(i)
EFFECT OF FERTILIZERS, SEED SIZE, PLANT AGE AND DIFFERENT BEAN (PHASEOLUS VULGARIS L.) CiJL TIVARS
l
,
ON HALO BLIGHT DE\)ELOPt~iENT.IJ
BY
/
I
I
ff
IPHILIP ODUOF:~
A THESIS SUBMITTED IN PARTIAL FULFILME~T FOR THE
DEGREE OF MASTER OF SCIENCE IN PLANT PATHOLOGY AT KENYATTA UNIVERSITY.
1989
Owino, Philip Oduor Effect of
fertilizers, seed
111111111111~III
i
M H~II~ 11111111111111111111111 92/200982DECLARATION BY THE CANDIDATE
THIS THESIS IS MY ORIGINAL WORK AND HAS NOT BEEN PRESENTED FOR A DEGREE IN ANY OTHER UNIVERSITY.
~,,;f
r.••:. ••••• e •••••••••• -.;"' ••~""••••••• c••••• PHILIP ODUCR 0l4INO
DE::I ARA TIr-lN BY THE <;1IPERVIS0RS
THIS THESIS HAS BEEN SUB!"IITTED FOR EXAt-1INATION J..-iITH OUR APPROVAL AS UNIIJERSIT"'{ SUPERVISORS •
l<»t Supervisot-
.
$~~~
..
.
DR. STANLEY WAMUKAVA WAUDO; SENIOR LECTURER,
BOTANY DEPARTMENT, I<ENYATTA UNIVERSITY.
2nd Supervisor
,
•••• ~~m
DR. A.I<. MISRA; SENIOR LECTURER.
(iii)
TABLE OF CONTENTS
Title (1)
Declaration (ii)
Table of Contents (iii>
List of tables (v)
List of figures
Dedication (viii>
Acknowledgement Summary
CHAPTER 1.0 INTRODUCTION 1
CHAPTER2.(; LITERATURE REVIEW "
~.
<2.1 The Pathogen 3
r-:;? Halo blight disease cycle "7
.<...- ...;.
2=3 Symptoms 4
2.4 Control 4
'""C' Host Resistance 5
..:...'-01 •....• '
Host Nutrition and Disease
L.b
Development 7
CHAPTER3.0 MATERIALS AND t"iETHODS 11
3.1 Isolation and Identification of
pc;eudomonas phac;.eolicola 11
~ •....•
Preparation of Pseudomonas .J•..::.
(av)
Inoculation Techniques 15
3.4 Disease assessment 15
3.5 Classification o-f bean cultivars
into reaction groups. 16
3.6 Field tests 1 and 2 17
3.6.1 Nitrate determination in lea-f tissue 20
3.7 Field tests 3 and 4 21
3.8 Data analysis 26
CHAPTER4.0 RESULTS 27
4.1 Field test 1 27
4.2 Field test 2 43
4.3 Field test 3 57
4.4 Field test 4 78
CHAPTER5.0 DISCUSSIONAND CONCLUSIONS 94
5.1 Discussion 94
5.2 Conclusion 105
LITERATURECITED 107
(v)
LIST OF TABLES Table
1. Classification scale used for grouping bean cultivars into reaction groups using leaf
reaction indices. 18
2. Classification scale used for grouping bean cultivars into reaction groups using pod
reaction indices~ 18
3. Bean cultivars used to investigate effects of Pseudomonas phaseolicola on seed germination and bean performance in field tests 3 and 4.
4. Hesponses of bean cLlltivar GLP-l004 supplied with 4, 8 or 12gmof single superphosphate .:sSP)" calcium ammoniumnitrate (CAN)or diammoniumphosphate (DAP)to Pseudomonas
phaseolicola= field test 1. 28
5. t"1eandisease rating on leaves (MDRL),mean number of lesions per leaflet (MNLU,mean disease rating on pods <t"lDRP)and nitr-ate concentration (NC)(mg/litre) in leaves of bean cultivar GLP-1004 treated "lith 4, 8 or 12gmof single superphosphate (SSP), calcium ammoniumnitrate (CAN)or diammoniumphosphate
(vi>
phaseolicola; field test 1.
6. Relationships between different amounts (gm)
of single superphosphate (SSP), calcium
ammoniumnitrate (CAN)or diammoniumphosphate
mAP) fertilizers and the performance of bean cultivar GLP-1004 inoculated with Pseudomonas
phaspolicola. field test 1.
7. Relationships between nitrate concentration
(NC)(mg/litre), different levels of single
superphosphate (SSP)~calcium ammoniumnitrate
(CAN)or diammoniumphosphate mAP) and the
responses of bean cultivar GLP-l004 to
Pseudomonas phaseolicola; field test 2.
8. Responses of bean cultivat- GLP-l004 supplied with 4, 8 or 12gm of single supet-phosphate
(SSP), calcium ammoniumnitrate (CAN)or
diammoniumphosphate mAP) fertilizers to
pc;eudomonas phaseolicola; field test 2.
9. Relationships between different amounts(gm) of
single superphosphate (SSP), calcium ammonium
nitrate (CAN)or diammoniumphosphate and the
performance of bean cultivar GLP-1004
inoculated with Pseudomonas phaseolicola
field test 2.
33
37
48
(vii)
10. Relationships between different amounts(gm) of single superphosphate (SSP), calcium ammonium nitrate (CAN)or diammoniumphosphate mAP) and the responses of bean cultivar GLP-1004 to
Pseudomonas phaseolicola~ field test 2. 54
11. Relationships between the performance of bean cultivar GLP-1004 treated with different
levels of single superphosphate (SSP), calcium ammoniumnitrate (CAN)or diammoniumphosphate (DAP)and its responses to Pseudomonas
phaseolicola;field test 2. 56
12. i"1eanpercent seed germination and disease incidence of 25 bean cultivars 10 days aftet-inoculation with Pseudomonas phaseolicola;
field test 3. 58
13. Reactions of 25 bean cultivars to Pseudomonas
phaseolicola; field test 3. 61
14. Mean stem diameters (em) and number of pods per plant of 25 bean cultivars 80 days after inoculation with Pseudomonas phaseolicola; field test 3.
15. l'"1eandisease rating on leaves (MDRUof a resistant bean cultivar GLP-X92, tolerant cultivars GLP-1800 and GLP-24 and the
(viii)
susceptible cultivar 6LP-I004 inoculated "lith
Pseudomonas Phaseolicola; field test 3.
16. Correlation among plant responses to
Pseudomonas phac;policola; field test 3.
17. Mean percent seed germination and disease
incidence of 25 bean cultivars 11 days after
inoculation with Pseudomonac;phaseolicola; field test 4.
18= Reactions of 25 bean cultivars to Pseudomonas phaseolicola; field test 4.
19. i1ean stem diameters (em)and the number of
pods per plant of 25 bean cultivars 82 days
after inoculation with pc;eudomonac;
phaseolirola; field test 4.
20. Mean disease rating on leaves (MDRUof a
resistant cultivar GLP-X92,tolerant cLlltivars GLP-1800and GLP-24, and a susceptible
cultivar GLP-I004 after inoculation with pc;eudomonasphaseolicola. field test 4.
21. Relationships among plant responses to
Pseudomonas phaseolieola; field test 4.
Page
67
77
79
82
84
88
(i.x)
LIST OF FIGURES
FiQ1tre
1. Regressions of mean disease rating on pods (MDRP)of bean cultivar GLP-I004 on amounts of single superphosphate (SSP), calcium ammonium nitrate (CAN)or diammoniumphosphate mAP) 69 days after inoculation with pc;.eudomonas phaseolicola; field test 1.
2. Regressions of mean disease rating on leaves (t-lDRUof bean cultivar GLP-1004 on amounts of single superphosphate (SSP),
calcium ammoniumnitrate (CAN)or diammonium phosphate mAP)16 days after inoculation with 'Pseudomonas phaseolicola; field test 1.
3. Regressions of mean number of lesions per leaflet on nitrate concentration (NC)
(mgllitre) in leaves of bean cultivar GLP-I004 treated with di-fferent levels(gm) of single supper phosphate (SSP}, calcium ammonium nitrate (CAN)or diammoniumphosphate mAP)16 days after inoculation with Pseudomonas
phaseolicola; field test 1.
4. Regressions of mean dry seed weight (MDWSHgm) on mean disease rating on leaves (MDRUof
bean cultivar GLP-I004 treated "'.lithdi-fferent
35
38
(x)
levels of single superphosphate (SSP), calcium ammonium nitrate (CAN) or diammonium phosphate (DAP) and inoculated with Pseudomonas
phaseolicola. field test 1.
5. Regressions of mean number of pods per plant (MNPP) on mean disease rating on leaves (MDRL) of bean cultivar 6LP-1004 treated with
different levels of single superphosphate (SSP), calcium ammonium nitrate (CAN) or
diammonium phosphate (DAP) and inoculated with pc;eudomonac:; phac:;eolicola; field test 1.
6. Regressions of mean stem diameters (MSD) on mean disease rating on leaves (MDRL)of bean cultivar 6LP-1004 treated with different
levels of single superphosphate (SSP), calcium ammonium nitrate (CAN)or diammonium phosphate mAP) and inoculated with Pseudomonas
phaseolicol."l; field test 1.
7. Regressions of mean dry seed weight <t-1DSW)on mean disease rating on pods (NDRP) of bean cultivar 6LP-1004 treated with different
levels of single superphosphate (SSP), calcium ammonium nitrate (CAN)or diammonium phosphate (DAP)and inoculated with Pseudomonas
phaseolicola. field test 1.
41
42
44
8. Regressions of mean disease rating on pods H1DRPion mean disease rating on leaves (MDRL) of bean cultivat- GLP-l004 supplied with
different levels of single superphosphate (SSP), calcium ammonium nitrate (CAN)or
diammonium phosphate WAP) and inoculated with
Pseudomonas phaseolicola; field test 1. 46
9. Progression of disease rating on leaves
(MDRUof a resistant bean cultivar GLP-X92, tolerant cultivars GLP-1800 and GLP-24, and a susceptible cultivar GLP-1004 ,,,,ith plant age (days) after inoculation with Pseudomonas
phaseclirola; field test 3. 70
10. Regression of mean disease rating on leaves {MDRUof a resistant bean cultivar GLP-X92, tolerant cultivars GLP-1800 and GLP-24, and a susceptible cultivar GLP-l004 on plant age (days) after inoculation ""ith Pseudomonas
phaseolicola; field test 3. 71
11. Regression of mean disease rating on pods
(MDRP)on mean disease rating on leaves (MDRL) of 25 bean cultivars inoculated with
Pseudomonas phaseolicola.; field test 3. 73
(xi i)
(86%) on seed size (58) for 25 bean cultivars 10 days after inoculation lo"JithPseudomonas
phaseolicola:; field test 3. 74
13. Regression of mean stem diameters <t18D)on mean disease rating on leaves (MDRL)of 25
bean cultivars inoculated with Pseudomonas
pnaspolicola; field test 3. 75
14. Regression of mean number of pods per plant
\t'1NPP)on mean disease rating on leaves (MDRU
of 25 bean cultivars inoculated with
Pseudomonac; phaseolicola:; field test 3. 76
15. Progression of disease rating on leaves (MDRL)
of a resistant bean cultivar 6LP-X92, tolerant
cultivars GLP-1800 and GLP-24, and a
sLlsceptible cultivar GLP-1004 with plant age (days) after inoculation with Pseudomonas
DEDICATION
This thesis is dedicated to my son, Victor Odida
DdLlOr; my wife, Evelyn Siundu Mudhune; my father,
Nelson OIFlinoOwuor and my mother, Rita Anyango
(;-:vi Summar-v
Field tests, l'lere conducted to investigate effects of single superphosphate 'tSSP) calcium ammonium nitrate (Cf-iN) and
,
-diammonium phosphate WAP) fertilizers applied at the rates of 4, 8 or 12gmper plant on responses of bean cultivar GLP-1004 to Pseudomonac::.syringae PV. phasen1icola (Synonym: E:..Phaseolicola {BurkDOlFlSJ, 'that causes halo bl:ight in beans, and to determine
effect of seed size, plant f\ge and different bean cultivars on the development of this disease. Influence of nitrate
concentration in GLP-1004 on halo blight development was also
'in.vestigated. A randomized complete block design v-las used in all
tests.
Fertilizer--s influenced disease development differently. For
instance, _'12gm-CAN-treated plants had signi ficantl y (P=O.05) the lowest mean number
0+
lesions per leaflet {t"iNLL} and mean\
disease, rating on_leaves (NDRU of 1.24 and 0.84, respectively. The i"lDRL v-Jas based on a 0-5 rating scale. The 12gm-CAN treated
plants, hONever, ~"ere among the shortest v-Jthi mean heights of \
19.29cm. Four gt-am-CAN treated plants had mean number of lesions
of 15.77 per leaflet, and mean heights of up to 34.07cm, that were signi ficantl y (P=O.05) higher th,an those oo'cained from other
I I , ,
plants.
/
Four grams of SSP had the second greatest inhibitdry Effee
t-on disease development. The second lO!'-Jest f'1NLL of 3.89 ~..,as
obtained;; +rom the -4gm-SSP-trea tea plants. Plants supplied with
8gm of SSP had s6me of the highest MNLL, MDRL, and mean disease
- rating on pdds _(MDRP)of up- to 17.96, 4.26 and 47.59,
respectivel y, among the SSP, CAN,or DAP-treat.ed plants. Plants
treated with 4gm of SSP had the highest mean number of pods per
plant of up to 21.22.
of lesions of 6.42, t-1DRLof 1.63 and r'1DF:pof 31.44, t-lhile planes treated with 89m of DAP had s6me of the highest MNLL, MDRL and
t-1DF:Pof up to 16.64, 4.09 a.nd 45.5:'::;,respectively~ among the
DAP-treated plants. pr~;!:lts treated vlith 8 or' 129m of DAP had the he aviest; seeds \.~ith me,:'u-{ seed weights of 0.40gm. Coni::r'ol plant;::~ were
tr.,~
most infected ~""ith up to 22.70 lesions per leaflet.There was a.n l.nvel~Se relationship bet~'Jeen nitrate contentul G.l-F-l004 and disease development a.s vlas indicated by slopes.~o:f_,_,. -52.328, -33.260 and 7'2:8.265 obtained from the regF"essjons o-.~
mean number of lesions per leaflet on nitrate concentration for
CAN~SSP and DAp-treated plants, ~espectively.
Cultivars GLP-1800, GLP-1841, GLF'-24 and GLF'-585 lrJere
tolerant to E..phac.polic01,3. The cultivars mainta.ined large stem diameters and number of pods per pIa.nt illspite of high disease
severity as shoNn by j-1DEL values of bet",.<een 2u32 and 4.32~ and the leaf susceptibility inde:-: (LSD values of oet"Jeen 3.00 and 5.0(i. The t1DF:Land LSI ~"ere based on a 0-5 rating scale.
CLlltivars GLF'-X92, GLF'-1800 and GLP-1841 had r'1DRL vd.lut.?S of h=s~.
~-~~
Eighteen cultivar"s IFJere good hosts of this pcd:.hogen.
ho=.ts had re1atively 10~"ier seed genninatiof'l pe!-c~ntages7 fewa -pods, 'smaller stem diame-cETs and higher disease incidE'rlces ·than
the corresponding non-inoculated plants.
cultivar-s, good hosi:.s, had t"lDRL values of HlCH"ethan 3.00.
, Thkre ~>Jasan inver-52 t-elationsnip betv4een seed SlZP .3nd
percenta.ge seed germination as indicated by the SJ.OPE' of -30.2/L
,;Disease development i!,}ct-eased with plant age in c u l tive..rs
'lGLP~X92, GLP-2·<;~GLP-1.800 and GLF-1004 ac. revealed by o o sibve
Disease hOv-Jever1 developed faster in GLP-l00i\ than ·1.11 the otfH">:--,-~,
- 1
-CHAPTER1
INTRODi iCTION
legu.mcs ~n Kenya (Acland, 1971; j-lukunya et.al., 1982). A
major constraint in the production of beans are bact~ri~l
dise'::ises that destroy the cr-op. Halo blight, causE!d by
Pspudomonac; Sy~-innap. pv; ph.=i.seolicoL::>(S, ynonym:
E'..phaseolicola (Burk) Do~",s), is one of thG major -bean
bacterial diseases in i<enya. The disease causes severe
leaf defoLiation and pod infections dur-ing rainy seasons.
Vield losses of up to 431. have been associat.ed with this
disease (SaE,ttler and Pottet-,1970; Schwar'tz and Galvez,
1980).
The current recommended coritrol measures such as the
use of ant:ibiotics5 host resis~:ance, crop roi::ation and
copper ·fungicides have not adequately minimized the 10s:ses.
Effects ~f copper fungicides such a~ bordeaux mixture
ag;::,inst E..ph.::;.senlicola ere usua.lly shor1.:-lived. This is~
in part~ due to the development of tolerant strains in the
pathogen'~".population. !~lso, hit;ih costs and environmental
ha'zards associated with chemiG:i3ls limit their: Llse. The use
of crop rO'cation is also undesirable because of land
scarcity. Effective long-term- control, of ,E.~D~_$~o1icola,
th2refol~e~ will depend upon development of cultiv~rs such
as GLP-X92 that are resistant to races 1 and 2 of this
.:;..g3.instE.phaseolico@ is controlled by a s.ingle recessive
gene (i<inyua. et.al.~ 1981J, its durability may be shot-t-lived~
particularly In environments v-Ji·thn2\o'Jphysiological races.
In view of the sbove shortcomings, there is great need
to s~arch for more resistant· lines from the b~an germplasm,
and to e};plGf~e the possibility of developing more effective
control measures against halo blight. The use of inorganic fet-tilizet-s~ as a cultLII~al practice, may provide such an
alternative. Althou.gh host nutt~it.:i.on has been used
effect.:ively to control many pLarit; dise3ses,; its suitability to contr-ol halo blight has only been partictlly studied
under gn?enhouse conditions (Patel and walket-, 1963).
Tests to determine the repeatabilii.:.y of results from
greenhouse experiments under field conditions have not been
done.
The for-mulation of a viable disease management
stt-ategy, however, requires a pr-·opet- understanding of
factors such as seed size and host age that influ.ence·
disease dev·elopment. Ti]e present study ~'JEtS therefore
designed tc,:
Ii.) investigate ef-fects of single super-·phosphate {f3SP}
calcium ammonium nitrate (CAN) and diammonium
phosphate (DAP)f,ertilizers on halo blight development
on the suscept.ible bean cultivar GLP-l(H)4,
(ii) deten(\:i.ne effE:~cts of seE:'d size, plant age and
·CHAFTER 2
L1TEHATUR'EREVIEi.<J -,.- ~,-
--,-\
pathogen that causes halo blight of beans in many countries
including Kenya (1'1ughogho, 1970; I"lukunya, 1974; i<aiser-~·
1975; I<inyua et.al., 1981 Stoetzer, 1983; Stoetzer and Omunyin, 1983). This foliar pathogen is rod shaped, gram
negative~ forms cream colonies on nutrient agat-~ and does
not reduce nitrates to nitrites O"1isaghl and Grogan, 1969;
lLjeber, 1973; Schaad, 1980). The bacterium utilizes
D-gluconate, L(+} arabinose, Leserine and StIerose (Sand's
eL at., 1970).
PSpudOfHonas phaseolirola
I !
hE.l.S a wide hos"'t range.
Its "'hlbsts include F'hAseollls vulqaris L~
E.
.
an d:i foEI 15,E:..anc.iL'l~ric.and P.·, rarliata (Z~umeyer and Thomas,1957). It I
i
is'disseminated by rainsplashl wind (WalkeF' and Patel~
1964) and through infected seeds (Guthrie e'{:.al., 1970;
.
-Schuster, 191'7; Katherman et.al.~ 1980 b), The pathogen
'doe~ not fonn resistant spor~'s (Schuster and Coyne1 1974)
tHH: over-comes adverse environmental conditions by remaining
dormant in seeds, plant debris and other non-host materials,
~ - 4
-c9ndi tions ar-e favour-able" it invades plant tissues tnt-oLlgh
wounds and natur-al openings (lsJalket- and Patel 1964), The invasion process is aided by moisture (l"Jilliams and Keen~
1967) and hemicellulase enzymes (i'laino, 1972).
The type of symptoms formed depends on bean
genotype (Qmer- and tiJood 1969; Hubbt2ling, 197::::;;Taylm'- i3_nd
Dudley 1978; Kinyua et.al. 1'1i811. Resistant bean cultivars
e:<h1bit l~eC\ctions that r-angE> from no visible symptoms to
sillallilecrotic',spots on the infected leaves (Hubbeling~
19T::;)~A susceptible cultivar- responds by developing £:01a11
I'Jatei--soai<ed lesions on sides of the 1n- ',.-tec-c
.
t?o.
leaves, 3--5 days after infection (Omer- et.al., 1969L i"Jhen \conditions ar-'e favoLirable~ a yenot~ zone (halo) may devt."'lop
around the water--soaked lesions (Coyne and Schuster 1974~
Under epidemic conditions, stems and pods get infected
\
for-ming or-own sunken lesions ~-.jhichenlarge longitudinally
and turn br-owil (Zaumeyer and Thomas, 1957}. Heavily
infected bean plants ar-e def:Dliated W:osuge~ 1978i and
i
chlorotic due to the pr-oduction of Phaseolotoxin (Coyne ana
(l'htcheH and Bif."leski~ 1977).
2.4 ,Cor.trcI
Halo blight of beans has been partly contr-oll(~cl
"!,o,]«-Use'o-f deep ploughing CZaumeyer andc:rhomas~ 1957}
streptcmycin (f,',::dph~197b; Tay-lor and Dudley, 1977) <;;,,,d
Agriculture. N.i-1.R.S~Thika, Bean Research Files). Seed treatment using streptomycin is unsafe because it reduces
pLarrt; emergence (P=i1ph~.1976). The use of various
formulations of copper have been inadequate in minimizing
crop loss {Schuster and Coyne, l,:;j81; Stoetzer and Ornunyin,
--"---'--~-"'--- -part~ to the
development of "tolerant strains in the pathogen's
satisfactor-y con'l:rol of halo bli qht; using copper spt-ays nas
been obtained (Laumeyer and Thomas, 1957).
and vJalker, 1965; Lyon and ;"'jood1975; Gnanamanickam and
Pat:i11 1977 a; Taylor- et~..:u., 1978~ t=:inyua et.alri"~ 19811. Resistant bean cultivC"lTS exhibit resistance c•ga. insi:
,E:..pi·E1SF'!olicolaby inoibi ting the grm~th of the pathogen and the production of Fhas201oto:·;in (Fussell, 1976,.
FhaseoJoto:·:in is responsible fol'- the production of a yeHo~..;
zone (flalo/ C"lround~•.a•.ter-sGaked 1e:,::.ionsth2.t is a typical
sympt.om of halo blight {Coyne and Schuster"lS'74). The
inhibition of pathogen grdwth and phaseolotoxin production
1977a.~ Taylor eLai., 1978.) The hypersensitive respor:se
'is a post-invasion pt-m::esst:hat inhibits the activity o-F
(Gnanamanick2.m <'-H10 Patil, '-l977b.~,,'ians-field, l'i'82). For
6
Patil~ 19T?b}. The 2,ntibacte;--ial activity of phytoale;-{ins,
hm-'J2Ver, has been inconsistent (Lyon and t';80d, 1975; Wyman
and Van Etb?n~ 1978; Gnanamanickam and Smith~ 1980). This
meciium used, inoculum density~ SL\bjecti vit't~in reading
1978).
Host resistance may also be influenced by age,
morphological_~J3nd physiological character-istics o-F a plant
Schuster 1974~ Valladares'---Sanchez et.aL, 1979~ Hoody eLaJ., 1980L For instance,; to-,-er-ance in cucumbet- against
aga.irist l...ar'"CJ10rnOnas vesicat!=,ri.3 (Doidge) DfJ~L.,!S'7? irlcreases
~'Jith ageJ ~<,jhilebeans are more susceptible to
E.phaseolicola at their primc\t-Y and pod initiation 5;taqes
and Walke\"'-1963}. High suscerj-:- tibility
0+
beans at their Primarv, and o.
od initiation c\ssociat:ed IF-Jithintensive nutrient flow ~t theSE stages of bean development
~.•i'th age (Van Gundy and l.oJali::E:t19- 57,.
Seed s:;ze and cOloLw a}~:;D influence dis-eas2
to seedborne pathe'J2ns than ;,:',ma_llon2'=:; U<•~.umeyer et.a1.,
7
-1957) This is because larg2 seeds ·il.2tVea large. raphe that
facilita-tE:s seed infection <Zaumeyer and Thomas 1957;
Schuster and Coyne, 1974). Similarly, seed coat colour
influences the response of beans to pathogens (Prasad and
t·J:?igh",F/'76; Moody et.al., 1980). Seeds ~..,ith black seed
CCldi..s i:'U-~' more resistant to Rhizoctonia c::.o1ani (\<uhn> than
produce phenolic compounds that inhibit seed infection
(Prasad and tJ.Jeigle 1976). {Hso, seed coats of black seeds
do not cr.3_ck as t-eadily as those of Nhite seeds during
germination. Because of this, cotyledons of black seeds are
protected against pathogen attack {Pt-asad and \.J.Jeigle 1976;.
Ho::=.tnut.riticm influences plant response LO
\ pathogens (Gaufftman, 1950; Na}'udu et.al.~ 1960; Fatel and
Walker, 1963; Kr-ikbyand t"lengel 1978:. Lee and, Zentmyer
1982; Pataky et.aL~ 1984. !<f~el et.aL~ 1989j. PO!:2-.ssium
for examele, enhances translocation of assimilates and I
.
.
-,..
-.'
.
.
narcens pLant scructu~es co 1nvaSlon oy pa~hogens
(Fen"·enouno, 1977; Krikby and Nengel 1978)~ In cereals,
excess potassium leads to the formation of thick layers of
potassium silicate ~".jhichact as a barrier to fungal
invasion (Gassner and Hassebrau.k., 19~31!. High levels of
potassium inhibit development of bacterial spot of tomato
'levels of -potBssium, however! favour- bi:l.cterial blight o-f
lima bf2ariS caused by
E:.~..
cL
.
.9.g!.
e V. Hall {Thaung and Walh8
(Van Gundy and t·jalker, .1957), and wildfire disease of
tobacco incited by P.tabaci U.Jolf and Foster) F.L. Stevens
P~osphorus has variable effects on disease development
(G,;'ILur:m :'n~ J95>:». High levels of phosphorus predispose
tCJb2CCOplants to...£:..·tabaci (Borrirvq, 1930) and toma-i::o to
bacterial spot of tomato caused by X.vesir-fltoria (Nayuou et.al.,.1960). On the other harrcl, high levels of
phosphorus if] _Hoaglemds solution inhibit bacterial blight
of lirfia-J?e.arf?,- bacterial SpClt of peach and halo blight
o( beans inci tf:?d by
E.
syrinqae, K.m-uni (E.F.) Dows~and E:..ohac;pnlicola, r-espectively (Bachelder et.a1., 1956; Thaung and ~·Jalker~ 1957; Patel and ~..Jalker 196:.5).
PJ:losphorus is also kriown to inhibit destructive effects
of the bean fly~ Ophiomycia phaseoli (Tryon) (Dipi.:era:
Agr-ornyzidaeJ (F:c}gers, 198c); Floor eteal., 1984} ..
Phosphot-Us, hO~'Jever~ has no effect on angular leafspot
of cucumber (Van Gundy and vJalh'?,- 1957).
, I
Ni,trogen in-HO.ences -the development 0-1- many p larrt;
diseases (Spencer, 1935~ Fatel. c~ndi'}alker }9,S3; Black~
1983; Pat_ai::y eLal-., 1984). For ex-:?-.mpl.eQLlindro~ cladiu_m black root o+ peanuts (f::n-achis hypoqaea L.}, caused by
decreases \f~ith incre<":\Sf:?in levels of nitrogen (Black, 1983;
Un th~ other hand, eXCESS nitrogen
w-ecij sposes bec.ns to tobacco l'iosaic virus (Spencer; J 935)~
tobacco to [.:tabaci (BDning, 193(i)~ Peach fruit to
- 9
-angular- I-j,t~.;: spot (Van Gundy and ~'Jalker 1957). ~. .- .
I
""~l
Diffe;.f:--ent forms of nitr-ogen may influence disease
development differ-ently (Huber- and vJatson~ 1974,. For
-Fi.':.t,:..fI. cc"tJ.S:20 by E'hycophthoca cinnamoni is increased and
deer-eased by nitrate' and ammonIum nitrogen~ respectively
(Lee and Zentmyer-, 1982:. Citrus root r-ot caused by F'. citr"ophthor.:? (f";:.E.Srf:and EaH.so.m) IS increased by ammonium
nitrogen~ .v·,hile nitr-e,te nitr-ogen has opposite effect=. "." t'- . " 1~58)
, 4;-1<·.10 z, ..E1:.aJ.;· ,.., •• Both and nitrate nitrogen,
hoit-lever-,. r:wedispose resistant tobacco cul·tiva.r 'Cooker 139'
The effects of ammonium and nitrate nitr-ogen on
disease development have been attr-ibuted to their
'differ-ential effect on pH of plant 1974
i<rikby and i'lenge1 1978).
plant Cf:'11s=. (Smiley~ 1974), while ammonium nitr-ogen
oeCt-ease.;.=:, 11:. and !""..nH]. .H,"L~' '.19~~'I! 1.
,
/
tissue pH influences host metabolism~ and subsequently, it=.
('."
..
.t'..lrI<DY The val~iB.tion In
The r-ole of calcium in disease development is var-ia.ble
Fusarium ~ilt of tomato caused by Fusarium oxysoorum f.sp
Ivcoper~iri. {sacc)· Snyd. and Hi3ns~is incr;:.?ased and
decreased by deficient and e:,cess calcium~ respectively
- 10
-(Lee andZentmyer, 1982)
Calcium influences disc~ase development by reacting
oectic substances
.
~ in plant cell walls to formca.lciL~m pectate that strengthen plant tissLles against
en::ym2"~l'-:- hydrolysis (Bateman~ and t-1iller 1966).
l
,- 11
-CHAPTER 3
MATERIALS AND METHODS
Field tests were conducted at Kenyatta University,
Nairobi, Kenya, between September, 1988 and June, 1989 to:
(i) investigate effects of single superphosphate
(N:P:i<:
=
0-20-(», calcium ammonium nitrate(N:P:i<
=
26-0-0) and diammonium phosphate(N:P:i<:
=
18-46-(n on responses of thesusceptible been cultivar GLP-l004 to
E._phaseolicola;
(ii) determine effects of seed size, plant age and
different bean cultivars on the development of
halo blight
3.1 Isolation and identificaTion of P.phaseolirola
Pc::.eudomonasphaspolicola used in aU tests ~..,as
isolated from infected bean leaves collected from National
Horticultural Research Station, Thika, Kenya. The infected
leaves !.o'Jeresurface sterilized with 0.5% sodium
hypochlorite and rinsed with sterile distilled water. The
surface sterilized leaves were aseptically transfered to a
test tube containing 10 ml of sterile distilled water and
cut into small pieces using a pair of scissorsQ Bacteria
- 12
-water for 5 minutes. The resulting bacterial suspension
was streaked onto nutrient agar (NA)using a sterile
platinum loop (Riker' et.al., 1939). The cultures were
incubated for 48 hours at 25 C. After the incubation
period, single colonies were transfered to fresh NA
using a sterHe. platinum loop. The single colonies were
used for identifica-tion.
-Identification tests v-Iere based on Gram Stain (Schaad,
1980), nitrate reduction <Misaghi et.al., 1969), citrate
utilization, colony colour on NA (Weber, 1973) and on
pathogenicity tests. Gram stain tests were conducted using
three different 48-hour-old colonies. Each of the three
colonies was aseptically transfered to a microscope slide
using a sterile platinum loop and smears were made. The
bacterial smears were fi>~ed by flaming the underside of the
slide for 3 seconds. The smear was then flooded with
crystal violet solution for 1 minute, washed with tap
water, blo-tted dry and flooded with iodine solution for 1
minute. The iodine was then washed with a decolourizing
solvent, ethyl alcohol, for 30 seconds. The alcohol "-Ias
washed off using tap water for 2 seconds before flooding
the smear with safr-anin, a counter stain, for 10 seconds.
The smear was then washed with tap water, dried and
examined under a microscope. Colonies with gram negative"
rod-shaped bacteria were separately maintained on NA for
fur-ther identification. Colonies of E,.phaseolicola were
later partially confirmed on the basis of colour (Weber,
- 13
-colonies li'Jerefurthet- subjected to cib-ate utilization,
nitrate reduction, and pathogenicity tests.
The citrate utilization test involved aseptic transfer
of 48-hour old bacterial colonies to sodium citrate agar
(SeA)slants. The SeAslants without bacteria served as
controls~ The Cl,dtures were incubated for 24 hours. The
~">Ihiteto cream bacterial colonies turned the SeA media to
blue~a characteristic reaction of E:..phaseolico1a.
In the nitrate reduction test, bacteria from a single
colony, one colony per medium;!fiere aseptically cultured on
sterile nitF-ate broth containing peptone, potassium nitrate
and sterile distilled water in the ratio of 10gm:19m:
100m1, respectively (i"!isaghiet.al.,l969), for 24 hours at
20 C. Three drops of sulphanilic acid and naphtylamine
solutions t">lereadded to the broth after the 24 -hour
incubation period. ;Thesulphanilic acid was prepared by
dissolving 8gm of sulphanilic acid in 1000mlof 5t"i acetic
acid. The naphtyl"';\minesolution contained 5gm of
naphtylamine dissolved in 1000mlof 51"1 acetic acid.. A
distinct red colour appeared, indicating lack of reduction
of nib-ate, a characteristic feature of E:..phaseolicola
(Misaghiet.alo, 1969).
The Pathogenicity test was carried out using the
susceptible bean cultivar GLP-l004. Spray inoculation
method (Schuster, 1955) was adopted. A low pressure
- 14
-and the lower sides of the primary leaves sprayed to
run-off. The inoculated plants were covered with moist
plastic bags for 48hours to facilitate infection (Williams
and Keen, 1967). Typical symptoms of halo blight developed
16 days after inoculation. Small water soaked lesions
surrounded by a yellow zone (halo) were observed.
After- the identification, pure colonies of the
bacterium were maintained on NA and agar slants in a
refrigerator.
3.2 Preparation of Pseudomonas phaseolicola inoculum
Twenty-four hour old cultures of E..phaseolicola were
used as inocula in all tests. Bacterial colonies from the
pure culture were aseptically transfered to sterile
distilled ~~ater using a sterile platinum loop. A ten-fold
dilution series was then made from the resulting bacterial
suspension. One ml aliquot of the various dilutions were
transfered to different NAplates, one ml aliquot per
plate, and spread using a sterile glass rod. The cultures
"Jere incubated for 24 hours at 25°C. After the incubation
period, colonies were counted using a colony counter. The
number of colonies per plate "lere multiplied by the
dilution factor to obtain the number of bacterial cells per
ml of distilled water. An inoculum density of 5 x 1(;'
bacterial cells/ml of distilled water was used in field
tests 1 and 2. Inoculum levels of 5 x 106 and 5 x lOB
cells/ml of water were used in tests 3 and 4, respectively.
- 15
-3.3 Inoculation Techniques
Two inoculation techniques, spray and soil infestation
methodstwere used. The spray inoculation method decribed
by .schuster (1955) was adopted. A 10\'1# pressure sprayer was filled with the appropt-iate bacterial suspension and the
lower sides of primary leaves sprayed to run-off. Sprayed
plants were covered with moist plastic bags for 48 hours to
facilitate infection (Williams and Keen, 1967).
The soil infestation method described by Prasad et.al
(1976) and Schuster et.al (1975) was used to determine
effects of E..phaseolicola on disease incidence and seed
germination. For inoculation, ten mls of an appropriate
bacterial suspension were pipetted into planting holes just
before planting. Seeds planted in the non-infested holes
served as controls.
3.4. Dic;ease asspc;sment
Assessment of disease on leaves and pods was based on 18
randomly selected plants per row (e>~perimental unit). The
disease inde}dng methods described by Patel and Walker
(1963) were adopted.
A rating scale of 0 - 5 was used to assess disease on
leaves where, (; = no visual symptoms;
1
=
leaflets with brown necrotic spots without halos;2
=
leaves with 1-3mm halos covering 1-25'l. of the entireleaflet;
- 16
-leaflet
4
=
leaves with 9-20mmhalos covering 52-72% of theleaflet; and
5
=
Large halos, 21mmand above occupying over 72% ofthe leaf area
The observed values were averaged to obtain a mean
disease rating on leaves (MDRUfor each treatment.
The 0 - 5 disease rating scale was also used in computing
leaf susceptibility inde}~{LSD. The LSI represented the
highest value observed for each treatment using the
o -
5 rating scale.The number of water-soaked lesions per leaflet (MNLL)
was recorded and also used in disease assessment.
A O - 100 rating scale was used to assess disease on
pods where, 0 = no symptoms; 25
=
1-25%; 50=
26-51%;75 =52-77%; and 100
=
78-100% of the pod covered with lesions.The observed values were averaged to obtain a mean
disease rating on pods (MDRP)for each treatment. Pod
susceptibility inde~{(PSI>was also computed using the 0-100
rating scale. The PSI represented the highest value
observed for each treatment using the 0-100 rating scale.
3.5 r:lassification of bean 'ultivars into reaction groups
The t"lDRL,LSI~PSI and I"1DRPvalues were used to
classify bean cultivars as resistant" highly tolerant,
~
- 17
-shown in tables 1 and 2.
3.6 Field tec;ts 1 and?
Field tests 1 and 2 were conducted between September
and November, 1988 and between April and June, 1989,
respectively, to investigate effects of single
super-phosphate (SSP), cp_lciumammoniumnitrate (CAN)and
diammoniumphosphate (DAP)fer'tilizers on responses of the
susceptible bean cultivar GLP-l004 to E:..phaseolicola.
Different sites wer-e used for the tests. Before
incorporation of fertilizers into soil, soil samples t-Jere
randomly taken from each site, air dried, screened to
remove debris~ and analysed for mineral elements, soil
te}~ture, percentage organic matter, and hydrogen ion
concentration using procedures described by Jackson (1958),
Day (1965), Graham(1948) and Peech (1965), respectively.
Field test 1 was conducted in a sandy-clay soil with
50% sand, 381. clay and 121. silt. The soil had 0.181.
nitr-ogen, 18.001. phosphorus, 1.78% potassium, 1.22% carbon,
3.80% magnesium, 10%calcium, 0.841. sodium, 0.701. manganese
and an average pH of 6.60. Field test 2 was carried out
in a clay-sandy soil with 0.1.2%nitrogen, 9.00% phosphorus,
1.001. potassium, 1.111. carbon, 4.86% magnesium,8.76% calcium,
0.44 % sodium, 0.85% manganese, 45% sand, 50.10%clay and
4.80i'~ silt. The soil had an average pH of 5.86.
- .LO
-Table 1: Classification scale used for grouping bean cultivars
into reaction groups using leaf reaction indices.
. .Le a'f 'indices
MDRL1 LS12 Reaction group
"'O~O
-
1.00 0 or 1 Resistant1
:
1
,
-
z..00 2 Highly tolerant2.1 --3.00 3 Tolerant
3:1
-
5.00 4 or 5 SusceptibleI
Mean disease rating on leaves
2
Leaf susceptihility index
TabLe 2: Classification scale used for grouping bean cultivars into reaction groups using pod reaction indices.
Pod indices
MDRp3 PS14
0.0 - 24.
9
c
l
025.0 - 49.99 25
50.0 - 74.99 50
75.0 - 100.00 75 or 100
Reaction group
Resistant
Highly tolerant
"
Tolerant
Susceptible
3
Mean disease rating on pods
4Pod susceptibility index.
SBased on a 0-100% rating scale,
where, 0
=
no symptoms- 19
-soil at the ,-ate of 4, 8 or 12gmper planting hole just
before sO~Jing.Non-treated soils, soils without
fertilizers~ served as contF"ols. A randomized complete
block design ~..,iththree replications per treatment was
used. Seeds used in these tests were surface-sterilized
~lith 0.51.sodium hypochlorite for 5 minutes. The seeds
were planted at the rate of two seeds per planting hole.
Plant spacings of 10cmwithin rows and 50cm between rows
~..,e~eused as recommendedby the t-linistry of Agriculture
(!<enyat"linistry of Agriculture, 1976 - 1979). Each
experimental unit, a row, was 380cm and 495 cm long in
tests 1 and 2, respectively.
Thinningwas done 11and 12 days after planting in
tests 1 and 2, respectively, to leave one plant per hole.
Inoculum of E.phaseolicola was pr-epared, and inoculumlevel
of 5 x 10' cells/ml of sterile distilled !t'Jater was used for
inoculation in both tests. Twelve-day old seedlings were
inoculated using a low pressure sprayer as described in
section 3.3. Plants were watered regularly using a
portable sprinkler.
Rating scales of 0-5 and 0-100 were used to assess
disease progress on leaves and pods, respectively, as
described in section 3.4. Leaf infections were assessed 16
and 17 days after inoculation in field tests 1 and 2,
respectively. Pod infections were assessed 69 days after
inoculation in test 1, and 65 days after inoculation in
- 20
-Plant heights (em), stem diameters (cm), the number of
seeds per pod, pods per plant, and dry seed weights (gm) of
10 randomly selected plants per e}~perimental unit, a row,
were used to evaluate plant performance. Plant heights and
stem diameters were determined 40 and 50 days after
inoculation in tests 1 and 2, respectively. Dry seed
weights were based on a sample of 100 randomly selected
seeds per e}~perimental unit. The seeds "'Jere dried at 80°C
for 72 hours. The average ItJeight per seed was calculated
by dividing the total dry weight of 100 seeds by one
hundred.
3=6.1 Nitrate deb::>rmination in leaf tissue
Nitrate content in leaves of bean cultivar GP-1004
was assayed in order to determine whether or not the
levels of nitrates in leaf tissues influence halo blight
development. Brucine method described by Nicholus and
Nason (957) was adopted.
The uppermost trifoliate leaves of five randomly
selected plants per experimental unit were harvested
using a pair of scissors, 16 days after inoculation. The
top parts of these leaves were cut into small pieces to
make a 3 gm test sample. Four test samples were made
for each treatment.
The cut leaf portions were ground in a mortar
- 21
-ccmc., sulphuric acid (ccmc, H2S04) and rinsed with
distilled water. The macerate was passed through cheese
cloth and the filtrate centrifuged at 20,000 rPm for 30
minutes at 2<:>C.Chlorophyll and other particles formed a
pellet at the bottom of the centrifuge tubes. One ml of
the crude e}~tract (supet-natant) was diluted to 5ml by O.U",!
phosphate buffer of pH 7.4. To this, 2ml of 4% Brucine in
chloroform h'J/v) was added. Five ml of concentrated
sulphuric acid was carefully added using a burrette. The
mixt.ur-e ~'Jasplaced in a water bath at 90cC for 10 minutes
until all the chloroform was removed. It was then allowed
to cool at room temperature.
The colour intensity <optical density) was read off
from a spectrophotometer (Spectronic-20) at 4BOnm. A
standard graph was prepared by running a set of potassium
nitrate O<NO::s} standards of concentrations 0.100" 0.125,
0.166, 0.200, 0.25, 0.33, 0.500 and 1..00mg/litre through
the same procedure. All the optical densities were
converted t.o mgllitre from the standard graph.
3.7 Field tesrs 3 and 4
Field t.ests 3 and 4 were conducted between November,
1988 and February, 1989 and betweeen i"larch and May, 1989,
respectively, to:
(i) investigate effects of E..phaseoliro1a on seed
germination and performance of 25 bean cu1tivars;
(ii) investiga.te effects of host age on responses of a
GLP-24, GLP-1800 and the susceptible cultivar
GLP-l004 to E.~phac:.eolicola.
The 25 bean cultivars used in these tests wet-e
obtained fr-om the National Horticultural Research Station;
Thika, Kenya. The cultivars are listed in table 3. The
cultivars GLP-X92~GLP-24 and GLP-1004 were included in
the tests because they are known resistant, tolerant and
susceptible to E:..phaseolicola; respectively <Kinyuaet.al.,
1981; Stoetzer, 1983, Stoetzer et.al., 1983; Stoetzer and
Omunyin1983).
Field test 3 ~Jas conducted in a sandy-clay soil
!f>Jith56.00% sand; 36.70% clay and 7.30% silt. The soil
had O.19~f.nitrogen, 16.80%phosphorus, 1.82%potassium,
1.30%carbon, 3.66i~magnesium, 9.8% calcium, 0.79% sodium,
0.68% man9anese and an average pH of 6.80. Field test 4
I#as conducted in a clay-sandy soil with 37.20/: sand 52.40%
clay, 10.40%silt, 0.68% nitrogen, 15.80%phosphorus, 1.10%
potassium, 1.21%carbon, 3.80% magnesium, 9.1%calcium,
0.61/: sodium, 0.72 manganese and a pH of 6.90.
Surface sterilised seeds rinsed "..."ithdistilled water
\...,ereused in all the tests. For inoculation, 10mls of a
bacterial suspension containing 5 x 106 and 5 x loa
cellsiml of water were pipetted into planting holes just
befot-e planting, in tests 3 and 4, respectively. Planting
was done at the rate of 3 seeds per planting hole. Seeds
- 24
-cultivars were arranged in a randomized complete block
design with two replications per experimental unit. A
plant spacing of 60cm between rows and 10cmwithin rows was
used. Inter-row spacings of 60cm were necessary to minimize
contamination of non-inoculated seeds and/or plants. Each
four-meter rolt-Jconstituted an e}!perimental unit.
Thinningwas done 13 days after planting in both
tests, to leave one plant per hole. Plants were watered
regularly as required using a portable sprinker. The
percentage seed germination and disease incidence were
determined in both tests using the formulas below:
Percentage seed germination =
total No.of seedlings per cultivar x100
total no. of seeds planted
Disease incidence
=
total No.nf inferted sepdlinQs per cultivar x 100
total No.of seedlings per cultivar
After the assessment of percentage seed germination
and disease incidence, plants from inoculated seeds were
inoculated using a low pressure sprayer to avoid the
formation of a typical symptomsthat are associated with
inadequate inoculum (Clement, 1968)and/or to ensure that
all the cultivars were subjected to the inoculum.
Seedlings from non-inoculated seeds were covered with
The numberof lesions per leaflet and halo size on
leaves of inoculated plants was assessed 15 and 13 days
.after inoculation in field test 3 and 4, respectively. Pod
infections ~'1Iereassessed 65 days after inoculation in field
test 3, and 60 days after inoculation in field test 4.
r10difiedPa-tel and ~Jalker (1963)rating scales were adopted
as described in section 3.4. Disease assessments were
based on 18 randomly selected plants per row.
Stem diameters and the number of pods per plant "."ere
determined to evaluate the performance of the 25 different
bean cultivars. The evaluation was based on 18 randomly
selected plants per experimental unit.
Effects of host age on halo blight development was
determined over a 40-day period using a resistant cultivar
\
GLP-X92~tolerant cultivars GLP-24 and GLP-1800,and a
susceptible cultivar GLP-l004. t1ean disease rating on
leaves (MDRL)a meas, ure of halo size, was determined
10,15,21,26,30,36, and 40 days after inoculation in field
test 3; and 12,16,20,25, 31,35 and 40 days after
inoculation in field test 4. A split plot design was used
to determine effects of plant age, different bean cultivars,
and the interaction of age and different bean cultivars on
disease development.
Effects of e.phaseolicola on seed germination and
- 26
-sizes (Table 3sJ Seeds were rated in size using a modified
Evans (1974) rating scale,~">¥hereseeds weighing:
0.148gmto 0.248gm were rated as very small
O.249gmto 0.348gm were rated as small
0.349gm to 0.448gm ~",ererated as medium
O.449gmand over were rated as large.
Data obtained from the 4 tests -were subjected to a
-two-way analysis of variance (ANOVA).Treatment means
were compared using Duncan's Multiple Range Test (Duncan
1955) and Least significant differenc~ (LSD)tests (Carmer
and Walker, 1982) at 5% (P=O.05)probability level. A
split plot analysis of variance was used to determine
effects of plant age, different bean cultivars and the
interaction of plant age and cultivars (age x cultivat-s) on
halo blight developmen"t (Appendi>~22 and 33). Regressions
and correlation statistics "'Jere also used as shown in
figures 1 to 8 and tables 9, 10, 11, 16, and 21>.
Appendixes 1 to 38 show statistical analyses that were
27
CHAPTER4
REsULTS
4.1 Field test 1
Field test 1 was carried out between September, 1988
and November, 1988 at I<enyatta University, Nairobi, I(enya,
to investigate responses of the susceptible bean cultivar
GLP-I004 supplied with 4,8 or 12gm of single superphosphate
(SSP), calcium ammoniumnitrate (CAN)or diammonium
phosphate mAP) fertilizers to P.phaseolicola.
The SSP, CANand DAPfertilizers had no significant
effect on stem diametet-s 40 days after inoculation (Table
4). Plants treated with 4gm of SSP" however, had the
largest stems, while those supplied with 12gm of SSP or DAP
had the smallest stems <Table 4).
Different levels of SSP, CANor DAPhad significant
(P=O.Ol;Appendix1) effect on plant height. Plants
treated with 8gm or 12 gmof CANwere significantly
(P=O.05)shorter than controls, plants supplied with 4,8 or
12gm of DAP, or SSP and those treated with 4gm of CAN
<Table 4}. Plants treated with 8gm of DAP, SSP and 4gm of
CANwere among the tallest with heights of 34.55, 34.38 and
34.07cm, respecti.vely <Table 4}.
Tab le 4: Responses of bean cultivar GLP-1004 supplied with 4, 8 or l2gm of single superphosphate (SSp)
calcium ammonium nitrate (CAN) or diammonium phosphate (DAP) to Pseudomonas phaseolicola
field test 1.
Mean Stern Mean Plant Number of Pods Number of seeds Mean Dry Seed Weight(gm)
Diameter(cm) Heigh t (cm) Per Plant (MNPP) Per Pod (MNSPP) (MDSW)
(MSD) (MPH)
I \
Treatment
(gm/plant)
Days after inoculation
40 40 66 71 71
Control 0.64 32.40b2 l2.83ab 3.32 0.36ab
SSP
4 0.72 32.45b 2l.22bc 3.05 0.43bc
8
o
~67 34.38b l6.72ab 3.07 0.35ab12 0.59 29.65b l5.40ab 2.72 0.39ab
I
co
CAN
N
4 0.69 34.07b l4.l0ab 3.01 0.38ab
8 0.63 23.04a l6.l7ab 2.75 0.39ab
12 0.61 19.29a l3.33ab 2.47 0.30a
_DAP
4 0.67 33.42b 13.22ab 3.05 0.37ab
8 0.66 34.55b l7.4lab 2.91 0.40ab
12 0.59· 32.77b l2.27a 2.60 0.40ab
NS NS
lEach treatment was replicated thrice
2Figures followed by different letters within a column are significantly different at P = 0.05 by
- 29
-the lowest and highest number of pods per plant of 12.27
and 21.22, respectively, that were significantly .• <P=O.05)
different 66 days after inoculation with E..phaseolicola.
(Table 4). An average of 12.83 pods per plant obtained
from control plants were not significantly different from
those obtained from plants treated with SSP, CAN or DAP
(Table 4)
Single superphosphate, CAN and DAP fertilizers had no
signi ficant effect on number of seeds per pod 71 days after
inoculation (Table 4). Plants treated with 12 gm of CAN
and control plants, however, had the lowest and highest
number of seeds per pod of 2.47 and 3.32, respectively
(Table 4).
Single superphosphate treCl_tment had significant
<P=O.Ol;Appendi}~2) effect on seed weight 71 days after
inoculation (Table 4). Plants supplied with 4gm of SSP
gave significantly (P=O.05) heavier seeds than those of
plants treated with 12gm of CAN <Table 4). Effects of
other treatments on seed weight were not significantly
different from the effects of 4gm of SSP or 12gm of CAN
(Table 4)
There was no significant difference in the mean
disease rating on leaves (MDRU16 days after inoculation
<Table 5). Plants treated with 8gm of SSP, DAP or 4gm of
CAN,however, had some of the highest MDRLvalues of 4.26,
Table 5: Mean disease rating on leaves (MDRL) , mean number of lesions per leaflet (MNLL) , mean
disease rating on pods (MDRP) , and nitrate concentration (NC) (mg/litre) in leaves of
bean cultivar GLP-1004 treated with 4, 8 or l2g~ of single superphosphate (SSp),
calcium ammonium nitrate (CAN) or diammonium phosphate (DAP) and inoculated with
Pseudomonas phaseolicola; field test 1.
I
Treatment
(gm/plant)
MDRL MNLL MDRP NC (mg/li tre)
Days after inoculation
16 16 69 16
4.01 22.70bc 2 50.55ab O:22a
3.10 3.89a 37.l6ab 0.38cd
4.26 l7.96b 47.59ab 0.44cd
3.55 13.57b 44.33ab 0.46cd
4.14 15.77bc 51.43bc 0.25a
3.94 13.80b 36.l9ab 0.40bcd
2.86 2.44a 26.43a 0.58e
3.91 l2.22b 43.72ab 0.32ab
4.09 l6.64b 45.53ab 0.42abcd
3.44 9.67b 34.45ab 0.48de
NS Control
SSP
4 8
.12
CAN
0 4
("1")
8
12
DAP
4 8
12
lEach treatment was replicated thrice
2Figures followed by different letters within a column are significantly different at
- 31
-with 12gmof CANhad the lowest MDRLvalue of 2.86 <Table
Mean number of lesions per leaflet H1NLL)obtained
from plants supplied with 12gmof CANor 4gm of SSP were
signi ficantl y (P=O.05)lower than those obtained from other
plants including controls <Table 5). Control plants and
those tr-°eat.ed with 8gm of SSP had the highest and second
highest MNLLof 22.70 and 17.96, respectively <Table 5).
The MNLLof 12.22, 16.64 and 9.67 obtained from plants
treated with 4,8 and 12gmof DAP, respectively, were not
significantly different from those obtained from plants
treated with 8gmor 12 gmof SSP, 4gm or 8gm of CANor from
control plants <Table 5)
The mean disease rating on pods (I'-1DRP)of 26.43
obtained from plants treated with 12gmof CANwas
significantly (P=(l.05) 10,,"lerthan those obtained from
differently treated plants including controls 69 days after
inoculation ~...,ithE..phaseolicola <Table 5). Plants treated
with 4gmof CANand control plants had the highest and
second highest MDRPvalues of 51.43 and 50.55, respectively
<Table 5)
Nitrate contents extracted from 3gm of leaf tissue
were significantly (P=O.Ol;Appendi>;3) different 16 days
after inoculation (Table 5). The lowest nitrate
concentration, 0.22 mg/litre, e}~tracted from control plants
from plants treated with 4; 8 or 12gmof SSP, 8 or 12gmof
CANand 12gmof DAP(Table 5). The highest nitrate
concentration, 0.58mg/litre, obtained from plants treated
with 12gmof CANwas not significantly different from those
extracted from plants treated with 12gmof DAP16 days
after inoculation (Table 5). The lowest number of lesions.,
2.44, was associated with the highest nitrate concentration
of 0.58mg/litre <table 5).
There were great variations among plant responses to
E..phaseolicnla under different fertilizer treatments.
Regt-ession coefficients and regression equations showing
relationships between amount of fertilizers and plant
performance are depicted in table 6. Regression
coefficents of-0.0051·, -0.1570 and -0.0440 were
obtained from the regressions of stem diameter, plant
height and number of seeds per pod on amount of SSP,
respectively (Table 6). The negative slopes revealed an
inverse relationship between the three plant parameters and
SSP levels, though the relationships were not significant
(Table 6). Regression coefficients of 0.08 and 0.00035
IfJere obtained from the regressions of mean number of pods
per plant and mean dry seed ~·~eighton SSP levels,
t-espectively, though the slopes were not significant
(Table 6).
Stem diameters, plant heights and number of seeds per
pod had inverse relationships with the amount of CANas
Table 6: Relationships between amountsI(gm) of single super-phosphate (SSp), calcium amonium nitrate (CAN) or diammonium phosphate (DAP) fertilizers and the performance of bean cultivar GLP-I004 inoculated with Pseudomona~ phaseolicola; field test 1
Dependant Variable Independant Regression Regression Days after
Variable Equation Co-efficient inoculation
(slope)
Stem diameter(cm) Amount of SSP Y
"
=
0.68 - 0.0051X -0.0051 40.Plant height(cm) Amount of SSP Y1\
=
33.16 -.0.1570X -0.1570 40Number of pods per plant Amount of SSP
Q
=
16.10+
0.0800X 0.0800 66Number of seeds per pod Amount of SSP
~ =
3.30 - 0.0440X -0.0440 71Dry seed weight (gm) Amount of SSP
Y
=
9.38+
0.00035X 0.00035 71Stem diameter(cm) Amount of CAN Y
"
=
0.66 - 0.0035X - 0.0035 40Plant height (em) Amoun t 0f CAN
t
=
34.78 - 1.2500X -1.2500 40Number of pods per plant Amoun t 0f CAN
~
=
13.57+
0.0890X 0.0890 66Number of seeds per pod Amount of CAN y
"
=
3.30 - 0.0069X -0.0069,h'( 71Dry seed weight(gm) Amoun t 0f CAN
Y
=
0.36 - 0.00036X -0.00036 71,...
CV') Stem diameter(cm) Amount of DAP Y
=
0.66 - 0.0038X -0.0038 40CV')
Plant height (cm~ Amount of DAP
t
,...=
32.95+
0.0560X 0.0560 40Number of pods per plant Amount of DAP Y
=
13.55+
0.0628X 0.0628 66Number of seeds per pod Amount of DAP
Q
=
3.31 - 0.057 OX -0.057,'(ok 71Dry seed weight (gm) Amount of DAP Y
"
=
0.36+
0.00395X 0.00395 71I
Amount of SSP, CAN or DAP used were 4, 8 or 12 gm/plant
- 34
-respectively (Table 6). These regression coefficients were
not significant e~{cept -0.0069, (P=O.Ol; Appendid 4). A
positive relationship was obtained from the regression of
mean number of pods per plant and mean dry seed weight (gm)
on amounts of CANas indicated by the non-significant
regression coefficients of 0.0890 and 0.00036, respectively
(Table 6)
Stem diameters and seeds produced per pod had inverse
relationships with the amounts of DAP as indicated by
regression coeffcients of -0.0038 and -0.057,
respecti vel y (Table 6). The slope of -0.057 was highly
significant (P=O.Ol; Appendix 5). Increase in DAP levels,
hO\l'Jever, was accompanied by an increase in plant height,
number of pods per plant and dry seed weight 40,66 and 71
days after inoculation, respectively (Table 6i This
relationship was revealed by the positive regression
coefficients of 0.056, 0.0628 and 0.00395, respectively
(Table 6).
Linear regressions were performed to determine
relationships between amount of fertilizers and mean disease
ra.ting on pods (MDRP)and between amount of fertilizers and
mean disease rating on leaves (MDRL).Regression coefficients
(slopes) of -1.17, -0.20, and -2.19 were obtained from the
regressions of t"iDRPon amounts of DAP, SSP and CAN
fertilizers, respectively (fig 1). The negative slopes
revealed an inverse relationship between MDRPand levels of
35
Fig~ 1
\
Regr"essions of mean disease rating on pods (t'1DRP) of
bean cultivar GLP-l004 on amount1 of single
super-phosphate (SSP), calcium ammonium nitrate {CAN}Dr
diammonium phosphate {DAP) 69 days after inoculation
with PSPI~domnna<=.pha<=.policola~ field test 1.
1Amounts of SSP, CAN or OAP used were 4,8 or 12gm!
0
-cr 30
0
X
L
20
X
CA
N
.55
P
0·0 A P
1
0
60
50
40
s
p
0,+
-
.-
.-
-
.
_
o
4
8
12
36
Increase in levels of DAP, SSP, and CAN suppressed MDRPby
80.20%, 3.42% and 88.00'1. as revealed by coefficients of
determination (R2) of 0.8020, 0.0342 and 0.880, respectively
(Table 7>.
Regression coefficients of -0.0057, -0.038 and
-0.091 were obtained from the regressions of mean disease
rating on leaves (i"!DRL)on amounts of SSP, DAF'and CAN,
respectively, 16 days after inoculation lo'Jith E..phaseolicola (fig.2). Although these slopes were not significant, they
revealed inverse relationships beh",een amount of fertilizers
and t1DRL..Coefficients of determination of 0.00327, 0.633 and 0.458 were obtained for relationships beh",een MDRLand
SSP, t1DRLand CAN and between MDRLand DAP, respectively
(Table 7>. Thus increase in levels of SSP, CAN and DAP
accounted for 0.327'1., 63.300'1. a.nd 45.880% of the reduction In t1DRL, respecti vel y <Table 7>.
A significant (P=O.Ol; Appendi}: 6) inverse
relationship (b=-52.328) was obtained from the regression
of mean numbet- of lesions per leaflet <t1NLL)on
concen-tra.tion of nitrates (NC) (mg/litre) obtained from 3 gm of
leaf tissue for plants treated with CAN (Fig.3). Slopes of
-33.260 and -38.265 lo'lere obtained from the regressions of
MNLLon nitrate concentration for SSP and DAP-treated
plants, respectively, 16 days after inoculation (Fig.3).
The negative slopes revealed inverse
relation-ships beh",een MNLLand nitrate concentration, though the
Relationships between nitrate concentration (NC) (mg/Tt tre ) , different amount sl-of single superphosphate (SSP), calcium ammonium nitrate (CAN) or diammonium phosphate (DAP) and the responses of bean cultivar GLP-1004 to Pseudomonas phaseolicola; field test 1
Tab le 7:
Dependant Variable
(i) Mean number of lesions per leaflet (MNLL)
(LL ) Mean disease rating on leaves (MDRL)
(iii) Mean disease rating on pods (MDRP)
(iv) (MDRL)
I"- (v) (MDRP)
0")
(vi) (MDRL)
(vii ) (MDRP)
Independant Days after
R R2 R2x ,100(%)
Variable inoculation
Nitrates in SSP-treated plants 16 -0.016 0.00025 0.025
Nitrates in CAN-treated plants 16 -0.997,'("k 0.9950 99.500
Nitrates in DAP-treated plants 16 -0.769 0.5920 59.200
Amount of SSP 16 -0.057 0.00327 0.327
Amount of SSP 69 -0.185 0.0342 3.420'
Amount of CAN 16 -0.796 0.6330 63.300
Amount of CAN 69 -0.938 0.8800 88.000
Amount of DAP 16 -0.677 0.4580 45.800
Amount of DAP 69 -0.895 0.8020 80.200
lAmountsof SSP, CAN or DAP used were 4, 8 or l2gm/plant
R = Correlation coefficient R2
=
Coefficient of determinationR2x 100
=
indicate percentage reduction of bean responses to Pseudomonas phaseolicola with increase in levels of SSP, CAN, DAP or nitrates.38
Fig. 2
Regressions of mean disease rating on leaves (MDRL)of
bean cultivar GLP-1004 on amounts1 of single super
-phosphate (SSP), calcium ammoniumnitrate (CAN)or
-,diammoniumphosphate mAP) 16 days after inoculation
with Pseudomonac:;phac:;policola; field test 1.
1Amounts of SSP, CANor DAP used were 4, 8 or 12gml
5
4
3
...J
0:
o
1:
2
x
o
•
".•
Y: 3·76
- O·0057X SSP
y-/
?::4'09
- ~ ·28
-
0·038
- 0-09
X
DAP
1X
x
•
CAN
x
SSP
1
o
DA P
O~__
--~,--~'----~I---o
4
8
12
39
Fig. 3
Regressions of mean number of lesions per leaflet
n'iNLU on nitrate concentration (NC) (mg/litre) in
leaves of bean cultivar GLP-1004 treated with
different levels1 of single superphosphate (SSP),
calcium ammonium (nitrate (CAN) or diammonium
phosphate mAP) 16 days after inoculation with
p""pudnmona"" phaseolicola; field test 1.
1Levels of SSP, CAN or DAF used were 4;8 or 12gml
3S
•
CAN
X
s
s
P
o
DAP
...J
20
25
x
z
:L 15
o
o
10
5
•
x
oJ
-
-,--, __
.-, __
.-, __
'-. __
-,-,__
-,- __
o
0·1
0-2
0-3
0
'
4
0·5
0·6
- 40
-the correlation between I'-INLLand NC for plants treated with
SSP, CANand DAP~..,ere0.00025, 0.9950, and 0.5920
respectively (Table Ts: Thus, 0.0251..,99.501..and 59.20% of
the reduction in number of lesions could be attributted to
increase in nitrate concentration in leaves of the
susceptible cultivar GLP-l004 treated with SSP, CANand
DAP,respectively <Table 7).
Regression coefficients of -0.06, 0.054 and -0.0392
were obtained from the regressions of mean dry seed weight
(MDSW)on mean disease rating on leaves (t1DRUfor SSP, CAN
and DAPtreated plants, respectively (Fig.4). Although the
slopes were not significant, slopes of -0.06 and -0.0392
indicated that increase in MDRLwas accompanied by a
reduction in seed weight for plants treated with SSP and
DAP. The positive slope, 0.054, indicated that increase in
t-1DRL,a measure of halo blight severity, had no destructive
effect on seed ~Jeight. The CANfertilizer appears to have
suppressed the adverse effect of halo blight on the seeds.
Regression of mean number of pods per plant <t1NPP)on
I'-mRLfor CANand DAP-treated plants gave non-significant
slopes of 0.682 and 4.80, respectively (Fig.5). These slopes
indicate that CANand DAPhad an inhibitory effect on the
development of halo blighL Plants supplied ~"'IithSSP,
however, showed &. negative relationship between MNPPand
MDRLas, indicated by the non-significant slope of -4.71
41
i29m/plant.
Fig. 4
Regressions of mean dry seed weight (t'IDSiij)1(gm)on mean
disease rating on leaves (MDRL)2of bean cultivar
GLP-l004 treated with different levels3 of single
superphosphate (SSP}, calcium ammonium nitrate (CAN) or
diammonium phosphate mAP) and inoculated with
Pseudomonas Phaseolicola= field test 1.
1MDSWdetermined 71 days after inoculation
2i'"1DRLassessed 16 days after inoculation
• CAN
X SSP
o
OAP
()
.
s
0-6
"C <11 OJ VI
-..
.
.
~0-4
-3
Vl
o
(}3L
o
1
2
3
M D R L
42
12gm/plant. Fig. 5
Regressions of mean numbet-of pods per plant (/1NPP)1
on mean disease rating on leaves (t<1DRL)2of bean
cultivar GLP-1004 treated \l'Jith different levels3 of
single superphosphate {SSP)"calcium ammoniumnitrate
(CAN)or diammoniumphosphate mAP) and inoculated with
Pseudomonasphaseolicola:; field test L,
/'
1t1NPP determined 66 days after- inoculation.
2MDRLassessed 16 days after inoculation
•
CA N
X
SS P
o
OAP
35
30
25
D-
20
D-z
L
15
(
•
~
~'O
~S
"x
A>, \,;.'0
-,
~~
