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Abstract Background Allorhizobium vitis (formerly named Agrobacterium vitis or Agrobacterium biovar 3) is the primary causative agent of crown gall disease of grapevine worldwide. We obtained and analyzed whole-genome sequences of diverse All. vitis strains to get insights into their diversification and taxonomy. Results Pairwise genome comparisons and phylogenomic analysis of various All. vitis strains clearly indicated that All. vitis is not a single species, but represents a species complex composed of several genomic species. Thus, we emended the description of All. vitis, which now refers to a restricted group of strains within the All. vitis species complex (i.e. All. vitis sensu stricto) and proposed a description of a novel species, All. ampelinum sp. nov. The type strain of All. vitis sensu stricto remains the current type strain of All. vitis, K309T. The type strain of All. ampelinum sp. nov. is S4T. We also identified sets of gene clusters specific to the All. vitis species complex, All. vitis sensu stricto and All. ampelinum, respectively, for which we predicted the biological function and infer the role in ecological diversification of these clades, including some we could experimentally validate. All. vitis species complex-specific genes confer tolerance to different stresses, including exposure to aromatic compounds. Similarly, All. vitis sensu stricto-specific genes confer the ability to degrade 4-hydroxyphenylacetate and a putative compound related to gentisic acid. All. ampelinum-specific genes have putative functions related to polyamine metabolism and nickel assimilation. Congruently with the genome-based classification, All. vitis sensu stricto and All. ampelinum were clearly delineated by MALDI-TOF MS analysis. Moreover, our genome-based analysis indicated that Allorhizobium is clearly separated from other genera of the family Rhizobiaceae. Conclusions Comparative genomics and phylogenomic analysis provided novel insights into the diversification and taxonomy of Allorhizobium vitis species complex, supporting our redefinition of All. vitis sensu stricto and description of All. ampelinum. Our pan-genome analyses suggest that these species have differentiated ecologies, each relying on specialized nutrient consumption or toxic compound degradation to adapt to their respective niche.

Bacterial canker, caused by Pseudomonas syringae pv. syringae and pv. morsprunorum, is one of the most important diseases of stone fruit trees (Prunus spp.). The pathogen infects buds, flowers, fruitlets, leaves and shoots, from which the disease spreads to the branches, boughs and trunks, causing necrosis and cankers. The efficacy of different chemical and biological products for the control of bacterial canker on stone fruit trees was tested in 2018–2021. The experiments were conducted in sour cherry, plum and sweet cherry orchards in central Poland. Foliar application of the tested preparations was performed three times a season. The biological efficacy of the tested products in the control of bacterial canker was evaluated on sour cherry on the basis of infected leaves and fruits and on plum and sweet cherry on the basis of infected leaves. The highest efficacy was observed for products containing various forms of copper—copper oxide, copper oxychloride and copper hydroxide—as well as fertilizers with copper gluconate and the fungicide Luna Care 71.6 WG (fluopyram and fosetyl-Al). However, the biological preparations were significantly less effective. The conducted studies showed that preparations based on copper gluconate can be a valuable alternative to typical copper fungicides.

Abstract Xanthomonas fragariae is a quarantine bacterial pathogen that causes angular leaf spot on strawberry. The aim of our study was to analyse the mechanism of interaction of this bacterium with its host plant at the transcriptome level. For this purpose, mRNAs of X. fragariae growing in Wilbrink’s medium and from infected strawberry cv. Elsanta plants were isolated and sequenced using the Illumina MiSeq platform. The expression profiles of the bacteria in Wilbrink’s medium and in planta were very diverse. Of the 3939 CDSs recorded, 1995 had significantly different expression in planta (966 and 1029 genes were down- and upregulated, respectively). Among the genes showing increased expression in planta, those with eggNOG/COG (evolutionary genealogy of genes: Non-supervised Orthologous Groups/Cluster of Orthologous Groups) categories associated with bacterial cell motility, signal transduction, transport and metabolism of inorganic ions and carbohydrates and transcription were overrepresented. Among the genes with the most increased expression in planta, genes primarily associated with flagella synthesis and chemotaxis were found. It is also interesting to note that out of the 31 genes localized on a plasmid, 16 were expressed differently in planta, which may indicate their potential role in plant–pathogen interactions. Many genes with differentiated expression that were localized on chromosome and plasmid encode proteins of unknown function.

For the years 2012–2018, we investigated the occurrence of storage diseases caused by fungi on four cultivars of apples grown in the central part of Poland. The fruits were picked in orchards managed with Integrated Pest Management (IPM) practices and stored in a cold room for 5–7 months. Depending on the season, apple cultivar and localization of orchard, the incidence of diseases was different. On apple cvs “Gala”, “Ligol” and “Golden Delicious”, bull’s eye rot (Neofabraea spp.) was the most frequently observed disease, whereas on apple cv. “Gloster”, gray mold (Botrytis cinerea) predominated. The blue mold (Penicillium expansum), brown rot (Monilinia spp.) and fungi of Alternaria spp. occurred at significantly lower intensity. We detected the occurrence of the new storage diseases of apples caused by Colletotrichum spp., Neonectria ditissima and Diaporthe eres.

Abstract Background Tracking newly emergent virulent populations in agroecosystems provides an opportunity to increase our understanding of the co-evolution dynamics of pathogens and their hosts. On the one hand host plants exert selective pressure on pathogen populations, thus dividing them into subpopulations of different virulence, while on the other hand they create an opportunity for secondary contact between the two divergent populations on one tree. The main objectives of the study were to explore whether the previously reported structure between two Venturia inaequalis population types, virulent or avirulent towards Malus x domestica cultivars carrying Rvi6 gene, is maintained or broken several years after the first emergence of new virulent strains in Poland, and to investigate the relationship between ‘new’ and ‘native’ populations derived from the same commercial orchards. For this purpose, we investigated the genetic structure of populations of the apple scab fungus, occurring on apple tree cultivars containing Rvi6, Rvi1 or Rvi17 resistance gene or no resistance at all, based on microsatellite data obtained from 606 strains sampled in 10 orchards composed of various host cultivars. Results Application of genetic distance inferring and clustering methods allowed us to observe clear genetic distinctness of the populations virulent towards cultivars carrying Rvi6 gene from the Rvi6-avirulent populations and substructures within the Rvi6-group as a consequence of independent immigration events followed by rare, long-distance dispersals. We did not observe such a structuring effect of other genes determining apple scab resistance on any other populations, which in turn were genetically homogenous. However, in two orchards the co-occurrence of strains of different virulence pattern on the same trees was detected, blurring the genetic boundaries between populations. Conclusions Among several resistance genes studied, only Rvi6 exerted selective pressure on pathogens populations: those virulent toward Rvi6 hosts show unique and clear genetic and virulence pattern. For the first time in commercial Malus x domestica orchards, we reported secondary contacts between populations virulent and avirulent toward Rvi6 hosts. These two populations, first diverged in allopatry, second came into contact and subsequently began interbreeding, in such way that they show unambiguous footprints of gene flow today.

Abstract Background Erwinia amylovora is generally considered to be a homogeneous species in terms of phenotypic and genetic features. However, strains show variation in their virulence, particularly on hosts with different susceptibility to fire blight. We applied the RNA-seq technique to elucidate transcriptome-level changes of the lowly virulent E. amylovora 650 strain during infection of shoots of susceptible (Idared) and resistant (Free Redstar) apple cultivars. Results The highest number of differentially expressed E. amylovora genes between the two apple genotypes was observed at 24 h after inoculation. Six days after inoculation, only a few bacterial genes were differentially expressed in the susceptible and resistant apple cultivars. The analysis of differentially expressed gene functions showed that generally, higher expression of genes related to stress response and defence against toxic compounds was observed in Free Redstar. Also in this cultivar, higher expression of flagellar genes (FlaI), which are recognized as PAMP (pathogen-associated molecular pattern) by the innate immune systems of plants, was noted. Additionally, several genes that have not yet been proven to play a role in the pathogenic abilities of E. amylovora were found to be differentially expressed in the two apple cultivars. Conclusions This RNA-seq analysis generated a novel dataset describing the transcriptional response of the lowly virulent strain of E. amylovora in susceptible and resistant apple cultivar. Most genes were regulated in the same way in both apple cultivars, but there were also some cultivar-specific responses suggesting that the environment in Free Redstar is more stressful for bacteria what can be the reason of their inability to infect of this cultivar. Among genes with the highest fold change in expression between experimental combinations or with the highest transcript abundance, there are many genes without ascribed functions, which have never been tested for their role in pathogenicity. Overall, this study provides the first transcriptional profile by RNA-seq of E. amylovora during infection of a host plant and insights into the transcriptional response of this pathogen in the environments of susceptible and resistant apple plants.

Abstract To study the expression of pathogenicity-related genes in Erwinia amylovora, seven candidate reference genes (ffh, glyA, gyrA, proC, pykA, recA, rpoB) were selected and validated with the following five different mathematic algorithms: geNorm, NormFinder, BestKeeper, the delta CT method and the RefFinder web-based tool. An overall comprehensive ranking output from each of the selected software programs revealed that proC and recA, followed by ffh and pykA, were the most stably expressed genes and can be recommended for the normalization of RT-qPCR data. A combination of the three reference genes, proC, recA and ffh, allowed for the accurate expression analysis of amsB and hrpN genes and the calculation of their fold change in E. amylovora after its infection of susceptible and resistant apple cultivars. To the best of our knowledge, this is the first study presenting a list of the most suitable reference genes for use in the relative quantification of target gene expression in E. amylovora in planta, selected on the basis of a multi-algorithm analysis.

Triggering the plant resistance induction phenomenon by chemical compounds, for example acibenzolar-S-methyl ester, has been known and described in scientific literature. Other benzothadiazole derivatives have been also described; however, their properties have not been sufficiently studied. The tested substance, N-methyl-N-methoxyamide-7-carboxybenzo(1.2.3)thiadiazole (BTHWA), is an amide derivative of benzothiadiazole, showing a stimulating effect on plant growth, apart from its plant resistance inducing activity. This article presents the impact of BTHWA, used solo and in the program with fungicides, on the strawberry plants development, fruits health, yield, and quality parameters of the crop. The results show that the combined use of BTHWA and fungicides had a positive impact on the plants health and fruit health and nutraceutical and nutritional composition of compounds when compared to the results obtained when strawberries were treated only with the BTHWA or the fungicide. As a result of BTHWA use, the partitioning of assimilates has changed, which directly translated into the results of the conducted experiments. A reduction in the respiration of the fruit during storage was also observed, possibly due to a reduced disease infestation and a lower dry matter content in the fruit. A correlation between the parameters determined during the experiment was found. The BTHWA mode of action was evidenced to be beneficial to strawberry plants and fruit.

The relationship between housekeeping and pathogenicity-related genes and virulence or avirulence towards the primary Malus resistance genes (R) has not been previously studied for Venturia inaequalis fungus, the causal agent of apple scab. In this study, the sequences of two housekeeping genes encoding elongation factor alpha (EF-1α) and β-tubulin and two previously unstudied effector genes of V. inaequalis from mannosidase and glucosidase families of 100 strains collected from apple cultivars with Rvi6, Rvi1, and Rvi17 and without known scab resistance genes were submitted to the analyses. Based on the phylogenetic and diversity data, as well as recombination analyses of the sequenced regions, we assessed the phylogenetic relationships and genetic structure of the pathogen within the species and the evolutionary forces that are currently acting upon this microorganism. The topology of the obtained phylograms demonstrates the lack of a relationship between the phylogenetic position of the strain and the host cultivar and the geographical origin or race of the strain. The isolates from different hosts were differentiated but did not form diagnosable, distinct phylogenetic groups. These results suggest that the analyzed genes may be too conserved to reflect the adaptation of pathogens to apple genotypes with different R genes; thus, they do not adequately reflect race discrimination. In contrast, based on variation and gene flow estimation, genetic divergence was observed among strains virulent to apple trees containing Rvi6. The results of this study confirmed a lack of free recombination between strains and demonstrated that the analyzed regions are in linkage disequilibrium and contain non-random polymorphisms associated with the strain.

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Strawberry is economically important and widely grown, but susceptible to a large variety of phytopathogenic organisms. Among them, Xanthomonas fragariae is a quarantine bacterial pathogen threatening strawberry productions by causing angular leaf spots. Using whole transcriptome sequencing, the gene expression of both plant and bacteria in planta was analyzed at two time points, 12 and 29 days post inoculation, in order to compare the pathogen and host response between the stages of early visible and of well-developed symptoms. Among 28,588 known genes in strawberry and 4046 known genes in X. fragariae expressed at both time points, a total of 361 plant and 144 bacterial genes were significantly differentially expressed, respectively. The identified higher expressed genes in the plants were pathogen-associated molecular pattern receptors and pathogenesis-related thaumatin encoding genes, whereas the more expressed early genes were related to chloroplast metabolism as well as photosynthesis related coding genes. Most X. fragariae genes involved in host interaction, recognition, and pathogenesis were lower expressed at late-phase infection. This study gives a first insight into the interaction of X. fragariae with its host. The strawberry plant changed gene expression in order to consistently adapt its metabolism with the progression of infection.

The effective and rapid detection of Fire Blight, an important bacterial disease caused by the quarantine pest E.amylovora, is crucial for today’s horticulture. This study explored the application of non-invasive proximal hyperspectral remote sensing (RS) in order to differentiate the healthy (H), infected (I) and dry (D) leaves of apple trees. Analysis of variance was employed in order to determine which hyperspectral narrow spectral bands exhibited the most significant differences. Spectral signatures for the range of 400–2500 nm were acquired with Thermo Scientific Evolution 220 and iS50NIR spectrometers. The selected spectral bands were then used to evaluate several RS indices, including ARI (Anthocyanin Reflectance Index), RDVI (Renormalized Difference Vegetation Index), MSR (Modified Simple Ratio) and NRI (Nitrogen Reflectance Index), for Fire Blight detection in apple tree leaves. Furthermore, a new index was proposed, namely QFI. The spectral indices were tested on apple trees infected by Fire Blight in a quarantine greenhouse. Results indicated that the short-wavelength infrared (SWIR) band located at 1450 nm was able to distinguish (I) and (H) leaves, while the SWIR band at 1900 nm differentiated all three leaf types. Moreover, tests using the Pearson correlation indicated that ARI, MSR and QFI exhibited the highest correlations with the infection progress. Our results prove that our hyperspectral remote sensing technique is able to differentiate (H), (I) and (D) leaves of apple trees for the reliable and precise detection of Fire Blight.

Standard program of plant protection against fire blight consists of use of management practices and chemical control method. Recently a new, non-conventional possibilities based on application of biocontrol agents (two biopreparations have been already introduced into practice: Bliteban A506 (Pseudomonas fluorescens) and BlossomBless (Pantoea agglomerans), plant extracts active against Erwinia amylovora (AkseBio containing extracts from Thymbra spicata and Biomit Plussz with extracts from various plant species and microelements) and resistance inducers (Regalis, Bion and plant extracts) are of great interest. Also plant transformation with resistance genes such as: hrpN (harpin), dpo (EPS depolymerase) and lytic protein genes (attacin E, cecropin SB-37, T4 lysozyme) is a promising perspective.

During the spring of 2003, gall symptoms on the roots and crowns of young raspberry plants cv. Vilamette were observed near Valjevo. Phytopathogenic bacteria were isolated from diseased plant samples. Based on the pathogenic, morphological, differential biochemicaland physiological characteristics, the isolated strains were identified as tumorigenic Agrobacterium radiobacter (biovar 1 Agrobacterium). In order to confirm the identity of isolated strains by polymerase chain reaction (PCR) primers complementary to tms2 genelocated on the Ti plasmid were used. In the first PCR protocol using a tms2F1 + tms2R2 primer pair, 617 bp products specific for tumorigenic Agrobacterium strains were amplified. The second PCR protocol, using a tms2F1 + tms2B primer pair, amplified the expected 458 bp products. On the basis of multiplex PCR with primers complementary to chromosomal gene coding for 23S rRNA, the isolated strains were classified as biovar 1 Agrobacterium (A. radiobacter). This is the first report of tumorigenic A. radiobacter on raspberry in Serbia.

Comparative genomics of several strains of Erwinia amylovora, a plant pathogenic bacterium causal agent of fire blight disease, revealed that its diversity is primarily attributable to the flexible genome comprised of plasmids. We recently identified and sequenced in full a novel 65.8 kb plasmid, called pEI70. Annotation revealed a lack of known virulence-related genes, but found evidence for a unique integrative conjugative element related to that of other plant and human pathogens. Comparative analyses using BLASTN showed that pEI70 is almost entirely included in plasmid pEB102 from E. billingiae, an epiphytic Erwinia of pome fruits, with sequence identities superior to 98%. A duplex PCR assay was developed to survey the prevalence of plasmid pEI70 and also that of pEA29, which had previously been described in several E. amylovora strains. Plasmid pEI70 was found widely dispersed across Europe with frequencies of 5-92%, but it was absent in E. amylovora analyzed populations from outside of Europe. Restriction analysis and hybridization demonstrated that this plasmid was identical in at least 13 strains. Curing E. amylovora strains of pEI70 reduced their aggressiveness on pear, and introducing pEI70 into low-aggressiveness strains lacking this plasmid increased symptoms development in this host. Discovery of this novel plasmid offers new insights into the biogeography, evolution and virulence determinants in E. amylovora.