First Report of Streptomyces brasiliscabiei Causing Common Scab on Potato in China.

Severe typical deep-pitted lesions of Potato Common Scab (PCS) disease were observed in two locations in China, Dingxi, Gansu Province, and Shuozhou, Shaanxi Province, in 2021. Potato farms in Dingxi growing cultivar Huangxin 226 (26 hectares) exhibited a scab disease incidence of 10%, while cultivar Jinshu 15# (4 hectares) in Shuozhou showed a disease incidence of 30% (Fig. 1). During harvest, tubers displaying PCS symptoms were collected for pathogen isolation. To obtain pathogen isolates, surface-sterilized tuber tissue with scab lesions was ground in sterile water, serially diluted, and plated onto ISP5 agar medium plates (Handique et al. 2022). Five pure colonies of Streptomyces isolate were obtained, designated as ZRIMU1503, ZRIMU1502, ZRIMU1320, ZRIMU1321and ZRIMU791. Genomic DNA was extracted and sequenced using Illumina technology. Sequencing data of the 5 isolates were uploaded to NCBI GenBank and annotated (Accession numbers: JBBAYL010000000, JBBAYM010000000, JBBAYN010000000, JBBAYO010000000 and JBBAYP010000000, respectively) using the PGAP pipeline (Tatusova et al. 2016). Average Nucleotide Identity (ANI) values (97.52 %, 97.53 %,97.54 %,97.57 % and 97.52 %, respectively) indicated the identity of the five isolates to the type strain S. brasiliscabiei IBSBF 2867T. Additionally, pairwise comparisons of Digital DNA Hybridization (DDH) value (76.2%, 76.3%, 76.4%, 76.4% and 76.2% respectively) of all the Streptomyces type strains show the highest identity to S. brasiliscabiei IBSBF 2867T. Twelve housekeeping genes (acnA, atpD, dnaN, gap, gyrA, gyrB , infB, mdh, recA, rplB, rpoB, and trpB) were extracted from the genome sequence of the five isolates to construct a multi-locus sequence analysis (MLSA) tree. The evolutionary distance of the five isolates was constructed using MEGAX software (Kumar et al., 1994), along with other Streptomyces strains that are known to cause PCS. The resulting cladogram demonstrated the isolated strains clustered together with S. brasiliscabiei IBSBF 2867T (Fig.2). Koch's postulates were fulfilled by inoculating a perlite potting mix with spore suspensions of each isolate (104 CFU/ml), planting tubers (cv. Favorita), and reproducing PCS symptoms at harvest after three months. Negative control received water treatment. The plants were kept in greenhouse with 12 h of light per day and irrigated regularly. The experiment was repeated twice, once in April 2022 and again in April 2023. On harvest, all five isolates exhibited development of severe symptoms of PCS (Fig.1), while the negative controls had no lesions. The pathogen was reisolated from the lesions and confirmed to be identical to the original isolate by 16S rRNA gene sequences. To our knowledge, this is the first report of S. brasiliscabiei causing PCS in China. S. brasiliscabiei was identified as a new species to cause PCS in Brazil and was identified based on morphology, pathogenicity, and genetic features (Corrêa et al. 2021). Multiple pathogen-causing PCS has been recognized in China and a surge of disease incidence in potato fields has been reported (Handique et al. 2022; Wu et al. 2023). S. brasiliscabiei causes severe symptoms which makes potatoes unmarketable. The disease epidemiology of this pathogen needs to be investigated.

Comparative genomic and transcriptome analyses of two Pectobacterium brasiliense strains revealed distinct virulence determinants and phenotypic features.

Potato soft rot caused by Pectobacterium spp. are devastating diseases of potato which cause severe economic losses worldwide. Pectobacterium brasiliense is considered as one of the most virulent species. However, the virulence mechanisms and pathogenicity factors of this strain have not been fully elucidated. Here, through pathogenicity screening, we identified two Pectobacterium brasiliense isolates, SM and DQ, with distinct pathogenicity levels. SM exhibits higher virulence compared to DQ in inducing aerial stem rot, blackleg and tuber soft rot. Our genomic and transcriptomic analyses revealed that SM encodes strain specific genes with regard to plant cell wall degradation and express higher level of genes associated with bacterial motility and secretion systems. Our plate assays verified higher pectinase, cellulase, and protease activities, as well as fast swimming and swarming motility in SM. Importantly, a unique endoglucanase S specific to SM was identified. Expression of this cellulase in DQ greatly enhances its virulence compared to wild type strain. Our study sheds light on possible determinants causing different pathogenicity of Pectobacterium brasiliense species with close evolutionary distance and provides new insight into the direction of genome evolution in response to host variation and environmental stimuli.

Isolation, characterization and application of noble bacteriophages targeting potato common scab pathogen Streptomyces stelliscabiei.

Bacteriophages have emerged as promising alternatives to pesticides for controlling bacterial pathogens in crops. Among these pathogens, Streptomyces stelliscabiei (syn. S. stelliscabiei) is a primary causative agent of potato common scab (PCS), resulting in substantial global economic losses. The traditional management methods for PCS face numerous challenges, highlighting the need for effective and environmentally friendly control strategies. In this study, we successfully isolated three novel bacteriophages, namely Psst1, Psst2, and Psst4, which exhibited a broad host range encompassing seven S. stelliscabiei strains. Morphological analysis revealed their distinct features, including an icosahedral head and a non-contractile tail. These phages demonstrated stability across a broad range of temperatures (20-50°C), pH (pH 3-11), and UV exposure time (80 min). Genome sequencing revealed double-stranded DNA phage with open reading frames encoding genes for phage structure, DNA packaging and replication, host lysis and other essential functions. These phages lacked genes for antibiotic resistance, virulence, and toxicity. Average nucleotide identity, phylogenetic, and comparative genomic analyses classified the three phages as members of the Rimavirus genus, with Psst1 and Psst2 representing novel species. All three phages efficiently lysed S. stelliscabiei in the liquid medium and alleviated scab symptom development and reduced pathogen abundance on potato slices. Furthermore, phage treatments of radish seedlings alleviated the growth inhibition caused by S. stelliscabiei with no disease symptoms. In soil potted experiments, phages significantly reduced disease incidence by 40%. This decrease is attributed to a reduction in pathogen density and the selection of S. stelliscabiei strains with reduced virulence and slower growth rates in natural environments. Our study is the first to report the isolation of three novel phages that infect S. stelliscabiei as a host bacterium. These phages exhibit a broad host range, and demonstrate stability under a variety of environmental conditions. Additionally, they demonstrate biocontrol efficacy against bacterial infections in potato slices, radish seedlings, and potted experiments, underscoring their significant potential as biocontrol agents for the effective management of PCS.

Species diversity of Pectobacterium spp. causing potato aerial stem rot in China.

Pectobacterium spp. are the primary causative agents of aerial stem rot in potatoes in China. A nationwide survey revealed the widespread occurrence of aerial stem rot in the northern, southern, and southwestern cultivation regions, with occurrence rates ranging from 1% to 60%. In total, 36 strains were isolated and identified at the species level using multi-locus sequence analysis of six housekeeping genes (rpoS, proA, gapA, icdA, gyrA, and mdh). Genome sequencing was conducted on one representative strain for each species, and further confirmation of their identities was achieved through ANI and isDDH analysis. Five Pectobacterium species were identified, namely Pectobacterium atrosepticum, Pectobacterium brasiliense, Pectobacterium carotovorum, Pectobacterium polaris and Pectobacterium punjabense, with P. atrosepticum and P. brasiliense being the most widely distributed. Pathogenicity tests demonstrated that, among the strains isolated in this study and those obtained from other studies, P. atrosepticum and P. brasiliense are also the most virulent species. To the best of our knowledge, this is the first nationwide study describing the diversity and distribution of Pectobacterium spp. affecting potatoes in China. The information gathered will be utilized for disease diagnosis and the development of pathogen-specific integrated pest management (IPM) strategies to protect potato production.

First Report of Streptomyces niveiscabiei Causing Potato Common Scab in Shanxi and Gansu, China.

Multiple species of Streptomyces cause common scab disease in potato (Solanum tuberosum) (Kämpfer et al. 1991). Potato tubers (cv. Jinshu1 #5 and Longshu #6) with severe pitted common scab symptoms were observed from two farms in Chaozhou in Shanxi Province and in Tianzhu in Gansu Province during the national disease survey of bacterial diseases of potatoes in 2021. The disease incidence was around 30% on the 6.7 ha of the Chaozhou farm and 10% on the 0.7 ha on the Tianzhu farm. Three tubers with scab symptoms were surface disinfested with 3% sodium hypochlorite for 1 min. The symptomatic tissue was then ground in sterile water. Serially diluted ground samples were cultured on Streptomyces ISP Medium 5 agar plates (Shirling and Gottlieb 1966) and incubated at 280C for 5 days. Eight pure Streptomyces colonies were obtained and sequenced for identification using the universal 16S rRNA gene primers 27F (5'-AGAGTTTGATCMTGGCTCAG-3') and 1492R (5'-TACGGYTACCTTGTTACGACTT-3') (Monciardini et al. 2002) by colony PCR. Blast results of the sequences against the NCBI GenBank for the eight isolates, ZRIMU1508, ZRIMU1510, ZRIMU1511, ZRIMU1512, ZRIMU1514, ZRIMU1515, ZRIMU1516 and ZRIMU1530 (Accession numbers: OP941573 - OP941580), showed more than 99% sequence identity to S. niveiscabiei NRRLB-24457T type strain. Additionally, 12 housekeeping gene sequences, acnA (OP997624 - OP997625), atpD (OP997622 - OP997623), dnaN (OP997620 - OP997621), gap (OP997618 - OP997619), gyrA (OP997614 - OP997615), gyrB (OP997612 - OP997613), infB (OP997610- OP997611), mdh (OP997608 - OP997609), recA (OP997602 - OP997603), rplB (OP997600 - OP997601), rpoB (OP997598- OP997599), and trpB (OP997594 - OP997595), were extracted from the genome sequences of two strains, ZRIMU1510 and ZRIMU1530, and uploaded to GenBank. Genes for pathogenicity, txtA (OP997593 - OP997594), tomA (OP997596 - OP997597) and Nec1(OP997606 - OP997607), were also identified from the genome sequence and uploaded to GenBank. The housekeeping genes and the pathogenicity genes showed over 98% identity with S. niveiscabiei. Phylogenetic trees were constructed using concatenated housekeeping gene sequences (Kumar et al. 1994) and the cladogram showed that the isolates ZRIMU1510 and ZRIMU1530 grouped with the type strain NRRLB-24457T. Pathogenicity tests were done by drench application of 100 ml spore suspensions (104 CFU/ml) of ZRIMU1530, ZRIMU1510, or phosphate buffer into pots with potato plants (cv. Favorita) grown in potting mix. Five tubers were planted and inoculated with each pathogen or phosphate buffer as the negative control. The plants were then placed in a greenhouse with 12 h of light per day, irrigated regularly, and harvested after 3 months. The newly formed tubers were checked for disease symptoms. Tubers from pots inoculated with ZRIMU1530 and ZRIMU1510 exhibited typical symptoms of common scab with raised corky lesions with deep pits, but the negative controls remained asymptomatic. The pathogens were reisolated from the lesions and confirmed to be identical to the original isolates by 16S rRNA gene sequences, thus completing Koch's postulates. The pot experiment was conducted twice: first in May 2022 and second in February 2023. To our knowledge, this is the first report of S. niveiscabiei causing common scab of potato in Shanxi and Gansu, China. S. niveiscabiei was first reported in Korea (Park et al. 2003) and this report will draw attention to the study and management of scab pathogens in China.

First Report of Pectobacterium parvum Causing Diseases on Potato in Inner Mongolia, China.

Soft rot enterobacterial plant pathogens Pectobacterium spp. and Dickeya spp. have caused devastating blackleg, aerial stem rot, and soft rot of potato tubers (Charkowski 2018). In August 2021, potato plants (cv. Xisen6# or Maiken1) with blackleg or aerial stem rot symptoms were observed in two commercial fields in Xinghe County, Ulanqab City, Inner Mongolia Autonomous Region, China. The plants were wilted, and the crown stem showed gradual degradation and browning. The disease incidence was around 3 to 7% and 20 to 25% in Xinghe Zhangyou Village (33 ha) and Bianjia Village (7 ha), respectively. Pathogens were isolated on crystal violet pectate agar (CVP) plates (Ge et al. 2018). Briefly, symptomatic stem tissues were surface sterilized in 75% ethanol, ground, then serial dilutions were cultured on CVP plates (Handique et al. 2022). The plates were incubated at 28oC for 2 days. Pure colonies of Pectobacterium spp. were obtained from the pits of CVP plates and sequenced for identification using the universal 16S rRNA gene primers 27F/1492R (Monciardini et al. 2002). Results of the comparison of 16S sequences against NCBI GenBank showed 100% sequence identity to P. parvum FN20211T (CP087392.1) type strain for the three colonies designated as ZRIMU1006 (1542/1542 bp), ZRIMU1019 (1542/1542 bp), and ZRIMU1020 (1542/1542 bp). Sequences were deposited under accession numbers OP941529, OP941525, OP941526, respectively. Additionally, six housekeeping gene sequences were used to confirm identification at the species level and were uploaded to GenBank: fusA (OP793177, OP793171, OP793172), gapA (OP793221, OP793216, OP793217), gyrB (OP793265, OP793259, OP793260), infB (OP793310, OP793304, OP793305), pgi (OP793355, OP793349, OP793350), and rplB (OP793400, OP793394, OP793395). Phylogenetic trees constructed using the MEGA X program of concatenated sequences of the housekeeping genes sequences show that the three isolates grouped with P. parvam FN20211T, confirming that they are P. parvam. Pathogenicity tests for stem rot were done by injecting a bacterial suspension into potato seedlings (cv. Favorita) grown from seed tubers. The tubers were planted in perlite potting mix and 3 weeks after emergence, a 100 µl bacterial suspension (105 CFU/ml) or sterile phosphate-buffered solution was injected at the stem base. The bacterial injection experiment was performed twice in a greenhouse with five plants inoculated per bacterial strain. Plants were covered with plastic bags to maintain 100% humidity at 25°C for 2 days. Seven days after inoculation, the inoculated area of the seedlings had rotted or turned black, while the controls remained symptomless. Symptomatic tissues from each strain were processed as above and placed on CVP plates to reisolate the Pectobacterium spp. 16S rRNA sequence analysis confirmed the bacteria to be similar in sequence to the inoculated strains, thus completing Koch's postulates. Soft rot development was performed by adding bacterial suspension (100 µl, 105 CFU/ml) on tuber slices. The infected tubers rotted, while the controls were symptomless. The vacuum infiltration method on tuber that is used to test pathogens for blackleg did not result in the development of blackleg symptoms. Briefly, five tubers were inoculated with the pathogen by vacuum infiltration and planted in potting mix. The plants showed wilting after emergence, but no blackleg symptoms were observed. Recently, multiple new Pectobacterium species including P. parmentieri, P. polaris, and P. punjabense, were identified to cause potato disease in different provinces of China (Cao et al. 2021; Handique et al. 2022). In China, P. parvum was first isolated from Brassica, and in the year 2022, it was reported to cause aerial stem-rot on potato in Hebei province in China (Wang et al. 2022). Inner Mongolia is a major potato-seed-producing province and the incidence of new strains of Pectobacterium in the province causing aerial stem rot and soft rot of tuber might cause a reduction in seed production. This report will draw attention to the management of P. parvum by the seed-producing companies in Inner Mongolia which distributes the seed throughout China.

Micro-computed tomography evaluation of the effects of orthodontic force on immature maxillary first molars and alveolar bone mineral density of Sprague-Dawley rats.

Objective: To investigate changes in the immature teeth of Sprague-Dawley rats during orthodontic treatment and to explore the changes in the peri-radicular alveolar bone through micro-computed tomography (CT). Methods: Twenty-five 26-day-old male Sprague-Dawley rats were included. The maxillary left first molar was moved mesially under a continuous force of 30 cN, and the right first molar served as the control. After orthodontic treatment for 7, 14, 21, 28, and 42 days, the root length, tooth volume, and alveolar bone mineral density (BMD) around the mesial root were measured through micro-CT. Results: The immature teeth continued to elongate after application of orthodontic force. The root length on the force side was significantly smaller than that on the control side, whereas the differences in the volume change between both sides were not statistically significant. Alveolar bone in the coronal part of the compression and tension sides showed no difference in BMD between the experimental and control groups. The BMD of the experimental group decreased from day 14 to day 42 in the apical part of the compression side and increased from day 7 to day 42 in the apical part of the tension side. The BMD of the experimental group decreased in the root apex part on day 7. Conclusions: The root length and volume of immature teeth showed continued development under orthodontic forces. Alveolar bone resorption was observed on the compression side, and bone formation was observed on the tension side.

First Report of Pectobacterium versatile Causing Diseases on Potato in Xinjiang Uygur Autonomous Region and Heilongjiang Province, China.

Pectobacterium spp. and Dickeya spp. cause aerial stem rot on potatoes worldwide (Charkowski, 2018). Potato plants (cv. Xisen6# or Youjin) with aerial stem rot or blackleg symptoms (Fig. S1) were observed in the commercial fields in Changji, Xinjiang Province in September 2021 and Harbin, Heilongjiang Province in August 2021, in China. The field disease incidences were 45-50% and 15-20% in Changji (2 ha) and Harbin (1 ha), respectively. Five diseased plants from each field site were collected to isolate the pathogen. Symptomatic stems were soaked in 75% ethanol for 2 min, rinsed, and ground in sterile distilled water (Handique et al. 2022). The suspension was plated onto a crystal violet pectate agar (CVP) plate (Ge et al. 2018). Three days after incubation at 28°C, bacterial colonies that developed pits on CVP plates were purified and sequenced for identification using the universal 16S rRNA gene primer set 27F/1492R (Monciardini et al. 2002). Amplified 16S rDNA sequences from two isolates designated as ZRIMU1267 and ZRIMU1366 showed more than 99% sequence identity to P. versatile CFBP6051T type strain and the sequences were submitted to GenBank (accession numbers: OP476349, OP476350). Additionally, six housekeeping genes sequences were uploaded to GenBank: proA (OP487826, OP487832), gyrA (OP487828, OP487834), icdA (OP487823, OP487829), mdh (OP487825, OP487831), gapA (OP487824, OP487830), and rpoS (OP487827, OP487833) (Ma et al. 2007; Waleron et al. 2008). A phylogenetic tree based on concatenated sequences (MLSA) of the housekeeping genes (Fig. S2) of the two isolates was constructed using MEGA X (Tamura et al. 2013).The phylogenetic tree of MLSA sequences shows that the sequences from isolates ZRIMU1267 and ZRIMU1366 clustered with P. versatile CFBP6051T indicating that these isolates are P. versatile at the species level. Koch's postulate were performed on 3-week-old potato seedlings (cv. Favorita) and tubers. The bacterial suspension (100 µl, 105 CFU/ml) or sterile phosphate-buffered solution was injected into the crown area of the seedlings for the development of aerial stem rot or drenched in the potting mix for the development of blackleg, and the plants were covered with polybags to keep 100% humidity at 25° for 2 days. Five seedlings were inoculated for each strain and the experiment was repeated twice. Seven days after stem injection, the infected area of the inoculated seedlings was rotten, turned black, or even lodged, while the controls were symptomless (Fig. S3a). Four days after drench application, the seedlings were wilted and lodged, while the controls were symptomless (Fig. S3 c). Tuber slice assay for soft rot development was performed by adding bacterial suspension (100 µl, 105 CFU/ml). One day after inoculation, the infected tubers rotted, while the controls were symptomless (Fig. S3 b). ZRIMU1267 and ZRIMU1366 were reisolated from infected tissues on CVP plates and identified by 16S rRNA sequences to complete Koch's postulate. Diseases on potato has been reported to be caused by P. atrosepticum, P. carotovorum, P. brasiliense, P. parmentieri, P. polaris, and P. punjabense in China (Zhao et al. 2018; Cao et al. 2021; Wang et al. 2021; Handique et al. 2022). P. versatile causing aerial stem rot on potatoes have been reported in Hebei province (Han et al. 2022), while our study reports P. versatile strains that are able to cause multiple diseases on potatoes in Xinjiang Uygur Autonomous Region and Heilongjiang provinces of China. The results indicate that P. versatile might be widely distributed in northern China, and it is necessary to include cropping season and post-harvest strategies to control diseases caused by P. versatile.

Three-dimensional analysis of root changes after orthodontic treatment for patients at different stages of root development.

INTRODUCTION: We investigated changes in the roots of maxillary incisors at different stages of root development after fixed-appliance treatment using cone-beam computed tomography. METHODS: Data from 52 subjects receiving fixed-appliance treatment were collected retrospectively. The subjects were divided into 3 groups: mixed dentition group (aged 7-10 years; root development stage: Nolla eighth-10th; n = 16), early permanent dentition group (aged 12-18 years; root development stage: Nolla 10th; n = 20), and adult group (aged 18-35 years; root development stage: Nolla 10th; n = 16). Changes in root lengths and volume of the maxillary central incisors were measured using pretreatment and posttreatment cone-beam computed tomography. RESULTS: The root lengths and volumes of maxillary central incisors in the mixed dentition group significantly increased after orthodontic treatment (P >0.05). No significant differences were found when comparing the final root length and volume of the mixed dentition group with the pretreatment maxillary incisor values of the early permanent dentition group (P >0.05). The early permanent dentition group showed a significant decrease in root length (P <0.05), and both the root length and volume of the adult group significantly decreased after treatment (P <0.05). The differences in root length and volume reduction between the 2 groups were not significant (P >0.05). CONCLUSIONS: Orthodontic treatment had no significant negative impact on the continued root development of incomplete roots with two-thirds root formation. Both the early permanent dentition and adult groups exhibited root resorption after orthodontic treatment. It seemed age was not a factor that resulted in significant root resorption during routine orthodontic leveling and alignment treatment once the roots were fully developed.

First Report of Streptomyces stelliscabiei Causing Potato Common Scab in Guizhou Province, China.

Potato (Solanum tuberosum L.) common scab can be caused by multiple pathogenic Streptomyces spp. worldwide. Potato tubers (cv. Favorita) with severe pitted common scab symptoms were observed at a small farm (2 hectares) during harvest in Anshun, Guizhou province in early May 2020. The disease incidence was around 10%, and symptomatic samples were collected to isolate the pathogen. Two isolates, ZR-IMU141 and ZR-IMU146 (Accession number MW995958 and MW995959 respectively), showed more than 99% sequence identity to S. stelliscabiei sequences (Accession No. HM018085). Five house-keeping genes for multi-locus sequence analyze (MLSA) of Streptomycetaceae were amplified, sequenced and uploaded to NCBI: atpD (MZ343164 and MZ343165), gyrB (MZ343162 and MZ343163), recA (MZ343166 and MZ343167), rpoB (MZ343168and MZ343169) and trpB (MZ343170 and MZ343171). All the genes show over 98% identity with S. stelliscabiei. Phylogenetic trees of 16S rRNA gene sequence and multi-locus sequence analysis (MLSA) were constructed. The two isolates contain pathogenicity genes txtAB, nec1, and tomA, which was confirmed by PCR. To complete Koch's postulates, 9 potato seedlings (cv. Favorita, 15 centimeters high), were transferred to new pots and inoculated with spore suspensions of ZR-IMU141 and ZR-IMU146 (104 CFU/ml), or water as a negative control. Two months later, potato tubers inoculated with either ZR-IMU141 or ZR-IMU146 exhibited typical symptoms of potato common scab, such as superficial or deep, raised, pitted, or polygonal lesions like the field symptoms, but the negative controls remained asymptomatic. The pathogens were reisolated from the lesions and confirmed identical to the original isolate by 16s rRNA gene sequences. To our knowledge, this is the first report of S. stelliscabiei causing potato common scab in Guizhou province, China. We believe that this report will draw attention to the study and management of the increased pool of scab pathogens in China.

First Report of Pectobacterium parmentieri Causing Blackleg on Potato in Inner Mongolia, China.

Blackleg on potato plants (Solanum tuberosum) is caused by Pectobacterium spp. and Dickeya spp. (Charkowski, 2018) worldwide. From June to August in both 2018 and 2019, cases of blackleg were investigated in potato-producing areas in Hulunbuir, Ulanqab, and Hohhot in Inner Mongolia, China. The total surveyed field area was about 200 hectares. The plants showed typical blackleg symptoms, such as black and stunted stems or curled leaves (Fig. S1), and the number of infected plants were counted. The disease showed an incidence of around 8%. Five diseased plants were collected from a 10 ha potato field with approximately 75,000 potato plants (cv. mainly Favorita and Xisen) per hectare. Two-centimeter-long samples of symptomatic stems were removed from the selected plants using a sterile scalpel. The surfaces of the samples were disinfected with 75% ethanol for 2 min. They were then rinsed with sterile distilled water and soaked in 5 ml sterile distilled water for 30 min. Aliquots of three tenfold dilutions of this solution were plated onto the crystal violet pectate agar (CVP) plate and incubated for 3 days at 28°C (Ge et al., 2018). A single bacterial colony that showed pitting on CVP plates (Fig. S2) was picked with a toothpick, streaked onto nutritional agar (She et al., 2013) to obtain pure colonies. Amplification of a 1.4-kb segment containing 16S rRNA gene was performed on the pure colonies using the universal primer set 27F/1492R (Monciardini et al., 2002). The amplicons were sequenced and submitted to the GenBank Nucleotide Basic Local Alignment Search Tool analysis. The 16S rRNA gene sequences of four isolates (GenBank accession numbers: MN626412, MN626449, MN625916, and MT235556) showed more than 99% sequence identity to Pectobacterium parmentieri type strain RNS 08-42-1A (NR_153752.1) (Fig. S3). Six housekeeping genes proA (MT427753-MT427756), gyrA (MT427757-MT427760), icdA (MT427761-MT427764), mdh (MT427765-MT427768), gapA (MT427769-MT427772), and rpoS (MT427773-MT427776) of these four isolates were amplified and sequenced (Ma et al., 2007, Waleron et al., 2008). All sequences showed 99% to 100% sequence identity with Pectobacterium parmentieri strains. Phylogenetic trees (Fig. S4) were constructed by multi-locus sequence analysis (MLSA) using MEGA 6.0 software (Tamura et al., 2013). The samples were tested against Koch's postulates on potato seedlings (cv. Favorita) by injecting 100 µl bacterial suspension (107 CFU/ml) or sterile phosphate buffered solution into the stems 2 cm above the base (Ge et al., 2018). The seedlings were incubated at 21°C and 80% humidity (She et al., 2013). Three to 5 days after inoculation, only infected seedlings showed similar symptoms as those observed in the field: the infected area turned black and rotten (Fig. S5). Bacterial colonies isolated from these symptomatic seedlings were identified using the same methods described above and were identified as inoculated Pectobacterium parmentieri strains. Blackleg on potato plants has been reported to be caused by Pectobacterium atrosepticum, Pectobacterium carotovorum subsp. carotovorum, and Pectobacterium carotovorum subsp. brasiliense in China (Zhao et al., 2018). To our knowledge, this is the first report of blackleg of potato caused by Pectobacterium parmentieri in Inner Mongolia, China. We believe that this report will draw attention to the identification of this pathogen, which is essential to disease management.

Evolution and expression of genes encoding TCP transcription factors in Solanum tuberosum reveal the involvement of StTCP23 in plant defence.

BACKGROUND: The plant-specific Teosinte branched1/Cycloidea/Proliferating cell factor (TCP) family of transcription factors is involved in the regulation of cell growth and proliferation, performing diverse functions in plant growth and development. In addition, TCP transcription factors have recently been shown to be targets of pathogenic effectors and are likely to play a vital role in plant immunity. No comprehensive analysis of the TCP family members in potato (Solanum tuberosum L.) has been undertaken, however, and whether their functions are conserved in potato remains unknown. RESULTS: To assess TCP gene evolution in potato, we identified TCP-like genes in several publicly available databases. A total of 23 non-redundant TCP transcription factor-encoding genes were identified in the potato genome and subsequently subjected to a systematic analysis that included determination of their phylogenetic relationships, gene structures and expression profiles in different potato tissues under basal conditions and after hormone treatments. These assays also confirmed the function of the class I TCP StTCP23 in the regulation of plant growth and defence. CONCLUSIONS: This is the first genome-wide study including a systematic analysis of the StTCP gene family in potato. Identification of the possible functions of StTCPs in potato growth and defence provides valuable information for our understanding of the classification and functions of the TCP genes in potato.

Silencing of transcription factor encoding gene StTCP23 by small RNAs derived from the virulence modulating region of potato spindle tuber viroid is associated with symptom development in potato.

Viroids are small, non-protein-coding RNAs which can induce disease symptoms in a variety of plant species. Potato (Solanum tuberosum L.) is the natural host of Potato spindle tuber viroid (PSTVd) where infection results in stunting, distortion of leaves and tubers and yield loss. Replication of PSTVd is accompanied by the accumulation of viroid-derived small RNAs (sRNAs) proposed to play a central role in disease symptom development. Here we report that PSTVd sRNAs direct RNA silencing in potato against StTCP23, a member of the TCP (teosinte branched1/Cycloidea/Proliferating cell factor) transcription factor family genes that play an important role in plant growth and development as well as hormonal regulation, especially in responses to gibberellic acid (GA). The StTCP23 transcript has 21-nucleotide sequence complementarity in its 3' untranslated region with the virulence-modulating region (VMR) of PSTVd strain RG1, and was downregulated in PSTVd-infected potato plants. Analysis using 3' RNA ligase-mediated rapid amplification of cDNA ends (3' RLM RACE) confirmed cleavage of StTCP23 transcript at the expected sites within the complementarity with VMR-derived sRNAs. Expression of these VMR sRNA sequences as artificial miRNAs (amiRNAs) in transgenic potato plants resulted in phenotypes reminiscent of PSTVd-RG1-infected plants. Furthermore, the severity of the phenotypes displayed was correlated with the level of amiRNA accumulation and the degree of amiRNA-directed down-regulation of StTCP23. In addition, virus-induced gene silencing (VIGS) of StTCP23 in potato also resulted in PSTVd-like phenotypes. Consistent with the function of TCP family genes, amiRNA lines in which StTCP23 expression was silenced showed a decrease in GA levels as well as alterations to the expression of GA biosynthesis and signaling genes previously implicated in tuber development. Application of GA to the amiRNA plants minimized the PSTVd-like phenotypes. Taken together, our results indicate that sRNAs derived from the VMR of PSTVd-RG1 direct silencing of StTCP23 expression, thereby disrupting the signaling pathways regulating GA metabolism and leading to plant stunting and formation of small and spindle-shaped tubers.

Mechanical strain triggers differentiation of dental mesenchymal stem cells by activating osteogenesis-specific biomarkers expression.

Dental stem cell biotechnology has been used as a potential method to treat the dental diseases. This study aimed to investigate effects of mechanical stimulation on osteogenic properties of rat dental mesenchymal stem cells (DMSCs). DMSCs were isolated from rat teeth root tissues and identified by detecting vimentin and keratin expression. Flexcell FX4K tension system that mediating cyclic strain was used to treat DMSCs. MTT assay was used to observe DMSCs viability. Alkaline phosphatase (ALP) staining and alizarin red staining were conducted. Osteogenesis-specific biomarkers, such as receptor activator for nuclear factor-kB ligand (RANKL), osteoprotegerin (OPG), dentin sialoprotein (DSP) and bone sialoprotein (BSP), were evaluated using RT-PCR, western blot and immunohistochemistry assay, respectively. Positive ALP staining and alizarin red staining confirmed DMSCs phenotype. There were no significant morphology differences between mechanical stimuli treated cells and normal control cells. MTT results showed no significant differences between normal control cells and mechanically stimulated DMSCs. RT-PCR, western blot and immunohistochemistry assay indicated that 10% cyclic strain could trigger an obvious change of mRNA and protein expression of RANKL, OPG, DSP and BSP, respectively. Mechanical stimulation could trigger relative higher levels of calcium deposition in DMSCs. Mechanical strain triggered bone formation mainly through activating RANKL gene expression. In conclusion, 10% cycle mechanical strain could stimulate higher amounts of ALP and calcium deposition by activating RNAKL, and could trigger dramatically changes of mRNA and protein expression of osteogenesis-specific biomarkers, such as OPG, BSP and DSP.

Discriminant analysis of principal components and pedigree assessment of genetic diversity and population structure in a tetraploid potato panel using SNPs.

The reported narrow genetic base of cultivated potato (Solanum tuberosum) can be expanded by the introgression of many related species with large genetic diversity. The analysis of the genetic structure of a potato population is important to broaden the genetic base of breeding programs by the identification of different genetic pools. A panel composed by 231 diverse genotypes was characterized using single nucleotide polymorphism (SNP) markers of the Illumina Infinium Potato SNP Array V2 to identify population structure and assess genetic diversity using discriminant analysis of principal components (DAPC) and pedigree analysis. Results revealed the presence of five clusters within the populations differentiated principally by ploidy, taxonomy, origin and breeding program. The information obtained in this work could be readily used as a guide for parental introduction in new breeding programs that want to maximize variability by combination of contrasting variability sources such as those presented here.

Use of easy measurable phenotypic traits as a complementary approach to evaluate the population structure and diversity in a high heterozygous panel of tetraploid clones and cultivars.

BACKGROUND: Diversity in crops is fundamental for plant breeding efforts. An accurate assessment of genetic diversity, using molecular markers, such as single nucleotide polymorphism (SNP), must be able to reveal the structure of the population under study. A characterization of population structure using easy measurable phenotypic traits could be a preliminary and low-cost approach to elucidate the genetic structure of a population. A potato population of 183 genotypes was evaluated using 4859 high-quality SNPs and 19 phenotypic traits commonly recorded in potato breeding programs. A Bayesian approach, Minimum Spanning Tree (MST) and diversity estimator, as well as multivariate analysis based on phenotypic traits, were adopted to assess the population structure. RESULTS: Analysis based on molecular markers showed groups linked to the phylogenetic relationship among the germplasm as well as the link with the breeding program that provided the material. Diversity estimators consistently structured the population according to a priori group estimation. The phenotypic traits only discriminated main groups with contrasting characteristics, as different subspecies, ploidy level or membership in a breeding program, but were not able to discriminate within groups. A joint molecular and phenotypic characterization analysis discriminated groups based on phenotypic classification, taxonomic category, provenance source of genotypes and genetic background. CONCLUSIONS: This paper shows the significant level of diversity existing in a parental population of potato as well as the putative phylogenetic relationships among the genotypes. The use of easily measurable phenotypic traits among highly contrasting genotypes could be a reasonable approach to estimate population structure in the initial phases of a potato breeding program.

Developing a bacteriophage cocktail for biocontrol of potato bacterial wilt.

Bacterial wilt is a devastating disease of potato and can cause an 80% production loss. To control wilt using bacteriophage therapy, we isolated and characterized twelve lytic bacteriophages from different water sources in Kenya and China. Based on the lytic curves of the phages with the pathogen Ralstonia solanacearum, one optimal bacteriophage cocktail, P1, containing six phage isolations was formulated and used for studying wilt prevention and treatment efficiency in potato plants growing in pots. The preliminary tests showed that the phage cocktail was very effective in preventing potato bacterial wilt by injection of the phages into the plants or decontamination of sterilized soil spiked with R. solanacearum. Eighty percent of potato plants could be protected from the bacterial wilt (caused by R. solanacearum reference strain GIM1.74 and field isolates), and the P1 cocktail could kill 98% of live bacteria spiked in the sterilized soil at one week after spraying. However, the treatment efficiencies of P1 depended on the timing of application of the phages, the susceptibility of the plants to the bacterial wilt, as well as the virulence of the bacteria infected, suggesting that it is important to apply the phage therapy as soon as possible once there are early signs of the bacterial wilt. These results provide the basis for the development of bacteriophagebased biocontrol of potato bacterial wilt as an alternative to the use of antibiotics.

Delivery of cytoplasmic and apoplastic effectors from Phytophthora infestans haustoria by distinct secretion pathways.

The potato blight pathogen Phytophthora infestans secretes effector proteins that are delivered inside (cytoplasmic) or can act outside (apoplastic) plant cells to neutralize host immunity. Little is known about how and where effectors are secreted during infection, yet such knowledge is essential to understand and combat crop disease. We used transient Agrobacterium tumefaciens-mediated in planta expression, transformation of P. infestans with fluorescent protein fusions and confocal microscopy to investigate delivery of effectors to plant cells during infection. The cytoplasmic effector Pi04314, expressed as a monomeric red fluorescent protein (mRFP) fusion protein with a signal peptide to secrete it from plant cells, did not passively re-enter the cells upon secretion. However, Pi04314-mRFP expressed in P. infestans was translocated from haustoria, which form intimate interactions with plant cells, to accumulate at its sites of action in the host nucleus. The well-characterized apoplastic effector EPIC1, a cysteine protease inhibitor, was also secreted from haustoria. EPIC1 secretion was inhibited by brefeldin A (BFA), demonstrating that it is delivered by conventional Golgi-mediated secretion. By contrast, Pi04314 secretion was insensitive to BFA treatment, indicating that the cytoplasmic effector follows an alternative route for delivery into plant cells. Phytophthora infestans haustoria are thus sites for delivery of both apoplastic and cytoplasmic effectors during infection, following distinct secretion pathways.

Overexpression of StNF-YB3.1 reduces photosynthetic capacity and tuber production, and promotes ABA-mediated stomatal closure in potato (Solanum tuberosum L.).

Nuclear factor Y (NF-Y) is one of the most ubiquitous transcription factors (TFs), comprising NF-YA, NF-YB and NF-YC subunits, and has been identified and reported in various aspects of development for plants and animals. In this work, StNF-YB3.1, a putative potato NF-YB subunit encoding gene, was isolated from Solanum tuberosum by rapid amplification of cDNA ends (RACE). Overexpression of StNF-YB3.1 in potato (cv. Atlantic) resulted in accelerated onset of flowering, and significant increase in leaf chlorophyll content in field trials. However, transgenic potato plants overexpressing StNF-YB3.1 (OEYB3.1) showed significant decreases in photosynthetic rate and stomatal conductance both at tuber initiation and bulking stages. OEYB3.1 lines were associated with significantly fewer tuber numbers and yield reduction. Guard cell size and stomatal density were not changed in OEYB3.1 plants, whereas ABA-mediated stomatal closure was accelerated compared to that of wild type plants because of the up-regulation of genes for ABA signaling, such as StCPK10-like, StSnRK2.6/OST1-like, StSnRK2.7-like and StSLAC1-like. We speculate that the acceleration of stomatal closure was a possible reason for the significantly decreased stomatal conductance and photosynthetic rate.