First Report of Pectobacterium versatile Causing Aerial Stem Rot of Potato in China

Pectobacterium species cause blackleg, soft rot and stem rot in potato and many other vegetable crops (Charkowski 2015). In July 2020, potato plants showing characteristic symptoms of aerial stem rot were observed in a field (cv. Xisen 6) in Fengning Manchu Autonomous County, Chengde, Hebei Province (North China). The disease incidence in that field (5 ha in size) was more than 50%. Putative pectolytic bacteria were obtained from symptomatic stem tissues (light brown and water-soaked stem sections) by culturing on the crystal violet pectate (CVP) medium. Bacterial colonies producing pits, were restreaked and purified on Luria-Bertani (LB) agar. The isolates causing stem rot were gram negative and rod shaped, negative for oxidase, urease, indole production, gelatin liquefaction and acid production from maltose and D-sorbitol. All isolates were catalase positive, produced acid from lactose, rhamnose, saccharose, raffinose and D-arabinose, and were tolerant to 5% NaCl, and able to utilize citrate. The bacterial gDNA was extracted using the EasyPure Bacteria Genomic DNA Kit (TransGen Biotech). The 16S rDNA region was amplified by PCR using the universal primer pair 27F/1492R and sequenced. Result of the Blastn analysis of the 16S rDNA amplicons (MZ379788, MZ379789) suggested that the isolates FN20111 and FN20121 belonged to the genus Pectobacterium. To determine the species of the stem rot Pectobacterium isolates, multi-locus sequence analysis (MLSA) was performed with six housekeeping genes acnA, gapA, icdA, mdh, proA and rpoS (MZ403781-MZ403792), and phylogenetic tree was reconstructed using RAxML v8.2.12 (https://github.com/stamatak/standard-RAxML). The result of phylogenetic analysis showed that the stem rot Pectobacterium isolates FN20111 and FN20121 clustered with P. versatile (syn. ‘Candidatus Pectobacterium maceratum’) strains CFBP6051T (Portier et al. 2019), SCC1 (Niemi et al. 2017) and F131 (Shirshikov et al. 2018). And the isolates FN20111 and FN20121 were more closely related to the type strain CFBP6051T than to strains SCC1 and F131. Potato seedlings (cv. Xisen 6 and Favorita) were inoculated with the isolates FN20111 and FN20121 by injecting 100 µl of bacterial suspensions (108 CFU·mL-1) into the upper parts of the stems of potato plants, or injected with 100 µl of 0.9% saline solution as control. The seedlings were grown at 28°C and 50% relative humidity. Three days post-inoculation, only the bacteria-inoculated seedlings showed diseased symptoms resembling to those observed in the field. Bacterial colonies were obtained from the infected stems and were identified using the same PCR primers of housekeeping genes as described above, fulfill Koch’s postulates. P. versatile causing soft rot and blackleg on potato plants has been reported in Finland (Niemi et al. 2017), Russia (Shirshikov et al. 2018), Netherlands (Portier et al. 2019), Poland (Waleron et al. 2019) and in New York State (Ma et al. 2021). To our knowledge, this is the first report of P. versatile causing aerial stem rot of potato in China.

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First Report of Pectobacterium polaris Causing Aerial Stem Rot of Potato in China

Plant Disease ◽

10.1094/pdis-06-21-1269-pdn ◽

2021 ◽

Author(s):

Jinhui Wang ◽

Wanxin Han ◽

Zheng Li ◽

Jianing Cheng ◽

Yang Pan ◽

...

Keyword(s):

16S Rdna ◽

Disease Incidence ◽

Housekeeping Genes ◽

Luria Bertani ◽

Stem Rot ◽

Universal Primers ◽

Management Options ◽

First Report ◽

Potato Plants ◽

Aerial Stem

In July 2020, potato plants (cv. Xisen 6) showing characteristic symptoms of aerial stem rot were observed in a field in Fengning Manchu Autonomous County, Chengde, Hebei Province (northern China). The disease incidence in that field (5 ha in size) was more than 50%. Aerial stem rot of potato has increased in prevalence over recent years in Chengde, it can cause significant yield loss on susceptible cultivars such as Xisen 6 and Huangxin 226. Affected stem (light brown and water-soaked stem sections) pieces ca. 0.5 cm in length were surface-sterilized by dipping them in 75% ethanol for one min and then three successive rinses with sterile distilled water. Then, the tissues were soaked in 200 µl 0.9% saline for 20 min. Aliquots (20 μl) of three tenfold dilutions of the tissue specimen soaking solution were plated onto the crystal violet pectate (CVP) medium. The CVP plates were incubated at 28°C for 48 h. Colonies producing pits were restreaked and purified on Luria-Bertani (LB) agar plates. The bacterial gDNA was extracted using the EasyPure Bacteria Genomic DNA Kit (TransGen Biotech, Beijing, China). The 16S rDNA region was amplified by PCR using the universal primers 27F/1492R (Weisburg et al. 1991) and sequenced. Results of the Blastn analysis of the 16S rDNA amplicons (MZ348607, MZ348608) suggested that the isolates FN20211 and FN20222 belonged to the genus Pectobacterium. Housekeeping genes including acnA, gapA, icdA, mdh, proA and rpoS were also amplified using a set of primers (Ma et al. 2007; Waleron et al. 2008) followed by sequencing (MZ356250-MZ356261). To determine the species of the stem rot Pectobacterium isolates, multi-locus sequence analysis (MLSA) was performed with six housekeeping genes, and phylogenetic tree was reconstructed using RAxML (github.com/stamatak/standard-RAxML). No sequence variation was observed at any MLSA locus between FN20211 and FN20222. The result of phylogenetic analysis showed that the isolates clustered with P. polaris type strain NIBIO1006T, which was isolated from potato (Dees et al. 2017). And the concatenated sequence of the six loci of isolate FN20211/FN20222 is 100% identical to those of the strains PZ1 (CP046377.1) and WBC1 (GCF_011378945.1), which were isolated from potato in South Korea and from Chinese cabbage in China, respectively. Potato seedlings (cv. Xisen 6 and Favorita) were inoculated with the isolates FN20211 and FN20222 by injecting 100 µl of bacterial suspensions (108 CFU·mL-1) into the upper parts of the stems of potato plants, or injected with 100 µl of 0.9% saline as control. The seedlings were grown at 25°C and 50% relative humidity. Three days after inoculation, only the bacteria-inoculated seedlings showed disease symptoms resembling to those observed in the field. Bacterial colonies were obtained from the infected stems and were identified using the same PCR primers as described above. Therefore, P. polaris isolates FN20211 and FN20222 fulfill Koch’s postulates for aerial stem rot of potato. P. polaris causing blackleg and soft rot on potato plants has been reported in European countries including Netherlands, Norway (Dees et al. 2017) and Poland (Waleron et al. 2019), and also in Pakistan (Sarfraz et al. 2019) and Russia (Voronina et al. 2021). To our knowledge, this is the first report of P. polaris causing aerial stem rot of potato in China. The stem rot poses a significant threat to the local potato industry, and further research on epidemiology and disease management options is needed.

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First Report of Rhizome Rot Caused by Pectobacterium brasiliense on Ginger in China

Plant Disease ◽

10.1094/pdis-10-21-2324-pdn ◽

2022 ◽

Author(s):

Jinhui Wang ◽

Yuxiang Lu ◽

Wanxin Han ◽

Lijun Fu ◽

Xiaoqing Han ◽

...

Keyword(s):

16S Rdna ◽

Disease Incidence ◽

Housekeeping Genes ◽

Zingiber Officinale ◽

Soft Rot ◽

Management Options ◽

Universal Primer ◽

First Report ◽

Wide Range ◽

Rhizome Rot

In August 2020, ginger (Zingiber officinale) rhizomes (cv. Mianjiang) showing soft rot symptoms were observed in a field in Tayang Village, Fengrun District, Tangshan, Hebei Province (North China). The disease incidence in that field (15 ha in size) was more than 20%. Symptomatic rhizomes (brown and water-soaked) were surface-sterilized in 75% ethanol for 60 sec and then three successive rinses with sterile distilled water. Rhizomes were cut into pieces ca. 0.5 cm in length, and then were soaked in 500 µl 0.9% saline for 20 min. Aliquots (20 μl) of three tenfold dilutions of the tissue specimen soaking solution were plated onto the lysogeny broth (LB) medium. And LB plates were incubated at 28°C for 24 h. Five single colonies were picked from each LB plate and restreaked three times for purity. Endophytic bacteria were also isolated from asymptomatic rhizomes as control. The bacterial gDNA was extracted using the EasyPure Bacteria Genomic DNA Kit (TransGen Biotech, Beijing, China). The 16S rDNA region was amplified by PCR using the universal primer pair 27F/1492R (Weisburg et al. 1991) and sequenced. The results of BLASTN against NCBI nr of the 16S rDNA amplicons suggested that the most isolates (8/10) obtained from the rotten rhizomes belonged to the genus Pectobacterium, and few isolates (2/10) were Enterobacter spp.. Only Enterobacter spp. were isolated from asymptomatic rhizomes. Since all Pectobacterium isolates showed identical 16S rDNA sequence, thus, only two isolates were selected for further analysis. Pectobacterium isolates TS20HJ1 and TS20HJ2 (MZ853520, MZ853521) represent isolates from two plant individuals. To determine the species of the rhizome rot Pectobacterium isolates, multi-locus sequence analysis (MLSA) was performed with five housekeeping genes acnA, icdA, mdh, proA and rpoS (MZ994717-MZ994726) (Ma et al. 2007; Waleron et al. 2008), and a phylogenetic tree was reconstructed using RAxML v8.2.12 (github.com/stamatak/standard-RAxML). No sequence variation was observed at any MLSA locus between the two isolates. The result of phylogenetic analysis showed that the ginger rhizome isolates clustered with P. brasiliense type strain IBSBF1692T (Duarte et al. 2004; Nabhan et al. 2012). Ginger seedlings (cv. Mianjiang) were inoculated with the isolate TS20HJ1 by injecting 10 µl of bacterial suspensions (108 CFU·mL-1) into the rhizomes, or injected with 10 µl of 0.9% saline solution as control. The seedlings were grown at 28°C and 50% relative humidity. Ten days after inoculation, only the bacteria-inoculated rhizomes showed diseased symptoms resembling to those observed in the field. Bacterial colonies were obtained from the infected rhizomes and were identified with MLSA gene sequencing, fulfilling Koch’s postulates. P. brasiliense causes soft rot of a wide range of economically important crops (Oulghazi et al. 2021). To our knowledge, this is the first report of P. brasiliense causing rhizome rot of ginger in China. The rhizome rot caused 20-25% yield loss on average in Tangshan region in 2020, which poses a significant threat to the local ginger farming. Further research on epidemiology and disease management options is needed.

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First Report of Fusarium asiaticum Causing Stem Rot of Ligusticum chuanxiong in China

Plant Disease ◽

10.1094/pdis-05-21-1026-pdn ◽

2021 ◽

Author(s):

Tingting Zhu ◽

Linxuan Li ◽

Antonios Petridis ◽

George Xydis ◽

Maozhi Ren

Keyword(s):

Disease Incidence ◽

Elongation Factor ◽

Stem Rot ◽

Post Inoculation ◽

Pathogenicity Test ◽

First Report ◽

Ligusticum Chuanxiong ◽

Fusarium Asiaticum ◽

Sterile Needle ◽

Yellow Pigments

Ligusticum chuanxiong (known as Chuanxiong in China) is a traditional edible-medicinal herb, which has been playing important roles in fighting against COVID-19 (Ma et al. 2020). In March 2021, we investigated stem rot of Chuanxiong in six adjacent fields (~100 ha) in Chengdu, Sichuan Province, China. The disease incidence was above 5% in each field. Symptomatic plants showed stem rot, watersoaked lesions, and blackening with white hyphae present on the stems. Twelve symptomatic Chuanxiong plants (2 plants/field) were sampled. Diseased tissues from the margins of necrotic lesions were surface sterilized in 75% ethanol for 45 s, and 2% NaClO for 5 min. Samples were then rinsed three times in sterile distilled water and cultured on potato dextrose agar (PDA) at 25ºC for 72 h. Fourteen fungal cultures were isolated from 18 diseased tissues, of which eight monosporic isolates showed uniform characteristics. The eight fungal isolates showed fluffy white aerial mycelia and produced yellow pigments with age. Mung bean broth was used to induce sporulation. Macroconidia were sickle-shaped, slender, 3- to 5-septate, and averaged 50 to 70 μm in length. Based on morphological features of colonies and conidia, the isolates were tentatively identified as Fusarium spp. (Leslie and Summerell 2006). To identify the species, the partial translation elongation factor 1 alpha (TEF1-α) gene was amplified and sequenced (O’Donnell et al. 1998). TEF1-α sequences of LCSR01, LCSR02 and LCSR05 isolates (GenBank nos. MZ169386, MZ169388 and MZ169387) were 100%, 99.72% and 99.86% identical to that of F. asiaticum strain NRRL 26156, respectively. The phylogenetic tree based on TEF1-α sequences showed these isolates clustered with F. asiaticum using Neighbor-Joining algorithm. Furthermore, these isolates were identified using the specific primer pair Fg16 F/R (Nicholson et al. 1998). The results showed these isolates (GenBank nos. MZ164938, MZ164939 and MZ164940) were 100% identical to F. asiaticum NRRL 26156. Pathogenicity test of the isolate LCSR01 was conducted on Chuanxiong. After wounding Chuanxiong stalks and rhizomes with a sterile needle, the wounds were inoculated with mycelia PDA plugs. A total of 30 Chuanxiong rhizomes and stalks were inoculated with mycelia PDA plugs, and five mock-inoculated Chuanxiong rhizomes and stalks served as controls. After inoculation, the stalks and rhizomes were kept in a moist chamber at 25°C in the dark. At 8 days post inoculation (dpi), all inoculated stalks and rhizomes exhibited water-soaked and blackened lesions. At 10 dpi, the stalks turned soft and decayed, and abundant hyphae grew on the exterior of infected plants, similar to those observed in the field. No disease symptoms were observed on the control plants. The pathogen was re-isolated from the inoculated tissues and the identity was confirmed as described above. Ten fungal cultures were re-isolated from the 10 inoculated tissues, of which nine fungal cultures were F. asiaticum, fulfilling Koch’s postulates. To our knowledge, this is the first report of F. asiaticum causing stem rot of Chuanxiong in China. Chuanxiong has been cultivated in rotation with rice over multiple years. This rotation may have played a role in the increase in inoculum density in soil and stem rot epidemics in Chuanxiong. Diseased Chuanxiong may be contaminated with the mycotoxins produced by F. asciaticum, 3-acetyldeoxynivalenol or nivalenol, which may deleteriously affect human health. Therefore, crop rotations should be considered carefully to reduce disease impacts.

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First Report of Pectobacterium aroidearum Causing Soft Rot in Olecranon Honey Peach (Prunus persica L.) in China

Plant Disease ◽

10.1094/pdis-10-21-2238-pdn ◽

2021 ◽

Author(s):

Zhibin Liang ◽

Huidi Liu ◽

Zeling Xu ◽

Lian-hui Zhang

Keyword(s):

16S Rdna ◽

Dna Sequences ◽

Prunus Persica ◽

Bacterial Species ◽

Guangdong Province ◽

Housekeeping Genes ◽

Soft Rot ◽

Accession Number ◽

First Report ◽

Conserved Region

Olecranon honey peach (Prunus persica L.) is a popular fruit tree cultivated in Guangdong Province of China. Due to its excellent economic values and popularity, it has recently been widely adopted and planted in several other southern Provinces and Autonomous Region in China, including Yunnan, Hunan, Jiangxi, Guizhou, and Guangxi. In Lianping County of Guangdong Province alone, the annual peach fruit production was about 78,800 tonnes (Xie et al. 2017). In July 2021, peach fruits showing soft rot symptoms were collected from an olecranon honey peach plantation in Lechang, Guangdong, China. Symptoms included tissue disintegration with bacterial oozes and rotting smells. To isolate the causal agent of soft rot in the peach fruits, the bacterial oozes from various rotted fruits were streaked on the modified YEB agar plate (Huang et al. 2021), and 21 bacterial colonies were selected for PCR amplification using the primers targeting the conserved region of 16S rDNA gene (Wei et al. 2020). A blastN analysis of the DNA sequences of the obtained PCR fragments in NCBI website indicated that 17 isolates named as ZL strains were potential bacterial species of Pectobacterium with about 99% similarity (Genbank accession number of ZL1: OK189602) to Pectobacterium aroidearum SCRI 109T (Genbank accession number: NR_159926). Three of them (ZL1, ZL2 and ZL3) were selected for assay of pathogenicity. The bacterial suspensions (10 μl, 1×106 CFU/ml) of strains ZL1, ZL2 and ZL3 were injected into olecranon honey peach fruits by using a syringe. A portion of peach fruits were similarly injected with sterile distilled water as the negative control. After 18 h incubation at 25 °C, the typical symptom of soft rot, i.e., tissue decay, became visible on the peach fruits inoculated with the bacterial suspensions. After inoculation for 42 h, bacterial oozes were exuded from rotting tissues. Peach fruits without injuries were also sprayed with the bacterial suspensions under the same conditions, but decay symptoms were not observed, suggesting that the bacterial infection needs the wounding or injuries. To fulfill the Koch’s postulates, bacterial colonies were re-isolated from bacterial oozes, and their conserved region of 16S rDNA fragments were amplified and sequenced. Bioinformatics analysis of the DNA sequence data confirmed that all the isolated colonies were Pectobacterium strains. Using the Biolog Gen III system, the representative strain ZL1 was identified as Pectobacterium (SIM 0.56). Transmission electron microscopy analysis showed that the bacterial cells of strain ZL1 were rod-shaped with peripheral flagella. To further determine the species of ZL strains, eight housekeeping genes (acnA, gapA, icd, mdh, mtlD, pgi, proA and rpoS) were analyzed by the methods described previously (Nabhan et al. 2013). The amplified DNA sequences analyzed by the blastN program in NCBI showed that the sequences of eight housekeeping genes from strains ZL1, ZL2 and ZL3 were identical to each other (Genbank accession number: OK274248 to OK274255), and most of the gene sequences shared over 99% similarity to their counterparts in P. aroidearum L6 (Genbank accession number: NZ_CP065044) (Xu et al. 2021), except that the acnA and proA genes showed about 98% and 96% similarity respectively to the corresponding genes of P. aroidearum L6. In addition, the multi-locus sequence analysis (MLSA) using DNA sequences of above eight housekeeping genes showed that ZL strains were grouped with other P. aroidearum strains. Taken together, the results of molecular and biochemical assays confirmed that ZL strains isolated from olecranon honey peach fruits were P. aroidearum. To our knowledge, this is the first report of P. aroidearum causing soft rot disease in olecranon honey peach in China. P. aroidearum is a relatively newly described soft rot pathogen (Nabhan et al. 2013). More recently, the pathogen was found causing soft rot infections in lettuce, Chinese cabbage, pepper (Capsicum annuum) fruits, konjac, carrot and Syngonium podophyllum (Barroso et al. 2019; Moraes et al. 2020; Sun et al. 2019; Tang et al. 2020; Xu et al. 2021). The results of this study add a new plant species to the host range of P. aroidearum.

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

Plant Disease ◽

10.1094/pdis-12-21-2731-pdn ◽

2022 ◽

Author(s):

Utpal Handique ◽

Yaning Cao ◽

Dekang Wang ◽

Ruofang Zhang ◽

Wensi Li ◽

...

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Housekeeping Genes ◽

Soft Rot ◽

Rrna Gene ◽

Pectobacterium Carotovorum ◽

Fujian Province ◽

First Report ◽

Potato Plants ◽

Carotovorum Subsp

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.

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First Report of Potato Blackleg Disease Caused by Pectobacterium atrosepticum in Guangdong China

Plant Disease ◽

10.1094/pdis-03-13-0275-pdn ◽

2013 ◽

Vol 97 (12) ◽

pp. 1652-1652

Author(s):

X. M. She ◽

Z. F. He ◽

Y. F. Tang ◽

Z. G. Du ◽

G. B. Lan

Keyword(s):

16S Rdna ◽

Vascular Tissue ◽

Guangdong Province ◽

Bacterial Suspension ◽

Post Inoculation ◽

Blackleg Disease ◽

Potato Blackleg ◽

Pectobacterium Atrosepticum ◽

First Report ◽

Potato Plants

Potato (Solanum tuberosum L.) is an important crop in China. In 2013, diseased potatoes exhibiting blackleg and soft rot symptoms were found in the winter potato growing areas of Huizhou city, Guangdong Province, China, with an incidence of approximately 20%. Initially, the stem bases of infected plants blackened and this symptom spread upward. Later, foliage of the diseased plants became yellow and the stem rotted with vascular discoloration. Twenty diseased plants with typical black leg symptoms were collected from a 10-ha potato field with approximately 60,000 potato plants per hectare. A bacterium with small, irregular, round, fluidal, white colonies was isolated from the vascular tissue of all diseased plants on nutrient agar at 26°C for 2 days. Ten strains were randomly selected for pathogenicity assays. Potato plants (cv. Favorita) at the five- to six-leaf stage were inoculated by injecting their stems with 1 ml of each strain in a bacterial suspension (3 × 108 CFU/ml). The inoculated potato plants were incubated at 16 to 21°C and 65 to 85% humidity, and exhibited the same symptoms as the diseased potato plants in the field by 3 to 5 days post inoculation (dpi). The bacterium was reisolated from the diseased tissue (stem) of the inoculated potato plants and produced characteristic pits on crystal violet pectate medium (1). The bacterium utilized a-methyl glucoside, glucose, lactose, maltose, cellobiose, raffinose, melibiose, and citrate, but not d-arabitol, sorbitol, or malonate. The bacteria also gave a positive reaction for catalase and production of reducing substances from sucrose, but gave a negative reaction for oxidase, production of phosphatase, and indole. Using the universal bacterial 16S rDNA primer set, 27f/1541R (4), 1,400-bp fragments were amplified from the 10 strains. The sequences of the 10 fragments (GenBank Accessions KC695819 to KC695828) were identical and had 100% sequence identity with 16S rDNA of Pectobacterium atrosepticum CFBP 1526 (JN600332). Further, the 438-bp and 690-bp fragments were respectively amplified from all 10 strains with the P. atrosepticum-specific primers Y45/Y46 (3) and ECA1f/ECA2r (2). To our knowledge, this is the first report of potato blackleg disease caused by P. atrosepticum (formerly named as Erwinia carotovora subsp. atroseptica) in Guangdong Province, China. References: (1) D. Cupples et al. Phytopathology 64:468, 1974. (2) S. H. De Boer et al. Phytopathology 85:854, 1995. (3) D. Frenchon et al. Potato Research 41:63, 1995. (4) M. Horita et al. J. Gen. Plant Pathol. 70:278, 2004.

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First Report of Stalk Rot of Celery Caused by Erwinia rhapontici in China

Plant Disease ◽

10.1094/pdis-07-21-1364-pdn ◽

2021 ◽

Author(s):

Jinhui Wang ◽

Wanxin Han ◽

Yang Pan ◽

Aiguo Guo ◽

Dai Zhang ◽

...

Keyword(s):

16S Rdna ◽

Prunus Persica ◽

Housekeeping Genes ◽

Soft Rot ◽

Pcr Primers ◽

Apium Graveolens ◽

Management Options ◽

Disease Symptoms ◽

First Report ◽

Stalk Rot

Species belonging to the genus Erwinia cause diseases in many economically important plants (Mansfield et al. 2012). In May 2021, celery plants (Apium graveolens var. dulce) showing soft rot symptoms were observed in greenhouses (cv. Queen of France) in Boye County, Baoding, Hebei Province (North China). Disease symptoms began with pinkish water-soaked lesions on the midrib of celery stalks, but at the same time the leaves and root did not show symptoms. The infected celery plants rapidly developed brownish rotten stalks and leaves turned dry and yellow, but root remained asymptomatic. The disease incidence in two greenhouses (0.15 ha in size) was more than 50%. Affected celery stalk tissues were cut into 0.5 cm pieces, followed by surface sterilization using 75% ethanol for 60 sec and then three successive rinses with sterile distilled water. Then, the tissues were immersed in 200 µl 0.9% saline for 15 min. Aliquots of two tenfold dilutions of the tissue specimen soaking solution were plated onto Luria-Bertani (LB) agar plates and incubated at 28°C for 24 h. Single colonies were picked and restreaked onto LB agar three times for purity. The bacterial gDNA was extracted using the EasyPure Bacteria Genomic DNA Kit (TransGen Biotech). The 16S rDNA region was amplified by PCR using the universal primers 27F/1492R and sequenced. Result of blastn analysis of the 16S rDNA amplicons (MZ489246-MZ489247) indicated that the bacterial isolates (BY21311 and BY21312) belonged to the genus Erwinia. Biolog analysis (GEN III Microplate) identified the two isolates BY21311 (SIM=0.668) and BY21312 (SIM=0.638) as E. rhapontici. Housekeeping genes including acnA, gapA, icdA, mdh and rpoS were also amplified using a set of PCR primers (Ma et al. 2007; Waleron et al. 2008) followed by sequencing (MZ463029-MZ463038). To determine the species of the Erwinia isolates BY21311 and BY21312, multi-locus sequence analysis (MLSA) was performed with five housekeeping genes, and phylogenetic tree was reconstructed using RAxML v8.2.12 (Stamatakis et al. 2005). No sequence variation was observed at any MLSA locus between BY21311 and BY21312. The result of phylogenetic analysis showed that the celery stalk rot isolates BY21311 and BY21312 were clustered with E. rhapontici isolates. These celery isolates are closely related to the cabbage (Brassica rapa) isolate MAFF311153 (AP024329.1) in Japan. When celery plants have eight to nine true leaves, plants (cv. Queen of France) were inoculated with the isolate BY21311 by injecting 20 µl of bacterial suspensions (106 CFU·mL-1) into the celery stalks, or injected with 20 µl of 0.9% saline as control. The seedlings were grown at 25 °C and 50% relative humidity. Three days after inoculation, only infected seedling showed disease symptoms resembled to those observed in greenhouses. Bacterial colonies were obtained from the infected stalks and were identified using the same PCR primers of housekeeping genes as described above, fulfill Koch’s postulates. E. rhapontici has been reported to cause pink seed, crown and stem rot, soft rot or leaf spot on many plant hosts including pea (Pisum sativum), chickpea (Cicer arietinum), lentil (Lens culinaris), common bean (Phaseolus vulgaris), lucerne (Medicago sativa), wheat (Triticum aestivum), hyacinth (Hyacinthus orientalis), onion (Allium cepa), kiwifruit (Actinidia chinensis) and peach (Prunus persica) (Huang et al. 2003; Wang et al. 2017; Zhang et al. 2018; Kovács et al. 2020). To our knowledge, this is the first report of E. rhapontici causing stalk rot in celery. Stalk rot of celery has increased in prevalence over recent years in the Baoding region, it can cause significant yield loss and no cultivar has been found to be resistant to this disease so far. The stalk rot poses significant threat to local celery production, and further research on epidemiology and disease management options is needed.

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First Report of Erwinia carotovora subsp. carotovora Causing Bacterial Root Rot of Sweetpotato (Ipomoea batatas) in Louisiana

Plant Disease ◽

10.1094/pdis.1998.82.1.129a ◽

1998 ◽

Vol 82 (1) ◽

pp. 129-129 ◽

Cited By ~ 4

Author(s):

C. A. Clark ◽

M. W. Hoy ◽

J. P. Bond ◽

C. Chen ◽

Y.-K. Goh ◽

...

Keyword(s):

Root Rot ◽

Ipomoea Batatas ◽

Disease Incidence ◽

Erwinia Carotovora ◽

Soft Rot ◽

Stem Rot ◽

Identification System ◽

Natural Occurrence ◽

Storage Roots ◽

First Report

Bacterial root and stem rot of sweetpotato (Ipomoea batatas (L.) Lam.) was first fully characterized in the U.S. in 1977 (2). It was thought to be caused exclusively by Erwinia chrysanthemi. Although a previous report described sweetpotato as a host for E. carotovora subsp. carotovora, based on artificial inoculations, others have reported that neither E. carotovora subsp. carotovora nor E. carotovora subsp. atroseptica decay sweetpotato storage roots (1). In October 1995, storage roots of sweetpotato cv. Beauregard were received from St. Landry Parish, LA, that displayed typical bacterial root rot. Isolations from these roots yielded bacteria that showed a similarity of 0.945 to E. carotovora subsp. carotovora with the Biolog GN Bacterial Identification System (version 3.50). This isolate (Ecc-LH) also differed from isolates of E. chrysanthemi (Ech) from sweetpotato and other hosts in that it was insensitive to erythromycin, did not produce phosphatase or lecithinase, and did not produce gas from glucose. Ecc-LH differed from known strains of E. carotovora subsp. atroseptica in that it did not produce reducing substances from sucrose or acid from palatinose. When Beauregard storage roots were inoculated by inserting micropipette tips containing 50 μl of 1.0 × 108 CFU/ml, both Ecc-LH and Ech-48 produced typical bacterial root rot symptoms. However, when they were compared by infectivity titrations at 28 to 32°C, Ecc-LH was less virulent than Ech-48. Ecc-LH had an ED50 of approximately 1.0 × 106 CFU/ml and did not cause appreciable disease below inoculum concentrations of 1.0 × 105, whereas Ech-48 had an ED50 of approximately 1.0 × 108 and caused soft rot at the lowest concentration tested, 1.0 × 103. Similar disease incidence was observed in infectivity titrations at 22 to 24°C, but Ech-48 caused less severe soft rot. E. carotovora subsp. carotovora was reisolated from inoculated storage roots and its identity was reconfirmed by Biolog. When terminal vine cuttings of Beauregard were dipped in 1.0 × 108 CFU/ml and planted in a greenhouse, bacterial stem rot symptoms developed on plants inoculated with Ech-48 at about 4 weeks postinoculation, or when new growth began. However, no symptoms developed on plants inoculated with Ecc-LH. This is the first report of natural occurrence of E. carotovora subsp. carotovora causing bacterial root rot of sweetpotato in Louisiana. E. chrysanthemi remains the most important pathogen causing bacterial soft rot in sweetpotato since it is widely associated with sweetpotato, is more virulent on storage roots and also causes a stem rot. E. carotovora subsp. carotovora can cause root rot, but has been isolated in only one location to date, is less virulent on storage roots, and apparently does not cause stem rot on the predominant cultivar in U.S. sweetpotato production, Beauregard. References: (1) C. A. Clark and J. W. Moyer. 1988. Compendium of Sweet Potato Diseases. American Phytopathological Society, St. Paul, MN. (2) N. W. Schaad and D. Brenner. Phytopathology 67:302, 1977.

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First Report of Erwinia persicina Causing Stalk Rot of Celery in China

Plant Disease ◽

10.1094/pdis-08-21-1723-pdn ◽

2021 ◽

Author(s):

Jinhui Wang ◽

Wanxin Han ◽

Yang Pan ◽

Dai Zhang ◽

Dongmei Zhao ◽

...

Keyword(s):

16S Rdna ◽

Acid Production ◽

Housekeeping Genes ◽

Soft Rot ◽

Pcr Primers ◽

Water Soluble ◽

Produce Water ◽

Disease Symptoms ◽

First Report ◽

Stalk Rot

Species belonging to the genus Erwinia cause diseases in many economically important plants. In May 2021, celery (Apium graveolens var. dulce) plants (cv. Queen of France) showing soft rot symptoms were observed in greenhouses in Boye County, Baoding, Hebei Province (North China). Disease symptoms began with pinkish water-soaked lesions on the midrib of celery stalks, but at the same time the leaves and root were asymptomatic; and the infected celery plants rapidly developed brownish rotten stalks. The disease incidence in two greenhouses (0.15 ha in size) was more than 50%. Affected celery stalk pieces ca. 0.5 cm in length were surface-sterilized by dipping them in 75% ethanol for one min and then three successive rinses with sterile distilled water. Then, the tissues were immersed in 200 µl 0.9% saline for 15 min. Aliquots (20 μl) of two tenfold dilutions of the tissue specimen soaking solution were plated onto Luria-Bertani (LB) medium and incubated at 28°C for 24 h. Single colonies were picked and restreaked onto LB agar three times for purity. The bacterial gDNA was extracted using the EasyPure Bacteria Genomic DNA Kit (TransGen Biotech, Beijing, China). The 16S rDNA region was amplified by PCR using the universal primers 27F/1492R and sequenced. Result of blastn analysis of the 16S rDNA amplicons (MZ614654, MZ614655) indicated that the bacterial isolates (BY21211 and BY21212) belonged to the genus Erwinia. Housekeeping genes including mdh, gapA, icdA, rpoS, acnA and proA were also amplified using a set of PCR primers (Ma et al. 2007) followed by sequencing (MZ643221-MZ643232). No sequence variation was observed at any MLSA locus between isolates BY21211 and BY21212. To determine the species of the Erwinia isolate BY21211 and BY21212, multi-locus sequence analysis (MLSA) was performed with six housekeeping genes, and phylogenetic tree was reconstructed using RAxML v8.2.12 (github.com/stamatak/standard-RAxML). The result of phylogenetic analysis showed that the celery stalk rot isolate BY21211 and BY21212 was clustered with E. persicina type strain NBRC102418T (Hao et al. 1990). When celery plants (cv. Queen of France) have eight to nine true leaves, plants were inoculated with the isolate BY21211 by injecting 20 µl of bacterial suspensions (107 CFU·mL-1) into the celery stalks, and negative controls were injected with 20 µl of 0.9% saline. The seedlings were grown at 25°C and 50% relative humidity. Three days after inoculation, only the bacterial-inoculated seedlings showed disease symptoms resembled to those observed in greenhouses. Bacterial colonies were obtained from the infected stalks and were identified using the same PCR primers of housekeeping genes as described above. Therefore, E. persicina isolate BY21211 fulfill Koch’s postulates for stalk rot of celery. Isolates BY21211 and BY21212 produce water-soluble pink pigment on sucrose-peptone agar. These isolates were gram negative and rod shaped, negative for oxidase, urease, indole production, gelatin liquefaction and acid production from xylose and glycerol. They were positive for catalase, citrate utilization, acid production from sorbitol, raffinose, glucose, arabinose, cellobiose, rhamnose, maltose, saccharose, inositol, lactose and esculin (hydrolysis). E. persicina has been reported to cause pink seed, crown and stem rot, soft rot or leaf spot on many plant hosts including pea (Pisum sativum), soybean (Glycine max), common bean (Phaseolus vulgaris), lucerne (Medicago sativa), barley (Hordeum vulgare), onion (Allium cepa), garlic bulbs (Allium sativum), tomato (Lycopersicon esculentum) and cucumber (Cucumis sativus) (Hao et al. 1990; González et al. 2005, 2007; Zhang and Nan 2014; Gálvez et al. 2015; Cho et al. 2019; Kawaguchi et al. 2021). To our knowledge, this is the first report of E. persicina causing stalk rot in celery. Stalk rot of celery has increased in prevalence over recent years in the Baoding region, it can cause significant yield loss and no cultivar has been found to be resistant to this disease so far. The stalk rot poses significant threat to local celery production, and further research on epidemiology and disease management options is needed.

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First Report of Pectobacterium punjabense causing potato soft rot and blackleg in Serbia

Plant Disease ◽

10.1094/pdis-06-21-1199-pdn ◽

2021 ◽

Author(s):

Marta Loc ◽

Dragana Milošević ◽

Maja Ignjatov ◽

Žarko Ivanović ◽

Dragana Budakov ◽

...

Keyword(s):

16S Rdna ◽

Potato Plant ◽

Soft Rot ◽

Economic Losses ◽

Bacterial Suspension ◽

Distilled Water ◽

Potato Tubers ◽

First Report ◽

Potato Plants ◽

16S Rdna Pcr

Soft rot and blackleg are common diseases affecting potato (Solanum tuberosum) production in Serbia. Pectinolytic plant pathogens belonging to the genera Pectobacterium cause soft rot and wilt diseases by plant cell wall degradation. These opportunistic phytopathogens lead to considerable economic losses in many potato-growing regions worldwide and are listed among top 10 plant pathogenic bacteria (Mansfield et al. 2012). Potato plants (cv. VR808) with symptoms of wilting, slow growth, stem blackening and tubers softening, were collected from a commercial potato field in Zobnatica (Serbia) in July 2019 and subjected to analysis. All symptoms occurred in the same field and the incidence of symptomatic plants was approximately 5%. Isolation was performed from 10 randomly chosen potato plant and tuber samples, expressing wilting and soft rot symptoms. Plant tissue was surface-disinfected and 1 cm length sections from the margins of lesions were macerated in sterile distilled water for 25 min and streaked on nutrient-agar medium. After 48 h of incubation at 26°C, predominant shiny, cream-colored, round colonies were obtained from all samples. Three representative isolates (MMZKVR1, MMZCVR2, and MMZKVR3) from independent samples were selected randomly and subjected to biochemical and pathogenicity tests. Isolates were gram-negative, nonfluorescent facultative anaerobes, exhibiting pectinolytic activity on potato tuber slices and hypersensitive response on tobacco leaves. They expressed catalase activity but did not express oxidase or acid phosphatase activity or produce indole. All strains grew at 37°C, in 5% NaCl, and reduced nitrate. Pathogenicity of the obtained isolates was tested on 3-week-old healthy potato plants (cv. VR808 and cv. Kiebitz) grown in commercial Baltic Tray Substrate (Hawita) in the greenhouse, as well as on potato tubers of the same varieties. Three potato plant stems per isolate were inoculated by the toothpick piercing method (Duarte et al. 2004) using bacterial suspension (approx. 1 × 108 CFU/ml). Inoculated plants were incubated under plastic bags in a greenhouse at 25 ± 2°C. Blackleg symptoms and stem wilting developed 48 hours after inoculation. No symptoms were observed on plants inoculated with sterile toothpicks dipped in sterile distilled water. The pathogen was re-isolated from symptomatic plants, fulfilling Koch's postulates and sequencing of 16S rDNA confirmed the originally isolated pathogen. Three potato tubers per isolate were inoculated by toothpicks dipped in bacterial suspension (approx. 1 × 108 CFU/ml). Inoculated tubers were placed in a sealed plastic container at 25 ± 2°C. Treatment with sterile distilled water was used as a negative control. Softening of the tissue around the inoculation point developed within 48 h from inoculation, and no symptoms developed on the control tubers. For molecular analyses, total DNA of the isolates was extracted using the DNeasy Plant Mini Kit (Qiagen). The isolates were not detected in diagnostic PCR assays using specific primers Br1F/L1R for the detection of P. brasiliense (Duarte et al. 2004) and primers EXPCCF/EXPCCR for P. catotovorum subsp. carotovorum (Kang et al. 2003). The 16S rDNA PCR amplification was performed using the universal PCR primer pair 27F/1492R (Fredriksson et al. 2013) and followed by Sanger sequencing (Macrogen Europe BV). The BLASTn analysis of sequences (GenBank Accession Numbers MZ048661, MZ048662, and MZ157274) revealed 100% query coverage and 100% identity to the sequences of Pectobacterium punjabense in NCBI (MT242589 and CP038498) isolated from potato in China and Pakistan (Sarfraz et al. 2018), respectively. All three obtained isolates were proposed to belong to Pectobacterium punjabense sp. nov. To further validate the identification, isolate MMZCVR2 of P. punjabense was selected for multilocus sequence analyses of 5 housekeeping genes (gyrA, recA, recN, rpoA and rpoS). The gyrA (MZ161817), recA (MZ161818), recN (MZ161819), rpoA (MZ161820) and rpoS (MZ161821) sequence analysis showed the highest nucleotide identity (99.44 to 100%) with P. punjabense strain SS95 (Sarfraz et al. 2018) previously deposited in NCBI GenBank database. To our knowledge, this is the first report of blackleg and soft rot caused by P. punjabense on potato in Serbia. Pectobacterium punjabense is a newly described species causing soft rot and blackleg disease in potato plants (Sarfraz et al. 2018). Its current geographic distribution is not well-described but important to know since soft rot bacteria are easily transported long distances in latently infected seed tubers and can cause significant economic losses in potato production worldwide.

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