Modification of a multiple‐locus variable number tandem repeat analysis (MLVA) for typing isolates of Erwinia amylovora

Erwinia amylovora, the causal agent of fire blight that affects economically important rosaceous plants, is reported among the most important plant pathogenic bacteria. The low genetic diversity within E. amylovora and the lack of simple and high‐resolution genotyping techniques make epidemiology and evolutionary studies challenging for this pathogen. A multiple‐locus variable number tandem repeat analysis (MLVA) based on a set of nine variable number tandem repeat loci was successfully used to type 46 E. amylovora isolates collected from different host plants in 16 countries, mainly Mediterranean. The nine polymorphic loci proved to have high discriminatory power and to increase the resolution of the MLVA. Thirty‐eight haplotypes clustered in seven clonal complexes. The results identified potentially useful genetic markers among the Mediterranean strains, particularly from the Balkan Peninsula and the Eastern Mediterranean countries. Different MLVA types were observed amongst Italian strains only, indicating the possibility of multiple introductions of the disease. MLVA can be used effectively as a fast, cheap, and simple tool to track E. amylovora infection sources, to gain insight into geographic diversity, and to understand the dynamic evolution of the pathogen.     

The undiscovered potential of dehydroproline as a fire blight control option

The non‐protein amino acid 3,4‐dehydro‐l ‐proline (DHP) significantly reduced the incidence of fire blight infection on immature pear fruits infected with wildtype Erwinia amylovora. DHP also inhibited biofilm formation in both streptomycin‐sensitive and ‐resistant strains of E. amylovora and induced dispersal of preformed biofilms in the streptomycin‐sensitive strain. The investigations shed light on the hitherto undiscovered ability of DHP to inhibit bacterial biofilms and its potential as a chemical control option for fire blight.     

Complete sequence of Erwinia piriflorinigrans plasmids pEPIR37 and pEPIR5 and role of pEPIR37 in pathogen virulence

Erwinia piriflorinigrans is a newly described pathogen causing necrosis of pear blossoms. Complete sequencing of the 37‐kb plasmid pEPIR37 common to 27 E. piriflorinigrans strains revealed homology to sequences of the ubiquitous plasmids pEA29 of the fire blight pathogen E. amylovora, plasmid pEP36 of E. pyrifoliae, plasmid pEJ30 of Erwinia sp. from Japan, and genomic regions of the related Rosaceae epiphytic Erwinia species E. tasmaniensis and E. billingiae. A second 5·5‐kb cryptic plasmid pEPIR5, found in 12 E. piriflorinigrans strains, was also sequenced revealing mobilization and replication proteins with similarities to many small ColE1‐type plasmids in Erwinia spp. and other enterobacteria. Functional analyses of pEPIR37 introduced into a strain of E. amylovora cured of pEA29 plasmid, which has a reduced virulence, showed a role in increasing symptom development similar to that observed in E. amylovora carrying plasmid pEA29.     

Silencing of Erwinia amylovora sy69 AHL‐quorum sensing by a Bacillus simplex AHL‐inducible aiiA gene encoding a zinc‐dependent N‐acyl‐homoserine lactonase

Quorum sensing in Gram‐negative bacteria is regulated by diffusible signal molecules called N‐acyl‐l ‐homoserine lactones (AHLs). These molecules are degraded by lactonases. In this study, six Bacillus simplex isolates were characterized and identified as a new quorum‐quenching species of Bacillus. An aiiA gene encoding an AHL‐lactonase was identified based on evidence that: (i) it showed high homology with other aiiA genes of Bacillus sp.; (ii) the deduced amino acid sequence contained two conserved regions, ¹⁰⁴SHLHFDH¹¹¹ and ¹⁶⁵TPGHTPGH¹⁷³, characteristic of the metallo‐β‐lactamase superfamily; and (iii) the protein had zinc‐dependent AHL‐degrading activity. Additionally, the expression of the aiiA gene was significantly up‐regulated by 3‐oxo‐AHL. The AHL‐lactonase inhibited multiplication of the 3‐oxo‐C6‐AHL‐producing plant pathogen Erwinia amylovora sy69 both in vitro and in planta. The results provide support for the use of the quorum‐quenching functionality of B. simplex in the integrated control of the devastating fire blight pathogen.     

Diversity of Avr‐vnt1 and AvrSmira1 effector genes in Polish and Norwegian populations of Phytophthora infestans

The oomycete Phytophthora infestans, the cause of late blight, is one of the most important potato pathogens. During infection, it secretes effector proteins that manipulate host cell function, thus contributing to pathogenicity. This study examines sequence differentiation of two P. infestans effectors from 91 isolates collected in Poland and Norway and five reference isolates. A gene encoding the Avr‐vnt1 effector, recognized by the potato Rpi‐phu1 resistance gene product, is conserved. In contrast, the second effector, AvrSmira1 recognized by Rpi‐Smira1, is highly diverse. Both effectors contain positively selected amino acids. A majority of the polymorphisms and all selected sites are located in the effector C‐terminal region, which is responsible for their function inside host cells. Hence it is concluded that they are associated with a response to diversified target protein or recognition avoidance. Diversification of the AvrSmira1 effector sequences, which existed prior to the large‐scale cultivation of plants containing the Rpi‐Smira1 gene, may reduce the predicted durability of resistance provided by this gene. Although no isolates virulent to plants with the Rpi‐phu1 gene were found, the corresponding Avr‐vnt1 effector has undergone selection, providing evidence for an ongoing ‘arms race’ between the host and pathogen. Both genes remain valuable components for resistance gene pyramiding.     

Host‐induced gene silencing in the necrotrophic fungal pathogen Sclerotinia sclerotiorum

Sclerotinia sclerotiorum is a necrotrophic fungus that causes a devastating disease called white mould, infecting more than 450 plant species worldwide. Control of this disease with fungicides is limited, so host plant resistance is the preferred alternative for disease management. However, due to the nature of the disease, breeding programmes have had limited success. A potential alternative to developing necrotrophic fungal resistance is the use of host‐induced gene silencing (HIGS) methods, which involves host expression of dsRNA‐generating constructs directed against genes in the pathogen. In this study, the target gene chosen was chitin synthase (chs), which commands the synthesis of chitin, the polysaccharide that is a crucial structural component of the cell walls of many fungi. Tobacco plants were transformed with an interfering intron‐containing hairpin RNA construct for silencing the fungal chs gene. Seventy‐two hours after inoculation, five transgenic lines showed a reduction in disease severity ranging from 55·5 to 86·7% compared with the non‐transgenic lines. The lesion area did not show extensive progress over this time (up to 120 h). Disease resistance and silencing of the fungal chs gene was positively correlated with the presence of detectable siRNA in the transgenic lines. It was demonstrated that expression of endogenous genes from the very aggressive necrotrophic fungus S. sclerotiorum could be prevented by host induced silencing. HIGS of the fungal chitin synthase gene can generate white mould‐tolerant plants. From a biotechnological perspective, these results open new prospects for the development of transgenic plants resistant to necrotrophic fungal pathogens.     

Distribution of streptomycin-resistant strainsof Erwinia amylovora in Israel and occurrence of blossom blight in the autumn

Following failure in control of fire blight with streptomycin, the distribution of streptomycin-resistant strains ofErwinia amylovora in Israel was surveyed. During 1994–1997 109 pear, apple, loquat and quince orchards were monitored. Streptomycin-resistant strains ofE. amylovora were recovered from flowers and from infected branches collected at 18 locations in the Sharon, Galilee and Golan Heights regions. In the Sharon region all the isolated strains ofE. amylovora were streptomycin-resistant, whereas in the Galilee and Golan Heights, resistant as well as sensitiveE. amylovora strains were recovered at different locations. In the southern coastal plain no resistance could be detected. Streptomycin-resistant strains ofE. amylovora did not hybridize with the DNA probe SMP3, and resistance could not be transferred by mating to a sensitive strain, suggesting that streptomycin resistance in Israel is not plasmid-mediated. Fire blight symptoms were observed, for the first time, on pear blossoms during the autumn of 1994. A high population of 2x 10⁶-6x 10⁷ CFU/flower in the autumn of 1995 and of 1996 was correlated with the appearance of blossom blight symptoms.     

Characterisation of naturally occurring <Emphasis Type="Italic">Erwinia amylovora</Emphasis> strains lacking the common plasmid pEA29 and their detection with real-time PCR

Erwinia amylovora, the causal agent of fire blight, carries the common plasmid pEA29 of 29 kb. To screen for occurrence of natural strains without plasmid pEA29, we applied PCR analysis with primers from the plasmid and the chromosomal ams region. In addition, a described TaqMan probe from pEA29 and newly designed primers from the ams-region were used for identification by real-time PCR. One strain isolated in Iran, one strain from Spain and two strains from Egypt lacked plasmid pEA29. From a recent screening series in southern Germany, in 123 E. amylovora strains from necrotic fire blight host plants, one strain was found without the common plasmid. The strains without pEA29 were virulent in assays with immature pears and on apple seedlings, but showed a reduced growth level in minimal medium without amino acids and thiamine. Transposon-labelled pEA29 was transformed into the plasmid-free strains resulting in restoration of this growth deficiency. The plasmid was stably maintained in these E. amylovora cells. The newly designed chromosomal primers for conventional and for real-time PCR identified E. amylovora strains in field samples lacking pEA29. These variants are apparently rare, but were detected in isolates from different regions in the world with fire blight.     

Histological observation of cucumber infected with <Emphasis Type="Italic">Corynespora cassiicola</Emphasis>

Erwinia amylovora is the causative agent of fire blight, which is a destructive bacterial disease of rosaceous plants. In Hungary Erwinia amylovora (Burrill) Winslow et al. was first detected in 1996. Since the appearance of fire blight, E. amylovora samples have been collected from different host plats from various geographic locations. A motif of eight nucleotides (ATTACAGA) is repeated 3–15 times in the PstI fragment of the pEa29 plasmid in Erwinia amylovora strains, and represents a valuable tool for strain classification. The number of short-sequence DNA repeats in plasmid pEa29 of 30 Hungarian isolates were determined by PCR assays and they ranged from five to ten. The SSR test is suitable for distinguishing the individual strains between the E. amylovora isolates. The examined isolates showed high pathogenicity on immature pear fruits. Several biochemical techniques, such as miniaturized API 20E, were applied on the samples. Differences were also revealed in microbiological assays like levan formation and colony morphology on semi-selective media. Examining the Hungarian Erwinia amylovora population by molecular analysis we can draw the conclusion that the population consists of different strains, which shows great diversity. E. amylovora is a widespread pathogen in Hungary, which is supported by the 30 strains isolated from various host plants from many parts of the country. The phenotypic diversity-evaluation of the E. amylovora strains showed, that they differ metabolically, like other plant pathogenic bacteria as reported by several authors. This is the first report on the diversity of E. amylovora strains isolated from Hungary.     

Resistance to Sclerotinia sclerotiorum in wild Brassica species and the importance of Sclerotinia subarctica as a Brassica pathogen

Brassica crops are of global importance, with oilseed rape (Brassica napus) accounting for 13% of edible oil production. All Brassica species are susceptible to sclerotinia stem rot caused by Sclerotinia sclerotiorum, a generalist fungal pathogen causing disease in over 400 plant species. Generally, sources of plant resistance result in partial control of the pathogen although some studies have identified wild Brassica species that are highly resistant. The related pathogen S. subarctica has also been reported on Brassica but its aggressiveness in relation to S. sclerotiorum is unknown. In this study, detached leaf and petiole assays were used to identify new sources of resistance to S. sclerotiorum within a wild Brassica ‘C genome’ diversity set. High‐level resistance was observed in B. incana and B. cretica in petiole assays, whilst wild B. oleracea and B. incana lines were the most resistant in leaf assays. A B. bourgeai line showed both partial petiole and leaf resistance. Although there was no correlation between the two assays, resistance in the detached petiole assay was correlated with stem resistance in mature plants. When tested on commercial cultivars of B. napus, B. oleracea and B. rapa, selected isolates of S. subarctica exhibited aggressiveness comparable to S. sclerotiorum indicating it can be a significant pathogen of Brassica. This is the first study to identify B. cretica as a source of resistance to S. sclerotiorum and to report resistance in other wild Brassica species to a UK isolate, hence providing resources for breeding of resistant cultivars suitable for Europe.     

Alternative inoculum sources for fire blight: the potential role of fruit mummies and non‐host plants

Fire blight is the most damaging bacterial disease in apple production worldwide. Cankers and symptomless infected shoots are known as sites for the overwintering of Erwinia amylovora, subsequently providing primary inoculum for infection in the spring. In the present work, further potential sources of inoculum were investigated. Real‐time PCR assays covering a 3‐year‐period classified 19·9% of samples taken from fruit mummies as positive. Bacterial abundance in fruit mummies during autumn, winter and spring was up to 10⁹ cells per gram of tissue and correlated well with later infection rates of blossoms. Blossoms of non‐host plants growing close to infected trees were also shown to be colonized by E. amylovora and to enable epiphytic survival and propagation of bacteria. The results indicate a potential role of fruit mummies and buds in overwintering and as a source of primary inoculum for dissemination of the pathogen early in the growing season. Non‐host blossoms may also serve as an inoculum source in the build‐up of the pathogen population. Both aspects may contribute significantly to the epidemiology of E. amylovora. The significance of infected rootstocks as an inoculum source is also discussed. Fruit mummies might be used to determine pathogen pressure in an orchard before the beginning of the blooming period.     

Evidence of early defence to Ascochyta lentis within the recently identified Lens orientalis resistance source ILWL180

In plant–pathogen interactions, strong structural and biochemical barriers may induce a cascade of reactions in planta, leading to host resistance. The kinetic speed and amplitudes of these defence mechanisms may discriminate resistance from susceptibility to necrotrophic fungi. The infection processes of two Ascochyta lentis isolates (FT13037 and F13082) on the recently identified ascochyta blight (AB)‐resistant Lens orientalis genotype ILWL180 and two cultivated genotypes, ILL7537 (resistant) and ILL6002 (susceptible), were assessed. Using histopathological methods, significant differences in early behaviour of the isolates and the subsequent differential defence responses of the hosts were revealed. Irrespective of virulence, both isolates had significantly lower germination, shorter germ tubes and delayed appressorium formation on the resistant genotypes (ILWL180 and ILL7537) compared to the susceptible genotype (ILL6002); furthermore, these were more pronounced on genotype ILWL180 than on genotype ILL7537. Subsequently, host perception of pathogen entry led to the faster accumulation and notably higher amounts of reactive oxygen species and phenolic compounds at the penetration sites of the resistance genotypes ILWL180 and ILL7537. In contrast, genotype ILL6002 responded slowly to the A. lentis infection and reaffirmed previous gross disease symptomology reports as highly susceptible. Interestingly, quantification of H₂O₂ was markedly higher in ILWL180 particularly at 12 h post‐inoculation compared to ILL7537, potentially indicative of its superior resistance capability. Faster recognition of A. lentis is likely to be a major contribution to the superior resistance observed in genotype ILWL180 to the highly aggressive isolates of A. lentis assessed.     

An assessment of the relative resistance of three hawthorn species to three strains of <Emphasis Type="Italic">Erwinia amylovora</Emphasis> using three different inoculation methods

Fire blight is an important disease of hawthorn plants. In this study, the level of susceptibility of three hawthorn species (Crataegus monogyna, Crataegus laevigata, Crataegus persimilis) to the bacterium Erwinia amylovora was investigated. The results showed that all species were highly susceptible to this pathogen. In addition, the relative virulence of three different E. amylovora strains on the above species was examined. Variability among the strains was found, with strain 3 being the most virulent and strain 1 the least.     

Quantitative trait loci associated with resistance to a potato isolate of Verticillium albo‐atrum in Medicago truncatula

Verticillium albo‐atrum is responsible for considerable yield losses in many economically important crops, among them alfalfa (Medicago sativa). Using Medicago truncatula as a model for studying resistance and susceptibility to V. albo‐atrum, previous work has identified genetic variability and major resistance quantitative trait loci (QTLs) to Verticillium. In order to study the genetic control of resistance to a non‐legume isolate of this pathogen, a population of recombinant inbred lines (RILs) from a cross between resistant line F83005.5 and susceptible line A17 was inoculated with a potato isolate of V. albo‐atrum, LPP0323. High genetic variability and transgressive segregation for resistance to LPP0323 were observed among RILs. Heritabilites were found to be 0·63 for area under the disease progress curve (AUDPC) and 0·93 for maximum symptom score (MSS). A set of four QTLs associated with resistance towards LPP0323 was detected for the parameters MSS and AUDPC. The phenotypic variance explained by each QTL (R²) was moderate, ranging from 4 to 21%. Additive gene effects showed that favourable alleles for resistance all came from the resistant parent. The four QTLs are distinct from those described for an alfalfa V. albo‐atrum isolate, confirming the existence of several resistance mechanisms in this species. None of the QTLs co‐localized with regions involved in resistance against other pathogens in M. truncatula.     

Characterization and functional analysis of clpB gene from Acidovorax avenae subsp. avenae RS‐1

The type VI secretion system (T6SS) has been reported to be highly associated with various cellular activities in strain RS‐1 of Acidovorax avenae subsp. avenae (Aaa), the pathogen of bacterial brown stripe of rice. However, the role of the clpB gene that presents in the T6SS gene cluster in Aaa pathogenicity has not been clarified. The aim of the current study was to characterize the function of clpB and to investigate its contribution to bacterial pathogenesis using insertional deletion mutation and complementation approaches. The results indicated that mutation of clpB significantly affected bacterial growth, virulence, exopolysaccharide (EPS) production, biofilm formation and expression of 13 other T6SS genes of Aaa RS‐1. The reduction of virulence may be also partially due to the change in EPS composition, which was characterized by the Fourier transform infrared (FTIR) spectra. Furthermore, analysis of protein homology modelling showed that the structure of ClpB is different from those of the other T6SS components. In addition, structural difference was observed between ClpB and Type IV pili (TFP) as well as Type IV pilus biogenesis proteins (PilP), whose functions are similar to ClpB. Taken together, this study demonstrated that the clpB gene plays a key role in Aaa bacterial virulence.     

Taxonomic Position of the Causal Pathogen of Bacterial Shoot Blight of Pear

Bacteriological properties and DNA-DNA homology values were compared among the pathogen causing bacterial shoot blight of pear (BSBP) isolated in 1994–1996, Erwinia amylovora isolated outside of Japan, and other Amylovora group bacteria. Bacteriological properties of BSBP strains were identical to those of E. amylovora in the majority of tests, but differed distinctively in several tests, including hydrolysis of esculin and acid production from salicin, etc. BSBP strains differed from the others in the Amylovora group in many other tests. DNA homology among the strains of BSBP ranged from 85 to 103% and from 83 to 110% among strains of E. amylovora. In contrast, the values between BSBP strains and E. amylovora strains were 55 to 81%, while those between BSBP strains and other Amylovora group strains were 42% or less. We consider, therefore, that the BSBP pathogen may well be included in E. amylovora at the species level. E. amylovora, including BSBP strains, however, can be classified into four biovars based on differences in nine tests such as growth factor requirements and crater formation on high sucrose medium. Namely, there are two biovars from Maloideae sources, one from Rubus idaeus, and one from the source of BSBP in Hokkaido. The presence of these biovars suggests a correlation with geographical, serological, and pathogenic differentiations in the species of E. amylovora. The BSBP pathogen in Hokkaido was identified as E. amylovora bv. 4 which is distinct from E. amylovora bv. 1, 2 and 3 isolated in countries outside of Japan. Received 29 July 1999/ Accepted in revised form 12 October 1999     

Molecular characterization,comparison of screening methods,and evaluation of cross‐pathogenicity of black rot (Xanthomonas campestris pv. campestris) strains from cabbage,choy sum,leafy mustard and pak choi from Taiwan

Choy sum (Brassica rapa var. parachinensis), leafy mustard (Brassica juncea) and pak choi (B. rapa var. chinensis) are highly nutritious components of diets in Taiwan and other Asian countries, and bacterial black rot caused by Xanthomonas campestris pv. campestris (Xcc) is a major biotic constraint in these crops. As very little was known about the Xcc strains from these crops in these regions, including their cross‐pathogenicity and aggressiveness on different hosts, Xcc strains were obtained from cabbage (Brassica oleracea var. capitata), choy sum, leafy mustard and pak choi crops in Taiwan. Two previously published PCR‐based assays reliably distinguished the Xcc strains from other Xanthomonas species and subspecies. Phylogenetic analysis based on repetitive sequence‐based PCR assays placed the Xcc strains in a clade distinct from other Xanthomonas species, and also showed host specificity. Although all of the Xcc strains from the different host species were pathogenic on all five Brassica test species in both a detached leaf assay and an intact plant assay, in the intact plant assay they showed differences in virulence or aggression on the different test hosts. The Xcc strains from leafy mustard and pak choi were consistently highly aggressive on all the test host genotypes, but the strains from choy sum and cabbage were less aggressive on leafy mustard and choy sum. The intact plant assay proved more discriminating and reliable than the detached leaf assay for comparing the aggressiveness of Xcc strains on different host genotypes, and so, with the new Xcc strains isolated in this study, will be useful for screening leafy brassica germplasm accessions for resistance to black rot.     

Extended longevity of Erwinia amylovora vectored by honeybees under in vitro conditions and its capacity for dissemination

Fire blight outbreaks in Korea were first reported in 2015. Regular outbreaks have occurred since, indicating a continuous cycle of the fire blight pathogen in Korea. We determined the role of Apis mellifera (honeybee) as a vector of Erwinia amylovora by verifying the following: (a) E. amylovora longevity in/on honeybees; (b) the most common body parts that carry the bacteria; (c) the rate of bacterial spread to healthy host organs; and (d) the relationship between dispersal of viable but nonculturable (VBNC) and virulent bacterial cells. E. amylovora survived for 15 days on the exterior of honeybee bodies and was most abundant on the abdomen in comparison to other areas such as the labellum, wings, and hind legs. In the digestive system of honeybees, E. amylovora survived for 7 days, and bacteria were found in faeces for 3 days after exposure. The bacteria are likely to be VBNC on honeybees. Honeybees that were contaminated with bacteria transferred E. amylovora to healthy immature apple fruit, shoots, and flowers for 10 days after exposure. E. amylovora was also transferred from inoculated plant parts to uncontaminated honeybees. In addition, bacteria moved from inoculated plant tissues to unexposed honeybees and then from these honeybees to healthy plant tissues. Therefore, E. amylovora can survive in/on honeybees for extended amounts of time, which contradicts previous reports. The bacteria moved to host tissues via honeybees, suggesting that honeybees are the vectors of E. amylovora and play a role in the development of new outbreaks of fire blight disease in the central regions of Korea.     

Necrotrophic behaviour of Erwinia amylovora in apple and tobacco leaf tissue

An important issue related to the epidemiology of fire blight, a devastating disease of apples and pears, is how its causal agent, the bacterium Erwinia amylovora, survives and disseminates in the environment. Almost no information is available on the possibility of this pathogen overwintering as a necrotroph. In this study, bacterial survival in dead apple and tobacco (a non‐host) leaf tissues was addressed. In necrotized leaves collected 5, 6, 7 and 8 months following shoot inoculation of apple trees, viable E. amylovora cells were present in over 50% of samples from the midrib and in over 10% of samples from lateral veins, but were never found in parenchyma. Using a PCR‐based method, pathogen DNA was detected in more than 50% of samples that were found to be free of viable cells by conventional plating out. However, PCR analysis was insufficient to distinguish between the DNA of viable and dead bacteria. Sugars appropriate for bacterial growth were found in dead apple leaves. In spot‐inoculated attached apple and tobacco leaves, a remarkable increase in the bacterial population was observed in lesions that developed as a hypersensitive response (HR). As in other necrotrophic interactions, bacterial proliferation was associated with massive hydrogen peroxide production and progression toward plant cell death. The results indicate that E. amylovora has an ability to survive as a semi‐necrotroph or necrotroph, which allows for overwintering in dead apple leaves.