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Characterization and in Plant Detection of Bacteria that Cause Bacterial Panicle Blight of Rice

Temesgen Mulaw1*, Yeshi Wamishe1 and Yulin Jia2

1Rice Research and Extension Center, University of Arkansas, Stuttgart 72160, Germany

2United States Department of Agriculture-Agricultural Research Service (USDA-ARS), Dale Bumpers National Rice Research Center (DB NRRC), Stuttgart, AR 72160, Germany

*Corresponding Author:
Temesgen Mulaw
Rice Research and Extension Center
University of Arkansas, Stuttgart 72160, Germany
E-mail: [email protected]

Received date: January 04, 2018; Accepted date: January 30, 2018; Published date: February 05, 2018

Citation: Mulaw T, Wamishe Y, Jia Y (2018) Characterization and in Plant Detection of Bacteria that Cause Bacterial Panicle Blight of Rice. Plant Pathology Vol 1:3.

Copyright: © 2018 Mulaw T. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

 
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Abstract

Burkholderia glumae (BPB) presumably induces a grain rot symptom of rice that is threatening to rice production in most rice producing states of the USA.  The present study was to identify the causal agent of BPB, virulence based on hypersensitive reactions and distribution of the pathogen within a plant. 178 rice panicles samples were analyzed with semi-selective media (CCNT), polymerase chain reaction (PCR) with bacterial DNA gyrase (gyrB) specific markers, and hypersensitive reactions on tobacco leaves.  A total of 73 samples out of 178 produced a yellow bacterial colony with similar morphology on CCNT medium suggesting they were bacterial panicle diseases.  However, with PCR reactions we only determined that 45 of 73 were due to B. glumae, and the causal agent for the remaining samples was undetermined.  Within the 45 samples, 31 highly, 6 moderately, and 5 weakly virulent isolates were grouped based on lesion sizes of the hypersensitive reactions.  Pathogenicity variability among the 45 B. glumae detected suggests that different degrees of host resistance exist.  To determine the existence of bacteria in different plant tissues, naturally infected plant parts were examined with CCNT media and PCR analysis.  B. glumae was again isolated from seeds followed by stems and sheaths from light yellow pigmented CCNT media. In contrast, roots and leaves show no visible yellow pigment on CCNT. Consistent PCR products were produced from the stem, sheath, and seed, but not from the root and leaves. These findings suggest that B. glumae is distributed in the stem, sheath, and seed, and not in the leaf and root.  Together this study demonstrated the usefulness of artificial culture media, tobacco reactions, and DNA test with PCR for characterization of BPB, and distribution of bacteria in plants.  These findings will help to understand the mechanism of bacteria translocation in plants.

Keywords

Burkholderia glumae; Bacterial anicle blight (Bpb); Hypersensitive reactions; In-plant detection

Introduction

Rice production in the southern United States has a long history of loss to panicle blighting of unknown etiology. The losses caused by bacterial panicle blight (BPB) could be as high as 70%, including reduced yield and poor milling [1]. Significant yield losses from BPB have been experienced in the rice-producing regions of the Southern United States, including Louisiana, Texas and Arkansas in 1996, 1997, 2000, and the most recently, in 2010 [2]. Currently, this disease has affected rice production in many countries of Asia, Africa, South and North America; it is a typical example of the shifting from a minor plant disease to a major disease due to the changes of environmental conditions [3]. The symposiums of BPB often appears during the rice heading stage and is pronounced when rice is grown under high night temperature and frequent rainfalls predisposing rice to diseases outbreak [4].

Rapid detection and accurate identification of pathogens in plant are critical steps to prevent pathogens dissemination. Pathogen identification based on colony morphology or disease symptoms is difficult, time-consuming and unreliable because of the secondary infection by necrotrphic fungi and the similarity among Burkholderia spp.  For example, B. glumae, B. plantarii, and B. gladioli were known to infect rice plants causing similar symptoms [5]. Additionally, proliferation of B. glumae and B. plantarii were found to suppress B. gladioli in rice seeds [5]. Interactions among B. glumae, B. gladioli, B. plantarii and other unknown microorganisms often result in different outcomes of crop damage. For example, B. glumae was found to be responsible for the decrease of grain weight, floret sterility, inhibition of seed germination and reduction of stands in rice seedlings dependent on the outcome of the interactions with other bacteria and the environmental factors such as temperature and drought [6,7].

Previous studies have identified abundance of strains of B. glumae including some highly virulent strains that caused 50 to 75% yield reduction [8,9].  Additionally, it was predicted that the B. glumae strains in different rice-production regions have some undefined differences in their genome and virulence [3]. Furuya et al. demonstrated that the extent of virulence of B. glumae strains can be accurately estimated by the use of hypersensitive cell death on tobacco [10].  However, virulence characteristics of B. glumae isolated from rice, and distribution of the causal agent of bacterial panicle blight (BPB) in rice plants have not been clearly demonstrated. Tobacco hypersensitivity is a fast and convenient way to screen bacterial cultures for pathogenicity. It works particularly well for Pseudomonas but can be variable for Xanthomonas and Ralstonia. Some Xanthomonads may require some tweaking of the environmental conditions the tobacco grown in [11,12], and the response may take up to four days [13,14]. Erwinia amylovora and some of the coryneform bacteria will also cause a hypersensitive response. Ralstonia solanacearum cause various results depending on the race. Race 1 results in chlorosis after two days, race 2 induces a typical hypersensitive response in one day and race 3 results in chlorosis after two to eight days [15].

The genetic identity of Burkholderia species has been analyzed by polymerase chain reaction (PCR) using 16S rRNA sequences [16,17]. The discriminatory power of 16S rRNA is too restricted to reveal the detailed phylogenetic relationships among B. plantarii, B. glumae and B. gladioli because of extremely slow rate of evolution of the 16S rRNA gene, it cannot discriminate closely related microorganisms [18]. On the other hand, the genes encoding the β-subunit polypeptide of DNA gyrase (gyrB) estimated to evolve much faster than the 16S rRNA gene that can be used to develop a specific and sensitive detection method [18] to distinguish among Burkholderia species [5]. Therefore, specific primers developed from the gyrB sequences should be reliable for specific detection and identification of B. glumae and B. gladioli in rice materials.

The aims of this study were to 1) isolate and identify the bacterial panicle blight (BPB) pathogen with culture media; 2) verify the causal agent of BPB with PCR; 3) evaluate virulence with tobacco plants; and 4) determine distribution of B. glumae in plants with PCR.

Material and Methods

Isolation and identification of the pathogen

During a 2015 cropping season, 178 naturally infected immature rice panicles with bacterial Panicle blight (BPB) symptoms were collected from growing counties of Arkansas (Supplemental Table S1 and Figure 1).  Seeds and florets with discoloration and blanked panicles were collected in paper bags and kept in a refrigerator at 4°C until processing. Seeds were disinfected with 10% sodium hypochlorite for 1 min and rinsed three times with sterile distilled water then left to dry on a sterile filer paper.  Disinfected seeds were directly plated on a semi-selective media of CCNT (containing 2 g of yeast extract, 1 g of polypepton, 4 g of inositol, 10 mg of cetrimide, 10 mg of chloramphenicol, 1 mg of novobiocin, 100 mg of chlorotharonil and 18 g of agar in 1000 ml of distilled water, and adjusted to pH 4.8). [19]. From each individual sample 30 seeds were planted on two petri dishes using 15 seeds per dish.  Those dishes were sealed using a Para film and incubated at 38°C for 3 to 5 days. The bacterial colonies on these dishes were examined for their morphological characteristics compared with our reference strains of B. glumae. The typical features for bacterial Panicle blight (BPB) on artificial detection media (CCNT) are yellowish white, round, smooth and swollen colonies with a diffusible yellow pigment [19]. Single colonies from each culture plate were collected with a flamed bacteriological loop and streaked on King B medium [20], incubated at 38°C for 48 h, and then stored in Cryo-vial tubes at -80°C in 30% glycerol. Each isolate was given a culture number.

plant-pathology-Arkansas-rice

Figure 1: Arkansas rice production county map showing the location of BPB samples collected. Samples collected from 9 rice producing counties of Arkansas as indicated by round cycle dots (please make better figure, it was unclear for me to me to read the name of counties).

Verification of the causal agent of bacterial panicle blight (BPB) with PCR

The presence of B. glumae was identified with PCR using a pair of primers to detect specific DNA fragments corresponding to the gyrB nucleotide sequences, glu-FW (5’-GAAGTGTCGCCGATGGAG-3’) and glu-RV (5’-CCTTCACCGACAGCACGCAT-3’) [5]. Similar primer pairs of gli-FW (5’-CTGCGCCTGGTGGTGAAG-3’) and gli-RV (5’- CCGTCCCGCTGCGGAATA -3’) were also used to amplify DNA fragments corresponding to the gyrB nucleotide sequences of B. gladioli [5]. PCR amplification was initiated for 20 µl containing 1 µl of template DNA with denaturation at 94°C for 2 min: followed by 35 cycles at 94°C for 1 min, 63°C for 1 min and 72°C for 1 min and 72°C for 10 min as final extension. Aliquots (10 µl) of each PCR products were loaded onto horizontal electrophoresis on a 2% Tris-acetate-EDTA (TAE) agarose gel (Promega) at 80 V for 90 min. Gels were stained with Syber safe for detection of 530 bp,  and 479 bp DNA fragments corresponding to the gryB nucleotide sequences of B. glumae and B. gladioli, respectively [5]. A 1-kb ladder (Invitrogen Co.) was used to predict the fragment size of PCR products. However B. plantarii  and other Burkholdria spp. are not included with this study because they are not detected as important disease causing pathogen in USA. 

Distribution of B. glumae in plants with PCR

To study distribution of B. glumae ten naturally infected rice plants were uprooted from the production fields and brought to a laboratory.  Root, stem, sheath, leaf, chuff and seed were collected individually and cleaned with water. These plant parts were disinfected with 1% sodium hypochlorite for 1 min, then rinsed three times with sterile distilled water, and left to dry on a sterile filer paper. Disinfected plant parts were cut to a 1 cm long piece except the seeds that were placed directly on artificial detection (CCNT) media in petri dishes in an incubator at 38°C for 3 to 5 days. DNA was extracted from these plant parts using a DNeasy Plant Mini Kit (Qiagen, Carlsbad, CA, USA), and from bacteria DNA grow on plate media using a UltraClean Microbial DNA Isolation Kit (MO BIO Laboratories, Qiagen, Carlsbad, CA, USA), respectively.

Virulence evaluation with tobacco plants

All forty-three isolates identified to be B. glumae were tested for their pathogenicity level with tobacco as described by Furuya et al. [10].  Specifically, tobacco plants (Nicotinaa bethanamiana) were grown to 8 to 9 leaves in approximately 4 weeks after sowing in the greenhouse with a day time temperature ranging between 37°C to 41°C and 75 to 90% relative humidity (RH). Inocula were placed on a King’s B agar (KBA) plates incubated at 38°C for 48 h, then harvested with a sterile cotton swab and suspended in a test tube containing 9 mL of sterile distilled water, and concentration of bacterial suspension were adjusted to be about 108 CFU/mL for inoculation. Three to five tobacco seedlings with the fully expanded leaves were inoculated by injecting at least 3 leaves with 0.5 ml of bacterial suspension using 1 mL sterile syringes and control leaves were injected with sterile distilled water.  The control with water did not cause any symptoms one week after injection.  The diameters of the lesion of cell death were measured one week after inoculation using four-category disease scale described in Table 1. Large area of necrosis is an indicator for highly virulent strains. After disease scoring, bacteria strains were re-isolated from the diseased tobacco plant to complete Koch’s postulates [21].

Scalea Virulentb Symptoms No. of isolates
0 Not No symptom produced after inoculation 0
1 Weakly Slightly browning around the injected site with less than 0.5 cm in diameter 5
2 Moderately Distinct lesions with 0.5–1 cm in diameter 9
3 Highly Lesion spreading from the injection with  diameter more than 1 cm lesion and even in some isolated cases the whole leaf get wilted completely 31
aAssigned rating based on necrosis on tabacco leaves one week after injection.
bPredicted pathogenicity based on lesion of necrosis on tabacco leaves one week after injection.

Table 1: Scoring system for tobacco seedling based on the level of hypersensitive reaction to each isolates of B. glumae in greenhouse inoculation tests and number of isolates falling into each category.

Results

Isolation and morphological Identification of the pathogen

Initial symptoms of the bacterial Panicle blight (BPB) caused by B. glumae were observed on the panicles of the rice plant. Infected panicles with a dark­ brown discoloration and heavily infected panicles with upright due to blanking were basic characteristics to collect panicle samples from 9 rice producing counties of Arkansas. A total of 178 rice panicle samples were collected (Supplemental Table S1). Seeds and Florets from each collected samples were plated on a semi-selective medium and incubated at a temperature range of 38°C to 40°C for 5 days. The colony characteristics of these samples were compared with our reference strains of B. glumae. About 41% (73 samples) showed similar morphological characteristics to reference strain (Supplemental-2) which is yellowish white, round, smooth and swollen colonies with a diffusible yellow pigment [19] as shown in Table 2. Isolates which have all other morphological characteristics but lacked pigment production also grow well on artificial detection media (CCNT) medium but excluded from this study since it has been reported that this types of strains are not pathogenic to rice [22].

County No. of Samples No. of bacteria Isolated No. of B. glumae isolates
Praire 4 0 0
Lincoln 13 4 3
Desha 3 0 0
St. Francis 1 1 1
Clay 6 3 1
Mississippi 4 1 1
Craighead 3 2 1
Jackson 11 7 2
Woodruff 20 0 0
Arkansas 120 54 34
  178 73 43

Table 2: Total numbers, their morphological and molecular identification of samples with respective of the counties.

Verification of the causal agent of bacterial panicle blight (BPB) with PCR

The identity of seventy-three isolate of bacterial was also confirmed using B. glumae and B. gladioli-specific PCR amplification [5]. An approximately 530 bp DNA fragments of gryB were amplified for 45 isolates indicating that only 62% out of 73 isolates belongs to B. glumae and the remaining twenty-eight isolates did not react with B. glumae-specific primers. On the contrary, no fragments were amplified using B. gladioli-specific primer pairs (Table 3).

Sample No. Variety Virulencea PCR Ampliconb
glu-FW/glu-RV gli-FW/gli-RV
Bg5 Wells 3 Yes No
Bg 6 Roy J 3 Yes No
Bg 7 13AR1021 3 Yes No
Bg 14 CL2134 3 Yes No
Bg 17 Mermantau 3 Yes No
Bg 20 CLX2008 3 Yes No
Bg 31 STG12P-23-168 1 Yes No
Bg 32 RU1401081 2 Yes No
Bg 34 RU1501133 2 Yes No
Bg 38 RU1501027 3 Yes No
Bg 41 STG-12-145 3 Yes No
Bg 44 RU1501087 3 Yes No
Bg 46 CL151 3 Yes No
Bg 49 RU1401161 1 Yes No
Bg 50 RU1502165 3 Yes No
Bg 53 RU1403129 3 Yes No
Bg 60 RU1501093 3 Yes No
Bg 61 Roy J 3 Yes No
Bg 62 CoDR 3 Yes No
Bg 64 Rex 3 Yes No
Bg 73 RU1501173 3 Yes No
Bg 77 RU1501133 3 Yes No
Bg 81 RU1502068 3 Yes No
Bg 87 RU1504122 3 Yes No
Bg 90 RU1203190 1 Yes No
Bg 91 RU1404194 3 Yes No
Bg 108 RU1301021 1 Yes No
Bg 111 RU1504186 3 Yes No
Bg 112 RU1501185 3 Yes No
Bg 113 RU1303184 3 Yes No
Bg 114 RU1504193 1 Yes No
Bg 118 RU1502152 3 Yes No
Bg 119 RU1501182 3 Yes No
Bg 121 CL172 3 Yes No
Bg 125 RU1003113 2 Yes No
Bg 127 RU1503110 3 Yes No
Bg 128 RU1502109 3 Yes No
Bg 129 RU1501148 3 Yes No
Bg 132 RU1303181 2 Yes No
Bg 135 RU1501102 2 Yes No
Bg 145 RU1501108 3 Yes No
Bg 151 RU1501111 2 Yes No
Bg 153 CL271 2 Yes No
Bg 155 CL 111 3 Yes No
Bg 157 CL 111 3 Yes No
Control DI Water H    
a H indicates no visible reaction, 1, weakly virulent, 2, moderately virulent, 3,highly virulent one week after injection respectively.
b glu-FW/glu-RV indicates primers specifically to B. glumae and gli-FW/gli-RV indicates primers specifically to B. gladioli, respectively.  Yes indicates PCR product produced and No indicates no PCR amplicon.

Table 3: Results of Virulence level tested by inoculation of Isolates into tobacco leaves to determine pathogenicity level and PCR reaction for two primer sets.

Virulence evaluation with tobacco plants

Reaction to tobacco revealed that all 45 isolates tested are pathogenic at different virulence level (Table 3). About 31 isolates (69%) of the 45 isolates tested were highly virulent (Figure 2A), while nine isolates (20%) moderately virulent. The remaining isolates categorized as weakly virulent isolates whereas plants injected with sterile distilled water remained healthy with no visible hypersensitivity reaction on the leaves (Figure 2B). Koch’s postulates were confirmed by re-isolating from inoculated tobacco leaves and then grow them on a semi-selective media (CCNT) for B. glumae (data not shown) Pathogenic B. glumae isolates produced a yellow pigment, identified as toxoflavin, while non-pathogenic strains did not [22]. Accordingly, all seventy-three isolates tested for their virulence level reislolated from tobacco, and all produced a yellow pigment, which indicated that they are still pathogenic B. glumae bacteria.

plant-pathology-hypersensitive-necrosis

Figure 2: Photographic presentation of hypersensitive necrosis on tobacco leaves caused by B. glumae. A. Typical necrosis one week after injection with B. glumae, B. One week after tobacco leaves infiltrated with sterile distilled water. W indicated that water was injected.

Distribution of B. glumae in a plant

Ten naturally infected rice plants were removed from a rice paddy and different plants were plated on semi-selective media (CCNT). B. glumae were isolated from seed followed by stem and sheath at low concentration level of yellow pigment. However, roots and leaves did not show any visible yellow pigment on semi-selective media (CCNT) (Figure 3). Pathogen identification was confirmed by PCR using B. glumae–specific primer pair with DNA extracted from individual plant parts (root, stem, leaf, sheath, chaff, and seed). PCR products with predicted sizes were obtained from DNA extracted from seed and chaff. No PCR products were amplified from roots and leaves of rice plant but low level of amplification observed for stem and sheath (Figure 4).

plant-pathology-specific-medium

Figure 3: Photographic presentation of morphology of rice plant parts on Semi specific medium (CCNT). RT indicates roots, ST from stem, CF from Chaff. LF from leaf, SH from sheath, SD from seed, respectively.

plant-pathology-Photographic-presentation

Figure 4: Photographic presentation of PCR amplification of 530-bp product for B. glumae using a pair of primers to detect specific DNA fragments corresponding to the gyrB nucleotide sequences. Samples collected from indicated from different parts of infected rice plants M indicates 1 kilobase ladder, Bg indicates PCR product from B. glumae DNA as positive control, RT indicates DNA from roots, LF from leaf, ST from stem, SH from sheath, CF from Chaff, SD from seed and Co indicates water, respectively.

plant-pathology-Supplemental

Figure S2: Supplemental

Discussion

Bacterial Panicle blight (BPB) is an emerging bacterial disease that causes significant crop loss worldwide. Characterization of the causal agent for BPB and in plant detection pathogen is an important prerequisite to manage BPB.  In the present study, 45 disease samples out of the total 178 from commercial rice fields in the state of Arkansas, USA were determined due to B. glumae. None of the disease samples were caused by B. gladioli suggesting that B. glumae is the causal agent for BPB in Arkansas. The fact that many non B. glumae were isolated from diseased tissue needs further exploration to see if any uncharacterized microorganism can contribute the development of the syndrome and potential ecological relationships with B. glumae. It is well known that the development of disease symptoms and severity of any plant disease not only depends on virulence of the strain, but also on environmental factors, particularly weather conditions. Symptoms typically caused by B. glumae were panicle blighting with floret discoloration (with a gray-brown color), usually on the lower half of the developing grain, with a clear deep brown border followed by sterility or partial filling of the florets causing the panicles to stand erect [23,24].  However, all samples examined in the present study were with these symptoms but some of samples found to be other microorganism but not B. glumae and/or B. gladioli. Our study clearly demonstrated that symptom of bacterial Panicle blight (BPB) was not sufficient to identify the causal agent of this disease.

Apparently, it is challenge to differentiate pathogens that are closely related physiologically and taxonomically by the symptoms they produce and by their growth on selective media. A good identification scheme depends not only on developing a satisfactory resolution level of methods, but also on the group of bacteria studied [25-27]. Semi specific medium (CCNT) is useful for rough screening for bacteria that cause BPB by visualization of unique yellow pigment as indicative of toxoflavin producing bacteria.  In the present study we showed that unknown bacteria other than B. glumae and B. gladioli producing similar yellow pigment suggesting that Semi specific medium (CCNT) alone was not sufficient for positive identification of both bacteria.  It is fully possible that other unknown bacteria in rice seeds can producing toxoflavin that needs to be further investigated in order to understand their pathogenicity and their bio-control potentials for managing bacterial Panicle blight (BPB) and other rice diseases. In the future, a defined culture medium specifically to B. glumae and B. gladiolia will need to be developed.

We have not found B. gladioli in all diseased samples from Arkansas except B. glumae.  To our knowledge, the present study provides the first experimental evidence of B. glumae as the major cause of bacterial Panicle blight (BPB) in Arkansas. This is consistent with that the major causal agent of BPB was B. glumae whereas B. gladioli was less virulent in other geographic regions [28].  In the present study, different isolates of B. glumae show different levels of pathogenicity based on different hypersensitive reaction patterns on tobacco leaves suggesting that there exist genomic and virulence levels variation in Arkansas B. glumae isolates.  Forty-five isolates had a hypersensitivity index ranging from weakly to highly sensitive reaction (Table 3). The majority of them caused large necrosis on tobacco suggest that these Arkansas isolates are highly virulent.

In summary, we showed that accurate identification of the causal agent for bacterial Panicle blight (BPB) is challenging, and cross-referencing among two or more detection methods is desirable to ensure that the causal agent can be positively identified. We learned that once you suspect the symptom of rice plant tissue damaged by BPB, the next plausible step is to examine seeds derived from diseased rice plants. If possible, the disease tissues should be obtained from vegetative stage before flowering to localize pathogen in stem and/or sheaths.  In contrast, because leaves and roots are not a favorable residences for the B. glumae as compared to seed, stem and sheath. Therefore, it will not be useful to detect pathogen in leaves and roots.  Additionally, we demonstrated that there exhibit difference in virulence among B. glumae and these characterized isolates can be used to screen genetic resistance to bacterial Panicle blight (BPB). Together, our findings are useful for plant quarantine and bacterial Panicle blight (BPB) pathogen identification, ultimately these new knowledge will be useful to manage this emerging agronomically important rice disease worldwide.

Note: Isolates that showed yellow pigment on semi-selective media (CCNT) indicated by Yes but if they did not detected using specific primers for B glumae and B. gladioli using PCR they will be indicated with No. That means morphologically similar but not B glumae and B. gladioli because PCR is more specific detection than morphology.

Acknowledgements

The authors thank Scott Belmar, Tibebu Gebremariam of Rice Research and Extension Center, UA, AR and Tracy Bianco of USDA Agriculture Research Service Dale Bumpers National Rice Research Center for their excellent technical supports. This project was supported by Arkansas Rice Research Promotion Board.

References

      Host rice B.glumae Species-specific  Primer sets
Sample No.  Location/County Date variety Morphological glu-FW/glu-RV  gli-FW/gli-RV
1 Praire county 8/17/2015 Roy J No    
2 Praire county 8/17/2015 CL163 No    
3 Praire county 8/17/2015 CLX2134 No    
4 Praire county 8/17/2015 Taggart No    
5 Arkansas county  8/14/2014 Wells Yes Yes No
6 Arkansas county  8/14/2014 Roy J Yes Yes No
7 Arkansas county  8/14/2014 13AR1021 Yes Yes No
8 Arkansas county  8/13/2014 CL151 Yes No No
9 Lincoln county 8/20/2015 CL271 No    
10 Lincoln county 8/20/2015 CL111 No    
11 Lincoln county 8/20/2015 CL151 No    
12 Lincoln county 8/20/2015 CL163 No    
13 Lincoln county 8/20/2015 RU1301084 Yes No No
14 Lincoln county 8/20/2015 CL2134 Yes Yes No
15 Lincoln county 8/20/2015 CL172 No    
16 Lincoln county 8/20/2015 CL151 No    
17 Lincoln county 8/20/2015 Mermantau Yes Yes No
18 Lincoln county 8/20/2015 RU1501102 No    
19 Lincoln county 8/20/2015 RU1301021 No    
20 Lincoln county 8/20/2015 CLX2008 Yes Yes No
21 Lincoln county 8/20/2015 CLX2008 No    
22 Desha County 8/20/2015 RU1501105 No    
23 Desha County 8/20/2015 Lakast No    
24 Desha County 8/20/2015 Roy J No    
25 Arkansas county  8/24/2015 STG-04-121 No    
26 Arkansas county  8/24/2015 RU1301084 No    
27 Arkansas county  8/24/2015 RU1501105 No    
28 Arkansas county  8/24/2015 STG-12-145 No    
29 Arkansas county  8/24/2015 STG-04-065 No    
30 Arkansas county  8/24/2015 RU1501173 No    
31 Arkansas county  8/24/2015 STG12P-23-168 Yes Yes No
32 Arkansas county  8/24/2015 RU1401081 Yes Yes No
33 Arkansas county  8/24/2015 RU1501185 No    
34 Arkansas county  8/24/2015 RU1501133 Yes Yes No
35 Arkansas county  8/24/2015 Wells No    
36 Arkansas county  8/24/2015 RU1401161 No    
37 Arkansas county  8/24/2015 RU1501102 No    
38 Arkansas county  8/24/2015 RU1501027 Yes Yes No
39 Arkansas county  8/24/2015 RU1501007 Yes No No
40 Arkansas county  8/24/2015 STG-23-168 No    
41 Arkansas county  8/24/2015 STG-12-145 Yes Yes No
42 Arkansas county  8/24/2015 RU1501093 No    
43 Arkansas county  8/24/2015 STG-06--61 No    
44 Arkansas county  8/24/2015 RU1501087  Yes Yes No
45 Arkansas county  8/24/2015 CLX2008 No    
46 St. Francis county 8/24/2015 CL151 Yes Yes No
47 Arkansas county  8/25/2015 RU1003123 No    
48 Arkansas county  8/25/2015 RU1504198 No    
49 Arkansas county  8/25/2015 RU1401161 Yes Yes No
50 Arkansas county  8/25/2015 RU1502165 Yes Yes No
51 Arkansas county  8/25/2015 RU1503169 No    
52 Arkansas county  8/25/2015 RU1502128 Yes No No
53 Arkansas county  8/25/2015 RU1403129 Yes Yes No
54 Arkansas county  8/25/2015 RU1501130 Yes No No
55 Arkansas county  8/25/2015 RU0901130 No    
56 Arkansas county  8/25/2015 RU1501087  No    
57 Arkansas county  8/25/2015 RU1402088 No    
58 Arkansas county  8/25/2015 RU1501090 No    
59 Arkansas county  8/25/2015 RU1502097 No    
60 Arkansas county  8/25/2015 RU1501093 Yes Yes No
61 Arkansas county  8/25/2015 Roy J Yes Yes No
62 Arkansas county  8/25/2015 CoDR Yes Yes No
63 Arkansas county  8/25/2015 RU1505056 Yes No No
64 Arkansas county  8/25/2015 Rex Yes Yes No
65 Arkansas county  8/25/2015 CHNR No    
66 Arkansas county  8/25/2015 RU1504083 Yes No No
67 Arkansas county  8/25/2015 RU1301084 No    
68 Arkansas county  8/25/2015 RU1501081 Yes No No
69 Arkansas county  8/25/2015 MM14 No    
70 Arkansas county  8/25/2015 RU1502131 Yes No No
71 Arkansas county  8/25/2015 RU1502094 No    
72 Arkansas county  8/25/2015 RU1503095 Yes No No
73 Arkansas county  8/25/2015 RU1501173 Yes Yes No
74 Arkansas county  8/25/2015 RU1502174 No    
75 Arkansas county  8/25/2015 RU1303174 Yes No No
76 Arkansas county  8/25/2015 RU1503132 No    
77 Arkansas county  8/25/2015 RU1501133 Yes Yes No
78 Arkansas county  8/25/2015 RU1502134 No    
79 Arkansas county  8/25/2015 CL271 No    
80 Arkansas county  8/25/2015 RU0901130 No    
81 Arkansas county  8/25/2015 RU1502068 Yes Yes No
82 Arkansas county  8/25/2015 RU1303153 No    
83 Arkansas county  8/25/2015 RU1502065 No    
84 Arkansas county  8/25/2015 RU1503069 No    
85 Arkansas county  8/25/2015 RU1401070 Yes No No
86 Arkansas county  8/25/2015 RU1303181 No    
87 Arkansas county  8/25/2015 RU1504122 Yes Yes No
88 Arkansas county  8/25/2015 RU1404191 No    
89 Arkansas county  8/25/2015 RU1501076 No    
90 Arkansas county  8/25/2015 RU1203190 Yes Yes No
91 Arkansas county  8/25/2015 RU1404194 Yes Yes No
92 Arkansas county  8/25/2015 CL151 Yes No No
93 Arkansas county  8/25/2015 RU1502192 No    
94 Arkansas county  8/25/2015 RU1502125 No    
95 Arkansas county  8/25/2015 RU1504122 No    
96 Arkansas county  8/25/2015 RU1503098 No    
97 Arkansas county  8/25/2015 Frances Yes No No
98 Arkansas county  8/25/2015 RU1401081 No    
99 Arkansas county  8/25/2015 RU1502137 No    
100 Arkansas county  8/25/2015 RU1502045 No    
101 Arkansas county  8/25/2015 RU1502048 No    
102 Arkansas county  8/25/2015 RU0903147 Yes No No
103 Arkansas county  8/25/2015 RU1402051 No    
104 Arkansas county  8/25/2015 RU1303181 No    
105 Arkansas county  8/25/2015 RU1303153 No    
106 Arkansas county  8/25/2015 RU1502031 Yes No No
107 Arkansas county  8/25/2015 RU1304156 No    
108 Arkansas county  8/25/2015 RU1301021 Yes Yes No
109 Arkansas county  8/25/2015 RU1404154 No    
110 Arkansas county  8/25/2015 RU1402008 No    
111 Arkansas county  8/26/2015 RU1504186 Yes Yes No
112 Arkansas county  8/26/2015 RU1501185 Yes Yes No
113 Arkansas county  8/26/2015 RU1303184 Yes Yes No
114 Arkansas county  8/26/2015 RU1504193 Yes Yes No
115 Arkansas county  8/26/2015 RU1502183 Yes No No
116 Arkansas county  8/26/2015 RU1502195 No    
117 Arkansas county  8/26/2015 RU1404154 No    
118 Arkansas county  8/26/2015 RU1502152 Yes Yes No
119 Arkansas county  8/26/2015 RU1501182 Yes Yes No
120 Arkansas county  8/26/2015 CL163 No    
121 Arkansas county  8/26/2015 CL172 Yes Yes No
122 Arkansas county  8/26/2015 RU1502189 Yes No No
123 Arkansas county  8/26/2015 JZMN2 No    
124 Arkansas county  8/26/2015 RU1504197 No    
125 Arkansas county  8/26/2015 RU1003113 Yes Yes No
126 Arkansas county  8/26/2015 RU1501111 No    
127 Arkansas county  8/26/2015 RU1503110 Yes Yes No
128 Arkansas county  8/26/2015 RU1502109 Yes Yes No
129 Arkansas county  8/26/2015 RU1501148 Yes Yes No
130 Arkansas county  8/26/2015 RU1502146 No    
131 Arkansas county  8/26/2015 RU1501188 No    
132 Arkansas county  8/26/2015 RU1303181 Yes Yes No
133 Arkansas county  8/26/2015 RU1501142 No    
134 Arkansas county  8/26/2015 RU1403104 No    
135 Arkansas county  8/26/2015 RU1501102 Yes Yes No
136 Arkansas county  8/26/2015 RU1501099 No    
137 Arkansas county  8/26/2015 RU1501096 No    
138 Arkansas county  8/26/2015 RU1502140 No    
139 Arkansas county  8/26/2015 RU1502137 No    
140 Arkansas county  8/26/2015 RU1505178 No    
141 Arkansas county  8/26/2015 RU1504100 No    
142 Clay county 9/2/2015 Antonio No    
143 Clay county 9/3/2015 CLX2134 Yes No No
144 Clay county 9/4/2015 CL163 Yes No No
145 Clay county 9/5/2015 RU1501108 Yes Yes No
146 Clay county 9/6/2015 14SIT891 No    
147 Clay county 9/7/2015 RU1501151 Yes No No
148 Mississippi County 9/15/2015 RU1501105 No    
149 Mississippi County 9/15/2015 RU15001108 No    
150 Mississippi County 9/15/2015 14SIT891 No    
151 Mississippi County 9/15/2015 RU1501111 Yes Yes No
152 Craighead County 9/15/2015 RU1301021 Yes No No
153 Craighead County 9/15/2015 CL271 Yes Yes No
154 Craighead County 9/15/2015 MM14 No    
155 Jackson County 9/11/2015 CL 111 Yes Yes No
156 Jackson County 9/11/2015 CL 111 Yes No No
157 Jackson County 9/11/2015 CL 111 Yes Yes No
158 Jackson County 10/8/2015 RU1301084 No    
159 Jackson County 10/8/2015 SIT664 No    
160 Jackson County 10/8/2015 RU1501130 No    
161 Jackson County 10/8/2015 RU1501148 Yes No No
162 Jackson County 10/8/2015 RU1501027 Yes No No
163 Jackson County 10/8/2015 RU1501030 Yes No No
164 Jackson County 10/8/2015 RU1501050 Yes No No
165 Jackson County 10/8/2015 RU 1501027 No    
166 Woodruff County 7/29/2015 RTCLXL729 No    
167 Woodruff County 7/29/2015 RTXL753 No    
168 Woodruff County 7/29/2015 RU1100477 No    
169 Woodruff County 7/29/2015 CLX2008 No    
170 Woodruff County 7/29/2015 Mermenta No    
171 Woodruff County 7/29/2015 RTCLXL745 No    
172 Woodruff County 7/29/2015 Taggart No    
173 Woodruff County 7/29/2015 RTXP760 No    
174 Woodruff County 7/29/2015 RU1301023 No    
175 Woodruff County 7/29/2015 CL163 No    
176 Woodruff County 7/29/2015 Roy J No    
177 Woodruff County 7/29/2015 CL271 No    
178 Woodruff County 7/29/2015 Jupiter No    

Supplemental Table-S1: List of samples used in this and results by morphological and conventional polymerase chain reaction assay.

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