Crystal Violet Pectate Agar Research Articles

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.

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.

First report of Dickeya dadantii causing bacterial soft rot of Scindapsus pictus in Taiwan.

Scindapsus pictus (satin pothos or silver vine) is an evergreen climbing plant belonging to the Araceae family, subfamily Monstereae (Bown, 2000), which is also cultivated as a foliage ornamental (Masnira et al. 2019). In September of 2022, soft rot symptoms were observed on potted S. pictus plants grown in a greenhouse in Nantun District, Taichung, Taiwan, in which soft rot of another aroid (philodendron) has also been reported (Wu et al. 2023). The symptoms appeared on the petioles and most of them tended to extend to the leaf blades; the colors of leaf lesions ranged from dark brown to gray (Fig. S1). Some 70% of the plants in the greenhouse showed similar symptoms and losses were estimated to be 15-30%. Four symptomatic plants were sampled. Macerated tissues from rotting petioles were soaked in 10 mM MgCl2 and observed under a light microscope (Nikon, Japan) at 400 x magnification. Motile, rod-shaped bacteria were observed, and 1-2 loopfuls of undiluted sample suspension were streaked onto nutrient agar (NA; Gibco, USA). After culturing at 28°C for 1 day, all samples yielded round, creamy-white colonies (0.9 mm in diameter) and from each of the four samples a pure culture was obtained (Spi1-Spi4). All isolates exhibited oxidative and fermentative metabolism of glucose (Schaad et al. 2001). They caused pitting on crystal violet pectate agar, induced maceration on potato tuber and were tested positive for phosphatase activity and indigoidine production (Lee and Yu 2006; Schaad et al. 2001). Polymerase chain reaction tests using Dickeya-specific primers 5A and 5B (Chao et al. 2006) amplified the expected amplicon (0.5 kb) in extracted DNA samples of all isolates. Identification of the strains was achieved by amplifying and sequencing fragments of the housekeeping genes gyrB, recN, dnaA, dnaJ, and dnaX (Marrero et al. 2013); the lengths of the five gene fragments analyzed were 822, 762, 720, 672, and 450 bp, respectively (accession nos. OP985528-OP985532). The five sequences were concatenated for every isolate; the resulting 3,426 bp sequences were aligned with ClustalW and found to be identical. A maximum-likelihood analysis was conducted using the 3,426-bp sequences and those of known Dickeya species' type strains. Spi1 to Spi4 clustered with D. dadantii subsp. dieffenbachiae NCPPB 2976T and D. dadantii subsp. dadantii CFBP 1269T (Fig. S2) with sequence identities of 98.4 and 98%, respectively. To fulfil Koch's Postulates, stab inoculations of the four isolates into the petioles of cutting propagated, 38-day-old S. pictus plants (3 plants per isolate) were conducted using sterile toothpicks. The amounts of bacteria used was approximately 106 cfu per toothpick; the bacterial loads were estimated by suspending the cells in 10 mM MgCl2 and spread-plating diluted suspensions on NA. Sterile toothpicks were used as control. All tested plants were sealed in plastic bags (containing wet paper towel) and kept in a growth chamber (28°C; 12-h photoperiod). After 1 day, all isolates induced soft rot symptoms resembling those observed under natural conditions in the greenhouse. Bacteria were re-isolated, and they all shared the same dnaX sequence with strains Spi1 to Spi4. This is the first report of S. pictus affected by D. dadantii in Taiwan. Further investigation is needed to determine whether Spi1-Spi4 belong to D. dadantii subsp. dieffenbachiae. Dickeya dadantii has been found infecting different aroids (Lee and Chen 2021; Lin et al. 2012). The species has also been reported in Taiwan on poinsettia (Wei et al., 2019) and philodendron (Wu et al. 2023). Because these plants are often grown closely in the same facilities, growers should be wary of D. dadantii's spread among these plants. Reduction of environmental humidity and avoiding overhead irrigation may be effective in preventing the pathogen's transmission.

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 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 polaris Causing Soft Rot and Black Leg of Potato in Russia.

Blackleg and soft rot of potato (Solanum tuberosum) were monitored in the Central European part of Russia within a period of 2012- 2019. Symptoms included decay of tubers, blackening of stem vascular bundles, and partial yellowing of leaves. The disease causes serious potato yield losses in the field and storage. Pectobacterium parmentieri, P. brasiliense, P. versatile (syn. Ca. Pectobacterium maceratum), P. carotovorum, P. atrosepticum, Dickeya dianthicola, and D. solani are considered as main causal agents of soft rot and blackleg disease in Russia (Voronina et al. 2019, Ngoc Ha et al., 2019, Shirshikov et al. 2018, Kornev et al. 2012). Potato plant samples collected in commercial fields in routine plant health assay were used for bacteria isolation on crystal violet pectate agar (CVP) (Helias et al. 2012) as described previously (Voronina et al. 2019). Bacterial colonies producing pitting on CVP were re-isolated and purified on nutrient broth yeast extract medium. DNA of bacterial isolates was extracted, and polymerase chain reaction (PCR) amplifications were performed using gapA primers (Cigna et al. 2017) followed by sequencing. DNA sequence alignment showed that the isolates F099, F100, F106, F109, and F118 were identical (deposited as part of NCBI Ref.Seq. for F109 NZ_RRYS01000004.1, locus KHDHEBDM_RS06360) and grouped together with the type strain Pectobacterium polaris NIBIO1006T (CP017481), a new species described as a potato pathogen (Dees et al. 2017). These strains were negative in diagnostic PCR assays using specific primers Y45/Y46 for the detection of P. atrosepticum, Br1f and L1r for P. brasiliense (Duarte et al. 2004), and ADE1/ADE2 for Dickeya sp. (Nassar et al. 1996). To further validate the identification, strain F109 of P. polaris was selected for genome sequencing. The genome of P. polaris strain F109, (NCBI Reference Sequence NZ_RRYS00000000.1) reveals >99% sequence similarity with type strain P. polaris IPO_1606 (GenBank accession GCA_902143345.1). The strain F109 was deposited to All-Russian Collection of Microorganisms under number VKM V-3420. Thus, the characterization of five isolates provided evidence that a previously unreported pathogen was present in the surveyed fields. The isolates were uniform in genetic and physiological properties; they were gram negative, facultative anaerobes with pectinolytic activity, negative for oxidase, urease, indole production, gelatin liquefaction. All isolates were catalase positive, produced acid from lactose, rhamnose, saccharose, xylose, and trehalose, and were tolerant to 5% NaCl, unable to utilize malonate and citrate. All the isolates grew at 37°C. All isolates caused soft rot symptoms on 10 inoculated potato tubers. They produced typical black leg rot symptoms in young potato plants inoculated with 107 CFU/ml of the pathogen by stem injection and incubated at 25°C for 48 h. The bacteria were re-isolated successfully from symptomatic potato and pathogen confirmed by gapA sequencing to complete Koch's postulates. To our knowledge, this is the first report of blackleg and soft rot caused by P. polaris on potato in the Russian Federation. According to the data of commercial diagnostic laboratory "PhytoEngineering" (Moscow region), P. polaris occurred in 5% potato seed stocks harvested in 2017-2019 in the Moscow region. This finding may indicate that new Pectobacterium strains have adapted to a diverse environment, which is consistent with widespread distribution of commercial seed potatoes. The author(s) declare no conflict of interest. Funding: This work was supported by Russian Science Foundation grant #16-16-00073.

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.

First Report of Pectobacterium carotovorum subsp. brasiliense Causing Blackleg and Stem Rot Disease of Potato in Russia

Blackleg and stem rot of potato (Solanum tuberosum) were observed in several commercial fields in the central part of Russia with up to 40% incidence during 2012 to 2017. Symptoms included decay of internal root and stem tissues, blackening of petiole vascular bundles, and partial yellowing of leaves. The disease can cause serious potato yield losses in the field and storage in Russia. Pectobacterium carotovorum subsp. carotovorum, P. atrosepticum, Dickeya dianthicola, and D. solani are the main causal agents of blackleg disease in Russia (Kornev et al. 2012). Surveys conducted in commercial fields produced multiple samples showing stem rot symptoms. Bacteria were isolated from symptomatic tissues by suspending 1-cm stem sections with lesions in sterile tap water for 10 min, followed by serial dilutions on crystal violet pectate agar (CVP). Bacterial colonies producing pitting on CVP were reisolated and purified on nutrient broth yeast extract medium. Characterization of three isolates provided evidence that a previously unreported blackleg/stem rot pathogen was present in the surveyed region. F126 (from potato ‘Lady Clare’, Samara Region), F128 (from potato ‘Sante’ from Kaluga Region), and F140 (from Moscow region) were gram negative, facultatively anaerobic, and able to grow at 37°C on nutrient agar. They produced typical stem rot symptoms in potato plants inoculated with 10⁷ CFU/ml of the pathogen by stem injection and incubated at 25°C for 48 h. The strains were reisolated successfully from symptomatic potato to complete Koch’s postulates. These strains were negative in polymerase chain reaction (PCR) analyses using specific primers Y45/Y46 for the detection of P. atrosepticum and ADE1/ADE2 for Dickeya (Czajkowski 2011). In contrast, all strains yielded an amplicon (322 bp) in a PCR with primers Br1f and L1r for P. carotovorum subsp. brasiliense (Duarte et al. 2004). 16S rRNA was amplified using universal primer set F27/R1492 (Monciardini et al. 2002). The strains F126, F128, and F140 (GenBank accessions MG570140, MG570141, and MG570142) had 99% sequence identify with 16S rRNA of P. carotovorum subsp. brasiliense type strain BC1 (CP009769.1). Additional loci representing two housekeeping genes were amplified and sequenced: gyrB (MG570137, MG570138, and MG570139) and recA (MG570134, MG570135, MG570136). Primer sets used were as reported by Young and Park (2007) and Dauga (2002). All strains showed 97 to 98% similarity with type strain P. carotovorum subsp. brasiliense BC1. To our knowledge, this is the first report of blackleg and stem rot caused by P. carotovorum subsp. brasiliense on potato in the Russian Federation. According to the data of several diagnostic laboratories, P. carotovorum subsp. brasiliense occurred in >20% potato seed stocks harvested in 2017 and was the predominant blackleg pathogen in fields in the Moscow region in 2018.

Pectobacterium and Dickeya Responsible for Potato Blackleg Disease in New York State in 2016.

Beginning in 2014, outbreaks of blackleg disease compromised potato (Solanum tuberosum) production in the northeastern United States. Disease severity was atypical for plantings with certified seed. During 2016, 43 samples with blackleg symptoms were analyzed, originating from more than 20 farms operating in New York State. A combination of techniques was employed to identify the blackleg pathogens: isolation in vitro, diagnostic PCR assays for Pectobacterium and Dickeya sp., pathogenicity assays, and DNA sequencing. Twenty-three bacterial isolates were obtained, the majority of which were designated D. dianthicola or P. parmentieri; two of the isolates were designated P. atrosepticum. All isolates were pathogenic in stem lesion and tuber soft rot assays and exhibited pectin degrading activity (pitting) in crystal violet pectate agar medium. Phylogenetic analyses of dnaX gene sequences placed all but one of the isolates into clades corresponding to D. dianthicola, P. parmentieri, or P. atrosepticum. One atypical isolate clustered with P. carotovorum subspecies. Data are consistent with the hypothesis that D. dianthicola from New York and the northeast are part of a single clade, and at least three different soft rot bacteria were associated with blackleg during 2016 in New York.

BACTERIAL SOFT ROT OF EGGPLANT CAUSED BY PECTOBACTERIUM CAROTOVORUM subsp. CAROTOVORUM IN CHINA

In December 2016, soft rot lesions on the stem and fruit of eggplants cv. Green Beauty were noted in greenhouses in Anshan, Liaoning Province, China. Disease incidence was estimated at 5-10% and resulted in a substantial yield reduction. Symptoms appeared as soft, dark green stem lesions that progressively turned brown with vascular bundle necrosis appearing. Fruits of infected plants initially had small water-soaked lesions that rapidly enlarged with a watery ooze. Infected tissues from stems and fruits of eggplant were macerated in sterile water, streaked onto nutrient agar (NA) and incubated at 28oC for 2 days. The resultant bacterial colonies were round, translucent, white, convex with smooth edges. All isolated bacteria were gram-negative rods, facultative anaerobes, non-fluorescent on King’s B media, produced a positive pectolytic response on crystal violet pectate agar and gave a positive hypersensitive response in tobacco plants. All were negative for sucrose reduction, sensitive to erythromycin (50 µg/ml), indole, oxidase and could grow at 37°C. PCR amplification of the pel gene using Y1/Y2 primers (Darrasse et al., 1994) produced a 430 bp fragment. Amplification of the 16S- 23SrRNA region by G1 and L1 primers (Toth et al., 2001) produced two main bands of 550 and 580 bp. 16S rRNA fragment of 1440 bp was amplified from bacterial isolates and the partial sequence deposited in GenBank (accession No. KY786125) showed 99% identity to the sequence of Pectobacterium carotovorum subsp. carotovorum strain Y57 (KP187518.1). No amplification products were produced for any isolate using P. atrosepticum and P. carotovorum subsp. brasiliensis specific primers (Eca1f/Eca2r and Br1f/L1r, respectively), whereas the P. carotovorum subsp. carotovorum primers EXPCCF/EXPCCR produced the expected 550 bp fragment (Kang et al., 2003). For pathogenicity testing, mature fruits and stems of Green Beauty were needle-stab inoculated with a 10 µl bacteria suspension (1×108 CFU/ml). Sterile water was used as a negative control. Inoculated plants were incubated at 28°C for 48 h, after which watersoaked and soft-rot symptoms were observed on all eggplant fruits and stems, but not on control plants. Bacteria with the same characteristics as those inoculated were reisolated from diseased tissues. To our knowledge, this is the first report of P. carotovorum subsp. carotovorum on eggplant in China.

FIRST REPORT OF BACTERIAL PITH SOFT ROT CAUSED BY PECTOBACTERIUM CAROTOVORUM subsp. BRASILIENSE ON ARTICHOKE IN ITALY

A severe soft rot disease was observed in some commercial artichoke crops (F1-hybrid cultivars) during 2014 in the Bari, Foggia (Apulia, Southern Italy) and Matera (Basilicata, Southern Italy) provinces. Symptoms were chlorosis and wilting of the older leaves accompanied by dark-green to dark-brown soft rotting of the pith. Several bacterial isolates were obtained from affected plants by culturing on semi-selective crystal violet pectate agar medium. Biochemical, physiological and molecular analyses and pathogenicity tests, performed using five selected isolates, identified three of them as Pectobacterium carotovorum subsp. brasiliense. To the best of our knowledge, this is the first report of a bacterial soft rot caused by P. carotovorum subsp. brasiliense in Italy and the first finding of this bacterial subspecies on artichoke.

First Report of Orostachys malacophyllus Soft Rot Caused by Pectobacterium carotovorum subsp. carotovorum in Korea

HomePlant DiseaseVol. 100, No. 1First Report of Orostachys malacophyllus Soft Rot Caused by Pectobacterium carotovorum subsp. carotovorum in Korea PreviousNext DISEASE NOTES OPENOpen Access licenseFirst Report of Orostachys malacophyllus Soft Rot Caused by Pectobacterium carotovorum subsp. carotovorum in KoreaY. S. Kim and Y. JeonY. S. KimSearch for more papers by this author and Y. JeonSearch for more papers by this authorAffiliationsAuthors and Affiliations Y. S. Kim Y. Jeon , Department of Bioresource Sciences, Andong National University, Andong 760-749, Korea. Published Online:28 Oct 2015https://doi.org/10.1094/PDIS-04-15-0413-PDNAboutSectionsSupplemental ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat Orostachys malacophyllus is a perennial herbaceous plant belonging to the family Crassulaceae that is found widely in Korea, China, and Japan. The plant is typically harvested from summer to autumn, and is used for medicinal purposes after removing its roots and then drying under sunlight (Park et al. 1991). It has been observed that the plant becomes more susceptible to infection when cultivated. For our survey, we collected plants from plantations near Uljin in Gyeongbuk Province during the summers of 2013 and 2014. The early symptoms of plant disease were water-soaked lesions on leaves, starting from base of the stem. Thereafter, the lesions expanded rapidly over the entire plant, resulting in a soft rot of the plant. From July to August, after rainfall, disease incidence rapidly increased from 30 to 80%. In order to isolate the causal agent of the water-soaked lesions, 1-cm leaf segments were surface sterilized in 70% ethanol and 1% NaOCl solution for 30 s, rinsed and macerated in sterile distilled water, and then plated onto nutrient agar medium. Bacterial colonies developed on all the plates after incubation for 48 h at 28°C. Isolated bacteria were gram-negative, oxidase-negative, facultatively anaerobic, positive for degradation of crystal violet pectate agar (CVP), nonfluorescent on King’s B medium, positive for hypersensitive response in tobacco plants, and caused soft rot of potato tubers. To further identify the bacterial strains, we performed Biolog, fatty acid methyl ester (FAME), and 16S rDNA sequence analyses. The Biolog GN2 microplate and the Release 4.20 system putatively found the strain to exhibit 85% similarity with the bacterium Pectobacterium carotovorum subsp. carotovorum. Likewise, analysis of FAME profiles using the Microbial Identification System (Sherlock version 3.1, Microbial Identification System, Newark, DE, USA) also characterized the representative bacterial strain as P. carotovorum subsp. carotovorum with 89% similarity. The isolated bacterium’s 16S rDNA was amplified by PCR using the primer set 27F (5′-TCCGTAGGTGAACCTGCGG-3′) and 1492R (5′-TTCTCCGCTTATTGATATGC-3′) (Weisburg et al. 1991; Cheon et al. 2014), and subsequently subjected to a similarity search using BLAST analysis of the NCBI database. The 16S rRNA gene sequence from the strain (GenBank Accession No. KT222000) showed 99% identity with sequence of P. carotovorum subsp. carotovorum previously deposited in GenBank (KC790305). Pathogenicity of the three strains was confirmed by dropping a bacterial suspension (20 µl per inoculation site, 1 × 108 CFU/ml) onto leaf surfaces in a growth chamber maintained at 28°C and 80% relative humidity. Two leaves were inoculated with each strain, and the experiment was conducted twice. Sterile distilled water was used as the control. Water-soaked lesions appeared on the inoculated leaves within 48 h of inoculation, in contrast to the control leaves, which showed no symptoms. The bacteria were reisolated from the leaf showing symptoms and confirmed as P. carotovorum subsp. carotovorum by Biolog. To our knowledge, this is the first report of P. carotovorum subsp. carotovorum as a pathogen of O. malacophyllus in Korea.

First Report of Pectobacterium carotovorum subsp. carotovorum Causing Soft Rot of Orostachys japonica in Korea.

Orostachys japonica (Maxim) A. Berger is an important traditional medicine in Korea. The extract of this plant has antioxidant activity and suppresses cancer cell proliferation (1). From summer through fall of 2012 and 2013, a high incidence (~10% to 30%) of disease outbreaks of all plants characterized by water-soaked lesions and soft rot with a stinky odor was observed in cultivated O. japonica around Uljin (36°59'35.04″N, 126°24'1.51″E), Korea. Water-soaked lesions were first observed on the stem base of plants. Subsequently, the plants collapsed, although the upper portion remained asymptomatic. Thereafter, the lesions expanded rapidly over the entire plant. To isolate potential pathogens from infected leaves, small sections (5 to 10 mm2) were excised from the margins of lesions. Ten bacteria were isolated from ten symptomatic plants. Three representative isolates from different symptomatic plants were used for identification and pathogenicity tests. Isolated bacteria were gram negative, pectolytic on crystal violet pectate agar, nonfluorescent on King's medium B, and elicited a hypersensitive response in tobacco plants. All isolates caused soft rot of potato tubers. These isolates also differed from isolates of Erwinia chrysanthemi (Ech) that they were insensitive to erythromycin and did not produce phosphatase. These isolates differed from known strains of E. carotovora subsp. atroseptica in that they did not produce reducing substances from sucrose (2). Use of the Biolog GN microplate and the Release 4.0 system identified the isolate as Pectobacterium carotovorum subsp. carotovorum with 81.2% similarity. The 16S rRNA of the isolated bacteria was amplified by PCR and sequenced as described by Weisburg et al. (3). A BLAST analysis for sequence similarity of the 16S rRNA region revealed 99% similarity with nucleotide sequences for P. carotovorum subsp. carotovorum isolates (KC790305, KC790280, JF926758, JX196705, and AB680074). The pathogenicity of three bacterial isolates was examined on three 2-year-old O. japonica plants by adding 50 μl of a bacterial suspension containing 108 CFU/ml when wounding the leaves with sterile needles. Ten control plants were inoculated with sterilized water. After inoculation, plants were maintained in a growth chamber at 25°C with relative humidity ranging from 80 to 90%. After 2 to 3 days, tissue discoloration, water-soaked lesions, and soft rot developed around the inoculation point. Severe symptoms of soft rot and darkening developed on leaves of inoculated plants within 3 to 5 days after inoculation. All controls remained healthy during these experiments. The bacterial strains re-isolated from the parts of the leaf showing the symptoms and identified as P. carotovorum subsp. carotovorum on the basis of the biochemical and physiological tests, as well as Biolog system. The results obtained for pathogenicity, Biolog analysis, and molecular data corresponded with those for P. carotovorum subsp. carotovorum. To our knowledge, this is the first report of the presence of P. carotovorum on O. japonica in Korea.

A SEVERE OUTBREAK OF BACTERIAL LETTUCE SOFT ROT CAUSED BY PECTOBACTERIUM CAROTOVORUM subsp. CAROTOVORUM IN APULIA (ITALY)

A severe bacterial disease was observed in lettuce plants during surveys carried out in the province of Bari (Apulia, Italy). Symptoms were chlorosis and wilting of older leaves, water-soaked and dark green soft rot of the pith. Several bacterial isolates were obtained from the affected plants by isolations on semi-selective crystal violet pectate agar medium. Biochemical, nutritional, and pathogenicity tests identified them as Pectobacterium carotovorum subsp. carotovorum. The nutritional profile obtained using the Biolog system and the 434 bp product amplified by PCR using primers designed on P. carotovorum pectate lyase-encoding gene sequence further confirmed the identity of the isolates. Some epidemiological aspects of the pathogen and the main measures to prevent and control the disease are reported.

First Report of Pectobacterium carotovorum subsp. carotovorum on Spathiphyllum wallisii in Argentina.

Peace lily (Spathiphyllum wallisii Regel) is a popular ornamental potted plant in Argentina. During May of 2008 (austral autumn), necrotic lesions of unknown etiology were observed on S. wallisii in a nursery in Pontevedra (34°45'6″S, 58°42'42″W). Plants first showed water-soaked areas starting from the leaf tips. Infected tissue became irregular, brown, dark-to-black lesions on leaves ~12 to 14 mm in diameter surrounded by yellowish haloes. Disease incidence approached 30%. Abundant bacterial streaming was observed from lesions when examined at ×100. Bacteria isolated from lesions formed white-to-cream, glistening, convex colonies on yeast dextrose calcium carbonate agar. Three bacterial strains isolated from different symptomatic plants were selected for comparative analysis with Pectobacterium carotovorum subsp. carotovorum type strain ATCC 15713. All were facultatively, anaerobic, gram-negative rods, pectolytic on crystal violet pectate agar, nonfluorescent on King's medium B, and elicited a hypersensitive response in tobacco plants. All strains were oxidase and arginine dihydrolase negative, fermented glucose, did not hydrolyze starch, did not produce lecithinase, indole or the blue pigment indigoidine, reduced nitrates, hydrolyzed gelatin and esculin, able to rot onion slices, caused soft rot of potato tubers, resistant to erythromycin, and grew at 37°C. Acid was produced from cellobiose, d-glucose, d-melibiose, d-mannitol, d-mannose, l-rhamnose, d-sucrose, and l-arabinose but not from inositol and d-sorbitol. Bacteria utilized N-acetyl-glucosamine and citrate but not tartrate, benzoate, or propionate. Their identity was confirmed by 16S rRNA gene sequencing of strain F402Pcc (GenBank Accession No. FJ717337) showing a 99% homology with that of strain ATCC 3326 (FJ 5958691). Pathogenicity was verified on S. wallisii, Dieffenbachia picta, Aglaonema commutatum, and Anthurium andraeanum within the Araceae family by spraying two plants per strain tested with bacterial suspensions (108 CFU/ml) in sterile distilled water with and without wounding the leaves with sterile needles. Controls were sprayed with sterile distilled water. After 48 h in a humidity chamber, inoculated plants and controls were maintained at 25 ± 3°C in a greenhouse. Water-soaked areas developed from 24 to 48 h after inoculation and became necrotic within 4 to 5 days. Lesions expanded to resemble natural infection in S. wallisii within 20 days, while in the rest of the hosts tested, lesions were smaller and remained brown surrounded by yellowish haloes. All strains were reisolated from each host tested. The original and all reisolated strains were compared by enterobacterial repetitive intergeneric consensus-PCR (4) confirming that DNA fingerprints of the reisolated strains were identical to those of the original strains. No lesions were observed on controls. The pathogen was identified as P. carotovorum subsp. carotovorum based on biochemical, physiological, pathogenicity tests, and 16S rRNA sequencing (1-3).To our knowledge, this is the first report of this pathogen on S. wallisii in Argentina although it has been reported as causing tomato pith necrosis (1) and soft rot of vegetables after harvest (3).

First Report of Bacterial Stem Rot Caused by Pectobacterium carotovorum subsp. carotovorum and P. carotovorum subsp. atrosepticum on Grafted Eggplant in Italy.

In December 1999, widespread dieback of eggplant (Solanum melongena L., hybrid Mission Bell), grafted onto the interspecific hybrid Beaufort (Lycopersicon esculentum × Lycopersicon hirsutum) and on tomato hybrid Energy, was observed during four rootstock evaluation trials in greenhouses in Sicily, Italy. Dark brown to black, firm but sometimes fissured lesions, 1 to 20 cm long, were observed just above the grafting point. Water-soaked, soft, dark green lesions that turned brown with age were observed on the upper stem. Extensive discoloration of vascular tissues and, in some cases, breakdown of the pith and stem hollowness occurred. Eggplant seedlings, present in the same experimental trials, did not show any symptoms. From symptomatic tissues, numerous bacterial colonies were obtained on nutrient dextrose agar. Bacteria from purified colonies were gram-negative, oxidase-negative, facultatively anaerobic pectolitic on crystal violet pectate agar, which is nonfluorescent on King's B medium. On the basis of biochemical and physiological tests (1), seven of 10 isolates were identified as Pectobacterium carotovorum subsp. carotovorum (Jones 1901) Hauben et al. 1999, comb. nov. (2), and the remaining three were identified as P. carotovorum subsp. atrosepticum (van Hall 1902) Hauben et al. 1999, comb. nov. (2). Four days after prick inoculation of the stems of eggplant and tomato plants, all isolates caused extensive collapse of stems and internal brown discoloration and hollowness, respectively. In a second pathogenicity test, basil leaves of grafted eggplants and eggplant seedlings were either removed or left intact. These plants were then sprayed with bacterial suspensions (108 CFU/ml) of one isolate of each pathogen. Plants were held at 100% relative humidity or in a low-moisture incubator. After 6 days incubation, stem lesions and vascular discoloration, typical of symptoms observed in greenhouses, were observed on plants held at 100% relative humidity. No symptoms were observed in unwounded plants or plants maintained in low relative humidity. Similar symptoms on grafted eggplant have been attributed to physiological disorders (3). To our knowledge, only P. carotovorum subsp. carotovorum has been reported on eggplant as causing fruit rot.

Occurrence of Bacterial Stem Rot, Caused by Erwinia chrysanthemi, on Field-Grown Tomato in Florida.

In September 1997, wilted 4-week-old tomato (Lycopersicon esculentum Mill.) plants were observed in a commercial production field in St. Lucie County, FL. Closer inspection of affected plants revealed hollow stems and petioles with dark, water-soaked lesions. Diseased tissue was macerated and streaked onto nutrient agar (NA) and crystal violet pectate (CVP) agar. After incubation for 2 days at 30°C, isolates produced pits on the CVP agar. Isolates were transferred onto NA and the incubation and transfer procedure was performed two additional times to obtain pure cultures. Suspensions of bacterial cells were injected into tomato and tobacco leaves to test for a hypersensitive or pathogenic reaction. Isolates produced collapsed necrotic tissue on tomato while no reaction was observed on tobacco. Tests for differentiating species and subspecies in the 'carotovora' group of Erwinia were conducted following the protocol of Dickey and Kelman (1). With known cultures of E. carotovora subsp. carotovora and E. chrysanthemi as controls, the isolate from tomato was determined to function as a facultative anaerobe, utilize asparagine as a sole source of carbon and nitrogen, and give positive reactions for pectate degradation, phosphatase, and growth at 37°C. Known cultures of E. carotovora subsp. carotovora, E. chrysanthemi, and the tomato isolate were grown on trypticase soy broth agar for 24 h at 28°C and their cellular fatty acids derivatized to fatty acid methyl esters (FAMEs). Statistical analyses of FAME profile data (MIDI Microbial Identification System, Newark, DE, version 3.60) identified the tomato isolate as Erwinia chrysanthemi. Pathogenicity was determined by inoculating 50-day-old tomato plants (cv. SunPride) with a suspension of E. chrysanthemi obtained from nutrient broth plates incubated at 24°C for 60 h. Three plants each were inoculated with the E. chrysanthemi identified from tomato, sterile water, and known cultures of E. chrysanthemi and E. carotovora subsp. carotovora by placing a drop at the junction of the petiole and stem and passing a sterile needle through the drop into the stem. Plants were maintained in a greenhouse. Dark, water-soaked cankers were observed on the stems of plants inoculated with E. chrysanthemi, including the tomato isolate and E. carotovora subsp. carotovora, after 7 days. No symptoms were observed on plants inoculated with sterile water. Reisolation of the pathogen and identification was performed with tissue from one of the symptomatic inoculated plants. Analyses of FAMEs confirmed E. chrysanthemi as the causal agent. This is the first report of E. chrysanthemi causing a vascular disease of field-grown tomato in Florida. Reference: (1) R. S. Dickey and A. Kelman. 1988. Pages 44-59 in: Laboratory Guide for Identification of Plant Pathogenic Bacteria. N. W. Schaad, ed. American Phytopathological Society, St. Paul, MN.

Quantitative assessment of tuber contamination by pectolytic erwinia and its possible use in the prediction and control of blackleg

A Most Probable Number Technique based on the use of carbohydrate-free McConkey broth and crystal violet pectate agar for the quantification of tuber-borne pectolytic erwinia has been developed. The population of the organisms on naturally contaminated tubers has been shown to be log-normally distributed. The possibility of using tuber contamination and standards for tuber quality for the prediction and control of blackleg is explored.