Novel approach toward the understanding of genetic diversity based on the two types of amino acid repeats in Erwinia amylovora.

Erwinia amylovora is a notorious plant pathogenic bacterium of global concern that has devastated the apple and pear production industry worldwide. Nevertheless, the approaches available currently to understand the genetic diversity of E. amylovora remain unsatisfactory because of the lack of a trustworthy index and data covering the globally occurring E. amylovora strains; thus, their origin and distribution pattern remains ambiguous. Therefore, there is a growing need for robust approaches for obtaining this information via the comparison of the genomic structure of Amygdaloideae-infecting strains to understand their genetic diversity and distribution. Here, the whole-genome sequences of 245 E. amylovora strains available from the NCBI database were compared to identify intraspecific genes for use as an improved index for the simple classification of E. amylovora strains regarding their distribution. Finally, we discovered two kinds of strain-typing protein-encoding genes, i.e., the SAM-dependent methyltransferase and electron transport complex subunit RsxC. Interestingly, both of these proteins carried an amino acid repeat in these strains: SAM-dependent methyltransferase comprised a single-amino-acid repeat (asparagine), whereas RsxC carried a 40-amino-acid repeat, which was differentially distributed among the strains. These noteworthy findings and approaches may enable the exploration of the genetic diversity of E. amylovora from a global perspective.

Genome sequencing provides new insights on the distribution of Erwinia amylovora lineages in northern Italy.

Erwinia amylovora is a Gram-negative bacterium that colonizes a wide variety of plant species causing recurrent local outbreaks of fire blight in crops of the Rosaceae family. Recent genomic surveys have documented the limited genomic diversity of this species, possibly related to a recent evolutionary bottleneck and a strong correlation between geography and phylogenetic structure of the species. Despite its economic importance, little is known about the genetic variability of co-circulating strains during local outbreaks. Here, we report the genome sequences of 82 isolates of E. amylovora, collected from different host plants in a period of 16 years in Trentino, a small region in the Northeastern Italian Alps that has been characterized by recurrent outbreaks of fire blight in apple orchards. While the genome isolated before 2018 are closely related to other strains already present in Europe, we found a novel subclade composed only by isolates that were sampled starting from 2018 and demonstrate that the endemic population of this pathogen can be composed by mixture of strains.

Cyclic nucleotide gated channel genes (CNGCs) in Rosaceae: genome-wide annotation, evolution and the roles on Valsa canker resistance.

KEY MESSAGE: In Rosaceae, tandem duplication caused the drastic expansion of CNGC gene family Group I. The members MdCN11 and MdCN19 negatively regulate Valsa canker resistance. Apple (Malus domestica) and pear (Pyrus bretschneideri and P. communis) are important fruit crops in Rosaceae family but are suffering from threats of Valsa canker. Cyclic nucleotide-gated ion channels (CNGCs) take crucial roles in plant immune responses. In the present study, a total of 355 CNGCs was identified from 8 Rosaceae plants. Based on phylogenetic analysis, 540 CNGCs from 18 plants (8 in Rosaceae and 10 others) could be divided into four groups. Group I was greatly expanded in Rosaceae resulted from tandem duplications. A large number of cis-acting regulatory elements (cis-elements) responsive to signals from multiple stresses and hormones were identified in the promoter regions of CNGCs in Malus spp. and Pyrus spp. Expressions of most Group I members were obviously up-regulated in Valsa canker susceptible varieties but not in the resistant ones. Furthermore, overexpression of the MdCN11 and MdCN19 in both apple fruits and 'Duli' (P. betulifolia) suspension cells compromised Valsa canker resistance. Overexpression of MdCN11 induced expression of hypersensitive response (HR)-related genes. In conclusion, tandem duplication resulted in a drastic expansion of CNGC Group I members in Rosaceae. Among these, MdCN11 and MdCN19 negatively regulate the Valsa canker resistance via inducting HR.

A novel mitovirus isolated from the phytopathogenic fungus Botryosphaeria dothidea.

In this study, we isolated and determined the complete genome sequence of a novel mitovirus, "Botryosphaeria dothidea mitovirus 2" (BdMV2), from the phytopathogenic fungus Botryosphaeria dothidea isolate DT-5. BdMV2 has a genome 2,482 nt in length with an A+U content of 67%. The genome of BdMV2 contains a single large open reading frame (ORF) encoding an RNA-dependent RNA polymerase (RdRp) of 717 amino acids (aa) with a molecular mass of 81.86 kDa. A BLASTp comparison of the RdRp sequence showed the highest identity (66.67%) with that of Alternaria arborescens mitovirus 1 (AbMV1). Sequence comparisons and phylogenetic analysis revealed that BdMV2 is a new member of the genus Mitovirus of the family Mitoviridae.

A novel mycovirus isolated from the plant-pathogenic fungus Botryosphaeria dothidea.

A novel virus, Botryosphaeria dothidea bipartite mycovirus 1 (BdBMV1), was isolated from the plant-pathogenic fungus Botryosphaeria dothidea strain HNDT1, and the complete nucleotide sequence of its genome was determined. BdBMV1 consists of two genomic segments. The first segment is 1,976 bp in length and contains a single open reading frame (ORF) encoding the RNA-dependent RNA polymerase (RdRp) (68.95 kDa). The second segment is 1,786 bp in length and also contains a single ORF encoding a hypothetical protein of 35.19 kDa of unknown function. Based on the sequence of its RdRp, BdBMV1 is phylogenetically related to several other unclassified dsRNA mycoviruses, including Cryphonectria parasitica bipartite mycovirus 1 (CpBV1), and has a distant relationship to members of the family Partitiviridae.

Penicillium expansum: biology, omics, and management tools for a global postharvest pathogen causing blue mould of pome fruit.

Blue mould, caused primarily by Penicillium expansum, is a major threat to the global pome fruit industry, causing multimillion-dollar losses annually. The blue mould fungus negatively affects fruit quality, thereby reducing fresh fruit consumption, and significantly contributes to food loss. P. expansum also produces an array of mycotoxins that are detrimental to human health. Management options are limited and the emergence of fungicide-resistant Penicillium spp. makes disease management difficult, therefore new approaches and tools are needed to combat blue mould in storage. This species profile comprises a comprehensive literature review of this aggressive pathogen associated with pomes (apple, pear, quince), focusing on biology, mechanisms of disease, control, genomics, and the newest developments in disease management. TAXONOMY: Penicillium expansum Link 1809. Domain Eukaryota, Kingdom Fungi, Phylum Ascomycota, Subphylum Pezizomycotina, Class Eurotiomycetes, Subclass: Eurotiomycetidae, Order Eurotiales; Family Trichocomaceae, Genus Penicillium, Species expansum. BIOLOGY: A wide host range necrotrophic postharvest pathogen that requires a wound (e.g., stem pull, punctures, bruises, shoulder cracks) or natural openings (e.g., lenticel, stem end, calyx sinus) to gain ingress and infect. TOXINS: Patulin, citrinin, chaetoglobosins, communesins, roquefortine C, expansolides A and B, ochratoxin A, penitrem A, rubratoxin B, and penicillic acid. HOST RANGE: Primarily apples, European pear, Asian pear, medlar, and quince. Blue mould has also been reported on stone fruits (cherry, plum, peach), small fruits (grape, strawberry, kiwi), and hazel nut. DISEASE SYMPTOMS: Blue mould initially appears as light tan to dark brown circular lesions with a defined margin between the decayed and healthy tissues. The decayed tissue is soft and watery, and blue-green spore masses appear on the decayed area, starting at the infection site and radiating outward as the decayed area ages. DISEASE CONTROL: Preharvest fungicides with postharvest activity and postharvest fungicides are primarily used to control decay. Orchard and packinghouse sanitation methods are also critical components of an integrated pest management strategy. USEFUL WEBSITES: Penn State Tree Fruit Production Guide (https://extension.psu.edu/forage-and-food-crops/fruit), Washington State Comprehensive Tree Fruit (http://treefruit.wsu.edu/crop-protection/disease-management/blue-mold/), The Apple Rot Doctor (https://waynejurick.wixsite.com/applerotdr), penicillium expansum genome sequences and resources (https://www.ncbi.nlm.nih.gov/genome/browse/#!/eukaryotes/11336/).

The role of plant growth-promoting rhizobacteria (PGPR) in improving iron acquisition by altering physiological and molecular responses in quince seedlings.

Due to insoluble iron (Fe) sources in soil, limited Fe availability leads to the disruption of the photosynthetic apparatus; this affects the growth and productivity of plants such as quince (Cydonia oblonga) that are very sensitive to low Fe content. Plant growth-promoting rhizobacteria (PGPR) play an important role in the regulation of Fe uptake under its limited availability. Therefore, in this research, two PGPR (Pseudomonas fluorescens and Microccucuce yunnanensis), at two Fe levels [50 µM (Fe-sufficiency) or 5 µM (Fe-deficiency)], were used to investigate the impact of the given bacteria on improving the acquisition of Fe in quince seedlings. Upon Fe-deficiency, the highest shoot and root biomass (7.14 and 6.04 g plant-1 respectively), the greatest chlorophyll concentration (0.89 mg g-1FW), and the largest Fe concentrations in roots and shoots (30% and 48.7%, respectively) were shown in the quince treated with M. yunnanensis. Both PGPR increased the root citric acid and the phenolic compound concentration. Two days after Fe-deficiency and PGPR treatments, a 1.5- fold increase, was observed in the expression of HA7. The highest PAL1 gene expression and the greatest PAL activity (95.76 µmol cinnamic acid g-1FW) were obtained from the M. yunnanensis treatment. The expression of the FRO2 gene was also affected by Fe-deficiency and PGPR treatments, resulting in an increase in the FCR activity and a surge in the Fe concentrations of leaves and roots. It could, therefore, be concluded that the PGPR modulated Fe acquisition in the quince seedlings upon Fe-deficiency by influencing the physico-chemical and molecular responses.

Naganishia floricola sp. nov., a novel basidiomycetous yeast species isolated from flowers of Sorbaria sorbifolia.

Two yeast strains representing a novel species in the basidiomycetous yeast genus Naganishia were isolated from flowers of Sorbaria sorbifolia collected in Beijing Olympic Forest Park, PR China. Results of multi-gene phylogenetic analysis indicated that the two strains were closely related to the type strains of Naganishia bhutanensis (CBS 6294T) and Naganishia antarctica (CBS 7687T). However, the new isolates differed from N. bhutanensis CBS 6294T by 1.79 % sequence divergence in the D1/D2 domain (11 nt substitutions and three indels), and 2.42 % (15 nt differences and one indel) to N. antarctica CBS 7687T. In the ITS region, the new isolates showed 1.15 % divergence (7 nt substitutions and one indel) to N. bhutanensis CBS 6294T and 0.92 % divergence (5 nt substitutions and no indels) to N. antarctica CBS 7687T. A phylogenetic analysis employing the sequences of six genes (D1/D2 domain of large subunit rDNA, ITS, small subunit rDNA, two subunits of the RNA polymerase II and elongation factor-1α) indicated that the novel species belonged to the genus Naganishia and formed a well-supported clade with N. bhutanensis, N. antarctica and N. indica. Moreover, the two strains differed from their closest relatives by the ability to grow on distinct carbon and nitrogen sources and ability to grow at 30 °C. On the basis of these findings, we propose a novel species in the genus Naganishia (Filobasidiales), Naganishia floricola sp. nov. (holotype CGMCC 2.5856).

Isolation and characterization of Hena1 - a novel Erwinia amylovora bacteriophage.

Fire blight, caused by plant pathogenic bacterium Erwinia amylovora, is one of the most important diseases of Rosaceae plants. Due to the lack of effective control measures, fire blight infections pose a recurrent threat on agricultural production worldwide. Recently, bacterial viruses, or bacteriophages, have been proposed as environmentally friendly natural antimicrobial agents for fire blight control. Here, we isolated a novel bacteriophage Hena1 with activity against E. amylovora. Further analysis revealed that Hena1 is a narrow-host-range lytic phage belonging to Myoviridae family. Its genome consists of a linear 148,842 bp dsDNA (48.42% GC content) encoding 240 ORFs and 23 tRNA genes. Based on virion structure and genomic composition, Hena1 was classified as a new species of bacteriophage subfamily Vequintavirinae. The comprehensive analysis of Hena1 genome may provide further insights into evolution of bacteriophages infecting plant pathogenic bacteria.

Highly Contiguous Genome Resource of Colletotrichum fructicola Generated Using Long-Read Sequencing.

Colletotrichum fructicola is a plant-pathogenic fungus with a broad host range. It causes significant losses to important crops, including apple, pear, strawberry, and other Rosaceae and non-Rosaceae species. To date, two short read-based C. fructicola genomes are publicly available, but both are fragmented. In this study, we re-sequenced the genome of C. fructicola using nanopore long-read technology and refined the assembly with Hi-C map data. The resulting high-quality assembly is an important resource for further comparative and experimental studies with C. fructicola.

Rhizospheric soil fungal community patterns of Duchesnea indica in response to altitude gradient in Yunnan, southwest China.

The magnitude of the impact of altitude gradient on microbial community and diversity has been studied in recent decades. Whereas bacteria have been the focus of most studies, fungi have been given relatively less attention. As a vital part of the macro- and microscopic ecosystem, rhizosphere fungi play a key role in organic matter decomposition and relative abundance of plant species and have an impact on plant growth and development. Using Duchesnea indica as the host plant, we examined the rhizosphere soil fungal community patterns across the altitude gradient in 15 sites of Yunnan province by sequencing the fungal ITS2 region with the Illumina MiSeq platform. We determined the fungal community composition and structure. We found that, surprisingly, rhizosphere soil fungal diversity of D. indica increased with altitudinal gradient. There was a slight difference in diversity between samples from high- and medium-altitude sites, with medium-altitude sites having the greater diversity. Furthermore, the rhizosphere soil fungal community composition and structure kept changing along the altitudinal gradient. Taxonomic results showed that the extent of phylum diversity was greatest at high-altitude sites, with Ascomycota, Basidiomycota, Zygomycota, and Glomeromycota as the most dominant fungal phyla.

Diversity of yeasts in the soil adjacent to fruit trees of the Rosaceae family.

Yeasts are common constituents of different types of soil. Their diversity depends on the season, the type and depth of the soil, the plant species, and the locality. In this study, diversity of yeasts isolated from the soil adjacent to five fruit trees (apple, appricot, peach, pear, and plum) in two localities (in Slovakia) in four sampling periods was examined. Our results demonstrated differences in the species richness and evenness among the yeast populations, which inhabited the soil beneath individual fruit tree species in both localities. Altogether, 32 ascomycetous and 27 basidiomycetous yeast species were discovered. The highest species richness was found in the soil adjacent to the apricot trees. Galactomyces candidum, Metschnikowia pulcherrima, Hanseniaspora uvarum, Schwanniomyces capriottii, and Tausonia pullulans, as well as the genus Apiotrichum, were present in soil samples in all samplings. Two species of the genus Holtermanniella (H. festucosa and H. takashimae) were exclusively isolated during Sampling IV in April. Cyberlindnera spp., Clavispora reshetovae, S. capriottii, and Trichosporon asahii were found only in one of two localities. Ascomycetous yeasts were present more frequently than their basidiomycetous counterparts in the three samplings (one in June and two in October); they formed from 65.6% to 70.8% of the total yeast population, whereas basidiomycetous yeasts prevailed in the April sampling (61.2%).

Alternative Molecular-Based Diagnostic Methods of Plant Pathogenic Fungi Affecting Berry Crops-A Review.

Increasing consumer awareness of potentially harmful pesticides used in conventional agriculture has prompted organic farming to become notably more prevalent in recent decades. Central European countries are some of the most important producers of blueberries, raspberries and strawberries in the world and organic cultivation methods for these fruits have a significant market share. Fungal pathogens are considered to be the most significant threat to organic crops of berries, causing serious economic losses and reducing yields. In order to ameliorate the harmful effects of pathogenic fungi on cultivations, the application of rapid and effective identification methods is essential. At present, various molecular methods are applied for fungal species recognition, such as PCR, qPCR, LAMP and NGS.

A genome-wide analysis of desferrioxamine mediated iron uptake in Erwinia spp. reveals genes exclusive of the Rosaceae infecting strains.

Erwinia amylovora is the etiological agent of fire blight, a devastating disease which is a global threat to commercial apple and pear production. The Erwinia genus includes a wide range of different species belonging to plant pathogens, epiphytes and even opportunistic human pathogens. The aim of the present study is to understand, within the Erwinia genus, the genetic differences between phytopathogenic strains and those strains not reported to be phytopathogenic. The genes related to the hydroxamate siderophores iron uptake have been considered due to their potential druggability. In E. amylovora siderophore-mediated iron acquisition plays a relevant role in the progression of Fire blight. Here we analyzed the taxonomic relations within Erwinia genus and the relevance of the genes related to the siderophore-mediated iron uptake pathway. The results of this study highlight the presence of a well-defined sub-group of Rosaceae infecting species taxonomically and genetically related with a high number of conserved core genes. The analysis of the complete ferrioxamine transport system has led to the identification of two genes exclusively present in the Rosaceae infecting strains.

Draft Genome Resources for the Phytopathogenic Fungi Monilinia fructicola, M. fructigena, M. polystroma, and M. laxa, the Causal Agents of Brown Rot.

Fungi in the genus Monilinia cause brown rot disease of stone and pome fruits. Here, we report the draft genome assemblies of four important phytopathogenic species: M. fructicola, M. fructigena, M. polystroma, and M. laxa. The draft genome assemblies were 39 Mb (M. fructigena), 42 Mb (M. laxa), 43 Mb (M. fructicola), and 45 Mb (M. polystroma) with as few as 550 contigs (M. laxa). These are the first draft genome resources publicly available for M. laxa, M. fructigena, and M. polystroma.

Structural and functional analysis of Erwinia amylovora SrlD. The first crystal structure of a sorbitol-6-phosphate 2-dehydrogenase.

Sorbitol-6-phosphate 2-dehydrogenases (S6PDH) catalyze the interconversion of d-sorbitol 6-phosphate to d-fructose 6-phosphate. In the plant pathogen Erwinia amylovora the S6PDH SrlD is used by the bacterium to utilize sorbitol, which is used for carbohydrate transport in the host plants belonging to the Amygdaloideae subfamily (e.g., apple, pear, and quince). We have determined the crystal structure of S6PDH SrlD at 1.84 Šresolution, which is the first structure of an EC 1.1.1.140 enzyme. Kinetic data show that SrlD is much faster at oxidizing d-sorbitol 6-phosphate than in reducing d-fructose 6-phosphate, however, equilibrium analysis revealed that only part of the d-sorbitol 6-phosphate present in the in vitro environment is converted into d-fructose 6-phosphate. The comparison of the structures of SrlD and Rhodobacter sphaeroides sorbitol dehydrogenase showed that the tetrameric quaternary structure, the catalytic residues and a conserved aspartate residue that confers specificity for NAD+ over NADP+ are preserved. Analysis of the SrlD cofactor and substrate binding sites identified residues important for the formation of the complex with cofactor and substrate and in particular the role of Lys42 in selectivity towards the phospho-substrate. The comparison of SrlD backbone with the backbone of 302 short-chain dehydrogenases/reductases showed the conservation of the protein core and identified the variable parts. The SrlD sequence was compared with 500 S6PDH sequences selected by homology revealing that the C-terminal part is more conserved than the N-terminal, the consensus of the catalytic tetrad (Y[SN]AGXA) and a not previously described consensus for the NAD(H) binding.

A complete structural characterization of the desferrioxamine E biosynthetic pathway from the fire blight pathogen Erwinia amylovora.

The Gram-negative bacterium Erwinia amylovora is the etiological agent of fire blight, a devastating disease which affects Rosaceae such as apple, pear and quince. The siderophore desferrioxamine E plays an important role in bacterial pathogenesis by scavenging iron from the host. DfoJ, DfoA and DfoC are the enzymes responsible for desferrioxamine production starting from lysine. We have determined the crystal structures of each enzyme in the desferrioxamine E pathway and demonstrate that the biosynthesis involves the concerted action of DfoJ, followed by DfoA and lastly DfoC. These data provide the first crystal structures of a Group II pyridoxal-dependent lysine decarboxylase, a cadaverine monooxygenase and a desferrioxamine synthetase. DfoJ is a homodimer made up of three domains. Each monomer contributes to the completion of the active site, which is positioned at the dimer interface. DfoA is the first structure of a cadaverine monooxygenase. It forms homotetramers whose subunits are built by two domains: one for FAD and one for NADP+ binding, the latter of which is formed by two subdomains. We propose a model for substrate binding and the role of residues 43-47 as gate keepers for FAD binding and the role of Arg97 in cofactors turnover. DfoC is the first structure of a desferrioxamine synthetase and the first of a multi-enzyme siderophore synthetase coupling an acyltransferase domain with a Non-Ribosomal Peptide Synthetase (NRPS)-Independent Siderophore domain (NIS).

Diversity of plant defense elicitor peptides within the Rosaceae.

BACKGROUND: Plant elicitor peptides (Peps) are endogenous molecules that induce and amplify the first line of inducible plant defense, known as pattern-triggered immunity, contributing to protect plants against attack by bacteria, fungi and herbivores. Pep topic application and transgenic expression have been found to enhance disease resistance in a small number of model plant-pathogen systems. The action of Peps relies on perception by specific receptors, so displaying a family-specific activity. Recently, the presence and activity of Peps within the Rosaceae has been demonstrated. Here we characterized the population of Pep sequences within the economically important plant family of Rosaceae, with special emphasis on the Amygdaleae and Pyreae tribes, which include the most relevant edible species such as apple, pear and peach, and numerous ornamental and wild species (e.g. photinia, firethorn and hawthorn). RESULTS: The systematic experimental search for Pep and the corresponding precursor PROPEP sequences within 36 Amygdaleae and Pyreae species, and 100 cultivars had a highly homogeneous pattern, with two tribe-specific Pep types per plant, i.e. Pep1 and Pep2 (Amygdaleae) or Pep3 and Pep4 (Pyreae). Pep2 and Pep3 are highly conserved, reaching identity percentages similar to those of genes used in plant phylogenetic analyses, while Pep1 and Pep4 are somewhat more variable, with similar values to the corresponding PROPEPs. In contrast to Pep3 and Pep4, Pep1 and Pep2 sequences of different species paralleled their phylogenetic relationships, and putative ancestor sequences were identified. The large amount of sequences allowed refining of a C-terminal consensus sequence that would support the protective activity of Pep1-4 in a Prunus spp. and Xanthomonas arboricola pv. pruni system. Moreover, tribe-specific consensus sequences were deduced at the center and C-terminal regions of Peps, which might explain the higher protection efficiencies described upon topic treatments with Peps from the same tribe. CONCLUSIONS: The present study substantially enhances the knowledge on Peps within the Amygdaleae and Pyreae species. It can be the basis to design and fine-tune new control tools against important plant pathogens affecting Prunus, Pyrus and Malus species.

Comparative genomics of Spiraeoideae-infecting Erwinia amylovora strains provides novel insight to genetic diversity and identifies the genetic basis of a low-virulence strain.

Erwinia amylovora is the causal agent of fire blight, one of the most devastating diseases of apple and pear. Erwinia amylovora is thought to have originated in North America and has now spread to at least 50 countries worldwide. An understanding of the diversity of the pathogen population and the transmission to different geographical regions is important for the future mitigation of this disease. In this research, we performed an expanded comparative genomic study of the Spiraeoideae-infecting (SI) E. amylovora population in North America and Europe. We discovered that, although still highly homogeneous, the genetic diversity of 30 E. amylovora genomes examined was about 30 times higher than previously determined. These isolates belong to four distinct clades, three of which display geographical clustering and one of which contains strains from various geographical locations ('Widely Prevalent' clade). Furthermore, we revealed that strains from the Widely Prevalent clade displayed a higher level of recombination with strains from a clade strictly from the eastern USA, which suggests that the Widely Prevalent clade probably originated from the eastern USA before it spread to other locations. Finally, we detected variations in virulence in the SI E. amylovora strains on immature pear, and identified the genetic basis of one of the low-virulence strains as being caused by a single nucleotide polymorphism in hfq, a gene encoding an important virulence regulator. Our results provide insights into the population structure, distribution and evolution of SI E. amylovora in North America and Europe.

Conservation of Erwinia amylovora pathogenicity-relevant genes among Erwinia genomes.

The Erwinia genus comprises species that are plant pathogens, non-pathogen, epiphytes, and opportunistic human pathogens. Within the genus, Erwinia amylovora ranks among the top 10 plant pathogenic bacteria. It causes the fire blight disease and is a global threat to commercial apple and pear production. We analyzed the presence/absence of the E. amylovora genes reported to be important for pathogenicity towards Rosaceae within various Erwinia strains genomes. This simple bottom-up approach, allowed us to correlate the analyzed genes to pathogenicity, host specificity, and make useful considerations to drive targeted studies.