May 4, 1992 - Culape, Lucban, Quezon (1). Tilib, Lucban, Quezon (1). Palawan (1). Casigan, Narra, Palawan (1). Malinao, Narra, Palawan (1). Isabela (1).
Vol. 58, No. 7
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, July 1992, p. 2188-2195
0099-2240/92/072188-08$02.00/0 Copyright © 1992, American Society for Microbiology
Assessment of Genetic Diversity and Population Structure of Xanthomonas oryzae pv. Oryzae with a Repetitive DNA Element J. E. LEACH,1* M. L. RHOADS,' C. M. VERA CRUZ,"12 F. F. WHITE,' T. W. MEW,2 AND H. LEUNG2'3 Department of Plant Pathology, Throckmorton Hall, Kansas State University, Manhattan, Kansas 66506-55021; Department of Plant Pathology, The Intemnational Rice Research Institute, Manila, Philippines2; and Department of Plant Pathology, Washington State University, Pullman, Washington 99164-64303 Received 9 January 1992/Accepted 4 May 1992
A repetitive DNA element cloned from Xanthomonas oryzae pv. oryzae was used to assess the population structure and genetic diversity of 98 strains of X. oryzae pv. oryzae collected between 1972 and 1988 from the Philippine Islands. Genomic DNA from X. oryzae pv. oryzae was digested with EcoRI and analyzed for restriction fragment length polymorphisms (RFLPs) with repetitive DNA element as a probe. Twenty-seven RFLP types were identified; there was no overlap of RFLP types among the six races from the Philippines. Most variability (20 RFLP types) was found in strains of races 1, 2, and 3, which were isolated from tropical lowland areas. Four RFLP types (all race 5) were found among strains isolated from cultivars grown in the temperate highlands. The genetic diversity of the total population ofX. oryzae pv. oryzae was 0.93, of which 42% was due to genetic differentiation between races. The genetic diversities of strains collected in 1972 to 1976, 1977 to 1981, and 1982 to 1986, were 0.89, 0.90, and 0.92, respectively, suggesting a consistently high level of variability in the pathogen population over the past 15 years. Cluster analysis based on RFLP banding patterns showed five groupings at 85% similarity. The majority of strains from a given race were contained within one cluster, except for race 3 strains, which were distributed in three of the five clusters.
populations and relate the diversity to previous characterizations of the pathogen population.
The host population in a plant pathogen system influences the genetic diversity and population structure of pathogens (5, 13, 15). In rice, single-gene resistance has been the primary means of control for bacterial blight, caused by Xanthomonas oryzae pv. oryzae (ex Ishiyama 1922) (27). There is concern that the widespread use of a few resistance genes might accelerate the selection of new pathogenic races and result in destabilization of crop production (4, 21). However, little is known regarding the genetic diversity and population dynamics of X. oryzae pv. oryzae. In previous studies, X. oryzae pv. oryzae populations in the Philippines have been characterized on the basis of pathogenicity patterns on five indica varieties of rice (16). Six races of X. oryzae pv. oryzae are presently recognized. While useful, race groupings provide little insight into the genetic structure of the bacterial population. Physiological traits provide little additional value in the assessment of X. oryzae pv. oryzae diversity since most strains are similar with regard to many traits (28). Recently, restriction fragment length polymorphism (RFLP) analysis, which exploits the abundant variation in the DNA sequence, has been used to generate a large number of markers for the measurement of genetic diversity in populations (9, 20). We previously described the existence of a repetitive sequence in the X. oryzae pv. oryzae genome that is in sufficient copy number and distribution to provide a useful probe to detect restriction polymorphisms in the X. oryzae pv. oryzae genome (12). In order to better understand the population structure of X. oryzae pv. oryzae, we have used the repetitive element to measure the genetic diversity of the X. otyzae pv. oryzae *
MATERIALS AND METHODS Bacterial strains and culture conditions. The origin, date of collection, source variety, and race grouping for the X. oryzae pv. oryzae strains used in this study are shown in Table 1. All strains were isolated from naturally infected leaves collected between 1972 and 1988 from different regions in the Philippines. Varietal names of the host plants were obtained whenever possible. The race of each strain was determined with a set of bacterial blight differential varieties (see below). Strains collected before 1982 were maintained on Wakimoto's medium [0.5 g of Ca(NO3)2. 4H20, 0.82 g of Na2HPO4, 5 g of peptone, 20 g of sucrose, 300 g of potato, 15 g of agar per liter of water (24)] at -20°C and revived yearly by transferring to fresh slants. Since 1983, all stock cultures and newly collected strains were kept either in 5% skim milk at -20°C or as lyophilized cultures. The strains were revived in fresh slants of modified Wakimoto's medium (Wakimoto's medium without potato and supplemented with 0.05 g of ferrous sulfate per liter) for subsequent pathogenicity tests and DNA isolation. Pathogenicity tests and race grouping. The pathogenicity of each culture of X. oryzae pv. oryzae was determined by inoculation of susceptible rice varieties IR8 or IR24. Race grouping was based on virulence tests with the differential varieties IR8, IR20, Cas 209, IR1545-339, and DV85, which contain the bacterial blight resistance genes Xa-11, Xa-4, Xa-10, xa-5, and both xa-5 and Xa-7, respectively (16). To test virulence, three plants per variety were grown in the greenhouse, and the two youngest fully expanded leaves (35
Corresponding author. 2188
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X. ORYZAE pv. ORYZAE POPULATION DIVERSITY, STRUCTURE
2189
TABLE 1. X oryzae pv. oryzae strains from the Philippines used in RFLP analysis Strain
Race groupa
RFLP typeb
Yr of collection
PXO14 PXO20 PX084 PXO1 PXO151 PXO61
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 5 5 5 5 S 5 5 S S
1 1 1 1 1 1 1 1 1 2 2 2 2 2 3 4
1972 1972 1976 1972 1985 1973 1982 1972 1982 1986 1979 1986 1986 1986 1972 1972 1974 1985 1984 1973 1981 1980 1985 1981 1977 1985 1975 1984 1985 1986 1986 1986 1984 1985 1986 1982 1976 1984 1975 1981 1986 1985 1984 1984 1986 1980 1982 1974 1974 1979 1986 1986 1986 1974 1974 1982 1978 1981 1974 1988 1988 1986 1975 1975 1978 1975 1976 1986
PXO165 PX039 PX0133 PX0187 PX0132 PX0188 PX0184 PX0192 PX035 PX036 PX068 PX0157 PXO170 PX063 PX0126 PXO103 IRN793
PX0134 PX086 PX0172 PX078 PX0169 PX0175 PXO190 PX0185 PX0186 PX0168 PX0173 PX0189 PX0137 PX083 PXO171
PX079 PX0142 PXO191 PX0148 PX0166 PX0146 PX0179 PXO141 PX0143 PX087 PX088 PX0164 PX0178 PX0176 PX0177 PX069 PXO70 PX0129 PXO113 PXO125 PXO71 PX0198 PX0197 PX0182 PXO80 PXO107 PXO112 PXO105 PxO110 PXO180
S 6 7 8 8 8 8 8 8 9 9 9 9 9 9 9 9 9 9 10 11 12 13 14 15 15 15 15 15 15 16 17 17 18 18 19 20 21 21 21 21 21 21 22 22 22 22 22 22 22 22 22
Geographic origin (grouping)c
Los Baflos, Laguna (1) Bay, Laguna (1) Los Bafios, Laguna (1) Los Bafios, Laguna (1) Casigan, Narra, Palawan (1) Los Banios, Laguna (1) Malicboy, Pagbilao, Quezon (1) Salvaci6n, Rosales, Pangasinan (1) Los Banios, Laguna (1) Barrio 11, Paoay, Ilocos Sur (1) Buguey, Cagayan (1) Bantay, Ilocos Sur (1) Atimonan, Quezon (1) Tapao, Sinait, Ilocos Sur (1) Culape, Lucban, Quezon (1) Tilib, Lucban, Quezon (1) Palawan (1) Casigan, Narra, Palawan (1) Malinao, Narra, Palawan (1) Isabela (1) Los Bafios, Laguna (1) Los Banios, Laguna (1) Mabitac, Laguna (1) Pototan, Iloilo (3) Los Bafios, Laguna (1) Talavera, Nueva Ecija (1) Ajuy, Iloilo (3) Camansihay, Palo, Leyte (3) Abuyog, Leyte (3) Iraan, Aborlan, Palawan (1) Santa Ignacia, Tarlac (1) Munioz, Nueva Ecija (1) Baliwag, Bulacan (1) Claveria, Misamis Oriental (4) Aborlan (site 3), Palawan (1) Midsayap, North Cotabato (4) Nueva Ecija (1) San Enrique, Negros Occidental (3) Davao (4) Panabo, Davao (4) Kagibikan, Pototan, Iloilo (3) Gines, Zarraga, Iloilo (3) Tramo, Nato Sangay, Camarines Sur (1) Goa, Camarines Sur (1) Moriones, Ocampo, Camarines Sur (1) BRCES,e Pili, Camarines Sur (1) Lopez, Bohol (3) Isabela (1) Isabela (1) Polangui, Albay (1) Bascaran, Daraga, Albay (1) Sorsogon, Sorsogon (1) Pawa, Tabaco, Albay (1) Palawan (1) Palawan (1) Los Bahios, Laguna (1) Los Banios, Laguna (1) Los Bafhos, Laguna (1) Palawan (1) Banaue, Ifugao (2) Banaue, Ifugao (2) Banaue, Ifugao (2) Banaue, Ifugao (2) Banaue, Ifugao (2) Banaue, Ifugao (2) Banaue, Ifugao (2) Banaue, Ifugao (2) Banaue, Ifugao (2)
Variety (grouping)d
IR841 (M) IR20(?) dwarf (M) Ketan Lemar (T) Dwarf (M) C22 (M)
Malagkit (T) C4-63G (M)
(T) IR5 (M) IR8 (M) Wagwag (T) IR20 (M) IR27301-62-2 (M) IR14875-98-5 (M) IR48 (M) IR36 (M) IR480-5-9-3/IR20 (M) IR30 (M) IR46 (M) IR60 (M) IR36 (M) Burik (T) IR54 (M) IR26 (M) B3 (T) IR30 (M) Malagkit (T) IR36 (M) IR50 (M)
IR42 (M) Mestiza (T) IR46 (M)
BPI76 (T) Benzar (T) Balaratawee (T)
IR8 (M) IR17525-150-2-2-1-2-3 (M) C4-63G (M) (M) (T) F2 materials (M)
JP5 (T) IR3155 (M) Kaosiong 68 (M) Continued on following page
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APPL. ENVIRON. MICROBIOL.
LEACH ET AL. TABLE 1-Continued
Strain
Race groupa
PX0195 PX0194 PX0196 PX0181 PX0163 PXO111 PX0161
5 5 5 5 5 5 5
RFLP
type'
Yr of collection
Geographic origin (grouping)'
Variety (grouping)d
Banaue, Ifugao (2) (T) Banaue, Ifugao (2) (T) Banaue, Ifugao (2) (T) Banaue, Ifugao (2) Tinawen (T) Poblacion, Banaue, Ifugao (2) Banaue, Ifugao (2) Banaue Development Center, Banaue, Tinawen (T) Ifugao (2) 5 PX0162 22 1985 Tinawen (T) Bacoor, Banaue, Ifugao (2) IRN394 5 23 1981 IRCTNf Banaue, Ifugao (2) Tinawen (T) IRN753 5 23 1985 IRCTN, Banaue, Ifugao (2) Dakpa (T) 5 23 PXO154 1981 C16486 (T) IRCTN, Banaue, Ifugao (2) 5 IRN392 23 1981 IRCTN, Banaue, Ifugao (2) Gaebyeo (M) 5 PX0145 24 1982 Bontoc, Mt. Province (2) (T) 5 PX0144 24 1982 Bontoc, Mt. Province (2) (T) 5 PXO130 24 1981 Banaue, Ifugao (2) IR line (IRCTN#9745) (M) PX0193 5 25 1988 Banaue, Ifugao (2) (T) PX0183 5 25 1986 Banaue, Ifugao (2) 6 PXO116 26 1979 Los Bafios, Laguna (1) CR-157-392-284 (M) PXO117 6 26 1979 Los Bafios, Laguna (1) B-2277C-MR-99-2 (M) PXO114 6 26 1979 Los Banios, Laguna (1) 6 PXO118 26 1979 Los Bafios, Laguna (1) B2540b-Pn-20-2 (M) PXO119 6 26 1979 Los Bafios, Laguna (1) 3782-79 (M) PXO115 6 27 1979 Los Bafios, Laguna (1) PX0123 6 27 1980 Los Bafios, Laguna (1) CR-157-392-4 (M) PX0127 6 27 1982 Los Bafios, Laguna (1) IR8 (M) 6 PX0128 27 1982 Los Bafios, Laguna (1) IR442 (M) PX099 6 27 1980 Los Banios, Laguna (1) Rexoro (T) PX0124 6 27 1980 Los Bafios, Laguna (1) IR20878-R (M) PX0122 6 27 1980 Los Bafios, Laguna (1) Dourado Precose (T) PXO121 6 27 1979 Los Bafios, Laguna (1) a Race was determined by inoculation to the rice differential cultivars IR8 (Xa-11), IR20 (Xa-4), IR1545-339 (xa-S), DV85 (xa-5 Xa-7), and Cas 209 (Xa-10), as 22 22 22 22 22 22 22
1988 1988 1988 1986 1985 1978 1985
described in Materials and Methods. b RFLP type was determined from Southern blot analysis of EcoRI-digested total DNA from strains of X. oryzae pv. oryzae with pJEL101 as a probe. c Geographic origins of strains were grouped according to island clusters as follows: 1, Luzon lowland; 2, Luzon highland; 3, Visayas; and 4, Mindanao. d Where available, the name of the variety is given. Modem varieties (M) include released varieties and crosses or lines made by the International Rice Research Institute and the University of the Philippines, Los Bafios, Philippines. Modem varieties are shorter (semidwarf), less leafy, more responsive to nitrogen, and more resistant to lodging than traditional varieties (T). -, variety unknown. e Bicol Rice and Corn Experiment Station. f International Rice Cold Tolerance Screening Nursery.
to 40 days after sowing) were clip inoculated as described elsewhere (24). Bacteria, grown for 72 h at 28°C on modified Wakimoto's medium, were suspended in distilled water, adjusted to 109 CFU/ml, and used as an inoculum. Disease reactions were scored by both percent lesion area and lesion length 14 days after inoculation (17). DNA analysis. The DNA probe was pJEL101, a pUC18 plasmid containing a 2.4-kb EcoRI-HindIII fragment derived from race 2 strain PX086 of X. oryzae pv. oryzae (12). The insert of pJEL101 contains a high-copy-number repetitive DNA element present in DNA from over 100 X. oryzae pv. oxyzae strains tested (12). The plasmid was maintained in Escherichia coli TB1 under selection of 50 ,g of carbenicillin per ml, extracted by alkaline lysis, and purified by cesium chloride centrifugation as described previously (14). Genomic DNA from strains of X. oryzae pv. oryzae was isolated by a lysozyme-sodium dodecyl sulfate (SDS) lysis procedure of Owen and Borman (25) modified as described elsewhere (12). Genomic DNA from X. oryzae pv. oryzae was digested to completion by EcoRI (2 U of enzyme per ptg of DNA) according to the manufacturer's direction (Bethesda Research Laboratories, Gaithersburg, Md., or Promega Biotech, Madison, Wis.). DNA was fractionated by gel electrophoresis (horizontal 0.7% agarose gel, 20 by 21.5
cm) in Tris-borate buffer (89 mM Tris-HCI, 89 mM boric acid, and 2 mM Na2EDTA, pH 8.0). A 1-kb ladder (Bethesda Research Laboratories) was included in gels as a size standard. Southern transfer onto a nylon membrane was done according to instructions of the manufacturer (GeneScreen Plus; Du Pont Co., Wilmington, Del.). The entire VJEL101 plasmid was labeled (specific activities between 10 and 108 cpm/,ug of DNA) with [32P]dCTP by using a nick translation kit (Bethesda Research Laboratories); vector (pUC18) DNA does not hybridize with X. oryzae pv. oryzae genomic DNA (12). Hybridization and washing conditions (12) were high stringency. Hybridization was performed at 65°C in a mixture of 0.1% SDS, 50 mM sodium phosphate buffer (pH 7.0), 1.0 M NaCl, and 300 ,ug of denatured salmon sperm DNA per ml for 18 h. After hybridization, the blots were washed three times (20 min each) at 65°C in 2x SSC (1x SSC is 0.15 M NaCl plus 0.015 sodium citrate) containing 0.1% SDS and 5 mM sodium phosphate buffer (pH 7.0) and three times in 0.5 x SSC containing 0.1% SDS and 3 mM sodium phosphate buffer. Autoradiography was done at -80°C with Cronex film (Du Pont) with Cronex Lightning-Plus intensifying screens (Du Pont). DNA from strains of one race group together with strains representing each of the six described races from the Philippines (16) were analyzed in the same
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VOL. 58, 1992
gel. Distinct RFLP types were analyzed in a single blot to confirm that each RFLP type was unique. Data analysis. To determine the genetic relationships among RFLP types, the presence or absence of bands at 38 different positions was converted into binary data; i.e., the presence or absence of a band was coded as 1 or 0, respectively. Density of the bands was not considered in the analysis. Band positions more than 2 mm apart were considered different. DNA from a set of four isolates (PX061, PX086, PX0112, and PX099), EcoRI-HindIII-digested pJEL101, and 1-kb ladder (Bethesda Research Laboratories) were included in each gel as standards. Each strain was tested on at least two blots done at different times to confirm differences. The number of bands scored per strain ranged from 17 for PX035 to 9 for PX078. Similarity coefficients were calculated for all pairwise combinations by using a simple matching coefficient (26). Cluster analysis of the similarity coefficients was done by both Ward's method (30) and an unweighted pair group method (26) using a Clustan 3.2/PC computer program (32). The haplotype diversity approach of Nei and Tajima (23) was used to analyze the population structure of X. oryzae pv. oryzae. Haplotypic diversity measures the probability of obtaining two randomly chosen strains with different RFLP patterns and therefore has the same biological meaning and statistical properties as genetic diversity. Hereafter, haplotypic diversity is referred to as genetic diversity in describing populations of X. oryzae pv. oryzae. The genetic diversity (H) for each grouping of strains, i.e., race (HR), time of collection (HY), and geographic origin (HG), and the genetic diversity for the total population of X. oryzae pv. oryzae (HT) were estimated by the following equation (22): H = [n/(n - 1)](1 - YXi7), where Xi is the frequency of the ith RFLP type in any one grouping (for determination of HR. HY, or HG), or the average frequency of the ith RFLP type in the entire population (for determination of HT), and n is the number of strains examined. The extent of genetic differentiation among groups was estimated by the coefficient of genetic differentiation G (7): G = (HT - HX)/HT, where HX is the average genetic diversity of strain groupings by race (HR), time of collection (HY), or geographic origin
(HG).
RESULTS RFLP analysis. We examined 98 strains of X. oryzae pv. oryzae for DNA polymorphisms with the repetitive DNA element in pJEL101. Twenty-seven distinct banding patterns were observed (Fig. 1), and on the basis of comparison with these patterns, each strain was assigned an RFLP type (1 to 27, Table 1). No single RFLP type was present in any two different races (Table 2). Within a race grouping, the RFLP types sometimes differed only at one banding position. For example, the race 1 strains which were grouped in RFLP type 1 varied from those in RFLP types 5 and 6 (Fig. lb) at single band positions. Other RFLP types within a race were distinct from one another at several banding positions; for example, 12 positions vary between RFLP type 1 (race 1) and RFLP type 3 (race 1). In a few cases, patterns between two races were similar; for example, RFLP type 9 (race 2) differed from RFLP type 23 (race 5) at one band position
(Fig. lc). On the basis of the occurrence of RFLP types, races 1 and 3 were the most heterogeneous groups, with seven and eight different types in 19 and 15 strains tested, respectively. Race
Rac
1 1 1 1 1 1 1 2 2 2 2 2 3 3 3 3 3 3 3 3 4 5 5I5 6 6 6
6
j5
5
1.6
1
5
a 8
1-
-
2452 B b_ w~~~~~~~~~~-o 5
5
4'*
5
5
5
2
am 4*444.5
4 _ -~~~ _ *
Aw ~
.i--c
_
4
1_
3-
W- W wM _
4
NE
_
FIG. 1. Southern blot analysis of total DNA from strains of X. o?yzae pv. oryzae representing the 27 RFLP types. The probe was 32P-labeled pJEL101. (A) X. oryzae pv. oryzae strains and RFLP type are as follows: PXO61, type 1; PX0132, type 2; PX035, type 3; PX036, type 4; PX068, type 5; PX0157, type 6; PXO170, type 7; PX063, type 8; PX078, type 9; PX0137, type 10; PX083, type 11; PXO171, type 12; PX079, type 13; PX0142, type 14; PXO141, type 15; PX0143, type 16; PX087, type 17; PX0164, type 18; PX0176, type 19; PX0177, type 20; PX0125, type 21; PXO80, type 22; PX0154, type 23; PX0183, type 25; PXO114, type 26; and PXO115, type 27. Lane J, EcoRI-HindIII-digested pJEL101; lanes k, 1-kb ladder. (B) Differences in banding between RFLP types 1 and 6 (fragment a) and types 9, 23, and 24 (fragments b and c).
4 was the most homogeneous, with one RFLP type among the six strains tested. To determine the genetic relationships among strains of races, the banding patterns were analyzed by cluster analysis. All strains of X. oryzae pv. oryzae were similar to each other at a level of greater than 70% with the exception of one race 1 strain, PX035 (similarity level of 63%). At 85% similarity, five clusters of strains were observed in the 27 RFLP types (Fig. 2). Races represented in the five clusters were as follows: cluster I, races 1, 3, and 4; cluster II, races 2, 3, and 5; cluster III, race 3; cluster IV, race 6; and cluster V. race 1. Race 3 strains were present in three of the five clusters. At this level of similarity (85%), only race 6 was found in a unique cluster; at the 88% level of similarity, race 4 strains formed a unique cluster. The majority of strains from a given race were contained within one cluster at 85% similarity, i.e., race 1 (18 of 19 strains) in cluster I, race 4 (6
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TABLE 2. Frequencies of RFLP types of X oryzae pv. oryzae according to race grouping and the estimated genetic diversity of each group Frequency of RFLP type in each race grouping"
Total
RFLP typea
Type strain
PXO61 PX0132 PXO35 PX036 PX068 PXO157 PXO170 PX063 PX078 PX0137 PX083 PXO171 PX079 PX0142 PXO141 PX0143 PX087 PX0164 PX0176 PX0177 PXO125 PXO80 PXO154 PXO145 PX0183 PXO114 PXO115
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27
Total no. of strains Genetic diversity'
population No. of strains
% of race
9 5 1 1 1 1 1 6 10 1 1 1 1 1 6 1 2 2 1 1 6 17 4 3 2 5 8
9.2 5.1 1.0 1.0 1.0 1.0 1.0 6.1 10.2 1.0 1.0 1.0 1.0 1.0 6.1 1.0 2.0 2.0 1.0 1.0 6.1 17.3 4.1 3.1 2.0 5.1 8.1
98 0.93
1 No. of strains
9 5 1 1 1 1 1
2 % of race
3
No. of strains
% of race
6 10 1 1 1
31.5 52.6 5.3 5.3 5.3
No. of strains
4 % of race
5
No. of strains
% of race
6
100.0
6
No. of strains
% of race
17 4 3 2
65.4 15.4 11.5 7.7
No. of strains
6.7 6.7 40.0 6.6 13.3 13.3 6.7 6.7
5 8
0.73
race
47.3 26.3 5.3 5.3 5.3 5.3 5.3
1 1 6 1 2 2 1 1
19
% of
19 0.65
15 0.84
6 0
26
0.55
38.5 61.5
13
0.51
a RFLP type was determined from Southern blot analysis of EcoRI-digested, total DNA from strains of X. oryzae pv. oryzae by using pJEL101 as a probe. b Race was determined by inoculation to the rice differentials IR8 (Xa-11), IR20 (Xa-4), IR1545-339 (xa-5), DV85 (xa-5Xa-7), and Cas 209 (Xa-10) as described in Materials and Methods. c Genetic diversity is calculated by the formula [n/(n - 1)1(1 - YXi2), where Xi is the proportion of the ith RFLP type within a group and n is the number of strains tested in each group.
of 6 strains) in cluster I, race 2 (19 of 19 strains) in cluster II, race 5 (26 of 26 strains) in cluster II, race 3 (8 of 15 strains) in cluster III, and race 6 (13 of 13 strains) in cluster IV. Genetic diversity. The genetic diversity (HT) of the total population of X. oryzae pv. oryzae in the Philippines was estimated to be 0.93. Genetic diversity calculated for each race treated as a subpopulation (HR) was highest for race 3 (0.84) and lowest for race 4 (0.0) (Table 2). The average genetic diversity (HR) of six race groups was 0.54, and the coefficient of genetic differentiation (GR) was 0.41. To examine the effect of varietal release over the past 15 years on the diversity of the population of X. oryzae pv. oryzae, genetic diversities of strains collected in three 5-year periods (1972 to 1976, 1977 to 1981, and 1982 to 1986) were calculated (Table 3). The genetic diversities of strains sampled between 1972 and 1976 (25% of the total samples), 1977 and 1981 (30% of the total), and 1982 and 1986 (44% of the total) were 0.89, 0.90, and 0.92, respectively (Table 3). The coefficient of genetic differentiation (Gy) based on the grouping of time periods was 0.01. On the basis of information collected at the time of sampling, the varietal origins were classified as either modern (semidwarf) varieties or traditional varieties (Table 1).
Rice breeders consider modern varieties shorter, more responsive to nitrogen, and more resistant to lodging than the traditional varieties. No difference was observed in the genetic diversities of strains recovered from traditional (0.90) or modern (0.91) varieties. When strains were grouped according to their geographic origin in the Philippines, a marked difference in genetic diversity (HG) was found between strains from the Luzon lowlands (0.93) and the Luzon highlands (0.55) (Table 3). Only four RFLP types were identified among 26 strains obtained from the highlands, whereas 19 RFLP types were found among 60 strains tested in the lowlands in Luzon Island (Table 2). The coefficient of genetic differentiation (GG) based on geographic locations was 0.20. DISCUSSION We have used a repetitive DNA element to characterize the population structure of X. oryzae pv. oryzae in the Philippines. Two factors aided the analysis. First, the random genomic distribution of the repetitive element pJEL101 (12) provided a single DNA probe to scan for DNA polymorphisms and saved considerable effort in typing the strains
VOL. 58, 1992
X. ORYZAE pv. ORYZAE POPULATION DIVERSITY, STRUCTURE
PXO 61 (1) PXO 68 (I) PX0 157 () PXOI170() PXO 176 (3) PX0132 (I) PXO 36 (I) PX0142 (3) PXO 164 (3) PXO 87(3) PXO 125 (4) PXO 63 (2) PX0137 (2) PXO171 (2) PX0145 (5)PXO 83 (2) PX0154 (5) PXO 78(2) PXO 79(3) PXO 80(5) PXO 183 (5) PXO 141 (3) PXO 143 (3) PX0177 (3)
J
PXOI 14(6) PXOI I 5 (6) PXO 35(I)
....L......... | 70 60 80 Similarity (N) FIG. 2. Dendrogram displaying the relationshif )s among the 27 RFLP types for strains of X. oryzae pv. oryzae aft er Southern blot analysis using 32P-labeled pJEL101 as a probe. The race designation for each strain is shown in parentheses. Similarity coefficients were 100
90
grouped by using the unweighted pair group metho'J with arithmetic means.
compared with the use of single-copy or lo, v-copy-number DNA probes. Second, the background inform;ation regarding the time, location of collection, and host origi:ns of 98 strains of X. oryzae pv. oryzae from the Philippines were available from the International Rice Research Inistitute. Using pJEL101 as a hybridization probe, we obse-rved a strong association between RFLP types and race groupings. No RFLP type was shared among the six races on the basis of the criteria used in this study (Table 2); ho'wever, several RFLP types were observed within some rac.e groups, and some hybridization patterns were very siimilar between strains of different races. In an asexual haplc)id population, nonrandom associations of genes can be du(e to chance or epistatic selection of the genotypes involve d (31). In the absence of epistatic selection, a fast-evolving phenotype would be found nested within a more static phienotype. In X. oryzae pv. oryzae, we observe much greate.r variations in molecular phenotypes than in pathogenic pat:terns. If avirulence loci and the genomic distribution of the repetitive element are not functionally related, our obs4ervation would suggest that the molecular phenotypes, a:s revealed by pJEL101 hybridization patterns, change at a hiigher rate than avirulence loci. However, comparisons betmveen molecular phenotype and avirulence loci must take int;o account that
2193
measurement of diversity at avirulence loci is limited by the number of rice differential cultivars available for defining race. Alternatively, the repetitive element may play a role in race differentiation. Although we have no evidence to suggest that the repetitive element might be functionally associated with avirulence, sequence analysis of pJEL101 showed that it contained structural features common in prokaryotic transposable elements (33). Kearney et al. (10) demonstrated that a transposable element from Xanthomonas campestns pv. vesicatoria could insert into and inactivate a plasmid-borne avirulence gene, thereby affecting the interaction of the pathogen with pepper plants containing resistance genes corresponding to the avirulence gene. Work is in progress to determine whether the repetitive element contained in pJEL101 could direct or influence the evolution of races of X. oryzae pv. oryzae. Although the functional relationship between avirulence and repetitive elements remains unknown, the dendrogram constructed on the basis of RFLP banding patterns provides new insights into the evolutionary relationships among strains of X. oryzae pv. oryzae (Fig. 2). Race 1 (cluster I) was the predominant race before the widespread use of the bacterial blight resistance gene Xa-4 (18). Races 3 and 4, which are virulent to Xa-4, are also contained in cluster I. PX035 (cluster V) is classified as race 1 on the basis of host reactions but is different from other race 1 strains with respect to RFLP patterns and serological reactions (3). It is possible that PX035 will be grouped as a different race when tested against new differential hosts. In fact, strains with very different RFLP patterns proved to be good candidates for a new race group and were useful in screening for new sources of resistance (29). Race 3, which includes the most diverse group of strains, is present in all clusters except IV (race 6 strains only) and V (single strain PX035). It is possible that race 3 phenotypes arose in separate, unrelated populations. Alternatively, it is possible that race 3 has existed long enough to accumulate a high degree of genetic variation. Nitrosoguanidine mutagenesis experiments yielded mutants which changed from race 2 to race 3 (1, 11), indicating that the differences between the two phenotypes may be controlled by a single avirulence locus. Additional DNA markers, including cloned avirulence genes (10a) and other repetitive DNA elements (2, 12), are being used to further elucidate the phylogenetic relationships between races. The genetic diversity (HT = 0.93) estimated for the total population of X. oryzae pv. oryzae indicates a high level of variability in the pathogen population. The high level of diversity among X. oryzae pv. oryzae strains has been confirmed with different DNA probes (23a). Such a high degree of variability has also been observed for other phytopathogenic bacteria. Denny et al. (8) examined 17 Pseudomonas synngae pv. tomato and six P. syningae pv. syringae strains by RFLP analysis and found each strain to have a unique RFLP. Using nine DNA probes, seven of which encode information required for virulence and the hypersensitive response, Cook et al. (6) identified 28 RFLP patterns among 62 strains of Pseudomonas solanacearum representing three races and five biovars. It would be of interest to determine whether the high level of diversity in the genomes of plant pathogenic bacteria is the consequence of host genotype variability. By partitioning genetic diversities within race groupings, time periods, and geographic locations of our limited sample of X. oryzae pv. oryzae strains, we gain clues to the relative significance of these factors in shaping the population structure of the pathogen. Of the three factors examined, time had
2194
APPL. ENVIRON. MICROBIOL.
LEACH ET AL.
TABLE 3. Frequencies of RFLP types and estimated genetic diversities for strains of X. oryzae pv. oryzae grouped according to time period of collection and geographic origin Frequency of RFLP by: Time periodb
RFLP
Type
typea
strain
1972-1976 No. of strains
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27
PXO61 PX0132 PXO35 PX036 PX068 PX0157 PXO170 PX063 PX078 PX0137 PX083 PXO171 PX079 PX0142
PXO141 PX0143 PX087 PX0164 PX0176 PX0177 PXO125 PXO80 PXO154 PXO145
%
6
27.2
1 1 1
4.5 4.5 4.5
1 1
4.5 4.5
1
4.5
1
2
3 4
1977-1981 No. of strains
1 1
4
%
No. of strains
%
3.6 3.6
2 4
4.7 9.5
14.2
1 1 1 9 1
2.3 2.3 2.3 21.4 2.3
1
2.3
Luzon
low-
Land No. of strains
%
9 5 1 1 1 1 1 5 6
15.0 8.3 1.6 1.6 1.6 1.6 1.6 8.3 10.0
1
1.6
Mindanao
Visayas
land
of strains
No. strains
%
1 3
12.5 37.5
1
12.5
4.5 1 1
3.6 3.6
1
3.6
2 2 3 1
7.1 7.1 10.7 3.6
5 6
17.8 21.4
5 1
11.9 2.3
1 1 1 1 6 1 2 1
2.3 2.3 2.3 2.3 14.2 2.3 4.7 2.3
2
4.7
9.1
13.6 18.2
PX0183 PXO114 PXO115
1982-1986
Geographic origin' Luzon high-
4
6.6
2 2 1 1 6
3.3 3.3 1.6 1.6 10.0
2 1
17 4 3 2
5 8
No. of strains
%
1 1
25.0 25.0
1 1
25.0 25.0
25.0 12.5
65.3 15.3 11.5 7.7
8.3 13.3
4 26 8 42 60 22 28 Total no. of strains 1.0 0.55 0.86 0.93 0.90 0.92 0.89 Genetic diversityd a RFLP type was determined by Southern analysis of EcoRI-digested total DNA from strains of X. oryzae pv. oryzae by using pJEL101 as a probe. h Only 92 of the 98 strains of X. oryzae pv. oryzae were grouped in the three time periods. Six strains were collected in 1988 and therefore not included in the 1982 to 1986 grouping. c Strains of X. oryzae pv. oryzae from the Philippines were grouped according to island clusters. Luzon lowland includes Ilocos Sur, Cagayan, Isabela, Pangasinan, Nueva Ecija, Tarlac, Bulacan, Laguna, Quezon, Camarines Sur, Albay, Sorsogon, and Palawan; Luzon highland includes Ifugao and Mt. Province; Visayas includes Iloilo, Leyte, Bohol, and Negros Occidental; Mindanao includes North Cotabato, Misamis Oriental, and Davao. Cropping intensity and rice varieties grown vary between the Luzon highlands (one to two crops per year; generally, traditional varieties) and the Luzon lowlands (two to three crops per year; generally, improved varieties). d Genetic diversity is calculated by the formula [nl(n - 1)1(1 - l;X/), where Xi is the proportions of the ith RFLP type within a group and n is the number of strains tested in each group.
the least effect on diversity. The coefficient of differentiation (Gy) partitioned under the three 5-year periods from 1972 to 1986 is low (0.01). The genetic diversities of strains grouped according to the three periods are similar (0.89, 0.90, and 0.92), indicating that the pathogen population has had a consistently high level of diversity over the past 15 years. Modem semidwarf varieties carrying the resistance gene Xa-4 were released in the early 1970s and accounted for over 90% of the total rice production in the Philippines by 1978. Vera Cruz (28) and Mew et al. (19) observed a shift in the frequencies of the six races in the pathogen population from race 1 (avirulent to Xa-4) to those which were virulent to Xa-4. It appears that although host varieties influence the relative frequency of races within a field, the standing genetic variability of X. oryzae pv. oryzae (i.e., the number of variants present at any one time) has not been reduced dramatically by the uniformity in host resistance.
When genetic diversities were partitioned according to race groupings, a high degree of differentiation between race groups (GR = 0.41) was observed. Since race groupings are defined by interactions with rice differential cultivars, a logical inference is that host genotypes play an important role in structuring the pathogen population. Geographic locations also appear to play a role in the differentiation ofX. oryzae pv. oryzae population (GG = 0.20). The geographic differentiation stems mainly from the differences between strains from the mountain and lowland areas. The rice ecosystems in the mountain and lowland areas differ in many respects. The mountain area has a temperate climate with one crop of traditional rice varieties grown per year. In the tropical lowland, two to three crops of modern semidwarf varieties are grown. Thus, climatic and ecological factors as well as cropping intensity can contribute to the observed differentiation. More intensive sampling of strains along a
VOL. 58, 1992
X. ORYZAE pv. ORYZAE POPULATION DIVERSITY, STRUCTURE
transect across the mountain and lowland areas is being made to investigate the fine-scale variation of X. otyzae pv. oryzae under different ecological conditions (23a). The ability to describe pathogen populations over time and space and to understand the impacts of host genotype, climate, and cropping intensity on pathogen variability can be of practical use for the deployment of host resistance. At present, the release of new varieties is based primarily on multilocation yield trials and on the observed level of disease and insect resistance under trial conditions. Detailed descriptions of race composition, genetic variability, and the genetic potential to change in the pathogen population have not been available to research scientists, farmers, or extension workers in planning varietal release. The availability of suitable probes such as the repetitive element in pJEL101 to characterize different pathogen populations from broad geographic areas will provide useful information for the testing and release of resistant germ plasm among rice-growing countries. ACKNOWLEDGMENTS This research is supported by grants from the Rockefeller Foundation (RF86058 #56 to KSU and RF86058 #44 to IRRI), the Kansas Agricultural Experiment Station (contribution no. 92-341-J) and Washington State Agricultural Research Center (contribution no. PPNS no. 0116). We thank Rebecca Nelson and Scot Hulbert for their helpful comments on the manuscript. REFERENCES 1. Ardales, E., N. Chua, J. Leach, and H. Leung. 1988. Isolation of chemically-induced mutants of Xanthomonas campestns pv. oryzae with changed race-specificity, p. 103. Fifth International Congress on Plant Pathology, Kyoto, Japan. (Abstr.) 2. Baraoidan, M. R., R. Nelson, J. E. Leach, T. W. Mew, and H. Leung. 1990. Use of putative transposable elements as probes for population studies of the bacterial blight pathogen of rice. Phytopathology 80:982. (Abstr.) 3. Benedict, A. A., A. M. Alvarez, J. Berestecky, W. Imanaka, C. Y. Mizumoto, L. W. Pollard, T. M. Mew, and C. F. Gonzalez. 1989. Pathovar-specific monoclonal antibodies for Xanthomonas campestns pv. oryzae and for Xanthomonas campestris pv. oryzicola. Phytopathology 79:322-328. 4. Browning, J. A., and K. J. Frey. 1969. Multiline cultivars as a means of disease control. Annu. Rev. Phytopathol. 7:355-382. 5. Burdon, J. J., and A. M. Jarosz. 1990. Disease in mixed cultivars, composites, and natural plant populations: some epidemiological and evolutionary consequences, p. 215-228. In A. H. D. Brown, M. T. Clegg, A. L. Kahler, and B. S. Weir (ed.), Plant population genetics, breeding, and genetic resources. Sinauer Associates Inc., Sunderland, Mass. 6. Cook, D., E. Barlow, and L. Sequeira. 1989. Genetic diversity of Pseudomonas solanacearum: detection of restriction fragment length polymorphisms with DNA probes that specify virulence and the hypersensitive response. Mol. Plant-Microbe Interact. 2:113-121. 7. Crow, J. F. 1986. Basic concepts in population, quantitative and evolutionary genetics. W. H. Freeman, San Francisco. 8. Denny, T. P., M. N. Gilmor, and R. K. Selander. 1988. Genetic diversity and relationships of two pathovars of Pseudomonas syringae. J. Gen. Microbiol. 134:1949-1960. 9. Gabriel, D. W., J. E. Hunter, M. T. Kingsley, J. W. Miller, and G. R. Lazo. 1988. Clonal population structure of Xanthomonas campestns and genetic diversity among citrus canker strains. Mol. Plant-Microbe Interact. 1:59-65. 10. Kearney, B., P. C. Ronald, D. Dahlbeck, and B. J. Staskawicz. 1988. Molecular basis for evasion of plant host defence in bacterial spot disease of pepper. Nature (London) 332:541-543. 10a.Leach, J. E., et al. Unpublished data.
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