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August 2011 ISSN 0257 -2605

Vol. 21 No. 1 /2

Indexed by AGRIS

Analyses o f D eterm inan ts of Farm Input (Chem ical Fertilize r)

Comm ercial O rien tation o f Sm allho lder Farm Househo lds in

D rough t-prone A reas o f the C entral R ift V alley of E thiopia

A dam B ekele, B elay K JsSJ and Be la ineh Legesse

Rura l Poverty and Incom e D istribution : The Case of

K alu D istric t, Sou th W ollo , E th iop ia

Fisseha legeye , Daw it A lem u and Ranjan S . KJrippa i 16

Genetic V ariability and A ssociation am ong C haracters in N avy B ean(P ha se o/u s v ulg aris /.) Germplasm

Getachew Erana, Sentayehu A lamerew and Setegn Gebeyehu 26

Charac terization o f E th iop ian Ch ickpea ( Cic er a rie tin um L.) Germplasm

Accessions fo r R esponse to In festa tion by A dzuk i B ean B eetle

(C a lfo sobru chu s c hin en sis L.) I. Perfo rm ance E valuation

G em echu Keneni, Endashaw Bekele, Em ana G etu, M uham mad lmtisz,

K iffe D agn e a nd Fassif A ssefa 41

Characterization of E thiopian Ch ickpea (C ic er a rie tin um L.) Germplasrn

A ccessions fo r R esponse to In festa tion by A dzuk i B ean Beetle

(C a lfo sob ru chus c hin ens is L.) II. Pheno typ ic D iversity

Gemechu Kenenf, Endashaw Bekele, Emana Getu, Muhamm ad lm tiaz,

K ifle D agn e a nd Fassif A sefa 66

Effect o f P lan ting M onth and Variety on Sugarcane Borers in E thiopia

Leul M eng istu , F irehun Y irefu and Y ohannes lekarias 84

Effect of Seed S ize , P lan ting D ensity and Phosphate Fertilizer on Y ield

and Y ield com ponents o f F aba B ean in the cen tra l H ighlands o f E th iop ia

Getscne« Agegnehu and Missa De tn is sie



Characterization of Root Nodule Bacteria, Rhizosphere Bacteria and

AMF from E ry th rin a b ru ce i and their Impact On the Host's Growth

and Production

S hasho M egersa and Fasil A se ffa rO B

Short C omm unic atio n

Occurrence and Distribution of Tomato Bacterial Wilt

( Ra ls to n ia s o la nac earum ) in Major Tomato C , iwing

Areas of Ethiopia

G etachew A yana , C hem eda F in in sa , S e id A hm ed and K ers tin W yd ra 119

Economic Evaluation of Castor Production for Export or

Biodiesel under Dry Land Condition

Y itayal Abebe , G etine t A lem aw and Dereje M ersha 13 1

Registration of Crop Varieties 136



Ethiop. J. A gric . S ci. 2 1 :1 08·1 18 (20 11 )

Characterization of Root Nodule Bacteria, Rhizosphere

Bacteria and AMF from Erythr;na bruce; and their

Impact On the Host's Growth and ProductionShasho Megersa! and Fasil Aseff'a-

lF orestn j R esearch Cen te r , E th iopian In stitu te of Agriculturn l R esea rch , P. 0. Box 2322,

Addis Abnbn, Eth iopia . Ennai]: s/11751z(l]I/I 'g( 'r~a@ lf i1llOo.C[111l

2Applied M icrobio logy, BIO logy Departnien t, Addis Ababn Uniuersi t i] , P.o. Box 1176, Addis

Ababa, Ethiopia. Enntn l : nsc[a[nsI12003'i j;1(I1Ii(JO.C(l1ll

Abstract

Iso la tio n o f m icro orga nism s a sso cia te d 71 'it71Erythrina brucei was conduc ted to a sse ss

how these Iso la tes a ffec t th e produciioi i i j o f th is tree spenes . Rhizosphere hacter ia!

c olo nie s (h ath C - p e and C+p'), fa st- a nd SI0 1I'-grO ll 'ln g ro ot n od ule b ac te ria a nd a rb usc ula r

m yco rrh iza l fungi spo re groups (Gigaspora, Glomus and Acaulospora) iocre

cha rac ter ized from so ils co llec ted unde r E. h ruce i tree s. Bo tli e ifrc /iuc and th e le ss e ffe c liuc

rh izoh ia l iso lutes w ere id en tifie d . A ll iso la II's w ere [ound to be e ffec tipc a lld th e d ij(£ 're IlC l'

in e ffec tiven ess o f th e iso la te s ta r ied a s compared to tlu: niirogen-Ieriilizcd c on tro l. III

b io lo gica l e ffe ctiven ess , in dun dua t ro ot n od ule b ac ter ia l iso la tes S /1 01 1'1 ,do aria iio ns up to45% in sh oo t d ry w eigh t h etll'CC Il tile h igh ly e ffcc ti7 'C a nd lea st c (fcd iue gro ups . S im ila rly ,

se lec tio e dua l and m ultiple Inocula tion s a lso showed oa riutious 011 th e d iffe re nt grouth

parameters . D ua l ino cula tion o f Bradyrhizobium ERS07 and P seudomona s P53F l

resul ted ill to tile h ighe st shoo t d ry weigh t fo llow ed hy multiple ino cula tion s con sis tillg o f

ER S07, PS3F 1, Gigaspora and BS3X . These trea tm en ts shouicd s ig llific a nt d iffe re n ce

bo th from dua l ino cula lion o f Bradyrhizobium plus AM F and single ino cula tion

Bradyrhizobium iso la tes . There is a 20 -45% d iffe ren ce In shoo t d ry io e igh ! h e tluee ll tlu:

ino cula ted plan ts and th e uri-ino cula ted plan ts . In gen era l, tile t oork s ho lP e d p os itiv e

respon se o f multiple ino cula tion o f th e ho s t b y rh izo spliere m icroo rgan ism s to induce

more nodula tion and shoa l dn ) m a iler a ccumula tion . T ile s tudy dem ons tra ted tha t

se lec ted rh izo sphere m icroo rgan ism s can be man ipula ted to rea lize th e full po ten tia l o f

th e se grow ing tree spec ies fo r d iffe ren t PUipOSfS.

Keywords: dual/ multiple inoculation, nodulation, mycorrhization, 111 u ltipurpose tree

Introduction

En jtllrllll7 b ruce i Schweinf. is an endemic leguminous tree that grow in open places of

disturbed forests or woodlands in different agro-ecological zones mainly in moist

Woina Dega of Ethiopia (Thulin, 1989). It is a fast growing multi-purpose tree used

for fuel wood, feed, constructional material, live fence, bee forage and it is used as an

important component in agroforestry practices in the country (Demel, ] 99-+).



Characterization of microorgcuusmsfrom E. brucei 11091

E. brucei is found to fix atmospheric nitrogen and improve soil fertility in an

endosymbiotic relationship with root nodule bacteria. Fassil (1993) evaluated the

nodulation pattern and nitrogen fixing capability of the endemic E. b ruce i using

acetylene reduction and found that it is among the best nitrogen fixing trees tested

and can initiate root nodules when treated with bacterial strains of the genus

Bmd!J r li izoh ium.

Nitrogen fixation in legumes is limited by many factors The most important

factors are deficiency of nutrients, mainly phosphorus, and lack or inefficiency of the

specific root nodule bacteria in the soil where the legume tree is growing. Apart from

the presence of specific and effective rhizobium, rhizosphere microorganisms that are

known as plant growth promoting microorganisms (PGPM), species like

P se ud on un uts , B ac illus , A spe rgilll ls , Peniciluuu, Arbuscular mycorrhizal fungi (AMF) and

others are also vital to the growth and establishment of plants (Duineveld et al.. 1998).

A vailable information showed that some root nodule bacteria could also behave asPGPM when they grow as free living microorganisms in the rhizosphere (Vinther and

Jensen, 2000).

AMF is another important PGPM and is known to stabilize soil, maintain plant

diversity and increase root absorption area. It is well known that AMF stimulate the

growth and nitrogen fixation of many tropical trees and shrubs by enhancing the

uptake of nutrient elements such as phosphorus and by increasing the resistance of

plants to drought and root pathogens (Michelsen and Rosendahl, 1990). Different

studies have established that effective symbiotic activity is also greater in legumes

that have mycorrhizal association than legumes with no mycorrhizal association

(Yonas and Fassil. 2007).

Michelsen (1993) showed that application of effective AMF from pure pot culture

is regarded as a way of increasing plant productivity on Acacia plants. The interaction

of the PGPM and plants also improves the plant nutrition and enhances the defense

system of the host against soil-borne parasites (Linderman, 1988). Generally, the

interactions of plant roots and rhizosphere microorganisms are based largely on

interactive modification of the soil environment. These include release of organic

chemicals to the soil by the plant roots, microbial production of growth factors, and

microbially mediation of availability of nutrients (Atlas and Bartha, 1993).

Although E. b ruce i is an important plant in agroforestry system, adequate study

has not been conducted on the role of rhizosphere microorganisms to enhance

growth and productivity in its habitat. In the present work, attempt has been made

to evaluate the effect of the associated and free living microbes on the growth and

nitrogen fixation of E . b ruc ei.

Materials and Methods

Nodule induction and isolation of root nodule bacteria

The soil sample used for the isolation of root nodule bacteria was collected from the

rhizosphere of E. bruce! trees grown in the campus of the science faculty, Addis Ababa



! 1101

Univc-rs itv Th,'st' <oi l ,'II~lples well' used to produce :';l'l'dlll':~" ( > I i.brucci, form

whic h rout nodules \\l'r\' c..ollected.

Seed:, were colll','I, Ii t rnrn F . b ruce, Irl"':' III the campus l he:«: C,l'l'lb were surface-

ster il ized with 95', l'~II"IIOI and 0.2"" nu-rcuric chloride solut iou-. I(Jr Iltll'l' mmuus

and washed several tim: s with sterile \\ ater (Vinccnt,1970). llt tlu-se seeds. three

seeds were planted in pots field with the soil obt, ined Irorn b"IIl'<l th l . h ruc ( 'I trees.These pots were h.l'~,t;n ,I greenhouse for 90 days at 12/ 12h pllOlojll'1 iud, .md 25/ 15"C

average day and night temperature until the seeds germinate ,l'lLl gr(l\\·.

ThE' sl'edlings gro\\'n in the pots were uprooted to lo!ll'd till' runt nodules

deve lope-d 011 th e routs. These nodules were surface steri l izc-d .uid thl'll macera t ed UP

gldSSslid" to form" ,'>li'>Pl'IISion(Vincent et ell ]970). The SU.., pl'Il'>I,lll.'>\\erl' inoculc1ll'd

011the Ye-ast Fxtr.irt :dcllllliiol ,\gar (YEi\IA) (Oxoid) and incubd;,:d at 28uC fur SL'Vl'n

davs In order to g:'ow t l : c - bacteria involved in the root nodule Iurn.at ion (Parkinson c - t

.il.. 1971). Felch ut 11lL'r," ult.uit bacterial colony was repeatedly suo ,'ulturL'cI, purified

.uiu pn",l'rvl'd cll-+"C for' urthcr characterization.

Authentication and eirectlveness of root nodule bacteria

TIll'~;l' i"(ll.ll,',, \\1'[, [,)( ul.iu«! \<11 YF\l bro th and incuba te -d Itl[ ~,\('Il dcl'·"'. l lu-

Ill)Cl'li.l "l'lI ::: 'll\ 11'1 lh.: broth vvcrc cullect('d and used to inorul.u« 111<'l.int-, gr()\\n

in the pot III If'dl'l II' .iu t lu-nticatc i t the isolates an' root nodu k- bclckricl or nut. This

\ld" tl'~tl'd on i./".ILt, ~l'l'dling grown on sand culture medium (\'lI1cl:l1tI970) Ior

th.s pL,rpl) c', .,uII,lLl' - tv rr lizo d L IJrII(ci seeds were a l lowed to gl'1111incltl' and gro\\l1

in f'ot'> fillll' \\,111,,1LId,\\ Islted sand culture medium (Vincent CI'f;\)) After one: \\'L'C", ) 1 ' ,;rL'" lit (,clell ""(',111,.;" \\,lS inoculated with 108 ccllsy m l obtclilll'd .ro rn YEr<.1broth .

l'I,lIlts (11' Illl' c onl r u ! I'"I~ w cr« fe rtilized w ith 0 .3°" KN03 (on . :« d \\L'L''') but not

inor u l.r} '.\ ..h b,llll' I,d isolates. The "L'cdlings in the sand (1IIIlIIl' \\'l'r(' feltilizl'd

with J<="l..,'·:, N-II"l' uu-chu m twice a \H'L'k for 60 days.

Alte-r ()() d,h'" sl'~dlillgS were uprooted an d evaluated 1 . " 1 ' Iltl' prL'sl'ncp of

nodule's. 'I h : apf"'clldllll of plants, nodule number and shoot \\1 :~~ltt uf ,>eedlings

were used to sclert sf..'l'dlinl:;s for the isolation of dfecti\l' ~I.ilin'> for turthor

cxperinu-ntation. FIII'l , 11('55 in nitrogen fixation of the isol.itc-, \1' [', ('valuc1ll'd Llsi'll;

the u-chniquc« f) f I ).tk l'l .r]. (IL)lj,o,).

Eff<>ctin'nes" (c.;h,lIll I ry weigh t -of inocula ted planls ,' c.;hoot dr\ \\eight ot

nitrogl'n-Il'rtilizl'd pl'lIl!'» 100

Characterization of root nodule bacteria

The ,wthentiGlt!'d ,(','l (,dltle bacteria were characterized ,)11 t h. h.1"i" llf cultural

and morpholog rc.il i h.u.u [('IS. The cultural characteristics of 11lL'I'>,",ll('s );ruI\'ing Oil

YEMA meduu n. fill IU,illl» the colonv morphology. color, .uu l ',i/, '.\l'rt' rl'cLlrLiL'd .md

evaluate-d (:\IiI11l'd 'I "I 1<11'-+).solates were also Cram-stained III L'L1Sl'rn' lilt' ~I/.e and

shape of the cells i I'l' .iluhtv of root nodule bacterial isolates til 11rudll"'~ c1Lidor Ll,l..,('

on U'I R-Yh\IA gr(l\\ II. IlWdlUI11was tested acrord ing to Jordan (llJl'\'+)



Charucterization ofmicroorganismsfrom E. brucei 1 1 1 1 1

Isolation and characterization of rhizosphere bacteria

Soil samples for this purpose were also collected from different E. bruce i trees at

science faculty campus of Addis Ababa University. The rhizosphere bacteria were

isolated using the general bacterial media of Parkinson et aI. (1971).

Ten grams of soil from each sample was suspended in 90ml of sterilized water

(1:10 dilution). The soil suspensions were mixed and serially diluted (10-2-10-6). From

the 10L 106 dilutions, O.lml of suspensions were transferred to the media and

incubated for 3 days at 30oC. From each dilution, colonies that grow separately from

other colonies were picked, purified and maintained on the slant agar medium. The

isolates were characterized to the respective genera using cultural and biochemical

tests (Anjena, 1993; Roberts et al.. 1995, Sundra and Sinha, 1963).

Isolation, characterization and mycorrhization of AMF

With regard to AMF, spore isolation was made from soil samples collected from therhizosphere of E. brucei trees found in science faculty campus (Addis Ababa

University). Spores were extracted using the wet sieving and decanting technique

(Hayman, 1982). Under microscope, individual spores were separated from the

surrounding organic matter using the needles and spores were characterized to

different groups.

Spore morphology including spore size, color, form of hyphae attachment to

spore were used to categorize the spores in to different groups (Hayman, 1982).

Spores of different types were transferred to separate watch glasses and thepopulation of spores per 100 gram of soil was counted for each group. About 50

spl":es belonging to similar spore category were separated and then inoculated to the

roots of onion (AlliulIl cepa L.) to induce establishment of AMF and to ensure its

presel vation.

After three months, small proportion of the soil from the rhizosphere of onion

plants was taken, wet sieved and the prevalence of spores was checked to confirm the

establishment of AMF. There after the mixture of the soil containing spores, mycelia

segments and root was used as inoculum in the follow up treatments.

Greenhouse experiment

The soil used for pot experiment at green house was brought from around Sebeta

Town, about 25km south and south west of Addis Ababa on the road to [imrna. The

soil was analyzed at the national soil laboratory following the soil analysis manual of

NSL (1994) and the physical and chemical characteristics of the soil had a clayey

texture; pH, 7.1; OIVI, 1.53; TN %, 0.2; Av.P, 37 ppm; EC, 0.99 dsy m: CEC, 2 8

Meq/100g; Exchangeable bases; Na, 0.12; K, 0.26; Ca, 13.56; Mg, 5.32.

The best performed root nodule bacteria during sand culture, the most

commonly encountered free living rhizosphere bacteria and the most common l y

encountered and picked AMF spore type were selected for the final experimentation

Root nodule bacterial isolate was coded as ERS07 (BmdyrliizobillIlI). The two free living

rhizosphere bacterial isolates were coded as PS3F1 (PSClitiOIIIOllllS) and BS3X (Bileilllls).

The dominant AMF spore type belonged to Cigaspor« spore type.

The soil obtained from Sebta was filled into alcohol-sterilized pots. Five surface

sterilized seeds of E. bruce i were planted in each pot. After germination, the number of



1 1 1 2 1 5ila 5/to I1 l1 d Fasi l

seedlings were thinned and reduced to three. Then these seedlings were inoculated

with one ml of each bacterial suspension that contained about 109 cells/ ml. Similarly,

20g of soil containing AMF spores, roots, and mycelia segments was inoculated in the

pots where the test plants were growing.

The experimental design of the green house experiment for the evaluation of E.

bruce i growth was according to the following seven treatments: Tl: Soil without

inoculation; T2: Soil inoculated with Bradurhizobium (ERS07); T3: Soil inoculated with

Brady rh i zob iun l (ERS07) + Ps eudomona s (PS3F1); T4: Brads j rh i zobium (ERS07) + Baci l lus

(BS3X); T5: Soil inoculated with Bradurhizobium (ERS07) + Pseudnionas (PS3F1) +

(Baci l lus) BS3X;T6: Soil inoculated with Bmdurhizobium (ERS07) + AMF (Gigaspora) ;

T7: Soil inoculated with all test microorganisms. All these treatments were inoculated

in three replicate pots each containing three seedlings.

The experiment was laid out in a completely randomized design with three

replications under greenhouse conaitions of 12/12h photoperiod, and 25/15°Caverage day and night temperature. The inoculated plants were kept in the

greenhouse for 90 days.

After 90 days of growth, the roots of the inoculated plants were carefully

removed to collect and count the nodules. The above ground plant parts were oven

dried at 70GC for two days to constant weights and these were used to determine the

dry weight of the seedlings from each treatments. The dry shoot weight was

measured using balance. Total nitrogen and phosphorus contents of the plants were

analyzed using methods the manual of the national soil laboratory (NSL, 1994).

Establishment of mycorrhizal mycelia on inoculated seedlings of E . bruce i , root

.surface was evaluated after roots were cleaned with 10% KOH and rinsed with 2%

HC!. Then tips of the roots were stained with 0.05% trypan blue in lactophenol and

evaluated microscopically (Hayman, 1982). The effectiveness of mycorrhizal infection

in the roots was computed as follows:

%AMF infection = (number of infected segments/ total number of segments

screened)100 (Hayman 1982).

The collected data were subjected to one-way analysis of variance. Means were

separated using Least Significance Difference (LSD) at 0.05 level of significance.

Results and Discussion

Both slow growing and fast growing root nodule bacteria were found associated with

root nodules of E. bruce i trees. Accordingly, six of the isolates identified as ERS01,

ERS03, ERS04, ERS06, ERS07, and ERS08 were categorized as slow-growing root

nodule bacteria, whereas two isolates, coded as ERS02 and ERS05, were grouped in to

fast growing root nodule bacteria (data not shown). Tentatively, the slow growing

root nodule bacteria were grouped into Bra durh izo bium (sen su s tr ic tu) and the fast

growing root nodule bacteria were grouped into Rhizobium (Jordan, 1984). Fassil

(1993), however, isolated only the slow growing Bradijrhizooium strains from nodules

of E. bruce i . The result of the current work indicated that the species has a wide

spectrum of nodulation. The implication here is that the host plant had a competitive



Churacterizauon ofmicroorganisms from E. brucei 1 1 1 3 1

advantage for its metabolic and ecological preference to establish itself in different

ecosystems.

Out of 94 rhizosphere microbial colonies collected in this study, 69% belong to the

gram negative rod shaped bacteria and 31% of the colonies belong to gram positive

rod shaped bacteria (data not shown). Based on the characteristics described in

Bergey's Manual of Systematic Bacteriology (Palleroni, 1984), most of the C°\'e groups

were classified into the genus Pseudomonas . whereas most of the C + v e bacteria were

grouped into the genus Baci l lus , respectively (Table 1). The sporulating C+ve bacteria

with other cultural characteristics are classified as B a cillu s. P se ud om o na s are oxidizers

of carbohydrates and this character differentiate them from Ae r omonae and enteric

bacilli which are fermenters of carbohydrates (Liu, 1952; Roberts et al., 1995; Hugh

and Leifson, 1953; Palleroni, 1984). The result of this study is in agreement with other

works reporting that Ps eudomona s and Baci l lus colonies co-occur around the roots of

different plants (Atlas and Bartha, 1993). In this work, two of the isolates (PS3FJ and

B~X) showed phosphate solubilization activity with a larger clear zone diameter on

modified Pikovskaya's medium (Sundra and Sinha, 1963).



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Characterization ofmicroorgamsmsfrom E. brucei 1 1 1 5 1

Three different groups of AMF spores were isolated frorn the rhizosphere soil of

E. brucei (Table 2). About 50% of the spore isolates were yellow to brown in color with

diameters of 400-600~lm and had bulbous stalk attached to them. This grou p belong

to the spore type (genus) Gignspom. The other groups had straight stalk with reddish

brown colored spores of diameters ranging between 50-225~lm. These represent 39%of the spore isolates and identified to belong to the spore type (genus) GlolIlll~.The

spores in the third group representing 10% of the isolates had spore diameter in a

range of 200-400~lm without stalks attached to them. This group belong to the spore

type (genus) Acaulospora (Hayman,1982; Trappe, 1982). This result was different in

the density and diversity of AMF from ACi7C/ilpOlljilCtJlltllil reported by Yonas and

Fassil (2007) These authors identified Glomus and Gigl7spon7 spore types with

percentage composition of 61 and 39, respectively. Though AMF species are usually

considered unspecific to different potential hosts, some authors have found that plant

species can influence the species composition of AMF communities (Dodd, 2000).

Tab le 2 . D ensity and som e characte ristics of A MF spore s iso la te d from E bruce i,

A ttache d Spore

G roup S pore size S pore co lo r sta lk type de nsity/100g so il S pore group

1 400-600~ lm ye llow brown B u lbous 205 Gigaspora

2 50-2251lm re d dis h b ro w n Stra ight 1 57 Glomus

3 200-400~ lm H one y S e ssile 42 Acaulospora

In the present study, root nodule bacterial isolates showed variations in symbiotic

effectiveness on sand culture (Table 3). Consequently, the isolates induced nodulation

with nodule numbers ranging from 91 nodules/plant by isolate ERS07

(Brndyrlllzohilllll) to 35 nodules/plant recorded by isolate ERS04 (Bmdljrlll:ll/7IIilllll).

Likewise, the highest shoot dry weight of 2.49g/ plant was recorded by LES07

(Brndyrlllzohill/ll) comparable to 2.41g/plant displayed by the nitrogen fertilized

plants (positive control). IsoiateERS06 had the second highest shoot dry weight nexl

to the isolate ERS07. The lowest shoot dry matter values of 1.48g/ plant and

1.35g/plant were obtained from the host plants inoculated by isolate ERS04

(Bmliyrhizohillll/) and ERSOl ((Bradljrhizol71UIll), respectively. This indicate the different

bacterial strains have different influence on the growth of the host plant and signih

the need to select for specific strain or to further evaluate how the host perform wh.-n

mix of these strains are inoculated the tree species.



1 1 1 6 1 S / / I l S / 1 O and F asil

Table 3 . The nodule num be r and shoot dry we ight of E. brucei se e dlin gs g ro wn in sand cultu re .

N odule num be r Shoot dry w e ight In te r-isolate diffe re nce

N o. Isolate Plant' ~Iant· l % e ffe ctiv e ne ss in e ffe ctiv e ne ss ( " ! o j

1 ER S07 (8) 91±3 a 2.49±0.05 a 1 00 (VE) 46

2 ER S06 (8) 82±5b 2.2±0.19 b 88 (VE) 39

3 ER S08 (8) 63±2c 1.82±005 d 73 (E) 26

4 ER S05 (R ) 54±3d 1.93±0.13c 78 (E) 30

5 ER S03 (8) 53±2d 1 .83±004d 73 (E) 26

6 ER S01 (8) 45±5e 1.35±005 f 54 (E)

7 ER S02 (R ) 39±5e 1.52±0.04e 61 (E) 11

8 ER S04 (8) 35±8 f 1.48±002 e 59 (E) 8

9 + C ontro l na 2.41±0.15 na na

Values fo llowed b y th e s am e le tte rs d id n ot sh ow sig nific an t d iffe re n ce a m on gst o ne a no th e r; B : Bradyrhizobium; R : Rhizobium,

na=not app licab le

Evaluation of the symbiotic effectiveness of the strains showed that there is

variation between the isolates. The data showed that isolates ERS07 and ERS06 were

very effective (80-100%), whereas the effectiveness rate of the other strains ranged

between 50-80% (Table 3) (Date et al., 1993). Although the isolates were within the

effectiveness groups, the data showed inter-isolate differences up to 46% equivalence

to the nitrogen fertilized control plants between the 1110steffective ERS07 and the least

effective isolate ERS01 (Table3). The effect of single, dual, and multiple inoculation of

the microorganisms to E. b ruc ei showed significant differences in shoot weight, nodule

number nodule weight, nitrogen content and phosphorus content compared to the

uri-inoculated control (Tl) (Table 4). Inoculation by different combinations of test

microorganisms (Treatments; T2-T7) showed prolific nodulation with nodule number

of 125-143/plant, and nodule fresh weight between 0.37-0.48g/plant, and increase in

nitrogen and phosphorus contents. But there is no any statistically significant

difference among the treatments in nodule number, nodule weight and phosphorus

and nitrogen contents.

Variations in shoot dry weight were more obvious in the different treatments(Table 4). The highest shoot dry weight of -+.O-l-g/plant was recorded from T3 (dual

inoculation of Brndyrlzizobil l I I I and Psciuunnonu: isolates), followed by T7 (multiple

inoculation of Bra dur ttiz ob iu n), P sc iu to in o uu- fiillillils and AMF). T4 (Brntiyrlii:obilllll+

Bacillus) and T5 (B rn dy rh izo billlll+ Ba cilllls + jJ:;llld uJ lIO I 1I7 S ) showed a slightly lower shoot

dry weight than the T3 and T7. However, the data showed a 20-45% difference in

shoot dry weight between the inoculated plants (T2-T7) and the uninocu lated control

plants (T1). It is also very interesting to note that inoculation of the host plant with

rhizosphere microbes and root nodule bacteria (Table 4) increased nodule number

and shoot dry weight by 14-37% and 30-48%, respectively, in the soil experiment than

with single inoculation of the host with root nodule bacteria in a sand culture (Table

2).



Characterization of microorganisms from E. brucei 1 1 1 7 1

Table 4: The e ffect of inoculation of diffe rent rhizosphe re m icroorganisms on nodule num be r, nodule we ight

(fresh) , shoot dry we ight, n itrogen and phosphorus contents of see dlings of E ry th rina b ruce i

Parameters T1 T2 T3 T4 T5 T6 T7

N odule number na 125a 143 a 128a 134 a 132a 141 a

N odule we ight (g/plant) na 0.37a 0.45a 0.38a O.4a 0.39a 0.48aShoot dry we ight (g/p lant) 278 c 3.25b 4.04a 3.45a 3.54a 3.34b 3.62a

T ota l n itro ge n (% ) 2.4 b 3.4a 4.2a 3.7" 3 .8a 3.7" 3.9 a

Total phosphorus (ppm) 1560b 1587a 1604a 1586a 1698a 1990a 2160a

n .a = n ot a pp lic ab le ( rh iz ob ia w e re n ot in oc ula te d), re su lts a re m e a n o f th e th re e

re p lic atio ns , V alu e s fo llo we d b y th e s am e le tte rs did n ot s ho w s ig nific an t diffe re n ce

a m on gst o ne a no th e r

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