Identification and Characterization of Pectobacterium carotovorum subsp. carotovorum and P. atrosepticum of Potato Pathogenic Bacteria in Northwest China

CHENG Liang

(1.Key Laboratory of Agricultural Integrated Pest Management in Qinghai Province, Institute of Plant Protection, Qinghai Provincial Academy of Agriculture and Forestry Sciences, Xining 810016, China; 2.State Key Laboratory of Plateau Ecology and Agriculture, Qinghai University, Xining 810016, China)

Abstract:Northwest China is an important potato producing area in China, so to identify tuber soft rot and stem black leg pathogens of potato (Solanum tuberosum L.) in Qinghai, Gansu, Ningxia, Shaanxi and Inner Mongolia, this paper constructed phylogenetic tree from 28 standard potato tuber soft rot and stem blackleg pathogen strains and 48 pathogenetic strains isolated from 5 provinces in China based on the 16S rDNA gene sequences. Pathogenic bacterial strains isolated from infected potato tuber and stem collected from 5 provinces were identified based on their morphological and cultural characteristics, biochemical and physiological methods, 16S rDNA sequences as well as subspecies/species region analysis. Pathogenicity difference among disease-causing isolates was analyzed by potato tuber inoculation method. The pel specific primers showed that all the 48 bacterial strains have pectolytic characteristics. 16 strains were identified as Pectobacterium carotovorum subsp. carotovorum with a frequency of 31.25% and 30 strains as Pectobacterium atrosepticum with a frequency of 64.50%. Phylogenetic analysis of the 16S rRNA gene sequence showed that Pectobacterium carotovorum subsp. carotovorum and Pectobacterium atrosepticum isolates clustered together with those identified in other parts of the world with 97% and 99% bootstrap support values, respectively. Pathogenicity assays using isolated representative strains showed varied levels of tuber maceration ability. Aggressive of Pectobacterium carotovorum subsp. carotovorum and Pectobacterium atrosepticum isolated strains were less than that of the standard strains. These results indicated that the causing agents of potatoes in Northwest region were identified as Pectobacterium carotovorum subsp. carotovorum and Pectobacterium atrosepticum.

Key words:Solanum tuberosum L.; identification; pathogenicity; Pectobacterium spp.; northwest region

Potato (Solanumtuberosum) is one of the most important food crops in China and in the world[1]. After the implementation of the potato staple food strategy in China in 2015, potato crop gradually become the staple food for human consumption[2]. According to the data from the Food and Agriculture Organization in 2010, the planting area of potatoes throughout the worldwide is about 1.87～1.92 million hm2, and the total output is from 320 to 340 million tons[3]. However, potato crop loss due to pathogens places pressure on the production of potato industry.Pectobacteriumspp. and other soft rot bacteria have been reported in the world[4-7]. However, although soft rot have been reported in other parts of Asia and elsewhere in the world, the identification or genetic diversity of soft rot pathogens infecting potatoes or other host plants have not been reported throughout the northwestern region of China. The genusPectobacteriumis currently divided into five distinct clades includingPectobacteriumatrosepticum(Pa),Pectobacteriumwasabiae(Pw),Pectobacteriumbetavasculorum(Pbv),Pectobacteriumcarotovorumsubsp.brasiliense(Pcb) andPectobacteriumcarotovorumsubsp.carotovorum(Pcc)[8-12].Pectobacteriumspecies has a wide range of hosts, probably due to its widespread in the natural environments and has been separated from different sources including soil, water, plant hosts and arthropods[13-14], while obligate host of Pcb, Pw and Pc is potato which usually leads to blackleg[15].

The northwest region is the main potato producing area in China, which is the largest area and the highest yield area of potato. At the same time, this region is also the concentrated place of the poor population in China. Potato production in northwest region is an important income to households but it is hampered by soft rot and blackleg diseases. All farmers in northwest regions encounter a very difficult task of maximizing yield when facing blackleg and soft rot diseases. Therefore, it is important to investigate the accurate identity and genetic diversity ofPectobacteriumspp. causing soft rot and blackleg in potatoes grown in northwest. This study isolated and established the identity and virulence of pectolytic bacteria associated with soft rot diseases of potatoes from northwest regions.

1 Materials and methods

1.1 Bacterial strains, media, isolation, mainten-ance and characterization

1.1.1DiseasedsamplecollectionPotato tubers showing symptoms of soft rot were obtained from the main potato producing areas in Qinghai, Gansu, Ningxia, Shanxi and Inner Mongolia during the 2015/2016 potato growing season. Bacterial strains were isolated from each sample according to standard protocols[16]. Type strains ofP.carotovorumsubsp.brasiliense(IBSBF1692),P.wasabiae(CFBP 3304),P.carotovorumsubsp.carotovorum(LMG2404),P.carotovorumsubsp.odoriferum(CFBP3259),P.betavasculorum(CFBP2122),P.atrosepticum(LMG2386) andD.dadantii(Ech25) from the Belgian Coordinated Collections of Microorganisms were used in this study for comparison.

1.1.2IsolationandmaintenancePotato tubers samples showing disease symptoms were cleaned, surface-sterilized with 0.5% sodium hypochlorite, washed with sterile distilled water, placed in sterile 0.85% saline solution and crushed using sterile mortar and pestle under aseptic conditions. The resulting suspension plated on crystal violet pectate (CVP) (Pectobacteriumselective medium) media according to the procedure[17-18], and incubated at 28 ℃ for 48 h. Individual colonies which formed pits on CVP media were picked up, resuspended in 0.85% saline and streaked on LB plates (Tryptone 10 g, Yeast extract 5 g, Sodium chloride 10 g, ddH2O 1 L) and then incubated at 28 ℃ for another 24 h. The procedure was repeated several times to obtain pure cultures. The purified colonies were stored in 20% glycerol (volume fraction) at -80 ℃.

1.1.3BiochemicalandphysiologicaltestsThe identification of 48Pectobacteriumstrains was carried out according to the method described by Schaadand associates[19], which was commonly used to differentiatePectobacteriumsubspecies: indol production, sucrose reducing substance, maltose, sorbitol, arabitol, cellobiose, raffinose, melibiose and α-methylglucoside acid-producing action, lactose fermentation, growth at 37 ℃, tolerance to 5% NaCl, utilization of citrate, phosphatase, catalase and oxidase activity, and sensitivity to erythromycin.

1.2 PCR identification

Extraction of total DNA from 48 isolated strains was carried out using Ezup?(SK8255) column genomic DNA extraction kit [Sangon Biotech (Shanghai) Co., Ltd.] according to the manufacturer’s recommendations. The purity of extracted DNA was monitored by using Nanodrop 2000 spectrophotometer (Thermo Scientific; Wilmington, DE, USA) before storage at -20 ℃.

The pectatelyase (pel) gene in the soft rot bacteria was amplified by ADE-1/ADE-2 primers[20]in all isolates and standard strains for identification of pectolytic bacteria. Furthermore, species and subspecies-specific primers (Sangon Biotech (Shanghai) Co., Ltd.) used to distinguish betweenPectobacteriumspp. were shown in Table 1. The species and subspecies-specific Eca1-F/Eca1-R[21],Br1-F/L1-R[22], EXPCC-F/EXPCC-R[23]and PhF/Ph-R[24]primers were used to identify the presence ofP.atrosepticum,P.carotovorumsubsp.carotovorum,P.carotovorumsubsp.brasilienseandP.wasabiae, respectively.

Table 1 Sequence of subspecies -specific primers used in this study

PCR reactions were performed in a total volume of 50 μL consisting of 50 ng template DNA, 200 μmol·L-1dNTPs, 0.4 μmol each forward and reverse primers, 0.5 UTaqDNA polymerase and 1×TaqDNA polymerase reaction buffer. PCR conditions were: 94 ℃ for 3 min, followed by 35 cycles of 94 ℃ for 30 s, 30～45 s at a primer-specific temperature (Table 1) and 72 ℃ for 90 s. A final elongation step was performed at 72 ℃ for 4 min. All amplification reactions were done using the My cyclerTMthermal cycler (Bio-rad, USA) and all primers were synthesized from Sangon Biotech (Shanghai) Co., Ltd.. Following PCR amplification, 5 μL of each reaction products were electrophoresed by using a 2% (mass volume fraction) agarose gel. The gel was run at 200 V for 30 min and observed under UV-illumination, Bio-rad gel docTMEZ imager (California, USA). 1 kb DNA ladder was used as a control.

1.3 Sequencing and phylogenetics

A special primer ofPectobacteriumspp. 16S rDNA gene was used for subsequent phylogenetic analysis. Total DNA previously extracted was used to perform PCR reactions by using primers 27F/1942R(5′-AGAGTTTGATCGGCTCAG-3′/5′-TACGGCTACCTTG-TTACGACTT-3′). PCR reactions were carried out in a final mixture of 50 μL consisting of 50 ng of template DNA, 200 μmol of NTPs, 0.4 μmol of each forward and reverse primers, 0.5 UTaqDNA polymerase and 1×TaqDNA polymerase reaction buffer. DNA amplification was performed in a My cyclerTMthermal cycler (Bio-rad, USA) under the following conditions: 95 ℃ for 5 min, followed by 30 cycles of 95 ℃ for 30 s, 52 ℃ for 30 s, and 72 ℃ for 90 s with a final extension at 72 ℃ for 4 min. All the PCR products were purified using the Wizard?SV Cleanup System (Promega, Corp.) according to the manufacturer’s recommendations. Direct bidirectional sequencing was done using the ABI PRISM 3730 genetic analyzer at Sangon Biotech (Shanghai) Co., Ltd. before the obtained raw sequences were checked and manually edited for base mismatch correction using chromas.exe v 2.4.1 software. The neighbor-joining (NJ) phylogenetic analyses were performed with MEGA v 5.05 software. Boot strapping was executed with 1 000 replications. The 16S rRNA gene sequence ofDickeyadadantiiEch586 (CP001836) was used outgroup.

1.4 Pathogenicity tests

The ability of 48Pectobacteriumstrains to macerate potato tubers (Solanumtuberosum) cv. Xiazhai 65 (Agricultural Technology Extension Station in Huzhu County of Qinghai Province) was determined using a potato tuber assay. Isolates and four standard strains in this study were cultured overnight in LB broth at 25 ℃ for at least 24 h (forP.atrosepticum) and 28 ℃ (forPectobacteriumcarotovorumsubsp.carotovorum,P.wasabiaeandP.carotovorumsubsp.brasiliense). Next, the OD600from each strain was adjusted to OD600= 1.0 (5× 108CFU·mL-1) after suspending the bacterial cells in 0.9% physiological saline[25]. Surface-sterilized potato tubers were inoculated by toothpick approximately 1 cm deep holes into each tuber with a sterile pipette tip, injecting 10 μL of the inoculum and sealing the spot with petroleum jelly. The tubers were then incubated at 28 ℃ and 95% relative humidity for 72 h before the diameter of the rotting tissue in each tuber was measured. The experiment was independently repeated three times. For the negative control, pipette tips were inserted into tubers contained 0.9% physiological saline rather than a bacterial suspension.

2 Results and analysis

2.1 Identification and characterization of pectolytic bacteria

2.1.1MorphologyandpathogenicityofbacterialisolatesPotato plants with soft rotted tubers or black-legged were obtained from Qinghai, Gansu, Ningxia, Shanxi, and Inner Mongolia potato growing zones of Northwest region in 2015 to 2016. A total of 48 pectolytic bacterial strains were obtained from 95 samples collected from the five potato growing areas by using selective CVP media. All of the isolated strains were able to grow at 28 and 37 ℃ and form pits on CVP, and their pectolytic characteristics was further confirmed usingpelspecific primers. Thepelgene specific primers ADE-1/ADE-2 produced an expected characteristic band of 434 bp.

As shown in Fig.1, the strains isolated from the soft rot disease were screened by CVP selective medium and produced a cup concave strain on the CVP medium. Detection and re-isolation from diseased stems validated Koch’s postulates for confirmation of strains correctness. The virulence ofPectobacteriumrepresentative isolates obtained in this study was tested for maceration ability using tuber and stem assays. In the tuber rotting assay, symptoms in tuber inoculated with strains belonging toPectobacteriumappeared as water soaked lesions with dark brown surrounding at the point of inoculation of potato tubers, developing to rot lesions and smelly. In the stem rotting assay, symptoms in the base of the stem inoculated with strains became brown obviously, then rapidly softened and decayed, and eventually the plant withered and died, but variation in symptoms severity were observed between replicate plants. Symptoms of tissue macerated by isolates which were consistent with the field basic symptoms, the incidence of inoculation of potato tuber tissue separation, obtained the same pathogens. There were no symptoms developed on mock-inoculated controls.

2.1.2PhysiologicalandBiochemicalCharacteristicsofbacterialisolatesRegarding the morphology of the bacterial colonies on LB media, the colonies appeared to be round, milky white, smooth surface, and middle raised after 48 h incubation at 28 ℃. Likewise, 48 strains were found to be gram-negative. According to biochemical tests (Table 2), some of 15 Pcc strains grew at 37 ℃ on LB containing 5% of NaCl, and did not produce acid from sucrose, and were not sensitive to erythromycin. The gelatin liquefication and catalase reactions were positive but oxidase, phosphatase activity and indole production test were negative. Strains of Pcc metabolized citrate, lactose, raffinose, D-(+)-cellobiose, and α-D-(+)-melibiose did not utilize maltose, sorbitol, D-(+)-arabinitol, α-methylglucoside.

Fig.1 Cavities formal on CVP agar and pathogenicity of the isolates

Pa strains could not grow at 37 ℃ which showed tolerance 5% NaCl, was not sensitive to erythromycin and produced acid from reducing substances from sucrose. Gelatin liquefaction and catalase test were positive, oxidase, indole and phosphatase activity test were negative. Pa strains could utilize lactose, maltose, raffinose, D-(+)-cellobiose, α-D-(+)-melibiose, citrate and α-methylglucoside and did not utilized sorbitol, D-(+)-arabinitol.

Table 2 Comparison of physiological and biochemical characteristics of northwest potato pectolytic bacteria with those of Pectobacterium spp. reference strains

P.wasabiae(QDD-5) strain was negative for indol production, phosphatase activity, reducing substances from sucrose and oxidase, and acid production from maltose, sorbitol, D-(+)-arabitol, lactose, raffinose, α-D-(+)-melibiose and α-methylglucoside. However, this strain isolated from potato did not grow on LB at 37 ℃ and was not able to tolerate 5% NaCl. Likewise, the strain was positive for gelatin liquefaction and catalase, and showed insensitivity to erythromycin, and utilized D-(+)-cellobiose and citrate.

2.2 Molecular identification of pectolytic bacteria

In this study, the species and subspecies-specific PCR was used to further identify characterization of pectolytic bacterial strains. The majority (64.58%) of the 31 isolated strains andP.atrosepticumstandard strain produced expected 690 bp amplification products by using subspecies-specific Eca1-F/ Eca2-R primer pair (Fig.2). Another group of 15 isolated strains (31.25%) andP.Carotovorumsubsp.carotovorumstandard strain produced the expected amplicon of 550 bp using subspecies-specific EXPCC-F/EXPCC-R primer pair. Only Qinghai strain (QDD-5) ofPectobacteriumspp. andP.wasabiaestandard strain produced 550 bp amplicon using species-specific primers Ph-F/Ph-R. Almost all isolated strains which obtained expected products by using subspecies-specific primers were preliminarily identified asP.atrosepticumandP.carotovorumsubsp.carotovorum.

2.3 Phylogenetic analysis of Pectobacterium isolates

Based on phylogenetic analysis of 16S rRNA gene, thePectobacteriumspp. was further classified in this study. A neighbor-joining (NJ) phylogram was constructed by comparing the partial sequence of 16S rRNA gene obtained from northwestern strains with the reference sequence of NCBI GenBank database (Fig.3). In the concatenated NJ tree, the fifteen isolated strains (QPB-4, QHX-3, QHL-2, QHS-4, QML-6, QLL-10, QDM-7, GDX-5, GJY-2, SJY-2, NXJ-1, GGH-4, SJY-1, NXN-3, NZD-2) identified asP.carotovorumsubsp.carotovorumusing specific primers had sequences with a high homology. The bootstrap support value between 15 strains and Pcb, Pco was high which reached 99%.

31 isolated strains were identified asP.atrosepticum(QHL-7, QHQ-1, QDM-1, QLZ-4, QLM-1, QHN-3, QHS-2, QMX-6, QML-4, QPS-4, GZS-7, GLF-3, GLG-6, GTQ-2, GJY-5, GDX-7, SJS-7, SJY-4, NCH-2, SXS-1, SLS-3, SDW-1, NWH-5, NZD-6, NZD-1, NXS-3, NXJ-7, NYH-4, NJZ-4, NYX-2, QLZ-2) by specific primers, and they were highly homologous. They had high homology with variousP.atrosepticumstrains from all over the world, forming 99% bootstrap support values. The Qinghai strain (QDD-5) identified asP.wasabiaehas high homology with theP.wasabiaestrains published with a 100% support rate of. Therefore, the phylogenetic groups among the isolates of the genusPectobacteriumspp in this study were based on the PCR-based molecular approach.

2.4 Pathogenicity analysis of Pectobacterium isolates

The ability ofP.carotovorumsubsp.carotovorum,P.atrosepticumandP.wasabiaetogether with (four) standard strains which caused soft rot of potato tubers was assessed. Potato tubers ofS.tuberosumcv Xiazhai 65 were inoculated and the macerated tissue diameter were measured. It was found that all isolates tested caused soft rot lesions at the point of inoculation of potato tubers. The isolated strains ofP.carotovorumsubsp.carotovorum,P.atrosepticumandP.wasabiaeshowed significantly lower tuber maceration ability compared to their standard strains (P<0.05). The diameter of maceration induced by theP.atrosepticumwas less than decay incited byP.carotovorumsubsp.carotovorumandP.wasabiaestrains (Table 3). The virulence of different subspecies in the same species was different. Similarly, the pathogenicity of different strains in the same region was also different. As expected, potato tubers inoculated with 0.9% physiological saline negative controls showed no decay symptoms. The characteristics of rot disease caused by inoculation of potato tubers with differentPectobacteriumspp. and subspecies were similar.

M: DNA molecular weight marker DL2 000; Lane 1: Sterile water used as negative control. Lane 2～32: QHL-7, QHQ-1, QDM-1, QLZ-4, QLM-1, QHN-3, QHS-2, QMX-6, QML-4, QPS-4, GZS-7, GLF-3, GLG-6, GTQ-2, GJY-5, GDX-7, SJS-7, SJY-4, NCH-2, SXS-1, SLS-3, SDW-1, NWH-5, NZD-6, NZD-1, NXS-3, NXJ-7, NYH-4, NJZ-4, NYX-2, QLZ-2, respectively.Fig.2 PCR products of the Pa with primers Eca1-F/Eca1-R amplification in the 19 strains

●—P. atrosepticum/Clade Ⅲ; ○—P. wasabiae/Clade Ⅱ; △—P. c. subsp. odoriferum; ■—P. c.subsp.carotovorum/CladeⅠ; ◇—outgroup. Bootstrap values supporting each clade have been indicated after 1 000 replicates. Partial nucleotide sequences from Dickeya spp. is included as an outgroups.Fig.3 A neighbor-joining trees based on 16S rRNA partial nucleotide sequences of Pectobacterium strains

3 Discussion

In this study, the occurrence ofPectobacteriumspp. on potato in northwest of China was checked by using several biochemical, physiological and molecular techniques. On the basis of the physiological and biochemical results, it was proved that 16 strains belong to Pcc. Their phenotype was consistent with those reported by Mahmoudi and associates[26]. 30 strains have similar classification to Pa. Our results were consistent with the description ofP.atrosepticumfeatures[15,27]. The biochemical properties of only 1 strain were in line with those of Maetal.[28], Pitmanetal.[29]and Sarehetal.[30].

The number of species and subspecies ofPectobacteriumhas increased in recent years. Therefore, identification onPectobacteriumby traditional microbiological tests has become more and more difficult. However, molecular techniques are not only helpful for simple and rapid identification of bacteria, but also as a diagnostic tool to improve the opportunity to assess multiple bacteria in complex environments. DNA markers, serology and DNA hybridization have been reported in pathogenic bacteria subspecies and pathogenic types of many plants[28, 31-33]. Among them, 16S rDNA sequence analysis is the main bacterial phylogenetic classification. In order to further clarify the subspecies classification status of pathogenic bacteria, the genome of the tested strains was analyzed. By analyzing the sequence of 16S rRNA gene and combining withPectobacteriumspp. subspecies sequence region specific primer amplification, the 31 strains were identified as Pa while 15 strains identified as Pcc.

Table 3 Collection sites and pathogenicity levels of the 48 strains used in this study

Phylogenetic analyses in this study based on the 16S rRNA genes accurately grouped the northwest region strains with Pcc and Pa strains that have been identified in other parts of the world. 16S rDNA phylogenetic analysis gathered the northwest region Pcc strains in clade I with other Pcc strains isolated (Pcc ICMP 5702, Pc CFBP2046, LMG2404 and ATCC15713) elsewhere in the world. Similarly, the northwest region strains identified as pagathered well in clade Ⅲ which include the Pa LMG2386, CFBP1526, JG10-08, ICMP1526, 9201, 9205, 21A, Ec32, PI, CA2G8 strains.

At present, it is not surprising that new strains reported recently are different from those currently known since the Pcc strain is still regarded as diversity. PCR with theP.carotovorumsubsp.carotovorumprimer pair did not show specificity because they could amplify the expected 550 bp product from some strains identified asP.wasabiaeandP.carotovorumsubsp.odoriferum[22]but not amplify DNA from other strains that were identified asP.carotovourmsubsp.carotovorumon. Therefore, 1 strain of Pw and 1 strain of Pco in this study may belong to the Pcc category. More accurate methods were needed to distinguish Pcc, Pco and Pw in further research.

The identification ofP.carotovorumsubsp.carotovorumandP.atrosepticumasPectobacteriumspp. in potatoes from the northwest regions provides more literature onPectobacteriumspp. in these regions since there is little known about the genetic diversity of potato soft rot disease from this region. The use of DNA-based typing in combination with other methods towards accurate identification of the northwest regions strains thus proved to be an effective approach.

Here, the results showed that Pcc and Pa are the causal agents of soft rot diseases in potatoes from the northwest regions, similar to other studies, and the Pcc and Pa isolated strains were also the main agents causing soft rot in potato tubers[21, 26-27]. However, it is worth noting that the maceration ability of isolates strains is significantly lower than that of standard strains. The variability in tuber maceration ability, as demonstrated by pathogenicity assays in this study needs to be tested further. Meantime, it is too early to draw a general conclusion because only a limited number of strains from five provinces have been analyzed and therefore the difference of pathogenicity among strains from five provinces must be confirmed in the future. In addition, as observed by Onkendi and associates[7],P.carotovorumsubsp.carotovorumis a more aggressivePectobacteriumspp. compared to their standard strains and therefore further studies was needed on the distribution, host range and route of dissemination in northwest region.

In the northwest potato region, a major problem for farmers is to grow disease-resistant tubers. Most farmers obtained potato seeds with disease from other farmers. Therefore, understanding the pathogens of soft rot disease in different growing areas can greatly improve the yield of potato. The analytical methods used in this study are quite accurate and can clearly identify the genetic diversity of isolates, with a wide host range. DNA classification based on molecular markers is reliable and accurate in the classification of bacterial strains.

This study provided the latest information on the occurrence and distribution of potato soft rot pathogens causing tuber soft rot and plant blackleg in five provinces’ major potato-producing areas.P.carotovorumsubsp.carotovorumandP.atrosepticumis the dominant pathogenic subspecies /species and distributed widely in these regions. At the same time, this is first time to monitorP.carotovorumsubsp.carotovorumandP.atrosepticumsimultaneously causing soft rot of potato in five northwest provinces China. The severity of disease in some provinces in northwest regions exceeded the damage threshold level. Therefore, the farmers, agronomists, and plant pathologists should cooperate to prevent important economic losses of potato yields caused byPectobacteriumspp..