Genetic Diversity of Indonesian Bacterial Leaf Blight Isolate (Xanthomonas oryzae pv. oryzae) Core Collection based on the VNTR and avrXa7 Molecular Markers

Bacterial leaf blight (BLB) is one of the major dis eases in rice caused by Xanthomonas oryzae pv. oryzae. This study aimed to identify and analyze the genetic diversity of 18 BLB isolates that consist of 7 races and 11 haplotypes from various locations in Indonesia. The genetic diversi ty analysis was conducted on the basis of the VNTR (Variable Number of Tandem Repeat) markers and the avrxa7 gene marker. The banding pattern of the amplificat on product was made into binary data as input for the construction of a dendogram. Based on the dendogram, three X. oryzae pv. oryzae genotype groups with different virulence levels wer e formed. The VII (IXO80_021) race of X. oryzae pv. oryzae genotype group I and the VIII-A (IXO 80_024) race o f genotype group II were avirulent, whereas the rac es and haplotypes of genotype group III were virulent.


Introduction
Bacterial leaf blight (BLB) is one of the major disease in rice caused by Xanthomonas oryzae pv. oryzae [1]. The disease can cause yield losses up to 80% of total rice production [2,3]. According to Naveed et al. [4], the BLB disease causes 90% reduction in grain weight. The BLB disease has a widespread distribution and is primarily destructive in the lowland rice area [5]. The BLB incursion reaches 14,178 ha [6] of the 7.4 million ha of rice areas in Indonesia [7]. Currently, the identification of X. oryzae pv. oryzae is conducted on the basis of the virulence level in the different varieties of rice. Therefore, many races or pathotypes have been determined in these pathogenic bacteria.
A total of 12 pathotypes of X. oryzae pv. oryzae have been identified in Indonesia [8]. Three of them, namely, pathotype groups III, IV, and VIII, are dominant in several rice production areas in Java. Pathotype groups I and II, initially found in Japan, were also found in Indonesia in 1991-1992. According to Yamamoto et al. [9], X. oryzae pv. oryzae pathotype group III, which has a low virulence level, is distributed in South Sulawesi, South Kalimantan, West Java, Central Java, East Java, Bali, and North Sumatera. Conversely, X. oryzae pv.
oryzae pathotype group IV, which has a high virulence level, is spread in South Sulawesi, West Java, Central Java, East Java, Bali, and North Sumatera [10]. Pathotype group V is found only in Bali, while pathotype groups VI and VII are found in West Java [11]. Pathotype group VIII has a medium virulence level and is found in South Kalimantan, West Java, and North Sumatera [10]. Some pathotype groups have shown to have high pathogenicity and virulence level in different rice varieties. Therefore, the development of a plant disease management strategy for X. oryzae pv. oryzae is required to reduce yield losses and to prevent the development of the BLB disease epidemic. The qualitative identification and characterization of major BLB resistance genes and polygenic factors to form the quantitative resistance system is considered to increase the success of resistant cultivar development and BLB disease control [12].
The identification of X. oryzae pv. oryzae pathogen isolates is conducted by utilizing several molecular markers, including the markers from the virulence gene [13] and multiple loci variable number of tandem repeat (VNTR) [14]. X. oryzae pv. oryzae is a plant pathogenic bacterium that belongs to phylum Protebacteria and is characterized by having the secretion system of the protein virulence effector (PVE) type III. Generally, the PVE of Xanthomonas spp. is coded by the AvrBs3/PthA gene group. Several molecular markers of this gene have been developed and used in the BLB pathogen virulence analysis [15]. The pathotype analysis using the VNTR marker has also been utilized in pathotype analysis as part of an epidemiological observation on the pathogen [16]. By utilizing the VNTR marker, several loci associated with the temporary and spatial diversity of the pathogen population can be identified and characterized [14]. In this study, the genetic diversity of X. oryzae pv. oryzae core collection isolated from various locations in Indonesia was determined using the VNTR markers and the avrxa7 gene marker.

Materials and Methods
Bacterial leaf blight (BLB) isolate. A total 18 BLB isolates consisting of 7 representative isolates from the race or pathotype group and 11 representative isolates from the haplotype group (Table 1) were used in this study. These isolates were included in the BLB core collection of the Indonesian Center for Agricultural Biotechnology and Genetic Resource Research and Development, Ministry of Agriculture, Bogor. These BLB collections belong to the 1974-1994 collection isolated from several BLB disease endemic locations in Indonesia. All these isolates were grown in the Wakimoto plus Ferrous sulfate or WF medium [17] consisting of 20 g sucrose, 5 g peptone, 0.5 g Ca(NO 3 GTGGTTCGCGTGCTTGGTTTT/ACGCCTGATCCGGTGTTTGAG  Table 2).
Rejuvenation and preparation of isolates. About 10 µL of the stock isolate preserved in the skimmed milk medium 10% (b/v), which contains 0.05% L-glutamic acid at 0 °C [16], was taken out and sinuously streaked in the WF petri dish agar medium by using a sterile skewer and incubated at 28-30 °C for two days. The colonies' characteristics are yellowish and slimy ( Figure   1). The single colony was further grown in the nutrient broth (NB) medium and shaken in an incubator overnight at 100 rpm-200 rpm for DNA extraction materials.
DNA extraction. About 2 mL of X. oryzae pv. oryzae from the NB medium was precipitated in Eppendorf tubes and centrifuged at 10,000 rpm for 10 min. The supernatant was discarded, and 250 µL Tris-EDTA (TE) buffer was added to the precipitates composed of bacterial cells and further vortexed until homogeneous. About 50 µL SDS 10% and 5 µL proteinase-K were A B C added to the homogenous solution, which was then incubated in a water bath at 37 °C for 1 h. About 65 µL NaCl 5M and 80 µL CTAB were further added, followed by dispersing by vortex. The solution was incubated in a water bath at 65 °C for 20 min. After incubation, the solution was diluted with the same volume of chloroform: isoamilalcohol (24:1) solution and gently shaken, followed by double centrifugation. The first centrifugation was conducted at 15,000 rpm for 5 min at room temperature and the second centrifugation at 8,000 rpm for 15 min. The cell suspension was gently poured into new Eppendorf tubes, added with cold isopropanol, and then inverted slowly. The whitish DNA was stored in a freezer (-20 °C) for 30 min. The DNA was purified by cold ethanol 70% and then centrifuged for 5 min at 14,000 rpm. The ethanol was discarded, and the DNA pellet was dissolved in 25 µL-50 µL TE [18].
DNA quality and quantity test. The quality of bacterial DNA was tested by 1% gel electrophoresis added with GelRed (GelRed TM Biotium). The TAE buffer 0.5x was used as a running buffer. About 2 µL of the DNA pathogen solution from each sample was mixed with 2 µL loading solution or BlueJuice, 2 µL DNA marker (bp), and 3 µL loading solution, and the solution was further homogenized with a micropipette. Each mixture was poured into the agarose gel wells. The electrophoresis apparatus was connected to the remaining electrical voltage at 90 v until the BlueJuice color travels to as far as 85% of the agarose length. The electrophoresis product was examined under ultraviolet (UV) ChemiDoc gel scanning from BioRad. The quantity of DNA was measured by a spectrophotometer at 260 nm and 280 nm based on the Sambrook method [19]. Concentration and purity of the DNA were calculated as follows: DNA concentration = OD 260 x 50 µg/mL x the dilution factor. This equation is based on the prediction that OD 260 = 1, and the DNA concentration is 50 µg/mL. The DNA purity = OD 260 /OD 280 . Dilution of the DNA was conducted to reach a 10 ng/µL concentration.  [14]. The DNA separation of the PCR product was conducted in an electrophoresis machine using 1.2% agarose gel immersed in 2 µL GelRed at an electrical voltage of 90 v for 45 min and visualized using a UV transilluminator ChemiDoc gel scanning (Bio Rad). The DNA bands were scored for the presence and absence of DNA, and the profile was further translated into binary data.

Data analysis.
Each SSR band in the gel that represents the DNA fragment from each race genotype or haplotype of the bacteria was scored based on the marker sized used (100 bp). The scores were analyzed using the TASEL program.

Results and Discussion
DNA extraction of X. oryzae pv. Oryzae isolates.
Extracted DNA from all X. oryzae pv. oryzae isolates in this study was almost uniform and ranges from 100 ng/µL to 150 ng/µL. Not all genomic DNA was in good quality ( Figure 2). The genomic DNA with good quality was clean, not degraded, and not contaminated. Contamination by Chisam and other organic materials could be observed from the appearance of the background that was smeared along the movement path of genomic DNA bands. Purity of the extracted DNA samples shows the purity value that ranges from 1.8 to 2.0. This purity level is the standard for DNA quality for molecular analysis [19].

Genotype profile of the BLB isolates based on the VNTR markers.
After screening with 20 VNTR primers and 3 primers that were compatible with the AvrXa7 gene, the result obtained did not vary. Figure 3 shows that X. oryzae pv. oryzae races and haplotypes amplified by B02 primer produced polymorphic bands with sizes ranging from 150 bp to 950 bp. This result showed that several races and haplotypes used in this study have high virulence level or endemic in a particular area. As an example race IVB (IXO80-004) and race VIIIB (IXO79-008) were outbreak in West Java with dominant virulence on local rice host. While haplotype AvrXa-10-C (IXO93-103) and haplotype AvrXa-10-V (IXO93-230) were dominant virulence onlocal and elite rice varieties, particularly in Central Java and Bali. So, each race or haplotype has a specific virulence to the host and location endemic.
The VNTR marker is the molecular marker that denotes the tandem repeated sequence variation in various pathogenic bacteria, including X. oryzae pv. oryzae. This marker is considered effective in detecting the genetic variation of bacteria population associated with the important factor in the pathogen epidemiology, which includes temporal and spatial geographical factors [14].
The genotypic diversity analysis of the BLB isolates in this study shows several variations among isolates collected in different years, host origins, and locations.
Some of them have similar genotype profiles. This finding indicates that certain BLB genotypes have high adaptability to a particular season, host cultivars, and different locations. Figure 3 shows that the highest genotype profile variation is exhibited using the B02 primer. Less genetic variations were detected using three other primers, namely, G01, B06, and B20. The specific variation was shown by sample number 4 (ID: IXO80-004). This isolate produced the amplicon fragments with sizes of 500, 450, and 400 bp using primer B02 (Figure 3a). The genetic profile that is the same as sample 4 is sample 9. This specific profile was detected in sample number 8 (ID: IXO79-008), which had an amplicon fragment of 800 bp in the B02 primer.
The polymorphic sample was also found in the profile generated from the B06 primer. Sample number 9 (IXO93-103) was specific to the amplicon fragment of 250 bp. The genetic specific variations were seen in sample numbers 13 (ID: IXO93-230) and 14 (IXO74-037-2) in the profile generated from the B02, G01, and B20 primers.
The genetic diversity of the BLB isolate based on the temporal and spatial geographical factors was shown as a genetic profile based on the B02 marker ( Figure 4). Figure 4 shows that three different genotype profiles, namely, types A, B, and C, were detected. Among them, the isolates belonging to type B were found from different years and locations. Genotype types A and C have a narrow distribution based on the time (year/ season) and/or location of the collection.
Genotype profile of the BLB isolates based on the avrXa7 gene marker. The genotype profile of the BLB isolates based on the avrxa7 gene marker (AvrXa7_1 primer) showed a variation with band size ranging from +150 bp to -750 bp; the AvrXa7_3 primer had a band size of +150 bp; and the AvrXa7_4 primer had a band size of +250 bp. This result shows that several BLB races and haplotypes possess the PthXo4, PthXoS, and AvrXa7-sacB50 genes that belong to the AvrBs3/PthA gene family [15].
Genotype diversity of the BLB isolates based on the VNTR markers and the avrXa7 gene marker. The dendogram of Figure 6 shows that several isolates have specific relationships with specific isolates. This finding is probably due to the genetic variation caused by mutation, which often occurs among X. oryzae pv. oryzae isolates. Three groups were detected from the dendogram ( Figure 6). Genotype group I contains a single isolate (race VII, IXO80_021) from the Pusaka Negara Subang isolate, which has the PthXo4 gene of Genotype group II also contains a single race VIII-A (IXO 80_024) from the Kalimantan isolate, which has the AvrBs3/PthA gene family. The PthXo4 gene has a dependent elicitor activity because it is associated with the loss of the PVE production activity [15].
the PthXoS gene. The AvrBs3/PthA gene of genotype groups I and II is compatible with the avrXa7 gene. The PthXo4 and PthXoS genes have a TGA base variation, which is a signal for the stop codon that causes the absence of the PVE transcriptional activity, and therefore the pathogen becomes avirulent [15]. In this study, the pathogens belonging to genotype groups I and II were avirulent, and thus these races could not be used again to screen for plant resistance. The avirulent reaction from these races could be caused by the time of preservation, as the isolate was collected in 1980. Therefore, the virulence activity could have been decreased. The loss of virulence activity could also have been caused by gene mutation.  [20]. The AvrXa7sacB50 gene has an NLS signal that functions in the process of protein virulent targeting into the host cell nucleus [21]. Genotype group III has a race group with a strong virulence activity. This virulence activity is shown in the pattern of race distribution and spread, which includes race I, race III-A, race III-B, race IV-B, race VIII-B, and all haplotypes used in this study. The races in group III are endemic to the X. oryzae pv. oryzae isolates from the BLB population in Kota Batu (Bogor, West Java), Jatisari (Cikampek), Cianjur (West Java), and Pusakanegara (Subang). This study revealed the possibility of a relationship between the virulence of the X. oryzae pv. oryzae pathotype and that of the X. oryzae pv. oryzae genotype.

Conclusion
The diversity of the X. oryzae pv. oryzae isolate core collection using the VNTR and avrXa7 gene markers produced three X. oryzae pv. oryzae genotype groups with different virulence levels. The avirulent X. oryzae pv. oryzae belongs to genotype group I (race VII, IXO80_021) and genotype group II (race VIII-A, IXO 80_024), while the majority of virulent isolates belong to genotype group III.