Characterisation of Pectobacterium wasabiae and Pectobacterium carotovorum subsp. carotovorum isolates from diseased potato plants in Finland

To identify bacteria causing soft rot and blackleg in potato in Finland, pectinolytic enterobacteria were isolated from diseased potato stems and tubers. In addition to isolates identified as Pectobacterium atrosepticum and Dickeya sp., many of the isolated strains were identified as Pectobacterium carotovorum subsp. carotovorum. Phylogenetic analysis and biochemical tests indicated that one of the isolates from potato stems resembled Pectobacterium wasabiae. Furthermore, two blackleg‐causing P. carotovorum strains recently isolated in Europe clustered with P. wasabiae, suggesting that at least some of these isolates were originally misidentified. All the other Finnish P. carotovorum isolates resembled the subsp. carotovorum type strain in biochemical tests but could be clustered into two distinct groups in the phylogenetic analysis. One of the groups mainly contained strains isolated from diseased tubers, whereas the other mainly included isolates from potato stems. In contrast to the tuber isolates, the stem isolates lacked genes in Type III secretion genes, were not able to elicit a hypersensitive response in tobacco leaves and produced only small amounts of autoinducers in the stationary phase in vitro. P. wasabiae isolate was able to cause similar amount of blackleg‐like symptoms as P. atrosepticum in a field experiment with vacuum‐infiltrated tubers, whereas both P. atrosepticum and P. carotovorum isolates reduced emergence and delayed growth more than P. wasabiae. Our findings confirm the presence of P. wasabiae in Finland and show that the Finnish P. carotovorum subsp. carotovorum isolates can be divided into two groups with specific characteristics and possibly also different ecologies.     

Salicylic and jasmonic acid pathways are necessary for defence against Dickeya solani as revealed by a novel method for Blackleg disease screening of in vitro grown potato

Potato is major crop ensuring food security in Europe, and blackleg disease is increasingly causing losses in yield and during storage. Recently, one blackleg pathogen, Dickeya solani has been shown to be spreading in Northern Europe that causes aggressive disease development. Currently, identification of tolerant commercial potato varieties has been unsuccessful; this is confounded by the complicated etiology of the disease and a strong environmental influence on disease development. There is currently a lack of efficient testing systems. Here, we describe a system for quantification of blackleg symptoms on shoots of sterile in vitro potato plants, which saves time and space compared to greenhouse and existing field assays. We found no evidence for differences in infection between the described in vitro‐based screening method and existing greenhouse assays. This system facilitates efficient screening of blackleg disease response of potato plants independent of other microorganisms and variable environmental conditions. We therefore used the in vitro screening method to increase understanding of plant mechanisms involved in blackleg disease development by analysing disease response of hormone‐ related (salicylic and jasmonic acid) transgenic potato plants. We show that both jasmonic (JA) and salicylic (SA) acid pathways regulate tolerance to blackleg disease in potato, a result unlike previous findings in Arabidopsis defence response to necrotrophic bacteria. We confirm this by showing induction of a SA marker, pathogenesis‐related protein 1 (StPR1), and a JA marker, lipoxygenase (StLOX), in Dickeya solani infected in vitro potato plants. We also observed that tubers of transgenic potato plants were more susceptible to soft rot compared to wild type, suggesting a role for SA and JA pathways in general tolerance to Dickeya.     

Pectolytic Bacteria Associated with Soft Rot of Calla Lily (Zantedeschia spp.) Tubers

Soft rot is the most important disease on calla lily in Poland. The isolation of the presumptive pathogen from symptomatic tubers on nutrient agar yielded bacteria with different colony morphology. Of 41 isolates collected, 10 showed pectolytic activity on crystal violet pectate medium and caused soft rot on potato slices. All pectolytic bacteria appeared to be Gram‐negative rods producing typical soft rot on inoculated leaf petioles of calla lily. Bacteria with colonies which morphologically resembled those used for inoculation were re‐isolated from diseased petioles. Their identification was based on phenotypic characters and sequence of the gene fragment coding 16S rRNA. It was found that, in addition to Pectobacterium carotovorum subsp. carotovorum, soft rot of calla lily can be caused by Pectobacterium carotovorum subsp. atrosepticum, Pseudomonas marginalis, Pseudomonas veronii and Chryseobacterium indologenes. The latter two are described for the first time as plant pathogens. The pectolytic activity of all identified bacteria, except that of P. carotovorum subsp. atrosepticum, was lower than that of P. carotovorum subsp. carotovorum, but strains of P. veronii showed a higher activity than P. marginalisand C. indologenes species.     

The use of fluorescent reporter protein tagging to study the interaction between Root‐Knot Nematodes and Soft Rot Enterobacteriaceae

The study of plant parasitic nematodes such as Meloidogyne spp. and their interactions with phytopathogenic bacteria remains underexplored. One of the challenges towards establishing such interactions is the dependence on symptom development as a measure of interaction. In this study, mCherry was employed as a reporter protein to investigate the interaction between the soft rot Enterobacteriaceae (SRE) Pectobacterium carotovorum subsp. brasiliensis (Pcb) and root‐knot nematode (M. incognita). Pectobacterium carotovorum subsp. brasiliensis was transformed with pMP7604 generating Pcb_mCherry strain. This strain was shown to attach to the surface coat of M.incognita J2 at the optimum temperature of 28°C. This suggests that RKN juveniles may play a role in disseminating Pcb in soils that are heavily infested with Pcb. The presence of RKN juveniles was shown to play a role in introducing Pcb_mCherry into potato tubers potentially acting as a source of latent tuber infections.

Significance and Impact of the Study

This study uses fluorescent reporter protein tagging as a tool to demonstrate the interaction between root‐knot nematode (Meloidogyne incognita) and the soft rot Enterobacteriacea (Pectobacterium carotovorum subsp. brasiliensis). Introduction of Pectobacterium through wounds generated by second‐stage juveniles (J2) into potato tubers was demonstrated. These results suggest that RKN juveniles can facilitate latent infection of potato tubers in the soil. These findings have important implications in the management of RKN and SRE in seed potato production. Furthermore, this tool can be used to study other nematode–bacteria interactions that have not been previously studied.     

Efficacy of potent antagonistic yeast Wickerhamiella versatilis against soft rot disease of potato caused by Pectobacterium carotovorum subsp. carotovorum

Abstract

In this study, an antagonistic yeast isolate, Wickerhamiella versatilis was considered as a promising biocontrol agent against Pectobacterium carotovorum subsp. Carotovorum (Pcc) the causal agent of soft rot disease of potato. Antagonistic yeast inhibited the growth of Pcc in vitro, and reducing the soft rot severity of infected potato tubers (cv. Diamant) under greenhouse conditions. Consequently, cellulase and pectinase hydrolytic activities in infected potato tubers with yeast?+?Pcc were decreased compared with infected tubers with Pcc. The histological characterization of treated potato tubers with antagonistic yeast W. versatilis using scanning electron microscope showed the accumulation of extracellular substances that may induce plant resistant and protects potato tubers from hydrolysis and damages. This study may introduce the possibility of using the antagonistic yeast isolate, as a biocontrol agent against soft rot of potato tubers.     

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.     

Host-induced gene silencing of Pectobacterium carotovorum quorum sensing gene enhances soft rot disease resistance in potato plants

Quorum sensing is a regulatory mechanism of gene expression in bacteria which regulated many physiological processes such as production of virulence factors in Pectobacterium carotovorum. In this research, the plant expression vector, pBI121, harbouring the anti-quorum sensing gene, aiiA, was transformed into Potato (Solanum tuberosum cv. Agria) explants by Agrobacterium mediated transformation procedure and putative transformants containing pBI121/aiiA were obtained. The results of bioassay test revealed that the expression of aiiA gene in potato plant induces the resistance to the early stage of bacterium pathogenesis. The soft rot symptoms such as wilting, yellowing and tissue maceration were not observed until 48?hr after inoculation of transgenic potatoes with P. carotovorum. Although in non-transgenic plants, the disease symptoms were appeared after 24?hr of plants infection. Hence, this study proves that the heterologous expression of aiiA gene enhances the resistance against potato soft rot disease.     

Comparison of temperature effects on the in vitro growth and disease development in potato tubers inoculated with bacteria Pectobacterium atrosepticum,P carotovorum subsp. carotovorum and Dickeya solani

The relationship between the rate of in vitro growth of bacterial isolates of Pectobacterium atrosepticum, P. carotovorum subsp. carotovorum and Dickeya solani and their pathogenicity was investigated in tubers of two potato cultivars at four temperatures ranging from 18°C to 30°C. The rate of in vitro growth was highly positively correlated with the number of rotted tubers (r ranged from 0.91 to 0.93) and with the weight of macerated potato tissue, which, however, was only found for P. carotovorum and D. solani (r = 0.76; r = 0.91, respectively) and not for P. atrosepticum. The weight of macerated tissue increased with the temperature, but significant differences between species of bacteria were observed only at 26°C and above, at which temperatures D. solani was the most aggressive, followed by P. carotovorum and P. atrosepticum. Almost all potato tubers inoculated with bacteria showed symptoms of soft rot at 26°C and 30°C, but the number of rotting tubers at lower temperatures (22°C and 18°C) decreased significantly. The lowest disease incidence, 11% of tubers with symptoms, was observed for the D. solani and cultivar Sonda at 18°C, what was also confirmed in a separate experiment with tubers from four potato cultivars inoculated with the highly aggressive isolate of D. solani. At temperatures from 18°C to 30°C, the differences in disease severity between potato cultivars with various resistance to bacteria increased in line with temperature, while the differences in disease incidence decreased.     

Characterization of Pectobacterium carotovorum subsp. carotovorum Causing Watermelon Soft Rot Disease in Iran

The aim of this study was characterized Pectobacterium carotovorum subsp. carotovorum (Pcc) the causal pathogen of watermelon soft rot disease in Iran. Of fifty bacterial isolates with white grey and convex colonies on nutrient agar obtained from symptomatic watermelon, ten isolates were selected for further tests. Pathogenicity tests results showed that all test isolates developed typical water‐soak symptoms after 2 days and signs of soft rot began 4 days after inoculation on watermelon fruits. Based on the phenotypic properties, the isolates were identified as Pectobacterium carotovorum subsp. carotovorum. The 16S rDNA sequences of isolates were 99% similar to the corresponding 16S rDNA sequence of the reference Pcc isolate. BOX and ERIC‐PCR analysis indicated that genetic diversity was present among the isolated Pcc isolates did not relate to the geographic location isolated from. To the best of our knowledge, this is the first study of biochemical and genotypic characterization of Pcc isolates the causal agents of soft rot disease on watermelon, in Iran.     

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.     

Transplastomic Nicotiana benthamiana plants expressing multiple defence genes encoding protease inhibitors and chitinase display broad‐spectrum resistance against insects,pathogens and abiotic stresses

Plastid engineering provides several advantages for the next generation of transgenic technology, including the convenient use of transgene stacking and the generation of high expression levels of foreign proteins. With the goal of generating transplastomic plants with multiresistance against both phytopathogens and insects, a construct containing a monocistronic patterned gene stack was transformed into Nicotiana benthamiana plastids harbouring sweet potato sporamin, taro cystatin and chitinase from Paecilomyces javanicus. Transplastomic lines were screened and characterized by Southern/Northern/Western blot analysis for the confirmation of transgene integration and respective expression level. Immunogold localization analyses confirmed the high level of accumulation proteins that were specifically expressed in leaf and root plastids. Subsequent functional bioassays confirmed that the gene stacks conferred a high level of resistance against both insects and phytopathogens. Specifically, larva of Spodoptera litura and Spodoptera exigua either died or exhibited growth retardation after ingesting transplastomic plant leaves. In addition, the inhibitory effects on both leaf spot diseases caused by Alternaria alternata and soft rot disease caused by Pectobacterium carotovorum subsp. carotovorum were markedly observed. Moreover, tolerance to abiotic stresses such as salt/osmotic stress was highly enhanced. The results confirmed that the simultaneous expression of sporamin, cystatin and chitinase conferred a broad spectrum of resistance. Conversely, the expression of single transgenes was not capable of conferring such resistance. To the best of our knowledge, this is the first study to demonstrate an efficacious stacked combination of plastid‐expressed defence genes which resulted in an engineered tolerance to various abiotic and biotic stresses.     

A quorum sensing‐defective mutant of Pectobacterium carotovorum ssp. brasiliense 1692 is attenuated in virulence and unable to occlude xylem tissue of susceptible potato plant stems

Pectobacterium carotovorum ssp. brasiliense 1692 (Pcb1692) is an important emerging pathogen of potatoes causing blackleg in the field and soft rot during post‐harvest storage. Blackleg diseases involve the bacterial colonization of vascular tissue and the formation of aggregates, also known as biofilms. To understand the role of quorum sensing in vascular colonization by Pcb1692, we generated a Pcb1692ΔexpI mutant strain. Inactivation of expI led to the reduced production of plant cell wall‐degrading enzymes (PCWDEs), the inability to produce acyl homoserine lactone (AHL) and reduced virulence in potato tubers and stems. Complementation of the mutant strain with the wild‐type expI gene in trans successfully restored AHL and PCWDE production as well as virulence. Transmission electron microscopy and in vitro motility assays demonstrated hyperpiliation and loss of flagella and swimming motility in the mutant strain compared with the wild‐type Pcb1692. Furthermore, we noted that, in the early stages of infection, Pcb1692 wild‐type cells had intact flagella which were shed at the later stages of infection. Confocal laser microscopy of PcbΔexpI‐inoculated plants showed that the mutant strain tended to aggregate in intercellular spaces, but was unable to transit to xylem tissue. On the contrary, the wild‐type strain was often observed forming aggregates within xylem tissue of potato stems. Gene expression analyses confirmed that flagella are part of the quorum sensing regulon, whereas fimbriae and pili appear to be negatively regulated by quorum sensing. The relative expression levels of other important putative virulence genes, such as those encoding different groups of PCWDEs, were down‐regulated in the mutant compared with the wild‐type strain.     

Jasmonate‐dependent modifications of the pectin matrix during potato development function as a defense mechanism targeted by Dickeya dadantii virulence factors

The plant cell wall constitutes an essential protection barrier against pathogen attack. In addition, cell‐wall disruption leads to accumulation of jasmonates (JAs), which are key signaling molecules for activation of plant inducible defense responses. However, whether JAs in return modulate the cell‐wall composition to reinforce this defensive barrier remains unknown. The enzyme 13–allene oxide synthase (13–AOS) catalyzes the first committed step towards biosynthesis of JAs. In potato (Solanum tuberosum), there are two putative St13–AOS genes, which we show here to be differentially induced upon wounding. We also determine that both genes complement an Arabidopsis aos null mutant, indicating that they encode functional 13–AOS enzymes. Indeed, transgenic potato plants lacking both St13–AOS genes (CoAOS1/2 lines) exhibited a significant reduction of JAs, a concomitant decrease in wound‐responsive gene activation, and an increased severity of soft rot disease symptoms caused by Dickeya dadantii. Intriguingly, a hypovirulent D. dadantii pel strain lacking the five major pectate lyases, which causes limited tissue maceration on wild‐type plants, regained infectivity in CoAOS1/2 plants. In line with this, we found differences in pectin methyl esterase activity and cell‐wall pectin composition between wild‐type and CoAOS1/2 plants. Importantly, wild‐type plants had pectins with a lower degree of methyl esterification, which are the substrates of the pectate lyases mutated in the pel strain. These results suggest that, during development of potato plants, JAs mediate modification of the pectin matrix to form a defensive barrier that is counteracted by pectinolytic virulence factors from D. dadantii.     

Molecular detection of Pectobacterium species causing soft rot of <Emphasis Type="Italic">Amorphophallus konjac</Emphasis>

Soft rot disease of Amorphophallus konjac is caused by Pectobacterium species. Infected corms are considered a primary and important source of inocula. Based on the 16S rDNA sequences of the soft rot pathogens, one pair of specific primers was designed to identify the soft rot disease by real-time PCR and the other two were used to identify the pathogens of Pectobacterium carotovorum subsp. carotovorum. and P. chrysanthemi respectively. According to the results, a single cell of Pectobacterium could be detected by real-time PCR with the designed primer pair, while at least 100 bacteria were required for conventional PCR. Moreover, the two special primers can directly and accurately authenticate to Pectobacterium carotovorum subsp. carotovorum and P. chrysanthemi by the conventional PCR system without testing the pathogenicity, biochemical and phenotypic characterizations and so on. In conclusion, the PCR-based techniques showed several significant advantages in identifying the soft rot pathogens from konjac, such as higher sensitivity, rapidness and precision, and it could be widely used in seed quarantine.     

Proteins Involved in the Interaction of Potato Tubers with Pectobacterium atrosepticum: a Proteomic Approach to Understanding Partial Resistance

Potato can be severely affected by various pathogens, including Pectobacterium atrosepticum, the cause of bacterial soft rot on tubers and of blackleg on stems. To date, no complete resistance to P. atrosepticum is available, so that only cultivars exhibiting partial resistance can be found. The mechanistic basis of this type of resistance is still poorly understood. A proteomic approach was thus developed to identify pathways specifically activated during the interaction between potato tubers and P. atrosepticum. Protein profiles on silver‐stained gels in the 5–8 pH range were obtained from healthy and infected tubers from two cultivars differing for resistance level and analyzed by 2‐DE and nano‐LC‐MS/MS. Thirteen proteins were differentially up‐regulated in the partially resistant cv. Kerpondy; by contrast, no significant differences in protein profiles of inoculated and control tubers were observed in the susceptible cv. Bintje. Mass spectrometry and database searching showed that these proteins are involved in energetic metabolism (glyceraldehyde‐3‐phosphate dehydrogenase, 2‐phosphoglycerate dehydratase or enolase, fructose biphosphate aldolase and ATPase α subunit), cytoskeleton structure (actin), protein catabolism (cysteine protease inhibitor) and patatins or patatin precursors. Their involvement in defence responses of cv. Kerpondy to P. atrosepticum is discussed. Proteomic appears as an efficient approach to have insight into the mechanisms and pathways leading to potato resistance against P. atrosepticum.     

Characterisation of Pectobacterium carotovorum causing soft rot on Kalanchoe gastonis-bonnierii in Malaysia

Soft rot disease can be found worldwide on fleshy storage tissues of fruits, vegetables and ornamentals. The soft rot Pectobacterium carotovorum subsp. carotovorum (Pcc) is an important pathogen of Kalanchoe spp. and other ornamental plants. The disease occurs on crops in the field, greenhouses and during transit, resulting great economic damages. The economic importance of crop loss by soft rot bacteria varies by severity of the disease and value of the crop. A destructive disease on Kalanchoe gastonis-bonnierii was observed in commercial ornamental plant greenhouses in Cameron highland and Melaka, Malaysia in 2011. Samples suspected to be infested with Pectobacterium spp. were brought to the laboratory. In pathogenicity test, a suspension of 10^6?CFU/ml of strains was able to cause soft rot on leaves and stems. A 434?bp banding pattern on 1% agarose gel was produced in polymerase chain reaction (PCR) amplification of pectate lyase encoding gene (Pel gene). PCR amplification of the intergenic transcribed spacer (ITS) (16S–23S rRNA) ITS region with G₁ and L₁ primers produced two main bands at about 540 and 570?bp. The ITS-PCR products were digested with RsaI restriction enzyme. For discrimination of the P. carotovorum subsp. carotovorum (Pcc) from P. carotovorum subsp. odoriferum (Pco), all isolates subjected to α-methyl glucoside test. All isolates were identified as Pcc based on phenotypic and molecular methods. This is the first report of soft rot disease caused by P. carotovorum subsp. carotovorum on K. gastonis-bonnierii, in Malaysia.     

Infection of potato tubers with soft rot bacteria

Stolons attached to developing potato tubers were inoculated with the soft rot bacterium Erwinia carotovora var. atroseptica. Almost all the stolons rotted, but soft rots developed in less than 10% of new tubers; the bacterium was isolated later from these tubers. No rots developed in the other tubers but the bacterium was later isolated from about half of them. It could not be isolated from tubers attached to inoculated stolons where the rot on them did not extend to the tuber or from tubers attached to stolons that were not inoculated though many of these rotted. The bacterium was reisolated from almost all arrested lesions in tubers inoculated 8 month earlier with E. carotovora var. atroseptica. Blackleg did not develop from plants grown fom these tubers under various soil conditions. It did develop in a large proportion of plants from tubers inoculated shortly before planting and grown in cool, wet soil. Less than 1% blackleg developed in plants grown from tubers from plants with blackleg or from plants immediately adjacent. The presence of pectolytic bacteria and E. caratovora var. atroseptica in seed and new tubers was investigated during June, July and August. Although E. caratovora var. atroseptica was obtained from c. 40% tubers, only c. 0·3% of c. 8400 plants developed blackleg. The bacterium was isolated from only three of 160 new tubers sampled during the summer.