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Occurrence and Identification of Pectobacterium carotovorum subsp. brasiliensis and Dickeya dianthicola Causing Blackleg in Some Potato Fields in Serbia
Abstract
Blackleg outbreaks were noticed on three fields (total c. 100 ha) during two consecutive years (2018, 2019) in one of the main potato growing areas in Serbia (Bačka region, Vojvodina). The percentage of infected plants reached 40-70% with 10.5% to 44.7% yield reductions. From the three fields out of 90 samples Pectobacterium carotovorum subsp. brasiliensis was most frequently identified and diagnosed as causal agent of potato blackleg in Serbia for the first time (29 isolates). Dickeya dianthicola was a less frequently causative bacterium, which was also noticed for the first time (nine isolates). A total of 38 isolates were characterized based on their phenotypic and genetic features, including a pathogenicity test on potato. The repetitive element Polymerase Chain Reaction (rep-PCR) using BOX, REP and ERIC primer pairs differentiated five genetic profiles among 38 tested isolates. Multilocus sequence analysis (MLSA) of four housekeeping genes, acnA, gapA, icdA and mdh, revealed the presence of three so far unknown P. c. subsp. brasiliensis multilocus genotypes and confirmed clustering into two main genetic clades as determined in other studies. MLSA also revealed the presence of a new genotype of D. dianthicola in Serbia.
... The Population Analysis with Reticulate Trees (PopART) software can use such data as input for the analysis of the available population genetic data and for constructing popular haplotype networks such as Templeton, Crandall, and Sing's (TCS), minimum spanning networks (MSNs), and medianjoining networks (MJNs), thus helping visualize intra-species genealogical relationships as well as advance our knowledge of biogeography and the history of populations [15]. The use of TCS haplotype networks for the determination of phylogeography of other soft-rotcausing bacteria (e.g., P. brasiliense and Dickeya dianthicola) was previously demonstrated in the work performed by Marković et al. [16] on strains isolated from potato in Serbia, based on concatenated sequences of four housekeeping genes (acnA, icdA, gapA, and mdh). Thus far, the TCS algorithm has been successfully applied for providing a phylogeographic insight into the population diversity of bacterial species belonging to different genera, such as Xanthomonas [17,18], Pseudomonas [19], Agrobacterium [20], Ralstonia [21], and Clavibacter [22]. ...
... In Serbia, different Pectobacterium spp.-P. atrosepticum, P. brasiliense, P. carotovorum, P. odoriferum, P. punjabense, P. zantedeschiae, and P. versatile-have been isolated from various hosts [13,16,[23][24][25][26][27][28][29][30]. Three of these species (P. ...
... Its benefit is further increased by the fact that, to the best of our knowledge, this is a pioneering study using the TCS haplotype network for exploring the phylogeography of P. carotovorum, as well as P. versatile and P. odoriferum. Thus far, only P. brasiliense strains from the Pectobacterium genus have been subjected to such analysis, which was based on a different combination of acnA, gapA, icdA, and mdh housekeeping genes [16]. The obtained findings indicate the existence of four haplotypes (PCB-1, PCB-2, PCB-3, and PCB-4) among twenty tested Serbian P. brasiliense strains from potato; however, no connection between the geographic origin and the genetic diversity was established [16]. ...
The aim of this study was to establish a link between genetic diversity and the geographic origin of Pectobacterium strains belonging to three species-P. carotovorum, P. versatile, and P. odor-iferum-isolated from cabbage in Serbia by comparing their sequences with those of strains sourced from different hosts and countries in Europe, Asia, and North America. Phylogeographic relatedness was reconstructed using the Templeton, Crandall, and Sing's (TCS) haplotype network based on concatenated sequences of the housekeeping genes dnaX, icdA, mdh, and proA, while pairwise genetic distances were computed by applying the p-distance model. The obtained TCS haplotype networks indicated the existence of high intra-species genetic diversity among strains of all three species, as reflected in the 0.2-2.3%, 0.2-2.5%, and 0.1-1.7% genetic distance ranges obtained for P. carotovorum, P. versatile, and P. odoriferum, respectively. Five new haplotypes (denoted as HPc1-HPc5) were detected among cabbage strains of P. carotovorum, while one new haplotype was identified for both P. versatile (HPv1) and P. odoriferum (HPo1). None of the TCS haplotype networks provided evidence of significant correlation between geographic origin and the determined haplotypes, i.e., the infection origin. However, as haplotype network results are affected by the availability of sequencing data in public databases for the used genes and the number of analyzed strains, these findings may also be influenced by small sample size.
... Bacterial soft rot is a disease that seriously affects plants, especially vegetables and ornamental plants, and can cause soft rot, blackleg, and wilt [7], such as soft rot of potato [8], blackleg of potato [9], tomato bacterial wilt [10]. Soft rot disease can cause serious economic losses to crops, for example, cucumber soft rot disease can cause 20% to 30% yield loss in China [11], and the percentage of infected potato plants has reached 40% to 70%, with 10.5% to 44.7% yield reductions in Serbia [12]. The bacteria in soft rot attack the plant cell walls by secreting pectin lyase, which causes the maceration of the tissue and gives a foul odor [13]. ...
... brasiliense, as evidenced by the rapid onset of stem and fruit rot, as well as necrosis, within a mere 48 h period post-inoculation. This bacterium, known to be highly virulent, has been found to inflict significant damage on a wide range of crops including potato [12], tomato [42], and pepper [19], among oth- ...
An outbreak of stem rot in eggplants was observed in Heshuo County, Xinjiang, during winter 2021–2022 in about 12–35% of the eggplants in the region (about 40 hm2). The infected tissues yielded a total of four bacterial strains, which were subsequently subjected to physiological and biochemical assays as well as molecular identification. Based on these analyses, the pathogen was identified as Pectobacterium carotovorum subsp. brasiliense. The pathogenicity was confirmed through the fulfillment of Koch’s postulates. The host range test confirmed the broad spectrum of species susceptible to infection by the strains. This study represents the first case of infection caused by P. carotovorum subsp. brasiliense resulting in stem rot in eggplant.
... The Republic of Serbia has areas suitable for seed and ware potato production, both in the main agricultural production areas in the Province of Vojvodina and in the mountainous region [65,66]. However, numerous Pectobacterium species are frequently and increasingly isolated from diseased potato originating from field and storage conditions [40][41][42][43][44]. In the highlight of recent climate changes, stressful environmental conditions that favour pathogen development-drought, followed with more frequent and heavy rainfall or floods, may be the cause of increased number of disease outbreaks and disease severity. ...
... This species has rapidly spread since its first appearance and has largely replaced Dickeya species [11,38]. In the present survey conducted during 2018-2020, no Dickeya species were obtained from any of soft rot and blackleg potato samples, however the presence of Dickeya in Serbian potato fields has recently been recorded [42]. The second most often isolated Pectobacterium species in this study is the rare and newly described pathogen P. punjabense [44] and species P. carotovorum. ...
Pectobacterium is a diverse genus which comprises of multiple destructive bacterial species which cause soft rot/blackleg/wilt disease complex in a wide variety of crops by employing high levels of virulence factors. During the 2018, 2019 and 2020 potato growing seasons, numerous outbreaks of bacterial wilt, stem blackleg and tuber soft rot were recorded, and symptomatic plant samples from ten localities in the Province of Vojvodina (Serbia) were collected and analysed. Bacterial soft-rot pathogens were detected in 63 samples using genus and species-specific primers. Through 16S rRNA Sanger sequencing of 19 representative isolates, the identity of P. brasiliense (73.7%), P. punjabense (15.8%), and P. carotovorum (10.5%) species were revealed. To further validate the identification, genotypic profiling of Pectobacterium strains using rep-PCR (ERIC, BOX, REP) was conducted for 25 selected isolates and the phylogenetic assessment based on four selected housekeeping genes (gyrA, recA, rpoA, and rpoS). Physiological and biochemical properties were analysed using basic microbiological tests and VITEK® 2 GN card, and pathogenicity was confirmed on cv. VR808 and cv. Desiree potato tubers and plants. This study confirmed the distinctiveness of the newly described P. punjabense in Serbia as well as the high diversity of Pectobacterium brasiliense and Pectobacterium carotovorum species in Serbia.
... carotovorum strain Pcc10 [38], and potato in case of strains of P. carotovorum subsp. brasiliensis Pcb62 and D. dianthicola Dd31 [39]. All strains were molecularly identified [38,39]. ...
... brasiliensis Pcb62 and D. dianthicola Dd31 [39]. All strains were molecularly identified [38,39]. For the experiments, strains were grown on Nutrient Agar (NA) for 48 h at 26 • C. The inhibitory effect was evaluated by Agar well diffusion method using holes in the medium [44]. ...
In this work by encapsulation technique we have synthetized three new clove bud essential oil (CEO) Emulsifiable Concentrate (EC) formulations depending on the carrier (synthetic zeolite- F-CSZ, nature zeolite- F-CNZ and gelatin- F-CG). The main idea was to develop an eco-friendly biopesticide that can find use in plant protection as an alternative to the use of conventional pesticides. By encapsulation we wanted to enable water solubility and ensure prolonged efficacy of the essential oil. Biological activity of designed CEO formulations was tested on potato tuber moth Phthorimaea operculella (fumigant mode of action), gray mold fungal pathogen Botrytis cinerea (preserver coatings), and soft rotting bacterial pathogens Pectobacterium carotovorum (subsp. carotovorum and brasiliensis) and Dickeya dianthicola (direct competition). CEO formulations evinced a prolonged action on mortality of P. operculella during the insects’ exposure to the concentration of the emulsions of 40 µL L⁻¹ air. The mortality gradually decreased from a probability of 100% after the first 24 h to 50% after 5 days for F-CSZ or after 4 days for F-CNZ and F-CG. The most promising formulation is F-CSZ enabling activity during 14 days of exposure, while the effect of the other two formulations lasted 10 days. All three formulations produced a strong fungicidal effect against B. cinerea by preventing infection and disease development. The best efficacy was evidenced with F-CSZ (synthetic zeolite as a carrier) showing 100% efficacy when it was used even at the lowest tested concentration of active CEO (1%). The results of in vitro testing against soft rot pathogens determined the MIC value of CEO formulations to be 1% of active CEO. By this research, we present a novel perspective on the use of essential oils as an alternative, environmental biopesticide. CEO formulations can be commercially exploited as a fumigant or preserver coatings to extend the shelf life of stored products or the fresh-fruit market.
... Dickeya dianthicola was first detected causing stunting and slow wilting of Dianthus in the early 1950s in Denmark, then in the Netherlands and the UK (Hellmers 1958;Parkinson et al. 2015). The presence of D. dianthicola strains has been reported on potato or ornamental plants in more than 10 countries, including USA, UK, Belgium, France, Morocco, Netherlands, Pakistan, Switzerland, Israel, Serbia etc. Marković et al. 2020;Nasaruddin et al. 2019;Oulghazi et al. 2017;Rosenzweig et al. 2016;Sarfraz et al. 2018;Toth et al. 2011). ...
... Pectobacteriaceae pathogens Dickeya and Pectobacterium are pectolytic bacteria that cause blackleg and soft rot (BSR) on potato. These pathogens also infect numerous other economically important vegetables, grain and ornamental crops(Adeolu et al. 2016;Dees et al. 2017;Duarte et al. 2004;Ekbataniamiri 2020;Gardan et al. 2003;Khayi et al. 2016;Ma et al. 2007;Parkinson et al. 2014;Perombelon and Kelman 1980;Samson et al. 2005;Sławiak et al. 2009;Tian et al. 2016;van der Wolf et al. 2014).Dickeya dianthicola has been reported in more than 10 countries in the regions of North America, Europe, North Africa, and the Middle East on potato, vegetable or ornamental plantsMarković et al. 2020;Nasaruddin et al. 2019;Oulghazi et al. 2017;Patel et al. 2019;Rosenzweig et al. 2016;Sarfraz et al. 2018;Toth et al. 2011). Dickeya dianthicola and D.solani have emerged in European countries in the past decades and have severely affected the European potato industriesvan der Wolf et al. 2014). ...
Potato blackleg and soft rot (PBSR), which can be caused by Dickeya spp. and Pectobacterium spp., is a serious problem worldwide. The recent outbreak of PBSR in the Northeastern USA, caused primarily by D. dianthicola, has resulted in significant economic losses since 2015. This seedborne disease is highly associated with and therefore spread by seed tuber distribution. To understand how the outbreak occurred and where the pathogen originated, a total of 1204 potato samples were collected from 11 northeastern states from 2015 to 2020. All the samples were processed for bacterial isolation and DNA extraction. Dickeya dianthicola and P. parmentieri were detected using conventional polymerase chain reaction (PCR). Dickeya dianthicola and P. parmentieri were found in 38.1% and 53.3% of the samples, respectively, and 20.6% of samples contained both D. dianthicola and P. parmentieri. Seventeen isolates of D. dianthicola were obtained from the samples and classified into three genotypes (Type I, II, III). Results based on 258 samples showed that Maine mainly had Type I but no Type III, while Type II appeared to be distributed throughout the Northeastern USA. By pan-genomic analysis, D. dianthicola strains collected worldwide were classified into eight distinguished clades. Type I strains had an extraordinarily high homogeneity and distinct discrimination from other countries, indicating a single-strain population. Virulence-related systems, such as plant cell-wall degrading enzymes, flagellar and chemotaxis related features, two-component regulatory system, and type I/II/III secretion systems were highly conserved, but type IV/VI secretion systems varied, in which type I strain had an additional set T4SS cluster, implying more aggressiveness and virulence. Thus, the PBSR outbreak was proposed to be associated with a new strain derived by mutation. Bacterial communities were analyzed on the samples using Illumina sequencing targeting on the V3-V4 region of 16S rRNA gene. Genera Dickeya or Pectobacterium prevailed in the microbial community when they each existed alone, while Dickeya surpassed Pectobacterium when they coexisted. Among the pathogen complex, D. dianthicola was the only species in the Dickeya genus, while species varied in the Pectobacterium genus, with P. parmentieri, P. polaris, P. carotovora subsp. carotovora, P. carotovora subsp. odoriferum being the most prevalent presumptive species. Isolates of the four presumptive species and P. c. subsp. brasiliensis were identified by sequencing the gapA gene and were confirmed to be pathogenic on potatoes. Thus, PBSR was caused by intergeneric or intrageneric species of Dickeya and Pectobacterium that contribute collectively to the disease complex. To further investigate the relationship between the two bacterial species and their interaction, field trials were established. Three varieties of potato seed pieces were inoculated with bacterial suspensions of D. dianthicola and planted in the field. Two-year results showed that there was a significant linear correlation (P < 0.01) between relative yield loss and percentage of inoculated seed pieces. Furthermore, D. dianthicola was more virulent than P. parmentieri in the field, but the co-inoculation of the two species resulted in increased disease severity compared to single-species inoculation with either pathogen.
... were most abundant in K3 (18%) and geocaulosphere samples Z1, Z2, and Z3 (19-21%). According to the whole dataset with singletons excluded, the Dickeya genus was not found, although bacteria belonging to the D. dianthicola were isolated and identi ed from the same eld (Marković et al. 2020). ...
... Using traditional isolation and molecular characterization in the same eld in Northern Serbia during 2018, combined infection of P. carotovorum subsp. brasiliensis and D. dianthicola was determined (Marković et al. 2020) while the Dickeya spp. was found at the limit of detection in metabarcoding samples. This suggests the possibility that Dickeya spp. is a secondary pathogen that participates in, or even just succeeds infection of the primary pathogen (Pectobacterium) rather than inducing it, as shown by Kõiv et al. (2015) for some endophytes. ...
The soft rot caused by Pectobacterium and Dickeya spp. is among the most important potato disease, responsible for outbreaks worldwide. In 2018, potato tubers (cultivar Lady Claire) with and without visible soft rot symptom, together with their geocaulospheres were sampled from the field in Bačka region (Serbia). The 16S rRNA Next Generation Sequencing (NGS) of tubers with and without soft rot symptom and their corresponding soils was performed to detect differences in microbial diversity and, using this data to predict causal agent(s) of disease and which samples are potentially best for isolating biocontrol strains. The ubiquitous soil bacteria from the phyla Proteobacteria, Firmicutes, Actinobacteria, and Bacteroidetes were dominant in all samples. The sequences identified as Pectobacterium aroidearum , P. carotovorum , and P. polaris were present in all tested samples and it can be hypothesized that they caused soft rot. The K3 sample showed the presence of genera with potential antimicrobial activity ( Pseudomonas, Arthrobacter, Chryseobacterium, Bacillus , and Exiguobacterium ). This result shows that diversity analysis could be used for checking for the presence of potential antagonistic bacteria at infected sites. Also, following the presence or absence of particular taxa could point out a capacity of soil to endure a growth season without an outbreak of soft or to maintain it without significant losses. This is a new approach to interpreting the results of the diversity of bacterial communities of tubers and can be useful for screening the health status of the soil.
... (Czajkowski et al. 2012;Adeolu et al. 2016;Lebecka et al. 2018). They are regulated non-quarantine pests of potato seed and potato consumption (Marković et al. 2021). Both diseases cause yield and quality losses in potato production in the field, during harvest, transportation, and storage (Czajkowski et al. 2015;Charkowski et al. 2020). ...
Pectobacterium species are one of the main bacterial agents of soft rot and blackleg diseases that cause economic damage to potato crops. Farmers around the world suffer losses from both diseases and use different strategies to control. In this study, local potato genotypes were evaluated for disease severity of soft rot and blackleg. We discovered putative genotypes with low disease severity for both diseases. In the tuber experiment, the lowest soft rot was determined for genotype 18 with 34% disease severity. The highest soft rot was found in the cultivars Natasha, Laperla and Sultanecem. In the plant experiment, the lowest lesion length was measured in the cultivars Sultannur, Orchestra and Marabel. The 33% disease severity for these cultivars was also calculated for genotypes 18 and 32. With regard to soft rot and blackleg of potato, this is the first study to determine disease severity of Turkish local potato genotypes.
... P. carotovorum subsp. brasiliensis, Pcb) are mostly found on potatoes (Duarte et al., 2004;Ma et al., 2007;Malko et al., 2019;Marković et al., 2021;Muturi et al., The Role of metC in Pectobacterium 63 2018; Portier et al., 2019;van der Merwe et al., 2010). ...
Plant pathogenic Pectobacterium species cause severe soft rot/blackleg diseases in many economically important crops worldwide. Pectobacterium utilizes plant cell wall degrading enzymes (PCWDEs) as the main virulence determinants for its pathogenicity. In this study, we screened a random mutant, M29 is a transposon insertion mutation in the metC gene encoding cystathionine β-lyase that catalyzes cystathionine to homocysteine at the penultimate step in methionine biosynthesis. M29 became a methionine auxotroph and resulted in growth defects in methionine-limited conditions. Impaired growth was restored with exogenous methionine or homocysteine rather than cystathionine. The mutant exhibited reduced soft rot symptoms in Chinese cabbages and potato tubers, maintaining activities of PCWDEs and swimming motility. The mutant was unable to proliferate in both Chinese cabbages and potato tubers. The reduced virulence was partially restored by a complemented strain or 100 µM of methionine, whereas it was fully restored by the extremely high concentration (1 mM). Our transcriptomic analysis showed that genes involved in methionine biosynthesis or transporter were downregulated in the mutant. Our results demonstrate that MetC is important for methionine biosynthesis and transporter and influences its virulence through Pcc21 multiplication in plant hosts.
... Nonetheless, P. aroidearum, P. brasiliense, P. odoriferum, P. polaris, and P. wasabiae species have also been reported to result in cabbage soft rot in different countries [28][29][30][31][32][33]. In Serbia, different Pectobacterium species (i.e., P. atrosepticum, P. brasiliense, P. carotovorum, P. punjabense, P. zantedeschiae, and P. versatile) have been isolated from broccoli, calla lily, carrot, celery, parsley, potato, squash, and watermelon [19,20,23,[34][35][36][37][38][39]. ...
The aim of this work was to identify and characterize the pectolytic bacteria responsible for the emergence of bacterial soft rot on two summer cabbage hybrids (Cheers F1 and Hippo F1) grown in the Futog locality (Bačka, Vojvodina), known for the five-century-long tradition of cabbage cultivation in Serbia. Symptoms manifesting as soft lesions on outer head leaves were observed during August 2021, while the inner tissues were macerated, featuring cream to black discoloration. As the affected tissue decomposed, it exuded a specific odor. Disease incidence ranged from 15% to 25%. A total of 67 isolates producing pits on crystal violet pectate (CVP) medium were characterized for their phenotypic and genotypic features. The pathogenicity was confirmed on cabbage heads. Findings yielded by the repetitive element palindromic-polymerase chain reaction (rep-PCR) technique confirmed interspecies diversity between cabbage isolates, as well as intraspecies genetic diversity within the P. carotovorum group of isolates. Based on multilocus sequence typing (MLST) using genes dnaX, mdh, icdA, and proA, five representative isolates were identified as Pectobacterium carotovorum (Cheers F1 and Hippo F1), while two were identified as Pectobacterium versatile (Hippo F1) and Pectobacterium odoriferum (Hippo F1), respectively, indicating the presence of diverse Pectobacterium species even in combined infection in the same field. Among the obtained isolates, P. carotovorum was the most prevalent species (62.69%), while P. versatile and P. odoriferum were less represented (contributing by 19.40% and 17.91%, respectively). Multilocus sequence analysis (MLSA) performed with concatenated sequences of four housekeeping genes (proA, dnaX, icdA, and mdh) and constructed a neighbor-joining phylogenetic tree enabled insight into the phylogenetic position of the Serbian cabbage Pectobacterium isolates. Bacterium P. odoriferum was found to be the most virulent species for cabbage, followed by P. versatile, while all three species had comparable virulence with respect to potato. The results obtained in this work provide a better understanding of the spreading routes and abundance of different Pectobacterium spp. in Serbia.
... This might explain the slighter abundance of Pectobacterium spp. in macerated samples from this study. The previous work of Marković et al. (2021) reported combined infection with P. carotovorum subsp. brasiliensis and D. dianthicola in July 2018 in this same potato field using traditional isolation and molecular characterization techniques. ...
Plant pathogenic bacteria from the genera Pectobacterium and Dickeya in association with potato cause blackleg and tuber soft rot disease. A metabarcoding was used to analyze the microbial communities of soft-rotted tubers and their geocaulospheres. In September 2018, samples collected from the blackleg-infected field (58 ha) in the Bačka region (Serbia) reached 45% disease incidence. Next-generation sequencing Illumina MiSeq platform was used to obtain 16S rRNA sequences from six tested tubers (with and without soft rot symptom) and the surrounding geocaulosphere. Proteobacteria, Bacteroidetes, Firmicutes, and Actinobacteria were present in all samples, with Proteobacteria being the most prevalent, especially in tubers without soft rot symptoms. In all tested samples, species of the Pectobacterium (P. aroidearum, P. atrosepticum, P. carotovorum, and P. polaris) were detected but were more represented in tubers without symptoms. However, the genus Dickeya was at the limit of detection or not detected at all. Acinetobacter was the most dominant in tubers with soft rot, while Pseudomonas and Enterobacter were the most abundant in tubers without symptoms. The genera Bacteroides and Dysgonomonas with starch-degradable features were almost exclusively present in soft rotted tubers and their corresponding geocaulospheres. The most represented genera in the geocaulosphere sample associated with no-symptom tubers were Gaiella, Sphingomonas, Sphingobium, Gemmatimonas, and Geminicoccus, which include species with confirmed biocontrol potential. This study indicates that the soft rot maceration process arises due to complex interactions between plant pathogens and other endophytic bacteria.
... Multilocus sequence analysis (MLSA) was performed with a total of 36 randomly selected isolates, with three per year of isolation, location, and potato cultivar (Table 1). Total DNA was extracted using the modified CTAB protocol described by Marković et al. (2021). ...
Since 2011, the outbreaks of brown rot caused by Ralstonia solanacearum race 3, biovar 2, phylotype IIB-1 (R3/B2/PIIB-1) have significantly compromised potato production in Serbia. During six years of monitoring (2013–2018) among 3,524 potato tuber samples, 344 were found positive for brown rot disease. R. solanacearum R3/B2/PIIB-1 was isolated from five cultivars among 12 monitored, and in seven localities among 17 monitored. Cultivar Lady Claire was found to have the highest disease frequency (31.98%). A total of 78 isolates were identified by R. solanacearum-specific primer pairs (PS-1/PS-2 and OLY-1/Y-2), as well as the following tests: RFLP analysis, biovar determination, immunofluorescence, biochemical analysis, and pathogenicity. The genetic composition of 36 selected isolates assessed using multilocus sequence analysis with seven genes (adk, gapA, gdh, gyrB, ppsA, hrpB, and fliC) showed that all isolates originating from Serbian potato are homogeneous. By using the TCS algorithm of concatenated sequences to get insight into the phylogeography of isolates and other R. solanacearum strains deposited in the NCBI database, we showed that their origin is undetermined. Peroxidase (POD) activity was measured in brown rotted potato tubers. A positive correlation was found between POD activity and disease severity rated on the analysed tubers. In general, POD activity increased by 2 to 22 times in vascular necrotic tissues compared to nonnecrotic ones, and depended on disease severity but not on cultivar. Native PAGE analysis of POD profiles resulted in a total of 10 distinct POD isoforms, of which PODs 3–5 were highly intensified in response to R. solanacearum.
Potato is ranked as one of the most important food crops. Bacterial wilt caused by Ralstonia solanacearum and blackleg and soft rot caused by different species from genera Pectobacterium and Dickeya are considered two of the most important disease of the potato. Biological control is the optimal strategy for controlling pathogens in crops generally, including bacteria. The present study aimed to evaluate the antagonistic effects of two Bacillus amyloliquefaciens strains, SS-12.6 and SS-38.4, against bacterial pathogens isolated from the potato in Serbia, such as R. solanacearum, Pectobacterium carotovorum, Pectobacterium brasiliense, and Dickeya dianthicola. The diameter of the inhibition zones formed by ethyl acetate extracts of SS-12.6 and SS-38.4 strains show much higher values than the inhibition zones of supernatants, which implies the main power of these antagonists' potential lies in lipopeptides. The effectiveness of the treatment (19.7-44.5%), based on the difference in weight of potato tubers on the fifth and 15th day after treatment (DAT), showed that the antagonistic strains were almost equally effective in the suppression of P. carotovorum, P. brasiliense, and D. dianthicola strains. Strains SS-12.6 and SS-38.4 exhibited the efficacy in the suppression of R. solanacearum wilt from 28.64 to 60.22%. The analysis of the area under the disease progress (AUDPC) confirmed differences among pathogen control treatments and biocontrol treatments with B. amyloliquefaciens strains in all trials. This study shows that the two B. amyloliquefaciens strains, SS-12.6 and SS-38.4, can potentially be used as biocontrol agents against potato pathogens.
Pectobacterium brasiliense is an important bacterial species that causes soft rot disease in a wide range of host plants including potatoes worldwide. In 2018, seed potato tubers were collected to verify the presence and characterize strains of P. brasiliense in Turkey. Bacterial isolation from soft-rotted seed tubers was performed on a CVP medium, and we found that 18 of 34 strains (55%) belonged to the P. brasiliense species. All of them produced 322 bp products with Br1f/L1r primers in the PCR assay. Partial nucleotide sequences of the housekeeping genes mdh and recA confirmed the result obtained with the species-specific primers. Phylogenetic analysis of the recA gene sequences allowed the strains to be grouped into a cluster divided into two clades distinct from other Pectobacterium species. There was a difference between the tested strains for their ability to macerate potato tubers tissue. Four Turkish P. brasiliense strains were determined to be highly virulent on potato tubers, which caused soft rot symptoms on plants of 17 species of 8 families. Multiple strains also caused soft rot on harvested plant organs (leaves, head, and bulb) and necrosis on sugar beet roots and eggplant fruits, except strain Pbr5, whose pathogenicity was limited. This is the first isolation of P. brasiliense from infected seed potato tubers in Turkey and the first detailed report showing the host range of domestic strains.
Pectobacterium spp. are causative agents of blackleg and soft rot of potato. However, little is known about the relationship between the pathogenicity of mixed infections of different Pectobacterium spp. at different temperatures. In this study, two pectinolytic strains of Pectobacterium spp. were isolated from the same potato plant with typical symptoms of blackleg and identified as P. brasiliense and P. carotovorum by multilocus sequence analysis (MLSA), whole-genome phylogenetic tree construction, average nucleotide identity (ANI) analysis and digital DNA–DNA hybridization (dDDH). Plant cell wall degrading enzyme, including pectinases, cellulases and proteases, as the most important virulence factors, as well as pathogenicity toward potato tuber, were compared between the strains P. brasiliense BL-2 and P. carotovorum BL-4 at 28 ℃. The results showed that P. carotovorum had higher cell wall-degrading enzyme activities and brought more severe disease symptoms to potato tubers than P. brasiliense. Moreover, the pathogenicity of P. carotovorum and P. brasiliense increased with increasing temperature (20, 25, 28, 32 ℃). The pathogenicity was more severe when P. carotovorum strain BL-4 was co-inoculated with P. brasiliense strain BL-2, especially when the former exhibited an advantage in bacterial number at the initial time. The results of this study provide new insight for understanding the pathogenicity caused by mixed infections with different species of Pectobacterium spp., and they may provide some guidance for controlling potato blackleg and soft rot.
Potato blackleg is frequently observed on the production fields in the Bačka region of Vojvodina province, which is one of the largest potato-growing areas in Serbia. This disease usually occurs during June and July. In July 2020, blackleg symptoms in the form of stem necrotic lesions, vascular discoloration, hollow stems, and wilting of whole plants were noted on potato cultivar VR808 on a field 28 ha in size located in Maglić village (GPS coordinates 45.349325 N, 19.542768 E). Disease incidence was estimated at 20−25%. Isolations were performed from 12 potato samples on Crystal Violet Pectate medium (CVP). Stem sections consisted of brown lesions and healthy tissue (c.10 cm) were surface sterilized with ethyl alcohol 70% (w/v) and rinsed with sterile distilled water. Small pieces of tissue were taken at the edges of stem lesions (between healthy and diseased tissue) were soaked in phosphate buffer saline for 20 min and plated using a standard procedure (Klement et al. 1990). Single colonies that formed pits after 48 hours at 26 °C were re-streaked onto Nutrient Agar (NA) where creamy white colonies with smooth surfaces were formed. A total of 30 isolates were selected and DNA isolated from the colonies was further analyzed by polymerase chain reaction (PCR) using the partial dnaX gene (DNA polymerase subunit III gamma/tau) with primer pair dnaXf/dnaXr for Pectobacterium and Dickeya species identification (Slawiak et al. 2009). A single characteristic band of 535 bp was amplified in all isolates (Slawiak et al. 2009). DNA sequence alignment showed two distinct groups of isolates (Fig.S1), which were genetically uniform within each group. Using BLASTn search, it was established that the dnaX sequence of the first group (consisting of 19 Serbian potato isolates) had 99.79% identity with NCBI-deposited Pectobacterium versatile strains 14A and 3-2 from potato from Belarus (Acc. No. CP034276 and CP024842, respectively) as well as SCC1 from Finland (Acc. No. CP021894). The remaining 11 dnaX sequences had 100% identity with Pectobacterium carotovorum subsp. carotovorum strain CFBP7081 originating from water in Spain (Acc. No. MK516961). The partial dnaX sequences of three Serbian P. versatile isolates (Pv1320, Pv1520, and Pv1620) and one P. carotovorum subsp. carotovorum (Pcc2520) were deposited in GenBank under Acc. No. MW839571, MW805306, MW839572, and MW805307, respectively. These results, indicating combined infection in the observed field, signify the first identification of P. versatile in Serbia. Multilocus sequence analysis (MLSA) performed with proA (proAF1/ proAR1) and mdh (mdh2/mdh4) genes (Ma et al. 2007; Moleleki et al. 2013) grouped three tested Serbian potato P. versatile isolates together with P. versatile strains from NCBI (Fig.S2). For both tested genes, BLASTn search revealed 100% homology with P. versatile strain SCC1 from Finland. Three Serbian P. versatile potato isolates were deposited under Acc. Nos. MZ682623-25 for proA and MZ682620-22 for mdh genes. According to the routine tests suggested for Pectobacteriaceae (Schaad et al. 2001), Serbian isolates possessed microbiological traits identical to P. versatile description (Portier et al. 2019). Pathogenicity was performed on potato cultivar VR808 with three selected P. versatile isolates (Pv1320, Pv1520, and Pv1620) in the following assays: (i) surface-sterilized tuber slices with holes in the center filled with 100 µL of bacterial suspensions (adjusted to 109 CFU mL-1) to test the isolates’ ability to cause soft rot, and (ii) young, four-week old plants with developed 3rd true leaf (c. 30 cm tall) were inoculated by injecting stems with bacterial suspension adjusted to 107 - 108 CFU mL-1 at a height 5 cm above the soil line. Negative controls were treated with sterile distilled water. Inoculated plants were kept under controlled conditions (25 °C temperature and >70% relative humidity). Each assay was replicated twice. Soft rot appeared on tuber slices 24 h after inoculation. On inoculated stems, initial symptoms manifested as greasy elongated spots at inoculation sites two days after inoculation (DAI), and subsequently extended along the vascular tissue and became necrotic. Whole plant's decay was recorded in five DAI, while negative controls remained healthy. To complete Koch's postulates, bacteria were re-isolated from symptomatic potato plants and confirmed by PCR and sequencing of dnaX. This first report of P. versatile in potato indicates that blackleg currently present in Serbia is caused by a diverse bacterial population. This pathogen was first identified in genome comparison as ‘Candidatus Pectobacterium maceratum’ (Shirshikov et al. 2018) and was later renamed as Pectobacterium versatile sp. nov. (Portier et al. 2019). Thus far, bacterium Pectobacterium carotovorum subsp. brasiliensis has been recognized as dominant pathogen on most of the infected fields in Vojvodina province, and was recently noted on one plot subjected to a combined infection with Dickeya dianthicola (Marković et al. 2021). Findings achieved in this study are highly relevant, as they point to the diversity in potato blackleg pathogens, likely due to the increasingly widespread distribution of imported seed potatoes.
Dickeya dianthicola has caused an outbreak of blackleg and soft rot of potato in the eastern half of the USA since 2015. To investigate genetic diversity of the pathogen, a comparative analysis was conducted on genomes of D. dianthicola strains. Whole genomes of 16 strains from the USA outbreak were assembled and compared to 16 previously sequenced genomes of D. dianthicola isolated from potato or carnation. Among the 32 strains, eight distinct clades were distinguished based on phylogenomic analysis. The outbreak strains were grouped into three clades, with the majority of the strains in clade I. Clade I strains were unique and homogeneous, suggesting a recent incursion of this strain into potato production from alternative hosts or environmental sources. Pangenome of the 32 strains contained 6693 genes, 3377 of which were core genes. By screening primary protein subunits associated with virulence from all USA strains, we found many virulence-related gene clusters, such as plant cell wall degrading enzyme genes, flagellar and chemotaxis related genes, two-component regulatory genes, and type I/II/III secretion system genes were highly conserved but type IV and type VI secretion system genes varied. The virulent clade I strains encoded two clusters of type IV secretion systems, while clade II and III strains encoded only one cluster. Clade I and II strains encoded one more VgrG/PAAR spike protein than clade III. Thus, we predicted that the presence of additional virulence-related genes may have enabled the unique clade I strain to become predominant source in the USA outbreak.
Bacteria from Enterobacteriaceae family (SRE) are significant problem in plant production, not only during vegetation, in the field, but also during storage and marketing of agricultural commodities. Species P. carotovorum subsp. brasiliense (Pcb) is a newly identified member of Enterobacteriaceae family. It causes soft rot of different plant species, including root vegetables. Pcb is described as a new subspecies of P. carotovorum due to differences in phenotypic and genotypic characteristic, more pronounced virulence and aggressiveness. Patohogenicity of this bacterium is based on the production of several enzymes: pectatliase, polygalacturonase, cellulase and proeteases. The aim of this study was to determine whether and at which rate Pcb isolates originating from potato plants exhibit pectolytic activity on root of different root vegetable species - carrot, radish, celery, kohlrabi and beetroot. The obtained data confirmed wide host range of the bacterium Pectobacterium carotovorum subsp. brasiliense, but pointed to significant differences in pectolytic activity on different species of root vegetables (carrot, radish, celery, kohlrabi), while on beetroot tested Pcb isolates did not exhibit pectolytic activity. Moreover, on same species of root vegetables different levels of pectolytic activity of tested Pcb isolates were recorded.
Bacterial diseases are one of the most important biotic constraints of potato production, especially in tropical and subtropical regions, and in some warm temperate regions of the world. About seven bacterial diseases affect potato worldwide and cause severe damages especially on tubers, the economically most important part of the plant. Bacterial wilt and back leg are considered the most important diseases, whereas potato ring rot, pink eye, and common scab are the minor. Knowledge about zebra chip is extremely rare, as it occurs in a very isolated area and is an emerging disease in New Zealand, Europe, the USA and Mexico. Potato crop losses due to bacterial diseases could be direct and indirect; and they have several dimensions, some with short-term consequences such as yield loss and unmarketability of the produce and others with long-term consequences such as economic, environmental, and social. Some of them are of national and international importance and are the major constraints to clean seed potato production, with considerable indirect effects on trade. This review focuses on Clavibacter spp., Ralstonia spp., Pectobacterium spp., Dickeya spp., Streptomyces spp., and Liberibacter spp. pathogenic to potato, and looks at the respective pathogen in terms of their taxonomy and nomenclature, host range, geographical distribution, symptoms, epidemiology, pathogenicity and resistance, significance and economic losses, and management strategies. Nevertheless, the information collected here deal more with diseases known in developed and developing countries which cause severe economic losses on potato value chain.
The Pectobacterium carotovorum species corresponds to a complex, including two subspecies with validly published names,
two proposed subspecies and two new species, Pectobacterium polaris and Pectobacterium aquaticum. Recent studies
suggested that this complex needed revision. We examined the taxonomic status of 144 Pectobacterium strains isolated from
a wide range of plant species, various geographical origins and waterways. Sequences of the leuS, dnaX and recA
housekeeping genes clustered 114 of these Pectobacterium strains together within a not yet described clade. We sequenced
eight strains of this clade and analysed them together with the 102 Pectobacterium genomes available in the NCBI database.
Phylogenetic analysis, average nucleotide identity calculation and in silico DNA–DNA hybridization allowed us to differentiate
seven clades. This led us to propose the elevation of Pectobacterium carotovorum subsp. odoriferum to species level as
Pectobacterium odoriferum sp. nov. (type strain CFBP 1878T=LMG 5863T=NCPPB 3839T=ICMP 11533T), the proposal of
Pectobacterium actinidiae sp. nov. (type strain KKH3=LMG 26003 T=KCTC 23131T) and Pectobacterium brasiliense sp. nov. (type
strain CFBP 6617T= LMG 21371T=NCPPB 4609T), to emend the description of Pectobacterium carotovorum (type strain CFBP
2046T=LMG 2404T=NCPPB 312T=ICMP 5702T), and to propose a novel species, Pectobacterium versatile sp. nov (type strain
CFBP6051T= NCPPB 3387T=ICMP 9168T) which includes the strains previously described as ‘Candidatus Pectobacterium
maceratum’. Phenotypic analysis performed using Biolog GENIII plates on eight strains of P. versatile sp. nov. and related
strains completed our analysis.
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.
Pectinolytic bacteria belonging to the genera Pectobacterium and Dickeya cause blackleg and soft rot diseases on several plants and crops including Solanum tuberosum (Pérombelon 2002). In Morocco, Pectobacterium carotovorum is described as the main causal agent of these diseases (Terta et al. 2010). In March 2016, blackleg symptoms were observed in commercial potato fields in the north of Morocco. Using molecular microbiology tools, these pathogenic isolates on potato plants have been characterized as belonging to Dickeya dianthicola. The isolation of pectinolytic bacteria (including species of the genera Dickeya and Pectobacterium) was performed on the standard medium crystal violet pectate (CVP) agar (Hélias et al. 2012). Identification of pectinolytic bacteria on CVP plates was carried out by observing the formation of a depression around bacterial colonies. All cultures were kept at 28 to 30°C for at least 3 days. Routine subcultures were performed on Luria-Bertani (LB) agar plates. To further determine the identity of the potential pectinobacteria, PCR was used with primer sets ADE1/ADE2 and Y1/Y2 targeting the pelADE genes (Nasser et al. 1996). Results from PCR screening showed that 69 isolates were identified as Pectobacterium spp. and 20 as Dickeya spp. Ten out of 20 Dickeya isolates were subjected to PCR amplification targeting the dnaX (Sławiak et al. 2009) and rrs 16S rDNA (with universal primers PA and 518r) genes. Nucleotide sequences of the PCR products were determined. The sequences were deposited under accession numbers KX985805–814 for dnaX and KX985795–804 for rrs. Both dnaX and rrs sequences showed 100 and 97% identity with published D. dianthicola sequences, respectively. A multilocus sequence analysis (MLSA) of the concatenated 16S rDNA and dnaX genes fragments of the Moroccan isolates in comparison with complete genomes from D. chrysanthemi NCPPB 516, D. dadantii 3937, D. dianthicola RNS049, D. solani 3337, D. zeae 1591, and P. atrosepticum CFBP6276 showed that all Moroccan Dickeya strains were clustered in the D. dianthicola clade. Koch’s postulates were completed on potato plants cv. Lusa in order to verify the pathogenicity of the 10 Moroccan D. dianthicola isolates. Potato tubers were planted in 2-liter pots and incubated at 25 to 28°C with regular watering in a greenhouse. Three weeks after stem emergence, 10 µl of a cell suspension at 107 cfu/ml of each Dickeya strain was injected into the lower part of stem with a sterile syringe. Six plants were infected by each of the 10 D. dianthicola isolates. Additional plants infiltrated by a sterile NaCl 0.85% solution were used as a control. Fourteen days after inoculation, all inoculated stems by D. dianthicola showed black and rotten stems, but control plants remained symptomless. Bacteria were isolated from the lesions and confirmed to be Dickeya sp. pathogens. In conclusion, combination of molecular and symptomatic analysis demonstrated the occurrence of D. dianthicola species from some potato fields in Morocco, and showed that D. dianthicola strains are virulent on potato plants. These findings will increase awareness and offer new technologies to producers and phytosanitary authorities in prophylactic measures to avoid the spread of Dickeya in potatoes. © 2017, American Phytopathological Society. All rights reserved.
Blackleg and stem rot of potato occur sporadically in New Zealand, causing economic damage under optimal temperature and humidity conditions for disease development. Both Pectobacterium atrosepticum ( Pba ) and P. carotovorum subsp.…
The soft rot Enterobacteriaceae (SRE) Pectobacterium and Dickeya species (formerly classified as pectinolytic Erwinia spp.) cause important diseases on potato and other arable and horticultural crops. They may affect the growing potato plant causing blackleg and are responsible for tuber soft rot in storage thereby reducing yield and quality. Efficient and cost-effective detection and identification methods are essential to investigate the ecology and pathogenesis of the SRE as well as in seed certification programmes. The aim of this review was to collect all existing information on methods available for SRE detection. The review reports on the sampling and preparation of plant material for testing and on over thirty methods to detect, identify and differentiate the soft rot and blackleg causing bacteria to species and subspecies level. These include methods based on biochemical characters, serology, molecular techniques which rely on DNA sequence amplification as well as several less-investigated ones.
Until recently Dickeya was regarded as a pathogen not established in Finland. As a result the blackleg symptom observed on potato was often associated with Pectobacterium atrosepticum. The occurrence of Dickeya spp. on potato in Finland was first reported in 2004. Since then the prevalence of Dickeya has been monitored through surveys and routine test of seed lots produced in the country. The results of monitoring of Dickeya spp. in seed lots produced in Finland between the years 2004 and 2008 indicated a steady increase in the incidence of Dickeya spp. The highest incidence was observed in samples from the 2006 growing season where about 37% were positive for Dickeya spp. The summer in 2006 was one of the warmest summers recorded in 100 years in Finland. The majority of infected lots were imported varieties. Since recently heavy blackleg outbreaks have occurred in production fields in the High Grade (HG) zone. A detailed study of these incidents of blackleg outbreaks in North Finland during the years 2008 and 2010 indicated that Dickeya spp. was the major component in the observed blackleg complex. It was detected and isolated from almost all symptomatic plants investigated. Repetitive sequences PCR (REP‐PCR) and Pulsed Field Gel Electrophoresis (PFGE) analysis of strains isolated in Finland showed identical pattern with those isolated recently in other European countries with a proposed name ‘Dickeya solani’. Moreover, the dnaX gene sequence of the representative strains isolated in Finland indicated 100% similarity to the dnaX sequences of ‘D. solani’. The study presents the first report of a detailed analysis of bacteria involved in potato blackleg complex from natural field outbreaks in North Finland HG zone and characterisation of the ‘D. solani’ strains playing the major role in the disease complex.
Pectolytic bacteria were isolated from potato tubers and stems showing tuber soft rot and blackleg symptoms. Approximately half (52 %) of the isolates could grow at both 27 and 37 °C while another half (48 %) failed to grow at 37 °C. All isolates could be amplified with primers specific to the pectate lyase (pel) gene. Carbon utilisation profiles could not con-clusively identify these isolates. PCR amplification using primers specific for Pectobacterium carotovo-rum subsp. brasiliensis was positive for all isolates that grew at 37 °C. However, the group that did not grow at 37 °C failed to amplify with P. atrosepticum specific primers. To characterise this group of isolates, the intergenic transcribed spacer region (ITS) was amplified and PCR products digested with two restric-tion enzymes (RsaI and CfoI) to generate ITS-PCR-RFLP profiles. The profiles of these new isolates were compared to those of the type strains of other pectolytic bacteria. Profiles of five of the selected atypical strains generated with the enzyme CfoI appeared to be most similar to those of P. wasabiae type strain. Phylogenetic analysis using concatenated partial gene sequences of housekeeping genes mdh and gapA clustered these isolates together with those of P. wasabiae reference strains thus confirming their identity. These strains were virulent on potato tubers and stems but did not elicit hypersensitive response on tobacco plants. This is the first report of P. wasabiae causing soft rot and blackleg of potatoes in South Africa.
We announce the release of an advanced version of the Molecular Evolutionary Genetics Analysis (MEGA) software, which currently contains facilities for building sequence alignments, inferring phylogenetic histories, and conducting molecular evolutionary analysis. In version 6.0, MEGA now enables the inference of timetrees, as it implements our RelTime method for estimating divergence times for all branching points in a phylogeny. A new Timetree Wizard in MEGA6 facilitates this timetree inference by providing a graphical user interface (GUI) to specify the phylogeny and calibration constraints step-by-step. This version also contains enhanced algorithms to search for the optimal trees under evolutionary criteria and implements a more advanced memory management that can double the size of sequence data sets to which MEGA can be applied. Both GUI and command-line versions of MEGA6 can be downloaded from www.megasoftware.net free of charge.
The species Pectobacterium carotovorum includes a diverse subspecies of bacteria that cause disease on a wide variety of plants. In Morocco, approximately 95% of the P. carotovorum isolates from potato plants with tuber soft rot are P. carotovorum subsp. carotovorum. However, identification of this pathogen is not always related to visual disease symptoms. This is especially true when different pathogen cause similar diseases on potato, citing as an example, P. carotovorum, P. atrosepticum and P. wasabiae. Numerous conventional methods were used to characterize Pectobacterium spp., including biochemical assays, specific PCR-based tests, and construction of phylogenetic trees by using gene sequences. In this study, an alternative method is presented using a gene linked to pathogenicity, in order to allow accuracy at subspecies level. The pmrA gene (response regulator) has been used for identification and analysis of the relationships among twenty nine Pectobacterium carotovorum subsp. carotovorum and other Pectobacterium subspecies.
Phylogenetic analyses of pmrA sequences compared to ERIC-PCR and 16S rDNA sequencing, demonstrated that there is considerable genetic diversity in P. carotovorum subsp. carotovorum strains, which can be divided into two distinct groups within the same clade.
pmrA sequence analysis is likely to be a reliable tool to identify the subspecies Pectobacterium carotovorum subsp. carotovorum and estimate their genetic diversity.
The nucleotide sequence for a DNA hybridization probe specific for Erwinia carotovora subsp. atroseptica was determined, and primers were selected for detection of the blackleg pathogen using the polymerase chain reaction (PCR). Primers ECA1f (5'-CGGCATCATAAAAACACG-3') and ECA2r (5'-GCACACTTCATCCAGCGA-3') specifically amplified a 690-bp DNA fragment of all E. carotovora subsp. atroseptica strains tested but not strains of other E. carotovora subspecies isolated from various hosts and geographic regions or other plant- and soil-associated bacteria. Visualization of the E. carotovora subsp. atroseptica-specific PCR product on ethidium bromide-stained agarose gels required a minimum of 250 to 500 CFU per ml. A total of 170 samples of potato stem and tuber tissue was tested by PCR and compared with reactions in enzyme-linked immunosorbent assay (ELISA) with a monoclonal antibody specific for the lipopolysaccharide of E. carotovora subsp. atroseptica. Although 50.6% of these samples were positive in PCR compared to 46.5% in ELISA, some of the ELISA-positive samples were negative in PCR. When E. carotovora subsp. atroseptica DNA was added to these samples, amplified products were obtained in all but two samples after repeating the PCR, indicating that in most cases the failure to obtain PCR amplification for some of the ELISA-positive samples was not due to the presence of inhibitors.
Pectobacterium atrosepticum, P. carotovorum subsp. brasiliensis, P. carotovorum subsp. carotovorum, and P. wasabiae were detected in potato stems with blackleg symptoms using species- and subspecies-specific polymerase chain reaction (PCR). The tests included a new assay for P. wasabiae based on the phytase gene sequence. Identification of isolates from diseased stems by biochemical or physiological characterization, PCR, and multi-locus sequence typing (MLST) largely confirmed the PCR detection of Pectobacterium spp. in stem samples. P. atrosepticum was most commonly present but was the sole Pectobacterium sp. detected in only 52% of the diseased stems. P. wasabiae was most frequently present in combination with P. atrosepticum and was the sole Pectobacterium sp. detected in 13% of diseased stems. Pathogenicity of P. wasabiae on potato and its capacity to cause blackleg disease were demonstrated by stem inoculation and its isolation as the sole Pectobacterium sp. from field-grown diseased plants produced from inoculated seed tubers. Incidence of P. carotovorum subsp. brasiliensis was low in diseased stems, and the ability of Canadian strains to cause blackleg in plants grown from inoculated tubers was not confirmed. Canadian isolates of P. carotovorum subsp. brasiliensis differed from Brazilian isolates in diagnostic biochemical tests but conformed to the subspecies in PCR specificity and typing by MLST.
Characteristics of forty strains from macerated potato tubers and water-soaked lesions of some ornamental plants were studied
in north parts of Iran. The causal organisms isolated from infected tissues were identified as Pectobacterium spp. based on their physiological and biochemical assays and confirmed by species and subspecies specific PCR and RFLP analysis
of 16S–23S intergenic transcribed spacer region. Artificial inoculation of isolates to their related hosts generated the same
symptoms on potato and ornamental plants, from which the same bacteria were isolated and identified. We detected two groups
of atypical isolates in this study. The first group from potato classified as Pectobacterium carotovorum subsp. carotovorum by phenotypic tests but was unable to elicit HR on tobacco leaves, to grow at 37°C and to amplify the pel gene relevant to this subspecies. The second one from ornamental plants which was again characterized as Pectobacterium carotovorum subsp. carotovorum in biochemical assays, produced a unique ITS-RFLP profile different from all of known Pectobacterium species and subspecies. Our findings based on phylogenetic analysis using concatenated partial sequences of housekeeping
genes mdh and gapA, indicated the occurrence of P. wasabiae as a novel species in potato storage in Iran. Furthermore we detected a distinct clade of Pectobacterium spp. from some ornamental plants including Schlumbergera bridgesii, Syngonium podophyllum and Iris spp.
KeywordsHousekeeping genes–Ornamental plants–Potato–
Pectobacterium strains–Soft-rot–Plant disease
It is well established that the pectinolytic bacteria Pectobacterium atrosepticum (Pca) and Dickeya spp. are causal organisms of blackleg in potato. In temperate climates, the role of Pectobacterium carotovorum subsp. carotovorum (Pcc) in potato blackleg, however, is unclear. In different western and central European countries plants are frequently
found with blackleg from which only Pcc can be isolated, but not Pca or Dickeya spp. Nevertheless, tubers vacuum-infiltrated with Pcc strains have so far never yielded blackleg-diseased plants in field
experiments in temperate climates. In this study, it is shown that potato tubers, vacuum-infiltrated with a subgroup of Pcc
strains isolated in Europe, and planted in two different soil types, can result in up to 50% blackleg diseased plants.
A new statistical method for estimating divergence dates of species from DNA sequence data by a molecular clock approach is developed. This method takes into account effectively the information contained in a set of DNA sequence data. The molecular clock of mitochondrial DNA (mtDNA) was calibrated by setting the date of divergence between primates and ungulates at the Cretaceous-Tertiary boundary (65 million years ago), when the extinction of dinosaurs occurred. A generalized leastsquares method was applied in fitting a model to mtDNA sequence data, and the clock gave dates of 92.311.7, 13.31.5, 10.91.2, 3.70.6, and 2.70.6 million years ago (where the second of each pair of numbers is the standard deviation) for the separation of mouse, gibbon, orangutan, gorilla, and chimpanzee, respectively, from the line leading to humans. Although there is some uncertainty in the clock, this dating may pose a problem for the widely believed hypothesis that the bipedal creatureAustralopithecus afarensis, which lived some 3.7 million years ago at Laetoli in Tanzania and at Hadar in Ethiopia, was ancestral to man and evolved after the human-ape splitting. Another likelier possibility is that mtDNA was transferred through hybridization between a proto-human and a protochimpanzee after the former had developed bipedalism.
In South Africa during the 2006/2007 potato growing season, outbreaks of blackleg occurred, causing severe economic losses
in commercial potato production fields. Symptoms were initially observed on only one stem per plant, on which the top leaves
rolled upwards, wilted and became necrotic. As symptoms progressed to the lower leaves with subsequent leaf desiccation, a
light to dark brown discolouration of the vascular system at the stem base developed, followed by external darkening. Under
prevailing wet and humid conditions stems became slimy and pale. In the stems, the pith became necrotic and hollow. These
symptoms were similar to those described in Brazil, where the causal agent was identified as a new subspecies, Pectobacterium carotovorum subsp. brasiliensis (Pbcb). Isolations from plants showing typical blackleg symptoms were made on CVP medium. Sequences and phylogenetic analysis of
the partial 16S–23S rDNA intergenic spacer region indicated that the isolates were Pbcb. Comparison of PCR-RFLP patterns of the 16S–23S rDNA of isolates to reference cultures confirmed the identity of the South
African blackleg strains as Pbcb, identical to strain 8 isolated in Brazil. This is the first report of Pbcb in South Africa and it appears to be the most important causal agent of blackleg in South Africa. The disease poses a major
potential threat to the South African potato industry especially in terms of seed exports, tuber quality and yield.
This paper presents PyElph, a software tool which automatically extracts data from gel images, computes the molecular weights of the analyzed molecules or fragments, compares DNA patterns which result from experiments with molecular genetic markers and, also, generates phylogenetic trees computed by five clustering methods, using the information extracted from the analyzed gel image. The software can be successfully used for population genetics, phylogenetics, taxonomic studies and other applications which require gel image analysis. Researchers and students working in molecular biology and genetics would benefit greatly from the proposed software because it is free, open source, easy to use, has a friendly Graphical User Interface and does not depend on specific image acquisition devices like other commercial programs with similar functionalities do.
PyElph software tool is entirely implemented in Python which is a very popular programming language among the bioinformatics community. It provides a very friendly Graphical User Interface which was designed in six steps that gradually lead to the results. The user is guided through the following steps: image loading and preparation, lane detection, band detection, molecular weights computation based on a molecular weight marker, band matching and finally, the computation and visualization of phylogenetic trees. A strong point of the software is the visualization component for the processed data. The Graphical User Interface provides operations for image manipulation and highlights lanes, bands and band matching in the analyzed gel image. All the data and images generated in each step can be saved. The software has been tested on several DNA patterns obtained from experiments with different genetic markers. Examples of genetic markers which can be analyzed using PyElph are RFLP (Restriction Fragment Length Polymorphism), AFLP (Amplified Fragment Length Polymorphism), RAPD (Random Amplification of Polymorphic DNA) and STR (Short Tandem Repeat). The similarity between the DNA sequences is computed and used to generate phylogenetic trees which are very useful for population genetics studies and taxonomic classification.
PyElph decreases the effort and time spent processing data from gel images by providing an automatic step-by-step gel image analysis system with a friendly Graphical User Interface. The proposed free software tool is suitable for researchers and students which do not have access to expensive commercial software and image acquisition devices.
Although there are adequate DNA sequence differences among plant-associated and plant-pathogenic bacteria to facilitate molecular approaches for their identification, identification at a taxonomic level that is predictive of their phenotype is a challenge. The problem is the absence of a taxonomy that describes genetic variation at a biologically relevant resolution and of a database containing reference strains for comparison. Moreover, molecular evolution, population genetics, ecology, and epidemiology of many plant-pathogenic and plant-associated bacteria are still poorly understood. To address these challenges, a database with web interface was specifically designed for plant-associated and plant-pathogenic microorganisms. The Plant-Associated Microbes Database (PAMDB) comprises, thus far, data from multilocus sequence typing and analysis (MLST/MLSA) studies of Acidovorax citrulli, Pseudomonas syringae, Ralstonia solanacearum, and Xanthomonas spp. Using data deposited in PAMDB, a robust phylogeny of Xanthomonas axonopodis and related bacteria has been inferred, and the diversity existing in the Xanthomonas genus and in described Xanthomonas spp. has been compared with the diversity in P. syringae and R. solanacearum. Moreover, we show how PAMDB makes it easy to distinguish between different pathogens that cause almost identical diseases. The scalable design of PAMDB will make it easy to add more plant pathogens in the future.
ABSTRACT Pectobacterium and Dickeya spp. are related broad-host-range entero-bacterial pathogens of angiosperms. A review of the literature shows that these genera each cause disease in species from at least 35% of angiosperm plant orders. The known host ranges of these pathogens partially overlap and, together, these two genera are pathogens of species from 50% of angiosperm plant orders. Notably, there are no reported hosts for either genus in the eudicots clade and no reported Dickeya hosts in the magnoliids or eurosids II clades, although Pectobacterium spp. are pathogens of at least one plant species in the magnoliids and at least one in each of the three eurosids II plant orders. In addition, Dickeya but not Pectobacterium spp. have been reported on a host in the rosids clade and, unlike Pectobacterium spp., have been reported on many Poales species. Natural disease among nonangiosperms has not been reported for either genus. Phylogenetic analyses of sequences concatenated from regions of seven housekeeping genes (acnA, gapA, icdA, mdh, mtlD, pgi, and proA) from representatives of these genera demonstrated that Dickeya spp. and the related tree pathogens, the genus Brenneria, are more diverse than Pectobacterium spp. and that the Pectobacterium strains can be divided into at least five distinct clades, three of which contain strains from multiple host plants.
Conserved regions about 420 bp long of the pelADE cluster specific to Erwinia chrysanthemi were amplified by PCR and used to differentiate 78 strains of E. chrysanthemi that were obtained from different hosts and geographical areas. No PCR products were obtained from DNA samples extracted from other pectinolytic and nonpectinolytic species and genera. The pel fragments amplified from the E. chrysanthemi strains studied were compared by performing a restriction fragment length polymorphism (RFLP) analysis. On the basis of similarity coefficients derived from the RFLP analysis, the strains were separated into 16 PCR RFLP patterns grouped in six clusters, These clusters appeared to be correlated with other infraspecific levels of E. chrysanthemi classification, such as pathovar and biovar, and occasionally with geographical origin. Moreover, the clusters correlated well with the polymorphism of pectate lyase and pectin methylesterase isoenzymes. While the pectin methylesterase profiles correlated with host monocot-dicot classification, the pectate lyase polymorphism might reflect the cell wall microdomains of the plants belonging to these classes.
jModelTest is a new program for the statistical selection of models of nucleotide substitution based on “Phyml” (Guindon and Gascuel 2003. A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. Syst Biol. 52:696–704.). It
implements 5 different selection strategies, including “hierarchical and dynamical likelihood ratio tests,” the “Akaike information
criterion,” the “Bayesian information criterion,” and a “decision-theoretic performance-based” approach. This program also
calculates the relative importance and model-averaged estimates of substitution parameters, including a model-averaged estimate
of the phylogeny. jModelTest is written in Java and runs under Mac OSX, Windows, and Unix systems with a Java Runtime Environment
installed. The program, including documentation, can be freely downloaded from the software section at http://darwin.uvigo.es.
In Western-Europe, Pectobacterium carotovorum subsp. brasiliense is emerging as blackleg causing agent. In field experiments in the Netherlands, the virulence of this pathogen was compared with strains of other Dickeya and Pectobacterium strains. In 2013 and 2014, seed potato tubers were vacuum-infiltrated with high densities of bacteria (106 cfu ml−1) and planted in clay soil. Inoculation with P. carotovorum subsp. brasiliense and Pectobacterium atrosepticum resulted in high disease incidences (75-95%), inoculation with D. solani and P. wasabiae led to incidences between 5% and 25%, but no significant disease development was observed in treatments with P. c subsp. carotovorum, D. dianthicola and the water control. Co-inoculations of seed potatoes with P. c. subsp. brasiliense and D. solani gave a similar disease incidence to inoculation with only P. c. subsp. brasiliense. However, co-inoculation of P. c. subsp. brasiliense with P. wasabiae resulted in a decrease in disease incidence compared to inoculation with only P. c subsp. brasiliense. In 2015, seed potatoes were inoculated with increasing densities of P. c. subsp. brasiliense, D. solani or P. atrosepticum (103 – 106 cfu ml−1). After vacuum-infiltration, even a low inoculum density resulted into a high disease incidence but if immersion without vacuum was applied, blackleg was only found at high densities. To detect the pathogens in progeny tubers, specific TaqMan assays were developed and/or evaluated for P. c. subsp. brasiliense, P. wasabiae and P. atrosepticum. The presence of the pathogens in progeny tubers of plants derived from vacuum-infiltrated seed tubers was confirmed by TaqMan assays. This article is protected by copyright. All rights reserved.
Amplified fragment length polymorphism (AFLP) markers and multilocus sequence analysis (MLSA) were used to analyse 63 bacterial strains, including 30 soft-rot-causing bacterial strains collected from Syrian potato fields and 33 reference strains. For the MLSA, additional sequences of 41 strains of Pectobacterium and Dickeya, available from the NCBI GenBank, were included to produce a single alignment of the 104 taxa for the seven concatenated genes (acnA, gapA, proA, icd, mtlD, mdh and pgi). The results indicate the need for a revision of the previously classified strains, as some potato-derived Pectobacterium carotovorum strains were re-identified as P. wasabiae. The strains that were classified as P. carotovorum during the analyses demonstrated high heterogeneity and grouped into five P. carotovorum highly supported clusters (PcI to PcV). The strains represented a wide range of host plants including potatoes, cabbage, avocados, arum lilies, sugar cane and more. Host specificity was detected in PcV, in which four of the six strains were isolated from monocotyledonous plants. The PcV strains formed a clearly distinct group in all the constructed phylogenetic trees. The number of strains phylogenetically classified as subspecies ‘P. c. subsp. brasiliensis’ in PcIV dramatically increased in size as a result of the characterization of new isolates or re-identification of previous P. carotovorum and P. atrosepticum strains. The P. carotovorum strains from Syria were grouped into PcI, PcII and PcIV. This grouping indicates a lack of correlation between the geographical origin and classification of these pathogens.
Productivity of crops grown for human consumption is at risk due to the incidence of pests, especially weeds, pathogens and animal pests. Crop losses due to these harmful organisms can be substantial and may be prevented, or reduced, by crop protection measures. An overview is given on different types of crop losses as well as on various methods of pest control developed during the last century.
Estimates on potential and actual losses despite the current crop protection practices are given for wheat, rice, maize, potatoes, soybeans, and cotton for the period 2001–03 on a regional basis (19 regions) as well as for the global total. Among crops, the total global potential loss due to pests varied from about 50% in wheat to more than 80% in cotton production. The responses are estimated as losses of 26–29% for soybean, wheat and cotton, and 31, 37 and 40% for maize, rice and potatoes, respectively. Overall, weeds produced the highest potential loss (34%), with animal pests and pathogens being less important (losses of 18 and 16%). The efficacy of crop protection was higher in cash crops than in food crops. Weed control can be managed mechanically or chemically, therefore worldwide efficacy was considerably higher than for the control of animal pests or diseases, which rely heavily on synthetic chemicals. Regional differences in efficacy are outlined. Despite a clear increase in pesticide use, crop losses have not significantly decreased during the last 40 years. However, pesticide use has enabled farmers to modify production systems and to increase crop productivity without sustaining the higher losses likely to occur from an increased susceptibility to the damaging effect of pests.
The concept of integrated pest/crop management includes a threshold concept for the application of pest control measures and reduction in the amount/frequency of pesticides applied to an economically and ecologically acceptable level. Often minor crop losses are economically acceptable; however, an increase in crop productivity without adequate crop protection does not make sense, because an increase in attainable yields is often associated with an increased vulnerability to damage inflicted by pests.
Pectolytic bacteria, including Pectobacterium spp. and Dickeya spp., are best isolated on crystal violet pectate (CVP), a semiselective medium containing pectin. The source of pectin is essential, because pectolytic bacteria are not able to degrade all of them. The aims of this study were to identify a new pectin source and to perfect formulations of semiselective CVP media to isolate the pectolytic bacteria Pectobacterium spp. and Dickeya spp. from different environmental compartments (plants, soil and water). The AG366 pectin, selected after screening six different formulations, was incorporated into single-layer (SL-CVPAG366) and double-layer (DL-CVPAG366) CVP media. Both media were compared with those based on Bulmer, Sigma-Aldrich and Slendid-Burger pectins, using 39 Pectobacterium and Dickeya strains. All strains formed deep cavities on AG366-CVPs, whereas nine did not produce cavities on Bulmer or Sigma-Aldrich media. Recovery rates were similar on DL-CVPAG366, Sigma-Aldrich and Bulmer CVPs for a given taxon, and did not differ significantly between SL- and DL-CVPAG366. Pectolytic bacteria were successfully isolated on both media from field samples of diseased potatoes, carrots, tobacco, onions, radishes and ornamentals. AG366 is thus a high-performance pectin source for the elaboration of CVP media suitable to isolate Dickeya and Pectobacterium. It is also efficient for enrichment purposes in liquid medium. The validation of AG366 as an improved source of pectin to recover the polyphagous Pectobacterium and Dickeya in different environmental compartments is essential given the current worldwide emergence and recrudescence of these bacteria.
Dickeya species (formerly Erwinia chrysanthemi) cause diseases on numerous crop and ornamental plants world-wide. Dickeya spp. (probably D. dianthicola) were first reported on potato in the Netherlands in the 1970s and have since been detected in many other European countries. However, since 2004–5 a new pathogen, with the proposed name ‘D. solani’, has been spreading across Europe via trade in seed tubers and is causing increasing economic losses. Although disease symptoms are often indistinguishable from those of the more established blackleg pathogen Pectobacterium spp., Dickeya spp. can initiate disease from lower inoculum levels, have a greater ability to spread through the plant’s vascular tissue, are considerably more aggressive, and have higher optimal temperatures for disease development (the latter potentially leading to increased disease problems as Europe’s climate warms). However, they also appear to be less hardy than Pectobacterium spp. in soil and other environments outside the plant. Scotland is currently the only country in Europe to enforce zero tolerance for Dickeya spp. in its potato crop in an attempt to keep its seed tuber industry free from disease. However, there are a number of other ways to control the disease, including seed tuber certification, on-farm methods and the use of diagnostics. For diagnostics, new genomics-based approaches are now being employed to develop D. dianthicola- and ‘D. solani’-specific PCR-based tests for rapid detection and identification. It is hoped that these diagnostics, together with other aspects of ongoing research, will provide invaluable tools and information for controlling this serious threat to potato production.
Pectobacterium carotovorum is a heterogeneous species consisting of two named subspecies, P. carotovorum subsp. carotovorum and P. carotovorum subsp. odoriferum. A third subspecies, P. carotovorum subsp. brasiliense, was previously proposed. The study aimed to confirm the subspecies status and validate the proposed name of P. carotovorum subsp. brasiliense using a novel and standard microbial taxonomy.
DNA-DNA hybridization confirmed that P. carotovorum subsp. brasiliense is a different species from P. wasabiae, P. betavasculorum and P. atrosepticum, with 28, 35 and 55% similarity values, respectively, but is a member of the P. carotovorum species with 73-77% similarity values. Sequencing the entire 16S rRNA gene of two polymorphic copies from strains of each of the P. carotovorum subspecies demonstrated that the average 16S rRNA gene sequence diversity between P. carotovorum subsp. brasiliense and P. carotovorum subsp. carotovorum was lower than the maximum genetic distances between two sequence types obtained from the same strain. Multilocus sequence analysis based on eight housekeeping genes (mtlD, acnA, icdA, mdh, pgi, gabA, proA and rpoS) differentiated the subspecies and delineated two P. carotovorum subsp. brasiliense clades.
Pectobacterium carotovorum subsp. brasiliense clade I was comprised of strains isolated from Brazil and Peru, while clade II included strains from Asia, North America and Europe. Strains in clade I but not clade II were phenotypically consistent with the original description of P. carotovorum subsp. brasiliense in that they produced reducing substances from sucrose and acid from α-methyl glucoside. The type strain for P. carotovorum subsp. brasiliense 212(T) (= LMG2137(T) = IBSBF1692(T) = CFBP6617(T) ) was previously designated. The GC mol content of the type strain is 51·7%.
the study introduces a full description for the strains belonging to the two different clades assigned to P. carotovorum subsp. brasiliense.
This paper briefly reviews research on the causative agents of blackleg and soft rot diseases of potato, namely Pectobacterium and Dickeya species, and the disease syndrome, including epidemiological and aetiological aspects. It critically evaluates control methods used in practice based on the avoidance of the contamination of plants, in particular the use of seed testing programmes and the application of hygienic procedures during crop production. It considers the perspective of breeding and genetic modification to introduce resistance. It also evaluates the application of physical and chemical tuber treatments to reduce inoculum load and examines the possibility of biocontrol using antagonistic bacteria and bacteriophages.
Sixty-five potato strains of the soft rot-causing plant pathogenic bacterium Dickeya spp., and two strains from hyacinth, were characterised using biochemical assays, REP-PCR genomic finger printing, 16S rDNA
and dnaX sequence analysis. These methods were compared with nineteen strains representing six Dickeya species which included the type strains. A group of twenty-two potato strains isolated between 2005-2007 in the Netherlands,
Poland, Finland and Israel were characterised as belonging to biovar 3. They were 100% identical in REP-PCR, dnaX and 16S rDNA sequence analysis. In a polyphasic analysis they formed a new clade different from the six Dickeya species previously described, and may therefore constitute a new species. The strains were very similar to a Dutch strain
from hyacinth. On the basis of dnaX sequences and biochemical assays, all other potato strains isolated in Europe between 1979 and 1994 were identified as D. dianthicola (biovar 1 and 7), with the exception of two German strains classified as D. dieffenbachia (biovar 2) and D. dadantii (biovar 3), respectively. Potato strains from Peru were classified as D. dadantii, from Australia as D. zeae and from Taiwan as D. chrysanthemi bv. parthenii, indicating that different Dickeya species are found in association with potato.
Detailed studies were conducted on the distribution of Pectobacterium carotovorum subsp. carotovorum and Dickeya spp. in two potato seed lots of different cultivars harvested from blackleg-diseased crops. Composite samples of six different
tuber sections (peel, stolon end, and peeled potato tissue 0.5, 1.0, 2.0 and 4.0cm from the stolon end) were analysed by
enrichment PCR, and CVP plating followed by colony PCR on the resulting cavity-forming bacteria. Seed lots were contaminated
with Dickeya spp. and P. carotovorum subsp. carotovorum (Pcc), but not with P. atrosepticum. Dickeya spp. and Pcc were found at high concentrations in the stolon ends, whereas relatively low densities were found in the peel and
in deeper located potato tissue. Rep-PCR, 16S rDNA sequence analysis and biochemical assays, grouped all the Dickeya spp. isolates from the two potato seed lots as biovar 3. The implications of the results for the control of Pectobacterium and Dickeya spp., and sampling strategies in relation to seed testing, are discussed.
Six Dickeya spp. strains representative of a larger group of bacteria isolated from potato, onion and irrigation water in Spain between
years 2003–2005, were characterised by biochemical, serological, molecular and pathogenicity assays. Biochemical and serological
differences, as well as pathogenic behaviour in host range and virulence levels, were observed among the strains. They were
classified into biovars 3 and 6. Phylogenetic analysis and comparison of the isolates with type strains of Dickeya species characterised to date were performed using concatenated partial sequences of the housekeeping genes gapA and mdh. One of the Spanish strains was identified as D. dieffenbachiae, whereas the other ones did not fit clearly into the previously described six Dickeya species, and may therefore constitute novel species. Isolation of dissimilar pathogenic strains in different rivers and irrigation
water sources supports the idea that Dickeya species is commonly present in such an environment, and contaminated water is a potential source of inoculum for the disease
in different crops.
Keywords
Dickeya species-Genetic relationship-Housekeeping genes-Pathogenicity
Suspected Dickeya sp. strains were obtained from potato plants and tubers collected from commercial plots. The disease was observed on crops
of various cultivars grown from seed tubers imported from the Netherlands during the spring seasons of 2004–2006, with disease
incidence of 2–30% (10% in average). In addition to typical wilting symptoms on the foliage, in cases of severe infection,
progeny tubers were rotten in the soil. Six strains were characterised by biochemical, serological and PCR-amplification.
All tests verified the strains as Dickeya sp. The rep-PCR and the biochemical assays showed that the strains isolated from blackleg diseased plants in Israel were
very similar, if not identical to strains isolated from Dutch seed potatoes, suggesting that the infection in Israel originated
from the Dutch seed. The strains were distantly related to D. dianthicola strains, typically found in potatoes in Western Europe, and were similar to biovar 3 D. dadanti or D. zeae. This is the first time that the presence of biovar 3 strains in potato in the Netherlands is described. One of the strains
was used for pathogenicity assays on potato cvs Nicola and Mondial. Symptoms appeared 2 to 3days after stem inoculation,
and 7 to 10days after soil inoculation. The control plants treated with water, or plants inoculated with Pectobacterium carotovorum, did not develop any symptoms with either method of inoculation. The identity of Dickeya sp. and P. carotovorum re-isolated from inoculated plants was confirmed by PCR and ELISA.
This compendium of techniques coverage of state-of-the-art developments in molecular biology, with over 600 pages of information contributed by a wide range of authorities.
Some simple formulae were obtained which enable us to estimate evolutionary distances in terms of the number of nucleotide substitutions (and, also, the evolutionary rates when the divergence times are known). In comparing a pair of nucleotide sequences, we distinguish two types of differences; if homologous sites are occupied by different nucleotide bases but both are purines or both pyrimidines, the difference is called type I (or "transition" type), while, if one of the two is a purine and the other is a pyrimidine, the difference is called type II (or "transversion" type). Letting P and Q be respectively the fractions of nucleotide sites showing type I and type II differences between two sequences compared, then the evolutionary distance per site is K = -(1/2) ln [(1-2P-Q) square root of 1-2Q]. The evolutionary rate per year is then given by k = K/(2T), where T is the time since the divergence of the two sequences. If only the third codon positions are compared, the synonymous component of the evolutionary base substitutions per site is estimated by K'S = -(1/2) ln (1-2P-Q). Also, formulae for standard errors were obtained. Some examples were worked out using reported globin sequences to show that synonymous substitutions occur at much higher rates than amino acid-altering substitutions in evolution.
Reconstructing phylogenies from intraspecific data (such as human mitochondrial DNA variation) is often a challenging task because of large sample sizes and small genetic distances between individuals. The resulting multitude of plausible trees is best expressed by a network which displays alternative potential evolutionary paths in the form of cycles. We present a method ("median joining" [MJ]) for constructing networks from recombination-free population data that combines features of Kruskal's algorithm for finding minimum spanning trees by favoring short connections, and Farris's maximum-parsimony (MP) heuristic algorithm, which sequentially adds new vertices called "median vectors", except that our MJ method does not resolve ties. The MJ method is hence closely related to the earlier approach of Foulds, Hendy, and Penny for estimating MP trees but can be adjusted to the level of homoplasy by setting a parameter epsilon. Unlike our earlier reduced median (RM) network method, MJ is applicable to multistate characters (e.g., amino acid sequences). An additional feature is the speed of the implemented algorithm: a sample of 800 worldwide mtDNA hypervariable segment I sequences requires less than 3 h on a Pentium 120 PC. The MJ method is demonstrated on a Tibetan mitochondrial DNA RFLP data set.
To determine the characteristics of bacteria associated with the blackleg disease of potato in Brazil and compare them with species and subspecies of pectolytic Erwinia.
Biochemical and physiological characteristics of 16 strains from blackleg-infected potatoes in State of Rio Grande do Sul, Brazil, were determined and differentiated them from all the E. carotovora subspecies and E. chrysanthemi. Pathogenicity and maceration ability of the Brazilian strains were greater than those of E. carotovora subsp. atroseptica, the causal agent of potato blackleg in temperate zones. Analyses of serological reaction and fatty acid composition confirmed that the Brazilian strains differed from E. carotovora subsp. atroseptica, but the sequence of 16S rDNA gene and the 16S-23S intergenic spacer (IGS) region confirmed the Brazilian strains as pectolytic Erwinia. Restriction analysis of the IGS region differentiated the Brazilian strains from the subspecies of E. carotovora and from E. chrysanthemi. A unique SexAI restriction site in the IGS region was used as the basis for a primer to specifically amplify DNA from the Brazilian potato blackleg bacterium in PCR.
The bacterium that causes the blackleg disease of potato in Brazil differs from E. carotovora subsp. atroseptica, the blackleg pathogen in temperate zones. It also differs from other subspecies of E. carotovora and from E. chrysanthemi and warrants status as a new subspecies, which would be appropriately named E. carotovora subsp. brasiliensis.
The blackleg disease of potato is caused by a different strain of pectolytic Erwinia in Brazil than in temperate potato-growing regions. The Brazilian strain is more virulent than E. carotovora subsp. atroseptica, the usual causal agent of potato blackleg.
Why is ( sp.) taking over? The ecology of a blackleg pathogen
- J V Van Der Wolf
- A Speksnijder
- H Velvis
- J V Haar
- J V Doorn