Pathotypic variation of Xanthomonas oryzae pv. oryzae in Bangladesh

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1 Archives of Phytopathology and Plant Protection ISSN: (Print) (Online) Journal homepage: Pathotypic variation of Xanthomonas oryzae pv. oryzae in Bangladesh Md. Samiul Alam, Md. Rashidul Islam, Ismail Hossain, M.R. Bhuiyan & M.A.I. Khan To cite this article: Md. Samiul Alam, Md. Rashidul Islam, Ismail Hossain, M.R. Bhuiyan & M.A.I. Khan (2016): Pathotypic variation of Xanthomonas oryzae pv. oryzae in Bangladesh, Archives of Phytopathology and Plant Protection, DOI: / To link to this article: Published online: 04 Mar Submit your article to this journal View related articles View Crossmark data Full Terms & Conditions of access and use can be found at Download by: [Bangladesh Agricultural University] Date: 04 March 2016, At: 07:01

2 Archives of Phytopathology and Plant Protection, Pathotypic variation of Xanthomonas oryzae pv. oryzae in Bangladesh Md. Samiul Alam a *, Md. Rashidul Islam a, Ismail Hossain a, M.R. Bhuiyan b and M.A.I. Khan b a Department of Plant Pathology, Bangladesh Agricultural University, Mymensingh, Bangladesh; b Plant Pathology Division, Bangladesh Rice Research Institute, Gazipur, Bangladesh (Received 16 October 2015; accepted 25 January 2016) Bacterial Blight (BB) caused by Xanthomonas oryzae pv. oryzae (Xoo), a destructive disease of rice. Altogether, 96 isolates of Xoo were collected from 19 rice growing districts of Bangladesh in irrigated and rainfed seasons during 2014 to assess pathotypic variation. Pathotypic analyses on a set of 12 Near Isogenic Lines (NILs) of rice containing resistance genes viz. Xa1, Xa2, Xa3, Xa4, Xa5, Xa7, Xa8, Xa10, Xa11, Xa13, Xa14 and Xa21 and two check varieties IR24 and TN1 by leaf clip-inoculation technique. A total of 24 pathotypes were identified based on their virulence patterns on NILs tested. Among these, pathotypes VII, XII, and XIV considered as major, containing maximum number of isolates, (9.38% each) frequently distributed in North to Mid-Eastern districts of Bangladesh. Most virulent pathotype I recorded from Habiganj and Brahmanbaria. This pathotypic variation explained the pathogenic relatedness of X. oryzae pv. oryzae populations from diverse geographic areas in Bangladesh. Keywords: Xanthomonas oryzae pv. oryzae; pathotypes; variation; Bangladesh Introduction Rice (Oryza sativa L.) is the staple food for over half of the world s population (Khush 2005). Around 90% of the total rice is grown in Asia (Salim et al. 2003). The low yield of rice is attributed to various factors. Among them, vulnerability of crop to pests and diseases is important one and sometimes leading to disastrous consequence (Hossain 2001). As many as 43 rice diseases are reported in Bangladesh (Fakir 2000). Among rice diseases, Bacterial Blight (BB) of rice caused by Xanthomonas oryzae pv. oryzae (Ishiyama 1922; Swings et al. 1990) is one of the most destructive diseases of rice throughout the world (Mew et al. 1982; Mew 1987). Xanthomonas is a large genus of gram-negative and yellow-pigmented bacteria (Ishiyama 1922). The bacterium consists of straight rods, with a single polar flagellum; and exists in singular form, in pairs and sometime in chains (Swings et al. 1990). X. oryzae pv. oryzae enters rice leaf typically through the hydathodes at the leaf margin, multiplies in the intercellular spaces of the underlying epithelial tissues and moves to the xylem vessels to cause systemic infection (Noda & Kaku 1999). BB has two types of symptoms: First one, systemic infection known as kresek (Reddy 1989), results in desiccation of leaves and death, particularly of young transplanted plants. The symptom of second kind is known as leaf blight, that produce tarnish grey to straw coloured lesions along the vein with inner wavy margin at *Corresponding author. rasha740177@yahoo.com 2016 Taylor & Francis

3 2 Md. S. Alam et al. the maximum tillering and reproductive stage (Rafi et al. 2013). BB is a seed borne disease (Srivastava & Rao 1963) as was first observed by the farmers of Japan in 1884 (Tagami & Mizukami 1962). All growth stages of rice are susceptible to BB and yield loss due to the disease ranges between 20 and 30% and in the case of severe infection can be reduced up to 80%, based on the crop stage, degree of susceptibility, and environmental conditions in which it occurs (Ou 1985; Shin et al. 1992; Mew et al. 1993). Yield losses due to BB may reach 65 95% during epiphytotic seasons (Reddy et al. 1979). The disease appears every year with different degrees of severity in Bangladesh (Jalaluddin et al. 2005), since the introduction of the high yielding varieties (HYV) obtained from the Philippines during mid 1960s (Adhikari & Mew 1994). An increased application of nitrogen fertiliser in HYVs of rice encourages the disease occurrence. Different physical, cultural, and chemical techniques had been used for disease control but no durable solution was available due to high pathogenic variability. Therefore, the use of varietal resistance is the most appropriate strategy for controlling the disease (Pinta et al. 2013). But changes in the pathotypic or genetic structure within the pathogen population break down the host resistance responsible for an alarming issue on rice production against BB devastating effects. High degree of genetic diversity and pathotypic variation of Xanthomonas oryzae pv. oryzae strains were reported from major rice growing countries of Asia such as Bangladesh, China, India, Indonesia, Korea, Malaysia, Nepal, Srilanka and Philippines (Adhikari et al. 1994; Ochiai et al. 2000; Khan et al. 2009). There are over 30 reported X. oryzae pv. oryzae races from several countries (Mew 1987; Adhikari, Mew, et al. 1999; Noda et al. 2001). A set of races identified in the Philippines using five differential rice cultivars has been used widely for identifying and classifying resistance to bacterial blight in other cultivars (Mew 1987; Lee et al. 2003). Most strains from South Asia (Nepal and India) were virulent to cultivars containing the bacterial blight resistance gene Xa5, while most strains from other countries were avirulent to Xa5. The regional differentiation of clusters of X. oryzae pv. oryzae (Xoo) in Asia and the association of some pathotypes of Xoo with single clusters suggested that strategies that target regional resistance breeding and gene deployment are feasible (Adhikari et al. 1995). Pathogenic diversity of X. oryzae pv. oryzae strains from Yunnan Province of China revealed that the strains were polymorphic for virulence to 12 Near Isogenic Lines (Noda et al. 2001). The assessment of genetic and pathogenic diversity of Xoo on high yielding local variety, in India revealed that all of them were compatible with the resistance genes, while these pathotypes were incompatible with the genes, Xa5, Xa10, Xa13 and Xa21 indicating the possibility of deploying them for enhancing the resistance (Reddy et al. 2009). Several pathotypes of X. oryzae pv. oryzae were identified based on the pathogenic reactions on NILs of rice from the major rice growing areas in Bangladesh (Khan et al. 2009). Intriguingly, comprehensive report on field-based assessment of BB and the pathotypic variation of BB pathogen collected from different locations of Bangladesh have not been reported yet. In view of the facts, the present research work has been carried out to know the pathotypic variation and to compare the virulence patterns of Xoo isolates collected from major rice growing districts of Bangladesh. Materials and methods Collection of BB infected rice leaf samples The bacterial blight infected plant samples showing typical symptom were collected randomly from 19 different districts of Bangladesh (Figure 1). Five to ten different

4 Archives of Phytopathology and Plant Protection 3 Figure 1. Pathotypic distribution of X. oryzae pv. oryzae in 19 districts of Bangladesh.

5 4 Md. S. Alam et al. locations of each district were selected for sampling. From each location, upper 3 leaves of 20 plants were collected which formed composite sample and a representative sample was taken for the isolation of X. oryzae pv. oryzae. After collection, the diseased plant samples were brought in the Laboratory and preserved in the refrigerator for isolation. A total of 96 isolates (from 127 samples) were purified. Isolation of X. oryzae pv. oryzae isolates Each diseased leaf sample was cut into small pieces, about 1 2 cm in length with the margin of typical lesions. The cut leaf pieces were sterilised by 10% Clorox solution for 1 min and then with 70% Ethanol for 1 min followed by rinsing with sterilised deionised water. Each leaf sample was then homogenised with 1 ml of sterile distilled water. The resulting suspension was diluted serially (10 5 to 10 6 ) and then spreaded on Nutrient Broth Yeast Extract (NBY) agar medium. The plates were then be incubated at 28 C for 3 4 days. Identification and purification of X. oryzae pv. oryzae cultures The bacterial growth obtained in the plates was recorded and those having morphological characteristics of Xanthomonas oryzae pv. oryzae were picked up with sterilised loop and purified cultures were obtained by streaking on both NBY and YDC (Yeast Dextrose Carbonate) agar media. Confirmation of X. oryzae pv. oryzae isolates by pathogenicity test The isolates of X. oryzae pv. oryzae were confirmed by the pathogenicity test using two susceptible rice cultivars TN1 and IR24. Seeds of the rice cultivars were sown in the plastic pot for raising the seedlings. The days old seedlings were then be transplanted in to the earthen pot in the net house for inoculation. The bacterial isolates were cultured in NBY agar medium at 28 C for h and then resuspended in sterile distilled water at cell density cells/ml measured by Spectrophotometer. The second or third youngest leaves in each plant were inoculated by scissors clip method. Briefly, the tips of the leaves were clipped by sterilised scissor dipped in the bacterial cell suspension. After inoculation with each isolates, the inoculated plants were kept in the net house for the development of symptoms. Determination of pathotypic variation of X. oryzae pv. oryzae isolates Pathotypic analyses of X. oryzae pv. oryzae with a set of Near Isogenic Lines (NILs) of rice viz., IRBB2, IRBB3, IRBB4, IRBB5, IRBB7, IRBB8, 0, 1, 3, 4 and IRBB21 carrying single known bacterial blight resistance gene (R-gene) in the background of susceptible cultivar IR24 and TN1 were carried out. Plants were raised in earthen pots and there were three hills in each pot. Three pots were maintained as replications for each isolate. Method of inoculation Plants (50 days old) were inoculated with 96 isolates of X. oryzae pv. oryzae followed by leaf-clipping method (Kauffman et al. 1973). Pairs of sterilised scissors were dipped

6 Archives of Phytopathology and Plant Protection 5 into the bacterial suspension ( cells/ml) and the tips of the leaves were clipped. A control of each variety was also maintained, for this, scissors dipped in sterile water was used for clipping off leaves. Individual isolate was used for inoculation on all the leaves of five plants which were of around total 20 leaves. The plants were cultivated in a net house under natural photoperiodic conditions. Twenty-one days after inoculation, the lesion length formed due to inoculation was measured with a ruler. Categorisation of pathoypes The lesion lengths were measured at 21 days after inoculation and averaged for each isolate in all the Near-Isogenic Lines (NILs). To categorise pathotypes of X. oryzae pv. oryzae isolate-based on the reactions against the NILs, a mean lesion length of <3 cm was considered as resistant and a mean lesion length greater 3 cm considered as susceptible (Adhikari et al. 1995). Results Determination of pathotypes of X. oryzae pv. oryzae isolates All 96 Xoo isolates induced the typical disease symptom on susceptible check varieties IR24 and TN1. Pathotypic analyses of all Xoo isolates were conducted based on their reactions against 12 NILs and a total of 24 pathotypes/races were recorded. All isolates formed yellow mucous colonies on both NBY and YDC media. Pathotype VI, XII and XIV consisted of maximum isolates (9) followed by Pathotype XIII (8), Pathotype II and XVI (7), Pathotype I, IV and XI (4), Pathotype V, XV, XVII, XX, XXI and XXII (3 isolates each), Pathotyes III, VI, VIII, IX, X, XVIII, IX and XXIII (2 isolates each). Pathotype XXIV represented by single isolate (Table 1). Pathotypic reactions against NILs and their distributions The overall reactions of all 24 pathotypes against 12 Near Isogenic Lines (NILs) are shown in Table 1. Among 24 pathotypes, pathotypes VII, XII and XIV considered as major, containing maximum number of isolates, (9.38% each) whereas Pathotype XXIV considered as minor, representing only 1.04% isolates. The major pathotype VII showed virulence reactions to Xa3, Xa4, Xa7, Xa8, Xa10, Xa13 and Xa14, whereas pathotypes XIV showed virulence reactions to Xa3, Xa4, Xa7 and Xa8 only. Another major pathotype XII showed virulence reactions to Xa1, Xa2, Xa4, Xa8 and Xa13. These three pathotypes exhibited avirulence reactions to others NILs. However, pathotype I showed highest virulence or aggressiveness compatible with all NILs whereas pathotype XVI showed lowest virulence incompatible to NILs except Xa8. Pathotype XXIV also exhibited avirulence reactions with almost all R-genes except Xa2 & Xa7. Similarly, all other pathotypes showed distinct virulence and avirulence reactions from each others with NILs tested. The frequent distributions of major pathotypes within each district along with total number of isolates are shown in Table 2. Maximum 14 pathotypes were recorded from greater Mymensingh district, whereas lowest single pathotype was recorded from Natore and Gaibandha districts. The districts represented by double pathotypes are Brahmanbaria, Jamalpur, Gaibandha, Sirajganj and Pabna. Remaining 11 districts represented by 3 6 pathotypes. Major pathotype VII was recorded from North to Mid-Eastern districts

7 6 Md. S. Alam et al. Table 1. Response of the NILs containing resistance genes to the major pathotypes of Xoo in Bangladesh. Pathotypes Near isogenic lines (NILs) with known resistance gene No. of isolates %of isolates (Xa1) (Xa2) (Xa3) (Xa4) (Xa5) (Xa7) (Xa8) (Xa10) (Xa11) (Xa13) (Xa14) (Xa21) I S S S S S S S S S S S S II S S S S S S S R R R S S III S S S S S S S S R R S R IV R R R S S S S S S S S S V S R S S R R S S S S S S VI S R S S S S S S S R R R VII R R S S R S S S S S R R VIII S S S S S S S R R R R R IX R S R R R S S R S S S R X R R R R R R S S S S S R XI S S S R R R S R R S R R XII S S R S R R S R R S R R XIII R R R S S R S R S R R S XIV R R S S R S S R R R R R XV R R R R R R S S R R S S XVI R R R R R R S R R R R R XVII S R S R R R R R R R S R XVIII S S R R R R R S S R S S XIX R R R S R S R R R R R R XX S R R S R S R S S R R R XXI S R R R R R R R R R S R XXII R R R S S R R S R R R R XXIII R R R R R R S R R S R S XXIV R S R R R S R R R R R R R resistant, S susceptible.

8 Archives of Phytopathology and Plant Protection 7 Table 2. Distribution of the major pathotypes of X. oryzae pv. oryzae in different rice growing districts of Bangladesh. Growing areas Pathotypes I II III IV V VI VII VIII IX X XI XII XIII XIV XV XVI XVII XVIII IXX XX XXI XXII XXIII XXIV Total isolates Panchagarh Thakurgaon Dinajpur Rangpur Gaibandha 1 1 Bogra Rajshahi Natore 1 1 Pabna Sirajganj Tangail Gazipur Netrokona Sherpur Jamalpur Kishoreganj Brahmanbaria Habiganj Mymensingh Note: The number of isolates of each pathotype is provided along with the total number of isolates and total number of pathotypes collected from different 19 rice growing districts of Bangladesh.

9 8 Md. S. Alam et al. of Panchagarh, Thakurgaon, Dinajpur, Rangpur, Bogra, Mymensingh and Habiganj. Pathotype XII was recorded from Thakurgaon, Rajshahi, Bogra, Mymensingh, Kishoreganj and Brahmanbaria, whereas pathotype XIV from Thakurgaon, Rangpur, Mymensingh, Jamalpur, Habiganj and Kishoreganj. However, most virulent pathotype I recorded from Habiganj and Brahmanbaria districts. Single isolate containing pathotype XXIV recorded from Mymensingh district. Others all pathotypes also frequently distributed in all surveyed 19 districts recorded from both irrigated and rain fed seasons 120 Susceptible Resistant Frequency of pathotypes (%) Xa 1 Xa 2 Xa 3 Xa 4 Xa 5 Xa 7 Xa 8 Xa 10Xa 11Xa 13Xa 14Xa 21 Resistance genes Figure 2. Performance of Xa genes against X. oryzae pv. oryzae pathotypes. The X-axis indicates resistance gene and the Y-axis indicates the frequency of pathotypes against which these genes confer resistance. Reaction showing lesion length < 3 cm was considered as resistant (R) and > 3 cm were considered as susceptible (S). Frequency of isolates (%) Susceptible Resistant 0 Xa1 Xa2 Xa3 Xa4 Xa5 Xa7 Xa8 Xa10 Xa11 Xa13 Xa14 Xa21 Resistance genes Figure 3. Performance of Xa genes against X. oryzae pv. oryzae isolates. The X-axis indicates resistance gene and the Y-axis indicates the frequency of isolates against which these genes confer resistance. Reaction showing lesion length < 3 cm were considered as resistant (R) and > 3 cm were considered as susceptible (S).

10 Archives of Phytopathology and Plant Protection The performance of Xa genes against X. oryzae pv. oryzae pathotypes and isolates are shown in Figures 2 and 3, respectively. Performance of Xa genes against X. oryzae pv. oryzae pathotypes Considering the performance of individual R-genes against the X. oryzae pv. oryzae pathotypes, R-genes Xa5 and Xa21 appeared as the most effective, conferring resistance 66.67% each, of the total pathotypes, followed by Xa2 and Xa13 (62.50%), Xa3 and Xa11 (58.33%), Xa10 and Xa14 (54.17%), Xa1 and Xa7 (50.00%), Xa4 (41.67%) and Xa8 having the lowest resistance value 29.17% (Figure 2). Performance of Xa genes against X. oryzae pv. oryzae isolates Considering the performance of individual R-genes against the X. oryzae pv. oryzae isolates, R-gene Xa5 performed better resistance (66.67%) followed by Xa2 and Xa21 (65.63%), Xa14 (63.54%), Xa10 (61.46%), Xa11 and Xa13 (59.38%), Xa1 (54.17%), Xa3 (53.13), Xa7 (51.04%), Xa4 (30.21%) and Xa8 (17.71%) containing lowest resistance value (Figure 3). Discussion The present study was designed to clarify the pathotypic variation of BB pathogen, X. oryzae pv. oryzae in Bangladesh. On the basis of virulence analysis of 96 isolates, 24 distinct pathotypes were determined. This pathotypic analyses explained the pathogenic relatedness of X. oryzae pv. oryzae populations from diverse geographic areas in Bangladesh. Most of the isolates of X. oryzae pv. oryzae tested in this study were collected from a single rice variety, Swarna, that is grown in different districts in Bangladesh during the 2014 rain fed season. The prevalence of the same pathotypes in different riceproducing zones could be due to the widespread cultivation of Swarna in different areas, and this cultivar may have served as a source of dissemination of the pathogen within the country through the seeds. Similar study has also been conducted by (Mew 1987; Adhikari, Mew, et al. 1999; Noda et al. 2001). They observed most of the X. oryzae pv. oryzae strains (70%) tested in their study were collected from a single rice variety, Mansuli, that is grown in different locations in Nepal. On the basis of prevalence they assumed, it could be due to the widespread cultivation of Mansuli in different zones, and this cultivar may have served as a source of dissemination of the pathogen within Nepal. Although all 96 strains in this study isolated from four different rice cultivars, Modern variety, Hybrid rice, Swarna and Bangladesh Rice-11 (BR-11), but host (rice) diversity had no effect on pathotypic diversity. Adhikari et al. (1999) conducted an experiment in Nepal and reported that diversities of X. oryzae pv. oryzae populations collected from traditional and improved rice cultivars were similar, suggesting that host diversity does not affect pathogen diversity. Based on a comparison of different agroecosystems and cultivars in the Philippines, Ardales et al. (1996) also suggested that host diversity did not strongly affect pathogen diversity. However, based on the virulence patterns of 96 X. oryzae pv. oryzae isolates against NILs, it was found the NILs containing R-genes Xa5 performed better resistance (66.67%) followed by Xa2 and Xa21 (65.63%). The Xa5 gene was found to confer resistance in Fangchenggang, China (Yang et al. 2012) and Xa21 to other different

11 10 Md. S. Alam et al. countries (Song et al. 1995; Wang et al. 1996). Li et al. (2009) confirmed that R gene Xa21 provides much broader resistance than the other 11 genes to strains across China. Thus, Xa5 and Xa21 genes may be recommended for breeding rice cultivars resistant to BB disease in Bangladesh. On the other hand, several studies have shown the degree of resistance conferred by Xa8 varies for X. oryzae pv. oryzae (Xoo) strains from different regions in China (Liu et al. 2007; Li et al. 2009). Among 33 strains of Xoo, majority (85%) were virulent on Xa8, suggesting that Xa8 cannot provide effective resistance against BB disease of rice in Fangchenggang, China. In these findings, Xa8 also performed as highly susceptible to Xoo isolates conferred resistance only 17.71%. Another important factor, the effects of host pathogen interactions through gene-forgene theory and changing the pathogenic structure through mutation process responsible for the high degree of pathotypic variation should be considered. Wolfe et al. (1976), Browder and Eversmeyer (1977) and Lebeda (1982) solely evaluated the genetic relationships by either the frequency of virulence factors or quantified the degree of association between the resistance factor in the host and the virulence factor in the pathogen population, in a manner similar to the gene-for-gene relationship. Nayak (1986) stated that widespread repeated use of a few resistance genes might accelerate the selection of new pathogenic races at a rate equivalent to 1.64 times the increase in specific virulence of the isolates following resistance host-plant selection pressure after a single crop cycle. This will lead to a change in pathogen population structure through either mutation or recombination to adapt itself to the new resistant host plant or environmental changes. Conclusions Total 24 pathogenic races of Xanthomonas oryzae pv. oryzae were recorded in Bangladesh during 2014 covering both irrigated and rain fed season. Their frequent distribution dominance varied across different rice growing districts. Pathotypes VII, XII and XIV were dominated and observed in different districts of Bangladesh. This addressed that bacterial blight disease is still a major threat to rice production in Bangladesh. However, these findings have practical implications in (i) understanding the pathogen population structure and their changes over time and space, (ii) selection of resistant sources in regional breeding programmes to control the severity of BB disease, (iii) understanding the impacts of variety mixing, cropping intensity and transport system on pathogenic variability. The present study may serve as a platform for refined characterisation of new isolates whose racial classification has not been determined. More isolates of all rice growing areas are needed to be analysed to fully understand the pathogen population structure in Bangladesh. The results of this study and further virulence analyses would be useful in the selection of strains for additional resistance screening. Disclosure statement No potential conflict of interest was reported by the authors. Acknowledgements This work was supported by International Foundation for Science (IFS) [grant number C/5035-1] to Dr. Md. Rashidul Islam, Department of Plant Pathology, BAU. The authors would like to acknowledge to Bangladesh Rice Research Institute (BRRI) for providing the IRBB NILs.

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