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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
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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
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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-+).
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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
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! 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'\'+)
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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
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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
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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.
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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).
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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
References
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