First Report of Pectobacterium punjabense Causing Blackleg and Soft Rot on Potato in Hebei and Fujian Province, China

Pectobacterium spp. and Dickeya spp. cause blackleg and soft rot on potato worldwide (Charkowski, 2018). Potato plants (cv. Favorita or Jizhang 8#) with blackleg symptoms (vascular browning of crown stems, Fig. S1) were observed in the field in Zhangjiakou, Hebei province in 2018, and in Ningde, Fujian Province in 2019, in China. The disease incidence was around 50% and 10% in Zhangjiakou (5 ha) and Ningde (4 ha), respectively. Diseased plants (3 from each site) were collected to isolate the pathogen. Blackleg symptomatic stems were soaked in 75% ethanol for 2 min, rinsed and ground in sterile distilled water. Serial tenfold dilutions of the above solution were plated onto the crystal violet pectate agar (CVP) plate (Ge et al., 2018). Two to 3 days after incubation at 28°C, 4 bacterial colonies in total which digested pectin from the media and developed pit on CVP plates were purified and sequenced for identification using the universal 16S rRNA gene primer set 27F/1492R (Monciardini et al., 2002). Two colony sequences that showed more than 99% sequence identity to Pectobacterium punjabense type strain SS95 (MH249622) were submitted to the GenBank ( accession numbers: OK510280, MT242589). Additionally, six housekeeping genes proA (OK546205, OK546199), gyrA (OK546206, OK546200), icdA (OK546207, OK546201), mdh (OK546208, OK546202), gapA (OK546209, OK546203), and rpoS (OK546210, OK546204) of these two isolates were amplified and sequenced (Ma et al., 2007, Waleron et al., 2008). All strains show 99% to 100% identity with MH249622T . Phylogenetic trees based on 16S rRNA gene sequences (Fig. S2) and concatenated sequences of the housekeeping genes (Fig. S3) of the 2 isolates were constructed using MEGA 6.0 software (Tamura et al., 2013). Koch’s postulate was performed on potato seedlings and potato tubers (cv. Favorita) by injecting 100 μl bacterial suspension (105 CFU/ml) or sterile phosphate-buffered solution into the crown area of the stems or the tubers and kept at 100% humidity and 21°C for 1 day. Four days after inoculation, the infected area of the inoculated seedlings rotten and turned black, while the controls were symptomless (Fig. S4). Two days after inoculation, the infected tubers rotten and turned black, while the controls were symptomless (Fig. S4). Bacterial colonies were reisolated from these symptomatic tissues and identified using the same methods described above. Blackleg on potato plants or soft rot on potato has been reported to be caused by Pectobacterium atrosepticum, Pectobacterium carotovorum subsp. carotovorum, Pectobacterium carotovorum subsp. brasiliense, Pectobacterium parmentieri, Pectobacterium polaris in China (Zhao et al., 2018; Cao et al., 2021; Wang et al., 2021). To our knowledge, this is the first report of blackleg/soft rot of potato caused by Pectobacterium punjabense in China. We believe that this report will draw attention to the management of this pathogen in China.

First report of soft rot of Aconitum carmichaelii caused by Pectobacterium brasiliense in China

Aconitum carmichaelii Debx. is a Chinese traditional medicine herb, and is widely planted in China. The processed lateral roots of A. carmichaelii is known as Fuzi, and is used for the treatment of pain and inflammation in the joints (Zhou et al., 2015). In July 2019, a high incidence (approximately 50-100%) of soft rot of A. carmichaelii was observed in several commercial fields in Jiangyou County of Szechuan Province of China. Soft rot brownish lesions developed on infected stems, leading to collapse and wilting of entire plants. From symptomatic plants, the margins between the diseased and healthy areas were cut into pieces (5 × 5 mm), which were surface sterilized using 75% ethanol for 30 s and 2% NaOCl for 1 min, followed by three rinses with sterile water. The sterilized sections were macerated in drops of sterile water, and the extract was streaked onto King’s B (KB) agar medium and incubated for 48 h at 30°C. Single colonies that are round, convex and creamy on the plates after 2 days were streaked on KB agar plates. Ten bacterial strains were isolated, and the strain Fuzi915 was chosen for further analyses. The 16S rDNA gene sequence (GenBank accession MZ881946) amplified by primer pair 27F/1492R (Monciardini et al., 2002) showed 99.85% identity to the sequence of Pectobacterium brasiliense (syn. Pectobacterium carotovorum subsp. brasiliense, Pcb) strain HNP201736 (MN393938.1) and P. carotovorum subsp. carotovorum strain PJP201706 (MN394020.1), respectively, and also showed 99.78% identity to P. brasiliense strain SX309 (CP020350.1). To further identify the Fuzi915 strain, the PCR assay was carried out using primer pairs Y1/Y2, EXPCCF/EXPCCR and BR1f/L1r (De Boer and Ward, 1995; kang et al., 2003; Duarte et al., 2004), specific to P. carotovorum, P. carotovorum subsp. carotovorum and P. carotovorum subsp. brasiliense (Pcb), respectively. Specific fragments of 434 bp and 322 bp were amplified by the Y1/Y2 and BR1f/L1r primer sets, receptively, but there was no amplification by the EXPCCF/EXPCCR primer set, indicating that the Fuzi915 strain belongs to Pcb (Onkendi and Moleleki, 2014). Additional phylogenetic trees based on two housekeeping genes mdh (MZ892962) and gapA (MZ892963) were constructed using Maximum-likelihood method with 1000 bootstraps. The Fuzi915 strain clustered with all P. brasiliense strains including type strain P. brasiliense BC1. Further, a pathogenicity test was conducted on healthy A. carmichaelii roots and seedlings maintained in a growth chamber at 25°C and 95% humidity. Root inoculation was followed by drenching 107 CFU/ml of the cell suspension of Fuzi915 strain in soil surrounding the A. carmichaelii roots. Ten roots were inoculated with cell suspension while 10 roots were drenching inoculated with sterile water as negative control. Stem inoculation was followed by injecting 103 CFU/ml of the cell suspension in the stem of 10 A. carmichaelii seedlings, while 10 were injected with sterile water as negative control. After 5 days, Pcb-inoculated roots became brown and soft, and Pcb-inoculated seedlings became wilted and water soaked and started to collapse, similar to symptoms observed in the field. No symptoms were observed on the control plants inoculated with sterile water. The strain was re-isolated successfully from symptomatic A. carmichaelii and was identified as P. brasiliense by using PCR with the same primers to complete Koch’s postulates. To our knowledge, this is the first report of the soft rot of A. carmichaelii caused by P. brasiliense in China.

First Report of Pectobacterium polaris Causing Blackleg on Potato in Inner Mongolia and Sichuan Province, China

Pectobacterium spp. and Dickeya spp. cause Blackleg on potato worldwide (Charkowski, 2018). Potato plants (cv. Innovator V4 or Favorita) with blackleg symptoms (vascular browning of crown stems or curled leaves, Fig. S1) were observed in the field in Xilingol League, Inner Mongolia in 2018, and in Chengdu, Sichuan Province in 2020, in China. The disease incidence were around 10% and 20% in Xilingol League (20 ha) and Chengdu (40 ha), respectively. Diseased plants (5 from Xilingol League, and 2 from Chengdu) were collected to isolate the pathogen. Blackleg symptomatic stems were soaked in 75% ethanol for 2 min, rinsed and ground in sterile distilled water. Serial tenfold dilutions of the above solution were plated onto the crystal violet pectate agar (CVP) plate (Ge et al., 2018). Two to 3 days after incubation at 28°C, the bacterial colonies which digested pectin from the media and developed pit on CVP plates were purified and sequenced for identification using the universal 16S rRNA gene primer set 27F/1492R (Monciardini et al., 2002). Three colony sequences that showed more than 99% sequence identity to Pectobacterium polaris type strain NIBIO1392 (NR_159086.1) were submitted to the GenBank ( accession numbers: MT242579, MT242580, and MZ489432). Additionally, six housekeeping genes proA (MZ39581–MZ395583), gyrA (MZ395569–MZ395571), icdA (MZ395572–MZ39574), mdh (MZ395575–MZ395577), gapA (MZ395578– MZ395580), and rpoS (MZ39584–MZ395586) of these three isolates were amplified and sequenced (Ma et al., 2007, Waleron et al., 2008). All strains show 99% to 100% identity with Pectobacterium polaris strain NIBIO1392. Phylogenetic trees based on 16S rRNA gene sequences (Fig. S2) and concatenated sequences of the housekeeping genes (Fig. S3) of the 3 isolates were constructed using MEGA 6.0 software (Tamura et al., 2013). Koch’s postulate was performed on potato seedlings (cv. Favorita) by injecting 100 μl bacterial suspension (107 CFU/ml) or sterile phosphate-buffered solution into the crown area of the stems and kept at 80% humidity and 21°C for 2 days. Seven days after inoculation, the infected area of the inoculated seedlings rotten and turned black or even lodged, while the controls were symptomless (Fig. S4). It was observed that isolate MZ489432 from Chengdu, Sichuan Province was more virulent than the isolates from Xilingol League (Fig. S4). Bacterial colonies were reisolated from these symptomatic seedlings and identified using the same methods described above. Blackleg on potato plants has been reported to be caused by Pectobacterium atrosepticum, Pectobacterium carotovorum subsp. carotovorum, Pectobacterium carotovorum subsp. brasiliense, and Pectobacterium parmentieri in China (Zhao et al., 2018; Cao et al., 2021). To our knowledge, this is the first report of blackleg of potato caused by Pectobacterium polaris in China. We believe that this report will draw attention to the management of this pathogen in China.

First report of Pectobacterium versatile causing blackleg of potato in Serbia

Potato blackleg is frequently observed on the production fields in the Bačka region of Vojvodina province, which is one of the largest potato-growing areas in Serbia. This disease usually occurs during June and July. In July 2020, blackleg symptoms in the form of stem necrotic lesions, vascular discoloration, hollow stems, and wilting of whole plants were noted on potato cultivar VR808 on a field 28 ha in size located in Maglić village (GPS coordinates 45.349325 N, 19.542768 E). Disease incidence was estimated at 20−25%. Isolations were performed from 12 potato samples on Crystal Violet Pectate medium (CVP). Stem sections consisted of brown lesions and healthy tissue (c.10 cm) were surface sterilized with ethyl alcohol 70% (w/v) and rinsed with sterile distilled water. Small pieces of tissue were taken at the edges of stem lesions (between healthy and diseased tissue) were soaked in phosphate buffer saline for 20 min and plated using a standard procedure (Klement et al. 1990). Single colonies that formed pits after 48 hours at 26 °C were re-streaked onto Nutrient Agar (NA) where creamy white colonies with smooth surfaces were formed. A total of 30 isolates were selected and DNA isolated from the colonies was further analyzed by polymerase chain reaction (PCR) using the partial dnaX gene (DNA polymerase subunit III gamma/tau) with primer pair dnaXf/dnaXr for Pectobacterium and Dickeya species identification (Slawiak et al. 2009). A single characteristic band of 535 bp was amplified in all isolates (Slawiak et al. 2009). DNA sequence alignment showed two distinct groups of isolates (Fig.S1), which were genetically uniform within each group. Using BLASTn search, it was established that the dnaX sequence of the first group (consisting of 19 Serbian potato isolates) had 99.79% identity with NCBI-deposited Pectobacterium versatile strains 14A and 3-2 from potato from Belarus (Acc. No. CP034276 and CP024842, respectively) as well as SCC1 from Finland (Acc. No. CP021894). The remaining 11 dnaX sequences had 100% identity with Pectobacterium carotovorum subsp. carotovorum strain CFBP7081 originating from water in Spain (Acc. No. MK516961). The partial dnaX sequences of three Serbian P. versatile isolates (Pv1320, Pv1520, and Pv1620) and one P. carotovorum subsp. carotovorum (Pcc2520) were deposited in GenBank under Acc. No. MW839571, MW805306, MW839572, and MW805307, respectively. These results, indicating combined infection in the observed field, signify the first identification of P. versatile in Serbia. Multilocus sequence analysis (MLSA) performed with proA (proAF1/ proAR1) and mdh (mdh2/mdh4) genes (Ma et al. 2007; Moleleki et al. 2013) grouped three tested Serbian potato P. versatile isolates together with P. versatile strains from NCBI (Fig.S2). For both tested genes, BLASTn search revealed 100% homology with P. versatile strain SCC1 from Finland. Three Serbian P. versatile potato isolates were deposited under Acc. Nos. MZ682623-25 for proA and MZ682620-22 for mdh genes. According to the routine tests suggested for Pectobacteriaceae (Schaad et al. 2001), Serbian isolates possessed microbiological traits identical to P. versatile description (Portier et al. 2019). Pathogenicity was performed on potato cultivar VR808 with three selected P. versatile isolates (Pv1320, Pv1520, and Pv1620) in the following assays: (i) surface-sterilized tuber slices with holes in the center filled with 100 µL of bacterial suspensions (adjusted to 109 CFU mL-1) to test the isolates’ ability to cause soft rot, and (ii) young, four-week old plants with developed 3rd true leaf (c. 30 cm tall) were inoculated by injecting stems with bacterial suspension adjusted to 107 - 108 CFU mL-1 at a height 5 cm above the soil line. Negative controls were treated with sterile distilled water. Inoculated plants were kept under controlled conditions (25 °C temperature and >70% relative humidity). Each assay was replicated twice. Soft rot appeared on tuber slices 24 h after inoculation. On inoculated stems, initial symptoms manifested as greasy elongated spots at inoculation sites two days after inoculation (DAI), and subsequently extended along the vascular tissue and became necrotic. Whole plant's decay was recorded in five DAI, while negative controls remained healthy. To complete Koch's postulates, bacteria were re-isolated from symptomatic potato plants and confirmed by PCR and sequencing of dnaX. This first report of P. versatile in potato indicates that blackleg currently present in Serbia is caused by a diverse bacterial population. This pathogen was first identified in genome comparison as ‘Candidatus Pectobacterium maceratum’ (Shirshikov et al. 2018) and was later renamed as Pectobacterium versatile sp. nov. (Portier et al. 2019). Thus far, bacterium Pectobacterium carotovorum subsp. brasiliensis has been recognized as dominant pathogen on most of the infected fields in Vojvodina province, and was recently noted on one plot subjected to a combined infection with Dickeya dianthicola (Marković et al. 2021). Findings achieved in this study are highly relevant, as they point to the diversity in potato blackleg pathogens, likely due to the increasingly widespread distribution of imported seed potatoes.

Punica granatum L. extract contributes to phytopathogens control and enhances Eruca vesicaria (L.) Cav. germination in vitro and in vivo

The study aimed to investigate antimicrobial activity of the hydroalcoholic crude extract from the fruit peel of Punica granatum (Pp) and punicalagin compound (Pg) on phytopathogenic bacterial isolates and its potential use as a sustainable alternative in treatment of vegetable seeds. The antimicrobial activity in vitro was tested by agar well diffusion assay and through viability tests in liquid medium. In vivo treatment with Pp was tested on Eruca vesicaria seeds infected with Xanthomonas campestris pv. campestris. Pp induced the formation of large inhibition zones to the growth of the tested pathogens (35.33 mm – 6.66 mm), with dose-dependent effect. Viability tests confirmed the antimicrobial activity of the Pp on X. campestris pv. campestris and P. carotovorum subsp. carotovorum with minimum inhibitory concentration (MIC) of 125 μg/mL. Punicalagin compound presented MIC of the 31.25 μg/mL. The seed treatment with Pp indicated control of pathogen-induced symptoms in seedlings of the E. vesicaria and positive effect in seed germination, emergence and in stomatal functionality. The results indicate strong potential of the extract from the fruit peel of P. granatum and Punicalagin for formulating botanical pesticides for plant disease control.