Pathogenic Diversity of Xanthomonas oryzae pv. oryzae Isolates in Togo
The present study aimed at determining Xanthomonas oryzae pv. oryzae pathogenic diversity. Therefore, the disease survey was conducted in three ecozones of Togo-Forest savanna transition, Forest and dry savanna zones and the diseased samples were collected, the bacteria isolated and characterized by testing their virulence on 21 rice genotypes. The results revealed the occurrence of the bacterial leaf blight in the three ecozones. High bacterial leaf blight incidence of 50-65% was observed in Forest savanna transition and Forest zones, while it was up to 70% in the dry savanna zone. The highest incidence (70%) was recorded in dry savanna zone and the lowest (25%) was frequently observed in Forest zone. Pathotyping analysis of 13 bacterial isolates from samples collected from the infected fields against 21 rice genotypes to isolate and characterize bacteria virulence was carried out. Thus, AMMI cluster analysis revealed the existence of 3 Pathotypes (Pt) among these isolates: PtA highly virulent has 1 isolate, PtB virulent was made up of 3 isolates and PtC moderately virulent was made up of 9 isolates. At ecozone the analysis revealed the presence of PtB and PtC in the Forest savanna transition zone, PtA and PtC in Forest zone and PtB and PtC in the dry savanna zone. The present results provided knowledge on the bacteria virulence and its structure across ecozones of Togo and provided useful information for selection of genotypes for durable resistance to the disease.
April 02, 2012; Accepted: July 30, 2012;
Published: October 10, 2012
Rice is one of the most important staple crops worldwide. Unfortunately, its
production is constrained by several diseases. Bacterial leaf Blight (BB), caused
by the vascular pathogen Xanthomonas oryzae pv. oryzae (Xoo) (Swings
et al., 1990), is one of the most serious diseases of rice. The disease
is widespread throughout Asia and also was reported from rice-growing areas
in Australia, united states and several countries in Latin America and Africa
(Mew et al., 1993; Sere
et al., 2005). Bacterial leaf blight may cause damage at seedling stage
resulting in complete wilting or death of affected tillers. The infection at
the maximum tillering stage results in blighting of leaves. Yield losses of
up to 80% due to BB have been reported (Ou, 1985). Recently,
BB disease was reported in several farmers fields across ecozones in Niger,
Burkina Faso, Nigeria, Benin and Mali, with high incidence of 70-80 and yield
losses ranging from 50-90% (Sere et al., 2005).
In Togo, BB was reported to occur in several rice-growing areas in the North
part of Togo, with high incidence and severity in most of the fields visited
(Dewa et al., 2011). The authors observed a wide
spread of BB in farmers fields in West African rice growing countries including
Togo. Investigation on the pathological characterization of some Xoo isolates
from these countries have been undertaken by testing the virulence of the bacteria
and revealed a high level of virulence on cultivated rice varieties (Sere
et al., 2005; Dewa et al., 2011). Bacterial
leaf blight is characterized by a high degree of race-cultivar specificity.
Pathotyping analysis of 50 Xoo isolates from seven West African countries against
18 rice cultivars revealed two major pathotypes of Xoo virulence (Onasanya
et al., 2009). Although, preliminary studies on BB survey in the
North of Togo and the virulence test of Xoo isolates, no investigation on the
diversity of the pathogen was carried out. However, investigation on the Xoo
virulent population structure using isolates from different ecozones is important
for selection of varieties for durable resistance to BB. Therefore, the present
study aimed at collecting bacterial leaf blight samples from infected farmers
fields in different ecozones of Togo and characterizing Xoo isolates virulence
for identification of pathotypes.
MATERIALS AND METHODS
Survey of bacterial leaf blight and collection of samples: BB disease survey was conducted from 26 June to 5 July 2010 across three of the four main ecozones of Togo. A total of eight localities were covered by the survey: Lome Kovie and Davie in the Forest savanna transition zone (Maritime Region), Kpele Atime, Kpele Tutu and Sodo in the Forest zone (Plateau Region) and Koumbeloti and Tantiegou in the dry savanna zone (Savanna Region). In each locality, fields were visited and the number of infected plants were assessed in the four corners (1 m2 per corner) and in the centre of the field in order to establish the incidence of the disease at field level. In the infected fields, diseased leaf samples were collected from rice and/or weed plants showing typical BB symptoms, conditioned in envelopes and conserved in cool plastic boxes for transport. Once in laboratory, the samples were kept into a refrigerator at 4°C for further process.
Bacteria isolation: Bacterial isolation was carried out using Nutrient
Agar (NA) medium and purified on Glucose Yeast Calcium Agar (GYCA) medium for
further investigations. In laboratory, leaf samples were gently washed under
running water, rinsed with sterile distilled water and superficially sterilized
in 1% hypochlorite solution and rinsed again with sterilized distilled water.
Then, leaves were immerged into sterile distilled water for 30 min to reactivate
bacteria cells in leaf tissues. Very small pieces in size of each leaf sample
were cut and incubated in some drops of sterile distilled water in tubes and
incubated for about 3 h. Thereafter loopfuls of the suspension were streaked
on NA medium in petri dishes, which were incubated at 28°C for 48-72 h for
bacterial growth. Then, typical Xoo-like colonies identified according to Agarwal
et al. (1994) were checked and purified on fresh GYCA medium. To
confirm the identity of the isolates obtained, biochemical tests were performed:
Gram test was carried out using KOH3 and the growth test on a selective
medium was conducted using the nutrient agar+copper nitrate (Cu(NO3)2)
0,001% (Sere et al., 2005).
Pathotyping of Xoo isolates
Bacterial isolates: For the pathotyping test 13 Xoo isolates from
different ecozones were used. These consisted of one or two isolates per locality
depending on the occurrence of BB disease on rice plants only or on both rice
and weeds plants.
Plant materials: Thirteen Near Isogenic Lines (NILs) with known resistance gene to bacteria leaf blight obtained from the International Rice Research Institute (IRRI), six improved genotypes from Africa Rice Center, Cotonou, Benin and two improved genotypes from Institut Togolais de Recherche Agronomique (ITRA), Togo, widely grown in Togo were used for X. oryzae pv. oryzae pathotyping test (Table 1).
Experimental design: The experiment was carried out using the split plot design with 3 replications. Thirteen isolates (Table 2) from different ecozones were used to screen 21 genotypes (Table 1) in the screen house at Africa Rice Center in Cotonou, Benin. Rice grains were first pre-germinated in sterilized petri dishes under sterile conditions for 5 days and were transplanted in plastic pots. One plastic pot per genotype per Xoo isolate in 3 replications was used.
|| Varieties and near isogenic lines used for pathotyping of
|aCultivated in Togo, NILs: Near isogenic lines
|| Xoo isolates, their host plants and their reaction to biochemical
|aRice genotypes, bNot identified rice
cultivars, cWeed species, FST: Forest savanna transition zone,
F: Forest zone, DS: Dry savanna zone
Fertilizer application: For fertilization, 1.0 g of NPK 15-15-15 per pot was applied 8 days after transplanting and 0.2 g of Urea 46% per pot was applied 15 days after transplanting.
Bacterial suspension and inoculation: Inoculum was prepared using a
48 h old culture of Xoo isolates produced on GYCA was harvested from agar plates
and suspended in sterile distilled water and adjusted to a concentration of
109 CFU mL-1 (OD650 = 0.5) prior to use. Inoculation was
by clipping method (Sere et al., 2005). The whole
leaves of each plant in the plastic pot were clip inoculated 21 days after transplanting.
Data assessment: Evaluation consisted on the measurement of the lesion
length due to bacterial leaf blight disease induced by inoculation with each
of the isolates and also the measurement of the total leaf length 14 days after
inoculation (Sere et al., 2005). From these data,
the percentage of lesion length was determined for each inoculated leaf.
Data analysis: Using the percentage lesion length, Analysis of Variance
(ANOVA) and Additive Main Effect and Multiplicative Interaction (AMMI) analysis
were performed using IRRISTAT software to Xoo isolates virulence and identify
eventual pathotypes (Aleong and Howard, 1985; Bruckner
and Slanger, 1986; Ebdon and Gauch, 2002; Zhu
and Kuljaca, 2005). AMMI analysis was shown to be effective in understanding
complex Genotype by Environment interactions trials that are difficult to understand
using ordinary ANOVA (Ebdon and Gauch, 2002).
Survey and bacteria isolates: The survey revealed the occurrence of BB in the three ecozones, Forest savanna transition zone, Forest zone and dry savanna zone with variable incidence ranging from 25-70%. BB incidence of 25% was recorded in Lome (Forest savanna transition zone), Kpele Atime and Kpele Tutu (Forest zone), while it was 50% in Davie (Forest savanna transition zone), 60% in Sodo and Tantiegou (dry savanna zone), 65% in Kovie (Forest savanna transition zone) and 70% in Koumbeloti (dry savanna zone). BB was observed on rice plants but also on some weed species in some localities.
A total of 134 isolates have been obtained as X. oryzae pv. oryzae (Xoo) after biochemical tests with the pathogens isolated. The identified Xoo bacteria were obtained not only from rice plants but also from some weed plants. A total of eight weed species of three families were found to be host plants of Xoo: Commelina benghalensis (Commelinaceae) at Davie, Digitaria horizontalis (Poaceae) at Davie and Koumbeloti, Echinochloa colona (Poaceae) at Davie and Tantiegou, Leersia hexandra (Poaceae) at Kovie, Kpele Atime and Kpele Tutu, L. oryzoides at Kovie, Koumbeloti and Tantiegou, Panicum maximum (Poaceae) at Kovie and Kpele Atime, Sorghum arundinaceum (Poaceae) at Kovie and Zizania latifolia (Poaceae) at Tantiegou.
Virulence of Xoo isolates and pathotyping: The results on the percentage
of lesion length due to bacterial leaf blight revealed considerable variability
in the reactions of the 13 Xoo isolates to 21 genotypes tested. Analysis of
Variance (ANOVA) for the percentage lesion length due to inoculation with Xoo
isolates from different ecozones revealed significant interaction (p<0.05)
between Xoo isolates and rice genotypes (Table 3). Differential
reaction was observed for Xoo isolates of the same ecozone or locality of origin
|| Analysis of variance for percentage lesion length induced
by Xoo isolates
|*,**Significant at 5 and 1% level, ns: Non significant
|| Means of percentage BB disease lesion length due to inoculation
with Xoo isolates from different ecozones
|Isolates; I1: KV4-2, I2: KV14-2, I3: IL23-1, I4: DV39-1, I5:
DV58-2, I6: KA63-2, I8: KT84-2, I9: SD94-1, I10: KM101-1, I11: KM129-2,
I12: TN135-2, I13: TN160-2, Rice genotypes; G1: TGR203 (WITA4), G2: IR841,
G3: NERICA4, G4: NERICA8, G5: NERICA14, G6: NERICA19, G7: Gigante, G8: TOG
5681, G9: IRBB1, G10: IRBB2, G11: IRBB3, G12: IRBB4, G13: IRBB5, G14: IRBB7,
G15: IRBB8, G16: IRBB10, G17: IRBB11, G18: IRBB13, G19: IRBB14, G20: IRBB21,
G21: IR24, Values within row with different letters are significantly different
Significant different lesion length was observed between I1 (KV4-2) and I2
(KV14-2) from Kovie, with 12.35 and 1.31% of lesion length, respectively, I4
(DV39-1) and I5 (DV58-2) from Davie, with 29.15 and 5.67%, respectively in the
Forest savanna transition zone, between I7 (KT83-2) and I8 (KT84-2) from Kpele
Tutu, with 4.20 and 49.38%, respectively in the Forest zone, between I12 (TN135-2)
and I13 (TN160-2) from Tantiegou, with 4.28 and 17.80%, respectively in the
dry savanna zone. Also, Differential reactions were observed for Xoo isolates
of different ecozones or localities. Significant differences in percentage of
lesion length (p<0.01) were observed between isolate I8 (KT84-2) (49.38%)
from the Forest zone and I2 (KV14-2) (1.78%) from Forest savanna transition
zone, between I11 (KM129-2) (2.43%) from the dry savanna zone and I4 (DV39-1)
(29.15%) from Forest savanna transition zone and between I8 (KT84-2) (17.94%)
from the Forest zone and I10 (KM101-1) (1.31%) from the dry savanna zone (Table
Isolate I8 (KT84-2) recorded the highest percentage of lesion length of 49.39% on genotype G8 (TOG5681) and induced more than 12% of lesion length on three other genotypes. However, this isolate induced less than 3% of lesion on 7 of the 21 genotypes tested. Isolates I12 (TN135-2) and I13 (TN160-2) induced more than 12% of lesion length on at least 3 genotypes and reached the percentage of about 25%. However, they caused less than 3% of lesion length on at least three genotypes. Also, isolate I4 (DV39-1) exhibited high virulence on the genotypes G6 (NERICA19) and G8 (TOG5681) with the lesion length of 18.93 and 29.15%, respectively but induced less than 3% of lesion length on eight genotypes (Table 4).
The results showed that the highest means percentage of disease lesion length due to inoculation was recorded by the isolates I8 (KT84-2) with 8.61%, while the lowest lesion length was recorded by isolate I11 (KM129-2) with 1.42%. Analysis of variance for the means percentage of disease lesion length due to inoculation revealed significant difference between isolates (p<0.01) and classified them into 3 groups: The first group was made up of isolates I8 (KT84-2), I13 (TN160-2) and I12 (TN135-2) which recorded the highest means lesion length of 8.61, 8.41 and 7.03%, respectively; the second group included isolates I4 (DV39-1), I1 (KV4-2) and I9 (SD94-1) with means lesion length of 5.59, 4.05 and 3.65%, respectively and the third group was made up of isolates I11 (KM129-2), I10 (KM101-1), I2 (KV14-2), I6 (KA63-2), I3 (IL23-1), I5 (DV58-2) and I7 (KT83-2) with means lesion length ranging from 1.42-2.86% (Table 4).
Additive Main effects and Multiplicative Interaction (AMMI) analysis of percentage lesion length revealed differential reactions of isolates on genotypes tested and the cluster allowed identifying 3 groups of pathotypes among the 13 isolates from different ecozones: Pathotype A (PtA) made up of one isolate I8 (KT84-2) highly virulent, Pathotype B (PtB) made up of 3 virulent isolates including I4 (DV39-1), I12 (TN135-2) and I13 (TN160-2) and Pathotype C (PtC) made up of 9 moderately virulent isolates I1 = KV4-2; I2 = KV14-2; I3 = IL23-1; I5 = DV58-2; I6 = KA63-2; I7 = KT83-2; I9 = SD94-1; I10 = KM101-1; I11 = KM129-2 (Fig. 1, Table 5). At ecozone level cluster analysis revealed the presence of PtB and PtC in the Forest savanna transition zone, PtA and PtC in the Forest zone and PtB and PtC in the dry savanna zone. This indicates variability of the pathogen within and across ecozones.
|| Xoo identified pathotypes, their virulence and distribution
|PtA: Pathotype A, PtB: Pathotype B, PtC: Pathotype C, ++++:
Highly virulent, +++: Virulent, ++: Moderately virulent, FST: Forest savanna
transition zone, FZ: Forest zone, DS: dry savanna zone
||Pathotyping dendrogram of X. oryzae pv. oryzae
isolates using AMMI analysis, coefficient: fusion level
Bacterial leaf blight incidence was generally higher in the dry savanna zone
than in the other ecozones. Previous survey on rice diseases in the North part
of Togo reported higher BB incidence of >75% in dry savanna zone than in
Wet savanna zone with incidence <50% (Dewa et al.,
2011). Recent research conducted in five West African countries including
Niger, Burkina Faso, Nigeria, Benin and Mali revealed frequent occurrence of
BB in farmers fields across these countries with incidence ranging from
70-85% (Sere et al., 2005). In the present study,
high incidence of 70% was found in some ecozones. This indicates a wide spread
of bacterial leaf blight in farmers fields across West African countries including
Togo. The present survey coupled with the earlier investigation (Dewa
et al., 2011) revealed that BB is widely spread in rice growing areas
The variability of the BB incidence observed across ecozones could be related
to the environmental conditions. Earlier observations on cassava bacterial blight
caused by X. axonopodis pv. manihotis reported that the disease incidence
was higher in dry savanna zone than in Forest savanna transition, Wet savanna
and Forest zones (Banito et al., 2007). Also,
cassava bacterial blight was found in various ecozones across four West African
countries, with generally higher incidences in the savanna zones than in the
transition Forest zones (Wydra and Verdier, 2002). The
generally lower incidence of bacterial disease in the Forest zone compared to
the savanna zones may be due to the climatic conditions, since great differences
in night versus day temperatures in the savanna zones were reported to promote
the disease (Lozano, 1986). This could explain the higher
BB incidence found in dry savanna zone than in Transition and Forest zones.
Bacterial leaf blight was found to occur on some weed species such as Poaceae,
Cyperaceae and Commelinaceae in six of the eight prefectures visited. The occurring
on weeds such as L. oryzoides (L.) Sw., Z. latifolia (Griesb)
Turcz. ex Stapf, Leptochloa chinensis (L.) Nees and Cyperus rotundus
L. has been reported (Webster and Gunnell, 1992).
Host plant resistance is the best mean to control bacterial diseases of plant.
Therefore, knowledge on the pathogen population structure is important to identify
and select representative strains for screening genotypes for durable resistance
to the disease (Nelson et al., 1994; Choi
et al., 1998). In the present study, the virulence of X. oryzae
pv. oryzae isolates and the genotype by isolate interaction were evaluated
by inoculating 21 genotypes with 13 isolates from different ecozones of Togo.
Virulence test of 11 Xoo isolates revealed most of the isolates virulent and
one isolate from the dry savanna zone was highly virulent, while 3 isolates
were moderately virulent (Dewa et al., 2011).
AMMI analysis revealed diversity among isolates tested and identified 3 pathotypes.
Diversity in bacterial population and its structure and identification of pathotypes
have widely been documented (Restrepo et al., 2004;
Wydra et al., 2004; Jeung
et al., 2006). Pathotyping analysis of 50 Xoo isolates from seven
West African countries against 18 rice cultivars was carried out and Xoo virulence
was identified and characterized and two main groups of pathotypes were found
and clustered under five subgroups of pathotypes (Onasanya
et al., 2009). Virulence analysis undertaken by Liu
et al. (2007) revealed nine Xoo races from China rice grown regions.
Also, Banito et al. (2010) revealed the existence
of pathotypes among highly virulent X. axonopodis pv. manihotis strains
from different African origins.
The spatial movement of X. oryzae pv. oryzae pathogens is an
important factor to be considered in controlling bacterial leaf blight disease.
In the present study, virulent PtB was found in the Forest savanna transition
zone and in the dry savanna zone, while the PtC moderately virulent was found
in the Forest savanna transition zone, the Forest zone and the dry savanna zone,
indicating a certain movement of Xoo pathogens across ecozones. This is likely
due to possible contaminated germplasm exchange between ecozones. Several investigations
on pathogen migration have been carried out; for example the European continental
movement of the Erysiphe graminis (Brown et al.,
1991) has been reported. Recently, the presence of two pathotypes PtA (virulent)
and PtB (middle virulent) was reported in 7 West African countries including
Mali, Nigeria, Benin, Burkina Faso, Niger, Guinea and Gambia and suggested the
possible movement of the pathogen across these countries (Onasanya
et al., 2009). The present results revealed important genotype by
pathotype interaction and knowledge on population structure of Xoo virulence
across ecozones of Togo and provided useful information for selection of genotypes
with durable resistance to bacterial leaf blight. However, experiments under
field conditions in different environments are also needed.
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