Characterisation of Erwinia carotovora subspecies and detection of Erwinia carotovora subsp. atroseptica in potato plants, soil and water extracts with PCR-based methods

A PCR-RFLP test based on a pectate-lyase encoding gene permits the detection of several Erwinia carotovora subspecies, but requires complete DNA extraction. This paper reports on the suitability of a simplified PCR-RFLP protocol to characterise E. carotovora strains and on the performance of PCR, using the same primers, to detect the atroseptica subspecies in substrates of epidemiological significance. A collection of 140 strains from various hosts and geographical origins was characterised for biochemical traits and PCR-RFLPs. PCR performed on boiled bacterial suspensions yielded an amplification product of 434 bp in 109 of the 140 strains. None of the E. carotovora subsp. betavasculorum strains was amplified, even after complete DNA extraction. RFLPs of the PCR product yielded 24 groups, 3 of which were new. Twenty one groups were specific to one subspecies. Several strains biochemically similar to E. carotovora subsp. atroseptica, but growing at 37 °C, showed PCR-RFLP profiles characteristic of E. carotovora subsp. carotovora. Phenetic and cladistic analyses gave three main domains, not strictly related to hosts or geographical origins. The atroseptica (RFLP groups 1 and 2) and wasabiae (group 21) subspecies constituted one of the domains, despite clustering distantly from one another. Host specialisation and molecular homogeneity suggest a clonal structure within these subspecies. Conversely, E. carotovora subsp. odorifera, despite its limited host range and geographical distribution, and E. carotovora subsp. carotovora showed great molecular diversity, spreading respectively across five and 19 RFLP groups. These two subspecies shared RFLP groups 4, 5 and 6. The tree nodes in the phenograms showed a low robustness when bootstrapping the data matrix. PCR coupled with a 48h enrichment step in a polypectate-rich medium improved detection thresholds of E. carotovora subsp. atroseptica (1.5.10²- 1.5.10³ bacteria/ml in leaves, stems, and tuber peel extracts to 4.10⁷ bacteria/ml in wash water) relative to either immunomagnetic separation coupled with PCR or DAS-ELISA (2.10⁵ in plant samples to 2.10⁷ bacteria/ml in wash water).     

Development of symptoms caused by Erwinia carotovora ssp. atroseptica under field conditions and their effects on the yield of individual potato plants

Infection of seed tubers by pectinolytic Erwinia species can lead to the development of various symptoms during vegetative growth of potato crops, including non-emergence of plants, chlorosis, wilting, haulm desiccation and typical blackleg. The relationships between types of symptoms and yield are poorly documented, and are investigated by following the development of symptoms in potato plants grown under field conditions from seed tubers artificially inoculated with E. carotovora ssp. atroseptica ( Eca ) , and measuring the yield of each plant. Symptoms were classified into five main types (non-emergence, wilting/chlorosis, blackleg, haulm desiccation and plant death). Each plant was scored for types of symptom on four successive dates; plants without visible symptoms were scored as healthy. The method of inoculation and inoculum concentration proved major factors for the subsequent development of symptoms. Disease development was more severe after vacuum infiltration of bacteria into seed tubers than after shaking tubers in contaminated sand. Disease usually progressed from chlorosis and/or wilting to partial or total desiccation on a given plant. Yield losses varied according to symptom type, but the relationship between symptoms recorded and yield also depended on scoring dates. The data suggest that the beginning of tuber growth might be the most suitable stage for predicting yield losses from symptom observations. In both cultivars studied (Bintje, highly susceptible, and Désirée, moderately resistant), the yield of symptomless plants growing from inoculated seed tubers was significantly less than that of control plants, indicating that the presence of bacteria on the seed tuber was detrimental, even in the absence of visible symptoms. Differences in symptom expression in the field between cultivars matched the level of visible infection of tubers at harvest, as Bintje tubers showed a higher incidence of rot than Désirée tubers.     

Methods of testing potato cultivars for resistance to soft rot of tubers caused by Erwinia carotovora subsp. atroseptica

The resistance of eight potato cultivars to tuber soft rot caused by E. carotovora subsp. atroseptica was assessed in 2 years using three different test methods. Similar cultivar resistance rankings were obtained for any one method within a year and between years for two methods (single site, infectivity titration), but not for the third (vacuum infiltration). However, the ranking of cultivars differed for the three methods. Ranking was not affected by inoculating the cortex or the more susceptible medullary tissue, or by assessing rotting in terms of infection frequency or lesion size, but it was affected by oxygen concentration during incubation. Differences among cultivars were greater when inoculated tubers were incubated anaerobically than when incubated with 5% oxygen. There was no relationship between the relative susceptibilities of cultivars to tuber soft rot in storage in January/February and those of mother tubers after planting.     

Influence of abiotic factors,inoculum source,and cultivar susceptibility on the potato tuber blemish diseases black dot (Colletotrichum coccodes) and silver scurf (Helminthosporium solani)

Black dot and silver scurf are potato blemish diseases whose economic impact has increased in recent years. Because their symptomatology on tubers is visually similar, disease assessment does not usually differentiate between the two pathogens, which share the same ecological niche. The epidemiology of black dot has been extensively studied, especially in the UK, but the factors that influence silver scurf have been less investigated. In this study, the influence of cultivar, source of inoculum, and environmental conditions on both diseases was studied in field trials over a three-year period (2016–2018) in Switzerland. Planting minitubers did not prevent either disease in daughter tubers, indicating the contribution of soil as an inoculum reservoir. An arbitrary threshold of Colletotrichum coccodes soil inoculum could be set to discriminate between low and high disease risk. For the first time, Helminthosporium solani DNA was detected in stolons, and infections appeared earlier in stolons than in tubers. H. solani stolon and tuber infections usually appeared later in the season than those of C. coccodes. Black dot severity correlated positively with precipitation, while silver scurf severity correlated positively with temperature. Table potato cultivars commonly grown in Switzerland exhibited significant differences in susceptibility to both diseases, and cultivars with low susceptibility to both silver scurf and black dot were identified. These results gave new insights into understanding the factors driving the epidemiology of potato blemish diseases and may contribute to building a risk assessment scheme to manage both diseases simultaneously.     

几种化学物质诱导彩色马蹄莲对软腐病抗性的研究

本试验测定了纳米硅、草酸、硅酸钠和硫酸亚铁4种化学物质在不同浓度下对彩色马蹄莲软腐病菌的室内抑菌活性和诱导抗病效果。结果表明,硫酸亚铁3种浓度对彩色马蹄莲软腐病菌均表现较强的抑制作用;1 g/L纳米硅、0.15 g/L草酸和0.2 g/L硅酸钠3种化学物质对软腐病菌无明显抑制作用且诱导抗病效果较好,分别达到62.28%、77.56%、88.46%;经3种化学物质诱导处理后再进行接种,诱导处理和接种期间彩色马蹄莲叶片组织内过氧化物酶(POD)、苯丙氨酸解氨酶(PAL)、多酚氧化酶(PPO)酶活性均高于对照,其中硅酸钠处理后彩色马蹄莲叶片组织内POD、PAL活性高于纳米硅和草酸处理,草酸处理后叶片组织内PPO活性高于纳米硅和硅酸钠处理。     

Volatile Metabolite Profiling for the Discrimination of Onion Bulbs Infected by Erwinia carotovora ssp. carotovora, Fusarium oxysporum and Botrytis allii

The volatile metabolites of the headspace gas of onion bulbs inoculated with three different pathogens, Erwinia carotovora ssp. carotovora, Fusarium oxysporum and Botrytis allii, were profiled using gas chromatography/mass spectrometry. Differences in the number and amount of volatile metabolites were observed. Two hundred and fifty three volatile metabolites were detected in bulbs inoculated with three pathogens or sterile distilled water. On day three, 202 volatile metabolites were observed, compared to 166 on day six. Of the 253 compounds, however, only 59 occurred relatively consistently over replications, of which 25 compounds were specific to one or more pathogens, including 10 that were unique to a pathogen. Metabolites such as 1-Oxa-4,6-diazacyclooctane-5-thione and 4-mercapto-3-(methylthio)--(thio-lactone)-crotonic acid were exclusive to onions inoculated with F. oxysporum. Acetone, acetic acid-hydrazide, propylcarbamate, 1-bromo-1-propene, thiirane, 1-(methylthio)-E-1-propene and 1-ethenyl-4-ethyl-benzene were specific to B. allii. 3-bromo-furan was specific to E. carotovora ssp. carotovora. Sterile water-inoculated bulbs produced 3,3-dioxy-1,2-propanediol-tetranitrate. Highest amount of sulfurs was found in pathogen-inoculated, while highest amounts of terpenes, aromatics and aliphatics were found in sterile distilled water-inoculated bulbs. The possible use of these differences in the volatile metabolites for detecting and discriminating diseases of onion in storage is discussed.     

A comparison of methods to evaluate the susceptibility of potato cultivars to blackleg (caused by Erwinia carotovora subsp. atroseptica) in the field at different sites

Potato seed tubers were inoculated either using a needle or using a high-pressure jet injector at a number of sites in the United Kingdom. Over 2 years, different cultivars showed different trends in blackleg symptoms. Comparisons with inoculation by infiltration of tubers under vacuum were made at two sites, and by damaging followed by dipping tubers in inoculum at one site. The relationship between plant vigour in July and yield was closer than that between blackleg symptoms and yield.     

利用CRISPR/Cas13a编辑技术获得马铃薯抗PVX植株

为探究利用CRISPR/Cas13a系统获得抗马铃薯X病毒（potato virus X,PVX）马铃薯的可行性,通过设计靶向PVX中TGBp1基因的小向导RNA（small guide RNA,sgRNA）,构建CRISPR/Cas13a基因编辑载体,以马铃薯栽培种Désirée为受体材料进行稳定遗传转化,并通过机械摩擦接种法鉴定转基因植株对PVX的抗性。结果显示,成功构建了靶向PVX的PVX-Cas13a载体,并获得了表达Cas13a的转基因马铃薯植株,对其中3个高表达Cas13a的株系进行PVX抗性鉴定,发现在接种PVX 10~20 d后,转基因植株系统叶上无明显发病症状,且PVX积累量均显著低于未接种PVX对照。表明靶向PVX的CRISPR/Cas13a系统能够有效抑制该病毒的积累,这为马铃薯抗PVX育种提供了一条有效策略。     

Characterization of Fusarium spp. responsible for causing dry rot of potato in Great Britain

A 3-year survey was undertaken to establish the relative frequency of different Fusarium spp. present as inoculum on potato tubers collected from four regions of Great Britain. A total of 219 samples (comprising 10 950 tubers) were collected from the 2000, 2001 and 2002 crops and processed to recover dry rot-producing isolates. In total, 228 isolates of Fusarium spp. were recovered. Most (94·7%) of these isolates were attributed to one of four Fusarium species: F. coeruleum, F. avenaceum, F. culmorum and F. sambucinum (formerly F. sulphureum) . The incidence of the combined Fusarium spp. increased the further south the crops had been grown. Fusarium coeruleum was the most commonly isolated species in each survey year, comprising 37 to 52% of the total Fusarium species. Selected isolates of each species were evaluated for their ability to produce rots in potato tubers. Fusarium sambucinum was a more aggressive pathogen than the other Fusarium species in eight out of 10 cultivars. Fusarium avenaceum and F. culmorum were relatively weaker pathogens. However, these species were aggressive on some cultivars, notably Hermes. The selected isolates were also assessed for their sensitivity to the fungicides thiabendazole and imazalil. Using in vitro tests, 65% of F. sambucinum isolates were resistant to thiabendazole and 7% of F. avenaceum isolates were resistant to imazalil. Tubers treated with imazalil yielded a higher proportion of isolates of F. avenaceum than those that were untreated. Similarly, a higher proportion of F. sambucinum isolates were recovered from tubers treated with thiabendazole than from those that were not treated.     

Evaluation Of Tests to Determine Resistance of Zantedeschia spp. (Araceae) to Soft Rot caused by Erwinia carotovora subsp. carotovora

Bacterial soft rot caused by Erwinia carotovora subsp. carotovora is a major disease in Zantedeschia spp., particularly in cultivars from the section Aestivae. The disease can be partly controlled by cultural measures, but by combining cultural methods with resistant plant material a promising strategy for control of soft rot can be developed. No tests are available for resistance testing in breeding Zantedeschia spp. Therefore, three tests developed for use in potato breeding were adapted for use on eight cultivars of Zantedeschia spp. Variation was found in all three tests. Resistant control cultivar Zantedeschia aethiopica Crowborough scored most resistant in all three tests. Within the section Aestivae, degrees of susceptibility were identified that were in agreement with each other and with field observations, indicating reliability of two of the methods in which tubers were used. The correlation coefficient of these two tests was high. A new non-destructive test method was developed for use on seedlings which involved immersion of leaf disks in a bacterial suspension. The percentage of decayed leaf area was a measure of resistance and results were in general agreement with the other tests. These methods will be useful for breeding for soft rot resistance and performing genetic analyses.     

The importance of the infected seed tuber and soil inoculum in transmitting Potato mop‐top virus to potato plants

Potato mop‐top virus (PMTV), the cause of spraing in potato tubers, is transmitted by Spongospora subterranea, the cause of powdery scab, and by planting infected seed tubers. This study was undertaken to determine the relative importance of these sources of infection in seed potato production in Scotland. The transmission of PMTV from tested seed tubers to daughter plants was examined over 2 years and six cultivars. The development of foliar symptoms varied with year and cultivar. Infection of daughter tubers derived from PMTV‐infected seed tubers was more prevalent on plants affected by foliar symptoms than those without symptoms. The rate of transmission of PMTV from infected seed tubers to daughter tubers ranged from 18 to 54%. Transmission was affected by cultivar and by origin of seed tubers used for a cultivar, but not by a cultivar's sensitivity to PMTV infection. The incidence of PMTV in daughter tubers of cv. Cara grown from seed potatoes from one source (common origin) by more than 25 seed producers was examined over two successive generations. The incidence of PMTV in daughter tubers was not correlated with that in the seed tubers but appeared to be strongly associated with soil inoculum. The incidence of PMTV was correlated with powdery scab in those crops in which both were present. There was some evidence from soil tests conducted in 2006 using a tomato bait plant and real‐time RT‐PCR that planting PMTV‐infected seed potatoes could increase the risk of introducing the virus into land not infested by PMTV.