Correlation between populations of xanthomonas axonopodis pv. vignicola builds up in vivo and symptoms manifestation in infected cowpea plant.

Journal of Natural Sciences Research www.iiste.org
ISSN 2224-3186 (Paper) ISSN 2225-0921 (Online)
Vol.3, No.7, 2013
29
Correlation Between Populations of Xanthomonas Axonopodis Pv.
Vignicola Builds Up In Vivo and Symptoms Manifestation in
Infected Cowpea Plant.
Umoru Simeon. Amodu1*
Nnennaya Ogechi Agbenin1
Edun Bolanle3
1. Department of Crop Protection, faculty of Agriculture/ Institute for Agricultural Research, P. M.
B. 1044 Ahmadu Bello University, Zaria, Nigeria.
2. Department of Crop Protection, Faculty of Agriculture Bayero University kano, Nigeria.
*amodusimeon@yahoo.co.uk, simeonumoru@gmail.com.
Abstract.
From the perspective of plant disease epidemiology, pathogen spread and multiplication is important in assessing
the likelihood of disease outbreaks. Many plant pathogenic bacteria multiply quite successfully in association
with susceptible tissues without causing lesions. In this paper we aimed to understand the dynamics of
Xanthomonas axonopodis pv. vignicola population sizes in regard to symptom development on cowpea plant.
Seven cowpea genotypes were use for the investigation. The seed lots were obtained from seed companies,
research institute and open markets within Zaria environment. These were so selected to reflect the various ways
farmers usually obtain their seeds. The seed samples were grouped into treated and inoculated and non-treated
inoculated seed lots. The treated seed lots were treated with three different types of fungicides (Apron star, Dress
force, and Team fungicides) before inoculation with bacteria suspension. Since Xanthomonas axonopodis pv.
vignicola is a seed-borne pathogen, the inherent bacteria were estimated from which the population builds up
were determined. The seeds were inoculated with bacteria suspension adjusted ca. 4.7 x 107
cfu/ml and were also
spray inoculated at 14 days after sowing. The result shows that there were no correlation between bacteria
disease incidence, severity and population with respect to foliar lesion development. The usual practice of
taking disease incidence and severity should be discouraged. I would recommend pathogen population
assessment should be undertaken in conjunction with disease incidence not disease severity. Fungicides used
had no significant effect on the pathogen’s growth and pathogenicity. Although, seed treatment has been
adjudged to be the best method of control soil and seed-borne diseases, seed treatment chemical must be pest
specific.
Keywords; correlation, population, pathogenicity, symptom.
1. Introduction
Production of cowpea (Vigna unguiculata L. Walp) is hampered by pests among which is the bacterial blight
induced by Xanthomonas axonopodis pv. vignicola (Xav). Both internally and externally infected seed have been
mentioned as important source of inoculums (Cafati and Saettler, 1980).When describing the epidemiology of
plant diseases, we often say that a disease has spread when new disease occurs. The occurrence of new disease
implies that the pathogen has successfully dispersed, to a new habitat, a susceptible host, and that growth on the
host has occurred, leading to disease (Upper et al., 2003). Much of the classical epidemiology of plant diseases
has been developed for disease in which disease development can be equal to pathogen population. Many
pathogenic bacteria multiply quite successfully in association with susceptible tissues without causing lesions
(Hirano and Upper, 1983). Attempt to fit classical epidemiological model to such diseases are often less
successful and clearly have less biological relevance (Upper et al., 2003). In this system, the likelihood of
disease occurring on a given leaf is a function of some other factors rather than merely pathogen population size
on that leaf (Rouse et al., 1985). Most gram-negative bacteria pathogens harbor a T3SS (type III secretion
system) that allow the delivery of effectors directly into the plant cytoplasm, leading to a hypersensitive reaction
(HR) in incompatible interactions and symptoms development in compatible ones (Bruning and Gabrial, 2003;
Gurlebeck et al., 2006). The plant immune system is based on the recognition of pathogen-associated molecular
patterns (PAMPs), resulting in the activation of defense responses (Niruberger and Lipka, 2005). From the
perspective of plant disease epidemiology, pathogen spread and multiplication is important in assessing the
likelihood of disease outbreaks. In this paper we aimed to understand the dynamics of Xav population sizes in
regard to symptom development on cowpea plant
2. Materials and methods
Three Ife-brown seed lots were collected from different cowpea seed companies and were already treated with
different fungicides; Alheri seed treated with Apron star (Alheri A), Premier seed treated with Dress force
(Premier D), Masalaha seed treated with Team (Masalaha T). Three seed lots of local varieties were purchased

Vol.3, No.7, 2013
30
from open markets within Zaria; these were Local Wusasa (Local W), Local Sabon-Gari (Local SG), and Local
Samaru Local S). Two seed lots, IT86D-721 and IT98-503-1 obtained from International Institute of Tropical
Agriculture (IITA). Five seed lots of SAMPEA-7 and one Local Ife-brown were obtained from Institute for
Agricultural Research and open market respectively. These were SAMPEA-7 treated with Apron star
(SAMPEA-7A), SAMPEA-7 treated with Team (SAMPEA-7T), SAMPEA-7 treated with Dress force
(SAMPEA-7D), untreated SAMPEA-7 as control (SAMPEA-7C), inoculated SAMPEA-7 (SAMPEA-7I), and
inoculated Ife-brown (Ifb I) making a total of fourteen seed lots, that were used in the trial. The initial
populations of Xav were quantified by ground up four hundred seeds each of the seed lot and plated on YDCA
media before sowing from which population build were monitored. The inoculated Ife-brown was as a result of
its moderate resistance to Xav, and inoculated SAMPEA-7 was as a result of its known susceptibility to Xav to
serve as positive control. These non treated but inoculated seeds and the control were first of all soaked in
ethanol for twenty minutes to remove inherent pathogens after which, the seeds were washed three times in
sterile distilled water. The different fungicides used by the seed companies were Apron-star (tiamethoam 20 % +
metalaxyl-m 20 % + difenocoazole 20 % w/w), Dress force (imidacloprids 20 % + metalaxyl-m 20 % +
tebuconazoles 20 %) and Team (carbendazin 12 % + mancozeb 63 %). These were carefully selected to reflect
the various sources farmers usually obtained seeds. Seeds were inoculated by soaking hundred seeds in 100 ml
of bacteria suspension adjusted to ca 4.7 x 107
cfu/ml for four hours before fungicide treatment. The fungicidal
treatment was done at the rate of 2 g/kg of seeds. Seeds from each seed lot were planted in plastic pots of 25 cm
diameter filled with sterile soil. Each seed lots were planted at the rate of 3 seeds per pot but thinned to 2 plants
per pot after seedling establishment, with 5 replications. The seeded pots were placed randomly in the screen
house and observed for germination. Disease incidences were taken by counting the number of infected plants
and severity were scored using a modified CIAT 1-9 scale (Opio et al., 1993).
After which the plants were observed for a typical blight symptoms on cotyledons, stem, leaves and general
seedling mortality for two weeks. Two weeks after, the plants in the pots were spray inoculated with the
suspension of bacterial blight pathogen adjusted to ca.4.5 x 107
cfu/ml. At 14, 28, 42, and 56 DAS, leaf and stem
discs measuring about 2 mm2
were taken from the plants using cork borer. The discs were teased in drops of
SDW and a loopful of the resulting suspensions was mixed in 10 ml of SDW from which a serial dilution of up
to 10-5
were prepared. Ten micro milligrams of the suspensions were placed on YDCA in Petri-dishes and spread
using glass spreader and incubated at 28 0
C for 48 h.
To determine the number of infected pods and consequently aborted pods, ten plants were randomly chosen in
pots and tagged accordingly at 42, 49, 56, 63 and 70 DAS.
The number of flower fallen without the pod initiation was counted (aborted flower). The total number of
flowers and pods produced by the tagged plants were recorded, from which, the percentage of infected aborted
flower and pods were calculated. The experiments were laid out using CRD. Data collected were analyzed
statistically using ANOVA and means were separated using New Duncan’s multiple Range Tests (NDMRT).
The trials were repeated once.
3. Results
Table 1; shows the incidence; severity and population build-up on germinating and emerging seedling at 14 DAS.
There was significant difference (P < 0.05) between the treatments in terms of germination though the control
treatments appeared to be the lowest (60 %) which was as a result of soaking the control seed in ethanol for 20
minutes. There was significant difference between the treatments in terms of seedling establishment. While the
control treatments were recording 100 % establishment Masalaha seed treated with Team fungicide had 50 %
seedling establishment. There was high disease incidence (67 %) in local variety obtained from Sabon Gari
market compared to the control seedlings (0.00). The disease severity was higher on all the SAMPEA-7 varieties
(both treated and untreated) which were statistically higher than other treatments and there was however,
significant difference between all other treatments and the control. The population build up in vivo was higher in
SAMPEA-7 seeds treated with Apron star (4.5x 108
) and was statistically difference from other SAMPEA-7
treatments, variety IT98K-503-IT and IT86D-721A. IT98K-503-I T Masalaha T, and Alheri A had lower in vivo
population build up (2.0x108
) but were statistically difference from Premier D, MasalahaT, IT86D-721A, local
W, Local SG and Local S. Although initial Xav population on the non-treated inoculated seeds was higher
(4.7x107
), there was no statistics difference in terms of the effect of pathogen on the seedling parts and the
population builds up in vivo compared to other initial populations and their effects on seedling parts and the
resultant population builds up in vivo. The result demonstrated the non significance effect of fungicides on Xav.
Fig.1. shows the disease incidence and severity after inoculation. At 14 DAS the disease incidence was higher in
SAMPEA-7I followed by SAMPEA-7D, SAMPEA-7T and SAMPEA-7A. There was no statistical difference
between all other treatments except the control. Similar results were also obtained in the disease severity, where
the severity was higher in all SAMPEA-7 varieties except the control, LIfbI which is statistically comparable to

Vol.3, No.7, 2013
31
IT98K-503-IT, IT86D-721A, MasalahaT, Premier D and Alheri A. There was no statistical difference between
Local W, Local SG, and Local S. At 28 DAS disease incidence was higher in SAMPEA-7I, SAMPEA-7 D and
SAMPEA-7T followed by SAMPEA-7A. There was no statistical difference between all other treatments except
the control. The disease severity was however high in SAMPEA-7A followed by Masalaha T, IT98K-503-IT
and Local W and was statistically comparable to all the other treatments except the control. At 42 DAS, there
was no statistical difference between all the treatments except Alheri A. In disease severity however, SAMPEA-
7D had the highest severity and were comparable to SAMPEA-7I, SAMPEA-7T, SAMPEA-7A, Local S,
MasalahaT, Premier D.f and Alheri A. The lowest severity was obtained in IT86D-721A but was comparable to
IT98K -503-IT, Local W, Local SG and LIfbI. At 56 DAS, the disease incidence was higher on all SAMPEA-7
variety. There was no statistical difference between all other treatments except Alheri A. The disease severity
was higher in SAMPEA-7A which was comparable to SAMPEA-7T, SAMPEA-7D and SAMPEA-7I followed
by AlheriA which was comparable to Premier D Masalaha T, Local W and Local SG. The lowest severity was
obtained in Local S and LIFbI.
Table 2 shows the severity of Xav on floral parts. The percentage of flower aborted at 42 DAS was higher on
IT98K-503-IT (29.85 %), the least was SAMPEA-7C which was the control. There was statistical difference
between all the treatments. There was statistical difference between the percentage of flower aborted on all the
treatments and the control at 49 DAS, similar trend of results were obtained at 56, and 63 DAS. At 63 DAS,
however, the highest pod abortion was recorded on IT86D-721A (46.40 %) and there was statistically difference
between all other treatments and the control (10.40 %). At 70 DAS, the severity on pod was higher (4.65 %) on
MasalahaT and there was statistical difference between all other treatments and the control.
4. Discussion
Cowpea bacterial blight is a seed-borne disease (Cafati and Saettler, 1980) and seed treatment is an effective
method of controlling seed-borne diseases (Sikirou, 1999). But the result of this work showed that all the
fungicides used for the treatment did not have significant effect on the pathogen. These results confirm the report
of McMullen and Lamey (2000) and Shenge (2007) that most fungicides do not control bacterial pathogens and
most will not control all types of fungal diseases. The seed treatment chemical must be pest specific. The
percentage germination of control treatments was lower (60 %) than the other treatments. This was as a result of
soaking control seeds in ethanol for 20 minutes. Gilbert and Peaden (1987) and Taormina et al. (1999) reported
that ethanol was very effective in killing naturally occurring microorganisms in/on seeds but it inhibits seed
germination. The variable effect of Xav on the seedling establishment may be partly due to variation in the
location of the pathogen on the seeds (Shoaga, 1996). The internally located pathogen (especially on the embryo
can cause seedling mortality while the pathogen located on the surface of the seed will contaminate the soil
(Murty and Devadath, 1984). Similarly the variability in disease incidence, severity and population of Xav in
vivo could be attributed to the differential susceptibility of the different varieties. Varietal susceptibility or other
wise of cowpea to bacterial blight have been reported (Singh and Monoz, 1999), while Shoaga (1996) reported
the variability in pathogenicity of different isolates of Xav and Okechukwu et al. (2010) reported the variability
in pathogenicity of different isolates of Xav due to different agro ecological differences. From (Table 1), there
was no clear correlation between initial pathogen population and disease incidence and severity. The result
corroborate the report Hirano and Upper, (1983) that many pathogenic bacteria multiply quite successfully in
association with susceptible tissues without causing lesions .Spread of the pathogen often occur independent of
lesion occurrence (Upper et al., 2003) and when pathogen population remain modest in size, host and pathogen
coexist without disease..
Lesions on the other hand, occur when extensive multiplications require the bacterial to rely on expression of
T3SS (Darsonval et al., 2003). In compatibles interactions, defense responses are suppressed by T3SS effectors
(DebRoy et al., 2004; Boureau et al., 2006). This result corroborate the report of Rouse et al. (1985) that as
population sizes increase, so does the probability of disease development. While the disease incidence remain
fairly constant, disease severity steadily declined at 28 and 42 DAS. There was general increase in flower
abortion from 42-49 DAS and there was no apparent varietal difference in CoBB effect on flower. Similar
results were also observed in pod abortion. Most yield losses, however, presumably result from high flower and
pod abortion (Goodwin, 1992). The result of this work is completely at variance with the report of Leith and
Reynolds (1988) that the effect of a phytophagous pest on plant growth was considered to be equivalent to the
amount of defoliation it caused, that is reduction in radiation interception (RI). However, this will not be
appropriate for bacterial blight of cowpea, because photosynthesis is reduced by more than merely a loss of
radiation interception (RI) due to blight lesions (visibly destroying green leaf tissue) but reduce radiation use
efficiency (RUE) (Goodwin, 1982; Upper et al., 2003). Similar to other bacterial leaf spot pathogens, Xav may
colonize cowpea plants without inducing visibly symptoms. Symptomless systemic infection of cowpea could
also result in seed contamination in absence of foliar symptoms (Barak et al., 2002). In such case, field

Vol.3, No.7, 2013
32
inspection of seed crops would fail to reveal this source of seed infection.
5. Conclusion
Fungicides used for the treatment of seeds had no significant effect on the pathogen growth and pathogenicity.
Although, seed treatment has been adjudged to be the best method of control soil and seed-borne diseases, seed
treatment chemical must be pest specific. Population build up did not correlate to foliar symptoms (lesion)
developments. Visibly lesion free cowpea field is not a guarantee to get bacterial free seed especially for the seed
companies. Thus, it appears advisable to perform seed assays to confirm that seed lots are not contaminated with
Xav particularly if such seed lots are to be planted in areas where conditions are favorable for development of
bacterial leaf blight disease. The usual practice of taking disease incidence and severity should be discouraged. I
would recommend pathogen population assessment should be undertaken in conjunction with disease incidence
not disease severity. Experiment to evaluate cowpea resistance genotype should be conducted on cowpea plant
from germinating to production stage since resistance varies from vegetative to reproduction stage (flower and
pod abortion).
Acknowledgement
We wish to sincerely thank Mrs. M. Haman for her role played in the collection of seed samples used in this
work. We acknowledge with thanks the technical support received from mall am A. Aliu and the statistical
analysis done by the staff of the data processing unit of the Institute for Agricultural Research, Samaru.
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Table 1: Incidence, Severity and Population build-up of Xav on Germinating Cowpea Seedling 14 DAS in
2011 Two Trials Combine Analysis
Means in a column followed by the same letter are not significantly different at p ≤ 0.05 level of significance
using NDMRT test.
Table 2: Flower and Pod aborted and Severity of Xav on Pod in 2011 Two Trails Combine Analysis
% Flower Aborted % Pod Aborted Severity on Pod
Source/ Variety 42 49 56 63 70 DAS
Alheri A 20.60l 30.65j 53.95j 43.65h 4.05g
Premier D 24.85h 30.65j 60.15a 42.95k 4.00h
Masalaha T 29.00c 33.60c 59.10b 45.90c 4.65a
IT86D-721A 23.85 i 32.10g 57.45d 46.40a 4.60b
IT98K-503-1T 29.85a 31.10i 54.45i 45.60d 4.20f
Local W 24.30k 32.20f 54.60h 43.05j 4.25d
Local SG 24.95h 31.40h 55.60g 45.10e 3.85j
Local S 25.45g 32.80d 55.60g 43.30j 4.05g
SAMPEA-7A 29.10b 34.10b 57.75c 44.35f 3.95i
SAMPEA-7T 26.20f 31.40h 55.60g 43.45i 4.21e
SAMPEA-7D 26.60e 33.60c 56.15e 46.20b 4.30c
SAMPEA-7I 27.10d 53.10a 55.75f 43.80g 4.00h
Ife-brown I 24.40j 32.45e 54.45i 42.25l 4.20f
SAMPEA-7C
S.E
3.50m
0.70
5.40k
0.67
10.10k
1.22
10.40m
1.34
0.00k
1.37
Means in a column followed by the same letter are not significantly different at 5 % level of significance using
NDMRT test.
Source/Variety Initial
Population
Germination % Establishment % Incidence
%
Severity Population
cfu/ml
Alheri A 1.3x103
65.0c 56.30f 27.7j 2.0i 2.0x108
j
Premier D 1.0x103
70.0b 58.40c 50.0i 2.4e 2.0x108
j
Masalaha T 1.3x103
65.0c 50.0h 50.0i 2.1h 2.9x108
g
IT86D-721A 1.3x103
70.0b 51.0g 49.8j 2.4e 3.2x108
f
IT98K-503-1T 1.0x103
75.0a 57.0e 52.1g 2.1h 3.4x108
e
Local W 1.9x103
70.0b 58.3d 64.0b 2.3f 3.5x108
d
Local SG 1.4x103
70.0b 57.0e 63.4c 2.3f 2.3x108
h
Local S 3.3 x103
70.0b 57.0e 67.3a 2.2g 2.2x108
i
SAMPEA-7A 2.7 x103
70.0b 43.0f 52.4f 4.6b 4.5x108
a
SAMPEA-7T 3.1 x103
70.0b 58.3d 50.0i 4.8a 3.3x108
e
SAMPEA-7D 1.0 x103
65.0c 57.0e 50.3h 4.3d 3.2x108
g
SAMPEA-7I 4.5 x107
65.0c 57.3e 62.4e 4.4c 3.8x108
b
Ife-brown I 4.5 x107
70.0b 64.0b 62.9d 4.3d 3.6x108
c
SAMPEA-7C 0.00 60.0d 100a 0.00k 0.0j 0.00k
S.E 0.74 0.93 0.84 0.92 0.91

Vol.3, No.7, 2013
34
0
10
20
30
40
50
60
70
80
90
100
Incidence Severity Incidence Severity Incidence severity Incidence Severity
Incidence/SeverityofCoBB
FIG 1:Disease Incidence and Severity at 14,28,42 and 56 DAS after spray
inoculation
Alheri A Premier D.F Mashlaha T IT86D-721A IT98K-503-1T
LOCAL W lOCAL SG lOCAL S SAMPEA-7A SAMPEA-7T

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