Role of certain potato tubers constituents in their resistance to bacterial soft rot caused by Erwinia carotovora pv. carotovora

Erwinia soft rot causes destructive and serious damages to many vegetable crops including potato, in the field, transit and storage periods. The role of certain potato tuber constituents in the physiology of disease resistance has been investigated. Pectin substances and calcium contents of potato tuber had a pronounced role in the physiology of disease resistance. Alpha and Santa (less susceptible cultivars) contained the higher amount of pectin and calcium compared by Mirkal, Diamant, Askort, Geganite and Nicola cultivars (more susceptible cultivars). Tubers extracts of all healthy tested potato tubers cultivars contained fructose except Santa cultivar and glucose except Alpha and Mirkal cultivars. Tuber extracts of the more susceptible cultivars (Nicola and Askort) contained a higher concentration of glucose and fructose than those of less susceptible cultivars (Geganite, Mirkal, Santa, Alpha and Diamant).     

Effect of certain cultural practices on susceptibility of potato tubers to soft rot disease caused by Erwinia carotovora pv. carotovora

Erwinia soft rot causes destructive and serious damage to many vegetable crops including potato in the field, transit and storage periods. The effect of certain cultural practices on the susceptibility of potato tubers to soft rot bacteria was studied and the results of this work can be summarised in the following: potato tubers harvested on 1 May first exhibited the highest disease incidence compared with those harvested on 15 May or 30 May. Harvesting on 15 June resulted in the lowest disease infection. The application of high levels of nitrogen fertiliser as urea (46.5%), ammonium nitrate (31%) and ammonium sulphate (20.5%) resulted in an increase of the susceptibility of potato tubers to bacterial soft rot disease. In contrast, the addition of phosphorous as superphosphate (15.5%) fertiliser caused the reverse effect. The addition of potassium as potassium sulphate (48%) alone at any of the tested levels showed no effect. The susceptibility of potato tubers to bacterial soft rotting disease was increased by increasing storage periods at 4°C for 1, 2, 3 and 4 months. Spraying copper sulphate exhibited the highest decrease in soft rot incidence disease followed by manganese, zinc and iron. However, spraying of boron increased susceptibility to the disease. Potato tubers obtained from plants sprayed with copper and stored for different periods showed the lowest susceptibility to disease incidence. Tuber sprayed with zinc, iron, manganese and finally boron came next.     

Characterization of blackleg and soft rot from potato in northern Thailand

Blackleg and soft rot of potato cause economic loss through reduced yield and quality. The causal agents of bacterial blackleg and soft rot of potato were identified based on biological data and sequence analyses of the 16S rDNA gene. Between 2016 and 2018, diseased potato stems and tubers were collected in Chai Prakan District, Chiang Mai Province, and Chiang Khum District, Pa Yao Province. The symptoms included black stem lesions, soft rot on tubers, wilting, break down of the stem vascular ring and foliar yellowing. Of 13 bacterial isolates, five were identified as Pectobacterium carotovorum subsp. brasiliense, four‐Dickeya dadantii, two‐Pseudomonas putida and two‐Bacillus altitudinis. Pathogenicity tests of P. carotovorum subsp. brasiliense and D. dadantii resulted in lower leaves turning yellow and wilting followed by blackleg symptoms on lower stems and maceration of tuber tissue. Symptoms caused by P. putida were yellowing and wilting of leaves. B. altitudinis caused yellowing of the lower leaves and wilting followed by drying of leaf tissue. This is a first report of these bacterial pathogens causing blackleg and soft rot of potato in Thailand.     

Inhibitory effects on microbial growth of Botrytis cinerea and Erwinia carotovora on potato using of a biopolymer chitosan at different molecular weights

The antimicrobial efficiency of chitosan at different molecular weights (5, 37, 57 and 290 kDa) against Botrytis cinerea and Erwinia carotovora on potato (Solanum tuberosum L.) was investigated. In vitro study showed that chitosan of 37 kDa was the most active against E. carotovora (minimum inhibitory concentration (MIC) = 950 mg/L), whereas 5 kDa chitosan was the most active against B.cinerea. Coating of potato tubers with 100, 250 and 500 mg/L significantly decreased the rate of weight loss and chitosan of 37 kDa showed the best effect. The in vivo antibacterial effect indicated that all treatments (500, 1000 and 2000 mg/L) significantly inhibited the growth of E. carotovora compared with the control. The lowest decay incidence was observed with 37 kDa chitosan. However, the antifungal activity against B. cinerea inoculated of leaves showed no decay incidence at 500 and 1000 mg/L with 57 kDa chitosan after 48 h.     

Differentiation of Erwinia carotovora subsp. atroseptica and carotovora by RAPD-PCR

Six different 10-mer oligonucleotide primers were used to differentiate Erwinia carotovora subsp. atroseptica (Eca) and carotovora (Ecc) using RAPD-PCR. All primers gave different banding patterns for Eca and Ecc indicating their value for identification. UPGMA clustering analysis clearly showed two separate clusters, one for Eca and the other for the Ecc group. Similarity within Eca strains was very high, over 85% among most isolates but within the Ecc group extensive genetic diversity was found and many of the Ecc strains were no more than 50% similar. Similarity between the 10 Eca and 10 Ecc strains was generally only 10–25% based on the results from six primers. Three RAPD fragments from Eca group, which were amplified by three different RAPD primers, were isolated and used as probes for Southern hybridisation to test, if homologous fragments were amplified from Ecc strains. All these probes hybridised only with Eca isolates indicating that these fragments could be useful in order to develop a PCR-based detection system for Eca strains.     

Characterization of Iranian Pectobacterium carotovorum Strains from Sugar Beet by Phenotypic Tests and Whole-cell Proteins Profile

Thirty isolates of Pectobacterium carotovorum from soft rot‐affected sugar beet plants in the Fars province of Iran were characterized phenotypically and by analysis of whole‐cell protein electrophoresis patterns. The isolates were found to be heterogeneous based on the results of physiological and biochemical tests and protein profiles. The results of numerical analysis of phenotypic characteristics and protein patterns showed that only 27% of the collected isolates (phenon 4) could be identified as P. betavasculorum when compared with reference strains. Strains of the first, second, third and fifth phenon shared similar characters with those of P. carotovorum subsp. carotovorum, P. betavasculorum and P. carotovorum subsp. odoriferum, but were distinct from these subspecies. Inoculation of phenon 4 isolates into wounded sugar beet petioles led to black streaking, root rot and vascular necrosis. Other isolates were incapable of causing systemic symptoms in inoculated plants.     

Bacterial Stem Rot in Greenhouse Pepper (Capsicum annuum L.) in Sardinia (Italy): Occurrence of Erwinia carotovora subsp. carotovora

An unusual bacterial disease was observed in pepper plants during research carried out in greenhouses in central‐north Sardinia. The characteristics were: the presence of lesions and exudates on stems, soft rot of the pith, and a brownish‐black colour in the petioles and leaf‐veins. Only two isolates of 21 were pathogens. One was obtained from exudate present on the stem and the other from pith. Experimental infections revealed that the bacterial isolates were particularly aggressive in the stems and fruit of pepper and tomato. Biochemical, physiological and serological tests in conjunction with fatty acid profile analysis confirmed that they were Erwinia carotovora subsp. carotovora (Jones) Bergey et al. The product of 434 bp polymerase chain reaction (PCR) enabled a preliminary identification of isolates to be made. Restriction fragment length polymorphism (RFLP) analysis of amplification products showed that the isolates DPP 23_ef and DPP 24_m, strain type CFBP 2046 and DPP 28₁, isolated from pepper fruit, belonged to the RFLP group 12, whereas DPP 29, also isolated from pepper fruit, was included in RFLP group 1. Measures to prevent and control this recently introduced disease are suggested in the conclusion of this paper.     

Specific detection of Pectobacterium carotovorum by loop‐mediated isothermal amplification

Potatoes are an important agroeconomic crop worldwide and maceration diseases caused by pectolytic bacterial pathogens result in significant pre‐ and post‐harvest losses. Pectobacterium carotovorum shares a common host range with other Pectobacterium spp. and other members of the Enterobacteriaceae, such as Dickeya spp. As these pathogens cannot be clearly differentiated on the basis of the symptoms they cause, improved methods of identification are critical for the determination of sources of contamination. Current standardized methods for the differentiation of pectolytic species are time consuming and require trained personnel, as they rely on traditional bacteriological practices that do not always produce conclusive results. In this growing world market, there is a need for rapid diagnostic tests that can differentiate between pectolytic pathogens, as well as separate them from non‐pectolytic enteric bacteria associated with soft rots of potato. An assay has been designed previously to detect the temperate pathogen Pectobacterium atrosepticum, but there is currently no recognized rapid assay for the detection of the tropical/subtropical counterpart, Pectobacterium carotovorum. This report describes the development of a loop‐mediated isothermal amplification (LAMP) assay that detects P. carotovorum with high specificity. The assay was evaluated using all known species of Pectobacterium and only showed positive reactions for P. carotovorum. This assay was also tested against 15 non‐target genera of plant‐associated bacteria and did not produce any false positives. The LAMP assay described here can be used as a rapid test for the differentiation of P. carotovorum from other pectolytic pathogens, and its gene target can be the basis for the development of other molecular‐based detection assays.     

Interactions of Ralstonia solanacearum and Pectobacterium carotovorum with Meloidogyne incognita on potato

Effect of interactions of Meloidogyne incognita with Ralstonia solanacearum and interaction of M. incognita with Pectobacterium carotovorum were studied in sequential and simultaneous inoculations on potato (Solanum tuberosum). Inoculation of M. incognita caused a lesser reduction in plant growth than caused by R. solanacearum. Inoculation of M. incognita plus R. solanacearum caused a greater reduction in plant growth than the damage caused by either pathogen. Inoculation of M. incognita prior to R. solanacearum resulted in a greater reduction in plant growth than R. solanacearum was inoculated prior to M. incognita. However, inoculation of M. incognita or P. carotovorum caused similar reduction in plant growth. Inoculation of P. carotovorum prior to M. incognita caused lesser reduction in plant growth than simultaneous inoculation of both pathogens. Inoculation of M. incognita caused galling in potato roots but the size of galls was small. Inoculation of P. carotovorum or R. solanacearum with M. incognita had adverse effect on galling and nematode multiplication. Wilting or soft rot index was 3 when R. solanacearum or P. carotovorum was inoculated alone. In other treatments, where R. solanacearum or P. carotovorum was inoculated with M. incognita, wilting or soft rot indices were 5.     

A new clade of Dickeya spp. plays a major role in potato blackleg outbreaks in North Finland

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.     

珠芽魔芋对细菌性软腐病的抗性鉴定研究

采用魔芋Amorphophallus spp.块茎点种、注射、灌根接种及田间调查等方法,对国内普遍栽种的珠芽红魔芋A. bulbifer、珠芽金魔芋A. muelleri、花魔芋A. konjac和白魔芋A. albus等12个种质材料进行抗软腐病鉴定、比较和评价,以分析珠芽魔芋对抗细菌性软腐病的抗病水平。结果表明,供试材料对软腐病抗性差异较大,珠芽金魔芋种质对细菌性软腐病均有免疫性(I),德宏及临沧珠芽红魔芋种质为高抗病品种(HR);缅甸珠芽红魔芋为抗病品种(R);富源花魔芋、楚雄花魔芋、日本农林2号、鄂魔芋1号、秦魔1号、昭通白魔芋、丽江白魔芋均属易感品种(S),与田间抗性调查情况基本相符。     

Multiplex detection and identification of bacterial pathogens causing potato blackleg and soft rot in Europe,using padlock probes

The objective of this study was to develop a multiplex detection and identification protocol for bacterial soft rot coliforms, namely Pectobacterium wasabiae (Pw), Pectobacterium atrosepticum (Pba) and Dickeya spp., responsible for potato blackleg and tuber soft rot. The procedures were derived from the phylogenetic relationships of these and other Enterobacteriaceae based on recA sequences. The group of Pw strains was highly homogeneous and could be distinguished from the other species. A ligation‐based method for detection of Pw was developed. Five padlock probes (PLPs) were designed, targeting recA sequences to identify the Pw, Pba or Dickeya spp., whereas a sixth probe recognised recA sequences of all soft rot coliforms including Pectobacterium carotovorum subsp. carotovorum (Pcc). Two PLP‐based applications were developed: one using real‐time PCR and one using universal microarrays. Assay sensitivity and specificity were demonstrated using 71 strains of Pw, Pcc, Pba and Dickeya spp. Both multiplex methods can be potentially used for seed testing and in ecological studies, but further validation is required.     

Simultaneous detection of major blackleg and soft rot bacterial pathogens in potato by multiplex polymerase chain reaction

A multiplex polymerase chain reaction (PCR) assay for simultaneous, fast and reliable detection of the main soft rot and blackleg potato pathogens in Europe has been developed. It utilises three pairs of primers and enables detection of three groups of pectinolytic bacteria frequently found in potato, namely: Pectobacterium atrosepticum, Pectobacterium carotovorum subsp. carotovorum together with Pectobacterium wasabiae and Dickeya spp. in a multiplex PCR assay. In studies with axenic cultures of bacteria, the multiplex assay was specific as it gave positive results only with strains of the target species and negative results with 18 non‐target species of bacteria that can possibly coexist with pectinolytic bacteria in a potato ecosystem. The developed assay could detect as little as 0.01 ng µL^–1 of Dickeya sp. genomic DNA, and down to 0.1 ng µL^–1 of P. atrosepticum and P. carotovorum subsp. carotovorum genomic DNA in vitro. In the presence of competitor genomic DNA, isolated from Pseudomonas fluorescens cells, the sensitivity of the multiplex PCR decreased tenfold for P. atrosepticum and Dickeya sp., while no change was observed for P. carotovorum subsp. carotovorum and P. wasabiae. In spiked potato haulm and tuber samples, the threshold level for target bacteria was 10¹ cfu mL^–1 plant extract (10² cfu g^–1 plant tissue), 10² cfu mL^–1 plant extract (10³ cfu g^–1 plant tissue), 10³ cfu mL^–1 plant extract (10⁴ cfu g^–1 plant tissue), for Dickeya spp., P. atrosepticum and P. carotovorum subsp. carotovorum/P. wasabiae, respectively. Most of all, this assay allowed reliable detection and identification of soft rot and blackleg pathogens in naturally infected symptomatic and asymptomatic potato stem and progeny tuber samples collected from potato fields all over Poland.     

Isolation and Preliminary Characterization of Cryptic Plasmids from Erwinia carotovora

Of the fifty-two Erwinia carotovorastrains studied, sixteen were found to contain extrachromosomal DNA (plasmids) from 2.5 to 129 kbp in size. Some E. carotovorastrains bore two to five different plasmids. Experiments showed that the cryptic plasmids of erwinia are not responsible for their resistance to antibiotics and are not involved in the synthesis of macromolecular colicin-like carotovoricins. At the same time, one of the E. carotovorastrains, 13A, augmented the production of carotovoricin after curing from one of its plasmids, the 47.7-kbp pCA 6-2. Three E. carotovorasubsp.carotovorastrains and one E. carotovorasubsp.atrosepticastrain contained large 129-kbp plasmids, which may play a role in the ecology of phytopathogenic pectinolytic erwinia.     

Susceptibility of eight potato cultivars to tuber infection by Phytophthora erythroseptica and Pythium ultimum and its relationship to mefenoxam-mediated control of pink rot and leak

In addition to cultural practices, the application of the fungicide mefenoxam is an important disease management tactic used to control both pink rot and leak on potato tubers grown in the USA. Mefenoxam resistance has been identified in many of the potato growing regions, and therefore resistance management strategies are very important for retaining this fungicide as a tool to manage these storage rot diseases. The relationship between mefenoxam efficacy and cultivar susceptibility to pink rot and leak was assessed in post‐harvest inoculation studies. Mefenoxam was applied to potato (Solanum tuberosum) cultivars known to express varying levels of susceptibility to pink rot and leak caused by Phytophthora erythroseptica and Pythium ultimum, respectfully. Tubers harvested from plants treated with in‐furrow and foliar applications of mefenoxam were inoculated with isolates sensitive to the fungicide. Incidence and severity of both diseases ranged widely among cultivars. Russet Norkotah was the most susceptible to infection by P. erythroseptica, while cvs Pike and Atlantic were the most resistant. Cultivars Dark Red Norland, Russet Norkotah, Goldrush and Russet Burbank were most susceptible to infection by P. ultimum whereas Snowden was most resistant. Control of pink rot differed significantly among cultivars following mefenoxam treatment, ranging from 28% (cv. Goldrush) to 67% (cv. Snowden) and generally provided the greatest level of disease control on susceptible and moderately susceptible cultivars such as Russet Norkotah and Snowden, respectively. In contrast, the impact of mefenoxam on leak development was minimal and disease control did not differ significantly among the cultivars. The fungicide failed to control leak in the susceptible cvs Atlantic and Pike and control ranged from 1.7% to 5.2% in cvs Goldrush, Russet Norkotah, Dark Red Norland, Russet Burbank and Kennebec. The greatest level of leak control was achieved with the moderately resistant cv., Snowden, at 12.7%. Cultivars most likely to benefit from mefenoxam treatments should be targeted as part of a pink rot management programme. Judicious use of the fungicide, when matched with the level of cultivar susceptibility, may prove to be an efficient and effective approach to reduce infection rates and possibly manage mefenoxam resistance thereby maintaining longevity of the compound.     

Pre-PCR Processes in the Molecular Detection of Blackleg and Soft Rot Erwiniae in Seed Potatoes

The polymerase chain reaction (PCR) based detection of blackleg and soft rot erwiniae involves pre‐PCR processing steps which may compromise the sensitivity of detection. The aim of this study was to standardize these various steps to develop reproducible diagnostic PCR protocol for the detection of the three known soft rot erwiniae as they occur in the tuber, singly or in combination. Comparison of tuber peel and stolon end tissue as a starting material for enrichment of the bacteria indicated that tuber peel samples resulted in more representative and sensitive detection of the strains than extract from stolon end tissues. Substances of potato origin in the peel extract were found to be highly inhibitory to the PCR. Addition of the antioxidant Dethiotreitol to the samples before enrichment did not have any significant effect on detection during the 24 h period incubation of the peel extract at room temperature. Bulk washing of tubers with one rotten tuber included with the working sample caused surface contamination on 67–91% of the healthy tubers. Washing tubers individually circumvents the problem. The optimum temperature for enrichment of all the three strains was 27°C. At 37°C, Pectobacterium carotovorum failed to be detected while PCR on Pectobacterium atrosepticum and isolates of Dickeya spp. always produced amplification of the specific DNA fragments. Viability test on Nutrient Agar showed that only Dickeya isolates were viable after 48 h of incubation at 37°C suggesting that the detection of P. atrosepticum at 37°C was from dead or non‐viable cells. Post cell death detection experiment further confirmed that DNA was amplified from dead cells of all the strains at 27°C and 33°C whereas at 37°C, only DNA from dead cells of isolates of Dickeya and P. atrosepticum were amplified. There was no amplification from the dead cells of all isolates of P. carotovorum following the 48 h post death incubation at 37°C. The reason for this difference in post death longevity is not clear at this stage.     

Detection,identification and differentiation of Pectobacterium and Dickeya species causing potato blackleg and tuber soft rot: a review

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.     

Characterization of the early events of potato tuber development

The early events of potato ( Solanum tuberosum L. cv. Superior) tuberization were examined by using a model system of axillary bud tuber development from petiole-leaf single-node cuttings. Both fresh weight and starch accumulation were monitored to establish a developmental framework for morphological changes. Fresh weight and starch content began to increase in axillary buds after 2 days. Visible changes in bud morphology could be detected 4 days after the start of incubation. Substantial increases in both total protein and total RNA were observed at the onset of tuber morphology. Immunoblot analysis showed that the major tuber protein, patatin, could be initially detected in day 4 buds and that a 22-kDa proteinase inhibitor could be initially detected at day 8. Northern blot analysis corroborated this pattern of accumulation at the RNA level for both protein types. Substantial accumulation of the two proteinase inhibitor mRNAs occurred later than patatin mRNA accumulation. The results of this study showed that there is considerable accumulation of both protein and mRNA occurring during the early stages of tuber development prior to the substantial accumulation of the major tuber storage proteins.