IDENTIFICATION OF PATHOTYPES OF XANTHOMONAS ORYZAE PV. ORYZAE CAUSING BACTERIAL BLIGHT OF RICE IN BANGLADESH

Authors: S. M. K. H. Chowdhury2, I. H. Mian1 and M.A.I. Khan3

Abstract

M. K. H. Chowdhury, I. H. Mian and M.A.I. Khan. 2013. Identification of pathotypes of Xanthomonas oryzae pv. oryzae causing bacterial blight of rice in Bangladesh. Bangladesh J. Plant Pathol. 29 (1&2):21-27.

An investigation was conducted to identify pathotypes or races of Xanthomonas oryzae pv. oryzae causing bacterial blight of rice depending on the reaction patterns to a set of nine near-isogenic lines (NILs) or differentials. Ten isolates of X. oryzae pv. oryzae collected from different locations of Bangladesh were tested on the NILs namely IRBB2, IRBB4, IRBB5, IRBB7, IRBB10, IRBB11, IRBB13, IRBB14 and IRBB21 harboring the resistance genes Xa2, Xa4, xa5, Xa7, Xa10, Xa11, xa13, Xa14 and Xa21, respectively. The resistance frequency was maximal in IRBB21 followed by IRBB4, IRBB5 and IRBB7.  Among the differentials (NILs), the resistance frequency was the lowest in IRBB13 followed by IRBB10, IRBB2, IRBB14 and IRBB11. Based on susceptibility of the differentials and virulence of the isolates in terms of lesion length and infection frequency the isolates were grouped into 8 different pathotypes or races. Of them three isolates (ISO3, ISO4 and ISO6) showed identical virulence on the differentials and they were grouped into pathotype 1. Other seven isolates were grouped into 7 different pathotypes (Pathotype 2 to 8). The most avirulent isolate was ISO-1 followed by ISO-5 and ISO-10. Only one differential, IRBB21 showed resistant reaction to all isolates whereas IRBB13 was susceptible to all isolates except ISO1.

 

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INTRODUCTION

Bacterial blight (BB) caused by Xanthomonas oryzae pv. oryzae is one of the major diseases of rice (Oryza sativa L.) in Bangladesh. The disease appears every year with different degree of severity (Jalaluddin et al. 2005). It is noted as the most destructive and wide spread bacterial disease in the tropics and sub-tropics (Mew 1989, Ezuka 2000). The average yield of rice in Bangladesh is lower compared to the other rice growing countries of the world. There are many constraints responsible for low yielding, among them disease are considerable as the most important one. Bacterial blight disease has been recognized as one of the most damaging disease of rice in Bangladesh. But, comprehensive report on the effects of BB on rice yield in Bangladesh is not available.  Reports from other rice growing countries reveal that yield loss due to BB varied from 2% to 74% depending on location, season, crop growth stage and genotype (Kumar et al. 2013).

    Present recommendations for the management of bacterial blight of rice are use of host resistance, modifications in cultural practices, use of biological control agents, botanical extracts, natural products, and conventional and non-conventional chemicals. Rice genotypes showing resistance to bacterial blight in different countries are also recorded (Ou 1985, Anon. 1988).  Use of resistant varieties is the most effective and economic management practice against bacterial blight of rice. When different strains of a bacterial pathogen are present, it is recommended to grow resistant varieties possessing field resistant genes to overcome development of new races in the pathogen populations (Khan et al. 2009). Khan et al. (2009) identified virulence differentiation of 41 isolates of X. oryzae pv. oryzae collected from different areas of Bangladesh, based on strain-cultivar interaction using 9 near isogenic lines of rice with different resistance genes. Leaves were inoculated following leaf clipping method. The virulence of the strains was differentiated distinctly. Weak interaction patterns were found between bacterial isolates and rice cultivars, but there were specific interactions. They suggested that X. oryzae pv. oryzae could be divided into 6 pathotypes.

 Environment, rice cultivation with new varieties, BB incidence/severity might cause a shift in pathogen-host interaction. Therefore, regular investigations especially before development of durable resistant rice varieties to BB, is necessary to identify the existing pathotypes or races of the causal pathogen. Considering the above point, the present investigation was undertaken to identify pathotypes or races of X. oryzae pv. oryzae (Xoo) isolates collected from selected areas of Bangladesh.

MATERIALS AND METHODS

Leaf samples of rice infected with Xanthomonas oryzae pv oryzae (BB) were collected from farmers’ fields of Gazipur, Comilla, Chittagong, Rangpur and Dinajpur districts in Bangladesh during 2010-2011. Ten isolates of X. oryzae pv. oryzae were established from the collected samples using peptone sucrose agar (PSA) medium (Jalaluddin et al. 2005). Single colony of pure culture of each isolate was prepared and maintained in PSA medium slants. Bacteria were mixed in sterilized clay suspension and stored under room condition for long term use (Hossain 2001).

The isolates were designated using an abbreviation ‘ISO’ for isolate and a serial number.  Pathogenicity of the isolates was tested on a set of nine near-isogenic lines (NILs) namely IRBB2, IRBB4, IRBB5, IRBB7, IRBB10, IRBB11, IRBB13, IRBB14 and IRBB21 harboring the resistance genes Xa2, Xa4, xa5, Xa7, Xa10, Xa11, xa13, Xa14 and Xa21, respectively (Huang et al. 1997). This set of NILs was developed by IRRI, Philippines using background of IR24.

Seeds of the NILs were collected from Bangladesh Rice Research Institute (BRRI). The BRRI obtained them from International Rice Research Institute (IRRI), Manila, Philippines. The experiment was conducted in the Experimental Farm of BRRI, Gazipur during T. Aman season, 2011. Seedlings of each differential were raised in earthen pots containing sandy loam soils mixed with well decomposed cow dung. Thirty days old seedling was transplanted in the field at one seedling per hill maintaining row to row and hill to hill distances of 20 cm. Ten sets of nine NILs were planted for ten isolates maintaining 40 cm set to set distance.

The isolates of X. oryzae pv. oryzae were grown on PSA slants separately for 72 hours at 30°C. Inoculum of each isolate was prepared by mixing the cultured bacteria in 10 ml sterile distilled water in a test tube. Before inoculation, the concentration of the bacterial suspension was adjusted to 108 CFU/ml using sterile distilled water. At the maximum tillering stage, 8-10 leaves/hill were inoculated by clipping with sterile scissors dipped in the bacterial suspensions. Three hills of each of the NILs were inoculated with individual isolate (Khan et al. 2009).

For data collection, a total of 20 leaves infected with BB were collected after 21 days of inoculation. Data on lesion length on each leaf was measured. Disease reactions of the NILs to the isolates were categorized based on lesion length, where less than 3 cm was considered as resistant (R) and more than 3 cm was rated as susceptible (S) according to Li et al. (2008). The isolates of X. oryzae pv. oryzae produced more than 3 cm long lesion on inoculated leaves were considered as virulent and the NILs were considered as susceptible (S), and those produced less than 3 cm long lesion were noted as avirulent and the NILs as resistant (R).

RESULTS AND DISCUSSION

Reactions of nine near-isogenic lines (NILs) of rice to X. oryzae pv. oryzae isolates

Mean lesion length of bacterial blight developed on nine near-isogenic lines (NILs) owing to inoculation with ten isolates of X. oryzae pv. oryzae varied greatly. Each of ten isolates produced a range of lesion size, showing the largest size lesion on a single NIL and the shortest on another NIL. These two extreme values were significantly different. The intermediate values some were significantly different from the highest value and some were different from the lowest value (Table 1).

Significantly the highest mean lesion length was recorded from NIL IRBB10. The second highest mean lesion length was found on IRBB11 followed by IRBB4 and IRBB2. The lesion length of three rice NILs was statistically similar but significantly higher compared to other NILs except IRBB10. The average lesion length on leaves of IRBB5, IRBB13 and IRBB7 was also statistically similar and significantly higher compared to only IRBB14 and IRBB21. The lowest lesion length was found in IRBB21 followed by IRBB14. The response of two NILs to the pathogen was significantly different (Fig. 1).

The mean lesion length on nine-rice NILs caused by isolates ISO4, ISO6 and ISO3 of X.  oryzae pv. oryzae was statistically similar and significantly higher compared to other seven isolates. The mean lesion length developed by ISO2, ISO8 and ISO9 was also statistically similar and significantly higher compared to ISO1, ISO5 and ISO10. The differences in average lesion length of later three isolates were not significant (Fig. 2).

Virulence diversity among ten isolates

The isolates of X. oryzae pv. oryzae were polymorphic for virulence to nine NILs of rice possessing the resistance genes Xa2, Xa4, xa5, Xa7, Xa10, Xa11, xa13, Xa14 and Xa21. Ten isolates collected from naturally BB infected rice plant grown in the districts of Rangpur, Dinajpur, Gazipur, Jhalokati, Comilla and Chittagong were classified into 8 pathogenic groups, which were tentatively designated as pathotype 1 to 8 on the basis of their pathogenicity on nine NILs. The reaction patterns of the 8 pathogenic groups were respectively SSSSSSSSR, SRRRSRSRR, RRRRSRSRR, RSSSSSSSR, SRRRSSSRR, RRRRSRSSR, RRRRRRSRR and RRRRRRRRR. None of the isolates showed virulence effect on all the NILs (Table 2).

identification-of-pathotypes-of-xanthomonas-oryzae-pv-oryzae-1 identification-of-pathotypes-of-xanthomonas-oryzae-pv-oryzae-2 identification-of-pathotypes-of-xanthomonas-oryzae-pv-oryzae-3 identification-of-pathotypes-of-xanthomonas-oryzae-pv-oryzae-4

All tested isolates were avirulent to only one differential (IRBB21). Isolates ISO3, ISO4 and ISO6 were virulent to all NILs except IRBB21 which were grouped into pathotype 1. The isolate, ISO1 was avirulent to all the NILs which was placed in another pathotype designated as pathotype 8. Isolate ISO7 was avirulent to IRBB2 and IRBB21 containing resistance genes Xa2 and Xa21 respectively. Other isolates (ISO2, ISO5, ISO7, ISO8, ISO9 and ISO10) showed variable reaction patterns on the NILs and they were classified into different pathotypes and designated as pathotype 2 to 7, respectively (Table 2 and Fig. 3).

Differential characteristics of near-isogenic lines

Ten isolates were tested to assess the differential characteristics of nine NILs. Genotype IRBB21 showed resistant reaction to all isolates showing lesion length of 0.55-2.71 cm with mean 1.57 cm. IRBB13 (xa13) showed resistant reaction to only ISO1 having lesion length of 1.60 cm and IRBB10 possessing resistant gene Xa10 was resistant to ISO1 and ISO10 showing lesion length of 1.18 cm and 1.20 cm, respectively. Three genotypes, IRBB4, IRBB5 and IRBB7 containing the resistant genes Xa4, xa5, Xa7 respectively were resistant to 6 isolates (ISO1, ISO2, ISO5, ISO8, ISO9 and ISO10). In other NILs, IRBB2, IRBB11 and IRBB14 showing resistant reaction against 5 different isolates and lesion length ranged 0.51-23.73, 0.67-21.21 and 0.82-13.37 cm with means of 7.95, 8.57 and 4.29 cm, respectively (Tables 1&2 and Fig. 1).

Resistance frequency of isolates on rice near-isogenic lines (NILs)

The resistance frequency of the isolates on the rice near-isogenic lines having specific gene for resistance is shown in Table 3 and Fig. 2. The BB resistance gene showed 10 to 100% resistant reaction to the isolates of X. oryzae pv. oryzae tested in the present study. The highest resistance frequency was found in IRBB21 (Xa21) followed by IRBB4 (Xa4), IRBB5 (xa5) and IRBB7 (Xa7). The lowest resistance frequency was found in IRBB13 (xa13) followed by IRBB10 (Xa10), IRBB2 (Xa2), IRBB14 (Xa14) and IRBB11 (Xa11).

identification-of-pathotypes-of-xanthomonas-oryzae-pv-oryzae-5

Table 3. Resistance genes of rice near-isogenic lines (NILs) and their resistance frequency to 10 Bangladeshi isolates of X. oryzae pv. oryzae

NILs Resistance gene Resistance frequency (%)a
IRBB2 Xa2 50
IRBB4 Xa4 60
IRBB5 xa5 60
IRBB7 Xa7 60
IRBB10 Xa10 20
IRBB11 Xa11 50
IRBB13 xa13 10
IRBB14 Xa14 50
IRBB21 Xa21 100

aResistance frequency was calculated as the ratio of isolates including resistance reaction vs. total isolates tested on each NILs.

Pathotypes or races of bacterial plant pathogens are generally grouped on the basis of virulence phenotypes on a set of differential varieties of rice (Jeung et al. 2006, Liu et al. 2007, Khan et al. 2009). In the present investigation, nine NILs of rice with distinct genes for bacterial blight resistance were tested in determining virulence of ten isolates of X. oryzae pv. oryzae collected from different locations of Bangladesh. The symptoms developed by those isolates were clear to distinguish virulent and avirulent isolates (Fig. 3). Based on susceptibility of the differentials and virulence of the isolates, in terms of lesion length and infection frequency, the isolates were grouped into 8 different pathotypes. Similar results were reported by other investigators who worked in other countries with other isolates of the pathogen (Noda et al. 2001, Khan et al. 2009). Khan et al. (2009) tested 41 Bangladeshi isolates of X. oryzae pv. oryzae using the same set of differentials and grouped them into 6 pathotypes. Pathotypes 1 contained 33 isolates, 2 contained 3 isolates and pathotype 3 contained 2 isolates. Each of pathotypes 4, 5 and 6 contained only one isolate. The BB resistance gene showed 9.09 to 63.64% resistant reaction to the isolates used by them. In the present study, 8 pathotypes were identified. Among them ISO3, ISO4 and ISO6 were most virulent. Among the differentials IRBB13 (xa13) and IRBB10 (Xa10) were susceptible to almost all the tested isolates. In China, xa5 and xa13 genes were found resistant to almost all the isolates tested by Noda et al. (2001). It indicates that Bangladeshi isolates tested in the present investigation are different from those in China (Li et al. 2008, Liu et al. 2007). The NIL IRBB21 (Xa21) was found resistant to all pathotypes tested in the present study.  IRBB5 and IRBB7 showed complete resistance to all isolates, and IRBB2 and IRBB11 were susceptible to all isolates in Vietnam, indicating that Bangladeshi isolates are different from those in Vietnam (Lai Van et al. 1999).

Based findings of te investigation it may be concluded that wide variations exist in the populations of X. oryzae pv. oryzae that cause bacterial blight of rice in Bangladesh. There are at least 8 different pathotypes in the populations of the BB pathogens occur in the country. The isolate ISO4, along with ISO3 and ISO6, was the most virulent whereas ISO1showed the most avirulent pathotype. All isolates were avirulent to only one differential, IRBB21 (Xa21) tested in the present study.

ACKNOWLEDGEMENTS

The authors express their gratefulness to Research Management Committee (RMC) of Bangabandhu Sheikh Mujibur Rahman Agricultural University (BSMRAU), Gazipur-1706, Bangladesh for providing fund to undertake the research work. The authors also express their indebtedness to Plant Pathology Division, Bangladesh Rice Research Institute (BRRI), Bangladesh for providing research materials and laboratory facilities to undertake the study.

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1Professor and 2Graduate Student, Department of Plant Pathology Bangabandhu Sheikh Mujibur Rahman Agricultural University, Gazipur-1706, Bangladesh, and 3Senior Scientific Officer, Bangladesh Rice Research Institute, Gazipur-1701, Bangladesh
(This is a part of MS thesis of first author)

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