of the 17 isolates from Papi 1 ionoideae, 4 w'ere fast grov/ers while 13 v/ere slow growers. It is thus seen that isolates from plant 51 species of one subfamily did not necessarily belonged to one group. If the species wise distribution in the Papili- onoideae is taken into consideration it becomes evident that while the isolates from all the 4 species of Crotalaria were slow growing, 2 from the species of Hrythrina were fast growing and one each from Tephrosia spp. was slow' and fast growing. It is thus seen that the isolates from different species of the same genus did not necessarily belong to the same group. Similar results were obtained by Lim and Ng (1977) with isolates from Phaseolus spp. It could further be seen tl'.a t none of the isolates were positive for Ketolactose test, they did not reduce Nile blue indicator as evidenced by the fact that there w'as no change in the colour of the dye and did not take up Congo red as evidenced by the fact that the colonies of the isolates on Congo red Yeast extract Mannitol agar were white, translucent, glistening and they were not stained. It thus becomes evident that none of the isolates belonged to genus Agrobacterium. As for the growth of the isolates on Glucose Peptone agar, it is seen that there was no growth on the medium nor there was a change in the pll of the medium after 2 days of incubation. The rhizobial growth on this medium, is usually poor except for some strains of Rhizobi uin m e 1 i 1 o t i. and 5 2 rhizobia from Lotononis bainesii (Dj/e 1979} and they cause very lit t le change in the pH. It thus becomes evident that a ll the isolates belonged to rhizobial group and there were no contaminants present. The data on the Koch's postulates studies show that all the isolates were able to nodulate their respective host;'., where as, plants in the control pots were devoid o£ nodules. The data thus prove that there is a symbiotic relationship between the isolate and its homologous host, confirming the authenticity of the isolate. According to Allen and Allen (1981) for building up of a rhizobial collection, Koch’s postulates become a vital requirement in which the a b ility of the isolate to form nodules on its homologous host is confirm.ed. This is further substantiated by Graham (1976) who has stated that the biochemical tests are having limited value in the identification of root nodule bacteria and the only technique by which presumptive root nodule bacteria can be screened is through their nodulation a b ility . He, however, has brought out difficulty in this test that seeds of appropriate hosts are not r e a d i^ ly available. Fortunately, the seeds of a ll the 19 leguminous species collected in the present studies were available, thus, authenticity on homologous host could easily proved. 53 The number of nodules formed on tap root ranged in between 0 to 21 while the number on lateral roots ranged from 0 to 18. In case of Crotalaria calycina, Mucuna monosperma and Priotropsis cystisoi des nodules v;ere found only on lateral roots whereas in case of Smithia salsuginea nodules appeared only on tap root. 5.3 Morphological and Cultural Characteristics 5. 3.1 Gram staining : It was observed that a ll the fast and slow growing isolates were Gram negative, non sporeforming, rod shaped bacteria. 5. 3.2 Cultural characteristics : The colonies of three fast growing isolates viz. Erst, Esbp and Prey on Yeast extract mannitol agar, had water clear appearance, with large amount of free flowing gum and thus they resembled the growth of R.leguminosarum, R .phaseoli and R .t r i f o l i i (Graham and Parker 1964]. The growth of remaining two fast growing isolates v iz . Cahk and Tpfk, on the other hand, was evenly opaque and with less gum, thus resembling the growth of R .m e lilo ti. These 2 types of colony morpliologies v/ere also observed by Trinick (1980) with fast growing rhizobia from Leucaena group. The colonies of slow growing isolates v;ere white in colour, circular with convex elevation and ^,’ith entire margin. They produced less gum which was dense and sticky. S4 5.3.3 Rate of growth in synthetic medium : The data on the growth, generation time, specific growth rate and fin al pli of tlie medium are given in Table 5. It could be seen from the table and F ig .9 that the fast growing rhizobia had a short lag period of 24 to 36 hours. This was followed by a logarithmic phase and maximum growth of these rhizobia reached after 72 to 96 hours. The strain Cakk required 96 hours to achieve maximum growth. In case of slow growing rhizobia the lag period rar.^ed between 36 to 72 hours and the maxiinum grov:th \vas obtained after 168 to 192 hours. Two slow growing strains v iz . Bumk and Smsg had maxim.um lag period (84 hours) and they required 192 hours to reach maximum growth. It could, also, be seen from the table that the fast growing rhizobia had generation time ranging between 3.6 and 4.2 hours. Esbp was the most fast growing strain , which had generation time of 3.6 hours. The generation time of slow growing rhizobia ranged between 7.9 to 10.2 hours. Bumk was the most slow growing strain , with a generation time of 10.2 hours. According to Vincent [1976) fast growing rhizobia are having mean generation time of 2 to 4 hours, on the other hand, slow growing rhizobia have mean generation time of 6 to 8 hours. Tan and Broughton (1981] obtained mean generation Table 5 ; Growth patterns of fast and slow growing strains • S r. Strain Lag T ime Genera Specific Final pH No. period required tion growth of the h. for maxi time rate medium mum gro h. Genera- (in it ia l wth tions/h. h. p^ 6. 75) Slow growing strains 1. Actm 60 180 7.98 0.125 7. 86 2. Alpb 48 180 8.52 0.117 7.92 3. Atsv 48 192 8.39 0.119 8.15 4. Bumlc 48 192 10. 26 0. 097 7.29 5. Cram 48 180 8.88 0.113 8.12 6. Cals 36 168 9. 84 0.101 8. 23 7. Clps 48 180 8. 80 0. 114 8.49 8. Ctrb 48 180 9.36 0.106 7.88 9. Dink 36 168 8. 66 0.115 8.25 10. Incs 72 168 8.91 0.112 7. 76 11 . Mump, 48 168 8.55 0„117 8. 56 12. Rhrt 60 180 8.09 0.124 7.93 13. Smsg 84 192 9.78 0.102 8.02 14. Tptb 48 180 9.06 0.110 8.18 Fast growing strains 1. Cakk 36 ,96 4. 06 0, 246 4.95 2. Esbp 24 84 3.66 0. 273 5.28 3. Erst 24 84 4.35 0.229 5.20 4. Prey 2 4 72 3.82 0.262 5.8 3 5. Tpfk 36 84 3.9 7 0 „ 2 51 5.73 A- A Prey- FAST GROWING STRAIN. «- o Bumk- SLOW GROWING STRAIN. fig: 9. GROWTH RATE OF FAST SLOW GROWING STRAINS IN THE SYNTHETIC MEDIUM. 5 6 time of 3.0 and 4.2 hours v:ith fast growing rliizobia and 8.6 hours with slow growing rhizobia from tropical legumes. According to Dreyfus and Dommergues (1981) the generation time of fast growing isolates from Acacia spp. ranged between 8 to 12 hours. Crow et al. (1981) designated strains with generation times of < S hours as fast growing and those v.'ith generation times > 5 hours as slov; grov.’ing. The values obtained in the present in^^estigation thus compare well with the figures obtained earlier by various workers. It could also be seen from the table that tlie specific growth rates of the fast growing isolates ranged between 0.23 to 0.27 generations per hour, whereas, those of slow growing isolates ranged in between 0,09 to 0.12 generations per hour. Martinez De Drets and Arias (197 2) have shown specific growth rates in the range of 0.21 to 0.29 generations per hour for fast growing rhizobia and 0.03 to 0.16 generations per hour for slow growing rhizobia. It could be seen from the results that the slow growing strains increased the pH of the medium while the fast grov;ing strains decreased it . The in it ia l pH of the medium was 6.75 and the fin al pH of the medium ranged inbetween 7.95 to 8.56 with slow growing strains and 4.95 to 5.83 with fast growing 5 7 strains. The maximum increase in the pll was shown by the slow growing strain f’ump (inal pll 8. 56) v.'hile maximum decrease in the pH was shown by the fast grov;ing strain Cakk (fin a l pH 4. 95). The results are cojuparable with those obtained by Bromfield and Kumar Rao (1983) with slow growing isolates from Caj an iis caj an (final pll 7. 5) and fast grov/ing isolates f rom _C . ca 1 a_i^ and C i c e r ari et inum (fin al pK 4. 4). The result of the daily cliange in tlie pH of the medium by the fast growing strain Cahh and tlie slow growing strain Mump are given in Fig. 10. It could be seen from the Fig. 10 that with the fast growing isolate the pH of the medium increased slig h tly during f ir s t 36 hours and then it rapidly decreased upto 4.9 within next 36 hours and remained constant during the subsequent period of incubation. The in it ia l increase in the pH of the medium by fast growing isolates compare well with the results of Tan and Broughton (1981) obtained with isolates from T r i f o1i um, Medi cago and Leucaena. With the slow growing isolate Mump, the pH of the medium increased gradually upto 8.5 within 7 days, v/hen the maximum growth of the organism was achieved. fig: 10 pH CHANGES BY FAST ^ SLOW GROW'ING STRAINS IN THE SYNTHETIC MEDIUM. '5. 4 Riochemical and Physiological c nr ante ri sties : The data on the bioclieir.ical and physiological chara of tlie rliizobial strains are given in Tables 6 to 17 as detailed below ; 5. 4.1 Production of acid or a lk a li in YF,MA ; It could be seen from Table 6 that 14 slow growing isolates viz. Actir, Alpb, Atsv, Rum]:, Cram, Cals, Clps, Ctrb, Dink, Incs, '4un;p, ^-’hrt, Smsg and Tptl) ]iroduced alkali uh.iJe 5 fast growing isolates \^J_z. Cakk, hi'SL , Esbp, Prey and Tpfk produced acid in tlie mediuii;. Fast growing acid producing rhizobia have been isolated froiT! some tropical legumes by different v/orkers e.g. Leucaena, Acacia, Mimosa and Sesbania (Trinick 1980), Cicer arietinum. (Gaur and Sen 1981], Soybean (Sadowsl:y et a j . 1 984), Calliandra, Mim.o^, Piptadenia, Adesmia and Astragalus (Rothschild 1981), Swainsoni a 1es sert i i fo1i a, Kennedi a prostrata, Acacia longifolia and A. mearnsi i (Lawrie 1983), Acacia pennatula and Hlyr ic idiurn sep ium (Roskoski and V.'ood 1984], Acacia, Desmodiurn, Glycine, Mimosa and Sesbania (Broughton et al. 1984] and Caj anus cai an (Bromfield and Kumar Rao 198 3). The data obtained in the present investigation, tlius, contradict the hypothesis of Norris (196B] that the rh.izobia Table 6 : Production of acid/alkali in YEMA and changes in the Litmus milk by root nodule bacteria. Sr. Strain Reaction in YEMA Litmus milk Reaction No o Serum Zone Reaction Slow growing strains lo Actm Alkaline Alkaline 2. Alpb 3. Atsv 4 o Bumk 5. Cram 6. Cals 7. Clps 8. Ctrb 9. Dink lOo Incs 11. Mump 12. Rhrt 13. Smsg 14. Tptb Fast growing strains 1. Cakk Acidic Acidic 2„ Erst " Alkaline 3. Esbp 4. Prey 5. Tp£k Acidic 60 a-y associated with tropical legumes produce alkali and.primitive forms of the microsymniont while those associated with tem.perate legumes produce acid and are evolved forms and sybstantiate finding of Eli (1977) that the cowpea type rhizobia are slow growing and a lk a li i)roducing bacteria , however, not a ll sym.bionts isolated from tropical legumes sliare these properties. 5. 4.2 Litmus milk reaction ; It could be seen from^ Table 6 that decolorization of litm.us and curdling of the milk was not sliown by any of the strain thus, confirm.ing the absence of nonrhizobial contaminants (Vincent 1970). It could further be seen that two fast growing strains viz. Cakk and Tpfk produced serum, zone (peptonization) w'ith an acid reaction, a reaction chara of Rh i zobium. mel ilo t i strains (Graham and Parker 1964 and Kleczkowska et _al_. 1968) indicating thereby tl'e resemblance of these strains witli R.m e lilo ti. The rem.aining 3 fast growing strains v iz . Erst, Esbp and Prey did produce serum zone in litm^us milk but reaction was alkaline, the reaction often given by R. 1 e gum i no s a rum,, R. phaseol i and R . t r i f o l i i (Graham, and Parker 1964) thus showing rescm.blance of the strains vdth these fast growing Rbizobium spp. Similar results were obtained, with fast gro'.cing rhizobia from, tropical legumes, by different workers. Trinick (1 980) 61 in his work with 52 strains from Leucaena, Acacia, Mimosa and Seshania reported that 12 strains produced serum zone with acidic reaction and 33 strains produced serum zone with alkaline reaction. He also reported 7 strains from Mimosa spp. not producing serum zone. Gaur and Sen (1981) in their work with 115 strains of Cicer rhizobia reported that 22 strains produced serum zone with alkaline reaction while 93 strains gave only alkaline reaction. In the studies carried out Sadowsky ^ (1983) fast growing soyabean rhizobia gave a variety of Litmus milk reactions. Four strains produced acid and 3 strains alkali with the formation of serum zone, 1 strain gave alkaline reaction without changing the pH. Vyas and Prasad (1959) reported that isolates from Zornia dip hylla, Crotalaria medicaginea and Sesbania aegyptica produced serum zone with alkaline reaction while isolates from. Vigna cat j ang, Crotalaria juncea and Desmodium rotundifolium produced acid. Lawrie (1983) reported that isolates from Kennedia prostrata produced serum zone with acidic reaction while isolates from Sv/ainsonia le ss e rtifo lia and Acacia longifolia produced it with alkaline reaction. According to Scholia and Elkan (1984) for fast growing Rhizobium fre d ii the litmus milk reaction is variable for acid and alkali production. It could, also be seen from the table 6 that all the 14 show growing isolates did not produce serum zone in litmus 62 milV. while they invariably gave an alkaline reaction. These results are in agreement with tl-'ose of Graham and Parker (1964) who found that none of the slow grov.'ing strain, out of 31 tested by them, produced serum zone or gave an acid reaction in litmus milk. Sadowsky £t £l. (1983) found that a ll the 21 slow growing soyabean rhizobia, v/ith only one exception, exhibited no peptonization (serum zone formation) but did exhibit alkaline pH changes. 5. 4.3 Carbohydrate u tiliza tio n : It could be seen from Table 7 that while all the isolates utilized arabinose, glycerol, fructose, galactose, glucose mannitol, m_annose and xylose, cellobiose, inositol, maltose, lactose, raffinose, sorbitol, sucrose and trehalose were u tilize d only by fast growing isolates and none could utilize dextrin, dulcitol, inulin, and starch. It is thus seen that fast growing isolates could u tiliz e as many as 16 carbohydrates whereas slow growing isolates could u tiliz e only 8. The data thus substantiate the findings of Allen and Allen C1950) and Graham (1964) who have shown the carbohydrate nutritional differences betv/een fast and slow grov;ing rhizobia. The ability of fast growing rhizobia to utilize large number of carbohydrates has been shown by various workers. Graham (1964] found that Rh i z ob ium t r i f o l i i and R . 1 et^umi nos arum u tilize d 20 different carbohydrates and tricarboxylic acid » o c CQ -H HH VI CD •H I t I 9 I > 8 9 9 9 I ) » I + + + + + u cd 1 o i/} rH O e? u to + + + + + + + + + + + + + + + + + + + c •H 9 u o CO o Jh r-H !/) DO 9 CO o a GO + + + + + + + + + + + IS + + + + + o r—1 1 t/) y 0) to 'V 5 ^ o p O +- c3 + + + + + + + + + + + + + + + + + + + 4-i o to u CO >N«—' rH O CJ ^ + + + + + + + + + + + + + + + + + + + rH (D V) » o N cycle intermediates. Trinick C19S0) studied 45 fast growing isolates from Leucaena, Mimosa, Acacia, Sesbania and Lablab and found that they were able to u tiliz e 16 of the 18 carbohydrates tested. Scholia and Elkan (1984) and Stov;ers and fiagleshain. (1984) showed that fast grov/ing soybean rhizobia C?^hi;?obium f re d ii) u tilize d IS of the 20 carbohydrates tested. According to Allen and Allen (1950) the slo\\f growing rhizobia are more specific in their carbohydrate requirements in every respect. The relative fastidiousness of these rhizobia has also been shown by various workers. Graham (1964) found that slow growing rhizobia utilize d only 8 of the 20 carbohydrates tested. Elkan and Kwik (1968) studied 36 strains of R.japonicum for their nutritional requirements and found that they u tilize d 8 of the 15 carbohydrates tested. Sadowsky £]_. C1383) found that slow growing soyabean rriizoBia were able to utilize 8 of the 20 carbohydrates tested, Martinez-de-Prets and Arias C1972} have proposed an enzymatic Base to explain the difference exhibited by fast and slow growing rhizobia in the utilization of different carbohydrates and that is the presence of NADP-6-phosphogluconate dehydro genase (TZC 1 .1 .1 . 43) only in the fast grow'ing rhizobia. This enzyme a ctiv ity was also shown in fast growing soybean rhizobia by Sadowsky et 0-983). The Entner-Doudoroff, the Em.bden-Meyerhof glycolytic and the oxidative pentose 65 phosphate pathways Iiave been reported in the fast growing rhizobia (^Tartinez-cle-Drets and Arias 1972) and only Entner Doudoroff pathway occurred in the slow growing R. japonicum (Elhan 1971). It could further be seen that the disacchrides viz. cellobiose, ----> lactose ^maltose, sucrose, and trelualose were u tilize d only by fast growing rhizobia. The results in tlie present investigation thus confirm observations of Sadowsky _^t jU . (1983) tjiat disacchrides were utilized only by fast growing rhizobia and of Shiiiyrcva and Plaksina (1972) v.'ho, in tlie studies with slow growing R.lupini and R. japonicum, have concluded that the in a b ility to u tiliz e disacchrides was one of the peculiarities of slow growing nodule bacteria. Martinez-de-Drets et a l. C1974) in their studies on growth on sucrose by 64 fast and slow growing Rhizobium strains detected an inducible p glucosidase (invertase) in only fast growing rhizobia, Sadowshy £t £]_. [1983] showed that only fast growing rhizobia were having appreciable /3 galactos’idase activities. According to Glenn and Dilworth C19811 slow growing rhizobia tend to lack both uptake systems and catabolic enzymes for disacchrides. It is interesting to note that Graham (1964)_ found that disacchrides were u tilize d by 36 out of 40 fast growing rhizobial strains and by only 9 of 5 5 slow growing strains. 66 It also becomes evident that dextrin, dulcitol, inulin and starch were not u tilize d by almost a ll tlie fast and slow growing strains. Inability to utilize these carbohydrates by fast growing rhizobia was s]iov/n by Sadowsky et al . flQSS) and Trinick (1980). Graham (1964) found tliat only 6 of the 108 fast and slow growing rliizobial strains tested utilized dextrin and only one strain utilized starch. According to Triniclc (1980) fast growing rhizobia from Leucaena, Mimosa group differed from the slow growing rhizobia of other tropical legumes in carbohydrate utilization and in this respect they are sim ilar to the fast growing Rhizobium t r i f o l i i R .m.el ilo t i , R. leguminosarum and _R. phaseoli. The data obtained in the present investigation substantiates the findings ox Trinick. 5. 4.4 Utilization of organic acids; It could be seen from Table 8.that oxalate and tartarate were u tilize d by alm.ost a ll tlie fast and slow growing strains, while the remaining 5 organic acids viz., c itra te , fumarate, malate, pyruvate and succinate were utilized by only fast growing strains Avith few exceptions of slow growers v iz ., Dink u tilize d fumarate, Atsv, Clps and Rhrt u tilize d malate, Atsv and Rhrt utilized pyruvate and Dink and Mum.p u tilized succ inate, Table 8 : Utilization of organic acids by fast and slow growing strain s. Isolate Sodium Sodium Sodium Sodium Sodium Sodium Sodium Citra- fuma- malate oxa- Pyru- Succi- ta rtra te rate late vate ' nate rate Slow growing strains Actm + Alpb - - - + - - + Atsv -_ + + + _ + Bumk + Cram - ~ - + - - + Cals “ - + - - + Clps + Ctrb - - - + D in k - + “ + - + + Incs - “ + - - + Mump - - - + - + + Rhrt - - + + + - + Smsg - - ■“ + Tptb - - » + - - + Fast growing strains Cakk + + + + + + + Esbp + + + Erst + + + + Prey + + + + Tpfk + + + + + : organic acid utilized - : organic acid not utilized 6 8 Graham and Parker (1964) sliowed that fumarate, malate, pyruvate and succinate were utilized by most of the strains of R .t r i f o l i i , P. leguminosarum, R.phaseoli and R.m eliloti where as fumarate and succinate were not u tilize d by slow growing cow^pea rhizobia. They also reported few slow growing rhizohia utilizing malate and pyruvate. flkan and Kwik (1968] showed that pyruvate and succinate were of limited use whereas acetate, propionate, m:alate and citrate were uniformly inadeauate as a carbon or energy source for Rhizobium japonicTim. Trinick (1 980) showed tliat a ll the fast grow'ing rhizobia from Leucaena group w^ere able to u tiliz e sodium citra te . Sadowsky _e_t £ l . (1983) showed that fumarate, malate and succinate were utilized by fast growing soybean rhizobia but not by slow growing ones. These results com.pare well with the results obtained in the present investigation. 5. 4.5 Vitamin requirements of root nodule bacteria : It could Be seen from Table 9 that the growth of a ll the strains in the control tubes without any Vitamin, except for two fast growing strains viz. Cakk and Tpfk, compared well with growth in the medium, containing biotin, calcium pantothenate and thiamine indicating clearly that a ll these strains did not require any Vitamin for their growth. Table 9 ; Effect of addition of vitamins on the growth (absorbance values) of the rhi zobial strains. Sr. Rhi zobial Control Bi otin Calcium Thiamine No. strain (without pantothenate vitamin) (100 (100 >ug/ml (200 Mg/ ml) ml) Slow growing strains lo Actm 0.59 0.64 0.66 0.60 2. Alpb 0.64 0.70 0.68 0.66 3. Atsv 0. 61 0. 73 0.63 0.70 4. Bumk 0. 56 0. 54 0.58 0.61 5. Cram 0. 71 0.67 0.80 0.75 6„ Cals 0.57 0. 59 0.61 0.60 7. Clps 0.56 0.60 0.63 0.59 8. Ctrb 0.68 0.73 0.66 0.75 9. Dink 0.60 0.68 0,70 0.63 10„ Incs 0.62 0.66 0.61 0.67 llo Mump 0.64 0.70 0.74 0.68 12. Rhrt 0.80 0.82 0.87 0.78 13. Smsg 0. 53 0.66 0. 58 0.55 14. Tptb 0. 73 0.79 0.70 0.72 Fast growing strains 1. Cakk 0.32 0.78 0.88 0.84 2. Erst 0.69 0.72 0,66 0.90 3. Esbp 0.74 0.79 0.72 0.76 4 . Prey 0.80 0.70 0.74 0.78 5. Tpfk 0.28 0. 81 0.80 0.7 3 Figures indicate absorbance values at 420 nm. 70 Non stimulation of slow growing strains with the Vitamins is in conformation with the results of Graham (1963) who showed that thiamine and calcium pantothenate were not required for the growth of slow growing rhizobia and only 7 out of 31 strains tested responded to biotin. Elkan and Kwik (1968) tested 10 vitamins and showed that 36 strains of R. j aponicum grev; independently of any vitam.ins except for 3 strains which were stimulated by biotin. The fact that two fast growing strains viz., Cakk and Tpfk show'ed poor grow'th in the control medium and tlieir growth was stimulated by all the 3 vitamins show that they are sim ilar to R. leguminosarum, R.phaseoli and R. t r if o 1i i since Graham and Parker (1964) showed that 26 out of 31 strains of this group responded to addition of one or more of biotin, thiamine and calcium pantothenate. The need of these 3 fast grow'ing species, particularly for biotin and thiamine, was also shown by Allen and Allen (1950). According to Biordii and Ertola (1985) some strains of R.phaseoli are having an absolute requirement for biotin. The growth of remaining 3 fast growing strains viz. Esbp, Erst and Prey independent of any vitamins substantiates findings of Stowers and Eaglesham (1984) who obtained sim ilar results with fast growing soyabean rhizobia. They have further stated that based on this characteristic i.e. growth independent of vitamins, the rl'.izobia lin k to slow growers rather than fast growers. 71 Elkan and Kwik (1968] have reported Inhibition of growth of 4 strains of R .j aponi cum by biotin. The data obtained in the present investigation, hoxvever, do not support this finding. 5. 4.6 Sensitivity to antibiotics : It could be seen from Table 10 and F ig .11 that out of 19 strains tested, slow growing strains viz Atsv and Dink v.'ere sensitive to 4 antibiotics, strains Actm, Atsv, Cram, Cals, Mump, Rhrt and Tptb to 5 antibiotics and strains Alpb, Clps, Ctrb, Incs and Smsg to 6 antibiotics while the fast growing isolates Cakk, Esbp and Tpfk were inhibited by 11 antibiotics and isolates Erst and Prey by 13 antibiotics The difference in the sensitivity of the isolates to different antibiotics compare well v/ith the results of Graham (1963b) who have shown that slow growing rhizobia are less susceptible to antibiotics as coinpared to fast growing ones. It could also be seen from the table that C arbenicillin and Chloramphenicol inhibited growth of only fast growing isolates and not of the slow growing ones, Trinick (1983) has shown that slow growing tropical legume rhizobia Avere resistant to chloramphen ical at 6Q xig/ml while the fast growing rhizobia were sensitive to it in as little a concentration as 5 Aig/ml, Poskoski and Wood (1984) haA'-e found that fast grooving rhizobia from Acacia pennatiila and G1 yricidium sepium r-^ cj CO ^ to 1 a r-~i K) . J C'J rs) rH rH fNj r-j to r: r - Cli p : V~J '■—' CO CO ^ CO ' P i CO ^ p : P i p : CO ^ P i p : CO ^ P i CO CO ^ CO a : CO r, >^ /•—\ /—V 'J r I -Tj- o VC 'd- rg o vO r j 00 vO + j r-H lO 1—< rH r-H r ' rH rH rvi rH, to (N rO 4-> o t-- vO ro r - a r4 r J rH CM CSJ H Cti cii P i P i CO ' p ; P i P i CO P i p ; P i P i P i CO PS P i CO P i CO to l/> CO oo CO Tj“ 00 E r-H tvl rH rH rH rH CO Oi CxL a : P i P i P i P i CO ^ CO ' CO P i CO ' p ; P i P i CO PS P i CO P i PS w /- -> Ol o e C4 vO ,-i Cl, r —i f—\ g o LO o CO fNj 3 r4 rsj »H rH z P i csi CJi p ; P i P i P i CO CO cc C/) CO P i P i P i CO ' PS p i P i P i P i (0 /—^ /—N u 00 K> o rsj o c r^j rH rvj to rH to to ►—1 C4 cs: cC P i p : CO P i CO CO CO ' P'. CO CO ^ P i P i P i 0£ P i to P i P i c: fNj o oo cri !-H r j f J rH rH Q cr: ct: p : P i a : CO P i P i P i CO ^ p : CO P i P i CO P i P i IX P i P i P i ta ,__ , C o m;5 O lO 00 LO rg V) 'rH 4-) r^j r-i rsi rH rH CM to C rt U o i Oi P i P i P i CO P i P i P i CO ^ P i CO ^ P i P i CO ^ CO ' os (X to p : to •r-< }-. n W — , ,— > S ,—» CO D. T-t LO UO in rj- rH 4-J r-i tH r^i rH rHo •V cd) O C u • H Bumk CO m z Oink IS) -j < Actm —I Atsv o Cram X Cals m :o Mump X N Rhrt o GO Tptb r—> Alpb (J) —t :xj Clps > 21 U-) Incs —I o Smsg Ctrb ro Cakk o Esbf) o U) Tpfk Erst Prey 73 were inhibited by chlorarnp'^en i co l, Sen sitivity of fast growing rbizol ia to chlorair'pVienicol was also shown in case of R.trifoli i by Abdel Wahab et a l . (1976). These results compare well \ ith those obtained in the present investigation. Gentamycin and Neomycin inhibited growth of 4 out of 5 fast growing isola es. Tliese results compare well witli results of Scholia and El ban (1 9 84 ) and Stowers and F.aglesham (19S4) wlio have showi: tliat a ll strains of fast growing Rh 1 zobiurn fre d ii were si seeptihie to these 2 antibiotics. It could further be seen from the table and F ig .12 that the frequency of sensitivity to different antibiotics was in the order of bede m.ycin > Tetracyline = Soframycin > Neomycin Vibramycin > . anamycin = Erythromycin > Gentamycin > Gantricin Polymyxin B = Sulphadiazine > Carbenicillin = Cliloramphenicol > Bacitracin = Oxytrtracycline > Ampicillin = Streptomycin> Amoxycillin = Penicillin = Triacetyl Oleandomycin . It is evident from tlie results that of the 21 antibiotics tested, Leder.'ycin was the most effective since it inhibited growth of 16 ;trains. bedermycin was shown to be m.ost effective antibiotic by Graham (1963b). Tetracycline inhibited gro /th of 13 strains as evident from the data collected in he present investigation. Skrdleta (1965) has shown tet -acyclinc as the most potent inhibitor of rhizobia. NUMBER OF ISOLATES SENSITIVE TO ANTIBIOTIC CD cn Cb <0 0 Xx cr, r 1______i _ 1 —I_ ho Amoxy cn rn Trlace. z: !S@ friPr Peni. < —I CO "n Ampi. O f— > —I o ui > :§ -• o Strepto. "ri z o CT C l XI :x) ''o’ ‘o' '^’=' ■•o'' '*’c :j3 Oxytetra. •i?-- -I?- 0 'IJ* 'S?-!* *..'1- ■I’j* ■*Pj« ‘u 2 Q X CD s I Baci. m N I-y, f'H '*Pj« f'-.« 'fc°-' °J* " o o 73 CD '/☆☆tVtVt-ViVtV^V ro Chloram. (7i > o r~ i— Carbeni. _ r~ CO in > —I m XI Sulpha. I— m m > Si > LO z m CD Poly.B in —I o Gantri • o GenlB. T| "n Erythro. m iggsgggi 7 0 ☆☆☆☆☆ m Kana. Vibra. > 2 : —I Neo. ro o Sofra. o Tetra. j •Si -"j ■yj !>y K! CO Uii Leder. > I*’;?' <*’/^** --- 74 It could further be seen that all the fast and slow growing strains under st idy v;ere resistant to tlirec antibiotics viz. Amoxyc i 1 ] ii:, Penicillin and Triacetyl Oleandomycin. Davis (1962) re] Drted that the rhizobia from all cross inoculation groi;ps were resistant to Penicillin. Inability of Penicillin to inhibit the growtli of rhizobia was also shown with R . t r i f ^ i i (Abdnl Kahab c_t a1.1 97 6), some temperate zone rhizobia (I'e’cskes and Mannirger 1962), and fast growini; P.hizobium frcdii (Scholia and Elkan 1984) . Thus the resistance of all the strains studied in the present investigation to Penicillin compare well with the above results» 5.4„7 Effect of the pH of the nutrient medium on the growth of root nodule bacteria : It could be seen from Table 11 that at pH 3.5 none of the strains was able to grow and only 4 slow growing strains viz.^ Dink, Clps, Ctrb and Incs could grow at pll 4.0 and the growth was very poor. This finding is contrary to that of Yadav and Vyas (197 ') \:ho showed that 5 out of 10 strains of cowpea rhizob a were able to grow at pH 3.5 and most strains grew at pH 4.0. At pH 4„5, only slow growing strains showed growth. Generally R.meliloti strains are more sensitive to low pH then Rotrifolii, R.phaseoli and R.leguminosarum, while slow Table 11 Effect of pH of the medium on the growtli of root nodule bacteria pH of the nutrient medium Strain 5.0 6.0 6.5 7.0 7,5 8.0 9.0 9.5 10 Slow growing strains Actm + + + + + + + + + + + + + Alpb + + + + + + + + + + + + + + + + + - - Atsv + + + + + + + + + + + ^ + + + + + -“ Bumk + + + + 4 + + + + + + -“ Cram + + + + + + + + + + + + -- - Cals + + H- + + + + + + -f+ -f - “ - Clps + + + + + + + + + + + + + + + " - Ctrb + + + + + + + + + + + + + + + - - Dink + + H- + + + + + + + + + + + + - - - Incs + + + + + + + + + + + + + + + 4- + -- Mump + + + + + + + + + + + + + + + - - - Rhrt + + + + + + + + + + + + + -“ - Smsg + + + + + + + + + + + + + --- Tptb + + + + + + + + + + + — “ Fast growing strains Cakk . - + + + + + + + + + + + + + + + + + Erst - - - + + + + -f + + + + + + + + -f + + - Esbp + + + + + + + + + + + + + + + - Prey - - - + + + + + + + + + + + + + - Tpfk - - _ + -f- + + -f + + + + + + + -t- + + Absorbance value at 4 20 nm No growth + 0.30 Poor growth ++ 0.30-0.60 Fair growth +++ 0.60 Good.growth 76 growing rhizobia are more tolerant (Graham and Parker 1964, Okafor and Alexander 19 75 , Munns 19 77). Sadowsky ^ . (1983) showed that the slow growing soybean rhizobia were able to grow at pH 4„5 b'Jt not the fast growing ones. It could also be seen from the table that only fast growing strains could grow at pH 9.0 and 9.5 whereas the slew growing strains could tolerate pH values upto 8.0 only. The results compare well with those of Sadowsky et aj_. (19S3) who showed growtli of fast growing soybean rhizobia at pH 9,0 and 9.5. Graham and Parker (1964) found that at pH 9.0, 18 fast growing rhi :obial strains could groAv while only one slov; growing strain tolerated this pH. They also reported growth of 10 R.m3liloti strains at pH 9.5. None of the rhizobial strains tested in this study could tolerate pH 10.0 of the nutrient medium. The results compare well with those of Graham and Parker (1964) who found that all the 97 rhizobial strains tested by them were not able to survive at pH 10„0. However the data contradict results of Yadav and Vyas (1973) and Bhardwaj (1975) who have reported survival of some Rhizobium strains at pH 10.0. The optimum pH was 6.0 to 6.5 for slow growing strains and 7„0 to 7.5 for fast growing strains. Generally optimum pH values are of thj crdc7- of 6.8 - 7.2 for clover rhizobia. 77 :7.Q-7„5 for medic rhizobia and 5,8-6„2 for slow growing rhizobia (Trinick 1982). According to Diatloff (1970) the optimum pH for the growth of R.japonicum is about 6.0. Pandher and Kahlon (1978) observed good growth of Roleguminosarum in the pll range 6.5-8.0. It is thus seen ' that the optimum pH values obtained in the present investi gation fall in the typical ranges of fast and slow grov;ing rhi zobia. 5.4.8 Sodium Chloride tolerance ; It could be seen from Table 12 and Fig.13 that fast growing rhizobial strains were more tolerant to NaCl as compared to slow growing strains o Fast grov/ing strains showed growth in the nutrient medium containing 1.5 and 2.0% NaCl concentration whereas slow growers could tolerate only 0„5 - lo0% NaCl concentration. These results are in agreement with those of Sadowsky _et al. (1983) who found that fast growing strains only and not slow growing ones from soybean were able to tolerate 1% NaCl concentration. Upchurch and Elkan (1977) showed that nonslimy and small colony types of R.j aponicum were sensitive to salt whereas the slimy and the large colony types were not sensitive. According to Yadav and Vyns (1971) fast grov/ing pea rhizobia were sensitive to 0.2o to 0.3% Sodium chloride while Pandher and Ka.hlon (1978) observed that R. 1 egumi nos arum Table 12 : Effect of salt concentration on growth of root nodule bacteria. Sr. Rhi zobial Nad concentration No. strain 0.5% 1.0% 1.5% 2.0% 2.5%2 Slow growing strains 1. Actm + + + + + --- 2. Alpb + + + + + -- - - 3. Atsv + + + + + + - - - 4. Bumk + + + + - --- 5. Cram + + + + + + ■ - - 6. Cals + + + + + + + - -- 7. Clps + + + + - - - - 8. Ctrb + + + + - - -- 9. Dink + + + + + + -- 10. Incs + + + + + + - -- llo Mump + + + + + --- 12„ Smsg + + + + + - - 13„ Tptb + + + + + + - - - - 14. Rhrt + + + + + + -- Fast growing strains 15. Cakk + + + + + + + + + + - 16. Erst + + + + + + + + + - 17. Esbp + + + + + + + + + + - 18„ Prey + + + + + + + + + + - 19. Rhrt + + + + + + + + + ■ “ Absorbance value at 4 2 0 nm No growth + 0.30 Poor growth + + 0.30 - 0.60 Fair growtli 0.60 Good growth FIG ; 13. EFFEC T OF SALT CONCENTRATION ON THE GROWTH OF FAST SLOW GROWING STRAINS. 79 strains could not grow at 1.8"o Salt concentration. According to Vincent (1977) out of four fast growing species of rhizobia, tolerance to 2.0"5 NaCl was shown by R.meliloti. It thus becomes clear that 5 fast growing strains obtained in the present study are siipilar to R.meliloti with respect to salt tolerance. None of the strains in this study was able to grow in the medium with above 2.0% salt concentration.- These data are in agreement with tliose of Graham and Parker (1964) who observed that most of the strains from all the six Rhizobium spp, were inhibited by 1% NaCl concentration. The toxicity of NaCl is ascribed to the chloride ion by Steinborn and Rougley (197 5) . 5.4„9 Crystal Violet sensitivity ; It could be seen from Table 13 that all the fast and slow growing strains were able to grow at 1:50,000 to 1:75,000 concentration of Crystal Violet in the nutrient medium. At 1:25,000 concentration 2 slow growing strains viz. Bumk and Smsg did not grow while at 1:10,000 concentration 7 slow growing strains vi z. Actm, Atsv, Cals, Ctrb, Dink, Rhrt and Tptb could grow. 1:5,000 concentration of Crystal Violet was not tolerated by any slow growing strain, however, all the fast growing strains, except Prey, showed growth at this concentration. Table 13 : Effect of crystal \''iolct on the growth of mot nodule bacteria. Sr. Rhizobial 1:5,000 1:10,000 1:25,000 1:50,000 1:75,000 No. strain Slow growing strains 1. Actm + + + + + + + 2. Alpb + 4- + + + 3. Atsv + + + + + + + 4. Bumk + + 5. Cram + + + 6. Cals + + + + + + + 7. Clps + + + 8. Ctrb + + + + + 9. Dink + + + + + 10. Incs + + + + + 11. Mump + 12. Rhrt + + + + + + + + 13. Smsg + + + 14. Tptb + + + + + Fast growing strains 1. Cakk + + + + + + + + + + + 2. Erst + + + + + + + + + 3. Esbp + + + + + + 4. Prey + + + + + + 5. Tpfk + + + + + + + - = No growth; + = Poor growtli; ++ = Fair growth; + + + - Good growth 81 Johnson and Allen (1952) stated that most of the fast growing strains from Sesbania spp. did not tolerate crystal violet concentration greater than 1:10,000 whereas none of the slov/ gro^/ing strainstolerated above 1:30, 000 concentration. Allen and Allen (1958) showed that acid producing fast growing rhizobia are less sensitive (1/1000) while all;:ali producing slow growers are more sensitive (1/1,50,000) to Crystal Violet. Konde (1975) found that cowpea rhizobia from Dolichos lablab could tolerate Crystal Violet concentration only upto 1:25,000 and that few strains showed negligible growth at 1:10,000. Rhizobium meli]oti on the other hand could tolerate 1:10,000 concentration and fcAv strains showed growth even at 1:5,000 concentration. These results compare well with those obtained in the present investigation. Tolerance of' fast growing strains to crystal violet may be due to the large amount of polysacchride produced by them as stated by Johnson and Allen (1952) who have further stated that a relationship exists between tolerance of rhizobial strains to crystal violet and their ability to produce gum. It was noticed that while growing on the medium containing Crystal Violet, the rliizobial strains did not absorb the dye. The non absorption of crystal violet was observed 82 in case of Cicer rhizobia by Gaur and Sen (1981). They found this characteristic to be useful as one of the presumptive tests in the identification of Rhizobium. 5.4.10 Penicillinase activity : It could be seen from Table 14 that all the fast growing strains and twelve of the fourteen slow growing strains produced penicillinase. The remaining two strains viz., Incs and Smsg did not show Penicillinase activity. The results in the present investigation compare v'ell with those of Sadowsky £t ^1 . (1983) ;;ho found penicillinase activity in all the fast grcwing and 711 of the slow growing strains from soylean. Graham and Parker (1964), how'ever, have observed tbiis activity in only 4 of the 24 strains of R»leguminosarum and R.meliloti and this activity was absent in R.trifolii and R.phaseoli. Of the 31 strains of slow growing rhizobia tested by them 24 strains showed penicillinase activity. 5.4.11 Oxidase activity ; It could be seen from Talle 14 that all the fast and slow growing strains under stridyshowed oxidase activity, substantiating the findiigs of Graham and Parker (1964) who observed this activity in 28 of the 48 isolates of fast grov;ing and 23 of the 31 isolates of slow growing rhizobia. Table 14 : Penicillinase, Gelatinase, Catalase, Urease and Oxidase activities o£ root nodule bacteria. Sr. Rhizobial Penicillinase Gelatinase Catalase Urease Oxidase N 0 o strain Slow growing strains 1. Actm + 2. Alpb + 3. Atsv + 4. Bumk + 5. Cram + 6. Cals + 7. Ctrb + 8„ Clps + 9. Dink + 10. Incs 11. Mump + 12. Rhrt + 13. Smsg 14. Tptb + Fast growing strains 1„ Cakk + + + 2. Erst + + + 3. Esbp + + 4. Prey + + 5. Tpfk + 84 Sadowsky _et _al. (1 983) have shown production of oxidase by all the fast and slow growing soybean rhizobia studied by them, 5.4.12 Urease activity : It could be seen from Table 14 that all the fast and slow growing strains (except for two slow growing strains viz. Cals and Tptb) produced Urease. These results compare well with those of Graham and Parker (19641, who liave shown this activity in 37 of the 48 strains of fast growing rhizobia and 25 of the 31 strains of slow growing rhizobia. Sadowsky _et (1983) have shown urease activity in all the fast and slow growing isolates from soybean. 5.4.13 Catalase activity : It could be seen from Table 14 that all the fast and slow growing strains under study, with the exception of two slow growing strains viz Actm and Mump, shr'/ed catalase activity, These results compare well with those of Graham and Parker (1964} who showed catalase activity in 43 of the 48 strains of fast growing Rhizobium spp. and 24 of the 31 strains of slow growing Rhizobium spp. Sadowsky ^ (1983) have observed this activity in all tlie fast and slow growing soybean rhizobia tested by them. 85 5„4.14 Gelatinasc activity : It could be seen from Table 14 that all the fast growing strains under study shoved gelatinase actr^ity v/hile the slow growing strains did not produce tliis enzyme. SimiJar results were UL^tained by Sadowsky ^ _a2- (1983) witli fast and slow growing strains from soybean. They also sliowed that gelatinase activity was absent in R.leguminosarum, R.phaseoli, R.t r i f o1i i and rhizobia from Sesbani a and Leucaena. lIoAvever one strain from Leuc n enn w'as positive for gelatinase. It, thus, becomes evident from the results that penicillinase, catalase, urease and oxidase were produ.ced by almost all the fast and slow growing strains under study and Gelatinase activity is the only activity, amongst these tests, which is helpful in distinguishing fast and slow growing groups. 5.4.15 Nitrate reductase activity : It could be seen from Table 15 that when the strains were grown in Yeast extract Mannitol medium all the 5 fast growing strains and 3 slow growing strains viz. Alpb, Cram and Incs showed nitrate reductase activity. Graham and Parker (1964) showed nitrate reductase activity in 44 out of 48 fast growing strains and in only 14 strains out of 31 slow Table 15 : Nitrate reductase activity o£ fast and slow growing strains Sr. Rhizobial YEMA Defined Medium No. strain Aerobic Anaerobic Aerobic Anaerobic Slow growing strains ri. Actm 0 0 0 0 2. Alpb 10.2 32.7 205.4 211 o6 3. Atsv 0 0 18.8 45.9 4. Bumk 0 0 24.0 49.0 5 . C r am 14.5 22.6 28.7 35.4 6. Cals 0 0 162„0 114.6 7. Ctrb 0 0 0 0 8. Clps 0 0 132.3 163 . 2 9. Dink 0 0 0 0 10. Incs 13.7 23„ 2 112.2 129. 2 11. Mump 0 0 0 0 12. Rhrt 0 0 21. 2 32.5 13. Smsg Q Q 0 0 14. Tptb 0 0 18.2 16.4 Fast growing trains 1. Cakk 4.7 6.2 12.6 14.7 2. Erst 8„6 5.3 280.8 296. 7 3. Esbp 72.8 54.6 152.5 195.5 4. Prey 13.7 20.2 191.2 204 .1 5. Tpkf 12.8 23.9 14.2 38.6 Figures indicate amount of nitrite produced n moles/mg of cell protein/hour. 87 growing strains. Gaur and Sen (1981) observed nitrate reductase activity in 29 out of 115 strains of Cicer rhizobia, Konde a d Moniz (1967) showed nitrate reduction by 5 out of 14 isolates from Trigonella and 3 out of 10 isolates from Polichos. Sadowsky ^ al. (1983) found nitrate reductase activity in all the fast and slow growing soybean rhizobia» It could also be seen from the table that when the strains were grown in the YEM medium, the amount of nitrite produced ranged between 8.6 to 72.8 n moles/mg of cell protein/hour under aerobic condition and 5.3 to 54.6 n moles/mg of cell protein/hour under anaerobic conditon. IVhen the strains were grown in the defined medium all the fast growing strains and 9 slow growing strains viz., Alpb, Atsv, Bumk, Cram, Cals, Clps, Incs, Rhrt and Tptb showed nitrate reductase activity. The amount of nitrite produced ranged between 12.6 to 280.8 n moles/mg of cell protein/hour when assayed aerobically and 14.7 to 296.7 n moles/mg o£ cell protein/hour when assayed anaerobically. The results in the present investigation thus confirm the findings of Manhart (1979) who stated that rhizobia synthesize little nitrate reductase in presence of yeast extract. He further showed that the amount of nitrite produced ranged l3etween 4.4 to 19g6. n n)oIes' /gin of cell protein/hour when cells were grown in YEM medium and from 5.5 - 340 n moles/gm of cell protein/hour when grown in defined iiiedium. He also shewed increase in the amount of nitr'te produced when the rhizobia v;ere assayed anaerobically for nitrate reductase activity, 5.4,16 Grov.'th and change in the pH of the medium containing combination of sugar and amino acid : It could be seen from Table 16 that all the strains grew well on four combinations of 2 sugars (fructose and galactosel and 2 amino acids Caspargine and glutamine). Slow grov/ing strains produced alkali in the medium with the exception of 3 strains viz, Mumpwhich gave a final pH 6.54 in the medium containing fructose + aspargine and Alpb and Incs which gave final pH values 6,67 and 6.59 respectively in the inedium containing fructose + glutamine. With the remaining slow growing strains final pH of the growth medium ranged between 6,94 to 8,02. Maximum increase in the pH was shown by the strain Tptb in the medium containing galactose + aspargine. It could also be seen from the table that all the fast growing strains gave acidic reaction with four combinations of sugars and amino acids. The final pH of the growth medium ranged between 4.94 to 6.23. ^1aximum decrease in Table 16 : Growth of fast and slow growing strains on 4 combina tions of sugars and amino acids Sr. Rhi zobial Fructose Fructose Galactose Galacto No. strain + . + + + Aspargine Glutamine Aspargine Glutami Slow growing strains 1. Actm 0,68 0.59 0.76 0,54 (7.56) (7.14) (7.49) (7.33) 2. Alpb 0.79 0.72 0.56 0.63 (7.09) (6.59) (7.12) (7.48) 3. Atsv 0.61 0.73 0.58 0.48 (7.39) (7.57) (7.84) (7.52) 4. Bumk 0„44 0.41 0.52 0.57 (7.01). (7.33) (7.95) (7.66) 5. Cram 0.57 0.48 0.61 0.70 (7.38) (7.44) (7.89) (7.22) 6. Cals Q.49 0.33 0.57 0.59 (8.15) (7.35) (7.98) (7.45) 7. Clps 0.77 0.56 0.85 0.66 C6„9 8) (7.21) (7.49) (7.26) 8. Ctrb 0.59 0.51 0.6 8 0.70 (7.40) C6.98) (7.23) (6.54) 9. Dink 0.52 0.70 0.61 0.48 (7.12) (7.30) C7.62) (7.54) 10. Incs 0„ 54 0.73 0.85 0. 56 (6.94) (6.67) (7.72) (7.12) 11. Mump 0.79 0.61 0.69 0.51 C6.541 (7„42)- (7.30) (7.05) 12. Rhrt 0.41 0.54 0.47 0.67 (6.95) (7ol3) (7.22) (7.01) 13. Smsg 0.62 0.36 0.68 0.42 (7.85) (7.52) (7.17) (7.40) 14. Tptb 0„ 71 0.49 0„54 0.65 (7.7 8) (7.48) C8.02) (7.69) Table 16 contd Sr. Rhi zobial ] ructose Fructose Galactose G a 1 a c 10 No. strain + + + + j ipargine Glutami ne Aspargi r-e Glut ami Fast growing t] lins 1. Cakk 0.83 0.78 0.6 5 0.94 (4.97) (5.89) (5.05) (5a4) 2. Esbp 0. 74 0.7 9 0.63 0„83 C5.25) (5.39) (5.50) (5.19) 3. Erst 0.68 0o75 0.98 0.7 0 (4.85) (5.36) (5.07) (5.20) 4. Prey 0„80 0.72 0 .92 0.96 (5.13) (5.41) (6.28) (5.55) 5. Tptk 0.78 0„88 0.95 0.91 (4.94) (5.52) (5.82) (5.12) Growth expressed ; 5 he absorbance value at 420 nm. values in the brackets show 'h final of the medium. Initial p^ of the medium - 6.80 91 the pH was shown by the strain Tp£k in the medium containing fructose + aspargine. It thus becomes evident from the results that reactions given by fast and slow growing strains on four combinations of sugars and amino acids are similar to classic reactions of fast and slow growing rhizobia on Yeast extract Mannitol medium. The four combinations of sugars and amino acids used in this study have been obtained by extensive preliminary experiments done by Tan and Broughton (1981)„ They showed production of acid by 3 fast growing strains from Leucaena leucocephala, Medicago sativa and Trifolium subterraneum and production of alkali by 3 slow growing strains from Centrosema pubescens, Glycine max and Crotalaria anagyroides. These results compare well with the results obtained in the present investigation„ According to Tan and Broughton (1981) the different direction of pH changes in defined medium of one sugar and one amino acid, by fast and slow growing rhizobia are probably due to preferential utilization of sugars by fast growing rhizobia and nitrogenous compounds by slow growing ones. Substances causing acidic pH could be various organic acids like isobu- tyrate, butyrate,acetate, propionate and pyruvate, which were found in the growth medium of R.trifolii by Holding and Lowe (1971), Alkaline end products may result from the breakdown of various nitrogenous compounds„ 92 5.4.17 Effect of Carbon source on the change in the pH of the medium : It could be seen from Table 17 that good growth of all the strains was obtained with 5 carbon sources tested. When xylose \v^as used in the medium all tlie fast and slow growing strains gave acidic reaction. The final pll of the growth medium of the slow growing strains ranged between 5.41 to 6.44 and that of the fast growing strains between 4.71 to 5„18. With arabinose also all the strains produced acid in the medium (final pH of the growth medium 5.20 to 6„74 for slow growing strains and 4.65 to 4.d6 for fast growing strains. It could also be seen from the table that when glucose was used as a carbon source, the slow growing strains gave alkaline reaction and fast growing strains acidic reaction (final pH of the growth medium 7.49 to 8.26 for slow growing strains and 4.90 to 5.66 for fast growing strains). With rhamnose as a carbon source, fast growing strains showed decrease in the pH (final pH 5.32 to 5.97) but slow growing strain:: did not show much change in the pH of the medium (final pH 6o35 to 7„10)o When sodium gluconate was used both fast and slow growing strains gave alkaline reaction, (final pH 7„26 to 8„30 for slow growing strains and 7.62 to 7„96 for fast growing strains). Lange (1961) has shown that 85 strains of slow growing Table 17 : Growth of fast and slow growing strains with 5 different Carbon sources. Sr. Rhi zobial Xylose Arabinose Rhamnose Sodium Glucos No. strain Gluconate Slow growing: strains 1. Actm 0.56 0.61 0. 51 0.69 0. 73 (6.18) (6.26) (6.6 2) (7.86) (8.09) 2. Alpb 0.49 Oo 70 0. 61 0. 74 0.67 (6.51) (6.08) (6.86) (7.63) (7.88) 3. Atsv 0.92 0.80 0.75 0.68 0. 89 (5.87) (5.29) (6.70) (8.02) (7.75) 4. Bumk 0„87 0.76 0.57 0.54 0.61 (6.41) (6.74) (6.9 3) (7.38) (7.49) 5. Cram 0.65 0. 81 0.69 0.65 0.72 (6.44) (6.29) (6.35) (7.93) (7.85) 6. Cals 0. 71 0.58 0.50 0.70 0. 76 (5.83) (6.6 0) (6.71) (8.30) (8.21) 7. Clps 0. 85 0.72 0.66 0.68 0. 74 (5.41) (5.20) (7.05) (7.77) (7.87) 8. Ctrb 0.80 0.65 0.72 0.58 0.82 (6.37) (6.51) (6.91) (7.72) (8.19) 9. Dink 0.68 0.91 0.50 0. 73 0.86 (6.29) (6.45) (6.67) (7.89) (8.26) 10„ Incs 0.69 0.52 0.60 0.70 0.72 (6.05) (5.48) (7.10) (7.88) (7.95) llo Mump 0.5 7 0.48 0. 62 0. 71 0.82 (6.38) (6.19) (6.50) (8.11) (8.20) 12. Rhrt 0.70 0.79 0 .85 0.58 0.83 (6.18) (6.00) (6.51) (7.75) (8.12) 13„ ■Smsg 0.44 0.56 0. 50 0.62 0.68 (6.22) (6.41) (6.70) (7.26) (7.56) 14. Tptb 0.77 0.82 0.61 0.73 0.80 (5.75) (6.37) (6.41) (7.92) (7.88) Table 17 contd. Sr. Rhizobial Xylose Arabinose Rhamnose Sodium Glucose No. strain Gluconate Fast growing strains 1. Cakk 0.78 0.74 0.80 0.72 0.89 (5.18) (4.65) (5.97) (7.71) (5.66) 2. Esbp 0.90 0.85 0.92 0.76 0.80 (5.10) (4.86), (5.22) (7.62) (5.29) 3. Erst 0.95 0.82 0.75 0.65 0.93 (5.07) (4.69) (5.56) (7.96) (5.07) 4» Prey 1.05 0.85 0.91 0.69 Oo 87 ,4.91) (4.76) (5.12) (7.86) (5.26) 5. Tpfk 1. 01 0.90 0.85 0„68 0.82 (4.71) (4.86) (5.32) (7.80) (4.90) Growth is expressed as the absorbance value at 4 20 nm. Values in the brackets ‘^how the final p H o£ the medium. Initial p H of the medium - 6»8 . □J FAST GROWING ISOLATE Tpfk llllil SLOW GROWING ISOLATE CIps 8 2- 8-0- 7-8- 7-6- El ny 7-4- m m 7.;>- Ej ii: p j 7.0- El lii I ej 6.8 lllll El I'iJ Hi" p] PI 6.6- PJ 3: r'i Q- !'’‘i I. ! 5.4- m \M. P i 6.2i p I [ I p n 6.0- p n n 1:1 r-j 5.8 pi" 1 i'l 5-6- I l.u I 5.4- P ! ^ ri« 11 PI [^11 5-2- (‘'I brfi liiJ 5-0- IT! m r:i 4- [Hi Glucose Xylose Arabinose Rhamnose Sodium gluconate f ig ; i/,. e f f e c t of c a r bo n so u r c e on the cnange in the pH of the m ed iu m . 95 rhizobia from native legumes of Western Aurtralia produced acid on arabinose and xylose and acid or no change of pH on rhamnose. Padmanabhan (1978) showed that fast groisring rhizobia from tropical legumes produced alkali in the medium containing sodium gluconate. Stowers and Eaglesham (1984) also showed production of alkali by 2 fast growing soybean rhizobia with gluconate as a carbon source. These results compare well with the data obtained in the present investigation, It becomes evident from the results that production of acid or alkali in the medium is having limited importance and according to Date and Halliday (1979) the results can be altered by minor changes in the test medium. According to Parker et (1977) the production of acid or alkali depends on the composition of the medium, the presence of various carbohydrates and organic nitrogen compounds and the prefer ential requirement of the particular strain^ The data thus show that based on the cultural characteristics the 19 rhizobial strains could be divided in two groups viz 5 in the fast growing group and 14 in the slow growing group„ It is further seen that fast growing strains are distinctly different from the slow growing ones with respect to their biochemical and physiological characteristics. 9 6 5.5 Serological Characteristics of root nodule bacteria 5.5.1 Agglutination The results on the agglutination reactions of 19 antigens against 19 antisera are given in Table 18. It could be seen from the table that tlie agglutination titers of the antisera against their homologous antigens ranged between 3200 to 6400^ In the heterologous reactions it could be seen that antisera of 3 slow growing strains vi z„ Rhrt, Cals and Alpb and 1 fast growing strain Cakk did not react with any other antiserum indicating their strain specifically. Maximum number of reactions, on the other-hand were given by antiserum of a slow growing strain Bumk since it reacted with 5 different antigens in addition to its homologous strain. Antisera of 3 slow growing strains viz Mump, Clps and Tptb reacted with 4 differ ent antigenSo Amongst antisera against fast growing strains, antiserum of Prey reacted with 3 different antigens in addition to homologous antigen^ As regards the reactions of antigens, the slow growing strain Clps showed broad spectrum reactions as it reacted with 6 different antisera in addition to its homologous antiserum. Three slow growing strains viz» Rhrt, Cals and Alpb and 1 fast growing strain Cakk, on the other hand, reacted only with ^c3 t I I I I I t ! t I ] ] i 1 i t U ) I ] 9 I } 3 1 to 'O I 4-> 'J1 9 II 5 I 1 I B I 5 I ) 5 3 to Cs] vO CNJ I }- U < I < I t i I ( I i I I 1 I Cn] ! I } CU U-( t I I I I I i t I I t 0 I I I I t o o CO cu t 0 I I I e I I I I I I I vO 9 I I I I rt I I I 1 I I I t I I I 9 I I I I I II 1 I I ^0 CnJ o I 9 ■M » I I s 1 I s t » c-j r-, c- O j I I to lO O r j ro I i I I 9 I I I I I o o :3 O OJ e:; t-H ro > 'Si in I c-J r-J t^ t I I I ! t I I I I I 9 a 4-> o < u u 9irtO'OtOIII9l‘ll9IIIJI o on c CNlhOT-Hr--|tJ5ltO!ll I I I I II O o •M hO Cxir--'^'?:j-|l«J I I I I I 9 I I II r; o ■M •H :3 +->03 CiO CNj to I I I I I I I I I- I I I ! I I W) •H 4-> D 4-j r- i_n -r to I I I 3 .1 I I 5 I i 9 i i j i to U • b/) < < I—( C- c/^ tn > B bO 4-> CO >N 4-) rO ■M c: g P- u e +J 03 l-H a '-M U f/J U r-H D T—1 c 4-) B X c3 (—1 p. to cc5 < Q u l-H < CQ H U U CO (X U <; H o- w U susati'^v 98 their lioiiio 1 ogous anti serum. It becomes evident from the results tlint 11 serogroups can be postulated for 19 rhizobial strains cn tho basis of somatic cross-agglutination reactions. They are as follows : Serogroup Rhizobial strains A Actm, Dink, B Clps, I n c s , C Atsv, Bumk D A tsv, Bumk, E C r am, Smsg F Rhrt G Gals H Alpb I Tpfk, Prey J Erst, Esbp K Cakk It becomes evident from the results that some strains were common to more than one serogroup e„g. Clps was common to serogroups A and B, Atsv to serogroups B, C and D, Bumk to serogroups C and D and Erst to I and J. Rhizobial strains Tnore th^n serogroup were observed with serolo gical studies on R.trjfolii by Sidliu e_t (1 977), Gram rhizobia by Chnhal ^ (1978) and _R. j aponicum by Date and Decker (1965) . 99 Agglutination reactions are used by various workers to accomo-*.. date rliizobia into different scrogroups. Date and Decker (1965) postulated 15 serogroups. for 28 strains of R.japonicum, Skrdleta (1969] grouped 11 strains of R.j aponicum into 2 sero groups. Dadarv/al e_t ]U..(1974) postulated 27 serogroups for 44 isolates of rhizobia. Gidhu et al.(1977) postulated 6 serogroups for 9 strains of Rotrifolii. Chahal et al.(1978) grouped 17 isolates from Gram into 7 i:L;rogroups. Ikram and Brougliton (1980] arranged 62 strains of Psophocarpus tetragono- lobus into 25 serogrou.ps„ Fuquay ^t £1.(1 984] placed 9 strains of R^meliloti into 5 serogroups,, Hariharan and Rangarajan (1985) categorized 17 rhizobial strains from 5 different hosts of cowpea miscellany into 11 serogroups. In the present study also agglutination method was found useful in accomodating the rhizobial strains into different serogroups^ • •• 5.5.2 Immunddiffusion The results on the immunodiffusion tests with 19 antigens against 19 antisera are given in Table 19„ In the immunodiffusion studies weak or indistinct precipitin bands were formed close to the antigen well in the homologous reaction when untreated cell suspensions were used. Similar results were obtained in the serological studies xv'ith fast and slow growing lotus rhizobia by Pankhurst (1979), with \oo v ^ ' i e e z - \ c_;C3 CO I + a + rt 5-c h uj 00 + I—I (U >N*—* u CIh CO ^fX h-H C3 >- ci in DO'-' t/) CO > CJ 00 Xj ^ +-< H C/2 t-H ( X CO (/) I—t u T ^ - ^ 6 6 Z 101 R.meliloti, by Dudman (1964), with R. i aponi cum by Dudman (1971) and with rhizobia from tropical legumes by Padmanabhan (1978). According to them the^insoluble or indiffusible nature of these, antigens is due to their ex tr acel lul ar po ly sacchr i de whi ch remain firmly bound to the cells and may therefore act as a barrier to the diffusion of macromolecules. Heat treatment of cell sus pensions of both fast and slow growing strains intensified the slow diffusing precipitin band formed near the antigen well. These bands were heat stable and concave^ to the antigen well (fig„ 15). According to Pankhurst (1979) this slow diffusing precipitin band is formed by somatic antigens in intact lipopo- lysacchrides. It becomes evident from the results that the somatic band was strain specific^ The strain specificity of somatic antigens was shown by Vincent et al. (1973), Vincent (1974) and Pankhurst (1979). The somatic band of 3 slow grov/ing strains vi z Dink, Mump and Clps got split into 2-4 bands after 5-6 days of incubationo The splitting up of somatic bands was observed with R.meliloti by Dudman (1964) and with rhizobia from Arachi s- species by Dadarwal et al. (1974) „ It could further be seen that the titers of tlie antiscra with the heated cell suspension required to form precipitin bands ranged between 1/4 to l/64„ In the immunodiffusion studies with R.meliloti Sinha and Peterson (1980) found the antiserum titers in the above range„ Fig.15: Slow diffusing somatic band. 102 Ultrasonic disruption i o£ the cells led to appearance of additional bands. These bands were formed either close to antiserum well or midway between antigens and antiserum wells Appearance of these fast diffusing, straight line bands with sonicated cell preparations v;as also sliown by Dudman (1964, 1971) and Sinha and Peterson (1980). According to Pankhursf (1979) these internal or subsurface antigens have cytoplasmic origin. It could be seen from tlie fig. 16 that 9 isolates vi z „ Actm, Alpb, Cakk, Cals, Erst, Incs, Smgs, Tptb and Tptk showed 2 precipitin bands, 8 isolates viz„ Atsv, Bumk, Clps, Ctrb, Cram, Esbp, Prey, and Rhrt showed 3 bands and 2 isolates viz. Dink, and Mump showed 4 bandso It could further be seen that 3 slow growing strains viz Alpb, Cram, and Rhrt and i fast growing strain Cakk formed precipitin bands only with their homologous reactions indicating strain specificity of the antiserao None of the antigens tested was able to cross react with all the different antisera. At best, the slow growing strain Clps showed broad spectrum reactions as it formed precipitin bands with antisera against 8 slow growing strains„ Antiserum against this strain reacted witli 7 different antigens. Antisera against 2 slow growing strains viz. Smsg and Cals and Esbp reacted with only one antigen in addition to their corresponding homologous strain. © Prey .0 . 0 9 CaUk Tpfk Alpb i & Esbp Cram Ctrb • e 0 Erst Cals CIps e 0 • Bumk Rhrt Dink \.n y ' 0 © ® Astv Tptb Mump 0 e Incs Smsg Actm ®-AntisGrum well O-Antigen well fig: 16 PATTERN OF PRECIPITIN BANDS WITH HOMOLOGUS ANTISERA 103 In the cross immunodiffusion reactions only internal bands were formed. The results are thus in agreement with those of Vincent and Humphrey (1 970), Vincent et n_l. (1973) and Pankhurst (1979) v.'ho sliowed that the internal antigens are widely shared amongst the rliizobia, 5.5.3 Comparison of agglutination and immunodiffusion reactions - U’lien the results of agglutination and immunodiffusion reactions are compared it becoines evident that rnizobial strains belon ging to particular group based on agglutination reactions did not necessarily form precipitin bands in immunodiffusion. e.g. Clps antigen of group A did not form precipitin band against the Actm and Dink antisera from the same group, Ctrb antigen from group D did not form precipitin band against Bumk anti- serum of same group, Prey antigen of group I did not form pre cipitin band against antiserum of Tpfk from the same group^ Production of precipitin bands in immunodiffusion reactions also was not always correlated with agglutination reactions. Thus e.g. Clps and Incs antigens forming precipitin bands with the antiserum of Ctrb from group D were accomodated in group B based on agglutination reactions, Dink antigen forming precipitin band with antiserum of Smsg from group E was accomo dated in group A on the basis of agglutination reactions. 104 These results are in agreement with those of Skrdleta (1969) and Ikram and Broughton (1980) who observed that the production o£ bands in immunodiffusion was not always correlated with agglutinating properties and isolates v;ith high agglutination titers occassionally gave negative gel diffusion reactions^ It becomes evident from the results that the fast and slow growing rhizobial strains are serologically distinct as no cross reactions between these 2 groups were observed in agglu tination as well as immunodiffusion reactions,, The absence of serological relationship between fast and slow growing rhizobia was previously shown by Vincent and Humphrey (1970) and Ikram and Broughton (1980)» Vincent ^ al. (1973) found at least one common antigen in 23 slow growing rhizobia tested against anti sera from 3 soybean strains, but 51 fast growing rhizobia failed to give any reaction with them. In the studies with 62 fast and 76 slow growing strains of Lotus rhizobia Pankhurst (1979) showed that there was no sharing of somatic or internal anti gens between fast and slow growing rhizobia. Recently, with the help of Enzyme Linked immunosorbent assay (ELISA) technique, Ahmed et al (1981) re-examined the possibility of some related ness between fast and slow growing cowpea rhizobia and confirmed the unrelatedness of these two groups. These results, thus support the data obtained in the present investigation^ 105 It could also be seen from the results that the isolates from one genus of leguminous plants were not serologically similar in agglutination as well as immunodiffusion reactions. The results are in agreement with those of Padmanabhan (1978) who in the study of rhizobia from tropical legumes noted lack of ser^ological similarities amongst the isolates of the different genera of legumes which indicated heterogeneity in the heat stable somatic agglutinating and soluble antigens. The data thus show that fast growing rhizobial strains are serologically distinct from slow growing strains and the 19 strains formed 11 serogroups on the basis of cross agglutination tests. 106 5.6 Symbiotic characteristics of root nod'jle bacteria 5.6.1 Nodulation tests on Siratro The results on the time required for initiation of nodules on siratro (Macropti 1 ium atropurpureum Urb.] by the rhizobial strains in Gibson's partly enclosed assembly and the number of nodules formed are given in Table 20. It could be seen from the table that the time required for initiaL^on of nodules on the roots ranged between 8 to 14 days. The fast growing strain Erst required 8 days for initia tion of nodules while the slow growing strain Smsg required 14 dayso Not much difference was, however, found in the time required for formation of nodules between fast and slow growing rhizobia ^ It could also be seen from the table that all the rhizobial strains could nodulate siratroplants. Nodules were seen on both tap and lateral roots (Fig. 17). The total number of nodules formed ranged between 8 to 19. The maximum nodules were formed by slow growing strains Cram and Smsg. Siratro is usually used as a test plant for cowpea cross inoculation group (Erockwell 1981). Padmanabhan (1978) used siratro in the nodulation test to confirm isolates from tro pical legumes as rhizobia„ Gonzalez Cu (1985) found that 40 Table 20 : Nodulation tests on Siratro (Macroptilium atropurpureum) Urb Sr. Rhizobial Time required Number of Nodules NOo strain for initiation ^ ^ ^ n r j On tap root On lateral Total of nodules(Days) ^ roots 1. Actm 12 5 4 9 2. Alpb 9 3 5 8 3. Atsv 9 5 10 15 4. Bumk 12 8 9 17 5. Cram 10 6 13 19 6. Cals 11 3 6 9 7. Clps 9 6 10 16 8. Ctrb 9 6 4 10 9. Dink 13 4 8 12 10. Incs 12 4 10 14 11. Mump 10 8 9 17 12. Rhrt 10 4 7 11 13. Smsg 14 7 12 19 14. Tptb 9 8 9 17 15. Cakk 12 4 6 10 16. Esbp 10 7 11 18 17. Erst 8 8 10 18 18. Prey 10 7 6 13 19. Tpfk 9 3 6 9 20. Control 0 0 0 Fig.17: Nodulation of Siratro in Gibson's asseinbly. 108 strains of Rhizobium spp. isolated from 14 different species of tropical legumes nodulated siratro. All the 5 fast growing rhizobial strains in this study were able to nodulate siratro plants. The results are in agreement with those of Lawrie (1983) who found that fast growing iso lates from Acacia longifolia and Kennedia prostrata could nodulate siratro. Fast growing soybean rhizobia (Rhizobium fredii) were also shown to nodulate siratro plants (Scholia and Elkan 1984). According to Rangarajan and Balaji (1985) use of siratro as a test host for certain tree legume rhizobia is invalid, because they found that two fast growing rhizobial strains from Acacia dealbata and A.melanoxylon did not nodulate siratro although they were able to nodulate their homologous hosts and cowpea plants. All the isolates obtained in the present study, however, could nodulate this plant. 5.6.2 Modulation test on Cowpea The results on the effect of inoculation with rhizobial strains under study on the growth of cowpea (Vigna unguiculata L.lValp.) plants in Leonard jars are given in Table 21. It could be seen from the table that all the rhizobial strains were able to nodulate cowpea plants. The number of nodules formed ranged between 14 to 48/plant on tap root and 16 to 49/ plant on side roots. Maximum number of nodules were formed by the slow growing strain Atsv. Pry weight of nodules ranged Fig.18: Effect of inoculation with the strain Mump on the growth of cov;pea plants in Leonard jars. a i-* u r-- o cn o rt cn o o to r- uO C7» rj L/> f-> CO in <71 vO rH CO rf o cn 'jD rvj LO o r-- OO ro t—( rH o a> o cn to CO LO 1-H LO 00 o rj lO rj r«. o r- vO o OO •M cr» vO to t-- cn cr» O Ln rH T-l rj (NJ to to o LO •M XI o «sj- rg to CO CO o LO C7> LO CM cn »—1 CM \0 T3- Oi o r'j C fcO-H «•••9•••••••••• 0 •«••• ci; <0 -rH +j r4 oo VO LO LO r-. C-- cn LO VO C7l CM cr> 00 CTi rH O 03 U CJ •H > 00 (M rH to LO r-i ro rH > O 00 LO CM cn t-H O O r-«. o o ^ LO vO CM CM Oi VO o r-- rH LO C3 :0 to o LO to rH O o o •H U rt DO *-» o cn Oi fsl LO o m \o OO rH lO 00 LO vO CM to o o C •H C LO CM vO vO LO o m rH r-4 ! - LO LO \o o\ LO vO rH r- *j vO W) O o3 cr> to rj CM CO 00 o to vO ■jO o to CM rH CO O CM CS CM • r* ^ rH {JO o CO o rH Ch r-- r-. Ln «rr ■jO LO to r-- to rH r>. to O Tj- o. 0••• ••••■•■ 0 ■• • • •• • •• • ^ »+-i OPU CM o to rj CM rH CM CM CM to rH (N) to o to to to rH o CM Q o G O 4-» Pu > o u OOrH LO rH to CNJ (M CT» r-- vO vO o CO LO CM 00 o LO c^ 00 CM C • rH 3 o CTl 1-H t'' to LO o LO rH LO o rH rH o o rH LO SO \o O 0) T3 CM CM \0 rH to cn r>- cn \o o lO CM LO CM O cn rs) ♦ to ^ o 4-> OO to rH CM CM CM rH CM rH tH CM to rH CM CM rH CM CM O'! rH CM to c c •••••••••••••••••••• c >s«4-l rHcd o O o O o O o o o O o o O o O o O o O o n3 OOP. Vh bO o iM 4-) CO •r< O to - a rH CO P i c 4 H P . r t O V> •M 0> V) 4> rH a X i 3 Mh S a , o OJ tH 3 o o o to T-i ^ Mh H D i o >N 0) bO'M 03 B XI > B I/) to X to bO X a X o rH U 4-1 p, to B a rH a u +-> X ■ t/) u Mh c rH CM +J U r-H •M X5 i-i cd rH 4-J C EP- 03 to Jh p. o 00 < < < CQ u U u Q 7-. ‘ CO H ■. O w w CU u > rt 5- O H in 2; 110 between 0.1262 g to 0.3205 g/plant. Dry weight of the plant top was used as acriterion to test the symbiotic effectiveness of the isolates. Since highly significant congelations were obtained between shoot dry weight and total nitrogen content of the plant (Ramaswaniy £t a_l-197‘;^, Padmanal)han 1 978, Kremer and Peterson 1 93 2)., It could be seen from the table that one fast growing isolate Cakk and one slow growing isolate Alpb ineffectively nodulated cowpea plants as there was no significant increase in the dry weight of plant tops over uninoculated control. Remaining 17 isolates and a homologous strain of cowpea (VKIO) showed effective nodulation. These results are in agreement with those of Srivastava and Tewa.'i (1981) Ramachandran £t (1980), and Lim and Ng (1978) who have shown effective nodulation of cowpea plants by rhizobia from wild legumes. The effective nodulation of cowpea plants byfast growing rhizobia was shown by Trinick (1968, 1980a) and Broughton et al (1984). Stowers and Eaglesham (1984) showed that fast growing soybean rhizobia nodulated pigeon pea, Mungbean and cowpea to varying d.egrees. Cowpea showed the best response to inocula tion, both in terms of nodule number and dry weight of plant ■. top which was significantly higher than uninoculated control. It could further be seen that tlie plant weight ratio compared to uninoculated control ranged from 2„299S to 9.5744 with the Ill slow growing rhizobia and from 2.1428 to 9.4371 with fast growing rliizobia. It thus becomes evident that both fast and slow growing isolates are equally effective. Similar results were obtained with fast and slow g-^owing rhizobia from Green gram by Dadarwal et (1979) and from Caj anus caj an and Cicer arietinum by Bromfield and Kumar Rao (1983). When compared with'the homologous strain of cowpea viz. VKIO, it was observed that 2 slow growing strains viz. Mump and Tptb and 2 fast growing strains vi z. Esbp and Prey gave significantly higher dry weights of plant tops. The symbiotic effectiveness of the isolates as compared to homologous strain ranged betwewn 31.67 to 131.891 with slow growing rhizobia and 29^52 to 130.01 with fast growing rhizobia. Maximum symbiotic effectiveness was shown by Mump amongst slow growing isolates and by Prey amongst fast growing ones. Basak Goyal (1980)found some rhizobial strains from tree legumes superior to specific rhizobia of Green gram on the basis of their performance in the pot culture experiment. Yanasugondha et al. (1977) also found some strains from wild legumes effective on Mungbean. These results compare well with the results obtained with cowpea in the present investigation. I'/hen symbiotic efficiency was determined by the formula of Herridge Roughley (1975) it ranged from 2,94 to 13.75 with maximum efficiency shown by a fast growing strain Erst. In the studies with C3756 culture Herridgeand Roughley (1975) found 112 symbiotic efficiency in the range of 5 to 12 witli axillaris and 4 to 12 with siratro. It, thus becomes evident from the results that root nodule bacteria from wild legumes can be effectively used to increase yield of cultivated plants like co\,pea. Grant and Purdom (1977) showed that bottle jar tests give reliable estimates of tlie relative efficiency of strains and the data can be used with confidence to select effective strains for commercial use. 5.6.3 Acetylene reduction l:L;st The data on the acetylene reduction activities of the cowpea (Vigna unguiculata L.V/alp.] plants inoculatcd with fast and slow growing rhizobial strains is given in Table 22. It could be seen from the table that the total nitrogenase activity of the plants inoculated v/ith slow growing strains ranged between 105--1 to 5441 n moles of ethylene produced/plant/ hour and that the maximum activity was seen with the plants inoculated with the strain Aclm. In case of plants inoculated with fast growing strains the activity ranged between 978 to 11"^68 n moles of ethylene produced/plant/hour and the maximum activity was shown by plants inoculated with the strain Esbp. Zalviotowicz and Foclit (1981) found tliat acctylcne reduction activities of cowpea ]:>lants inoculatcd v.ith 1 fast and 3 slow grov.'i.ng rhizobia were in the range of (>100 to 13000 n moles of Table 22 : Nitrogenase (acetylene reduction) activiti cowpea plants inoculated with different rh strains. Sr„ Nodules Total nitrogenase Specific nit No. formed by activity activity the strain n mol/plant/hour n mol/g dry ’ of nodule/hoi 1. Actm 5441 17164 2o Alpb 1205 6409 3. Atsv 3332 18213 4. Bumk 5421 18628 5. Cram 1129 8240 6. Cals 4810 16996 7. Clps 4913 20820 8. Ctrb 1807 8288 9. Dink 3463 18036 10. Incs 2259 14031 11. Mump 4995 21718 12. Rhrt 1054 8046 13. Smsg 2862 11540 14. Tptb 5421 18628 15. Cakk 978 7951 16. Esbp 11068 29514 17. Pecy 8583 24177 18. Erst 7905 33496 19. Tpfk 2067 8471 2280 10604 20. VKlo o 3N31AH13 S3 70W tv CD O o CD O O CD O o O O o CD CD CD to CD C 3 I n CD r n f N _J ____ _J ____ —U_ X 1— -k-^'k'k'k'k'k'k'k'k'k'k'k'k'k-ki dujn^^j ?@000SS!!SK:SK!3K:t in z ADJd < cn k'k'k'k'k'k'k'k'k'k'k'k -k-k-k k^‘r'k■k'k ■k'k-k'k'kic'k'k'k'k'k'k'i dqs3 I— 0^ qidi _J 300550S@S0ii:Ei0SS0000000g50SS!g?0Sg5&5S0g50g5Si0Si < m >-• tsj3 1— o N > t— AJSV X u CE sd|3 < X JSi0g5SSSiSgi0SigJS:S UJ U1 m Q < z ; > - 'k'k'k-k-; K-k'kk^'k'k'k'k'kic'kTk'k'k'k'k qjJ3 < L l ) G0C0SSS0i50SS0SK0g@000S0SgiS:Si5sS450 LU CD f e ''!'<★★ iV ; — f - ’ — I— — r— -T' L n C 3 CD t o o r n f N c S u _ 1HD13A\ AdG lOOHS 114 ethylene produced/plant/hour. These results compare well with the results obtained in the present investigation. It could also be seen irom the table that the specific nitrogenase activity of the plants inoculated with slow grow ing rhizobial strains ranged between 6409 to 21718 n moles of etliylene produced/g dry weight of nodules/hour. The maximum activity was shown by plants inoculated with Mump„ In case of plants inoculated with fast growing strains the activity ranged between 7951 ta'334-96 n moles of ethylene produced/g dry weight of nodules/hour. Maximum activity was shown by plants inoculated with the strain Erst. Dart and Day (1971) showed nitrogenase activity of cowpea plants in the range of 8o9 to 43.5 yU molo of ethylene/g dry weight of nodule/hour. Bal et al (1982) showed tliat acetylene reduction activity of polebean ranged between 0.30 to 15.9 mol of ethylene/g weight of nodule/hour. The values of acetylene reduction activity in the range of 12000 to 36000 n mol C^H^/g weight of nodule/ hour are often obtained for effectively nodulated legumes. (Hardy et aj.-1973, Trinick e_t ^ . 1976, Trinick 1980b)., These data compare well with the results obtained in the present investigation. It becomes evident from the results that plants inoculated with 9 slow growing strains viz<, Actm, Bumk, Cals, Clps, Dink, Incs, Mump, Smsg and Tptb and 3 fast growing strains viz. Esbp, Erst and Prey showed higher nitrogenese activity than the plants 115 inoculated with homologous strain of cowpea viz. VKIO. - , 5.6.4 Effect of inoculation on the growth of cowpea plants in unsterilized soil The results of the effect on the growth of cowpea plants through the inoculation with 8 rhizobial strains and nodule occupancy ' of these strains in unsterilized soil are given in Table 23, It could be seen from the Table that the number of nodules/ plant ranged between 56 to 96. The maximum nodules were formed on plants inoculated with fast growing strain F.sbp. Control plants grown without application of any inoculum also showed good number of nodules indicating thereby the presence of native rhizobia in the soil. Fresh weight of nodules ranged between 0.9256 to 1.8557 g/plant. Significant increase in the dry weight of plant top over un inoculated control was shown by 3 slow growing strains viz., Actm, Dink and Mump and 3 fast growing strains viz., Erst, Esbp and Prey. Maximum increase in the dry weight was shown by the slow growing strain Mump. The plant dry weight ratio, as compared to uninoculated control ranged from 1.0411 to 2.3564. As regards the competition with the indigenous rhizobia in the soil to form nodules on the plants, the competitive ability of Table 23 : Effectivity of rhizobial strains on cowpea (Vigna unguiculata L.Walp) in unsterilized soil. Sr. Rhizobial No. of Fresh weight Dry weight Plant Nodule No. strain nodules/ of nodules/ of plant weight occupancy plant plant top ratio % g g Slow growing strains 1. Ctrb 66 1.8084 3.8766 1.2331 33.3 2. Dink 78 1.7576 5.0555* 1.6081 66.7 3. Incs 68 1.3613 3.2730 1.0411 39. 2 4, Mump 82 1 . 7954 7.4077* 2.3 5 64 65.8 5. Tptb 84 1„ 2956 4.1285* 1.3133 44.1 Fast growing strains A 6. Erst 72 1.6219 5.8358 1.8 564 44.2 7. Esbp 96 1.8357 6.4005* 2.0360 61.7 8. Prey 75 1.8005 5.1886* 1.6505 52.5 9. Control 56 0.9256 3.1436 0 C.D. at 5% 0.9828 * Treatment showing significant difference over uninoculated control. Vo ADNVdDDDO 31f)00N C5 O >- CO Lo vr Q o r s ) I CD rs o ^ dOl INVld J0IHDI3M Ada U- 117 the strains ranged between 33„3 to 66„7%. Maximum nodule occupancy was shown by the slow growing strain Dink. It also becomes evident from the results that the rhizobial strains showing higher increase in the dry weight of plant top showed good nodule occupancy e.g„ Mump was having 65.8"o and Esbp 61.7'^. nodule occupancyo The results are in agreement with those of Padmanabhan (1978) who showed that dry weight of plant tops corelated with the increase in the number of nodules formed by more competitive strain. In the competition studies on ^hIng bean, Dadarwal e_t al. (1979) found that slow growing strain formed more number of nodules/ plant but in competitiveness fast growing strain was superior. Studies on the fast growing Gram rhizobia by Chahal et _al (1978) showed the competitive ability of these rhizobia in the range of 16 to 851. Competition studies with fast and slow growing soyabean rhizobia by Dowdle and Bohlool (1985) showed that the fast growing rhizobia were highly competitive and they formed 86% of the nodules. According to Trinick ^ aJ. (1983) at lower temperature fast growing rhizobia were superior competitors for nodule sites on Vigna unguiculata. In the present study however this kind of clear difference was not observed between fast and slow growing rhizobia. The results are in agreement with those of Thomas e_t aj.,(1 985) who showed that faster growth rate of soybean rhizobia on laboratory media 118 did not result in superior competitiveness in the rhizosphere They have further stated’ that faster growth rate in laboratory media is having little advantage because Bohlool et al, (1984) ' have shown that fast and slow growing Rhizobium species have the same doubling time in the rhizosphere^ Zablotowicz and Focht (1981) also shov/ed that there was no relation between growth rate in vitro and the capability of rhizobia to compete for nodule forming sites. The data thus show that 3 fast and 3 slow growing strains showed effective nitrogen fixation and competitiveness in the unsterilized soil. So they I.ave got a potential to enhance the yield of cultivated pulse legumes like cowpea.